Smac/DIABLO与肿瘤治疗的研究进展

靶向性治疗一直是肿瘤治疗的研究热点,肿瘤细胞与正常细胞区别主要在于:肿瘤细胞具有无限增殖,逃避凋亡,组织侵袭性以及转移的能力.如何在治疗过程中只杀死肿瘤细胞而不影响正常细胞是治疗肿瘤的难点.Smac/NIABLO是2000年首先由Wang等[1]发现并报道,命名为第2个线粒体衍生的胱天蛋白酶激活剂(second mitochondria-derived activator of caspase,Smac),确定其位于线粒体中,在凋亡过程中与细胞色素C一同由线粒体中释放出来,与细胞凋亡密切相关.     

促凋亡蛋白Smac及其下游通路在内毒素诱导L02细胞凋亡中的作用

近年研究表明,细胞凋亡是导致肝脏损伤的一个重要细胞机制,在重型肝炎发生发展过程中发挥重要作用.在正常情况Smac位于线粒体膜问隙,在抗癌药物、化学或物理的凋亡信号、DNA损伤等作用下,可释放入胞质,与凋亡抑制蛋白(IAPs)结合并解除其对天冬氨酸特异性半胱氨酸蛋白酶(Caspases)的抑制作用,从而使Caspase9活性增强;而Caspase3可以剪切Caspase9前体(procaspase9),促使染色质固缩,DNA片段化、细胞起泡,促进细胞凋亡发生[1].本实验采用内毒素(LPS)诱导正常肝细胞凋亡,探讨其对促凋亡分子Smac从线粒体释放及对Caspase9和Caspase3活化的影响,为重型肝炎内毒素血症的防治提供新思路.     

线粒体凋亡通路在肝细胞癌发生、发展中作用的研究进展

线粒体介导的凋亡通路,主要是由于其上游信号分子作用于线粒体膜,导致线粒体膜的通透性改变,从而使线粒体内的Cyt C、AIF、Smac等凋亡相关因子释放到线粒体外,引起下游caspase家族蛋白活化或者直接作用于相应底物,启动细胞凋亡程序。在正常肝细胞的发育和再生过程中,细胞凋亡可清除异常的肝细胞。若异常的肝细胞凋亡不足,可导致肝癌的发生;而肝癌细胞凋亡不足,会诱导癌细胞的恶性化演进。线粒体凋亡通路可诱导肝癌细胞凋亡,而线粒体膜的通透性改变在线粒体凋亡通路中起枢纽作用,外源性或内源性的应力刺激激活Bcl-2家族或caspase家族,启动线粒体膜的通透性改变并诱导肝癌细胞的凋亡。通过靶向干预该通路中的相关分子,可为肝癌的临床治疗提供新策略。     

阻断细胞内凋亡抑制剂可以清除HBV

<正>【据《Proc Natl Acad Sci U S A》2015年5月报道】题:阻断细胞内凋亡抑制剂可以清除HBV(作者Ebert G等)来自墨尔本大学的Ebert G等已经发现细胞内凋亡蛋白抑制剂(c IAPs)可以通过阻止肿瘤坏死因子(TNF)介导的对感染细胞的杀伤/死亡作用,削弱HBV的清除。对于治疗的重大意义是,这一发现能够转化为治疗药物从而清除感染的细胞。c IAPs抑制剂作为抗癌药物来促进TNF介导的肿瘤杀伤作用。这些药物叫做Smac的模拟物,因为它们可以模拟内源性蛋白Smac/Diablo的作用来拮抗c IAP的功能。通过具有免疫能力的慢性HBV感染小鼠发现,birinapant和其他的Smac模拟物可以快速的降低血清HBV DNA和HBc Ag,并且他们可以     

凋亡抑制蛋白XIAP与消化系肿瘤关系的研究进展

X-连锁凋亡抑制蛋白(XIAP)是近年来发现的一种具有细胞凋亡抑制作用的新蛋白。XIAP可以与caspase-9、caspase-7和caspase-3直接结合,抑制caspase活性,而且XIAP还可以通过NF-κB途径和参与信号转导途径抑制细胞调亡。同时XIAP受XIAP相关因子(XAF1)和Smac/DIABLO(Direct IAP binding protein with low pI)等线粒体结合蛋白的负性调节。本文对XIAP的分子结构、表达调控、作用机制及其与消化系肿瘤的关系等方面的研究进展进行综述。以XIAP为靶点的抗肿瘤治疗为癌症治疗提供一个新的方向。     

线粒体损伤与神经元缺血死亡

神经元缺血电灌注时由于线粒体损伤,能量代谢衰竭,导致神经元因能量耗竭而死亡。另一方面,线粒体又是细胞凋亡调控网络中最重要的一种细胞器,凋亡调控过程中的一些重要因子,如细胞色素c、Bcl-2家族、caspase前体等都可定位于线粒体,线粒体损伤导致细胞色素c释放和caspase前体释放与激活,从而介导细胞凋亡。因此,线粒体损伤与神经元缺血性死亡有着极其密切的关系。     

凋亡抑制蛋白家族在食管癌分子靶向治疗中的研究进展

细胞凋亡与抗细胞凋亡信号通路在胚胎及淋巴细胞发育、免疫系统调控、组织稳定及肿瘤发生中发挥着重要作用.研究发现凋亡抑制蛋白(inhibitor of apoptosis proteins,IAPs)在多种恶性肿瘤组织中高表达,并与肿瘤的发生、发展、放化疗抵抗性及预后密切相关,使其成为肿瘤分子靶向治疗的研究热点.近年来,多种靶向IAPs药物正在进行临床试验评估,并在试验中显示出较好的治疗前景.同样,IAPs在食管癌发生发展中有着重要的地位,使其成为食管癌分子靶向治疗潜在的靶点.本文主要对IAPs在食管癌分子靶向治疗的研究进展进行综述.     

线粒体损伤与神经元缺血死亡

神经元缺血再灌注时由于线粒体损伤，能量代谢衰竭，导致神经元因能量耗竭而死亡，另一方面，线粒体又是细胞凋亡调控网络中最重要的一种细胞器，凋亡调控过程中的一些重要因子，如细胞色c、Bcl-2家族、caspase前体等都可定位于线粒体，线粒体损伤导致细胞色素c释放和caspase前体释放与激活，从而介导细胞凋亡。因此，线粒体损伤与神经元缺血性死亡有着其密切的关系。     

线粒体与细胞凋亡的研究进展

线粒体在细胞凋亡和肿瘤的发生及耐药中有重要作用,线粒体信号转导通路在细胞能量代谢、细胞凋亡启动中的特殊地位,及线粒体DNA(mtDNA)突变与肿瘤耐药的密切关系,使探讨线粒体与肿瘤细胞凋亡的相关性研究具有重要的理论和临床意义。     

Smac/DIABLO过表达对人结肠癌细胞凋亡的影响

Smac／DIABLO是一种线粒体促凋亡蛋白，研究表明其在结肠癌细胞中表达降低。目的：研究Smac／DIABLO对人结肠癌细胞生长的影响及其可能的促凋亡机制。方法：将pcDNA3.1-Smac质粒或pcDNA3.1空质粒以脂质体转染人人结肠癌细胞株LoVo,以未转染细胞作为空白对照。以逆转录聚合酶链反应（RT-PCR）检测Smac mRNA表达，蛋白质印迹法检测Smac蛋白表达，Hoechst33258染色观察凋亡细胞核形态学变化，Annexin V-FITC／PI双染流式细胞仪分析检测细胞凋亡率,PI染色流式细胞仪分析检测细胞周期。Caspase-3分光光度法检测试剂盒检测Caspase-3活性。结果：pcDNA3.1-Smac质粒转染12h后，kVo细胞Smac mRNA表达增加；转染48h后，Smac蛋白表达增加。转染48h后,Smac组LoVo细胞呈现明显凋亡细胞核形态学特征。凋亡率显著高于hVo细胞组和空质粒组，GO／G1期细胞增多,Caspase-3活性亦显著高于空质粒组（P〈0．05）。结论：Smac／DIABLO可激活caspase-3途径，抑制人结肠癌细胞生长,促进细胞凋亡。使细胞周期阻滞于G0／G1期。     

Pgp-positive leukaemic cells have increased mtDNA but no increased rate of proliferation

Cells of solid tumours tend to rely on glycolysis for energy. On the other hand, increased glycolysis in solid tumour cells expressing the multidrug resistance protein MDR-1 has been associated with increased malignancy in tumours. We have previously shown that cells of the MDR-1-positive CEM/VLB100 leukaemic cell line have increased mitochondrial electron transport chain (mtETC) activity compared with parental CEM cells. In the present study we used infrared (IR) spectroscopy to demonstrate that the mitochondrial DNA (mtDNA) content in the CEM/VLB100 cell line was significantly increased compared to that in the parental CEM cells. The increase in mtDNA was not accompanied by an increase in mitochondrial protein as both lipid and protein levels were decreased in CEM/VLB100 mitochondria. The ATP content was similar in these two cell lines. However, the ATP-dependent membrane efflux pump function in CEM/VLB100 cells was significantly reduced when mitochondrial ATP synthesis was inhibited by oligomycin, a specific inhibitor of mitochondrial F0F1-ATPase. Proliferation of CEM/VLB100 cells was significantly decreased compared to parental CEM cells, and was independent of p53 expression. Thus, we conclude that: (1) IR spectroscopy is a potential powerful technique for detecting mtDNA, protein and lipid contents simultaneously; (2) leukaemic cells mainly rely on mtDNA for energy; (3) increased expression of an ATP-dependent membrane efflux pump such as Pgp may up-regulate ATP generation and mtDNA content. These metabolic perturbations may exist merely to serve the efflux pump and do not result in an increase in leukaemic cell proliferation. In addition, the associated reduction in mitochondrial lipid and protein may contribute to sensitize the cells to cytochrome c release.     

Hepatitis C virus core protein, cytochrome P450 2E1, and alcohol produce combined mitochondrial injury and cytotoxicity in hepatoma cells

BACKGROUND & AIMS: Alcohol consumption exacerbates liver injury in chronic hepatitis C, and enhanced mitochondrial oxidative stress is one possible mechanism. The aim of this study was to determine whether hepatitis C virus core protein and alcohol-inducible cytochrome P450 2E1 contribute to reactive oxygen species production and cytotoxicity in human hepatoma cells. METHODS: Huh-7 cells expressing core protein, cytochrome P450 2E1, or both were exposed to 0.1 mmol/L tertiary butyl hydroperoxide, tumor necrosis factor alpha, and/or 25 mmol/L ethanol. Cytotoxicity, reactive oxygen species production, glutathione content, and mitochondrial membrane potential were measured. RESULTS: Expression of core/cytochrome P450 2E1 synergistically enhanced cell death induced by either tertiary butyl hydroperoxide or tumor necrosis factor alpha. After tertiary butyl hydroperoxide treatment, total reactive oxygen species production was increased more than 3-fold compared with cells that did not express core and cytochrome P450 2E1. Mitochondrial depolarization and reduced glutathione depletion occurred as well, and cell death was prevented by inhibition of mitochondrial permeability transition or caspase activity. Confocal microscopy showed that the mitochondria themselves were the origin of the reactive oxygen species. In the absence of core/cytochrome P450 2E1 expression, mitochondrial changes and cell death did not occur. Ethanol treatment further decreased mitochondrial reduced glutathione content and exacerbated mitochondrial reactive oxygen species production, depolarization, and cell death. All these effects were prevented by the antioxidant N -acetylcysteine. CONCLUSIONS: Mitochondrial reactive oxygen species production is induced by hepatitis C virus core and cytochrome P450 2E1, resulting in a reduction of mitochondrial antioxidant capacity and sensitivity to oxidants and tumor necrosis factor alpha. Alcohol further depletes mitochondrial reduced glutathione, which exacerbates depolarization and cell death. Sensitization of mitochondria to oxidative insults is thus a potential mechanism for alcohol-related exacerbation of liver injury in chronic hepatitis C.     

线粒体及其蛋白质质量控制失调与动脉粥样硬化的关系

线粒体是哺乳动物细胞内重要的细胞器,作为细胞能量代谢和细胞死亡的调控中心,其功能异常会导致多种疾病的发生与发展。 线粒体功能依赖于线粒体蛋白质组的完整性和稳态,因此线粒体蛋白质质量控制系统对于维持线粒体稳态和机体健康十分重要。当线粒体及其蛋白质质量控制系统出现异常时,会直接损伤线粒体并出现异常线粒体蛋白堆积,发生细胞内环境紊乱,甚至细胞功能障碍,进而影响动脉粥样硬化性疾病的发生与发展。文章回顾了线粒体及其蛋白质质量控制系统在动脉粥样硬化性疾病发生发展中的作用,并对该领域未来的发展前景和挑战进行展望,以期为寻找与动脉粥样硬化性疾病密切相关的特异性线粒体蛋白提供线索。     

Increasing uncoupling protein-2 in pancreatic beta cells does not alter glucose-induced insulin secretion but decreases production of reactive oxygen species

Aims/hypothesis Levels of uncoupling protein-2 (UCP2) are regulated in the pancreatic beta cells and an increase in the protein level has been associated with mitochondrial uncoupling and alteration in glucose-stimulated insulin secretion. However, it is not clear whether an increase in uncoupling protein-2 per se induces mitochondrial uncoupling and affects ATP generation and insulin secretion. Materials and methods Transgenic mice with beta cell-specific overexpression of the human UCP2 gene and INS-1 cells with doxycycline-inducible overproduction of the protein were generated and the consequences of increased levels of UCP2 on glucose-induced insulin secretion and on parameters reflecting mitochondrial uncoupling were determined. Results In transgenic mice, an increase in beta cell UCP2 protein concentration did not significantly modify plasma glucose and insulin levels. Glucose-induced insulin secretion and elevation in the ATP/ADP ratio were unaltered by an increase in UCP2 level. In INS-1 cells, a similar increase in UCP2 level did not modify glucose-induced insulin secretion, cytosolic ATP and ATP/ADP ratio, or glucose oxidation. Increased levels of UCP2 did not modify the mitochondrial membrane potential and oxygen consumption. Increased UCP2 levels decreased cytokine-induced production of reactive oxygen species. Conclusion/interpretation The results obtained in transgenic mice and in the beta cell line do not support the hypothesis that an increase in UCP2 protein per se uncouples the mitochondria and decreases glucose-induced insulin secretion. In contrast, the observation that increased UCP2 levels decrease cytokine-induced production of reactive oxygen species indicates a potential protective effect of the protein on beta cells, as observed in other cell types. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorised users. N. Produit-Zengaffinen and N. Davis-Lameloise contributed equally to this work.     

Disruption of ATP-binding cassette B8 in mice leads to cardiomyopathy through a decrease in mitochondrial iron export

Mitochondrial iron levels are tightly regulated, as iron is essential for the synthesis of Fe/S clusters and heme in the mitochondria, but high levels can cause oxidative stress. The ATP-binding cassette (ABC) transporter ABCB8 is a mitochondrial inner membrane protein with an unknown function. Here, we show that ABCB8 is involved in mitochondrial iron export and is essential for baseline cardiac function. Induced genetic deletion of ABCB8 in mouse hearts resulted in mitochondrial iron accumulation and cardiomyopathy, as assessed by echocardiography and invasive hemodynamics. Mice with ABCB8 deletion in the heart also displayed mitochondrial damage, and higher levels of reactive oxygen species and cell death. Down-regulation of ABCB8 in vitro resulted in decreased iron export from isolated mitochondria, whereas its overexpression had the opposite effect. Furthermore, ABCB8 is needed for the maturation of the cytosolic Fe/S proteins, as its deletion in vitro and in vivo led to decreased activity of cytosolic, but not mitochondrial, iron-sulfur-containing enzymes. These results indicate that ABCB8 is essential for normal cardiac function, maintenance of mitochondrial iron homeostasis and maturation of cytosolic Fe/S proteins. In summary, this report provides characterization of a protein involved in mitochondrial iron export.     

Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset neurological disease resulting from mutations in the SACS gene encoding sacsin, a 4,579-aa protein of unknown function. Originally identified as a founder disease in Québec, ARSACS is now recognized worldwide. Prominent features include pyramidal spasticity and cerebellar ataxia, but the underlying pathology and pathophysiological mechanisms are unknown. We have generated an animal model for ARSACS, sacsin knockout mice, that display age-dependent neurodegeneration of cerebellar Purkinje cells. To explore the pathophysiological basis for this observation, we examined the cell biological properties of sacsin. We show that sacsin localizes to mitochondria in non-neuronal cells and primary neurons and that it interacts with dynamin-related protein 1, which participates in mitochondrial fission. Fibroblasts from ARSACS patients show a hyperfused mitochondrial network, consistent with defects in mitochondrial fission. Sacsin knockdown leads to an overly interconnected and functionally impaired mitochondrial network, and mitochondria accumulate in the soma and proximal dendrites of sacsin knockdown neurons. Disruption of mitochondrial transport into dendrites has been shown to lead to abnormal dendritic morphology, and we observe striking alterations in the organization of dendritic fields in the cerebellum of knockout mice that precedes Purkinje cell death. Our data identifies mitochondrial dysfunction/mislocalization as the likely cellular basis for ARSACS and indicates a role for sacsin in regulation of mitochondrial dynamics.     

Cytochrome P450-generated reactive metabolites cause mitochondrial permeability transition, caspase activation, and apoptosis in rat hepatocytes

Although cytochrome P-450 (CYP)-generated reactive metabolites can cause hepatocyte apoptosis, the mechanism of this effect is incompletely understood. In the present study, we assessed the hepatotoxicity of skullcap, a diterpenoid-containing herbal remedy. Male rat hepatocytes were incubated for 2 hours with skullcap diterpenoids (100 microg/mL). This treatment decreased cell glutathione and protein thiols and increased cell [Ca(2+)]. This activated Ca(2+)-dependent tissue transglutaminase, forming a cross-linked protein scaffold, and also opened the mitochondrial permeability transition pore, causing outer mitochondrial membrane rupture, increased cytosolic cytochrome c, activation of procaspase 3, internucleosomal DNA fragmentation, and ultrastructural features of apoptosis. Cell death was increased by a CYP3A inducer (dexamethasone) or a sulfur amino acid-deficient diet increasing glutathione depletion. In contrast, cell death was prevented by decreasing CYP3A activity (with troleandomycin), preventing glutathione depletion (with cysteine or cystine), blocking Ca(2+)-modulated events (with calmidazolium), preventing mitochondrial permeability transition (with cyclosporin A), or inhibiting caspase 3 (with acetyl-Asp-G u-Va-Asp-a dehyde). Both calmidazolium and cyclosporin A also prevented the increase in cytosolic cytochrome c and procaspase 3 activation. In conclusion, CYP3A activates skullcap diterpenoids into reactive metabolites that deplete cellular thiols and increase cell [Ca(2+)]. This activates Ca(2+)-dependent transglutaminase and also opens the mitochondrial permeability transition pore, causing outer mitochondrial membrane rupture, cytochrome c release, and caspase activation. Preventing mitochondrial permeability transition pore opening and/or caspase activity blocks apoptosis, showing the fundamental role of these final events in metabolite-mediated hepatotoxicity.     

Ian4 is required for mitochondrial integrity and T cell survival

Apoptosis is a regulated cell death program controlled by extrinsic and intrinsic signaling pathways. The intrinsic pathway involves stress signals that activate pro-apoptotic members of the Bcl-2 family, inducing permeabilization of mitochondria and release of apoptogenic factors. These proteins localize to the outer mitochondrial membrane. Ian4, a mitochondrial outer membrane protein with GTP-binding activity, is normally present in thymocytes, T cells, and B cells. We and others have recently discovered that a mutation in the rat Ian4 gene results in severe T cell lymphopenia that is associated with the expression of autoimmune diabetes. The mechanism by which Ian4 controls T cell homeostasis is unknown. Here we show that the absence of Ian4 in T cells causes mitochondrial dysfunction, increased mitochondrial levels of stress-inducible chaperonins and a leucine-rich protein, and T cell-specific spontaneous apoptosis. T cell activation and caspase 8 inhibition both prevented apoptosis, whereas transfection of T cells with Ian4-specific small interfering RNA recapitulated the apoptotic phenotype. The findings establish Ian4 as a tissue-specific regulator of mitochondrial integrity.     

Antioxidant dysfunction: potential risk for neurotoxicity in ethylmalonic aciduria

Mitochondrial dysfunction and oxidative stress are central to the molecular basis of several human diseases associated with neuromuscular disabilities. We hypothesize that mitochondrial dysfunction also contributes to the neuromuscular symptoms observed in patients with ethylmalonic aciduria and homozygosity for ACADS c.625G>A-a common variant of the short-chain acyl-coenzyme A (CoA) dehydrogenase (SCAD) enzyme in the mitochondrial fatty acid oxidation pathway. This study sought to identify the specific factors that initiate cell dysfunction in these patients. We investigated fibroblast cultures from 10 patients with neuromuscular disabilities, elevated levels of ethylmalonic acid (EMA) (>50 mmol/mol creatinine), and ACADS c.625G>A homozygosity. Functional analyses, i.e., ACADS gene and protein expression as well as SCAD enzyme activity measurements, were performed together with a global nano liquid chromatography tandem mass spectroscopy (nano-LC-MS/MS)-based screening of the mitochondrial proteome in patient fibroblasts. Moreover, cell viability of patient fibroblasts exposed to menadione-induced oxidative stress was evaluated. Loss of SCAD function was detected in the patient group, most likely due to decreased ACADS gene expression and/or elimination of misfolded SCAD protein. Analysis of the mitochondrial proteome in patient fibroblasts identified a number of differentially expressed protein candidates, including reduced expression of the antioxidant superoxide dismutase 2 (SOD2). Additionally, patient fibroblasts demonstrated significantly higher sensitivity to oxidative stress than control fibroblasts. We propose that reduced mitochondrial antioxidant capacity is a potential risk factor for ACADS c.625G>A-associated ethylmalonic aciduria and that mitochondrial dysfunction contributes to the neurotoxicity observed in patients.     

Helicobacter pylori vacuolating cytotoxin A (VacA) engages the mitochondrial fission machinery to induce host cell death

A number of pathogenic bacteria target mitochondria to modulate the host's apoptotic machinery. Studies here revealed that infection with the human gastric pathogen Helicobacter pylori disrupts the morphological dynamics of mitochondria as a mechanism to induce host cell death. The vacuolating cytotoxin A (VacA) is both essential and sufficient for inducing mitochondrial network fragmentation through the mitochondrial recruitment and activation of dynamin-related protein 1 (Drp1), which is a critical regulator of mitochondrial fission within cells. Inhibition of Drp1-induced mitochondrial fission within VacA-intoxicated cells inhibited the activation of the proapoptotic Bcl-2-associated X (Bax) protein, permeabilization of the mitochondrial outer membrane, and cell death. Our data reveal a heretofore unrecognized strategy by which a pathogenic microbe engages the host's apoptotic machinery.