莱菔硫烷抑制肺癌干细胞增殖的体外实验

Sulforaphane Suppressed Proliferation of Lung Cancer Stem Cells in vitro

目的研究莱菔硫烷(sulforaphane,SFN)对肺癌干细胞增殖的影响及其机制。方法采用MTT法分析SFN对H460细胞增殖的影响;应用流式细胞术(FACS)检测SFN对细胞凋亡和侧群细胞比例的影响;肿瘤球培养法分析SFN对肿瘤球生长的影响;使用sh RNA慢病毒载体构建β-catenin低表达细胞株,并应用蛋白电泳法检测在β-catenin正常或低表达情况下莱菔硫烷对β-catenin、Oct4、Sox2、c-Myc、Nanog等基因表达的影响。结果 SFN有效抑制H460细胞增殖,IC50为11.2μmol/L。SFN处理72 h后,细胞凋亡呈剂量依赖性增高。SFN有效抑制原代肿瘤球和2代肿瘤球的生长,在较低浓度(1.0μmol/L)下即有明显的抑制作用。FACS检测提示侧群细胞比例随SFN浓度增高而减少。SFN浓度依赖性地抑制β-catenin、Oct4、Sox2、c-Myc和Nanog等蛋白表达。在低表达β-catenin情况下,Oct4、Sox2、c-Myc、Nanog等基因表达水平与SFN浓度相关。结论 SFN通过β-catenin和干性相关基因(Sox2、c-Myc、Nanog和Oct4)特异地抑制肺癌干细胞的增殖。

Objective To investigate the effect of sulforaphane（SFN） on regulating proliferation of lung cancer stem cells and its mechanism.Methods The effect of SFN on the proliferation of H460 cells was measured by MTT method;cell apoptosis and side population rate were analyzed by fluorescence activated cell sorting（FACS）;the change of tumor sphere growth was detected by sphere formation experiment;H460 cells with low expression of β-catenin gene was established by sh RNA lentivirus transfection,and the effect of SFN on the expression of β-catenin,Oct4,Sox-2,c-Myc and Nanog was examined with or without β-catenin expression by Western blot.Results SFN efficiently suppressed the proliferation of H460 cells in vitro and the IC50 was 11.2μmol/L.The number of apoptotic cells was dose-dependently increased after the treatment of SFN for 72 h.In tumor sphere formation experiment,SFN dose-dependently suppressed the growth of 1st and 2nd passage of tumor sphere,even in a very low dose（1.0 μmol/L） of SFN.The proportion of side population cells detected by FACS was decreased as increment of SFN concentration.Western blot experiment showed that SFN dose-dependently suppressed the expression of several stemness-related genes,such as β-catenin,Oct4,Sox2,c-Myc and Nanog,changed as well as side population,even in absence of β-catenin.Conclusion SFN specially suppresses the proliferation of lung cancer stem cells via β-catenin signaling and several stemness-related genes,such as Oct4,Sox2,c-Myc and Nanog.