| Backgroud:Leukemia is a group of diseases which arise from the malignant cloning of hematopoietic stem cells, characterized by the disregulated proliferation as well as the blockage of differentiation and apoptosis of abnormal leukemia cells. In recent years, even if great improvement in its treatment was made by new drugs, optimized chemptherapy scheme and allo-genetic hematopoietic transplantation (Allo-HSCT) which greatly improved the survival of the leukemia patients, clinical practice is always handicapped by primary or secondary drug-resistance of leukemia cells to chematherapy which needs to be solved urgently. Since there is a high rate of relapse in patients with pirmary or secondary drug-resistance, deeply exploration to some abnormal molecules and signal pathways associatted with leukemia drug-resistance, is of great importance and significance for early diagnosis and overcoming drug-resistance of such kind of disease.The Eph receptors are the largest family of receptor tyrosine kinases.They are divided into an EphA and an EphB class, which respectively bind the glycosylphosphatidylinositol-linked ephrin-A ligands and the transmembrane ephrin-B ligand. Eph receptor binding with the ephrin ligands on the other cell surface, which occurs at sites of cell-cell contact, stimulates tyrosine kinase activity of both Eph receptor and Ephrin ligand called biodirectional signaling. Some reports demonstrated that Eph receptors are closely related with neural development, angiogenesis and tissue boundary formation in physiologic process, whereas recent researches found overexpression of Eph in many types of tumor which, as an important moderator of drug-resistance, could greatly enhance the drug-resistance capability of leukemia cells. Since the related mechanism is till largely unknown, the first "Eph/Ephrins and Cancer" meeting was held in Winston-Salem, North Carolina on June 25 to 26,2008 indicating Eph/Ephrin has become an importitant research focus for researchers all over the world.So far, it is the EphB4 class that has been studied at the most detailed extent. The tyrosine kinase activity on the intracellular domain of EphB4, induced by binding with the specific ligand EphrinB2, could activates proteins like Ras, Rac, Rho, Rapl and FAK et.al in order to deliver the regulatory signals for tissue boundary formation, angiogenesis, cell migration, neural axon direction and osteogenesis balance. EphB4 also takes part in some pathologic process such as tumorigenesis, for many types of tumor have been found with abnormal upregulated expression or activity of EphB4 in recent years,such as mammary adenocarcinoma, colon carcinoma, ovarian cancer, prostatic carcinoma, melanoma, mesothelioma, uterine cervix cancer and so on., EphB4 could also trigger some cell signal cascades to affect biological behavior of tumor cells, which is closely related to prognosis, but the mechanism is still unclear. Moreover, there is controversies among the discoveries about the role of EphB4 in cancer, just as EphB4 could play both the oncogene and tumor suppressor roles in different tumor contexts.However, few studies about EphB4 in myeloid leukemia have been taken so far, and our previous work found that expression of EphB4 was increased in relapse or refractory patients, compared with it in normal or incipient ones. Above all, investigation of the expression and activity of EphB4 in myeloid leukemia would give us a new molecule target for reversing and overcoming drug-resistance.Objective:We chose leukemia cell lines K562,HL60,HL60/ADM,U937 and KGla for our study, the drug-resistance to adriamycin(ADM), the expression and phosphorylation level of which were tested in order to explore the possible relationship between EphB4 and drug-resistance phenomenon of these cells.Then we upregulated the phosphorylation level of EphB4 in cells to clarify the influence of its phosphorylation activity on drug-resistance capacity of myeloid leukemia cells and the related mechanism.Finally bioinformatic method was use to explore the principle signal pathways involved in the drug-resistance of acute myeloid leukemia cells which probably was induced by disregulated overexpression of EphB4.Methods:1. The drug-resistance capacity to adriamycin of K562,HL60,HL60/ADM,U937 and KGla was tested by CCK-8 analysis. The IC50 value of each kind of cell lines was calculated by inhibition rates of cells.2. RT-PCR and Western blot were analyzed by extraction of total RNA and protein from K562,HL60,HL60/ADM,U937 and KGla cell lines in order to detect the expression of EphB4 in both translational and transcriple levels which was then compared with drug-resistant capacity.3. phosphorylated proteins were extracted from K562,HL60,HL60/ADM,U937 and KG1a cells, and the phosphorylation level of EphB4 in each type of cells was detected by phosphorylation ELISA.4. Clustered solube recombinant EphrinB2-Fc chimeral ligand protein was used at different concentrations to stimulate EphB4 on HL60/ADM cells. And the change of phosphorylation level of EphB4 after stimulation in cells was tested either.6. We upregulated the phosphorylation level of EphB4 in HL60/ADM cells with solube recombinant EphrinB2-Fc chimeral ligand protein at 4ug/ml, cells with 0.4ug/ml IgG Fc or nothing were also taken as negative controls.The drug-resistance capacity to ADM of the three groups was also tested.7. HL60/ADM cells were stimulated with solube recombinant EphrinB2/Fc chimeral ligand protein for 30min, 1PANTHERour,24hour respectively, phosphorylation and expression level of EphB4 at each time point were tested.8. STRING (Search Tool for the Retrieval of Interacting Genes Protein) and PANTHER database were used to explore proteins which could interact whth EphB4,and some related signal parthways which may induce the drug resistance of acute myeloid leukemia cells.9. Statistic analysis was performed with the statistical software package SPSS 13.0, and the significance was set at P value less than 0.05 in our tests.Results:l.The results of CCK-8 analysis for drug-resistance to ADM of K562,HL60,HL60/ADM,U937 and KGla cell lines were HL60/ADM had the most strong capacity of drug resistance, K562 and KGla are less than it but more than U937 and HL60.2.Both Western blot analysis and RT-PCR found high expression level of EphB4 in K562, HL60/ADM and KGla, whereas extremly low level in HL60 and U937, which indicated that the EphB4 protein was probablely related with the drug-resistance of leukemia cells.3. phosphorylation ELISA found low phosphorylation levels in all of the five cell lines and low significant difference was seen. 4..Base on the previous references and discoveries in our own research, we used solube recombinant EphrinB2-Fc chimeral ligand protein at gradient concentrations of 1ug/ml,2ug/ml,and 4ug/ml to stimulate HL60/ADM cells which has the highest expression of EphB4, the phosphorylation level was increaced in a dose depended manner after stimulaiton.5 We used solube recombinant EphrinB2-Fc chimeral ligand protein at 4ug/ml for stimulation of HL60/ADM cells to investigate the change of drug-resistance after phosphorylation of EphB4 increased. And we finally found that the decreasing of the drug-resistance capacity to ADM of HL60/ADM cells after EphrinB2-Fc stimulation, while there is no change in the IgG Fc group or blank group.6 When HL60/ADM cells were stimulated with solube recombinant EphrinB2/Fc chimeral ligand protein for 30min,1 hour,8hour,24hour respectively, we found the phosphorylation level of EphB4 started to decrease at 1 hour and come to the minimum at 24 hour. And so did the change of the EphB4 protein expression level.7.Use pSTRING and PANTHER, we deduce that desregulated EGFR and VEGF signal pathways would be the key mechanism which accounts for the drug resistance enhancement capacity of EphB4, and EphB4 interact with this two signal pathways independent of its tyrosine kinase activity.Conclusion:The expression of EphB4 protein was upregulated at different extent in myeloid leukemia cell lines with high drug-resistance capacity to ADM, but the phosphorylation levels were low in-all of the cell lines.Stimulation of EphB4 in leukemia cells with its solube ligand protein EphrinB2-Fc could increase its phosphorylation activity in a dose dependent manner. And phosphorylation activity at myeloid leukemia cells could impair the drug-resistance to ADM which indicated that there would be other functional domains in EphB4 protein which promotes drug-resistant phenomenon of myeloid leukemia cells. the phosphorylation level of EphB4 started to decrease at 1 hour and come to the minimum at 24 hour. And so did the change of the EphB4 protein expression level. And bioinformatic analysis indicated that EphB4 would enhance drug resistance of acute myeloid leukemia cells through two key signal pathways EGFR and VEGF signal pathways, which interact with EphB4 independent of its tyrosine kinase domain. |
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