Combined dual effect of modulation of human neutrophils’ oxidative burst and inhibition of colon cancer cells proliferation by hydroxycinnamic acid derivatives

Article abstract of DOI:10.1016/j.bmc.2016.05.065

Colon cancer is one of the most incident cancers in the Western World. While both genetic and epigenetic factors may contribute to the development of colon cancer, it is known that chronic inflammation associated to excessive production of reactive oxygen and nitrogen species by phagocytes may ultimately initiate the multistep process of colon cancer development. Phenolic compounds, which reveal antioxidant and antiproliferative activities in colon cancer cells, can be a good approach to surpass this problem. In this work, hydroxycinnamic amides and the respective acid precursors were tested in vitro for their capacity to modulate human neutrophils’ oxidative burst and simultaneously to inhibit growth of colon cancer cells. A phenolic amide derivative, caffeic acid hexylamide (CAHA) (4) was found to be the most active compound in both assays, inhibiting human neutrophils’ oxidative burst, restraining the inflammatory process, inhibiting growth of colon cancer cells and triggering mitochondrial dysfunction that leads cancer cells to apoptosis. Altogether, these achievements can contribute to the understanding of the relationship between antioxidant and anticancer activities and based on the structure–activity relationships (SAR) established can be the starting point to find more effective phenolic compounds as anticancer agents.

Full text of DOI:10.1016/j.bmc.2016.05.065

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Combined dual effect of modulation of human neutrophils’ oxidative
burst and inhibition of colon cancer cells proliferation by
hydroxycinnamic acid derivatives
a,b,
⇑
a
b,c,d
, João C. Encarnação c,d,
Elisiário J. Tavares-da-Silva
, Carla L. Varela , Ana S. Pires
b,c,d
b,c,d
b,e
f
f
Ana M. Abrantes
, Maria F. Botelho
Eduarda Fernandes , Fernanda M. F. Roleira
Pharmaceutical Chemistry Group, Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
CNC.IBILI, University of Coimbra, Portugal
Biophysics Institute, IBILI-Faculty of Medicine, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
CIMAGO, FMUC-Faculty of Medicine, University of Coimbra, Portugal
, Rui A. Carvalho , Carina Proença , Marisa Freitas ,
f
a,b,
⇑
a
b
c
d
e
f
Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Portugal
UCIBIO, REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Colon cancer is one of the most incident cancers in the Western World. While both genetic and epigenetic
factors may contribute to the development of colon cancer, it is known that chronic inflammation
associated to excessive production of reactive oxygen and nitrogen species by phagocytes may ultimately
initiate the multistep process of colon cancer development. Phenolic compounds, which reveal antioxi-
dant and antiproliferative activities in colon cancer cells, can be a good approach to surpass this problem.
In this work, hydroxycinnamic amides and the respective acid precursors were tested in vitro for their
capacity to modulate human neutrophils’ oxidative burst and simultaneously to inhibit growth of colon
cancer cells. A phenolic amide derivative, caffeic acid hexylamide (CAHA) (4) was found to be the most
active compound in both assays, inhibiting human neutrophils’ oxidative burst, restraining the inflamma-
tory process, inhibiting growth of colon cancer cells and triggering mitochondrial dysfunction that leads
cancer cells to apoptosis. Altogether, these achievements can contribute to the understanding of the
relationship between antioxidant and anticancer activities and based on the structure–activity
relationships (SAR) established can be the starting point to find more effective phenolic compounds as
anticancer agents.
Received 30 March 2016
Revised 26 May 2016
Accepted 29 May 2016
Available online xxxx
Keywords:
Hydroxycinnamic acid derivatives
Synthesis
Human neutrophils
Oxidative burst
Colon cancer cells
Antiproliferative
Ó 2016 Elsevier Ltd. All rights reserved.
1
. Introduction
Colon cancer is one of the most incident cancers in the Western
system to fight opportunistic infections as well as cancer cells.
Taking this knowledge into account, the search for new therapeutic
options for the chemoprevention and/or the treatment of colon
cancer is a matter of interest.
1
World and nowadays it is also spreading into Asian countries,
probably due to the adoption of Western diet. In spite of relevant
improvement in survival over the past decade, a significant num-
ber of patients relapse after surgical and conventional therapies
and do not respond to metastatic cancer treatment.2 On the other
hand, it is well known that cytotoxic drugs used in chemotherapy
protocols have several adverse effects namely the weakening of
patients’ immune system, also destroying peripheral blood
mononuclear cells, which are critical components of the immune
In addition, it has been assumed that excessive production of
reactive oxygen (ROS) and nitrogen (RNS) species by phagocytes,
namely neutrophils, may lead to chronic inflammation and
ultimately initiate the multistage process of colon cancer develop-
,3
4
,5
ment. Further, it has been described that ROS are involved, not
only in the initiation of the carcinogenesis process, but also in cancer
6
,7
promotion and progression. In particular, the inflammatory bowel
disease, namely the ulcerative colitis, has been linked to an
increased risk of colorectal cancer being the oxidative reactions an
8
,9
important part of the inflammatory response.
⇑
Corresponding authors. Tel.: +351 239 488 400; fax: +351 239 488 503.
E-mail addresses: etavares@ff.uc.pt (E.J. Tavares-da-Silva), froleira@ff.uc.pt
A large amount of scientific evidence reported in the literature
suggests that phenolic compounds present in diet or consumed
(
F.M.F. Roleira).
http://dx.doi.org/10.1016/j.bmc.2016.05.065
968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
0
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2
E. J. Tavares-da-Silva et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
alone can act as chemopreventive and/or chemotherapeutic
million (ppm) downfield from tetramethylsilane as an internal
standard. All J values are given in Hz. Caffeic (1) and ferulic (2)
acids were purchased from Sigma–Aldrich (Schnelldorf, Germany)
and 3,4,5-trihydroxycinnamic acid (3) to Apin Chemicals Limited
(Abingdon, Oxon, United Kingdom). Reagents and solvents were
used as obtained from suppliers without further purification.
1
0
agents. Among these, hydroxycinnamic acid derivatives consti-
tute a major group of antioxidant compounds with inhibitory
activity in proliferation of several cancer cell lines. Particularly,
caffeic acid (CA) showed a protective effect on paclitaxel induced
antiproliferation and apoptosis of lung cancer cells1 however it
1
1
2,13
also presented antiproliferative effects against colon,
fibrosar-
1
8
4
15,16
16
15,17
coma,
breast,
cervical,
liver,
and leukemia cancer
cells. Ferulic acid (FA) is described as an antiproliferative agent
against breast and liver cancer cells and revealed to delay the cell
cycle progression, specifically in the S and G2/M phases of colon
2.2. General procedure to obtain the cinnamic acid hexylamides
4, 5 and 6
1
15
To synthesize the amides CAHA (4), FAHA (5) and OHCAHA (6),
cinnamic acids CA (1), FA (2) and OHCA (3), respectively (Scheme 1)
were dissolved in dimethylformamide (DMF) and triethylamine
(TEA). The solution was then cooled in an ice-water bath and hexy-
lamine was added, followed by a solution of (benzotriazol-1-yloxy)
tris(dimethylamino)phosphonium hexafluorophosphate (BOP) in
dichloromethane. The mixture was stirred at 0 °C for 30 min and
then at room temperature for specific periods of time. Dichloro-
methane was removed under reduced pressure and the remaining
solution was diluted with water (100 mL). The mixture was then
extracted with ethyl acetate (2 ꢀ 100 mL). The extracts were
1
9
cancer cells.
3,4,5-Trihydroxycinnamic acid (OHCA) showed
antiproliferative activities in cervical, colon, prostate and oral cav-
2
0
ity cancer cell lines. Recently, new lipophilic caffeic and ferulic
acid derivatives were synthesized and their cytotoxicity was com-
pared with that of the parent compounds showing increased cyto-
toxicity towards breast cancer cell lines. These results indicated
that the new compounds inhibited cell proliferation and induced
2
1
cell cycle alterations and cell death in the referred cancer cells.
Based on the above-mentioned considerations, we aimed to find
new and more effective agents suitable for chemoprevention
and/or chemotherapeutic purposes against colon cancer which will
be able to simultaneously modulate human neutrophils’ oxidative
burst, restraining the inflammatory process, and to inhibit growth
of colon cancer cells. For this purpose, n-hexylamide derivatives of
caffeic, ferulic and 3,4,5-tryhydroxycinnamic acids with superior
lipophilicity and consequently with improved ability to cross cell
membranes were synthesized (Scheme 1). Subsequently, they
were screened along with their parent acids, to test their anti-
inflammatory activity against human neutrophils’ oxidative burst
as well as in terms of cytotoxicity, on two colon adenocarcinoma
cell lines with different genetic background and origin localization,
WiDr (rectosigmoid) and C2BBe1 (descending colon). Finally, some
structure–activity considerations were inferred.
washed with 1 N HCl (2 ꢀ 100 mL), water (2 ꢀ 100 mL), NaHCO
5% (3 ꢀ 100 mL) and finally with water (2 ꢀ 100 mL), dried over
anhydrous MgSO filtered and concentrated. The obtained
3
4
,
residues were purified by column chromatography yielding the
corresponding hexylamides (4, 5 and 6).
2.2.1. N-Hexyl-3-(3,4-dihydroxyphenyl)-2-propenamide (4)
2
2
As described before.
2.2.2. N-Hexyl-3-(4-hydroxy-3-methoxyphenyl)-2-propenamide
(5)
As described before.22
2
.2.3. N-Hexyl-3-(3,4,5-trihydroxyphenyl)-2-propenamide (6)
Compound (250.0 mg, 1.27 mmol); DMF (2.9 mL); TEA
0.18 mL); hexylamine (0.17 mL, 1.27 mmol); BOP (561.7 mg,
3
2
2
. Materials and methods
(
1
0
.27 mmol); CH
2 2
Cl (3 mL). The reaction mixture was stirred at
.1. Chemistry
°C for 30 min and then at room temperature for 5 h 30 min.
The residue obtained after work-up was purified by silica gel
column chromatography (hexane/ethyl acetate) giving the pure
compound 6 in 39% yield. Mp(hexane/ethyl acetate) 93–97 °C. IR
Reactions were controlled by thin layer chromatography (TLC)
using silica gel 60 F254 plates. Column chromatography was
performed using silica gel 60 (0.063–0.200 mm). Melting points
ꢁ1
(
(
(
NaCl plates, CHCl
CAO phenolic alcohol). H NMR (600 MHz, DMSO-d
3
) mmax cm : 3267 (NAH), 1642 (C@O), 1315
(
MPs) were determined on a Reichert Thermopan hot block
1
) d: 0.85
apparatus and were not corrected. IR spectra were recorded on a
6
Jasco 420 FT/IR spectrometer. The 1H NMR and 13C NMR spectra
0
0
0
3
3H, m, –CH ), 1.28 (6H, m, –CH
2
(3 –5 )), 1.41 (2H, m, –CH
2
(2 )),
0
3
.12 (2H, dd, J = 12.9, J = 6.9, –CH
2
(1 )), 6.26 (1H, d, J = 15.6, –CH
were recorded at 600 MHz and 150 MHz, respectively on a Varian
Unity 600. Chemical shifts were recorded in d values in parts per
(a)), 6.47 (2H, s, –CH(2 and 6)), 7.11 (1H, d, J = 15.6, –CH(b)),
O
O
R
R
1
2
R
R
1
2
(
i)
OH
N
H
+
NH
2
R
3
R
3
(
1) R
1
1
1
= R
= OCH
= R = R
2
= OH; R
; R = OH; R
= OH
3
= H
(4) R
(5) R
(6) R
1
1
1
= R
= OCH
= R = R
2
= OH; R
; R
= OH
3
= H
(
2) R
= H
3
2
= OH; R
3
= H
3
2
3
(3) R
2
3
2
3
(
1) caffeic acid (CA)
2) ferulic acid (FA)
3) 3,4,5-trihydroxycinnamic acid (OHCA)
4) caffeic acid hexylamide (CAHA)
5) ferulic acid hexylamide (FAHA)
6) 3,4,5-trihydroxycinnamic acid hexylamide (OHCAHA)
(
(
(
(
(
Scheme 1. Synthesis and structures of hydroxycinnamic acid n-hexylamide derivatives. Reagents and conditions: (i) dimethylformamide (DMF), triethylamine (TEA),
benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), dichloromethane, rt.
(
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E. J. Tavares-da-Silva et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
7
.94 (1H, t, J = 5.7, –NH), 8.15 (1H, s, –OH(4)), 9.03 (2H, s, –OH(3
USA) and grown in Dulbecco’s Modified Eagle’s Medium supple-
mented with 100 M sodium pyruvate (Life Technologies), 5%
heat-inactivated fetal bovine serum and 1% antibiotic/antimycotic
(100 U/mL penicillin and 10 g/mL streptomycin) at 37 °C with
95% air and 5% CO . Unless specified otherwise all cell culture
reagents were [Sigma–Aldrich Co. LLC (St. Louis, USA)].
1
3
and 5)). C NMR (150 MHz, DMSO-d
6
0
) d: 13.9 (CH
3
), 21.9, 26.1,
l
0
0
2
1
9.1, 30.9 (C-2 )–(C-5 ), 38.5 (C-1 ), 106.6 (C-2 and C-6),
18.6 (C-
a), 125.3 (C-1), 135.0 (C-4), 139.1 (C-b), 146.0 (C-3 and
l
C-5), 165.1 (C = O).
2
2
.3. Isolation of human neutrophils
2
.7. Effects of compounds on colon cancer cell protein synthesis
Neutrophils were isolated from blood collected from human
volunteers, following approval by the Ethical Committee of Hospi-
tal Geral de Santo António, CHP, Porto, and all procedures comply
with Helsinki Declaration. The design and execution of the exper-
iment were thoroughly explained to the participants, and informed
consent was obtained for blood collection. The following assays
were carried out using blood from n P 8 individuals. Venous blood
was collected by antecubital venipuncture, from each human
The effect of the compounds on colon cancer cells proliferation
was evaluated using the colorimetric Sulphorhodamine B (SRB)
assay. This assay relies on the uptake of the negatively charged
pink aminoxanthine dye, SRB by basic amino acids in the cells,
giving a measure of protein synthesis which gives information
2
6
about cell proliferation. For each experiment, cells were seeded
in 48 multiwells in a concentration of 50,000 cells/mL. Cells were
then treated with increasing concentrations of the compounds
3
healthy volunteer into vacuum tubes with K EDTA. The isolation
of human neutrophils was performed by the density gradient cen-
(0–200 lM) and after 24, 48, 72 and 96 h of cell incubation SRB
trifugation method as previously reported.23 Tris–glucose (25 mM
assay was performed as described before. Culture medium was
removed, cells were washed twice with phosphate buffered saline
(PBS), and then incubated with a frozen solution of 1% acetic acid
(Panreac) in methanol for 1 h at 4 °C. The fixation solution was
discarded and cells were incubated for 1 h with SRB 0.4%, at room
temperature and kept in the dark. After this period, multiwells
were washed with water and 10 mM TrisꢂNaOH (pH = 10) was
added. The content of each well was transferred to a 96 multiwells
plate and the absorbance was quantified at 540 nm with a
27
Tris, 1.26 mM CaCl
NaCl, and 5.55 mM
the evaluation of neutrophils’ oxidative burst, as previously
2
ꢂ2H
2 4
O, 5.37 mM KCl, 0.81 mM MgSO , 140 mM
-Glucose) was the incubation media used in
D
2
4
recommended. All of the reagents used in isolation of human
neutrophils were [Sigma–Aldrich Co. LLC (St. Louis, USA)].
2
.4. Human neutrophils’ viability
Ò
Cell viability was determined by the trypan blue exclusion
reference filter of 690 nm in a spectrophotometer (Biotek Synergy
assay. Neutrophils were incubated with all the tested compounds
HT). Unless specified otherwise all chemicals were [Sigma–Aldrich
Co. LLC (St. Louis, USA)].
(
at the maximum concentration tested, 200 M) for 1 h at 37 °C.
l
Twenty microliter of neutrophil suspension were added to an equal
volume of trypan blue solution 0.4% (Sigma–Aldrich Co., St. Louis,
USA) in a microtube and gently mixed. After 2 min on ice, neu-
trophil number and viability (viable cells excluding trypan blue)
were counted. Assays were performed in triplicate.
2.8. Effects of CAHA (4) on colon cancer cells viability and
oxidative stress
Subsequent studies were performed with the amide CAHA (4),
given the most promising results obtained with it.
2
.5. Measurement of human neutrophils’ oxidative burst
In order to perform several tests by flow cytometry, colon
cancer human cell lines were treated with different CAHA (4)
concentrations for 48 h. Based on cell proliferation results,
The chemiluminescent probe luminol has been thoroughly
studied and used for monitoring the production of reactive species
C2BBe1 cells were treated with 22 M and 50
l
lM of CAHA (4)
Åꢁ
by neutrophils, namely the superoxide anion radical (O
2
), hydro-
and WiDr cells with 35 M and 50 lM of CAHA (4).
l
Å
gen peroxide (H
O
2 2
), hydroxyl radical (HO ), hypochlorous acid
In order to evaluate the influence of CAHA (4) on cell cycle, pro-
Å
ꢁ 25
(
HOCl), nitric oxide ( NO) and peroxynitrite anion (ONOO ). The
pidium iodide (PI) was used. For this purpose, a cell suspension of
6
measurement of neutrophils’ oxidative burst was undertaken by
chemiluminescence, by monitoring reactive species-induced oxi-
dation of luminol [Fluka Chemie GmbH (Steinheim, Germany)]
using a microplate reader (BiotekÒ Synergy HT). The reaction mix-
1 ꢀ 10 cells for each condition was centrifuged at 1300g for 5 min
and the supernatant discarded. For fixation, 200 lL of 70% ethanol
was added and incubated for 30 min at 4 °C. Then, cells were
washed with PBS and the supernatant discarded. Finally, it was
added 200 lL of PI/RNase (Immunostep) and incubated for
15 min in dark, at room temperature. Detection was performed
6
tures contained neutrophils (2 ꢀ 10 cells/mL) and the following
reagents at the indicated final concentrations (in a final volume
of 250
lL): tested compounds at various concentrations, luminol
with an excitation wavelength of 488 nm.
(
500 M) and phorbol myristate acetate (PMA) [Sigma–Aldrich
l
Cell viability was analyzed using annexin-V/propidium iodide
6
Co. LLC (St. Louis, USA)] (160 nM). Cells were pre-incubated with
luminol and the tested compounds for 5 min before the addition
of PMA, and the measurements were carried out at 37 °C, under
continuous soft shaking. Kinetic readings were initiated immedi-
ately after cell stimulation. Measurements were taken at the peak
of the curve. This peak was observed at around 15 min. Effects are
expressed as the percent inhibition of luminol oxidation. Each
study corresponds to, at least, eight individual experiments,
performed in triplicate in each experiment.
(AV/PI) incorporation assay. For that, 1 ꢀ 10 cells were incubated
during 15 min in binding buffer with 2.5 lL of AV (Kit Immuno-
tech) and 1 lL of PI (Kit Immunotech). Subsequently, cells were
excited at a wavelength of 525 nm for AV and 640 nm for PI. In
order to assess the percentage of viable, early apoptotic, late
4
apoptotic/necrotic and necrotic cells, 10 events were collected.
To evaluate the effect of CAHA (4) on mitochondrial membrane
0
0
potential (
D
w
m
), cells were labeled with 5,5 ,6,6 -tetrachloro-
1,1 ,3,3 -tetraethylbenzimidazolocarbocyanine iodide (JC-1). An
estimate of is given by the ratio between the intensities of
red and green fluorescence. Intracellular ROS production was
0
0
Dw
m
2
.6. Colon cancer cell culture
0
0
analyzed using two probes: 2 ,7 -dichlorodihydrofluorescein diac-
etate (DCFH -DA, Invitrogen) to quantify intracellular peroxides
C2BBe1 [clone of Caco-2] (ATCCÒ CRL-2102 ) and WiDr (ATCCÒ
TM
2
TM
CCL-218 ), two colorectal adenocarcinoma cell lines, were
acquired from American Type Culture Collection (Rockville, MD,
and, dihydroethidium probe (DHE) to quantify intracellular
Åꢁ
O
2
production. Expression of intracellular reduced glutathione
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4
E. J. Tavares-da-Silva et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
(
GSH) was evaluated with mercury orange. Assays were performed
As shown in Table 1, compounds CA (1) and FA (2) were
noticeably less effective than the other tested compounds. Besides,
it was not possible to achieve the IC50 value up to the highest
according to
a
method previously described.28 Results are
presented as mean fluorescence intensity (MFI). Unless specified
otherwise all chemicals were [Sigma–Aldrich Co. LLC (St. Louis,
USA)].
tested concentration (200
clear that compound CAHA (4) was the most effective, presenting
an IC50 = 6.6 ± 0.8 M, followed by FAHA (5) (IC50 = 14 ± 2 M)
lM) of the compound CA (1). It is also
l
l
2
.9. Statistical analysis
and OHCA (3) (IC50 = 19 ± 4), being the effect dependent on the
concentration.
The results of the assays with human neutrophils were analyzed
TM
using GraphPad Prism (version 5.0; GraphPad Software). Results
are expressed as mean ± standard error of the mean (SEM) (from
at least eight individual experiments, performed in triplicate in
each experiment). Cell proliferation results were analyzed and
processed in software OriginPro v. 8.0, in order to determine the
concentration of the compound that inhibits cell proliferation in
3.4. Cytotoxic effect on colon cancer cell lines
The effect of the compounds on colon cancer cells proliferation
was evaluated through the SRB assay, after subjecting the cells to a
range of compound concentrations from 0 to 200 lM for 24, 48, 72
and 96 h of exposure. Generally, as it can be seen in Figure 2, for
both cell lines, as the concentration of the compound increases, cell
proliferation decreases, except for FA (2). Therefore, a dose-depen-
dent response is observed, but not always accompanied by a time-
dependent response (Table 2). In fact, a time-dependent inhibitory
effect was observed following exposure of C2BBe1 cells to OHCA
(3) and OHCAHA (6) compounds (p = 0.003 and p = 0.002,
respectively).
5
0% (IC50), through sigmoid fitting (Boltzman function). Flow
cytometry statistical analysis was accomplished using the software
Ò
Ò
IBM SPSS Statistics, v. 20.0 (IBM Corporation, Armonk, New York,
EUA). Differences between therapeutic conditions or cell lines were
performed by one-factor ANOVA test followed by Bonferroni post
hoc analysis for multiple comparisons. A statistical significance
level of 5% was settled.
The data obtained revealed that the parent compounds FA (2)
and CA (1) (data not shown) do not have any antiproliferative
effect on the colon cancer cells under study, however, the cytotoxic
effect of its amide derivatives is evident. According to Figure 2 and
Table 2, both cell lines revealed to be more sensitive to CAHA (4),
with statistical differences at 48 and 72 h of incubation time for
C2BBe1 cells (p <0.03 and p <0.02, respectively) comparing with
all other compounds. Regarding the effect of OHCA (3), it should
be noted the difference between the two cell lines response,
wherein the IC50 values obtained for C2BBe1 cells were statistically
lower than those obtained for WiDr cells (p <0.015), whose lower
3
3
. Results
.1. Chemistry
The synthesis of the hexylamides 4 to 6 (Scheme 1) was
performed through a single-step reaction involving the cinnamic
acids to with hexylamine, in dimethylformamide and
triethylamine, in the presence of the coupling agent BOP, at room
temperature. This is a specially appropriate procedure to allow
the direct amidation of
desired amides in good yields (from 39% to 70%).
1
3
a,b-unsaturated acids, leading to the
29
IC50 value obtained was 113.7 lM after 72 h. On the other hand,
it is noteworthy that WiDr cells revealed a higher sensitivity to
OHCAHA (6) than C2BBe1 cells, with statistically significant
differences for 72 h of exposure to the compound (p = 0.009).
Given the most promising results obtained with CAHA (4),
subsequent studies were performed with this compound to deepen
the knowledge of the cytotoxic effect.
3
.2. Effect of compounds on human neutrophils’ viability
Cell viability of human neutrophils was maintained over 98%,
after 1 h of exposure to the tested compounds, at the maximum
concentration tested, 200 M.
l
To study CAHA (4) effect on cell cycle of both cell lines, PI/RNase
staining assay was performed (Fig. 3). Cell cycle evaluation
3
.3. Evaluation of compounds on human neutrophils’ oxidative
burst
indicates that higher CAHA (4) concentrations (50
cycle arrest of C2BBe1 cells on G2/M phase, with an increase of
.8% of cells in this phase compared to control (p = 0.013). In
lM) induce cell
The tested compounds exhibited different effects on the
suppression of human neutrophils’ oxidative burst, being the order
of potencies: CAHA (4) > FAHA (5) > OHCA (3) > FA (2) > CA (1)
8
contrast for WiDr cell line, higher CAHA (4) concentrations induce
cell cycle arrest on S phase, with an increase of 9.3% of cells in this
(Fig. 1).
phase compared to control (p = 0.007). Moreover,
apoptotic peak was observed when WiDr cells are exposed to
M of CAHA (4) (p = 0.035).
Given cell proliferation and cell cycle results, it was of great
a pre-G1
100
50 l
interest to study the effect of CAHA (4) on viability and types of
induced cell death of both cell lines. So, for this purpose, the double
7
5
2
5
0
5
0
CA
FA
OHCA
CAHA
FAHA
OHCAHA
Table 1
Inhibition of phorbol myristate acetate (PMA)-induced neutrophils’ oxidative burst by
the compounds under study (IC50
lM, mean ± SEM)
Compound
IC50 (lM)
*
CA (1)
FA (2)
OHCA (3)
CAHA (4)
FAHA (5)
OHCAHA (6)
44% 200
ffi200
lM
Concentration (µM)
Figure 1. Effect of the compounds under study (0–200
oxidative burst stimulated with phorbol myristate acetate (PMA), as measured by
luminol-amplified chemiluminescence. The values are given as the mean ± SEM
19 ± 4
6.6 ± 0.8
14 ± 2
36 ± 3
l
M) on human neutrophils’
*
(
n P 8).
Scavenging effect (mean%) at the highest tested concentration (in superscript).
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E. J. Tavares-da-Silva et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
C2BBe1
WiDr
1
20
00
120
100
80
60
40
20
0
2
4
72h
4h
8h
24h
48h
72h
96h
1
9
6h
8
6
4
2
0
0
0
0
0
0
,8
,8
,8
,8
,8
1,0
1,0
1,0
1,0
1,0
1,2
1,2
1,2
1,2
1,2
1,4
1,6
1,8
M))
2,0
2,0
2,0
2,0
2,0
2,2
2,2
2,2
2,2
2,2
2,4
0,8
1,0
1,0
1,0
1,0
1,0
1,2
1,2
1,2
1,2
1,2
1,4
1,6
1,8
M))
2,0
2,0
2,0
2,0
2,0
2,2
2,2
2,2
2,2
2,2
2,4
log([CAHA]) (log(
µ
log([CAHA]) (log(
µ
2
4h
1
20
00
120
100
80
60
40
20
0
2
4
4h
8h
72h
48h
72h
96h
1
9
6h
8
6
4
2
0
0
0
0
0
0
1,4
1,6
1,8
M))
2,4
0,8
1,4
1,6
1,8
M))
2,4
log([OHCA]) (log(
µ
log([OHCA]) (log(
µ
1
20
00
120
100
80
60
40
20
0
2
4
4h
8h
72h
6h
24h
48h
72h
96h
1
9
8
6
4
2
0
0
0
0
0
0
1,4
1,6
1,8
2,4
0,8
1,4
1,6
1,8
2,4
log([OHCAHA]) (log(
µM))
log([OHCAHA]) (log(µM))
1
20
00
120
100
80
60
40
20
0
1
8
6
4
2
0
0
0
0
0
2
4
7
9
4h
8h
2h
6h
24h
4
7
9
8h
2h
6h
0
1,4
1,6
1,8
M))
2,4
0,8
1,4
1,6
1,8
M))
2,4
log([FA]) (log(
µ
log([FA]) (log(
µ
1
20
00
120
100
80
60
40
20
0
2
4
4h
8h
72h
6h
24h
48h
72h
96h
1
9
8
6
4
2
0
0
0
0
0
0
1,4
1,6
1,8
M))
2,4
0,8
1,4
1,6
1,8
M))
2,4
log([FAHA]) (log(
µ
log([FAHA]) (log(
µ
Figure 2. Effect of compounds ferulic acid (FA) (2), 3,4,5-trihydroxycinnamic acid (OHCA) (3), caffeic acid hexylamide (CAHA) (4), ferulic acid hexylamide (FAHA) (5) and
3
4
,4,5-trihydroxycinnamic acid hexylamide (OHCAHA) (6) on C2BBe1 and WiDr cells proliferation. Cell proliferation was evaluated by colorimetric Sulforhodamine B assay
8 h after cells treatment. Dose–response curves were obtained after a sigmoid fitting adjust (r > 0.9) that was performed in order to calculate the concentration needed to
2
inhibit cell proliferation in 50% (IC50 values).
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6
E. J. Tavares-da-Silva et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 2
Half maximal inhibitory concentrations (IC50 values) obtained for the two colorectal cancer cell lines after incubation with compounds ferulic acid (FA) (2), 3,4,5-
trihydroxycinnamic acid (OHCA) (3), caffeic acid hexylamide (CAHA) (4), ferulic acid hexylamide (FAHA) (5) and 3,4,5-trihydroxycinnamic acid hexylamide (OHCAHA) (6) for 24,
4
8, 72 and 96 h
Cell line
Incubation time (h)
24
CAHA (4)
OHCA (3)
IC50 M)
OHCAHA (6)
IC50 M)
FA (2)
IC50
FAHA (5)
IC50 M)
IC50
(lM)
(
l
(l
(
lM)
(l
C2BBe1
WiDr
37.3
22.0
23.4
22.6
131.2
87.9
58.9
39.1
114.8
114.7
99.8
>200
>200
>200
>200
132.3
122.6
89.5
4
7
9
8
2
6
50.9
60.6
24
56.1
34.6
35.5
30.8
>200
>200
113.7
136.4
71.4
82.4
37.7
31.7
>200
>200
>200
>200
84.9
91.0
77.4
83.3
4
7
9
8
2
6
staining with AV and PI was performed. As shown in Figure 4,
increasing concentrations of CAHA (4) induced a decrease on cell
viability in both colorectal cancer cell lines under study. WiDr cells
revealed to be the most sensitive to CAHA (4) with a decrease on
cell viability of 32.8% and 43.9% compared to control, after
2.63 ± 0.25 comparing to control (p <0.001). Regarding intracellular
production of peroxides, a slight increase was observed in WiDr
cells with increasing CAHA (4) concentrations. The production of
the antioxidant peptide GSH was also influenced by the presence
of CAHA (4). With 50
lM it was observed an increase of
incubation with 35
respectively. In C2BBe1 cells it was observed a significant decrease
of 11.4% on cell viability after incubation with 22 M (IC50) and a
significant decrease of 14.7% with 50 M of CAHA (4) (p = 0.006
lM (IC50) and 50 lM of CAHA (4) (p <0.001),
1.47 ± 0.08 for C2BBe1 cells (p = 0.004).
l
4
. Discussion
l
and p = 0.003, respectively) comparing to control. The decrease of
WiDr cells viability was accompanied by an increase of different
types of cell death, mainly early apoptosis, however with no
statistical significance. This result corroborates the presence of a
pre-G1 apoptotic peak. In C2BBe1 cells, an increase of early
apoptosis was observed with 50 lM of CAHA (4) (p = 0.033).
Apoptosis can be closely related to mitochondrial dysfunction,
which is associated with the decrease of mitochondrial membrane
It is well known that inflammation is intrinsically involved in the
promotion and progression of almost all cancers. In fact, the link
between inflammation and cancers, rather than a recent concern,
was noticed ꢃ150 years ago. The involvement of the inflammatory
process in the development of cancers might be a process driven
3
0
by inflammatory cells. At the very early stage of inflammation,
neutrophils are the first cells to migrate to the inflammatory sites
and defend the organism by the production of an array of reactive
pro-oxidant species. As such, it was important to understand if
the compounds under study were able to modulate human neu-
trophils’ oxidative burst at non-toxic concentrations. First of all
we used the trypan blue method in order to study the toxicity of
the compounds under study. Our results showed that none of the
tested compounds affect neutrophils viability at the maximum con-
potential (
When both cell lines are treated with 50
ing aggregates/monomers ratio, there is a significant increase,
D
w
m
). CAHA (4) also interferes with
m
Dw (Fig. 5-A).
l
M of CAHA (4), concern-
1
.27 ± 0.04 times, relative to control (p <0.001 for WiDr cells).
3
.5. Oxidative stress on colorectal cancer cells
centration tested (200 lM). In order to mimic the inflammatory
In order to understand whether CAHA (4) influences oxidative
process, we used PMA that activates protein kinase C, which results
Åꢁ
environment of colorectal cancer cells, intracellular production of
in NADPH oxidase activation with the production of O
2
and other
Åꢁ
3
1
O
2
(Fig. 5-B), peroxides (Fig. 5-C) and GSH (Fig. 5-D) was assessed
reactive species through a cascade reaction. Luminol was the
by flow cytometry.
probe used to detect the neutrophils’ oxidative burst since it is able
Åꢁ
2
Åꢁ
CAHA (4) provoked a statistical significant increase of O
to react unspecifically with several reactive species as O
HO , HOCl, nitric oxide ( NO) and ONOO .
2
, H
2
O
2
,
Å
Å
ꢁ 25
production in a dose-dependent manner for both cells lines. In
All the tested
WiDr cells, 50
lM
of CAHA (4) induced an increment of
compounds demonstrated ability to decrease human neutrophils’
C2BBe1
WiDr
8
6
4
2
0
0
0
0
0
80
Control
Control
*
22 µM
0 µM
3
5 µM
0 µM
60
40
20
0
5
5
*
*
*
*
*
*
*
*
*
*
Ap Peak
G0/G1
S
G2/M
Ap Peak
G0/G1
S
G2/M
Figure 3. Cell cycle evaluation after incubation of C2BBe1 and WiDr cells with caffeic acid hexylamide (CAHA) (4) for 48 h. Cell cycle was evaluated by flow cytometry, using
propidium iodide staining. Data represent mean ± SE of cells at each phase of cell cycle (Ap Peak, G0/G1, S and G2/M) of at least three independent experiments per cell line.
Statistically significant differences are shown with *p <0.05, p <0.01.
**
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E. J. Tavares-da-Silva et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
C2BBe1
WiDr
1
00
100
Control
Control
8
6
4
2
0
0
0
0
0
22 µM
50 µM
80
60
40
20
0
35 µM
*
*
**
50 µM
*
**
*
**
*
V
EA
LA/N
N
V
EA
LA/N
N
Figure 4. Cell viability analysis after incubation of C2BBe1 and WiDr cells with caffeic acid hexylamide (CAHA) (4) for 48 h. Cell viability and death was assessed by flow
cytometry using dual-staining with annexin-V/propidium iodide. Results are expressed as the mean ± SE of viable (V), in early apoptosis (EA), in late apoptosis and/or necrosis
LA/N) and necrotic cells (N) of at least four independent experiments in duplicate. Statistically significant differences are shown with *p <0.05, p <0.01 and p <0.001.
**
***
(
A
B
1
1
0
0
.5
.0
.5
.0
4.0
***
*
*
3
2
1
.0
.0
.0
***
*
***
*
**
**
0.0
22 µM 50 µM
35 µM 50 µM
22 µM 50 µM
35 µM 50 µM
C
1
1
0
0
.5
.0
.5
.0
D
2.5
**
*
2
1
1
0
.0
**
.5
.0
.5
0.0
22 µM 50 µM
35 µM 50 µM
22 µM 50 µM
35 µM 50 µM
C2BBe1
WiDr
m
Figure 5. Analysis of mitochondrial membrane potential (Dw ) (A) and intracellular production of anion radical superoxide (B), peroxides (C) and reduced glutathione (GSH)
(
D), after incubation of C2BBe1 and WiDr cells with caffeic acid hexylamide (CAHA) (4) for 48 h. These parameters were assessed by flow cytometry. Results are expressed as
*
**
***
the mean ± SE relative to control, of at least three independent experiments in duplicate. Statistically significant differences are shown with p <0.05, p <0.01 and p <0.001.
oxidative burst, being the most effective the amide derivative CAHA
4). Interestingly, as discussed below, this was also the compound
be involved affecting the final effectiveness of carboxylic acid 3
and its amide 6. The substitution of a hydroxyl group of the CAHA
(4) by a methoxyl group, as in FAHA (5), or even an introduction
of another hydroxyl group, as in OHCAHA (6), did not shown to be
advantageous to the modulatory effect of human neutrophils’
oxidative burst. The parent carboxylic acids CA (1) and FA (2) were
undoubtedly the less effective in inhibiting production of reactive
(
that shown to be the most cytotoxic against the colon cancer cell
lines. Amide derivatives CAHA (4) and FAHA (5) were more effective
than the correspondent parent carboxylic acids CA (1) and FA (2)
but, on the contrary, the amide derivative OHCAHA (6) is less effec-
tive than the parent carboxylic acid OHCA (3). We speculate that the
superior lipophilicity of the amides 4 and 5 allows its entrance in the
cell favoring its inhibitory effect on neutrophils’ oxidative burst.
The same argument cannot be used for the amide 6. In this case,
other forms of cell penetration rather than passive diffusion must
species by neutrophils. The inhibitory activity of the CA (1) and FA
Åꢁ
(2) on PMA-induced O
2
generation in human neutrophils was
3
2
already reported. Interestingly, the concentration of 100
CA (1) inhibited 84% and FA (2) had no effect on PMA-induced O
l
M of
Åꢁ
2
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8
E. J. Tavares-da-Silva et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
generation. These results are in accordance with our findings. The
less activity of CA (1) in our system can be related to the selectivity
of the probe, as luminol detect several reactive species and in the
apoptosis.36 In a previous study, CAHA (4) compound proved to
increase P53 levels in breast cancer cells. Thus, in WiDr cells, a
possible mutant P53 increment may led to cell cycle arrest in
S phase, since G1 checkpoint is not activated.
2
1
3
2
Åꢁ
2
reported work the authors only studied the O . It is important
to stress that, to the best of our knowledge, there are no reports in
literature about the effect of the synthesized amide derivatives on
human neutrophils.
A cytotoxic effect induced by CAHA (4) was also observed in a
dose-dependent manner, with a decrease on cell viability followed
by an increase of early apoptotic cell population in both cell lines.
This fact corroborates the presence of the apoptotic peak in WiDr
cells. Moreover, the increment of aggregates/monomers ratio indi-
cates a decrease of mitochondrial membrane potential, showing
that CAHA (4) induces mitochondrial dysfunction. A reduction of
mitochondrial membrane potential is an important event in the
Concerning the cytotoxicity in colon cancer cells, it was possible
to infer that some of the studied compounds present effective
antiproliferative activity against C2BBe1 and WiDr cells. As
observed in the inhibition of human neutrophils’ oxidative burst,
the amide CAHA (4) was the most active in both colon cancer cells
and amides CAHA (4), FAHA (5) and OHCAHA (6) (in WiDr cells)
were more cytotoxic than the correspondent parent carboxylic
acids CA (1) (data not shown), FA (2) and OHCA (3) towards the
referred cell lines. Actually, the parent carboxylic acids CA (1)
and FA (2) did not demonstrate any cytotoxic activity against the
studied colon cancer cells. This was also observed in another study
3
7
activation of apoptosis intrinsic pathway.
Interestingly, the
cytotoxic effect was most evident in WiDr cells, allowing us to infer
that P53 may play an important role in the apoptotic mechanism
induced by CAHA (4).
Several studies demonstrated recently the anti-tumor effect of
3
8
39
several phenolic compounds, like quercetin,
resveratrol,
2
1
40
13,17,21,41
from the authors, in which these compounds were tested in a
breast cancer cell line (MCF-7). The explanation for this may lie
in the increased lipophilicity of the amides, conferred by the
additional alkyl chain, when compared to that of the parent acids,
which is expected to favor the intracellular accumulation of the
compounds due to their ease of crossing cell membranes. Regard-
ing the parent carboxylic acid OHCA (3), and unlike the other acids,
in C2BBe1 cells, after 48, 72 and 96 h, it demonstrates a better
antiproliferative activity than the correspondent amide OHCAHA
curcumin
and caffeate derivatives.
Although several
mechanisms underlying their anticancer effect have been pro-
posed, a common denominator exists, their anti- and prooxidant
2
1,42,43
activity.
Regarding hydroxycinnamic acid derivatives, it is
of paramount importance taking into account that their antioxi-
dant or prooxidant nature depends on the concentration used.44
In order to understand the anti-cancer mechanism of CAHA (4),
Åꢁ
intracellular production of O
2
, peroxides and GSH was evaluated.
Data obtained revealed an increase of intracellular production of
peroxides that is accompanied by an increase of GSH production.
(6). Again, a similar result was observed for this compound in
the inhibition assay of human neutrophils’ oxidative burst. In this
case, the lower lipophilicity of the acid does not justify the
observed result and, therefore, other factors must be involved,
probably the different genetic background and protein profile of
both cell lines. In fact, P53-null CaCo-2 cells, cell line from which
C2BBe1 cells are cloned, do not express endogenous ABCB1 trans-
GSH detoxifies any H
reactive peroxides to glutathione,
2 2
O formed by transferring the energy of the
4
5
reason why significant
differences in peroxides may not be seen. In turn, intracellular
Åꢁ
O
2
analysis after CAHA (4) treatment revealed an increment of
its production in a dose-dependent manner, reaching almost 3-fold
higher levels than control in WiDr cells. Given these results, it
33
Åꢁ
porter (known to be a multidrug efflux transporter), in contrast
appears that O
2
could be the main ROS implicated in cell death
3
4
Åꢁ
with WiDr cells. This may be related with the observed inhibitory
effect of OHCA (3).
mechanism of CAHA (4). The most pronounced production of O
2
observed in WiDr cells can be related with the presence of P53
protein, which is absent in C2BBe1 cells. Budanov et al. described
the direct correlation between P53 overexpression and ROS
In relation to other structure–activity relationships (SAR), the
presence of a catechol group, as in CAHA (4), seems to be the best
structural feature for achieving effective antiproliferative activity
in the studied colon cancer cells as well as in inhibition of human
neutrophils’ oxidative burst. The substitution of a hydroxyl group
of the CAHA (4), by a methoxyl group, as in FAHA (5), led to a
decrease in the antiproliferative activity, although for the higher
concentrations, FAHA (5) was very effective in reducing cell prolif-
eration. The addition of a supplementary hydroxyl group, as in
OHCAHA (6), also led to a decrease in the anti-proliferative activity,
particularly in C2BBe1 cells, although this decrease was lower than
that caused by hydroxyl group substitution by a methoxyl group.
As amide CAHA (4) revealed to be the most cytotoxic com-
pound, further studies were carried out in order to better under-
stand the mechanisms of colon cancer cell death. Compound
CAHA (4) revealed that its antiproliferative effect on C2BBe1 cells
was related to a cell cycle blockade in G2/M phase. It is known that
P53-null cancer cells lack G1 checkpoint, depending on checkpoint
kinases for G2/M checkpoint.35 Considering that C2BBe1 cells do
not express P53 protein (data not shown), a possible DNA damage
induced by CAHA (4) could retain cells in G2/M phase for DNA
repair or apoptosis pathways activation. On the other hand, in
WiDr cells CAHA (4) induced cell cycle arrest in S phase. The muta-
tion of the TP53 gene in WiDr cells is described by ATCC to prevent
them from regulating P21 protein following DNA damage. P21 is a
cyclin dependent kinase inhibitor responsible for the regulation of
cell cycle G1 arrest. Generally, DNA damages induce an increase in
P53 levels and a consequent P21 transcription, in order to activate
G1 checkpoint and allow cells to repair damages or proceed to
46
accumulation in cancer cells in response to genotoxic stress.
Åꢁ
O
2
is a by-product of mitochondrial respiratory chain, which can
produce oxidative damage in mitochondria itself, but it can also
diffuse away from its origin site and cause damages in many differ-
ent macromolecules, like DNA, phospholipids and proteins.
45,47
Åꢁ
Thus, the accumulation of O
2
induced by CAHA (4) could be an
oxidative trigger for DNA damage and mitochondrial dysfunction,
demonstrated by mitochondrial membrane potential decrease,
leading cells to apoptosis.
5. Conclusion
In summary, new structure–activity relationships on hydrox-
ycinnamic acid derivatives were achieved concerning inhibition
of human neutrophils’ oxidative burst and inhibition of colon can-
cer cells proliferation, showing that the presence of a catechol
group is very important for both activities and that, generally,
amides are more potent than the corresponding acids. A phenolic
amide derivative CAHA (4) was found to be the most active
compound in all the performed biological activities capable of
inhibiting human neutrophils’ oxidative burst, restraining the
inflammatory process, and inhibiting growth of colon cancer cells,
acting as antioxidant in conditions of excessive ROS and RNS
production and prooxidant in the intracellular medium of colon
cancer cells, triggering mitochondrial dysfunction that leads cells
to apoptosis.
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E. J. Tavares-da-Silva et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
Altogether, these achievements can contribute to the under-
standing of the relationship between antioxidant and anticancer
activities and based on the SAR established, can be the starting
point to find more effective phenolic compounds as anticancer
agents.
14. Rajendra Prasad, N.; Karthikeyan, A.; Karthikeyan, S.; Reddy, B. V. Mol. Cell.
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Acknowledgements
We would like to thank FCT (Fundação para a Ciência e
Tecnologia, Portugal) for the financial support to IBILI-FMUC
through PEst-C/SAU/UI3282/2013, to CNC.IBILI through strategic
project UID/NEU/04539/2013 and to Applied Molecular Biology
Unit, Institute of Immunology and University Clinic of Hematology,
Faculty of Medicine of Coimbra, for the data acquisition of flow
cytometry studies. UCIBIO, REQUIMTE also acknowledges European
Union (FEDER funds POCI/01/0145/FEDER/007728) and National
Funds [FCT/MEC (Ministério da Educação e Ciência)] under the
Partnership Agreement PT2020 UID/MULTI/04378/2013.
Marisa Freitas and Ana Salomé Pires acknowledge FCT the
financial support for the Pos-doc grant (SFRH/BPD/76909/2011)
and for the PhD grant (SFRH/BD/75300/2010), respectively, in the
ambit of ‘QREN e POPH e Tipologia 4.1 e Formação Avançada’,
cosponsored by FSE and by national funds of MCTES.
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Please cite this article in press as: Tavares-da-Silva, E. J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.065