Akt激酶抑制剂MK-2206诱导U2OS人骨肉瘤细胞凋亡与自噬

 目的：研究Akt抑制剂MK-2206对U2OS细胞凋亡和自噬的影响。方法：采用MTT法检测MK-2206对U2OS细胞活力的影响,DNA片段末端标记试剂盒检测细胞凋亡的变化,免疫印迹法检测细胞内蛋白的表达,用LC3-II的表达量用来确定细胞的自噬水平。结果：MK-2206剂量依赖性地降低了U2OS细胞的活力;MK-2206能够促进caspase-9、caspase-3和PARP的活化切割而诱导U2OS细胞发生凋亡;MK-2206给药后可促进细胞内LC3-II的表达,氯喹阻断自噬后明显增强了MK-2206对U2OS细胞活力的抑制作用。结论：Akt 抑制剂MK-2206能够诱导U2OS细胞发生凋亡和自噬;抑制自噬可促进MK-2206对U2OS细胞的毒性。     

CUDC-907诱导胶质瘤细胞DNA损伤与自噬的实验研究

目的:探讨新型组蛋白去乙酰化酶(histone deacetylase,HDAC)/磷脂酰肌醇3-激酶(phosphatidyl-inositol 3-kinase,PI3K)双靶点抑制剂CUDC-907对人胶质瘤U251细胞DNA损伤、细胞周期及自噬的影响。方法:采用不同浓度的CUDC-907处理U251细胞24 h,MTT法检测细胞活力的变化;激光共聚焦显微镜观察DNA损伤标志物γ-H2AX在细胞内的分布;流式细胞术分析CUDC-907对细胞周期的影响;Western blot实验检测细胞内相关蛋白表达水平的变化。结果:CUDC-907能够抑制U251细胞活力。在CUDC-907处理的细胞中,蛋白激酶B(PKB)/Akt和p70核糖体蛋白S6激酶(p70s6K)的磷酸化水平降低,γ-H2AX的焦点数量与蛋白表达显著升高(P 0.05);U251细胞经CUDC-907作用后,G2/M期细胞数量增多;Western blot实验结果表明,CUDC-907促进p21的表达,同时抑制细胞周期素B1(cyclin B1)的蛋白表达和细胞分裂周期蛋白2(Cdc2)的磷酸化水平(P 0.05);另外,CUDC-907能够诱导细胞自噬,抑制自噬可促进CUDC-907诱导的DNA损伤。结论:CUDC-907能够显著抑制PI3K/Akt信号通路,诱导胶质瘤细胞发生DNA损伤,并阻滞细胞于G_2/M期,同时可诱导胶质瘤细胞发生保护性自噬。     

细胞自噬在Aβ_(25-35)损伤SH-SY5Y细胞中的作用

目的:探究β-淀粉样蛋白(Aβ)诱导的神经细胞损伤作用是否与调节细胞自噬相关,并基于Akt/mTOR通路对其作用机制进行初步探究。方法:5μmol/L、10μmol/L、15μmol/L、20μmol/L和25μmol/L的Aβ_(25-35)与人神经母细胞瘤SH-SY5Y细胞共同孵育24 h,MTT法检测细胞活力,Western blot检测细胞内微管相关蛋白1轻链3-I(LC3-I)、微管相关蛋白1轻链3-II(LC3-II)、Akt、p-Akt、mTOR和p-mTOR蛋白表达情况。选用合适浓度的Aβ_(25-35),观察自噬诱导剂雷帕霉素(Rapa)和自噬抑制剂3-甲基腺嘌呤(3-MA)分别与Aβ_(25-35)联用后上述指标的变化。结果:各浓度Aβ_(25-35)均能引起SH-SY5Y细胞损伤,使细胞活力下降。Aβ_(25-35)能够增加自噬标志蛋白LC3-II蛋白表达,提高LC3-II/LC3-I水平,下调Akt和mTOR蛋白磷酸化水平(P0.05)。与自噬诱导剂Rapa联用,细胞活力未见明显变化,而细胞内LC3-II蛋白表达升高,LC3-II/LC3-I明显增加,p-mTOR/mTOR明显降低(P0.05);与自噬抑制剂3-MA联用,LC3-II蛋白表达和LC3-II/LC3-I水平有下降趋势,p-Akt/Akt水平明显下降(P0.05)。结论:Aβ_(25-35)可能通过下调Akt和mTOR蛋白磷酸化水平而诱导SH-SY5Y细胞自噬状态及损伤。     

microRNA-424-5p对人胃癌细胞SGC-7901自噬的影响及机制

目的研究microRNA-424-5p(miR-424-5p)对人胃癌细胞SGC-7901自噬的影响及可能机制。方法培养人正常胃黏膜上皮细胞GES和人胃癌细胞SGC-7901,应用实时定量PCR方法检测miR-424-5p的表达水平;应用Lipofectamine LTX试剂将化学合成的miR-424-5p agomir和miR-424-5p antagomir转染人胃癌细胞SGC-7901,验证转染效率后应用MTT方法检测细胞活力;应用免疫荧光法检测自噬标记物微管相关蛋白轻链-3(LC3)和自噬降解底物p62/SQSTM1在SGC-7901细胞的分布;应用Western blot方法检测LC3-Ⅱ、p62/SQSTM1和自噬相关蛋白Beclin1的蛋白表达水平。结果 miR-424-5p在人胃癌细胞SGC-7901中呈低表达,miR-424-5p过表达显著抑制人胃癌细胞SGC-7901的细胞活力,miR-424-5p表达沉默的作用效果与之相反。miR-424-5p过表达后自噬标记物LC3在SGC-7901细胞的胞浆内呈点状分布,荧光表达显著增强,并且LC3-II的蛋白表达水平显著上调,同时伴有自噬降解底物p62/SQSTM1在细胞浆内荧光表达显著减弱,蛋白表达下调,此外,自噬相关蛋白Beclin1的蛋白表达水平显著上调;miR-424-5p表达沉默的作用效果与之相反。结论 miR-424-5p过表达明显抑制人胃癌细胞SGC-7901的细胞活力,诱导细胞发生自噬,其机制之一可能与自噬相关蛋白Beclin1上调有关。     

紫草素通过PI3K/Akt/mTOR信号通路诱导人宫颈癌HeLa细胞凋亡和自噬

目的:观察紫草素(shikonin)对人宫颈癌HeLa细胞凋亡(apoptosis)和自噬(autophagy)的影响,并初步探讨PI3K/Akt/mTOR信号通路在其中的可能作用。方法:以紫草素作用于HeLa细胞后,采用CCK-8检测细胞活力;Annexin V/PI双染法检测细胞凋亡;GFP-LC3质粒转染HeLa细胞后观察自噬小体;紫草素分别与自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)和凋亡抑制剂Z-DEVD-FMK共同作用后,Western blot分析检测细胞内自噬和凋亡相关蛋白微管相关蛋白1轻链3 (LC3)和cleaved caspase-3表达的变化,并检测PI3K/Akt/mTOR信号通路中磷酸化PI3K(p-PI3K)、磷酸化Akt(p-Akt)和磷酸化mTOR(p-mTOR)蛋白表达的变化。结果:紫草素显著抑制HeLa细胞活力(P0.05)。与对照组比较,紫草素可诱导HeLa细胞凋亡(P0.05)。GFP-LC3质粒转染分析结果显示,HeLa细胞经紫草素作用后,细胞质中出现绿色点状聚集的自噬小体,而对照组细胞中极少观察到点状聚集的自噬小体形成。与紫草素组比较,紫草素+3-MA组中LC3-II/LC3-I显著降低,而cleaved caspase-3表达显著升高(P0.05);与紫草素组比较,紫草素+Z-DEVD-FMK组LC3-II/LC3-I显著升高,而cleaved caspase-3表达显著降低(P0.05)。与对照组比较,紫草素可使p-PI3K、p-Akt和p-mTOR表达明显降低(P0.05)。结论:论紫草素能诱导HeLa细胞凋亡和自噬,且其凋亡及自噬具有协同作用,其机制可能与抑制PI3K/Akt/mTOR信号通路有关。     

AZD5363诱导肝癌细胞凋亡与自噬的实验研究

目的:研究新型Akt抑制剂AZD5363对人肝癌HepG2和Huh7细胞活力、凋亡和自噬的影响,并探讨其抗肿瘤活性的分子机制。方法:采用不同浓度AZD5363作用于体外培养的HepG2和Huh7细胞,MTT法检测细胞活力;TUNEL标记法检测肝癌细胞凋亡的变化;Western blot实验分析细胞凋亡相关蛋白多腺苷二磷酸核糖聚合酶[poly(ADP-ribose) polymerase,PARP]及自噬标志蛋白LC3-II的表达水平;细胞转染GFP-LC3绿色荧光蛋白融合表达质粒检测细胞自噬。结果:AZD5363能够剂量依赖性地抑制HepG2和Huh7细胞活力,并通过促进PARP的切割而诱导肝癌细胞发生凋亡;肝癌细胞给予AZD5363后,细胞内GFP-LC3融合蛋白斑点增多,同时LC3-II的表达水平增加(P 0.05);当用氯喹阻断自噬后,AZD5363对肝癌细胞凋亡的诱导作用明显增强。结论:AZD5363可促进HepG2和Huh7细胞发生凋亡和保护性自噬。抑制自噬促进了AZD5363诱导的肝癌细胞凋亡。     

PI3K/Akt信号通路在脓毒症肾脏损伤诱导的自噬中的调节作用*

 目的：研究脓毒症造成肾脏损伤时的自噬情况以及磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B(Akt)信号通路的调节作用。方法：对大鼠盲肠进行结扎与穿刺（CLP）,对肾脏组织切片进行HE染色,并测定血清尿素氮和肌酐。通过Western blotting定量分析CLP大鼠肾脏损伤发生后不同时点自噬相关分子微管相关蛋白轻链3（LC3）Ⅰ/Ⅱ、beclin-1和Akt蛋白磷酸化的表达情况;体外用LPS诱导人近端肾小管上皮细胞株HK-2发生自噬,检测不同浓度LPS和不同刺激时间自噬相关分子LC3Ⅰ/Ⅱ和Akt蛋白磷酸化的表达情况;进一步使用PI3K抑制剂、Akt抑制剂和LPS刺激HK-2细胞观察自噬相关蛋白的表达情况及细胞的凋亡水平。结果：同对照组相比,CLP大鼠显微镜下可见肾损伤的典型病理改变,血清尿素氮和肌酐均有上升。CLP肾脏损伤发生后,自噬相关蛋白LC3Ⅰ/Ⅱ、beclin-1含量及Akt磷酸化水平均有上升。LPS刺激HK-2细胞后,随着刺激浓度的增加,p-Akt(308)表达量逐渐提高,而LC3Ⅰ/Ⅱ及p-Akt(472)的表达量在10 mg/L LPS刺激组最高。随着刺激时间的延长,p-Akt(308)表达量逐渐提高;LC3Ⅰ/Ⅱ表达量同p-Akt(472)在刺激8 h时最高;使用PI3K抑制剂及Akt抑制剂后,LPS诱导的LC3表达显著下调,HK-2细胞凋亡明显增加。结论：CLP肾脏损伤发生时可以诱导自噬发生, PI3K／Akt信号通路在其中发挥重要调节作用。     

GDC-0032抑制U251胶质瘤细胞活力并诱导DNA损伤

目的:探讨磷脂酰肌醇3-激酶(PI3K)抑制剂GDC-0032对人胶质瘤U251细胞活力、细胞周期和DNA损伤的影响。方法:GDC-0032处理U251细胞,MTT实验检测细胞活力;流式细胞术分析GDC-0032对细胞周期的影响;Western blot检测细胞内蛋白表达的变化;免疫荧光检测组蛋白γ-H2AX在细胞内的表达与分布。结果:GDC-0032剂量依赖性地抑制U251细胞活力;U251细胞经GDC-0032作用后,处于G1期的细胞数量明显增多,同时p27的表达水平增加,而细胞分裂周期蛋白2(Cdc2)的表达则受到抑制;在GDC-0032作用的细胞中,γ-H2AX焦点的生成和表达水平明显升高;另外,GDC-0032上调胶质瘤细胞中丝裂原活化蛋白激酶家族成员细胞外信号调节激酶(ERK)和c-Jun氨基末端激酶(JNK)的磷酸化水平,而survivin的表达则受到抑制。结论:GDC-0032能够通过抑制细胞活力和阻滞细胞于G1期而抑制U251细胞的增殖,并且诱导胶质瘤细胞发生DNA损伤;GDC-0032的抑癌活性与其调控ERK和JNK的活性及下调survivin的表达相关。     

聚ADP-核糖聚合酶-1在喉鳞状细胞癌细胞DNA氧化损伤及凋亡中的作用

目的 探究聚ADP-核糖聚合酶-1(PARP1)在喉鳞状细胞癌（LSCC）中的表达及其对LSCC细胞DNA氧化损伤与凋亡的影响。方法 收集40例患者的LSCC组织及癌旁组织样本,采用免疫组化染色和qRT-PCR检测LSCC组织与癌旁组织中PARP1的表达。采用不同浓度Menadione诱导LSCC细胞系Hep-2,MTT法检测不同浓度诱导下细胞增殖抑制率。将Hep-2细胞随机分为对照组（正常培养）、Men组（20μmol/L Menadione处理）、Men+PARP1抑制剂组（20μmol/L Menadione+10μmol/L PARP1抑制剂Olaparib共处理）。MTT法计算各组细胞增殖抑制率;流式细胞术检测各组细胞内活性氧（ROS）水平;细胞免疫荧光染色观察各组细胞DNA损伤标记分子γ-H2AX焦点生成情况;Western blot测定各组细胞γ-H2AX、DNA-PKcs、BRCA1、LIG4、Caspase-3及Caspase-9蛋白表达;Hoechst33258染色观察各组细胞凋亡情况。结果 LSCC组织中PARP1阳性表达率及PARP1 mRNA表达均明显高于癌旁...     

虫草素通过ERK1/2、Ezrin和Akt信号通路抑制胆囊癌细胞SNU-308的增殖和迁移

目的:探讨虫草素对胆囊癌细胞SNU-308增殖和迁移的影响及其分子机制。方法:MTT法和平板集落形成实验检测不同浓度虫草素对胆囊癌SNU-308细胞活力和集落形成能力的影响;Annexin V/PI双染法检测细胞凋亡率;Western blot法检测细胞凋亡和自噬相关蛋白以及Akt、ERK1/2和Ezrin蛋白的磷酸化水平;免疫荧光染色法检测细胞内LC3的表达水平;划痕愈合实验和Transwell实验检测虫草素对胆囊癌细胞迁移能力的影响;划痕实验检测Akt抑制剂和ERK1/2抑制剂及Ezrin基因沉默对细胞迁移能力的影响。结果:虫草素可显著抑制胆囊癌细胞的活力和集落形成能力(P0.05)。流式细胞术结果显示,虫草素可诱导胆囊癌细胞凋亡(P0.05)。Western blot结果显示,虫草素处理后Bcl-2表达降低,Bax、细胞色素C(Cyto C)、Fas、Fas L和cleaved caspase-3蛋白水平升高,自噬标识蛋白LC3-II/I比例和beclin 1表达上调(P0.05)。免疫荧光染色结果显示,虫草素处理后SNU-308细胞胞浆中LC3荧光颗粒的数量明显增多。划痕实验和Transwell实验结果显示虫草素可抑制细胞迁移(P0.05)。虫草素明显抑制Akt、ERK1/2和Ezrin蛋白的磷酸化水平(P0.05)。Ezrin基因沉默及Akti-1/2和GDC-0994均可抑制胆囊癌细胞的迁移作用(P0.05)。结论:虫草素通过诱导凋亡和自噬抑制胆囊癌细胞的增殖和迁移,其机制可能与调控ERK1/2,Ezrin和Akt信号通路有关。     

MCPH1在电离辐射诱导食管癌细胞DNA损伤通路中的作用

目的:研究MCPH1在电离辐射诱导食管癌细胞DNA损伤通路中的作用。方法:应用已构建的沉默MDC1的食管癌ECA109细胞株接受8 Gy电离辐射后1 h,检测相关因子核内斑点形成情况。构建沉默MCPH1的食管癌ECA109细胞株,检测此细胞株接受同样照射条件后相关因子核内斑点的形成情况。结果:成功构建沉默MCPH1的食管癌ECA109细胞;电离辐射使MDC1、MCPH1与γ-H2AX蛋白相互作用。沉默MDC1不影响γ-H2AX和MCPH1核内斑点的形成;沉默MCPH1使电离辐射导致的MDC1核内斑点减少,不影响γ-H2AX核内斑点的形成。结论:MCPH1在电离辐射诱导的DNA损伤通路中可能位于H2AX下游、MDC1上游,可以调控MDC1核内斑点形成。     

1Ubiquitylation of histones at sites of DNA damage

A compromised DNA damage response leads to genomic instability, which is one of the hallmarks of cancer. Conversely, the DNA damage response is activated in cancer cells subjected to chemo- and radio therapy. One of the most striking features of the cellular response to DNA double strand breaks (DSBs) is the accumulation of proteins into large and microscopically discernible structures in the vicinity of the lesions – the so-called ionizing radiation induced foci (IRIF). This accumulation process requires post-translational modifications of chromatin, such as the phosphorylation of the histone variant H2AX by the ATM kinase (γ-H2AX). Recently, we discovered an additional de novo histone modification at sites of DNA damage, namely the ubiquitylation of the core histones H2A and H2AX. This modification is carried out by a complex of proteins containing among other the Mdc1 adaptor protein and the RNF8 ubiquitin ligase. The complex is both assembled and targeted to the sites of DNA damage by multiple ATM phosphorylation events.
We have previously shown that proteins accumulate in IRIF in two distinct kinetic waves. The first wave comprises the γ-H2AX binding protein Mdc1 and proteins that physically interact with Mdc1, such as RNF8 and Nbs1. During the second wave, initiated 1–2 minutes after the first wave, proteins such as 53BP1 and BRCA1 accumulate. We can now show that histone ubiquitylation couples these two waves and that the presence of active RNF8 at sites of DNA damage is required for the accumulation of late factors such as 53BP1 and BRCA1.
The ability of cells to locally ubiquitylate histones determines the survival of cells to ionizing radiation and thus the outcome of the DNA damage response. Given the potential to pharmacologically inhibit this activity, our findings may eventually provide a means to modulate the ability of cancer cells to respond to chemo- and radio therapy.     

Machine learning extracts oncogenic-specific γ-H2AX foci formation pattern upon genotoxic stress

H2AX is a histone H2A variant that becomes phosphorylated upon genotoxic stress. The phosphorylated H2AX (γ-H2AX) plays an antioncogenic role in the DNA damage response and its foci patterns are highly variable, in terms of intensities and sizes. However, whether characteristic γ-H2AX foci patterns are associated with oncogenesis (oncogenic-specific γ-H2AX foci patterns) remains unknown. We previously reported that a defect in the acetyltransferase activity of TIP60 promotes cancer cell growth in human cell lines. In this study, we compared γ-H2AX foci patterns between TIP60 wild-type cells and TIP60 HAT mutant cells by using machine learning. When focused solely on the intensity and size of γ-H2AX foci, we extracted the TIP60 HAT mutant-like oncogenic-specific γ-H2AX foci pattern among all datasets of γ-H2AX foci patterns. Furthermore, by using the dimensionality reduction method UMAP, we also observed TIP60 HAT mutant-like oncogenic-specific γ-H2AX foci patterns in TIP60 wild-type cells. In summary, we propose the existence of an oncogenic-specific γ-H2AX foci pattern and the importance of a machine learning approach to extract oncogenic signaling among the γ-H2AX foci variations.     

Kinetics of gamma-H2AX induction and removal in bone marrow and testicular cells of mice after X-ray irradiation

Male germ cells have been shown to differ in their DNA damage response (DDR) with respect to somatic cells. In addition, DDR pathways are modulated along spermatogenesis, accompanying profound chromatin modifications. Histone H2AX phosphorylation is a fundamental step of DDR. Few data are available on the long-term kinetics of phosphorylated H2AX (γ-H2AX) after in vivo irradiation. We have investigated, by microscopic and flow cytometric immunochemistry, γ-H2AX induction and removal in testicular cells of irradiated mice, in comparison with bone marrow cells. In unirradiated testicular cells, much higher levels of γ-H2AX were measured by flow cytometry with respect to bone marrow cells. Irradiation induced a redistribution of γ-H2AX into discrete foci detectable by microscopy. In irradiated bone marrow, the percentage of labelled cells peaked at 1 h and rapidly declined, in agreement with data on in vitro cell lines. In contrast, spermatocytes and round spermatids showed persistent labelling until 48 h. During this time, in spermatids, topological changes were observed in γ-H2AX foci from a pattern of many uncountable dots to a pattern of few large spots. Observations of testicular sections confirmed this trend in the reduction of foci number in spite of substantially invariable percentages of labelled cells in the analysed timeframe. To assess whether γ-H2AX persistence in testicular cells was due to unrepaired DNA breaks, we performed comet assay and immunofluorescence analysis of Mdc1, a marker of DDR different from γ-H2AX. Comet assay showed that most breaks were repaired within 2 h. Forty-eight hours after irradiation, contrary to γ-H2AX foci that remained detectable in 80% of initially labelled cells, Mdc1 foci were observed in only 20-30% of cells. These data suggest that, at long times after irradiation, mechanisms additional to impairment of DNA break repair may account for the long persistence of γ-H2AX foci in male germ cells.     

Preferential localization of γH2AX foci in euchromatin of retina rod cells after DNA damage induction

DNA damage may lead to cell transformation, senescence, or death. Histone H2AX phosphorylation, immunodetected as γH2AX foci, is an early response to DNA damage persisting even after DNA repair. In cycling mammalian cells with canonical nuclear architecture, i.e., central euchromatin and peripheral heterochromatin, γH2AX foci map preferentially to euchromatin. Mice retina rods are G0 cells displaying an inverted nuclear architecture 28 days after birth (P28). Rod nuclei exhibit one or two central constitutive heterochromatin chromocenters encircled by facultative heterochromatin. Euchromatin resides at the nuclear periphery, extending to the equator in cells with two chromocenters. To assess the impact of chromatin relocation in the localization of DNA damage, γH2AX and TUNEL foci induced ex vivo by radiomimetic bleomycin were mapped in H3K4me3 immunolabeled P28 rod nuclei. A preferential localization of γH2AX foci in euchromatin was detected together with foci clustering. Besides, a decay of H3K4me3 signal at γH2AX foci sites was observed. TUNEL and γH2AX foci exhibited similar localization patterns in BLM-treated rod cells thus excluding curtailed access of anti-γH2AX antibodies to heterochromatin. Lack of γH2AX foci in rod chromocenters appears to be unrelated to the occurrence of mid-range foci movements. Foci clusters may arise through DNA double-strand break proximity, local non-directional chromatin movements or chromatin relaxation. H3K4me3 signal reduction at γH2AX foci could stem from local chromatin decondensation or downregulation of histone H4 methylation. The observed topology of DNA damage in retina-differentiated rods indicates that euchromatin is damage-prone, regardless of the canonical or inverted nuclear architecture of mammalian cells.     

阿帕替尼通过阻断VEGF通路增强胃癌放疗疗效

目的:探讨血管内皮生长因子(VEGF)受体2酪氨酸激酶抑制剂阿帕替尼对胃癌细胞株SGC-7901放疗疗效的影响及其可能机制。方法:试验设对照组、阿帕替尼组、单纯放疗组与联合组。CCK-8法检测细胞活力,流式细胞术分析细胞凋亡比例与细胞周期,免疫荧光染色观察细胞核内γ-H_2AX的表达,Western blot法检测细胞增殖和凋亡相关蛋白。结果:与阿帕替尼组或单纯放疗组相比,阿帕替尼联合X射线显著降低SGC-7901细胞的生长活力(P0.01),增殖相关蛋白p-PLCγ1和p-ERK1/2的水平下降;细胞凋亡比例明显升高(P0.01),凋亡相关蛋白PARP、cleaved caspase-9和cleaved caspase-3蛋白水平上调,Bcl-2表达下降;SGC-7901细胞核内γ-H_2AX焦点淬灭延迟,表明阿帕替尼干扰放射线诱导的DNA双链断裂的修复;SGC-7901 G_2期细胞比例显著增高(P0.01)。结论:阿帕替尼通过阻断VEGF通路增加胃癌细胞对X射线照射的敏感性。