G-CSF Upregulates the Expression of Aquaporin-9 through CEBPB to Enhance the Cytotoxic Activity of Arsenic Trioxide to Acute Myeloid Leukemia Cells

Background: Arsenic trioxide (ATO) is widely used for acute promyelocytic leukemia (APL) treatment, but it can’t show satisfactory efficacy in non-APL leukemia. Aquaporin-9 (AQP9), a transmembrane transporter, is required for ATO uptake. Acute myeloid leukemia (AML) other than APL cells express relatively lower levels of AQP9, which limits the cytotoxic activity of ATO in those types of leukemia cells. Recently, we found that granulocyte colony stimulating factor (G-CSF) can upregulate the expression of AQP9. We hypothesized that the combination of G-CSF with ATO may enhance the antitumor effect of ATO in non-APL leukemia cells. We also aimed to elucidate the underlying mechanisms by which G-CSF upregulates the expression of AQP9. Methods: : AML cell lines including THP-1 and HL-60 were pretreated with or without G-CSF (100ng/ml) for 24 hours, followed by the treatment with ATO (2μM) for 48 hours. Cell morphology was observed under microscope after Wright-Giemsa staining. Flow cytometry was performed to evaluate the cell apoptosis levels. The intracellular concentrations of ATO were determined by the atomic fluorescence spectrometry. The mRNA and protein expression were respectively measured by quantitative reverse transcription PCR (RT-qRCR) and western blotting. Target genes were knocked down by transfection with small interfering RNA (siRNA), or overexpressed by transfection with overexpression plasmids. The cell line derived xenograft mouse model was established to confirm the results of the in vitro experiments. Results: : Compared with using ATO alone, the combination of ATO with G-CSF induced apoptosis in AML cells more dramatically. G-CSF upregulated the expression of AQP9 and enhanced the intracellular concentrations of ATO in AML cells. When AQP9 was overexpressed, it markedly enhanced the cytotoxic activity of ATO. On the other hand, when AQP9 was knocked down, it dramatically attenuated the combinational effect. Moreover, we found that the upregulation of AQP9 by G-CSF depends on the transcription factor CCAAT enhancer binding protein beta (CEBPB). We also demonstrated that ATO and G-CSF combination significantly inhibited the tumor growth in xenograft mouse model. Conclusions: : ATO and G-CSF combination may be a potential therapeutic strategy for AML patients.