Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming, 650500, China.
Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming, 650500, China.
Toxicology. 2018 Dec 1;410:231-246. doi: 10.1016/j.tox.2018.08.013. Epub 2018 Aug 25.
Breast cancer is the most diagnosed diseases and the second-leading cause of death in females, among which the estrogen receptor positive (ER+) patients are more common of all cases. In present study, we selected MCF-7 as an in vitro model and investigated the combinatorial anti-proliferative effects of tamoxifen and naringenin on ER+ breast cancer, and then explored the potential mechanisms involved in mitochondrial dysfunction and oxidative stress mediated by several estrogen receptor subtypes. Six assessment endpoints including cell viability, cell migration, cell cycle, cell apoptosis, mRNA, and protein expression were estimated. Tamoxifen and naringenin were shown to inhibit the cell growth of MCF-7 cells at higher concentrations, and co-exposure with them significantly inhibited cell proliferation in an additive manner. Results from a wound healing assay indicated that the combined treatment of tamoxifen and naringenin markedly suppressed cell migration compared with the single exposure by downregulating the expression of MMP-9 and MMP-2. Flow cytometry analysis revealed that the combined treatment of tamoxifen and naringenin blocked cell cycle in G2/M phase through suppressing the transcription of cell cycle regulation proteins. Simultaneously, co-treatment with them also induced cell apoptosis by regulating the expression of mitochondrial apoptotic proteins as well as by simulating the production of reactive oxygen species (ROS). Real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB) analysis results further demonstrated that the two nuclear estrogen receptors-ERα66 and ERβ, as well as the two membrane estrogen receptors-ERα36 and GPR30 were downregulated when cells were exposed to single tamoxifen, whereas naringenin treatment group not only downregulated the expression of ERα66 and GPR30 but also upregulated ERβ and ERα36. Interestingly, co-treatment group resulted in significant downregulation of ERα66, ERα36 and GPR30 but upregulation of ERβ. Taken together, co-treatment of tamoxifen and naringenin could inhibit cell proliferation more effectively in ER+ breast cancer cells, which was associated with a modulation of the expression levels of several estrogen receptors.
乳腺癌是女性中最常见的诊断疾病和第二大死亡原因，其中雌激素受体阳性（ER+）患者更为常见。在本研究中，我们选择 MCF-7 作为体外模型，研究了他莫昔芬和柚皮素对 ER+乳腺癌的联合抗增殖作用，并探讨了几种雌激素受体亚型介导的线粒体功能障碍和氧化应激的潜在机制。评估了包括细胞活力、细胞迁移、细胞周期、细胞凋亡、mRNA 和蛋白质表达在内的六个评估终点。结果表明，较高浓度的他莫昔芬和柚皮素均可抑制 MCF-7 细胞的生长，两者联合暴露可显著以相加方式抑制细胞增殖。划痕愈合试验结果表明，与单一暴露相比，联合治疗显著下调 MMP-9 和 MMP-2 的表达，从而显著抑制细胞迁移。流式细胞术分析表明，联合治疗通过抑制细胞周期调节蛋白的转录，阻断细胞周期进入 G2/M 期。同时，通过调节线粒体凋亡蛋白的表达以及模拟活性氧（ROS）的产生，联合治疗也诱导细胞凋亡。实时定量聚合酶链反应（RT-qPCR）和蛋白质印迹（WB）分析结果进一步表明，当细胞暴露于单一他莫昔芬时，两种核雌激素受体-ERα66 和 ERβ 以及两种膜雌激素受体-ERα36 和 GPR30 下调，而柚皮素处理组不仅下调 ERα66 和 GPR30 的表达，还上调 ERβ 和 ERα36 的表达。有趣的是，联合治疗组导致 ERα66、ERα36 和 GPR30 的表达显著下调，而 ERβ 的表达上调。总之，他莫昔芬和柚皮素的联合治疗可更有效地抑制 ER+乳腺癌细胞的增殖，这与几种雌激素受体表达水平的调节有关。
