Danya Ben-Hail

VDAC, a multi-functional mitochondrial protein as a pharmacological target.

Regulation of mitochondrial physiology requires an efficient exchange of molecules between mitochondria and the cytoplasm via the outer mitochondrial membrane (OMM). The voltage-dependent anion channel (VDAC) lies in the OMM and forms a common pathway for the exchange of metabolites between the mitochondria and the cytosol, thus playing a crucial role in the regulation of metabolic and energetic functions of mitochondria. VDAC is also recognized to function in mitochondria-mediated apoptosis and in apoptosis regulation via interaction with anti-apoptotic proteins, namely members of Bcl-2 family, and the pro-survival protein, hexokinase, overexpressed in many cancer types. Thus, VDAC appears to be a convergence point for a variety of cell survival and cell death signals, mediated by its association with various ligands and proteins. In this article, we review mammalian VDAC, specifically focusing on VDAC1, addressing its functions in cell life and the regulation of apoptosis and its involvement in several diseases. Additionally, we provide insight into the potential of VDAC1 as a rational target for novel therapeutics.

Mediation of the Antiapoptotic Activity of Bcl-xL Protein upon Interaction with VDAC1 Protein

Background: Bcl-xL is overexpressed in cancer, contributing to resistance to chemotherapy. Results: Bcl-xL directly interacts with VDAC1 to mediate its antiapoptotic activity, activity that can be prevented by VDAC1-based peptides. Conclusion: Bcl-xL regulates apoptosis through direct interaction with VDAC1. Significance: Interfering with the interaction of Bcl-xL with VDAC1 can lead to apoptosis and potentiate the efficacy of conventional chemotherapeutics. The mitochondrial protein, the voltage-dependent anion channel (VDAC), is implicated in the control of apoptosis, including via its interaction with the pro- and antiapoptotic proteins. We previously demonstrated the direct interaction of Bcl2 with VDAC, leading to reduced channel conductance. VDAC1-based peptides interacted with Bcl2 to prevent its antiapoptotic activity. Here, using a variety of approaches, we show the interaction of the antiapoptotic protein, Bcl-xL, with VDAC1 and reveal that this interaction mediates Bcl-xL protection against apoptosis. C-terminally truncated Bcl-xL(Δ21) interacts with purified VDAC1, as revealed by microscale thermophoresis and as reflected in the reduced channel conductivity of bilayer-reconstituted VDAC1. Overexpression of Bcl-xL prevented staurosporine-induced apoptosis in cells expressing native VDAC1 but not certain VDAC1 mutants. Having identified mutations in VDAC1 that interfere with the Bcl-xL interaction, certain peptides representing VDAC1 sequences, including the N-terminal domain, were designed and generated as recombinant and synthetic peptides. The VDAC1 N-terminal region and two internal sequences were found to bind specifically, and in a concentration- and time-dependent manner, to immobilized Bcl-xL(Δ21), as revealed by surface plasmon resonance. Moreover, expression of the recombinant peptides in cells overexpressing Bcl-xL prevented protection offered by the protein against staurosporine-induced apoptosis. These results point to Bcl-xL acting as antiapoptotic protein, promoting tumor cell survival via binding to VDAC1. These findings suggest that interfering with Bcl-xL binding to the mitochondria by VDAC1-based peptides may serve to induce apoptosis in cancer cells and to potentiate the efficacy of conventional chemotherapeutic agents.

The mitochondrial voltage-dependent anion channel 1 in tumor cells ☆

VDAC1 is found at the crossroads of metabolic and survival pathways. VDAC1 controls metabolic cross-talk between mitochondria and the rest of the cell by allowing the influx and efflux of metabolites, ions, nucleotides, Ca2+ and more. The location of VDAC1 at the outer mitochondrial membrane also enables its interaction with proteins that mediate and regulate the integration of mitochondrial functions with cellular activities. As a transporter of metabolites, VDAC1 contributes to the metabolic phenotype of cancer cells. Indeed, this protein is over-expressed in many cancer types, and silencing of VDAC1 expression induces an inhibition of tumor development. At the same time, along with regulating cellular energy production and metabolism, VDAC1 is involved in the process of mitochondria-mediated apoptosis by mediating the release of apoptotic proteins and interacting with anti-apoptotic proteins. The engagement of VDAC1 in the release of apoptotic proteins located in the inter-membranal space involves VDAC1 oligomerization that mediates the release of cytochrome c and AIF to the cytosol, subsequently leading to apoptotic cell death. Apoptosis can also be regulated by VDAC1, serving as an anchor point for mitochondria-interacting proteins, such as hexokinase (HK), Bcl2 and Bcl-xL, some of which are also highly expressed in many cancers. By binding to VDAC1, HK provides both a metabolic benefit and apoptosis-suppressive capacity that offer the cell a proliferative advantage and increase its resistance to chemotherapy. Thus, these and other functions point to VDAC1 as an excellent target for impairing the re-programed metabolism of cancer cells and their ability to evade apoptosis. Here, we review current evidence pointing to the function of VDAC1 in cell life and death, and highlight these functions in relation to both cancer development and therapy. In addressing the recently solved 3D structures of VDAC1, this review will point to structure-function relationships of VDAC as critical for deciphering how this channel can perform such a variety of roles, all of which are important for cell life and death. Finally, this review will also provide insight into VDAC function in Ca2+ homeostasis, protection against oxidative stress, regulation of apoptosis and involvement in several diseases, as well as its role in the action of different drugs. We will discuss the use of VDAC1-based strategies to attack the altered metabolism and apoptosis of cancer cells. These strategies include specific siRNA able to impair energy and metabolic homeostasis, leading to arrested cancer cell growth and tumor development, as well VDAC1-based peptides that interact with anti-apoptotic proteins to induce apoptosis, thereby overcoming the resistance of cancer cell to chemotherapy. Finally, small molecules targeting VDAC1 can induce apoptosis. VDAC1 can thus be considered as standing at the crossroads between mitochondrial metabolite transport and apoptosis and hence represents an emerging cancer drug target. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

Structure-based analysis of VDAC1: N-terminus location, translocation, channel gating and association with anti-apoptotic proteins.

Structural studies place the VDAC1 (voltage-dependent anion channel 1) N-terminal region within the channel pore. Biochemical and functional studies, however, reveal that the N-terminal domain is cytoplasmically exposed. In the present study, the location and translocation of the VDAC1 N-terminal domain, and its role in voltage-gating and as a target for anti-apoptotic proteins, were addressed. Site-directed mutagenesis and cysteine residue substitution, together with a thiol-specific cross-linker, served to show that the VDAC1 N-terminal region exists in a dynamic equilibrium, located within the pore or exposed outside the β-barrel. Using a single cysteine-residue-bearing VDAC1, we demonstrate that the N-terminal region lies inside the pore. However, the same region can be exposed outside the pore, where it dimerizes with the N-terminal domain of a second VDAC1 molecule. When the N-terminal region α-helix structure was perturbed, intra-molecular cross-linking was abolished and dimerization was enhanced. This mutant also displays reduced voltage-gating and reduced binding to hexokinase, but not to the anti-apoptotic proteins Bcl-2 and Bcl-xL. Replacing glycine residues in the N-terminal domain GRS (glycine-rich sequence) yielded less intra-molecular cross-linked product but more dimerization, suggesting that GRS provides the flexibility needed for N-terminal translocation from the internal pore to the channel face. N-terminal mobility may thus contribute to channel gating and interaction with anti-apoptotic proteins.

The role of calcium in VDAC1 oligomerization and mitochondria-mediated apoptosis

The voltage-dependent anion channel (VDAC), located at the outer mitochondria membrane (OMM), mediates interactions between mitochondria and other parts of the cell by transporting anions, cations, ATP, Ca(2+), and metabolites. Substantial evidence points to VDAC1 as being a key player in apoptosis, regulating the release of apoptogenic proteins from mitochondria, such as cytochrome c, and interacting with anti-apoptotic proteins. Recently, we demonstrated that VDAC1 oligomerization is a general mechanism common to numerous apoptogens acting via different initiating cascades and proposed that a protein-conducting channel formed within a VDAC1 homo/hetero oligomer mediates cytochrome c release. However, the molecular mechanism responsible for VDAC1 oligomerization remains unclear. Several studies have shown that mitochondrial Ca(2+) is involved in apoptosis induction and that VDAC1 possesses Ca(2+)-binding sites and mediates Ca(2+) transport across the OMM. Here, the relationship between the cellular Ca(2+) level, [Ca(2+)]i, VDAC1 oligomerization and apoptosis was studied. Decreasing [Ca(2+)]i using the cell-permeable Ca(2+) chelating reagent BAPTA-AM was found to inhibit VDAC1 oligomerization and apoptosis, while increasing [Ca(2+)]i using Ca(2+) ionophore resulted in VDAC1 oligomerization and apoptosis induction in the absence of apoptotic stimuli. Moreover, induction of apoptosis elevated [Ca(2+)]i, concomitantly with VDAC1 oligomerization. AzRu-mediated inhibition of mitochondrial Ca(2+) transport decreased VDAC1 oligomerization, suggesting that mitochondrial Ca(2+) is required for VDAC1 oligomerization. In addition, increased [Ca(2+)]i levels up-regulate VDAC1 expression. These results suggest that Ca(2+) promotes VDAC1 oligomerization via activation of a yet unknown signaling pathway or by increasing VDAC1 expression, leading to apoptosis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Expression of a truncated active form of VDAC1 in lung cancer associates with hypoxic cell survival and correlates with progression to chemotherapy resistance

Resistance to chemotherapy-induced apoptosis of tumor cells represents a major hurdle to efficient cancer therapy. Although resistance is a characteristic of tumor cells that evolve in a low oxygen environment (hypoxia), the mechanisms involved remain elusive. We observed that mitochondria of certain hypoxic cells take on an enlarged appearance with reorganized cristae. In these cells, we found that a major mitochondrial protein regulating metabolism and apoptosis, the voltage-dependent anion channel 1 (VDAC1), was linked to chemoresistance when in a truncated (VDAC1-ΔC) but active form. The formation of truncated VDAC1, which had a similar channel activity and voltage dependency as full-length, was hypoxia-inducible factor-1 (HIF-1)-dependent and could be inhibited in the presence of the tetracycline antibiotics doxycycline and minocycline, known inhibitors of metalloproteases. Its formation was also reversible upon cell reoxygenation and associated with cell survival through binding to the antiapoptotic protein hexokinase. Hypoxic cells containing VDAC1-ΔC were less sensitive to staurosporine- and etoposide-induced cell death, and silencing of VDAC1-ΔC or treatment with the tetracycline antibiotics restored sensitivity. Clinically, VDAC1-ΔC was detected in tumor tissues of patients with lung adenocarcinomas and was found more frequently in large and late-stage tumors. Together, our findings show that via induction of VDAC1-ΔC, HIF-1 confers selective protection from apoptosis that allows maintenance of ATP and cell survival in hypoxia. VDAC1-ΔC may also hold promise as a biomarker for tumor progression in chemotherapy-resistant patients.

Ca2 +-mediated regulation of VDAC1 expression levels is associated with cell death induction

VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c (Cyto c) release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release. As VDAC1 transports Ca(2+) across the OMM and because Ca(2+) has been implicated in apoptosis induction, we addressed the relationship between cytosolic Ca(2+) levels ([Ca(2)(+)]i), VDAC1 oligomerization and apoptosis induction. We demonstrate that different apoptosis inducers elevate cytosolic Ca(2+) and induce VDAC1 over-expression. Direct elevation of [Ca(2+)]i by the Ca(2+)-mobilizing agents A23187, ionomycin and thapsigargin also resulted in VDAC1 over-expression, VDAC1 oligomerization and apoptosis. In contrast, decreasing [Ca(2+)]i using the cell-permeable Ca(2+)-chelating reagent BAPTA-AM inhibited VDAC1 over-expression, VDAC1 oligomerization and apoptosis. Correlation between the increase in VDAC1 levels and oligomerization, [Ca(2+)]i levels and apoptosis induction, as induced by H2O2 or As2O3, was also obtained. On the other hand, cells transfected to overexpress VDAC1 presented Ca(2+)-independent VDAC1 oligomerization, cytochrome c release and apoptosis, suggesting that [Ca(2+)]i elevation is not a pre-requisite for apoptosis induction when VDAC1 is over-expressed. The results suggest that Ca(2+) promotes VDAC1 over-expression by an as yet unknown signaling pathway, leading to VDAC1 oligomerization, ultimately resulting in apoptosis. These findings provide a new insight into the mechanism of action of existing anti-cancer drugs involving induction of VDAC1 over-expression as a mechanism for inducing apoptosis. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy

The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.

Novel Compounds Targeting the Mitochondrial Protein VDAC1 Inhibit Apoptosis and Protect against Mitochondrial Dysfunction

Apoptosis is thought to play a critical role in several pathological processes, such as neurodegenerative diseases (i.e. Parkinsons and Alzheimers diseases) and various cardiovascular diseases. Despite the fact that apoptotic mechanisms are well defined, there is still no substantial therapeutic strategy to stop or even slow this process. Thus, there is an unmet need for therapeutic agents that are able to block or slow apoptosis in neurodegenerative and cardiovascular diseases. The outer mitochondrial membrane protein voltage-dependent anion channel 1 (VDAC1) is a convergence point for a variety of cell survival and death signals, including apoptosis. Recently, we demonstrated that VDAC1 oligomerization is involved in mitochondrion-mediated apoptosis. Thus, VDAC1 oligomerization represents a prime target for agents designed to modulate apoptosis. Here, high-throughput compound screening and medicinal chemistry were employed to develop compounds that directly interact with VDAC1 and prevent VDAC1 oligomerization, concomitant with an inhibition of apoptosis as induced by various means and in various cell lines. The compounds protected against apoptosis-associated mitochondrial dysfunction, restoring dissipated mitochondrial membrane potential, and thus cell energy and metabolism, decreasing reactive oxidative species production, and preventing detachment of hexokinase bound to mitochondria and disruption of intracellular Ca2+ levels. Thus, this study describes novel drug candidates with a defined mechanism of action that involves inhibition of VDAC1 oligomerization, apoptosis, and mitochondrial dysfunction. The compounds VBIT-3 and VBIT-4 offer a therapeutic strategy for treating different diseases associated with enhanced apoptosis and point to VDAC1 as a promising target for therapeutic intervention.

A New Fungal Diterpene Induces VDAC1-dependent Apoptosis in Bax/Bak-deficient Cells

Background: VDAC1 functions in both cellular metabolism and mitochondria-mediated apoptosis. Results: New compounds were identified that induce apoptosis by promoting VDAC1 oligomerization and apoptosis in a Bak- and Bak-independent manner. Conclusion: Bax and Bak are dispensable for VDAC1-mediated apoptosis, revealing a novel mechanism of apoptosis involving VDAC1 oligomerization. Significance: In cancers with Bax/Bak down-regulated, VDAC1-induced apoptosis offers a novel approach for tumor therapies. The pro-apoptotic Bax and Bak proteins are considered central to apoptosis, yet apoptosis occurs in their absence. Here, we asked whether the mitochondrial protein VDAC1 mediates apoptosis independently of Bax/Bak. Upon screening a fungal secondary metabolite library for compounds inducing apoptosis in Bax/Bak-deficient mouse embryonic fibroblasts, we identified cyathin-R, a new cyathane diterpenoid compound able to activate apoptosis in the absence of Bax/Bak via promotion of the VDAC1 oligomerization that mediates cytochrome c release. Diphenylamine-2-carboxilic acid, an inhibitor of VDAC1 conductance and oligomerization, inhibited cyathin-R-induced VDAC1 oligomerization and apoptosis. Similarly, Bcl-2 overexpression conferred resistance to cyathin-R-induced apoptosis and VDAC1 oligomerization. Silencing of VDAC1 expression prevented cyathin-R-induced apoptosis. Finally, cyathin-R effectively attenuated tumor growth and induced apoptosis in Bax/Bak-deficient cells implanted into a xenograft mouse model. Hence, this study identified a new compound promoting VDAC1-dependent apoptosis as a potential therapeutic option for cancerous cells lacking or presenting inactivated Bax/Bak.