Effect And Mechanisms Of Chlorambucil Combined With Ibrutinib On Mantle Cell Lymphoma Cell Line Jeko-1

Objective This paper mainly studies the inhibitory and apoptosis effect of Chlorambucil and Ibrutinib on Mantle cell lymphoma(MCL)cell line Jeko-1,explores whether there is a synergistic effect on the combination of the two drugs,and investigates the mechanism of their apoptosis-inducing effects.It is hoped that the two drug combination may provide a potentially feasible new therapeutic option for MCL,especially for the patients with relapsed and refractory MCL,which may further improve its prognosis and overall survival.Methods Jeko-1 cell line was cultured in T25 cell culture flasks.When the cell density in the culture flasks grew to 80%,they were centrifuged and resuspend,and then seeded evenly in 6-well plates or 96-well plates for different experimental interventions according to different experimental purposes.The experiments were divided into three parts: Part Ⅰ: Inhibition and induction of apoptosis of Jeko-1 cells by different concentrations of Chlorambucil and different concentrations of Ibrutinib Jeko-1 cells were treated with different concentrations of Chlorambucil(3.125μmol/L,6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L)and different concentrations of Ibrutinib(3.125μmol/L,6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L).A blank control group was set up,and each group was cultured for 24 h,48h and 72 h respectively.The CCK-8 method was used to detect the proliferation of the cells in each group and calculate their inhibition rate,and the apoptosis rate was detected by flow cytometry.Part Ⅱ: The inhibitory and apoptosis-inducing effect of the combination of different concentrations of Chlorambucil and Ibrutinib on Jeko-1 cells According to the results of part Ⅰ,using the effective lower doses of the two drugs for joint applications(single drug inhibition rate<50%),four experimental groups were set up: a blank control group,a Chlorambucil single drug group,an Ibrutinib single drug group and a Chlorambucil + Ibrutinib combination group.The main experimental interventions were different concentrations of Chlorambucil(3.125μmol/L,6.25μmol/L,12.5μmol/L)combined with the same concentrations of Ibrutinib(3.125μmol/L,6.25μmol/L,12.5μmol/L)which were applied to Jeko-1 cells.After 72 hours of culture,the cell activity was detected by CCK-8 assay,and the apoptosis rate was detected by flow cytometry.Part Ⅲ: Study of the mechanism of combined application of Chlorambucil and Ibrutinib on Jeko-1 cells Western Blot assay was used to detect the expression levels of CASPASE-3,BCL-2,PI3 K,p-AKT,AKT in the blank control group,Chlorambucil single drug group,Ibrutinib single drug group and Chlorambucil + Ibrutinib combination group.Results Part I: Inhibition and induction of apoptosis of Jeko-1 cells by different concentrations of Chlorambucil and different concentrations of Ibrutinib The activity of Jeko-1 cells treated with either Chlorambucil or Ibrutinib alone for 24 h,48h,and 72 h was reduced.The inhibition rate tended to increase with increasing concentration of the intervening drug and the duration of the intervention and the rate of apoptosis increased with the increase of the concentration of intervention drugs.Among them,after the Jeko-1 cells treated by different concentrations of Chlorambucil(3.125μmol/L,6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L)for 72 h,the inhibition rates of cells were(17.41±1.14)%,(35.91±1.66)%,(51.76±1.98)%,(64.48 ± 2.09)%,(82.86 ± 2.88)%,and apoptosis rates were(10.07 ± 0.84)%,(22.13 ± 3.01)%,(44.43 ± 5.01)%,(62.77 ± 3.95)%,(77.17 ± 2.35)%,respectively.While the Jeko-1 cells treated by different concentrations of Ibrutinib(3.125μmol/L,6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L)for 72 h,the inhibition rate of cells were(7.71 ± 1.06)%,(15.28 ± 2.20)%,(41.32 ± 3.17)%,(79.70±3.43)%,(97.25±2.69)%,and apoptosis rates were(7.60±0.44)%,(13.43±1.63)%,(35.10±2.21)%,(55.67±1.53)%,(98.57±1.70)%,respectively.Compared with the control group,the differences were statistically significant(all p values < 0.05).When the concentration of both drugs was greater than 12.5μmol/L,the apoptosis rate and inhibition rate were able to more than 50%.Part II: The inhibitory and apoptosis-inducing effect of the combination of different concentrations of Chlorambucil and Ibrutinib on Jeko-1 Cells Using the effective lower doses of the two drugs for joint applications,which is intervention with the combination of Chlorambucil(3.125μmol/L,6.25μmol/L,12.5μmol/L)and the equal concentration of Ibrutinib(3.125μmol/L,6.25μmol/L,12.5μmol/L)increased the inhibition rate of Jeko-1 cells compared with single drug to(23.37±3.12)%,(62.58±3.94)%,(93.51±2.50)%,and the apoptosis rate of Jeko-1 cells was increased to(15.95±0.78)%,(54.75±0.07)%,(85.05±1.20)% respectively.The inhibition rate and apoptotic rate in the combined drug treatment group were significantly increased compared with the single drug treatment group(all p values < 0.05).Part III: Study of the mechanism of combined application of Chlorambucil and Ibrutinib on Jeko-1 cells When the Jeko-1 cells were treated with Chlorambucil monotherapy,the expression of BCL-2,PI3 K and p-AKT/AKT was down-regulated,while the expression of CASPASE-3 was up-regulated.The trend of Ibrutinib monotherapy on the expression of the above proteins was similar to the results of Chlorambucil.In contrast,the two drug combination group could decrease the expression of BCL-2,PI3 K and p-AKT/AKT and further increase the expression of Caspase-3 significantly.The differences were all statistically significant in the combination group compared with the single drug group(all p values < 0.05)Conclusion Both Chlorambucil and Ibrutinib can induce the apoptosis of Jeko-1 cells.At high concentration,Ibutinib is superior to Chlorambucil alone,and the combination of the two drugs can produce synergistic effect,which is mainly through the regulation of Akt signaling pathway.This provides experimental and theoretical basis for a new therapeutic regimen of MCL.