N‐terminal cleavage of Bax by calpain generates a potent proapoptotic 18‐kDa fragment that promotes Bcl‐2‐independent cytochrome C release and apoptotic cell death

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase‐activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N‐terminus, generating a potent proapoptotic 18‐kDa fragment (Bax/p18). Both the calpain‐mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane‐enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase‐3, cleavage of poly(ADP‐ribose) polymerase, and fragmentation of DNA. Unlike the full‐length Bax, Bax/p18 did not interact with the antiapoptotic Bcl‐2 protein in the mitochondrial fraction of drug‐treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide‐induced calpain activation, Bax cleavage, cytochrome c release, and caspase‐3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase‐3‐mediated apoptosis that was not blocked by overexpression of Bcl‐2 protein. Therefore, Bax/p18 has a cytochrome c–releasing activity that promotes cell death independent of Bcl‐2. Finally, Bcl‐2 overexpression inhibited etoposide‐induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution. J. Cell. Biochem. 80:53–72, 2000. © 2000 Wiley‐Liss, Inc.     

p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release

The p53 tumor suppressor gene is critically involved in cell cycle regulation, DNA repair, and programmed cell death. Several lines of evidence suggest that p53 death signals lead to caspase activation; however, the mechanism of caspase activation by p53 still is unclear. Expressing wild type p53 by means of an adenoviral expression vector, we were able to induce apoptotic cell death, as characterized by morphological changes, phosphatidylserine externalization, and internucleosomal DNA fragmentation, in p53(null) Saos-2 cells. This cell death was accompanied by caspase activation as well as by cleavage of caspase substrates and was preceded by mitochondrial cytochrome c release. The addition of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) directly after transduction almost completely prevented p53-induced apoptotic cell death but did not inhibit mitochondrial cytochrome c release. In contrast, N-acetylcysteine, even at high concentrations, could not prevent induction of programmed cell death by p53 expression. Cytosolic extracts from Saos-2 cells transduced with p53, but not from Saos-2 cells transduced with the empty adenoviral vector, contained a cytochrome c-releasing activity in vitro, which was still active in the presence of zVAD-fmk. When Bax was immunodepleted from the cytosolic extracts of p53-expressing cells before incubation with isolated mitochondria, the in vitro cytochrome c release was abolished. Thus, we could demonstrate in cells and in vitro that p53 activates the apoptotic machinery through induction of the release of cytochrome c from the mitochondrial intermembrane space. Furthermore, we provide in vitro evidence for the requirement of cytosolic Bax for this cytochrome c-releasing activity of p53 in Saos-2 cells.     

Loss of Bif-1 suppresses Bax/Bak conformational change and mitochondrial apoptosis

Bif-1, a member of the endophilin B protein family, interacts with Bax and promotes interleukin-3 withdrawal-induced Bax conformational change and apoptosis when overexpressed in FL5.12 cells. Here, we provide evidence that Bif-1 plays a regulatory role in apoptotic activation of not only Bax but also Bak and appears to be involved in suppression of tumorigenesis. Inhibition of endogenous Bif-1 expression in HeLa cells by RNA interference abrogated the conformational change of Bax and Bak, cytochrome c release, and caspase 3 activation induced by various intrinsic death signals. Similar results were obtained in Bif-1 knockout mouse embryonic fibroblasts. While Bif-1 did not directly interact with Bak, it heterodimerized with Bax on mitochondria in intact cells, and this interaction was enhanced by apoptosis induction and preceded the Bax conformational change. Moreover, suppression of Bif-1 expression was associated with an enhanced ability of HeLa cells to form colonies in soft agar and tumors in nude mice. Taken together, these findings support the notion that Bif-1 is an important component of the mitochondrial pathway for apoptosis as a novel Bax/Bak activator, and loss of this proapoptotic molecule may contribute to tumorigenesis.     

Subcellular localization and physiological consequences of introducing a mitochondrial matrix targeting signal sequence in bax and its mutants

Bax, a proapoptotic member of the Bcl-2 family of proteins, resides in the cytosol and translocates to the mitochondrial membrane upon induction of apoptosis. It has been proposed that Bax does not translocate to mitochondria under normal physiological conditions, due to interaction between amino (ART) and carboxy (TM) terminal domains. Here, we report the physiological consequences of introducing a matrix targeting mitochondrial signal sequence (Su9) at the amino terminus of Bax and its mutants lacking ART, TM, or both segments. In vitro mitochondrial protein import assays of the fusion proteins suggests localization to the mitochondrial matrix. When expressed in Cos-1 cells, Su9 could target Bax to mitochondria in the absence of an apoptotic stimulus. However, mitochondrial localization did not result in apoptosis. When ART, TM, or both segments of Bax were deleted, expression of fusion proteins containing Su9 resulted in apoptosis via cytochrome c release. Cell death was inhibited by the pan-caspase inhibitor zVAD-fmk. We thus demonstrate that an effective mitochondrial matrix targeting signal can override the inhibition of import of Bax to the organelle, presumed to arise as a result of interaction between ART and TM segments, in the absence of apoptotic stimulus. We also demonstrate the ability of truncated variants of Bax to cause apoptosis when targeted to mitochondria by cytochrome c release from an ectopic environment.     

Overexpression of mitochondrial uncoupling protein-3 does not decrease production of the reactive oxygen species, elevated by palmitate in skeletal muscle cells

Fatty acids induced an increase in reactive oxygen species (ROS) and enhanced NF-kappaB activation in L6 myotubes differentiated in culture. Palmitate proved more effective than oleate in eliciting these effects. The induction of uncoupling protein-3 (UCP3) at levels similar to those occurring in vivo, attained through the use of an adenoviral vector, led to a reduction of mitochondrial membrane potential in L6 myotubes. However, the capacity of palmitate to increase ROS was not reduced but, quite the opposite, it was moderately enhanced due to the presence of UCP3. The presence of UCP3 in mitochondria did not modify the expression of genes encoding ROS-related enzymes, either in basal conditions or in the presence of palmitate. However, in the presence of UCP3, UCP2 mRNA expression was down-regulated in response to palmitate. We conclude that UCP3 does not act as a protective agent against palmitate-dependent induction of ROS production in differentiated skeletal muscle cells.     

Activation of mitochondrial-driven apoptosis in skeletal muscle cells is not mediated by reactive oxygen species production

While the acquisition of apoptosis resistance is part of the differentiation program of skeletal muscle cells, differentiated muscle cells can undergo apoptosis in response to physiological or pathological stimuli. The generation of reactive oxygen species by mitochondria plays a major role in the control of apoptosis in many cell types. Indeed their involvement in controlling apoptosis in differentiated muscle cells, or in generating resistance to apoptosis remains unknown. Moreover, differentiated muscle cells specifically express the uncoupling protein-3, a mitochondrial protein potentially involved in controlling reactive oxygen species production. To study the role of mitochondrial reactive oxygen species in the control of apoptosis in skeletal muscle cells, L6E9 myoblasts and myotubes were exposed to staurosporine, an inducer of apoptosis via mitochondrial pathways. Staurosporine activated apoptotic pathways (i.e. caspase-3 and caspase-9) increasing reactive oxygen species in myoblasts and, to a minor extent, in myotubes. However, the increase in reactive oxygen species was not needed to induce apoptosis nor was it involved in the differential sensitization of myoblasts and myotubes to apoptosis. Moreover, expression of uncoupling protein-3 in myotubes did not affect reactive oxygen species production, although it produced a slight sensitization for staurosporine-induced apoptosis. Results indicate that apoptotic activation in skeletal muscle cells mainly involves reactive oxygen species-independent mechanisms and that mitochondrial uncoupling protein-3 is not protective either for reactive oxygen species production or for apoptotic activation in muscle cells.     

Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells

Expression of HSV-1 genes leads to the induction of apoptosis in human epithelial HEp-2 cells but the subsequent synthesis of infected cell protein prevents the process from killing the cells. Thus, viruses unable to produce appropriate prevention factors are apoptotic. We now report that the addition of either a pancaspase inhibitor or caspase-9-specific inhibitor prevented cells infected with an apoptotic HSV-1 virus from undergoing cell death. This result indicated that HSV-1-dependent apoptosis proceeds through the mitochondrial apoptotic pathway. However, the pancaspase inhibitor did not prevent the release of cytochrome c from mitochondria, implying that caspase activation is not required for this induction of cytochrome c release by HSV-1. The release of cytochrome c was first detected at 9 hpi while caspase-9, caspase-3 and PARP processing were detected at 12 hpi. Finally, Bax accumulated at mitochondria during apoptotic, but not wild type HSV-1 infection. Together, these findings indicate that HSV-1 blocks apoptosis by precluding mitochondrial cytochrome c release in a caspase-independent manner and suggest Bax as a target in infected human epithelial cells.     

Trail-induced apoptosis in Type I leukemic cells is not enhanced by overexpression of bax

We have previously shown that Bax translocation was crucial in TNFalpha or etoposide-induced apoptosis. Overexpression of Bax sensitized chronic myeloid leukemic K562 cells to etoposide-induced apoptosis. Treatment with TNF-related apoptosis-inducing ligand (TRAIL) induces a loss of mitochondrial membrane potential (DeltaPsim), cytochrome c release from mitochondria, activation of caspases-8, -9, and -3, and cleavage of Bid in the K562 cell line. Bax failed to sensitize K562 cells to TRAIL-induced apoptosis. TRAIL did not induce Bax expression and/or translocation from cytosol to mitochondria in the K562 cell line. However, 100 microM Z-VAD.fmk, a pan caspase inhibitor, completely blocked TRAIL-initiated mitochondrial alterations and cleavages of caspases and Bid. We propose that TRAIL-induced apoptosis in K562 cells is via Type I apoptotic signal pathway. Bax translocation is not essential for TRAIL-induced cytochrome c release and DeltaPsim collapse in the Type I cells.     

Short interfering RNA against the PDCD5 attenuates cell apoptosis and caspase-3 activity induced by Bax overexpression

The programmed cell death 5 (PDCD5) protein plays an important apoptosis-accelerating role in cells undergoing apoptosis. Decreased expression of PDCD5 has been detected in various human carcinomas. Here we describe that one potent short interfering RNA (siRNA) against the PDCD5 (siPDCD5) specifically inhibits the expression of PDCD5 at both the mRNA and protein level. Cells with decreased PDCD5 expression displayed reduced sensitivity to an apoptotic stimulus induced by Bax overexpression in HeLa, HEK293 and 293T cell lines. Furthermore, we also show that siPDCD5 inhibited both caspase-3 activity and procaspase-3 cleavage. Suppressed expression of PDCD5 attenuates the release of cytochrome c from mitochondria to cytosol induced by Bax overexpression. This phenomenon is accompanied by the reduced translocation of Bax from the cytosol to mitochondria. MTT assay shows that targeted suppression of PDCD5 expression markedly promoted cell proliferation in Hela and HEK293 cell lines. Our data suggests that PDCD5 may exert its effects through pathway of mitochondria by modulating Bax translocation, cytochrome c release and caspase 3 activation directly or indirectly, and that decreased PDCD5 expression may be one of the mechanisms by which tumor cells achieve resistance to apoptotic stimulus induced by anticancer drugs.     

The permeability transition pore triggers Bax translocation to mitochondria during neuronal apoptosis

Cerebellar granule neurons (CGNs) require depolarization for their survival in culture. When deprived of this stimulus, CGNs die via an intrinsic apoptotic cascade involving Bim induction, Bax translocation, cytochrome c release, and caspase-9 and -3 activation. Opening of the mitochondrial permeability transition pore (mPTP) is an early event during intrinsic apoptosis; however, the precise role of mPTP opening in neuronal apoptosis is presently unclear. Here, we show that mPTP opening acts as an initiating event to stimulate Bax translocation to mitochondria. A C-terminal (alpha9 helix) GFP-Bax point mutant (T182A) that constitutively localizes to mitochondria circumvents the requirement for mPTP opening and is entirely sufficient to induce CGN apoptosis. Collectively, these data indicate that the major role of mPTP opening in CGN apoptosis is to trigger Bax translocation to mitochondria, ultimately leading to cytochrome c release and caspase activation.     

Withanolide induces apoptosis in HL-60 leukemia cells via mitochondria mediated cytochrome c release and caspase activation

The present study is on the growth inhibitory effect of Withania somnifera methanolic leaf extract and its active component, withanolide on HL-60 promyelocytic leukemia cells. The decrease in survival rate of HL-60 cells was noted to be associated with a time dependent decrease in the Bcl-2/Bax ratio, leading to up regulation of Bax. Both the crude leaf extract and the active component activated the apoptotic cascade through the cytochrome c release from mitochondria. The activation of caspase 9, caspase 8 and caspase 3 revealed that caspase was a key mediator in the apoptotic pathway. DNA fragmentation analysis revealed typical ladders as early as 12h indicative of caspase 3 role in the apoptotic pathway. Flow cytometry data demonstrated an increase of sub-G1 peak upon treatment by 51% at 24h, suggesting the induction of apoptotic cell death in HL-60 cells.     

Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells

The pro-apoptotic protein, Bax, has been reported to translocate from cytosol to mitochondria following exposure of cells to apoptotic stresses including cytokine withdrawal and treatment with glucocorticoids and cytotoxic drugs. These observations, coupled with reports showing that Bax causes the release of mitochondrial cytochrome c, implicate Bax as a central mediator of the apoptotic process. In this report we demonstrate by subcellular fractionation a significant shift in Bax localization from cytosol to cellular membranes in two human tumor cell lines exposed to staurosporine or etoposide. Immunofluorescence studies confirmed that Bax specifically relocalized to the mitochondria. This redistribution of Bax occurred in concert with, or just prior to, proteolytic processing of procaspase-3, activation of DEVD-specific cleavage activity and degradation of poly(ADP-ribose) polymerase. However, Bax membrane translocation was independent of caspase activity as determined using the broad-range caspase inhibitor z-VAD-fmk. High level overexpression of the anti-apoptotic protein Bcl-2 prevented Bax redistribution to the mitochondria, caspase activation and apoptosis following exposure to staurosporine or etoposide. These data confirm the role of Bax in mitochondrial cytochrome c release, and indicate that prevention of Bax translocation to the mitochondrial membrane represents a novel mechanism by which Bcl-2 inhibits drug-induced apoptosis.     

Effect of thymol on peripheral blood mononuclear cell PBMC and acute promyelotic cancer cell line HL-60

Thymol, a naturally occurring phenolic compound, has been known for its antioxidant, anti microbial, and anti inflammatory activity. Thymol has also been reported as anti-cancer agent, but its anti-cancer mechanism has not yet been fully elucidated. Thus, we aimed to investigate anticancer activity of thymol on HL-60 (acute promyelotic leukemia) cells. In our study, thymol demonstrated dose dependent cytotoxic effects on HL-60 cells after 24 h of exposure. However, thymol did not show any cytotoxic effect in normal human PBMC. The cytotoxic effect of thymol on HL-60 cells appears to be associated with induction of cell cycle arrest at sub G0/G1 phase, and apoptotic cell death based on genomic DNA fragmentation pattern. Thymol also showed significant increase in production of reactive oxygen species (ROS) activity, increase in mitochondrial H₂O₂ production and depolarization of mitochondrial membrane potential. On performing Western Blot analysis, thymol showed increase in Bax protein level with a concomitant decrease in Bcl2 protein expression in a dose dependent manner. Our study also showed activation of caspase -9, -8 and -3 and concomitant PARP cleavage, which is the hallmark of caspase-dependent apoptosis. Moreover, to rule out the involvement of other mechanisms in apoptosis induction by thymol, we also studied its effect on apoptosis inducing factor (AIF). Thymol induced AIF translocation from mitochondria to cytosol and to nucleus, thus indicating its ability to induce caspase independent apoptosis. We conclude that, thymol-induced apoptosis in HL-60 cells involves both caspase dependent and caspase independent pathways.     

Cellular death linked to irreversible stress in the sarcoplasmic reticulum: the effect of inhibiting Ca(2+) -ATPase or protein glycosylation in the myocardiac cell model H9c2

Experimental sarcoplasmic reticulum damage induced by 3 microM thapsigargin or 1 microg/ml tunicamycin provoked viability loss of the cell population in approximately 72 h. Release of cytochrome c from mitochondria was an early event and Bax translocation to the mitochondria preceded or was simultaneous with cytochrome c release. The release of cytochrome c was not related with mitochondria depolarization or caspase activation. Irreversible stress in the sarcoplasmic reticulum, detected by the early activation of caspase 12, was functionally linked to the mitochondrial apoptotic pathway. Caspase 3 processing was blocked by cells preincubation with a selective inhibitor of either caspase 9 or caspase 8 whereas caspase 8 activation was inhibited by a selective caspase 9 inhibitor. This was consistent with the involvement of caspase 8 in a positive feedback loop leading to amplify the caspase cascade. Caspase inhibition did not protect against cell death indicating the existence of alternative caspase-independent mechanisms.     

Kaempferol induces apoptosis in two different cell lines via Akt inactivation,Bax and SIRT3 activation,and mitochondrial dysfunction

Kaempferol (3,4′,5,7‐tetrahydroxyflavone) is a flavonoid with anti‐ and pro‐oxidant activity present in various natural sources. Kaempferol has been shown to posses anticancer properties through the induction of the apoptotic program. Here we report that treatment of the chronic myelogenous leukemia cell line K562 and promyelocitic human leukemia U937 with 50 µM kaempferol resulted in an increase of the antioxidant enzymes Mn and Cu/Zn superoxide dismutase (SOD). Kaempferol treatment induced apoptosis by decreasing the expression of Bcl‐2 and increasing the expressions of Bax. There were also induction of mitochondrial release of cytochrome c into cytosol and significant activation of caspase‐3, and ‐9 with PARP cleavage. Kaempferol treatment increased the expression and the mitochondria localization of the NAD‐dependent deacetylase SIRT3. K562 cells stably overexpressing SIRT3 were more sensitive to kaempferol, whereas SIRT3 silencing did not increase the resistance of K562 cells to kaempferol. Inhibition of PI3K and de‐phosphorylation of Akt at Ser473 and Thr308 was also observed after treating both K562 and U937 cells with kaempferol. In conclusion our study shows that the oxidative stress induced by kaempferol in K562 and U937 cell lines causes the inactivation of Akt and the activation of the mitochondrial phase of the apoptotic program with an increase of Bax and SIRT3, decrease of Bcl‐2, release of cytochrome c, caspase‐3 activation, and cell death. J. Cell. Biochem. 106: 643–650, 2009. © 2009 Wiley‐Liss, Inc.     

Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis

Activation of p53 induces apoptosis in various cell types. However, the mechanism by which p53 induces apoptosis is still unclear. We reported previously that the activation of a temperature-sensitive mutant p53 (p53(138Val)) induced activation of caspase 3 and apoptosis in Jurkat cells. To elucidate the pathway linking p53 and downstream caspases, we examined the activation of caspases 8 and 9 in apoptotic cells. The results showed that both caspases were activated during apoptosis as judged by the appearance of cleavage products from procaspases and the caspase activities to cleave specific fluorogenic substrates. The significant inhibition of apoptosis by a tetrapeptide inhibitor of caspase 8 and caspase 9 suggested that both caspases are required for apoptosis induction. In addition, the membrane translocation of Bax and cytosolic release of cytochrome c, but not loss of mitochondrial membrane potential, were detected at an early stage of apoptosis. Moreover, Bax translocation, cytochrome c release, and caspase 9 activation were blocked by the broad-spectrum caspase inhibitor, Z-VAD-fmk and the caspase 8-preferential inhibitor, Ac-IETD-CHO, suggesting that the mitochondria might participate in apoptosis by amplifying the upstream death signals. In conclusion, our results indicated that activation of caspase 8 or other caspase(s) by p53 triggered the membrane translocation of Bax and cytosolic release of cytochrome c, which might amplify the apoptotic signal by activating caspase 9 and its downstream caspases.     

Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

Bax and Bak play a redundant but essential role in apoptosis initiated by the mitochondrial release of apoptogenic factors. In addition to their presence at the mitochondrial outer membrane, Bax and Bak can also localize to the ER. Agents that initiate ER stress responses can induce conformational changes and oligomerization of Bax on the ER as well as on mitochondria. In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells. In bax-/-bak-/- cells, introduction of Bak mutants selectively targeted to either mitochondria or the ER can induce apoptosis. However, ER-targeted, but not mitochondria-targeted, Bak leads to progressive depletion of ER Ca2+ and induces caspase 12 cleavage. In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak. These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.     

The role of p38 MAPK and JNK in Arsenic trioxide-induced mitochondrial cell death in human cervical cancer cells

Previously, we have shown that the release of AIF from mitochondria is required for As2O3-induced cell death in human cervical cancer cells, and that reactive oxygen species (ROS) is necessary for AIF release from mitochondria. In this study, we further investigated the role of MAPKs in ROS-mediated mitochondrial apoptotic cell death triggered by As2O3. As2O3-induced apoptotic cell death in HeLa cells was associated with activation and mitochondrial translocation of Bax, a marked phosphorylation of Bcl-2, reduction of Bcl-2 and Bax interaction, dissipation of mitochondrial membrane potential. Using small interfering RNA, reduced Bax expression effectively attenuated As2O3-induced mitochondrial membrane potential loss and apoptotic cell death. Moreover, the phosphorylation of Bcl-2 induced by As2O3 diminished its ability to bind to Bax. Treatment of cells with As2O3 activated both the p38 MAPK and JNK pathways. Mitochondrial translocation of Bax was completely suppressed in the presence of p38 MAPK inhibitor PD169316 or si-p38 MAPK. The As2O3-induced Bcl-2 phosphorylation was attenuated largely by JNK inhibition using SP600125 or si-JNK and to some extent by p38 MAPK inhibition with PD169316 or si-p38 MAPK. In addition, N-acetyl-L-cystein (NAC), a thiol-containing anti-oxidant, completely blocked As2O3-induced p38 MAPK and JNK activations, mitochondria translocation of Bax, and phosphorylation of Bcl-2. These results support a notion that ROS-mediated activations of p38 MAPK and JNK in response to As2O3 treatment signals activation of Bax and phosphorylation of Bcl-2, resulting in mitochondrial apoptotic cell death in human cervical cancer cells.