ER stress triggers apoptosis induced by Nogo-B/ASY overexpression

Nogo-B/ASY has been characterized as a novel human apoptosis-inducing protein without any known apoptosis-related motifs. However, the validity of Nogo-B/ASY as a physiological apoptotic protein was recently questioned. In present research, we demonstrate that ASY overexpression contributes to ER stress and induces apoptosis through ER Ca2+ depletion and ER-specific pathways. ER stress and the disorder of intracellular calcium trigger the apoptosis induced by ASY overexpression. At the same time, stable transfectants overexpressing high levels of ASY are resistant to ER-stress-associated stimuli, which implies that ASY overexpression activates protective response in response to ER stress. Our results provide a direct apoptotic pathway that ASY overexpression induces apoptosis through ER stress and ER-specific signal pathways.     

柯萨奇病毒A组16型研究进展

柯萨奇病毒A组16型(CA16)是引起手足口病(HFMD)的主要病原体,与肠道病毒71型(EV71)交替或共同流行;特别是近年在西太平洋地区呈现流行强度高、重症和死亡人数多的特点,已成为该地区的重大公共卫生问题。研发安全有效的疫苗是控制HFMD流行的有效手段。由于EV71所致疾病在重症和死亡病例中所占比例高,对其疫苗研发得到了广泛关注,全病毒灭活疫苗已进入III期临床,有望即将应用于婴幼儿HFMD的防控。EV71疫苗的顺利研发随之也增加了对CA16疫苗研发的迫切性。近年来日本、新加坡以及中国台湾地区逐渐开始关注CA16相关的研究,我国也有多家企业开展CA16疫苗的研发。本文就CA16的病原学,流行病学,实验室诊断,治疗和预防等方面进行了综述。     

Active mixture of serum-circulating small molecules selectively inhibits proliferation and triggers apoptosis in cancer cells via induction of ER stress

Genetic and epigenetic regulation as well as immune surveillance are known defense mechanisms to protect organisms from developing cancer. Based on experimental evidence, we proposed that small metabolically active molecules accumulating in cancer cells may play a role in an alternative antitumor surveillance system. Previously, we reported that treatment with a mixture of experimentally selected small molecules, usually found in the serum (defined ‘active mixture’, AM), selectively induces apoptosis in cancer cells and significantly inhibits tumor formation in vivo. In this study, we show that the AM elicits gene expression changes characteristic of endoplasmic reticulum (ER) stress in HeLa, MCF-7, PC-3 and Caco-2 cancer cells, but not in primary human renal epithelial cells. The activation of the ER stress pathway was confirmed by the upregulation of ATF3, ATF4, CHAC1, DDIT3 and GDF15 proteins. Mechanistically, our investigation revealed that eIF2α, PERK and IRE1α are phosphorylated upon treatment with the AM, linking the induction of ER stress to the antiproliferative and proapoptotic effects of the AM previously demonstrated. Inhibition of ER stress in combination with BBC3 and PMAIP1 knockdown completely abrogated the effect of the AM. Moreover, we also demonstrated that the AM induces mIR-3189-3p, which in turn enhances the expression of ATF3 and DDIT3, thus representing a possible new feedback mechanism in the regulation of ATF3 and DDIT3 during ER stress. Our results highlight small molecules as attractive anticancer agents and warrant further evaluation of the AM in cancer therapy, either alone or in combination with other ER stress inducing agents.     

柯萨奇病毒 A16在恒河猴外周血单个核细胞中的增殖研究

柯萨奇病毒A16（CA16）感染灵长类动物模型显示该病毒在猴体内可形成明显的病毒血症期,在此基础上,本研究进一步分析病毒血症形成与CA16在外周血单个核细胞（PBMC ）不同组分中增殖的关系。首先用CA16感染恒河猴,检测病毒在PBMC组分CD4＋、CD8＋、CD20＋、CD11c＋、CD16＋和CD14＋细胞中的增殖情况和感染动力学,发现病毒仅在部分CD14＋细胞中有增殖表现。然后通过刺激CD14＋细胞产生一定量的树突细胞（DC）,用CA16感染DC ,发现 CA16能在DC中形成具有动力学意义的增殖过程,增殖峰值出现在感染后12～36 h。这些结果可能为CA16感染发病机制提供重要信息。     

Simultaneous inhibition of the ubiquitin-proteasome system and autophagy enhances apoptosis induced by ER stress aggravators in human pancreatic cancer cells

In contrast to normal tissue, cancer cells display profound alterations in protein synthesis and degradation. Therefore, proteins that regulate endoplasmic reticulum (ER) homeostasis are being increasingly recognized as potential therapeutic targets. The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. However, interactions between autophagy, the proteasome, and ER stress pathways in cancer remain largely undefined. This study demonstrated that withaferin-A (WA), the biologically active withanolide extracted from Withania somnifera, significantly increased autophagosomes, but blocked the degradation of autophagic cargo by inhibiting SNARE-mediated fusion of autophagosomes and lysosomes in human pancreatic cancer (PC) cells. WA specifically induced proteasome inhibition and promoted the accumulation of ubiquitinated proteins, which resulted in ER stress-mediated apoptosis. Meanwhile, the impaired autophagy at early stage induced by WA was likely activated in response to ER stress. Importantly, combining WA with a series of ER stress aggravators enhanced apoptosis synergistically. WA was well tolerated in mice, and displayed synergism with ER stress aggravators to inhibit tumor growth in PC xenografts. Taken together, these findings indicate that simultaneous suppression of 2 key intracellular protein degradation systems rendered PC cells vulnerable to ER stress, which may represent an avenue for new therapeutic combinations for this disease.     

Molecular epidemiology of Coxsackievirus A16 strains isolated from children in Yamagata,Japan between 1988 and 2011

To clarify the longitudinal molecular epidemiology of coxsackievirus A16, phylogenetic analysis based on the VP1 region of 220 isolates in Yamagata, Japan was performed. The resultant phylogenetic tree indicates that the Yamagata isolates and reference strains can be readily genotyped into three genogroups, and 0, 12 and 208 isolates belonged to the first, second, and third genogroups, respectively. The first genogroup includes only the prototype strain, the second strains that had disappeared by the end of the 20th century and the third comprises those that have been circulating since then in local communities, such as Yamagata.     

Development and characterization of a clinical strain of Coxsackievirus A16 and an eGFP infectious clone

Coxsackievirus A16(CA16) is one of the major causes of hand, foot, and mouth disease(HFMD) worldwide, which is a common illness that affects children. The frequent occurrence of HFMD outbreaks has become a serious public health problem in Asia. Therefore, it is important to understand the pathogenesis and replication of CA16. In this study, a stable infectious c DNA clone of an epidemic strain of Coxsackievirus A16(CA16) was assembled, and subsequently a reporter virus(e GFP-CA16) was constructed by inserting the e GFP gene between the 5'-UTR and the N-terminus of VP4, with the addition of a 2A protease cleavage site(ITTLG) at its C-terminus. This was transfected into Vero cells to generate infectious recombinant viruses. The growth characteristics and plaque morphology, in vitro, in mammalian cells were found to be indistinguishable between the parental and recombinant viruses. Although the e GFP-CA16 showed smaller plaque size as compared to recombinant CA16, both were found to exhibit similar growth trends and EC50 of NITD008. In summary, this stable infectious c DNA clone should provide a valuable experimental system to study CA16 infection and host response. The e GFP-CA16 is expected to provide a powerful tool to monitor e GFP expression in infected cells and to evaluate the antiviral activity of potential antiviral agents in the treatment of CA16 infections.     

ER stress contributes to ischemia-induced cardiomyocyte apoptosis

Myocardial ischemia is a severe stress condition that leads to loss of cardiomyocytes. The cell loss is attributed to apoptosis, although the exact mechanisms involved are only partially defined, which limits therapeutic opportunities. Here, we show caspase activation and apoptosis in neonatal rat cardiomyocyte cultures subjected to simulated ischemia by serum, glucose, and oxygen deprivation (SGO). Caspase activation was preceded by endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR), detected by the induction of Grp78, induction and splicing of XBP1, and phosphorylation of eukaryotic initiation factor 2-alpha (eIF2alpha). At a later time the ER stress response switched from UPR and cytoprotective response to a pro-apoptotic response as demonstrated by the upregulation of CHOP and processing of pro-caspase-12. Thus, we provide evidence that the ER can generate and propagate apoptotic signals in response to ischemic stress and this pathway is therefore a novel target for prevention of ischemia-mediated cardiomyocyte loss.     

CD437 induces apoptosis in ovarian adenocarcinoma cells via ER stress signaling

A synthetic retinoid, CD437, has been shown to exert potent anti-tumor activity against various types of cancer cell lines, regardless of their sensitivities to natural retinoids. We herein demonstrate that CD437 induces endoplasmic reticulum (ER) stress, including the up-regulation of CHOP, BIP and GADD34 mRNA through ER stress transducer (PERK and IRE1α) activation in an ovarian adenocarcinoma cell line, SKOV3. It was also shown that CD437 induced the CHOP and GADD34 expressions in another four ovarian adenocarcinoma cell lines, indicating that CD437 functions as an ER stress inducer in these cell lines. Moreover, the siRNA-mediated knockdown of inducible CHOP expression prevented the cytotoxic effect of CD437. These results suggest that ER stress plays an important role in the mechanism by which CD437 induces apoptosis in ovarian adenocarcinoma cells.     

Anticancer compound Oplopantriol A kills cancer cells through inducing ER stress and BH3 proteins Bim and Noxa

Oplopantriol-A (OPT) is a natural polyyne from Oplopanax horridus. We show here that OPT preferentially kills cancer cells and inhibits tumor growth. We demonstrate that OPT-induced cancer cell death is mediated by excessive endoplasmic reticulum (ER) stress. Decreasing the level of ER stress either by inactivating components of the unfolded protein response (UPR) pathway or by expression of ER chaperone protein glucose-regulated protein 78 (GRP78) decreases OPT-induced cell death. We show that OPT induces the accumulation of ubiquitinated proteins and the stabilization of unstable proteins, suggesting that OPT functions, at least in part, through interfering with the ubiquitin/proteasome pathway. In support of this, inhibition of protein synthesis significantly decreased the accumulation of ubiquitinated proteins, which is correlated with significantly decreased OPT-induced ER stress and cell death. Finally, we show that OPT treatment significantly induced the expression of BH3-only proteins, Noxa and Bim. Knockdown of both Noxa and Bim significantly blocked OPT-induced cell death. Taken together, our results suggest that OPT is a potential new anticancer agent that induces cancer cell death through inducing ER stress and BH3 proteins Noxa and Bim.     

Bosellia serrata-induced apoptosis is related with ER stress and calcium release

It has been reported that the gum resin of Boswellia serrata (BS), which has been shown to have antiinflammatory properties, might also have anticancer effects. This study examined the potential of BS as an anticancer agent. The BS extract induces apoptosis in HeLa human cervical carcinoma cells, as confirmed by two apoptosis analyses, Hoechst staining and Annexin V/PI assay. Among the apoptosis pathways, the ER stress-associated mechanism was examined to determine its role in BS-induced apoptosis. The expression of GRP78 and CHOP, which are representatives of the ER stress proteins, and the calcium-binding protein-calpain were determined. The results showed significantly higher levels of both GRP78 and CHOP, and stronger calpain activity in the BS-treated cells than in the control cells. This shows that there is a correlation between ER stress signaling and apoptosis, which suggests the possibility of the BS-ER stress initiator as an anticancer therapeutic agent in human cervical carcinoma.     

Acrolein induces a cellular stress response and triggers mitochondrial apoptosis in A549 cells

Acrolein is a highly reactive, α,β-unsaturated aldehyde that is an omnipresent environmental pollutant. Humans are exposed to acrolein in food, vapors of overheated cooking oil, cigarette smoke and by combustion of organic products. Acrolein is a toxic by-product of lipid peroxidation resulting from oxidative stress, which is implicated in pulmonary, cardiac and neurodegenerative diseases. Low dose exposure to toxic compounds often leads to adaptive responses. If the adaptive response does not counteract the adverse exposure, death processes such as apoptosis will eliminate the cell. This study investigates the activation of antiapoptosis survival factors in relation to the induction of cell death by apoptosis, following exposure to low doses of acrolein, in A549 human lung cells. Exposure to acrolein (<15 μM, 30 min) activated the survival factor AKT, which led to phosphorylation of Bad and induction of antiapoptosis proteins cIAP1/2. Acrolein (10–50 μM, 30–60 min) increased reactive oxygen species and caused mitochondrial membrane hyperpolarisation. Inhibition by the antioxidants catalase, polyethylene glycol-catalase, sodium pyruvate and MnTBAP showed that acrolein-induced reactive oxygen species were responsible for mitochondrial membrane hyperpolarisation. Acrolein (3–27 μM, 30–60 min) activated early stage processes in the mitochondrial pathway of apoptosis, such as Bax translocation to mitochondria, cytochrome c release, caspase-9 activation, and translocation of apoptosis-inducing factor to the nucleus. Acrolein (10–50 μM) triggered later stage processes such as activation of caspases-3, -7 and -6, phosphatidylserine externalization and cleavage of poly(ADP)ribose polymerase after longer times (2 h). These events were inhibited by polyethylene glycol-catalase, showing that apoptosis was mediated by overproduction of reactive oxygen species by acrolein. The novel findings show that antiapoptosis processes dominate at low dose (<15 μM)/shorter exposure times to acrolein, whereas proapoptotic processes dominate at higher dose (10–50 μM)/longer exposure times. Acrolein induced apoptosis through the mitochondrial pathway that was mediated by reactive oxygen species.     

Differential interferon pathway gene expression patterns in Rhabdomyosarcoma cells during Enterovirus 71 or Coxsackievirus A16 infection

Exposure of cells to type I interferon (IFN) induces an antiviral state that prevents viral infection, but viruses can utilize multiple tactics to antagonize the host immune system. Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are two major pathogens that cause hand, foot, and mouth disease (HFMD), which is prevalent among children. We found that both EV71 and CA16 have different reactions to type I IFN pretreatment and induction patterns of type I IFN on Rhabdomyosarcoma (RD) cells. Further, a human-α and β IFN PCR array was employed to analyze the expressions of 84 genes related to the type I IFN pathway. We found significant up-regulation of multiple genes in the presence of type I IFN and differential regulation patterns during EV71 or CA16 infection in RD cells. For instance, EV71 infection repressed the JAK-STAT signaling pathway and interferon-stimulated gene (ISG) expression, whereas CA16 infection normally triggers the JAK-STAT pathway, leading to the expression of ISGs. Taken together, this study provides a comprehensive view of the differential impacts of EV71 and CA16 infection on 84 genes in the IFN pathway, shedding light on the different resistances of these viruses to type I IFN treatment and cytotoxic effects in RD cells.     

ER stress and autophagy contribute to CsA-induced death of malignant glioma cells

Cyclosporine A (CsA), which revolutionized transplantology due to its ability to block the activation of lymphocytes and other immune system cells, triggers autophagy in malignant glioma cell lines via stimulation of endoplasmic reticulum (ER) stress. We also found that autophagy serves as a protective mechanism against CsA toxicity.     

SelK is a novel ER stress-regulated protein and protects HepG2 cells from ER stress agent-induced apoptosis

Selenoprotein K (SelK), an endoplasmic reticulum (ER) resident protein, its biological function has been less-well studied. To investigate the role of SelK in the ER stress response, effects of SelK gene silence and ER stress agents on expression of SelK and cell apoptosis in HepG2 cells were studied. The results showed that SelK was regulated by ER stress agents, Tunicamycin (Tm) and β-Mercaptoethanol (β-ME), in HepG2 cells. Moreover, the SelK gene silence by RNA interference could significantly aggravate HepG2 cell death and apoptosis induced by the ER stress agents. These results suggest that SelK is an ER stress-regulated protein and plays an important role in protecting HepG2 cells from ER stress agent-induced apoptosis.