Glutathionylation of Adenine Nucleotide Translocase Induced by Carbon Monoxide Prevents Mitochondrial Membrane Permeabilization and Apoptosis

The present work demonstrates the ability of CO to prevent apoptosis in a primary culture of astrocytes. For the first time, the antiapoptotic behavior can be clearly attributed to the inhibition of mitochondrial membrane permeabilization (MMP), a key event in the intrinsic apoptotic pathway. In isolated non-synaptic mitochondria, CO partially inhibits (i) loss of potential, (ii) the opening of a nonspecific pore through the inner membrane, (iii) swelling, and (iv) cytochrome c release, which are induced by calcium, diamide, or atractyloside (a ligand of ANT). CO directly modulates ANT function by enhancing ADP/ATP exchange and prevents its pore-forming activity. Additionally, CO induces reactive oxygen species (ROS) generation, and its prevention by β-carotene decreases CO cytoprotection in intact cells as well as in isolated mitochondria, revealing the key role of ROS. On the other hand, CO induces a slight increase in mitochondrial oxidized glutathione, which is essential for apoptosis modulation by (i) delaying astrocytic apoptosis, (ii) decreasing MMP, and (iii) enhancing ADP/ATP translocation activity of ANT. Moreover, CO and GSSG trigger ANT glutathionylation, a post-translational process regulating protein function in response to redox cellular changes. In conclusion, CO protects astrocytes from apoptosis by preventing MMP, acting on ANT (glutathionylation and inhibition of its pore activity) via a preconditioning-like process mediated by ROS and GSSG.     

LINGO-1-Fc蛋白对低钾诱导小脑颗粒神经元凋亡的保护作用

髓鞘抑制因子Nogo-A、MAG和OMgp通过共同的受体信号复合物NgR/p75NTR(或者TROY)发挥对中枢神经纤维再生的抑制作用.新近克隆的跨膜蛋白LINGO-1是该信号途径的另一个重要组成成分和调节分子.LINGO-1特异表达于中枢神经系统,神经元上的LINGO-1被证明参与调节中枢神经再生的抑制信号,而少突胶质细胞表达的LINGO-1分子参与负调节少突胶质细胞的髓鞘化过程.为探讨LINGO-1分子在神经元凋亡过程中的作用,利用包含LINGO-1分子胞外段LRR和IgC2结构域的Fc融合蛋白作为功能性拮抗剂,研究LINGO-1对低钾诱导的小脑颗粒神经元凋亡的保护作用.利用成熟的Hoechst标记凋亡细胞的方法,观察到经LINGO-1-Fc蛋白预处理2h能够显著阻止小脑颗粒神经元的凋亡.仅包括LRR结构域的GST-LINGO-1与LINGO-1-Fc蛋白,虽同样具有与颗粒神经元的结合活性,但是GST-LINGO-1不能有效地阻止低钾诱导的细胞凋亡.这些结果提示,LINGO-1-Fc蛋白能够阻止低钾诱导的小脑颗粒神经元凋亡,并且这种作用可能是IgC2结构域依赖的.     

Arsenic Trioxide and Paclitaxel Induce Apoptosis by Different Mechanism

Arsenic trioxide (ATO) and paclitaxel (TAXOL) are effective in the treatment of various types of cancers. Both drugs induce G2/M arrest. We have previously shown that ATO is a potent inducer of apoptosis in myeloma cells expressing mutant p53 engaging both the intrinsic and extrinsic apoptotic pathways. Here we compared the effect of ATO and TAXOL on myeloma cells expressing mutant p53 and varying levels of Bcl-2. ATO rapidly induced Apo2/TRAIL, activation of caspase 8, cleavage of BID, depolarization of mitochondrial membrane (MM) and release of AIF from mitochondria in a Bcl-2 independent fashion. Apoptosis was associated with early formation of ring-like perinuclear condensed chromatin co-localized with AIF. In contrast, paclitaxel-induced apoptosis and MM depolarization, cytochrome C release and activation of caspase 9 was blocked by Bcl-2. Apoptosis was associated with a random chromatin condensation and nuclear fragmentation with no early involvement of AIF.     

Myc Potentiates Apoptosis by Stimulating Bax Activity at the Mitochondria

The ability of the c-Myc oncoprotein to potentiate apoptosis has been well documented; however, the mechanism of action remains ill defined. We have previously identified spatially distinct apoptotic pathways within the same cell that are differentially inhibited by Bcl-2 targeted to either the mitochondria (Bcl-acta) or the endoplasmic reticulum (Bcl-cb5). We show here that in Rat1 cells expressing an exogenous c-myc allele, distinct apoptotic pathways can be inhibited by Bcl-2 or Bcl-acta yet be distinguished by their sensitivity to Bcl-cb5 as either susceptible (serum withdrawal, taxol, and ceramide) or refractory (etoposide and doxorubicin). Myc expression and apoptosis were universally associated with Bcl-acta and not Bcl-cb5, suggesting that Myc acts downstream at a point common to these distinct apoptotic signaling cascades. Analysis of Rat1 c-myc null cells shows these same death stimuli induce apoptosis with characteristic features of nuclear condensation, membrane blebbing, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation; however, this Myc-independent apoptosis is not inhibited by Bcl-2. During apoptosis, Bax translocation to the mitochondria occurs in the presence or absence of Myc expression. Moreover, Bax mRNA and protein expression remain unchanged in the presence or absence of Myc. However, in the absence of Myc, Bax is not activated and cytochrome c is not released into the cytoplasm. Reintroduction of Myc into the c-myc null cells restores Bax activation, cytochrome c release, and inhibition of apoptosis by Bcl-2. These results demonstrate a role for Myc in the regulation of Bax activation during apoptosis. Moreover, apoptosis that can be triggered in the absence of Myc provides evidence that signaling pathways exist which circumvent Bax activation and cytochrome c release to trigger caspase activation. Thus, Myc increases the cellular competence to die by enhancing disparate apoptotic signals at a common mitochondrial amplification step involving Bax activation and cytochrome c release.     

RTN3与细胞凋亡

RTN3是RTN家族的成员之一,因其主要定位于内质网,所以用reticulon来命名.RTN3与RTN4B是RTN家族中目前已知的、唯一一对具有凋亡诱发功能的基因.过表达的RTN3介导了真核细胞的三大凋亡信号转导通路:死亡受体途径、线粒体途径、内质网途径,并使之交联形成凋亡调控网络;RTN3可与RTN4B相互作用,形成同源或异源二聚来调控细胞凋亡.另外,过表达RTN3还参与了细菌的类凋亡作用.RTN3广泛表达于多种组织,其过表达诱发凋亡的机制的总结将让人们更好的了解RTN3及其家族,完善细胞凋亡的信号转导研究.     

昆虫杆状病毒细胞凋亡抑制基因

杆状病毒感染过程中,可能会诱导产生一条导致细胞凋亡的路径,细胞凋亡是一种程序性细胞死亡。宿主细胞的凋亡可以导致细胞的提前死亡或感染的终止,因此细胞凋亡可以限制病毒在被感染机体中的扩散或限制感染机体的发病。家蚕的杆状病毒拥有两种对抗细胞凋亡死亡的基因,p35和iap(inhibitorofapoptosis),它们可能通过阻止病毒感染引起的细胞凋亡或存在于大量生物体内的各种诱导信号引起的细胞凋亡。     

The Influence of Lentinan on the Capacity of Repair of DNA Damage and Apoptosis Induced by Paclitaxel in Mouse Bone Marrow Cells

The ability of the flavonoid lentinan (LAN) to enhance the repair of paclitaxel (PAC)‐induced DNA damage and apoptosis in mouse bone marrow cells was investigated. Moreover, the possible mechanism underlying this modulation was determined. LAN was neither genotoxic nor apoptogenic at doses equivalent to 1 or 2 mg/kg/day. Pretreatment of mice with LAN significantly enhances the repair of PAC‐induced DNA damage and bone marrow suppression in a dose dependent manner. Moreover, LAN affords significant protection against PAC‐induced apoptosis. A significant increase of reactive oxygen species and a decrease in reduced glutathione levels were observed after PAC treatment and prior administration of LAN before PAC challenge ameliorated these oxidative stress markers. Conclusively, our study provides, for the first time, that LAN enhances the repair of PAC‐induced DNA damage and apoptosis that resides, at least in part, on its ability to modulate the cellular antioxidant levels and consequently protect bone marrow cells from PAC genotoxicity. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:370‐377, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21499     

TAK1 Prevents Endothelial Apoptosis and Maintains Vascular Integrity

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ASPP2过表达可增强DRAM介导的自噬对HepG2细胞的促调亡作用

p53凋亡刺激蛋白2（apoptosis stimulating protein 2 of p53, ASPP2）能特异性地与p53蛋白结合并增强其促凋亡功能,进而发挥抗肿瘤作用.最近文献提示,自噬对肿瘤发生、发展及肿瘤细胞对抗肿瘤药物的反应都具有重要作用.在本研究中,甲基磺酸(MMS)处理HepG2细胞24 h后,用calcein AM/PI和M30染色检测细胞凋亡,可引起早期（M30免疫组化阳性）和晚期细胞凋亡（PI染色阳性）. 给HepG2细胞转染GFP-LC3质粒后,发现MMS处理24 h可引起自噬的发生. ASPP2腺病毒（rAd-ASPP2）感染HepG2细胞引起ASPP2过表达后,再用MMS处理24 h,能引起更明显的早期、晚期细胞凋亡和自噬. 荧光定量PCR检测发现,rAd-ASPP2诱导了更高的BCL-2相关X蛋白基因（BAX）和p53蛋白的目的基因p53诱导的自噬调节蛋白（p53-induced modulator of autophagy,DRAM）的表达. 但仅用rAd-ASPP2处理HepG2细胞不能引起自噬和凋亡.利用2条DRAM特异性的siRNA下调DRAM的表达,发现rAd-ASPP2引起的自噬被完全抑制, 早期和晚期凋亡均部分被抑制,同时BAX 的mRNA水平也明显下降. 以上结果说明,ASPP2可通过上调BAX和DRAM基因的转录而促进MMS引起的HepG2细胞凋亡; 另外,DRAM介导的自噬是ASPP2促进MMS引起的肿瘤细胞凋亡的机制之一. 该研究可为肝癌的基因治疗提供新的思路.     

Crocin Prevents Patulin‐Induced Acute Toxicity in Cardiac Tissues via the Regulation of Oxidative Damage and Apoptosis

Patulin (PAT) is a mycotoxin produced by several species of the genera of Penicillium, Aspergillus, and Byssochlamys principally by Penicillium expansum. This mycotoxin is suspected to affect several organs including kidney and liver. However, its toxic effect on heart remains unknown. The present study investigated for the first time the cardiotoxic effect of PAT in mice. We demonstrated that PAT increased creatinin phosphokinase (CPK) level, induced lipoperoxydation and protein oxidation, and triggered the antioxidant enzymes such as superoxide dismutase and catalase activities. We also demonstrated that acute administration of PAT triggers apoptosis via P53 overexpression and caspase 3 activation. We further investigated the antioxidant efficiency of crocin (CRO), a carotenoid pigment, against PAT‐induced cardiotoxicity. We found that pretreatment with CRO prevents cardiac impairment by reducing CPK levels, restoring the redox statute and suppressing apoptosis. Collectively, our data provide new preventive effect of CRO toward PAT‐induced cardiotoxicity in mice.     

Melatonin Prevents Oxidative Stress and Hepatocyte Cell Death Induced by Experimental Cholestasis

The induction of oxidative stress precedes liver injury during experimental obstructive jaundice (OJ). In this sense, different evidences suggest that melatonin (MEL), as antioxidant, may be useful in the protection against apoptosis and necrosis during experimental cholestasis. In addition, we will also assess if MEL-dependent protection is related to a recovery of antioxidant status disturbances induced by OJ. Cholestasis was achieved by double ligature and sectioning of the principal bile duct. MEL was injected intraperitoneally (500?μg/kg/day). Lipid peroxidation was evaluated by the measurement of malondialdehyde (MDA) content in liver. Different parameters related to antioxidant status, such as reduced glutathione (GSH), glutathione peroxidase (GPx), catalase and superoxide dismutase (SOD) were determined in liver. Liver injury was assessed by alanine aminotransferase (ALT) in serum, histological examination, DNA fragmentation and TUNEL assay. The activation of perisinusoidal stellate cells was evaluated by immunohistochemical measurement of α-smooth muscle actin in liver sections. The induction of OJ increased all the parameters related to apoptosis and necrosis in liver. The induction of liver injury was associated with stellate cell activation, as well as an increase in MDA (p<0.0001) and a reduction in GSH, GPx, catalase and SOD content (p<0.0001) in liver. MEL reduced hepatic apoptosis and necrosis (p<0.004) with a significant improvement in all oxidative stress markers. In conclusion, our results showed that MEL recovered the antioxidant status and reduced apoptosis and necrosis induced by experimental cholestasis.     

Neuronal Apoptosis Induced by Endoplasmic Reticulum Stress

Apoptosis is a conserved active cellular mechanism occurring under a range of physiological and pathological conditions. In the nervous system, apoptosis plays crucial roles in normal development and neuronal degenerating diseases. Various deleterious conditions, including accumulation of the mutant proteins in the endoplasmic reticulum (ER) and inhibition of ER to Golgi transport of proteins, may result in apoptosis. In this study, we examined the downstream events of apoptosis in differentiated PC 12 cells under ER stress induced by brefeldin A, an inhibitor of ER to Golgi protein transport. Activation of NF-B and degradation of I-B were observed within 2 hours, followed by up-regulation of GRP78 protein level in treated cells. Caspase-12 only appeared around 24 hours after brefeldin A treatment, coincident with cell nuclei fragmentation. These results suggest that neuronal apoptosis may be induced by ER stress through a NF-B and caspase related pathway.     

Protease Activation in Apoptosis Induced by MAL

The proteolytic caspase cascade plays a central role in the signaling and execution steps of apoptosis. This study investigated the activation of different caspases in apoptosis induced by MAL (a folding variant of human alpha-lactalbumin) isolated from human milk. Our results show that the caspase-3-like enzymes, and to a lesser extent the caspase-6-like enzymes, were activated in Jurkat and A549 cells exposed to MAL. Activated caspases subsequently cleaved several protein substrates, including PARP, lamin B, and alpha-fodrin. A broad-range caspase inhibitor, zVAD-fmk, blocked the caspase activation, the cleavage of proteins, and DNA fragmentation, indicating an important role for caspase activation in MAL-induced apoptosis. Since an antagonistic anti-CD95 receptor antibody, ZB4, did not influence the MAL-induced killing, we conclude that this process does not involve the CD95-mediated pathway. While MAL did not directly activate caspases in the cytosol, it colocalized with mitochondria and induced the release of cytochrome c. Thus, these results demonstrate that caspases are activated and involved in apoptosis induced by MAL and that direct interaction of MAL with mitochondria leads to the release of cytochrome c, suggesting that this release is an important step in the initiation and/or amplification of the caspase cascade in these cells.     

Genistein Enhances the Cisplatin‐Induced Inhibition of Cell Growth and Apoptosis in Human Malignant Melanoma Cells

Genistein, a naturally occurring isoflavone found chiefly in soybeans, has been reported to be a potent antitumor agent. Genistein is presumed to exert multiple effects related to the inhibition of cancer growth. Metastatic melanoma is a chemotherapy‐refractory neoplasm. The present study was designed to explore the possible activity of genistein to inhibit the aberrant proliferation and to induce apoptosis of human malignant melanoma cells in cooperation with cisplatin treatment. Five human melanoma cell lines were utilized for these experiments. Genistein at physiologic concentrations (20 μM) did not induce apoptosis by itself but did enhance cisplatin‐induced apoptosis in all five human melanoma cell lines tested. The enhanced susceptibility among the cell lines was diverse. Changes in the expression of two anti‐apoptotic proteins, bcl‐2 and bcl‐xL, and one pro‐apoptotic protein, apoptotic protease activating factor‐1 (Apaf‐1), were examined. Genistein alone or cisplatin alone generally did not alter bcl‐2 expression or bcl‐xL expression, but slightly increased Apaf‐1 in some cell lines. The combined treatment with genistein and cisplatin significantly reduced bcl‐2 and bcl‐xL protein and increased Apaf‐1 protein expression. These data suggest that genistein therapy may enhance the chemosensitivity of melanoma patients.     

Praziquantel Synergistically Enhances Paclitaxel Efficacy to Inhibit Cancer Cell Growth

The major challenges we are facing in cancer therapy with paclitaxel (PTX) are the drug resistance and severe side effects. Massive efforts have been made to overcome these clinical challenges by combining PTX with other drugs. In this study, we reported the first preclinical data that praziquantel (PZQ), an anti-parasite agent, could greatly enhance the anticancer efficacy of PTX in various cancer cell lines, including PTX-resistant cell lines. Based on the combination index value, we demonstrated that PZQ synergistically enhanced PTX-induced cell growth inhibition. The co-treatment of PZQ and PTX also induced significant mitotic arrest and activated the apoptotic cascade. Moreover, PZQ combined with PTX resulted in a more pronounced inhibition of tumor growth compared with either drug alone in a mouse xenograft model. We tried to investigate the possible mechanisms of this synergistic efficacy induced by PZQ and PTX, and we found that the co-treatment of the two drugs could markedly decrease expression of X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptotic protein. Our data further demonstrated that down-regulation of XIAP was required for the synergistic interaction between PZQ and PTX. Together, this study suggested that the combination of PZQ and PTX may represent a novel and effective anticancer strategy for optimizing PTX therapy.     

C-Peptide Prevents Hippocampal Apoptosis in Type 1 Diabetes

To explore mechanisms underlying central nervous system (CNS) complications in diabetes, we examined hippocampal neuronal apoptosis and loss, and the effect of C-peptide replacement in type 1 diabetic BB/W rats. Apoptosis was demonstrated after 8 months of diabetes, by DNA fragmentation, increased number of apoptotic cells, and an elevated ratio of Bax/Bcl-x_L, accompanied by reduced neuronal density in the hippocampus. No apoptotic activity was detected and neuronal density was unchanged in 2-month diabetic hippocampus, whereas insulin-like growth factor (IGF) activities were impaired. In type 1 diabetic BB/W rats replaced with C-peptide, no TdT-mediated dUTP nick-end labeling (TUNEL)- positive cells were shown and DNA laddering was not evident in hippocampus at either 2 or 8 months. C-peptide administration prevented the preceding perturbation of IGF expression and reduced the elevated ratio of Bax/Bcl-x_L. Our data suggest that type 1 diabetes causes a duration-dependent programmed cell death of the hippocampus, which is partially prevented by C-peptide.