The Effects And Mechanisms Of Btk And NFκB Inhibitors On Acute B Lymphocytic Leukemia Mice

Objective: To explore the effects and mechanisms of Btk and NFκB inhibitors on acute B lymphoblastic leukemia-NOD/SCID(B-ALL-NOD/SCID) mice.Methods:1. Establish B-ALL-NOD/SCID mouse model: 4~5 weeks old NOD/SCID mice were injected with 5×106Nalm-6 cells(0.2ml) via tail vein. Clinical manifestation were observed, and the percentage of CD10+CD19+cells in peripheral blood were analysed.2. The effects of Bortezomib, PCI-32765 and Ara-c on B-ALL-NOD/SCID mice :The mice were divided into seven groups: control group(no drug), single drug group(PCI-32765, Bortezomib, Ara-c) 、 two drug combination group(PCI-32765 and Bortezomib), and three drug combination group(PCI-32765, Bortezomib and Ara-c).The mice were feeded in sterile laminar tank, and were treated at different time point(3days, 7days or 15 days after Nalm-6 cells injection). Clinical manifestation,lifespan, spleen size and pathomorphology were investigated. Btk, NFκB, caspase-3,BCL-XL, CIAP-1 were detected by western blot and immunohistochemical assay.Results:1. Establishment of B-ALL-NOD/SCID mice:1.1. 4~5 weeks old NOD/SCID mice were injected with 5×106 Nalm-6 cell via tail vein, 7 days post injection, more than 1% CD10+CD19+cells in peripheral blood were detected by flow cytometry, indicating the biological onset of B-ALL.1.2. Hind limb paralysis 、 hunched back and anorexia appeared on 15 days,indicating clinical onset of B-ALL.1.3. The lifespan of B-ALL-NOD/SCID mice was18.40±0.89 days.2. The effects of Bortezomib, PCI-32765 and Ara-c on B-ALL-NOD/SCID mice.2.1. The lifespan of the mice:A. Treatment start at 3 days post injection, the lifespan were(22.70±0.37),(22.30±0.52) and(21.33±0.67) days respectively in Bortezomib, PCI-32765 and Ara-c(10mg/kg) single drug groups, as compared with control group(18.67±0.42)days, the difference is signicant(p<0.05). The lifespan of Bortezomib and PCI-32765 combination group is(28.00±0.60) days, and Bortezomib, PCI-32765 and Ara-c combination group(31.50±1.23) days, compared with control group, the difference is signicant(p<0.05).B. Treatment start at 7 days post injection, the lifespan were( 21.83±0.70 and(21.67±0.42) days in Bortezomib and PCI-32765 single drug groups, and(24.17±0.80)days in Bortezomib and PCI-32765 combination group respectively, compared with control group(18.67±0.42) days, the difference is signicant(p<0.05).C. Treatment start at 15 days post injection, the mice live longer than control mice in Bortezomib and PCI-32765 single drug groups, but there were no significant difference. The lifetspan were(21.83±0.48) days in Ara-c single drug group, and(23.67±0.72) days in Bortezomib, PCI-32765 and Ara-c combination group, as compared with control group and Bortezomib, PCI-32765 single drug groups, the difference is signicant(p<0.05).2.2. The expression level of Btk, NFκB, BCL-XL and CIAP-1 in the spleen tissue were decreased, and caspase-3 was increased in treated mice as detected by western blot. The IOD in control group, Bortezomib, PCI-32765 single drug group and combination group were Btk:( 120348.30±9881.02 、 73890.18±1911.34 、74300.38±1516.14 、 36039.81±2894.98) vs control group( 120348.30±9881.02)(p<0.05). NFκB:(109770.90±6849.65、73379.62±2121.27、74184.62±2208.24、33857.30±2789.34)vs control group(109770.90±6849.65)(p<0.05).Conclusions:1. B-ALL-NOD/SCID mice were successfully established by injecting5×106 Nalm-6 cells in 4~5 weeks old NOD/SCID micevia tail vein. The biological onset timewas 7 days and the clinical onset time was 15 days post cells injection. The lifespan of B-ALL-NOD/SCID mice was(18.40±0.89)days. The method was simple, feasible,and repeatable.2. Bortezomib and PCI-32765 have synergistic effects on B-ALL-NOD/SCID mice,they can prolong the survival. The underlying mechanisms were decrease the expression level of Btk, NFκB, BCL-X and CIAP-1, and incrcase the expression of Caspase-3.3. Bortezomib and PCI-32765 have synergistic effects with Ara-c. These results indicatin that chemotherapy-drug and target-drug can be used together in the treatment of ALL, so as to enhance the therapeutic effect and prolong the survival.