• Molecules and Cells
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• v.26 no.2
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• pp.152-157
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• 2008

Caspases play critical roles in the execution of apoptosis. Caspase-3 and caspase-7 are closely related in sequence as well as in substrate specificity. The two caspases have overlapping substrate specificities with special preference for the DEVD motif. However, they are targeted to different subcellular locations during apoptosis, implying the existence of substrates specific for one or other caspase. To identify new caspase-7 substrates, we digested cell lysates obtained from the caspase-3-deficient MCF-7 cell line with purified recombinant caspase-7, and analyzed spots that disappeared or decreased by 2-DE (we refer to this as the caspase-7 degradome). Several proteins with various cellular functions underwent caspase-7-dependent proteolysis. The substrates of capase-7 identified by the degradomic approach were rather different from those of caspase-3 (Proteomics, 4, 3429-3435, 2004). Among the candidate substrates, we confirmed that Valosin-containing protein (VCP) was cleaved by both capspase-7 and caspase-3 in vitro and during apoptosis. Cleavage occurred at both $DELD^{307}$ and $DELD^{580}$. The degradomic study yielded several candidate caspase-7 substrates and their further analysis should provide valuables clues to the functions of caspase-7 during apoptosis.

• Journal of Life Science
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• v.27 no.2
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• pp.164-171
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• 2017

Our previous study suggested that S-allylcysteine (SAC) inhibits the proliferation of the human cervical cancer cell line, HeLa, at least in part through the induction of apoptosis and cell cycle arrest. To further analyze the specific molecular mechanism(s) by which SAC mediates its antiproliferative effects, this study examined the role of SAC in regulating the protein expression of initiator caspase (caspase-9), effector caspases (caspase-3 and caspase-7), and poly-ADP-ribose polymerase (PARP) in HeLa. Western blot analysis showed that when cells were treated with 50 mM SAC for 48 hr, the expression of procaspase-3, -7, and -9 and PARP was reduced by 94%, 38%, 95%, and 64%, respectively, as compared to the untreated control. In contrast, the expression of caspase-3, -7, and -9 and cleaved-PARP was markedly increased by SAC treatment. The SAC-mediated changes in the expression of these proteins were correlated with the concomitant inhibition of cellular proliferation by SAC. The cell proliferation assay showed that HeLa treatment with more than 20 mM SAC for 6-48 hr resulted in both concentration- and time-dependent inhibition of cellular proliferation. These results indicate that the SAC-induced antiproliferative effect in HeLa may be mediated at least in part through the activation of caspase-9, followed by the activation of caspase-3 and caspase-7 as well as the inactivation of PARP, thus leading to cellular apoptosis.

• Toxicological Research
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• v.17 no.3
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• pp.215-223
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• 2001

Cadmium is an ubiquitous toxic metal and chronic exposure to cadmium results in the accumulation of cadmium in the liver and kidneys. In contrast, acute exposure leads to damage mainly in the liver. Apoptosis induced by cadmium has been shown in many tissues in vivo and in cultured cells in vitro. However, the molecular mechanism of cadmium-induced apoptosis is not clear in hepatocyte. To investigate the induction of apoptosis in the hepatocyte, we used mouse hepatoma cell line, Hepalclc7 cells, and analysed the molecules that involved in cadmium-induced apoptosis. Cadmium induced the genomic DNA fragmentation, PARP cleavage, and activation of caspase-3 like protease. Caspase-9 cysteine protease was activated in a time-dependent manner but caspase-8 cysteine protease was not significantly activated in cadmium-treated Hepalclc7 cells. Cadmium also induced mitochondrial dysfunction including cytochrome c release from mitochondria, change oj mitochondrial membrane potential tranition, and tranlocation of Bax Protein into mitochondria. These results strong1y indicated that the signal Pathway of apoptotic death in cadmium-treated Hepalclc7 cells is modulated by caspase cascade via mitochondria.

• Molecules and Cells
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• v.38 no.4
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• pp.349-355
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• 2015

EphA7 has been implicated in the regulation of apoptotic cell death in neural epithelial cells. In this report, we provide evidence that EphA7 interacts with caspase-8 to induce apoptotic cell signaling. First, a pull-down assay using biotinylated ephrinA5-Fc showed that EphA7 co-precipitated with wild type caspase-8 or catalytically inactive caspase-8 mutant. Second, co-transfection of EphA7 with caspase-8 significantly increased the number of cleaved caspase-3 positive apoptotic cells under an experimental condition where transfection of EphA7 or caspase-8 alone did not affect cell viability or apoptosis. EphA4 also had a causative role in inducing apoptotic cell death with caspase-8, whereas EphA8 did not. Third, caspase-8 catalytic activity was essential for the apoptotic signaling cascade, whereas tyrosine kinase activity of the EphA4 receptor was not. Interestingly, we found that kinase-inactive EphA4 was well co-localized at the plasma membrane with catalytically inactive caspase-8, suggesting that an interaction between these mutant proteins was more stable. Finally, we observed that the extracellular region of the EphA7 receptor was critical for interacting with caspase-8, whereas the cytoplasmic region of EphA7 was not. Therefore, we propose that Eph receptors physically associate with a transmembrane protein to form an apoptotic signaling complex and that this unidentified receptor-like protein acts as a biochemical linker between the Eph receptor and caspase-8.

• BMB Reports
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• v.50 no.10
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• pp.510-515
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• 2017

Triglyceride (TG) accumulation causes macrophage cell death, which affects the development of atherosclerosis. Here, we examined whether caspase-2 is implicated in TG-induced macrophage cell death. We found that caspase-2 activity is increased in TG-treated THP-1 macrophages, and that inhibition of caspase-2 activity drastically inhibits TG-induced cell death. We previously reported that TG-induced macrophage cell death is triggered by caspase-1, and thus investigated the relationship between caspase-2 and caspase-1 in TG-induced macrophage cell death. Inhibition of caspase-2 activity decreased caspase-1 activity in TG-treated macrophages. However, caspase-1 inhibition did not affect caspase-2 activity, suggesting that caspase-2 is upstream of caspase-1. Furthermore, we found that TG induces activation of caspase-3, -7, -8, and -9, as well as cleavage of PARP. Inhibition of caspase-2 and -1 decreased TG-induced caspase-3, -7, -8, and -9 activation and PARP cleavage. Taken together, these results suggest that TG-induced macrophage cell death is mediated via the caspase-2/caspase-1/apoptotic caspases/PARP pathways.

• Molecules and Cells
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• v.24 no.2
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• pp.261-267
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• 2007

Apoptotic signals are typically accompanied by activation of aspartate-specific cysteine proteases called caspases, and caspase-3 and -7 play crucial roles in the execution of apoptosis. Previously, using the proteomic approach, protein disulfide isomerase (PDI) was found to be a candidate substrate of caspase-7. This abundant 55 kDa protein introduces disulfide bonds into proteins (via its oxidase activity) and catalyzes the rearrangement of incorrect disulfide bonds (via its isomerase activity). PDI is abundant in the ER but is also found in non-ER locations. In this study we demonstrated that PDI is cleaved by caspase-3 and -7 in vitro. In addition, in vivo experiment showed that it is cleaved during etoposide-induced apoptosis in HL-60 cells. Subcellular fractionation showed that PDI was also present in the cytosol. Furthermore, only cytosolic PDI was clearly digested by caspase-3 and -7. It was also confirmed by confocal image analysis that PDI and caspase-7 partially co-localize in both resting and apoptotic MCF-7 cells. Overexpression of cytosolic PDI (ER retention sequence deleted) inhibited cell death after an apoptotic stimulus. These data indicate that cytosolic PDI is a substrate of caspase-3 and -7, and that it has an anti-apoptotic action.

• Biomedical Science Letters
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• v.25 no.1
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• pp.66-74
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• 2019

Hyperlipidemia is defined as conditions of the accumulation of lipids such as free fatty acids (FFA), triglyceride (TG), cholesterol and/or phospholipid in the bloodstream. Hyperlipidemia can cause lipid accumulation in non-adipose tissue, which is lipid-cytotoxic effects in many tissues and mediates cell dysfunction, inflammation or programmed cell death (PCD). TG is considered to be a major cause of atherosclerosis through inflammatory necrosis of vascular endothelial cells. Recently, TG have also been shown to exhibit lipid-cytotoxicity and induce PCD. Therefore, we investigated the effect of TG on the cytotoxic effect of various cell types. When exposed to TG, the cell viability of U937 monocytes and Jurkat T lymphocytes, as well as the cell viability of MCF-7, a non-immune cell, decreased in time- and dose-dependent manner. In U937 cells and Jurkat cells, caspase-9, an intrinsic apoptotic caspase, and caspase-8, an extrinsic apoptotic caspase, were increased by exposure to TG. However, in TG-treated MCF-7 cells, caspase-8 activity increased only without caspase-9 activity. In addition, the reduction of cell viability by TG was recovered when all three cell lines were treated with pan-caspase inhibitor. These results suggest that activation of apoptotic caspases by TG causes lipotoxic effect and decreases cell viability.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.3
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• pp.1499-1506
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• 2016

Background: Crocus sativus and its major constituent crocin are well established to have anti-cancer properties in breast cancer cells (MCF-7). However the role of C. sativus extract (CSE) and crocin on caspase signaling mediated MCF-7 cell death at molecular level is remains unclear. In this study, we tried to unravel role of CSE and crocin on caspase mediated MCF-7 cells death and their in vivo preclinical toxicity profiling and immune stimulatory effect. Materials and Methods: CSE extract was fractionated by HPLC and crocin was isolated and characterized by NMR, IR, and MS. MCF-7 cells were treated with both CSE and crocin and expression of Bcl-2 and Bax was assessed after 24 and 36 hours. Furthermore, caspase 3, caspase 8 and caspase 9 expression was determined by Western blotting after 24 hours of treatment. DNA fragmentation analysis was performed for genotoxicity of CSE and crocin in MCF-7 cells. The in vivo toxicity profile of CSE (300 mg/kg of b.wt) was investigated in normal Swiss albino mice. In addition, peritoneal macrophages were collected from crocin (1, 1.5 and 2 mg/kg body weight) treated mice and analyzed for ex vivo yeast phagocytosis. Results: Immunoblot analysis revealed that there was time dependent decline in anti-apoptotic Bcl-2 with simultaneous upregulation of Bax in CSE and crocin treated MCF-7 cells. Further CSE and crocin treatment downregulated caspase 8 and 9 and cleaved the caspase 3 after 24 hours. Both CSE and crocin elicited considerable DNA damage in MCF-7 cells at each concentration tested. In vivo toxicity profile by histological studies revealed no observable histopathologic differences in the liver, kidney, spleen, lungs and heart in CSE treated and untreated groups. Crocin treatment elicited significant dose and time dependent ex vivo yeast phagocytosis by peritoneal macrophages. Conclusions: Our study delineated involvement of pro-apoptotic and caspase mediated MCF-7 cell death by CSE and crocin at the molecular level accompanied with extensive DNA damage. Further we found that normal swiss albino mice can tolerate the maximum dose of CSE. Crocin enhanced ex vivo macrophage yeast phagocytic ability.

• Journal of Integrative Natural Science
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• v.7 no.1
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• pp.25-38
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• 2014

Amyloid-${\beta}$-peptide ($A{\beta}$) is important in the pathogenesis of Alzheimer's disease (AD). Calpain ($Ca^{2+}$-dependent protease) and caspase-8 (the initiating caspase for the extrinsic, receptor-mediated apoptosis pathway) have been implicated in $AD/A{\beta}$ toxicity. We found that $A{\beta}$ promoted degradation of calpastatin (the specific endogenous calpain inhibitor); calpastatin degradation was prevented by inhibitors of either calpain or caspase-8. The results implied a cross-talk between the two proteases and suggested that one protease was responsible for the activity of the other one. In neuron-like differentiated PC12 cells, calpain promotes active caspase-8 formation from procaspase-8 via the $A{\beta}$ and CD95 pathways, along with degradation of the procaspase-8 processing inhibitor caspase-8 (FLICE)-like inhibitory protein, short isoform (FLIPS). Inhibition of calpain (by pharmacological inhibitors and by overexpression of calpastatin) prevents the cleavage of procaspase-8 to mature, active caspase-8, and inhibits FLIPS degradation in the $A{\beta}$-treated and CD95-triggered cells. Increased cellular Ca2+ per se results in calpain activation but does not lead to caspase-8 activation or FLIPS degradation. The results suggest that procaspase-8 and FLIPS association with cell membrane receptor complexes is required for calpain-induced caspase-8 activation. The results presented here add to the understanding of the roles of calpain, caspase- 8, and CD95 pathway in $AD/A{\beta}$ toxicity. Calpain-promoted activation of caspase-8 may have implications for other types of CD95-induced cell damage, and for nonapoptotic functions of caspase-8. Inhibition of calpain may be useful for modulating certain caspase-8-dependent processes.

• Journal of Integrative Natural Science
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• v.7 no.2
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• pp.87-91
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• 2014

Amyloid beta ($A{\beta}$) peptide has been implicated in the pathogenesis of Alzheimer's disease and has been reported to induce apoptotic death in cell culture. Cysteine Proteases, a family of enzymes known as caspases, mediate cell death in many models of apoptosis. In the present study, we examined the caspase activity and cell death in $A{\beta}$-treated SHSY5Y cells, as an attempt to elucidate the relationship between the type of caspase and $A{\beta}$-induced cell death. $A{\beta}$ at 20 ${\mu}M$ induce activation of caspase-3, 8 and 9 activity, but not the caspase-1. Caspase-3, 8 and 9 were processed by Ab treatment, consistent with the activity assay. Inhibition of the caspase activities by the selective inhibitors, however, marginally affected the cell death induced by $A{\beta}$. Taken together, the results indicate that $A{\beta}$-induced cell death may be independent of caspase activity and rather, the enzymes might be activated as a result of the cell death.