Caspase- and p53-dependent apoptosis in breast carcinoma cells induced by a synthetic selenadiazole derivative

Selenadiazole derivative is one kind of synthetic organoselenium compounds with potent and broad-spectrum antitumor activity. In this study, we showed that anthrax [1,2-c] [1,2,5] selenadiazolo-6,11-dione (ASDO), an novel selenadiazole derivative, induced time- and dose-dependent apoptotic cell death in MCF-7 human breast carcinoma cells, as indicated by accumulation of sub-G1 cell population, DNA fragmentation, nuclear condensation, caspase activation and PARP cleavage. ASDO-induced apoptosis was significantly inhibited by a general caspase inhibitor z-VAD-fmk, demonstrating the important role of caspases in ASDO-induced apoptotic pathway. Treatment of MCF-7 cells with ASDO resulted in a rapid depletion of mitochondrial membrane potential and release of cytochrome c and Smac/Diablo through up-regulation of Bax, Bad and PUMA expression and down-regulation of Bcl-xl expression. Moreover, ASDO treatment up-regulated the expression levels of total p53 and its target gene p21Waf1. Silencing of p53 activation with RNA interference effectively blocked the ASDO-induced cell PARP cleavage, DNA fragmentation and caspase activation. Furthermore, ASDO-induced apoptosis was interestingly found to be independent of reactive oxygen species production. Taken together, we conclude that ASDO induces MCF-7 cell apoptosis through a p53-dependent and mitochondria-mediated pathway.     

PUMA与心肌细胞凋亡的研究进展

p53上调的凋亡调节物(p53 upregulated modulator of apoptosis,PUMA)是新近发现的一种具有促凋亡作用的p53靶基因.与以往发现的其他p53靶基因比较,PUMA在促凋亡作用中有两个重要的特点:一是PUMA几乎介导p53依赖的所有凋亡信号;二是PUMA不仅介导p53依赖的凋亡信号,而且还可以介导p53非依赖的凋亡信号.也就是说,尽管PUMA是p53靶基因,但是其在p53非依赖细胞凋亡中也发挥重要作用.由此可见,PUMA是一个强大的促凋亡因子.在心肌细胞,PUMA参与缺血/再灌注、内质网应激、阿霉素等多种刺激诱导的细胞凋亡.因此,PUMA在心肌细胞凋亡中发挥重要作用.     

PUMA在结肠癌细胞中的表达及诱导细胞凋亡中的作用

目的:通过5-Fu诱导携带有野生型p53基因的HCT116和携带有突变性p53基因的HT-29两种结肠癌细胞系,比较两株细胞在各时间点凋亡水平和PUMA mRNA表达情况的差异,探讨PUMA对细胞凋亡的作用及诱导其表达的基本途径。方法:用逆转录聚合酶链反应(RT—PCR)检测不同结肠癌细胞株HT-29、HCT116在抗肿瘤药物5-Fu作用下不同时间点结肠癌细胞株内PUMA mRNA表达水平的差异,用吖啶橙/溴化乙啶(AO/EB)荧光染色检测各时间点细胞的凋亡水平,分析其与PUMAmRNA表达水平之间的关系。结果:携带有野生型P53基因的结肠癌细胞株HCT116在5-Fu作用下6h即可出现PUMA mRNA的表达,随着药物作用时间的延长其表达强度增加,并且与细胞凋亡水平呈正相关;含有突变型P53基因的结肠癌细胞株HT-29在5-Fu作用下无PUMA的表达。结论:通过5-Fu诱导细胞凋亡出现的PUMA表达需要野生型P53基因,突变型P53基因无法诱导PUMA的表达。PUMA与结肠癌细胞凋亡水平呈正相关,是一种促凋亡蛋白。     

c-Abl is involved in high glucose-induced apoptosis in embryonic E12.5 cortical neural progenitor cells from the mouse brain

Hyperglycemia causes direct apoptosis of neural progenitor cells (NPCs) in diabetic-induced neural tube defects in embryos. However, the underlying mechanisms are poorly understood. The present study is aimed to investigate the specific cellular proteins that may be involved in NPCs apoptosis as well as mechanisms by which the proteins regulate the oxidative stress-induced NPCs apoptosis. Our present results have shown that the expression of c-Abl was up-regulated in NPCs exposed to high glucose in vitro . The increased c-Abl was localized mainly in the nucleus. High glucose also induced an increase in nuclear p53 protein levels and the p53-c-Abl complex in NPCs. Administration of reactive oxygen species scavengers decreased the protein level of c-Abl, p53 and NPCs apoptosis. Inhibition of c-Abl reduced NPCs apoptosis and the nuclear protein level of p53 in response to high glucose. These results demonstrate that c-Abl is involved in the reactive oxygen species-activated apoptotic pathways in NPCs apoptosis. Inhibition of c-Abl may protect NPCs against insults induced by high glucose via the modulation of NPCs apoptotic machinery.     

Neuroprotective cytokines repress PUMA induction in the 1-methyl-4-phenylpyridinium (MPP(+)) model of Parkinson's disease

The hematopoietic cytokines erythropoietin (Epo) and granulocyte-colony stimulating factor (G-CSF) provide neuroprotection in several in vitro and in vivo models of Parkinson’s disease (PD). The molecular mechanism by which Epo and G-CSF signals reduce the neuronal death in PD is not clear. Here, we show that in rat pheochromocytoma PC12 cells, Epo and G-CSF efficiently repressed the 1-methyl-4-phenylpyridinium (MPP⁺)-induced expression of the proapoptotic protein PUMA (p53 up-regulated modulator of apoptosis). Accordingly, Epo and G-CSF treatment reduced the PC12 cell fraction that underwent apoptosis by MPP⁺ treatment and thus improved cell viability. Downregulation of PUMA expression by Epo and G-CSF in MPP⁺-treated PC12 cells seems to be mediated by repression of p53, as the expression of p53 was increased by MPP⁺-treatment and reduced by Epo and G-CSF. Together, these results suggest that the neuroprotective activities of Epo and G-CSF in an experimental model of PD involve the repression of the apoptosis-inducing action of PUMA.     

p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation

p73, an important developmental gene, shares a high sequence homology with p53 and induces both G(1) cell cycle arrest and apoptosis. However, the molecular mechanisms through which p73 induces apoptosis are unclear. We found that p73-induced apoptosis is mediated by PUMA (p53 up-regulated modulator of apoptosis) induction, which, in turn, causes Bax mitochondrial translocation and cytochrome c release. Overexpression of p73 isoforms promotes cell death and bax promoter transactivation in a time-dependent manner. However, the kinetics of apoptosis do not correlate with the increase of Bax protein levels. Instead, p73-induced mitochondrial translocation of Bax is kinetically compatible with the induction of cell death. p73 is localized in the nucleus and remains nuclear during the induction of cell death, indicating that the effect of p73 on Bax translocation is indirect. The ability of p73 to directly transactivate PUMA and the direct effect of PUMA on Bax conformation and mitochondrial relocalization suggest a molecular link between p73 and the mitochondrial apoptotic pathway. Our data therefore indicate that PUMA-mediated Bax mitochondrial translocation, rather than its direct transactivation, correlates with cell death. Finally, human DeltaNp73, an isoform lacking the amino-terminal transactivation domain, inhibits TAp73-induced as well as p53-induced apoptosis. The DeltaNp73 isoforms seem therefore to act as dominant negatives, repressing the PUMA/Bax system and, thus, finely tuning p73-induced apoptosis. Our findings demonstrate that p73 elicits apoptosis via the mitochondrial pathway using PUMA and Bax as mediators.     

Nimbolide sensitizes human colon cancer cells to TRAIL through reactive oxygen species- and ERK-dependent up-regulation of death receptors, p53, and Bax

TNF-related apoptosis-inducing ligand (TRAIL) shows promise as a cancer treatment, but acquired tumor resistance to TRAIL is a roadblock. Here we investigated whether nimbolide, a limonoid, could sensitize human colon cancer cells to TRAIL. As indicated by assays that measure esterase activity, sub-G(1) fractions, mitochondrial activity, and activation of caspases, nimbolide potentiated the effect of TRAIL. This limonoid also enhanced expression of death receptors (DRs) DR5 and DR4 in cancer cells. Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. Using pharmacological inhibitors, we found that activation of ERK and p38 MAPK was required for DR up-regulation by nimbolide. Gene silencing of ERK abolished the enhancement of TRAIL-induced apoptosis. Moreover, our studies indicate that the limonoid induced reactive oxygen species production, which was required for ERK activation, up-regulation of DRs, and sensitization to TRAIL; these effects were mimicked by H(2)O(2). In addition, nimbolide down-regulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and up-regulated the pro-apoptotic proteins p53 and Bax. Interestingly, p53 and Bax up-regulation by nimbolide was required for sensitization to TRAIL but not for DR up-regulation. Overall, our results indicate that nimbolide can sensitize colon cancer cells to TRAIL-induced apoptosis through three distinct mechanisms: reactive oxygen species- and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of p53 and Bax.     

P53 activation plays a crucial role in silibinin induced ROS generation via PUMA and JNK

Silibinin is an active constituent extracted from blessed milk thistle (Silybum marianum). Our previous study demonstrated that silibinin induced autophagy and apoptosis via reactive oxygen species (ROS) generation in HeLa cells. In this study, we investigated whether the autophagy- and apoptosis-associated molecules also involved in ROS generation. Silibinin promoted the expression phosphorylated-p53 (p-p53) in a dose-dependent manner. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced ROS production and reversed silibinin's growth-inhibitory effect. The ROS scavenger N-acetyl cysteine (NAC) attenuated silibinin-induced up-regulation of p-p53 expression, suggesting that p53 might be regulated by ROS and forms a positive feedback loop with ROS. On the other hand, silibinin dose-dependently promoted the expression of phosphorylated-c-Jun N-terminal kinase (p-JNK). Inhibition of JNK by SP600125 decreased ROS generation. NAC down-regulated the expression of p-JNK, indicating that JNK could be activated by ROS. Activation of p53 was suppressed by SP600125 and expression of p-JNK was inhibited by PFT-α, therefore silibinin might activate a ROS-JNK-p53 cycle to induce cell death. Silibinin up-regulated the PUMA and Bax expressions and down-regulated the mitochondrial membrane potential (MMP) level. PFT-α reduced the expression of PUMA and Bax. These results showed that p53 could interfere with mitochondrial functions such as MMP via PUMA pathways, thus resulting in ROS generation. In order to elucidate the functions of p53 in silibinin induced ROS generation, we have chosen the A431 cells (human epithelial carcinoma) because they lack p53 activity (p53His273 mutation). Interestingly, silibinin did not up-regulate the ROS level in A431 cells but lower the ROS level. PFT-α had no influence on ROS level in A431 cells. p53 activation plays a crucial role in silibinin induced ROS generation.     

P53 activation plays a crucial role in silibinin induced ROS generation via PUMA and JNK

Silibinin is an active constituent extracted from blessed milk thistle (Silybum marianum). Our previous study demonstrated that silibinin induced autophagy and apoptosis via reactive oxygen species (ROS) generation in HeLa cells. In this study, we investigated whether the autophagy- and apoptosis-associated molecules also involved in ROS generation. Silibinin promoted the expression phosphorylated-p53 (p-p53) in a dose-dependent manner. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced ROS production and reversed silibinin's growth-inhibitory effect. The ROS scavenger N-acetyl cysteine (NAC) attenuated silibinin-induced up-regulation of p-p53 expression, suggesting that p53 might be regulated by ROS and forms a positive feedback loop with ROS. On the other hand, silibinin dose-dependently promoted the expression of phosphorylated-c-Jun N-terminal kinase (p-JNK). Inhibition of JNK by SP600125 decreased ROS generation. NAC down-regulated the expression of p-JNK, indicating that JNK could be activated by ROS. Activation of p53 was suppressed by SP600125 and expression of p-JNK was inhibited by PFT-α, therefore silibinin might activate a ROS-JNK-p53 cycle to induce cell death. Silibinin up-regulated the PUMA and Bax expressions and down-regulated the mitochondrial membrane potential (MMP) level. PFT-α reduced the expression of PUMA and Bax. These results showed that p53 could interfere with mitochondrial functions such as MMP via PUMA pathways, thus resulting in ROS generation. In order to elucidate the functions of p53 in silibinin induced ROS generation, we have chosen the A431 cells (human epithelial carcinoma) because they lack p53 activity (p53His273 mutation). Interestingly, silibinin did not up-regulate the ROS level in A431 cells but lower the ROS level. PFT-α had no influence on ROS level in A431 cells. p53 activation plays a crucial role in silibinin induced ROS generation.     

Ubiquitination and degradation of the anti-apoptotic protein ARC by MDM2

Current evidence shows that cardiomyocyte apoptosis plays a central role in the pathogenesis of myocardial disease and that reactive oxygen species is critically responsible for mediating cardiomyocyte apoptosis in both ischemia-reperfusion injury and dilated cardiomyopathy. ARC (Apoptosis Repressor with Caspase recruitment domain) is an anti-apoptotic protein that is found abundantly in terminally differentiated cells such as cardiomyocytes. The ARC knock-out mouse developed larger infarct in response to ischemia-reperfusion and transitioned more rapidly and severely to dilated cardiomyopathy following aortic constriction. In addition, ARC protein levels are decreased in human dilated cardiomyopathy and when cardiomyocytes are exposed to oxidative stress in vitro, but the mechanisms regulating ARC protein levels are not known. Here we show that degradation of ARC is dependent on the p53-induced ubiquitin E3 ligase, MDM2. Oxidative stress reduced ARC levels and up-regulated MDM2. MDM2 directly accelerated ARC protein turnover via ubiquitination and proteasomal-dependent degradation. This activity requires a functioning MDM2 ring finger domain because the MDM2(C464A) mutant was unable to direct ARC degradation. Furthermore, ARC degradation requires MDM2, because MDM2 knock-out fibroblasts showed defective ARC degradation that could be rescued by MDM2. Proteasomal inhibitors rescued both MDM2 and H(2)O(2)-induced degradation of ARC and inhibited cardiomyocyte apoptosis. Dilated cardiomyopathic hearts from mice that have undergone transverse aortic banding have increased MDM2 levels associated with decreased ARC levels. We conclude that MDM2 is a critical regulator of ARC levels in cardiomyocytes. Prevention of MDM2-induced degradation of ARC represents a potential therapeutic target to prevent cardiomyocyte apoptosis.