Anti-tumor Effect And Mechanism Of Artemether On Tongue Squamous Cell Carcinoma In Vitro

[Objective]Tongue Squamous Cell Carcinoma is one of the most common malignant tumors in oral cavity.TSCC is characterized high degree of malignancy,high invasiveness and easy relapse.Current treatments for patients with TSCC are radiotherapy,surgery and chemotherapy,but the five-year survival rate of patients has not significantly improved.Artemether which is methyl ether derivative of artemisinin can inhibit the growth of a variety of cancer cells and has potential antitumor effect.However,the molecular mechanism of its antitumor effect on TSCC is still unknown.The purpose of this study was to explore the antitumor Effect and mechanism of artemether on TSCC in vitro.We focused on whether the HSP90/AKT pathway and miRNA were related to the influence artemether on the growth and apoptosis of Cal-27 cell,to provide theoretical basis and experimental basis for the treatment of tongue squamous cell carcinoma[Methods](1)CCK8 assay was used to detect the percent inhibition of Cal-27 cells and SCC-15 cells before and after artemether treatment.(2)Flow cytometry was used to measure cell cycle of Cal-27 cells and analyze apoptosis rates of Cal-27 cells and SCC-15 cells.(3)Western blotting was used to detect the expression of apoptosis-related proteins(Bad,Bax,Bcl-2 and cleaved-caspase3).(4)The mRNA expression of HSP90α and AKT were analyzed by qRT-PCR and the protein expression were analyzed by Western blotting before and after treatment with artemether.(5)Co-immunoprecipitation(Co-IP)and Duolink in situ proximity ligation assay were used to detect the interaction between HSP90α and AKT protein.(6)We analyzed and chose miRNAs which targeted at AKT through bioinformatics website(microRNA.org,TargetScan and miRTarBase)and by combined with literature reports.The expression of miRNAs was detected by qRT-PCR before and after treatment with artemether,miR-29a-3p was increased after the treatment with artemether.(7)Dual luciferase reporter assay was used to identifying the targeting-inhibition of AKT2 and AKT3 by miR-29a-3p.(8)Stable miR-29a-3p knockdown Cal-27 cell line(Cal-27-hsa-miR-29a-3p inhibitor sponge)and negative control Cal-27 cell line(Cal-27-shNC)were constructed by lentiviral vectors were established.After treated with 0.10 mg/ml artemether for 24 hours,the cell proliferation activity was detected by CCK8 assay,the apoptosis rate was detected by flow cytometry,and the expression of apoptosis related proteins was detected by Western blot.(9)Statistical software SPSS 25.0 was used for statistical analysis of the experimental data,and statistical graphs were drawn with GraphPad prism 5.0 software.[Results](1)Artemether can inhibit the proliferation of Cal-27 cells and SCC-15 cells and induce G2/M cell cycle arrest of Cal-27 cells.As the cell proliferation activity of each group detected by CCK8,artemether has an inhibitory effect on the proliferation activity of Cal-27 cells and SCC-15 cells.The percent inhibition of cell proliferation increased with the increase of artemether concentration and had a concentration dependence(p<0.01).Flow cytometry of cell groups in each group showed that artemether induced G2/M cell cycle arrest(p<0.01)of Cal-27 cells.(2)Artemether can induce apoptosis of Cal-27 and SCC-15 cells,up-regulate the expression of cleaved caspase 3 and down-regulate the expression of Bcl-2 of Cal-27 cells.Flow cytometry showed that the apoptosis rate of artemether treated groups with different concentrations was significantly higher than the control group,and the difference was statistically significant(p<0.01)of Cal-27 and SCC-15 cells.Further detection of apoptosis-related proteins of Cal-27 cells by Western blotting revealed that compared with the control group,the expression of Bax and Bad in the artemether treatment group had no significant changes(p>0.05).However,artemether can up-regulate the expression of cleaved caspase 3 and significantly down-regulate the expression of Bcl-2 in Cal-27 cells(p<0.01).(3)Artemether down-regulates mRNA and protein expression of HSP90α in Cal-27 cells.The detection by qRT-PCR revealed that artemether can down-regulate HSP90AA1 mRNA expression in a concentration-and time-dependent manner.The expression of HSP90a protein detected by Western blotting was similar to the expression of HSP90AA1 mRNA.(4)Artemether can down-regulate AKT and p-AKT protein expression in Cal-27 cells.qRT-PCR showed that artemether had no significant effect on AKT mRNA expression in Cal-27 cells(p>0.05).Western blotting showed that the expression of AKT and p-AKT decreased in artemether treated groups at different concentrations compared with the control group(p<0.05).(5)Artemether can inhibit protein-protein interaction between HSP90α and AKT in Cal-27 cells.It was found that protein-protein interaction between HSP90α and AKT existed in Cal-27 cells of tongue squamous cell carcinoma,and the combination of the two decreased after artemether treatment by Co-immunoprecipitation test.Further verification using Duolink in situ PLA also obtained similar results.(6)Artemether can up-regulate miR-29a-3p expression in Cal-27 cells and negatively regulate AKT3.Using qRT-PCR detection,artemether increased the expression of miR-29a-3p in Cal-27 cells.Double luciferase reporter analysis revealed that in 293T cells,miR-29a-3p and AKT2 did not bind;both targets of AKT3 and miR-29a-3p had binding effects.(7)miR-29a-3p dysfunction can reduce the antitumor effect of artemether on Cal-27 cells in vitro.A stable knockdown of miR-29a-3p Cal-27 cell line was constructed.CCK8 assay showed that knockdown miR-29a-3p reduced the inhibitory effect of artemether on Cal-27 cell proliferation;flow cytometry revealed that knockdown miR-29a-3p can attenuate the apoptosis rate of Cal-27 cells which artemether induced:western blotting detection showed that knockdown miR-29a-3p reduced the effect of up-regulate expression of cleaved caspase3 and reduced the effect of down-regulate expression of Bcl-2,AKT3 and p-AKT3 in Cal-27 cells.[Conclusions](1)Artemether can inhibit the proliferation of Cal-27 cells and SCC-15 cells,artemether can induce G2/M cell cycle arrest of Cal-27 cells;artemether can down-regulate the expression of apoptosis-related protein Bcl-2 and promote the activation of Caspase 3,induced apoptosis and exerts anti-tumor effects.(2)Artemether may down-regulate the expression of HSP90 and AKT,and inhibit the protein-protein interaction between the two.At the same time,it may down-regulate AKT3 expression by up-regulating miR-29a-3p,inhibit the AKT signaling pathway,and exert anti-tongue squamous cell carcinoma effect.