线粒体，活性氧和细胞凋亡

在能量代谢和自由基代谢中,线粒体均占据着十分重要的地位.通过呼吸链电子漏途径,线粒体产生大量超氧阴离子,并通过链式反应形成对机体有损伤作用的活性氧.通过呼吸链电子漏,氧化磷酸化解偶联,线粒体内膜产生通透性转变孔道（PTP）及Box-和/或PTP-介导的细胞色素c向胞质的转移等种种因素,线粒体参与一般抗氧化防御及细胞凋亡等重要生理过程的调控.在与线粒体相关的细胞凋亡中,活性氧的信号作用是十分明显的.     

C-myc-siRNA通过调控ROS诱导子宫内膜癌细胞凋亡

为探讨原癌基因C-myc-siRNA对子宫内膜癌细胞凋亡的影响,本研究利用细胞计数盒(cell counting Kit-8, CCK-8)法分别检测C-myc-siRNA转染组和空载体转染组(Vector-NC)的子宫内膜癌细胞活力;蛋白免疫印迹法(Western blotting)分别检测C-myc-siRNA转染组和空载体转染组(Vector-NC)的子宫内膜癌细胞凋亡蛋白Bax以及抗凋亡蛋白Bcl-2的变化;流式细胞仪分别检测C-myc-siRNA转染组和空载体转染组(Vector-NC)活性氧簇(reactive oxygen species, ROS)的变化;蛋白免疫印迹法(Western blotting)分别检测预处理NAC (ROS抑制剂)后,C-myc-siRNA转染组和空载体转染组(Vector-NC)的子宫内膜癌细胞凋亡相关蛋白Bax以及Bcl-2的变化。结果表明:C-myc-siRNA转染子宫内膜癌细胞后,促凋亡相关蛋白Bax的表达显著高于空载体转染组,且抗凋亡相关蛋白Bcl-2的表达明显低于空载体转染组(p<0.05);C-myc-siRNA转染子宫内膜癌细胞后,细胞ROS水平明显增加(p<0.05);NAC预处理显著减弱C-myc-siRNA对子宫内膜癌细胞凋亡的促进作用(p<0.05)。本研究结论表明,C-myc-siRNA能够通过调控ROS诱导子宫内膜癌细胞凋亡。     

活性氧、线粒体通透性转换与细胞凋亡

线粒体是真核细胞中非常重要的细胞器,细胞中的活性氧等自由基主要来源于此,线粒体膜的通透性转换(mitochondrial permeability transition,MPT)及其孔道(mitochondrialpermeability transition pore,MPTP)更是在内源性细胞凋亡中发挥了关键作用。持续性的线粒体膜通透性转换在凋亡的效应阶段起决定性作用,可介导细胞色素c等促凋亡因子从线粒体释放到胞浆中,进一步激活下游的信号通路,导致细胞不可逆地走向凋亡。瞬时性的线粒体膜通透性转换及其偶联的线粒体局部的活性氧爆发同样具有促凋亡的作用。线粒体通透性孔道的开放释放出大量活性氧,这些活性氧又能够进一步激活该孔道,以正反馈的形式进一步加剧孔道的打开,放大凋亡信号。活性氧、线粒体通透性转换与细胞凋亡之间具有密不可分的联系,本文根据已知的研究结果集中讨论了这三者的关系,并着重论述了该领域中的最新发现和成果。     

用PCR技术克隆细小病毒H－1部分非结构蛋白基因

应用ＰＣＲ技术定向克隆了细小病毒Ｈ－１的非结构蛋白（ＮＳ）部分基因片段。自行设计并合成了ＰＣＲ引物△Ｐ３和△Ｐ４,在两个引物中分别引入两个突变碱基,使扩增后的ＤＮＡ片段的两端含有限制性核酸内切酶ＨｉｎｄⅢ或ＢａｍＨＩ的酶切位点,经双酿切法把该ＤＮＡ片段重组到ｐＵＣ１１８质粒中。对插入片段的ＤＮＡ序列测定和分析结果证实该片段为Ｈ－１ＮＳ－１基因序列。以此重组质粒为探针,采用分子杂交的方法,分别测定了Ｈ－１及ＭＶＭＤＮＡ在细胞内的复制水平。这一基因的克隆为制备Ｈ－１的质量监测、Ｈ－１及ＭＶＭＮＳ－１蛋白抑瘤作用机理及其在肿瘤细胞及正常组织中的转录表达等研究奠定了基础。     

Docosahexaenoic Acid Induces Apoptosis in MCF-7 Cells In Vitro and In Vivo via Reactive Oxygen Species Formation and Caspase 8 Activation

Background

The present study sought to further investigate the in vitro and in vivo anticancer effects of a representative omega-3 fatty acid, docosahexaenoic acid (DHA), with a focus on assessing the induction of oxidative stress and apoptosis as an important mechanism for its anticancer actions.

Methodology/Principal Findings

In vitro studies showed that DHA strongly reduces the viability and DNA synthesis of MCF-7 human breast cancer cells in culture, and also promotes cell death via apoptosis. Mechanistically, accumulation of reactive oxygen species and activation of caspase 8 contribute critically to the induction of apoptotic cell death. Co-presence of antioxidants or selective inhibition or knockdown of caspase 8 each effectively abrogates the cytotoxic effect of DHA. Using athymic nude mice as an in vivo model, we found that feeding animals the 5% fish oil-supplemented diet for 6 weeks significantly reduces the growth of MCF-7 human breast cancer cells in vivo through inhibition of cancer cell proliferation as well as promotion of cell death. Using 3-nitrotyrosine as a parameter, we confirmed that the fish oil-supplemented diet significantly increases oxidative stress in tumor cells in vivo. Analysis of fatty acid content in plasma and tissues showed that feeding animals a 5% fish oil diet increases the levels of DHA and eicosapentaenoic acid in both normal and tumorous mammary tissues by 329% and 300%, respectively.

Conclusions/Significance

DHA can strongly induce apoptosis in human MCF-7 breast cancer cells both in vitro and in vivo. The induction of apoptosis in these cells is selectively mediated via caspase 8 activation. These observations call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of human breast cancer.     

Mitochondrial NLRP3 Protein Induces Reactive Oxygen Species to Promote Smad Protein Signaling and Fibrosis Independent from the Inflammasome

Nucleotide-binding domain and leucine-rich repeat containing PYD-3 (NLRP3) is a pattern recognition receptor that is implicated in the pathogenesis of inflammation and chronic diseases. Although much is known regarding the NLRP3 inflammasome that regulates proinflammatory cytokine production in innate immune cells, the role of NLRP3 in non-professional immune cells is unclear. Here we report that NLRP3 is expressed in cardiac fibroblasts and increased during TGFβ stimulation. NLRP3-deficient cardiac fibroblasts displayed impaired differentiation and R-Smad activation in response to TGFβ. Only the central nucleotide binding domain of NLRP3 was required to augment R-Smad signaling because the N-terminal Pyrin or C-terminal leucine-rich repeat domains were dispensable. Interestingly, NLRP3 regulation of myofibroblast differentiation proceeded independently from the inflammasome, IL-1β/IL-18, or caspase 1. Instead, mitochondrially localized NLRP3 potentiated reactive oxygen species to augment R-Smad activation. In vivo, NLRP3-deficient mice were protected against angiotensin II-induced cardiac fibrosis with preserved cardiac architecture and reduced collagen 1. Together, these results support a distinct role for NLRP3 in non-professional immune cells independent from the inflammasome to regulate differential aspects of wound healing and chronic disease.     

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 4 Induces Apoptosis Dependent on Its 3C-Like Serine Protease Activity

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease in pigs caused by PRRS virus (PRRSV). Although PRRSV infection-induced cell apoptosis has been established, the related viral protein is still unknown. Here, we reported that PRRSV nonstructural protein 4 (nsp4) was a critical apoptosis inducer. Nsp4 could activate caspase-3, -8, and -9. Using truncated constructs without different domains in nsp4, we demonstrated that the full-length of nsp4 structure was required for its apoptosis-inducing activity. Furthermore, using site-directed mutagenesis to inactivate the 3C-like serine protease activity of nsp4, we showed that nsp4-induced apoptosis was dependent on its serine protease activity. The ability of nsp4 to induce apoptosis was significantly impaired by His39, Asp64, and Ser118 mutations, suggesting that His39, Asp64, and Ser118 were essential for nsp4 to trigger apoptosis. In conclusion, our present work showed that PRRSV nsp4 could induce apoptosis in host cells and might be partially responsible for the apoptosis induced by PRRSV infection. PRRSV 3C-like protease-mediated apoptosis represents the first report in the genus Arterivirus, family Arteriviridae.     

活性氧参与一氧化氮诱导的神经细胞凋亡

采用激光共聚焦成像技术，用氧化还原敏感的特异性荧光探针(DCFH-DA和DHR123)直接研究了一氧化氮供体S-亚硝基-N-乙酰基青霉胺(SNAP)诱导未成熟大鼠小脑颗粒神经元凋亡过程中的细胞胞浆、线粒体中活性氧水平的变化，发现神经细胞经0.5 mmol/L SNAP处理1 h后，细胞胞浆及线粒体中活性氧水平大大增加.一氧化氮清除剂血红蛋白能够有效抑制细胞胞浆、线粒体中活性氧的产生，防止细胞凋亡.外源性谷胱甘肽对细胞也具有良好的保护作用，而当细胞中谷胱甘肽的合成被抑制后，一氧化氮的神经毒性大大增强.实验结果表明一氧化氮通过促进神经细胞产生内源性活性氧而启动细胞凋亡程序，而谷胱甘肽可能是重要的防止一氧化氮引发神经损伤的内源性抗氧化剂.     

Involvement of Reactive Oxygen Species in Capsaicinoid-induced Apoptosis in Transformed Cells

Some varieties of sweet pepper accumulate non-pungent isosters of capsaicin, a type of compounds exemplified by capsiate. The only structural difference between capsaicin and capsiate is the link between the vanillyl and the acyl moieties, via an amide bond in the former and via an ester bond in the latter. By flow cytometry analyses we have determined that nor-dihydrocapsiate, a simplified analogue of capsiate, is a pro-oxidant compound that induces apoptosis in the Jurkat tumor cell line. The nuclear DNA fragmentation induced by nor-dihydrocapsiate is preceded by an increase in the production of reactive oxygen species and by a subsequent disruption of mitochondria transmembrane potential. Capsiate-induced apoptosis is initiated at the S phase of the cell cycle and is mediated by a caspase-3-dependent pathway. The accumulation of intracellular reactive oxygen species in capsiate-treated cells is greatly prevented by the presence of ferricyanide, suggesting that capsiates target a cellular redox system distinct from the one involved in the mitochondrial electron-chain transport. Methylation of the phenolic hydroxyl of nor-dihydrocapsiate completely abrogated the ability to induce reactive oxygen species and apoptosis, highlighting the relevance of the presence of a free phenolic hydroxyl for the pro-oxidant properties of capsaicinoids.     

The 18-kDa Translocator Protein Inhibits Vascular Cell Adhesion Molecule-1 Expression via Inhibition of Mitochondrial Reactive Oxygen Species

Translocator protein 18 kDa (TSPO) is a mitochondrial outer membrane protein and is abundantly expressed in a variety of organ and tissues. To date, the functional role of TSPO on vascular endothelial cell activation has yet to be fully elucidated. In the present study, the phorbol 12-myristate 13-acetate (PMA, 250 nM), an activator of protein kinase C (PKC), was used to induce vascular endothelial activation. Adenoviral TSPO overexpression (10–100 MOI) inhibited PMA-induced vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) expression in a dose dependent manner. PMA-induced VCAM-1 expressions were inhibited by Mito-TEMPO (0.1–0.5 μM), a specific mitochondrial antioxidants, and cyclosporin A (1–5 μM), a mitochondrial permeability transition pore inhibitor, implying on an important role of mitochondrial reactive oxygen species (ROS) on the endothelial activation. Moreover, adenoviral TSPO overexpression inhibited mitochondrial ROS production and manganese superoxide dismutase expression. On contrasts, gene silencing of TSPO with siRNA increased PMA-induced VCAM-1 expression and mitochondrial ROS production. Midazolam (1–50 μM), TSPO ligands, inhibited PMA-induced VCAM-1 and mitochondrial ROS production in endothelial cells. These results suggest that mitochondrial TSPO can inhibit PMA-induced endothelial inflammation via suppression of VCAM-1 and mitochondrial ROS production in endothelial cells.     

Receptor-interacting Protein 1 Increases Chemoresistance by Maintaining Inhibitor of Apoptosis Protein Levels and Reducing Reactive Oxygen Species through a microRNA-146a-mediated Catalase Pathway

Although receptor-interacting protein 1 (RIP1) is well known as a key mediator in cell survival and death signaling, whether RIP1 directly contributes to chemotherapy response in cancer has not been determined. In this report, we found that, in human lung cancer cells, knockdown of RIP1 substantially increased cytotoxicity induced by the frontline anticancer therapeutic drug cisplatin, which has been associated with robust cellular reactive oxygen species (ROS) accumulation and enhanced apoptosis. Scavenging ROS dramatically protected RIP1 knockdown cells against cisplatin-induced cytotoxicity. Furthermore, we found that, in RIP1 knockdown cells, the expression of the hydrogen peroxide-reducing enzyme catalase was dramatically reduced, which was associated with increased miR-146a expression. Inhibition of microRNA-146a restored catalase expression, suppressed ROS induction, and protected against cytotoxicity in cisplatin-treated RIP1 knockdown cells, suggesting that RIP1 maintains catalase expression to restrain ROS levels in therapy response in cancer cells. Additionally, cisplatin significantly triggered the proteasomal degradation of cellular inhibitor of apoptosis protein 1 and 2 (c-IAP1 and c-IAP2), and X-linked inhibitor of apoptosis (XIAP) in a ROS-dependent manner, and in RIP1 knockdown cells, ectopic expression of c-IAP2 attenuated cisplatin-induced cytotoxicity. Thus, our results establish a chemoresistant role for RIP1 that maintains inhibitor of apoptosis protein (IAP) expression by release of microRNA-146a-mediated catalase suppression, where intervention within this pathway may be exploited for chemosensitization.     

Mitochondrial Dysfunction Promotes Breast Cancer Cell Migration and Invasion through HIF1α Accumulation via Increased Production of Reactive Oxygen Species

Although mitochondrial dysfunction has been observed in various types of human cancer cells, the molecular mechanism underlying mitochondrial dysfunction mediated tumorigenesis remains largely elusive. To further explore the function of mitochondria and their involvement in the pathogenic mechanisms of cancer development, mitochondrial dysfunction clones of breast cancer cells were generated by rotenone treatment, a specific inhibitor of mitochondrial electron transport complex I. These clones were verified by mitochondrial respiratory defect measurement. Moreover, those clones exhibited increased reactive oxygen species (ROS), and showed higher migration and invasive behaviors compared with their parental cells. Furthermore, antioxidant N-acetyl cysteine, PEG-catalase, and mito-TEMPO effectively inhibited cell migration and invasion in these clones. Notably, ROS regulated malignant cellular behavior was in part mediated through upregulation of hypoxia-inducible factor-1 α and vascular endothelial growth factor. Our results suggest that mitochondrial dysfunction promotes cancer cell motility partly through HIF1α accumulation mediated via increased production of reactive oxygen species.     

Simvastatin Inhibited the Apoptosis of PC12 Cells Induced by 1-Methyl-4-Phenylpyridinium Ion via Inhibiting Reactive Oxygen Species Production

In the present study, we investigated the neuroprotection of simvastatin in PC12 cells following 1-methyl-4-phenylpyridinium ion (MPP+) neurotoxicity. Simvastatin inhibited the decrease of cell viability induced by MPP+ in PC12 cells. The damage of PC12 cells in morphology was alleviated and the apoptotic rates were decreased due to simvatatin pretreatment against MPP+ cytotoxicity. The reactive oxygen species production exposure to MPP+ was inhibited by simvatatin in PC12 cells. So simvastatin may be of therapeutic benefit for PD patients.     

NS1 Protein of Influenza A Virus Down-Regulates Apoptosis

Wild-type (WT) influenza A/PR/8/34 virus and its variant lacking the NS1 gene (delNS1) have been compared for their ability to mediate apoptosis in cultured cells and chicken embryos. Cell morphology, fragmentation of chromatin DNA, and caspase-dependent cleavage of the viral NP protein have been used as markers for apoptosis. Another marker was caspase cleavage of the viral M2 protein, which was also found to occur in an apoptosis-specific manner. In interferon (IFN)-competent host systems, such as MDCK cells, chicken fibroblasts, and 7-day-old chicken embryos, delNS1 virus induced apoptosis more rapidly and more efficiently than WT virus. As a consequence, delNS1 virus was also more lethal for chicken embryos than WT virus. In IFN-deficient Vero cells, however, apoptosis was delayed and developed with similar intensity after infection with both viruses. Taken together, these data indicate that the IFN antagonistic NS1 protein of influenza A viruses has IFN-dependent antiapoptotic potential.