Oxidant-induced autophagy and ferritin degradation contribute to epithelial–mesenchymal transition through lysosomal iron

Abstract

PurposeTransforming growth factor (TGF)-β1 triggers epithelial–mesenchymal transition (EMT) through autophagy, which is partly driven by reactive oxygen species (ROS). The aim of this study was to determine whether leaking lysosomes and enhanced degradation of H-ferritin could be involved in EMT and whether it could be possible to prevent EMT by iron chelation targeting of the lysosome.Materials and methodsEMT, H-ferritin, and autophagy were evaluated in TGF-β1-stimulated A549 human lung epithelial cells cultured in vitro using Western blotting, with the additional morphological assessment of EMT. By using immunofluorescence and flow cytometry, lysosomes and ROS were assessed by acridine orange and 6-carboxy-2′,7′-dichlorodihydrofluorescein acetate assays, respectively.ResultsTGF-β1-stimulated cells demonstrated a loss of H-ferritin, which was prevented by the antioxidant N-acetyl-L-cysteine (NAC) and inhibitors of lysosomal degradation. TGF-β1 stimulation generated ROS and autophagosome formation and led to EMT, which was further promoted by the additional ROS-generating cytokine, tumor necrosis factor-α. Lysosomes of TGF-β1-stimulated cells were sensitized to oxidants but also completely protected by lysosomal loading with dextran-bound deferoxamine (DFO). Autophagy and EMT were prevented by NAC, DFO, and inhibitors of autophagy and lysosomal degradation.ConclusionThe findings of this study support the role of enhanced autophagic degradation of H-ferritin as a mechanism for increasing the vulnerability of lysosomes to iron-driven oxidant injury that triggers further autophagy during EMT. This study proposes that lysosomal leakage is a novel pathway of TGF-β1-induced EMT that may be prevented by iron-chelating drugs that target the lysosome.

DpdtC-Induced EMT Inhibition in MGC-803 Cells Was Partly through Ferritinophagy-Mediated ROS/p53 Pathway

“…Normally, NCOA4 acts as a transcriptional coactivator of several nuclear receptors, and its additional function was recently identified as an inhibitor in the activation of DNA replication origins [48]; the downregulation of snail, slug, and vimentin in DpdtCtreated cells might be due to the upregulation of NCOA4. In addition, it has been shown that ROS involved TGF-β1induced EMT, and the decrease of ferritin heavy chain (FHC) contributed to EMT transition [49][50][51]. However, the ferritin degradation induced by DpdtC achieved EMT inhibition in our study; the difference might be attributed to a difference in manner of ferritin downregulation achieved; the knockdown of ferritin by sh-RNA was at translation level, while induced ferritinophagy by DpdtC at posttranslation modification.…”

Laser-captured spinal cord motorneurons from ALS subjects show increased gene expression in vacuolar ATPase networks

“…Recent studies in non-CNS cells have demonstrated that EMT can occur with activation of autophagy stimulated by reactive oxygen species [48]. We found that expression of two EMT markers were increased in ALS motor neurons, vimentin (ALS/CTL = 2.6) and N-cadherin (CDH2, ALS/CTL = 2.9).…”

“…In terms of additional investigations, it might be worthwhile to pursue the possibility that the "prolonged autophagic death" described by Martin [49] and increased autophagy markers in sporadic ALS MN's described by Sasaki [45] might arise as a result of oxidative stress-driven autophagy [48] through activation of epithelial-mesenchymal transition (EMT) of motor neurons. EMT is not known to occur normally in non-malignant tissues except during developmental neuronal polarization and migration [50], amniotic membrane rupture [51], organ fibrosis [52] and cardiac cells in culture [53].…”

Conservative and Atypical Ferritins of Sponges

“…Ferritin synthesis is stimulated during the development and cell differentiation, inflammation, and tumorigenesis. A decrease in H-ferritin can induce epithelial-to-mesenchymal transition of mammalian tumor cells through the TGF-β1 pathway [12][13][14]. Hypoxia inducible factor A (HIFA) can directly bind with hypoxia response element (HRE) in the promoter region of human L-ferritin (HuLF) to enhance its expression thus regulating epithelial-to-mesenchymal transition of glioma [15].…”