Dong Xinmin, Zhang Jian, Yang Fan, Wu Jing, Cai Rui, Wang Tian, Zhang Jiren
Department of Oncology, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510280, P.R. China.
Department of Oncology, The Affiliated People's Hospital of Inner Mongolia Medical University, Hohhot, Inner Mongolia 010010, P.R. China.
Exp Ther Med. 2018 Oct;16(4):3186-3194. doi: 10.3892/etm.2018.6526. Epub 2018 Jul 26.
The present study aimed to investigate the effect of luteolin on the methylation of opioid binding protein/cell adhesion molecule (OPCML) in breast cancer cells, as well as its underlying mechanism of action. Human breast cancer cell lines BT474 and MCF-7 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. The cells were treated with 0-30 µmol/l luteolin prior to investigation. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to determine the mRNA and protein expression, respectively. High performance liquid chromatography and electrosprary ionization-mass spectrometry was used to analyze the methylation of the OPCML promoter region and whole genome. The methylation activity in the cell nucleus was determined using a DNA methyltransferase catalytic test. ELISA analysis was used to detect changes in the activity of transcription factors Sp1 and nuclear factor (NF)-κB. An MTT assay was performed to determine cell proliferation, while flow cytometry was used to detect cell cycle stage and apoptosis. Luteolin effectively upregulated the expression of OPCML in breast cancer cells. Luteolin activated OPCML by reducing intracellular methylation levels. Luteolin downregulated intracellular methylation levels by decreasing Sp1 and NF-κB activities. Luteolin affected the expression of DNMT1 and OPCML by downregulating Sp1 activity. Luteolin inhibited the proliferation and induced the apoptosis of BT474 and MCF-7 cells. The results of the present study suggest that luteolin inhibits the growth of breast cancer cells by decreasing the methylation and upregulating the expression of the OPCML gene.
本研究旨在探讨木犀草素对乳腺癌细胞中阿片样物质结合蛋白/细胞粘附分子（OPCML）甲基化的影响及其潜在作用机制。人乳腺癌细胞系BT474和MCF-7在补充有10%胎牛血清的RPMI-1640培养基中培养。在研究前，用0 - 30µmol/l的木犀草素处理细胞。分别采用逆转录定量聚合酶链反应和蛋白质印迹分析来测定mRNA和蛋白质表达。采用高效液相色谱和电喷雾电离质谱分析OPCML启动子区域和全基因组的甲基化情况。使用DNA甲基转移酶催化试验测定细胞核中的甲基化活性。采用ELISA分析检测转录因子Sp1和核因子（NF）-κB活性的变化。进行MTT试验以测定细胞增殖，同时使用流式细胞术检测细胞周期阶段和凋亡情况。木犀草素可有效上调乳腺癌细胞中OPCML的表达。木犀草素通过降低细胞内甲基化水平来激活OPCML。木犀草素通过降低Sp1和NF-κB活性来下调细胞内甲基化水平。木犀草素通过下调Sp1活性影响DNMT1和OPCML的表达。木犀草素抑制BT474和MCF-7细胞的增殖并诱导其凋亡。本研究结果表明，木犀草素通过降低甲基化并上调OPCML基因的表达来抑制乳腺癌细胞的生长。
