2nd International Conference on Environment, Agriculture and Food Sciences (ICEAFS'2013) August 25-26, 2013 Kuala Lumpur (Malaysia)

Protective Effects of [6]-Shogaol on Histological and Immunohistochemical Gene Expression in DMBA Induced Hamster Buccal Pouch Carcinogenesis

Suresh Kathiresan1*, Arokia Vijayaanand Mariadoss1, Rajasekar Muthusamy1, and Sivakumar Kathiresan2

Proteins of the bcl-2 family together with caspases have among Abstract- The present study designed to investigate the protective others been identified as essential components of the effects of [6]-shogaol on 7,12-Dimethylbenz (a)anthracene (DMBA) intracellular apoptotic signaling pathways [4]. Caspases are the induced buccal pouch carcinogenesis in male golden Syrian hamsters. heart of the apoptotic machinery. Several caspases have been Oral squamous cell carcinoma was developed in left buccal pouch of shown to be key executioners of apoptosis, among the family hamster by painting with 0.5% of DMBA, three times in a week. We observed an altered status of apoptotic associated gene expression of caspase; Caspase-3, a key factor in apoptosis execution and (P53, bcl-2, Caspase-3 and TNF-α) were observed in the DMBA alone the activation of caspase-3 is crucial for mitochondrial painted hamsters as compared to control hamsters. Oral dependent and independent apoptotic pathways [5]. TNF-α has administration of [6]-shogaol at a dose of 20 mg/kg b.wt to DMBA been described as a powerful anti-cancer effectors and cytokine treated animals on alternative days for 14 weeks significantly reduced produced by immune cells such as macrophages and the neoplastic changes and improved the status of apoptosis lymphocytes. It mediates tumor cell killing primarily by associated gene expression. These observations confirmed that [6]- shogaol not only suppress the neoplastic changes but also effectively apoptosis [6]. enhance the apoptotic associated gene expression in DMBA treated Many medicinal plants based chemopreventive agents are animals. rich in antioxidant phytochemicals, recognized to exert their anti-carcinogenic effects by inhibiting cell proliferation, Keywords- [6]-shogaol, Apoptosis, DMBA, Hamster. inducing cell differentiation and apoptosis [7]. The pungent phenolic constituents derived from are believed to INTRODUCTION possess many interesting pharmacological and physiological POPTOSIS has become a major research area in the activities. [6]-shogaol [1-4-hydroxy-3-methoxyphenyl-4-decen- Abiomedical sciences. It is also termed programmed cell 3- one) is one of the major biologically active compounds death, plays diverse roles in embryogenesis and normal found in the rhizome of Zingiber officinale [8]. The available homeostasis, as well as in tumorigenesis [1]. The apoptotic literature also documented that the [6]-shogaol shows it anti- processes are executed by a number of factors, which have hepatotoxic effect against galactosamine induced cytotoxicity inhibitory or stimulatory effects [2]. p53, tumor suppressor in primary cultured rat hepatocytes and protects against LPS gene, product is a multifunctional molecule that influences the induced inflammation in macrophage cells [9]. Pan et al., cell cycle, DNA repair and apoptosis by regulating (2008) reported that [6]-shogaol inhibits the growth of human transcription and interacting directly with other proteins [3]. cancer cells and induces apoptosis in COLO 205 cells through modulation of mitochondrial functions regulated by reactive oxygen species [10]. However, no scientific reports were Suresh Kathiresan1 is with the Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar-608 002, Tamilnadu, available on literature about apoptosis associated genes of p53, India. (Tel.: +91-4144-239141(Extn.*209);Telefax:+91-4144-238080;e-mail: bcl-2, caspase-3 and TNF-α gene regulation of [email protected] [6]-shogaol on DMBA induced carcinogenesis in male golden Arokia Vijayaanand Mariadoss1 is with the Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar-608 002, Syrian hamsters. The present study is designed to screen the Tamilnadu, India. (e-mail:[email protected]) protective role of [6]-shogaol assessed by histological Rajasekar Muthusamy1 is with the Department of Biochemistry and examinations and immunohistochemical techniques on DMBA Biotechnology, Annamalai University, Annamalai Nagar-608 002, Tamilnadu, India. (e-mail:[email protected]) induced carcinogenesis. Sivakumar Kathiresan2 is with the Department of Botany, Annamalai University, Annamalai Nagar-608 002, Tamilnadu, India. (e- mail:[email protected])

77 2nd International Conference on Environment, Agriculture and Food Sciences (ICEAFS'2013) August 25-26, 2013 Kuala Lumpur (Malaysia)

II. MATERIALS AND METHODS [1994] as follows: +++=strong staining, more than 50% of cells were stained; ++=moderate staining, between 20 and 50% A. Animals And Experiential Design of cells were stained; +=week staining, between 1 and 20% of Male golden Syrian hamsters (80 ± 120 g), were obtained cells were stained; 0=negative, less than 1% of cell staining. from National Institute of Nutrition, Hyderabad, India. The experiments were designed and conducted in accordance with III. RESULTS the institutional guidelines. Hamsters were divided into 4 Fig. 1 and Table 1 show the tumor incidence, tumor volume groups of 10 each. Group I, served as an untreated control. The and tumor burden of hamsters in all experimental groups along Group II and Group III were painted with 0.5% DMBA in with the histopathological features. In Group II animals liquid paraffin three times a week for 14 weeks on the left (DMBA alone treated), we have observed 100% tumor buccal pouches using (No. 4 brush) to induce the buccal pouch formation with mean volume (98.12 ± 9.43), tumor burden carcinogenesis. Group II received no other treatment. Group (1601±161.1). For histopathological confirmation; we noticed III animals were orally administered with [6]-shogaol (20 severe keratosis, hyperplasia, dysplasia and oral neoplasm. In mg/kg b.wt; dissolved in 0.5% DMSO) starting one week Group III (DMBA+ [6]-shogaol) we noticed mild before the exposure to the carcinogen and continuing on preneoplastic lesions like mild dysplasia, keratosis and alternate days of the DMBA painting until the animals were hyperplasia which indicated the key role of [6]-shogaol in sacrificed. regaining the antioxidant status. The buccal pouch of Group I B. Sample Collections (Control) and Group IV ([6]-shogaol alone) animals resembles the same with no tumor formation and preneoplastic lesions. At the end of the experimental period, the hamsters were sacrificed by cervical decapitation under anesthetic conditions (Ketamin 30 mg/kg, i.p.). The control and experimental animal buccal tissues were immediately removed, washed using ice- cold phosphate buffer solution (pH 7.4), and then the buccal tissues were divided for assessment of histological and immunohistochemical studies. C. Tumor Study The left sides of the control and experimental animal’s buccal pouch was excised and flattened on a transparency. Visible tumors in the oral cavity were counted and the diameter Fig.1 Gross appearance of oral squamous cell carcinoma in DMBA of each tumor was measured with a vernier caliper. painted hamsters

D. Histopathological Evaluation TABLE I TUMOR INCIDENCE, VOLUME, BURDEN AND HISTOPATHOLOGICAL Histological slides were prepared by according to the CHANGES IN THE BUCCAL POUCHES OF DMBA PAINTED GOLDEN SYRIAN method of Ramos Vara, (2005). The buccal pouches of the HAMSTERS hamsters were fixed in 10% buffered and Parameters Control DMBA DMBA + [6]-shogaol dehydrated in rising concentrations of ethanol and embedded [6]-shogaol alone in paraffin. 5 µm thickness of tissue sections were mounted on Tumour incidence 0 100% 14% 0 frosted glass slides and dried for overnight. The sections were Total no of then deparaffinized with xylene, rehydrated with alcohol and tumour/animal 0 27(6) 3(2) 0 Tumour volume (mm3) 0 98.4±9.43 71.4±6.33 0 water. The rehydrated sections were stained using H&E and Tumour burden (mm3) 0 1601.7± 17.6 743.1±67.1 0 viewed under the microscope 40X magnifications (Olympus Keratosis Not observed Severe Moderate Not observed BX51 microscope, Tokyo, Japan). Hyperplasia Not observed Severe Moderate Not observed Dysplasia Not observed Severe Mild Not observed Squamous cell Not observed Well --- Not observed E. Immunohistochemical Studies carcinoma differentiated Monoclonal anti-mouse p53 antibody, monoclonal anti- mouse bcl-2 antibody and monoclonal anti-mouse caspases-3 antibody (Sigma Aldrich Inc., Santa Cruz, CA) and the anti- mouse -peroxidase conjugated secondary antibodies were obtained from Biogenex, San Ramon, CA. To examine the expression patterns and inter correlation between apoptosis related molecular markers p53, bcl-2, caspase-3 and TNF-α were prepared by according to the method of Cotran et al., (1994) [11].

F. Statistical Analysis The percentage of positive cells in immunohistochemical were scored according to the method of Nakagawa et al., Fig. 2 Histopathological changes observed in the control and experimental animals

78 2nd International Conference on Environment, Agriculture and Food Sciences (ICEAFS'2013) August 25-26, 2013 Kuala Lumpur (Malaysia)

(A). Microphotograph of untreated control animals (Group I), picturising normal Hamster buccal pouch carcinogenesis (HBP) is an excellent epithelium in buccal mucosa. (B). Microphotograph of DMBA alone treated animals (Group II), picturising well defined squamous cell carcinoma with hyper chromatic nuclei model to study oral carcinogenesis, for the reason, that the containing epithelial and keratin pearls. (C). Microphotograph of DMBA+ [6]-shogaol development of DMBA induced squamous cell carcinoma in treated animals (Group III), picturising mild to moderate dysplasia and hyperplasia. (D). Microphotograph of [6]-shogaol alone treated animals (group IV), picturising normal HBP simulates many of the structural and biological alterations epithelium in buccal mucosa. that occur in its human counterpart [13]. These evaluations are likely to be helpful in identifying intermediate biomarkers that TABLE II would help in monitoring clinical trials or evaluating drug THE INTENSITY OF STAINING OF P53, BCL-2, CASPASE-3 AND TNF-Α EXPRESSION IN THE BUCCAL POUCH OF CONTROL AND EXPERIMENTAL effect measurements. This study was designed to investigate ANIMALS the ability of [6]-shogaol to modulate expression of the apoptosis related proteins which are implicated in DMBA induced hamster buccal pouch carcinogenesis, including p53, bcl-2 caspase-3 and TNF-α. Recent study have been documented that p53 most frequent genetic alteration observed in carcinomas. In particular, a high incidence of p53 mutation has been demonstrated in many experimental carcinogenesis including oral cancer [14]. In the

present investigation, we noticed significant expression of Fig.3 and Table 2 show the immunohistochemial gene mutant p53 in buccal tissues of DMBA treated hamsters which expression of p53, bcl-2, caspase-3 TNF-α in control and may be due to the significant molecular alterations at p53 gene. experimental animals of each group. The presence of cells with Moreover, p53 is implicated in the induction of two distinct clear and unequivocal staining identified in p53 positive cells. apoptotic signaling pathways that lead to the activation of the Bcl-2 cells were identified as diffused golden yellow colour in caspases which mediate apoptosis. The extrinsic pathway cytoplasmic staining and caspase-3 were identified as red involves engagement of particular death receptors that belong brown in color. The positive cells of TNF-α predominantly to the TNF receptor family and, leads to a cascade of activation were located with brown yellow color in cytoplasm. We of caspases, including caspase-3, which in turn induce observed a significant and sequential raise in protein levels by apoptosis [15]. The finding of this study conclude that oral p53, bcl-2, TNF-α and down regulation of caspase-3 gene administration of [6]-shogoal decreased p53 level to improve expression were observed in DMBA alone treated hamsters as apoptosis process. compared to control hamsters. Oral administration of [6]- There are various signaling pathways of apoptosis within an shogaol at a dose of 20mg/kg b.wt effectively restored the organism, of which the mitochondrial pathway is one of the expression pattern of p53, bcl-2, caspase-3 and TNF-α. No most important. Bcl-2 family proteins are key regulatory significant changes were observed in expression of p53, bcl-2, factors of the mitochondrial pathway. Several studies have TNF-α, and caspase-3 in control and [6]-shogaol alone treated shown that overexpression of bcl-2 prevents the mitochondrial animals. release of cytochrome c, thereby inhibiting the activation of caspases cascade and apoptosis [16]. Over expression of antiapoptotic proteins such as bcl-2 may contribute to tumor formation in DMBA-treated animals. We monitored well defined expression of bcl-2 gene in buccal tissues of DMBA treated hamsters which may be due to the enhanced inhibition of release of cytochrome C by bcl-2 gene. Oral administration of [6]-shogaol reduced the expression of bcl-2 in DMBA painted hamsters which may be due to the reduced release of cytochrome C from mitochondria and increasing the expression of apoptotic gene. The results of the present study validate our conviction that DMBA causes carcinogenesis via up regulation Fig. 3 Immunohistochemical expression pattern of P53, bcl-2, of p53, down regulation of bcl-2 levels and inducing Caspase-3 and TNF-α in buccal mucosa of control and experimental mitochondrial damage. Further, [6]-shogaol at a dose of 20 animals in each group mg/kg b.wt prevented the up regulation of Bax and maintained (A). Shows the normal expression of p53 bcl-2, caspase-3 and TNF-α genes in buccal tissues of control hamsters. (B). Shows the well defined oral squamous cell carcinoma with mitochondrial integrity hence, protected the DMBA induced the altered expression of p53, bcl-2, caspase-3 and TNF-α in buccal tissues of DMBA carcinogenic process in experimental animals. Thus, these treated hamsters. (C). Improved the expression of p53, bcl-a, caspase-3 and TNF-α genes observations hold promise for further molecular target oriented in [6]-shogaol and DMBA treated hamsters. (D). Normal expression of p53, bcl-2, caspase-3 and TNF-α in buccal tissues of [6]-shogaol alone treated hamsters. studies. Caspase is an important apoptosis signaling molecule that IV. DISCUSSION triggers a cascade of molecular interaction leading to Molecular and immunohistochemical markers have been apoptosis. Studies have shown that the extrinsic activation found to be useful in characterizing the progression of human triggers the hallmark Caspase cascade of apoptotic pathway, in cancer. These markers include changes in several genes that which caspase-3 plays a dominant role [17]. By inducing the encode the expression of cell proliferation regulators [12]. release of mitochondrial cytochrome c, p53 may be able to

79 2nd International Conference on Environment, Agriculture and Food Sciences (ICEAFS'2013) August 25-26, 2013 Kuala Lumpur (Malaysia) activate effector caspases, including caspase-3. Activation of [5] S.A. Lakhani, A. Masud, K. Kuida, G.A. Porter, C.J. Booth, W.Z. caspase-3 subsequently leads to apoptotic cell death through Mehal, I. Inayat, R.A Flavell, “Caspases 3 and 7: key mediators of mitochondrial events of apoptosis”, Science, vol. 311, pp. 847-851, Feb cleavage of a broad spectrum of target proteins [18]. 2006. [6]-shogaol may induce apoptosis mediated by p53 through the [6] M.E. Peter, P.H. Krammer, “The CD95(APO-1/Fas) DISC and beyond”, down regulation of bcl-2 and up regulation of Bax. In this Cell Death Differ, vol. 10, pp. 26-35, Jan 2003. study we were seen a significant increase in caspase-3 [7] Y. Shukla, M. Singh,“Cancer preventive properties of ginger: a brief review”, Food Chem Toxicol, Vol. 45, pp. 683-690, May 2007. expression in [6]-shogaol treated animals. 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Dixit, “Death receptors: signaling and modulation”, compound extracted from cruciferous vegetables suppresses Science, vol. 281, pp. 1305-1308, 1998. [16] T. Kuwana, D.D. Newmeyer, “Bcl-2-family proteins and the role of TNF-a-induced NF-kB activity and induces apoptosis through mitochondria in apoptosis” Curr Opin Cell Biol, vol. 15, pp. 691-699, activation of ROS-dependent caspase-3[22]. Our findings was Dec 2003. agreed this observation. Oral administration of [6]-shogaol [17] M. Leist, M. Jaattela, “Four deaths and a funeral: from caspases to significantly reduced the expressions of TNF-α in DMBA alternative mechanisms” Nat Rev Mol Cell Biol, vol. 2, pp.589-598, Aug 2001. induced hamster and shows its anti-inflammsatory activity. [18] P.U. 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