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Chemopreventive and chemotherapeutic mechanisms of
sphingoilpid metabolites in human colon cancer cells and
breast stem, normal, and tumorigenic cells

presented by

Eun Hyun Ahn

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PhD. degree in Human Nutrition- Environmental
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6/01 c:/ClRC/DateDua.p65-p.15

Chemopreventive and chemotherapeutic mechanisms of sphingolipid metabolites
in human colon cancer cells and breast stem, normal, and tumorigenic cells

By

Eun HyunAhn

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

in
Human Nutrition-Environmental Toxicology
Department of Food Science & Human Nutrition

and
Institute of Environmental Toxicology

2003

ABSTRACT
CHEMOPREVENTIVE AND CHEMOTHERAPEUTIC MECHANISMS OF

SPHINGOLIPID METABOLIT ES IN HUMAN COLON CANCER CELLS AND
BREAST STEM, NORMAL, AND TUMORIGENIC CELLS

By
Eun Hyun Ahn

The effects of the sphingolipid metabolites (sphingosine, sphinganine, ceramide,
and dihydroceramide) on proliferation and differentiation in human normal epithelial
cells versus tumorigenic cells and stem cells have not previously been compared. This
dissertation research addressed these questions using following cell culture models: HT -
29 and HCT-l 16 human colon cancer epithelial cells, human breast epithelial cells
(HBEC) developed from tissues of healthy women obtained during reduction
mammoplasty (type I HBEC with stem cell characteristics as target cells for breast
carcinogenesis and type H HBEC with basal epithelial cell phenotypes as breast normal
cells) and transformed tumorigenic type I HBEC as breast cancer cells.

The objectives of the present study were to: I) investigate the effects of sphingoid
bases (sphingosine and sphinganine), ceramide, and dihydroceramide on proliferation,
cell cycle, and apoptosis of HT-29 and HGT-116 cells; 2) determine the effects of
sphinganine on major isoforms of mitogen activated protein kinases (ERK, JNK, and
p38) and AKT (protein kinase B) in HT-29 cells; 3) evaluate the effects of sphingoid
bases on proliferation, cell cycle, and apoptosis of tumorigenic type I HBEC and type I
HBEC compared to type II HBEC; and 4) determine the effects of sphingoid bases on the

ability of type I HBEC to differentiate to type H HBEC.

Data fiom HT—29 and HGT-116 cells indicate that sphingosine, sphinganine, and
Cz-ceramide inhibited the growth and arrested cell cycle at Gz/M phase. Sphingosine,
sphinganine, and Cz-ceramide at 20—50 uM induced apoptosis, with sphinganine being
the most potent of the metabolites studied. In contrast, Cz-dihydroceramide had no
effect, suggesting that the 4,5-trans double bond is necessary for the inhibitory effects of
Cz-ceramide, but not for inhibitory effects of sphingoid bases. Sphinganine at an
apoptosis-inducing concentration (35 uM) caused early and strong activation of
JNKZ/JNKI and p38 and early inhibition of AKT, with minimal effects on activation of
ERKl/ERKZ in HT-29 cells.

Results fi'om HBEC show that sphinganine more potently inhibited the growth
and induced apoptosis of tumorigenic type I I-[BEC than sphingosine (ICso for
sphinganine 4 uM; sphingosine 6.4 pM). Sphingoid bases (8-10 uM) arrested cell cycle
at G2/M with reductions in S and 60/6. phases. Sphinganine also more potently inhibited
the growth and caused death of type I HBEC than sphingosine, while the same
concentrations of both sphinganine and sphingosine had minor effects on type II HBEC.
At concentrations (0.05-0.5uM), which are below the growth inhibitory range, sphingoid
bases induced differentiation of type I HBEC to type II HBEC, as detected with
concomitant expression of the novel tumor suppressor protein, maspin, in type II HBEC.

In conclusion, sphingoid bases and ceramide might possess chemotherapeutic
properties against colon cancer, with sphinganine being the most potent. Activation of
JNK and p38 and inhibition of AKT might mediate sphinganine-induced apoptosis of
colon cancer cells. Data indicate the potential for sphingoid bases to be employed as

chemotherapeutic and chemopreventive agents against human breast cancer.

Dedicated to
my mother Sun Yul Yu, my late father Byung Hyun Ahn and my older brothers Suk Yul
Ahn and Hyoung You] Ahn who show endless love toward me and God who created the

world and human beings and continuously teaches us to love one another.

iv

ACKNOWLEDGEMENT

I sincerely thank my precious advisor Dr. Joseph J. Schroeder for his capable
advise, warm encouragement, guidance, and patience throughout my Ph.D. program. He
is always willing to spend his time and share his life for his graduate students. He has
shown me what it means to walk with maturity and to show genuine care toward others
and mutual respect under any circumstances. He has taught me to work professionally
while pursuing a team spirit and practicing kindness and hospitality. All of these nice
and excellent aspects are the attributes I wish to pass unto the next generation.

I am grateful to my mentor and Ph. D. guidance committee Dr. Chia-Cheng
Chang for his excellent and clear advice, insights on science, faithfulness, thoughtfulness,
composure, and patience. I was fortunate to research with human breast epithelial cells
(stem cells, normal cells, and tumorigenic cells) Dr. Chang’s group has developed.
While I worked at Dr. Chang’s lab for nine months, I finished the last part of my second
research project of colon cancer and started and completed the last breast cancer project.
Dr. Chang was available day and night to provide close and professional advice. I was
honored to learn how to culture human breast epithelial cells directly from Dr. Chang.

I thank my academic advisor Dr. Dale R. Romsos for his timely advice, support,
patience, and wisdom. In the midst of his busy schedule, he spent time with me on a
regular basis during the last stage of my Ph.D. program, which helped me to focus on my
research and to plan my career after the graduation. He provided helpful suggestions and
objective perspectives on my breast cancer project. I thank Drs. Maurice R. Bennink and

Leslie D. Bourquin who were members of my Ph. D. guidance committee for helpful

comments and encouragement. I am grateful to Dr. Brad Upham for participating in my
Ph.D. dissertation defense evaluation process and for his helpful suggestions and warm
smile. My appreciation extends to Dr. James E. Trosko for his encouragement and
support. It was a great opportunity for me to get to know Dr. Trosko during the last
period of my Ph.D. program.

I thank for their technical advice in various areas the following: Drs. Louis King
and Zahidul Islam on flow cytometry analysis, Dr. Youngjoo Chung on western blotting,
Dr. Ludmila Roze and Rebecca Uzarski on fluorescence microscopic detection of
apoptotic cells, and Therese Soderdahl on immunofluorescence staining. Drs. Chung and
Islam’s friendship made my journey of Ph.D. program more joyful. I thank Drs. James
Pestka and John Linz for providing access to their experimental instruments. I thank
Michelle Komosinski and Mark Hagerty for excellent technical assistance on quantitation
of colonies of human breast epithelial cells.

I am grateful to fellow graduate students Min Sun Kim, Eun Hee Lee, Chi Zhang,
and Hong Yang for their companionship, fiiendship, and enthusiasm of science while we
worked together in the same laboratory during my Ph.D. and MS. program. I thank
people (Maki Satoh, Gina Brooks, Therese Soderdahl, Dr. Mei-Hui Tai, Chad Coe,
Shauna Matlen, Andrea Satoh, and short-term visiting scholars) at Drs. Chang, Trosko,
and Upham’s lab for their companionship and fi'iendship. I thank previous and/or current
staff at our dept (Mary Schneider, Debie Klein, Shelli Pfeifer, and Sharon Hart for their
service.

I thank Dr. Won 0. Song for her loving encouragement and advice since I came

to MI, USA. I appreciate Dr. Mun Shig Son at University of Vermont for his father-like

vi

encouragement. I thank fellow graduate students at our department (Dr. loo-Won Lee,
Seung-Yeon Lee, Dr. Micheal Miller, Dr. Soc-Young Kang, Yikyung Park, Chia-F en
Chung, Jean Kerver, and Sungyong Hong, Dr. Hanseung Shin, Dr. Sangjin Chung, Saori
Obayashi, Dr. Miyoung Jang, and many others) for fi'iendship.

I thank my best fiiends J ungae Nam and Mia Chung, my relatives (J ieun Kim,
Bockyul You, Woosung Lee, Ingoo Lee, Hyungoo Lee, Keumyul you, Youhae Kim,
J ihoon Kim, Soonyul You, and many others), my precious and close fiiends Dr. Hae-
Sook Park and Susan Park Song, and sincere fi'iends at University Reformed Church in
East Lansing, MI (Dr. Irvine Widders, Romelia Widders, Gary Brinkman, Jaeun Joo,
Kisoon Kim, Stacey Bieler, Dr. Tom Bieler, and pastor Tom Stark), and nice and old
fi'iends Yeonsook Lim, Yoonyoung Yang, and Oakja Back for encouragement. I am
grateful to my American host family (Alice, Ian, and Stanely Morgan and Tory, Dale,
Benett Foster) for their prayer in love. I thank the beautiful children with special needs at
the Lansing Area Parent’s Respite Center, MI for the time we shared together. I thank Dr.
David A. Talmage at Columbia University for his great patience, encouragement, and

support during my final stage of dissertation.

vii

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS

CHAPTER 1. Introduction
Rationale of this study
Objective and hypothesis

CHAPTER 2. Literature review

A. Cancer
A.1. Cancer as a disease of proliferation and differentiation
A.2. Cancer through mutations
A.3. Environmental factors and cancer

B. Colon cancer

8.]. Colon cancer incidence

B.2. Colon cancer etiology and risk factors
32.]. Colon carcinoma development
B.2.2. Family history as a risk factor for colon cancer
B.2.3. Genes associated with high risk for colon cancer
B.2.4. Other risk factors for colon cancer

33. Dietary factors in colon cancer
B.3.l . Dietary fat
B.3.2. Caloric restriction
B.3.3. Dairy products
B.3.4. Soy products

C. Breast cancer

C. l . Breast cancer incidence

C.2. Breast cancer etiology and risk factors
C.2.1. Family history as a risk factor for breast cancer
C.2.2. Genes associated with high risk for breast cancer
C.2.3. Other risk factors for breast cancer

C.3. Dietary factors in breast cancer
C.3.1. Dietary fat
C.3.2. Caloric restriction
C.3.3. Dairy products
C.3.4. Soy products

viii

page
xiii
xiv
xix

UuNtd

10
11
11
12
14

14

14
14

18

22
22
22

26

D. Cancer chemotherapy and chemoprevention

D. l . Chemotherapy
D.1.l. Definition of chemotherapy
D. 1 .2. Goals for use of chemotherapy
D.2. Chemoprevention
D.2.l . Definition of chemoprevention
D.2.2. Characteristics of the chemopreventive agents
D.2.3. Defense mechanisms for damaged cells
D.3. Induction of apoptosis by chemotherapeutic and/or chemopreventive
agents
D.3.1. Removal of unwanted cells via apoptosis
D.3.2. Induction of apoptosis by chemotherapeutic agents
D.3.3. Induction of apoptosis by chemopreventive agents
D.4. Roles of stem cell differentiation for cancer prevention
D.4.l . Characteristics of stem cells
D.4.2. Differentiation genes
D.4.3. A mechanism of CAMP-induced differentiation
D.5. Suppression of telomerase activity as a novel chemotherapy
D.5.1. Telomeres
D.5.2. Telomerase
D.5.3. High telomerase activity in cancers
D.5.4. Roles of telomerase activity on proliferation and differentiation
D.5.5. Suppression of telomerase activity as a novel chemotherapy
D.5.6. Regulation of telomerase

E. Sphingolipids as possible chemotherapeutic and chemopreventive agents

E.l. Sphingolipids structures
E.2. Sphingolipid signaling pathway: Ceramide generation caused by
sphingomyelinase activation
E.3. Sphingolipids occurrence and dietary sphingolipids
E.4. Prevention of colon carcinogenesis by dietary sphingomyelin
E.4.1. Uptake and metabolism of sphingolipids
E.4.2. Inhibition of colon carcinogenesis by sphingolipids in rodent
models
E.5. Inhibition of human breast cancer cell growth by sphingolipids
E.6. Ceramide and sphingoid bases as mediators for various anticarcinogenic
agents
E.7. Induction of apoptosis by ceramide and sphingoid bases
E.8. Regulation of apoptosis by Bel-2 family genes
E.9. Regulation of apoptosis by mitogen activated protein kinases (MAPKs)
E.9.l. Major isoforms of MAPKs (ERK, JNK, and p38)
E.9.2. Roles of ERK, JNK, and p38
E.9.3. Activations of ERK, JNK, and p38
E.9.4. Apoptosis triggered by imbalance between ERK, JNK, and p38
E.9.5. Regulation of MAPKs by sphingolipids

3O
30

31

33

35

37

41
41
42

45
45

47
48

49
49
5 l

E.10. Regulation of apoptosis by AKT
E.10.1. AKT activation as cell survival and anti-apoptotic signal
E.10.2. Activation/phosphorylation of AKT at thr308 and ser473
E.10.2. Regulation of AKT by sphingolipids
E.11. Involvements of calcium or calmodulin in proliferation and apoptosis
E.12. Summary: Mechanisms of actions of sphingolipids in proliferation,
apoptosis, and differentiation

F. Cellular models for chemotherapy and chemoprevention studies
F .1. Human colon cancer cells
RI .1. HT-29 cells
F.1.2. HCT-116 cells
F .1 .3. HT-29 and HCT-l 16 cells for chemotherapy studies
F.2. Human breast epithelial cells (HBEC)
F .2.]. Type I HBEC as normal stem cells
F.2.2. Transformed tumorigenic type I HBEC
F.2.3. Type II HBEC as normal cells
F.2.4. HBEC for chemotherapy and chemoprevention studies
F.2.5. Criteria for identification of chemotherapeutic and
chem0preventive agents in HBEC

56

59
61

62
62

CHAPTER 3. Sphingoid bases and ceramide induce apoptosis in HT-29 and
HCT-ll6 human colon cancer cells

A. ABSTRACT
B. INTRODUCTION
C. MATERIALS AND METHODS
C. l . Chemicals and reagents
C.2. Sphingolipid treatment
C.3. Cell culture
C.4. Assessment of cell proliferation
C.5. Fluorescence microscopic detection of apoptotic cells
C.6. Isolation and quantitation of fiagmented DNA
C.7. Flow cytometric analysis of cell cycle and population
D. RESULTS
D. l . Sphingosine, sphinganine, and Cz-ceramide inhibit growth and
cause death of HT-29 and HCT-116 human colon cancer cells
D.2. Fluorescence microscopic detection of apoptotic cells in HT-29 and
HCT-l 16 cells
D.3. Quantitation of fragmented DNA using diphenylamine assay
D.4. Flow cytometric analysis of cell cycle of HT—29 and HGT-116 cells
E. DISCUSSION

CHAPTER 4. Sphinganine causes early activation of ERK, JNK, and p38
and early inhibition of AKT activation in HT-29 human colon cancer cells

A. ABSTRACT
B. INTRODUCTION
C. MATERIALS AND METHODS
C.1. Sphinganine preparation
C.2. Cell culture
C.3. Isolation and quantitation of cellular protein
C.4. Western blot analysis for protein expression of ERKl/ERKZ,
JNK2/JNK1, p3 8, and AKT and phosphorylated forms of
ERKl/ERKZ, JNK2/JNK1, p38, AKT-ser473, and AKT-thr308
C.5. Densitometry
D. RESULTS
D. 1 . Sphinganine causes early activation of ERKl/ERK2
D.2. Sphinganine causes early activation of JNK2/JNK1
D.3. Sphinganine causes early activation of p38 MAP kinase
D.4. Sphinganine causes early inhibition of AKT-ser473 and AKT-
thr308
E. DISCUSSION

xi

72

73
75
79

84

95

103

104
105
108

111

117

CHAPTER 5.

Evaluation of sphinganine and sphingosine as breast cancer 121

chemotherapeutic and chemopreventive agents using human breast stem,
normal, and tumorigenic cell models

A. ABSTRACT 122
B. INTRODUCTION 124
C. MATERIALS AND METHODS 129

CI.
C2.
C3.
C.4.

C5.

C6.

C7.
Q8.
Q9.

Sphingolipids

HBEC culture media

Development of human breast epithelial cells (HBEC) from
reduction mammoplasty tissues of healthy females

Separation of type I HBEC with stem cell characteristics and type
II HBEC

Derivation of in vitro neoplastically transformed HBEC lines
from HBEC, normal human mammary epithelial culture fiom
reduction mamoplasty tissues

Culture of type I HBEC, tumorigenic type I HBEC, and type H
HBEC

Assessment of cell proliferation

Flow cytometry of cell cycle and population

Assessment of type I HBEC differentiation

C.10. Immunofluorescence staining
C.11. Statistical analyses

D. RESULTS
D.1.

D2.

D3.

D4.

D5.

D6.

137
Sphinganine more potently inhibits the growth and causes death of
tumorigenic type I HBEC than sphingosine
Sphinganine more potently induces apoptosis of tumorigenic type I
HBEC than sphingosine. Both sphingoid bases arrest cell cycle at
Gle with reductions in S and Go/Gl phases.
Sphinganine more potently inhibits the growth and causes death of
type I HBEC than sphingosine
Type II HBEC are less sensitive to growth inhibitory effects of
sphinganine and sphingosine than tumorigenic type I HBEC and
type I HBEC
Sphinganine and sphingosine induce differentiation of type I HBEC
to type H HBEC
Sphinganine increases the expression of a tumor suppressor protein,
maspin during induction of differentiation of type I stem HBEC to
type II HBEC

E. DISCUSSION 154

CHAPTER 6.
APPENDIX

Conclusion and significance of the study 162
168

REFERENCES l 82

xii

LIST OF TABLES

Page
CHAPTER 2
Table 2.1. Modes of cell death: Distinct features of necrosis and 34
apoptosis

Table 2.2. Different effects of W7 on apoptosis induced by various agents 60
in a variety of cells

Table 2.3. The differences in gene mutation status in HT-29 and HCT- 65

116 cells
Table 2.4. Phenotypic differences between type I and type H human 71
breast epithelial cells (HBEC)
Table 5.1. Sphingoid bases induce differentiation of type I HBEC 151
(HME29-A) to type II HBEC
APPENDIX
Table A. 1 . Mobility of sphingolipids on Thin-Layer Chromatography 169
Table A2. Rfvalues of sphingolipids on 2-Dimemsional Thin-Layer 169

Chromatography

xiii

Figure 2.1.

Figure 2.2.

Figure 2.3.

Figure 2.4.

Figure 2.5.

Figure 2.6.

Figure 2.7.

LIST OF FIGURES

CHAPTER 2
Multi-step model for the colon carcinoma development

Sphingolipids signaling pathway: Ceramide and sphingosine as
mediators for protective effects of sphingomyelin against
cancers

Mitogen Activated Protein (MAP) Kinases signaling pathways
The cell survival and anti-apoptotic actions of AKT

HT-29 and HGT-116 human colon cancer cells. Type I normal
human breast epithelial cells (HBEC), tumorigenic type I
HBEC, and type H HBEC (photographs)

Differentiation of type I HBEC with stem cell characteristics
to type H HBEC and formation of ductal and terminal end bud-
like structure fiommixture oftypelandtype H HBEC

(photographs)
Derivation of tumorigenic type I HBEC and type 11 normal

human breast epithelial cells (HBEC) from type I HBEC with
stem cell characteristics

xiv

Page

55

58

69

70

Figure 3.1.

Figure 3.2.

Figrue 3.3.

Figure 3.4.

Figure 3.5.

Figure 3.6.

Figure 3.7.

Figure 3.8

CHAPTER 3
Structures of sphingosine, sphinganine, ceramide, Cz-ceramide,
and Cz-dihydroceramide

Effects of sphingoid bases and ceramides on growth and death
of HT-29 and HOT-116 human colon cancer cells

Effects of sphingoid bases and ceramides on chromatin and
nuclear condensation in HT-29 and HCT -1 16 human colon
cancer cells (photographs)

Effects of sphingoid bases and ceramide on fragmented low
molecular DNA in HT -29 and HCT-116 human colon cancer
cells

Sphingosine increases the number of sub-Go/G. cells indicative
of apoptosis (one representative histogram)

Effects of sphingoid bases and ceramides on HT-29 and HCT-
116 apoptotic cell number

Effects of sphingoid bases and ceramides on cell cycle
distribution of HT-29 and HGT-116 human colon cancer cells

Regulation of cell growth, cell cycle, and apoptosis by
sphingoid bases and ceramides in PIT-29 and HCT-l 16 hrnnan
colon cancer cells

Page
78

85

87

89

92

93

101

Figure 4.1.

Figure 4.2.

Figure 4.3.

Figure 4.4.

Figure 5.1.

Figure 5.2.

Figure 5.3.

Figure 5.4.

Figure 5.5.

Figure 5.6.

Figure 5.7.

Figure 5.8.

CHAPTER 4
Effects of sphinganine on activation of extracellular signal-
regulated kinase 1/2 (ERKl/ERKZ) in HT-29 human colon

cancer cells

Sphinganine causes early activation of c-Jun NH2-terminal
kinase-2/1 (JNK2/JNK1) in HT-29 human colon cancer cells

Sphinganine causes early activation of p38 mitogen activated
protein kinase in HT-29 human colon cancer cells

Sphinganine causes early inhibition of AKT-ser473 and AKT-
thr308 in HT-29 human colon cancer cells.

CHAPTER 5
Structures of sphingosine, sphinganine, ceramide, C2-
ceramide, and Cz-dihydroceramide

Derivation of tumorigenic type I HBEC and type H normal
human breast epithelial cells (HBEC) from type I HBEC with
stem cell characteristics

Sphinganine more potently inhibits the growth and causes
death of tumorigenic type I HBEC than sphingosine

Sphinganine more potently inhibits the growth and causes
death of tumorigenic type I HBEC than sphingosine at 24 h
(photographs)

Sphinganine increases the number of A0 (sub-GO/Gl) cells,
indicative of apoptosis, and arrests cell cycle at G2/M phase in
tumorigenic type I HBEC (one representative histogram)

Sphinganine more potently induces apoptosis of tumorigenic
type I HBEC than sphingosine. Both sphingoid bases at high
concentrations (8-10 uM) arrest cell cycle at G2/M phase with
reductions in S and Go/G1 phases

Effects of sphinganine and sphingosine on growth and death of
type I HBEC (HME29-A) and type H HBEC (HME29-A)

Sphinganine inhibits the growth and causes death of type I
HBEC (HME30-4A) (photographs)

xvi

113

114

115

116

126

127

138

139

142

143

145

146

Figure 5.9.

Figure 5.10.

Figure 5.11.

Figure 5.12.

Figure A.1.

Figure A.2.

Figure A.3.

Figure A.4.

Figure A.5.

Figure A.6.

Figure A.7.

Type H HBEC (HME29-A) are less sensitive to growth
inhibitory effects of sphinganine and sphingosine than
tumorigenic type I HBEC and type I HBEC at 6 (1
(photographs)

Sphinganine and sphingosine induce differentiation of type I
HBEC (HME29-A) to type II HBEC (Representative
photographs)

Sphinganine increases the expression of a tumor suppressor
protein maspin during induction of differentiation of type I
HBEC (HME29-A) to type H HBEC (photographs)

Chemopreventive properties of sphinganine and sphingosine in
type I and type H HBEC and chemotherapeutic effects of
sphinganine and sphingosine against tumorigenic type I HBEC
and type I HBEC

APPENDIX

Effects of exogenous Crceramide and Cz-dihydroceramide on
intracellular concentrations of free sphingoid bases in HCT-

1 16 human colon cancer cells

Effects of sphinganine (SA) on gap junctional intercellular
communication on HOT-116 human colon cancer cells
(photographs)

Effects of sphinganine on gap junctional intercellular
commrmication on HT-29 human colon cancer cells

(photographs)

Effects of sphingosine, sphinganine, Cz-ceramide, and C2-
dihydroceramide on DNA fragmentation, indicative apoptosis,
in HT-29 human colon cancer cells

Effects of sphingosine, sphinganine, Cz-ceramide, and C2—
dihydroceramide on DNA fragmentation, indicative apoptosis,
in HGT-116 human colon cancer cells

Effects of sphingoid bases on apoptotic cell number in HCT-
116 human colon cancer cells

Effects of sphingoid bases on cell cycle distribution of HCT-
116 human colon cancer cells

xvii

147

152

153

171

171

172

173

174

175

175

Figure A.8. Effects of a calmodulin antagonist W7 on growth inhibitory 176
effects of sphinganine (SA) in HCT-116 human colon cancer
cells

Figure A.9. Effects of sphinganine and sphingosine on gap junctional 178
intercellular communication on trunorigenic type I HBEC at 24

h (photographs)

Figure A.10. Sphinganine (SA) more potently inhibits the growth and 179
causes death of tumorigenic type I HBEC than sphingosine at
2 d (photographs)

Figure A.11. Effects of sphinganine and sphingosine on cell cycle 179
distribution in tumorigenic type I HBEC at 12h

Figure A.12. Effects of bovine serum albumin (BSA) on growth of 180
tumorigenic type I HBEC and type H HBEC

Figure A.13. Fibroblasts and the third type of cells are found in human 180
breast epithelial cells (HME23A, HME29A, HME30-4A,
HME3 l-L2A) (photographs)

Figure A. 14. Type II HBEC (HME29A) are less sensitive to growth 181
inhibitory effects of sphinganine and sphingosine than
tumorigenic type I HBEC and type I HBEC at 3 (1
(photographs)

xviii

HBEC
PHSZ
COX2
FBS
HNPCC
APC
FAP
AFAP
FCC
PCR
AP-l
CRE
CREB
PKA
NSAID
EGF
PDGF
TNFor
IL-l
RTK

MEK
MEKK
INK/SAPK

cPLA2

MSKI

CHOP
p90RSK
MAPKs

ERK

p38
BMK-l/ERKS

MEK kinase
PBK
PPARr

LISTS OF ABBREVIATIONS

3’, 5’-cyclic Adenosine Monophosphate

Human Breast Epithelial Cells

Prostaglandin H synthase 2

Cyclooxygenase 2

Fetal Bovine Serum

Hereditary Nonpolyposis Colorectal Cancer
Adenomatous Polyposis Coli

Familial Adenomatous Polyposis

Attenuated form of Familial Adenomatous Polyposis
Familial Colorectal Cancer

Polymerase Chain Reaction

Activator protein-1

c-AMP response element

c-AMP response element binding protein

Protein kinase A

NOnsteroidal anti-inflammatory drugs

Eidermal Gowth Factor

Patelet Derived Growth Factor

Tumor Necrosis Factor-or

Interleukin-1

Receptor Tyrosine Kinase

MAP kinase kinase

MAP/ERK kinase

MAP/ERK kinase kinase

c-Jun NHz-terminal Kinase/Stress Activated Protein Kinase
JNK kinase

cytosolic phopholipase A2

mitogen-and stress-activated protein kinase-1
cAMP response element binding protein homologous protein
Ribosomal p90 S6 kinase

Mitogen Activated Protein kinases

Extracellular signal-Regulated Kinase

p3 8/RK/C SBP

Big Mitogen-activated protein Kinase-l/ERKS
Phosphatidylinositol 3-kinase

Peroxisome Proliferator Activated Receptor gamma

xix

CHAPTER 1. INTRODUCTION

Rationale of this study

In recent years, sphingolipids have emerged as components of the diet which may
help to protect against development of colon carcinogenesis (for review: Vesper et al.
1999, Dillehay et al. 1994, Schmelz et al. 1994, Schmelz et al. 1996, Schmelz et al. 1997,
Schmelz et al. 1998, Schmelz et al. 2000, Schmelz et al. 2001). Complex sphingolipids
are important components of dairy and soy products (Ahn and Schroeder, 2002a), two
classes of foods that appear to have the potential to prevent carcinogenesis and some
other diseases.

Sphingolipid digestion products such as ceramide and sphingoid bases
(sphingosine and sphinganine) play important roles in regulating cell proliferation,
differentiation, and apoptosis and have been implicated as putative second messengers in
signaling pathways (Merrill et al. 1997). This raises the possibility that complex
sphingolipids may protect against carcinogenesis via digestion to ceramide or sphingoid
bases. Schmelz et al (1994) reported that ~88% of dietary sphingomyelin is digested and
absorbed as ceramides and sphingoid bases in the small intestine and small amounts of
dietary sphingolipids appear in lymph (Nilsson, 1968, Nilssorr, 1969) and serum
(Imaizumi et al. 1992). Thus, the colon and other tissues could be exposed to ceramide
and sphingoid bases via the circulation. Alternatively, ~12% of dietary sphingomyelin is
not absorbed and passes directly into the colonic lumen where it may be metabolized by
colonic bacteria. This provides a more direct route by which colonic tissues may be
exposed to ceramide and sphingoid bases.

Evidence is emerging that bioactive digestion products of sphingolipids also may

have the potential to both prevent and treat human breast cancer. Yang et al (submitted)

reported that sphingosine kills tumorigenic breast cells by inducing apoptosis
(programmed cell death) while not affecting type H normal human breast epithelial cells
(HBEC). These findings strengthen the potential use of sphingolipids as
chemotherapeutic agents since this form of programmed cell death can kill target tumor
cells without influencing adjacent normal cells (Sweeney et al. 1996). In addition, Yang
et al (Submitted) found that low concentrations of sphingosine caused differentiation of
type I HBEC (having stem cell characteristics) to type H HBEC suggesting that
sphingosine may also have chemopreventive properties by reducing the number of stem
cell targets of carcinogenesis.

Although recent studies suggest that dietary sphingolipids may protect against the
development of colon cancer and possibly breast cancer, the mechanism by which
sphingolipids kill colon and breast cancer cells is not clear. One possibility is that
sphingolipids alter the expression of genes that modulate apoptosis. For example,
ceramide induces apoptosis in PC-12 cells via the activation of caspase-3 and these
effects were blocked by overexpression of bcI-2 suggesting that ceramide may activate
caspase-3 by down-regulating bcl-Z (Yoshimura et al. 1998). Alternatively, apoptosis
induced by sphingosine and ceramide in other cell-types is accompanied by an altered
balance between extracellular signal-regulated kinase (ERK), c-Jun NHz-terminal
kinase/stress activated protein kinase (JNK/SAPK), and p38 mitogen activated protein
kinases (MAPKs) (Xia et al. 1995, Kummer et al. 1997, Jarvis et al. 1997). In addition,
the AKT pathway is a probable target because ceramide (Schubert et a1, 2000) and
sphingosine inhibit phosphorylation (activation) of AKT (a cell survival signal) to induce

apoptosis in human liver cancer cells (Chang et al, 2001b).

The mechanism by which sphingoid bases cause differentiation of type I HBEC to
type II HBEC (which are more resistant to carcinogenesis) is unclear. Other agents that
trigger this differentiation elevate cAMP. Therefore, sphingoid bases could activate
adenylate cyclase, inhibit phosphodiesterase activity, or act via a cAMP-independent
mechanism.

In the present studies, HT-29 and HCT-116 human colon cancer cells and
transformed tumorigenic type I HBEC will be used to examine mechanisms by which
sphingolipids inhibit growth and cause death of colon and breast cancer cells. Also,
effects of sphingolipids on growth of type H HBEC representing normal breast cells will
be tested. Type I HBEC will be used to investigate the mechanism by which sphingoid

bases stimulate differentiation of breast cells to type H HBEC.

Objective and hypothesis

The overall objective of this proposed research is to investigate roles of
sphingolipid metabolites sphingoid bases (sphingosine and sphinganine), Cz-ceramide,
and Cz-dihydroceramide on cell growth, apoptosis (a programmed cell death), cell cycle,
differentiation, and cell signaling pathways in HT—29 and HGT-116 human colon cancer
cells and type I human breast epithelial cells (HBEC), type H HBEC, and transformed
tumorigenic type I HBEC.

The specific aims of the research are to:

Study 1: Sphingoid bases and ceramide induce apoptosis in HT-29 and HCT-
116 human colon cancer cells (CHAPTER 3)
Aim 1. Determine effects of sphingosine, sphinganine, Cz-ceramide, and C2-
dihydroceramide on growth and death of HT-29 and HGT-116 human colon cancer cells.
Aim 1 Hypothesis. Since complex sphingolipids inhibit colon carcinogenesis in rodent
models, sphingosine, sphinganine, and Cz-ceramide will inhibit growth and cause death
of HT-29 and HCT-116 human colon cancer cells.
Aim 1 Null Hypothesis. Cz-dyhydroceramide will inhibit growth and cause death of HT-
29 and HCT-116 human colon cancer cells.
Aim 2. Determine whether sphingosine, sphinganine, Cz-ceramide, and C2-
dihydroceramide cause death of HT-29 and HCT-l 16 human colon cancer cells by
inducing apoptosis.
Aim 2 Hypothesis. Sphingosine, sphinganine, and Cz-ceramide will cause death of

HT-29 and HCT-116 human colon cancer cells by inducing apoptosis.

Aim 2 Null Hypothesis. Cz-dyhydroceramide will cause death of HT-29 and HCT-116
human colon cancer cells by inducing apoptosis.

Aim 3. Determine whether sphingosine, sphinganine, Cz-ceramide, and C2-
dihydrocerarrride inhibit growth of HT-29 and HGT-116 human colon cancer cells by
arresting the cell cycle at a specific phase.

Aim 3 Hypothesis. Sphingosine, sphinganine, and Cz-ceramide will inhibit growth of
HT-29 and HGT-116 human colon cancer cells by arresting the cell cycle at a specific
phase.

Aim 3 Null Hypothesis. Cz-dihydroceramide will inhibit growth of HT-29 and

HCT-116 human colon cancer cells by arresting the cell cycle at a specific phase.

Study 2: Sphinganine causes early activation of JNK and p38 and early
inhibition of AKT in HT-29 human colon cancer cells (CHAPTER 4)
Aim 4. Determine effects of sphinganine on mitogen activated protein kinases
(MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun NHz-terminal
kinase/stress-activated protein kinase (JNK/SAPK), and p3 8, and AKT that are
responsible for regulating cell proliferation and apoptosis in HT-29 human colon cancer
cells.
Aim 4.A. Determine the time required for an apoptotic concentration of sphinganine
to affect active phosphorylated forms of MAPKs (ERKl/ERK2, JNK2/JNK1, and p38)
and the expression levels of MAPKs in HT-29 cells.
Aim 4.A. Hypothesis. Since MAPKs play significant roles in regulating cell
proliferation and apoptosis, sphinganine will alter phosphorylation status of MAPKs
(ERKl/ERK2, JNK2/JNK1, and p38) in HT-29 cells.
Aim 4.B. Determine the time required for an apoptotic concentration of sphinganine
to affect activation of AKT by phosphorylation at ser473 and thr3 08 and the expression
levels of AKT in HT-29 cells.
Aim 4.B. Hypothesis. Since AKT plays significant roles in regulating cell
proliferation and apoptosis, sphinganine will alter phosphorylation of AKT at ser473 and

thr308 in HT-29 cells.

Study 3: The study of chemotherapeutic and chemopreventive roles of
sphinganine and sphingosine in breast carcinogenesis using human breast stem,
normal, and'tumorigenic cell models (CHAPTER 5)

Type I human breast epithelial cells (HBE C) with stem cell characteristics
Aim 5.A. Determine effects of sphinganine on growth and death of type I HBEC in
comparison to sphingosine.

Aim 5.A. Hypothesis. Sphinganine will inhibit growth and cause death of type I
HBEC to a similar degree as the known cytotoxic effects of sphingosine.
Aim 5.B. Determine whether sphinganine induces differentiation of type I HBEC to
type H HBEC.
Aim 5.8. Hypothesis. Sphinganine will induce differentiation of type I HBEC to
type H HBEC to a similar degree as the known differentiating effect of sphingosine.
Type II HBEC

Aim 6. Determine effects of sphinganine on growth of type H HBEC in
comparison to sphingosine.
Aim 6. Hypothesis. Sphinganine will mimic sphingosine by having no or little effect
on growth of type II HBEC.

Transformed tumorigenic type I HBE C (M13S V1R2N1)
Aim 7. Determine effects of sphinganine on growth and death of tumorigenic type
I HBEC in comparison to sphingosine.
Aim 7. Hypothesis. Sphinganine and sphingosine inhibit growth and cause death of

tumorigenic type I HBEC.

Aim 8. Determine whether sphinganine causes death of tumorigenic type I HBEC
by inducing apoptosis in comparison to sphingosine.

Aim 8 Hypothesis. Sphinganine and sphingosine will cause death of tumorigenic
type I HBEC by inducing apoptosis.

Aim 9. Determine whether sphinganine and sphingosine inhibit growth of
tumorigenic type I HBEC by arresting the cell cycle at a specific phase.

Aim 9 Hypothesis. Sphinganine and sphingosine will inhibit growth of tumorigenic

type I HBEC by arresting the cell cycle at a specific phase.

CHAPTER 2. LITERATURE REVIEW

10

A. Cancer

A. 1. Cancer as a disease of proliferation and differentiation. A major
characteristic of all higher eukaryotes is the defined life span of the organism. Normal
cells appear to be "mortal", with highly regulated growth and division, and the potential
to become terminally differentiated. Once cells are terminally differentiated, they can no
longer proliferate (Trosko and Ruch, 1998). In contrast, cancer cells lose their usual
growth control and proliferate indefinitely if provided adequate nutrients (Lewin, 1997)
suggesting that cancer cells may be immortal (Kimball, 1983). Thus, a cancer can be
described as an uncontrolled proliferation of cells.

Cancer cells appear to originate from tissue precursor cells (stem cells), that are
normally in a period of rapid mitosis and not yet fully differentiated. A common
characteristic of cancer cells is that they are partially, but not terminally differentiated.
Cancer cells are usually less differentiated than the cells of the tissue from which they
arose. Thus, a malignancy also can be characterized by loss of differentiation called
anaplasia (Markert, 1968) and cancer can be described as deregulation of differentiation
or oncogeny as blocked or partially blocked ontogeny (Potter, 1978, Potter, 1987).

Most cancers (~80%) originate fiom epithelial cells. Epithelial cells form the
outer covering of tissues and surface cells of the skin and line the intestines and lungs.
The surface cells are more likely to be exposed to dangerous chemicals and harmful
radiation. The underlying layer must continue to divide as the outlayers are constantly
worn away. Cells fi-om tissues, such as nerve and muscle, where cell division is rare,

only occasionally become cancerous (Kimball, 1983, Lodish et al. 1999).

11

A.2. Cancer through mutations. Progression to cancer (carcinogenesis)
occurs through multiple-stages including initiation, promotion, and progression phases.
During the initiation phase, a single cell in a tissue acquires a mutation in an oncogene or
tumor suppressor gene and stem cells are irreversibly altered. During the promotion
stage, the initiated stem cells undergo clonal proliferation generating many descendants
and these descendants are prone to further mutations in other genes that can lead to
neoplasia. As the rate of mitosis in that clone increases, the chances of further DNA
damage increase and many (perhaps 6-8) mutations may arise until the growth of that
clone becomes completely unregulated. During the progression phase, cells within a
tumor acquire further mutations that allow the angiogenic induction of vacuolization in
the tumor. At this stage, cancer cells break away from the primary tumor, travel in blood
and lymph, and establish metastases in other locations of the body. Metastases is
normally a lethal stage of cancer in the patient (Kimball, 1983, Lodish et al. 1999, Trosko
and Ruch, 1998).

Mutations leading to cancer often occur in the following genes (Kimball, 1983,
Lewin, 1997, Van Noorden et al. 1998, Lodish et al. 1999):

1) Cell cycle regulatory genes that regulate mitosis. These include proto-
oncogenes (also called cellular genes) and tumor suppressor genes. Mutations or
overexpression of prom-oncogenes (eg. erbB, the gene encoding the receptor for
epidermal growth factor) stimulate mitosis although normal growth signals are
absent. Oncogenes act as dominants meaning that only one of the pair needs to be
mutated to predispose the cell to cancer. Tumor suppressor genes normally

inhibit mitosis. For example, the p53 gene product normally senses DNA damage

12

and either halts the cell cycle until it can be repaired or, if the damage is too
massive, triggers apoptosis. Tumor suppressor genes are recessive meaning that
either both copies must be mutated for their function to be lost, or more
commonly, the healthy copy of the gene has been lost.

2) Genes that regulate apoptosis (for details: see D.3. Induction of apoptosis by
chemotherapeutic and/or chemopreventive agents). Mutations in these genes
direct the cell to ignore signals telling it that it is irreparably damaged and should
comnrit suicide.

3) Genes that maintain telomeres (for details: see D.5. Suppression of telomerase
activity as a novel chemotherapy).

4) Genes that stimulate angiogenesis. Like other tissue, tumors require a blood
supply to provide nutrients and oxygen and to remove waste. As a developing
cancer grows, it must be able to stimulate the growth of new and normal blood
vessels into itself. This is aided by the release of angiogenesis stimulants such as
vascular endothelial growth factor. This may be the result of additional mutated
oncogenes or tumor suppressor genes.

5) Metastasis genes'. These genes include: a) Genes whose products normally
keep the cells of a tissue adhering to one another. For example, the E-cadherin
genes help to hold epithelial cells together. b) Genes whose products normally
keep the cells adhering to their extracellular substrate. For example, elevated
expression of integrin genes encode proteases, which can break down the

proteinaceous extracellular material that normally holds cells in place.

 

1 Genes that enable the tumor to separate from the primary tumor and migrate to other parts of the
body

13

A.3. Environmental factors and cancer. Cancer is a disease which may be
strongly influenced by environmental factors including diet, smoking, body mass,
physical activity, and life style. Among many forms of cancers, colon and breast cancers
appear to be particularly strongly affected by diet (World Cancer Research Fund and
American Institute of Cancer Research, 1997). Thus colon and breast cancers might be
preventable by appropriate diet, activity patterns, and reduction of other environmental
exposures (World Cancer Research Fund and American Institute for Cancer Research,

1997)

B. Colon cancer

B. I . Colon cancer incidence. Colon cancer is the second leading cause
of cancer mortality in the United States (American Cancer Society, 1996) and the fourth
most common cause of cancer mortality in the world (World Cancer Research Fund and
American Institute of Cancer Research, 1997). North America, Europe and Australia are
areas of high incidence of colon cancer; whereas, Central and South America, Asia and
Africa are areas of low incidence. Men and women have similar incidence rates of colon
cancer (World Cancer Research Fund and American Institute of Cancer Research, 1997).

8.2. Colon cancer etiology and risk factors.

8.2.1. Colon carcinoma development. The adenoma-carcinoma hypothesis
proposed by Hill et a1 (1978) is a widely accepted description of the pathogenesis of
colorectal cancer. The target cells of colon carcinogenesis are crypt epithelial cells (Hill
et al. 1978). According to this model, the initial colorectal lesion arises as a benign

adenomatous polyp that later undergoes further disorganization of cellular and tissue

14

phenotype. Bird et a1 (1987) suggested that hyperproliferation of the upper crypt cells
leads to the formation of aberrant crypt foci (putative precancerous lesions of the colon)
and microadenomas. Increased proliferation and decreased differentiation of colonic
epithelial cells are considered to be biomarkers for an increased risk for developing colon
cancer (Lipkin, 1990).

A molecular model for the adenoma-carcinoma sequence (development of colon
carcinoma) can be described by the multi-step process in which cells accumulate
alterations of multiple genes that control cell growth and differentiation, resulting in the
neoplastic phenotype (Figure 2.1) (V ogelstein et al. 1989, Fearon et al. 1990, Fearon and
Vogelstein, 1990, Kinzler et al. 1991). The genes involved in colon carcinogenesis are:
mutations or loss of the adenomatous polyposis coli (APC) gene (a tumor-suppressor
gene) (Aaltonen et al. 1993), mutation of K-ras (a prom-oncogene), early disorganization
of DNA methylation (Feinberg and Vogelstein, 1983), and late loss of p53 (a tumor-
suppressor gene). DNA hypomethylation is an early event in colon carcinogenesis
(F einberg and Vogelstein, 1983).

DNA methylation is genetically controlled and the expression of the methyl
transferase gene seems to be increased in the normal mucosa of cancer patients and
further increased in polyp and cancer tissue (El-Deiry et al. 1991). Several animal
studies showed that isothiocyanates, which mainly are present in cruciferous vegetables,
inhibited both carcinogenesis and DNA methylation (W attenberg, 1977, Wattenberg,
1987, Steinmetz and Potter, 1991a, & Steinmetz and Potter, 1991b). Although these
observations are contradictory, both hypo- and hypermethylation of DNA are at least

partially responsible for early stages of the carcinogenesis.

15

Normal epithelium

Loss of APC
(tumor suppressor gene)

Abnormal cell division
Hyperproliferation

$4— DNA hypomethylation

Adenoma
4— K-ras activation

(oncogene)

+— Loss of DCC

(tumor suppressor gene)
4— Loss of p53

(tumor suppressor gene)

Carcinoma
¢¢—— More mutations

Metastasis

Figure 2.1. Muti-step model for the colon carcinoma development
(Adapted from Fearon and Volgetstein, 1990)

16

B.2.2. Family history as a risk factor for colon cancer. A family history of
colon cancer is correlated with increased risk of colon cancer (Burt et a1. 1985, Bufill,
1990). One of the earliest studies of family history of colorectal cancer examined Utah
families and reported a higher number of deaths from colorectal cancer (3.9%) among the
first-degree relatives of patients who had died from colorectal cancer, compared to sex-
and age-matched controls (1.2%) (Woolf, 195 8). Numerous studies which also have
shown this difference have consistently established that first-degree relatives of affected
cases are themselves at a two- to three-fold increased risk of colorectal cancer. The
magnitude of colon cancer risk among family history groups is consistent in spite of the
various study designs (case-control, cohort), sampling fiames, sample sizes, methods of
data analysis, and countries where the studies originated (Duncan and Kyle, 1982, Rozen
et al. 1987, St. John et al. 1993, Fuchs et al. 1994, Slattery and Kerber, 1994, Negri et al.
1998).

8.2.3. Genes associated with high risk for colon cancer. Several genes
associated with colon cancer risk have been identified. All gene mutations recognized to
predispose to colon cancer are inherited in an autosomal dominant fashion (Burt and
Petersen, 1996). Thus, the family characteristics that implicate autosomal dominant
inheritance of cancer predisposition are important indicators of high risk and of the
possible occurrence of a cancer-predisposing mutation. For example, hereditary
colorectal cancer has two forms: familial adenomatous polyposis (F AP, including an
attenuated form of familial adenomatous polyposis, AP AP) and hereditary nonpolyposis

colorectal cancer (HNPCC).

17

People with F AP possess gerrnline mutations in the APC gene and carry nearly
100 percent risk of colon adenocarcinoma (Aaltonen et al. 1993). HNPCC, in which the
affected individuals have gerrnline mutations in DNA mismatch repair genes, is a
syndrome which is not easily distinguished fiom sporadic polyposis and cancer on
physical examination. This syndrome accounts for a larger proportion of colon cancer
cases than familial adenomatous polyposis (Lynch and Lynch, 1985). Other families
displaying aggregation of colorectal cancer and/or adenomas, but without any apparent
association with an identifiable hereditary syndrome are known collectively as familial
colorectal cancer (FCC) (Burt and Petersen, 1996).

B. 2. 4. Other risk factors for colon cancer. Putative risk factors for
development of colon cancer include smoking (Potter et a1. 1993) and alcohol
consumption (Potter et al. 1982, Tuyns et al. 1988, Longnecker, 1990, Choi and Kahyo,
1991). Other factors which appear to affect risk of colon cancer include level of physical
activity (Garabrant et al. 1984, Gerhardsson et al. 1986, Slattery et al. 1988), age, and
diet. Lee et a1 (1991) showed that individuals with high levels of physical activity were
at lower risk for developing colon cancer and this observation partially accounted for the
international differences in cancer rates (Garabrant et al. 1984, Gerhardsson et al. 1986,
Slattery et al. 1988, Willett, 2001).

3.3. Dietary factors in colon caner. A large proportion of colon cancer
deaths might be preventable by appropriate diets and behaviors (World Cancer Research
Fund and American Institute of Cancer Research, 1997). In recent years, identification of
specific beneficial food components which protect against chronic diseases such as colon

cancer has been an area of focus in nutrition research. In this literature review, roles of

18

dietary fat and dairy and soy products on colon cancer are discussed since previously we
found that bioactive compounds called sphingolipids are important components of dairy
and soy products (Ahn and Schroeder, 2002a).

B.3.1. Dietary fat. A hypothesis that a high-fat diet is responsible for colon,
breast, and possibly prostate cancers in Western countries is rooted in the ecological
correlations between national per capita fat consumption and rates of these major cancers
(Armstrong and Doll, 1975, for review: Reddy 1993, Giovannucci and Goldin 1997,
Lipkin et a1. 1999). This hypothesis also has been supported by animal studies (Cohen et
al. 1986 Zevenbergen et a1. 1992, Tang et al. 1996, for review: Reddy 1993, Lipkin et al.
1999). It is noteworthy that both epidemiological studies and animal experiments have
provided evidence that not only the amount of fat but also different types of fat play
important roles in affecting colon cancer risk (for review: for review: Reddy 1993,
Giovannucci and Goldin 1997, Lipkin et al. 1999). For example, saturated fat and/or
animal fat has been shown to increase colon cancer risk, whereas fish products rich in w-
3 fatty acids, including docosahexanoic acid (DHA) and eicosapentanoic acid (EPA), has
been associated with lowering colon cancer risk.

Possible mechanisms of action of dietary fat and types of fat in colon
carcinogenesis include: 1) diets high in beef tallow, lard, or corn oil increase the
concentration of colonic luminal/fecal secondary bile acids, which promote colonic
epithelial cell proliferation and function as promoters of colon cancer; 2) high dietary fat
might alter membrane phospholipid turnover and prostaglandin synthesis by activating
phospholipase PLA2, which enhances colon tumor promotion; and 3) w-3 fatty acids rich

in fish oil have been shown to inhibit the cyclooxygenase (COX) pathway and

19

arachidonic acid (released fiom phospholipids) metabolism, which result in inhibiting
prostaglandin synthesis. At least two isoforms of COX (COXl and COX2) are reported.
Overexpression of COX2, but not COXl has been found in colonic mucosa and tumors
of rats.

On the other hand, the hypothesis of a high-fat diet as a risk factor for colon
cancer has been argued with findings from prospective studies. For example, Slattery et
a1 (1997) collected detailed dietary intake data flour a population of 1,993 colon cancer
cases and 2,410 controls in 3 areas of the United States. Data indicated that neither total
dietary fat nor specific fatty acids were associated with risk of colon cancer, after
adjusting for total energy intake, physical activity, and body size (Slattery et al. 1997).

In summary, ecological (correlational) studies and animal experiments support the
association of a high-fat diet and colon cancer risk; whereas no association is seen in
prospective cohort studies. A clear conclusion for dietary fat as a risk factor for colon
cancer remains elusive. Dietary recommendations should not only consider total dietary
fat intake but also types of fat, consumption of other nutrients and dietary components,
and lifestyle factors that may influence colon cancer.

3.3.2. Caloric restriction. Energy or caloric restriction has gained much
attention for its inhibitory effect on cancers. In addition, caloric restriction seems to be
the only dietary treatment to improve longevity in laboratory animals. A case-control
study indicated that increase of total calories is associated with risk of colon cancer
(Graham et al, 1988). In rats treated with azoxymethane, caloric restriction reduced
colon tumor growth (Pollard et al, 1984, Reddy et al, 1987). This cancer inhibitory effect

appears to be independent of the effect of dietary fat because in chemically induced

20

models of colon and mammary cancers, 40% caloric-restricted rats showed reduced
carcinogenesis even as they were fed increased percentage of dietary fat (Klurfeld et al,
1987)

B.3.3. Dairy products. Studies have shown that dairy products appear to
protect against colon carcinogenesis. Epidemiological studies indicate that regular
consumption of fermented milk products such as yogurt may be protective against some
forms of cancer (Peters et al. 1992, Kampman et al. 1994). In addition, some lactic acid
bacteria present in fermented milk, especially Lactobacilli, are components of intestinal
microflora (Bianchi Salvadori, 1986) and have protective effects against pathogenic
microorganisms (Mutai and Tanaka, 1987).

For example, a dietary supplement of Lactobacillus acidophilus reduced the
incidence of 1,2-dimethylhydrazine-induced colon cancer in F344 rats (Goldin and
Gorbach, 1980). Also, epidemiological studies have reported that, despite the high fat
intake in Finland, colon cancer incidence is lower than in other countries (Malhorta,
1977, International Agency for Research on Cancer, 1983). This may be due to Finland’s
high consumption of milk, yogurt, and other dairy products.

B.3.4. Soy producm. Several investigations suggest that soy products are another
group of functional foods which contribute to the lower rates of colon cancer as well as
breast and prostate cancers in China and Japan (Setchell et al. 1984, Barnes et a1. 1990,
Adlercreutz, 1990). The isoflavone genistein, which is rich in soybeans and considered
one of the most significant plant estrogens, has been reported to protect against
development of colon carcinogenesis (Akiyama et al. 1987, Ogawara et al. 1989, Teraoka

et a1. 1989, Thiagarajan et al. 1998).

21

C. Breast Cancer

CI. Breast cancer incidence. Breast cancer is the most common cancer
among women in the United States as well as among women worldwide and it is
responsible for almost 30% of newly diagnosed cancers and 17% of cancer-related deaths
(Parkin et al. 1992, Miller et al. 1993, Am. Inst. Cancer Res., 1997). Incidence rates are
highest in North America and Northern Europe. Asia and Africa are the areas of the
lowest incidence, although rates have been increasing markedly in several Asian and
developing countries (Parkin et al. 1992).

Among Caucasian women in the United States, age-adj usted breast cancer
incidence rates have increased slowly for many decades, whereas age-adj usted breast
cancer mortality rates have remained fairly constant since 1930 (Kelsy, 1993, Kelsey and
Horn, 1993, Miller et al. 1993). The incidence of estrogen receptor-positive tumors has
also risen over time (Glass and Hoover, 1990). The major factor accounting for the
increase in incidence rates is improved breast cancer detection by screening of
premenopausal women using mammography. Several factors might partially explain the
increased incidence with no apparent increase in mortality: detection of early stage
tumors by mammography; better prognosis associated with estrogen receptor-positive
compared with estrogen receptor-negative tumors; and advances in medicine (Kelsy,
1993, Kelsey and Horn, 1993, Miller et al. 1993).

C.2. Breast cancer etiology and risk factors.

612.]. Family history as a risk factor for breast cancer. Genetic contribution

to breast cancer risk is found in the increased incidence of breast cancer among women

22

with a family history of breast cancer, and in the observation of rare families where
multiple family members are affected with breast cancer, in a compatible pattern with
autosomal dominant inheritance (genetic conditions that occur when a mutation is present
in one copy of a given gene meaning that the person is heterozygous). Cross-sectional
studies of adult populations reported that 5-10% of women have a mother or sister with
breast cancer, and approximately 10-20% of women have either a first-degree or a
second-degree relative with breast cancer.

Taken together, epidemiological studies have clearly established that family
history is an important risk factor for breast cancer. After gender and age, a positive
family history is the strongest known predictive risk factor for breast cancer (American
Cancer Society, 1999, Yang et al. 1998a, Colditz et al. 1993).

C.2.2. Genes associated with high risk for breast cancer. Studies of large
kindreds with multiple individuals affected by breast cancer led to the identification of
several susceptibility genes including BRCAl, BRCA2, p53, and PTEN/MMACI.
Women with gerrnline mutations in BRCAI or BRCA2 have a very high lifetime risk of
developing breast cancer. Most of these germ-line mutations are predicted to generate a
truncated protein product (For review: Sellers 1997).

Furthermore, in all cancer studies from mutation carriers, the wild-type allele is
deleted which strongly suggests that BRCA1 and BRCA2 belong to a class of tumor
suppressor genes (Smith et al. 1992, Collins et al. 1995). The transcription of both
BRCA1 and BRCA2 genes is induced late in the G1 phase of the cell cycle and remains
elevated during the S phase, demonstrating a role in DNA synthesis (Gudas et al. 1996,

Rajan et al. 1996, Vaughn et al. 1996). Interestingly, both BRCA1 and BRCA2 knockout

23

mice can be partially rescued by crossing with a p53 knockout strain, indicating that these
genes may interact with the p5 3-mediated DNA damage checkpoint (Brugarolas and
Jacks, 1997). Zhang et a1 (1998) provided further evidence that both BRCA1 and
BRCA2 hereditary susceptibility genes function as " gate keepers" (similar to tumor
suppressor gene p53) by serving to maintain genomic integrity. The loss of this function
allows for the accumulation of other genetic defects that cause cancer.

Breast cancer is a constituent of the rare Li-Fraumeni syndrome (LF S), where
germline mutations of the p53 gene on chromosome 17p have been documented (Garber
et al. 1991). The Li-Fraumeni syndrome is characterized by premenopausal breast cancer
in association with childhood sarcoma, brain tumors, leukemia, and adrenocortical
carcinoma (Bottomley et al. 1968, Malkin et al. 1993. Over 50% of families exhibiting
this syndrome have shown a gerrnline mutation in the p53 gene. The germ line mutation
of p53 is inherited with a penetrance of at least 50% by age 50 (for review: National
Cancer Institute, 2002).

It is known that individuals homozygous for ATM gene are at increased risk of
malignancies, especially hematologic. Epidemiologic studies have suggested that female
heterozygote carries of ATM gene might have an increased risk of breast cancer (Swift et
al. 1987, Easton et al. 1994, Olsen et al. 2001). Ataxia telangiectasia is an autosomal
recessive disorder described by neurologic deterioration, telangiectasias,
immunodeficiency states, and hypersensitivity to ionizing radiation. By estimation,
approximately 1% of the general population may be heterozygote carriers of the ATM
gene (Savitsky et al. 1995). ATM proteins appear to play a role in cell cycle control

(Uhrhammer et al. 1998) (for review: National Cancer Institute, 2002).

24

In summary, breast cancer occurs as a result of accumulation of mutations in
critical genes that regulate cell proliferation, cell cycle, and differentiation. Most breast
cancers appear as adenocarcinomas (a malignant tumor originating in epithelial cells in a
glandular or gland-like pattern). Thus terminal end buds, which are rich in highly
proliferating mammary epithelial stem cells, are considered to be the target cells of
mammary neoplastic transformation (Russo and Russo, 1987).

C.2.3. Other risk factors for breast cancer. Other risk factors that affect breast
cancer among women include age, previous breast disease, reproductive and menstrual
history, estrogen therapy, radiation exposure, alcohol intake (Davis et al. 1993), and diet
(Marshall. et al. 1993). For example, cumulative risk of breast cancer increases with age
and most breast cancers occur after the age of 50 (Feuer et al. 1993). In the case of
women with a genetic susceptibility, breast cancer tends to arise at an earlier age than in
sporadic cases.

Breast cancer risk increases with early menarche and late menopause, whereas
early first full term pregnancy decreases the risk. The Nurses' Health Study reported that
these factors of reproductive and menstrual history influenced breast cancer risk only
among women who did not have a mother or sister with breast cancer (Colditz et al.
1996)

Conflicting data exist regarding the association between breast cancer and
postmenopausal hormone replacement therapy. A meta-analysis of data from 51 studies
showed a relative risk of breast cancer of 1.35 (95% CI 1.21-1.49) for women who had
received hormone replacement therapy for 5 or more years afier menopause. The excess

risk is reduced with termination of hormone replacement therapy and largely vanishes

25

within 5 years (Collaborative Group on Hormonal Factors in Breast Cancer, 1997). A
recent study by Writing Group for the Women’s Health Initiative Investigators (2002)
demonstrated that overall health risks exceeded benefits from use of combined estrogen
plus progestin for an average 5.2 year follow-up among healthy postmenopausal US
women. This study reported that women on hormone replacement therapy showed a
higher rate of developing invasive breast cancer than the control group (Writing Group
for the Women’s Health Initiative Investigators 2002).

(3.3. Dietary factors in breast cancer. A large proportion of breast cancer
deaths might be preventable by appropriate diets and behaviors (World Cancer Research
Fund and American Institute of Cancer Research, 1997). In recent years, identification of
specific beneficial food components which protect against chronic diseases such as breast
cancer has been an area of focus in nutrition research. In this literature review, roles of
dietary fat and dairy and soy products on breast cancer are discussed since previously we
found that bioactive compounds called sphingolipids are important components of dairy
and soy products (Ahn and Schroeder, 2002a).

C.3.1. Dietary fat. The association between a high-fat diet and breast cancer
has been an active area of research since early 1950. Tannenbaum and Silverstone
(1953) demonstrated that a high-fat diet (12 %) stimulated mammary tumor development
in mice compared to a low-fat diet (3 %). Ecological correlations exist between national
per capita fat consumption and age-adj usted incidence and mortality from breast cancer
(Armstrong and Doll, 1975; for review: Wynder et al. 1997, Willet 1997, Rogers, 1997,
Lee and Lin 2000). Although correlational/ecological studies used food disappearance

data, which do not reflect actual food-consumption habits due to differences in reporting

26

and wasted foods, the different fat intakes between countries have been confirmed by
dietary surveys within national populations (for review: Wynder et al. 1997, Lee and Lin
2000). In addition, as Asian women with low fat diets increased their fat intake or
migrated to western countries with higher fat diets, incidence of breast cancer increased
(Osteen et al. 1986).

Animal studies conducted over the past 50 years also support the hypothesis that a
high-fat diet stimulates mammary turnorigenensis. A meta-analysis of 100 animal
experiments showed an independent tumor-promoting effect of dietary fat (Freedman et
al. 1990). Interestingly, several animal studies (Cohen et al. 1986, Zevenbergen et al.
1992, Tang et al. 1996) suggested a possibility that the tumor promoting effect of dietary
fat intake may have a threshold level. For example, Tang et a1 (1996) examined effects
of dietary fat ranging from 5 to 45% of calories, where fat was isocalorically substituted
for carbohydrates. Data indicated that colon and mammary tumors rapidly increased
when dietary fat level increased from 15 to 30 % of calories and no additional significant
effects were seen on colon and mammary tumorigenesis above 30 % of calories fi'om fat
(Tang et al. 1996). The mechanisms by which dietary fat promotes mammary
tumorigenesis include: 1) conversion of essential fatty acid to eicosanoids; 2) generation
of reactive oxygen species by reactions of oxygen and the conjugated double bonds in
polyunsaturated fatty acids (PUFA); and 3) interaction between fatty acids and genomic
DNA which alters gene expression (for review: Wynder et al. 1997, Lee and Lin 2000).

Furthermore, it is important to note that different types of fat have different
effects on breast cancer risk. Epidemiological studies showed that Mediterranean

countries where olive oil is a staple showed lower breast cancer incidence compared to

27

most Western countries (for review: Esteve et al. 1993). For example, the incidence of
breast cancer in Spain is about 40 % lower than that of North America or Northern
Europe (for review: Esteve et al. 1993). Greek women consumed 42 % of calories from
fat (mainly from olive oil) showed significantly lower rates of breast cancer than US.
women who consumed 35 % of calories from fat (for review: Wynder et al. 1997, Lee
and Lin 2000). Animal studies also indicated that high levels of olive oil and fish oil did
not promote mammary tumorigenesis.

However this concept of dietary fat as a risk factor for breast cancer based on
correlational studies and animal experiments has been challenged. In a pooled anlysis of
the six prospective studies with more than 200 cases of breast cancer (Hunter et al. 1996)
did not find an association for total fat intake ranging < 20 to > 45 % of calories from fat
with breast cancer. A large prospective cohort study of postmenopausal women (V elie et
al. 2000) found little association between total fat (ranging < 28 to > 42 %) or fat
subtypes and breast caner risk. A possible threshold effect of dietary fat observed in
animal models (Cohen et al. 1986, Zevenbergen et al. 1992, Tang et al. 1996) may at
least be partially related to this lack of association in cohort studies.

In summary, correlational (ecological) studies and animal experiments support the
association of a high-fat diet and breast cancer risk; whereas no association is observed in
prospective cohort studies. A clear conclusion for dietary fat as a risk factor for breast
cancer remains unsolved. Dietary recommendations should not only consider total
dietary fat intake but also types of fat, consumption of other nutrients and dietary

components, and lifestyle factors that may influence breast cancer.

28

C3. 2. Caloric restriction. Energy or caloric restriction has gained much
attention for its inhibitory effect on cancers. In addition, caloric restriction seems to be
the only dietary treatment to improve longevity in laboratory animals. Animal studies
demonstrated that caloric restriction inhibits breast cancer incidence (Klurfeld et al.
1991). It is proposed that obese postmenopausal women have a higher risk of breast
cancer (Cleary and Maihle, 1997) and overweight breast cancer patients have a poorer
prognosis (Bastarrachea et al. 1994).

613.3. Dairy producm. Beneficial effects of fermented milk products are
documented in epidemiological, animal, and cell culture studies. A case control study in
the Netherlands showed a reduced risk of breast cancer among women who consume
fermented milk products (Veer et al. 1989). Conjugated linoleic acid, butyric acid, ether
lipids, and sphingomyelin found in milk fat components demonstrated anticarcinogenic
effects in animal models (Parodi, 1997). Antiproliferative activity of fermented milk was
demonstrated in MCF-7 breast cancer cells (Biffi et al. 1997).

C.3.4. Soy products. Soy intake has gained much attention as an effective cancer
fighter for years. Messina et a1 (1994) demonstrated that soy intake was inversely
correlated with breast cancer risk among 142,857 women in Japan over a period of 17
years. Furthermore, studies of women in Singapore and China indicated that those who
consume a traditional diet high in soy products had a low incidence of breast cancer (Lee
et al. 1991, Wu et al. 1996). However, afier Asian women immigrated to the United
States, the second generation (Ziegler et al. 1993), but not the first, loses this protection.
These findings imply a possible mechanism for breast cancer protection fi'om early and

specific nutritional exposure. Animal studies indicated that early exposure to estrogen

29

and progesterone caused differentiation of mammary tissue and reduced subsequent
susceptibility to chemically induced mammary cancer (Russo et al. 1979, Grubbs et al.
1985).

Genistein is an isoflavone found in soy and acts as a weak agonist to estrogen
receptor. It also shows a property of an antioxidant, inhibits topoisomerase II and
angiogenesis, and induces cell differentiation (Lamartiniere et al. 1997). Lamartiniere’s
group proposes that breast cancer protection in Asian women consuming a traditional
soy-containing diet is gained fiom early exposure to soybean products containing

genistein (for review: Lamartiniere, 2000).

D. Cancer chemotherapy and chemoprevention

D.1. Chemotherapy.

D. 1.1. Definition of chemotherapy. Chemotherapy is defined as the treatment
of cancer with drugs that can destroy cancer cells by inhibiting their growth/proliferation.
These drugs often are called “anticancer” drugs (also known as “anticarcinogens”). Side
effects may occur when drugs affect healthy normal cells. Ofien two or more drugs are
given at the same time because some drugs are more effective together than alone. This
is referred to as combination chemotherapy (National Cancer Institute, 1999).

D. 1.2. Goals for use of chemotherapy. Depending on the type of cancer and
severity of cancer progression, chemotherapy is used for different goals:

1) Removal of cancer cells. When the patient remains free of evidence of cancer

cells, cancer is considered cured.

30

2) Regulation of cancer cells. This is achieved by preventing cancer cells fiom
spreading, by slowing growth, and by killing cancer cells.
3) Alleviation of symptoms caused by cancer. This can help patients live more

comfortably (National Cancer Institute, 1999).

Chemotherapy is often used in addition to surgery, radiation therapy, and/or
biological therapy2 for the following reasons:

1) To shrink a tumor before surgery or radiation therapy. This is called neo-adjuvant
therapy.

2) To help destroy any cancer cells that may remain after surgery and/or radiation
therapy. This is called adjuvant chemotherapy.

3) To improve radiation therapy and biological therapy.

4) To help destroy cancer if it recurs or has spread to other parts of the body from

the original tumor (National Cancer Institute, 1999).

Some chemotherapy drugs might be used for just one or two types of cancer,
while others are used for many different types of cancer. A chemotherapy treatment plan
by a doctor is based on: 1) the type of cancer a patient has; 2) the location the cancer is
found; 3) the effect of the cancer on normal body functions; and 4) the patient’s overall
health (National Cancer Institute, 1999).

D.2. Chemoprevention. Possible strategies to reduce the mortality and
morbidity of cancer include: 1) prevention; 2) early diagnosis and intervention; 3)

successful treatment of localized cancer; and 4) improved management of nonlocalized

 

2 Biological therapy, also called irnmunotherapy, is a treatment to stimulate or restore the ability of the
immune system to fight against infection and disease.

31

cancer. Among these 4 strategies, the most practical approach for cancer management
seems to be the prevention (Mukhtar and Ahmad, 1999).

D.2. 1. Definition of chemoprevention. Chemoprevention can be defined as
a means of cancer management in which the occurrence of the disease can be entirely
prevented, slowed, or reversed by the administration of one or more naturally occurring
and/or synthetic compounds (Ahmad et al. 1998). In addition, the definition of
chemoprevention can be expanded as the use of agents (ofien called drugs) to prevent the
occurrence of precancerous lesions or markers or to retard or reverse progression of
premalignancy to malignancy (Kaityar and Mukjtar, 1996, Lippman et al. 1998).
Chemoprevention is different from the treatment of established cancer. For example,
when an established cancer is treated by chemotherapy, chemoprevention could prevent
its recurrence and further development of new cancers.

D. 2. 2. Characteristics of the chemopreventive agents. Ideal characteristics
of the chemopreventive compounds (also known as anticancer drugs or anticarcinogens)
for human usages are: 1) little or no adverse effects; 2) high efficacy against multiple
sites; 3) effectiveness at achievable dose levels; 4) activity following oral consumption;
5) a known mechanism of action; 6) low cost; 7) history of use by the human
population; and 8) general human acceptance (Mukhtar and Ahmad, 1999).

D.2.3. Defense mechanism for damaged cells. Many environmental, dietary,
and genetic factors impair the normal cell by different mechanisms at different targets. It
is known that most humans have many initiated cells in the target tissues at all times
(Jonason et al. 1996). These damaged cells, as a part of the defense mechanism, can react

in different manners: 1) differentiate to other cell types without harboring the damages;

32

2) undergo apoptosis (programmed cell death) which eliminates the cell from the living
organism; and 3) enter cell cycle arrest which prolongs the normal length of the cell
cycle, thereby giving more time to repair damage.

D. 3. Induction of apoptosis by chemotherapeutic and/or chemopreventive
agents.

D.3.1. Removal of unwanted cells via apoptosis. Apoptosis is a type of
programmed cell death or cell suicide with distinctive morphological and biochemical
changes that allows deletion of unwanted cells fiom an organism (V aux et al. 1996).
Unlike tissue oncotic necrosis which occurs in response to severe insults and injury to
cells, apoptosis involves an orderly breakdown of cells. In apoptosis, the nuclear
chromatin is condensed and aggregates under the nuclear membrane. Then, activation of
endonucleases causes fiagmentation of DNA into multiples of 200 base pair nucleosome-
sized pieces. Cells shrink, exhibit cytoplasmic budding, and fragment into membrance-
bound vesicles of cytosol and organelles called apoptotic bodies (Michaelson, 1991).
Normally, apoptotic bodies are phagocytosed and degraded without eliciting an
inflammatory response in the surrounding tissue (Walker et al. 1988). The presence of
apoptosis can be determined by alterations in morphological appearance of nucleus and
membrane and presence of endonucleosomal DNA fragments. The distinctive features of

oncotic necrosis and apoptosis are represented in Table 2.1.

33

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D. 3. 2. Induction of apoptosis by chemotherapeutic agent. Data indicate
that many successful chemotherapeutic agents selectively kill target tumor cells without
influencing adjacent normal cells by inducing apoptosis (Sweeney et al. 1996, Hannun,
1997). Susceptibility to apoptotic induction varies by cell type suggesting that
chemotherapeutic agents may induce apoptosis in tumor cells, while arresting the cell
cycle in normal cell counterparts (David, 1994). Disruption of the apoptosis pathway in
tumor cells may lead to a survival advantage or resistance to chemotherapeutic treatment
(David, 1994). Alternatively, inappropriate stimulation of proliferation may cause
apoptosis since prom-oncogenes including c-myc (Green, 1997), c-fos and c-jun (Preston
et al. 1996), which stimulate cell proliferation, can also induce apoptosis.

B.3.3. Induction ofapoptosis by chemopreventive agena. In recent years,
apoptosis has gained considerable interest in biomedical research as the rate of apoptosis
significantly affects the life span of both normal and cancer cells within a living system.
Chemopreventive agents that can modulate apoptosis may be able to affect the steady-
state cell population. Therefore, the chemopreventive agents, which have proven effects
in animal tumor bioassay systems and/or human epidemiology and induce apoptosis in
cancer cells, have great implications for cancer chemotherapy and chemoprevention.

D. 4. Roles of stem cell ditferentiation for cancer prevention.

D.4.I. Characteristics of stem cells. Stem cells are undifferentiated cells that
are capable of proliferation and self-renewal, giveing rise to both differentiated
descendants and more stem cells (Potten, 1997). Totipotent stem cells have unlimited
capacity to specialize into extraembryonic membranes and tissues, the embryo, and all

postembryonic tissues and organs. Pluripotent stem cells are capable of giving rise to

35

many types of cells but not all types of cells necessary for fetal development. (National
Institute of Health, 2001, Trosko and Ruch, 1998). The relatively undifferentiated nature
of tumor cells could be attributed to the blocked differentiation in stem cells or de-
differentiation of differentiated cells that lead to cancer cells (V armur and Weinberg,
1993).

As described in C. 2. Breast cancer etiology and risk factors, early full-term
pregnancy decreases risk for breast cancer. This might be linked to stem cell
multiplication that develops beginning at the time of puberty and during each ovarian
cycle until the first pregnancy, but not after the first pregnancy (Cairns, 1975).
Alternatively, pregnancy may induce full differentiation of the mammary gland (Russo et
al. 1990), reduce the number of stem cells, and thus decrease the susceptibility for
carcinogenesis. In a similar pattern, dietary components may decrease the risk of breast
cancer by reducing the number of stem cells.

D. 4.2. Differentiation genes. Although it is proposed that differentiation plays
an important role in carcinogenesis (Prasad, 1991, Prasad et al. 1998), the specific genes
that induce terminal differentiation in normal or transformed cells have not been
identified yet. Prasad et a] (2001) emphasized the role of differentiation genes (genes
that induce terminal differentiation in normal cells.

Briefly, downregulation of a differentiation gene may be an initiating event to
cause immortalization of normal cells. Such a gene can be referred to as a tumor-
initiating gene in carcinogenesis. Secondary events may include mutations (point
mutation or overexpression) in cellular oncogenes, growth regulatory genes, and tumor

suppressor genes that may convert immortalized cells into transformed cells. Such genes

36

could be called tumor-promoting genes. Prasad et al’s differentiation gene hypothesis for
carcinogenesis predicts the following concepts (for review: Prasad et al. 2001):

“Tumor cells can be induced to differentiate terminally by appropriate agents if
the differentiation gene is not mutated; Conversely, tumor cells with the mutated
differentiation gene can be resistant to terminal differentiation”. 1) An elevation of 3’,
5’-cyclic adenosine monophosphate (cAMP) induces terminal differentiation in
neuroblastoma cells despite the presence of extensive chromosomal anomalies and
mutations in vitro and in vivo (Prasad, 1991, Prasad et al. 1994). This indicates that a
differentiation gene is down-regulated due to alterations in the regulatory genes, rather
than to a mutation in a differentiation gene, and thus a differentiation gene can be
activated by an appropriate agent. 2) The existence of cAMP-resistant neuroblastoma
cells suggests that such cells may carry a mutated differentiation gene.

D.4.3. A mechanism of cAMP-induced differentiation. A possible mechanism
of cAMP-induced differentiation is that an elevated cAMP level may activate a wild-type
tumor suppressor gene (Li et al. 1998) or inactivate a differentiation suppressor gene.
Hovland et al (2001) have identified some candidate “differentiation suppressor genes”
that are down-regulated (N-myc, cyclin B 1, and protease nexin-I), and some candidate
“differentiation genes” (c-fos, c-fes, and RAG-I gene activator) that are upregulated
during cAMP-induced terminal differentiation in neuroblastoma cells (Hovland et al.
2001, for review: Prasad et al. 2001).

D. 5. Suppression of telomerase activity as a novel chemotherapy.

D.5. 1. Telomeres. Telomeres are repetitive TTAGGG sequences at eukaryotic

chromosomal ends that are shortened by 50-200 base pairs after each cell division as a

37

result of the incomplete replication of the 5’ ends of DNA molecules (Shay et a1. 1993).
Telomeres are needed for proper chromosome segregation during mitosis by preventing
nuclease degradation and for end-to-end fusion of chromosomes during replication (Kirk
et al. 1997). Telomere shortening afier each division is correlated with cell senescence in
vitro and cell aging in vivo. The telomere hypothesis suggests that telomere length serves
as a mitotic clock for timing cellular replicative life span (Autexier et al. 1996, Chin and
Harley, 1997).

D. 5.2. Telomerase. Telomerase is a specialized ribonucleoprotein polymerase
that adds hexonucleotides (TTAGGG) onto human chromosomal ends thereby preventing
the replication-dependent shortening of DNA. The human telomerase enzyme is
composed of at least three components including a RNA (hTERC, human telomerase
RNA component), a telomerase-associated protein (TEPl), and a telomerase catalytic
subunit (hTERT, human telomerase reverse transcriptase) with sequence similarity to
reverse transcriptase enzyme (for review: Prasad et al. 2001).

D. 5.3. High telomerase activity in cancers. A high level of telomerase activity
is readily detected in most tumor cells. For example, telomerase activity has been shown
in 98% of bladder cancers (Kyo et al. 1997, Lin et al. 1996), 95% of colorectal cancers
(Tahara et al. 1995a), 94% of neuroblastomas (Hiyama et al. 1995a), 93% of breast
cancers (Hiyama et al. 1996), 85% of liver cancers (Tahara et al. 1995b), 84% of prostate
cancers (Sommerfeld et al. 1996), 80% of lung cancers (Hiyama et al. 1995b), and 75%
of oral carcinomas (Kannan et al. 1997).

In contrast, the majority of studies have demonstrated that most adult normal

human somatic cells show little or no telomerase activity. Several studies have shown

38

that telomerase activity is expressed in human germ line cells (testes and ovaries) (Wright
et al. 1996), human embryonic stem cells (Thomson et al. 1998), human lymphocytes and
hematopoietic progenitor cells (Hiyama et al. 1995, Norrback et al. 1996, Weng et al.
1996, Hohaus et al. 1997), candidate stem cells from the fetal liver (Yui et al. 1998),
human epidermal cells expressing a basal cell marker (Y asumoto et al. 1996), and from
the basal layer (Harle et al. 1996), and in human endothelial (Hsiao et al. 1997) and
uroepithelial cells (Belair et al. 1997). In addition, Sun et al (1999) reported that normal
human breast epithelial cells (HBEC) (type I and type H HBEC) have a low level of
telomerase activity. Type H HBEC show even lower level of telomerase activity than
type I HBEC with stem cell characteristics (Sun et al. 1999).

The presence of high levels of telomerase in most tumor types and immortalized
cells supported the hypothesis that increased telomerase activity is a critical event for
continuous cell proliferation of immortalized cells (Bryan et al. 1997, Counter et al. 1994,
Kim et al. 1994, Rhyu, 1995, Kyo et al. 1997, Lin et al. 1996, Tahara et al. 1995a,
Hiyama et al. 1995a, Hiyama et al. 1996, Tahara et al. 1995b, Sommerfeld et al. 1996,
Hiyama et al. 1995b, Kannan et al. 1997). Kiyono et a1 (1998) have proposed that both
telomerase activity and inactivation of Rb/Pl 6 INK4a are required to immortalize human
epithelial cells.

D.5.4. Roles of telomerase activity on proliferation and differentiation. Belair
et a1 (1997) have proposed that telomerase activity can serve as an indicator for levels of
cell proliferation rather than transformation. High telomerase activity can also be used to
classify the degrees of colorectal carcinomas (Okayasu et al. 1998), esophageal cancer

(Asai et al. 1998), prostate cancer (Uemura et al. 1998) and leukemia (Zhang et al. 1996).

39

Moreover, Sun et a1 (1999) reported that human breast epithelial cells with stem cell
features are more sensitive to telomerase activation and immortalization by SV40 large T
antigen than those with a more differentiated phenotype. However activation of
telomerase does not arise until middle [~60 cumulative population doubling level (cpdl)]
or late (~100 cpdl) passages. This suggests that increased telomerase activity is acquired
subsequent to immortalization.

D. 5. 5. Suppression of telomerase activity as a novel chemotherapy. Researchers
propose that suppression of telomerase activity may be a novel ‘adjuvant therapy’
(anticancer drugs or hormones given after surgery and/or radiation to help prevent the
cancer from coming back) for the treatment of human cancer and detection of telomerase
activity may be a significant tool to diagnose cancer (Schalken, 1998, Holt and Shay,
1999). Data suggest that telomerase activity is associated with aggressiveness of breast
tumors and reflects anti-proliferative effects of chemotherapy. For example, Hooes et a1.
(1998) reported that telomerase activity was detected in all breast cancer patients. A
significant correlation was found between enzyme activity, and tumor size, lymph node
status and stage. Telomerase activity appeared to increase in primary carcinomas with
ongoing tumor progression, while all chemotherapy-treated tumors showed lower
telomerase activity.

D. 5. 6. Regulation of telomerase. The regulation of telomerase in cancer cells
is not clearly understood. Ku et al (1997) demonstrated that a protein kinase C inhibitor,
sphingosine inhibited telomerase activity in NFC-076 human nasopharyngeal cancer
cells. In human breast cancer, telomerase activity is associated with cell cycle regulatory

defects including overexpression of cyclin D1 and/or cyclin E (Landberg et al. 1997).

40

Absence of telomerase activity was seen in Gz/M-synchronized MCF-7 and MBA-435
breast cancer cells (Zhu et al. 1996). Kang et al (1999) reported that AKT (also called
protein kinase B), which is known as anti-apoptotic, enhanced the telomerase activity in
SK-MEL 28 human melanoma cells. Recently, Yang et a1 (Submitted) indicatated that
sphingosine inhibited telomerase activity in transformed tumorigenic type I human breast
epithelial cells (HBEC) suggesting a possible role of sphingolipids in regulation of

telomerase in cancers.

E. Sphingolipids as possible chemotherapeutic and chemopreventive agents

E. 1. Sphingolipids structures. Sphingolipids include ceramides,
sphingomyelin, cerebrosides, sulfatides, and gangliosides, all of which are elaborations of
a long-chain (sphingoid) base, the most common of which is an 18-carbon compound
called sphingosine (Figure 2.2). Sphingosine (trans-4-sphingenine) and sphinganine
(without the 4,5-trans double bond) are the most prevalent free long-chain bases of most
mammalian tissues, but the cellular concentration of sphinganine is usually lower than
that of sphingosine (Menill, 1991).

Sphingomyelin (N—acylsphingosine- l-phosphocholine or ceramide
phosphocholine) is mainly composed of sphingosine as a sphingoid base backbone and
contains an amide-linked fatty acid and a phosphorylcholine as a polar head group
(Figure 2.2). Except for sphingosine l-phosphate, sphingoid bases, ceramides,
sphingosylphosphorylcholine, and sphingomyelin, all other sphingolipids are designated

glycosphingolipids since they contain carbohydrate head groups.

41

Glycosphingolipids include neutral glycosphingolipids and acidic
glycosphingolipids. Neutral glycosphingolipids contain from one (cerebroside) to 20 or
more glucose units (Makita and Taniguchi, 1985, Hakomori, 1983). Acidic
glycosphingolipids contain one or more sialic acid residues (gangliosides) or monoester
groups (sulfatides) (Wiegandt, 1985).

E. 2. Sphingolipid signaling path ways: Ceramide generation caused by
sphingomyelinase activation. Ceramides and sphingoid bases (sphingosine and
sphinganine) fimction in cells as second messengers in signal transduction pathways
which regulate cell proliferation, apoptosis, and differentiation. Extracellular agonists
including cytokines, growth factors, and hormones, stimulate their cell surface receptors
to activate a sphingomyelinase which cleaves sphingomyelin to generate cellular
ceramide (Figure 2.2.).

For example, 10L,25-dihydroxyvitamin D3 (Okazaki et al. 1989), tumor necrosis
factor-a (TNF-oc) (Kim et al. 1991, Mathias et al. 1991), and y—interferon (Kim et al.
1991, Dressler et al. 1992), which are inducers of differentiation of HL-60 human
leukemia cells, cause hydrolysis of sphingomyelin to form ceramide. In addition,
interleukin-1 (Ballou et al. 1992, Mathias et al. 1993), dexamethasone (Rarnachandran et
al. 1990), complement components (N iculescu et al. 1993), and fimgal macrolide
brefeldin A (Linardic et al. 1992) also may generate ceramide as a second messenger.

Cz-ceramide and other short-chain water-soluble analogues of ceramide induced
differentiation in HL-60 leukemia cells which mimicked the effects of 1a,25-
dihydroxyvitamin D3, tumor necrosis factor alpha (TNF-a), and y-interferon on HL-60

cells (Okazaki et al. 1990). Treatment of U937 human myeloid leukemia cells with TNF-

42

0: caused sphingomyelin hydrolysis and resulted in elevation of level of ceramide. In
addition, ceramide analogs or TNF-a caused intemucleosomal DNA fragmentation, a

halhnark of apoptosis in myeloid and lymphoid cells (Obeid et al. 1993).

43

H
WCHZOPO3'CH20H2N+(CH3)3

NH
Sphingomyelin 3

 

NSAID

H
WHICH
W"
Ceramide 3

Ceramidase

H
MCHZOH
Sphingosine "3+

Figure 2.2. Sphingolipids signaling pathway: Ceramide and
sphingosine as mediators for protective effects of sphingomyelin
against cancers. Abbreviations used are: 1,25-(OH)2 D3, 1a,25—
dihydroxyvitamin D3; NSAID, nonsteroidal anti-inflammatory drugs.

1.2540le D: $ Sphingomyelinase

Apoptosis

E3. Sphingolipids occurrence and dietary sphingolipids. Sphingolipids are
found in all eukaryotic and some prokaryotic cells and are located primarily in plasma
membranes and related organelles including the Golgi apparatus, endosomes, and
lysosomes which are functionally associated with cellular responses to external agents
such as growth factors, cytokines, extracellular matrix proteins, neighboring cells and
microbial toxins and receptors (Merrill et al. 1995b). For example, sphingomyelin is a
phospholipid located mainly in the outer leaflet of the plasma membrane and accounts for
20% of plasma membrane lipid (Kolesnick et al. 1991) in most mammalian cells (Parodi,
1997). Furthermore, complex sphingolipids, as components of food, are found in dairy
and soy products at relatively high concentrations (Vesper et al. 1999, Ahn and
Schroeder, 2002a)

E. 4. Prevention of colon carcinogenesis by dietary sphingomyelin.

E. 4. 1. Uptake and metabolism of sphingolipids. Recently, sphingolipids have
emerged as another component of the diet which may help to protect against development
of colon carcinogenesis. Imaizumi et al (1992) reported that dietary sphingomyelin
increased serum sphingomyelin in a concentration-dependent manner implying that
dietary sphingolipids can also reach other organs via the circulation. Furthermore, using
rodents, Schmelz et al (1994) showed that about 12% of dietary sphingolmyelin passes
through the small intestine to the colon. These findings raised the possibility that
consumption of sphingomyelin may provide bioactive sphingolipid metabolites such as

ceramide and sphingoid bases which could inhibit the development of colon cancer

(Figure 2.2.).

45

15.4.2. Inhibition of colon carcinogenesis by sphingolipids in rodent models.
The hypothesis that sphingomyelin metabolites ceramide and sphingoid bases could
inhibit colon carcinogenesis was tested in initiation-promotion studies conducted by
Merrill’s group using sphingomyelin isolated from nonfat dry milk (Dillehay et al. 1994,
Schmelz et al. 1996). The results showed that sphingomyelin at 0.05% of the diet
inhibited formation of aberrant colonic crypts in CF-l mice treated with 1,2-
dimethylhydrazine (Dillehay et al. 1994). A subsequent longer term (34 weeks) study
has shown that sphingomyelin at 0.1% of the diet does not reduce the number of tumors
but causes a higher percentage of adenomas (benign tumors) and lower percentage of
adenocarcinomas (malignant tumors) (Schmelz et al. 1996).

Also, the potential of synthetic sphingomyelins with saturated or unsaturated
sphingoid base backbones to suppress the number of aberrant crypt foci was investigated
using CF—l mice treated with 1,2-dimethylhydrazine (Schmelz et al. 1997). In this study,
the reduction of the number of aberrant colonic foci by synthetic dihydrosphingomyelin
(N-palmitoyldihydrosphingomyelin) (70%, p < 0.0001) was significantly greater than by
synthetic sphingomyelin (N-palmitoylsphingomyelin) (52%, p = 0.002) and milk
sphingomyelin (54%, p = 0.002). This indicates that the 4,5-trans double bond is not
required for the suppression of colon carcinogenesis since synthetic
dihydrosphingomyelin, which lacks the 4,5-trans double bond of the sphingoid base
backbone, efliciently reduced the number of aberrant colonic foci (Schmelz et al. 1997).

Major milk glycosphingolipids including glucosylceramide, lactosylceramide, and

ganglioside GD; effectively reduced aberrant colonic foci in CFl mice treated with colon

46

carcinogen 1,2-dimethylhydrazine (Schmelz et al. 2000). Therefore, the inhibitory
effects of sphingolipids are not altered by the complexity of the sphingolipid head groups.

In addition, diets supplemented with ceramide, sphingomyelin, glucosylceramide,
lactosylceramide, and ganglioside GD3 reduced the number of tumors in all regions of the
intestine in Multiple Intestinal Neoplasia (Min) mice, which have a mutation in the APC
gene resulting in the expression of a truncated Adenomatous Polyposis Coli (APC) gene
product (Schmelz et al. 2001). Therefore, the inhibitory effects of sphingolipids are not
limited to the chemically induced colon cancer animal model.

E. 5. Inhibition of human breast cancer cell growth by sphingolipids. Yang
et a1 (Submitted) tested the potential of sphingolipids to serve as chemotherapeutic and
chemopreventive agents for human breast cancer. The results indicated that both
sphingosine and Cz-ceramiade (a short chain ceramide analog) inhibited proliferation and
caused death of tumorigenic breast cells (in vitro neoplastically transformed type I human
breast epithelial cells (HBEC) and MCF-7 human breast cancer cells) via apoptosis
indicating that both sphingosine and ceramide can function as potential chemotherapeutic
agents. Furthermore, concentrations of sphingosine cytotoxic to tumorigenic breast cells
had no effect on normal type II HBEC. Sphingosine activated the tumor suppressor
retinoblastoma protein by dephosphorylation and inhibited telomerase activity in
transformed tumorigenic type I HBEC. Interestingly, sphingosine induced differentiation
of type I HBEC stem cells to type H normal HBEC, which is more resistant to
carcinogenesis; whereas, Cz-ceramide did not affect differentiation, suggesting that
sphingosine might serve as a potential chemopreventive agent by reducing the number of

target stem cells for neoplastic transformation.

47

E. 6. Ceramide and sphingoid bases as mediators for various anticarcinogenic
agents. It has been reported that anticarcinogenic effects of various agents are
associated with sphingolipid metabolism including the generation of ceramide via
activation of sphingomyelinase (Figure 2.2.). For example, Chan et al. (1998) reported
that sulindac, one of the most extensively investigated nonsteroidal antiinflarnmatory
drugs (NSAH)), induced apoptosis in HCT-116 and SW-480 human colon cancer cells
through increasing the level of arachidonic acid followed by stimulating conversion of
sphingomyelin to ceramide. Furthermore, tamoxifen, widely used for breast cancer
treatment, induced apoptosis in both estrogen receptor positive and negative human
breast cancer cells (Frankfirrt et a1. 1995, Perry et a1. 1995, Gelmann et al. 1996,
Mandlekar et al. 2000). The chemotherapeutic effect of tamoxifin is reduced when
generation of acylated-sphingoid base is impaired, but is enhanced when the degradation
of acylated-sphingoid base is blocked (for review: Senchenkov et al. 2001).

B—sitosterol (the main phytosterol in the diet) inhibited HT-29 human colon
cancer cell growth and caused a 50% reduction in membrane sphingomyelin suggesting
that the conversion of sphingomyelin to simpler sphingolipids such as ceramide and
sphingoid bases might be involved in the protective effects of B—sitosterol (Awad et a1.
1996, 1998). Also, Holtz et al. (1998) reported that B—sitosterol induced apoptosis in
LNCaP human prostate cancer cells by increasing the concentration of ceramide. Cheng
et al. ( 1999) indicated that ursodeoxycholic acid inhibits the development of rat colon
carcinoma with increases in the activities of alkaline sphingomyelinase and caspase-3, a

key regulatory protease in apoptosis.

48

Generation of ceramide was observed in photodynamic treatment mediated
apoptosis of L5178Y mouse lymphoma cells (Separovic et al. 1997). Photodynamic
therapy (PDT) is a novel cancer treatment which can destroy selectively malignant,
premalignant, and benign lesions in patients and is initiated by a harmless
photosensitizer. Perpheophorbide-a methyl ester (a photosensitizer) induced slow and
sustained NF-kB activation by activating the acidic sphingomyelinase that released
intracellular ceramide (Matroule et al. 1999). The tumor killing effect of ionizing
radiation was enhanced by chelerythnine, a PKC inhibitor, via activating
sphingomyelinase (Chmura et al. 1997), while acidic sphingomyelinase-deficient human
lymphoblasts and mice are defective in radiation induced apoptosis (Santana et al. 1996).

E. 7. Induction of apoptosis by ceramide and sphingoid bases. Although
evidence is now emerging which suggests that dietary sphingolipids may protect against
the development of colon cancer and possibly breast cancer, little is known about the
mechanism. Protective effects of sphingolipids against some cancers may be the result of
digestion of complex sphingolipids such as sphingomyelin to ceramide and sphingoid
bases (sphingosine and sphinganine) which play important roles in regulation of cell
growth, differentiation, and apoptosis. For example, ceramide induces apoptosis in JB6
tumor cells (Davis et al. 2000), while sphingosine and a methylated derivative, N,N-
dimethyl sphingosine, induce apoptosis in a variety of human cancer cells including
CMK-7, HL-60, U-937, HRT-18, MKN-74, and COLO-205 cells (Sweeney et al. 1996).

E. 8. Regulation of apoptosis by Bcl-2 family genes. One possible
protective mechanism of sphingolipids is that sphingolipids alter the expression of genes

which modulate apoptosis. For example, Bcl-2 family genes (Reed, 1995) encode for

49

proteins which either can inhibit (e. g. Bcl-Z, BcI-xL) or promote (e. g. Bax, Bak) apoptosis
(Shimizu et al. 2000). For example, in PC-12 cells ceramide induces apoptosis via the
activation of caspase-3 and these effects were blocked by overexpression of bcI-2
suggesting that ceramide may activate caspase-3 by down-regulating bcl-2 (Yoshimura et

al. 1998).

50

E. 9. Regulation of apoptosis by mitogen activated protein kinases MAPKs).

E. 9.1. Major isoforms of MAPKs (ERK, JNK, and p38). Mammalian protein
kinases are categorized into two major families that phosphorylate either serine and/or
threonine residues (serine/threonine kinases) or tyrosine residues (tyrosine kinases). A
major role for cellular kinases is to participate in signal transduction pathways through
which cells respond functionally to external messages or to extracellular stresses. A
family of serine/threonine mitogen-activated protein kinases (MAPKs) are activated by
diverse stimuli (Cohen, 1997, Fanger et al. 1997, Ferrell, 1996, Marshall, 1995). Major
isoforms of MAPKs include: Extracellular signal-regulated kinase—l (ERK1) and ERK2,
also known as p44 and p42 MAP kinases, c-Jun NHz-terminal kinase/stress-activated
protein kinase (JNK/SAPK), p38/RK/CSBP (p38), and big mitogen-activated protein
kinase-l/ERKS (BMK-l/ERKS) (Cohen, 1997, Fanger et a1. 1997, Ferrell, 1996,
Marshall, 1995, Kato et al. 1998) (Figure 2.3).

[5.9.2. Roles of ERK, JNK, and p38. ERK] and ERK2 represent the
prototypical mammalian MAPKs. ERK catalytic activation requires phosphorylation by
MAP kinase kinase/ERK kinase kinase (MEK), exemplified by MEK-l (a dual
specificity kinase), which in turn is phosphorylated and activated by a MAPK kinase
kinase (also called MEK kinase) of the Raf family. Among these, c-Raf-l has been
studied mostly and is activated at the plasma membrane after binding to the GTP-bound
form of the low molecular weight G-protein p21ras (Cohen, 1997, Fanger et al. 1997,
Ferrell, 1996, Marshall, 1995). As with ERK, JNK/SAPK, p38, and BMK-I/ERKS
kinases are activated by phosphorylation that occurs at a specific threonine and tyrosine

residue (Figure 2.3).

51

Substrate proteins that MAPKs phosphorylate include: additional ‘downstream’
serine/threonine kinases (which themselves become activated), cytoskeletal elements, cell
death regulators, a number of nuclear receptors and transcription factors (for review:
Camps et a1. 1999, Wilkinson and Millar, 2000). For example, ERK is linked to cellular
chemotaxis, cell cycle progression, mitogenesis, oncogenic transformation, metastasis,
neuronal differentiation and survival, and processes underlying memory and learning
(Cohen, 1997, Fanger et a1. 1997, Ferrell, 1996, Marshall, 1995, Komhauser and
Greenberg, 1997, Atkins et a1. 1998, Mansour et al. 1994, Cowley et al. 1994, Dudley et
al. 1995, Reszka et al. 1997, Pages et al. 1993, Xia et a1. 1995). Emerging data suggest
that the ERK pathway functions mainly to positively regulate the cell cycle dming G1 at
the level of cyclin D synthesis, assembly of cyclin D-dependent kinase complexes and
subsequent phosphorylation of retinoblastoma protein (Wilkinson and Millar, 2000).

On the contrary, JNK/SAPK and p38 play important roles in pathways regulating
T cell differentiation, production of inflammatory cytokines and eicosanoids, and
apoptotic cell death (Cohen, 1997, Yang et al. 1997a, Yang et al. 1998b, Dong et a1.
1998, Xia et al. 1995, Saklatvala et al. 1996, Ichijo et al. 1997, Yang et al.
l997b,Verheij et al. 1996, Zanke et a1. 1996). JNK/SAPK may oppose mitogenic
functions of ERK and evoke a stress-induced cell cycle arrest. Accumulative studies
have focused pathway identifications in immortalized cell lines using dominant negative
or interfering mutants of JNK and p38. However, transformation of normal primary cells
requires additional alterations in oncogenes or tumor suppressor genes (Figure 2.3).

15.9.3. Activations of ERK, JNK, and p38. Diverse cell stimuli preferentially

activate distinct MAPKs. Growth factors, G-protein linked receptors, cell adhesion,

52

phorbol esters (known tumor promoters), and some oncogenes activate the ERKs,
whereas inflammatory cytokines, trophic factor deprivation, and cell stresses
preferentially activate JNK/SAPK and p38 (Cohen, 1997, Fanger et al. 1997, Ferrell,
1996, Marshall, 1995). Little is known about BMK-l/ERKS responsiveness, although
Kato et a1 (1998) reported that both growth factors and stressful stimuli may activate the
BMK-l/ERKS (Figure 2.3).

E. 9. 4. Apoptosis triggered by imbalance between ERK, JNK, and p38. MAP
kinases pathways do not function disj ointedly, instead they are integrated into networks
of cellular signaling that affect upon and are influenced by MAP kinases. Furthermore,
cells receive diverse stimuli that lead to integrated responses and cross-talk between
multiple signaling pathways. Imbalance of these signals can alter transcription, cell cycle
progression/arrest, and switch of cell survival versus apoptosis (Figure 2.3).

E. 9. 5. Regulation of MAPKs by sphingolipids. Studies have indicated potential
connections between sphingolipid signaling and MAP kinases pathways. For example,
Cz-ceramide stimulated JNK activity and increased c-Jun mRNA levels, while C2-
dihydroceramide had no effect in HL-60 human leukemia cells (W estwick et al. 1995).
Jarvis et al (1997) showed that ceramide-mediated lethality is predominantly associated
with strong stimulation of JNK2/JNK1 and weak inhibition of ERKl/BRKZ, whereas
sphingosine-mediated lethality is largely associated with weak stimulation of
JNK2/JNK1 and strong inhibition of ERKl/ERK2 in U937 human monoblastic leukemia
cells.

Sakakura et al (1997) reported that sphingosine and N,N-dirnethylsphingosine

induced apoptosis and significantly inhibited ERK activity in tumor cell lines with high

53

ERK activity. In contrast, untransfonned cells and those tumor cell lines with low ERK
activity displayed no significant change in activity and no apoptosis. Furthermore, high
concentrations of Cz-ceramide, which induced apoptosis in the solid tumor cells, did not
demonstrate significant effect on ERK activity. In an additional study, Sakakura et al.
(1998) showed that D-erythro—sphingoinse was the most potent stimulator for ERK
activity among D-erythro-sphingosine, L-threo-sphingosine, and DL-erythro-
dihydrosphingosine tested, while DL-erythro-dihydrosphingosine was completely
inactive. In summary, an altered balance between ERK, JNK, and p38 (Xia et al. 1995)

may trigger induction of apoptosis by sphingosine and ceramide.

54

Growth factors (EGF, PDGF) Stress, UV, DNA damaging agent, Oxidant

Mitogens, Hormones Osmotic and heat shock, Lipopolysaccharide
Phorbol ester Inflammatory cytokines (T NFa, lL—1)
./ ‘s ,x
\ if

RTK / \
Ras / \
Raf (MKKK, MEKK) MEKK1,2,3 MEKK1,4

MKK1,2 (MEK1,2) MKK4,7(JNKK1,2) MKK3,6

l

ERK1,2 JNK1,2,3 p38
Elk-1 1 1
Elk-1 Elk-1
cPLA2 ATF2 ATF2
MSK1 cJun cPLA2
PPARr MSK1

Proliferation, Apoptosis

Figure 2.3. Mitogen Activated Protein Kinases signaling pathway.
Abbreviations used are: EGF, epidermal growth factor; PDGF. platelet derived
growth factor; TNFa, tumor necrosis factor-a; lL-1, interleukin-1; RTK,
receptor tyrosine kinase; MKKK, MAP kinase kinase kinase; MKK, MAP kinase
kinase; MEK, MAP/ERK kinase; MEKK, MAP/ERK kinase kinase; ERK.
extracellular signal regulated kinase; JNK, c-Jun NH2-terminal kinase; JNKK,
JNK kinase; cPLA2, cytosolic phopholipase A2; MSK1, mitogen-and stress-
activated protein kinase-1; PPARr, peroxisome proliferator activated receptor
gamma; CHOP. cAMP response element binding protein homologous protein;
p90RSK, ribosomal p90 S6 kinase.

55

E.10. Regulation of apoptosis by AK T.

E.10.1. AK T activation as cell survival and anti-apoptotic signal. AKT (also
referred to as protein kinase B) is a serine/threonine kinase which plays a significant role
in regulating the balance between cell survival and apoptosis (Zhou et al. 2000). An anti-
apoptotic role of AKT is rooted fiom studies demonstrating that growth factors such as
insulin, insulin-like growth factor-1, and nerve grth factor, which block apoptosis,
increase activity of AKT by expressing either its constitutively active forms or its
upstream regulators. Furthermore, enhanced AKT activity is associated with cancer
progression in various tissues (Graff et al. 2000, Krasilnikov, 2000, Tsatasanis and
Spandidos, 2000, Yuan et al. 2000).

AKT appears to promote cell survival and to inhibit apoptosis by its ability to
phosphorylate and inactivate several pro-apoptotic targets, including Bad (Cardon et al.
1998), caspase-9 (Rommel et al. 1999), and Forkhead transcription factors (Brunet et al.
1999, Kos et al., 1999). The cell survival and anti-apoptotic actions of AKT are
presented in Figure 2.4.

E] 0.2. Activation/phosphorylation of AK T at thr308 and serl 73. AKT has an
amino-terminal pleckstrin homology (PH) domain that binds phosphorylated lipids at the
membrane in response to activation of P13 kinases. AKT is activated via binding of
phospholipids and phosphorylation of activation loop at thr308 by 3-phosphoinositide—
dependent kinase (PDK) (Alessi et al. 1996) and also within the C-terminus at ser473
(Figure 2.4).

E] 0.3. Regulation of AK T by sphingolipids. Recent studies evolve potential

connections between sphingolipid signaling and AKT pathways. For example, a study in

56

Hep3B human hepatoma cell line by Chang et al. (2001b) demonstrated that sphingosine
induced apoptosis and suppressed phosphorylated/active forms of AKT. Moreover,
sphingosine-induced apoptosis was attenuated in cells transfected with constitutively
active AKT.

In HMNl motor neuron cells, Zhou et al. (1998) indicated that Cz-ceramide
induced apoptosis and decreased both basal and insulin- or serum- stimulated AKT
kinase activity. Furthermore, constitutively active AKT kinase reduced Cz-ceramide-
induced cell death of HM] cells as well as COS-7 cells. Taken together, the AKT
pathway is a probable target for actions of ceramide- (Zhou et al., Schubert et al, 2000)

and sphingosine-induced (Chang et al, 2001b) apoptosis.

57

EGF, PDGF

Insulin
\,
PI3K
AKT l’\ Apaf-1
\ Iliad \ I
74 x ° "
14-3-3 , > '
, ,, z \ Caspase 9

Apoptosis
(Death)

 

Cell survival

Figure 2.4. The cell survival and anti-apoptotic actions of AKT.

Abbreviations used are: EGF, epidermal growth factor; PDGF, platelet
derived growth factor; PI3K, phosphatidylinositol 3-kinase. Images in this
dissertation are presented in color.

58

E.11. Involvements of Ca or calmodulin in proliferation and apoptosis
pathways. Studies have shown that calcium and/or calmodulin play significant roles
in DNA synthesis and cell proliferation (Chafouleas et al. 1982, Durha and Walton 1982,
Parsons et al. 1983, Means 1988). There are conflicting data regarding the association
between calcium channel blockers or calmodulin antagonists and cancer risk or cancer
cell growth.

For example, a cohort study by Sorensen et al. (2000) reported that there is no
evidence of increased mortality of colon or breast cancer with administration of Ca
channel blockers (verapamil, dihydropyridines, diltiazem, and mixed uses of these).
Batra and Alenfall (1991) tested the effects of Ca antagonists (perhexilene, prenylamine,
bepridil), Ca channel blockers (verapamil, diltiazem, nifedipine), calmodulin antagonists
(pirnozide, R245 71, Trifluoroperazine), other compounds having a mixed Ca and
calmodulin inhibitory effect on proliferation of HT-29 human colon cancer cells. The
data suggested that in general, Ca antagonists had a moderate to strong inhibitory growth
effects. Ca channel blockers were less effective and calmodulin antagonism seemed to be
a common feature of compounds inhibiting cell proliferation in HT-29 cells.

Recently Kim et a1 (2001) reported that fumonisin Bl, which blocks sphinganine
(sphingosine) N-acyltransferase (also called ceramide synthase) and causes sphinganine
accumulation, induced apoptosis by increasing the calmodulin expression at the levels of
mRN A and protein. A calmodulin antagonist W7 attenuated fumonisin Bl-induced
apoptosis in LLC-PKl pig renal epithelial cells (Kim et al. 2001). Different effects of
W7 on apoptosis induced by various agents in a variety of cells are represented in Table

2.2.

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E. 12. Summary: Mechanisms of actions of sphingolipids in proliferation,
apoptosis, and differentiation. Cumulative data suggest that sphingolipids have
potential as chemotherapeutic and chemopreventive agents based on their abilities to: 1)
inhibit proliferation; 2) arrest cell cycle; 3) induce apOptosis in a variety of cancer cells;
and 4) sphingolipids may induce differentiation of stem cells. Mechanisms by which
sphingolipids inhibit colon and breast carcinogenesis are not clearly understood. Possible
targets affected by sphingolipids to inhibit carcinogenesis include mitogen activated

protein kinases (MAPKs), AKT, or Bel-2 family genes.

61

F. Cellular models to study chemotherapy and chemoprevention

FJ. Human colon cancer cells. HT-29 and HCT-116 human colon cancer
cells provide useful models to study cancer chemotherapy. Both HT-29 and HCT-l 16
cells possess adherent growth property and epithelial morphology and are tumorigenic in
nude mice GBrattain et a1. 1981) (Figure 2.5-Photographs). HT-29 and HCT-l 16 cells
show different mutation status in genes related to carcinogenesis and apoptosis (Table
2.3).

F.1.I. H T-29 cells. HT-29 cells derived from colorectal adenocarcinoma
harbor an APC tumpr suppressor gene mutation (Liu et al. 1997) and over-express a
mutated nonfunctional p53 tumor suppressor gene (Rodrigues et a1. 1990, Redman et al.
1997), but carry normal B-catenin (Liu et al. 1997) and ras (K-ras, H-ras, N-ras) proto-
oncogenes (Olivier et al. 1999). Also, HT-29 cells express c-myc, Myb, sis, fos
oncogenes, and receptors for urokinase and vitamin D, but do not express N-myc
oncogene (American Type Culture Collection, 2001). HT -29 cells express catalytically
inactive cyclooxygenase 2 (COX2), which is also called prostaglandin H synthase
2 (PHSZ) (Hsi et al. 2000) (for review regarding COX2: Levy, 1997) (Figure 2.5, Table
2.3).

F.1.2. HGT-116 cells. In contrast, HCT-116 cells derived from human
colorectal carcinoma carry functional/normal APC (Groden et al. 1995, Narayan and
Jaiswal, 1997), p53 (Zhang et al. 2000), box (pro-apoptotic gene) (Zhang et al. 2000).
However HCT-116 cells have mutated B-catenin (Ilyas et al. 1997) and carry a mutation

in codon 13 of the ras proto-oncogene and can be used as a positive control for

62

polymerase chain reaction (PCR) assays of mutation in this codon (Schroy et al. 1995).
HCT-116 cells do not express COX2 (Kutchera et al. 1996, Hsi et al. 2000) (Table 2.3).
F.1.3. H T -29 and HGT-116 cells for chemotherapy studies. HT-29 and
HCT-116 human colon cancer cells can be good models to study chemotherapy as they
show epithelial morphology and epithelial cells are the target cells of colon
carcinogenesis (Hill et al. 1978) and are tumorigenic in viva. Furthermore, it is useful to
study the gene targets of actions of sphingolipids as the two cells possess different
mutation status in genes related to colon carcinogenesis including APC, ,B-catenin, ras,
p53, and COX2. The differences in gene mutation status in HT-29 and HCT-116 cells are

summarized in Table 2.3.

63

HT-29 . Her-116

   
 

  

-.'._ ’4 f'
, ‘ ‘ “id"

Human Colon Cancer Cells

Type I Tumorigenic type I Type II

   

Human Breast Epithelial Cells

Figure 2.5. HT-29 and HGT-116 human colon cancer cells. Type I
normal human breast epithelial cells (HBEC), tumorigenic type I HBEC,

and type II HBEC (photographs). Images in this dissertation are
presented in color.

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F.2. Human breast epithelial cells for chemoprevention study models. Human
breast epithelial cells (HBEC) used in the current study were developed from tissues of
healthy women obtained during reduction mammoplasty (Kao et al. 1995) (Type I and
type H HBEC) (Figure 2.5; Figure 2.6; Figure 2.7; Table 2.4). Type I HBEC show stem
cell characteristics and type H HBEC display basal epithelial cell phenotypes. In vitro
neoplastically transformed type I HBEC (M13SV1R2N1) shows characteristics of
tumorigenic breast cells (Figure 2.5; Figure 2.6; Figure 2.7). Derivation of type I HBEC,
type II HBEC and neoplastically transformed type I HBEC are represented in Figure 2.7.
Phenotypic differences between type I and type II HBEC are summarized in Table 2.4.

F.2.1. T ype I HBEC as normal stem cells. Type I normal human breast
epithelial cells (HBEC) display stem cell characteristics and are able to differentiate to
type II HBEC (Figure 2.6), (Kao et al. 1995, Kang et al. 1997). Type I HBEC show
variable shapes with smooth boundary, lack gap junctional intercellular communication,
form budding/ductal structures on matri-gel (extracellular matrix), express estrogen
receptor (Kang et a1. 1997) and luminal epithelial markers (e.g. epithelial membrane
antigen, cytokeratin-IS, cytokeratin-19). Type I HBEC are more sensitive to telomerase
activation and to immortalization after SV40 transformation (Kao et al. 1995, Sun et al.
1999). Therefore, type I HBEC appear to be the major target cells for breast
carcinogenesis. Fetal bovine serum (FBS) stimulates type I HBEC growth. Type I
HBEC and these simian virus 40 (SV 40) transformed cells can grow in an anchorage
independent fashion (Kao et al. 1995). Phenotypic differences between type I and type II

HBEC are summarized in Table 2.4.

66

F.2.2. Transformed tumorigenic type I HBE C. Derivation of in vitro
neoplastically transformed HBEC lines fiom type I HBEC has been previously reported
(Kao et al. 1995, Kang et a1 1998, for review: Chang et al. 2001a) (Figure 2.5; Figure
2.7). Type I HBEC are sequentially transformed to immortalized/non-tumorigenic cells
(M13SV1), weakly tumorigenic cells (M13SV1R2), and highly tumorigenic cells
(M138V1R2N1) by transfections with SV-40, x—ray, and neu oncogene (Figure 2.7).
Transformed tumorigenic type I HBEC (M13SV1R2N1) are similar to breast carcinomas
and possess many phenotypes of type I HBEC (eg. expression of epithelial membrane
antigen, cytokeratin 18 and estrogen receptors, and deficiency in gap junctional
intercellular communication) (Kao et al. 1995, Kang et al. 1997) (Table 2.4.). In the
current study, transformed tumorigenic type I HBEC (MIBSVIRZNI) were used to
represent a breast cancer cell model.

F.2.3. Type II HBE C as normal cells. Type H HBEC do not express
estrogen receptors. Type H HBEC express basal epithelial cell markers (e. g. a-6 integrin
and cytokeratin-14), have gap junction genes (e.g. connexin 26 and 43), grow in an
anchorage dependent fashion, and rarely become immortal afier SV40 transformation
(Kao et al. I995, Sun et al. 1999). Fetal bovine serum inhibits type H HBEC growth
(Table 2.4.).

F.2.4. HBE C for chemotherapy and chemoprevention studies. Type I HBEC
carry stem cell characteristics and are more susceptible to neoplastic formation indicating
that type I HBEC are an excellent cell model for chemopreventive study since stem cells

appear to be the targets of breast carcinogenesis. Chemotherapeutic potential of the

67

compounds can be studied in transformed type I HBEC as breast tumor cells in
comparison to type H HBEC as normal breast cells.

E2. 5. Criteria for identification of chemotherapeutic and chemopreventive
agents in HBEC. The following criteria can be applied to identify the
chemotherapeutic and chemopreventive properties of dietary compounds in Type I,
transformed Type I, and Type H human breast epithelial cells:

1) Successful chemopreventive agents inhibit proliferation and induce apoptosis

and differentiation in type I HBEC, but do not affect growth of type II HBEC.

This will decrease the target stem cells for neoplastic transformation.

2) Successful chemotherapeutic agents inhibit proliferation and induce apoptosis

in transformed type I HBEC.

68

Type II HBE

Type I HBEC

    
 

 

 

Type I HBEC

 

After 24 h, After 2 weeks,
mammary gland-like organoid ductal & terminal end
bud-like structures

Figure 2.6. Differentiation of type I HBEC with stem cell characteristics to
type II HBEC and formation of ductal and terminal end bud-like structure
from mixture of type I and type II HBEC (photographs). Type | HBEC are
differentiated to type II HBEC over the course of time. Type I or a mixture
of type I and type II HBEC have a unique ability to form mammary gland-
like organoids after 24 h and ductal and terminal end bud-like structures
after 2 weeks on Matrigel. Top panel: Magnification 10X plus 4X. The
second and third photos are used with Dr. C.-C. Chang’s permission.
Bottom panel: A photo on the left is obtained from Chang et al. 2001a and
a photo on the right is from Sun et al. 1999, which are used with Dr. C.-C.
Chang’s permission. Images in this dissertation are presented in color.

Type I HBEC (stem cells) Type II HBEC

Differentiation \
r

t— SV40 transfection —-i

, M13SV1_ lmmortalized at
Immortallzed at '0“, fre uenc
high frequency q y

 

X-ray

‘\\\‘\\\-‘\“- ~\

\.

\

V

T \.
a \__

M1 3SV1 R2
Weakly tumorigenic

C-erbBZ/neu oncogene

‘T

M13SV1R2N1
Highly tumorigenic

 

Tumorigenic type I HBEC

Figure 2.7. Derivation of tumorigenic type I HBEC and type II normal
human breast epithelial cells (HBEC) from type I HBEC with stem cell
characteristics. Abbreviation used: SV40, simian virus 40.

70

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71

CHAPTER 3

Sphingoid bases and ceramide induce apoptosu in [IT-29 and HCT-ll6
human colon cancer cells1

Running title: Sphingolipids and colon cancer cell apoptosis
Key words: sphingosine; sphinganine; ceramide; colon cancer cells; apoptosis

1This chapter is published in Experimental Biology and Medicine (2002) 227:345-353
with authors Eun Hyun Ahn and Joseph J. Schroeder.

72

ABSTRACT
Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been
reported to inhibit development of colon cancer. This protective role may be the result of
turnover to bioactive metabolites including sphingoid bases (sphingosine and
sphinganine) and ceramide which inhibit proliferation and stimulate apoptosis. The
purpose of the present study was to investigate the effects of sphingoid bases and
ceramides on growth, death, and cell cycle of HT-29 and HGT-116 human colon cancer
cells. The importance of the 4,5-trans double bond present in both sphingosine and C2-
ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of
these lipids to those of sphinganine and Cz-dihydroceramide (a short chain analog of
dihydroceramide) which lack this structural feature. Sphingosine, sphinganine, and C2-
ceramide inhibited growth and caused death of colon cancer cells in time- and
concentration-dependent manners; whereas, Cz-dihydroceramide had no effect. These
findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of
Cz-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via
fluorescence microscopy and quantitation of fragmented low molecular weight DNA
using the diphenylamine assay demonstrated that sphingoid bases and Cz-ceramide cause
chromatin and nuclear condensation as well as fragmentation of DNA suggesting these
lipids kill colon cancer cells by inducing apoptosis. Flow cytometric analyses confirmed
that sphingoid bases and Cz-ceramide increased the number of cells in the A0 peak
indicative of apoptosis and demonstrated that sphingoid bases and ceramide in HT29
cells and sphinganine and ceramide in HCT-116 cells arrest the cell cycle at Gle phase.

These findings establish that sphingoid bases and ceramide induce apoptosis in colon

73

cancer cells and implicate them as potential mediators of the protective role of more

complex dietary sphingolipids in colon carcinogenesis.

74

INTRODUCTION

Recently, dietary sphingolipids have gained attention for their potential to protect
against the development of colon cancer. Sphingolipids are a family of compounds
which have a long-chain (sphingoid) base backbone and include free sphingoid bases
(sphingosine and sphinganine), ceramides, sphingomyelins, cerebrosides, sulfatides, and
gangliosides (Merrill et al. 1997). Several studies have been conducted with milk
sphingomyelin, synthetic sphingomyelin, synthetic dihydrosphingomyelin, as well as
milk glycosphingolipids to determine if the sphingolipids protect CFl mice from 1,2-
dimethylhydrazine (DMID-induced colon cancer (Dllehay et al. 1994; Schmelz et al.
1996, 1997, 2000). The results showed that sphingomyelin at 0.1% of the diet caused a
higher percentage of adenomas and lower percentage of the more advanced
adenocarcinomas (Schmelz et al. 1996). Interestingly, synthetic dihydrosphingomyelin
(N-palmitoyldihydrosphingomyelin) more potently reduced the number of aberrant
colonic foci than synthetic sphingomyelin (N-palmitoylsphingomyelin) and milk
sphingomyelin, suggesting that the 4,5-trans double bond, which is absent in
dihydrosphingomyelin, is not required for the suppression of colon carcinogenesis
(Schmelz et al. 1997). Furthermore, major milk glycosphingolipids including
glucosylceramide, lactosylceramide, and ganglioside GD3 effectively reduced aberrant
colonic foci in CF] mice treated with DMH (Schmelz et al. 2000). In addition, diets
supplemented with ceramide, sphingomyelin, glucosylceramide, lactosylceramide, and
ganglioside GD3 reduced the number of tumors in all regions of the intestine in Multiple
Intestinal Neoplasia (Min) mice with a truncated Adenomatous Polyposis Coli (APC)

gene product (Schmelz et al. 2001). Therefore, the inhibitory effects of sphingolipids are

75

not altered by the complexity of the sphingolipid head groups and are not limited to the
chemically induced colon cancer animal model.

Although evidence is now emerging which suggests that dietary sphingolipids
may protect against the development of colon cancer, little is known about the
mechanism. Sphingolipid metabolites such as ceramide and sphingoid bases play
important roles in regulating cellular behavior and have been implicated as putative
second messengers in signaling pathways (Merrill et al. 1997). For example, ceramide
induces apoptosis in JB-6 tumor cells (Davis et al. 2000) and HT-29 human colon cancer
cells (V eldman et al. 1998), while sphingosine and its methylated derivative N,N-
dimethyl sphingosine induce apoptosis in a variety of human cancer cells including
CMK—7, HL-60, U-937, HRT-18, MKN-74, and COLD-205 cells (Sweeney et al. 1996).
Therefore, a possible mechanism by which complex dietary sphingolipids may reduce
colon carcinogenesis is via digestion to bioactive metabolites such as ceramide and
sphingoid bases.

The purpose of the present study was to systematically examine the effects of
sphingoid bases and ceramides on growth, apoptotic cell death, and cell cycle of PIT-29
and HGT-116 human colon cancer cells. Both cell lines show epithelial morphology, are
tumorigenic in vivo, and are commonly used to study chemotherapy (Hill et al. 1978), but
have different genetic mutations. HT-29 cells carry mutations in APC (Liu et al. 1997)
and p53 (Rodrigues et al. 1990; Redman et al. 1997), but have normal ,B-catenin (Liu et
al. 1997) and ms proto-oncogene (Olivier et al. 1999) and express catalytically inactive
COX2 (His et al. 2000). In contrast, HCT-116 cells carry mutations in fl-catem'n (Ilyas et

al. 1997) and ms (Schroy et al. 1995), but have normal APC (Groden et al. 1995) and p53

76

(Zhang et al. 2000) and do not express COX2 (Kutchera et al. 1996). In order to evaluate
the significance of the 4,5-trans double bond, the effects of sphingosine and the cell
permeable ceramide analog Cz-ceramide were compared to those of sphinganine and C2-
dihydroceramide which lack this structural feature (Figure 3.1). Morphological and
biochemical features of apoptosis were assessed via fluorescence microscopy,
quantitation of fiagmented low molecular weight DNA using the diphenylamine assay,

and flow cytometry.

77

OH

WCHZOH

NH3+ Sphingosine

OH

NH3+ Sphinganine

OH
WCHZOH
\

8

OH

\

H3C\,NH C2-Ceramide

3

OH
WCHZOH

H3CJH Cz-dihydroceramide

3

Figure 3.1. Structures of sphingosine, sphinganine, ceramide, Cz-ceramide,
and Cz-dihydroceramide.

78

MATERIALS AND METHODS

Chemicals and reagents. Sphingolipids were obtained from Matreya
(Pleasant Gap, PA). All other chemicals were from Sigma (St. Louis, MO) unless
otherwise indicated.

Sphingolipid treatment. Sphingosine and sphinganine were prepared as 1:1
complexes with bovine serum albumin (BSA) dissolved in phosphate buffered saline
(PBS) while Cz-ceramide and Cz-dihydroceramide (cell permeable short chain analogs of
naturally occurring ceramide and dihydroceramide, Figure 3.1) were dissolved in ethanol.

Cell culture. HT-29 and HGT-116 human colon cancer cell lines were
purchased from American Type Culture Collection (Rockville, MD). Stock cultures of
HT-29 and HCT-116 cells were cultured in 100 mm dishes (Corning, Cambridge, MA)
containing Dulbeco’s Modified Eagle Medium (DMEM) (Gibco BRL, Life
Technologies, Gaithersburg, 1WD) supplemented with 10% fetal bovine serum (F BS)
(GibcoBRL, Life Technologies, Grand Island, NY), 3.5 g glucose/L and 2.5 mL
penicillin-streptomycin/L (GibcoBRL, Life Technologies, Grand Island, NY.) at 37°C
and 5% C02. All experiments were performed using cells with from passage 20 or less.

Assessment of cell proliferation. To assess the effects of sphingolipids on cell
growth and death, total nucleic acids were measured as previously described (Li et al.
1990) and used as an index of cell number. Briefly, HT-29 or HGT-116 cells were
seeded at a density of 3 x 105 cells/mL (i.e. 6.3 x 104/cm2 growth area) in 6-well dishes
and cultured with 2 mL of DMEM with 10% FBS for 24 or 36 h to insure that cells were
in log phase before treatment with sphingolipids. Then the medium was replaced with

DMEM supplemented with 1% FBS. Various concentrations of sphingolipids were

79

added directly to each dish and the cells were cultured for up to 48 h. Following
treatment, the medium and dead floating cells were removed, the viable attached cells
were rinsed with 1 mL of phosphate-buffered saline (PBS), and then the cells were lysed
with 1 mL of 0.1M NaOH. Total nucleic acid concentration was determined by
measuring absorbance of the cell lysate at 260 nm using Gene Quant-RNA/DNA
Calculator (Pharmacia Biotech, Piscataway, NJ).

Fluorescence microscopic detection of apoptotic cells. To determine
whether sphingolipids kill cells via apoptosis, cells were seeded at a density of 2 x 105
cells/mL (i.e. 4.2 x 104/cm2 growth area) in 6-well dishes and cultured with 2 mL of
DMEM with 10% FBS for 24 h to insure that cells were in log phase before treatment
with sphingolipids. Then the medium was replaced with DMEM supplemented with 1%
FBS and cells were cultured with sphingolipids at 20 M for 24 h. Both attached and
detached cells were collected, stained with acridine orange and ethidium bromide and
examined via fluorescence microscopy (Mishell et al. 1980). Acridine orange
intercalates into DNA and causes the DNA appear to green. It also binds to RNA but
cannot intercalate so that the RNA stains red-orange. Ethidium bromide is only taken up
by nonviable cells and intercalates into DNA, causing it to appear to orange, but binds
only weakly to RNA. Thus, dead cells will have bright orange chromatin because the
ethidium bromide overwhelms the acridine orange. Cells were photographed using a
fluorescence microscope equipped with a camera under 10X plus 40X magnification with
400/490nm excitation and 520nm emission (Nikon Labophoto, Nikon Inc. Instrument

Group, Garden City, NY.) Apoptotic and non-apoptotic cells were classified by the

80

differences in their chromatin organization as previously described (Martin and Lenardo,
1991)

Isolation of fragmented low molecular weight DNA. Cells were seeded at
a density of 2 x 105 cells/mL (i.e. 1.8 x 104/cm2 growth area) in 100 mm dishes and
cultured with 5 mL of DMEM with 10% FBS for 24 h to insure that cells were in log
phase before treatment. Then the medium was replaced with DMEM supplemented with
1% F BS and cells were cultured with sphingolipids at 50 W for 48 h. Afier treatments,
both attached and detached cells were collected and pelleted via centrifugation (150xg,
5min) and the supernatant was removed. Lysis buffer at 200 uL was added and the cells
were incubated for 10 min at 4°C. Afler centrifugation (14,000xg, 30 min, 4°C) the
supernatant containing fiagmented DNA was transferred into a new 1.5 mL Eppendorf
tube. Pellets were resuspended in 150 uL of TE buffer and were incubated with 2 uL of
40 mg/mL RNAase A at 37°C for l h and 4 uL of 40 mg/mL Proteinase K (Life
Technologies-Gibco BRL, Rockville, MD) at 37°C for 1 h.

Quantitation of fragmented low molecular weight DNA using the
diphenylamine assay. Fragmented DNA was quantitated via the diphenylamine
assay (Burton 1968). Briefly, 108 1.1L of 5 M perchloric acid was added to samples (final
concentration of perchloric acid was 1 M) and samples were heated at 70°C for 15 min.
Then diphenylamine reagent (1.5 g diphenylamine, 1.5 mL concentrated sulfiiric acid and
0.5 mL 1.6% aqueous acetaldehyde in 100 mL glacial acetic acid) at 1076 uL was added
and the samples were incubated at 30°C for 15-18 h for color development. Salmon

Sperm DNA (GibcoBRL, Grand Island, NY.) was used as a standard. Absorbance was

81

measured at 600 nm and the percentage of fragmented DNA was calculated as:
(Fragmented DNA/Total DNA) x 100.

F low cytometric analysis of cell cycle and population. Flow cytometric
analysis was performed as described by Telford et al. (1994) with some modifications.
Cells were seeded at a density of 2 x 105 cells/mL (i.e. 1.8 x 104/cm2 growth area) in 100
mm dishes and cultured with 5 mL of DMEM with 10% FBS for 24 h to insure that cells
were in log phase before treatment. Then the medium was replaced with DMEM
supplemented with 1% FBS and cells were cultured with sphingolipids at 35 M for 24 h.
After treatments, both attached and detached cells were collected, trypsinized, and
filtered through 40 um nylon sieve. Following centrifugation (150xg, 5 min), the
supernatant was removed and PBS (pH 7.4) containing 50% FBS was added. Cells at 106
to 2x10‘5 cells/tube were prepared and fixed with 70% cold ethanol at a final

concentration of 50-53%. After cells were stored at 4°C for 1-5 h, cells were pelleted by

centrifugation (150xg, 5 min) and resuspended in 500 uL of PBS containing 4% FBS
(Darzynkiewcz et al. 1980). Then, 1 mL DNA staining solution [4% FBS, 0.1% Triton
X—100, 100 M EDTA, 0.05 mg/mL RNase A (50 units/mg), 50 ug/mL propidium iodide
in PBS (pH 8.0)] was added and the cells were kept at 4°C for 1-2 h before reading using
a FACS Vantage Flow Cytometer (Becton Dickinson, San Jose, CA). The total number
of cells analyzed for each sample was 5000.

The A0 peak represents apoptotic cells and is the hypo-diploid area (sub-Go/G 1) to
the left of the Gn/Gl diploid peak. The percentage of cells in the A0 peak was estimated
with the PCS Express version 1.0 software (De Novo Software, Thomhill, Ontario,

Canada). The percentages of cells in Go/Gt, S, and szM phases were determined using

82

‘Win cycle’, a Multi-cycle DNA content & cell cycle analysis software (Phoenix Flow
Systems, Inc., San Diego, CA). The Win cycle software program does not include any
cell populations outside of standard cell cycle populations when calculating areas, thus
excluding sub-Go/G1 data.

Statistical analyses. Data for total nucleic acids assay were analyzed by
two-way factorial analysis of variance (AN OVA). After application of ANOVA, the
significance of differences in the means between control and treatment groups at specific
culture periods were evaluated by multiple comparisons using the Bonferroni method.
Data for quantitation of fragmented DNA using the diphenylamine assay were analyzed

by one-way AN OVA. Differences were considered significant at p<0.05.

83

RESULTS

Sphingosine, sphinganine, and Cz-ceramide inhibit growth and cause death of
H T-29 and HGT-116 human colon cancer cells. To determine the effects of
sphingolipids on growth and death of human colon cancer cells, subconfluent HT-29 and
HCT-116 cells were cultured with sphingosine, sphinganine, Cz-ceramide, or C2-
dihydroceramide and then total nucleic acids were determined as an index of cell number
(Li et al. 1990). For HT-29 cells, total nucleic acids in control cultures doubled over 36-
48 h (Figure 3.2). Sphingosine, sphinganine, and Cz-ceramide each caused
concentration- and time-dependent decreases in proliferation of HT-29 cells.
Specifically, addition of sphingosine (Figure 3.2) at 20 and 50 M significantly reduced
total nucleic acid concentrations within 24 h by 47 and 62%, respectively (p<0.05)
compared to the corresponding control. Sphinganine (Figure 3.2) at 10, 20, or 50 M
significantly (p<0.05) reduced total nucleic acid concentrations within 24 h by 20, 28,
and 80%, respectively compared to the corresponding control. Cz-ceramide (Figure 3.2)
also caused a significant (p<0.05) reduction in the total nucleic acid concentrations at 20
and 50 M within 48 h. Culture with Cz-ceramide at 50 W for 48h completely killed all
cells. In contrast to the sphingoid bases and Cz-ceramide, Cz-dihydroceramide had no
effect on proliferation of HT-29 cells at concentrations as high as 50 M (Figure 3.2).

Total nucleic acids in control cultures of HGT-116 cells doubled within 24 to 36
h, indicating slightly more rapid growth than for HT-29 cells (Figure 3.2). Similar to
their effects on HT-29 cells, sphingosine, sphinganine, and C2-ceramide caused
concentration- and time-dependent decreases in proliferation of HGT-116 cells.

Specifically, addition of sphingosine (Figure 3.2) at 20 and 50 M significantly (p<0.05)

84

 

 

HT-29 m HCT-1 16

250
200 -
150 -
100 - o

 

 

 

 

150 r-
100 r

 

 

 

 

 

 

 

 

Total Nucleic Acid (% of time zero)

 

 

 

 

 

 

m _Dh-cer m _Dh-cer
150 150
100 100
50 50-
o a L n a n o l A 4 n 1
012243648 012243648
Hours

Figure 3.2. Effects sphingoid bases and ceramides on growth and death of
HT-29 and HGT-116 human colon cancer cells. Subcontinent cells were
cultured with sphingosine (SO), sphinganine (SA), Cz-ceramide (Cer), and
Cz-dihydroceramide (Dh—cer) at O (a), 1 (O), 5 (V), 10 (V), 20 (Cl) and 50 (I)
uM for 3, 12, 24, or 48 h. Total nucleic acids were measured as an index of
cell number. Results are expressed as a percentage of the control value at
the 0 h. Data are from two experiments and represent mean 1 SEM (n=8).
Where an error bar is not shown, it lies within the dimensions of the symbol.
Means at each culture period with an asterik (*) are significantly different (P <
0.05) from the corresponding control.

85

reduced total nucleic acid concentrations within 24 h by 65 and 68%, respectively
compared to the corresponding control. Sphinganine (Figure 3.2) at 10, 20, or 50 M
significantly (p<0.05) reduced total nucleic acid concentrations within 24 h by 46, 77,
and 91%, respectively compared to the corresponding control. Cz-ceramide at 20 “M
also caused a significant (p<0.05) reduction in total nucleic acid concentrations within 24
h by 36% and Cz-ceramide at 50 M killed all of the HCT-116 cells within 24 b (Figure
3.2). Unlike the sphingoid bases and Cz-ceramide, Cz-dihydroceramide did not inhibit
proliferation at concentrations as high as 50 M (Figure 3.2).

Fluorescence microscopic detection of apoptotic cells in H T -29 and HGT-116
cells. The proliferation studies demonstrated that the higher concentrations of sphingoid
bases and Cz-ceramide not only inhibited proliferation, but also caused death of the colon
cancer cells. To determine whether the sphingolipids kill the cells by inducing apoptosis,
chromatin organization of HT-29 and HCT-116 cells was evaluated by staining with
acridine orange and ethidium bromide using fluorescence microscopy (Mishell et al.
1980; Martin and Lenardo, 1991). Sphingosine, sphinganine, and Cz-ceramide at 20 M
for 24 h each caused characteristic apoptotic morphological changes in both FIT-29 and
HCT-116 cells (Figure 3.3). These included viable cells with apoptotic nuclei which
contain bright green chromatin with highly condensed or fragmented structure as well as
nonviable cells with apoptotic nuclei which contain orange chromatin with highly
condensed or fragmented structure. The condensed and fragmented material is uniformly
stained by the acridine orange and shows small bright circles with the overall cytoplasm

not as bright as the cytoplasm of non-apoptotic control cells. In comparison, C2-

86

HT-29 HCT-1 16

Control

SO

SA

Cer

Dh-cer

 

Figure 3.3. Effects of sphin oid bases and ceramides on chromatin and
nuclear condensation in HT— 9 and HGT-116 human colon cancer cells
(photographs). Subconfluent cells were cultured in the absence (control) or
presence of sphingosine (SO), sphinganine (SA), Cz-eeramide (Cer). and
Cz—dihydroceramide (Dh-cer) at 20 uM for 24 h. Cells were stained with
acridine orange and ethidium bromide and photographed under
fluorescence microscope. Bar=20 um. images in this dissertation are
presented in color.

87

dihydroceramide had no effect and viable cells with normal nuclei contained bright green
chromatin similar to control cells (Figure 3.3).

Quantitation of fragmented low molecular weight DNA using the
diphenylamine assay. To assess the percentage of cells that undergo apoptosis in
response to sphingolipids, both attached and detached cells were collected 48 h afier
treatment with each sphingolipid at 50 M and intemucleosomal fragmented low
molecular weight DNA was quantitated using the diphenylamine assay (Burton 1968).
For PIT-29 cells, control cultures had ~13% fragmented DNA (Figure 3.4). Sphingosine,
sphinganine, and Cz-ceramide caused significantly greater DNA fragmentation at 21, 31,
and 27%, respectively (p<0.05). In contrast, DNA fiagmentation in cells cultured with
Cz-dihydroceramide was 17% which was comparable to controls (Figure 3.4).

Results were similar for HCT-116 cells, although the control cultures had a higher
level of DNA fragmentation at ~22% (Figure 3.4). Sphingosine, sphinganine, and C2-
ceramide significantly increased DNA fragmentation to 37, 47, and 38%, respectively
(p<0.05). In contrast, DNA fragmentation in cells cultured with Cz-dihydroceramide was
24% which was similar to controls (Figure 3.4).

Flow cytometric analysis of cell cycle and apoptotic H T -29 and HGT-116 cells.
The effects of sphingoid bases and ceramides at 35 M for 24 h on the populations of
HT-29 and HCT-116 cells in each phase of the cell cycle were evaluated via flow
cytometry (Telford et al. 1994; Darzynkiewicz et al. 1980). As shown in a representative
histogram in Figure 3.5, sphingosine potently reduced the ntunber of HCT-116 cells with
diploid DNA content (Go/G1 region) compared to control and caused a corresponding

increase in the number of cells with hypo-diploid DNA content (sub-Go/Gl region)

88

HT-29 HCT-1 16

50 b
40 a

30

20

10

 

 

 

 

 

 

 

 

40
30.
20.
10..

0 * I .
Cont Cer Dh-ce oCont Cer Dh-cer

u
D
I
a:
r
N

% DNA Fragmentation
N
O

J
O
T

 

 

 

 

Figure 3.4. Effects of sphingoid bases and ceramides on fragmented low
molecular DNA in HT-29 and HOT-116 human colon cancer cells.
Subconfluent cells were cultured in the absence (control, Cont) or presence
of sphingosine (SO), sphinganine (SA), Cz-ceramide (Cer), and C,-
dihydroceramide (Dh-cer) at 50 uM for 48h. Fragmented DNA were isolated
and quantitated using the diphenylamine assay. Data are mean 1 SEM
(n=3 for HT-29 cells; n=4 for HOT-116 cells). Means with different letters
are significantly different (P <0.05).

89

100 "

3° " Control

 

Sphingosine

Cell Number

 

o 160 320 480 640 800
DNA content

Figure 3.5. Sphingosine increases the number of A0 (sub-GOIG1) cells
indicative of apoptosis (one representative histogram). Subconfluent HCT-
116 cells were cultured in the absence (control) or presence of sphingosine
at 35 uM for 24 h and stained with propidium iodide. The cell cycle and
population were determined via flow cytometric analysis.

90

indicative of apoptosis (called A0). To quantify the degree of apoptosis, cells in the A0
peak were counted and expressed as a percentage of the total cell population. For HT-29
cells, only about 0.8% of the control cells were in the A0 apoptotic peak, while
sphingosine, sphinganine, and Cz-ceramide increased the number of apoptotic cells to 2,
3, and 8%, respectively (Figure 3.6). In comparison, HT-29 cells cultured with C2-
dihydroceramide were similar to control cultures having only ~1% in the A0 apoptotic
peak.

Similar but more profound effects were seen in HGT-116 cells. Control cultures
had 1-5% of the cells in the A0 apoptotic peak, while sphingosine, sphinganine, and C2-
ceramide increased the number of apoptotic cells to 39, 45, and 27%, respectively (Figure
3.6). In contrast, only ~2% of the cells cultured with Cz-dihydroceramide were apoptotic
which was similar to control.

The percentages of HT—29 cells in Go/Gl, S, and Gz/M (mean j; SEM) (Figure 3.7)
for each treatment were: control for sphingoid bases (65.9 i 0.4; 25.6 i 1.5; 8.6 i 1.1),
sphingosine (41.2 i 6.3; 45.7 i 4.7; 13.2 i 1.7), sphinganine (31.5 i 0.6; 55.8 i 0.8; 12.7
_+_ 0.3), control for ceramides (83.2 i 2.5; 11.0 i 1.5; 5.9 i 1.0), Cz-ceramide (40.5 i 6.2;
19.7 i 2.7; 39.9 i 3.5), and Cz-dihydroceramide (84.8 i 1.5; 9.4 i 1.4; 5.9 i 0.2).

The percentages of HGT-116 cells in Go/Gl, S, and Gz/M (mean 1; SEM) (Figure
3.7) for each treatment were: control for sphingoid bases (72.3 j; 4.1; 16.3 :I; 2.3; 11.5 1;
1.8), sphingosine (48.8 i 2.3; 45.0 i 1.0; 6.2 1; 1.3), sphinganine (19.9 i 4.7; 36.3 i 5.7;
43.9 i 10.3), control for ceramides (73.0 i 1.0; 19.5 i 0.5; 7.6 i 0.4), Cz-ceramide (25.8

j; 1.6; 17.2 ft 0.6; 57.1 i 2.1), and Cz-dihydroceramide (69.0 j; 4.0; 23.0 i 1.0; 8.0 i 0.5).

91

HT-29 HOT-1 16

 

 

     

 

 

 

 

 

 

 

   

3.5. 50.
2.8L 4o.
2_1_ 30..
=-"_’1.4. 20.
80.7.. 13.
.3 0 Cont 80 SA
3 10 30
g s. 2
o. 6. 18
f 4. 12
o\ 2. 6
0 0

 

 

 

Cont Cer Dh-cei Cont Cer Dh-cer

Figure 3.6. Effects of sphingoid bases and ceramides on apoptotic cell
number in HT-29 and HGT-116 human colon cancer cells. Subconfluent
cells were cultured in the absence (control, Cont) or presence of
sphingosine (SO), sphinganine (SA), Cz-ceramide (Cer). and C2-
dihydroceramide (Dh-cer) at 35 111M for 24 h. DNA was then stained with
propidium iodide, the cell cycle was examined via flow cytometric
analysis, and the percentage of cells in the A0 (sub-Go/G1) region was
estimated with the PCS express version 1.0 software. Data are mean 1
SEM (n=2).

92

 

 

HT-29 HGT-1 16

 

 

 

 

 

 

 

 

 

 

 

100
75
50
25

% Cells

 

 

 

 

oCont Cer Dh-Cel 0 Cont Cer Dh-cer

 

 

 

 

 

Figure 3.7. Effects of sphingoid bases and ceramides on cell cycle
distribution of HT-29 and HOT-116 human colon cancer cells. Subconfluent
cells were cultured in the absence (control, Cont) or presence of
sphingosine (SO), sphinganine (SA), Cz-ceramide (Cer), and C,-
dihydroceramide (Dh-cer) at 35 pM for 24 h. DNA was then stained with
propidium iodide, the cell cycle was examined via flow cytometric analysis,
and the percentage of cells in each stage of the cell cycle, including Go/G1
phase, //,; S phase, 0; and (3le phase, I, was determined using the Multi-
cycle DNA content & cell cycle analysis software. The A0 (sub-6016,) cell
population was not included in calculation of cell population. Data are mean
1 SEM (n=2).

93

Sphingoid bases and ceramide in HT-29 cells and sphinganine in HGT-116 cells
caused a greater percentage of cells in the Gle and S phase compared to controls
(Figure 3.7). This increase in S phase cell population was accompanied by decreased
Go/Gl cells (Figure 3.7). Cz-ceramide caused accumulation of cells at G2/M with
reductions in S and Go/G1 phases. These findings demonstrate that sphingoid bases and
ceramide in HT-29 cells and sphinganine and Cz-ceramide arrested the cell cycle at the

G2/M phase.

94

DISCUSSION

Complex dietary sphingolipids including sphingomyelin and glycosphingolipids
have gained attention for their potential to protect against the development of colon
cancer. These compounds reduce the number of aberrant colonic foci and the percentage
of adenocarcinomas in chemically initiated mice (Dllehay et al. 1994; Schmelz et al.
1996, 1997, 2000) and decrease the number of tumors in Multiple Intestinal Neoplasia
(Min) mice (Schmelz et al. 2000). The mechanism by which sphingolipids inhibit colon
cancer is not clear; however, it is known that suppression of colon carcinogenesis by
complex sphingolipids is not dependent on the complexity. of the sphingolipid head
groups (Schmelz et al. 2000, Schmelz et al. 2001). This raises the possibility that their
bioactive digestion products ceramide and sphingoid bases may mediate the inhibitory
effects of sphingolipids. These molecules are putative second messengers in cells and
have been shown to reduce proliferation and induce apoptosis in a variety of cancer cells
(Merrill et al. 1997; Davis et al. 2000; Veldrnan et al. 1998; Sweeney et al. 1996). This
study demonstrates that both of the sphingoid bases sphingosine and sphinganine, as well
as Cz-ceramide inhibit proliferation and cause death of both HT-29 and HGT-116 human
colon cancer cell lines at 10-50 M. This concentration range of sphingoid bases and
ceramide is similar to that previously shown to inhibit growth and/or cause differentiation
or death in a variety of tumor cells (Davis et al. 2000; Veldman et al. 1998; Sweeney et
al. 1996; Okazaki et al. 1990; Stevens et al. 1990a; Stevens et al. 1990b; Goldkorn et al.
1991; Endo et al. 1991; Obeid et al. 1993). The finding that a portion of the cell
population survived the insult fiom sphingoid base and ceramide treatments and

subsequently grew slowly between 24 and 48 h is probably due to metabolism of these

95

compounds either via incorporation into more complex sphingolipids or phosphorylation
and degradation (Smith et al. 1995; Smith et al. 1997; Warden et al. 1999).

Presently, it is not clear whether colonic tissue is normally exposed to the
concentrations of sphingoid bases and/or ceramide used in this study. Schmelz et a1
(1994) reported that ~88% of dietary sphingomyelin is digested and absorbed as
ceramides and sphingoid bases in the small intestine and small amounts of dietary
sphingolipids appear in lymph (Nilsson 1968) and serum (Nilsson 1969). Therefore,
colonic tissue could be exposed to ceramide and sphingoid bases via the circulation.
Alternatively, ~12% of dietary sphingomyelin is not absorbed and passes directly into the
colonic lumen (Schmelz et al. 1994) where it may be metabolized by colonic bacteria.
This would provide a more direct route by which colonic tissues may be exposed to
ceramide and sphingoid bases.

Another important finding of this study is that Cz-dihydroceramide, which lacks
the 4,5-rrans double bond present in Cz-ceramide, had no effect on proliferation or death
of either HT-29 or HOT-116 human colon cancer cells. This result establishes that the
4,5-trans double bond is necessary for the growth inhibitory and cytotoxic properties of
Cz-ceramide in human colon cancer cells, but is not required by sphingoid bases. This is
consistent with the results of previous studies which showed that short-chain ceramides
caused apoptosis in many systems, while dihydroceramides lacking the 4,5-trans double
bond had no biological effects (Obeid et al. 1993; Tepper et al. 1995; Sawai et al. 1995;
Brugg et al. 1996; Karasavvas et al. 1996; Xu et al. 1998). In contrast, both sphingosine
and sphinganine inhibit growth and induce apoptosis in a variety of cell lines and tumor

xenografis (Sweeney et al. 1996; Endo et al. 1991; Ohta et al. 1995; Jarvis et al. 1996).

96

Interestingly, previous studies have indicated that synthetic dihydrosphingomyelin (N-
palmitoyldihydrosphingomyelin) which lacks the 4,5-trans double bond was even more
effective than synthetic sphingomyelin (N-palmitoylsphingomyelin) at reducing the
number of aberrant colonic foci in CF-l mice treated with DMH (Schmelz et al. 1997).
Similarly in the present study, sphinganine (which lacks the 4,5-trans double bond) more
potently increased DNA fragmentation than did sphingosine. Thus, complex
sphingolipids may inhibit colon carcinogenesis via turnover to the free sphingoid bases
(sphinganine and/or sphingosine) rather than to ceramide and dihydroceramide; however,
fiom a practical perspective most complex sphingolipids present in food contain the 4,5-
trans double bond (Ahn and Schroeder 2002). Jarvis et al. (1996) showed that a
combination of ceramide and a sublethal concentration of sphingosine or sphinganine
was more effective at causing death of two human myeloid leukemia cell lines HL-60 and
U-937 than ceramide alone. Therefore, another way in which complex sphingolipids may
inhibit colon carcinogenesis is via turnover to a mixture of ceramide, sphingosine, and
sphinganine.

The present study demonstrates that sphingosine, sphinganine, and C2-ceramide
kill HT-29 and HOT-116 human colon cancer cells by inducing apoptosis. These
findings are consistent with those of Veldman et al (1998) who showed that Cz-ceramide
induces apoptosis in HT-29 cells. Our findings underscore the possibility of utilizing
sphingoid bases as well as ceramide or synthetic analogs as chemopreventive or
chemotherapeutic agents for human colon cancer. Many chemotherapeutic agents have
been shown to kill susceptible cells via apoptosis. For example, nonsteroidal anti-

inflammatory drugs (NSAID) such as aspirin (Qiao et al. 1998), sulindac (Piazza et al.

97

1997; Waddell et al. 1998), piroxicam (Waddell et al. 1998), and indomethacin (Erickson
et al. 1999) inhibit colon carcinogenesis via induction of apoptosis. Also, it is
noteworthy that the anticarcinogenic effects of many chemotherapeutic agents are
associated with activation of sphingomyelinase and generation of ceramide (and possibly
sphingosine). Chan et al (1998) reported that sulindac, the most extensively investigated
NSAID, induced apoptosis in HOT-116 and SW-480 human colon cancer cells by
increasing the level of arachidonic acid which stimulated conversion of sphingomyelin to
ceramide. Chemotherapeutic agents which induce apoptosis appear to have the advantage
of targeting individual cells without eliciting an inflammatory response in the
surrounding normal tissue. Sphingosine may also have this property since it has
previously been shown to induce ap0ptosis in neoplastically transformed HUVEC and rat
mesangial cells (Sweeney et al. 1996) as well as human breast epithelial cells (Hong et al.
unpublished data) but not in their primary culture counterparts.

To our knowledge, this is the first study to demonstrate that sphingoid bases and
ceramide in HT-29 cells and sphinganine in HGT-116 cells arrest the cell cycle at the
G2/M phase and cause accumulation of cells in the S phase. Apoptosis triggered under
these conditions is often found in response to suppression of DNA replication or DNA
repair in drug-induced cell death and appears to involve inhibition of topoisomerase I or
H (Del Bino et al. 1991). For example, DNA topoisomerase inhibitor I (camptothecin) or
H (teniposide) induced apoptosis in HL-60 cells with S phase accumulation (Del Bino et
al. 1991; Gorczyca et al. 1993). In addition, the anticancer drug 5-fluorouracil induced
apoptosis by arresting the cell cycle at G2 phase in human breast cancer grated in nude

mice (Okarnoto et al. 1996) and mitomycin C and etoposide induced apoptosis with G2/M

98

phase arrest and S phase accumulation in HGT-116 human colon cancer cells (Olivier et
al. 1998). Folate deficiency-induced ap0ptosis was found to coincide with a block at
62M and accumulation of cells in the S phase (Huang et al. 1999). Also, diallyl
disulfide isolated from garlic (which is believed to reduce tumor incidence and suppress
tumorigenesis) arrested HCT-15 cells in the G2/M phase and increased the percentage of
cells in the S phase (Knowles et al. 1998; Knowles et al. 2001).

The mechanism by which sphingoid bases and ceramide induce apoptotic cell
death is an area of active investigation, but is still not clearly understood. Though the
present study did not examine targets of ceramide and sphingoid bases, there does not
appear to be a role for p53 as a mediator of ceramide or sphingoid bases induced
apoptosis in HT-29 cells because these cells over-express a mutated nonfunctional p5 3
(Rodrigues et al. 1990; Redman et al. 1997). Furthermore, Dbaibo et al. (1998) have
shown that ceramide inhibits cell growth, arrests the cell cycle, and induces apoptosis, but
does not increase p53 expression in HL-60 and U937 human leukemia cells lacking
functional p53. In addition, Bax (a pro-apoptotic member of the Bcl-2 family genes)
probably does not play a major role in ceramide-induced apoptosis in HT -29 and HCT-
116 cells. Though HT-29 cells do not appear to have mutations in Bax (Carethers and
Pham 2000) and HGT-116 cells are heterozygous for Bax (Bax +/+, +/-, -/-) (Carethers
and Pham 2000; Zhang et al. 2000), Kim et al (1999) found that Bax protein expression
did not change in Cz-cerarnide-induced apoptosis of HT-29 cells and Zhang et al (2000)

reported that Cz-ceramide-induced apoptosis was effected very little in HGT-116 cells

carrying only Bax (-/-).

99

In summary, this study systematically evaluated the effects of the sphingoid
bases, sphingosine and sphinganine, as well as Cz-cerarnide and Cz-dihydroceramide on
growth, death, and the cell cycle of HT-29 and HGT-116 human colon cancer cells. The
sphingoid bases and Cz-ceramide were found to kill colon cancer cells by inducing
apoptosis; whereas, Cz-dihydroceramide was not effective (Figure 3.8). These data
suggest that ceramide and the sphingoid bases have the potential to mediate the protective
effects of more complex dietary sphingolipids and raise the possibility that they
themselves may be effective chemopreventive and chemotherapeutic agents for human
colon cancer. Sphingoid bases appear to specifically arrest the cell cycle at G2/M and
cause accumulation of cells in the S phase similar to apoptosis induced by several other

anticancer drugs.

100

 

Colon cancer cells \

- - ~L Proliferation
Sp hlngosme T Apoptosis 8: Arrest at GZIM
. . Jr Proliferation
Sphinganine TT Apoptosis 8. Arrest at 6le
Ceramide ~L Proliferation
T Apoptosis & Arrest at 62m
Dihyd roceramide No effect

 

J

Figure 3.8. Regulation of cell growth, cell cycle, and apoptosis by sphingoid
bases and ceramides in HT-29 and HOT-116 human colon cancer cells.

101

Acknowledgements

We would like to express our thanks to Dr. Louis King and Dr. Zahidul Islam for the
technical advice on flow cytometry analysis and to Dr. Ludmila Roze and Rebecca
Uzarski for advice on fluorescence microscopic detection of apoptotic cells. We also
express our appreciation to Dr. James Pestka and Dr. John Linz for providing access to
their fluorescence microscope.

102

CHAPTER 4

Sphinganine causes early activation of JNK and p38 mitogen activated protein
kinase andearly inhibition of AKT in HT -29 human colon cancer cells1

Running title: Regulation of ERK, JNK, p3 8, and AKT by sphinganine
Key words: sphinganine; ERK; JNK; p38; AKT; apoptosis

1This chapter is prepared for publication
with authors Eun Hyun Ahn and Joseph J. Schroeder.

103

ABSTRACT
The sphingoid base sphinganine inhibits growth and induces apoptosis in HT—29 human
colon cancer cells more potently than other bioactive sphingolipid metabolites
sphingosine and Cz-ceramide tested in our previous study (Ahn and Schroeder, 2002b).
The objectives of the current study were to investigate the effects of sphinganine at a
concentration-inducing apoptosis on the mitogen activated protein kinases (MAPKs) and
AKT (protein kinase B), which regulate cell proliferation and apoptosis. Major isoforms
of MAPKs include extracellular signal-regulated kinase (ERKl/ERKZ), c-Jun NH;-
terminal kinase (JNK2/JNK1, also called stress-activated protein kinase (SAPK)), and
p38 mitogen activated protein kinase. HT-29 cells were cultured with sphinganine at 35
M and the protein expression and/or phosphorylation status of ERKI/ERKZ (p44/p42),
JNK2/JNK1 (p54/p46), and p38 mitogen activated protein kinase, AKT-ser473, and
AKT-thr308 were determined using Western blot analysis. Sphinganine had little or no
effect on the protein expression level of any of the kinases. In contrast, sphinganine
clearly increased the active phosphorylated forms of JNK2/JNK1, and p38 mitogen
activated protein kinase at 15, 30, and 60 min, with minimal effects on activation of
ERKllERK2. Sphinganine inhibited phosphorylation of AKT at ser473 at 60 min and
thr308 at 15 and 60 min. The findings are consistent with a mechanism by which
sphinganine induces apoptosis in HT-29 human colon cancer cells via early inhibition of

AKT and early activation of JNK and p38 mitogen activated protein kinase.

104

INTRODUCTION

Sphingolipids are dietary constituents (Ahn and Schroeder, 2002a) that have
gained much attention for their potential to protect against the development of colon
cancer (for review: Vesper et al. 1999). Complex dietary sphingolipids including
sphingomyelin, dihydrosphingomyelin, glucosylceramide, lactosylceramide, and
ganglioside GD3 reduced aberrant colonic foci in CF 1 mice treated with DMH (Schmelz
et al. 1996, 1997, 1998, 2000) and the number of tumors in all regions of the intestine in
Multiple Intestinal Neoplasia (Min) mice with a truncated Adenomatous Polyposis Coli
(APC) gene product (Schmelz et al. 2001).

This protective role of sphingolipids against colon carcinogenesis may be the
result of conversion of complex sphingolipids to bioactive metabolites including
sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation
and induce apoptosis (programmed cell death) in various cancer cells (Sweeney et al.
1996, Ahn and Schroeder, 2002b). For example, sphingoid bases and ceramide inhibit
growth and arrest the cell cycle at 02M phase in HT-29 and HGT-116 human colon
cancer cells (Ahn and Schroeder, 2002b). Furthermore sphingoid bases and ceramide
cause chromatin/nuclear condensation and DNA fiagmentation and increase the number
of cells in the sub-Go/Gl phase, which are indicative of apoptosis, with sphinganine being
the most potent among Cz-ceramide and sphingoid bases tested (Ahn and Schroeder,
2002b)

A possible mechanism by which sphinganine could inhibit proliferation and
induce apoptosis is by altering the balance of anti-apoptotic and pro-apoptotic signaling

through mitogen activated protein kinases (MAPKs) and AKT. MAPKs are a family of

105

serine/threonine kinases and major isoforms include: extracellular signal-regulated kinase
(ERKl/ERK2, p44/p42), c-Jun NHz-terminal kinase (JNK2/JNK1, p54/p46, also called
stress-activated protein kinase (SAPK)), and p38 mitogen activated protein kinase
(Cohen, 1997, Fanger et a1. 1997, Ferrell, 1996, Marshall, 1995, Kato et al. 1998). JNK
and p38 mitogen activated protein kinase appear to have pro-apoptotic roles. Although
ERK activation has most often been thought of as anti-apoptotic and pro-mitogenic,
recent studies showed that ERK activation is not always required for mitogenic
(Tharnilselvan et al. 2002) and anti-apoptotic (Chang et al, 2001b) effects.

Sphingoid bases could induce apoptosis by altering the balance between ERK,
JNK, and p38 mitogen activated protein kinase activation. For example, Jarvis et al
(1997) reported that sphingosine-mediated lethality is largely associated with weak
stimulation of JNKZ/JNKI, strong inhibition of ERKl/ERKZ, and enhanced p38 mitogen
activated protein kinase activity in U937 human monoblastic leukemia cells.

Alternatively, sphingosine could induce apoptosis by inhibiting AKT (protein
kinase B). AKT is a cytoplasmic serine/threonine kinase that promotes cell survival and
inhibits apoptosis (for review: Testa and Bellacosa, 2001, Talapatra and Thompson,
2001). Enhanced AKT activity is associated with cancer progression in various tissues
(Graff et al. 2000, Krasilnikov, 2000, Tsatasanis and Spandidos, 2000, Yuan et al. 2000,
Sun et al. 2001). Chang et al (2001b) demonstrated that sphingosine induced apoptosis
and suppressed phosphorylated/active forms of AKT. Moreover, apoptosis induced by
sphingosine was attenuated in cells transfected with constitutively active AKT.

The hypothesis of the present study is that altered activation of MAPKs and AKT

may regulate inhibition of proliferation and induction of apoptosis by sphinganine

106

observed in HT-29 human colon cancer cells. The influence of a concentration-inducing
apoptosis of sphinganine was examined for effects on the protein expression and/or
phosphorylation of MAPKs (ERKl/ERKZ, JNK2/JNK1, p38) and AKT (AKT-ser473

and AKT-thr308).

107

MATERIALS AND METHODS

Sphinganine preparation. Sphinganine (D-erythro—dihydro-sphingosine) was
obtained from Matreya Co. (cat. no. 1831, State College, PA). Stock solutions of
sphinganine were prepared by dissolving sphinganine in ethanol at 50 mM and storing at
4°C. A working solution of sphinganine was prepared as a 1:1 complex with 1 mM
bovine serum albumin (BSA) dissolved in phosphate buffered saline (PBS) and stored at
4°C up to one month. Prior to use of sphinganine for experiments, sphinganine was
incubated at 37°C overnight and sonicated for 2 h to ensure sphinganine-BSA complex
formation.

Cell culture. HT-29 human colon cancer cells were purchased from American
Type Culture Collection (Rockville, MD). HT-29 cells were cultured in 100 mm dishes
(Corning, Cambridge, MA) containing Dulbeco’s Modified Eagle Medium (DMEM)
(Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum
(FBS) (Invitrogen Life Technologies, Carlsbad, CA), 3.5 g glucose/L and 2.5 mL
penicillin-streptomycin/L (Invitrogen Life Technologies, Carlsbad, CA) at 37°C supplied
with 5% C02 and humidified air. All experiments were performed with cell passage $20.

Isolation and quantitation of cellular protein. Confluent HT-29 cells were
trypsinizedl and seeded at 2 x 105 cells/mL (i.e. 1.8 x 104/cm2 growth area) in 100 mm
dish and cultured in 5 mL of DMEM supplemented with 10 % FBS and 3.5 g glucose/L
and 2.5 mL penicillin-streptomycin/L for 24 h to insure that cells were in log phase
before treatment with sphinganine. Then the medium was replaced with DMEM
supplemented with l % FBS, 3.5 g glucose/L, and 2.5 mL penicillin-streptomycin/L and

sphinganine was added directly to each dish for various culture periods. Cells on ice

108

were extracted with lysis buffer (1.5 M Tris-Cl, pH 8.8, 100mM sodium vanadate, 20%
SDS) and cell lysates were sonicated for 5-10 sec with a probe sonicator and stored at -
80°C until use. The cellular protein was quantitated via the Bradford assay (Bio-Rad
Laborotories, Hercules, CA) (Bradford 1976).

Western blot analysis for protein expression of ERKI/ERKZ, JNKZ/JNKI, and
p38, and phosphorylated forms of ERKI/ERKZ, JNKZ/flVKI, p38, AK T-ser4 73, and
AK T -tlrr308. Protein (20 pg for both phospho-ERK, JNK, p38 and ERK, JNK, p38; 60
pg for phospho-AKT-ser473; 40 pg for phospho-AKT-thr308) was applied and separated
on 10% SDS-PAGE gel under reducing conditions and transferred to PVDF membranes
at 80 V for 20 min and 120 V for 1.5 h. The blots were washed three times and blocked
with Tris buffered solution-T (TBST, 100mM Tris-Cl pH 7.5, 0.9% (=150mM) NaCl,
0.1% Tween-20) containing 5% BSA for 8 h at 4°C and washed twice with TBST. The
blots were incubated with primary antibodies (Cell signaling/New England Biolabs,
Beverly, MA), (1:10,000 for anti-rabbit IgG polyclonal antibodies against phospho-
ERKl/ERKZ, phospho-JNKZ/JNKI, phospho-p38, ERKl/ERKZ, JNK2/JNK1, p38;
1:1,000 for phospho-AKT-ser473 and phospho-AKT-thr308) in blocking buffer (5 %
BSA in TBST) with gentle shaking overnight (~18 h) at 4°C. The primary antibodies
were removed and the membranes were washed 4 times with TBST and incubated with
horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Cell signaling,
Beverly, MA), (1 :20,000 for phospho-ERKI/ERK2, phospho-JNKZ/JNKI, phospho-p38
and ERKl/ERKZ, JNK2/JNK1, p38; 1:2,000 for phospho-AKT-ser473, phospho-AKT-
thr308) for 1 h at room temperature and detected with western blotting

chemiluminescence luminol reagent (Santa Cruz Biotechnology Inc., Santa Cruz, CA)

109

and/or Supersignal West Dura Extended Duration Chemiluminescent Substrate (Pierce
Biotechnology Inc., Rockford, IL). Membranes were exposed to X-ray film for 1-10 min.

Densitometry. Density of bands was estimated using the GelExpert (San
Mateo, CA) or Kodak ID 3.6 computer software program (Kodak Scientific Imaging
System, New Haven, CT) and are expressed in % of time zero. Then each value for
Phosphorylated forms of ERKl, ERK2, JNK2, JNKl, and p38 was divided by the

corresponding control for total ERKl, ERK2, JNK2, JNKl , and p3 8, respectively.

110

RESULTS

To determine effects of sphinganine on the status of protein expression and/or
phosphorylation/activation of ERKl/ERKZ, JNKZ/INKl, p38, AKT-ser473, AKT-
thr308, sub-confluent HT-29 cells were cultured with sphinganine at 35 pM, which
promotes apoptosis (Ahn and Schroeder, 2002b). Cellular protein was isolated and
kinases were evaluated using Western blot analysis. The density of bands was calculated
using densitometry and expressed in % of time zero and then each value for
phosphorylated forms of ERK], ERK2, JNK2, JNK], and p38 was divided by the
corresponding control for total ERK], ERK2, JNK2, JNK], and p3 8, respectively.

Sphinganine has small efl'ects on early activation of ERK] and ERKZ.

Sphinganine at 35 pM has small effects on the increase of active phosphorylated
forms of ERKl/ERK2 (p44/p42) at 15, 30, and 60 min compared to the corresponding
control, and had little or no effect on protein expression levels of ERKl/ERKZ (Figure
4.1).

Sphinganine causes early activation of .flVKZ and JNK]. Phosphorylated forms
of JNK2/JNK1 were virtually undetectable in controls (Figure 4.2). Sphinganine at 35
pM clearly increased the active phosphorylated forms of JNK2/JNK1 (p54/p46) in time-
dependent manner compared to the corresponding control, while having little or no effect
on protein expression levels of JNK2/JNK1. The strongest activation/phosphorylation of
JNK2/JNK1 was observed at 60 min (Figure 4.2).

Sphinganine causes early activation of p38 mitogen activated protein kinase.

Phosphorylated forms of p38 mitogen activated protein kinase were virtually

undetectable in controls (Figure 4.3). Sphinganine at 35 pM clearly increased the active

111

phosphorylated forms of p38 at 15, 30, and 60 min compared to the corresponding
control, but had little or no effect on protein expression levels of p38. The strongest
activation/phosphorylation of p38 was observed at 60 min Giigure 4.3).

Sphinganine causes early inhibition of AK T-ser4 73 and AK T -thr308.
Sphinganine at 35 pM (an apoptotic concentration) inhibited the active phosphorylated
forms of AKT-ser473 at 60 min and AKT-thr308 at 15 and 60 min compared to the

corresponding control (Figure 4.4).

112

 

 

Minutes 0 15 15 30 30 60 60
Sphinganine - — + — + — +
- ._.. _— .._. ~.. P-ERK1(p44)
8.. - -
"" "' P-ERK2(p42)
Minutes 0 15 15 30 30 60 60
Sphinganine — .— + — + — +
""‘ W “H - ERK1(p44)
.. ERK2(p42)
a: e a: g
E 3 1-8 ‘3 8 t8 SA
5 o 3 0
:2 g 12 g g 1.2
‘- ... u-
:1“ a: o" ' control g 3: 0'6 control
0'. v o 1 . i v o

 

 

o 15 30 45 so
Minutes

 

 

 

 

3 1‘5 3b is so
Minutes

Figure 4.1. Effects of sphinganine on activation of extracellular signal-
regulated kinase 1/2 (ERK1/ERK2) in HT -29 human colon cancer cells.
Sub-confluent cells were treated without (O) or with sphinganine (0, SA)
at 35 pM for 0, 15, 30, and 60 min. Cellular proteins were isolated,
quantitated via Bradford assay, and analyzed via Western blotting. The
density of bands was calculated using Kodak ID 3.6 computer software
program and expressed in % of time zero and then each value for P-ERK1
and P-ERK2 was divided by the corresponding control for total ERK1 and
ERK2.

113

Time(min) 0 15 15 30 30 60 60

 

 

 

 

 

 

 

Sphinganine - - + - + — +
f - -- -- P-JNKZ (p54)
L ' P-JNK1 (p46)
Time (min) 0 15 15 30 30 6O 6O
Sphinganine - -__-_ + - * + .. +
..... = ~ 9 #- .. "r M JNK2 (p54)
. ~ JNK1 (p46)
a -m- s J“-
z 0 Z 2
'1 '5 150 - 2 9’ 150 * SA
3 N a 3
o
0 g 100 E 5 “Mr
E “5 5o .. g '3 50 ..
§ >5 7 23 control
a o o. o
0 15 30 45 60 0 15 30 45 60
Minutes Minutes

Figure 4.2. Sphinganine causes early activation of c-Jun NHz-tenninal
kinase-2H (JNK2/JNK1) in HT-29 human colon cancer cells. Sub-
confluent cells were treated without (O) or with sphinganine (0, SA) at 35
pM for 0, 15, 30, and 60 min. Cellular proteins were isolated, quantitated
via Bradford assay, and analyzed via Western blotting. The density of
bands was calculated using Gel Expert and Kodak D 3.6 computer
software program, respectively and expressed in % of time zero. Then
each value for P-JNKZ and P-JNK1 was divided by the corresponding
control for total JNK2 and JNK1.

114

Time (min) 0 15 15 30 30 60 60

Sphinganine. _ _ + _ + .. -r-
' _m" P-p38

0 15 15 30 30 60 60

Time (min) — — + — f — +
Sphinganine _ 1 -..... ¢ . - Q . p38

 

 

P-p3BITotal p38
(“lo of time zero)
a s E E E ‘2‘

 

 

 

0 15 30 45 60
Minutes

Figure 4.3. Sphinganine causes early activation of p38 mitogen
activated protein kinase in HT-29 human colon cancer cells. Sub-
confluent cells were treated without (0) or with sphinganine (0, SA) at 35
pM for O, 15, 30, and 60 min. Cellular proteins were isolated, quantitated
via Bradford assay, and analyzed via using Western blotting. The density
of bands for P-p38 and total p38 was calculated using Gel Expert and
Kodak ID 3.6 computer software program, respectively and expressed in
% of time zero. Then each value for P-p38 was divided by the
corresponding control for total p38.

115

Time (min)

Sphinganine -
1...... .§--_-— P-AKT-cer473

Time (min)

Sphinganine -
t .

P-AKT-ser473

(% of time zero)
a s s ii § 5

Figure 4.4. Sphinganine causes early inhibition of AKT-ser473 and AKT-
thr308 in HT-29 human colon cancer cells. Sub-confluent cells were
treated without (0) or with sphinganine (0, SA) at 35 pM for 0, 15, 30, and
60min. Cellular proteins were isolated, quantitated via Bradford assay, and
analyzed using Western blotting. The density of bands was calculated
using Kodak ID 3.6 computer software program and expressed in % of time

zero.

15
+

15

0

15

15
«1-

0

i ii

 

l

P

)-

 

contro

SA

 

1 l J

 

c 15 so 45 eh
Minutes

116

30

30

30

+

30

P-AKT-thr308
(% of time zero)

1-

60

60 60
-— 'l-
are? P-AKT-thr308
150
12° ' control
90 r
so .-
3“ ’ SA
0 l L l L L
o 15 30 45 60
Minutes

60
4.

 

 

 

 

DISCUSSION

Sphingoid bases (sphingosine and sphinganine) and Cz-cerarnide inhibit growth
and induce apoptosis in HT-29 and HCT—l 16 human colon cancer cells, with sphinganine
being the most potent (Ahn and Schroeder, 2002b). One possible mechanism by which
sphinganine inhibits proliferation and induces apoptosis is by altering the activation of
MAPKs and AKT. The current study is the first to demonstrate that sphinganine at an
apoptosis-inducing concentration (35 pM) causes early inhibition of AKT and early and
strong activation of JNK, and p38 and in HT-29 human colon cancer cells. Activation of
JNK and p38 is observed as early as 15 min and is maintained up to 60 min.

Though ERK activation has most often thought of as mitogenic and anti-
apoptotic, recent evidences suggest that ERK activation is not always required for
mitogenic (Thamileselvan et al. 2002) and anti-apoptotic (Chang et al. 2001b) effects. In
the present study, sphinganine had a small effect on ERK activation. The relatively
greater activation of ERKl/ERKZ at 15 min for both control cultures and sphinganine-
treated cultures compared to other groups at 0, 30, and 60 min might be associated with
the “burst” of sphinganine and sphingosine that occurs when cells in culture are changed
to flesh medium (Smith et al, 1995). Our results contrast with those previously reported
by Kim et a1 (1999) who found that 50 pM Cz-ceramide, an acylated sphingoid base,
inhibited ERK activity in HT-29 cells as early as 15 min and inhibition was sustained for
up to 3 h. Differing culture conditions (e.g. 1% F BS in the present study versus 10 %
FBS in the earlier study by Kim et al. 1999) may have contributed to the disparity in

results between the studies.

117

We found that sphinganine causes strong activation of JNK at 15, 30, and 60 min
in HT-29 human colon cancer cells. Our results are consistent with a previous study that
showed that Cz-ceramide increases JNK activity in HT-29 cells (Kim et al. 1999). Our
study demonstrated that sphinganine causes maximum activation of JNK at ~60 min;
whereas, the previous study showed Cz-ceramide causes maximum activation of JNK at
~15 min (Kim et al. 1999). Jarvis et al (1997) showed that sphingosine-mediated
lethality of U937 human monoblastic leukemia cells is largely associated with weak
stimulation of JNK2/JNK1 and enhanced p38 mitogen activated protein kinase activity
(Jarvis et a1. 1997).

It is noteworthy that several chemotherapeutic drugs including indomethacin (a
nonsteroidal anti-inflammatory drug) (Kim et al. 1999) and tamoxifen (Mandlekar et al.
2000) cause turnover of complex sphingolipids to bioactive sphingolipid metabolites,
such as ceramide and/or sphingoid bases, and induce apoptosis by stimulating JNK
activity. Our findings might suggest that the sphingoid base sphinganine might mediate
the apoptotic effect of chemotherapeutic drugs in cancer cells, in part, by activating JNK.

Alternatively, sphingosine could induce apoptosis by inhibiting AKT (protein
kinase B). AKT is a cytoplasmic serine/threonine kinase that promotes cell survival and
inhibits apoptosis (for review: Testa and Bellacosa, 2001, Talapatra and Thompson,
2001). Enhanced AKT activity is associated with cancer progression in various tissues
(Graff et al. 2000, Krasilnikov, 2000, Tsatasanis and Spandidos, 2000, Yuan et al. 2000,
Sun et al. 2001). AKT activation is achieved by phosphorylations at ser473 and thr308
residues and thr-308 phosphorylation is necessary for AKT activation and ser473

phosphorylation is only required for maximal activity (Testa and Bellacosa, 2001).

118

However most studies have examined phosphorylation status of AKT only at ser473.
The present study demonstrated that sphinganine causes early inhibition of the
phosphorylated/active forms of AKT-ser473 and AKT-thr308 in HT-29 human colon
cancer cells. Our findings are consistent with those of Chang et al (2001b) who showed
that sphingosine induced apoptosis and suppressed activation of AKT. Moreover,
apoptosis induced by sphingosine was attenuated in cells transfected with constitutively
active AKT (Chang et al. 2001b). Thus, sphinganine and/or sphingosine might induce
apoptosis, in part, by inhibiting AKT activation in human cancer cells.

In conclusion, this is the first study to demonstrate that sphinganine at a
concentration-inducing apoptosis causes early and strong activation of JNK and p3 8, with
minimal effect on ERK activation, and early inhibition of AKT in HT-29 human colon
cancer cells. The findings suggest that JNK and p38 mitogen activated protein kinase
and AKT might mediate the action of sphinganine to initiate apoptosis of colon cancer

cells.

119

Acknowledgement

We express our thanks to: Dr. Yon-co Chung for technical advice on western blot
analysis; Mr. Chris Salvadore at Cell Signaling Co. for providing a sample of phopho-
JNK antibody; Drs. Dale R. Romsos and Chia—Cheng Chang for their encouragement; Dr.
James Pestka for providing access to his experimental instruments.

120

CHAPTER 5

Evaluation of sphinganine and sphingosine as breast cancer chemotherapeutic and
chemopreventive agents using human breast stem, normal, and tumorigenic cell
models

Running title: Sphingoid bases and breast cancer
Key words: sphinganine; sphingosine, cancer, stem cells, apoptosis, differentiation

1This chapter is prepared for publication with authors Eun Hyun Ahn, Chia-Cheng
Chang, and Joseph J. Schroeder.

121

ABSTRACT
Sphingolipids have gained attention for their potential as chemotherapeutic or
chemopreventive agents based on in vitro cancer cell and animal studies. However, no
comparative study of the effects of the sphingolipid metabolites sphingosine and
sphinganine on proliferation and differentiation in normal human epithelial cells versus
tumorigenic cells and epithelial stem cells has been reported. The purpose of this study
was to evaluate the chemotherapeutic and chemopreventive potential of sphingoid bases
using a novel cell culture system of normal human breast epithelial cells (HBEC)
developed from tissues of healthy women obtained during reduction mammoplasty (type
I HBEC with stem cell characteristics and type II HBEC with basal epithelial cell
phenotypes) and transformed tumorigenic type I HBEC. The results show that
sphinganine more potently inhibited the growth and induced apoptosis of tumorigenic
type I HBEC than sphingosine (ICso for sphinganine 4 pM; sphingosine 6.4 pM). High
concentrations of either sphingoid base (8-10 pM) arrested the cell cycle at G2/M with
reductions in S and Go/Gl phases, while sphingosine showed a threshold effect.
Sphinganine also more potently inhibited the growth and caused death of type I HBEC
than sphingosine, thus sphingoid bases may reduce target cells for breast carcinogenesis.
In comparison, type II HBEC were less sensitive to the growth inhibitory effects of
sphingoid bases than tumorigenic type I HBEC and type I HBEC, suggesting that
sphingoid bases may serve as chemotherapeutic agents. At concentrations (0.05-0.5 pM),
which are below the growth inhibitory ranges, sphingoid bases induced differentiation of
type I HBEC to type H HBEC, as detected morphologically and via expression of a novel

tumor suppressor protein maspin in type II HBEC. The current study is the first to

122

systematically compare the chemotherapeutic and chemopreventive properties of
sphingoid bases in normal, stem, and tumorigenic breast cells. The data support the
potential for sphingoid bases to be employed as chemotherapeutic and chemopreventive

agents against human breast cancer.

123

INTRODUCTION

Breast cancer is the most common cancer among women in the United States and
worldwide (American Cancer Society 1999). Although the etiology of breast cancer is
not well understood, the cumulative exposure of breast tissue to estrogen appears to be a
major factor in the development of breast cancer risk (Henderson et al. 1993, Jordan and
Morrow 1999). Recently the identification of protective dietary compounds that prevent
the development of diseases including breast cancer has gained much attention. In
particular, the search for and development of chemotherapeutic and/or chemopreventive
agents that do not exert side effects and cause drug resistance has been an active area of
research.

Sphingolipids are constituents of dairy and soy products at relatively high
concentrations (Ahn and Schroeder, 2002a). These dietary sphingolipids are digested and
absorbed as ceramide and sphingoid bases (sphingosine and sphinganine) (Schmelz et al.
1994). Ceramide and sphingoid bases function as second messengers in signal
transduction pathways, inhibiting cell proliferation and inducing apoptosis (programmed
cell death) in a variety of human cancer cells (Ahn and Schroeder 2002b, Sweeney et al.
1996) including breast cancer cells (Gill et al. 1997, Cai et al. 1997). For example, in
HT-29 and HGT-116 human colon cancer cells sphinganine more potently induces
apoptosis than sphingosine (Ahn and Schroeder 2002b). One of the mechanisms by
which many clinically important drugs, including antiestrogens such as tamoxifen and the
novel synthetic retinoid N—(4-hydroxyphenyl) retinamide (4-HPR) that inhibit
proliferation and induce apoptosis of cancer cells, is through activation of sphingolipid-

mediated pathways (for review: Senchenkov et al. 2001).

124

Most studies examining sphingolipids have focused on the pro-apoptotic actions
of ceramide (acylated form of sphingoid bases) and fewer studies have examined the
actions of sphingosine and sphinganine (Figure 5.1). Furthermore, it is uncertain whether
the sphingosine or sphinganine backbones of ceramides mediate anti-proliferative and
pro-apoptotic activities of ceramide on cancer cells (Sakakura et al. 1998, Back et al.
2001, for review: Goswami and Dawson, 2000, Radin, 2001). In addition, no study has
compared the effects of sphingoid bases on proliferation and differentiation in human
normal epithelial cells to that in tumorigenic cells and epithelial stem cells.

Previously, we have reported the characterization of two types of morphologically
distinguishable and phenotypically different normal human breast epithelial cells (HBEC)
from reduction mammoplasty tissues of healthy females (type I and type II HBEC) (Kao
et al. 1995, for review: Chang et al. 2001a) (Figure 5.2). Type I HBEC display stem cell
characteristics based on observations that type I HBEC differentiate to type II HBEC and
mixture of type I and type II HBEC form budding and ductal organoids on Matrigel (Kao
et al. 1995, Chang et al. 2001a). Type I HBEC express estrogen receptors (Kang et al.
1997) and are more susceptible to neoplastic transformation, telomerase activation, and
immortalization (Kao et al. 1995, Kao et al. 1997, Kang et al. 1998, Sun et a1. 1999).
Therefore type I HBEC appear to be the major target cells for breast carcinogenesis.
Type H HBEC do not express estrogen receptors, rarely become immortal after simian
virus 40 transformation and, unlike type I HBEC, do not have the capability of anchorage
independent growth (Kao et al. 1995, Sun et al. 1999, Chang et al. 2001a).

In the current study, this novel culture system of type I and type H HBEC was

used as a cell model system to assess the chemopreventive potential of sphinganine in

125

OH

"'13" Sphingosine

H
cuon

NH3+ Sphinganine

OH

/W\/\/\/\/\/J\/CH20H
\

\/\/\/\/\/\/\/\/\/NH Ceramide
3

OH

MCI-'20“

H3C\/NH C2-Ceramide

41

OH

H3C\,NH Cz-dihydroceramide

ll

Figure 5.1. Structures of sphingosine, sphinganine, ceramide, Cz-ceramide,
and Cz-dihydroceramide.

126

Type I HBEC (stem cells) Type II HBEC

Differentiation

 

7

f— SV40 transfection ——i

”133‘", lmmortalized at
Immortallzed at
. low frequency

3 high frequency

X-ray

7

M13SV1 R2
Weakly tumorigenic

(— C-erbBZ/neu oncogene

 

N

V

M13SV1R2N1

Highly tumorigenic

 

Tumorigenic type I HBEC

Figure 5.2. Derivation of tumorigenic type I HBEC and type ll normal
human breast epithelial cells (HBEC) from type i HBEC with stem cell
characteristics. Abbreviation used: SV40, simian virus 40.

127

comparison to sphingosine. The chemotherapeutic potential of sphinganine and
sphingosine was also evaluated by examining the effects of the sphingoid bases on
tumorigenic type I HBEC representing breast tumor cells in comparison to type II HBEC
representing normal breast cells. The following criteria were applied to determine the
chemotherapeutic and chemopreventive properties of sphingoid bases in tumorigenic type
I HBEC, and type I and type II HBEC: 1) A chemotherapeutic agent is expected to inhibit
the proliferation and induce apoptosis of tumorigenic type I HBEC; 2) A
chemopreventive agent is expected to preferentially inhibit the proliferation and induce
the differentiation of type I HBEC; thereby, decreasing the target stem cells for neoplastic
transformation.

The objectives of this study were to determine: 1) whether sphingoid bases
preferentially inhibit proliferation of tumorigenic type I HBEC and type I HBEC,
compared to type H HBEC; 2) the effects of sphingoid bases on apoptosis and the cell
cycle in tumorigenic type I HBEC; and 3) whether sphingoid bases induce the

differentiation of type I HBEC to type II HBEC.

128

MATERIALS AND METHODS

Sphingolipids. Sphinganine (D-erythro-dihydro-sphingosine) was obtained
from Matreya Co. (cat. no. 1831, State College, PA) and D-erythro-sphingosine was from
Sigma-Aldrich Co. (S-6879, St. Louis, MO). Stock solutions of sphinganine and
sphingosine were prepared by dissolving the sphingoid base in ethanol at 50 mM and
storing at 4°C. Working solutions of sphingoid bases were prepared as 1:1 complexes
with 1 mM bovine serum albumin (BSA) in phosphate buffered saline (PBS) and stored
at 4°C up to one month. Prior to experiments, sphingoid bases were incubated at 37°C
overnight and sonicated for 2 h to ensure sphingoid bases-BSA complex formation.

HBEC culture media. The MSU-1 medium (Kao et al. 1995) with various
supplements was used to culture type I HBEC, tumorigenic type I HBEC, and type H
HBEC. The MSU-1 medium is a 1:1 mixture of a modified Eagle’s MEM (called D
medium) (Invitrogen Co., Carlsbad, CA) (Chang et al. 1981) and a modified MCDB 153
(M-7403, Sigma-Aldrich Co., St. Louis, MO) (Pittelkow et al. 1986), supplemented with
human recombinant epidermal growth factor (0.5 ng/mL, E-9644, Sigma-Aldrich Co.),
insulin (5 pg/mL, I-1882, Sigma-Aldrich Co.), hydrocortisone (5 pg/mL, H-0888, Sigrna-
Aldrich Co.), human transferrin (5 pg/mL, T-7786, Sigma-Aldrich Co.), l7-B-estradiol
(10'8M, 13-2257, Sigma-Aldrich Co.), Gentamicin (50 pg/mL, 15710-064, Invitrogen Co.)
(Kao et al. 1995).

Development of human breast epithelial cells (HBE C) from reduction
mammoplasty tissues of healthy females. Breast tissues of healthy women at 23, 26,
and 23 years of age were obtained during reduction mammoplasty at a hospital in

Lansing, MI. These initial primary HBEC cultures developed from breast tissues were

129

designated as HME23, HME29, and I-IME30, respectively. Kao et a1 (1995) and Chang
et al (2001a) developed HBEC from these specimens. Procedure for the development of
type I HBEC with stem cell characteristics and type II HBEC from reduction
mammoplasty tissues has been described (Kao et al. 1995, Trosko et al. 2000). HBEC
were stored in liquid nitrogen until use.

Separation of type I HBE C with stem cell characteristics and type II HBEC.
HBEC stored at liquid nitrogen were thawed at 37°C and plated in the MSU-1 medium
supplemented with 5 % PBS in a 100 mm dish, designated as “Plate A”. After 2 h
incubation, the unattached cells were transferred to a 15 mL tube and the MSU-1 medium
supplemented with 0.4 % bovine pituitary extracts was added to the attached cells in
Plate A. After centrifugation (1000 rpm for 8 min) of the unattached cells, the
supernatant was removed and the cell pellet was suspended in the MSU-1 medium
supplemented with 0.4 % bovine pituitary extracts and plated in a dish labeled “Plate B”
with the MSU-1 medium supplemented with 0.4 % bovine pituitary extracts. Both plates
of cells were cultured overnight at 37 °C in incubators supplied with 5% C02 and
humidified air.

The next day, the unattached cells in the medium in plate B were collected in a 15
mL tube and MSU-l medium supplemented with 0.4 % bovine pituitary extracts was
added to the attached cells in plate B. After centrifugation (1000 rpm for 8 min) of the
medium containing the unattached cells from plate B, the supernatant was removed and
the cell pellet was suspended in the MSU-l medium supplemented with 5 % F BS and
plated in the MSU-1 medium supplemented with 5 % F BS in a dish labeled “Plate C”.

Plate A contained fibroblasts and was not used for experiments. Plate B contained

130

mainly type II HBEC. Plate C contained mainly type I HBEC and a few fibroblasts
(which, after forming colonies, were marked and removed by scraping with a rubber
policeman. Appendix L: Figure A.13-photographs).

Derivation of in vitro neoplastically transformed HBEC lines from HBEC,
normal human mammary epithelial culture from reduction mamoplasty tissues.
Previously Kao et a1 (1995) and Kang et a1 (1998) derived in vitro neoplastically
transformed HBEC lines (Ml3SVl, M13SV1R2, and M13SV1R2N1) from type I HBEC
(for review: Chang et al. 2001a). These transformed type I HBEC were stored in liquid
nitrogen until use. Type I HBEC with stem cell characteristics developed from reduction
mammoplasty tissues were sequentially transformed to immortalized/non-tumorigenic
cells (MlBSVl), weakly tumorigenic cells (Ml3SV1R2), and highly tumorigenic cells
(M13SV1R2N1) by transfections with SV-40, X-ray, and neu oncogene (Kao et al. 1995,
Kang et al. 1998). In the current study, transformed tumorigenic type I HBEC
(M13SV1R2N1) were used as the breast cancer cell model (Figure 5.2).

Culture of type I HBEC, tumorigenic type I HBEC, and type II HBEC. Type I
HBEC and tumorigenic type I HBEC were cultured in the MSU-1 medium supplemented
with 5 % FBS (Kao et al., 1995). Type H HBEC were cultured in the MSU-1 medium
supplemented with 0.4 % bovine pituitary extracts (prepared from bovine pituitary
glands, Pel-Freez Biologicals, Rogers, AR) (Kao et al. 1995). Cells were cultured at 37
°C in incubators supplied with 5% C02 and humidified air.

Assessment of cell proliferation. To assess the effects of sphingoid bases on
cell growth and death, total nucleic acids were measured as previously described (Li et al.

1990) and used as an index of cell number. Briefly, confluent cells were trypsinized and

131

cells were seeded at a density of 6 x 104 cells per well in 6-well dishes (i.e. 6.3 x 103
cells/cm2 growth area) and cultured in 2 mL of the MSU-1 medium supplemented with 5
% FBS for 24 h for tumorigenic type I HBEC to insure that cells were in log phase before
treatment with sphingoid bases. For type I HBEC, cells were cultured in the MSU-1
medium supplemented with 5 % F BS for 3 d since type I HBEC have lower cell plating
efficiency and proliferation rate. For type II HBEC, cells were cultured in the MSU-1
medium supplemented with 5 % F BS for 1 d and then switched to the MSU-1 medium
supplemented with 0.4 % bovine pituitary extracts for 2 d since type II HBEC are
sensitive to trypsinization and have lower cell plating efficiency after re-plating in the
MSU-1 medium supplemented with 0.4 % bovine pituitary extracts. Then these media
were replaced with FBS-free MSU-l medium. Various concentrations of sphinganine
and sphingosine were added directly to each dish and the cells were cultured for up to 6
days. Fresh FBS-free MSU-1 medium and treatments were renewed after 3 (1. Following
treatments, the media and floating dead cells were removed, the viable attached cells
were rinsed with 1 mL of PBS, and the cells were lysed with 1 mL of 0.1 M NaOH.
Total nucleic acids concentrations were determined by measuring the absorbance of the
cell lysate at 260 nm using Beckman DU-7400 spectrophotometer (Beckman Coulter
Inc., Fullerton, CA).

Flow cytometry of cell cycle and population. Flow cytometric analysis was
performed as described by Telford et al. (1994) with modifications. Confluent
tumorigenic type I HBEC were trypsinized, seeded at a density of 3.5 x 105 cells/ 100 mm
dish (i.e. 6.3 x 103 cells/cm2 growth area), and cultured with 5 mL of the MSU-1 medium

supplemented with 5 % FBS for 24 h to ensure that cells were in log phase before

132

treatment with sphingoid bases. Then the medium was replaced with FBS-free MSU-l
medium. Various concentrations of sphinganine and sphingosine were added directly to
each dish and the cells were cultured for 12 or 24 h. After treatments, floating cells were

collected and 1.5 mL PBS with 8% F BS (heat inactivated at 50°C for 30 min and filtered

with 0.22 pm filter syringe) was added to each tube. Attached cells were trypsinized for
10 min in the incubator at 37°C and supplied with 5 % C02 and humidified air.
Trypsinization was terminated by adding 3 mL of PBS containing 4 % FBS to each dish
and detached cells were transferred to the previous tubes containing floating cells. After
centrifugation (1200 rpm, 4 min), the supernatant was removed and PBS (pH 7.4)
containing 50 % FBS was added. Cells were fixed with 70 % cold ethanol at a final
concentration of 50-53 %. After storage at 4°C for 6-8 h, cells were pelleted by
centrifugation (1200 rpm, 4 min) and resuspended in 500 pL of PBS containing 4 % FBS.
Then, 300 pL DNA staining solution [4 % FBS, 0.1 % Triton X-100, 100 pM EDTA (pH
8.0), 0.05 mg/mL RNase A (50 units/mg), 50 pg/mL propidium iodide in PBS (pH 7.4)
containing 4% FBS] was added and the cells were kept at 4°C for 2 h and then filtered
through 40 pm nylon mash (Small Parts Inc., Miami Lakes, FL) and collected into Falcon
tubes. The cells were kept at 4°C for 3-5 h before reading using a FACS Vantage Flow
Cytometer (Becton Dickinson, San Jose, CA). The total number of cells analyzed for
each sample was 7000.

The percentages of cells in Go/Gl, S, and G2/M phases were determined using
Mod-fit LT version 3.1 software program (Verity Software House, Topsham, ME). The
Mod-fit software program does not include any cell populations outside of standard cell

cycle populations when calculating areas, thus excluding sub-Go/Gl cells. The F CS

133

Express version 2.0 software (De Novo Software, Thomhill, Ontario, Canada) was used
to determine the percentage of apoptotic cells represented by the A0 peak in the hypo-
diploid (DNA content less than 2N) sub-Go/01 area to the left of the Go/Gl diploid (DNA
content 2N) peak.

Assessment of type I HBE C difierentiation. The effects of sphingoid
bases on the ability of type I HBEC to differentiate to type H HBEC were evaluated by
counting morphologically distinguishable colonies of type I, type H, and type I
surrounded by type H HBEC. Type I HBEC at cell seeding density of 5000 cells/60 mm
dish with grids (i.e. 238.1 cells/cm2 growth area, 1250 cells/mL) were grown in 4 mL of
the MSU-1 medium supplemented with 5 % FBS (which supports the growth of type I
HBEC, but not type H HBEC) for 2 d. On the third day, cells were cultured in FBS-free
MSU-1 medium (which supports growth of both type I and type H HBEC) and then
sphingoid bases at various concentrations and cholera toxin (C-8052, Sigma-Aldrich Co.)
as a positive control (Kao et al., 1995) were directly added to each dish. Cells were
cultured in FBS-free MSU-1 medium for 7 days with changing of fresh FBS-free MSU-1
medium and renewed treatments on day 3 and day 5. At the end of the experiment, the
medium containing floating cells was removed. Cells were washed once with PBS and
stained with 1% crystal violet (Sigma-Aldrich Co.) for 2 min and then rinsed with water.
Stained colonies in dishes were air-dried and kept at room temperature.

The number of colonies of type I, type H, and type I surrounded by type H HBEC
(the cell culture started from single cell plating of pure type I HBEC) were visually
identified and counted via microscopy. To ensure objectivity, the identities of the

treatment for each dish were unknown (blinded) to two people who were instructed to

134

score colonies and did not participate in other parts of the differentiation experiment.
The same dishes were independently counted by two people with the same criteria and
the results were consistent to each other.

Immunofluorescence staining. Type I HBEC at cell seeding density of 1250
cells per well in 4-well chamber slide (i.e. 735.3 cells/cm2 growth area) were grown in 1
mL of the MSU-1 medium supplemented with 5 % FBS (which supports the growth of
type I HBEC, but not type H HBEC) for 2 d. On the third day, cells were cultured in
FBS-free MSU-l medium (which supports growth of both type I and type H HBEC) and
then sphingoid bases at various concentrations were directly added to each dish. Cells
were cultured in FBS-free MSU-1 medium for 7 days with changing of flesh FBS-free
MSU-1 medium and renewed treatments on day 3 and day 5. At the end of the
experiment, the medium containing floating cells was removed. After fixation of cells
with 4 % paraformaldehyde in PBS for 30 min at room temperature with gentle shaking,
cells were rinsed with PBS and permeabilized with 0.5 % triton X-100, 2% BSA, 0.05 %
NaN3 in PBS. Permeabilization reagent is removed and cells were incubated with a
mouse anti-human maspin monoclonal antibody (1:100 dilution, cat no. 554292, BD
Biosciences-Pharmingen, San Diego, CA) for 20 h at 4°C with gentle shaking. After
rinsing cells with PBS, cells were incubated with FITC-conjugated sheep anti-mouse IgG
(1:200 dilution, cat no. 115-095-166, Jackson ImmunoResearch Laboratories Inc., West
Grove, PA) for 45 min at 4°C. After rinsing cells four times with PBS, cells were
mounted with Aqua-Poly mount (Polysciences Inc., Wanington, PA) and photographed
using a fluorescence/phase contrast microscope equipped with a digital camera

(Diagnostic Instruments, Sterling Heights, MI).

135

Statistical analyses. Data for total nucleic acids assay at various concentrations
and culture periods were analyzed by two-way factorial analysis of variance (AN OVA).
After application of two-way AN OVA, the significance of differences in the means
between control and treatment groups at specific culture periods was evaluated by
multiple comparisons using the Bonferroni method. Data for total nucleic acids at
various concentrations and a single culture period were analyzed by one-way factorial
AN OVA. After application of one-way AN OVA, the significance of differences in the
means between control and treatment groups at a single culture period was evaluated by
multiple comparisons using the Student-Newman-Keuls method. Differences were

considered significant at p < 0.05.

136

RESULTS

Sphinganine more potently inhibits the growth and causes death of tumorigenic
type I HBE C than sphingosine. To determine the effects of sphinganine on growth
and death of tumorigenic type I human breast epithelial cells (HBEC) in comparison to
sphingosine, sub-confluent cells were cultured with sphinganine and sphingosine at
various concentrations and total nucleic acids were determined as an index of cell number
(Figme 5.3; Appendix K: Figure AID—photographs). Total nucleic acids in control
cultures doubled within 1 day and increased 10 fold in 5 days (Figure 5.3-Lefi panel).
Sphinganine caused concentration- and time-dependent decreases in proliferation of
tumorigenic type I HBEC. Specifically, addition of sphinganine at 3.5 and 5 pM
significantly reduced total nucleic acids at l d by 53 and 89 %, at 3 d by 49 and 89 %,
and at 5 d by 66 and 95 %, respectively compared to the corresponding controls (p<0.05).
In contrast, sphingosine at 5 pM significantly reduced total nucleic acids at 1 d by 51 %,
at 3 d by 35 %, and at 5 d by 36 % compared to the corresponding controls (Figure 5.3-
Lefi panel). These data suggest that sphinganine more potently inhibited the growth and
caused death of tumorigenic type I HBEC than sphingosine.

The effects of sphingoid bases on death of tumorigenic type I HBEC were further
examined at concentrations ranging from 2 to 10 pM for 24 b (Figure 5.3-Right panel;
Figure 5.4-Photographs). Sphinganine at 2, 3.5, and 5 pM decreased total nucleic acids
by 24, 37, and 85 %, respectively compared to the control. Sphinganine at 8 and 10 pM
completely killed cells. In contrast, sphingosine at 3.5, 5, and 8 pM decreased total

nucleic acids concentrations by 7, 23, and 78 %, respectively compared to the control.

137

Tumorigenic type I HBEC

 

 

    
  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A 180

2 100° Sphinganine 150

o 800 g

N 120

., 600 1-

.§ 400 3 A 9°

: zoo a 2 6°

2 0 fl 3 3°

0 0

i; o 1 3 5 “‘5’ ° _ 8_

1: Days .2 '5 pM Sphinganine

E 1000's hin osine ‘7', 3 18°

.2 300- p g E :35150

2 , - 120

3 60° * ‘3 so

: 400) l- 60

E 200» so

0 o l I I 4 1 1

'— o 1 3 5 ”o 2 4 e 3 1o
Days pM Sphingosine

Figure 5.3. Sphinganine more potently inhibits the growth and causes the
death of tumorigenic type I HBEC than sphingosine. Lefl panel: Sub-
confluent cells were cultured with sphinganine at 0 (e), 0.5 (O), 2 (V), 3.5
(V), and 5 M (D) and with sphingosine at O (c) and 5 pM (D) for 1, 3, and 5
days. Right panel: Sub-confluent cells were cultured with sphinganine and
sphingosine at O, 2, 3.5, 5, 8, and 10 pM for 24 hours. Total nucleic acids
were measured as an index of cell number. Results are expressed as a
percentage of the zero hour. Data shown are mean 1 SEM (n=3). Where an
error bar is not shown, it lies within the dimensions of the symbol. Means at
each culture period with an asterisk (*) are significantly different (P < 0.05)
from the corresponding control.

138

Sphinganine

  
  

 

Tumorigenic
type I HBEC

at 24 h
Control

 

.. . , 10pM

Figure 5.4. Sphinganine more potently inhibits the growth and causes
death of tumorigenic type | HBEC than sphingosine at 24 h (photographs).
Sub-confluent cells were cultured with sphinganine and sphingosine at 0, 2,
3.5, 5, 8, and 10 pM for 24 hours. Magnification 10X plus 10X. Images in

this dissertation are presented in color.

139

Sphingosine at 10 pM completely killed cells (Figure 5.3-Right panel). The ICso for
sphinganine was approximately 4 pM, while that for sphingosine was ~ 6.4 pM.

Sphinganine more potently induces apoptosis of tumorigenic type I HBE C than
sphingosine. Both sphingoid bases arrest the cell cycle at G/M with reductions in S
and 61/62 phases. The effects of sphingoid bases on cell cycle and apoptosis of
tumorigenic type I HBEC were analyzed via flow cytometry using concentrations of
sphingosine and sphinganine found to be growth inhibitory and cytotoxic in total nucleic
acids assays (Figure 5.3). As shown in Figure 5.5, sphinganine at 8 pM increased the
number of cells in the hypo-diploid sub-Go/Gl region, indicative of apoptosis (called A0).
To quantify the degree of apoptosis, cells in the A0 peak were quantitated and expressed
as a percentage of the total cell population. Specifically, about 4.3 % of the control cells
for sphinganine were in the A0 apoptotic peak, while sphinganine at 3.5, 5, and 8 pM
increased the number of apoptotic cells to 7.9, 8.6, and 10.9 %, respectively (Figure 5.6).
About 2.5 % of the control cells for sphingosine were in the A0 apoptotic peak. In
comparison, sphingosine at 5, 8, 10 pM increased the number of apoptotic cells to 3.4,
4.3, and 7.8 %, respectively (Figure 5.6).

The effects of sphingoid bases on the cell cycle distribution of tumorigenic type I
HBEC were also examined (Figure 5.6). The percentages of cells in Go/Gl, S, G2/M after
24 h exposure to each treatment were: control for sphinganine (34; 39; 27), 3.5 pM
sphinganine (41; 40; 20), 5 pM sphinganine (39; 37; 24), 8 pM sphinganine (29; 24; 47),
control for sphingosine (36; 38; 26), 5 pM sphingosine (36; 36; 28), 8 pM sphinganine
(29; 35; 37), 10 pM sphingosine (27; 36; 37). Sphinganine at 8 pM and sphingosine at 8

and 10 W for 24 h increased the percentages of cells in G2/M and decreased the

140

percentages of cells at S and Go/Gl phases compared to the corresponding controls
(Figure 5.6). Sphingosine at 8 pM for 12 h also caused accumulation of cells at 02M
and reductions at S and Go/Gl phases (Appendix K-Figure A.11).

Interestingly sphingosine at both 8 and 10 pM caused similar degrees of
accumulations of cells in G2/M, although 10 pM sphingosine caused a much greater
increase of apoptotic cells than 8 pM sphingosine (Figure 5.6). This implies that
sphingosine has a threshold greater than 5 but less than 8 pM which causes accumulation

of cells in G2/M phase (Figure 5.6).

141

120 Tumorigenic type I HBEC

100 1 Control

 

 

 

 

 

120
100 Sphinganine
sol

car/e,
60
I A9 I W
20 l '
11W ““111wa
0 160 320 480 640

0
800

Cell Number

 

 

 

DNA content

Figure 5.5. Sphinganine increases the number of A0 (sub-GOIG,) cells,
indicative of apoptosis, and arrests cell cycle at Gle phase in tumorigenic
type I HBEC (one representative histogram). Sub—confluent cells were
cultured in the absence (control) or presence of sphinganine at 8 pM for 24
hours and DNA were stained with propidium iodide. The cells in sub-GOIG1
area, indicative of apoptosis, and each cell cycle of 60/6,, 8, and GZIM
phases were examined via flow cytometric analysis and calculated using FCS
Express version 2.0 and Mod fit cell cycle analysis software as described in
Materials and Methods.

142

 

 

 

Tumorigenic 12 . 12-
type I HBEC a . a.
at 24 h e / s
3 . 3 ,
o

 

A! lllllll

 

0245810

 

 

SS 383
rrfi 6""C
N N
L «I
O O

‘
G

 

 

 

 

 

 

 

 

 

 

 

 

 

 

% Cells at 6le % Cells at S % Cells at 60/61 % Apoptotic cells

2o an.
10 1o.
Ja‘i‘i‘é‘é 523543513
40 4o
so so
zob zo
1o- 10
o ........ 0

pM Sphinganine pM Sphingosine

Figure 5.6. Sphinganine more potently induces apoptosis of tumorigenic
type I HBEC than sphingosine. Both sphingoid bases at high concentrations
(8-10 pM) arrest cell cycle at Gle phase with reductions in S and 60/6,
phases. Sub-confluent cells were cultured with sphinganine at 0, 3.5, 5, and
8 pM and with sphingosine at 0, 5, 8, and 10 pM for 24 h and DNA were
stained with pmpidium iodide. The cells in sub-GOIG1 area, indicative of
apoptosis, and each cell cycle of 60/6,, S, and Gle phases were examined
via flow cytometric analysis and wiculated using FCS Express version 2.0
and Mod fit cell cycle analysis software as described in Materials and
Methods. Where an error bar is not shown, it lies within the dimensions of
the symbol. Data are mean 1 SEM (n=2, except n=6 for sphingosine at 0).

143

Sphinganine more potently inhibits the growth and causes death of type I
HBE C than sphingosine. To determine the effects of sphinganine on growth and
death of type I HBEC with stem cell characteristics in comparison to sphingosine, sub-
confluent cells were cultured with sphinganine and sphingosine at various concentrations
and culture periods and total nucleic acids were determined as an index of cell number
(Figure 5.7-Lefi panel and Figure 5.8-Photographs).

Total nucleic acids in control cultures increased more than 3 fold in 6 days,
indicating that type I HBEC (Figure 5.7-Lefl panel) grew much slower than tumorigenic
type I HBEC (Figure 5.3-Lefi panel). Sphinganine caused concentration- and time-
dependent decreases in proliferation of type I HBEC. Specifically, addition of
sphinganine at 3.5 and 5 pM reduced total nucleic acids at 3 d by 60 and 90 % and at 6 d
by 78 and 94 %, respectively compared to the corresponding controls. Sphinganine at 8
pM completely killed cells. In contrast, sphingosine at 5 pM reduced total nucleic acids

at 3 d by 51 % and at 6 d by 74 % (Figure 5.7-Lefi panel and Figure 5.8-Photographs).

144

Total nucleic acids (% of time zero)

Figure 5.7. Effects of sphinganine and sphingosine on growth and death
of type I HBEC (HME29-A) and type II HBEC (HME29-A). Sub-confluent
cells were cultured with sphinganine at 0 (O), 3.5 (V), 5 (V), and 8 (El)
pM and with sphingosine at O (O) and 8 (D) pM for 3 and 6 days. Total
nucleic acids were measured as an index of cell number.
expressed as a percentage of the zero hour. Data are mean 1 SEM (Left
panel: n=2, except n=5 for 0 11; Right panef. Data are n=3, except n=6 for
0 d. Means at each culture period with an asterisk (*) are significantly
different (P < 0.05) from the corresponding control). Where an error bar is

Type I HBEC

 

400
300
200
1 00

. Sphinganine

p

 

400
300
200
1 00

 

 

Sphingosine

j

 

l L 4 J J L

 

 

0 3
Days

Type II HBEC

 

400
300
200
1 00

_ Sphinganine

  

 

L414 kg. 1

 

400 Sphingosine

300

200 '

100

 

  

l I

l l l l l

 

0 3
Days

not shown, it lies within the dimensions of the symbol.

145

 

 

Results are

 

. Control

. Sphinganine

3.5mm

Sphinganine

5PM

Sphinganine

811M

 

3 days 6 days

Figure 5.8. Sphinganine inhibits the growth and causes deeds of type I
HBEC (HME30-4A) (photographs). Sub-confluent cells were cultured with
sphinganine at 0, 3.5, 5. and 8 M for 3 and 6 days. Magnification 10X
plus 10X. Images in this dissertation are presented in color.

146

Type II HBEC

Control

' - Sphinganine
-'1 3.5pM

Sphinganine

5n"

 

Sphinganine
81M

 

     

Sphingosine
. sum

6 days
Figure 5.9. Type II HBEC (HME29-A) are less sensitive to growth inhibitory
effects of sphinganine and sphingosine than tumorigenic type I HBEC and
type I HBEC at 6 d (photographs). Sub-confluent cells were cultured with
sphinganine at 0, 3.5, 5, and 8 pM and with sphingosine at 0 and 8 W for
6 days. Left panel: Areas where the growth of cells are not affected by
treatments with sphinganine and sphingosine. Right panel: Amas where
the growth of cells are inhibited by treatments with sphinganine and
sphingosine. Magnification 10X plus 10X. Images in this dissertation are
presented in color.

147

Type II HBE C are less sensitive to growth inhibitory effects of sphinganine and
sphingosine than tumorigenic type I HBE C and type I HBE C. To examine the
effects of sphingoid bases on grth of type H HBEC (representing normal human breast
epithelial cells), sub-confluent cells were cultured with sphinganine and sphingosine at
various concentrations and total nucleic acids were determined as an index of cell number
(Figure 5.7-Right panel; Figure 5.9-Photographs; Appendix 13: Figure A.14-
Photographs).

Total nucleic acids in control cultures of type H HBEC increased more than 4 fold
in 6 days, indicating that type H HBEC (Figure 5.7-Right panel) grew much slower than
tumorigenic type I HBEC (Figure 5.3-Lefi panel) but grew slightly faster than type I
HBEC (Figure 5.7-Lefi panel). Sphinganine at 3.5, 5, and 8 pM decreased total nucleic
acid concentrations at 3 d by 26, 28, and 61 % and at 6 d by 21, 23, and 53 %,
respectively compared to the corresponding controls. Sphingosine at 8 pM reduced total
nucleic acids at 3 d by 24 % and at 6 d by 24 % compared to the corresponding control
(Figure 5.7-Right panel). These data showed that type H HBEC are less sensitive to
growth inhibitory effects of sphingoid bases than tumorigenic type I HBEC (Figure 5.3)
and type I HBEC (Figure 5.7-Lefi panel).

Sphinganine and sphingosine induce differentiation of type I HBEC to type II
HBEC. Type I HBEC were cultured with sphinganine and sphingosine at non-
growth inhibitory concentrations for 7 d and the ability of type I HBEC to differentiate to
type H HBEC were evaluated by counting morphologically distinguishable colonies of
type I, type H, and type I surrounded by type H HBEC (Table 5.1; Figure 5.10-

Photographs).

148

Controls (not treated with any chemicals) had ~ 6.1 % differentiation rate and low
colony forming efficiency by 7 d. Control vehicles (0.1 % (v/v) of lmM BSA) had ~ 5.4
% differentiation rate. Cells treated with 0.5 % (v/v) of 1 mM BSA increased the
differentiation rate to 12.5 % and enhanced colony forming efficiency rate, as shown by
the increase in the number of total colonies. This suggests that concentration of BSA
higher than that present as the control vehicle might induce the differentiation of type I
HBEC to type H HBEC.

Sphinganine at 0.05, 0.1, and 0.5 pM increased the differentiation rate to ~ 12.1,
13.3, and 12.4 %, respectively. Sphingosine also increased the differentiation rate to
10.9, 13.5, and 9.8 %, respectively. Cholera toxin (0.1 ng/mL and 1 ng/mL), a positive
control known to induce differentiation of type I HBEC to type H HBEC (Kao et al.
1995), increased the number of type H HBEC containing colonies (to 23 and 18 %,
respectively for 0.1 and 1 ng/mL), while reduced colony forming efficiency, as shown by
the decrease in the number of total colonies. Ethanol at 0.01 and 0.1 % increased the
differentiation rate to ~ 13 and 11 %, while reduced colony forming efficiency (Table
5.1).

Sphinganine increases the expression of a tumor suppressor protein, maspin
during induction of drflerentiation of type I HBE C to type II HBE C. Type I HBEC
were cultured with sphinganine at 0.05 and 0.5 pM for 7 d and the expression of a tumor
suppressor protein, maspin was determined using immunofluorescence staining (Figure
5.11). Recently our laboratory demonstrated that maspin is expressed in type H HBEC,
but not in type I HBEC and in vitro transformed HBEC (immortalized type I I-IBEC,

weakly tumorigenic type I, highly tumorigenic type I HBEC) (Saitoh and Chang,

149

unpublished data). Therefore, maspin expression may serve as a biomarker for
differentiation of type I HBEC to type H HBEC. As shown in Figure 5.11, maspin
expression is not detected in type I HBEC colonies, whereas type H HBEC containing \
colonies show maspin expression. Type H HBEC were more readily detected in

sphinganine treated cells compared to the control (Figure 5.11).

150

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151

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5"“ ‘- ~_*—~~a "cl—d

   

.-T___T.

    

SPH 0.1 pM: 1type ll colony SPH 0.5 ”M: 1 type II colonies.
1 type I differentiating to type II

Figure 5.10. Sphinganine and sphingosine induce differentiation of type I
HBEC (HME29-A) to type II HBEC (Representative photographs). Type I
HBEC were seeded at 5000 cells per 60 mm dish with or without grids and
grown for 2 days and then cultured with sphinganine (SA) and sphingosine
(SPH) at 0, 0.05, 0.1, and 0.5 pM for 7 days. Fresh medium with and
without chemicals were renewed on day 3 and 5. The number of colonies
of type I, type II, and type I surrounded by type II HBEC containing colonies
were quantitated after 7 d treatments. Magnification 10X plus 4X. Images
in this dissertation are presented in color.

152

Control
Type I HBEC

Control
Type I HBEC

SA 0.05 pH
Type II HBEC

SA 0.5 pm
Type II HBEC

 

Figure 5.11. Sphinganine increases the expression of a tumor suppressor
protein, maspin during induction of differentiation of type I HBEC (HME29-
A) to type II HBEC (photographs). Type I HBEC were seeded at 1250
cells per well in 4-well chamber and grown for 2 days and then cultured
with sphinganine (SA) at O. 0.05, and 0.5 uM for 7 days. Fresh medium
with and without chemicals were renewed on day 3 and 5. On day 7, cells
were immunostained with an antibody against maspin. Left panel: Phase
contrast; Right panel: Fluorescence. Magnification 10X plus 20X. Images
in this dissertation are presented in color.

153

DISCUSSION

Sphingolipids have gained much attention for their potential as chemotherapeutic
or chemopreventive agents based on in vitro cancer cell and animal studies. For example,
in rodent models, both dietary and synthetic sphingolipids inhibit colon carcinogenesis
(Dillehay et al. 1994, Schmelz et a1 1996, 1997, 2000, 2001). In addition, a number of
studies have demonstrated that the sphingolipid metabolites ceramide and sphingoid
bases inhibit growth and induce apoptosis of cancer cells (Ahn and Schroeder 2002b, for
review: Merrill et al. 1997, Vesper et al. 1999). However, no study has compared the
anti-proliferative and pro-apoptotic efl’ects of sphingoid bases (sphingosine and
sphinganine) on human tumorigenic cells to that of human stem and normal cells.

The present study is the first to evaluate the chemotherapeutic and
chemopreventive potentials of sphingoid bases using the unique normal human breast
epithelial cell culture system (type I human breast epithelial cells (HBEC) as normal stem
cells; type II HBEC as normal cells; tumorigenic type I HBEC as cancer cells) (Figure
5.2). Since type I HBEC with stem cell characteristics are more susceptible to neoplastic
transformation (Kao et al. 1995, Sun et al. 1999), type I HBEC seem to be the target cells
for breast carcinogenesis, while type II HBEC may not be the major target cells for
carcinogenesis.

A major finding of this study is that both sphingosine and sphinganine potently
inhibit the growth and induce apoptosis of tumorigenic type I HBEC. Apoptosis was
induced in tumorigenic type I HBEC with sphingoid bases ranging from 5-10 M which
is lower than the concentrations of ceramide and sphingoid bases necessary to induce

apoptosis in other cancer cells (~10-50 pM). This might suggest that breast cancer cells

154

may be more sensitive to sphingoid bases than cancer cells originating in other tissues. It
is noteworthy that many other drugs currently used in cancer chemotherapy selectively
kill target cancer cells by inducing apoptosis. For example, tamoxifen, widely used for
breast cancer treatment, induced apoptosis in both estrogen receptor positive and negative
human breast cancer cells (Frankfurt et a1. 1995, Perry et al. 1995, Gelmann et al. 1996,
Mandlekar et al. 2000). Moreover, the anticarcinogenic efficacy of many
chemotherapeutic agents is associated with sphingolipid metabolism. For example, the
chemotherapeutic effect of tamoxifin is reduced when generation of acylated-sphingoid
base is impaired, but is enhanced when the degradation of acylated-sphingoid base is
blocked (for review: Senchenkov et al. 2001).

To our knowledge, this is the first study to show that sphingoid bases at
concentrations-inducing apoptosis arrested cell cycle at Gle with reductions in S and
Go/Gl phases in human breast tumorigenic cells. The ability to arrest the cell cycle at
G2/M appears to be an important characteristic of cancer chemotherapeutic agents. For
example, podophyllotoxin, a drug used clinically for cancer treatment (Aisner and Lee,
1990), arrested cell cycle at G2/M with reductions in S and Go/G1 phases and induction of
apoptosis in HT-29 human colon cancer cells (Tseng et al. 2002). Interestingly our data
suggest that sphingosine has a threshold (greater than 5 but smaller than 8 M4) to cause
accumulations of cells in Gz/M phase. Therefore, the Gz/M arrest may not be a good
predictor of the degree of apoptosis because sphingosine at both 8 and 10 1.1M caused

similar degrees of cell accumulations at G2/M. However, 10 W sphingosine caused a

much greater increase of apoptotic cells than 8 uM sphingosine. Furthermore,

155

sphinganine at lower apoptotic concentrations (3.5 and 5 pM) did not arrest cell cycle at
G2/M.

Another important finding of this study was that both sphingosine and
sphinganine inhibited the growth and caused the death of type I HBEC. Type I HBEC
display stem cell characteristics and are more susceptible to ne0plastic transformation,
telomerase activation, and immortalization (Kao et al. 1995, Kao et al. 1997, Kang et al.
1998, Sun et al. 1999). Thus, sphingoid bases might reduce target cells for breast
carcinogenesis.

The present study demonstrated that sphinganine is a more potent apoptotic agent
for both normal type I HBEC and tumorigenic type I HBEC than sphingosine. This result
is consistent with our previous findings in human colon cancer cells (Ahn and Schroeder
2002b) and suggests that the disparity in potency is maintained across cell types. The
reason for the greater apoptotic potency of sphinganine than sphingosone is not clear, but
is probably related to the relative sensitivity of the molecular targets of the sphingoid
bases and/or the activities of the enzymes responsible for sphingoid base metabolism
(Sakakura et al. 1998, Baek et al. 2001, for review: Goswami and Dawson, 2000, &
Radin, 2001). From a dietary perspective, most complex sphingolipids naturally present
in foods contain sphingosine as the backbone.

Another important finding of this study is that sphingoid bases seem to meet the
criteria for chemotherapeutic agents. That is, sphingoid bases at concentrations that were
growth inhibitory and cytotoxic for normal type I HBEC and tumorigenic type I HBEC
(sphinganine at 3.5 to 5 M and sphingosine at 8 uM) showed only minor growth

inhibitory effects for type II HBEC. This is consistent with those of Sweeney et a1 (1996)

156

who reported that sphingosine induced apoptosis in simian virus 40-transformed rat
mesangial cells, but not in their primary culture counterparts (normal rat mesangial cells).

This study is the first to evaluate the chemopreventive potential of sphinganine by
comparing the effects of sphingoid bases on type I HBEC to those on type II HBEC.
Data indicated that sphingoid bases at non-growth inhibitory concentrations (0.05 to 0.5
pM) induced differentiation of type I HBEC to type II HBEC and no dose-response effect
was observed within the rage of sphingoid bases concentrations tested. The mechanisms
by which sphingoid bases induced differentiation of type I HBEC remains unknown.
Ceramide, the acylated form of sphingosine, was first recognized as a differentiating
agent for HL-6O human leukemia cells (Okazaki et al. 1989, Okazaki et al. 1990, Kim et
al. 1991). Vitamin D3 (Okazaki et al. 1989) and TNFa (Kim et al. 1991) induced this
undifferentiated HL-60 cells to become monocytic/macrophage-like cells by increasing
ceramide formation. Also, exogenous ceramide mimicked vitamin D3- or TNFa-induced
monocytic differentiation of HL-60 cells (Okazaki et al. 1990, Kim et al. 1991). TNFa-
induced differentiation of HL-60 cells caused down-regulation of c-myc and the time
required to affect c-myc corresponded to the time required for ceramide formation. This
suggests that ceramide induced differentiation by regulating c-myc expression. Further
studies of ceramide-induced differentiation have been mostly done in neuronal cells and
keratinocytes (epidermal epithelial cells) (for review: Huwiler et al. 2000, Luberti et al.
2002). Alternatively, sphingoid bases might increase cellular cAMP to induce the
differentiation since cholera toxin (used as a positive control in the current study), a well-
known inducer of cAMP, effectively increased the differentiation rate of type I HBEC to

type 11 HBEC (Kao et al. 1995).

157

Interestingly, ethanol at 0.01 and 0.1 % also induced differentiation of type I
HBEC to type H HBEC. Roles of ethanol on proliferation and differentiation are not
clearly understood. Effects of ethanol on proliferation and differentiation might vary
depending on different concentrations of ethanol, different cells from different tissue
origins, and different culture conditions. For example, ethanol enhanced neural
differentiation of PC12 cells with involvement of protein kinase C (for review: Messing
1993). Ethanol inhibited growth and induced apoptosis of hypothalamic beta-endorphin
neurons (De et al. 1994). Ethanol inhibited skeletal muscle cell proliferation and delays
its differentiation in cell culture (Garriga et al. 2000). Ethanol at 0.3 % stimulated
proliferation of MCF-7 human breast epithelial cells, while ethanol at higher than 0.3 %
inhibited the proliferation (Izevbigie et al. 2002). This stimulation of proliferation by
ethanol was mediated by elevated activity of extracellular signal-regulated kinase-l/Z
(ERKl/ERKZ) (Izevbigie et al. 2002). Ethanol at 10 mM increased proliferation and
reduced differentiation of head and neck squamous cell carcinoma cells (Komfehl et al.
1999)

The current study is the first to show the detection of maspin expression during
sphinganine induced differentiation of type I HBEC to type II HBEC. Maspin (mammary
serpin) is a novel tumor suppressor gene and an inhibitor of serine proteases. Maspin was
originally isolated from normal breast epithelial cells and its expression was decreased
during tumor progression (Maass et al. 2002, Shi et al. 2002, Shi et al. 2003). Recently
Saitoh and Chang (unpublished data) demonstrated that maspin is expressed in type H
HBEC, but not in type I HBEC and in vitro neoplastically transformed HBEC

(immortalized type I HBEC, weakly tumorigenic type I, highly tumorigenic type I

158

HBEC). This suggests that maspin expression can serve as a biomarker for status of
differentiation of type I HBEC to type H HBEC. Further studies are needed to elucidate
the effects of sphinganine on maspin status during differentiation of type I HBEC.

In summary, this is the first study that evaluated chemotherapeutic and
chemopreventive potentials of sphinganine and sphingosine using a novel cell culture
system of HBEC (type I HBEC with stem cell characteristics, type II HBEC, and
transformed tumorigenic type I HBEC). The data support the potential for sphingoid
bases to be employed as chemotherapeutic and chemopreventive agents against human

breast cancer (Figure 5.12).

159

Type I HBEC (stem cells) Type II HBEC

 

Maspin <-> I ‘ ”35”” H
I Differentiation '
:4».
Sphinganine
" Sphingosine
" Apoptosis

 

Arrest at GZIM

 

Tumorigenic type I HBEC

Figure 5.12. Chemopreventive properties of sphinganine and
sphingosine in type | and type II HBEC and chemotherapeutic effects of

sphinganine and sphingosine against tumorigenic type I HBEC and type
I HBEC.

160

Acknowledgement

We express our thanks to: Dr. Dale R. Romsos for his advice and support; Michelle
Komosinski and Mark Hagerty for excellent technical assistance on quantitation of
colonies of HBEC; Dr. Louis King for advice on flow cytometry analysis; Drs. James E.
Trosko and Brad Upham for providing access to their experimental instruments; and Dr.
Won 0. Song for her encouragement.

161

CHAPTER 6. CONCLUSION AND SIGNIFICANCE OF THE STUDY

162

CONCLUSION & SIGNIFICANCE OF THE STUDY

This study evaluated chemotherapeutic and chemopreventive roles of four major
sphingolipid metabolites (sphingosine, sphinganine, ceramide, and dihydroceramide) in
colon and breast cancers. Conclusions and significance of the current study are as
follows:

Conclusion and significance pertaining to colon cancer

Data fi'om HT-29 and HGT-116 human colon cancer epithelial cells suggested
that sphingoid bases (sphingosine and sphinganine) and Cz-ceramide might possess a
potential as chemotherapeutic agents, based on their abilities of inhibiting proliferation,
arrest cell cycle, and induce apoptosis within concentration ranges of 20-50 M (Figure
3.8).

I examined the importance of the 4,5-trans double bond present in both
sphingosine and Cz-ceramide by comparing the effects of these lipids to those of
sphinganine and Cz-dihydroceramide, which lack the 4,5-trans double bond (Figure 3.1).
My finding of no effect of Cz-dihydroceramide, and a more potent effect of sphinganine
than of the other metabolites to induce apoptosis of HT-29 and HGT-116 cells suggest: I)
that the 4,5-trans double bond is necessary for the inhibitory effects of Cz-ceramide, but
not for inhibitory effects of sphingoid bases; and 2) the more potent effect of synthetic
dihydrosphingomyelin (lacks 4,5-trans double bond) than synthetic and dietary
sphingomyelin at reducing aberrant colonic foci in CF -1 mice treated with DMH
(Schmelz et al. 1997) might be via conversion to sphinganine and/or sphingosine rather

than to dihydroceramide and/or ceramide (Figure 2.2).

163

It is unclear why Cz-dihydroceramide lacks biological effects on growth and death
of colon cancer cells. One possible explanation is that Cz-dihydroceramide may exhibit
limited conversion to flee sphingoid bases. I found that cells exposed to C2-
dihydroceramide over 3 to 48 h showed lower intracellular concentrations of free
sphingoid bases than those of cells treated with Cz-ceramide (Appendix C, Figure A.l).
Quantitation of intracellular concentrations of ceramide as well as free sphingoid bases
after sphingoid bases and ceramides treatments would help to clarify possible roles of the
4,5-trans double bond.

Both HT-29 and HGT-116 cells responded similme to effects of sphingoid bases
and ceramides on growth, cell cycle, and apoptosis. This suggests that several genes
showing different mutation status (APC, p53, B-catenin, ras, COX2) between these two
cell lines (Table 2.3) do not appear to play major roles for mediating the anti-proliferative
and pro-apoptotic actions of sphingoid bases and ceramide.

Sphinganine may induce apoptosis of HT-29 cells by altering the balance between
pro-apoptotic and anti-apoptotic signaling pathways through JNK2/JNK1, p3 8, and AKT
(Figure 2.3; Figure 2.4). My study indicated that sphinganine at an apoptotic
concentration (35uM) caused early activation of JNK2/JNK1 and p38, which may be
responsible for inhibiting proliferation and stimulating apoptosis. Sphinganine also
caused early inhibition of AKT. AKT activation functions in promoting proliferation and
inhibiting apoptosis and its activity is enhanced with cancer progression.

Conclusion and significance pertaining to breast cancer
My study was the first to systemically evaluate the chemotherapeutic and

chemopreventive potentials of sphinganine in comparison to sphingosine using a novel

164

normal human breast epithelial cells (HBEC) culture system (Figure 2.7; Table 2.4). The
HBEC were developed from mammary tissues of healthy females (type I HBEC with
stem cell characteristics and type II HBEC with basal epithelial cell phenotypes). Since
type I HBEC are more susceptible to neoplastic transformation, type I HBEC seem to be
the target cells for breast carcinogenesis. Tumorigenic type I HBEC were transformed
from type I HBEC by a series of oncogenic treatments (Figure 2.7; Table 2.4).

The current study advocates that sphingoid bases seem to meet the criteria for
chemotherapeutic agents based on following observations (Figure 5.11): 1) Sphingoid
bases inhibited proliferation, arrested cell cycle at Gz/M with reductions in S and Go/G1
phases, and induced apoptosis of tumorigenic type I HBEC, with sphinganine being more
potent (IC50 for sphinganine 4 uM; sphingosine 6.4 1.1M); 2) Sphingoid bases inhibited the
growth and caused the death of type I HBEC with sphinganine being more potent; and 3)
Type II HBEC are less sensitive to growth inhibitory effects of sphingoid bases than
tumorigenic type I HBEC and type I HBEC.

Sphingoid bases might function as chemopreventive agents since sphingoid bases
at non-growth inhibitory concentrations (0.05-0.5uM), increased the differentiation rate
of type I HBEC to type II HBEC, as detected with expression of a novel tumor
suppressor protein maspin in type H HBEC. The mechanisms by which sphingoid bases
induced difi‘erentiation of type I HBEC remain unknown. Future study might determine
whether c-myc mRN A and cAMP levels mediate actions of sphingoid bases since cholera
toxin used as a positive control (Kao et al. 1995) in the current study is known to increase
cAMP and c-myc levels were altered during ceramide-induced differentiation of HL-60

cells (Kim et al. 1991).

165

The present study has raised an interesting question of why sphinganine is more
potent than sphingosine in inhibiting the growth and causing the death of tumorigenic
type I HBEC and type I HBEC as well as HT-29 and HGT-116 cells. The only structural
difference between sphinganine and sphingosine is that sphinganine lacks the 4,5-trans
double bond (Figure 3.1). Up to now, it is unclear why sphinganine is more potent than
sphingosine and ceramide and why 4,5-trans double bond is responsible for the different
effect of ceramide and sphingoid bases. Further metabolic studies of ceramide and
sphingoid bases including determinations of cellular concentrations of ceramide and
sphingoid bases and of activities of enzymes responsible for metabolizing ceramides and
sphingoid bases might answer this question. However, fi'om a practical perspective most
complex sphingolipids present in foods contain the 4,5-trans double bond.
Considerations should be made to compare the efficacy of synthetic sphingoid bases and
dietary sphingoid bases to achieve effective doses in vivo.

It still remains unclear whether colonic and/or breast tissues are normally exposed
to the concentrations of sphingoid bases and/or ceramide used in this study. To use
sphingoid bases clinically, further studies are needed to determine digestion, absorption,
and excretion of dietary and synthetic sphingolipids in vivo. In the current study, the
apoptotic concentration range of ceramide and sphingoid bases for HT-29 and HCT-l 16
human colon cancer cells were 20-50 M and for tumorigenic type I HBEC sphingoid
bases at 3.5-10 uM induced apoptosis. Most previous studies reported about 10-50 M
as apoptotic concentrations of ceramide and sphingosine, suggesting that breast
tumorigenic cells might be more sensitive to sphingoid bases than to cancer cells

originated from other tissues.

166

From a clinical view, individual variations in sensitivity of normal breast cells to
sphingoid bases could not be excluded. I examined type I and type II HBEC developed
from three Caucasian young healthy females (23-26 years old). Here I found a small
growth inhibitory effects of sphingoid bases on type II HBEC, however Yang et al
(Submitted) did not observe any effect of sphingosine on growth of type II HBEC,
suggesting that different individuals might respond differently to sphingoid bases
chemotherapy for breast cancer. For future study, it is possible to study the effects of
sphingoid bases on the development of human mammary gland in vitro system since type
I HBEC together with type II HBEC are able to form terminal end-bud-like structures on
Matrigel.

Overall, the current study contributes to a better understanding of
chemotherapeutic and chemopreventive roles for the sphingolipid metabolites
sphingosine, sphinganine, ceramide, and dihydroceramide in colon and breast cancers.
My study suggests that sphingoid bases and ceramide might possess chemotherapeutic
property against colon cancer, with sphinganine being the most potent. Early activation
of JNK and p38 and early inhibition of AKT might mediate sphinganine-induced
apoptosis of colon cancer cells. Moreover, the data from HBEC support the potential for
sphingoid bases to be employed as chemotherapeutic and chemopreventive agents against

human breast cancer.

167

APPENDIX

168

APPENDIX A

Table A.l. Mobility of sphingolipids on Thin-Layer Chromatography.

 

Rfvalues
Solvent system (v/v) Cer SM SO SPC Gm PhfloSO
Ch/Me/Ace/W ND 0.50 0.76 0.19 0.23 0.86
(25:15:43.5)
Ch/Me/0.22 % CaClz ND 0.34 0.33 0.11 0.21 ND
(60:35:8)

Ch/Me/TA/P/0.25% KC] ND ND ND ND ND ND
(30:9:25:18z6)

Ch/Me/Ace/W 0.97 0.12 0.73 0.03 0.03 0.72
(56:30:42)

E/Me (99: 1) 0.57 0.00 0.00 0.00 0.00 0.00
Ch/Me/NI‘LOH 0.94 0.15 0.80 0.04 0.00 0.55
(13:7:1)

Ch/Hexane/Me 0.63 0.00 0.05 0.00 0.00 0.04
(10:10:3)

Ch/Me/2N NH40H 0.84 0.03 0.20 0.00 0.00 0.10
(40: 10: 1)

Ch/Me/Formic acid/W 0.97 0.24 0.76 0.09 0.05 0.73
(56:30:42)

 

Abbreviations used are: ND, nondetectable when same concentrations of sphingolipids
were applied; Ch, chloroform; Me, methanol; Ace, acetic acid; TA, triethylamine; P, 2-
propanol; W, water; E, diethylether; Cer, Ceramide; SM, sphingomyelin; SO,
sphingosine; SPC, sphingophosphorylcholine; Gm, ganglioside Gm; PhytoSO,
phytosphingosine.

Table A.2. Rf values of sphingolipids on 2-Dimemsional Thin-Layer Chromatography.

 

Rf values
Solvent system (Viv) Cer SM ISO SPC Gm
lst Diethylether/Methanol 0.57 0.00 0.00 0.00 0.00
(99:1)
2nd Chloroform/Methanol/Acet 0.92 0.13 0.73 0.04 0.04
ic acid/Water (56:30:422)

 

169

APPENDIX 8
METHODS

Quantification of free sphingoid bases. Mass measurements of long-chain
(sphingoid) bases were conducted as previously described (Merrill et al. 1988, Riley et al.
1994). Briefly, Czo-sphingosine was added as an internal standard only to samples for the
purpose of measuring intracellular fiee sphingoid bases in HGT-116 cells. The sphingoid
bases were then extracted with chloroform and methanol and treated with base to remove
interfering glycerolipids. After preparation of the o-phthalaldehyde derivatives, the long-
chain bases were separated by reverse-phase high-performance liquid chromatography
(l-IPLC) using a C13 column eluted isocratically with methanol, 5 mM potassium
phosphate, pH 7.0 (91 :9, v/v). The concentrations of sphingoid bases were determined
by reference to the Czo-sphingosine, internal standard.

Analysis of intemucleosomal DNA fragmentation by gel elctrophoresis. The
fragmented DNA was detected using the Genomix cells small scale kit purchased from
Talent. The procedure was based on Goruppi et al. (1994). After treatment of cells with
sphingoid bases and ceramides for 12 or 24 h, cells were harvested in 150 uL of
phosphate buffered saline (PBS). Cells were lysed and aqueous phase was separated
using chloroform, acidfication solution, and gel barriers. Cells were precipitated and
centrifuged and liquid phase was removed. The DNA pellet was resuspended in distilled
water followed by electrophoresis in a 2% agarose gel at 100 V for 70~90 min. The
DNA bands stained with ethidium bromide was photographed under UV light.

Quantitation of cellular protein using Lowry method. Cellular protein was
determined using Lowry method as described (Lowry et al. 1951).

The scrape loading/dye transfer assay. Effects of sphingoid bases on gap
junctional intercellular communications were determined by the scrape loading/dye
transfer assay as described (El-Fouly et al. 1987 and Trosko et al. 2000). Cells were
grown until confluency in 35 mm dish and treated with sphingoid bases at various
concentrations for 24 and 48 h. Cells were rinsed twice with CaMg-PBS [137 mM NaCl
(8g/L), 2.68 mM KCl (0.2g/L), 8.1 mM NazHPO4 (1.15g/L), 1.47 nM KHzPO4 (0.2g/L),
0.68 mM CaClz, 0.49 mM MgClz]. The Lucifer yellow reagent [0.5 mg/mL Lucifer
yellow CH lithium salt (Molecular Probes, Eugene, OR), 0.5 mg/mL rhodamine-dextran
(Molecular Probes) in CaMg-PBS] was added to cell monolayer and the cell monolayer
was wounded in a straight line with a sterile scalpel blade. After incubation of the
wounded monolayer in dark room for 3 min, Lucifer yellow was removed fi'om cells and
cells were rinsed four times with CaMg-PBS and 4 % (v/v) formalin in CaMg-PBS was
added to cells. The distance of dye (Lucifer yellow) transfer from the cells along the
scrape line into neighbor cells were observed under fluorescence microscope.

170

APPENDIX c
HCT-1 1 6 goells

- Dihydroceramide r
so Ceramide so

 

 

40

20 20

 

Free sphingoid bases
(nmollmg protein)
8

012243648 0012243648
Hours Hours

Figure A.1. Effects of exogenous Cz—ceramide and Cz-dihydroceramide on
intracellular concentrations of free sphingoid bases in HGT-116 human colon
cancer cells. Subconfluent cells were cultured with Cz-ceramide and C2-
dihydroceramide at 0 (O), 20 (A), and 50 (0) p.M for 3, 12. 24, or 48 h and
cellular concentrations of free sphingoid bases (sphingosine and sphinganine)
were quantitated via HPLC. Data represent mean 1 SEM (n=4). Where an
error bar is not shown, it lies within the dimensions of the symbol.

HGT-116 cells

   

Control 48h SA 20 pM, 48h SA 35 pM, 48h

Figure A.2. Effects of sphinganine (SA) on gap junctional intercellular
communication on HGT-116 human colon cancer cells (photographs).
Confluent cells were cultured with sphinganine at 0, 20. and 35 uM for 48 h.
Top panel: Florescence; Bottom panel: Phase contrast. Magnification 10X
plus 10X. Images in this dissertation are presented in color.

171

APPENDIX D
HT-29 cells :=.- . .

  

   

' Control 24h

._ Sphinganine
20 pM, 24h

Sphinganine
35 pM, 24h

Control 48h

Sphinganine
_ 20 pM, 48h

‘ r1 Sphinganine

r: 35 pM, 48h
Figure A.3. junctional intercellular
communication on HT—29 human colon cancer cells (photographs). Confluent
cells were cultured with sphinganine at 0, 20. and 35 uM for 24 and 48 h. Left
panel: Florescence; Right panel: Phase contrast. Magnification 10X plus 20X.
images in this dissertation are presented in color.

172

APPENDIX E

HT-29 cells
Cont SA M

 

Cont Cer M Cont Dh-cer M

 

Figure A.4. Effects of sphinganine, Cz-ceramide, and Cz-dihydroceramide
on DNA fragmentation, indicative apoptosis, in HT—29 human colon cancer
cells. Subconfluent HT—29 cells were cultured without sphingolipids
(control, cont) or with sphinganine (SA) at 10 M for 12h and Cz-ceramide
(Cer) and Cz-dihydroceramide (Dh—cer) at 20 uM for 24h. DNA was
extracted and analyzed using gel electrophoresis on a 2 % agarose gel at
100V. Abbreviation used: M, DNA marker (123 bp).

173

APPENDIX F

HGT-116 cells
Cont SD M Cont SA M

Hi

 

Cont Cer M Cont Dh-cer M

 

Figure A.5. Effects of sphingosine, sphinganine, Cz-ceramide, and C2-
dihydroceramide on DNA fragmentation, indicative apoptosis, in HCT-
116 human colon mncer cells. Subconfluent HOT-116 cells were
cultured without sphingolipids (control, cont) or with sphingosine (SO) at
40 l1 M for 24h, sphinganine (SA) at 10 uM for 12h, Cz-ceramide (Cer)
and Cz-dihydroceramide (Dh-cer) at 20 uM for 24h. DNA was extracted
and analyzed using gel electrophoresis on a 2 % agarose gel at 100V.
Abbreviation used: M, DNA marker (123 bp).

174

APPENDIX G

 

 

 

a HGT-1 1 6 cells
=3 1

g 12

:3: 9

a

o

O.

< 0

.\° Cont 80 SA

Figure A.6. Effects of sphingoid bases on apoptotic cell number in HCT-
116 human colon cancer cells. Subconfluent cells were cultured in the
absence (control, Cont) or presence of sphingosine (SO) and sphinganine
(SA) at 35 uM for 12 h. DNA was then stained with propidium iodide, the
cell cycle was examined via flow cytometric analysis, and the percentage
of cells in the A0 (sub-Go/G1) region was estimated with the FCS express
version 1.0 software. Data are mean 1 SEM (n=2).

 

HGT-116 cells
E 100
:3: 75
E": 50
$9.. 25
o 0

 

 

 

Cont 80 SA

Figure A.7. Effects of sphingoid bases on cell cycle distribution of HOT-116
human colon cancer cells. Subconfluent cells were cultured in the absence
(control, Cont) or presence of sphingosine (SO) and sphinganine (SA) at 35
uM for 12 h. DNA was then stained with propidium iodide, the cell cycle was
examined via flow cytometric analysis, and the percentage of cells in each
stage of the cell cycle, including GalG1 phase, /4; S phase, D; and GZIM
phase, I, was determined using the Multi—cycle DNA content & cell cycle
analysis software. The A0 (sub-6016,) cell population was not included in
calculation of cell population. Data are mean (n=2).

175

APPENDIX H

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

HGT-116 cells
A 125 ‘5.
e 100 - 13 .
a 15 - ‘I' '
0 75 -
E 50 _ 56 _
f ”r as SA
0 ' 0 : ‘
°\° 13° ' 151 -
V _ 125 ,
'3 90 10% CA
'5 3' : 75)
< _ s-
_2 3. M as: W7
0 o k ‘ 0 a l a L
_5 ‘5‘ 166
g 126 126 -
_ 106 -
5 ’°
0 6° w
" so SA+W7 w SA+W7
0 . . . . .
o 3 6 s 12 ' o s a 9 12
hours hours

Figure A.8. Effects of a calmodulin antagonist W7 on growth inhibitory effects
of sphinganine (SA) in HOT-116 human colon cancer cells. Lelt panel.
Subconfluent HGT-116 cells were cultured with SA at 0 (O), 35 uM (O), with
W7 at 0 (e), 1 (O), 3 (V), 10 uM (0) and simultaneously with both sphinganine
and W7 at control (0), 35 pM SA + 1 uM W7 (0), 35 M SA + 3 M (V), 35 uM
SA + 10 pM W7 (0) for 0, 3, and 12 h. Right panel: Subconfluent HGT-116
cells were cultured with SA at 0 (IO), 10 (O), 15 uM (V), with W7 at 0 (e), 1
(O), 3 (V), 10 M (O) and simultaneously with both sphinganine and W7 at
control (C), 10 uM SA +1 pM W7 (0), 1O uM SA + 3 uM (V), 10 uM SA +10
uM W7 (6), 15 pM SA +1 pM W7 (Cl), 15 uM SA + 3 uM (I), 15 uM SA +10
pM W7 (I) for 0, 3, and 12 h. Total nucleic acids were determined as an index
of cell number. Data represent mean 1 SEM (n=3). Where an error bar is not
shown, it lies within the dimensions of the symbol.

176

APPENDIX I
RESULTS

Efl‘ects of a calmodulin antagonist W7 on growth inhibiton ejfects of
sphinganine in HGT-116 human colon cancer cells. HGT-116 cells were treated
with a calmodulin antagonist W7 at 1, 3, 5, 10, and 20 M to find the non-growth
inhibitory concentration range of W7. Data indicated that W7 at 1, 3, and lOuM did not
affect the growth of HOT-116 cells (Figure A.8). HOT-116 cells were treated
simultaneously with sphinganine at 35 M and W7 at l, 3, and 10 M for O, 3, and 12 h.
The result showed that a calmodulin antagonist W7 did not attenuate the growth
inhibitory effect of sphinganine (Figure A.8). However we speculated that this
concentration of sphinganine at 35 M was too high and cells were overwhelmed by
sphinganine growth inhibitory effect (Figure A.8). As a follow-up experiment, cells were
simultaneously treated with sphinganine at lower concentrations of 10 and 15 11M and
W7 at 1, 3, 10 M for 0, 3, and 12 h and the similar result was observed (Figure A.8).
This finding suggests that the growth inhibitory effect of sphinganine may not be related
to an action of calmodulin in HOT-116 human colon cancer cells.

177

APPENDIX J
Tumorigenic type I HBEC

Control

Sphinganine
3.5pM

Sphinganine

5PM

Sphingosine

5PM

Sphingosine
BuM
Figure A.9. Effects of sphinganine and sphingosine on gap junctional
intercellular communication on tumorigenic type I HBEC at 24 h
(photographs). Confluent cells were culturedwithsphinganine etc, 3. 5, and
5M and with sphingosine at 0, 5, and 8 uM for 24 h. Left panel.
Florescence; Right panel Phase contrast. Magnification 10X plus 10X.
Images in this dissertation are presented in color.

 

APPENDIX K
Tumorigenic type I HBEC

 

SA 3.5 uM SA 5 uM Sphingosine 5 pM
Figure A.10. Sphinganine (SA) more potently inhibits the growth and
causes death of tumorigenic type I HBEC than sphingosine at 2 d
(photographs). Sub—confluent cells were cultured with sphinganine at 0, 0.5,
2, 3.5, and 5 uM and with sphingosine at 0 and 5 uM for 2 days. 100x
magnification. Images in this dissertation are presented in color.

Tumorigenic type I HBEC

 

 

pm 0 1.; 5 10
SA SO

Figure A.11. Effects of sphingosine and sphinganine on cell cycle distribution
in tumorigenic type | HBEC at 12h. Sub-confluent cells were cultured with
sphinganine (SA) at 0 and 3.5 uM and with sphingosine ($0) at O, 5, and 10
uM for 12 h and DNA were stained with propidium iodide. The cells in sub-
Go/G1 area, indicative of apoptosis, and the cell cycle were examined via flow
cytometric analysis. The percentage of cells in each cell cycle of GolG1 phase,
2; S phase, El; and GZIM phase, I, was determined using the Mod fit cell
cycle analysis software. The A0 (sub-6016,) cell population was not included
in calculation of cell population. Data are mean 1 SEM (n=2 ).

179

APPENDIX L

 

.C

a Tumorigenic type I HBEC 3 Type II HBEC
a 73450 g ‘5 300

8 0120 E a

T; g 90 g o 200

o g 50 2 -,§. 100

s '5 30 5 *5

Es ° 3 -\° °

_ .. o 0.5 1 5 g .. 0 0-8
g BSA (%) ,2 BSA (%1

Figure A.12. Effects of bovine serum albumin (BSA) on growth of
tumorigenic type I HBEC and type II HBEC. Sub-confluent cells were
cultured with bovine serum albumin at 0, 0.5, 1, and 5% for 24 hours in
tumorigenic type I HBEC and at 0 and 0.8% for 3 days in type II HBEC.
Total nucleic acids were measured as an index of cell number. Results
are expressed as a percentage of the zero hour. Data Shown are mean 1
SEM (n=3). Where an error bar is not shown, it lies within the dimensions
of the symbol.

. Fibbtsrolasv

*

Third type cells

      

Figure A.13. Fibroblasts and the third type of cells are found in human
breast epithelial cells (HME23A, HME29A, HME30-4A, HME31-L2A)

(photographs). Top panel. 40X magnification. Bottom panel. 100x
magnification. Images in this dissertation are presented in color

180

 

 

IIIIIIIIII‘ I|

APPENDIX M

Type II HBEC

 
 
   
   
 
   

Control

I ‘ Sphinganine

‘ 3.5pM

Sphinganine

5PM

3 days
Figure A.14. Type II HBEC (HME29A) are less sensitive to growth
inhibitory effects of sphinganine and sphingosine than tumorigenic type |
HBEC and type I HBEC at 3 d (photographs). Sub-confluent cells were
cultured with sphinganine at 0, 3.5, 5,, and 8 uM and with sphingosine at 0

and 8 uM for 3 days. Magnification 100x. Images in this dissertation are
presented in color

181

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