Modulation of mRNA expression and activities of xenobiotic metabolizing enzymes , CYP 1 A 1 , CYP 1 A 2 , CYP 2 E 1 , GPx and GSTP 1 by the Salicornia freitagii extract in HT-29 human colon cancer cells

Phase I-II detoxification and antioxidant enzymes are responsible for the detoxification and elimination of activated carcinogens, acting as important biomarkers for chemoprevention. Among them, cytochrome P450s plays a prominent role in the metabolic activation of xenobiotics. The herb Salicornia freitagii (SF) (Amaranthaceae) is known for its anticancer, antioxidant, antidiabetic and antiinflammatory activities. In this study, we determined the bioactive phenolics in the SF methanol extract and investigated its antiproliferative potential in HT-29 human colon cancer cells. We also investigated the modulation of some phase I and II enzyme (CYP 1A1, 1A2, 2E1, GSTP1 and GPx) mRNA expression and enzymatic activities by the SF extract and its major bioactive phenolic compounds. LC/MS-MS analysis showed that the main phenolic compounds of the methanolic SF extract are vanillic acid (48 μg/100g) and p-coumaric acid (10.8 μg/100g). SF extract, vanillic acid and p-coumaric acid exhibited high antiproliferative activities in HT-29 cells, with IC50 values of 81.79μg/mL, 98.8 μM and 221.6 μM, respectively. The mRNA expression levels of CYP1A2 and CYP2E1 were decreased, while those of GSTP1 and GPx in HT-29 cells were increased after application of either the SF extract or vanillic acid. The SF extract by itself also increased the activities of GPx and GSTP1 enzymes 1.68and 1.49-fold, respectively. Our data indicate that the SF extract and its major bioactive compound, vanillic acid, could exert a modulatory effect on the expression of enzymes that are involved in xenobiotic activation and detoxification pathways in the gastrointestinal tract. For this reason, SF can be considered as a natural source of chemopreventive agents.


INTRODUCTION
Plant phytochemicals have gained importance in recent years due to their diverse biological properties, including antioxidant, antimicrobial, antifungal and anticancer activities.Glassworts, a group of succulent halophytic plants, belong to the Amaranthaceae family.They are consumed as vegetables and used as medicinal plants around the world.They have been classified in two different genera as Salicornia and Sarcocornia.Many epidemiological studies have revealed a relation between health benefits and the consumption of glassworts [1][2][3].Because they are rich sources of protein, vitamins, dietary fiber, essential fatty acids and minerals, they have become essential for human nutrition [4][5][6][7].Glassworts have been reported to be rich sources of antioxidant compounds [8][9][10][11].Therefore, the significance of glassworts is not only due to their nutritive values but also because they are an excellent source of bioactive compounds that possess potentially important therapeutic effects, such as anticancer, antioxidant, antihyperlipidemic, antidiabetic and antiinflammatory activities [12][13][14][15].Glassworts are used for the treatment of intestinal ailments, nephropathy, cancer, asthma, arthritis, hepatitis, hypertension and hemorrhoids [16,17].Colorectal cancer, which is the third most common cancer type worldwide and the fourth leading cause of cancer-related death, is diagnosed in over one million new cases per year [18].As most gastrointestinal system cancers are highly linked to dietary habits, they are considered to be preventable [19].
By bioactivating a variety of xenobiotics, including drugs, food additives, industrial solvents and pollutants, to highly reactive and mutagenic metabolites, the cytochrome P450 (CYP) monooxygenase system assumes a key role in toxicity and carcinogenesis [20].Although the main function of CYP enzymes is detoxification of xenobiotics, many CYP isoforms catalyze the metabolic activation of procarcinogens to their ultimate carcinogenic forms [21].The induction of some CYP1A isozymes that metabolize a variety of polycyclic aromatic hydrocarbons (PAHs), is mediated by the aryl hydrocarbon receptor (AhR), which is involved in various biological processes [22].Benzo[a] pyrene (BaP) plays a major role in lung cancer development.Conversion of inactive Benzo[a]pyrene (BaP) to the mutagenic metabolite BaP-7,8-diol-9,10epoxide, and the subsequent formation of DNA adducts mainly with deoxyguanosine is an example of the catalytic activity of CYP1A1 [23].CYP2E metabolizes many drugs, toxicants and carcinogens, such as acetaminophen, nitrosamines, phenol, benzene, 4-nitrophenol, carbon tetrachloride, chloroform, pyrazole and vinyl chloride [24,25].The most prominent CYP isoforms expressed in colon tissue are CYP 1A1, 1A2, 2E1 and 3A4 [26].
Phase II drug-metabolizing enzymes convert xenobiotics or active chemical carcinogens to less toxic or inactive metabolites that are readily eliminated from the body [20].Glutathione S-Transferases (GSTs) are phase II detoxifying enzymes that metabolize electrophilic substrates containing carbon, nitrogen and sulfur atoms by conjugation reaction with the endogenous tripeptide glutathione (GSH), which results in the formation of less reactive and more water soluble products [27].Some drugs, including cisplatin and carmustine, are substrates of GSTP1 and GSTM1, respectively, and are excreted by conjugation with glutathione [28,29].The enzyme glutathione peroxidase (GPx), catalyzes the reduction of hydrogen peroxides to water and alcohols at the expense of GSH [30].It protects the organism from oxidative damage by re-ducing fatty acid hydroperoxides, H 2 O 2 , phospholipid hydroperoxides and cholesterol hydroperoxides [31].It was reported that GPx knockout mice were highly susceptible to oxidative stressors [32].Cardiovascular diseases and stroke incidences were found to be highly increased in the absence of GPx [33].
In this context, the inhibition of CYP1A1, CYP1A2 and CYP2E1 enzymes and the activation of GSTP1 and GPx enzymes could represent a promising cancer chemoprevention strategy.Therefore, in this study we aimed to determine the major phenolic compounds of SF, to examine their antiproliferative effects and to elucidate their effects on several phase I, phase II and antioxidant enzymes in HT-29 cells.

Plant material and extract preparation
Naturally growing samples of the glasswort SF were collected from Şereflikoçhisar, Ankara Province, Turkey, in 2014.Voucher specimens are kept in the Laboratory of Seed Plant Systematic, Department of Biology, Ankara University, Turkey.The aerial parts of the plants were dried at room temperature and protected from direct exposure to sunlight.About 30 g of dried leaves were mixed with 250 mL of methanol and shaken overnight.This process was performed three times.To obtain ultra-dry powders, the methanol extract was concentrated under vacuum in a rotary evaporator (Heidolph, Germany) and then freezedried (Christ, Germany).The extracted powder was weighed and stored at -20°C in a brown bottle until further use.

Identification and quantification of phenolic compounds by LC-MS/MS
The methanol extract of SF was examined for its phenolic content by liquid chromatography (Agilent 1200 equipped with a micro degasser, autosampler and diode array detector (DAD), connected to an Agilent 6460 triple ion trap mass spectrometer via an electrospray interface).Separation was achieved on a Zorbax SB-C18 column (2.1x50 mm x 1.8 µm) at a flow rate of 0.3 mL/min at 35 o C, using solvent A (0.05% formic acid, 5 mM ammonium formate) and solvent B (methanol) as a mobile phase.A gradient system was performed and running time was 13 min.The DAD recorded the spectra from 180 to 800 nm.Standards were prepared in the range of 0.031 to 10 ppm.The extract was dissolved in methanol and filtered (0.22 µm) to obtain a final concentration of 1mg/mL.The injection volume into the LC-MS/MS instrument was 5 µL.Agilent G3793AA MassHunter Optimizer software was used for data evaluation.

Cell culture
HT-29 human colon adenocarcinoma cells were obtained from ATCC (American Type Culture Collection, LGC Promochem, UK).Cancer cells were grown in McCoy's 5A medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen Strep) solution.Cultures were incubated at 37°C with 5% CO 2 and 95% humidity.Cell culture studies were performed in a Metisafe Class II Safety Cabinet.

Cytotoxicity assay
Antiproliferative activity tests were performed by measuring the cellular metabolic activity by the colorimetric 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H tetrazolium-5-carboxanilide (XTT) assay.Cells were plated at a concentration of 1x10 5 cells/mL in 96-well culture plates and allowed to adhere overnight at 37°C.After 24 h, the cells were treated for 48 h with differ-ent concentrations of extract (20-150 µg/mL), vanillic acid (50-200 µM) and p-coumaric acid (50-400 µM).After the incubation, the antiproliferative effects of the agents were evaluated using the XTT cell proliferation kit (Biological Industries, Israel).Absorbance was measured at 415 nm with a Multiscan GO microplate reader (Thermo Scientific, USA).Controls were treated with DMSO alone (0.2%).The results were expressed as IC 50 (µg/mL) for the extract and IC 50 (µM) for the phenolic compounds, and all are presented as means±SEM of three independent experiments, each performed in triplicate.

RNA isolation, cDNA synthesis and quantitative real time qPCR
HT-29 cells (25x10 4 cells/mL) were seeded in 6-well plates and incubated for 24 h in a CO 2 incubator at 37 o C. The following day, the medium was refreshed and the cells were treated with SF extract and its main bioactive components, vanillic and p-coumaric acid, at the concentrations of the determined IC 50 values and incubated for 48 h.After incubation, isolation of total RNA was performed according to the Gene-Jet RNA Purification Kit (Thermo Scientific USA).The isolated RNA was quantified by measuring the absorbance at 260 nm and its purity and quality were evaluated by measuring the 260/280 nm ratio.Reverse transcription of RNA to cDNA was performed using the RevertAid™ First Strand cDNA Synthesis Kit (Thermo Scientific USA) according to the manufacturer's protocol.The synthesized cDNA was stored at -80 o C until further use.
The effects of SF extract, vanillic acid and p-coumaric acid on the expression of selected genes in HT-29 cells were studied by quantitative real-time PCR (qRT-PCR) using Corbett Rotor Gene 6000 (Corbett Life Science, Concorde, NSW, Australia).Ten µL of the final reaction mixture contained 1.6 µL cDNA (50-100 ng), 0.2 µL of 10 µM of the forward primer, 0.2 µL of 10 µM of the reverse primer, 5 µL of Maxima®SYBR Green qPCR Master Mix (Fermentas, Glen, Burnie, MD) and 3 µL of RNase-free distilled water.In order to check for genomic DNA contamination, NTC (no template control) was used during the reaction.As an internal standard, GAPDH was used.The qRT-PCR program consisted of the following cycling profile: ini-tial melting at 95°C for 10 min; the amplification and quantification program was repeated 40 times; melting: 95°C for 20 s, annealing: 58-62°C (depending on the gene), as shown in Table 1, for 30 s; extension: 72°C for 20 s.In order to confirm the PCR product, melting curve analysis of the amplification product was carried out at the end of each amplification reaction.

Protein extraction
HT-29 cells (25x10 4 cell/ mL) were seeded in tissue culture plates for protein extraction.After 24 h, the cells were treated with the SF extract at a concentration corresponding to the IC 50 (mg/mL) value and incubated for 48 h.Protein extraction was performed with 400 µL of 1X RIPA buffer containing1 mM phenylmethanesulfonylfluoride (PMSF).The cells were scraped and lysed by sonication for 5 min and centrifuged at 14000xg for 10 min.The collected supernatant was stored at -80°C until further use.The protein concentration was determined according to the Lowry method [34].

Measurement of enzyme activities
GST activity was determined by monitoring thioether formation between 1-chloro-2,4-dinitrobenzene (CDNB), which served as the substrate, and GSH as a cofactor, at 340 nm [35].Briefly, to a single well of a 96-well plate, 162.5 μL dH 2 O, 50 μL 500 mM phosphate buffer pH 7.4, 10 μL 25 mM GSH and 15 μL enzyme source (20-50 μg protein) were added.The reaction was started after the addition of 12.5 μL of 20 mM CDNB and measured for 5 min.GST activity was expressed as nmol/min per mg protein; the extinction coefficient was 6.29 mM -1 cm -1 .
GPx activity was measured according to [36], with some modifications.In a single well of a 96-well plate, 105 μL of 0.1 M Tris-HCl buffer (pH:8.0), 25 μL of 3 mM GSH, 25 μL of 0.5 unit/mL glutathione reductase (GR) and 20 μL of the cytosolic fraction were added and incubated for 3 min.The reaction was started after the addition of 25 μL H 2 O 2 .Reactions were measured for each well every 10 s for 4 min at 240 nm with a Multiskan™ GO spectrophotometer (Thermo Scientific, USA).GPx activity was expressed as nmol/min per mg protein; the extinction coefficient was 0.00373 mM -1 cm -1 .

Statistical analysis
Statistical analyses were performed by Student's t-test using the GraphPad Prism ver.6 statistical software package for Windows.All results were expressed as means with their standard deviation (SD); p<0.05 was chosen as the minimum level for significance.

Characterization of SF extract by LC-MS/MS
Fig. 1A shows the chromatogram used to identify and quantify ten phenolic compounds, namely: vanillic acid, p-coumaric acid, caffeic acid, gallic acid, syringic acid, chlorogenic acid, rosmarinic acid, (-)-epicatechin (EC), epigallocatechin gallate (EGCG) and rutin trihydrate in the SF methanolic extract.LC-MS/MS analysis revealed that the SF methanol extract contained high concentrations of vanillic acid (48 µg/100 g extract) and p-coumaric acid (10.8 µg/100g extract), which were therefore by far the predominant phenolic compounds of all of the phenolics (Fig. 1B).All other phenolics were below the limit of quantification.

Cytotoxic activities
The cell-growth inhibitory effects of the SF methanol extract and its bioactive components on HT-29 human adenocarcinoma cell lines were investigated by the XTT assay.This has not been examined previously.
The findings from the present study clearly demonstrate that the extract, vanillic acid and p-coumaric acid can inhibit the proliferation of human cancer cells in a time-and concentration-dependent manner (Fig. 2).The XTT assays revealed the absence of any antiproliferative effects of the SF extract on HT-29 cells up to 24 h incubation.On the other hand, a 48-h treatment showed that proliferation of the cells decreased critically in the presence of the SF extract in the range of 25 to 200 µg/mL.At 200 µg/mL, the extract displayed maximum antiproliferative activity, observed as a significant decrease in cell viability.Additionally, vanillic acid and p-coumaric acid exhibited their cytotoxic activities against HT-29 cancer cells in the range of 25 to 500 µM.The IC 50 values of SF extract, vanillic and p-coumaric acid were determined as 81.79 µg/mL, 98.8 µM and 221.6 µM, respectively.

Effects of SF extract on enzyme activities of GPx and GSTP1 in HT-29 Cells
Fig. 5 shows the effects of the SF extract on GPx (Fig. 5A) and GSTP1 (Fig. 5B) enzyme activities in HT-29 cells.The SF extract at the concentration corresponding to IC 50 increased cytosolic GPx activity 1.68-fold (p<0.005).The SF extract also increased the activity of cytosolic GSTP1 1.49-fold (p<0.005).

DISCUSSION
In recent years, traditional medicine such as herbal remedies or dietary supplements, have gained increasing popularity, encouraging investigators to examine the actions of phytochemicals on xenobiotic metabolism and their antioxidant, antitumor, anticarcinogenic and antimutagenic effects.The major phenolic compounds in SF extracts have been reported to be  vanillic acid, p-coumaric acid, ferulic acid, caffeic acid, kaempferol and galangin [37].Our SF methanol extract showed close similarities with the literature, displaying high amounts of vanillic and p-coumaric acid.
It has been reported that some CYP1A and CYP2E enzymes are expressed at high levels in extrahepatic tissues, such as the colon and intestine, and deemed to be responsible for the formation of unique extrahepatic metabolites and resulting tissue-specific consequences in cellular toxicity and organ pathology [26].The expression of CYPs, phase II and antioxidant en-zymes is controlled at the transcriptional and translational levels.The mRNA levels of CYP genes were shown to be affected by the expression of micro RNAs [38,39].To examine aspects of the control mechanism of these enzymes after exposure to SF extract and its major phenolic compound vanillic acid at the transcriptional and translational levels, we examined changes in mRNA expression and enzyme activities.
CYP1A1and CYP1A2 are the principal members of the CYP1A family that are induced by PAHs and are also involved in the metabolic activation of PAHs and heterocyclic amines [40].The metabolism of PAHs by members of the CYP1A family generates reactive products that irreversibly bind to protein and DNA, producing toxic and carcinogenic events [41].It was reported that certain phenolic compounds/flavonoids have roles in reducing cancer susceptibility,  especially by preventing the induction of CYP1A isozymes [42,43].In light of the role of CYP1A in the formation of reactive products, the observed inhibition of CYP1A mRNA expression by the SF extract and its major phenolic component vanillic acid could lead to the reduction or prevention of the onset of many diseases, including cancer.In some cases, the modulation of CYPs has been attributed to other minor bioactive phenolic compounds and/or their synergistic effects in the extract [44].Our results demonstrated that the induction of CYP1A mRNA expression by the SF extract can be attributed to this event since vanillic acid, the major phenolic compound of the extract, suppressed CYP1A mRNA expression.Xenobiotic responsive elements (XRE) are located in the promoter regions of xenobiotic responsive genes encoding for some CYP isozymes.The expression of these genes can be regulated through the aryl hydrocarbon receptor (AhR), which is a cytosolic protein that can be activated by PAH.The activated AhR then translocates to the nucleus and dimerizes with the AhR nuclear translocator (ARNT) and finally interacts with XRE [45].The induction of CYP1A isozymes by phenolic compounds occurs through various mechanisms, including direct stimulation of gene expression via specific receptors [46].Some flavonoids induce CYPs by binding to AhR by mimicking 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [47].Although a variety of flavonoids are dietary ligands of the AhR, they can exert different effects on CYP expression [48].Suppression of CYP isozymes by the SF extract and vanillic acid could be attributed to their binding to the AhR by blocking the binding of other AhR ligands, such as TCDD.In this context, the SF extract and vanillic acid behave as antagonists of AhR.The reason why p-coumaric acid did not have any considerable effect on CYP450 enzymes may be due to its differential structure (it does not optimally fit into the binding site on AhR) from vanillic acid.
Similarly, CYP2E1 has a wide range of exogenous substrates, such as industrial solvents, protoxins and procarcinogens, and some drugs such as acetaminophen, chlorzoxazone and methoxyflurane are metabolized by CYP2E1 [49].Thus, inhibition of CYP2E1 can prevent the conversion of such procarcinogens to their carcinogenic forms such as acrylamide, nitrosamine, phenol and benzene.Consequently, in the present study, the inhibitory mechanisms of the SF extract and its major phenolic component vanillic acid on CYP2E1 might be regarded as a protective effect due to the potential suppression of tumor formation, induced by PAHs, drugs and other carcinogens.
Flavonoids in dietary foods were shown to be effective in the induction of many phase II enzymes, including GPx, GSTs and NQO1 [50,51].The importance of GST in chemical carcinogen inactivation was reported in a study on mice deficient in transcription factor Nrf2, which is required for Phase II enzyme induction [52].In our study, the SF extract and its major phenolic compounds, vanillic acid and p-coumaric acid, induced the GSTP1 mRNA expression.It seems that both phenolic compounds had a major impact on the modulation of GSTP1 enzyme.Therefore, the induction of GST by the vanillic acid-and p-coumaric acid-rich SF extract could help eliminate toxic metabolites from cells and would therefore have an important role in early defense against carcinogenesis.
GPx has been long been known to play a role in the first step of defense against reactive oxygen species (ROS).Increased levels of GPx activity could enhance the resistance against ROS.It was reported that GPx1-overexpressing mice were more resistant to paraquat-induced lethality than GPx1 knockout mice [53].Furthermore, increased levels of GPx have been shown to have a protective role in cardiovascular diseases, as ROS cause significant changes in vascular tone and structure [54].The SF extract and vanillic acid increased the expression of GPx mRNA, while p-coumaric acid did not.Therefore, the combined induction of GPx and GSTP1 by the SF extract and/or vanillic acid could represent a remarkable enhancement of the defense against the toxicity of various agents.
The examination of enzyme activities showed that the levels of GPx and GSTP1 activities were significantly increased by the SF extract.These results demonstrate that the modulation of the investigated enzymes by the SF extract occurred at gene and enzyme activity levels.Moreover, cross analyses of gene expression vs. enzyme activity revealed a strict correlation between the mRNA expression levels and corresponding enzyme activities in SF-treated and control groups.

CONCLUSIONS
The current state of our knowledge indicates that the selective induction of carcinogen-detoxifying enzymes and suppression of enzymes responsible for xenobiotic activation could be a useful approach in chemoprevention and carcinogenesis inhibition.The present study demonstrated that the SF methanol extract and its major bioactive compound, vanillic acid, exert modulatory effects on the expression of the enzymes involved in xenobiotic activation and detoxification pathways.However, necessary precautions should be taken before this plant is consumed in replacement treatments because of its possible interactions with drugs and dietary foods.

Fig. 2 .
Fig. 2. Concentration-dependent cytotoxic effects against HT-29 cell lines after 48 h of treatment.SF extract (a); vanillic acid (b); p-coumaric acid (c).Each point represents the average of three independent measurements, each done in triplicate, with the means±SD.

Fig. 5 .
Fig. 5. Effects of SF extract on the activities of antioxidant enzymes in HT-29 cells.A -GPx; B -GSTP1.Enzyme activities were determined as described in the Materials and Methods section.Values are means±SD for triplicate determinations (n≥3).Significantly different from the control by two-tailed Student's t-test -*p<0.05,** p<0.001.

Table 1 .
Primer pairs used for amplification, with annealing temperatures and sizes of the PCR products.