EphB4 Expression and Biological Significance in Drug Resistance of Myeloid Leukemia

Abstract 4725

Chemotherapy is widely used in treatment of myeloid leukemia, the efficancy of which, however, is often hampered by the development of intrinsic and acquired multidrug resistance(MDR), the exact mechanism of which is still unclear. Overexpression and deregulated activation of protein tyrosine kinase(PTK) are frequently observed in several types of hematologic malignancies, the abnormally signal cascade transducted by which has been demonstrated to play an important role in antiapoptosis, differentiation block, enhancing autonomous proliferation, and also in inducing drug resistance. EphB4 is also a protein tyrosine kinase, a member of the largest family of receptor tyrosine kinase, which has been found with abnormally upregulated expression or activity in many types of cancer especially solid tumor types, such as mammary adenocarcinoma, colon carcinoma, ovarian cancer and prostatic carcinoma. Here the aim of our study was to examine the expression and biological role of EphB4 in drug resistance of myeloid leukemia. Using RT-PCR assay and western blot, we tested the mRNA level in 35 myeloid leukemia patients including 4 cases of acute myeloid leukemia(AML) with primary multiple drug-resistance(MDR), 3 cases of AML which have relapsed, 5 cases of newly diagnosed AML, 5 cases of AML which got (complete remission) CR at the first chemotherapy treatment, 9 cases of chronic myeloid leukemia in chronic phase(CML-CP), and 9 cases of CML in blast crisis(CML-BC). The results showed the EphB4 mRNA level was significantly upregulated in AML bearing relapse(EphB4/β-actin ratio:0.962±0.114) or MDR(EphB4/β-actin ratio: 0.993±0.047) and also in CML-BC(EphB4/β-actin ratio: 1.001±0.060) compared with newly diagnosed AML(EphB4/β-actin ratio: 0.332±0.014), AML with CR(EphB4/β-actin ratio:0.401±0.015) and CML-CP(EphB4/β-actin ratio: 0.432±0.020). Subsequently, we examined both the transcriptional and translational level of EphB4 in several myeloid leukemia cell lines including K562, HL60,U937,KG1α and an adriamycin-resistant cell line—HL60/ADM, and drug-resistant capacity of the five cell lines was also tested by CCK-8 assay. Finally EphB4 protein expression is found to be upregulated at both transcriptional and translational level in K562, KG1αand HL60/ADM, which showed stronger capacity of resistance to gradient concentrations of adriamycin(IC50 of K562:0.451±0.037ug/ml,KG1α:0.217±0.017ug/ml, HL60/ADM: 2.663±0.102ug/ml) compared with that of HL60 and U937(IC50 of HL60:0.040±0.001ug/ml, U937:0.040±0.005ug/ml) in which little or no expression of EphB4 mRNA or protein was observed. And the most noteworthy is that HL60/ADM, which shows the strongest drug resistant capability. also bears the most amount of EphB4 at both transcriptional level (EphB4 /β-actin ratio: 1.002±0.017), and translational level (EphB4 /β-actin ratio: 0.975±0.051). These data supports a role for EphB4 in inducing drug resistance and raise the possibility that therapeutic intervention to EphB4 expression or signaling might inhibit or even reverse drug resistande in meyloid leukemia.