Establishment Of Nasopharyngeal Carcinoma CDDP-resistant Cell Lines And Proteomics Research Of Drug-resistant

BackgroundNasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China. Majority of NPC are poorly differentiated and highly malignant, and usually have early cervical lymph node and distant metastasis. Now, the mainly therapy methods are radiotherapy and chemotherapy. And the5-year survival rate are50%-70%. There were poor curative effect on middle and advanced stage NPC, and there were often multi-drug resistance (MDR) during the therpy of NPC and caused a failure of chemotherapy in NPC. Therefore, it is necessary to study the mechanism of MDR of NPC.MDR is a phenomenon where human tumors that acquire resistance to one type of drug are found to be resistant to several other drugs that are often quite different in both structure and mode of action, which are one of reasons in the failure of chemocherapy and recurence in malignant tumor, and involved in many complicated molecule mechanism. Above all, the role of multi-drug resistance protein (MRP) was thought to be important.By comparing the differential expression proteins between the drug resistance tumor cells and the parent plant tumor cells, we could find the drug resistance proteins, and offer a new direction for research on biomarkers of drug resistance and targeted therapy of tumor.Part I Establishment of CNE2/cDDPObjective:In this study, we established the cis-Diammine-Dichloro-Platinum (cDDP)-resistant NPC cell lines (CNE2/cDDP) by cDDP concentration progressively increased method. And the biological behavior was studied. Methods:CNE2/cDDP was established in three months by cDDP concentration progressively increased method. Morphology study was performed by invert microscope, and drug resistance was detected by MTT.Results:MTT showed that there was significant increase of IC50and resitance index in CNE2/cDDP compared with CNE2, after dealed with cDDP, Vincristin, Carboplatin, Taxotere, and5-fluorouracil. And the drug resistance of cDDP in CNE2/cDDP was the highest, RI (resistance index) was increased to200.6-folds. While the lowest drug resistance was in5-Fu, and RI was increased to200.6-folds. And there was clone aggregation and slowly growth speed in CNE2/cDDP. And there was more longer doubling time18h in CNE2/cDDP increased to24h in CNE2.There was a much smaller volume in CNE2/cDDP compared with CNE2. and clone aggregation always appeared in CNE2/cDDP. Conclusin:In this study, we established CNE2/cDDP by cDDP concentration progressively increased method. Those data showed that there was drug resistance not only to cDDP but also to others chemecherapy drugs in CNE2/cDDP. And the characters of CNE2/cDDP were stable, Which could be stably transfered and preserved, and CNE2/cDDP may be an ideal cell line used for study of drug resistance of NPC. These data provided the foundation for further proteomics study.Part II Proteomics research of drug resistance in CNE2/cDDP and function study of key proteinsObjective:In this study, the differential expression proteins between the drug resistance tumor cells and the parent plant tumor cells were obtained by proteomics methods. The key proteins keratin I (KRT1) were studied to reveal its role in drug resistance of NPC.Methods:The differential expression maps between CNE2/cDDP and CNE2were obtained by2-DE+MALDI-TOF-MS. And the expression level of three key proteins (KRT1, CTSD, ANXA5) were detected by western blot. After siRNA was performed to downregulate the expression level of KRT1, and the relationship between keratin1and drug resistance of NPC were study. Results:The differential expression maps between CNE2/cDDP and CNE2were obtained and17differential expression proteins were identified. These proteins include KRTl, KRT18, Annexin A5, Cathepsin D, cDNA FLJ52723, NME2,3,2-trans-enoyl-CoA isomerase, Histone H2B, Heat shock protein beta-1,3-hydroxyacyl-CoA dehydrogenase type-2, Mercaptopyruvate sulfurtransferase, Glutamate dehydrogenase1, Transitional endoplasmic reticulum ATPase,Alpha-enolase,Heat shock protein HSP90-alpha,NADH dehydrogenase,Heterogeneous nuclear ribonucleoproteins C1/C2. Their functions were involved in cytoskeleton, metabolic enzyme, metastasis pathway, chaperones, membrane transport pathway, protein synthesis, and so on. The expression of three proteins (KRT1, CTSD and ANXA5) were upregulated in CNE2/cDDP than CNE2. And the results were coincident compared with proteomics methods. Those data showed that the results obtained by proteomics methods were reliable. The expression of KRT1was higher in CNE2/cDDP than CNE2. After downregulated by siRNA, the IC50of five chemotherapy drugs was significantly decreased in CNE2/cDDP-siRNA than CNE2/cDDP, and the sensitivity to chemotherapy drug was increased. After down regulation of keratin1, the expression of MRP was also decreased, which showed that there was relationship between the expression of KRT1and MRP. The down-regulation of KRT1could cause decreased drug resistance in NPC.Conclusions:1.The differential expression maps between CNE2/cDDP and CNE2were obtained by2-DE+MALDI-TOF-MS.2.17differential expression proteins were obtained, and their function involved in cytoskeleton, metabolic enzyme, metastasis pathway, chaperones, membrane transport pathway, protein synthesis, and so on.3.There was relationship among KRT1,CTSD, ANXA5and drug resisitance of NPC.4.Low expression of KRT1could decrease the drug resistance to cDDP, Vincristin, Carboplatin, Taxotere, and5-fluorouracil, and down regulation of KRT1could result in decreasing of drug resistance to drugs.5.The mechanism of drug resistance of NPC may have relationship with down regulation of MRP caused by KRT1decreasing.