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Effects of serum containing natural cerebrolysin on glucose-regulated protein 78 and CCAAT enhancer-binding protein homologous protein expression in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress

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Neural Regeneration Research 2009年第2期4卷 92-97页

作者： Zhengzhi Wu Ming Li Andrew C.J. Huang O Xiuqing Jia Yinghong Li Manyin Chen Shenzhen Hospital Southern Medical University Shenzhen 518033 Guangdong Province China Larry Hiflblom Islet Research Center David Geffen School of Medicine UCLA Los Angeles CA 90024 USA

BACKGROUND： Glucose-regulated protein 78 （GRP78）, a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein （CHOP）, a transcription factor specific for endoplasmic reticulum stress, can cause cell cycle arrest and cell apoptosis. OBJECTIVE： To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions. DESIGN, TIME AND SETTING： A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008. MATERIALS： Adult Sprague-Dawley rats were perfused with natural Cerebrolysin aqueous extract （0.185 g/kg/d） to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. tunicamycin was provided by Sigma （St. Louis, USA）, and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf （1：2：2）, by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine. METHODS： PC12 cells were treated with DMEM culture media containing 10% blank serum （normal control group）, tunicamycin （1 μg/mL; model group）, and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin （1 μ g/mL; low-, moderate-, and high-dose serum containing natural cerebrotysin groups）, for 2 hours. MAIN OUTCOME MEASURES： PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR. RESULTS： The apoptotic index and CHOP mRNA expression were in th

关键词： natural cerebrolysin PC12 cell strain endoplasmic reticulum stress tunicamycin glucose regulated protein 78 CCAAT/enhancer-binding protein homologous protein

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Low levels of Bax inhibitor-1 gene expression increase tunicamycin-induced apoptosis in human neuroblastoma SY5Y cells

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Neural Regeneration Research 2012年第17期7卷 1331-1337页

作者： Dan Wu Peirong Wang Shiyao Wang Department of Medical Genetics School of Basic Medical SciencesPeking UniversityBeijing 100191China Institute of Biophysics Chinese Academy of SciencesBeijing 100190China

A human SH-SY5Y neuroblastoma cell line with a low level of Bax inhibitor-1 expression was established by lentivirus-mediated RNA interference and fluorescence-activated cell sorting. In control SH-SY5Y cells, tunicamycin treatment induced endoplasmic reticulum stress-mediated apoptosis; however, after Bax inhibitor-1 gene knockdown, cell survival rates were significantly decreased and the degree of apoptosis was significantly increased following tunicamycin treatment In addition, chromatin condensation and apparent apoptotic phenomena, such as marginalization and cytoplasmic vesicles, were observed. Our findings indicate that Bax inhibitor-1 can delay apoptosis induced by endoplasmic reticulum stress.

关键词： Bax inhibitor-1 RNA interference SH-SY5Y endoplasmic reticulum stress tunicamycin apoptosis neural regeneration

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Characterization of the tunicamycin gene cluster unveiling unique steps involved in its biosynthesis

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Protein & Cell 2010年第12期1卷 1093-1105页

作者： Wenqing Chen Dongjing Qu Lipeng Zhai Meifeng Tao Yemin Wang Shuangjun Lin Neil P.J.Price Zixin Deng Laboratory of Microbial Metabolism and School of Life Sciences&BiotechnologyShanghai Jiao Tong UniversityShanghai 200030China National Center for Agricultural Utilization Research(NCAUR) USDA-ARS1815 North University StreetPeoriaIllinois 61604USA College of Pharmacy Wuhan UniversityWuhan 430072China

tunicamycin,a potent reversible translocase I inhibitor,is produced by several Actinomycetes *** tunicamycin structure is highly unusual,and contains an 11-carbon dialdose sugar and anα,β-1″,11′-glycosidic *** we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression(HHE)strategy combined with a *** of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains,demonstrating the role of the genes for the biosynthesis of *** disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes(tunA–tunL).Amongst these is a putative radical SAM enzyme(Tun B)with a potentially unique role in biosynthetic carbon-carbon bond ***,a seven-step novel pathway is proposed for tunicamycin ***,two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM *** data provide clarification of the novel mechanisms for tunicamycin biosynthesis,and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.

关键词： tunicamycin biosynthetic gene cluster high-throughput heterologous expression bioassay combinatorial biosynthesis

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IRE1α knockdown rescues tunicamycin-induced developmental defects and apoptosis in Xenopus laevis

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The Journal of Biomedical Research 2014年第4期28卷 275-281页

作者： Li Yuan Jin Yu Xinxin Li Jiaojiao Feng Chenyang Yin Xinru Wang Department of Biochemistry and Molecular Biology Nanjing Medical University Department of Hematology Friendship hospital of yili kazakh prefecture Key Laboratory of Reproductive Medicine Institute of ToxicologyNanjing Medical University

Inositol requiring enzyme-1 （IRE1） is highly conserved from yeasts to humans. Upon endoplasmic reticulum （ER） stress, IRE1 activates X-box-binding protein 1 （XBP1） by unconventional splicing of XBP1 mRNA, which activates unfolded protein response （UPR） to restore ER homeostasis. In mice, IRE1α plays an essential role in extraembryonic tissues. However, its precise action during the early stage of development is unknown. In this study, the gain and loss-of-function analyses were used to investigate the function of Xenopus IRE1α （xIRE1α）. The effects of xIRE1α during embryo development were detected with RT-PCR and whole mount in situ hybridization. ER stress was induced by tunicamycin. The apoptofic cells were measured by TUNNEL assays. Although both gain and loss of xlRE1α function had no significant effect on Xenopus embryogenesis, knockdown of xIRE1α could rescue tunicamycin-induced developmental defects and apoptosis. The finding indicates that xIRE1α is not required for embryogenesis but is required for tunicamycin-induced developmental defects and apoptosis in Xenopus laevis.

关键词： IRE1α Xenopus laevis tunicamycin developmental defects

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Endoplasmic Reticulum Stress Induced by tunicamycin and Antagonistic Effect of Tiantai No.1(天泰1号) on Mesenchymal Stem Cells

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Chinese Journal of Integrative Medicine 2010年第1期16卷 41-49页

作者：吴正治李映红 Andrew C.J.Huang 李明张晓丽王济国杨敏陈嫚茵 Shenzhen Hospital of Southern Medical University Larry Hillblom Islet Research Center David Geffen School of MedicineUCLALos AngelesCA 90024USA

Objective： Changes of the internal and external cellular environments can induce calcium homeostasis disorder and unfolded protein aggregation in the endoplasmic reticulum （ER）. This ER function disorder is called endoplasmic reticulum stress （ERS）. Severe long-term ERS can trigger the ER apoptosis signaling pathway, resulting in cell apoptosis and organism injury. Recent researches revealed that ERS-induced cell death was involved in the neurocyte retrogradation in the progress of neuron degenerative diseases, such as Alzheimer＇s disease （AD）, Parkinson＇s disease and so on. Therefore, the protection effect of the traditional Chinese drug Tiantai No. 1 （天泰1号） on the ERS injury of AD was investigated at the molecular gene level in this study with a view to explore the gene pharmacodynamic actions and mechanisms of this drug. Methods： Primarily cultured marrow mesenchymat stem cells （MSCs） of rats were treated by tunicamycin （TM） in order to induce ERS. RT-PCR, fluorescence immunocytochemistry and Western blot techniques were used to determine the mRNA and protein expression levels of the protective stress protein-ER molecular chaperones GRP78 and GRP94 （which would assist cells to resist cellular stress injury）, and to determine the mRNA and protein expression levels of apoptosis promoting molecule Caspase-12 on the membrane of the ER, respectively. Results： Protein expression levels of GRP78 and GRP94 were significantly increased in the TM-induced MSCs, and the mRNA level of Caspase-12 was also remarkably increased in the TM-induced MSCs （P〈0.05）. All these proved that the ERS model was successfully established by TM in MSC. Meanwhile, the mRNA and protein levels of GRP78 and GRP94 were all significantly increased compared with the model group （P〈0.05 or P〈0.01） after MSCs were treated with Tiantai No.1 while the mRNA and protein expression levels of Caspase-12 were significantly decreased compared with the model group （P〈0.05 or P〈0.01）. This effect showed a dose d

关键词： endoplasmic reticulum stress Alzheimer's disease mesenchymal stem cell tunicamycin Tiantai No.1

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氯离子通道阻断剂对tunicamycin诱导心肌细胞凋亡的影响

氯离子通道阻断剂对Tunicamycin诱导心肌细胞凋亡的影响

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作者：沈明志第四军医大学

学位级别：硕士

凋亡(apoptosis),也可以称为细胞程序性死亡(programmed cell death, PCD)。诱导凋亡的途径包括死亡受体和线粒体途径;而内质网应激(endoplasmic reticulum stress, ERS)诱导的细胞凋亡逐渐被认识,其在多种心血管疾病的发生和发展过程... 详细信息

凋亡(apoptosis),也可以称为细胞程序性死亡(programmed cell death, PCD)。诱导凋亡的途径包括死亡受体和线粒体途径;而内质网应激(endoplasmic reticulum stress, ERS)诱导的细胞凋亡逐渐被认识,其在多种心血管疾病的发生和发展过程中发挥着重要作用。因此抑制ERS,减轻凋亡可能是治疗心血管疾病的新策略。凋亡的早期阶段伴随着细胞持续性的皱缩,即凋亡性容积减小(apoptotic volume decrease,AVD)。大量的研究表明,Cl-外流以及伴随的水外流导致的AVD是细胞凋亡的重要前提,并且早于caspases的激活;而阻断氯离子通道减轻了AVD,并且可以抑制细胞凋亡。本实验拟在衣霉素通过ERS途径诱导心肌细胞凋亡的模型上,观察氯通道阻断剂DIDS对心肌细胞凋亡的影响,并初步探讨其机制。研究目的 1.原代培养SD大鼠乳鼠心肌细胞,应用经典的内质网应激凋亡诱导剂tunicamycin(Tm)构建心肌细胞凋亡模型。 ***诱导心肌细胞凋亡过程中,观察氯离子通道阻断剂DIDS对心肌细胞存活及凋亡的影响。 3.初步探讨氯离子通道阻断剂DIDS对Tm诱导的心肌细胞凋亡的影响机制。实验方法 1.实验分为阴性对照组,阳性对照组(Tm组)及实验组(Tm+DIDS),以及单独应用DIDS组。 2.四唑盐(MTT)比色法观察心肌细胞的存活率;流式细胞术(Anexin v/PI)反映心肌细胞凋亡程度。 3.免疫印迹方法测定Tm诱导心肌凋亡过程中靶分子GRP78、CHOP蛋白水平的变化。 4.氯离子通道阻断剂DIDS干预对Tm处理的心肌细胞存活率、细胞凋亡率及伴侣分子GRP78、信号分子CHOP水平的变化。实验结果 1.与阴性对照组相比,衣霉素(Tm)组心肌细胞存活率显著下降,并呈现与Tm的剂量与作用时间正相关(P<0.05,n=12);Tm的最佳诱导浓度为100ng/ml,作用时间为72h。 2.通过MTT比色法和流式细胞术来判断心肌细胞死亡的性质。当Tm浓度为100ng/ml,作用72h时,心肌细胞存活率和凋亡率分别为57.4±3.2%(n=12),25.9±5.8%(n=6)。提示Tm导致心肌细胞死亡的主要方式是凋亡性死亡。 ***作用于心肌细胞后,内质网应激蛋白GRP78和CHOP表达于6h开始增加,24h达到峰值,随后呈下降趋势。 4.与Tm组相比,Tm加DIDS组的细胞存活率较Tm组显著增加,二者分别是57.4±3.2%和80.4%±4.2%(P<0.05,n=12)。 5.与Tm组相比,Tm加DIDS组的细胞凋亡率较Tm组显著降低,二者分别是25.9±5.8%和14.3%±2.9%(P<0.05,n=6)。 ***处理心肌细胞24h后,细胞内的GRP78、CHOP水平分别为阴性对照组的3倍和6.5倍,有显著的统计学差异(P<0.05,n=6)。而Tm加DIDS共同处理心肌细胞时,细胞内的GRP78、CHOP表达水平显著降低(P<0.05,n=6)。结论 1.成功构建Tm诱导心肌细胞内质网应激凋亡模型,Tm的最佳诱导浓度为100ng/ml,作用时间为72h。 2.氯离子通道阻断剂DIDS能够显著抑制Tm诱导的心肌细胞凋亡的发生,具有明显的减轻内质网应激性心肌细胞的损伤。 3.氯离子通道阻断剂DIDS明显抑制Tm诱导心肌细胞凋亡的主要机制可能与抑制GRP78、CHOP水平的表达,减轻了内质网应激发生有关。

关键词：心肌细胞凋亡氯离子通道 tunicamycin GRP78 CHOP

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Ricinus communis agglutinin I functionalisation of poly(methyl methacrylate) (PMMA) as a substrate for microfluidic device

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Science China Chemistry 2012年第4期55卷 537-542页

作者： ZHU DeRong LIU Xia WANG ZhenXin Department of Medicinal Chemistry and Pharmaceutical analysis Guangdong Medical CollegeDongguan 523808China State Key Laboratory of Electroanalytical Chemistry Changchun Institute of Applied ChemistryChinese Academy of SciencesChangchun 130022China

We report a functionalisation strategy which is able to generate Ricinus communis agglutinin 1 （RCA 120） modified PMMA microfluidic device for binding and culturing living cells. The functionalisation is achieved by standard aminealdehyde （Schiff base） reaction through the crosslinker, glutaraldehyde. To prove the ability of the RCA 120 modified PMMA surface, the PC 12 cell line （rat pheochromocytoma ceils） has been captured and cultured by the microfluidic device. In the presence of tunicamycin, the dose/timedependence on decreasing of binding affinity of RCA 120 modified device with PC 12 cell is also observed. The experimental results demonstrate that the lectin-functionalized microfluidic device can be employed as efficient cell culturing platform, and has a great promise of being used as a powerful tool for monitoring the interaction of drug with living cell.

关键词： poly(methyl methacrylate) (PMMA) microfluidic device Ricinus communis agglutinin I (RCA 120) tunicamycin PC 12 cell line

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Lectin Conjugated Gold Nanoparticle-based Colorimetric Assay for Studying the Interactions of Antibiotic with Living Cell

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Chemical Research in Chinese Universities 2011年第2期27卷 193-197页

作者： WANG Jin-e WANG Cheng-ke LIU Dian-jun WANG Zhen-xin State Key Laboratory of Electroanalytical Chemistry Changchun Institute of Applied Chemistry Chinese Academy of Sciences Changchun 130022 P. R. China Graduate School of Chinese Academy of Sciences Beijing 100049 P. R. China

The interactions of antibiotic with living cells were studied by lectin conjugated gold nanoparticles（GNPs） based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNPs are able to employ as indicators for monitoring the antibiotic induced changes of glycosyl complexes. The interactions of a well known antibiotic, tunicamycin, with two different cell lines, HeLa and SHG-44, were selected to establish this assay. In the presence of tunicamycin, the dose- and time-dependence on the decreasing of binding affinity of lectin conjugated GNPs with living cells were demonstrated by conventional microscopic and UV-Vis spectroscopic studies. The experimental result demonstrates that our approach can be used to identify antibiotic induced expression difference of glycosyl complexes on different cellular surfaces and determine drug activity quantitatively. For further confirming the capability of the GNP-based assay, the system was also studied by confocal laser scanning microscopy（CLSM） and classic flow cytometry（FCM） assay, and satisfactory results were obtained.

关键词： Lectin-conjugated gold nanoparticle tunicamycin Colorimetric assay Living cell

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Endoplasmic reticulum stress sensitizes human esophageal cancer cell to radiation

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World Journal of Gastroenterology 2013年第11期19卷 1736-1748页

作者： Xue-Li Pang Gang He Yang-Bo Liu Yan Wang Bo Zhang Department of Oncology Southwest HospitalThird Military Medical University Department of Medical Genetics Third Military Medical University

AIM:To investigate the role of endoplasmic reticulum(ER) stress in cancer radiotherapy and its molecular ***:tunicamycin(TM) was applied to induce ER stress in human esophageal cancer cell line EC109,and the radiosensitization effects were detected by acute cell death and clonogenic survival *** cycle arrest induced by TM was determined by flow cytometric analysis after the cellular DNA content was labeled with propidium *** of EC109 cells induced by TM was detected by annexin V staining and Western blotting of caspase-3 and its substrate poly ADP-ribose *** response was determined by acridine orange(AO) staining and Western blotting of microtubule-associated protein-1 light chain-3(LC3) and autophagy related gene 5(ATG5).In order to test the biological function of autophagy,specific inhibitor or Beclin-1 knockdown was used to inhibit autophagy,and its effect on cell apoptosis was thus ***,involvement of the phosphatidylinositol-3 kinase(PI3K)/Akt/mammalian target of the rapamycin(mTOR) pathway was also detected by Western ***,male nude mice inoculated subcutaneously with EC109 cells were used to confirm cell model ***:Our results showed that TM treatment enhanced cell death and reduced the colony survival fraction induced by ionizing radiation(IR),which suggested an obvious radiosensitization effect of ***,TM and IR combination treatment led to a significant increase of G2/M phase and apoptotic cells,compared with IR *** also observed an increase of AO positive cells,and the protein level of LC3-II and ATG5 was induced by TM treatment,which suggested an autophagic response in EC109 ***,inhibition of autophagy by using a chemical inhibitor or Beclin-1 silencing led to increased cell apoptosis and decreased cell viability,which suggested a cytoprotective role of autophagy in stressed EC109 ***,TM treatment also activated mTORC1,and

关键词： Endoplasmic reticulum stress tunicamycin Esophageal cancer Radiosensitivity Autophagy Apoptosis

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Inhibition of KL-6/MUC1 glycosylation limits aggressive progression of pancreatic cancer

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World Journal of Gastroenterology 2014年第34期20卷 12171-12181页

作者： Huan-Li Xu Xin Zhao Ke-Ming Zhang Wei Tang Norihiro Kokudo Department of Pharmacology School of Basic Medical SciencesCapital Medical University Department of Hepatobiliary Surgery 302 Hospital of Chinese People’s Liberation Army Hepato-Biliary-Pancreatic Surgery Division Department of SurgeryGraduate School of MedicineUniversity of TokyoTokyo 113-8654Japan

AIM: To evaluate the significance of KL-6/MUC1 (a type of MUC1) glycosylation in pancreatic cancer progression.

关键词： Benzyl-N-acetyl-alpha-galactosaminide Glycosylation KL-6/MUC1 Pancreatic carcinoma tunicamycin

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