Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sulfur mustard (SM) induces vesication via poorly understood pathways. The blisters that are formed result primarily from the detachment of the epidermis from the dermis at the level of the basement membrane. In addition, there is toxicity to the basal cells, although no careful study has been performed to determine the precise mode of cell death biochemically. We describe here two potential mechanisms by which SM causes basal cell death and detachment: namely, induction of terminal differentiation and apoptosis. In the presence of 100 microM SM, terminal differentiation was rapidly induced in primary human keratinocytes that included the expression of the differentiation-specific markers K1 and K10 and the cross-linking of the cornified envelope precursor protein involucrin. The expression of the attachment protein, fibronectin, was also reduced in a time- and dose-dependent fashion. Features common to both differentiation and apoptosis were also induced in 100 microM SM, including the rapid induction of p53 and the reduction of Bcl-2. At higher concentrations of SM (i.e., 300 microM), formation of the characteristic nucleosome-sized DNA ladders, TUNEL-positive staining of cells, activation of the cysteine protease caspase-3/apopain, and cleavage of the death substrate poly(ADP-ribose) polymerase, were observed both in vivo and in vitro. Both the differentiation and the apoptotic processes appeared to be calmodulin dependent, because the calmodulin inhibitor W-7 blocked the expression of the differentiation-specific markers, as well as the apoptotic response, in a concentration-dependent fashion. In addition, the intracellular Ca2+ chelator, BAPTA-AM, blocked the differentiation response and attenuated the apoptotic response. These results suggest a strategy for designing inhibitors of SM vesication via the Ca2+-calmodulin or caspase-3/PARP pathway.
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PMID:Sulfur mustard induces markers of terminal differentiation and apoptosis in keratinocytes via a Ca2+-calmodulin and caspase-dependent pathway. 966 88

Sulfur mustard is cytotoxic to dermal fibroblasts as well as epidermal keratinocytes. We demonstrated that poly(ADP-ribose) polymerase (PARP) modulates Fas-mediated apoptosis, and other groups and we have shown that PARP plays a role in the modulation of other types of apoptotic and necrotic cell death. We have now utilized primary dermal fibroblasts, immortalized fibroblasts, and keratinocytes derived from PARP(-/-) mice and their wildtype littermates (PARP(+/+)) to determine the contribution of PARP to sulfur mustard toxicity. Following sulfur mustard exposure, primary skin fibroblasts from PARP-deficient mice demonstrated increased internucleosomal DNA cleavage, caspase-3 processing and activity, and annexin V positivity, compared to those derived from PARP(+/+) animals. Conversely, propidium iodide staining, PARP cleavage patterns, and random DNA fragmentation revealed a dose-dependent increase in necrosis in PARP(+/+) but not PARP(-/-) cells. Using immortalized PARP(-/-) fibroblasts stably transfected with the human PARP cDNA or with empty vector alone, we show that PARP inhibits markers of apoptosis in these cells as well. Finally, primary keratinocytes were derived from newborn PARP(+/+) and PARP(-/-) mice and immortalized with the E6 and E7 genes of human papilloma virus. In contrast to fibroblasts, keratinocytes from both PARP(-/-) and PARP(+/+) mice express markers of apoptosis in response to sulfur mustard exposure. The effects of PARP on the mode of cell death in different skin cell types may determine the severity of vesication in vivo, and thus have implications for the design of PARP inhibitors to reduce sulfur mustard pathology.
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PMID:PARP determines the mode of cell death in skin fibroblasts, but not keratinocytes, exposed to sulfur mustard. 1188 24

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are neoplasms leading to death in most cases. Patients with Neurofibromatosis type 1 have an increased risk of developing this malignancy. The metabolites of the inactive prodrug Sulindac, Sulindac Sulfide and Sulindac Sulfone (Exisulind) are new chemopreventive agents that show promising results in the treatment of different cancer types. In this study we examined the antineoplastic effect of these compounds on primary cells derived from two MPNSTs of Neurofibromatosis type 1 patients. RESULTS: Exisulind and Sulindac Sulfide showed a dramatic time- and dose-dependent growth inhibitory effect with IC50-values of 120 microM and 63 microM, respectively. The decrease in viability of the tested cells correlated with induction of apoptosis. Treatment with 500 microM Exisulind and 125 microM Sulindac Sulfide for a period of 2 days increased the rate of apoptosis 21-27-fold compared to untreated cells. Reduced expression of RAS-GTP and phosphorylated ERK1/2 was detected in treated MPNST cells. Moreover, elevated levels of phosphorylated SAPK/JNK were found after drug treatment, and low activation of cleaved caspase-3 was seen. CONCLUSIONS: Our results suggest that this class of compounds may be of therapeutic benefit for Neurofibromatosis type 1 patients with MPNST.
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PMID:Sulindac derivatives inhibit cell growth and induce apoptosis in primary cells from malignant peripheral nerve sheath tumors of NF1-patients. 1514 81

The endogenous production of hydrogen sulfide (H2S) and its physiological functions, including membrane hyperpolarization and smooth muscle cell relaxation, position this gas well in the family of gasotransmitters together with nitric oxide (NO) and carbon monoxide (CO). In this study, we demonstrate that H2S at physiologically relevant concentrations induced apoptosis of human aorta smooth muscle cells (HASMCs). Exposure of HASMCs to H2S did not induce necrosis as verified with Trypan blue exclusion and LDH release analysis. After inhibiting endogenous H2S production, exogenous H2S induced much more significant apoptosis, which was not altered by the presence of albumin or glutathione. H2S treatment increased the activities of ERK and p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal kinase activity. Suppression of extracellular signal-regulated kinase (ERK) activity, but not of p38 activity, inhibited the H2S-induced apoptosis of HASMCs. The activation of ERK by H2S in HASMCs was accompanied by increased caspase-3 activity. Inhibition of caspase-3 by AC-DEVD-CHO attenuated the H2S-induced cell apoptosis. Inhibition of ERK by U0126 decreased caspase-3 activity, whereas AC-DEVD-CHO did not alter ERK activity. In conclusion, exogenous H2S induces apoptosis of HASMCs, which is significantly affected by the endogenous H2S level. Of the three investigated MAPKs, only ERK played an active role in mediating H2S-induced apoptosis of HASMCs by activating caspase-3. These findings may help reveal novel mechanisms for many diseases linked to H2S-related abnormal cellular proliferation and apoptosis.
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PMID:Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of mitogen-activated protein kinases and caspase-3. 1537 30

Sulfur mustard [SM; bis-(2-chloroethyl) sulfide], which causes skin blistering or vesication [(1991). Histo- and cytopathology of acute epithelial lesions. In: Papirmeister, B., Feister, A. J., Robinson, S. I., Ford, R. D., eds. Medical Defense Against Mustard Gas: Toxic Mechanisms and Pharmacological Implications. Boca Raton: CRC Press, pp. 43-78.], is a chemical warfare agent as well as a potential terrorism agent. SM-induced skin blistering is believed to be due to epidermal-dermal detachment as a result of epidermal basal cell death via apoptosis and/or necrosis. Regarding the role of apoptosis in SM pathology in animal skin, the results obtained in several laboratories, including ours, suggest the following: 1) cell death due to SM begins via apoptosis that proceeds to necrosis via an apoptotic-necrotic continuum and 2) inhibiting apoptosis decreases SM-induced microvesication in vivo. To study the mechanisms of SM-induced apoptosis and its prevention in vitro, we have established a convenient fluorometric apoptosis assay using monolayer human epidermal keratinocytes (HEK) adaptable for multiwell plates (24-, 96-, or 384-well) and high-throughput applications. This assay allows replication and multiple types of experimental manipulation in sister cultures so that the apoptotic mechanisms and the effects of test compounds can be compared statistically. SM affects diverse cellular mechanisms, including endoplasmic reticulum (ER) Ca2+ homeostasis, mitochondrial functions, energy metabolism, and death receptors, each of which can independently trigger apoptosis. However, the biochemical pathway in any of these apoptotic mechanisms is characterized by a pathway-specific sequence of caspases, among which caspase-3 is a key member. Therefore, we exposed 80-90% confluent HEK cultures to SM and monitored apoptosis by measuring the fluorescence generated due to hydrolysis of a fluorogenic caspase-3 substrate (acetyl- or benzyl oxycarbonyl-Asp-Glu-Val-Asp-fluorochrome, also designated as AC-or Z-DEVD- fluorochrome) added to the assay medium. Fluorescence was measured using a plate reader. We used two types of substrates, one (Sigma-Aldrich, CASP-3-F) required cell disruption and the other (Beckman-Coulter CellProbe HT Caspase-3/7 Whole Cell Assay Kit) was cell permeable. The latter substrate was useful in experiments such as determining the time-course of apoptosis immediately following SM exposure without disruption (e.g., due to cell processing). In SM-exposed HEK, fluorescence generated from the fluorogenic caspase-3 substrate hydrolysis increased in a time (0-24 h) and concentration (0.05, 0.1, 0.15, 0.2, 0.3, 0.5 mM) dependent manner. SM caused maximum fluorescence at about 0.5 mM. However, at 2 mM SM, fluorescence decreased compared with 0.5 mM, which remains to be explained. Following 0.3 mM SM exposure, which is considered to be the in vitro equivalent of a vesicating dose in vivo (Smith, W. J., Sanders, K. M., Ruddle, S. E., Gross, C. L. (1993). Cytometric analysis of DNA changes induced by sulfur mustard. J. Toxicol.-Cut. Ocular Toxicol. 12(4):337-347.), a small fluorescence increase was observed at 6 to 8 h, which was markedly higher at 12 h. At 24 h, all SM concentrations increased fluorescence. Fluorescence increase due to SM was prevented 100% by a caspase-3-specific peptide inhibitor AC-DEVD-CHO (acetyl-Asp-Glu-Val-Asp-aldehyde, 0.1 mM), but less effectively by a general caspase inhibitor Z-VAD-FMK (benzyl oxycarbonyl-Val-Ala-Asp-fluoromethylketone, 0.01 mM), indicating that the fluorescence increase was due to caspase-3-mediated apoptosis. These results suggest potential applications of this method to study apoptosis mechanisms involving caspase-3 substrates and possibly those involving other caspase substrates.
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PMID:A convenient fluorometric method to study sulfur mustard-induced apoptosis in human epidermal keratinocytes monolayer microplate culture. 1572 39

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). The CSE/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we overexpressed CSE in human aorta smooth muscle cells (HASMCs) using a recombinant defective adenovirus containing CSE gene (Ad-CSE). Infection of HASMCs with Ad-CSE resulted in a significant increase in the expression of CSE protein and H2S production. Ad-CSE transfection inhibited cell growth and stimulated apoptosis, as evidenced by cell viability assay, Hoechst 33258 staining, TUNEL, and caspase 3 activation. CSE-mediated apoptosis was associated with an increased ERK and p38 MAPK activation, up-regulation of p21(Cip/WAK-1), and down-regulation of cyclin D1 expression. After inhibiting endogenous background CSE gene expression, direct administration of H2S at 100 microM induced apoptosis of HASMCs. The other two endproducts of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, failed to do so. These results demonstrate that overexpression of CSE stimulates SMC apoptosis due to an increased endogenous production of H2S. Adenovirus-mediated transfer of CSE gene may provide a novel therapeutic approach in treating vascular diseases linked to abnormal cellular proliferation and vascular remodeling.
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PMID:Pro-apoptotic effect of endogenous H2S on human aorta smooth muscle cells. 1650 67

Sulfur mustard (SM) causes blisters in the skin through a series of cellular changes that we are beginning to identify. We earlier demonstrated that SM toxicity is the result of induction of both death receptor and mitochondrial pathways of apoptosis in human keratinocytes (KC). Because of its importance in apoptosis in the skin, we tested whether calmodulin (CaM) mediates the mitochondrial apoptotic pathway induced by SM. Of the three human CaM genes, the predominant form expressed in KC was CaM1. RT-PCR and immunoblot analysis revealed upregulation of CaM expression following SM treatment. To delineate the potential role of CaM1 in the regulation of SM-induced apoptosis, retroviral vectors expressing CaM1 RNA in the antisense (AS) orientation were used to transduce and derive stable CaM1 AS cells, which were then exposed to SM and subjected to immunoblot analysis for expression of apoptotic markers. Proteolytic activation of executioner caspases-3, -6, -7, and the upstream caspase-9, as well as caspase-mediated PARP cleavage were markedly inhibited by CaM1 AS expression. CaM1 AS depletion attenuated SM-induced, but not Fas-induced, proteolytic processing and activation of caspase-3. Whereas control KC exhibited a marked increase in apoptotic nuclear fragmentation after SM, CaM1 AS cells exhibited normal nuclear morphology up to 48h after SM, indicating that suppression of apoptosis in CaM1 AS cells increases survival and does not shift to a necrotic death. CaM has been shown to activate the phosphatase calcineurin, which can induce apoptosis by Bad dephosphorylation. Interestingly, whereas SM-treated CaM1-depleted KC expressed the phosphorylated non-apoptotic sequestered form of Bad, Bad was present in the hypophosphorylated apoptotic form in SM-exposed control KC. To determine if pharmacological CaM inhibitors could attenuate SM-induced apoptosis via Bad dephosphorylation, KC were pretreated with the CaM-specific antagonist W-13 or its less active structural analogue W-12. Following SM exposure, KC exhibited Bad dephosphorylation, which was inhibited in the presence of W-13, but not with W-12. Consequently, W-13 but not W-12 markedly suppressed SM-induced proteolytic processing and activation of caspase-3, as well as apoptotic nuclear fragmentation. Finally, while the CaM antagonist W-13 and the calcineurin inhibitor cyclosporin A attenuated SM-induced caspase-3 activation, inhibitors for CaM-dependent protein kinase II (KN62 and KN93) did not. These results indicate that CaM, calcineurin, and Bad also play a role in SM-induced apoptosis, and may therefore be targets for therapeutic intervention to reduce SM injury.
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PMID:Calmodulin mediates sulfur mustard toxicity in human keratinocytes. 1693 4

Sulfur is commonly used in Asia as an herbal medicine to treat inflammation and cancer, and potent chemopreventive effects have been demonstrated in various in vivo and in vitro models for sulfur-containing compounds found in naturally occurring products. Here, we report the growth inhibitory and apoptosis-related effects of a newly developed highly purified sulfur (HPS) on immortalized human oral keratinocytes (IHOKs) and on oral cancer cells representing two stages of oral cancer (HN4, HN12) based on a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Western blotting, cell cycle analysis, and nuclear staining. The purity of the sulfur preparation was verified by high-performance liquid chromatography. HPS inhibited the proliferation of immortalized and malignant oral keratinocytes in a dose- and time-dependent manner. FITC-annexin V staining, DNA fragmentation testing, and Hoechst 33258 staining revealed that HPS inhibited cell growth via apoptosis. HPS increased the sub-G1 cell cycle fraction, with decreased expression of cyclins D1, D2, and E and their activating partners cdk2, cdk4, and cdk6, and a concomitant induction of p53 and p21/WAF1. Furthermore, HPS treatment increased the cytosolic level of cytochrome c and resulted in caspase-3 activation; this effect was correlated with Bax up-regulation and Bcl-2 down-regulation. Thus, these data suggest that HPS is a potential candidate for anti-cancer therapy in oral cancer.
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PMID:Anti-cancer activity of highly purified sulfur in immortalized and malignant human oral keratinocytes. 1792 Feb 32

Sulfur mustard (SM) is a bifunctional alkylating agent. Its primary toxic consequence is severe skin damage with blisters, occurring after skin contact. These vesicant properties of SM have been linked to cell death of proliferating keratinocytes in the basal layer of the skin. Catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) has been demonstrated to be a major event in response to high levels of DNA damage, and PARP-1 activation may be part of apoptotic signaling. In other contexts, overstimulation of PARP-1 triggers necrotic cell death because of rapid consumption of its substrate, beta-nicotinamide adenine dinucleotide (NAD+) and the consequent depletion of ATP. These findings prompted us to evaluate whether SM induces apoptosis in keratinocytes like HaCaT cells and to determine whether blocking of PARP enzyme activity with 3-aminobenzamide (3AB) can influence the mode of cell death. HaCaT cells were exposed to SM (10-1,000 microM; 30 min) and then cultivated in SM-free medium with or without 3AB for up to 48 h. This treatment resulted in a time and SM dose-dependent increase of apoptotic cell death characterized by PARP-1 cleavage and DNA fragmentation during the experimental period. After just 45 min of exposure to 1 mM SM, we observed a significant increase in PARP-1 activity in HaCaT cells. About 6 h after exposure, intracellular ATP levels were diminished by 22%, which seemed to be completely prevented by the addition of 3AB directly after exposure. However, 18 h later, this 3AB effect on the SM concentration-dependent loss of ATP was no longer detectable. Interestingly, the effect of SM on total cell viability was not changed by 3AB. However, the mode of cell death was influenced by 3AB exhibiting an increase of apoptotic cells and a concomitant decrease of necrotic HaCaT cells during the first 24 h after SM exposure. Our results indicate that SM concentrations of 1 mM or higher induce a prominent PARP activation leading to ATP depletion and necrosis. In contrast, lower concentrations of SM cause minor PARP activation and, especially, PARP-1 cleavage by caspase 3 without ATP depletion. Because ATP is required for apoptosis, we suggest that ATP acts as an early molecular switch from apoptotic to necrotic modes of SM-induced cell death, at least at high concentrations (> or =1 mM). Thus, the observed early proapoptotic effect of 3AB at lower SM concentrations may point to the influence of ATP-independent cell-death regulating mechanisms.
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PMID:Inhibition of poly(ADP-ribose) polymerase (PARP) influences the mode of sulfur mustard (SM)-induced cell death in HaCaT cells. 1804 40

Recent global events have focused attention on the potential threat of international and domestic chemical terrorism, as well as the possibility of chemical warfare proliferation. Sulphur mustard (SM) is one of the potent chemical warfare agents (CWA), which initiates a cascade of events that converge on the redox mechanisms common to brain injury. The present study was designed to examine the effects of chronic SM exposure on neurobehavioral impairments, mitochondrial oxidative stress in male Swiss Albino mice and its role in inducing apoptotic neuronal cell death. The animals were divided into four groups (control, low, medium and high dose) of 5 animals each. Exposure to SM was given percutaneously daily for 12 weeks. The results demonstrated impairment in neurobehavioral indices viz. rota rod, passive avoidance and water maze tests in a dose dependent manner. There was a significant increase in lipid peroxidation and protein carbonyl content whereas, decrease in the activity of manganese superoxide dismutase (MnSOD), glutathione reductase and glutathione peroxidase suggesting impaired antioxidant defense system. Immunoblotting of cytochrome c, Bcl-2, Bax and activation of caspase-3 suggest induction of apoptosis in a dose dependent manner. Finally, increased p53 expression suggests that it may target the mitochondrial pathway for inducing apoptosis in response to DNA damage signals. In conclusion, chronic SM exposure may have the potential to generate oxidative stress which may trigger the release of cytochrome c as well as caspase-3 activation in neurons leading to cell death by apoptosis in a dose dependent manner which may in the end be responsible for the disruption of cognitive functions in mice.
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PMID:Neurobehavioral impairments, generation of oxidative stress and release of pro-apoptotic factors after chronic exposure to sulphur mustard in mouse brain. 1956 Apr 81


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