Melatonin induces reactive oxygen species generation and changes in glutathione levels and reduces viability in human pancreatic stellate cells

  • Matias Estaras
  • Noelia Moreno
  • Patricia Santofimia-Castaño
  • Salome Martinez-Morcillo
  • Vicente Roncero
  • Gerardo Blanco
  • Diego Lopez
  • Miguel Fernandez-Bermejo
  • Jose M. Mateos
  • Juan L. Iovanna
  • Gines M. Salido
  • Antonio GonzalezEmail author
Original Article


In this study, the effects of pharmacological concentrations of melatonin (1 μM–1 mM) on human pancreatic stellate cells (HPSCs) have been examined. Cell type–specific markers and expression of melatonin receptors were analyzed by western blot analysis. Changes in intracellular free Ca2+ concentration were followed by fluorimetric analysis of fura-2–loaded cells. Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined by fluorescence techniques. Production of reactive oxygen species (ROS) was monitored following 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester and MitoSOX™ Red–derived fluorescence. Cell viability was studied using the AlamarBlue® test. Cultured cells expressed markers typical of stellate cells. However, cell membrane receptors for melatonin could not be detected. Thapsigargin, bradykinin, or melatonin induced changes in intracellular free Ca2+ concentration. In the presence of the indole, a decrease in the GSH/GSSG ratio was observed that depended on the concentration of melatonin used. Furthermore, the indole evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Finally, melatonin decreased HPSC viability in a time and concentration-dependent manner. We conclude that melatonin, at pharmacological concentrations, induces changes in the oxidative state of HPSC. This might regulate cellular viability and could not involve specific plasma membrane receptors.


Melatonin Calcium Glutathione Cell viability Human pancreatic stellate cells 



Intracellular free Ca2+ concentration


5-(and-6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester


Endoplasmic reticulum


Fura-2-acetoxymethyl ester


Reduced glutathione


Oxidized glutathione


Hank’s balanced salts


Human pancreatic stellate cells


Hydrogen peroxide


Reactive oxygen species


Rat pancreatic stellate cells


Sarcoendoplasmic reticulum Ca2+-ATPase




Alpha-smooth muscle actin


Funding information

This study was partly funded by the Ministerio de Economía y Competitividad (BFU2016-79259-R; UNEX13-1E-1608) and Junta de Extremadura-FEDER (IB16006). The funding sources had no role in the study design; in the collection, analysis, and interpretation of the data; in the writing of the report; and in the decision to submit the paper for publication.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© University of Navarra 2019

Authors and Affiliations

  • Matias Estaras
    • 1
  • Noelia Moreno
    • 1
  • Patricia Santofimia-Castaño
    • 2
  • Salome Martinez-Morcillo
    • 3
  • Vicente Roncero
    • 4
  • Gerardo Blanco
    • 5
  • Diego Lopez
    • 5
  • Miguel Fernandez-Bermejo
    • 6
  • Jose M. Mateos
    • 6
  • Juan L. Iovanna
    • 2
  • Gines M. Salido
    • 1
  • Antonio Gonzalez
    • 1
    Email author
  1. 1.Department of Physiology, Institute of Molecular Pathology BiomarkersUniversity of ExtremaduraCáceresSpain
  2. 2.Centre de Recherche en Cancérologie de Marseille, INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de LuminyMarseilleFrance
  3. 3.Unit of Toxicology, Veterinary FacultyUniversity of ExtremaduraCáceresSpain
  4. 4.Unit of Histology and Pathological Anatomy, Veterinary FacultyUniversity of ExtremaduraCáceresSpain
  5. 5.Hepatobiliary-Pancreatic Surgery and Liver Transplant UnitInfanta Cristina HospitalBadajozSpain
  6. 6.Department of GastroenterologySan Pedro de Alcantara HospitalCáceresSpain

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