沙利霉素对葡萄膜恶性黑色素细胞的侵袭和增殖的抑制作用机制研究

Inhibition effect and mechanism of salinomycin on the invasion and proliferation of uveal malignant melanocytes

目的研究沙利霉素对葡萄膜恶性黑色素瘤92-1细胞的侵袭和增殖及对β-连环蛋白(β-catenin)/转录因子4(TCF4)表达水平的影响。方法用不同浓度沙利霉素(1,2.5,5,10,20,50,100,150,200μmol·L-1)处理葡萄膜恶性黑色素瘤92-1细胞,48 h后以CCK-8实验检测细胞活力。将92-1细胞随机分为4组:空白对照组和低、中、高3个浓度实验组,空白对照组给予0.9%NaCl,低、中、高浓度实验组分别以终浓度为5,10,20μmol·L-1沙利霉素处理。Transwell实验检测92-1细胞侵袭,克隆形成实验检测92-1细胞增殖,以蛋白质印迹法检测β-catenin和TCF4蛋白表达水平。结果给药48 h后,空白对照组和低、中、高3个浓度实验组侵袭细胞数量分别为76±14,56±12,28±15和10±6;这4组克隆形成率分别为(77±11)%,(54±8)%,(27±9)%和(11±6)%;这4组的β-catenin蛋白相对表达量分别为1.26±0.14,0.57±0.21,0.16±0.08和0.02±0.01;这4组的TCF4蛋白相对表达量分别为0.95±0.12,0.42±0.14,0.12±0.06,0.03±0.02。上述指标:低、中、高3个浓度实验组与空白对照组比较,差异均有统计学意义(均P<0.05)。结论沙利霉素可有效抑制葡萄膜恶性黑色素92-1细胞侵袭及增殖,且具有显著的浓度依赖性。这可能与沙利霉素调节β-catenin/TCF4信号通路有关。

Objective To investigate the effects of salinomycin on invasion and proliferation of uvular malignant melanoma cells 92-1 and expression of β-catenin/transcription factor 4(β-catenin/TCF4). Methods The 92-1 cells of uvular malignant melanoma were treated with different doses of salinomycin(1, 2.5, 5, 10, 20, 50, 100, 150, 200 μmol·L-1), and the cell viability was detected by CCK-8 assay at 48 h after treatment. The 92-1 cells were randomly divided into four groups: blank control group and low, medium and high doses experimental groups. The blank control group was given 0.9% NaCl, and the low, medium and high dose experimental groups were treated with final concentration of 5, 10, 20 μmol·L-1 salinomycin. The 92-1 cell invasion capacity was detected by Transwell assay. The proliferation of 92-1 cells was detected by clone formation assay. The expression of β-catenin and TCF4 proteins were detected by Western blot. Results After 48 h of administration,the number of invasion cells in blank control group,low,medium and high doses experimental groups were 76 ± 14,56 ± 12,28 ± 15 and 10 ± 6,respectively;the clone formation rates in the four groups were(77 ± 11) %,(54 ± 8) %,(27 ± 9) %,and(11 ± 6) %,respectively;the expression of β-catenin protein in the four groups were 1. 26 ± 0. 14,0. 57 ± 0. 21,0. 16 ± 0. 08 and 0. 02 ± 0. 01,respectively;the expression of TCF4 protein in the four groups were 0. 95 ± 0. 12,0. 42 ± 0. 14,0. 12 ± 0. 06 and 0. 03 ± 0. 02. Compared between three doses experimental groups and blank control group,the difference of the factors were significant(all P < 0. 05).Conclusion Salinomycin can effectively inhibit the invasion and proliferation of uvular malignant melanin 92-1 cells in a significant dose-dependent manner. This may be related to the regulation of β-Catenin/TCF4 signaling pathway by salinomycin.