CAJ | 학술논문

目的研究氯硝柳胺对人三阴性乳腺癌MDA-MB-231细胞的放射增敏作用及其与Wnt/β-连环蛋白信号通路相关的作用机制.方法四甲基偶氮唑蓝(MTT)法检测不同浓度的氯硝柳胺对MDA-MB-231细胞生长的抑制效应,求得IC50值.将细胞分为空白对照组、氯硝柳胺单纯给药组、单纯照射组和氯硝柳胺+照射组,氯硝柳胺预处理的浓度为1.0和1.5 μmol/L,”7Cs γ射线的照射剂量为0、2、4和6 Gy;克隆形成试验检测氯硝柳胺对MDA-MB-231细胞的放射增敏作用;免疫荧光法检测辐射诱导的γH2AX焦点形成;流式细胞仪检测细胞周期的变化;Western blot法检测磷酸化和非磷酸化β-连环蛋白和Cyclin D1蛋白表达.结果氯硝柳胺抑制MDA-MB-231细胞生长24 h的IC50值为13.63 μmol/L;1.0和1.5 μmol/L氯硝柳胺对MDA-MB-231细胞均有较好的放射增敏作用,放射增敏比SER分别为1.37和1.62,随给药剂量的增大而增强;氯硝柳胺预处理使受照MDA-MB-231细胞的γH2AX焦点形成率较单纯照射组显著增加(t=3.91,P＜0.05),G2/M期阻滞明显减少(t=8.05,P＜0.01),并显著抑制了辐射诱导的磷酸化β-连环蛋白(S675)、非磷酸化β-连环蛋白和Cyclin D1蛋白表达的升高.结论氯硝柳胺对MDA-MB-231细胞具有显著的放射增敏作用,其作用机制与抑制Wnt/β-连环蛋白信号通路,从而抑制DNA DSBs修复和减少辐射诱导的G2/M期阻滞有关.Wnt/β-连环蛋白信号通路可能是有效的三阴性乳腺癌放射增敏的新靶点.
목적 연구록초류알대인삼음성유선암MDA-MB-231세포적방사증민작용급기여Wnt/β-련배단백신호통로상관적작용궤제.방법 사갑기우담서람(MTT)법검측불동농도적록초류알대MDA-MB-231세포생장적억제효응,구득IC50치.장세포분위공백대조조、록초류알단순급약조、단순조사조화록초류알+조사조,록초류알예처리적농도위1.0화1.5 μmol/L,”7Cs γ사선적조사제량위0、2、4화6 Gy;극륭형성시험검측록초류알대MDA-MB-231세포적방사증민작용;면역형광법검측복사유도적γH2AX초점형성;류식세포의검측세포주기적변화;Western blot법검측린산화화비린산화β-련배단백화Cyclin D1단백표체.결과 록초류알억제MDA-MB-231세포생장24 h적IC50치위13.63 μmol/L;1.0화1.5 μmol/L록초류알대MDA-MB-231세포균유교호적방사증민작용,방사증민비SER분별위1.37화1.62,수급약제량적증대이증강;록초류알예처리사수조MDA-MB-231세포적γH2AX초점형성솔교단순조사조현저증가(t=3.91,P＜0.05),G2/M기조체명현감소(t=8.05,P＜0.01),병현저억제료복사유도적린산화β-련배단백(S675)、비린산화β-련배단백화Cyclin D1단백표체적승고.결론 록초류알대MDA-MB-231세포구유현저적방사증민작용,기작용궤제여억제Wnt/β-련배단백신호통로,종이억제DNA DSBs수복화감소복사유도적G2/M기조체유관.Wnt/β-련배단백신호통로가능시유효적삼음성유선암방사증민적신파점.
Objective To investigate the radiosensitization effect of antihelminthic niclosamide on human triple-negative breast cancer MDA-MB-231 cells and the potential mechanism related to Wnt/β-catenin signaling pathway.Methods Four methyl thiazolyl tetrazolium(MTT) assay was used to measure the effect of niclosamide on cell viability at different concentrations and 50％ inhibitory concentration(IC50)value was calculated.MDA-MB-231 cells were divided into 4 groups:untreated control,niclosamide treatment alone group,radiation alone group and niclosamide plus radiation treatment group.The cells with or without 1.0 and 1.5 μmol/L niclosamide pre-treatment were irradiated with 137Cs γ-rays at doses of 0,2,4 and 6 Gy.Cell survival was assayed with the colony formation method,radiation-induced γH2AX foci was analyzed with immunofluorescence,cell cycle progression was assayed with flow cytometry,and the changes of phospho-and non-phospho-β-catenin and Cyclin D1 protein expressions were measured with Western blot.Results Niclosamde obviously inhibited the viability of MDA-MB-231 cells in a dosedependent manner with a IC50 value of 13.63 μmol/L.Pretreatment of cells with 1.0 and 1.5 μmol/L niclosamide evidently enhanced the radiosensitivity of MDA-MB-231 cells to γ-rays,and the values of SER were 1.37 and 1.62,respectively.Niclosamide pretreatment significantly increased radiation-induced γH2AX foci formation(t =3.91,P ＜0.05),diminished the radiation-induced G2/M arrest(t =8.05,P ＜0.01),and inhibited radiation-induced expressions of phospho-β-catenin (S675),non-phospho-β-catenin and Cyclin D1 proteins in MDA-MB-231 cells.Conclusions Niclosamide significantly can enhance the sensitivity of MDA-MB-231 cells to γ-ray irradiation through inhibiting Wnt/β-catenin signaling pathway,which results in the inhibition of DNA DSBs repair and the reduction of radiation-induced G2/M arrest.Wnt/β-catenin signaling pathway may serve as an ideal molecular target for radiosensitization of triplenegative breast cancer.