Promote Apoptotic Protease Caspase Inhibitors Enhanced Oridonin-induced L929 Cell Death Mechanism

As we have known cysteinyl aspartate-specific proteinase caspase played a very important role in the cell apoptosis. When the activities of caspases are inhibited, the progress of cell apoptosis is blocked. Our previous studies showed that oridonin significantly induced apoptosis in several cancer cell lines, which was pronouncedly blocked by caspase inhibitors. However caspase inhibitors rendered murine fibrosarcoma L929 cells more sensitive to the oridonin. Therefore, in this research we focused on the role of caspase in oridonin-treated L929 cellsThe studies demonstrated that oridonin significantly inhibited proliferation of murine fibrosarcoma L929cells in a time- and dose-dependent manner (IC50: 65.1±1.5μmol/L). MTT assay, photomicroscopical observation, and LDH activity-based assay showed that oridonin induced both apoptotic and necrotic cell death in L929 cells. Pan-caspase inhibitor, z-VAD-fmk and caspase-3 inhibitor, z-DEVD-fmk made L929 cells more sensitive to oridonin instead of inhibition of apoptosis. Inhibitor of reactive oxygen species ROS-NAC not only significantly blocked oridonin-alone-treated cell death in L929 cells, but also suppressed the cell death induced by combined treatment of oridonin and caspase inhibitors.Treatment with oridonin in L929 cells for 2 h, the generation of ROS was markedly increased. Western blot analysis showed that ROS up-regulated the expression of tumor suppression factor p53 and proapoptotic protein Bax. In addition, the expression of antiapoptosis protein Bcl-2 was down-reguladted. The presence of z-DEVD-fmk and z-VAD-fmk resulted in a more significant increase of oridonin-mediated production of ROS. Therefore, L929 cells became even more sensitive to oridonin. Although caspase-dependent apoptotic pathway was blocked, the cell death form might be switched to caspase-independent apoptosis or necrosis. However, the cytotoxic sensitization by z-VAD-fmk was reversed by PARP inhibitor 3-AB. It suggest that PARP may play a very important role after treatment with z-VAD-fmk, however, the exactly mechanism still unclear.