BTK Regulate P-gp-mediated Drug-resistance In Acute Lymphoblastic Leukemia

Objective:To explore the role and mechanisms of BTK-P-gp signaling pathway in the drug resistance of Acute lymblastic leukemia（ALL）and the cell biologies in ALL resistant cell lines.Methods:1、Construction of ALL drug-resistant cell lines:the drug-resistant cell lines SUP-B15/ADR and RS4;11/ADR were derived by exposing the parental cells[SUP-B15（Ph⁺）and RS4;11（Ph^-）]to the increasing concentrations of Adriamycin（ADR）.2、Biological characteristics of drug-resistant ALL cells:the cell viabilities were assayed by CCK-8 method.The expression of P-gp was examined by Western blot,RT-q PCR was performed to detect the expression of MDR1.3、Effects of Ibrutinib on MDR1 and P-gp expression and the cell biological characteristics:ALL cells and drug-resistant ALL cells were treated with Ibrutinib,the RNA and protein expression levels of BTK,NFκB and MDR1（P-gp）were measured by RT-q PCR and Western blot,respectively,the cell proliferation and apoptosis were detected by CCK-8 method and flow cytometry assay.Results:1、Construction and characteristics of drug-resistant ALL cells:the drug resistant cell lines SUP-B15/ADR and RS4;11/ADR were successfully established,the resistance indexes（RI）were 14.088±0.763 and 10.473±1.024,respectively.After the cryopreserved SUP-B15/ADR and RS4;11/ADR cells were revived,the recovery rates were 88.4±1.2%and 89.3±1.6%,while the resistance indexes were13.976±0.967 and 10.342±0.846,respectively（P﹥0.05）.When the drug resistant cells were cultured without ADR for 1 month,the resistance indexes decreased to12.893±1.255 and 9.327±0.321,respectively（P﹤0.05）.ADR-resistant cells were cross-resistant to Cytarabine and Etoposide as well.2、The expression of P-gp and MDR1 in drug resistant cells was significantly higher than that of wild-type cells.3、Ibrutinib regulated the expression of NFκB and P-gp in ALL and drug-resistant ALL cells:after being treated with Ibrutinib,the RNA and protein expression levels of NFκB,MDR1 and P-gp in SUP-B15,RS4;11 and SUP-B15/ADR decreased drmatically as compared with control group（P<0.05）.4、Ibrutinib affected the cell proliferation and apoptosis upon the treatment of Ara-C or ADR:the survival rate of Ibrutinib treated group were significantly lower than that of the control group in the same concentration of Ara-C and ADR（P<0.05）.Conclusion:1、Two drug-resistant ALL cell lines were successfully established by being exposed to increasing concentration of ADR.The over expression of MDR1 and P-gp may be the important mechanisms underlying the drug resistance.2、Ibrutinib could downregulate the expression of MDR1 and P-gp by inhibiting the expression of NFκB in ALL cells.Ibrutinib may increase the sensitivity of ALL cells to Ara-C and ADR chemotherapy.