Overexpression of catalase in the mitochondrial or cytosolic compartment increases sensitivity of HepG2 cells to tumor necrosis factor-alpha-induced apoptosis

The sensitivity of HepG2 cells overexpressing catalase in either the cytosolic or mitochondrial compartment to tumor necrosis factor-alpha (TNF-alpha) and cycloheximide was studied. Cells overexpressing catalase in the cytosol (C33 cells) and especially in mitochondria (mC5 cells) were more sensitive to TNF-alpha-induced apoptosis than were control cells (Hp cells). The activities of caspase-3 and -8 were increased by TNF-alpha, with the highest activities found in mC5 cells. Sodium azide, an inhibitor of catalase, reduced the increased sensitivity of mC5 and C33 cells to TNF-alpha to the level of toxicity found with control Hp cells. Azide also decreased the elevated caspase-3 activity of mC5 cells. A pan-caspase inhibitor prevented the TNF-alpha-induced apoptosis and toxicity produced by catalase overexpression. Addition of H(2)O(2) prevented TNF-alpha-induced apoptosis and caspase activation, an effect prevented by simultaneous addition of catalase. TNF-alpha plus cycloheximide increased ATP levels, with higher levels in C33 and mC5 cells compared with Hp cells. TNF-alpha did not produce apoptosis in mC5 cells maintained in a low energy state. TNF-alpha signaling was not altered by the overexpression of catalase, as activation of nuclear factor kappaB and AP-1 by TNF-alpha was similar in the three cell lines. These results suggest that catalase, overexpressed in the cytosolic or especially the mitochondrial compartment, potentiates TNF-alpha-induced apoptosis and activation of caspases by removal of H(2)O(2).     

TNF-alpha/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species

Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.     

Mechanisms of the induction of apoptosis in human hepatoma cells by tumour necrosis factor-alpha.

Tumour necrosis factor alpha (TNF-alpha) at 20 ng/ml induced apoptosis in human hepatoma cells in vitro. The effect of TNF-alpha-induced apoptosis was exacerbated by the hypoxanthine-xanthine oxidase (HX/XO) system and cycloheximide (CHX), but alleviated by superoxide dismutase (SOD), suggesting that TNF-alpha-induced apoptosis may be due to oxidative stress, and independent of protein synthesis. TNF-alpha elevated free Ca(2+)concentration, triggered lipid peroxidation and decreased the expression of bcl-2 protein. The findings suggest that TNF-alpha-induced apoptosis may be involved in stimulating Ca(2+)-dependent endonuclease activity and increasing membrane lipid peroxidation. Bcl-2 may play a pivotal role in serving as a Ca(2+)regulator or antioxidant, preventing lipid peroxidation in the process.     

Inhibition of phosphatidylinositol 3-kinase sensitizes vascular endothelial cells to cytokine-initiated cathepsin-dependent apoptosis

In the presence of cycloheximide, tumor necrosis factor or interleukin-1 initiates caspase activation, loss of mitochondrial membrane potential (DeltaPsi), DNA degradation, and nuclear condensation and fragmentation characteristic of apoptotic cell death in human vascular endothelial cells (EC). Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002, but not inhibition of Akt by dominant-negative mutation, also sensitizes EC to cytokine-initiated apoptosis. Cytokine-initiated caspase activation is slower and comparatively less with LY294002 than with cycloheximide. Cycloheximide but not LY294002 decreases expression of c-FLIP (cellular FLICE inhibitory protein), an inhibitor of caspase-8 activation. The caspase inhibitor zVADfmk completely blocks caspase activation, DNA degradation, and nuclear fragmentation in both cases but only prevents loss of DeltaPsi and cell death for cytokine plus cycloheximide treatment. In contrast, overexpression of Bcl-2 protects EC treated with cytokine plus LY294002 but not EC treated with cytokine plus cycloheximide. The cathepsin B inhibitor CA-074-Me prevents loss of DeltaPsi, caspase activation, and cell death for EC treated with cytokine plus LY294002 but has no effect on EC treated with cytokine plus cycloheximide. Cathepsin B translocates from lysosomes to cytosol following treatment with LY294002 prior to the activation of caspases. These results suggest that inhibition of PI3K allows cytokines to activate a cathepsin-dependent, mitochondrial death pathway in which caspase activation is secondary, is not inhibited by c-FLIP, and is not essential for cell death.     

Synergistic perturbation of phosphatidylcholine/sphingomyelin bilayers by diacylglycerol and cholesterol

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity partially prevented hypoxia-induced apoptosis. 2-Deoxy-D-ribose inhibits a number of components of the caspase-mediated hypoxia-induced apoptotic pathway. It inhibits hypoxia-induced caspase 3 activation, mitochondrial cytochrome c release, downregulation of Bcl-2 and Bcl-x(L), upregulation of hypoxia-inducible factor (HIF)-1 alpha, and loss of mitochondrial transmembrane potential in human leukemia HL-60 cell line. These findings suggest a molecular mechanism by which 2-deoxy-d-ribose confers the resistance to apoptosis. Thus 2-deoxy-D-ribose-modulated suppression of HIF-1 alpha expression could prevent the hypoxia-induced decrease of the anti-apoptotic Bcl-2 and Bcl-x(L) on the mitochondria. 2-Deoxy-L-ribose and its analogs may enhance apoptosis and suppress the growth of tumors by competitively inhibiting the activities of 2-deoxy-d-ribose and thus these analogs show promise for anti-tumor therapy.     

Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis

The Bcl-2 family of proteins are involved in regulating the redox state of cells. However, the mode of action of Bcl-2 proteins remains unclear. This work analyzed the effects of Bcl-x(L) on the cellular redox state after treatment with tumor necrosis factor alpha (TNF-alpha) or exogenous oxidants. We show that in cells that undergo TNF-alpha-induced apoptosis, TNF-alpha induces a partial decrease in mitochondrial membrane potential (DeltaPsi(m)) followed by high levels of reactive oxygen species (ROS). ROS scavengers delay the progression of mitochondrial depolarization and apoptotic cell death. This indicates that ROS are important mediators of mitochondrial depolarization. However, ROS scavengers fail to prevent the initial TNF-alpha-induced decrease in DeltaPsi(m). In contrast, expression of Bcl-x(L) prevents both the initial decrease in DeltaPsi(m) following TNF-alpha treatment and the subsequent induction of ROS. Bcl-x(L) itself does not act as a ROS scavenger. In addition, Bcl-x(L) does not block the initial decrease in DeltaPsi(m) following treatment with the oxidant hydrogen peroxide. However, unlike control-transfected cells, Bcl-x(L)-expressing cells can recover their mitochondrial membrane potential following the initial drop in DeltaPsi(m) induced by hydrogen peroxide. These data suggest that Bcl-x(L) plays a regulatory role in controlling the membrane potential of and ROS production by mitochondria rather than acting as a direct antioxidant.     

Hyperthermia and tumour necrosis factor-alpha induced apoptosis via mitochondrial damage

Hyperthermia is a potential anti-cancer regimen but the mode of action is far from clear. Based on the flow cytometric analysis with FITC-annexin V and propidium iodide, apoptosis was found to be the major form of cell death after the treatment with hyperthermia (43 degrees C, 3 h) and/or recombinant murine tumour necrosis factor-alpha (TNF-alpha, 50 ng/ml) in L929 cells. Since mitochondria are thought to play a key role in apoptosis, experiments were done to assess their role in the hyperthermia-mediated apoptosis. Our results indicate that hyperthermia was able to depolarize the mitochondrial membrane potential (delta psi m) and release cytochrome c to the cytoplasm, in a way very similar to the action of TNF-alpha. With the use of cyclosporin A to inhibit the delta psi m dissipation, the cytotoxicity mediated by hyperthermia or TNF-alpha was suppressed. Taken together, our results indicate that hyperthermia and TNF-alpha can induce apoptosis in L929 cells and the mitochondrial dysfunction plays a key role in the cell death process.     

In vitro exposure of human lymphocytes to 900 MHz CW and GSM modulated radiofrequency: studies of proliferation, apoptosis and mitochondrial membrane potential

The aim of this study was to investigate the nonthermal effects of radiofrequency (RF) fields on human immune cells exposed to a Global System for Mobile Communication (GSM) signal generated by a commercial cellular phone and by a sinusoidal non-modulated signal. To assess whether mobile phone RF-field exposure affects human immune cell functions, peripheral blood mononuclear cells (PBMCs) from healthy donors were exposed in vitro to a 900 MHz GSM or continuous-wave (CW) RF field 1 h/day for 3 days in a transverse electromagnetic mode (TEM) cell system (70-76 mW/kg average specific absorption rate, SAR). The cells were cultured for 48 or 72 h, and the following end points were studied: (1) mitogen-induced proliferation; (2) cell cycle progression; (3) spontaneous and 2-deoxy-D-ribose (dRib)-induced apoptosis; (4) mitochondrial membrane potential modifications during spontaneous and dRib-induced-apoptosis. Data obtained from cells exposed to a GSM-modulated RF field showed a slight decrease in cell proliferation when PBMCs were stimulated with the lowest mitogen concentration and a slight increase in the number of cells with altered distribution of phosphatidylserine across the membrane. On the other hand, cell cycle phases, mitochondrial membrane potential and susceptibility to apoptosis were found to be unaffected by the RF field. When cells were exposed to a CW RF field, no significant modifications were observed in comparison with sham-exposed cells for all the end points investigated.     

Emodin inhibits TNF-alpha-induced human aortic smooth-muscle cell proliferation via caspase- and mitochondrial-dependent apoptosis

Vascular smooth-muscle cell (VSMC) proliferation plays a vital role in hypertension, atherosclerosis and restenosis. It has been reported that emodin, an active component extracted from rhubarb, can stop the growth of cancer cells; however, it is not known if emodin exerts similar anti-atherogenic effects in TNF-alpha treated human aortic smooth-muscle cells (HASMC). In this study, emodin treatment showed potent inhibitory effects in TNF-alpha-induced HASMC proliferation that were associated with induced apoptosis, including the cleavage of poly ADP-ribose polymerase (PARP). Moreover, inhibitors of caspase-3, -8 and -9 (Ac-DEVD-CHO, Z-IETD-FMK and Z-LEHD-FMK) efficiently blocked emodin-induced apoptosis in TNF-alpha treated HASMC. Therefore, emodin-induced cell death occurred via caspase-dependent apoptosis. Emodin treatment resulted in the release of cytochrome c into cytosol and a loss of mitochondrial membrane potential (DeltaPsi(m)), as well as a decrease in the expression of an anti-apoptotic protein (Bcl-2) and an increase in the expression of an a pro-apoptotic protein (Bax). Emodin-mediated apoptosis was also blocked by a mitochondrial membrane depolarization inhibitor, which indicates that emodin-induced apoptosis occurred via a mitochondrial pathway. Taken together, the results of this study showed that emodin inhibits TNF-alpha-induced HASMC proliferation via caspase- and a mitochondrial-dependent apoptotic pathway. In addition, these results indicate that emodin has potential as an anti-atherosclerosis agent.     

Different sensitivity to apoptosis in cells of monocytic or lymphocytic origin chronically infected with human immunodeficiency virus type-1

Apoptotic death of CD4+ T lymphocytes is a major cause of the immunodeficiency caused by human immunodeficiency virus (HIV), but it is still unclear how this process precisely occurs. To characterize a potentially useful cellular model, we have analyzed the tendency of chronically HIV-infected CD4+ human cell lines of different origin to undergo apoptosis. We studied ACH-2 and U1 lines, derived from the CD4+ T-cell A301 and the promonocytic U937 cell lines, respectively, and induced apoptosis via several stimuli that trigger different pathways. Their capacity to regulate plasma membrane CD95 expression and to produce soluble CD95 was also analyzed. Using staurosporine, TNF-alpha plus cycloheximide, and gamma-radiations, we observed that ACH-2 were more sensitive to programmed cell death than A301, while U1 were less sensitive than U937. Both infected cell types had a lower sensitivity to CD95-induced apoptosis; the analysis of changes in mitochondrial membrane potential corroborated these observations. Plasma membrane CD95 was similarly regulated in all cell types, which, however, presented a different capacity to produce soluble CD95 molecules. Our in vitro results may offer a new perspective for developing further studies on the pathogenesis of HIV infection. A chronically infected cell line of lymphocytic origin is more susceptible to apoptosis than its parental cell type, while infected monocytic cells are less sensitive than their uninfected counterpart. Thus, it is possible to hypothesize that one of the reasons by which circulating monocytes survive and represent a viral reservoir is the capacity of HIV to decrease the sensitivity to apoptosis of this cell type. However, further studies on ex-vivo collected fresh cells, as well as on other cell lines, are urgently needed to confirm such hypothesis.     

Heat-shock response protects peripheral blood mononuclear cells (PBMCs) from hydrogen peroxide-induced mitochondrial disturbance

The present study was designed to investigate ex vivo the protective mechanisms of heat-shock response against H₂O₂-induced oxidative stress in peripheral blood mononuclear cells (PBMCs) of rats. Twenty-four hours later, heat-shock treatment was executed in vivo; rat PBMCs were collected and treated with H₂O₂. The accumulation of reactive oxygen species and the mitochondrial membrane potential were evaluated by intracellular fluorescent dHE and JC-1 dye staining, respectively, and expression of HSP72 and cytochrome c was detected by Western blot analysis. Cellular apoptosis was assayed by TUNEL staining and double staining of Annexin V and PI. The results showed that H₂O₂-induced oxidative stress leads to intracellular superoxide accumulation and collapse of the mitochondrial membrane potential in rat PBMCs. Moreover, cellular apoptosis was detected after H₂O₂ treatment, and the release of mitochondrial cytochrome c from mitochondria to cytosol was significantly enhanced. Heat-shock pretreatment decreases the accumulation of intracellular superoxide in PBMCs during H₂O₂-induced oxidative stress. Moreover, heat-shock treatment prevents the collapse of the mitochondrial membrane potential and cytochrome c release from mitochondria during H₂O₂-induced oxidative stress. In conclusion, mitochondria are critical organelles of the protective effects of heat-shock treatment. Cellular apoptosis during H₂O₂-induced oxidative stress is decreased by heat-shock treatment through a decrease in superoxide induction and preservation of the mitochondrial membrane potential.     

Peroxiredoxin III, a mitochondrion-specific peroxidase, regulates apoptotic signaling by mitochondria

Various proapoptotic stimuli increase the production of superoxide and H(2)O(2) by mitochondria. Whereas superoxide impairs mitochondrial function and is removed by Mn(2+)-dependent superoxide dismutase, the role and metabolism of mitochondrial H(2)O(2) during apoptosis have remained unclear. The effects on apoptotic signaling of depletion of peroxiredoxin (Prx) III, a mitochondrion-specific H(2)O(2)-scavenging enzyme, have now been investigated by RNA interference in HeLa cells. Depletion of Prx III resulted in increased intracellular levels of H(2)O(2) and sensitized cells to induction of apoptosis by staurosporine or TNF-alpha. The rates of mitochondrial membrane potential collapse, cytochrome c release, and caspase activation were increased in Prx III-depleted cells, and these effects were reversed by ectopic expression of Prx III or mitochondrion-targeted catalase. Depletion of Prx III also exacerbated damage to mitochondrial macromolecules induced by the proapoptotic stimuli. Our results suggest that Prx III is a critical regulator of the abundance of mitochondrial H(2)O(2), which itself promotes apoptosis in cooperation with other mediators of apoptotic signaling.     

Mitochondrial membrane potential is reduced in copper-deficient C2C12 cells in the absence of apoptosis

Mitochondrial membrane potential is reduced in copper-deficient rat hearts, but it is uncertain if this will lead to the onset of apoptosis. To determine if copper deficiency per se leads to apoptosis, C2C12 cells were made copper deficient by treatment with the copper chelator tetraethylenepentamine (TEPA). In TEPA-treated cells, the activity of Cu, Zn-superoxide dismutase and cytochrome-c oxidase decreased dramatically. The protein levels of nuclear-encoded subunits of the cytochromie-c oxidase decreased, but the mitochondrial-encoded subunits remained unchanged. Decreased mitochondrial membrane potential was indicated in TEPA-treated cells, but further investigation of the potential induction of apoptosis by measuring caspase-3 activity, protein concentrations of Bcl-2 and Bax, and DNA fragmentation suggested that apoptosis is not induced in TEPA-treated C2C12 cells. Cells with decreased mitochondrial membrane potential were not destined to apoptosis as a result of copper deficiency.     

NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells

The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G(1) phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-kappaB (NF-kappaB) in this process. Although depletion of polyamines by alpha-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-alpha (TNF-alpha) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-kappaB proteins, induced NF-kappaB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-kappaB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-alpha in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-alpha-induced cell death, 2) polyamine depletion induces NF-kappaB activation, and 3) disruption of NF-kappaB function is associated with altered susceptibility to apoptosis induced by STS or TNF-alpha. These findings suggest that increased NF-kappaB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.     

Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.     

Crambene induces pancreatic acinar cell apoptosis via the activation of mitochondrial pathway

We investigated the apoptotic pathway activated by crambene (1-cyano-2-hydroxy-3-butene), a plant nitrile, on pancreatic acinar cells. As evidenced by annexin V-FITC staining, crambene treatment for 3 h induced the apoptosis but not necrosis of pancreatic acini. Caspase-3, -8, and -9 activities in acini treated with crambene were significantly higher than in untreated acini. Treatment with caspase-3, -8, and -9 inhibitors inhibited annexin V staining, as well as caspase-3 activity, pointing to an important role of these caspases in crambene-induced acinar cell apoptosis. The mitochondrial membrane potential was collapsed, and cytochrome c was released from the mitochondria in crambene-treated acini. Neither TNF-alpha nor Fas ligand levels were changed in pancreatic acinar cells after crambene treatment. These results provide evidence for the induction of pancreatic acinar cell apoptosis in vitro by crambene and suggest the involvement of mitochondrial pathway in pancreatic acinar cell apoptosis.     

Immobilized alpha2,6-linked sialic acid suppresses caspase-3 activation during anti-IgM antibody-induced apoptosis in Ramos cells

In Ramos cells, a human Burkitt's lymphoma cell line, stimulation of the B cell antigen receptor with anti-IgM antibody (Ab) induces apoptosis as indicated by a decrease in cell viability and an increase in DNA fragmentation and cell surface exposure of phosphatidylserine. Furthermore, these changes are suppressed by incubating the cells in alpha(1)-acid glycoprotein (AGP)-coated tissue culture plates. Here, we found that, during Anti-IgM Ab-induced apoptosis in Ramos cells, caspase-3 is activated downstream of caspase-8 and the mitochondrial pathway is activated, as indicated by a loss of mitochondrial membrane potential, an increase in the release of cytochrome c to the cytoplasm, and enhanced Bax expression. Anti-IgM Ab-induced apoptosis of neuraminidase-treated Ramos cells was suppressed by incubating the cells on plates coated with AGP, which contains a high concentration of alpha2,6-linked sialic acid. The incubation on plates coated with AGP also suppressed anti-IgM Ab-stimulated caspase-3 activity and increased the level of X-linked inhibitor of apoptosis protein (XIAP), but it did not affect caspase-8 activity, the mitochondrial membrane potential, cytochrome c release, or Bax expression. The results indicate that the interaction of Ramos cells with immobilized alpha2,6-linked sialic acid enhances XIAP expression, directly or indirectly suppressing caspase-3 activity and inhibiting anti-IgM Ab-induced apoptosis.     

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.