INTRODUCTION

Obesity is a risk factor for many cancers and obese cancerpatients have a poorer prognosis (1). A positive associationbetween body mass index (BMI), visceral obesity and risk forgastrointestinal cancers is well documented (2-4). Colorectalcancer (CRC) is the third leading cause of cancer relatedmortality in USAand Europe (5). A number of mechanisms havebeen proposed for the adverse effect of obesity on colorectalcancer risk including the distribution of body fat, level of obesityrelated inflammation and oxidative stress, metabolicdisturbances as well as alternation in fat derived hormonalpatterns (6). Oxidative stress is also considered as a probablemechanism in the pathogenesis and development of manydiseases, including obesity and cancer (7).

White adipose tissue (WAT) is currently recognized as animportant endocrine organ. The physiological functions of

adipose tissue are changed in obesity, leading to an alteredsecretion of adipocytokines such as: visfatin, leptin, adiponectin,resistin, which may influence cancer pathogenesis andprogression (8). These adipokines may significantly influence thegrowth and proliferation of tumor stroma and malignant cellswithin primary tumor (9). Adipose tissue represents an importantsource of ROS, which may contribute to the development ofobesity-associated cancer. The increased levels of systemicoxidative stress that occur in obesity may contribute to theobesity-associated development of the insulin resistance and type2 diabetes, hypertension, atherosclerosis, and cancer (7, 10).

Visfatin/PBEF/Nampt is predominantly expressed in visceraland subcutaneous fat (11). This protein is a recently described asan adipocytokine with diverse and complex functions. Initiallynamed PBEF (pre-B cell colony enhancing factor) (12), it waslater found to have similarities with the nicotinamidephosphoribosilotransferasenadh gene from prokaryote

JOURNALOF PHYSIOLOGYAND PHARMACOLOGY 2015, 66, 4, 557-566

www.jpp.krakow.pl

R.J.BULDAK 1, M. GOWARZEWSKI1, L. BULDAK 2, M. SKONIECZNA3, M. KUKLA 4, R.POLANIAK 5, K. ZWIRSKA-KORCZALA1

VIABILITY AND OXIDATIVE RESPONSE OFHUMAN COLORECTAL HCT-116 CANCER CELLS TREATED WITH VISFATIN/ENAMPT IN VITRO

1Medical University of Silesia, School of Medicine with the Division of Dentistry, Department of Physiology in Zabrze, Zabrze, Poland; 2Medical University of Silesia, School of Medicine, Department of Internal Medicine and Clinical Pharmacology,

Katowice, Poland; 3Silesian University of Technology, Institute of Automatic Control, Biosystems Group, Department of Automatics, Electronics, and Informatics, Gliwice, Poland; 4Medical University of Silesia, School of Medicine,Department of Gastroenterology and Hepatology, Katowice, Poland; 5Medical University of Silesia, School of Public Health,

Department of Human Nutrition, Katowice, Poland.

Visfatin/eNampt is a novel adipokine, secreted by visceral and subcutaneous fat, which could be involved in thedevelopment of obesity-associated cancer. Only few studies revealed reactive oxygen species (ROS)-dependent actionof visfatin in endothelial cells, myotubes and melanoma cells. The potential pro-apoptotic properties of visfatin/eNamptin human colorectal HCT-116 cells remain unknown. The aim of the study was to examine the effects of visfatin/eNampton cell viability along with the determination of apoptosis/necrosis extent and ROS level in HCT-116 cells. Additionallyantioxidant enzymes’activities (i.e catalase (CAT), gluthatione peroxidase (GSH-Px)), and lipid peroxidation intensityin HCT-116 cells line was evaluated. Viability of HCT-116 cells was decreased after visfatin/eNampt treatment for 24hours. The number of apoptotic cells in tested cells treated with increasing visfatin/eNampt concentrations (10, 100, 250ng/ml) was elevated compared to untreated cells (6.4%, 9.7%, 16% vs. 3.2%; respectively). After 24 hours in thevisfatin/eNampt treated group (10 – 100 ng/ml) CAT and GSH-Px activities significantly increased and this observationwas accompanied by the decrease of ROS level when compared to the control group. Interestingly ROS level (usingDCF detection technique) and lipid peroxidation ratio were increased in cells stimulated by visfatin/eNampt inconcentration of 250 ng/ml along with the decreased activity of selected antioxidant enzymes when compared toremaining study groups, including control. We concluded that visfatin/eNampt induces decrease of cell viability andapoptosis boost in human colorectal cancer HCT-116 cells line. Visfatin/eNampt affected the level of ROS as well asantioxidant capacity, however the association of ROS level and apoptosis rate was not linear. The role forvisfatin/eNampt in cancer redox status in vitro may provide a greater insight into the association between fat derivedvisfatin/eNampt and its endocrine action in colorectal carcinoma cells.

K e y w o r d s: adipokine, visfatin/eNampt, HCT-116 cells, reactive oxygen species, apoptosis, necrosis, catalase, gluthationeperoxidase GSH-Px, lipid peroxidation, colorectal cancer

Haemophilus ducreyi, Nampt (in eukaryote cells) (13). In humans,serum levels of visfatin are elevated in subjects with visceral fataccumulation and type 2 diabetes mellitus (11, 14). Visfatinstimulates glucose uptake by adipocyte and muscle cells in vitroand decreases blood glucose levels in mice (15). Visfatin/iNamptexpression promotes cell growth and survival, angiogenesis, whatis more it is highly expressed in gastric and colorectal carcinomasas well as malignant glioblastomas (16-20). It is more highlyexpressed in primary colorectal cancer than in non-neoplasticmucosa (19). It has been shown that visfatin/iNampt may beoverexpressed by factors such as: hypoxia, serum deprivation andmethylmethane sulfonate in cell culture in vitro (21).Epidemiological studies also revealed that visfatin/eNampt levelsin human serum may be a good biomarker of colorectal malignantpotential, independently from BMI, and also stage of progressionof colorectal (22) and gastric cancer patients (17).

Visfatin/eNampt also induces ROS-reactive oxygen speciesgeneration in human umbilical endothelial cells (23),differentiated mice myotubes (24) as well as human malignantMe45 melanoma cells (25) in vitro. Recently, Wang et al.,reported that visfatin had no anti-apoptotic effect on normalcultured VSMCs, and it exerted an anti-apoptotic effect onlyduring cell apoptosis induced by H2O2, (26). Our previous studyalso revealed protective role of visfatin in hydrogen peroxide-induced DNAdamage in human melanoma Me45 cells (27).Recently, Jacques et al., demonstrated that visfatin inducedapoptosis in murine articular chondrocytes cultured in vitrothrough unknown pathway (28).

Various metabolic pathways produce ROS, includingaerobic metabolism in the mitochondrial respiratory chain. Itplays a critical role in the initiation and progression of varioustypes of cancers. ROS affect different signaling pathways,including growth factors and mitogenic pathways, they controlmany cellular processes, such as cell proliferation and apoptosis(29). Investigation of effects of higher level of intracellular ROSin cancer cells showed the occurrence of temporary growtharrest, senescence, apoptosis or necrosis (30). Apoptosis may beconnected to generation of hydrogen peroxide (H2O2) andcaspase activation, while necrosis is attributed to reduced ATPlevel and energy failure in conjunction with thiol depletion (31).The primary defenses of normal and cancer cells againstoxidative stress include antioxidant enzymes such as: superoxidedismutase (SOD) and glutathione peroxidase (GSH-Px).

Using cell model, we tried to verify the hypothesis ifvisfatin/eNampt derived from fat tissues influences on cellviability and apoptosis/necrosis extent along with oxidativestatus of colorectal carcinoma HCT116 cells cultured in vitro. Inthis case we measured intracellular ROS level and antioxidantenzymes activities in cell lysates. Because ROS level is alsoconnected with cell viability, HCT-116 cells viability and thelevel of apoptosis/necrosis cells in culture upon visfatin/eNamptstimulation were also studied. Changes in cellular ROS level andantioxidant enzymes activity in control cells were compared tothat which was stimulated by visfatin/eNampt at differentconcentrations (10, 100 and 250 ng/ml) which were also used inother in vitro studies (11, 24-27, 32, 33).

MATERIAL AND METHODS

Cell culture

Human colorectal carcinoma HCT-116 cells were obtainedfrom Silesian University of Technology, as a kind gift from drMagdalena Skonieczna. Originally cells were derived fromATCC: CCL-247 collection. Cells after recovery from frozenstock were seeded, at a density of 1 × 106 per 25 cm2 dishes, and

cultured with McCoy’s 5A modified medium (Sigma-Aldrich,MA, USA), supplemented with 10% fetal bovine serum (Gibco,North Androver, Mass), and antibiotics (0.1 mg/mLstreptomycin, 100 U/mLpenicillin; PAA Laboratories), as wellas: fungicide; amphotericin B (2.5 µg/ml); (PAA Laboratories).Cell cultures were led on standard sterile condition, under anatmosphere of 95% air and 5% CO2 at 37°C in HERAcellincubator (Thermo scientific). Afterwards, cells were harvestedby trypsinization, and the viable cells were counted in anautomated cell counter (model no. TC 20; Bio-Rad) using 0.4%trypan blue solution. The amount of dead cells in cell culture didnot exceed 4%. HCT116 cell line was free of bacteria species,mycoplasma and yeast-like fungi. Cultures were maintained forno longer than four weeks after recovery from frozen stocks.Fungizone was added to cell culture but only in the first passageof cells obtained from frozen stock, functional tests wereassayed without fungicide.

Drug preparation and treatment regimens

Visfatin/eNampt (Enzo Life-Science, Plymouth, PA, USA)was dissolved in phosphate buffered saline PBS, without Mg2+,Ca2+ (Sigma-Aldrich, St Louis, Mo). Solutions were preparedfresh, were protected from light exposure, and were added to theincubation medium in concentrations: 10 ng/ml, 100 ng/ml, and250 ng/ml. The purity of the visfatin was in the range of 96% -97% (SDS-PAGE analysis) and contained <0.01 ng µg–1 LPS asdetermined by the Limulusamebocyte lysate method. HCT116cells were incubated with or without different concentration ofvisfatin for 24 hours. During incubation period media from cellsculture were not removed. After this time period cells weretrypsinised (1% trypsin solution; PAA Laboratories) andcollected by centrifugation (2000 rpm for 3 min).

MTT assay

Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide salt (MTT, 5 mg/ml, Sigma-Aldrich, MA, USA). The cultures were incubated in 96-well platesat a density of 2 × 103 cells per well. After 24 h incubation, cellswere treated with various concentrations of visfatin/eNampt (10,100 and 250 ng/ml) and cultured for 24 h. After incubation, 10 mlof MTT reagent was added to each well and incubated for 4 h at37°C in the dark. The supernatant was aspirated and formazancrystals were dissolved in 200 ml of DMSO at 37°C for 15 minwith gentle agitation. The absorbance was measured at 540 nmusing the microplate reader model no. ELx800 (BioTec Ins).

Data was normalized to the absorbance of wells containingmedia only (0%) and untreated cells (100%). Cell viability (%)was expressed as a percentage compared to the controls usingthe following equation: Cell viability [X] = Ns × 100/Nc; whereNs and Nc are the absorbance of surviving cells treated withsample solution and the absorbance of control, respectively.Each experiment was repeated eight times for each sample.

Flow cytometry apoptosis/necrosis quantification

Cells were seeded at a density of 1 × 104/well in 24-well platesfor 24 h before treatment of visfatin/eNampt (10, 100, 250 ng/ml)for 24 hours. Apoptosis was quantified 24 h after visfatin/eNampttreatment using a two-parameter fluorescence-activated cellsorting (FACS) analysis with annexin V/propidium iodidedetection kit, according to manufacturer’s instructions(Invitrogen). Briefly, cells were detached with trypsin (1%),washed with 1 × PBS and then resuspended in 1 × binding buffer(10 mM HEPES; 140 mM NaCl; 2,5 mM CaCl2; pH 7,4), at aconcentration of 1 × 104 cells/ml. Afterwards, samples were

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stained with 5 mLannexin V conjugated with fluoresceinisothiocyanate (FITC) and 5 mLpropidum iodide (100 ng/ml);(PI), at room temperature for 15 min in the dark. They were thendiluted in 400 mLof binding buffer (samples on ice) and analyzedwithin 1 h using a flow cytometer Aria III, (BD Biosciences); withparameter setting for FITC-Annexin V conjugates (488 nm laserline, LPmirror 503, BPfilter 530/30), and for PI (488 nm laserline, LPmirror 566, BPfilter 585/42). Four experiments repeatedin triplicate were performed in this assay (n = 12).

Detection of reactive oxygen species using fluorimetric methodwith dichlorofluroescein

To monitor the intracellular ROS, we utilized cell-permeableoxidation sensitive fluorescent probes 5,6-carboxy-2’,7’-dichlorofluoresceindiacetate (DCFH-DA); (Molecular Probes,Leiden, Netherlands) using a fluorescent measurement system(Model Astroscan Cytofluor 2300/2350, Applied Biosystems,Millipore, Billerica, MA) as previously described. Non-fluorescent DCFH-DA, hydrolyzed to DCFH inside of cells,yields highly fluorescent DCF in the presence of intracellularhydrogen peroxide (H2O2). Therefore, the dichlorofluroescein(DCF) fluorescence intensity is proportional to the amount ofintracellular ROS. Samples of 2 × 104 cells were placed onCorning 6-well plates (Sigma-Aldrich, MA, USA) and were pre-incubated with 5 mM H2DCF-DAfor 1 h at 37°C. The plateswere centrifuged at 1200 rpm for 10 min and the fluorescence ofcontrol and treated cells was read in the Cytofluor reader(excitation at 504 nm, emission at 526 nm). The background ofdeacetylated, oxidized 2’,7’-dichlorofluorescein (DCF) wasapproximately 60 – 75 relative fluorescent units (RFU). Eachexperiment was repeated eight times for each sample.

Detection of reactive oxygen species using flow cytometry andCellROX® Green reagent

Samples of 5 × 105 cells were placed on Corning 6-wellplates (Sigma-Aldrich, MA, USA) and were incubated withvisfatin/eNampt (10, 100, 250 ng/ml) for 24 h in completegrowth medium. After this period, CellROX Green Reagent(Molecular Probes, Poland) mixed with DMSO (MolecularProbes, Poland) was added to cells to a final concentration of 5µM and incubated at 37°C in a 5% CO2 humidified incubator for30 min. Then cells were harvested, washed in PBS and analyzedon an Aria III flow cytometer (BD Biosciences). Fluorescencewas read in the flow cytometry; on the FITC configuration (488nm laser line, LPmirror 503, BPfilter 530/30). Each experimentwas repeated eight times for each sample.

Enzymes activity assays

Antioxidant enzyme activities: GSH-Px, CAT, and the levelof malondialdehyde (MDA) were measured in cell supernatants.Cells were collected after 24 h of incubation with different dosesof visfatin/eNampt (10, 100 and 250 ng/ml) and werecentrifuged (2000 rpm, 5 min) and supernatants were kept at–80°C until analysis.

Cell lysates preparation

Control and visfatin/eNampt-treated HCT116 cells werewashed twice in ice-cold PBS and lysed at 4°C in 200 ml of lysisbuffer (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris- HCl, 1%Triton X-100; all chemicals obtained from POCH Gliwice,Poland) and then sonicated for 10 s. Cell lysates were obtainedby centrifugation at 17.000 × g for 30 min at 4°C; proteinconcentration in the supernatant was determined by Bio-Rad

protein reagent (Bio-Rad Laboratories, Hercules, CA, USA),and lysates were adjusted to equivalent concentrations with lysisbuffer. Aliquots of 60 mg of total cell lysate were then frozen(–20°C) until antioxidant enzyme assays were performed.

GSH-Px activity assay

The method of Paglia and Valentine (34) was used withminor modifications as described previously (27) to measuredGSH-Px activity in HCT116 cell lysates. Firstly, humancolorectal HCT116 cells were pooled to a concentration of 5 ×106 cells/µl. After centrifugation, the cell pellet was mixed withcell lysis buffer and then sonicated for 10 s (as described above).The protein concentration was measured using Bio-Rad proteinreagent (Bio-Rad Laboratories, Hercules, CA, USA). Equalvolumes of each sample, containing 30 µg of protein were mixedwith 2.68 ml of 0.05 M phosphate buffer (pH 7.0), containing0.005 M ethylenediaminetetraacetic acid (EDTA). The followingsolutions were then added sequentially: 0.010 ml of glutathionereductase (GR), 0.100 ml of 0.0084 M NADPH, 0.01 ml of1.125 M sodium nitrate (NaNO3), and 0.1 ml of 0.15 M reducedglutathione (GSH). The enzymatic reaction was initiated by theaddition of 0.1 ml of 0.0022 M H2O2. The conversion of NADPHto oxidised NADP+ was followed by continuous recording of thechange in absorbency at 340 nm between 2 min and 4 min afterthe initiation of the reaction. The control measurements wererecorded in a simultaneous assay where the sample was replacedby an equal volume of cell lysis buffer. 1 IU of GSH-Px enzymeactivity is defined as 1mM NADPH converted to NADP+ permg of protein (IU/mg p). Analysis was carried out in duplicaterepeated six times; (n = 12).

Catalase activity

Catalase activity was measured spectrophotometrically asdescribed by Aebi et al. (35). Direct disappearance of 10 mMhydrogen peroxide in 50 mM potassium phosphate buffer (pH7.0), 1 mM EDTA was measured at 240 nm over 30 s on aBeckman DU-70 spectrophotometer. Enzyme activity wascalculated based on the molar extinction coefficient of hydrogenperoxide at 240 nm (ε = 39.4 M–1 cm–1) and reported as µmolhydrogen peroxide decomposed per minute, recalculated to kIUper mg of protein (kIU/mg p.). Analysis was carried out induplicate. Sample size in all experiments was 12.

Malondialdehyde assay

The measurement of MDA, a product of lipid peroxidation,was determined by the thiobarbituric acid (TBA) reaction asdescribed by Placer et al. (36) and Ohkawa et al. (37) with minormodifications. Aliquots of reaction buffer (1.5 ml) that included50 µg of protein from each sample and 1.4 ml of 0.2 M Tris -0.16 M KCl (pH 7.4) were incubated at 37°C for 30 min. Next,1.5 ml of TBA reagent were added, and the mixture was heatedin a boiling water bath for 10 min. After cooling with ice, 3.0 mlof pyridine:n-butanol (3:1, v/v) and 1.0 ml of 1 M NaOH wereadded and mixed by shaking, and the absorbance was read at 548nm. The blank control contained the same reaction mixture butwas not incubated. The level of MDAwas expressed as µmolMDA per mg of protein (µmol MDA/mg p.). Analysis wascarried out in duplicate. Sample size in all experiments was 12(four experiments repeated in triplicate).

Statistical analysis

Results are expressed as the mean +/– standard deviation(S.D.). The normality of distribution was checked by means of

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Shapiro-Wilk’ s test. The statistical analysis of the data wasperformed using one-way ANOVA followed by the post hocTukey honestly significant difference test or Kruskal-Wallis testwith Mann-Whitney tests according to variables distribution.The Bonferroni adjustment was applied for multiplecomparisons. Differences were considered significant for P<0.05. Statistical analysis was performed using a Statistica 7.0software (Statsoft, Poland). According to experiments, samplesize was 8 or 12; four experiments were performed in duplicate(n = 8) or in triplicate (n = 12), respectively.

RESULTS

The evaluation of the viability of human colon carcinoma cellsHCT-116 using MTTassay

According to MTTassay, a decrease in viability of humancolorectal HCT116 cancer cells line was observed after treatmentwith visfatin/eNampt for 24 hours. The percentage of live cellsafter the treatment with visfatin/eNampt (10, 100, 250 ng/ml) was89.2%, 87.7% and 83.8% compared to 97% in control cells;respectively (Fig. 1). The statistical significance was observed inall study groups when compared to control (untreated) cells.

The evaluation of the number ofapoptotic andnecrotic cellsafter thetreatment ofhuman colon carcinoma cells HCT-116with visfatin/Nampt

The treatment of HCT-116 cells with visfatin/eNampt for 24 hat various concentrations (10, 100, 250 ng/ml) resulted in thestatistically significant increase in the percentage of dead cellsreaching respectively 6.6%, 9.8% and 16.3% compared to controlcells - 3.6% (all P< 0.05). The apoptosis was responsible for themajority of the effect resulting in the percentage of apoptotic cellsreaching 6.4%, 9.7%, 16%, in cultures with visfatin/eNampt at 10,100 and 250 ng/ml, vs. 3.2% in control cells, P< 0.05. Both earlyapoptosis (4.5%, 6.7%, 10.2% vs. 2.3% in control cells; P< 0.05)and late apoptosis (1.9%, 3.0%, 5.8% vs. 0.9% in control cells; P< 0.05) were affected by increasing concentrations ofvisfatin/eNampt. Visfatin/eNampt did not change necrosis rate incells compared to control. Representative FITC-staining flowcytometry analysis for the visfatin-treated HCT-116 cells andcontrol shows Fig. 2.

The detection of reactive oxygen species using fluorimetricmethod with dichlorofluroescein

We tried to verify the hypothesis that occurring decrease inviability as well as increases apoptosis of HCT-116 cells treatedwith visfatin/eNampt was caused by changing the level of ROSfollowing visfatin/eNampt treatment.

Mean fluorescence of untreated HCT-116 cells after 24 hoursof culture resulted in 169.23 ± 5.3 RFU (relative fluorescenceunits). Visfatin/eNampt at 10 and 100 ng/ml reduced the ROSlevel 125.25 ± 10.72 RFU and 154.57 ± 5.47 RFU, respectively,compared to control cells 169.23 ± 5.3 RFU (both P< 0.05). Onthe other hand, visfatin/eNampt at 250 ng/ml resulted in asignificant, 18% increase in ROS level after 24 h culture (199.57± 5,47 RFU vs. 169.23 ± 5.30 RFU; (P< 0.05)) (Fig. 3).

The detection of reactive oxygen species using flow cytometryand CellROX® Green reagent

Compared to control cells (771.3 ± 5.6 RFU), visfatin/eNamptat 10 and 100 ng/ml for 24 hours reduced ROS level (725 ± 9.6RFU and 671.4 ± 1.5 RFU, respectively; both P< 0.05 vs. controlcells). ROS level after visfatin/eNampt at 250 ng/ml remainedunaffected compared to control cells (Fig. 4).

Activities of selected antioxidant enzymes andmalondialdehyde concentration in cell lysates derived fromcultures of HCT-116 cells

We tried to verify the hypothesis that ROS level in HCT-116cells was dependent on antioxidant capacity of tested cellsexposed to visfatin/eNampt, therefore we analyzed activity ofselected antioxidant enzymes (CAT, GSH-Px) in HCT-116 cellsexposed to visfatin/eNampt after 24 hours.

Catalase activity

Visfatin/eNampt at 10 and 100 ng/ml increased the catalaseactivity in HCT-116 cells compared to control cells (28.67 ±3.53 kIU/mg and 25.18 ± 3.89 kIU/mg, respectively vs. 18.22± 1.05 kIU/mg in control cells; both P< 0.05). Interestingly,visfatin/eNampt at 250 ng/ml diminished the activity ofcatalase (14.14 ± 0.36 kIU/mg vs. 18.22 ± 1.05 kIU/mg; P<0.05). (Fig. 5A).

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Fig. 1. Viability of human colorectalHCT-116 cells line aftervisfatin/eNampt treatment for 24hours. Cells were treated with variousconcentrations of visfatin/eNampt(10, 100 and 250 ng/ml) for 24 hours.Cell viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay.Results represent the mean ± S.D. offour independent experimentsrepeated in duplicate (n = 8); *P<0.05 vs. control.

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Fig. 2. A representative FITC-staining flow cytometryanalysis histogram for thevisfatin-treated HCT-116 cells.For each FITC report, the lowerleft quadrant represents thedetection of living cells, thelower right quadrant representsearly apoptotic cells, the upperright quadrant represents lateapoptosis cells, and the upperleft quadrant represents necroticcells after 24 h incubation withor without visfatin/eNampt (10,100, 250 ng/ml).

Fig. 3. (A) Effects of visfatin/eNampt treatment (10, 100 and 250 ng/ml) for 24 hours on intracellular ROS level in HCT-116 cellsusing DCF-loaded cells (A). DCF-detectable ROS were measured in all study groups and control (untreated cells). Values wereexpressed as a relative fluorescence units (R.F.U) and were analyzed with one-way ANOVA test and post-hoc Bonferroni correction.Data are expressed as means ± S.D. (n.=.12); a) P< 0.05 vs. control, b) P< 0.05 vs. visfatin (10 ng/ml)-treated group, c) P< 0.05 vs.visfatin (100 ng/ml)-treated group, d) P< 0.05 vs. visfatin (250 ng/ml)-treated group. (B) A representative merged images recorded bya confocal laser scanning microscopy (FluoViewTM FV1000, Olympus, Japan), from ROS signals stained with DCF (FITC channel,green) and nucleus stained with DAPI (DAPI channel, blue), magnification × 40.

Glutathione peroxidase activity (GSH-Px)

Visfatin/eNampt at 10 ng/ml did not affect GSH-Pxactivity, but the increased concentration of visfatin/eNamptreaching 100 ng/ml resulted in an increase in GSH-Px activitycompared to control cells (270.03 ± 8.72 IU/mg vs. 224, 07 ±4.89 IU/mg; P < 0.05). Nevertheless further increase invisfatin/eNampt concentration (250 ng/ml) caused a reductionin GSH-Px activity (197.02 ± 6.43 IU/mg vs.224.07 ± 4.89IU/mg; P< 0.05) (Fig. 5B).

The concentration of malondialdehyde

After 24 h of the treatment of HCT-116 cells withvisfatin/eNampt at 10 and 100 ng/ml significant reductions inMDA levels were observed (1.42 ± 0.1 µmol MDA/mg and1.01 ± 0.1 µmol MDA/mg vs. 1.76 ± 0.1 µmol MDA/mg incontrol group; P < 0.05). Contrary to the lowerconcentrations, visfatin/eNampt at 250 ng/ml increased MDAlevel in HCT-116 after 24 h treatment compared to control

cells (2.36 ± 0.3 µmol MDA/mg vs. 1.76 ± 0.1 µmolMDA/mg; P< 0.05) (Fig. 5C).

DISCUSSION

In our current paper we showed a decrease of viability ofhuman colorectal HCT116 cells after visfatin/eNampt treatment.In MTT a linear increase in the percentage of dead cells (10.6%,12.2% and 16.1%) was associated with increased concentrationsof visfatin/eNampt (10, 100 and 250 ng/ml, respectively). Incontrols the percentage of dead cells reached 3%. Similar resultswere obtained using flow cytometry (6.6%, 9.8% and 16.3%,respectively). Cells were dying due to increased apoptosis rate(6.4%, 9.7%, 16.0%). Similar level of necrosis was seen in allstudied groups and control cells. Recently, Jacques et al.,showed that visfatin/eNampt together with 1 mM APO866treatment (inhibitor of iNampt enzyme activity) inducedapoptosis in 4.5% of murine articular chondrocytes, whereasvisfatin/eNampt added solely to cell culture also induced

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Fig. 4. Effects of visfatin/eNampttreatment (10, 100 and 250 ng/ml) for24 hours on intracellular ROS level inHCT 116 cells using flow cytometryanalysis with CellROX green reagent(A). CellROX-detectable ROS weremeasured in all study groups andcontrol (untreated cells). Values wereexpressed as relative fluorescenceunits (R.F.U.) and were analyzed withone-way ANOVA test and post-hocBonferroni correction. Data areexpressed as means ± S.D. (n = 12); aP < 0.05 vs. control, bP < 0.05 vs.visfatin (10 ng/ml)-treated group, cP < 0.05 vs. visfatin (100 ng/ml)-treated group, dP < 0.05 vs. visfatin(250 ng/ml)-treated group.

Fig. 5. (A) Catalase (CAT), (B) Glutathione peroxidase (GSH-Px)enzymes’ activities and (C) malondialdehyde (MDA)concentration in the cell supernatants ofhuman colorectal carcinoma HCT116cell line upon visfatin/eNampttreatment (10, 100 and 250 ng/ml) for24 hours. Data represent means ± S.D.;n = 12 and were analyzed with one-wayANOVA; aP < 0.05 vs. control, bP< 0.05 vs. visfatin (10 ng/ml)-treatedgroup, cP < 0.05 vs. visfatin (100ng/ml)-treated group, dP < 0.05 vs.visfatin (250 ng/ml)-treated group.

apoptosis in 0.5% of chondrocytes in vitro via unknownmechanism (28). The effects of visfatin/eNampt onviability/proliferation are dependent on cellular model used instudy. Patel et al. showed visfatin-induced increases in cellproliferation in androgen-insensitive PC3 cells but not inandrogen-sensitive LNCaPcells, suggesting that circulatingvisfatin can exert differing effects based on cell characteristics.They also showed that in LNCaPcells visfatin/iNampt proteinwas detected more abundantly in nucleus than cytosol whencompared to PC-3 cells (33). Our previous findings also markedan influence of subcellular distribution of intracellularvisfatin/iNampt (nucleus vs. cytosol) on proliferative status ofHCT-116 cells cultured in vitro (38). It should be kept in mindthat others authors have reported that visfatin/eNampt expressesanti-apoptotic effects in HUVECs (32), neutrophils (39),hepatocytes (40) and beta pancreatic isles cells (41). Cheng et al.showed that visfatin/eNampt reduces the percentage of apoptoticbeta pancreatic cells induced by palmitic acid. This observationwas associated by increased expression of Bcl-2 and reducedexpression of caspase-3. PI3K/Akt and Erk1/2 kinases wereresponsible for the intracellular signaling in the above mentionedexperiments (41).

We tried to verify the hypothesis that visfatin-induceddecrease of viability of HCT-116 cells was dependent onchanging ROS level followed visfatin treatment of tested cells.

Reactive oxygen species (ROS) are side products of oxidativemetabolism in living cells. They play important role in cellphysiology, e.g. in intracellular signal transduction, proliferation,cell cycle control, cellular differentiation and aging (42–44). ROSat low concentrations tend to promote cell differentiation, whereashigh concentrations induce cell death (apoptosis or necrosis) (45).Currently ROS are assayed in cell supernatants using fluorescence(fluorescence microscopy, flow cytometry, fluorymetry) orconfocal microscopy when a spacial distribution of ROS in cellsis evaluated (46). In our paper quantitative analysis of ROS inhuman colorectal cells HCT-116 treated with variousconcentrations of visfatin was performed. Two different methodswere employed in the study: (i) fluorymetric analysis of thereaction between ROS and a H2DCFDA producing a specific dyeand (ii) flow cytometry with specific reagent for the detection ofROS - CellROX® Green reagent.

We showed that visfatin/eNampt at 10 and 100 ng/ml led toa decrease in ROS concentration in HCT-116 cells. Fluorescentmethod showed 26% and 23% in respective concentrations,whereas flow cytometry showed 6% and 13% reduction. On theother hand, visfatin/eNampt at 250 ng/ml was associated with asignificant rise in ROS concentration (18%) that was detected influorescent method but it was absent in flow cytometry.Quantitative measurement of ROS in cells requiressophisticated research techniques. This is a consequence ofshort half-lives of ROS and activity of enzymatic and non-enzymatic pathways degrading ROS in living cells. DCFmethod is predominantly specific for the detection of hydrogenperoxide (H2O2). This is a result of a propensity of hydrogenperoxide to oxide H2DCFDA, which leads to the generation offluorescent dye DCF. Hydroxyl radicals (OH–•) do notparticipate in the transformation of H2DCFDA into DCF.Therefore results derived from DCF-based methods ofassessment of oxidative stress should be taken with caution. Onthe other hand Le Bel et al. stated that H2DCFDA may betransformed into DCF by other oxide-reductive reactions andconcluded that DCF method of ROS detection detects also otherthan H2O2 ROS in cells (47).

A positive association between oxidative damage (lipidperoxidation, DNAdamage) and the number of apoptotic cellsis seen. The accumulation of DNAand lipid damage lead toprogrammed cell death - apoptosis. Cai et al. (48) noted

increased susceptibility to irradiation in colon cancer cell lines(LS1747 and HT-29) after stimulation with docosahexaenoicacid (DHA) and eicosapentaenoic acid (EPA). LS1747Tcoloncancer cell line is much more susceptible to ionizing radiationthan HT-29 cell line. Addition of EPA and DHA increasedapoptosis rate after irradiation of cell cultures, which may be aresult of increased ROS generation, lipid peroxidation andDNA damage (48). In our current study we have shown thatvisfatin/eNampt administration to HCT116 cell culturesresulted in a decrease of viability of these cells after 24 hours.However this effect was not linearly associated with ROS level.Visfatin/eNampt at 10 ng/ml even reduced ROS generation, butstill a significant rise in apoptosis rate was observed. On theother hand high visfatin/eNampt concentration (250 ng/ml)resulted in the highest apoptosis rate (16% vs. 3.2% in controlcells) which was accompanied by a substantial rise in ROS level(18% using DCF method) and MDAlevel (33%). Thesephenomena may stem from insufficiency in antioxidativepathways of HCT-116 cells when they are challenged with highconcentration of visfatin/eNampt (250 ng/ml). At lowerconcentrations of visfatin the oxidative burst was blunted by theincreased antioxidant enzymes (CAT, GSH-Px) activity.According to our findings we cannot state that visfatin/eNamptin HCT-116 cells induces apoptosis as a result of increased ROSgeneration. Others have also reported that apoptosis may not beconnected with increased ROS level in dexamethasone-inducedapoptosis of murine thymocytes (49). It should be also stressedout that our research methods were focused on the detection ofH2O2. Other ROS or RNS (reactive nitrogen species) mightindeed be responsible for the additional oxidative damage thatis not caused by hydrogen peroxide only, which finally maylead to increased apoptosis (50).

There are few studies exploring the in vitro generation ofROS in cells stimulated with visfatin/eNampt. Other researchersshowed elevated levels of ROS after the administration ofvisfatin in culture medium in vascular smooth muscle cells(VSMC) (26), murine myocytes (C2C12) (24), and humanmelanoma cells (Me45) (25). The level of ROS was increased2.5-fold in C2C12 cells treated with visfatin/eNampt (100ng/ml) for 18 h and 3-fold in Me45 cells treated withvisfatin/eNampt at the same concentration for 24 h; respectively(24, 25). Contrary to the above-mentioned studies we showed amodest (13%) decrease in ROS in similar culture conditions inhuman colorectal HCT-116 cells line. The increase in ROS wasseen only when visfatin/eNampt at 250 ng/ml was used and wasdetectable solely in DCF method. This observation was notconfirmed in flow cytometry using CellROX® green reagent,because it gave stronger fluorescence after intracellularmacromolecule binding (DNA-specific dye), and then enhancesoxidized signal from ROS - the first signal comes only fromnucleus and mitochondria (51). Moreover, It is not excluded thatother ROS scavenging enzymes such as: SOD isoenzymes andTRX enzyme may alters ROS level detected by CellROX inHCT-116 cells exposed to visfatin/eNampt.

These findings were followed by changes in MDAlevel aftervisfatin/eNampt treatment. In order to assess the changes in ROSand MDAlevel we evaluated the activity of selected antioxidantenzymes activitiy such as: CAT and GSH-Px. The diverseinfluence on H2O2 level in HCT-116 cells may result from theinfluence of visfatin/eNampt on antioxidant enzymes (catalaseand glutathione peroxidase) that degrade hydrogen peroxide.Catalase (CAT) changes hydrogen peroxide into water andoxygen: 2H2O2 › 2H2O + O2. Glutathione peroxidase (GSH-Px)deals not only with hydrogen peroxide but also with lipidperoxides (52). Decreased concentrations of ROS may beattributable to the increased activity of CAT and GSH-Px duringcell culture with 10 and 100 ng/ml of visfatin/eNampt. For

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instance culture with visfatin/eNampt for 24 hours resulted in38% increase in CAT activity and 20% increase in GSH-Pxactivity, which was associated with reduced ROSconcentrations. Interestingly in cells treated withvisfatin/eNampt at 250 ng/ml a significant rise in ROS wasconnected with the decrease in CAT and GSH-Px activities. Ourprevious study showed increased proliferation rate in melanomaMe45 cells that were treated with visfatin/eNampt. Wespeculated that the influence of visfatin/eNampt on cellproliferation depends on sufficient antioxidative response, whichdeals with ROS excess. Visfatin/eNampt (100 and 250 ng/ml)greatly increased the CAT activity compared to control cells(32.1 kIU/mg p and 35.66 kIU/mg p vs. 7.55 kIU/mg p in controlcells), which might reflect decrease in ROS level (27). In ourcurrent study on HCT-116 cells visfatin/eNampt at 100 ng/mlslightly increased the activity of CAT (25.18 kIU/ mg p vs. 18.22kIU/ mg p; P< 0.05) but at higher concentration (250 ng/ml)induced a drop in CAT activity (14.14 kIU/mg p vs. 18.22kIU/mg p; P< 0.05).

The difference in the effects of visfatin/eNampt on Me45and HCT-116 cells may be a result of differentproliferative/apoptotic reaction. Me45 cells showed increasedproliferation rate after visfatin/eNampt, while HCT-116 cellsshowed cytotoxic features and increased apoptosis. The finaleffect is therefore depending on ROS generation rate and theactivity of antioxidant enzymes.

GSH-Px was similarly affected by visfatin/eNampt in bothMe45 and HCT-115 cells. Visfatin/eNampt at 100 ng/ml doubledthe GSH-Px activity in Me45 (210% of control) and significantlyrised it in HCT-116 (120% of control). However when avisfatin/eNampt concentration was increased to 250 ng/ml thenGSH-Px activity was lowered in both Me45 and HCT-116 (60%and 80% of control, respectively). GSH-Px reduces not onlyhydrogen peroxide but also lipid peroxides. In HCT-116 cellsvisfatin/eNampt at 100 ng/ml was associated with a reducedlipid peroxides assessed by MDAlevel, but 250 ng/ml ofvisfatin/eNampt resulted in a 33% increase in MDAconcentration. Buldak et al. (53) made similar observations inMe45 cells treated with visfatin/eNampt. Difference in MDAlevel may reflect divergent intensity of oxide-reductiveprocesses in cultured control cells, which may affect furtheractions of visfatin/eNampt on cells.

In summary, we showed that visfatin/eNampt inducesdecrease of cell viability and apoptosis in human colon cancerHCT-116 cells. Visfatin/eNampt affected the level of ROS level,however the association of ROS level and apoptosis rate wasnot linear. Moreover, elevation of antioxidant enzyme activitiesprobably explains the drug resistance phenotype of somecancers cells (53), in this case, HCT-116 cells may cannotadaptive enough to ROS stress and apoptosis was occurs in cellculture.

Our study has a few limitations. Its in vitro setting may notfully reflect more complex relationships in organisms as a whole.The in vitro setting of the study must be kept in mind. The resultmay not be identical to those observed in living subjects.However authors put much effort on the exploration of oxidativestress, including two distinct methods of ROS assays and a broadspectrum of the experiments on activities of antioxidant enzymes.

Visfatin induced cytotoxic effects and promoted apoptosis inhuman colon cancer cells HCT-116 in vitro. Visfatin at lowerconcentrations (10, 100 ng/ml) reduced oxidative stress, but athigh concentration (250 ng/ml) it showed pro-oxidative featuresin HCT-116 cells.

Visfatin at 100 ng/ml induced the activity of selectedantioxidant enzymes (CAT, GSH-Px) in HCT-116 cell in vitro.However at the concentration of 250 ng/ml it reduced antioxidantcellular potential.

Acknowledgements: Biological experiments were performedin the Biotechnology Center of the Silesian University ofTechnology and Medical University of Silesia in Katowice usingequipment financed by the “Silesian Biofarma” program; M.S.was also supported by grant No SUT-BK 256/Rau1/2014 t.3from Polish Ministry of Science.

Conflict of intersts: None declared.

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R e c e i v e d :October 13, 2014A c c e p t e d :April 22, 2015

Author’s address: Dr. Rafal J. Buldak, Medical University ofSilesia, School of Medicine with the Division of Dentistry,Department of Physiology, 19 Jordana Street, 41-808 Zabrze,PolandE-mail: rbuldak@gmail.com

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