Differentiation-inducing Effects And Molecular Mechanisms Of Icaritin On K562 Leukemia Cells

Background:Chronic myelogenous leukemia(CML) arises from the neoplastic transformation of a pluripotent hematopoietic stem cell carrying the balanced translocation t(9;22)(q34;q11),which cytogenetically results in the Philadelphia chromosome and molecularly gives rise to the Bcr/Abl hybrid gene.The protein kinase Bcr/Abl,a constitutively active tyrosine kinase,encoded by the Bcr/Abl oncogene is central to the pathogenesis of CML.At present,the clinically available treatment strategies for CML,including the front-line tyrosine kinase inhibitor(TKI)-Imatinib,hydroxyurea,interferon-α(IFNα) and allogeneic hematopoietic stem cell transplantation,can significantly prolong life in CML patients.However,screening novel therapeutic medicine for CML is still necessary and important.In resent years,several extracts of traditional Chinese medicine (TCM) have shown excellent effects against leukemia in relative studies. It's reported that Matrine,Curcumin and Oridonin,derived from TCM, could be able to inhibit growth,induce apoptosis and differentiation of various leukemic cells in vitro.Simultaneously,few of adverse events were detected in animal tests.The human CML cell line K562 was established from the pleural extravasation of a patient with CML in blast crisis,and as such expresses the chimeric Bcr-Abl tyrosine kinase whose high constitutive activity is thought to play a crucial role in the genesis of CML.In addition,K562 cells can undergo differentiation in megakaryocytic,erythroid or monocytic lineages depending on the stimulus.Thus,it has been used extensively as model for the study of leukemia differentiation.Icariin is a constituent of Epimedium which is a traditional Chinese herbal medicine.Recently,a limited data showed that Icariin could induce HL-60 cells differentiation.Furthermore,Icariin has been shown to be able to induce cardiomyocyte differentiation of murine embryonic stem (ES) cells in vitro,and the mechanism is associated with P38MAPK activation.Icaritin,a hydrolytic product of Icariin,has the ability of anti-estrogen receptor.In the early stage of our studies,we have found that Icaritin was able to inhibit growth and induce apoptosis in a dose-dependent manner,in K562 cell line.Icaritin has potential as an agent for the treatment of CML,but the precise modes of its effects on anti-leukemia require further elucidation.In this study,we focus on utilizing Icaritin to induce differentiation in K562 cell line,and discuss the effects and molecular mechanisms of Icaritin on differentiation in K562 cells.PartⅠDifferentiation-inducing effects of Icaritin on K562 cellsObjective:To determine the differentiation-inducing potentiality of Icaritin on K562 cells.Methods:Differentiation induction experiment was performed by culturing K562 cells with the presence of Icaritin(0～64μmol/L) for 6 days.The changes of morphology,cytochemisrty and cytophyletic characteristic markers(CD7 and CD235a) of K562 cells were observed before and after the treatment with Icaritin.,by the methods of wright's-Giemsa staining,benzidine staining and flow-cytometry separately.Results:K562 cells treated with 8μmol/L Icaritin were found differentiated and morphologically changed to polychromatic normoblasts and orthrochromatic normoblasts.Treatment of K562 cells with Icaritin increased hemoglobin content, benzidine-positive cells from 3.97±2.18%(0μmol/L) to 11.46±2.29% (8μmol/L) and up-regulated expressions of CD7 and CD235a. Conclusion:Icaritin can induce K562 cells differentiation.PartⅡMolecular mechanisms of the differentiation-inducing effects of Icaritin on K562 cellsObjective:To explore the molecular mechanisms of differentiation-inducing effects of Icaritin on K562 cells.Methods:the changes of protein expression in K562 cell line,including JNK,P-JNK,ERK,P-ERK,P38 and P-P38,were detected by Western blot before and after the treatment with Icaritin.Results:Western blot results showed that the phosphorylation of P38 was up-regulated for 4-8 days on K562 cells after administration of Icaritin;the phosphorylation of JNK was up-regulated for 2-6 days and down-regulated on 8th day;the expression of ERK was up-regulated mildly;the expressions of JNK,P38 and ERK were not altered.Conclusion:the activation of P38 and JNK and the inhibition of ERK could be involved in Icartin-promoted erythroid differentiation of K562 cell line.