Gene transfer of constitutively active caspase-3 induces apoptosis in a human hepatoma cell line

BACKGROUND: The caspase-3 gene is expressed at significantly lower levels in human hepatocellular carcinomas than in normal hepatocytes. Gene transfer technologies offer the possibility to restore caspase-3 gene expression. We explored the interest for cancer gene therapy of a constitutively active recombinant caspase-3 (RevCasp3) obtained by rearranging its subunits. METHODS: An amphotropic retroviral vector was used to express the RevCasp3 gene. HuH7 cells were infected 1 and 2 days after plating. Caspase-3 activity was measured every 24 h for the following 6 days. The level of poly (ADP-ribose) polymerase cleavage induced by caspase-3 was measured by Western blot. The percentage of apoptotic cells was estimated after Hoechst-acridine orange and TUNEL stainings. RESULTS: Caspase-3 activity significantly increased from days 4 to 7 after infection. Caspase-3 activity peaked on day 7, and was 5.4-fold higher in RevCasp3-transduced HuH7 cells than in control cells. Poly (ADP-ribose) polymerase cleavage was first detected 6 days after the first infection. Hoechst-acridine orange and TUNEL stainings showed that most infected HuH7 cells were apoptotic. CONCLUSIONS: Apoptosis was selectively induced following infection of HuH7 cells with RevCasp3, demonstrating that retroviruses expressing RevCasp3 are of potential interest for the treatment of hepatocellular carcinomas and other tumour types.     

TNF-alpha-mediated cardiomyocyte apoptosis involves caspase-12 and calpain

Following ischemia-reperfusion, there is a sustained increase of TNF-alpha both locally in the heart as well as in circulating levels in blood. While TNF-alpha has been implicated in cardiomyocyte apoptosis which occurs in several cardiomyopathies, the molecular pathways by which TNF-alpha induces apoptosis in these cells are not fully elucidated. We investigated the role of the two families of cysteine proteases, caspases and calpains, which are known to participate in apoptotic cell death. The effect of the highly specific calpain inhibitor, Z-LLY-fmk, and the caspase pathways involved in TNF-alpha-mediated apoptosis of the HL-1 cardiomyocyte cell line were examined. Activation of the downstream caspase-3, and the cleavage of poly ADP-ribose polymerase (PARP) were observed in a time-dependent manner upon treatment with TNF-alpha. Caspase-12, but not caspase-9, was activated in response to TNF-stimulation, indicating that an endoplasmic reticulum (ER)/calcium-dependent pathway may be involved. In HL-1 cardiomyocytes, TNF-alpha-induced apoptosis appears to be mediated by calpain as apoptotic changes were abrogated in the presence of the highly specific calpain inhibitor, Z-LLY-fmk. In conclusion, our results suggest that TNF-alpha-mediated apoptosis in HL-1 cardiomyocytes follows the caspase-12 apoptotic pathway that involves calpain.     

1,25-Dihydroxyvitamin D3 induces Ca-mediated apoptosis in adipocytes via activation of calpain and caspase-12

Induction of apoptotic cell death is emerging as a promising strategy for prevention and treatment of obesity because removing of adipocytes via apoptosis may result in reducing body fat and a long-lasting maintenance of weight loss. However, the mechanisms controlling adipocyte apoptosis are unknown and even the ability of adipocytes to undergo apoptosis has not been conclusively demonstrated. We have shown previously that the specific Ca²⁺ signal, sustained increase in intracellular Ca²⁺, triggers apoptotic cell death via activation of Ca²⁺-dependent proteases and that the apoptosis-inducing effect of the hormone 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is mediated through Ca²⁺ signaling. Here, we report that 1,25(OH)₂D₃ induces apoptosis in mature mouse 3T3-L1 adipocytes via activation of Ca²⁺-dependent calpain and Ca²⁺/calpain-dependent caspase-12. Treatment of adipocytes with 1,25(OH)₂D₃ induced, in concentration- and time-dependent fashion, a sustained increase in the basal level of intracellular Ca²⁺. The increase in Ca²⁺ was associated with induction of apoptosis and activation of μ-calpain and caspase-12. Our results demonstrate that Ca²⁺-mediated apoptosis can be induced in mature adipocytes and that the apoptotic molecular targets activated by 1,25(OH)₂D₃ in these cells are Ca²⁺-dependent calpain and caspase-12. These findings provide rationale for evaluating the role of vitamin D in prevention and treatment of obesity.     

Involvement of caspase-3 activation in squamocin-induced apoptosis in leukemia cell line HL-60

Annonaceous acetogenins have potent antitumor effect in vitro and in vivo. Squamocin is one of the annonaceous acetogenins and has been reported to have antiproliferative effect on cancer cells. Our results from this study showed that squamocin inhibited proliferation of HL-60 cells with IC50 value of 0.17 microg/ml and induced apoptosis of HL-60 cells. Investigation of the mechanism of squamocin-induced apoptosis revealed that treatment of HL-60 cells with squamocin resulted in extensive nuclear condensation. DNA fragmentation, cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and induction of caspase-3 activity. Pretreatment of HL-60 cells with caspase-3 specific inhibitor DEVD-CHO prevented squamocin-induced DNA fragmentation, PARP cleavage and cell death. The expression levels of protein bcl-2, bax have no change in response to squamocin treatment in HL-60 cells, whereas stress-activated protein kinase (SAPK/JNK) was activated after treatment with squamocin in HL-60 cells. These results suggest that apoptosis of HL-60 cells induced by squamocin requires caspase-3 activation and is related to SAPK activation.     

Fas-mediated apoptosis in a rat acinar cell line is dependent on caspase-1 activity

Apoptosis plays an important role in the dysfunction of exocrine glands. Fas is a death-inducing receptor found on many types of cells including epithelial acinar cells. To elucidate the intracellular mechanism of Fas-mediated cell death in exocrine glands, an epithelial acinar cell line, SMG-C6, was studied. Caspase-1, -3, -8, and -9 activities were elevated in SMG-C6 cells after the induction of apoptosis by soluble Fas ligand (FasL). The activation of caspase-1 and -8 occurred prior to caspase-3 and -9 activation. The caspase-1 inhibitor, zYVAD-fmk, was effective in preventing cell death, whereas the caspase-3 and -8 inhibitors (ac-DEVD-CHO and ac-IETD-CHO, respectively) were not. zYVAD-fmk was able to inhibit caspase-3 activation indicating that caspase-1 is upstream to caspase-3. Furthermore, kinetic studies show that caspase-1 is an early event in the Fas apoptotic pathway. This study shows that caspase-1 participates in Fas-mediated apoptosis of epithelial cells by initiating the caspase cascade.     

Rac1 mediates intestinal epithelial cell apoptosis via JNK

Apoptosis plays a key role in the maintenance of a constant cell number and a low incidence of cancer in the mucosa of the intestine. Although the small GTPase Rac1 has been established as an important regulator of migration of intestinal epithelial cells, whether Rac1 is also involved in apoptosis is unclear. The present study tested the hypothesis that Rac1 mediates TNF-alpha-induced apoptosis in IEC-6 cells. Rac1 is activated during TNF-alpha-induced apoptosis as judged by the level of GTP-Rac1, the level of microsomal membrane-associated Rac1, and lamellipodia formation. Although expression of constitutively active Rac1 does not increase apoptosis in the basal condition, inhibition of Rac1 either by NSC-23766 (Rac1 inhibitor) or expression of dominant negative Rac1 protects cells from TNF-alpha-induced apoptosis by inhibiting caspase-3, -8, and -9 activities. Inhibition of Rac1 before the administration of apoptotic stimuli significantly prevents TNF-alpha-induced activation of JNK1/2, the key proapoptotic regulator in IEC-6 cells. Inhibition of Rac1 does not modulate TNF-alpha-induced ERK1/2 and Akt activation. Inhibition of ERK1/2 and Akt activity by U-0126 and LY-294002, respectively, increased TNF-alpha-induced apoptosis. However, inhibition of Rac1 significantly decreased apoptosis in the presence of ERK1/2 and Akt inhibitors, similar to the effect observed with NSC-23766 alone in response to TNF-alpha. Thus, Rac1 inhibition protects cells independently of ERK1/2 and Akt activation during TNF-alpha-induced apoptosis. Although p38 MAPK is activated in response to TNF-alpha, inhibition of p38 MAPK did not decrease apoptosis. Rac1 inhibition did not alter p38 MAPK activity. Thus, these results indicate that Rac1 mediates apoptosis via JNK and plays a key role in proapoptotic pathways in intestinal epithelial cells.     

顺铂对小鼠耳蜗螺旋神经节细胞凋亡及caspase-3表达的影响

目的:建立小鼠顺铂(CDDP)耳毒性模型,研究不同剂量顺铂对小鼠耳蜗螺旋神经节细胞凋亡及caspase-3表达的影响。方法:采用末端脱氧核苷酸转移酶介导的缺口末端标记(TUNEL)技术检测螺旋神经节细胞的凋亡;应用免疫组织化学Envision法检测caspase-3在螺旋神经节中的表达;同时结合听脑干反应(ABR)测试,观察用药前后小鼠听力的变化。结果:不同剂量顺铂组小鼠体重和听力明显下降,与对照组比较均有显著性差异(P0.05,P0.01);并且随着顺铂给药剂量的增加,小鼠耳蜗螺旋神经节中TUNEL阳性细胞数增多,以及caspase-3表达明显增强。结论:应用小鼠能建立可靠的顺铂耳毒性模型;顺铂可导致耳蜗螺旋神经节细胞凋亡,而且此凋亡过程中有caspase-3的参与,进一步证实了凋亡可能是顺铂耳毒性机制之一。     

Cytoskeletal disruption induces T cell apoptosis by a caspase-3 mediated mechanism

T cell apoptosis can be triggered by different mechanisms that lead to distinctive features such as cell shrinkage, membrane blebbing, phosphatidylserine externalization, and internucleosomal DNA fragmentation. Prevailing models for the induction of apoptosis place the cytoskeleton as a distal target of the death effector molecules ('executioners'). However, the cytoskeleton can also play a role in the induction of apoptosis as suggested by the finding that cytoskeletal disruption can induce apoptosis. The mechanism by which this occurs is unknown. Here, we report that T cell apoptosis by cytoskeletal disruption involves a protein synthesis-independent mechanism leading to up-regulation of caspase-3 protease activity and increased accessibility of active caspase-3 to its substrate. Thus, cytoskeleton integrity may regulate the subcellular compartmentalization of death effector molecules.     

IL-3 induces apoptosis in a ras-transformed myeloid cell line

Growth factors promote cell survival and proliferation. Homeostasis is maintained by programmed cell death which occurs when the growth stimulus is withdrawn, in response to negative growth regulators such as interferons, TNF- and CD95 ligand, or following differentiation. Although acutely-transforming oncogenes often overcome the need for growth factors, growth regulatory cytokines can influence proliferative responses of transformed cells. In this study we investigated the effects of IL-3 on the proliferative responses of parental bone marrow-derived 32D cells and cells transformed with ras and abl oncogenes. We show that treatment of ras-transformed 32D cells with IL-3 reduced proliferative responses and decreased colony-forming ability. These effects were exacerbated in the absence of serum and associated with inhibition of tyrosine kinase activity, down-regulation of RAS and MYC expression, and induction of apoptosis as indicated by DNA fragmentation. In contrast, treatment of parental 32D cells with IL-3, which is obligatory for cell survival and proliferation, increased tyrosine kinase activity, upregulated MYC and RAS expression and maintained DNA integrity. With abl-transformed cells, proliferation and colony-forming ability were also inhibited by IL-3. Tyrosine kinase activity and MYC expression were reduced, but early apoptosis was not evident. Calcium uptake however, was stimulated by IL-3 in both parental and oncogene-transformed cells. These results suggest that threshold levels of tyrosine kinase activity are necessary for cell survival and proliferation and that with ras-transformed cells, IL-3 treatment may result in this threshold being breached. We conclude that in some situations, growth-promoting cytokines can inhibit proliferation of transformed cells and induce cell death by apoptosis.     

MPA increases idarubicin-induced apoptosis in chronic lymphatic leukaemia cells via caspase-3

The caspase family of protease is speculated to have a crucial role in apoptosis. The effect of treatment with Idarubicin (IDA) and Medroxyprogesterone acetate (MPA), used alone or in combination, on the activation of Caspase-3 in canine Chronic Lymphatic Leukaemia (CLL) cells was investigated, in order to clarify the mechanism of chemo- and hormone-therapy mediated apoptosis. Caspase activity was determined by a quantitative fluorimetric assay. Apoptosis was monitored by propidium iodide (PI) and nucleosomes assay. Treatment of CLL cells for 24 h with MPA 5 microM did not significantly activate caspase-3 but its activity was increased almost 5-fold more with IDA 1 microM (P < 0.05) than control. Treatment of CLL cells with IDA 1 microM in equimolecular association with MPA was able to increase the activation of caspase-3 induced by IDA of the 61.2% (P < 0.05) in comparison with IDA alone. The activation of caspase-3 was confirmed evaluating apoptosis by PI and nucleosomes assay. Furthermore, both caspase-3 activation and apoptosis triggered by IDA alone or in combination with MPA were significantly inhibited by specific caspase-3 inhibitor AC-DEVD-CMK. These findings provide an explanation for IDA and MPA induced-apoptosis mechanism.     

Trichomonas vaginalis: reactive oxygen species mediates caspase-3 dependent apoptosis of human neutrophils

There are many neutrophils in the vaginal discharge from women infected with Trichomonas vaginalis. The aim of our study was to determine whether human neutrophil apoptosis may be regulated by reactive oxygen species (ROS) in response to trichomonads infection. Incubation of human neutrophils with live trichomonads caused marked receptor shedding of CD16, decrease of mitochondrial membrane potential (MMP) and caspase-3 activation in human neutrophils. These proapoptotic effects of T. vaginalis on neutrophils were inhibited by pretreatment of neutrophils with an inhibitor of NADPH oxidase, diphenyleneiodonium chloride (DPI), suggesting an important role of intracellular ROS accumulation in T. vaginalis-triggered apoptosis. Indeed, large amounts of ROS levels were detected in neutrophils incubated with live trichomonads, and were also effectively inhibited by DPI. However, pan-caspase inhibitor z-VAD-fmk or caspase-3 inhibitor z-DEVD-fmk did not affect T. vaginalis-induced ROS generation in neutrophils. These results suggest that ROS-dependent caspase-3 activation plays an important role in apoptosis of human neutrophils induced by T. vaginalis.     

Butyrate mediates Caco-2 cell apoptosis via up-regulation of pro-apoptotic BAK and inducing caspase-3 mediated cleavage of poly-(ADP-ribose) polymerase (PARP).

Butyrate exerts potent anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. Using the Caco-2 cell line, a well established model of colon cancer cells, our data show that butyrate induced apoptosis (maximum 79%) is mediated via activation of the caspase-cascade. A key event was the proteolytic activation of caspase-3, triggering degradation of poly-(ADP-ribose) polymerase (PARP). Inactivation of caspase-3 with the tetrapeptide zDEVD-FMK completely inhibited the apoptotic response to butyrate. In parallel, butyrate potently up-regulated the expression of the pro-apoptotic protein bak, without changing Caco-2 cell bcl-2 expression. Butyrate-induced Caco-2 cell apoptosis was completely blocked by the addition of cycloheximide, indicating the necessity of protein synthesis. However, when this inhibitor was added at a time point where bak expression was already enhanced (12 - 16 h after butyrate stimulation), it failed to protect Caco-2 cells against apoptosis. Taken together, these data provide evidence that the molecular events involved in butyrate induced colon cancer cell apoptosis include the caspase-cascade and the mitochondrial bcl-pathway.     

Proteolysis of the human DNA polymerase epsilon catalytic subunit by caspase-3 and calpain specifically during apoptosis

Human DNA polymerase epsilon (pol ) normally contains a 261-kDa catalytic subunit (p261), but from some sources it is isolated as a 140-kDa catalytic core of p261. This shortened form possesses normal or somewhat enhanced polymerase activity and its significance is unknown. We report here that caspase-3 and calpain can form p140 from p261 in vitro and in vivo and that during early stages of apoptosis induced in Jurkat cells by staurosporine or anti-Fas-activating antibody, p261 is cleaved into p140 by caspase-3. At later stages, activated calpain might also contribute to this conversion. The sites of cleavage by caspase-3 have been identified, and mutations at these ‘DEAD boxes’ resulted in cleavage-resistant enzyme. Cleavage at these sites separates the ‘N-terminal catalytic core’ from the ‘C-terminal’ regions described for p261. Cleavage does not occur during necrosis or following exposure to H₂O₂ or methanesulfonic acid methyl ester. p140 is unlikely to be able to functionally replace p261 in vivo, since it does not bind to PCNA or the other pol subunits.     

Zearalenone induces apoptosis and necrosis in porcine granulosa cells via a caspase-3- and caspase-9-dependent mitochondrial signaling pathway

Zearalenone is a mycotoxin produced mainly by Fusarium. There are numerous incidences of mycotoxicosis in laboratory and domestic animals, especially in pigs. However, little is known about molecular mechanisms of zearalenone toxicity. Granulosa cells are the maximal cell population in follicles, and they play an essential role in the development and maturation of follicles. The objective of this study was to explore the effect of zearalenone at high concentrations on proliferation and apoptosis of porcine granulosa cells and uncover signaling pathway underlying the cytotoxicity of zearalenone. We found that zearalenone reduced the proliferation of porcine granulosa cells in a dose-dependent manner as shown by the MTT assay and zearalenone resulted in an obvious apoptosis and necrosis in porcine granulosa cells as determined by the TUNEL analysis and flow cytometry. In addition, TUNEL assay with caspase inhibitors showed that zearalenone triggered a caspase-3- and caspase-9-dependent apoptotic process in porcine granulosa cells. Fluorescence spectrophotometer displayed that zearalenone led to a loss of mitochondrial transmembrane potential of porcine granulosa cells but enhanced reactive oxygen species (ROS) levels of the cells. Notably, Western blots revealed that caspase-3 and caspase-9 were activated by zearalenone in porcine granulosa cells. Collectively, our results suggest that zearalenone induces apoptosis and necrosis of porcine granulosa cells in a dose-dependent manner via a caspase-3- and caspase-9-dependent mitochondrial pathway. This study thus offers a novel insight into molecular mechanisms by which zearalenone has adverse cytotoxicity on reproduction.     

Caspase-7 mediates caspase-1-induced apoptosis independently of Bid

Inflammasomes are innate immune mechanisms that activate caspase-1 in response to a variety of stimuli, including Salmonella infection. Active caspase-1 has a potential to induce two different types of cell death, depending on the expression of the pyroptosis mediator gasdermin D (GSDMD); following caspase-1 activation, GSDMD-sufficient and GSDMD-null/low cells undergo pyroptosis and apoptosis, respectively. Although Bid, a caspase-1 substrate, plays a critical role in caspase-1 induction of apoptosis in GSDMD-null/low cells, an additional mechanism that mediates this cell death independently of Bid has also been suggested. This study investigated the Bid-independent pathway of caspase-1-induced apoptosis. Caspase-1 has been reported to process caspase-6 and caspase-7. Silencing of caspase-7, but not caspase-6, significantly reduced the activation of caspase-3 induced by caspase-1, which was activated by chemical dimerization, in GSDMD/Bid-deficient cells. CRISPR/Cas9-mediated depletion of caspase-7 had the same effect on the caspase-3 activation. Moreover, in the absence of GSDMD and Bid, caspase-7 depletion reduced apoptosis induced by caspase-1 activation. Caspase-7 was activated following caspase-1 activation independently of caspase-3, suggesting that caspase-7 acts downstream of caspase-1 and upstream of caspase-3. Salmonella induced the activation of caspase-3 in GSDMD-deficient macrophages, which relied partly on Bid and largely on caspase-1. The caspase-3 activation and apoptotic morphological changes seen in Salmonella-infected GSDMD/Bid-deficient macrophages were attenuated by caspase-7 knockdown. These results suggest that in addition to Bid, caspase-7 can also mediate caspase-1-induced apoptosis and provide mechanistic insights into inflammasome-associated cell death that is one major effector mechanism of inflammasomes.     

BMP-dependent activation of caspase-9 and caspase-8 mediates apoptosis in pulmonary artery smooth muscle cells

Germ line mutations in the bone morphogenetic protein (BMP) receptor type II (BMPRII) gene have been found in >50% of familial idiopathic pulmonary arterial hypertension (IPAH) patients and in 30% of sporadic cases of IPAH. Mutations of BMPRII occur in the extracellular ligand-binding domain, in the cytoplasmic serine/threonine kinase domain, or in the long carboxy terminus domain of unknown function. In this study, we demonstrate that BMPs promote apoptotic cell death in normal human pulmonary artery smooth muscle cells (PASMCs) by activation of caspases-3, -8, and -9, cytochrome c release, and downregulation of Bcl-2. Normal PASMCs expressing a kinase domain mutant or a carboxy-terminal domain deletion mutant of BMPRII identified in IPAH patients are resistant to BMP-mediated apoptosis. This dominant-negative effect may act in heterozygous patients and lead to the development of the pulmonary vascular medial hypertrophy found in IPAH patients. Our study also demonstrates an essential role of the carboxy terminus domain of BMPRII in the activation of the apoptotic signaling cascade.     

Codeine-induced hepatic injury is via oxido-inflammatory damage and caspase-3-mediated apoptosis

Molecular Biology Reports - Codeine (3-methylmorphine) is a known analgesic, antitussive, and antidiarrheal drug that is often abused for recreational purposes. It is metabolized in the liver via...     

Light-induced photoreceptor apoptosis in vivo requires neuronal nitric-oxide synthase and guanylate cyclase activity and is caspase-3-independent

Apoptosis is the mode of photoreceptor cell death in inherited and induced retinal degeneration. However, the molecular mechanisms of photoreceptor cell death in human cases and animal models of retinal dystrophies remain undefined. Exposure of Balb/c mice to excessive levels of white light results in photoreceptor apoptosis. This study delineates the molecular events occurring during and subsequent to the induction of retinal degeneration by exposure to white light in Balb/c mice. We demonstrate an early increase in intracellular calcium levels during photoreceptor apoptosis, an event that is accompanied by significant superoxide generation and mitochondrial membrane depolarization. Furthermore, we show that inhibition of neuronal nitric-oxide synthase (nNOS) by 7-nitroindazole is sufficient to prevent retinal degeneration implicating a key role for neuronal nitric oxide (NO) in this model. We demonstrate that inhibition of guanylate cyclase, a downstream effector of NO, also prevents photoreceptor apoptosis demonstrating that guanylate cyclase too plays an essential role in this model. Finally, our results demonstrate that caspase-3, frequently considered to be one of the key executioners of apoptosis, is not activated during retinal degeneration. In summary, the data presented here demonstrate that light-induced photoreceptor apoptosis in vivo is mediated by the activation of nNOS and guanylate cyclase and is caspase-3-independent.