The Effects And Mechanisms Of Aminosteroid H42649 On K562 And CML Leukemia Cells

Objective:To explore the proliferation and differentiation-inducing effects of aminosteroid H42649 on human chronic myelogenous leukemia K562 cell line and CML cells.Methods:The effects of H42649 on K562 and CML cell proliferation were studied by cell count,colony count and MTT assay. Differentiation was measured by morphology,benzidine stain,NBT stain and flow cytometry.The changes of intracellular[Ca²⁺]i concentration were verified by fluorescence spectrophotometer after using H42649 on K562 cells and CML cells for 30min.Real-time PCR was used to detect the effect on calcium channel protein mRNA in K562 cells and CML cells treated by H42649.The bcr/abl fusion gene mRNA expression in K562 cells and CML cells after treatment with H42649 was checked by Realtime PCR.Results1.The effects of aminosteroid H42649 on proliferation of K562 cells and CML cells.The growth of K562 cells and CML cells were inhibited after treatment with 10^-9～10^-5mol/L H42649 for 5 days.The inhibition rates for K562 cells in MTT assay was(55.26±2.54)%after treatment by 10^-6 mol/L H42649 for 5 days and the inhibition rates for CML cells was (63.53±2.36)%respectively.The results also demonstrated that H42649 inhibited the proliferation of K562 cells and CML cells in a dose-dependent manner.2.The effects of aminosteroid H42649 on K562 and CML cell differentiationH42649 also induced K562 cells toward erythroid differentiation.It was verified by bezindine stain that the A value at the dose of 10^-6mol/L H42649 for 5 days was increased(0.2701±0.1960)%compare with the control.H42649 could induced CML alls toward macrophage-cike cell differentiation.It was verified by NBT stain that A value was increased (1.7539±0.0280)%comare with the control.The morphology showed the differentiation tendency after treatment of K562 cells.Data from flow cytometry showed that after treatment with 10^-6mol/L H42649 for 4 days, the percentage of positive CD71 expression in differentiated K562 cells was 84%,while positiveCD14 in CML cells 87%.3.The mechanisms of the proliferation and differentiation-inducing effects of H42649 on K562 and CML cell.3.1 The effects of H42649 on intracellular[Ca²⁺]i ofK562 and CML cell.K562 cells preloaded with Fura-2/AM were treated with 10^-6 mol/L H42649 for 30rain.It showed that H42649 induced decrease of A₃₄₀(P <0.01)compared with the control.CML cells preloaded with Fura-2/AM were treated with 10^-6 mol/L H42649 for 30min.It showed that H42649 induced a decrease of A₃₄₀(P<0.01) compared with the control.3.2 The calcium channel gene CACNA2D1 mRNA expression in K562 and CML cell after treatment with 10^-6 mol/L H42649 for 3 days by Realtime PCR.The relative quantity of K562 cell calcium channel gene CACNA2D1 mRNA from control group and test group were 2.05×10^-3 and 5.94×10^-3 respectively,test/control=2.90,while CML cells were 3.48×10^-3and 5.94×10^-3 respectively,test/control=1.70.Calcium channel mRNA of K562 and CML cells were up regulated by aminosteroid H42649.3.3 The bcr/abl fusion gene mRNA expression in K562 and CML cells after treatment with 10^-6 mol/L H42649 for 3 days by Realtime PCR.The relative quantity of K562 cells bcr/abl fusion gene mRNA from control group and test group were 5.30×10^-2 and 2.13×10^-2 respectively, test/control=0.40,while CML cells were 6.42×10^-2 and 2.37×10^-2 respectively,test/control=0.37,bcr/abl fusion gene mRNA of K562 and CML cells were down regulated by aminosteroid H42649.Conclusion:1.Aminosteroid H42649 inhibited the proliferation of K562 and CML cells in a dose-dependent manner.2.Aminosteroid H42649 induced K562 toward erythroid differentiation.and induced CML cells toward macrophage-cike diferentiation.3.H42649 induced decrease of free intracellular[Ca²⁺]after treatment ofK562 and CML cell for 30min.4.Calcium channel mRNA of K562 and CML cell were up regulated by aminosteroid H42649.5.bcr/abl fusion gene mRNA of K562 and CML cell were down regulated by aminosteroid H42649.