Reductive carboxylation supports growth in tumour cells with defective mitochondria

Mitochondrial metabolism provides precursors to build macromolecules in growing cancer cells. In normally functioning tumour cell mitochondria, oxidative metabolism of glucose- and glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, which is required for tumorigenesis. Yet some tumours harbour mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function, and it is unknown how cells from such tumours generate precursors for macromolecular synthesis. Here we show that tumour cells with defective mitochondria use glutamine-dependent reductive carboxylation rather than oxidative metabolism as the major pathway of citrate formation. This pathway uses mitochondrial and cytosolic isoforms of NADP(+)/NADPH-dependent isocitrate dehydrogenase, and subsequent metabolism of glutamine-derived citrate provides both the acetyl-coenzyme A for lipid synthesis and the four-carbon intermediates needed to produce the remaining CAC metabolites and related macromolecular precursors. This reductive, glutamine-dependent pathway is the dominant mode of metabolism in rapidly growing malignant cells containing mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with mutations in fumarate hydratase, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. Our findings reveal the novel induction of a versatile glutamine-dependent pathway that reverses many of the reactions of the canonical CAC, supports tumour cell growth, and explains how cells generate pools of CAC intermediates in the face of impaired mitochondrial metabolism.     

Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC.

During transduction of an apoptotic (death) signal into the cell, there is an alteration in the permeability of the membranes of the cell's mitochondria, which causes the translocation of the apoptogenic protein cytochrome c into the cytoplasm, which in turn activates death-driving proteolytic proteins known as caspases. The Bcl-2 family of proteins, whose members may be anti-apoptotic or pro-apoptotic, regulates cell death by controlling this mitochondrial membrane permeability during apoptosis, but how that is achieved is unclear. Here we create liposomes that carry the mitochondrial porin channel (also called the voltage-dependent anion channel, or VDAC) to show that the recombinant pro-apoptotic proteins Bax and Bak accelerate the opening of VDAC, whereas the anti-apoptotic protein Bcl-x(L) closes VDAC by binding to it directly. Bax and Bak allow cytochrome c to pass through VDAC out of liposomes, but passage is prevented by Bcl-x(L). In agreement with this, VDAC1-deficient mitochondria from a mutant yeast did not exhibit a Bax/Bak-induced loss in membrane potential and cytochrome c release, both of which were inhibited by Bcl-x(L). Our results indicate that the Bcl-2 family of proteins bind to the VDAC in order to regulate the mitochondrial membrane potential and the release of cytochrome c during apoptosis.     

线粒体外膜通道蛋白VDAC与靶向肿瘤细胞凋亡

VDAC(Voltage-dependent anion channel)是位于线粒体外膜上的一种主要通道蛋白,参与线粒体内外物质和能量的运输,在线粒体与细胞其它部位的通讯中起重要调节作用.近年来研究发现,VDAC也是线粒体与其它蛋白质相互作用的功能结合位点,可与多种凋亡调节蛋白(如HK-Ⅰ/Ⅱ、Bcl-2家族蛋白、tubulin、MAP2/4等)以及非蛋白调节因子相互作用,参与调控细胞凋亡.因此,VDAC成为线粒体凋亡通路中一种关键的靶蛋白.本文对近年来VDAC在肿瘤细胞凋亡中的作用机制进行简要综述.     

BRAF mutation predicts sensitivity to MEK inhibition

The kinase pathway comprising RAS, RAF, mitogen-activated protein kinase kinase (MEK) and extracellular signal regulated kinase (ERK) is activated in most human tumours, often through gain-of-function mutations of RAS and RAF family members. Using small-molecule inhibitors of MEK and an integrated genetic and pharmacologic analysis, we find that mutation of BRAF is associated with enhanced and selective sensitivity to MEK inhibition when compared to either 'wild-type' cells or cells harbouring a RAS mutation. This MEK dependency was observed in BRAF mutant cells regardless of tissue lineage, and correlated with both downregulation of cyclin D1 protein expression and the induction of G1 arrest. Pharmacological MEK inhibition completely abrogated tumour growth in BRAF mutant xenografts, whereas RAS mutant tumours were only partially inhibited. These data suggest an exquisite dependency on MEK activity in BRAF mutant tumours, and offer a rational therapeutic strategy for this genetically defined tumour subtype.     

Selective killing of cancer cells by a small molecule targeting the stress response to ROS

Malignant transformation, driven by gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes, results in cell deregulation that is frequently associated with enhanced cellular stress (for example, oxidative, replicative, metabolic and proteotoxic stress, and DNA damage). Adaptation to this stress phenotype is required for cancer cells to survive, and consequently cancer cells may become dependent upon non-oncogenes that do not ordinarily perform such a vital function in normal cells. Thus, targeting these non-oncogene dependencies in the context of a transformed genotype may result in a synthetic lethal interaction and the selective death of cancer cells. Here we used a cell-based small-molecule screening and quantitative proteomics approach that resulted in the unbiased identification of a small molecule that selectively kills cancer cells but not normal cells. Piperlongumine increases the level of reactive oxygen species (ROS) and apoptotic cell death in both cancer cells and normal cells engineered to have a cancer genotype, irrespective of p53 status, but it has little effect on either rapidly or slowly dividing primary normal cells. Significant antitumour effects are observed in piperlongumine-treated mouse xenograft tumour models, with no apparent toxicity in normal mice. Moreover, piperlongumine potently inhibits the growth of spontaneously formed malignant breast tumours and their associated metastases in mice. Our results demonstrate the ability of a small molecule to induce apoptosis selectively in cells that have a cancer genotype, by targeting a non-oncogene co-dependency acquired through the expression of the cancer genotype in response to transformation-induced oxidative stress.     

细胞应激条件下嗜热四膜虫线粒体膜电位变化研究

单细胞线虫纲原生动物四膜虫(Tetrahymena),在H2O2的诱导下可发生凋亡样死亡.另外,最新研究表明细胞中活性氧簇(ROS)的积累可有效地诱导细胞自噬途径的发生.通过流式细胞技术和荧光显微技术,检测了经饥饿和ROS诱导剂处理后,线粒体内膜电位变化及细胞内ROS的积累.此外,应用两种抗氧化剂氮乙酰半胱氨酸(NAC)和过氧化氢酶(Catalase)分别对四膜虫细胞进行处理,检测了在氧化应激条件下的抗氧化作用效果.结果表明,Oligomycin和Menadione可有效抑制线粒体膜电位的维持,从而导致细胞内ROS积累.同时,H2O2处理和饥饿处理可以导致嗜热四膜虫细胞线粒体膜电位丧失以及细胞质ROS积累.另外,N-acetylcystine和Catalase可有效地降低四膜虫细胞内ROS的积累,以及保持四膜虫细胞线粒体内膜电位,维持通透性.     

人参皂甙Rh2和桦木酸协同诱导A549细胞凋亡的研究

我们已经发现,天然成分人参皂甙Rh2(G-Rh2)和桦木酸(Bet A)能够协同作用诱导人源肺癌A549细胞死亡.通过细胞形态观察、流式细胞检测分析、酶活性测定、免疫印记分析等实验技术,从细胞和分子水平提供证据,证明了G-Rh2和Bet A协同诱导A549细胞死亡的过程为细胞凋亡作用,且涉及线粒体途径.     

Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death

Mitochondria play a critical role in mediating both apoptotic and necrotic cell death. The mitochondrial permeability transition (mPT) leads to mitochondrial swelling, outer membrane rupture and the release of apoptotic mediators. The mPT pore is thought to consist of the adenine nucleotide translocator, a voltage-dependent anion channel, and cyclophilin D (the Ppif gene product), a prolyl isomerase located within the mitochondrial matrix. Here we generated mice lacking Ppif and mice overexpressing cyclophilin D in the heart. Ppif null mice are protected from ischaemia/reperfusion-induced cell death in vivo, whereas cyclophilin D-overexpressing mice show mitochondrial swelling and spontaneous cell death. Mitochondria isolated from the livers, hearts and brains of Ppif null mice are resistant to mitochondrial swelling and permeability transition in vitro. Moreover, primary hepatocytes and fibroblasts isolated from Ppif null mice are largely protected from Ca2+-overload and oxidative stress-induced cell death. However, Bcl-2 family member-induced cell death does not depend on cyclophilin D, and Ppif null fibroblasts are not protected from staurosporine or tumour-necrosis factor-alpha-induced death. Thus, cyclophilin D and the mitochondrial permeability transition are required for mediating Ca2+- and oxidative damage-induced cell death, but not Bcl-2 family member-regulated death.     

Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7

Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.     

Progression from lymphoid hyperplasia to high-grade malignant lymphoma in mice transgenic for the t(14; 18)

Follicular lymphoma, the most common human lymphoma, characteristically has a t(14; 18) interchromosomal translocation. It is typically an indolent disease comprised of small resting B cells, but frequently develops into a high-grade lymphoma. The t(14; 18) translocates the Bcl-2 gene, generating a deregulated Bcl-2-immunoglobulin fusion gene. Bcl-2 is a novel inner mitochondrial membrane protein that extends the survival of certain cells by blocking programmed cell death. To determine the oncogenic potential of the t(14; 18) translocation, we produced transgenic mice bearing a Bcl-2-immunoglobulin minigene that structurally mimicked the t(14; 18). An indolent follicular hyperplasia in these transgenic mice progressed to a malignant diffuse large-cell lymphoma. The long latency, progression from polyclonal to monoclonal disease, and histological conversion, are all suggestive of secondary changes. Half of the immunoblastic high-grade lymphomas had a rearranged c-myc gene. Our transgenic mice provide an animal model for tumour progression in t(14; 18) lymphoma and show that prolonged B-cell life increases tumour incidence.     

Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells

Little is known about metabolic regulation in stem cells and how this modulates tissue regeneration or tumour suppression. We studied the Lkb1 tumour suppressor and its substrate AMP-activated protein kinase (AMPK), kinases that coordinate metabolism with cell growth. Deletion of the Lkb1 (also called Stk11) gene in mice caused increased haematopoietic stem cell (HSC) division, rapid HSC depletion and pancytopenia. HSCs depended more acutely on Lkb1 for cell-cycle regulation and survival than many other haematopoietic cells. HSC depletion did not depend on mTOR activation or oxidative stress. Lkb1-deficient HSCs, but not myeloid progenitors, had reduced mitochondrial membrane potential and ATP levels. HSCs deficient for two catalytic α-subunits of AMPK (AMPK-deficient HSCs) showed similar changes in mitochondrial function but remained able to reconstitute irradiated mice. Lkb1-deficient HSCs, but not AMPK-deficient HSCs, exhibited defects in centrosomes and mitotic spindles in culture, and became aneuploid. Lkb1 is therefore required for HSC maintenance through AMPK-dependent and AMPK-independent mechanisms, revealing differences in metabolic and cell-cycle regulation between HSCs and some other haematopoietic progenitors.     

AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress

Overcoming metabolic stress is a critical step for solid tumour growth. However, the underlying mechanisms of cell death and survival under metabolic stress are not well understood. A key signalling pathway involved in metabolic adaptation is the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Energy stress conditions that decrease intracellular ATP levels below a certain level promote AMPK activation by LKB1. Previous studies showed that LKB1-deficient or AMPK-deficient cells are resistant to oncogenic transformation and tumorigenesis, possibly because of the function of AMPK in metabolic adaptation. However, the mechanisms by which AMPK promotes metabolic adaptation in tumour cells are not fully understood. Here we show that AMPK activation, during energy stress, prolongs cell survival by redox regulation. Under these conditions, NADPH generation by the pentose phosphate pathway is impaired, but AMPK induces alternative routes to maintain NADPH and inhibit cell death. The inhibition of the acetyl-CoA carboxylases ACC1 and ACC2 by AMPK maintains NADPH levels by decreasing NADPH consumption in fatty-acid synthesis and increasing NADPH generation by means of fatty-acid oxidation. Knockdown of either ACC1 or ACC2 compensates for AMPK activation and facilitates anchorage-independent growth and solid tumour formation in vivo, whereas the activation of ACC1 or ACC2 attenuates these processes. Thus AMPK, in addition to its function in ATP homeostasis, has a key function in NADPH maintenance, which is critical for cancer cell survival under energy stress conditions, such as glucose limitations, anchorage-independent growth and solid tumour formation in vivo.     

Oncogene-induced senescence as an initial barrier in lymphoma development

Acute induction of oncogenic Ras provokes cellular senescence involving the retinoblastoma (Rb) pathway, but the tumour suppressive potential of senescence in vivo remains elusive. Recently, Rb-mediated silencing of growth-promoting genes by heterochromatin formation associated with methylation of histone H3 lysine 9 (H3K9me) was identified as a critical feature of cellular senescence, which may depend on the histone methyltransferase Suv39h1. Here we show that Emicro-N-Ras transgenic mice harbouring targeted heterozygous lesions at the Suv39h1, or the p53 locus for comparison, succumb to invasive T-cell lymphomas that lack expression of Suv39h1 or p53, respectively. By contrast, most N-Ras-transgenic wild-type ('control') animals develop a non-lymphoid neoplasia significantly later. Proliferation of primary lymphocytes is directly stalled by a Suv39h1-dependent, H3K9me-related senescent growth arrest in response to oncogenic Ras, thereby cancelling lymphomagenesis at an initial step. Suv39h1-deficient lymphoma cells grow rapidly but, unlike p53-deficient cells, remain highly susceptible to adriamycin-induced apoptosis. In contrast, only control, but not Suv39h1-deficient or p53-deficient, lymphomas senesce after drug therapy when apoptosis is blocked. These results identify H3K9me-mediated senescence as a novel Suv39h1-dependent tumour suppressor mechanism whose inactivation permits the formation of aggressive but apoptosis-competent lymphomas in response to oncogenic Ras.     

新生儿坏死性小肠结肠炎中细胞死亡机制的研究进展

坏死性小肠结肠炎(Necrotizing Enterocolitis, NEC)是由多因素作用导致的肠道急性炎症性疾病,是早产儿主要的死亡原因之一。以往的研究认为,细胞凋亡是NEC中肠上皮细胞最主要的死亡形式。但近年来的研究发现,程序性坏死(Necroptosis)、细胞焦亡(Pyroptosis)及铁死亡(Ferroptosis)等非凋亡形式的程序性细胞死亡(Programmed Cell Death, PCD)也可能参与到NEC的发病机制中,不同形式的细胞死亡的信号调节通路不同,可能会相互影响或存在共同的调节机制如细胞广泛凋亡小体(PANoptosome)等。本文综述了非凋亡形式的不同类型程序性细胞死亡方式及其信号调节通路,以及其在NEC中的作用机制,并提出NEC诊断生物标志物或防治的新靶点,以期为临床NEC的预防和管理提供思路。     

Calpain的激活与鱼藤酮导致的细胞凋亡

在缺氧或呼吸链抑制剂存在条件下,细胞的呼吸链受到抑制,线粒体的功能受到直接干扰,细胞色素C通过线粒体的外膜特异性通道进入细胞浆内,启动了procaspase-3等一系列凋亡因子,细胞发生与线粒体相关的凋亡。另一方面,因线粒体的功能被抑制,细胞内的钙离子浓度升高,calpain被激活并裂解细胞膜蛋白及细胞内的生物化学分子,促进了细胞凋亡的发生。鱼藤酮作为线粒体呼吸链complexI的抑制剂可导致细胞凋亡,其凋亡途径不仅与caspase介导的机制有关,还有可能与calpain有关。     

Induction by E1A oncogene expression of cellular susceptibility to lysis by TNF

Tumour necrosis factor alpha (ref. 1), synthesized primarily by monocytes in response to various invasive agents, induces a wide variety of biological effects relevant to regulating cell growth and differentiation, including the selective killing of some tumour cells and the growth stimulation of some normal fibroblasts. As tumour necrosis factor (TNF) appears to kill tumour cells preferentially, we asked whether TNF sensitivity correlates with the expression of specific oncogene(s). If so, by examining the cellular target(s) of the oncogene product, it might be possible to identify specific factor(s) which mediate TNF action. By using an in vitro cytotoxicity assay with NIH 3T3 and Fisher BRK-derived cells expressing exogenously introduced oncogenes, we found that adenovirus E1A proteins induce susceptibility to TNF killing.     

Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants

Caenorhabditis elegans homologues of the retinoblastoma (Rb) tumour suppressor complex specify cell lineage during development. Here we show that mutations in Rb pathway components enhance RNA interference (RNAi) and cause somatic cells to express genes and elaborate perinuclear structures normally limited to germline-specific P granules. Furthermore, particular gene inactivations that disrupt RNAi reverse the cell lineage transformations of Rb pathway mutants. These findings suggest that mutations in Rb pathway components cause cells to revert to patterns of gene expression normally restricted to germ cells. Rb may act by a similar mechanism to transform mammalian cells.     

新型杀虫剂的作用机制和选择毒性

作用机制的研究对于开发新型害虫防治药剂是非常重要的。本文综述了一系列具有新颖作用机制的新型杀虫剂,包括神经毒性杀虫剂、昆虫生长调节剂、几丁质合成抑制剂以及作用于氧化磷酸化的杀虫剂,并讨论了它们对非靶标生物的选择毒性。