金合欢素通过AKT/GSK-3β/β-catenin信号通路发挥抗骨肉瘤作用

Acacetin shows anti-osteosarcoma effect through AKT/GSK-3β/β-catenin signaling pathway

目的:探究金合欢素(acacetin)对人骨肉瘤细胞增殖、凋亡、迁移和侵袭能力的影响,及可能的作用机制。方法:体外培养人骨肉瘤细胞143B和MG63,分别给予不同浓度(0、20、25、30和35μmol/L)的金合欢素处理;随后,分别采用结晶紫染色和MTT法检测细胞的增殖能力,Hoechst33258染色法和FCM法检测细胞的凋亡情况,细胞划痕愈合实验和Transwell小室实验检测细胞的迁移和侵袭能力;最后,采用蛋白质印迹法检测细胞内增殖相关蛋白[增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、细胞周期蛋白D1(Cyclin D1)和c-Myc]、凋亡相关蛋白[Bcl-2、Bax和剪切型Caspase-3(cleaved-Caspase-3)]以及迁移和侵袭相关蛋白[基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)、Snail和波形蛋白(Vimentin)和E-钙黏蛋白(E-cadherin)]的表达水平,并进一步检测AKT、磷酸化-AKT(phospho-AKT,p-AKT)、糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)、p-GSK-3β(ser9)和β-连环蛋白(β-catenin)的表达情况。结果:结晶紫染色和MTT法检测结果表明,金合欢素可抑制骨肉瘤细胞143B和MG63的增殖,且蛋白质印记实验结果表明金合欢素处理可降低143B细胞内增殖相关蛋白(PCNA、Cyclin D1和c-Myc)的表达水平(P均<0.05)。Hoechst33258染色和FCM法检测结果表明,金合欢素可促进骨肉瘤细胞143B的凋亡,且蛋白质印记实验结果显示金合欢素处理可使143B细胞内抗凋亡蛋白Bcl-2的表达水平下降,促凋亡蛋白Bax、凋亡标志蛋白cleaved-Caspase-3的表达水平升高(P均<0.01)。细胞划痕愈合实验和Transwell小室实验结果表明金合欢素可抑制骨肉瘤细胞143B的迁移和侵袭,且蛋白质印记实验结果显示金合欢素处理可使143B细胞内MMP-2、Vimentin的表达水平下降,E-cadherin的表达水平升高(P均<0.05)。同时,进一步蛋白质印记实验结果表明,金合欢素处理可使143B细胞内的p-AKT、p-GSK-3β(ser9)和β-catenin蛋白表达水平下降(P均<0.05)。结论:金合欢素可抑制人骨肉瘤细胞的增殖、迁移和侵袭,并促进其凋亡,其可能是通过阻断AKT/GSK-3β/β-catenin信号通路发挥作用。

Objective:To investigate the effects of acacetin on the proliferation,apoptosis,migration and invasion of human osteosarcoma cells and its underlying mechanism.Methods:Human osteosarcoma cells 143B and MG63 were cultured in vitro and treated with different concentrations(0,20,25,30 and 35μmol/L)of acacetin for the following analyses.The proliferative activity of 143B and MG63 cells was analyzed by crystal violet staining and MTT assay.The apoptosis of 143B cells was examined by Hoechst33258 staining and flow cytometry(FCM)assay.The migration and invasion abilities of 143B cells were assessed by wound healing assay and Transwell assay respectively.The expression levels of proliferationrelated proteins[proliferating cell nuclear antigen(PCNA)and Cyclin D1],apoptosis-related proteins[Bax,Bcl-2 and cleaved-Caspase-3],as well as migration-and invasion-related proteins[matrix metalloproteinase-2(MMP-2),Snail,Vimentin and E-cadherin]in 143B cells were examined by Western blotting.Furthermore,the expression levels of AKT,p-AKT(ser473),GSK-3β,p-GSK-3β(ser9)andβ-catenin in 143B cells after acacetin treatment were also checked by Western blotting for mechanism study.Results:Acacetin inhibited the proliferation of 143B and MG63 cells as indicated by crystal violet staining and MTT assay results,and the expression levels of proliferationrelated proteins(PCNA,Cyclin D1 and c-Myc)in 143B cells were decreased(P<0.05)correspondingly according to Western blotting results.Acacetin could also induce apoptosis in 143B cells as shown by Hoechst33258 staining and FCM assay results,and the expression level of anti-apoptotic protein Bcl-2 was decreased while those of pro-apoptotic protein Bax and apoptotic marker protein cleaved-Caspase-3 were increased(all P<0.05)correspondingly in 143B cells according to Western blotting results.Acacetin inhibited the migration and invasion of 143B cells as revealed by wound healing assay and Transwell assay results,and the expression levels of MMP-2,Snail and Vimentin proteins were decreased while that of E-cadherin was increased(P<0.05)correspondingly in 143B cells according to Western blotting results.Furthermore,the expression levels of p-AKT(ser473),p-GSK-3β(ser9)andβ-catenin proteins were all decreased(P<0.05)in 143B cells after acacetin treatment as evidenced by Western blotting results.Conclusion:Acacetin can inhibit the proliferation,migration and invasion while promote the apoptosis of osteosarcoma cells,possibly through blocking the AKT/GSK-3β/β-catenin signaling pathway.