Effects Of Artesunate On The Proliferation,invasion And Metastasis Of Human Adenoid Cystic Carcinoma SACC-83 Cell Line

Objective:To detect the effects of artesunate（ART）on the proliferation,invasion and migration of human adenoid cystic carcinoma SACC-83 cell line in vitro,and the effects of ART on the expression of related markers of SACC-83 cell line.Methods:1.The effects of different concentrations of ART on the proliferation and apoptosis of human adenoid cystic carcinoma SACC-83 cells cultured in vitro for 24 h,48 h and 72 h were detected by CCK-8 assay,and the drug concentration in subsequent experiments was screened based on the results.In order to minimize the inhibitory effect of ART on SACC-83 cells,follow-up experiments were conducted with 1/4 IC₀₅5 and 1/2 IC₅₀0 as reference values?for the concentration range of in vitro experiments.2.The effect of ART on clonogenesis ability of SACC-83 cell was detected by clone formation experiment.3.Scratch wound healing assay was utilized to detect the inhibitory effect of different concentrations of ART on migration of SACC-83 cell.4.The effect of ART on the invasion and metastasis of SACC-83 cells were detected with transwell invasion and metastasis assay.5.The expression of E-cadherin and VEGFA protein in SACC-83 cells after intervention of ART for 48 h were detected by immunocytochemical staining.6.After drug induced cells had been interfered with different concentrations of ART for 48 h.The expression of E-cadherin,COX-2 and VEGFA mRNA were detected by real-time q PCR assay.Results:1.CCK8 assay showed that different concentrations of ART can significantly inhibit the proliferation of SACC-83 cells,and the inhibition rate increased with the increase of drug concentration over time.Calculation and analysis showed that the median lethal concentration(IC₅₀)of ART to SACC-83cells is 462.60±35.15μM after 48 hours of ART intervention,and IC₀₅5 is59.28±39.86μM.In order to minimize the killing effect of ART on SACC-83cells,follow-up experiments were conducted with 1/4 IC₀₅5 and 1/2 IC₅₀0 as reference values for the concentration range of in vitro experiments.15,30,60,120 and 240μM were selected for the scratch wound healing assay and invasion experiments.2.The result of clone formation experiment indicated that ART can inhibit the clonogenesis ability of SACC-83 cell compared with control group,and the inhibitory effect of ART enhanced with the increase of drug concentration（P<0.05）.3.The scratch wound healing assay results showed that ART can effectively suppress the the migration ability of SACC-83 cells（P<0.05）.And the migration ability of SACC-83 cells significantly decreased with the increase of ART concentration（F=54.586,P<0.001）.4.The results of transwell invasion and migration experiment indicated that compared with blank control group,the invasion and migration of SACC-83cells were inhibited by ART at 15μM,and with the increase of concentration,the inhibitory effect appeared more significant（P<0.05）.5.The average optical density of VEGFA antibody-stained area in SACC-83 cells was gradually decreased as the concentration of ART drug increased（F=326.054,P<0.01）,while the protein expression of E-cadherin wasup-regulatedwiththeincreaseofARTconcentration（F=1503.760,P<0.01）.6.The result of Real-time PCR showed that the expression of VEFGA and COX-2 mRNA in SACC-83 cells could be effectively inhibited by ART in vitro（F=319.580,P<0.01）,（F=185.828,P<0.01）.Additionally,the mRNA expression of E-cadherin in SACC-83 cells cells was up-regulated after ART intervention（F=2513.935,P<0.01）.The difference between each drug group and 0μM blank control group was statistically significant（P<0.01）.Conclusion:1.ART can effectively inhibit the proliferation and clonogenesis ability of human salivary adenoid cystic carcinoma cell line SACC-83 in vitro.2.ART has the potential to reduce the invasion and migration of SACC-83cells in vitro.3.ART can down-regulate the expression of VEGFA and COX-2 and up-regulate the expression of E-cadherin in SACC-83 cells.