DHPG对食管鳞癌细胞KYSE-450增殖的作用

摘要

目的 探讨Ⅰ组代谢型谷氨酸受体激动剂3,5二羟基苯甘氨酸(DHPG)对食管鳞癌细胞KYSE-450增殖的影响及其可能的机制.方法 体外常规培养KYSE-450,采用Western印迹检测Ⅰ组代谢型谷氨酸受体在食管鳞癌细胞的表达情况,应用四甲基偶氮唑盐(MTT)法检测不同浓度DHPG对KYSE-450细胞增殖的影响,通过Western印迹检测药物作用后KYSE-450细胞中AKT、mTOR、4EBP1蛋白及其磷酸化水平的变化.结果 mGluR1和mGluR5在KYSE-450中均有表达.50、100、150 μmol/L DHPG对KYSE-450细胞增殖均有抑制作用,且呈剂量和时间依赖性.空白对照组细胞胞质透亮,折光性强,细胞呈梭形、不规则星形.100 μmol/L DHPG加药组细胞形态随药物浓度增加及作用时间的延长出现皱缩,细胞核固缩成点状,有时出现空泡状.与对照组相比,100μmol/L DHPG加药72 h后,p-AKT、p-mTOR、p-4EBP1水平均明显降低.结论 Ⅰ组代谢型谷氨酸受体激动剂DHPG能够抑制KYSE-450的增殖,其机制可能与抑制AKT/mTOR信号通路相关.%Objective To study the effect of DHPG on the proliferation of esophageal squamous carcinoma KYSE-450 cell line and the possible mechanisms.Methods KYSE-450 cells were cultured and MTT assay was performed to investigate the effect of DHPG on the proliferation of KYSE-450 cells.Western blot was utilized to detect the protein expression of group Ⅰ metabotropic glutamate receptors both in Het-1A cells derived from normal esophageal epithelia tissue and KYSE-450 cells derived from ESCC tissue.The alteration of phosphorylation of AKT,mTOR as well as 4EBP1 induced by DHPG treatment was examined by Western blot.Results Both mGluR1 and mGluR5 were expressed in KYSE-450 cells.Application of DHPG obviously repressed KYSE-450 cell proliferation in a dose and time dependent manner.Phase contrast imaging showed that untreated control KYSE-450 cells were epithelial-like adherent cells,with a flat and polygonal shape.When treated with 100μmol/L DHPG,the cells gradually appeared to shrink and nuclei were progressively condensed into spots,and sometimes vacuolated,following the duration of drug treatment.When compared with the control group,the protein levels of p-AKT,p-mTOR and p-4EBP1 were significantly decreased at 72 h after 100 μmol/L DHPG administration.Conclusions DHPG could inhibit KYSE-450 cell proliferation and the mechanism might be related to AKT/mTOR signaling pathway.