Gastric irritant-induced apoptosis in guinea pig gastric mucosal cells in primary culture

When the gastric mucosa is exposed to various irritants, apoptosis and subsequent gastric mucosal lesion can result in vivo. We here show that gastric irritants induced apoptosis in gastric mucosal cells in primary culture and examined its molecular mechanism. Ethanol, hydrogen peroxide, and hydrochloric acid all induced, in a dose-dependent manner, cell death, apoptotic DNA fragmentation, and chromatin condensation, suggesting that each of these gastric irritants induced apoptosis in vitro. Since each of these irritants decreased the mitochondrial membrane potential and stimulated the release of cytochrome c from mitochondria, gastric irritant-induced apoptosis seems to be mediated by mitochondrial dysfunction. Caspase-3, caspase-8, and caspase-9-like activities were all activated simultaneously by each of these irritants and the activation was concomitantly with cell death and apoptotic DNA fragmentation. Furthermore, pre-treatment of gastric mucosal cells with an inhibitor of caspase-8 suppressed the onset of cell death as well as the stimulation of caspase-3- and caspase-9-like activities caused by each of these gastric irritants. Based on these results, we consider that caspase-8, an initiator caspase, plays an important role in gastric irritant-induced apoptosis.     

Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death

Glutamate receptor overactivation contributes to neuron death after stroke, trauma, and epileptic seizures. Exposure of cultured rat hippocampal neurons to the selective glutamate receptor agonist N-methyl-d-aspartate (300 microm, 5 min) or to the apoptosis-inducing protein kinase inhibitor staurosporine (300 nm) induced a delayed neuron death. In both cases, neuron death was preceded by the mitochondrial release of the pro-apoptotic factor cytochrome c. Unlike staurosporine, the N-methyl-d-aspartate-induced release of cytochrome c did not lead to significant activation of caspase-3, the main caspase involved in the execution of neuronal apoptosis. In contrast, activation of the Ca(2+)-activated neutral protease calpain I was readily detectable after the exposure to N-methyl-d-aspartate. In a neuronal cell-free apoptosis system, calpain I prevented the ability of cytochrome c to activate the caspase cascade by inhibiting the processing of procaspase-3 and -9 into their active subunits. In the hippocampal neuron cultures, the inhibition of calpain activity restored caspase-3-like protease activity after an exposure to N-methyl-d-aspartate. Our data demonstrate the existence of signal transduction pathways that prevent the entry of cells into a caspase-dependent cell death program after the mitochondrial release of cytochrome c.     

Synergistic induction of apoptosis in murine hepatoma Hepa1-6 cells by IFN-gamma and TNF-alpha

In this study, we examined the susceptibility of murine hepatoma Hepa1-6 cells to undergo IFN-gamma- and/or TNF-alpha-induced apoptosis. IFN-gamma or TNF-alpha alone had no demonstrable cytotoxic effects, whereas IFN-gamma and TNF-alpha in combination induced apoptosis drastically in Hepa1-6 cells. During this apoptosis, an increase in caspase-3- and -8-like protease activities and activation of caspase-3, identified by the appearance of its p17 fragment, were observed. Moreover, the cytotoxic induction and caspase-3 activation were effectively inhibited by Z-Asp-CH(2)-DCB (Z-Asp), a caspase inhibitor. Further, an elevation of cytochrome c in the cytosol, in a parallel to activation of caspase-3, was observed in a time-dependent manner. Concurrently, up-regulation of caspase-11 gene expression and processing of procaspase-11 were detected during this apoptosis. These results suggest that the caspase-3 activation, the release of cytochrome c from mitochondria, and increased caspase-11 gene expression involve in synergistic induction of apoptosis in Hepa1-6 cells by IFN-gamma and TNF-alpha.     

Involvement of protein kinase C-regulated ceramide generation in inostamycin-induced apoptosis

Activation of caspases is commonly involved in the apoptosis induced by various anticancer drugs. However, the upstream events leading to the activation of caspases seem to be specific to each anticancer drug. In the present study, we examined the possible involvement of protein kinase C (PKC) and ceramide generation in caspase-3(-like) protease activation induced by inostamycin, a phosphatidylinositol synthesis inhibitor. Treatment of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, suppressed the release of cytochrome c from mitochondria and the activation of caspase-3(-like) proteases in inostamycin-treated cells, but not in other anticancer drug-treated cells. Inostamycin induced the elevation of intracellular ceramide levels, and fumonisin B1, an inhibitor of ceramide synthase, inhibited inostamycin-induced cytochrome c release, caspase-3(-like) protease activation, and apoptosis. Moreover, TPA also inhibited inostamycin-induced ceramide synthesis. Taken together, our results suggest that inostamycin-induced apoptosis is mediated by PKC-regulated ceramide generation, leading to the activation of a caspase cascade.     

Molecular mechanism of indomethacin-induced gastropathy

The probable cross talk among large numbers of inflammatory and angiogenic parameters in indomethacin (IND)-induced gastropathy and the associated signaling mechanism were studied in a mouse model. A single dose of IND (18 mg/kg, po) produced robust gastric ulceration in mice without any mortality, which peaked on the third day, but started healing from the fifth day onward. The ulceration was associated with increased myeloperoxidase activity and expression of proinflammatory (TNF-α, adhesion molecules, COX-2) and antiangiogenic (endostatin) parameters. The levels of proangiogenic factors such as COX-1, prostaglandin E, VEGF, and von Willebrand factor VIII were downregulated by IND. Our results revealed that although the maximal and minimal levels of these parameters were attained sequentially at different time points, TNF-α upregulation was the primary event to initiate and induce gastric ulceration. IND also activated NF-κB and all the MAP kinases, but only the inhibitors of TNF-α, NF-κB, and JNK MAP kinase could abrogate the IND-induced damages. Further TNF-α inhibition also reduced the IND-mediated activation of NF-κB and JNK MAP kinase. All this evidence strongly suggests that mitigation of TNF-α may offer a potential solution to IND-mediated gastropathy.     

Mouse mammary gland involution is associated with cytochrome c release and caspase activation

At weaning, milk producing mammary epithelial cells undergo apoptosis and are removed by phagocytosis. Here, we show that mouse mammary gland involution is associated with mitochondrial cytochrome c release and processing of numerous caspases, including caspase-1, -3, -7, -8 and -9. Induction of caspase-3-like activity paralleled cleavage of poly-(ADP--ribose) polymerase. Dexamethasone inhibited processing of caspase-3, -7 and -8 and apoptosis, but had no effect on caspase-1 accumulation and cytochrome c release. In Bcl-2 transgenic animals, cytochrome c release, caspase activation and apoptosis were impaired. Thus, the pro-apoptotic signaling pathway in mammary epithelial cells during involution involves the release of cytochrome c and activation of caspases. It is inhibited by Bcl-2 at the mitochondrial level and by dexamethasone at a post-mitochondrial level.     

Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system.

In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redox-inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors.     

Release of mitochondrial cytochrome c and activation of cytosolic caspases induced by myocardial ischaemia

It has previously been shown that apoptosis is increased in ischaemic/reperfused heart. However, little is known about the mechanism of induction of apoptosis in myocardium during ischaemia. We investigated whether prolonged myocardial ischaemia causes activation of caspases and whether this activation is related to cytochrome c release from mitochondria to cytosol during ischaemia. Using an in vitro model of heart ischaemia, we show that 60 min ischaemia leads to a significant accumulation of cytochrome c in the cytosol and a decrease in mitochondrial content of cytochrome c but not cytochrome a. The release of cytochrome c from mitochondria was accompanied by activation of caspase-3-like proteases (measured by cleavage of fluorogenic peptide substrate DEVD-amc) and a large increase in number of cells with DNA strand breaks (measured by TUNEL staining). Caspase-1-like proteases (measured by YVAD-amc cleavage) were not activated during ischaemia. Addition of 14 microM cytochrome c to cytosolic extracts prepared from control hearts induced ATP-dependent activation of caspase-3-like protease activity. Our data suggest that extended heart ischaemia can cause apoptosis mediated by release of cytochrome c from mitochondria and subsequent activation of caspase-3.     

Cytochrome c is dispensable for fas-induced caspase activation and apoptosis.

Cytochrome c is thought to play an important role in the initiation of apoptosis following its release from mitochondria. It is controversial whether such release is also involved in caspase activation and apoptotic cell death after ligation of the cell surface molecule Fas. We addressed this issue by investigating cells from the human cell lines Jurkat and SKW6 which had been treated with the inhibitor of the mitochondrial F0/F1-ATPase, oligomycin. Oligomycin-treatment led, over a wide range of concentrations, to ATP-depletion and, at similar concentrations, abrogated the appearance of caspase-3-like activity caused by stauroporine. Electroporation of cytochrome c protein into intact cells induced caspase activation in both cell lines and significant nuclear apoptosis in Jurkat cells. In ATP-depleted cells, electroporation of cytochrome c induced neither caspase activation nor nuclear fragmentation. Fas-induced caspase activation and nuclear apoptosis, however, were unaffected by the depletion of ATP. Thus, cytochrome c is unlikely to be an important factor in Fas-induced cell death.     

The temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochrome c. Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochrome c in apoptosis induced by the Fas receptor. We demonstrate that cytochrome c is released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochrome c. The protein phosphatase inhibitor calyculin A prevented cytochrome c release and apoptosis induced by both agents, suggesting that release of cytochrome c is required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochrome c. The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochrome c and apoptosis induced by both insults.     

Curcumin induces Apaf-1-dependent,p21-mediated caspase activation and apoptosis

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase-3 activation, which is required for apoptosis as confirmed using the pan caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role of caspase-8 in curcumin-induced caspase-3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase-3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of proapoptotic proteins such as Bax, Bak, Bid, and Bim. Crosslinking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid, and Bim causes Bax-channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation, and apoptosis. Importantly, p21-deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement of p21 in Apaf-1 dependent caspase activation and apoptosis. Together, our findings demonstrate that Apaf-1, Bax, and p21 as novel potential targets for curcumin or curcumin-based anticancer agents.     

Inhibition of nitric-oxide-mediated apoptosis in Jurkat leukemia cells despite cytochrome c release

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.     

Activation of caspases and cleavage of Bid are required for tyrosine and phenylalanine deficiency-induced apoptosis of human A375 melanoma cells

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.     

Caspase-6 is the direct activator of caspase-8 in the cytochrome c-induced apoptosis pathway: absolute requirement for removal of caspase-6 prodomain

Caspase activation resulting from cytochrome c release from the mitochondria is an essential component of the mechanism of apoptosis initiated by a range of factors. The activation of Bid by caspase-8 in this pathway promotes further cytochrome c release, thereby completing a positive feedback loop of caspase activation. Although the identity of the caspases necessary for caspase-8 activation in this pathway are known, it is still unclear which protease directly cleaves caspase-8. In order to identify the factor responsible we undertook a biochemical purification of caspase-8 cleaving activity in cytosolic extracts to which cytochrome c had been added. Here we report that caspase-6 is the only soluble protease in cytochrome c activated Jurkat cell extracts that has significant caspase-8 cleaving activity. Furthermore the caspase-6 that we purified was sufficient to induce Bid dependent cytochrome c releasing activity in cell extracts. Inhibition of caspase-6 activity in cells significantly inhibited caspase-8 cleavage and apoptosis, therefore establishing caspase-6 as a major activator of caspase-8 in vivo and confirming that this pathway can have a critical role in promotion of apoptosis. We also show that caspase-6 is inactive until the short prodomain is removed. We suggest that the requirement for two distinct cleavage steps to activate an effector caspase may represent an effective mechanism for restriction of spontaneous caspase activation and aberrant entry into apoptosis.     

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.     

Lansoprazole protects and heals gastric mucosa from non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy by inhibiting mitochondrial as well as Fas-mediated death pathways with concurrent induction of mucosal cell renewal

We have investigated the mechanism of antiapoptotic and cell renewal effects of lansoprazole, a proton pump inhibitor, to protect and heal gastric mucosal injury in vivo induced by indomethacin, a non-steroidal anti-inflammatory drug (NSAID). Lansoprazole prevents indomethacin-induced gastric damage by blocking activation of mitochondrial and Fas pathways of apoptosis. Lansoprazole prevents indomethacin-induced up-regulation of proapoptotic Bax and Bak and down-regulation of antiapoptotic Bcl-2 and Bcl(xL) to maintain the normal proapoptotic/antiapoptotic ratio and thereby arrests indomethacin-induced mitochondrial translocation of Bax and collapse of mitochondrial membrane potential followed by cytochrome c release and caspase-9 activation. Lansoprazole also inhibits indomethacin-induced Fas-mediated mucosal cell death by down-regulating Fas or FasL expression and inhibiting caspase-8 activation. Lansoprazole favors mucosal cell renewal simultaneously by stimulating gene expression of prosurvival proliferating cell nuclear antigen, survivin, epidermal growth factor, and basic fibroblast growth factor. The up-regulation of Flt-1 further indicates that lansoprazole activates vascular epidermal growth factor-mediated controlled angiogenesis to repair gastric mucosa. Lansoprazole also stimulates the healing of already formed ulcers induced by indomethacin. Time course study of healing indicates that it switches off the mitochondrial death pathway completely but not the Fas pathway. However, lansoprazole heals mucosal lesions almost completely after overcoming the persisting Fas pathway, probably by favoring the prosurvival genes expression. This study thus provides the detailed mechanism of antiapoptotic and prosurvival effects of lansoprazole for offering gastroprotection against indomethacin-induced gastropathy.     

Involvement of Akt in mitochondria-dependent apoptosis induced by a cdc25 phosphatase inhibitor naphthoquinone analog

Vitamin K-related analogs induce growth inhibition via a cell cycle arrest through cdc25A phosphatase inhibition in various cancer cell lines. We report that 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDN), a naphthoquinone analog, induces mitochondria-dependent apoptosis in human promyelocytic leukemia HL-60 cells. DDN induced cytochrome c release, Bax translocation, cleavage of Bid and Bad, and activation of caspase-3, -8, -9 upon the induction of apoptosis. Cleavage of Bid, the caspase-8 substrate, was inhibited by the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), whereas cytochrome c release was not affected, suggesting that activation of caspase-8 and subsequent Bid cleavage occur downstream of cytochrome c release. DDN inhibited the activation of Akt detected by decreasing level of phosphorylation. Overexpression of constitutively active Akt protected cells from DDN-induced apoptosis, while dominant negative Akt moderately enhanced cell death. Furthermore, Akt prevented release of cytochrome c and cleavage of Bad in DDN-treated HL-60 cells. Taken together, DDN-induced apoptosis is associated with mitochondrial signaling which involves cytochrome c release via a mechanism inhibited by Akt.     

Activation of caspase-3 by lysosomal cysteine proteases and its role in 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells

We previously reported that in addition to mitochondrial cytochrome c dependent activation, lysosomal cysteine proteases were also involved in the activation of caspase-3. In this study, we have separately obtained the lysosomal and mitochondrial caspase-3 activating factors in a crude mitochondrial fraction and characterized their ability to activate pro-caspase-3 in the in vitro assay system. When a rat liver crude mitochondrial fraction containing lysosomes (ML) was treated with a low concentration of digitonin, lysosomal factors were selectively released without the release of a mitochondrial factor (cytochrome c, Cyt.c). Treatment of ML with Ca(2+) in the presence of inorganic phosphate (P(i)), in contrast, released mitochondrial Cyt.c without the release of lysosomal factors. The obtained lysosomal and mitochondrial factors activated caspase-3 in different manners; caspase-3 activation by lysosomal and mitochondrial factors was specifically suppressed by E-64, a cysteine protease inhibitor, and caspase-9 inhibitor, respectively. Thus, the activation of caspase-3 by lysosomal factors was found to be distinct from the activation by mitochondrial Cyt.c dependent formation of the Apaf-1/caspase-9 complex. To further determine whether or not the activation of caspase-3 by lysosomal cysteine proteases is involved in cellular apoptosis, the effect of E-64-d, a cell-permeable inhibitor of cysteine protease, on 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells was investigated. As a result, DNA fragmentation induced by AAPH was found to be remarkably (up to 50%) reduced by pretreatment with E-64-d, indicating the participation of lysosomal cysteine proteases in AAPH-induced apoptosis in HL-60 cells.     

Endoplasmic reticulum stress-induced cell death mediated by the proteasome

Cells exposed to sustained endoplasmic reticulum (ER) stress undergo programmed cell death and display features typical of apoptosis, such as cysteine aspartyl protease (caspase) activation, cytochrome c release, and DNA fragmentation. Here, we show that the execution of cell death induced by ER stress is mediated via the proteasome. Inhibition of the proteasome by lactacystin prevented ER stress-induced degradation of Bcl-2, release of cytochrome c, processing of effector caspase-3, and exposure of phosphatidylserine. Owing to the ability of lactacystin to inhibit cytochrome c release, we propose that the pro-apoptotic activity of the proteasome lies upstream of mitochondrial activation. Thus, the proteasome serves as a principal mediator of ER stress-induced cell death in this system.     

Possible involvement of cytochrome c release and sequential activation of caspases in ceramide-induced apoptosis in SK-N-MC cells

Ceramide is characterized as a second messenger of apoptosis induced by various agents such as tumor necrosis factor (TNF-alpha), Fas ligand, hydrogen peroxide, heat shock and ionizing radiation. In this study, we investigated the mechanism of ceramide-induced apoptosis using a human neuroblastoma cell line, SK-N-MC. N-Acetyl-sphingosine (C2-ceramide), a cell-permeable ceramide analogue, was able to induce apoptosis in SK-N-MC cells as estimated by DNA fragmentation and chromatin condensation. C2-ceramide-induced DNA fragmentation was blocked by caspase inhibitor (Z-Asp-CH(2)-DCB). An increase in caspase-3 (CPP32)-like protease activity was evident during C2-ceramide-induced apoptosis, suggesting that caspases are involved in this apoptosis. Moreover, enzymatic cleavage of VDVAD-AFC and LEHD-AFC (specific substrates for caspase-2 and -9, respectively) was increased by treatment with C2-ceramide. To elucidate which types of caspase are activated in C2-ceramide-treated cells, we performed Western blot analysis using antibodies against each isoform. Both proforms of caspase-2 and -3 were decreased in response to C2-ceramide in a time-dependent manner. Mitochondrial cytochrome c is also time-dependently released into the cytosol in response to treatment with C2-ceramide. Addition of cytochrome c into the S-100 fractions prepared from SK-N-MC cells could activate caspase-2 in cell-free systems. These results suggest the possibility that cytochrome c released to the cytosol can activate caspases (caspase-9, -3, and -2) during C2-ceramide-induced apoptosis of SK-N-MC cells.