Pro-caspase-3 overexpression sensitises ovarian cancer cells to proteasome inhibitors

The ubiquitin-proteasome pathway plays a critical role in the degradation of several proteins involved in the cell cycle. Dysregulation of this pathway leads to inhibition of cellular proliferation and the induction of apoptosis. Ubiquitination and its downstream consequences have been investigated intensively as targets for the development of drugs for tumour therapy. Here we have investigated the mechanism of apoptosis induced by the proteasome inhibitors MG-132, lactacystin and calpain inhibitor I (ALLN), in the HEK 293 cell line and the ovarian cancer cell lines SKOV3 and OVCAR3. We have found strong caspase-3-like and caspase-6-like activation upon treatment of HEK 293 cells with MG-132. Using a tricistronic expression vector based on a tetracycline-responsive system we generated stable SKOV3 nd OVCAR3 cell lines with inducible expression of pro-caspase-3. Induction of pro-caspase-3 expression in normally growing cells does not induce apoptosis. However, in the presence of the proteasome inhibitors MG-132, lactacystin or ALLN we found that cells overexpressing pro-caspase-3 are rapidly targeted for apoptosis. Our results demonstrate that pro-caspase-3 can sensitise ovarian cancer cells to proteasome inhibitor-induced apoptosis, and a combination of these approaches might be exploited for therapy of ovarian and other cancers.     

Involvement of redox events in caspase activation in zinc-depleted airway epithelial cells

Airway epithelial cells (AEC) contain both pro- and anti-apoptotic factors but little is known about mechanisms regulating apoptosis of these cells. In this study we have examined the localization of pro-caspase-3 and Zn(2+), a cellular regulator of pro-caspase-3, in primary sheep and human AEC. Zn(2+) was concentrated in both cytoplasmic vesicles and ciliary basal bodies, in the vicinity of both pro-caspase-3 and the antioxidant Cu/Zn superoxide dismutase (Cu/Zn SOD). Depletion of intracellular Zn(2+) in sheep AEC, using the membrane permeant Zn(2+) chelator TPEN, increased lipid peroxidation in the apical cell membranes (as assessed by immunofluorescence with anti-hydroxynonenal) as well as increasing activated pro-caspase-3 and apoptosis. There were smaller increases in caspase-2 and -6 but not other caspases. Activation of caspase-3 in TPEN-treated AEC was inhibited strongly by N-acetylcysteine and partially by vitamin C and vitamin E. These findings suggest that cytoplasmic pro-caspase-3 is positioned near the lumenal surface of AEC where it is under the influence of Zn(2+) and other anti-oxidants.     

Iron toxicity in organotypic cultures of hippocampal slices: role of reactive oxygen species

Free iron has been assumed to potentiate oxygen toxicity by generating reactive oxygen species (ROS) via the iron-catalyzed Haber-Weiss reaction, leading to oxidative stress. ROS-mediated iron cytotoxicity may trigger apoptotic cell death. In the present study, we used iron treatment of organotypic cultures of hippocampal slices to study potential mechanisms involved in iron-induced neuronal damage. Exposure of mature hippocampal slices to ferrous sulfate resulted in concentration- and time-dependent cell death. After iron treatment, markers of ROS formation and lipid peroxidation, i.e. intensity of dichlorofluorescein (DCF) fluorescence and levels of thiobarbiturate reactive substances (TBARS), were significantly increased. Levels of cytochrome c were increased while levels of pro-caspase-9 and pro-caspase-3 were decreased in cytosolic fractions of iron-treated hippocampal slice cultures. Treatment of cultured slices with a synthetic catalytic ROS scavenger, EUK-134, provided between 50 and 70% protection against various parameters of cell damage and markers of oxidative stress. In addition, inhibition of caspase-3 activity by Ac-DEVDcho partially protected cells from iron toxicity. The combination of EUK-134 and Ac-DEVDcho resulted in an almost complete blockade of iron-induced damage. These results indicate that iron elicits cellular damage predominantly by oxidative stress, and that ROS-mediated iron toxicity may involve cytochrome c- and caspase-3-dependent apoptotic pathways.     

Antiapoptotic property of human alpha-synuclein in neuronal cell lines is associated with the inhibition of caspase-3 but not caspase-9 activity

Abnormalities of alpha-synuclein (alpha-Syn) are mechanistically linked to Parkinson's disease (PD) and other alpha-synucleinopathies. To gain additional insights into the relationships between alpha-Syn expression and cell death, we examined the effects of expressing human alpha-Syn (Hualpha-Syn) variants on the cellular vulnerability to apoptotic stimuli. We show that the expression of wild-type (WT) and A30P mutant, but not A53T mutant, Hualpha-Syn leads to the protection of neuronal cell lines from apoptosis but not necrosis. Significantly, Hualpha-Syn did not protect non-neuronal cell lines from apoptosis. We also show that A53T mutant is a loss of function in regards to the antiapoptotic property since the expression of WT Hualpha-Syn with an excess of A53T mutant Hualpha-Syn leads to protection of the cells from apoptosis. The antiapoptotic property is specific to human alpha-Syn as neither beta-Syn nor mouse alpha-Syn protected cells from apoptosis, and the carboxy-terminal 20 amino acids are required for the antiapoptotic property. Analyses of capase-3 and caspase-9 activation reveal that the antiapoptotic property of Hualpha-Syn in neuronal cell lines is associated with the attenuation of caspase-3 activity without affecting the caspase-9 activity or the levels of cleaved, active caspase-3. We conclude that Hualpha-Syn modulates the activity of cleaved caspase-3 product in neuronal cell lines.     

Silencing of cellular prion protein (PrPC) expression by DNA-antisense oligonucleotides induces autophagy-dependent cell death in glioma cells

Malignant gliomas are the most common and lethal primary central nervous system neoplasms. Several intriguing lines of evidence have recently emerged indicating that the cellular prion protein (PrPC) may exert neuro- and cyto-protective functions: PrPC overexpression protects cultured neurons and also tumor cell lines exposed to various pro-apoptotic stimuli while, on the contrary, PrPC silencing sensitizes Adriamycin-resistant human breast carcinoma cells to TRAIL-mediated cell death. In order to determine if PrPC is involved in the resistance of glial tumors to cell death, the effects of cellular prion protein downregulation by antisense approach were investigated in different human malignant glioma cell lines. PrPC downregulation induced profound morphological changes and significant cell death. In addition, a significant tumor volume reduction was noted after PrPC silencing in a EGFP-GL261 glioma murine model. Investigations of the molecular effects induced by PrPC silencing were carried out on T98G human glioma cells by analysing autophagic as well as typical apoptotic markers (nuclear morphology, caspase-3/7, p53 and PARP-1). The results indicated that apoptosis was not induced after PrPC downregulation while, on the contrary, electron microscopy analysis, and an accumulation of GFP-LC3-II in autophagosomal membranes of GFP-LC3 transfected cells, indicated a predominant activation of autophagy. PrPC silencing also led to induction of LC3-II, increase in Beclin-1 and a concomitant decrease in p62, Bcl-2 and in the phosphorylation of 4E-BP1, a target of mTOR autophagy signaling. In conclusion, our results show for the first time that interfering with the cellular prion protein expression could modulate autophagy-dependent cell death pathways in glial tumor cells.     

Silencing of cellular prion protein (PrPC) expression by DNA-antisense oligonucleotides induces autophagy-dependent cell death in glioma cells

Malignant gliomas are the most common and lethal primary central nervous system neoplasms. Several intriguing lines of evidence have recently emerged indicating that the cellular prion protein (PrPC) may exert neuro- and cyto-protective functions: PrPC overexpression protects cultured neurons and also tumor cell lines exposed to various pro-apoptotic stimuli while, on the contrary, PrPC silencing sensitizes Adriamycin-resistant human breast carcinoma cells to TRAIL-mediated cell death. In order to determine if PrPC is involved in the resistance of glial tumors to cell death, the effects of cellular prion protein downregulation by antisense approach were investigated in different human malignant glioma cell lines. PrPC downregulation induced profound morphological changes and significant cell death. In addition, a significant tumor volume reduction was noted after PrPC silencing in a EGFP-GL261 glioma murine model. Investigations of the molecular effects induced by PrPC silencing were carried out on T98G human glioma cells by analysing autophagic as well as typical apoptotic markers (nuclear morphology, caspase-3/7, p53 and PARP-1). The results indicated that apoptosis was not induced after PrPC downregulation while, on the contrary, electron microscopy analysis, and an accumulation of GFP-LC3-II in autophagosomal membranes of GFP-LC3 transfected cells, indicated a predominant activation of autophagy. PrPC silencing also led to induction of LC3-II, increase in Beclin-1 and a concomitant decrease in p62, Bcl-2 and in the phosphorylation of 4E-BP1, a target of mTOR autophagy signaling. In conclusion, our results show for the first time that interfering with the cellular prion protein expression could modulate autophagy-dependent cell death pathways in glial tumor cells.     

Identification of an alternative form of caspase-9 in human gastric cancer cell lines: a role of a caspase-9 variant in apoptosis resistance

Overcoming apoptosis resistance to chemotherapy and radiation may lead to a reduction in gastric cancer death. We hypothesize that the apoptotic machinery in gastric cancer cells is dependent upon specific cellular conditions. In the course of our study of the expression of apoptosis-related genes in human gastric cancer cell lines, we have identified a cDNA clone which predicts an alternative form of caspase-9. The caspase-9 variant, which we designated as caspase-9 beta, retained a truncated structure of native caspase-9 without its catalytic domain and was expressed in seven cell lines from human gastric cancer. Among the cell lines examined, MKN-28 cells, which exhibited the most resistance against apoptotic stimuli, expressed the highest level of caspase-9 beta. The induction of apoptosis by staurosporine or actinomycin D was markedly suppressed in caspase-9 beta-transfected HeLa cells. These results are consistent with our hypothesis that the caspase-9 beta may be an endogenous dominant-negative molecule which attenuates apoptotic activity in human gastric cancer cells.     

Taxol-induced apoptosis in human SKOV3 ovarian and MCF7 breast carcinoma cells is caspase-3 and caspase-9 independent

Taxol is used in chemotherapy regimens against breast and ovarian cancer. Treatment of tumor model cell lines with taxol induces apoptosis, but exact mechanism is not sufficiently understood. Our results demonstrate that in response to taxol, various cell types differentially utilize distinct apoptotic pathways. Using MCF7 breast carcinoma cells transfected with caspase-3 gene, we showed that taxol-induced apoptosis occurred in the absence of caspase-3 and caspase-9 activation. Similar results were obtained with ovarian SKOV3 carcinoma cells, expressing high level of endogenous caspase-3. In contrast, staurosporine-induced apoptosis in these cells was accompanied by proteolytic cleavage of pro-caspase-3 and induction of caspase-3 enzymatic activity. The effect of taxol appears to be cell type-specific, since taxol-induced apoptosis in leukemia U937 cells involved caspase-3 activation step. We conclude that a unique caspase-3 and caspase-9 independent pathway is elicited by taxol to induce apoptosis in human ovarian and breast cancinoma cells.     

Epoxycyclohexenone inhibits Fas-mediated apoptosis by blocking activation of pro-caspase-8 in the death-inducing signaling complex

Death receptors belong to the tumor necrosis factor receptor family. They can induce apoptosis following engagement with specific ligands and are known to play an important role in the regulation of the immune system. Here we report that epoxycyclohexenone (ECH) inhibits apoptosis induced by anti-Fas antibody, Fas ligand (FasL), or tumor necrosis factor-alpha but not by staurosporine, MG-132, C2-ceramide, or UV irradiation. These results suggest that ECH specifically blocks death receptor-mediated apoptosis. Neither the surface expression of Fas nor the Fas-FasL interaction was influenced by ECH. However, ECH did block the activation of pro-caspase-8 in the death-inducing signaling complex, although recruitment of Fas-associating death domain (FADD) and pro-caspase-8 was not affected. ECH inhibited the enzymatic activity of recombinant active caspase-8 at slightly lower concentrations than it did for active caspase-3 and active caspase-9 in vitro. However, in FasL-treated cells, ECH was only able to inhibit the activation of pro-caspase-8, and it had no effect on the already activated caspase-8 at a concentration that is effective at inhibiting Fas-induced apoptosis. ECH directly bound the large subunit of active caspase-8 that contains the active center cysteine and had a relatively higher affinity to pro-caspase-8. Moreover, compared with pro-caspase-3 and pro-caspase-9, pro-caspase-8 was predominantly depleted by biotinylated ECH with avidin beads in the cell lysates, suggesting that ECH preferentially affects pro-caspase-8. Thus, our results suggest that ECH blocks the self-activation of pro-caspase-8 in the death-inducing signaling complex and thus selectively inhibits death receptor-mediated apoptosis.     

Hydrogen peroxide-induced apoptosis in human gastric carcinoma MGC803 cells

Hydrogen peroxide (H(2)O(2)), a representative ROS, has been used to study the apoptosis of cancer cells to oxidative stress. In this study, we exploited the cellular and molecular mechanisms involved in H(2)O(2)-induced apoptosis in human gastric carcinoma MGC803 cells. Exposure of cells to H(2)O(2) might cause significant viability loss and the increase in apoptotic rate. Treatment with 0.4 mmol/L H(2)O(2) up-regulated Bax but down-regulated Bcl-2 in a time-dependent manner, while Bcl-xL expression remained unchanged. Our results also showed that the levels of Fas and Fas-L were increased, the pro-caspase-3 and pro-caspase-9 were down-regulated in H(2)O(2)-treated MGC803 cells. Under H(2)O(2) stress, we found that the protein p53 also participated in MGC803 cells apoptosis. Taken together, the present study indicated that Fas-mediated cell surface death receptor pathway and mitochondria-mediated pathway may participate in regulating the MGC803 cells apoptosis under oxidative stress.     

Primary hepatocyte apoptosis is unlikely to relate to caspase-3 activity under sustained endogenous oxidative stress

We previously showed that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in endogenous oxidative stress and apoptosis. For the present study, we determined whether this apoptosis involved activation of caspase-3, which is known to execute apoptosis in many cell types. ATZ and MS increased levels of reactive oxygen species (ROS) from 3-9 h, just before the onset of chromatin condensation (apoptosis) and decreases in protein thiols. Pretreatment with either SKF, a cytochrome P450 inhibitor, or L-ascorbic acid, an antioxidant, completely suppressed the increase in ROS levels and apoptosis, suggesting that the sustained ROS increases may cause the apoptosis. SKF also abolished the decrease in protein thiol content, further supporting the contribution of the P450 system to increased ROS levels. DEVD-CHO, a caspase-3 inhibitor, even at 1 mM had no effect on apoptosis. Caspase-3 activity remained unchanged and pro-caspase-3 processing was not detected during 18 h incubation with ATZ and MS. Moreover, the amount of unoxidized pro-caspase-3 decreased even below the level of untreated hepatocytes. These findings suggest that the sustained oxidative stress is a major cause for the hepatocyte apoptosis, which occurs independently of the caspase-3 related pathway.     

Intracellular K+ concentration decrease is not obligatory for apoptosis

K(+) efflux is observed as an early event in the apoptotic process in various cell types. Loss of intracellular K(+) and subsequent reduction in ionic strength are suggested to release the inhibition of proapoptotic caspases. In this work, a new K(+)-specific microelectrode was used to study possible alterations in intracellular K(+) in Xenopus laevis oocytes during chemically induced apoptosis. The accuracy of the microelectrode to detect changes in intracellular K(+) was verified with parallel electrophysiological measurements. In concordance with previous studies on other cell types, apoptotic stimuli reduced the intracellular K(+) concentration in Xenopus oocytes and increased caspase-3 activity. The reduction in intracellular K(+) was prevented by dense expression of voltage-gated K (Kv) channels. Despite this, the caspase-3 activity was increased similarly in Kv channel-expressing oocytes as in oocytes not expressing Kv channels. Thus, in Xenopus oocytes caspase-3 activity is not dependent on the intracellular concentration of K(+).     

Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopolysaccharide and interferon-gamma

Based on high sequence homology, there are six members in the caspase-1 subfamily: caspases 1, 4, 5, and 13 in humans and caspases 1, 11, and 12 in mice. Only caspase-1 is known to activate interleukin-1beta and interleukin-18, and caspase-11 activates pro-caspase-1 in vivo. Almost nothing is known about caspases 4, 5, and 13. Here we report a sensitive and specific polymerase chain reaction system to analyze closely related genes. We employed this system to analyze the gene expression and regulation of human caspases 1, 4, 5, and 13, demonstrating that they have different expression patterns in normal tissues and cell lines. Interferon-gamma strongly induced CASP1 and CASP5 but not CASP4 or CASP13 gene expression in HT-29 colon carcinoma cells. In contrast to the mRNA, interferon-gamma up-regulated caspase-1 but not caspase-5 protein. In the monocytic cell line THP-1, CASP1 mRNA and caspase-1 protein are expressed constitutively, and their levels were not increased by lipopolysaccharide, whereas both CASP5 mRNA and caspase-5 protein were induced by lipopolysaccharide. Caspase-1 subfamily members displayed different in vitro activities toward pro-caspases 1 and 3 and pro-interleukin-1beta. Our results demonstrate that caspase-1 and caspase-5 levels are modulated by interferon-gamma and lipopolysaccharide, respectively, and suggest that caspase-1 subfamily members are differentially regulated and may have distinct functions.     

Bax and p53 are differentially involved in the regulation of caspase-3 expression and activation during neurodegeneration in Lurcher mice

Intrinsic Purkinje cell death in heterozygous Lurcher (Grid2Lc/+) mice is accompanied by the target-related death of granule cells and olivary neurons. The expression of pro-caspase-3 is increased in Grid2Lc/+ Purkinje cells and activated caspase-3 is detected in all three cell types before their death. Bax inactivation in Grid2Lc/+ mutants rescues granule cells but not Purkinje cells. Here, we show that, while Bax inactivation inhibits caspase-3 activation in both cell types, p53 inactivation does not affect caspase-3 activation and neuronal loss in Grid2Lc/+ mice. The up-regulation of pro-caspase-3 in Grid2Lc/+ Purkinje cells is Bax and p53 independent. These results suggest that Grid2Lc/+ granule cell death is dependent on Bax and caspase-3 activation, whereas several pathways can mediate Grid2Lc/+ Purkinje cell death.     

羊栖菜多糖通过激活Caspase途径诱导Lovo细胞凋亡

研究了羊栖菜多糖（Sargassum Fusiforme Polysaccharides,SFPS）诱导人大肠癌lovo细胞凋亡及凋亡过程中caspase-3、caspase-8、caspase-9的活性变化。MTT法检测SFPS对lovo细胞增殖的抑制率;通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡;应用Western印迹法测定caspase-3酶原和caspase-9的变化;RToPCR检测caspase-3 mRNA表达;caspase-3,caspase-8、caspase-9活性检测试剂盒观察caspase-3、caspase-8、caspase-9的活性改变。结果显示,SFPS对lovo细胞增殖有显著抑制作用,经形态变化、DNA条带和流式细胞分析,可见明显的细胞凋亡特征。SFPS处理lovo细胞后,发现caspase-3酶原蛋白表达降低,caspase-3 mRNA高表达,并具有剂量和时间的依赖性。而在检测蛋白中,也发现caspase-9被激活进而形成具有活性的片段。另外,caspase的活性检测也进一步发现caspase-3、caspase-9的活性逐步增高。实验结果提示SFPS在体外诱导lovo胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一,并且是通过激活启动caspase-9,进而激活下游效应caspase-3的级联反应来实现的。     

Phloroglucinol derivative MCPP induces cell apoptosis in human colon cancer

This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (3,6-bis(3-chlorophenylacetyl)phloroglucinol; MCPP) in human colon cancer cells. MCPP induced cell death and antiproliferation in three human colon cancer, HCT-116, SW480, and Caco-2 cells, but not in primary human dermal fibroblast cells. MCPP-induced concentration-dependent apoptotic cell death in colon cancer cells was measured by fluorescence-activated cell sorter (FACS) analysis. Treatment of HCT-116 human colon cancer cells with MCPP was found to induce a number of signature endoplasmic reticulum (ER) stress markers; and up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein (GRP)-78, phosphorylation of eukaryotic initiation factor-2α (eIF-2α), suggesting the induction of ER stress. MCPP also increased GSK3α/β(Tyr270/216) phosphorylation and reduced GSK3α/β(Ser21/9) phosphorylation time-dependently. Transfection of cells with GRP78 or CHOP siRNA, or treatment of GSK3 inhibitor SB216163 reduced MCPP-mediated cell apoptosis. Treatment of MCPP also increased caspase-7, caspase-9, and caspase-3 activity. The inhibition of caspase activity by z-DEVE-FMK or z-VAD-FMK significantly reduced MCPP-induced apoptosis. Furthermore, treatment of GSK3 inhibitor SB216763 also dramatically reversed MCPP-induced GRP and CHOP up-regulation, and pro-caspase-3 and pro-caspase-9 degradation. Taken together, the present study provides evidences to support that GRP78 and CHOP expression, and GSK3α/β activation in mediating the MCPP-induced human colon cancer cell apoptosis.     

The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex

Death receptors, such as Fas and tumor necrosis factor-related apoptosis-inducing ligand receptors, recruit Fas-associated death domain and pro-caspase-8 homodimers, which are then autoproteolytically activated. Active caspase-8 is released into the cytoplasm, where it cleaves various proteins including pro-caspase-3, resulting in apoptosis. The cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein long form (FLIP(L)), a structural homologue of caspase-8 lacking caspase activity because of several mutations in the active site, is a potent inhibitor of death receptor-induced apoptosis. FLIP(L) is proposed to block caspase-8 activity by forming a proteolytically inactive heterodimer with caspase-8. In contrast, we propose that FLIP(L)-bound caspase-8 is an active protease. Upon heterocomplex formation, a limited caspase-8 autoprocessing occurs resulting in the generation of the p43/41 and the p12 subunits. This partially processed form but also the non-cleaved FLIP(L)-caspase-8 heterocomplex are proteolytically active because they both bind synthetic substrates efficiently. Moreover, FLIP(L) expression favors receptor-interacting kinase (RIP) processing within the Fas-signaling complex. We propose that FLIP(L) inhibits caspase-8 release-dependent pro-apoptotic signals, whereas the single, membrane-restricted active site of the FLIP(L)-caspase-8 heterocomplex is proteolytically active and acts on local substrates such as RIP.     

Apoptosis: checkpoint at the mitochondrial frontier.

Apoptosis, an evolutionarily conserved form of cell death, requires a regulated program. Central to the apoptotic program is a family of cysteine proteases, known as caspases, that cleave a subset of cellular proteins, resulting in the stereotypic morphological changes of apoptotic cell death. In living cells caspases are present as inactive zymogens and become activated in response to pro-apoptotic stimuli. Mitochondria participate in the activation of caspases by releasing cytochrome c into the cytosol where it binds to the adaptor molecule Apaf-1 (apoptotic protease activating factor 1) and causes its oligomerization. This renders Apaf-1 competent to recruit and activate the cell death initiator caspase, pro-caspase-9. Once caspase-9 is activated, it cleaves and activates downstream cell death effector caspases. Bcl-2, an apoptosis inhibitor localized to mitochondrial outer membranes, prevents cytochrome c release, caspase activation and cell death. This review discusses recent advances on the role of mitochondria and cytochrome c in the central pathway leading to apoptotic cell death.