The depolarization of mitochon drial membrane potential increased with time upon in cubation with the indicated concentrations of fungal taxol and baccatin III. In contrast, control and vehicle treated samples did not exhibit significant loss in mitochondrial membrane potential. Thus, it can be con cluded that activation of the mitochondrial apoptotic machinery AZD9291 astrazeneca occurs in Jurkat cells upon fungal taxol and baccatin III exposure. Role of caspases in fungal taxol and baccatin III induced apoptosis It is well known that a family of cysteinyl proteases, called caspases, is involved in apoptotic cell death. To check the involvement of caspase 8 of extrinsic pathway in fungal taxol or baccatin III induced apoptosis, caspase 8 deficient Jurkat cells were treated with these compounds.

Both compounds induced apoptosis in 60 80% of cells after 48 h of treatment, suggesting that caspase 8 may not be involved in taxol or baccatin III induced apoptosis of Jurkat cells. We then tested whether fungal taxol or baccatin III could induce apoptosis in J16 Jurkat cells that over express the anti apoptotic protein Bcl 2. Fungal taxol or baccatin III did not induce significant apoptosis in J16 Jurkat cells even after 48 h, which suggests that Bcl 2 overexpression rescues taxol and baccatin III induced apoptosis. A comparison of percentage of apoptosis induced by fungal taxol and baccatin III in JR4 Jurkat, J16 Jurkat and caspase 8 deficient Jurkat cells is also shown. Both the compounds in duced 55 60% apoptosis in JR4 Jurkat cells, 60 70% apoptosis in caspase 8 deficient Jurkat cells, while no significant apoptosis was observed in J16 Jurkat cells.

This confirms the involvement of intrinsic mitochondrial pathway of apoptosis. To determine which of the caspases are involved in the taxol and baccatin induced apoptosis, the effect of specific inhibitors of these enzymes were examined using Jurkat cells. Cells were pre treated for 1 h with pan caspase inhibitor, caspase 3 inhibitor, caspase 2 inhibitor, caspase 9 inhibitor or caspase 10 in hibitor at various concentrations and then treated with either fungal taxol or baccatin III for 24 or 48 h in the continued presence of the respective caspase inhibitor. The cells were then stained with PI and subjected to FACS analysis. The pan caspase inhibi tor at 100 uM concentration showed complete rescue of Jurkat cells from fungal taxol and baccatin III induced apoptosis.

None of the inhibitors for caspase 3, caspase 2 or caspase 9 could siginificantly rescued Jurkat cells from apoptosis which indicated that these caspases are not involved Batimastat in fungal taxol and baccatin III induced apoptotic mechanism. But we observed a siginificant rescue of these cells at 100 uM of caspase 2 inhibitor treatment. Interestingly, caspase 10 inhibitor exhibited complete rescue of JR4 Jurkat cells from apoptosis.