摘要
目的研究松油烯4醇体内外抑制人非小细胞肺癌细胞株A549增殖及诱导自噬的作用。方法不同浓度松油烯4醇作用A549细胞后,应用MTT法检测抑制率及IC50;流式细胞仪检测细胞周期、凋亡、细胞内钙离子浓度变化;透射电镜观察作用前后细胞超微结构的变化。A549细胞移植瘤裸鼠药物连续腹腔注射10 d后分别测量各组移植瘤的平均体积和质量,观察药物的体内抑制肿瘤生长作用。用实时荧光定量PCR和Western blot法检测自噬相关基因BECN1和LC3的体内外表达情况。结果 MTT结果显示松油烯4醇作用于A549细胞24 h后,随着药物浓度升高抑制率逐渐升高,呈浓度依赖性(P<0.05),其24 h的IC50为0.067%(体积分数)。FCM检测结果显示,实验组细胞周期被阻滞在S期,实验组细胞总凋亡率均高于对照组(P<0.05),各实验组细胞内钙离子荧光强度均高于对照组(P<0.05)。电镜结果显示经松油烯4醇处理的A549细胞内自噬泡数量明显增加。实验组移植瘤平均瘤体积和平均瘤质量均低于对照组(P<0.05),高、低剂量实验组抑瘤率分别为77.46%和53.33%。实时荧光定量PCR及Western blot结果显示BECN1和LC3蛋白及mRNA表达量上调(P<0.05)。结论松油烯4醇在体内外可诱导人非小细胞肺癌细胞株A549自噬并抑制其增殖。
Objective To assay the role of terpinen-4-ol in inducing autophagy and inhibiting proliferation in human lung adenocarcinoma A549 cells in vitro and explore its possible mechanism related to autophagy pathway. Methods A549 cells were incubated with various concentrations of terpinen-4-ol. MTT assay was used to observe the inhibitory effect of terpinen-4-ol on the growth of A549 cells and determine its IC50. The changes of cell cycle,apoptosis and intracellular free calcium concentration were measured by flow cytometry. The ultrastructure of A549 cells were observed by transmission electron microscopy before and after terpinen-4-ol treatment. After xenograft transplantation tumor of A549 cells（ 1 × 106,with the diameter over0. 5 cm） were established in nude mice,terpinen-4-ol was injected intra-peritoneally for 10 d and then the nude mice were killed. Transplantation tumor were taken and then detected. Real-time fluorescent quantitative PCR and Western blotting were performed to detect the expression of BECN1 and LC3 in the A549 cells and the transplantation tumor. Results MTT assay showed that the viability of A549 cells was in a dose-dependent manner after the cells were treated with terpinen-4-ol for 24 h,with a IC50 value of 0. 067%. The cell cycle was arrested in S phase according to the results of flow cytometry,and cell apoptosis was induced by terpinen-4-ol and the intracellular calcium concentrations were significantly higher in treatment groups than the control group（ P〈 0.05）. Transmission electron microscopy displayed that there were more autophagic vacuoles in the A594 cells treated by terpinen-4-ol. Low and high-dose treatment resulted in the growth of the transplantation tumor obviously restrained（ tumor inhibitory rate of 53. 33% and 77. 76% respectively）. Real-time PCR and Western blot analysis showed that terpinen-4-ol inhibited the mRNA and protein levels of BECN1 and LC3（ P〈 0.05）.Conclusion Terpinen-4-ol induces autophagy and inhibits cell proliferation in A549 cells in vitro and in vivo.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2014年第21期2201-2206,共6页
Journal of Third Military Medical University
基金
重庆市科委科技攻关计划项目(CSTC2012ggB10002)~~
