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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 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on melanoma cells after directly inhibiting the expression of extracellular signal-regulated kinase (ERK) Influence and mechanism of action. METHODS: A375 melanoma cells were infected with lentivirus carrying ERK-specific or control shRNA. After 48 h, recombinant TRAIL protein was added to a final concentration of 100 μg / ml for 6 h. Cells were harvested and stained with propidium iodide for flow cytometry The cell cycle, apoptosis and the expression of TRAIL receptor were detected by immunohistochemistry. The expression of related protein was detected by Western blotting. Results: Subtype-specific ERK shRNA silence ERK1 or ERK2, respectively, resulting in G 1 phase arrest in A375 cells (71% of G 1 cells in control shRNA group, 85% in ERK1, ERK2 and ERK1 + 2 shRNA groups, 90%, and 81%, P <0.01). The number of cells in S phase decreased from 11% to 2% (P <0.001) in control group and from 17% to 3% in control group (P < 0.01). Cell cycle changes accompanied by P21, P27 protein upregulation and Cyclin D1 protein decreased, but no obvious apoptosis. The control shRNA and TRAIL treated cells, only 15% of the apoptotic rate, and ERK shRNA and TRAIL combined treatment rate of apoptosis reached 40% to 60% (P <0.01). Inhibition of ERK protein expression significantly upregulated the expression of TRAIL receptor DR4 on the cell surface (from 32% to 75% -80% in the control group, P <0.01), but DR5 did not change significantly. ERK inhibition also significantly decreased the protein expression of glucose transporter 1 (Glut 1) and hexokinase II (HK-Ⅱ) in A375 cells. CONCLUSION: Direct inhibition of ERK can not only inhibit tumor cell cycle progression, but also enhance the killing effect of TRAIL on A375 melanoma cells. The mechanism is related to the up-regulation of DR4 expression and the interference of tumor cell glucose metabolism.