Abstract：
BACKGROUND: Breast cancer (BC), a common tumor in women, has high morbidity and mortality. Formononetin, an active ingredient in red clover and Astragalus membranaceus, has a wide range of pharmacological applications, including as an anticancer agent. Since immunotherapy is a hot topic in the treatment strategy of BC, it was dedicated to appraising the specific mechanism of formononetin in BC immunotherapy in this research.
METHODS: Different formononetin concentrations (0, 20, 40, 60, 80, 100 μM) were used to treat BC cells transfected with pcDNA3.1-Programmed death ligand 1 (PD-L1) or Short-hairpin RNA (sh)-PD-L1. Cells were separated into four subgroups: CTRL, pcDNA3.1-PD-L1, sh-CTRL, and sh-PD-L1. Cell viability and cell cycle were assessed through Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and flow cytometry. Programmed death ligand 1 (PD-L1) mRNA concentration was validated via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Cell metastasis was evaluated via cloning assay and transwell assay. The p-STING/stimulator of interferon genes (STING), p-p65/p65, and PD-L1 concentrations were determined by western blot.
RESULTS: Formononetin restrained the proliferation of MCF-7 and MDA-MB-468 cells, and reduced PD-L1 mRNA, p-STING/STING, and p-p65/p65 protein concentrations. Whereas PD-L1 inhibition restrained the viability of BC cells, pcDNA3.1-PD-L1 intervention had the opposite result. STING pathway inhibitor C-176 combined with formononetin treatment further restrained cell proliferation, colony formation, and cell invasion, in contrast to cells treated with formononetin alone.
CONCLUSIONS: Formononetin can restrain the proliferation of BC cells, which may be mediated through the interference of PD-L1 and suppression of the activation of the STING-NF-κB pathway.
METHODS: Different formononetin concentrations (0, 20, 40, 60, 80, 100 μM) were used to treat BC cells transfected with pcDNA3.1-Programmed death ligand 1 (PD-L1) or Short-hairpin RNA (sh)-PD-L1. Cells were separated into four subgroups: CTRL, pcDNA3.1-PD-L1, sh-CTRL, and sh-PD-L1. Cell viability and cell cycle were assessed through Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and flow cytometry. Programmed death ligand 1 (PD-L1) mRNA concentration was validated via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Cell metastasis was evaluated via cloning assay and transwell assay. The p-STING/stimulator of interferon genes (STING), p-p65/p65, and PD-L1 concentrations were determined by western blot.
RESULTS: Formononetin restrained the proliferation of MCF-7 and MDA-MB-468 cells, and reduced PD-L1 mRNA, p-STING/STING, and p-p65/p65 protein concentrations. Whereas PD-L1 inhibition restrained the viability of BC cells, pcDNA3.1-PD-L1 intervention had the opposite result. STING pathway inhibitor C-176 combined with formononetin treatment further restrained cell proliferation, colony formation, and cell invasion, in contrast to cells treated with formononetin alone.
CONCLUSIONS: Formononetin can restrain the proliferation of BC cells, which may be mediated through the interference of PD-L1 and suppression of the activation of the STING-NF-κB pathway.
摘要：
背景：乳腺癌（BC），女性常见的肿瘤，有很高的发病率和死亡率。Formononetin,红三叶草和黄芪中的活性成分，具有广泛的药理应用，包括作为抗癌剂。由于免疫疗法是BC治疗策略中的热门话题，在这项研究中，它致力于评估Formonoetin在BC免疫治疗中的具体机制。
方法:使用不同浓度(0、20、40、60、80、100μM)处理用pcDNA3.1-程序性死亡配体1(PD-L1)或短发夹RNA(sh)-PD-L1转染的BC细胞。细胞分为四个亚组：CTRL，pcDNA3.1-PD-L1,sh-CTRL,和sh-PD-L1.通过甲基噻唑基二苯基-溴化四唑（MTT）测定和流式细胞术评估细胞活力和细胞周期。通过定量实时逆转录聚合酶链反应(qRT-PCR)验证程序性死亡配体1(PD-L1)mRNA浓度。通过克隆测定和transwell测定评估细胞转移。干扰素基因的p-STING/刺激因子（STING），通过蛋白质印迹测定p-p65/p65和PD-L1浓度。
结果：Formononetin抑制MCF-7和MDA-MB-468细胞的增殖，减少PD-L1mRNA，p-STING/STING,和p-p65/p65蛋白浓度。而PD-L1抑制抑制了BC细胞的活力，pcDNA3.1-PD-L1干预具有相反的结果。STING通路抑制因子C-176联合海蒙素处置进一步克制细胞增殖,菌落形成,和细胞入侵，与单独用海蒙素处理的细胞相反。
结论：Formononetin能抑制BC细胞的增殖，这可能是通过干扰PD-L1和抑制STING-NF-κB通路的激活来介导的。
方法:使用不同浓度(0、20、40、60、80、100μM)处理用pcDNA3.1-程序性死亡配体1(PD-L1)或短发夹RNA(sh)-PD-L1转染的BC细胞。细胞分为四个亚组：CTRL，pcDNA3.1-PD-L1,sh-CTRL,和sh-PD-L1.通过甲基噻唑基二苯基-溴化四唑（MTT）测定和流式细胞术评估细胞活力和细胞周期。通过定量实时逆转录聚合酶链反应(qRT-PCR)验证程序性死亡配体1(PD-L1)mRNA浓度。通过克隆测定和transwell测定评估细胞转移。干扰素基因的p-STING/刺激因子（STING），通过蛋白质印迹测定p-p65/p65和PD-L1浓度。
结果：Formononetin抑制MCF-7和MDA-MB-468细胞的增殖，减少PD-L1mRNA，p-STING/STING,和p-p65/p65蛋白浓度。而PD-L1抑制抑制了BC细胞的活力，pcDNA3.1-PD-L1干预具有相反的结果。STING通路抑制因子C-176联合海蒙素处置进一步克制细胞增殖,菌落形成,和细胞入侵，与单独用海蒙素处理的细胞相反。
结论：Formononetin能抑制BC细胞的增殖，这可能是通过干扰PD-L1和抑制STING-NF-κB通路的激活来介导的。
