Anti-tumor Effect And Mechanisms Of Icaritin On CML Leukemic Cells In Vitro And In Vivo

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 results in bcr/abl fusion gene and encodes 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-α,allogeneic hematopoietic stem cell transplantation and newly tyrosine kinase inhibitors-Imatinib,have improved greatly,there was still no good effect on CML blast crisis patients.Therefore,searching for new treatment methods for CML is still the research object.In recent years,it has made great progress which screens the anti-leukemia drugs from the traditional Chinese medicine extracts.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 no adverse reactions were found in animal test. Recent studies of Institute of Hematology of Rui Jin Hospital Affiliated to Shanghai Jiao Tong University have shown 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 was established 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.Icaritin is a simple compound extracted from Herba Epimdii after hydrolyzation,which has anti-estrogen receptor effect and Wt is 368.The former study of our laboratory showed that Icaritin had proliferation-inhibiting and apoptosis-inducing effects on K562 cells in a concentration-dependent way.To observe the definite anti-CML effect of Icaritin in vivo and in vitro,we treated CML patient's primary marrow leukemic cells(CML cells) with Icaritin,and observed whether Icaritin could inhibit proliferation,induce apoptosis of CML cells and explored its molecular mechanisms.Finally we treated CML NOD-SCID mice model with Icaritin,and observed whether Icaritin could prolong life span,reduced k562 leukemic cells infiltration in mice bone marrow,spleen and liver. On the whole,we aim to ascertain the anti-leukemia effect and its mechenism of Icaritin by experiments in vitro and in vivo.Partâ… Proliferation-inhibiting and apoptosis-inducing effects of Icaritin on CML cellsObjective:To observe the proliferation-inhibiting and apoptosis-inducing capability of Icaritin on CML primary cells.Methods: (1) CML primary cells were treated with different concentrations of Icaritin,and then cell viability was analyzed with trypan blue exclusion test and MTT assay;(2) The percentage of earlier apoptosis cell was analyzed by flow cytometry using Annexin V-FITC/PI staining;(3) The molecular character of apoptosis was analyzed by detecting Caspase-9,Caspase-3,Bax,Bcl-2,Cyt-c protein expression using Western blot. Results:(1) Icaritin effectively inhibited the proliferation of CML primary cells in a concentration-dependent way,and the IC₅₀s were 13.5μmol/L(CP) and 18μmol/L(BC);(2)Icaritin could induce the apoptosis of CML primary cells in a concentration-dependent way;(3) The apoptosis of CML cells induced by Icaritin accompanied the ratio changement between Bcl-2 and Bax protein expressions,Cyt-c releasing,cleavage of Caspase-3 and Caspase-9.Conclusion:Icaritin can inhibit proliferation and induce apoptosis of CML primary cells effectively in a concentration-dependent manner.The apoptosis of CML cells,which was induced by Icaritin,companied with regulation of Bcl-2 and Bax expression,Cyt-c releasing,cleavage of Caspase-3 and Caspase-9.These datas suggested that mitochondrion-mediated cell apoptosis pathway played an important role in the apoptosis-inducing capability of Icaritin on CML primary cells.Partâ…¡Molecular mechanisms of the proliferation-inhibiting and apoptosis-inducing effects of Icaritin on K562 cellsObjective:To explore the molecular mechanism of proliferation-inhibiting and apoptosis-inducing effects of Icaritin on K562 cells.Methods:After K562 cells were incubated with different concentrations of Icaritin,(1) The protein expression of ER-36,ERK,P-ERK,JNK,P-JNK,P38,P-P38,c-Jun,P-c-Jun and Bcr/Abl in K562 cells were detected by Western blot;(2) The mRNA expression levels of Bcr/Abl were detected with Real-time PCR;Results:(1) Icaritin down-regulated ER-36,P-ERK,P-P38,and up-regulated P-JNK,P-c-Jun protein expression in a concentration-dependent way of K562 cells;(2) There were no difference in mRNA and protein expressions of Bcr/Abl between Icaritin-treated and Icaritin-untreated K562 cells;Conclusion: The anti-leukemic effect of Icaritin is related to down-regulated ER activation and modulation of MAPK signal transduction pathway.Partâ…¢The anti-leukemic effects of Icaritin in vivo on NOD-SCID mice loading CML cells model.Objective:To observe the anti-leukemic effect of Icaritin in vivo on NOD-SCID mice loading CML cells model.Methods:NOD-SCID mice model of K562 cells were treated with different concentrations of Icaritin, using Imatinib as positive control.(1) Living situations and life spans of mices were observed continuously;(2) Mice marrow pathological examinations were observed by Wight-Gimesa staining,and liver and spleen pathological examinations were observed by hematoxylin-eosin staining.(3) White blood cells(WBC) and human WBC antigen(CD45)⁺ cell percentages in peripheral blood were counted weekly.(4) ER-36 espressions of CML cells in mice spleen were observed by immunohischemistry.Results:(1) The life span of mice bearing K562 leukemic cells was prolonged by Icaritin;(2) The infiltration of K562 leukemic cells in mice bone marrow,spleen and live was reduced by Icaritin;(3) WBC count and human WBC antigen(CD45)⁺ cell percentages in peripheral blood of mice bearing K562 cells were reduced by Icaritin;(4) CML cells ER-36 expression of mice spleen significantly was inhibited by Icaritin;and almost no markedly toxicity reactions were observed in animal test.Conclusion:Icaritin exhibited good effects on K562 cells NOD-SCID mice model in vivo and showed similar anti-CML effect with Imatinib.