International Medical Journal Vol. 26, No. 1, pp. 2 - 6 , February 2019

GENETICS

Effect of Reserpine on the Expression of Tumour Promoter, Tumour Suppressor and Ulcer Healing Genes in the

Stomach of Female Sprague-Dawley Rats

Isyraqiah Faizatul1), Methil Kannan Kutty1), Damayanthi Durairajanayagam1), Norita Salim1), Sergey Gupalo4), Norizan Kamal Basah1), Harbindar Jeet Singh1,2,3)

ABSTRACTIntroduction: Reserpine is often used to induce gastric ulcers in animal models but has also been shown to induce tumours of

adrenal and mammary glands. However, its effect on the expression of tumour promoter, suppressor, and ulcer healing genes in the stomach is unknown.

Aims: This study examined the expression of these genes in the stomach of rats 24 hours after reserpine treatment. Materials and Methods: After an overnight fast, sixteen, 6-week old female rats were divided equally into control and experi-

mental groups (20 mg/kg of reserpine intraperitoneally). All rats were euthanized after 24 hours. Body weights were measured before treatment and 24 hours after treatment. Following euthanization, their stomachs were collected for histopathological examination, and expressions of tumour promoter (L-myc, PGC), tumour suppressor (ARID1A, E-Cadherin, FAT4, APC), and ulcer healing genes (PPARG, TFF1, TFF2) using RT-qPCR. Data were analysed using one-way ANOVA.

Results: Haemorrhagic gastric ulcer developed after 24 hours of reserpine treatment. Microscopy showed superficial ulcer-ation with degenerated necrotic mucosa and inflammatory cells. Expression of E-cadherin, APC, FAT4, PGC, Lmyc, and TFF1 genes was significantly lower, whereas ARID1A and TFF2 expressions were significantly higher in reserpine-treated rats. No changes were observed in the expression of PPARG. Body weight of reserpine-treated rats was significantly lower compared to that in controls.

Conclusions: The decreased expression of E-cadherin, APC, FAT4, and TFF1 suggests that reserpine might promote gastric carcinogenesis. However, other tumour promoter genes need to be examined to ascertain the effect of reserpine on gastric car-cinogenesis.

KEY WORDSreserpine, gastric ulcer, tumour promoter, tumour suppressor, ulcer healing

Received on November 8, 2017 and accepted on March 6, 20181) Faculty of Medicine, Universiti Teknologi MARA 47000 Sungai Buloh, Selangor, Malaysia2) IMMB, Faculty of Medicine, Universiti Teknologi MARA 47000 Sungai Buloh, Selangor, Malaysia3) I-PPerFORM, Universiti Teknologi MARA Malaysia4) Faculty of Medicine, Universiti Sultan Zainal Abidin 20400 Kuala Terengganu, Terengganu, MalaysiaCorrespondence to: Harbindar Jeet Singh(e-mail: hjsingh@salam.uitm.edu.my)

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INTRODUCTION AND LITERATURE REVIEW

Reserpine is well-known for its role as an anti-psychotic1) and anti-hypertensive drug2,3), although it is rarely used today to treat hyper-tension due to its many side effects4). Reserpine has however been used successfully to treat patients with psychosis and mania5,6). As a mono-amine depletor, reserpine has been used as a model to induce depression in studies investigating anti-depressant drugs7,8). Repeated low dose of reserpine administration leads to changes in motor functions accompa-nied by higher striatal oxidative stress, and has therefore been suggested as a viable rat model for the neuroprogression of Parkinson's disease9,10).

However, administration of high doses of reserpine has also been shown to cause adverse reactions, particularly in inducing gastric ulcers4). Studies on reserpine-induced gastric ulcers have been well documented, and it has been proven to be a good toxic chemical for studies related to gastric ulcer11-13).

Long-term exposure to low doses of reserpine was found to increase the incidence of adrenal medullary pheochromocytomas, undifferentiat-ed carcinomas of seminal vesicles, and mammary cancers in rats and mice14). Although reserpine is often used as an ulcer causing agent, no reports exists on its effects on tumour related genes. It is generally known that tumorigenesis involves the down-regulation of tumour sup-pressor genes and up-regulation of tumour promoter genes. Many stud-

C 2019 Japan Health Sciences University & Japan International Cultural Exchange Foundation

Faizatul I. et al. 3

ies have depicted the role of E-cadherin (Epithelial Cadherin), APC (Adenomatous Polyposis Coli), ARID1A (AT Rich Interactive Domain 1A), and FAT4 (FAT Atypical Cadherin 4) genes as tumour suppressors in gastric cancers15-18). PGC (pepsinogen C) and L-myc (Myelo-cytomatosis) genes that function as tumour promoters are also described in gastric cancer studies19,20). In addition, TFF1 (Trefoil factor 1), TFF2 (Trefoil factor 2), and PPARG (Peroxisome Proliferator-activated recep-tor gamma) have been reported to play an important role in the healing of gastric ulcers21-23). Hence, the aim of this study was to evaluate the effect of reserpine on the expressions of tumour promoter, tumour sup-pressor, and ulcer healing genes in normal rats.

MATERIALS AND METHODS

Animals and diets

Sixteen, six-week old female Sprague-Dawley rats (135 - 145 g) were obtained from the Laboratory Animal Care Unit (LACU), Faculty of Medicine, Universiti Teknologi MARA (UiTM) Sungai Buloh, (Selangor, Malaysia). The rats were housed in polypropylene cages, with a stainless steel wire grid as cover, in the animal room of LACU. Ambient temperature and relative humidity were maintained at 23 - 25℃ and 50 - 55 % respectively, with a 12-hour light/dark cycle. Their beddings were changed twice a week. The rats had access ad libitum to vegetarian diet pellet (Special Feeds, Australia) and reverse-osmosis (RO) drinking water. Animal handling and experimental treatment pro-cedures were done in accordance with the Guidelines for Care and Use of Laboratory Animals prepared by the Animal Care and Users

Committee (ACUC, UiTM Shah Alam, Selangor, Malaysia). This study was approved by the Animal care and users Committee of the Faculty of Medicine, UiTM.

Experimental animals and grouping

Briefly, sixteen female rats were divided into two groups (Group 1 and 2), consisting of 8 rats per group. All rats were fasted 24 hours before the start of the experiment. This was labelled as day 0. Group 1 was kept as controls, while Group 2 was administered with a stat dose of 0.1 mL of 20 mg/kg reserpine suspended in 0.5% acetic acid solution via intraperitoneal route24). This was labelled as day 1. All rats were again fasted for another 24 hours (day 2). Then, they were lightly anes-thetized with diethyl ether and euthanized using a small guillotine. The rat treatment groups are summarised in Figure 1.

Histological Study

Stomachs of rats were collected and sectioned longitudinally, divid-ing the lesser and greater curvatures. One part of the stomach was fixed in 10% neutral buffered formalin, processed and then embedded in par-affin. Thin sections were made and stained with haematoxylin and eosin (HE). The slides were then viewed under a light microscope at 200x and 400x magnifications.

RT-qPCR Analysis

The other part of the stomach was stored at -80℃ immediately after dissection. InnuPREP_RNA Mini Kit (Analytik Jena, Germany) was used to extract RNA from the stomach tissues and were stored immedi-ately at -20℃. The RNA extract was then converted to cDNA using Maxima First Strand cDNA synthesis kit (Thermo Scientific, USA) and incubation was performed using Mastercycler pro thermal-cycler (Eppendorf, Germany). The cDNA was then stored at -20℃. Reverse-Transcriptase quantitative PCR (RT-qPCR) was performed to determine the expressions of tumour suppressor genes (FAT4, ARID1A, APC, E-Cadherin), tumour promoter genes (PGC and L-myc), and ulcer heal-ing genes (PPARG, TFF1, and TFF2) using Luminaris Colour HiGreen qPCR Master Mix (2X) (Thermo Scientific, USA). RT-qPCR was run using PCR thermal cycler CFX 96 (Bio-Rad, USA). GAPDH and RPL29 was used as reference genes to normalize the relative gene expressions.

Figure 1. Experimental Design

Figure 2. Mean body weight of control and reserpine-treated rats at day 1 and 2.

Figure 3. Control and reserpine treated rats after 24 hours

Figure 4. Macroscopic examination of reserpine-treated rats showed haemorrhagic ulcer at the greater curvature of the stomach (B) compared to control (A).

Figure 5. Microscopic examination of reserpine-treated rats showed acute superficial gastric ulcer with degenerated necrotic mucosa (B) (x200). Further examination of the degenerated necrotic mucosa showed presence of inflammatory cells (D) (x400).

Expression of Genes in the Stomach4

Statistical analysis

Body weight was analysed using two-way ANOVA, while gene expression was analysed using one-way ANOVA. A 'p' value of less than 0.05 was considered statistically significant.

RESULTS

No significant differences were evident in body weight between the two groups at day 1. Body weight of reserpine-treated rats was signifi-cantly lower compared to that of the control group at day 2 (p < 0.001) (Figure 2). Twenty-four hours after reserpine administration rats were found to bleed from the nostrils and eyes (Figure 3). Reserpine-treated rats developed haemorrhagic ulcers in the stomachs (Figure 4). Microscopic examination of the ulcerated stomach showed degenerated necrotic mucosa and presence of inflammatory cells (Figure 5). In the reserpine-treated group, the expressions of E-Cadherin (p < 0.05), FAT4 (p < 0.001), and APC (p < 0.001) were significantly lower, while expression of ARID1A was significantly higher (p < 0.001) when com-pared to those in the controls (Figure 6.1). Reserpine-treated rats had significantly lower expressions of L-myc and PGC in the stomach when compared to that in the controls (Figure 6.2). Reserpine-treated rats had significantly lower expression of TFF1 (p < 0.001) and significantly higher expression of TFF2 (p < 0.001) when compared to that in the controls. There was no significant difference in the expression of PPARG in reserpine-treated rats compared to that in the controls (Figure 6.3).

DISCUSSION

Reserpine induced acute haemorrhagic ulceration of the gastric mucosa at the greater curvature near the antral region (Figure 4). Histopathological examination of the ulcer revealed an area of degener-ated necrotic mucosa (Figure 5). Inflammatory cells were also observed near the ulcerated area (Figure 5). Induction of gastric ulcer by reserpine

has been reported before, where it becomes evident from as early as 18 hours after its administration, provided the animals were fasted for 24 or 36 hours prior its administration25). Reserpine most probably triggers gastric ulceration through indirect over-stimulation of the vagus nerve. The direct effect of reserpine as a monoamine depletor might be at the sympathetic neurons first, which then causes an increase in the activity of the parasympathetic neurons, including the vagus nerve. Reserpine is reported to prevent the accumulation of catecholamines in the synaptic vesicles of sympathetic neurons by competing with catecholamines for the binding site on the vesicular membrane transporter 2 (VMAT2) receptor; binding irreversibly to the receptor26). The inhibitory effect of reserpine in the sympathetic neurons results in an over-stimulation of the vagus nerve in the stomach, which then releases acetylcholine that binds to its receptors at the parietal cells. Diffusion of calcium ions increases, which then activates phosphorylation of kinases. The proton pump is then activated, secreting hydrogen ions into the stomach. Excessive gastric acid secretion then induces the development of gastric mucosal lesions and ulcers27,28).

Reserpine-treated rats had significantly lower expression of tumour suppressor E-cadherin, FAT4, and APC compared to control (Figure 6.1). To our knowledge, this is the first study reporting the effect of reserpine on the expressions of tumour promoter, tumour suppressor, and ulcer healing genes. E-cadherin plays a role in cell-to-cell adhesion between epithelial cells. Each cadherin forms an intersection with a cad-herin on an adjacent cell, which forms an adheren junction29). Inactivation of E-cadherin reduces cellular adhesion, promoting metas-tasis of malignant tumour cells30). E-cadherin expression has been reported to be significantly lower in patients with gastric adenocarcino-ma15). Low expression of E-cadherin might promote tumorigenesis in reserpine-treated rats. FAT4 gene plays a role in regulating planar cell polarity of epithelial cells31,32). It synthesizes transmembrane proteins that arrange and align specialized cells; like body hair in one specific direction through asymmetric localization32,33). Loss of FAT4 protein function promotes the development of hyperplastic epithelial cancer cells34-36). Patients with gastric cancer with poor 5-year disease survival rate have been shown to have significantly lower FAT4 expression16). Low FAT4 expression might facilitate gastric cancer in reserpine-treated rats. APC gene is an important tumour suppressor, and about 80% of colon cancers have APC mutations. It regulates the ubiquitination and disintegration of oncogene B-catenin. APC inactivation leads to accu-

Figure 6-1. Expressions of E-Cadherin, FAT4, APC, and ARID1A gene in the stomach of female rats after 24 hours of reserpine administration.

Figure 6-2. Expressions of L-myc and PGC gene in the stomach of female rats after 24 hours of reserpine administra-tion.

Figure 6-3. Expressions of TFF1, TFF2 and PPARG gene in the stomach of female rats treated after 24 hours of reser-pine administration.

Faizatul I. et al. 5

mulation of B-catenin, which subsequently triggers transcription of cyclin D1 and C-myc37,38). Patients with intestinal type gastric cancer exhibited mutations in the APC gene17). Low expression of APC in reser-pine-treated rats might promote gastric cancer. However, reser-pine-treated rats had significantly higher expression of ARID1A com-pared to control. ARID1A plays a role in regulating expressions of genes through remodelling of chromatin structure. High expression of ARID1A causes chromatin to bind tightly, hence lowering the activities of gene transcriptions, replications, methylations, and repairs. Silencing of ARID1A gene causes unnecessary transcription of genes that are involved in stimulating proliferation of cells18). Patients with gastric can-cer had lower expression of ARID1A compared to their matched non-tu-mour tissues39). Reserpine might increase ARID1A expression to sup-press the development of gastric cancer. Reserpine-induced ulcer might promote gastric carcinogenesis by lowering the expression of tumour suppressors, which perhaps might explain the increase risk of develop-ing gastric cancer among patients with gastric ulcers.

The expressions of tumour promoter L-myc and PGC were signifi-cantly lower in reserpine-treated rats (Figure 6.2). PGC is an inactive precursor of pepsin, which is activated by the acidity of the stomach40,41). PGC might play a role in triggering proliferation and development of breast cancer cells42). PGC was also highly expressed in basal cell and squamous cell carcinomas of the eyelids43). However, PGC expression in the stomach was significantly lower in patients with gastric cancer, but higher in patients with gastric ulcer44). This is somewhat similar to that in this study, where the expression of PGC was significantly lower in reserpine-treated rats. It is possible that PGC might be involved in the development of other cancer types but not that of the stomach. PGC might alternatively be used as a biomarker for gastric cancer45,46). Myc gene is an oncogene that encodes a transcription factor protein47). It binds to specific genes and activates its transcription, which may be involved in cell cycle, apoptosis and cell differentiation48). Mutation of Myc gene is found in about 50% of Burkitt's lymphoma patients49). It upregulates Bcl-2, which inhibits apoptosis of B-cell lymphomas50). Myc protein was found over-expressed in gastric cancer patients, and the pro-moter Myc gene was hypo-methylated19). It is possible that L-myc, a variant of Myc, might not be involved in gastric cancer development. L-myc is highly expressed in small cell lung carcinomas, but C-myc; another variant of Myc is highly expressed in solid tumours. C-myc was found highly expressed in gastric cancer cells, and may involve in pro-moting its growth51). Reserpine might lower the expression of L-myc to inhibit lung carcinoma development, but the role of C-myc in promoting gastric carcinogenesis in reserpine-treated animals needs to be investi-gated. Gastric carcinogenesis may not involve tumour promoter L-myc and PGC in reserpine-treated rats.

No significant difference was evident in the expression of PPARG in reserpine-treated rats compared to that in the control (Figure 6.3). PPARG is a ligand-activated transcription factor found in genes involved in energy homeostasis and lipid metabolism52). Activation of PPARG by its agonist troglitazone inhibits the proliferation of gastric cancer cells, possibly by favouring apoptosis and causing the arrest of G1 cell cycle53). However, in reserpine-treated rats, no significant chang-es were evident in PPARG expression, which probably indicates that PPARG does not play a role in gastric carcinogenesis in reserpine-treat-ed rats. Perhaps if the treatment duration were longer, the expression of PPARG would be significantly lower, hence indicating that reserpine might lower the expression of PPARG to promote gastric carcinogene-sis. In addition, the expression of TFF1 was significantly lower in reser-pine-treated rats (Figure 6.3). TFF1 proteins are secreted by the pit cells of stomach, and plays a role with mucin in maintaining the stomach's integrity by forming the mucous barrier54). Expression of TFF1 was sig-nificantly higher in patients with gastric ulcers, and was related to the gastric ulcer healing process21). However, TFF1 expression was down-regulated in gastric cancer cell lines due to hyper-methylation of its pro-moter gene55). Reserpine may promote gastric carcinogenesis by lower-ing the expression of TFF1 in the stomach. The expression of TFF2 was significantly higher in the reserpine-treated group compared to that in the control group (Figure 6.3). TFF2 is secreted by mucous neck cells of gastric mucosa and is important in the recovery of damaged gastric mucosa54). TFF2 gene was hypo-methylated in patients with gastric ulcer56). TFF2-deficient mice secreted higher levels of gastric acid, low number of mucous cells, and had thin mucosal layer, which might be due to lower proliferative rate of the mucosa22). However, hyper-methyl-ation of TFF2 promoter gene was found to significantly lower the expression of TFF2 in gastric cancer cell lines57). Reserpine may increase the expression of TFF2 to promote healing of the ulcerated mucosa based on the presence of inflammatory cells. However, TFF2 may not be involved in gastric carcinogenesis in reserpine-treated rats.

Reserpine-treated rats had significantly lower body weight com-pared to that of the controls (Figure 2). The rats lost about 35% of their body weight after 24 hours of reserpine administration. Reserpine-treated rats have been shown to lose about 10% body weight after 24 hours of 10 mg/kg reserpine administration58). The precise reason for this is not apparent but it might be associated with diarrhoea and hypophagia following reserpine administration. The diminished appetite may be correlated to the sedative effect of reserpine.

CONCLUSION

In conclusion, it appears that reserpine might promote gastric car-cinogenesis by lowering the expressions of tumour suppressors (E-cadherin, APC, FAT4) and ulcer healing gene (TFF1). However, fur-ther studies on the expressions of other tumour promoter genes in reser-pine-treated rats might clarify the role of reserpine in promoting the development of gastric cancer.

ACKNOWLEDGEMENT

This study was supported by 600-RMI / DANA 5/3/RIF (492/2012) research grant. Special thanks to all the members of the Adipokine Interest Group, Faculty of Medicine, UiTM for their comments and their continuous support. The authors wish to acknowledge the staff of Laboratory Animal Care Unit (LACU) and Institute of Medical Molecular Biotechnology (IMMB) for their technical help and support.

REFERENCES

1) Bisong SA, Brown RE, Osim EE. Comparative extrapyramidal effects of Rauwolfia vomitoria, chlorpromazine and reserpine in mice. J. Nat. Med. 2013;67:107-12.

2) May E, Heyblon R. The treatment of arterial hypertension with Rauwolfia serpentina. Bull.Soc.Med.Hop 1953;69:958-66.

3) McGregor M, Segel N. The Rauwolfia alkaloids in the treatment of hypertension. Br. Heart J. 1955;17:391-6.

4) Bulpitt CJ, Dollery CT. Side effects of hypotensive agents evaluated by a self-adminis-tered questionnaire. Br. Med. J. 1973;3:485-90.

5) Chen F-E, Huang J. Reserpine: a challenge for total synthesis of natural products. Chem. Rev. 2005;105:4671-706.

6) Eloff I, Esterhuysen W. Reserpine for the treatment of refractory mania. South African J. Psychiatry 2014;20:31-2.

7) Antkiewicz-Michaluk L,WąsikA, Możdżeń E, Romańska I , Michaluk J .Antidepressant-like Effect of Tetrahydroisoquinoline Amines in the Animal Model of Depressive Disorder Induced by Repeated Administration of a Low Dose of Reserpine: Behavioral and Neurochemical Studies in the Rat. Neurotox. Res. 2014;26:85-98.

8) Lee M-J, Wei J-W. The influences of reserpine and imipramine on the 5-HT2 receptor binding site and its coupled second messenger in rat cerebral cortex. Chin. J. Physiol. 2013;56:199-208.

9) Reckziegel P, Peroza LR, Schaffer LF, Ferrari MC, de Freitas CM, B ger ME, et al. Gallic acid decreases vacuous chewing movements induced by reserpine in rats. Pharmacol. Biochem. Behav. 2013;104:132-137.

10) Fernandes VS, Santos JR, Leao AHFF, Medeiros AM, Melo TG, Izidio GS, et al. Repeated treatment with a low dose of reserpine as a progressive model of Parkinson's disease. Behav. Brain Res. 2012;231:154-63.

11) Nedelcu L, Grapa D, Sandor V, Orbai P, Dumitrascu DL. Inhibition of Reserpine-InducedUlcersbyCalcitonininRats.Bull.Transilv.Univ.Braşov2011;4:21-6.

12) Okokon JE, Antia BS, Umoh EE. Antiulcerogenic activity of ethanolic leaf extract of Lasianthera africana. African J. Tradit. Complement. Altern. Med. 2009;6:150-4.

13) Zhou Y, Wang RUI, Feng XIA, Zhao XIN. Preventive effect of insect tea against reser-pine-induced gastric ulcers in mice. Exp. Ther. Med. 2014;8:1318-24.

14) Prejean C, Fitzhugh O., Belzer J, Brown I, Prejean CA, Thompson RB, et al. Bioassay of Reserpine for Possible carcinogenecity. Natl. Cancer Inst. Carcinog. 1982;1-121.

15) Anbiaee R, Mojir Sheibani K, Torbati P, Jaam H. Abnormal Expression of E-Cadherin in Gastric Adenocarcinoma, and Its Correlation With Tumor Histopathology and Helicobacter Pylori Infection. Iran. Red Crescent Med. J. 2013;15:218-22.

16) Jung HY, Cho H, Oh M, Lee J, Lee HJ, Jang S-H, et al. Loss of FAT Atypical Cadherin 4 Expression Is Associated with High Pathologic T Stage in Radically Resected Gastric Cancer. J. Gastric Cancer 2015;15:39-45.

17) Ebert MP, Fei G, Kahmann S, Muller O, Yu J, Sung JJ, et al. Increased beta-catenin mRNA levels and mutational alterations of the APC and beta-catenin gene are present in intestinal-type gastric cancer. Carcinogenesis 2002;23:87-91.

Expression of Genes in the Stomach6

18) Wang D, Chen Y, Pan K, Wang W, Chen S, Chen J, et al. Decreased Expression of the ARID1A Gene Is Associated with Poor Prognosis in Primary Gastric Cancer. PLoS One 2012;7:e40364.

19) de Souza CRT, Leal MF, Calcagno DQ, Costa Sozinho EK, Borges B do N, Montenegro RC, et al . MYC Deregulation in Gastric Cancer and Its Clinicopathological Implications. PLoS One 2013;8.

20) He C, Sun L, Xu Q, Liu J, Jiang J. PGC TagSNP and Its Interaction with H . pylori and Relation with Gene Expression in Susceptibility to Gastric Carcinogenesis. 2014;1-17.

21) Saitoh T, Mochizuki T, Suda T, Aoyagi Y, Tsukada Y, Narisawa R, et al. Elevation of TFF1 gene expression during healing of gastric ulcer at non-ulcerated sites in the stomach: Semiquantification using the single tube method of polymerase chain reac-tion. J. Gastroenterol. Hepatol. 2000;15:604-9.

22) Farrell JJ, Taupin D, Koh TJ, Chen D, Zhao CM, Podolsky DKM, et al. TFF2/SP-deficient mice show decreased gastric proliferation, increased acid secretion, and increased susceptibility to NSAID injury. J. Clin. Invest. 2002;109:193-204.

23) Konturek PC, Brzozowski T, Burnat G, Szlachcic A, Koziel J, Kwiecien S, et al. Gastric ulcer healing and stress-lesion preventive properties of pioglitazone are atten-uated in diabetic rats. J. Physiol. Pharmacol. 2010;61:429-36.

24) Ji CX, Fan DS, Li W, Guo L, Liang ZL, Xu RM, et al. Evaluation of the anti-ulcerogen-ic activity of the antidepressants duloxetine, amitriptyline, fluoxetine and mirtazapine in different models of experimental gastric ulcer in rats. Eur. J. Pharmacol. 2012;691:46-51.

25) Ma X, Lu G, Song S, Liu W, Wen Z, Zheng X, et al. The features of reserpine-induced gastric mucosal lesions. Acta Pharmacol. Sin. 2010;31:938-43.

26) Slotkin TA, Edwards K. Effects of reserpine on the content and properties of rat adrenal medullary storage vesicles. Biochem. Pharmacol. 1973;22:549-60.

27) Yamaguchi I, Hiroi J, Fuke H, Kumada S. Mechanism of gastric secretogogue effect of reserpine in rats. J. Pharmacol. Exp. Ther. 1978;205:71-7.

28) Salim AWSS. Mechanism of reserpine-induced acute gastric mucosal injury in the rat: role of endogenous 5-hydroxytryptamine. Clin. Sci. 1991;80:59-64.

29) Gumbiner BM. Cell adhesion: The molecular basis of tissue architecture and morpho-genesis. Cell1996;84:345-57.

30) Tycko B. Epigenetic gene silencing in cancer. J. Clin. Invest. 2000;105:401-7. 31) Strutt H, Strutt D. Nonautonomous planar polarity patterning in Drosophila: dishev-

elled-independent functions of frizzled. Dev. Cell 2002;3:851-63. 32) Yang C, Axelrod JD, Simon MA. Regulation of Frizzled by fat-like cadherins during

planar polarity signaling in the Drosophila compound eye. Cell 2002;108:675-88. 33) Axelrod JD. Progress and challenges in understanding planar cell polarity signaling.

Semin. Cell Dev. Biol. 2009;20:964-71. 34) Reddy BVVG, Irvine KD. The Fat and Warts signaling pathways: new insights into

their regulation, mechanism and conservation. Development 2008;135:2827-38. 35) Pan D. The hippo signaling pathway in development and cancer. Dev. Cell

2010;19:491-505. 36) Genevet A, Tapon N. The Hippo pathway and apico-basal cell polarity. Biochem. J.

2011;436:213-24. 37) Noubissi FK, Elcheva I, Bhatia N, Shakoori A, Ougolkov A, Liu J, et al. CRD-BP

mediates stabilization of [beta]TrCP1 and c-myc mRNA in response to [beta]-catenin signalling. Nature 2006;441:898-901.

38) Tetsu O, McCormick F. [beta]-Catenin regulates expression of cyclin D1 in colon car-cinoma cells. Nature 1999;398:422-6.

39) Wang K, Kan J, Yuen ST, Shi ST, Chu KM, Law S, et al. Exome sequencing identifies

frequent mutation of ARID1A in molecular subtypes of gastric cancer. Nat. Genet. 2011;43:1219-23.

40) Foltmann B. Structure and function of proparts in zymogens for aspartic proteinases. Biol. Chem. Hoppe. Seyler. 1988;369 Suppl:311-4.

41) Moore SA, Sielecki AR, Chernaia MM, Tarasova NI, James MN. Crystal and molecular structures of human progastricsin at 1.62 A resolution. J. Mol. Biol. 1995;247:466-85.

42) Sanchez LM, Freije JP, Merino AM, Vizoso F, Foltmann B, Lopez-Otin C. Isolation and characterization of a pepsin C zymogen produced by human breast tissues. J. Biol. Chem. 1992;267:24725-31.

43) Alvarez ML, Barbon JJ, Gonzalez LO, Lamelas ML, Vazquez J, Vizoso FJ. Expression of two androgen-induced proteins (pepsinogen C and apolipoprotein d) in epithelial skin cancers of the eyelids. Ophthalmol. J. Int. d'ophtalmologie. Int. J. Ophthalmol. Zeitschrift fur Augenheilkd. 2004;218:115-9.

44) Ning P-F, Liu H-J, Yuan Y. Dynamic expression of pepsinogen C in gastric cancer, pre-cancerous lesions and Helicobacter pylori associated gastric diseases. World J. Gastroenterol. 2005;11:2545-8.

45) Melle C, Ernst G, Schimmel B, Bleul A, Kaufmann R, Hommann M, et al . Characterization of pepsinogen C as a potential biomarker for gastric cancer using a histo-proteomic approach. J. Proteome Res. 2005;4:1799-804.

46) Kon OL, Yip T-T, Ho MF, Chan WH, Wong WK, Tan SY, et al. The distinctive gastric fluid proteome in gastric cancer reveals a multi-biomarker diagnostic profile. BMC Med. Genomics 2008;1:54.

47) Tansey WP. Mammalian MYC Proteins and Cancer. New J. Sci. 2014;2014:1-27. 48) Nesbit CE, Tersak JM, Prochownik E V. MYC oncogenes and human neoplastic dis-

ease. Oncogene 1999;18:3004-16. 49) Li Z, Van Calcar S, Qu C, Cavenee WK, Zhang MQ, Ren B. A global transcriptional

regulatory role for c-Myc in Burkitt's lymphoma cells. Proc. Natl. Acad. Sci. U. S. A. 2003;100:8164-9.

50) Otake Y, Soundararajan S, Sengupta TK, Kio EA, Smith JC, Pineda-Roman M, et al. Overexpression of nucleolin in chronic lymphocytic leukemia cells induces stabiliza-tion of bcl2 mRNA. Blood 2007;109:3069-75.

51) Zhang L, Hou Y, Ashktorab H, Gao L, Xu Y, Wu K, et al. The impact of C-MYC gene expression on gastric cancer cell. Mol. Cell. Biochem. 2010;344:125-35.

52) Schoonjans K, Staels B, Auwerx J. Role of the peroxisome proliferator-activated recep-tor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J.Lipid Res. 1996;37:907-25.

53) Sato H, Ishihara S, Kawashima K, Moriyama N, Suetsugu H, Kazumori H, et al. Expression of peroxisome proliferator-activated receptor (PPAR)gamma in gastric can-cer and inhibitory effects of PPARgamma agonists. Br. J. Cancer2000;83:1394-400.

54) Hoffmann W. Trefoil factors TFF (trefoil factor family) peptide-triggered signals pro-moting mucosal restitution. Cell. Mol. Life Sci. 2005;62:2932-8.

55) Feng G, Zhang Y, Yuan H, Bai R, Zheng J, Zhang J, et al. DNA methylation of trefoil factor 1 (TFF1) is associated with the tumorigenesis of gastric carcinoma. Mol. Med. Rep. 2014;9:109-17.

56) Hong SJ, Oh JH, Jung YC, Kim YH, Kim SJ, Kang SJ, et al. DNA methylation patterns of ulcer-healing genes associated with the normal gastric mucosa of gastric cancers. J. Korean Med. Sci. 2010;25:405-17.

57) Jiang P, Yu G, Zhang Y, Xiang Y, Zhu Z, Feng W, et al. Promoter hypermethylation and downregulation of trefoil factor 2 in human gastric cancer. Oncol. Lett. 2014;1525-31.

58) Halaris AE, Freedman DX. Loss of body weight as a predictor of reserpine-induced amine depletion. Eur. J. Pharmacol. 1975;32:93-101.