Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rat: a role for gender

Hypoxia-ischaemia in the developing brain results in brain injury with prominent features of apoptosis. In the present study, a third generation dipeptidyl broad-spectrum caspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh), was tested in a model of unilateral focal ischaemia with reperfusion in 7-day-old rats. Q-VD-OPh (1 mg/kg, i.p.) reduced cell death, resulting in significant neuroprotection at 48 h of recovery (infarct volume of 12.6 +/- 2.8 vs. 24.3 +/- 2.2%, p = 0.006). The neuroprotective effects observed at 48 h post-ischaemia hold up at 21 days of survival time and attenuate neurological dysfunction. Analysis by gender revealed that females were strongly protected (6.7 +/- 3.3%, p = 0.006), in contrast to males in which there was no significant effect, when Q-VD-OPh was given after clip removal on the left common carotid artery. Immunoblot analysis demonstrated that Q-VD-OPh inhibits caspase 3 cleavage into its p17 active form and caspase 1 up-regulation and cleavage in vivo. Following ischaemia in P7 rats, males and females displayed different time course and pattern of cytochrome c release and active p17 caspase 3 during the first 24 h of recovery. In contrast, no significant difference was observed for caspase 1 expression between genders. These results indicate that ischaemia activates caspases shortly after reperfusion and that the sex of the animal may strongly influences apoptotic pathways in the pathogenesis of neonatal brain injury. The specificity, effectiveness, and reduced toxicity of Q-VD-OPh may determine the potential use of peptide-derived irreversible caspase inhibitors as promising therapeutics.     

The role of caspases in Alzheimer’s disease; potential novel therapeutic opportunities

Although apoptosis plays a critical role in molding the CNS into its final appearance and function, inappropriate activation of this pathway in the aging brain may contribute to neurodegeneration. In Alzheimer’s disease (AD), an overwhelming body of evidence supports the activation of apoptosis in general, and caspases specifically as an early event that may not only contribute to neurodegeneration but also promote the underlying pathology associated with this disease. Therefore, caspase inhibitors may provide an effective strategy for treating AD. However, despite the compelling evidence indicating a role for caspases in disease progression, chronic treatment with caspase inhibitors in animal models of AD has never been undertaken. In this review the role of caspases in AD will be addressed, including recent studies utilizing in vivo transgenic mouse models of tauopathies. In addition, a discussion of the therapeutic value and dangers of targeting caspase inhibition in the treatment of AD using caspase inhibitors such as Q-VD-OPh will be evaluated.     

Dose-dependent effects of the caspase inhibitor Q-VD-OPh on different apoptosis-related processes

The effects of the pan-caspase inhibitor Q-VD-OPh on caspase activity, DNA fragmentation, PARP cleavage, 7A6 exposition, and cellular adhesivity to fibronectin were analyzed in detail in three different apoptotic systems involving two cell lines (JURL-MK1 and HL60) and two apoptosis inducers (imatinib mesylate and suberoylanilide hydroxamic acid). Q-VD-OPh fully inhibited caspase-3 and -7 activity at 0.05 μM concentration as indicated both by the measurement of the rate of Ac-DEVD-AFC cleavage and anti-caspase immunoblots. Caspase-8 was also inhibited at low Q-VD-OPh concentrations. On the other hand, significantly higher Q-VD-OPh dose (10 μM) was required to fully prevent the cleavage of PARP-1. DNA fragmentation and disruption of the cell membrane functionality (Trypan blue exclusion test) were both prevented at 2 μM Q-VD-OPh while 10 μM inhibitor was needed to inhibit the drug-induced loss of cellular adhesivity to fibronectin which was observed in JURL-MK1 cells. The exposition of the mitochondrial antigen 7A6 occurred independently of Q-VD-OPh addition and may serve to the detection of cumulative incidence of the cells which have initiated the apoptosis. Our results show that Q-VD-OPh efficiency in the inhibition of caspase-3 activity and DNA fragmentation in the whole-cell environment is about two orders of magnitude higher than that of z-VAD-fmk. This difference is not due to a slow permeability of the latter through the cytoplasmic membrane.     

Motexafin gadolinium induces mitochondriallymediated caspase-dependent apoptosis

Motexafin gadolinium (MGd, Xcytrin^®) is a tumor-localizing redox mediator that catalyzes the oxidation of intracellular reducing molecules including NADPH, ascorbate, protein and non-protein thiols, generating reactive oxygen species (ROS). MGd localizes to tumors and cooperates with radiation and chemotherapy to kill tumor cells in tissue culture and animal models. In this report, we demonstrate that MGd triggers the mitochondrial apoptotic pathway in the HF-1 lymphoma cell line as determined by loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspase-9 prior to caspase-8, cleavage of PARP and annexin V binding. There was minimal effect on MGd-induced apoptosis by the caspase inhibitor z-VAD-fmk, even though caspase-3 activity (as measured by DEVD-cleavage) was completely inhibited. However, MGd-induced apoptosis was reduced to baseline levels by the more potent caspase inhibitor Q-VD-OPh, demonstrating that MGd-induced apoptosis is indeed caspase-dependent. Apoptosis induced by dexamethasone, doxorubicin and etoposide (mediated through the mitochondrial pathway) was also more sensitive to inhibition by Q-VD-OPh than z-VAD-fmk. Our results demonstrating differential sensitivity of drug-induced apoptosis to caspase inhibitors suggest that the term “caspase-independent apoptosis” cannot be solely defined as apoptosis that is not inhibited by z-VAD-fmk as has been utilized in some published studies.     

The effect of specific caspase inhibitors on TNF-alpha and butyrate-induced apoptosis of intestinal epithelial cells

Tumour necrosis factor-alpha (TNF-alpha)-induced intestinal epithelial cell apoptosis may contribute to mucosal injury in inflammatory bowel disease. Inhibition of TNF-alpha-induced apoptosis, using specific caspase inhibitors could, therefore, be of benefit in the treatment of disease. In vitro, CaCo-2 colonic epithelial cells are refractory to apoptosis induced by TNF-alpha alone; however, TNF-alpha can act synergistically with the short-chain fatty acid (SCFA) and colonic fermentation product, butyrate, to promote apoptosis. TNF-alpha/butyrate-induced apoptosis was characterised by nuclear condensation and fragmentation and caspase-3 activation. Inhibitors of caspase-8 (z-IETD.fmk) and caspase-10 (z-AEVD.fmk) significantly reduced TNF-alpha/butyrate-induced apoptosis, based on nuclear morphology and terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling (TUNEL), although caspase inhibition was associated with a significant increase in cells demonstrating atypical nuclear condensation. Inclusion of atypical cells in calculations of total cell death, still demonstrated that z-IETD.fmk and z-AEVD.fmk (in combination) significantly reduced cell death. Reduction in cell death was associated with maintenance of viable cell number. Transmembrane resistance was also used a measure of the ability of caspase inhibitors to prevent TNF-alpha/butyrate-mediated damage to epithelial monolayers. TNF-alpha/butyrate resulted in a significant fall in transmembrane resistance, which was prevented by pre-treatment with z-IETD.fmk, but not z-AEVD.fmk. In conclusion, synthetic caspase inhibitors can reduce the apoptotic response of CaCo-2 colonic epithelial cells to TNF-alpha/butyrate, improve the maintenance of viable cell numbers and block loss of transmembrane resistance. We hypothesise that caspase inhibition could be a useful therapeutic goal in the treatment of inflammatory bowel conditions, such as ulcerative colitis.     

Function of caspases in regulating apoptosis caused by erythropoietin deprivation in erythroid progenitors

Erythropoietin (EP) is required by late stage erythroid progenitor cells to prevent apoptosis. In a previous study (Gregoli and Bondurant, 1997, Blood 90:630-640), it was shown that rapid proteolytic conversion of procaspase 3 to the fully activated enzyme occurred when erythroblasts were deprived of EP for as little as 2 h. In the present study, protein and mRNA analyses of erythroblasts indicated the presence of the proenzyme precursors of caspases 1, 2, 3, 5, 6, 7, 8, and 9. The effects of various caspase inhibitors on caspase 3 processing and on apoptosis were examined. These inhibitors were benzyloxycarbonyl (z-) and fluoromethyl-ketone (FMK) derivatives of peptides that serve as substrates for selected caspases. z-VAD-FMK, t-butoxycarbonyl-aspartate-FMK (Boc-D-FMK), and z-IETD-FMK blocked the initial cleavage of procaspase 3, while z-DEVD-FMK, z-VEID-FMK, and z-VDVAD-FMK did not block the initial cleavage but had some effect on blocking apoptosis. The peptide inhibitor z-FA-FMK, which inhibits cathepsins B and L but is not known to inhibit caspases, altered caspase 3 processing to a final 19 kDa large subunit that appeared to retain enzymatic activity. The action of z-FA-FMK in preventing the usual conversion to a 1 7 kDa subunit suggests the possibility that a noncaspase protease may be involved in caspase 3 processing. Studies with the peptide inhibitors and EP were done to determine the short- and long-term effectiveness of the caspase inhibitors in protecting EP-deprived cells from apoptosis. Although several of the inhibitors were effective, z-IETD-FMK was studied most extensively because of its specificity for enzymes which cleave procaspase 3 at aspartate 175 (IETD175). Large percentages of EP-deprived erythroblasts treated with z-IETD-FMK appeared morphologically normal and negative by a DNA strand breakage (TUNEL) assay at 24 h (75%) compared to EP-deprived controls (10%) which were not treated with inhibitor. However, inhibitor-treated erythroid progenitors deprived of EP for 24 h and then resupplied with EP showed only a modest improvement in long-term survival compared to cells which did not receive the caspase inhibitor during the 24 h EP deprivation. Thus, while the manifestations of apoptosis were delayed in most cells by inhibiting caspase activity, the processes initiating the loss of cell viability due to EP deprivation were irreparablein the majority of the cells and eventually led to their deaths.     

Partial cleavage of RasGAP by caspases is required for cell survival in mild stress conditions

Tight control of apoptosis is required for proper development and maintenance of homeostasis in multicellular organisms. Cells can protect themselves from potentially lethal stimuli by expressing antiapoptotic factors, such as inhibitors of apoptosis, FLICE (caspase 8)-inhibitory proteins, and members of the Bcl2 family. Here, we describe a mechanism that allows cells to survive once executioner caspases have been activated. This mechanism relies on the partial cleavage of RasGAP by caspase 3 into an amino-terminal fragment called fragment N. Generation of this fragment leads to the activation of the antiapoptotic Akt kinase, preventing further amplification of caspase activity. Partial cleavage of RasGAP is required for cell survival under stress conditions because cells expressing an uncleavable RasGAP mutant cannot activate Akt, cannot prevent amplification of caspase 3 activity, and eventually undergo apoptosis. Executioner caspases therefore control the extent of their own activation by a feedback regulatory mechanism initiated by the partial cleavage of RasGAP that is crucial for cell survival under adverse conditions.     

Chlamydia trachomatis‐infected host cells resist dsRNA‐induced apoptosis

Human pathogenic Chlamydia trachomatis have evolved sophisticated mechanisms to manipulate host cell signalling pathways in order to prevent apoptosis. We show here that host cells infected with C. trachomatis resist apoptosis induced by polyI:C, a synthetic double‐stranded RNA that mimics viral infections. Infected cells displayed significantly reduced levels of PARP cleavage, caspase‐3 activation and a decrease in the TUNEL positive population in the presence of polyI:C. Interestingly, the chlamydial block of apoptosis was upstream of the initiator caspase‐8. Processing of caspase‐8 was reduced in infected cells and coincided with a decrease in Bid truncation and downstream caspase‐9 cleavage. Moreover, the enzymatic activity of caspase‐8, measured by a luminescent substrate, was significantly reduced in infected cells. Caspase‐8 inhibition by Chlamydia was dependent on cFlip as knock‐down of cFlip decreased the chlamydial block of caspase‐8 activation and consequently reduced apoptosis inhibition. Our data implicate that chlamydial infection interferes with the host cell's response to viral infections and thereby influences the fate of the cell.     

XIAP activity dictates Apaf-1 dependency for caspase 9 activation

The current model for the intrinsic apoptotic pathway holds that mitochondrial activation of caspases in response to cytotoxic drugs requires both Apaf-1-induced dimerization of procaspase 9 and Smac/Diablo-mediated sequestration of inhibitors of apoptosis proteins (IAPs). Here, we showed that either pathway can independently promote caspase 9 activation in response to apoptotic stimuli. In drug-treated Apaf-1(-/-) primary myoblasts, but not fibroblasts, Smac/Diablo accumulates in the cytosol and sequesters X-linked IAP (XIAP), which is expressed at lower levels in myoblasts than in fibroblasts. Consequently, caspase 9 activation proceeds in Apaf-1(-/-) myoblasts; concomitant ablation of Apaf-1 and Smac is required to prevent caspase 9 activation and the onset of apoptosis. Conversely, in stimulated Apaf-1(-/-) fibroblasts, the ratio of XIAP to Smac/Diablo is high compared to that for myoblasts and procaspase 9 is not activated. Suppressing XIAP with exogenous Smac/Diablo or a pharmacological inhibitor can still induce caspase 9 in drug-treated Apaf-1-null fibroblasts. Thus, caspase 9 activation in response to intrinsic apoptotic stimuli can be uncoupled from Apaf-1 in vivo by XIAP antagonists.     

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.     

Apoptosis-inducing factor is a major contributor to neuronal loss induced by neonatal cerebral hypoxia-ischemia

Nine-day-old harlequin (Hq) mice carrying the hypomorphic apoptosis-inducing factor (AIF)(Hq) mutation expressed 60% less AIF, 18% less respiratory chain complex I and 30% less catalase than their wild-type (Wt) littermates. Compared with Wt, the infarct volume after hypoxia-ischemia (HI) was reduced by 53 and 43% in male (YX(Hq)) and female (X(Hq)X(Hq)) mice, respectively (P<0.001). The Hq mutation did not inhibit HI-induced mitochondrial release of cytochrome c or activation of calpain and caspase-3. The broad-spectrum caspase inhibitor quinoline-Val-Asp(OMe)-CH(2)-PH (Q-VD-OPh) decreased the activation of all detectable caspases after HI, both in Wt and Hq mice. Q-VD-OPh reduced the infarct volume equally in Hq and in Wt mice, and the combination of Hq mutation and Q-VD-OPh treatment showed an additive neuroprotective effect. Oxidative stress leading to nitrosylation and lipid peroxidation was more pronounced in ischemic brain areas from Hq than Wt mice. The antioxidant edaravone decreased oxidative stress in damaged brains, more pronounced in the Hq mice, and further reduced brain injury in Hq but not in Wt mice. Thus, two distinct strategies can enhance the neuroprotection conferred by the Hq mutation, antioxidants, presumably compensating for a defect in AIF-dependent redox detoxification, and caspase inhibitors, presumably interrupting a parallel pathway leading to cellular demise.     

Cadmium Induces Thymocyte Apoptosis via Caspase‐Dependent and Caspase‐Independent Pathways

Based on our recent findings that 25  µ M cadmium triggers oxidative stress–mediated caspase‐dependent apoptosis in murine thymocytes, this study is designed to explore whether Cd also induces caspase‐independent apoptosis. We found that pretreatment with caspase inhibitors fails to prevent Cd‐induced apoptosis completely, suggesting the possibility of an additional pathway. Western blot and flow cytometry techniques indicated marked expression of apoptosis‐inducing factor and endonuclease G in nuclear fraction, signifying their translocation from mitochondria to nucleus. Intracellular Ca²⁺ and reactive oxygen species (ROS) levels significantly raised by Cd were restored by ruthenium red, which had no influence on mitochondrial membrane depolarization and caspase activity and apoptosis. Using cyclosporin A, ROS formation and mitochondrial membrane depolarization were completely abolished, whereas apoptosis was partly attenuated. These results clearly demonstrate more than one apoptotic pathway in thymocytes and support the role of mitochondrial permeability transition pore in the regulation of caspase‐independent cell death triggered by Cd. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:193‐203, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21468     

The effect of Bcl‐2, YAMA,and XIAP over‐expression on apoptosis and adenovirus production in HEK293 cell line

Many viruses induce cell death and lysis as part of their replication and dissemination strategy, and in many cases features of apoptosis are observed. Attempts have been made to further increase productivity by prolonging cell survival via the over‐expression of anti‐apoptotic genes. Here, we extend the study to investigate the association between virus replication and apoptosis, pertinent to large‐scale vector production for gene therapy. Infection of an HEK293 cell line with a replication defective type‐5‐adenovirus expressing a GFP reporter (Ad5GFP) resulted in rapid decline in viability associated with increased virus titer. The over‐expression of bcl‐2 resulted in improved cell resistance to apoptosis and prolonged culture duration, but reduced virus specific and total productivity. In contrast, the over‐expression of pro‐caspase‐3 (Yama/CPP32/apopain) resulted in reduced cell survival but increased virus productivity. The treatment of infected cells with caspase inhibitors support the preposition that caspase‐3 dependent apoptosis, and to a lesser degree caspase‐9 dependent apoptosis, represent important steps in virus production, thus implicating the intrinsic apoptosis pathway in the production of adenovirus from HEK293 cells. The suppression of apoptosis by the over‐expression of XIAP (inhibitors of caspase family cell death proteases) further shows that caspase‐mediated activation plays an important role in virus infection and maturation. Biotechnol. Bioeng. 2009; 104: 752–765 © 2009 Wiley Periodicals, Inc.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

A DEVD-Inhibited Caspase Other than CPP32 Is Involved in the Commitment of Cerebellar Granule Neurons to Apoptosis Induced by K+ Deprivation

Abstract: Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K⁺ (25 mM KCI) to medium containing lower levels of K⁺ (5 mM KCI). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp·(O-methyl)·fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp·chloromethyl ketone (YVAD·cmk), an interleukin-1β converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp·fluoromethyl ketone (DEVD·fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin-1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K⁺-deprived granule neurons.     

Protein kinase Cδ and caspase‐3 modulate TRAIL‐induced apoptosis in breast tumor cells

This report describes that protein kinase C delta (PKCδ) overexpression prevents TRAIL‐induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL‐induced apoptosis was significantly inhibited in PKCδ overexpressing MCF‐7 (MCF7/PKCδ) cells. Our data reveal that PKCδ inhibits caspase‐8 activation, a first step in TRAIL‐induced apoptosis, thus preventing TRAIL‐induced apoptosis. Inhibition of PKCδ using rottlerin or PKCδ siRNA reverses the inhibitory effect of PKCδ on caspase‐8 activation leading to TRAIL‐induced apoptosis. To determine if caspase‐3‐induced PKCδ cleavage reverses its inhibition on caspase‐8, we developed stable cell lines that either expresses wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) or caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδ mut) utilizing MCF‐7 cells expressing caspase‐3. Cells that overexpress caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδmut) significantly inhibited TRAIL‐induced apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. In MCF‐7/cas‐3/PKCδmut cells, TRAIL‐induced caspase‐8 activation was blocked leading to inhibition of apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. Together, these results strongly suggest that overexpression of PKCδ inhibits caspase‐8 activation leading to inhibition of TRAIL‐induced apoptosis and its inhibition by rottlerin, siRNA, or cleavage by caspase‐3 sensitizes cells to TRAIL‐induced apoptosis. Clinically, PKCδ overexpressing tumors can be treated with a combination of PKCδ inhibitor(s) and TRAIL as a new treatment strategy. J. Cell. Biochem. 111: 979–987, 2010. © 2010 Wiley‐Liss, Inc.     

Apoptosis and Its Suppression in Hepatocytes Culture

In order to achieve the goal of developing extracorporeal liver support devices, it is necessary to optimise bioprocess environment such that viability and function are maximised. Optimising culture medium composition and controlling the constitution of the cellular microenvironment within the bioreactor have for many years been considered vital to achieving these aims. Coupled to this is the need to understand apoptosis, the prime suspect in the demise of animal cultures, including those of hepatocytes. Results presented here show that absent nutrients including glucose and amino acids play a substantial part in the induction of apoptosis. The use of chemical apoptosis inhibitors was utilised to investigate key components of hepatic apoptosis where caspases, predominantly caspase 8, were implicated in staurosporine (STS)-induced HepZ apoptosis. Caspase 9 and 3 activation although recorded was of less significance. Interestingly, these results were not consistent with those of mitochondrial membrane depolarisation where inhibition of caspase activation appeared to drive depolarisation. Inhibition of mitochondrial permeability transition and use of anti-oxidants was unsuccessful in reducing apoptosis, caspase activation and mitochondrial membrane depolarisation. In further studies, the anti-apoptotic gene bcl-2 was over-expressed in HepZ, resulting in a cell line that was more robust and resistant to death induced by glucose and cystine deprivation and treatment with STS. Bcl-2 did not however show significant cytoprotectivity where apoptosis was stimulated by deprivation of glutamine and serum. Overall, results indicated that although apoptosis can be curbed by use of chemical inhibitors and genetic manipulation, their success is dependent on apoptotic stimuli.     

Caspase inhibitors: viral, cellular and chemical

Caspases, key mediators of apoptosis, are a structurally related family of cysteine proteases that cleave their substrates at aspartic acid residues either to cause cell death or to activate cytokines as part of an immune response. They can be controlled upstream by the regulation of signals that lead to zymogen activation, or downstream by inhibitors that prevent them from reaching their substrates. This review specifically looks at caspase inhibitors as distinct from caspase regulators: those produced by the cell itself; those whose genes are carried by viruses; and artificial caspase inhibitors used for research and potentially as therapeutics.     

The Temporal Relationship between Protein Phosphatase, Mitochondrial CytochromecRelease, and Caspase Activation in Apoptosis

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochromec.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 cytochromecin apoptosis induced by the Fas receptor. We demonstrate that cytochromecis 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 cytochromec.The protein phosphatase inhibitor calyculin A prevented cytochromecrelease and apoptosis induced by both agents, suggesting that release of cytochromecis 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 cytochromec.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 cytochromecand apoptosis induced by both insults.     

Staurosporine-induced cell death in salmonid cells: the role of apoptotic volume decrease, ion fluxes and MAP kinase signaling

Apoptotic cell death in mammalian models is frequently associated with cell shrinkage. Inhibition of apoptotic volume decrease (AVD) is cytoprotective, suggesting that cell shrinkage is an important early event in apoptosis. In salmonid hepatoma and gill cells staurosporine induced apoptosis, as assessed by activation of effector caspases, nuclear condensation, and a decrease of mitochondrial membrane potential (MMP), and these changes were accompanied by cell shrinkage. The Cl⁻ transport inhibitor DIDS and the K⁺ channel inhibitor quinidine prevented AVD, but only DIDS inhibited apoptosis. Other Cl⁻ flux inhibitors, as well as a pan-caspase inhibitor, did not prevent cell shrinkage, but still prevented caspase activation. Furthermore, regulatory volume decrease (RVD) under hypotonic conditions was not facilitated, but diminished in apoptotic cells. Since all transport inhibitors used blocked RVD, but only DIDS and quinidine inhibited AVD, the ion transporters involved in both processes are apparently not identical. In addition, our data indicate that inhibition of Cl⁻ fluxes rather than blocking cell shrinkage or K⁺ fluxes is important for preventing apoptosis. In line with this, inhibition of MAP kinases reduced RVD and not AVD, but still diminished caspase activation. Finally, we observed that MAP kinases were activated upon staurosporine treatment and that at least activation of ERK was prevented when AVD was inhibited.