CAJ | 학술논문

背景与目的:Akt通路是细胞内一条重要的信号通路,与细胞生长、凋亡密切相关,本研究旨在探讨Akt通路在热化疗诱导人小细胞肺癌细胞凋亡中的作用,进而探讨热疗抑制肺部肿瘤细胞增殖可能的机制.方法:参考临床常用剂量,采用43℃加热联合120μg/L紫杉醇(热化联合组)、43℃加热联合120μg/L紫杉醇并加入1 μmol/L Akt特异抑制剂Wortmannin(Wortmannin组)、43%加热联合120μg/L紫杉醇并加入30 μmol/L活性氧(reactive oxygen species,ROS)特异抑制剂NAC(NAC组)、单纯使用120μg/L紫杉醇(单纯化疗组)处理H446细胞,以未处理的H446细胞作为对照组,应用噻唑蓝比色法(MTT法)检测各处理方式下细胞增殖率,流式细胞术(FCM)检测细胞凋亡率,荧光法检测细胞内ROS,通过蛋白免疫印迹法(Western blot)检测Akt磷酸化水平和caspase-3的表达,采用SPSS 13.0统计软件对数据进行统计分析.结果:热化联合组细胞增殖率(59.83±3.36)%低于对照组(100.00±0.00)%和单纯化疗组(69.16±2.95)%(P<0.05);热化联合组的细胞凋亡率最高(27.59±5.47)%(P<0.05);热化联合组的ROS表达增高(102.14±18.34)(P<0.05),NAC可抑制其表达(28.01±1.19),Wortmannin对其无影响(99.87±8.35);Akt磷酸化水平在热化联合组降低(0.69±0.03)(P<0.05),Wortmannin可抑制其磷酸化(0.00±0.00),NAC使其磷酸化水平升高(1.05±0.29)(P<0.05);热化联合组的caspase-3表达(1.07±0.08)高于其他各组(P<0.05).结论:热化疗联合应用可以明显抑制H446细胞的生长,这种抑制作用可能是通过诱导ROS的产生,继而抑制Akt通路活化,并通过caspase途径导致细胞凋亡.
배경여목적:Akt통로시세포내일조중요적신호통로,여세포생장、조망밀절상관,본연구지재탐토Akt통로재열화료유도인소세포폐암세포조망중적작용,진이탐토열료억제폐부종류세포증식가능적궤제.방법:삼고림상상용제량,채용43℃가열연합120μg/L자삼순(열화연합조)、43℃가열연합120μg/L자삼순병가입1 μmol/L Akt특이억제제Wortmannin(Wortmannin조)、43%가열연합120μg/L자삼순병가입30 μmol/L활성양(reactive oxygen species,ROS)특이억제제NAC(NAC조)、단순사용120μg/L자삼순(단순화료조)처리H446세포,이미처리적H446세포작위대조조,응용새서람비색법(MTT법)검측각처리방식하세포증식솔,류식세포술(FCM)검측세포조망솔,형광법검측세포내ROS,통과단백면역인적법(Western blot)검측Akt린산화수평화caspase-3적표체,채용SPSS 13.0통계연건대수거진행통계분석.결과:열화연합조세포증식솔(59.83±3.36)%저우대조조(100.00±0.00)%화단순화료조(69.16±2.95)%(P<0.05);열화연합조적세포조망솔최고(27.59±5.47)%(P<0.05);열화연합조적ROS표체증고(102.14±18.34)(P<0.05),NAC가억제기표체(28.01±1.19),Wortmannin대기무영향(99.87±8.35);Akt린산화수평재열화연합조강저(0.69±0.03)(P<0.05),Wortmannin가억제기린산화(0.00±0.00),NAC사기린산화수평승고(1.05±0.29)(P<0.05);열화연합조적caspase-3표체(1.07±0.08)고우기타각조(P<0.05).결론:열화료연합응용가이명현억제H446세포적생장,저충억제작용가능시통과유도ROS적산생,계이억제Akt통로활화,병통과caspase도경도치세포조망.
Background and Objective: Akt pathway plays an important role in cell growth and apoptosis.This study was to characterize the role of Akt in the synergistic effects of thermo-chemotherapy on lung cancer cell growht and its underlying mechanisms.Methods: H446 cells were subjected to different thermo-chemotherapy schemes: 43℃+paclitaxel(120 μg/L)(thermo-chemotherapy group),43℃+paclitaxel(120 μg/L)+Wortmannin(1 μmol/L,PI3K/Akt pathway inhibitor)(Wortmannin group),43℃+paclitaxel(120 μg/L)+N-acety-L-cysteine(NAC)(30 μmol/L,reactive oxygen species,ROS inhibitor)(NAC group),and paclitaxel(120 μg/L)group.The cells without any treatment were used as the control.MTT assay was conducted to measure the cell proliferation rate.Cell apoptosis was analyzed by flow cytometry(FCM).ROS was detected with fluorescence.Phosphorylation of Akt and the expressions of Caspase-3 were determined by Western blot.Results: The cell proliferation rate was significantly lower in the thermo-chemotherapy group than in the control and the chemotherapy groups[(59.83±3.36)% vs.(100.00±0.00)% and(69.16±2.95)%,P<0.05].The rate of cell apoptosis was the highest in the thermo-chemotherapy group(27.59±5.47)%(P<0.05).The ROS expression level was higher in the cells of thermo-chemotherapy group(102.14±18.34)than in the other groups(P<0.05),which could be inhibited by NAC(28.01±1.19),but not by the PI3K inhibitor Wortmannin(99.87±8.35).Phosphorylation of Akt significantly decreased in the thermo-chemotherapy group(0.69±0,03)(P<0.05),which could be blocked by Wortmannin(0.00±0.00),but increased by NAC(1.05±0.29)(P<0.05).The expression level of Caspase-3 was higher in the thermo-chemotherapy group(1.07±0.08)than in other groups(P<0.05).Conclusion: Thermo-chemotherapy has a stronger inhibitory effect than chemotherapy alone in lung tumor cell growth,probably through induction of ROS production and subsequent inhibition of Akt pathway activation and Caspase pathway-induced cancer cell apoptosis.