Effects Of CAL-101 In Combination With BTZ On Mantle Lymophma Cells And Exploration Of Its Relative Mechanism

Objective:To investigate the effect of CAL-101, a selective inhibitor of PI3Kδ, in combination with bortezomib on the proliferation and apoptosis of human mantle cell lymphoma cell lines Z138, HBL-2 and Jeko-1 in vitro, and to explore its mechanisms and provide the foundation for effective treatment strategies against mantle cell lymphoma.Methods:1. Z138, HBL-2 and Jeko-1 cells were separately treated by differrent concentrations of CAL-101( 10μmol/L 、 20μmol/L 、 40μmol/L 、 80μmol/L 、100μmol/L) for 24, 48 and 72 hours. MTT assay was applied to detect the inhibitory effects of different concentrations of CAL-101;2. The values of IC50 of these two drugs in three different MCL cell lines were evaluated by 13.0 SPSS software;3. Synergistic experiments: Cells were divided into four groups: control group(did not add no medication), CAL-101 group( 20%、40%、60%、80% and 100.% of IC50), BTZ group(20%、40%、60%、80% and 100% of IC50) and CAL-101 / BTZ group(the concentrations were accorded with monotherapy group) and then treated three cell lines for 48hours; Calcusyn software(Biosoft, Ferguson, MO America)analyses were preformed to detect synergistic cytoxicity(CI);4. Western Blot detected the expression of PI3K-p110σ in three different MCL cell lines.5. Then the expression level of AKT、ERK、p-AKT and p-ERK was analysed by Western Blot after treated by different concentrations of CAL-101;6. Flow cytometry was employed to assay the apoptosis rates of CAL-101 group,BTZ group and combination group in HBL-2 and Z138 cells7. NF-κB Kit was performed to study the location changes of NF-κB p65 of four groups in three groups;8. Western Blot was applied to detect the level of caspase-3 and thephosphorylation of AKT in different group.Results:1. Exposed to 10μmol/L、20μmol/L、40μmol/L、80μmol/L、100μmol/L of CAL-101 for 48 hours, the inhibitory rates in Z138 cells were:(17±1.2)%,(27±3.6)%,(41±3.6)%,(55±0.2)%,(62±3.5)%,in HBL-2 cells were:(20±0.4)%,(35±0.2)%,(43±0.3)%,(53±0.6)%,(59±0.2)%,and in Jeko-1 cells were(15±1.4)%,(27±2.7)%、(38±3.8)%,(47±0.7)%,(59±0.1)%;2. After treatment of single-agent CAL-101 or BTZ for 48 hours in Z138,HBL-2 and Jeko-1 cells, the IC50 values of CAL-101 accordingly were: 56.957 ±0.6364, 62.99 ± 0.57003, 77.82 ± 0.65339, and the IC50 values of BTZ were: 0.73 ±0.84902, 1.433 ± 0.34312, 1.5 ± 0.3579;3. Synergistic experiments: CAL-101 group(added 18, 36, 54,72 and 90 μmol/L of CAL-101), BTZ group(added 0.06, 0.12, 0.18, 0.24 and 0.30 μmol/L of BTZ) and CAL-101/BTZ conbination group(the concentrations were accorded with monotherapy group) treated three cell lines for 48 hours and and 80% of IC50 of concentrations were choosed in three cell lines. The inhibitory rates of CAL-101 group 、 BTZ group and CAL-101 conbined BTZ group in Z138 cells were(38.5±1.4)%,(35.6±5.1)%, and(58.1±6.1)%; in HBL-2 cells were(42.3±1.2)%,(35.7±1.6)% and(67.3±3.3)%;in Jeko-1 cells were(33.2±1.2)%,(36.0±1.1)%and(56.5±2.9)%;4. CAL-101/BTZ combination induced significantly synergistic cytotoxicity in MCL cells by Calcu Syn software calculation(CI<1);5. PI3K-p110σ is expressed in these threel MCL cells;6. After treatment with varing concentrations of CAL-101, phosphorylation of AKT and ERK were inhibited in a dose-dependent pattern and there were no difference in the expression level of AKT or ERK;7. Co-administration of CAL101 and Bortezomib synergistically increased apoptosis in MCL cells: Z138 cells were exposed to drugs for 48 h, the apoptosis rates of the control group, CAL-101 group, BTZ group and CAL-101/BTZ conbination group were:(2.6±1.8)%,(40.0±3.0)%,(34.0±1.0)%,(67.4±1.0)%, andwhen drug treatment was given to HBL-2 cells over 96 h, apoptosis rates were:(7.4 ±0.6)%,(30.7±5.7)%,(12.0±1.0)%,(85.0±4.0)%. So CAL-101 combined with BTZ induced pronounced apoptosis(P<0.001);8. Co-exposure of MCL cells to CAL101 and bortezomib contributed to enhanced inactivity of nuclear factor-κB(NF-κB):like-ELISA assay suggested cells were exposed to these four groups, the level of NF-κB expression in Z138 were 1.0,(43.3±1.2)%,(43.2±1.2)%,(22.2±0.6)%; In HBL-2 cells, were 1.0,(35.7±0.4)%,(36.8±1.0)%,(23.1±1.3)%; and in Jeko-1 cells, NF-κB were 1.0,(46.1±1.0)%,(54.2±0.5)%,(22.5±0.5)%(P<0.01);9. By Western Blot, Combinded tretment induced marked downregulation of p-AKT in Z138, HBL-2, Jeko-1 cells; cleaved Caspase-3 was upregulated after combined treatment in MCL cell lines.Conclusion:We have known that PI3 K / AKT and ERK signaling pathway were aberrantly activated in MCL cells, which have positive effects on cell proliferation, invation and drug-resistance. Therefore, dysregulation of this pathway is important in the etiology of human malignancies. Thus blocking PI3 K / AKT and ERK signaling pathway by targeting some critical protein molecules, ultimately can protect the tumor cells from growing and invating.This study demonstrated that CAL-101 was cytotoxic to human MCL cells and potently enhanced the activity of proteasome inhibitor bortezomib by targeting NF-κB and AKT activity. In view of emerging or established evidence of activity of these agents in MCL, further exploration of this regimen in vivo setting appears justified, and is currently underway. Taken together, we hope to provide a platform for clinical trials of CAL-101 in MCL and a rationale for its use in combination therapy with bortezomib.