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This is to certify that the
dissertation entitled
STUDIES OF GASTRIN PRODUCTION AFTER
CISPLATIN TREATMENT IN RATS
presented by

YING WANG

has been accepted towards fulﬁllment
of the requirements for

Ph.D. degree in Zoology

Major professor

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STUDIES OF GASTRIN PRODUCTION AFTER CISPLATIN TREATMENT IN
RATS
By

YING WANG

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Zoology

2000

ABSTRACT
STUDIES OF GASTRIN PRODUCTION AFTER CISPLATIN TREATMENT IN
RATS
By

YING WANG

Cisplatin administration (9 mg/kg) causes stomach bloating, decreased pH of
stomach contents, ulceration, and hypocalcemia in rats. Gastric acid production, which is
one of the key factors that regulate the pH in stomach, is mainly stimulated by gastrin in
rats. Gastrin both directly stimulates parietal cells to produce gastric acid and indirectly
acts on ECL-cells to release histamine, which is a strong acid production stimulant. In
rats, somatostatin is the main factor that inhibits the gastrin production, while
extracellular calcium concentration is one of the key factors that regulate gastrin
production. We tested the hypothesis that the decreased pH of stomach contents and
gastric ulceration due to Cisplatin treatment is caused by increased gastrin production.
Also we checked the changes of the somatostatin levels in order to correlate these to the
changes of gastrin after Cisplatin administration.

As measured by immunocytochemistry, in Situ hybridization, northern blot, and
dot blot techniques, gastrin was found to be below detectable limits just 1 day after
Cisplatin treatment. However, 10-15 days after the last injection of Cisplatin, the levels of
both gastrin and its mRN A gradually returned to normal. Northern blot studies showed
that a decrease in somatostatin mRNA paralleled the changes of gastrin and its mRNA.

Immunocytochemistry test showed that inducible nitric oxide synthase levels were also

decreased. Supplements of vitamin D to rats, receiving cisplatin treatment, counteracted
the inhibition of gastrin production. This effect of vitamin D on gastrin production,
through the maintenance of the serum calcium levels, was conﬁrmed by the studies of
Rat Insulinoma B6 (RIN B6) cell lines under different concentrations of extracellular
calcium with or without cisplatin treatment.

In conclusion, the cisplatin-induced decreased pH of stomach contents is not
caused by gastrin. The inhibition of gastrin production is not due to the inhibitory effects
of somatostatin; otherwise the somatostatin level should increase after cisplatin treatment.
However, supplements of vitamin D can counteract this inhibition of gastrin production
due to cisplatin in rats. Recently it is also proven that gastrin, mediated by the nitric oxide
pathway, also maintains the gastric blood flow and mucosal integrity, which are keys to
protect gastric mucosa from injuries by aggressive factors, such as pepsin and gastric
acid. Cisplatin-induced lowered gastrin and nitric oxide production may be part of the

reason that leads to gastrin ulceration.

ACKOWNLEDGEMENTS

I would like to express my most sincere gratitude to my advisor, Dr. Surinder
Aggarwal for giving me this wonderful opportunity to work in his lab. His guidance,
enthusiasm and support in both my research and daily life are greatly appreciated. I also
thank my committee members, Drs. Will Kopachik, Neal Band, and James Mayle for
their kind help through my graduate study, and their insightful ideas and technical
support of my research project.

Thanks to the Dr. Aggarwal’s laboratory (Brad Johnson, Dale Telgenhoff, Heather
Muenchen, Travis Multhaupt and Dan Meara) for their support and love. Thanks to Eric
Olle who helped me on the molecular biology techniques. Thanks to Sonya Lawrence and
Anthony D’Angelo for offering me the ﬁrst assistantship at MSU, which helped me to go
through the ﬁrst tough year. Thanks to Zoology Ofﬁce (Judy, Lisa, Chris and Jan) for
their kind helps. Thanks to all my friends at MSU who made the life outside the lab
wonderful.

Special thanks to Blue Cross Blue Shield Michigan Foundation for their kind

ﬁnancial support (352-SAP/99) of my research project.

iv

TABLE OF CONTENTS

Page

LIST OF TABLES ............................................................................... VII
LIST OF FIGURES ............................................................................ VIII
Chapter One: Introduction ................................................................ 1
References ................................................................. 6

Chapter Two: Effects of Cisplatin and Taxol on Inducible Nitric Oxide Synthase,
Gastrin and Somatostatin in Gastrointestinal Toxicity . . 8

Abstract ..................................................................... 9
Introduction .............................................................. 10
Materials and Methods ................................................ 12
Results .................................................................... 14

Discussion ............................................................... 17

References ............................................................... 22

Chapter Three: Irnmunocytochemical and In Situ Hybridization Studies of Gastrin

After Cisplatin Treatment .............................................. 25
Abstract ................................................................... 26
Introduction .............................................................. 27
Materials and Methods ................................................. 28
Results ..................................................................... 33

Discussion ............................................................... 41

References ................................................................ 43

Chapter Four: Role of Vitamin D on The Inhibition of Gastrin Production
Aﬁer Cisplatin Treatment ............................................. 45
Abstract ................................................................... 46
Introduction ............................................................. 47
Materials and Methods ................................................. 48
Results ................................................................... 51
Discussion ............................................................... 56
References ................................................................ 58

Chapter Five: Summary And Perspectives ........................................... 59
References ................................................................. 62

COPYRIGHT PERMISSION LETTERS ...................................................... 63

vi

LIST OF TABLES

Table 1 Number of viable and dead cells from cultures with different
concentrations of calcium, with or without cisplatin and/or
calcijex ............................................................................ 53

vii

LIST OF FIGURES

Chapter Two:
Figure 1 Cross section of the muscularis mucosa from the pyloric region

of a normal rat ....................................................................
Figure 2 Pyloric region of a rat stomach after cisplatin treatment .
Figure 3 Random section through the pancreatic islet from a normal

rat showing a sparse distribution of iNOS positive cells . . . . ..
Figure 4 Pancreatic islet from a cisplatin-treated rat Showing an increase in

the number and staining intensity of cells due to iNOS . . . . ..
Figure 5 Gastrin positive cells (g) in the pyloric villi .................................
Figure 6 Pyloric villi (V) from a cisplatin-treated rat
Figure 7 Distribution of gastrin positive cells both in the villi (arrows)

and the lamina propria (curved arrows) aﬁer taxol treatment ..............
Figure 8 Somatostatin positive cells (arrows) in the pancreatic islets of

a normal rat .......................................................................
Figure 9 Increased number of somatostatin positive cells after cisplatin

Treatment (arrows) ................................................................

Chapter Three:

Figure 1A Immunocytochemical localization of gastrin in the normal rat
stomach ...........................................................................

Figure 1B Immunocytochemical study showing complete absence of gastrin-
positive cells from the pylorus of the rat gastric mucosa 1-6 days
after cisplatin (9 mg/kg) treatment

Figure 1C Light micrograph showing the immunocytochemical distribution of

gastrin positive cells 10 days after cisplatin treatment as viewed aﬁer
immunocytochemical method ..................................................

viii

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15

15

15

15

15

15

15

35

35

35

Figure 1D ISH study demonstrating the presence of gastrin mRNA-positive
cells (arrows) in the basal portions of the pylorus
of the normal rat stomach ...................................................... 35

Figure 1B In situ hybridization study showing complete absence of
gastrin mRNA positive cells from the gastric mucosa
1-6 days after cisplatin treatment .............................................. 35

Figure 1F Light microgragh showing the gastrin mRNA-positive

cells 15 days after the last cisplatin treatment .............................. 35
Figure 2 Graph Showing the numbers of gastrin positive cells after

immunocytochemical study (broken line) vs. gastrin mRNA-

positive cells after ISH study (solid line) ..................................... 37
Figure 3 Northern blot analysis ofgastrin mRNA 39
Figure 4 Northern blot analysis ofsomatostatin mRNA . 39
Figure 5 Dot blot analysis of gastrin after cisplatin treatment . .. 39
Chapter Four:

Figure 1 Northern blot analysis of gastrin mRNA from the RIN B6 cells
cultured in different concentrations of calcium with or
without cisplatin .................................................................. 54

Figure 2 Northern blot analysis of gastrin mRNA from the RIN B6
cells showing the effects of calcium and/or vitamin D on

the inhibition of gastrin production after cisplatin administration 54
Figure 3 Northern blot analysis of gastrin mRNA from normal, cisplatin
and cisplatin plus vitamin D treated animal stomachs . .. 54

ix

CHAPTER ONE

INTRODUCTION

INTRODUCTION

Cisplatin (cis-diamminedichloroplatium II) was ﬁrst shown by Rosenberg et a]
(1965) to possess antibiotic and antimitotic activity. Subsequent study demonstrated its
antitumor activity. It was introduced into clinical trials by the National Cancer Institute in
1972. Cisplatin is a water-soluble square planar coordination complex containing a
central platinum atom surrounded by two chloride atoms and two ammonia moieties in
cis conﬁguration. The antitumor activity of the complex is much greater when the
chloride and ammonia moieties are in the cis position rather than trans position. The
success of cisplatin in cancer chemotherapy is mainly ﬁ'om its ability to crosslink DNA
and alter its structure. Most cisplatin-DNA adducts are intrastrand d (GpG) and d (ApG)
crosslinks, which unwind and bend the duplex, thus to facilitate the binding of proteins
that contain one or more high-mobility group (HMG) domains. When HMG—domain
proteins bind cisplatin intrastrand crosslinks, they can shield the adducts from excision
repair. It sensitizes cells to cisplatin and contributes to its cytotoxic properties.

Cisplatin is currently one of the most commonly used anticancer drugs. Although
it has proven effective in the treatment of a variety of human malignancies, including
bladder, lung, ovarian 1» 2, head and neck 3, testicular 4 and breast cancers 5' 6, it has severe
side effects. One of the major side effects of this drug in clinical studies is its
gastrointestinal toxicity, which causes nausea, vomiting 7 and peptic ulcers 3' 9. Peptic
ulcers in the stomach and duodenum can lead to perforation and subsequent death 10:12. In
rats cisplatin induces lowering of the pH in the gastric contents, bloating of the stomach

and ulceration. The low pH can be detected as early as 2 days aﬁer cisplatin

administration. Gastric lesions occur in the cardiac portion of the stomach in Wistar rats
within 3 days. Use of cisplatin at a chemotherapeutic dose of 9 mg/kg body weight leads
to hemorrhagic gastric ulcers 5 days after treatment '3’ ‘4.

No single pathogenic mechanism responsible for peptic ulcers has been identiﬁed.
The pathogenesis of peptic ulceration appears to be multifactorial and involves an
imbalance between "aggressive" factors (e. g., acid and pepsin) and "mucosal defensive"
factors (e. g., prostaglandin production, mucus/mucosa] barrier establishment, bicarbonate
production, blood ﬂow, and cell regeneration) 15.

The dictum "no acid, no ulcer" was formulated more that 80 years ago and is still
valid today. Acid plays an important role in peptic ulceration, and control of gastric acid
secretion has been central to therapeutic strategies for the management of peptic ulcers ‘6’
17. In vivo experiments suggest that a histamine-containing cell in the gastric mucosa, the
enterochromafﬁn-like (ECL) cell, is the cell stimulated by gastrin and/or acetylcholine to
release histamine. This histamine stimulates gastric acid secretion ‘3. In some cases,
vagotomy is effective in controlling the acid secretion and mitigating the gastric ulcers 9’
19. However, the role of gastrin in the gastric ulceration due to cisplatin treatment has not
been explored.

Gastrin is released from the G cells, which are located in the epithelium of gastric
antrum and duodenum, but are more abundant in the former. The main gastric peptides
are gastrin-34 (G34) and its C-terminal fragment gastrin-17 (G17). G34 and G17 have
similar agonist activity on the gastrin receptor. However, G34 is cleared more slowly
from the circulation. About 95 percent of antral gastrin are G17, while duodenal gastrin is

about 60 percent G34 20’ 2‘. Gastrin acts through blood stream to increase acid secretion.

It does this both directly by stimulating parietal cells and indirectly by stimulating ECL-
cells to release histamine. The latter accounts for most of the stimulation of histamine
release from the ECL cell in vivo, and therefore, the G cells play a vital role in
stimulation of acid secretion 2‘.

Gastrin release in rats appears to be more strongly dependent upon paracrine
regulation of G cells by somatostatin than it does in several other mammals 22.
Somatostatin peptides are released from D-cells located throughout the gastrointestinal
tract. There are two main forms of somatostatin, $28 and $14. Somatostatin has
widespread inhibitory effects on endocrine and exocrine cells, including G-cells, ECL-
cells and parietal cells 23. Somatostatin inhibits gastrin mRN A transcription and decreases
the stability of gastrin mRNA in G cells 24. 25. Therefore, usually the antral somatostatin
mRNA concentrations change in opposite directions ﬁom gastrin mRNA 21.

Neurotransmitters such as gastrin-releasing peptides (GRP), acetylcholine, and B-
adrenergic agonist stimulate G-cells by receptor-mediated release of intracellular
mediators, which in turn stimulate gastrin secretion from G—cells 26, 27. Besides these
neurotransmitters, in-vitro experiments show that extracellular calcium concentration
could also alter the gastrin transcription and secretion. In the B6 rat insulinoma (RIN) cell
line, which mimic the functions of G-cells, gastrin mRNA levels were 2-fold higher in
cells grown in a medium with higher calcium concentration (40 uM) compared to the
medium with lower calcium concentration (less than 10 M) 23.

Recently, both exogenous and endogenous gastrin have been shown to exert
gastroprotective action 29’ 30. Gastrin plays an important physiological role in the

maintenance of gastric mucosa] integrity. Mediated by the nitric oxide pathway, gastrin

also maintains the gastric mucosal blood flow, which plays a crucial role in
gastroprotection 3‘. Nitric oxide is produced by nitric oxide synthase, which occurs in
three forms: neuronal (nNOS), epithelial (eNOS), and inducible (iNOS).

Production of NO from nN OS is calcium dependent. Calcium and calrnodulin
complex is necessary for the activation of nN OS 32. Cisplatin hydrolyzes into monoaqua
and diaqua species inside the cell under low chloride ion concentration. In the diaqua
conﬁguration, it competitively binds to the calcium binding sites of calrnodulin, which
prevents the formation of calcium and calrnodulin complex 3’ 33. Hypocalcemia is another
side effect caused by cisplatin, and might also be the reason for this lowered NO
production after cisplatin treatment.

Using rats as a model, the correlation between gastrin and lowered pH of gastric
contents and peptic ulcers due to cisplatin treatment was investigated. Gastrin levels after
the cisplatin administration would be measured and monitored for a period of 15 days.
iNOS and somatostatin would be tested using immunocytochemical method and northern

blot respectively in order to correlate their changes to the change of gastrin.

REFERENCES

1.Comis R L. Seminars in Oncology 1994; 21:109.

2. 02018 R F and Young R C. Seminar in Oncology 1984; 11:251.

3. Choksi A and Hong W K. Chemotherapy of Head and Neck Cancer. In: Nicolini M,
ed. Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy.
Padua: Martinus Nijhoff; 1987.375.

4. Einhorn L H and Williams S D. Cancer 1980; 46:1339.

5. Fan S, Smith M L, Rivet D J, Duba D, Zhan Q, Kohn K W, Fomace A J J and
O'Connor P M. Cancer Research 1995; 55:1649.

6. Jurga L, Misurova E, Kovac V and Sevcikova L. Neoplasma 1994; 41:347.

7. Hacker M P and Roberts J D. Cisplatin Efﬁcacy and Toxicity: Are They Separable? In:
Nicolini M, ed. Platinum and Other Metal Coordination Compounds in Cancer
Chemotherapy. Padua: Martinus Nijhoff; 1987.375.

8. Aggarwal S K. J Histochem Cytochem 1993; 41:1053.

9. Aggarwal S K, Antonio J D S, Sokhansanj A and Miller C M. Anti-Cancer Drugs
1994; 5:177.

10. Liaw C C, Huang J S, Wang H M and Wang C H. Cancer 1993; 72:1382.

11. Saito Y, Mori K, Tominaga K, Yokoi K and Miyazawa N. Cancer Chemother
Pharmacol 1992; 31:81.

12. Sartori S, Nielsen I, Maestri A, Beltrami D, Trevisani L and Pazzi P. Oncology 1991;
48:356.

13. Aggarwal S K and F adool J M. Effects of Cisplatin and Carboplatin on
Neurohypophysis, Parathyroid and Their Role in Nephrotoxicity. In: Nicolini M, ed.
Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy.
Padua: Martinus Nijhoff; 1987.375.

14. Aggarwal S K and Fadool J M. Anti-Cancer Drugs 1993; 4: 149.

15. Peura D A. Am J Gastroenterol 1997; 92:88.

16. 8011 A H. JCIin Gastroenterol 1989; 11:81.

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Freston M S and Freston J W. Geriatrics 1990; 45:39.

Hakanson R, Bottcher G, Ekblad E, Panula P, Simonsson M, Dohlsten M, Hallberg T
and Sundler F. Histochemistty 1986; 86:5.

Valen B, Dregelid E, Tonder B and Svanes K. Surgery 1991; 110:824.

Calam J, Dockray G J, Walker R, Tracy H J and Owens D. Eur J Clin Invest 1980;
10:241.

Walsh J H. Gastrin. In: Walsh J H, Graham GJ, eds. Gut Peptides: Biochemistry and
Physiology. New York: Raven Press; 1994.75.

Azuma T, Taggart R T and Walsh J H. Gastroenterology 1987; 93:322.

Chiba T and Yamada T. Somatostatin. In: Walsh J H, Graham GJ, eds. Gut Peptides:
Biochemistry and Physiology. New York: Raven Press; 1994.7 5.

Kamik P S, Monahan S J and Wolfe M M. J Clin Invest 1989; 83:367.

Kamik P S and Wolfe M M. J Biol Chem 1990; 265:2550.

Sugano K, Park J, 8011 A H and Yamada T. J Clin Invest 1987; 79:935.

Giraud A S, 8011 A H, Cuttitta F and Walsh J H. Am J Physiol 1987; 252:G413.
Brand S J and Wang T C. JBiol Chem 1988; 263:16597.

Konturek S J, Brzozowski T, Bielanski W and Schally A V. Eur J Pharmacol 1995;
278:203.

Stroff T, Plate S, Respondek M, Muller K M and Peskar B M. Gastroenterology
1995; 109:89.

Leung F W, Robert A and Guth P H. Gastroenterology 1985; 88:1948.
Jarve R K and Aggarwal S K. Cancer Chemother Pharmacol 1997; 39:341.

Jones M M, Basinger M A, Beaty J A and Holscher M A. Cancer Chemother
Pharmacol 1991; 29:29.

Chapter Two

Effects of Cisplatin and Taxol on Inducible Nitric Oxide Synthase, Gastrin and

Somatostatin in Gastrointestinal Toxicity

This chapter was published on:

Anti-Cancer Drugs. Volume 8. 1997. P 853

ABSTRACT

Cisplatin (9 mg/kg) or Taxol (20 mg/kg) treatment of Wistar rats produced a sharp
decrease in inducible nitric oxide synthase (iNOS) and gastrin in the pyloric region of the
stomach, and an increase in iNOS and somatostatin in the pancreatic islets. Nitric oxide
(NO) functions as a relaxation factor in the smooth muscle of the muscularis mucosa
while gastrin plays an important role in the gastroprotection of the mucosa through NO. It
is proposed that a decline of the iNOS and gastrin after the cisplatin or taxol treatments is
related to distention of the stomach, and possibly nausea and vomiting. Hyperglycemia
and glucose intolerance after cisplatin treatment may be caused by increases of
somatostatin and iNOS in the pancreatic islets. Combination therapy with cisplatin and

taxol seems to ameliorate various toxicities due to these two individual drugs.

INTRODUCTION

Cisplatin [cis-diamminedichloroplatinum(II)], a broad-spectrum anticancer drug,
has proven effective in the treatments of bladder, lung, ovarian 1’2, head and neck 3,
testicular 4, breast 5’6 cancers and certain types of leukemia 7. Drawbacks of this
chemotherapeutic drug are its severe toxic side effects, which include nausea, vomiting,
hyperglycemia 3, stomach distention and gastric ulceration 9’10. Nausea and vomiting are
the dose-limiting factors in some cases. Distention of the stomach in rats 1‘ has been
shown to parallel nausea and vomiting in other animals ‘2, and seems to be a prerequisite
for ulceration ‘0. Nitric Oxide (N O) has been shown to induce relaxation of the pyloric
sphincter in the stomach 13. NO and gastrin have been shown to generate gastroprotective
effects against acid injury W5. Somatostatin and NO suppress the secretion of insulin ‘6.
NO is regarded as one of the main mediators to induce insulin-dependent diabetes 17.

Taxol (Paclitaxel) is another antineoplastic drug with a taxane ring that shows
signiﬁcant anticancer activity. It is proven effective in the treatments of ovarian 13,19,
breast 20, lung 2', head and neck 22 cancers. The adverse effects of this drug include
nausea, vomiting, neutropenia and neurotoxicity. Nausea and vomiting are again the
dose-limiting factors in some cases. Cisplatin and taxol in combination have proven to be
much more effective and seemingly less toxic compared to either of the two drugs when
administered alone. Based on the actions of NO and gastrin on the stomach and the
functions of NO and somatostatin on the secretion of insulin from the pancreatic islets,
the present study was undertaken to see the changes of gastrin and iNOS after cisplatin

and taxol administration, and how these changes inﬂuence stomach motility and mucosa

10

to cause nausea, vomiting and ulceration. Also, changes in somatostatin and iNOS after
the cisplatin treatment and what inﬂuence these have on the pancreatic islets and insulin

secretion were explored.

11

MATERIALS AND METHODS

Animals

Male Wistar rats (Charles Rivers Laboratories, Wilmington, MA) weighing 100-
150 g were kept on a 12 h light/ 12 h dark cycle with free access to laboratory animal feed
and water. Cisplatin (9 mg/kg), taxol (20 mg/kg) and cisplatin plus taxol (9 mg/kg+20
mg/kg) were administered as IP injections in ﬁve divided dosages over a period of 5
days. The control animals received injection of the vehicle (0.85% NaCl) only. Twenty
four hours following the last injection, the rats were sacriﬁced and various tissues

excised.

Tissues
Stomach (cardia, body, pylorus) and pancreas were excised and ﬁxed in Bouin’s
solution or 4% paraforrnaldehyde for 48 h. Fixed tissues were dehydrated and embedded

in parafﬁn at 56°C. Sections (7pm) were prepared for immunocytochemical studies.

Antisera

A monoclonal antibody speciﬁc for iNOS (Transduction Laboratories, Lexington,
KY) was used in a dilution of 1 :1000. The gastrin-speciﬁc (Dako Corporation,
Carpinteria, CA) and somatostatin-speciﬁc (Incstar Corporation, Stillwater MN)

antibodies were used in a dilution of 1:500.

12

Immunocytochemistry

Irnmunocytochemical staining was performed using the avidin-biotin-peroxidase
complex method 23. iNOS was demonstrated by the Vectastain Elite ABC Kit (Vector,
Burlingame, CA), while somatostatin and gastrin were demonstrated using the Vectastain

ABC-AP kit.

iNOS, Somatostatin and Gastrin

Parafﬁn sections (7 mm) were treated with 3% H202 in dHZO for 20 min to block
any endogenous peroxidase activity and incubated in normal serum supplied in the kits
for 20 min. These sections were incubated in the speciﬁc primary antibody overnight at
4°C and in the secondary antibody for 45 min at room temperature. Sections were
subsequently incubated in the avidin—biotin-peroxidase complex for 45 min at room
temperature. Peroxidase activity was revealed by incubation in 0.03%
3,3’diaminobenzidine (DAB) in 0.1M sodium acetate/acetic buffer (pH 6.0), containing
2.5% nickel ammonium sulfate, 0.2% B-D-glucose, 0.04% ammonium chloride and
0.001% glucose oxidase 24. Sections were rinsed in tap water for 5 min and 0.1M
acetate/acetic buffer (pH 6.0) before being mounted in permount through various
dehydration series. For somatostatin and gastrin, alkaline phosphatase activity was

revealed by using the Vector Red Substrate Kit (Vector Laboratories).

13

RESULTS

High levels of iNOS activity were observed in the macrophagic cells at the base of
the gastric villi and in the smooth muscle of the pyloric muscularis mucosa of the normal
rat stomach (Figure 1). After cisplatin, taxol or cisplatin plus taxol treatments, iNOS
positive cells were absent in the smooth muscle of the pyloric muscularis mucosa (Figure
2). However, at the base of the villi in the lamina propria, there were some iNOS positive
cells, but their number was greatly reduced compared to the normal (Figure 2).

In the pancreas, i-NOS positive cells were sparsely distributed throughout the
islets (Figure 3). Cisplatin treatments induced an increase in their number and the
intensity of their staining (Figure 4).

There were a large number of gastrin positive cells in the pyloric villi of the
normal rat stomach (Figure 5), that were negative after cisplatin treatment (Figure 6).
Taxol treatment induced a reduction in their number but maintained their presence
(Figure 7). However, there were some gastrin positive cells at the base of the villi after
cisplatin, taxol and cisplatin plus taxol not visible in the normal rat stomach (Figure 5). In
their morphology and distribution these cells are similar to macrophagic cells.

In the pancreas, somatostatin positive cells were located in the peripheral regions
of the islets (Figure 8). After cisplatin treatment there was an increase in the number of
such cells (Figure 9). Taxol treatment did not show any change from the normal.
Cisplatin plus taxol treatment demonstrated an intermediate situation between the normal

and cisplatin treatment.

14

Figure 1.Cross-section of the muscularis mucosa from the pyloric region of a normal rat.

It shows the presence of macrophagic cells (arrows) at the bottom of the gastric villi (gv)

in the lamina propria and among the smooth muscle ﬁbers (sm) of the muscularis mucosa
(double arrows). Original magniﬁcation: X 262. Bar=0.1mm

Figure 2.Pyloric region of a rat stomach after cisplatin treatment. Note the absence of

macrophages from the muscularis mucosa (sm) and a much reduced number at the base
of the villi (gv) in the lamina propria (lp). Original magniﬁcation: X 262. Bar=0.1mm

Figure 3.Random section through the pancreatic islet from a normal rat showing a sparse
distribution of iNOS positive cells (arrows). Original magniﬁcation: X 1050. Bar=20 um

Figure 4.Pancreatic islet from a cisplatin-treated rat showing an increase in the number
and staining intensity of cells due to iNOS. Original magniﬁcation: X 1050. Bar=20 um

Figure 5. Gastrin positive cells (g) in the pyloric villi. Note the absence of gastrin positive
cells from the lamina propria (1p). Original magniﬁcation: X 2625. Bar=10 urn

Figure 6. Pyloric villi (V) from a cisplatin-treated rat. Note the absence of gastrin positive

cells from the villi. However, there are some gastrin positive (arrow) cells at the bottom
of the villi in the lamina propria (1p). Original magniﬁcation: X 2625. Bar=10 um

Figure 7. Distribution of gastrin positive cells both in the villi (arrows) and the lamina
propria (curved arrows) after taxol treatment. Original magniﬁcation: X 1050. Bar=20um

Figure 8. Somatostatin positive cells (arrows) in the pancreatic islets of a normal rat.
Original magniﬁcation: X 262. Bar=0.1 mm

Figure 9. Increased number of somatostatin positive cells after cisplatin treatment

(arrows). Original magniﬁcation: X 262. Bar=0.l mm

Images in this chapter are presented in color.

15

 

DISCUSSION

Various chemotherapeutic drugs, such as cisplatin and taxol, are known to induce
nausea and vomiting, while cisplatin has also been shown to induce gastric lesions and
ulceration in rats 25. In order to combat these adverse effects, it is essential to understand
the cause or mechanism of induction of these effects. Distention of the stomach has been
shown to parallel the nausea and vomiting that are associated with the clinical use of
cisplatin ”. In rats, bloating of the stomach has been demonstrated to be due to the
retention of food in the stomach with lowered pH leading to ulceration 10. Thus,
alleviation of stomach distention may be the key point to the symptoms of nausea,
vomiting and ulceration.

Normal stomach emptying depends on the contractility of the stomach and
relaxation of the pyloric sphincter. The former is under the control of acetylcholine, while
the later is under N0 control. A major part of the autonomic innervation of the
gastrointestinal tract is by non-adrenergic non-cholinergic (NAN C) neurons. In the
sphincter region of the gut, this NAN C innervation is mainly inhibitory and is
physiologically important in the relaxation of the sphincters for the passage of the
ingested food. NO and ATP contribute to the NAN C inhibitory response of the rat pyloric
sphincter 13. Unlike the other neurotransmitters, NO can easily diffuse through the cell
membrane 26. NO produced in the pyloric smooth muscle can difﬁrse into the nearby
sphincter muscle to cause its relaxation.

Release of acetylcholine from the nerve ending and NO production by the

neuronal NO synthase (nNOS) are calcium dependent 27. Cisplatin hydrolyzes into

17

monoaqua and diaqua (divalent) species inside the cell under low chloride ion
concentrations. In the diaqua conﬁguration, it competitively binds to the calcium binding
sites. Thus nNOS activation and acetylcholine vesicle release are prevented by a
competitive inhibition of the calmodulin molecule. NO production and acetylcholine
release are lowered after cisplatin treatment resulting in prolonged relaxation of the
stomach smooth muscle ‘0, and pyloric sphincter smooth muscle hypercontractility 28,
that in turn probably causes stomach distention.

In the normal rat, pyloric sphincter smooth muscle and at the bottom of the pyloric
villa, there are a large number of iNOS immunoreactive macrophages. However, after 2
days of cisplatin and taxol treatments, there are no iNOS immunoreactive macrophages in
the pyloric smooth muscle. NO production from nNOS is calcium-calrnodulin dependent,
whereas NO production from iNOS is independent of calcium-cahnodulin. Considering
the different character of the iNOS from the nN OS, we can safely say that NO production
by the iNOS is much less in cisplatin-treated rats compared to normal rats.

The main cause of gastric ulceration is the weakened gastroprotection, which
includes maintenance of normal mucosal blood ﬂow, mucosa integrity, mucosa growth
and mucus secretion. Administration of the L-NMMA (NC-monomethyl-L-arginine), an
inhibitor of NO synthase, induces gastric mucosal injury over a 45 min period in rats
pretreated with indomethacin, in dosages of either agent that alone do not provoke
mucosal injury. Likewise in rats chronically pretreated with capsaicin, L-NMMA induces
extensive hemorrhagic mucosal injury. Such ﬁndings indicate that endogenous NO at
least partly appears to serve as a modulator in the regulation of gastric mucosal

integrity29'30. Endogenous NO may also play a role in offsetting the musosal

18

microcirculatory actions of local vasoconstrictor mediator such as norepinephrine,
neuropeptide or the endothelins. Through the increase of intracellular mediator (cGMP),
NO induces mucus secretion without evidence of cellular damage 3‘. Mucus secreted by
these epithelial cells plays a local protective role, since it forms a continuous viscoelastic
layer that prevents access of pepsin to mucosa. Furthermore, secretion of bicarbonate into
the unstirred layer of mucus can generate a gradient of increasing pH from the luminal
bulk phase to the epithelial cell 32. Lowered NO production after cisplatin treatment,
which may be either from lowered iNOS or inhibited nNOS, probably accounts for the
decrease of basal gastroprotection.

Gastrin, a kind of enteric peptide hormone, is released from the stomach and
proximal portion of the gut upon normal ingestion of nutrients, and affects various
physiological functions such as gastric secretion and mucosal growth 33. It plays a
physiological role in maintaining gastric mucosal integrity '5. The protection induced by
gastrin is accompanied by a signiﬁcant elevation of gastric mucosal blood ﬂow ‘5 which
plays a crucial role in gastroprotection by prostaglandins 34 and endogenous NO release
constitutively 35.35. The pretreatment with Ng-nitro-L-argininemethyl ester signiﬁcantly
reduces the gastric blood ﬂow induced by gastrin and prevents its protective activity.
These effects of Ng-nitro-L-argininemethyl ester are reversed by concurrent
administration of L-arginine, a substrate for NO synthase. NO plays a crucial role in
mucosal hyperemia and gastroprotection by gastrin 15. In cisplatin-treated rat stomachs,
there was a sharp decline of gastrin-secreting G cells in the pyloric villi, compared to
normal. This decline of gastrin may also be responsible for ulceration after cisplatin

treatment. However, after taxol treatment there were some gastrin-secreting G cells along

19

the pyloric villi. The number of such cells was small but sufﬁcient to protect the mucosa,
as no ulceration was observed after taxol treatment.

There are no gastrin-immunoreactive cells in the pyloric region of the normal rat
stomach other than the G cells along the villi. In the cisplatin and taxol treated rat
stomach, there are gastrin immunoreactive cells in the interstitial region along the bottom
of the villi. These gastrin immunoreactive cells in the interstitial area of the pyloric
region may be compensatory for the sharp decline of gastrin-secreting G cells after
cisplatin and taxol treatments.

Similar to other heavy divalent metal ions, i.e. cadmium 37, cobalt 38, zinc 39 and
nickel 40, cisplatin administration impairs glucose tolerance, which is indicated by
marked hyperglycemia 3. Administration of cisplatin results in hyperglucagonemia and a
relative deﬁciency of insulin secretion. Timed interval studies of glucose intolerance
following cisplatin treatment have been demonstrated to be related to a deﬁciency in
insulin secretiongv41 ’42.

Somatostatin -14 and somatostatin-28 are the two known bioactive peptides, and
share the ability to inhibit the release of various peptides, including insulin”, which is
mediated by a pertussis toxic—sensitive GTP-binding protein '6. Thus, in the cisplatin-
treated rat pancreatic islets, the increased somatostatin-immunoreactive cells may be
responsible for the suppression of insulin.

There is a marked increase in the iNOS immunoreactive cells in the rat pancreatic
islets after cisplatin treatment. Both [3 cells and macrophages in pancreatic islet may be

sources of iNOS 44. In insulin-dependent diabetes, a large amount of NO produced by the

20

increased number of iNOS in pancreatic islets suppresses insulin secretion and
participates in the destruction of B-cells 45.

In conclusion, cisplatin or taxol depress iNOS and gastrin levels in the stomach
while increasing iNOS and somatostatin in the pancreatic islets. In combination they

seem to modulate these effects probably through their different mechanism of action.

21

9.

10

11.

12.

13.

14.

15.

16.

17.

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Murphy W, Fossella F, Winn R, Shin D, Hynes H, Gross H, Davilla E, Dhingra H
and Raber M. J Natl Cancer Institute 1993; 85:384.

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Appleton and Lange; 1991.

J arve R and Aggarwal S K. Cancer Chemother Pharmarcol 1997; 39:341.

Huang P L, Dawson T M, Bredt D S, Snyder S H and Fishman M C. Cell 1993;
75:1273.

Whittle B J R. Br J Pharmacol 1993; 110:3.

Whittle B J R, Lopez-Belmonte J and Moncada S. Br. J. Pharmacol 1990; 99:607.
Brown J, Keates A, Hanson P and Whittle B. Amer J Physiol 1993; 265:G418.

Allen A, Hunter A C, Leonard A J, Pearson J P and Sellers L A. Peptic Activitiy and
The Mucus-Bicarbonate Barrier. In: Garner A, Whittle B J R, eds. Advances in Drug
Therapy of Gastrointestinal Ulceration. Chichester, UK: John Wiley & Sons; 1989.
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Walsh J H. Gastrin. In: Walsh J H, Graham GJ, eds. Gut Peptides: Biochemistry and
Physiology. New York: Raven Press; 1994.75.

34. Leung F N, Robert A and Guth P H. Gastroenterology 1985; 88:1948.

35.

Palmer R M J, Ashton D S and Moncada S. Nature 1988; 333:664.

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36. Brown J F, Teppennan B L, Hanson P J, Whittle B J R and Moncada S. Biophys Res
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37. Ghafghazi T and Mennear J H. T oxicol Appl Pharmacol 1973; 26:231.
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41. Goldstein R S, Mayor G H, Rosenbaum R W, Hook J B, Santiago J V and Bond J T.
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50:698.

43. Patel Y C. Somatostatin. In: Becker K L, ed. Principles and Practice of
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44. Rabinovitch A, Suarez-Pinzon W L, Sorensen O and Bleackley R C. Endocrinology
1996; 13722039.

45. Mandrup-Poulsen T, Helqvist S, Wogensen L D, MOlvig J, Pociot F, Johannesen J
and Nerup J. Curr Top Microbial Immuno 1990; 164:169.

24

Chapter Three

Irnmunocytochemical And In Situ Hybridization Studies Of Gastrin After Cisplatin

Treatment

This chapter was published on:

The Journal of Histochemistry and Cytochemistry Volume 47 (8). 1999. P 1057

25

ABSTRACT

Cisplatin treatment (9 mg/kg) causes bloating of the stomach, an increase in
gastric acid, and ulceration in rats. Gastrin, a gut peptide, plays an important role in
regulating gastric acid production. To study the role of gastrin in this increased gastric
acid production after cisplatin treatment, male Wistar rats (100-150g) were treated with
cisplatin (9 mg/kg) in ﬁve divided doses over 5 consecutive days. The rats were
sacriﬁced 1, 6, 10 or 15 days after the last treatment. As measured by
immunocytochemistry, in situ hybridization, Northern blot and dot-blot techniques,
gastrin was found to be below detectable limits just 1 day after cisplatin treatment.
However, 10-15 days after the last injection, the levels for both gastrin and its mRNA
gradually recovered to normal. Northern blot studies showed that decreased somatostatin
mRN A parallels the changes of gastrin and its mRNA. These results suggest that after
cisplatin treatment the increased gastric acid production in rat stomach is independent of
gastrin. This decrease of gastrin production is not under the inﬂuence of somatostatin,

which also decreased after cisplatin treatment.

26

INTRODUCTION

Cisplatin (cis-dichlorodiammineplatinum II; CDDP), a broad-spectrum
chemotherapeutic drug, has been proved effective in the treatment of bladder, lung,
ovarian 1’ 2, head and neck 3, testicular 4 and breast 5 cancers. One of the major side
effects of this drug in clinical studies is its gastrointestinal toxicity, which includes severe
nausea and vomiting. In rats it induces bloating of the stomach and gastric ulceration 6.

Under low chloride ion concentrations inside the cell, cisplatin hydrolyzes into
monoaqua (monovalent) and diaqua (divalent) species. The diaqua form of cisplatin with
its divalent charge has been demonstrated to bind to calrnodulin and inhibit its binding to
calcium. Without calcium-calrnodulin complex, acetylcholine release is inhibited,
resulting in bloating of the stomach and an increase in its acid content 7.

Gastrin, one of the gut peptides, which is primarily produced and secreted in the
stomach and proximal duodenum, is a potent stimulant of gastric acid secretion and
proliferation of the acid-secreting oxyntic cells of the gastric mucosa. It acts both directly
on gastrin receptors of the parietal cells and indirectly on gastrin receptors of the
enterochromafﬁn-like (ECL) cells to produce histamine which, in turn, stimulates the
gastric acid production from parietal cells. Somatostatin is known to inﬂuence gastrin
production in rat stomach through a paracrine action 3. This study was undertaken to
investigate the changes in gastrin and somatostatin aﬁer cisplatin treatment and to

correlate the changes to the lowered pH in rat stomach.

27

MATERIALS AND METHODS

Animals and Tissue Preparation

Male Wistar rats (Charles Rivers Laboratory; Wilmington, MA) weighing 100-
150 g were housed on a 12-hr light /12-hr dark cycle with free access to food and water.
Cisplatin at a clinical dosage of 9 mg/kg in physiological saline was injected (IP) in ﬁve
divided doses over 5 days. The controls received only the injection vehicle. The rats were
anaesthetized with equithesin and either perfused with buffered 4% paraformaldehyde
(0.1 M phosphate buffer, pH 7.4) or stomach tissues were excised and frozen (-700 C) 1,
6, 10, or 15 days after last cisplatin treatment. Each interval had a minimum of three
animals. Perfused stomach tissues were postﬁxed with buffered 4% paraformaldehyde
(0.1 M phosphate buffer, pH 7.4) for 12 hr, dehydrated through ethanol gradients, and
embedded in parafﬁn at 56°C. Sections (10 um) were cut and placed on gelatin-coated

slides for in situ hybridization (ISH) and immunocytochemical studies.

Immunocytochemistry

Irnmunocytochemical study for gastrin was performed using the avidin—biotin-
peroxidase complex (ABC) 9 with a Vectastain ABC-AP Kit (Vector Laboratories;
Burlingame, CA). Parafﬁn sections were deparafﬁnized in xylene, hydrated, and
sequentially incubated in gastrin-speciﬁc primary antibody (1 :1000) (Dako; Carpinteria,
CA ) at 4°C overnight and the biotinylated antibody for 45 min at room temperature (RT).
Sections were then incubated in ABC- alkaline phosphatase (AP) complex for 45 min at

RT. AP activity was revealed by using the Vector Red Substrate Kit. After rinsing in tap

28

water and proper dehydration, sections were mounted in Perrnount and viewed for
intensity differences on a visual basis. The tissues were directly stained with Vector Red
Substrate to check the effect of the endogenous AP activity. The liver tissue served as a

negative control.

Oligonucleotide Probes

5 ’-GACCTTGGGGCCCCAGCTGTCTCCGAT-3 ’, a 27-mer hybridization
sequence complementary to the coding sequence of rat gastrin mRNA (position 212-238)
‘0 and 5 ’-CCAGAAGAAGTTCTTGCAGCCAGCTTTGCGTTCCCGGGGTGCCAT-3 ’,
a 45-mer hybridization sequence complementary to the coding region of rat somatostatin
mRNA (bases 286-330) 11, were synthesized using the ABI 3948 Synthesis and
Puriﬁcation System. The sense probe was synthesized at the same time to serve as
control. Both sense and anti-sense oligonucleotides were labeled with the digoxigenin
(DIG) Oligonucleotide Tailing Kit (Boehringer Mannheim; Indianapolis, IN) according
to the manufacture’s instructions. A 100 pmol probe was incubated with tailing buffer,
CoClz solution, DIG-dUTP, dATP, and terminal transferase solutions at 37°C for 18 min,
then placed on ice and the reaction was stopped by adding 4 ul of stop solution. The
tailed probe was precipitated in Sul 4M LiCl and 150 pl prechilled (-20°C) ethanol at —
20C for 3 hr and then centriﬁrged at 12,000 X g for 15 min. The supernatant was

discarded and the pellet was allowed to air dry and kept at -70°C till use.

29

In Situ Hybridization

The protocols for in situ hybridization were followed according to Larsson and
Hougaard (1993). Deparafﬁnized sections (10 um) were soaked in chloroform
(Mallinckrodt; Paris, KY), and hydrated through descending ethanol gradients.
Proteolytic treatment was carried out with 0.015% pepsin (Boehringer Mannheim;
Indianapolis, IN) in 0.2 M HCl for 20 min at RT. Sections were ﬁxed with buffered 4%
paraformaldehyde (0.1 M phosphate buffer, pH 7.4) at 4°C for 5 min. After washing
twice with PBS, the sections were soaked in freshly made 0.25% acetic anhydride in
triethanolarnine (TEA) buffer (pH 8.0). The sections were pretreated for 30 min at 42°C
in the prehybridization buffer which is identical to the hybridization buffer but without
the probe. Hybridization was performed at 42°C overnight with 50 ul hybridization buffer
covering each slide and containing 5 ng of the labeled probe, 50% fonnamide, 1x
Denhardt’s solution , 10% dextran sulfate, 10 mM dithiothreitol (DTT) (Sigma; St Louis,
MO), 150 ug/ml tRNA (Boehringer Mannheim), 100 ug/ml denatured sheared salmon
sperm DNA (GIBCO BRL; Gaithersburg, MD) and 3 x standard saline citrate (SSC).
Stringency wash was canied out in 0.1 SSC (four changes x 20 min) at 42°C.
Immunocytochemical detection of DIG was performed with DIG Nucleic Acid Detection
Kit (Boehringer Mannheim). Sections were washed in maleic acid buffer (pH 7.4) for 10
min, then sequentially incubated in blocking buffer in the kit for 30 min and 1:1000 anti-
DIG antibody for 45 min. After washes in PBS and IOOmM Tris-HCl buffer (pH 9.5),

sections were incubated in color-substrate solution, nitroblue

tetrazolium/bromochloroindolyl phosphate (NBT/BCIP) overnight. Finally, the sections

30

were dehydrated and mounted in Permount. All the H20 used in above study was diethyl

pyrocarbonate (DEPC)-treated double distilled water.

Statistical Analysis

Numbers of gastrin and its mRNA-staining positive cells were counted in 15
random visual ﬁelds of ﬁve different tissue sections from each group. The data was
averaged and plotted. All data were statistically analyzed by the Student’s t-test when

comparison between control and cisplatin treatment was made 12.

Northern Blot Analysis

RNA was extracted ﬁ'om the stomach tissues by a guanidinium isothiocyanate
solution and puriﬁed by the CsCl cushion method 13. Final RNA recovered from each
sample was quantiﬁed by its UV absorbance at 260 nm. Intensity of ribosomal RNA
bands was studied after ethidium bromide staining.

The RNA (20 ug) was size-separated by electrophoresis in a 1.3% agarose gel
containing formaldehyde and electroblotted onto nylon membrane (GeneScreen; New
England Nuclear, Boston, MA). The blots were prehybridized with the hybridization
buffer without probes for 3 hr at 42° C. Blots were incubated with fresh hybridization
buffer in the presence of labeled probes (40 ng/ml) at 42° C for 2 days. The hybridization
buffer for the gastrin mRNA contained 50% formarnide, 10% dextran sulfate, 50 mM
Tris (pH 6.8), 3 X SSC, 100 ug/ml sonicated salmon sperm DNA and 5 X Denhardt’s
solution. The buffer for the somatostatin was the same as that for gastrin except without

dextran sulfate. The blots were washed with two changes of 2 X SSC/0. 1% SDS at 42° C

31

for 30 min each and 0.1 X SSC/0. 1% SDS at 42° C for 15 min. The probes were detected

by the same kit for in situ hybridization and following the same procedure.

Dot-blot Analysis

Stomach tissues were homogenized 14 and the protein was quantiﬁed by its UV
absorbance at 280 mm”. Total denatured protein sample (100 pg) was dot-blotted on the
nitrocellulose membrane (ImmunoSELECT; GIBCO BRL) by Hybri-Slot Manifold (
Bethesda Research Laboratories; Bethesda, MD). The blot was incubated in the primary
antibody to gastrin at 4° C overnight and was detected by the same method used in the

immunocytochemical study described above. Liver tissue served as negative control.

32

RESULTS

Immunocytochemistry and In Situ Hybridization

Using a gastrin-speciﬁc primary antibody, immunocytochemical study showed
that gastrin-positive cells were mostly localized in the basal portion of the pylorus of rat
stomach (Fig. 1A). Control sections stained only with Vector Red substrate showed no
signiﬁcant endogenous AP activity. The liver tissue was also negative. At 1 and 6 days
after the last cisplatin treatment, no gastrin-positive cells were observed in the gastric
mucosa (Fig. 1B). However, 10 days after cisplatin treatment, gastrin-positive cells were
evident (Fig. 1C). The intensity and distribution of staining were back to normal on Day
15 after the last cisplatin treatment.

Gastrin-speciﬁc primary antibody cross reacts with choleocystokinin (CCK)
octopeptide, so ISH and Northern blot tests were applied to the adjacent tissues for
immunocytochemical and dot—blot studies to conﬁrm the results from these studies. The
intensity and distribution of gastrin mRNA-positive cells were similar to those observed
after immunocytochemical study, mostly in the basal portion of the pylorus of the rat
stomach, with intense positive cytoplasmic staining (Fig. 1D). The number of positively
stained cells was less than that after the immunocytochemical study. This is probably
because the CCK-positive cells did not stain after ISH. No gastrin mRNA-positive cells
were noted 1 and 6 days after cisplatin treatment in the gastric mucosa (Fig. 1E).
However, 15 days after cisplatin treatment, the intensity and distribution of gastrin-

positive cells were similar to those of the normal tissues (Fig. 1F). The numbers of

33

gastrin and gastrin mRNA-positive cells in each group were signiﬁcantly different (Fig

2), p< 0.01.

34

Figure 1A. Immunocytochemical localization of gastrin in normal rat stomach (arrows),
basal portion of gastric mucosa. *: basal lamina. Bar = 40 um

Figure 1B. Immunocytochemical study showing complete absence of gastrin-positive
cells from the pylorus of the rat gastric mucosa 1-6 days after cisplatin (9 mg/kg)
treatment. Bar = 20 um

Figure 1C. Light micrograph showing the immunocytochemical distribution of gastrin-
positive cells 10 days after cisplatin treatment. Note the appearance of the gastrin-
positive cells. *: basal lamina. Bar = 10 um

Figure 1D. ISH study demonstrating the presence of gastrin mRNA-positive cells
(arrows) in the basal portions of the pylorus of normal rat stomach. The cytoplasm of
these cells is positively stained, whereas the nuclei are negative. The distribution pattern
of gastrin mRNA-positive cells is the same as in immunocytochemistry study shown in
A. *: basal lamina. Bar = 20 um

Figure 1B. In situ hybridization study showing complete absence of gastrin mRNA-
positive cells from the gastric mucosa 1-6 days after cisplatin treatment. Bar = 20 um

Figure 1F. Light microgragh showing the gastrin mRNA-positive cells 15 days after the
last cisplatin treatment. Staining appears similar in intensity and distribution to that of
normal tissues. *: basal lamina. Bar = 20 um

Images on this page are presented in color.

35

 

36

 

mRNA

 

Positive cells for gastrin and

 

 

-5 l t l l
-1 4 9 14 19
Days after cisplatin treatment

 

 

 

Figure 2. Numbers of gastrin-positive cells after immunocytochemical study (broken
line) vs gastrin mRNA-positive cells after ISH (solid line). Gastrin and its mRNA drop to
negligible levels 1 day after the last cisplatin injection. These levels remain depressed for
about 6 days and then gradually rise to normal after 15 days (p <0.01).

37

Northern Blot and Dot-blot Analysis

Total RNA from rat stomach tissues was isolated and size-separated by
electrophoresis on formaldehyde 1.3% agarose gels. The RNA blot was hybridized with
DIG-labeled Oligonucleotide probes for either gastrin mRNA or somatostatin mRNA. At
the corresponding RNA molecular marker level, gastrin mRNA was detected (Fig 3). At
1 and 6 days after the cisplatin treatment the gastrin mRNA bands were almost negative.
However, 10 days after the last cisplatin treatment the gastrin mRNA levels became
signiﬁcant, reaching normal levels 15 days after treatment. Somatostatin (Fig. 4) mRNA
followed the similar patterns as described for gastrin mRNA.

For dot-blot studies, 100-ug protein samples were dot-blotted onto the
nitrocellulose membrane. The membrane was incubated with rabbit anti-gastrin antibody
and detected by the ABC method (Fig. 5). At 1 and 6 days after cisplatin treatment,
gastrin levels were undetectable, as in case of gastrin mRNA levels described above.
However, 10-15 days after cisplatin treatment the gastrin levels showed a gradual
increase. The rats in the same groups demonstrated a similar change in Northern and dot-

blot tests.

38

Figure 3. Northern blot analysis of gastrin mRNA. Total rat stomach RNA (20 pg) from
different groups was fractionated by electrophoresis on a 1.3% agarose gel, electroblotted
onto a nylon membrane, and hybridized with rat gastrin probe, which was detected by a
nonradioactive method. Gastrin mRNA levels were hardly detectable at Day 1 and Day 6
after cisplatin treatment. However, the levels gradually returned to normal 10-15 days
after the last cisplatin treatment. 28S ribosomal RNA served as control for any variations
in sample size. C, control; 1, 6, 10 and 15 represent days after the last treatment.

Figure 4. Northern blot analysis of somatostatin mRNA. Total rat stomach RNA (20 ug)
from different groups was fractionated by electrophoresis on a 1.3% agarose gel,
electroblotted onto a nylon membrane and hybridized with somatostatin probe, which
was detected by a nonradioactive method. The somatostatin mRNA level dramatically
decreased 1 and 6 days after cisplatin treatment, and gradually returned to normal 10-15
days after cisplatin treatment. 28S ribosomal RNA served as control for any variation in
sample size. C, control; 1, 6, 10 and 15 represent days after the last treatment.

Figure 5. Dot-blot analysis of gastrin after cisplatin treatment. One hundred ug protein
sample was dot-blotted onto the nitrocellulose membrane and incubated with rabbit anti—
gastrin antibody (1 :1000). The antibody was detected by the ABC method. Gastrin was
negative 1 and 6 days after the last cisplatin treatment. Note a gradual increase of gastrin
10 days after the cisplatin treatment. C, control; 1, 6, 10 and 15 represent days after the
last treatment.

39

Fig 3

 

10

c" 1’ 6 ”I? ‘
n94 .men‘OEKB

 

Fig 5

 

 

40

DISCUSSION

Gastrin regulates the secretion of gastric acid through its action on its receptors on
both the parietal cells and ECL cells. ECL cells produce histamine, which is a potent
stimulant of gastric acid production. The half-life for rat gastrin in circulation is around
10 min, so the circulating gastrin level is basically maintained by the continuous
production and secretion of gastrin from G-cells 8. Gastrin mRNA transcription and
gastrin production were inhibited after the ﬁrst day of cisplatin treatment, and the acid
content of the stomach builds up soon after Day 2 6. Gastrin release in rats is strongly
dependent on paracrine regulation by somatostatin—secreting cells (D-cells) in the
stomach 3. Northern blot studies show that somatostatin mRNA was hardly detectable at
1 and 6 days after the cisplatin treatment, but gradually recovered 10 days after the last
treatment. This change in the somatostatin mRNA level is parallel to that of gastrin
mRNA. Therefore, gastrin inhibition after cisplatin treatment is independent of
somatostatin. Otherwise, we would have observed an increase in somatostatin mRNA.

The cellular toxicity of cisplatin is mainly caused by its ability to bind covalently
to DNA to form intrastrand and/or interstrand crosslinks, which in turn prevents DNA
replication and transcription 16. Suppressed DNA replication and transcription of DNA
after cisplatin treatment might represent a nonspeciﬁc inhibition of gastrin and
somatostatin mRN A production.

Ulceration of the stomach is probably caused by an imbalance between gastric
protection against injury and the erosive acid/peptic factors that exist in the normal

stomach content 17. Recently, both exogenous and endogenous gastrin have been shown

41

to exert gastroprotective action 18’ 19. Gastrin plays an important physiological role in the
maintenance of gastric mucosal integrity. Mediated by the nitric oxide pathway, gastrin
also maintains the gastric mucosal blood ﬂow, which plays a crucial role in
gastroprotection 20. It appears that after cisplatin treatment there is a signiﬁcant
decreased gastrin.

Normal stomach motility involves contraction of the stomach smooth muscle and
relaxation of the pyloric sphincter, both of which are controlled by the release of
acetylcholine from the nerve terminals. Acetylcholine release is dependent on calcium-
calrnodulin. Under the low chloride ion concentrations inside the cell, cisplatin
hydrolyzes into monoaqua (monovalent) and diaqua (divalent) species. The diaquatic
form interferes with binding of calmodulin to calcium, which is further affected by
hypocalcemia induced by cisplatin 21. Lack of calcium-calrnodulin complex inhibits
acetylcholine release from the synaptic vesicles, which, in turn, causes bloating of the
stomach and increased production of gastric acid. It has been demonstrated that stomach
bloating and a signiﬁcant increase in gastric acid in rats can be prevented by
administration of calcium 6, 7, 22, whereas gastrin and somatostatin have no direct
involvement in the gastric acid increase responsible for ulceration. However, it is

probably that gastroprotective effects are compromised in the absence of gastrin.

42

REFERENCES

1. Comis R L. Seminars in Oncology 1994; 21:109.

2. 02018 R F and Young R C. Seminar in Oncology 1984; 11:251.

3. Choksi A and Hong W K. Chemotherapy of Head and Neck Cancer. In: Nicolini M,
ed. Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy.
Padua: Martinus Nijhoff; 1987.375.

4. Einhom L H and Williams S D. Cancer 1980; 46:1339.

5. Jurga L, Misurova E, Kovac V and Sevcikova L. Neoplasma 1994; 41:347.

6. Aggarwal S K, Antonio J D S, Sokhansanj A and Miller C M. Anti-Cancer Drugs
1994; 5:177.

7. Jarve R K and Aggarwal S K. Cancer Chemother Pharmacol 1997; 39:341.

8. Walsh J H. Gastrin. In: Walsh J H, Graham GJ, eds. Gut Peptides: Biochemistry and
Physiology. New York: Raven Press; 1994.75.

9. Hsu S M, Raine L and Fanger H. J Histochem Cytochem 1981; 29:577.
10. Larsson L I and Hougaard D M. J Histochem Cytochem 1993; 41:157.

11. Rage F, Lazaro J B, Benyassi A, Arancibia S and Tapia-Arancibia L. J
Neuroendocrinol 1994; 6:19.

12. Steele R and Torrie J. Principles and Procedures of Statistics, A Biochemical
Approach. New York: McGraw-Hill; 1980.

13. Chirgwin J M, Przybyla A E, MacDonald R J and Rutter W J. Biochemistry 1979;
18:5294.

l4. Sambrook J, Fritsch E and Maniatis T. Molecular Cloning, 2nd ed. Cold Spring
Harbor: Cold Spring Harbor Laboratory Press; 1989.

15. Deutscher M. Methods in Enzymology: Guide to Protein Purification. New York:
Academic Press; 1990.

16. Andrews P and Howell S. Cancer Cells 1990; 2:35.

17. Mertz H R and Walsh J H. Med Clin North Am 1991; 75:799.

43

18. Konturek S J, Brzozowski T, Bielanski W and Schally A V. EurJ Pharmacol 1995;
278:203.

19. Stroff T, Plate S, Respondek M, Muller K M and Peskar B M. Gastroenterology
1995; 109:89.

20. Leung F W, Robert A and Guth P H. Gastroenterology 1985; 88: 1948.
21. Blachley J D and Hill I B. Ann Intern Med 1981; 95:628.

22. Aggarwal S K and Fadool J M. Anit-Cancer Drugs 1993; 4:149.

44

Chapter Four

Role of Vitamin D on The Inhibition of Gastrin Production After Cisplatin Treatment

45

ABSTRACT

In rats cisplatin induces hypocalcemia, bloating of the stomach, and ulceration
ameliorated through calcium supplements. This study was undertaken to test the role of
calcium on the gastrin mRNA production in vitro and in vivo. RIN B6 cells were cultured
in medium with calcium (1.8, 3.6 and 7.2 mM) and the active form of vitamin D
(calcijex). Cisplatin was added (10 ug/ml) for 12 hrs and cells were harvested for RNA
from various treatment groups. Male Wistar rats were treated with cisplatin (9 mg/kg),
before and after vitamin D (0.3 mg/ 100g/week). The rats were killed and stomach tissues
excised on days 1, 6, 10 and 15 after cisplatin treatment. RNA from the stomach was
analyzed using the northern blot technique. Gastrin mRNA was suppressed after cisplatin
treatment both in vitro and in vivo. In vitro calcium but note vitamin D additions partially
prevented the gastrin mRNA. In vivo, however, vitamin D and calcium were equally

effective in preventing gastrin mRN A loss.

46

INTRODUCTION

Cisplatin (cis-dichlorodiammineplatinum H; CDDP), a broad-spectrum anticancer
drug, is effective in the treatment of a variety of cancers. One drawback is severe toxic
side effects including nausea, vomiting, and the induction of peptic ulcers in the
gastrointestinal tract 1. Ulceration of the stomach is due to the erosive acid/peptic factors,
which exist in normal stomach content 2. Both exogenous and endogenous gastrin show a
gastroprotective effect by maintenance of gastric mucosal integrity 3» 4. There is however,
a signiﬁcant suppression of gastrin production after cisplatin treatment, suggesting
gastrin loss might be one factor leading to ulceration 5.

In rats given cisplatin a lack of vomiting reﬂexes, causes bloating of the stomach
and ulceration 1. The bloating of stomach is associated with hypocalcemia due to
cisplatin 6. Calcium plays a role in release of acetylcholine from the nerve ﬁbers, inducing
relaxation of the pyloric sphincter and contraction of the gastric smooth muscles 1: 7. In
vitro, fundal strips from cisplatin-treated rats are hypercontractile to acetylcholine in
calcium-free Tyrode solution, but contract normally in Tyrode solution with calcium.
Thus there is a clear role for calcium in cisplatin-treated smooth muscle contraction 3.
Pretreatment with calcium prevents the bloating of the stomach due to cisplatin 1’ 9.
Hypocalcemia is also associated with inhibition of ATP synthesis and various membrane
transport enzymes. Again pretreatment with calcium has a protective effect on these
enzymes 6' 9.

The present study was undertaken to test for a direct role of vitamin D in cell

cultures ‘0 in comparison to the indirect effect through calcium homeostasis in the rats.

47

MATERIALS AND METHODS

Cell Culture

RIN B6 cells (rat insulinoma cell line) 1°, a generous gift from Dr. Loyal Tillotson,
Department of Medicine, University of North Carolina, were cultured at 37°C in
Dulbecco's modiﬁed Eagle's medium (DMEM): 4.5 g/ml glucose with 10% fetal bovine
serum, 100 mg/ml streptomycin and 100 units/ml penicillin in an atmosphere of 5% C02.
Calcium chloride stock solution (1 M) was added to achieve the calcium concentrations
of 3.6 mM or 7.2 mM. EDTA (1.5mM) was used to chelate the calcium in the culture
medium. Some cultures were treated with calcijex (1,25-dihydrox-Vitamin D3, from
Abbott Laboratories, Chicago, IL) at a 50 pg/ml concentration, calcij ex plus calcium (3.6
mM), or calcijex plus calcium and cisplatin (IOug/ml). Cells were also cultured in
cisplatin containing medium with calcij ex or calcium alone. Untreated cultures served as
controls. Cisplatin (IOug/ml) was added to various cultures for 12 h. The cells were
trypsinized and stained with trypan blue (GIBCO BRL, Grand Island, NY). Cell counts
were taken and RNA was extracted in all the experiments by methods previously

described 1 1.

Animals and Tissue Preparation

Male Wistar rats (Charles River Laboratory; Wilmington, MA) weighing 100-150
g were kept in a 12-h light/12-h dark cycle with free access to food and water. The rats
were divided into 3 groups. The ﬁrst group of rats was injected (IP) with cisplatin (9

mg/kg), in 0.9% normal physiological saline in ﬁve divided doses over 5 days. Rats in the

48

second group were given vitamin D (0.3 mg/ 100g) (Banner Phannacaps Inc., Elizabeth,
NJ) once a week for the duration of the experiment. And given cisplatin (9 mg/kg) after
one week after the ﬁrst vitamin D administration. The third group of rats received the
injection of vehicle only. Three rats from each group were sacriﬁced and stomach tissues
excised 1, 6, 10 and 15 days after the last cisplatin injection and frozen (70°C) until use.

The experiment was repeated at least 3 times.

Northern Blot Analysis

A 27-mer sequence 5’-GACCTTGGGGCCCCAGCTGTCTCCGAT-3’,
complementary to the coding sequence of rat gastrin mRNA (position 212-238) ‘2, was
synthesized using the ABI 3948 Synthesis and Puriﬁcation System. The gastrin
Oligonucleotide probe was labeled with the digoxigenin (DIG) Oligonucleotide tailing kit
(Boehringer Mannheim Corp. Indianapolis, IN). A 100 pmol probe was incubated with
tailing buffer, CoCl2 solution, DIG-dUTP, dATP and terminal transferase solutions at
37°C for 18 minutes, then placed on ice and the reaction was stopped by adding 4ul stop
solution. The 28s rRNA probe was labeled using DIG high prime kit (Boehringer
Mannheim Corp. Indianapolis, IN). The rRNA probe (50ng) in 16 ul water was denatured
and incubated with 4 ul DIG high prime mix for 20 hours. The reaction was stopped by
adding 2 ul 0.5 M EDTA. The labeled probes were precipitated in 5 ul 4M LiCl and 150
pl prechilled (-20°C) ethanol at —20°C for 3 hours and then centrifuged at 12,000 g for 15
minutes. The supernatant was discarded and the pellet was air-dried.

RNA was extracted from the whole stomach tissues or cells by guanidinium

isothiocyanate solution and puriﬁed through a CsCl cushion 1‘. The RNA (20 ug) was

49

size-separated by electrophoresis in a 1.3% agarose gel containing formaldehyde and
electroblotted onto nylon membrane (GeneScreen, New England Nuclear, Boston, MA).
The blots were prehybridized with the buffer containing 50% fonnamide, 10% dextran
sulfate, 50 mM Tris (pH 6.8), 3 X standard saline citrate (SSC), 100 mg/ml sonicated
salmon sperm DNA and 5 X Denhardt’s solution for 3 hours at 42°C. Blots were then
incubated with fresh hybridization buffer in the presence of labeled probes, 40 ng/ml for
gastrin and 5 ng/ml for 285 rRNA, at 42°C for 2 days. The blots were washed with two
changes of 2 X SSC/0.1% sodium dodecyl sulfate (SDS) at 42°C for 30 min each and 0.1
X SSC/0.1% SDS at 42°C for another 15 min. The DIG in the probes was detected using
a DIG nucleic acid detection kit (Boehringer Mannheim; Indianapolis, IN). The blots
were washed in maleic acid buffer (pH 7.4) for 10 min, then sequentially incubated in
blocking buffer for 30 min and followed by 1/ 1000 anti-DIG antibody for 45 min. After
washes in PBS and IOOmM Tris-HCl buffer (pH 9.5), the blots were incubated in
nitroblue tetrazolium-bromochloroindolyl phosphate (NBT/BCIP) solution overnight.
RNA extracts from liver tissue served as control for the absence of gastrin. All the H20

used in the above study was diethyl pyrocarbonate (DEPC) treated double distilled water.

Statistical Analysis
The number of viable versus dead cells ﬁom cultures with different concentrations
of calcium, with or without cisplatin and/or calcijex were counted. The data was

statistically analyzed by the One-way AN OVA test '3.

50

 

RESULTS

RNA extracted from RIN B6 cells was subjected to electrophoresis and
hybridization with DIG labeled probe, speciﬁc for gastrin mRNA (Fig. 1). In the absence
of calcium in the culture medium, the cells had negligible levels of gastrin mRNA.
However, when supplemented with calcium (1.8mM) the gastrin mRNA levels were
equivalent to those in normal stomach tissue (Fig 1). Cisplatin treatment of RIN B6 cells
in presence of calcium (1.8 mM) signiﬁcantly inhibited gastrin mRNA levels. Cisplatin
treatment, however, with 3.6 mM or 7.2 mM of calcium restored near normal levels of
gastrin mRN A. Vitamin D supplementation did not affect the control of gastrin mRNA
level (Fig. 2/lanes 2 and 7). Cisplatin treatment of RIN B6 cells inhibited gastrin mRNA
levels with or without vitamin D (lanes 5 and 6). In contrast, the calcium supplementation
maintained the gastrin mRNA levels in both cell cultures with or without vitamin D
(lanes 3 and 8). In order to monitor the amount and integrity of RNA sample loading in
each lane, rRNA (28s) served as a control. The rat stomach RNA served as a positive
control for gastrin mRNA, whereas liver RNA served as a negative control (data not
shown).

To ensure that cell viability was unaffected by the various treatment regimes the
percentage dead cells was determined (Table 1). We conclude that there are insigniﬁcant
differences in cell viability which could account for the loss of gastrin mRNA in cells
after cisplatin treatment. The cell counts are depicted in Table l.

Rats were treated with cisplatin and cisplatin plus vitamin D. RNA from the rat

stomach tissues was analyzed by the same methods to detect gastrin mRNA (Fig. 3).

51

After 1 and 6 days since cisplatin treatment, the gastrin mRNA bands were almost
undetectable, compared to the control. However, 10 days after cisplatin treatment the
gastrin mRNA levels started to increase reaching levels found in the untreated animal
around day 15. When vitamin D was given before cisplatin treatment gastrin mRNA
levels were not reduced compared to the group that only received cisplatin. Interestingly
the gastrin mRNA increased to greater levels by day 15. The equal amount of loading

was shown by the equal intensity of the 28s bands.

52

Table 1. Number of viable and dead cells from cultures with different concentrations of

calcium, with or without cisplatin and/or calcijex. (p=0.27)

 

 

Calcium concentration (mM) 1.8 3.6 3.6 1.8 1.8 1.8 3.6 7.2
Cisplatin (9ug/ml) -- + -- + + -- + +
Calcijex (50pg/ml) -- + + + -- + -- --
Time of cisplatin treatment (hr) 12 12 12 12 12 12 12
Viable cells (10°/ml) 4.6 4.7 4.4 3.4 3.6 3.2 3.9 3.3
Dead cells (105/ml) 2.6 2.7 2.5 2.7 1.4 1.7 1.5 1.8
Percentage of dead celﬂ%) 5.7 5.4 5.7 7.9 3.9 5.3 3.8 5.1

 

 

+, treatment; --, no treatment

53

Figure 1. Northern blot analysis of gastrin mRNA from the RIN B6 cells cultured in
different concentrations of calcium with or without cisplatin. Note the negligible levels of
gastrin mRNA in the absence of calcium (EDTA) from the culture medium (lane 5).

After supplementing calcium (1.8mM) the gastrin mRN A levels were maintained to
normal (lane 4). Cisplatin treatment inhibited gastrin mRN A production (lane 3),
however, in the presence of 3.6 mM or 7.2 mM of calcium gastrin mRNA levels were
close to normal (lanes 2, 1). Gastrin mRNA from the rat stomach served as a molecular
size marker for gastrin mRNA. Equal amounts of 28s ribosomal RNA were detected in

all samples.

Figure 2. Northern blot analysis of gastrin mRNA from the RIN B6 cells showing the
effects of calcium and/or vitamin D on the inhibition of gastrin production after cisplatin
administration. Note the suppression of gastrin mRNA after cisplatin (lane 6) or cisplatin
plus calcijex (lane 5) treatments. Calcijex by itself has no effect (lane 7). However,
calcium (3.6mM) seems to counteract the suppression of gastrin mRN A after cisplatin
treatment (lane 8), as does calcijex and calcium (lane 3). Gastrin mRNA from the rat
stomach served as a molecular size marker for gastrin mRNA (lane 1). Equal ribosomal
RNA (28s) was detectable in all samples.

Figure 3. Northern blot analysis of gastrin mRNA from normal, cisplatin and cisplatin
plus vitamin D treated animal stomachs. Note gastrin mRNA level was almost
undetectable on day 1 and day 6 after cisplatin treatment but restored to normal levels
around day 10. In the animals pretreated with vitamin D, however, the gastrin mRNA
levels were not lower after cisplatin treatment. The gastrin mRN A started to increase
after day 10 of cisplatin treatment reaching above normal levels by day 15. Ribosomal
RNA (28$) served as control for possible variation in sample RNA amount or integrity.

54

 

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55

DISCUSSION

Currently there is no dependable method available to isolate the functional gastrin-
producing cells from the stomach. Therefore RIN B6 cells were used as a model to
analyze the regulation of gastrin gene expression ‘0. Although EDTA treatment inhibited
the expression of gastrin mRNA, exogenous calcium restored it to normal levels“).
Higher levels of extracellular calcium might be expected to increase the amount of
calcium transported through the calcium channels into the cell. Gastrin gene expression
may be maintained in the RIN B6 cells by elevation of intracellular calcium"). The
mechanism of how cisplatin decreases, and calcium increases gastrin mRNA levels is
unknown. Calcium may counteract lowered transcription rates or increased mRNA
degradation. Cisplatin has been shown to bind covalently with DNA in intra- and inter-
strand crosslinks preventing its replication or transcription '4. In order for us to interpret
the decrease in the gastrin mRNA levels in cultures speciﬁcally due to cisplatin induced
cell death, we counted the number of dead cells after each treatment. Our results
documented no signiﬁcant changes in number of viable cells amongst different
treatments. Therefore, we infer that decreased levels of gastrin mRNA are not due to the
loss of cells. Vitamin D, in order to be effective as a regulator of calcium homeostasis, is
hydrolyzed into an active form of vitamin D (1,25-dihydrox-Vitamin D3) in the liver and
kidney. The active form of vitamin D did not Show any demonstrable changes in the
mRNA levels of gastrin. Vitamin D acts via a nuclear receptor to affect gene
transcription”. It is conceivable that the RIN B 6 cells lack this receptor and therefore fail

to respond. Alternatively vitamin D may act by increasing calcium only in vivo.

56

However, exogenous calcium demonstrated a profound protective effect on gastrin
mRNA after cisplatin treatments. In support of this mechanism cisplatin treatments have
been shown to cause severe hypocalcemia in rats 1’ °. However, pretreatment with vitamin
D is able to maintain the serum calcium levels within the normal range ‘5. In vitro,
vitamin D or calcij ex did not show any protective effect against cisplatin suppression of
gastrin mRN A, however, calcium supplement demonstrated a signiﬁcant protective
effect. In vivo, vitamin D was just as effective in maintaining the gastrin mRNA levels

possibly by prevention of hypocalcemia.

57

REFERENCES

1. Aggarwal S K, Antonio J D S, Sokhansanj A and Miller C M. Anti-Cancer Drugs
1994; 5:177.

2. Mertz H R and Walsh J H. Med Clin North Am 1991; 75:799.

3. Stroff T, Plate S, Respondek M, Muller K M and Peskar B M. Gastroenterology 1995;
109289.

4. Konturek S J, Brzozowski T, Bielanski W and Schally A V. Eur J Pharmacol 1995;
278:203.

5. Wang Y, Aggarwal S K and Painter C L. J Histochem Cytochem 1999; 47 :1057.
6. Aggarwal S K. J Histochem Cytochem 1993; 4121053.

7. Jarve R K and Aggarwal S K. Cancer Chemother Pharmacol 1997; 39:341.

8. San Antonio J D and Aggarwal S K. J Clin Hematol Oncol 1984; 14:55.

9. Aggarwal S K and Fadool J M. Anit-Cancer Drugs 1993; 4:149.

10. Brand S J and Wang T C. JBiol Chem 1988; 263:16597.

11. Chirgwin J M, Przybyla A E, MacDonald R J and Rutter W J. Biochemistry 1979;
18:5294.

12. Larsson L I and Hougaard D M. J Histochem Cytochem 1993; 41:157.

13. Campﬁeld T, Braden G, F lynn-Valone P and Powell S. Pediatrics 1997; 99:814.
14. Andrews P and Howell S. Cancer Cells 1990; 2:35.

15 . Brown A J, Dusso A and Slatopolsky E. Am J Physiol 1999; 277:F157.

16. Minghetti P P and Norman A W. FASEB J 1988; 2:3043.

58

Chapter Five

Summary And Perspectives

59

SUMMARY AND PERSPECTIVES

Cisplatin treatment (9 mg/kg) causes bloating of the stomach, lowered pH,
ulceration, and hypocalcemia in rats ”’3. Gastrin, controlled mainly by somatostatin and
extracellular calcium concentration in rats, plays an important role in regulating gastric
acid production 4’5. Through the nitric oxide pathway, gastrin also maintains the gastric
blood ﬂow, which plays an important role in gastroprotection against acid and pepsin in
the stomach content °.

Gastrin and its mRNA productions are severely inhibited after cisplatin treatment
in rats, and start to recover on day 10 after the last cisplatin treatment. Somatostatin and
iNOS productions are also inhibited. The lowered pH, which happens two days after the
cisplatin administration, is not related to the gastrin. The inhibition of gastrin production
is not because of the somatostatin action, for the somatostatin itself is severely inhibited
alter cisplatin treatment. Nitric oxide production ﬁ'om either nNOS and iNOS are
suppressed alter the cisplatin treatment. We infer that the normal gastric blood ﬂow from
the action of gastrin can’t not be maintained.

Vitamin D supplementation in the rats received cisplatin, counteracts the
inhibition of gastrin production. This counteraction is believed through the maintenance
of the serum calcium levels by the vitamin D. The results is further conﬁrmed by the in
vitro study using gastrin producing RIN B6 cells. It is the increased calcium
concentration in the culture media that stimulates the gastrin production in stead of

vitamin D itself.

60

The cellular toxicity of cisplatin occurs mainly through its ability to covalently
bind to DNA to form intrastrand and/or interstrand crosslinks, which in turn prevents
DNA replication and transcription 7. Suppressed DNA replication and transcription of
DNA after cisplatin treatment might be a nonspeciﬁc inhibition of the gastrin,
somatostatin mRNA, and iNOS productions.

It is of great interest to run the nuclear run-on essay to further conﬁrm that the
inhibition of gastrin mRNA production is due to the suppressed transcription. If so,
further investigation can be done to locate if the transcription factors and/or the promoter
region of the gastrin gene is inhibited after cisplatin treatment.

Gene screen technology, such as differential display, can be done to see how many
genes transcriptions are changed, inhibited or stimulated, after cisplatin treatment.
Among these altered genes transcriptions, how many can really be restored by the
vitamin D mediated serum calcium levels.

Stomach mucosal prostaglandins play key roles in maintaining the gastrin mucosal
integrity against the aggressive factors, such as acid and pepsin 8’9’10. It is also of great
interest to test the prostaglandin levels in the stomach in order to see their affect on the

ulcer formation in the cisplatin treated rats.

61

REFERENCES

l. Aggarwal S K. J Histochem Cytochem1993; 41 : 1053.

2. Aggarwal S K, Antonio J D S, Sokhansanj A and Miller C M. Anti-Cancer Drugs
1994; 52177.

3. Aggarwal S K and Fadool J M. Anit-Cancer Drugs 1993; 4: 149.

4. Walsh J H. Gastrin. In: Walsh J H, Graham GJ, eds. Gut Peptides: Biochemistry and
Physiology. New York: Raven Press; 1994.75.

5. Brand S J and Wang T C. JBiol Chem 1988; 263216597.
6. Leung F W, Robert A and Guth P H. Gastroenterology 1085; 88:1948.
7. Andrews P and Howell S. Cancer Cells 1990; 2:35.

8. Brzozowski T, Konturek S J, Drozdowicz D, Dembinski A and Stachura J. Digestion
1995; 56:463.

9. Peura D A. Am J Gastroenterol 1997; 92:88.

10. Kang J Y. Ann Acad Med Singapore 1995; 24:218.

62

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