(A) The iron concentration in DFO-treated or untreated Hs578T cells with 5?g/ml DMT1, TfR1 neutralizing antibody and both was measured by using ICP-MS. and DMT1 expressed on cell Ketorolac membrane were involved in high iron uptake in TNBCs under DFO-induced iron deficient condition. For the possible regulatory mechanism, we found that DFO treatment could promote a high expression level of IL-6 in aggressive MDA-MB-231 cells. The activated IL-6/PI3K/AKT pathway upregulated the expression of iron-uptake related proteins, TfR1 and DMT1, leading to increased iron uptakes. Conclusion: We demonstrated that DFO could upregulate expression of TfR1 and DMT1 , which enhanced?iron uptake via activating?IL-6/PI3K/AKT signaling pathway in aggressive TNBCs. IL-6/PI3K/AKT pathway after DFO treatment, thus we suggested that both IRP1 and IRP2 responded to DFO-induced iron deficiency in mediating the regulation of DMT1 and TfR1.20,39 It is noteworthy that iron metabolism pathways are closely related to inflammatory stressors.23 Pro-inflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor- (TNF-) or IL-6 influence the posttranscriptional control of iron homeostasis by modulating the binding affinity of IRP1 and IRP2 to in human monocytic cells and neuron cells.40C42 However, the role of IL-6 in mediating iron uptake in tumor cells remained to be elucidated. Under the iron deficient condition induced by DFO, triple-negative MDA-MB-231 cells were triggered to up-regulate the expression level of IL-6, but the situation in ER-positive MCF-7 cells was just on the contrary. As an inflammatory cytokine, IL-6 is higher expressed in highly aggressive TNBCs, whereas is almost not expressed in non-aggressive Ketorolac ER-positive breast cancer cells.34,35 Meanwhile, IL-6 were associated with iron homeostasis.43,44 After DFO treatment, the activation of IL-6/PI3K/AKT pathway led to increase expression of IRP1 and IRP2 in MDA-MB-231 cells. IRPs regulates TfR1 and DMT1 mRNA stability, ultimately increasing protein levels of TfR1 and DMT1 to promote iron uptake in TNBC cells.38 The present results were suggested that IL-6 involved in iron uptake through the activated PI3K/AKT pathway under the iron-deficient condition induced by DFO. In this study, we suggested that both TfR1 and DMT1 were involved Alcam in increasing iron uptake in triple-negative MDA-MB-231 cells under DFO-induced iron-deficient condition, but the intracellular iron transport and iron storage remained unsolved. The further studies were in process to elucidate the route of the intracellular iron transport, and intracellular iron storage in aggressive TNBCs under the iron-deficient condition induced by DFO. Collectively, our study suggested that aggressive TNBCs exhibited the activated IL-6/PI3K/AKT signaling to up-regulate the expression of TfR1 and DMT1 leading to increased iron uptake under the iron-deficient condition induced by DFO. Our study also suggested that when DFO was applied to treat breast cancer cells, it should be considered that DFO has different effects on iron metabolism in breast cancer cells with different phenotype leading to distinct biological outcomes. Acknowledgments This work was supported by the National Natural Science Foundation of China (U1532116 and 81571729;), Ketorolac the National Key Research and Development Program (2016YFC0106201;), and the Shanghai Science and Technology Commission of Shanghai Municipality (11DZ2211000). Disclosure The authors report no conflicts of interest in this work. Supplementary materials Open in a separate window Figure S1 Effects of DFO treatment on the expression of iron-uptake and iron-storage proteins in Hs578T and BT549 cell lines. (A) Hs578T cells were treated with or without 200?M DFO for 24?h. Proteins from cell lysates were analyzed using Western blotting. (B) TfR1 and Ketorolac DMT1 on cell membrane were detected by Western blotting. (C) BT549 cells were treated with or without 200?M DFO for 24?h. Proteins from cell lysates were analyzed using Western blotting. (D) TfR1 and DMT1 on cell membrane in BT549 cells were detected by western blotting. Western blotting quantification with anti- actin antibody: values were the means of three independent experiments SD. ** em p /em 0.01, *** em p /em 0.001. Abbreviations: DFO, deferoxamine; TfR1, transferrin receptor 1; DMT1, divalent metal transporter 1. Open in a separate window Figure S2 The expression of iron-uptake and iron-storage proteins in (left) Hs578T and (right) BT549 cell lines after 200 M DFO treatment was observed using immunofluorescence staining. * em p /em 0.05, ** em p /em 0.01, *** em p /em 0.001. Abbreviations: dFO, Deferoxamine; TfR1, transferrin receptor 1; DMT1, divalent metal transporter 1. Open in a separate window Figure S3 Effects of DFO treatment on the expression of.