PO-062 BAX and BAK interaction with the mitochondrial permeability transition pore (MPTP) is required for taxol-mediated apoptosis

Introduction Microtubule Interfering Agents (MIA’s), such as Taxol (Paclitaxel) are used in the treatment of cancers such as breast, ovarian and non–small cell lung cancer. MIA’s arrest cells in mitosis by activating the mitotic checkpoint. Prolonged mitotic arrest results in apoptotic cell death although the precise mechanism of Taxol-induced cell death is unclear. Previous studies have shown that Taxol activates the pro-apoptotic effector proteins, Bak and Bax, which accumulate at the mitochondrial outer membrane. At the mitochondrion Bax and Bak are thought to oligomerize and/or interact with the Mitochondrial Permeability Transition Pore (MPTP) to increase the permeability of the mitochondrial outer membrane which then leads to cytochrome c release and apoptosis. In this study we have examined the requirement for Bax and Bak in Taxol-induced apoptosis and their possible interaction with components of the MPTP. Material and methods Human cervical carcinoma (HeLa) cells were treated with either Bax, Bak or both Bax and Bak siRNA, synchronised and treated with Taxol (60 nM) for varying times (0–24 hour). Apoptosis was assessed using either cytokeratin 18 cleavage (M30 antibody), poly (ADP-ribose) polymerase (PARP) cleavage or activation of pro-caspases (3 and 9). Bak and Bax were immunoprecipitated using anti-active Bak (N-20) and anti-active Bax (6A7) antibodies in CHAPS lysis buffer. To identify interacting proteins, Bak and Bax IPs were subjected to peptide mass fingerprint analysis by mass spectrometry. Confocal microscopy was used to examine the intracellular localization of Bax and Bak following Taxol treatment. Results and discussions The results of our siRNA studies indicate that although Bax and Bak can form homo-oligomers in the absence of each other, both proteins are required for Taxol-induced apoptosis. Our immunofluorescence study shows that Bak and Bax co-localise at mitochondria in the Taxol-arrested cells. Our proteomic and co-IP analyses indicate that Bak and Bax form a complex specifically in the Taxol-arrested cells that also includes components of the MPTP such as the voltage dependent anion channel (VDAC), the adenine nucleotide translocator (ANT2) and Bcl-2. Conclusion We conclude that the oligomerisation of Bax and Bak is insufficient for Taxol-mediated apoptosis. However, the interaction of activated Bax and Bak with the MPTP appears to be necessary for Taxol-induced apoptosis.