Phenolic compounds as selective antineoplasic agents against multidrug-resistant human cancer cells

Article abstract of DOI:10.1055/s-0029-1240892

Twelve phenolic compounds, including three stilbenes, two flavonoids, two coumarins, one neolignan, and four lignans, isolated from Euphorbia and Pycnanthus species or obtained by derivatization, were assayed for their potential antineoplastic efficacy in three human cancer cell lines: gastric (EPG85-257), pancreatic (EPP85-181), and colon (HT-29) carcinomas as well as derived multidrug-resistant sublines. In each case, two different multidrug-resistant variants, i.e., cell lines with classical and atypical MDR phenotype, were used. The majority of the MDR cancer sublines showed increased sensitivities to the studied compounds when compared to the parental sublines. The most active compound was the flavonoid naringenin, found to be 15-fold more effective against the atypical MDR subline of gastric carcinoma than in parental drug-sensitive cells. Furthermore, the stilbene trans-3,5,3,4- tetramethoxypiceatannol and the lignans 4-hydroxy-3,3,4-trimethoxylignan and heliobuphthalmin also exhibited high antineoplasic activities against the classical MDR subline derived from gastric carcinoma. The results of this study suggest that some phenolic compounds might be valuable for the treatment of multidrug-resistant cancer cells. Georg Thieme Verlag KG Stuttgart - New York.

Full text of DOI:10.1055/s-0029-1240892

Original Articles 975
Phenolic Compounds as Selective Antineoplasic Agents
against Multidrug-resistant Human Cancer Cells
1
2
1
1
Authors
Noélia Duarte , Hermann Lage , Marta Abrantes , Maria-José U. Ferreira
1
Affiliations
Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal
Charité Campus Mitte, Institute of Pathology, Berlin, Germany
2
Key words
Abstract
compounds when compared to the parental sub-
lines. The most active compound was the flavo-
noid naringenin, found to be 15-fold more effec-
tive against the atypical MDR subline of gastric
carcinoma than in parental drug-sensitive cells.
Furthermore, the stilbene trans-3,5,3′,4′-tetrame-
thoxypiceatannol and the lignans 4′-hydroxy-
3,3′,4-trimethoxylignan and heliobuphthalmin
also exhibited high antineoplasic activities
against the classical MDR subline derived from
gastric carcinoma. The results of this study sug-
gest that some phenolic compounds might be val-
uable for the treatment of multidrug-resistant
cancer cells.
"
l antiproliferatives
!
"
l multidrug resistance
Twelve phenolic compounds, including three stil-
benes, two flavonoids, two coumarins, one neo-
lignan, and four lignans, isolated from Euphorbia
and Pycnanthus species or obtained by derivatiza-
tion, were assayed for their potential antineoplas-
tic efficacy in three human cancer cell lines: gas-
tric (EPG85-257), pancreatic (EPP85-181), and co-
lon (HT-29) carcinomas as well as derived multi-
drug-resistant sublines. In each case, two differ-
ent multidrug-resistant variants, i.e., cell lines
with classical and atypical MDR phenotype, were
used. The majority of the MDR cancer sublines
showed increased sensitivities to the studied
"
l stilbenes
"
l lignans
"
l piceatannol
"
l naringenin
Introduction
metabolites, due to the presence of chirality and
functionality represents an extremely rich bioge-
netic resource, providing also pointers for rational
drug design [3,4]. Natural product derived drugs
represent 60% of the chemotherapeutic agents
currently in use for all types of cancers [3]. In re-
cent years, there is a growing interest in the study
of lignans because some of them exhibit a re-
markable antineoplastic activity. In particular,
the lignan podophyllotoxin, isolated from Podo-
phyllum sp, was used as a lead structure for its
semisynthetic derivatives etoposide and tenipo-
side, widely employed in the treatment of a varie-
ty of malignant diseases [5]. Stilbenes have as well
been considered attractive candidates in the ther-
apeutic development of anticancer drugs [6]. An
important example is the stilbene piceatannol
that was first isolated as an antileukemic com-
pound from Euphorbia lagascae seeds and, later
on, identified as a potent tyrosine kinase inhibitor
[7,8]. There are also various reports on the anti-
proliferative and apoptosis induction activities of
piceatannol and other stilbenes against several
cell lines and animal models [6,9]. On the other
hand, flavonoids have been described as exerting
!
The major clinical obstacle to the successful che-
motherapy of neoplastic diseases is the develop-
ment of drug resistance. Combination chemother-
apy was supposed to overcome this phenomenon
by treating with multiple agents that exert their
effects by different mechanisms. However, cancer
cells respond by development of multidrug resis-
tance (MDR), i.e., simultaneous resistance to a
panel of mechanistically and structurally diverse
drugs [1]. When multidrug-resistant phenotype
is depending on the enhanced activity of the drug
extrusion pump MDR1/P-gp, the MDR is referred
as classical MDR; in the case of independence
from MDR1/P-gp, the drug-resistant phenotype
is designated as atypical MDR [2]. One possible
approach to overcome MDR is to find out new
anticancer drugs without cross-resistance in can-
cer cells exhibiting a multidrug-resistant pheno-
type. Consequently, there is an urgent need to
identify new cancer chemotherapeutic leads. Nat-
ural products have long been regarded as excel-
lent sources for drug discovery and development.
In fact, the wide structural diversity of secondary
received
revised
accepted
Nov. 13, 2009
January 12, 2010
January 24, 2010
Bibliography
DOI http://dx.doi.org/
0.1055/s-0029-1240892
Published online February 15,
010
Planta Med 2010; 76: 975–980
Georg Thieme Verlag KG
1
2
©
Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Maria-José U. Ferreira
Faculty of Pharmacy
University of Lisbon
Av. das Forças Armadas
1
600-083 Lisbon
Portugal
Phone: + 351217946475
Fax: + 351217946470
mjuferreira@ff.ul.pt
Duarte N et al. Phenolic Compounds As… Planta Med 2010; 76: 975–980

9
76 Original Articles
Fig. 1 Chemical sructures of the twelve tested
compounds.
a wide range of biochemical and pharmacological effects. Their
antitumor and cancer-preventing properties have been some of
the most investigated and have been attributed to a wide variety
of mechanisms, including antiproliferative activity [10,11].
Continuing our search for anticancer agents from plant sources,
in this work we studied the potential antineoplasic activity of
several phenolic compounds, previously isolated from Euphorbia
and Pycnanthus species (1, 4–9, and 12), or obtained by derivati-
zation (2, 3), together with the new acetylated (10) and methy-
lated (11) derivatives of compound 9. These compounds were
evaluated in sensitive gastric, pancreatic, and colon human can-
cer cells and in their classical and atypical multidrug-resistant
variants.
Tested compounds
"
Twelve compounds, whose structures are presented in l Fig. 1,
were tested for their potential antiproliferative activity in sensi-
tive and multidrug-resistant cancer cell lines: the stilbenes picea-
tannol (1), trans-3,5,3′,4′-tetraacetoxypiceatannol (2), and trans-
3,5,3′,4′-tetramethoxypiceatannol (3), the flavonoids naringenin
(4) and aromadendrin (5), the coumarins scopoletin (6) and escu-
letin (7), the neolignan dehydrodiconiferyl diacetate (8), and the
lignans (−)-dihydroguaiaretic acid (4,4′-dihydroxy-3,3′-dime-
thoxylignan, 9), 4,4′-diacetyl-3,3′-dimethoxylignan (10), 4′-hy-
droxy-3,3′,4-trimethoxylignan (11), and heliobuphthalmin (12).
The purity of the compounds was more than 95% (HPLC). All the
compounds were dissolved in DMSO. Compounds 1, 7, and 6, 8
were isolated from the methanol extracts of E. lagascae defatted
seeds and aerial parts, respectively [12–14]. Compounds 4 and 5
were isolated from the methanol extract of E. tuckeyana aerial
parts [15]. Compounds 2 and 3 were obtained by acetylation and
methylation reactions of piceatannol [12,16]. Compounds 9 and
Materials and Methods
!
General experimental procedures
The NMR spectra were recorded on a Bruker Avance 400 NMR
12 were isolated from the CH Cl2 extract of P. angolensis stem
2
1
13
spectrometer ( H 400 MHz; C 100.61 MHz) using CDCl as sol-
bark [17]. Compounds 10 and 11 were obtained by acetylation
and methylation reactions of (−)-dihydroguaiaretic acid (9), as
described below.
3
vent. MS was taken on a Kratos MS25RF mass spectrometer
(
70 eV). TLC was performed on SiO F
plates (Merck 5554 and
2
254
5744) and visualized under UV radiation by spraying with sul-
phuric acid-acetic acid-water (1:20:4) followed by heating.
Duarte N et al. Phenolic Compounds As… Planta Med 2010; 76: 975–980

Original Articles 977
Acetylation of (−)-dihydroguaiaretic acid
Results and Discussion
!
50 mg of (−)-dihydroguaiaretic acid (9) were suspended in Ac O
2
(
1.0 mL) and pyridine (1.0 mL). After stirring at room temperature
In our search for bioactive compounds from plant sources, several
phenolic compounds were isolated from Euphorbia lagascae [12–
14], Euphorbia tuckeyana [15], and Pycnanthus angolensis [17], or
obtained by derivatization reactions [12,16]. These compounds
include the stilbenes piceatannol (1), trans-3,5,3′,4′-tetraacetoxy-
piceatannol (2), and trans-3,5,3′,4′-tetramethoxypiceatannol (3),
the flavonoids naringenin (4) and aromadendrin (5), the couma-
rins scopoletin (6) and esculetin (7), the neolignan dehydrodico-
niferyl diacetate (8), as well as the lignans (−)-dihydroguaiaretic
acid (4,4′-dihydroxy-3,3′-dimethoxylignan, 9) and heliobuph-
for 24 hours, the excess of reagents were eliminated with N . Pu-
2
rification was made by preparative chromatography (CHCl3-
MeOH, 39:1) yielding 52 mg of compound 10 (4,4′-diacetyl-3,3′-
dimethoxylignan).
4
,4′-diacetyl-3,3′-dimethoxylignan (10): EIMS, m/z (rel. int.): 414
+
1
[M] (2), 372 (28), 330 (51), 137 (100). H RMN (400 MHz, CDCl ):
3
δ 6.92 (d, J = 8.0 Hz, H-5 and H-5′), 6.67 (dd, J = 1.6 and 8.0 Hz, H-6
and H-6′), 6.66 (br s, H-2 and H-2′), 3.78 (s, OMe-3 and OMe-3′),
2
.61 (dd, J = 6.8 and 13.6 Hz, H-7a and H-7′a), 2.46 (dd, J = 8.0 and
3.6 Hz, H-7b and H-7′b), 2.33 (s, OCOMe-4 and OCOMe- 4′), 1.81
"
1
thalmin (12, l Fig. 1).
(
m, H-8 and H-8′), and 0.87 (d, J = 6.4 Hz, H-9 and H-9′).
Continuing in this study our search for antineoplasic agents, the
lignan (−)-dihydroguaiaretic acid (9) was derivatized to afford
the new compounds 4,4′-diacetyl-3,3′-dimethoxylignan (10) and
4′-hydroxy-3,3′,4-trimethoxylignan (11). The structure elucida-
tion of these compounds was based on a comparison of their MS
and NMR spectra with those of (−)-dihydroguaiaretic acid (9).
The twelve phenolic compounds listed above were investigated
for their potential antiproliferative activity in several human can-
cer cell lines which were derived from three different tumor en-
tities: gastric (EPG85-257), pancreatic (EPP85-181), and colon
cancer cells (HT-29). Furthermore, in each case two different
multidrug-resistant variants of these cells were also investigated:
cell lines with a classical MDR phenotype (associated with the
overexpression of MDR1/P-gp, EPG85-257RDB, EPP85-181RDB,
and HT-29RDB) and cell lines with an atypical MDR phenotype
(no enhanced expression of MDR1/P-gp, EPG85-257RNOV,
EPP85-181RNOV, and HT-29RNOV). For assessment of cytotoxic-
ity of the twelve tested compounds, the IC50 values of each agent
were determined by proliferation assays in each of the different
cell variants after continuous exposure for 5 days. The etoposide
and cisplatin-specific IC50 values were considered as positive
controls for maintenance of the drug-resistant phenotype. Rela-
tive resistance (RR) values were also determined as the relation
between the IC50 of the resistant cell line and the IC50 of the pa-
rental drug-sensitive cell line. When the sensitivity against a giv-
en compound was less than 10% of the corresponding parental
cell line, the compound was assessed to be highly efficient in this
drug-resistant cell line [18,19].
Methylation of (−)-dihydroguaiaretic acid
5
by-drop to a solution of compound 9 (50 mg in 2 mL of anhydrous
ether). The reaction occurred overnight and then was followed by
TLC. The purification of the compounds was performed by prep-
arative chromatography (CH Cl -acetone, 19:1) to yield 14.5 mg
mL of an ethereal solution of diazomethane was added drop-
2
2
of compound 11 (4′-hydroxy-3,3′,4-trimethoxylignan).
′-hydroxy-3,3′,4-trimethoxylignan (11): EIMS, m/z (rel. int.): 344
4
+
1
[M] (35), 152 (30), 151 (100), 137 (75), 28 (17). H RMN
(400 MHz, CDCl ): δ 6.82 (d, J = 8.0 Hz, H-5′), 6.78 (d, J = 8.0 Hz,
3
H-5), 6.65 (dd, J = 2.0 and 8.0 Hz, H-6), 6.61 (dd, J = 2.0 and 8.0 Hz,
H-6′), 6.59 (d, J = 2.0 Hz, H-2), 6.56 (d, J = 2.0 Hz, H-2′), 5.49 (s, OH-
4
and 13.6 Hz, H-7a and H-7′a), 2.41 (dd, J = 6.8 and 13.6 Hz, H-7b
and H-7′b), 1.77 (m, H-8 and H-8′), and 0.85 (d, J = 7.2 Hz, H-9
and H-9′).
′), 3.88 (s, OMe-4), 3.83 (s, -OMe-3 and -OMe-3′), 2.56 (dd, J = 6.8
Cell lines, cell culture, and cell proliferation assay
Human cancer cell lines and drug-resistant sublines were grown
in modified Leibovitz L-15 medium (Biowhittaker) as described
previously [15,18,19]. Drug-resistant cell lines were established
from parental cell lines by continuous exposure of the cells to
stepwise increasing concentrations of antineoplastic agents [20].
For maintenance of drug-resistant phenotypes, the medium of
drug-resistant sublines was supplemented with the selection
agent. Resistance to etoposide, cisplatin (both from Gry-Pharma),
and tested compounds was assessed using a proliferation assay
based on sulforhodamine B (SRB) staining. Briefly, 800 cells per
well were seeded in 96-well plates in triplicate. After 24-h attach-
ment, the particular agent was added in a dilution series for 5
days incubation. Cells were fixed by chilled 10% trichloroacetic
acid for 1 h at 4°C and washed five times with tap water before
staining was performed with 0.4% SRB (Sigma) in 1% acetic acid
for 10 min at room temperature. After washing with 1% acetic ac-
id, drying, and resolubilization in 20 mM Tris-HCl (pH 10), ab-
sorbance was measured at 562 nm against the reference wave-
length of 690 nm. Mean IC50 values and standard deviations were
calculated from at least two independent experiments in tripli-
cate for each cell line by using the Prism software (GraphPad Soft-
ware, Inc.). Relative resistance (RR) values were also determined
as:
The IC50 and RR values of drug-resistant cell variants in compari-
son to the drug-sensitive parental cell lines are summarized in
"
l Tables 1, 2 and 3 for the three EPG85-257 gastric, EPP85-181
pancreatic, and HT-29 colon carcinomas, respectively. As it can
be observed, in parental drug-sensitive cell lines, all the tested
compounds (1–12) showed a moderate/weak antiproliferative ef-
fect or were inactive. In contrast, when comparing with the pa-
rental sublines, some of the multidrug-resistant variants showed
increased sensitivities to the studied compounds, particularly the
EPG85-257RDB subline.
When considering the results obtained for the stilbenes (1–3), it
can be observed that in the drug-resistant subline EPG85-
257RDB (associated with the classical MDR phenotype) derived
from gastric carcinoma, piceatannol (1) and its tetramethylated
derivative (3) were found to be as effective as the positive control
etoposide (IC50 = 6.2 µM) and slightly less effective than cisplatin
(IC50 = 4.0 µM), showing IC50 values of 5.6 and 6.3 µM, respective-
RR = (IC50 resistant cell line)/(IC50 parental-drug sensitive cell
line)
"
ly (l Table 1). Furthermore, trans-3,5,3′,4′-tetramethoxypicea-
tannol (3) exhibited a RR value of 0.18 and thus can be consid-
ered very effective against this cell line. On the other hand, when
compared to piceatannol, its acetoxy derivative (2) showed just a
Duarte N et al. Phenolic Compounds As… Planta Med 2010; 76: 975–980

9
78 Original Articles
Table 1 Cytotoxicity of phenolic compounds 1–12 in parental, drug-sensitive, and in different multidrug-resistant EPG85-257 gastric carcinoma cells.
Compound
EPG85-257Pa
IC50 (µM)
EPG85-257RDBb
IC50 (µM)
EPG85-257RNOVc
IC50 (µM)
RRd
0.53
RRd
2.48
Stilbenes
"
"
"
1
2
3
10.5 ± 1.8
61.7 ± 6.4
34.7 ± 3.3
5.6 ± 1.1
14.9 ± 2.6
6.3 ± 0.6
26.0 ± 3.2
16.7 ± 3.3
48.8 ± 4.2
0.24
0.18
0.27
1.40
Flavonoids
"
4
5
95.0 ± 8.6
27.0 ± 2.4
64.0 ± 6.6
0.28
6.2 ± 0.8
0.06
"
> 100
< 0.64
29.0 ± 2.5
< 0.29
Coumarins
"
6
7
> 100
17.0 ± 2.3
> 100
n.c
62.0 ± 8.1
16.0 ± 2.4
< 0.62
0.94
"
12.5 ± 2.0
0.74
Lignans
"
"
"
"
"
8
> 100
20.0 ± 2.3
77.2 ± 7.8
7.7 ± 1.7
15.5 ± 2.1
4.7 ± 0.6
3.5 ± 0.4
6.2 ± 0.3
4.0 ± 0.3
< 0.77
0.38
0.32
0.19
0.12
59
> 100
n.c
9
8.8 ± 1.6
34.2 ± 2.8
6.3 ± 0.8
0.44
0.71
0.25
0.84
14.8
0.6
10
11
12
48.3 ± 4.9
24.6 ± 2.1
27.9 ± 2.6
0.105 ± 0.008
4.4 ± 0.4
23.3 ± 2.5
1.55 ± 0.09
2.6 ± 0.2
Etoposide
Cisplatin
1
a
EPG85-257P: parental, drug-sensitive gastric carcinoma cell line; b EPG85-257RDB: gastric carcinoma cell line with classical MDR phenotype; c EPG85-257RNOV: gastric carcinoma
cell line with atypical MDR phenotype; d RR: relative resistance in relation to the parental, drug sensitive cell line EPG85-257P; n.c: not calculated
Table 2 Cytotoxicity of phenolic compounds 1–12 in parental, drug-sensitive, and in different multidrug-resistant EPP85-181 pancreatic carcinoma cells.
Compound
EPP85-181Pa
IC50 (µM)
EPP85-181RDBb
IC50 (µM)
EPP85-181RNOVc
IC50 (µM)
RRd
1.33
RRd
0.74
Stilbenes
"
"
"
1
2
3
27.0 ± 2.4
76.6 ± 8.2
67.2 ± 8.2
36.0 ± 2.9
65.1 ± 7.3
60.8 ± 7.8
20.0 ± 2.1
58.6 ± 6.9
64.6 ± 8.5
0.85
0.90
0.76
0.96
Flavonoids
"
4
65.0 ± 4.5
14.5 ± 2.3
73.0 ± 4.6
12.5 ± 1.6
1.12
0.86
88.0 ± 4.9
8.0 ± 0.7
1.35
0.55
Coumarins
"
7
Lignans
"
"
"
"
9
44.0 ± 5.2
66.4 ± 7.6
15.7 ± 1.8
35.3 ± 3.8
0.58 ± 0.03
0.08 ± 0.01
28.0 ± 4.3
49.3 ± 5.5
14.8 ± 1.7
19.4 ± 2.5
62.0 ± 4.2
0.09 ± 0.01
0.63
0.74
0.94
0.55
37.0 ± 4.6
63.2 ± 6.9
27.4 ± 3.2
28.9 ± 3.1
4.5 ± 0.7
0.84
0.95
1.74
0.81
7.8
10
11
12
Etoposide
Cisplatin
106.9
1.2
2.6 ± 0.2
34
Compounds 5, 6, and 8 were ineffective in the sensitive and resistant variants of pancreatic carcinoma cells (IC50 > 100 µM); a EPP85-181P: parental, drug-sensitive pancreatic car-
cinoma cell line; b EPP85-181RDB: pancreatic carcinoma cell line with classical MDR phenotype; c EPP85-181RNOV: pancreatic carcinoma cell line with atypical MDR phenotype;
d
RR: relative resistance in relation to the parental, drug sensitive cell line EPP85-181P; n.c: not calculated
moderate effect against this multidrug-resistant subline
suggesting that the presence of an extra hydroxyl group at C-3 in
aromadendrin (5) may be responsible for its smaller activity.
Concerning the set of lignans (8–12), 4′-hydroxy-3,3′,4-trime-
thoxylignan (11) and heliobuphthalmin (12) showed significant
antiproliferative activities against the subline EPG85-257RDB
that overexpresses MDR1/P-gp (IC50 = 4.7 and 3.5 µM and RR =
"
(IC50 = 14.9 µM, l Table 1).
Regarding the results obtained for flavonoids (4, 5) and couma-
rins (6, 7), it should be observed that except for compound 4, all
the compounds showed a moderate activity or were inactive
against EPG85-257P gastric cancer cells and its drug-resistant cell
variants. Nevertheless, the best result was obtained in the multi-
drug-resistant EPG85-257RNOV subline associated with no
MDR1/P-gp expression, for the flavanone naringenin (4), which
"
0.19 and 0.12, respectively, l Table 1). In fact, this multidrug re-
sistance subline is 5-fold and 8-fold more sensitive than the pa-
rental drug-sensitive cells and therefore, lignans 11 and 12 could
be considered very effective against EPG85-257RDB cells.
"
exhibited a significant IC50 (6.2 µM, l Table 1) and a RR value of
0
.06, being 15-fold more effective in this resistant cell line than in
Although the structures of dibenzylbutane-type lignans (9–11)
are very similar, it could be observed that they considerably differ
in their activities. As shown by the results, acetylation of com-
pound 9 resulted in a decrease of antiproliferative activity for
parental drug-sensitive cells. Compounds 4 and 5 only differ in
the substitution pattern of the six-membered heterocyclic ring,
Duarte N et al. Phenolic Compounds As… Planta Med 2010; 76: 975–980

Original Articles 979
Table 3 Cytotoxicity of phenolic compounds 1–12 in parental, drug-sensitive, and in different multidrug-resistant HT-29 colon carcinoma cells.
Compound
HT-29Pa
HT-29RDBb
IC50 (µM)
HT-29RNOVc
IC50 (µM)
IC50 (µM)
RRd
0.98
RRd
Stilbenes
"
"
"
1
2
3
56.0 ± 4.8
77.8 ± 12.3
84.5 ± 9.2
55.0 ± 4.8
96.0 ± 14.8
89.4 ± 10.6
38.0 ± 4.3
70.0 ± 10.9
78.0 ± 8.7
0.68
0.90
0.92
1.23
1.06
Flavonoids
"
4
> 100
> 100
n.c
73.0 ± 10.5
17.0 ± 2.0
< 0.73
0.70
Coumarins
"
7
24.0 ± 3.4
19.0 ± 2.2
0.79
Lignans
"
"
"
"
9
54.0 ± 3.8
51.9 ± 4.4
45.6 ± 3.7
70.0 ± 8.2
2.3 ± 0.3
54.0 ± 4.1
69.7 ± 5.7
67.9 ± 5.2
1.0
1.34
1.49
< 1.43
11.3
0.7
55.0 ± 3.6
71.0 ± 5.9
66.4 ± 6.1
1.0
1.37
1.46
< 1.43
15.2
1
10
11
12
> 100
> 100
Etoposide
Cisplatin
26.0 ± 1.7
2. 7 ± 0.1
35.0 ± 2.6
3.8 ± 0.1
3.8 ± 0.1
Compounds 5, 6, and 8 were ineffective in the sensitive and resistant variants of colon carcinoma cells (IC50 > 100 µM). a HT-29P: parental, drug-sensitive colon carcinoma cell line;
b
HT-29RDB: colon carcinoma cell line with classical MDR phenotype; c HT-29RNOV: colon carcinoma cell line with atypical MDR phenotype; d RR: relative resistance in relation to the
parental, drug sensitive cell line HT-29P; n.c: not calculated
compound 10, against all the three sublines of EPG85-257 gastric
carcinoma cells (l Table 1). On the other hand, compound 11,
drugs against resistant human cancer cells since they appear not
to be extruded by ABC-transporters.
"
which differs from compound 9 only by the presence of a me-
thoxyl group at C-4 instead of a hydroxyl group, showed an in-
creasing activity particularly against the EPG85-257RDB subline.
Besides, both compounds 9 and 11 showed a moderate activity
Acknowledgements
!
(
IC50 = 8.8 µM, RR = 0.44 and IC50 = 6.3 µM, RR = 0.25, respectively)
This work was supported by The Science and Technology Foun-
dation, Portugal (FCT) and COST B16 Action of the European
Commission. The authors thank Prof. Madureira for the collection
of P. angolensis.
in multidrug-resistant EPG85-257RNOV cells, suggesting that the
antiproliferative activity is dependent from the individual drug
resistance phenotype.
With regard to the activity against the three sublines of pancreat-
ic carcinoma cells (EPP85-181), most of the tested compounds
exhibited just a moderate antiproliferative activity (l Table 2).
In this cell line, the lowest IC50 values were obtained for the cou-
marin-type compound esculetin (7) against the two multidrug-
References
"
1 O’ Connor R, Clynes M, Dowling P, O’ Donovan N, Driscoll L. Drug resis-
tance in cancer – searching for mechanisms, markers and therapeutic
agents. Expert Opin Drug Metab Toxicol 2007; 3: 805–817
2
Lage H. An overview of cancer multidrug resistance: a still unsolved
problem. Cell Mol Life Sci 2008; 65: 3145–3167
resistant sublines EPP85-181RDB and EPP85-181RNOV (IC50
=
1
2.5 and 8.0 µM). Similar results were obtained for the colon car-
3 Newman D, Cragg G. Natural products as sources of new drugs over the
last 25 years. J Nat Prod 2007; 70: 461–477
"
cinoma cells (HT-29, l Table 3). Actually, these cell lines (EPP85-
81 and HT-29) appeared markedly more resistant than the gas-
4
Jones W, Chin Y, Kinghorn D. The role of pharmacognosy in modern
medicine and pharmacy. Curr Drug Targets 2006; 7: 247–264
Saleem M, Kim H, Ali M, Lee Y. An update on bioactive plant lignans. Nat
Prod Rep 2005; 22: 696–716
1
tric carcinoma cells, which may indicate a tissue-dependence.
In previous studies we have reported that trans-3,5,3′,4′-tetra-
methoxystilbene (3) strongly modulates the efflux pump P-gly-
coprotein on the human MDR1 gene-transfected mouse lympho-
ma cells in a dose-dependent manner. Additionally, when tested
in combination with doxorubicine, compound 3 showed an addi-
tive antiproliferative effect [12], while in other assays it was
found to be an effective inhibitor of the activity of voltage-gated
potassium Kv 1.3 channels, which are important to inhibit the
proliferation of prostate, breast, and colon cancer cells [21]. Fur-
thermore, the stilbenes piceatannol (1) and trans-3,5,3′,4′-tetra-
acetoxypiceatannol (2), as well as the flavonoids naringenin (4)
and aromadendrin (5) were found to be effective inhibitors of
the multidrug-resistance associated protein 1 (MRP1) [16].
In summary, together with our previous data, the results ob-
tained lead us to conclude that some phenolic compounds, par-
ticularly the stilbene trans-3,5,3′,4′-tetramethoxypiceatannol
5
6 Roupe KA, Remsberg C, Yánez J, Davies N. Pharmacometrics of stibenes:
seguing towards the clinic. Curr Clin Pharmacol 2006; 1: 81–101
7
Ferrigni NR, McLaughlin JL, Powell RG, Smith CR. Use of potato disc and
brine shrimp bioassays to detect activity and isolate piceatannol as the
antileukemic principle from the seeds of Euphorbia lagascae. J Nat Prod
1984; 4: 347–352
8 Geahlen RL, McLaughlin JL. Piceatannol (3,4,3′,5′-tetrahydroxy-trans-
stilbene) is a naturally occurring protein-tyrosine kinase inhibitor. Bio-
chem Biophys Res Commun 1989; 165: 241–245
9
Rimando A, Suh N. Biological/chemopreventive activity of stilbenes and
their effect on colon cancer. Planta Med 2008; 74: 1635–1643
10 Crozier A, Jaganath I, Clifford N. Dietary phenolics: chemistry, bioavail-
ability and effects on health. Nat Prod Rep 2009; 26: 1001–1043
1
1 Moon Y, Wang X, Morris M. Dietary flavonoids: effects on xenobiotic
and carcinogen metabolism. Toxicol In Vitro 2006; 20: 187–210
2 Ferreira MJU, Duarte N, Gyémánt N, Radics R, Chepernev G, Varga A, Mol-
nár J. Interaction between doxorubicine and a resistance modifier stil-
bene in the antiproliferative effect on multidrug resistant mouse lym-
phoma and human breast cancer cells. Anticancer Res 2006; 26: 3541–
1
(
3), the flavonoid naringenin (4), and the lignans 4′-hydroxy-
,3′,4-trimethoxylignan (11) and heliobuphthalmin (12), may be
interesting as potential scaffolds for the development of new
3
546
3
Duarte N et al. Phenolic Compounds As… Planta Med 2010; 76: 975–980

9
80 Original Articles
1
3 Duarte N, Kayser O, Abreu P, Ferreira MJU. Antileishmanial activity of
piceatannol isolated from Euphorbia lagascae seeds. Phytother Res
18 Lage H, Aki-Sener E, Yalcin I. High antineoplastic activity of new hetero-
cyclic compounds in cancer cells with resistance against classical DNA
Topoisomerase II-targeting drugs. Int J Cancer 2006; 119: 213–220
19 Lage H, Duarte N, Coburger C, Hilgeroth A, Ferreira MJU. Antitumor ac-
tivity of terpenoids against classical and atypical multidrug resistant
cancer cells. Phytomedicine, advance online publication 13 August
2009; DOI: 10.1016/j.phymed.2009.07.009
2
008; 22: 455–457
1
1
4 Duarte N, Ferreira MJU. Lagaspholones A and B: two new jatropholane-
type diterpenes from Euphorbia lagascae. Org Lett 2007; 9: 489–492
5 Duarte N, Lage H, Ferreira MJU. Three new jatrophane diterpene poly-
esters and other constituents from Euphorbia tuckeyana. Planta Med
2
008; 74: 61–68
20 Lage H. Proteomic approaches for investigation of therapy resistance in
cancer. Proteomics Clin Appl 2009; 3: 883–911
21 Teisseyre A, Duarte N, Ferreira MJU, Michalak K. Influence of the multi-
drug transporter inhibitors on the activity of Kv1.3 voltage-gated po-
tassium channels. J Physiol Pharmacol 2009; 60: 69–76
1
1
6 Wesolowska O, Wiśniewski J, Duarte N, Ferreira MJU, Michalak K. Inhibi-
tion of MRP1 transport activity by phenolic and terpenic compounds
isolated from Euphorbia species. Anticancer Res 2007; 27: 4127–4134
7 Abrantes M, Mil-Homens T, Duarte N, Lopes D, Cravo P, Madureira M,
Ferreira MJU. Antiplasmodial activity of lignans and extracts from Pyc-
nanthus angolensis. Planta Med 2008; 74: 1408–1412
Duarte N et al. Phenolic Compounds As… Planta Med 2010; 76: 975–980