Abstract
In this work we have studied the effects of pharmacological concentrations of melatonin (1 µM–1 mM) on pancreatic stellate cells (PSC). Cell viability was analyzed by AlamarBlue test. Production of reactive oxygen species (ROS) was monitored following CM-H2DCFDA and MitoSOX Red-derived fluorescence. Total protein carbonyls and lipid peroxidation were analyzed by HPLC and spectrophotometric methods respectively. Mitochondrial membrane potential (ψm) was monitored by TMRM-derived fluorescence. Reduced (GSH) and oxidized (GSSG) levels of glutathione were determined by fluorescence techniques. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Determination of SOD activity and total antioxidant capacity (TAC) were carried out by colorimetric methods, whereas expression of SOD was analyzed by Western blotting and RT-qPCR. The results show that melatonin decreased PSC viability in a concentration-dependent manner. Melatonin evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Oxidation of proteins was detected in the presence of melatonin, whereas lipids oxidation was not observed. Depolarization of ψm was noted with 1 mM melatonin. A decrease in the GSH/GSSG ratio was observed, that depended on the concentration of melatonin used. A concentration-dependent increase in the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 was detected in cells incubated with melatonin. Finally, decreases in the expression and in the activity of superoxide dismutase were observed. We conclude that pharmacological concentrations melatonin modify the redox state of PSC, which might decrease cellular viability.
Highlights
It is nowadays increasing the focus of research on the role of pancreatic stellate cells (PSC) in the physiology and the pathophysiology of the pancreas
Activated PSC are responsible for the progressive fibrosis and for the accumulation of extracellular matrix that occurs in severe pancreatic disorders such as chronic pancreatitis and pancreatic cancer[1,2]
We have shown that melatonin modulates proliferation of murine[17] and human PSC18
Summary
It is nowadays increasing the focus of research on the role of pancreatic stellate cells (PSC) in the physiology and the pathophysiology of the pancreas. It is thought that activated PSC are involved in tumor progression and chemoresistance In this regard, PSC contribute to stromal or fibrotic reaction by the release of matrix components, release signaling molecules that act on neighboring cells to modulate their proliferation and tissue growth within cancer[3]. PSC depict an important role as components of the tumor microenvironment and have emerged as key modulators in the context of tissue injury. In this regard, we have shown that melatonin modulates proliferation of murine[17] and human PSC18. We have continued our former studies to further investigate the ways by which melatonin could exert its effects on PSC to control their proliferation
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