The Effect Of Artesunate On Human Glandular Cystic Carcinoma Cisplatin Resistance NACC/DDP Cell Lines Invasion And Angiogenesis Chemotaxis

Objective:To establish the drug resistant cell line of human salivary adenoid cystic carcinoma and observe the effect of artesunate(ART) on the ability of invasion and on human umbilical vein endothelial cells' chemotaxis.And to detect the angiogenesis in nude mice parotid gland tumorMethods:1 A dichlorodiamineplatinum (DDP) resistance adenoid cystic carcinoma cell line was induced by culturing the NACC cell line with a constant concentration of DDP periodically. In order to verify the drug resistance of DDP induced cells, the degree of tolerance of drug induced cells to DDP was detected by MTT method. And the difference of the invasion ability of drug induced cells and NACC parental cells was detected by Transwell under the intervention of DDP2 The inhibitory effect of ART on the proliferation of drug induced cells was detected by MTT method. Results are used to screen the following experimental drug concentration.3 Inhibitory effects of ART on the invasion and migration of drug induced cells were determined by Transwell.4 The inhibitory effect of conditioned medium from drug induced cells after treated with ART on the mobile ability of HUVEC cells was detected by wound healing assay.5 Effect of ART on the invasion and migration of drug induced cells chemotaxis HUVEC cells under the co-culture condition by Transwell assay.6 After drug induced cells had been interfered with different concentrations of ART. The expression of E-cadherin, MMP-9, COX-2 and VEGFA mRNA were detected by real-time qPCR assay.7 Parotid gland tumor model was established in nude mice with drug induced cells. After different concentrations of ART have been injected into the abdominal cavity of nude mice, the expression of E-cadherin, MMP-9 and VEGFA in parotid gland of nude mice was detected by immunohistochemistry.8 The blood vessels in the tumor tissue were marked and counted by CD34 antibody.Results:1. After 7 months of DDP induction, a drug resistant adenoid cystic carcinoma cell line with stable DDP resistance cell line was established. The volume enlargement of DDP resistance cells was observed under an inverted optical microscope. The number of nucleolus was increased and the shape of the cell was irregular.2. The result of MTT assay showed that the resistance index of cells to DDP was 2.16. The result of transwell assay indicated that under the intervention of 1.5 ?M DDP. The number of cell invasion and migration of drug induced cells is much more than that of NACC cells (P<0.05). The drug induced cell line was named as NACC/DDP.3. The result of MTT assay showed that ART could effectively inhibit the proliferation of NACC/DDP cells, with the increase of drug concentration,NACC/DDP cell proliferation was inhibited more significantly. Calculation and analysis showed that the median lethal concentration (IC50) of ART to NACC/DDP cells was 1258.52 + 36.87?M, IC05 was 166.86 + 49.84 ?M.In order to exclude the effect of ART on the invasion and migration of NACC/DDP cells, we chosed 37.5, 75,150,600?M as the experimental concentration.4. The result of transwell assay showed that the invasion and migration of NACC/DDP cells were inhibited by ART, with the increase of concentration, the inhibitory effect appeared more significant (P<0.05).5. Cell wound healing assay showed that the locomotivity of HUVEC cells were inhibited by conditioned medium from drug induced cells cultured with ART. The inhibition of the invasion ability of HUVEC cells effected with the conditioned medium related with the concentration of ART (P<0.05). In the experiment of co-culture of NACC/DDP cells and HUVEC cells, the ability of NACC/DDP cells to chemotaxise the invasion and migration of HUVEC cells was inhibited by different degreeswith the increase of ART concentration(P<0.05).6. The result of invasion and migration assay of NACC/DDP cells and experiment of 37.5 ?MART group and 75 ?M ART group in NACC/DDP+HUVEC co-culture experiment showed no statistical difference.So group 75?M ART was removed from Real-time PCR assay. The result of Real-time PCR showed that the expression of MMP-9, VEFGA and COX-2 gene mRNA in NACC/DDP cells in vitro could be effectively inhibited by ART(P<0.05). Additionally, the E-cadherin mRNA expression in NACC/DDP cells was up-regulated after ART intervention (P<0.05).7. The positive rate of VEFGA and MMP-9 expression in the nude mice subcutaneous xenograft model with NACC/DDP cells was decreased with the increase of ART concentration (P<0.05). The positive rate of E-cadherin expression was increased with the increase of ART concentration P<0.05). The microvessels in the nude mice parotid gland tumor tissue were marked and counted by CD34, the result showed that after abdominal injection,the number of new vessels in tumor was decreased compared with the saline group(P<0.05).Conclusion:1. The NACC/DDP cell line has the stable fundamental characteristics of a drug resistance.Under the same concentration of DDP, the invasion and metastasis ability of NACC/DDP cells was stronger than that of NACC cells.2 ART can effectively inhibit the invasion and migration of NACC/DDP cells in vitro, it can also inhibit the invasion and migration of NACC/DDP cells chemotaxis HUVEC cells effectively.ART can inhibit the angiogenesis of the parotid gland in nude mice subcutaneous xenograft model, and the mechanism may be related to the down-regulation of MMP-9, VEGFA, COX-2 gene expression and the up-regulation of E-cadherin gene expression.