防己诺林碱诱导人乳腺癌细胞MDA-MB-231凋亡的作用机制

目的 探讨防己诺林碱(FAN)对三阴性乳腺癌(TNBC)的抗肿瘤机制.方法 体外细胞培养人乳腺癌细胞MDA-MB-231,Alamar-Blue法检测FAN对人乳腺癌细胞MDA-MB-231的半抑制浓度(IC50);6孔板检测细胞迁移情况;细胞流式技术检测细胞凋亡情况;Western Blot检测磷脂酰肌醇-3羟基激酶(PI3K)、蛋白激酶B(AKT)、哺乳类动物雷帕霉素靶蛋白(mTOR)及磷酸化PI3K、AKT、mTOR蛋白表达.结果 FAN可抑制人乳腺癌细胞MDA-MB-231的活力(IC50为6.25μmol/L),抑制人乳腺癌细胞MDA-MB-231的迁移能力,且随着FAN浓度升高,抑制作用明显.FAN可以诱导人乳腺癌细胞MDA-MB-231凋亡,且随着FAN浓度升高,细胞凋亡率增高,同时FAN还可以下调PI3K、AKT、mTOR及磷酸化PI3K、AKT、mTOR蛋白的表达,随药物浓度的升高,其蛋白表达降低.结论 FAN可通过下调TNBC MDA-MB-231细胞凋亡PI3K/AKT/mTOR信号通路,抑制TNBC细胞的增殖、迁移,诱导细胞凋亡,可能具有抗肿瘤作用.     

小分子抑制剂Torin-2抑制小细胞肺癌NCI-H446细胞增殖和促进细胞凋亡的作用和分子机制

目的:研究哺乳动物雷帕霉素（mammalian target of rapamycin,mTOR）新型小分子抑制剂Torin-2对人小细胞肺癌NCI-H446细胞增殖及凋亡的影响并探讨其机制。方法:体外培养并取对数生长期人小细胞肺癌NCI-H446细胞,分别给予不同浓度Torin-2或1 μmol/L处理不同时间,然后于镜下观察细胞离巢凋亡,通过克隆形成实验检验细胞生长抑制,采用MTS比色法测定吸光度值并计算相对细胞活性,流式细胞术检测细胞周期,免疫印迹法检测mTOR、p-mTOR（Ser2448）、p-Akt（Ser473）、Cyclin-B1、Cyclin-D1、Cyclin-E1、Cleaved Caspase-3等相关蛋白表达。结果:与对照组相比,Torin-2可有效抑制小细胞肺癌NCI-H446细胞增殖及相对细胞活性（P＜0.05）;诱导细胞周期阻滞（P＜0.05）;抑制p-mTOR、p-Akt磷酸化激活,降低Cyclin-D1、Cyclin-E1等细胞周期蛋白表达并诱导Cleaved Caspase-3蛋白激酶切割激活。结论:Torin-2可能通过抑制Akt-mTOR相关信号通路和激活凋亡相关蛋白诱导人小细胞肺癌NCI-H446细胞增殖抑制并促进其凋亡。     

山柰酚通过Met/PI3K/Akt/mTOR通路诱导非小细胞肺癌NCI-H1650细胞发生自噬进而影响其增殖

目的：探讨山柰酚诱导人非小细胞肺癌（NSCLC）NCI-H1650细胞发生自噬及其机制。方法：常规培养NCI-H1650细胞，用不同浓度山柰酚处理细胞，用CCK-8法、MTT法以及EdU法检测山柰酚对NCI-H1650细胞增殖能力的影响，用自噬双标记腺病毒mCherry-EGFR-LC3感染实验检测山柰酚对NCI-H1650细胞发生自噬的影响，用WB法检测山柰酚处理后NCI-H1650细胞中自噬相关蛋白及Met/PI3K/Akt/mTOR信号通路相关蛋白的表达，用qPCR法检测山柰酚处理后NCI-H1650细胞中Met mRNA的表达。采用荧光素酶标记A549-luc细胞建立裸鼠移植瘤模型后用山柰酚进行处理，用活体动物成像技术观察移植瘤生长情况，用WB法检测移植瘤组织中自噬相关蛋白以及Met/PI3K/Akt/mTOR信号通路相关蛋白的表达。结果：山柰酚能显著抑制NCI-H1650细胞的增殖能力（P<0.05），山柰酚处理后NCI-H1650细胞内的自噬小体数量明显增加（P<0.05）、自噬标志蛋白LC3B和beclin1表达均明显上调（均P<0.05）、P62表达明...     

胃癌细胞SGC7901凋亡的PI3K/Akt通路介导作用及雷帕霉素的作用机制分析

目的：分析雷帕霉素对胃癌细胞中PI3K/Akt蛋白表达的影响作用,以为临床治疗提供参考。方法将生长状态良好的胃癌细胞SGC7901随机分为4组：对照组,雷帕霉素低剂量组、中剂量、高剂量组（n=8）。采用DAPI染色法分析各组细胞的凋亡情况;采用免疫印迹法和Real-time PCR检测PI3K/AKT信号通路中相关蛋白PI3K、AKT、mTOR蛋白及mRNA的表达。结果雷帕霉素低、中、高剂量组均可抑制细胞的增殖,差异具有统计学意义（P﹤0.05）;雷帕霉素下调了胃癌细胞中PI3K、AKT、mTOR蛋白及mRNA的表达,且与对照组比较,差异均有统计学意义（P﹤0.05）。结论雷帕霉素主要通过抑制PI3K、AKT及mTOR的过表达发挥肿瘤抑制作用。     

8-溴-7-甲氧基白杨素诱导人小细胞肺癌NCI-H446细胞凋亡及其机制的探讨

目的:观察8-溴-7-甲氧基白杨素(BrMChR)诱导人小细胞肺癌(NCI-H446)细胞凋亡作用,并探讨其与Noxa/Mcl-1比值增高的关系.方法:以体外培养的NCI-H446细胞和正常小鼠肺上皮CHL细胞为研究对象.MTT比色法测定NCI-H446细胞和CHL细胞活性;PI染色流式细胞术(FCM)分析NCI-H446细胞凋亡率;FITC标志间接免疫荧光FCM检测NCI-H446细胞NOXA, Mcl-1和Caspase-3蛋白表达水平.结果:MTT分析结果显示BrMChR明显抑制NCI-H446细胞活性,呈浓度依赖性,而对CHL细胞活性的抑制作用弱,其选择指数为46;PI染色FCM分析显示0.3、3.0和30.0 μmol/L的BrMChR作用于NCI-H446细胞48 h后,其凋亡率分别为8.83%、23.7%和34.9%, 其中BrMChR 3.0和30.0 μmol/L组较ChR 30.0 μmol/L (19.7%)高;间接免疫荧光FCM分析结果显示,凋亡相关基因Noxa表达上调和Mcl-1表达下调,Noxa/Mcl-1比值升高,Caspase-3活化.结论:BrMChR具有诱导人小细胞肺癌细胞凋亡的作用,其作用机制可能与其增高Noxa/Mcl-1比值,激活Caspase-3蛋白表达有关.     

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。     

青藤碱诱导肺癌NCI-H460细胞凋亡及其机制的实验研究

目的:探究青藤碱(SIN)诱导肺癌NCI-H460细胞凋亡的作用机制。方法:四甲基偶氮唑蓝(MTT)法测定SIN对肺癌NCI-H460细胞生长的影响;Western blot测定Bcl-2,Bax蛋白的表达;用SIN、PI3K/Akt和MAPK/ERK信号通路抑制剂干预NCI-H460细胞,流式细胞术检测细胞的凋亡率。结果:SIN对肺癌NCI-H460细胞生长有抑制作用,呈时间、浓度依赖性;SIN可以使NCI-H460细胞Bcl-2表达减低,Bax增强;SIN与PI3K/Akt和MAPK/ERK信号通路抑制剂联用后可协同增加NCI-H460细胞凋亡(P<0.05)。结论:SIN可以抑制肺癌NCI-H460细胞的生长,能改变其Bax,Bcl-2蛋白的表达,与PI3K/Akt和MAPK/ERK信号通路抑制剂联用可协同发挥促进肺癌细胞凋亡作用,可望成为新的肺癌治疗药物。     

青藤碱诱导肺癌NCI-H460细胞凋亡及其机制的实验研究

目的：探究青藤碱（SIN）诱导肺癌NCI-H460细胞凋亡的作用机制。方法：四甲基偶氮唑蓝（MTT）法测定SIN对肺癌NCI-H460细胞生长的影响;Western blot测定Bcl-2,Bax蛋白的表达;用SIN、PI3K/Akt和MAPK/ERK信号通路抑制剂干预NCI-H460细胞,流式细胞术检测细胞的凋亡率。结果：SIN对肺癌NCI-H460细胞生长有抑制作用,呈时间、浓度依赖性;SIN可以使NCI-H460细胞Bcl-2表达减低,Bax增强;SIN与PI3K/Akt和MAPK/ERK信号通路抑制剂联用后可协同增加NCI-H460细胞凋亡（P〈0.05）。结论：SIN可以抑制肺癌NCI-H460细胞的生长,能改变其Bax,Bcl-2蛋白的表达,与PI3K/Akt和MAPK/ERK信号通路抑制剂联用可协同发挥促进肺癌细胞凋亡作用,可望成为新的肺癌治疗药物。     

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。     

非小细胞肺癌大分割放疗敏感性与PI3K/AKT/mTOR信号通路研究进展

常规分割放疗在中晚期非小细胞肺癌治疗中疗效有限,大分割放疗地位越来越重要,但仍有相当部分肿瘤细胞具有放射线抗拒,其分子机制未明。PI3K/AKT/mTOR信号通路与非小细胞肺癌常规分割放疗抗拒有关,但与大分割放疗抗拒关系尚未明确。调控PI3K/AKT/mTOR信号通路的基因表达及蛋白磷酸化水平有望增加NSCLC肿瘤细胞对大分割放疗的敏感性,并可能达到逆转放疗抗拒的效果;在PI3K/AKT/mTOR信号通路的诸多基因中,有望从临床样本中筛选获得预测NSCLC大分割放疗疗效的分子标记。      

Inhibition of PI3K/Akt/mTOR signaling pathway enhances the sensitivity of the SKOV3/DDP ovarian cancer cell line to cisplatin in vitro

The activation of the PI3K/AKT/m TOR pathway plays a key role in ovarian cancer tumorigenesis, progression and chemotherapy resistance. This study aimed to explore the possible mechanism that PI-103, a dual inhibitor of phosphatidylinositide 3-kinase and m TOR, enhances the sensitivity of SKOV3/DDP ovarian cancer cell line to cisplatin chemotherapy. The results showed that PI-103 could significantly increase the sensitivity of SKVO3/DDP cells to cisplatin through inhibiting the activation of PI3K/Akt/m TOR signaling pathway and inducing cell cycle arrest and apoptosis.     

Targeting the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway: An emerging treatment strategy for squamous cell lung carcinoma

Squamous cell lung carcinoma accounts for approximately 30% of all non-small cell lung cancers (NSCLCs). Despite progress in the understanding of the biology of cancer, cytotoxic chemotherapy remains the standard of care for patients with squamous cell lung carcinoma, but the prognosis is generally poor. The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is one of the most commonly activated signaling pathways in cancer, leading to cell proliferation, survival, and differentiation. It has therefore become a major focus of clinical research. Various alterations in the PI3K/AKT/mTOR pathway have been identified in squamous cell lung carcinoma and a number of agents targeting these alterations are in clinical development for use as single agents and in combination with other targeted and conventional treatments. These include pan-PI3K inhibitors, isoform-specific PI3K inhibitors, AKT inhibitors, mTOR inhibitors, and dual PI3K/mTOR inhibitors. These agents have demonstrated antitumor activity in preclinical models of NSCLC and preliminary clinical evidence is also available for some agents. This review will discuss the role of the PI3K/AKT/mTOR pathway in cancer and how the discovery of genetic alterations in this pathway in patients with squamous cell lung carcinoma can inform the development of targeted therapies for this disease. An overview of ongoing clinical trials investigating PI3K/AKT/mTOR pathway inhibitors in squamous cell lung carcinoma will also be included.     

吉西他滨对肺癌细胞凋亡及PI3K/AKT信号通路的影响

目的探讨吉西他滨对肺癌细胞凋亡及PI3K/AKT信号通路的影响。方法选取对数生长期的肺癌NCI-H292细胞随机分为吉西他滨组、阿霉素组和对照组,分别采用7μmol/L的吉西他滨、阿霉素与生理盐水处理,采用CCK-8法检测细胞增殖,Transwell小室检测细胞迁移与侵袭,Annexin V-PI双标记法检测细胞凋亡,Western blot实验检测PI3K和AKT蛋白表达。结果细胞处理后24h和48h,吉西他滨组和阿霉素组的细胞增殖指数均低于对照组,吉西他滨组低于阿霉素组,差异均有统计学意义(均P <0.05)。细胞处理后24h和48h,吉西他滨组和阿霉素组的细胞迁移与侵袭指数均低于对照组,吉西他滨组低于阿霉素组,差异均有统计学意义(均P <0.05)。细胞处理后24h和48h,吉西他滨组和阿霉素组的细胞凋亡指数均高于对照组,吉西他滨组高于阿霉素组,差异均有统计学意义(均P <0.05)。细胞处理后24h和48h,吉西他滨组和阿霉素组PI3K和AKT蛋白表达水平均低于对照组,吉西他滨组低于阿霉素组,差异均有统计学意义(均P <0.05)。结论吉西他滨可促进肺癌...     

细胞角蛋白13通过PTEN抑制PI3K/AKT/mTOR通路增强鼻咽癌HNE1细胞放疗敏感性

目的:探讨细胞角蛋白13(cytokeratin13,CK13)对鼻咽癌HNE1细胞放疗敏感性的影响及其作用机制.方法:将HNE1细胞分为对照组、anti-CK13#a组及anti-CK13#b组(敲减CK13)、对照组+西罗莫司处理组(100nmol/L的西罗莫司处理1 h)、anti-CK13#a+西罗莫司处理组(...     

Combination of PTEN and γ-Ionizing Radiation Enhances Cell Death and G2/M Arrest Through Regulation of AKT Activity and p21 Induction in Non–Small-Cell Lung Cancer Cells

PURPOSE: To identify the role of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) during gamma-ionizing radiation (gamma-IR) treatment for non-small-cell lung cancer cells. METHODS AND MATERIALS: Wild-type PTEN or mutant forms of PTEN plasmids were transfected to construct stable transfectants of the NCI-H1299 non-small-cell lung cancer cell line. Combined effects of PTEN expression and IR treatment were tested using immunoblot, clonogenic, and cell-counting assays. Related signaling pathways were studied with immunoblot and kinase assays. RESULTS: At steady state, stable transfectants showed almost the same proliferation rate but had different AKT phosphorylation patterns. When treated with gamma-IR, wild-type PTEN transfectants showed higher levels of cell death compared with mock vector or mutant transfectants, and showed increased G(2)/M cell-cycle arrest accompanied by p21 induction and CDK1 inactivation. NCI-H1299 cells were treated with phosphosinositide-3 kinase (PI3K)/AKT pathway inhibitor (LY29002), resulting in reduced AKT phosphorylation levels. Treatment of NCI-H1299 cells with LY29002 and gamma-IR resulted in increased cell-cycle arrest and p21 induction. Endogenous wild-type PTEN-containing NCI-H460 cells were treated with PTEN-specific siRNA and then irradiated with gamma-IR: however reduced PTEN levels did not induce cell-cycle arrest or p21 expression. CONCLUSIONS: Taken together, these findings indicate that PTEN may modulate cell death or the cell cycle via AKT inactivation by PTEN and gamma-IR treatment. We also propose that a PTEN-PI3K/AKT-p21-CDK1 pathway could regulate cell death and the cell cycle by gamma-IR treatment.     

奥沙利铂诱导人小细胞肺癌NCI-H446细胞凋亡的研究

目的：研究奥沙利铂对人肺癌细胞株NCI-H446生长抑制及诱导凋亡的作用并探讨其机制。方法：以不同浓度的奥沙利铂作用于NCI-H446细胞,应用采用噻唑蓝（MTT）法检测奥沙利铂对NCI-H446细胞生长的抑制作用。Hoechst33258荧光染色法观察细胞凋亡形态学变化;流式细胞术检测细胞周期和凋亡;分光光度法检测caspase-3、caspase-8、caspase-9的活性。结果：奥沙利铂可抑制NCI-H446细胞的生长并具有时间和剂量依赖性,Hoechst 33258荧光染色可见典型的细胞凋亡特征,流式细胞仪检测到细胞阻滞于S期和sub-G1峰。经过奥沙利铂诱导,caspase-3、caspase-9活性被上调,较对照组明显升高（P〈0.05）,caspase-8活性未见明显改变（P〉0.05）。结论：奥沙利铂具有抑制肺癌细胞株NCI-H446增殖及诱导凋亡作用,此作用可能通过激活内源性凋亡途径实现。     

奥沙利铂诱导人小细胞肺癌NCI-H446细胞凋亡的研究

目的:研究奥沙利铂对人肺癌细胞株NCI-H446生长抑制及诱导凋亡的作用并探讨其机制。方法:以不同浓度的奥沙利铂作用于NCI-H446细胞,应用采用噻唑蓝(MTT)法检测奥沙利铂对NCI-H446细胞生长的抑制作用。Hoechst33258荧光染色法观察细胞凋亡形态学变化;流式细胞术检测细胞周期和凋亡;分光光度法检测caspase-3、caspase-8、caspase-9的活性。结果:奥沙利铂可抑制NCI-H446细胞的生长并具有时间和剂量依赖性,Hoechst 33258荧光染色可见典型的细胞凋亡特征,流式细胞仪检测到细胞阻滞于S期和sub-G1峰。经过奥沙利铂诱导,caspase-3、caspase-9活性被上调,较对照组明显升高(P<0.05),caspase-8活性未见明显改变(P>0.05)。结论:奥沙利铂具有抑制肺癌细胞株NCI-H446增殖及诱导凋亡作用,此作用可能通过激活内源性凋亡途径实现。     

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.