Pancreatic stellate cells exhibit adaptation to oxidative stress evoked by hypoxia

Abstract

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% O₂ ) on pancreatic stellate cells physiology was investigated. Changes in intracellular free-Ca²⁺ concentration, mitochondrial free-Ca²⁺ 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-Ca²⁺ 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 NH₂ -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.

Keywords: Calcium; Glutathione; Hypoxia; Pancreatic stellate cells; Reactive oxygen species.