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Background: VDAC1 mediates the transfer of pro-apoptotic Ca 2ϩ signals into mitochondria. Results: The BH4 domain of Bcl-XL, but not that of Bcl-2, targets VDAC1 and suppresses its pro-apoptotic Ca 2ϩ -flux properties. N-terminal VDAC1 peptide alleviates this effect of BH4-Bcl-XL. Conclusion: Bcl-XL via its BH4 domain inhibits VDAC1 activity. Significance: Bcl-2 and Bcl-XL differ in their BH4 domain biology by regulating ER and mitochondrial Ca 2ϩ -transport systems, respectively.

Anti-apoptotic Bcl-2 proteins are upregulated in different cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), enabling survival by inhibiting pro-apoptotic Bcl-2-family members and inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R)-mediated Ca 2+ -signaling. A peptide tool (Bcl-2/IP 3 R Disruptor-2; BIRD-2) was developed to abrogate the interaction of Bcl-2 with IP 3 Rs by targeting Bcl-2′s BH4 domain. BIRD-2 triggers cell death in primary CLL cells and in DLBCL cell lines. Particularly, DLBCL cells with high levels of IP 3 R2 were sensitive to BIRD-2. Here, we report that BIRD-2-induced cell death in DLBCL cells does not only depend on high IP 3 R2-expression levels, but also on constitutive IP 3 signaling, downstream of the tonically active B-cell receptor. The basal Ca 2+ level in SU-DHL-4 DLBCL cells was significantly elevated due to the constitutive IP 3 production. This constitutive IP 3 signaling fulfilled a prosurvival role, since inhibition of phospholipase C (PLC) using U73122 (2.5 µM) caused cell death in SU-DHL-4 cells. Milder inhibition of IP 3 signaling using a lower U73122 concentration (1 µM) or expression of an IP 3 sponge suppressed both BIRD-2-induced Ca 2+ elevation and apoptosis in SU-DHL-4 cells. Basal PLC/IP 3 signaling also fulfilled a pro-survival role in other DLBCL cell lines, including Karpas 422, RI-1 and SU-DHL-6 cells, whereas PLC inhibition protected these cells against BIRD-2-evoked apoptosis. Finally, U73122 treatment also suppressed BIRD-2-induced cell death in primary CLL, both in unsupported systems and in co-cultures with CD40L-expressing fibroblasts. Thus, constitutive IP 3 signaling in lymphoma and leukemia cells is not only important for cancer cell survival, but also represents a vulnerability, rendering cancer cells dependent on Bcl-2 to limit IP 3 R activity. BIRD-2 seems to switch constitutive IP 3 signaling from pro-survival into pro-death, presenting a plausible therapeutic strategy.

Calcium ions (Ca2+) are crucial, ubiquitous, intracellular second messengers required for functional mitochondrial metabolism during uncontrolled proliferation of cancer cells. The mitochondria and the endoplasmic reticulum (ER) are connected via “mitochondria-associated ER membranes” (MAMs) where ER–mitochondria Ca2+ transfer occurs, impacting the mitochondrial biology related to several aspects of cellular survival, autophagy, metabolism, cell death sensitivity, and metastasis, all cancer hallmarks. Cancer cells appear addicted to these constitutive ER–mitochondrial Ca2+ fluxes for their survival, since they drive the tricarboxylic acid cycle and the production of mitochondrial substrates needed for nucleoside synthesis and proper cell cycle progression. In addition to this, the mitochondrial Ca2+ uniporter and mitochondrial Ca2+ have been linked to hypoxia-inducible factor 1α signaling, enabling metastasis and invasion processes, but they can also contribute to cellular senescence induced by oncogenes and replication. Finally, proper ER–mitochondrial Ca2+ transfer seems to be a key event in the cell death response of cancer cells exposed to chemotherapeutics. In this review, we discuss the emerging role of ER–mitochondrial Ca2+ fluxes underlying these cancer-related features.

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The inositol 1,4,5-trisphosphate (IP) receptor (IPR) is a ubiquitously expressed Ca-release channel localized in the endoplasmic reticulum (ER). The intracellular Ca signals originating from the activation of the IPR regulate multiple cellular processes including the control of cell death versus cell survival via their action on apoptosis and autophagy. The exact role of the IPRs in these two processes does not only depend on their activity, which is modulated by the cytosolic composition (Ca, ATP, redox status, …) and by various types of regulatory proteins, including kinases and phosphatases as well as by a number of oncogenes and tumor suppressors, but also on their intracellular localization, especially at the ER-mitochondrial and ER-lysosomal interfaces. At these interfaces, Ca microdomains are formed, in which the Ca concentration is finely regulated by the different ER, mitochondrial and lysosomal Ca-transport systems and also depends on the functional and structural interactions existing between them. In this review, we therefore discuss the most recent insights in the role of Ca signaling in general, and of the IPR in particular, in the control of basal mitochondrial bioenergetics, apoptosis, and autophagy at the level of inter-organellar contact sites.

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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.