毛壳素靶向Hsp90抑制食管鳞癌细胞Eca109生长的机制研究

Mechanism of chaetocin targeting Hsp90 to inhibit Eca109 proliferation in esophageal squamous carcinoma cells

目的:探讨毛壳素对食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)细胞增殖、迁移、侵袭、细胞周期和凋亡的影响及潜在作用机制。方法:采用不同浓度毛壳素处理Eca109细胞,CCK-8法、3D肿瘤成球实验、克隆形成实验检测毛壳素对食管鳞癌细胞增殖活力的影响,流式细胞仪检测毛壳素对食管鳞癌细胞周期和凋亡的影响,划痕愈合实验和Transwell实验检测毛壳素对迁移、侵袭的影响。分子对接、细胞热位移实验验证毛壳素与Hsp90的相互作用。蛋白免疫印迹法检测周期、凋亡、迁移侵袭及分子机制相关蛋白的表达变化。结果:毛壳素能显著抑制Eca109细胞的增殖活性,抑制成球能力和集落形成能力(P<0.01)。流式细胞仪检查结果显示,与对照组相比,毛壳素处理组G 2/M期的比例明显增加(P<0.001),凋亡率显著增加(P<0.01)。划痕愈合实验和Transwell实验结果表明,毛壳素处理组Eca109细胞的迁移和侵袭能力均显著降低(P<0.01)。分子对接和细胞热位移实验显示毛壳素与Hsp90具有较强的结合能力。Western blot结果显示,毛壳素处理细胞后,周期相关分子CDK1表达下调,p-Histone H3上调,凋亡相关蛋白Cleaved-PARP、Cleaved-caspase-3上调,迁移侵袭相关蛋白E-Cadherin上调,N-Cadherin和Snail下调,Hsp90客户蛋白p-EGFR、p-STAT3、p-AKT、p-ERK1/2表达下降。结论:毛壳素可能通过靶向Hsp90抑制其客户蛋白活性,从而抑制食管鳞癌细胞增殖。

Objective:To investigate the effects and mechanisms of chaetocin on cell proliferation,migration,invasion,cell cycle and apoptosis in esophageal squamous cell carcinoma(ESCC).Methods:After traeting Ecalog cells with different concentrations of chaetocin treatment Eca109 cells,CCK-8 assay,3D tumor spheroid formation assay,clone formation assay were applied to detect the influence of chaetocin on ESCC cells proliferation vitality.Flow cytometry was used to detect the influence of chaetocin on ESCC cell cycle and apoptosis.Wound healing assay and Transwell assay were applied to detect the influence of chaetocin on migration and invasion.Molecular docking and cellular thermal shift experiments verified the interaction between chaetocin and Hsp90.Cell cycle,apoptosis,migration and invasion and molecular mechanisms related protein were detected by immunoblotting.Results:Chaetocin significantly inhibited Eca109 cell survival and inhibited sphere-forming capacity and colony-forming capacity(P<0.01).Flow cytometry results showed that the percentage of G 2/M phase increased(P<0.001)and apoptosis cells increased in chaetocin group,compared with control group(P<0.01).The results of wound healing assay and Transwell showed that the migration and invasion of Eca109 cells were significantly reduced by chaetocin(P<0.01).Molecular docking and cellular thermal shift experiments suggested that chaetocin has strong binding capacity to Hsp90.Western blot results showed that the cell cycle-related proteins CDK1 was decreased,p-Histone H3 was increased,apoptosis-related proteins Cleaved-PARP and Cleaved-caspase-3 were increased,migration and invasion-related proteins E-Cadherin was decreased,N-Cadherin and Snail were decreased,Hsp90 client proteins p-EGFR,p-STAT3,p-AKT and p-ERK1/2 were decreased.Conclusion:Chaetocin cloud suppress the proliferation of ESCC cell by targeting Hsp90 inhibit the activity of its client proteins.