Formononetin Restrains Tumorigenesis of Breast Tumor by Restraining STING-NF-κB and Interfering with the Activation of PD-L1.

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. CONCLUSION: 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.