| Objective: To establish a drug-resistant human salivary adenoid cystic carcinoma(SACC-83/DDP)from the parental cell line SACC-83 by cisplatin and detect the drug resistance index of SA CC-83/DDP.Study the possible mechanism of artesunate in killing and reversing the drug resistance of SACC-83/DDP.Method: Established a cisplatin-resistant human salivary adenoid cystic carcinoma cell line SACC-83/DDP from the parental cell line SACC-83 by short-term exposing to high dose of DDP and long-term maintenance of low of DDP periodically.The multidrug resistance index of SACC-83/DDP was detected by CCK8 method.The cell growth curve was drawn by the results of CCK8 method.Compared the clone ability between SACC-83/DDP and SACC-83 cells by plate cloning experiment.Compared the expression of MDR1 and survivin protein in SACC-83/DDP and SACC-83 cells by immunohistochemistry.Compared the expression of GST? and Bcl-2 protein in SACC-83/DDP and SACC-83 cells by immunofluorescence staining.Compared the expression of MDR1 and GST? genes in SACC-83/DDP and SACC-83 cells by real-time fluorescence quantitative PCR.The expression of MDR1,GST?,survivin and Bcl-2 protein in SACC-83/DDP and SACC-83 cells was detected by Western Blot method.Detected the proliferation and apoptosis of SACC-83/DDP after exposing to ART for 48 h in vitro.Detected the reversal effect of ART on cisplatin-resistance of SACC-83/DDP by CCK8 assay.The expression of MDR1 and survivin in SACC-83/DDP cells 48 hours after ART intervention was detected by immunohistochemistry.The expression of GST? and Bcl-2 protein in SACC-83/DDP cells after treated with ART for 48 h was detected by immunofluorescence staining.Real-time fluorescence quantitative PCR was used to detect the expression of MDR-1,GST?,survivin and Bcl-2 in SACC-83/DDP cells after treated with ART for 48 h.Western Blot assay was used to detect the expression of MDR1,GST?,survivin and Bcl-2 in SACC-83/DDP cells after treated with ART for 48 h.Result: A stable adenoid cystic carcinoma drug-resistant cell line SACC-83/DDP was established after treated with cisplatin for 7 times.Compared with parent cell SACC-83,the growth rate and clone formation rate of drug-resistant cell SACC-83/DDP had no significant difference.CCK8 results showed that SACC-83/DDP had certain resistance to DDP,and the resistance index was 2.38.At the same time,SACC-83/DDP also produced resistance to BLM and VCR.Immunohistochemical results showed that the expression of MDR1 and survivin protein in SACC-83/DDP cells was higher than that in SACC-83 cells,and the difference was statistically significant.Immunofluorescence results showed that the expression of GST? and Bcl-2 in SACC-83/DDP cells was higher than in SACC-83 cells,and the difference was statistically significant.The results of PCR showed that the expression of MDR1,GST?,survivin and Bcl-2 mRNA in SACC-83/DDP cells was higher than that in SACC-83 cells,and the difference was statistically significant.Western Blot results showed that the expressions of MDR1 and GST? protein in SACC-83/DDP cells were higher than those in SACC-83 cells,and the difference was statistically significant.CCK8 results showed that ART can inhibit the proliferation of SACC-83/DDP cells,and the inhibition rate increased with the increase of drug concentration.CCK8 results showed that ART had a certain effect on reversing SACC-83/DDP resistance.When ART concentration was 30 ?mol/L,the reversal index of resistance was 1.76.Immunohistochemical results showed that,comparing with the control group,the expression of MDR1 and survivin protein in SACC-83/DDP cells was down-regulated after ART intervention at 30,60,120,240,480 ?mol/L for 48 h,and the difference was statistically significant.Immunofluorescence results showed that,compared with the control group,the expression of GST? in SACC-83/DDP cells was down-regulated after ART intervention at 30,60,120,240 and 480 ?mol/L for 48 h,and the difference was statistically significant.The results of PCR showed that,compared with the control group,the expression of MDR1,GST?,survivin and B-cl2 mRNA in SACC-83/DDP cells was down-regulated after ART intervention of 30,60,120,240,480 ?mol/L for 48 h,and the difference was statistically significant.Western Blot results showed that,compared with the control group,the expressions of MDR1,GST? and B-cl2 in SACC-83/DDP cells were down-regulated after 30,60,120,240,480 ?mol/L ART intervention for 48 h,and the differences were statistically significant.Conclusion:1.The SACC-83/DDP cells established in this study showed a certain drug resistance compared with their parent cells SACC-83,and the drug resistance index was 2.38.There was no significant difference in clone formation rate and cell growth rate between SACC-83/DDP and SACC-83.The expression of MDR1,GST?,Bcl-2 and survivin in SACC-83/DDP cells was higher than that in SACC-83 cells,which may be related to the drug resistance of SACC-83/DDP.2.Artesunate can effectively inhibit the proliferation of SACC-83/DDP cells and reverse the drug resistance of SACC-83/DDP.The expression of MDR1,GST?,Bcl-2 and survivin in SACC-83/DDP cells were significantly decreased after 48 h treatment with artesunate at different concentrations,and the expression was significantly decreased with the increase of drug concentration,which may be related to the reverse of drug resistance by artesunate in SACC-83/DDP cells. |
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