Targeting procaspase‐3 with WF‐208, a novel PAC‐1 derivative,causes selective cancer cell apoptosis

Caspase‐3 is a critical effector caspase in apoptosis cascade, and is often over‐expressed in many cancer tissues. The first synthesized procaspase‐3 activator, PAC‐1, induces cancer cell apoptosis and exhibits antitumour activity in murine xenograft models. To identify more potent procaspase‐3 activators, a series of compounds were designed, synthesized and evaluated for their ability of inducing cancer cell death in culture. Among these compounds, WF‐208 stood out by its high cytotoxicity against procaspase‐3 overexpressed HL‐60 cells. Compared with PAC‐1, WF‐208 showed higher cytotoxicity in cancer cells and lower toxicity in normal cells. The further investigation described herein showed that WF‐208 activated procaspase‐3, degraded IAPs (The Inhibitors of apoptosis proteins) and leaded to caspase‐3‐dependent cell death in tumour cells, which possibly because of the zinc‐chelating properties. WF‐208 also showed greater antitumour activity than PAC‐1 in murine xenograft model. In conclusion, we have discovered WF‐208 as a promising procaspase‐3 activating compound, with higher activity and higher cell selectivity than PAC‐1.     

Suppression of ERK signaling evokes autocrine Fas‐mediated death in arachidonic acid‐treated human chronic myeloid leukemia K562 cells

Arachidonic acid (AA)‐induced apoptotic death of K562 cells (human chronic myeloid leukemic cells) was characteristic of reactive oxygen species (ROS) generation and mitochondrial depolarization. N‐Acetylcysteine pretreatment rescued viability of AA‐treated cells and abolished mitochondrial depolarization. In contrast to no significant changes in phospho‐JNK and phospho‐ERK levels, AA evoked notable activation of p38 MAPK. Unlike that of JNK and p38 MAPK, ERK suppression further reduced the viability of AA‐treated cells. Increases in Fas/FasL protein expression, caspase‐8 activation, the production of tBid and the loss of mitochondrial membrane potential were noted with K562 cells that were treated with a combination of U0126 and AA. Down‐regulation of FADD attenuated U0126‐evoked degradation of procaspase‐8 and Bid. Abolition of p38 MAPK activation abrogated U0126‐elicited Fas/FasL up‐regulation in AA‐treated cells. U0126 pretreatment suppressed c‐Fos phosphorylation but increased p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun protein expression by siRNA suggested that c‐Fos counteracted the effect of c‐Jun on Fas/FasL up‐regulation. Taken together, our data indicate that AA induces the ROS/mitochondria‐dependent death pathway and blocks the ERK pathway which enhances the cytotoxicity of AA through additionally evoking an autocrine Fas‐mediated apoptotic mechanism in K562 cells. J. Cell. Physiol. 222: 625–634, 2010. © 2009 Wiley‐Liss, Inc.     

Direct interaction of GD3 ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability transition, cytochrome c release, and caspase activation.

Glycosphingolipids, including gangliosides, are emerging as signaling intermediates of extracellular stimuli. Because mitochondria play a key role in the orchestration of death signals, we assessed the interaction of GD3 ganglioside (GD3) with mitochondria and the subsequent cascade of events that culminate in cell death. In vitro studies with isolated mitochondria from rat liver demonstrate that GD3 elicited a burst of peroxide production within 15-30 min, which preceded the opening of the mitochondrial permeability transition, followed by cytochrome c (cyt c) release. These effects were mimicked by lactosylceramide and N-acetyl-sphingosine but not by sphinganine or sphingosine and were prevented by cyclosporin A and butylated hydroxytoluene (BHT). Reconstitution of mitochondria pre-exposed to GD3 with cytosol from rat liver in a cell-free system resulted in the proteolytic processing of procaspase 3 and subsequent caspase 3 activation. Intact hepatocytes or U937 cells selectively depleted of glutathione in mitochondria by 3-hydroxyl-4-pentenoate (HP) with the sparing of cytosol reduced glutathione (GSH) were sensitized to GD3, manifested as an apoptotic death. Inhibition of caspase 3 prevented the apoptotic phenotype of HP-treated cells caused by GD3 without affecting cell survival; in contrast, BHT fully protected HP-treated cells to GD3 treatment. Treatment of cells with tumor necrosis factor increased the level of GD3, whereas blockers of mitochondrial respiration at complex I and II protected sensitized cells to GD3 treatment. Thus, the effect of GD3 as a lipid death effector is determined by its interaction with mitochondria leading to oxidant-dependent caspase activation. Mitochondrial glutathione plays a key role in controlling cell survival through modulation of the oxidative stress induced by glycosphingolipids.     

Reactive oxygen species‐induced cell death of rat primary astrocytes through mitochondria‐mediated mechanism

Astrocytes, the most abundant glial cell population in the central nervous system (CNS), play physiological roles in neuronal activities. Oxidative insult induced by the injury to the CNS causes neural cell death through extrinsic and intrinsic pathways. This study reports that reactive oxygen species (ROS) generated by exposure to the strong oxidizing agent, hexavalent chromium (Cr(VI)) as a chemical‐induced oxidative stress model, caused astrocytes to undergo an apoptosis‐like cell death through a caspase‐3‐independent mechanism. Although activating protein‐1 (AP‐1) and NF‐κB were activated in Cr(VI)‐primed astrocytes, the inhibition of their activity failed to increase astrocytic cell survival. The results further indicated that the reduction in mitochondrial membrane potential (MMP) was accompanied by an increase in the levels of ROS in Cr(VI)‐primed astrocytes. Moreover, pretreatment of astrocytes with N‐acetylcysteine (NAC), the potent ROS scavenger, attenuated ROS production and MMP loss in Cr(VI)‐primed astrocytes, and significantly increased the survival of astrocytes, implying that the elevated ROS disrupted the mitochondrial function to result in the reduction of astrocytic cell viability. In addition, the nuclear expression of apoptosis‐inducing factor (AIF) and endonuclease G (EndoG) was observed in Cr(VI)‐primed astrocytes. Taken together, evidence shows that astrocytic cell death occurs by ROS‐induced oxidative insult through a caspase‐3‐independent apoptotic mechanism involving the loss of MMP and an increase in the nuclear levels of mitochondrial pro‐apoptosis proteins (AIF/EndoG). This mitochondria‐mediated but caspase‐3‐independent apoptotic pathway may be involved in oxidative stress‐induced astrocytic cell death in the injured CNS. J. Cell. Biochem. 107: 933–943, 2009. © 2009 Wiley‐Liss, Inc.     

Corilagin inhibits breast cancer growth via reactive oxygen species‐dependent apoptosis and autophagy

Corilagin is a component of Phyllanthus urinaria extract and has been found of possessing anti‐inflammatory, anti‐oxidative, and anti‐tumour properties in clinic treatments. However, the underlying mechanisms in anti‐cancer particularly of its induction of cell death in human breast cancer remain undefined. Our research found that corilagin‐induced apoptotic and autophagic cell death depending on reactive oxygen species (ROS) in human breast cancer cell, and it occurred in human breast cancer cell (MCF‐7) only comparing with normal cells. The expression of procaspase‐8, procaspase‐3, PARP, Bcl‐2 and procaspase‐9 was down‐regulated while caspase‐8, cleaved PARP, caspase‐9 and Bax were up‐regulated after corilagin treatment, indicating apoptosis mediated by extrinsic and mitochondrial pathways occurred in MCF‐7 cell. Meanwhile, autophagy mediated by suppressing Akt/mTOR/p70S6K pathway was detected with an increase in autophagic vacuoles and LC3‐II conversion. More significantly, inhibition of autophagy by chloroquine diphosphate salt (CQ) remarkably enhanced apoptosis, while the caspase inhibitor z‐VAD‐fmk failed in affecting autophagy, suggesting that corilagin‐induced autophagy functioned as a survival mechanism in MCF‐7 cells. In addition, corilagin induced intracellular reactive oxygen species (ROS) generation, when reduced by ROS scavenger NAC, apoptosis and autophagy were both down‐regulated. Nevertheless, in SK‐BR3 cell which expressed RIP3, necroptosis inhibitor Nec‐1 could not alleviate cell death induced by corilagin, indicating necroptosis was not triggered. Subcutaneous tumour growth in nude mice was attenuated by corilagin, consisting with the results in vitro. These results imply that corilagin inhibits cancer cell proliferation through inducing apoptosis and autophagy which regulated by ROS release.     

The Apaf‐1•procaspase‐9 apoptosome complex functions as a proteolytic‐based molecular timer

During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that apoptosome‐mediated cleavage of procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector procaspase‐3, procaspase‐9 cannot be processed by the apoptosome as a typical substrate. Indeed, procaspase‐9 possessed higher affinity for the apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase‐9 per se contributed little to the activation of procaspase‐3. Thus, the Apaf‐1 apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of procaspase‐9 sets the overall duration of the timer, procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate procaspase‐3) dictates how fast the timer ‘ticks’ over.     

Requirement of Apaf-1 for mitochondrial events and the cleavage or activation of all procaspases during genotoxic stress-induced apoptosis

Sequential activation of caspases is critical for the execution of apoptosis. Recent evidence suggests caspase 2 is a significant upstream caspase capable of initiating mitochondrial events, such as the release of cytochrome c. In particular, in vitro studies using recombinant proteins have shown that cleaved caspase 2 can induce mitochondrial outer membrane permeabilization directly or by cleaving the BH3-only protein BID (BH3 interacting domain death agonist). However, whether interchain cleavage or activation of procaspase 2 occurs prior to Apaf-1-mediated procaspase 9 activation under more natural conditions remains unresolved. In the present study, we show that Apaf-1-deficient Jurkat T-lymphocytes and mouse embryonic fibroblasts were highly resistant to DNA-damage-induced apoptosis and failed to cleave or activate any apoptotic procaspase, including caspase 2. Significantly, drug-induced cytochrome c release and loss of mitochondrial membrane potential were inhibited in cells lacking Apaf-1. By comparison, procaspase proteolysis and apoptosis were only delayed slightly in Apaf-1-deficient Jurkat cells upon treatment with anti-Fas antibody. Our data support a model in which Apaf-1 is necessary for the cleavage or activation of all procaspases and the promotion of mitochondrial apoptotic events induced by genotoxic drugs.     

Mitochondrial accumulation under oxidative stress is due to defects in autophagy

Mitochondrial dynamics maintains normal mitochondrial function by degrading damaged mitochondria and generating newborn mitochondria. The accumulation of damaged mitochondria influences the intracellular environment by promoting mitochondrial dysfunction, and thus initiating a vicious cycle. Oxidative stress induces mitochondrial malfunction, which is involved in many cardiovascular diseases. However, the mechanism of mitochondrial accumulation in cardiac myoblasts remains unclear. We observed mitochondrial dysfunction and an increase in mitochondrial mass under the oxidative conditions produced by tert‐butyl hydroperoxide (tBHP) in cardiac myoblast H9c2 cells. However, in contrast to the increase in mitochondrial mass, mitochondrial DNA (mtDNA) decreased, suggesting that enhanced mitochondrial biogenesis may be not the primary cause of the mitochondrial accumulation. Therefore, we investigated changes in a number of proteins involved in autophagy. Beclin1, Atg12–Atg5 conjugate, Atg7 contents decreased but LC3‐II accumulated in tBHP‐treated H9c2 cells. Moreover, the capacity for acid hydrolysis decreased in H9c2 cells. We also demonstrated a decrease in DJ‐1 protein under the oxidative conditions that deregulate mitochondrial dynamics. These results reveal that autophagy became defective under oxidative stress. We therefore suggest that defects in autophagy mediate mitochondrial accumulation under these conditions. J. Cell. Biochem. 114: 212–219, 2012. © 2012 Wiley Periodicals, Inc.     

The octadecaneuropeptide ODN prevents 6‐hydroxydopamine‐induced apoptosis of cerebellar granule neurons through a PKC‐MAPK‐dependent pathway

Oxidative stress, induced by various neurodegenerative diseases, initiates a cascade of events leading to apoptosis, and thus plays a critical role in neuronal injury. In this study, we have investigated the potential neuroprotective effect of the octadecaneuropeptide (ODN) on 6‐hydroxydopamine (6‐OHDA)‐induced oxidative stress and apoptosis in cerebellar granule neurons (CGN). ODN, which is produced by astrocytes, is an endogenous ligand for both central‐type benzodiazepine receptors (CBR) and a metabotropic receptor. Incubation of neurons with subnanomolar concentrations of ODN (10^?18 to 10^?12 M) inhibited 6‐OHDA‐evoked cell death in a concentration‐dependent manner. The effect of ODN on neuronal survival was abrogated by the metabotropic receptor antagonist, cyclo_1–8[DLeu⁵]OP, but not by a CBR antagonist. ODN stimulated polyphosphoinositide turnover and ERK phosphorylation in CGN. The protective effect of ODN against 6‐OHDA toxicity involved the phospholipase C/ERK MAPK transduction cascade. 6‐OHDA treatment induced an accumulation of reactive oxygen species, an increase of the expression of the pro‐apoptotic gene Bax, a drop of the mitochondrial membrane potential and a stimulation of caspase‐3 activity. Exposure of 6‐OHDA‐treated cells to ODN blocked all the deleterious effects of the toxin. Taken together, these data demonstrate for the first time that ODN is a neuroprotective agent that prevents 6‐OHDA‐induced oxidative stress and apoptotic cell death.     

Mitochondrial regulation of cell death: mitochondria are essential for procaspase 3-p21 complex formation to resist Fas-mediated cell death

Death receptor Fas transduces cell death signaling upon stimulation by Fas ligand, and this death signaling is mediated by caspase. Recently, we reported that the cell cycle regulator p21 interacts with procaspase 3 to resist Fas-mediated cell death. In the present study, the molecular characterization and functional region of the procaspase 3-p21 complex was further investigated. We observed the p21 expression in the mitochondrial fraction of HepG2 cells and detected Fas-mediated cell death only in the presence of actinomycin D. However, mitochondrial-DNA-lacking HepG2 (MDLH) cells showed this effect even in the absence of actinomycin D. Both p21 and procaspase 3 were expressed in MDLH cells, but the procaspase 3-p21 complex formation was not observed. Interestingly, the resistance to Fas-mediated cell death in the MDLH cells without actinomycin D was recovered after microinjection of HepG2-derived mitochondria into the MDLH cells. We conclude that mitochondria are necessary for procaspase 3-p21 complex formation and propose that the mitochondrial role during cell death is not only death induction but also death suppression.     

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.     

Overexpression of Bcl‐2 prevents neomycin‐induced hair cell death and caspase‐9 activation in the adult mouse utricle in vitro

Mechanosensory hair cells of the inner ear are especially sensitive to death induced by exposure to aminoglycoside antibiotics. This aminoglycoside‐induced hair cell death involves activation of an intrinsic program of cellular suicide. Aminoglycoside‐induced hair cell death can be prevented by broad‐spectrum inhibition of caspases, a family of proteases that mediate apoptotic and programmed cell death in a wide variety of systems. More specifically, aminoglycoside‐induced hair cell death requires activation of caspase‐9. Caspase‐9 activation requires release of mitochondrial cytochrome c into the cytoplasm, indicating that aminoglycoside‐induced hair cell death is mediated by the mitochondrial (or “intrinsic”) cell death pathway. The Bcl‐2 family of pro‐apoptotic and anti‐apoptotic proteins are important upstream regulators of the mitochondrial apoptotic pathway. Bcl‐2 is an anti‐apoptotic protein that localizes to the mitochondria and promotes cell survival by preventing cytochrome c release. Here we have utilized transgenic mice that overexpress Bcl‐2 to examine the role of Bcl‐2 in neomycin‐induced hair cell death. Overexpression of Bcl‐2 significantly increased hair cell survival following neomycin exposure in organotypic cultures of the adult mouse utricle. Furthermore, Bcl‐2 overexpression prevented neomycin‐induced activation of caspase‐9 in hair cells. These results suggest that the expression level of Bcl‐2 has important effects on the pathway(s) important for the regulation of aminoglycoside‐induced hair cell death. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 89–100, 2004     

Taiwan cobra phospholipase A2‐elicited JNK activation is responsible for autocrine fas‐mediated cell death and modulating Bcl‐2 and Bax protein expression in human leukemia K562 cells

Phospholipase A₂ (PLA₂) from Naja naja atra venom induced apoptotic death of human leukemia K562 cells. Degradation of procaspases, production of tBid, loss of mitochondrial membrane potential, Bcl‐2 degradation, mitochondrial translocation of Bax, and cytochrome c release were observed in PLA₂‐treated cells. Moreover, PLA₂ treatment increased Fas and FasL protein expression. Upon exposure to PLA₂, activation of p38 MAPK (mitogen‐activated protein kinase) and JNK (c‐Jun NH₂‐terminal kinase) was found in K562 cells. SB202190 (p38 MAPK inhibitor) pretreatment enhanced cytotoxic effect of PLA₂ and led to prolonged JNK activation, but failed to affect PLA₂‐induced upregulation of Fas and FasL protein expression. Sustained JNK activation aggravated caspase8/mitochondria‐dependent death pathway, downregulated Bcl‐2 expression and increased mitochondrial translocation of Bax. SP600125 (JNK inhibitor) abolished the cytotoxic effect of PLA₂ and PLA₂‐induced autocrine Fas death pathway. Transfection ASK1 siRNA and overexpression of dominant negative p38α MAPK proved that ASK1 pathway was responsible for PLA₂‐induced p38 MAPK and JNK activation and p38α MAPK activation suppressed dynamically persistent JNK activation. Downregulation of FADD abolished PLA₂‐induced procaspase‐8 degradation and rescued viability of PLA₂‐treated cells. Taken together, our results indicate that JNK‐mediated autocrine Fas/FasL apoptotic mechanism and modulation of Bcl‐2 family proteins are involved in PLA₂‐induced death of K562 cells. J. Cell. Biochem. 109: 245–254, 2010. © 2009 Wiley‐Liss, Inc.     

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

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

RNAi‐mediated silencing of insulin receptor substrate‐4 enhances actinomycin D‐ and tumor necrosis factor‐α‐induced cell death in hepatocarcinoma cancer cell lines

Insulin receptor substrate‐4 (IRS‐4) transmits signals from the insulin‐like growth factor receptor (IGF‐IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS‐4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS‐4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS‐4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor‐α (TNF‐α). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial‐dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF‐α amplifies the effect of Act D on HepG2 cell apoptosis increasing c‐jun N‐terminal kinase (JNK) activity, IκB‐α proteolysis and glutathione depletion. IRS‐4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS‐4 action is independent of Akt, IκB kinase and JNK. IRS‐4 down regulation, however, decreased γ‐glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF‐α. These results suggest that IRS‐4 protects HepG2 cells from oxidative stress induced by drug treatment. J. Cell. Biochem. 108: 1292–1301, 2009. © 2009 Wiley‐Liss, Inc.     

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     

N‐(3‐Oxo‐acyl)‐homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts

N‐(3‐Oxododecanoyl)‐l ‐homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum‐sensing molecule for bacteria–bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12‐triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in “initiator” caspases or “effector” caspases. Our data indicate that C12 selectively induces the mitochondria‐dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both “initiator” and “effector” caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis.     

H. pylori‐Induced Apoptosis in Human Gastric Cancer Cells Mediated via the Release of Apoptosis‐Inducing Factor from Mitochondria

Background and Aim: Our previous study of Helicobacter pylori‐induced apoptosis showed the involvement of Bcl‐2 family proteins and cytochrome c release from mitochondria. Here, we examine the release of other factors from mitochondria, such as apoptosis‐inducing factor (AIF), and upstream events involving caspase‐8 and Bid. Methods: Human gastric adenocarcinoma (AGS) cells were incubated with a cagA‐positive H. pylori strain for 0, 3, 6, and 24 hours and either total protein or cytoplasmic, nuclear, and mitochondrial membrane fractions were collected. Results: Proteins were immunoblotted for AIF, Bid, polyadenosine ribose polymerase (PARP), caspase‐8, and β‐catenin. H. pylori activated caspase‐8, caused PARP cleavage, and attenuated mitochondrial membrane potential. A time‐dependent decrease in β‐catenin protein expression was detected in cytoplasmic and nuclear extracts, coupled with a decrease in β‐actin. An increase in the cytoplasmic pool of AIF was seen as early as 3 hours after H. pylori exposure, and a concomitant increase was seen in nuclear AIF levels up to 6 hours. A band corresponding to full‐length Bid was seen in both the cytoplasmic and the nuclear fractions of controls, but not after H. pylori exposure. Active AIF staining was markedly increased in gastric mucosa from infected persons, compared to uninfected controls. Conclusion: H. pylori might trigger apoptosis in AGS cells via interaction with death receptors in the plasma membrane, leading to the cleavage of procaspase‐8, release of cytochrome c and AIF from mitochondria, and activation of subsequent downstream apoptotic events, as reported previously for chlorophyllin. This is consistent with AIF activation that was found in the gastric mucosa of humans infected with H. pylori. Hence, the balance between apoptosis and proliferation in these cells may be altered in response to injury caused by H. pylori infection, leading to an increased risk of cancer.     

Neuromelanin selectively induces apoptosis in dopaminergic SH-SY5Y cells by deglutathionylation in mitochondria: involvement of the protein and melanin component

Parkinson's disease (PD) is characterized by selective depletion of nigral dopamine (DA) neurons containing neuromelanin (NM), suggesting the involvement of NM in the pathogenesis. This study reports induction of apoptosis by NM in SH-SY5Y cells, whereas protease-K-treated NM, synthesized DA- and cysteinyl dopamine melanin showed much less cytotoxicity. Cell death was mediated by mitochondria-mediated apoptotic pathway, namely collapse of mitochondrial membrane potential, release of cytochrome c , and activation of caspase 3, but Bcl-2 over-expression did not suppress apoptosis. NM increased sulfhydryl content in mitochondria, and a major part of it was identified as GSH, whereas dopamine melanin significantly reduced sulfhydryl levels. Western blot analysis for protein-bound GSH demonstrated that only NM reduced S -glutathionylated proteins in mitochondria and dissociated macromolecular structure of complex I. Reactive oxygen and nitrogen species were required for the deglutathionylation by NM, which antioxidants reduced significantly with prevention of apoptosis. These results suggest that NM may be related to cell death of DA neurons in PD and aging through regulation of mitochondrial redox state and S -glutathionylation, for which NM-associated protein is absolutely required. The novel function of NM is discussed in relation to the pathogenesis of PD.