Study On The Leukemia Target Therapy And The Mechanism Of Multidrug Resistance Reversing

Objective The interleukin-3 receptor alpha chain (IL-3Ra or CD 123) was strongly expressed in AML stem cells. Conversely, normal bone marrow derived CD34+/CD38-cells showed virtually no detectable expression of the CD123 antigen. Lidamycin (LDM) is a new member of enediyne antitumor antibiotics family that can be separated and reconstituted. It consists of a labile active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). LDM is now in phaseâ…¡clinical trials. Here, we described the antitumor features of a fusion protein of mIL3-LDM, targeted to CD 123 expressed by AML stem cells.Methods Utilizing overlap PCR and PCR to construct the IL3-LDM expressing vector Pet28aIL3-LDM.Rreforming the fusion protein with proteomics and molecular cloning to achieve another higher stable fusion pretein mIL3-LDM. Expressed in soluble form as mIL3-LDM fusion protein by IPTG inducing and identified the protein by SDS-PAGE and western blot. To examine the protein yield by BCA assay and determine the purity by IL3 ELISA assay.Identified the binding activity by FACS,fluorescence microscopy and the confocal laser scanning microscope. Investigated the effect of mIL3-LDM fusion protein against cell lines expressed CD 123 by CCK-8 detection. Investigated the effect of mIL3-LDM fusion protein against AML clinic samples by clone forming test.Result mIL3-LDM was constructed by a series of structural modification to improve the protein stability. Purity of recombinant protein was above 90% and the yield of fusion proteins is over 1mg per liter. Protein binding activity is directly proportional to the positive rate of CD 123 expression on cell surface. It is observed by CCK-8 that mIL3-LDM had stronger cytotoxicity than LDM. Exerted potent cytotoxicity against CD123+ cell lines in vitro (IC50:0.9-1.2nM).The IC50 value of mIL3-LDM was only 1/300 of that of LDM for TF-1 cells and 1/200 for M07e cells respectively.Fusion protein had cytotoxicity mechanism by apoptosis and cell cycle test which be consistent with the result in LDM.Conclusion m IL3-LDM could serve as a new technology platform for making a series of highly potent antineoplastic agents. Objective Multidrug resistance (MDR) mediated by the overexpression of the drug efflux protein P-glycoprotein is one of the major obstacles to successful cancer chemotherapy.The development of safe and effective MDR-reversing agents is an important approach to addressing this problem clinically. In this study, we evaluated the P-gp-modulatory potential of O-(4-ethoxyl-butyl)-berbamine (EBB), a novel calmodulin antagonist and derivative of bisbenzylisoquinoline alkaloid.Method MTT assay display the EBB reversing effect on multi-drug resistance in MCF-7/ADR cell line.FACS indicate that the P-gp expression decline on the cell surface after treated with EBB which cause the drug and Rh123 accumulation in the cells. We detected the relation between the Ca2+level and EBB effect in the cells.The change of cell apoptosis and cell cycle are also find out after the EBB treatment.The level of cell cycle relation protein expression was detected by western blot.Result EBB significantly improved the chemosensitivity of P-glycoprotein mediated multidrug-resistant cells to doxorubicin compared with the efficacy of a conventional P-glycoprotein inhibitor, verapamil.EBB not only blocked the function of P-glycoprotein confirmed by the fact that EBB increased intracellular accumulation of rhodamine 123 and doxorubicin but also inhibited the expression of P-glycoprotein actualized by downregulating P-glycoprotein. Furthermore, our results showed that cotreatment with EBB and doxorubicin resulted in marked G2/M arrest and apoptosis of MCF-7/ADR cells, accompanied by downregulation of the proteins cdc2/p34 and cyclin B1 and increased the levels of calcium ions.Conclusion Taken together, these results suggest that cotreatment with EBB and doxorubicin could strongly potentiate the antitumor activity of doxorubicin, thus may have significant clinical application in cancer chemotherapy.