Apigenin inactivates Akt to trigger apoptosis in human prostate cancer cells

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Phosphatidylinositol 3-kinase (PI3K) and serine/threonine protein kinase B (PKB or Akt) pathways regulate important cellular processes such as cell growth, survival, proliferation, migration and differentiation. Inappropriate activation of PI3K/Akt signaling pathway has been associated with the development of a number of human pathologies including autoimmunity, cancer and resistance to chemotherapy. Modulating Akt activity is now a commonly observed endpoint in various chemopreventive and chemotherapeutic regimens. Apigenin (4', 5, 7,-trihydroxyflavone), a naturally occurring plant flavonoid, abundantly present in common fruits and vegetables has been shown to possess cancer preventive and therapeutic properties. Earlier studies from our laboratory have demonstrated that apigenin treatment resulted in reduced proliferation and induction of apoptosis in human prostate cancer cells. However, the molecular mechanism has not been elucidated. In the present study, we explored the mechanism(s) of apigenin action on human prostate cancer PC-3 cells which possess constitutively active Akt. Treatment of PC-3 cells with apigenin (5-40 μM) resulted in significant dose and time dependent decrease in Ser (473) phosphorylation of Akt leading to inhibition of its kinase activity. Reduced Akt kinase activity was further confirmed by decreased phosphorylation of proapoptotic protein BAD and Glycogen synthase kinase-3 by apigenin treatments, which are downstream targets of Akt. Lower phosphorylation level of BAD resulted in reduced interaction of BAD with 14-3-3 beta protein after 24 h exposure of PC-3 cells with 20 μM apigenin. Apigenin-mediated inactivation of Akt was associated with downregulation of insulin-like growth factor receptor 1 protein level and inhibition of its autophosphorylation. Treatment with apigenin significantly induced caspase-9 activity and decreased the survival of PC-3 cells in a dose-dependent manner. Ectopic expression of Ser (473) phosphorylation in DU145 cells, which possess constitutively active Akt, was significantly reduced upon 20 μM apigenin treatment. The apigenin-induced apoptosis in both cell lines was attenuated in the presence of pan caspase inhibitor zVAD-fmk and caspase-9 specific inhibitor zLEHD-fmk. Furthermore, apigenin administration to mice bearing PC-3 prostate cancer xenografts inhibited the growth of tumors and proteins relevant to Akt signaling essentially confirming the observations in vitro. Downregulation of constitutive Akt kinase activity by apigenin provide the rationale to explore its role as a single chemotherapeutic agent or in combination to overcome therapeutic resistance to chemotherapy.