Effects of chlorogenic acid, epicatechin gallate, and quercetin on mucin expression and secretion in the Caco‐2/HT29‐MTX cell model

Abstract Mucins are a family of large glycoproteins that represent the major structural components of the mucus and are encoded by 20 different mucin genes. Mucin expression can be modulated by different stimuli. In this study, we analyzed four mucins (MUC2, MUC3, MUC13, and MUC17) in coculture of Caco‐2/HT29‐MTX cells to demonstrate the variation in gene expression in the presence of antioxidant compounds like chlorogenic acid, epicatechin gallate, and quercetin (apple, tea, and coffee polyphenols, respectively). coculture of Caco‐2/HT29‐MTX cells was treated with polyphenols, and the expression of four mucins was determined by reverse‐transcriptase PCR. In addition, the secretion levels of MUC2 were established by enzyme‐linked immunoassay (ELISA) analysis. The results showed that each polyphenol compound induces different expression patterns of the mucin genes. Statistically significant up‐regulation of MUC17 was observed following incubation with epicatechin gallate and quercetin. ELISA results did not prove any significant differences in protein levels of MUC2 after treatment by the polyphenol compounds. The polyphenols considered in this study may influence mucin secretion and act on diverse salivary substrates to change the barrier properties of mucins for mucus secretion in different ways.

Transmembrane mucins play important roles in preventing infection at mucosal surfaces, but also contribute to the development, progression, and metastasis of adenocarcinomas. They seem to have evolved to monitor and repair damaged epithelia, whereas this function can be "hijacked" by cancer cells (van Putten & Strijbis, 2017). Secreted mucins are either produced by mucosal cells that are present in the submucosal glands, or by specialized cells from apical surface epithelium, generally called Goblet cells (Tarang, Kumar, & Batra, 2012). Secreted mucins include MUC2, MUC5AC, MUC5B, MUC6, MUC7, MUC8, and MUC19, and the membrane-bound mucins are MUC1, MUC3, MUC4, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, and MUC20 (Tarang et al., 2012). In the human intestine, MUC2 is the major secreted mucin of the mucosal layer (Hews et al., 2017). Mucins are characterized by a defined pattern of expression that can be modified by environmental factors and thereby involve an alteration of gene expression (Hollingsworth & Swanson, 2004). Recently, therapeutic approaches have focused on mucin regulation during inflammation and cancer in order to use mucins as therapeutic targets (Macha et al., 2015).
Previous studies demonstrated that dietary compounds, which interact with Goblet cells, could modify the secretion and composition of mucins. Some fibers, like sulfated polymers, and major short-chain fatty acids present in the colon may increase mucin secretion (Barcelo et al., 2000;Deplancke & Gaskins, 2001;Sharma, Schumacher, Ronaasen, & Coates, 1995).
Polyphenols are the main class of plant secondary metabolites that show efficacy in the prevention of certain diseases, such as cancer, type II diabetes, and cardiovascular disease (Rothwell et al., 2013). They are characterized by the presence of several phenol rings, which are associated with generally complex structures of high molecular weight with one or more attached hydroxyl groups (Biasi et al., 2013). Recently, these polyphenols have gained considerable interest because of their potential health benefits; as such, they are likely the most studied class of molecules with nutritional interest in mind (Calani et al., 2012). Polyphenols are largely metabolized in tissues, such as the colon, small intestine, and liver, where they can exert several pharmacological effects, such as antioxidative and anticarcinogenic (Yang, Wang, Lu, & Picinich, 2009). The bioavailability of polyphenols in humans is abundantly discussed. The maximum concentration of parent compound in human plasma rarely exceeds 1 μM after the consumption of 10-100 mg of a single phenolic compound (Karakaya, 2004;Scalbert & Williamson, 2000). Following the ingestion of flavonoids as part of a normal diet, they undergo hydrolysis in the small intestine but are mostly poorly absorbed (Havlik & Edwards, 2018). After entering the proximal colon, they are often (but not always) transformed into simple phenolic compounds by the resident microbiota and may be absorbed for hepatic transformation and enter circulation (Havlik & Edwards, 2018;Selma, Espin, & Tomas-Barberan, 2009).
In the upper and lower digestive tract, epithelial cells are probably exposed to low, but physiologically relevant, concentrations of free polyphenols. It has been suggested that luminal concentrations of flavonoids, for example after consumption of 20 mg of quercetin-rich food may peak at ~100 μM in the ceacum, and such low concentrations appear relevant for diet-based studies (Havlik & Edwards, 2018).
In our study, we investigated how the gene expression of four mucins is affected by the presence of the three representative dietary plant polyphenols, in order to examine the effect of antioxidant compounds on mucin alteration in a coculture of intestine cancer cells: Caco-2 and HT29-MTX.
The four mucins in question were selected for several reasons. MUC2 is a major secreted mucin (Han, Deglaire, Sengupta, & Moughan, 2008), MUC3 is the most studied mucin of the adhering membrane class (Tarang et al., 2012), and MUC13 and MUC17 have only been recently discovered (Pelaseyed et al., 2014). were grown in Dulbecco's Modified Eagle medium (DMEM) supplemented with 20% (v/v) fetal bovine serum, 1% nonessential amino acids, 100 U/ml Penicillin, and 100 μg/ml Streptomycin. All media and reagents were purchased from Sigma-Aldrich (Saint-Louis, USA). Cell cultures were incubated at 37°C in the presence of 5% (v/v) CO 2 , and the cell culture medium was replaced every 2 days. The cell lines were seeded in two NUNC 24-well culture plates at concentrations of 3.6 × 10 4 Caco-2 cells and 0.4 × 10 4 HT29-MTX cells in 1 ml of DMEM for mucin expression and ribonucleic acid (RNA) studies. Cells were grown for 14 ± 1 days until 80% cell monolayer confluence. All culture plastic materials were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA).

| Cell cultures and reagents
Chlorogenic acid, epicatechin gallate, and quercetin were selected as representatives of the most abundant and well-characterized dietary phenolics, since they occur naturally in apples, tea leaves, and coffee. They were purchased from Extrasynthese (Genay Cedex, France). Stock solutions of the compounds in 80% ethanol were serially diluted in DMEM to a final concentration of 10 μM, keeping the ethanol concentration <1%.

| MTT assay
Prior to the experiment, appropriate concentrations of the polyphenols were selected based on the results of cytotoxicity screenings (Biasi et al., 2013;Lee, Ji, & Sung, 2010). Cell viability was measured using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT) assay. Caco-2 cells, HT29-MTX cells, and their cocultures were pre-incubated in a NUNC 96-well plate at a density of 2.5 × 10 3 cells of Caco-2 and HT29-MTX cells/well and for coculture at a density of 3.6 × 10 3 Caco-2 cells and 0.4 × 10 3 HT29-MTX cells/well for 24 hr at 37°C in a 5% CO 2 humidified atmosphere. Cells were treated with a twofold serial dilution of the compounds in the concentration range of 40-1,280 μmol/ml for 72 hr. Subsequently, MTT reagent (1 mg/ml) in DMEM was added to each well, and the plates were incubated for an additional 2 hr at 37°C. Culture supernatants were aspirated, and 100 μl of dimethylsulfoxide was added to each well. The absorbance was measured at 555 nm using the Tecan Infinite M200 reader (Tecan Austria GmbH, Grödig, Austria).
The percentage of mortality in the presence of each concentration of extracts was plotted and used to determine the 50% inhibitory concentration (IC 50 ).

| Mucin expression and RNA assays
The old medium in a confluent 24-well plate was aspirated, 1 ml of medium with 10-μM concentration of the selected compound was added, and the plate was incubated for 48 hr. As a control, DMEM without the compound was added. All samples were tested in six replicates; three adjoining wells were used for RNA studies and three for mucin expression.
For RNA extraction and follow-up reverse transcriptase PCR (RT-PCR) analysis, the medium was aspirated after the treatment. Nonadhered cells were removed by washing the plate three times with sterile phosphate buffered saline (PBS), before treatment with 300 μl of 1% trypsin (Sigma-Aldrich, Saint-Louis, USA). Finally, the solution was suspended in PBS. To determine the amount of mucin secreted using ELISA, wells were released by scrubbing cells with pipette tips. The total contents of the wells were transferred into 15-ml polypropylene tubes and centrifuged (2,000 × g, 10 min).
Supernatants were kept at −18°C for later analysis.

| RNA extraction and Real-time RT-PCR analysis
The total RNA from cells was extracted using RNeasy ® Mini Kit The products were stored at −20°C until analysis. β-Actin was used as a reference gene after confirmation of its transcriptional stability in our experimental conditions. This reference gene was selected out of the three considered genes and amplified using specific primers for β-actin (forward 5′-CTTCCTGGGCATGGAGTC-3′ and reverse 5′-GCAATGATCTTGATCTTCATTGTG-3′) (Johansson et al., 2008), and GAPDH (forward, 5′-AGCCACATCGCTCAGACAC-3′ and reverse, 5′-GCCCAATACGACCAAATCC-3′) (Tamagawa et al., 2012) with three technical replicates. The relative abundance of transcripts was calculated as per the report by Livak and Schmittgen (2001).
Statistical analysis (treated vs. control) was performed by the nonparametric Wilcoxon signed-rank test by using GraphPad InStat v.

| Statistical analysis
Statistical analysis was performed using the statistics software GraphPad Prism version 6.0 (GraphPad Software, USA). All experiments were performed in a minimum of three replicates; results are represented as the mean ± standard deviation (SD) and standard error of measurement (SEM). Differences were considered significant when p < 0.05.

| Toxicity of polyphenols in the intestinal model
The MTT assay, which measures mitochondrial activity in viable cells, was evaluated in the Caco-2/HT29-MTX coculture and Caco-2 and HT29-MTX cultures after 72 hr treatment with chlorogenic acid, epicatechin gallate, and quercetin. A twofold dilution series of 1,280, 640, 320, 160, 80, and 40 μmol/ml were used for the analysis. The percentage of mortality for each concentration was plotted and fit with a sigmoidal curve in order to determine the 50% growth inhibitory concentration (IC 50 ). The MTT assay was also carried out for the individual cell lines, in order to establish that the decrease of cell viability was not because of cocul-  Thus, expression of MUC17 was elevated after all treatments; however, the other mucins showed compound-specific patterns.

| ELISA assay of polyphenols
To evaluate whether the modulation of mucin mRNA abundance translated to altered mucin protein abundance, the relative level of

Polyphenols are ingested by humans as complex mixtures immersed
in a food matrix as part of the normal diet. Approximately 90-95% of dietary polyphenols are not absorbed by the small intestine and therefore reach the colon (Clifford, 2004). We chose to study three phenolic compounds that are ordinarily present in the human diet.
Each of the polyphenolic components was examined at a concentration of 10 μM, since this concentration is close to the amount that is expected to be present in the gut and involved in polyphenol-gut interactions as per our toxicity measurements (Deprez, Mila, Huneau, Tome, & Scalbert, 2001;Peng & Kuo, 2003). Our data are also in good agreement with the data reported by O'Hara et al. (2006) published recently, which showed that the cells may induce mucin expression.  Kim et al., 2008). As mentioned above, in the present study, we found that each phenolic compound induces differential expression patterns of mucin genes. The effect of polyphenols on mucin barrier might be complex as previous work indicated that polyphenols from tea (epigallocatechin gallate and epicatechin) act as crosslinkers of intestinal mucins (Georgiades, Pudney, Rogers, Thornton, & Waigh, 2014). Polyphenols, such as epigallocatechin gallate and epicatechin, may exert beneficial effects on human health, not only as simple antioxidants acting as free-radical scavengers, but also by indirectly interfering with specific signaling proteins, which mediate gene regulation in response to oxidative stress and inflammation (Biasi et al., 2013). In the study by Kim et al. (2008), it was found that The aforementioned findings are in agreement with the literature available on this topic (Shimamura et al., 2005). Our results also indicate that the polyphenols used in this study upregulated the expression of MUC17, which is consistent with a previous finding that the expression of MUC17 increases in the presence of polyphenols (Senapati et al., 2010). Our results, in accordance with prior investigations, confirm that the examined polyphenols play an important role in regulating mucin expression, which may also be related to cancer development.

| CON CLUS ION
In conclusion, our study demonstrated that polyphenols modulate mucin expression, which may affect bacteria that utilize mucin to protect the digestive tract from pathogens, and may be linked to cancer progression, although a causal link has not yet been established. The use of micronutrients found in certain foods appears to aid in the prevention and treatment of gastrointestinal tract

E TH I C A L S TATEM ENT
I testify on behalf of all coauthors that this article is original and has not been published and will not be submitted for publication elsewhere.
The manuscript does not contain experiments using animals or human.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflict of interest.