1
Phenethyl Isothiocyanate Inhibited Tumor Migration and
2
Invasion via Suppressing Multiple Signal Transduction
3
Pathways in Human Colon Cancer HT29 Cells
4
K
UANG-C
HIL
AI,
†,‡S
HU-C
HUNH
SU,
§C
HAO-L
INK
UO,
)S
IU-W
ANI
P,
^ 5Y
UAN-M
ANH
SU,
#H
UI-Y
INGH
UANG,
^S
HIN-H
WARW
U,
rANDJ
ING-G
UNGC
HUNG*
,#,O6 †Department of Surgery, China Medical University Beigang Hospital, Yunlin, Taiwan,‡School of
7 Medicine, China Medical University, Taichung, Taiwan,§Department of Chemical Engineering,
8 Hsiuping Institute of Technology, Taichung, Taiwan, )School of Chinese Medicine Resources,
9 China Medical University, Taichung, Taiwan,^Department of Nutrition, China Medical University,
10 Taichung, Taiwan,#Department of Biological Science and Technology, China Medical University,
11 Taichung, Taiwan,rDivision of Critical Care Medicine, Department of Internal Medicine,
12 Changhua Christian Hospital, Changhua, Taiwan, andODepartment of Biotechnology,
13 Asia University, Wufeng, Taichung, Taiwan
14 Phenethyl isothiocyanate (PEITC), one of the major compounds from dietary cruciferous vegetables, 15 has been found to have antitumor properties and therefore could generate special interest for the 16 development of chemopreventive and/or chemotherapeutic agent for human cancers. In the primary
17 studies, we found that PEITC induced cytotoxic effect (decreased the percentage of viable cells) in
18 human colon cancer HT29 cells. Here, in this study, we are the first to report the antimetastatic
19 effect of PEITC in HT29 human colon cancer cells. The results show that PEITC exhibited an
20 inhibitory effect on the abilities of adhesion, migration, and invasion by Boyden chamber assay.
21 Western blotting examination indicated that PEITC exerted an inhibitory effect on the SOS-1, PKC,
22 ERK1/2 and Rho A for causing the inhibitions of MMP-2 and -9 then followed by the inhibition of
23 invasion and migration of HT29 cells in vitro. PEITC also affected Ras, FAK, PI3K or inhibited
24 GRB2, NF-κB, iNOS and COX-2 for causing the inhibition of cell proliferation in HT29 cells. Real-25 time PCR also showed that PEITC inhibited the gene expressions of MMP-2, -7, -9, FAK and Rho A
26 after PEITC treatment for 48 h in HT29 cells. PEITC also inhibited the activities of AKT, ERK, JNK
27 and PKC. Our results provide a new insight into the mechanisms and functions of PEITC which
28 inhibit migration and invasion of HT29 human colon cancer cells. These results suggest that
29 molecular targeting of NF-κB led to the inhibition of MMP-2, -7, and -9 and it might be a useful 30 strategy for the inhibition of migration and invasion on human colon cancer.
31 KEYWORDS: PEITC; migration; invasion; MMP-2; MMP-9; human colon cancer HT29 cells
32 INTRODUCTION
33
Colon cancer is the second leading cause of human cancer death
34
in the US (1). In Taiwan, about 18.5 persons per 100 thousand die
35
annually from colon cancer, based on reports from the People’s
36
Health Bureau of Taiwan in year 2008. Currently, the treatment
37
of colon cancer includes surgery, radiation, chemotherapy, or
38
combination of radiotherapy with chemotherapy. However, the
39
mortality in colon cancer patients remains high. Epidemiologic
40
studies have demonstrated that dietary intake of cruciferous
41
vegetables may decrease the risk of various types of
malignan-42
cies (2) including colon cancer (3). The anticarcinogenic effect of
43
cruciferous vegetables is attributed to organic isothiocyanates
44
(ITCs) in edible cruciferous vegetables including broccoli (2).
45
Phenethyl ITC (PEITC) is one of the ITC family of compounds
46
which exhibits cancer chemopreventive activity (4). ITCs inhibit
47
cancer formation including lung, esophagus, mammary gland,
48
liver, small intestine and bladder (5).
49
It was reported that PEITC induces apoptosis in HT-29 cells in
50
a time and dose-dependent manner via the mitochondria caspase
51
cascade, and the activation of JNK (6). PEITC was shown to
52
inhibit cytochrome P450 (CYP) enzymes and to induce phase II
53
detoxification enzymes (7). Furthermore, PEITC was shown to
54
inhibit 4-(methylnitrosamino)-1-(3-pyridyl)-1-butone-induced
pul-55
monary neoplasia in rats and mice (8, 9) and azoxymethane-induced
56
colonic aberrant crypt foci formation in rats (10). However, there
57
is no available information to address the effects of PEITC on
58
invasion and migration of cancer cells.
59
It is well-documented that invasion and migration are
funda-60
mental properties of malignant cancer cells. The formation of
meta-61
static nodules of colon cancer involves multiprocessing cascades
62
such as cell adhesion, migration, and proteolysis of the extracellular
*Corresponding author. Tel:þ886 4 2205 3366, int 2161. Fax: þ886 4 2205 3764. E-mail: [email protected].
JFood | 3b2 | ver.9 | 15/9/010 | 21:47 | Msc: jf-2010-02384n | TEID: tms00 | BATID: 00000 | Pages: 7.14
J. Agric. Food Chem. XXXX, XXX, 000–000
A
DOI:10.1021/jf102384n
pubs.acs.org/JAFC © XXXX American Chemical Society
63
matrix (ECM). The matrix metalloproteinases (MMPs) (a family
64
of zinc-dependent endopeptidases) are deeply involved in the
65
invasion and metastasis of various tumor cells (11-13). About
66
24 kinds of MMPs have been identified. However, MMP-2
67
(gelatinase-A) and MMP-9 (gelatinase-B) are most associated
68
with tumor migration, invasion and metastasis for various human
69
cancers (14
-16). Therefore, agents from natural products which
70
can suppress the expressions of MMP-2 or -9 may be considered
71
worthy of development for anticolon cancer invasion and
72
metastasis.
73
Although many studies have shown that PEITC can be used
74
as an inducer of apoptosis (anticancer activities), there are no
75
reports to show that PEITC inhibited the migration and invasion
76
of colon cancer cells. Therefore, in the present study, we focused
77
on the effect of PEITC on the migration and invasion of HT29
78
human colon cancer cells in vitro.
79 MATERIALS AND METHODS
80 Chemicals and Reagents. Phenethyl isothiocyanates (PEITC),
81 dimethyl sulfoxide (DMSO), propidium iodide, potassium phosphates,
82 Triton X-100 and trypan blue were obtained from Sigma Chemical Co.
83 (St. Louis, MO). RPMI-1640 medium,L-glutamine, fetal bovine serum,
84 penicillin-streptomycin, and trypsin-EDTA were obtained from
Invitro-85 gen (Carlsbad, CA). Primary antibodies used for Western analysis were
86 obtained as follows: antibodies for PI3K, PKC, Ras, GRB2, SOS1,
87 P-ERK, ERK1/2, MMP-2, MMP-9, Rho A, FAK, iNOS, COX-2 and
88 NF-κB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA)
89 and diluted in cell culture medium before use.
90 HT29 Cell Line. The HT29 human colon cancer cell line was
91 purchased from the Food Industry Research and Development Institute
92 (Hsinchu, Taiwan). Cells were cultured in 88% RPMI-1640 medium with
93 1.5 mML-glutamine supplemented with 10% fetal bovine serum (Gibco
94 BRL, Grand Island, NY), and 2% penicillin-streptomycin (100 units/mL
95 penicillin and 100μg/mL streptomycin) and were cultured in a humidified
96 atmosphere of 5% CO2and 95% air at 37C.
97 Effects of PEITC on the Percentage of Viable HT29 Cells. The
98 HT29 cells (2 105cells/well) were placed in 12-well plates and incubated
99 at 37C for 24 h before each well was cotreated with 0, 0.01, 0.25, 0.5, 1.0,
100 2.5, 5.0, 7.5, and 10μM PEITC for 24 h. 0.5% DMSO (solvent) was used
101 through the whole study. Cells were harvested by centrifugation before
102 being used for determining cell viability, and the flow cytometric protocol
103 was used, as previously described (17, 18).
104 Wound Healing Assay. HT-29 cells were grown on 6-well dish plates
105 to 100% confluent monolayer and then scratched to form a 100μm “wound”
106 using sterile pipet tips. The cells were then cultured in the presence or
107 absence of PEITC (0.01, 0.25μM) in serum-free media for 24 h. The
108 images were recorded at 24 and 48 h after scratch using an Olympus
109 photomicroscope (19).
110 In Vitro Migration Assay. The migration of HT29 cells was also 111 measured by chemotactic directional migration which was determined
112 using a 24-well Transwell insert. Briefly, 8μm pore filters (Millipore, MA)
113 were coated with 30μg of type Ι collagen (Millipore, MA) for 1 h. The
114 HT29 cells (104cells/0.4 mL of RPMI-1640) were plated in the upper
115 chamber with or without PEITC (0.01 or 0.25μM) and allowed to undergo
116 migration for 24/48 h. In the upper chamber, the nonmigrated cells were
117 removed with a cotton swab and the filters were stained with 2% crystal
118 violet. Migrated cells adherent to the underside of the filter were counted
119 and photographed under a light microscope at200 (20, 21). Each
120 treatment was assayed twice, and three independent experiments were
121 performed.
122 In Vitro Invasion Assay. The invasion of HT29 cells was measured 123 using Matrigel-coated Transwell cell culture chambers (8μm pore size)
124 as described previously (21, 22). Cells were maintained in serum-free
125 RPMI-1640 medium for 24 h before being trypsinized and resuspended in
126 serum-free medium and placed in the upper chamber of the Transwell
127 insert (5 104cells/well) and treated with 0.5% DMSO or PEITC (0, 0.01,
128 or 0.25μM). RPMI-1640 medium containing 10% FBS was placed in
129 the lower chamber. Cells were incubated for 24 or 48 h in a humidified
130 atmosphere with 95% air and 5% CO2at 37C. Invasive cells were fixed
131
with 4% formaldehyde in PBS and stained with 2% crystal violet in 2%
132
ethanol. The noninvasive cells in the upper chamber were removed by
133
wiping with a cotton swab. The cells in the lower surface of the filter which
134
penetrated through the Matrigel were counted under a light microscope
135
at200.
136
Western Blotting Analysis. HT29 cells were cultured in 6-well tissue
137
culture plates and grown for 24 h. PEITC was added to cells at a final
138
concentration of 2.5μM, while DMSO (solvent) alone was added to
139
control cells. Cells were incubated with PEITC in 90% RPMI-1640
140
medium with 1% FBS at 37C for 0, 6, 12, 24, and 48 h. The cells were
141
then harvested and resuspended in ice-cold 50 mM potassium phosphate
142
buffer (pH 7.4) containing 2 mM EDTA and 0.1% Triton X-100. The cells
143
were sonicated and centrifugated at 13000g for 10 min at 4C to remove
144
cell debris. The supernatant was collected and total protein
concentra-145
tion of each sample was determined using a Bio-Rad protein assay kit
146
(Hercules, CA) with bovine serum albumin (BSA) as the standard . SDS
147
gel electrophoresis and Western blotting were conducted as described
148
previously (23,24). Western blotting was performed to determine effects of
149
PEITC on protein levels of PI3K, PKC, Ras, GRB2; SOS1, p-ERK,
150
ERK1/2, MMP-2, MMP-9, Rho A, FAK, iNOS, COX-2 and NF-κB p65.
151
Real-Time PCR of MMP-2, -7, and -9, FAK and RhoA. HT29
152
cells were cultured in 6-well plates and grown for 24 h. PEITC was added
153
to cells in each well for a final concentration of 2.5μM for 24 h. Cells were
154
then harvested and total RNA was extracted using the Qiagen RNeasy
155
Mini Kit as described previously (21, 25). RNA samples were
reverse-156
transcribed at 42C with High Capacity cDNA Reverse Transcription Kit
157
for 30 min according to the protocol of the supplier (Applied Biosystems).
158
Quantitative PCR conditions were as follows: 2 min at 50C, 10 min at
159
95C, and 40 cycles of 15 s at 95 C; 1 min at 60 C using 1 μL of the cDNA
160
reverse-transcribed as described above, 2X SYBR Green PCR Master Mix
161
(Applied Biosystems) and 200 nM forward and reverse primers as shown
162
in Table 1. Applied Biosystems 7300 Real-Time PCR system was used for T1 163
each assay in triplicate, and expression fold-changes were derived using the
164
comparative CTmethod.
165
AKT, ERK, JNK and PKC Activity Assay. The inhibitory activity
166
of PEITC (1000, 500, 250, 125, 62.50, 31.25, 15.63, 7.81, and 3.71μM in
167
DMSO) was measured in kinase assays. To measure AKT, ERK, JNK
168
and PKC activity specifically, specific substrate peptide (AKT, ERK, JNK
169
and PKC substrates (Crosstide, MBP, ATF2 and Histone H1þ Lipid
170
Activator, respectively)) and 10μCi/μL of33P-ATP were mixed in a
Tris-171
HCl buffer (pH 7.5), 1.5 mM CaCl2; 16μg/mL calmodulin; 2 mM MnCl2
172
in Base Reaction Buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM
173
EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT,
174
1% DMSO). Human AKT, ERK, JNK and PKC protein samples (20μL)
175
were added to the reaction mix and incubated for 120 min at room
176
temperature. Reactions were spotted onto P81 ion exchange paper. Filters
177
were washed extensively in 0.1% phosphoric acid followed by
count-178
ing (26, 27).
179
Statistical Analysis. Data are presented as the mean( SEM for the
180
indicated number of separate experiments. Statistical analyses of data were
181
done by one-way ANOVA, and *P < 0.05 were considered significant. Table 1. The DNA Sequence Was Evaluated Using the Primer Expressiona
primer name primer sequence
homo MMP-2-F CCCCAGACAGGTGATCTTGAC homo MMP-2-R GCTTGCGAGGGAAGAAGTTG homo MMP-7-F GGATGGTAGCAGTCTAGGGATTAACT homo MMP-7-R AGGTTGGATACATCACTGCATTAGG
homo FAK-F TGAATGGAACCTCGCAGTCA
homo FAK-R TCCGCATGCCTTGCTTTT
homo RhoA-F TCAAGCCGGAGGTCAACAAC homo RhoA-R ACGAGCTGCCCATAGCAGAA
homo ROCK1-F ATGAGTTTATTCCTACACTCTACCACTTTC homo ROCK1-R TAACATGGCATCTTCGACACTCTAG homo GAPDH-F ACACCCACTCCTCCACCTTT homo GAPDH-R TAGCCAAATTCGTTGTCATACC
a
RNA samples were reverse-transcribed for 30 min at 42C with High Capacity cDNA Reverse Transcription Kit according to the standard protocol of the supplier (Applied Biosystems). Each assay was run on an Applied Biosystems 7300 Real-Time PCR system at least twice to ensure reproducibility.
182
RESULTS
183
Effects of PEITC on the Percentage of Viable WEHI-3 Cells.
184
To verify the effect of PEITC on cell viability, HT29 cells were
185
exposed to different concentrations of PEITC for 24 h, and cells
186
were examined under microscope. They then were collected for
187
propidium iodine staining for viability analysis. The results are
188
present in Figure 1
and indicated that a significant loss of viability
F1189
was detected at 2.5, 1, and 5
μM PEITC in a dose-dependent
190
manner (Figure 1). Cell viability by PEITC was further confirmed
191
by trypan blue dye exclusion method. Based on the decreased
192
percentage of viable HT29 cells following PEITC treatment, we
193
investigated the functional effects of PEITC on cell migration and
194
invasion.
195
Effects of PEITC on Migration and Invasion of HT29 Cells.
196
HT29 cells have an ability to migrate a 24-well Transwell insert.
197
Treatment of PEITC for 24 and 48 h exhibited significant
inhibi-198
tion of cell migration in a dose-dependent manner. PEITC also
199
inhibited cancer cell migration in the wound healing test at
200
concentrations of 0.01 and 0.25
μM (Figure 2
A). Results from
F2201
migration assay are shown in Figures 2B and 2C, which indicate
202
that PEITC had a significant inhibitory effect on cell migration at
203
concentrations between 0.01 and 0.25
μM. Data in Figure 2C
Figure 1. Effect of PEITC on cell viability in human colon cancer HT29 cells. HT29 cells were incubated with various concentrations (0, 0.01, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, and 10.0μM) for 24 h. Cells were directly photographed (200) and then were harvested and stained with PI; then the percentage of viable HT29 cells was determined as described in Materials and Methods. A: Percentage of viable cells. B: The migration of cells. Data represents mean( SD of three experiments. *p < 0.05 compared with the untreated control (dose 0).
Figure 2. Effect of PEITC on migration and invasion of HT29 cells. HT29 cells were treated with various concentrations (0, 0.01, and 0.25μM) of PEITC for 24 and 48 h. (A) Cell motility was determined by wound healing assay after PEITC treatment for 24 and 48 h. (B) Cell migration was measured in a Boyden chamber for 12 and 24 h with polycarbonate filters (pore size, 8μm). (D) Cell invasion was measured in a Boyden chamber for 12 and 24 h; polycarbonate filters (pore size, 8μm) were precoated with Matrigel. Migration (C) and invasion (E) ability of HT29 cells were quantified by counting the number of cells that invaded the underside of the porous polycarbonate membrane under microscopy and represent the average of three experiments. *p < 0.05 compared with the untreated control (dose 0). *p < 0.05 compared with the untreated control (dose 0). Scale bar, 40μm.
204
indicate that the inhibition was at 57-64% and 61-69% when
205
cells were incubated with PEITC for 24 and 48 h treatment,
respec-206
tively. HT29 cells have an ability to invade through
Matrigel-207
coated Transwell cell culture chambers. Treatment of PEITC for
208
24 and 48 h exhibited the significant inhibition of cell invasion in a
209
dose-dependent manner. Results from invasion assay are shown
210
in Figures 2D and 2E. Figure 2D shows that HT29 cells moved
211
from the upper chamber to the lower chamber in the absence
212
of PEITC (control group). However, the penetration of the
EHS-213
coated filter by HT29 cells was inhibited in the presence of
214
PEITC. The percentage inhibition at 0.01 was 18
-58%, and at
215
0.25
μM inhibition, it ranged from 29 to 54% (Figure 2E).
216
Effects of PEITC on Levels of Proteins Associated with
Migra-217
tion and Invasion in HT29 Cells. Results from Western blotting
218
assay are shown in Figure 3
F3
A
-D, which indicates that PEITC
219
reduced levels of PERK, FAK, ERK1/2 and JNK (Figure 3A),
220
GRB2, Rho A, RCK1, SOS1, pI3K and PKC (Figure 3B), iNOS,
221
NF-
κB p65 and COX-2 (Figure 3C), and MMP-2 and MMP-7
222
(Figure 3D), but increased protein levels of MEEK3 (Figure 3A)
223
in examined HT29 cells. These effects may lead to the inhibition
224
of migration and invasion of HT29 cells.
225
Effects of PEITC on MMP-2, MMP-7, MMP-9, FAK and Rho
226
A mRNA Expressions in HT29 Cells. To further investigate whether
227
or not PEITC affected migration- and invasion-associated gene
228
expression in HT29 cells, cells were treated with PEITC (2.5
μM)
229
for 0, 24, and 48 h. Total RNA was isolated from control and
230
PEITC treatment, and gene expressions were examined by
real-231
time PCR. The results are shown in Figure 4
, and they indicate
F4232
that the expression levels of MMP-2, MMP-7 and MMP-9 were
233
inhibited during PEITC treatment for 48 h but only MMP-7 was
234
inhibited in 24 h treatment (Figure 4A). However, FAK and Rho
235
A mRNA were decreased at 48 h treatment, but it did not show in
236
24 h treatment of PEITC (Figure 4B).
237
Effects of PEITC on AKT, ERK, JNK and PKC Activities.
238
Results from Western blotting indicate that PEITC decreases the
239
protein levels of AKT, ERP, JNK and PKC, and we further
240
investigated whether PEITC also affected the activities of AKT,
241
ERK, JNK and PKC; the results are shown in Figure 5
A-D.
F5Figure 3. Effects of PEITC on the protein levels of associated proteins for migration and invasion in HT29 cells. HT29 cells were treated with 2.5μM PEITC for 6, 12, 24 and 48 h. The total proteins were collected from each sample, and the protein levels (A, PERK, MKK3, FAK, ERK1/2 and p38; B, Ras, GRB2, Rho A, ROCK1 and SOS1; C, iNOS, NF-κB p65, COX-2 and uPA; D, MMP-2, MMP-7 and MMP-9) were measured by SDS-PAGE and Western blotting as described in Materials and Methods.
242
Figure 5 indicated that PEITC inhibited the activities of AKT,
243
ERK, JNK and PKC in a dose-dependent manner. However, the
244
initial inhibition concentrations were different such as AKT from
245
15.63 to 100
μM, ERK from 62.5 to 100 μM, JNK from 7.81 to
246
100
μM, and PKCa from 3.71 to 100 μM of PEITC.
247 DISCUSSION
248
Tumor invasion requires degradation of basement membranes,
249
proteolysis of ECM, pseudopodial extension, and cell
migra-250
tion (28). A number of proteolytic enzymes, including MMPs and
251
serine proteinases, are involved in these tumor host interactions,
252
such as degradation of underlying basement membrane. Of these
253
basement membrane degrading enzymes, MMPs, especially
acti-254
vated forms of MMP-2 and MMP-9, are thought to play an
255
important role in its degradation because of their ability to cleave
256
the type IV collagen. MMPs are produced by cancer cells or
257
through the induction of surrounding stromal cells. Several studies
258
indicate that inhibition of MMP expressions or enzyme activities
259
can be used as early targets for preventing cancer metastasis
260
(29
-31). It is well-known that cell migration is a multicomplex
261
process which provides many molecular targets for the
develop-262
ment of therapeutic agents to inhibit cancer metastasis
263
Although PEITC was reported to possess anticancer potential
264
against several cancer cell lines (6-9), the role of PEITC against
265
the migration and invasion of HT29 cells and associated protein
266
levels and gene expressions is still unclear. Our results showed that
Figure 4. Effects of PEITC on MMP-2, MMP-7, MMP-9, FAK and Rho A mRNA expression in HT29 cells. The total RNA was extracted from each treatment of PEITC (2.5μM) on HT29 cells for 0, 24, and 48 h, and RNA samples were reverse-transcribed with cDNA then for real-time PCR as described in Materials and Methods. The ratios of MMP-2, MMP-7 and MMP-9 (A), FAK and Rho A (B) mRNA/GAPDH are presented in panels A and B. Data represents mean ( SD of three experiments. *P < 0.05, ***P < 0.001 compared with the untreated control (dose 0).
Figure 5. Effects of PEITC on AKT, ERK, JNK and PKC activities. AKT, ERK, JNK and PKC substrates (Crosstide, MBP, ATF2 and Histone H1þ Lipid Activator, respectively) in Base Reaction Buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA,
0.1 mM Na3VO4, 2 mM DTT, 1% DMSO) with cofactors (1.5 mM CaCl2;
16 ug/mL calmodulin; 2 mM MnCl2). Then kinase was added to the
sub-strate solution and gently mixed and PEITC (1000, 500, 250, 125, 62.50, 31.25, 15.63, 7.81, and 3.71μM in DMSO) was added to the kinase reaction mixture. Then the33P-ATP (specific activity 0.01μCi/μL final) was
added into the reaction mixture before being spotted onto P81 ion exchange paper. Washing of filters extensively in 0.1% phosphoric acid and counting were as described in Materials and Methods. Data represents mean( SD of three experiments. *P < 0.05 were considered significant.
267
PEITC induced cytotoxicity and inhibited the migration and
268
invasion in HT29 cells and that these effects are dose dependent
269
(Figure 1). Furthermore, we found that PEITC decreased the
270
migration and invasion associated protein levels such as PERK,
271
FAK, ERK1/2, JNK, p38 (Figure 3A, Ras, GrB2, Rho A,
272
ROCK1, SOS1, PI3K and PKC (Figure 3B, iNOS, NF-κB p65,
273
COX-2 and uPA (Figure 3C) MMP-2, MMP-7 and MMP-9
274
(Figure 3D) cells. PEITC also inhibited the activities of AKT,
275
ERK, JNK and PKC. Our results also showed that PEITC
276
suppressed MMP-9 gene expression via suppressing the PKCs/
277
MAPK and PI3K/AKT/NF-
κB cascades with consequent
sup-278
pression of colony formation, tumor migration and invasion by
279
human colon cancer HT29 cells. The activities of MMP-2 and
280
u-PA have been shown to play a critical role in degrading the
281
basement membrane in cancer invasion and migration. We also
282
found that PEITC tremendously reduced MMP-2 activity in a
283
dose-dependent manner, whereas uPA activity was also inhibited
284
by PEITC (data not shown).
285
It was reported that MMP-2 overexpressed in highly metastatic
286
tumors, and that MMP-9 can be stimulated by TNF-
R (32) or a
287
growth factor such as VEGF, EGF and TGF-
β (33-35), or Ras
288
oncogene (36, 37) through activation of different intracellular
289
signaling pathways. It was also reported that the activation of
290
PKC led to the translocation of the protein to membranes and led
291
to control the expression of MMP-9 through modulating the
292
activation of transcription factors such as AP-1, NF-
κB or Sp-1
293
through MAPK and PI3K signaling pathways (38
-40).
294
It was reported that the activation of NF-
κB is involved in the
295
induction of the MMP-9 gene associated with the invasion and
296
metastasis of tumor cells by different agents including TPA,
297
growth factors such as EGF, VEGF, platelet-derived growth
298
factor, transforming growth factor-b, and inflammatory
cyto-299
kines (32, 41). Therefore, in the present study, the regulation of
300
NF-
κB, and the downstream of the PI3K/Akt and MAPK
301
(ERK1/2, p38 and JNK) pathways, might be involved in PEITC
302
suppressed MMP-9 expression and invasion in HT29 cells. We
303
also found that HT29 cells were treated with PEITC which led to
304
decrease the protein levels of PI3K, Akt, MMP-2 and MMP-9.
305
It was reported that PI3K activation leads to activate the
306
downstream main target Akt which plays various important roles
307
in regulating cellular growth, differentiation, adhesion, the
inflam-308
matory reaction, and invasion (33, 42). We also found that
309
PEITC decreased the JNK and PKC levels (Figure 5). It was
310
reported that resveratrol suppresses MMP-9 expression in
TPA-311
induced human Ca Ski cells by blocking JNK and PKC
δ signal
312
transduction (43). So far, there is no report to show the receptor in
313
cells for PEITC. However, there may be other possible
mecha-314
nisms in which PEITC penetrates cells, probably to compete with
315
coenzymes or ATP to inhibit the activity of PKC.
316
Other factors also play an important role in migration and
317
invasion such as 52-kDa uPA which plays a major role in the
318
decomposition of basement membranes. This enzyme is highly
319
expressed in solid tumors. It was reported that the activation of
320
the uPA/uPAR/plasmin proteolytic network has been shown to
321
play key roles in tumor invasion and dissemination of various
322
malignancies (44, 45). The presence of uPA in tumor tissues has
323
been proposed as a potential prognostic factor, and the levels of
324
uPA and uPAR expression serve as prognostic markers in various
325
malignancies. However, high levels of expression are often
326
associated with a poor prognosis (46). We then examined whether
327
PEITC blocks the expressions of MMP-2, -7, and -9 and uPA
328
which are closely associated with tumor invasion, and the results
329
confirmed this hypothesis.
330
The present study provides proof that, through a molecular
331
mechanism, PEITC promotes a strong anti-invasive and
332
antimigration effect through downregulation of PKC and then
333
blocking MAPK and PI3K/Akt signaling pathways, NF-κB, and
334
uPA which then led to the inhibition of MMP-2 and MMP-9
335
(Figure 6
). Therefore, we conclude that PEITC may have a
F6336
potential for inhibiting the migration and invasion of human colon
337
cancer in future.
338
ABBREVIATIONS USED
339
ERK, extracellular signal-regulated kinases; FAK, focal
adhe-340
sion kinase; JNK, c-Jun NH2-terminal kinase; MMPs, matrix
341
metalloproteinase; NF-κB, nuclear factor kappaB; PEITC,
phen-342
ethyl isothiocyanate; PKC, protein kinase C; RhoA, ras
homo-343
logue gene family, member A; GRB2, growth factor
receptor-344
bound protein 2; Cox-2, cyclooxygenase-2; INOS, inducible nitric
345
oxide synthase; PI3K, phosphoinositide 3-kinases; SOS1, son of
346
sevenless homologue 1; AP-1, activator protein 1; MAPK,
mitogen-347
activated protein kinase.
348
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538 Received for review June 21, 2010. Revised manuscript received
539 August 31, 2010. Accepted September 5, 2010. This work was supported by
540 Grant CMU98-ASIA-10 from China Medical University, Taichung, Taiwan.
