PD98059抑制MAPK/ERK信号通路对胃癌细胞生物学功能的影响
|
摘要
目的:研究MAPK/ERK信号通路中关键信号分子MEK和ERK在胃癌SGC-7901细胞中的表达及PD98059抑制MAPK/ERK通路对胃癌细胞生物学功能的影响。方法:体外培养胃癌细胞株SGC-7901,不同浓度(0、25、50、100、200、300和400 mmol/L)PD98059处理24 h后CCK-8法检测细胞增殖率变化;再用0、25、50和100μmol/L PD98059处理24 h后采用实时荧光定量PCR(qPCR)检测MEK和ERK m RNA的表达量;Western blot检测MEK和ERK蛋白的表达;流式细胞术检测细胞周期和凋亡变化。同时设正常胃黏膜上皮GES-1细胞为对照。结果:与正常胃黏膜上皮GES-1细胞相比,胃癌SGC-7901细胞中MEK和ERK mRNA的表达升高,差异具有统计学意义(P<0.05);p-MEK、p-ERK蛋白的表达亦显著升高,差异具有统计学意义(P<0.05)。0～200μmol/L PD98059处理SGC-7901细胞后,细胞增殖率随着抑制剂浓度的升高而降低(P<0.05)。当PD98059浓度处于200～400μmol/L时抑制作用逐渐趋于平稳。0～100μmol/L PD98059作用后MEK、ERK m RNA的表达量低于对照组(P<0.05),随着PD98059浓度升高,ERK mRNA表达量逐渐降低(P<0.05)。Western blot检测结果显示50和100μmol/L PD98059作用后p-MEK1/2、p-ERK1/2蛋白表达降低(P<0.05)。且抑制剂PD98059使胃癌SGC-7901细胞发生G0/G1期阻滞,可诱导细胞凋亡。结论:MAPK/ERK信号通路在胃癌细胞中激活,PD98059通过抑制MAPK/ERK信号通路的活性可影响胃癌细胞的生物学功能。
OBJECTIVE:To investigate expression of key signaling molecules MEK and ERK in theMAPK/ERK signaling pathway and effect of PD98059 on MAPK/ERK pathway's biological function in thegastric cancer SGC-7901 cells.METHODS:Gastric cancer cell line SGC-7901 was cultured in vitro.Cellproliferation rates were detected by the CCK-8 method after treatment with different concentrations of PD98059 at 0,25,50,100,200,300 and 400 μmol/L for 24 h.Expression of MEK and ERK mRNA was detectedby real-time quantitative PCR(qPCR) after treatment with 0,25,50 and 100 μmol/L PD98059 for 24 h.Expression of MEK and ERK proteins was detected by Western blot.Cell cycle and apoptosis were detected byflow cytometry.The normal gastric mucosal epithelial GES-1 cells were used as controls.RESULTS:Comparedwith the normal cells,expression of MEK and ERK mRNA and p-MEK and p-ERK proteins in the gastriccancer cells was significantly increased(P<0.05).After treatment with PD98059,the cell proliferation rateswere decreased with increasing concentration and in a dose-dependent manner.When the concentration ofPD98059 was between 200 and 400 μmol/L,the inhibition became stabilized.Expression of the MEK andERK m RNA was lower than that of the control group after treatment with 0-100 μmol/L PD98059(P<0.05).With increasing concentrations of PD98059,expression of ERK mRNA was gradually reduced(P<0.05).Western blot analyses show that expression of p-MEK1/2 and p-ERK1/2 protein decreased after treatment with50 and 100 μmol/L PD98059(P<0.05).Moreover,PD98059 caused G0/G1 phase arrest and induced apoptosis.CONCLUSION:MAPK/ERK signaling pathway was active in the gastric cancer cells.PD98059 inhibitedactivities in the MAPK/ERK signaling pathway and affected the biological function of the gastric cancer cells.
OBJECTIVE:To investigate expression of key signaling molecules MEK and ERK in theMAPK/ERK signaling pathway and effect of PD98059 on MAPK/ERK pathway's biological function in thegastric cancer SGC-7901 cells.METHODS:Gastric cancer cell line SGC-7901 was cultured in vitro.Cellproliferation rates were detected by the CCK-8 method after treatment with different concentrations of PD98059 at 0,25,50,100,200,300 and 400 μmol/L for 24 h.Expression of MEK and ERK mRNA was detectedby real-time quantitative PCR(qPCR) after treatment with 0,25,50 and 100 μmol/L PD98059 for 24 h.Expression of MEK and ERK proteins was detected by Western blot.Cell cycle and apoptosis were detected byflow cytometry.The normal gastric mucosal epithelial GES-1 cells were used as controls.RESULTS:Comparedwith the normal cells,expression of MEK and ERK mRNA and p-MEK and p-ERK proteins in the gastriccancer cells was significantly increased(P<0.05).After treatment with PD98059,the cell proliferation rateswere decreased with increasing concentration and in a dose-dependent manner.When the concentration ofPD98059 was between 200 and 400 μmol/L,the inhibition became stabilized.Expression of the MEK andERK m RNA was lower than that of the control group after treatment with 0-100 μmol/L PD98059(P<0.05).With increasing concentrations of PD98059,expression of ERK mRNA was gradually reduced(P<0.05).Western blot analyses show that expression of p-MEK1/2 and p-ERK1/2 protein decreased after treatment with50 and 100 μmol/L PD98059(P<0.05).Moreover,PD98059 caused G0/G1 phase arrest and induced apoptosis.CONCLUSION:MAPK/ERK signaling pathway was active in the gastric cancer cells.PD98059 inhibitedactivities in the MAPK/ERK signaling pathway and affected the biological function of the gastric cancer cells.
引文
[1]CHEN W,ZHENG R,BAADE P D,et al.Cancer statistics in China,2015[J].Ca Cancer J Clin,2016,66(2):115-132.
[2]TALMADGE J E,FIDLER I J.AACR centennial series:the biology of cancer metastasis:historical perspective[J].Cancer Res,2010,70(14):5649-5669.
[3]DANG Y,WANG Y C,HUANG Q J.Microarray and next-generation sequencing to analyse gastric cancer[J].Asian Pac J Cancer Prev,2014,15(19):8033-8039.
[4]KOHNO M,POUYSSEGUR J.Targeting the ERKsignaling pathway in cancer therapy[J].Ann Med,2006,38(3):200-211.
[5]BURKHARD K,SMITH S,DESHMUKH R,et al.Development of extracellular signal-regulated kinase inhibitors[J].Curr Top Med Chem,2009,9(8):678.
[6]CHAPPELL W H,STEELMAN L S,LONG J M,et al.Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR inhibitors:rationale and importance to inhibiting these pathways in human health[J].Oncotarget,2011,2(3):135-164.
[7]CAI L,WANG Z,MEYER J M,et al.Macrophage SR-BI regulates LPS-induced pro-inflammatory signaling in mice and isolated macrophages[J].J Lipid Res,2012,53(8):1472.
[8]ARTHUR J S,LEY S C.Mitogen-activated protein kinases in innate immunity[J].Nat Rev Immunol,2013,13(9):679-692.
[9]KOHNO M,POUYSSEGUR J.Pharmacological inhibitors of the ERK signaling pathway:application as anticancer drugs[J].Prog Cell Cycle Res,2008,5(5):219-224.
[10]陈惠,渠景连,龚婕宁.现代医学对恶性肿瘤转移相关机制的研究进展[J].中国中药杂志,2014,39(15):2823.
[11]KIM E K,CHOI E J.Pathological roles of MAPKsignaling pathways in human diseases[J].Biochim Biophys Acta,2010,1802(4):396-405.
[12]ASATI V,MAHAPATRA D K,BHARTI S K.PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways inhibitors as anticancer agents:Structural and pharmacological perspectives[J].Eur J Med Chem,2016,109:314-341.
[13]REDDY K B,NABHA S M,ATANASKOVA N.Role of MAP kinase in tumor progression and invasion[J].Cancer Metast Rev,2003,22(4):395-403.
[14]SUN Y,LIU W Z,LIU T,et al.Signaling pathway of MAPK/ERK in cell proliferation,differentiation,migration,senescence and apoptosis[J].J Recept Signal Transduct Res,2015,35(6):600-604.
[15]CALVO N,MARTíN M J,DE BOLAND A R,et al.Involvement of ERK1/2,p38 MAPK,and PI3K/Akt signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells[J].Biochem Cell Biol,2014,92(4):305-315.
[16]MCCUBREY J A,MILELLA M,TAFURI A,et al.Targeting the Raf/MEK/ERK pathway with small-molecule inhibitors[J].Curr Opin Investig Drugs,2008,9(6):614.
[17]WANG X,SUN D R,CHEN Z,et al.RAF may induce cell proliferation through hypermethylation of tumor suppressor gene promoter in gastric epithelial cells.[J].Cancer Sci,2010,100(1):117-125.
[18]NORMANNO N,LUCA A D,MAIELLO M R,et al.The MEK/MAPK pathway is involved in the resistance of breast cancer cells to the EGFR tyrosine kinase inhibitor gefitinib[J].J Cell Physiol,2010,207(2):420-427.
[19]宋霆婷,姜玉华,李妙玉,等.PD98059对肝癌细胞RasMAPK信号转导的作用[J].实用医学杂志,2005,21(5):446-448.
[20]FUJIMORI Y,INOKUCHI M,TAKAGI Y,et al.Prognostic value of RKIP and p-ERK in gastric cancer[J].J Exp Clin Cancer Res,2012,31(1):30.
[21]LIANG B,WANG S,ZHU X G,et al.Increased expression of mitogen-activated protein kinase and its upstream regulating signal in human gastric cancer[J].World J Gastroenterol,2005,11(5):623-628.
[22]CORONA G,DEIANA M,INCANI A,et al.Hydroxytyrosol inhibits the proliferation of human colon adenocarcinoma cells through inhibition of ERK1/2 and cyclin D1[J].Mol Nutr Food Res,2010,53(7):897-903.
[23]SUN C H,CHANG S H,SUN C Y,et al.Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells[J].J Vet Sci,2009,10(1):23-28.
[24]MOON D O,PARK C,HEO M S,et al.PD98059triggers G1 arrest and apoptosis in human leukemic U937cells through downregulation of Akt signal pathway[J].Int Immunopharmacol,2007,7(1):36-45.
[25]WU R,LI H,LI T,et al.Myostatin regulates miR-431expression via the Ras-Mek-Erk signaling pathway[J].Biochem Biophys Res Commun,2015,461(2):224-229.
[26]李楠.小鼠巨噬细胞S100A9/NF-κB/SAA3/mir-204环路在炎症相关性结肠癌发展中的作用[D].长沙:中南大学,2013.
[2]TALMADGE J E,FIDLER I J.AACR centennial series:the biology of cancer metastasis:historical perspective[J].Cancer Res,2010,70(14):5649-5669.
[3]DANG Y,WANG Y C,HUANG Q J.Microarray and next-generation sequencing to analyse gastric cancer[J].Asian Pac J Cancer Prev,2014,15(19):8033-8039.
[4]KOHNO M,POUYSSEGUR J.Targeting the ERKsignaling pathway in cancer therapy[J].Ann Med,2006,38(3):200-211.
[5]BURKHARD K,SMITH S,DESHMUKH R,et al.Development of extracellular signal-regulated kinase inhibitors[J].Curr Top Med Chem,2009,9(8):678.
[6]CHAPPELL W H,STEELMAN L S,LONG J M,et al.Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR inhibitors:rationale and importance to inhibiting these pathways in human health[J].Oncotarget,2011,2(3):135-164.
[7]CAI L,WANG Z,MEYER J M,et al.Macrophage SR-BI regulates LPS-induced pro-inflammatory signaling in mice and isolated macrophages[J].J Lipid Res,2012,53(8):1472.
[8]ARTHUR J S,LEY S C.Mitogen-activated protein kinases in innate immunity[J].Nat Rev Immunol,2013,13(9):679-692.
[9]KOHNO M,POUYSSEGUR J.Pharmacological inhibitors of the ERK signaling pathway:application as anticancer drugs[J].Prog Cell Cycle Res,2008,5(5):219-224.
[10]陈惠,渠景连,龚婕宁.现代医学对恶性肿瘤转移相关机制的研究进展[J].中国中药杂志,2014,39(15):2823.
[11]KIM E K,CHOI E J.Pathological roles of MAPKsignaling pathways in human diseases[J].Biochim Biophys Acta,2010,1802(4):396-405.
[12]ASATI V,MAHAPATRA D K,BHARTI S K.PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways inhibitors as anticancer agents:Structural and pharmacological perspectives[J].Eur J Med Chem,2016,109:314-341.
[13]REDDY K B,NABHA S M,ATANASKOVA N.Role of MAP kinase in tumor progression and invasion[J].Cancer Metast Rev,2003,22(4):395-403.
[14]SUN Y,LIU W Z,LIU T,et al.Signaling pathway of MAPK/ERK in cell proliferation,differentiation,migration,senescence and apoptosis[J].J Recept Signal Transduct Res,2015,35(6):600-604.
[15]CALVO N,MARTíN M J,DE BOLAND A R,et al.Involvement of ERK1/2,p38 MAPK,and PI3K/Akt signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells[J].Biochem Cell Biol,2014,92(4):305-315.
[16]MCCUBREY J A,MILELLA M,TAFURI A,et al.Targeting the Raf/MEK/ERK pathway with small-molecule inhibitors[J].Curr Opin Investig Drugs,2008,9(6):614.
[17]WANG X,SUN D R,CHEN Z,et al.RAF may induce cell proliferation through hypermethylation of tumor suppressor gene promoter in gastric epithelial cells.[J].Cancer Sci,2010,100(1):117-125.
[18]NORMANNO N,LUCA A D,MAIELLO M R,et al.The MEK/MAPK pathway is involved in the resistance of breast cancer cells to the EGFR tyrosine kinase inhibitor gefitinib[J].J Cell Physiol,2010,207(2):420-427.
[19]宋霆婷,姜玉华,李妙玉,等.PD98059对肝癌细胞RasMAPK信号转导的作用[J].实用医学杂志,2005,21(5):446-448.
[20]FUJIMORI Y,INOKUCHI M,TAKAGI Y,et al.Prognostic value of RKIP and p-ERK in gastric cancer[J].J Exp Clin Cancer Res,2012,31(1):30.
[21]LIANG B,WANG S,ZHU X G,et al.Increased expression of mitogen-activated protein kinase and its upstream regulating signal in human gastric cancer[J].World J Gastroenterol,2005,11(5):623-628.
[22]CORONA G,DEIANA M,INCANI A,et al.Hydroxytyrosol inhibits the proliferation of human colon adenocarcinoma cells through inhibition of ERK1/2 and cyclin D1[J].Mol Nutr Food Res,2010,53(7):897-903.
[23]SUN C H,CHANG S H,SUN C Y,et al.Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells[J].J Vet Sci,2009,10(1):23-28.
[24]MOON D O,PARK C,HEO M S,et al.PD98059triggers G1 arrest and apoptosis in human leukemic U937cells through downregulation of Akt signal pathway[J].Int Immunopharmacol,2007,7(1):36-45.
[25]WU R,LI H,LI T,et al.Myostatin regulates miR-431expression via the Ras-Mek-Erk signaling pathway[J].Biochem Biophys Res Commun,2015,461(2):224-229.
[26]李楠.小鼠巨噬细胞S100A9/NF-κB/SAA3/mir-204环路在炎症相关性结肠癌发展中的作用[D].长沙:中南大学,2013.