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Archives of Toxicology

, Volume 91, Issue 2, pp 827–837 | Cite as

Epigallocatechin gallate (EGCG) inhibits adhesion and migration of neural progenitor cells in vitro

  • Marta Barenys
  • Kathrin Gassmann
  • Christine Baksmeier
  • Sabrina Heinz
  • Ingrid Reverte
  • Martin Schmuck
  • Thomas Temme
  • Farina Bendt
  • Tim-Christian Zschauer
  • Thomas Dino Rockel
  • Klaus Unfried
  • Wim Wätjen
  • Sivaraj Mohana Sundaram
  • Heike Heuer
  • Maria Teresa Colomina
  • Ellen Fritsche
In vitro Systems

Abstract

Food supplements based on herbal products are widely used during pregnancy as part of a self-care approach. The idea that such supplements are safe and healthy is deeply seated in the general population, although they do not underlie the same strict safety regulations than medical drugs. We aimed to characterize the neurodevelopmental effects of the green tea catechin epigallocatechin gallate (EGCG), which is now commercialized as high-dose food supplement. We used the “Neurosphere Assay” to study the effects and unravel underlying molecular mechanisms of EGCG treatment on human and rat neural progenitor cells (NPCs) development in vitro. EGCG alters human and rat NPC development in vitro. It disturbs migration distance, migration pattern, and nuclear density of NPCs growing as neurospheres. These functional impairments are initiated by EGCG binding to the extracellular matrix glycoprotein laminin, preventing its binding to β1-integrin subunits, thereby prohibiting cell adhesion and resulting in altered glia alignment and decreased number of migrating young neurons. Our data raise a concern on the intake of high-dose EGCG food supplements during pregnancy and highlight the need of an in vivo characterization of the effects of high-dose EGCG exposure during neurodevelopment.

Keywords

Food supplements Extracellular matrix Adhesion Migration Neurospheres DNT 

Notes

Acknowledgments

M.B. received a Leibniz—German Academic Exchange Service (DAAD) Research Fellowship. K.G. was supported by the Deutsche Forschungsgemeinschaft (DFG—GRK1427). This work was supported by the German Ministry of Education and Research (BMBF Grant 16V0899).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

204_2016_1709_MOESM1_ESM.docx (21 kb)
Supplementary material 1 (DOCX 21 kb)
204_2016_1709_MOESM2_ESM.tif (797 kb)
Online Resource 2: Lack of significant effects of EGCG on proliferation and intracellular signaling in NPCs and lack of significant effects of integrin-β4 antibody on hNPC migration. (a) Proliferation of rat and human neurospheres cultured with increasing concentrations of EGCG was measured by means of diameter increase (black circles; 7 d in culture) or BrdU incorporation (blue triangles; 3 d in culture), in six neurospheres/concentration in three (diameter increase) and seven (BrdU incorporation) independent experiments. Results are presented as mean ± SEM in % of solvent control (DMSO 0.1%) and fitted to a nonlinear sigmoidal concentration–response curve with GraphPad Prism software. Data were analyzed with one-way ANOVA and Bonferroni’s post hoc test. (b) Human neurospheres were exposed to increasing concentrations of trolox and vitamin C for 48 h. Data represent mean migration in % control ± SEM of three independent experiments (with five spheres/condition each). Statistical analysis: one-way ANOVA and Bonferroni’s post hoc test. (c) Human neurospheres were exposed to 10 µM EGCG or solvent control (DMSO 0.1%) for 24 h. Whole cell lysates were analyzed for ERK1/2 activation by Western blot. P-ERK1/2 analyses were normalized to GAPDH expression and expressed relative to control levels as mean ± SEM. of three independent experiments. (d and e) Human neurospheres were exposed to increasing concentrations of integrin-β4 antibody or isotype control antibody, RGD or RAD as control peptide, for 1 h prior differentiation and during differentiation on laminin-coated slides for further 48 h (five neurospheres/concentration). Data represent the mean ± SEM of at least three independent experiments as % of respective control. Statistical analysis was performed with two-way ANOVA and Bonferroni post hoc test. (TIFF 797 kb)
204_2016_1709_MOESM3_ESM.tif (3.2 mb)
Online Resource 3: EGCG and structure-related catechins inhibit hNPCs adhesion to laminin. (a) Human neurospheres were mechanically dissociated and exposed for 1 h to increasing concentrations of EGCG (six wells/concentration). Representative microscopic phase-contrast images showing the remaining adherent cells after 1 h of exposure and washing step (scale bar = 100 µm). (b) Adhesion was measured by means of Alamar Blue metabolization after 1 h of exposure at increasing concentrations of EGCG and other flavonoids. Adhesion expressed in % of sc and presented as mean ± SEM of at least three independent experiments (six wells/concentration). *: p<0.05 versus respective sc; #: p<0.05 versus respective EGCG concentration. EGCG: epigallocatechin gallate; ECG: epicatechin gallate; EGC: epigallocatechin; EC: epicatechin; H: hesperetin; K: kaempherol. (c) Human neurospheres were exposed to increasing concentrations of EGCG for 18 h. Representative images of four time points of a neurosphere exposed to 10 µM EGCG showing cell movement (black arrows). See Online Resource 4 movie covering the complete incubation period (scale bar = 200 µm). (TIFF 3238 kb)
204_2016_1709_MOESM4_ESM.avi (3.9 mb)
Online Resource 4: Representative movie of a human neurosphere cultured for 18 h under differentiation conditions in the presence of EGCG (10 µM) showing NPC motility. (AVI 3994 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Marta Barenys
    • 1
  • Kathrin Gassmann
    • 1
  • Christine Baksmeier
    • 1
  • Sabrina Heinz
    • 1
  • Ingrid Reverte
    • 2
  • Martin Schmuck
    • 1
  • Thomas Temme
    • 1
  • Farina Bendt
    • 1
  • Tim-Christian Zschauer
    • 1
  • Thomas Dino Rockel
    • 1
  • Klaus Unfried
    • 1
  • Wim Wätjen
    • 3
  • Sivaraj Mohana Sundaram
    • 1
  • Heike Heuer
    • 1
  • Maria Teresa Colomina
    • 2
  • Ellen Fritsche
    • 1
  1. 1.IUF – Leibniz Research Institute for Environmental MedicineDüsseldorfGermany
  2. 2.Laboratory of Toxicology and Environmental Health/NEUROLAB, Department of PsychologyUniversitat Rovira i VirgiliTarragonaSpain
  3. 3.Institute of Agricultural and Nutritional SciencesMartin-Luther-Universität Halle-WittenbergHalle/SaaleGermany

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