Effects Of Btk On Acute Lymphoblastic Leukemia And Its Molecular Mechanisms

Objective:By observing the effects of Btk inhibitor, NFκB inhibitor and Bcr-abl tyrosine kinase inhibitor on tumor B cells, including lymphoma cells (Raji, Ramos) and acute lymphoblastic leukemia (Sup-B15, RS4; 11), to explore the role of Btk on the cells proliferation, apoptosis and the possible mechanism.Methods:1、Raji、Ramos cells were treated with PCI-32765 and Bortezomib, the cell proliferation and apoptosis were detected by CCK-8 and flow cytometry assay respectively, and proteins were detected by Western blot.2、RS4;11、Sup-B15 cells were treated with PCI-32765 and Bortezomib, the cell proliferation and apoptosis were detected, the RNA expression level of Btk、 NFκB were detected by RT-PCR, NFκB and other apoptotic proteins were detected by Western blot.3、RS4;11、Sup-B15 cells were treated with PCI-32765 and Dasatinib, the cell proliferation and apoptosis were detected, the RNA expression level of Btk、NFκB were detected by RT-PCR, Btk and other apoptotic proteins were detected by Western blot.Results:1、The role and the mechanisms of PCI-32765 and Bortezomib on Raji and Ramos cells:1.1 Both Bortezomib (10nmol/L、20nmol/L、30nmol/L、40 nmol/L 50 nmol/L、60 nmol/L、80 nmol/L) and PCI-32765 (0.5μmol/L、1.0μmol/L、 2.0μmol/L、3.0μmol/L、4.0μmol/L、5.0μmol/L、6.0μmol/L) can inhibit the proliferation of Raji and Ramos cells, the effects shows dosage and time dependent manner, and PCI-32765 and Bortezomib have synergy effects.1.2 The Raji and Ramos cells were treated with PCI-32765 and Bortezomib alone and/or combination for 48 hours.1.2.1 The cell apoptosis rate in drug treated group was increased as compared with control group, and the cell apoptosis rate in drug combination group was markedly increased as compared with single drug group (p<0.05).1.2.2 Compared with the control group, the expression level of Btk, NFkB, c-IAP1 and BCL-XL were decreased in the drug treated groups, but the expression of Caspase-3 is in the different way.2、The role and the mechanisms of PCI-32765 and Bortezomib on RS4;11 and Sup-B15 cells:2.1 Both PCI-32765(0.5μmol/L、1.0μmol/L、2.0μmol/L、 4.0μmol/L、6.0μmol/L、8.0μmol/L、10.0μmol/L) and Bortezomib (2.5nmol/L、5.0 nmol/L、7.5nmol/L、10.0nmol/L、12.5nmol/L、15.0nmol/L、17.5nmol/L、20.0 nmol/L) can inhibit the proliferation of RS4;11 and Sup-B15, the effects shows dosage dependent manner, and PCI-32765 and Bortezomib have synergy effects.2.2 The RS4;11 and Sup-B15 cells were treated with PCI-32765 and Bortezomib alone and/or combination for 48 hours.2.2.1 In RS4;11 cells, the cell apoptosis rate in the control group, Bortezomib group, PCI-32765 group and PCI-32765 and Bortezomib combination group were (5.21±2.18)%、(40.97±4.09)%、(37.21±6.98)%、and (78.30±3.39)% respectively, the differences was significant (p<0.05).2.2.2 In Sup-B15 cells, the cell apoptosis rate in the control group, Bortezomib group, PCI-32765 group and PCI-32765 and Bortezomib combination group were (9.37±1.08)%、(41.03±5.29)%、(32.14±6.18)% and (73.09±4.32)% respectively, the differences was significant(p< 0.05).2.3 The mRNA expression level of Btk、NFκB in RS4;11 and Sup-B15 cells treated with Bortezomib and PCI-32765 alone and/or combination.2.3.1 In RS4;11 cells, the mRNA expression level of Btk、NFκB in drug treated group was decreased as compared with control group, and markedly decreased in drug combination group.2.3.2 In Sup-B15 cells, the mRNA expression level of Btk、NFκB in drug treated group was decreased as compared with control group, and markedly decreased in drug combination group,2.4 Compared with control group, the nuclear expression level of NFκB was decreased in Bortezomib and PCI-32765 single drug group and combination group(p<0.05).2.5 Compared with the control group, the expression level of Btk, NFκB and anti-apoptosis protein BCL-XL are lower in the single drug and combination groups, but the expression of Caspase-3 is in the different way (p< 0.05).3^ The role and the mechanisms of PCI-32765 and Dasatinib on RS4;11 and Sup-B15 cells:3.1 The effect of cell biological characteristics:3.1.1 PCI-32765 (0.5μmol/L、1μmol/L、2μmol/L、4μmol/L、6μmol/L、8μmol/L、10μmol/L) could inhibit the proliferation of RS4; 11 and Sup-B15 cells in a dose-dependent manner, Sup-B15 cells were more sensitive to PCI-32765 than RS4;11 cells. Their IC50 were 3μmol/L and 8μmol/L respectively, the difference between them was statistically significant (P<0.05).3.1.2 Dasatinib (1.0μmol/L、2.0μmol/L、3.0μmol/L、 4.0μmol/L、5.0μmol/L、6.0μmol/L、7.0μmol/L、8.0μmol/L、9.0μmol/L、10.0μmol/L) could inhibit the proliferation of RS4; 11 cells, and Dasatinib(1.0nmol/L、2.0nmol/L、 3.0nmol/L、4.0nmol/L、5.0nmol/L、6.0nmol/L、7.0nmol/L、8.0nmol/L、9.0nmol/L、 10.0nmol/L) could inhibit the proliferation of Sup-B15 cells in a dose-dependent manner. The inhibitory effects of Dasatinib on Sup-B15 cells was more stronger than RS4; 11. Their IC50 were 5 nmol/L and 5μmol/L respectively, with the ratio 1:1000, the difference between them was statistically significant (P<0.05), and the inhibitory effect was enhanced by combined with the PCI-32765(P<0.05).3.1.3 The cell survival rate decreased gradually in PCI-32765 and Dasatinib monotherapy group and the combination group at different time-point (8h,12h,24h,36h,48h and 72h), the two drugs have a synergistic effect on cells in a time-dependent manner.3.1.4 After RS4; 11 and Sup-B15 cells were treated with PCI-32765 and Dasatinib, Liu’s stain shows that Cell shrinkage, increased cytoplasmic density, nuclear pyknosis, deviation and karyorrhexis, and the apoptotic cells increased in combination group, while the pro-apoptotic effects of low dosage Dasatinib on RS4; 11 cells was not strong.3.2 PCI-32765 and Dasatinib decrease the expression and activity of Bcr-abl, Btk, Lyn, Src in Sup-B15 and RS4;11 cells.Conclusion:1、PCI-32765 and Bortezomib have synergistic effects on the proliferation and apoptosis of Raji and Ramos cells, the mechanism were down-regulation the expression level of NFκB, c-IAP1 and BCL-XL and up-regulation the expression of Caspase-3.2、PCI-32765 and Bortezomib have synergistic effect in the proliferation and a poptosis of ALL cell lines RS4;11 and Sup-B15. The mechanism was to inhibit the activity of Btk、NFκB and inhibit NFκB nuclear translocation to prevent the continuous activation of NFκB, and were down-regulation the expression level of BCL-XL and up-regulation the expression of Caspase-3.3、At human body tolerated concentration range, Dasatinib could inhibit the proliferation of Ph+ Sup-B15 cells, and it has the synergy effects with PCI-32765. But to Ph-RS4; 11 cells, at human body tolerated concentration range, Dasatinib could not inhibit its proliferation, however, PCI-32765 can enhance the Dasatinib (at human body tolerated concentration) effects. The possible mechanisms could be by inhibition the activity of Btk, Bcr-abl and Src family kinases, to block the B Cell Receptor (BCR) signal pathway, thereby inhibit the proliferation and promote apoptosis of malignant B cells. These results provide the evidence that the Dasatinib and PCI-32765 combination therapy not only effective in Ph+ cells, but also effective in Ph- cells, indicate that these drugs could be effectively used in clinical ALL patients.