Antiproliferative and apoptotic effects of the oxidative dimerization product of methyl caffeate on human breast cancer cells

Article abstract of DOI:10.1016/j.bmcl.2012.11.009

Caffeic acid derivatives are increasingly regarded as potential oncoprotective that could inhibit both the initiation and progression of cancer. Here we have synthesized seven 1-arylnaphthalene lignans and related compounds and tested their impact on breast cancer cell growth in tissue culture. The product of the oxidative dimerization of methyl caffeate, 1-phenylnaphthalene lignan, was found to induce a strong decrease in breast cancer cell number (IC₅₀ ～1 μM) and was selected for further investigation. Flow cytometry analysis revealed a decrease in cell proliferation and an increase in apoptosis in both MCF-7 and MDA-MB-231 breast cancer cell lines that are representative of the two main categories of breast tumors. The 3,4-dihydroxyphenyl group probably induced the biological activity, as the control compounds lacking it had no effect on breast cancer cells. Together, our data indicate that the oxidative dimerization product of methyl caffeate can inhibit breast cancer cell growth at a concentration adequate for pharmacological use.

Full text of DOI:10.1016/j.bmcl.2012.11.009

Bioorganic & Medicinal Chemistry Letters 23 (2013) 574–578
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiproliferative and apoptotic effects of the oxidative dimerization product
of methyl caffeate on human breast cancer cells
Fabrice Bailly a, Robert-Alain Toillon b, Olympe Tomavo a,b, Nathalie Jouy c, Hubert Hondermarck d,
Philippe Cotelle a,
⇑
a Université Lille 1, EA4478, F-59650 Villeneuve d’Ascq, France
b Inserm U 908, IFR-147, Université Lille 1, F-59650 Villeneuve d’Ascq, France
c Flow Cytometry Facility of BiCEL, Campus Lille 2-Imprt, Lille, France
d School of Biomedical Sciences & Pharmacy, Faculty of Health, University of Newcastle, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Caffeic acid derivatives are increasingly regarded as potential oncoprotective that could inhibit both the
initiation and progression of cancer. Here we have synthesized seven 1-arylnaphthalene lignans and
related compounds and tested their impact on breast cancer cell growth in tissue culture. The product
of the oxidative dimerization of methyl caffeate, 1-phenylnaphthalene lignan, was found to induce a
Received 2 October 2012
Revised 3 November 2012
Accepted 6 November 2012
Available online 22 November 2012
strong decrease in breast cancer cell number (IC50 ꢀ1
lM) and was selected for further investigation.
Flow cytometry analysis revealed a decrease in cell proliferation and an increase in apoptosis in both
MCF-7 and MDA-MB-231 breast cancer cell lines that are representative of the two main categories of
breast tumors. The 3,4-dihydroxyphenyl group probably induced the biological activity, as the control
compounds lacking it had no effect on breast cancer cells. Together, our data indicate that the oxidative
dimerization product of methyl caffeate can inhibit breast cancer cell growth at a concentration adequate
for pharmacological use.
Keywords:
Breast cancer
Methyl caffeate dimerization
Apoptosis
Cell proliferation
1-Phenyl-1,2-dehydronaphthalenes
Ó 2012 Elsevier Ltd. All rights reserved.
Food derived polyphenol anti-oxidants have been shown to
have an oncoprotective effect.1,2 This is particularly well illustrated
with propolis that was used for the treatment of tumors in tradi-
tional oriental medicine, and has been shown to have the ability
to inhibit breast cancer cell growth.3,4 Several studies5–10 have re-
ported the antiproliferative effect of caffeic acid derivatives
(including caffeic acid phenylethyl ester (CAPE), a beehive propolis
component) on human breast cancer cells. Caffeic acid itself has
been found to exert an antiproliferative and anti-apoptotic activity
at low concentrations on some breast cancer cell lines such as
T47D, whereas others such as MCF-7, MDA-MB-231 and HS578T
were insensitive.3 Several synthetic caffeic acid esters were found
to inhibit the growth of MCF-7 cells5,8,9 and the reduction of the
double bond improved this antiproliferative activity.5 In addition,
(Chart 1), obtained by oxidative dimerization of methyl caffeate
by iron(III) chloride, was previously tested as topoisomerase inhib-
itor.13 It presented a 100% topoisomerase II inhibition at 50
lM.
In the present study, we explored the effect on breast
cancer cells of a series of 1-phenylnaphthalenes issued from the
oxidative dimerization of methyl caffeate and ferulate by iron tri-
chloride14–18 or by a procedure described by Yvon et al.19 Amongst
a series of seven selected compounds that have been tested, the
best tumor cell growth inhibitory effect was obtained with com-
pound 1. Subsequently, the antiproliferative and apoptotic activity
of
1
was evaluated on MCF-7 and MDA-MD-231 cells.20–22
OH
R1O
HO
COOMe
COOMe
MeO
HO
COOMe HO
COOR1
COOR2
CAPE induced apoptosis of MCF-7 cells by inhibiting NF-
jB and
activating Fas.7
HO
COOMe
Lignans are formed in nature by the oxidative dimerization of
C6C3 phenols and are defined as those compounds in which the
two C6C3 units are linked by a bond connecting the central carbon
atom of each side chain. Many lignans, such as podophylotoxin or
enterolactone, exhibit important antitumor activities11 and are
regarded as phytoestrogens.12 The 1-phenylnaphthalene lignan 1
R2
R2
OMe
MeO
OR1
OMe
OH
OH
OH
5: R1 = H; R2 = H
1: R1 =H; R2 = H
6: R1 = C2H5; R2 = C2H5
7: R1 = C2H5; R2 = H
4
2: R1 = CH3; R2 = H
3: R1 = CH3; R2 = CH3
⇑
Corresponding author.
E-mail address: philippe.cotelle@univ-lille1.fr (P. Cotelle).
Chart 1.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.009

F. Bailly et al. / Bioorg. Med. Chem. Lett. 23 (2013) 574–578
575
Compound 1 revealed an efficient inhibition of both proliferation
and survival of breast cancer cells.
Compounds 1,14 2,15,16 3 and 4,17,18 (Chart 1) were synthesized
according to known procedures using iron(III) chloride as oxidizing
agent. The purity and spectral data of these derivatives were as re-
ported in the literature.14–18 Compounds 5, 6 and 7 (Scheme 1) were
obtained in the following four-step procedure. 3,4-Dimethoxybenz-
aldehyde was condensed on diethyl succinate in the presence of
sodium tert-butoxide in tert-butyl alcohol to give ethyl (E)-2-[(3,4-
dimethoxyphenyl)-methylene]succinic acid 1-methylester (I) in
56% yield. This monoester was esterified using thionyl chloride in
ethanol to give II. A second Stobbe condensation was accomplished
using 4-methoxybenzaldehyde in the presence of LDA in THF and
the intramolecular cyclization was obtained in trifluoroacetic acid
at room temperature yielding trans ethyl 1,2-dihydro-6,7-dime-
thoxy-1-(40-methoxyphenyl)-naphthalene-2,3-dicarboxylate (III)
in 40% yield. Finally, the total demethylation of the methoxy groups
was accomplished using boron tribromide. Whatever the reaction
conditions (temperature, equivalent number, time, temperature
and time of hydrolysis) we always obtained a mixture of 5 and 7. Un-
der particular conditions, small amounts of 6 were also isolated. The
best results were obtained when the reaction was conducted with 4
equivalents of boron tribromide in dichloromethane at room tem-
perature during 30 min. With a 1 h hydrolysis at reflux, 5 and 7 were
isolated in 54 and 18% yield, respectively. With a 30 min hydrolysis
at room temperature, 7 was obtained in 28% yield and traces of 6
could be isolated.
Figure 1. Effect of compounds 1–7 (10
EMEM with 10% FCS.
l
M) on the growth of MCF-7 cells in basal
growth of both cell lines. IC50 was not significantly different in
presence or absence of FCS. It has been estimated to ꢀ1 M in
M in MDA-MB-231 cells.
l
MCF-7 and to ꢀ5
l
As revealed by Hoechst staining, cancer cells entered apoptosis
(Fig. 3A and B) when treated with compound 1. In these experi-
ments, apoptosis was found to be concentration-dependent and
depended on the medium. When treated with 10 lM of 1 in the
presence of FCS, percentage of apoptotic cells was 25% higher,
independently of the breast cancer cell line.
The effect of compound 1 at 5 lM on breast cancer cell cycle
was then evaluated. Typical feature of flow cytometry are shown
in Figure 5A and B. Cell populations were reported on Figure 4A
and B for MCF-7 and MDA-MB-231 cell line, respectively. The ab-
sence of FCS induced a G0/G1 phase cell arrest, as evidenced by
accumulation in the G0/G1 phase, from 35.6% (MCF-7) and 31.7%
(MDA-MB-231) with FCS to 70.9% (MCF-7) and 73.9% (MDA-MB-
231) without FCS with a concomitant decrease in cell accumulation
in the S and G2/M phases. In the presence of FCS, 1 induced differ-
ent effects on cell cycle of MCF-7 and MDA-MB-231. Whereas 1 in-
duced a slight decrease in MCF-7 cell accumulation in the G0/G1, S
The synthesized molecules 1–7 were first tested on the growth
of MCF-7 breast cancer cells at the standard concentrations of
10 lM (Fig. 1). 1 was found to be the best compound of the series
with 82% of inhibition.
Compounds 2 and 3 had no effect on breast cancer cell growth,
indicating that the catechol moieties may support biological activ-
ity. The biological activities of compounds 5, 6 and 7 show the rel-
ative impact of the two catechol moieties of 1 on the inhibition of
the MCF-7 cell growth. The 3,4-dihydroxyphenyl group probably
carried the biological properties of 1. This result can be compared
to the difference of activities of caffeic esters and dihydrocaffeic es-
ters previously reported.5 Dihydrocaffeic esters presented, in all
cases, higher activities than their caffeic esters analogues. Com-
pounds 4 and 5 presented moderate activities, but due to chemical
instabilities (dehydration of 4 and decarboxylation of 5) they were
not selected for further investigation.
and G2/M phases (from 35.6% (without 1) to 29.4% (1 at 5 lM) for
G0/G1 phase, from 46.6% to 34.7% for S phase and from 17.8% to
17.0% for S/G2 phase), 1 promoted a S-phase cell cycle arrest as evi-
denced by the accumulation of MDA-MB-231 cells in the S-phase
(from 40.4% (without 1) to 56.1% (1 at 5 lM), with a concomitant
decrease in cell accumulation in the G0/G1 and the G2/M phases.
As revealed by Propidium Iodide staining (PI staining), cell
exhibited apoptosis when treated with compound 1. MDA-MB-
In order to characterize the growth inhibitory effect of 1 on
breast cancer cells, we extended our study to MDA-MB-231 cells.
231 and MCF-7 cells treated with 1 at 5 lM exhibited a sub-G0
We first tested compound 1 (0.05–10
l
M) in the absence or pres-
population. The sub-G0 peak corresponded to about 20% of total
ence of FCS. As depicted in Figure 2, compound 1 inhibited the
count whatever the cell lines or culture conditions.
CHO
CO2Et
MeO
CO2Et
CO2Et
MeO
MeO
CO2Et
CO2H
MeO
MeO
CO2Et
CO2Et
SOCl2, EtOH 0 °C
then reflux
(CH3)3COH, Na
Reflux
(1) LDA, THF, - 78 °C
(2)
+
OMe CO2Et
MeO
II
MeO
CHO
OH
OMe
I
OMe
TFA, rt
MeO
MeO
CO2Et
CO2Et
(1) BBr3, CH2Cl2
(2) H2O
5, 6 or 7
III
OMe
Scheme 1. Synthesis of the target compounds 5–7.

576
F. Bailly et al. / Bioorg. Med. Chem. Lett. 23 (2013) 574–578
Figure 3. (A) Characteristic nuclear morphologies of apoptosis in MDA-MB-231
cells observed after Hoechst 33258 staining; (B) Apoptotic MDA-MB-231 or MCF-7
cells in the presence of different concentrations of 1.
Figure 2. Effect of 1 on the growth of MDA-MB-231 cells (A) and MCF-7 cells; (B) in
basal EME.
Several studies on the antiproliferative and antiapoptotic prop-
erties of caffeic acid and corresponding esters (including CAPE and
methyl caffeate) have been published. Caffeic acid exhibits antipro-
liferative and apoptotic effects on T47D human breast cancer cells
at a submicromolar level (IC50 2 nM)6 whereas it has no effect on
MCF-7, MDA-MB-231 or HS578T human breast cancer lines.7
Caffeic acid esters are usually more active than caffeic acid as
exemplified by the comparison of cytotoxicity of hexylcaffeate
and caffeic acid.7 Other caffeic acid esters have been described to
have antiproliferative activities on several cancer cells including
breast cancer5,9,10 with decamicromolar IC50
.
Caffeic acid was shown to have antiproliferative and apoptotic
effects on T47D human breast cancer cells via the inhibition of
AhR-induced CYP1A enzyme6 whereas CAPE inhibits MDA-MB-
231 and MCF-7 growth by inducing apoptosis via the inhibition
of NF-
tor of NF-
j
B and activation of Fas.8,23 CAPE is also an effective inhibi-
jB in PC-3 cells, but the mechanism of apoptosis in this
case was described as caspase-dependent.24
Caffeic acid and esters are known to dimerize chemically
(FeCl3,15 Ag2O11 or MnO214) or enzymatically (horseradish peroxi-
dase,25 catechol oxidase,26 polyphenol oxidase27). It has also been
shown that caffeic acid underwent a dimerization in isolated per-
fused rat liver to yield 1-phenylnaphthalene derivatives.28 Sinapic
and ferulic acid dimers (compounds structurally related to com-
pounds 2 and 3) that have been recently identified in cereal dietary
fibers may also contribute to decrease breast cancer risk.29
Caffeic, sinapic and ferulic acid methyl esters were therefore
submitted to dimerization conditions and the resulting 1-phenyl-
naphthalene lignans were tested as breast cancer cell proliferation
inhibitors. We also synthesized three 1-phenylnaphthalene lignans
Figure 4. MCF-7 (A) and MDA-MB-231; (B) cells in the presence of 1 (5 lM)
measured by flux cytometry.

F. Bailly et al. / Bioorg. Med. Chem. Lett. 23 (2013) 574–578
577
Figure 5. Typical flow cytometric analysis of the cell cycle: MCF-7 cells with FCS in the absence (A) or presence; (B) of 1 (5 lM).
with only one hydroxyl group on the phenyl ring as diacid 5,
diethyl ester 6 and monoethyl ester 7. At 10 M concentration, 1
presented the best activity of the series and was selected for fur-
ther experiments on the two breast cancer cell lines MDA-MB-
231 (estradiol receptor negative) and MCF-7 (estradiol receptor po-
sitive). Compound 1 was found to inhibit cell proliferation with
pharmacological use. Several polyphenols have already entered
clinical trials involving patients with premalignant diseases and
cancers,34 and it is clear that the large chemical variety of polyphe-
nols offers opportunities for further developments. A recent study
of a large prospective cohort found no impact of coffee intake in
breast carcinogenesis,35 but an equivalent investigation to assess
the impact of coffee consumption on the progression of the disease
has never been conducted. The present study indicated that some
products of the oxidative dimerization of methyl caffeate can inhi-
bit breast cancer cell survival and proliferation, and that should be
taken under consideration when assessing the impact of food de-
rived polyphenol oxidant for human health. Compound 1 was iso-
lated as a racemic mixture of the trans isomer. Our efforts will now
be focused on the chemical synthesis of the cis isomer and on the
separation and purification of each enantiomer.
l
IC50 of ꢀ1
l
M (MCF-7) and ꢀ 5
lM (MDA-MB-231) that is a
1000-fold more active than methyl caffeate on MCF-75 and 10–
100-fold more active than more lipophilic caffeic acid ester7,9
but it had similar inhibitory activity than CAPE.23
,
Flow cytometry analysis showed several strong differences be-
tween the culture conditions whatever the cell lines. MDA-MB-
231 and MCF-7 cells treated with 1 at 5 lM exhibited a sub-G0
peak corresponded to about 20% of total count slightly superior
to those found with cell staining by Hoechst 33258 at the same
concentration of 1. No difference was observed between MDA-
MB-231 and MCF-7 cell lines in spite of the lack of caspase-3
expression in MCF-7 cells which is known to decrease sub-G0 peak
formation.30 As expected, cell growth in serum free medium
showed an increase of G0/G1 phase cells indicating that prolifera-
tion was inhibited. In MCF-7 cells treated with 1, the percentage of
non-cycling cell (G0/G1 phase) was increased. This was accompa-
nied by a decrease of cycling cells (S+G2/M phase). Compound 1
had a similar inhibitory effect on MDA-MB-231 cells with an in-
crease of the percentage of cell in G0/G1.
Acknowledgments
This work was financially supported by grants from la Région
Nord Pas-de-Calais, le Centre National de la Recherche Scientifique
(CNRS) and the University of Newcastle Australia. The Mass Spec-
trometry facility used in this study was funded by the European
Community (FEDER), the Région Nord Pas-de-Calais (France), the
CNRS, and the Université des Sciences et Technologies de Lille.
Thus, it seems that 1 induced apoptosis of MCF-7 and MDA-MB-
231 cells via different pathways. Compared to CAPE, apoptosis in-
Supplementary data
duced by 1 could be due to NF-
other proteins.23,31 Differences between MCF-7 and MDA-MB-231
could be due to the absence of constitutive NF-
B activation32 or
j
B inhibition,8 caspases24 or several
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.11.
009.
j
to the lack of caspase-3 expression30 in the estrogen receptor posi-
tive MCF-7 cells. However, one can only speculate and a more in-
depth exploration would be necessary to determine the molecular
targets of compound 1 that is responsible for the antiproliferative
and apoptotic effects. Of note, proteomic analysis now offers new
possibilities for the identification of proteins targeted by small
chemical compounds and is therefore increasingly used in cancer
oriented pharmacological investigations.33
References and notes
1. Ajila, C. M.; Brar, S. K.; Verma, M.; Tyagi, R. D.; Godbout, S.; Valéro, J. R. Crit. Rev.
Biotechnol. 2011, 31, 227.
2. Vauzour, D.; Rodriguez-Mateos, A.; Corona, G.; Oruna-Concha, M. J.; Spencer, J.
P. Nutrients 2010, 2, 1106.
3. Jung, B.; Kim, M.; Kim, H.; Kim, D.; Yang, J.; Her, H.; Song, Y. S. Phytother. Res.
2010, 24, 295.
4. Omene, C. O.; Wu, J.; Frenkel, K. Invest. New Drugs 2011. http://dx.doi.org/
10.1007/s10637-011-9667-8.
In conclusion, our study revealed that the product of the oxida-
tive dimerization of methyl caffeate, 1-phenylnaphtalene lignan,
inhibited breast cancer cell growth at concentration adequate for
5. Etzenhouser, B.; Hansch, C.; Kapur, S.; Selassie, C. D. Bioorg. Med. Chem. 2001, 9,
199.

578
F. Bailly et al. / Bioorg. Med. Chem. Lett. 23 (2013) 574–578
6. Kampa, M.; Alexaki, V.; Notas, G.; Nifli, A.; Nistikaki, A.; Hatzoglou, A.;
22. Vergote, D.; Cren-Olivé, C.; Chopin, V.; Toillon, R. A.; Rolando, C.; Hondermarck,
H.; Le Bourhis, X. Breast Cancer Res. Treat. 2002, 76, 195.
23. Wu, J.; Omene, C.; Karkoszka, J.; Bosland, M.; Eckard, J.; Klein, C. B.; Frenkel, K.
Cancer Lett. 2011, 308, 43.
24. McEleny, K.; Coffey, R.; Morrissey, C.; Fitzpatrick, J. M.; Watson, R. W. BJU Int.
2004, 94, 402.
Bakogeorgou, E.; Kouimtzoglou, E.; Blekas, G.; Boskou, D.; Gravanis, A.;
Castanas, E. Breast Cancer Res. 2004, 6, R63.
7. Serafim, T. L.; Carvalho, F. S.; Marques, M. P. M.; Calheiros, R.; Silva, T.; Garrido,
J.; Milhazes, N.; Borges, F.; Roleida, F.; Silva, E. T.; Holy, J.; Oliveira, J. Chem. Res.
Toxicol. 2011, 24, 763.
8. Watabe, M.; Hishikawa, K.; Takayanagi, A.; Shimizu, N.; Nakati, T. J. Biol. Chem.
2004, 279, 6017.
25. Matsumoto, K.; Takahashi, H.; Miyake, Y.; Fukuyama, Y. Tetrahedron Lett. 1999,
40, 3185.
9. Jayaprakasam, B.; Vanisree, M.; Zhang, Y.; Dewitt, D. L.; Nair, M. G. J. Agric. Food
Chem. 2006, 54, 5375.
26. Rompel, A.; Fischer, H.; Meiwes, D.; Büldt-Karentzopoulos, K.; Eicken, C.;
Gerdemann, C.; Krebs, B. FEBS Lett. 1999, 445, 103.
10. Xia, C.; Li, H.; Liu, F.; Hua, W. Bioorg. Med. Chem. Lett. 2008, 16, 6553.
11. Ionkova, I. Mini-Rev. Med. Chem. 2011, 11, 843.
27. Cheynier, V.; Moutounet, M. J. Agric. Food Chem. 1992, 40, 2038.
28. Gumbinger, H. G.; Vahlensieck, U.; Winterhoff, H. Planta Med. 1993, 59, 491.
29. Bunzel, M.; Ralph, J.; Kim, H.; Lu, F.; Ralph, S. A.; Marita, J. M.; Hatfield, R. D.;
Steinhart, H. J. Agric. Food Chem. 2003, 51, 1427.
30. Kagawa, S.; Gu, J.; Honda, T.; McDonnell, T. J.; Swisher, S. G.; Roth, J. A.; Fang, B.
Clin. Cancer Res. 2001, 7, 1474.
31. Lin, H. P.; Jiang, S. S.; Chuu, C. P. PLoS One 2012, 7, e31286.
32. Biswas, D.; Iglehart, J. D. J. Cell. Physiol. 2006, 209, 645.
33. Hondermarck, H.; Tastet, C.; El Yazidi-Belkoura, I.; Toillon, R. A.; Le Bourhis, X. J.
Proteome Res. 2008, 4, 1403.
34. Thomasset, S. C.; Berry, D. P.; Garcea, G.; Marczylo, T.; Steward, W. P.; Gescher,
A. J. Int. J. Cancer 2007, 120, 451.
12. Virk-Baker, M. K.; Barnes, S. Planta Med. 2010, 76, 1132.
13. Kashiwada, Y.; Bastow, K. F.; Lee, K. H. Bioorg. Med. Chem. Lett. 1995, 5, 905.
14. Daquino, C.; Rescifina, A.; Spatafora, C.; Tringali, C. Eur. J. Org. Chem. 2009, 6289.
15. Ahmed, R.; Lehrer, M.; Stevenson, R. Tetrahedron 1973, 29, 3753.
16. Cartwright, N. J.; Haworth, R. D. J. Chem. Soc. 1944, 535.
17. Wallis, A. F. A. Aust. J. Chem. 1973, 26, 1571.
18. Ahmed, R.; Lehrer, M.; Stevenson, R. Tetrahedron Lett. 1973, 15, 747.
19. Yvon, B. L.; Datta, P. K.; Le, T. N.; Charlton, J. L. Synthesis 2001, 1556.
20. Toillon, R. A.; Adriaenssens, E.; Wouters, D.; Lottin, S.; Boilly, B.; Hondermarck,
H.; Le Bourhis, X. Mol. Cell Biol. Res. Commun. 2000, 3, 338.
21. Demont, Y.; Corbet, C.; Page, A.; Ataman-Önal, Y.; Choquet-Kastylevsky, G.;
Fliniaux, I.; Le Bourhis, X.; Toillon, R. A.; Bradshaw, R. A.; Hondermarck, H. J.
Biol. Chem. 1923, 2012, 287.
35. Gierach, G. L.; Freedman, N. D.; Andaya, A.; Hollenbeck, A. R.; Park, Y.;
Schatzkin, A.; Brinton, L. A. Int. J. Cancer 2012, 131, 452.