Caffeic acid derivatives inhibit the growth of colon cancer:
Involvement of the PI3-K/Akt and AMPK signaling pathways
En-Pei Isabel Chiang 1, 2,†, Shu-Yao Tsai 3, †,Yueh-Hsiung Kuo 4,5, Man-Hui Pai 6, Hsi-Lin Chiu 4,5, Raymond L. Rodriguez 7,and Feng-Yao Tang 8,§
1 Department of Food Science and Biotechnology and 2 NCHU-UCD Plant and
Food Biotechnology Program and Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan, Republic of China;
3 Department of Health and Nutrition Biotechnology, Asia University, Taichung
41354, Taiwan, Republic of China;
4 Department of Chinese Pharmaceutical Sciences and Chinese Medicine
Resources, China Medical University, Taichung 40402, Taiwan, Republic of China;
5 Department of Biotechnology, Asia University, Taichung 413, Taiwan,
Republic of China;
6 Department of Anatomy, Taipei Medical University, Taipei 11031, Taiwan,
Republic of China;
7 Department of Molecular and Cellular Biology, University of California, Davis,
CA 95616, USA
8 Biomedical Science Laboratory, Department of Nutrition, China Medical
University, Taichung 40402, Taiwan, Republic of China
Author information Corresponding author 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
§ Tel: (886-4) 22060643, Fax: (886-4) 22062891, E-mail:
vincenttang@mail.cmu.edu.tw
Present address
§ Biomedical Science Laboratory, Department of Nutrition, China Medical
University, 91 Hsueh-Shih Road, Taichung 40402,Taiwan, Republic of China
Notes
†: These authors equally contributed to this work.
Abstract
Background: The aberrant regulation of phosphatidylinositide 3-kinases (PI3-K)/Akt , AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (m-TOR) signaling pathways in cancer has prompted significant 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
interest in the suppression of these pathways to treat cancer. Caffeic acid (CA) has been reported to possess important anti-inflammatory actions. However, the molecular mechanisms by which CA derivatives including caffeic acid phenethyl ester (CAPE) and caffeic acid phenylpropyl ester (CAPPE), exert inhibitory effects on the proliferation of human colorectal cancer (CRC) cells have yet to be elucidated.
Methodology/Principal Findings: CAPE and CAPPE were evaluated for their ability to modulate these signaling pathways and suppress the proliferation of CRC cells both in vitro and in vivo. Anti-cancer effects of these CA derivatives were measured by using proliferation assays, cell cycle analysis, western blotting assay, reporter gene assay and immunohistochemical (IHC) staining assays both in vitro and in vivo. This study demonstrates that CAPE and CAPPE exhibit a dose-dependent inhibition of proliferation and survival of CRC cells through the induction of G0/G1 cell cycle arrest and augmentation of apoptotic pathways. Consumption
of CAPE and CAPPE significantly inhibited the growth of colorectal tumors in a mouse xenograft model. The mechanisms of action included a modulation of PI3-K/Akt , AMPK and m-TOR signaling cascades both in vitro and in vivo. In conclusion, the results demonstrate novel anti-cancer mechanisms of CA derivatives against the growth of human CRC cells.
Conclusions: CA derivatives are potent anti-cancer agents that augment AMPK activation and promote apoptosis in human CRC cells. The structure of CA derivatives can be used for the rational design of novel inhibitors that target human CRC cells.
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
Keywords: Caffeic acid phenylpropyl ester; cell cycle arrest; Akt; AMP-activated protein kinase; human colorectal cancer cells
Introduction
Colorectal cancer (CRC) is one of the leading causes of cancer and cancer
mortality in many countries [1,2]. In the United States alone, approximately
50,000 deaths are attributed to this cancer annually [1,2]. Many studies have
indicated that mutations of the phosphatidylinositide 3-kinase (PI3-K)/Akt and 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91
mitogen-activated protein kinase (MAPK)/ extracellular-signal-regulated
kinase (ERK) molecules are commonly observed in various types of cancer
[3,4]. For example, oncogenic activation of PI3-K/Akt molecules enhances cell
proliferation by increasing the cyclin D1 level [5,6]. It is well known that the
aberrant expression of the cyclin D1 and Cdk4 proteins is involved in the
proliferation of CRC cells [7]. Suppression of the PI3-K/Akt and MAPK/ERK
signaling pathways leads to the blockade of cell proliferation and
demonstrates the importance of these signaling cascades in the control of
both cell cycle progression and cell growth during cancer development [4,8].
Therefore, the PI3-K/Akt and MAPK/ERK signaling pathways play
predominant roles in determining the fate of tumor growth. Malignant cancer
cells detach from the primary tumor and migrate across structural barriers,
including basement membranes and the surrounding stromal extracellular
matrix (ECM) [9]. Tumor invasion and metastasis both require an increase in
the expression of matrix metalloproteinases (MMPs) and the degradation of
ECM [9,10]. MMPs are zinc-dependent endopeptidases capable of degrading
ECM components [11]. Enzymes such as MMP-9 degrade ECM and create a
microenvironment that maintains tumor development [10,11].
AMP-activated protein kinase (AMPK) is a fuel-sensing molecule that 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
functions as a regulator of energy balance [12]. AMPK has been shown to be
ubiquitously expressed in mammalian cells and to be involved in energy
homeostasis [13]. An increased adenosine monophosphate (AMP)/adenosine
triphosphate (ATP) ratio, reflecting a decrease in the cell's energy state, leads
to the activation of the AMPK protein by phosphorylation [14]. The
augmentation of AMPK activation is thought to be inversely correlated with
cancer risk [15]. Recent studies have further suggested that the activation of
the PI3-K/Akt and MAPK/ERK signaling molecules is associated with a
decreased level of phosphorylated (activated) AMPK in the course of tumor
progression [16,17]. Additional studies concluded that AMPK agonists are
effective in the treatment of cancer [15,18], while other studies showed that
the lipogenic enzyme fatty acid synthase (FASN) is regulated by energy intake
and plays a crucial role in carcinogenesis [19]. One recent study reported that
FASN expression is correlated with the growth and progression of CRC [20].
The phosphorylation (i.e. activation) of Akt was shown to induce the
expression of FASN and to trigger aggressive malignancy in cancer cells [21].
In contrast, treatment with an AMPK agonist, leading to the activation of
AMPK, suppressed the expression of FASN and blocked the growth of
colorectal tumor [22-24]. Moreover, epidemiological studies further indicated 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129
that AMPK (PRKAG2) single-nucleotide polymorphism (SNP) is associated
with risk of human CRC [25]. Thus, AMPK-mediated energy homeostasis has
attracted interest in this pathway as a means of treating human colon cancer.
Many studies have demonstrated that phenolic acid compounds function as
potent antioxidants [26]. Among them, caffeic acid (CA) is a non-vitamin
phenolic compound found largely in vegetables and fruit. In addition to its
antioxidant activity, CA exerts anti-inflammatory effects in several kinds of
cells [27,28]. Recent studies indicated that caffeic acid phenethyl ester
(CAPE), a CA derivative naturally isolated from honeybee propolis, also exerts
its beneficial effects through antioxidant and anti-inflammatory activities
[29,30]. Furthermore, it has been demonstrated that CAPE inhibits the
proliferation of cancer cells and act as a potential anti-cancer agent [31,32].
However, there is no report of the inhibitory effects of CA derivatives on the
AMPK pathway and /or FASN expression during the progression of CRC.
Moreover, the lack of consistent results across numerous studies and the
failure to determine the mechanism of action of the CA derivatives may
explain the difficulty in demonstrating the in vivo benefits of CA derivative
supplementation against CRC. We investigated, therefore, the inhibitory 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148
effects of various CA derivatives on human CRC cells both in vitro and in vivo.
The results demonstrated that CA derivatives such as CAPE and caffeic acid
phenylpropyl ester (CAPPE) significantly inhibited cellular proliferation in
human CRC cells. CAPE and CAPPE induced cell cycle arrest through the
suppression of the PI3-K/Akt and mTOR signaling pathways. Furthermore, CA
derivatives reduced cellular ATP levels and suppressed FASN expression.
The mechanism of action was associated in part with an augmentation of the
AMPK pathway. The results of this study suggest that CA derivatives act as
chemopreventive agents against human CRC by modulating the PI3-K/Akt,
mTOR and AMPK signaling pathways both in vitro and in vivo.
Materials and Methods
Reagents and antibodies
Human colon cancer cells HCT-116 and SW-480 were purchased from
American Type Culture Collection (Walkersville, MD). The following
monoclonal antibodies were purchased from Cell Signaling Technology, Inc.:
Anti- N-cadherin (#4061), PTEN (#9559), anti-phosphorylation PDK1 (Ser241;
#3061), total-PDK1(#3062), anti-phosphorylation Akt (S473; #4060), total-Akt
(#9272), phosphorylation GSK3α (S21; #9327), total- GSK3α (4337), anti-149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167
phosphorylation GSK3β(S9; #9323), total- GSK3β(#9315),
anti-phosphorylation FOXO3 (T32; #9464), total- FOXO3 (#12829), total- TSC1
(#6935), total- TSC2 (#3990), total- LKB1 (#3047), total- 14-3-3
(#8312),phosphorylation ERK 1/2 (T202/Y204; #9101), total-ERK 1/2 (#9102),
phosphorylation AMPKα (T172; #2535), total-AMPKα(#5832),
phosphorylation m-TOR (S2448; #5536), total-m-TOR (2983),
FASN(#3180), NF-B (p65) (#3033), Cdk4(#2906),
anti-p21waf/cip1(#2947), anti-cyclin E(#4132), anti-cyclin D1(#2978), anti-c-myc
(#9402) and anti-Lamin A (#2032) (Danvers, MA). The anti- β-actin (# A2066)
antibody and compound C (specific inhibitor of AMPK) were purchased from
Sigma (St Louis, MO). The active Akt (Myr-Akt1, Addgene plasmid # 9008)
and control empty vector (pcDNA3, Addgene plasmid # 10792) were obtained
from Addgene. The tumor necrosis factor-α (TNF-α) recombinant protein was
from R&D System (Minneapolis, MN). The nuclear Protein Extract Reagent Kit
was purchased from Pierce Biotechnology Inc. (Lackford, IL). The
luminescence ATP detection assay kit (ATPlite kit) was purchased from
Perkin Elmer Life Science (Boston, MA). The NF-κB response element
(NF-κB-RE) plasmid and Dual-Luciferase Reporter Assay kit were purchased from
Promega (Madison, WI). PI (propidium Iodine) and anti- proliferating cell 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
nuclear antigen (PCNA) (#610664) monoclonal antibodies were purchased
from BD Biosciences Inc. (Franklin Lakes, NJ). CA derivatives, including
CAPE and CAPPE (Figure 1) were provided by Dr. Y. H. Kuo (China Medical
University). These CA derivatives were dissolved in dimethyl sulfoxide
(DMSO) at a concentration of 200 mM stock solution and stored at -20oC.
Immediately before the experiment, the stock solution was added to the cell
culture medium, as described previously.
Cell culture
Briefly, human CRC cells were cultured in a 37oC humidified incubator with
5% CO2 and grown to confluency using fetal bovine serum (FBS)
supplemented RPMI-1640 media. The cells used in the different experiments
have the same passage number. RPMI-1640 medium was supplemented with
10% heat-inactivated FBS, 2 mM L-glutamine and 1.5 g/L sodium
bicarbonate.
Supplementation with CA derivatives
Human CRC cells were incubated with different concentrations (0, 5, 10, 20,
50, and 100 μM) of the CA derivatives for 2h or 24 h. For efficient uptake of
the CA derivatives by human colon cancer cells, these compounds were 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206
incorporated into FBS for 30 min and mixed with the medium. In control
groups, cells were incubated with an equivalent volume of solvent DMSO
(final concentration: 0.05% v/v) as a carrier vehicle.
Assessment of cell proliferation
The MTT (3-[4,5-dimethhylthiaoly]- 2,5-diphenyltetrazolium bromide) assay
was conducted to detect the cell proliferation. Human CRC cells were seeded
in 24-well plates, each well containing 1x 105 cells. After 24 h, the culture
medium was replaced by media containing CA derivatives at one of five
concentrations (i.e.,0, 5, 10, 20, 50 and 100 µM) in the presence or absence
of compound C. Transfections of constitutively active Akt (Myr-Akt1, Addgene
plasmid 9008) and empty vector (pcDNA3, Addgene plasmid 10792) were
conducted by using Lipofectamine LTX transfection reagent. Each
concentration was tested in triplicate. At the end of the experiment, one of the
plates was taken out and fresh MTT (final concentration 0.5 mg/mL in PBS)
was added to each well. After 2 hr incubation, the culture media were
discarded, 200 μL of acidic isopropanol were added to each well and vibrated
to dissolve the depositor. The optical density was measured at 570 nm with a
microplate reader. 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225
Quantitative analysis of cell cycle by flow cytometry
Human colon cancer CRC cells were cultured into 6-well plates at a density of
1x106 cells per well. Before the experiment, cells were synchronized by
culturing them in 0.05 % FBS supplemented RPMI-1640 media overnight until
CAPE or CAPPE treatment. To measure the distribution of the cell cycle, cells
were treated with CAPE or CAPPE (0, 10, 50, 100 μM) for an additional 24 h.
Cells were harvested after treatment with a solution of trypsin and
ethylenediaminetetraacetic acid (EDTA) and suspended with the binding
buffer (1x105 cells/mL). Human CRC cells were stained with PI and analyzed
following the manufacturer’s protocol. Briefly, five microliters of PI were added
to the suspended cells and incubated at room temperature in the dark and
analyzed by BD FACSCanto flow cytometry (BD Biosciences Inc., Franklin
Lakes, NJ). The PI-stained cells were analyzed using accessory software.
Xenograft implanation of tumor cells
To establish the mouse xenograft model, subconfluent cultures of colon
cancer HCT-116 cells were given fresh medium 24h before being harvested
by a brief treatment with 0.25% trypsin and 0.02% EDTA. Trypsinization was 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244
stopped with medium containing 10% FBS, and the cells were washed twice
and resuspended in serum-free RPMI 1640 medium. Only single-cell
suspensions with a viability of > 90% were used for the injections.
Animals, Diet and CA Derivative Supplementation
Adult (3-4 week old) BALB/C AnN-Foxn1 nude mice (19-22 g) were obtained
from the National Laboratory Animal Center (Taipei, Taiwan). Mice were
maintained under specific pathogen-free conditions in facilities approved by
the National Laboratory Animal Center in accordance with current regulations
and standards (animal protocol no. 102-142-N). The animal use protocol listed
above has been reviewed and approved by the Institutional Animal Care and
Use Committee at China Medical University. The animal study was conducted
according to the national guideline and the approved animal protocol in order
to maintain animal welfare and ameliorate suffering in the experimental
animals. During the entire experimental period, mice were fed a standard Lab
5010 Diet purchased from LabDiet Inc. (St. Louis, MO, USA). The standard
diet contains crude fat (13.5 % total dietary energy), protein (27.5%) and
carbohydrate (59%), and had no detectable CA derivatives, as indicated by
the supplier. Mice that had been anesthetized with an inhalation of isofluorane 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263
were placed in a supine position. The mice were subcutaneously (s.c.)
injected with human colon cancer HCT-116 cells (1 x 106/0.1 ml medium) into
the right flank of each BALB/C AnN-Foxn1 nude mouse. A well-localized bleb
was considered to be a sign of a technically satisfactory injection.
After the inoculation, mice were divided into three subgroups (n=6 per group).
CA derivatives were given to the experimental animals by gavage once a day
at a total volume 0.15 mL. The CAPE and CAPPE groups each received a
daily oral dose of CA derivatives dissolved in corn oil (4% w/w) at 50 nmol/kg
of BW once per day. The tumor control group received corn oil (4% w/w) once
per day only. Normal mice without tumor- inoculation were used as the
negative control. Tumor volume was calculated by the following formula:
0.524 L1(L2)2, where L1and L2 represent the long and short axis of the tumor,
respectively. BW was determined once weekly. No significant differences of
food intake or body weight were found in this study. At the end of the
experimental period, the animals were euthanized by CO2 inhalation; tumor
tissues were then excised, weighed, and frozen immediately. These tumor
tissues were sectioned and stained with Mayer’s hematoxilin– eosin (H&E) for
examination by light microscopy. The remaining tissues of the liver, lung,
spleen, pancreas and intestine were also excised, weighed and frozen for 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282
further experiments. Blood samples were collected from the heart in a 1-ml
vacutainer tube in the presence or absence of heparin and centrifuged for 10
min at 1000g to obtain plasma or serum, respectively.
Histopathological and immunohistochemical staining of tumor
tissues
Frozen tumor tissues were cut in 5 μm sections and immediately fixed with 4%
paraformaldehyde. Sections were stained with Meyer’s Hematoxylin-Eosin
(H&E) for light microscopy. Negative controls did not exhibit any staining.
Three hot spots were examined in a blinded manner per tumor section (high
power field 200X) from six different tumors in each group. For
immunohistochemical staining, frozen tissue sections were treated with 0.3%
hydrogen peroxide to block the endogenous peroxide activity. Non-specific
protein binding was blocked with 10% normal goat serum (NGS) for 1hr
followed by incubation with either anti-FASN or anti-PCNA primary antibodies
(1:300). Tissue sections were washed with 0.1 M phosphate buffer saline
(PBS) and incubated with biotinyated immunoglobin G (1:300 secondary
antibody) at room temperature for 1 hr. Tissue sections were stained with
Avidin-Biotin complex (ABC), diaminobenzidine (DAB) and hydrogen 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301
peroxide. Cell nuclei were stained with hematoxylin. Imaging was performed
at 200 X magnifications. Images of tumor sections were acquired on an
Olympus BX-51 microscope using an Olympus DP-71 digital camera and
imaging system (Olympus, Tokyo, Japan).
Preparation of protein extraction
Human CRC HCT-116 cells were cultured in 10% FBS culture media in the
presence of CAPE or CAPPE for 2h or 24h. Cell lysates (cytoplasmic and
nuclear proteins) from colon cancer cells were prepared using the Nuclear
Protein Extract Reagent Kit containing a protease inhibitor and phosphatase
inhibitors according to the manufacturer’s instructions. After centrifugation for
10 minutes at 12,000 xg to remove cell debris, the supernatants were retained
as a cytoplasmic extract. Cross contamination between nuclear and
cytoplasma fractions was not detected (data not shown).
Detection of Plasma MMP-9 by Enzyme-Linked Immunosorbent
Assay (ELISA)
The MMP-9 plasma level was measured by ELISA according to the
manufacturer’s instructions (R&D Systems Inc.). Briefly, a 100 μL diluted
plasma sample (1:8 dilution) from each group was added to each well and 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321
analyzed. Upon completion of the ELISA process, the plate was read at
450/570 nm wavelength using a microplate reader (Tecan Inc., Mannedorf,
Switzerland).
Analysis of cellular ATP levels
Human CRC cells were cultured for 24h in 96-well plates, each well containing
1x 104 cells in the presence of CAPE or CAPPE. Measurements of cellular
ATP were analyzed following the manufacturer’s protocol. Briefly, cell lysates
were prepared using cell lysis buffer directly. Total cell lysate (100 μL) were
mixed with substrate solution and vibrated to dissolve the deposits according
to the manufacturer’s instructions. The optical density was measured with a
Synergy HT Multi-Mode Microplate Reader (BioTek, Winooski, VT).
Western Blotting Analysis
Cellular proteins (70 μg) were fractionated on 10% SDS-PAGE, transferred to
a nitrocellulose membrane, blotted with anti-phosphorylation Akt monoclonal
antibody, and performed with chemiluminescence based assay. Protein
phosphorylation of PDK1, phosphorylation of GSK3α, phosphorylation of
GSK3βphosphorylation of FOXO3, phosphorylation of AMPK,
phosphorylation of m-TOR, PTEN, N-cadherin, PDK1, Akt, GSK3α, GSK3β, 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341
FOXO3, TSC1, TSC2, mTOR, LKB1, 14-3-3, AMPK, FASN, NF-κB (p-65),
cyclin D1, Cdk4, PCNA, p21CIP1/WAF1, cyclin E and c-myc in the cell lysates
were measured using the same procedure described above. The blots were
stripped and reprobed with eitherβ-actin or lamin A antibodies as the loading
control.
Reporter gene assay
Transfection of NF-κB response element (NF-κB-RE) plasmid was carried out
in human CRC HCT-116 and SW-480 cells according to the manufacturer's
instruction. Human CRC HCT-116 and SW-480 cells were then plated at a
density of 2 X105 cells per well in 12-well plates in 2 mL of media and
incubated overnight. Cells were treated with either CAPE or CAPPE at
different concentrations for 24 h before the analysis of reporter gene activities.
The reporter gene assay was performed by using Dual- Luciferase Reporter
Assay kit. Luciferase intensities were measured using with a Synergy HT
Multi-Mode Microplate Reader (BioTek, Winooski, VT).
Statistical analysis
A quantitative methodology was used to determine whether there was any
significant difference in the cell viability as well as protein expression between 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361
experimental sets and control sets of colon cancer cells. In brief, statistical
analyses of the differences in cell viability among triplicate sets of the
experimental conditions were performed using SYSTAT software.
Confirmation of a difference in cell viability as significant requires rejection of
the null hypothesis of no difference between the mean indices obtained from
the replicate sets of experimental and control groups at the P=0.05 level,
utilizing the one way ANOVA model. The Bonferroni post hoc test was used to
determine differences among the different groups.
Results
CA derivatives significantly inhibited the proliferation of human
CRC cells in vitro
The inhibitory effects of CA derivatives on the proliferation of human CRC
cells (HCT-116 and SW-480 cells) were investigated in vitro. As shown in
Figure 2-3, CA derivatives (at the concentrations of 5, 10, 20, 50 and 100μM)
significantly inhibited the proliferation of human CRC HCT-116 and SW-480
cells. At the concentrations of 5, 10, 20, 50 and 100μM, CAPE and CAPPE
each significantly suppressed the proliferation of human CRC HCT-116 cells,
respectively. (Inhibitory effects of CAPE: 4, 31, 47, 54, and 58%; CAPPE: 5, 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380
45, 56, 59 and 64%) (Figure. 2A). The IC50s for CAPE and CAPPE in human
CRC HCT-116 cells are 44.2 μM and 32.7 μM, respectively. At the
concentrations of 5, 10, 20, 50 and 100μM, CAPE and CAPPE significantly
suppressed the proliferation of human CRC SW-480 cells, respectively.
(Inhibitory effects of CAPE: 0.5, 8.9, 14, 19 and 32%; CAPPE: 6, 15, 22, 26
and 47%) (Figure. 3A). The IC50s for CAPE and CAPPE in human CRC
SW-480 cells are 132.3 μM and 130.7 μM, respectively. These results
demonstrate that CAPE and CAPPE are each able to significantly inhibit the
proliferation of human CRC cells in a dose-dependent manner. CAPPE
seems to inhibit the proliferation of human CRC HCT-116 cells more
effectively than CAPE. For this reason, CAPE and CAPPE were selected for
further study of their potential anti-cancer effects on human CRC cells. The
role of signalling molecules on cell proliferation in human CRC cells treated
with CA derivatives was investigated. In these cells, Akt was either
over-expressed by transfection with a constitutively active Myr-Akt1 plasmid, or
AMPK activity was inhibited by compound C. As shown in Figure. 2A, both
over-expression of Akt and suppression of AMPK activity rescued cell
proliferation inhibited by CAPE or CAPPE treatments in human CRC HCT-116
cells. The effects of Akt over-expression or reduced AMPK activity on 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399
rescuing cell proliferation were less significant, however, in SW-480 cells
treated with CA-derivative (Figure 3A). Expression levels of p-Akt and t-Akt
proteins by the overexpression of a constitutively active form of Akt in human
CRC HCT-116 and SW-480 cells were shown in Figure 2B and Figure 3B,
respectively. Expression levels of p-AMPK and t-AMPK proteins by the
treatment of compound C in human CRC HCT-116 and SW-480 cells were
shown in Figure 2C and Figure 3C, respectively. The results suggested that
CA derivatives act as chemopreventive agents against human CRC through a
modulation of the PI3-K/Akt and AMPK signaling pathways
CAPE and CAPPE each induced G
0/G
1cell cycle arrest in CRC
cells
To determine whether CA derivative-mediated suppression of cell proliferation
was due to an arrest at a certain stage of the cell cycle, the effects of CAPE
and CAPPE were studied further in HCT-116 and SW-480 cells. Cells treated
with CAPE or CAPPE were subjected to flow cytometric analysis after their
DNA was stained with PI. Histograms of the flow cytometric data are shown in
Figure 4A. CAPE and CAPPE significantly induced cell cycle arrest at the
G0/G1 phase in a dose-dependent manner (P<0.05). At a concentration of 50
400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418
μM, CAPE and CAPPE significantly increased cell cycle arrest of HCT-116
cells during the G0/G1 phase by up to 56% and 61 %, respectively, whereas in
the control group the percentage of cells in the G0/G1 phase was only 34%
(Figure. 4B). At a concentration of 50 μM, CAPE and CAPPE significantly
increased cell cycle arrest of SW-480 cells during the G0/G1 phase by up to
44% and 57 %, respectively, whereas in the control group the percentage of
cells in the G0/G1 phase was only 37% (Figure. 4C). These increases in G0/G1
arrest were mostly at the expense of the S and G2/M phase cell populations.
CAPPE seems to induce G0/G1 cell cycle arrest more effectively than CAPE in
human CRC cells. Thus, it is plausible that CAPE and CAPPE inhibited cell
proliferation of human CRC cells through a cell cycle arrest at the G0/G1
phase.
To determine the molecular mechanisms underlying these effects, we further
investigated the chemopreventive effects of CA derivatives on human CRC
cells. A previous study had indicated that the cell cycle progression through
G1 phase is primarily regulated by cyclinD1/Cdk4 proteins [33]. To investigate
the possible inhibitory effects of CA derivatives on cell cycle regulatory
proteins, HCT-116 and SW-480 cells were treated with the aforementioned
concentrations of CAPE and CAPPE and the expression of nuclear proteins 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437
were measure by western blot analysis. As shown in Figure. 4D (HCT-116
cells) and 4E (SW-480 cells), CAPE and CAPPE each significantly inhibited
the expression of the cyclin D1 protein in a dose-dependent manner. CAPE
and CAPPE also suppressed the expression of proliferating cell nuclear
antigen (PCNA) protein in CRC cells. These results indicated that CAPE and
CAPPE significantly induced cell cycle arrest of CRC cells at the G0/G1 phase
through suppression of the nuclear cyclin D1 and PCNA proteins.
CAPE and CAPPE inhibited the proliferation of human CRC cells
through the modulation of the PI3K/Akt, AMPK and mTOR
signaling pathways
Previous studies indicated that the PI3-K/ Akt, mTOR and AMPK signaling
pathways play important roles in the growth and progression of human CRC
[18,34-37]. To explore the molecular mechanisms by which inhibition of
signaling cascades might induce cell cycle arrest, we investigated the
inhibitory effects of CAPE and CAPPE on the PI3-K/ Akt, mTOR and AMPK
signaling pathways. As shown in Figure 5 (HCT-116 cells) and 6 (SW-480
cells), CAPE and CAPPE significantly inhibited the phosphorylation of the
PDK1, Akt and mTOR signaling molecules compared to untreated control 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456
cells. Moreover, treatment with CAPE or CAPPE significantly augmented the
expression of the 14-3-3 protein and the phosphorylation of the FOXO3
proteins. Previous studies showed that the upregulation of N-cadherin is
associated with the progression of carcinoma cells [38]. Here, the results
demonstrated that CAPPE significantly inhibited the expression of N-cadherin
in CRC cells. These results suggested that CAPE and CAPPE each
significantly inhibited cell proliferation and progression through the modulation
of PI-3K/Akt and mTOR cascades, as well as the downstream target
molecules, in HCT-116 cells.
Recent studies suggested that the AMPK signaling pathway is involved in
FASN expression and the progression of human CRC cells via crosstalk with
the PI3-K/Akt signaling cascades [21-24]. Therefore, we further examined the
effects of CAPE and CAPPE on the AMPK signaling pathway. As shown in
Figure 5A and 6A, CAPE and CAPPE each significantly augmented the
phosphorylation (i.e. activation) of AMPK molecule in CRC cells. Moreover,
the results also showed that CAPPE- mediated activation of AMPK pathway is
associated with the suppression of FASN expression (Figure. 5A and 6A) and
decreased ATP levels in CRC cells (Figure. 5B and 6B). The results
suggested that CAPE and CAPPE suppressed the expression of FASN in 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475
human CRC cells, in part through the augmentation of AMPK signaling
molecules. These results further suggest that CAPPE inhibits the PI3-K/Akt,
AMPK and mTOR signaling pathways in CRC cells more effectively than
CAPE (Figure. 5A and 6A). Moreover, the inhibitory effect of CAPPE on the
cellular ATP levels is also more significant than CAPE in CRC cells (Figure.
5B and 6B).
CAPE and CAPPE inhibited the proliferation of CRC cells
independently of NF-κB signaling pathway
Previous study showed that CAPE is a well know NF-κB inhibitor in U937 cells
[39]. To investigated whether CAPE and CAPPE inhibited the proliferation of
human CRC cells through NF-κB pathway, the expression of NF-κB (p65;
RelA) by Western blotting assay and reporter gene assay were performed in
this study. The results demonstrated that CAPE or CAPPE moderately
inhibited the expression of NF-κB (p65; RelA) protein in HCT-116 cells at 2 h
time point (Figure 7A). Moreover, the expression of NF-κB (p65) protein was
only inhibited by the treatment of CAPPE rather than CAPE in SW-480 cells at
2 h time point (Figure 7B). However, CAPE and CAPPE did not suppress the
reporter gene activities of NF-κB response element (NF-κB-RE) in HCT-116 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494
(Figure 7C) or SW-480 cells (Figure 7D) at 24 h time point. To determine
whether NF-κB inhibition is important for cell proliferation, tumor necrosis
factor-α (TNF-αa NF-κB activator) was utilized in this study. The results
showed that CAPE and CAPPE had differential effects on the suppression of
cell growth in HCT-116 (Figure 7E) or SW-480 cells (Figure 7F) in the
presence of TNF-α(1 ng/mL). These results suggested that CAPE and
CAPPE mediated-suppression of cell growth was independent of NF-κB
pathway in human CRC cells.
Consumption of CAPE or CAPPE suppressed the growth of
colorectal tumor in a mouse xenograft model
To verify these in vitro findings, we further examined the respective effects of
CAPE and CAPPE on the growth of human colon cancer HCT-116 cells in a
mouse xenograft model. As shown in Figure. 8A, consumption of CAPE and
CAPPE (at dosages of 50 nmol /kg of BW per day) significantly inhibited the
growth of colorectal tumors in a mouse xenograft model (P<0.05). By the end
of the 6-week study period, CAPE or CAPPE significantly reduced tumor
weights (P<0.05) compared to the tumor control group (Figure. 8B).
Histopathological staining results indicated that consumption of either CAPE 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513
or CAPPE inhibited the growth of colorectal tumor in these experimental
animals (Figure. 8C). Moreover, consumption of CAPE or CAPPE also
suppressed the expression of malignant biomarker proteins, such as PCNA
(Figure. 8 D) and FASN in tumor tissues (Figure. 8 E). Previous studies had
suggested that the expression of MMP-9 was associated with tumor invasion
and progression of CRC [11,40]. In the current study, we investigated whether
consumption of CAPE or CAPPE modulated the expression of plasma MMP-9
proteins in these experimental animals. By the end of the study, the basal
MMP-9 plasma levels in the tumor-free mice were approximately 11.3 ng/mL.
Mice inoculated with colon cancer HCT-116 cells had high plasma levels of
MMP-9 (mean ± SD : 125.6 ± 14 ng/mL). The consumption of CAPE or
CAPPE, however, significantly decreased the MMP-9 plasma level in these
tumor-bearing mice. The MMP-9 plasma levels decreased from 125.6 ng/mL
in the tumor control group to 43.1ng/mL and 32.8 ng/mL in the CAPE and
CAPPE–fed groups, respectively (Figure. 8F). No hepatoxicity was induced by
CAPE or CAPPE at doses of 50 nmol /kg of BW in this study (data not
shown). These results show that consumption of CAPE or CAPPE
significantly inhibited tumor growth of CRC in a mouse xenograft model. The
chemopreventive effects of CAPE and CAPPE were in part associated with 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532
the suppression of the PCNA, FASN and MMP-9 proteins in these
tumor-bearing animals.
CAPE- or CAPPE-mediated suppression of tumor growth was
associated with the modulation of the PI3-K/Akt, AMPK and mTOR
signaling pathways in experimental animals
The results described above clearly show the inhibitory effects of CAPE and
CAPPE on the growth of CRC cells in a mouse xenograft model. We also
demonstrated the molecular mechanisms of action of the CA derivatives in
vitro. To verify these mechanistic findings, we further examined the molecular
effects of CAPE and CAPPE in these tumor-bearing mice. As shown in
Figure. 9A, CAPE and CAPPE consumption each significantly inhibited the
expression of cyclin D1, Cdk4, cyclin E and c-myc proteins in vivo. Moreover,
the in vivo chemopreventive effects of CAPE and CAPPE were associated
with the upregulation of the p21CIP1/WAF1 protein.
It is well known that the PI3-K/Akt and MAPK/ERK signaling cascades play an
important role in tumor growth and progression [4,41]. Suppression of the
PI3-K/Akt and MAPK/ERK signaling cascades leads to down-regulation of
downstream target proteins such as cyclin D1/Cdk4 and a blockade of the cell 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551
cycle [4,36,41-45]. Therefore, we further investigated the inhibitory effects of
CAPE and CAPPE on the PI3-K/ Akt and MAPK/ ERK signaling pathways. As
shown in Figure. 9B, consumption of CAPE or CAPPE effectively inhibited the
activation of the Akt, mTOR and ERK 1/2 signaling molecules. CA
derivative-mediated suppression of the Akt, mTOR and ERK 1/2 signaling cascades was
associated with an up-regulation of E-cadherin as well as a suppression of
N-cadherin. Moreover, CAPE and CAPPE -mediated suppression of FASN
protein was associated with the augmentation of the AMPK cascade in
tumor-bearing mice (Figure. 9B). These results show that CAPE or
CAPPE-mediated suppression of PI3-K/Akt and MAPK/ ERK signaling cascades, as
well as an augmentation of the AMPK signaling pathway is associated with
the suppression of tumor growth at least in small laboratory animals.
Discussion
Previous studies suggest that CAPE has potential as a chemopreventive and
therapeutic agents [46-49]. Many studies demonstrated that CAPE could
inhibit tumor angiogenesis and suppress the growth of several types of cancer
[47-52]. The aberrant PI3K/Akt pathway has been shown to be the
predominant pathway in the tumorigenesis of many types of cancer including 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570
colon cancer [53]. Studies suggested that suppression of the PI3K/Akt and
integrin-mediated signaling pathways by CAPE could effectively inhibit the
tumor growth [50,54]. To date, the effects of CAPPE on the proliferation and
survival of human CRC cells have not been convincingly demonstrated. In the
current study, we demonstrate the inhibitory effects of CA derivatives (CAPE
and CAPPE) on the proliferation of human colon cancer cells both in vitro and
in vivo. The results show that CAPE and CAPPE each effectively suppressed
the proliferation of human colon cancer cells in a dose-dependent manner.
CAPE and CAPPE effectively suppressed the proliferation of human CRC
cells through the induction of cell cycle arrest at the G0/G1 phase. Previous
studies suggested that the overexpression of cell cycle-related proteins, such
as D1 and Cdk4, is correlated with the proliferation of human cancer cells [33].
In this study, the results showed that CAPE or CAPPE significantly inhibited
the expression of cyclin D1 protein. Recently, cyclin D1 was identified as a
target of the PI3-K/Akt pathways in CRC cells [44]. We further confirmed that
the molecular effects of CAPE and CAPPE were carried out through the
inhibition of the PI3-K/Akt and mTOR signaling pathways in human CRC cells.
Moreover, CAPE and CAPPE inhibited the expression of FASN through an
augmentation of the AMPK cascade. A recent study reports that the activation 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589
of AMPK is associated with an increased cellular AMP/ATP ratio [55]. A low
energy status leads to the phosphorylation (i.e. activation ) of AMPK and the
suppression of mTOR activity through the effect on the LKB1 protein [55]. The
current study suggested than CAPPE may suppress the activity of mTOR
protein in a LKB-independent manner. In contrast, CAPPE-mediated
activation of the AMPK molecule was more significantly correlated with the
decreased ATP levels in CRC cells. Therefore, it is probable that the
respective CAPE- and CAPPE- mediated augmentation of the AMPK cascade
and suppression of mTOR protein are in part associated with a decreased
level of ATP in these CRC cells. There are several possible scenarios to
explain why CAPPE is a more effective anti-cancer compound than CAPE.
One explanation might be that CAPPE has a cell membrane solubility higher
than that of CAPE. This possibility is consistent with the findings of an earlier
toxicity study [56]. Previous studies demonstrated that the inhibitory effect of
CA derivatives on nitric oxide (NO) production is correlated with the increasing
length of the alkyl chain (i.e. CAPPE> CAPE) [56]. A recent study showed that
the L-arginine -mediated NO reaction is also associated with AMPK activation
[57]. These findings suggest that the upregulation of AMPK activation is
dependent on the increasing length of the CA derivatives. It is to be expected, 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608
therefore, that CAPPE would be more effective in AMPK activation than
CAPE. This may explain why CAPPE is a more effective regulator of AMPK
activation and the suppression of cell proliferation than CAPE. The
anti-proliferation effect of CAPPE could be achieved by increasing the dosage
levels of CAPE (Figure. 2,3). The current study also showed an inverse
correlation between AMPK and mTOR activity in vivo. These results are
consistent with AMPK -mediated downregulation of mTOR activity [22,23].
This suggests that CAPE and CAPPE may act through this pathway as
effective anti-cancer agents against human CRC cells. Moreover, the results
suggested that CAPE and CAPPE mediated- suppression of cell proliferation
was independent of NF-κB pathway in human CRC cells.
To verify these in vitro findings, we further examined the respective inhibitory
effects of CAPE and CAPPE on the growth of colorectal tumor in a xenograft
mouse model. As shown in Figure. 8, consumption of CAPE or CAPPE
significantly inhibited tumor growth in vivo. We also examined the actions of
these bioactive compounds on multiple signaling pathways including
PI3-K/Akt, MAPK/ERK and AMPK signaling cascades (Figure. 9). The results
demonstrated that CAPE and CAPPE also effectively induced the activation of
the AMPK cascade and suppressed the activation of both the PI3-K/Akt and 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627
MAPK/ERK signaling cascades. CAPE and CAPPE further significantly
inhibited the expression of FASN , cyclin D1 , cyclin E, Cdk4 and c-myc
proteins of tumor tissues in an in vivo animal study. We further examined
whether the consumption of CAPE or CAPPE would help prevent tumor
progression in tumor-bearing mice. The results demonstrated that CAPE or
CAPPE significantly inhibited the expression of plasma MMP-9 in vivo (Figure.
8F). These results are consistent with the in vitro findings.
In conclusion, this is the first demonstration of the inhibitory effects of CA
derivatives (CAPE and CAPPE) on the proliferation of human colon cancer
cells both in vitro and in vivo. The directional changes in protein expression
produced by CAPE and CAPPE are in relevant pathways and consistent with
the properties of a chemopreventive agent. Whether CAPPE is a more potent
chemopreventive agent than CAPE will require further preclinical studies.
Figure Legends
Figure 1 Chemical structure of the CA derivatives
The CA derivatives are depicted in Fig.1. (A) CAPE and (B) CAPPE differ in
the elongation of the alkyl side chain of the caffeic acid ester. 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647
Figure 2 CA derivatives significantly inhibited the proliferation of human CRC HCT-116 cells in vitro
(A) Human CRC HCT-116 cells were cultured in RPMI-1640 medium with
CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 μM) in the
presence or absence of compound C (10 μM) for 24 h. Transfections of
constitutively active Akt (Myr-Akt1) and empty vector (pcDNA3) were
conducted before the treatment of CA derivatives . The cell proliferation was 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668
measured by MTT assay as described in Materials and Methods. Data are the
mean SD (standard deviation) of three independent experiments. The
different symbols ( for CAPE and for CAPPE) represent a statistically ※ △
significant difference compared to the CA derivative -untreated control group
in each group, respectively, at P<0.05. The different symbols (＃ for
CAPE_Akt, § for CAPE_compound C, ▲ for CAPPE_Akt, and ■ for
CAPPE_compound C) represent a statistically significant difference compared
to each corresponding CA derivative- treated control group in each dosage
subgroup, respectively, at P<0.05.
(B-C) Cytoplasmic proteins were prepared for Western blotting analysis using
monoclonal antibodies against phosphorylation Akt (S473), total-Akt,
anti-phosphorylation AMPKα (T172) and total-AMPKα
Figure 3 CA derivatives significantly inhibited the proliferation of human CRC SW-480 cells in vitro
(A) Human CRC SW-480 cells were cultured in RPMI-1640 medium with
CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 μM) in the
presence or absence of compound C (10 μM) for 24 h. Transfections of
constitutively active Akt (Myr-Akt1) and empty vector (pcDNA3) were
conducted before the treatment of CA derivatives . The cell proliferation was 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687
measured by MTT assay as described in Materials and Methods. Data are the
mean SD (standard deviation) of three independent experiments. The
different symbols ( for CAPE and for CAPPE) represent a statistically ※ △
significant difference compared to the CA derivative -untreated control group
in each group, respectively, at P<0.05. The different symbols (＃ for
CAPE_Akt, § for CAPE_compound C, ▲ for CAPPE_Akt, and ■ for
CAPPE_compound C) represent a statistically significant difference compared
to each corresponding CA derivative- treated control group in each dosage
subgroup, respectively, at P<0.05.
(B-C) Cytoplasmic proteins were prepared for Western blotting analysis using
monoclonal antibodies against phosphorylation Akt (S473), total-Akt,
anti-phosphorylation AMPKα (T172) and total-AMPKα
Figure 4 CAPE and CAPPE each induced G0/G1 cell cycle arrest in CRC
cells
Human CRC cells were synchronized in RPMI-1640 medium with 0.05 % FBS
in tissue culture dishes overnight. To measure the distribution of the cell cycle,
cell were cultured in the presence or absence of CAPE and CAPPE (0, 10, 50
and 100 μM) cultured in 10% FBS RPMI-1640 medium for another 24 h. (A)
The measurement of the cell population at different cell cycle phases was 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706
performed using flow cytometry analysis, as described under Materials and
Methods. The data indicate the (B) HCT-116 cell (C) SW-480 cell population
percentage at different cell phases under the treatment of CAPE or CAPPE in
human CRC cells.
Human CRC (D) HCT-116 cells (E) SW-480 cells were treated with either
CAPE or CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 μM) in 10%
FBS RPMI-1640 for 24h. Nuclear proteins were prepared for Western blotting
analysis using monoclonal antibodies against cyclin D1, Cdk4, PCNA, and
lamin A antibodies, as described under Materials and Methods. The levels of
detection represent the amounts of cyclin D1, Cdk4 and PCNA in the nuclei of
human CRC cells. The results (mean ± SD) represent the folds change of
control group and are representative of three different experiments. The
immunoreactive bands are noted with an arrow. The mean integrated
densities of these proteins adjusted with the internal control lamin A protein
are shown in bottom row. The standard deviation (SD) of each measured
protein was indicated in the parenthesis. A single asterisk indicates a
significant difference compared to the CAPE- or CAPPE-untreated control
group, respectively (P<0.05). 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725
Figure 5 CAPE and CAPPE inhibited the proliferation of human CRC HCT-116 cells through the modulation of the PI3K/Akt, AMPK and mTOR signaling pathways
Human CRC HCT-116 cells were treated with either CAPE or CAPPE (at
concentrations of 0, 5, 10, 20, 50 and 100 μM) in 10% FBS RPMI-1640 for
24h.
(A) Cytoplasmic proteins were prepared for Western blotting analysis using
monoclonal antibodies against N-cadherin, PTEN, anti-phosphorylation PDK1
(S241), total-PDK1, phosphorylation Akt (S473), total-Akt, anti-726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746
phosphorylation GSK3α (S21), total- GSK3α, anti-phosphorylation
GSK3β(S9), total- GSK3β, anti-phosphorylation FOXO3 (T32), total- FOXO3,
total- TSC1, total- TSC2, total- LKB1, total- 14-3-3, anti-phosphorylation
AMPKα (T172), total-AMPKα, anti-phosphorylation m-TOR (S2448),
total-m-TOR, anti-FASN and β-actin as described under Materials and Methods. The
levels of detection represent the amounts of each protein in the cytoplasm of
HCT-116 cells. The results (mean ± SD) represent the folds change of control
group and are representative of three different experiments. The
immunoreactive bands are noted with an arrow. The mean integrated
densities of these proteins adjusted with the control protein are shown in
bottom row. The standard deviation (SD) of each measured protein was
indicated in the parenthesis. A single asterisk indicates a significant difference
compared to the CAPE- or CAPPE-untreated control group, respectively
(P<0.05).
(B) The measurement of cellular ATP was performed as described under
Materials and Methods. Data represent the percentage of cellular ATP levels
in the CAPE- or CAPPE-treated human CRC HCT-116 cells. A single or
double asterisk indicates a significant difference compared to the CAPE- or
CAPPE-untreated control group, respectively (P<0.05). 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765
Figure 6 CAPE and CAPPE inhibited the proliferation of human CRC SW-480 cells through the modulation of the PI3K/Akt, AMPK and mTOR signaling pathways
Human CRC SW-480 cells were treated with either CAPE or CAPPE (at
concentrations of 0, 5, 10, 20, 50 and 100 μM) in 10% FBS RPMI-1640 for
24h.
(A) Cytoplasmic proteins were prepared for Western blotting analysis using
monoclonal antibodies against N-cadherin, PTEN, anti-phosphorylation PDK1
(S241), total-PDK1, phosphorylation Akt (S473), total-Akt,
anti-phosphorylation GSK3α (S21), total- GSK3α, anti-anti-phosphorylation 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786
GSK3β(S9), total- GSK3β, anti-phosphorylation FOXO3 (T32), total- FOXO3,
total- TSC1, total- TSC2, total- LKB1, total- 14-3-3, anti-phosphorylation
AMPKα (T172), total-AMPKα, anti-phosphorylation m-TOR (S2448),
total-m-TOR, anti-FASN and β-actin as described under Materials and Methods. The
levels of detection represent the amounts of each protein in the cytoplasm of
HCT-116 cells. The results (mean ± SD) represent the folds change of control
group and are representative of three different experiments. The
immunoreactive bands are noted with an arrow. The mean integrated
densities of these proteins adjusted with the control protein are shown in
bottom row. The standard deviation (SD) of each measured protein was
indicated in the parenthesis. A single asterisk indicates a significant difference
compared to the CAPE- or CAPPE-untreated control group, respectively
(P<0.05).
(B) The measurement of cellular ATP was performed as described under
Materials and Methods. Data represent the percentage of cellular ATP levels
in the CAPE- or CAPPE-treated human CRC SW-480 cells. A single or double
asterisk indicates a significant difference compared to the CAPE- or
CAPPE-untreated control group, respectively (P<0.05). 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805
Figure 7 CAPE and CAPPE inhibited the proliferation of CRC cells independently of NF-B signaling pathway
(A-B) Human CRC cells were treated with either CAPE or CAPPE (at
concentrations of 0, 5, 10, 20, 50 and 100 μM) in 10% FBS RPMI-1640 for 2h. Nuclear proteins were prepared for Western blotting analysis using
monoclonal antibodies against NF-κB (p65) and lamin A as described under
Materials and Methods. The levels of detection represent the amounts of each
protein in the nuclei of HCT-116 cells (A) or SW-480 cells (B). The results
(mean ± SD) represent the folds change of control group. The mean
integrated densities of these proteins adjusted with the control protein are 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825
shown in bottom row. The standard deviation (SD) of each measured protein
was indicated in the parenthesis. Human CRC HCT-116 cells (C) or SW-480
cells (D) were transfected with NF-κB-RE plasmid and then treated with either
CAPE or CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 μM) in 10%
FBS RPMI-1640 for 24h. The relative light units (R.L.U) were measured by the
manufacturer's instruction as described under Materials and Methods. A
single or double asterisk indicates a significant difference compared to the
CAPE- or CAPPE-untreated control group, respectively (P<0.05).
Human CRC HCT-116 cells (E) or SW-480 cells (F) were cultured in
RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50
and 100 μM) in the presence or absence of TNF-α (1 ng/mL) for 24 h. The cell
proliferation was measured by MTT assay as described in Materials and
Methods. Data are the mean SD (standard deviation) of three independent
experiments. The different symbols ( for CAPE and for CAPPE) represent※ △
a statistically significant difference compared to the CA derivative -untreated
control group in each group, respectively, at P<0.05. The different symbols (▲
for CAPE_TNF-α and ■ for CAPPE_TNF-α) represent a statistically significant
difference compared to each corresponding CA derivative- treated control
group in each dosage subgroup, respectively, at P<0.05. 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844
Figure 8 Consumption of CAPE or CAPPE suppressed the growth of colorectal tumor in a mouse xenograft model
Xenograft nude mice (n=6 for each group) were divided into three groups (the
tumor group, tumor with CAPE, tumor with CAPPE) and given CAPE or
CAPPE (at a dosage of 50 nmol /kg of body weight (BW)/day) for 6 weeks.
Data (mean ± SD) represent the change in the tumor volume (A) or tumor
weight (B) among the tumor group (i.e. the control group), tumor with CAPE
and tumor with CAPPE. The different letters at the same time point represent
a statistically significant difference, (P<0.05).
Tumor tissues were formalin-fixed, embedded in paraffin, sectioned and
subjected to hematoxylin-eosin (H&E) staining (C) as described under
Materials and Methods. Blue spots represent the nuclei stained with 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864
hematoxylin. The red spots represent cytoplasm stained with eosin. For
immunohistochemical (IHC) staining, tumor tissues (at week 6) were frozen ,
sectioned and subjected to either anti- PCNA (D) or anti-FASN (E) antibodies.
The intense dark brown color indicates the distribution of the PCNA or FASN
proteins in HCT-116 cells stained with a monoclonal antibody. The blue area
represents the localization of the cell nuclei. Imaging was documented at
200X magnification. (F) The plasma levels of MMP-9 were determined using
an ELISA Kit (R&D systems). Upon completion of the ELISA process,
fluorescence intensities were read using a wavelength of 450/570 nm. The
results presented are representative of six different experiments and are
presented as plasma MMP-9 levels. The different letters represent a
significant difference in a comparison of normal mice, tumor control mice,
CAPE-treat mice and CAPPE-treated mice, P<0.05. 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884
Figure 9 CAPE- or CAPPE-mediated suppression of tumor growth was associated with the modulation of the PI3-K/Akt, AMPK and mTOR signaling pathways in the experimental animals
(A) Nuclear proteins from tumor tissues were prepared for Western blotting
analysis using monoclonal antibodies against p21CIP1/WAF1, cyclin D1, cyclin E,
Cdk4 and c-myc as described under Materials and Methods. The results
(mean ± SD) represent the folds change of control group and are
representative of three different experiments. The immunoreactive bands are
noted with an arrow. The levels of detection represent the amount of these
proteins in the nuclei of CRC cells in the experimental animals. The mean
integrated densities of these proteins are adjusted with the control protein
and shown in bottom row. The standard deviation (SD) of each measured
protein was indicated in the parenthesis. A single asterisk represent a
statistically significant difference compared to the control group, P<0.05. 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904
(B) Cytoplasmic proteins from tumor tissues were prepared for Western
blotting analysis using monoclonal antibodies against E-cadherin, N-cadherin,
p-Akt, p-mTOR, p-ERK 1/2, p-AMPK, FASN and actin, as described under
Materials and Methods. The results (mean ± SD) represent the folds change
of control group and are representative of three different experiments. The
levels of detection represent the amount of these proteins in the cytoplasm of
CRC cells in the experimental animals. The mean integrated densities of
these proteins are adjusted with the control protein and shown in bottom row.
The standard deviation (SD) of each measured protein was indicated in the
parenthesis. A single asterisk represent a statistically significant difference
compared to the control group, P<0.05. 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924
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