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Chemopreventive Potential of Powdered Red Wine Pomace Seasonings against Colorectal Cancer in HT-29 Cells. Raquel Del Pino-García, Maria Dolores Rivero-Perez, Maria L. GonzálezSanJosé, Miriam Ortega-Heras, Javier García-Lomillo, and Pilar Muñiz J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04561 • Publication Date (Web): 13 Dec 2016 Downloaded from http://pubs.acs.org on December 14, 2016

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Journal of Agricultural and Food Chemistry

Chemopreventive Potential of Powdered Red Wine Pomace Seasonings against Colorectal Cancer in HT-29 Cells.

Raquel Del Pino-García a, María D. Rivero-Pérez a, María L. González-SanJosé a, Miriam OrtegaHeras a, Javier García Lomillo a, and Pilar Muñiz a,*.

a

Department of Food Biotechnology and Science, Faculty of Sciences, University of Burgos, Plaza

Misael Bañuelos s/n, 09001, Burgos, Spain.

Corresponding Author: *(P.M.) Phone: +34-947258800, ext. 8210 . Fax: +34-947258831. E-mail: [email protected].

Email addresses: Raquel Del Pino-García ([email protected]), María D. Rivero-Pérez ([email protected]), María L. González-SanJosé ([email protected]), Miriam Ortega-Heras ([email protected]), Javier García Lomillo ([email protected]), Pilar Muñiz ([email protected]).

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ABSTRACT

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This study evaluates the anti-proliferative and anti-genotoxic actions of powdered red wine pomace

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seasonings (Sk-S:seedless, W-S:whole, Sd-S:seeds). In vitro gastrointestinal digested and colonic

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fermented fractions of the seasonings were used as cell treatments. Phenolic acids from Sk-S

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showed the highest bioaccessibility in the small intestine whereas polyphenols contained in Sd-S

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might be the most fermentable in the colon. Dietary fiber from Sk-S was the best substrate for short

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chain fatty acids production by gut microbiota. Colon cancerous (HT-29) cell viability was inhibited

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by 50% (IC50 values) at treatment concentrations ranging from 845 (Sk-S) to 1085 (Sd-S) µg/mL

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prior digestion, but all digested fractions exhibited similar anti-proliferative activities (mean

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IC50=814 µg/mL). Oxidative DNA damage in cells was also attenuated by the treatments (200

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µg/mL, 24-h preincubation), with all colonic fermented fractions displaying similar genoprotective

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action. These results suggest the potential of red wine pomace seasonings as chemopreventive

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agents in colorectal cancer.

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KEYWORDS

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Anti-proliferative activity; Chemoprevention; Colon cancer; Genoprotection; Oxidative stress;

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Wine pomace.

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INTRODUCTION

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Oxidative stress is undoubtedly associated with colorectal cancer. The colonic epithelium is greatly

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expose to several oxidants incorporated with the diet and reactive oxygen/nitrogen species (RONS)

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generated in fecal materials, which has been suggested to play a significant role in the etiology of

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colon cancer.1,2 In addition, inflammatory cells and cancer cells themselves overproduce free

25

radicals and soluble mediators which lead to further RONS production. All the exogenous and

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endogenous deleterious reactive species generated can directly oxidize DNA and interfere with

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mechanisms of DNA repair, triggering DNA chain breaks, base modification, and other oxidative

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DNA lesions.3 Permanent alteration of genetic material as a result of such oxidative damage is

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involved in mutagenesis, carcinogenesis, and ageing.4

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In this framework, attenuation of RONS-induced DNA damage is included as a first line of defense

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against carcinogenesis as it could prevent the initiation phase of this multistage process (primary

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cancer prevention). In addition, other important anti-cancer mechanisms consist on inhibiting

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cancerous cell proliferation, which could arrest cell promotion and progression (secondary cancer

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prevention).5,6 These effects can be achieved by the chronic administration of synthetic, natural or

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biological agents to reduce or delay the occurrence of cell malignancy, strategy that is known as

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cancer chemoprevention.7

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Numerous studies support the beneficial effects of some dietary constituents as chemopreventive

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agents against colorectal cancer. Consistent evidence indicates that high intake of dietary fiber from

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fruit, vegetables and whole grains is inversely associated with the risk of this type of cancer.8 Such

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benefits have been partly attributed to the production of short chain fatty acids (SCFAs) in the large

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intestine during fiber fermentation.9 Also the presence of significant amounts of phenolic

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antioxidants in fruits and vegetables is believed to largely contribute to their chemopreventive

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effects in the colon.10–12

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Previous research in polyphenol-rich extracts from wines and grapes has demonstrated the ability of

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their phytochemicals to modulate colonocyte mutagenesis and prevent tumor initiation and ACS Paragon Plus Environment

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promotion.13 Beneficial effects of wine pomace upon the large intestinal mucosa have been also

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suggested 14 and mainly related to the high content of both polyphenolic molecules and dietary fiber

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in this winemaking residue.15,16

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Recently, new seasonings obtained directly from different parts of red wine pomace have been

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developed, avoiding any extractive processes during its manufacture. These powdered red wine

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pomace seasonings (RWPSs) represent an innovative strategy to reduce/replace salt added to foods

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while increasing the intake of dietary fiber and natural antioxidants, mainly polyphenols.17 Such

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high contents in sources of bioactive compounds further suggest possible health benefits of RWPSs

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in the gastrointestinal tract.

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On the basis of these considerations, the major aim of this study was to investigate the potential of

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different RWPSs in chemoprevention by assessing their anti-proliferative and anti-genotoxic effects

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against oxidative stress in human colorectal adenocarcinoma (HT-29) cells, taking into account the

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biaccessibility and metabolism of the RWPS polyphenols and dietary fiber along the gut, until

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reaching the colon.

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MATERIALS AND METHODS

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Chemicals. Ammonium bicarbonate (NH4HCO3), calcium chloride dihydrate (CaCl2.2H2O),

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cobalt(II) chloride hexahydrate (CoCl2.6H2O), L-cysteine hydrochloride, hydrochloric acid (HCl),

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iron(III) chloride hexahydrate (FeCl3.6H2O), Low-Melting-point agarose, Normal-Melting-point

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agarose, menadione, Dulbecco's Modified Eagle Medium (DMEM), Fetal bovine serum (FBS),

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Tritton X-100, magnesium sulfate heptahydrate (MgSO4.7H2O), manganese(II) chloride

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tetrahydrate (MnCl2.4H2O), 10,000 U/mL penicillin and 100 mg/mL streptomycin solution (P/S),

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phosphoric acid solution (H3PO4), porcine bile extract, porcine pancreas pancreatin, potassium

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chloride (KCl), resazurin sodium salt, sodium bicarbonate (NaHCO3), sodium hydroxide (NaOH),

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sodium-lauryl-sarcosineate, sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic

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(NaH2PO4), sodium sulfide nonahydrate (Na2S.9H2O), thiazolyl blue tetrazolium bromide (MTT), ACS Paragon Plus Environment

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Tris hydrochloride (Tris), tryptone, enzymes used in enzymatic digestion (α-amylase (EC 3.2.1.1),

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amyloglucosidase (EC 3.2.1.3), lipase (EC 3.1.1.3), and pepsin (E.C 3.4.23.1)), pure phenolic acid

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standards (caffeic acid, caftaric acid, coutaric acid, ellagic acid, ethyl gallate, fertaric acid, ferulic

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acid, gallic acid, p-coumaric acid, p-OH-benzoic acid, protocatechuic acid, salicylic acid, syringic

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acid, and vallinic acid), analytical standard solutions of SCFAs (acetic acid, butyric acid, propionic

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acid, and valeric acid) and cellulose membrane dialysis tubing (12,000 Da molecular weight cut-

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off) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Ethylenediaminetetraacetic acid

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(EDTA), and sodium carbonate (Na2CO3) were purchased from Panreac Química S.L.U.

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(Barcelona, Spain). Dimethyl sulfoxide (DMSO) and phosphate buffered saline (PBS) tablets were

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purchased from Merck (Darmastadt, Germany).

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Red wine pomace seasonings (RWPSs). The seasonings used in this study were made in the pilot

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plant of the Food Technology Department of University of Burgos (Spain) as previously

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described.17,18 Red wine pomace (Vitis vinifera L. cv. Tempranillo) was supplied by several

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wineries located at Burgos (Spain). All wine pomace was mixed, dehydrated (final water content
0.96) for all the treatments. The concentration of each treatment that inhibited

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50% of cell viability (IC50) was determined, expressing the IC50 values for each fraction in µg/mL

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of medium.

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Anti-genotoxic activity assessment (Comet assay). Single-cell gel electrophoresis under alkaline

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conditions, also known as the comet assay, was performed as previously reported 24 to determine

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the effect of RWPS and their derived fractions on colonocyte DNA damage, using menadione as

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oxidation agent. HT-29 cells were seeded in 25 cm2 cell culture flasks (1x104 cells/cm2, flask filled

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with 5 mL of medium), pre-incubated with basal DMEM for 24 h, and exposed for other 24 h to

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each fraction at a final concentration of 200 µg/mL of medium. Non-oxidised control (C) and

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oxidised control (O C) cells were incubated just with DMEM. Two types of experiments were

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performed:

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a) O-RWPS: the treatments were first removed and oxidation was then induced by adding 5 mL of

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DMEM containing menadione (0.2 µM of final concentration) and incubating for 5 h.

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b) O+RWPS: an aliquot of 50 µL of menadione (20 µM, diluted in DMEM) was directly added to

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the flasks containing the treatments and oxidation was maintained for 5 h.

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The possible genotoxic effect of the fractions was also evaluated by treating pre-incubated cells

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with PBS instead of menadione. Following oxidation, HT-29 cells were scraped, centrifuged (3,000

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g, 5 min, 4 ºC), resuspended in pre-heated 1% Low-Melting-point agarose, and added to frosted

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microscope slides precoated with 1% Normal-Melting-point agarose. After the agarose solidified,

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slides were immersed in cold lysing solution (2.5 M NaCl, 100 mM EDTA, 100 mM Tris, 1%

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sodium-lauryl-sarcosineate, 1% Tritton X-100, 10% DMSO, pH 10) overnight at 4 ºC. The next

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day, all slides were placed in an electrophoresis tank containing alkaline electrophoresis buffer (1

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mM EDTA, 0.3 N NaOH, pH 10) and the DNA allowed to unwind for 40 min. Electrophoresis was

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conducted at 4 ºC, 25 V, 500 mA and 150 W. The slides were subsequently wash three times for 2

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min with neutralizing buffer (0.4 M Tris, pH 7.5), stained with ethidium bromide (20 µg/mL,

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20µL/slide), observed using a ZEISS axioplan fluorescence microscope (Carl Zeiss A.G., ACS Paragon Plus Environment

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Oberkochen, Germany), and photographed with a Nikon CoolPix L31 16MP digital camera (Nikon

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Europe B.V., Amsterdam, The Netherlands). The images were analyzed using the program

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CometScoreTM Version 1.5 (TriTek, Corp., Sumerduck, VA, USA). For each analysis, 60 randomly

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selected individual cells were calculated and their tail length evaluated. Each experiment was

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repeated independently three times. DNA damage was expressed as mean % tail length in relation

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to the oxidized control ([T/O C]%).

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Statistical analysis. Analysis was performed using Statgraphics® Centurion XVI, version 16.2.04

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(Statpoint Technologies Inc., Warranton, VA, USA). Statistical significance of the data was tested

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by one-way analysis of variance (ANOVA), using the Fisher's least significant difference (LSD)

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test to compare the means that showed significant variation (p < 0.05).

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RESULTS

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Phenolic acid contents of RWPSs and derived digested fractions. The sum of phenolic acids

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determined in the UD, GIDD and CF fractions derived from the RWPSs is presented in Figure 1. In

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the three types of RWPSs, higher concentrations of phenolic acids were detected in the digested

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than the UD fractions, but important differences between the seasonings were detected. For Sk-S

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the most concentrated fraction was GIDD, showing about three- and two-folds higher (p < 0.001)

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contents than UD and CF, respectively. For W-S both digested fractions were around three times

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more concentrated than the UD fraction, with GIDD showing slightly but significantly higher

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values than CF. The opposite trend was observed for Sd-S where the CF fraction contained the

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highest quantity of phenolic acids, being almost five- and four-folds more concentrated (p < 0.001)

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than UD and GIDD, respectively. Comparing between the RWPSs, the sum of phenolic acids was

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significantly higher in Sk-S than Sd-S for both the UD and the GIDD fractions whereas the contrary

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was observed for the CF fractions, with W-S obtaining intermediate values in all cases. Further

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information about the contents in the individual phenolic acids can be seen as Supporting ACS Paragon Plus Environment

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Information Table S1, which reflects the higher contribution of hydroxybenzoic than

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hydroxycinnamic acids to the sum of phenolic acids, as well as the most representative compounds

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determined for each fraction.

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SCFAs levels of CF fractions. The highest levels of SCFAs were detected in Sk-S, and the lowest

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in Sd-S, both evaluating the individual (butyric, acetic and propionic acids) and the total SCFA

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contents (Figure 2). Acetic acid was the main SCFA produced in the three RWPSs, and butyric

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acid the lowest. The molar SCFA ratio obtained for each seasoning was different, with Sk-S

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showing the highest relative production of butyric acid, W-S of propionic acid, and Sd-S of acetic

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acid.

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Anti-proliferative effects of RWPSs and their respective digested fractions. Following

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incubation of HT-29 cells with different concentrations of the treatments for 24 h, a dose-dependent

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inhibition of cell viability was observed for all the RWPSs and derived digested fractions (data not

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shown), allowing to calculate the IC50 value of each fraction (Figure 3). The concentration of

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treatment capable to inhibit cell viability by 50% was used to compare the anti-proliferative actions

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between the different fractions and seasonings. For Sk-S, all fractions elicited similar anti-

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proliferative activities (mean IC50 around 833 µg of fraction/mL of medium). For W-S the GIDD

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fraction was the most effective, showing around 25% higher (p < 0.05) capacity to reduce the

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number of viable cells than the respective UD treatment. As regards Sd-S, both of its digested

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fractions obtained similar IC50 values and displayed around 23% more potent anti-proliferative

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effects than prior digestion. Comparing the same type of fractions, differences between the RWPSs

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were observed only for the UD treatments where Sk-S exhibited the highest ability to reduce cell

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viability and Sd-S the lowest.

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Anti-genotoxic effects of RWPSs and their respective digested fractions. The protection of the

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treatments against oxidative DNA damage in HT-29 cells was assessed using the comet assay. ACS Paragon Plus Environment

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When the treatments were absents during the oxidative insult (Figure 4, A) the genopreventive

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effect of Sk-S before in vitro digestion was preserved after the whole process, while the capacity to

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prevent oxidative DNA damage for the other RWPSs increased progressively along digestion,

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showing significant differences from UD to CF fractions for W-S, and between all the fractions for

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Sd-S (p < 0.05). Significant differences in the anti-genotoxic effect observed between the RWPSs

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were only found before digestion, with Sk-S showing the highest efficacy and Sd-S the lowest. In

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the presence of the treatments during the oxidation (Figure 4, B) the trends between the fractions

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derived from both Sk-S and Sd-S were similar as described above in their absence. However, the

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tendency observed for W-S changed, not detecting significant differences between its fractions in

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the O+RWPS experience. The seasoning showing the highest anti-genotoxic action was Sk-S

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preceding colonic fermentation (UD and GIDD fractions) and Sd-S after the action of gut

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microbiota (CF fraction). The treatments alone were not genotoxic towards the HT-29 cells at the

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concentrations used.

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The table accompanying Figure 4 shows the increase in the anti-genotoxic effect from O-RWPS to

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O+RWPS. The differences ranged around 20% higher (p < 0.001) protection due to the presence of

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the treatments during the oxidation for most types of fractions and seasonings. Nonetheless, the %

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change detected for the UD fraction derived from W-S was the highest, both in comparisons

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between the different fractions of this seasoning and between the UD fractions of the three RWPSs.

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DISCUSSION

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There is increasing interest in strategies for cancer prevention, with a number of dietary constituents

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being regarded as promising chemopreventive agents.6 In this study, three new powdered

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seasonings derived from different parts of red wine pomace (seedless: Sk-S, whole: W-S, seeds: Sd-

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S) have been investigated for their anti-genotoxic and anti-proliferative effects in cancerous

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colonocytes as surrogate indexes of their potential in primary and secondary cancer

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chemoprevention.5,7,25 ACS Paragon Plus Environment

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Our results showed that Sk-S and W-S contained high levels of weakly bound or free phenolic acids

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that were readily liberated during the gastrointestinal phase of digestion. In the organism, some of

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these phenolic acids will pass through the epithelial cells of the small intestine and reach the blood

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stream whereas non-absorbed ones will reach the large bowel. Part of these compounds are

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represented by the phenolic acids contained in the GIDD fractions,26 which were found at

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substantial increased concentrations than before digestion in both Sk-S and W-S.

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Once compounds bound to dietary fiber of wine pomace reach the colon, they may be liberated

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from non-digested matrices and degraded by colonic microbiota. Monomeric flavonoids and soluble

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proanthocyanidins of high molecular weight are susceptible to extensive microbial fermentation,

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suffering de-polymerization and degradation of monomers and resulting in the accumulation of

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simple phenolic acids and metabolites.27,28 Hence, the high concentration in phenolic acids,

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predominantly gallic acid, detected in the CF fractions derived from W-S and especially from Sd-S

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was probably due to the large contents in mono- and oligomeric flavan-3-ols and galloylated

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molecules of wine pomace seeds,17,18,29 which can be degraded to gallic acid in the colon.30

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The colonic microbiota can also break down dietary fiber contained in RWPSs,17 leading to the

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production of SCFAs which are chiefly represented by acetic, propionic and butyric acids.27

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Qualitative and quantitative differences were detected in the pattern of SCFAs resultant from

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colonic fermentation of the different RWPSs. The differences in the type of carbohydrates

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constituting dietary fibers and the peptides retained to the insoluble matrices, as well as the

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interactions between them and their influence in gut microbiota,31 might partly explained the

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different SCFA levels and molar proportion found for each RWPS. In addition, the higher contents

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of proanthocyanidins in wine pomace seeds than skins may well contribute to the lower SCFA

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contents detected in colonic fermented fractions derived from Sd-S than from Sk-S, as these

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complex phenolic compounds can inhibit SCFA formation.32

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The potential of the RWPS-derived bioactive compounds to exert beneficial actions against colon

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cancer were assessed in the HT-29 cell line, which is a widely employed model of transformed

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neoplastic colorectal cells in studies of cell cycle events and genotoxicity.33 It should be also noted ACS Paragon Plus Environment

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that the total phenolic contents of all cell treatments (Supporting Information Table S2; values

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estimated from the Folin-Ciocalteu indexes of each fraction)20 represented physiologically relevant

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doses accordingly to previous studies where total phenolic concentrations below 50 µg GAE/mL of

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medium were established as achievable in the colon.47

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The chemopreventive effects described for some phenolic compounds 11,34 and SCFAs 9,25 can be

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mediated by either their blocking activities, which are mostly related to primary cancer prevention,

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or their suppressing activities, which can provide secondary cancer prevention.5 Exerting one type

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of activity or the other depends greatly on the concentration of such bioactive compounds.

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The IC50 values obtained for RWPS and their respective fractions showed that, before digestion, Sk-

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S may display the highest anti-proliferative activity in colon cancerous cells. However, the

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transformations taking place along the digestive process appear to increase the overall capacity to

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inhibit cell viability of the compounds derived from W-S and Sd-S, but not from Sk-S. Therefore,

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our results are consistent with the previous studies reporting anti-proliferative effects of grape

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phytochemicals in numerous cancer cells, which have been associated with molecular mechanisms

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that result in the arrest of the cell cycle, and the induction of apoptosis, among other cancer

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suppressing effects.35,36

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Considering the phenolic composition of the undigested RWPSs,17 the highest efficacy of Sk-S to

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inhibit HT-29 cell growth preceding digestion can be explained by its high content in anthocyanins,

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whose cytotoxic and anti-proliferative effects in colon cancerous cells have been reported by

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several authors.37,38 In fact, previous research in extracts obtained from grapes has demonstrated

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that those fractions rich in anthocyanins, followed by flavonols and tannins, displayed the highest

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anti-proliferative activity in HT-29 cells, while the phenolic acid-enriched fractions were the least

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effective.39 In the fractions collected just after gastrointestinal digestion (GIDD fractions), our

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results suggested that some phenolic acids such as salicylic, syringic, and protocatechuic acids

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could contribute to the observed anti-proliferative effects, particularly in Sk-S and W-S, where

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these hydroxybenzoic acids are present at high concentrations. Salicylic could be particularly

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responsible bearing in mind that its pro-apoptotic effects on colon carcinoma cells have been ACS Paragon Plus Environment

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previously demonstrated,40 whereas none or low anti-proliferative and cytotoxic activities have been

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seen for protocatechuic, syringic and vallinic acids,37,41 In addition, non-absorbed flavonoids

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liberated from the RWPS matrices may also play a substantial cancer suppressing role,36 mainly in

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Sd-S where phenolic acids were poorly bioaccessible until reaching the colon.

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After colonic fermentation, gallic acid could be implicated in the observed reduction of HT-29 cell

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viability, mainly in W-S and Sd-S, as this phenolic acid has been described as a potent inhibitor of

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cancerous cell proliferation.37 The CF fraction derived from Sk-S retained its capacity to decrease

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cell viability to a similar extent as prior colonic fermentation and as other CF fractions, which

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evidenced the contribution of either colonic metabolites or non-degraded native compounds (such

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as anthocyanins)41 to the anti-proliferative effects of this treatment. Furthermore, the SCFAs formed

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in the large bowel may also protect against colorectal cancer.42 Previous studies have proposed that

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both butyrate and propionate, but not acetate, are able to counteract HT-29 cells proliferation.43 In

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particular, the ability of butyrate to promote growth arrest, differentiation, and apoptosis in

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cancerous cells has been extensively studied,44,45 As the HT-29 cells carry a mutant form of the p53

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gene, which is often mutated in colorectal tumors, butyrate might efficiently induce the

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programmed death of these cancerous cells because this SCFA has been suggested to activate p53-

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independent mechanisms of apoptosis.2

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The anti-genotoxic effects in HT-29 cells of the RWPSs and their respective digested fractions were

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assessed by the comet assay. As phenolics can themselves be genotoxic and act in a pro-oxidant or

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an antioxidant way over different dose ranges,46 treatment concentrations that may exert beneficial

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instead of putative detrimental effects in cells must be investigated to evaluate the potential of

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phenolic-rich agents in primary cancer prevention. Thus, based on the anti-proliferation results and

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considering concentrations lower than the IC20 as non-cytotoxic, 200 µg of fraction per mL of

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culture medium was selected as a non-cytotoxic concentration for all treatments.

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The RWPS-derived bioactive compounds were able to decreased DNA breakage under oxidative

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stress when they were both absent (O-RWPS) and present (O+RWPS) in the medium during the

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oxidative insult. As expected, the presence of the fractions during the oxidation increased their ACS Paragon Plus Environment

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capacity to protect DNA, showing that the extent of this increase was rather similar for most of the

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treatments. Indirect antioxidant mechanisms leading to these anti-genotoxic effects may be

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mediated by the RWPS-derived bioactive compounds up-taken by the cells 28,48 and also by those in

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contact with the colonocytes. Both polyphenols from grapes and wine 13,14,39,49 and certain SCFAs

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such as butyrate and propionate 25,50,51 have previously demonstrated their ability to inhibit cellular

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processes associated with genotoxic events in colorectal cancer through a broad spectrum of

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indirect mechanisms that involve the modulation of several signaling pathways. On the other hand,

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phenolic compounds retained within cells and those present in the medium in the O+RWPS

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experience may also exhibit direct antioxidant activities that help to decrease RONS levels and

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contribute to reduce oxidative DNA damage in colon cells.52

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The anti-genotoxic action of Sk-S preceding digestion, which was the highest between the RWPSs

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in both experiences, was not altered by the digestion, which agrees with previous studies using

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digested and fermented anthocyanin-rich extracts from different berries, also in HT-29 cells.47 On

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the other hand, a progressive higher anti-genotoxic potential was observed for Sd-S with the

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advance of the digestive process, which was parallel to the increased release/generation of phenolic

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acids in this RWPS. These results support the great contribution of phenolic compounds to the

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potential anti-genotoxicity of Sd-S, which is in accordance with the high genoprotective effects

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reported for gallic acid by other authors.53 With regard to W-S, certain synergisms may occur

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before digestion between its compounds, derived from both grape skins and seeds,20,54 but these

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synergistic interactions might be lost, or at least not so noticeable, after the digestive process.

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Taken together, our data suggest the chemopreventive properties of seasonings directly obtained

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from winemaking residues against colorectal cancer. Dose-dependent anti-proliferative effects in

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cancerous colon cells for all the seasonings tested and their respective digested fractions, which

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might help to attenuate cancer progression, as well as anti-genotoxic effects in their presence or

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their absence during an oxidative insult, which could prevent mutagenesis and block tumor

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initiation and advance, were detected.


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The seasoning derived from seedless wine pomace (Sk-S) was the most effective prior digestion,

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but the seasonings containing wine pomace seeds (Sd-S and W-S) were more susceptible to

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increase their chemopreventive capacity during digestion, with all of the seasonings tested showing

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similar effects once in the colon.

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Finally, it should be noted that the potential suppressing and blocking activities against colorectal

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cancer demonstrated in vitro (HT-29 cells) in this study should be further assessed in other cell lines

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and in vivo to confirm the interest of these seasonings as primary and secondary chemopreventive

403

agents in colorectal cancer. However, these dual protective role of the RWPSs, along with the

404

synergistic or antagonistic effects that chemoprevention might have with other chemotherapies,

405

must be taking into account when designing strategies with therapeutic purposes.

406 407


17


408

ABBREVIATIONS USED

409

CF, colonic fermented; GIDD, gastrointestinal digested+dialyzed; HT-29, human colorectal

410

adenocarcinoma; IC50, concentration of each treatment that inhibited 50% of cell viability; RONS,

411

reactive oxygen/nitrogen species; RWPS, red wine pomace seasoning; SCFAs, short chain fatty

412

acids; Sd-S, seasoning obtained from the seeds isolated from wine pomace; Sk-S, seasoning

413

obtained from seedless red wine pomace, in which grape skins are the main component; UD,

414

undigested; W-S, seasoning obtained from whole red wine pomace.

415 416

SUPPORTING INFORMATION

417

Supporting Information available: Table S1) Phenolic acid contents (µg/g) of the red wine pomace

418

seasonings (RWPSs) in the fractions obtained during simulated digestion; Table S2) Total phenolic

419

contents of the cell treatments. (These materials is available free of charge via the Internet at

420

http://pubs.acs.org).

421


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AUTHOR INFORMATION

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Funding

580

Authors thank the financial support of the Autonomous Government of Castilla y León

581

(Research project BU282U13). R. Del Pino-García and J. García-Lomillo are holders of a FPU

582

grant from the Spanish Ministry of Education, Culture and Sports (AP2010-1933 and

583

FPU12/05494, respectively).

584

Notes

585

The authors declare no competing financial interest.


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FIGURE CAPTIONS

Figure 1. Phenolic acid contents of the red wine pomace seasonings (RWPSs) and their respective digested fractions. RWPS obtained from seedless wine pomace (Sk-S), whole wine pomace (W-S), and isolated seeds (Sd-S). Fractions analyzed during simulated digestion: undigested (UD), gastrointestinal digested and dialyzed (GIDD), and colonic fermented (CF). Results expressed in µg/g of fraction and represented as the mean value ± standard deviation (n = 3) of the total sum of phenolic acids quantified in each fraction. Roman letters indicate significant differences (p < 0.05) between the RWPSs (Sk-S, W-S, Sd-S). Greek letters show significant differences (p < 0.05) between the fractions (UD, GIDD, CF).

Figure 2. Short chain fatty acid (SCFA) contents of the colonic fermented (CF) fractions. CF fractions derived red wine pomace seasonings (RWPSs) obtained from seedless wine pomace (SkS), whole wine pomace (W-S), and isolated seeds (Sd-S). Results expressed as mM of total SCFAs and represented as the mean value ± standard deviation (n = 3), also indicating the mean values of each individual SCFA. CFA ratio (B:P:A) represents the molar ratio of butyric (B), propionic (P) and acetic (A) acids in each CF fraction. Roman letters indicate significant differences (p < 0.05) between the RWPSs (Sk-S, W-S, Sd-S).

Figure 3. Anti-proliferative activity of the RWPSs and their respective digested fractions in HT-29 cells. Red wine pomace seasonings (RWPSs) obtained from seedless wine pomace (Sk-S), whole wine pomace (W-S), and isolated seeds (Sd-S). Fractions analyzed: undigested (UD), gastrointestinal digested and dialyzed (GIDD), and colonic fermented (CF). Results expressed as the concentration of the treatments giving 50% inhibition (IC50) relative to the viability of control cells and represented as the mean value ± standard deviation (n = 3). Roman letters point out significant differences (p < 0.05) between the RWPSs (Sk-S, W-S, Sd-S). Greek letters indicate significant differences (p < 0.05) between the fractions (UD, GIDD, CF). ACS Paragon Plus Environment

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Figure 4. Anti-genotoxic effects of the RWPSs and their respective digested fractions in HT-29 cells. Red wine pomace seasonings (RWPSs) obtained from seedless wine pomace (Sk-S), whole wine pomace (W-S), and isolated seeds (Sd-S). Fractions analyzed: undigested (UD), gastrointestinal digested and dialyzed (GIDD), and colonic fermented (CF). These treatments were used at a non-cytotoxic concentration (200 µg of fraction/mL of medium). Protective effects against oxidative DNA damage induced by menadione were assessed when the treatments were absent (A, O-RWPS) and present (B, O+RWPS) during the oxidation. DNA migration evaluated by the comet tail length (n = 60 cells for each sample). The results are expressed as [T/OC]% = % of relative tail length with respect to the oxidised control and represented as mean values ± standard deviation (n = 3). The differences between the anti-genotoxic effects of O-RWPS and O+RWPS experiences are presented as % in the table below the comet test results. Roman letters show significant differences (p < 0.05) between the RWPSs (Sk-S, W-S, Sd-S). Greek letters refer to significant differences (p < 0.05) between the fractions (UD, GIDD, CF). C) Photographs obtained during the comet tests using the fractions derived from Sd-S and analyzed using the CometScoreTM program.


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Figure 1.


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Figure 3.


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TABLE OF CONTENTS GRAPHIC


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Chemopreventive Potential of Powdered Red Wine ... - ACS Publications

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