Induction of apoptosis and inhibition of PI3K/Akt pathway in PC-3 and LNCaP prostate cancer cells by ethanolic neem leaf extract.

Abstract Aim of the study The present study is aimed to delineate the effect of ethanolic neem leaf extract on PI3K/Akt and apoptotic pathway in prostate cancer cell lines (PC-3 and LNCaP). Materials and methods To test the hypothesis, two different prostate cancer cell lines LNCaP (androgen dependent) and PC-3 (androgen independent) were taken. Cells were exposed to various concentrations of ethanolic neem leaf extract (ENLE) (25–125 μg/ml). The doses were fixed by cell viability (MTT) assay. For apoptotic detection in situ apoptosis assay, caspase-3 activity and protein expression of cytochrome c and Poly-ADP Ribose Polymerase (PARP) were analysed as well as mRNA expression of Bcl-2 family proteins was studied by RT-PCR. The phosphoinositide 3-kinase (PI3K) and p-Akt were analysed by western blotting and mRNA expression of Akt 1 and 2, PTEN was performed by RT-PCR. Immunoblotting of cyclin D1 and p21 was done to access the inhibition of cell proliferation. Results ENLE gives 50% inhibition at a dose of 100 μg/ml in both PC-3 and LNCaP cells and considered as effective dose. ENLE decreased the protein expression of PI3K as well as p-Akt and the mRNA expression of Akt 1and 2 in both the cells. There was a significant decrease in mRNA expression of PTEN in LNCaP cells. ENLE induced apoptosis and inhibited cell proliferation by inhibiting PI3K/Akt pathway. Decrease in p-Akt leads to increase in the protein level of Bad, p21 and decrease in the cyclin D1, respectively. ENLE treatment increased the cytochrome c expression and caspase-3 activity as well as regulated the mRNA expression of Bcl-2 family proteins thereby inducing apoptosis to both the cell lines. In situ apoptosis assay showed increased red fluorescence in 100 μg/ml of ENLE in both PC-3 and LNCaP cell lines. Conclusions The results suggested that ENLE induces apoptosis and inhibits cell proliferation through inhibiting PI3K/Akt pathway in both PC-3 and LNCaP cells.