蛋白酶体抑制剂MG 132对诱导激活的肝星状细胞凋亡的影响

目的观察蛋白酶体抑制剂MG 132对诱导体外培养激活的肝星状细胞(HSC)凋亡的影响.方法大鼠肝星状细胞分离采用胶原酶原位灌注法,用流式细胞仪和琼脂糖凝胶电泳法检测MG 132对诱导激活的HSC凋亡的影响.结果1、2、3μmol/L MG132培养HSC 24 h后,细胞周期分析发现S期细胞减少,G2/M期细胞显著增加(P＜0.01),呈现一个剂量依赖性的关系;流式细胞术检测到明显的亚G1峰,各组的凋亡指数(%)分别是12.70±1.7、17.52±2.3、22.60±3.4,与对照组(1.9±0.6)相比,差异有显著性(P＜0.01);3 μmol/L MG 132作用12、24、36、48 h,凋亡指数(%)分别是16.43±2.2、22.60±2.7、29.80±1.7和36.30±1.4,与对照组相比,差异有显著性(P＜0.01),呈现一个时间依赖性的关系;琼脂糖凝胶电泳可以看到明显的DNA梯带的形成.结论MG 132能够诱导激活的HSC发生凋亡,且在发生凋亡之前有一个明显的G2/M期的阻滞.     

蛋白酶体抑制剂MG132诱导人巨噬细胞THP-1凋亡

泛素-蛋白酶体途径是生物体内进行蛋白质选择性降解的重要途径之一，广泛参与细胞周期调控、DNA修复、细胞信号转导、细胞凋亡等多种生理过程。为了研究调控泛素一蛋白酶体途径表达对巨噬细胞THP-1凋亡和细胞内载脂蛋白B的蓄积的影响，本实验采用蛋白酶体抑制剂MG132(5／μmol／L)处理THP-1细胞，流式细胞术检测细胞凋亡率和细胞内载脂蛋白B含量，用半定量逆转录一聚合酶链反应检测细胞内UPP途径相关基因的表达。结果发现：MG132可以诱导THP-1细胞凋亡；实验组细胞内载脂蛋白B蓄积增加；实验组细胞内泛素活化酶、泛素缀合酶和泛素-蛋白连接酶mRNA表达均降低，而26S蛋白酶体mRNA无显著改变。结果提示，蛋白酶体抑制荆MG132能够诱导THP-1细胞凋亡，其机制可能与MG132抑制泛素-蛋白酶体途径中泛素活化酶、泛素缀合酶和泛素一蛋白连接酶活性，使细胞内载脂蛋白B经泛素-蛋白酶体途径降解减少，在细胞内蓄积而使细胞凋亡率增加。     

hFRNK基因对胃泌素诱导的人结肠癌细胞侵袭力的影响

目的 观察腺病毒介导hFRNK基因对胃泌素所诱导的人结肠癌Colo320WT细胞侵袭力的影响.方法 试验分为胃泌素组、hFRNK组和对照组,胃泌素组用100 μmol/L胃泌素诱导结肠癌Col0320WT细胞12 h;hFRNK组,首先用脂质体瞬时转染腺病毒受体pCR3.1-CAR于Col0320WT细胞48 h,然后用100 μmol/L胃泌素干预结肠癌Colo320WT细胞12 h,再用重组腺病毒(pAdhFRNK)感染细胞;对照组为未经处理的Colo320WT细胞.用免疫印迹检测hFRNK基因黏着斑激酶(FAK)397位酪氨酸(FAKTyr397)的磷酸化表达,激光共聚焦显微镜观察FAKTyr397在细胞板状伪足的表达情况,免疫共沉淀检测hFRNK基因对四联信号复合物FAK-Src-Doek180一p130Cas形成的影响,Pull-down法检测hFRNK对Rac蛋白活性的影响.结果 胃泌素诱导后,磷酸化FAKTyrr397明显增强;与胃泌素组相比,hFRNK组中FAKTyr397表达下降,FAKTyr397定位到细胞板状伪足的量明显减少,FAK、Src、Dockl80和p130Cas 四联信号复合物没有形成,Rac的活性降低.结论 hFRNK基因可阻断胃泌素引起的FAK的磷酸化,阻断FAKTyr397摹积到细胞的板状伪足,阻止四联信号复合物FAK-Src-Dock180-p130Cas的形成以及Rac的活化,为hFRNK基因防治肿瘤的侵袭和转移提供理论依据.     

泛素蛋白酶体抑制剂MG-132与心力衰竭

心力衰竭是各种心脏疾病发展的严重阶段,发病率的上升是心血管医学面临的最大挑战之一,已成为心脏病治疗的最后战场。MG-132是一种泛素蛋白酶体抑制剂,其在心力衰竭发展过程中扮演重要的角色,但其具体作用尚未完全阐明。现综述MG-132与心力衰竭关系及相关分子机制。     

蛋白酶体抑制剂MG132对球囊损伤后血管狭窄的影响

目的 观察蛋白酶体抑制剂MG132对球囊损伤后血管狭窄的影响.方法 将新西兰白兔40只随机分成正常对照组、高脂组、球囊损伤组和MG132组.球囊损伤组和MG132组兔采用球囊拉伤颈总动脉复制血管损伤后狭窄模型;MG132组在损伤血管局部应用蛋白酶体抑制剂MG132.喂养2周、4周和8周后取颈总动脉损伤段血管制成病理切片行HE染色,测定内膜厚度、中膜厚度和管腔面积,评价颈总动脉血管狭窄.结果 球囊损伤组颈总动脉发生明显狭窄、管腔减小、内膜增厚,有泡沫细胞和大量血管平滑肌细胞增殖,而MG132组的病变明显减轻.结论 用球囊拉伤新西兰白兔颈总动脉复制的血管狭窄模型,颈总动脉发生明显狭窄,局部应用蛋白酶体抑制剂MG132能够抑制血管内膜增生,抑制损伤后血管狭窄.     

纤维连接蛋白对人结肠癌细胞侵袭力的影响

目的研究纤维连接蛋白（fibronectin，FN）对人结肠癌细胞侵袭力的影响，并探讨其中的信号传导机制。方法以递增浓度的FN刺激结肠癌细胞株Colo320，以免疫沉淀和蛋白质印迹法检测结肠癌细胞内黏着斑激酶（focal adhesion kinase，FAK）第397位酪氨酸（tyr-397）磷酸化的状况．以改良Boyden小室法检测相应的细胞侵袭力变化。设计反义寡核苷酸阻断FAK蛋白质表达，再次观察接受FN刺激后．结肠癌细胞内FAK tyr-397磷酸化状况及细胞侵袭力的改变。结果FN能够促进Colo320 FAK tyr-397磷酸化，在一定范围内具有剂量依赖性。FN浓度达10nmol／L时．此作用最显著，继续增加FN浓度．FAK tyr-397磷酸化不再呈现继续增强趋势．当浓度达到100nmol／L时，磷酸化程度反而有所下降；FN可以增强细胞侵袭力，同样在一定范围内具有剂量依赖性；反义寡核苷酸干预后．结肠癌细胞内FAK tyr-397磷酸化及细胞侵袭力均有显著降低（P〈0.01）。结论FN可以有效增强结肠癌细胞的侵袭力，其作用是通过FN—FAK信号通路实现的，FAK的活性形式是其酪氨酸位点的磷酸化．阻断FAK的表达町以减弱FN促进细胞侵袭的作用。     

姜黄素对结肠癌细胞SW480 FasL基因表达和侵袭能力的影响

目的研究姜黄素对人结肠癌SW480细胞FasL mRNA表达及对其侵袭能力的影响,为中药抗肿瘤提供实验依据。方法根据MTT法得到姜黄素对SW480细胞的半数有效抑制浓度（IC50）,确定药物作用浓度。SW480细胞分别经姜黄素不同浓度（0．5 IC50、IC50）作用后,应用逆转录-聚合酶链反应（RT-PCR）法检测姜黄素作用前后人结肠癌SW480细胞FasL mRNA的变化;应用Transwell细胞侵袭试验检测姜黄素对SW480细胞侵袭能力的影响。结果姜黄素处理后SW480细胞FasL mRNA表达水平均明显高于对照组（P〈0．01）;而且FasL mRNA表达水平随姜黄素作用浓度增加显著上调,姜黄素不同浓度组比较均有显著性差异（P〈0．01）;随姜黄素作用浓度升高,SW480细胞侵袭能力明显增强,不同浓度组比较均有显著性差异（P〈0．01）。结论姜黄素在一定时间内均可上调人结肠癌SW480细胞FasL mRNA的表达,而且这种上调作用在一定范围内呈剂量依赖性,可使结肠癌细胞的侵袭能力增强。     

蛋白酶体抑制剂MG132抑制糖尿病肾脏疾病机制的研究

目的探讨蛋白酶体抑制剂MG132是否抑制糖尿病肾脏疾病(DKD)的发展及其可能的机制。方法 STZ复制糖尿病大鼠,随机分为糖尿病组(DM组,n=9)、MG132治疗组(MG132组,n=9),MG132组从第9周起予MG 132[0.1mg/(kg·d)]腹腔注射治疗糖尿病大鼠。同时设对照(Con组,n=9)组,检测生化指标,观察肾组织病理改变;Western blot检测肾组织SnoN、第10号染色体缺失的磷酸酶和张力蛋白同源基因(PTEN)、钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-SMA)的表达。结果与Con组比较,DM组血糖[(5.85±0.86)vs(17.49±1.21)mmol/L,P=0.00]、尿微量白蛋白/尿肌酐比值(UACR)[(1.11±0.29)vs(16.36±3.06)mg/mmol,P=0.00]、肾脏指数(KW/BW)[(6.32±0.49)vs(14.54±1.49)mg/g,P=0.00]明显增加,且肾小管间质纤维化病变明显,α-SMA[(0.18±0.03)vs(0.33±0.02),P=0.002)增多,而SnoN[(0.87±0.10)vs(0.32±0.11),P=0.007)、PTEN[(2.06±0.09)vs(1.26±0.06),P=0.00]、E-cadherin[(1.32±0.06)vs(0.50±0.03),P=0.00]减少;MG132治疗后,UACR[(6.74±3.47)mg/mmol,P=0.003]、KW/BW[(12.43±1.11)mg/g,P=0.01]降低,纤维化病变减轻,α-SMA[(0.19±0.05),P=0.003]减少,SnoN[(0.55±0.09),P=0.033]、PTEN[(1.73±0.15),P=0.002]、E-cadherin[(1.11±0.10),P=0.00]蛋白的表达增加。结论 MG132可能通过恢复SnoN、PTEN蛋白水平抑制DKD的发展。     

蛋白酶体抑制剂MG132联合顺铂诱导肺癌A549细胞凋亡的机制

目的探讨蛋白酶体抑制剂(MG132)与顺铂(DDP)联合诱导肺癌A549细胞凋亡的机制及作用。方法采用四甲基偶氮唑盐(MTT)法检测不同浓度MG132、DDP对肺癌A549细胞的最适作用时间以及抑制率;通过Hochest33342染色法检测细胞形态变化;应用流式细胞术(FCW)检测MG132、DDP及两药联合作用于肺癌A549细胞的凋亡率;采用Western印迹检测药物干预前后细胞中促凋亡蛋白半胱氨酸蛋白酶(caspase)9、核转录因子(NF-κB)的表达情况。结果 MTT结果显示MG132、DDP能有效抑制细胞的增殖,呈浓度及时间依赖性;FCW结果显示MG132与DDP联合用药组作用于肺癌A549细胞凋亡率为(68.27±0.31)%,与MG132(22.13±0.36)%、DDP(25.76±0.25)%单用药组相比,细胞凋亡率明显提高(P<0.05)。Western印迹结果显示,与单用药组相比较,联合用药组凋亡相关蛋白caspase9表达明显增强,NF-κB表达显著减少。结论 MG132与DDP联合应用可明显提高细胞凋亡率,其机制可能是通过提高促凋亡蛋白caspase9的表达,并降低细胞NF-κB的表达而实现的。     

蛋白酶体抑制剂MG132对外周血内皮祖细胞数量与功能的影响

目的观察蛋白酶体抑制剂MG132对外周血内皮祖细胞(EPCs)数量与功能的影响。方法采用Ficoll密度梯度离心法从外周血获得单个核细胞,接种于包被人纤维连接蛋白的培养板,收集贴壁细胞,加入不同浓度MG132(20nmol/l,50nmol/l,100nmol/l,200nmol/l)培养12、24、48h。激光共聚焦显微镜鉴定,倒置荧光显微镜计数。分别采用MTT比色法、改良的Boyden小室、黏附能力测定观察EPCs的增殖、迁移、黏附能力。结果低剂量范围内,EPCs数量随MG132浓度与作用时间增加而减少,其增殖、迁移、黏附能力亦随MG132浓度与作用时间增加而减少。200nmol/l浓度MG132作用48h对EPC数量、增殖功能及黏附能力的影响最为显著。结论低剂量MG132减少EPCs数量并抑制EPCs的增殖、黏附、迁移能力。     

Effect of MG132 on proteasome activity and prooxidant/antioxidant status of rat liver subjected to ischemia/reperfusion injury.

Aim: Previous studies have shown that proteasome inhibitors exerted protective effects against ischemia/reperfusion injury (IRI) of brain, heart, kidney and intestine. The aim of the present study was to investigate: (i) whether the proteasome inhibitor MG132 protects rat liver against IRI; and (ii) whether MG132 modulates prooxidant/antioxidant status of rat liver subjected to warm IRI. Methods: The left lateral and medial lobes (approximately 70% of the total liver volume) of livers of male Wistar rats were subjected to 30-min ischemia followed by 60-min reperfusion. Lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were measured in the plasma. Proteasome chymotryptic-like (ChT-L) activity, levels of thiobarbituric acid-reactive substances (TBARS), protein carbonyls (PC) and glutathione (GSH), as well as superoxidase dismutase (SOD), catalase (CAT), glutathionine peroxidase and glutathionine reductase activities were measured in liver fractions. Results: Thirty-min ischemia followed by 60-min reperfusion increased liver TBARS and PC, CAT and SOD activities, but decreased GSH level. Ischemia/reperfusion-induced oxidative stress was exacerbated in mitochondria, indicating that these organelles are the preferential target of IRI. Plasma LDH and AST levels were decreased by MG132 during both ischemia and reperfusion, while ALT values were decreased only after 30 min of reperfusion. MG132 did not significantly affect liver TBARS and GSH levels, but it increased PC and decreased ChT-L activity; the activities of CAT and SOD were also decreased. Conclusions: MG132 exerts a protective effect during the early phase of reperfusion and it modulates prooxidant/antioxidant status of rat liver subjected to warm IRI.     

miR-132 inhibits colorectal cancer invasion and metastasis via directly targeting ZEB2

AIM:To investigate the biological role and underlying mechanism of miR-132 in colorectal cancer(CRC)progression and invasion.METHODS:Quantitative RT-PCR analysis was used to examine the expression levels of miR-132 in five CRC cell lines(SW480,SW620,HCT116,HT29 and LoVo)and a normal colonic cell line NCM460,as well as in tumor tissues with or without metastases.The KaplanMeier method was used to analyze the prognostic significance of miR-132 in CRC patients.The biological effects of miR-132 were assessed in CRC cell lines using the transwell assay.Quantitative RT-PCR and western blot analyses were employed to evaluate the expression of miR-132 targets.The regulation of ZEB2 by miR-132was confirmed using the luciferase activity assay.RESULTS:miR-132 was significantly down-regulated in the CRC cell lines compared with the normal colonic cell line(P<0.05),as well as in the CRC tissues withdistant metastases compared with the tissues without metastases(10.52±4.69 vs 23.11±7.84)(P<0.001).Down-regulation of miR-132 was associated with tumor size(P=0.016),distant metastasis(P=0.002),and TNM stage(P=0.020)in CRC patients.Kaplan-Meier survival curve analysis indicated that patients with low expression of miR-132 tended to have worse diseasefree survival than patients with high expression of miR-132(P<0.001).Moreover,ectopic expression of miR-132 markedly inhibited cell invasion(P<0.05)and the epithelial-mesenchymal transition(EMT)in CRC cell lines.Further investigation revealed ZEB2,an EMT regulator,was a downstream target of miR-132.CONCLUSION:Our study indicated that miR-132 plays an important role in the invasion and metastasis of CRC.     

miR-132 inhibits lung cancer cell migration and invasion by targeting SOX4

Background

Multiple MicroRNAs (miRNAs) have been identified in the development and progression of lung cancer. However, the expression and roles of miR-132 in non-small cell lung cancer (NSCLC) remain largely undefined. The aim of this study is to investigate the biological functions and its molecular mechanisms of miR-132 in human lung cancer cells.

Methods

miR-132 expression was measured in human lung cancer cell lines by quantitative real-time PCR (qRT-PCR). The cells migration and invasion ability were measured by wound healing assay and transwell assay. The influence of miR-132 on tumor progression in vivo was monitored using NSCLC xenografts in nude mice. The target gene of miR-132 was determined by luciferase assay and western blot.

Results

The expression level of miR-132 was dramatically decreased in examined lung cancer cell lines. Then, we found that introduction of miR-132 significantly suppressed the migration and invasion of lung cancer cells in vitro. Besides, miR-132 overexpression could also inhibit tumor growth in the nude mice. Further studies indicated that the sex determining region Y-box 4 (SOX4) is a target gene of miR-132. SOX4 re-introduction could reverse the anti-invasion role of miR-132.

Conclusions

Our finding provides new insight into the mechanism of NSCLC progression. Therapeutically, miR-132 may serve as a potential target in the treatment of human lung cancer.     

Fas通路的激活促进结肠癌细胞迁移侵袭

目的探讨Fas通路激活对结肠癌细胞SW480和DLD1产生的非凋亡效应。方法流式细胞学检测结肠癌SW480及DLD1细胞系的Fas、FasL、抗凋亡蛋白c-FLIP、Bcl-2、Bcl-xl、XIAP等在细胞膜上的表达水平;对结肠癌SW480及DLD1细胞系予以梯度浓度（0ng/ml、12.5ng/ml、25ng/ml、50ng/ml）的FasL刺激,计数并比较各组细胞的增殖速率变化;对结肠癌SW480及DLD1细胞系予以低剂量FasL处理,分别对实验组和对照组进行细胞增殖速率及迁移侵袭能力测试,探讨低剂量FasL刺激对结肠癌细胞活力的影响;稳定敲除SW480和DLD1细胞的Fas基因表达,重复上述实验,以明确低剂量FasL刺激对结肠癌细胞活力的影响是否依赖于Fas通路的激活。结果 SW480及DLD1细胞系中均可检测到中等程度的Fas表达及抗凋亡蛋白FLIP、Bcl-2的表达,未检测到FasL表达,在SW480中可测到Bcl-xl表达;低剂量FasL不影响结肠癌SW480和DLD1细胞的增殖速率,但可促进两种细胞的迁移侵袭能力;稳定敲除Fas基因后,低剂量FasL对结肠癌细胞迁移侵袭能力的促进作用受到抑制。结论低剂量FasL可激活Fas通路的非凋亡途径从而增强结肠癌SW480和DLD1细胞的迁移侵袭能力。     

Therapeutic implications of colon cancer stem cells

Colorectal cancer is the second most common cause of cancer-related death in many industrialized countries and is characterized by a heterogenic pool of cells with distinct differentiation patterns. Recently, the concept that cancer might arise from a rare population of cells with stem cell-like properties has received support with regard to several solid tumors, including colorectal cancer. According to the cancer stem cell hypothesis, cancer can be considered a disease in which mutations either convert no...