雷帕霉素对人淋巴瘤Raji细胞株增殖影响及其机制的探讨

目的:探讨雷帕霉素(宜欣可)对人淋巴瘤细胞株Raji细胞体外增殖及细胞周期的影响,并分析其分子作用机制.方法:采用MTT法检测不同浓度(0、1、5、10、20、40、50和100 nmol/L)雷帕霉素作用不同时间(24、48和72 h)对Raji细胞增殖的影响.用流式细胞仪测定雷帕霉素对Raji细胞周期分布和凋亡的影响.应用蛋白质印迹法检测雷帕霉素对Raji细胞周期蛋白Cyclin D1、Cyclin E、Cyclin A和p27及凋亡蛋白抑制因子Survivin蛋白表达的影响.结果:雷帕霉素浓度＞5 nmol/L对Raji细胞增殖有明显的抑制作用(P＜0.05).且呈现明显的剂量-效应和时间-效应依赖关系.雷帕霉素处理后,可明显抑制Raji细胞周期发展(P＜0.05),随着药物浓度的加大、作用时间的延长,处于G0/G1期细胞逐渐增多,S期和G2/M期细胞则逐渐减少.但Raji细胞没有发生明显的凋亡现象(P＞0.05).50 nmol/L雷帕霉素处理后,Raji细胞的Cyclin D1、Cyclin E、p27表达水平没有明显变化(P＞0.05),但Cyclin A和Survivin表达水平被明显抑制(P＜0.05).结论:雷帕霉素通过阻滞细胞周期发展抑制Raji细胞增殖,其作用机制可能与下调细胞Cyclin A和Survivin表达有关.     

雷帕霉素对食管鳞癌裸鼠移植瘤生长及mTOR/p70S6K信号通路的影响

目的:探讨雷帕霉素(rapamycin, RAPA)对荷人食管鳞癌裸鼠移植瘤生长及mTOR/p70S6K信号通路的影响.方法:建立人食管鳞癌细胞株EC9706的裸鼠移植瘤模型,观察经RAPA和RAPA联合顺铂治疗后荷瘤裸鼠的肿瘤生长情况;应用TUNEL、RT-PCR及Western 印迹法观察肿瘤组织中的细胞凋亡、mTOR和p70S6K mRNA以及mTOR、p70S6K和p-p70S6K的蛋白表达.结果:RAPA和顺铂均使肿瘤生长速度减慢(P<0.05),并且2者联合应用效果更好(P<0.01);TUNEL检测发现,RAPA在体内能引起EC9706细胞的凋亡(P<0.05);RT-PCR和Western 印迹法检测结果表明,RAPA在体内降低了mTOR、p70S6K mRNA和mTOR、p-p70S6K的蛋白表达,但可提高p70S6K的蛋白表达水平.结论:RAPA在体内通过抑制mTOR/p70S6K信号通路的活性促进细胞凋亡,从而抑制人食管鳞癌细胞EC9706裸鼠移植瘤的生长.     

雷帕霉素对人淋巴瘤Raji细胞增殖、VEGF-A及HIF-1α表达的影响

目的:探讨雷帕霉素对人淋巴瘤细胞株Raji细胞体外增殖、VEGF-A及HIF-1α表达的影响及其临床意义.方法:不同浓度(0、10、50、100、250、500nmol/L)雷帕霉素对Raji细胞作用不同时间(24、48、72h)后,使用CCK8法检测细胞增殖的变化;采用蛋白质印记法(Western blot,WB)检测72h组不同浓度mTOR、VEGF-A、HIF-1α蛋白变化;采用反转录聚合酶链式反应(RT-PCR)检测其mRNA的变化.结果:雷帕霉素对Raji细胞增殖具有显著抑制作用(P＜0.01);雷帕霉素处理72h后的Raji细胞,随着药物浓度增加,mTOR、VEGF-A和HIF-1 α蛋白明显减少(P＜0.01);雷帕霉素处理后的Raji细胞,随着药物浓度和作用时间的增加,mTOR、VEGF-A和HIF-1α的mRNA明显减少(P＜0.01);mTOR、VEGF-A及HIF-1α的mR-NA表达两两之间呈正相关性.结论:雷帕霉素通过mTOR通路,能明显抑制人淋巴瘤Raji细胞增殖过程,表现出明显的时间、剂量效应依赖关系;同时也可明显减少mTOR、VEGF-A、HIF-1α的mRNA的转录表达,减少p-mTOR、VEGF-A、HIF-1α等蛋白的翻译表达,从而抑制Raji细胞的生长及其血管新生.     

干扰mTOR表达增强食管鳞癌EC9706细胞对雷帕霉素的敏感性

目的：观察哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR)特异性小干扰RNA（mTOR-siRNA）干扰mTOR表达后,食管鳞癌EC9706细胞对雷帕霉素(rapamycin)敏感性的变化。方法：mTOR-siRNA转染EC9706细胞, RT-PCR检测干扰效果。mTOR-siRNA转染前后的EC9706细胞用雷帕霉素处理,Western blotting检测EC9706细胞中mTOR及其下游p70S6K蛋白的表达;流式细胞术检测EC9706细胞的周期及凋亡,CCK-8试剂盒检测EC9706细胞的增殖。结果：mTOR-siRNA下调EC9706细胞中mTOR mRNA的表达（P<0.05或P<0.01);雷帕霉素抑制EC9706细胞中mTOR和p-p70S6K蛋白的表达（P<0.05),并促进p70S6K蛋白表达(P<0.01),且mTOR-siRNA转染后此作用更明显（P<0.05）。雷帕霉素可诱导EC9706细胞凋亡(P<0.01)、抑制EC9706细胞增殖(P<0.05或P<0.01)、使EC9706细胞阻滞于G1期(P<0.01),且mTOR-siRNA转染后这些作用更强（P<0.05）。结论：mTOR-siRNA能特异性下调食管鳞癌EC9706细胞中mTOR表达,提高EC9706细胞对雷帕霉素的敏感性。     

雷帕霉素抑制胆囊癌GBC-SD细胞的生长和转移

目的：研究雷帕霉素 (rapamycin) 对胆囊癌GBCSD细胞生长和转移的影响,探讨雷帕霉素治疗胆囊癌的临床应用前景。方法:采用MTT法检测不同浓度（12.5、25、50 nmol/L）的雷帕霉素对胆囊癌细胞增殖的影响,以流式细胞术检测不同浓度的雷帕霉素对细胞凋亡和细胞周期的变化,Transwell小室检测雷帕霉素对细胞迁移能力的影响,利用Western blotting检测胆囊癌细胞中雷帕霉素哺乳动物靶标（mammalian target of rapamycin,mTOR）及其磷酸化pmTOR水平。结果:雷帕霉素可显著抑制胆囊癌GBCSD细胞中mTOR的磷酸化,但对mTOR表达无影响。雷帕霉素显著抑制胆囊癌细胞的生长,并呈剂量依赖性抑制(P<0.01)。雷帕霉素可引起胆囊癌细胞周期G1/S阻滞和细胞凋亡;可显著抑制胆囊癌细胞的转移(P<001)。结论:雷帕霉素能显著抑制胆囊癌细胞生长及转移,其机制可能与抑制pmTOR通路、诱导细胞周期阻滞和细胞凋亡有关。     

雷帕霉素下调基质金属蛋白酶表达抑制伯基特淋巴瘤细胞转移的作用和机制研究

目的:研究雷帕霉素(Rapamycin,RAPA)对伯基特淋巴瘤细胞株Raji中基质金属蛋白酶(MMP-2,MMP-9)表达水平的影响,探讨雷帕霉素在伯基特淋巴瘤细胞株Raji转移方面的作用.方法:以伯基特淋巴瘤细胞株Raji为实验对象,应用Transwell迁移实验、Western blot、qPCR技术来检测雷帕霉素对Raji细胞迁移能力,MMP-2、MMP-9的蛋白和mRNA表达水平的抑制情况.结果:雷帕霉素可显著抑制Raji的迁移能力,且呈浓度依赖性,当雷帕霉素浓度为1 nmol/L时抑制作用最强(P<0.05);Raji细胞中MMP-2、MMP-9蛋白及mRNA的表达水平有明显的下降趋势,且呈浓度依赖性,mRNA的表达水平在作用48 h时最低.结论:雷帕霉素可以显著抑制伯基特淋巴瘤细胞株Raji的迁移能力,降低MMP-2、MMP-9的蛋白及mRNA的表达,抑制淋巴瘤细胞的转移.     

雷帕霉素对人非霍奇金淋巴瘤Raji细胞生物学行为的影响及其机制研究

目的 探讨不同浓度雷帕霉素作用不同时间对人非霍奇金淋巴瘤Raji细胞生物学行为的影响及其相关机制.方法采用0、10、50、100、250、500 nmol/L雷帕霉素分别作用Raji细胞24、48、72 h,采用CCK-8法测定Raji细胞增殖抑制率;采用流式细胞术测定Raji细胞凋亡及细胞周期;采用Caspase-3、Caspase-9活性检测试剂盒检测Raji细胞中Caspase-3、Caspase-9的酶活性;采用蛋白印迹法及反转录聚合酶链反应(RT-PCR)检测Raji细胞中bcl-2、p53蛋白及其mRNA表达情况.结果作用24 h后,随着雷帕霉素浓度由0 nmol/L逐渐增加至500 nmol/L,Raji细胞增殖抑制率由(23.7±4.2)％升高至(51.7±3.7)％(P<0.01);细胞凋亡率由(4.9±1.9)％升高至(20.5±1.5)％(P<0.01);G0/G1期细胞比例由(40.8±1.4)％增加至(63.6±1.7)％(P<0.01);Caspase-3酶活性由0.16±0.05增加至1.08±0.04(P<0.01);Caspase-9酶活性由0.19±0.04增加至1.34±0.06(P<0.01);bcl-2 mRNA表达量由0.90±0.03减少至0.46±0.03,p53 mRNA表达量由2.51±0.41增加至5.85±0.21,并且bcl-2蛋白表达降低,p53蛋白表达增高.Raji细胞48 h和72 h实验结果与24 h实验结果趋势一致.结论雷帕霉素可能通过Caspase-3、Caspase-9、bcl-2、p53途径抑制Raji细胞增殖,并诱导Raji细胞凋亡.     

Ghrelin促进乳腺癌细胞MDA-MB-231增殖的分子机制

目的:探讨生长激素释放肽（Ghrelin）促进乳腺癌细胞MDA-MB-231增殖的分子机制。方法:乳腺癌细胞MDA-MB-231经Ghrelin、Ghrelin受体（growth hormone secretagogue receptor,GHSR)抑制剂［D-Lys3］-GHRP-6或哺乳动物雷帕霉素靶蛋白(mammalian target of Rapamycin,mTOR)抑制剂雷帕霉素（Rapamycin）处理后,MTT或BrdU实验检测MDA-MB-231细胞的增殖能力;Western blot检测MDA-MB-231细胞GHSR表达及mTOR、p70S6K和S6磷酸化水平。结果:增殖实验结果表明Ghrelin增强MDA-MB-231细胞增殖能力;Western blot检测发现Ghrelin激活MDA-MB-231细胞mTOR、p70S6K和S6磷酸化,［D-Lys3］-GHRP-6或Rapamycin消除Ghrelin促进MDA-MB-231细胞增殖效应,同时抑制Ghrelin诱导的mTOR、p70S6K和S6磷酸化。结论:Ghrelin通过与GHSR结合激活MDA-MB-231细胞mTOR/p70S6K/S6信号途径促进MDA-MB-231细胞增殖。     

雷帕霉素对食管鳞癌细胞系EC9706的mTOR/p70S6K信号通路的影响

目的：观察雷帕霉素（rapamycin，rapa）对食管鳞癌细胞系EC9706的mTOR／p70S6K信号通路的影响。方法：采用免疫细胞化学证实mTOR／p70S6K信号通路的存在，然后通过DNALadder、RT—PCR、Westernblot及流式细胞术分别从DNA、RNA、蛋白及细胞水平研究rapa对细胞凋亡和信号通路的影响。结果：免疫细胞化学结果显示，在细胞核及细胞质中mTOR均呈阳性；rapa处理后有明显DNALadder产生，且mTOR的mRNA水平及蛋白水平下调。但是，mTOR下游的直接靶点p70S6K的mRNA水平及蛋白水平则升高，二者的变化程度均与rapa剂量的相关；流式细胞术检测结果表明，rapa可使细胞停滞于G1期。结论：食管鳞癌细胞系EC9706中存在mTOR／p70S6K信号通路并且处于激活状态，rapa能明显促进细胞凋亡并抑制该通路激活，从而间接抑制翻译的进行。     

雷帕霉素对肺癌细胞mTOR信号通路相关蛋白表达的作用

目的探讨mTOR信号通路的功能状态与肺癌对雷帕霉素敏感度的关系。方法Western blot检测mTOR信号通路中AKT、S6K1、eIF4E、4E-BP1、P-S6K1蛋白在4种人肺癌细胞株的表达;MTT法检测雷帕霉素对肺癌细胞增殖的抑制情况,将肺癌细胞分为雷帕霉素敏感组和不敏感组,找出与敏感度相关的差异蛋白,用En Vision法检测患者肺癌组织中差异蛋白的表达。结果AKT、S6K1、eIF4E、4E-BP1蛋白在4种肺癌细胞株中均有表达。而P-S6K1在不敏感株中未检测到, 在敏感株中呈高表达,为雷帕霉素差异蛋白。P-S6K1在正常肺组织中的阳性表达率为15％（3/20例),在肺癌组织中的阳性表达率为62%(62/100例),其中在小细胞肺癌组织中的阳性表达率(81.0%,17/21例)高于非小细胞肺癌组织（59%,45/79例)。结论在mTOR信号通路相关蛋白中,仅P-S6K1在一定程度上可预测肺癌细胞株对雷帕霉素的敏感度,为一种mTOR靶向抑制剂——雷帕霉素治疗肺癌的差异蛋白。P-S6K1在小细胞肺癌中表达较非小细胞肺癌高,提示在P-S6K1表达率高的小细胞未分化肺癌中应用mTOR靶向抑制剂——雷帕霉素的疗效可能更佳。     

哺乳动物雷帕霉素靶蛋白小分子干扰RNA对食管鳞癌细胞中mTOR-p70S6K信号通路及裸鼠移植瘤生长的影响

目的 检测哺乳动物雷帕霉素靶蛋白(mTOR)小分子干扰RNA(siRNA)对食管鳞癌中mTOR-p70S6K信号通路的阻断作用以及对裸鼠移植瘤生长的影响.方法 以食管鳞癌细胞株EC9706为研究对象,采用siRNA干扰、逆转录聚合酶链反应(RT-PCR)、Western blot、流式细胞术及CCK-8等方法,观察mTOR-p70S6K信号通路被阻断后,通路中各因子蛋白及mRNA表达的变化,及其对细胞增殖和凋亡的影响.进行裸鼠成瘤实验,观察mTOR siRNA对肿瘤生长的影响.结果 与未转染细胞相比,转染mTOR siRNA的细胞中mTOR和磷酸化p70S6K的表达水平降低(P<0.05),而pTOS6K的表达水平升高(P<0.05).转染mTOR siRNA后,细胞凋亡增加,细胞增殖能力降低,且对顺铂的敏感性增高,细胞被阻滞在G,期(P<0.05).mTOR siRNA能明显抑制裸鼠移植瘤的生长,siRNA组和siRNA+顺铂组的肿瘤抑制率分别为50.9%和62.3%.结论 mTOR siRNA能有效抑制mTOR-pTOS6K信号通路,抑制细胞增殖,促进细胞凋亡,在裸鼠体内能显著抑制肿瘤的生长.mTOR-pVOSSK信号通路在食管鳞癌发生发展中起重要作用.     

哺乳动物雷帕霉素靶蛋白小分子干扰RNA对食管鳞癌细胞中mTOR-p70S6K信号通路及裸鼠移植瘤生长的影响

目的 检测哺乳动物雷帕霉素靶蛋白(mTOR)小分子干扰RNA(siRNA)对食管鳞癌中mTOR-p70S6K信号通路的阻断作用以及对裸鼠移植瘤生长的影响.方法 以食管鳞癌细胞株EC9706为研究对象,采用siRNA干扰、逆转录聚合酶链反应(RT-PCR)、Western blot、流式细胞术及CCK-8等方法,观察mTOR-p70S6K信号通路被阻断后,通路中各因子蛋白及mRNA表达的变化,及其对细胞增殖和凋亡的影响.进行裸鼠成瘤实验,观察mTOR siRNA对肿瘤生长的影响.结果 与未转染细胞相比,转染mTOR siRNA的细胞中mTOR和磷酸化p70S6K的表达水平降低(P<0.05),而pTOS6K的表达水平升高(P<0.05).转染mTOR siRNA后,细胞凋亡增加,细胞增殖能力降低,且对顺铂的敏感性增高,细胞被阻滞在G,期(P<0.05).mTOR siRNA能明显抑制裸鼠移植瘤的生长,siRNA组和siRNA+顺铂组的肿瘤抑制率分别为50.9%和62.3%.结论 mTOR siRNA能有效抑制mTOR-pTOS6K信号通路,抑制细胞增殖,促进细胞凋亡,在裸鼠体内能显著抑制肿瘤的生长.mTOR-pVOSSK信号通路在食管鳞癌发生发展中起重要作用.     

哺乳动物雷帕霉素靶蛋白小分子干扰RNA对食管鳞癌细胞中mTOR-p70S6K信号通路及裸鼠移植瘤生长的影响

目的 检测哺乳动物雷帕霉素靶蛋白(mTOR)小分子干扰RNA(siRNA)对食管鳞癌中mTOR-p70S6K信号通路的阻断作用以及对裸鼠移植瘤生长的影响.方法 以食管鳞癌细胞株EC9706为研究对象,采用siRNA干扰、逆转录聚合酶链反应(RT-PCR)、Western blot、流式细胞术及CCK-8等方法,观察mTOR-p70S6K信号通路被阻断后,通路中各因子蛋白及mRNA表达的变化,及其对细胞增殖和凋亡的影响.进行裸鼠成瘤实验,观察mTOR siRNA对肿瘤生长的影响.结果 与未转染细胞相比,转染mTOR siRNA的细胞中mTOR和磷酸化p70S6K的表达水平降低(P<0.05),而pTOS6K的表达水平升高(P<0.05).转染mTOR siRNA后,细胞凋亡增加,细胞增殖能力降低,且对顺铂的敏感性增高,细胞被阻滞在G,期(P<0.05).mTOR siRNA能明显抑制裸鼠移植瘤的生长,siRNA组和siRNA+顺铂组的肿瘤抑制率分别为50.9%和62.3%.结论 mTOR siRNA能有效抑制mTOR-pTOS6K信号通路,抑制细胞增殖,促进细胞凋亡,在裸鼠体内能显著抑制肿瘤的生长.mTOR-pVOSSK信号通路在食管鳞癌发生发展中起重要作用.

Abstract：

Objective To investigate the effects of mTOR siRNA on mTOR-p70S6K signaling pathway in esophageal squamous cell carcinoma ( ESCC) cells in vitro, and growth and apoptosis in transplanted tumor in nude mice. Methods mTOR siRNA was transfected into ESCC cell line EC9706 cells. The expressions of factors of the mTOR/p70S6K signaling pathway were detected by RT-PCR and Western blot. DNA contents and cell apoptosis were determined by flow cytometry, and cell proliferation was measured by CCK-8 assay. The effects of mTOR siRNA on the transplanted tumor growth were assessed in nude mice. Results The levels of mTOR and p-p70S6K were significantly decreased ( P < 0.05 ) while the level of p70S6K was increased (P<0.05) in the cells transfected with mTOR siRNA, compared with that in untransfected cells and cells transfected with control siRNA. After being interfered by mTOR siRNA, the number of apoptotic cells was increased, cell proliferation became slower and cell cycle was arrested in G, phase compared with that in control cells. Also, mTOR siRNA inhibited the growth of transplanted tumor in vivo. Conclusions mTOR siRNA can effectively interfere in mTOR-p70S6K signaling pathway, induce cell apoptosis and inhibit cell proliferation and tumor growth, suggesting that mTOR-p70S6K signaling pathway plays an important role in the carcinogenesis and development of esophageal squamous cell carcinoma.     

Rapamycin provides a therapeutic option through inhibition of mTOR signaling in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a neoplasm of myeloid progenitor cells expressing Bcr-Abl fusion protein. However, some patients with CML are less likely to respond to imatinib, the inhibitor of Bcr-Abl kinase. Recent studies showed that mTOR pathway can increase responses to imatinib. The analysis of mTOR pathway in CML may provide new insights into possible targets of novel therapies. Therefore, we examined the expression of mTOR pathway molecules in bone marrow cells from CML patients and effect of rapamycin on K562 cells in?vitro. Western blot analysis showed the visibly higher phosphorylation of mTOR (70.6%), 4E-BP1 (76.5%) and p70S6K (73.5%) in bone marrow cells from CML patients. Moreover, treatment of CML cell line (K562) with rapamycin resulted in a decrease of phosphorylation of mTOR, 4E-BP1 and p70S6K. In?vitro, the cell viability in groups with rapamycin treatment displayed a significant decrease in a dose-dependent manner by MTT. The data presented an increase of G0/G1 phase cells and decrease of S phase cells after rapamycin treatment, and the decreased expression of cyclinD1, higher expression of p21 at mRNA level was also detected in K562 with rapamycin. Treatment with 20 nmol/l or more rapamycin could increase apoptotic cells, decrease expression of bcl-2 and activate caspase-3. In conclusion, the mTOR pathway might be involved in chronic myelogenous leukemia. Inhibition of mTOR pathway could interfer with cell proliferation and increase cell apoptosis in K562 cells. It suggested that mTOR might be an important therapeutic target for myelogenous leukemia.     

Longitudinal inhibition of PI3K/Akt/mTOR signaling by LY294002 and rapamycin induces growth arrest of adult T-cell leukemia cells

This study found that phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling was activated in human T-cell lymphotropic virus type I (HTLV-1)-infected leukemia cells. Rapamycin (1-100 nM, 48h), the inhibitor of mTOR and its analog RAD001 (1-100 nM, 48 h)-induced growth inhibition and G0/G1 cell cycle arrest of these cells in association with de-phosphorylation of p70S6K and 4E-BP-1, although IC50 was not achieved. Paradoxically, rapamycin-stimulated phosphorylation of Akt at Ser473. Blockade of Akt signaling by the PI3K inhibitor LY294002 (1-20 microM, 48 h) also resulted in the growth inhibition and G0/G1 cell cycle arrest of HTLV-1-infected cells, with IC50 ranging from 5 to 20muM, and it caused de-phosphorylation of p70S6K and 4E-BP-1. Of note, when rapamycin was combined with LY294002, rapamycin-induced phosphorylation of Akt was blocked, and the ability of rapamycin to induce growth arrest of HTLV-1-infected T-cells and suppress the p-p70S6K and p-4E-BP-1 proteins was potentiated. Moreover, both LY294002 and rapamycin down-regulated the levels of c-Myc and cyclin D1 proteins in these cells, and their combination further decreased levels of these cell cycle-regulating proteins. Taken together, longitudinal inhibition of PI3K/Akt/mTOR signaling represents a promising treatment strategy for individuals with adult T-cell leukemia.     

An activated mTOR/p70S6K signaling pathway in esophageal squamous cell carcinoma cell lines and inhibition of the pathway by rapamycin and siRNA against mTOR

mTOR/p70S6K pathway is considered a central regulator in various malignant tumors, but its roles in esophageal squamous cell carcinoma (ESCC), which is a common cause of mortality in China, remain unknown. Here, we identify that the mTOR/p70S6K pathway is activated in ESCC; rapamycin and siRNA against mTOR rapidly inhibited expression of mTOR and the phosphorylation of its major downstream effectors, p70S6K and 4E-BP1, arrested cells in the G(0)/G(1) phase and induced apoptosis of ESCC cells. The findings may lay a foundation for making further investigations on the mTOR/p70S6K pathway as a potential target for ESCC therapy.     

Erufosine simultaneously induces apoptosis and autophagy by modulating the Akt-mTOR signaling pathway in oral squamous cell carcinoma

We investigated the anticancer activity of erufosine in oral squamous carcinoma cell lines in terms of cell proliferation, colony formation, induction of autophagy/apoptosis, cell cycle and mTOR signaling pathway. Erufosine showed dose-dependent cytotoxicity in all cell lines, it induced autophagy as well as apoptosis, G2 cell cycle arrest and modulation of cyclin D1 expression. Further erufosine downregulated the phosphorylation of major components of mTOR pathway, like p-Akt at Ser473 and Thr308 residues, p-Raptor, p-mTOR, p-PRAS40 and its downstream substrates p-p70S6K and p-4EBP1 in a dose-dependent manner. The pre-treatment of tumor cells with p-mTOR siRNA increased cytotoxic effects of erufosine comparable to cisplatin but higher than rapamycin.     

Fibronectin stimulates non-small cell lung carcinoma cell growth through activation of Akt/mammalian target of rapamycin/S6 kinase and inactivation of LKB1/AMP-activated protein kinase signal pathways

The Akt/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K) pathway is considered a central regulator of protein synthesis and of cell proliferation, differentiation, and survival. However, the role of the Akt/mTOR/p70S6K pathway in lung carcinoma remains unknown. We previously showed that fibronectin, a matrix glycoprotein highly expressed in tobacco-related lung disease, stimulates non-small cell lung carcinoma (NSCLC) cell growth and survival. Herein, we explore the role of the Akt/mTOR/p70S6K pathway in fibronectin-induced NSCLC cell growth. We found that fibronectin stimulated the phosphorylation of Akt, an upstream inducer of mTOR, and induced the phosphorylation of p70S6K1 and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), two downstream targets of mTOR in NSCLC cells (H1792 and H1838), whereas it inhibited the phosphatase and tensin homologue deleted on chromosome 10, a tumor suppressor protein that antagonizes the phosphatidylinositol 3-kinase/Akt signal. In addition, treatment with fibronectin inhibited the mRNA and protein expression of LKB1 as well as the phosphorylation of AMP-activated protein kinase (AMPKalpha), both known to down-regulate mTOR. Rapamycin, an inhibitor of mTOR, blocked the fibronectin-induced phosphorylation of p70S6K and 4E-BP1. Akt small interfering RNA (siRNA) and an antibody against the fibronectin-binding integrin alpha5beta1 also blocked the p70S6K phosphorylation in response to fibronectin. In contrast, an inhibitor of extracellular signal-regulated kinase 1/2 (PD98095) had no effect on fibronectin-induced phosphorylation of p70S6K. Moreover, the combination of rapamycin and siRNA for Akt blocked fibronectin-induced cell proliferation. Taken together, these observations suggest that fibronectin-induced stimulation of NSCLC cell proliferation requires activation of the Akt/mTOR/p70S6K pathway and is associated with inhibition of LKB1/AMPK signaling.