UV radiation in HCT 116 cells influences intracellular H 2 O 2 and glutathione levels, antioxidant expression, and protein glutathionylation

UV radiation influences cellular levels of hydrogen peroxide (H 2 O 2 ) and glutathione (GSH) and alters the ex- pression of antioxidant genes in the human colorectal cancer cell line HCT116. In this study, cells were irradi- ated with UV light of different wavelength (A, B, or C). A surge in H 2 O 2 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 West- ern 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 exhib-ited 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 glu- tathionylation 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 pro- tection.


INTRODUCTION
Oxidative stress is one of the external factors that influence human health (Cieślar-Pobuda et al., 2017). Maintaining a similar level between the oxidation and reduction processes in tissues and cells is called a redox equilibrium (Buldak et al., 2015). Intracellular redox potential affects cellular functions and its dysregulation is associated with disease (Finkel, 2011;Skonieczna et al., 2017).
Reactive oxygen species (ROS) are radical or nonradical forms of oxygen, which are characterized by high reactivity. In healthy cells, ROS participate in many cellular processes like hormone secretion, drug removal, detoxification, and stimulation of the immune system, and their concentration is physiological. ROS are mainly produced by mitochondrial complexes. The superoxide anion radical (O 2 •-) and hydrogen peroxide (H 2 O 2 ) are the most common ROS (Puzanowska-Tarasiewicz et al., 2010;Finkel, 2011;Buldak et al., 2013). Overproduction of ROS may lead to oxidative stress, which may result in permanent changes in cells, leading to loss of function of proteins, which in turn can cause disease (Kowalska, 2008;Buldak et al., 2013).
Oxidative stress is the result of an imbalance between ROS and antioxidant activity (Rzeszowska-Wolny et al., 2009). UV radiation is classified as one of the factors causing oxidative stress (Krzywon et al., 2018). It disrupts the redox balance, resulting in irreversible changes in the cell, and causing severe disturbances within the entire metabolic pathways (Widel et al., 2014). Such UVinduced oxidative stress and imbalance may lead to diseases such as diabetes, atherosclerosis, or cancer (Valko et al., 2005;Karpińska & Gromadzka, 2013;Kasperczyk et al., 2013;Buttke & Sandstrom, 2016;Widel, 2016).
Antioxidants are substances that inhibit the oxidation of molecules and cause the conversion of radicals into inactive derivatives (Schmatz et al., 2012;Azqueta et al., 2013;Modrzejewska et al., 2016). Their main task is to protect the cell against oxidative stress and maintain the optimal redox state (Schafer & Buettner, 2001;Czajka, 2006;Kowalska, 2008). Among the main antioxidants that form the antioxidant barrier are glutathione and glutathione peroxidase (Czajka, 2006). Glutathione is a substrate for the S-glutathionylation reaction, which protects proteins from the irreversible effects of oxidation (Peskin et al., 2016). There are two forms of glutathione in the cell: reduced (GSH, about 90%) and oxidized (GSSG, about 10%) (Gómez-Cambronero, 2000). Glutathionylation is a reversible reaction in which glutathione attach- es to cysteine residues of proteins via a disulfide bond (Gómez-Combronero, 2000;Żmijewski et al., 2009). Glutathione reductase (GSR) is involved in maintaining the balance between the oxidized and reduced glutathione. The expression of GSR increases in cells with an excess of GSSG (Bilska et al., 2007). GSR is an NADPHdependent enzyme of cytosolic origin that catalyzes the reduction reaction of GSSG to two reduced GSH molecules (Formula 1) (Bilska et al., 2007;Zhu et al., 2018).
NADPH + H + + GSSG  NADP + + 2GSH Formula 1. Reaction of reduction of oxidized form of glutathione GSSG to GSH with the participation of GSR.
Another important antioxidant is glutathione peroxidase (GPX), an enzyme that catalyzes the reduction of hydrogen peroxide to water using reduced glutathione as a substrate (Formula 2). GPX plays a protective role against oxidative stress in the cells (Łukaszewicz-Hussain, 2003).
Hydrogen peroxide reduction reaction involving glutathione.
UV radiation, which causes oxidative stress, has a wavelength range of approx. 100 to 400 nm and is not visible to humans. UV radiation is classified as UVA (400-329nm), UVB (320-290nm) or UVC (<290nm) (Diffey, 2002;Grimes, 2015). 90% of the solar UVA radiation reaches the Earth surface. It is the mildest form of UV radiation, yet, it can still penetrate deep into the skin. UVB can penetrate through the outer layer of the epidermis, however, most of the solar UVB is blocked in the atmosphere and only 10% of the radiation reaches the Earth surface. Out of the three types of UV radiation, UVC is the most harmful because at the wavelength range (<290 nm) it can be absorbed by RNA and DNA (245-290 nm) (Soehnge et al., 1997;Skórska, 2016), yet it is also mostly blocked in the Earth atmosphere. In the presented study, we investigated the impact of UV radiation on human HCT116 cancer cells, via the ROS-induced glutathione target and measurement of its recovery potential.

MATERIALS AND METHODS
Cell cultures. The colorectal cancer cell line, HCT116 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM-F12 medium (PAA, Poland) supplemented with 10% FBS (EURx, Poland) and Antibiotic-Antimycotic Solution (100×; Sigma-Aldrich, Germany). All cultures were carried out in 75 ml flasks (Sarstedt, Germany) under standard conditions (37°C and 5% CO 2 , at 80% humidity). Twenty-four hours before irradiation, the cells were seeded in 6-well plates (Sarstedt, Germany) at 1×10 5 cells/well in 2 ml of fresh medium. UV irradiation was carried out using UV crosslinkers (model CL-1000, UVP, Upland, CA, USA) with the following settings: UVA (365 nm), UVB (302 nm), and UVC (254 nm) at doses of 10 kJ/m 2 , 5 kJ/m 2 and 100 J/m 2 , respectively. After irradiation, cells were collected at five time points (1, 3, 6, 12, and 24 h) and either frozen at -20ºC for further tests (PCR, Western Blot), or stained directly with specific dyes for fluorimetric or cytometric assays. Intracellular ROS levels. ROS levels were measured by flow cytometry using the specific H 2 O 2 -detecting dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA; Thermo Scientific), a cell-permeable non-fluorescent probe, which, when oxidized by ROS, converts into a highly green-fluorescent form thanks to removal of its acetate groups by intracellular esterases. Three hundred μl of cell suspension in 1×PBS (prepared in triplicates) were stained with carboxy-H2DCF-DA (30 μM). After incubation in darkness (30 min at 37°C), the cells were washed with PBS and kept on ice for 15 minutes. Then, the fluorescence was measured for at least 10 000 cells using a flow cytometer Aria III Beckton Dickinson with the FITC configuration (488nm excitation; emission: LP mirror 503, BP filter 530/30). The results from three experiments with three technical repeats each were analyzed using the FlowingSoftware 2.5.0 (Perttu Terho, Turku Centre for Biotechnology, University of Turku, Finland) and presented as mean fluorescence.
RNA Isolation. Cells were collected from plates and suspended in Fenozol (203-100, A&A Biotechnology, Gdynia, Poland). RNA was isolated using a total RNA isolation kit (A&A Biotechnology, Gdynia, Poland) following the producer's protocol. RNA purity and concentration were measured using NanoDrop 2000 (Thermo Fisher Scientific). Next, reverse transcription was performed using a dART RT kit (EURx, Gdansk, Poland) to produce cDNA.
Results from qPCR were analyzed using the 2 -ΔΔCt method (Livak & Schmittgen, 2001, Tyburski et al., 2008. Assay of intracellular GSH. Cells were seeded in a 96-well plate (Sarstedt, Germany). Monochlorobimane (MCB; Sigma, Germany), which penetrates cells and reacts with GSH to form the fluorescent compound GSH-monochlorobimane (Webb et al., 2006), was added to the culture medium. The samples were incubated with the compound for 30 min, after which the fluorescence at 490 nm (ex 394 nm/em490 nm) was measured using a plate reader (Infinite 200 PRO, Tecan, Männedorf, Switzerland).
Quantitation of glutathionylated proteins. Cells were seeded in a 96-well plate. BioGEE (178 mM) was added to the culture medium, followed by streptavidin-FITC (Sigma S3762, 1:200 dilution) after 30 minutes of incubation. After 10 min the medium was aspirated, 50 μl of PBS was added to the cells, and fluorescence was measured at 535 nm (ex 485nm/em 535nm) on a plate reader (Infinite 200 PRO, Tecan). GSR GPX Separation of glutathionylated proteins by SDS-PAGE and Western Blot One μl of BioGEE (Life Technologies), a biotinylated glutathione analogue (178 mM solution) was added to cells 30 minutes before harvesting, to saturate the cells with reagent. The cells were collected and resuspended in 1% SDS, and protein concentration was measured using Bradford reaction (Bio-Rad). Samples containing the same amount of protein (10 μg) were separated on polyacrylamide gradient gels (10%) together with molecular mass markers (Precision Plus Protein™ Dual Xtra Standards, Bio-Rad). Semi-dry transfer to a nitrocellulose membrane was carried out using the Trans-Blot® Tur-boTM system (Bio-Rad). The membrane was blocked with 5% milk solution (milk dissolved in distilled water) overnight, washed with PBS-Tween, incubated with Streptavidin-HRP conjugate (GE HealthCare) for 1, washed with PBS-Tween 3 times, 10 minutes each, at room temperature. Immunodetection was performed using ECL reagents from the Western Bright Quantum kit (Advansta, San Jose, CA, USA). After air-drying on the membrane, pictures were taken using a G: BOX chemiXX6 (Syngene, Frederick, MD, USA). GAPDH was detected with primary rabbit anti-human antibody (1:5000) followed by HRP-conjugated secondary mouse anti-rabbit antibody (1:10 000) (both from Santa Cruz).
Statistical analysis. Experimental data are presented as mean from at least three independent experiments, with three technical repeats each, ± standard deviation (± S.D.). Outliers were discarded using Dixon's Q test. Statistical significance was calculated using a T-test. The asterisk * in the figures denotes a p-value of 0.05 in a comparison of a sample to untreated control from first hour time point.

Intracellular level of reactive oxygen species
Hydrogen peroxide is one of the major reactive oxygen species in cells (Lennicke et al., 2015;Sies, 2017;Jones & Sies, 2015). 1 hour after irradiation with UVA, the level of H 2 O 2 was below the control level. Six hours after irradiation, a surge in H 2 O 2 concentrations appeared, and this effect was seen in all the UV wavelength treatments. After 24 hours, H 2 O 2 in the irradiated cells returned to the control levels ( Fig. 1).

Expression levels of antioxidant genes
UVB irradiation caused a significant increase (p=0.05) in expression of GPX4 gene in HCT116 cells 24 h after the treatment. However, in UVC irradiated cells, the expression was down-regulated over the 24-hours of observations (beyond 6 h, Fig. 2A).
The level of GSR mRNA was down-regulated directly after UVB and UVC exposure until 12 h post-irradiation. UVA stimulated GSR expression but only at 6 and 24 hour post-irradiation time points (Fig. 2.B).

Level of intracellular glutathione
Intracellular glutathione levels were assayed using monochlorobimane, which is essentially nonfluorescent until conjugated with glutathione, or another low molecular weight thiols (Kamencic et al., 2000;Webb et al., 2006). The levels of reduced glutathione increased in irradiated cells at all measuring points up to 24 h after treatment (Fig. 3).

Plate-reader fluorometric measurements
Incorporation of glutathione into proteins can be detected using biotinylated glutathione ethyl ester (BioGEE), a cell-permeant biotinylated glutathione ana-  log which is incorporated into proteins (García-Giménez et al., 2013). Cells exposed to UVA, UVB and UVC responded with over-production of glutathionylated antioxidant proteins, which could be the first protective reaction against protein damage and oxidation (Fig. 4) (Yang et al., 2010). The highest level of glutathionylation occurred in the first hour after irradiation. After that time, the level of glutathionylated proteins oscillated around the control level (Fig. 4). The activation of GSH at 1 hour (Fig. 3), together with a glutathionylation process (Fig. 4), protected the proteins from the irreversible effects of oxidation. The glutathionylation increased for a second time at 12 h, in correlation with an increase in GSH.

Western Blot analysis
Total cell proteins conjugated with biotinylated-BioGEE were detected with Western Blot following SDS-PAGE (Fig. 5). In control HCT116 cells a set of   glutathionylated proteins was detected, especially noticeable for the first 12 h, with a small amount of proteins detected at the 24th hour of observation (Fig. 5A).
In the case of UVA-irradiated cells (Fig. 5B), a similar amount of proteins was glutathionylated at each of the time-points of our observation, except for 12h post-irradiation, where more bands could be seen. The glutathionylation process was specific to proteins with characteristic molecular weight: 60 kDa and 72 kDa, relating to NOX oxidases, which was confirmed using antibodies against NOX-4 (not shown).
UVB radiation resulted in an increase in the number of different glutathionylated proteins at 3, 6 and 24 hours after irradiation (Fig. 5C). The glutathionylated proteins had different molecular weights, which became clearly visible at the timepoints of 3+ hours and was still noticeable after 24 hours. In control cells, at the same 24 h timepoint, almost no glutathionylated proteins were detected (Fig. 5A).
UVC exposure results were similar to UVA (Fig. 5B). Signals for glutathionylated proteins in each lane for all time points were similar, and showed specific glutathionylation for ~60 and ~72 kDa sized NOX proteins (Fig. 5D). These results may indicate the same profile of glutathionylation for analogous proteins (the same mass and bands from UVA and UVB membranes). However, at 12 h timepoint, additional proteins of a lower mass were observed. GAPDH (Fig. 6) was made as a reference to check that the same amount of protein was applied.

DISCUSSION
Cells possess natural protective mechanisms against oxidation and rapid damage of proteins, DNA, lipids and other macromolecules (Jones & Sies, 2015). The natural "shield" consists of antioxidants, responsible for protecting the cells from action of free radicals (Modrzejewska et al., 2016;Dziaman et al., 2018). The most important antioxidant found in the cells is glutathione, which protects proteins against the irreversible oxidation (Peskin et al., 2016). The mechanism of action for GSH is to participate in the reversible S-glutathionylation reaction, which creates disulfide bonds between protein's cysteines and forms glutathione and a protected protein (Drozd et al., 2016). Measurements of hydrogen peroxide levels in cells after UV exposure (Fig. 1) showed a significant increase of this radical at the sixth hour after irradiation. The response to the increased H 2 O 2 was prevalent at twelve hours post-irradiation. We observed higher levels of glutathionylated proteins (Fig. 5) and an increase in total glutathione (GSH and GSSG) in the cells (Fig. 2). After the expected reduction of hydrogen peroxide to water within the next measured timepoint (24h), we no-ticed an increase in the expression of glutathione reductase (GSR) and glutathione peroxidase (GPX) (Ciesielska et al., 2019). These enzymes are responsible for the reduction of glutathionylation products. GSR restores the level of reduced glutathione (GSH), and in the cell more than 90% of the GSH pool comes from the reduction of its oxidized form (GSSG) (Sabens Liedhegner et al., 2012;Mieyal & Chock 2012). In contrast, GPX reduces glutathione attached to proteins. Analysis of glutathione-related products after UV exposure showed the presence of NOX-family proteins, which were involved in the glutathione target that provided protection to HCT116 cells against oxidative stress. The reaction of glutathionylation is reversible, which allows the reduced/ oxidized pool of glutathione to remain balanced via activation/deactivation of antioxidative enzymes -crucial players in protective feedback loops: glutathione reductases and glutathione peroxidases (Sullivan et al., 2002;Yang et al., 2010;Mailloux et al., 2011;Mailloux et al., 2013). Expression levels for both enzymes were measured on mRNA. Additionally, a pool of reduced glutathione, together with glutathionylation process were estimated on the post-translational process. Obtained results showed important roles of all the investigated elements in UV-induced oxidative stress prevention in HCT116 cancer cells. These findings can be used further research aimed at weakening the antioxidative barrier in cancer cells in future UV-based therapies.