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

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(3)Rs) 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(3)R 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(3)R2 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.