Cytotoxic effects of new trans-2,4-diaryl-r-3-methyl-1,2,3,4- tetrahydroquinolines and their interaction with antitumoral drugs gemcitabine and paclitaxel on cellular lines of human breast cancer

Article abstract of DOI:10.1016/j.cbi.2010.11.010

Two tetrahydroquinoline compounds, called DM8 and DM12, from a new series of the cis-2,4-diaryl-r-3-methyl-1,2,3,4-tetrahydroquinolines, were selected for cytotoxic effects studies on cellular lines of human breast cancer. The synergistic, additive and an

Full text of DOI:10.1016/j.cbi.2010.11.010

Chemico-Biological Interactions 189 (2011) 215–221
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Cytotoxic effects of new
trans-2,4-diaryl-r-3-methyl-1,2,3,4-tetrahydroquinolines and their interaction
with antitumoral drugs gemcitabine and paclitaxel on cellular lines of human
breast cancer
Arturo Mun˜oza, Felipe Sojob, Diego R. Merchan Arenasc,
Vladimir V. Kouznetsovc,∗, Francisco Arvelob,a,∗∗
a Laboratorio de Cultivo de Tejidos y Biología de Tumores, Instituto de Biología Experimental, Universidad Central de Venezuela, código postal 1041, Caracas, Venezuela
b Centro de Biociencias, Fundación Instituto de Estudios Avanzados-IDEA, Sartenejas, Venezuela
c Laboratorio de Química Orgánica y Biomolecular, Escuela de Química, Universidad Industrial de Santander, A.A. 678, Bucaramanga, Colombia
a r t i c l e i n f o
a b s t r a c t
Article history:
Two tetrahydroquinoline compounds, called DM8 and DM12, from a new series of the cis-2,4-diaryl-
r-3-methyl-1,2,3,4-tetrahydroquinolines, were selected for cytotoxic effects studies on cellular lines of
human breast cancer. The synergistic, additive and antagonistic effects in combination of these com-
pounds with anticancer drugs, such as paclitaxel and gemcitabine, were studied. The isobolograms and
their analysis demonstrated models of synergism, additivity and antagonism of these tetrahydroquino-
lines in the presence of paclitaxel and gemcitabine. Results showed that compounds DM8 and DM12
individually induced growth inhibition on breast cancer cell lines MCF-7 and SKBR3, and the addition of
paclitaxel and gemcitabine intensified their cytotoxic activity on both cell lines at conc. below 1 ␮g/mL.
During these studies the compound DM12 was identified as new, perspective and safe agent for adjuvant
therapy.
Received 21 September 2010
Received in revised form
29 November 2010
Accepted 29 November 2010
Available online 4 December 2010
Keywords:
Tetrahydroquinolines
Breast cancer
Synergism
© 2011 Elsevier Ireland Ltd. All rights reserved.
Adjuvant therapy
1. Introduction
ease. Currently, taxanes (paclitaxel) and gemcitabine stand out
among a plethora of useful drugs utilized in systemic cancer ther-
Breast cancer is the main cause of death among European
women [1]. In Latin America and the Caribbean, the highest inci-
dence rates of the disease correspond to Colombia, Peru, Chile,
Uruguay and Argentina. Even though the incidence of breast can-
cer has decreased worldwide, this pathology still continues to be a
great risk to women’s health [2]. In spite of the availability of cur-
rent clinical tools, such as hormonal therapies and the application
of certain chemotherapeutical agents, breast cancer continues to be
cureless, with less than 10% of the patients overcoming the disease
in less than 5 years [3,4].
apy. However, in pre-clinical studies it has been demonstrated that
the combined administration of paclitaxel and gemcitabine pro-
duces a cytotoxic effect, which is independent on the pattern of
application of the drugs [5]. It was found in breast cancer treatment
that the administration of paclitaxel previous to that of gemcitabine
is associated with an increase in the intracellular concentration
of the 2ꢀ2ꢀ-difluorodeoxycytidine-5ꢀ-triphosphate, the gemcitabine
active metabolite [6]. Thus, there is a need to developments new
compounds that would be safe agents for adjuvant therapy for
cancer. Among them, the quinoline derivatives are attractive, and
easily available models showing a wide range of biological activ-
ities, including antimalarial [7], antitumoral [8], antioxidant [9]
and anti-mycobacterial [10,11]. Polyfunctionalized quinolines have
been used as potential anti-carcinogens [12], exhibiting pharma-
cological activity as anti-angiogenic agents [13] and as telomerase
inhibitors in different types of human tumoral cells [14].
Breast cancer treatment is an integral process, using local
(surgery, radiotherapy) and systemic (chemotherapy and hormonal
therapy) treatments, according to the degree and stage of the dis-
∗
Corresponding author. Fax: +57 76 349069.
Corresponding author at: Laboratorio de Cultivo de Tejidos
∗∗
Keeping in view the above mentioned facts, we selected the
tetrahydroquinolines DM8 and DM12, to test their usefulness
as anti cancer agents in combination with taxanes (paclitaxel)
and nucleoside analogs (gemcitabine). First we determined the
y Biología de
Tumores, Instituto de Biología Experimental, Universidad Central de Venezuela,
código postal 1041, Caracas, Venezuela. Fax: +58 02127535897.
E-mail addresses: vkuznechnik@gmail.com (V.V. Kouznetsov),
franarvelo@yahoo.com (F. Arvelo).
0009-2797/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2010.11.010

216
A. Mu˜noz et al. / Chemico-Biological Interactions 189 (2011) 215–221
Table 1
IC50 values of the drugs studied on growth inhibition and SI (selectivity index) of human breast cancer and human dermic fibroblast. They are presented as mean SD (n = 3).
Camp.
IC10 of breast cancer cell tines and control cells
Selectivity index (SI)
MCF-7
MCF-7
SKBR3
Human dermic fibroblast
SKBR3
0.74
30.81 1.00 ␮g/mL
73.89 1.00 ␮M
25.77 ␮g/mL
67.33 0.02 ␮M
19.12 ␮g/mL
52.56 1.01 ␮M
0.62
11.9
43
4.92 1.14 ␮g/mL
11.30 1.14 ␮M
6.77 0.06 ␮g/mL
15.46 0.06 ␮M
58.55 0.51 ␮g/mL
134.46 0.51 ␮M
8.64
0.47
0.03 0.01 ␮g/mL
0.10 0.01 ␮M
2.76 0.02 ␮g/mL
9.21 0.02 ␮M
1.29 0.01 ␮g/mL
4.30 0.01 ␮M
0.14 0.03 ␮g/mL
0.16 0.03 ␮M
1.03 0.22 ␮g/mL
l.21 0.22 ␮M
0.50 0.06 ␮g/mL
0.59 0.06 ␮M
3.68
0.48
cytotoxic activity of DM8 and DM12, and then we compared their
effects on cellular lines of human breast cancer MCF-7 and SKBR3
in combinations with paclitaxel and gemcitabine. We here report
the interesting pharmacological effects of the studied tetrahydro-
quinolines when used in combinations with taxanes (paclitaxel)
and nucleoside analogs (gemcitabine). However, at present pre-
liminary in vitro studies have been conducted, an extrapolating our
result to clinical studies an adjuvant therapy would be premature.
tion was maintained at 1%. The DM8 and DM12 compounds were
also dissolved in DMSO and the evaluated concentrations were 1, 5,
15, 25, 50, 80 and 100 ␮g/mL. The reported IC50 values represented
the average obtained from at least three independent experiments.
Cultures were carried out at 37 ◦C in a humidified atmosphere with
5% CO2. Cells were incubated with the drugs for 72 h in 96-well
plates.
2.2.1. Extraction and purification of DM8 and DM12 compound
The DM8 and DM12 compounds are part of a new series of trans-
2,4-diaryl-r-3-methyl-1,2,3,4-tetrahydroquinolines prepared from
isoeugenol, benzaldehyde (p-nitrobenzaldehyde) and aniline (o-
nitroaniline). The two new DM8 and DM12 compounds were
prepared, purified by column chromatography and fully charac-
terized by all spectroscopic methods as synthetic protocols work
[15]. Chemical purity of the two prepared compounds was con-
firmed by analysis of GC–MS, as well as spectroscopic methods
(1H NMR and 13C NMR). The first analysis indicated that com-
pound DM8 has a high degree of purity (99.5%), while the sample
DM12–98.9% with the respective retention times, tR = 39.4 and
75.1 min. The nuclear magnetic resonance method, especially 13C
NMR, also confirmed the chemical purity and molecular struc-
ture of both compounds. Procedure of 1,2,3,4-tetrahydroquinoline
DM12: a mixture of o-nitroaniline (0.30 g, 2.17 mmol) and o-
nitrobenzaldehyde (0.36 g, 2.38 mmol) in anhydrous MeCN (15 mL)
was stirred at room temperature for 20 min. One eq. of BF3·OEt2
(0.31 g, 2.17 mmol) was added into the mixture. Finally, a solu-
tion of MeCN (15 mL) and trans-isoeugenol (0.43 g, 2.60 mmol) was
added to the mixture at constant drip. The temperature was kept
to 70 ◦C at inert atmosphere of nitrogen for 10 h. The reaction was
treated with a satured solution of Na2CO3 and the organic phase
was removed with ethyl acetate (2× 30 mL). The resulting prod-
uct crude was purified by chromatography column for yield the
comp. DM12, 8-nitro-4-(4-hydroxy-3-methoxyphenyl)-3-methyl-
2. Materials and methods
2.1. Materials
Cell culture. MCF-7 (breast carcinoma, without over-expression
of the HER2/c-erb-2 gene) and SKBR3 (breast carcinoma, in which
the HER2/c-erb-2 gene is over-expressed, moreover HER-2 over-
expressing mammary tumors are commonly indicative of a poor
prognosis in patients) were used. Human breast cancer cell lines
were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco)
supplemented with 10% (v/v) heat inactivated fetal bovine serum
(FBS; Gibco), 2 mM glutamax (Gibco), 100 units/mL penicillin and
100 ␮g/mL streptomycin. Human dermis fibroblasts, used as con-
trol cells, were obtained from primary cultures and grown in DMEM
supplemented with 10% heat inactivated FBS, 2 mM glutamax,
100 units/mL penicillin and 100 ␮g/mL streptomycin.
2.2. Drugs, synthetic compounds and treatment
The following drugs were studied: taxol (Bristol-Myers Squibb)
and gemcitabine® (Eli Lilly and Company). Concentrations of the
studied drugs ranged from 0.001 to 100 ␮g/mL. Drugs stock were
prepared in 100% dimethylsulfoxide (DMSO; Sigma), and dilutions
were done with cell culture media. Control cells were incubated
with DMSO alone. For all drug preparations DMSO final concentra-

A. Mu˜noz et al. / Chemico-Biological Interactions 189 (2011) 215–221
217
Fig. 1. Effects of gemcitabine, paclitaxel and tetrahydroquinolines (DM8 and DM12) on survival of breast cancer cells MCF-7 and SKBR3, and human dermic fibroblasts (used
as control). Drugs were applied individually or in combination for 72 h. Values are mean SD (n = 3).
2-(2-nitrophenyl)-1,2,3,4-tetrahydro-quinoline;Yellow solid. Mp
the organic phase was removed with ethyl acetate (2× 30 mL).
The resulting product crude was purified by chromatography
column for yield the 8-nitro-4-(4-hydroxy-3-methoxyphenyl)-3-
methyl-2-phenyl-1,2,3,4-tetrahydroquinoline (DM1); Red solid.
187–192 ◦C. Yield 48%. IR (KBr): ꢀ 3363, 3525, 2966, 1254, 1612,
1520 cm−1 1H NMR (CDCl3, 400 MHz): ı 8.40 (1H, s, NH), 8.02
.
(1H, d, J = 8.5 Hz, 3-HAr), 7.82 (1H, d, J = 8.1, 7-H), 7.66, (1H, d
J = 7.7 Hz, 5-H), 7.64 (1H, t, 7.4 Hz, 5-HAr), 7.49 (1H, t, J = 7.6 Hz,
4-HAr), 6.88 (1H, d, J = 8.0 Hz, 6-HAr), 6.66 (1H, dd, J = 7.5, 1.0 Hz,
6ꢀ-HAr), 6.84 (1H, d, J = 7.4 Hz, 5ꢀ-HAr), 6.61 (1H, br. s, 2ꢀ-HAr), 6.51
(1H, t, J = 7.7 Hz, 6-H),5.64 (1H, br. s, OH), 5.51 (1H, d, J = 9.7 Hz, 2-
H), 3.85 (1H, s, OMe), 3.77 (1H, d, J = 10.9 Hz, 4-H), 2.34-2.22 (1H,
m, 3-H), 0.65 (3H, d, J = 6.5 Hz, 3-Me); 13C NMR (CDCl3, 100 MHz): ı
150.5, 146.9, 144.7, 142.6, 136.4, 133.7, 133.4, 131.3, 129.1, 128.7,
124.9, 124.2, 122.2, 115.4, 114.6, 110.9, 56.0, 55.9, 51.4, 39.4, 16.0.
MS m/z (EI): 435 (M+). Found: C, 63.53; H, 4.97; N, 9.86. Anal. calcd
for C24H22N2O2: C, 63.44; H, 4.86; N, 9.65. Procedure of 1,2,3,4-
tetrahydroquinoline DM8: (a) a mixture of o-nitroaniline (0.40 g,
2.89 mmol) and benzaldehyde (0.34 g, 3.19 mmol) in anhidrous
MeCN (15 mL) was stirred at room temperature for 20 min. One
eq. of BF3·OEt2 (0.41 g, 2.89 mmol) was added into the mixture.
Finally, a solution of MeCN (15 mL) and trans-isoeugenol (0.57 g,
3.47 mmol) was added to the mixture at constant drip. The tem-
perature was kept to 70 ◦C at inert atmosphere of nitrogen for 10 h.
The reaction was treated with a satured solution of Na2CO3 and
Mp 241–242 ◦C. Yield 90%. IR (KBr): ꢀ 3448, 2927, 1514, 1282 cm−1
.
1H NMR (CDCl3, 400 MHz): ı 8.47 (1H, s, NH), 8.01 (1H, dd, J = 8.6,
1.0 Hz, 7-H), 7.43-7.33 (5H, m, all HPh), 6.91 (1H, d, J = 8.0 Hz, 5ꢀ-HAr),
6.78 (1H, br. d, J = 8.6 Hz, 5-H), 6.72 (1H, dd, J = 8.0, 1.9 Hz, 6ꢀ-HAr),
6.60 (1H, d, J = 1.9 Hz, 2ꢀ-HAr), 6.45 (1H, t, J = 7.4 Hz, 6-H), 5.58 (1H,
br. s, OH), 4.32 (1H, d, J = 10.1 Hz, 2-H), 3.85 (3H, s, OMe), 3.72 (1H, d,
J = 11.5 Hz, 4-H), 2.40 (1H, m, 3-H), 0.62 (3H, d, J = 6.5 Hz, 3-Me); 13
C
NMR (CDCl3, 100 MHz): ı 147.0, 144.6, 143.0, 141.2, 136.1, 134.1,
130.9, 129.5, 128.9, 128.6, 127.6, 124.8, 122.6, 121.5, 114.7, 110.6,
110.7, 63.4, 56.0, 51.8, 39.1, 16.4. MS m/z (EI): 390 (M+). Found:
C, 70.54; H, 5.86; N, 7.21. Anal. calcd forC23H22N2O4: C, 70.75; H,
5.68; N, 7.17. (b) A mixture of 0.20 g (0.49 mmol) of compound DM1
and 0.04 g (0.38 mmol) of Pd/C in 20 mL of MeOH was leave stirred
overnight in H2 atmosphere. The reaction crude was filtered and
concentrated to vacuum for yield without previous purification
the compound DM8, 8-amino-4-(4-hydroxy-3-methoxyphenyl)-
3-methyl-2-phenyl-1,2,3,4-tetrahydroquinoline; Green solid. Mp
237–238 ◦C. Yield 100%. IR (KBr): ꢀ 3387, 3611, 3058, 1235 cm−1. 1H

218
A. Mu˜noz et al. / Chemico-Biological Interactions 189 (2011) 215–221
NMR (DMSO-d6, 400 MHz): ı 7.50–7.30 (5H, m, all HPh), 6.71 (1H, d,
J = 7.9 Hz, 5ꢀ-HAr), 6.68 (1H, d, J = 1.7 Hz, 2ꢀ-HAr), 6.66 (1H, s, NH),6.58
(1H, dd, J = 8.0, 1.8 Hz, 6ꢀ-HAr), 6.24 (1H, t, J = 7.6 Hz, 6-H), 5.79 (1H, d,
J = 7.7 Hz, 5-H), 4.45 (2H, br. s, NH2), 4.05 (1H, d, J = 9.9 Hz, 2-H), 3.69
(3H, s, OMe), 3.65 (1H, d J = 10.9 Hz, 4-H), 2.15-2.01 (1H, m, 3-H),
0.44 (3H, d, J = 6.4 Hz, 3-Me); 13C NMR (DMSO-d6, 100 Hz): ı 147.4,
144.8, 144.7, 143.3, 135.8, 133.8, 132.8, 128.1, 128.0, 127.8, 127.3,
125.1, 121.6, 118.5, 116.5, 115.1, 112.7, 112.3, 63.1, 55.6, 51.5, 40.5,
16.4.MS m/z (EI): 360 (M+). Found: C, 76.52; H, 6.94; N, 7.61. Anal.
calcd for C23H24N2O2: C, 76.64; H, 6.71; N, 7.77.
2.3. Methods
MTT assay. This assay relies on the ability of viable cells to
metabolically reduce a yellow tetrazolium salt (MTT; Sigma) to
a purple formazan product. This reaction takes place when mito-
chondrial reductases are active [16]. Cells were grown in 96-well
plates (5 × 103 cells/well). After incubation with the drugs and com-
pounds the medium was removed, and the cells were treated with
100 ␮L MTT for 3 h at 37 ◦C. Subsequently, 100 ␮L DMSO were
added to the mixture. The solubilized formazan product was quan-
tified with the help of a microtiter plate reader TECAN-sunrise at
570 nm.
2.4. Selectivity index
The selectivity index (SI) was calculated as the ratio IC50 (control
cells)/IC50 (tumoral cell line). A selectivity index > 1 indicates that
the cytotoxicity on tumoral cells surpassed the one on the healthy
non-tumoral cells [17].
2.5. Analysis of drug–synthetic compounds interactions
Cells of both breast cancer cell lines were simultaneously incu-
bated for 72 h with taxol or gemcitabine, combined with either
DM8 or DM12. The nature of the interactions between the drugs
here studied was analyzed with the help of an isobologram [18]
and median effect methods described by Chou and Talalay [19–21].
The isobolograms were constructed by determining the doses of
different drugs causing a defined fractional cell kill effect (Fa).
The obtained doses, for each drug alone, were plotted on the
x- and y-axes, and the line of additivity was created by joining
both data points. The observed dose combination of two agents,
giving the defined Fa, located near or on the line of additivity
(achieved for a particular effect, e.g. 50%) represents an additive
drug–drug interaction, whereas dose combinations falling below
or above this line represent synergism or antagonism, respectively
[18].
The combination index (CI), a quantitative mathematical evalu-
ation of a pharmacological interaction between two drugs [22], was
calculated over a range of Fa levels from 0.01 to 1 (1–100% cell kill),
using data from the cytotoxicity experiments and CalcuSyn ver. 2.0
software (Biosoft, Cambridge, UK), values of CI < 1 indicate syner-
gism (the smaller the value, the greater the degree of synergism),
CI = 1 indicates additivity, and CI > 1 indicate antagonism. Each CI
ratio is represented as the average from at least three independent
experiments.
Fig. 2. Combination index (CI) at the affected fractions of 50% (IC50), of gemcitabine
or paclitaxel combined with the tetrahydroquinolines DM8 and DM12 on MCF-
7, SKBR3 (breast cancer cell lines and human dermic fibroblasts (control). Mean
IC50 SD (n = 3), (*) indicates significant differences among treatments at p < 0.05.
3. Results
3.1. Studies of cellular growth
The drugs and synthetic compounds used in this study were able
to inhibit the growth of breast cancer cells, as well as that of control
cells analyzed by the MTT assay (Table 1). In all cases, the inhibitory
effect of cellular growth showed a dose- and time-dependent
behavior (Fig. 1). Compounds DM8 and DM12 showed an inhibitory
activity on cellular growth, with a range of 20–100 ␮g/mL on
tumoral cell lines as well as on control cells. Gemcitabine and taxol
showed an inhibitory effect 50% higher than that of the synthetic
compounds, in spite of the fact that their concentrations were one
hundred times lower than those of the synthetic compounds. The
latter did not show an inhibitory activity at concentrations below
1 ␮g/mL.
Compound DM12 presented cytotoxic activity on both tumoral
cell lines, with IC50 values within the range of 4.92–6.77 ␮g/mL.
However, MCF-7 cells showed a small increase on their sensitiv-
ity to this compound when compared to SKBR3 cells. An IC50 of
58.55 ␮g/mL was obtained in control cells, evidencing a differ-
ence in DM12 cytotoxic activity, which was found to be up to
ten times higher than on breast cancer cell lines. Regarding DM8,
2.6. Statistical analysis
All experiments were done in triplicate. The results are
expressed as the average SD. The values of IC50 were determined
by a non-linear regression of individual experiments using the pro-
gram Graph Pad Prism (Intuitive Software for Science, San Diego,
CA, USA).

A. Mu˜noz et al. / Chemico-Biological Interactions 189 (2011) 215–221
219
Fig. 3. Isobolograms describing the interaction of gemcitabine and paclitaxel with the tetrahydroquinolines DM8 or DM12. The isobolograms were constructed by connecting
the IC50 values of gemcitabine and paclitaxel with the IC50 of DM8 or DM12. The solid lines indicate the theoretical additivity line. Results below the additivity line indicate
synergism and those above it antagonism. Mean IC50 SD (n = 3).
this compound presented cytotoxic activity from 25.77 ␮g/mL to
30.81 ␮g/mL for both breast cancer cell lines, which was lower than
the IC50 values found for DM12.
compounds DM8 and DM12 enhanced the inhibitory effects on
cancer cell lines and control cells. In all evaluated cell lines,
the inhibitory effect due to the combination of a cytotoxic
drug with DM8 or DM12 presented a dose-dependent behav-
ior.
The nature of the interaction of DM8 and DM12 with cytotoxic
drugs on cancer cell survival was realized with the help of the
isobolograms and the median effect method proposed by Chou and
Talalay [19–21]. The isobolograms (Fig. 3) described an interaction
between DM8 and DM12 with cytotoxic drugs. The interactions
between DM8 and DM12 with both cytotoxic drugs were strongly
antagonistic on MCF-7 cells. On SKBR3 cells, all tested combina-
tions among the cytotoxic drugs and compounds DM8 and DM12
presented a synergistic effect. On control cells, there was an antag-
onistic effect with DM8, a behavior observed on MCF-7 cells as well,
whereas combinations DM12-Gemcitabine and DM12-Taxol where
synergistic on both cell lines.
Valuesof SI are shownin Table1. Inthecase ofMCF-7, compound
DM12 presented a highly selective cytotoxic action, given that their
SI values were greater than 1. The activity of DM12 was up to 12
times more cytotoxic on cell lines than on the controls.
Unlike MCF-7 on SKBR3 cells three out of the four compounds
(DM8, taxol and gemcitabine) presented SI values below 1, indi-
cating a greater cytotoxic activity on control cells than on this cell
line.
3.2. Interaction of the gemcitabine and taxol with compounds
DM8 and DM12
Fig. 1 represents the survival of MCF-7 and SKBR3 in the pres-
ence of gemcitabine or taxol and DM8 or DM12, applied either
individually or combined in a 1:1 proportion. Gemcitabine and
taxol in the presence of an equal proportion of the synthetic
The analysis carried out with the aid of the median effect
method, revealed that combinations between DM8 and DM12 with

220
A. Mu˜noz et al. / Chemico-Biological Interactions 189 (2011) 215–221
the gemcitabine and taxol drugs resulted in either synergistic or
antagonistic effects, depending on the level of cell killing.
value for MCF-7 was higher than one, indicating an antagonism pos-
sibly due to competition among drugs. This is a different situation
from fibroblasts, in which value was lower than one (synergism).
The cytotoxicity of most antitumor compounds is thought to be
mediated by their capacity to induce apoptosis [27], other mech-
anisms of cytotoxicity could be by massive damage associated to
oxidative stress [28], and by telomerase expression in cancer cells,
but not in normal cells [29]. Investigations concerning the eluci-
dation of the mechanism of the action of DM12 are in progress.
The major interest is the discovery of antitumor products with an
original mechanism of action to increase the range of anticancer
agents. Such treatment would limit side-effects against normal cells
but remain far more effective against cancer cells than classical
cytostatics alone
The synergistic effect was statistically significant on SKBR3 cells,
reaching an inhibition level of 50% for the four possible combina-
tions, with CI values ranging from 0.02 to 0.65. For MCF-7 breast
cancer cell line, the combinations tested displayed an antagonistic
interaction, with CI values between 1.30 and 56.70 and an inhibi-
tion level of 50%. The assays performed on fibroblasts showed two
different patterns: (1) an antagonistic behavior of the DM8 gem-
citabime and taxol combination with CI values of 3.35 and 3.15,
respectively and (2) the combination of DM12 with gemcitabine
and taxol showed a synergistic behavior with CI values of 0.53 and
0.44, respectively, for an inhibition level of 50% (Fig. 2).
Finally, DM12 synthetic compound has a greater safety margin
as far as its toxicity on normal cells, although additional studies
must be conducted with in vivo models, and later clinical validation
to corroborate is efficacy as an agent in cancer therapeutics.
4. Discussion
The most relevant finding in the present study was that
the addition of a new series of cis-2,4-diaryl-r-3-methyl-1,2,3,4-
tetrahydroquinolines to cytotoxic drugs-based chemotherapy
enhanced the inhibitory effects of these drugs on breast cancer cell
lines MCF-7 and SKBR3. Individually, compounds DM8 and DM12
were able to induce growth inhibition on both cell lines, but the
addition of taxol and gemcitabine intensified the cytotoxic activ-
ity at concentrations below 1 ␮g/ml, level at which, when acting
individually, these drugs are generally inactive.
The isobolograms, constructed on the basis of IC50 values, indi-
cated the synergistic or antagonistic interactions between the DM8
and DM12 compounds and the cytotoxic drugs on MCF-7, SKBR3
and control cell line. There were synergistic interactions between
the tetrahydroquinolines and the cytotoxic drugs on SKBR3 cells;
a similar effect was observed for the combination of DM12 with
either taxol or gemcitabine on control cells.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
Financial support for FA by the Project PG 03-00-6569-2006
CDCH-UCV is gratefully acknowledged. VK and DM thank COLCIEN-
CIAS (CENIVAM, grant no. 432-2004) for financial support.
References
[1] F. Bray, R. Sankila, J. Ferlay, D.M. Parkin, Estimates of cancer incidence and
mortality in Europe in 1995, Eur. J. Cancer 38 (2002) 99–166.
These findings are supported by the quantification method pro-
posed by Chou and Taladay, which is based at an inhibition level of
50% and establishes synergism with values of CI lower than 1 [23].
DM12 compound showed synergistic effects with cytotoxic
drugs, with a relative low cytotoxicity towards normal fibroblasts
(as shown by the SI for IC50 control cell/IC50 SKBR3 cell line, see
Table 1) and SKBR3 cells. However, its effects towards MCF-7 cells
were antagonic, with relatively low cytotoxicity on control cells.
Meanwhile, DM8 compound showed antagonistic effects on MCF-
7 and control cell, and synergistic effects on SKBR3. These results
indicate that DM12 could be a more promising candidate for adju-
vant therapy than DM8. However, until now, DM12 compound has
been used only in in vitro assays with drugs and cell lines utilized in
this work. Clinical and pre-clinical studies have not been conducted.
Compounds DM8 and DM12 have a similar chemical structure,
with differences on substitutions of specific chemical groups. In
terms of cytotoxic effectiveness, it was demonstrated that the pres-
ence of an amino group (NH2) in the DM8 structure generates a low
cytotoxic activity on breast cancer cell lines, in comparison with
DM12, which presents an ortho substitution of two nitro groups.
Consequently, DM12 cytotoxic activity on MCF-7 cells was up to
6 times greater with respect to DM8 and up to 4 times greater on
SKBR3.
Based on the obtained SIs, we hypothesize that the differences
found in the assays on SKBR3 are due to the intrinsic characteristic
of encoding a nonfunctional p53 protein [24,25], which represents
an advantage on cell proliferation [26]. Thus, the cytotoxic activity
on tumoral cells is less pronounced than on control cells.
Observing the CIs of DM12 on MCF-7 and SKBR3 cells and com-
paring them to the indices obtained by taxol and gemcitabine, it
is demonstrated that DM12 cytotoxic activity is more selective for
tumoral cells. Cytotoxic selectivity was also found for taxol on MCF-
7 cells, and gemcitabine on SKBR3 cells. On the other hand, the CI
[2] C. Mettlin, Global breast cancer mortality statistics, CA Cancer J. Clin. 49 (1999)
138–144.
[3] P.A. Greenberg, G.N. Hortobagy, T.L. Smith, L.D. Ziegler, D.K. Frie, A.U. Buzdar,
Long-term follow-up of patients with complete remission following combi-
nation chemotherapy for metastatic breast cancer, J. Clin. Oncol. 14 (1996)
2197–2205.
[4] G. Falkson, D.C. Tormey, P. Carey, R. Witte, H.C. Falkson, Long-term survival of
patients treated with combination chemotherapy for metastatic breast cancer,
Eur. J. Cancer 27 (1991) 973–977.
[5] J.R. Kroep, G. Giaccone, C. Tolis, D.A. Voorn, W.J. Loves, C.J. Groeninger, H.M.
Pinedo, G.J. Peters, Sequence dependent effect of paclitaxel on gemcitabine
metabolism in relation to cell cycle and cytotoxicity in non-small-cell lung
cancer cell lines, Br. J. Cancer 83 (2000) 1069–1076.
[6] J. Carmichael, The role of gemcitabine in the treatment of other tumours, Br. J.
Cancer 78 (Suppl. 3) (1998) 21–25.
[7] I. Jacquemond-Collet, F. Benoit, C. Mustofa, A. Valentin, E. Stanislas, M. Mallié, I.
Fourasté, Antiplasmodial and cytotoxic activity of galipinine and other tetrahy-
droquinolines from Galipea officinalis, Planta Med. 68 (2002) 68–69.
[8] O. Wallace, K. Lauwers, S. Jones, J. Dodge, Tetrahydroquinoline-based selec-
tive estrogen receptor modulators (SERMs), Bioorg. Med. Chem. Lett. 13 (2003)
1907–1910.
[9] G. Dorey, B. Lockhart, P. Lestage, P. Casara, New quinolinic derivatives as cen-
trally active antioxidants, Bioorg. Med. Chem. Lett. 10 (2000) 935–939.
[10] S. Vangapandu, M. Jain, S. Kaur, P. Singh, Ring-substituted quinolines as poten-
tial anti-tuberculosis agents, Bioorg. Med. Chem. 12 (2004) 2501–2508.
[11] V. Monga, A. Nayyar, B. Vaitilingam, P. Palde, S. Jhamb, S. Kaur, P. Singh, Ring-
substituted quinolines. Part 2: Synthesis and antimycobacterial activities of
ring-substituted quinolinecarbohydrazide and ring-substituted quinolinecar-
boxamide analogues, Bioorg. Med. Chem. 12 (2004) 6465–6472.
[12] M. Khan, Quinoline analogs as antiangiogenic agents and telomerase inhibitors,
Top. Heterocycl. Chem. 11 (2007) 213–229.
[13] J. Isaacs, R. Pili, D. Qian, S. Dalrymple, J. Garrison, N. Kyprianou, A. Björk, Iden-
tification of ABR-215050 as lead second generation quinoline-3-carboxamide
anti-angiogenic agent for the treatment of prostate cancer, Prostate 66 (2006)
1768–1778.
[14] V. Caprio, B. Guyen, Y. Opoku-Boahen, J. Mann, S. Gowan, L. Kelland, M. Read, S.
Neidle, A novel inhibitor of human telomerase derived from 10H-indolo[3,2-
b]quinoline, Bioorg. Med. Chem. Lett. 10 (2000) 2063–2066.
[15] V.V. Kouznetsov, D.R. Merchan Arenas, F. Arvelo, J.S. Bello Forero, F.
Sojo, A. Mun˜oz, New 4-hydroxy-3-methoxyphenyl substituted 3-methyl-
tetrahydroquinoline derivatives as potential antitumoral agents obtained
through imino Diels–Alder reactions, Lett. Drug Design Discov.
632–639.
7 (2010)

A. Mu˜noz et al. / Chemico-Biological Interactions 189 (2011) 215–221
221
[16] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: appli-
cation to proliferation and cytotoxicity assay, J. Inmunol. Methods 65 (1983)
55–63.
[23] G.J. Peters, C.L. Van Der Wilt, C.J. Van Moorsel, J.R. Kroep, A.M. Bergman,
S.P. Ackland, Basis for effective combination cancer chemotherapy with
antimetabolites, Pharmacol. Therap. 87 (2000) 227–253.
[17] D. Callacondo, A. Quispe, S. Lindo, A. Vaisberg, Actividad citotóxica del extracto
etanólico de Gnaphalium spicatum “Keto Keto” en cultivos de líneas celulares
tumorales humanas, Rev. Peru Med. Exp. Salud Pública 25 (2008) 380–385.
[18] P.K. Gessner, Isobolographic analysis of interactions: an update on applications
and utility, Toxicology 105 (1995) 161–179.
[24] M.V. Blagosklonny, J. Toretsky, L. Weckers, Geldanamycin selectively destabi-
lizes and conformationally alters mutated p53, Oncogene 11 (1995) 933–939.
[25] J. Kovach, R. McGovern, I. Cassady, S. Swason, L. Wald, B. Vogelsteim, S. Som-
mer, Direct sequencing from touch preparations carcinomas: analysis of p53
mutation in breast carcinomas, J. Natl. Cancer Inst. 83 (1991) 1004–1009.
[26] J. Cossman, R. Schlegel, p53 in the diagnosis of human neoplasia, J. Natl. Cancer
Inst. 83 (1991) 980–981.
[19] T.C. Chou, P. Talalay, Analysis of combined drug effects: a new look at a very
old problem, Trends Pharmacol. Sci. 4 (1983) 445–450.
[20] T.C. Chou, P. Talalay, Quantitative analysis of dose–effect relationships: the
combined effects of multiple drugs or enzyme inhibitors, Adv. Enzyme Regul.
22 (1984) 27–55.
[21] J.C. Chou, Quantitation of synergism and antagonism of two or more drug by
computerized analysis, in: T.C. Chou, D.C. Rideout (Eds.), Synergism and Antag-
onism in Chemotherapy, Academic Press, New York, 1991, pp. 223–244.
[22] T.C. Chou, N. Martin, A computer program for quantitation of synergism and
antagonism in drug combinations, and the determination of IC50 and ED50
and LD50 values, in: CompuSyn for Drug Combinations: PC Software and User’s
Guide, ComboSyn, Paramus, NJ, 2005.
[27] I. Tamm, F. Schriever, B. Dörken, Apoptosis: implications of basic research for
clinical oncology, Lancet 2 (2001) 33–42.
[28] Q. Li, S. Peng, Z. Sheng, Y. Wan, Ofloxacin induce oxidative damage to join chon-
drocytes of juvenile rabbits: excessive production of reactive oxygen species,
lipid peroxidation and DNA damage, Eur. J. Pharmacol. 626 (2010) 146–153.
[29] U. Ghosh, N. Bhattacharyya, Benzamide and 4-amino 1,8 naphthalamide
treatment inhibe telomerase activity by down-regulation the expression
of telomerase associated protein and inhibiting the poly ADP-ribosylation
of telomerase reverse transcriptase in cultured cells, FEBS J. 272 (2005)
4237–4424.