| Background and Objective of the study:Esterase play an important role in the substance metabolism and transport, cell signaling and gene expression in vivo.There are a series of non-specific esterases which can be inhibited by sodium fluoride (NaF) in monouclear cells. At present, It is reported by the domestic and foreign scholars that various systems of the body are affected by fluoride. Some study s have concluded that fluoride could induce apoptosis. In this study,we treated human mononuclear leukemia THP-1cells with NaF, and observed the inhibtion of a-naphthol acetate esterase activity and the induction of apoptosis.We also investigated the mechanisms underlying the induction of apoptosis,with a particular focus on the expression of Bcl-2and Bax and the activation of a-NAE.It will provide a new idea for targeted therapy of acute monocytic leukemia.Methods:(1) Human acute monocytic leukemia THP-1cells were cultured with varying concentrations of NaF (0、0.5、1、2、4、8mM),0mM as a control group.(2) Wright’s stain was used to observe apoptosis morphology of THP-1cells treated with NaF.(3) a-naphthol acetate esterase staining and chromogenic method were used to detect the activation of a-NAE qualitatively and quantitatively.(4) Proliferation of the THP-1cells with NaF were detected by the method of MTT assay.(5) Flow cytometry was used to detect apoptosis of THP-1cells.(6)RT-PCR was used for investigating the expression of apoptosis-related genes such as Bax and Bcl-2.Results:(1)Morphology:The cultured cells of control and experimental group were observed by inverted phase contrast microscope.The morphology of the control group cells showed that:the cells were grown in suspension,cell morphology was round, the size was uniform,the cell body was refractive and the background was clear.After being treated with NaF for24h, the morphology of cells changed:cell body shrinkaged, the cell size was of variety, the membrane damaged, finally,the cells dissolved,the background was turbid. Wrighe stain:The cells, incubated with NaF for24h, showed a typical apoptotic morphology with different forms, membrane fragmentation, coarse edges, concentrated cytoplasm, chromatin condensation, karyopyknosis and apoptotic bodies emerged.(2) the activation of a-NAE:After treated by NaF for24h, the cells observed by a-NAE stain, showed that:with the increase of NaF concentration, the a-NAE reaction positive intensity and positive rate decreased(P<0.05). The results of chromogenic method were accord with the a-NAE stain.(3)MTT:At lower concentration(0.5mM or1mM),no significant inhibition of cell proliferation was observed in response to NaF treatment. However, at higher concentrations(2-8mM), NaF inhbit cell proliferation in a time-, concentration-dependent manner(P<0.05). The24h,48h,72h IC50of NaF on THP-1cell were4mM,2.75mM,2.4mM.(4)The results of apoptosis rate:NaF can induce THP-1cell apoptosis. With the increase of concentration of NaF, the rates of apoptosis increase, and the apoptosis rates of each experimental group was significant different with control group except the group of0.5mM and1mM(P<0.05).(5)RT-PCR:In the presence of2-8mM NaF for24h, human THP-1cells showed a down-regulation of Bcl-2and up-regulation of Bax (P<0.05).Conclutions:(1)NaF can inhibit THP-1cells proliferation and induce THP-1cells apoptosis in a concentration-dependent manner at concentrations of2-8mM,maybe due to down-regulation of Bcl-2and up-regulation of Bax.(2) The apoptosis mediated by NaF in THP-1cells was accompanied by a decline in the activation of a-NAE, suggesting that NaF specifically inhibiting the activation of a-NAE may have played a certain role in the apoptosis; It will provide a new idea for targeted therapy of acute monocytic leukemia. |
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