神经酰胺诱导肿瘤细胞凋亡的机制研究进展

神经酰胺(ceramide,Cer)作为一种神经鞘磷脂分子,不仅是细胞膜的组成成分,而且可以作为各种信号转导途径的第二信使,参与细胞增殖、分化、衰老和凋亡等生命活动的调节。Cer的合成、代谢及信号转导在肿瘤发生发展甚至耐药和抵抗放射治疗中有着密切的关系。Cer可以被诸如肿瘤坏死因子α(Tumor necrosis factorα,TNF-α)、激素、电离辐射和化疗药物等细胞外信号和受体激活,其主要可以通过内源性凋亡途径和外源性凋亡途径诱导肿瘤细胞凋亡的发生。在肿瘤细胞凋亡发生过程中,Cer通过激活Jun氨基末端激酶(JNKs)、有丝分裂原活化蛋白激酶/细胞外信号调节蛋白激酶(MAPK/ERK)和P38等信号通路以及蛋白激酶、组织蛋白酶D、蛋白磷酸酶1(Protein phosphatase1,PP1)和蛋白磷酸酶2A(Protein phosphatase2A,PP2A)等效应分子介导肿瘤细胞凋亡。本文综述近年来有关Cer在应激反应级联以及肿瘤细胞凋亡中的作用的研究进展。     

封面故事

<正>肿瘤坏死因子受体超家族(TNFRSF)的成员是机体非常重要的调节分子,参与细胞的生长、分化、凋亡与存活等过程。已有研究表明,TNFRSF家族成员可以通过在细胞质膜和内吞体上结合不同的接头分子,启动截然不同的下游信号通路。例如,TNF结合细胞膜表面的TNFR1,能够招募TRADD,RIP1和TRAF2组成信号复合物Ⅰ,激活经典的NF-κB通路;而内化的TNF-TNFR1复合物则能够招募TRADD、FADD和Caspase-8组成信号复合物Ⅱ,即招募死亡诱导信号复合物介导细胞凋亡。死亡受体6(DR6)属于肿瘤坏死因子受体超家族的一员,它在免疫系统和神经系统中发挥着重要功     

引发细胞凋亡受体的死亡结构域

死亡结构域是存在于肿瘤于死因因子受体Ⅰ型和Ｆａｓ等能引起细胞凋亡的细胞膜表面受包浆区的一段氨基酸序列,它通过聚合针这些膜表面受体与胞浆信号蛋白联系起来,成为引起细胞凋亡或活化的信号转导通路中重要的一个环节。本文综述了死亡结构域及其在细胞信号转导过程中所起作用的最新进展。     

Bcl-2蛋白家族调节凋亡和自噬信号通路的研究进展

自噬和凋亡是哺乳动物清除体内自身物质的两种重要生理过程,不同之处在于前者利于细胞生存,后者促进细胞死亡,它们在组织稳态、发育和疾病中起主要作用。Bcl-2蛋白家族对自噬和凋亡的信号通路存在交叉调控,使细胞的生死抉择具有可控性的同时形成复杂的信号转导网络,导致人们对其机制不甚清楚。该文首先总结了Bcl-2蛋白家族通过调节线粒体外膜通透和钙信号进而调控凋亡的分子机制,然后讨论了该家族成员的相互作用及其对钙信号的影响在自噬信号通路中的关键作用,最后提出了Bcl-2蛋白家族通过调节凋亡和自噬决定细胞命运的观点。     

Hippo信号通路在乳腺癌中的调控机制及作用

Hippo信号通路是调控器官大小和肿瘤发生发展的关键通路,近年来受到广泛的关注。TAZ/YAP作为哺乳动物中Hippo信号通路两个核心下游效应分子,通过Hippo信号通路依赖性和非依赖性的机制受到细胞内外信号的严密调控。除了参与正常乳腺组织发育,Hippo信号通路还在人乳腺癌细胞的增殖、分化、凋亡、迁移、侵袭、上皮-间质转化和干性维持等多个过程中起着关键性作用。本文总结了Hippo信号通路的调控机制和调节信号,阐述了Hippo信号通路异常在乳腺癌发生发展中的作用,并讨论了其在乳腺癌中作为治疗靶点的临床策略。     

Notch信号:一个新型代谢调节因子

Notch信号通路进化上非常保守,广泛表达于脊椎动物和无脊椎动物中。Notch信号通路由受体、配体、CSL-DNA结合蛋白等组成,在细胞的增殖、分化和凋亡中发挥重要的调控作用。近年来发现,Notch信号作为一个新型的代谢调节因子,在骨骼肌细胞和脂肪细胞稳态、脂肪肝、糖尿病及糖尿病肾病中发挥重要的调节作用。本文就Notch信号在细胞的稳态及一些代谢性疾病中的调节作用及其分子机制作一综述。     

TGF-β和PI3K/Akt信号通路共同维持组织稳态

转化生长因子-β(TGF-β)超家族分子通过跨膜受体和胞浆内信号转导分子Smad进行信号转导,调节细胞的增殖、分化和凋亡。许多生长因子和激素通过其受体激活磷脂酰肌醇3-激酶(PI3K),PI3K可以使肌醇环上的3位羟基磷酸化,磷酸化的肌醇脂可招募和激活许多信号通路分子,促进细胞增殖、细胞迁移和细胞存活。近几年来的研究表明这两条信号通路通过多水平的相互作用共同调节细胞增殖、分化及凋亡,在维持组织稳态的过程中发挥重要的作用。     

microRNA调控细胞凋亡的研究进展

microRNA(miRNA)是一类非编码小RNA,通过基因转录后调控来调节细胞的各生理过程。其中,细胞凋亡作为细胞自主有序的死亡过程,在维持内环境稳态中起重要作用。同时,miRNA作为细胞凋亡信号通路的关键调节因子,已成为生命科学研究的热点之一。本文综述了miRNA对细胞凋亡相关通路(线粒体通路,死亡受体通路和内质网通路)调控的研究进展,总结了不同组织及细胞中miRNA对凋亡通路的调节作用,为癌症等疾病治疗提供理论指导和新的思路。     

MAPK信号通路在辐射应答中的作用

丝裂原活化蛋白激酶（MAPK）超家族是介导细胞反应的重要信号系统,主要由MAPK、MAPK激酶（MAPKK）、MAPKK激酶（MAPKKK）等3类保守的蛋白激酶组成,通过级联反应不断磷酸化下游靶蛋白而参与细胞的增殖、分化、衰老、凋亡。辐射损伤使细胞膜受体和其他感应分子激活细胞内的MAPK信号通路,产生一系列应答反应。简要介绍MAPK家族中各条通路在辐射应答中的作用。     

Ceramide channels and their role in mitochondria-mediated apoptosis

A key, decision-making step in apoptosis is the release of proteins from the mitochondrial intermembrane space. Ceramide can self-assemble in the mitochondrial outer membrane to form large stable channels capable of releasing said proteins. Ceramide levels measured in mitochondria early in apoptosis are sufficient to form ceramide channels in the outer membrane. The channels are in dynamic equilibrium with non-conducting forms of ceramide in the membrane. This equilibrium can be strongly influenced by other sphingolipids and Bcl-2 family proteins. The properties of ceramide channels formed in a defined system, planar phospholipid membranes, demonstrate that proteins are not required for channel formation. In addition, experiments in the defined system reveal structural information. The results indicated that the channels are barrel-like structures whose staves are ceramide columns that span the membrane. Ceramide channels are good candidates for the protein release pathway that initiates the execution phase of apoptosis.     

线粒体神经酰胺对细胞凋亡的诱导作用

凋亡诱导期,线粒体内神经酰胺水平升高,当每纳摩尔线粒体膜磷脂内含4～6皮摩尔神经酰胺时,神经酰胺即在线粒体外膜形成稳定的跨膜通道,从而使外膜通透性增加,线粒体膜间蛋白释放,启动细胞凋亡.神经酰胺通道只能在线粒体外膜形成,它是由神经酰胺柱组成的桶装结构,神经酰胺的反式双键具有增加通道的稳定性的作用.     

The targeting of plasmalemmal ceramide to mitochondria during apoptosis

Ceramide is a key lipid mediator of cellular processes such as differentiation, proliferation, growth arrest and apoptosis. During apoptosis, ceramide is produced within the plasma membrane. Although recent data suggest that the generation of intracellular ceramide increases mitochondrial permeability, the source of mitochondrial ceramide remains unknown. Here, we determine whether a stress-mediated plasmalemmal pool of ceramide might become available to the mitochondria of apoptotic cells. We have previously established annexin A1--a member of a family of Ca(2+) and membrane-binding proteins--to be a marker of ceramide platforms. Using fluorescently tagged annexin A1, we show that, upon its generation within the plasma membrane, ceramide self-associates into platforms that subsequently invaginate and fuse with mitochondria. An accumulation of ceramide within the mitochondria of apoptotic cells was also confirmed using a ceramide-specific antibody. Electron microscopic tomography confirmed that upon the formation of ceramide platforms, the invaginated regions of the plasma membrane extend deep into the cytoplasm forming direct physical contacts with mitochondrial outer membranes. Ceramide might thus be directly transferred from the plasma membrane to the mitochondrial outer membrane. It is conceivable that this "kiss-of-death" increases the permeability of the mitochondrial outer membrane thereby triggering apoptosis.     

Biological aspects of ceramide-enriched membrane domains

Ceramide has been shown to be critically involved in many aspects of cellular responses to receptor-dependent and -independent stimuli. For instance, ceramide was demonstrated to be a central component of the signaling cascades mediating apoptosis after death receptor stimulation, treatment with chemotherapy or exposure to gamma-irradiation or UV-A light. Further studies indicated the importance of ceramide for the infection of mammalian cells with bacterial, viral and parasitic pathogens. Ceramide is released by the activity of acid, neutral or alkaline sphingomyelinases or de novo synthesized. A concept unifying the diverse biological functions of ceramide indicates that ceramide forms distinct membrane domains, named ceramide-enriched membrane domains or platforms. These domains serve the clustering of receptor molecules, the re-organization of signaling proteins, the exclusion of inhibitory signals and, thus, initiate and greatly amplify a primary signal. In addition, ceramide directly interacts with and stimulates intracellular enzymes that may act together with signals initiated in ceramide-enriched membrane domains to transmit signals into a cell.     

Mechanisms of ceramide‐induced COX‐2‐dependent apoptosis in human ovarian cancer OVCAR‐3 cells partially overlapped with resveratrol

Ceramide is a member of the sphingolipid family of bioactive molecules demonstrated to have profound, diverse biological activities. Ceramide is a potential chemotherapeutic agent via the induction of apoptosis. Exposure to ceramide activates extracellular‐signal‐regulated kinases (ERK)1/2‐ and p38 kinase‐dependent apoptosis in human ovarian cancer OVCAR‐3 cells, concomitant with an increase in the expression of COX‐2 and p53 phosphorylation. Blockade of cyclooxygenase‐2 (COX‐2) activity by siRNA or NS398 correspondingly inhibited ceramide‐induced p53 Ser‐15 phosphorylation and apoptosis; thus COX‐2 appears at the apex of the p38 kinase‐mediated signaling cascade induced by ceramide. Induction of apoptosis by ceramide or resveratrol was inhibited by the endocytosis inhibitor, cytochalasin D (CytD); however, cells exposed to resveratrol showed greater sensitivity than ceramide‐treated cells. Ceramide‐treated cells underwent a dose‐dependent reduction in trans‐membrane potential. Although both ceramide and resveratrol induced the expressions of caspase‐3 and ‐7, the effect of inducible COX‐2 was different in caspase‐7 expression induced by ceramide compared to resveratrol. In summary, resveratrol and ceramide converge on an endocytosis‐requiring, ERK1/2‐dependent signal transduction pathway and induction of COX‐expression as an essential molecular antecedent for subsequent p53‐dependent apoptosis. In addition, expressions of caspase‐3 and ‐7 are observed. However, a p38 kinase‐dependent signal transduction pathway and change in mitochondrial potential are also involved in ceramide‐induced apoptosis. J. Cell. Biochem. 114: 1940–1954, 2013. © 2013 Wiley Periodicals, Inc.     

Ceramide synthesis in the endoplasmic reticulum can permeabilize mitochondria to proapoptotic proteins

Increased mitochondrial ceramide levels are associated with the initiation of apoptosis. There is evidence that ceramide is causal. Thus, the conversion of the precursor, dihydroceramide, to ceramide by the enzyme dihydroceramide desaturase may be important in preparing the cell for apoptosis. Ceramide can initiate apoptosis by permeabilizing the mitochondrial outer membrane to apoptosis-inducing proteins. However, the mitochondrion's ability to produce ceramide may be limited by its proteome. Here, we show that ceramide synthesized in isolated mammalian endoplasmic reticulum (ER) vesicles from either C8-dihydroceramide or sphingosine to produce long-chain ceramide can transfer to isolated mitochondria. The rate of transfer is consistent with a simple collision model. The transfer of the long-chain ceramide is faster than expected for an uncatalyzed process. Sufficient ceramide is transferred to permeabilize the outer membrane to cytochrome c and adenylate kinase. The mitochondria-associated membranes, ER-like membranes that are tightly associated with isolated mitochondria, can produce enough ceramide to permeabilize the outer membrane transiently. Thus, this ceramide exchange obviates the need for a complete ceramide de novo pathway in mitochondria to increase ceramide levels to the critical value required for functional changes, such as ceramide channel self-assembly followed by protein release.     

Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations

Recent evidence suggests that the ability of ceramides to induce apoptosis is due to a direct action on mitochondria. Mitochondria are known to contain enzymes responsible for ceramide synthesis and hydrolysis and mitochondrial ceramide levels have been shown to be elevated prior to the mitochondrial phase of apoptosis. Ceramides have been reported to induce the release of intermembrane space proteins from mitochondria, which has been linked to their ability to form large channels in membranes. The aim of this study was to determine if the membrane concentration of ceramide required for the formation of protein permeable channels is within the range that is present in mitochondria during the induction phase of apoptosis. Only a very small percentage of the ceramide actually inserts into the mitochondrial membranes. The permeability of the mitochondrial outer membrane correlates directly with the level of ceramide in the membrane. Importantly, the concentration of ceramide at which significant channel formation occurs is consistent with the level of mitochondrial ceramide that occurs during the induction phase of apoptosis (4 pmol ceramide/nanomole phospholipid). Similar results were obtained with short- and long-chain ceramide. Ceramide channel formation is specific to mitochondrial membranes in that no channel formation occurs in the plasma membranes of erythrocytes even at concentrations 20 times higher than those required for channel formation in mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway by which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis.     

Ceramide in bacterial infections and cystic fibrosis

Ceramide is formed by the activity of sphingomyelinases, by degradation of complex sphingolipids, reverse ceramidase activity or de novo synthesized. The formation of ceramide within biological membranes results in the formation of large ceramide-enriched membrane domains. These domains serve the spatial and temporal organization of receptors and signaling molecules. The acid sphingomyelinase-ceramide system plays an important role in the infection of mammalian host cells with bacterial pathogens such as Neisseria gonorrhoeae, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium and Pseudomonas aeruginosa. Ceramide and ceramide-enriched membrane platforms are also involved in the induction of apoptosis in infected cells, such as in epithelial and endothelial cells after infection with Pseudomonas aeruginosa and Staphylococcus aureus, respectively. Finally, ceramide-enriched membrane platforms are critical regulators of the release of pro-inflammatory cytokines upon infection. The diverse functions of ceramide in bacterial infections suggest that ceramide and ceramide-enriched membrane domains are key players in host responses to many pathogens and thus are potential novel targets to treat infections.     

Mitochondrial Ceramide and the Induction of Apoptosis

In most cell types, a key event in apoptosis is the release of proapoptotic intermembrane space proteins from mitochondria to the cytoplasm. In general, it is the release of these intermembrane space proteins that is responsible for the activation of caspases and DNases that are responsible for the execution of apoptosis. The mechanism for the increased permeability of the mitochondrial outer membrane during the induction phase of apoptosis is currently unknown and highly debated. This review will focus on one such proposed mechanism, namely, the formation of ceramide channels in the mitochondrial outer membrane. Ceramides are known to play a major regulatory role in apoptosis by inducing the release of proapoptotic proteins from the mitochondria. As mitochondria are known to contain the enzymes responsible for the synthesis and hydrolysis of ceramide, there exists a mechanism for regulating the level of ceramide in mitochondria. In addition, mitochondrial ceramide levels have been shown to be elevated prior to the induction phase of apoptosis. Ceramide has been shown to form large protein permeable channels in planar phospholipid and mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway with which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis.     

Fas-mediated apoptosis and sphingomyelinase signal transduction: the role of ceramide as a second messenger for apoptosis

In this article, we review the role of sphingomyelinases and ceramide in the Fas-mediated apoptosis signal transduction cascade. Several stimuli, including ligation of Fas, have been shown to enhance either neutral and/or acidic sphingomyelinase activity and increase ceramide content in intact cells or cell membrane preparations. Ceramide seems to have different functions, including induction of apoptosis, growth arrest, and/or differentiation, depending on cell type or location of sphingomyelin hydrolysis within the cell. Several putative targets for ceramide activity, including a kinase and a phosphatase, have also been identified. While ceramide and acidic sphingomyelinase activity appear to be involved in apoptotic signalling for Fas and other members of the tumour necrosis factor receptor family, it is clear that other signals and mechanisms are necessary for Fas-mediated apoptosis.