Effects And Mechanism Of IC162 On CML Leukemic Cells In Growth Inhibition Or Apoptosis Inducement, And Enhancing Chemotherapic Sensitivity

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. The characteristic chromosome t (9; 22) (q34; q11) of CML result in bcr/abl fusion gene and encode fusion protein P²¹⁰, which plays an important role in the pathogenesis of CML by promoting leukemia cells proliferation and resisting apoptosis. Although the traditional therapic methods of CML, including hydroxyurea, interferon-a, allogeneic hematopoietic stem cell transplantation and newly tyrosine kinase inhibitors, have improved greatly patient's prognosis, there are still some limitations for the CML patients with blastic crisis. Therefore, searching for new drugs in CML therapy is the main objective. In recent years, the studies of traditional Chinese medicine extracts for the treatment of leukemia have been extensively investigated,such as Matrine, Curcumin and Oridonin are proved in vitro that these drugs can suppress leukemia cells proliferation and induce cells apoptosis, and no adverse reactions are found in animal model. Recent studies from Institute of Hematology,Shanghai Rui Jin Hospital show that Oridonin could induce apoptosis on acute myeloid leukemia M₂ (AML- M₂) cell line and primary leukemic cells with t (8; 21) positive both in vitro and in vivo with little side effects. The research on the anti-leukemia effect of Chinese medicine and their clinical application have become an important issue. K562 cell line originated from human CML cells with blastic transformation.. Because of the character of acute leukemia cells and the specific essence of CML cells, K562 cells are a good study model for researching anti-leukemia drug. IC162 is a pure compound extracted from a Chinese herb medicine, which has phytoestrogen-like effect and Wt is 368. In present study, we try to determine whether IC162 may inhibit CML cells proliferation, induce CML cells apoptosis and enhance the chemotherapic sensitivity to Daunomycin and Homoharringtonine and explore its molecular mechanisms.Partâ… Proliferation-inhibiting effect of IC162 on CML cellsObjective: To determine proliferation-inhibiting effect of IC162 on K562 and primary CML cells from CML patients with blast crisis. Methods: K562 cells and primary CML cells were treated with different concentrations of IC162, and then cells viability were analyzed with trypan blue exclusion test and MTT assay colony formation test. Cell cycle was checked by flow cytometry. Results: (1) IC162 effectively inhibited the proliferation of K562 and primary CML cells in a concentration-dependent way, and the IC50 was S.2μmol/L. (2) IC162 significantly inhibited colony formation of K562 cells in a dose-dependent way. (3) IC162 increased the percentage of G0/G1 phase K562 cells and decreased percentage of S phase K562 cells. Conclusion: IC162 can inhibit proliferation of K562 and primary CML cells by arresting 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 primary CML cells from CML patients with blast crisis. 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 apoptotic cells were analyzed by flow cytometry; (3) the molecular character of apoptosis was assayed by Western blot for detecting Caspase-3 expression. Results: (1) Hochest33258 staining and flow cytometry detection testify IC162 can induce the apoptosis of primary CML cells in a concentration-dependent way; (2) IC162-induced CML cells apoptosis is associated with up-regulation and cleavage of Caspase-3. Conclusion: IC162 can induce the apoptosis of primary CML cells with a concentration -dependent manner; The molecular mechanisms of apoptosis induction was related to up-regulated caspase-3 expression and activation. Partâ…¢Effect of IC162 on enhancing the chemotherapic sensitivity of Daunomycin or Homoharringtonine on K562 cellsObjective: To determine whether IC162 can enhance the anti-proliferative effect and apoptosis-inducing effect on K562 cells to Daunomycin and Homoharringtonine. Methods: K562 cells were treated with low dose IC162 along,Daunomycin or Homoharringtonine along,and IC162 (2μmol/L) combined with Daunomycin or Homoharringtonine interfered with K562 cells for 48 h. The effects of the drugs on K562 cell growth inhibitation, apoptosis inducement were analyzed by MTT assay, AnnexinV-FITC/ PI assay. Results: The treatment of IC162 (2μmol/L) combined with Daunomycin(320μg/L) or Homoharringtonine (320μg/L) could significantly inhibit the K562 cell viability and induce the K562 cell apoptosis than those of treatments for IC162, Daunomycin or Homoharringtonine alone. Conclusion: IC162 can enhance the anti-proliferative effect and Apoptosis effect of Daunomycin or Homoharringtonine on K562 cells.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 COX-2 in K562 cells were detected by Western blot; (2) The mRNA expression levels of COX-2,Survivin,CyclinD2,CyclinE,p27,MMP-2,MMP-9 and VEGF were detected with Real-time PCR; Results: (1) IC162 could down-regulate the expression of COX-2 proteins and mRNA in a concentration- dependent way on K562 cells; (2) IC162 could down-regulate mRNA expression levels of Survivin,CyclinD2, and CyclinE,and up-regulate mRNA expression levels of p27 in a concentration-dependent way on K562 cells; (3) IC162 could down-regulate mRNA expression levels of MMP-2,MMP-9 and VEGF in a concentration-dependent way on K562 cells. Conclusion: The anti-leukemic effets of IC162 is related to inhibition of signal transduction pathway involved in Cyclins, COX-2 and MMPs family...