Anticancer activity and cDNA microarray studies of a (RS)-1,2,3,5- tetrahydro-4,1-benzoxazepine-3-yl]-6-chloro-9H-purine, and an acyclic (RS)-O,N-acetalic 6-chloro-7H-purine

Article abstract of DOI:10.1016/j.ejmech.2011.05.047

Completing a SAR study, a series of (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7H or 9H-purines was previously prepared. The most potent antiproliferative agent against the MCF-7 adenocarcinoma cell line that belongs to the ben

Full text of DOI:10.1016/j.ejmech.2011.05.047

European Journal of Medicinal Chemistry 46 (2011) 3802e3809
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anticancer activity and cDNA microarray studies of a (RS)-1,2,3,5-tetrahydro-4,
1-benzoxazepine-3-yl]-6-chloro-9H-purine, and an acyclic (RS)-O,N-acetalic
6-chloro-7H-purine
Octavio Caba a, Mónica Díaz-Gavilán b, Fernando Rodríguez-Serrano a, Houria Boulaiz a,
,
b,
**
Antonia Aránega a, Miguel A. Gallo b, Juan A. Marchal a , Joaquín M. Campos
*
a Instituto de Biopatología y Medicina Regenerativa (IBIMER), Departamento de Anatomía y Embriología Humana, Facultad de Medicina, Avenida de Madrid s/n,
18071 Granada, Spain
b Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, c/ Campus de Cartuja s/n, 18071 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Completing a SAR study, a series of (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-
7H or 9H-purines was previously prepared. The most potent antiproliferative agent against the MCF-7
adenocarcinoma cell line that belongs to the benzoxazepine O,N-acetalic family is (RS)-9-[1-(9H-fluorenyl-
9-methoxycarbonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl]-6-chloro-9H-purine (16, IC50 ¼ 0.67 ꢀ
Received 15 February 2011
Received in revised form
11 May 2011
Accepted 19 May 2011
Available online 27 May 2011
0.18
mM), whilst (RS)-7-{2-(N-hydroxymethylphenyl)-2-nitrobenzenesulfonamido]-1-methoxyethyl}-6-
chloro-7H-purine (37) shows the lowest IC50 value between the family of acyclic O,N-acetals
M). Moreover, 16 showed the better in vitro Therapeutic Index in breast cell lines (3.19),
(IC50 ¼ 3.25 ꢀ 0.23
m
Keywords:
whilst 37 was found to be 3.69-fold more active against HT-29 human colon cancer cell line than versus IEC-6
normal rat intestinal epithelial cell line. The global apoptotic cells caused by 16 and 37 against MCF-7 were
80.08% and 54.85% of cell population after 48 h, respectively. cDNA microarray technology reveals potential
drug targets, which are mainly centred on positive apoptosis regulatory pathway genes, and the repression of
genes involved in carcinogenesis, proliferation and tumour invasion.
Acyclic Purine O,N-acetals
Anti-tumour activity
Apoptosis
cDNA microarray
Invasion
1,2,3,5-Tetrahydro-4,1-benzoxazepines
MCF-7
Ó 2011 Elsevier Masson SAS. All rights reserved.
Therapeutic index
1. Introduction
tumour specimens. Since the technology allows for the simulta-
neous analysis of many thousands of individual genes in cell or
In recent years, microarrays have been extensively used to study
molecular differences between different types of cancer. One of the
most intensely studied tumour types in this context are breast and
colon cancers. Characteristic patterns of gene expression have
emerged, reflecting molecular differences between previously
known, as well as newly defined, subtypes of breast and colon
cancers. These molecular differences have been shown to correlate
with clinical features such as survival, prognosis, and treatment
sensitivity, as well as traditional histopathological parameters.
Array-based molecular profiling represents a technological
breakthrough in how biological samples can be analyzed such as
tissue samples, the complex biology of cancer may now be studied
much more comprehensively than was previously possible. Present
technology allows for microarrays features to be genome-
representative (the human genome sequence suggests that the
total number of genes is approximately 20,000e25,000 [1]). Hence,
it is nowadays possible to monitor gene expression or copy number
levels of almost all known human genes in a biological specimen in
a single experiment. The impact of this technology defines a revo-
lution in basic biological science, and once fully standardized and
accurately validated it is likely to revolutionize the practice of
medicine.
In a short communication we reported [2] the anticancer
activities against the MCF-7 human breast cancer cell line of ben-
zoxazepine O,N-acetals 1e26 (Chart 1) and acyclic O,N-acetals
27e37 (Chart 2). We have selected the two most active compounds
of both families (16 for the cyclic O,N-acetals, and 37 for the acyclic
O,N-acetals) and in this manuscript we report the anticancer
* Corresponding author. Tel.: þ34 958 249321; fax: þ34 958 246296.
** Corresponding author. Tel.: þ34 958 243848; fax: þ34 958 243845.
E-mail addresses: jmarchal@ugr.es (J.A. Marchal), jmcampos@ugr.es
(J.M. Campos).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.047

O. Caba et al. / European Journal of Medicinal Chemistry 46 (2011) 3802e3809
3803
R1
N
R1
N
R2
O
N
O
NH
N
O
O
NH
O
O
R2
1
2
3
4
5
6
7
R1 = H, R2 = F
8
9
R1 = H, R2 = F
R1 = SO2-C6H4-pNO2; R2 = F
R1 = SO2-C6H4-pNH2; R2 = F
R1 = SO2-C6H4-oNO2; R2 = F
R1 = SO2-C6H4-oNH2; R2 = F
R1 = SO2-C6H4-pNO2; R2 = H
R1 = SO2-C6H4-oNO2; R2 = H
R1 = SO2-C6H4-pNO2; R2 = F
R1 = SO2-C6H4-oNO2; R2 = F
R1 = SO2-C6H4-oNH2; R2 = F
R1 = SO2-C6H4-pNH2; R2 = H
R1 = SO2-C6H4-oNO2; R2 = H
10
11
12
13
R1
N
R1
N
N
N
N
N
O
R3
O
N
N
R3
N
N
14 R1 = SO2-C6H4-pNO2; R3 = Cl
15 R1 = SO2-C6H4-oNO2; R3 = Cl
16 R1 = Fmoc; R3 = Cl
17
R1 = SO2-C6H4-pNO2; R3 = Cl
R1 = SO2-C6H4-oNO2; R3 = Cl
R1 = SO2-C6H4-oNH2; R3 = Cl
R1 = SO2-C6H4-pNH2; R3 = Cl
R1 = SO2-C6H4-pNO2; R3 = OH
R1 = SO2-C6H4-oNO2; R3 = OH
R1 = Fmoc; R3 = OH
R1 = H; R3 = SPh
R1 = SO2-C6H4-pNO2; R3 = SPh
R1 = SO2-C6H4-oNO2; R3 = SPh
18
19
20
21
22
23
24
25
26
Chart 1. Benzoxazepine O,N-acetals containing pyrimidine (1e13) and purine (14e26) rings.
activities against the MCF-7 human breast and the HT-29 human
colon cancer cell lines, and the corresponding antiproliferative
activities against the corresponding non-tumour cell lines (the
MCF-10A breast cell line, and the IEC-6 epithelial cells from rat
jejunum) in order to establish the in vitro therapeutic indexes (TIs).
Moreover, the cell cycle, apoptosis and microarray studies are
reported herein.
(IC50 ¼ 3.25 ꢀ 0.23
mM) [2]. Additionally, we used a non-cancerous
human mammary epithelial cell line (MCF-10A), in order to study
the in vitro TI against breast cancer. These two compounds were
selected to be further assayed on the human colon cancer cell line
HT-29, and on the non-tumour rat small intestine epithelial cell line
IEC-6 to define the TI in intestinal lineage cells. IEC-6 Cells have
been used as a valid experimental model for toxicity studies of anti-
tumour drugs [6,7]. The improvement of the relationship between
desired and undesired effects of therapy, the TI, is a major goal of
cancer therapy. The in vitro TI of a drug is defined as the ratio of the
toxic dose to the therapeutic dose. Table 1 shows the anti-
proliferative activities for 5-FU (as a reference drug [8]), 16 and 37
against the cell lines MCF-7, MCF-10A, HT-29 and IEC-6, and the
corresponding in vitro TIs. Compound 16 is the most selective
compound against the cancerous breast cancer cell line (TI ¼ 3.19)
in relation to the normal one, as 37 is against the cancerous colon
cell line in relation to normal intestinal cells (TI ¼ 3.69). The
unnatural pyrimidine 5-FU is not selective at all, with TI < 1 in both
pairs of epithelial, non-cancerous and normal cells [in vitro TI
(breast) ¼ 0.17, and in vitro TI (intestinal) ¼ 0.61].
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of derivatives
Compounds 1e13 were described by Díaz-Gavilán et al. [3].
Compounds 14e15 [4], 16 [2], 17e19 [4], 20 [2], 21e22 [4], 23 [2],
24e27 [4], 29e30 [4], 32e34 [4], and 35e37 [5] were previously
reported by us. Compounds 28 and 38 are described in the
Supporting Information Section.
2.2. Biological activities
2.2.1. Cytotoxic activities
2.2.2. Cell cycle analysis
The most potent antiproliferative agent against the MCF-7
adenocarcinoma cell line that belongs to the benzoxazepine O,N-
In order to determine if the anti-tumour effects of the drugs
involve changes in cell cycle distribution, the breast cancer cell line
MCF-7and the human colon cancercell line HT-29 weretreatedwith
compounds 16 and 37 at doses of their IC50 values during 48 h and
acetalic family is 16 (IC50 ¼ 0.67 ꢀ 0.18
mM), whilst 37 shows the
lowest IC50 value between the family of acyclic O,N-acetals

3804
O. Caba et al. / European Journal of Medicinal Chemistry 46 (2011) 3802e3809
O
R1 OMe
R1 OMe
R2
O
R2
N
N
N
N
O
N
H
O
N
H
OH
OH
27 R1 = H, R2 = F
28 R1 = CO-C6H5; R2 = F
34 R1 = SO2-C6H4-pNO2; R2 = H
29 R1 = SO2-C6H4-pNO2; R2 = F
30 R1 = SO2-C6H4-oNO2; R2 = F
31 R1 = SO2-C6H4-oNH2; R2 = F
32 R1 = SO2-C6H4-pNO2; R2 = H
33 R1 = SO2-C6H4-oNO2; R2 = H
R1 OMe
N
R1 OMe
N
N
N
N
N
R3
OH
N
N
R3
OH
N
N
35 R1 = SO2-C6H4-pNO2; R3 = Cl
36 R1 = SO2-C6H4-oNO2; R3 = Cl
37
R1 = SO2-C6H4-oNO2; R3 = Cl
Chart 2. Acyclic O,N-acetals containing pyrimidine (27e34) and purine (35e37) moieties.
then analyzed by flow cytometry (Tables 2 and 3). In relation to the
MCF-7 cell line, both compounds (16 and 37) were able to induce an
accumulation in both G0/G1 and G2/M phases with the consequently
significant decrease in the S phase in comparison with control-
DMSO-treated cells (Table 2). Therefore, 16 and 37 slowly
decreased the progression of the cell cycle as demonstrated by the
percentage of cells in the S phase and its increase of the G0/G1 phase.
In this way, the mitotic index (MI ¼ S þ G2/M/G1) is reduced in MCF-7
cells due to the blockage of the cells in the G1 phase [9]. A cell
blockage in the G1 phase serves to prevent the DNA replication,
damaged by the action of the various drugs.
were treated with the IC50 values of compounds and stained using
Annexin V and propidium iodide (PI) at 48 h’ post-drug treatment.
Apoptosis assays were accomplished in the MCF-7 human breast
cancer cell line, where the demonstration of programmed cell
death by known apoptosis-inducing agents has been proved diffi-
cult and only few cytotoxic agents act preferentially through an
apoptotic mechanism in human breast cancer cells [11,12]. Pacli-
taxel (TaxolÒ) induced programmed cell death of up to 43% of the
cell population [13]. Simultaneous staining with annexin V-FITC
and the PI non-vital dye made it possible to distinguish between
early apoptosis (stained positive for annexin V-FITC and negative
for PI) and late apoptosis or cell death (stained positive for both
annexin V-FITC and PI).
In MCF-7 control-DMSO cultures neither early nor late apoptosis
were detected after 48 h. A high percentage of necrotic cells were
observed due to the intense proliferative activity and the great
aggressiveness shown by this tumour cell line. MCF-7 cells treated
during 48 h with 16 and 37 showed a significant increase of late
apoptotic cells in relation to the control culture with percentages
varying from 72.81% in cells treated with 16e48.49% after treat-
ment with 37, in comparison with control-DMSO-treated cells
(Table 4, Fig. 1A). The global (early þ late) apoptotic cells caused by
16 and 37 were 80.08% and 54.85%, respectively. These high
apoptotic percentages are consistent with the G1 and G2/M arrest
In relation to the HT-29 cell line, both compounds halted the
cells in the S phase and provoked a decrease in the G0/G1 phase, and
a variable effect on the G2/M phase (Table 3).
2.2.3. Apoptosis assay
After analyzing the effects of the drugs at the level of the
cell cycle, our interest was centred on explaining the mechanism
of action ofsuch drugs. According toSwanton [10], the mechanism of
action of anti-tumour agents has a great manipulative potential of
the cell cycle in promoting the growth arrest, differentiation or even
apoptosis.
To determine if the observed growth inhibition was in part due
to apoptosis, flow cytometry studies were carried out. The cells
Table 1
Antiproliferative activitiesa against several cancerous and normal cell lines for 5-FU, 16 and 37.
Compd.
IC50 MCF-7
IC50 MCF-10A
In vitro TIb (breast)
IC50 HT-29
IC50 IEC-6
In vitro TIc (intestinal)
5-FU [8]
16
37
4.32 ꢀ 0.02
0.67 ꢀ 0.18
3.25 ꢀ 0.23
0.73 ꢀ 0.12
2.14 ꢀ 1.10
6.23 ꢀ 1.90
0.17
3.19
1.92
1.08 ꢀ 0.15
3.38 ꢀ 0.61
3.28 ꢀ 0.80
0.66 ꢀ 0.10
10.1 ꢀ 1.67
12.1 ꢀ 1.20
0.61
2.99
3.69
a
All experiments were conducted in duplicate and gave similar results. The data are means ꢀ SEM of three independent determinations. The treatment time was 6 days.
b
c
In vitro TI ¼ IC50 MCF-10A/IC50 MCF-7.
In vitro TI ¼ IC50 IEC-6/IC50 HT-29.

O. Caba et al. / European Journal of Medicinal Chemistry 46 (2011) 3802e3809
3805
Table 2
Table 4
Percentage of accumulated cells in the several phases of the cell cycle, together with
their respective standard deviations, in the MCF-7 cell line.
Percentage of viable, necrotic and apoptotic cells in the cell line MCF-7 after 48 h of
induction.
GO/G1 (%)
S (%)
G2/M (%)
Viable (%) Early Apoptosis (%) Late Apoptosis (%) Necrosis (%)
CONTROL
16
37
57.26 ꢀ 5.13
64.39 ꢀ 0.34
65.09 ꢀ 0.11
37.25 ꢀ 6.35
22.43 ꢀ 2.69
23.94 ꢀ 4.87
5.50 ꢀ 0.67
13.18 ꢀ 2.35
10.98 ꢀ 2.76
CONTROL 53.52
1.24
7.27
6.36
1.57
72.81
48.49
43.67
14.85
24.79
16
37
5.07
20.36
since cells exposed to specific agents typically enter apoptosis from
a given phase of the cell cycle [12,14].
The global apoptotic cells caused by 16 and 37 in the HT-29 cell
line (31.7% and 36.74%, respectively) were high; however, these
percentages were less accentuated than in the MCF-7 one (Table 5,
Fig. 1B).
closely related to cancer, suggesting an important role in tumour
progression and response to treatment [22]. Another important
repressed gene was TDGF1, a gene used as a predictive marker for
metachronous metastasis in colorectal patients. Moreover, it has
been proved that the siRNA inhibition of this gene resulted in
a significant reduction in cell growth and invasion [23]. Further-
more it has been recently showed that the bradykinin receptor,
BDKRB1, maintains tumour growth, induces the proliferation of
oestrogen-sensitive breast cancers, and its antagonism may be
a valuable alternative for the treatment of breast cancer [24,25].
Another example is the gene codifying for the molecule CD72,
previously associated with kidney cancer [26]. More important was
the down-regulation of TMPRSS6 up to 2.03 fold. Recent studies
suggest that TMPRSS6 gene is associated with breast cancer risk in
the eastern Finnish population [27]. In this case we also found
down-regulated genes, such as the guanylate binding protein 1
(GBP1) gene which is related with another cellular mechanism that
collaborates in tumour progression. Over-expression of GBP1 is
associated with resistance to several drugs including paclitaxel
[28]. Finally, we have to highlight the decrease in the expression of
the p53 mediator P53AIP1 [29]. This data indicates that the mech-
anism of apoptosis previously observed in the MCF-7 cell with the
compound 16 may not be mediated by the activation of this pro-
apoptotic way.
2.2.4. Analysis of the gene expression with microarrays
Up to some years ago one of the main difficulties that
researchers found in the fight against cancer was the high number
of genes that underlie the mechanisms of malign transformation.
Nowadays the microarray technique allows the simultaneous study
of an abundant number of genes with the corresponding advan-
tages. Moreover, this technique permits the study of markers that
may predict the response to a certain treatment. This riposte is
mediated by the expression of a high number of genes that,
nevertheless, may be studied by the DNA microarrays. Sorlie et al.
[15] and van’t Veer et al. [16] have carried out such studies in breast
cancer. Hybridization of microarrays was done before and after
treatment in both cell lines (MCF-7 and HT-29), and 2-fold down-
or up-regulated genes were selected in order to investigate the
mechanism of action of 16 and 37. Then selected genes were
grouped based on their biological functions [17].
In both compounds the number of repressed genes was higher
than those over-expressed. Compound 16 induced the over-
expression of 289 genes and the down-regulation of 351 genes in
the MCF-7 cell line. Compound 37 provoked less change (249
induced and 257 repressed genes) in these breast cancer cells.
A deep analysis demonstrated that 16 in the MCF-7 cell line
increase the expression several apoptotic genes (Table 6,
Supporting Information). The MYCN gene has been shown to syn-
ergize with anticancer drugs to induce apoptosis along several
different pathways, e.g. by increasing expression levels of Bax
mRNA and protein or cooperating in the activation of the CD95
system [18,19]. ERN1 was another pro-apoptotic gene activated but
this gene has a different way of action since it promotes a potent
unfolded-protein response because it senses unfolded proteins in
the lumen of the endoplasmic reticulum, triggering growth arrest
and apoptosis [20]. Very important was the activation of the
GPR132 gene, which encodes a subfamily member of the G-protein
couple receptor (GPCR) superfamily and is involved in apoptosis
and at the G2/M checkpoint delaying mitosis [21].
On the other hand, treatment with 16 resulted in the repression
of several genes that have been reported as related with the
progression and invasion of different types of cancer (Table 6,
Supporting Information). So, we observed that SPINK2 was one of
the most suppressed genes, up to 3.01 fold. The expression of
SPINK2, a serine peptidase inhibitor that can also inhibit trypsin, is
Table 3
Fig. 1. Apoptosis induction in both MCF-7 human breast cancer (A) and HT-29 human
colon cancer (B) cell lines after treatment for 48 h with the compounds. Representative
panels of cytometry analysis for control cells treated with 0.5% DMSO alone, 16 and 37.
In each panel, lower left quadrant (LL) shows viable cells which are negative for both
annexin V-FITC and PI, lower right (LR) shows annexin V positive cells which are in the
early stage of apoptosis, upper left (UL) shows only PI positive cells which are dead,
and upper right (UR) shows both annexin V and PI positive, which are in the stage of
late apoptosis.
Percentage of accumulated cells in the several phases of the cell cycle, together with
their respective standard deviations, in the HT-29 cell line.
GO/G1 (%)
S (%)
G2/M (%)
CONTROL
16
37
77.36 ꢀ 1.45
74.68 ꢀ 0.64
71.58 ꢀ 0.86
7.96 ꢀ 2.64
14.69 ꢀ 4.09
10.46 ꢀ 0.98
15.73 ꢀ 4.76
14.87 ꢀ 1.63
12.71 ꢀ 3.90

3806
O. Caba et al. / European Journal of Medicinal Chemistry 46 (2011) 3802e3809
Table 5
Another important fact was the repression in HT-29 cells of the gene
AKT2 in which its over-expression plays a critical role in the estab-
lishment of colorectal cancer metastasis. Several authors have
proposed the selective targeting of the PI3K/AKT2 pathway to
provide a novel treatment strategy for colorectal cancer metastasis
[42]. Also remarkable was the high repression in several genes with
a role in the maintenance of genomic integrity, such as PRP19, FUS
and POLE. The first one is related to tumourogenesis in lung cancer
Percentage of viable, necrotic and apoptotic cells in the cell line HT-29 after 48 h of
induction.
Viable (%) Early Apoptosis (%) Late Apoptosis (%) Necrosis (%)
CONTROL 67.18
16
37
0.58
18.07
21.10
3.76
13.63
15.64
28.50
8.61
8.33
59.69
54.93
Also in the MCF-7 cell line with 37 (Table 7, Supporting
Information) we observed the up-regulation of several important
genes. ZNF141, a gene involved in transcriptional regulation as
a repressor, appeared up-regulated up to 4.04 fold. The gene NPAT is
required for progression through the G1 and S phases of the cell
cycle [30]. The increase in the expression of this gene, up to 2.68
fold, may be responsible for the significant decrease in the S phase
in comparison with control-DMSO-treated cells. Also important
was the up-regulation of the transcription factor ERG that regulates
angiogenesis and endothelial apoptosis through VE-cadherin [31].
On the contrary, we found that 37 induced the down-regulation
of several pro-tumour genes, such as RAC2. Several authors have
described the increased expression of this gene in a malignant
squamous cell cancer and premalignant dysplastic cell lines [32].
ADAMTS3 are metalloproteinases characterized by the presence of
[43], FUS,
a mediator of the androgen-dependent cell cycle
progression to prostate cancer growth [44]. POLE is another gene
related to the maintenance of genomic integrity, participating in
DNA repair and in chromosomal DNA replication. This gene acts as
a sensor of DNA replication that coordinates the transcriptional and
cell cycle responses to replication blocks, linking the DNA replication
machinery to the S phase checkpoint [45]. The down-regulation of
POLE could explain the accumulation of HT-29 cells in the S phase of
cell cycle induced by this compound. Another important down-
regulated gene was PRPS1 that catalyzes the phosphoribosylation
of ribose 5-phosphate to 5-phosphoribosyl-1-pyrophosphate, and is
necessary for purine metabolism and nucleotide biosynthesis [46].
Finally, also remarkable was the repression of several genes related
with the transcriptional regulation and pre-mRNA processing such
as HNRNPA2B1, ZNF482, TSR2 and ZNF768 [47]. The negative action of
the compound against the expression of these genes could explain
the cytotoxicity in tumour cells.
additional thrombospondin type
I (TSP-I) motifs in their
C-terminal. These proteinases have different effects in the cancer
progression and act by the regulation of growth factor activities and
integrin functions [33]. Similarly to 16, we observed that in the
MCF-7 cell line, 37 induced the down-regulation of two important
genes, P53AIP1 and GBP1. These data suggest that both compounds
have very similar mechanisms of action and support the previously
obtained conclusions.
The most important fact for 37 against the HT-29 cells was the
down-regulation of the pro-apoptotic gene P53AIP1 (Table 9,
Supporting Information), as in the MCF-7 cell line, confirming the
same mechanism of action previously commented. This effect also
appeared when we studied the expression of GBP1, previously
reported as down-regulated in the MCF-7 cell line in connection
with a phenomenon of resistance [27]. Also remarkable was the
increase with 37 in the expression of PDE4B, a phosphodiesterase
that inactivates the second messenger cyclic adenosine 30,50
monophosphate (cAMP). This inactivation limits the cAMP associ-
ated to a PI3K/AKT2-dependent apoptosis previously explained and
that was also reduced by the down-regulation of AKT2 by 16 [48].
YPEL1 was the gene most up-regulated by 37. This gene encodes
a potential zinc-binding protein that may play a role in the regu-
lation of cellular morphology. The expression of YPEL1 is very
common in pancreatic cancer cell lines and tissues, and reduced
expression of YPEL1 in cancer has been related with greater power
of invasion and poor prognosis [49]. It is worth noting that the last
gene up-regulated by this compound is PMP22, an integral
membrane protein involved in growth regulation whose over-
expression has been related with apoptotic phenotype [50].
More important were the genes repressed by 37 in the HT-29
cell line. The most repressed was AK5, a gene which encodes
a member of the adenylate kinase family which is involved in
regulating the adenine nucleotide composition within a cell by
catalyzing the reversible transfer of phosphate groups among
adenine nucleotides [51]. In a similar mechanism of action to
previous gene, SLC28A2 appeared down-regulated up to 3.32 fold.
SLC28A2 is a purine-selective transporter that plays a critical role in
the specific uptake of and salvage of purine nucleosides. Altogether
it seems that 37 acts interfering the normal cycle of purines halting
the production of tumour cells. Compound 37 also acts repressing
the expression of several genes related to different pro-oncogenic
processes, such as, a) the transcription factor POU6F1, important
for the proliferation of several tumour cells and represents
a potential new molecular target [52]; b) NFATC1 a gene that exerts
strong oncogenic properties through induction of the G1-S phase
promoting genes [53]; and c) HAS2, hyaluronan synthase, whose
abnormal over-expression stimulate cell growth, proliferation,
angiogenesis and metastasis in cancer cells [54].
Several reports have been recently published in relation to
microarrays in HT-29 cells [34e36]. Compounds 16 and 37 in the
HT-29 cell line did not halt the cells in the G0/G1 phase, and this fact
can be explained by the fact that the drugs did not show any effects
on the expression levels of the encoded genes of CDK2 and CDK4.
Moreover, the induction of apoptosis was substantial. When we
studied the gene expression with 16 (Table 7, Supporting
Information) we observed that the main promoter of this
phenomenon was the gene TP53INP which activates in response to
the breaking of the double helix of the DNA [18]. Although the
apoptosis mechanism induced by p53 is not completely known, it
seems that the phosphorylation of Ser-46 is essential, promoted by
this gene on activating a series of specific serine kinases [19]. This
process is not triggered in the absence of this protein, and the
activation of the p53 mediator p53AIP1 is not produced [20]. For
this compound the pro-apoptotic gene UNC5B, which is induced by
p53-mediated cell stress, is also increased. In fact, the repression of
this gene inhibits the apoptosis induction by adriamycin,
a compound that provokes cell death via p53 [21]. This gene
codifies a protein that possesses a cytoplasmic death terminus and
seems to be one of the main ways of induction of p53-mediated
apoptosis [37], and it has been reported that the down-regulation
of UNC5B promotes cell survival and tumour formation [38,39].
EGLN3 was another pro-apoptotic gene that was up-regulated by
the compound 16 in the HT-29 cells. This gene is specifically
required among the three EglN family members for apoptosis,
promoting cell death through a caspase-dependent mechanism,
a down-stream of c-Jun stimuli [40].
With 16 the most repressed gene was MDGA1 (up to 3.92 fold)
(Table 8, Supporting Information). This gene is related with cell
motility and cell-to-cell adhesion, reducing adhesion to extracel-
lular matrix proteins in MDCK cells [41]. In another important anti-
tumour therapy the down-regulation of MDGA1 could reduce the
mobilization of HT-29 cells and so the possibility of metastasis.

O. Caba et al. / European Journal of Medicinal Chemistry 46 (2011) 3802e3809
3807
3. Conclusion
4.1.6. Analysis of the gene expression by the microarray technique
4.1.6.1. Extraction of the total RNA. Compounds 16 and 37 were
selected to study the modification of the gene expression pattern.
Culture of 8 ꢄ 106 cells was induced 48 h with the IC50 concen-
tration in the MCF-7 and HT-29 cell lines. The RNA extraction was
carried out by using the kit RNeasy Midi (QIAGEN) according to the
following protocol: Cells were detached from the culture flasks
with a PBS-EDTA solution. Cells were gathered together in 30 mL
universal (Iwaki microplate, Japan), adding to them the same
amount of PBS. They were centrifuged at 600g for 5 min. Cell
buttons were re-suspended in 2 mL of trizol, and were homoge-
The anti-carcinogenic potency of the target molecules (16 and
37) is reported against two tumour cell lines and two non-
cancerous ones. cDNA microarray technology reveals potential
drug targets, which are mainly centred on positive apoptosis
regulatory pathway genes, and the repression of genes involved in
carcinogenesis, proliferation and tumour invasion. Breast and colon
cancers are the most intensely studied human malignancies in the
genomic era. Given the grossly heterogeneous nature of both
neoplasms and the lack of robust conventional markers for disease
prediction, prognosis, and response to treatment, the notion that
a transcriptional profile comprising multiple genes will be more
predictive of tumour behaviour is both appealing and reasonable.
nized by passing them through a 2 mL needle 8e10 times. 800 mL of
chloroform were added to the samples after 3 min at room
temperature, everything was thoroughly mixed with vortex and
was left to settle 10 min at room temperature. Samples were
transferred to 15 mL tubes containing Phase Lock Gel (PLG,
Eppendor Corp., Hamburg, Germany), which optimizes the sepa-
ration of the DNA phase and proteins of that of RNA, and then were
centrifuged at 2000g for 15 min. After centrifugation the super-
natant was transferred to 50 mL universal tubes (Iwaki microplate,
Japan) and the same volume of 70% alcohol was added drop by
drop. The samples were layered on top of the Rneasy columns and
were centrifuged for 5 min at 3000g. The eluted phase was
collected, placed again in the column and centrifuged again 5 min
at 3000g. The eluted phase was then decanted and 4 mL of RW1
buffer (washing buffer) were added to the column, centrifuging at
3000g for 5 min. The phase that went through the column was
4. Experimental protocols
4.1. Biology
4.1.1. Cell culture
MCF-7, MCF-10A, HT-29 and IEC-6 cells were grown at 37 ꢁC in
an atmosphere containing 5% CO2, with Dubelcco’s modified Eagle
Medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10%
heat-inactivated foetal bovine serum (FBS) (Gibco), 2% L-glutamine,
2.7% sodium bicarbonate, 1% Hepes buffer, 40 mg/L gentamicin and
500 mg/L ampicillin.
decanted and 160 mL of DNase were added and left to act for 15 min
4.1.2. Drugs and drug treatments
at room temperature. Then 2 mL of RW1 buffer were added and
after waiting for 5 min, it was centrifuged at 3000g for 5 min. After
this, the eluted phase was discarded and 3 mL of RPEr (washing
buffer to which ethanol was previously added) were added and the
sample was centrifuged for 5 min at 3000g. The eluted phase was
decanted and after adding another 3 mL of RPEr, was centrifuged at
3000g another 5 min. The mini-column was carefully taken from
The drugs were dissolved in DMSO or water and stored
at ꢂ20 ꢁC. For each experiment, the stock solutions were further
diluted in medium to obtain the desired concentrations. The final
solvent concentration in cell culture was ꢃ0.1% v/v of DMSO,
a concentration without effect on cell replication. Parallel cultures
of cells in medium with DMSO were used as controls.
the collector and transferred to a new tube by adding 150 mL of
4.1.3. Cytotoxicity assays in vitro
RNase-free water directly over the membrane, and later on
centrifuged for 5 min at 3000g. This last process was repeated and
the eluted phase was kept in Eppendorf tubes (Eppendorf Corp.,
Hamburg, Germany).
The effect of anticancer drugs on cell viability was assessed
using the sulforhodamine-B colorimetric assay [55]. Aliquots of
MCF-7 cells suspension (1 ꢄ 103 cells/well) were seeded onto 24-
well plates and incubated for 24 h. The cells were then treated
with different concentrations of drugs in the culture medium.
Three days later, the wells were aspirated, fresh medium and
treatment were added, and cells were maintained for 3 additional
days. Thereafter, cells were processed as described previously [20],
using a Titertek Multiscan apparatus (Flow, Irvine, California) at
492 nm. We evaluated the linearity of the SRB assay with a cell
number for each cell line before each cell growth experiment. The
IC50 values were calculated from semilogarithmic doseeresponse
curves by linear interpolation. All of the experiments were plated in
triplicate wells and were carried out at least twice.
4.1.6.2. Quantifying RNA. It was carried out at a wave length
between 260 nm and 280 nm by means of the spectrophotometer
Ultrospec 2000 of Pharma Biotech. The lecture in this interval
allowed us to calculate the concentration of nucleic acids in the
sample through the ratio between O.D. 260 nm/O.D. 280 nm),
keeping in mind that 1 unity of O.D. at 260 nm is equal to 40 mg/mL
of RNA.
4.1.6.3. Determination of the RNA integrity. 1 mg of RNA was checked
by electrophoresis in 0.8% agarose gel for 2 h at 80 V. Visualization
of the RNA by UV trans-illumination was carried out in a Molecular
Imager Gel Doc XR System densitometre by using the software
Quantity One 4.5.2 (Bio-Rad Laboratories Inc., USA), and allowed us
to prove that it was not degraded.
4.1.4. Cell cycle distribution analysis
The cells at 70% confluence were treated with either DMSO
alone or with concentrations of the compounds determined by
their IC50 values. FACS analysis was performed after 48 h of treat-
ment as described [14]. All experiments were performed in tripli-
cate and yielded similar results.
4.1.6.4. Analysis of the samples. Once the integrity of the RNA was
quantified and checked, 10 mg diluted in 3 volumes of pure ethanol
(Sigma Chemical Co., St. Louis, MO, USA) was sent to the Genomic
Unity of the Centro Nacional de Investigaciones Oncológicas (CNIO)
in Madrid for its analysis.
4.1.5. Apoptosis detection by staining with annexin V-FITC and
propidium iodide
The annexin V-FITC apoptosis detection kit I (Pharmingen, San
Diego, CA, USA) was used to detect apoptosis by flow cytometry
according to Marchal et al. [55]. All experiments were performed in
triplicate and yielded similar results.
Briefly, the protocol was the following: NaAc pH5 (Sigma
Chemical Co., St. Louis, MO, USA) was added to the received RNA up
to 0.3 M and 1 mL of the LPA carrier (Ambion Inc., Austin, USA). This
mixture was incubated at ꢂ80 ꢁC for at least 1 h. It was centrifuged

3808
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Acknowledgements
This study was supported by the Instituto de Salud Carlos III
(Fondo de Investigación Sanitaria, FEDER) through projects PI10/
00592 and PI10/02295, and the Ministerio de Ciencia e Innovación
through the project SAF-2010-18263.
Appendix. Supplementary material
The supplementary data associated with this article can be
found in the on-line version at doi:10.1016/j.ejmech.2011.05.047.
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