Studies on the molecular mechanisms involved in thymoquinone induced growth inhibition of prostate cancer cells: Role of reactive oxygen species
Description
Thymoquinone (TQ), an active ingredient of black seed (Nigella sativa) oil, has been shown to possess chemopreventive and anti-neoplastic activity against a variety of experimental tumors. Previous reports have shown that the action of TQ in prostate cancer (PCa) cells is dependent upon down-regulating the expression of androgen receptor (AR) and transcription factor E2F-1. However, the precise mechanism of action of TQ remains unexplored, especially in AR-independent (C4-2B) and AR naive (PC-3) PCa cells. Our results demonstrated that treatment with TQ (50 muM and 100 muM) significantly inhibits growth, induces ROS generation and depletes glutathione levels in both PC-3 and C4-2B cells, which was inhibited by pretreatment with N-acetylcysteine (4 mM). TQ induced growth inhibition of PC3 cells was independent of action of JNK and caspases. Apoptotic array analysis of PC-3 cells upon treatment with TQ (50 muM) for 2 h showed significant changes in the expression of various apoptotic and antiapoptotic genes; especially growth arrest and DNA damage inducible gene (GADD45alpha). Treatment of PC-3 cells with TQ (100 muM) for 6 h resulted in significant up-regulation of GADD45alpha and apoptosis inducing factor (AIF) and significant down-regulation of anti-apoptotic Bcl-2 family of proteins: Bcl-2, Bcl2A1 and BAG-1. We also analyzed the effects of TQ on AR expression and activation in C4-2B cells. Our data demonstrated that TQ inhibited expression, transactivation and transcriptional activity of AR. However, the protective effects of NAC against TQ induced cytotoxicity were not coupled with an increase in the expression or activation of AR in C4-2B cells suggesting that growth inhibition of C4-2B cells is primarily due to generation of ROS by TQ. To test the effects of other anti-oxidants on TQ induced cytotoxicity, TEMPOL was used. Contrary to our predictions, we observed that the combination of TQ and TEMPOL is more cytotoxic than their individual treatments due to synergistic increase in the levels of ROS generation. Also, pretreatment with NAC protected PCa cells against the combination of TQ and TEMPOL. This study offers a novel insight towards the mechanism of growth inhibition induced by TQ and the combination of TQ and TEMPOL in PCa cells