Anti-tumor Effect Of IC162 On K562 Leukemic Cells And Molecular Mechanisms

Background:Chronic myeloid leukemia(CML)is a malignant hematopoietic stem cell disease characterized with the Philadelphia chromosome,and over-proliferation and apoptosis tolerance of malignant clone lead to formation and development of CML.The characteristic chromosome t(9;22)(q34;q11)of CML result in bcr/abl fusion gene and encode protein P210,which plays an important role in the incidence of CML by promoting leukemia cells proliferation and delaying apoptosis. Although the traditional treatment methods of CML,including hydroxyurea,interferon-a,allogeneic hematopoietic stem cell transplantation and newly tyrosine kinase inhibitors,have improved greatly,there are still some limitations.Therefore,searching for new treatment methods for CML is still the research objective.In recent years, the use of traditional Chinese medicine extracts on the treatment of leukemia has been made very good efficiency.The anti-leukemia drugs such as Matrine,Curcumin and Oridonin are proved in vitro that suppress various leukemia cells proliferation and induce cell apoptosis,and almost found no adverse reactions in animal test.Recent studies of Institute of Hematology of Rui Jin Hospital Affiliated to Shanghai Jiao Tong University show that Oridonin could induce apoptosis on acute myeloid leukemia M₂(AML-M₂)cell line and primary leukemic cells with t(8; 21)positive in vitro and vivo with little side effects.The research on the anti-leukemia mechanism of Chinese medicine effective components and its clinical application has become an important issue.K562 cell line comes from acute leukemia which changed from human CML.Because of the character of acute leukemia cell and the specific essence of CML cell,K562 cell line becomes good study model of cell for the drug research in vitro.IC162 is simple compound extracted from some plants, which have phytoestrogen-like effect and Wt is 368.Because of commercial confidentiality,the specific element is secrecy.In our study, we observe whether IC162 inhibits proliferation,induce apoptosis and induce differentiation of K562 cells and CML patient's primary marrow leukemic cells(CML cells)and explore its molecular mechanisms.Partâ… Proliferation-inhibiting effect of IC162 on CML cellsObjective:To determine proliferation-inhibiting capability of IC162 on K562 and CML primary cells.Methods:K562 and CML primary cells were treated with different concentrations of IC162,and then cell viability and formation of colony were analyzed with trypan blue exclusion test,MTr assay and colony culture test.Cell cycle was tested by flow cytometry.Results:(1)IC162 effectively inhibited the proliferation of K562 and CML primary cells in a concentration-dependent way,and the IC50 was 8.2μmol/L.(2)IC162 significantly inhibited colonic formation of K562 cells in a dose-dependent way.(3)IC162 increased the percentage of G0/G1 phase and decreased K562 cells of S phase.Conclusion:IC162 can inhibit proliferation of K562 and CML primary cells effectively by blocking cells in G0/G1 phase on a concentration-dependent manner.Partâ…¡Apoptosis-inducing effect of IC162 on CML cellsObjective:To ensure whether IC162 can induce the apoptosis of CML cells.Methods:After CML cells were incubated with different concentrations of IC162,(1)morphologic character of apoptosis was evaluated by Hochest33258 staining;(2)the percentage of earlier apoptosis cell was analyzed by flow cytometry;(3)the molecular character of apoptosis was analyzed by Western blot detecting Caspase-3 expression.Results:(1)Hochest33258 staining and flow cytometry detection testify IC162 can induce the apoptosis of CML cells in a concentration-dependent way;(2)The apoptosis of CML cells,which was induced by IC162,kept company with up-regulation and cleavage of Caspase-3.Conclusion:IC162 can induce the apoptosis of CML cells with a concentration-dependent manner;The molecular mechanisms of apoptosis induction was related to up-regulated caspase-3 expression and activation.Partâ…¢Differentiation-inducing effects of IC162 on K562 cellsObjective:To determine the differentiation-inducing potentiality of IC162 on K562 cells.Methods:Differentiation-inducing effects of IC162 (1～12μmol/L)were evaluated by morphology and function of K562 cells.We evaluated the effects of IC162 on K562 cells morphology by Wight-Gimesa staining and the function by detecting intracellular hemoglobin concentration and benzidine dihydrochloride staining of hemoglobin,analyzing CD71 and HIR2 antigen through flow cytometry. Results:IC162 increased hemoglobin content in K562 cells and enhanced the expression of CD71 and HIR2 antigen on K562 cells.After the drug treatment,K562 cells showed differentiation morphology of erythroid cells.Conclusion:Low dose of IC162 can induce K562 cells to erythroid cells differentiation.Partâ…£Molecular mechanisms of the proliferation-inhibiting and apoptosis-inducing effects of IC162 on K562 cellsObjective:To explore the mechanism of proliferation-inhibiting and apoptosis-inducing effects of IC162 on K562 cells.Methods:After K562 cells were incubated with different concentrations of IC162,(1)The protein expression of NF-κB,IκB,STAT3,STAT5b,JAK2,P-NF-κB, P-STAT1 and P-STAT5 in K562 cells were detected by Western blot;(2) The mRNA expression levels of MMP-9 were detected with Real-time PCR;(3)The subcellular distribution and changes of NF-κB in K562 cells were detected with indirect immunofluorescence technique.Results: (1)IC162 down-regulated NF-κB,STAT3,STAT5b,P-NF-κB and P-STAT5 proteins,and up-regulated Iκ3 protein in a concentration-dependent way in K562 cells;but no JAK2 and P-STAT1 protein changes were observed;(2)IC162 down-regulated mRNA expression levels of MMP-9 in a concentration-dependent way in K562 cells;(3)NF-κB was located in cytoplasm of K562 cells.Conclusion: The anti-leukemic effet of IC162 is related with inhibition of signal transduction pathway of NF-κB and STATs.