Mir-30a抑制自噬及上皮细胞间质化增强肺腺癌细胞对克唑替尼的敏感性

目的探索上调肺腺癌细胞中Mir-30a的表达水平能否增强细胞对克唑替尼的敏感性,观察细胞增殖率的变化及克唑替尼IC50等的变化,并探索相关的机制。方法使用Lipofectamine2000携带Mir-30a对H3122细胞进行瞬时转染,并与克唑替尼共同作用。将肺腺癌H3122细胞分为对照组、克唑替尼与联合组。MTT法检测各组细胞增殖率变化,Transwell检测细胞侵袭性。Western blot检测ALK、c-MET、Beclin-1、E-cadherin及Vimentin蛋白的表达。结果在H3122细胞中Mir-30a转染与克唑替尼共同作用较克唑替尼单独作用有更显著的细胞杀伤作用。转染后的细胞较单独应用克唑替尼组侵袭力减弱。转染与克唑替尼联合组Beclin1、ALK、c-MET及Vimentin的蛋白的表达有明显下降,E-cadherin稍有增强。结论 Mir-30a增强肺腺癌细胞对克唑替尼的敏感性,可能与过量表达的Mir-30a抑制癌细胞的自噬及上皮细胞间质化相关。     

Autophagy mediates neutrophil responses to bacterial infection

Neutrophils constitute the first line of cellular defense against pathogens and autophagy is a fundamental cellular homeostasis pathway that operates with the intracellular degradation/recycling system. Induction of the autophagic process in neutrophils, in response to invading pathogens, constitutes a crucial mechanism in innate immunity. Exploration of autophagy has greatly progressed and diverse strategies have been reported for studying this molecular process in different biological systems; especially in infectious and inflammatory diseases. Furthermore, the role of autophagy in neutrophils, during pathogenic infection, continues to be of interest, due to the role of the cell in immunity function, its recruitment to the site of infection and its implication in inflammatory diseases. This review focuses on the known role of autophagy in neutrophils defence against pathogenic infections. A more detailed discussion will concern the recent findings highlighting the role of autophagy in inflammation and cell death in infected neutrophils.     

Xenophagy in Helicobacter pylori‐ and Epstein–Barr virus‐induced gastric cancer

Helicobacter pylori and Epstein–Barr virus (EBV) account for roughly 80% and 10%, respectively, of gastric carcinomas worldwide. Autophagy is an evolutionarily conserved and intricately regulated cellular process that involves the sequestration of cytoplasmic proteins and organelles into double‐membrane autophagosomes that eventually fuse with lysosomes for degradation of the engulfed content. Emerging evidence indicates that xenophagy, a form of selective autophagy, plays a crucial role in the pathogenesis of H. pylori‐ and EBV‐induced gastric cancer. Xenophagy specifically recognizes intracellular H. pylori and EBV and physically targets these pathogens to the autophagosomal–lysosomal pathway for degradation. In this connection, H. pylori or EBV‐induced dysregulation of autophagy may be causally linked to gastric tumourigenesis and therefore can be exploited as therapeutic targets. This review will discuss how H. pylori and EBV infection activate autophagy and how these pathogens evade recognition and degradation by the autophagic pathway. Elucidating the molecular aspects of H. pylori‐ and EBV‐induced autophagy will help us better understand the pathogenesis of gastric cancer and promote the development of autophagy modulators as antimicrobial agents. Published by John Wiley & Sons, Ltd     

Autophagy Activity in Pulmonary Metastatic Tumor Tissues from Colorectal Cancer: A Pilot Study

Purpose

Autophagy has been reported to be involved in treatment failure in tumor. We aimed to evaluate autophagy activity in tumor tissue and compare them between the recurrence and non-recurrence groups.

Materials and Methods

We analyzed expressions of autophagy-related proteins in tumor tissues which were obtained from pulmonary metastases of colorectal cancer patients by Western blot. We also analyzed autophagosomes by transmission electron microscopy.

Results

Tumor tissues from recurrence group showed increased levels of LC3B-II, decreased levels of p62/SQSTM1, and also a marked accumulation of autophagosomes compared with tissues from non-recurrence group.

Conclusion

The present study suggests that autophagy may be associated with treatment failure of metastatic colorectal cancer.     

柯萨奇病毒B组3型2B蛋白诱导细胞自噬及其基序鉴定

目的 探讨柯萨奇病毒B组3型(CVB3) woodruff病毒2B蛋白对细胞自噬的诱导作用,及其自噬相关基序的鉴定.方法 分别构建增强型绿色荧光蛋白(EGFP)与CVB3 woodruff病毒2B蛋白及其9种截短蛋白的融合蛋白(EGFP-2B、EGFP-2B1-249、EGFP-2B1-201、EGFP-2B1-153、EGFP-2B1-105、EGFP-2B1-57、EGFP-2B106.201、EGFP-2B106-249、EGFP-2 B205-297、EGFP-2B106-201)真核表达载体,红色荧光蛋白(mCherry)与微管相关蛋白轻链3(LC3)的融合蛋白真核表达载体pmCherry-LC3;应用激光共聚焦与蛋白质免疫印迹(Western blot)检测病毒2B蛋白对宫颈癌细胞(HeLa) LC3表达的影响;荧光显微镜观察9种截短蛋白在HeLa细胞中的表达;Western blot检测pEGFP-2B和pEGFP-2B106-249转染细胞后LC3的表达;观察自噬抑制剂3-甲基腺苷(3-MA)处理后,pEGFP-2B和pEGFP-2B106-249诱导细胞自噬的情况.结果 CVB3攻击HeLa细胞后,mCherry-LC3呈现细胞核周点状聚集表达,Western blot检测出现清晰LC3-Ⅱ条带;pEGFP-2B与pmCherry-LC3共转染后也可见细胞核周围绿色荧光与红色荧光均成点状聚集并相互重叠,且LC3-Ⅱ条带明显;9种截短融合蛋白质粒分别转染HeLa细胞后,其中可见细胞核周围绿色荧光点状聚集的最短截短蛋白为EGFP-2B106-249;pEGFP-2B和pEGFP-2B106-249转染细胞后可检测出现清晰LC3-Ⅱ条带;3-MA处理后,pEGFP-2B和pEGFP-2B106-249分别与pmCherry-LC3共转染的细胞均未见绿色和红色荧光的核周点状聚集.结论 CVB3 woodruff病毒2B蛋白可以诱导宿主细胞发生自噬,其中截短蛋白2B106-249是病毒蛋白2B诱导HeLa细胞发生自噬的功能基序.     

Cholesterol crystallization in human atherosclerosis is triggered in smooth muscle cells during the transition from fatty streak to fibroatheroma

Recent studies have shown that in addition to being major constituents of the atheromatous core, solid cholesterol crystals (CCs) promote atherosclerotic lesion development and rupture by causing mechanical damage and exerting cytotoxic and pro‐inflammatory effects. These findings suggest that targeting CCs might represent a therapeutic strategy for plaque stabilization. However, little is known about how cholesterol crystallization is initiated in human atherothrombotic disease. Here, we investigated these mechanisms. We performed a thorough immunohistological analysis of non‐embedded, minimally processed human aortic tissues, combining polarized light and fluorescence microscopy. We found that CC formation was initiated during the fatty streak to fibroatheroma transition in tight association with the death of intralesional smooth muscle cells (SMCs). Cholesterol‐loaded human SMCs were capable of producing CCs in vitro, a process that was enhanced by type I collagen and by inhibition of autophagy and cholesterol esterification. The fibrous transition, which was characterized by increased type I collagen expression, was associated with changes in the expression of autophagy and cholesterol flux‐related genes, including a decrease in the autophagic adapter p62 and an increase in the cholesterol intracellular transporter Niemann–Pick C1. Collagen was identified as a potent inducer of these changes in SMCs. Collagen‐induced changes in cholesterol metabolism and autophagy flux in smooth muscle foam cells at the fibrolipid transition likely contribute to initiate cholesterol crystallization in human atherosclerosis. Also, our data are in support of a protective role of autophagy against CC formation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

抑制自噬增强MCF-7乳腺癌细胞对依托泊苷的敏感性

目的探讨自噬抑制剂(Baf)在乳腺癌MCF-7细胞对依托泊苷(VP-16)的药物敏感性的影响。方法实验分为对照组(NC)、Baf组、VP-16和VP-16+Baf组。用MTT法检测细胞生存活力、GFP-LC3质粒转染后荧光显微镜检测绿色荧光的分布、Western blot检测蛋白表达和流式细胞仪检测细胞凋亡。结果 15μmol/L的VP-16使细胞活力降低,而在收集细胞前12 h时加入10 nmol/L的Baf进一步降低了细胞活力(P0.01);VP-16明显增加MCF-7细胞的LC3Ⅱ的表达和GFP-LC3绿色荧光斑点的聚集,而减少了P62的蛋白表达;与VP-16组比较,VP-16+Baf组细胞P62的蛋白表达增多,凋亡蛋白cleaved-PARP的表达和细胞凋亡比例也明显增多(P0.01)。结论 VP-16抑制乳腺癌细胞增殖的过程中诱导了保护性自噬,抑制自噬促进了凋亡性死亡,可以增加癌细胞对VP-16的敏感性。     

Detachment‐Based Equilibrium of Anoikic Cell Death and Autophagic Cell Survival Through Adaptor Protein p66Shc

Anoikis (detachment‐induced cell death) confers a tumor‐suppressive function in metastatic cancer cells. Autophagy, a conserved self‐degradative process, enhances the anoikis resistance of detached cancer cells by maintaining cellular homeostasis. However, the mechanism of regulating cell fate‐decision by balancing anoikis and autophagy has been poorly understood. Our previous studies have shown that the adaptor protein p66^Shc mediates anoikis through RhoA activation and inhibits tumor metastasis in vivo. We also found that p66^Shc depletion mitigates nutrient‐deprivation‐induced autophagy. These findings suggest p66^Shc may coordinately regulate these two processes. To verify this hypothesis, we investigated the effect of p66^Shc on the cell death of detached lung cancer cells, and measured autophagy markers and autophagic flux. Results showed that p66^Shc depletion significantly inhibited anoikis, and reduced the formation of LC3B‐II and the degradation of Sequestosome 1 (SQSTM1, p62) in detachment‐induced cells. Using monodansylcadaverine (MDC)‐LysoTracker double staining and monomeric Cherry (mCherry)‐GFP‐LC3 assay, we found that the autophagic flux was also mitigated by p66^Shc depletion. In addition, p66^Shc knockdown increased the formation of full‐length X‐linked inhibitor of apoptosis (XIAP)‐associated factor 1 (XAF1), which enhances anoikis sensitivity. In conclusion, p66^Shc plays an essential role in detachment‐based equilibrium of anoikic cell death and autophagic cell survival. Anat Rec, 299:325–333, 2016. © 2015 Wiley Periodicals, Inc.     

阻塞性睡眠呼吸暂停低氧对脑缺血大鼠海马区神经细胞自噬的影响

目的:探讨阻塞性睡眠呼吸暂停(obstructive sleep apnea hypoxia hypoxia,OSAH)低氧对脑缺血大鼠海马区神经细胞自噬的影响。方法:雄性Wistar大鼠采用数字随机法随机分成假手术组(SO组)、单纯脑缺血再灌注组(I/R组)、OSAH低氧7 d脑缺血再灌注组(IH7+I/R组)、OSAH低氧21 d脑缺血再灌注组(IH21+I/R组)。造模前,IH7+I/R组、IH21+I/R组分别给予间歇性低氧7、21 d。采用改良的Pulsinelli四血管阻断法制备脑缺血再灌注模型,术后24 h,H-E染色观察海马区神经细胞形态变化,RT-PCR、免疫组织化学检测大鼠脑组织海马区mTOR、beclin--1表达情况。结果:与假手术组比较,I/R组大鼠在各时间点均神经元结构损伤;免疫组织化学显示mTOR、beclin--1蛋白免疫阳性细胞数增加;RT-PCR示mTOR、beclin-mRNA表达增强。与I/R组比较,OSAH低氧各组大鼠在各时间点,H-E染色示神经元结构损伤加重;免疫组织化学示mTOR、beclin--1蛋白免疫阳性细胞数增加;RTPCR示mTOR、beclin-1 mRNA表达增强;上述变化在IH21+I/R组更为显著。结论:OSAH低氧加重脑缺血后海马神经元的自噬性损伤。