"Withaferin A suppresses the expression of vascular endothelial growth factor in Ehrlich ascites tumor cells via Sp1 transcription factor Prasanna Kumar S., Shilpa P. and Bharathi P. Salimath"
Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 Withaferin A suppresses the expression of vascular endothelial growth factor in Ehrlich ascites tumor cells via Sp1 transcription factor Prasanna Kumar S., Shilpa P. and Bharathi P. Salimath* Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore-570006, India. For Correspondent : Salimathuom@rediffmail.com Abstract order to exert its antiangiogenic activity. These In the ayurvedic system of medicine, the results clearly indicate the antiangiogenic potential medicinal plant, Withania somnifera Dunal of withaferin A in modulating antitumor activity. (Solanaceae) finds application for numerous Keywords: Ehrlich ascites tumor; Withaferin ailments including cancer. This herbal plant yields A; Angiogenesis; Sp1, VEGF. a host of steroidal lactones called withanolides, some of which have shown growth inhibition of Introduction human tumor cell lines. Withaferin A amongst Several natural compounds are recognized these withanolides reportedly is very active in as cancer chemo preventive agents. Withanalides impairing antitumor activity. However; the are especially well known to suppress tumor cell underlying molecular mechanisms of this activity growth via cell-cycle arrest and by the induction remains still unclear. In the present study, we have of apoptosis in several tumor cell lines (1-3). shown that withaferin A inhibited vascular Moreover, withaferin A inhibits endothelial cell endothelial cell growth factor (VEGF) -induced proliferation and angiogenesis in vitro (4). tube formation by human umbilical vein endothelial Angiogenesis is essential for the growth, cells (HUVECs) and angiogenesis in chick progression and metastasis of solid tumors (5). chorioallantoic membrane (CAM) assay. In Withaferin A, a member of the withanalides family Ehrlich ascites tumor (EAT) model, the animals that is present at high levels in roots and leaves treated with withaferin A suppressed in vivo, the of Withania somnifera plant has been found to peritoneal angiogenesis and microvessel density. possess antioxidant and antitumor activity (6-9). When compared to the untreated animals, the However, the mechanism by which withaferin A withaferin A treated tumor bearing mice showed suppresses angiogenesis has not been fully elucidated. a decrease in the volume of ascites and tumor cell number. Quantitation of VEGF levels in ascites Vascular endothelial growth factor (VEGF) from withaferin A untreated or treated tumor is a major angiogenic factor that facilitates tumor bearing mice indicated decreased secretion of growth and metastasis. Hypoxia is known to VEGF in ascites from treated mice, as measured induce the expression of VEGF gene (10, 11). by ELISA. Studies at molecular level revealed VEGF promoter analysis has revealed several that withaferin A inhibits binding of Sp1 potential transcription factor-binding sites, such transcription factor to VEGF-gene promoter, in as hypoxia-inducible factor-1(HIF-1), activator Sp1 transcription factor Current Trends in Biotechnology and Pharmacy 139 Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 protein (AP)-1, AP-2, early growth response- Materials and methods 1(Egr-1) and Sp1 (12). Materials The GC box-binding protein, Sp1 is a Ehrlich ascites tumor (EAT) cells were ubiquitous transcription factor that belongs to the routinely maintained in Swiss albino mice in the Sp family of transcription factors, consisting of animal house, University of Mysore, Mysore, Sp1, Sp2, Sp3, and Sp4 (13). Sp1 has been India. Endothelial growth medium (EGM-2) was implicated in the transcription of large number of procured from Cambrex Biosciences, genes, including housekeeping genes, tissue- Walkersville, USA. Dulbecco’s modified Eagle’s specific genes and genes involved in growth medium (DMEM), fetal bovine serum (FBS), regulation (13-15). Sp1 activities are regulated penicillin-streptomycin and trypsin-EDTA were by a variety of stimuli. Most of these regulations purchased from Invitrogen, USA.T4 occur through either post-translational polynucleotide kinase kit was obtained from modification or alteration of Sp1 protein Amersham biosciences. The Sp1 oligonucleotides abundance. (5’-d (ATTCGA TCG GGG CGG GGCGAG C)- 3’) for gel shift assays were obtained from The principal known post-translational Promega. Radioacive ã-[32P] ATP was obtained modifications are phosphorylation and from Bhabha Atomic Research Centre (BARC), glycosylation through the O-linkage of the Mumbai, India. RNA isolation kit was procured monosaccharide, N-acetylglucosamine (O- from Qiagen, USA. Secondary antibodies GlcNAc) (16). conjugated to alkaline phosphatase and proteinase Expression level of the VEGF mRNA is inhibitors were obtained from Bangalore Genei, tightly regulated by both transcriptional and post- Bangalore, India. The rest of the chemicals were transcriptional mechanisms. Recent studies have of analytical grade of purity and were procured demonstrated that intracellular signaling pathways locally. and genetic elements are involved in controlling its expression. VEGF promoter activity is Methods preceded by the activation of transcription factor Isolation of withaferin A from Withania Sp1 (17). Therefore it is clear that a constitutive somnifera roots Sp1 activation is essential for the differential over expression of VEGF, which in turn plays an Withania somnifera roots were collected important role in angiogenesis and the progression locally from Mysore, India. The plant specimens of cancer. It has also been shown that Sp1 in were identified and authenticated at the herbarium particular, plays an important role in tumor of the Department of Botany, University of angiogenesis and contributes to the aggressive Mysore, Mysore, India. The roots were washed, nature of human pancreatic adenocarcinoma (18). shade dried and powdered. One hundred grams of the root powder was extracted in 500ml of In this study, we tested the hypothesis methanol overnight. Withaferin A was isolated that the antiangiogenic effect of withaferin A on from the methanol extract of Withania somnifera EAT cells involves a reduction in secretion of roots as previously described (4). The compound ascites fluid and expression of VEGF, which is Withaferin A (10mg) was dissolved in 100ìl of regulated by Sp1 transcription factor. Moreover, DMSO and diluted 100 times with sterile distilled we investigated the molecular mechanism by water to make final concentration 1ìg/ìl and used which withaferin A inhibits angiogenesis in vivo. for subsequent experiments. Prasanna et al Current Trends in Biotechnology and Pharmacy 140 Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 Human Umbilical Vein Endothelial Cells Tube formation assay (HUVECs) culture Tube formation of HUVECs was HUVECs were purchased from conducted for the assay of in vitro angiogenesis. Cambrex Biosciences, Walkersville, USA. The The assay was performed as described in earlier cells were cultured in 25 cm3 tissue culture flask report (20). Briefly, a 96-well plate was coated (NUNC, Genetix Biotech Asia, Bangalore, India) with 50µl of Matrigel (Becton Dickinson Labware, and grown using EGM-2 medium and endothelial Bedford, MA), which was allowed to solidify at cell basal medium according to the 370C for 1 hour. HUVECs (5x 103 cells per well) manufacturer’s protocol. Incubation was carried were seeded on the Matrigel and cultured in EGM out in a humidified atmosphere of 5% CO2 in air medium containing withaferin A (3.5-14µg) for 8 at 370C. When cells reached confluency, they hours. After incubation at 370C and 5% CO2, the were passaged after trypsinization. HUVECs of enclosed networks of complete tubes from five passages 2-5 were used for the experiments. randomly chosen fields were counted and Animals and in vivo tumor generation photographed under an Olympus inverted Six to eight weeks old mice were microscope (CKX40; Olympus, New York, NY) acclimated for one week while caged in groups connected to a digital camera at 40X of five. Mice were housed and fed a diet of animal magnification. chow and water ad libitum throughout the Chick chorioallantoic membrane (CAM) experiment. All experiments were conducted assay according to the guidelines of the Committee for the Purpose of Control and Supervision of CAM assay was carried out according Experiments on Animals (CPCSEA), to the detailed procedure as described by Gururaj, Government of India. EAT cells (5×106cells/ A.E. et al. (21, 22). In brief, fertilized chicken mouse) injected intraperitoneally grow in mice eggs were incubated at 370C in a humidified peritoneum forming an ascites tumor with massive incubator. On the 11th day of development, a abdominal swelling. The animals show a dramatic rectangular window was made in the egg shell increase in body weight over the growth period and glass cover slips (6-mm diameter) saturated and the animals succumb to the tumor burden with 25ng/ml vascular endothelial growth factor 15-16 days after implantation. The number of cells (VEGF) and VEGF + withaferin A (7ìg) was increased over the 14 days of growth with placed on the CAM and the window was closed formation of 7-8 ml of ascites fluid with extensive using sterile wrap. The windows were opened neovascularization in the inner lining of peritoneal after 48h of incubation and were inspected for wall. EAT cells from fully grown tumor bearing changes in the microvessel density in the area mice were harvested from the peritoneal cavity below the cover slip and photographed using a of mice (19). The ascites fluid was collected in Nikon digital camera. isotonic saline solution containing 3.8% sodium In vivo withaferin A treatment inhibits EAT citrate. The cells were pelleted by centrifugation growth (3000 rpm for 10 min at 40C). Contaminating red blood corpuscles if any were lysed with 0.8% To determine whether withaferin A ammonium chloride. Cells were resuspended in inhibits tumor growth and angiogenesis in EAT 0.9% saline. These cells or their aliquots were cells in vivo, withaferin A (7mg/kg/day/mouse) used either for transplantation or for further and vehicle control (0.1% of DMSO) was injected experiments. into the EAT bearing mice every alternate day Sp1 transcription factor Current Trends in Biotechnology and Pharmacy 141 Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 after 6th day of tumor transplantation and growth Preparation of nuclear extracts of the tumor was monitored by taking the body Nuclear extracts were prepared weight of the animals. Animals were sacrificed according to the method previously described (25). on the 14th day and the EAT cells along with Briefly, cells (5X106) treated either with or without ascites fluid were harvested into the beaker and withaferin A in complete HBSS for different time centrifuged at 3000 rpm for 10 min at 40C. The intervals were washed with cold phosphate pelleted cells were counted by Trypan blue dye buffered saline and suspended in 0.5 ml of lysis exclusion method using a haemocytometer. A buffer (20mM HEPES, pH 7.9, 350 mM NaCl, measure of the supernatant gave the volume of 20% Glycerol, 1% NP-40, 1 mM MgCl2, 0.5 mM ascites fluid. EGTA, 0.5 mM DTT, 1 mM Pefablock, 1µg/ml Aprotinin, 1µg/ ml Leupeptin). The cells were Peritoneal angiogenesis and micro vessel allowed to swell on ice for 10 min; the tubes were density then vigorously mixed on a vortex mixer for 1 After harvesting the EAT cells from min and centrifuged at 10,000 rpm for 10 min at control and withaferin A-treated animals, the 40C. The supernatant was immediately stored at peritoneum was cut open and the inner lining of -200C. the peritoneal cavity was examined for extent of Electrophoretic Mobility Shift Assay (EMSA) neovasculature and photographed. Formaldehyde Nuclear proteins were extracted from fixed and paraffin embedded tissues of peritoneum EAT cells treated either with or without withaferin from EAT bearing mice either treated or untreated A for 60,120 and 180 min respectively. The EMSA with withaferin A were taken and 5ìm sections was performed as described in earlier report (26, were prepared using automatic microtome (SLEE 27). The double stranded Sp1 consensus Cryostat) and stained with hematoxylin and eosin. oligonucleotide probes [5’-d (ATT CGA TCG The images were photographed using Leitz- GGG CGG GGC GAG C)-3’] were end-labeled DIAPLAN microscope with CCD camera and with ã-[32P] ATP. Nuclear proteins (40ìg) were the blood vessels were counted. incubated with 40fmoles of ã-[32P]-labeled Sp1 consensus oligonucleotides for 30min in binding Quantitation of VEGF buffer containing 100mM HEPES (pH 7.9),10mM EAT bearing mice were treated with or MgCl 2, 125 mM KCl, 0.5mM EDTA, 4% without withaferin A (7mg/kg/day) for 5 doses glycerol,0.5% NP-40,1ìg of poly [dI-dC] and on 6th, 8th, 10th and 12th day of tumor 1mg/ml BSA. The samples were electrophoresed transplantation. The animals were sacrificed and in 4% non denaturing polyacrylamide gel in 0.5% ascites fluid was collected after 24h of each dose. TBE at room temperature for 2 hr at 200V. The VEGF-ELISA was carried out using the ascites gel was dried, transferred to imaging plate (IP) fluid (21, 23, 24). In brief, 100µl of ascites from and the image was scanned by image analyzer tumor bearing mice either with or without Fujifilm (FLA-5000). withaferin A treatment, was coated using coating Results buffer (50 mM carbonate buffer pH 9.6) at 40C overnight. Subsequently, wells were incubated Withaferin A inhibits tube formation of with anti-VEGF 165 antibodies, followed by HUVECs induced by VEGF incubation with secondary antibodies tagged to In order to verify if withaferin A interferes alkaline phosphatase and detection using p-nitro- directly with the formation of blood vessels by phenyl phosphate (pNPP) as a substrate. HUVECs, we performed tube formation assay Prasanna et al Current Trends in Biotechnology and Pharmacy 142 Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 in vitro. The HUVECs were plated on the Withaferin A inhibits VEGF induced neovas- Matrigel. The HUVECs in the basal medium could cularization on chick chorioallantoic membrane not form tubes and VEGF was used to induce CAM assay is one of the widely used the tube formation. In the positive control group validation assays for formation of new blood stimulated with VEGF (10ng), HUVECs rapidly vessels. In order to further verify if withaferin A aligned with one another and formed tube-like is an inhibitor of new blood vessel formation, structures resembling a capillary plexus within 8 withaferin A was applied on chorioallantoic hours, after VEGF treatment. However, membrane of chick embryo to test its in vivo treatment with withaferin A prevented VEGF - antiangiogenic activity. In the CAM assay model stimulated tube formation of HUVECs in a withaferin A induced avasculature zone formation concentration (3.5-14µg) - dependent manner in the developing embryos. Notably newly formed (Fig. 1). Meanwhile, no cytotoxicity was observed microvessels were regressed around the area of under this concentration range of withaferin A withaferin A treated CAM (Fig. 2). used in the assay. Withaferin A was shown to interfere with the ability of HUVECs to form the in vitro vessel-like tubes, one of the important traits of the endothelial cells. Fig. 2: Effect of withaferin A on neovascularization in the chick CAM Withaferin A was applied on CAM of 11-day- old chicken embryo. After 48h of incubation, the applied area was inspected for changes in neovascularization. The arrows indicate the applied area. The data shown represents the result of an experiment, which was done using maximum six eggs in each group. All photographs were taken at same magnification. Withaferin A inhibits growth of EAT cells and secretion of ascites in vivo Initially, proliferation of tumor cells in mice was monitored by measuring the weight of Fig. 1: Effect of withaferin A on VEGF induced HUVECs the animals every day. A decrease in body weight tube formation in withaferin A treated animals was observed when compared to the increased body weight of HUVECs (5X103 cells) cultured in EGM medium the untreated tumor bearing mice. It was also with 3.5µg, 7µg and 14 µg withaferin A was added to the Matrigel coated 96 wells plate. After incubation observed that withaferin A lessened the tumor for 8 hours at 37 0 C, capillary networks were burden considerably in a dose dependent manner photographed and quantified (Magnification: X40). showing the optimum activity at 7mg/kg/dose. Cell Concentration dependent inhibition of tube formation number was counted after each dose of withaferin by withaferin A was recorded. All datas are presented A treatment. In control group, which is tumor as mean from three different experiments with triplicates bearing mice not treated with withaferin A, the and means of ± S.E.M. P<0.05 vs control. number of EAT cells increased exponentially. In Sp1 transcription factor Current Trends in Biotechnology and Pharmacy 143 Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 contrast, in the withaferin A treated group, the Withaferin A inhibits angiogenesis in vivo number of cells were drastically decreased (Fig. Sprouting of new blood vessels is evident 3A). This implied that withaferin A inhibit tumor in the inner peritoneal lining of EAT bearing mice. cell growth in vivo. The volume of ascites was The peritoneal lining of EAT bearing animals and also measured using ascites from EAT bearing withaferin A treated mice was inspected for the as well as withaferin A treated EAT bearing effect on tumor-induced peritoneal animals. The results indicated that withaferin A neovascularization. EAT bearing mice treated with treatment reduces the secretion of ascites fluid withaferin A showed decreased peritoneal (Fig. 3B). It is indicative from this data that angiogenesis when compared to the extent of withaferin A is probably capable of inhibiting the peritoneal angiogenesis in untreated EAT bearing proliferation of tumor cell growth in vivo. mice (Fig. 4A). Histopathological staining of peritoneum sections from the EAT bearing group appeared well vascularized. In contrast withaferin A treated peritoneum sections were characterized by a pronounced decrease in micro vessel density and the caliber of detectable vascular channels. While tumor bearing peritoneum sections showed 17 ± 1.2 blood vessels, withaferin A treated peritoneum showed 6.8 ± 1.3 blood vessels (Fig. 4B). Fig. 4: Withaferin A inhibits angiogenesis and microvessel density A) Inhibition of peritoneal angiogenesis. EAT bearing mice were treated with and without Fig. 3: Effect of withaferin A on EAT cell number and withaferin A for four doses (7mg/kg/animal). The mice ascites volume in vivo were sacrificed and the peritoneal lining was observed for extent of neovascularization. We presented EAT cells (5X106 cells/animal, i.p.) were injected into representative photograph of peritoneum. mice. After 6 days of tumor transplantation, withaferin A (7mg/kg/animal) was injected on days 7th, 9th, 11th and B) Reduction in microvessel density (MVD) 13th. The animals were sacrificed on days 8th, 10th, 12th The peritoneum of control as well as withaferin A and 14th. EAT cells were collected along with ascites fluid. treated EAT bearing mice was embedded in paraffin Cells were counted in haemocytometer (A) and ascites and 5ìm sections were taken using microtome. The volume was measured (B). At least five mice were used in sections were stained with hematoxyline and eosine each group and the results obtained are an average of three and observed for microvessel density. Arrows indicate individual experiments and means of ± S.E.M. the microvessels. Prasanna et al Current Trends in Biotechnology and Pharmacy 144 Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 Withaferin A inhibits VEGF secretion in performed. The results indicated that withaferin ascites fluid of EAT bearing mice A inhibits binding of Sp1 transcription factor to In order to verify whether withaferin A the proximal promoter region of the VEGF gene. induced inhibition of neovascularization and In contrast, there was prominent band showing microvessel density is due to decreased secretion the binding of Sp1 to the proximal promoter region of VEGF by EAT cells, we have quantified the (Fig. 6) when nuclear extract from tumor bearing secreted VEGF in ascites fluid of control and mice was used. different doses of withaferin A treated animals by ELISA. It is evident in Fig. 5 that withaferin A inhibits production of VEGF in EAT cells. In EAT bearing mice, 1280ng of VEGF was detected in ascites. However in case of withaferin A treated animal ascites 220ng of VEGF was detected per mouse. Fig. 6: Effect of withaferin A on Sp1-DNA binding activity Nuclear extracts were prepared from EAT cells untreated and treated with withaferin A. Sp1-DNA binding activity was assayed by EMSA using Sp1 Fig. 5: Effect of withaferin A on in vivo production oligonucleotides. and expression of VEGF EAT bearing mice were injected with withaferin A (7mg/ Discussion kg/animal) for four doses and ascites fluid was Plant and dietary products contribute to collected after sacrificing the animal every alternate about one-third of potential anticancer drugs and day after each dose of treatment. ELISA was carried the preventive effects of plant-based diets on out to quantitate VEGF in ascites fluid using anti- tumorigenesis and other chronic diseases have VEGF165 antibodies. Animals bearing EAT cells not treated with withaferin A was used as control. been well documented (28). Withania somnifera (L.) Duna1 commonly known as Ashwagandha Withaferin A inhibits Sp1 DNA binding (family Solanaceae) is extensively used in many activity in EAT cells indigenous preparations. W. somnifera is To verify for the involvement of reported to have anti-inflammatory (29), anti- transcription factor Sp1 in withaferin A induced arthritic (30) and anti-tumor (31) activities. inhibition of VEGF gene expression, an Withaferin A, a withanolide was isolated and electrophoretic mobility shift assay was reported to be antiangiogenic and anti-tumor active Sp1 transcription factor Current Trends in Biotechnology and Pharmacy 145 Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 principle from Withania somnifera. A recent expression. Withaferin A is already reported as a study demonstrated that the anti-angiogenic effect potent inhibitor of NF-êB and AP-1 DNA binding of withaferin A was due to the inhibition of activity (4, 36, 37). endothelial cell proliferation (4). However, the detailed molecular mechanisms involved in the Recent studies indicated that Sp1 anti-angiogenic effect of withaferin A were not transcription factor plays an important role in clearly understood. In this study we investigated VEGF expression and tumor angiogenesis. A the anti-angiogenic effects of withaferin A both region between nucleotide-109 and -61 of the in vitro and in vivo model. Withaferin A VEGF promoter and its intact Sp1-binding sites suppressed human endothelial cell- tube formation were required for the inhibition of VEGF promoter which is one of the hallmarks of angiogenesis activity. In this study, we found that withaferin A indicating that withaferin A inhibits endothelial cell treatment reduced Sp1 DNA binding activity to proliferation. This may be due to the induction of the proximal promoter region of VEGF gene in a HUVECs apoptosis by withaferin A (4). Further, time dependent manner. It was shown recently in Ehrlich ascites tumor bearing mice and also by that celecoxib inhibits VEGF expression and using several ex-vivo and cell based validation reduces angiogenesis and metastasis of human assays, we observed that withaferin A besides pancreatic cancer via suppression of Sp1 (38). inhibiting the growth of the tumor suppressed Sp1 suppression was closely correlated with peritoneal angiogenesis and microvessel density reduced VEGF level. Withaferin A has been by down regulating VEGF gene expression and reported as potent inhibitor of PKC and TNF VEGF secretion into the ascites of tumor bearing dependent IêB kinase â, which subsequently mice. It also inhibited neoangiogenesis induced blocks NF-êB nuclear translocation (3, 37). PKC by VEGF in CAM assay indicating that withaferin isoforms are also involved in the activation of Sp1, A targets both tumor and endothelial cell to exert NF-êB and AP-1 in B16F1 murine melanoma its anti proliferative and antiangiogenic activities. cells (39, 40). In summary, our experiments have Increased VEGF expression is closely shown that inhibition of VEGF secretion and tumor associated with an increase in microvessel density microvessel formation is one of the potential (32). VEGF being a permeability factor plays mechanisms by which withaferin A suppresses fundamental role in the fluid accumulation and the growth of Ehrlich ascites tumor. Additionally, tumor growth in ascites tumor. By secreting the suppression of VEGF secretion appears to VEGF, ascites tumor enhances the permeability be as a consequence of altered Sp1 of preexisting microvessel lining of peritoneal transactivation and inhibition of VEGF gene cavity to stimulate ascites formation thereby expression. The data clearly indicates a novel tumor progression. Inhibition of fluid accumulation, mechanism for the antiangiogenic activity of tumor growth and microvessel density by withaferin A and also substantiate the important neutralization of VEGF has been demonstrated role of Sp1 in tumor biology and the biological underlying the importance of VEGF in malignant basis for the development of new Sp1-targeting ascites formation (33-35). Our results indicated agents for cancer treatment. that there was decrease in the VEGF secretion in withaferin A treated animals. Inhibition of VEGF Acknowledgements secretion could be due to inhibition of activity of The authors thank Department of Science transcription factors NF-êB, AP-1 or Sp1 which and Technology (FIST), Government of India, are involved in the regulation of VEGF gene India, University Grants Commission, Prasanna et al Current Trends in Biotechnology and Pharmacy 146 Vol. 3 (2) 138-148, Apirl 2009. ISSN 0973-8916 Government of India, India, and Department of brain frontal cortex and striatum. Indian Atomic Energy (BNRS), for financial support to Journal of Experimental Biology, 35: 236- perform this work. We thank Dr. H.N. Yejurvedi, 239. In-charge, animal facility, Department of Zoology, 7. Bhattacharya, A., Ghosal, S. and University of Mysore for providing animals for Bhattacharya, S.K. (2001). Anti-oxidant the experiment. effect of Withania somnifera glycowithanolides in chronic footshock References stress-induced perturbations of oxidative 1. Srinivasan, S., Ranga, R.S., Burikihanov, R., free radical scavenging enzymes and lipid Han, S.S. and Chendil, D. (2007). Par-4- peroxidation in rat frontal cortex and Dependent Apoptosis by the Dietary striatum. Journal of Ethnopharmacology, 74: Compound Withaferin A in Prostate Cancer 1-6. Cells. Cancer Research, 67: 246-53. 8. Mishra, L.C., Singh, B.B. and Dagenais, S. 2. Malik, F., Kumar, A., Bhushan, S., Khan, (2000). 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