CAJ | 학술논문

目的研究表皮生长因子受体(EGFR)信号通路调控人黑色素瘤A375细胞发生紫杉醇耐药和逆转耐药的分子机制.方法以不同浓度的紫杉醇、20 μmol/L EGFR阻断剂AG1478、40μmol/L细胞外信号调节激酶(ERK) 1/2阻断剂PD98059和10 μmol/L PI3K抑制剂LY294002单独或联合处理A375细胞,采用Western blot法检测不同处理组A375细胞中p-EGFR、p-ERK和p-AKT蛋白的表达,采用四甲基偶氮唑蓝(MTT)法检测不同处理组A375细胞的增殖抑制情况,采用流式细胞仪检测不同处理组A375细胞的细胞周期和凋亡率.结果 0.001、0.01和0.1μmol/L紫杉醇以时间和浓度依赖方式抑制A375细胞的增殖,AG1478可促进紫杉醇的细胞增殖抑制作用.0.01 μmol/L紫杉醇通过降低G0/G1期细胞含量和延长有丝分裂进程促使A375细胞发生凋亡;而0.1 μmol/L紫杉醇则使G2/M期细胞发生阻滞实现细胞凋亡.当A375细胞发生DNA损伤时,可使细胞中p-EGFR、p-ERK和p-AKT蛋白表达均升高;当阻断EGFR信号时,紫杉醇激活MAPK和PI3 K/AKT信号通路的作用消失,且不影响紫杉醇对A375细胞周期的影响.20μmol/L AG1478阻断EGFR信号后,可使0.001和0.01 μmol/L紫杉醇诱导A375细胞的早期凋亡率分别提高1.73和1.80倍;40μmol/L PD98059阻断ERK信号后,可使0.001和0.01 μmol/L紫杉醇诱导A375细胞的早期凋亡率分别提高2.73和2.25倍;10 μmol/L LY294002阻断AKT信号后,可使0.001和0.01 μmol/L紫杉醇诱导A375细胞的早期凋亡率分别提高2.02和1.46倍.结论紫杉醇通过激活MAPK细胞增殖信号通路和PI3 K/A KT抗凋亡信号通路使黑色素瘤A375细胞产生耐药,阻断EGFR、ERK和AKT 3个信号通路靶点可明显增敏紫杉醇的抗黑色素瘤作用.
목적 연구표피생장인자수체(EGFR)신호통로조공인흑색소류A375세포발생자삼순내약화역전내약적분자궤제.방법 이불동농도적자삼순、20 μmol/L EGFR조단제AG1478、40μmol/L세포외신호조절격매(ERK) 1/2조단제PD98059화10 μmol/L PI3K억제제LY294002단독혹연합처리A375세포,채용Western blot법검측불동처리조A375세포중p-EGFR、p-ERK화p-AKT단백적표체,채용사갑기우담서람(MTT)법검측불동처리조A375세포적증식억제정황,채용류식세포의검측불동처리조A375세포적세포주기화조망솔.결과 0.001、0.01화0.1μmol/L자삼순이시간화농도의뢰방식억제A375세포적증식,AG1478가촉진자삼순적세포증식억제작용.0.01 μmol/L자삼순통과강저G0/G1기세포함량화연장유사분렬진정촉사A375세포발생조망;이0.1 μmol/L자삼순칙사G2/M기세포발생조체실현세포조망.당A375세포발생DNA손상시,가사세포중p-EGFR、p-ERK화p-AKT단백표체균승고;당조단EGFR신호시,자삼순격활MAPK화PI3 K/AKT신호통로적작용소실,차불영향자삼순대A375세포주기적영향.20μmol/L AG1478조단EGFR신호후,가사0.001화0.01 μmol/L자삼순유도A375세포적조기조망솔분별제고1.73화1.80배;40μmol/L PD98059조단ERK신호후,가사0.001화0.01 μmol/L자삼순유도A375세포적조기조망솔분별제고2.73화2.25배;10 μmol/L LY294002조단AKT신호후,가사0.001화0.01 μmol/L자삼순유도A375세포적조기조망솔분별제고2.02화1.46배.결론 자삼순통과격활MAPK세포증식신호통로화PI3 K/A KT항조망신호통로사흑색소류A375세포산생내약,조단EGFR、ERK화AKT 3개신호통로파점가명현증민자삼순적항흑색소류작용.
Objective To study the molecular mechanism ot epidermal growth factor receptor (EGFR) signaling pathway in mediating paclitaxel-resistance and improving paclitaxel sensitivity in human melanoma A375 cells.Methods Human melanoma cell line A375 cells were treated with different concentrations of paclitaxel with or without 20 μmol/L AG1478 (EGFR inhibitor),40 μmol/L PD98059 (extracellular signal conditioning kinase (ERK) 1/2 blockers) or 10 μmol/L LY294002 (PI3K inhibitor).MTT method was used to measure the proliferation of A375 cells.Flow cytometry was used to detect cell cycle and apoptosis in the A375 cells.The expressions of P-EGFR,P-ERK and P-AKT proteins were determined by Western blot analysis.Results Paclitaxel (0.001 μ mol/L to 0.1 μmoL/L) inhibited the growth of A375 cells (P ＜ O.01) and induced apoptosis (P ＜ 0.05) in a dose-and time-dependent manner.AG1478 (20 μmol/L) increased the 0.01 μmol/L paclitaxel-induced inhibition rate from 38.5％ to 62.6％ at 72 h.Different doses of paclitaxel induced apoptosis in A375 cells by different ways,in which G0/G1 phase cells were decreased and mitotic phase was prolonged at O.O1 μ mol/L,and cell cycle arrest at G2/M phase by 0.1 μmol/L paclitaxel.When DNA damage occurred in A375 cells exposed to paclitaxel,expression of P-EGFR,P-ERK and P-AKT proteins was increased.When EGFR signaling pathway was blocked,paclitaxel did not activate MAPK signaling pathway or PI3K/AKT signaling pathway and did not change its effect on cell cycle in vitro.When EGFR was inhibited by 20 μmol/L tyrophostin AG1478,the 0.001 and 0.01 μmol/L paclitaxel-induced early apoptosis rate in A375 cells was increased by 1.73-and 1.80-fold,respectively.When the ERK signaling was blocked by 40 μmol/L PD98059,the 0.001 and 0.01 μmol/L paclitaxel-induced early apoptosis rate in A375 cells was increased by 2.73-and 2.25-fold,respectively.When the AKT signaling was blocked by 10 μmol/L LY294002,the 0.001 and 0.01 μmol/L paclitaxel-induced early apoptosis rate in A375 cells was increased by 2.02-and 1.46-fold,respectively.Conclusions Human melanoma A375 cells produce resistance to paclitaxel (0.001 to O.1 μmol/L) by activating MAPK signaling and PI3K/AKT signaling pathways.Targeting EGFR,ERK and AKT signaling pathways significantly enhances the cytotoxic effect of paclitaxel on human melanoma cells.