Induction of caspase-dependent, p53-mediated apoptosis by apigenin in 22Rv1 athymic nude mice tumor xenograft

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Apigenin, a common plant flavonoid has recently gained interest because of its low intrinsic toxicity and non-mutagenicity compared with other structurally-related flavonoids. Apigenin has shown to possess remarkable cancer preventive and some cancer therapeutic effects. The mechanisms underlying the cancer-protective effect remains elusive. Inactivation of p53-dependent pathways occur at any stage of carcinogenesis that leads to deregulation of cell cycle control and evasion of apoptosis. Our previous studies have shown that apigenin causes cell growth inhibition and induces apoptosis in prostate cancer cells and has potential to increase wild-type p53 stability, therefore, we hypothesized the involvement of p53-signaling during these events. We used androgen-sensitive human prostate carcinoma 22Rv1 tumor xenograft (harboring wild-type p53), subcutaneously implanted in athymic male nude mice. Apigenin feeding at 20- and 50- μg/mouse/day in 0.2 ml of a vehicle containing 0.5% methyl cellulose and 0.025% Tween 20, beginning 2 weeks after tumor implantation, resulted in significant reduction in tumor volume by 39% and 53% (p<0.01 and 0.002) and wet weights of tumor by 31 and 42% (p<0.05), respectively. Dietary intake of apigenin resulted in significant increase in apoptosis in tumor xenograft. The tumor inhibitory and apoptotic effects of apigenin were associated with increased accumulation of p53 protein and p53 phosphorylation on Ser-15; and p53-induced gene products p21/WAF1 and Bax. Furthermore, apigenin intake resulted in release of cytochrome c and induction of Apaf-1 along with activation of caspases 9 and 3 in tumor lysates. Importantly, tumor growth inhibition, induction of apoptosis and accumulation of p53 correlated with increasing serum apigenin levels. No toxic symptoms were observed after apigenin feeding whereas a modest loss in body weight was observed in animals receiving higher dose of apigenin. In cell culture studies, apigenin at 20- and 40- μM concentration resulted in cell growth inhibition and induction of apoptosis which correlated with transactivation and stabilization of p53 with significant increase in pSer15-p53. These results were further confirmed by p53 promoter-Luc transfected cells where apigenin treatment leads to activation of p53-Luc reporter activity. Apigenin treatment to cells also resulted in significant increase in caspase 9 and 3 along with increase in p21/WAF1 and Bax. Furthermore, apigenin-induced apoptosis was attenuated by incubation of cells with zVAD-fmk, a general caspase inhibitor, conferring chemopreventive effects of apigenin on caspase-mediated p53 signaling. Taken together, this study presents the first evidence that the in vitro and in vivo growth inhibitory and apoptotic effects of apigenin are mediated by caspase activation leading to transactivation and stabilization of p53 in prostate cancer.