Chemopreventive Potential of Powdered Red Wine Pomace

Subscriber access provided by University of Otago Library

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

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]).

ACS Paragon Plus Environment

1


1

ABSTRACT

2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

agents in colorectal cancer.

14 15

KEYWORDS

16

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

17

Wine pomace.

18


2

Page 2 of 32

Page 3 of 32

19


INTRODUCTION

20 21

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

22

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

23

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

24

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

26

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

27

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

28

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

29

involved in mutagenesis, carcinogenesis, and ageing.4

30

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

31

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

32

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

33

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

34

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

35

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

36

cancer chemoprevention.7

37

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

38

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

39

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

40

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

41

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

42

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

43

effects in the colon.10–12

44

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

45

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

3


46

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

47

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

48

in this winemaking residue.15,16

49

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

50

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

51

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

52

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

53

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

54

in the gastrointestinal tract.

55

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

56

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

57

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

58

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

59

reaching the colon.

60 61

MATERIALS AND METHODS

62 63

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

64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

4

Page 4 of 32

Page 5 of 32


73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

purchased from Merck (Darmastadt, Germany).

83 84

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

85

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

86

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

87

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

182

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

183

of medium.

184 185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

performed:

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

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

207

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

9


208

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

209

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

210

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

211

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

212

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

213

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

214 215

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

216

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

217

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

218

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

219 220

RESULTS

221 222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233

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

234

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

10

Page 10 of 32

Page 11 of 32


235

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

236

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

237

determined for each fraction.

238 239

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

240

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

241

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

242

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

243

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

244

acid.

245 246

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

247

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

248

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

249

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

250

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

251

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

252

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

253

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

254

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

255

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

256

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

257

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

258

viability and Sd-S the lowest.

259 260

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

261

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

11


262

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

263

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

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

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

272

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

273

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

274

concentrations used.

275

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

276

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

277

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

278

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

279

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

280 281

DISCUSSION

282 283

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

284

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

285

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

286

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

287

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

288

chemoprevention.5,7,25 ACS Paragon Plus Environment

12

Page 12 of 32

Page 13 of 32


289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

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

299

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

300

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

301

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

302

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

303

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

304

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

305

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

306

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

307

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

308

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

309

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

310

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

311

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

312

complex phenolic compounds can inhibit SCFA formation.32

313

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

314

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

315

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

13


316

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

317

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

318

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

319

medium were established as achievable in the colon.47

320

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

321

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

322

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

323

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

324

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

325

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

326

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

327

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

328

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

329

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

330

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

331

suppressing effects.35,36

332

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

333

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

334

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

335

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

336

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

337

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

338

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

339

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

340

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

341

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

342

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

14

Page 14 of 32

Page 15 of 32


343

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

344

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

345

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

346

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

347

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

348

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

349

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

350

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

351

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

352

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

353

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

354

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

355

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

356

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

357

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

358

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

359

independent mechanisms of apoptosis.2

360

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

361

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

362

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

363

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

364

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

365

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

366

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

367

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

368

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

369

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

15


370

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

371

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

372

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

373

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

374

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

375

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

376

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

377

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

378

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

379

contribute to reduce oxidative DNA damage in colon cells.52

380

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

381

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

382

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

383

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

384

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

385

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

386

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

387

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

388

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

389

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

390

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

391

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

392

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

393

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

394

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

395

initiation and advance, were detected.


16

Page 16 of 32

Page 17 of 32


396

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

397

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

398

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

399

similar effects once in the colon.

400

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

401

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

402

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


18

Page 18 of 32

Page 19 of 32


422

REFERENCES

423

(1)

424 425

Babbs, C. F. Free radicals and the etiology of colon cancer. Free Radic. Biol. Med. 1990, 8, 191–200.

(2)

Hague, A.; Manning, A.; Hanlon, K.; Huschtscha, L.; Hart, D.; Paraskeva, C. Sodium

426

butyrate induces apoptosis in human colonic tumour cell lines in a p53-independent pathway:

427

implications for the possible role of dietary fibre in the prevention of large-bowel cancer. Int.

428

J. Cancer 1993, 55, 498–505.

429

(3)

430 431

Valko, M.; Rhodes, C.; Moncol, J.; Izakovic, M.; Mazur, M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 2006, 160, 1–40.

(4)

Khansari, N.; Shakiba, Y.; Mahmoudi, M. Chronic inflammation and oxidative stress as a

432

major cause of age-related diseases and cancer. Recent Pat. Inflamm. Allergy Drug Discov.

433

2009, 3, 73–80.

434

(5)

Manson, M. M.; Gescher, A.; Hudson, E. A.; Plummer, S. M.; Squires, M. S.; Prigent, S. A.

435

Blocking and suppressing mechanisms of chemoprevention by dietary constituents. Toxicol.

436

Lett. 2000, 112–113, 499–505.

437

(6)

438 439

Growth Metastasis 2014, 7, 19–25. (7)

440 441

Landis-Piwowar, K. R.; Iyer, N. R. Cancer chemoprevention: Current state of the art. Cancer

Steward, W.; Brown, K. Cancer chemoprevention: a rapidly evolving field. Br. J. Cancer 2013, 109, 1–7.

(8)

Murphy, N.; Norat, T.; Ferrari, P.; Jenab, M.; Bueno-de-Mesquita, B.; Skeie, G.; Dahm, C.

442

C.; Overvad, K.; Olsen, A.; Tjønneland, A.; et al. Dietary fibre intake and risks of cancers of

443

the colon and rectum in the European prospective investigation into cancer and nutrition

444

(EPIC). PLoS One 2012, 7, e39361.

445

(9)

446 447 448

Scheppach, W.; Bartram, H. P.; Richter, F. Role of short-chain fatty acids in the prevention of colorectal cancer. Eur. J. Cancer 1995, 31A, 1077–1080.

(10)

Araújo, J. R.; Gonçalves, P.; Martel, F. Chemopreventive effect of dietary polyphenols in colorectal cancer cell lines. Nutr. Rsearch 2011, 31, 77–87. ACS Paragon Plus Environment

19


449

(11)

450 451

Ramos, S. Cancer chemoprevention and chemotherapy: Dietary polyphenols and signalling pathways. Mol. Nutr. Food Res. 2008, 52, 507–526.

(12)

Santiago-Arteche, R.; Muñiz, P.; Cavia-Saiz, M.; Garcia-Giron, C.; García-Gonzalez, M.;

452

Llorente-Ayala, B.; Coma-Del Corral, M. Cancer chemotherapy reduces plasma total

453

polyphenols and total antioxidants capacity in colorectal cancer patients. Mol. Biol. Rep.

454

2012, 39, 9355–9360.

455

(13)

Dolara, P.; Luceri, C.; De Filippo, C.; Femia, A. Pietro; Giovannelli, L.; Caderni, G.;

456

Cecchini, C.; Silvi, S.; Orpianesi, C.; Cresci, A. Red wine polyphenols influence

457

carcinogenesis, intestinal microflora, oxidative damage and gene expression profiles of

458

colonic mucosa in F344 rats. Mutat. Res. 2005, 591, 237–246.

459

(14)

López-Oliva, M. E.; Agis-Torres, A.; Goñi, I.; Muñoz-Martínez, E. Grape antioxidant dietary

460

fibre reduced apoptosis and induced a pro-reducing shift in the glutathione redox state of the

461

rat proximal colonic mucosa. Br. J. Nutr. 2010, 103, 1110–1117.

462

(15)

463 464

function. J. Agric. Food Chem. 2011, 59, 43–49. (16)

465 466

Saura-Calixto, F. Dietary fiber as a carrier of dietary antioxidants: An essential physiological

Zhu, F.; Du, B.; Zheng, L.; Li, J. Advance on the bioactivity and potential applications of dietary fibre from grape pomace. Food Chem. 2015, 186, 207–212.

(17)

García-Lomillo, J.; González-Sanjosé, M. L.; Del Pino-García, R.; Rivero-Pérez, M. D.;

467

Muñiz, P. Antioxidant and antimicrobial properties of wine by-products and their potential

468

uses in the food industry. J. Agric. Food Chem. 2014, 62, 12595–12602.

469

(18)

Del Pino-García, R.; González-Sanjosé, M. L.; Rivero-Pérez, M. D.; García-Lomillo, J.;

470

Muñiz, P. The effects of heat treatment on the phenolic composition and antioxidant capacity

471

of red wine pomace seasonings. Food Chem. 2016. Available at:

472

http://dx.doi.org/10.1016/j.foodchem.2016.10.113

473

(19)

474 475

Saura-Calixto, F.; Serrano, J.; Goñi, I. Intake and bioaccessibility of total polyphenols in a whole diet. Food Chem. 2007, 101, 492–501.

(20)

Del Pino-García, R.; González-Sanjosé, M. L.; Rivero-Pérez, M. D.; García-Lomillo, J.; ACS Paragon Plus Environment

20

Page 20 of 32

Page 21 of 32


476

Muñiz, P. Total antioxidant capacity of new natural powdered seasonings after

477

gastrointestinal and colonic digestion. Food Chem. 2016, 211, 707–714.

478

(21)

Pérez-Magariño, S.; Ortega-Heras, M.; Cano-mozo, E. Optimization of a solid-phase

479

extraction method using copolymer sorbents for isolation of phenolic compounds in red

480

wines and quantification by HPLC. J. Agric. Food Chem. 2008, 58, 11560–11570.

481

(22)

Zhao, G.; Nyman, M.; Jönsson, J. Å. Rapid determination of short-chain fatty acids in

482

colonic contents and faeces of humans and rats by acidified water-extraction and direct-

483

injection gas chromatography. Biomed. Chromatogr. 2006, 20, 674–682.

484

(23)

485 486

Twentyman, P.; Luscombe, M. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br. J. Cancer 1987, 56, 279–285.

(24)

Del Pino-García, R.; González-Sanjosé, M. L.; Rivero-Pérez, M. D.; Muñiz, P. Influence of

487

the degree of roasting on the antioxidant capacity and genoprotective effect of instant coffee:

488

Contribution of the melanoidin fraction. J. Agric. Food Chem. 2012, 60, 10530–10539.

489

(25)

Scharlau, D.; Borowicki, A.; Habermann, N.; Hofmann, T.; Klenow, S.; Miene, C.; Munjal,

490

U.; Stein, K.; Glei, M. Mechanisms of primary cancer prevention by butyrate and other

491

products formed during gut flora-mediated fermentation of dietary fibre. Mutat. Res. 2009,

492

682, 39–53.

493

(26)

Saura-calixto, F.; Pérez-Jiménez, J.; Touriño, S.; Serrano, J.; Fuguet, E.; Torres, J. L.; Goñi,

494

I. Proanthocyanidin metabolites associated with dietary fibre from in vitro colonic

495

fermentation and proanthocyanidin metabolites in human plasma. Mol. Nutr. Food Res.

496

2010, 54, 939–946.

497

(27)

498 499

Bravo, L.; Saura-Calixto, F. Characterization of dietary fibre and the in vitro indigestible fraction of grape pomace. Am. J. Enol. Vitic. 1998, 49, 135–141.

(28)

Goodrich, K. M.; Neilson, A. P. Simultaneous UPLC-MS/MS analysis of native catechins

500

and procyanidins and their microbial metabolites in intestinal contents and tissues of male

501

Wistar Furth inbred rats. J. Chromatogr. B 2014, 958, 63–74.

502

(29)

Yu, J.; Ahmedna, M. Functional components of grape pomace: their composition, biological ACS Paragon Plus Environment

21


503 504

properties and potential applications. Int. J. Food Sci. Technol. 2013, 48, 221–237. (30)

505 506

Selma, M. V; Espín, J. C.; Tomás-Barberán, F. A. Interaction between phenolics and gut microbiota: Role in human health. J. Agric. Food Chem. 2009, 57, 6485–6501.

(31)

Taciak, M.; Barszcz, M.; Tusnio, A.; Bachanek, I.; Pastuszewska, B.; Skomial, J. The effects

507

of type of protein and fibre fermented in vitro with different pig inocula on short-chain fatty

508

acids and amines concentrations. J. Anim. Feed Sci. 2015, 24, 235–243.

509

(32)

Aura, A. M.; Mattila, I.; Hyötyläinen, T.; Gopalacharyulu, P.; Cheynier, V.; Souquet, J. M.;

510

Bes, M.; Le Bourvellec, C.; Guyot, S.; Orešič, M. Characterization of microbial metabolism

511

of Syrah grape products in an in vitro colon model using targeted and non-targeted analytical

512

approaches. Eur. J. Nutr. 2013, 52, 833–846.

513

(33)

Coates, E. M.; Popa, G.; Gill, C. I. R.; McCann, M. J.; McDougall, G. J.; Stewart, D.;

514

Rowland, I. Colon-available raspberry polyphenols exhibit anti-cancer effects on in vitro

515

models of colon cancer. J. Carcinog. 2007, 6, 4.

516

(34)

517 518

Russo, G. L. Ins and outs of dietary phytochemicals in cancer chemoprevention. Biochem. Pharmacol. 2007, 74, 533–544.

(35)

Derry, M.; Raina, K.; Agarwal, R.; Agarwal, C. Differential effects of grape seed extract

519

against human colorectal cancer cell lines: The intricate role of death receptors and

520

mitochondria. Cancer Lett. 2014, 334, 69–78.

521

(36)

Jara-Palacios, M. J.; Hernanz, D.; Cifuentes-Gómez, T.; Escudero-Gilete, M. L.; Heredia, F.

522

J.; Spencer, J. P. E. Assessment of white grape pomace from winemaking as source of

523

bioactive compounds, and antiproliferative activity. Food Chem. 2015, 183, 78–82.

524

(37)

Forester, S. C.; Waterhouse, A. L. Gut metabolites of anthocyanins, gallic acid, 3-O-

525

methylgallic acid, and 2,4,6-trihydroxybenzaldehyde, inhibit cell proliferation of caco-2

526

cells. J. Agric. Food Chem. 2010, 58, 5320–5327.

527

(38)

Jing, P.; Bomser, J.; Schwartz, S.; He, J.; Magnuson, B.; Giusti, M. Structure-function

528

relantionships of anthocyanidins from various anthocyanidin-rich extracts on the inhibition of

529

colon cancer cell growth. J. Agric. Food Chem. 2008, 56, 9391–9398. ACS Paragon Plus Environment

22

Page 22 of 32

Page 23 of 32

530


(39)

531 532

Yi, W.; Fischer, J.; Akoh, C. C. Study of anticancer activities of Muscadine grape phenolics in vitro. J. Agric. Food Chem. 2005, 53, 8804–8812.

(40)

Zitta, K.; Meybohm, P.; Bein, B.; Huang, Y.; Heinrich, C.; Scholz, J.; Steinfath, M.;

533

Albrecht, M. Salicylic acid induces apoptosis in colon carcinoma cells grown in vitro:

534

Influence of oxygen and salicylic acid concentration. Exp. Cell Res. 2012, 318, 828–834.

535

(41)

Correa-Betanzo, J.; Allen-Vercoe, E.; McDonald, J.; Schroeter, K.; Corredig, M.; Paliyath,

536

G. Stability and biological activity of blueberry (Vaccinium angustifolium) polyphenols

537

during simulated in vitro gastrointestinal digestion. Food Chem. 2014, 165, 522–531.

538

(42)

Hague, A.; Elder, D.; Hicks, D.; Paraskeva, C. Apoptosis in colorectal tumour cells:

539

induction by the short chain fatty acids butyrate, propionate and acetate and by the bile salt

540

deoxycholate. Int. J. Cancer 1995, 60, 400–406.

541

(43)

Gamet, L.; Daviaud, D.; Denis-Pouxviel, C.; Remesy, C.; Murat, J.-C. Effects of short chain

542

fatty acids on growth and differentiation of the human colon cancer cell line HT29. Int. J.

543

Cancer 1992, 52, 286–289.

544

(44)

545 546

resistant starch and nonstarch polysaccharides. Physiol. Rev. 2001, 81, 1031–1064. (45)

547 548

Wong, J.; de Sauza, R.; Kendall, C.; Emam, A.; Jenkins, D. Colonic health: fermentation and short chain fatty acids. J. Clin. Gastroenterol. 2006, 40, 235–243.

(46)

549 550

Topping, D. L.; Clifton, P. M. Short-chain fatty acids and human colonic function: roles of

Cemeli, E.; Baumgartner, A.; Anderson, D. Antioxidants and the comet assay. Mutat. Res. 2009, 681, 51–67.

(47)

Brown, E. M.; McDougall, G. J.; Stewart, D.; Pereira-Caro, G.; González-Barrio, R.;

551

Allsopp, P.; Magee, P.; Crozier, A.; Rowland, I.; Gill, C. I. R. Persistence of anticancer

552

activity in berry extracts after simulated gastrointestinal digestion and colonic fermentation.

553

PLoS One 2012, 7, 3–12.

554

(48)

Nuñez-Sánchez, M. A.; García-Villalba, R.; Monedero-Saiz, T.; García-Talavera, N. V.;

555

Gómez-Sánchez, M. B.; Sánchez-Álvarez, C.; García-Albert, A. M.; Rodríguez-Gil, F. J.;

556

Ruiz-Marín, M.; Pastor-Quirante, F. A.; et al. Targeted metabolic profiling of pomegranate ACS Paragon Plus Environment

23


557

polyphenols and urolithins in plasma, urine and colon tissues from colorectal cancer patients.

558

Mol. Nutr. Food Res. 2014, 58, 1199–1211.

559

(49)

Nguyen, A. V; Martinez, M.; Stamos, M. J.; Moyer, M. P.; Planutis, K.; Hope, C.;

560

Holcombe, R. F. Results of a phase I pilot clinical trial examining the effect of plant-derived

561

resveratrol and grape powder on Wnt pathway target gene expression in colonic mucosa and

562

colon cancer. Cancer Manag. Res. 2009, 1, 25–37.

563

(50)

Abrahamse, S. L.; Pool-Zobel, B. L.; Rechkemmer, G. Potential of short chain fatty acids to

564

modulate the induction of DNA damage and changes in the intracellular calcium

565

concentration by oxidative stress in isolated rat distal colon cells. Carcinogenesis 1999, 20,

566

629–634.

567

(51)

Hamer, H. M.; Jonkers, D.; Venema, K.; Vanhoutvin, S.; Troost, F. J.; Brummer, R. J.

568

Review article: The role of butyrate on colonic function. Aliment. Pharmacol. Ther. 2008,

569

27, 104–119.

570

(52)

571 572

nutraceuticals. Nutrients 2014, 6, 391–415. (53)

573 574 575

Georgiev, V.; Ananga, A.; Tsolova, V. Recent advances and uses of grape flavonoids as

Fan, P.; Lou, H. Effects of polyphenols from grape seeds on oxidative damage to cellular DNA. Mol. Cell. Biochem. 2004, 267, 67–74.

(54)

Yang, J.; Xiao, Y.-Y. Grape phytochemicals and associated health benefits. Crit. Rev. Food Sci. Nutr. 2013, 53, 1202–1225.

576 577


24

Page 24 of 32

Page 25 of 32


578

AUTHOR INFORMATION

579

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.


25


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

26

Page 26 of 32

Page 27 of 32


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.


27


Figure 1.


28

Page 28 of 32

Page 29 of 32


Figure 2.


29


Figure 3.


30

Page 30 of 32

Page 31 of 32


Figure 4.


31


TABLE OF CONTENTS GRAPHIC


32

Page 32 of 32

Chemopreventive Potential of Powdered Red Wine Pomace

Recommend Documents