番茄红素通过ROS/NF-κB调控乳腺癌干细胞干性和化疗敏感性的研究

Study on Lycopene Regulates Stemness and Chemosensitivity of Breast Cancer Stem Cell Through ROS/NF-κB Signaling

关键词:  番茄红素  乳腺癌  肿瘤干细胞  化疗敏感性  活性氧

Key words:  lycopene  breast cancer  cancer stem cell  chemosensitivity  reactive oxygen species

摘要:

目的 探讨番茄红素对乳腺癌干细胞(breast cancer stem cell,BCSC)干性和化疗敏感性的影响及其可能的作用机制。方法 体外培养乳腺癌MCF-7细胞,通过悬浮培养和流式细胞术筛选CD44⁺MCF-7干细胞(BCSC)进行后续实验;BCSC分为对照组和番茄红素组(5 μmol·L^-1),给药24 h后,球体形成试验检测BCSC球体形成大小和数量;对照组和番茄红素组BCSC经不同浓度顺铂(0,5,10,20,40,80 μmol·L^-1)处理24 h,CCK8试验检测BCSC细胞增殖,流式细胞术检测细胞凋亡情况;流式细胞术检测对照组和番茄红素组BCSC中活性氧(reactive oxygen species,ROS)水平;BCSC经10 μmol·L^-1活性氧清除剂N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)或10 μmol·L^-1 NF-κB抑制剂BAY 11-7085(BAY)处理24 h后,Western blotting检测细胞中p NF-κB和干性相关蛋白LSD1及SOX9的表达水平。结果 CD44⁺细胞中LSD1和SOX9蛋白表达水平显著高于CD44^-细胞;番茄红素明显降低BCSC球体形成数量和大小;番茄红素显著提高顺铂诱导的BCSC细胞凋亡率;番茄红素显著降低BCSC中ROS水平;番茄红素与NAC均降低细胞中p NF-κB蛋白表达水平;番茄红素、NAC和BAY均抑制细胞中LSD1、SOX9蛋白表达水平,且分别经番茄红素、NAC和BAY处理后,BCSC对顺铂敏感性明显升高。结论 番茄红素可能通过ROS/NF-κB信号通路抑制乳腺癌干细胞干性,增强顺铂治疗的敏感性。

Abstract:

OBJECTIVE To investigate the effect of lycopene on the stemness and chemotherapy sensitivity of breast cancer stem cells(BCSC) and its possible mechanism. METHODS Breast cancer cells MCF-7 were cultured in vitro, CD44⁺ MCF-7 stem cells(BCSC) were screened by suspension culture and flow cytometry for follow-up experiments. BCSC were divided into control group and lycopene group(5 μmol·L^–1). After administration for 24 h, sphere formation assay was used to detect the size and number of spheroid BCSC cells. BCSC in control group and lycopene group were treated cisplatin at different concentrations(0, 5, 10, 20, 40 and 80 μmol·L^–1) for 24 h. CCK8 assay was used to detect BCSC cell proliferation and flow cytometry was used to detect apoptosis. Reactive oxygen species(ROS) levels were detected by flow cytometry in BCSC of control group and lycopene group. BCSC were treated with 10 μmol·L–1 N-acetyl-L-cysteine(NAC) or 10 μmol·L^–1 BAY11-7085 (BAY) for 24 h. Western blotting was used to detect the protein expression of p NF-κB and stemness-related proteins LSD1 and SOX9. RESULTS The protein expression levels of LSD1 and SOX9 in CD44⁺ cells were significantly higher than those in CD44^- cells. Lycopene significantly reduced the number and size of BCSC spheres. Lycopene significantly increased the apoptosis rate of BCSC cells induced by cisplatin. Furthermore, lycopene significantly reduced ROS levels in BCSC. Lycopene and NAC both reduced the expression level of p NF-κB protein in BCSC cells. Importantly, lycopene, NAC and BAY all inhibited the expression levels of LSD1 and SOX9 proteins in cells, and all increased the sensitivity to cisplatin of BCSC. CONCLUSION Lycopene may inhibit the stemness and enhance the sensitivity to cisplatin of breast cancer stem cells through the ROS/NF-κB signaling pathway.