CAJ | 학술논문

目的：研究二氢青蒿素（ dihydroartemisinin， DHA）诱导NSCLC细胞毒性的分子机制。方法：将不同浓度的DHA作用NSCLC细胞系A549和NCI－H1650细胞不同时间，运用MTT法、克隆形成实验、Annexin V染色和流式细胞术测定DHA对细胞活力、克隆形成能力和细胞凋亡的影响；同时测定DHA对细胞中葡萄糖水平、ATP和乳酸含量的影响；Western blot 检测PI3K 通路活性和GLUT2表达变化；通过细胞转染实现葡萄糖转运体2（GLUT2）和Rheb在A549和NCI－H1650细胞中高表达，测定和分析DHA作用下细胞活力、细胞凋亡、葡萄糖水平、ATP含量和PI3K通路活性的变化；分析葡萄糖缺乏对DHA诱导NSCLC细胞毒性的影响。结果：与对照组比较， DHA显著抑制A549和NCI－H1650细胞活力和克隆形成能力以及诱导细胞凋亡，同时降低ATP和乳酸含量以及抑制细胞对葡萄糖的摄取，具有时间和剂量依赖效应。 Western blot结果显示，DHA能抑制PI3K通路活性和GLUT2的表达。上调GLUT2的表达和激活PI3K通路能减弱DHA对NSCLC细胞的毒性作用；葡萄糖缺乏能增强DHA对NSCLC细胞的毒性；相反，高浓度葡萄糖则抑制DHA对NSCLC细胞的毒性。结论： DHA能抑制NSCLC细胞活力和克隆形成，诱导细胞凋亡，该作用是通过降低PI3K活性和GLUT2的表达进而抑制细胞糖酵解代谢实现的。
목적：연구이경청호소（ dihydroartemisinin， DHA）유도NSCLC세포독성적분자궤제。방법：장불동농도적DHA작용NSCLC세포계A549화NCI－H1650세포불동시간，운용MTT법、극륭형성실험、Annexin V염색화류식세포술측정DHA대세포활력、극륭형성능력화세포조망적영향；동시측정DHA대세포중포도당수평、ATP화유산함량적영향；Western blot 검측PI3K 통로활성화GLUT2표체변화；통과세포전염실현포도당전운체2（GLUT2）화Rheb재A549화NCI－H1650세포중고표체，측정화분석DHA작용하세포활력、세포조망、포도당수평、ATP함량화PI3K통로활성적변화；분석포도당결핍대DHA유도NSCLC세포독성적영향。결과：여대조조비교， DHA현저억제A549화NCI－H1650세포활력화극륭형성능력이급유도세포조망，동시강저ATP화유산함량이급억제세포대포도당적섭취，구유시간화제량의뢰효응。 Western blot결과현시，DHA능억제PI3K통로활성화GLUT2적표체。상조GLUT2적표체화격활PI3K통로능감약DHA대NSCLC세포적독성작용；포도당결핍능증강DHA대NSCLC세포적독성；상반，고농도포도당칙억제DHA대NSCLC세포적독성。결론： DHA능억제NSCLC세포활력화극륭형성，유도세포조망，해작용시통과강저PI3K활성화GLUT2적표체진이억제세포당효해대사실현적。
[ ABSTRACT ] AIM: To investigate the molecular mechanisms of cytotoxicity induced by dihydroartemisinin ( DHA) in non-small cell lung cancer ( NSCLC) cells.METHODS:NSCLC cell lines A549 and NCI-H1650 were treated with various concentrations of DHA for indicated time.Subsequently, the effects of DHA on the cell activity, colony forma-tion ability and apoptosis were determined by MTT assay, colony formation assay, Annexin V-FITC/PI staining and flow cy-tometry, respectively.At the same time, the effects of DHA on glucose, ATP and lactate levels were assessed, and the PI3K pathway activation and glucose transporter 2 ( GLUT2) expression were detected by Western blot in the A549 cells and NCI-H1650 cells.Overexpression of GLUT2 and Rheb was established in A549 and NCI-H1650 cells by transfection with GST-GLUT2 and GST-Rheb plasmids, respectively, and the effects of DHA on cell activity, apoptosis, glucose level, ATP content and PI3K pathway activation were analyzed in A549 cells and NCI-H1650 cells.The effect of glucose depriva-tion on the cytotoxicity triggered by DHA in NSCLC cells was also determined.RESULTS:Compared with control group, DHA significantly inhibited cell activity and colony formation ability, and induced remarkable cell apoptosis in the A549 cells and NCI-H1650 cells.At the same time, DHA reduced ATP and lactate contents, and hindered glucose uptake in a time-and dose-dependent manner in A549 cells and NCI-H1650 cells.The activity of PI3K pathway and GLUT2 expression were downregulated, while upregulated GLUT2 expression and activated PI3K pathway reduced the cytotoxicity induced by DHA in NSCLC cells.Glucose deprivation increased DHA-mediated cytotoxicity in NSCLC cells.On the contrary, high levels of glucose inhibited DHA-mediated cytotoxicity in NSCLC cells.CONCLUSION: DHA restrains cell activity and colony formation, and induces apoptosis.DHA induces cytotoxicity via inhibiting PI3K pathway activation and GLUT2 ex-pression, leading to inhibit glycolytic metabolism in NSCLC cells.