摘要
目的研究白杨素(Chrysin,CHR,5,7-二羟基黄酮)通过AKT/mTOR通路在肺癌细胞中诱导自噬的机制。方法培养肺癌细胞A549及PC9并通过行细胞活力实验、克隆形成实验检测白杨素对肺癌细胞增殖能力的影响;通过行透射电镜、免疫荧光实验、蛋白质印记、活性氧实验等探究AKT/mTOR通路在肺癌细胞中诱导自噬的机制。结果1.细胞增殖实验、克隆形成实验结果表明,白杨素可以以时间浓度依赖性的方式抑制肺癌细胞增殖。2.透射电镜的结果显示,白杨素能够增加肺癌细胞中自噬小体和自噬溶酶体数量。3.免疫荧光的实验结果表明,在白杨素作用后的肺癌细胞中,检测到LC3Ⅱ的细胞内定位和丰富的LC3Ⅱ斑点,而对照组细胞没有表现出明显的荧光强度。4.一定浓度的白杨素上调了肺癌细胞中LC3Ⅱ和Beclin 1蛋白的表达,下调了P62蛋白的表达。联合使用氯喹(CQ)后,LC3Ⅱ的表达进一步升高。5.细胞增殖实验结果表明,在加入氯喹后,白杨素对肺癌细胞增殖的抑制的能力进一步增强。6.白杨素可以促进肺癌细胞活性氧(ROS)集聚,在加入N-乙酰-L-半胱氨酸(NAC)后,LC3Ⅱ的表达下调。7.白杨素抑制了AKT/mTOR通路相关蛋白的表达,在加入NAC后,通路相关蛋白的表达升高。结论在肺癌中,活性氧介导的AKT/mTOR通路的失活参与了白杨素诱导的自噬,自噬抑制剂可以进一步增强白杨素的抗癌效果。
Objective To investigate the mechanism of autophagy induced by Chrysin(CHR,5,7-dihydroxyflavone)in lung cancer cells via the AKT/mTOR pathway.Methods Lung cancer cells A549 and PC9 were cultured,and the effects of poplar on the proliferation of lung cancer cells were detected by cell viability test and clonal formation test.The mechanism of autophagy induced by the AKT/mTOR pathway in lung cancer cells was investigated by transmission electron microscopy.Results 1.The results of cell proliferation experiments and clone formation experiments indicated that Chrysin could inhibit lung cancer cell proliferation in a time-dependent manner.2.The results of transmission electron microscopy showed that chrysin could increase the number of autophagosomes and autophagosomes in lung cancer cells.3.The experimental results of immunofluorescence showed that LC3Ⅱintracellular localization and abundant LC3Ⅱspots were detected in lung cancer cells treated with chrysin,while the control group cells did not show significant fluorescence intensity.4.Chrysin upregulated the expression of LC3Ⅱand Beclin 1 proteins in lung cancer cells,while downregulated the expression of P62 protein.After the combined use of chloroquine,the expression of LC3Ⅱfurther increased.5.The results of cell proliferation experiments indicated that after the addition of chloroquine,the inhibitory effect of chrysin on lung cancer cell proliferation was further enhanced.6.Chrysin could promote ROS aggregation in lung cancer cells,and after the addition of NAC,the expression of LC3Ⅱwas downregulated.7.Chrysin inhibited the expression of AKT/mTOR pathway-related proteins,and after adding NAC,the expression of pathway-related proteins increased.Conclusion In lung cancer,the inactivation of the AKT/mTOR pathway mediated by ROS is involved in the autophagy induced by Chrysin,and autophagy inhibitors can further enhance the anticancer effect of Chrysin.
作者
项莹
韩军
武前枝
XIANG Ying;HAN Jun;WU Qianzhi(Department of Respiratory and Critical Care Medicine,The Luan Hospital affiliated with Anhui Medical University(Luan People′s Hosptial),Luan,Anhui 237000,China)
出处
《临床肺科杂志》
2024年第7期1055-1065,共11页
Journal of Clinical Pulmonary Medicine
基金
六安市人民医院科研课题面上项目(No.2022kyke23)。
