Carcinogenesis vol.35 no.10 pp.2314–2320, 2014
doi:10.1093/carcin/bgu173
Advance Access publication August 14, 2014
A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and
proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells
Leonarda D’Angelo1,2,†, Giulia Piazzi1,2,†, Annalisa Pacilli3,
Anna Prossomariti1,2, Chiara Fazio1,2, Lorenzo Montanaro3,
Giulia Graziani4, Vincenzo Fogliano5, Alessandra Munarini1,2,6,
Francesca Bianchi1,7, Andrea Belluzzi8, Franco Bazzoli1 and
Luigi Ricciardiello1,2,*
1
Department of Surgical and Medical Sciences, University of Bologna, 40138
Bologna, Italy, 2Center for Applied Biomedical Research (CRBA), S.OrsolaMalpighi Hospital, University of Bologna, 40138 Bologna, Italy, 3Department
of Experimental, Diagnostic and Specialty Medicine, University of Bologna,
40138 Bologna, Italy, 4Department of Food Science, University of Naples
“Federico II”, 80055 Portici (NA), Italy, 5Food Quality & Design group,
Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands,
6
Endocrinology Unit, Department of Medical and Surgical Sciences, S.OrsolaMalpighi Hospital, 40138 Bologna, Italy, 7Laboratory of Molecular Biology
and Stem Cell Engineering, National Institute of Biostructures and Biosystems,
S.Orsola-Malpighi Hospital, University of Bologna, 40138 Bologna, Italy
and 8Gastroenterology Unit, Department of Medical and Surgical Sciences,
S.Orsola-Malpighi Hospital, 40138 Bologna, Italy
*To whom correspondence should be addressed. Department of Medical and
Surgical Sciences, University of Bologna, Via Massarenti 9, PAD11, Bologna
40138, Italy. Tel /Fax: +39-051-6363381;
Email: [email protected]
Colorectal cancer (CRC) is one of the major causes of cancer
death worldwide. The development of novel anti-CRC agents able
to overcome drug resistance and/or off-target toxicity is of pivotal
importance. The mammalian target of rapamycin (mTOR) plays
a critical role in CRC, regulating protein translation and controlling cell growth, proliferation, metabolism and survival. The aim of
this study was to explore the effect of a combination of three natural compounds, eicosapentaenoic acid-free fatty acid (EPA-FFA),
epigallocatechin-3-gallate (EGCG) and proanthocyanidins (grape
seed [GS] extract) at low cytotoxic concentrations on CRC cells and
test their activity on mTOR and translational regulation. The CRC
cell lines HCT116 and SW480 were treated for 24 h with combinations of EPA-FFA (0–150 µM), EGCG (0–175 µM) and GS extract
(0–15 µM) to evaluate the effect on cell viability. The low cytotoxic
combination of EPA-FFA 150 µM, EGCG 175 µM and GS extract
15 µM completely inhibited the mTOR signaling in HCT116 and
SW480 cells, reaching an effect stronger than or comparable to
that of the mTOR inhibitor Rapamycin in HCT116 or SW480 cells,
respectively. Moreover, the treatment led to changes of protein
translation of ribosomal proteins, c-Myc and cyclin D1. In addition,
we found a reduction of clonal capability in both cell lines, with
block of cell cycle in G0G1 and induction of apoptosis. Our data
suggest that the low cytotoxic combination of EPA-FFA, EGCG
and GS extract, tested for the first time here, inhibits mTOR signaling and thus could be considered for CRC treatment.
Introduction
Colorectal cancer (CRC) is one of the leading causes of death worldwide in both men and women (1). Antineoplastic drugs are widely
used for CRC treatment but drug resistance and/or off-target toxicity
limit their efficiency (2,3). Therefore, the development of novel antiCRC agents is strikingly important.
The mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine kinase that regulates protein translation and
controls key cellular processes such as cell metabolism, growth,
proliferation, angiogenesis and survival, by integrating signals
from growth factors, nutrients and energy status (4,5). It is known
that mTOR, acting downstream of the phosphatidylinositol-4,
5-bisphosphate 3-kinase (PI3K)/Akt signaling, forms two multiprotein complexes: mTORC1, sensitive to Rapamycin, and mTORC2,
resistant to Rapamycin, which have distinct physiological functions. In particular, mTORC1 regulates messenger RNA (mRNA)
expression, ribosome biogenesis, cellular growth, proliferation and
survival (4,6). In response to mitogenic stimuli or nutrient availability, mTORC1 is activated, leading to phosphorylation of two
well-characterized downstream effectors: the 70 kDa ribosomal
protein S6 kinase (p70S6K) and the eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4EBP1), which together
control protein synthesis (7).
mTOR is an oncogene that plays a pivotal role in several human
cancers (8–10). Emerging data have shown that components of the
mTOR signaling pathway are frequently activated or overexpressed in
CRC, representing a promising therapeutic target (5,11,12).
Rapamycin and its analogs (rapalogs) like RAD001 (everolimus Novartis), AP23573 (Ariad Pharmaceuticals) and CCI779
(temsirolimus) are the most well-studied mTORC1 inhibitors.
One problem regarding the use of this class of drugs for cancer
treatment is related to a distinct spectrum of dose-limiting toxicities (13) such as skin reactions, mucositis and myelosuppression
(11,14).
In nature, fruits and vegetables are an important source of phytochemicals which have chemopreventive activities; however, they can
also be suitable for cancer treatment (15). In particular, epigallocatechin-3-gallate (EGCG), the most important polyphenol from green
tea, inhibits cell growth and proliferation in vitro, inducing apoptosis
on CRC cells (16,17).
Another important source of phytochemicals, in particular proanthocyanidins, is grape seed (GS) extract. Data indicate that GS
extract has strong growth inhibitory and apoptosis-inducing effects
in CRC cell lines (18,19) and xenografts (20). Fish oil is a source of
omega-3 (ω-3) polyunsaturated fatty acids, such as eicosapentaenoic
acid (EPA). EPA inhibits cell proliferation and tumor growth in murine
and rodent models (21,22). Moreover, EPA has an effect in reducing
the size of liver metastases in animal models (23–25). Overall, literature data show that each of the three compounds mentioned above,
tested as single molecules, have strong effects on CRC cells (26–28),
and EGCG and EPA have been found to act on the PI3K/Akt/mTOR
pathway (29,30). However, to achieve anticancer effects, dosages are
usually high. This would make natural compounds toxic rather than
beneficial.
To minimize the toxic effects produced by the use of single
agents at high concentrations and to develop a strategy more suitable in humans, in this study, we tested for the first time a combination of EPA as free fatty acid (FFA), EGCG and GS extract
at low toxicity dosage, on mTOR signaling and on translational
regulation in CRC cell lines. We found that this combination completely inhibited the mTOR signaling, led to changes of protein
translation, reduced cell proliferation and induced apoptosis in
CRC cells.
Materials and methods
Abbreviations: CRC, colorectal cancer; EPA-FFA, eicosapentaenoic acidfree fatty acid; EGCG, epigallocatechin-3-gallate; GS, grape seed; mRNA,
messenger RNA; mTOR, mammalian target of rapamycin.
†
These authors equally contributed to the work.
Cell lines and treatments
The human CRC cell lines HCT116 (PIK3CA mutant, Rapamycin sensitive,
p53 wild-type) and SW480 (PIK3CA wild-type, Rapamycin resistant, p53
mutant) were obtained from ATCC (Manassas, VA) and cultured in IMDM
© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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Effect of EPA-FFA, EGCG and GS on mTOR signaling in CRC
supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml
streptomycin and 2 mM glutamine (Euroclone, Milan, Italy). Cells were maintained at 37°C and 5% CO2. In our laboratory, all cells are tested and authenticated every year using known genetic and epigenetic markers.
Eicosapentaenoic acid-free fatty acid (EPA-FFA, ALFA, SLA Pharma AG,
Switzerland), EGCG (purity ≥95%, Sigma-Aldrich, Milan, Italy), GS (purchased at a local market) extract and Rapamycin (Cell Signaling Technology,
Milan, Italy) were used for cell culture treatments.
Preparation of GS extracts
One hundred grams of Aglianico red GSs were freeze dried and finely ground in
a blender. They were then extracted three times with 200 ml of methanol obtaining
the crude extract that was analyzed by liquid chromatography coupled with tandem
mass spectrometry, as described previously (31). The main compounds identified
were catechins, dimers and trimers, although significant amounts of polymers, up
to 8 units, were also detected. To obtain the molar concentration of GS extract, the
sum of all peaks present in the chromatogram were considered and quantified using
the calibration curve of procyanidin B2. Therefore, the GS amount was expressed
in procyanidin B2 equivalents. In particular, GS extract 10 mmol/l procyanidin B2
equivalents were prepared and opportunely diluted for cell treatments.
Cell viability assay
Cells were seeded into 96-well plates (3 × 103 cells/well) and treated with different concentrations of EPA-FFA (0–300 µM), EGCG (0–400 µM) and GS
(0–200 µM) in combination. After 24–96 h treatment, the number of viable
cells compared with the control was measured using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay (MTT, Sigma-Aldrich)
following the manufacturer’s instructions. Each experiment was performed in
quintuplicate and repeated independently twice.
Colony-forming assay
Cells were seeded in six-well plates (103 cells/well) and treated with a combination of EPA-FFA 150 µM, EGCG 175 µM and GS 15 µM or Rapamycin
20 nM for 24 h. After colony formation, the plates were washed with phosphate-buffered saline and fixed with 0.5% crystal violet in paraformaldehyde.
The total number of visible colonies was counted using ImageJ software
(rsbweb.nih.gov/ij/). Each experiment was performed in triplicate and repeated
independently three times.
Cell cycle analysis
Upon treatment with EPA-FFA 150 µM, EGCG 175 µM and GS 15 µM or
Rapamycin 20 nM for 24 h, cells were trypsinized, pelleted and resuspended in
the Nusse 1 solution (NaCl 584 mg/l, trisodium citrate 1139 mg/l, ribonuclease
10 mg/l and Nonidet P40 300 µg/l) to obtain a final concentration of 2 × 106
cells/ml and incubated for 45 min on ice. An equal amount of Nusse 2 solution
(sucrose 85.5 g/l and citric acid 15 g/l) was then added to the mixture to obtain
a final concentration of 106 cells/ml. Samples were stained with propidium
iodide (50 µg/ml) for 15 min and analyzed by flow cytometry (FACSAria, BD
Biosciences, Milan, Italy).
Apoptosis analysis
Cells were treated for 24 h with the combination of EPA-FFA 150 µM, EGCG
175 µM and GS 15 µM or Rapamycin 20 nM. Apoptosis was evaluated with
the DeadEnd fluorometric TUNEL System (Promega, Milan, Italy), according
to the manufacturer’s recommendations. The apoptotic index was expressed as
the number of apoptotic cells/total cells.
Western blot analysis
Cells were treated with EPA-FFA, EGCG and GS as single compounds or in
combination or with Rapamycin 20 nM for 4–8 h. These time points were chosen because in the literature the effects on mTOR targets are studied between
1 and 8 h of treatment (32). Fifty micrograms of proteins for each sample
were separated on a 4–12% sodium dodecyl sulfate–polyacrylamide gel (Life
Technologies, Monza, Italy) and transferred onto nitrocellulose membrane.
After blocking, membranes were incubated overnight at 4°C with primary
antibodies: phosphorylated p70S6K (Thr389) (1:1000), total p70S6K (1:800)
and 4E-BP1 (1:600) (Cell Signaling Technology), phosphorylated 4E-BP1
(Ser65/Thr70) (1:1000), cyclin D1 (1:500) and c-Myc (1:500) (Santa Cruz
Biotechnology, CA). After incubation with the secondary antibody, the signal
was detected with a luminol enhancer solution (Chemiluminescent Sensitive
Plus HRP, BioFx), and images were acquired with the ChemidocTM XRS+
(Biorad). β-Actin (Sigma-Aldrich) was used as housekeeping protein. Each
experiment was repeated three times.
Polysome analysis
Cells were treated with the combination of EPA-FFA 150 µM, EGCG 175µM
and GS 15 µM or Rapamycin 20 nM for 4 h. Because mTOR regulates mRNAs
actively translated, we chose to analyze the polysome profile at the same time
as when we studied the effect of the combination, or of Rapamycin, on mTOR
targets. Cells were then washed with phosphate-buffered saline containing
100 µg/ml cycloheximide (Sigma-Aldrich), pelleted at 1500 rpm for 5 min at
4°C and lysed in buffer containing 100 µg/ml cycloheximide and ribonuclease
inhibitor 0.04 U/µl (Promega). Lysates were then centrifuged at 14 000 rpm for
10 min at 4°C. Supernatants were loaded onto 11 ml 15–50% sucrose gradient
and centrifuged at 36 000 rpm for 2 h at 4°C. After centrifugation, gradients
were fractionated manually (150 µl fractions) and the optical density of each
fraction was measured with Nanodrop (Thermo Fischer Scientific, Illkirch
Cedex, France).
RNA extraction and quantitative RT-PCR
Total and polysomal RNA were isolated using Trizol reagent according to
the manufacturer’s instructions and reverse transcribed using GoScriptTM
Reverse Transcriptase Kit (Promega, Milan, Italy). Quantitative RT-PCR (qRTPCR) was performed using TaqMan Gene Expression Assays for c-Myc (Hs
00153408-m1) and cyclin D1 (Hs 00154737-m1) or Sybr Select Master mix for
L5, L11, L13, β-actin (Life Technologies). Primer sequences for Sybr Green
assays are reported in Supplementary Table I, available at Carcinogenesis
Online. qRT-PCR was performed on iCycler (Bio-Rad) and data were analyzed
with the 2−ΔΔCt method using β-actin as housekeeping gene, except for mRNA
associated with polysomes. For each sample, three replicates were analyzed.
Statistical analysis
Data were analyzed using Statistics 9.0 Software and Graphpad 5.0 Software
(Graphpad Software, CA). When necessary, values were transformed using
the function Y = log(Y) to stabilize variances. For continuous variables, the
unpaired t-test and one sample t-test were used to evaluate the mean differences between two unmatched groups and a reference value, respectively. Oneway analysis of variance followed by Tukey’s or Dunnett’s post hoc tests were
used for pairwise comparisons. Data are shown as means ± SEM. P values less
than 0.05 were regarded as statistically significant.
Results
Treatment with EPA-FFA, EGCG and GS decreases mTOR activity
in CRC cells
To investigate the effect of EPA-FFA, EGCG and GS on cell viability, we treated HCT116 and SW480 cells with different concentrations of the compounds in combination for 24 h. We found that the
treatment with the combination of EPA-FFA 150 µM, EGCG 175 µM
and GS 15 µM (hereafter named as EPA-FFA+EGCG+GS) caused
a decrease in the number of viable cells compared with the control
in HCT116 (P < 0.01) and SW480 (P < 0.001) cells, respectively
(Figure 1A and B left). In addition, we tested the effect of the combination of EPA-FFA+EGCG+GS in HCT116 and SW480 cells at 48,
72 and 96 h, finding a significant decrease at all the time points tested
compared with the control (P < 0.001 for both HCT116 and SW480).
Importantly, the inhibitory effect on cell viability persisted for 24 h in
the absence of the combination (Supplementary Figure 1, available at
Carcinogenesis Online).
We then evaluated the phosphorylation status of two well-known
mTOR downstream effectors, p70S6K and 4EBP1, in treated cells
at 4 h (Figure 1) and 8 h (Supplementary Figure 2, available at
Carcinogenesis Online). As shown in Figure 1A (right), the treatment
for 4 h on HCT116 cells with EPA-FFA and EGCG used as single
compounds decreased the phosphorylation of p70S6K, whereas GS
increased or did not change the levels of this protein. However, the
phosphorylation of 4EBP1 did not seem to be significantly affected
when all three natural substances were used as single compounds.
Importantly, the treatment with the combination of EPA-FFA 150 µM,
EGCG 175 µM and GS 15 µM at 4 h significantly reduced the levels
of P-p70S6K (P < 0.05) and p-4EBP1 (P < 0.05) compared with the
control, achieving a stronger effect than the one obtained by using
the mTOR inhibitor Rapamycin at 20 nM (P = n.s. control versus
Rapamycin for both P-p70S6K and p-4EBP1). Because of these
effects, we decided to perform all the experiments on p70S6K and
4EBP1 phosphorylation at 4 h.
We also tested the effect of the different combinations of EPA-FFA,
EGCG and GS in SW480 cells at 4 h and we found that the combined
treatment with EPA-FFA 150 µM, EGCG 175 µM and GS 15 µM
resulted in a significant reduction of P-4EBP1 (P < 0.001 control
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L.D’Angelo et al.
Fig. 1. Combined treatment for 4 h with EPA-FFA, EGCG and GS in CRC cells had effects on mTOR pathway. (A) HCT116 cells were treated with EPAFFA (0–150 µM), EGCG (0–175 µM), GS (0–15 µM) and the number of viable cells compared with the control (%) was assessed with MTT assay (ANOVA
P = 0.0016, **P < 0.01 Dunnett’s test, n = 3) (left). mTOR downstream targets P-p70S6K, p70S6K, P-4EBP1, 4EBP1 were detected by western blotting on
cells treated with compounds alone or in combinations or with Rapamycin. Statistical significance was tested only on HCT116 cells treated with EPA-FFA
150 µM, EGCG 175 µM, GS 15 µM or Rapamycin compared with the control (untreated cells) using one-way ANOVA (P = 0.0285 for P-p70S6K on logarithmic
transformed data and P = 0.0347 for P-4EBP1) followed by Tukey’s test, n = 4 (right). (B) SW480 cells were treated with EPA-FFA, EGCG and GS and the
number of viable cells compared with the control was evaluated with MTT assay (ANOVA P = 0.0004, *P < 0.05; ***P < 0.001 Dunnett’s test, n = 3) (left).
mTOR downstream targets P-p70S6K, p70S6K, P-4EBP1, 4EBP1 were assessed upon treatment. Statistical significance was tested on SW480 cells treated
with EPA-FFA 150 µM, EGCG 175 µM, GS 15 µM or Rapamycin 20 nM compared with the control (untreated cells) using one-way ANOVA (P = 0.0294 for
P-p70S6K on logarithmic transformed data and P = 0.0008 for P-4EBP1) followed by Tukey’s test, n = 4 (right).
versus combination) comparable to Rapamycin (P < 0.01 control versus Rapamycin) and a decrease of P-p70S6K compared with control,
even though this latter did not reach statistical significance (Figure 1B
right).
In addition, we tested whether the inhibitory effect of EPAFFA+EGCG+GS at 4 h on mTOR targets persisted after the removal
of the treatment. Both in HCT116 and SW480 cells, the modulation of p70S6K and 4EBP1 phosphorylation was lost 20 h after the
removal of the combination (Supplementary Figure 3, available at
Carcinogenesis Online).
These results indicate that the combined treatment with EPA-FFA
150 µM, EGCG 175 µM and GS 15 µM at low toxicity concentrations
for 4 h inhibits mTOR signaling in HCT116 and SW480 cell lines, as
shown by inhibition of its downstream effectors.
EPA-FFA, EGCG and GS treatment reduces translation of ribosomal
proteins, c-Myc and cyclin D1 in CRC cells
Because mTOR regulates the initiation of translation, we next examined the effects of EPA-FFA+EGCG+GS for 4 h on translational
regulation through the analysis of polysome profiles in HCT116
(Figure 2A). The sum of absorbance at 260 nm of polysomal fractions was lower in the treated sample compared with the control,
but this difference was not statistically significant. However, when
we evaluated the levels of selected mRNAs known to be translationally regulated by mTOR, such as those encoding for ribosomal
proteins (L5, L11, L13), c-Myc and cyclin D1, we found a significant decrease of L5 in total RNA (P = 0.03), L11 (P = 0.03) and
cyclin D1 (P = 0.005) in RNA associated with polysomal fractions
(Figure 2B) (33,34). In addition, the same analysis was performed in
HCT116 cells treated with Rapamycin 20 nM for 4 h. No statistically
significant difference was observed between the polysomal profiles
of control and Rapamycin-treated cells (Supplementary Figure 4A,
available at Carcinogenesis Online). We then evaluated total and polysomal levels of selected mRNAs encoding for ribosomal proteins in
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Rapamycin-treated cells finding a significant decrease only for L13
in total RNA (P = 0.03 control versus Rapamycin, Supplementary
Figure 4B, available at Carcinogenesis Online). To identify the
mRNAs that were subjected to transcriptional and/or translational
regulation after the treatment with EPA-FFA+EGCG+GS, the relative
translatability of mRNA was determined by normalizing the change
in abundance in polysomal RNA to the change in abundance in total
RNA for each mRNA (Poly/Total) (Table I). As reported previously
(35), mRNAs with a Poly/Total ratio of 0.65 or lower were considered
as translationally decreased whereas those mRNAs with a Poly/Total
ratio equal or higher to 1.5 were considered translationally increased.
Our data suggest that the treatment with EPA-FFA+EGCG+GS significantly decreased the total mRNA abundance of L5 and reduced the
translation of L11, L13, c-Myc and cyclin D1.
We then investigated whether the observed translational decrease of
c-Myc and cyclin D1 mRNA might affect their protein levels. Compared
with the control, in treated HCT116 cells, we found suppression of c-Myc
(P < 0.05) and a decrease of cyclin D1 that, however, did not reach statistical significance. Notably, the treatment with Rapamycin did not affect
the level of c-Myc and cyclin D1 compared with the control (P = n.s.),
(Figure 3A). In addition, we assessed the effect of EPA-FFA+EGCG+GS
or Rapamycin on c-Myc and cyclin D1 proteins in SW480 cells, without
finding any difference from the control (P = n.s.) (Figure 3B).
EPA-FFA, EGCG and GS treatment inhibits cell proliferation and
increases apoptosis in CRC cells
To understand whether the reduction of c-Myc and cyclin D1 could
cause alterations in cell proliferation, we examined the effect of EPAFFA+EGCG+GS treatment for 24 h on the clonogenic capability of
HCT116 and SW480 cells (Figure 4A). We found a significant reduction of the number of colonies in HCT116 and SW480 cells treated
with the combination (P < 0.001 and P < 0.01, respectively). No significant reduction in the number of colonies was obtained by treating
cells with Rapamycin 20 nM for 24 h.
Effect of EPA-FFA, EGCG and GS on mTOR signaling in CRC
Fig. 2. Effect of EPA-FFA, EGCG and GS on mRNA translation in HCT116 cells. (A) Polysomal profiles on control and treated cells are shown. Statistical
significance was assessed using unpaired t-test (P = n.s., n = 2) (B) mRNA levels of L5, L11, L13, c-Myc and cyclin D1 were assessed by qRT-PCR both in total
RNA and in polysomal associated RNA. Statistical significance was assessed using one sample t-test, n = 2, using 100 (control) as the reference value.
Table I. Relative translatability in EPA-FFA+EGCG+GS-treated cells: fold
change in polysomal RNA/fold change in total RNA
Gene
Poly/total
L5 (60S ribosomal protein L5, RPL5)
L11 (60S ribosomal protein L11, RPL11)
L13 (60S ribosomal protein L13, RPL13)
c-Myc
cyclin D1
1.1
0.4
0.57
0.25
0.43
In addition, we performed a cell cycle analysis in both cell lines after
treatment with the combined EPA-FFA+EGCG+GS mixture or with
Rapamycin 20 nM for 24 h. As shown in Figure 4B, in HCT116, the
combined mixture significantly increased cells in the G0G1 phase and
reduced cells in the S and G2M phases (P < 0.01 control versus combination), whereas Rapamycin did not change the distribution of cells
in the different cell cycle phases (P = n.s. control versus Rapamycin).
Conversely, the treatment with either the combination or Rapamycin
did not cause alteration of the cell cycle in SW480 cells (P = n.s.).
We then evaluated whether treatment with EPA-FFA+EGCG+GS
affects apoptosis, and we found a significant increase in the apoptotic index both in HCT116 (P < 0.05 control versus combination)
(Figure 4C) and SW480 cells (P < 0.001 control versus combination). Furthermore, we assessed apoptosis on HCT116 and SW480
cells treated with Rapamycin, finding no differences compared with
untreated control cells (P = n.s.).
Our data indicate that this low cytotoxic combination has antiproliferative and proapoptotic effects in CRC cell lines. In particular,
EPA-FFA+EGCG+GS treatment was more effective than Rapamycin
in the tested CRC cell lines and this is in line with the effects observed
at the molecular levels described above.
Discussion
In this study, we have shown that the treatment with a combination of
three natural compounds, EPA-FFA, EGCG and GS, at low cytotoxic
concentrations, decreased mTOR activity in CRC cell lines leading to
a reduced translation of the c-Myc and cyclin D1 cancer genes. These
effects were stronger than those obtained with the selective mTOR
inhibitor Rapamycin. In addition, the EPA-FFA, EGCG and GS treatment affected proliferation with a block of cells in G0G1 phase and
increased apoptosis.
The mTOR pathway is a central regulator of many cellular
events, such as proliferation, growth, metabolism and survival and
is frequently activated in CRC, due to a gain of function mutation
of the PI3K catalytic subunit gene (36–38). With its biological
role and its constitutive activation in CRC, mTOR has become a
key target of cancer research and drug development. Several studies have shown that pharmacological inhibitors of mTOR reduce
the growth of CRC cells in vitro and in vivo, but clinical studies
highlight drawbacks related to toxicity that could lead to treatment failure (11,13,14). Moreover, mutations in the PI3K catalytic subunit that confer resistance to some inhibitors have been
identified (39).
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Fig. 3. Effect of EPA-FFA, EGCG and GS on c-Myc and cyclin D1. c-Myc and cyclin D1 proteins levels were assessed in cell extracts treated with EPAFFA+EGCG+GS or Rapamycin from (A) HCT116 or (B) SW480 cells. Analyses were performed on logarithmic transformed data for HCT116. After the
ANOVA global test (P = 0.0190, n = 4, HCT116; P = n.s., n = 4), Tukey’s post hoc test was used for pairwise comparisons.
Fig. 4. Effect of EPA-FFA, EGCG and GS on cell proliferation and apoptosis in HCT116 and SW480 cells. (A) Clonogenic assay on HCT116 and SW480 cells
treated with EPA-FFA+EGCG+GS or Rapamycin (ANOVA P = 0.0012 and P = 0.0017 for HCT116 and SW480, respectively; Tukey’s test was applied as post
hoc test, n = 3). (B) Cell cycle analysis (ANOVA P = 0.0059 for HCT116 and P = n.s. for SW480, n = 3, Dunnett’s test) (C) Tunnel assay: representative pictures
and quantification. Statistical significance was tested using one-way ANOVA (P = 0.0447 for HCT116 and P = 0.0011 for SW480, followed by Dunnett’s test for
comparison with the control cells, n = 2). Ctrl = control; Comb = Combination; Rapa = Rapamycin.
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Effect of EPA-FFA, EGCG and GS on mTOR signaling in CRC
Phytochemicals have been widely used in chemoprevention. They
could overcome off-target toxicity and drug resistance when used as
anticancer agents and might represent a cheaper strategy compared
with the development of synthetic drugs (40,41). However, to obtain
antineoplastic effects, dosages are usually increased. A recent study
showed that EGCG in combination with curcumin inhibits the PI3K/
mTOR pathway in leiomyosarcoma cells, although the concentrations
used in that study were higher than ours (42,43). It had also been
reported that EPA downregulates PI3K/mTOR signaling in CRC cells
in combination with lycopene (30). In the search for new approaches
to minimize toxic effects due to the use of single agents at high concentrations, we tested for the first time a low cytotoxic combination of
EPA-FFA 150 µM, EGCG 175 µM and GS 15 µM on mTOR pathway
in CRC cells. Using a combination of natural agents at lower concentrations might be relevant for future clinical approaches. Interestingly,
by combining three bioactive compounds, we found a complete abrogation of p70S6K and 4EBP1 phosphorylation in HCT116 cells at
4 h that we did not obtain using each agent individually. However,
because EGCG and EPA-FFA had moderate effects on p70S6K and
4EBP1, whereas GS had more limited effects, our results suggest
a synergic inhibition obtained with the combination. Importantly,
the combined treatment for 4 h also reduced the phosphorylation of
p70S6K and 4EBP1 in SW480 cells. These effects were stronger for
HCT116, and comparable for SW480, with respect to those obtained
with the pharmacological inhibition of mTOR by Rapamycin. Even
though the effect of the combined treatment on mTOR targets at 4 h
was lost after combination removal for 20 h, the decrease in the number of viable cells persisted even after media shift.
It is known that phosphorylated p70S6K further phosphorylates
the 40S ribosomal protein S6, leading to enhancement of the translation of mRNAs encoding for ribosomal proteins and translation
factors (44). In this study we found a consistent reduction of phosphorylated p70S6K and 4EBP1 in association with a decrease of
polysomal actively translated mRNA in HCT116 cells treated with
EPA-FFA+EGCG+GS. Indeed, we observed an increase of free ribosomal subunits and a decrease of RNA in polysomal fractions in the
treated sample. In particular, the treatment decreased the total amount
of ribosomal protein L5 together with the translatability of L11 and
L13. Moreover, phosphorylated 4EBP1 promotes dissociation of
4EBP1 from eIF4E, thereby increasing the translation of cyclin D1
and c-Myc mRNAs (4,6). According to this hypothesis, our results
show a reduction in the translatability of cyclin D1 and c-Myc mRNA
in association with a decrease in protein expression in HCT116treated cells with respect to controls. These data suggest that EPAFFA+EGCG+GS treatment has a bigger effect on translation than
on transcription in the subset of genes analyzed. A previous study
that evaluated the effect of EPA alone in an AOM rodent model suggested that fish oil mainly causes transcriptional alterations (45). We
can speculate that EPA-FFA used alone act mainly on transcriptional
regulation, whereas in combination with EGCG and GS they could
cause translational alterations.
Furthermore, the decrease in cyclin D1 and c-Myc induces a significant reduction of clonogenic potential with a block of HCT116
cells in G0G1 phase and induction of apoptosis in treated cells. In
addition, a reduction of c-Myc and cyclin D1 proteins was observed
in SW480-treated cells with a concomitant decrease of clonogenic
potential. However, SW480 did not undergo a block of cell cycle in
G0G1 phase. This lack of block could be due to the mutation of the
cell cycle regulator p53 in SW480 (20). Moreover, the treatment with
Rapamycin had no effect on c-Myc, cyclin D1, clonogenic potential,
cell cycle and apoptosis. The failure of Rapamycin to inhibit cell proliferation could be due to the administration as a single agent at a low
concentration as previously reported (46).
Overall, our findings suggest that in CRC the combination of EPAFFA, EGCG and GS could be used at a concentration with lower
toxicity than mTOR inhibitors. Because of the central role of mTOR
signaling in CRC, the combined treatment with EPA-FFA, EGCG
and GS may be considered a promising anti-CRC therapy and further
investigations are thus warranted.
Supplementary material
Supplementary Figures 1–4 and Table I can be found at http://carcin.
oxfordjournals.org/
Funding
This work was supported by the Italian Association for Cancer
Research (IG10216 to L.R., Fellowship “David Raffaelli” 13837 to
An.Pr.) and the European Community’s Seventh Framework Program
(Pathway-27 311876 to L.R.).
Acknowledgements
We thank Pasquale Chieco for his support on statistical analyses.
Conflict of Interest Statement: L.R. has received an unrestricted scientific grant
from SLA Pharma AG.
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Received February 4, 2014; revised July 18, 2014; accepted August 6, 2014