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thesis entitled

SPHINGOLIPIDS IN FOODS AND DIFFERENTIAL SENSITIVITY OF HUMAN
COLON CANCER CELLS TO SPHINGOID BASES AND CERAMIDES

presented by

Eun-Hyun Ahn

 

has been accepted towards fulfillment
of the requirements for

M.S. degree in Human Nutrition

 

 

 

7221283535?”

Joseph J. Schroeder

Date December 10, 1998

 

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use W14

SPHINGOLIPIDS IN FOODS AND DIFFERENTIAL SENSITIVITY OF HUMAN
COLON CANCER CELLS TO SPHINGOID BASES AND CERAMIDES

by

Eun-Hyun Ahn

A THESIS

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

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1998

ABSTRACT
SPHINGOLIPIDS IN FOODS AND DIFFERENTIAL SENSITIVITY OF HUMAN
COLON CANCER CELLS TO SPI;I)NGOID BASES AND CERAMIDES
Eun-Hyun Ahn

Complex sphingolipids have been shown to protect against development of colon
cancer. Sphingoid bases and ceramides are bioactive metabolites of sphingomyelin and
other more complex sphingolipids found in foods. The purpose of the present study was
to quantitate sphingolipids in some common foods and investigate the effects of
Sphingoid bases and ceramides on growth and death of HT-29 and HGT-116 human
colon cancer cells. The concentrations of total sphingolipids and free Sphingoid bases
(nmng of dry weight) were approximately: nonfat dry milk (203, 146); yogurt (138,
1.2); Swiss cheese (167, 6.5); full fat soy flakes (609, 2.6); soy flour (610, 1.6); isolated
soy protein (210, 2.8). Most sphingolipids in foods were present as complex
sphingolipids and sphingosine was the predominant Sphingoid base backbone of total
sphingolipids in foods. Sphingosine, sphinganine, and Cz-ceramide inhibited grth
and caused death of HT-29 and HCT-l 16 cells in concentration- and time-dependent
manners; whereas, Cz-dihydroceramide had no effect suggesting that the 4,5-trans
double bond was necessary for the inhibitory effect of ceramide. Sphingosine and
sphinganine killed cells by inducing apoptosis; whereas, Cz-ceramide did not induce
apoptosis. The results indicate that sphingolipids are significant constituents of soy
fractions and dairy products and that the colonic concentrations of Sphingoid bases
and/or ceramide which may be achieved after consumption of the foods are sufficient to

inhibit growth and cause death of human colon cancer cells.

ACKNOWLEDGEMENTS

I would like to express very special thanks to my major advisor, Dr. Joseph J.
Schroeder for his kind support, endless understanding, warm encouragement and wise
guidance. I was lucky enough to be his first graduate student. I also would like to extend
my sincere gratitude to my committee, Dr. Dale R. Romsos and Dr. Leslie D. Bourquin
for their guidance.

I would like to thank my fellow graduate students-Chi Zhang, Hong Yang, and
Min-Sun Kim and Dr. Do-Yle Lee, Julie Arnold, Stacey Tremp, and Jason Wiesinger for
their companionship. All of them contributed to various parts of my pleasant memories
while we worked at the same laboratory.

I cannot forget to give my appreciation to Dr. Won 0. Song for her loving
encouragement. My thanks also go to Dr. James J. Pestka and Dr. John E. Linz who
allowed me to use their instruments.

Finally, I would like to give very sincere thanks to my precious family for their
unlimited love, measureless understanding, patience, and support. Their presence have
always brought me happiness and strength. I also thank my special friends in Seoul,

Korea and USA for their friendship and encouragement.

I dedicate this fruit of my sweat to my parent who gave me a life, my older

brothers, relatives, special friends, and God who created the world.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES v
LIST OF FIGURES vi
I. LITERATURE REVIEW 1
A. Colon cancer 1
l. Colon cancer incidence
2. Colon cancer etiology
3. Colon cancer and diet
B. Dietary sphingolipids might reduce colon carcinogenesis 5
1. Sphingolipids regulate cell behavior
2. Milk sphingomyelin might prevent the progression
of adenomas to adenocarcinomas in colon cancer
3. Ceramide and sphingosine may mediate the protective
effect of sphingomyelin against colon cancer
C. Knowledge of the types and concentrations of sphingolipids 14
in foods is limited
II. OBJECTIVES 17
III. MATERIALS AND METHODS 18
IV. RESULTS 28
A. Sphingolipid concentrations in foods 28
B. Differential sensitivity of human colon cancer cells to 33
Sphingoid bases and ceramides
V. DISCUSSION 41

VI. REFERENCES 47

iv

Table 1.
Table 2.

Table 3.

LIST OF TABLES

Total lipids and moisture content in foods.
Sphingolipid concentrations in foods.
Percentage of free Sphingoid bases and Sphingoid base

backbones of food complex sphingolipids that are
sphingosine and sphinganine.

Page
29
3O

32

Figure 1.
Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

LIST OF FIGURES

Structures of sphingolipids.
Bioactive products of Sphingolipid hydrolysis.

Dietary sphingomyelin and its turnover products can
reach the colon.

Elution profiles of sphingosine, sphinganine,
Czo-sphingosine on C1; reverse-phase HPLC.

Structures of Sphingoid bases and ceramides with or without
the 4,5-trans double bond.

Total Sphingolipid concentrations in foods.

Sphingosine, sphinganine, and ceramide inhibit growth and
cause death of HT-29 human colon cancer cells.

Sphingosine, sphinganine, and ceramide inhibit growth and
cause death of HT-29 human colon cancer cells (photographs).

Sphingosine, sphinganine, and ceramide inhibit growth and
cause death of HCT-l 16 human colon cancer cells.

Sphingosine, sphinganine, and ceramide inhibit growth and

cause death of HCT-l 16 human colon cancer cells (photographs).

Sphinganine kill HT-29 human colon cancer cells by
inducing apoptosis (photographs).

vi

Page

11

12

22

25

31

34

35

38

39

4o

I. LITERATURE REVIEW

A. Colon cancer

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

2. Colon cancer etiology. The adenoma-carcinoma hypothesis proposed by
Hill et al (1978) is now 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
phenotype. Bird er al (1987) suggested that hyperproliferation of the upper crypt cells
leads to the formation of aberrant crypt foci 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 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 (Vogelstein et al., 1989, Fearon er al., 1990,

Fearon and Vogelstein, 1990, Kinzler et al., 1991). The genes involved in colon

carcinogenesis are: mutations or loss of the APC gene (a tumor-suppressor gene)
(Aaltonen er al., 1993), mutation of K-ras (a prom-oncogene) and early disorganization
of DNA methylation (Feinberg and Vogelstein, 1983) and late loss of p53 (a tumor-
suppressor gene). People having familial adenomatous polyposis possess a mutation in
the APC gene and carry an almost 100 percent risk of colon adenocarcinoma (Aaltonen et
al., 1993). Hereditary nonpolyposis colorectal cancer is a syndrome which is not easily
distinguished from 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). 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 (EI-Deiry et al., 1991). Several animal studies showed that isothiocyanates,
which mainly are present in cruciferous vegetables, inhibited both carcinogenesis and
DNA methylation (Wattenberg, 1977, Wattenberg, 1987, Steinmetz and Potter, 1991a, &
Steinmetz and Potter, 1991b). Although these observations are contradictory, both hypo-
and hypermethylation of DNA are considered to be hallmarks of the early stages of the
carcinogenesis.

3. Colon cancer and diet. Epidemiological studies of diet, nutrient intake and
colon cancer indicate that meat consumption which reflects fat and protein intake is
associated with high incidence of colon cancer (Wynder and Shigematsu, 1967, Wynder
et al., 1969, Drasar and Irving, 1973, Haenszel et al., 1973, Bjelke, 1973, Armstrong and

Doll, 1975, Hirayama, 1981, Heaton, 1982, Phillips and Snowdon, 1985, Potter, 1989)

 

and fruit and vegetable consumption reflecting fiber intake is associated with a protective
effect against development of colon cancer (Burkitt, 1969, Manousos et al., 1983, Stubs,
1985, Trock er al., 1990, Potter, 1990, Steinmetz and Potter, 1991a, & Steinmetz and
Potter, 1991b). Bile acid metabolism and volatile fatty acids might account for the
association of fat and fiber consumption with colon cancer. Fat increases bile acid
production and then bowel mucosa is more exposed to toxic bile acids (Hill, 1971). In
contrast, fiber binds bile acids, reduces transit time, increases stool bulk, and increases
bile acids fermentation to non-toxic volatile fatty acids (Stephen and Cummings, 1983,
Cummings, 1983).

Other factors which might affect risk of colon cancer include physical activity
(Garabrant et al., 1984, Gerhardsson er al., 1986, Slattery et al., 1988), family history
(Burt er al., 1985, Bufill, 1990), and alcohol consumption (Potter et al., 1982, Tuyns er
al., 1988, Longnecker, 1990, Choi and Kahyo, 1991). Lee et al (1991) showed that
individuals with high levels of physical activity were at the low risk for developing colon
cancer (Garabrant er al., 1984, Gerhardsson et al., 1986, Slattery et al., 1988). A family
history of colon cancer is related with increased risk of colon cancer (Burt et al., 1985,
Bufill, 1990). Although some studies showed that alcohol consumption (mostly beer)
was related with increased risk of colon cancer, cautious interpretation of these results is
recommended (Potter et al., 1982, Tuyns er al., 1988, Longnecker, 1990, Choi and
Kahyo, 1991).

Epidemiological studies suggest that dietary fat and specific dietary fatty acids are
associated with colon cancer (Potter et al., 1993); however, this association is

controversial since dietary fat is highly correlated with total energy intake. Slattery et aI

 

r—_.__—_- d"; .

(1997) evaluated these potential associations using detailed dietary intake data collected
in a population-based study of 1,993 colon cancer cases and 2,410 controls in three areas
of the United States. In this study population, fats added in the preparation of foods such
as fried foods or bakery products or fats added to other foods at the table were responsible
for one-third of the total diet fat intake. Interestingly, neither total dietary fat nor specific
fatty acids were associated with risk of colon cancer afier adjusting for total energy
intake, physical activity, and body size. However, fats from food preparation were
associated with increased risk of colon cancer among older women, while fats from foods
themselves or from additions to other foods were not. Taken together, it seems that the
percentage of energy fi'om fat in the diet is not a major indicator of colon cancer risk.
Several investigations suggested that soy consumption may contribute to the
lower rates of breast, prostate, and colon cancer in China and Japan (Setchell er al., 1984,
Barnes et a1, 1990, Adlercreutz, 1990). The isoflavone genistein, which is rich in
soybeans and considered as one of the most significant plant estrogens, has been reported
to protect against development of colon carcinogenesis (Akiyama et al., 1987, Ogawara
er al., 1989, Teraoka et al., 1989, Bourquin er al., 1996, Thiagarajan er al., submitted).
Other epidemiological studies indicate that regular consumption of fermented
milk products such as yogurt may be protective against some forms of cancer (Peters er
al., 1992, Karnpman et al., 1994). 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-dirnethylhydrazine-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. Furthermore, yogurt
fractions obtained by membrane dialysis on cultured mammalian intestinal cells
decreased cell proliferation in both IEC-6 and Caco-2 cells and reduced the number of

IEC-6 cells in the initial growth phase (Ganjarn et al., 1997).

B. Dietary sphingolipids might reduce colon carcinogenesis.

l. Sphingolipids regulate cell behavior. 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
termed sphingosine (Figure 1). Sphingosine (trans-4-sphingenine) and Sphinganine
(without the 4,5-trans double bond) are the most prevalent fi'ee long-chain bases of most
mammalian tissues (Merrill, 1991). Except for Sphingoid bases, ceramides, and
sphingomyelin, all other sphingolipids are designated glycosphingolipids since they
contain carbohydrate head groups. 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).

Sphingolipids are located mainly in plasma membranes and related organelles

including the Golgi apparatus, endosomes, and lysosomes which are functionally

 

H
\ CHZOH
I"'3‘, Sphingosine
H
CHZOH
\

W\/\/\/\/\/\/\/NH Ceramide
g .

 

H

WWNH Sphlngomyelin

ll

NANA

GLU(4<-1)GAL(4<-1)GALNAC(3<-1)GAL

H |
\ H20

W" Ganglloside 6""

Figure 1. Structures of sphingolipids. Abbreviations used are: NANA, N-acetyl neuraminic
acid; GAL, galactose; N AC, N-acetyl; GLU, glucose.

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). Sphingolipids can regulate cell behavior at the surface of the cell, where
they bind to extracellular ligands, such as bacterial toxins, lectins from neighboring cells,
and antibodies. For instance, ganglioside GMl binds to cholera toxin and mediates cholera
toxin’s effects on cells by activating adenylate cyclase (F ishman, 1982) and gangliosides
GD“, Gm, and Go“, function as natural receptors for Sendai virus in host cells (Markwell
er al., 1981). In addition, carbohydrate sequences present in gangliosides are recognized
by monoclonal antibodies raised against murine teratocarcinomas (Solter and Knowles,
1978, Gooi et al., 1981, Kannagi er al., 1982), carcinomas of the pancreas, lung, colon and
stomach (Brockhaus er al., 1982) and myeloid leukemia cells and granulocytes (Skubitz er
al., 1983, Urdal et al., 1983, Magnani er al., 1984). Sphingolipids might act to form a
classical receptor-ligand interaction, or to define regions of the membrane with surface
characteristics that aid in receptor binding and responses, internalization, recycling, or in
anchoring proteins to the cell surface (Menill er al., 1993). An example of a sphingolipid
acting at the surface of the cell is the interaction between ganglioside GM, and epidermal
growth factor receptor (Bremer er al., 1986, Hanai et al., 1988a, Hanai er al., 1988b).
Ganglioside GM3 inhibits epidermal growth factor-stimulated growth of human epiderrnoid
carcinoma cells KB and A431. The mechanism involves inhibition by ganglioside GM3 of
epidermal growth factor-stimulated phosphorylation of the epidermal growth factor
receptors (Bremer et al., 1986).
Sphingolipids can regulate cell behavior by changes that take place in the plasma

membrane and/or endosomes, where complex sphingolipids and their turnover products

 

can stimulate or inhibit receptor kinases, responses of G proteins, protein kinases, protein
phosphatases, and ion transporters (Merrill, 1991). Some sphingolipid hydrolysis
products could influence both the external and internal leaflets of the plasma membrane.
For example, Hope and Cullis (1987) showed that ceramides are relatively nonpolar and
can cross membranes readily. In addition, sphingosine seems to undergo rapid movement
across membranes in the neutral form and the hydroxyls at position 2 of the Sphingoid
base backbone gives a pKa near physiological pH (Merrill et al., 1989).

Sphingolipids can regulate cell behavior at intracellular areas that are sensitive to
products of sphingolipid turnover. Some sphingolipids, such as lysosphingolipids, N-
deacylation products of sphingolipids, and free long-chain (Sphingoid) bases, can move
rapidly among membranes and might affect targets at sites distant from their locations of
formation. For example, sphingosine might be liberated in the plasma membrane but
affect protein kinase C in the nucleus (Hannun er al., 1986a, Hannun et al., 1986b).
Sphingolipids. are also able to be converted to other bioactive compounds, such as
ceramides, ceramide l-phosphate, Sphingoid bases, sphingosine l-phosphate, N-
methylated sphingosines, and lysosphigolipids which can affect multiple intracellular
targets (Zhang et al., 1991, Merrill et al., 1993).

2. Milk sphingomyelin might prevent the progression of adenomas to
adenocarcinomas in colon cancer. Recently, sphingolipids have emerged as
another component of the diet which may help to protect against development of colon
carcinogenesis. Using rodents, Schmelz et a1 (1994) showed that about 12% of dietary
sphingolmyelin passes through the small intestine to the colon. This finding 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 and Figure 3). This hypothesis 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 foci in CF -1 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 and lower percentage of the more
advanced adenocarcinomas (Schmelz er al., 1996). Also, the potential of synthetic
sphingomyelins with saturated or unsaturated sphingoid base backbones to suppress the
number of aberrant colonic foci was investigated using CF -1 mice treated with 1,2-
dimethylhydrazine (Schmelz er 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—palrnitoylsphingomyelin) (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, efficiently reduced the number of aberrant colonic foci (Schmelz et al., 1997).
3. Ceramide and sphingosine may mediate the protective effect of
sphingomyelin against colon cancer. The ability of dietary sphingomyelin to
reduce colon carcinogenesis may be the result of turnover of sphingomyelin to bioactive

metabolites such as ceramides and sphingoid bases which play important roles in signal

transduction and cell regulation (Figure 2 and Figure 3). Extracellular agonists, such as
certain cytokines, growth factors, and hormones, stimulate their cell surface receptors to
activate a sphingomyelinase which cleaves sphingomyelin to generate cellular ceramide
(Figure 2). For example, 1a,25-dihydroxyvitamin D3 (Okazaki et al., 1989), tumor
necrosis factor alpha (TNF-or) (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), dexarnethasone (Ramachandran
er al., 1990), complement components (Niculescu er al., 1993), fungal macrolide
brefeldin A (Linardic er al., 1992), and B-sitosterol, the main phytosterol in the diet
(Awad er al., 1998), were found to induce hydrolysis of sphingomyelin in other cell lines.

Cz-ceramide and other short-chain water-soluble analogues of ceramide induced
differentiation in HL-60 leukemia cells which mimicked the effects of lor,25-
dihydroxyvitarnin D3, tumor necrosis factor alpha (TNF-a), and y—interferon on HL-60
cells (Okazaki er al., 1990). Treatment of U937 human myeloid leukemia cells with
TNF-a caused sphingomyelin hydrolysis and resulted in elevation of level of ceramide.
In addition, ceramide analogs or TNF-a caused intemucleosomal DNA fragmentation, a
hallmark of apoptosis in myeloid and lymphoid cells (Obeid et al., 1993). Unlike tissue
necrosis, which occurs in response to severe insults and injury to cells, apoptosis involves
an orderly breakdown of cells. Apoptosis is characterized with chromatin condensation
followed by DNA fragmentation via the activation of an endonuclease, cytoplasmic

blebbing, and condensation, and finally disintegration into dense particles called

10

H
WCHZOPO3-CH2N+(CH3)3

W” Sphingomyelin

Diacylglycerol Sphingomyelinase
Phosphocholine

MCI-I20“
\

V\/\/\/\/\/\/\/\/NH ceramide

ll

Ceramidase
Ceramide synthase

Fatty acid

H
CH20H

+
H3 Sphingosine

Sphingosine kinase

H
CH20PO3H2

+-
H3 Sphingosine-1phoephate

Figure 2. Bioactive products of sphingolipid hydrolysis.

      
   
   
 
  

Intestinal Lumen Colonic Cell

Sphingomyelin

l

 
 

Ceramide/ Ceramide!
Dihydroceramide Dihydroceramide
Sphingosine/ Sphingosine!
Sphinganine Sphinganine

    

Figure 3. Dietary sphingomyelin and its turnover products can reach the colon.

12

apoptotic bodies (Michaelson, 1991). Ceramide serves as a second messenger for the
action of extracellular agonists by transmitting the signal to the nucleus through multiple
downstream targets such as protein kinase C (zeta isofonn), ceramide-activated protein
phosphatase (CAPP), and ceramide-activated protein kinase. For example, ceramide
induced early down-regulation of c-myc protooncogene (Kim et al., 1991). Ceramide
activated the nuclear factor kappa B (N F-kB) in permeabilized (Schutze et al., 1992) but
not in intact Jurkat T cells (Dbaibo er al., 1993). NF-kB (Schutze er al., 1992) is known
to participate in the control of cell proliferation, in the dephosphorylation of the
retinoblastoma gene product (pr), a tumor suppressor gene that plays an important role
in cell-growth suppression and regulation of cell-cycle progression. Ceramide can act as
an activator of transcription of the cyclooxygenase gene (Ballou er al., 1992).
Sphingosine (F aucher et al., 1988) caused phosphorylation of the epidermal growth factor
receptor on threonine 669 which was independent of protein kinase inhibition and this
action of sphingosine might be associated with the conversion of sphingosine to ceramide
(Goldkorn er al., 1991). Furthermore, this phosphorylation is mediated by a membrane
kinase (ceramide-activated protein kinase) which is activated by TNF-or in intact and in
cell-free systems (Mathias er al., 1991). Ceramide-activated protein kinase shares the
substrate specificity of mitogen-activated protein kinase (MAP kinase) (Joseph et al.,
1993). Ceramide activates MAP kinase (Raines er a1, 1993). Ceramide-activated protein
phosphatase (CAPP) acts as a mediator of the action of ceramide. Ceramide-activated
protein phosphatase (CAPP) serves as a serine/threonine protein phosphatase which is

activated directly and specifically by ceramide but not by dihydrocerarnide (Dobrowsky

l3

and Hannun, 1992) and is inhibited by okadaic acid (Dobrowsky er al., 1993), a tumor
promotor.

Sphingoid bases present in the colon may also help to protect against the
development of colon cancer. Schmelz et a1 (1998) reported that both sphinganine and
fumonisin B,, a mycotoxin which blocks de novo sphingolipid biosynthesis and causes
accumulation of sphinganine, induce apoptosis in HT-29 human colon cancer cells.
Sphingosine inhibits protein kinase C (Hannun et al., 1986a) which has been related with
tumor promotion (W einstein, 1988) and blocks induction of ornithine decarboxylase by
phorbol esters (Gupta et al., 1988, Enkvetchakul et al., 1989). Sphingosine also inhibits
the transformation of C3H10T1/2 cells (Borek et al., 1991). Sphingosine and N-
methylated derivatives inhibit growth of various human tumor cell lines in vitro (Stevens
et al., 1990a) and in nude mice (Endo et al., 1991) and reduce the metastatic potential of
a murine melanoma cell line (Okoshi er al., 1991). In addition, Sweeney et al (1996)
showed that sphingosine and its methylated derivative N,N-dimethyl sphingosine induce
apotosis in a variety of human cancer cells including CMK-7, HL-60, U-937, HRT-l8,
MKN-74, and COLD-205. In contrast, primary cell cultures seem to be less susceptible
to sphingosine than cancer cells. Sphingosine did not induce apoptosis in normal
epithelial cells such as HUVECs or rat mesangial cells, but caused apoptosis in their
transformed counterparts (Sweeney et al., 1996). Thus, sphingolipids have the possibility

to inhibit carcinogenesis through many mechanisms.

C. Knowledge of the types and concentrations of sphingolipids in foods is

limited. Although evidence is now emerging which suggests that dietary

l4

sphingolipids may protect against the development of disease, knowledge of the types
and concentrations of sphingolipids in foods is limited. Sphingolipids are found in all
eukaryotic and some prokaryotic cells (Merrill, 1991). However little is known about
their concentrations in foods. Sphingomyelin (N-acylsphingosine-1-phosphocholine or
ceramide phosphocholine) is a phospholipid located mainly in the outer leaflet of the
plasma membrane of most mammalian cells (Parodi, 1997). The levels of sphingomyelin
have been measured in the following foods: milk (100~200 nmol/ml) (Zeisel et al.,
1986), salmon (160 nmol/g), pork and beef tissues (350~390 nmol/g) and chicken (530
nmol/g) (Blank et al., 1992). Kamath and Charles (1997) reported that the major
phospholipid components of Swiss cheese whey lipid fractions were phosphatidylcholine,
phosphatidylethanolamine, and sphingomyelin.

Total lipids in milk are composed of 0.2 to 1.0% phospholipids. Sphingomyelin
represents about 30% of total milk phospholipids and the concentration of sphingomyelin
in milk was found to be about 100~200 nmol/mL (Zeisel er al., 1986). However, the
concentration of sphingomyelin in milk is affected by season and the cow's stage of
lactation (Parodi, 1997). Lactic acid bacteria, particularly Lacrobacillus and
Bifidobacteria, are commonly used in the production of fermented milk. A study of the
lipid composition of dairy starters reported that sphingomyelin is present in Lactobacillus
bulgaricus and Lactobacillus acidophilus (Chand et aI. , 1992). Therefore, fermented
milk products may be rich in sphingolipids. Furthermore, lactic acid bacteria such as
Lactobacilli and certain components of fermented milk products have been shown to
protect against the development of diseases (Malhorta, 1977, Goldin and Gorbach, 1980,

International Agency for Research on Cancer, 1983, Bianchi Salvadori, 1986, Mutai and

15

Tanaka, 1987, & Ganjam er al., 1997). Since sphingosine and ceramide can induce
apoptosis, the protective effect of fermented milk products against diseases may be
related, in part, to the dairy products’ sphingolipid content.

Many studies reported that soy foods and certain components in soy products such
as genistein protect against development of colon carcinogenesis (Akiyama et al., 1987,
Ogawara er al., 1989, Teraoka et al., 1989, Bourquin et al., 1996, Thiagarajan et al,
submitted). Ohnishi and Fujino (1982) reported that the amount of cerebroside in
soybean was about 2 umol/g. The protective effect of soy products against colon cancer

may also be related to sphingolipid concentration.

16

II. OBJECTIVES

The specific objectives of this research are to:

A. Determine the concentrations of total sphingolipids and free sphingoid bases in soy
fractions, nonfat dry milk, yogurt, and Swiss cheese.

B. Determine the effects of exogenous sphingolipids on growth and death of human

colon cancer cells.

17

III. MATERIALS AND METHODS

Chemicals. Sphingolipids were purchased from Sigma (St. Louis, MO) and
Matreya (Pleasant Gap, PA) and the other chemicals were obtained from Sigma (St.
Louis, MO).

Foods. Yogurt (Low fat yogurt: custard style-thick & creamy, strawberry
and vanila) was obtained from Yoplait Inc. (Minneapolis, MN). Swiss cheese was from
Amish Country Cheese (Linwood, MI), nonfat dry milk and ultra-pasteurized coffee
cream were from Kroger Co. (Cincinnati, OH). Full fat soy flakes, soy flour, and soy
protein concentrate were from Central Soya (Fort Wayne, IN). Isolated soy protein with
isoflavones was from Archer Daniels Midland Co. (Decatur, IL). The yogurt, Swiss
cheese, and coffee cream were lyophilized in a freeze drier (Freeze Dry/Shell Freeze
System, Labconco, Kansas City, Missouri). Nonfat dry milk and full fat soy flakes
(whole soybeans) and the freeze-dried pellets from yogurt, Swiss cheese, and coffee
cream were powdered using a mortar and pestle.

Colon cancer cell lines. The human colon adenocarcinoma cell line
HT-29 and the human colon carcinoma cell line HCT-116 were purchased from
American Type Culture Collection (Rockville, MD).

Determination of total lipids in foods. Total lipids in nonfat dry milk,
yogurt, Swiss cheese, coffee cream, full fat soy flakes (whole soybeans), soy flour,
isolated soy protein, and soy protein concentrate were extracted using

Chloroforrn/methanol (2:1, v/v). Chloroform/methanol (2:1, v/v) at 100 mL was added to

18

5g of dry materials and fat was solublized at 50 °C for 3 hours. Solvents and solubilized
lipids were removed from substrates by Whatrnan filter paper and residues were dried at
60 °C for 24 h. The amounts of lipids were determined by gravimetric analysis.

Yogurt, Swiss cheese, and coffee cream which contain large amounts of moisture
were freeze-dried to remove water. The freeze-dried materials of the foods were
powdered using a mortar and pestle for determination of total lipids and sphingolipids.
To measure moisture content in foods, raw food materials were dried at 60 °C for 24 h in
VWR Scientific 1330 F Constant Temperature Oven (Forced air or gravity) (V WR
Company, Philadelphia, PA) and the results were presented in Table 1.

Extraction of sphingolipids from foods for the determination of total
sphingolipids. Sphingolipids were isolated by the one phase extraction method
(Smith and Merrill 1995). The volume of 3 mL of chloroform/methanol (1:2, v/v) was
added to the sample to be extracted. C20 sphingosine at 300 pmol as an intemal standard
was added to each sample to determine the concentrations of total sphingolipids.
Samples were incubated at 37°C for l h in the shaker water bath (Shaker bath, Lab-Line
Instrumentals, Inc., Melrose Park, IL) at 100g. After 1 h incubation, the sample was
centrifuged at 800g for 10 min (Beckrnan Instruments, Inc., Fullerton, CA) and the clear
supernatant was transferred to a new tube. This step was repeated for twice.

Three mL chloroform/methanol (2:1, v/v) was added to the pellet. After one hour
incubation at 37°C in the shaker water bath, this extract was centrifuged and the clear

supernatant was collected in the tube from the first step. This step was repeated for twice.

19

The pooled chloroform/methanol extracts from the above two steps were dried in
a speed vacuum concentrator (AES 2000 Automatic Environmental Speed Vac® with
Vapornet, Savant Instruments, Inc., Holbrook, NY). The dried extracts were dissolved in
1 mL of chloroform. After 1 h incubation at 37°C in the shaker water bath, the extracts
were centrifuged at 800g for 10 min and clear supernatant was transferred to a new tube.

One mL of chloroform was added to the pellet. After 1 h incubation at 37°C in
the shaker water bath, this pellet in chloroform was centrifuged and the clear supernatant
was transferred to the supernatant collected in the tubes from the last step. The pooled
chloroform extracts were dried in a speed vacuum concentrator.

Quantitation of total sphingolipids. After the one-phase extraction step, 2
mL of 0.5 N hydrochloric acid in methanol was added and the samples were incubated at
65°C in a dry bath (Barnstead/Thermolyne, Dubuque, IA) to hydrolyze the acyl groups.
After 18 h incubation, samples were neutralized with 200 uL of 5 N ammonium
hydroxide.

The samples were extracted with 1 mL of chloroform and 5 mL of water. This
resulted in two phases of the samples: the top phase consisted of water and methanol and
the lower phase consisted of lipids in chloroform. After the samples were centrifuged,
the upper aqueous phase was removed (This step was repeated for twice). The
chloroform layer was passed through a column filled with sodium sulfate to remove
residual water. The chloroform was evaporated using a Speed Vacuum Concentrator for

about 1 h. The samples were saponified by adding 1 mL of 0.1 M potassium hydroxide

20

in methanol/chloroform (4:1, v/v) and incubating the samples for 1 h in a shaker water
bath at 37°C.

After the 1 h saponification, the samples were extracted with 1 mL of chloroform
and 5 mL of water. This resulted in two phases of the samples: the top phase consisted of
water and methanol and the lower phase consisted of lipids in chloroform. After the
samples were centrifuged, the upper aqueous phase was removed (This step was repeated
twice). The chloroform layer was passed through a column filled with sodium sulfate to
remove residual water. The chloroform was evaporated using a speed vacuum
concentrator for about 45 min. The samples were redissolved in 400 uL of mobile phase
(methanol: 5 mM potassium phosphate, 87:13, v/v). The sphingoid bases in samples were
derivatized with the addition of 200 uL of o-phthaldehyde in 3% borate buffer at pH 10.5.
The samples were centrifuged at 10,000g for 10 min (Microcentrifuge, IEC/Micromax®)
and the clear upper fraction was transferred to HPLC vials and the sphingoid bases were
quantitated using High-Performance Liquid Chromatography (HPLC) (Merrill et al.,
1988, Riley et al., 1994). The absolute amounts of total sphingolipids were determined
by the reference to the internal standard, Czo-sphingosine.

The elution profiles of sphingosine, sphinganine, and Czo-sphingosine on Cl8
reverse-phase HPLC with the solvent of methanol: 5 mM potassium phosphate, pH 7.4
(87:13) were shown in Figure 4. Sphingosine, sphinganine, and Czo-sphingosine
appeared at 12.5, 17.8, and 23.1 min respectively.

Quantitation of free sphingoid bases- sphingosine and sphinganine.

Chloroform/methanol (l :2, v/v) at 1.9 mL was added to samples. Czo-sphingosine at 300

21

 

 

 

23. 125
Czo-Sphingosine

1 7.846
Sphinganine
12.535
Sphingosine

 

 

 

 

 

 

 

Retention Time (min)

Figure 4. Elution profiles of sphingosine, sphinganine, and Czo-sphingosine on C13
reverse-phase HPLC.

22

pmol was added as an internal standard to samples and samples were mixed. After the
addition of 100 uL of 2 N ammonium hydroxide, the samples were incubated at 37°C in a
shaker water bath for 1 h. After 1 h saponification, the samples were extracted with 1 mL
of chloroform and 5 mL of water. This resulted in two phases of the samples: the top
phase consisted of water and methanol and the lower phase was a mixture of lipids in
chloroform. After the samples were centrifuged at 800g, the upper aqueous phase was
removed (This step was repeated for twice). The chloroform layer was passed through a
column filled with sodium sulfate to remove residual water. The chloroform was
evaporated using a speed vacuum concentrator for about 45 min. The samples were
redissolved in 400 uL of mobile phase (methanol: 5 mM potassium phosphate, 87:13,
v/v). The sphingoid bases in samples were derivatized with the addition of 200 uL of o-
phthaldehyde in 3% borate buffer at pH 10.5. The samples were centrifuged at 10,000g
for 10 min (Microcentrifuge, IEC/Micromax®) and the clear upper fraction was
transferred to HPLC vials and the sphingoid bases were quantitated using High-
Perforrnance Liquid Chromatography (HPLC) (Merrill er al., 1988, Riley et al., 1994).
The absolute amounts of free sphingoid bases were determined by the reference to the
internal standard, Czo-sphingosine.

The elution profiles of sphingosine, sphinganine, and Czo-sphingosine on Cl8
reverse-phase HPLC with the solvent of methanol: 5 mM potassium phosphate, pH 7.4
(87 :13) were shown in Figure 4. Sphingosine, sphinganine, and Czo-sphingosine were

appeared at 12.5, 17.8, and 23.1 min respectively.

23

Culture of human colon cancer cells. Stock cultures of PIT-29 and HCT-
116 human colon cancer cells were grown in 100 mm culture dishes (Coming,
Cambridge, MA) containing Dulbeco’s Modified Eagle Medium (DMEM) supplemented
with 10% fetal bovine serum at 37°C and 5% C02. Dulbeco’s Modified Eagle Medium
was purchased from Gibco BRL (Life Technologies, Gaithersburg, MD).

Sphingolipid stock. Sphingosine and sphinganine were prepared as a
complex with bovine serum albumin at concentrations of 104 M, 5x10‘4 M, and 10'3 M.
Cz-cerarnide and Cz-dihydroceramide (cell permeable and short chain analog of naturally
occurring ceramide and dihydroceramide) (Figure 5) were dissolved in ethanol as a stock
solution at concentrations of 10" M, 5x104 M, 10'3 M, 2x10'3 M, and 5x10‘3 M.

Total nucleic acid assay. 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. Cells were seeded at a density of 3.0 x 10’ cells/mL in
6-well dishes. HT-29 and HCT-116 cells were grown in 2 mL of Dulbeco’s Modified
Eagle Medium (DMEM) with 10% fetal bovine serum for 24 or 36 h to insure that cells
were in log phase before treatment with sphingolipids. After 24 or 36 h, the medium was
replaced with DMEM supplemented with 1% fetal bovine serum and 20 uL of various
concentrations of sphingolipids were added directly to the cell culture medium.

Subconfluent cells were incubated with sphingosine, sphinganine, Cz-cerarnide, or
Cz-dihydroceramide at final concentrations of 1, 5, 10, 20 and 50 uM for 0, 3, 12, 24, and
48 h. At each culture period of time, floating dead cells were removed by aspiration and

the remaining live cells were rinsed with 1 mL of phosphate-buffered saline (PBS). Then

24

H
CHZOH
\

NI'I3"' Sphingosine

H
CHZOH

NH3+ Sphinganine

H
WCHZOH
\

WWW” Ceramide
ll

H
CHZOH
\

H3c\,NH Cz-Ceramide

H

H3CVNH Cz-dihydroceramide

8

Figure 5. Structures of sphingoid bases and ceramides with or without
the 4,5-trans double bond.

25

the cells were lysed with 1 mL of 0.1 M sodium hydroxide and the total nucleic acid
concentration was determined by reading absorbance of the cell lysate at 260 nm using
Gene Quant-RNA/DNA Calculator (Pharmacia Biotech, Piscataway, NJ).

Examination of cellular morphology. Cells were treated with sphingolipids
as described above and the cells were viewed and photographed using a Nikon inverted
microscope (Nikon, Garden City, NY.) fitted with a Polaroid Micro Camera (Polaroid
Corporation, Cambridege, MA).

Microscopic detection of apoptotic cells" To determine whether sphingoid
bases and ceramide kill cells via apoptosis or via a non-apoptotic pathway, cells were
incubated with sphingoid bases and ceramide at 10, 20, and 50 uM for 0, 15, 30, 60, 120,
or 180 min and 12, or 24 h. Cells were collected at each time and stained with acridine
orange and ethidium bromide (Mishell et al., 1980). Cells were photographed using a
fluorescence microscope equipped with a camera under 10X plus 40X magnification with
400/490 nm excitation and 520 nm emission (Nikon Labophoto, Nikon Inc. Instrument
Group, Garden City, N. Y.). Apoptotic and non-apoptotic cells were classified by the
differences in their chromatin organization (Martin and Lenardo, 1998). Viable cells with
normal nuclei appeared as containing bright green chromatin with organized structure.
Viable cells with apoptotic nuclei appeared as containing bright green chromatin with
highly condensed or fragmented structure. Nonviable cells with normal nuclei appeared
as containing orange chromatin with organized structure. Nonviable cells with apoptotic

nuclei appeared as containing orange chromatin with highly condensed or fragmented

Structure.

26

Statistical analysis. Data for cell grth as influenced by sphingolipids
were analyzed by two-way factorial analysis of variance (ANOVA). After application of
ANOVA to the data, the significance of differences in the means of total nucleic acid
content in HT-29 and HCT-116 cells between control and treatment groups at specific
culture periods were evaluated by multiple comparisons using the Bonferroni method.

Differences were considered significant at p < 0.05.

27

IV. RESULTS

A. Sphingolipid concentrations in foods.

Determination of total lipids in foods. Total lipids in foods were expressed
as percent of lipids on a dry weight basis (Table 1). Coffee cream, Swiss cheese, full fat
soy flakes, and yogurt were relatively high in fat (54.5, 36.7, 23.1, 16.2 % respectively).
Nonfat dry milk, soy flour, isolated soy protein, and soy protein concentrate contained
1.2, 2.7, 2.2, and 0.2 % fat, respectively on a dry weight basis.

Determination of sphingolipids in foods. The present study showed that most
sphingolipids in foods are present as complex sphingolipids and little are present as flee
sphingoid bases (Table 2). The concentrations of total sphingolipids and flee sphingoid
bases (nmng of dry weight) were: nonfat dry milk (203 3: 75, 146 j; 78); yogurt (138 i
55, 1.2 i 0.3); Swiss cheese (167 i 45, 6.5 1; 7.6); full fat soy flakes (609 i 627, 2.6 i
1.1); soy flour (610 i 509, 1.6 j; 0.3); isolated soy protein (210 i 112, 2.8 i- 1.7) (mean j;
SD, n=4) (Table 2 and Figure 6). Sphingosine is the predominant free sphingoid base and
long-chain base backbone of total sphingolipids in foods. The percentage of flee
sphingoid bases and sphingoid base backbones of food complex sphingolipids that are
sphingosine and sphinganine is shown in Table 3. Sphingosine accounts for 90, 74, 91,
85, 98, and 96% of the sphingoid base backbone of total sphingolipids in nonfat dry milk,
yogurt, Swiss cheese, full fat soy flakes, soy flour, and isolated soy protein, respectively
(I able 3). Sphingosine accounts for 97, 69, 90, 64, 77, and 73% of flee sphingoid bases
in nonfat dry milk, yogurt, Swiss cheese, full fat flake, soy flour, and isolated soy protein,

respectively (Table 3).

28

Table 1. Total lipids and moisture content in foods. Lipids are expressed

on a dry weight basis. Data are mean 1; SD (n=2).

 

 

 

 

 

 

 

 

 

 

 

 

Foods % of lipids Water (%)
Nonfatdryrnilk 1.2 1; 0.07 5.4 i 0.3
Yogurt 16.2 3; 0.21 72.9 i 0.002
Swiss cheese 36.7 i 0.16 32.8 i 6.7
Coffee cream 54.5 3; 1.35 72.2 i 0.2
Full fat soy flakes 23.1 i 0.14 3.4 i 0.4
Soy flour 2.7 j; 0.14 4.5 i 0.07
Isolated soy protein 2.2 i 0.12 7.6 i 0.02
Soy protein concentrate 0.2 i 0.14 4.4: 0.04

 

29

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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800

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(nmollg of dry weight)
‘5
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Total Sphingolipids

N
O
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Dry Yogurt Swiss Soy Soy Soy
milk cheese flakes flour isolate

Figure 6. Total sphingolipid concentrations in foods. Data are espressed on a dry weight
basis and are mean 1 SD (n=4).

31

Table 3. Percentage of flee sphingoid bases and sphingoid base backbones of food
complex sphingolipids that are sphingosine and sphinganine.

Data are expressed on a dry weight basis and are mean -_i-_ SD (n=4).

 

 

 

 

 

 

 

 

 

 

 

 

 

Total Sphingolipids Free sphingoid bases
Foods (%) (%)
Sphingosine Sphinganine Sphingosine Sphinganine
backbone backbone
Nonfat
Dry Milk 89.8 i 10.7 10.2 i 10.7 96.7 i 2.7 3.3 j; 2.7
Yogurt 74.0 i 9.1 26.0 1- 9.1 69.4 3; 7.5 30.6 i 7.5
Swiss
Cheese 91.2 i 4.8 8.8 _-I; 4.8 90.0 i 7.7 10.4 i 7.7
Full fat soy
flake 84.5 i 12.1 15.5 i 12.1 64.4 i 22.2 35.6 i 22.2
Soy flour 98.4 j; 81.6 3.2 i 2.2 76.7 1; 4.8 23.3 -_I-_ 4.8
Isolated soy
protein 96.1 i 2.0 3.9 i 2.0 73.2 i 8.0 26.8 i 8.0

 

32

 

B. Differential sensitivity of human colon cancer cells to sphingoid bases and
ceramide.

Sphingosine, sphinganine, and ceramide inhibit growth and cause death of H T-
29 human colon cancer cells. Based upon the concentrations of total
sphingolipids that were found in milk products in this study (~138-203 nmol/g) and
assuming that the average American consumes ~730g of dairy products per day (USDA,
1994, Putnam and Allhouse, 1996), the sphingolipid concentration in the colonic lumen
could reach >20 uM. Cells were incubated with sphingolipids at the concentrations
where may be achieved after consumption of the foods based on this estimation.

To determine the effects of sphingolipids on growth and death of HT-29 human
colon cancer cells, subconfluent cells were treated with sphingosine, sphinganine, C2-
ceramide, or Cz-dihydroceramide and the concentration of total nucleic acids was
determined as an index of cell number. The concentration of total nucleic acids in control
cultures doubled over 24 to 36 hours. Sphingosine, sphinganine, and ceramide caused
concentration- and time-dependent decreases in total nucleic acids of HT-29 cells.
Specifically, addition of sphingosine (Figure 7A) at 20 and 50 uM significantly reduced
total nucleic acid concentrations within 24 hours by 50 and 66%, respectively (p <0.05)
compared to corresponding controls. Sphinganine (Figure 7B) at 10, 20 or 50 uM
significantly reduced total nucleic acid concentrations within 24 hours by 55, 65, and
80%, respectively (p < 0.05) compared to corresponding controls. Cz-ceramide (Figure
7C) also caused a significant reduction in the total nucleic acid concentrations at 20 and

50 uM within 48 hours (p < 0.05). Incubation with Cz-ceramide at 50 uM for 48 h killed

33

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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‘5 a

8 8 200

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35' i 150

3 2

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12 24 36 12 24 36 48
Culture Period (h) Culture Period (h)

2

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2 150 8 20°

° '5

5 £150 ..

p

E 100 E

2 .2 100 I-

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E - J J

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12 24 36 12 24 36 48
Culture Period (h) Culture PGflOd (h)

' . S hin osine, s ' anine, and ceramide inhibit growth and cause death of HT-
12:39:6qu cpblong cancer 2:111: Subconfluent cells were cultured with sphingosrne (A),
sphinganine (B), Cz-ceramide (C), and Cz-dihydrocerarnide (D) at 0 (O), 1 (O), 5 (V), 10
(V), 20 (CI), and 50 (I) uM for 3, 12, 24, or 48 h and total nucleic acrd was deterrnrned
as an index of cell number. Data are flom two experiments and represent mean 3; SEM
(n=8). Means at each culture period with an asterik (*) are significantly different (P <
0.05) flom the corresponding controls.

34

Control 20 uM Sphingosine 50 uM Sphingosine

--

 

 

 

Control 20 IIM Sphinganine 50 uM Sphinganine
Control 20 uM Cz-ceramide 50 uM Cz-ceramide
Control 20 uM Cz-dihydroceramide 50 uM Cz-dihydroceramide

 

Figure 8. Sphingosine, sphinganine, and ceramide inhibit growth and cause death of HT-
29 human colon cancer cells (photographs). Subconfluent cells were cultured with
sphingosine (A), sphinganine (B), Cz-ceramide (C), and Cz-dihydroceramide (D) at 0, 20,
and 50 uM for 24 h.

 

35

all cells. Unlike Cz-ceramide and the sphingoid bases, Cz-dihydroceramide did not
reduce total nucleic acid concentrations (Figure 7D). Changes in cellular morphology
caused by treatment of the cells with the sphingolipids for 24 h are shown in Figure 8.
Sphingosine, sphinganine, and ceramide inhibit growth and cause death of
[JCT-116 human colon cancer cells. Additional studies were conducted to
exaxnine the effects of sphingolipids on HGT-116 human colon cancer cells. The
concentration of total nucleic acids in control cultures of HCT-l 16 cells doubled over 18
to 3 6 hours. Similar to the effects on HT-29 cells, sphingosine, sphinganine, and
ceratxlide caused concentration- and time-dependent decreases in total nucleic acids of
HCT—ll6 cells. Specifically, addition of sphingosine (Figure 9A) at 20 and 50 11M
. sig11ificantly reduced total nucleic acid concentrations within 24 h by 62 and 71%,
respectively (p <0.05) compared to corresponding controls. Sphinganine (Figure 9B) at
10, 20 or 50 uM significantly reduced total nucleic acid concentrations within 24 h by 55,
77, and 93%, respectively (p < 0.05) compared to corresponding controls. Cz-cerarnide at
20 RM also caused a significant reduction in total nucleic acid concentrations within 24
hours by 33% and Cz-ceramide at 50 uM for 24 h killed all cells (Figure 9C). Unlike C2-
Ceramide and the sphingoid bases, Cz-dihydroceramide did not reduce total nucleic acid
c("1'23etltrations (Figure 9D). Changes in cellular morphology caused by treatment of the
cells With the sphingolipids for 24 h are shown in Figure 10.
Sphingoid bases and ceramide difl'erentially afl'ect death of human colon
“nee!" cells. To determine whether sphingoid bases and ceramide kill cells via

apoptosis or via a non-apoptotic pathway, cells were stained with acridine orange and

36

ethidium bromide and chromatin organization was examined under fluorescent light
(Martin and Lenardo, 1998). Apoptotic cells were found in HT-29 and HCT-116 cells

cultured with sphingosine at 20 11M and sphinganine at 10 and 20 uM for 12 and 24 h

(Figure 11).

37

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Culture Period (h) Culture Period (h)
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3 €-
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<
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o 12 24 36 4a 0 12 24 36 48
Culture Period ('1) Culture Period (h)

Figure 9. Sphingosine, sphinganine, and ceramide inhibit grth and cause death of
HCT-116 human colon cancer cells. Subconfluent cells were cultured with sphingosine
(A), sphinganine (B), Cz-cerarnide (C), and Cz-dihydroceramide (D) at 0 (O), 1 (O), 5
(V), 10 (V), 20 (C1), and 50 (I) uM for.3, 12, 24, or 48 h and total nucleic acid was
determined as an index of cell number. Data are from two experiments and represent
mean 1 SEM (n=8). Means at each culture period with an asterik (*) are significantly
different (P < 0.05) flom the corresponding controls.

38

 

Control 20 uM Sphingosine 50 uM Sphingosine

Control 20 uM Sphinganine 50 uM Sphinganine

Control 20 uM Cz-ceramide 50 uM Cz-ceramide

Control 20 uM Cz-dihydroceramide 50 uM Cz-dihydroceramide

.-

Figure 10. Sphingosine, sphinganine, and ceramide inhibit growth and cause death of
HGT-116 human colon cancer cells (photographs). Subconfluent cells were cultured with
sphingosine (A), sphinganine (B), Cz-ceramide (C), and Cz-dihydroceramide (D) at 0, 20,
and 50 uM for 24 h.

 

 

 

 

39

 

Figure 11. Sphinganine kill PIT-29 cells by inducing apoptosis (photographs).
Subconfluent cells were cultured either without sphinganine (A), or with sphinganine (B)
at 10 uM for 12 h. Cells were stained with acridine orange and ethidium bromide. The
right panels show cells under phase contrast and the lefi represents cells under florescent
light.

40

V. DISCUSSION

Many studies suggest that specific foods and their components may protect
against development of colon cancer. For example, soy foods (Akiyama et al., 1987,
Ogawara et al., 1989, Teraoka et al., 1989, Bourquin et al., 1996, Hughes et al., 1997,
Thiagarajan et al, submitted) as well as milk and fermented milk products (Peters et al.,
1992, Karnpman et al., 1994) appear to protect against the development of colon
carcinogenesis. Studies are underway to identify the component of these foods which
provide the protective effects and their mechanisms of action. Recently dietary
sphingolipids have gained attention for their potential to protect against the development
of colon cancer (Dillehay et al., 1994, Schmelz et al., 1996, Schmelz et al., 1997);
however, little is known about the types and concentrations of sphingolipids in foods. In
the present study sphingolipids were extracted fi'om soy fiactions, milk, and fermented
milk products and quantitated via HPLC. The concentrations of total sphingolipids in full
fat soy flakes, soy flour, and isolated soy protein were ~609, 610, and 210 nmng of dry
weight, respectively. Although these concentrations are lower than those previously
estimated for cerebroside (2 pmol/g) in soybeans (Ohnishi and Fujino, 1982), they
indicate that sphingolipids are significant constituents of soy fi'actions. Furthermore, the
data suggest that the processing steps to generate soy flour from full fat soy flakes using
hexane extraction do not significantly influence the concentration of total sphingolipids.
The present studies also show that the concentrations of total sphingolipids in nonfat dry

milk, yogurt, and Swiss cheese are ~203, 138, and 167 nmng of dry weight,

41

respectively. Assuming that sphingomyelin is the major complex sphingolipid in milk,
the concentration of total sphingolipids in nonfat dry milk (203 nmol/g) is comparable
with the value reported for sphingomyelin in milk (100-200 nmol/mL) (Zeisel et al.,
1986). The concentrations of total sphingolipids in the fermented milk products are
similar to that in nonfat dry milk suggesting that dairy starter cultures which contain
sphingomyelin (Tamine and Deeth, 1980, Banwart, 1989, Chand et al., 1992, Marshall,
1993) do not contribute significantly to the total sphingolipid concentration of the
fermented foods. Sphingolipids are present in soy fractions and milk products primarily
as complex sphingolipids; whereas, the simpler free sphingoid bases are present at low
concentrations. Moreover, sphingosine is the predominant long-chain base accounting
for ~64-97% of the free sphingoid bases and ~74-98% of the sphingoid base backbone of
complex sphingolipids with sphinganine accounting for the balance.

Presumably dietary sphingolipids must reach the colonic tissue in order to inhibit
the development of colon cancer. Schmelz et a! (1994) reported that ~88% of dietary
sphingomyelin is absorbed via the small intestine. Therefore, one route that dietary
sphingolipids may reach the colon is via the blood. Alternatively, the remaining 12% of
dietary sphingomyelin that is not absorbed passes directly into the colonic lumen
(Schmelz et al., 1994). This provides another more direct route by which dietary
sphingolipids may reach the colonic cells. Based upon the concentrations of total
sphingolipids that were found in milk products in this study (~138-203 nmng) and

assuming that the average American consumes ~730g of dairy products per day (USDA,

42

1994, Putnam and Allhouse, 1996), the sphingolipid concentration in the colonic lumen
could reach >20 uM.

The mechanism by which sphingolipids inhibit colon cancer is not clear. One
possibility is that complex sphingolipids such as sphingomyelin are hydrolyzed to
bioactive metabolites including sphingoid bases and/or ceramides. Assuming complete
hydrolysis of complex sphingolipids which reach the colon, the concentration of
sphingoid bases and/or ceramides that colonic cells may be exposed to via the lumen
could reach >20 uM. This concentration of sphingoid bases and/or ceramides has been
shown to inhibit growth and induce differentiation and/or apoptosis in a variety of tumor
cells (F aucher et al, 1988, Okazaki et al, 1990, Stevens et al, 1990a, Stevens et al, 1990b,
Michaelson, 1991, Goldkom et al, 1991, Endo et al, 1991, Obeid et al, 1993, Sweeney et
a1, 1996).

The present study showed that sphingosine and ceramide at 20 and 50 uM and
sphinganine at 10, 20, or 50 uM significantly inhibit growth and caused death of HT-29
and HGT-116 human colon cancer cells. In contrast, Cydihydroceramide which lacks the
4,5-trans double bond has no effects. These results suggest that the 4,5-trans double
bond is necessary for inhibition of growth and induction of death by ceramide, but not by
sphingoid bases (Figure 5). This finding is consistent with the results of previous studies
which showed that short-chain ceramides caused apoptosis in many systems, while
dihydroceramides which lack the 4,5-trans double bond were ineffective (Bielawska et
al., 1993, Obeid, et al., 1993, Tepper et al., 1995, Sawai et al., 1995, Brugg et al., 1996,

Karasavvas et al., 1996). In contrast, both sphingosine and sphinganine inhibit growth

43

and induce apoptosis in a variety of cell lines and tumor xenografis (Endo et al., 1991,
Ohta et al., 1995, Jarvis et al., 1996a, Sweeney et al., 1996). Interestingly, previous
studies have indicated that synthetic dihydrosphingomyelin (N-
palmitoyldihydrosphingomyelin) (70%, p < 0.0001) was even more effective than
synthetic sphingomyelin (N-palmitoylsphingomyelin) (52%, p < 0.002) at reducing the
number of aberrant colonic foci in CF -1 mice treated with 1,2-dimethylhydrazine
(Schmelz et al., 1997). Taken together with our results, this suggests that synthetic
dihydrosphingomyelin (and perhaps sphingomyelin) inhibits colon carcinogenesis via
turnover in the colonic lumen to the free sphingoid base (sphinganine and/or sphingosine)
which may in turn inhibit growth and cause death of colon cancer cells. Alternatively,
Jarvis er al (1996a) showed that a combination of ceramide and sublethal concentrations
of sphingosine or sphinganine was more effective at inducing DNA fi'agmentation in
human myeloid leukemia cell lines HL-6O and U937 than ceramide alone. Therefore,
complex sphingolipids may inhibit colon carcinogenesis via turnover to a mixture of
sphingosine, sphinganine, and ceramide.

Incubation of cells with sphingosine at 20 uM and sphinganine at 10 and 20 uM
for 12 h induced apoptosis; whereas, Cz-ceramide did not induce apoptosis. This finding
raised a possibility of the utilization of sphingoid bases as a chemotherapeutic agent since
many chemotherpeutic agents have been shown to kill susceptible cells by apoptosis
(Kaufmann, 1989, Walker et al., 1991, Shinomiya et al., 1994, Havrilesky et al., 1995,
Huschtscha, et al., 1996). Apoptosis is a form of programmed cell death which is either

launched in response to specific stimuli such as cytokines, tumor necrosis factor a, and

the fas ligand (Nagata, 1992, Smith et al., 1994), or activated in response to cell injury or
stress (Gerschenson and Rotello, 1992, Michaelson, 1991). Cancer cells have been known
to circumvent the normal apoptotic mechanisms to prevent their self-destruction due to
their many mutations (Kerr et al., 1994, Williams, 1991). Chemotherapeutic agents
which induce apoptosis appear to have less side effects since the agents can selectively
kill cancer cells with no cytotoxic effects on neighboring normal tissue (Kaufmann, 1989,
Walker et al., 1991, Shinomiya et al., 1994, Havrilesky et al., 1995, Huschtscha, et al.,
1996). The mechanisms by sphingoid bases induce apoptosis in human colon cancer
cells has not well established. Wild-type p53 protein and Bel-2 oncogene were identified
as two major endogenous regulators of apoptosis (V ogelstein and Kinzler, 1992, Oren,
1992, Lane, 1992, Reed, 1995, Korsmeyer et al., 1995). Wild-type p53 protein induces
cell death especially in response to DNA damaging events (V ogelstein and Kinzler, 1992,
Oren, 1992, Lane, 1992). In contrast, Bel-2 oncogene show anti-apoptotic function
(Reed, 1995, Korsmeyer et al., 1995). Sphingosine might affect various systems to
induce apoptosis. For example, sphingosine inhibits protein kinase C (Hannun et al.,
1986b), Na*/K+-A'I'Pase (Oishi et al., 1990), and phospatidic acid phosphohydrolase
(Lavie and Liscovitch, 1990, Jamal et al., 1991, Mullmann et al., 1991). Sphingosine
activates the epidermal growth factor receptor kinase (F aucher et al., 1988, Wedegaertner
and Gill, 1989) and other sphingosine-specific kinases (Pushkareva et al., 1992).
Sakakura et al (1996) showed that sphingosine induce apoptosis via down-regulation of
Bcl-2 oncogene in HL-60 cells. Thus, sphingoid bases might induce apoptosis by

affecting multiple downstream targets.

45

Taken together, the results indicate that sphingolipids are significant constituents
of dairy products and soy fractions and that the colonic concentrations of sphingoid bases
and/or ceramide which may be achieved after consumption of soy fractions and
fermented milk products are sufficient to inhibit growth and cause death of human colon

cancer cells.

46

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