The voltage-dependent anion channel-1 modulates apoptotic cell death.

The role of the voltage-dependent anion channel (VDAC) in cell death was investigated using the expression of native and mutated murine VDAC1 in U-937 cells and VDAC inhibitors. Glutamate 72 in VDAC1, shown previously to bind dicyclohexylcarbodiimide (DCCD), which inhibits hexokinase isoform I (HK-I) binding to mitochondria, was mutated to glutamine. Binding of HK-I to mitochondria expressing E72Q-mVDAC1, as compared to native VDAC1, was decreased by approximately 70% and rendered insensitive to DCCD. HK-I and ruthenium red (RuR) reduced the VDAC1 conductance but not that of E72Q-mVDAC1. Overexpression of native or E72Q-mVDAC1 in U-937 cells induced apoptotic cell death (80%). RuR or overexpression of HK-I prevented this apoptosis in cells expressing native but not E72Q-mVDAC1. Thus, a single amino-acid mutation in VDAC prevented HK-I- or RuR-mediated protection against apoptosis, suggesting the direct VDAC regulation of the mitochondria-mediated apoptotic pathway and that the protective effects of RuR and HK-I rely on their binding to VDAC.

The voltage-dependent anion channel (VDAC): function in intracellular signalling, cell life and cell death.

Research over the last decade has extended the prevailing view of mitochondria to include functions well beyond the critical bioenergetics role in supplying ATP. It is now recognized that mitochondria play a crucial role in cell signaling events, inter-organelle communication, aging, many diseases, cell proliferation and cell death. Apoptotic signal transmission to the mitochondria results in the efflux of a number of potential apoptotic regulators to the cytosol that trigger caspase activation and lead to cell destruction. Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) is involved in this release of proteins via the outer mitochondrial membrane. VDAC in the outer mitochondrial membrane is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. VDAC has been recognized as a key protein in mitochondria-mediated apoptosis since it is the proposed target for the pro- and anti-apoptotic Bcl2-family of proteins and due to its function in the release of apoptotic proteins located in the inter-membranal space. The diameter of the VDAC pore is only about 2.6-3 nm, which is insufficient for passage of a folded protein like cytochrome c. New work suggests pore formation by homo-oligomers of VDAC or hetero-oligomers composed of VDAC and pro-apoptotic proteins such as Bax or Bak. This review provides insights into the central role of VDAC in cell life and death and emphasizes its function in the regulation of mitochondria-mediated apoptosis and, thereby, its potential as a rational target for new therapeutics.

Inhibition of Ca2+ accumulation in isolated sarcoplasmic reticulum by thyroid hormones.

Thyroid hormones inhibit Ca2+ accumulation and ATPase activity of isolated sarcoplasmic reticulum vesicles. Half-maximal inhibition was obtained by about 2.5 microM. The ATP hydrolysis activity of the purified (Ca2+ + Mg2+)-ATPase or of the SR vesicles, in the presence of the Ca2+ ionophore A23187, is not inhibited by T3 or T4. Modification of T3 or T4 in the ring portion, but not in the amino portion, of the molecules results in T4 and T3 analogues which are unable to inhibit Ca2+ accumulation. T3 and T4 have no significant effect on various partial reactions of the transport cycle such as: the binding of ATP and Ca2+, or ADP-ATP exchange and E-P formation from ATP, but they inhibit the E-P formation from inorganic phosphate (Pi) and ATP-Pi exchange. The inhibition of both Ca2+ accumulation and ATPase activity by T3 or T4 is increased in the presence of Pi. Binding sites for [125I]T3 and for [125I]T4 in SR proteins were demonstrated using either equilibrium dialysis or gel overlay techniques. The results suggest that the thyroid hormones inhibit the ATP-dependent Ca2+ accumulation, probably by inhibiting the transport of anions which act as the Ca2+ precipitating anion.

The interaction of spermine with the ryanodine receptor from skeletal muscle.

The effect of polyamines on ryanodine binding activity of junctional sarcoplasmic reticulum membranes is described. Spermine stimulated the binding of ryanodine to its receptor up to 5-fold, with half-maximal stimulation obtained with 3.5 mM. Spermidine and putrescine also stimulated ryanodine binding, but they were about 12-fold less potent. The degree of stimulation is dependent on the NaCl concentration present in the assay medium. Spermine has no effect on the Ca(2+)-dependency of ryanodine binding but it increases the ryanodine binding affinity (Kd) by about 5.6-fold. Both the rate of ryanodine association with, and dissociation from, its binding site were affected by spermine. Spermine also stimulates the photoaffinity labelling by 3-O-(4-benzoyl)benzoyl[alpha-32P]ATP ([alpha-32P]BzATP) of the ryanodine receptor, increasing the BzATP binding affinity. We suggest that the stimulatory effect of spermine on ryanodine binding is due to its specific interaction with the ryanodine receptor. This spermine interaction enabled us to develop a new, one-step, fast and with high yield method for the purification of ryanodine receptor (Shoshan-Barmatz, V. and Zarka, A. (1992) Biochem. J. 284, in press).

Modulation of the skeletal muscle ryanodine receptor by endogenous phosphorylation of 160/150-kDa proteins of the sarcoplasmic reticulum.

This paper demonstrates and characterizes the inhibition of ryanodine binding caused by the phosphorylation of the 160/150-kDa proteins in skeletal muscle sarcoplasmic reticulum (SR). Inhibition of ryanodine binding was obtained by preincubation of SR membranes with ATP + NaF . The inhibition was characterized by the following findings: (a) If ATP was replaced by AdoPP[NH]P, inhibition of ryanodine binding activity was not observed. (b) The inhibitory effect of preincubation with ATP + NaF, like the phosphorylation of 150/160-kDa proteins, was Ca2+ dependent. (c) Inhibition of ryanodine binding, as the protein phosphorylation, was not observed if NaF (> 30 mM) was replaced with okadaic acid. (d) The optimal pH for the inhibition and the phosphorylation was about 7.0. (e) Both the phosphorylation of the 160/150-kDa proteins and inhibition of ryanodine binding were prevented by dichlorobenzimidazole riboside and hemin, inhibitors of casein kinase II. (f) Dephosphorylation of the 160/150-kDa proteins prevented the inhibition of ryanodine binding. (g) The presence of NP-40 during the phosphorylation prevented both the 160/150-kDa phosphorylation and the inhibition of ryanodine binding. Furthermore, a linear relationship was obtained between the degree of ryanodine binding inhibition and the level of phosphorylation of the 160/150-kDa proteins, as controlled by ATP or NaF concentrations. The binding affinity for Ca2+ of the ryanodine receptor (RyR) was modified by phosphorylation of the 160/150-kDa proteins, decreasing by up to 100-fold. The phosphorylation of the SR membranes resulted in an elimination of ryanodine binding sites with slight effect on the ryanodine binding affinity. These results suggest the modulation of the properties of the RyR by phosphorylation/dephosphorylation of the 160/150-kDa proteins. The identification of the phosphorylated 160/150-kDa proteins, their kinase, and the structural interactions between them and the RyR are presented in the accompanying paper.

Ryanodine binding to sarcoplasmic reticulum membrane; comparison between cardiac and skeletal muscle.

[3H]Ryanodine binding to skeletal muscle and cardiac sarcoplasmic reticulum (SR) vesicles was compared under experimental conditions known to inhibit or stimulate Ca2+ release. In the skeletal muscle SR, ryanodine binds to a single class of high-affinity sites (Kd of 11.3 nM). In cardiac SR vesicles, more than one class of binding sites is observed (Kd values of 3.6 and 28.1 nM). Ryanodine binding to skeletal muscle SR vesicles requires high concentrations of NaCl, whereas binding of the drug to cardiac SR is only slightly influenced by ionic strength. In the presence of 5'-adenylyl imidodiphosphate (p[NH]ppA), increased pH, and micromolar concentration of Ca2+ (which all induce Ca2+ release from SR) binding of ryanodine to SR is significantly increased in skeletal muscle, while being unchanged in cardiac muscle. Ryanodine binding to skeletal but not to cardiac muscle SR is inhibited in the presence of high Ca2+ or Mg2+ concentrations (all known to inhibit Ca2+ release from skeletal muscle SR). Ruthenium red or dicyclohexylcarbodiimide modification of cardiac and skeletal muscle SR inhibit Ca2+ release and ryanodine binding in both skeletal and cardiac membranes. These results indicate that significant differences exist in the properties of ryanodine binding to skeletal or cardiac muscle SR. Our data suggest that ryanodine binds preferably to site(s) which are accessible only when the Ca2+ release channel is in the open state.

Cross-linking of the ryanodine receptor/Ca2+ release channel from skeletal muscle.

The relationship between the tetrameric organization of the ryanodine receptor (RyR) and its activity in binding of ryanodine was approached through cross-linking studies using several bifunctional reagents, differing in their linear dimensions and flexibility, as well as in the reactivity of the active groups. Cross-linking with: 1,5-difluoro-2,4-dinitrobenzene (DFDNB); di(fluoro-3-nitrophenyl)sulfone (DFNPS), 1-ethyl-3-(3-dimethylamino)propyl)carbodiimide (EDC); dimethyl suberimidate (DMS); ethylene glycol bis(succinimidylsuccinate) (EGS); and glutaraldehyde resulted in the disappearance of the, 470 kDa, RyR monomer protein band with concomitant appearance of additional bands of molecular masses higher than the monomer. At the relatively low concentrations of the reagents and the conditions used, RyR is the only cross-linked protein of SR membranes. The 'new' protein bands cross-react with antibodies against the RyR and correspond to dimers and tetramers of the RyR subunits while trimers were not detectable. DFDNB and DFNPS produced also a 560 kDa protein band which probably represents an intramolecular cross-linked monomer. The SDS-electrophoretic patterns of the cross-linked purified RyR resemble those of the membrane-bound receptor. Ryanodine binding to the high-affinity site was inhibited by modification of SR membranes with DFDNB and DFNPS, but not with DMS, EDC, EGS and glutaraldehyde, although RyR was completely cross-linked. The inhibition by DFDNB and DFNPS is due to modification of a specific lysyl residue which is also involved in the control of Ca2+ release. On the other hand, cross linking of the RyR with glutaraldehyde or EGS resulted in inhibition of ryanodine binding to the low-affinity, but not to the high-affinity binding sites. Thus, the cross-linking of two or more sites in each monomer (which lead to fixation of dimers or tetramers) did not prevent the conformational changes involved in the binding and occlusion of ryanodine at the high-affinity site, but inhibited its binding to the low-affinity sites.

The identification of the phosphorylated 150/160-kDa proteins of sarcoplasmic reticulum, their kinase and their association with the ryanodine receptor.

In the present work we studied the relationship between the phosphorylated 150- and 160-kDa proteins and other SR proteins in the 150,000-170,000 range of molecular masses. on SDS-PAGE, the identification of their kinase, as well as the purification and structural interactions between these proteins and the rynodine receptor (RyR). The phosphorylated 150-kDa protein was identified as sarcalumenin based on: (a) its cross-reactivity with three different monoclonal antibodies specific for sarcalumenin. (b) its mobility in SDS-PAGE which was modified upon digestion with endoglycosidase H, (c) its elution from lentil-lectin column by alpha-methyl mannoside, (d) its resistance to trypsin, (e) its ability to bind Ca2+ and to stain blue with Stains-All. The phosphorylated 160-kDa protein was identified as the histidine-rich Ca2+ binding protein (HCP) based on: (a) its Ca(2+)-binding property and staining blue with Stains-All, (b) phosphorylation with the catalytic subunit of cAMP-dependent kinase. (c) its increased mobility in SDS-PAGE in the presence of Ca2+ (d) its heat stability and (e) its partial amino acid sequence. The endogenous kinase was identified as casein kinase II (CK II) based on the inhibition of the endogenous phosphorylation 160/150-kDa proteins by heparin, 5.6-dichlorobenzimidazole riboside, polyaspartyl peptide and hemin, and its ability to use [gamma-32P]GTP as the phosphate donor. The association of CK II with SR membranes, was demonstrated using specific polyclonal anti-CK II antibodies. The luminal location of CK II is suggested because CK II was extracted from the SR by l M NaCl only after their treatment with hypotonic medium, and CK II activity was inhibited with the charged inhibitors heparin and polyaspartyl peptide only after their incubation with the SR in the presence of NP-40. The 160- and 150-kDa proteins were purified on spermine-agarose column, and were phosphorylated by CK II. Like the endogenous phosphorylation of the 150/160-kDa proteins in SR. the phosphorylation of the purified proteins by CK II was inhibited by La3+ (Cl50 = 4 microM) and hemin. The results suggest the phosphorylation of the luminally located sarcalumenin and HCP with CK II.

The structure, function, and cellular regulation of ryanodine-sensitive Ca2+ release channels.

The fundamental biological process of Ca2+ signaling is known to be important in most eukaryotic cells, and inositol 1,2,5-trisphosphate and ryanodine receptors, intracellular Ca2+ release channels encoded by two distantly related gene families, are central to this phenomenon. Ryanodine receptors in the sarcoplasmic reticulum of skeletal and cardiac muscle have a predominant role in excitation-contraction coupling, but the channels are also present in the endoplasmic reticulum of noncontractile tissues including the central nervous system and the immune system. In all, three highly homologous ryanodine receptor isoforms have been identified, all very large proteins which assemble as (homo)tetramers of approximately 2 MDa. They contain large cytoplasmically disposed regulatory domains and are always associated with other structural or regulatory proteins, including calmodulin and immunophilins, which can have marked effects on channel function. The type 1 isoform in skeletal muscle is electromechanically coupled to surface membrane voltage sensors, whereas the remaining isoforms appear to be activated solely by endogenous cytoplasmic second messengers or other ligands, including Ca2+ itself ("Ca(2+)-induced Ca2+ release"). This review concentrates on ryanodine receptor structure-function relationships as probed by a variety of methods and on the molecular mechanisms of channel modulation at the cellular level (including evidence for the regulation of gene expression and transcription). It also touches on the relevance of ryanodine receptors to complex cellular functions and disease.

High affinity ryanodine binding sites in rat liver endoplasmic reticulum.

The binding of [3H]ryanodine to liver microsomal subfractions was investigated. The smooth microsomal membranes were enriched with ryanodine binding sites and also with a polypeptide of 360 kDa. Caffeine completely inhibited [3H]ryanodine binding. Ryanodine also affected the membrane Ca2+ permeability. At low concentrations (less than 10 microM) ryanodine stimulated Ca2+ efflux and at higher concentrations (greater than 50 microM) it blocked Ca2+ efflux. These results suggest that hepatic microsomes contain ryanodine binding sites which can modify the membrane permeability for Ca2+.

High-affinity Ca2+-binding site inhibiting Ca2+ release from sarcoplasmic reticulum.

Ca2+ release from sarcoplasmic reticulum membranes, activated by alkaline pH occurs only when EGTA is present in the release medium. Addition of very low concentrations of Ca2+ to the medium inhibits Ca2+ release. The concentration of free Ca2+ required for 50% inhibition ranges from between 5 and 20 nM in different experiments and/or membrane preparations, irrespective of whether the free Ca2+ concentration is controlled by EGTA or CDTA. Other divalent cations such as Mn2+, Ba2+, Cu2+, Cd2+ and Mg2+ also exert an inhibitory effect on Ca2+ release, with higher or lower potency than that of Ca2+. The inactivation of Ca2+ release by Ca2+ is reversible. We suggest the involvement of high-affinity Ca2+-binding sites in the control of Ca2+ release.

The effect of local anaesthetics on the ryanodine receptor/Ca2+ release channel of brain microsomal membranes.

The effects of various local anaesthetics (LAs) on ryanodine binding of the sheep brain ryanodine receptor were tested. Tetracaine and dibucaine inhibit the binding with half-maximal inhibition (CI50) of 0.12 mM and 0.7 mM, respectively. Lidocaine and its analog QX-314, on the other hand, stimulate the binding up to 3-fold with half-maximal stimulation occurring with about 2 mM of the drugs. Lidocaine increases both the receptor affinity for ryanodine by about 5-fold and the rate of ryanodine association with its binding site by about 6-fold. Tetracaine and lidocaine also interact with the purified brain ryanodine receptor and produce inhibitory and stimulatory effects similar to those obtained with the membrane-bound receptor. The interaction of the LAs with the brain ryanodine receptor, as well as with the skeletal muscle receptor [J. Memb. Biol. 133 (1993) 171-182], suggest that ryanodine receptor possesses intrinsic binding site(s) for LAs.

Characterization of sheep brain ryanodine receptor ATP binding site by photoaffinity labeling.

Two high Mr protein bands (440 and 420 kDa) in sheep brain microsomal membranes were labeled with the photoaffinity ATP analog, O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz2ATP). The 420 kDa band is labeled by [alpha-32P]-Bz2ATP with about 1000-fold higher affinity than the 440 kDa band. The heavily labeled 420 kDa band is identified as dynein heavy chain based on its partial amino acid sequence, and cross-reactivity with anti-dynein antibodies. The 440 kDa protein is immunologically identified as the type-2 RyR. Bz2ATP binding is obtained in the absence of divalent cations. Bz2ATP and ATP increased the binding of ryanodine to its receptor up to 3-fold, and increased the binding affinity up to 6-fold. Other nucleotides stimulate ryanodine binding with decreasing effectiveness: Bz2ATP > ATP > ADP > AMP > AMP-PNP > GTP > cAMP. With respect to nucleotide specificity, this binding site is similar to the skeletal muscle RyR (type 1). However, the brain RyR may have additional one or more sites with lower affinity with inhibitory effect on ryanodine binding. These results suggest that the major RyR isoform in sheep brain corresponds to the type-2 isoform, and that modulation of ryanodine binding by ATP involves its binding to the RyR protein. The association of dynein with brain microsomal membranes may reflect a linkage of RyR to the cytoskeleton.

VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle.

In this study we demonstrate the existence of a protein with properties of the voltage-dependent anion channel (VDAC) in the sarcoplasmic reticulum (SR) using multiple approaches as summarized in the following: (a) 35 and 30 kDa proteins in different SR preparations, purified from other membranal systems by Ca2+/oxalate loading and sedimentation through 55% sucrose, cross-react with four different VDAC monoclonal antibodies. (b) Amino acid sequences of three peptides derived from the SR 35 kDa protein are identical to the sequences present in VDAC1 isoform. (c) Similar to the mitochondrial VDAC, the SR protein is specifically labeled by [14C]DCCD. (d) Using a new method, a 35 kDa protein has been purified from SR and mitochondria with a higher yield for the SR. (e) Upon reconstitution into a planar lipid bilayer, the purified SR protein shows voltage-dependent channel activity with properties similar to those of the purified mitochondrial VDAC or VDAC1/porin 31HL from human B lymphocytes, and its channel activity is completely inhibited by the anion transport inhibitor DIDS and about 80% by DCCD. We also demonstrate the translocation of ATP into the SR lumen and the phosphorylation of the luminal protein sarcalumenin by this ATP. Both ATP translocation and sarcalumenin phosphorylation are inhibited by DIDS, but not by atractyloside, a blocker of the ATP/ADP exchanger. These results indicate the existence of VDAC, thought to be located exclusively in mitochondria, in the SR of skeletal muscle, and its possible involvement in ATP transport. Together with recent studies on VDAC multicompartment location and its dynamic association with enzymes and channels, our findings suggest that VDAC deserves attention and consideration as a protein contributing to various cellular functions.

Modulation of the voltage-dependent anion channel (VDAC) by glutamate.

The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is a large channel permeable to anions, cations, ATP, and other metabolites. VDAC was purified from sheep brain synaptosomes or rat liver mitochondria using a reactive red-agarose column, in addition to the hydroxyapatitate column. The red-agarose column allowed further purification (over 98%), concentration of the protein over ten-fold, decreasing Triton X-100 concentration, and/or replacing Triton X-100 with other detergents, such as Nonidet P-40 or octylglucoside. This purified VDAC reconstituted into planar-lipid bilayer, had a unitary maximal conductance of 3.7 +/- 0.1 nS in 1 M NaCl, at 10 mV and was permeable to both large cations and anions. In the maximal conducting state, the permeability ratios for Na(+), acetylcholine(+), dopamine,(+) and glutamate(-), relative to Cl(-), were estimated to be 0.73, 0.6, 0.44, and 0.4, respectively. In contrast, in the subconducting state, glutamate(-) was impermeable, while the relative permeability to acetylcholine(+) increased and to dopamine(+) remained unchanged. At the high concentrations (0.1-0.5 M) used in the permeability experiments, glutamate eliminated the bell shape of the voltage dependence of VDAC channel conductance. Glutamate at concentrations of 1 to 20 mM, in the presence of 1 M NaCl, was found to modulate the VDAC channel activity. In single-channel experiments, at low voltages (+/-10 mV), glutamate induced rapid fluctuations of the channel between the fully open state and long-lived low-conducting states or short-lived closed state. Glutamate modification of the channel activity, at low voltages, is dependent on voltage, requiring short-time (20-60 sec) exposure of the channel to high membrane potentials. The effect of glutamate is specific, since it was observed in the presence of 1 M NaCl and it was not obtained with aspartate or GABA. These results suggest that VDAC possesses a specific glutamate-binding site that modulates its activity.

Effects of ryanodine on calcium sequestration in the rat liver.

Ryanodine, a highly toxic alkaloid known to react specifically with the Ca2+ release channels in sarcoplasmic reticulum (SR), was employed to study Ca2+ sequestration in the liver. Ryanodine at a 200 microM concentration increased cytosolic free Ca2+ levels and phosphorylase a activity in isolated hepatocytes. These effects may involve microsomal Ca2+ sequestration, because ryanodine, in the presence of inhibitors of mitochondrial Ca2+ uptake, at concentrations of 1 nM, 1 microM, 50 microM and 100 microM decreased 45Ca2+ retention in permeabilized hepatocytes. This inhibition of Ca2+ retention by ryanodine was not due to inhibition of the microsomal Ca(2+)-ATPase. Dantrolene, a compound shown previously to inhibit ryanodine binding in the liver, also decreased 45Ca2+ retention in permeabilized hepatocytes, and activated phosphorylase a. These results show that ryanodine administration alters calcium sequestration in liver. The possibility of the existence of a ryanodine-sensitive Ca(2+)-release channel in liver is discussed.

VDAC1-based peptides: novel pro-apoptotic agents and potential therapeutics for B-cell chronic lymphocytic leukemia.

The voltage-dependent anion channel 1 (VDAC1), localized in the outer mitochondrial membrane, mediates metabolic cross-talk between the mitochondrion and the cytoplasm and thus serves a fundamental role in cell energy metabolism. VDAC1 also plays a key role in mitochondria-mediated apoptosis, interacting with anti-apoptotic proteins. Resistance of cancer cells to apoptosis involves quenching the mitochondrial apoptotic pathway by over-expression of anti-apoptotic/pro-survival hexokinase (HK) and Bcl-2 family proteins, proteins that mediate their anti-apoptotic activities via interaction with VDAC1. Using specifically designed VDAC1-based cell-penetrating peptides, we targeted these anti-apoptotic proteins to prevent their pro-survival/anti-apoptotic activities. Anti-apoptotic proteins are expressed at high levels in B-cell chronic lymphocytic leukemia (CLL), an incurable disease requiring innovative new approaches to improve therapeutic outcome. CLL is characterized by a clonal accumulation of mature neoplastic B cells that are resistant to apoptosis. Specifically, we demonstrate that the VDAC1-based peptides (Antp-LP4 and N-Terminal-Antp) selectively kill peripheral blood mononuclear cells (PBMCs) obtained from CLL patients, yet spare those obtained from healthy donors. The cell death induction competence of the peptides was well correlated with the amount of double positive CD19/CD5 cancerous CLL PBMCs, further illustrating their selectivity toward cancer cells. Moreover, these VDAC1-based peptides induced apoptosis by activating the mitochondria-mediated pathway, reflected in membrane blebbing, condensation of nuclei, DNA fragmentation, release of mitochondrial cytochrome c, loss of mitochondrial membrane potential, decreased cellular ATP levels and detachment of HK, all leading to apoptotic cell death. Thus, the mode of action of the peptides involves decreasing energy production and inducing apoptosis. Over 27 versions of cell-penetrating VDAC1-based peptides were designed and screened to identify the most stable, short and apoptosis-inducing peptides toward CLL-derived lymphocytes. In this manner, three optimized peptides suitable for in vivo studies were identified. This study thus reveals the potential of VDAC1-based peptides as an innovative and effective anti-CLL therapy.

Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death.

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD.

Mitochondrial VDAC1: function in cell life and death and a target for cancer therapy.

Found at the outer mitochondrial membrane, the voltage-dependent anion channel, VDAC, assumes a crucial position in the cell, serving as the main interface between mitochondrial and cellular metabolisms by mediating transport of ions and metabolites. VDAC thus functions as a gatekeeper, controlling cross-talk between mitochondria and the rest of the cell. Moreover, its location at the boundary between the mitochondria and the cytosol enables VDAC to interact with proteins that mediate and regulate the integration of mitochondrial functions with other cellular activities. Here, we review current knowledge related to the roles played by VDAC in the regulation of cell life and cell death, with relation to cancer. The current concepts of altered metabolism in cancer cells are presented with specific emphasis on mitochondrial, more specifically VDAC1-bound hexokinase (HK), facilitating and promoting the high glycolytic tumor phenotype. In this respect, the up-regulation of HK expression in tumor cells and its binding to VDAC provide both a metabolic benefit and apoptosis-suppressive capacity that offers the cell a growth advantage and increases its resistance to chemotherapy. VDAC has also been recognized as a key protein in mitochondria-mediated apoptosis since it is the proposed target for the pro- and antiapoptotic Bcl-2-family of proteins, as well as due to its function in the release of apoptotic proteins located in the inter-membranal space. These and other functions point to VDAC1 as being a rational target for the development of a new generation of therapeutics.

Methyl jasmonate binds to and detaches mitochondria-bound hexokinase.

Cellular bio-energetic metabolism and mitochondria are recognized as potential targets for anticancer agents, due to the numerous relevant peculiarities cancer cells exhibit. Jasmonates are anticancer agents that interact directly with mitochondria. The aim of this study was to identify mitochondrial molecular targets of jasmonates. We report that jasmonates bind to hexokinase and detach it from the mitochondria and its mitochondrial anchor-the voltage-dependent anion channel (VDAC), as judged by hexokinase immunochemical and activity determinations, surface plasmon resonance analysis and planar lipid bilayer VDAC-activity analysis. Furthermore, the susceptibility of cancer cells and mitochondria to jasmonates is dependent on the expression of hexokinase, evaluated using hexokinase-overexpressing transfectants and its mitochondrial association. Many types of cancer cells exhibit overexpression of the key glycolytic enzyme, hexokinase, and its excessive binding to mitochondria. These characteristics are considered to play a pivotal role in cancer cell growth rate and survival. Thus, our findings provide an explanation for the selective effects of jasmonates on cancer cells. Most importantly, this is the first demonstration of a cytotoxic mechanism based on direct interaction between an anticancer agent and hexokinase. The proposed mechanism can serve to guide development of a new selective approach for cancer therapy.