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Bioactive Constituents, Metabolites, and Functions

Vanillin Ameliorated the Development of Azoxymethane/Dextran Sodium Sulfate-Induced Murine Colorectal Cancer: the Involvement of Proteasome/Nuclear Factor-#B/Mitogen-Activated Protein Kinase Pathways Jung-Miao Li, Yu-Chen Lee, Chia-Cheng Li, Hsin-Yi Lo, FengYuan Chen, Yi-Siou Chen, Chien-Yun Hsiang, and Tin-Yun Ho J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01582 • Publication Date (Web): 23 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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Journal of Agricultural and Food Chemistry

1

Vanillin Ameliorated the Development of Azoxymethane/Dextran Sodium

2

Sulfate-Induced

3

Proteasome/Nuclear Factor-κB/Mitogen-Activated Protein Kinase Pathways

Murine

Colorectal

Cancer:

the

Involvement

of

4 5

Jung-Miao Li,†,‡ Yu-Chen Lee,§ Chia-Cheng Li,† Hsin-Yi Lo,† Feng-Yuan Chen,†

6

Yi-Siou Chen,ǁ Chien-Yun Hsiang*,ǁ and Tin-Yun Ho,*,†,⊥

7

†

Taiwan

8 9

‡

§

14

Graduate Institute of Acupuncture Science, China Medical University, Taichung 40402, Taiwan

12 13

Department of Chinese Medicine, Show Chwan Memorial Hospital, Changhua 50008, Taiwan

10 11

Graduate Institute of Chinese Medicine, China Medical University, Taichung 40402,

ǁ Department of Microbiology, China Medical University, Taichung 40402, Taiwan ⊥

Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan

15 16 17

*

18

Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan. Tel.: +886 4

19

22053366

Corresponding author. Prof. Chien-Yun Hsiang, Department of Microbiology, China

x

2163.

Fax:

+886

4

22053764.

1

ACS Paragon Plus Environment

E-mail

address:


20

[email protected]

21

*

22

China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan. Tel.: +886

23

4 22053366 x 3302. Fax: +886 4 22032295. E-mail address: [email protected]

Corresponding author. Prof. Tin-Yun Ho, Graduate Institute of Chinese Medicine,

2


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ABSTRACT

25

Vanillin is a natural dietary flavoring widely used in food industry. Colorectal cancer

26

(CRC) is one of the common malignancies in the world. Chronic intestinal

27

inflammation is a risk factor for the development of CRC. We have previously found

28

that vanillin improves and prevents colitis in mice. Here we evaluated the inhibitory

29

activities of vanillin on a mouse model of colitis-induced CRC. Mice were challenged

30

intraperitoneally with azoxymethane (AOM) and orally with dextran sodium sulfate

31

(DSS). Various dosages of vanillin were orally administered for 13 consecutive weeks.

32

Vanillin alleviated the development of tumors in AOM/DSS-induced mice. The total

33

number of tumors in 100 mg/kg vanillin group was significantly reduced by

34

57.14±7.67%, compared with sham group. Gene expression analysis showed that

35

vanillin down-regulated the expression levels of proteasome genes in colon tissues.

36

Moreover, vanillin at 10 mM significantly suppressed proteasome activities in

37

HCT-116

38

phosphorylation of mitogen-activated protein kinases (MAPK) and reduced the

39

number of p65-positive cells, proliferating cells, and granulocytes in colon tissues

40

with statistical significance. In conclusion, our data suggested that vanillin was a

41

bioactive compound that ameliorated the development of AOM/DSS-induced colon

42

cancer in mice. Moreover, the amelioration of vanillin might be associated with the

cells

by

41.27±0.41%.

Furthermore,

3


vanillin

diminished

the


43

downregulation of proteasome, nuclear factor-κB, and MAPK pathways.

44 45

KEYWORDS: vanillin, colorectal cancer, proteasome, mitogen-activated protein

46

kinases, nuclear factor-κB

4


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INTRODUCTION

48

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the natural component isolated from

49

vanilla beans. It is commonly used in food, beverage, cosmetic, and pharmaceutical

50

industries as a flavoring agent. Previous studies suggested that vanillin exhibits

51

pharmacological effects via its anti-inflammatory activities in vivo. For example,

52

vanillin protects against cancer chemotherapeutics-induced renal injuries in rats by

53

inhibiting inflammation, oxidative stress, and apoptosis.1 Vanillin ameliorates

54

psoriatic skin inflammation in mice by the downregulation of interleukin-17

55

(IL-17)/IL-23 expression.2 Vanillin displays neuroprotective effects in mice by

56

anti-inflammatory and anti-oxidant activities.3 Vanillin suppresses ethanol-induced

57

gastric ulcer in rats via the modulation of inflammation, gastric secretion, and

58

oxidative stress.4 Vanillin also improves and prevents trinitrobenzene sulfonic acid

59

(TNBS)-induced colitis in mice by the downregulation of proinflammatory

60

cytokines.5 Nevertheless, less studies report the effects of vanillin on the

61

inflammation-mediated cancers.

62

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and

63

the fourth leading cause of cancer death worldwide.6 According to the cancer

64

statistical data from Surveillance, Epidemiology, and End Results Program, CRC

65

represents 8.0% of all new cancer cases and 8.4% of all cancer deaths in the U.S. in 5



66

2017.7 CRC is a complex disease caused by genetic background and environmental

67

risk factors, such as diabetes, cholecystectomy, obesity, and high fat diet.6 In the U.S.,

68

up to 47% of CRC cases can be prevented by staying physically active, maintaining a

69

healthy body weight, and eating a healthy diet.8 Uptake of dietary phytochemicals and

70

microbiota affords CRC protection or reduces CRC risk, also suggesting the

71

association between obesity/diet and CRC.9,10 In addition, patients with long-standing

72

inflammatory bowel disease (IBD) are at a higher risk of developing CRC. The

73

cumulative risk for developing CRC in extensive IBD is 19-fold increase when

74

compared with the general population, also suggesting that chronic intestinal

75

inflammation is a predisposing condition to CRC.11

76

The ubiquitin-proteasome system (UPS) plays a crucial role in the regulation of

77

cellular function and homeostasis by the degradation of the majority of intracellular

78

proteins. Several studies indicate that UPS regulates cell cycle progression, tumor

79

suppression, inflammation, and apoptosis.12 Proteasome is the enzymatic core engine

80

of UPS, which recognizes polyubiquitinated proteins and hydrolyzes them into short

81

peptide fragments. Inhibition of proteasome activities blocks the degradation of

82

proteins and causes the accumulation of misfolded proteins, which in turn triggers

83

heat shock response, cell death, and apoptosis.13 In addition, proteasome inhibitors,

84

such as bortezomib, carfilzomib and ixazomib, have been used as anti-cancer drugs 6


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against multiple myeloma. These findings suggested the proteasome is a valid target

86

for chemotherapy.14

87

In previous study, we have found that vanillin is able to improve and prevent

88

TNBS-induced colitis in mice, a model resembling IBD in human.5 Since IBD is a

89

risk factor for developing CRC, we wondered whether vanillin displayed inhibitory

90

activities against CRC. A mouse model of colitis-induced colon cancer was

91

established by intraperitoneally injecting with azoxymethane (AOM) and orally

92

giving with dextran sodium sulfate (DSS).15 The improvement of colon cancer by

93

vanillin was evaluated by macroscopic and microscopic lesions. The anti-cancer

94

mechanism of vanillin was analyzed by transcriptomic tools and further confirmed by

95

immunohistochemical (IHC) staining and Western blot. Our data showed that vanillin

96

ameliorated the development of cancers in mice with AOM/DSS-induced

97

colitis-associated colon cancer. Moreover, the improvement of vanillin might be

98

associated with proteasome inhibition.

99 100 101

MATERIALS AND METHODS

102

Chemicals. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO),

103

unless indicated. The purities of 5-aminosalicylic acid (5-ASA) and vanillin are ≥ 7



104

99% and 99%, respectively. DSS (36,000-50,000 Da) was purchased from MP

105

Biomedicals (Santa Ana, CA). Proteasome β5 substrate N-succinyl-Leu-Leu-Val-Tyr

106

7-amido-4-methylcoumarin (Suc-LLVY-AMC) was dissolved in dimethyl sulfoxide

107

(DMSO) at 10 mM. Proteasome inhibitor MG-132 was purchased from Enzo

108

(Farmingate, NY) and dissolved in DMSO at 25 mg/ml. Rabbit polyclonal antibodies

109

against inhibitory κB-α (IκB-α), phospho-IκB-α, IκB kinase α/β (IKKα/β), phospho-

110

IKKα/β,

111

N-terminal kinases (JNKs), phospho-JNKs, p38, and phospho-p38 were purchased

112

from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antibody against

113

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and rabbit polyclonal antibody

114

against proliferating cell nuclear antigen (PCNA) were purchased from Santa Cruz

115

(Dallas, TX). Rabbit monoclonal antibody against CD11b and rabbit polyclonal

116

antibody against proteasome β5 were purchased from abcam (Cambridge, MA).

117

Mouse monoclonal antibody against p65 was purchased from Millipore (Temecula,

118

CA).

extracellular signal-regulated kinases (ERKs), phospho-ERKs, c-Jun

119 120

Cell Culture. Human colon cancer cells (HCT-116) were purchased from

121

Bioresource Collection Research Center (Hsinchu, Taiwan). HCT-116 cells were

122

maintained in McCoy's 5A medium (Hyclone, Logan, UT) supplemented with 10% 8


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fetal bovine serum (Hyclone, Logan, UT). Cells were incubated at 370C in a

124

humidified atmosphere containing 5% CO2.

125 126

Animal Experiments. BALB/c mice (6-week old, female) were purchased from

127

National Laboratory Animal Center (Taipei, Taiwan). Mouse experiments were

128

conducted under ethics approval from China Medical University Animal Care and Use

129

Committee (Permit No. 2016-034). Mice were maintained under a 12:12 light-dark

130

cycle with free access to water and standard diet (#5001, LabDiet, St Louis, MO).

131

A total of 60 mice was randomly divided into 6 groups of 10 mice: (1) mock, (2)

132

sham, (3) 5-aminosalicylic acid (5-ASA), (4) V10, (5) V50, and (6) V100.

133

Colitis-associated colon cancer was induced as described previously.15 Briefly, on

134

Day 1, mice were injected intraperitoneally with either phosphate-buffered saline

135

(PBS) (137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, 2.7 mM KCl, pH 7.2)

136

(mock group) or 12.5 mg/kg AOM diluted in PBS (sham group). On Day 8, mice

137

receiving the AOM injection were treated with 2.5% DSS in supplemented drinking

138

water for 5 consecutive days, followed by normal drinking water for 16 days. DSS

139

treatment

140

AOM/DSS-induced mice were orally given with 75 mg/kg 5-ASA three times a week

141

for 13 consecutive weeks. For vanillin groups, AOM/DSS-induced mice were orally

was

repeated

for

three

additional

cycles.

9


For

5-ASA

group,


142

given with 10, 50, and 100 mg/kg vanillin resuspended in distillted water for 13

143

consecutive weeks (Figure 1A). Body weight was measured weekly. On Day 91, mice

144

were sacrificed. Colons were removed and opened longitudinally for observation. The

145

size and the number of macroscopic tumors were measured and recorded. Colon

146

tissues were then collected for histological examination, IHC staining, microarray

147

analysis, and Western blot analysis. Blood samples were collected for the

148

measurement of cytokines.

149 150

Histological Examination, IHC Staining, and Cytokine Enzyme-Linked

151

Immunosorbent Assay (ELISA). Histological examination was performed on

152

hematoxylin/eosin (H&E)-stained colon sections. For IHC staining, colon tissue

153

sections were incubated with antibodies against PCNA, p65 active form, and CD11b

154

(1:200 dilution) at 40C overnight. Sections were then stained with biotinylated

155

secondary antibody, avidin-biotin complex reagent, and 3,3'-diaminobenzidine

156

according to manufacturer's manual (Histostain®-Plus, Invitrogen, Camarillo, CA).

157

Slides were scanned using Aperio ScanScope (Leica Microsystems, Wetzlar, Germany)

158

and analyzed using ImageJ (Media Cybernetics, Bethesda, MD). The proportions of

159

PCNA-, p65-, and CD11b-positive cell (%) were calculated as (the number of brown

160

cells/the total number of cells) × 100. One hundred cells were counted in each view. 10


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For cytokine ELISA, the levels of IL-1β and tumor necrosis factor-α (TNF-α) were

162

quantified using mouse IL-1β and TNF-α ELISA kits (Thermo Fisher, Waltham,

163

MA).16

164 165

Microarray Analysis. Total RNA was extracted from 30 mg of colon tissues using

166

RNeasy Mini kit (Qiagen, Valencia, CA). The amount and the integrity of RNA

167

samples were evaluated using spectrophotometer (Beckman Coulter, Fullerton, CA)

168

and Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively.

169

Microarray

170

fluorescence-labeled RNA was hybridized to Mouse Whole Genome OneArray

171

(Phalanx Biotech Group, Hsinchu, Taiwan). The fluorescent intensity was scanned by

172

an Axon 4000 scanner (Molecular Devices, Sunnyvale, CA) and analyzed by genepix

173

4.1 software (Molecular Devices, Sunnyvale, CA). All microarray data are MIAMI

174

compliant database (Gene Expression Omnibus accession number GSE109602).

analysis

was

performed

as

described

previously.17

Cy5

175

Microarray data were normalized by R program in the limma package using

176

quantile normalization. The number of replicates was three. The threshold of gene

177

expression intensity was set at 500 to filter out genes that were lowly expressed. The

178

differentially expressed genes were determined based on their absolute intensity

179

values and p-values. Intensity change of gene in sham group was calculated by 11



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subtracting gene intensity in sham group from one in mock group, while intensity

181

change of gene in V100 group was calculated by subtracting gene intensity in V100

182

group from one in sham group. The differentially expressed genes were further

183

selected for biological pathway analysis using DAVID Bioinformatics Resources 6.7

184

(https://david-d.ncifcrf.gov/home.jsp). The gene ontology enrichment analysis was

185

performed and displayed using the Gene Ontology Enrichment Analysis Software

186

Toolkit (http://omicslab.genetics.ac.cn/GOEAST/).

187 188

Quantitative real-time polymerase chain reaction (qPCR)

189

The expression levels of proteasome genes (Psma4, Psmb5, and Psmb10) were

190

validated by qPCR. Briefly, total RNA was reverse-transcribed at 370C for 120 min

191

using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster

192

City, CA). qPCR was performed by the following condition: 10 min at 950C; 40

193

cycles of 15 sec at 950C and 1 min at 600C. Each assay was run on an Applied

194

Biosystems 7300 Real-Time PCR system in triplicates. Fold changes were calculated

195

using the comparative CT method.18 Gapdh gene was used as an endogenous control.

196

The

197

5'-TCAGCTCTATCAGAGTGACCCAAGT-3'

198

5'-GCCTTCTTTGTAGTCTTGTTTCAACA-3'

primer

set

for each

gene

is as follows.

Psma4

and (GenBank

12


forward

reverse Accession

primer primer No.

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199

NM_011966); Psmb5 forward primer 5'-GAAAGTGGAGGAGGCCTATGATC-3'

200

and reverse primer 5'-GACTGCCCCTCCGGAGTAG-3' (GenBank Accession No.

201

NM_011186); Psmb10 forward primer 5'-TGCAGCCGTGGCACTGT-3' and reverse

202

primer

203

NM_013640); Gapdh forward primer 5'-GTTGTCTCCTGCGACTTCA-3' and

204

reverse primer 5'-GGTGGTCCAGGGTTTCTTA-3' (GenBank Accession No.

205

NM_011966.3).

5'-CACTCAGGATCCCTGCTGTGA-3'

(GenBank

Accession

No.

206 207

Proteasome Activity Assay. Proteasome activity assay was performed as described

208

previously with a slight modification.19 Briefly, HCT-116 cells (5×105 cells/well) were

209

cultured in 6-well plates and incubated at 370C. After a 16-h incubation, medium was

210

removed, and cells were washed once with PBS and treated with 10 µM MG-132 or

211

various amounts of vanillin for 2 h at 370C. Cells were then washed with ice-cold PBS

212

twice, lyzed with NP-40 lysis buffer (50 mM HEPES, pH 7.5, 1% NP-40, 150 mM

213

NaCl, 5 mM EDTA), and scraped using cell scrapers. After a centrifugation at 14,000

214

rpm for 10 min at 40C, the supernatant was collected and the amount of protein in

215

supernatant was determined by a Bradford method (Bio-Rad, Hercules, CA). A 100-µl

216

reaction mixture containing 10 µg protein, 50 µM Suc-LLVY-AMC, 25 mM HEPES

217

(pH 7.5), 0.5 mM EDTA, 0.05 % NP-40, 0.001% sodium dodecyl sulfate (SDS), and 13



218

5 mM MgCl2 was incubated at 370C for 2 h. The fluorescent compound

219

7-amino-4-methylcoumarin released from Suc-LLVY-AMC after proteasome cleavage

220

was measured using a fluorometer (Fluoroskan Ascent FL, Thermo Fisher, Waltham,

221

MA) with an excitation wavelength at 380 nm and an emission wavelength at 460 nm.

222

Proteasome β5 activity (%) was calculated as (the fluorescence intensity of MG-132-

223

or vanillin-treated cells / the fluorescence intensity of solvent-treated cells)×100.

224 225

Western Blot Analysis. Colon tissues were lyzed with RIPA buffer (50 mM

226

Tris-HCl, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl,

227

2 mM EDTA, 50 mM NaF) containing protease/phosphatase inhibitor cocktail (Cell

228

Signaling Technology, Boston, MA). Protein samples (50 µg) were separated by 10%

229

SDS-polyacrylamide gel electrophoresis and transferred electrophoretically to

230

nitrocellulose membranes. After blocking the nonspecific sites with 5% skimmed milk

231

for 1 h, membranes were incubated with primary antibodies at 40C overnight and

232

horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h,

233

visualized by chemiluminescence (Phototope®-HRP Western detection kit, New

234

England Biolabs, Ipswich, MA), and then exposed by autoradiography. Protein bands

235

on the X-ray films were measured using Gel-Pro® Analyzer (Media Cybernetics,

236

Silver Spring, MD). Quantitative data were normalized by internal control (GAPDH) 14


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and further expressed as relative protein expression, which was presented as the

238

comparison with the amount relative to mock.20

239 240

Statistical Analysis. Data were presented as mean ± standard error. Data were

241

analyzed by one-way ANOVA and post hoc Bonferroni test using SPSS Statistics

242

version 20 (IBM, Armonk, NY). A p-value < 0.05 was considered as statistically

243

significant.

244 245 246

RESULTS

247

Vanillin Ameliorated the Development of Tumors in AOM/DSS-Induced Mice.

248

To analyze whether vanillin attenuated the formation of colon cancers, we established

249

an AOM/DSS-induced colitis-associated colon cancer model in BALB/c mice.

250

Vanillin was orally given for 13 consecutive weeks. Body weight was measured

251

weekly. As shown in Figure 1B, the body weight of AOM/DSS-treated mice was

252

reduced when mice were supplemented with 2.5% DSS drinking water. However, the

253

body weight loss was alleviated when they received tap water without DSS. Mice

254

received 5-ASA or vanillin also had a reduced body weight loss, while the reduced

255

body weight was recovered more quickly than that in sham group. No statistical 15



256

differences were observed in the body weight of mice treated with vehicle (mock

257

group), 5-ASA, or vanillin during the experimental period.

258

Mice were sacrificed on Day 91, and colons were opened longitudinally for

259

observation. The length of colon in sham group (7.64±0.51 cm) was significantly

260

shorter than that in mock group (8.76±0.57 cm) (Figure 2A). However, 5-ASA

261

(8.38±0.40 cm) and vanillin (8.51±0.60 cm at 100 mg/kg) treatments significantly

262

reduced the colon shortening. Moreover, the alleviative effects of vanillin displayed a

263

dose-dependent manner. Histological examination showed that colon sections in mock

264

group displayed a normal microvilli architecture with intact surface epithelia, crystal

265

glands, and submucosa (Figure 2B). AOM/DSS treatment showed distorted crypt and

266

epithelia, inflammatory cell infiltration, and tubular adenocarcinoma. 5-ASA and

267

vanillin treatment showed improved crypt structures and histological features. The

268

incidence of tumors was 100% in sham group. 5-ASA and vanillin administration

269

failed to reduce the incidence of tumor, while the total number and the size of tumors

270

were significantly reduced by 5-ASA and vanillin. The total number of tumors in

271

sham group was 36±10.3 per mouse and the number of large tumors (diameter > 4

272

mm) was 19.29±4.96 (Figure 2B). Approximately 54%, 32%, and 14% of the tumors

273

were > 4 mm, 2-4 mm, and < 2 mm in diameter, respectively. However, the total

274

numbers of tumors in 5-ASA and V100 groups were reduced by 57.78±7.19% and 16


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57.14±7.67%, respectively, compared with sham group. The number of large tumors

276

was reduced by 54.95±5.91% and 37.77±11.87% in 5-ASA and V100 groups,

277

respectively. Approximately 37%, 33%, and 30% of the tumors in V100 group were >

278

4 mm, 2-4 mm, and < 2 mm in diameter, respectively. These findings suggested that

279

vanillin ameliorated the development of tumors in AOM/DSS-induced mice.

280

Moreover, the amelioration of vanillin displayed a dose-dependent manner.

281 282

Vanillin

Affected

Gene

Expression

Profiles

of

Colon

Tissues

in

283

AOM/DSS-Induced Mice. To elucidate the mechanisms of vanillin on the alleviation

284

of colon cancers, we extracted RNA samples from mock, sham, and V100 groups, and

285

the gene expression profiles were analyzed by microarray. Normal colon tissues in

286

mock group and tumor mass tissues in both sham group and V100 group were used

287

for RNA and protein extraction. Tissues in relative anatomical colon locations were

288

selected in each group to avoid the interferences resulting from different colon

289

sections. The expression of genes in sham group was compared with that in mock

290

group to analyze the carcinogenic mechanism of AOM/DSS, while the expression

291

levels of genes in V100 group were compared with those in sham group to evaluate

292

the anti-carcinogenic mechanism of vanillin. The transcripts of 1,437 genes and 915

293

genes were up-regulated and down-regulated, respectively, with an intensity change ≥ 17



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294

500 or ≤ -500 in sham group. However, a total of 1,244 AOM/DSS-upregulated genes

295

was down-regulated by 100 mg/kg vanillin. Genes with intensity changes ≥ 500 or ≤

296

-500 in vanillin group were further selected for gene ontology analysis and biological

297

pathway analysis. Gene ontology analysis showed that 471 biological process

298

categories were affected (data not shown). About a third of categories belonged to

299

biological regulation, while a third of categories belonged to metabolic process.

300

Pathway analysis further showed that six biological pathways involved in protein

301

folding and degradation (proteasome and ER-associated degradation), transcription

302

(spliceosome), immune system (Fcγ-mediated phagocytosis), cell motility (regulation

303

of actin cytoskeleton), and glycan metabolism (N-glycan biosynthesis) were

304

significantly affected by vanillin (Table 1).

305 306

Vanillin

Reduced

Proteasome

Expressions

in

Colon

Tissues

of

307

AOM/DSS-Induced Mice and Inhibited Proteasome Activities in HCT-116 Cells.

308

Proteasome is a

309

damaged proteins by proteolysis. Proteasome is involved in several essential cellular

310

functions, including cell cycle, cell differentiation, signal transduction pathways,

311

antigen processing, inflammatory responses, and apoptosis.12 Proteasome inhibitors,

312

such as bortezomib, carfilzomib and ixazomib, have been used as anti-cancer drugs

protein-destroying apparatus that degrades unneeded or

18


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313

against multiple myeloma.14 In this study, we found that vanillin significantly altered

314

the proteasome biological pathway in colon tissues of AOM/DSS-induced mice. The

315

expression levels of nine proteasome genes were up-regulated in sham group,

316

compared with mock (Figure 3A) by microarray analysis. However, the induced

317

expressions were significantly down-regulated by vanillin. Proteasome consists of a

318

20S multicatalytic core and two 19S regulatory subunits. 20S proteasome is a

319

chambered structure that consists of α subunits and β subunits. The expression levels

320

of 20S core α subunit gene (Psma4) and β subunit genes (Psmb5 and Psmb10) were

321

further validated by qPCR. Table 2 shows that AOM/DSS treatment up-regulated the

322

expression levels of Psma4, Psmb5, and Psmb10 genes, while vanillin treatment

323

down-regulated the expression of these genes. In addition, Western blot was

324

performed to analyze the protein level of proteasome β5. Figure 3B shows that, in

325

comparison with mock, the level of proteasome β5 was increased by 1.73 ± 0.23 fold

326

in sham group. However, in comparison with sham, vanillin decreased the protein

327

level of proteasome β5 by 0.81 ± 0.13 fold. The consistency between microarray data,

328

qPCR, and Western blot suggested that vanillin suppressed the expression of

329

proteasome genes. Proteasomen β5 exhibits the chymotrypsin-like activity.13

330

Proteasome activity assay using β5 substrate (Suc-LLVY-AMC) was therefore

331

performed. Human colon cancer HCT-116 cells were treated with MG-132 or various 19



Page 20 of 50

332

amounts of vanillin. Proteasome activities were analyzed by detecting the fluorescent

333

intensity released from Suc-LLVY-AMC after proteasome cleavage. As shown in

334

Figure 3C, MG-132, a proteasome inhibitor, significantly reduced the proteasome

335

activity by 80%, compared with mock. Vanillin also significantly suppressed

336

proteasome activities in a dose-dependent manner. The maximal inhibition was

337

achieved at 10 mM vanillin. No cytotoxic effect was observed (data not shown).

338

These findings suggested that vanillin suppressed the expression of proteasome genes

339

and consequently led to the reduction of proteasome activities.

340 341

Vanillin Altered the Activation of Nuclear Factor-κB (NF-κB) and

342

Mitogen-Activated

Protein

Kinase

(MAPK)

343

AOM/DSS-Induced Mice. Proteasome is responsible for the activation of NF-κB by

344

the degradation of phosphorylated IκB-α.21 NF-κB interacts with MAPK and plays

345

important roles in inflammation and carcinogenesis.22 We wondered whether vanillin

346

suppressed NF-κB activation and altered MAPK pathways via the inhibition of

347

proteasome activity, IHC staining and Western blots on colon tissue samples were

348

therefore performed. Sections of colon tissues were stained with antibody against the

349

nuclear localization signal of p65. Compared with mock, the number of p65-positive

350

cells in the colons was significantly increased in sham group (Figure 4A). However, 20


in

Colon

Tissues

of

Page 21 of 50


351

vanillin and 5-ASA reduced the number of p65-positive cells. Western blot showed

352

that the levels of MAPK proteins were similar in all groups (Figure 4B). The

353

phosphorylations of ERKs, JNKs, and p38 were induced in sham group. However,

354

vanillin and 5-ASA significantly diminished the phosphorylation of ERKs, JNKs, and

355

p38. These findings suggested that NF-κB and MAPKs were activated in colon tissues

356

of AOM/DSS-induced mice, while activated NF-κB and MAPKs were suppressed by

357

vanillin.

358 359

Vanillin Suppressed Cell Proliferation, Granulocyte Infiltration, and Cytokine

360

Production in Mice with AOM/DSS-Induced Colitis-Associated Colon Cancer.

361

NF-κB and MAPK pathways are associated with inflammation and various cellular

362

processes, such as cell proliferation and cell survival. Activation of NF-κB has been

363

implicated in the development and the progression of various human malignancies,

364

such as colon cancer and breast cancer.23 Constitutive activation of MAPK has also

365

been correlated with malignancies in various types of cancers.24 We found that NF-κB

366

and MAPK were activated in colon tissues of AOM/DSS-induced mice. However,

367

induced activities were reduced by vanillin. Therefore, we further analyzed whether

368

vanillin suppressed cell proliferation and inflammation in colon tissues by IHC

369

staining. PCNA has been found in the nuclei of cells that undergo cell proliferation. 21



370

CD11b is implicated in various adhesive interactions of monocytes, macrophages and

371

granulocytes. As shown in Figure 5A, the numbers of PNCA-positive cells and

372

CD11b-positive cells in the colons were significantly increased in sham group,

373

compared with mock. However, vanillin and 5-ASA reduced the number of

374

PCNA-positive cells and CD11b-positive cells. These findings suggested that vanillin

375

suppressed the proliferation of cells and the infiltration of immune cells in colon

376

tissues. In addition to IHC staining, we performed cytokine ELISA to measure the

377

amount of proinflammatory cytokines in sera. Figure 5B shows that the amounts of

378

IL-1β and TNF-α in sera were significantly increased in sham group, compared with

379

mock. However, the increased amounts of IL-1β and TNF-α were significantly

380

decreased by vanillin and 5-ASA. These data suggested that vanillin suppressed the

381

proliferation of cells and the infiltration of immune cells via NF-κB and MAPK

382

pathways, leading to the amelioration of AOM/DSS-induced colitis-associated colon

383

cancer. Moreover, the suppression of vanillin on the development of tumors in

384

AOM/DSS-induced mice might be associated with proteasome inhibition.

385 386 387

DISCUSSION

22


Page 22 of 50

Page 23 of 50


388

Clinical studies have shown that patients with extensive IBD have a 19-fold risk of

389

developing CRC, compared to the general population. Therefore, AOM/DSS-induced

390

colitis-associated colon cancer model was established in BALB/c mouse strain in this

391

study. DSS has been applied to induce colitis in rodent models. Administration of DSS

392

in drinking water leads to the ulceration of colonic mucosa and the development of

393

chronic colitis in mice, which resembles to the phenotypic features of human IBD.25

394

AOM is a carcinogen that causes the formation of O6-methylguanine after metabolic

395

activation and induces the development of tumors in the colon of rodents.26 We found

396

that the incidence of colon cancer induced by AOM and DSS was 100% and the

397

tumors were detected in both the distal and the proximal colons in BALB/c mice.

398

However, previous study showed that the higher tumor burden is observed in the

399

distal colon/rectum, while little tumor growth is detected in the proximal colon.15 The

400

differences might be due to the sensitivity of mouse strains to AOM/DSS treatment

401

because Suzuki et al. (2006) reported that BALB/c mice are extremely sensitive to

402

AOM/DSS-induced colon carcinogenesis with the highest tumor incidence and

403

multiplicity, followed by C57BL/6N, C3H/HeN, and DBA/2N mice.27 In addition,

404

pathological findings and gene expression observed in this study were consistent with

405

those in other studies. For example, histological examination showed distorted crypt

406

and epithelia, and tubular adenocarcinoma, which were similar to human 23



407

adenocarcinomas of the colon. Moreover, the expression of β-catenin gene (CRC

408

molecular biomarker gene) was significantly upregulated in sham group (an intensity

409

of 33,201), compared with mock group (an intensity of 20,791) in this study.

410

In previous study, we have found that vanillin improves and prevents the

411

TNBS-induced colitis in mice.5 However, no study reports the anti-cancer activities of

412

vanillin on the colitis-associated colon cancer so far. Ho et al. (2012) investigated the

413

effect of vanillin on AOM-induced aberrant crypt foci (ACF)-bearing rats.28 ACF are

414

early pre-neoplastic lesions of adenocarcinoma that appears on the surface of rodents

415

after the treatment of AOM.29 The carcinogenesis of AOM-induced ACF is through

416

colonotropic mutagenicity of AOM, which is totally different from that of

417

colitis-related colon cancer induced by AOM and DSS. They found that oral

418

administration of vanillin has no effect on ACF density and multiplicity in rats.

419

Previous studies indicated that vanillin is an antimutagenic dietary flavoring that

420

induces DNA damage and elicits recombinational DNA repair, which in turn reduces

421

spontaneous mutations in HCT-116 cells.30 However, vanillin has no effect on the

422

expression of Xpa (nucleotide excision repair), protein kinase C (nonhomologous end

423

joining), Mgmt (direct reversal), and Apex1 (base excision repair) genes in rats treated

424

with AOM.27 In addition, our study found that the expression of aforementioned genes

425

was not affected or was slightly downregulated by vanillin (data not shown). These 24


Page 24 of 50

Page 25 of 50


426

findings might explain why vanillin did not display efficacies on chemical

427

mutagen-induced ACF. Additionally, previous study indicated that DSS complexes to

428

medium-chain-length fatty acids that are present in the colonic lumen, forming

429

nanometer-sized vesicles that can fuse with colonocyte membranes, entry into the

430

cytoplasm, and activate intestinal inflammatory signaling pathways.31 Other study

431

indicated that vanillin binds covalently to the amino groups of proteins via Schiff base

432

formation under a liquid or high-moisture food environment.32 Because of the lack of

433

amino groups in DSS-associated molecules (free glucose, sodium sulfate solution, and

434

free dextran), we speculated that vanillin won't interact with DSS and inhibited

435

DSS-induced inflammation although both vanillin and DSS were given on Day 1.

436

Effects of vanillin on human CRC cell lines have been analyzed previously. Deb et

437

al. (2011) reported that vanillin exhibits relatively limited toxicity against SW480

438

cells, with an IC50 value of 2.5 mM.33 Ho et al. (2009) reported that vanillin induces

439

apoptosis and arrests cell cycle in HT-29 cells at different checkpoints.34 G0/G1 arrest

440

is achieved at a lower concentration (1.3 mM) of vanillin, while G2/M arrest occurs at

441

a higher concentration (6.6 mM) of vanillin. In this study, we found that vanillin

442

significantly reduced the total number and the size of tumors in AOM/DSS-induced

443

mice. Gene ontology analysis showed that vanillin affected 471 biological process

444

categories, including cell proliferation, cell death, and programmed cell death (data 25



445

not shown). We further found that pathways involved in protein folding, protein

446

degradation, and inflammation (FcγR-mediated phagocytosis) were significantly

447

affected by vanillin. These findings suggested that, in addition to altering cell cycle,

448

vanillin affected inflammation and protein conformational stability in mice with

449

colitis-related colon cancer.

450

Proteasome is a cellular multiprotein complex that modulates or degrades cellular

451

proteins and plays a crucial role on the regulation of proliferation, apoptosis,

452

motility/metastasis, tumor suppression, and inflammation.12 26S proteasome complex

453

has two 19S regulatory particles flanking a barrel-shaped 20S core particle. Proteins

454

carrying polyubiquitinated chains are recognized by the 19S subunit and degraded

455

into small peptides by the 20S subunit. 20S core particle consists of α subunits and β

456

subunits, where β subunits are predominantly catalytic and comprise three proteolytic

457

sites, β1 (caspase-like), β2 (trypsin-like), and β5 (chymotrypsin-like).13 In this study,

458

we found that the expression of 20S core α subunit (Psma1 and Psma4), 20S core β

459

subunit (Psmb2, Psmb5, Psmb9, and Psmb10), 26S proteasome ATPase (Psmc4), and

460

26S proteasome non-ATPase (Psmd3 and Psmd8) genes was upregulated in sham

461

group. Indeed, the alteration of proteasome pathway plays a crucial role on the

462

development of CRC.35 The induced expression of proteasome genes was

463

down-regulated in vanillin group. Moreover, vanillin significantly suppressed 26


Page 26 of 50

Page 27 of 50


464

proteasome activities of HCT-116 cells. Recent study showed that the ubiquitination

465

of Akt is elevated in response to vanillin treatment prior to proteasomal degradation,

466

suggesting that vanillin can inhibit cancer stem cell-like behavior in NCI-H460 cells

467

through the induction of Akt-proteasomal degradation.36 Nevertheless, no study

468

indicates that vanillin is capable of suppressing the expression and activities of

469

proteasome. Therefore, our findings suggested that vanillin suppressed the expression

470

of proteasome genes and consequently led to the reduction of proteasome activities.

471

Moreover, because the association between proteasome pathway and CRC, our

472

findings suggested that the suppression of vanillin on the development of tumors in

473

AOM/DSS-induced mice might be associated with proteasome inhibition.

474

Proteasome is the enzymatic core engine of ubiquitin-proteasome system, which

475

recognizes polyubiquitinated proteins and hydrolyzes them into short peptide

476

fragments. Although the degradation of ubiquitinated proteins by proteasome leads to

477

the decreased levels of ubiquitinated protein, the cleavage of ubiquitin from proteins

478

by deubiquitinating enzymes might affect the levels of ubiquitinated proteins in

479

cells.37 The crosstalk mechanisms of proteasome and deubiquitinating enzymes affect

480

the levels of ubiquitinated proteins, so we did not analyze the amounts of

481

ubiquitinated proteins in this study. Nevertheless, IHC staining shows that the number

482

of p65-positive cells in the colons was significantly increased in sham group, while 27



483

vanillin reduced the number of p65-positive cells. These findings suggested that the

484

degradation of ubiquitinated IκB-α by proteasome resulted in the nuclear translocation

485

of p65 in sham group, while the suppression of proteasome led to the inhibition of

486

translocation of p65 in vanillin group.

487

Proteasome inhibitors, such as bortezomib, carfilzomib and ixazomib, have been

488

used as anti-cancer drugs against hematologic cancers, such as multiple myeloma and

489

lymphoma.14 Proteasome inhibition also leads to the inhibition of proliferation in a

490

variety of solid tumors, including CRC.38 NF-κB and MAPK pathways are prominent

491

mechanisms proposed to explain the inhibitory effects of proteasome inhibitors.

492

NF-κB and MAPK pathways are associated with inflammation, cell proliferation, and

493

cell survival. NF-κB up-regulation is observed during colorectal carcinogenesis,

494

suggesting that activation of NF-κB plays a crucial role on the development and the

495

progression of CRC.23 Constitutive activation of MAPK has also been correlated with

496

malignancies in various types of cancers.24 We found that NF-κB and MAPK were

497

activated in colon tissues of AOM/DSS-induced mice, while induced activities were

498

reduced by vanillin. Therefore, we proposed that vanillin suppressed the expression of

499

proteasome and subsequently altered NF-κB and MAPK pathways, which in turn

500

suppressed the proliferation of cells and the infiltration of immune cells. The

28


Page 28 of 50

Page 29 of 50


501

suppression by vanillin further led to the alleviation of tumors in mice with

502

AOM/DSS-induced colitis-associated colon cancer.

503

In conclusion, vanillin is a dietary flavoring with a low toxicity. Oral

504

administration of vanillin at 300 mg/kg in rats (equivalent to 600 mg/kg in mice)

505

displays no toxicity in rats.39 In this study, we first identified that vanillin affected the

506

expression of proteasome genes. Moreover, oral administration of vanillin at 100

507

mg/kg ameliorated the development of tumors in AOM/DSS-induced mice. Because it

508

is well known that CRC cases can be prevented by eating natural healthy diet, our

509

findings suggested that the consumption of vanillin might have a beneficial effect on

510

CRC.

511 512

FUNDING SOURCES

513

This work was supported by grants from Ministry of Science and Technology

514

(MOST104-2320-B-039-018-MY3 and MOST105-2320-B-039-017-MY3) and China

515

Medical University (CMU104-H-01 and CMU104-H-02).

516 517

NOTES

518

The authors declare no competing financial interest.

519 29



Page 30 of 50

520

ABBREVIATIONS USED

521

5-ASA, 5-aminosalicylic acid; ACF, aberrant crypt foci; AOM, azoxymethane; CRC,

522

colorectal cancer; DMSO, dimethyl sulfoxide; DSS, dextran sodium sulfate; ELISA,

523

enzyme-linked immunosorbent assay; ERKs, extracellular signal-regulated kinases;

524

GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, hematoxylin/eosin; IBD,

525

inflammatory bowel disease; IHC, immunohistochemical; IKKα/β, IκB kinase α/β;

526

IL-17, interleukin-17; IκB-α, inhibitory κB-α; JNKs, c-Jun N-terminal kinases;

527

MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; PBS,

528

phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; qPCR,

529

quantitative real-time polymerase chain reaction; SDS, sodium dodecyl sulfate;

530

Suc-LLVY-AMC, N-succinyl-Leu-Leu-Val-Tyr 7-amido-4-methylcoumarin; TNBS,

531

trinitrobenzene

532

ubiquitin-proteasome system

sulfonic

acid;

TNF-α,

tumor

30


necrosis

factor-α;

UPS,

Page 31 of 50


533

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534

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37



655

FIGURE CAPTIONS

656

Figure 1. Effect of vanillin on the body weight of AOM/DSS-induced mice. (A)

657

Scheme of the experiments. AOM/DSS-induced mice were orally given with 75

658

mg/kg 5-ASA (three times a week) or various amounts of vanillin (daily). On Day 91,

659

mice were sacrificed for further analysis. (B) Body weight was measured weekly for

660

the 13-week treatment period. Arrows indicate the supplementation of 2.5% DSS in

661

drinking water. Values are mean ± standard error (n=10/group). #p < 0.05, ##p < 0.01,

662

compared with mock group. *p < 0.05, compared with sham group.

663 664

Figure 2. Effect of vanillin on the macroscopic and microscopic lesions of colon

665

cancers. (A) Macroscopic observation of colons. AOM/DSS-induced mice were orally

666

given with 75 mg/kg 5-ASA or various amounts of vanillin. On Day 91, mice were

667

sacrificed and colons were opened longitudinally. The length of colons were

668

measured and shown on the bottom. (B) Microscopic observation of colon tissues.

669

Colon sections were stained with H&E (original magnification 200×). Black arrow

670

indicates the inflammatory cells. Yellow arrow indicates the tubular adenocarcinoma.

671

Photos are representative images (n=10/group). Scale bar = 100 µm. The number and

672

the size of tumors were measured and shown on the bottom. Values are mean ±

673

standard error (n=10/group). #p < 0.05, ##p < 0.01, ###p < 0.001, compared with mock 38


Page 38 of 50

Page 39 of 50


674

group. *p < 0.05, **p < 0.01, ***p < 0.001, compared with sham group.

675 676

Figure 3. Effect of vanillin on the expressions and the activities of proteasome. (A)

677

Expression levels of proteasome genes. AOM/DSS-induced mice were orally given

678

with 100 mg/kg vanillin for 13 consecutive weeks. RNA samples were collected from

679

colon tissues and proteasome gene expression was analyzed by microarray. Data are

680

expressed as gene intensity. Values are mean ± standard error (n=3). (B) Western blot.

681

Proteins samples from colon tissues were separated by SDS-PAGE. The expression

682

levels of proteasome β5 were detected by Western blot and visualized by

683

chemiluminescence. Photos are representative images (n=3). Quantitative data were

684

normalized by internal control (GAPDH) and further expressed as relative protein

685

expession, which is presented as the comparison with the amount relative to mock.

686

Values are mean ± standard error (n=3). (C) Proteasome activity assay. HCT-116 cells

687

were treated with 10 µM MG-132 or various amounts of vanillin for 2 h at 37℃.

688

Proteasome activity was performed by incubating cell lysates with Suc-LLVY-AMC.

689

7-Amino-4-methylcoumarin released from Suc-LLVY-AMC after proteasome

690

cleavage was measured using a fluorometer. Data are expressed as proteasome

691

activity (%). Values are mean ± standard error (n=6).

692

compared with mock. 39


**

p < 0.01,

***

p < 0.001,


Page 40 of 50

693 694

Figure 4. Effect of vanillin on NF-κB and MAPK activities. AOM/DSS-induced mice

695

were orally given with 100 mg/kg vanillin or 75 mg/kg 5-ASA for 13 consecutive

696

weeks. Colon sections were analyzed by IHC staining and colon tissues were

697

extracted for Western blot analysis. (A) IHC staining. Colon sections were stained

698

with antibody against p65. Photos are representative images (n=10/group).

699

Quantification of p65-positive cell (%) is shown at the bottom. Values are mean ±

700

standard error (n=10-12). (B) Western blot. Proteins samples from colon tissues were

701

separated

702

phosphorylated JNKs (p-JNKs), p38, and phosphorylated p38 (p-p38) were detected

703

by Western blot and visualized by chemiluminescence (top panel). Photos are

704

representative images (n=3). Quantitative data were normalized by internal control

705

(GAPDH) and further expressed as fold, which is presented as the comparison with

706

the amount relative to mock (bottom panel). Values are mean ± standard error (n=3).

707

#

708

compared with sham group.

by

SDS-PAGE.

ERKs,

phosphorylated

ERKs

(p-ERKs),

JNKs,

p < 0.05, ##p < 0.01, ###p < 0.001, compared with mock group. *p < 0.05, **p < 0.01,

709 710

Figure 5. Effect of vanillin on cell proliferation, immune cell infiltration, and

711

cytokine production. AOM/DSS-induced mice were orally given with 100 mg/kg 40


Page 41 of 50


712

vanillin or 75 mg/kg 5-ASA for 13 consecutive weeks. Colon sections were analyzed

713

by IHC staining and sera were collected for cytokine ELISA. (A) IHC staining. Colon

714

sections were stained with antibody against PCNA or CD11b. Photos are

715

representative images (n=10/group). Quantification of PCNA-positive cell (%) and

716

CD11b-positive cells (%) is shown at the bottom. (B) Cytokine ELISA. The amounts

717

of IL-1β and TNF-α in sera were quantified using mouse IL-1β and TNF-α ELISA kits.

718

Values are mean ± standard error (n=10).

719

mock group. *p < 0.05, **p < 0.01, ***p < 0.001, compared with sham group.

##

p < 0.01,

###

41


p < 0.001, compared with


Table 1

Biological pathway significantly affected by vanillin in colon tissues

Pathway

Proteasome

Counta

9

%b

p-value

19.2

6.41×10

-5

Affected genes Psma1, Psma4, Psmb2, Psmb5, Psmb9, Psmb10, Psmc4, Psmd3, Psmd8

Spliceosome

14

11.3

7.57×10-5

Loc100045848, Eftud2, Hnrnpu, Sf3a3, Sf3b5, Snrpb, Snrpc, Snrpd1, Snrpd2, Snrpd3, Snrpe, Snrpg, Tra2b, U2af1

FcγR-mediated phagocytosis

10

10.2

2.50×10-3

Akt1, Arpc1b, Arpc3, Arpc5, Cfl2, Gsn, Marcks, Vasp, Wasf3, Wasl

6.45

-2

Arpc1b, Arpc3, Arpc5, Cfl2, Fgfr2, Gng12, Gsn, Hras1, Myh9, Myl12b,

Regulation of actin cytoskeleton

14

ER-associated degradation pathway

4

25

3.39×10-2

Ganab, Mogs, Rbx1, Sec61a1

N-Glycan biosynthesis

5

10.9

4.79×10-2

Dad1, Dpagt1, Ganab, Mogs, Rpn1

a

b

1.29×10

Myl9, Mylk, Tiam1, Wasl

Number of affected genes in the pathway. Percentage = (the number of altered genes / the total number of genes in the pathway ) × 100

42


Page 42 of 50

Page 43 of 50


Table 2

Verification of proteasome gene expression by qPCR

target

∆CTa

∆∆CTb

Relative to mock

Mock

21.8±0.20

17.8±0.12

4.02±0.20

0±0.2

1.00

Sham V100

21.1±0.08 21.4±0.26

17.7±0.06 17.8±0.12

3.38±0.08 3.59±0.26

-0.65±0.08 -0.43±0.26

1.56 1.35

Mock Sham

23.9±0.18 23.1±0.07

17.8±0.12 17.7±0.06

6.10±0.18 5.40±0.07

0±0.18 -0.69±0.07

1.00 1.62

V100

23.3±0.17

17.8±0.12

5.43±0.17

-0.67±0.17

1.59

Mock Sham V100

22.7±0.17 21.1±0.08 22.0±0.24

17.8±0.12

4.91±0.17 3.40±0.08 4.13±0.24

0±0.17 -1.52±0.08 -0.78±0.24

1.00 2.86 1.72

Sample

Psma4

Psmb5

Psmb10

a

Average CT of

Average CT of Gapdh

Gene

17.7±0.06 17.8±0.12

The ∆CT value is determined by subtracting the average CT value of Gapdh gene from the average CT value of target gene. The standard deviation of the difference is calculated from the standard deviations of target gene and Gapdh gene.

b

The ∆∆CT value is determined by subtracting the ∆CT value of mock group from the ∆CT value of treatment group. This is a subtraction of an arbitrary constant, so the standard deviation of ∆∆CT is the same as the standard deviation of ∆CT value.

43



Page 44 of 50

Figure 1

(A) Week

1

2 DSS

3

4

5

6

7

DSS

8

9

DSS

10

11

12

13

DSS

Sacrifice

AOM Daily oral administration of vanillin

(B)

44


Page 45 of 50


Figure 2 (A)

(B) Mock Sham 5-ASA V10

Mock

Sham

5-ASA

V100

V50 V100

< 2 mm3 2-4 mm3 >4 mm3

45



Figure 3 (A)

(B) Proteasome β5 Mock

Sham

GAPDH

V100

Mock

Sham

(C)

46


V100

Page 46 of 50

Page 47 of 50


Figure 4 (A) Mock

Sham

V100

5-ASA

5.83±1.63

55.93±2.53###

6.70±1.76***

6.09±0.86***

p65

(B) Mock

Sham

V100

5-ASA

Mock

ERKs

p-ERKs

JNKs

p-JNKs

p38

p-p38

GAPDH

ERKs

JNKs

47


Sham

V100

5-ASA


Page 48 of 50

Figure 5 (A) Mock

Sham

5-ASA

PCNA

CD11b

(B)

48


V100

Page 49 of 50


49



Table of Contents

Vanillin

Proteasome

NF-κB pathway

MAPK pathway

1 ACS Paragon Plus Environment

Page 50 of 50

Dextran

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