Thymoquinone induced apoptosis in PC3 Cells involves generation of mitochondrial reactive oxygen species (ROS) and inhibition of Akt phosphorylation

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Thymoquinone (TQ), an active ingredient of black seed oil, has been shown to possess chemopreventive and antineoplastic 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). However, the precise mechanism of action of TQ remains unexplored especially in AR-independent and AR naive PCa cells as models of hormone refractory disease. To this end, we examined if TQ’s tumor growth inhibitory effects are mediated via PI3K/Akt, an important signaling pathway involved in survival, proliferation, and up-regulation of AR transcripts in PCa cells. Our results demonstrated that TQ inhibits growth of PCa cells that were AR responsive (LNCaP), AR independent (C4-2B), and AR naive (PC3). The IC50 values of TQ determined by MTT assay at 72 h against these cells were 78, 118 and 198 µM, respectively. TQ exposure of PC3 cells at sub-lethal concentrations (50 µM) initiated apoptosis at 6 h as observed by annexin-V staining assay and at 24 h by DNA fragmentation assay. Caspase-8 activity was also induced in PC3 cells by 34% at 6 h under these conditions. Our mechanistic studies demonstrated that the TQ growth inhibitory and apoptotic effects may be attributed to inhibition of Akt phosphorylation and generation of ROS in the AR naive PC3 cells. Mitochondria have a key role in the survival of cells by producing ATP but could also contribute to cellular damage by producing ROS. We have shown that the antioxidant effects of TQ are observed only at lower concentrations (<20 μM), which are non-toxic to PCa cells. At cytotoxic concentrations (50 μM), TQ induced mitochondrial oxidative stress as observed by accumulation (>50%) of ROS in PC3 cells at 24 h. At higher concentrations TQ induced a concentration dependent increase in ROS by 164% at 100 μM within 2 h. Human apoptotic microarray analysis of PC3 cells, upon treatment with TQ (50 µM) only for 2 h, indicated a 2-fold decrease in the mRNA levels of Akt1 and Bcl2L1, whereas the levels of BAX and GADD45 mRNA were increased by 2-fold. Furthermore, TQ inhibited pAkt formation at 25 and 50 µM concentrations in PC3 cells in a concentration dependent manner as observed by western blot analysis suggesting a direct action of TQ at this step since the expression of Akt itself was not abrogated. This was further confirmed by monitoring the levels of p-GSK3α formation that were significantly inhibited in cell-free lysates of PC3 cells pretreated (24 hr) with TQ in the presence of GSK3α and ATP. Under these conditions, TQ also inhibited the PMA stimulated nuclear p65 translocation by 50% in PC3 cells, suggesting its role in inhibiting NF-κB activation. Overall, these results suggest that TQ induced cell death in the AR naive PC3 cells occurs through generation of ROS and inhibition of Akt phosphorylation.