Apigenin-mediated modulations of PI3K-Akt and MAPK signaling pathways causes growth Inhibition and cell cycle arrest in human prostate cancer cells

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Protein kinases play a critical role in signal transduction pathways regulating cellular proliferation, differentiation and apoptosis. Inhibition of these growth-related kinases might be significant in providing therapeutic modalities against cancer. Development of small molecules protein kinase inhibitors provide a new rational to combat cancer, however, highly selective or specific blocking of only one of the kinases has been associated with limited or sporadic responses during clinical trials. Therefore, agents which could regulate cellular growth by modulating kinases may be developed as effective anticancer agent. Apigenin, a dietary plant flavonoid has shown anti-proliferative and anticancer properties. Potential targets for apigenin include PI3K, Akt, MAPK, CDKs, and CK2. We therefore studied the molecular events of apigenin action in human prostate cancer cells. Treatment of asynchronized androgen-responsive LNCaP and androgen-refractory PC-3 cell lines with apigenin (1-40 μM) causes increase arrest of cells in G0-G1 phase of the cell cycle. Treatment of cells with apigenin decreased both retinoblastoma (Rb) protein phosphorylation at Ser780 and Ser807/811 and total Rb protein in dose- and time- dependent fashion. Apigenin treatment caused an increased phosphorylation of ERK1/2 and JNK1/2. This sustained activation of ERK1/2 and JNK1/2 resulted in decrease in ELK-1 phosphorylation and c-FOS expression thereby inhibiting cell survival. Use of cyclin-dependent kinase inhibitors and MEK1/2 inhibitor induced ERK1/2 phosphorylation albeit at different levels and did not contribute to cell cycle arrest in comparison to apigenin treatment. Despite activation of MAPK pathway, apigenin caused a significant decrease in cyclin D1 protein expression, an effect that occurred simultaneously with the loss of Rb phosphorylation and inhibition of cell cycle progression. The reduced expression of cyclin D1 protein correlated with decrease in protein expression and phosphorylation of p38 and PI3K-Akt, which are regulators of cyclin D1 protein. Interestingly, apigenin caused a marked reduction in cyclin D1, D2 and E and their regulatory partners CDK 2, 4 and 6 which are operative in G0-G1 phase of the cell cycle. This was accompanied by a loss of RNA polymerase II phosphorylation, suggesting the effectiveness of apigenin in inhibiting transcription of these proteins. This provides a possible mechanism by which apigenin can cause modulation in MAPK pathways and inhibition in Akt and Rb protein phosphorylation at multiple sites along with decrease in cyclin D1 causing arrest of cells in the G1→S phase transition. Taken together, this study provides an insight into the molecular mechanism of apigenin in modulating various tyrosine kinases and perturbs cell cycle progression, suggesting its future development and use as anticancer agent in humans.