Establishment Of Drug Resistance Cell Line Of Adenoid Cystic Carcinoma ACC-M/DDP And Research Into Its Mutidrug Resistance Mechanism

Part 1 Establishment of drug resistance cell line of human adenoid cystic carcinoma—ACC-M/DDPObjective: In view of the current problems with chemotherapy resistance of head and neck cancer, to establish drug resistance cell line of human adenoid cystic carcinoma ACC-M/DDP and study the biological characteristics, for providing the experimental basis of clinical treatment.Methods: Adenoid cystic carcinoma cell line ACC-M was induced by Cisplatin, with high-dose impact and gradual increase in dose, and resistant cell line—ACC-M/DDP was established which grew well in the 1μg / ml Cisplatin. Sensitivity of ACC-M/DDP cells and parental cells to different chemotherapeutic drugs was detected by MTT assay, cell growth curve drawn to the results of cell counting, cell cycle determined by flow cytometry.Results: Resistant cell line ACC-M/DDP was established successfully, and the results of MTT of drug susceptibility testing showed it had different degrees of tolerance to CDDP, Taxol,5-FU and CTX, with resistance index 7.26, 3.32, 4.18 and 4.82 respectively. Results of cell counting showed that the doubling time of ACC-M and ACC-M/DDP cells was 21.3, 28.7 hours respectively. Results of flow cytometry showed that S phase drug-resistant cells ACC-M/DDP were more than ACC-M cells (P < 0.05).Conclusion: The acquired multi-drug resistant cell line ACC-M/DDP with stable characters significantly different from that of parental cells was a reliable cell model of human adenoid cystic carcinoma of multidrug resistance and likely to apply to the study of multi-drug resistance mechanisms.Part 2 Expression of MDR1 gene in multidrug resistance cell line ACC-M/DDP and its clinical significanceObjective: To explore the expression of MDR1 gene in multidrug resistance cell line of human adenoid cystic carcinoma ACC-M/DDP and its clinical significance for providing theoretical basis for the study of tumor multidrug resistance and reversal agents.Methods: Accumulation of rhodamine 123 in ACC-M and ACC-M/DDP cells was detected by flow cytometry, intracellular expression of MDR1 mRNA by RT-PCR, and P-gp expression in nude model by immunohistochemistry after the establishment of parotid gland tumor model in nude mice.Results: Rhodamine 123 results showed that the relative fluorescence intensity of ACC-M/DDP and ACC-M cells were 1.4±0.2 and 38.9±0.4 (P <0.01); RT-PCR results showed that MDR1 gene was not expressed in ACC-M cells, but highly expressed in ACC-M/DDP cells. The time of occurrence of xenograft tumors was (8. 00±1. 76) d in Group ACC-M and (14. 17±1.17) d in Group ACC-M/DDP. Tumors in the latter group grew slowly with less invasiveness. Immunohistochemical results showed P-gp was expressed intensely in ACC-M/DDP, but not expressed in ACC-M.Conclusion: Multi-drug resistance of ACC-M/DDP cell line induced by Cisplatin was closely related with the expression of MDR1/P-gp which could maintain drug-resistance stabily in vivo. This study provides a theoretical basis for increasing the sensitivity to chemotherapy of adenoid cystic carcinoma by using MDR1 reversal agents.Part 3 The role of Survivin on inducing multidrug resistance and apoptosis of adenoid cystic carcinoma cellObjective: To observe the effects of Paclitaxel on the biological characteristics of ACC-2, ACC-M and ACC-M/DDP, to detect the variation of Survivin expression during the process, and to investigate the effects of Paclitaxel on adenoid cystic carcinoma cells and the role of Survivin in inducing multi-drug resistance.Methods: The apoptosis and cell cycle of ACC-2, ACC-M and ACC-M/DDP cell with different concentration of Paclitaxel were observed on flow cytometry; the cells of apoptosis were stained by AO / EB fluorescent; mRNA expression of survivin gene was detected by RT-PCR and survivin protein was observed by immunohistochemistry.Result:2μg /ml Paclitaxel could block ACC-2,ACC-M and ACC-M/DDP cells into G2/M phase. Treated by 5μg/ml Paclitaxel for 24 hours, ACC-M/DDP cells of G2/M phase accounted for (65.4±1.1)%, this percentage significantly higher than that of ACC-M. Annexin V/PI results showed that Paclitaxel could inhibit the growth of ACC-2, ACC-M and ACC-M/DDP to different extents; low-dose Paclitaxel could caused effects with significant difference (P<0.05). The result of RT-PCR demonstrated that Survivin was expressed in each kind of cells; expression of Survivin mRNA was significantly stronger in cells with 5μg /ml paclitaxel than that in cells with 2μg /ml paclitaxel. Immunohistochemistry showed that Survivin protein expressed in ACC-M/DDP cells was obviously higher than that in ACC-M.Conclusion: Survivin played a critical role in inhibition of tumor cell apoptosis, and its over-expression may contribute to the occurrence of multi-drug resistance of adenoid cystic carcinoma.