Subscriber access provided by Kaohsiung Medical University
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
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 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 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.
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 50
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:
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 2 of 50
Page 3 of 50
Journal of Agricultural and Food Chemistry
24
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
ACS Paragon Plus Environment
vanillin
diminished
the
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 4 of 50
Page 5 of 50
Journal of Agricultural and Food Chemistry
47
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 6 of 50
Page 7 of 50
Journal of Agricultural and Food Chemistry
85
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 8 of 50
Page 9 of 50
Journal of Agricultural and Food Chemistry
123
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
ACS Paragon Plus Environment
For
5-ASA
group,
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 10 of 50
Page 11 of 50
Journal of Agricultural and Food Chemistry
161
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 12 of 50
180
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
ACS Paragon Plus Environment
forward
reverse Accession
primer primer No.
Page 13 of 50
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 14 of 50
Page 15 of 50
Journal of Agricultural and Food Chemistry
237
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 16 of 50
Page 17 of 50
Journal of Agricultural and Food Chemistry
275
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 18 of 50
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
ACS Paragon Plus Environment
Page 19 of 50
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
in
Colon
Tissues
of
Page 21 of 50
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 22 of 50
Page 23 of 50
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 24 of 50
Page 25 of 50
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 26 of 50
Page 27 of 50
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 28 of 50
Page 29 of 50
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
necrosis
factor-α;
UPS,
Page 31 of 50
Journal of Agricultural and Food Chemistry
533
REFERENCES
534
(1) Elseweidy, M. M.; Askar, M. E.; Elswefy, S. E.; Shawky, M. Vanillin as a new
535
modulator candidate for renal injury induced by cisplatin in experimental rats.
536
Cytokine 2017, 99, 260-265.
537
(2) Cheng, H. M.; Chen, F. Y.; Li, C. C.; Lo, H. Y.; Liao, Y. F.; Ho, T. Y.; Hsiang,
538
C. Y. Oral Administration of vanillin improves imiquimod-induced psoriatic skin
539
inflammation in mice. J. Agric. Food Chem. 2017, 65, 10233-10242.
540
(3) Yan, X.; Liu, D. F.; Zhang, X. Y.; Liu, D.; Xu, S. Y.; Chen, G. X.; Huang, B.
541
X.; Ren, W. Z.; Wang, W.; Fu, S. P.; Liu, J. X. Vanillin protects dopaminergic
542
neurons against inflammation-mediated cell death by inhibiting ERK1/2, p38 and the
543
NF-κB signaling pathway. Int. J. Mol. Sci. 2017, 18, 389.
544
(4) Al Asmari, A.; Al Shahrani, H.; Al Masri, N.; Al Faraidi, A.; Elfaki, I.;
545
Arshaduddin, M. Vanillin abrogates ethanol induced gastric injury in rats via
546
modulation of gastric secretion, oxidative stress and inflammation. Toxicol. Rep. 2015,
547
3, 105-113.
548
(5) Wu, S. L.; Chen, J. C.; Li, C. C.; Lo, H. Y.; Ho, T. Y.; Hsiang, C. Y. Vanillin
549
improves and prevents trinitrobenzene sulfonic acid-induced colitis in mice. J.
550
Pharmacol. Exp. Ther. 2009, 330, 370-376.
31
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
551
(6) Arnold, M.; Sierra, M. S.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray,
552
F. Global patterns and trends in colorectal cancer incidence and mortality. Gut 2017,
553
66, 683-691.
554
(7) Howlader, N.; Noone, A. M.; Krapcho, M.; Miller, D.; Bishop, K.; Kosary, C.
555
L.; Yu, M.; Ruhl, J.; Tatalovich, Z.; Mariotto, A.; Lewis, D. R.; Chen, H. S.; Feuer, E.
556
J.; Cronin, K. A., Eds. SEER cancer statistics review, 1975-2014, National Cancer
557
Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2014/, based on November
558
2016 SEER data submission, posted to the SEER web site, April 2017.
559
(8) World Cancer Research Fund/American Institute for Cancer Research.
560
Continuous update project report: diet, nutrition, physical activity and colorectal
561
cancer. (wcrf.org/colorectal-cancer-2017).
562
(9) Brown, E. M.; Latimer, C.; Allsopp, P.; Ternan, N. G.; McMullan, G.;
563
McDougall, G. J.; Stewart, D.; Crozier, A.; Rowland, I.; Gill, C. I. In vitro and in vivo
564
models of colorectal cancer: antigenotoxic activity of berries. J. Agric. Food Chem.
565
2014, 62, 3852-3866.
566 567 568 569
(10) Macdonald, R. S.; Wagner, K. Influence of dietary phytochemicals and microbiota on colon cancer risk. J. Agric. Food Chem. 2012, 60, 6728-6735. (11) Chen, J.; Pitmon, E.; Wang, K. Microbiome, inflammation and colorectal cancer. Semin. Immunol. 2017, 32, 43-53. 32
ACS Paragon Plus Environment
Page 32 of 50
Page 33 of 50
Journal of Agricultural and Food Chemistry
570 571
(12) Glickman, M. H.; Ciechanover, A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 2002, 82, 373-428.
572
(13) Manasanch, E. E.; Korde, N.; Zingone, A.; Tageja, N.; Fernandez de Larrea,
573
C.; Bhutani, M.; Wu, P.; Roschewski, M.; Landgren, O. The proteasome: mechanisms
574
of biology and markers of activity and response to treatment in multiple myeloma.
575
Leuk. Lymphoma 2014, 55, 1707-1714.
576 577
(14) Manasanch, E. E.; Orlowski, R. Z.; Proteasome inhibitors in cancer therapy. Nat. Rev. Clin. Oncol. 2017, 14, 417-433.
578
(15) Thaker, A. I.; Shaker, A.; Rao, M. S.; Ciorba, M. A. Modeling
579
colitis-associated cancer with azoxymethane (AOM) and dextran sulfate sodium
580
(DSS). J. Vis. Exp. 2012, 67, e4100.
581
(16) Ho, T. Y.; Li, C. C.; Lo, H. Y.; Chen, F. Y.; Hsiang, C. Y. Corn silk extract
582
and its bioactive peptide ameliorated lipopolysaccharide-induced inflammation in
583
mice via nuclear factor-κB signaling pathway. J. Agric. Food Chem. 2017, 65,
584
759-768.
585
(17) Lo, H. Y.; Ho, T. Y.; Lin, C.; Li, C. C.; Hsiang, C. Y. Momordica charantia
586
and its novel polypeptide regulate glucose homeostasis in mice via binding to insulin
587
receptor. J. Agric. Food Chem. 2013, 61, 2461-2468.
33
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
588 589
(18) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆CT method. Methods 2001, 25, 402-408.
590
(19) Meng, L.; Mohan, R.; Kwok, B. H.; Elofsson, M.; Sin, N.; Crews, C. M.
591
Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo
592
antiinflammatory activity. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 10403-10408.
593
(20) Lo, H. Y.; Li, C. C.; Chen, F. Y.; Chen, J. C.; Hsiang, C. Y.; Ho, T. Y.
594
Gastro-resistant insulin receptor-binding peptide from Momordica charantia
595
improved the glucose tolerance in streptozotocin-induced diabetic mice via insulin
596
receptor signaling pathway. J. Agric. Food Chem. 2017, 65, 9266-9274.
597
(21) Magnani, M.; Crinelli, R.; Bianchi, M.; Antonelli, A. The ubiquitin-dependent
598
proteolytic system and other potential targets for the modulation of nuclear factor-kB
599
(NF-κB). Curr. Drug Targets 2000, 1, 387-399.
600
(22) Nair, S.; Doh, S. T.; Chan, J. Y.; Kong, A. N.; Cai, L. Regulatory potential for
601
concerted modulation of Nrf2- and Nfkb1-mediated gene expression in inflammation
602
and carcinogenesis. Br. J. Cancer. 2008, 99, 2070-2082.
603 604 605 606
(23) Zubair, A.; Frieri, M. Role of nuclear factor-κB in breast and colorectal cancer. Curr. Allergy Asthma Rep. 2013, 13, 44-49. (24) Dhillon, A. S.; Hagan, S.; Rath, O.; Kolch, W. MAP kinase signalling pathways in cancer. Oncogene 2007, 26, 3279-3290. 34
ACS Paragon Plus Environment
Page 34 of 50
Page 35 of 50
Journal of Agricultural and Food Chemistry
607 608
(25) Perše, M.; Cerar, A. Dextran sodium sulphate colitis mouse model: traps and tricks. J. Biomed. Biotechnol. 2012, 2012, 718617.
609
(26) Bissahoyo, A.; Pearsall, R. S,; Hanlon, K.; Amann, V.; Hicks, D.; Godfrey, V.
610
L.; Threadgill, D. W. Azoxymethane is a genetic background-dependent colorectal
611
tumor initiator and promoter in mice: effects of dose, route, and diet. Toxicol. Sci.
612
2005, 88, 340-345.
613
(27) Suzuki, R.; Kohno, H.; Sugie, S.; Nakagama, H.; Tanaka, T. Strain differences
614
in the susceptibility to azoxymethane and dextran sodium sulfate-induced colon
615
carcinogenesis in mice. Carcinogenesis 2006, 27, 162-169.
616
(28) Ho, K. L.; Chong, P. P.; Yazan, L. S.; Ismail, M. Vanillin differentially affects
617
azoxymethane-injected rat colon carcinogenesis and gene expression. J. Med. Food
618
2012, 15, 1096-1102.
619 620
(29) Bird, R. P. Role of aberrant crypt foci in understanding the pathogenesis of colon cancer. Cancer Lett. 1995, 93, 55-71.
621
(30) King, A. A.; Shaughnessy, D. T.; Mure, K.; Leszczynska, J.; Ward, W. O.;
622
Umbach, D. M.; Xu, Z.; Ducharme, D.; Taylor, J. A.; Demarini, D. M.; Klein, C. B.
623
Antimutagenicity of cinnamaldehyde and vanillin in human cells: Global gene
624
expression and possible role of DNA damage and repair. Mutat. Res. 2007, 616,
625
60-69. 35
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
626
(31) Laroui, H.; Ingersoll, S. A.; Liu, H. C.; Baker, M. T.; Ayyadurai, S.; Charania,
627
M. A.; Laroui, F.; Yan, Y.; Sitaraman, S. V.; Merlin, D. Dextran sodium sulfate (DSS)
628
induces colitis in mice by forming nano-lipocomplexes with medium-chain-length
629
fatty acids in the colon. PLoS One 2012, 7, e32084.
630 631
(32) Weerawatanakorn, M.; Wu, J. C.; Pan, M. H.; Ho, C. T.; Reactivity and stability of selected flavor compounds. J. Food Drug Anal. 2015, 23, 176-190.
632
(33) Deb, J.; Dibra, H.; Shan, S.; Rajan, S.; Manneh, J.; Kankipati, C. S.; Perry, C.
633
J.; Nicholl, I. D. Activity of aspirin analogues and vanillin in a human colorectal
634
cancer cell line. Oncol. Rep. 2011, 26, 557-565.
635
(34) Ho, K.; Yazan, L. S.; Ismail, N.; Ismail, M. Apoptosis and cell cycle arrest of
636
human colorectal cancer cell line HT-29 induced by vanillin. Cancer Epidemiol. 2009,
637
33, 155-160.
638 639
(35) Voutsadakis, I. A. The ubiquitin-proteasome system in colorectal cancer. Biochim. Biophys. Acta 2008, 1782, 800-808.
640
(36) Srinual, S.; Chanvorachote, P.; Pongrakhananon, V. Suppression of cancer
641
stem-like phenotypes in NCI-H460 lung cancer cells by vanillin through an
642
Akt-dependent pathway. Int. J. Oncol. 2017, 50, 1341-1351.
36
ACS Paragon Plus Environment
Page 36 of 50
Page 37 of 50
Journal of Agricultural and Food Chemistry
643
(37) Gupta, I.; Singh, K.; Varshney, N. K.; Khan, S. Delineating crosstalk
644
mechanisms of the ubiquitin proteasome system that regulate apoptosis. Front. Cell
645
Dev. Biol. 2018, 6, 11.
646
(38) Potts, B. C.; Albitar, M. X.; Anderson, K. C.; Baritaki, S.; Berkers, C.;
647
Bonavida, B.; Chandra, J.; Chauhan, D.; Cusack, J. C. Jr.;, Fenical, W.; Ghobrial, I.
648
M.; Groll, M.; Jensen, P. R.; Lam, K. S.; Lloyd, G. K.; McBride, W.; McConkey, D.
649
J.; Miller, C. P.; Neuteboom, S. T.; Oki, Y.; Ovaa, H.; Pajonk, F.; Richardson, P. G.;
650
Roccaro, A. M.; Sloss, C. M.; Spear, M. A.; Valashi, E.; Younes, A.; Palladino, M. A.
651
Marizomib, a proteasome inhibitor for all seasons: preclinical profile and a framework
652
for clinical trials. Curr. Cancer Drug Targets 2011, 11, 254-284.
653
(39) Ho, K.; Yazan, L. S.; Ismail, N.; Ismail, M. Toxicology study of vanillin on
654
rats via oral and intra-peritoneal administration. Food Chem. Toxicol. 2011, 49, 25-30.
37
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 38 of 50
Page 39 of 50
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
**
p < 0.01,
***
p < 0.001,
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 41 of 50
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
p < 0.001, compared with
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 42 of 50
Page 43 of 50
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 45 of 50
Journal of Agricultural and Food Chemistry
Figure 2 (A)
(B) Mock Sham 5-ASA V10
Mock
Sham
5-ASA
V100
V50 V100
< 2 mm3 2-4 mm3 >4 mm3
45
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 3 (A)
(B) Proteasome β5 Mock
Sham
GAPDH
V100
Mock
Sham
(C)
46
ACS Paragon Plus Environment
V100
Page 46 of 50
Page 47 of 50
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Sham
V100
5-ASA
Journal of Agricultural and Food Chemistry
Page 48 of 50
Figure 5 (A) Mock
Sham
5-ASA
PCNA
CD11b
(B)
48
ACS Paragon Plus Environment
V100
Page 49 of 50
Journal of Agricultural and Food Chemistry
49
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Table of Contents
Vanillin
Proteasome
NF-κB pathway
MAPK pathway
1 ACS Paragon Plus Environment
Page 50 of 50