Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways

Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.     

Bcl-2-regulated apoptosis and cytochrome c release can occur independently of both caspase-2 and caspase-9

Apoptosis in response to developmental cues and stress stimuli is mediated by caspases that are regulated by the Bcl-2 protein family. Although caspases 2 and 9 have each been proposed as the apical caspase in that pathway, neither is indispensable for the apoptosis of leukocytes or fibroblasts. To investigate whether these caspases share a redundant role in apoptosis initiation, we generated caspase-2(-/-)9(-/-) mice. Their overt phenotype, embryonic brain malformation and perinatal lethality mirrored that of caspase-9(-/-) mice but were not exacerbated. Analysis of adult mice reconstituted with caspase-2(-/-)9(-/-) hematopoietic cells revealed that the absence of both caspases did not influence hematopoietic development. Furthermore, lymphocytes and fibroblasts lacking both remained sensitive to diverse apoptotic stimuli. Dying caspase-2(-/-)9(-/-) lymphocytes displayed multiple hallmarks of caspase-dependent apoptosis, including the release of cytochrome c from mitochondria, and their demise was antagonized by several caspase inhibitors. These findings suggest that caspases other than caspases 2 and 9 can promote cytochrome c release and initiate Bcl-2-regulated apoptosis.     

Targeting neonatal ischemic brain injury with a pentapeptide-based irreversible caspase inhibitor

Brain protection of the newborn remains a challenging priority and represents a totally unmet medical need. Pharmacological inhibition of caspases appears as a promising strategy for neuroprotection. In a translational perspective, we have developed a pentapeptide-based group II caspase inhibitor, TRP601/ORPHA133563, which reaches the brain, and inhibits caspases activation, mitochondrial release of cytochrome c, and apoptosis in vivo. Single administration of TRP601 protects newborn rodent brain against excitotoxicity, hypoxia–ischemia, and perinatal arterial stroke with a 6-h therapeutic time window, and has no adverse effects on physiological parameters. Safety pharmacology investigations, and toxicology studies in rodent and canine neonates, suggest that TRP601 is a lead compound for further drug development to treat ischemic brain damage in human newborns.     

Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation

Dysregulation of apoptosis contributes to the pathogenesis of many human diseases. As effectors of the apoptotic machinery, caspases are considered potential therapeutic targets. Using an established in vivo model of Fas-mediated apoptosis, we demonstrate here that elimination of certain caspases was compensated in vivo by the activation of other caspases. Hepatocyte apoptosis and mouse death induced by the Fas agonistic antibody Jo2 required proapoptotic Bcl-2 family member Bid and used a Bid-mediated mitochondrial pathway of caspase activation; deficiency in caspases essential for this pathway, caspase-9 or caspase-3, unexpectedly resulted in rapid activation of alternate caspases after injection of Jo2, and therefore failed to protect mice against Jo2 toxicity. Moreover, both ultraviolet and gamma irradiation, two established inducers of the mitochondrial caspase-activation pathway, also elicited compensatory activation of caspases in cultured caspase-3(-/-) hepatocytes, indicating that the compensatory caspase activation was mediated through the mitochondria. Our findings provide direct experimental evidence for compensatory pathways of caspase activation. This issue should therefore be considered in developing caspase inhibitors for therapeutic applications.     

IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases.

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.     

Delineation of the caspase-9 signaling cascade

In the intrinsic apoptosis pathway, mitochondrial disruption leads to the release of multiple apoptosis signaling molecules, triggering both caspase-dependent and -independent cell death. The release of cytochrome c induces the formation of the apoptosome, resulting in caspase-9 activation. Multiple caspases are activated downstream of caspase-9, however, the precise order of caspase activation downstream of caspase-9 in intact cells has not been completely resolved. To characterize the caspase-9 signaling cascade in intact cells, we employed chemically induced dimerization to activate caspase-9 specifically. Dimerization of caspase-9 led to rapid activation of effector caspases, including caspases-3, -6 and -7, as well as initiator caspases, including caspases-2, -8 and -10, in H9 and Jurkat cells. Knockdown of caspase-3 suppressed caspase-9-induced processing of the other caspases downstream of caspase-9. Silencing of caspase-6 partially inhibited caspase-9-mediated processing of caspases-2, -3 and -10, while silencing of caspase-7 partially inhibited caspase-9-induced processing of caspase-2, -3, -6 and -10. In contrast, deficiency in caspase-2, -8 or -10 did not significantly affect the caspase-9-induced caspase cascade. Our data provide novel insights into the ordering of a caspase signaling network downstream of caspase-9 in intact cells during apoptosis.     

Caspase-dependent apoptotic pathways in CNS injury

Recent studies have suggested a role for neuronal apoptosis in cell loss following acute CNS injury as well as in chronic neurodegeneration. Caspases are a family of cysteine requiring aspartate proteases with sequence similarity to Ced-3 protein of Caenorhabditis elegans. These proteases have been found to contribute significantly to the morphological and biochemical manifestations of apoptotic cell death. Caspases are translated as inactive zymogens and become active after specific cleavage. Of the 14 identified caspases, caspase-3 appears to be the major effector of neuronal apoptosis induced by a variety of stimuli. A role for caspase-3 in injury-induced neuronal cell death has been established using semispecific peptide caspase inhibitors. This article reviews the current literature relating to pathways regulating caspase activation in apoptosis associated with acute and chronic neurodegeneration, and suggests that identification of critical upstream caspase regulatory mechanisms may permit more effective treatment of such disorders.     

Antimycin A-induced killing of HL-60 cells: apoptosis initiated from within mitochondria does not necessarily proceed via caspase 9.

BACKGROUND: Antimycin A (AMA) inhibits mitochondrial electron transport, collapses the mitochondrial membrane potential, and causes the production of reactive oxygen species. Previous work by me and my colleagues has demonstrated that AMA causes an array of typical apoptotic phenomena in HL-60 cells. The hypothesis that AMA causes HL-60 apoptosis by the intrinsic apoptotic pathway has now been tested. METHODS: Z-LEHD-FMK and Z-IETD-FMK were used as specific inhibitors of the initiator caspases 9 and 8, respectively. Caspase 3 activation, DNA fragmentation, and cellular disintegration were measured by flow cytometry. Cytochrome c release, chromatin condensation, and nuclear fragmentation were measured by microscopy. RESULTS: AMA caused mitochondrial cytochrome c release and neither Z-LEHD-FMK nor Z-IETD-FMK inhibited that. In the absence of caspase inhibition there was a very close correlation between cytochrome c release and caspase 3 activation. Z-LEHD-FMK blocked caspase 3 activation but enhanced DNA fragmentation and failed to stop nuclear or cellular disintegration. Z-IETD-FMK also blocked caspase 3 activation but, in contrast to Z-LEHD-FMK, delayed DNA fragmentation and disintegration of the nucleus and the cell. CONCLUSIONS: The hypothesis to explain AMA-induced HL-60 apoptosis was clearly inadequate because: (a) caspase 9 inhibition did not prevent DNA fragmentation or cell death, (b) apoptosis proceeded in the absence of caspase-3 activation, (c) the main pathway leading to activation of the executioner caspases was by caspase-8 activation, but caspase 8 inhibition only delayed apoptosis, and (d) activation of caspases 8 and 9 may be necessary for caspase-3 activation. Thus, in this cell model, apoptosis triggered from within the mitochondria does not necessarily proceed by caspase 9, and caspase 3 is not critical to apoptosis. The results provide further evidence that, when parts of the apoptotic network are blocked, a cell is able to complete the program of cell death by alternate pathways.     

The proteolytic procaspase activation network: an in vitro analysis

In general, apoptotic stimuli lead to activation of caspases. Once activated, a caspase can induce intracellular signaling pathways involving proteolytic activation of other caspase family members. We report the in vitro processing of eight murine procaspases by their enzymatically active counterparts. Caspase-8 processed all procaspases examined. Caspase-1 and -11 processed the effector caspases procaspase-3 and -7, and to a lesser extent procaspase-6. However, vice versa, none of the caspase-1-like procaspases was activated by the effector caspases. This suggests that the caspase-1 subfamily members either act upstream of the apoptosis effector caspases or else are part of a totally separate activation pathway. Procaspase-2 was maturated by caspase-8 and -3, and to a lesser extent by caspase-7, while the active caspase-2 did not process any of the procaspases examined, except its own precursor. Hence, caspase-2 might not be able to initiate a wide proteolytic signaling cascade. Additionally, cleavage data reveal not only proteolytic amplification between caspase-3 and -8, caspase-6 and -3, and caspase-6 and -7, but also positive feedback loops involving multiple activated caspases. Our results suggest the existence of a hierarchic proteolytic procaspase activation network, which would lead to a dramatic increase in multiple caspase activities once key caspases are activated. The proteolytic procaspase activation network might allow that different apoptotic stimuli result in specific cleavage of substrates responsible for typical processes at the cell membrane, the cytosol, the organelles, and the nucleus, which characterize a cell dying by apoptosis.     

An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus

Respiratory syncytial virus (RSV) infection induced programmed cell death or apoptosis in the cultured lung epithelial cell line, A549. The apoptotic cells underwent multiple changes, including fragmentation and degradation of genomic DNA, consistent with the activation of the DNA fragmentation factor or caspase-activated DNase (DFF or CAD). The infection led to activation of FasL; however, a transdominant mutant of FAS-downstream death domain protein, FADD, did not inhibit apoptosis. Similarly, modest activation of cytoplasmic apoptotic caspases, caspase-3 and -8, were observed; however, only a specific inhibitor of caspases-3 inhibited apoptosis, while an inhibitor of caspase-8 had little effect. No activation of caspase-9 and -10, indicators of the mitochondrial apoptotic pathway, was observed. In contrast, RSV infection strongly activated caspase-12, an endoplasmic reticulum (ER) stress response caspase. Activation of the ER stress response was further evidenced by upregulation of ER chaperones BiP and calnexin. Antisense-mediated inhibition of caspase-12 inhibited apoptosis. Inhibitors of NF-kappa B had no effect on apoptosis. Thus, RSV-induced apoptosis appears to occur through an ER stress response that activates caspase-12, and is uncoupled from NF-kappa B activation.     

FLIP inhibits endothelial cell apoptosis during hyperoxia by suppressing Bax

High oxygen tension (hyperoxia) causes pulmonary cell death, involving apoptosis, necrosis, or mixed death phenotypes, though the underlying mechanisms remain unclear. In mouse lung endothelial cells (MLEC) hyperoxia activates both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) apoptotic pathways. We examined the hypothesis that FLIP, an inhibitor of caspase-8, can protect endothelial cells against the lethal effects of hyperoxia. Hyperoxia caused the time-dependent downregulation of FLIP in MLEC. Overexpression of FLIP attenuated intracellular reactive oxygen species generation during hyperoxia exposure, by downregulating extracellular-regulated kinase-1/2 activation and p47(phox) expression. FLIP prevented hyperoxia-induced trafficking of the death-inducing signal complex from the Golgi apparatus to the plasma membrane. Furthermore, FLIP blocked the activations of caspase-8/Bid, caspases -3/-9, and inhibited the mitochondrial translocation and activation of Bax, resulting in protection against hyperoxia-induced cell death. Under normoxic conditions, FLIP expression increased the phosphorylation of p38 mitogen-activated protein kinase leading to increased phosphorylation of Bax during hyperoxic stress. Furthermore, FLIP expression markedly inhibited protein kinase C activation and expression of distinct protein kinase C isoforms (alpha, eta, and zeta), and stabilized an interaction of PKC with Bax. In conclusion, FLIP exerted novel inhibitory effects on extrinsic and intrinsic apoptotic pathways, which significantly protected endothelial cells from the lethal effects of hyperoxia.     

Caspase inhibition and N6-benzyladenosine-induced apoptosis in HL-60 cells.

As an extension of our recently published work (Mlejnek and Kuglík [2000] J. Cell. Biochem. 77:6-17), the role of caspases in N(6)-benzylaminopurine riboside (BAPR)-induced apotosis in HL-60 cells was evaluated in this study. Here, BAPR-induced apoptosis was accompanied by activation of caspase-3 and caspase-9. However, when these caspases were selectively inhibited, the progression of BAPR-induced apoptosis was not markedly affected. Besides that, activation of caspase-3 and caspase-9 was found to be rather late event in apoptotic process. These results suggested that other caspases might be critically implicated. Indeed, pan-specific caspase inhibitor, Z-VAD-FMK, completely prevented DNA cleavage and apoptotic bodies formation. However, Z-VAD-FMK failed to prevent cell death and it was incapable to fully counteract the main apoptotic hallmark-chromatin condensation. Finally, our data indicate that cellular decision between apoptosis and necrosis is made upon the availability of both caspase proteases and intracellular ATP.     

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

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

In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock-induced apoptosis

Activation of 'initiator' (or 'apical') caspases-2, -8 or -9 (refs 1-3) is crucial for induction of apoptosis. These caspases function to activate executioner caspapses that, in turn, orchestrate apoptotic cell death. Here, we show that a cell-permeable, biotinylated pan-caspase inhibitor (bVAD-fmk) both inhibited and 'trapped' the apical caspase activated when apoptosis was triggered. As expected, only caspase-8 was trapped in response to ligation of death receptors, whereas only caspase-9 was trapped in response to a variety of other apoptosis-inducing agents. Caspase-2 was exclusively activated in heat shock-induced apoptosis. This activation of caspase-2 was also observed in cells protected from heat-shock-induced apoptosis by Bcl-2 or Bcl-xL. Reduced sensitivity to heat-shock-induced death was observed in caspase-2(-/-) cells. Furthermore, cells lacking the adapter molecule RAIDD failed to activate caspase-2 after heat shock treatment and showed resistance to apoptosis in this setting. This approach unambiguously identifies the apical caspase activated in response to apoptotic stimuli, and establishes caspase-2 as a proximal mediator of heat shock-induced apoptosis.     

Caspase-1 promiscuity is counterbalanced by rapid inactivation of processed enzyme

Members of the caspase family of cysteine proteases coordinate the highly disparate processes of apoptosis and inflammation. However, although hundreds of substrates for the apoptosis effector caspases (caspase-3 and caspase-7) have been identified, only two confirmed substrates for the key inflammatory protease (caspase-1) are known. Whether this reflects intrinsic differences in the substrate specificity of inflammatory versus apoptotic caspases or their relative abundance in vivo is unknown. To address this issue, we have compared the specificity of caspases-1, -3, and -7 toward peptide and protein substrates. Contrary to expectation, caspase-1 displayed concentration-dependent promiscuity toward a variety of substrates, suggesting that caspase-1 specificity is maintained by restricting its abundance. Although endogenous concentrations of caspase-1 were found to be similar to caspase-3, processed caspase-1 was found to be much more labile, with a half-life of ~9 min. This contrasted sharply with the active forms of caspase-3 and caspase-7, which exhibited half-lives of 8 and 11 h, respectively. We propose that the high degree of substrate specificity displayed by caspase-1 is maintained through rapid spontaneous inactivation of this protease.     

Differential expression of rat brain caspase family proteins during development and aging.

It is well recognized that caspases are essential effector molecules for carrying out apoptosis in eukaryotic cells. The expression of rat brain caspase family proteins (caspase-2, -3, -6, -7, -8, -9, 10) in development and aging was assessed using immunochemical detection. All of these caspases were expressed in the rat brain. Immunoblot analysis of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that cytosolic caspase-3, -7, -8, and -10 were highly expressed at E19, and decreased after birth. In contrast, cytosolic caspase-2, -6, and -9 were constitutively expressed from the early stages to 96 weeks of age. These results show that the expression of rat brain caspase family proteins is differentially regulated during the development and aging.     

Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death.

Caspase activation and apoptotic volume decrease are fundamental features of programmed cell death; however, the relationship between these components is not well understood. Here we provide biochemical and genetic evidence for the differential involvement of initiator caspases in the apoptotic volume decrease during both intrinsic and extrinsic activation of apoptosis. Apoptosis induction in Jurkat T lymphocytes by Fas receptor engagement (intrinsic) or ultraviolet (UV)-C radiation (extrinsic) triggered the loss of cell volume, which was restricted to cells with diminished intracellular K(+) ions. These characteristics kinetically coincided with the proteolytic processing and activation of both initiator and effector caspases. Although the polycaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone completely inhibited the Fas-mediated apoptotic volume decrease and K(+) efflux, it was much less effective in preventing these processes during UV-induced cell death under conditions whereby caspase activities and DNA degradation were blocked. To define the roles of specific initiator caspases, we utilized Jurkat cells genetically deficient in caspase-8 or stably transfected with a dominant-negative mutant of caspase-9. The results show that the activation of caspase-8, but not caspase-9, is necessary for Fas-induced apoptosis. Conversely, caspase-9, but not caspase-8, is important for UV-mediated shrunken morphology and apoptosis progression. Together, these findings indicate that cell shrinkage and K(+) efflux during apoptosis are tightly coupled, but are differentially regulated by either caspase-8 or caspase-9 depending on specific pathways of cell death.     

Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes

Caspases are cysteine proteases that are key effectors in apoptotic cell death. Currently, there is a lack of tools that can be used to monitor the regulation of specific caspases in the context of distinct apoptotic programs. We describe the development of highly selective inhibitors and active site probes and their applications to directly monitor executioner (caspase-3 and -7) and initiator (caspase-8 and -9) caspase activity. Specifically, these reagents were used to dissect the kinetics of caspase activation upon stimulation of apoptosis in cell-free extracts and intact cells. These studies identified a full-length caspase-7 intermediate that becomes catalytically activated early in the pathway and whose further processing is mediated by mature executioner caspases rather than initiator caspases. This form also shows distinct inhibitor sensitivity compared to processed caspase-7. Our data suggest that caspase-7 activation proceeds through a previously uncharacterized intermediate that is formed without cleavage of the intact zymogen.     

Endoplasmic reticulum stress-induced death of mouse embryonic fibroblasts requires the intrinsic pathway of apoptosis

Members of the caspase family are essential for many apoptotic programs. We studied mouse embryonic fibroblasts (MEFs) deficient in caspases 3 and 7 and in caspase 9 to determine the role of these proteases in endoplasmic reticulum (ER) stress-induced apoptosis. Both caspase 3(-/-)/caspase 7(-/-) and caspase 9(-/-) MEFs were resistant to cytotoxicity induced via ER stress and failed to exhibit apoptotic morphology. Specifically, apoptosis induced by increased intracellular calcium was shown to depend only on caspases 3 and 9, whereas apoptosis induced by disruption of ER function depended additionally on caspase 7. Caspase 3(-/-)/caspase 7(-/-) and caspase 9(-/-) MEFs also exhibited decreased loss of mitochondrial membrane potential, which correlated with altered caspase 9 processing, increased induction of procaspase 11, and decreased processing of caspase 12 in caspase 3(-/-)/caspase 7(-/-) cells. Furthermore, disruption of ER function was sufficient to induce accumulation of cleaved caspase 3 and 7 in a heavy membrane compartment, suggesting a potential mechanism for caspase 12 processing and its role as an amplifier in the death pathway. Caspase 8(-/-) MEFs were not resistant to ER stress-induced cytotoxicity, and processing of caspase 8 was not observed upon induction of ER stress. This study thus demonstrates a requirement for caspases 3 and 9 and a key role for the intrinsic pathway in ER stress-induced apoptosis.     

Trichosanthin induced apoptosis in HL-60 cells via mitochondrial and endoplasmic reticulum stress signaling pathways

Trichosanthin (TCS), a traditional Chinese medicine, exerts antitumor activities by inducing apoptosis in many different tumor cell lines. However, the mechanisms remain obscure. The present study focused on various caspase pathways that may be involved in TCS-induced apoptosis in leukemia HL-60 cells. Key caspases in both intrinsic and extrinsic pathways including caspase-8, -9 and -3 were activated upon TCS treatment. Additionally, TCS treatment induced upregulation of BiP and CHOP and also activated caspase-4, which for the first time strongly supported the involvement of endoplasmic reticulum stress pathway in TCS-induced apoptosis. Interestingly, although caspase-8 was activated, Fas/Fas ligand pathway was not involved as evidenced by a lack of induction of Fas or Fas ligand and a lack of inhibitory effect of anti-Fas blocking antibody on TCS-induced apoptosis. Instead, caspase-8 was activated in a caspase-9 and -4 dependent manner. The involvement of mitochondria was demonstrated by the reduction of mitochondrial membrane potential and release of cytochrome c and Smac besides the activation of caspase-9. Further investigation confirmed that caspase-3 was the major executioner caspase downstream to caspase-9, -4 and -8. Taken together, our results suggested that TCS-induced apoptosis in HL-60 cells was mainly mediated by mitochondrial and ER stress signaling pathways via caspase-3.