title: Berberine induces neuronal differentiation through inhibition of cancer stemness and epithelial-mesenchymal transition in neuroblastoma cells.
abstract: BACKGROUND
  Berberine, a plant alkaloid, has been used since many years for treatment of gastrointestinal disorders. It also shows promising medicinal use against metabolic disorders, neurodegenerative disorders and cancer; however its efficacy in neuroblastoma (NB) is poorly explored.
  
  
  OBJECTIVE
  EMT is important in cancer stemness and metastasis resulting in failure to differentiate; thus targeting EMT and related pathways can have clinical benefits.
  
  
  METHODS
  Potential of berberine was investigated for (i) neuronal differentiation and cancer stemness inhibition, (ii) underlying molecular mechanisms regulating cancer-stemness and (iii) EMT reversal.
  
  
  METHODS
  Using neuro2a (N2a) neuroblastoma cells (NB); we investigated effect of berberine on neuronal differentiation, cancer-stemness, EMT and underlying signalling by immunofluorescence, RT-PCR, Western blot. High glucose-induced TGF-β mediated EMT model was used to test EMT reversal potential by Western blot and RT-PCR. STRING analysis was done to determine and validate functional protein-interaction networks.
  
  
  RESULTS
  We demonstrate berberine induces neuronal differentiation accompanying increased neuronal differentiation markers like MAP2, β-III tubulin and NCAM; generated neurons were viable. Berberine attenuated cancer stemness markers CD133, β-catenin, n-myc, sox2, notch2 and nestin. Berberine potentiated G0/G1 cell cycle arrest by inhibiting proliferation, cyclin dependent kinases and cyclins resulting in apoptosis through increased bax/bcl-2 ratio. Restoration of tumor suppressor proteins, p27 and p53, indicate promising anti-cancer property. The induction of NCAM and reduction in its polysialylation indicates anti-migratory potential which is supported by down regulation of MMP-2/9. It increased epithelial marker laminin and smad and increased Hsp70 levels also suggest its protective role. Molecular insights revealed that berberine regulates EMT via downregulation of PI3/Akt and Ras-Raf-ERK signalling and subsequent upregulation of p38-MAPK. TGF-β secretion from N2a cells was potentiated by high glucose and negatively regulated by berberine through modulation of TGF-β receptors II and III. Berberine reverted mesenchymal markers, vimentin and fibronectin, with restoration of epithelial marker E-cadherin, highlighting the role of berberine in reversal of EMT.
  
  
  CONCLUSIONS
  Collectively, the study demonstrates prospective use of berberine against neuroblastoma as elucidated through inhibition of fundamental characteristics of cancer stem cells: tumorigenicity and failure to differentiation and instigates reversal in the EMT.
authors.affiliation: Laboratory of Immunology, School of Biological Sciences and Biotechnology, Indian Institute of Advanced Research, Gandhinagar (382 007), Gujarat, India.
authors.forename: C R
authors.initials: CR
authors.lastname: Naveen
authors.affiliation: Laboratory of Immunology, School of Biological Sciences and Biotechnology, Indian Institute of Advanced Research, Gandhinagar (382 007), Gujarat, India.
authors.forename: Sagar
authors.initials: S
authors.lastname: Gaikwad
authors.affiliation: Laboratory of Immunology, School of Biological Sciences and Biotechnology, Indian Institute of Advanced Research, Gandhinagar (382 007), Gujarat, India.. Electronic address: reena@iiar.res.in.
authors.forename: Reena
authors.initials: R
authors.lastname: Agrawal-Rajput
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001599:Berberine / Q000494:pharmacology*; D014408:Biomarkers, Tumor / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D051379:Mice; D014411:Neoplastic Stem Cells / Q000187:drug effects*; D009447:Neuroblastoma / Q000188:drug therapy* / Q000473:pathology; D009474:Neurons / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D014408:Biomarkers, Tumor; D016212:Transforming Growth Factor beta; D001599:Berberine
keywords: Berberine; Cancer stemness; Cell cycle; Differentiation; EMT; Neuroblastoma