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.     

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.     

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.     

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。     

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.     

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.     

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.     

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.     

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.     

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.     

线粒体参与介导MOPDO抑制HepG2细胞增殖、诱导其凋亡的作用

研究新合成的一氧化氮供体--核苷衍生物6-甲基-4-(2-氟-3,5-二-O-苯甲酰基-1-β-D-呋喃阿拉伯糖)-[1,2,5]噁二唑[3,4-d]嘧啶-5(4H),7(6H)-二酮1-氧化物(MOPDO)抑制人肝癌细胞HepG2增殖、诱导凋亡的作用.以不同浓度的MOPDO作用于人肝癌细胞HepG2,MTT检测与相差显微镜观察相结合,分析MOPDO对HepG2细胞增殖的影响;膜联蛋白-V/PI双染检测细胞凋亡率;JC-1染色检测线粒体膜电位(△ψm)变化;硝酸酶法测定细胞培养液中NO的含量.结果发现,MOPDO以时间和剂量依赖性方式抑制HepG2细胞的增殖,细胞形态出现相应的变化;细胞凋亡率的增加呈时-效和量-效关系;JC-1染色显示,线粒体膜电位随着MOPDO剂量的增加降低;MOPDO释放的NO量呈剂量依赖性增加.表明线粒体可能参与介导MOPDO抑制HepG2细胞的增殖、诱导其凋亡的作用.     

Investigation of Calcium Accumulation in Mitochondria in Cells Undergoing Apoptosis

One of the earliest features of apoptosis is the induction of the mitochondrial permeability transition (MPT) due to opening of a pore in the mitochondrial membrane. We estimated the Ca²⁺ capacity of mitochondria (a threshold level of Ca²⁺ that induces the release of this cation from mitochondria) during apoptosis. Incubation of thymocytes at 37°C for 4 h equally decreased the mitochondrial Ca²⁺ capacity both in the presence and the absence of dexamethasone, an inducer of apoptosis. At the same time, dexamethasone significantly stimulated internucleosomal DNA fragmentation, which is one of the manifestations of apoptosis. Cyclosporin A prevented the time-dependent decrease in the Ca²⁺ capacity of mitochondria but did not affect internucleosomal DNA fragmentation. Therefore, induction of apoptosis assessed by internucleosomal DNA fragmentation is not mediated by the mitochondrial permeability transition.     

Circadian Rhythms in Neurospora crassa: The Role of Mitochondria

Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Numerous examples of inhibitors that affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms. Analogous studies on the role of mitochondria in the Neurospora circadian rhythm mechanism have also been reported and summarized. This communication differs from previous studies on other organisms in that it will focus on two lines of evidence derived from studies on Neurospora strains carrying mutations affecting the mitochondria, (a) Strains whose growth rate is resistant to oligomycin (oli^t) owing to an altered protein in the F₀ sector of the mitochondrial ATPase, showed no phase shifts when pulsed with oligomycin. Control strains (oli⁸) showed large phase shifts when pulsed with oligomycin. This indicates that the phase-shifting effect of oligomycin is due to the direct inhibition of the mitochondrial ATPase and not some side effect of this inhibitor, (b) In Neurospora, many different strains are known that carry mutations in the nuclear or mitochondrial genome that affect mitochondrially localized proteins. Some of these, such as oli', [MI-3], or cya-5, showed shorter (≥ 19-h) periods compared with the normal (21.5-h) period. Others showed little or no change in period. Those mutant strains exhibiting shorter periods also contained ≥60% more mitochondrial protein per gram total protein in extracts compared with the normal strains. Assays of the level of a mitochondrial-specific protein, acyl carrier protein, showed that the cellular content of this protein was approximately doubled. A parallel set of studies on the effects of antimycin or chloramphenicol on Neurospora demonstrated that these inhibitors also produced shorter periods as well as increased amounts of mitochondrial proteins. These two new lines of evidence may be interpreted to indicate that in Neurospora either some part of the oscillator is localized to the mitochondria and/or that mitochondria exert their effect on the clock mechanism through their effects on biosynthetic pathways or by their contribution in determining ion gradients.     

Circadian Rhythms in Neurospora crassa: The Role of Mitochondria

Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Numerous examples of inhibitors that affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms. Analogous studies on the role of mitochondria in the Neurospora circadian rhythm mechanism have also been reported and summarized. This communication differs from previous studies on other organisms in that it will focus on two lines of evidence derived from studies on Neurospora strains carrying mutations affecting the mitochondria, (a) Strains whose growth rate is resistant to oligomycin (oli^t) owing to an altered protein in the F₀ sector of the mitochondrial ATPase, showed no phase shifts when pulsed with oligomycin. Control strains (oli⁸) showed large phase shifts when pulsed with oligomycin. This indicates that the phase-shifting effect of oligomycin is due to the direct inhibition of the mitochondrial ATPase and not some side effect of this inhibitor, (b) In Neurospora, many different strains are known that carry mutations in the nuclear or mitochondrial genome that affect mitochondrially localized proteins. Some of these, such as oli', [MI-3], or cya-5, showed shorter (≥ 19-h) periods compared with the normal (21.5-h) period. Others showed little or no change in period. Those mutant strains exhibiting shorter periods also contained ≥60% more mitochondrial protein per gram total protein in extracts compared with the normal strains. Assays of the level of a mitochondrial-specific protein, acyl carrier protein, showed that the cellular content of this protein was approximately doubled. A parallel set of studies on the effects of antimycin or chloramphenicol on Neurospora demonstrated that these inhibitors also produced shorter periods as well as increased amounts of mitochondrial proteins. These two new lines of evidence may be interpreted to indicate that in Neurospora either some part of the oscillator is localized to the mitochondria and/or that mitochondria exert their effect on the clock mechanism through their effects on biosynthetic pathways or by their contribution in determining ion gradients.     

Pluripotent Stem Cells for Uncovering the Role of Mitochondria in Human Brain Function and Dysfunction

Mitochondrial dysfunctions are a known pathogenetic mechanism of a number of neurological and psychiatric disorders. At the same time, mutations in genes encoding for components of the mitochondrial respiratory chain cause mitochondrial diseases, which commonly exhibit neurological symptoms. Mitochondria are therefore critical for the functionality of the human nervous system. The importance of mitochondria stems from their key roles in cellular metabolism, calcium handling, redox and protein homeostasis, and overall cellular homeostasis through their dynamic network. Here, we describe how the use of pluripotent stem cells (PSCs) may help in addressing the physiological and pathological relevance of mitochondria for the human nervous system. PSCs allow the generation of patient-derived neurons and glia and the identification of gene-specific and mutation-specific cellular phenotypes via genome engineering approaches. We discuss the recent advances in PSC-based modeling of brain diseases and the current challenges of the field. We anticipate that the careful use of PSCs will improve our understanding of the impact of mitochondria in neurological and psychiatric disorders and the search for effective therapeutic avenues.