Inhibitory effect of cytotoxic nitrogen-containing heterocyclic stilbene analogues on VEGF protein secretion and VEGF, hTERT and c-Myc gene expression

Article abstract of DOI:10.1039/c5md00197h

A group of 21 nitrogen-containing heterocyclic stilbene derivatives, prepared by means of Heck coupling reactions, has been investigated for their cytotoxicity as well as their ability to inhibit the production of the vascular endothelial growth factor (VEGF) and the activation of telomerase. The ability of these compounds to inhibit proliferation of two tumoral cell lines (HT-29 and MCF-7) and one non-tumoral cell line (HEK-293) was first determined. Subsequently, we determined the capacity of the compounds to inhibit the secretion of VEGF in the aforementioned cell lines and downregulate the expression of the VEGF, hTERT and c-Myc genes, of which the latter two are involved in controlling the activation of telomerase.

Full text of DOI:10.1039/c5md00197h

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Inhibitory effect of cytotoxic nitrogen-containing
heterocyclic stilbene analogues on VEGF protein
secretion and VEGF, hTERT and c-Myc gene
expression†
Cite this: DOI: 10.1039/c5md00197h
a
a
a
a
*
*
Rosa Martí-Centelles, Juan Murga, Eva Falomir, Miguel Carda
and J. Alberto Marcob
A group of 21 nitrogen-containing heterocyclic stilbene derivatives, prepared by means of Heck coupling
reactions, has been investigated for their cytotoxicity as well as their ability to inhibit the production of the
vascular endothelial growth factor (VEGF) and the activation of telomerase. The ability of these compounds
to inhibit proliferation of two tumoral cell lines (HT-29 and MCF-7) and one non-tumoral cell line (HEK-
293) was first determined. Subsequently, we determined the capacity of the compounds to inhibit the sec-
retion of VEGF in the aforementioned cell lines and downregulate the expression of the VEGF, hTERT and
c-Myc genes, of which the latter two are involved in controlling the activation of telomerase.
Received 7th May 2015,
Accepted 10th August 2015
DOI: 10.1039/c5md00197h
www.rsc.org/medchemcomm
chromosomal telomeres.12 Telomeres are the terminal zones
of chromosomes and display a special structure that fulfills
Introduction
It is widely known that cancer, which is one of the leading
causes of death in developed countries,1 may be induced by a
plethora of both external and internal factors, including
genetic mutations. Accordingly, a number of types of thera-
peutic attack have been investigated.2,3 One of these involves
the use of cytotoxic drugs, which exert their effect in many
cases by means of inducing cell apoptosis.4
The second type of therapeutic strategy against cancer is
based on the use of compounds with vascular-targeting prop-
erties. These may be due to their ability to either inhibit the
formation of new blood vessels (anti-angiogenic agents) or
promote the destruction of existing ones (anti-vascular
agents).5,6 Tumor angiogenesis is a very complex process and
involves the tight interplay of many factors.7 Vascular endo-
thelial growth factor (VEGF) is an inducer of angiogenesis
and promotes endothelial cell survival, proliferation and
migration while increasing vascular permeability.8 In fact,
overexpression in the production of VEGF has been reported
to occur in various types of tumors.9 Not unexpectedly, VEGF
has become one further key target molecule in cancer ther-
apy.10,11 The third therapeutic line follows the path of
at least two essential functions: (a) they must be recognized
as functional domains, thus distinguishing them from ran-
dom chromosomal breaks that would stimulate the onset of
repair mechanisms and (b) they must prevent these DNA
ends from fusing with other DNA ends.13 To comply with
these functions, an appropriate amount of repetitive DNA
sequences (telomeres) must be added to the ends of the chro-
mosomes. This task is fulfilled by a special type of ribonu-
cleoprotein complex called telomerase, an enzyme with
reverse transcriptase activity. The expression of this enzyme
is restricted or absent in normal human somatic cells and so
telomeres progressively shorten during a cell's lifespan.12
This triggers a DNA damage response which culminates
in cellular senescence or apoptosis.14 Cancer cells are able
to avoid senescence upon telomerase activation.15 This
enzyme has been detected in about 90% of all malignant
tumors,16 thus drugs with the ability to inhibit telomerase
activity are potentially useful weapons in the fight against
cancer, aging and other diseases related to premature
telomere shortening.17
When aiming for a more efficient anticancer therapy, com-
pounds exhibiting not only high cytotoxic activity but also
the ability to suppress tumoral angiogenesis and gene expres-
sion related to telomerase activation would be highly desir-
able.18 In our ongoing research on the anticancer properties
of synthetic analogues derived from natural products, we
have recently reported the inhibition of VEGF formation in
cancer cells and on endothelial cell differentiation promoted
a Depart. de Química Inorgánica y Orgánica, Univ. Jaume I, E-12071 Castellón,
Spain. E-mail: efalomir@qio.uji.es; Fax: +34 964728214; Tel: +34 964728240
b Depart. de Química Orgánica, Univ. Valencia, C/Dr. Moliner 50, E-46100
Burjassot, Spain
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5md00197h
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by synthetic stilbene derivatives.19 Our study has established
that the naturally occurring stilbene resveratrol20,21 (Fig. 1)
and several synthetic stilbene derivatives bring about a
decrease in the production of VEGF by HT-29 cells. Further-
more, it has been reported that resveratrol downregulates the
telomerase activity of MCF-7 cells and the nuclear levels of
hTERT, the reverse transcriptase subunit of the telomerase
complex.18
In view of these precedents, we decided to evaluate the
influence of the new synthetic stilbene analogues not only on
the production of VEGF, but also on the expression of the
hTERT gene, which is involved in the process of telomerase
activation. In addition, we further decided to measure the
inhibition of expression of c-Myc, an oncogene which
encodes the c-Myc protein that acts as a transcription factor
for hTERT expression.
scarce. Representatives of this class of compounds have been
found to display protein kinase inhibitory,23 anti-inflamma-
tory24 and antiproliferative25 activities, as well as other vari-
ous biological properties.26
The structures of the synthetic nitrogen-containing stil-
bene derivatives which are the subject of this work are
depicted in Fig. 2. These compounds are classified into two
types; one of them corresponds to stilbenes displaying one
nitrogen atom (compounds 1–15 in Fig. 2), whereas the other
(compounds 16–21) includes stilbenes carrying two nitrogen
atoms in one of the aromatic rings.
Results and discussion
Chemistry
Azastilbene derivatives 1–21 were prepared by means of
palladium-catalyzed Heck-type cross-coupling reactions
(Scheme 1).27 In the case of compounds 1–9 and 16–21,
methoxylated styrenes were used as the nucleophilic partners
with the appropriate bromopyridine or bromopyrimidine
playing the role of the electrophilic component and
PdIJNH3)2Cl2 being the catalyst (conditions A in Scheme 1). For
compounds 10–15, 4-vinylpyridine was allowed to react with the
appropriate bromophenol or bromoaniline under slightly differ-
ent conditions (conditions B in Scheme 1). These reactions were
performed under microwave irradiation, as these conditions
have often been found to accelerate this kind of coupling reac-
tion28 (for experimental details, see the ESI†).
While studies on the pharmacological properties of natu-
ral and synthetic stilbene derivatives are numerous,22 reports
related to their heterocyclic aza analogues are comparatively
Fig. 1 Structure of the natural stilbene resveratrol.
Biological studies
Cytotoxicity of the azastilbene derivatives
We carried out measurement of the cytotoxic activity of our
synthetic azastilbenes using two tumoral cell lines, the
Scheme 1 Synthesis of the heterocyclic azastilbene derivatives by means
of palladium-catalyzed Heck coupling reactions (for more detailed
descriptions of the reaction conditions and yields, see the ESI†).
Fig.
2 Structures of the nitrogen-containing heterocyclic stilbene
analogues investigated in this study.
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human colon HT-29 and the breast adenocarcinoma MCF-7
cell lines, as well as one non-tumoral cell line, the human
embryonic kidney cell line, HEK-293.29 Table 1 shows the
cytotoxicity values for azastilbenes 1–21, expressed as the
compound concentration (μM) that causes 50% inhibition of
cell growth (IC50). Resveratrol has been included for the pur-
pose of comparison. Table 1 further shows the α and β coeffi-
cients, obtained by dividing the IC50 values of the normal cell
line by those of either the HT-29 or the MCF-7 cell lines,
respectively (see footnote in Table 1). The higher the value of
either coefficient, the higher the therapeutic safety margin of
the compound in the corresponding cell line.
The cytotoxicity of compounds 1–21 was in general low or
moderate. With regard to the HT-29 cell line, only com-
pounds 7–9 have IC50 values below 100 μM. For MCF-7, com-
pound 1 proved to be highly cytotoxic but only compounds 6,
8, 11 and 17 have IC50 values below 100 μM. Compound 1 is
noteworthy as it has high cytotoxicity towards the MCF-7 cell
line but only weak activity towards the HT-29 cell line.30
HT-29 cell line were used. Accordingly, the concentrations
were always 10 μg mL−1 (ca. 50 μM) except for compound 8,
which was used at a concentration of 5 μg mL−1 (ca. 25 μM)
because of its higher cytotoxicity.
The secretion of the vascular endothelial growth factor
(VEGF) was first determined by means of the ELISA proce-
dure,31 as described in the Experimental section. The results
for the selected compounds are depicted in Fig. 3, which
shows the percentage of VEGF secreted to the culture
medium after 72 h of incubation in the presence of DMSO
(control experiment) and each of the compounds investigated
at a concentration of 10 μg mL−1 (lower than their IC50
values).
Compounds 7 and 9 show an appreciable ability to inhibit
the production of the VEGF protein (reduction to less than
50% of the control value). Interestingly, this value indicates a
much stronger activity than that shown by resveratrol (78%).
Compound 8, which has the highest cytotoxicity (Table 1),
also shows noticeable inhibitory activity on VEGF production
(52% of the control value), which is still stronger than that of
resveratrol.
Effect of azastilbene derivatives 1–21 on VEGF protein
While the results depicted in Fig. 3 point out that the
investigated compounds cause inhibition of VEGF produc-
tion, they do not permit immediate conclusions about the
precise phase of the VEGF generation process with which
they interfere. In order to further investigate the mechanism
of action of the compounds under study, we proceeded to
determine whether they were able to control protein produc-
tion at the transcriptional level. The VEGF production we
have determined with the ELISA procedure is that secreted to
the culture medium and corresponds mainly to the lighter
VEGFA-165 isoform.32 Therefore, in order to evaluate the abil-
ity of our compounds to inhibit the expression of the VEGF
gene, we incubated HT-29 cells for 48 h with several selected
stilbene derivatives at a concentration of 10 μg mL−1, again
secretion and VEGF gene inhibition
The action of the stilbene derivatives on VEGF secretion and
VEGF gene inhibition was conducted on the HT-29 tumoral
cell line. For these measurements, compounds 3, 5, 7, 8, 9,
14 and 19 were selected because they had α coefficients
greater than 1. In addition, compound 15 was also selected
because it had a lower IC50 value than resveratrol. For these
assays, concentrations lower than the IC50 values towards the
Table 1 IC50 values (μM) and selectivity coefficients for azastilbenes
1–21a
Compd
HT-29
150
MCF-7
71
HEK-293
31
αb
βc
Resveratrol
6
5
4
0.2
0.4
1
2
3
4
5
6
7
8
274 14
223 24
1.7 0.7
204 33
147 47
190 14
128 19
175 28
68 10
199 33
0.6
0.3
1.7
0.7
1.2
0.7
1.1
1.3
1.9
<0.5
0.1
<0.1
<0.1
>3
0.4
<0.6
0.7
0.4
1.1
>100
0.3
1.3
0.6
0.9
3.4
0.9
0.6
0.8
<0.5
0.6
0.8
<0.1
>2
~2
<0.6
2.6
0.4
<0.3
<0.4
<0.5
114
3
142 19
104 24
299 38
76 14
107
3
123 28
213 28
85 14
62
3
100 19
88 11
142 14
>500
44
61
4
2
56
8
9
114 24
10
11
12
13
14
15
16
17
18
19
20
21
>500
243
45
13
6
310 41
168 20
255 20
168 20
100 11
>500
170 57
267 38
146 19
302 52
>500
71
6
4
2
9
16 0.1
383 102
235 15
20.6 0.26
>500
44.1 1.0
274 52
>500
13
>500
41 15
274 47
113 24
118 38
160 28
198 33
259 14
Fig. 3 VEGF protein secretion from the HT-29 cells determined by
means of the ELISA procedure. At least three measurements were
performed in each case. The concentration of all compounds was 10
μg mL−1 (for 8, 5 μg mL−1). Bars represent mean values of VEGF secre-
tion (percentage values related to control) and error bars indicate stan-
dard errors of the mean. The statistical significance was evaluated
using one-sample t-tests (P < 0.001).
>500
>500
~0.7
<0.5
a
Values are the average ( s.d.) of three different measurements
b
performed as described in the Experimental section. α = IC50 (HEK-
293)/IC50 (HT-29). β = IC50 (HEK-293)/IC50 (MCF-7). The values of α
and β have been rounded off to a decimal figure.
c
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with DMSO as the control set-up. For this purpose, the
reverse transcription quantitative PCR (RT-qPCR) methodol-
ogy33 was used as described in the Experimental section. The
results for all the selected compounds are shown in Fig. 4 as
the percentage of VEGF gene expression. All values were stan-
dardized (100%) relative to the control (DMSO) and to
β-actin.
It is worth noting that compounds 7 and 9, which had the
strongest inhibitory activity on the production of the VEGF
protein, now display a markedly lower ability to inhibit the
expression of the corresponding VEGF gene. In contrast, com-
pound 14, which was only weakly active in inhibiting VEGF
production, now shows the strongest ability to inhibit the
expression of the VEGF gene (48% of the control value). This
suggests that these compounds exert control of VEGF produc-
tion at a phase different from that of gene transcription, per-
haps during the post-translational stage, during which lighter
isoforms32 such as VEGFA-165 are formed and secreted to the
medium.
subunit is the rate-limiting factor of the enzyme complex. For
the expression of the hTERT gene, several transcriptional fac-
tors such as c-Myc, a proto-oncoprotein,34 have been found
to play an important role through upregulation of the mRNA
encoding the hTERT protein subunit.35 Thus, as an initial
study of the potential activity of these stilbene derivatives as
inhibitors of telomerase activation, we have investigated their
ability to inhibit the expression of the hTERT and c-Myc
genes, which are responsible for the production of the corre-
sponding proteins.
In order to determine whether the studied compounds
were able to regulate the expression of the two aforemen-
tioned genes, we have performed RT-qPCR analysis using
again HT-29 tumoral cells. The cells were incubated for 48 h
in the presence of DMSO (control) and, same as above, 10 μg
mL−1 of each of the studied compounds (for 8, 5 μg mL−1).
The results are depicted in Fig. 5 (hTERT gene expression)
and Fig. 6 (c-Myc gene expression).
Compounds 3, 5 and 7 display a similar ability to inhibit
the expression of the hTERT gene (reduction to about 50% of
the control value, a value comparable with that of resvera-
trol). The remaining compounds, including 8, only show mar-
ginal activities, a little below that of the control. However, the
profile of activities with regard to the inhibition of expression
of the c-Myc gene (Fig. 6) proved to be markedly different.
For instance, compounds 3, 5 and 7, which had an apprecia-
ble inhibitory activity on hTERT gene expression (Fig. 5), are
much less active in inhibiting c-Myc gene expression. In con-
trast, compounds 8, 9, 14 and 15, which had a very weak
inhibitory activity on hTERT gene expression, are now much
more active in inhibiting c-Myc gene expression, most partic-
ularly 8 and 15, which have an activity similar to that of
resveratrol.
Effect of the stilbene derivatives on inhibition of the hTERT
and c-Myc genes
Human telomerase contains an RNA component (hTERC)
that serves as a template for the addition of the repeated
nucleotide sequences and also a reverse transcriptase subunit
(hTERT) which catalyzes the nucleotide polymerization pro-
cess. In addition, there are other associated protein factors,
the roles of which have not yet been completely elucidated.
Human telomerase is regulated during development and dif-
ferentiation, mainly through transcriptional control of the
hTERT gene, the expression of which is restricted to cells that
exhibit telomerase activity. This indicates that the hTERT
Fig.
4
Expression percentage of the VEGF gene after 48
h
of
Fig. 5 Expression percentage of the hTERT gene after 48 h of
incubation of the HT-29 cells determined by means of the RT-qPCR
methodology. At least three measurements were performed in each
case. The concentration of all compounds was 10 μg mL−1 (for 8, 5 μg
mL−1). Error bars indicate standard errors of the mean. The statistical
significance was evaluated using one-sample t-tests (P < 0.001).
incubation of the HT-29 cells determined by means of the RT-qPCR
methodology. At least three measurements were performed in each
case. The concentration of all compounds was 10 μg mL−1 (for 8, 5 μg
mL−1). Error bars indicate standard errors of the mean. The statistical
significance was evaluated using one-sample t-tests (P < 0.001).
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F i g . 8 IC 5 0 valu es toward s th e HT-29 c ell line f or IJ E) -
IJmethoxystyryl)pyridines and IJE)-IJmethoxystyryl)pyrimidines.
Fig.
6 Expression percentage of the c-Myc gene after 48 h of
derivatives compared to that of the 2-pyridine, 3-pyridine and
pyrimidine rings, regardless of the position of the methoxy
group in the other ring. It can be deduced from this figure
that the most cytotoxic activity is again retained by the IJE)-4-
IJmethoxystyryl)pyridines (compounds 7–9).
incubation of the HT-29 cells determined by means of the RT-qPCR
methodology. At least three measurements were performed in each
case. The concentration of all the compounds was 10 μg mL−1 (for 8, 5
μg mL−1). Error bars indicate standard errors of the mean. The statisti-
cal significance was evaluated using one-sample t-tests (P < 0.001).
Moreover, some of the compounds displayed noticeable
inhibitory activities in the production of the angiogenic pro-
tein factor VEGF, as well as in the expression of the
telomerase-related hTERT and c-Myc genes. For example,
compound 7 (Fig. 9) was able to reduce VEGF protein secre-
tion down to 37% of the control value, and hTERT gene
expression to 54% of the control value. This compound there-
fore features better than resveratrol (78% of VEGF secretion
and 57% of hTERT gene expression at the same concentra-
tion). In addition, the concentration at which compound 7
exhibits this activity is clearly below its IC50 value towards
the non-tumoral HEK-293 cells. This opens the possibility of
such a compound having a potential anticancer activity
through a dual mechanism at doses that are non-toxic for
non-tumoral cells. Another compound which deserves men-
tion because of its potential multiple mode of action is com-
pound 8 (Fig. 9). Besides having the highest cytotoxicity
towards HT-29 (Table 1), it reduces VEGF production down to
52% of the control value and c-Myc gene expression to 50%
of the control value. As in the case of 7, it behaves similarly
or better than resveratrol with regard to these biological
properties. Thus, the two azastilbene derivatives that feature
best in the aforementioned properties have in common a pyr-
idine ring substituted at C-4 and an ortho or meta methoxy
group in the benzene ring. Other substitution patterns, how-
ever, have apparently a detrimental effect.
Summary and conclusions
In conclusion, heterocyclic azastilbene derivatives 1–21 have
been synthesized and evaluated for their biological activity.
While their cytotoxicities were not high, some of these deriva-
tives (compounds 3, 5, 7, 8 and 9) exhibited lower IC50 values
than resveratrol towards HT-29 and higher IC50 values
towards a non-tumoral cell line such as HEK-293. In this
respect, they compare favorably with resveratrol. In Fig. 7, the
IC50 values of the IJE)-4-IJsubstituted styryl)pyridines (com-
pounds 7–15) towards HT-29 cells are presented. As seen in
this figure, the most active compounds were those bearing
methoxy substituents on the phenyl ring (compounds 7–9).
In Fig. 8, the IC50 values of IJE)-4-IJmethoxystyryl)pyridines
as well as the remaining methoxy derivatives towards HT-29
cells are presented. As deduced from Fig. 8, the presence of
the 4-pyridine ring improves the cytotoxic activity of these
Fig. 7 IC50 values towards the HT-29 cell line for IJE)-4-IJsubstituted
Fig. 9 Structures of two azastilbene analogues with potential multiple
styryl)pyridines.
modes of action.
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Experimental
Chemical procedures
Assays: Hs99999903-m1 (β-actin), Hs00900055-m1 (VEGF),
Hs00972646-m1 (hTERT) and Hs00153408-m1 (c-Myc).
Detailed descriptions of the reaction conditions, yields and
physical data are given in the ESI.†
Acknowledgements
Financial support has been granted by the Spanish Govern-
ment (MINECO, Ministerio de Economía y Competitividad,
project CTQ2011-27560), the Consellería d'Empresa,
Universitat i Ciencia de la Generalitat Valenciana (projects
PROMETEO/2013/027 and ACOMP/2014/272) and the
Universitat Jaume I IJPI-1B-2011-37). R. M.-C. thanks the
Universitat Jaume I for a pre-doctoral fellowship.
Biological procedures
Cell culture. The cell culture media were purchased from
Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was
a product of Harlan Sera-lab (Belton, UK). Supplements and
other chemicals not listed in this section were obtained from
Sigma Chemicals Co. (St. Louis, MO, USA). Plastics for cell
culture were supplied by Thermo Scientific™ BioLite. All
tested compounds were dissolved in DMSO at a concentra-
tion of 10 μg mL−1 and stored at −20 °C until use.
The cell lines were maintained in Dulbecco's modified
Eagle's medium (DMEM) containing glucose (1 g L−1), gluta-
mine (2 mM), penicillin (50 IU mL−1), streptomycin (50 μg
mL−1) and amphotericin (1.25 μg mL−1), supplemented with
10% FBS.
Cytotoxicity assays. The 3-IJ4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT; Sigma Chemical Co., St.
Louis, MO) dye reduction assay in 96-well microplates was
used, as previously described.36 Some 5 × 103 cells of HT-29,
MCF-7 or HEK-293 cell lines in a total volume of 100 μL of
their respective growth media were incubated with serial dilu-
tions of the tested compounds. After 3 days of incubation (37
°C, 5% CO2 in a humid atmosphere), 10 μl of MTT (5 mg
ml−1 in PBS) was added to each well and the plate was incu-
bated further for 4 h (37 °C). The resulting formazan was
dissolved in 150 μL of 0.04 N HCl/2-propanol and read at 550
nm. All determinations were carried out in triplicate.
ELISA analysis. HT-29 cells at 70–80% confluence were col-
lected after serum starvation for 24 h. The cells were incu-
bated with the selected stilbene derivatives in DMSO for 72 h
(Fig. 3). Culture supernatants were collected and VEGF sec-
reted by HT-29 cells was determined using an Invitrogen
human vascular endothelial growth factor ELISA kit
according to the manufacturer's instructions.
RT-qPCR analysis. HT-29 cells at 70–80% confluence were
collected after serum starvation for 24 h. The cells were incu-
bated with 10 μg mL−1 of the corresponding drugs in DMSO
for 48 h (for 8, 5 μg mL−1). The cells were collected and the
total cellular RNA from the HT-29 cells was isolated using an
Ambion RNA extraction kit according to the manufacturer's
instructions. The cDNA was synthesized by MMLV-RT with 1–
21 μg of extracted RNA and oligoIJdT)15 according to the man-
ufacturer's instructions.
Amplification of the genes was performed by the use of a
StepOnePlus™ thermal cycler. A TaqMan fast gene expres-
sion master mix containing the appropriate buffer for the
amplification conditions, dNTPs, a thermostable DNA poly-
merase enzyme and a passive reference probe was used. Each
of the genes was amplified using the following predesigned
primers by Life Technologies TaqMan® Gene Expression
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