The Potential Role Of Icat On Human Leukemia Cells HL-60 In NSC67657 Treatment

Objective To construct monocytic differentiation model, and to explore the effects of NSC67657 on biological behavior of HL-60 cells.To investigate the potential role of ICAT on HL-60 cells in NSC67657 treatment.Methods The experimental cells were treated by 10μmol/L NSC67657(dissolved by DMSO) for 5 days, and the control cells were treated by DMSO. Wright’s staining, α-NBE staining and NaF inhibit staining were used to identify the morphological changes of HL-60 cells. Flow Cytometry(FCM) was carried out to detect surface antigen CDl4,cell cycle and apoptosis. We used Lactate dehydrogenase(LD) detection kit to identify cellular activity.RT-PCR and Western blot were used to investigate the mRNA and protein expressions of ICAT, β-catenin, TCF-4 and Wnt signaling pathway’s target genes(c-myc, cyclinD1, TCF-1). Immunofluorescence(IF) was used to analysis location of β-catenin and TCF-4 in HL-60 cells.Result The monocytic differentiation model was successfully constructed. The result of Wright’s staining showed that the cell morphology was changed in experimental cells. The ratio of nucleus and cytoplasm was obviously decreased. Cellular nucleolus reduced or disappeared. The nucleus presented circular or irregular shape. The result of α-NBE staining showed that cytoplasm had red solids precipitation in experimental cells. At the same time, the red solids precipitation can be inhibited by NaF, and the inhibition rate >50%. There had no change in control cells. The number of CD14+ HL-60 cells reached(94.37±2.84)%.It was significantly much more than that of the control group[(1.31±0.09)%](P<0.01).The cell cycle was arrest at G1/G0 phase with a percentage of( 76.46±2.83) %,significant higher than that of the control group [(59.4±5.42)%](P<0.05). At the same time, The number of HL-60 cells at S phase(18.76±0.98)% was significant lower than the control group(34.38±2.61)%(P<0.05). The cell apoptosis and LD expression level had no significant difference(P>0.05).By RT-PCR and Western Blot we found that the mRNA and protein level of ICAT were up-regulated(P<0.01), β-catenin was down-regulated at the level of mRNA, total protein, and nucleoprotein(P<0.01), TCF-4 expression level of mRNA, total protein and nucleoprotein had no obvious change(P>0.05). Immunofluorescence staining showed that β-catenin protein was decreased in nuclear. The location of TCF-4 had no obviously change. By RT-PCR and Western Blot we found that the mRNA and protein levels of the Wnt signaling pathway’s target genes(c-myc, cyclinD1 and TCF-1) were down-regulated(P<0.05).Conclusion NSC67657 could induce HL-60 cells to differentiate into monocytes without toxic side effect. It inhibited cell proliferation while had no effect on apoptosis. ICAT expression was up-regulated, β-catenin and Wnt signaling pathway’s downstream genes expressions were downregulated, suggesting that ICAT may be involved in the process of HL-60 cells monocyte differentiation through inhibiting Wnt/β-catenin signaling pathway.