Purpose-Gallic acid, a natural agent present wide-range of fruits and vegetables, has been of potential interest as anti-cancer agent; herein, we evaluated its efficacy in androgen-independent DU145 and androgen-dependent-22Rv1 human prostate cancer (PCa) cells Materials and Methods-Cell viability was determined by MTT and apoptosis by Annexin V-PI assays. In vivo anti-cancer efficacy was assessed by DU145 and 22Rv1 xenograft growth in nude mice given normal drinking water or one supplemented with 0.3% or 1% (w/v) gallic acid. PCNA, TUNEL and CD31 immunostaining was performed in tumor tissues for in vivo anti-proliferative, apoptotic and anti-angiogenic effects of gallic acid.Results-Gallic acid decreased cell viability in a dose-dependent manner in both DU145 and 22Rv1 cells largely via apoptosis induction. In tumor studies, gallic acid feeding inhibited the growth of DU145 and 22Rv1 PCa xenografts in nude mice. Immunohistochemical analysis revealed significant inhibition of tumor cell proliferation, induction of apoptosis, and reduction of microvessel density in tumor xenografts from gallic acid-fed mice as compared to controls in both DU145 and 22Rv1 models Conclusion-Taken together, our findings show the anti-PCa efficacy of gallic acid providing a rationale for additional studies with this naturally-occurring agent for its efficacy against PCa.
Mutations in APC/beta-catenin resulting in an aberrant activation of Wnt/beta-catenin pathway are common in colorectal cancer (CRC), suggesting that targeting the beta-catenin pathway with chemopreventive/anticancer agents could be a potential translational approach to control CRC. Using human CRC cell lines harboring mutant (SW480) versus wildtype (HCT116) APC gene and alteration in beta-catenin pathway, herein we performed both in vitro and in vivo studies to examine for the first time whether silibinin targets beta-catenin pathway in its efficacy against CRC. Silibinin treatment inhibited cell growth, induced cell death, and decreased nuclear and cytoplasmic levels of beta-catenin in SW480 but not in HCT116 cells, suggesting its selective effect on the beta-catenin pathway and associated biologic responses. Other studies, therefore, were performed only in SW480 cells where silibinin significantly decreased beta-catenin-dependent T-cell factor-4 (TCF-4) transcriptional activity and protein expression of beta-catenin target genes such as c-Myc and cyclin D1. Silibinin also decreased cyclin-dependent kinase 8 (CDK8), a CRC oncoprotein that positively regulates beta-catenin activity, and cyclin C expression. In a SW480 tumor xenograft study, 100- and 200-mg/kg doses of silibinin feeding for 6 weeks inhibited tumor growth by 26% to 46% (P < .001). Analyses of xenografts showed that similar to cell culture findings, silibinin decreases proliferation and expression of beta-catenin, cyclin D1, c-Myc, and CDK8 but induces apoptosis in vivo. Together, these findings suggest that silibinin inhibits the growth of SW480 tumors carrying the mutant APC gene by down-regulating CDK8 and beta-catenin signaling and, therefore, could be an effective agent against CRC.
Chemoprevention is a practical and translational approach to reduce the risk of various cancers including colorectal cancer (CRC), which is a major cause of cancer-related deaths in the United States. Accordingly, here we assessed chemopreventive efficacy and associated mechanisms of long-term silibinin feeding on spontaneous intestinal tumorigenesis in the APC min/+ mice model. Six-week-old APC min/+ mice were p.o.fed with vehicle control (0.5% carboxymethyl cellulose and 0.025% Tween 20 in distilled water) or 750 mg silibinin/kg body weight in vehicle for 5 d/wk for 13 weeks and then sacrificed. Silibinin feeding strongly prevented intestinal tumorigenesis in terms of polyp formation in proximal, middle, and distal portions of small intestine by 27% (P < 0.001), 34% (P < 0.001), and 49% (P < 0.001), respectively. In colon, we observed 55% (P < 0.01) reduction in number of polyps by silibinin treatment. In size distribution analysis, silibinin showed significant decrease in large-size polyps (>3 mm) by 66% (P < 0.01) and 88% (P < 0.001) in middle and distal portions of small intestine, respectively. More importantly, silibinin caused a complete suppression in >3 mm sized polyps and 92% reduction in >2 to 3 mm sized polyps in colon. Molecular analyses of polyps suggested that silibinin exerts its chemopreventive efficacy by inhibiting cell proliferation, inflammation, and angiogenesis; inducing apoptosis; decreasing β-catenin levels and transcriptional activity; and modulating the expression profile of cytokines. These results show for the first time the efficacy and associated mechanisms of long-term p.o. silibinin feeding against spontaneous intestinal tumorigenesis in the APC min/+ mice model, suggesting its chemopreventive potential against intestinal cancers including CRC.
Colorectal cancer is one of the leading causes of cancerrelated morbidity and mortality. The use of nontoxic phytochemicals in the prevention and intervention of colorectal cancer has been suggested as an alternative to chemotherapy. Here we assessed the anticancer efficacy of silibinin against advanced colorectal cancer LoVo cells both in vitro and in vivo. Our results showed that silibinin treatment strongly inhibits the growth of LoVo cells (P < 0.05-0.001) and induces apoptotic death (P < 0.01-0.001), which was associated with increased levels of cleaved caspases (3 and 9) and cleaved poly (ADP-ribose) polymerase. Additionally, silibinin caused a strong cell cycle arrest at G 1 phase and a slight but significant G 2 -M-phase arrest at highest concentration (P < 0.01-0.001). Molecular analyses for cell cycle regulators showed that silibinin decreases the level of cyclins (D1, D3, A and B1) and cyclin-dependent kinases (1, 2, 4, and 6) and increases the level of cyclin-dependent kinase inhibitors (p21 and p27). Consistent with these results, silibinin treatment also decreased the phosphorylation of retinoblastoma protein at Ser 780 , Ser 795, and Ser 807
Chemoprevention is a practical approach to control colorectal cancer, which is one of the major causes of cancer mortality in the United States. Based on our recent silibinin efficacy studies in human colorectal cancer cells, we investigated the effects of its dietary feeding on azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation and associated biomarkers in male Fisher 344 rats. Five-week-old male Fisher 344 rats were fed control or silibininsupplemented (0.033%, 0.1%, 0.33%, or 1%, w/w) diet. After 2 weeks, AOM was injected once a week for 2 weeks while silibinin treatments were continued. In another protocol, identical silibinin treatments were done but started 2 weeks post-AOM initiation. All rats were sacrificed at 16 weeks of age, and colon samples were evaluated for ACF, followed by proliferation, apoptosis, and inducible nitric oxide synthase and cyclooxygenase-2, by immunohistochemistry and/or immunoblotting. Silibinin significantly (P < 0.001) reduced dose-dependently the number and multiplicity of AOM-induced ACF formation. Silibinin feeding in pre-and post-AOM initiation decreased mean number of ACF by 39% to 65% and in post-AOM initiation by 29% to 55%. Silibinin dose-dependently decreased AOMinduced colonic cell proliferation, evidenced by proliferative cell nuclear antigen and cyclin D1 immunohistochemical staining, and induced apoptosis in these colon tissues, evidenced by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining and cleaved poly(ADP-ribose) polymerase. Furthermore, silibinin significantly decreased AOMinduced inducible nitric oxide synthase-and cyclooxygenase-2-positive cells in colon tissues. The present findings show possible beneficial activity of silibinin at least in early stage of colon tumorigenesis, suggesting that silibinin might be an effective natural agent for colorectal cancer chemoprevention.
Chemoprevention by dietary agents/supplements has emerged as a novel approach to control various malignancies, including colorectal cancer (CRC). This study assessed dietary grape seed extract (GSE) effectiveness in preventing azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation and associated mechanisms in Fischer 344 rats. Six-week old rats were injected with AOM, and fed control diet or the one supplemented with 0.25% or 0.5% (w/w) GSE in preand post-AOM or only post-AOM experimental protocols. At 16 weeks of age, rats were sacrificed and colons were evaluated for ACF formation followed by cell proliferation, apoptosis and molecular analyses by immunohistochemistry. GSE-feeding caused strong chemopreventive efficacy against AOM-induced ACF formation in terms of upto 60% (P<0.001) reduction in number of ACF and 66% (P<0.001) reduction in crypt multiplicity. Mechanistic studies showed that GSE-feeding inhibited AOM-induced cell proliferation but enhanced apoptosis in colon including ACF, together with a strong decrease in cyclin D1, COX-2, iNOS and survivin levels. Additional studies showed that GSE-feeding also decreased AOM-caused increase in β-catenin and NF-κB levels in colon tissues. Compared to control animals, GSE alone treatment did not show any considerable change in these biological and molecular events in colon, and was nontoxic. Together, these findings show the chemopreventive efficacy of GSE against the early steps of colon carcinogenesis in rats via likely targeting of β-catenin and NF-κB signaling, and suggest its potential usefulness for the prevention of human CRC.
Purpose-Here we assessed whether silibinin, a nontoxic chemopreventive agent, inhibits spontaneous intestinal tumorigenesis in APC min/+ mouse model, a genetically predisposed animal model of human familial adenomatous polyposis (FAP).Materials and Methods-Six-week old APC min/+ mice were divided into four groups and orally gavaged with 0.2 ml vehicle, or 250, 500 and 750 mg silibinin/kg body weight in 0.2 ml vehicle for five days/week. After 6 weeks, polyp burden was analyzed and tissues examined for molecular alterations.Results-Silibinin treatments decreased total number of intestinal polyps by 34% (P<0.01), 42% (P<0.01) and 55% (P<0.001), respectively. Immunohistochemical analysis showed that silibinin dose-dependently decreases (P<0.001) proliferation and induces (P<0.001) apoptosis only in intestinal polyps without any considerable effects on normal crypt-villi in APC min/+ or wild-type mice. Further analysis of polyps showed that silibinin decreases β-catenin, cyclin D1, c-Myc and phospho-glycogen synthase kinase-3β expression. Silibinin treatment also decreased phospho-Akt, cyclooxygenase-2, inducible nitric oxide synthase, nitrotyrosine and nitrite levels in polyps, the wellknown mediators of intestinal/colon carcinogenesis.Conclusion-Together, these results establish silibinin efficacy in a well-established genetic model of FAP, APC min/+ mouse, and suggest that this natural agent modulates various molecular pathways including β-catenin in its overall chemopreventive efficacy against intestinal carcinogenesis.
Purpose: Colorectal cancer is the second leading cause of cancer-associated deaths, which suggests that more effort is needed to prevent/control this disease. Herein, for the first time, we investigate in vivo the efficacy of silibinin against azoxymethane-induced colon tumorigenesis in A/J mice.Experimental Design: Five-week-old male mice were gavaged with vehicle or silibinin (250 and 750 mg/kg) for 25 weeks starting 2 weeks before initiation with azoxymethane (pretreatment regime) or for 16 weeks starting 2 weeks after the last azoxymethane injection (posttreatment regime). The mice were then sacrificed, and colon tissues were examined for tumor multiplicity and size, and molecular markers for proliferation, apoptosis, inflammation, and angiogenesis.Results: Silibinin feeding showed a dose-dependent decrease in azoxymethane-induced colon tumorigenesis with stronger efficacy in pretreatment versus posttreatment regimen. Mechanistic studies in tissue samples showed that silibinin inhibits cell proliferation as evident by a decrease (P < 0.001) in proliferating cell nuclear antigen and cyclin D1, and increased Cip1/p21 levels. Silibinin also decreased (P < 0.001) the levels of inducible nitric oxide synthase, cyclooxygenase-2, and vascular endothelial growth factor, suggesting its anti-inflammatory and antiangiogenic potential in this model. Further, silibinin increased cleaved caspase-3 and poly(ADP-ribose) polymerase levels, indicating its apoptotic effect. In other studies, colonic mucosa and tumors expressed high levels of β-catenin, insulin-like growth factor-1 receptorβ, phospho Glycogen synthase kinase-3β, and phospho protein kinase B/pAkt proteins in azoxymethane-treated mice, which were strongly lowered (P < 0.001) by silibinin treatment. Moreover, azoxymethane reduced insulinlike growth factor binding protein-3 protein level, which was enhanced by silibinin.Conclusions: Silibinin targets β-catenin and IGF-1Rβ pathways for its chemopreventive efficacy against azoxymethane-induced colon carcinogenesis in A/J mice. Overall, these results support the translational potential of silibinin in colorectal cancer chemoprevention. Clin Cancer Res; 16(18); 4595-606. ©2010 AACR.
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