Involvement of calcium-mediated apoptotic signals in H2O2-induced MIN6N8a cell death

Reactive oxygen species are believed to be the central mediators of beta-cell destruction that leads to type 1 and 2 diabetes, and calcium has been reported to be an important mediator of beta cell death. In the present study, the authors investigated whether Ca2+ plays a role in hydrogen peroxide (H2O2)-induced MIN6N8a mouse beta cell death. Treatment with low concentration H2O2 (50 μM) was found to be sufficient to reduce MIN6N8a cell viability by 55%, largely via apoptosis. However, this H2O2-induced cell death was near completely blocked by pretreatment with BAPTA/AM (5 μM), a chelator of intracellular Ca2+. Moreover, the intracellular calcium store channel blockers, such as, xestospongin c and ryanodine, significant protected cells from 50 μM H2O2-induced cell death and under extracellular Ca2+-free conditions, 50 μM H2O2 elicited transient [Ca2+]i increases. In addition, pharmacologic inhibitors of calpain, calcineurin, and calcium/calmodulin-dependent protein kinase II were found to have a protective effect on H2O2-induced death. Moreover, H2O2-induced apoptotic signals, such as c-JUN N-terminal kinase activation, cytochrome c release, caspase 3 activation, and poly (ADP-ribose) polymerase cleavage were all down-regulated by the intracellular Ca2+ chelation. These findings show that [Ca2+]i elevation, possibly due to release from intracellular calcium stores and the subsequent activation of Ca2+-mediated apoptotic signals, critically mediates low concentration H2O2-induced MIN6N8a cell death. These findings suggest that a breakdown of calcium homeostasis by low level of reactive oxygen species may be involved in beta cell destruction during diabetes development.