顺铂通过抑制PI3K/AKT/mTOR信号通路诱导子宫内膜癌Ishikawa细胞自噬

目的：探讨顺铂对子宫内膜癌Ishikawa细胞自噬的影响,并初步探讨PI3K/AKT/mTOR信号通路在其中的作用。方法：透射电镜观察自噬泡形成,免疫荧光检测微管相关蛋白1轻链3融合蛋白II（microtubule-associated protein 1 light chain 3II,LC3II） 的荧光聚集情况。采用Westerblot检测mTOR通路中的PI3K、AKT及mTOR蛋白的表达。结果：顺铂（20μg/mL）能诱导Ishikawa细胞发生自噬,其中24h组明显高于12h组（P<0.05）;与对照组（20μg/mL-0h组）相比较,自噬相关蛋白-LC3的表达随着时间延长表达增加（P<0.05）。磷酸化AKT1、磷酸化mTOR及PI3Kp85蛋白表达水平随着时间延长表达下降。胰岛素样生长因子-1（insulin-like growth factors-1,IGF-1）与顺铂共培养组自噬小体及LC3蛋白表达少于顺铂组,但高于对照组。结论：顺铂可能通过抑制PI3K/AKT/mTOR信号通路,从而诱导子宫内膜癌细胞发生自噬。     

异莲心碱通过PI3K/Akt/mTOR信号通路影响结肠癌SW480细胞增殖、 凋亡和自噬

目的：探讨异莲心碱（Iso）通过PI3K/Akt/mTOR信号通路对结肠癌SW480细胞增殖、凋亡和自噬的影响。方法：用 10、20和40 μmol/L的Iso处理结肠癌SW480细胞,CCK-8法、流式细胞术和WB法分别检测Iso对细胞增殖活力、凋亡和自噬相关 蛋白LC3Ⅰ、LC3Ⅱ、p62表达的影响。然后,用20 μmol/L的Iso和25 μmol/L的PI3K激活剂740 Y-P分别处理SW480细胞,将细 胞分为对照组、740 Y-P组、Iso组和Iso+740 Y-P组,流式细胞术、WB法检测Iso和740 Y-P对各组细胞凋亡及细胞中LC3Ⅰ、LC3Ⅱ、 p62、PI3K、p-PI3K、 mTOR和p-mTOR蛋白表达的影响。结果：10、20和40 μmol/L的Iso处理后,SW480细胞增殖活力均显著下 降（均P<0.05）,细胞凋亡率均显著升高（均P<0.05）,LC3Ⅱ/LC3Ⅰ表达均显著上调（均P<0.05）,p26蛋白表达显著下调（P<0.05）。 Iso和740 Y-P处理后,与对照组相比,740 Y-P 组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达均显著下降（均P<0.05）,p26、p-PI3K/PI3K和 p-mTOR/mTOR 表达均显著升高（均 P<0.05）;Iso 组 细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达升高（均 P<0.05）,p26、p-PI3K/PI3K和 p-mTOR/mTOR 表达均显著下降（均 P<0.05）;与 740 Y-P 组相比,Iso+740 Y-P 组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达升高（P<0.05）, p26、p-PI3K/PI3K和p-mTOR/mTOR表达均显著下降（均P<0.05）;与Iso组相比,Iso+740 Y-P组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达下 降（均P<0.05）,p26、p-PI3K/PI3K和p-mTOR/mTOR表达均显著升高（均P<0.05）。结论：Iso通过抑制PI3K/Akt/mTOR信号通路 抑制结肠癌SW480细胞增殖并诱导细胞凋亡和自噬。     

Prucalopride通过促进自噬抑制胶质瘤细胞U251增殖

目的:研究Prucalopride对胶质瘤U251细胞增殖、自噬、凋亡的影响,并探讨其相关信号通路。方法:通过CCK8检测细胞增殖的变化;Transwell检测迁移和侵袭的变化;细胞流式实验、Western blot检测细胞凋亡的变化;Western blot检测自噬相关蛋白LC3、Beclin1、p62的表达;AKT/mTOR通路相关蛋白的变化。结果:CCK8显示Prucalopride显著抑制U251细胞的增殖（P＜0.05）;Transwell侵袭实验显示Prucalopride可以抑制脑胶质瘤细胞的迁移和侵袭（P＜0.05）;细胞流式实验显示Prucalopride促进U251细胞的凋亡（P＜0.05）,Prucalopride处理后细胞凋亡相关蛋白Bax、Active Caspase3水平升高,Bcl-2表达降低;自噬相关蛋LC3、Beclin1表达上调,p62表达下调;p-AKT蛋白和p-mTOR蛋白水平显著降低。结论:Prucalopride通过抑制AKT/mTOR信号通路激活自噬抑制U251细胞增殖和迁移,促进其凋亡。     

三七皂苷R1调控PI3K/AKT/mTOR信号通路诱导人下咽鳞状细胞癌FaDu细胞凋亡和自噬北大核心

目的:探讨三七皂苷R1(notoginsenoside R1,NGR1)对人下咽鳞状细胞癌(hypopharyngeal squamous cell carcinoma,HSCC)FaDu细胞凋亡以及自噬的影响,并对其涉及的信号通路进行研究。方法:75μmol/L、150μmol/L、300μmol/L NGR1作用于FaDu细胞24 h后,采用MTT检测细胞增殖能力;流式细胞术检测细胞凋亡;自噬双标腺病毒检测自噬流;Western blot检测自噬相关蛋白LC3Ⅱ/LC3Ⅰ以及PI3K/AKT/mTOR信号通路相关蛋白表达水平。结果:NGR1能够抑制FaDu细胞的增殖并促进细胞凋亡;NGR1可诱导FaDu细胞自噬,并呈一定浓度依赖性;Western blot结果显示,NGR1作用于Fa Du细胞24 h后,LC3Ⅱ表达明显增加,而p-PI3K、p-AKT、p-m TOR表达相较于Control组明显下降。结论:NGR1可抑制Fa Du细胞增殖,诱导细胞凋亡与自噬,其机制可能与抑制PI3K/AKT/m TOR信号通路有关。     

鸦胆子油乳剂对食管鳞状细胞癌TE-1细胞增殖、凋亡和自噬的影响及其可能的机制

目的：探讨鸦胆子油乳剂（BJOE）对食管鳞状细胞癌TE-1细胞增殖、凋亡和自噬的影响及其可能的机制。方法：按干预措施的不同,将TE-1细胞分为对照组、RAPA（自噬激动剂）组、740Y-P（PI3K激活剂）组、BJOE 组、BJOE+RAPA组和BJOE+740Y-P 组。采用FCM、克隆形成、Transwell 实验检测细胞凋亡、增殖、迁移和侵袭能力,qPCR 法检测细胞中PI3K、Akt、mTOR、LC3Ⅰ、LC3Ⅱ、p62、Beclin 1、caspase-3的mRNA表达,WB法检测PI3K、Akt、mTOR及其磷酸化、LC3Ⅱ/Ⅰ、p62、Beclin 1、caspase-3的蛋白表达。结果: 与对照组比较,RAPA组和BJOE 组细胞凋亡率均显著升高（均P<0.01）,细胞克隆形成率、迁移和侵袭能力均显著降低（均P<0.01）,细胞中PI3K、Akt、mTOR 的mRNA 和蛋白磷酸化水平均显著降低（均P<0.05）,p62的mRNA 和蛋白水平显著降低（均P<0.01）,LC3Ⅱ/Ⅰ、Beclin 1和caspase-3的mRNA和蛋白水平显著升高（均P<0.05）,740Y-P 组的结果则相反（均P<0.05）;与RAPA组或740Y-P 组比较,BJOE+RAPA组或BJOE+740Y-P 组细胞凋亡率显著升高（均P<0.01）,克隆形成率、细胞侵袭和迁移能力显著降低（均P<0.01）,PI3K、Akt、mTOR的mRNA和蛋白磷酸化水平均显著降低（均P<0.05）,p62 的mRNA和蛋白水平均显著降低（均P<0.05）,LC3Ⅱ/Ⅰ、Beclin 1 和caspase-3 mRNA和蛋白水平显著升高（均P<0.05）。结论: BJOE显著抑制TE-1细胞增殖、迁移、侵袭并促进细胞凋亡与自噬,其机制可能与抑制PI3K/Akt/mTOR信号通路的激活有关。     

柴胡皂苷d通过AKT/mTOR信号通路调控胶质瘤C6细胞自噬

目的 探讨柴胡皂苷d在胶质瘤C6细胞自噬中的作用及其机制。方法 分别以终浓度为0 μmol/L（Control组）、2 μmol/L、4 μmol/L、6 μmol/L、8 μmol/L、10 μmol/L、12 μmol/L的柴胡皂苷d作用胶质瘤C6细胞后,用CCK⁃8检测细胞增殖能力,后续分别采用克隆形成实验、划痕实验检测柴胡皂苷d（8 μmol/L）对胶质瘤C6细胞增殖能力和细胞迁移能力的影响,Western blot和qRT⁃PCR检测胶质瘤C6细胞中自噬相关蛋白Beclin1、LC3和AKT/mTOR信号通路相关蛋白及基因的表达情况。结果 与Control组比较,各浓度柴胡皂苷d均能抑制胶质瘤C6细胞增殖且增殖抑制率随着药物浓度的升高而升高（均P<0.01）。8 μmol/L柴胡皂苷d作用24 h后,与Control组比较,胶质瘤C6细胞的克隆形成数目显著减少［（479.33±30.66）个 vs （258.66±73.35）个,P<0.01］,细胞迁移率显著降低［（70.83±4.29）% vs （19.47±1.71）%,P<0.001］,自噬相关蛋白Beclin1和LC3的蛋白及mRNA表达水平均显著升高（均P<0.01）,而AKT和mTOR信号蛋白及mRNA表达水平均降低（均P<0.01）。结论 柴胡皂苷d可能通过抑制AKT/mTOR信号通路诱导胶质瘤C6细胞发生自噬并抑制其增殖与迁移。     

CHKA基因沉默对胶质瘤细胞生物学行为的影响及其机制

背景与目的：胶质瘤是颅内常见的原发性恶性肿瘤之一,但发病机制不明,胆碱激酶A（choline kinase A,CHKA）与胶质瘤的恶性程度呈正相关,但具体作用途径尚不清楚。探讨下调CHKA基因的表达对胶质瘤细胞系U87和U251增殖、凋亡和迁移的影响及其作用机制。方法：使用慢病毒感染胶质瘤细胞系U87和U251,同时设置阴性对照组（shNC组）和空白对照组（control组）。为研究磷脂酰肌醇3-激酶（phosphoinositide 3-kinase,PI3K）/蛋白激酶B（protein kinase B,AKT）信号转导通路与CHKA的相互作用,另设DMSO组和LY294002组。采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）检测CHKA基因mRNA的表达水平,采用细胞计数试剂盒-8（cell counting kit-8,CCK-8）法检测细胞增殖能力,采用流式细胞术检测细胞周期和凋亡,采用transwell实验检测细胞侵袭能力,采用划痕实验检测细胞迁移能力,采用蛋白质印迹法（Western blot）检测胶质瘤细胞中CHKA、PI3K、p-PI3K、AKT和p-AKT蛋白的水平。结果：Western blot结果显示,与control组和shNC组相比,shCHKA组胶质瘤细胞中p-PI3K、p-AKT和CHKA蛋白水平同步降低（P＜0.05）,PI3K、AKT蛋白水平无明显改变。在使用通路抑制剂后CHKA的表达量在control组和shNC组之间差异无统计学意义（P＞0.05）。与此同时,shCHKA组胶质瘤细胞的侵袭、迁移能力均弱于control组和shNC组（P＜0.05）,细胞凋亡率明显增加,细胞周期主要停滞于G ₂ 期,细胞增殖明显降低（P＜0.05）。结论：CHKA基因可能是通过调控PI3K/AKT信号转导通路,从而影响胶质瘤细胞增殖、凋亡、迁移和侵袭等生物学行为,且CHKA对PI3K/AKT信号转导通路的调控是单向的,PI3K/AKT信号转导通路中蛋白水平的降低对CHKA的表达无反馈作用。     

内质网激动剂调控PI3K/AKT/mTOR通路诱导人小细胞肺癌NCI-H446细胞凋亡

目的 检测内质网应激能否通过PI3K/AKT/mTOR通路对人小细胞肺癌NCI-H446细胞凋亡产生作用.方法 采用MTT法检测不同浓度衣霉素对人小细胞肺癌NCI-H446的细胞毒性,Annexin V/PI检测药物作用下人小细胞肺癌NCI-H446细胞凋亡情况,Western Blot检测PI3K/AKT/mTOR通路相关蛋白的表达.结果 衣霉素可抑制人小细胞肺癌NCI-H446细胞的活性,且呈时间和浓度依赖性.衣霉素能够激活内质网应激,抑制PI3K/AKT/mTOR信号通路,使PI3K、AKT、mTOR蛋白磷酸化下调,诱导细胞凋亡.结论 内质网激动剂能够调控PI3K/AKT/mTOR通路诱导人小细胞肺癌NCI-H446细胞凋亡.     

芹菜素诱导胃癌SGC-7901细胞自噬及其对凋亡 的影响

目的：探讨芹菜素（API）是否诱导胃癌SGC-7901细胞发生自噬及其对细胞凋亡的影响。方法：采用不同浓度（0、20、40、60、80 μmol/L）的API处理胃癌SGC-7901细胞24 h,MTT法检测API对细胞生长的抑制情况。分别采用不同浓度（0、20、40、60、80 μmol/L）API处理细胞24 h,及60 μmol/L的API处理不同时间（0、12、24、36、48 h）,Western blot检测自噬标志蛋白微管相关蛋白l轻链3（LC3）、Beclin-1和磷脂酰肌醇-3-激酶（PI3K）/Akt/mTOR信号通路中关键分子p-Akt、p-mTOR的表达;电子显微镜下观察细胞自噬情况。细胞经自噬抑制剂氯喹（CQ）或3-甲基腺苷（3-MA）预处理后再加入API,Western blot检测聚二磷酸腺苷-核糖体聚合酶（PARP）的表达;流式细胞术流检测细胞凋亡率。结果：API对胃癌SGC-7901细胞生长具有明显抑制作用并呈剂量-效应关系。随着API浓度和作用时间的增加,SGC-7901细胞LC3Ⅱ与Beclin-1蛋白表达均增加,各剂量组与对照组相比较差异均具有统计学意义（P < 0.05）;API处理后的细胞逐渐出现自噬囊泡和自噬溶酶体;p-Akt、p-mTOR蛋白表达降低。采用自噬抑制剂抑制细胞自噬后,除API组LC3Ⅱ与Beclin-1蛋白表达明显高于对照组外（P < 0.05）,各组蛋白表达无显著差异。API+CQ组和API+3-MA组的PARP蛋白剪切片段表达及凋亡率均高于对照组和API组（P < 0.05）。结论：API可以诱导胃癌SGC-7901细胞发生自噬,其机制可能与抑制PI3K/Akt/mTOR信号通路关键分子p-Akt、p-mTOR活性有关;抑制API诱导的自噬能够增强API的凋亡诱导效应,从而增强对胃癌SGC-7901细胞的杀伤作用。     

熊果酸对胃癌细胞株MGC-803 凋亡和自噬的调控及其作用机制

[摘要] 目的:观察熊果酸(UA)对胃癌细胞株MGC-803 自噬和凋亡的调控作用,并探讨UA基于PI3K/AKT/mTOR信号通路诱发MGC-803 细胞自噬的机制。方法:体外培养人胃癌细胞株MGC-803,分为空白对照组、UA干预组和UA+3-MA组。采用流式细胞术检测各组细胞凋亡水平,双荧光mRFP-eGFP-LC3 质粒转染细胞法检测各组细胞自噬发生情况,qPCR实验检测各组细胞LC3B、BAX、Bcl-2 mRNA的表达水平,WB实验检测各组细胞Ⅰ型PI3K、p-AKT、p-mTOR、ULK1、LC3B、BAX、Bcl-2 蛋白的表达水平。结果: 与空白对照组相比,UA干预组细胞凋亡率显著增加（P<0.05）;与UA干预组相比,UA+3-MA组细胞凋亡率显著降低（P<0.05）。双荧光mRFP-eGFP-LC3 质粒转染法显示,与空白对照组相比,UA 干预组绿色和红色荧光亮点数均显著增加（P<0.05）;与UA干预组相比,UA+3-MA组绿色和红色荧光亮点数均显著减少（P<0.05）。qPCR和WB实验结果显示,与空白对照组相比,UA干预组BAX和LC3B mRNA和蛋白以及ULK1 蛋白表达水平均显著上调,Bcl-2 基因和蛋白表达水平以及Ⅰ型PI3K、p-AKT、p-mTOR蛋白表达水平均显著下调（均P<0.05）;与UA干预组相比,UA+3-MA组BAX、LC3B mRNA和蛋白表达水平显著下调,Bcl-2 基因和蛋白表达水平显著上调（均P<0.05）,Ⅰ型PI3K、p-AKT、p-mTOR 和ULK1 蛋白表达水平无显著差异（P>0.05）。结论: UA诱导的自噬可以促进胃癌细胞MGC-803 的凋亡,其机制可能与UA参与调控PI3K/AKT/mTOR信号通路相关蛋白表达有关。     

Effects of 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione on autophagy and the PI3K/AKT/mTOR signaling pathway in human cholangiocarcinoma QBC939 cells

Background2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) has been reported to have good antitumor effects. The aim of this study was to investigate whether DMDD induces apoptosis and autophagy in human cholangiocarcinoma (CCA) QBC939 cells and determine its effect on the PI3K/AKT/mTOR signaling pathway.MethodsQBC939 cells were cultured in vitro and changes in cell viability were detected by the Cell Counting Kit (CCK8) assay after treatment with different concentrations of DMDD for 24, 48, and 72 h. The cells were divided into control and DMDD-treated groups (treated concentrations were 10, 15, and 20 µM/L), and the cell cycle, apoptosis, and autophagic vesicles were assessed. The expression levels of PI3K, AKT, mTOR, microtubule-associated protein 1 light chain 3 beta (LC3-II)/I, Beclin-1, and P62 were detected by Western blot. A xenograft mouse model was constructed to detect the effect of DMDD on CCA.ResultsThe experimental results showed that DMDD was able to inhibit proliferation, migration, and invasion and induce cell cycle arrest and autophagy of QBC939 cells. In addition, DMDD decreased the protein expression of PI3K, AKT, and mTOR and increased the expression of LC3-II/I, Beclin-1, and P62. In mice, DMDD was able to inhibit the growth of tumors.ConclusionsDMDD inhibits CCA cell viability and induces cell cycle arrest and autophagy by a mechanism that may be related to the downregulation of the PI3K/AKT/mTOR signaling pathway.     

Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors

The mammalian target of rapamycin (mTOR) is a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway and a central modulator of cell proliferation in malignant gliomas. Therefore, the targeting of mTOR signaling is considered a promising therapy for malignant gliomas. However, the mechanisms underlying the cytotoxic effects of a selective mTOR inhibitor, rapamycin, on malignant glioma cells are poorly understood. The purpose of this study was thus to elucidate how rapamycin exerts its cytotoxic effects on malignant glioma cells. We showed that rapamycin induced autophagy but not apoptosis in rapamycin-sensitive malignant glioma U87-MG and T98G cells by inhibiting the function of mTOR. In contrast, in rapamycin-resistant U373-MG cells, the inhibitory effect of rapamycin was minor, although the phosphorylation of p70S6 kinase, a molecule downstream of mTOR, was remarkably inhibited. Interestingly, a PI3K inhibitor, LY294002, and an Akt inhibitor, UCN-01 (7-hydroxystaurosporine), both synergistically sensitized U87-MG and T98G cells as well as U373-MG cells to rapamycin by stimulating the induction of autophagy. Enforced expression of active Akt in tumor cells suppressed the combined effects of LY294002 or UCN-01, whereas dominant-negative Akt expression was sufficient to increase the sensitivity of tumor cells to rapamycin. These results indicate that rapamycin exerts its antitumor effect on malignant glioma cells by inducing autophagy and suggest that in malignant glioma cells a disruption of the PI3K/Akt signaling pathway could greatly enhance the effectiveness of mTOR inhibitors.     

Radix Tetrastigma Extracts Enhance the Chemosensitivity in Triple-Negative Breast Cancer Via Inhibiting PI3K/Akt/mTOR-Mediated Autophagy

ObjectiveDrug resistance in tumors is one of the major factors that leads to chemotherapy failure. This study aims to investigate the effect of Radix Tetrastigma extracts (RTEs) on Taxol-induced autophagy and the chemosensitivity against drug resistance in triple-negative breast cancer (TNBC).MethodsTaxol-resistant MDA-MB-468 (MDA-MB-468/Taxol) cells were induced and treated with RTEs and/or Taxol. Mice were subcutaneously inoculated with MDA-MB- 468/Taxol cells to establish xenograft models. The associated protein levels were measured by western blotting. Flow cytometry, CCK-8 and EdU assay were performed to detect cell apoptosis, viability, and proliferation, respectively.ResultsIn MDA-MB-468/Taxol cells, RTEs & Taxol treatment increased cell apoptosis, reduced cell viability and proliferation, up-regulated anti-autophagy marker LC3I/LC3II ratio, and enhanced mTOR level. With RTEs & Taxol treatment, mTOR silencing downregulated LC3I/LC3II ratio, increased cell viability and proliferation, and reduced cell apoptosis, while mTOR overexpression showed the opposite results. PI3K inhibitor reduced AKT and mTOR levels, and the effects on cell activities were similar to the results of mTOR silencing. After RTEs & Taxol injection, xenograft tumor was smaller, and AKT, mTOR, LC3I/LC3II ratio and apoptotic marker cleaved caspase-3 were increased.ConclusionRTEs enhanced the chemosensitivity of resistant TNBC cells to Taxol through inhibiting PI3K/Akt/mTOR-mediated autophagy.MicroRTEs exerted anti-tumor effects in various cancers, and this study determined its role in TNBC. Taxol-resistant MDA-MB-468 cells were induced and xenograft models were established. We found that RTEs inhibited autophagy of MDA-MB-468/Taxol cells and reduced tumor growth. Inhibition of PI3K/Akt/mTOR pathway promoted autophagy of MDA-MB-468/Taxol cells. We may provide a new potential strategy for TNBC treatment.     

马鞭草总黄酮通过AKT/mTOR信号通路调控自噬抑制肝癌细胞增殖

目的:分析马鞭草总黄酮(total flavonoids of verbena officinalis L.,TFV)对肝癌细胞自噬和增殖的影响,并探讨其机制。方法:MDC染色法检测自噬泡水平,Western印迹检测自噬相关蛋白水平,运用3-甲基腺嘌呤(3-MA)、雷帕霉素(RAPA)研究TFV对自噬、细胞增殖的影响,MTT法检测细胞存活率。裸鼠成瘤实验验证TFV对裸鼠体内瘤体的瘤重、瘤体积及自噬相关蛋白水平的影响。结果:TFV能增加HepG2、Huh-7细胞中MDC染色标记的荧光颗粒,并伴随浓度的上调明显增加,促进HepG2、Huh-7细胞中自噬蛋白Beclin-1、LC3 II/LC3 I水平表达,降低p-AKT/AKT、p-mTOR/mTOR水平(P<0.05)。与TFV组比较,TFV+3-MA组抑制率、LC3 II/LC3 I明显降低,p-mTOR/mTOR明显增加(P<0.05);与TFV组比较,TFV+RAPA组抑制率、LC3 II/LC3 I明显增加,p-mTOR/mTOR明显降低(P<0.05);与TFV组比较,TFV+3-MA组裸鼠瘤体LC3 II/...