自噬对Folliculin基因缺陷的肾癌细胞放疗敏感性的影响

摘要

Objective To investigate the radiosensitivity of FLCN-deficient renal caner cells usingγ-irra-diation. ,and determine the impact of autophagy on irradiation-mediated FLCN-deficient cell death. Methods To determine the impact of FLCN on radiosensitivity of renal caner cells,we used Human renal cancer cell lines ACHN,786-O,769-P,Caki-1 and UOK257 for Clonogenic assay. To investigate the impact of autophagy on irradiation-mediated FLCN-deficient cell death,we used two pairs of cell lines:FLCN siRNA-silenced ACHN-5968 cell line and scrambled ACHN line (ACHN-sc);FLCN-null UOK257 cell line and UOK257-2 line restored with ectopic expression of FLCN. Cell apoptosis assay was carried out using TUNEL methods. Irradiation-induced autophagy was measured by LC3 western blot and GFP-LC3 assays. Results Clonogenic assay results indicated that the FLCN-deficient RCC cell UOK257 is relatively more sensitive to IR comparing with other renal cancer cell lines. In situ colorimetric TUNEL assay results indicated that FLCN-deficient renal cancer cells exhibited less irradation-induced apoptosis. Western blot and GFP-LC3 assay results indicated that autophagy occurred in these FLCN-deficient RCC cells after irradiation,and induction of autophagy could further increase radiosensitivity of FLCN-de?cient renal cancer cells. Conclusions Our results indicated that FLCN-deficient renal cancer cells exhibited higher radiosensitivity. Irradation induced autophagy in FLCN-deficient renal cancer cells,and induction of autophagy could further increase radiosensitivity of FLCN -de?cient renal cancer cells. γ-irradiation combined with autophagy inducer might be a potential therapeutic treat-ment for BHD associated tumors.%目的：利用不同剂量γ线分别照射Folliculin（FLCN）缺失或下调的肾癌细胞与高表达FL-CN的肾癌细胞，探讨FLCN蛋白对肾癌细胞γ线放射敏感性的影响，并进一步探讨联合应用自噬激活剂和γ线放射治疗BHD伴发肾癌的有效性和可行性。方法利用克隆存活分析实验测定细胞对γ线照射的敏感性，利用TUNEL法测定细胞凋亡，利用GFP－LC3荧光分析以及LC3蛋白印迹分析方法检测细胞自噬，并在抑制或激活细胞自噬后，通过克隆存活分析实验比较自噬改变后细胞的生存指数曲线。利用Western blot检测ERK信号通路的关键蛋白ERK／P－ERK，以及自噬调节蛋白Bceclin 1。结果克隆存活分析实验结果显示，无FLCN表达的UOK257肾癌细胞对γ线放射较敏感。GFP－LC3荧光分析以及LC3蛋白Western blot结果显示，相对于高表达FLCN的细胞，γ线放射可以在FLCN缺失或下调的肾癌细胞中诱导更明显的自噬反应。克隆存活分析实验结果显示，应用自噬诱导剂雷帕霉素可以增强γ线放射对UOK257和ACHN5968细胞的杀伤作用。结论 FLCN蛋白可以降低肾癌细胞对γ线放射的敏感性，同时增加γ线放射在肾癌细胞内诱导的凋亡。相对于高表达FLCN的细胞，γ线放射可以在FLCN缺失或下调的肾癌细胞中激活ERK信号通路并上调Beclin－1蛋白从而诱导更明显的自噬反应。联合应用自噬激活剂和γ线放射可能成为治疗BHD伴发肾癌的潜在有效途径。