BH3 mimetic-elicited Ca(2+) signals in pancreatic acinar cells are dependent on Bax and can be reduced by Ca(2+)-like peptides

BH3 mimetics are small-molecule inhibitors of B-cell lymphoma-2 (Bcl-2) and Bcl-xL, which disrupt the heterodimerisation of anti- and pro-apoptotic Bcl-2 family members sensitising cells to apoptotic death. These compounds have been developed as anti-cancer agents to counteract increased levels of Bcl-2 proteins often present in cancer cells. Application of a chemotherapeutic drug supported with a BH3 mimetic has the potential to overcome drug resistance in cancers overexpressing anti-apoptotic Bcl-2 proteins and thus increase the success rate of the treatment. We have previously shown that the BH3 mimetics, BH3I-2′ and HA14-1, induce Ca(2+) release from intracellular stores followed by a sustained elevation of the cytosolic Ca(2+) concentration. Here we demonstrate that loss of Bax, but not Bcl-2 or Bak, inhibits this sustained Ca(2+) elevation. What is more, in the absence of Bax, thapsigargin-elicited responses were decreased; and in two-photon-permeabilised bax(−/−) cells, Ca(2+) loss from the ER was reduced compared to WT cells. The Ca(2+)-like peptides, CALP-1 and CALP-3, which activate EF hand motifs of Ca(2+)-binding proteins, significantly reduced excessive Ca(2+) signals and necrosis caused by two BH3 mimetics: BH3I-2′ and gossypol. In the presence of CALP-1, cell death was shifted from necrotic towards apoptotic, whereas CALP-3 increased the proportion of live cells. Importantly, neither of the CALPs markedly affected physiological Ca(2+) signals elicited by ACh, or cholecystokinin. In conclusion, the reduction in passive ER Ca(2+) leak in bax(−/−) cells as well as the fact that BH3 mimetics trigger substantial Ca(2+) signals by liberating Bax, indicate that Bax may regulate Ca(2+) leak channels in the ER. This study also demonstrates proof-of-principle that pre-activation of EF hand Ca(2+)-binding sites by CALPs can be used to ameliorate excessive Ca(2+) signals caused by BH3 mimetics and shift necrotic death towards apoptosis.