Apigenin-induced prostate cancer cell death is initiated by reactive oxygen species and p53 activation

Abstract

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Cancer prevention is profoundly dependent on the p53 tumor suppression protein as it functions in regulating growth and protecting cells from malignant transformation. The ability of p53 in providing prevention against cancer lies in the elimination of damaged or infected cells by causing cell cycle arrest and/or apoptosis. Several studies have demonstrated the anti-tumor activity of p53 and in fact, p53 is the most frequently altered protein in human cancer. Approximately, 50% of all human malignancies harbor mutations or deletions in the TP53 gene that disable the tumor suppressor function of the encoded protein. The other half of human tumors expresses wild type protein and encodes further defects in the p53 signaling pathway playing critical role in tumorigenesis. Agents which can differentially activate wild-type p53 in cancer cells could be develop for chemoprevention. Apigenin (4’,5,7-trihydroxyflavone) is one such agent that potentially activates wild-type p53 and induces apoptosis in cancer cells. We conducted detailed studies to understand its mechanism of action. Exposure of human prostate cancer 22Rv1 cells, harboring wild type p53, to growth suppressive concentration (10-80 μM) of apigenin resulted in i) stabilization of p53 by phosphorylation on critical serine sites, ii) p14/ARF-mediated downregulation of murine double minute (MDM) 2 protein, and iii) induction of WAF1/p21 in dose- and time- dependent manner. Apigenin, at these doses resulted in reactive oxygen species (ROS) generation which was accompanied by disruption of mitochondrial membrane potential, cytosolic release of cytochrome C and apoptosis. Interestingly, we observed accumulation of p53 to the mitochondria which was rapid and occurred between 1-3 h after apigenin treatment. All these effects were significantly blocked by pretreatment with antioxidant, N-acetylcystiene, and a well known p53 inhibitor, pifithrin. Apigenin-mediated p53 activation and apoptosis was further attenuated by p53 anti-sense treatment. Exposure of cells to apigenin further led to decrease in Bcl-2 expression and increase in Bax thereby shifting the Bax/Bcl-2 in favor of apoptosis. This altered expression of Bcl-2 family members triggered activation of the initiator caspases 9 and 8 followed by activation of effector caspase 3. Treatment of 22Rv1 cells with general caspase inhibitor, Z-VAD-FMK and a specific caspase-3 inhibitor, DEVD-CHO partially rescued these cells from apigenin-induced apoptosis. Taken together, the present study indicates that apigenin-induced apoptosis in 22Rv1 cells is mediated by ROS-dependent disruption of the mitochondrial membrane potential and stabilization of p53.