Abstract
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.
Highlights
BH3 mimetics are small-molecule inhibitors of B-cell lymphoma-2 (Bcl-2) and Bcl-xL, which disrupt the heterodimerisation of antiand pro-apoptotic Bcl-2 family members sensitising cells to apoptotic death
We have previously shown that the BH3 mimetics, BH3 inhibitors (BH3Is)-2′ and HA14-1, induce Ca2+ release from intracellular stores followed by a sustained elevation of the cytosolic Ca2+ concentration
Ca2+ responses to BH3 mimetics in pancreatic acinar cells are dependent on Bax
Summary
BH3 mimetics are small-molecule inhibitors of B-cell lymphoma-2 (Bcl-2) and Bcl-xL, which disrupt the heterodimerisation of antiand pro-apoptotic Bcl-2 family members sensitising cells to apoptotic death These compounds have been developed as anticancer agents to counteract increased levels of Bcl-2 proteins often present in cancer cells. This study 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. Disrupted regulation of apoptosis is the hallmark of carcinogenesis allowing accumulation of further genetic mutations and acquisition of metastatic properties.[1] Cancer cells often express increased levels of anti-apoptotic Bcl-2 (B-cell lymphoma-2) proteins, which provides additional protection against cell death signals[2,3] correlating with chemotherapy resistance and poor prognosis for patients.[4]. Pharmacological suppression of anti-apoptotic Bcl-2 members leading to activation of Bax and Bak should, in principle, be capable of recovering the programmed cell death.[7,8]
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