Suppression of Constitutive and Tumor Necrosis Factor -Induced Nuclear Factor (NF)- B Activation and Induction of Apoptosis by Apigenin in Human Prostate Carcinoma PC-3 Cells: Correlation with Down-Regulation of NF- B-Responsive Genes

Purpose: Development of androgen independence and resistance to apoptosis in prostate cancer are often correlated with high levels of serum tumor necrosis factor (TNF)in these patients. The loss of sensitivity to TNF-induced apoptosis in androgen-insensitive prostate carcinoma cells is due in part to constitutive activation of Rel/nuclear factor (NF)B transcription factors that regulate several cell survival and antiapoptotic genes. Our previous studies have demonstrated growth inhibitory and apoptotic effects of apigenin, a common plant flavonoid, in a variety of human prostate carcinoma cells. Here we examined whether apigenin is effective in inhibiting NFB expression in androgen-insensitive human prostate carcinoma cells exhibiting high constitutive levels of NFB. Experimental Design: Using androgen-insensitive human prostate carcinoma PC-3 cells, the effect of apigenin was assessed on NFB activation by electrophoretic mobility shift assay and reporter gene assay. Expression of NFB subunits p65 and p50, I B , p-I B , in-beads kinase assay and NFB-regulated genes were determined by Western blot analysis. Apoptosis was determined by annexin V/propidium iodide staining after fluorescence-activated cell-sorting analysis. Results: Treatment of cells with 10–40M doses of apigenin inhibited DNA binding and reduced nuclear levels of the p65 and p50 subunits of NFB. Apigenin inhibited I B degradation and I B phosphorylation and significantly decreased IKK kinase activity. Apigenin also inhibited TNF-induced activation of NFB via the I B pathway, thereby sensitizing the cells to TNF-induced apoptosis. The inhibition of NFB activation correlated with a decreased expression of NFB-dependent reporter gene and suppressed expression of NFB-regulated genes [specifically, Bcl2, cyclin D1, cyclooxygenase-2, matrix metalloproteinase 9, nitric oxide synthase-2 (NOS-2), and vascular endothelial growth factor]. Conclusions: Our results indicate that inhibition of NFB by apigenin may lead to prostate cancer suppression by transcriptional repression of NFB-responsive genes as well as selective sensitization of prostate carcinoma cells to TNF-induced apoptosis.