UV radiation in HCT 116 cells influences intracellular H2O2 and glutathione levels, antioxidant expression, and protein glutathionylation

Abstract

UV radiation influences cellular levels of hydrogen peroxide (H2O2) and glutathione (GSH) and alters the expression of antioxidant genes in the human colorectal cancer cell line HCT116. In this study, cells were irradiated with UV light of different wavelength (A, B, or C). A surge in H2O2 concentration and total glutathione (level occurred 6 hours later. Consequently, protein glutathionylation increased above control levels. Expression of the antioxidant enzymes: glutathione peroxidase (GPX) and glutathione reductase (GSR), assessed by real-time quantitative PCR, increased by 1.5–2 times after 24 hours post-irradiation, in comparison to the untreated controls. Glutathionylation of proteins was enhanced after UV radiation and the set of biotinylated glutathione ethyl ester (BioGEE) tagged proteins was detected by Western Blot procedure. This specific glutathione analogue is conjugated with antioxidant proteins during glutathionylation especially under oxidized conditions in cells. A pool of glutathionylated proteins in the treated cells showed peculiar characteristics. These proteins exhibited varying molecular weights. For UVA-irradiated cells, 24 hours after the treatment we observed two additional ~60 and ~72 kDa bands of glutathionylated proteins from NADPH oxidases (NOX family). Total glutathione level in the UV-irradiated HCT116 cells was higher than in the control. This correlates with the detection of glutathionylation in UV-irradiated cells in the first and twelfth hour of post-irradiation, and can be defined as a specific antioxidant element activation for cellular protection.

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