Sensitization for γ-Irradiation-Induced Apoptosis by Smac.

Abstract

Smac is released from mitochondria during the onset of apoptosis and promotes apoptosis via abrogating the binding of Inhibitor of Apoptosis Proteins (IAPs) to caspases. γ-irradiation is one of the most commonly used therapeutic approaches in clinical oncology, which triggers cell death in tumors via DNA and/or membrane damage. Since we recently found that Smac agonists sensitized even resistant tumors for apoptosis induced by death receptor ligation or anticancer drugs, we investigated the effect of Smac agonists on apoptosis following γ-irradiation in the present study. Here, we report for the first time that overexpression of mitochondrial or cytosolic Smac significantly increased radiosensitivity of various cancers.Transfection-enforced expression of Smac strongly enhanced apoptosis upon γ-irradiation in SH-EP neuroblastoma cells, which were resistant to g-irradiation in the absence of Smac. Importantly, Smac overexpression also reduced clonogenic tumor cell survival following γ-irradiation. Analysis of signaling pathways revealed that overexpression of Smac resulted in more rapid and more potent activation of caspase pathways, e.g caspase-2, -3,- 8, -9. The broad range caspase inhibitor zVAD.fmk abrogated apoptosis upon γ-irradiation indicating that apoptosis was mediated by caspases. In addition, overexpression of Smac promoted loss of mitochondrial membrane potential and cytochrome c release upon γ-irradiation. Interestingly, γ-irradiation-induced mitochondrial perturbations were blocked in the presence of the caspase inhibitor zVAD.fmk suggesting that caspase activity was required for mitochondrial alterations in response to γ-irradiation. Notably, cell cycle alterations and activation of NF-κB occured in a similar manner in vector control and Smac-transfected cells suggesting that Smac did not significantly alter the initial cellular stress response upon γ-irradiation. Importantly, Smac overexpression sensitized various tumor cell lines for γ-irradiation-induced apoptosis, indicating that the sensitization effect of Smac for γ-irradiation was not restricted to a particular cell type. By demonstrating that Smac can sensitize various tumor cells towards γ-irradiation-induced cell death, our findings provide for the first time evidence that Smac agonists may be a useful tool to enhance radiosensitivity in a variety of human cancers.