沉默ERK增强A375黑素瘤细胞对TRAIL诱导细胞凋亡的敏感性

Silencing ERK enhances susceptibility of A375 melanoma cells to cell apoptosis induced by TRAIL

目的：探讨直接抑制细胞外信号调节激酶（extra cellular signal-regulated kinase,ERK）表达后,对肿瘤坏死因子相关的凋亡诱导配体（TNF related apoptosis inducing ligand,TRAIL）对黑素瘤细胞杀伤作用的影响及其作用机制。方法：用携带ERK特异或对照shRNA的慢病毒感染A375黑素瘤细胞,48 h后加入终浓度为100μg/ml基因重组TRAIL蛋白继续培养6h,收获细胞,碘化丙啶染色,用流式细胞术检测细胞周期、细胞凋亡和TRAIL受体的表达;Western blotting检测相关蛋白的表达水平。结果：亚型特异的ERK shRNA分别沉默ERK1或ERK2,导致A375细胞出现G_1期阻滞（对照shRNA组的G_1期细胞比例为71%,ERK1、ERK2、ERK1＋2 shRNA组分别增加到85%、90%和81%,P〈0.01）;S期细胞从对照组的11%减少到2%左右（P〈0.001）、G_2/M期细胞也从对照组的17%减少到3%（P〈0.01）。细胞周期改变伴有P21、P27蛋白上调和Cyclin D1蛋白降低,但未见明显的细胞凋亡。经对照shRNA和TRAIL处理的细胞,仅有15%的凋亡率,而ERK shRNA和TRAIL联合处理则使凋亡率达到40%~60%（P〈0.01）。抑制ERK蛋白表达能显著上调细胞表面TRAIL受体DR4的表达（从对照组32%增加到75%~80%,P〈0.01）,但DR5变化不明显。ERK抑制还明显降低A375细胞的葡萄糖转运受体1（Glut 1）和己糖激酶Ⅱ（HK-Ⅱ）的蛋白表达水平。结论：直接抑制ERK不仅可以抑制肿瘤细胞周期的进展,而且可以增强TRAIL对A375黑素瘤细胞的杀伤作用,其机制与上调细胞表面DR4的表达和干扰肿瘤细胞糖代谢有关。

Objective： To investigate the effect of extra cellular signal-regulated kinase（ ERK） knockdown on melanoma cell apoptosis mediated by TNF related apoptosis inducing ligand（ TRAIL） and the underlying mechanisms. Methods： A375 cells were infected with lentivirus carrying either scramble shRNA（ SC shRNA） or ERK（ shRNA） for 48 hours and then treated with recombinant TRAIL protein（ 100 μg / ml） for another 6 hours. Cells were harvested and stained with PI; Flow cytometry was used to detect cell cycle,apoptosis,and the expression of TRAIL receptor; Western blotting was used to measure the expression level of relevant proteins. Results： Knockdown of either ERK1 or ERK2 by isotype specific shRNA resulted in G_1-phase growth arrest of A375 cells（ cell percentageat G_1-phase in control group was 71%,compared to 85%,90%,81% respectively in ERK1,ERK2 and ERK1 ＋ 2 shRNA groups,P〈0. 01）. Accordingly,the A375 cell percentage at S-phase was 11% in control group,compared to around 2% in various ERK shRNA groups（ P 0. 001）; the G_2/ M cell percentage was 17% in control group,compared to the around 3% in various ERK shRNA groups（ P〈0. 01）. The change of cell cycle was accompanied by up-regulation of P21 and P27 proteins,and down-regulation of cyclin D1 level,however no obvious cell apoptosis was observed. Treatment of scramble shRNA together with TRAIL caused about 15% of cell apoptosis,in contrast,the combined treatment of ERK shRNA and TRAIL increased cell apoptosis rate up to 40% ~ 60%（ P〈0. 01）. Silencing of ERK enhanced DR4 expression on TRAIL receptor（ from 32% to 75% ~ 80%,P〈0. 01）,but not DR5 expression. Furthermore,directly silencing ERK resulted in inhibited expression of Glut 1 and hexokinase Ⅱ,which are involved in the unique glucose metabolism of tumor cells. Conclusion： Directly silencing ERK with specific shRNA inhibited tumor cell growth and enhanced tumor cell apoptosis mediated by TRAIL. A combination of increased DR4 expression and impaired glucose metabolism is likely to contribute to the synergistic effect seen with TRAIL and ERK shRNA treatment in melanoma cells.