01 M to 5 M. Since the observed results for these and some subsequent experiments were relatively similar with all the doses and cell lines, and for the sake of brevity, we opted to show only data for PC3 cells treated with relatively low and high concentrations. These correspond to plasma DTX concen trations achieved in treated PCa patients during the initial hours post infusion. Both concentrations of DTX induced gradual activation of caspases 2 and 3 during treatment of PC3 cells for up to 48 h. STS and TRAIL were used as controls for caspase 2 and caspase 3 activation, respectively. Caspase 2 activity was abolished by pre treatment of cells with the specific inhibitor Ac VDVAD CHO Regorafenib , whereas both caspases were completely inhibited with the broad cas pase inhibitor Z VAD FMK. In light of this, most subsequent experiments were conducted only in the pres ence of Z VAD FMK. We observed that the caspase sub strates PARP and LEDGF p75 were cleaved into their signature apoptotic fragments of p85 and p65, respec tively, during DTX induced PC3 cell death. How ever, no cleavage was observed in the presence of Z VAD FMK, consistent with inhibition of caspase activity. To further highlight the importance of the caspase dependent pathway in DTX induced PC3 cell death we analyzed the effect of DTX on mitochondrial membrane potential. Loss of MMP was induced at both con centrations of DTX after 48 h of exposure to the drug, and was inhibited by Z VAD FMK, confirming its dependence on caspase activation. We also measured DNA fragmentation by flow cytometric analysis of DNA con tent. As observed in Fig. 1D, treatment with either 0. 1 or 3. 0 M DTX for 48 h led to significant increase in the subG1 cell population. In the presence of Z VAD FMK there was a decrease in the subG1 cell population con comitant with increase in the G2 M fraction. Taken together, the data presented in Fig. 1 indicate that both low and high doses of DTX induce caspase activation, cas pase substrate cleavage, loss of MMP, and DNA fragmen tation. In addition to these events, we also observed that DTX induced reactive oxygen species in PC3 cells, consistent with a previous report. To explore the possibility that DTX induced cell death in PCa cells may also involve a caspase independent path way, we first determined whether caspase inhibition would prevent the death of drug treated PC3 cells. Both low and high concentrations of DTX reduced cell viability as measured by crystal violet staining. However, this reduction was only partially abrogated in the presence of the caspase inhibitors Z VAD FMK and Ac VDVAD CHO, suggesting that a caspase independent pathway was also activated by DTX. Similar results were observed in DU145 and RWPE 2 cells.