瑞戈非尼逆转肝癌细胞对索拉非尼耐受及作用机制
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摘要
目的探讨瑞戈非尼逆转肝癌细胞对索拉非尼耐受及作用机制。方法培养人肝癌细胞株SMMC-7721,通过长时间渐进加量刺激的方法诱导构建对索拉非尼耐受的7721-SR细胞,用0、2、4μmol/L索拉非尼处理SMMC-7721和7721-SR细胞,检测细胞活力和细胞凋亡。Transwell和划痕实验检测细胞侵袭和迁移能力,Western blot实验检测E钙黏蛋白(E-cadherin)、波形蛋白、磷酸化MET(P-MET)、磷酸化细胞外调节蛋白激酶(P-ERK)和磷酸化蛋白激酶B(P-Akt)表达情况。用瑞戈非尼0.5μmol/L预处理7721-SR细胞6h,检测细胞活力、细胞凋亡、细胞侵袭及相关蛋白表达情况。结果用0、2、4μmol/L索拉非尼培养SMMC-7721、7721-SR细胞,发现7721-SR细胞活力高于SMMC-7721细胞,7721-SR细胞凋亡少于SMMC-7721细胞(P<0.05)。与SMMC-7721细胞比较,7721-SR细胞侵袭和迁移能力增强,E-cadherin表达下降,波形蛋白、P-MET、P-ERK和P-Akt表达上调(P<0.05)。用MET抑制剂克唑替尼、ERK抑制剂U0126和Akt抑制剂MK-2206 2HCl处理7721-SR细胞,发现细胞活力降低(P<0.05),E-cadherin表达上调,波形蛋白表达下调(P<0.05)。用瑞戈非尼预处理的7721-SR细胞活力降低,细胞凋亡增加,P-ERK、波形蛋白表达下调,E-cadherin表达上调(P<0.05),细胞侵袭和迁移能力受到了抑制(P<0.05)。结论对索拉菲尼耐受的肝癌细胞中MET/ERK和MET/Akt通路被激活,发生了上皮间质转化(EMT);瑞戈非尼能够通过抑制ERK通路和阻断EMT的发生,来逆转肝癌细胞对索拉菲尼耐受。
Objective To investigate the effect and mechanism of regorafenib on reversing the sorafenib resistance in hepatocellular carcinoma cells.Methods Human hepatocellular carcinoma cells(SMMC-7721)were cultured and 7721-SR cells tolerant to sorafenib was constructed by long term incremental stimulation.After treated with sorafenib 0,2 and 4μmol/L,the cell viability and apoptosis of SMMC-7721 and 7721-SR cells were detected.The cell invasion and migration were detected by Transwell and scratch test and the expressions of E-cadherin,vimentin,phosphorylation of MET(P-MET),phosphorylation of extracellular regulated protein kinase(P-ERK)and phosphorylation of protein kinase B(P-Akt)were detected by Western blot analysis.7721-SR cells were pretreated with regorafenib 0.5μmol/L and the cell viability,cell apoptosis,cell invasion and protein expressions were detected.Results The cell viability was higher and cell apoptosis was lower in 7721-SR cells than those in SMMC-7721 cells after treated with different concentrations of sorafenib(P<0.05).Compared to SMMC-7721 cells,7721-SR cells had higher cell invasion and migration ability,lower expression of E-cadherin and higher expressions of vimentin,P-MET,P-ERK and P-Akt(P<0.05).The cell viability was lower,E-cadherin expression was higher and vimentin expression was lower after the 7721-SR cells were treated with MET inhibitor crizotinib,ERK inhibitor U0126 and Akt inhibitor MK-2206 2 HCl(P<0.05).The cell viability was lower,cell apoptosis was higher,expressions of P-ERK and vimentin were lower,E-cadherin was higher and cell invasion and migration were inhibited after the 7721-SR cells were pretreated with regorafenib.Conclusion The MET/ERK and MET/Akt pathways are activated and epithelial-mesenchymal transition(EMT)occurrs in sorafenib-tolerant hepatocellular carcinoma cells.Regorafenib can reverse the tolerance of liver cancer cells to sorafenib by inhibiting the ERK pathway and blocking the occurrence of EMT.
Objective To investigate the effect and mechanism of regorafenib on reversing the sorafenib resistance in hepatocellular carcinoma cells.Methods Human hepatocellular carcinoma cells(SMMC-7721)were cultured and 7721-SR cells tolerant to sorafenib was constructed by long term incremental stimulation.After treated with sorafenib 0,2 and 4μmol/L,the cell viability and apoptosis of SMMC-7721 and 7721-SR cells were detected.The cell invasion and migration were detected by Transwell and scratch test and the expressions of E-cadherin,vimentin,phosphorylation of MET(P-MET),phosphorylation of extracellular regulated protein kinase(P-ERK)and phosphorylation of protein kinase B(P-Akt)were detected by Western blot analysis.7721-SR cells were pretreated with regorafenib 0.5μmol/L and the cell viability,cell apoptosis,cell invasion and protein expressions were detected.Results The cell viability was higher and cell apoptosis was lower in 7721-SR cells than those in SMMC-7721 cells after treated with different concentrations of sorafenib(P<0.05).Compared to SMMC-7721 cells,7721-SR cells had higher cell invasion and migration ability,lower expression of E-cadherin and higher expressions of vimentin,P-MET,P-ERK and P-Akt(P<0.05).The cell viability was lower,E-cadherin expression was higher and vimentin expression was lower after the 7721-SR cells were treated with MET inhibitor crizotinib,ERK inhibitor U0126 and Akt inhibitor MK-2206 2 HCl(P<0.05).The cell viability was lower,cell apoptosis was higher,expressions of P-ERK and vimentin were lower,E-cadherin was higher and cell invasion and migration were inhibited after the 7721-SR cells were pretreated with regorafenib.Conclusion The MET/ERK and MET/Akt pathways are activated and epithelial-mesenchymal transition(EMT)occurrs in sorafenib-tolerant hepatocellular carcinoma cells.Regorafenib can reverse the tolerance of liver cancer cells to sorafenib by inhibiting the ERK pathway and blocking the occurrence of EMT.
引文
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[15]Firtina Karagonlar Z,Koc D,Iscan E,et al.Elevated hepatocyte growth factor expression as an autocrine c-Met activation mechanism in acquired resistance to sorafenib in hepatocellular carcinoma cells[J].Cancer Sci,2016,107(4):407-416.
[16]Zhang PF,Li KS,Shen YH,et al.Galectin-1induces hepatocellular carcinoma EMT and sorafenib resistance by activating FAK/PI3K/AKT signaling[J].Cell Death Dis,2016,7:e2201.
[17]Huang XY,Ke AW,Shi GM,et al.αB-crystallin complexes with 14-3-3ζto induce epithelial-mesenchymal transition and resistance to sorafenib in hepatocellular carcinoma[J].Hepatology,2013,57(6):2235-2247.
[18]Bae JS,Noh SJ,Kim KM,et al.Serum response factor induces epithelial to mesenchymal transition with resistance to sorafenib in hepatocellular carcinoma[J].Int J Oncol,2014,44(1):129-136.
[19]Fan LC,Teng HW,Shiau CW,et al.Regorafenib(Stivarga)pharmacologically targets epithelial-mesenchymal transition in colorectal cancer[J].Oncotarget,2016,7(39):64136-64147.
[20]Fan LC,Teng HW,Shiau CW,et al.SHP-1is a target of regorafenib in colorectal cancer[J].Oncotarget,2014,5(15):6243-6251.
[2]EL-Serag HB.Hepatocellular carcinoma[J].N Engl J Med,2011,365(12):1118-1127.
[3]Yeo W,Mok TS,Zee B,et al.A randomized phaseⅢstudy of doxorubicin versus cisplatin/interferon alpha-2b/doxorubicin/fluorouracil(PIAF)combination chemotherapy for unresectable hepatocellular carcinoma[J].J Natl Cancer Inst,2005,97(20):1532-1538.
[4]Bruix J,Gores GJ,Mazzaferro V.Hepatocellular carcinoma:clinical frontiers and perspectives[J].Gut,2014,63(5):844-855.
[5]Llovet JM,Ricci S,Mazzaferro V,et al.Sorafenib in advanced hepatocellular carcinoma[J].N Engl J Med,2008,359(4):378-390.
[6]Cheng AL,Kang YK,Chen Z,et al.Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma:aphaseⅢrandomised,doubleblind,placebo-controlled trial[J].Lancet Oncol,2009,10(1):25-34.
[7]Bruix J,Qin S,Merle P,et al.Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment(RESORCE):a randomised,double-blind,placebocontrolled,phase 3trial[J].Lancet,2017,389(10064):56-66.
[8]陈卫波,蔡辉华,杨岳,等.瑞戈非尼治疗肝细胞癌的研究进展[J].临床肝胆病杂志,2017,33(12):2425-2428.
[9]Carr BI,Cavallini A,Lippolis C,et al.Fluoro-Sorafenib(Regorafenib)effects on hepatoma cells:growth inhibition,quiescence,and recovery[J].J Cell Physiol,2013,228(2):292-297.
[10]Tai WT,Chu PY,Shiau CW,et al.STAT3 mediates regorafenib-induced apoptosis in hepatocellular carcinoma[J].Clin Cancer Res,2014,20(22):5768-5776.
[11]Mir N,Jayachandran A,Dhungel B,et al.Epithelial-to-mesenchymal transition:a mediator of sorafenib resistance in advanced hepatocellular carcinoma[J].Curr Cancer Drug Targets,2017,17(8):698-706.
[12]Han P,Li H,Jiang X,et al.Dual inhibition of Akt and c-Met as a second-line therapy following acquired resistance to sorafenib in hepatocellular carcinoma cells[J].Mol Oncol,2017,11(3):320-334.
[13]Hardin H,Guo Z,Shan W,et al.The roles of the epithelialmesenchymal transition marker PRRX1and miR-146b-5p in papillary thyroid carcinoma progression[J].Am J Pathol,2014,184(8):2342-2354.
[14]Cecchi F,Rabe DC,Bottaro DP.Targeting the HGF/Met signalling pathway in cancer[J].Eur J Cancer,2010,46(7):1260-1270.
[15]Firtina Karagonlar Z,Koc D,Iscan E,et al.Elevated hepatocyte growth factor expression as an autocrine c-Met activation mechanism in acquired resistance to sorafenib in hepatocellular carcinoma cells[J].Cancer Sci,2016,107(4):407-416.
[16]Zhang PF,Li KS,Shen YH,et al.Galectin-1induces hepatocellular carcinoma EMT and sorafenib resistance by activating FAK/PI3K/AKT signaling[J].Cell Death Dis,2016,7:e2201.
[17]Huang XY,Ke AW,Shi GM,et al.αB-crystallin complexes with 14-3-3ζto induce epithelial-mesenchymal transition and resistance to sorafenib in hepatocellular carcinoma[J].Hepatology,2013,57(6):2235-2247.
[18]Bae JS,Noh SJ,Kim KM,et al.Serum response factor induces epithelial to mesenchymal transition with resistance to sorafenib in hepatocellular carcinoma[J].Int J Oncol,2014,44(1):129-136.
[19]Fan LC,Teng HW,Shiau CW,et al.Regorafenib(Stivarga)pharmacologically targets epithelial-mesenchymal transition in colorectal cancer[J].Oncotarget,2016,7(39):64136-64147.
[20]Fan LC,Teng HW,Shiau CW,et al.SHP-1is a target of regorafenib in colorectal cancer[J].Oncotarget,2014,5(15):6243-6251.
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