Zhang Xiao-jing, Zhang Liang, Liu Yun-peng, Xu Hui-mian, Sun Ping, Song Jin-gang, Luo Ya-hong
Department of Bone and Soft Tissue Tumors, Liaoning Tumor Hospital, Clinical Cancer Institute, Dalian Medical University, Shenyang 110042, China.
Zhonghua Zhong Liu Za Zhi. 2013 Mar;35(3):181-6. doi: 10.3760/cma.j.issn.0253-3766.2013.03.005.
To study the molecular mechanism of epidermal growth factor receptor (EGFR) signaling pathway in mediating paclitaxel-resistance and improving paclitaxel sensitivity in human melanoma A375 cells.
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.
Paclitaxel (0.001 µmol/L to 0.1 µmol/L) inhibited the growth of A375 cells (P < 0.01) 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 0.01 µmol/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.
Human melanoma A375 cells produce resistance to paclitaxel (0.001 to 0.1 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.
研究表皮生长因子受体（EGFR）信号通路在介导人黑色素瘤A375细胞对紫杉醇耐药及提高紫杉醇敏感性中的分子机制。
用不同浓度的紫杉醇处理人黑色素瘤细胞系A375细胞，同时加入或不加入20 μmol/L AG1478（EGFR抑制剂）、40 μmol/L PD98059（细胞外信号调节激酶（ERK）1/2阻断剂）或10 μmol/L LY294002（PI3K抑制剂）。采用MTT法检测A375细胞的增殖情况。用流式细胞术检测A375细胞的细胞周期和凋亡情况。通过蛋白质免疫印迹分析检测P-EGFR、P-ERK和P-AKT蛋白的表达。
紫杉醇（0.001 μmol/L至0.1 μmol/L）以剂量和时间依赖性方式抑制A375细胞的生长（P < 0.01）并诱导凋亡（P < 0.05）。AG1478（20 μmol/L）使72小时时0.01 μmol/L紫杉醇诱导的抑制率从38.5%提高到62.6%。不同剂量的紫杉醇通过不同方式诱导A375细胞凋亡，其中0.01 μmol/L时G0/G1期细胞减少，有丝分裂期延长，0.1 μmol/L紫杉醇使细胞周期阻滞于G2/M期。当暴露于紫杉醇的A375细胞发生DNA损伤时，P-EGFR、P-ERK和P-AKT蛋白的表达增加。当EGFR信号通路被阻断时，紫杉醇在体外未激活MAPK信号通路或PI3K/AKT信号通路，且对细胞周期的影响未改变。当用20 μmol/L酪氨酸磷酸化抑制剂AG1478抑制EGFR时，0.001和0.01 μmol/L紫杉醇诱导的A375细胞早期凋亡率分别提高了1.73倍和1.80倍。当用40 μmol/L PD98059阻断ERK信号时，0.001和0.01 μmol/L紫杉醇诱导的A375细胞早期凋亡率分别提高了2.73倍和2.25倍。当用10 μmol/L LY294002阻断AKT信号时，0.001和0.01 μmol/L紫杉醇诱导的A375细胞早期凋亡率分别提高了2.02倍和1.46倍。
人黑色素瘤A375细胞通过激活MAPK信号通路和PI3K/AKT信号通路对紫杉醇（0.001至0.1 μmol/L）产生耐药。靶向EGFR、ERK和AKT信号通路可显著增强紫杉醇对人黑色素瘤细胞的细胞毒性作用。
