The Establishment Of Human Nasopharyngeal Carcinoma Multidrug-resistant Cell Line CNE2/ADM And Research Of Cepharanthine Reverse Multidrug Resistance

Nasopharyngeal carcinoma(NPC) is the most common malignant tumor in the head and neck region. It has a high incidence rate in the south of China, which seriously endangers the health of the people. Chemotherapy is one of the important methods for the treatment of NPC, but the production of multidrug resistance of tumor cells often leads to the failure of chemotherapy. Therefore, it has become one of the hot spots in the field of cancer research, to study the mechanism of multidrug resistance in cancer, to find the multidrug resistance reversal agents, and to improve the efficacy of chemotherapy. Over the years, many compounds with a reversal of tumor multidrug resistance in vitro have been found, butmost of them have greater toxic and side effects, and were limited in clinical applications. Cepharanthine is a natural bisbenzylisoquinoline alkaloid extracted from the roots of Stephania cepharantha Hayata, which has various strong biologicalactivities in vitro and in vivo and lesser toxic and side effects. In recent years, the study found that cepharanthine can reverse multidrug resistance of many tumours including leukemia, breast cancer, liver cancer. But whether it can reverse the multidrug resistance of NPC and the possible mechanism of reversal, there is no relevant reports at present. Therefore, this research based on the construction of multidrug resistant cell model of NPC, to investigate the reversal effect of cepharanthine on multidrug resistance of NPC and the possible reversal mechanism. ObjectiveTo establish a human NPC multidrug resistant cell line CNE2/ADM and to investigate its biological features. To detect the reversal effect of cepharanthine on multidrug resistant cell line CNE2/ADM and investigate its possible mechanism. Materials and Methods(1)Adriamycin resistance was induced in human NPC cell line CNE2 by continuous exposure to gradually increasing doses of adriamycin. Cell morphology, drug sensitivity, cell growth curves and the population doubling time, cell cycle distribution, the function and expression level of P-gp were investigated to determine the biological features of CNE2/ADM cells.(2)MTT assay was used to detect the reversal effect of cepharanthine on CNE2/ADM cells. The effects of cepharanthine on P-gp function in CNE2/ADM cells were detected by Rho123 accumulation experiment. The effects of cepharanthine on P-gp expression in CNE2/ADM cells were detected by Western blot. Results(1)CNE2/ADM cell line was established after 6 months, which had a resistance index of 12.98 to adriamycin.The CNE2/ADM cells showed cross-resistance to cisplatin, vincristine and 5-fluorouracil. Compared with CNE2, the CNE2/ADM cell line was significantly heteromorphosis, the population doubling time was longer(P<0.05), and the cell number of the G0/G1–phase was increased, whereas that of the S-phase and G2/M-phase was decreased(P<0.05), the function of P-gp was enhanced and the expression level of P-gp was up-regulated.(2)Cepharanthine could reverse drug resistance of CNE2/ADM cells to adriamycin, the reversal index of 2、4、8nmol/m L of cepharanthine on adriamycin resistance in CNE2/ADM cells were 1.49 、2.16、3.19, and presented concentration dependent manner(P<0.05). Cepharanthine increased Rho123 accumulation and inhibited the function of P-gp in CNE2/ADM cells in a concentration dependent manner. The inhibition effect of cepharanthine on P-gp expression in CNE2/ADM cells is in a time and concentration dependent manner. Conclusion(1)The MDR cell line CNE2/ADM was established successfully. CNE2/ADM cell line shows the typical multidrug resistance phenotype. It is a good model for exploring the mechanism of MDR and the selection of reversal agents.(2)The drug resistance of CNE2/ADM cells to adriamycin can be reversed by cepharanthine. The reversal mechanism of cepharanthine is possibility related to inhibition of P-gp function and expression.