Role of Mitochondria in Human Aging

Mitochondria are the major intracellular source and target sites of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in animal and human cells. It has been demonstrated that mitochondrial respiratory function declines with age in various human tissues and that a defective respiratory chain results in enhanced production of ROS and free radicals in mitochondria. On the other hand, accumulating evidence now indicates that lipid peroxidation, protein modification and mitochondrial DNA (mtDNA) muutation are concurrently increased during aging. On the basis of these observations and the fact that the rate of cellular production of superoxide anions and hydrogen peroxide increases with age, it has recently been postulated that oxidative stress is a major contributory factor in the aging process. A causal relationship between oxidative modification and mutation of mtDNA, mitochondrial dysfunction and aging has emerged, although some details have remained unsolved. In this article, the role of mitochondria in the human aging process is reviewed on the basis of recent findings gathered from our and other laboratories.     

Lipocalin-2 Induces Apoptosis in Human Hepatocellular Carcinoma Cells Through Activation of Mitochondria Pathways

Lipocalin 2 (LCN2) is a secreted, iron-binding glycoprotein that is abnormally expressed in some malignant human cancers. However, the roles of LCN2 in hepatocellular carcinoma (HCC) cells are unknown. In this study, we suggested the LCN2 and LCN2R were weak detected in the HCC cell lines, LCN2 and LCN2R were found to be down-regulated in tumor tissues in 16 HCC patients. MTT, DAPI, TUNEL, and flow cytometry analyses revealed that LCN2 overexpression dramatically inhibited cell viability, induced apoptosis features of cell-cycle arrest in sub-G1 phase, in DNA fragmentation, and in condensation of chromatin in Huh-7 and SK-Hep-1 cells. Western blots were used to detect the activation of caspase, pro-apoptosis, and anti-apoptosis protein expression in overexpress-LCN2 HCC cells. LCN2-induced apoptosis was characterized by cleavage of caspase-9, -8, -3, and PARP protein, and a reduction in the mitochondrial membrane potential (MMP). Furthermore, LCN2 also enhanced the down-regulated Bcl-2 and up-regulated the expression of Bax. In addition, our experiments with caspase inhibitors LEHD-FMK and IETD-FMK prevent LCN2-induced apoptosis. We also demonstrated that treatment of overexpress-LCN2 HCC cells with the LCN2 neutralized antibody also significantly attenuated LCN2-induced cell apoptosis. These findings indicate that LCN2 overexpression can effectively induce apoptosis of HCC cells and may be used as a potent therapy against human HCC.     

Mitochondria in Ca2+ Signaling and Apoptosis

Cellular Ca²⁺ signals are crucial in the control of most physiological processes, cell injuryand programmed cell death; mitochondria play a pivotal role in the regulation of such cytosolicCa²⁺ ([Ca²⁺]_c) signals. Mitochondria are endowed with multiple Ca²⁺ transport mechanismsby which they take up and release Ca²⁺ across their inner membrane. These transport processesfunction to regulate local and global [Ca²⁺]_c, thereby regulating a number of Ca²⁺-sensitivecellular mechanisms. The permeability transition pore (PTP) forms the major Ca²⁺ effluxpathway from mitochondria. In addition, Ca²⁺ efflux from the mitochondrial matrix occursby the reversal of the uniporter and through the inner membrane Na⁺/Ca²⁺ exchanger. Duringcellular Ca²⁺ overload, mitochondria take up [Ca²⁺]_c, which, in turn, induces opening of PTP,disruption of mitochondrial membrane potential (_m) and cell death. In apoptosis signaling,collapse of ;_m and cytochrome c release from mitochondria occur followed by activationof caspases, DNA fragmentation, and cell death. Translocation of Bax, an apoptotic signalingprotein from the cytosol to the mitochondrial membrane, is another step during thisapoptosis-signaling pathway. The role of permeability transition in the context of cell death in relationto Bcl-2 family of proteins is discussed.     

The in Vitro Growth of Human Chondrocytes

Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects has been described by other workers, however, relatively few details of the in vitro growth of the cells have been published. Here we describe the release of cells from adult human articular cartilage and their growth characteristics in vitro.Cultures were successfully established from 29 of 30 biopsies taken from patients aged 20–72 year. No significant relationship was found between donor age and initial cell yield following cartilage digest, however, the time to primary confluence increased in direct proportion to age. Thereafter the kinetics of cell proliferation was independent of donor age.The proportion of apoptotic or necrotic cells in the cartilage digest was low and increased with time in culture only in those cells which remained non-adherent. Conversely, entry into cell cycle was restricted to those cells which had become adherent.These results illustrate that previously reported techniques for isolating and culturing chondrocytes are reproducible, that adherent chondrocytes have considerable proliferative potential, and that concern about cell growth and viability need not, in itself, limit the clinical application of ACI to younger patients.     

Copper Oxide Nanoparticles Induced Mitochondria Mediated Apoptosis in Human Hepatocarcinoma Cells

Copper oxide nanoparticles (CuO NPs) are heavily utilized in semiconductor devices, gas sensor, batteries, solar energy converter, microelectronics and heat transfer fluids. It has been reported that liver is one of the target organs for nanoparticles after they gain entry into the body through any of the possible routes. Recent studies have shown cytotoxic response of CuO NPs in liver cells. However, the underlying mechanism of apoptosis in liver cells due to CuO NPs exposure is largely lacking. We explored the possible mechanisms of apoptosis induced by CuO NPs in human hepatocellular carcinoma HepG2 cells. Prepared CuO NPs were spherical in shape with a smooth surface and had an average diameter of 22 nm. CuO NPs (concentration range 2–50 µg/ml) were found to induce cytotoxicity in HepG2 cells in dose-dependent manner, which was likely to be mediated through reactive oxygen species generation and oxidative stress. Tumor suppressor gene p53 and apoptotic gene caspase-3 were up-regulated due to CuO NPs exposure. Decrease in mitochondrial membrane potential with a concomitant increase in the gene expression of bax/bcl2 ratio suggested that mitochondria mediated pathway involved in CuO NPs induced apoptosis. This study has provided valuable insights into the possible mechanism of apoptosis caused by CuO NPs at in vitro level. Underlying mechanism(s) of apoptosis due to CuO NPs exposure should be further invested at in vivo level.     

Apoptosis of Terminally Differentiated Chondrocytes in Culture

During the process of endochondral ossification chondrocytes progress through stages of terminal differentiation culminating in apoptotic death. We have developed a serum-free suspension culture that allows terminal differentiation and facilitates the investigation of factors affecting chondrocyte apoptosis. We have found that chondrocytes not committed to terminal differentiation, i.e., those from the caudal region of chick embryo sterna, a region that remains cartilaginous for some months after the chick hatches, maintained high viability in serum-free suspension culture. A strong dependence of viability on culture density and sensitivity to induction of apoptosis with the protein kinase inhibitor, staurosporine, was consistent with the proposal that these chondrocytes, like nearly all cells, require intercellular communication for survival. Chondrocytes that were committed to terminal differentiation, i.e., those from the cephalic region of chick embryo sterna, a region that is replaced by bone before the chick hatches, expressed the hypertrophic phenotype but maintained their viability in culture for only approximately 6 days. Subsequent cell death was very consistent between cultures and shown to occur by an apoptotic process by analysis of DNA fragmentation and cell morphology. Short-term viability of hypertrophic chondrocytes was independent of culture density and relatively resistant to treatment with staurosporine. Induction of the hypertrophic phenotype in immature chondrocytes committed them to cell death and prevention of expression of the hypertrophic phenotype prevented cell death. We conclude that commitment of chondrocytes to terminal differentiation is associated with a commitment to apoptosis and apoptosis of hypertrophic chondrocytes in growth cartilage does not require initiation by external signals.     

microRNA-15a模拟物对人关节软骨细胞增殖与凋亡的影响

目的：近年来研究表明,关节软骨细胞凋亡在骨关节炎发病过程中起到了重要的作用,本文旨在探讨microma-15a模拟物对于原代人膝关节软骨细胞增殖与凋亡的影响。方法：取人外伤性截肢后的膝关节软骨,采用双酶消化法分离获得人膝关节软骨细胞,并进行体外培养,通过甲苯胺蓝染色和II型胶原免疫细胞化学染色进行软骨细胞鉴定。将培养的软骨细胞传代后取第l代细胞,分为实验组和对照组,实验组采用mir．15a模拟物（has．mir-15amimics）转染软骨细胞,上调软骨细胞内mir-15a的表达量;对照组分为阴性对照组、空白对照组。采用MTT法测定细胞增殖曲线,流式细胞仪测定细胞凋亡率。结果：原代细胞中细胞呈多角形、圆形与梭型,贴壁生长;甲苯胺蓝染色胞质呈深蓝色,II型胶原染色胞质呈黄褐色,为特异性染色。经统计学分析,实验组与对照组相比增殖速率明显下降（P〈0．05）。实验组凋亡率（7．13％±0．57）与阴性对照组凋亡率（2．66％±0．15）相比明显增高（P〈0．05）。结论：采用双酶消化法成功分离并培养具有生物学特性的原代人膝关节软骨细胞,通过转染mir-15a模拟物外源性增加关节软骨细胞内mir．15a表达量可显著促进其凋亡并抑制其增殖,为阐明骨关节炎发病机制提供了新的理论依据,为’临床治疗提供了新的靶点。     

The Role of Mitochondria in Glioma Pathophysiology

It has long been recognised that malignant tumours favour aerobic glycolysis to generate ATP and contain abnormalities of the intrinsic, mitochondria-dependent, apoptotic pathway, suggesting the involvement of dysfunctional mitochondria in tumour pathophysiology. However, the mechanisms underlying such processes in gliomas are poorly understood. Few recent studies have evaluated mitochondrial ultrastructure and proteomics in the pathophysiology of malignant gliomas. However, aberrant energy metabolism has been reported in gliomas and mitochondrial dysfunction links to glioma apoptotic signalling have been observed. Mitochondrial structural abnormalities and dysfunction in malignant gliomas is a neglected area of research. Definition of abnormalities in mitochondrial proteomics, membrane potential regulation, energy metabolism and intrinsic apoptotic pathway signalling in gliomas may open novel therapeutic opportunities.     

Role of Siglec-7 in Apoptosis in Human Platelets

Background

Platelets participate in tissue repair and innate immune responses. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are well-characterized I-type lectins, which control apoptosis.

Methodology/Principal Findings

We characterized the expression of Siglec-7 in human platelets isolated from healthy volunteers using flow cytometry and confocal microscopy. Siglec-7 is primarily expressed on α granular membranes and colocalized with CD62P. Siglec-7 expression was increased upon platelet activation and correlated closely with CD62P expression. Cross-linking Siglec-7 with its ligand, ganglioside, resulted in platelet apoptosis without any significant effects on activation, aggregation, cell morphology by electron microscopy analysis or secretion. We show that ganglioside triggered four key pathways leading to apoptosis in human platelets: (i) mitochondrial inner transmembrane potential (ΔΨm) depolarization; (ii) elevated expression of pro-apoptotic Bax and Bak proteins with reduced expression of anti-apoptotic Bcl-2 protein; (iii) phosphatidylserine exposure and (iv), microparticle formation. Inhibition of NAPDH oxidase, PI3K, or PKC rescued platelets from apoptosis induced by Siglec-7 recruitment, suggesting that the platelet receptors P2Y1 and GPIIbIIIa are essential for ganglioside-induced platelet apoptosis.

Conclusions/Significance

The present work characterizes the role of Siglec-7 and platelet receptors in regulating apoptosis and death. Because some platelet pathology involves apoptosis (idiopathic thrombocytopenic purpura and possibly storage lesions), Siglec-7 might be a molecular target for therapeutic intervention/prevention.     

FGF-2 在细胞凋亡中的作用机制

成纤维细胞生长因子2(fibroblast growth factor 2,FGF-2)具有多种细胞生物学功能。FGF-2在肿瘤组织中呈高水平表达状态,且可抑制多种化疗药物的促凋亡作用,从而曾为肿瘤细胞存活的重要刺激因素。但也有研究表明FGF-2可诱导部分细胞的分化和凋亡。鉴于FGF-2在肿瘤的发生发展中发挥的重要作用,FGF-2与细胞凋亡的关系及其相应的调节机制成为有待于深入研究和迫切需要解决的问题。本文主要阐述在细胞凋亡通路中,FGF-2关键分子的作用机制及其最新研究进展。     

Apoptosis of Chondrocytes in Transgenic Mice Lacking Collagen II

Previous work demonstrated that collagen fibrils were not detectable in the cartilage of transgenic mice homozygous for targeted inactivation of the collagen II gene. In the present work, we used the same mice to show that chondrocytes undergo apoptosis in the absence of collagen II, the major component of the extracellular matrix of cartilage. The chondrocytes in the homozygous mice had condensed nuclei, fragmentation of nuclear DNA, and decreased levels of the Bcl-2 protein. These results provide direct evidence that cartilage extracellular matrix lacking collagen II cannot support the survival of chondrocytes. In addition, the results suggest that apoptosis may play a role in degenerative connective tissue diseases such as osteoarthritis in which there is extensive tissue loss.     

The Role of Mitochondria in Stem Cell Biology

This mini-review summarizes the current literature on the role of mitochondrial DNA mutations and mitochondrial metabolism in stem cell biology. The possible uses of stem cells as a therapeutic tool in mitochondrial disorders are also reported.     

Study on the Apoptosis Mechanism Induced by T-2 Toxin

T-2 toxin is known to induce apoptosis in mammalian cells. The mechanism of apoptosis induced by T-2 toxin has been proposed to be linked with oxidative stress and mitochondrial pathway. In the current study, the toxic effect of T-2 on Hela, Bel-7402, and Chang liver cells was examined in dose-dependent and time-dependent manner by MTT assay. Caspase-3 was found to be up-regulated under T-2 toxin stress, which suggested that T-2 toxin induced cell apoptosis. Endogenous GSH and MDA levels in all three cell lines were found down- and up-regulated respectively, which indicated the link between toxic effect of T-2 toxin and intracellular oxidative stress. It was also found by MTT assay that NAC, which maintained the level of GSH in cells, could protect cells from death. Western-blot result showed that the level of both activated Caspase-8 and Caspase-9 increased when cells were treated by T-2 toxin. Caspase-9 was found to be activated earlier than Caspase-8. It was also found that p53 was up-regulated under T-2 toxin stress in the study. These results implied that the effect of T-2 toxin on cells was apoptosis rather than necrosis, and it was probably induced through mitochondrial pathway. To the best of our knowledge, the present study is the first to show that JunD is down-regulated in T-2 toxin induced apoptosis. By construction of an over-expression vector for the JunD gene, we observed that the survival ratio of JunD over-expressed cells obviously increased under T-2 toxin stress. These results suggested that the mechanism of T-2 induced cell death was closely connected with oxidative stress, and that JunD plays an important role in the defensive process against T-2 toxin stress.     

Role of Mitochondria in Ferroptosis

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抑瘤基因NGX6对人结肠癌细胞凋亡的影响

NGX6基因是中南大学肿瘤研究所分子遗传室在对鼻咽癌研究中筛选克隆出的一个新基因．以稳定转染并表达NGX6基因的HT-29细胞为实验组,转染空白质粒的HT－29细胞以及HT－29细胞为对照组,利用PI／Annexin—V双染流式细胞仪检测结肠癌细胞的凋亡情况,通过EMSA分析体外培养的结肠癌细胞及种植瘤细胞内核转录因子－κB（NF－κB）的激活情况．研究结果表明：在裸鼠结肠癌种植瘤中,转染了NGX6基因的与未转染及空白载体组比较,结肠癌细胞凋亡率明显增高;EMSA（electrophoretic mobility shif tassay）分析体外培养的结肠癌细胞及种植瘤细胞内核转录因子－κB（NF—κB）的激活情况,发现转染了NGX6基因的细胞NF—κB的激活均明显受到抑制．此研究说明,NGX6基因只有诱导结肠癌细胞凋亡的能力,其可能的机制是抑制了结肠癌细胞NF—κB的激活．     

Apoptosis in the developing mouse embryos from T-2 toxin-inoculated dams.

T-2 toxin (3 mg/kg b.w.) was orally inoculated to pregnant mice at 11 days of gestation to examine the effect of T-2 toxin on the developing embryos. At 24 hours after T-2 toxin-inoculation, moderate pyknosis or karyorrhexis was generally observed in some layers of the central nervous system, caudal sclerotomic segment, caudal region of the tongue to pharyngeal- to laryngeal-mesenchyma, trachea and facial mesenchyma. These pyknotic or karyorrhectic nuclei were strongly stained by the modified TUNEL method widely used for the in situ detection of apoptotic nuclei and also showed ultrastructural changes characteristic for apoptosis. This is the first report of mycotoxin-induced apoptosis in embryos.     

Role of Mitochondria in Parvovirus Pathology

Proper functioning of the mitochondria is crucial for the survival of the cell. Viruses are able to interfere with mitochondrial functions as they infect the host cell. Parvoviruses are known to induce apoptosis in infected cells, but the role of the mitochondria in parvovirus induced cytopathy is only partially known. Here we demonstrate with confocal and electron microscopy that canine parvovirus (CPV) associated with the mitochondrial outer membrane from the onset of infection. During viral entry a transient depolarization of the mitochondrial transmembrane potential and increase in ROS level was detected. Subsequently, mitochondrial homeostasis was normalized shortly, as detected by repolarization of the mitochondrial membrane and decrease of ROS. Indeed, activation of cell survival signalling through ERK1/2 cascade was observed early in CPV infected cells. At 12 hours post infection, concurrent with the expression of viral non-structural protein 1, damage to the mitochondrial structure and depolarization of its membrane were apparent. Results of this study provide additional insight of parvovirus pathology and also more general information of virus-mitochondria association.     

Glucocorticoid-Induced Apoptosis: Revisited: A Novel Role for Glucocorticoid Receptor Translocation to the Mitochondria

Recent data cast new light on the mechanisms by which glucocorticoids (GCs)elicit apoptosis of thymocytes and leukemia cells. Here we attempt to integrate recentstudies by others and us, which provide a novel insight to this apoptotic process. In thelast few years it was made clear that there is a tight cooperation between genomic andnon-genomic effects exerted by GC receptors (GRs). GC invokes major alterations in thegene expression profile through GR-mediated transactivation and transrepression, whichultimately tip the balance between pro-survival and pro-apoptotic proteins. Althoughessential in shaping the cell’s proteome, these genomic effects are insufficient to elicitapoptotic death and additional signals are required for activating the pro-apoptoticproteins. Several non-genomic effects have been described that occur immediatelyfollowing exposure to GC, which are imperative for the induction of apoptosis. We haverecently observed that GC induces instant GR translocation to the mitochondria in GCsensitive,but not in GC-resistant, T lymphoid cells. This response contrasts the nucleartranslocation of GR occurring in both cell types. We propose that the sustained elevationof GR in the mitochondria following GC exposure is crucial for triggering apoptosis.