PURPOSE Despite adequately expressing functional receptors for tumor necrosis factor receptor apoptosis-inducing ligand (TRAIL), many cultured tumor cells are refractory to the cytotoxic effect of this ligand. Cytotoxic chemotherapeutic drugs have been shown to synergize with Apo2L/TRAIL to mediate apoptosis in cancer cells. The main goal of this study was to evaluate the effect of either cisplatin or paclitaxel, two common used chemotherapeutic agents for solid tumors, on enhancing Apo2L/TRAIL cytotoxicity in a panel-cultured thoracic cancer cells and to examine the role of the mitochondria-dependent caspase activation cascade in mediating apoptosis of combination-treated cells. METHODS Cultured thoracic cancer cells were treated with cisplatin/Apo2L/TRAIL or paclitaxel/Apo2L/TRAIL sequential combinations in vitro. Cell viability and apoptosis were determined by 4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays. Stable transfectants expressing high levels of Bcl-2 were created by retroviral gene transfer. Specific proteolytic activity of caspases 3, 6, 8, and 9 were measured by commercially available kits using fluorescent substrates. RESULTS All cell lines preferentially expressed high levels of DR4 and/or DR5 and low levels of DcR1/DcR2; all of which were not altered by chemotherapeutic drug treatments. Pretreatment of these cancer cells with sublethal concentrations of either cisplatin or paclitaxel increased their susceptibility to Apo2L/TRAIL by twofold to > 20-fold. Profound synergistic induction of apoptosis was observed in combination-treated cells. Viability of primary normal cells was affected by neither Apo2L/TRAIL nor the combinations of chemotherapy and Apo2L/TRAIL. Overexpression of Bcl-2 or inhibition of caspase 9 activity completely abrogated combination-induced cytotoxicity and apoptosis, indicating the essential role of the mitochondria-dependent death signaling cascade in this process. Robust activation of caspase 8 in combination-treated cells was completely suppressed either by Bcl-2 overexpression or by blocking of the activity of the mitochondria-regulated caspase 9, thus identifying the amplification feedback loop as the source of elevated caspase 8 activity. Finally, mitochondria-mediated amplification of caspase 8 activity was indispensable for complete caspase activation and full execution of apoptosis, because suppression of its activity using the selective caspase 6 inhibitor (located downstream of the caspase 3 but upstream of the caspase 8 in the feedback loop) resulted in profound suppression of not only caspase 8 activity but also those of caspases 9 and 3, as well as complete protection of cancer cells from combination-induced cytotoxicity. CONCLUSION Cisplatin or paclitaxel synergistically interacts with Apo2L/TRAIL to mediate profound induction of apoptosis. The mitochondria-dependent caspase activation cascade and the amplification feedback loop are essential for the complete execution of the cell death program. Furthermore, our data identify mitochondria as the direct target for the development of more refined strategies to enhance the therapeutic effect of Apo2L/TRAIL as an anticancer agent.
