Effects Of Iron On The Mitochondrion-mediated Apoptotic Cell Death In Human Leukemia Hl-60 Cells

Iron is an essential element for vital cells to proliferate and to maintain their function and activities. It has already been reported that rich iron can inhibit apoptosis and accelerate tumor cells' growth by certain mechanism , and that, in the contrary, iron-deprivation can restrain the synthesis of nuclear DNA and activate cell apoptotic procedure. This suggests that iron is in a close relation to the occurrenc and development of neoplasm .Recently it has been discoved that the passway of mitochondrion-mediated cell death is the chief cellular apoptotic way. During apoptotic process, it has been detected that mitochondrial membrane potential ( Δ Ψ m) descended or even lost, and then apoptotic factors, such as cytochrome C (Cyt c), Smac/Diable and apoptosis inducing factor (AIF), were released from mitochondrion, which activated Caspase enzyme cascade reaction to lead to structural protein and nuclear DNA being splitted . Ultimately, apoptosis was performed. At present, there were few reports regarding the mechanism of the influence of iron on mitochondrion-mediated apoptosis in human Leukemia cell.In our study, human Leukemia HL-60 cells were used as object, and after being incubated with deferoxamine(DFO) and iron sesquichloride(FeCl3) respectively for some time, the chang of the markers correlating mitochondrion-mediated apoptotic way was observed. Atthe same time, we discussed the influence of iron on the apoptotic process, tring to make clear the mechanism of how iron affecting human Leukemia HL-60 cells'apoptotic process, and tring to provide experimental evidence and theory for clinic reasonably to adopt iron chelator dose or iron dose to treat Leukemia and other neoplasm.Method: HL-60 cells were co-cultured with different concentration of DFO and FeCl3 for certain time, resulting in status of intracellular iron deprivation or rich iron .Then we detected the cell vital force by MTT method, observed morphologic chang of apoptotic cell with phase contrast microscope, examined apoptotic cell ratio by TUNEL assay and flow cytometer(FCM), measured mitochondrial membrane potential by FCM after Rhodamine-123 (Rh-123)staining cells, investigated the variance of Caspase 3's expression and activity and of transcriptional level of apoptotic gene —bax by methods of immunocytochemistry and hybridization in situ. The study groups were divided as following: ICh 50> 10(h 50QMmol/L DFO groups and l(k lOQumol/L FeCb groups, and the control group, which was added the same volum saline, when HL-60 cells were incubated with different concentrations of DFO at 241u 481k 72h, APO%was higher than that of control group (P<0.05) at each point, especially at the concentration of lOQamol/L DFO.Results: (1) the results of cell vital force: after HL-60 cells being treated with aniso-concentration DFO, the values of cell vital force detected by MTT method showed a descending tendency in a time and dosage-dependent manner. With the culture time prolonging, cell vital force decreased because of the dead cell number increasing, especially at the concentration of lOQamol/LN 500fimo\/h DFO groups (/?<0.01); contrarily, cell vital force of FeCb groups showed an increasing tendency. At the same time point, FeCli—100 group's rised more markedly than the control's(/?<0.01). (2) the results of cell apoptosis percentage (APO%): the results of both TUNEL assay and FCM confirmed that DFO could induce HL-60 cells cultured in vitro apoptosis in a time and concentration-dependent manner. At the same time, classic character of morphologic changs of apoptotic cell was observed with phase contrast microscope. After cells being cultured with 10 > 50 ^ 100 u mol/L DFO for 72h, the APO% were respectively (15.87+2.8)%, (20.58 + 2.53)%? (44.27+4.03)%, and 100 u mol/LDFO for 24,48.72h, the APO% were respectively (22.60+2.53)%, (30.52±2.40)%, (44.27 ±4.03)%, the difference between experimental groups and the control group was significant (p<0.01). (3) the results of changes of mitochondrial membrane potential (A Wm) : after HL-60 being incubated with DFO, FCM detected A ^m in decreasing or even in losing, moreover, the decreasing degree was dependent on time and dosage. The effect of 100 u mol/L DFO group was more obvious, cells incubated with 100 u mol/L DFO for 12, 24, 48, 72h, the rates of A Wm were respectively (14.79+1.83)%, (21.28+2.44)%, (37.13 + 5.24) %, (44.77±5.63)%, being compared with that of the control, the difference was of statistic significance(p<0.01); while there was no significant difference between FeCl3 groups and the control group(p>0.05). (4) the results of Caspase 3 examination: when HL-60 cells being cultured with 100 U mol/L DFO for 24, 48, 72h, the rates of Caspase 3 expression positive cells were respectively (8.63±0.47)%, (13.67 + 1.07)%, (919.05 +1.34)%, being compared with that of the control group, the difference was of statistic significance(p<0.01); while the difference between the rates of FeCb -goups and that of the control group was of no statistic significance (p>0.05); (5) the results of the transcriptional level of apoptotic gene— bax: after 100j?mol/L DFO acted on cells for 24h, 48h, the rates of bax transcription positive cells was respectively (6.11 + 1.52)% and (11.78±2.91)%, and being compared with the control group's (2.49±0.50)% and (3.89±0.93)%, the difference was significant (U =9.704,p0.05).Conclusion:1. Iron had an obviouse influence on the proliferation and apoptosis of human Leukemia HL-60 cells cultured in vitro.2. Iron deprivation could induce apoptosis of HL-60 cells and restrain their proliferation; mitochondrion-mediated apoptotic cell death way was its possible mechanism. In addition, iron deprivation could also up-regulate the transcription of promoting apoptotic gene—bax , so to regulate the mitochondrion-mediated apoptotic cell death way upstream.3. Rich iron could promote the growth of HL-60 cells and inhibit their apoptosis, but had an little influence on the mitochondrion-mediated apoptotic cell death way.4. The confirmation of iron deprivation inducing apoptosis in Leukemia cells, provided an novel strategy for the clinical treatment of Leukemia.