| Leukemia is a malignant clonal disorder of the hematopoietic stem cells,which ranked the sixth place of varieties tumors in China.Just as other tumors,leukemia is characterized by uncontrolled proliferation of hematopoietic cells,maturation blocked and the unnormal process of apoptosis.Currently,there are various methods for the treatment of leukemia,such as radiation therapy,chemotherapy,targeted therapy,immunotherapy,and stem cells transplantation.However,there are advantages and disadvantages in current treatment methods.In recent years,traditional Chinese medicine as well as its active ingredient which have been in the anti-tumor activity of growing concern.Evodiamine(Evo)which extracting from Evodia medicine has many functions,such as anti-hypertension,hypoglycemic,analgesia,slimming,anti-tumor and so on.Research reports indicated that histone deacetylases(HDACs)had a very close relationship with the development of tumor as well as the occurrence of abnormal activation of expression.Many researchers believed that HDACs was a target for the cancer therapy.Histone deacetylase inhibitors(HDACi)were regarded as a novel anti-cancer drugs and had been used in clinical treatment.We assume that the anti-tumor effect of Evo may be associated with HDACs.In this study,human leukemia K562 cells were used as experimental subjects to investigate the effect of Evo on proliferation apoptosis and epigenetic modification in K562 cells,expectly to provide experimental evidence for the clinical application of traditional Chinese medicine.ObjectiveTo investigate the effects of Evo on the proliferation inhibition,apoptosis,epigenetic modification of human erthroleukemia cell line K562 and to explore the effects of Evo on histone acetylation of K562 cells from the aspects of epigenetic modification and the regulatory mechanisms.MethodsEvo(1,2,4,8,16 ?mol/L)induced human leukemia K562 cells line 24 h,48 h,and 72 h respectively in vitro.The activity of K562 cells was detected by CCK-8 assay;the growth of K562 cells was observed by optical microscopic;the changes of cell cycle distribution and apoptosis were examined by flow cytometry;chemical colorimetry assay was used to examine the activity of histone modification enzymes in K562;the changes of histone acetylation level,expressions of histone deacetylase protein and the key enzymes on signaling pathway,which is associated with HDACs,were examined by western blot analysis.Results1.Evo could effectively inhibit the proliferation of K562 cells,which exhibits a dose-dependent manner at the range of 1~16 ?mol/L.Besides,with the extension of time,the inhibition of Evo were gradually increased(within 72 h).2.Morphological observation under inverted microscope: The growth of K562 cells was good in the control group,but the number of K562 cells were significantly reduced after induced by Evo(2,4,8 ?mol/L).3.The results of flow cytometry indicated that Evo could arrest K562 cells in G0/G1 phase.The apoptosis rate of K562 cells were(11.47±1.05)%,(12.77±0.79)% and(18.58±1.37)% after induced by 2,4,8 ?mol/L Evo,which showed statistically significant difference(P<0.01)compared with the control group(2.79±1.0)%.4.Chemical colorimetry assay showed that Evo could reduce the activity of HDACs in K562 cells and improve HATs' activity.5.Western blot assay indicacted that Evo could enhance the acetylation levels of histone H3 in K562 cells.The expressions of Bcl-2,Cyclin D1,CDK4,HDAC1-3,HDAC6,JNK and p-ERK proteins were decreased in a dose-dependent manner after induced by Evo.However,the expressions of Bax,Caspase-3(activation type),p21,p16,p-p38,p38 and p-JNK proteins were increased.But,the expression of ERK protein had no significant difference(P > 0.05).Conclusion1.Evodiamine could effectively inhibit the Growth and proliferation of K562 cells in a dose-and time-dependent manner in vitro.2.Evodiamine could induce cell cycle arrest in G0/G1 phase through regulating the expression of cyclin proteins.3.Evodiamine could increase the levels of histone acetylation and activate HDACs,and through activating the HDACs' downstream signaling pathways to influence the proliferation and apoptosis of K562 cells. |
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