丹参酮Ⅱ_(A)通过调控PI3K/Akt/mTOR信号通路对食管癌细胞放疗敏感性的影响  

Effect of tanshinone II_(A)on radiotherapy sensitivity of esophageal cancer cells by regulating PI3K/Akt/mTOR signaling pathway

作　　者：张倩 刘艳娇[1] 贾晓龙 何龙英 史志杰 马丽 张楠楠 李晓鹏 ZHANG Qian;LIU Yan-jiao;JIA Xiao-long;HE Long-ying;SHI Zhi-jie;MA Li;ZHANG Nan-nan;LIXiao-peng(Handan First Hospital,Handan 056002,China)

机构地区：[1]邯郸市第一医院,河北邯郸056002

出　　处：《现代药物与临床》2023年第11期2645-2654,共10页Drugs & Clinic

基　　金：邯郸市科学技术研究与发展计划项目(22422083056ZC)。

摘　　要：目的研究丹参酮Ⅱ_(A)对人食管癌细胞放疗敏感性的影响,并基于磷脂酰肌醇3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路探讨其潜在机制。方法以人食管癌Eca-109细胞为受试细胞,分别采用不同浓度丹参酮Ⅱ_(A)和不同放射剂量处理Eca-109细胞,48 h后MTT法检测细胞增殖活力,计算丹参酮Ⅱ_(A)半数抑制浓度(IC_(50))和放射的IC_(50),分别作为后续实验丹参酮Ⅱ_(A)浓度和放射剂量。取对数生长期Eca-109细胞,设对照组、丹参酮Ⅱ_(A)组、放射组、丹参酮Ⅱ_(A)+放射组、丹参酮Ⅱ_(A)+放射+PI3K抑制剂(LY294002)组。通过平板克隆实验、MTT法、流式细胞术、荧光质粒转染法检测细胞克隆形成能力、增殖活力、凋亡率和自噬状况,RT-PCR、Western blotting法检测细胞中PI3K/Akt/mTOR信号通路相关mRNA和蛋白表达。结果丹参酮Ⅱ_(A)和放射对人食管癌Eca-109细胞增殖活力的抑制作用均呈现剂量相关性,丹参酮Ⅱ_(A)IC_(50)为8.75 mmol/L,放射量IC_(50)为4.63 Gy。与对照组相比,丹参酮Ⅱ_(A)组、放射组、丹参酮Ⅱ_(A)+放射组、丹参酮Ⅱ_(A)+放射+LY294002组细胞克隆形成能力和增殖活力明显降低,凋亡率和自噬体数量明显升高(P<0.05);PI3K、Akt、mTOR mRNA和蛋白表达量明显降低(P<0.05);Bcl-2、Bax、cleaved Caspase-3、LC3-I、LC3-II蛋白表达量及Bax/Bcl-2、Cleaved Caspase-3/Caspase-3、LC3-II/LC3-I明显升高(P<0.05)。与丹参酮Ⅱ_(A)组或放射组相比,丹参酮Ⅱ_(A)+放射组和丹参酮Ⅱ_(A)+放射+LY294002组对各检测指标的调控作用明显增强(P<0.05)。与丹参酮Ⅱ_(A)+放射组相比,丹参酮Ⅱ_(A)+放射+LY294002组对各指标的调控作用明显增强(P<0.05)。结论丹参酮Ⅱ_(A)可能通过下调PI3K/Akt/mTOR信号通路,抑制细胞增殖并促进其凋亡与自噬,进而增强人食管癌细胞放疗敏感性。Objective To investigate the effect of tanshinone IIA on radiotherapy sensitivity of human esophageal carcinoma cells,and explore its potential mechanism based on the PI3K/Akt/mTOR signaling pathway.Methods The human esophageal cancer Eca-109 cells was used as test cells,which were treated with different concentrations of tanshinone IIA and different radiation doses respectively.48 h later,the cell proliferation activity was detected by MTT method,the half-inhibitory concentration of IC_(50)tanshinone IIA(as the concentration of tanshinone IIA for follow-up experiments)and IC_(50)radiation(as the radiation dose for follow-up experiments)were calculated respectively.Take the Eca-109 cells in logarithmic growth period and set blank group,tanshinone IIA group,radiation group,tanshinone IIA+radiation group,tanshinone IIA+radiation+PI3K inhibitor(LY294002)group.The cloning ability,proliferation activity,apoptosis rate,and autophagy were detected by plate cloning assay,MTT assay,flow cytometry or fluorescent plasmid transfection.The PI3K/Akt/mTOR signaling pathway related mRNA and protein expression were detected by RT-PCR or Western blotting method.Results Both tanshinone IIA and radiation showed dose-dependent inhibitory effects on the proliferation of human esophageal cancer Eca-109 cells.The IC_(50)tanshinone IIA was 8.75 mmol/L,and the IC_(50)radiation was 4.63 Gy.Compared with the control group,the clonogenesis ability and proliferation activity of tanshinone IIA group,radiation group,tanshinone IIA+radiation group and Tanshinone IIA+radiation+LY294002 group were significantly decreased,the apoptosis rate and autophagosome number were significantly increased(P<0.05).The mRNA expressions of PI3K,Akt,mTOR were significantly decreased(P<0.05).The protein expression of PI3K,Akt,mTOR were significantly decreased.The protein expression of Bcl-2,Bax,Cleaved Caspase-3,LC3-I,LC3-II,and the expression ratios of Bax/Bcl-2,Cleaved Caspase-3/Caspase-3,LC3-II/LC3-I were significantly increased(P<0.05).Compared with tanshinone

关 键 词：丹参酮Ⅱ_(A) 食管癌 放疗 磷脂酰肌醇3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路 凋亡 自噬 

分 类 号：R285.5[医药卫生—中药学]