Effects Of 2-methoxyestradiol On The Proliferation, Apoptosis And Adhesion Of K562 Cells And Its Mechanisms

Chronic myelocytic leukemia (CML) is a malignant disease of the hematopoietic stem cell. More than 90% of CML patients have Philadelphia (Ph) chromosome which produces a specific oncoprotein known as bcr/abl fusion protein. This protein has strong tyrosine kinase activity, can disturb the normal signal transduction pathways inside the hematopoietic progenitors. This is the reason that ph+ cells proliferate excessively and escape apoptosis.Decreased cellular adhesion may enhance cell cycling and egress of leukemia cells out of the bone marrow into peripheral blood.CML is characterized by increased proliferation, resistance to apoptosis and aberrant adhesion and migration.2-methoxyestradiol (2-ME) is a physiological metabolite of the endogenous estrogen which fails to bind the estrogen receptor and has little action by affecting estrogen receptor ways. Several studies show that 2-ME have anticarcinogenic activitis, including anti-proliferation, anti-angiogenic, induction of apoptosis and cell cycle arrest. 2-ME can also enhance the radiation effects on cancer cells but has little side-effects on normal cells and organs. So it may be a promising candidate for the treatment of cancers. In the present study, we investigated the effect and mechanisms of 2-ME on the proliferation, cell cycle, apoptosis and adhesion of bcr/abl+ K562 cells, a leukemia cell line derived from a CML patient in blast crisis, in order to provide some useful experimental data for clinical application of 2-ME. This study includes the following four parts.Part1 Effects of 2-methoxyestradiol on the proliferation and cell cycle of K562 cellsObjective: To investigate the effects of 2-ME on the proliferation and cell cycle of K562 cells. Methords: (1) The cultured K562 cells were devided into two grupes:①The control group: K562 cells were cultured without 2-ME treatment.②The experimental group: K562 cells were cultured in the medium containing different concentrations of 2-ME (1μmol/L, 2μmol/L, 4μmol/L, 8μmol/L and16μmol/L) for different time periods (24h, 36h, 48h and 60h). (2)The morphological changes of these cultured K562 cells were observed and recorded by photographing when these cells had been treated with 2-ME for 36h and the cell morphological changes became obvious. (3) MTT assay was performed to evaluate the proliferation inhibition effect of 2-ME on K562 cells. (4) FCM assay was performed to evaluate the effect of 2-ME on cell cycle distribution of K562 cells.Results:(1)Cell morphological changes:No obverious morphological change of K562 cells treated with 1μmol/L 2-ME was observed. Along with increasing of 2-ME concentration and prolonging of culturing time, K562 cells displayed morphological changes, including cell nuclear condensation, congregate and putrescence. (2) Effects of 2-ME on proliferation of K562 cells:The proliferation of K562 cells treated with 1μmol/L~8μmol/L of 2-ME was inhibited, and the growth inhibition rate was signifcantly higher than that of the control group, but the increasing of growth inhibition rate was not significant when K562 cells were treated with 2-ME at the concentration of 16μmol/L;The growth inhibition rate of K562 cells treated with 2-ME from 24h to 48h was gradually increased along with the prolonging of culture time, but the increasing of growth inhibition rate was not significant when K562 cells were treated with 2-ME for 60h.These results showed that the proliferation of K562 cells was inhibited by 2-ME in a concentration-and time-dependent manner.(3) Effects of 2-ME on cell cycle of K562 cells:After treated with 0μmol/L~4μmol/L of 2-ME, the increasing of G0/G1 phase K562 cells was accompanied with the decreasing of S phase cells, while the percentage of G0/G1 phase K562 cells was not increased when the cells was treated with 2-ME at concentrations of 4μmol/L, 8μmol/L and 16μmol/L. However, when the cells were treatred with 2-ME at concentrations of 4μmol/L~16μmol/L, the percentage of G0/G1 phase cells was signifcantly higher than that in the control group.Conclusion: 2-ME inhibited the growth of K562 cells in a concentration-and time-dependent manner, increased the percentage of G0/G1 phase cells and induced cell morphological changes.Part2 Effects of 2-ME on the expression of apoptosis-related molecules and apoptosis of K562 cellsObjective: To investigate the effects of 2-ME on the expression of related-related molecules, Caspase-3 and XIAP, and apoptosis of K562 cells. Methods: (1) The apoptosis rate of K562 cells treated with or without 2-ME was detected by TUNEL methods.(2) The apoptosis rate of K562 cells treated with or without 2-ME was detected flow cytometry (FCM) methods. (3) The expression of apoptosis- related protein of Caspase-3 and XIAP of K562 cells was performed by FCM. (4) The mRNA expression of apoptosis-related genes Caspase-3 and XIAP of K562 cells was detected by half-quantitative reverse transcription-polymerase chain reaction (RT-PCR).(5)The activity of superoxide dismutase (SOD) and reactive oxygen species (ROS) was detected by xanthenes oxidized enzyme assay.Results:(1) Effects of 2-ME on the apoptosis of K562 cells detected by TUNEL and FCM:After treated with 2-ME for 36 hours at all the tested concentrations, except for the concentration of 16μmol/L, the apoptosis rate of K562 cells was significantly higher than that of the control group (P<0.05). The apoptosis rate of K562 cells detected by FCM was almost the same as that detected by TUNEL method.The result detected by TUNEL methods was positively correlated to that detected by FCM (γ=0.845,P=0.034).(2) Effects of 2-ME on the expression of Caspase-3 and XIAP protein in K562 cells:After treated with 2-ME at all the tested concentrations for 36 hours, the expression of Caspase-3 protein increased in a concentration-dependent manner, the expression level of it in the experimental group was higher than that in the control group. The expression of XIAP protein decreased along with the increasing of 2-ME concentration. However, the difference between the experimental group and the control group was not statistically significant (P>0.05).(3) Effects of 2-ME on the expression of Caspase-3 and XIAP mRNA in K562 cells : The expression of Caspase-3 mRNA was higher in the experimental group than that in the control group (P<0.01), and the expression of XIAP mRNA was lower in the experimental group than that in the control group (P<0.05). The expression level of Caspase-3 mRNA was inversely correlated with that of XIAP (γ=-0.966, P=0.000).(4) Effects of 2-ME on SOD and ROS activity in K562 cells:The SOD activity of K562 cells was lower in the experimental group than that in the control group (P<0.05), and the ROS activity of K562 cells was higher in the experimental group than that in the control group (P<0.05). The SOD activity was converse correlation with ROS activity in K562 cells (γ=-0.462, P=0.054).Conclusion: (1) In the specified concentration range, 2-ME induced apo- ptosis of K562 cells in a concentration-dependent manner. (2) The possible ac- tion mechanisms of 2-ME were up-regulating Caspase-3 but down- regulating XIAP mRNA expression, and increasing ROS activity but decreasing SOD activity.Part3 Effects of 2-ME on the expression of L-selectin mRNA and protein of K562 cellsObjective: To investigate the effects of 2-ME on the expression of L-selectin and its gene in K562 cells.Methods: (1)The expression of L-selectin protein in K562 cells was detected by flow cytometry (FCM) method. (2) The expression of L-selectin mRNA in K562 cells was performed by half-quantitative reverse transcription-polymer- ase chain reaction (RT-PCR).Results: (1) Effects of 2-ME on the expression of L-selectin protein in K562 cells:After treated with 2-ME at different concentrations for 36 hours, the expression of L-selectin protein increased in a concentration-dependent manner, the expression level of L-selectin protein in the experimental group (four groups of 2μmol/L,4μmol/L,8μmol/L,16μmol/L) of 2-ME was higher than that in the control group(P<0.05).(2) Effects of 2-ME on the expression of L-selectin mRNA in K562 cells:After treated with 2-ME at different concentrations for 36 hours, the expression of L-selectin mRNA increased in a concentration-dependent manner, the expression level of L-selectin mRNA in the experimental group was higher than that in the control group (P<0.01).(3) The relation between expression level of L-selectin mRNA and protein in K562 cells:The expression levels of L-selectin mRNA were positively correlated with that of L-selectin protein (γ=0.758,P=0.004).(4) The relation between expression level of L-selectin protein and P210 bcr/abl protein in K562 cells: The expression levels of L-selectin protein were converse correlated with that of P210 bcr/abl protein (γ=0.-976,P=0.001).Conclusion: 2-ME increased the expression of mRNA and protein of L-selectin, which may probably improve or restore the aberrant adhesion of K562 cellsPart4 Effects of 2-ME on the expression of bcr/abl fusion mRNA and protein of K562 cellsObjective: To investigate the effects of 2-ME on the expression of bcr/abl fusion gene and protein in K562 leukemia cells.Methods: (1)The expression of bcr/abl mRNA in K562 cells was performed by real-time fluorescence quantitative polymerase chain reaction (RQ-PCR) with SYBR Green I. (2)The bcr/abl fusion protein (P210) expression in K562 cells was detected by Western blot.Results: (1)The sensitivity of real-time fluorescence quantitative PCR with SYBR Green I:The 10-fold serial diluted cDNA of K562 cells were amplified by RQ-PCR with SYBR Green I, the sensitivity of the method was 10pg cDNA of K562 cells. The linearity and relativity of the quantitative standard curve were satisfactory, and correlation coefficient was 0.997.(2) The stability and repetition of real-time fluorescence quantitative PCR with SYBR Green I: The 105 cDNA of K562 cells was amplified by RQ-PCR with SYBR Green I for six times, six Ct (cycle threshold) value were obtained and the average value was 14.95±0.54,the coefficient variation(CV) was3.6%.(3) Effects of 2-ME on the expression of bcr/abl fusion mRNA in K562 cells:The quantitative standard curve were performed with originated copies of standard cDNA of K562 cells as the abscissa and corresponding Ct value as the y-axis, the copies of bcr/abl mRNA and GAPDH mRNA were achieved by the quantitative standard curve, and the expression level of bcr/abl mRNA was ratio of copies of bcr/abl and GAPDH genes. The results indicated that the expression of bcr/abl mRNA decreased gradually along with the increase of 2-ME concentration, however, the difference between the experimental group and the control group was not statistically significant(P>0.05).(4)Effects of 2-ME on the expression of bcr/abl fusion protein (P210) in K562 cells:The P210 protein expression in K562 cells decreased gradually along with the increase of 2-ME concentration, and the difference between the experimental group and the control group were statistically significant (P<0.01).(5)The correlation between the P210 protein expression and the apoptosis of K562 cells: The P210 protein expression in K562 cells detected by Western blot was conversely correlation with the apoptosis rate detected by TUNEL and FCM (γ1=-0.821,P1=0.045;γ2=-0.821,P2=0.045,respectively).Conclusion: 2-ME did not affect the expression of bcr/abl fusion gene in K562 cells, however, it affected the expression of bcr/abl fusion protein (P210), and the protein expression level was converse correlation with the apoptosis rate of K562 cells, which was probably one of action mechanisms through which 2-ME induced apoptosis of K562 cells.In summary, the characteristics of CML are increased proliferation, resistance to apoptosis and aberrant adhesion and migration. The present study was designed to explore the effects and mechanisms of 2-ME on cell proliferation, cell cycle, apoptosis and adhesion of bcr/abl+ K562 cells, which were derived from a CML patient in blast crisis phase, the results indicated that 2-ME could inhibit the cell proliferation,arrest cell cycle progression,induce apoptosis,improve adhesive function and reduce the expression of bcr/abl fusion protein in K562 cells.These experimental data indicate that 2-ME has promising potential in the treatment of CML patients.