Melatonin modulates red-ox state and decreases viability of rat pancreatic stellate cells

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.

Melatonin Modulates the Antioxidant Defenses and the Expression of Proinflammatory Mediators in Pancreatic Stellate Cells Subjected to Hypoxia

“…This effect has been related with a putative modulation of cell viability, majorly of tumor cells [ 30 , 44 , 45 , 46 , 47 ]. In a former work, we showed that melatonin stimulated ROS production in the cytosol in PSC under normoxia [ 29 ]. Additionally, previous results of our laboratory showed that hypoxia induced a prooxidant environment in PSC, without detection of cytosolic ROS generation [ 7 ].…”

“…Interestingly, a previous work showed that treatment of PSC with melatonin under normoxic conditions reduced the expression of these key enzymes, which are critical for the detoxification of ROS. This could explain why protein oxidation in PSC treated with melatonin was observed under normoxia, but not under hypoxia [ 29 ].…”

“…After treatment, cells were washed with standard phosphate saline solution and were lysed for analysis. Protein carbonyls were determined following the methods described by Villaverde et al [ 28 ], with slight modifications [ 29 ]. Five hundred µL of each sample were dispensed in 2 mL microtubes and treated with cold 10% trichloroacetic acid (TCA) solution.…”

“…Total antioxidant capacity (TAC) was determined using a commercially available kit, following manufacturer’s directions, as described previously [ 29 ]. In brief, a working solution containing Cu 2+ , provided with the assay kit, was added to all standard and sample wells.…”

Targeting Fibrosis: The Bridge That Connects Pancreatitis and Pancreatic Cancer

“…For example, the ROS induced by H 2 O 2 promotes the activation of PSCs, while resveratrol prevents the activation of PSCs by reducing the production of ROS [ 126 ]. Moreover, melatonin, at pharmacological concentrations, has shown a concentration-dependent decrease in cell viability in rat [ 127 ] and human [ 128 ] PSCs by modifying the redox state of the cells. Dimethyl fumarate (DMF) promotes the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and the expression of downstream antioxidant genes, eliminating intracellular ROS, inhibiting the activation of PSCs, and reducing the pancreatic fibrosis level [ 129 ].…”