Study On Epor Mechanisms Of Proliferation、Differentiation And Survival In Leukemic Cell Lines (TF-1/EPO And K562) Induced By Ginsenoside Rg1

TSPG(Total Saponins of Panax Ginseng), one of the components intraditional Chinese herb Ginseng radix, has the pharmacological effect ofanti-tumor. In the present study, we summerize TSPG is suggested tofunction as an adaptogen medicine. TSPG could not only promote theproliferation and differentiation of hematopoietic stem/progenitor cells butalso inhibit the proliferation of K562cells, and induce their apoptosis ordifferentiation toward the erythroid-lineage cells. We investigated anddiscussed the mechanism of TSPG and found it promotes proliferation,differentiation, or survival of normal (Epo-dependent) and abnormal bloodcells K562(Epo-independent) through different EpoR mechanisms, whichrevealed the different effects of Epo on different types of hematopoieticcells. Furthermore, more and more researches have suggested that Epo／EpoR were also expressed in nonhematopoietic tissues and cells. That isone of the potential mechanisms proffered to explain whyerythropoiesis-stimulating agents directly stimulated tumor growth through inducing cell proliferation and angiogenesis. In this study, the Epo-dependent leukemia cell line TF1/Epo and Epo-independent leukemia cellline K562were used as the research objects，to investigate the effect of Rg1on EpoR and p-EpoR，to explore the different EpoR signal mechanismsduring the proliferation, differentiation, and survival in different types oferythroid-lineage cells induced by ginsenoside Rg1. This study will providethe proofs and a new way for therapy strategies of different types ofhematopoietic diseases (especially hemopathy characterized by red bloodcells that develop abnormally) and even some non-hematopoietic cellstumors with Rg1.PART1: EFFECT OF RG1ON PROLIFERATIONDIFFERENTIATION IN DIFFERENT TYPESOF LEUKEMIA CELL LINESObjective: To study the effects of Rg1on different types of leukemiacellsMethods: TF-1/Epo, K562cell were cultured in conventional. Settingcontrol group and10μmol/L,20μmol/L,50μmol/L,100μmol/L,200μmol/L Rg1groups, TF-1/Epo and K562cells in logarithmic phasewere respectively exposed to various concentrations of Rg1for24,48,72h;The cell viabilities were measured by CCK-8chronometry. The change ofcell cycle and the SmCD, such as CD14, CD15, CD235a on these cells treated with Rg1(optimum concentration and time) were measured by flowcytometry.Results: The results showed that ginsenoside Rg1significantlyinhibited the proliferation of TF-1/Epo and K562cell at the concentrationof50-200μmol/L. The optimal concentration was100μmol/L and48h wasthe best reaction time; Compared with the control group, experimentalgroup cells were significantly arrested at G1phase; The levels of CD15,CD235a except CD14were increased by100μmol/L Rg1for48h.Conclusion: Rg1has the effects of inhibiting proliferation andinducing differentiation of TF1/Epo and K562cells towards erythrocyteand granulocyte.PART2: EFFECT OF GINSENOSIDE RG1ONTHE EXPRESSION AND ACTIVATION OF EPORObjective: To study the effects of Rg1on the expression and activationof EpoR in different types of leukemia cell.Methods: TF-1/Epo and K562cell were cultured in conventional andcells in logarithmic phase were used in experiment. TF-1/Epo and K562cells from all groups (0,100μmol/L Rg1) at24,48h were collected. Theextracted total RNA was firstly identified by agarose gel electrophoresis(AGE) and ultraviolet spectrophotometry, and investigated the expressionof EPOR mRNA by Real-time fluorescent quantitative PCR. Flow cytometry and laser scanning confocal microscopy were used to detect thequantity of EpoR on cell surface; Western blot was applied to determine theexpression of EpoR in total protein, cytomembrane protein and p-EpoR.Results: Real-time PCR results showed that the expression of EPORmRNA of24,48h Rg1groups reduced significantly compared with controlgroup. The positive ratio and the intensity of fluorescence of EpoR proteinin cell membrane on TF-1/Epo and K562cells are higher than thoseinduced by Rg1. Compared with control groups, the expression of EpoRprotein in total protein and total cellular membrane protein extracts ofTF-1/Epo and K562cells induced by Rg1are weakened. In addition, in theearlier phase of Rg1induced TF-1/Epo cells, no matter they werestimulated or not by Epo，the expression of p-EpoR of experimental groupdecreased significantly than that of control group. However the efficacy ofRg1in suppressing EpoR activation is not obvious in K562cells.Conclusion: The expressions of EpoR are downregulated and thequantity of EpoR on membrane decreased after inducing by Rg1in bothTF-1/Epo and K562cells. Before Rg1change the expression of EpoR inwhole cell extracts of TF-1/Epo cells, it inhibits its downstream signaltransduction pathways. While the down-regulation of activation of EpoR isunconspicuous in K562cells induced by Rg1.