Biochanin A Regulating PD-L1 Expression And Its Anti-Colon Cancer Mechanisms

Background: The role of Programmed Cell Death Ligand-1(PD-L1)in suppressing anti-tumor immune response has been extensively documented.Recent studies found a distinct tumor-intrinsic role for PD-L1 in modulating tumor proliferation,invasion,angiogenesis and resistance to therapy.Biochanin A,an O-methylated isoflavone mainly found in red clover,exhibits anti-inflammatory and anti-cancer properties.However,the mechanistic basis of its anti-tumor effects remains poorly understood.Purpose: This study was designed to investigate the efficiency of Biochanin A in suppressing colon cancer and to explore whether PD-L1 acts as an important mediator in this process.Methods: 1)Molecular docking,MTT assay,western blotting assay,RT-PCR,and flow cytometry were conducted to examine the efficiency of Biochanin A in regulating PD-L1 expression.2)The impact of Biochanin A on Ras/RAF/MEK/ERK pathway and on the accumulation of c-Myc protein was verified by IF(immunofluorescence assay),western blotting and molecular docking.3)Via IF,western blotting and molecular docking,we also investigated the effect of Biochanin A on HIF-1α and its molecular mechanism.4)IF,immunoprecipitation and other experiments were performed to explore the the manner whereby Biochanin A impacts the interaction between c-Myc and HIF-1α to regulate PD-L1 protein level.5)Colony formation assay was performed to assess cell proliferation rate using culture medium with or without Biochanin A.6)The effect of Biochanin A on angiogenesis was detected by invasion assay,migration assay and tube formation.Results:1)Biochanin A downregulated PD-L1 protein level in various tumor cell lines;2)Biochanin A inhibited PD-L1 protein level by suppressing PD-L1 protein synthesis;3)Biochanin A suppressed c-Myc expression via RAS/RAF/MEK/ERK signaling pathway;Biochanin A inhibited HIF-1α expression via m TOR/p70S6K/4EBP1 signaling pathway.4)By blocking the crosstalk between c-Myc and HIF-1α,Biochanin A inhibited the expression of PD-L1;5)Via suppressing PD-L1,Biochanin A inhibited tumor cell proliferation and cell cycle-related protein expression;6)PD-L1 mediated the inhibitory effect of Biochanin A on angiogenesis.Conclusion: Biochanin A inhibits PD-L1 expression by preventing HIF-1 and Myc from interacting.Biochanin A also inhibits cancer proliferation and angiogenesis by inhibiting PD-L1 expression.Our current research focuses on anti-tumor properties of Biochanin A,providing insight into the development of cancer therapeutics based on PD-L1 inhibition.