Study On Inhibitory Effect And Underlying Mechanisms Of Ampelopsin In Human Cervical Cancer Cell Line SiHa

Background: Cervical carcinoma is the most common gynecological malignancy.About 85% of cervical cancer cases and deaths occur in developing countries.At present,with the development of cervical cancer cytology detection technology,this cancer has been detected more earlier.However,the incidence of cervical carcinoma tends to be younger,and women’s health has been affected seriously.Human cervical squamous carcinoma cell line Si Ha is a human papillomavirus(HPV)-positive cervical cancer cell line.Si Ha cells belong to the high-risk type of HPV-16 and can cause squamous epithelial proliferation of human skin and mucosa.Modern research shows that the Chinese medicine and their activity extracts play multiple advantages in treating cervical cancer in humans.Ampelopsin(AMP),also known as dihydromyricetin,is an important natural flavonoid compound extracted from Ampelopsis megalophylla Diels et.Gilg.In recent years,studies have shown that AMP has a wide range of antitumor effects.However,its mechanism of inducing apoptosis and autophagy of cervical cancer cells has not been clear.Thus,AMP has not been widely developed and applied.Objectives: Study the effects of AMP on the proliferation inhibition,cycle and apoptosis of human cervical cancer cell line Si Ha,and further explore the mechanism of action of its inhibition on Si Ha cells.Methods: Different concentrations of AMP were applied to Si Ha cells,and the effect of AMP on the proliferation of Si Ha cells was measured using MTT assay.The effect of AMP on the apoptotic morphology of Si Ha cells was observed using the Hoechst 33258 method.Cell cycle,apoptotic rate and mitochondrial membrane potential were observed by flow cytometer.Transmission electron microscopy was used to observe the effects of AMP on the ultrastructure of Si Ha cells.Western blot was used to detect the expression levels of apoptosis and autophagy-related proteins with treatment of AMP.Results: Compared with the control group,AMP had a significant inhibitory effect on Si Ha cells proliferation,and it is time-and dose-dependent(p<0.05);Hoechst 33258 staining and Annexin V-FITC/PI double staining results showed that with the drug concentration increased,increased apoptotic cells were observed,so AMP could induce its apoptosis(p<0.01);PI single staining method detected cell cycle results showed that AMP at concentration of 0~40 μM the distribution ratio of the G0/G1 phase is increasing,AMP at concentration of 40~80 μM,the distribution ratio of the G0/G1 phase is decreased,and the distribution ratio of the S phase is increased.The cell cycle arrested in S and G2/M phases in time dependent;Rh-123 staining detection with the increase of drug concentration,mitochondrial transmembrane potential(ΔΨm)decreased,fluorescence intensity increased;western blot results showed that with drug concentration increased,apoptosis-related protein Bax/Bcl-2 ratio and cleaved-Caspase-3 expression levels increased(p<0.01),autophagyrelated proteins LC3-Ⅱ/LC3-Ⅰ ratio,Beclin1 expression levels increased(p<0.01),p62 expression levels decreased(p<0.01),The expression of phosphorylated protein in PI3K/Akt/m TOR signaling pathway were all downregulated(p<0.01).Conclusion: AMP has an inhibitory effect on Si Ha cells proliferation,AMP arrests cell cycle and induces its apoptosis,thereby reducing mitochondrial membrane potential.At the same time,AMP can regulate the expression level of Si Ha cells apoptosis-related proteins Bax,Bcl-2,cleavedCaspase-3 and autophagy-related proteins LC3,Beclin1,p62,also,AMP inhibits the phosphorylation of PI3 K,Akt,m TOR,and inhibits the activation of PI3K/Akt/m TOR signaling pathway to promote apoptosis and autophagy of Si Ha cells.