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 Ca2+-mediated apoptosis by suppressing the function of inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) located at the endoplasmic reticulum (ER). A peptide tool, called Bcl-2/IP3 receptor disruptor-2 (BIRD-2), was developed to disrupt Bcl-2/IP3R complexes, triggering pro-apoptotic Ca2+ signals and killing Bcl-2-dependent cancer cells. In DLBCL cells, BIRD-2 sensitivity depended on the expression level of IP3R2 channels and constitutive IP3 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 Ca2+ and more particularly on store-operated Ca2+ entry (SOCE), a Ca2+-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 Ca2+ 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 Ca2+ contributed to BIRD-2 sensitivity in DLBCL, since the extracellular Ca2+ buffer ethylene glycol tetraacetic acid (EGTA) blunted BIRD-2-triggered apoptosis. The protective effects observed with DPB162-AE are likely due to ER Ca2+-store depletion, since a similar protective effect could be obtained using the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. Thus, both the ER Ca2+-store content and extracellular Ca2+, but not SOCE, are critical factors underlying BIRD-2-provoked cell death.
Highlights
Cell death and survival is regulated by the Bcl-2-protein family, which consists of pro-apoptotic and anti-apoptotic family members[1]
We explored this hypothesis by focusing on the BIRD-2sensitive diffuse large B-cell lymphoma (DLBCL) cell model, SU-DHL-416, in which we studied the impact of the store-operated Ca2+ entry (SOCE) inhibitor DPB162-AE on the Bcl-2/IP3 receptor disruptor-2 (BIRD-2)-induced Ca2+ rise and cell death response
The BIRD-2-provoked cytosolic Ca2+ rise is quantified by measuring the peak amplitude (Δ F340/F380), shown in g, and the time constant τ (s), which is shown in h. i Representative scatter plots from flow cytometry analysis detecting apoptosis in SU-DHL-4 cells stained with Annexin V-FITC and 7-aminoactinomycin D (7-AAD)
Summary
Cell death and survival is regulated by the Bcl-2-protein family, which consists of pro-apoptotic and anti-apoptotic family members[1]. The anti-apoptotic protein Bcl-2 is upregulated in a large number of cancer cells, including B-cell lymphomas like chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL)[2,3]. Bcl[2] prevents apoptotic cell death by neutralizing proapoptotic family members, including the executioner proteins Bak and Bax and the BH3-only protein Bim, at the mitochondria[4,5]. BH3-mimetic compounds, like venetoclax, disrupt the binding between Bcl-2 and proapoptotic BH3-only proteins, thereby triggering apoptotic cell death in cancer cells that depend on Bcl-2's function at the mitochondria for their survival[6,7]. There, Bcl-2 binds with its Bcl-2 homology 4 (BH4) domain to the central, modulatory domain of the inositol 1,4,5-
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