Evaluation Of Anticancer Function And Mechanism Of Formononetin

Formononetin(FMN)is a kind of isoflavone compound,which has become a hot research topic over the years.In recent years,formononetin has anti-inflammatory,anti-lipid peroxidation,lowering blood lipid and other effects.It can also remove oxygen free radicals,improve atherosclerosis,can prevent a variety of cancers,such as breast cancer,prostate cancer,colorectal cancer and so on.In this study,human colorectal cancer cell line HCT116 was used as the test target,and formononetin was used as the test tool.MTS method,hoechst staining,scar test,flow cytometry,RT-qPCR,Western blot and other methods were used to explore the anti-colorectal cancer effect of formononetin.This research provides a scientific theoretical basis for the development of functional food with anti-cancer effects,and provides potential theoretical evidence for improving the utilization rate of formononetin,a crop by-product.This study showed that the concentration of formononetin set in this study did not significantly affect the activity of HUVEC in human umbilical vein endothelial cells.However,formononetin could inhibit the proliferation activity of human colorectal cancer HCT116 at concentration gradient in this study,resulting in morphological changes of cells in a concentration-dependent manner.Flow cytometry showed that 50 μg/mL formononetin could arrest 56.2%of the cell cycle in G1 phase,an increase of 5.3%compared with the control group.Among the genes related to cell cycle,formononetin could promote the mRNA expression of p53,p21,p27,p 16 and p18,and significantly increased the protein level,which were 2.25,3.08,7.13,4.25 and 3.24 times in the 50 μg/mL formononetin group compared with control group.At the protein level,Cyclin D1 and Cyclin E1 were inhibited by 0.17 and 0.66 times,respectively,in the 50 μg/mL formononetin group compared with the blank control group,resulting in cell cycle stagnation.To determine whether formononetin interferes with the HCT116 cell cycle by targeting p53,Pifithrin-α hydrobromide(PFT-α)has been used to intervene the p53 activity induced by formononetin in HCT116 cells.Western blot results showed that after inhibiting the transcriptional activity of p53 in HCT116 cells,the activation of p21 by formononetin was reversed,and the expression of formononetin decreased 3.32 times compared with the group treated with formononetin alone.The inhibition of formononetin on Cyclin D1 and Cyclin E1 was also reversed,and the protein expression levels of Cyclin D1 and Cyclin E1 increased 5.21 times and 1.57 times when PFT-α was combined with formononetin compared with the group treated with formononetin alone.These results suggest that formononetin inhibits proliferation of HCT116 cells by targeting p53.Afterwards,the effects of formononetin on JNK and ERK signals in MAPK signaling pathway were also investigated.Western blot results showed that compared with the blank control group,the expression levels of P-ERK1/2 and P-JNK1/2 decreased by 0.34 and 0.12 times at 50 μg/mL,respectively.Formononetin increased the expression of E-cadherin and decreased the expression of N-cadherin,MMP-2,MMP-9 and TGF-β1 at the mRNA level of cell invasions related genes.This study also confirmed that formononetin can induce the apoptosis of HCT116.Hoechst staining preliminarily proved that formononetin could induce apoptosis of HCT116 cells.The cell apoptosis induced by formononetin was quantitatively analyzed by flow cytometry.It was found that the number of HCT116 premature cells was 2.5%,3.4%,4.2%and 7.5%under the intervention of formononetin at the concentration of 0 μg/mL,12.5μg/mL,25 μg/mL and 50 μg/mL,respectively.It can be shown that formononetin can effectively promote the early metastasis of cancer cells.In terms of apoptosis-related genes,formononetin can reduce the ratio of Bcl-2 to Bax and enhance the expression of Cyto-C at protein and mRNA levels.The expression of Bid was increased by 2.91 times in the 50μg/mL formononetin group compared with the blank control group.The activity of Caspase 3 and Caspase 8 proteins in the Caspase family was upregulated 12.6 and 1.79 times in the 50 μg/mL formononetin group compared with the blank control group,which catalyzed the decomposition of PARP and ultimately induced apoptosis.In order to confirm that formononetin can target p53 signal to induce apoptosis of HCT116 cells,PFT-α was used to intervene the p53 activity induced by formononetin in HCT116 cells.Western blot results showed that the ratio of Bcl-2/Bax increased by 8.90 times after PFT-α and formononetin treatment compared with the group treated with formononetin alone,which confirmed that formononetin promoted apoptosis of HCT116 cells through targeted regulation of p53 signal.To further investigate the effect of formononetin on HCT116 apoptosis,the effect of formononetin on p38 MAPK phosphorylation expression was studied.Compared with the blank control group,the expression of p-p38 protein increased 3.78 times after 50 μg/mL formononetin treatment.In order to further study how formononetin affects the anticancer effect of HCT116 cells,transcriptome sequencing was used to study the changes of formononetin on thr RNA level of HCT116 cells,and 149 genes with significant changes(FC(FMN/CON)>2 or FC(FMN/CON)<0.5,P<0.05),in which 85 genes were significantly up-regulated and 64 genes were significantly down-regulated compared with blank control group.GO analysis and KEGG analysis showed that these genes were involved in a variety of biological processes and signal responses related to cell growth and angiogenesis.These genes were predicted to form a p53-centered interaction network,among which CFTR,ALK and MMP9 had strong relationships.Conclusion:Formononetin can inhibit the proliferation and invasion of colorectal cancer,and can promote the apoptosis of colorectal cancer.In-depth study of its anticancer molecular mechanism can provide scientific theoretical basis for the prevention of cancer functional food,so as to improve the standard of healthy living of people.