Proteomic Analysis Of Multidrug Resistance Related Proteins In Chronic Myelogenous Leukemia Cell Lines And Study On The Mechanism Of MDR

Multidrug resistance (MDR) describes a phenomenon ofcross-resistance of tumor cells to several structurally unrelatedchemotherapeutic agents after exposing to a single cytotoxic drug.Resistance to anticancer drugs is a major obstacle for an effectivetreatment on tumors. Recent years several mechanisms have been foundto contribute to MDR, involved proteins including P-glycoprotein (P-gp),multidrug resistance related protein (MRP), lung resistance relatedprotein (LRP), breast cancer multidrug resistance related protein (BCRP),topoisomeraseâ…¡(TOPâ…¡) and glutathione-S-transferase (GST) and so on.The phenomenon of MDR is also known to be a multifactodal event inwhich several mechanisms play an important role simultaneously. On theone hand, tumor cells can greatly down-regulate the intracellularconcentration of cytotoxic drugs by the ATP driven efflux pumpfunctions of P-gp, MRP and BCRP. On the other hand, anticancer drugscannot arrive at their targets because of transportation of the intracellularcytotoxic drugs to other subcellular structures by LRP. Additionally,structural alterations in the drug target enzymes and proteins mayenhance their detoxification and alterations in cellular metabolism, whichcan strengthen the ability of tumor cells for DNA damage repair andapoptosis resistance. Although the pathogenesis on MDR of tumors hasbeen extended investigated, the mechanisms of MDR remain intricate and require further study.Chronic myelogeneous leukemia (CML), one of the commonhematological malignant tumors, is taken up 15-25 percent of allleukemia. Clinically, the disease can be individed into three phases: thechronic phase, the accelerated phase and the blast phase. In blast phase,patients often die in a few months because of the insensitivity tochemotherapeutic drugs and MDR. The MDR mechanisms in CML cellshave been broadly explored. However, exact mechanism is still unclear.Adriaraycin-resistant K562/A02, which derived from human CML cellline K562 by stepwise selection in vitro using adriaraycin can alsocross-resist on other anticancer drugs such as VCR and HHT. What's thepathogenesis of MDR in K562/A02 cells? Are there some new associatedproteins with MDR in K562/A02 cells? In the program we applyproteomic tools to search new MDR related proteins and uncover themechanism of MDR in adriaraycin -resistant K562/A02 cell line.At first, two-dimensional gel electrophoresis (2-DE) technology wasperformed to separate the total protein of adriaraycin-resistant K562/A02cell line and its counterpart K562 cell line, respectively. Thewell-resolved, reproducible 2-DE patterns of K562/A02 and K562 wereestablished. Then, PDQuest software was applied to analyze 2-DE images,and the 17 differential expression proteins between the two cell lines wereidentified by peptide mass fingerprint (PMF) based on Matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). We found that compared with K562 cells GST wasover-expressed in K562/A02 cells, while mitochondrial ATPase, a newprotein related to MDR, was down regulated.To confirm the results deriving from proteomic analysis,western-blot analysis and real-time quantitative RT-PCR technology wereused to determine the differential expression levels of the partial proteins.The results showed that GST protein was higher expressed in K562/A02cells than in K562 cells and mitochondrial ATPase protein was lowerexpressed by western-blot analysis. Through real-time quantitativeRT-PCR we foundthat the GST mRNA was 3.72 folds increased inK562/A02 cells than in K562 cells and mitochondrial ATPase mRNA was7.07 folds decreased. The results were consistant with the proteomeanalysis both in protein level and in mRNA level.To identify the relationship between mitochondrial ATPase andadriaraycin-resistant K562/A02 cells and explore the mechanism of lowerexpression of mitochondrial ATPase, methylation-specific PCR (MSP)was carded out to determine the methylation status of mitochondrialATPase in both K562 and K562/A02 cells and methylation inhibitor(5-Azacytidine) was applied. Our results showed that the methylation.status of mitochondrial ATPase was higher in K562/A02 cells than inK562 cells. The inhibition of methylation in mitochondrial ATPase could enhance adriaraycin chemosensitivity in K562/A02 cells and reverseMDR of K562/A02 cells. The mitochondrial ATPase was identified as amultidrug resistance related protein in adriaraycin-resistant cell lineK562/A02.Above all, 17 differential expressional proteins between adriaraycin-resistant cell line K562/A02 and its parental cell line K562 wereidentified by 2-DE with MALDI-TOF-MS. Some of these proteins havebeen known to be relative with the development of tumor MDR such asGST. Our reseaches found that mitochondrial ATPase was associatedwith MDR of K562/A02. The lower expression of mitochondrial ATPasein K562/A02 cells was related to the methylation of CpG. The inhibitionof methylation in mitochondrial ATPase gene could reverse MDR ofK562/A02 cells partly. The regulation of methylation for mitochondrialATPase might be a new target to reverse MDR of CML.