伏立诺他抑制骨肉瘤细胞增殖、迁移、侵袭作用及其与雷帕霉素联用的抗肿瘤机制研究

A study on suberoylanilide hydroxamic acid inhibiting the proliferation, migration and invasion of osteosarcoma cells and the synergistic anti-tumor mechanism for combined use of it and rapamycin

目的探讨伏立诺他(SAHA)抑制骨肉瘤细胞U-2OS增殖、迁移、侵袭作用及其与雷帕霉素(Rapa)联用的抗肿瘤机制。方法分别加入SAHA 0、2.5、5、10、20和40μmol/L处理U-2OS细胞24和48 h,采用CCK-8法检测细胞增殖情况。分别加入SAHA 0、 5、10和20μmol/L处理U-2OS细胞,采用细胞集落实验检测细胞的集落形成情况,细胞迁移实验和Transwell实验检测细胞迁移和侵袭情况。另将U-2OS细胞分别加入SAHA 20μmol/L、Rapa 5μmol/L以及SAHA 20μmol/L+Rapa 5μmol/L联合处理后,采用CCK-8法检测细胞增殖情况,流式细胞术检测细胞活性氧(ROS)水平,Western blot检测内质网应激相关蛋白C/EBP同源蛋白(CHOP)、转录激活因子6(ATF6)和X-盒结合蛋白1(XBP1)的表达水平。结果 SAHA呈浓度和时间依赖性地抑制U-2OS细胞增殖(P<0.05),且呈浓度依赖性抑制细胞集落形成、细胞迁移和侵袭(P<0.05)。SAHA和Rapa单用均可抑制U-2OS细胞增殖,且使ROS水平和CHOP、ATF6和XBP1的表达升高(P<0.05),两者联用上述变化更显著(P<0.05)。结论 SAHA能够有效抑制U-2OS细胞增殖、迁移和侵袭,并与Rapa联用后起到协同抗肿瘤作用;其机制可能与升高ROS水平和激活内质网应激相关蛋白CHOP、ATF6和XBP1的表达有关。

Objective To explore the mechanism of suberoylanilide hydroxamic acid(SAHA) inhibiting the proliferation, migration and invasion of osteosarcoma cells U-2 OS and the synergistic anti-tumor mechanism for combined use of it and rapamycin.Methods The U-2 OS cells were treated with SAHA 0,2.5,5,10,20 and 40 μmol/L for 24 and 48 h, respectively, and the cell proliferation was detected by CCK-8 method.The U-2 OS cells were treated with SAHA 0,5,10 and 20 μmol/L,and the colony forming ability of the cells was detected by cell colony assay, the cell migration and Transwell assay were used to detect cell migration and invasion.In addition, the U-2 OS cells were treated with SAHA 20 μmol/L,rapamycin 5 μmol/L,and SAHA 20 μmol/L+rapamycin 5 μmol/L,and the cell proliferation and cellular ROS levels were detected by CCK-8 method and cell flow cytometry, the expression of endoplasmic reticulum stress-related proteins C/EBP homologous protein(CHOP),activator of transcription 6(ATF6) and X-box binding protein 1(XBP1) were detected by Western blot.Results SAHA inhibited the proliferation of U-2 OS cells in the concentration-and time-dependent manners(P<0.05),and inhibited the cell colony formation, cell migration and invasion in a concentration-dependent manner(P<0.05).The treatment with SAHA or rapamycin alone inhibited the U-2 OS cells proliferation and increased the ROS level and the expressions of CHOP,ATF6 and XBP1 in U-2 OS cells, and the changes of above indicators were more significant when combined use of SAHA and rapamycin(P<0.05).Conclusion SAHA can effectively inhibit the proliferation, migration and invasion of U-2 OS cells.Combined use of SAHA and rapamycin has a synergistic anti-tumor effect.The mechanism for that may be related to the increase of ROS level and the activation of the expressions of endoplasmic reticulum stress-related proteins CHOP,ATF6 and XBP-1.