Extracellular and ER-stored Ca ²⁺ contribute to BIRD-2-induced cell death in diffuse large B-cell lymphoma cells

Abstract

The anti-apoptotic protein Bcl-2 is upregulated in several cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). In a subset of these cancer cells, Bcl-2 blocks Ca ²⁺-mediated apoptosis by suppressing the function of inositol 1,4,5-trisphosphate (IP ₃) receptors (IP ₃Rs) located at the endoplasmic reticulum (ER). A peptide tool, called Bcl-2/IP ₃ receptor disruptor-2 (BIRD-2), was developed to disrupt Bcl-2/IP ₃R complexes, triggering pro-apoptotic Ca ²⁺ signals and killing Bcl-2-dependent cancer cells. In DLBCL cells, BIRD-2 sensitivity depended on the expression level of IP ₃R2 channels and constitutive IP ₃ signaling downstream of the B-cell receptor. However, other cellular pathways probably also contribute to BIRD-2-provoked cell death. Here, we examined whether BIRD-2-induced apoptosis depended on extracellular Ca ²⁺ and more particularly on store-operated Ca ²⁺ entry (SOCE), a Ca ²⁺-influx pathway activated upon ER-store depletion. Excitingly, DPB162-AE, a SOCE inhibitor, suppressed BIRD-2-induced cell death in DLBCL cells. However, DPB162-AE not only inhibits SOCE but also depletes the ER Ca ²⁺ store. Treatment of the cells with YM-58483 and GSK-7975A, two selective SOCE inhibitors, did not protect against BIRD-2-induced apoptosis. Similar data were obtained by knocking down STIM1 using small interfering RNA. Yet, extracellular Ca ²⁺ contributed to BIRD-2 sensitivity in DLBCL, since the extracellular Ca ²⁺ buffer ethylene glycol tetraacetic acid (EGTA) blunted BIRD-2-triggered apoptosis. The protective effects observed with DPB162-AE are likely due to ER Ca ²⁺-store depletion, since a similar protective effect could be obtained using the sarco/endoplasmic reticulum Ca ²⁺-ATPase inhibitor thapsigargin. Thus, both the ER Ca ²⁺-store content and extracellular Ca ²⁺, but not SOCE, are critical factors underlying BIRD-2-provoked cell death.