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

Estarás, Matías; Moreno, Noelia; Santofimia‐Castaño, Patricia; Martínez-Morcillo, Salomé; Roncero, V.; Blanco, Gerardo; López, D.; Fernandez-Bermejo, Miguel; Mateos, José; Iovanna, Juan; Salido, Ginés M.; González, Antonio

doi:10.1007/s13105-019-00671-x

Cited by 21 publications

(25 citation statements)

References 33 publications

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“…Our results show that hypoxia induced an increase in [Ca 2+ ] i in PSC. Former works showed that PSC exhibit Ca 2+ signals when stimulated with certain agonists (Gryshchenko et al., 2016; Estaras et al., 2019b). This observation points out that Ca 2+ may be a major signalling molecule in this cell type.…”

Section: Discussionmentioning

confidence: 99%

“…Cultures of PSC were prepared following previously used methods. With this procedure, cultures of activated PSC can be prepared (Estaras et al., 2019b; Santofimia‐Castaño et al., 2015). Pancreatic tissues were obtained from Wistar rats pups (3–5 days after birth).…”

Section: Methodsmentioning

confidence: 99%

“…The changes in the levels of reduced (GSH) and oxidized (GSSG) glutathione were determined using methods described previously (Estaras et al., 2019b). Cells were incubated during 4 h with the different stimuli assayed.…”

Section: Methodsmentioning

confidence: 99%

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Pancreatic stellate cells exhibit adaptation to oxidative stress evoked by hypoxia

Estarás

Martínez-Morcillo

García

et al. 2020

Biology of the Cell

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Background information. Pancreatic stellate cells play a key role in the fibrosis that develops in diseases such as pancreatic cancer. In the growing tumour, a hypoxia condition develops under which cancer cells are able to proliferate. The growth of fibrotic tissue contributes to hypoxia. In this study, the effect of hypoxia (1% O 2) on pancreatic stellate cells physiology was investigated. Changes in intracellular free-Ca 2+ concentration, mitochondrial free-Ca 2+ concentration and mitochondrial membrane potential were studied by fluorescence techniques. The status of enzymes responsible for the cellular oxidative state was analyzed by quantitative reverse transcriptionpolymerase chain reaction, high-performance liquid chromatography, spectrophotometric and fluorimetric methods and by Western blotting analysis. Cell viability and proliferation were studied by crystal violet test, 5-bromo-2deoxyuridine cell proliferation test and Western blotting analysis. Finally, cell migration was studied employing the wound healing assay. Results. Hypoxia induced an increase in intracellular and mitochondrial free-Ca 2+ concentration, whereas mitochondrial membrane potential was decreased. An increase in mitochondrial reactive oxygen species production was observed. Additionally, an increase in the oxidation of proteins and lipids was detected. Moreover, cellular total antioxidant capacity was decreased. Increases in the expression of superoxide dismutase 1 and 2 were observed and superoxide dismutase activity was augmented. Hypoxia evoked a decrease in the oxidized/reduced glutathione ratio. An increase in the phosphorylation of nuclear factor erythroid 2-related factor and in expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 were detected. The expression of cyclin A was decreased, whereas expression of cyclin D and the content of 5-bromo-2-deoxyuridine were increased. This was accompanied by an increase in cell viability. The phosphorylation state of c-Jun NH 2-terminal kinase was increased, whereas that of p44/42 and p38 was decreased. Finally, cells subjected to hypoxia maintained migration ability. Conclusions and Significance. Hypoxia creates pro-oxidant conditions in pancreatic stellate cells to which cells adapt and leads to increased viability and proliferation.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Pancreatic stellate cells exhibit adaptation to oxidative stress evoked by hypoxia

Estarás

Martínez-Morcillo

García

et al. 2020

Biology of the Cell

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“…Of major interest for the conclusions obtained in this study is the absence of melatonin MT1 and MT2 type receptors in PSCs. Previous work carried out in our laboratory showed that PSCs do not exhibit classical melatonin membrane receptors MT1 and MT2 (Estaras, Moreno, et al, 2019; Santofimia‐Castaño et al, 2015). Bearing in mind the existing relationship between Ca 2+ mobilization, ROS production, impairment of ER physiology, activation of MAPKs and onset of cell damage, we provided evidence that signal luzindole was a potentially injurious compound to the cells.…”

Section: Discussionmentioning

confidence: 87%

“…Finally, cells were seeded on polystyrene plates for cell culture (6 and 12 multiwell plates) and grown in a humidified incubator at 37°C and 5% CO 2 . With this procedure it is possible to obtain cultures of a population of PSCs as demonstrated by the expression of PSC‐specific markers (Estaras et al, 2019; McCarroll et al, 2014; Modi et al, 2014; Santofimia‐Castaño et al, 2015; Zha, Li, Xu, Chen, & Sun, 2014). Cultures were maintained in a humidified incubator at 37°C and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

The melatonin receptor antagonist luzindole induces the activation of cellular stress responses and decreases viability of rat pancreatic stellate cells

Estarás

Marchena

Fernandez–Bermejo

et al. 2020

J of Applied Toxicology

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In this study, we have examined the effects of luzindole, a melatonin receptor-antagonist, on cultured pancreatic stellate cells. Intracellular free-Ca 2+ concentration, production of reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPK), endoplasmic reticulum stress and cell viability were analyzed. Stimulation of cells with the luzindole (1, 5, 10 and 50 μM) evoked a slow and progressive increase in intracellular free Ca 2+ ([Ca 2+ ] i) towards a plateau. The effect of the compound on Ca 2+ mobilization depended on the concentration used. Incubation of cells with the sarcoendoplasmic reticulum Ca 2+-ATPase inhibitor thapsigargin (1 μM), in the absence of Ca 2+ in the extracellular medium, induced a transient increase in [Ca 2 + ] i. In the presence of thapsigargin, the addition of luzindole to the cells failed to induce further mobilization of Ca 2+. Luzindole induced a concentration-dependent increase in ROS generation, both in the cytosol and in the mitochondria. This effect was smaller in the absence of extracellular Ca 2+. In the presence of luzindole the phosphorylation of p44/42 and p38 MAPKs was increased, whereas no changes in the phosphorylation of JNK could be noted. Moreover, the detection of the endoplasmic reticulum stress-sensor BiP was increased in the presence of luzindole. Finally, viability was decreased in cells treated with luzindole. Because cellular membrane receptors for melatonin have not been detected in pancreatic stellate cells, we conclude that luzindole could exert direct effects that are not mediated through its action on melatonin membrane receptors.

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Melatonin modulates proliferation of pancreatic stellate cells through caspase-3 activation and changes in cyclin A and D expression

et al. 2020

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

Cited by 21 publications

References 33 publications

Pancreatic stellate cells exhibit adaptation to oxidative stress evoked by hypoxia

Pancreatic stellate cells exhibit adaptation to oxidative stress evoked by hypoxia

The melatonin receptor antagonist luzindole induces the activation of cellular stress responses and decreases viability of rat pancreatic stellate cells

Melatonin modulates proliferation of pancreatic stellate cells through caspase-3 activation and changes in cyclin A and D expression

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