Caspase-3 is Involved in IFN-γ- and TNF-α-Mediated MIN6 Cells Apoptosis via NF-κB/Bcl-2 Pathway

TNF-alpha and IFN-gamma are the major pro-inflammatory cytokines in the beta-cell destruction. However, the underlying mechanism remains unclear. The present study used a murine insulinoma cell line MIN6 for further investigation of the effect of Caspase-3 on the cytokines-induced pancreatic beta-cell apoptosis and analyzed the mechanisms involved in the activation of Caspase-3. It was showed that the combination of IFN-gamma and TNF-alpha significantly reduced the viability of MIN6 cells and the observed cells growth inhibition was due to cell apoptosis as judged by the morphological changes under a confocal laser scanning microscopy and FACS assay of Annexin-V/7-AAD double staining. Accompanying with NF-kappa B activation and Bcl-2 downregulation, both the cleaved Caspase-3 and PARP, a known substrate of Caspase-3 in vivo, were observed at 24 and 12 h, respectively, after cells exposure to IFN-gamma and TNF-alpha treatment. Pretreatment of Caspase-3 inhibitors remarkably attenuated IFN-gamma- and TNF-alpha-induced cells apoptosis. Inhibition of NF-kappa B activation led to the increase in Bcl-2 expression, a significant attenuation in Caspase-3 activity, and an obvious amelioration in cells viability in IFN-gamma- and TNF-alpha-treated MIN6 cells. Taken together, our results indicate that Caspase-3 is critical for the induction of MIN6 cells apoptosis and it's activation is further confirmed to be related to the NF-kappa B-mediated Bcl-2 downregulation, which may be the underlying mechanism of IFN-gamma- and TNF-alpha-mediated MIN6 cells apoptosis.