The prodomain of caspase-1 enhances Fas-mediated apoptosis through facilitation of caspase-8 activation

Caspase-1 (interleukin-1beta converting enzyme) is produced in the form of a latent precursor, which is cleaved to yield a prodomain in addition to the p20 and p10 subunits. It has been established that the (p20/p10)(2) heterotetramer processes the latent precursor of interleukin-1beta into an active form during apoptosis, but the function of the residual prodomain of caspase-1 (Pro-C1) has not been established. To evaluate the involvement of Pro-C1 in apoptosis, a Pro-C1 expression vector was transfected into the HeLa cell line, which is susceptible to Fas-mediated apoptosis. Expression of recombinant Pro-C1 in HeLa cells enhanced apoptosis mediated by Fas, but not etoposide-induced apoptosis. This enhancement of Fas-mediated apoptosis was abolished by inhibitors of caspase-8 (Ile-Glu-Thr-Asp-fluoromethyl ketone) and caspase-3 (Asp-Glu-Val-Asp-aldehyde) but was only slightly diminished by an inhibitor of caspase-1 (acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone). During apoptosis induced by an agonistic anti-Fas antibody, the activation of caspase-8 and caspase-3 was more pronounced and occurred more rapidly in HeLa/Pro-C1 cells than in the empty vector transfectant (HeLa/vec) cells; in contrast, caspase-1 was not activated in either HeLa/Pro-C1 or HeLa/vec cells. These results demonstrate an additional and novel function for caspase-1 in which Pro-C1 acts to enhance Fas-mediated apoptosis, most probably through facilitation of the activation of caspase-8.     

High glucose enhances MMP-2 production in adventitial fibroblasts via Akt1-dependent NF-kappaB pathway

To understand the role of adventitial fibroblasts (AF) in diabetic vascular diseases, the importance of high glucose (HG, 25mM) on matrix metalloproteinase-2 (MMP-2) production in AF was determined. HG enhanced mRNA, protein and gelatinolytic activity of MMP-2. The enhanced MMP-2 activity was significantly attenuated not only by a PI3K inhibitor but also by an Akt inhibitor. These HG-induced MMP-2 responses were markedly reduced in Akt1-deficient (1KO) cells. The diminished HG-induced MMP-2 responses were completely restored by re-expression of Akt1. Both the reporter activity and electrophoretic mobility shift assay for activator protein-1 and nuclear factor-kappa B (NF-kappaB) were enhanced by HG, but NF-kappaB were not increased in 1KO cells. Furthermore, HG-induced MMP-2 responses were markedly suppressed by NF-kappaB decoy oligodeoxynucleotides. Based on these results, it is suggested that HG augments MMP-2 production via PI3K/Akt1/NF-kappaB pathway.     

Autophagy inhibition enhances ursolic acid-induced apoptosis in PC3 cells

The phosphoinositol 3-kinase/Akt pathway plays a critical role in oncogenesis and the dysregulation of this pathway through loss of PTEN is a particularly common phenomenon in aggressive prostate cancers. Several recent studies have indicated that ursolic acid (UA), a pentacyclic triterpenoid, and its derivatives inhibit the growth of cancer cells by cell cycle arrest and the stimulation of apoptosis. In the present study, we report a novel autophagic response of UA in PTEN-deficient PC3 prostate cancer cells. As one of the major types of programmed cell death, autophagy has been observed in response to several anticancer drugs and demonstrated to be responsible for cell death. UA-induced autophagy in PC3 cells is associated with the reduced cell viability and the enhanced expression of LC3-II, an autophagosome marker in mammals, and monodansylcadaverine incorporation into autolysosomes. Furthermore, we found that UA exhibited anti-proliferative effects characterized by G1 phase arrest and autophagy at an early stage that precedes apoptosis. We also show that UA-induced autophagy in PC3 cells are mediated through the Beclin-1 and Akt/mTOR pathways. Inhibition of autophagy by either 3-methyladenine or Beclin-1/Atg5 small interfering RNA enhanced UA-induced apoptosis. Taken together, our data suggest that autophagy functions as a survival mechanism in PC3 cells against UA-induced apoptosis and a rational for the use of autophagy inhibitors in combination with UA as a novel modality of cancer therapy.     

HLJ1 is a novel caspase-3 substrate and its expression enhances UV-induced apoptosis in non-small cell lung carcinoma

Carcinogenesis is determined based on both cell proliferation and death rates. Recent studies demonstrate that heat shock proteins (HSPs) regulate apoptosis. HLJ1, a member of the DnaJ-like Hsp40 family, is a newly identified tumor suppressor protein closely related to relapse and survival in non-small cell lung cancer (NSCLC) patients. However, its role in apoptosis is currently unknown. In this study, NSCLC cell lines displaying varying HLJ1 expression levels were subjected to ultraviolet (UV) irradiation, followed by flow cytometry. Interestingly, the percentages of apoptotic cells in the seven cell lines examined were positively correlated with HLJ1 expression. Enforcing expression of HLJ1 in low-HLJ1 expressing highly invasive cells promoted UV-induced apoptosis through enhancing JNK and caspase-3 activation in NSCLC. Additionally, UV irradiation led to reduced levels of HLJ1 predominantly in apoptotic cells. The pan-caspase inhibitor, zVAD-fmk and caspase-3-specific inhibitor, DEVD-fmk, prevented UV-induced degradation of HLJ1 by the late stage of apoptosis. Further experiments revealed a non-typical caspase-3 cleavage site (MEID) at amino acid 125–128 of HLJ1. Our results collectively suggest that HLJ1 is a novel substrate of caspase-3 during the UV-induced apoptotic process.     

GSK-3beta inhibition enhances sorafenib-induced apoptosis in melanoma cell lines

Glycogen synthase kinase-3beta (GSK-3beta) can participate in the induction of apoptosis or, alternatively, provide a survival signal that minimizes cellular injury. We previously demonstrated that the multikinase inhibitor sorafenib induces apoptosis in melanoma cell lines. In this report, we show that sorafenib activates GSK-3beta in multiple subcellular compartments and that this activation undermines the lethality of the drug. Pharmacologic inhibition and/or down-modulation of the kinase enhances sorafenib-induced apoptosis as determined by propidium iodide staining and by assessing the mitochondrial release of apoptosis-inducing factor and Smac/DIABLO. Conversely, the forced expression of a constitutively active form of the enzyme (GSK-3beta(S9A)) protects the cells from the apoptotic effects of the drug. This protective effect is associated with a marked increase in basal levels of Bcl-2, Bcl-x(L), and survivin and a diminution in the degree to which these anti-apoptotic proteins are down-modulated by sorafenib exposure. Sorafenib down-modulates the pro-apoptotic Bcl-2 family member Noxa in cells with high constitutive GSK-3beta activity. Pharmacologic inhibition of GSK-3beta prevents the disappearance of Noxa induced by sorafenib and enhances the down-modulation of Mcl-1. Down-modulation of Noxa largely eliminates the enhancing effect of GSK-3 inhibition on sorafenib-induced apoptosis. These data provide a strong rationale for the use of GSK-3beta inhibitors as adjuncts to sorafenib treatment and suggest that preservation of Noxa may contribute to their efficacy.     

Alpha-tocopherol-mediated caspase-3 up-regulation enhances susceptibility to apoptotic stimuli

Although alpha-tocopherol is known as an essential micronutrient involved in various oxidative stress-related processes, its non-antioxidant activities have only been characterized in recent years. In this study, we reveal that (+)-alpha-tocopherol [RRR-alpha-tocopherol] enhances cellular susceptibility to both oxidative and non-oxidative apoptosis-inducing stimuli through up-regulation of caspase-3/CPP32 expression in several human cell lines. Exposure of (+)-alpha-tocopherol pretreated cells to known apoptosis-inducing stimuli, such as Fas, H(2)O(2), or etoposide, resulted in an increase in cellular apoptotsis. In addition, (+)-alpha-tocopherol also elevated the pro-caspase-3 protein level and mRNA expression in a time- and dose-dependent manner, while other tocopherol analogues showed no effect. Experiments using a GC-specific DNA binding agent, mithramycin A, and an electrophoretic mobility shift assay demonstrated that Sp1 might mediate the enhanced expression of caspase-3. Our results also confirmed that (+)-alpha-tocopherol promotes the expression, but not the activation, of caspase-3 in various human cell lines. These findings provide biological evidence showing that (+)-alpha-tocopherol can amplify the apoptotic response by up-regulating the expression of pro-caspase-3.     

Akt kinase activation blocks apoptosis in intestinal epithelial cells by inhibiting caspase-3 after polyamine depletion

Apoptosis plays a critical role in the maintenance of gut mucosal homeostasis and is regulated by numerous factors including polyamines. Although the exact roles of polyamines in apoptotic pathway are still unclear, inhibition of polyamine synthesis promotes the resistance of intestinal epithelial cells to apoptosis. Akt is a serine-threonine kinase that has been established as an important intracellular signaling in regulating cell survival. The current studies test the hypothesis that polyamines are involved in the control of Akt activity in normal intestinal epithelial cells (IEC-6 line) and that activated Akt mediates suppression of apoptosis following polyamine depletion. Depletion of cellular polyamines by alpha-difluoromethylornithine induced levels of phosphorylated Akt and increased Akt kinase activity, although it had no effect on expression of total Akt, pERK, p38, and Bcl-2 proteins. This activated Akt was associated with both decreased levels of active caspase-3 and increased resistance to tumor necrosis factor-alpha/cycloheximide-induced apoptosis. Inactivation of Akt by either treatment with LY294002 or ectopic expression of a dominant negative Akt mutant (DNMAkt) not only enhanced the caspase-3 activation in polyamine-deficient cells but also prevented the increased resistance to tumor necrosis factor-alpha/cycloheximide-induced apoptosis. Phosphorylation of glycogen synthase kinase-3, a downstream target of Akt, was also increased in alpha-difluoromethylornithine-treated cells, which was prevented by inactivation of Akt by LY294002 or DNMAkt overexpression. These results indicate that polyamine depletion induces the Akt activation mediating suppression of apoptosis via inhibition of caspase-3 in normal intestinal epithelial cells.     

Csnk1a1 inhibition modulates the inflammatory secretome and enhances response to radiotherapy in glioma

Glioblastoma multiforme (GBM), a fatal brain tumour with no available targeted therapies, has a poor prognosis. At present, radiotherapy is one of the main methods to treat glioma, but it leads to an obvious increase in inflammatory factors in the tumour microenvironment, especially IL-6 and CXCL1, which plays a role in tumour to resistance radiotherapy and tumorigenesis. Casein kinase 1 alpha 1 (CK1α) (encoded on chromosome 5q by Csnk1a1) is considered an attractive target for Tp53 wild-type acute myeloid leukaemia (AML) treatment. In this study, we evaluated the anti-tumour effect of Csnk1a1 suppression in GBM cells in vitro and in vivo. We found that down-regulation of Csnk1a1 or inhibition by D4476, a Csnk1a1 inhibitor, reduced GBM cell proliferation efficiently in both Tp53 wild-type and Tp53-mutant GBM cells. On the contrary, overexpression of Csnk1a1 promoted cell proliferation and colony formation. Csnk1a1 inhibition improved the sensitivity to radiotherapy. Furthermore, down-regulation of Csnk1a1 reduced the production and secretion of pro-inflammatory factors. In the preclinical GBM model, treatment with D4476 significantly inhibited the increase in pro-inflammatory factors caused by radiotherapy and improved radiotherapy sensitivity, thus inhibiting tumour growth and prolonging animal survival time. These results suggest targeting Csnk1a1 exert an anti-tumour role as an inhibitor of inflammatory factors, providing a new strategy for the treatment of glioma.     

Akt, MAPK (Erk1/2), and p38 act in concert to promote apoptosis in response to ErbB receptor family inhibition

The ErbB receptor family is implicated in the malignant transformation of several tumor types and is overexpressed frequently in breast, ovarian, and other tumors. The mechanism by which CI-1033 and gemcitabine, either singly or in combination, kill tumor cells was examined in two breast lines, MDA-MB-453 and BT474; both overexpress the ErbB-2 receptor. CI-1033, a potent inhibitor of the ErbB family of receptor tyrosine kinases, reduced levels of activated Akt in MDA-MB-453 cells. This effect alone, however, did not induce apoptosis in these cells. Gemcitabine treatment resulted in a moderate increase in the percentage of apoptotic cells that was accompanied by activation of p38 and MAPK (ERK1/2). CI-1033 given 24 h after gemcitabine produced a significant increase in the apoptotic fraction over treatment with either drug alone. During the combined treatment p38 remained activated, whereas Akt and activated MAPK were suppressed. Substitution of CI-1033 with the phosphatidylinositol 3-kinase inhibitor LY294002 and the MAPK/ERK kinase inhibitor PD 098059 in combination with gemcitabine produced the same results as the combination of CI-1033 and gemcitabine. p38 suppression by SB203580 prevented the enhanced cell kill by CI-1033. In contrast to MDA-MB-453, BT474 cells exhibited activated p38 under unstressed conditions as well as activated Akt and MAPK. Treatment of BT474 cells with CI-1033 inhibited both the phosphorylation of Akt and MAPK and resulted in a 47% apoptotic fraction. Gemcitabine did not cause apoptosis in the BT474 cells. These data indicate that suppression of Akt and MAPK in the presence of activated p38 results in cell death and a possible mechanism for the enhanced apoptosis produced by the combination of CI-1033 and gemcitabine in MDA-MB-453 cells. Furthermore, tumors that depend on ErbB receptor signaling for survival and exhibit activated p38 in the basal state may be susceptible to apoptosis by CI-1033 as a single agent.     

Autophagy inhibition enhances celecoxib-induced apoptosis in osteosarcoma

Osteosarcoma (OS) is the most prevalent bone malignancy in childhood and adolescence, with highly aggressive and early systemic metastases. Here, we reported that celecoxib, a selective COX-2 inhibitor in the NSAID class, exhibits strong antitumor activity in dose dependent manner in two OS cell lines-143B and U2OS. We showed that celecoxib inhibits OS cell growth, causes G0/G1-phase arrest, modulates apoptosis and autophagy and reduces migration in OS cells. In addition, the results of fluorescent mitochondrial probe JC-1 test indicated that the mitochondrial pathway mediates celecoxib-induced apoptosis. Significantly, the autophagy inhibitor CQ combined with celecoxib causes greater cell proliferation inhibition and apoptosis. Pharmacologic inhibition of autophagy with another potent autophagy inhibitor SAR405 also enhances celecoxib-mediated suppression of cell viability. These results were confirmed with shRNAs targeting the autophagy-related gene Atg5. In OS tumor xenografts in vivo, celecoxib also presents antitumor activity. Taken together, our results shed light on the function and mechanism of antitumor action of celecoxib for treatment of OS patients.     

Emerging roles of caspase-3 in apoptosis

Caspases are crucial mediators of programmed cell death (apoptosis). Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins. However, the specific requirements of this (or any other) caspase in apoptosis have remained largely unknown until now. Pathways to caspase-3 activation have been identified that are either dependent on or independent of mitochondrial cytochrome c release and caspase-9 function. Caspase-3 is essential for normal brain development and is important or essential in other apoptotic scenarios in a remarkable tissue-, cell type- or death stimulus-specific manner. Caspase-3 is also required for some typical hallmarks of apoptosis, and is indispensable for apoptotic chromatin condensation and DNA fragmentation in all cell types examined. Thus, caspase-3 is essential for certain processes associated with the dismantling of the cell and the formation of apoptotic bodies, but it may also function before or at the stage when commitment to loss of cell viability is made.     

Parthenolide protects human lens epithelial cells from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and caspase-9

The apoptosis of lens epithehal cells has been proposed as the common basis of cataract formation, with oxidative stress as the major cause. This study was performed to investigate the protective effect of the herbal constituent parthenolide against oxidative stress-induced apoptosis of human lens epithelial （HLE） cells and the possible molecular mechanisms involved. HLE cells （SRA01-04） were incubated with 50 μM H2O2 in the absence or presence of different doses of parthenolide （10, 20 and 50 μM）. To study apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining flow cytometry; to investigate the underlying molecular mechanisms, the expression of caspase-3 and caspase-9 were assayed by Western blot and quantitative RT-PCR, and the activities of caspase-3 and caspase-9 were measured by a Chemicon caspase colorimetric activity assay kit. Stimulated with H202 for 18 h, a high fraction of riLE cells underwent apoptosis, while in the presence ofparthenolide of different concentrations, dose-dependent blocking of HLE cell apoptosis was observed. The expression of caspase-3 and caspase-9 induced by H202 in HLE cells was significantly reduced by parthenolide both at the protein and mRNA levels, and the activation ofcaspase-3 and caspase-9 was also suppressed by parthenolide in a dose-dependent manner. In conclusion, parthenolide prevents HLE cells from oxidative stress-induced apoptosis through inhibition of the activation ofcaspase-3 and caspase-9, suggesting a potential protective effect against cataract formation.     

Eriocalyxin B induces apoptosis of t(8;21) leukemia cells through NF-kappaB and MAPK signaling pathways and triggers degradation of AML1-ETO oncoprotein in a caspase-3-dependent manner

Diterpenoids isolated from Labiatae family herbs have strong antitumor activities with low toxicity. In this study, Eriocalyxin B (EriB), a diterpenoid extracted from Isodon eriocalyx, was tested on human leukemia/lymphoma cells and murine leukemia models. Acute myeloid leukemia cell line Kasumi-1 was most sensitive to EriB. Significant apoptosis was observed, concomitant with Bcl-2/Bcl-XL downregulation, mitochondrial instability and caspase-3 activation. AML1-ETO oncoprotein was degraded in parallel to caspase-3 activation. EriB-mediated apoptosis was associated with NF-kappaB inactivation by preventing NF-kappaB nuclear translocation and inducing IkappaBalpha cleavage, and disturbance of MAPK pathway by downregulating ERK1/2 phosphorylation and activating AP-1. Without affecting normal hematopoietic progenitor cells proliferation, EriB was effective on primary t(8;21) leukemia blasts and caused AML1-ETO degradation. In murine t(8;21) leukemia models, EriB remarkably prolonged the survival time or decreased the xenograft tumor size. Together, EriB might be a potential treatment for t(8;21) leukemia by targeting AML1-ETO oncoprotein and activating apoptosis pathways.     

Tolerance of mice to lipopolysaccharide is correlated with inhibition of caspase-3-mediated apoptosis in mouse liver cells

Bacterial endotoxin lipopolysaccharide (LPS) often results in multiple organ failure.However,pre-exposure of mice to a sublethal dose of LPS renders the animal tolerant to a lethal dose of LPS.Thisstudy was designed to determine whether pre-exposure of a small dose of LPS was able to suppressapoptosis in mice when challenged with LPS in combination with D-galactosamine,and to investigate theexpression changes of the apoptosis-associated molecules.The results showed that a characteristic apoptoticDNA fragmentation existed in mouse livers of the LPS-naive group,but not in control groups;and the miceof the LPS-naive group were all dead after 2 d.However,in the LPS-tolerance groups,both the lethal rateand apoptotic DNA fragmentation were suppressed after the mice were challenged with LPS/D-galactosamine,and the protection against the lethality and apoptotic reaction could be maintained for up to 7 d.In thisperiod, significantly lower levels of caspase-3 and its mRNA appeared in LPS-tolerant groups compared tothose of the LPS-naive group (P<0.05),and the caspase-3 activities gradually recovered as the observationwas prolonged.Our findings suggest that LPS tolerance could suppress apoptosis in mouse liver cells,andthe expression and activity of caspase-3 could be down-regulated.     

sMEK1 enhances gemcitabine anti-cancer activity through inhibition of phosphorylation of Akt/mTOR

Recently, we reported that sMEK1 is down-regulated in cancer cells and tissues, and that it enhances the pro-proliferative effect as a novel pro-apoptotic protein. However, the biological mechanism of the sMEK1 tumor suppressor in the cellular signal pathway has not been well understood. In our current work, we examined whether sMEK1 could promote the cytotoxic activity of gemcitabine in the human ovarian carcinoma system. Initially, we attempted to use a treatment of gemcitabine traditional chemotherapeutic agent and over-expression of sMEK1 in OVCAR-3 cancer cells. The combined treatment of sMEK1 and gemcitabine was more effective at inhibiting cell proliferation than either chemotherapeutic agent treatment alone. In addition, sMEK1 actively contributes to cell migration through its ability to promote gemcitabine-inhibited cell migration in tumorigenesis. Cell cycle-related proteins are highly associated with the down-regulation of cyclin D1 and CDK4, and the promotion of p16 and p27 as a cyclin-dependent kinase inhibitor. At the same time, sMEK1 arrests cell cycle progression in the G(1)-G(0) phase, and activates p53 and p21 expression, whereas Bcl-2 and Bcl-xL protein expression is reduced. Additionally, sMEK1 and gemcitabine suppresses the phosphorylation of signaling modulators downstream of PI3K, such as PDK1 and Akt. The p53 and p21 promoter luciferase activities were promoted by either sMEK1 or gemcitabine, and sMEK1 and gemcitabine combined additively activated the promoter further. Furthermore, as expected, sMEK1 plus gemcitabine markedly reduced the phosphorylation of p70S6K and the phosphorylation of 4E-BP1, which is one of the best characterized targets of the mTOR complex cascade. Taken together, these results provide evidence that sMEK1 can effectively regulate the pro-apoptotic activity of gemcitabine through the up-regulation of p53 expression.     

Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis

Autophagy and apoptosis are both highly regulated biological processes that play essential roles in tissue homeostasis, development and diseases. Autophagy is also described as a mechanism of death pathways, however, the precise mechanism of how autophagy links to cell death remains to be fully understood. Beclin 1 is a dual regulator for both autophagy and apoptosis. In this study we found that Beclin 1 was a substrate of caspase-3 with two cleavage sites at positions 124 and 149, respectively. Furthermore, the autophagosome formation occurred, followed by the appearance of morphological hallmarks of apoptosis after staurosporine treatment. The cleavage products of Beclin 1 reduced autophagy and promoted apoptosis in HeLa cells and the cells in which Beclin 1 was stably knocked down by specific shRNA. In addition, the cleavage of Beclin 1 resulted in abrogating the interaction between Bcl-2 with Beclin 1, which could be blocked by z-VAD-fmk. Thus, our results suggest that the cleavage of Beclin 1 by caspase-3 may contribute to inactivate autophagy leading towards augmented apoptosis.