Dihydroartemisinin Downregulats Transferrin Receptor Expression And Inhibits Vascular Endothelial Growth Factor Autocrine/Paracrine In Iron Overload Myeloid Leukemia Cells

Iron is fundamental for life as it is a cofactor of enzymes such as cytochrome c and ribonucleotide reductase,which are essential for ATP production and DNA synthesis,respectively.The uptake of iron from transferfin(Tf) is controlled by its receptor TfR expression which is modulated by intracellular iron levels.As erythroid precursors and malignant cells,specially leukemia,lymphoma and myeloma are highly dependent upon iron to sustain their characteristic high levels of proliferation, therefore transferrin receptor(TfR) is expressed at a greater level.Particularly,the aggressiveness and the proliferation index of leukaemic cells are positively correlated with TfR content.Thus,several groups have tried to target TfR in order to induce apoptosis or to reduce proliferation of leukaemic cells.Artemisinin,is the active principle of the Chinese plant qinghao(Artemisia annua L.) originally distinguished itself as a new generation of anti-malarial drug with low toxicity.It contains an endoperoxide bridge which is essential for its anti-malarial activity,can react with ferrous iron to generate free radicals.The lactone can easily be reduced,resulting in the formation of dihydroartemisinin(DHA),the most active intermediate.Recently,several studies have revealed that DHA also has a preferentially cytotoxic effect on cancer cells.Furthermore,it has been reported that DHA kills cancer cells via an iron-mediated mechanism.In our previous report,we have shown that DHA inhibits the vascular endothelial growth factor(VEGF) expression in solid tumor xenograft and exhibits the potent antiangiogenic effect in solid tumors.However,to our knowledge,the down-regulation effect of DHA on TfR and VEGF expression in iron overload leukaemic cells as well as the inhibitory effect of DHA on proliferation of iron overload leukaemic cells have not yet been reported.Hence,the aim of the our present study was to investigate the effect of DHA on TfR expression both in iron normal and iron overload human myeloid leukemia cells and to evaluate whether or not any relationship exist between TfR expression,VEGF expression and angiogenesis.Results:1.Effect of DHA on TfR content in leukaemic cellsAfter treatment with various concentrations of DHA(2.5-10μM) for 48 h,the fluorescence intensity levels of TfR in K562 cells were reduced by 31.6%(5μM, P＜0.05) and 48.5%(10μM,P＜0.01),versus the solvent control groups.Whereas after treatment with various concentrations of DHA(0.25-1μM) for 48 h,the fluorescence intensity levels of TfR in HL60 cells was reduced by 33.2%(1μM,P＜0.01),versus the solvent control groups.Moreover,cells pretreated with 20nM holotransferrin 1 h prior to administration of 10μM DHA showed significantly reduction of the fluorescence intensity levels of TfR in K562 cells,by 30.2%(P＜0.05),as compared to iron normal cells treated DHA with same concentration.2.Effect of DHA on TfR expression in leukaemic cellsWestern blotting showed that untreated cells expressed high levels of TfR protein whereas in cells treated with DHA,TfR protein expression was down-regulated in a dose-dependent manner.Furthermore,when treated with 1μM or 10μM DHA for 0,6,12,24,48h,the expression of TfR protein in HL60 or K562 cells were also down-regulated in a time-dependent manner,respectively.Consistent with the result of flow cytometry analysis,cells pretreated with 20nM holotransferrin 1 h prior to administration of 10μM DHA showed significantly decrease of the TfR protein expression in K562 cells,by 28.1%(P＜0.01),as compared to iron normal cells treated DHA with same concentration.Furthermore,to investigate whether DHA could regulate TfR expression at the post-transcriptional level,the level of mRNA for TfR was tested using a RT-PCR assay.DHA significantly decreased the mRNA level of TfR in K562 cells by 22.3%(10μM,P＜0.01),versus the solvent control groups. Whereas cells pretreated with 20nM holotransferrin 1 h prior to administration of 10μM DHA showed significantly suppression of the mRNA levels of TfR in K562 cells, by 26.1%(P＜0.05),as compared to iron normal cells treated DHA with same concentration.3.Result of iron level by atomic absorption spectrophotometric analysesAfter the treatment of iron normal K562 cells with 2.5,5,and 10μM DHA for 48 h,Iron content of K562 cells was diminished by DHA in a dose-dependent manner. In detail,DHA at a low concentration(5μM) effectively reduced iron content of K562 cells by 48.3%(P＜0.05),compared to solvent control groups.However,after pretreatment of K562 cells with 20 nM holotransferrin,DHA exerted more potent effect than in iron normal conditions.In detail,the iron content was decreased 27.7% (P＜0.05) by 10μM DHA in iron overload K562 cells,as compared to iron normal cells treated DHA with same concentration.4.Effect of DHA on VEGF expression in iron overload K562 cellsRT-PCR showed that 10μM DHA significantly decreased the mRNA level of VEGF in K562 cells by 46.1%(10μM,P＜0.01),versus the solvent control groups. However,after treated with 20 nM holotransferrin,the mRNA levels of VEGF in iron overload K562 cells reduced by 16.4%,as compared to iron normal cells treated DHA with same concentration,but no obvious significance was observed(P＞0.05).Western blotting showed that DHA significantly reduced the VEGF expression in iron overload K562 cells,by 36.3%(P＜0.01),compared to iron normal cells treated with 10μM DHA.ELISA analysis was performed to determine the amount of secreted VEGF protein in conditioned media.Compared to solvent control groups,K562 cells pretreated with 5μM DHA showed a 30.6%decrease of the secreted VEGF level (P＜0.01).Whereas cells pretreated with 20nM holotransferrin 1 h prior to administration of 10μM DHA showed significantly reduction of the secreted VEGF level in K562 cells,by 68.7%(P＜0.001),as compared to iron normal cells treated DHA with same concentration.Furthermore,the in vivo effects of DHA were investigated in an angiogenesis model of chorioallantoic membrane of the chick embryo(CAM).Compared with CM from solvent control groups,the number of microvessels stimulated by CM from K562 cells pretreated with 2.5,5,and 10μM DHA was decreased by 28.2,44.2 and 56.7%,respectively.Whereas cells pretreated with 20nM holotransferrin 1 h prior to administration of DHA(5,10μM/5μl per egg) showed even more significantly suppression of new vessel growth(P＜0.05),as compared to iron normal cells treated DHA with same concentrations.5.Effect of DHA on K562 cell proliferation in iron normal and iron overload conditionsDHA reduced HL60 and K562 cells survival in a dose-dependent manner.The IC50 values on HL60 and K562 cell lines were 1.74μM and 11.33μM,and their 95% confidence interval were 0.86-2.60μM and 9.38-13.68μM.To further determine whether DHA was capable of inhibiting cell proliferation,trypan blue exclusion assays using an inverted phase-contrast microscope were performed on HL60 and K562 cells.Concentrations of DHA from 0.25-1μM or 2.5-10μM were tested and shown to suppress the growth of HL60 and K562 cells significantly in a time-dependent fashion.Since DHA becomes more cytotoxic in the presence of ferrous iron,we first investigated the effect of holotransferrin on the effect of K562 cells proliferation.The result showed that holotransferrin promoted the cell growth at maximal levels of approximately 20 nM Then we detected the effect of holotransferrin on the sensitization effect of DHA on K562 cells.The iron overload K562 cell lines showed even low IC50 values(3.67μM),and its 95%confidence interval was 3.20-4.21μM. And consistent result was found in trypan blue exclusion assays,the growth inhibitory activity of the DHA was also increased by pretreating holotransferrin.6.Correlation between TfR expression and VEGF expressionIron and its regulated factors such as TfR may have intricate interplay in leukaemic cells progression.Hence,to understand the relative significance of TtR,we examined its correlation with another leukaemic cell growth promoting factor VEGF. In iron normal and iron overload K562 cells,high level of TfR protein expression positively correlated with VEGF protein expression(r=0.902,P＜0.01).Consistent result was found in RT-PCR assay,level of TfR mRNA expression also positively correlated with VEGF mRNA expression(r=0.671,P＜0.05).In addition,the ELISA assay showed that there is a positive correlation exist between TfR expression and VEGF secretion(r=0.846,P＜0.01).Further,we assessed the relationship between TfR expression and angiogenesis detected by CAM.High expression of TfR protein and mRNA expression positively correlated with angiogenesis(r=0.916,P＜0.01;r=0.860, P＜0.01,respectively).Conclusion:Above all,the results of the current study demonstrate that DHA decreased cell growth of K562 cells more striking by raising the iron concentration,and it may raise the attractive possibility that leukaemic cells that express more TfR than normal cells are preferentially affected by the DHA/holotransferrin treatment.Moreover,a positive correlation exist between TfR expression,VEGF expression and angiogenesis in K562 cells was observed.This is the first study showing a significant association between TfR expression and abundant expression of VEGF in chronic myeloid leukemia.As there is a general agreement that molecules interfering simultaneously with multiple targets might be more effective than single target agents,the positive correlation exist between TfR expression,VEGF expression and angiogenesis might be a clue for future leukemia treatment strategies.