Inhibition of HIF-1 alpha and VEGF expression by the chemopreventive bioflavonoid apigenin is accompanied by Akt inhibition in human prostate carcinoma PC3-M cells

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Hypoxia initiates signaling pathways leading to expression of the hypoxia-inducible factor-1 (HIF-1). HIF-1 is a heterodimeric transcription factor composed of alpha and beta subunits. Expression of HIF-1 alpha is regulated by degradation and protein synthesis. Intracellular levels of alpha subunit are increased under chronic low oxygen conditions found in solid tumors, including prostate carcinoma. The involvement of serine/threonine kinase Akt in degradation/protein expression of HIF-1 remains controversial and seems to be cell type specific. HIF-1 activates the expression of many genes including vascular endothelial growth factor (VEGF) and thus plays an important role in tumor angiogenesis and growth. We have investigated the expression of HIF-1 alpha in the human prostate carcinoma cell line PC3-M. Western blot analysis indicated that PC3-M cells displayed very low levels of HIF-1 alpha protein under normoxic conditions (20% oxygen) and exposure to hypoxia (1.5% oxygen) for as little as 30 minutes resulted in a dramatic elevation of HIF-1 alpha level which remained elevated for up to 24 hrs of treatment. HIF-1 beta protein level was not affected by hypoxia. Expression of VEGF was analyzed by ELISA assay and was found to be elevated in PC3-M cells exposed to hypoxia for 24 hrs compared to cells maintained under normoxic conditions. Hypoxic conditions also dramatically increased the phosphorylation of Akt. Apigenin (4’, 5, 7,-trihydroxyflavone) is a common dietary flavonoid that has been shown to possess chemopreventive properties. Treatment of PC3-M cells with apigenin inhibited hypoxia-induced HIF-1 alpha levels in a concentration- and time-dependent manner. Apigenin down-regulated HIF-1 alpha by approximately 50 % at 50 uM. HIF-1 beta protein level remained unaffected even at the highest concentration of apigenin used (100 uM). We also examined the effect of apigenin on VEGF expression. Apigenin was found to inhibit VEGF expression by PC3-M cells in a dose-dependent manner. We next investigated the effect of apigenin on activation of Akt under hypoxic conditions and observed significant inhibition of Akt phosphorylation. These findings provide an insight into the potential mechanism of chemopreventive properties of apigenin.