The Effect Of Iron-deprivation On Related Genes Expression Of Apoptosis And Caspase-3 In Leukemia Cells

Iron is required for the all live cells, including tumor cells. The number and diversity of enzymes that contain or depend on the presence of iron for activity is extensive in human body. A few research found that iron was associated with tumour, the iron-depletion can inhibite tumor growth and induce apoptosis of tumor cells. Expression of multiple cell cycle-regulatory and apoptosis-related genes were modulated by cellular iron status. Recent studies have proved that both occurrence of hematopoietic malignancies and treatment of chemotherapeutic drugs are associated with apoptosis of tumor cells. Apoptosis is an active cell death form that is strictly controlled by genes. Although some studies have suggested that iron can affect the process of tumor cells apoptosis, the molecular mechanisms remain unclear. To investigate the molecular basis, we evaluated expression of genes involved in the regulation of cell apoptosis and caspase-3 activity. So as to elucidate the mechanisms of leukemia occurrence and development and to provide new rationale for further studies of iron-deprivation as a form of leukemiatreatment.Methods HL-60 and K562 cells were incubated at 37℃ in RPMI 1640 containing 10% heat-inactived fetal bovine serum in an water-saturated 5% CO₂ incubator.We used exponentially growing HL-60 and K562 cells (1 × 10⁶/ml) in experiment. The study groups were divided as following: DFO group, iron+DFO and control group. Following indices were detected which included viability by typanblue, apoptosis by morphological study, flow cytometry (FCM) assay, expression of rb, c-myc, bax, p21 by RT-PCR, caspase 3 activity was detected by melorimetry. The intracellular LIP was measured with a fluorimetric assay using the metalsensitive probe calcein-AM.Results (1) When HL-60 cells were incubated with different concentrations of DFO, viability assay was lower than that of control group at 12h, 24h and 48h (P<0.05). (2) cells were incubated with different concentration of DFO, APO% showed dose-time-dependent and was much higher than that of control group (P<0.01). Apoptotic peak appeared slowly even if at higher concentration of DFO group. Compared with that of control group, there was significant difference (P<0.01). FCM assay showed that apoptosis of cells occurred mainly in the S-spase. (3) Expression of bax, c-myc, rb, p21 in HL-60 and K562 cells was higher than that of control group when cells were incubated with 50μmol/L,100μmol/L DFO 121k 24h and 48h (P<0.05). The caspase-3 activity was significantly higher in the apoptotic cells than in control cells. The correlation was a negative between cellular LIP and expression of apoptosis-related and caspase-3 activity of cells.Conclusions(1) Iron-deprivation could inhibited proliferation and induced apoptosis in leukemia cells.(2) DFO induced apoptosis of Ieukemic cells may be through upregulated expression of apoptosis-related genes and activation of caspase-3.(3) The iron deprivation could have a place in the treatment of leakemia in conjuncting with other anticancer agents.