H2O2 accumulation is a crucial step for the cytotoxicity of Paclitaxel

5607

The cytotoxicity of anticancer chemotherapy is largely conditioned by the molecular pathways involved following DNA damage, especially induction of apoptosis. Various anticancer agents, such as cisplatin and 5-FU, acting through independent molecular targets, were previously found able to enhance the production of reactive oxygen species (ROS). We hypothesized that the formation of ROS might play a critical role in the cellular response to taxanes-induced damages, downstream from microtubule assembly. We studied the cytotoxicity of paclitaxel (PCX) in presence and in absence of ROS modulators. The A549 human lung cancer cell line was exposed to various concentrations of PCX for 48h. Cell survival was measured using MTT assay. The IC50 of PCX was 5μM. The variations in H2O2 and O2^.- production were monitored by oxidation of fluorescent probes H2DCFDA and DHE, respectively. Glutathione (GSH) concentration was determined by colorimetric micro-GSH assay. PCX induced a concentration-dependent ROS production : at IC50, a 1.8- and 1.3-fold increase in O2^.- and H2O2 levels, respectively, were detected, while the GSH intracellular pool was depleted to 20% of baseline levels. The co-exposure to either 1.6 mM N-acetyl-cysteine (NAC), a GSH reductase mimetic, or 12 mM reduced GSH, resulted in a 2-fold decrease in H2O2 levels resulting in a 2-fold increase in PCX resistance. The protection of NAC was concentration-dependent and correlated with the extent of H2O2 depletion. Conversely, Buthionine Sulfoximine (1.1 μM), a GSH-reductase inhibitor, enhanced H2O2 accumulation and increased by 2 the sensitivity to PCX. CuDIPS (0.4 mM), a Superoxyde-Dismutase mimetic, which catalyses O2^.- transformation into H2O2, induced 1.6-fold decrease in O2- concentration and enhanced PCX sensitivity, with a 1.6 fold- reduction in PCX IC50. The catalase inhibitor Amino-Triazole (6 mM), also increased by 2 PCX sensitivity. The intracellular location of PCX-induced ROS production was studied by measuring O2^.- levels by FACS in presence or in absence of either inhibitors of mitochondrial respiratory chain (rotenone and antimycin), or an inhibitor of xanthine oxydase, (allopurinol), or an inhibitor of the cytoplasmic NADPH oxydase (DPI). DPI induced a 20% decrease in ROS levels in A549 cells exposed to PCX while the other inhibitors had no detectable effect. Experiments in mice with xenograft are ongoing to study the role of ROS production in vivo. In conclusion, PCX induces an intracytoplasmic production of O2^.-, resulting in H2O2 accumulation, which appears as a crucial step for its cytotoxicity.