目的 研究萝卜硫素(SFN)及其衍生物 (BSFN) 对SH-SY5Y细胞的增殖抑制活性及通过PI3K/Akt途径探讨其抗肿瘤作用机制。方法 四甲基偶氮唑蓝比色法测定其对SH-SY5Y细胞增殖抑制率;通过免疫荧光法、流式细胞术、蛋白质印迹法对其进行抗癌机制研究。结果 四甲基偶氮唑蓝结果显示,萝卜硫素和萝卜硫素衍生物处理细胞72 h的IC50值分别为13.27和5.21 μmolL-1,且呈现时间剂量依赖性;Hoechst33258染色荧光显微镜观察发现,萝卜硫素、萝卜硫素衍生物能诱导SH-SY5Y细胞核形态学改变,部分细胞呈现典型的凋亡形态学特征;Annexin Ⅴ-FITC/PI荧光双染结果亦证实萝卜硫素、萝卜硫素衍生物可以诱导SH-SY5Y细胞发生凋亡;DAPI和DCFH-DA染色流式细胞术分析显示,萝卜硫素衍生物诱导SH-SY5Y细胞S期阻滞、细胞活性氧产生增加,且较萝卜硫素作用明显;蛋白质印迹分析发现萝卜硫素和萝卜硫素衍生物能调节PI3K/Akt途径中凋亡相关蛋白的表达,同时检测细胞内Nrf2与Keap-1蛋白表达,发现Nrf2蛋白表达增加,Keap-1蛋白表达减少。结论 萝卜硫素衍生物通过PI3K/Akt信号途径诱导SH-SY5Y细胞凋亡和S期阻滞,且作用优于萝卜硫素,可能成为一种新型的抗癌药物。
Abstract
OBJECTIVE To study the effect of sulforaphane (SFN) and its derivative 1-((4-isothiocyanatobutylsulfinyl)methyl)benzene (BSFN), a PI3K/Akt inhibitor, on the proliferation and apoptosis of SH-SY5Y cells and the possible action mechanism. METHODS The anti-proliferative effects of SFN and BSFN on SH-SY5Y cells were measured by MTT spectrophotometry method. The anticancer mechanism of SFN and BSFN on SH-SY5Y cells was investigated by immunofluorescence, flow cytometry and Western blot. RESUTLS MTT assay revealed that the IC50 values of SFN and BSFN on SH-SY5Y cells respectively were 13.27 and 5.21 μmolL-1 respectively after 72 h of treatment,and SFN and BSFN could inhibit the growth of SH-SY5Y cells in a concentration-and time-dependent manner. In SH-SY5Y cells treated by SFN and BSFN, topical morphological changes of apoptotic body formation were observed by Hoechst 33258 staining. The cell apoptosis induced by SFN and BSFN was further confirmed by Annexin Ⅴ-FITC/PI double staining assay. FCM analysis showed that BSFN could induce S phase arrest and increase the production of ROS in SH-SY5Y cells and which was better than SFN. By using Western blot, it was that SFN and BSFN could regulate the apoptosis-related protein expressions in PI3K/Akt pathway. Meanwhile, the increase of Nrf2 protein and the decrease of Keap1 protein expressions were detected by Western blot. XONCLUSION The results indicate that BSFN with the better ability to cause cell cycle S arrest and induce cell apoptosis in PI3K/Akt pathway, has potential becoming a novel antitumor agent.
关键词
萝卜硫素 /
萝卜硫素衍生物 /
SH-SY5Y细胞 /
细胞凋亡 /
PI3K/Akt信号途径
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Key words
sulforaphane /
1-[(4-isothiocyanatobutylsulfinyl)methyl] benzene /
SH-SY5Y cells /
apoptosis /
PI3K/Akt
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中图分类号：
R965
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参考文献
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脚注
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基金
国家自然科学基金资助项目(21202012);2012江苏省“青蓝工程”和常州市基础研究计划(应用基础)资助(CJ20130021)
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