依维莫司增强阿霉素抑制骨肉瘤干细胞的作用

Everolimus enhances the ability of adriamycin against the proliferation and invasion of osteosarcoma stem cells

背景:阿霉素能够抑制骨肉瘤细胞的增殖,但是阿霉素对骨肉瘤干细胞的作用以及依维莫司联合阿霉素对骨肉瘤干细胞的作用未见报道。目的:研究mTOR通路抑制剂依维莫司联合骨肉瘤化疗一线药物阿霉素对骨肉瘤干细胞增殖及侵袭的影响。方法:用免疫磁珠分选法分离人骨肉瘤细胞株MG63中的CD133^+骨肉瘤干细胞。MTT检测0,0.1,0.2,0.4 mg/L阿霉素以及0.2 mg/L阿霉素+20μmol/L依维莫司对骨肉瘤干细胞增殖的影响。Transwell小室检测0.2 mg/L阿霉素和0.2 mg/L阿霉素+20μmol/L依维莫司对骨肉瘤干细胞侵袭的影响。Western Blot检测0.2 mg/L阿霉素和0.2 mg/L阿霉素+20μmol/L依维莫司对骨肉瘤干细胞增殖细胞核抗原、基质金属蛋白酶蛋白表达的影响。结果与结论:(1)阿霉素能在一定质量浓度下抑制骨肉瘤干细胞的增殖,且依维莫司联合阿霉素较单独使用阿霉素时明显抑制骨肉瘤干细胞的增殖,说明依维莫司能够增强阿霉素对骨肉瘤干细胞增殖的抑制作用;(2)与单独使用阿霉素比较,依维莫司联合阿霉素作用于骨肉瘤干细胞后侵袭细胞数明显减少;(3)依维莫司联合阿霉素显著抑制骨肉瘤干细胞增殖细胞核抗原、基质金属蛋白酶蛋白的表达,提示依维莫司能够增强阿霉素抑制骨肉瘤干细胞增殖与侵袭的能力。

BACKGROUND:Adriamycin can inhibit the proliferation of osteosarcoma cells,but the effects of adriamycin alone or combined with everolimus on osteosarcoma stem cells are not well documented.OBJECTIVE:To explore the effects of adriamycin combined with everolimus,a mTOR inhibitor,on the proliferation and invasion of osteosarcoma stem cells.METHODS:CD133+osteosarcoma stem cells were separated from MG63cells by flow cytometer.We first used MTT method to detect the proliferation of osteosarcoma stem cells treated with adriamycin at the concentrations of0,0.1,0.2,0.4mg/L.Next,we chose an appropriate concentration of adriamycin(0.2mg/L)combined with20μmol/L everolimus to act on CD133+osteosarcoma stem cells.MTT and Transwell method were then used to detect the cell proliferation and invasion after intervention with0.2mg/L adriamycin alone or combined use of adriamycin(0.2mg/L)and everolimus(20μmol/L),respectively.Meanwhile,the expression levels of proliferating cell nuclear antigen and matrix metalloproteinase9in the osteosarcoma stem cells were detected by western blot.RESULTS AND CONCLUSION:MTT results showed that adriamycin and especially adriamycin combined with everolimus could inhibit the proliferation of CD133+osteosarcoma stem cells.Transwell assay results showed that adriamycin combined with everolimus could inhibit the invasion of osteosarcoma stem cells.Western blot results showed that adriamycin combined with everolimus significantly decreased the levels of proliferating cell nuclear antigen and matrix metalloproteinase9compared with the control group.These findings suggest that everolimus can enhance the ability of adriamycin against the proliferation and invasion of osteosarcoma stem cells.