The Study On The Effect And Mechanism Of Formononetin On The Proliferation Of Cervical Cancer Cells

Background: Cervical cancer, the second biggest threat to women’slives malignant disease, is the most common gynecologic malignancy,and become a major social problem. The most common treatment forcervical cancer is surgical treatment and radiotherapy. Combined therapyhas been become a new trend in the treatment of cervical cancer,including chemotherapy with radiotherapy, chemotherapy and surgery,radiation therapy combined with surgery. Astragalus (Astragalusmongholicus Bunge) belongs to leguminous plants of the genusAstragalus, and is an important traditional medicine. Modernpharmacological studies have shown that astragalus has a variety ofbiologically active immunomodulatory effects of astragalus extract andits main component, cardiovascular regulation, and cancer research hasmade great progress. Literature studies have shown that the chemicalcomposition of the diversity of Astragalus led to its diversity of biologicalactivity. Astragalus saponin I plays an important role in the treatment ofdiabetes. Astragalus saponin Ⅳ has protective effects of ischemic braininjury, anti-inflammation. Astragalus polysaccharide and saponins haveimmunomodulatory and cardiovascular-protective effects. Astragalus totalflavonoids possess antioxidant function and were used to study theanti-mutagenic, anti-aging and other aspects. As one of the activeingredients of Astragalus, formononetin can inhibit a variety of cancercells proliferation, induce apoptosis of various cancers, such as breast cancer, prostate cancer. But the effect of formononetin on cervical cancerhas not been reported.Objective: In this study, Hela cells were selected to investigate theeffects of formononetin on cervical cancers cells proliferation.Furthermore, xenograft transplanted tumor experiment was used todemonstrate the inhibitory effects in vivo.Methods: Hela cells were incubated with different concentrations offormononetin and MTT assay was used to measure the inhibitory effecton cells. Flow cytomety and Hoechst/PI staining were applied to detectthe apoptotic rate of cells. Xenografts transplanted tumor was establishedto confirm the inhibitory effects in vivo. Western blot was used to detectthe molecular changes including bax, bcl-2, caspases and P13K/Akt.Results:1) MTT assay showed that, drug treatment had no significant effectson cell proliferation at the concentration of1μmol/L. But formononetininhibited cell proliferation in a dose-dependent manner from theconcentration of5μmol/L.2) Flow cytometry was used to measure the effect of formononetinon cell apoptosis, Hela cells were cultured in different concentrations offormononetin (1μmol/L,5μmol/L,10μmol/L,25μmol/L,50μmol/L) for24h. Then cells were collected and performed V-FITC/PI staining. Resultsshowed that the drug had no significant effect on the rate of apoptosis atthe concentration of1μmom/l/. However, formononetin can significantlypromote apoptosis rate of tumor cells at the concentration of5μmom/l ina dose-dependent manner. Hoechst/PI staining was further used tomeasure the effect of formononetin on cell apoptosis. The results showedthat formononetin treatment of cervical cancer cells can significantlypromote cell apoptosis rate. 3) ATP content was detected after administration of differentconcentrations of formononetin (1μmol/L,5μmol/L,10μmol/L,25μmol/L)on Hela cells. The results showed that the intracellular ATP content wassignificantly lower at the concentration of5μmol/L in a dose dependentmanner. Next, we examined the effect of different concentrations offormononetin on the oxygen consumption (5μmol/L,10μmol/L,25μmol/L) for different time points (1min,2min,5min and15min),results showed that the oxygen consumption was decreased in a time-anddose-dependent fashion.4) All mice survived during the experiment, formononetinadministration (10mg/kg and20mg/kg) can significantly reduce theweight and volume of the tumors.5) Western blot showed that the formononetin administrationsignificantly increased the expression of bax in a dose-dependent mannerfrom the concentration of5μmol/L.6) Western blot showed that the formononetin treatment significantlyreduced the expression of bcl-2in a dose-dependent manner.7) Cells were treated with different concentrations of formononetin(1μmol/L,5μmol/L,10μmol/L) acting on Hela cells,24h post-apoptoticprotein caspase-3expression levels change detection. The results showedthat formononetin can significantly promote the expression of caspase-3.8) We further test formononetin P13K/Akt changes in cellularsignaling pathways. Different concentrations of formononetin (1μmol/L,5μmol/L,10μmol/L) acting on Hela cells,24h after P13K/Aktexpression levels change detection. The results showed that formononetincan significantly inhibit the phosphorylation levels of AKT.Conclusion: Our study found that formononetin treatmentsignificantly inhibited cell proliferation and promoted cell apoptosis. Xenografts also showed that formononetin could reduce the growth oftumor cells. In addition, formononetin regulated the expression of cellapoptos-related proteins, such as formononetin can significantly increasebax, caspase3expression, and reduce bcl-2expression. Furthermore, weexplored the molecular mechanisms of formononetin regulation of cellproliferation, and found that formononetin regulate cell growth andproliferation by inhibiting PI3K-Akt pathway. Through the interpretationof these results, we preliminary show the effect of formononetin againstcervical cancer growth and proliferation and its underline molecularmechanisms, so as to provide a solid foundation for target discovery andscreening of such tumors intervention, has important scientificsignificance and social significance.