银杏双黄酮对未分化型甲状腺癌细胞顺铂耐药性的影响与机制研究

目的:探究银杏双黄酮对未分化型甲状腺癌（ATC）细胞顺铂（DDP）耐药性的影响与分子机制。方法:体外培养人ATC细胞8505C，并构建8505C DDP耐药细胞株（8505C/DDP）。用不同浓度的DDP（1.25、2.5、5、10、20 μg/mL）或银杏双黄酮（1.25、2.5、5、10、20 μmol/L）处理后，CCK-8法检测DDP、银杏双黄酮对8505C、8505C/DDP细胞的毒性作用以及两者联合对8505C/DDP细胞的毒性作用。将8505C/DDP细胞分为DDP组、DDP+银杏双黄酮组、DDP+AMPK抑制剂组、DDP+银杏双黄酮+AMPK抑制剂组，CCK-8法检测细胞增殖活力；Annexin V-FITC/PI双染法检测细胞凋亡情况；划痕愈合实验检测细胞迁移能力；Transwell实验检测细胞侵袭能力；Western Blot检测AMPK/mTOR信号通路相关蛋白表达。结果:DDP对8505C/DDP细胞的半数抑制浓度（IC50）高于8505C细胞（P＜0.05）；银杏双黄酮对8505C/DDP、8505C细胞的IC50无明显差异（P>0.05）；银杏双黄酮可增强DDP对8505C/DDP细胞的敏感性（P＜0.05）。与8505C细胞比较，8505C/DDP细胞中p-AMPK/AMPK比值降低，p-mTOR/mTOR比值升高（P＜0.05）；银杏双黄酮可上调8505C细胞和8505C/DDP细胞中p-AMPK/AMPK比值，降低p-mTOR/mTOR比值（P＜0.05）。与DDP组比较，DDP+银杏双黄酮组8505C/DDP细胞增殖活力、划痕愈合率、侵袭数量、p-mTOR/mTOR比值降低，凋亡率、p-AMPK/AMPK比值升高（P＜0.05）；与DDP+银杏双黄酮组比较，DDP+银杏双黄酮+AMPK抑制剂组细胞增殖活力、划痕愈合率、侵袭数量、p-mTOR/mTOR比值升高，凋亡率、p-AMPK/AMPK比值降低（P＜0.05）。结论:银杏双黄酮可降低8505C/DDP细胞对DDP的耐药性，增强DDP对8505C/DDP细胞增殖、迁移和侵袭的抑制作用以及对细胞凋亡的诱导作用，其作用机制可能与激活AMPK/mTOR信号通路有关。

Effect and mechanism of ginkgetin on cisplatin resistance of anaplastic thyroid cancer cells

Objective:To investigate the effect and molecular mechanism of ginkgetin on cisplatin (DDP) resistance in anaplastic thyroid cancer (ATC) cells.Methods:Human ATC cells 8505C were cultured in vitro and 8505C DDP resistant cell lines (8505C/DDP) were constructed and treated with DDP (1.25,2.5,5,10,20 μg/mL) or ginkgetin (1.25,2.5,5,10,20 μmol/L).CCK-8 assay was used to detect the toxicity of DDP and ginkgetin on 8505C and 8505C/DDP cells and the toxicity of the combination of DDP and ginkgetin on 8505C/DDP cells.The 8505C/DDP cells were separated into DDP group,DDP+ginkgetin group,DDP+AMPK inhibitor group,and DDP+ginkgetin+AMPK inhibitor group.CCK-8 assay was applied to detect cell proliferative viability.Annexin V-FITC/PI double staining was applied to detect cell apoptosis.Scratch-healing assay was applied to examine cell migration ability.Transwell assay was used to detect cell invasiveness.Western Blot was applied to detect expression of proteins related to AMPK/mTOR signaling pathway.Results:The IC50 of DDP on 8505C/DDP cells was higher than that in 8505C cells (P＜0.05).There was no obvious difference in IC50 of ginkgetin on 8505C/DDP and 8505C cells (P>0.05).Ginkgetin could enhance the sensitivity of DDP to 8505C/DDP cells (P＜0.05).Compared with 8505C cells,the p-AMPK/AMPK ratio in 8505C/DDP cells was decreased,while the p-mTOR/mTOR ratio was increased (P＜0.05).Ginkgetin could increase p-AMPK/AMPK ratio and decrease p-mTOR/mTOR ratio in 8505C cells and 8505C/DDP cells (P＜0.05).Compared with DDP group,the proliferation activity,scratch healing rate,invasion number and p-mTOR/mTOR ratio of 8505C/DDP cells in DDP+ginkgetin group were decreased,while the apoptosis rate and p-AMPK/AMPK ratio were increased (P＜0.05).Compared with DDP+ginkgetin group,cell proliferation activity,scratch healing rate,invasion number and p-mTOR/mTOR ratio were increased in DDP+ginkgetin+AMPK inhibitor group,while apoptosis rate and p-AMPK/AMPK ratio were decreased (P＜0.05).Conclusion:Ginkgetin can reduce the resistance of 8505C/DDP cells to DDP,and enhance the inhibition of DDP on proliferation,migration and invasion of 8505C/DDP cells,as well as the induction of apoptosis,and the mechanism of action may be related to the activation of AMPK/mTOR signaling pathway.