COX-2抑制剂Mavacoxib对骨肉瘤干细胞增殖、凋亡及相关信号通路的影响

Effects of COX-2 inhibitor Mavacoxib on proliferation,apoptosis and related signaling pathways of human osteosarcoma stem cells

目的探讨COX-2抑制剂Mavacoxib对骨肉瘤干细胞增殖、凋亡及相关信号通路的影响。方法体外培养人骨肉瘤MG-63细胞以获取骨肉瘤干细胞,经Mavacoxib（0、1、10、50、100μmol/L）处理后采用四甲基偶氮唑盐（MTT）比色法检测增殖抑制率的变化,同时采用流式细胞术Annexin-FITC/PI双染法及PI单染法检测不同浓度Mavacoxib处理24、48 h的凋亡情况（早、晚期凋亡率）及48 h的细胞周期情况,Western blotting检测不同浓度Mavacoxib处理48 h的骨肉瘤干细胞中PI3K/Akt及Wnt/β-catenin信号通路的影响。结果在10~100μmol/L范围内,Mavacoxib可呈剂量和时间依赖的方式升高骨肉瘤干细胞的增殖抑制率,差异均有统计学意义（P〈0.05）;与0μmol/L相比,除1μmol/L 24 h的晚期凋亡率无统计学差异,10~100μmol/L的早晚期凋亡率、G0/G1期细胞比例均升高,S期、G2/M期细胞比例均降低（P〈0.05）。Mavacoxib处理后的PI3K/Akt通路中的PTEN水平升高,Akt水平降低;Wnt/β-catenin通路中β-catenin、C-myc和Cyclin D1水平均降低（P〈0.05）。结论 Mavacoxib对骨肉瘤干细胞有毒性作用,且可诱导其凋亡及细胞周期阻滞并抑制PI3K/Akt和Wnt/β-catenin信号通路的激活。

Objective To explore the effects of COX-2 inhibitor Mavacoxib on proliferation,apoptosis and related signaling pathways of human osteosarcoma stem cells. Methods The MG-63 cells were cultured in vitro to obtain osteosarcoma stem cells. The MTT assay was used to detect the proliferation inhibition rates after treatment with Mavacoxib（ 0,1,10,50,100 μmol/L）. Meanwhile,Annexin-FITC/PI double staining and PI staining were used to detect the apoptotic rates at 24,48 h and cell cycle at 48 h after different concentrations of Mavacoxib. The effects of Mavacoxib on the Wnt/β-catenin and PI3 K/Akt signaling pathway were measured by Western blotting. Results In the range of 10-100 μmol/L,Mavacoxib could improve the proliferation inhibition rates of osteosarcoma stem cells in a dose and time dependent manner with statistical significant difference（ P〈0. 05）. Compared with 0 μmol/L,the apoptotic rates and the percentage of G0/ G1 phase cells were increased,the proportions of S phase and G2/ M phase were decreased in other concentrations（ P〈0. 05）. After the treatment of Mavacoxib,the levels of PTEN in the PI3 K/Akt pathway were increased,the levels of Akt in the PI3 K/Akt pathway and the levels of β-catenin,C-myc and Cyclin D1 in the Wnt/β-catenin pathway were decreased（ P〈0. 05）. Conclusion Mavacoxib has toxic effect on human osteosarcoma stem cells,and can induce apoptosis and cell cycle arrest and inhibit the activation of Wnt/β-catenin and PI3 K/Akt signaling pathway.