Publications by authors named "Daniel Vara"

6 Publications

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Melatonin Induces Apoptosis and Modulates Cyclin Expression and MAPK Phosphorylation in Pancreatic Stellate Cells Subjected to Hypoxia.

Int J Mol Sci 2021 May 24;22(11). Epub 2021 May 24.

Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain.

In certain diseases of the pancreas, pancreatic stellate cells form an important part of fibrosis and are critical for the development of cancer cells. A hypoxic condition develops within the tumor, to which pancreatic stellate cells adapt and are able to proliferate. The consequence is the growth of the tumor. Melatonin, the product of the pineal gland, is gaining attention as an agent with therapeutic potential against pancreatic cancers. Its actions on tumor cells lead, in general, to a reduction in cell viability and proliferation. However, its effects on pancreatic stellate cells subjected to hypoxia are less known. In this study, we evaluated the actions of pharmacological concentrations of melatonin (1 mM-1 µM) on pancreatic stellate cells subjected to hypoxia. The results show that melatonin induced a decrease in cell viability at the highest concentrations tested. Similarly, the incorporation of BrdU into DNA was diminished by melatonin. The expression of cyclins A and D also was decreased in the presence of melatonin. Upon treatment of cells with melatonin, increases in the expression of major markers of ER stress, namely BIP, phospho-eIF2α and ATF-4, were detected. Modulation of apoptosis was noticed as an increase in caspase-3 activation. In addition, changes in the phosphorylated state of p44/42, p38 and JNK MAPKs were detected in cells treated with melatonin. A slight decrease in the content of α-smooth muscle actin was detected in cells treated with melatonin. Finally, treatment of cells with melatonin decreased the expression of matrix metalloproteinases 2, 3, 9 and 13. Our observations suggest that melatonin, at pharmacological concentrations, diminishes the proliferation of pancreatic stellate cells subjected to hypoxia through modulation of cell cycle, apoptosis and the activation of crucial MAPKs. Cellular responses might involve certain ER stress regulator proteins. In view of the results, melatonin could be taken into consideration as a potential therapeutic agent for pancreatic fibrosis.
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http://dx.doi.org/10.3390/ijms22115555DOI Listing
May 2021

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

Antioxidants (Basel) 2021 Apr 8;10(4). Epub 2021 Apr 8.

Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain.

Pancreatic stellate cells (PSC) play a major role in the formation of fibrotic tissue in pancreatic tumors. On its side, melatonin is a putative therapeutic agent for pancreatic cancer and inflammation. In this work, the actions of melatonin on PSC subjected to hypoxia were evaluated. Reactive oxygen species (ROS) generation reduced (GSH) and oxidized (GSSG) levels of glutathione, and protein and lipid oxidation were analyzed. The phosphorylation of nuclear factor erythroid 2-related factor (Nrf2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), and the regulatory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (IκBα) was studied. The expression of Nrf2-regulated antioxidant enzymes, superoxide dismutase (SOD) enzymes, cyclooxygenase 2 (COX-2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also studied. Total antioxidant capacity (TAC) was assayed. Finally, cell viability was studied. Under hypoxia and in the presence of melatonin generation of ROS was observed. No increases in the oxidation of proteins or lipids were detected. The phosphorylation of Nrf2 and the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1, heme oxygenase-1, SOD1, and of SOD2 were augmented. The TAC was increased. Protein kinase C was involved in the effects of melatonin. Melatonin decreased the GSH/GSSG ratio at the highest concentration tested. Cell viability dropped in the presence of melatonin. Finally, melatonin diminished the phosphorylation of NF-kB and the expression of COX-2, IL-6, and TNF-α. Our results indicate that melatonin, at pharmacological concentrations, modulates the red-ox state, viability, and the expression of proinflammatory mediators in PSC subjected to hypoxia.
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http://dx.doi.org/10.3390/antiox10040577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070371PMC
April 2021

Pancreatic stellate cells exhibit adaptation to oxidative stress evoked by hypoxia.

Biol Cell 2020 Oct 12;112(10):280-299. Epub 2020 Jul 12.

Institute of Molecular Pathology Biomarkers, University of Extremadura, Caceres, Spain.

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 ) on pancreatic stellate cells physiology was investigated. Changes in intracellular free-Ca concentration, mitochondrial free-Ca 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 transcription-polymerase 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-2-deoxyuridine 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 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 -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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http://dx.doi.org/10.1111/boc.202000020DOI Listing
October 2020

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

J Appl Toxicol 2020 11 22;40(11):1554-1565. Epub 2020 Jun 22.

Institute of Molecular Pathology Biomarkers, University of Extremadura, Caceres, Spain.

In this study, we have examined the effects of luzindole, a melatonin receptor-antagonist, on cultured pancreatic stellate cells. Intracellular free-Ca 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 ([Ca ] ) towards a plateau. The effect of the compound on Ca mobilization depended on the concentration used. Incubation of cells with the sarcoendoplasmic reticulum Ca -ATPase inhibitor thapsigargin (1 μm), in the absence of Ca in the extracellular medium, induced a transient increase in [Ca ] . In the presence of thapsigargin, the addition of luzindole to the cells failed to induce further mobilization of Ca . 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 . 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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http://dx.doi.org/10.1002/jat.4018DOI Listing
November 2020

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

J Physiol Biochem 2020 May 2;76(2):345-355. Epub 2020 May 2.

Institute of Molecular Pathology Biomarkers, University of Extremadura, Avenida de las Ciencias s/n, E-10003, Cáceres, Spain.

In this study, the effects of melatonin (1 μM-1 mM) on pancreatic stellate cells (PSC) have been examined. Cell viability and proliferation, caspase-3 activation, and the expression of cyclin A and cyclin D were analyzed. Our results show that melatonin decreased PSC viability in a time- and concentration-dependent manner. This effect was not inhibited by treatment of cells with MT1, MT2, calmodulin, or ROR-alpha inhibitors prior to melatonin addition. Activation of caspase-3 in response to melatonin was detected. The expression of cyclin A and cyclin D was decreased in cells treated with melatonin. Finally, changes in BrdU incorporation into the newly synthesized DNA of proliferating cells were also observed in the presence of melatonin. We conclude that melatonin, at pharmacological concentrations, modulates proliferation of PSC through activation of apoptosis and involving crucial regulators of the cell cycle. These actions might not require specific melatonin receptors. Our observations suggest that melatonin, at high doses, could potentially exert anti-fibrotic effects and, thus, could be taken into consideration as supportive treatment in the therapy of pancreatic diseases.
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http://dx.doi.org/10.1007/s13105-020-00740-6DOI Listing
May 2020

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

Sci Rep 2020 04 14;10(1):6352. Epub 2020 Apr 14.

Institute of Molecular Pathology Biomarkers, University of Extremadura, Caceres, Spain.

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-HDCFDA and MitoSOX Red-derived fluorescence. Total protein carbonyls and lipid peroxidation were analyzed by HPLC and spectrophotometric methods respectively. Mitochondrial membrane potential (ψ) 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 ψ 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.
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http://dx.doi.org/10.1038/s41598-020-63433-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156707PMC
April 2020