Cardiolipin oxidation sets cytochrome c free.
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Apoptosis↗
Biomedical subjects
Boris Zhivotovsky
Publications and source records attributed to Boris Zhivotovsky.
The HPV-16 E7 oncogene sensitizes malignant cells to IFN-alpha-induced apoptosis.
Interferons (IFNs) exert antitumor effects in several human malignancies, but their mechanism of action is unclear. There is a great variability in sensitivity to IFN treatment depending on both tumor type and the individual patient. The reason for this variable sensitivity is not known. The fact that several IFN-induced anticellular effects are exerted through modulation of proto-oncogenes and tumor suppressor genes may indicate that the malignant genotype may be decisive in the cell's sensitivity to IFN. To determine if a deregulated oncogene could alter the cellular response to IFN, a mouse lymphoma cell line (J3D) was stably transfected with the viral human papillomavirus-16 (HPV-16) E7 oncogene. The E7-transfected cells and their respective mock-transfected sister clones were treated with IFN-alpha and examined for possible IFN-induced anticellular effects. We found that the E7-transfected clones were greatly sensitized to IFN-alpha-induced apoptosis compared with their mock-transfected counterparts. Induction of apoptosis in the transfected cells correlated with the ability of IFN to activate parts of the proapoptotic machinery specifically in these cells, including activation of caspases and the proapoptotic protein Bak. In summary, our data suggest that transfection of malignant cells with the E7 oncogene can sensitize them to IFN-alpha-induced apoptosis. This demonstrates that an oncogenic event may alter the cellular sensitivity to IFN and might also have implications for treatment of HPV-related diseases with IFN.
Animals↗
Caspase-2 permeabilizes the outer mitochondrial membrane and disrupts the binding of cytochrome c to anionic phospholipids.
Caspases are cysteine proteases that play a central role in the execution of apoptosis. Recent evidence indicates that caspase-2 is activated early in response to genotoxic stress and can function as an upstream modulator of the mitochondrial apoptotic pathway. In particular, we have shown previously that fully processed caspase-2 can permeabilize the outer mitochondrial membrane and cause cytochrome c and Smac/DIABLO release from these organelles. Using permeabilized cells, isolated mitochondria, and protein-free liposomes, we now report that this effect is direct and depends neither on the presence or cleavage of other proteins nor on a specific phospholipid composition of the liposomal membrane. Interestingly, caspase-2 was also shown to disrupt the interaction of cytochrome c with anionic phospholipids, notably cardiolipin, and thereby enhance the release of the hemoprotein caused by treatment of mitochondria with digitonin or the proapoptotic protein Bax. Combined, our data suggest that caspase-2 possesses an unparalleled ability to engage the mitochondrial apoptotic pathway by permeabilizing the outer mitochondrial membrane and/or by breaching the association of cytochrome c with the inner mitochondrial membrane.
Caspase 2↗
Apoptosis-inducing factor determines the chemoresistance of non-small-cell lung carcinomas.
Non-small-cell lung carcinomas (NSCLCs) are resistant to the induction of apoptosis by conventional anticancer treatment. However, NSCLC cell lines are sensitive to the action of the broad protein kinase inhibitor, staurosporine (STS). In the NSCLC cell line U1810, STS induced the mitochondrial release of apoptosis-inducing factor (AIF) and cytochrome c (Cyt c) followed by activation of caspases, nuclear condensation, DNA fragmentation and finally cell death. Although preincubation of U1810 cells with the broad-spectrum caspase inhibitor z-VAD.fmk delayed the occurrence of nuclear apoptosis induced by STS, it did not impede mitochondrial alterations (such as the release of Cyt c and AIF) and cell death to occur. Moreover, the microinjection of neither Cyt c nor recombinant active caspase-3 into the cytoplasm promoted nuclear apoptosis-related changes in U1810 cells. Evaluation of the role of the caspase-independent factor AIF in STS-mediated death revealed that, upon immunodepletion of AIF, cytosols from STS-treated U1810 lost their capacity to induce nuclear condensation when incubated with isolated nuclei. In addition, microinjection of an anti-AIF antibody prevented AIF from translocating to the nuclei of STS-treated U1810 cells and reduced STS-induced cell death. Finally, although the transfection-enforced overexpression of AIF was not sufficient to induce cell death, it did enhance STS-mediated cell killing. Altogether, these results indicate that activation of caspases is not sufficient to kill U1810 cells and rather suggests an important role for the AIF-mediated mitochondrial-mediated death pathway.
Apoptosis Inducing Factor↗
Downregulation of peroxiredoxin V stimulates formation of etoposide-induced double-strand DNA breaks.
Antioxidant protein Peroxiredoxin V (PrxV) is located in mitochondria and peroxisomes but is also present in the nucleus. Here, we show that nuclear PrxV associates with coilin-containing bodies suggesting possible interaction of this protein with transcription complexes. We also studied etoposide-induced phosphorylation of histone H2AX (gamma-H2AX) in human cells in which PrxV activity was downregulated (knockdown, KD-clones) or compromised by overexpression of redox-negative (RD) protein. In KD clones, but not in RD-clones, formation of etoposide-induced gamma-H2AX was increased, indicating that PrxV inhibits conversion of topoisomerase II cleavage complexes into double-strand DNA breaks but this inhibition is not caused by its antioxidant activity.
Base Sequence↗
Endogenously released Smac is insufficient to mediate cell death of human lung carcinoma in response to etoposide.
Cytotoxic agents eliminate tumor cells via different mechanisms including apoptosis, although this process is not equally efficient in all kinds of cancer cells. Thus, small cell lung carcinomas (SCLCs) are more sensitive than non-small cell lung carcinomas (NSCLCs) to therapy-induced killing. During apoptosis, several apoptogenic proteins release from the mitochondria. Among these proteins is Smac/DIABLO. Overexpression of Smac effectively potentiates apoptosis by neutralizing the caspase-inhibitory function of the inhibitors of apoptosis proteins (IAPs). However, the physiological relevance of endogenously released Smac in the promotion of malignant cell death is still unclear. Analysis of a panel of human lung cancer cell lines revealed that there is no altered Smac expression in NSCLC and SCLC that might initially impair the drug-induced cell death. Upon engagement of the mitochondrial pathway of apoptosis, etoposide provoked cytosolic accumulation of Smac along with cytochrome c and loss of the mitochondrial membrane potential. Most of these events as well as nuclear apoptotic changes required caspase activation in SCLC, but not in NSCLC. Unexpectedly, pan-caspase inhibition had no effect on Smac release. Co-treatment of SCLC with the IAP-binding peptide Smac-N7 enhanced etoposide-induced apoptosis in a concentration-dependent manner, whereas Smac downregulation by small interfering RNA (siRNA) did not influence caspase-3/-7 activities, nuclear morphological changes, DNA fragmentation, and plasma membrane integrity. Release of cytochrome c and mitochondrial protease Omi/HtrA2 is still detectable at these conditions. These data suggest that Smac deficiency may be compensated for by action of redundant determinants to kill cancer cells. Thus, translocation of endogenous Smac into cytosol does not play a critical role in cell death of human lung carcinoma after etoposide treatment.
Antineoplastic Agents, Phytogenic↗
Processed caspase-2 can induce mitochondria-mediated apoptosis independently of its enzymatic activity.
The mechanism by which caspase-2 executes apoptosis remains obscure. Recent findings indicate that caspase-2 is activated early in response to DNA-damaging antineoplastic agents and may be important for the engagement of the mitochondrial apoptotic pathway. We demonstrate here that fully processed caspase-2 stimulates mitochondrial release of cytochrome c and Smac/DIABLO, but not apoptosis-inducing factor (AIF). This event occurs independently of several Bcl-2 family proteins, including Bax, Bak and Bcl-2, and inactivation experiments reveal that the proteolytic activity of caspase-2 is not required for the effect. Further, functional studies of mitochondria indicate that processed caspase-2 stimulates state 4 respiration and decreases the respiratory control ratio as a result of, in large part, an uncoupling effect. Combined, our data suggest that caspase-2 retains a unique ability to engage directly the mitochondrial apoptotic pathway, an effect that requires processing of the zymogen but not the associated catalytic activity.
Apoptosis↗
Endothelial cell surface ATP synthase-triggered caspase-apoptotic pathway is essential for k1-5-induced antiangiogenesis.
We have recently reported the identification of kringle 1-5 (K1-5) of plasminogen as a potent and specific inhibitor of angiogenesis and tumor growth. Here, we show that K1-5 bound to endothelial cell surface ATP synthase and triggered caspase-mediated endothelial cell apoptosis. Induction of endothelial apoptosis involved sequential activation of caspases-8, -9, and -3. Administration of neutralizing antibodies directed against the alpha- and beta-subunits of ATP synthase to endothelial cells attenuated activation of these caspases. Furthermore, inhibitors of caspases-3, -8, and -9 also remarkably blocked K1-5-induced endothelial cell apoptosis and antiangiogenic responses. In a mouse tumor model, we show that caspase-3 inhibitors abolished the antitumor activity of K1-5 by protecting the tumor vasculature undergoing apoptosis. These results suggest that the specificity of the antiendothelial effect of K1-5 is attributable, at least in part, to its interaction with the endothelial cell surface ATP synthase and that the caspase-mediated endothelial apoptosis is essential for the angiostatic activity of K1-5. Thus, our findings provide a mechanistic insight with respect to the angiostatic action and signaling pathway of K1-5 and angiostatin.
ATP Synthetase Complexes↗
Metacaspase-dependent programmed cell death is essential for plant embryogenesis.
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Apoptosis↗
DNA damage-induced apoptosis.
Unicellular organisms respond to the presence of DNA lesions by activating cell cycle checkpoint and repair mechanisms, while multicellular animals have acquired the further option of eliminating damaged cells by triggering apoptosis. Defects in DNA damage-induced apoptosis contribute to tumorigenesis and to the resistance of cancer cells to a variety of therapeutic agents. The intranuclear mechanisms that signal apoptosis after DNA damage overlap with those that initiate cell cycle arrest and DNA repair, and the early events in these pathways are highly conserved. In addition, multiple independent routes have recently been traced by which nuclear DNA damage can be signalled to the mitochondria, tipping the balance in favour of cell death rather than repair and survival. Here, we review current knowledge of nuclear DNA damage signalling, giving particular attention to interactions between these nuclear events and apoptotic processes in other intracellular compartments.
Adaptor Proteins, Signal Transducing↗
The transcriptosomal response of human A549 lung cells to a hydrogen peroxide-generating system: relationship to DNA damage, cell cycle arrest, and caspase activation.
Intracellular oxidative stress is a dynamic situation characterized by the accumulation of reactive oxygen metabolites, such as hydrogen peroxide. This is traditionally associated with both macromolecular damage and adaptive changes in gene expression, aimed at preventing cellular demise. However, the overall extent of such genetic changes is not well characterized. Here we present a comprehensive analysis of altered mRNA profiles in human A549 type II lung epithelial cells in response to hydrogen peroxide, at concentrations failing to induce necrotic toxicity. The results of an Affymetrix-based screen of the steady-state levels of mRNAs for several thousand genes revealed a complex pattern of transcriptional and/or posttranscriptional response to oxidative stress, which can be functionally related to both the oxidation and repair of damaged DNA, the induction and permanency of cell cycle arrest, and caspase-3 activation. Many of the genetic events can be related to activation of the p53/p21 pathway, but many other novel inductions and suppressions were detected, revealing the intricacy of the response. The data also disclosed a potential interaction between hydrogen peroxide treatment and increased sensitivity to cell killing by TRAIL, which could be functionally confirmed at the level of induction of caspase-3 activity.
8-Hydroxy-2'-Deoxyguanosine↗
Interferon alpha-induced apoptosis in tumor cells is mediated through the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway.
Interferon (IFN) alpha induces a caspase-dependent apoptosis that is associated with activation of the proapoptotic Bak and Bax, loss of mitochondrial membrane potential, and release of cytochrome c. In addition to the onset of the classical Jak-STAT pathway, IFNalpha also induced phosphoinositide 3-kinase (PI3K) activity. Pharmacological inhibition of PI3K activity by Ly294002 disrupted IFN-induced apoptosis upstream of mitochondria. Inhibition of mTOR by rapamycin or by overexpression of a kinase dead mutant of mTOR, efficiently blocked IFNalpha-induced apoptosis. A PI3K and mTOR-dependent phosphorylation of p70S6 kinase and 4E-BP1 repressor was induced by IFNalpha treatment of cells and was strongly inhibited by Ly294002 or rapamycin. The activation of Jak-STAT signaling upon IFNalpha stimulation was not affected by abrogating PI3K/mTOR pathway. Neither was the expression of several IFNalpha target genes affected, nor the ability of IFNalpha to protect against virus-induced cell death affected by inhibition of the PI3K/mTOR pathway. These data demonstrate that an intact PI3K/mTOR pathway is necessary for the ability of IFNalpha to induce apoptosis, whereas activation of the Jak-STAT pathway alone appears to be insufficient for this specific IFNalpha-induced effect.
Adaptor Proteins, Signal Transducing↗
Guarding the Bcl-2 army.
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Antimetabolites, Antineoplastic↗
Adiponectin-induced antiangiogenesis and antitumor activity involve caspase-mediated endothelial cell apoptosis.
Obesity is a risk factor for the development of many severe human diseases such as cardiovascular disorders, diabetes, and cancer, which are tightly linked to angiogenesis. The adipose tissue produces several growth factors/hormones including leptin, tumor necrosis factor alpha, and adiponectin. It has been found that adiponectin levels are reduced in obesity. Here, we report a unique function of adiponectin as a negative regulator of angiogenesis. In vitro, adiponectin potently inhibits endothelial cell proliferation and migration. In the chick chorioallantoic membrane and the mouse corneal angiogenesis assays, adiponectin remarkably prevents new blood vessel growth. Further, we demonstrate that the antiendothelial mechanisms involve activation of caspase-mediated endothelial cell apoptosis. Adiponectin induces a cascade activation of caspases-8, -9, and -3, which leads to cell death. In a mouse tumor model, adiponectin significantly inhibits primary tumor growth. Impaired tumor growth is associated with decreased neovascularization, leading to significantly increased tumor cell apoptosis. These data demonstrate induction of endothelial apoptosis as an unique mechanism of adiponectin-induced antiangiogenesis. Adiponectin, as a direct endogenous angiogenesis inhibitor, may have therapeutic implications in the treatment of angiogenesis-dependent diseases.
Adiponectin↗
Mitochondrial cytochrome c release may occur by volume-dependent mechanisms not involving permeability transition.
The mechanisms regulating mitochondrial outer-membrane permeabilization and the release of cytochrome c during apoptosis remain controversial. In the present study, we show in an in vitro model system that the release of cytochrome c may occur via moderate modulation of mitochondrial volume, irrespective of the mechanism leading to the mitochondrial swelling. In contrast with mitochondrial permeability transition-dependent release of cytochrome c, in the present study mitochondria remain intact and functionally active.
Animals↗
RasGTPase-activating protein is a target of caspases in spontaneous apoptosis of lung carcinoma cells and in response to etoposide.
p120 RasGTPase-activating protein (RasGAP), the main regulator of Ras GTPase family members, is cleaved at low caspase activity into an N-terminal fragment that triggers potent anti-apoptotic signals via activation of the Ras/PI-3 kinase/Akt pathway. When caspase activity is increased, RasGAP fragment N is further processed into two fragments that effectively potentiate apoptosis. Expression of RasGAP protein and its cleavage was assessed in human lung cancer cells with different histology and responsiveness to anticancer drug-induced apoptosis. Here we show that therapy-sensitive small lung carcinoma cell (SCLC) lines have lower RasGAP expression levels and higher spontaneous cleavage with formation of fragment N compared to therapy-resistant non-small cell lung carcinoma cell (NSCLC) lines. The first RasGAP cleavage event strongly correlated with the increased level of spontaneous apoptosis in SCLC. However, generation of protective RasGAP fragment N also related to the potency of SCLC to develop secondary therapy-resistance. In response to etoposide (ET), RasGAP fragment N was further cleaved in direct dependence on caspase-3 activity, which was more pronounced in NSCLC cells. Caspase inhibition, while effectively preventing the second cleavage of RasGAP, barely affected the first cleavage of RasGAP into fragment N that was always detectable in NSCLC and SCLC cells. These findings suggest that different levels of RasGAP and fragment N might play a significant role in the biology and different clinical course of both subtypes of lung neoplasms. Furthermore, constitutive formation of RasGAP fragment N can potentially contribute to primary resistance of NSCLC to anticancer therapy by ET but also to secondary therapy-resistance in SCLC.
Apoptosis↗
Apoptosis and genomic instability.
Genomic instability is intrinsically linked to significant alterations in apoptosis control. Chromosomal and microsatellite instability can cause the inactivation of pro-apoptotic pathways. In addition, the inhibition of apoptosis itself can be permissive for the survival and ongoing division of cells that have failed to repair DNA double-strand breaks, experience telomere dysfunction or are in an abnormal polyploid state. Furthermore, DNA-repair proteins can regulate apoptosis. So, genomic instability and apoptosis are intimately linked phenomena, with important implications for the pathophysiology of cancer.
Animals↗
Apoptosis, necrosis and between.
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Animals↗