Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Proteasome inhibition activates the mitochondrial pathway of apoptosis in human CD4+ T cells

We have previously shown that inhibition of the proteolytic activity of the proteasome induces apoptosis and suppresses essential functions of activated human CD4⁺ T cells, and we report now the detailed mechanisms of apoptosis following proteasome inhibition in these cells. Here we show that proteasome inhibition by bortezomib activates the mitochondrial pathway of apoptosis in activated CD4⁺ T cells by disrupting the equilibrium of pro‐apoptotic and anti‐apoptotic proteins at the outer mitochondrial membrane (OMM) and by inducing the generation of reactive oxygen species (ROS). Proteasome inhibition leads to accumulation of pro‐apoptotic proteins PUMA, Noxa, Bim and p53 at the OMM. This event provokes mitochondrial translocation of activated Bax and Bak homodimers, which induce loss of mitochondrial membrane potential (ΔΨm). Breakdown of ΔΨm is followed by rapid release of pro‐apoptotic Smac/DIABLO and HtrA2 from mitochondria, whereas release of cytochrome c and AIF is delayed. Cytoplasmic Smac/DIABLO and HtrA2 antagonize IAP‐mediated inhibition of partially activated caspases, leading to premature activation of caspase‐3 followed by activation of caspase‐9. Our data show that proteasome inhibition triggers the mitochondrial pathway of apoptosis by activating mutually independent apoptotic pathways. These results provide novel insights into the mechanisms of apoptosis induced by proteasome inhibition in activated T cells and underscore the future use of proteasome inhibitors for immunosuppression. J. Cell. Biochem. 108: 935–946, 2009. © 2009 Wiley‐Liss, Inc.     

TCR-independent and caspase-independent apoptosis of murine thymocytes by CD24 cross-linking

CD24, also referred to as the heat-stable Ag, is a T cell differentiation Ag that is highly expressed on both CD4-CD8- double negative and CD4+CD8+ double positive thymocytes. Here, we report that CD24 ligation by a new anti-CD24 Ab, mT-20, induced the apoptosis of both double negative and double positive thymocytes, as well as the Scid.adh thymic lymphoma cell line, in the absence of TCR/CD3 engagement. CD24-mediated apoptosis of mouse thymocytes and its signaling pathway appeared not to be associated with p53, CD95, TNFR, or caspases. Furthermore, we found that cell death was blocked by the addition of scavengers of reactive oxygen species or by Bcl-2 overexpression, implying the role of CD24 signaling in the mitochondrial regulation. In this study, we suggest that CD24 ligation induced the apoptosis of immature thymocytes independently of both caspase and TCR.     

Nitric oxide protects thymocytes from gamma-irradiation-induced apoptosis in correlation with inhibition of p53 upregulation and mitochondrial damage

Apoptosis plays a crucial role in clonal deletion in the thymus, and NO has been shown to prevent apoptosis in some cell types. Therefore, we examined the effect of NO on gamma-irradiation-induced thymocyte apoptosis. Treatment of 5 Gy gamma-irradiated thymocytes with 1 mM SNAP reduced cell death from 78 to 49% after 8 h incubation (spontaneous cell death in medium control cells was 26%). Coincubation with ZVAD blocked both the spontaneous cell death and the cell death induced by SNAP or gamma-irradiation. The gamma-irradiation-induced increase in caspase 3 and 6 activities was inhibited in the presence of SNAP. The increase in cytosolic cytochrome c as well as the decrease in mitochondrial membrane potential after gamma-irradiation was inhibited in the presence of SNAP. SNAP treatment also decreased the p53 upregulation in gamma-irradiated cells. In summary, we found that NO exerts a protective effect on mouse thymocyte apoptosis induced by gamma-irradiation. The mechanism of this protective effect may involve inhibition of p53 upregulation and reduction in mitochondrial damage, with subsequent inhibition of downstream caspase activation.     

Blocking granule-mediated death by primary human NK cells requires both protection of mitochondria and inhibition of caspase activity

Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.     

Proteasome inhibitor-induced apoptosis of glioma cells involves the processing of multiple caspases and cytochrome c release

The proteasome is a multiprotein complex that is involved in the intracellular protein degradation in eukaryotes. Here, we show that human malignant glioma cells are susceptible to apoptotic cell death induced by the proteasome inhibitors, MG132 and lactacystin. The execution of the apoptotic death program involves the processing of caspases 2, 3, 7, 8, and 9. Apoptosis is inhibited by ectopic expression of X-linked inhibitor of apoptosis (XIAP) and by coexposure to the broad-spectrum caspase inhibitor, benzoyl-VAD-fluoromethyl ketone (zVAD-fmk), but not by the preferential caspase 8 inhibitor, crm-A. It is interesting that specific morphological alterations induced by proteasome inhibition, such as dilated rough endoplasmic reticulum and the formation of cytoplasmic vacuoles and dense mitochondrial deposits, are unaffected by zVAD-fmk. Apoptosis is also inhibited by ectopic expression of Bcl-2 or by an inhibitor of protein synthesis, cycloheximide. Further, cytochrome c release and disruption of mitochondrial membrane potential are prominent features of apoptosis triggered by proteasome inhibition. Bcl-2 is a stronger inhibitor of cytochrome c release than zVAD-fmk. XIAP and crm-A fail to modulate cytochrome c release. These data place cytochrome c release downstream of Bcl-2 activity but upstream of XIAP- and crm-A-sensitive caspases. The partial inhibition of cytochrome c release by zVAD-fmk indicates a positive feedback loop that may involve cytochrome c release and zVAD-fmk-sensitive caspases. Finally, death ligand/receptor interactions, including the CD95/CD95 ligand system, do not mediate apoptosis induced by proteasome inhibition in human malignant glioma cells.     

Inhibition of urokinase-type plasminogen activator receptor induces apoptosis in melanoma cells by activation of p53

The urokinase-type plasminogen activator receptor (uPAR) is involved in several biological processes, including proteolysis, adhesion, migration and inflammation. Increased expression of uPAR is associated with metastasis in several tumor types. We studied the biological role of uPAR in melanoma and found that inhibition of uPAR via RNA interference induced massive death in three different metastatic cell lines. Annexin-V staining and caspase activation analysis revealed induction of the mitochondrial apoptotic pathway. The expression of members of the Bcl-2 family (Bax, Bcl-2, Bak and Bcl-x(L)) was changed in a pro-apoptotic manner. uPAR inhibition induced the expression of the tumor suppressor p53 and of its downstream target gene p21. Inhibition of p53 rescued cells from apoptosis indicating that p53 was critical for apoptosis induction. Apoptosis was observed in melanoma cells carrying activating BRAF mutations and occurred in the presence of extracellular signal-regulated kinase (ERK) phosphorylation. uPAR can activate focal adhesion kinase (FAK), which is implicated in adhesion-dependent tumor cell survival. However, inhibition of FAK did not induce apoptosis. Our data suggest a new function of uPAR acting as a survival factor for melanoma by downregulating p53. Inhibition of uPAR induces a pro-apoptotic signalling pathway via p53 that is independent of ERK or FAK signalling. These findings may offer new treatment strategies for metastatic melanoma.     

Apoptosis of macrophages induced by Trichomonas vaginalis through the phosphorylation of p38 mitogen-activated protein kinase that locates at downstream of mitochondria-dependent caspase activation

Trichomonas vaginalis, a flagellated protozoan parasite, is the causative organism of trichomoniasis. We have recently demonstrated that T. vaginalis induces apoptotic cell death via a Bcl-x(L)-dependent pathway in RAW264.7 macrophages. In this study, we attempted to characterize in detail the signaling cascades resulting in T. vaginalis-induced macrophage apoptosis, focusing particularly on mitochondrial changes and the role of p38 mitogen-activated protein kinase (p38 MAPK) activation. We found that T. vaginalis induced mitochondrial changes including the release of cytochrome c and the serial activation of caspases, leading to the activation of p38 MAPK in macrophages. These biochemical changes culminated in the apoptosis of the host cells. Caspase inhibitors induced a significant inhibition of T. vaginalis-induced nuclear damage, as well as the activation of p38 MAPK. Treatment with the p38 MAPK inhibitor, SB203580, or the overexpression of kinase-inactive p38 MAPK, induced an attenuation of T. vaginalis-induced apoptosis but not cytochrome c release, the activation of caspase-9 and caspase-3, or PARP cleavage. Furthermore, SB203580 treatment to human macrophages consistently blocked T. vaginalis-induced apoptosis. Collectively, our findings indicate that p38 MAPK signaling cascade is requisite to apoptosis of T. vaginalis-infected macrophage, and this apoptotic process occurs via the phosphorylation of p38 MAPK, which is located downstream of mitochondria-dependent caspase activation, conferring insight into the plausible molecular mechanism of T. vaginalis-immune evasion from macrophage attack.     

Proteasome inhibition can induce an autophagy-dependent apical activation of caspase-8

Antiapoptotic Bcl-2 family proteins are often highly expressed in chemotherapy-resistant cancers and impair mitochondrial outer membrane permeabilisation (MOMP), an important requirement for caspase activation via the intrinsic apoptosis pathway. Interestingly, although Bcl-2 overexpression in HeLa cervical cancer cells abrogated caspase processing in response to intrinsic apoptosis induction by staurosporine, tunicamycin or etoposide, residual caspase processing was observed following proteasome inhibition by bortezomib ([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid), epoxomicin (N-acetyl-N-methyl-lisoleucyl-L-isoleucyl-N-[(1S)-3-methyl-1-[[(2R)-2-methyloxiranyl]carbonyl]butyl]-L-threoninamide) or MG-132 (N-(benzyloxycarbonyl)leucinylleucinylleucinal). Similar responses were found in Bcl-2-overexpressing H460 NSCLC cells and Bax/Bak-deficient mouse embyronic fibroblasts. Mild caspase processing resulted in low DEVDase activities, which were MOMP independent and persisted for long periods without evoking immediate cell death. Surprisingly, depletion of caspase-3 and experiments in caspase-7-depleted MCF-7-Bcl-2 cells indicated that the DEVDase activity did not originate from effector caspases. Instead, Fas-associated death domain (FADD)-dependent caspase-8 activation was the major contributor to the slow, incomplete substrate cleavage. Caspase-8 activation was independent of death ligands, but required the induction of autophagy and the presence of Atg5. Depletion of XIAP or addition of XIAP-antagonising peptides resulted in a switch towards efficient apoptosis execution, suggesting that the requirement for MOMP was bypassed by activating the caspase-8/caspase-3 axis. Combination treatments of proteasome inhibitors and XIAP antagonists therefore represent a promising strategy to eliminate highly resistant cancer cells, which overexpress antiapoptotic Bcl-2 family members.     

Staphylococcus aureus alpha-toxin-induced cell death: predominant necrosis despite apoptotic caspase activation

Recent data suggest that alpha-toxin, the major hemolysin of Staphylococcus aureus, induces cell death via the classical apoptotic pathway. Here we demonstrate, however, that although zVAD-fmk or overexpression of Bcl-2 completely abrogated caspase activation and internucleosomal DNA fragmentation, they did not significantly affect alpha-toxin-induced death of Jurkat T or MCF-7 breast carcinoma cells. Caspase inhibition had also no effect on alpha-toxin-induced lactate dehydrogenase release and ATP depletion. Furthermore, whereas early assessment of apoptosis induction by CD95 resulted solely in the generation of cells positive for active caspases that were, however, not yet permeable for propidium iodide, a substantial proportion of alpha-toxin-treated cells were positive for both active caspases and PI. Finally, electron microscopy demonstrated that even in the presence of active caspases, alpha-toxin-treated cells displayed a necrotic morphology characterized by cell swelling and cytoplasmic vacuolation. Together, our data suggest that alpha-toxin-induced cell death proceeds even in the presence of activated caspases, at least partially, in a caspase-independent, necrotic-like manner.     

Involvement of FADD and caspase-8 signalling in detachment-induced apoptosis.

Detachment of most untransformed adherent cells from the extracellular matrix promotes apoptosis, in a process termed anoikis [1] [2]. The death signalling mechanisms involved in this process are not known, although adhesion or transformation by ras oncogenes have been shown to protect epithelial cells from apoptosis through activation of phosphatidylinositol 3-kinase and protein kinase B (PKB/Akt) [3]. Here we show that detachment-induced apoptosis (anoikis) is blocked by the expression of a dominant-negative form of FAS-associated death domain protein (FADD) in a number of untransformed epithelial cell lines. Because the soluble extracellular domains of the death receptors CD95, DR4 and DR5 failed to block anoikis, we conclude that ligand-dependent activation of these death receptors is not involved in this process. Detachment induced strong activation of caspase 8 and caspase 3. Detachment-induced caspase-8 activation did not require the function of downstream caspases but was blocked by overexpression of the anti-apoptotic proteins Bcl-2 or Bcl-X(L). We propose that caspase-8 activation is the initiating event in anoikis, which is subsequently subject to a positive-feedback loop involving mitochondrial events.     

Persistent mitochondrial dysfunction and oxidative stress hinder neuronal cell recovery from reversible proteasome inhibition

Oxidative stress, proteasome impairment and mitochondrial dysfunction are implicated as contributors to ageing and neurodegeneration. Using mouse neuronal cells, we showed previously that the reversible proteasome inhibitor, [N-benzyloxycarbonyl-Ile-Glu (O-t-bytul)-Ala-leucinal; (PSI)] induced excessive reactive oxygen species (ROS) that mediated mitochondrial damage and a caspase-independent cell death. Herein, we examined whether this insult persists in neuronal cells recovering from inhibitor removal over time. Recovery from proteasome inhibition showed a time and dose-dependent cell death that was accompanied by ROS overproduction, caspase activation and mitochondrial membrane permeabilization with the subcellular relocalizations of the proapoptotic proteins, Bax, cytochrome c and the apoptosis inducing factor (AIF). Caspase inhibition failed to promote survival indicating that cell death was caspase-independent. Treatments with the antioxidant N-acetyl-cysteine (NAC) were needed to promote survival in cell recovering from mild proteasome inhibition while overexpression of the antiapoptotic protein Bcl-xL together with NAC attenuated cell death during recovery from potent inhibition. Whereas inhibitor removal increased proteasome function, cells recovering from potent proteasome inhibition showed excessive levels of ubiquitinated proteins that required the presence of NAC for their removal. Collectively, these results suggest that the oxidative stress and mitochondrial inhibition induced by proteasome inhibition persists to influence neuronal cell survival when proteasome function is restored. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A role of the mitochondrial apoptosis-inducing factor in granulysin-induced apoptosis

Granulysin is a cytolytic molecule released by CTL via granule-mediated exocytosis. In a previous study we showed that granulysin induced apoptosis using both caspase- and ceramide-dependent and -independent pathways. In the present study we further characterize the biochemical mechanism for granulysin-induced apoptosis of tumor cells. Granulysin-induced death is significantly inhibited by Bcl-2 overexpression and is associated with a rapid (1-5 h) loss of mitochondrial membrane potential, which is not mediated by ceramide generation and is not inhibited by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide generation induced by granulysin is a slow event, only observable at longer incubation times (12 h). Apoptosis induced by exogenous natural (C(18)) ceramide is truly associated with mitochondrial membrane potential loss, but contrary to granulysin, this event is inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide-induced apoptosis is also completely prevented by Bcl-2 overexpression. The nuclear morphology of cells dying after granulysin treatment in the presence of caspase inhibitors suggested the involvement of mitochondrial apoptosis-inducing factor (AIF) in granulysin-induced cell death. We demonstrate using confocal microscopy that AIF is translocated from mitochondria to the nucleus during granulysin-induced apoptosis. The majority of Bcl-2 transfectants are protected from granulysin-induced cell death, mitochondrial membrane potential loss, and AIF translocation, while a small percentage are not protected. In this small percentage the typical nuclear apoptotic morphology is delayed, being of the AIF type at 5 h time, while at longer times (12 h) the normal apoptotic morphology is predominant. These and previous results support a key role for the mitochondrial pathway of apoptosis, and especially for AIF, during granulysin-induced tumoral cell death.     

Characterization of Puma-dependent and Puma-independent neuronal cell death pathways following prolonged proteasomal inhibition

Proteasomal stress and the accumulation of polyubiquitinated proteins are key features of numerous neurodegenerative disorders. Previously we demonstrated that stabilization of p53 and activation of its target gene, puma (p53-upregulated mediator of apoptosis), mediated proteasome inhibitor-induced apoptosis in cancer cells. Here we demonstrated that Puma also contributed to proteasome inhibitor-induced apoptosis in mouse neocortical neurons. Although protection afforded by puma gene deletion was incomplete, we found little evidence indicating contributions from other proapoptotic BH3-only proteins. Attenuation of bax expression did not further reduce Puma-independent apoptosis, suggesting that pathways other than the mitochondrial apoptosis pathway were activated. Real-time imaging experiments in wild-type and puma-deficient neurons using a fluorescence resonance energy transfer (FRET)-based caspase sensor confirmed the involvement of a second cell death pathway characterized by caspase activation prior to mitochondrial permeabilization and, more prominently, a third, caspase-independent and Puma-independent pathway characterized by rapid cell shrinkage and nuclear condensation. This pathway involved lysosomal permeabilization in the absence of autophagy activation and was sensitive to cathepsin but not autophagy inhibition. Our data demonstrate that proteasomal stress activates distinct cell death pathways in neurons, leading to both caspase-dependent and caspase-independent apoptosis, and demonstrate independent roles for Puma and lysosomal permeabilization in this model.     

Decrease in intracellular superoxide sensitizes Bcl-2-overexpressing tumor cells to receptor and drug-induced apoptosis independent of the mitochondria

At least two mechanisms of early cytosolic acidification during apoptotic signaling have been described, one that involves caspase 8 activation downstream of receptor ligation and another dependent on mitochondria-derived hydrogen peroxide during merocil-induced apoptosis. Here, we show that Bcl-2 inhibits both mechanisms of acidification. Moreover, Bcl-2 overexpression resulted in a slightly elevated constitutive level of superoxide anion and pH in CEM leukemia cells. Interestingly, decreasing intracellular superoxide concentration with an inhibitor of the beta-nicotinamide adenine dinucleotide phosphate oxidase or by transient transfection with a dominant-negative form of the guanosine triphosphate-binding protein Rac1 resulted in a significant increase in the sensitivity of CEM/Bcl-2 cells to CD95- or merocil-induced apoptosis. This increase in sensitivity was a direct result of a significant increase in caspase 8 activation and caspase 8-dependent acidification in the absence of caspase 9 activity or cytochrome c release. These findings suggest a mechanism of switching from mitochondria-dependent to mitochondria-independent death signaling in the same cell, provided the intracellular milieu is permissive for upstream caspase 8 activation, and could have implications for favorably tailoring tumor cells for drug treatment even when the mitochondrial pathway is compromised by Bcl-2.     

p38 mitogen-activated protein kinase mediates the Fas-induced mitochondrial death pathway in CD8+ T cells

The p38 mitogen-activated protein kinase (MAPK) signaling pathway can be activated by a variety of stress stimuli such as UV radiation and osmotic stress. The regulation and role of this pathway in death receptor-induced apoptosis remain unclear and may depend on the specific death receptor and cell type. Here we show that binding of Fas ligand to Fas activates p38 MAPK in CD8+ T cells and that activation of this pathway is required for Fas-mediated CD8+ T-cell death. Active p38 MAPK phosphorylates Bcl-xL and Bcl-2 and prevents the accumulation of these antiapoptotic molecules within the mitochondria. Consequently, a loss of mitochondrial membrane potential and the release of cytochrome c lead to the activation of caspase 9 and, subsequently, caspase 3. Therefore, the activation of p38 MAPK is a critical link between Fas and the mitochondrial death pathway and is required for the Fas-induced apoptosis of CD8+ T cells.     

Activation of caspase pathways during iron chelator-mediated apoptosis

Iron chelators have traditionally been used in the treatment of iron overload. Recently, chelators have also been explored for their ability to limit oxidant damage in cardiovascular, neurologic, and inflammatory disease as well as to serve as anti-cancer agents. To determine the mechanism of cell death induced by iron chelators, we assessed the time course and pathways of caspase activation during apoptosis induced by iron chelators. We report that the chelator tachpyridine sequentially activates caspases 9, 3, and 8. These caspases were also activated by the structurally unrelated chelators dipyridyl and desferrioxamine. The critical role of caspase activation in cell death was supported by microinjection experiments demonstrating that p35, a broad spectrum caspase inhibitor, protected HeLa cells from chelator-induced cell death. Apoptosis mediated by tachpyridine was not prevented by blocking the CD95 death receptor pathway with a Fas-associated death domain protein (FADD) dominant-negative mutant. In contrast, chelator-mediated cell death was blocked in cells microinjected with Bcl-XL and completely inhibited in cells microinjected with a dominant-negative caspase 9 expression vector. Caspase activation was not observed in cells treated with N-methyl tachpyridine, an N-alkylated derivative of tachpyridine which lacks an ability to react with iron. These results suggest that activation of a mitochondrial caspase pathway is an important mechanism by which iron chelators induce cell death.     

MDA-7/IL-24 induces Bcl-2 denitrosylation and ubiquitin-degradation involved in cancer cell apoptosis

MDA-7/IL-24 was involved in the specific cancer apoptosis through suppression of Bcl-2 expression, which is a key apoptosis regulatory protein of the mitochondrial death pathway. However, the underlying mechanisms of this regulation are unclear. We report here that tumor-selective replicating adenovirus ZD55-IL-24 leads to Bcl-2 S-denitrosylation and concomitant ubiquitination, which take part in the 26S proteasome degradation. IL-24-siRNA completely blocks Bcl-2 ubiquitination via reversion of Bcl-2 S-denitrosylation and protects it from proteasomal degradation which confirmed the significant role of MDA-7/IL-24 in regulating posttranslational modification of Bcl-2 in cancer cells. Nitric oxide (NO) is a key regulator of protein S-nitrosylation and denitrosylation. The NO donor, sodium nitroprusside (SNP), down-regulates Bcl-2 S-denitrosylation, attenuates Bcl-2 ubiquitination and subsequently counteracts MDA-7/IL-24 induced cancer cell apoptosis, whereas NO inhibitor 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (PTIO) shows the opposite effect. At the same time, these NO modulators fail to affect Bcl-2 phosphorylation, suggesting that NO regulates Bcl-2 stability in a phosphorylation-independent manner. In addition, Bcl-2 S-nitrosylation reduction induced by ZD55-IL-24 was attributed to both iNOS decrease and TrxR1 increase. iNOS-siRNA facilitates Bcl-2 S-denitrosylation and ubiquitin-degradation, whereas the TrxR1 inhibitor auranofin prevents Bcl-2 from denitrosylation and ubiquitination, thus restrains the caspase signal pathway activation and subsequent cancer cell apoptosis. Taken together, our studies reveal that MDA-7/IL-24 induces Bcl-2 S-denitrosylation via regulation of iNOS and TrxR1. Moreover, denitrosylation of Bcl-2 results in its ubiquitination and subsequent caspase protease family activation, as a consequence, apoptosis susceptibility. These findings provide a novel insight into MDA-7/IL-24 induced growth inhibition and carcinoma apoptosis.