Study On The Effect Of Total Saponins Of Panax Ginseng+/-EPO On The Proliferation And Differentiation Of KG1-A Cells And Its Mechanism

BackgroundTotal saponins of Panax Ginseng (TSPG) is one of the main effectivecomponents of Panax ginseng; studies showed that it has anti-canceractivity especially for hematological malignancy. Because TSPG containsnumerous monomer which structures are complicated, it makes TSPG havecharacteristic "dialectical" pharmacological action of traditional Chinesemedicine. Our previous research showed that TSPG could not only promotethe proliferation and differentiation of normal hematopoietic stem/progenitor cells, but also inhibit the proliferation of leukemia cells, induceits apoptosis and erythroid differentiation. Therefore, TSPG is an adaptivesource drugs; its mechanism may be based on the different signalingpathway mediated by erythropoietin receptor, thus there is a different effecton normal blood (EPO dependent) and abnormal blood cells (EPOindependent). Our previous studies showed that Rg1, one of monomer ofTSPG, could inhibit the proliferation of TF1/EPO (EPO dependent cell)and K562cells (EPO independent cell), and the differentiation mechanismwas related to the signal pathway mediated by EPOR, and there are obviousdifferences between the two pathways[1]. In this study, human leukemia KG1-cells as the research object, toobserve the effect of TSPG±EPO on the expression and activity of EPORin KG1-α cells; comparative study the mechanism of EPOR on theproliferation and differentiation or apoptosis of leukemia cells with orwithout EPO; to establish a "model system" to provide experimental basisand useful clues for the clinical application of TSPG in treatment of tumor.PART1：STUDY ON THE EFFECT OF TSPG+/-EPO ON THEPROLIFERATION AND DIFFERENTIATION OF KG1-ΑCELLSObjective: Study on the effect of TSPG+/-EPO on the proliferationand differentiation of KG1-α cellsMethods: KG1-α cells were cultured in vitro. The KG1-α cells inlogarithmic growth phase were used in the experiment. Control group:routine training; TSPG group: respectively incubated with25,50,100,200,400mg/L TSPG; TSPG+EPO group: the amount of TSPG, while adding0.5U/L EPO at the same time; and set the EPO in the EPO+control group.CCK-8assay was utilized to examine the effects of TSPG+/-EPO on theproliferation of KG1-α cells after cultured for3d,7d,14d. The changes incell cycle distribution and cell surface differentiation antigen CD14, CD15,CD235a were detected by flow cytometry (FCM);Results:1. TSPG can significantly inhibit the proliferation of KG1-α cells invitro,100μmol/L was the optimal concentration and7days was theoptimum time; in the presence of EPO, its optimal concentration wasdecreased to75mg/L;2. Compared with the control group, in the presence of EPO, TSPG could significantly arrest the cell cycle in G0/G1phase, but without theEPO, TSPG had no effect on cell cycle;3. The surface differentiation antigen, only the expression of CD235a(erythrocytic surface marker) increased after induced by TSPG+/-EPO for7days, and the expressions of CD14（monocytic surface marker）, CD15（granulocytic surface marker）had no changes.Conclusion: TSPG could inhibit the proliferation of KG1-α cells andinduce their erythroid differentiation, the addition of EPO can achieve thesame effect at low concentration; in the presence of EPO, TSPG couldarrest the cell cycle in in G0/G1phase, but without the EPO, TSPG had nothe same effect. PART2: TO STUDY THE MECHANISM OF TSPG+/-EPOEFFECT ON HUMAN LEUKEMIA KG1-αObjective: To study the mechanism of TSPG+/-EPO effect onhuman leukemia KG1-αcellsMethods: The KG1-α cells in logarithmic phase were chosen for theexperiment. The KG1–α cells of the control group, EPO+control group,the TSPG100mg/L group, the TSPG200mg/L group, TSPG50mg/L+EPOgroup, TSPG75mg/L+EPO group were collected separately. Realtime-PCR technique was employed to detect the expression of EPO/EPORpathway related genes, such as EPOR, STAT5, JAK2, and AKT; theexpression changes of EPOR before and after induced by TSPG and EPOwere observed by immunofluorescence; the expression of apoptosis related proteins, the expression and activity of EPOR protein were examined bywestern blot analysis; the cellular senescence was detected byβ-galactosidase staining, and immunohistochemical staining was used toobserve the expression of cellular senescence-related proteins.Results:1. Real time-PCR analysis showed that the expression of related genesin JAK-STAT signaling pathway mediated by EPOR, such as EPOR、Jak2、STAT5genes were decreased with the increasing concentration ofTSPG in KG1-α cells, and the high concentration had significantdifference (P <0.05);2. Under the laser scanning confocal microscope, the fluorescence intensity ofEPOR in KG1-α cells induced by TSPG alone was lower than the blank control group;the fluorescence intensity was increased after EPO treated, but did not significantlyincrease compared with the control group; while the use of EPO and TSPG,compared with blank control group the fluorescence intensity was noobvious changes, but compared with TSPG groups it enhanced obviously.3. Western Blot showed that the expressions of EPOR, P-EPOR, Jak2,STAT5, P-STAT5and Bcl-2proteins were down regulated, but theexpressions of Bax and Cleaved Caspases-3proteins were increased inKG1-αcells treated by TSPG for7days; however in the addition of the EPOtreatment groups, the expressions of Jak2, P-EPOR and P-STAT5Significant increased, no changes in expression of Bcl-2, Bax and CleavedCaspases-3.4. β-galactosidase staining showed that the senescent cells ratio inTSPG groups were less than10%, and no difference between the TSPGgroups; in the addition of the EPO treatment groups, the senescent cellsincreased with the increasing concentration of TSPG.Immunohistochemical staining found the expression of P53and P16were up-regulated obviously in TSPG+EPO (75mg/kg) group.Conclusion: TSPG could down-regulate the expressions of EPOR andrelated proteins in JAK-STAT pathway mediated by EPOR, and decreasedthe activity of EPOR, then decreased the expression of Bcl-2protein,increased the expressions of Bax and Cleaved Caspases-3proteins topromote the apoptosis of KG1-αcells; while adding EPO, this effect wasantagonistic, the inhibition of cell proliferation was induced by increasingthe expression of P53and P16and promoting cell senescence.