Novel Polyamine-Naphthalene Diimide Conjugates Targeting Histone Deacetylases and DNA for Cancer Phenotype Reprogramming

Article abstract of DOI:10.1021/acsmedchemlett.7b00289

A series of hybrid compounds was designed to target histone deacetylases and ds-/G-quadruplex DNAs by merging structural features deriving from Scriptaid and compound 1. Compound 6 binds different DNA arrangements, inhibits HDACs both in vitro and in cells, and is able to induce a reduction of cell proliferation. Moreover, compound 6 displays cell phenotype-reprogramming properties since it prevents the epithelial to mesenchymal transition in cancer cells, inducing a less aggressive and migratory phenotype, which is one of the goals of present innovative strategies in cancer therapies.

Full text of DOI:10.1021/acsmedchemlett.7b00289

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Letter
Novel Polyamine-Naphthalene Diimide Conjugates Targeting Histone
Deacetylases and DNA for Cancer Phenotype Reprogramming
Alice Pasini, Chiara Marchetti, Claudia Sissi, Marilisa Cortesi,
Emanuele Giordano, Anna Minarini, and Andrea Milelli
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00289 • Publication Date (Web): 24 Oct 2017
Downloaded from http://pubs.acs.org on October 27, 2017
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ACS Medicinal Chemistry Letters
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Novel Polyamine-Naphthalene Diimide Conjugates Targeting His-
tone Deacetylases and DNA for Cancer Phenotype Reprogramming
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‡,
∇
§,
∇
‡
‡,^,¥
§
∥
Alice Pasini, Chiara Marchetti, Claudia Sissi, Marilisa Cortesi, Emanuele Giordano,
Anna Minarini, Andrea
#*
Milelli
‡
Department of Electrical, Electronic and Information Engineering “Guglielmo Marconi” (DEI), Alma Mater Studiorumꢀ
University of Bologna, Via Venezia 52, 47521 Cesena (FC), Italy.
§
Department of Pharmacy and Biotechnology, Alma Mater StudiorumꢀUniversity of Bologna, Via Belmeloro 6, 40126 Boloꢀ
gna, Italy.
∥
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Itaꢀ
ly.
^
Health Sciences and Technologies ꢀ Interdepartmental Center for Industrial Research (HSTꢀICIR), Alma Mater Studiorumꢀ
University of Bologna, Via Tolara di Sopra 41/E, 40064 Ozzano dell’Emilia (BO), Italy.
¥
Advanced Research Center on Electronic Systems (ARCES), Alma Mater StudiorumꢀUniversity of Bologna, Via Vincenzo
Toffano 2/2, 40125 Bologna, Italy.
#
Department for Life Quality Studies, Alma Mater StudiorumꢀUniversity of Bologna, Corso d’Augusto 237, 47921 Rimini,
Italy.
KEYWORDS. Multiple ligands, Histone Deacetylase, Gꢀquadruplex, EpithelialꢀMesenchymal Transition.
ABSTRACT: A series of hybrid compounds were designed to target histone deacetylases and dsꢀ / Gꢀquadruplex DNAs by mergꢀ
ing structural features deriving from Scriptaid and compound 1. Compound 6 binds different DNA arrangements, inhibits HDACs
both in vitro and in cells, and is able to induce a reduction of cell proliferation. Moreover, compound 6 displays cell phenotypeꢀ
reprogramming properties, since it prevents the epithelial to mesenchymal transition in cancer cells, inducing a less aggressive and
migratory phenotype, which is one of the goals of present innovative strategies in cancer therapies.
The postꢀchemotherapy occurrence of relapse and metastasis in
many cancer types has encouraged to research novel tumor cell
phenotype reprogramming strategies in order to improve patient
response and survival. Since the expression and/or activity of epiꢀ
genetic modulator enzymes are often deregulated in cancer, conꢀ
sistently with patient prognosis, chromatin modifier enzymes, inꢀ
fluencing the chromatin transcriptional layout associated with a
neoplastic phenotype, are gaining considerable interest as targets
silenced tumor suppressor genes (TSGs). The following modulaꢀ
tion of cancer cell behavior determines cell reprogramming toꢀ
wards a less aggressive phenotype, consistently with a recovered
cell response to standard chemotherapeutic treatment ꢀ such as
3
DNAꢀalkylating agents and topoisomerases inhibitors. This obꢀ
servation highlights the concept that, as cancer cells exploit mulꢀ
tiple and redundant biochemical pathways to ensure their survival,
their treatment with multiple agents hitting distinct targets inꢀ
volved in the neoplastic development may represent a successful
therapeutic approach. However, concomitant administration of
different drugs with different pharmacokinetic and pharmacodyꢀ
namic properties might not result in a true synergistic effect. The
design of Multiple Ligands (MLs) appears as a promising alternaꢀ
1
of these approaches.
Several molecules have been used either alone or in combination
with other anticancer agents to target the chromatinꢀmediated
transcriptional control of gene expression, showing promising reꢀ
sults in preclinical studies. Just few of them however have gained
actual clinical significance, certified by the Food and Drug Adꢀ
ministration (FDA) approval for the treatment of specific cancer
subtypes. Among these epigenetic drugs, molecules targeting hisꢀ
tone deacetylases (HDACs) are extensively studied as therapeutic
agents in different diseases. As an example, Scriptaid, a naphꢀ
thalimideꢀbased HDAC inhibitor (HDACi), showed promising
4
tive to combination therapy, since it displays several advantages.
In particular, HDACis are extensively explored as component of
MLs due to their aboveꢀmentioned synergy with chemotherapeuꢀ
tics. Furthermore, the binding site of HDACs allows the accomꢀ
modation of a diverse array of chemical structures. In this respect,
the development of MLs able to bind both HDACs and DNA
seems to be a promising approach. Interesting results have been
achieved with HDAC/topoisomerase inhibitors and with
HDACi/Pt complexes, both of them being classes of compounds
interacting with double strand DNA (dsDNA). It is worth noting
2
anticancer activities.
HDAC inhibition has been reported to affect cancer cells mainly
via a global relaxation of chromatin structure driving the unlock
of promoter regions typically controlling relevant epigeneticallyꢀ
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Page 2 of 6
that, in addition to dsDNA, other secondary structures with releꢀ
vant biological roles exist, such as the Gꢀquadruplex DNA. Gꢀ
the reference compound 1. In particular, among the derivatives
containing the longer polyamine chains, an interesting trend can
5
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quadruplex appears to play a critical role in a range of biological
processes, since quadruplexꢀforming sequences are located not
only in telomeric regions of the human genome, but also at the
be defined: 3 < 4 ≈ 5 << 6. This suggests the importance of the
length of the spacers separating the amine groups. Indeed, by inꢀ
creasing the number of methylene groups (see 3 vs 4 and 5 vs 6)
the interaction is implemented with a leveling off occurring movꢀ
ing from compound 4 to compound 5. Likely, this reflects the
flexibility required to properly localize the amine groups in order
to optimize the ionic contacts with the phosphate backbone. When
compared to compound 1, compound 6 is more efficient in stabiꢀ
lizing dsDNA, whereas the substitution of the oꢀmethoxybenzylꢀ
propyl group impacts negatively on the telomeric Gꢀquadruplex
recognition. Indeed, with the exception of compound 6, all the
new derivatives turned out to be less efficient than compound 1 as
Gꢀquadruplex binders. Compound 2, characterized by two nitroꢀ
gen atoms in the side chain, is less active than compounds 3-6
characterized by three nitrogen atoms. No clear indications of the
role of the distance between the inner nitrogen atoms could be
obtained for this series of compounds. Indeed, similar to dsDNA
compound 4 (characterized by a three methylenes spacer) is the
most active on Gꢀquadruplex among compounds 3, 4 and 5 as reꢀ
ducing or increasing the number of methylenes leads to a drop in
the ꢁTm. Surprisingly, concerning Gꢀquadruplex recognition acꢀ
tivity, compound 6 shows a ꢂTm value comparable with that of
compound 1 (Table 1, ꢂTm at 2.5 ꢁM: compound 1, 21.4 °C,
compound 6, 20.4 °C). In summary, all new derivatives are
generally less specific for Gꢀquadruplex in comparison to 1 or
to the Gꢀquadruplex binder Bracoꢀ19, used as a reference.
Consistently, all our new derivatives retain the ability to imꢀ
pair the activity of DNA processing enzymes (i. e. Taqꢀ
polymerase, Fig. 1SI). The same analysis has also been perꢀ
formed using Scriptaid to exclude any direct interaction of this
HDACi with DNA.
6
promoter region of several oncogenes. None of the HDACꢀbased
MLs developed so far displays the ability to interact with both dsꢀ
and Gꢀquadruplex DNA. Recently, our research group developed
a series of Naphthalene diimide (NDI)–based Gꢀquadruplex bindꢀ
7
, 8
ers. Among these agents, the asymmetric NDI 1 emerged as the
most promising antiproliferative agent. Compound 1 is characterꢀ
ized by two different chains mounted on the NDI scaffold: in deꢀ
tail, an oꢀmethoxybenzylaminoꢀpropyl moiety on one side, and a
spermine tail on the other side (Fig. 1). NDI derivative 1 is enꢀ
dowed with a good affinity towards both human Gꢀquadruplex
and dsDNA, together with good antiproliferative and proapoptotic
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activities in different cancer cell lines. Motivated by these conꢀ
siderations and by the structural similarity between compound 1
and the HDACi Scriptaid, hybrid compounds 2-6 were rationally
designed by merging the structural features of the parent comꢀ
pounds. Indeed, we envisage that this combination may create
compounds able to both bind DNAs and inhibit HDACs. The new
compounds 2-6 are characterized by the presence of a) a classic
2
+
hydroxamic acid, as Zn ꢀbinding group in place of the oꢀ
methoxybenzyl group, b) a NDI moiety, allowing DNA interacꢀ
tion by stacking of its large heteroaromatic surface and c) a polyꢀ
amine chain varying in the methylene groups separating the amine
functions. The cationic charge of the polyamine chain is intended
to improve the molecular interaction with the negatively charged
nucleotides and aminoacids, as it has been already shown for othꢀ
1
0, 11
er polyamineꢀbased anticancer agents.
Herein, we report the
evaluation of the potential multitarget profiles of compounds 2-6
by assessing their ability to bind both dsꢀ and Gꢀquadruplex DNA,
to inhibit HDAC activity and to have a consistent functional effect
over cancer cell phenotype.
Table 1. ꢂTm Values (°C) for a human Telomeric Quadruplex
sequence (hTel22) and duplex DNA (dsDNA), IC50 values for in
vitro HDAC inhibition in HeLa nuclear extract.
b
O
ꢁTm (° C)
H
N
HO
N
O
a
dsDNA
hTel22
HDAC
O
Compounds
c
IC50 (ꢁM)
0
.75
2.5
0.75
2.5
Scriptaid
ꢁM
0.6
1.0
2.0
5.0
3.5
7.5
0.5
0.1
ꢁM
ꢁM
ꢁM
O
N
O
O
N
O
OCH
3
H
N
1
2
3
4
5
6
6.7
3.3
3.4
8.5
8.1
14.0
5.3
0.1
1.5
21.4
6.3
n.d.
NH
2
N
N
H
H
2.4
0.41 ± 0.03
0.55 ± 0.02
0.37 ± 0.04
0.52 ± 0.03
0.56 ± 0.04
n.d.
4.4
7.8
1
9.9
14.5
10.9
20.4
28.3
0.1
4.5
O
N
O
O
dual
HDACis
ds/G-quadruplex binders
H
H
N
N
13.3
11.0
0.1
HO
N
O
N
R
H
n
O
BRACOꢀ19
Scriptaid
2
: n = 2, R = H
4: n = 2, R = -(CH
: n = 3, R = -(CH
3: n = 1, R = -(CH
2
)
3
NH
NH
2
2
0.56 ± 0.03
2
2
)
)
3
NH
NH
2
5: n = 3, R = -(CH )
2 3
6
4
2
a
b
c
compounds 1-6 as hydrochloride salts. Errors were 0.4 °C.
IC50 values represent the concentration causing 50% of HDAC
activity
Figure 1. Drug design strategy leading to compounds 2-6.
The in vitro ability of compounds 2ꢀ6 to inhibit HDACs has been
preliminarily screened in HeLa nuclear extract (0.2, 0.5, 1, 5 and
Detailed descriptions of the synthetic sequences are reported in
the SI (Schemes 1SIꢀ4SI).
2
5 µM concentrations) (Fig. 2SI) and further explored in different
Fluorescence melting experiments on a human telomeric quadruꢀ
plex sequence (hTel22) and on a duplex DNA sequence (dsDNA)
were used to evaluate target compounds 2ꢀ6 ability to bind both
dsꢀ and Gꢀquadruplex DNA secondary structures. Thermal stabiliꢀ
zation induced by tested compounds at 0.75 and 2.5 ꢁM concenꢀ
trations are reported in Table 1. With the exception of compounds
cancer cell lines. Compounds 2-6 show IC50 values in the submiꢀ
cromolar range of concentrations, similar to the reference comꢀ
pound Scriptaid (IC50 = 0.56 ± 0.03 ꢁM, Table 1) when HeLa puꢀ
rified nuclear HDAC enzymes are tested. To verify HDAC isoꢀ
form selectivity, the inhibitory capacity of compound 6 has been
evaluated with purified human HDAC1, 2, 4 and 6, representative
2
and 3, all new compounds bind dsDNA more efficiently than
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ACS Medicinal Chemistry Letters
of three HDAC classes (Table 2). Compound 6 displayed nanoꢀ
molar activity against HDAC6 (IC50 = 0.05 ± 0.01 ꢁM), whilst its
activity against HDAC1, 2 and 4 were significantly lower.
Compounds 4 and 6 resulted significantly more active than Scripꢀ
1
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taid in inhibiting HDAC activity in U87 and HCT116 cell lines (p
< 0.05), while showing comparable effect to Scriptaid in the A549
cell line (p < 0.05). It is worth to note that compounds 2ꢀ6 and
Scriptaid only slightly affect HDAC activity in MCF7 cells (Fig.
On the other hand, testing HDAC inhibition in intact viable cells
implies test compounds internalization, thus their effective conꢀ
centration is expected to be affected (reduced) throughout their
transport within the cell. The dose that completely inhibits
HDACs in HeLa nuclear extract (5 ꢁM) was used to evaluate the
in vitro HDAC inhibitory activity of test compound 2ꢀ6 in intact
cells upon a 24 h treatment (Fig. 2A). Cancer cell lines representaꢀ
tive of different tissue types, i.e. glia (U87), breast (MCF7), colon
2
A). To further investigate the HDAC inhibitory activity of comꢀ
pound 6, the global levels of histone H3 and H4 lysine acetylation
acH3 and acH4) were detected by Western Blotting (WB) in
(
A549 cells treated for 24 h at 5 µM concentration. The effect of
compound 6 on histone acetylation was compared to that of comꢀ
pound 1, which is defective of the hydroxamic acid moiety. The
overall levels of acH3 and acH4 induced by compound 6 were
two times higher (p <0.05) with respect to control condition and
compound 1, confirming the HDAC inhibition capacity of comꢀ
pound 6 (Fig. 2BꢀC).
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(HCT116) and lung (A549) were used. Compounds 4 and 6 were
able to inhibit at least about 40% of HDAC activity in U87,
HCT116 and A549 cell lines with comparable effects, despite a
different IC50 (compound 4, 0.37 ± 0.04 ꢁM, compound 6, 0.56 ±
0
.04 ꢁM) scored in HeLa nuclear extract.
Cell growth inhibition was evaluated at 0.2, 1 and 5 µM comꢀ
pounds 4 and 6 scalar concentrations (Fig. 3SI) in the responsive
U87, HCT116 and A549 cell lines. The effects were compared
with that of compound 1. Despite the observed comparable levels
of HDAC inhibition in these distinct cancer cell lines (Fig. 2A),
compounds 4 and 6 induced different levels of reduction of cell
proliferation, as witnessed by the GI50 values reported in Table 3
and determined by the inhibitory doseꢀresponse curve (Fig. 3SI).
Although Scriptaid also shows slightly higher antiproliferative
effect than compound 6 (data not shown), gene expression data
Table 2. IC50 Values (ꢁM) of compound 6 against the HDAC1, 2,
4 and 6 isoforms.
b
IC50 vs. HDAC (ꢁM)
a
Compound
HDAC1
HDAC2
10.80 ± 0.10
HDAC4
HDAC6
6
4.12 ± 0.58
117.00 ± 7.00
0.05 ± 0.01
a
b
compound as hydrochloride salts. IC50 values represent the
concentration of inhibitor required to decrease enzyme activity by
0% and are the mean of two independent measurements.
(
see below) suggests that survivor cells are not reprogrammed to
a less aggressive phenotype, as it happens with compound 6 (Fig.
SI).
5
4
Table 3. GI50 values for compounds 1, 4 and 6 in U87, HCT116
and A549 cancer cell lines.
b
GI50 (ꢁM)
a
Compounds
U87
4.85 ± 0.05
> 5
HCT116
4.39 ± 0.07
2.13 ± 0.07
3.08 ±0.09
b
A549
> 5
1
4
6
> 5
> 5
> 5
a
compounds as hydrochloride salts GI50 values represent the
concentrations causing 50% cellular growth inhibition. They were
determined by the inhibitory doseꢀresponse curve, Log (inhibitor)
®
vs. response with variable slope in GraphPad Prism 6 . n.d.
Modulating histone acetylation is likely to produce nonspecific
effects in a cell phenotype, since it substantially relaxes chromatin
architectures, allowing DNA consensus regulatory sequences to
be accessed by their relevant transcriptional regulators. Epithelialꢀ
mesenchymal transition (EMT) has been recognized as one
among the adaptive changes known for being associated with canꢀ
Figure 2. A. HDAC inhibitory activity of compounds 2-6 comꢀ
pared to Scriptaid was tested with an in vitro cellular assay at a
concentration of 5 µM along 24 h of treatment in U87, MCF7,
HCT116, and A549 cell lines. The data arenormalized with reꢀ
spect to the condition of absence of inhibitor and reported as mean
value ± SEM of three independent experiments (*p <0.05 vs. conꢀ
trol; ^p <0.05 vs. Scriptaid;). B. Global histone H3 and H4 acetyꢀ
lation (acH3, acH4) detected by WB in A549 cells treated with
compounds 1 and 6 (5 µM) for 24 h. WB bands are shown out of
one representative experiment reporting the signal relative to
acH3 and acH4 and the total amount of proteins per lane detected
via a stainꢀfree gel system (BioꢀRad). C. Optical densitometry of
acH3 and acH4 normalized with the total amount of proteins per
lane. Reported data are the mean value ± SEM of three independꢀ
ent experiments versus control conditions (*p <0.05 vs. control;
°p<0.05 vs. 1).
12
cer progression and metastasis formation. EMT is a reversible
process, which determines the phenotype transition of epithelial
cells to a mesenchymalꢀlike state responsible for the increased
cellular motility, proliferation and metastasis formation. Many
studies showed that epigenetics is involved in the control of
13
EMT, and epigenetic drugs have been used for cell phenotype
14
reprogramming. The therapeutic potential of compound 6 has
thus been evaluated studying its effect in reprogramming gene
expression of cancer cells, monitoring the modulation of genes
involved in the EMT, such as Eꢀcadherin (CDH1), marker of the
epithelial phenotype, and Vimentin (VIM), specific for the mesenꢀ
chymal transition. The gene expression analysis of these two recꢀ
ognized EMT marker genes was performed upon a 48 hꢀlong
treatment with 5 µM 6 in HCT116 and A549 cell lines (Fig. 3).
Compound 6 upregulates CDH1 and downregulates VIM expresꢀ
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sion in both cancer cell lines. CDH1 level of expression was inꢀ
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creased 1.32 and 1.28 fold, while VIM expression was consistentꢀ
ly reduced 0.44 and 0.68 fold compared to the control condition in
HCT116 and A549 respectively (p<0.01). This effect credits
compound 6 of being able of cell reprogramming towards an epiꢀ
thelial phenotype, which is one of the goals of present innovative
strategies in cancer therapies. It is worth to note that neither comꢀ
pound 1 nor Scriptaid induced effects comparable to compound 6
on CDH1 and VIM expression in A549 cells (Fig. 4SI). To further
corroborate the role of compound 6 in the modulation of EMT
transition, its effects were evaluated in A549 cells stimulated with
transforming growth factorꢀβ1 (TGFꢀβ1) since the A549 cell line
has been reported as an epithelial cellular model, switching to the
mesenchymal phenotype when stimulated with TGFꢀβ1.
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Figure 4. A. Cell growth inhibition evaluated by compound 6 (48
hꢀlong 5 µM treatment) in adherent A549 cell lines stimulated
with TGFꢀβ1. The percentage of growth was evaluated according
to the NCI screening procedures, as mean value ± SEM of three
independent experiments performed in triplicate. B. Gene expresꢀ
sion analysis of compound 6 effects on CDH1 and VIM genes in
A549 cell line stimulated with TGFꢀβ1. Data are reported as mean
value ± SEM of three independent experiments. * p<0.05; **
p<0.01; *** p<0.001.
Figure 3. Gene expression analysis of CDH1 and VIM genes in
HCT116 and A549 cell lines treated with 5 µM of compound 6
along 48 h. The data are reported as mean value ± SEM of four
independent experiments (** p<0.01).
Cell growth inhibition, gene expression analysis and cell migraꢀ
tion capacity were measured to this aim. As expected, A549 cells
treated with TGFꢀβ1 showed increased proliferation compared to
unstimulated cells, as suggested by the increased amount of viable
cells (Fig. 4A). On the other hand, compound 6 significantly supꢀ
pressed the TGFꢀβ1ꢀinduced cell proliferation, with an 85% reꢀ
duction of cellular growth that was comparable to what was obꢀ
served with compound 6 alone (Fig. 4A). Consistently, the level
of expression of the EMT marker genes CDH1 and VIM, which
are respectively downregulated and upregulated by TGFꢀβ1 stimꢀ
ulation compared to control condition, were reverted towards the
control values when compound 6 was administered in combinaꢀ
tion with TGFꢀβ1. In detail CDH1 expression resulted 10 times
downregulated by TGFꢀβ1 compared to control condition. The
administration of compound 6 in TGFꢀβ1ꢀtreated cells induced a
3
.66 fold CDH1 upregulation (vs. TGFꢀβ1 alone). On the other
hand, VIM showed the opposite behavior: TGFꢀβ1 induced a 1.53
fold increase compared to control condition, and compound 6 adꢀ
ministered in combination with TGFꢀβ1 prevented this upregulaꢀ
tion with a significant 2.61 fold reduction of the level induced by
TGFꢀβ1 alone (Fig. 4B). Finally, in order to study how compound
6 modulated cell migration, a scratch wound healing assay was
carried out in A549 cells stimulated with TGFꢀβ1. As expected,
TGFꢀβ1 induced a significant increase of cell migration, determinꢀ
ing 65% repopulation of the wound area at 24 h postꢀscratch. On
the other hand, unstimulated cells, and cells treated with comꢀ
pound 6 alone, maintained more that 80% of wound area at 24 h.
Interestingly, when compound 6 was administered in combination
with TGFꢀβ1, cells migration and proliferation were significantly
Figure 5. Scratch wound healing assay of the effect of compound
6
in TGFꢀβ1 induced A549 cells. A. An example of image proꢀ
cessing comparing two different time points (t = 0 and t = 30 h):
(a) raw image; (b) local entropy image; (c) final segmented image
(see Mat & Meth). B. Cell free surface area was calculated over
time and fitted with a linear regression. Data are reported as mean
value ± SEM of three independent experiments. * p<0.05.
A new drug design strategy is emerging based on the assumption
that complex diseases, such as cancer, may be better faced by
Multiple Ligands (MLs). Due to their relevant roles in cancer deꢀ
velopment, progression and response to therapy, HDACs, have
been identified as interesting candidates to be targets of such
MLs. In this preliminary report, we have developed hybrid comꢀ
pounds that merge HDAC inhibition properties with DNA recogꢀ
nition competence ꢀ via hydroxamic acid and NDI moieties, reꢀ
spectively. Compound 6 emerges among these new NDI derivaꢀ
(1.38 fold) reduced compared to TGFꢀβ1 alone, confirming that
compound 6 displays a promising ability of selection and/or reꢀ
programming of cancer cells to a less aggressive phenotype (Fig.
5
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tives as the most promising hybrid, able to both bind DNA and
inhibit HDACs. The multiꢀtarget effects of compound 6, comꢀ
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pared with the individual effects of its parental compounds, is
emphasized by the its ability of inducing a decrease of cell viabilꢀ
ity, as showed by both its parental compounds 1 and Scriptaid.
Since compound 1 and Scriptaid affect cell proliferation, the efꢀ
fect induced by compound 6 is likely to be related to its double
action of DNA binder and HDACi. Compound 6 displays also cell
phenotype reprogramming properties, not exhibited by neither
compound 1 nor Scriptaid. This is evident by the reduction of cell
migration consistent with the downregulation of the mesenchymal
marker VIM, and the upregulation of the epithelial marker CDH1.
This effect credits compound 6 of being able to induce cell reproꢀ
gramming towards a less aggressive epithelial phenotype, which
is one of the goals of present innovative strategies in cancer theraꢀ
py. The observed effects of compound 6 are clearly related to the
combined interaction with HDAC and DNAs that can produce
synergistic effects. Indeed, HDAC inhibition, by inducing hyperꢀ
acetylation of histones, relaxes chromatin and exposes both dsꢀ
and telomeric GꢀquadruplexꢀDNAs to damaging molecules.
Worth of mention, in addition to telomeres, Gꢀquadruplex may
form in additional genomic sites, i. e. at promotors of oncogenes,
and again HDAC inhibition may facilitate their stabilization by Gꢀ
quadruplex binders and alter gene expression profile. In this reꢀ
spect, further investigations are currently ongoing in our laboratoꢀ
ries and will be report in due course.
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Milelli, A.; Tumiatti, V.; Micco, M.; Rosini, M.; Zuccari,
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B.; Trigili, C.; Barasoain, I.; Musetti, C.; Toniolo, M.; Sissi, C.;
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ASSOCIATED CONTENT
Supporting Information
8.
Marchetti, C.; Minarini, A.; Tumiatti, V.; Moraca, F.;
Parrotta, L.; Alcaro, S.; Rigo, R.; Sissi, C.; Gunaratnam, M.;
Ohnmacht, S. A.; Neidle, S.; Milelli, A., Macrocyclic naphthalene
diimides as Gꢀquadruplex binders. Bioorg Med Chem 2015, 23 (13),
3819ꢀ30.
The Supporting Information is available free of charge on the
ACS Publications website.
Synthetic procedures and characterization of compounds, biologiꢀ
cal procedures (PDF).
9.
Milelli, A.; Marchetti, C.; Greco, M. L.; Moraca, F.; Costa,
G.; Turrini, E.; Catanzaro, E.; Betari, N.; Calcabrini, C.; Sissi, C.;
Alcaro, S.; Fimognari, C.; Tumiatti, V.; Minarini, A., Naphthalene
diimideꢀpolyamine hybrids as antiproliferative agents: Focus on the
architecture of the polyamine chains. Eur J Med Chem 2017, 128,
107ꢀ122.
AUTHOR INFORMATION
Corresponding Author
*
+
Dr. Andrea Milelli, Phone: +390541434610. Fax:
390541434608. Eꢀmail: andrea.milelli3@unibo.it
.
10.
Pasini, A.; Caldarera, C. M.; Giordano, E., Chromatin reꢀ
Author Contributions
modeling by polyamines and polyamine analogs. Amino Acids 2014,
46 (3), 595ꢀ603.
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
11.
Palermo, G.; Minniti, E.; Greco, M. L.; Riccardi, L.; Siꢀ
∇
script. These authors contributed equally.
moni, E.; Convertino, M.; Marchetti, C.; Rosini, M.; Sissi, C.; Miꢀ
narini, A.; De Vivo, M., An optimized polyamine moiety boosts the
potency of human type II topoisomerase poisons as quantified by
comparative analysis centered on the clinical candidate F14512.
Chem Commun (Camb) 2015, 51 (76), 14310ꢀ3.
Funding Sources
This work was supported by the University of Bologna (RFO), the
Italian Ministry for Education, Universities and Research
(MIUR).
12.
mesenchymal transition. J Clin Invest 2009, 119 (6), 1420ꢀ8.
13. Kiesslich, T.; Pichler, M.; Neureiter, D., Epigenetic control
Kalluri, R.; Weinberg, R. A., The basics of epithelialꢀ
ACKNOWLEDGMENT
Authors thank Laura Fantini and Valentina Bondi for technical
support.
of epithelialꢀmesenchymalꢀtransition in human cancer. Mol Clin Onꢀ
col 2013, 1 (1), 3ꢀ11.
ABBREVIATIONS
14.
Liao, W.; Jordaan, G.; Srivastava, M. K.; Dubinett, S.;
Sharma, S., Effect of epigenetic histone modifications on Eꢀcadherin
splicing and expression in lung cancer. Am J Cancer Res 2013, 3 (4),
CDH1, Eꢀcadherin; dsDNA, double strand DNA; EMT, epiꢀ
thelialꢀmesenchymal transition; FDA, FoodꢀandꢀDrug Adminꢀ
istration; HDAC, histone deacetylase; HDACi, histone
deacetylase inhibitor; ML, multiple ligands; NDI, naphthalene
diimide; TGFꢀβ1, transforming growth factor beta 1; TSGs,
tumor suppressor genes; VIM, Vimentin.
3
74ꢀ89.
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