氯硝柳胺对肾上腺皮质癌SW-13细胞增殖和凋亡的影响

Effects and mechanism of niclosamide on proliferation and apoptosis of adrenocortical carcinoma SW-13 cells

目的研究氯硝柳胺对人肾上腺皮质癌SW-13细胞株细胞增殖、细胞周期、细胞凋亡和细胞迁移力、侵袭力的影响。方法通过体外培养SW-13细胞,采用CCK8法和i CELLigence实时无标记细胞增殖监测仪检测不同浓度氯硝柳胺对细胞增殖的影响;设对照组和氯硝柳胺组,流式细胞技术检测氯硝柳胺作用后SW-13细胞周期的变化,AO/EB染色法和Annexin V/PI染色流式细胞技术测定细胞凋亡率的变化,Transwell实验和细胞划痕实验检测细胞侵袭、迁移能力的变化,Western blot(WB)法检测β-catenin/LRP6表达的变化。结果不同浓度的氯硝柳胺分别作用SW-13细胞24、48、72、96、120 h后,呈时间和浓度依赖性抑制SW-13细胞增殖(F=157.93,P<0.001);流式细胞仪周期检测结果显示24、48、72 h后,与对照组相比氯硝柳胺组将SW-13细胞周期阻滞在G0/G1期(P=0.001、0.005、0.008);AO/EB染色法和Annexin V/PI染色法结果均显示氯硝柳胺组的凋亡率与对照组相比明显升高(P<0.001);Transwell实验结果示氯硝柳胺组SW-13细胞的侵袭力降低(P<0.001),细胞划痕实验结果显示氯硝柳胺组SW-13细胞的迁移能力下降(P=0.002),WB结果示氯硝柳胺组SW-13细胞β-catenin/LRP6的表达明显降低(P<0.001)。结论氯硝柳胺抑制人肾上腺皮质癌SW-13细胞增殖,阻滞细胞周期,抑制侵袭及迁移能力,促进细胞凋亡;可能与其抑制Wnt/β-catenin信号通路有关。

Objective To study the effects of niclosamide on cell proliferation,cell cycle,cell apoptosis,cell migration and invasion of human adrenal cortical carcinoma cells. Methods SW-13 cells were cultured in vitro. CCK-8 and i CELLigence real-time label free cell proliferation monitor were applied to detect the cell proliferation under different concentrations of niclosamide. There were control and niclosamide group. Flow cytometry was used to detect the cell cycle changes; AO/EB staining assay and annexin V/PI staining flow cytometry were applied to assess the apoptosis rate;and trans-well assay and cell scratch assay were used to assess cell invasion and migration abilities. The content changes of β-catenin/LRP6 were assessed using Western-Blotting. Results After treated with different concentrations of niclosamide for 24,48,72,96,and 120 hours,the proliferation of SW-13 cells were significantly inhibited（F=157. 93,P〈0.001）,and following time and concentration dependent manners. The results of flow cytometry cycle test showed that SW-13 cell cycle were blocked in G0/G1 phase after treated with niclosamide for 24,48 and 72 hours（P=0.001,0.005 and 0.008）,compared with the control group. AO/EB staining and Annexin V/PI staining showed that,compared with the control group,the apoptosis had significant difference after treated with niclosamide for 24 and 48 hours（P〈0.001）. Transwell experimental results showed niclosamide decreased SW-13 cell invasion（P〈0.001）,and cell scratch experiments showed that niclosamide had significant effect on the migration ability（P=0.002）,and down-regulation in β-catenin/LRP6 of SW-13 cells（P〈0.001）. Conclusion Niclosamide can inhibit the proliferation,cell cycle,invasion and migration of human adrenal cortical carcinoma SW-13 cells,and promote apoptosis,which may be related to its Wnt/beta-catenin signaling pathway. It may be a new drug for the treatment of adrenal cortical carcinoma.