Upon drug-induced apoptosis expression of prostate-apoptosis-response-gene-4 promotes cleavage of caspase-8, bid and mitochondrial release of cytochrome c

Par-4 functions as a tumor suppressor antagonizing the transforming capacity and the resistance of malignant cells towards apoptotic stimuli. After demonstrating that par-4 promotes apoptosis by activating signaling of the intrinsic pathway of apoptosis, we hypothesized that par-4 also impacts on key molecules of the extrinsic pathway without the requirement of a receptor/ligand interaction. Here, we provide first evidence, that expression of par-4 increases cleavage of caspase-8, truncation of Bid and its translocation to the mitochondria, resulting in an augmentation of cytochrome c and AIF efflux into the cytosol, effects par-4-positive cells are able to retain to a higher extent than par-4-negative cells upon inhibition of caspase-3 activation.     

Prostate apoptosis response gene-4 sensitizes neoplastic lymphocytes to CD95-induced apoptosis

Evaluating the functional consequences of prostate apoptosis response gene-4 (par-4) expression in CD95-induced apoptosis of neoplastic lymphocytes, we demonstrate that par-4 increases apoptosis by upregulating the CD95 receptor on the cell surface and—with a concomitant decrease of the FLICE-like inhibitory protein (FLIP)—by promoting cleavage of the initiator caspases-8 and -10. This results in an enforced activation of the executioner caspases-6, -7, and -3 as well as in an activation of the mitochondrial pathway. Upon inhibition of caspase-8, overexpression of par-4 enables Jurkat cells to maintain a higher sensitivity to CD95-induced apoptosis by downregulating cIAP-2 and XIAP and by enforcing activation of the initiator caspase-10 as well as of the executioner caspases-6, -7, and -3.     

Cytochrome c release is upstream to activation of caspase-9, caspase-8, and caspase-3 in the enhanced apoptosis of anaplastic thyroid cancer cells induced by manumycin and paclitaxel

We previously demonstrated that the combination of a farnesyltransferase inhibitor, manumycin A, and paclitaxel had a synergistic antineoplastic effect on anaplastic thyroid cancer. In this study we investigated the apoptosis pathway involved. In ARO and KAT-4 cells, manumycin- plus paclitaxel-induced DNA fragmentation was blocked by the inhibitors of caspase-9, caspase-8, and caspase-3. The drug combination enhanced the activation of caspase-9, caspase-8, and caspase-3 and cytochrome c release into the cytosol. Cytochrome c release was not affected by the inhibitors of caspase-9, caspase-8 and caspase-3. In a cell-free reconstitution assay, DNA fragmentation occurred after incubating nuclei purified from untreated KAT-4 cells with deoxy-ATP, exogenous cytochrome c and S-100 extracts from control KAT-4 cells, and also after incubation of purified KAT-4 nuclei with S-100 extracts from KAT-4 cells treated with manumycin-plus-paclitaxel. In both cases, the DNA fragmentation was blocked by the inhibitors of caspase-9, caspase-8 and caspase-3. We concluded that the cytochrome c release was upstream of the activation of caspase-9, caspase-8, and caspase-3 in the enhanced apoptosis of anaplastic thyroid cancer cells treated with manumycin plus paclitaxel, and that the interaction between manumycin and paclitaxel occurred at or upstream of cytochrome c in the apoptosis regulatory pathway in anaplastic thyroid cancer cells.     

复方丹参注射液对H2O2诱导的PC12细胞凋亡的保护作用

目的　探讨复方丹参注射液 (ISM)对 H2 O2 诱导的 PC1 2细胞凋亡的保护作用机制。方法　在 H2 O2 诱导 PC1 2细胞凋亡模型的基础上 ,采用 MTT比色分析测定细胞存活率 ,Hoechst- PI荧光染色和流式细胞仪检测分析细胞凋亡情况 ,RT- PCR检测 par- 4和 caspase- 3基因 m RNA表达 ,Western blot检测 par- 4蛋白表达和 caspase- 3P2 0活性片段 ,比色法检测 caspase- 3相对活性。结果　不同剂量 ISM预处理 1 h可提高 PC1 2细胞存活率 ,降低 par- 4和 caspase- 3的 m RNA表达以及 Par- 4的蛋白表达 ,caspase- 3 P2 0活性片段和 caspase- 3相对活性减少 ,但变化不明显。结论　 ISM可剂量依赖性地对抗 H2 O2的神经毒性作用 ,其机制可能与凋亡基因 par- 4表达有关 ,与 caspase- 3的关系尚需进一步探讨。     

Prostate apoptosis response gene-4 (par-4) abrogates the survival function of p185(BCR-ABL) in hematopoietic cells

OBJECTIVE: Prostate apoptosis response gene-4 (par-4) is deregulated in acute and chronic lymphatic leukemia. Given its pro-apoptotic role in neoplastic lymphocytes and evidence that par-4 antagonizes oncogenic Ras in solid tumors, we hypothesized that par-4 may act as a tumor suppressor impairing transformation induced by p185(BCR-ABL). MATERIALS AND METHODS: The capacity of par-4 to interfere with factor independence induced by p185(BCR-ABL) and V12ras was evaluated by analysis of factor-independent growth of p185(BCR-ABL)/ par-4 and V12ras/par-4 transduced cells. The expression of par-4 and p185(BCR-ABL) by the respective constructs was controlled by Western blot analysis. Activated Ras was detected by pull-down assay in the cell clones expressing p185(BCR-ABL) in the absence and presence of par-4. RESULTS: Expression of p185(BCR-ABL) causes factor independence, signifying a conversion toward a transformed phenotype in hematopoietic precursors. We demonstrate that par-4 completely abolishes factor independence induced by p185(BCR-ABL) and partially abrogates factor independence caused by activated V12ras. Evaluating the underlying molecular mechanisms, we show that par-4 hinders activation of oncogenic Ras and causes concomitant disruptions of p185(BCR-ABL)-mediated signaling. CONCLUSION: We provide the first evidence that par-4 exhibits an antitransforming capacity by antagonizing p185(BCR-ABL)-induced factor-independent proliferation in hematopoietic cells.     

Apaf-1 and caspase-9 are required for cytokine withdrawal-induced apoptosis of mast cells but dispensable for their functional and clonogenic death

Cytokines promote survival of mast cells by inhibiting apoptotic pathways regulated by the Bcl-2 protein family. We previously showed that lymphocyte apoptosis can proceed via a Bcl-2-inhibitable pathway independent of the canonical initiator caspase, caspase-9, and its adaptor, Apaf-1. Here we report that mast cells lacking caspase-9 or Apaf-1 are refractory to apoptosis after cytotoxic insults but still lose effector function and ability to proliferate. In response to cytokine deprivation or DNA damage, fetal liver-derived mast cells lacking Apaf-1 or caspase-9 failed to undergo apoptosis. Nevertheless, the cytokine-starved cells were not functionally alive, because, unlike those overexpressing Bcl-2, they could not degranulate on Fcepsilon receptor stimulation or resume proliferation on re-addition of cytokine. Furthermore, mast cells lacking Apaf-1 or caspase-9 had no survival advantage over wild-type counterparts in vivo. These results indicate that the Apaf-1/caspase-9-independent apoptotic pathway observed in lymphocytes is ineffective in cytokine-deprived mast cells. However, although Apaf-1 and caspase-9 are essential for mast cell apoptosis, neither is required for the functional or clonogenic death of the cells, which may be due to mitochondrial dysfunction.     

Stable expression of human immunodeficiency virus type 1 Nef confers resistance against Fas-mediated apoptosis

To investigate the effect of Nef on Fas-mediated apoptosis, we compared T cells, both population and subclones stably expressing Nef from HIV-1(NL432), with Nef(-) control cells. Fas-mediated apoptosis was significantly delayed in Nef(+) cells as determined by annexin V staining and the percentage of apoptotic cells was lower in all Nef-expressing cells than in the control cells by a maximum of 10-fold. Next we measured cell surface levels of Fas to test whether the delayed apoptosis in Nef(+) cells was due to reduced cell surface expression of Fas. We found that there was no significant difference in the surface level of Fas between the Nef(+) and Nef(-) cells. To further define the steps affected by Nef in the Fas signaling pathway, the activation of caspase-3 and caspase-8 was investigated. A reasonable correlation was found between the magnitude of apoptosis measured by annexin V staining and the enzymatic activity of caspase-3. The overall level of caspase-8 activity in Nef(+) cells was also lower than in Nef(-) cells, although the extent of inhibition was not as significant as seen for caspase-3. Overall, our results indicate that long-term stable expression of Nef, which mimicks persistent or latent infection in vivo, confers resistance against anti-Fas Ab-induced apoptosis through inhibition of caspase-3 and caspase-8 activation.     

Activation of caspase-8 during N-(4-hydroxyphenyl)retinamide-induced apoptosis in Fas-defective hepatoma cells.

We observed that N-(4-hydroxyphenyl)retinamide (4HPR), a chemopreventive and chemotherapeutic agent, effectively induced apoptosis in hepatoma cells. Interestingly, Fas-negative (Hep 3B and PLC/PRF/5) hepatoma cells were shown to be more susceptible to apoptosis induced by 4HPR than were Fas-positive (Hep G2 and SK-HEP-1) hepatoma cells. Thus, we explored the mechanisms underlying 4HPR-induced apoptosis in Fas-defective hepatoma cells. Hep 3B cells stably expressing the dominant-negative Fas-associated death domain (dnFADD) showed no alteration in 4HPR drug susceptibility, but when stably expressing E1B19K, Crm A, or dominant-negative FLICE (dnFLICE), Hep 3B cells were resistant, suggesting that 4HPR-induced apoptosis was mediated by caspase-8 activation. Furthermore, apoptosis could be completely blocked by Z-VAD-FMK (a general caspase inhibitor) or by IETD-CHO (a caspase-8 inhibitor), but was only partially blocked by Ac-DEVD-CMK (a caspase-3 inhibitor), by N-acetyl-L-cysteine (NAC) (an antioxidant), by N-acetyl-leucyl-leucyl-norleucinal (ALLN) (a calpain inhibitor I), or by Z-LEHD-FMK (a caspase-9 inhibitor). Time-sequence analysis of the induction of apoptosis by 4HPR revealed that an initial caspase-8 activation was followed by late mitochondrial cytochrome c release and minor caspase-9 activation, which suggested that caspase-8 activation is the primary upstream regulatory point. Activation of Bid or induction of proapoptotic Bax was not observed during apoptosis. In contrast, Bcl-xL expression was decreased during 4HPR-induced apoptosis. Taken together, these results indicate that 4HPR may be a potential chemotherapeutic drug, which is able to induce apoptosis in Fas-defective hepatoma cells through caspase-8 activation.     

Apaf-1 and caspase-9 deficiency prevents apoptosis in a Bax-controlled pathway and promotes clonogenic survival during paclitaxel treatment

Taxane derivatives such as paclitaxel elicit their antitumor effects at least in part by induction of apoptosis, but the underlying mechanisms are incompletely understood. Here, we used different cellular models with deficiencies in key regulators of apoptosis to elucidate the mechanism of paclitaxel-induced cell death. Apoptosis by paclitaxel was reported to depend on the activation of the initiator caspase-10; however, we clearly demonstrate that paclitaxel kills murine embryonic fibroblasts (MEFs) devoid of caspase-10 as well as human tumor cell lines deficient in caspase-10, caspase-8, or Fas-associating protein with death domain. In contrast, the lack of Apaf-1 or caspase-9, key regulators of the mitochondrial pathway, not only entirely protected against paclitaxel-induced apoptosis but could even confer clonogenic survival, depending on the cell type and drug concentration. Thus, paclitaxel triggers apoptosis not through caspase-10, but via caspase-9 activation at the apoptosome. This conclusion is supported by the fact that Bcl-2-overexpressing cells and Bax/Bak doubly-deficient MEFs were entirely resistant to paclitaxel-induced apoptosis. Interestingly, also the single knockout of Bim or Bax, but not that of Bak or Bid, conferred partial resistance, suggesting a particular role of these mediators in the cell-death pathway activated by paclitaxel.     

Tauroursodeoxycholic acid inhibits apoptosis induced by Z alpha-1 antitrypsin via inhibition of Bad

Z alpha-1 antitrypsin (AAT) deficiency is a genetic disease associated with accumulation of misfolded AAT in the endoplasmic reticulum (ER) of hepatocytes. ZAAT-expressing cells display ER stress responses including nuclear factor kappaB activation and apoptosis. Using an in vitro model of ZAAT ER accumulation, we investigated the mechanism of ZAAT-mediated ER-induced apoptosis and evaluated methods to inhibit this process. Here we demonstrate that expression of ZAAT, but not normal MAAT, in HEK293 cells leads to cleavage and activation of caspase-4 and induces apoptosis that is characterized by activation of caspase-3 and caspase-7 and DNA fragmentation. Similar effects are also induced using the ER agonist thapsigargin. A caspase-4-specific short interfering RNA (siRNA) does not impair ZAAT-induced caspase-3/7 activation or cell death in these cells. However, inhibition studies performed using tauroursodeoxycholic acid (TUDCA) demonstrate its ability to inhibit caspase-4 and caspase-3/7 activation, mitochondrial cytochrome c release, and caspase-3 cleavage induced by ZAAT and to promote cell survival. The mechanism by which TUDCA (tauroursodeoxycholic acid) promotes cell survival in ZAAT-expressing cells involves phosphorylation and inactivation of the proapoptotic factor Bad. TUDCA is unable to rescue cells from apoptosis or phosphorylate Bad in the presence of LY294002, a selective P-I-3-kinase inhibitor. CONCLUSION: These data show that caspase-4 is not essential for ZAAT-induced apoptosis in HEK293 cells and implicates P-I-3-kinase and Bad as potential therapeutic targets for the liver disease associated with ZAAT deficiency.     

Caspase-3 activation downstream from reactive oxygen species in heat-induced apoptosis of pancreatic carcinoma cells carrying a mutant p53 gene

In the present study we investigated the intracellular signaling pathway leading to p53-independent activation of caspase-3 during heat-induced apoptosis of pancreatic carcinoma cells. Induction of mutant p53 protein, but not p21/WAF-1, was observed after heat treatment of both heat-resistant (PANC-1) and heat-sensitive (MIAPaCa-2) cells. A specific inhibitor of caspase-3 (Ac-DMQD-CHO) caused 84% and 92% inhibition of apoptosis in MIAPaCa-2 and PANC-1 cells, respectively. Caspase-3 mRNA expression was increased in both cell lines after heat treatment. Further, heat-induced caspase-3 activity detected by fluorogenic assay in MIAPaCa-2 cells was almost completely inhibited by addition of the antioxidant N-acetyl-L-cysteine. In contrast, Ac-DMQD-CHO had no inhibitory effect on amounts of reactive oxygen species in heat-treated MIAPaCa-2 cells. These results suggest a possible pathway by which reactive oxygen species lead to caspase-3 activation to cause heat-induced death of pancreatic carcinoma cells carrying mutant p53.     

表没食子儿茶素没食子酸酯诱导细胞凋亡蛋白酶途径依赖的人胃癌细胞株MKN45凋亡

背景：表没食子儿茶素没食于酸酯（EGCG）可诱导人胃癌细胞株MKN45凋亡，但其凋亡信号的传导途径尚不清楚。目的：研究EGCG诱导人胃癌细胞株MKN45凋亡的作用是否通过细胞凋亡蛋白酶（caspsse-3）依赖途径，为其临床应用提供进一步的理论依据。方法：采用四甲摹偶氮唑蓝（MTT）比色法检测EGCG和caspase-3抑制剂z-DEVD-fmk作用后MKN45细胞的存活率：采用Annexin V-FITC＋PI双染色法检测EGCG和caspase-3抑制剂作用后MKN45细胞的凋亡率：采用酶联免疫吸附测定（ELISA）检测EGCG和caspase-3抑制剂作用后MKN45细胞内caspase-3活性的改变。结果：EGCG可诱导MKN45细胞凋亡，且细胞内caspase-3活性显著升高。而caspase-3抑制剂干预后，EGCG抑制MKN45细胞生长的作用明显减弱，细胞凋亡率下降，caspase-3活性显著下降。结论：EGCG可诱导MKN45细胞凋亡，该作用可被caspase-3抑制剂显著抑制，提示EGCG诱导MKN45细胞凋亡的作用是通过caspase-3依赖途径的。     

CD95-mediated apoptosis in naïve, central and effector memory subsets of CD4+ and CD8+ T cells in aged humans

Aging is associated with a decrease in naïve (T_N) and central memory (T_CM), and an accumulation of effector memory (T_EM and T_EMRA) T cell subsets. Previously, we have demonstrated an increased sensitivity of T_N and T_CM CD4+ and CD8+ T cells in aging to TNF-α-induced apoptosis. In this investigation, we examined whether similar differential sensitivity is applicable to CD95-mediated apoptosis. We show that T_N and T_CM CD4+ and CD8+ T cells from aged subjects are significantly more sensitive to CD95-mediated apoptosis. Increased apoptosis is associated with increased activation of caspase-8 and caspase-3. Both caspase-8 and caspase-3 inhibitors blocked CD95-mediated apoptosis and activation of caspase-8 and caspase-3 in T_N and T_CM CD4+ and CD8+ T cells. No significant difference was observed in apoptosis or in activation of caspase-8 and caspase-3 in T_EM and T_EMRA CD4+ and CD8+ T cells between young and aged subjects; both populations were relatively and comparably resistant to CD95-mediated apoptosis and caspase activation. No correlation was observed between the sensitivity/resistance of any of the subsets of CD4+ or CD8+T cells to CD95-mediated apoptosis and the expression of CD95. Our data suggest that increased CD95-mediated apoptosis of T_N and T_CM CD8+ and CD4+ T cells may play a role in their decline in human aging.     

系统性红斑狼疮患者CD4+ CD8+T细胞caspase-3及TNF相关凋亡诱导配体受体的表达

目的探求系统性红斑狼疮（SLE）患者CD4^＋,CD8^＋T淋巴细胞caspase-3及肿瘤坏死闪子相关凋亡诱导配体（TRAIL）受体的表达。方法采用免疫磁珠的方法对20例系统性红斑狼疮患者及10名正常人外周血CD4^＋、CD8^T淋巴细胞进行分离，采用反转录聚合酶链反应（RT—PCR）对CD4^＋、CD8^＋T淋巴细胞caspase-3及TRAIL受体的表达进仃分析。结果TRAIL—R3及TRAIL—R4在两符之间的表达差异无统计学意义，但SLE患者CD8^＋T细胞caspase-3（P=0．003）及TRAIL-R2（P=0．024）的表达增加显著高于正常对照。结论TRAIL—R2介导的通路可能在TRAIL诱导SLE患者T淋巴细胞凋亡中发挥重要的作用。     

Dexamethasone induces apoptosis in proliferative chondrocytes through activation of caspases and suppression of the Akt-phosphatidylinositol 3'-kinase signaling pathway

Although glucocorticoids are known to induce apoptosis in chondrocytes, the mechanisms for this effect and the potential antiapoptotic role of IGF-I are unknown. To address this, we studied the effects of dexamethasone (Dexa) on apoptosis in the HCS-2/8 chondrocytic cell line. Dexa (25 microm) increased apoptosis (cell death ELISA) by 39% and 45% after 48 and 72 h, respectively (P < 0.01 and P < 0.05, respectively). IGF-I (100 ng/ml) decreased Dexa-induced apoptosis to levels similar to control cells. Apoptosis was associated with cleavage of poly-ADP-ribose polymerase (PARP) and alpha-fodrin and activation of caspases-8, -9, and -3 (Western), an effect that was counteracted when chondrocytes were cocultured with Dexa + IGF-I. Inhibitors for caspases-8, -9, and -3 (50 microm each) equally suppressed Dexa-induced apoptosis (P < 0.01). Time-response experiments showed that caspase-8 was activated earlier (at 12 h) than caspase-9 (at 36 h). We studied the phosphatidylinositol 3'-kinase (PI3K) pathway to further investigate the mechanisms of Dexa-induced apoptosis. Dexa decreased Akt phosphorylation by 93% (P < 0.001) without affecting total Akt and increased the p85alpha subunit 4-fold. The Akt inhibitor SH-6 (10 microm) increased apoptosis by 54% (P < 0.001). When combining Dexa with SH-6, apoptosis was not further increased, showing that Dexa-induced apoptosis is mediated through inhibition of the PI3K pathway. Addition of IGF-I to SH-6- or Dexa + SH-6-treated cells decreased apoptosis by 21.2% (P < 0.001) and 20.6% (P < 0.001), respectively. We conclude that Dexa-induced apoptosis is caspase dependent with an early activation of caspase-8. IGF-I can rescue chondrocytes from Dexa-induced apoptosis partially through the activation of other pathways than the PI3K signaling pathway. Based on our in vitro data, we speculate that in vivo treatment with glucocorticoids may diminish longitudinal growth by increasing apoptosis of proliferative growth plate chondrocytes.     

Increased sensitivity of T lymphocytes to tumor necrosis factor receptor 1 (TNFR1)- and TNFR2-mediated apoptosis in HIV infection: relation to expression of Bcl-2 and active caspase-8 and caspase-3

The destruction of CD4 T cells in human immunodeficiency virus (HIV) infection is associated with activation of apoptotic programs, partly mediated by death receptors. The role of CD95L/CD95 in depletion of patients' CD4 T cells is well documented, but the possible contribution of the tumor necrosis factor/tumor necrosis factor receptor (TNF/TNFR) pathway has not been examined. In this study, we found that both TNFR1 and TNFR2 induced marked apoptosis in peripheral T cells from HIV-infected persons, involving both CD4 and CD8 T cells. Longitudinal follow-up of HIV(+) patients suggests an association between the in vivo evolution of CD4 T-cell numbers and variations in susceptibility to TNFR-induced apoptosis. Analysis of molecular mechanisms involved showed that it was not related to altered ex vivo expression of TNFR1-associated death domain, receptor interacting protein, or TNFR-associated factor 2. Susceptibility to TNFR-mediated apoptosis was rather related to Bcl-2 expression, because patients' T cells expressing high levels of Bcl-2 were completely protected from TNFR1- and TNFR2-induced cell death, whereas T cells expressing normal levels of Bcl-2 were not protected in patients in contrast to controls. Early recruitment of caspase-8 and caspase-3 is needed to transduce the apoptotic signals, and expression of both caspases in their active form was detected in blood T cells from HIV(+) patients, whereas it was hardly detected in controls. Moreover, ligation of TNFRs induced increased activation of both caspases in patients' T cells. Together these data demonstrate that exacerbated TNFR-mediated cell death of T cells from HIV-infected individuals is associated with both alteration of Bcl-2 expression and activation of caspase-8 and caspase-3 and may contribute to the pathogenesis of acquired immunodeficiency syndrome.