67.Modulatory effects of black tea polyphenols on oxidant-antioxidant profile and

J Gastroenterol 2007; 42:352–361DOI 10.1007/s00535-007-2018-zReceived: October 31, 2006 / Accepted: January 29, 2007Reprint requests to:S. NaginiModulatory effects of black tea polyphenols on oxidant–antioxidant profi le and expression of proliferation, apoptosis, and angiogenesis-associated proteins in the rat forestomach carcinogenesis modelR amalingam S enthil M urugan 1, K urapathy V enkata P oorna C handra M ohan 1, K oji U chida 2,Y ukihiko H ara 3, D uvuru P rathiba 4, and S iddavaram N agini 11 Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India 2Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan 3Mitsui Novin Co., Ltd., Tokyo, Japan 4Department of Pathology, Sri Ramachandra Medical College and Research Institute, Chennai, Indiadence that Polyphenon-B exerts multifunctional inhibi-tory effects on MNNG-induced gastric carcinogenesis and suggests that it can be developed as a potential chemopreventive agent.Key words: apoptosis, angiogenesis, antioxidants, black tea polyphenols, cell proliferation, MNNGIntroductionGastric cancer, the second leading cause of cancer death worldwide, is associated with alterations in oxidant–antioxidant status, increased cell proliferation and angiogenesis, and dysregulation of apoptosis.1,2 Scaven-gers of reactive oxygen species and proteins that play an important role in cell proliferation, angiogenesis, and apoptosis are considered surrogate end-point biomark-ers of carcinogenesis as well as of chemoprevention.Chemoprevention is a practical strategy to control gastric cancer. Many dietary agents are recognized to exert their anticarcinogenic effects by modulating the cellular redox status, inhibiting cell proliferation and angiogenesis, and inducing apoptosis.3 However, it is important to establish chemoprevention in an experi-mental model of gastric carcinogenesis before embark-ing on clinical trials. Gastric cancer induced by N -methyl-N ′-nitro-N -nitrosoguanidine (MNNG) in Wistar rats is an ideal model for chemoprevention studies. We have used this model to document the che-mopreventive effi cacies of several medicinal plants anddietary agents.4,5Several studies have demonstrated a positive correla-tion between consumption of vegetables, fruits, and beverages with reduced risk of cancer.3,6 Recent re-search interest has focused on tea made from the leaves of Camellia sinensis , one of the most popular beverages consumed worldwide.6 About 3 billion kilograms of teaBackground. Chemoprevention by dietary constitu-ents has emerged as a novel approach to control stomach cancer incidence. We therefore evaluated the chemopreventive effects of black tea polyphenols (Polyphenon-B) on oxidant–antioxidant status, cell proliferation, apoptosis, and angiogenesis during N -methyl-N ′-nitro-N -nitrosoguanidine (MNNG)-induced gastric carcinogenesis. Methods. Male Wistar rats were divided into four groups. Rats in group 1 and 2 were given MNNG (150 mg/kg body weight) by intragastric intubation three times at 2 week intervals and followed for 26 weeks. Rats in group 2 received in addition a basal diet containing 0.05% Polyphenon-B. Group 3 animals were given 0.05% Polyphenon-B alone. Group 4 animals served as controls. The status of lipid peroxi-dation and antioxidants and the expression of the lipid peroxidation marker 4-hydroxy nonenal (4-HNE), proliferating cell nuclear antigen (PCNA), glutathiones-transferase (GST)-π, Bcl-2, Bax, cytochrome C, caspase 3, cytokeratins, and vascular endothelial growth factor (VEGF) were used as biomarkers. Results. Intragastric administration of MNNG induced well-differentiated squamous cell carcinomas that showed diminished lipid and protein oxidation and an increase in antioxidant status. This was associated with increased cell prolifera-tion, angiogenesis, and invasive potential coupled with apoptosis evasion as revealed by upregulation of PCNA, GST-π, Bcl-2, cytokeratins, and VEGF and downregu-lation of Bax, cytochrome C, and caspase 3 protein expression. Dietary administration of Polyphenon-B effectively suppressed MNNG-induced gastric carcino-genesis, as evidenced by modulation of oxidant–antioxidant status, inhibition of cell proliferation and infi ltration, and angiogenesis associated with apoptosis induction. Conclusions.The present study provides evi-are produced and consumed annually, of which black tea accounts for 78%. Both green and black tea have been documented to protect against chronic diseases such as cardiovascular disease and cancer.7Of late, black tea has attracted the focus of attention as a potential chemopreventive agent. Black tea has been reported to exhibit a wide range of pharmacologi-cal effects, including antioxidant, antiproliferative, anti-infl ammatory, and immunomodulatory properties.8 Recently, we have demonstrated the greater effi cacy of black tea compared with green tea polyphenols in the hamster buccal pouch carcinogenesis model.9 The pres-ent study was designed to evaluate the chemopreven-tive effi cacy of black tea polyphenols (Polyphenon-B) on MNNG-induced gastric carcinogenesis. The extent of lipid and protein oxidation and the status of the antioxidants superoxide dismutase (SOD),catalase (CAT), reduced glutathione (GSH), and GSH-dependent enzymes in the stomach were used to bio-monitor chemoprevention. In addition, the expression of markers of lipid peroxidation [4-hydroxy nonenal (4-HNE)]; proliferation [proliferating cell nuclear anti-gen (PCNA) and glutathione S-transferase (GST)-π]; invasion [cytokeratins (CKs)]; angiogenesis [vascular endothelial growth factor (VEGF)]; and apoptosis [Bcl-2, Bax, cytochrome C, and caspase 3] were analyzed in the stomach tissue by immunohistochemical localiza-tion, and the activity of caspase 3 was assayed by a colo-rimetric method.Materials and methodsChemicalsBovine serum albumin, 2-thiobarbituric acid, 2,4-dini-trophenylhydrazine, GSH, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), nicotinamide adenine dinucleotide phosphate reduced (NADPH), and 3,3′-diaminobenzi-dine were purchased from Sigma (St. Louis, MO, USA). MNNG was obtained from Fluka-Chemika-Biochemika (Buchs, Switzerland). Polyphenon-B was kindly pro-vided by Mitsui Norin (Tokyo, Japan). The composition of Polyphenon-B is the same as described previously.9,10 It is a mixture of epicatechin (0.4%), epigallocatechin-3-gallate (1.4%), epicatechin-3-gallate (0.1%), gallo-catechin-3-gallate (0.2%), free theafl avins (0.32%), theafl avinmonogallate-A (0.14%), theafl avinmonogal-late-B (0.15%), theafl avindigallate (0.21%), tannin (35.6%), and caffeine (4.9%). All other reagents used were of analytical grade.AnimalsAll of the experiments were carried out with male Wistar rats, aged 6–8 weeks and weighing 90–110 g, ob-tained from the Central Animal House, Rajah Muthiah Institute of Health Sciences, Annamalai University, In-dia. They were housed fi ve to a polypropylene cage and provided food and water ad libitum. The animals were maintained in a controlled environment under standard conditions of temperature and humidity with an alter-nating 12-h light/dark cycle. The animals were main-tained in accordance with the guidelines of the Indian Council of Medical Research and approved by the ethi-cal committee of Annamalai University. Experimental diet was prepared every day by mixing Polyphenon-B into preweighed standard pellet diet (Mysore Snack Feed, Mysore, India) at a concentration of 0.05%. The diet was replenished everyday, and food consumption was recorded.Treatment scheduleThe animals were randomized into experimental and control groups and divided into four groups of ten ani-mals each. Rats in group 1 were given MNNG (150 mg/ kg body weight) by intragastric intubation three times at 2-week intervals.11Rats in group 2 administered MNNG as in group 1 and received in addition 0.05% Polyphenon-B in the diet, starting on the day following the fi rst exposure to MNNG and continued until the end of the experimental period. Group 3 animals were administered 0.05% Polyphenon-B alone in the diet as in group 2 but without MNNG. Group 4 received the basal diet and tap water throughout the experiment and served as untreated controls. The dose of Polyphenon-B used in the present study corresponds to the daily dietary intake of four cups of tea (30–40 mg of tea polyphenols per kilogram body weight for humans).12The experiment was terminated at 26 weeks, and all animals were killed by cervical dislocation after an overnight fast. The stomach tissues were subdivided and variously processed for distribution to each experiment.Preparation of tissue homogenateFresh tissues were used for biochemical estimations. The stomach tissues after weighing were homogenized in an all glass homogenizer with Tefl on pestle and stored on ice until use.HistopathologyTissues were immediately fi xed in 10% neutral buffered formalin, embedded in paraffi n, sectioned, mounted on polylysine-coated slides, and stained with hematoxy-lin and eosin. Basal cell hyperplasia, dysplasia, and squamous cell carcinoma (SCC) were diagnosed.Hyperplasia of the forestomach epithelium was indi-cated by an increased number of basal cells. Irregular epithelial stratifi cation, increased number of mitotic fi gures, increased nuclear-to-cytoplasmic ratio, and loss of polarity of basal cells characterized the dysplastic lesions. SCC was diagnosed by the invasion of underly-ing tissues, nuclear pleomorphism, and increased mitoses.ImmunohistochemistryThe tissue sections were deparaffi nized by heat at 60°C for 10 min, followed by three washes in xylene. After gradual hydration through graded alcohol, the slides were incubated in citrate buffer (pH 6.0) for two cycles of 5 min each in a microwave oven for antigen retrieval. The sections were allowed to cool for 20 min and then rinsed with Tris-buffered saline (TBS). The sectionswere treated for 15 min with 3% H2O2in distilled waterto inhibit endogenous peroxidase activity. Nonspecifi c antibody binding was reduced by incubating the sec-tions with normal goat serum for 25 min. The sections were then incubated with PCNA, cytokeratin AE1/ AE3, or Bcl-2 mouse monoclonal antibodies (Dako, Carpinteria, CA, USA); caspase3, Bax, or GST-π rabbit polyclonal antibodies or cytochrome C mouse monoclo-nal antibody (NeoMarkers, Fremont, CA, USA and BioGenex, San Ramon, CA, USA); 4-HNE mouse monoclonal antibody (generously provided by Dr. Koji Uchida, Laboratory of Food and Biodynamics, Gradu-ate School of Bioagricultural Sciences, Nagoya Univer-sity, Nagoya, Japan), or VEGF mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. The slides were washed with TBS and then incubated with anti-rabbit and anti-mouse biotin-labeled secondary antibody (Dako) followed by streptavidin-biotin-peroxidase for 30 min at room tem-perature. The immunoprecipitate was visualized by treating with 3,3′-diaminobenzidine and counterstain-ing with hematoxylin. For negative controls, the primary antibody was replaced with TBS. A positive control was also processed simultaneously for each antibody. The labeling index for PCNA and the data for GST-π, 4-HNE, CKs, Bcl-2, Bax, cytochrome C, caspase 3, and VEGF were expressed as the number of cells with posi-tive staining per 100 counted cells.Colorimetric estimation of caspase 3 activityDEVD-specifi c caspase 3 activity was assayed using a CASP-3-C colorimetric kit (Sigma) according to the manufacturer’s instructions. Cytosolic extracts were prepared by homogenizing tissues in lysis buffer con-taining 50 mM HEPES (pH 7.4), 5 mM CHAPS, and 5 mM dithiothreitol. The supernatant was collected as an enzyme source. The caspase 3 colorimetric assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-nitroanilide (Ac-DEVD-pNA) by caspase 3, resulting in release of the p-nitroaniline (pNA) moiety. The concentration of pNA released from the substrate was calculated from the absorbance values at 405 nm or from a calibration curve prepared with defi ned pNA solutions.Biochemical assaysLipid peroxidation was estimated as evidenced by the formation of thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH), and conju-gated dienes (CD). TBARS were assayed in the stomach tissue by the method of Ohkawa et al.13 LOOH were estimated by the method of Jiang et al.14 and CD by the method of Rao and Recknagel.15 Protein oxida-tion was measured by the method of Levine et al.16 based on the reaction of the carbonyl group with 2,4-dinitrophenylhydrazine to form 2,4-dinitrophenylhy-drazone. Total SOD and Mn-SOD activities were assayed as described by Oberley and Spitz17based on the half-maximal inhibition of nitroblue tetrazolium (NBT) reduction. Cu-Zn SOD activity was calculated by deducting the activity of Mn-SOD from total SOD activity. The activity of CAT was assayed by the method of Sinha,18 based on the utilization of hydrogen peroxide by the enzyme. GSH was determined by the method of Anderson19by measurement of the yellow color that develops when DTNB is added to compounds contain-ing sulfhydryl groups. Oxidized glutathione (GSSG) was estimated following oxidation of NADPH by glu-tathione reductase at 340 nm according to the method of Anderson.19 Selenium-dependent glutathione peroxi-dase (Se-GPx) activity was assayed by following the utilization of hydrogen peroxide according to the meth-od of Rotruck et al.20 Se-independent GPx activity was assayed following the method described by Lawrence and Burk21using cumene peroxide as substrate. The activity of GST was determined as described by Habig et al.22The protein content was estimated by the method of Lowry et al.23 with bovine serum albumin as standard.Statistical analysisThe data are expressed as means ± standard deviation (SD). The data for body weight, tumor burden, immu-nohistochemical analysis, colorimetric assay of caspase 3, and biochemical assays were analyzed using analysis of variance (ANOVA) followed by the least signifi cant difference test (LSD). The results were considered sta-tistically signifi cant if the P value was <0.05.R.S. Murugan et al.: Chemopreventive potential of black tea polyphenols355ResultsTumor incidence and histopathological observations Table 1 shows the mean body weight, food consump-tion, and preneoplastic and neoplastic lesions in experi-mental and control animals. Rats in group 1 showed atendency to have less body weight gain during the ex-periment, and the mean fi nal body weights were signifi -cantly decreased compared with controls (group 4). Treatment with Polyphenon-B of MNNG-treated ani-mals signifi cantly increased the mean fi nal body weight in group 2 animals compared with those in group 1. No signifi cant differences in body weight were observed between groups 3 and 4. The amount of diet consumed by groups 1 to 4 was not signifi cantly different. In rats administered MNNG alone (group 1), the incidence of gastric tumors was 100% (10/10 animals) with a mean tumor burden of 95.57 mm 3. No tumors were observed in groups 2 to 4. Forestomach tumors induced by MNNG were SCCs, with a number of epithelial keratin pearls and extensive infi ltration. Tumor cells showed an in-creased nuclear/cytoplasmic ratio, nuclear pleomor-phism, and hyperchromatism. Two of the ten animals treated with MNNG and Polyphenon-B (group 2) showed moderate keratosis and mild dysplasia, while the remainder exhibited normal keratinized and strati-fi ed squamous epithelial with mild to moderate hyper-plasia of the epithelium lining. The forestomach of rats in groups 3 and 4 showed normal lining of keratinized stratifi ed squamous epithelium. Representative photo-micrographs of histopathological changes observed in the forestomach of control and experimental animals are shown in Fig. 1.Immunohistochemical fi ndingsTable 2 and Fig. 2 show the effect of Polyphenon-B on the PCNA labeling index and expression of GST-π, 4-HNE, Bcl-2, Bax, cytochrome C, caspase 3, CKs, and VEGF in the stomach mucosa of experimental and con-trol animals. In animals administered MNNG (group 1), the expression of PCNA, GST-π, CKs, Bcl-2, and VEGF was signifi cantly higher and that of 4-HNE, Bax, cyto-chrome C, and caspase 3 signifi cantly lower compared with control animals (group 4). Administration of Polyphenon-B (group 2) signifi cantly decreased PCNA, GST-π, Bcl-2, CKs, and VEGF expression and signifi -cantly increased the expression of 4-HNE, Bax, cyto-chrome C, and caspase 3 compared with group 1. No signifi cant changes in the expression of these proteins were observed in group 3 animals compared with controls. All markers used in the present study are ex-pressed only in epithelial cells, and none of them showed expression in mesenchymal cells. While immunostain-T a b l e 1. B o d y w e i g h t , f o o d c o n s u m p t i o n , t u m o r i n c i d e n c e , a n d h i s t o p a t h o l o g i c a l c h a n g e s i n c o n t r o l a n d e x p e r i m e n t a l a n i m a l s (m e a n ± S D ; n = 10)F o o d T u m o r S q u a m o u sB o d y w e i g h t (g ) c o n s u m e d T u m o r T u m o r b u r d e n b c e l l G r o u p T r e a t m e n t I n i t i a l F i n a lg /r a t p e r d a y i n c i d e n c e m u l t i p l i c i t y a (m m 3) K e r a t o s i s H y p e r p l a s i a D y s p l a s i a c a r c i n o m a1. M N N G 99.99 ±2.62 121.93 ± 9.57* 10.90 ± 1.95 10/10 (100) 2.90 ± 1.19 95.57 ± 22.38 +++ (10/10) +++ (10/10) +++ (10/10) 10/10 (100)2. M N N G +P -B 96.95 ± 2.46 139.07 ± 8.09** 10.63 ± 1.82 — — — ++ (2/10) + t o ++ (8/10) + (2/10) —3. P -B 95.33 ± 3.17 145.17 ± 7.85 11.50 ± 1.93 — — — — — — —4. C o n t r o l 95.54 ± 3.28 143.64 ± 7.27 12.01 ± 2.10 — — — — — — —N o . o f a n i m a l s i s i n p a r e n t h e s e sM N N G , N -m e t h y l -N ′-n i t r o -N -n i t r o s o g u a n i d i n e ; P -B , P o l y p h e n o n -B ; +, m i l d ; ++, m o d e r a t e ; +++, s e v e r e ; —, n o c h a n g e ; A N O V A , a n a l y s i s o f v a r i a n c e ; L S D , l e a s t s i g n i fi c a n t d i f f e r e n c e a N u m b e r o f t u m o r s p e r r a t b C a l c u l a t e d b y m u l t i p l y i n g t h e m e a n t u m o r v o l u m e (4/3πr 3) b y t h e m e a n n u m b e r o f t u m o r s (r = 1/2 t u m o r d i a m e t e r i n m m )* S i g n i fi c a n t l y d i f f e r e n t f r o m g r o u p 4 (P < 0.05), A N O V A f o l l o w e d b y L S D t e s t ** S i g n i fi c a n t l y d i f f e r e n t f r o m g r o u p 1 (P < 0.05), A N O V A f o l l o w e d b y L S D t e s t356R.S. Murugan et al.: Chemopreventive potential of black tea polyphenolsB CADFig. 1A–D.Photomicrographs of histopathological changes in the stomach tissues of control and experimental animals (hema-toxylin and eosin, ×10). A Squamous cell carcinoma with extensive infi ltration in group 1 rats. B Mild dysplasia in the forestomach epithelium of rats in group 2. C Stomach epithelium showing normal histology in group 3 rats. D Stomach tissue showing nor-mal keratinized stratifi ed squamous epithelium in group 4 rats. Group 1, MNNG; group 2, MNNG + Polyphenon-B; group 3, Polyphenon-B; group 4, controling of PCNA showed nuclear localization, GST-π, 4-HNE, CKs, Bcl-2, Bax, cytochrome C, caspases 3, and VEGF were found in the cytoplasmic region.Caspase 3 activityFigure 3 illustrates the Bax/Bcl-2 ratio and activity of DEVD-specifi c caspase 3 in the stomach of control and experimental animals. In MNNG-treated animals (group 1), the Bax/Bcl-2 ratio and caspase 3 activities were signifi cantly reduced compared with controls (group 4). Treatment with Polyphenon-B signifi cantly increased the Bax/Bcl-2 ratio and enzyme activity in group 2 animals compared with group 1. In animals ad-ministered Polyphenon-B alone (group 3), the Bax/Bcl-2 ratio and activity of caspase 3 were not signifi cantly different from those in controls.Biochemical fi ndingsTable 3 shows the effect of treatment with Polyphenon-B on lipid peroxidation (as evidenced by TBARS, LOOH, and CD) and protein oxidation (as evidenced by formation of protein carbonyl) in the stomach. The extent of lipid and protein oxidation was signifi cantly lower in MNNG-treated rats (group 1) than in controls. Treatment with Polyphenon-B (group 2) signifi cantly decreased MNNG-induced lipid and protein oxidation in the stomach compared with group 1. Dietary admin-istration of Polyphenon-B alone signifi cantly reduced lipid and protein oxidation in group 3 animals compared with controls.The activities of the antioxidant enzymes SOD (total SOD, MnSOD, and Cu-Zn SOD), catalase, and GSH-dependent enzymes (GPx and GST), as well as the GSH/GSSG ratio in the stomach tissue of experimental and control animals, are presented in Fig. 4. In MNNG-treated animals (group 1), the activities ofACEEEEEEEEEPCNA (x10)CAGST-π (x40)CABcl-2 (x40)ACCytokeratins (x20)ACVEGF (x40)DB4-HNE (x40)BDBax (x40)DBCytochrome C (x40)BDCaspase 3 (x40)Fig. 2A–E. Representative photomicrographs of immunohistochemical staining of proliferating cell nuclear antigen (PCNA ), glutathione S -transferase (GST )-π, 4-hydroxy nonenal (4-HNE ), Bcl-2, Bax, cytochrome C, caspase 3, cytokeratins, and vascular endothelial growth factor (VEGF ) expression in experimental and control animals (mean ± SD; n = 10). A Overexpression of PCNA, GST-π, Bcl-2, cytokeratins, and VEGF in animals administered N -methyl-N ′-nitro-N -nitrosoguanidine (MNNG) (group 1). B Downregulation of 4-HNE, Bax, cytochrome C, and caspase 3 in animals administered MNNG (group 1). C Downregula-tion of PCNA, GST-π, Bcl-2, cytokeratins, and VEGF in animals administered MNNG + PB (group 2). D Overexpression of 4-HNE, Bax, cytochrome C, and caspase 3 in animals administered MNNG + PB (group 2). E PCNA, GST-π, 4-HNE, Bcl-2, Bax, cytochrome C, caspase 3, cytokeratins, and VEGF expression in control animals 357358R.S. Murugan et al.: Chemopreventive potential of black tea polyphenolsSODs and catalase and the GSH/GSSG ratio andGSH-dependent enzymes were signifi cantly increasedcompared with controls (group 4). Treatment withPolyphenon-B signifi cantly increased the antioxidantsin group 2 animals compared with group 1. Dietary ad-ministration of Polyphenon-B alone (group 3) signifi -cantly enhanced the antioxidant status compared withgroup 4.DiscussionIn the present study, administration of MNNG inducedwell-differentiated SCC and a very high tumor burden.Administration of Polyphenon-B for 26 weeks signifi -cantly reduced the incidence of MNNG-induced gastrictumors as well as preneoplastic lesions. These resultssubstantiate the anticarcinogenic effects of black teademonstrated in vitro as well as in experimental animaltumor models.24,25In particular, black tea polyphenolshave been shown to inhibit MNNG-induced precancer-ous gastric lesions in rats.26Rat forestomach tumors induced by MNNG exhib-ited increased proliferative, infi ltrative, and angiogenicpotential coupled with apoptosis evasion. The chemo-preventive effects of Polyphenon-B on MNNG-inducedgastric tumors was associated with modulation ofcellular redox status, decreased cell proliferation, andangiogenesis and induction of apoptosis as evidencedby downregulation of PCNA, GST-π, CKs, VEGF, andBcl-2, with upregulation of 4-HNE, Bax, cytochrome C,and caspase 3 expression.Lipid peroxides are recognized to prolong the G1phase of the cell cycle, whereas antioxidants such asGSH are required for cell cycle progression.27,28 Rapidlyproliferating tumor cells show resistance to lipid peroxi-dation and overexpress antioxidant enzymes.28Over-expression of PCNA and GST-π, reliable markers ofcell proliferation, has been documented in severaltumors.29,30 Thus diminished lipid and protein oxidationassociated with enhanced antioxidant defenses andoverexpression of GST-πand PCNA may confer asurvival advantage to MNNG-induced stomach tumors,creating a permissive environment for apoptosisevasion.Polyphenon-B reversed the susceptibility of MNNG-induced stomach tumors to lipid and protein oxidationwhile simultaneously increasing the activities of antioxi-dant enzymes. The results of the present study are inline with the antioxidant and antiproliferative proper-ties of tea and its constituents documented in literature.Black tea polyphenols have been demonstrated to exerttheir antioxidative properties by chelating metal ions,preventing free radical generation, and inhibiting lipidand protein oxidation.31,32Tea has been reported to Table2.PCNAlabelingindexandexpressionofGST-π,4-HNE,Bcl-2,Bax,cytochromeC,caspase3,cytokeratinandVEGFinexperimentalandcontrolanimals(n=1)GroupTreatmentPCNAGST-π4-HNEBcl-2BaxCytochromeCCaspase3CytokeratinsVEGF1.MNNG85.43±26.35*87.56±22.36*1.13±7.2*78.23±18.25*8.12±2.13*9.23±1.89*8.15±2.4*72.41±19.5*88.14±16.32*2.MNNG+P-B62.3±13.43**6.14±14.62**84.62±14.65**46.53±11.23**92.69±19.25**85.23±17.68**81.25±2.35**43.15±1.16**7.54±13.26**3.P-B23.54±2.1221.35±1.982.12±3.1222.25±1.3219.32±8.9519.65±9.212.36±6.2118.4±1.4343.62±2.864.Control2.13±1.819.23±1.6919.2±1.2517.13±1.217.5±6.8518.23±7.2319.68±5.1216.71±1.2741.25±2.98PCNA,proliferatingcellnuclearantigen;GST,glutathioneS-transferase;4-HNE,4-hydroxynonenal;VEGF,vascularendothelialgrowthfactor*Significantlydifferentfromgroup4(P<.5),ANOVAfollowedbyLSD**Significantlydifferentfromgroup1(P<.5)。