ABSTRACT
<p><b>OBJECTIVE</b>To study the molecular mechanism of apoptosis of leukemic cells (K562 cells) induced by iron chelating agent deferoxamine (DFO).</p><p><b>METHODS</b>The exponentially growing K562 cells were used (1×10(6)/mL) in this study. The K562 cells were treated with different concentrations of DFO (10, 50 and 100 mmol/L), DFO+FeCl3 (10 μmol/L each) or normal saline (blank control). The cellular labile iron pool was measured with a fluorimetric assay using the metalsensitive probe calcein-AM. The viable count and cell viability were determined by typanblue assay. Cell apoptosis was determined by morphological study and flow cytometry assay. Caspase-3 activity in K562 cells was detected by colorimetry.</p><p><b>RESULTS</b>After DFO treatment, the cellular labile iron pool and the viability of K562 cells were reduced and the cell apoptosis increased in a time- and dose-dependent manner compared with the blank control group. The apoptosis rate of K562 cells in the DFO+FeCl3 treatment group was not significantly different from that in the blank control group. The caspase-3 activity in K562 cells increased significantly 24 hrs after 50 and 100 μmmol DFO treatment when compared with the blank control group (P<0.01). There was a negative correlation between cellular labile iron pool and caspase-3 activity of K562 cells (r=-0.894, P<0.05).</p><p><b>CONCLUSIONS</b>DFO induces apoptosis of leukemic cells possibly through decreasing cellular labile iron pool and increasing caspase-3 activity of the cells.</p>
Subject(s)
Humans , Apoptosis , Caspase 3 , Metabolism , Deferoxamine , Pharmacology , Flow Cytometry , Iron Chelating Agents , Pharmacology , K562 CellsABSTRACT
To explore a rapid and easy method to detect labile iron of pool (LIP) in cells, HL-60 and K562 cells were cultured at a concentration 1 x 10(6)/ml in RPMI 1640 containing 10% heat-inactivated fetal bovine serum. The iron deprivation was induced by adding desferrioxamine (DFO) 10 - 100 micromol/L for 0 - 48 hours. The intracellular LIP was measured by probe calcein-AM. Calcein fluorescence was monitored in 1420 multilabel counter. The results indicated that when HL-60 and K562 cells were incubated with different concentrations of DFO, the calcein fluorescence intensity was higher than that of control group at 12, 24 and 48 hours (P < 0.05). Fluorescence value of representing LIP in DFO groups was lower than that in the control group. In conclusion, DFO can decrease LIP in leukemia cells. The approach used in this study may provide a simple and reliable method for detection of intracellular iron homeostasis.
Subject(s)
Humans , Cation Transport Proteins , Metabolism , Deferoxamine , Pharmacology , Fluoresceins , Fluorescent Dyes , HL-60 Cells , Iron , Metabolism , Iron Chelating Agents , Metabolism , Iron-Regulatory Proteins , Metabolism , K562 CellsABSTRACT
Objective To observe the relationship between apoptosis of K562 cell induced by iron-deprivation and activation of Caspase-3.Methods K562 cells were treated with desferrioxamine(DFO) in different dosages were collected at different time points.K562 cells were labelled with Annexin V/PI,and then the rate of apoptosis was measured by flow cytometry;The activation of Caspase-3 were detected by colorimetric method with pAN labelled substrate;The active protein of Caspase-3 were analyzed by Western blot.Results When K562 cells were treated with different concentrations of DFO,the apoptosis rate and the activity of Caspase-3 increases gradually.When K562 cells were incubated with DFO(50 ?mol/L and 100 ?mol/L) 24 h later,the enzymatic activity of Caspase-3 increases dramatically more than that of control group,and the difference was significantly(P0.05).All those effect above can be counteracted by equal mole concentration of FeCl_3.Conclusion Iron-deprivation maybe induce the apoptosis of K562 cell by chelating intracellular iron and activing Caspase-3.
ABSTRACT
<p><b>OBJECTIVE</b>12-0-tetradecanoylphorbol-13 acetate (TPA) plays an important role in precipitating cell differentiation for various tumor cells, especially leukemic cells. Changes of many genes may be involved in this process. The purpose of this study was to observe the relationship between the EGR1mRNA expression and cell differentiation during TPA-induced K562 cell differentiation.</p><p><b>METHODS</b>Incubation of human K562 cells in vitro was applied to cultivate K562 cells. The cells were treated in two different ways. K562 cells of experiment group were treated with TPA and those of control group were treated without TPA. Using morphology (Wright's staining and NSE staining) and flow cytometry (FCM), the investigators observed the differentiation characteristics of K562 cells, cell-cycle and the differentiation antigen expressions of CD33 and CD14 on cell membranes. RT-PCR was carried out to assay EGR1 mRNA expression.</p><p><b>RESULTS</b>After treated with TPA for 7 d, the morphology of K562 cells obviously tended to mature differentiation, like monocytes. The differentiation rate of induced K562 cells was up to 95% in experiment group and 4.5% in control group, respectively. Using SPSS software, the above result showed statistical significance (P < 0.01). Using NSE staining, K562 cells showed positive reaction. Some of them were densely stained. The positive rate was up to 86%. More than half of the positive cells could be inhibited by NaF. The inhibiting rate of NaF was up to 58.72%, showing statistical difference when compared with that of control group. FCM analysis showed that most of K562 cells stimulated by TPA underwent G1/S phase cell-cycle arrest. The composing rate of cell-cycle in TPA-treated group showed that (53.7 +/- 1.25)% of cells were at G0 + G1 phase and (44.3 +/- 1.32)% were at S phase (P < 0.05). The level of CD33 expression on cell membranes was mildly decreased from 0.997% to 0.893% (P > 0.05). However, the level of CD14 expression was significantly increased from 0.049% to 0.387% (P < 0.05).</p><p><b>CONCLUSION</b>K562 cells could express EGR1mRNA during TPA-induced differentiation, which suggested that EGR1mRNA might participate in the process of K562 cells differentiating into monocyte/macrophages, and might play an important role in precipitating and maintaining cell differentiation for leukemic cells.</p>
Subject(s)
Humans , Antigens, CD , Metabolism , Antigens, Differentiation, Myelomonocytic , Metabolism , Carcinogens , Pharmacology , Cell Cycle , Genetics , Cell Differentiation , Genetics , Cell Division , Genetics , Cell Membrane , Chemistry , DNA-Binding Proteins , Genetics , Early Growth Response Protein 1 , Flow Cytometry , Gene Expression Regulation, Neoplastic , Immediate-Early Proteins , Genetics , K562 Cells , Lipopolysaccharide Receptors , Metabolism , RNA, Messenger , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sialic Acid Binding Ig-like Lectin 3 , Tetradecanoylphorbol Acetate , Pharmacology , Transcription Factors , GeneticsABSTRACT
Objective To explore the effect of iron chelators on labile iron pool and expression of apoptosis associated genes in cells of K562, an erythroleukemia cell line.Methods K562 cells were incubated at 37 ℃ in RPMI 1640 containing 10% heat-inactived fetal bovine serum in an saturated humidity and 5% CO_2 incubator. K562 cells were incubated with different concentrations of desferro-(xamine(DFO)). The study groups were divided as following: DFO group, iron+DFO group and the control group. Following indices were detected which included apoptosis by flow cytometry (FCM) assay, expression of Rb, c-myc, bax mRNA by RT-PCR. The intracellular LIP was measured with a fluorimetric assay using the metalsensitive probe calcein-AM.Results 1. The viability of K562 cells incubated with different concentrations of DFO was lower than that of control group at 12 h,24 h and 48 h (P