| Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in south China and southeast Asian countries. The highest incidence of NPC in this district may reach 50/100, 000, and its mortality rate is 2. 81% among all the malignancies. In comparing to other malignant tumors, NPC has its unique properties, which are: (1) unique geographical distribution; (2) specific relationship with EB virus; (3) most cases of NPC belong to non or low differentiate type. With its notorious properties of very low differentiation and rapid invasive growth, most cases are inclined to distant metastasis and not ready to be completely cured by local therapies, so chemotherapy constitutes one of the chief methods in our treatment of this disease. However, the appearance of drug resistance often causes failure of chemotherapy. For overcoming drug resistance, it is of great importance to establish a drug-resistant cell line in vitro and to screen drug-resistant associated genes. To search for the key molecular alteration in genomic DNA level and in gene expression level, and to identify potential molecular targets should be of great significance in our understanding of drug-resistance mechanism and in our struggle against this cancer.The mechanisms of drug-resistance have been videly investigated ever since the application of chemotherapy in the treatment of cancers. The multidrug resistance (MDR) is defined that the cancer cells may establish the ability to resist different drugs whose structure and pharmacodynamics are not alike, after they are exposed to only one drug. MDR is one of the most common causes of chemotherapy failure. However,from a reasonable view, its essence is an adaptive process of tumor cells under an unfavorable survival circumstance. Many mechanisms might contribute to the appearance of MDR, such as the expression of pump protein, the P-glycoprotein in the membrane, the enhanced detoxifcation ability, the enhanced repair ability of damaged DNA, the quantity or activity alteration of drug target proteins such as topoisomerases and the alteration of activity and expression of protein kinase C. However, many obstacles still confront us in our struggle against drug resistance when we try to use these molecules as potental targets. Are there any other molecular mechanisms of drug resistance? Furthermore, considering the unique characteristics of NPC, are there any other unique mechanisms of drug resistance in NPC?This study was designed to establish a drug-resistant cell line from a human nasopharyngeal carcinoma cell line CNE-2, and to screen human nasopharyngeal carcinoma drug-resistant genes by combining a new strategy based on improved subtractive hybridization with the technique of comparative genomic hybridization.This project includes three parts:1. Establishment of a Human Nasopharyngeal Carcinoma Drug-Resistant Cell Line CNE-2/CDDPThe drug-resistant cell line was established by a program of treating the human nasopharyngeal carcinoma cells CNE-2 in the medium with repeated sharp high and then low but gradually increasing concentration of cisplatin. Drug sensitivity was measured by MTT assay of CNE2/CDDP and its parent cells to different drugs. Fluorescence activated cell analysis (FACS) was employed for determining the concentration of fluorescence dye rhodamine 123 within the cells. Also measured and observed including cell growth curve, doubling time and cell morphology. It was shown that the resistance indexes of CNE-2/CDDP to cisplatin (cDDP), fluorouracil (5-FU) and vincristine (VCR) were 27.9, 227.9 and 55.5 respectively, indicating its multi-drug resistant property. FACS analysis showed that the concentration of rhodamine 123 was much lowerin CNE-2/CDDP cells than that of CNE-2 cells (12.98 vs. 243.62). The CNE-2/CDDP cells appear smaller, more regularly round, with longer doubling time (26h vs. 19h) than CNE-2 cells.From the above bioloical characters we conclude that a stable cell line of multi-drug resistance, which is defined CNE2/CDDP was established. It...
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