Caspase-3 protease activation during the process of genistein-induced apoptosis in TM4 testicular cells

The role of caspase-3 (CPP32) protease in the molecular pathways of genistein-induced cell death in TM4 cells was investigated. Fluorescence microscopy with Hoechst-33258-PI nuclear stain was used to distinguish between apoptosis and necrosis pathways of cell death. The viability of the test cells was assessed with both the trypan blue exclusion and MTT tetrazolium (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetralzolium bromide, 2.5 mg/mL) assays. Caspase-3 enzymatic activity was determined using CasPASE Apoptosis Assay Kit. The overall results from all the data demonstrated that: i) genistein exerts dose- and time-dependent effects on TM4 testis cells; ii) apoptosis is induced by lower concentrations of genistein and necrosis induced by higher concentrations of genistein; iii) genistein induced activation caspase-3 enzymatic activity; iv) genistein-induction of apoptosis and necrosis was significantly inhibited by the caspase-3 inhibitor, z-DEV-FMK; v) sodium azide induced necrosis without activation of CPP32 enzymatic activity, and induction of apoptosis; and vi) genistein-induced apoptosis was associated with activation of CPP32 enzymatic activity in the cells. The overall results indicate a strong evidence of caspase-3 (CPP332) mediation in the molecular pathways of genistein-induced apoptosis in testicular cells. Apoptosis is the physiologically programmed cell death in which intrinsic mechanisms participate in the death of the cell, in contrast to necrosis, which induces inflammatory response in the affected cell. The fact that the chemopreventive role of several cancer drugs is due to induction of apoptosis augments the biotherapeutic potential of genistein for the treatment of malignant diseases including prostate and testicular cancers. It is therefore inevitable that identification of the apoptotic pathways and the points at which regulation occurs could be instrumental in the design of genistein biotherapy for such diseases.     

Immunohistochemical study of cell proliferation, Bcl-2, p53, and caspase-3 expression on preneoplastic changes induced by cadmium and zinc chloride in the ventral rat prostate.

This work was directed to evaluate immunoexpression of markers for apoptosis, resistance to apoptosis, and cell proliferation, as well as estimates of nuclear size in ventral prostate of rats treated with cadmium chloride and cadmium+zinc chloride because a possible protective effect of zinc has been postulated. The following variables were studied: volume fraction (VF) of Bcl-2 immunostaining, percentage of cells immunoreactive to proliferating cell nuclear antigen (LIPCNA) and p53 (LIp53), numerical density of caspase-3 immunoreactive cells (NV caspase-3), and estimates of volume-weighted mean nuclear volume (upsilonV). The LIPCNA and VF of Bcl-2 were significantly increased in the treated animals. The dysplasias (independent of their origin) showed a significant increase of the LIp53, NV caspase-3, and upsilonV in comparison with normal acini from treated and control animals. It can be concluded that cell proliferation is enhanced in long-term cadmium-exposed rats, and exposure to zinc combined with cadmium had no effect on any of the variables studied when comparing with normal acini. The increase of nuclear upsilonV could indicate a more aggressive behavior for pretumoral lesions.     

Lysophosphatidic acid inhibits anti-Fas-mediated apoptosis enhanced by actin depolymerization in epithelial ovarian cancer

Conflicting reports exist on the effect of actin depolymerization in anti-Fas-induced apoptosis. Lysophosphatidic acid (LPA) has been found to inhibit apoptosis in variable cell types. In this study, we evaluated LPA's protective effects on anti-Fas-induced apoptosis enhanced by actin depolymerization and possible mechanisms in epithelial ovarian cancer. OVCAR3 cells were pretreated with vehicle or LPA, then treated with Cytochalasin D (Cyto D), followed with anti-Fas mAb to induce apoptosis. Cells were stained with apoptotic markers and analyzed by flow cytometry. We report that LPA inhibited anti-Fas-induced apoptosis enhanced by actin depolymerization. Immunoprecipition of Fas death-inducing signaling complex (DISC) and Western blot suggested that the actin depolymerization accelerated caspase-8 activation, while LPA inhibited the association and activation of caspase-8 at the DISC. LPA inhibited caspase-3 and 7 activation induced by anti-Fas and/or Cyto D in cytosols. Phosphorylation of ERK and Bad112 by LPA may play a role in preventing caspase-3 activation through mitochondrial pathway induced by Cyto D. Our investigation found that LPA inhibited anti-Fas-induced apoptosis enhanced by actin depolymerization, and LPA may protect epithelial ovarian cancer from immune cell attack and cytoskeleton disrupting reagents induced apoptosis through multiple pathways.     

Genistein induces apoptosis and autophagy in human breast MCF-7 cells by modulating the expression of proapoptotic factors and oxidative stress enzymes

Breast cancer is one of the common tumors occurring in woman and despite treatment, the prognostic is poor. Genistein, a soy isoflavone, has been reported to have chemopreventive\chemotherapeutic potential in multiple tumor types. Here, we investigated the genistein antiproliferative effect in MCF-7 breast cancer, underlying the molecular mechanisms involved in this effect. MCF-7 cancer and CCD1059sK fibroblast cells were treated with estradiol (10 nM) or genistein (0.01–100 μM) for 24, 48, and 72 h and the cell proliferation was investigated by MTT; membrane cell permeability was evaluated by LDH and PI incorporation; apoptosis was investigated by externalization of phosphatidylserine by FACS; and presence of autophagy was detected by LC3A/B immunostaining. The expression of apoptotic proteins and antioxidant enzymes was evaluated by qPCR. The results demonstrate that genistein (100 μM) for 72 h of treatment selectively reduced MCF-7 cell proliferation independent of estrogen receptor activation, while no cytotoxicity was observed in fibroblast cells. Further experiments showed that genistein induced phosphatidylserine externalization and LC3A/B immunopositivity in MCF-7 cells, indicating apoptosis and autophagy cell death. Genistein increased in three times proapoptotic BAX/Bcl-2 ratio and promoted a parallel downregulation of 20 times of antiapoptotic survivin. In addition, genistein promoted a decrease of 5.5, 9.3, and 3.6 times of MnSOD, CuZnSOD, and TrxR mRNA expression, respectively, while the GPx expression was increased by 6.5 times. These results suggest that the antitumor effect of genistein involved the modulation of antioxidant enzyme and apoptotic signaling expression, which resulted in apoptosis and progression of autophagy.     

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。     

Celastrol inhibits tumor cell proliferation and promotes apoptosis through the activation of c-Jun N-terminal kinase and suppression of PI3 K/Akt signaling pathways

Celastrol, a plant triterpene has attracted great interest recently, especially for its potential anti-inflammatory and anti-cancer activities. In the present report, we investigated the effect of celastrol on proliferation of various cancer cell lines. The mechanism, by which this triterpene exerts its apoptotic effects, was also examined in detail. We found that celastrol inhibited the proliferation of wide variety of human tumor cell types including multiple myeloma, hepatocellular carcinoma, gastric cancer, prostate cancer, renal cell carcinoma, head and neck carcinoma, non-small cell lung carcinoma, melanoma, glioma, and breast cancer with concentrations as low as 1 μM. Growth inhibitory effects of celastrol correlated with a decrease in the levels of cyclin D1 and cyclin E, but concomitant increase in the levels of p21 and p27. The apoptosis induced by celastrol was indicated by the activation of caspase-8, bid cleavage, caspase-9 activation, caspase-3 activation, PARP cleavage and through the down regulation of anti-apoptototic proteins. The apoptotic effects of celastrol were preceded by activation of JNK and down-regulation of Akt activation. JNK was needed for celastrol-induced apoptosis, and inhibition of JNK by pharmacological inhibitor abolished the apoptotic effects. Overall, our results indicate that celastrol can inhibit cell proliferation and induce apoptosis through the activation of JNK, suppression of Akt, and down-regulation of anti-apoptotic protein expression.     

Genistein enhances TRAIL-induced apoptosis through inhibition of p38 MAPK signaling in human hepatocellular carcinoma Hep3B cells

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some carcinoma cancer cells. However, it was found that treatment with TRAIL in combination with nontoxic concentrations of genistein sensitized TRAIL-resistant human hepatocellular carcinoma Hep3B cells to TRAIL-mediated apoptosis. Combined treatment with genistein and TRAIL-induced chromatin condensation and sub-G1 phase DNA content. These indicators of apoptosis were correlated with the induction of caspase activity that resulted in the cleavage of poly(ADP-ribose) polymerase (PARP). Both cell viability and the cleavage of PARP induced by combined treatment were significantly inhibited by caspase-3, -8 and -9 inhibitors, which demonstrates the important roles of caspases in the observed cytotoxic effects. Genistein treatment also triggered the inhibition of p38-β mitogen-activated protein kinase (MAPK) activation. Pretreatment with SB203580 resulted in significantly increased sub-G1 population and loss of mitochondrial membrane potential (MMP) in TRAIL-induced apoptosis. By contrast, overexpression of p38 MAPK protected apoptosis by co-treatment with genistein and TRAIL, suggesting that the p38 MAPK act as key regulators of apoptosis in response to treatment with a combination of genistein and TRAIL in human hepatocellular carcinoma Hep3B cells.     

The mechanisms of lipoxygenase inhibitor-induced apoptosis in human breast cancer cells

Previous experimental studies have shown that high dietary fat intake is associated with mammary carcinogenesis. In the current study, the effect of 5-LOX or 12-LOX inhibitors on human breast cancer cell proliferation and apoptosis, as well as the possible mechanisms were investigated. The LOX inhibitors, NDGA, Rev-5901, and baicalein all inhibited proliferation and induced apoptosis in MCF-7 (ER+) and MDA-MB-231 (ER-) breast cancer cell in vitro. In contrast, the LOX products, 5-HETE and 12-HETE had mitogenic effects, stimulating the proliferation of both cell lines. These inhibitors also induced cytochrome c release, caspase-9 activation, as well as downstream caspase-3, caspase-7 activation, and PARP cleavage. LOX inhibitor treatment also reduced the levels of anti-apoptotic proteins Bcl-2 and Mcl-1 and increased the levels of the pro-apoptotic protein bax. In conclusion, blockade of both 5-LOX and 12-LOX pathways induces apoptosis in breast cancer cells through the cytochrome c release and caspase-9 activation, with changes in the levels of Bcl-2 family proteins.     

Pro-apoptotic effect and cytotoxicity of genistein and genistin in human ovarian cancer SK-OV-3 cells

We investigated the effects of genistein and genistin on proliferation and apoptosis of human ovarian SK-OV-3 cells and explored the mechanism for these effects. SK-OV-3 cells were treated with genistein and genistin at various concentrations (ranging from 1 to 100 muM) either alone or in combination for 24 and 48 h. Cell proliferation was estimated using an MTT assay, and cell cycle arrest was evaluated using FACS. Caspase-3 activity and annexin-based cell cycle analysis were used as measures of apoptosis. In addition, genistein- and genistin-induced cytotoxicity was determined by measuring release of LDH. Genistein treatment for 24 or 48 h substantially inhibited SK-OV-3 cell proliferation in a dose-dependent manner, and genistin treatment for 48 h also inhibited cell proliferation. Genistein caused cell cycle arrest at G2/M phase in dose- and time-dependent manner, and genistin caused cell cycle arrest not only at G2/M phase but also at G1 phase. Genistein markedly induced apoptosis and significantly increased LDH release, whereas genistin did not affect LDH release. Moreover, exposure to both genistein and genistin in combination for 48 h induced apoptosis without increasing LDH release. Genistein and genistin inhibit cell proliferation by disrupting the cell cycle, which is strongly associated with the arrest induction of either G1 or G2/M phase and may induce apoptosis. Based on our findings, we speculate that both genistein and genistin may prove useful as anticancer drugs and that the combination of genistein and genistin may have further anticancer activity.     

Thyroid hormone is a MAPK-dependent growth factor for thyroid cancer cells and is anti-apoptotic

Thyroid hormone (l-thyroxine, T(4), or 3,5,3'-triiodo-l-thyronine, T(3)) treatment of human papillary and follicular thyroid cancer cell lines resulted in enhanced cell proliferation, measured by proliferating cell nuclear antigen (PCNA). Thyroid hormone also induced activation of the Ras/MAPK (ERK1/2) signal transduction pathway. ERK1/2 activation and cell proliferation caused by thyroid hormone were blocked by an iodothyronine analogue, tetraiodothyroacetic acid (tetrac), that inhibits binding of iodothyronines to the cell surface receptor for thyroid hormone on integrin alphaVbeta3. A MAPK cascade inhibitor at MEK, PD 98059, also blocked hormone-induced cell proliferation. We then assessed the possibility that thyroid hormone is anti-apoptotic. We first established that resveratrol (10 microM), a pro-apoptotic agent in other cancer cells, induced p53-dependent apoptosis and c-fos, c-jun and p21 gene expression in both papillary and follicular thyroid cancer cells. Induction of apoptosis by the stilbene required Ser-15 phosphorylation of p53. Resveratrol-induced gene expression and apoptosis were inhibited more than 50% by physiological concentrations of T(4). T(4) activated MAPK in the absence of resveratrol, caused minimal Ser-15 phosphorylation of p53 and did not affect c-fos, c-jun and p21 mRNA abundance. Thus, plasma membrane-initiated activation of the MAPK cascade by thyroid hormone promotes papillary and follicular thyroid cancer cell proliferation in vitro.     

Serum deprivation increases ceramide levels and induces apoptosis in undifferentiated HN9.10e cells.

Sphingolipid metabolites have been involved in the regulation of proliferation, differentiation and apoptosis. While cellular mechanisms of these processes have been extensively analysed in the post-mitotic neurons, little is known about proliferating neuronal precursors. We have taken as a model of neuroblasts the embryonic hippocampal cell line HN9.10e. Apoptosis was induced by serum deprivation and by treatment with N-acetylsphingosine (C2-Cer), a membrane-permeant analogue of the second messenger ceramide. Following C2-Cer addition, cytochrome c was released from mitochondria, [Ca(2+)](i) and caspase-3-like activity increased. Both cytochrome c release and rise of [Ca(2+)](i) occurred before caspase-3 activation and nuclear condensation. The intracellular levels of ceramide peaked at 1h following the serum deprivation. These results indicate that the serum deprivation induces a rise in the intracellular ceramide level, and that increased ceramide concentration leads to calcium dysregulation and release of cytochrome c followed by caspase-3 activation. We show that cytochrome c is released without a loss of mitochondrial transmembrane potential.     

Isoflavone genistein protects human vascular endothelial cells against tumor necrosis factor-α-induced apoptosis through the p38β mitogen-activated protein kinase

Isoflavone genistein may have beneficial effects on vascular function, but the mechanism is unclear. Here, we investigated whether genistein protects vascular endothelial cells against apoptosis induced by tumor necrosis factor-α. We show that genistein significantly inhibited TNF-α-induced apoptosis in human aortic endothelial cells as determined by caspase-3 activation, 7-amino actinomycin D staining, in situ apoptotic cell detection and DNA laddering. The anti-apoptotic effect of genistein was associated with an enhanced expression of Bcl-2 protein and its promoter activity. Inhibition of extracellular signal-regulated kinase 1/2, protein kinase A, or estrogen receptors had no effect on the cytoprotective effect of genistein. However, inhibition of p38 mitogen-activated protein kinase (p38) completely abolished this genistein effect. Accordingly, stimulation of HAECs with genistein resulted in rapid activation of p38β, but not p38α. These findings provide the evidence that genistein acts as a survival factor for vascular ECs to protect cells against apoptosis via activation of p38β. Preservation of the functional integrity of the endothelial monolayer may represent an important mechanism by which genistein exerts its vasculoprotective effect.     

Fas-mediated apoptosis of proliferating, transiently growth-arrested, and senescent normal human fibroblasts

Previous studies suggest that apoptotic signaling may require proteins that are critical to cellular proliferation and cell cycle regulation. To further examine this question, proliferating, transiently growth-arrested, and senescent normal human fibroblasts were induced to undergo apoptosis in response to two distinct mediators of apoptosis-Fas (APO-1/CD95) death receptor and staurosporine. Ligation of the Fas receptor in the presence of cycloheximide or actinomycin D resulted in apoptosis of proliferating cells, cells transiently growth arrested by gamma-irradiation or serum starvation (i.e., G(0) arrest), and permanently growth-arrested senescent fibroblasts. Proliferating and G(0)-arrested cells were also susceptible to staurosporine-mediated apoptosis. Surprisingly, gamma-irradiated cells did not undergo staurosporine-mediated apoptosis, and remained viable for a prolonged time. Fas-mediated apoptosis of senescent fibroblasts was evidenced by chromosome condensation and by activation of caspase-8 and -3, proteases crucial for the execution of the Fas apoptosis pathway. In addition, ligation of the Fas receptor in G(0)-arrested cells did not result in the activation of p34(cdc2) kinase, arguing that activation of this kinase is not essential in this apoptotic process. From these studies we conclude that proliferating, transiently growth-arrested, and senescent normal human fibroblasts are susceptible to apoptotic signals and that apoptosis is not necessarily dependent upon cell cycle or proliferative state of the cell.     

Low dose leflunomide activates PI3K/Akt signalling in erythroleukemia cells and reduces apoptosis induced by anticancer agents

Rheumatoid arthritis (RA) is characterized by persistent joint synovial tissue inflammation. Leflunomide is an immunomodulatory agent that has been approved for treatment of active RA. In the past few years, uses other than RA treatment have appeared. Leflunomide has been reported to show antitumor potential through inhibition of cancer cell proliferation. We thus tested the antiproliferative potential of leflunomide on HEL and K562 erythroleukemia cells. The findings summarized in this report demonstrate for the first time that low dose leflunomide prolonged survival and reduced apoptosis induced by several anticancer agents in erythroleukemia cells. We showed that in treated cells, leflunomide reduced the signalling pathways involved in promoting apoptosis by reducing p38 MAPK and JNK basal activity. On the other hand, leflunomide transiently activated the ERK signalling pathway and induced a sustained activation of Akt. We also showed that leflunomide reduced caspase-3 activity and DNA fragmentation induced by anticancer agents. By using an inhibitory strategy, we showed that inhibition of Akt activation but not ERK abolished the protective effect of leflunomide. Thus our findings suggested that leflunomide reduced apoptosis induced by anticancer agents through PI3K/Akt signalling activation.     

Dysregulation of apoptosis by benzene metabolites and their relationships with carcinogenesis

Benzene is a widely recognized human carcinogen, the effect of which is attributed to the production of reactive oxygen species (ROS) from its metabolites. Although there have been many reports on the relationship between DNA damage induced by benzene metabolites and carcinogenesis, only a report approached the subject by examining the benzene-induced dysregulation of apoptosis. Inhibition of apoptosis, aberrantly prolonging cell survival, may contribute to cancer by facilitating the insurgence of mutations and by creating a permissive environment for genetic instability. In this study, we examined the mechanism of antiapoptotic effects by benzene metabolites, p-benzoquinone (BQ) and hydroquinone (HQ), and their relationships with carcinogenesis. BQ and HQ inhibited the apoptotic death of NIH3T3 cells induced by both serum starvation and lack of an extracellular matrix (ECM). An antioxidant agent, N-acetylcysteine, significantly inhibited the antiapoptotic effects induced by benzene metabolites, indicating that the effects were mainly due to the production of ROS. Furthermore, BQ and HQ inhibited the in vitro caspase-3 activation, suggesting that the inhibition of caspase-3 activation due to ROS produced by BQ- and HQ-treatment was related to the suppression of apoptosis. The cells that escaped apoptosis could survive with the addition of serum and attachment to the ECM. Levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine were higher in the cells which survived after BQ- and HQ-treatment than in the normal cells. Furthermore, the cells treated with BQ and HQ showed greater proliferation than normal cells under low-serum conditions and anchorage-independent growth in soft agar. These findings suggested that benzene metabolites induced dysregulation of apoptosis due to caspase-3 inhibition, which contributes to carcinogenesis.     

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.