Biological Effect Of Gemcitabine On CD34~+CD38~-Myeloid Leukemia Stem Cells

BackgroundLeukemia is a malignant disease in hematopoietic system because of gene mutation in the cloning-forming cell of pluripotent stem cells or more early progenitor cells on some developing stage.A large number of leukemia cells proliferation in bone marrow or other hematopoietic tissues,inhibition of normal hematopoietic function,and extensive infiltration in other tissues and organs.It is bring about hematopoietic system tumors.Which occurs four-five per hundred thousand people every year in China.The Mortality rate of Leukemia was No.6(male) and8(female) in malignanttumor and in children and adults under the age of thirty-five was highest.moreover,At present,acute myelogenous leukemia is a major type of leukemia in adults,and accounted for about60%in various leukemia of adults. AML is a serious and often lethal disease. Although the develop-ment of the better therapy methods has improved induction remission and overall survival,15-25%of patients fail to achieve complete remission because of resistance to treatment or death, and>40%of CR patients will relapse within2years. It is believed that AML was initiated and maintained by a rare population of Leukemic stem cells. Leukemia stem cells play the central role in the relapse and Drug resistance of AML, How to eliminate the resistance of immunity and drug resistance of leukemia stem cells is the key to reduce the leukemia relapse and cure leukemia. So the study of leukemia stem cells is of capital importance. and.Leukemia stem cells in Hematopoietic stem cells are usually present in very small number it is in the starting phase of cell clone, with a self-renewal, multipotential differentiation similar to normal cells, but not the self-regulation ability of the normal stem cells.KG1a cell line was derived from a male Acute Myelocytic Leukemia patient,and these cells do not respond to colony stimulating factor of granulocyte and macrophage. The previous study on the phenotype of KG la cells showed that its high expression of CD34+antigen, but not expressed CD38-antigen.the studies found that the leukemia stem cell phenotype characterized is CD34+CD38-cell.these results suggest that KG1a cells can be used as a relevant cellular model for researching the leukemia stem cell.CD34+CD38-cells had self-renewal capacities,moreover, they were resistant to conventional chemotherapy and natural killer cell-madiated cytotoxicity,Gemcitabine (2’-deoxy-2’,2’-difluorocytidine monohyddrochloride(β-isomer)) is a deoxycytidine and pyrimidine analogue and similar to cytarabine in structure, but phosphorylation efficiency is powerful than cytarabine. Gemcitabine is an anti-metabolism anticancer and cell cycle-specific cytotoxic drug. Gemcitabine exhibits the progression of cell through the S-phase boundary and primarily killing cells undergoing DNA synthesis (S-phase).At present,it has been widely applied in the treatment of patients with non-small cell lung cancer, pancreatic cancer and metastatic breast cancer. Abroad,the research of clinical trials for the treatment of refractory lymphoma and multiple myeloma has achieved good results in malignant system disease. But the study of gemcitabine in treatment of leukemia is still in the fundamental and clinical trials researches.Therefore,in this study with CD34+CD38-early acute myeloid leukemia KGla cells including leukemia stem cells as targeted cells,Gemcitabine as intervention factors,combined weigh immune cells activated by cytokines according to indicators of cell apoptosis,colony-forming cells,proliferation, cell cycle and so on,the sensitivities of KG1a cells to cytotoxicity of activated immune cells will be estimated, we propose that NKGZD ligand involve in KG1a attacked form NK-mediated,promote the development of novel therapeutic strategies to target leukemic stem cells. Part1Biological Effect of Gemcitabine on CD34+CD38-Myeloid Leukemia Stem CellsObjective in this study with CD34+CD38-KG1a cell self-renewal capacity and differentiation stages by stem cell phenotype, cell cycle, soft agar colony formation semisolid medium, CD34+CD38" early acute myeloid leukemia KGla cells as targeted cells,to investigate the effects of Gemcitabine (GEM) on colony formation cell cycle and apoptosis of myeloid leukemia stem cells (LSCs) of CD34+CD38-KG1a cells.Biological effect of gemcitabine on CD34+CD38-myeloid leukemia stem cells.Methods Expression of CD34and CD38on the surface of acute myeloid leukemia KGla cells was detected by flow cytometry. Effects of various concentration(0.05mg/L,0.1mg/L and0.5mg/L) of gemcitabine and cytosine on proliferation at24hour、48hour、72hour. Observe gemcitabine and cytosine pretreatment24hour group and direct action group on KG1a cell colony-formed and self-renewal ability by Soft agar colony formation semisolid medium.The colony-formed of KG1a cells were analyzed at14day and21day. The changes of cell cycle percentage of KG1a cells treated with various concentration (0.05mg/L,0.1mg/L and0.5mg/L) of GEM were tested by flow cytometry. The changes of apoptosis of KG1a cells treated with various concentration (0.05mg/L,0.1mg/L and0.5mg/L) of GEM were checked by flow cytometry through the staining of AnnexinV-FITC and propidium iodide (PI).Results Percentage of CD34+CD38-acute myeloid leukemia KGla cells was (98.02±0.72)%.,clone formation rate are (21.52+1.75)%and (23.97+0.64)%at14d and21d, and shows the characteristics of KGla cell leukemia stem cell phenotype and self-renewal capacity. After treated with0.1mg/L and0.5mg/L GEM for24h, the numbers of colony at14d were (15.06±1.15)%�'�(3.75±0.38)%,lowed to the saline control group (21.52±1.75)%(P<0.05).the numbers of colony at21d were (15.06±1.15)%�'�(3.75±0.38)%,lowed to the saline control group (23.97±0.64)% (P<0.05), Whereas treated with0.05mg/L GEM, the numbers of colony at14d and21d, the numbers of colony at21d were (20.28±1.11)%、(18.23±15.73)%compared to saline control group (21.52±1.75)%�'�(23.97±0.64)%, the differences were not statistically significant (P>0.05). After sustained medication treated with0.05mg/L,0.1mg/L and0.5mg/L GEM and Ara-C the numbers of colony at14d and21d decreased to0, lowed to the saline control group (P<0.05). The inhibitory effect of KGla cells, treated with0.05mg/L,0.1mg/L and0.5mg/L GEM for24h,48h and72h, with dose and time-dependant manners. The inhibitory effect is superior to cytarabine group with same concentration and time. KG1a cell survival rate after treated with0.5mg/L GEM for24h is (22.93+5.73)%,(88.10+1.97)%of KG1a cells of survival which is in the Go/G1phase, compared with saline control group (52.2+7.18)%, increased by35.9%(P<0.05), S phase and G2/M phase of the control group decreased by31.9%and31.9%(P<0.05),And0.05mg/L GEM group for24h and0.1mg/L GEM group for24h were similar to the saline control group in cell cycle distribution of KGla cells.Treated with0.5mg/L GEM for24h,(22.93±5.73)%of KG1a cells of survival which is the apoptosis rate of KG1a cells was (20.70±3.24)%higher than the saline control group(7.59±1.80)%and Ara-C group (14.22±1.41)%(P<0.05), while0.05mg/L GEM group for24h and0.1mg/L GEM group for24h were similar to the saline control group in cell apoptosis of KG1a cells,(P>0.05).Statistical analysis The analysis was performed using SPSS13.0software package. The data was represented as the mean±standard deviation. Comparisons of means among groups were performed using factorial design analysis of variance.Comparisons of means between two groups ware performed using T-text,P <0.05were considered to be statistically significant.summaryConclusion1. This experiment used KGla cells with stem cell phenotype and biological properties.2. In different concentrations of GEM can inhibit myeloid leukemia cells CD34+CD38-KGla cells proliferation, inhibit proliferation capability with dose and time-dependant manners.3. Gemcitabine can arrest the CD34+CD38-KG1a cells cycle at G0/G1phase.4. Gemcitabine induced the CD34+CD38-KG1a cells to apoptosis.5. Gemcitabine aignificantly inhibit the cloning formation ability of the CD34+CD38-KGla cells,for which cells which fast self-renewal and high potential proliferation,and continuously and steadily drug concentration enhance the inhibition effect.6. Gemcitabine inhibition the CD34+CD38-KGla cells proliferation and clone ability may associate with apoptosis and the arrestion of cell cycle at Go/G1phase.Part II The cytolytic sensitivities effect of peripheral blood mononuclear cell for Gemcitabine on CD34+CD38KGla cellsObjective To explore the cytolytic sensitivties of CD34+CD38-human acute myeloid leukemia KG1a cells after treated by Gemcitabineand and cytolytic by preliminary mechanism of unstimulated PBMC and the PBMC stimulated by IL-2,IL-5.Methods Applied Trypan blue assay to detect the inhibiting rate on peripheral blood mononuclear cells (PBMC),in which concentration of gemcitabine(GEM)(0.05mg/L、0.1mg/L、0.5mg/L). LDH texted untreated PBMC and the PBMC stimulated by IL-2,IL-15against CD34+CD38-KG1a cells which before or not treated by GEM(0.1mg/L) were analyzed by LDH releasing assay at different effector-to-target cell ratios(10:120:1). The expression of NKG2D (MICA、MICB、 ULBP1、ULBP2、ULBP3) ligands was assayed by Flow cytometry before and after treated by GEM for48h.Statistical analysis The analysis was performed using SPSS13.0software package. The data was represented as the mean±standard deviation.Comparisons of means among groups were performed using factorial design analysis of variance.Comparisons of means between two groups ware performed using T-text,P <0.05were considered to be statistically significant.Results We found that GEM cannot inhibit the proliferation of PBMC Effects of various concentration(0.05mg/L,0.1mg/L and0.5mg/L) of GEM on proliferation and cytotoxicity.Which is not time-dependant maners and no statistically significant difference (P>0.05).In E:T10:1,20:1the cytolytic rates of untreat PBMC to KG1a cells were only (4.87±0.99)%and (6.31±0.46)%,but the rates in KG1a cells after treat by GEM(0.1mg/L) were (17.88±3.76)%and (36.60±4.27)%,Respectively,increasing by13.01%,30.29%(P<0.05).In E:T10:1,20:l,the cytolytic rates of PBMC stimulated by IL-2,IL-15to KG1a cells were (25.63±2.95)%and (36.14±4.84)%,and the rates in KG1a cells after treat by GEM were (35.30±4.99)%and (53.74±7.74)%,Respectively,increasing by9.67%,17.6%(P<0.05).KG1a cells after treat by GEM for48hours, the cell surface of NKG2D ligands expressed not seen significant change(P>0.05).Conclusion1.In different concentrations of GEM cannot inhibit PBMC proliferation.2.PBMC surface receptor NKG2D expression increased when induced by IL-2and IL-15.3.GEM can enhance the cytotoxic activity sensitivity of KGla cell by PBMC.Article conclusion1.Gemcitabine can arrest the cycle, induce apoptosis and inhibit the cloning formation ability of CD34+CD38-KG1a cells.2.GEM cannot inhibit PBMC proliferation.3. GEM can enhance the cytotoxic activity sensitivity of KG1a cell by PBMC.4.the experimental basis of clinical myeloid leukemia treatments by GEM is provided.