title: Multi-walled carbon nanotubes directly induce epithelial-mesenchymal transition in human bronchial epithelial cells via the TGF-β-mediated Akt/GSK-3β/SNAIL-1 signalling pathway.
abstract: Multi-walled carbon nanotubes (MWCNT) are currently under intense toxicological investigation due to concern on their potential health effects. Current in vitro and in vivo data indicate that MWCNT exposure is strongly associated with lung toxicity (inflammation, fibrosis, granuloma, cancer and airway injury) and their effects might be comparable to asbestos-induced carcinogenesis. Although fibrosis is a multi-origin disease, epithelial-mesenchymal transition (EMT) is recently recognized as an important pathway in cell transformation. It is known that MWCNT exposure induces EMT through the activation of the TGF-β/Smad signalling pathway thus promoting pulmonary fibrosis, but the molecular mechanisms involved are not fully understood. In the present work we propose a new mechanism involving a TGF-β-mediated signalling pathway.
  
  
  
  Human bronchial epithelial cells were incubated with two different MWCNT samples at various concentrations for up to 96 h and several markers of EMT were investigated. Quantitative real time PCR, western blot, immunofluorescent staining and gelatin zymographies were performed to detect the marker protein alterations. ELISA was performed to evaluate TGF-β production. Experiments with neutralizing anti-TGF-β antibody, specific inhibitors of GSK-3β and Akt and siRNA were carried out in order to confirm their involvement in MWCNT-induced EMT. In vivo experiments of pharyngeal aspiration in C57BL/6 mice were also performed. Data were analyzed by a one-way ANOVA with Tukey's post-hoc test.
  
  
  
  Fully characterized MWCNT (mean length < 5 μm) are able to induce EMT in an in vitro human model (BEAS-2B cells) after long-term incubation at sub-cytotoxic concentrations. MWCNT stimulate TGF-β secretion, Akt activation and GSK-3β inhibition, which induces nuclear accumulation of SNAIL-1 and its transcriptional activity, thus contributing to switch on the EMT program. Moreover, a significant increment of nuclear β-catenin - due to E-cadherin repression and following translocation to nucleus - likely reinforces signalling for EMT promotion. In vivo results supported the occurrence of pulmonary fibrosis following MWCNT exposure.
  
  
  
  We demonstrate a new molecular mechanism of MWCNT-mediated EMT, which is Smad-independent and involves TGF-β and its intracellular effectors Akt/GSK-3β that activate the SNAIL-1 signalling pathway. This finding suggests potential novel targets in the development of therapeutic and preventive approaches.
authors.affiliation: Department of Oncology, University of Turin, via Santena 5/bis, 10126, Turin, Italy.|Interdepartmental Centre Scansetti for Studies on Asbestos and Other Toxic Particulates, University of Turin, Turin, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Polimeni
authors.affiliation: Department of Oncology, University of Turin, via Santena 5/bis, 10126, Turin, Italy.|Interdepartmental Centre Scansetti for Studies on Asbestos and Other Toxic Particulates, University of Turin, Turin, Italy.
authors.forename: Giulia Rossana
authors.initials: GR
authors.lastname: Gulino
authors.affiliation: Department of Oncology, University of Turin, via Santena 5/bis, 10126, Turin, Italy.|Interdepartmental Centre Scansetti for Studies on Asbestos and Other Toxic Particulates, University of Turin, Turin, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Gazzano
authors.affiliation: Department of Oncology, University of Turin, via Santena 5/bis, 10126, Turin, Italy.
authors.forename: Joanna
authors.initials: J
authors.lastname: Kopecka
authors.affiliation: Interdepartmental Centre Scansetti for Studies on Asbestos and Other Toxic Particulates, University of Turin, Turin, Italy.|Department of Chemistry, University of Turin, via Pietro Giuria 7, 10125, Turin, Italy.
authors.forename: Arianna
authors.initials: A
authors.lastname: Marucco
authors.affiliation: Interdepartmental Centre Scansetti for Studies on Asbestos and Other Toxic Particulates, University of Turin, Turin, Italy.|Department of Chemistry, University of Turin, via Pietro Giuria 7, 10125, Turin, Italy.|NIS - Nanostructured Interfaces and Surfaces, University of Turin, via Pietro Giuria 7, 10125, Turin, Italy.
authors.forename: Ivana
authors.initials: I
authors.lastname: Fenoglio
authors.affiliation: Department of Chemistry, University of Turin, via Pietro Giuria 7, 10125, Turin, Italy.|NIS - Nanostructured Interfaces and Surfaces, University of Turin, via Pietro Giuria 7, 10125, Turin, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Cesano
authors.affiliation: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Campagnolo
authors.affiliation: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Magrini
authors.affiliation: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Pietroiusti
authors.affiliation: Department of Oncology, University of Turin, via Santena 5/bis, 10126, Turin, Italy.|Interdepartmental Centre Scansetti for Studies on Asbestos and Other Toxic Particulates, University of Turin, Turin, Italy.
authors.forename: Dario
authors.initials: D
authors.lastname: Ghigo
authors.affiliation: Department of Oncology, University of Turin, via Santena 5/bis, 10126, Turin, Italy. elisabetta.aldieri@unito.it.|Interdepartmental Centre Scansetti for Studies on Asbestos and Other Toxic Particulates, University of Turin, Turin, Italy. elisabetta.aldieri@unito.it.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Aldieri
mesh_terms: D000818:Animals; D001980:Bronchi / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D015197:Carcinogenicity Tests; D002460:Cell Line; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D000071679:Glycogen Synthase Kinase 3 beta / Q000378:metabolism; D006801:Humans; D019570:Inhalation Exposure / Q000009:adverse effects; D008297:Male; D008810:Mice, Inbred C57BL; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D037742:Nanotubes, Carbon / Q000737:chemistry / Q000633:toxicity* / Q000648:ultrastructure; D010316:Particle Size; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011658:Pulmonary Fibrosis / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D015398:Signal Transduction / Q000187:drug effects*; D000071250:Snail Family Transcription Factors / Q000378:metabolism; D013499:Surface Properties; D016212:Transforming Growth Factor beta / Q000819:agonists* / Q000378:metabolism
chemical_list: D037742:Nanotubes, Carbon; C000604407:SNAI1 protein, human; D000071250:Snail Family Transcription Factors; D016212:Transforming Growth Factor beta; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D051057:Proto-Oncogene Proteins c-akt
keywords: Carbon nanotubes hazard; Epithelial cells; Epithelial-mesenchymal transition; Fibrogenic potential; Lung fibrosis; SNAIL-1 signalling pathway; TGF-β