Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.22.56 (caspase-3)
 35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rottlerin is widely used as a protein kinase C-delta inhibitor. Recently, several reports have shown the possible apoptosis-inducing effect of rottlerin in some cancer cell lines. Here we report that rottlerin induces not only apoptosis but also autophagy via a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells. Rottlerin treatment induced a dose- and time-dependent inhibition of cell growth, and cytoplasmic vacuolations were markedly shown. These vacuoles were identified as acidic autolysosomes by electron microscopy, acidic vesicular organelle (AVO) staining and transfection of green fluorescent protein-LC3. The LC3-II protein level also increased after treatment with rottlerin. Prolonged exposure to rottlerin eventually caused apoptosis via loss of mitochondrial membrane potential and translocation of AIF from mitochondria to the nucleus. However, the activities of caspase-3, -8 and -9 were not changed, and PARP did not show signs of cleavage. Interestingly, the pretreatment of cells with a specific inhibitor of autophagy (3-methyladenine) accelerated rottlerin-induced apoptosis as revealed by an analysis of the subdiploid fraction and TUNEL assay. Nevertheless, the knockdown of PKC-delta by RNA interference neither affected cell growth nor acidic vacuole formation. Similarly, rottlerin-induced cell death was not prevented by PKC-delta overexpression. Taken together, these findings suggest that rottlerin induces early autophagy and late apoptosis in a PKC-delta-independent manner, and the rottlerin-induced early autophagy may act as a survival mechanism against late apoptosis in HT1080 human fibrosarcoma cells.
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PMID:Rottlerin induces autophagy and apoptotic cell death through a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells: the protective role of autophagy in apoptosis. 1842 13

p-Anilinoaniline (pAA) is an aromatic amine that is widely used in hair dying applications. It is also a metabolite of metanil yellow, an azo dye that is commonly used as a food coloring agent. Concentrations of pAA between 10 and 25 microM were cytostatic to cultures of the normal human mammary epithelia cell line MCF10A. Concentrations >or=50 microM were cytotoxic. Cytostatic concentrations induced transient G(1) and S cell cycle phase arrests; whereas cytotoxic concentrations induced protracted arrests. Cytotoxic concentrations of pAA caused DNA damage, as monitored by the alkaline single-cell gel electrophoresis (Comet) assay, and morphological changes consistent with cells undergoing apoptosis and/or autophagy. Enzymatic and western blot analyses, and binding analyses of fluorescent labeled VAD-FMK, suggested that caspase family members were activated by pAA. Western blot analyses documented the conversion of LC3-I to LC3-II, a post-translational modification involved in the development of the autophagosome. Suppression of autophagosome formation, via knockdown of ATG7 with shRNA, prevented pAA-induced vacuolization, enhanced the activation of pro-caspase-3, and increased susceptibility of ATG7-deficient cells to the cytostatic and cytotoxic activities of markedly lower concentrations of pAA. Cells stably transfected with a nonsense shRNA behaved like parental MCF10A cells. Collectively, these data suggest that MCF10A cultures undergo autophagy as a pro-survival response to concentrations of pAA sufficient to induce DNA damage.
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PMID:Suppression of autophagy enhances the cytotoxicity of the DNA-damaging aromatic amine p-anilinoaniline. 1865 2

Areca nut (AN) is recognized as a human carcinogen; however, few studies of the cytotoxic effects of AN ingredients on cells have been reported. In Taiwan, AN, lime and inflorescence of Piper betle are the common components of betel quid (BQ). We recently noticed that extract of AN (ANE), but not those of lime and inflorescence of Piper betle, induces rounding cell morphology and nuclear shrinkage in different types of carcinoma cells. In this study, the rounding cell activity was first traced to the partially purified >or=10 kDa fraction (ANE >or= 10 K) and subsequently to the 30-100 kDa fraction (ANE 30-100 K). ANE and ANE >or=10 K stimulated nuclear shrinkage (P < 0.001 in both cases) and the clearance of the cytoplasm. ANE, ANE >or= 10 K, and ANE 30-100 K induced the cleavage of LC3-I (P < 0.05, 0.01, and 0.05, respectively) and the emergence of autophagic vacuoles (AVs) and acidic vesicles. On the other hand, arecoline (Are, the major alkaloid of AN) triggered caspase-3 activation, peri-nuclear chromatin condensation, and micronucleation. Meanwhile, ANE 30-100 K, but not Are, inhibited the phosphorylation of the mammalian target of rapamycin (mTOR)-Ser(2448). In conclusion, this study demonstrates that different AN ingredients exerting differential impact on mTOR-Ser(2448) phosphorylation are capable of triggering apoptosis and autophagy.
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PMID:Arecoline and the 30-100 kDa fraction of areca nut extract differentially regulate mTOR and respectively induce apoptosis and autophagy: a pilot study. 1866 45

Cigarette smoke-induced cell death contributes to the pathogenesis of chronic obstructive pulmonary disease, though the relative roles of apoptosis and autophagy remain unclear. The inducible stress protein heme oxygenase-1 (HO-1) confers cytoprotection against oxidative stress. We examined the relationships between these processes in human bronchial epithelial cells (Beas-2b) exposed to cigarette smoke extract (CSE). CSE induced morphological and biochemical markers of autophagy in Beas-2b cells and induced autophagosome formation as evidenced by formation of GFP-LC3 puncta and electron microscopic analysis. Furthermore, CSE increased the processing of microtubule-associated protein-1 light chain-3 (LC3B-I) to LC3B-II, within 1 hr of exposure. Increased LC3B-II was associated with increased autophagy, since inhibitors of lysosomal proteases and of autophagosome-lysosome fusion further increased LC3B-II levels during CSE exposure. CSE concurrently induced extrinsic apoptosis in Beas-2b cells involving early activation of death-inducing-signaling-complex (DISC) formation and downstream activation of caspases (-8,-9,-3). The induction of extrinsic apoptosis by CSE was dependent in part on autophagic proteins. Reduction of Beclin 1 levels with beclin 1 siRNA inhibited DISC formation and caspase-3/8 activation in response to CSE. LC3B siRNA also inhibited caspase-3/8 activation. The stress protein HO-1 protected against CSE-induced cell death by concurrently downregulating apoptosis and autophagy-related signaling. Adenoviral mediated expression of HO-1 inhibited DISC formation and caspase-3/9 activation in CSE-treated epithelial cells, diminished the expression of Beclin 1, and partially inhibited the processing of LC3B-I to LC3B-II. Conversely, transfection of Beas-2b with ho-1 siRNA augmented CSE-induced DISC formation and increased intracellular reactive oxygen species formation. HO-1 expression augmented CSE-induced phosphorylation of NFkappaB p65 in Beas-2b cells. Consistently, expression of IkappaB, the inhibitor of NFkappaB, increased CSE-induced DISC formation. LC3B siRNA also enhanced p65 phosphorylation. In fibroblasts from beclin 1 heterozygous knockout mice, p65 phosphorylation was dramatically upregulated, while CSE-induced DISC formation was inhibited, consistent with an anti-apoptotic role for NFkappaB and a pro-apoptotic role for Beclin 1. These studies demonstrated an interdependence of autophagic and apoptogenic signaling in CSE-induced cell death, and their coordinated downregulation by HO-1. An understanding of the regulation of cell death pathways during smoke exposure may provide therapeutic strategies in smoke-related illness.
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PMID:Autophagic proteins regulate cigarette smoke-induced apoptosis: protective role of heme oxygenase-1. 1922 62

The present investigation was undertaken to measure the relative abilities of pro-death versus pro-survival proteases in degrading each other and to determine how this might influence cellular susceptibility to death. For this, we first carried out in vitro experiments in which recombinant pro-death proteases (caspase-3 or cathepsin D) were incubated with the pro-survival protease (cathepsin L) in their respective optimal conditions and determined the effects of these reactions on enzyme integrity and activity. The results indicated that cathepsin L was able to degrade cathepsin D, which in turn cleaves caspase-3, however the later enzyme was unable to degrade any of the cathepsins. The consequences of this proteolytic sequence on cellular ability to undergo apoptosis or other types of cell death were studied in cells subjected to treatment with a specific inhibitor of cathepsin L or the corresponding siRNA. Both treatments resulted in suppression of cellular proliferation and the induction of a cell death with no detectable caspase-3 activation or DNA fragmentation, however, it was associated with increased accumulation of cathepsin D, cellular vaculolization, expression of the mannose-6-phosphate receptor, and the autophagy marker LC3-II, all of which are believed to be associated with autophagy. Genetic manipulations leading either to the gain or loss of cathepsin D expression implicated this enzyme as a key player in the switch from apoptosis to autophagy. Overall, these findings suggest that a hierarchy between pro-survival and pro-death proteases may have important consequences on cell fate.
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PMID:Role of the proteolytic hierarchy between cathepsin L, cathepsin D and caspase-3 in regulation of cellular susceptibility to apoptosis and autophagy. 1877 51

Although capsaicin, a pungent component of red pepper, is known to induce apoptosis in several types of cancer cells, the mechanisms underlying capsaicin-induced cytotoxicity are unclear. Here, we showed that dihydrocapsaicin (DHC), an analog of capsaicin, is a potential inducer of autophagy. DHC was more cytotoxic than capsaicin in HCT116, MCF-7 and WI38 cell lines. Capsaicin and DHC did not affect the sub-G(1) apoptotic peak, but induced G(0)/G(1) arrest in HCT116 and MCF-7 cells. DHC caused the artificial autophagosome marker GFP-LC3 to redistribute and upregulated expression of autophagy-related proteins. Blocking of autophagy by 3-methyladenine (3MA) as well as siRNA Atg5 induced a high level of caspase-3 activation. Although pretreatment with zVAD completely inhibited caspase-3 activation by 3MA, it did not prevent cell death. DHC-induced autophagy was enhanced by zVAD pretreatment, as shown by increased accumulation of LC3-II protein. DHC attenuated basal ROS levels through catalase induction; this effect was enhanced by antioxidants, which increased both LC3-II expression and caspase-3 activation. The catalase inhibitor 3-amino-1,2,4-triazole (3AT) abrogated DHC-induced expression of LC3-II, overexpression of the catalase gene increased expression of LC3-II protein, and knockdown decreased it. Additionally, DHC-induced autophagy was independent of p53 status. Collectively, DHC activates autophagy in a p53-independent manner and that may contribute to cytotoxicity of DHC.
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PMID:Dihydrocapsaicin (DHC), a saturated structural analog of capsaicin, induces autophagy in human cancer cells in a catalase-regulated manner. 1881 25

The flank organ of the Syrian hamster, which shows a biodynamic response to androgen stimulation, is considered a good model for studying the androgen effect on sebaceous gland and hair. This organ is susceptible to programmed cell death (PCD), a prominent feature associated with sexual organ adjustment. We have recently shown the first direct evidence of a relationship between autophagy and morphological changes in androgen-dependent organs. In this work, animals were stabulated (housed) for two months under control conditions and, after sacrifice, autophagy markers, Beclin 1 and LC3-II, were evaluated. The results revealed autophagic processes in the flank organ of both sexes, especially in females, and no detectable caspase-3 activity. Therefore, these data indicated that macroautophagy, and not apoptosis, was the main mechanism by which the flank organ responds to androgen. Here, we present additional data on the flank organ, showing that the intensity of autophagy between genders depends on the length of stabulation. These data could indicate a role for autophagy in response to behavioral influences. A possible explanation and its implication in Syrian hamster social defeat are discussed in this addendum.
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PMID:Potential role of autophagy in behavioral changes of the flank organ. 1910

Upon nutrient deprivation during culture, recombinant Chinese hamster ovary (rCHO) cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. To investigate the effect of Bcl-x(L) overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)-producing rCHO cell line with regulated Bcl-x(L) overexpression (EPO-off-Bcl-x(L)) was established using the Tet-off system. The expression level of Bcl-x(L) in EPO-off-Bcl-x(L) cells was tightly regulated by doxycycline in a dose-dependent manner. Bcl-x(L) overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl-x(L) overexpression suppressed apoptosis by inhibiting the activation of caspase-3 and -7. Simultaneously, Bcl-x(L) overexpression also delayed autophagy, characterized by LC3-II accumulation. Immunoprecipitation analysis with a Flag-tagged Bcl-x(L) revealed that Bcl-x(L) interacts with Bax and Bak, essential mediators of caspase-dependent apoptosis, as well as with Beclin-1, an essential mediator of autophagy, and may inhibit their pro-cell death function. Taken together, it was found that Bcl-x(L) overexpression inhibits both apoptosis and autophagy in rCHO cell culture.
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PMID:Effect of Bcl-xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient-deprived condition. 1926 76

Changes in the structure and function of aging non-locomotor muscles remains understudied, despite their importance for daily living. Extraocular muscles (EOMs) have a high incidence of age-related mitochondrial defects possibly because of the metabolic stress resulting from their fast and constant activity. Apoptosis and autophagy (type I and II cell death, respectively) are linked to defects in mitochondrial function and contribute to sarcopenia in hind limb muscles. Therefore, we hypothesized that apoptosis and autophagy are altered with age in the EOMs. Muscles from 6-, 18-, and 30-month-old male Fisher 344-Brown Norway rats were used to investigate type I cell death, caspase-3, -8, -9, and -12 activity, and type II cell death. Apoptosis, as measured by TUNEL positive nuclei, and mono- and oligo-nucleosomal content, did not change with age. Similarly, caspase-3, -8, -9, and -12 activity was not affected by aging. By contrast, autophagy, as estimated by gene expression of Atg5 and Atg7, and protein abundance of LC3 was lower in EOMs of aged rats. Based on these data, we suggest that the decrease in autophagy with age leads to the accumulation of damaged organelles, particularly mitochondria, which results in the decrease in function observed in EOM with age.
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PMID:Age-related changes of cell death pathways in rat extraocular muscle. 1934 88

Dopamine at 100-500 microM has toxic effects on human SH-SY5Y neuroblastoma cells, manifested as apoptotic cell loss and strong autophagy. The molecular mechanisms and types of dopamine-induced cell death are not yet well known. Their identification is important in the study of neurodegenerative diseases that specifically involve dopaminergic neurons. We looked for changes in expression and content of proteins involved in apoptosis and autophagy after dopamine treatment. All the changes found were prevented by avoiding dopamine oxidation with N-acetylcysteine, indicating a key role for the products of dopamine oxidation in dopamine toxicity. As early as 1-2h after treatment we found an increase in hypoxia-inducible factor-1alpha (HIF-1alpha) and an accumulation of ubiquitinated proteins. Proteins regulated by HIF-1alpha and involved in apoptosis and/or autophagy, such as p53, Puma and Bnip3, were subsequently increased. However, apoptotic parameters (caspase-3, caspase-7, PARP) were only activated after 12h of 500muM dopamine treatment. Autophagy, monitored by the LC3-II increase after LC3-I linkage to autophagic vacuoles, was evident after 6h of treatment with both 100 and 500 microM dopamine. The mTOR pathway was inhibited by dopamine, probably due to the intracellular redox changes and energy depletion leading to AMPK activation. However, this mechanism is not sufficient to explain the high LC3-II activation caused by dopamine: the LC3-II increase was not reversed by IGF-1, which prevented this effect when caused by the mTOR inhibitor rapamycin. Our results suggest that the aggregation of ubiquitinated non-degraded proteins may be the main cause of LC3-II activation and autophagy. As we have reported previously, cytosolic dopamine may cause damage by autophagy in neuroblastoma cells (and presumably in dopaminergic neurons), which develops to apoptosis and leads to cell degeneration.
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PMID:Effects of dopamine on LC3-II activation as a marker of autophagy in a neuroblastoma cell model. 1941 Jun 1


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