BH3 mimetic-elicited Ca2+ signals in pancreatic acinar cells are dependent on Bax and can be reduced by Ca2+-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 CA²⁺ release from intracellular stores followed by a sustained elevation of the cytosolic CA²⁺ concentration. Here we demonstrate that loss of Bax, but not Bcl-2 or Bak, inhibits this sustained CA²⁺ elevation. What is more, in the absence of Bax, thapsigargin-elicited responses were decreased; and in two-photon-permeabilised Bax^-/- cells, CA²⁺ loss from the ER was reduced compared to WT cells. The CA²⁺-like Peptides, CALP-1 and CALP-3, which activate EF hand motifs of CA²⁺-binding proteins, significantly reduced excessive CA²⁺ 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 CA²⁺ signals elicited by ACh, or cholecystokinin. In conclusion, the reduction in passive ER CA²⁺ leak in Bax^-/- cells as well as the fact that BH3 mimetics trigger substantial CA²⁺ signals by liberating Bax, indicate that Bax may regulate CA²⁺ leak channels in the ER. This study also demonstrates proof-of-principle that pre-activation of EF hand CA²⁺-binding sites by CALPs can be used to ameliorate excessive CA²⁺ signals caused by BH3 mimetics and shift necrotic death towards Apoptosis.