The Effects And Mechanisms Of Anti-leukemia Inducer Aminosteroids On K562 Leukemia Cells

Objective To explore the proliferation and differentiation-inducing effects and potencial mechanisms of aminosteroid on humam chronic myelogenous leukemia K562 cell line.Methods Proliferation was detected by MTT assay and by cell counting both in liquid and semisolid cultures.Differentiation was measured by morphology,benzidine staining and flow cytometry.The changes of intracellular and extracellular[Ca²⁺]were verified by atomic absorption spectrometer,fluorescence spectrophotometer and confocal laster scanning microscopy.We took cytosine arabinoside as the positive control.To search for the relationship between the effects of aminosteroid on K562 cell line and[Ca²⁺],we treated K562 cells with indomethacin, BayK8644,and EGTA on K562 as controls.Results1 The effects of aminosteroid on proliferation and differentiation of K562 cellsProliferation:The growth of K562 cells was inhibited except aminosteroid G after treatment with 10^-5mol/L of 8 different aminosteroids for 5d.The inhibition rates for K562 cells in MTT assay were respectively following.A(61.21±10.03)%,B(63.03±2.28)%,C(43.38±2.96)%,D(54.49±1.14)%,E(24.43±1.26)%,F(52.96±8.74)%,H (79.88±8.88)%。Differentiation:All 8 different aminosteroids induced K562 cells toward erythroid differentiation at 10^-5mol/L after culture for 5d.It was verified by benzidine staining and the differentiation effects increased as times compared with the control respectively:A 1.96,B 1.92,C 2.28, D2.55,E2.30,F1.99,G2.17,H2.30.We took aminosteroid C(code name H51817)to further explore the potential mechanisms of aminosteroid on humam chronic myelogenous leukemia K562 cell line.2 The effects of aminosteroid H51817 on proliferation and differentiation of K562 cellsProliferation:aminosteroid H51817 inhibited proliferation of K562 cells.Inhibition rate was(38.36±10.54)%and(45.92±11.15)%respectively at 10^-5mol/L after culture for 3d and 5d in liquid culture;The inhibition rates for K562 cells in colony assay was(77.98±6.98)%at 10^-5mol/L after culture for 8d.Differentiation:The morphlogy showed the differentiation tendency after treatment of K562 cells.Date from flow cytometry showed that aminosteroid H51817 induced K562 cells toward erythroid differentiation, in which the antigen CD71 expression rate was 67.74%at 10^-5mol/L after culture for 5d.3 The effects of EGTA,BayK8644,indomethacin,cytosine arabi- noside on proliferation and differentiation of K562 cellsProliferation:BayK8644,indomethacin,cytosine arabinoside inhibited proliferation of K562 cells.Inhibition rate was(14.94±0.01)%, (18.30±0.02)%,(50.10±0.12)%respectively at 0.01mmol/L after culture for 5d in MTT assay;EGTA showed little effect on K562 cells at 0.01～1 mmol/L after culture for 5d in MTT assay(p＞0.05)Differentiation:BayK8644,indomethacin,cytosine arabinoside induced K562 cells toward erythroid differentiation at 0.01mmol/L after culture for 5d,and the differentiation verified by benzidine staining increased 1.98,2.94 and 3.29 times respectively as compared with the control.However,EGTA showed little effect on K562 cells at 0.01～1 mmol/L after culture for 5d(p＞0.05)4 The effects of aminosteroid H51817 on intracellular and extracellular[Ca²⁺]in K562 cellsWe found that the intracellular[Ca²⁺]in K562 treated by 10^-5mol/L H51817 cells was subdued with the control by confocal laster scanning microscopy,fluorescence spectrophotometer,and atomic absorption spectrometer.The total intracellular[Ca²⁺]in K562 cells in DMEM decreased by(32.01±2.36)%at 10^-5mol/L H51817 treatment for 30min compared with the control,but the extracellular[Ca²⁺]did not change.In contrast,both the intracellular and extracellular[Ca²⁺]in K562 cells treated with 10^-5mol/L H51817 in 0.9%saline did not change at all. 5 The effects of EGTA,BayK8644,indomethacin,cytosine arabinoside on total intracellular[Ca²⁺]in K562 cellsAfter treatment with EGTA,BayK8644,indomethacin,cytosine arabinoside at 0.01 mmol/L on K562 cells for 30min,the total intracellular [Ca²⁺]decreased respectively.Conclution1 Almost aminosteroids inhibited the proliferation of K562 cells, but the sensitivity is different for different aminosteroids.2 All aminosteroids induced K562 cells toward erythroid differentiation,but the differentiation degree varies in K562 cells depending on different aminosteroids.3 Aminosteroid H51817 decreased intracellular[Ca²⁺]in K562 cells,but its action depended on the existence of extracellular Ca²⁺.4 Aminosteroids,indomethacin and cytosine arabinoside all decreased total intracellular[Ca²⁺]in K562 cells,and all of them inhibited the proliferation of K562 cells and induced them toward erythroid differentiation,it was indicated that the decrement of intracellular[Ca²⁺]in K562 cells maybe associated with the mechanism underlying the effects of aminosteroid H51817 on K562 cells.5 BayK8644 is an agonist for L-type calcium channels,but surprisingly,it decreased intracellular[Ca²⁺]in K562 cells,and it inhibited the proliferation of K562 cells and induced them toward erythroid differentiation also.Probably,there is other calcium channels rather than the classical L-type calcium channels existed on K562 cells, and this kind of channel maybe the target for the action of aminosteroid H51817 on K562 cells.6 EGTA is a calcium chelating agent.We found that it decreased intracellular[Ca²⁺]in K562 cells,but it neither inhibited the proliferation of K562 cells nor induced them toward erythroid differentiation,It was indicated that the decrement of intracellular[Ca²⁺]in K562 cells maybe just one of the mechanisms in prolifration inhibition and differentiation induction and the effects of aminosteroid H51817 on K562 cells may be through the other mechanisms beside the intracellular[Ca²⁺]decrement. Further research is needed on this issue.