PI3K/Akt/mTOR和MAPK/ERK信号通路在胃癌细胞生长中协同作用及机制
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摘要
目的初步探讨PI3K/Akt/m TOR和MAPK/ERK信号通路在胃癌细胞生长中的协同作用。方法采用不同浓度的PI3K/Akt/m TOR和MAPK/ERK信号通路抑制剂rapamycin与PD98059分别单独及联合作用于胃癌细胞SGC-7901。采用CCK8法检测SGC-7901细胞增殖情况;实时荧光定量PCR检测关键基因m RNA的表达;蛋白印迹法(WB)检测相关蛋白的表达;流式细胞术检测细胞周期和凋亡的变化。结果 Rapamycin(12.5、25、50、100、150、200 nmol/L)与PD98059(25、50、100、200、300、400μmol/L)单独作用可抑制SGC-7901细胞增殖活性,联合作用也可抑制其活性,且联合组的抑制作用强于单独作用(P<0.05)。与rapamycin、PD98059单独作用相比,联合组对AKT、m TOR、MEK、ERK基因m RNA表达和p-AKT、p-m TOR、p-MEK1/2、p-ERK1/2蛋白表达的抑制作用明显增强(P<0.05)。联合组阻滞于G0/G1期的细胞比例显著增多,且具有更强的促凋亡作用。结论 PI3K/Akt/m TOR和MAPK/ERK信号通路在胃癌细胞生长中具有一定的协同调控作用。
Objective To investigate the synergistic effect of phosphalyl inositol 3-kinase protein kinase B/mammalian target of rapamycin(PI3 K/Akt/m TOR) and mitogen activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK) signaling pathways on gastric cancer cell growth. Methods Different concentrations of PI3 K/Akt/m TOR and MAPK/ERK signaling pathways inhibitor rapamycin and PD98059 were used alone and in combination to SGC-7901 cells,respectively. The proliferation of SGC-7901 cells was detected with cholecystokinin-8(CCK8). The m RNA expressions of key genes were detected with real time quantitative polymerase chain reaction(RT-q PCR). The expressions of related proteins were detected with Western blot. Cell cycles and apoptosis were detected with flow cytometry. Results Both the single administration of rapamycin(at dosages of 12.5, 25, 50, 100, 150, and 200 nmol/L) and PD98059(at dosages of 25,50, 100, 150, 200, 300, and 400 μmol/L) could inhibit the growth of SGC-7901 cells; so did the combined administration of the two agents and the inhibitory effect of the combined administration was stronger than that of the single agent(P < 0.05).The combined administration of rapamycin and PD98059 exerted a much stronger inhibitive effect on the m RNA expressions of AKT, m TOR, mitogen activated protein kinase kinase(MEK), ERK and the protein expressions of p-AKT, p-m TOR,p-MEK1/2 and p-ERK1/2 than the single administration of one of the two agents(P < 0.05 for all); higher proportions of cells arrested at G0/G1 phase and apoptosis cells were also observed for the SGC-7901 cells with combined administration than those for the SGC-7901 cells with the single administration. Conclusion PI3 K/Akt/m TOR and MAPK/ERK signaling pathways have a synergistic effect on the growth of gastric cancer cells.
Objective To investigate the synergistic effect of phosphalyl inositol 3-kinase protein kinase B/mammalian target of rapamycin(PI3 K/Akt/m TOR) and mitogen activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK) signaling pathways on gastric cancer cell growth. Methods Different concentrations of PI3 K/Akt/m TOR and MAPK/ERK signaling pathways inhibitor rapamycin and PD98059 were used alone and in combination to SGC-7901 cells,respectively. The proliferation of SGC-7901 cells was detected with cholecystokinin-8(CCK8). The m RNA expressions of key genes were detected with real time quantitative polymerase chain reaction(RT-q PCR). The expressions of related proteins were detected with Western blot. Cell cycles and apoptosis were detected with flow cytometry. Results Both the single administration of rapamycin(at dosages of 12.5, 25, 50, 100, 150, and 200 nmol/L) and PD98059(at dosages of 25,50, 100, 150, 200, 300, and 400 μmol/L) could inhibit the growth of SGC-7901 cells; so did the combined administration of the two agents and the inhibitory effect of the combined administration was stronger than that of the single agent(P < 0.05).The combined administration of rapamycin and PD98059 exerted a much stronger inhibitive effect on the m RNA expressions of AKT, m TOR, mitogen activated protein kinase kinase(MEK), ERK and the protein expressions of p-AKT, p-m TOR,p-MEK1/2 and p-ERK1/2 than the single administration of one of the two agents(P < 0.05 for all); higher proportions of cells arrested at G0/G1 phase and apoptosis cells were also observed for the SGC-7901 cells with combined administration than those for the SGC-7901 cells with the single administration. Conclusion PI3 K/Akt/m TOR and MAPK/ERK signaling pathways have a synergistic effect on the growth of gastric cancer cells.
引文
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[16]陈晓伟,徐余超,孙海翔,等.Rapamycin抑制PI3K/AKT/m TOR信号通路对胃癌MGC-803细胞影响[J].中国公共卫生,2017,33(4):602-606.
[17]Zhang YJ,Tian XQ,Sun DF,et al.Combined inhibition of MEKand m TOR signaling inhibits initiation and progression of colorectal cancer[J].Cancer Investigation,2009,27(3):273-285.
[18]Patrick J,Roberts JE,Usary D.Combined PI3K/m TOR and MEKinhibition provides broad antitumor activity in faithful murine cancer models[J].Clinical Cancer Research An Official Journal of the American Association for Cancer Research,2012,18(19):5290-5303.
[19]Zhong H,Sanchez C,Spitrzer D,et al.Synergistic effects of concurrent blockade of PI3K and MEK pathways in pancreatic cancer preclinical models[J].PLo S One,2013,8(10):e77243.
[2]Mccubrey JA,Steelman LS,Chappell WH,et al.Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/m TOR cascade inhibitors:how mutations can result in therapy resistance and how to overcome resistance[J].Oncotarget,2012,3(10):1068-1111.
[3]Chang F,Lee JT,Navolanic PM,et al.Involvement of PI3K/Akt pathway in cell cycle progression,apoptosis,and neoplastic transformation:a target for cancer chemotherapy[J].Leukemia,2003,17(3):590-603.
[4]Carracedo A,Pandolfi PP.The PTEN-PI3K pathway:of feedbacks and cross-talks[J].Oncogene,2008,27(41):5527-5541.
[5]Aksamitiene E,Kiyatkin A,Kholodenko BN.Cross-talk between mitogenic Ras/MAPK and survival PI3K/Akt pathways:a fine balance[J].Biochemical Society Transactions,2012,40(1):139-46.
[6]Kathryn B,Simon DC,Gillings AS,et al.Intrinsic resistance to the MEK1/2 inhibitor AZD6244(ARRY-142886)is associated with weak ERK1/2 signalling and/or strong PI3K signalling in colorectal cancer cell lines[J].International Journal of Cancer Journal International Du Cancer,2009,125(10):2332-2341.
[7]Meng J,Peng H,Dai B,et al.High level of AKT activity is associated with resistance to MEK inhibitor AZD6244(ARRY-142886)[J].Cancer Biology and Therapy,2009,8(21):2073.
[8]Hausenloy DJ,Mocanu MM,Yellon DM.Cross-talk between the survival kinases during early reperfusion:its contribution to ischemic preconditioning[J].Cardiovascular Research,2004,63(2):305-312.
[9]Grant S.Cotargeting survival signaling pathways in cancer[J].Journal of Clinical Investigation,2008,118(9):3003-3006.
[10]She QB,Solit DB,Ye Q,et al.The BAD protein integrates survival signaling by EGFR/MAPK and PI3K/Akt kinase pathways in PTEN-deficient tumor cells[J].Cancer Cell,2005,8(4):287-297.
[11]Wang J,Yuan Y,Zhou Y,et al.Protein interaction data set highlighted with human Ras-MAPK/PI3K signaling pathways[J].Journal of Proteome Research,2008,7(9):3879.
[12]Guo RX,Zhang RF,Wang XY,et al.Effects of PD98059 and LY294002 on subcutaneous xenograft of human endometrial carcinoma in nude mice[J].Zhonghua Fu Chan Ke Za Zhi,2011,46(6):446-452.
[13]Ma XM,Blenis J.Molecular mechanisms of m TOR-mediated translational control[J].Nature Reviews Molecular Cell Biology,2009,10(5):307-318.
[14]Carracedo A,Ma L,Teruyafeldstein J,et al.Inhibition of m TORC1leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer[J].Journal of Clinical Investigation,2008,118(9):3065.
[15]Hoeflich KP,O'Brien C,Boyd Z,et al.In vivo antitumor activity of MEK and phosphatidylinositol 3-kinase inhibitors in basal-like breast cancer models[J].Clinical Cancer Research An Official Journal of the American Association for Cancer Research,2009,15(14):4649.
[16]陈晓伟,徐余超,孙海翔,等.Rapamycin抑制PI3K/AKT/m TOR信号通路对胃癌MGC-803细胞影响[J].中国公共卫生,2017,33(4):602-606.
[17]Zhang YJ,Tian XQ,Sun DF,et al.Combined inhibition of MEKand m TOR signaling inhibits initiation and progression of colorectal cancer[J].Cancer Investigation,2009,27(3):273-285.
[18]Patrick J,Roberts JE,Usary D.Combined PI3K/m TOR and MEKinhibition provides broad antitumor activity in faithful murine cancer models[J].Clinical Cancer Research An Official Journal of the American Association for Cancer Research,2012,18(19):5290-5303.
[19]Zhong H,Sanchez C,Spitrzer D,et al.Synergistic effects of concurrent blockade of PI3K and MEK pathways in pancreatic cancer preclinical models[J].PLo S One,2013,8(10):e77243.