The Role of Cytochrome c on Apoptosis Induced by Anagrapha falcifera Multiple Nuclear Polyhedrosis Virus in Insect Spodoptera litura Cells

There are conflicting reports on the role of cytochrome c during insect apoptosis. Our previous studies have showed that cytochrome c released from the mitochondria was an early event by western blot analysis and caspase-3 activation was closely related to cytochrome c release during apoptosis induced by baculovirus in Spodoptera litura cells (Sl-1 cell line). In the present study, alteration in mitochondrial morphology was observed by transmission electron microscopy, and cytochrome c release from mitochondria in apoptotic Sl-1 cells induced with Anagrapha falcifera multiple nuclear polyhedrosis virus (AfMNPV) has further been confirmed by immunofluoresence staining protocol, suggesting that structural disruption of mitochondria and the release of cytochrome c are important events during Lepidoptera insect cell apoptosis. We also used Sl-1 cell-free extract system and the technique of RNA interference to further investigate the role of cytochrome c in apoptotic Sl-1 cells induced by AfMNPV. Caspase-3 activity in cell- free extracts supplemented with exogenous cytochrome c was determined and showed an increase with the extension of incubation time. DsRNA-mediated silencing of cytochrome c resulted in the inhibition of apoptosis and protected the cells from AfMNPV-induced cell death. Silencing of expression of cytochrome c had a remarkable effect on pro-caspase-3 and pro-caspase-9 activation and resulted in the reduction of caspase-3 and caspase-9 activity in Sl-1 cells undergoing apoptosis. Caspase-9 inhibitor could inhibit activation of pro-caspase-3, and the inhibition of the function of Apaf-1 with FSBA blocked apoptosis, hinting that Apaf-1 could be involved in Sl-1 cell apoptosis induced by AfMNPV. Taken together, these results strongly demonstrate that cytochrome c plays an important role in apoptotic signaling pathways in Lepidopteran insect cells.     

Involvement of caspase activation through release of cytochrome c from mitochondria in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans

We previously reported that infection with the periodontopathic bacterium Actinobacillus actinomycetemcomitans induced apoptosis in a mouse macrophage cell line J774.1. In the present study, we examined the involvement of cytochrome c and caspases in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells. Following infection, the expression levels of cytochrome c, and cleaved forms of caspase-3 and caspase-9 in the cells were examined using immunoblot analysis. Cytochrome c was released from mitochondria into the cytoplasm after A. actinomycetemcomitans-infected J774.1 cells were cultured for 6 h, and caspase-3 and caspase-9 were found to be cleaved forms in the cells. Further, caspase-9 activity was markedly increased, and phosphorylated p53 was detected in the cells 30 h following infection. These results suggest that apoptosis in A. actinomycetemcomitans-infected J774.1 cells is regulated by the release of cytochrome c from mitochondria into cytoplasm and the subsequent activation of caspases through phosphorylation of p53.     

Ca(2+)-induced inhibition of apoptosis in human SH-SY5Y neuroblastoma cells: degradation of apoptotic protease activating factor-1 (APAF-1)

During apoptotic and excitotoxic neuron death, challenged mitochondria release the pro-apoptotic factor cytochrome c. In the cytosol, cytochrome c is capable of binding to the apoptotic protease-activating factor-1 (APAF-1). This complex activates procaspase-9 in the presence of dATP, resulting in caspase-mediated execution of apoptotic neuron death. Many forms of Ca(2+)-mediated neuron death, however, do not lead to prominent activation of the caspase cascade despite significant release of cytochrome c from mitochondria. We demonstrate that elevation of cytosolic Ca(2+) induced prominent degradation of APAF-1 in human SH-SY5Y neuroblastoma cells and in a neuronal cell-free apoptosis system. Loss of APAF-1 correlated with a reduced ability of cytochrome c to activate caspase-3-like proteases. Ca(2+) induced the activation of calpains, monitored by the cleavage of full-length alpha-spectrin into a calpain-specific 150-kDa breakdown product. However, pharmacological inhibition of calpain activity indicated that APAF-1 degradation also occurred via calpain-independent pathways. Our data suggest that Ca(2+) inhibits caspase activation during Ca(2+)-mediated neuron death by triggering the degradation of the cytochrome c-binding protein APAF-1.     

Role of the mitochondrial signaling pathway in murine coronavirus-induced oligodendrocyte apoptosis

A previous study demonstrated that infection of rat oligodendrocytes by mouse hepatitis virus (MHV) resulted in apoptosis, which is caspase dependent (Y. Liu, Y. Cai, and X. Zhang, J. Virol. 77:11952-11963, 2003). Here we determined the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis. We found that caspase-9 activity was 12-fold higher in virus-infected cells than in mock-infected cells at 24 h postinfection (p.i.). Pretreatment of cells with a caspase-9 inhibitor completely blocked caspase-9 activation and partially inhibited the apoptosis mediated by MHV infection. Analyses of cytochrome c release further revealed an activation of the mitochondrial apoptotic pathway. Stable overexpression of the two antiapoptotic proteins Bcl-2 and Bcl-xL significantly, though only partially, blocked apoptosis, suggesting that activation of the mitochondrial pathway is partially responsible for the apoptosis. To identify upstream signals, we determined caspase-8 activity, cleavage of Bid, and expression of Bax and Bad by Western blotting. We found a drastic increase in caspase-8 activity and cleavage of Bid at 24 h p.i. in virus-infected cells, suggesting that Bid may serve as a messenger to relay the signals from caspase-8 to mitochondria. However, treatment with a caspase-8 inhibitor only slightly blocked cytochrome c release from the mitochondria. Furthermore, we found that Bax but not Bad was significantly increased at 12 h p.i. in cells infected with both live and UV-inactivated viruses and that Bax activation was partially blocked by treatment with the caspase-8 inhibitor. These results thus establish the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis.     

Vibrio vulnificus cytolysin induces superoxide anion-initiated apoptotic signaling pathway in human ECV304 cells

Previous studies showed that exposure to Vibrio vulnificus cytolysin (VVC) caused characteristic morphologic changes and dysfunction of vascular structures in lung. VVC showed cytotoxicity for mammalian cells in culture and acted as a vascular permeability factor. In this study, the underlying mechanisms of VVC-induced cytotoxicity was investigated on ECV304 cell, a human vascular endothelial cell line. When cells were exposed to 0.4 hemolytic units (HU) of VVC, consecutive apoptotic events were observed; the elevation of superoxide anion (O (-.)(2)), the release of cytochrome c, the activation of caspase-3, the cleavage of poly(ADP-ribose) polymerase, and the DNA fragmentation. The pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), O(-.) 2) scavenger, completely abolished O(-.)(2) levels and downstream apoptotic events. Moreover, pretreatment with cyclosporin A (CsA), a mitochondrial permeability transition inhibitor, was capable of attenuating O(-.)(2)-mediated cytochrome c release and caspase-3 activation, and consequent apoptosis. Apoptosis, as demonstrated by oligonucleosomal DNA fragmentation and fluorescence microscopy, was induced 24 h after VVC treatment, which was also prevented by caspase-3 inhibitor, Ac-DEVD-CHO. Caspase-1 inhibitor, Ac-YVAD-CHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where VVC-induced apoptosis is triggered by the generation of O(-.)(2), release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation. The induction of apoptosis in endothelial cells by VVC may provide a pivotal mechanism for understanding the pathophysiology of septicemia.     

Mitochondrial cytochrome c release and caspase-3-like protease activation during indomethacin-induced apoptosis in rat gastric mucosal cells

Indomethacin (IND), a nonsteroidal anti-inflammatory drug, has been known to cause gastric mucosal injury as a side effect. Using a rat gastric mucosal cell line, RGM1, we determined whether apoptosis is involved in IND-mediated gastropathy, and whether caspase activation and mitochondrial cytochrome c release play an important role in producing apoptosis of IND-treated RGM1 cells in the presence of serum. IND caused caspase-3-like protease activation followed by apoptosis in a dose- and time-dependent manner. Caspase-1-like protease activity did not change during IND-induced apoptosis. IND also increased mitochondrial cytochrome c release in a time-dependent fashion. Mitochondrial cytochrome c efflux occurred just before or at the same time as caspase-3-like protease activation, and preceded the increase in apoptotic cell numbers. Z-VAD-FMK, a caspase inhibitor, inhibited both the increase in caspase-3-like protease activity and apoptosis in IND-treated RGM1 cells but did not affect caspase-1-like protease activity or mitochondrial cytochrome c release. These observations suggest that the apoptosis of gastric mucosal cells could be involved in IND-induced gastropathy, that cytochrome c is released from mitochondria into the cytosol during the early phase of IND-mediated apoptosis, and that subsequent activation of caspase-3-like protease, but not caspase-1-like protease, is required for the execution of apoptosis.     

The early phase of programmed cell death in Caco-2 intestinal cells exposed to PTH

The regulation of apoptosis is critical for ensuring the homeostasis of an organism. As such, the cell has derived various mechanisms to precisely control the balance between survival and apoptotic signaling. Parathyroid hormone (PTH) function as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. Depending on the cell type involved, PTH also inhibits or promotes the apoptosis. In a previous work we found that PTH promotes the apoptosis of human Caco-2 intestinal cells. In the current study, we demonstrate, for the first time, that stimulation of Caco-2 cells with PTH (10(-8) M) results in the dephosphorylation and translocation of pro-apoptotic protein Bad from the cytosol to mitochondria and release of cytochrome c and Smac/Diablo. The hormone also triggers mitochondria cellular distribution to the perinuclear region, morphological features consistent with apoptosis. PTH increases the enzymatic activity of caspase-3 (48 h) that is also evidenced from the appearance of its cleaved fragments in western blot experiments. Moreover, active caspase-3 is present in nucleus after PTH treatment. In addition, a caspase-3 substrate, poly (ADP-ribose) polymerase (PARP), is degraded by 48 h of PTH treatment. Taken together, our results suggest that, in Caco-2 cells, the induction of apoptosis in response to PTH is mediated by translocation of mitochondria to the perinuclear region, dephosphorylation of Akt, dephosphorylation of Bad and its movement to the mitochondria and subsequent release of cytochrome c and Smac/Diablo which result in activation of downstream caspase-3.     

Polyamine depletion prevents camptothecin-induced apoptosis by inhibiting the release of cytochrome c

We have shown previously that depletion of polyamines delays apoptosis induced by camptothecin in rat intestinal epithelial cells (IEC-6). Mitochondria play an important role in the regulation of apoptosis in mammalian cells because apoptotic signals induce mitochondria to release cytochrome c. The latter interacts with Apaf-1 to activate caspase-9, which in turn activates downstream caspase-3. Bcl-2 family proteins are involved in the regulation of cytochrome c release from mitochondria. In this study, we examined the effects of polyamine depletion on the activation of the caspase cascade, release of cytochrome c from mitochondria, and expression and translocation of Bcl-2 family proteins. We inhibited ornithine decarboxylase, the first rate-limiting enzyme in polyamine synthesis, with alpha-difluoromethylornithine (DFMO) to deplete cells of polyamines. Depletion of polyamines prevented camptothecin-induced release of cytochrome c from mitochondria and decreased the activity of caspase-9 and caspase-3. The mitochondrial membrane potential was not disrupted when cytochrome c was released. Depletion of polyamines decreased translocation of Bax to mitochondria during apoptosis. The expression of antiapoptotic proteins Bcl-x(L) and Bcl-2 was increased in DFMO-treated cells. Caspase-8 activity and cleavage of Bid were decreased in cells depleted of polyamines. These results suggest that polyamine depletion prevents IEC-6 cells from apoptosis by preventing the translocation of Bax to mitochondria, thus preventing the release of cytochrome c.     

Apo cytochrome c inhibits caspases by preventing apoptosome formation

Caspases are cysteine proteases and potent inducers of apoptosis. Their activation and activity is therefore tightly regulated. There are several mechanisms by which caspases can be activated but one key pathway involves release of holo cytochrome c from mitochondria into the cytoplasm. Cytoplasmic holo cytochrome c binds to apoptotic protease activating factor-1 (Apaf-1), driving the formation of an Apaf-1 oligomer (the apoptosome) which in turn binds and activates caspase-9. Previously we showed that the apo form of cytochrome c (lacking heme) can bind Apaf-1 and block both holo-dependent caspase activation in cell extracts and Bax-induced apoptosis in cells. Here we tested the ability of apo cytochrome c to inhibit caspase-9 activation induced by recombinant Apaf-1. Furthermore, using purified proteins and size exclusion chromatography we show that apo cytochrome c prevents holo cytochrome c-dependent apoptosome formation.     

LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway

We previously showed (Gastroenterology 123: 206-216, 2002) that lysophosphatidic acid (LPA) protects and rescues rat intestinal epithelial cells (IEC-6) from apoptosis. Here, we provide evidence for the LPA-elicited inhibition of the mitochondrial apoptotic pathway leading to attenuation of caspase-3 activation. Pretreatment of IEC-6 cells with LPA inhibited campothecin-induced caspase-9 and caspase-3 activation and DNA fragmentation. A caspase-9 inhibitor peptide mimicked the LPA-elicited antiapoptotic activity. LPA elicited ERK1/ERK2 and PKB/Akt phosphorylation. The LPA-elicited antiapoptotic activity and inhibition of caspase-9 activity were abrogated by pertussis toxin, PD 98059, wortmannin, and LY 294002. LPA reduced cytochrome c release from mitochondria and prevented activation of caspase-9. LPA prevented translocation of Bax from cytosol to mitochondria and increased the expression of the antiapoptotic Bcl-2 mRNA and protein. LPA had no effect on Bcl-xl, Bad, and Bak mRNA or protein expression. These data indicate that LPA protects IEC-6 cells from camptothecin-induced apoptosis through G(i)-coupled inhibition of caspase-3 activation mediated by the attenuation of caspase-9 activation due to diminished cytochrome c release, involving upregulation of Bcl-2 protein expression and prevention of Bax translocation.     

Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria

Nitric oxide (NO) can trigger either necrotic or apoptotic cell death. We have used PC12 cells to investigate the extent to which NO-induced cell death is mediated by mitochondria. Addition of NO donors, 1 mM S-nitroso-N-acetyl-DL-penicillamine (SNAP) or 1 mM diethylenetriamine-NO adduct (NOC-18), to PC12 cells resulted in a steady-state level of 1-3 microM: NO, rapid and almost complete inhibition of cellular respiration (within 1 min), and a rapid decrease in mitochondrial membrane potential within the cells. A 24-h incubation of PC12 cells with NO donors (SNAP or NOC-18) or specific inhibitors of mitochondrial respiration (myxothiazol, rotenone, or azide), in the absence of glucose, caused total ATP depletion and resulted in 80-100% necrosis. The presence of glucose almost completely prevented the decrease in ATP level and the increase in necrosis induced by the NO donors or mitochondrial inhibitors, suggesting that the NO-induced necrosis in the absence of glucose was due to the inhibition of mitochondrial respiration and subsequent ATP depletion. However, in the presence of glucose, NO donors and mitochondrial inhibitors induced apoptosis of PC12 cells as determined by nuclear morphology. The presence of apoptotic cells was prevented completely by benzyloxycarbonyl-Val-Ala-fluoromethyl ketone (a nonspecific caspase inhibitor), indicating that apoptosis was mediated by caspase activation. Indeed, both NO donors and mitochondrial inhibitors in PC12 cells caused the activation of caspase-3- and caspase-3-processing-like proteases. Caspase-1 activity was not activated. Cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) decreased the activity of caspase-3- and caspase-3-processing-like proteases after treatment with NO donors, but was not effective in the case of the mitochondrial inhibitors. The activation of caspases was accompanied by the release of cytochrome c from mitochondria into the cytosol, which was partially prevented by cyclosporin A in the case of NO donors. These results indicate that NO donors (SNAP or NOC-18) may trigger apoptosis in PC12 cells partially mediated by opening the mitochondrial permeability transition pores, release of cytochrome c, and subsequent caspase activation. NO-induced apoptosis is blocked completely in the absence of glucose, probably due to the lack of ATP. Our findings suggest that mitochondria may be involved in both types of cell death induced by NO donors: necrosis by respiratory inhibition and apoptosis by opening the permeability transition pore. Further, our results indicate that the mode of cell death (necrosis versus apoptosis) induced by either NO or mitochondrial inhibitors depends critically on the glycolytic capacity of the cell.     

Pro-caspase-8 is predominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation

The recruitment and cleavage of pro-caspase-8 to produce the active form of caspase-8 is a critical biochemical event in death receptor-mediated apoptosis. However, the source of pro-caspase-8 available for activation by apoptotic triggers is unknown. In human fibroblasts and mouse clonal striatal cells, confocal microscopy revealed that pro-caspase-8 immunofluorescence was colocalized with cytochrome c in mitochondria and was also distributed diffusely in some nuclei. Biochemical analysis of subcellular fractions indicated that pro-caspase-8 was enriched in mitochondria and in nuclei. Pro-caspase-8 was found in the intermembrane space, inner membrane, and matrix of mitochondria after limited digestion of mitochondrial fractions, and this distribution was confirmed by immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were treated with an inhibitor of the ADP/ATP carrier atractyloside, which opens the mitochondria permeability transition pore. Release was blocked by the mitochondria permeability transition pore inhibitor cyclosporin A (CsA). After clonal striatal cells were exposed for 6 h to an apoptotic inducer tumor necrosis factor-alpha (TNF-alpha), mitochondria immunoreactive for cytochrome c and pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and cytochrome c levels decreased in mitochondrial fractions and increased, along with pro-caspase-8 cleavage products, in the cytoplasm of the TNF-alpha-treated striatal cells. CsA blocked the TNF-alpha-induced release of pro-caspase 8 but not cytochrome c. Internucleosomal DNA fragmentation started at 6 h and peaked 12 h after TNF-alpha treatment. These results suggest that pro-caspase-8 is predominantly localized in mitochondria and is released upon apoptotic stimulation through a CsA-sensitive mechanism.     

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.     

In vivo application of mitochondrial pore inhibitors blocks the induction of apoptosis in axotomized neonatal facial motoneurons

Axotomy induces apoptosis in motoneurons of neonatal rodents. To identify the key players in motoneuron apoptosis, we assessed the progression of apoptosis at 4 h intervals following facial motoneuron axotomy. The mitochondrial release of cytochrome c, caspase-3 activation and nuclear condensation were first observed in the motoneuron cell bodies 16 h postaxotomy. In vivo application of inhibitors of the mitochondrial permeability transition pore, Bongkrekic acid and cyclosporin A prevented cytochrome c release as well as caspase-3 activation and attenuated motoneuron apoptosis. Similarly, in vivo application of RU360, an inhibitor of the mitochondrial calcium uniporter, also protected axotomized motoneurons from apoptosis. Taken together, our results show that cytochrome c release and subsequent caspase-3 activation are critical events that precipitate the apoptotic death of axotomized neonatal motoneurons in vivo. In addition, these results provide evidence that application of mitochondrial pore inhibitors in vivo can block the induction of apoptosis following motoneuron axotomy.     

Mitochondrially localized active caspase-9 and caspase-3 result mostly from translocation from the cytosol and partly from caspase-mediated activation in the organelle. Lack of evidence for Apaf-1-mediated procaspase-9 activation in the mitochondria

Active caspase-9 and caspase-3 have been observed in the mitochondria, but their origins are unclear. Theoretically, procaspase-9 might be activated in the mitochondria in a cytochrome c/Apaf-1-dependent manner, or activated caspase-9 and -3 may translocate to the mitochondria, or the mitochondrially localized procaspases may be activated by the translocated active caspases. Here we present evidence that the mitochondrially localized active caspase-9 and -3 result mostly from translocation from the cytosol (into the intermembrane space) and partly from caspase-mediated activation in the organelle rather than from the Apaf-1-mediated activation. Apaf-1 localizes exclusively in the cytosol and, upon apoptotic stimulation, translocates to the perinuclear area but not to the mitochondria. In most cases, the mitochondrially localized procaspase-9 and -3 are released early during apoptosis and translocate to the cytosol and/or perinuclear area. Cytochrome c and the mitochondrial matrix protein Hsp60 are also rapidly released to the cytosol early during apoptosis. Both the early release of proteins like cytochrome c and Hsp60 from the mitochondria as well as the later translocation of the active caspase-9/-3 are partially inhibited by cyclosporin A, an inhibitor of mitochondrial membrane permeabilization. The mitochondrial active caspases may function as a positive feedback mechanism to further activate other or residual mitochondrial procaspases, degrade mitochondrial constituents, and disintegrate mitochondrial functions.     

Arginine antimetabolite L-canavanine induces apoptotic cell death in human Jurkat T cells via caspase-3 activation regulated by Bcl-2 or Bcl-xL

L-Canavanine, a natural L-arginine analog, is known to possess cytotoxicity to tumor cells in culture and experimental tumors in vivo. In this study, we first show that apoptotic cell death is associated with antitumor activity of L-canavanine against human acute leukemia Jurkat T cells. When Jurkat T cells were treated with 1.25-5.0mM L-canavanine for 36 h, apoptotic cell death accompanying several biochemical events such as caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), and apoptotic DNA fragmentation was induced in a dose-dependent manner; however, cytochrome c release from mitochondria was not detected. Under these conditions, the expression of Bcl-2 and its functional homolog Bcl-xL was markedly upregulated. The L-canavanine-induced caspase-3 activation, degradation of PARP, and apoptotic DNA fragmentation were suppressed by ectopic expression of Bcl-2 or Bcl-xL, both of which are known to play roles as anti-apoptotic regulators. These results demonstrate that the cytotoxic effect of L-canavanine on Jurkat T cells is attributable to the induced apoptosis and that L-canavanine-induced apoptosis is mediated by a cytochrome c-independent caspase-3 activation pathway that can be interrupted by Bcl-2 or Bcl-xL.     

Involvement of cytochrome c and caspases in apoptotic cell death of human submandibular gland ductal cells induced by concanamycin A

In the present study, we found that a specific inhibitor of vacuolar type H+-ATPase (V-ATPase), concanamycin A, induced apoptosis in a human submandibular gland ductal cancer cell line, HSG. Immunoblot analysis revealed that cytochrome c was released from mitochondria into the cytoplasm when HSG cells were cultured with concanamycin A for 6 h. The maximum activities of caspase-3 and -9 were reached in HSG cells after 18 and 12 h culture of concanamycin A, respectively. Both caspase-3 and -9 were cleaved to an active form in HSG cells cultured with concanamycin A. Interestingly, concanamycin A decreased the level of heat shock protein 27 (HSP27) in HSG cells. Taken together, these findings suggest that apoptosis in HSG cells induced by concanamycin A is regulated by cytochrome c released from mitochondria into cytoplasm and the subsequent activation of caspases, and that HSP27 may interfere with caspase-dependent apoptotic cell death induced by concanamycin A.     

Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1DeltaN, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.     

PHAPI, CAS, and Hsp70 promote apoptosome formation by preventing Apaf-1 aggregation and enhancing nucleotide exchange on Apaf-1

During apoptosis, cytochrome c is released from mitochondria to the cytosol, where it binds Apaf-1. The Apaf-1/cytochrome c complex then oligomerizes either into heptameric caspase-9-activating apoptosome, which subsequently activates caspase-3 and caspase-7, or bigger inactive aggregates, depending on the availability of nucleotide dATP/ATP. A tumor suppressor protein, PHAPI, enhances caspase-9 activation by promoting apoptosome formation through an unknown mechanism. We report here the identification of cellular apoptosis susceptibility protein (CAS) and heat shock protein 70 (Hsp70) as mediators of PHAPI activity. PHAPI, CAS, and Hsp70 function together to accelerate nucleotide exchange on Apaf-1 and prevent inactive Apaf-1/cytochrome c aggregation. CAS expression is induced by multiple apoptotic stimuli including UV irradiation. Knockdown of CAS by RNA interference (RNAi) in cells attenuates apoptosis induced by UV light and causes endogenous Apaf-1 to form aggregates. These studies indicated that PHAPI, CAS, and Hsp70 play an important regulatory role during apoptosis.     

Mitochondria from TRAIL-resistant prostate cancer cells are capable of responding to apoptotic stimuli

TNFalpha-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in prostate cancer cells. However, some prostate cancer cells, such as LNCaP are resistant to TRAIL. In addition to the involvement of several pathways in the TRAIL-resistance of LNCaP, it has been shown that mitochondrial response to TRIAL is low in these cells. Therefore, in this study, using in vitro cell free and reconstitution models, we have demonstrated that mitochondria from these cells are capable of responding to apoptotic stimuli. Furthermore, experiments to determine the influence of cytochrome c on apoptotic response noted that incubation of cytosol with exogenous cytochrome c induced truncation of Bid. We have demonstrated that truncation of Bid by exogenous cytochrome c is mediated through the activation of caspases-9 and -3. Incubation of cytosol with recombinant caspases-9 and -3 in the absence or presence of inhibitors showed that activation of caspase-9, leading to the activation of caspase-3 was necessary for the truncation of Bid. Published results indicate that in apoptotic cells cytochrome c is released from the mitochondria in two installments, an early small amount and a late larger amount. Our results suggest that the initial release of cytochrome generates tBid that is capable of translocation into the mitochondria causing further release of cytochrome c. Thus, in addition to providing functional explanation for the biphasic release of cytochrome c from mitochondria, we demonstrate the presence of a feedback amplification of mitochondrial apoptotic signal.