Conjugation with polyamines enhances the antitumor activity of naphthoquinones against human glioblastoma cells

Article abstract of DOI:10.1097/CAD.0000000000000619

Glioblastoma multiform (GBM) is the most common and devastating type of primary brain tumor, being considered the deadliest of human cancers. In this context, extensive efforts have been undertaken to develop new drugs that exhibit both antiproliferation and antimetastasis effects on GBM. 1,4-Naphthoquinone (1,4-NQ) scaffold has been found in compounds able to inhibit important biological targets associated with cancer, which includes DNA topoisomerase, Hsp90 and monoamine oxidase. Among potential antineoplastic 1,4-NQs is the plant-derived lapachol (2-hydroxy-3-prenyl-1,4-naphthoquinone) that was found to be active against the Walker-256 carcinoma and Yoshida sarcoma. In the present study, we examined the effect of polyamine (PA)-conjugated derivatives of lapachol, nor-lapachol and lawsone on the growth and invasion of the human GBM cells. The conjugation with PA (a spermidine analog) resulted in dose-dependent and time-dependent increase of cytotoxicity of the 1,4-NQs. In addition, in-vitro inhibition of GBM cell invasion by lapachol was increased upon PA conjugation. Previous biochemical experiments indicated that these PA-1,4-NQs are capable of inhibiting DNA human topoisomerase II-α (topo2α), a major enzyme involved in maintaining DNA topology. Herein, we applied molecular docking to investigate the binding of PA-1,4-NQs to the ATPase site of topo2α. The most active molecules preferentially bind at the ATP-binding site of topo2α, which is energetically favored by the conjugation with PA. Taken together, these findings suggested that the PA-1,4-NQ conjugates might represent potential molecules in the development of new drugs in chemotherapy for malignant brain tumors.

Full text of DOI:10.1097/CAD.0000000000000619

Preclinical report
1
Conjugation with polyamines enhances the antitumor activity
of naphthoquinones against human glioblastoma cells
Luciana Romãoa,b, Vanessa P. do Cantoc, Paulo A. Netzc, Vivaldo Moura-Netob,
Ângelo C. Pintod,✠ and Cristian Follmere
Glioblastoma multiform (GBM) is the most common and
devastating type of primary brain tumor, being considered
the deadliest of human cancers. In this context, extensive
efforts have been undertaken to develop new drugs that
exhibit both antiproliferation and antimetastasis effects on
GBM. 1,4-Naphthoquinone (1,4-NQ) scaffold has been
found in compounds able to inhibit important biological
targets associated with cancer, which includes DNA
topoisomerase, Hsp90 and monoamine oxidase. Among
potential antineoplastic 1,4-NQs is the plant-derived
lapachol (2-hydroxy-3-prenyl-1,4-naphthoquinone) that was
found to be active against the Walker-256 carcinoma and
Yoshida sarcoma. In the present study, we examined the
effect of polyamine (PA)-conjugated derivatives of lapachol,
nor-lapachol and lawsone on the growth and invasion of the
human GBM cells. The conjugation with PA (a spermidine
analog) resulted in dose-dependent and time-dependent
increase of cytotoxicity of the 1,4-NQs. In addition, in-vitro
inhibition of GBM cell invasion by lapachol was increased
upon PA conjugation. Previous biochemical experiments
indicated that these PA-1,4-NQs are capable of inhibiting
DNA human topoisomerase II-α (topo2α), a major enzyme
involved in maintaining DNA topology. Herein, we applied
molecular docking to investigate the binding of PA-1,4-NQs
to the ATPase site of topo2α. The most active molecules
preferentially bind at the ATP-binding site of topo2α, which
is energetically favored by the conjugation with PA. Taken
together, these findings suggested that the PA-1,4-NQ
conjugates might represent potential molecules in the
development of new drugs in chemotherapy for malignant
brain tumors. Anti-Cancer Drugs 00:000–000 Copyright ©
2018 Wolters Kluwer Health, Inc. All rights reserved.
Anti-Cancer Drugs 2018, 00:000–000
Keywords: glioblastoma, lapachol, naphthoquinones, polyamine-conjugates,
topoisomerase
aCampus Xerém, bInstitute of Biomedical Sciences, cInstitute of Chemistry,
dDepartment of Organic Chemistry and eDepartment of Physical Chemistry,
Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Correspondence to Cristian Follmer, PhD, Department of Physical Chemistry,
Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-
909, Brazil
Tel: + 55 21 2562 7752; fax: + 55 21 2562 7265; e-mail: follmer@iq.ufrj.br
✠Deceased.
Received 6 November 2017 Revised form accepted 23 February 2018
Introduction
Quinones have been associated with a broad range of
biological properties, and many clinically important anti-
neoplastic drugs, including anthracyclines, daunorubicin,
mitomycin, mitoxantrones, doxorubicin and saintopin, dis-
play a quinone moiety [5,6]. 1,4-Naphthoquinone (1,4-NQ)
in particular is found in many inhibitors of biological targets
associated with cancer, which include DNA topoisomerase
[7], Hsp90 [8] and monoamine oxidase (MAO) [9]. Among
plant-derived 1,4-NQs with potential anticancer properties
are lapachol, a compound isolated from the tree Tabebuia
avellandedae, plumbagin, shikonin and juglone. These
molecules exhibit cytotoxic activities against different types
of tumor including carcinomas, sarcomas, breast cancer,
melanoma, pancreatic cancer, and prostate cancer [10–13].
Vitamin K is also a group of biologically active 1,4-NQs that
is believed to have important anticancer properties [14].
Glioblastoma multiform (GBM) are malignant tumors of
the central nervous system, with a very weak response to
current chemotherapies, notably because of a high level
of intrinsic drug resistance [1]. In addition, this type of
tumor is difficult to be removed surgically because of its
tendency to diffusely invade the local brain [2]. The
current standard treatment is based on maximal surgical
resection, radiotherapy and administration of the drug
temozolomide [3]. However, little success has been
obtained in extending the life expectancy of patients
diagnosed with GBM. As a result, this type of cancer is
associated with a very poor prognosis, with a mean overall
survival of 14 months [4]. In this context, the develop-
ment of new therapeutic strategies to treat GBM requires
the identification of compounds capable of inhibiting not
only the tumor growth but also its ability of invasion.
A possible strategy to enhance the antineoplastic activity
of chemotherapeutic agents is based on the conjugation
of these molecules with a polyamine (PA) backbone
[15,16]. The conjugation with PA may be an effective
way of selectively delivering cytotoxic molecules into
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DOI: 10.1097/CAD.0000000000000619
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2
Anti-Cancer Drugs 2018, Vol 00 No 00
human cancer cells by exploiting the highly active PA
transporters in tumors [17]. In addition, PA moiety
offers additional binding sites to the antitumor agents.
For instance, PA might recognize the ionic surface of
mitochondria and penetrate these organelles, an impor-
tant property for drugs targeting cancer [18]. Using
this approach, antitumor activities of 2-spermidine-3-R-
1,4-NQs were demonstrated for cancer cell lines such
as human promyelocytic leukemia, lung cancer, Burkitt
lymphoma, and mouse breast tumor [16]. The antitumor
activity of spermidine-1,4-NQs is attributed to the inhibition
of DNA human topoisomerase II-α (topo2α), an enzyme
involved in maintaining DNA topology, as demonstrated
by biochemical assays using purified ATPase domain of
topo2α [19].
Methods
Synthesis of spermidine-1,4-naphthoquinone
Spermidine-1,4-NQs were synthesized and characterized
as described by Cunha et al. [19] and Coelho-Cerqueira
et al. [9] The synthesis steps are indicated in Fig. 1, in
which PA (spermidine analog) was conjugated to lapachol
(a), lawsone (b) or nor-lapachol (c). The steps of the
synthesis were as follows: (i) the methylation of a and c
with dimethylsulphate in acetone and potassium carbo-
nate to yield 1a and 1c, and synthesis of methoxylawsone
(1b) from the sodium salt of 1,2-naphthoquinone-4-
sulfonic acid; (ii) preparation of the protected derivative
of spermidine, 2; (iii) nucleophilic displacement of
the methoxyquinones 1a, 1b, and 1c with 2, to yield the
compounds 3a, 3b and 3c (Fig. 1). To remove the pro-
tecting group BOC from 3b, a solution of TFA (0.20 ml;
2.6 mmol) in CH2Cl2 (5 ml) was slowly added to a
solution of 3b (62.7 mg; 0.13 mmol) in methanol (20 ml)
at 0°C. After 10 min, the reaction was taken to room
temperature and kept under stirring for 4 h until total
consumption of the starting material. The solvent was
removed under reduced pressure; addition of 10%
KHCO3 (10 ml) was followed by extraction with CH2Cl2.
The organic phase was dried with anhydrous Na2SO4 and
concentrated under reduced pressure to give the free
amine, 4b (50.3 mg, 99%), as a red oil that was purified
by flash chromatography (ethyl acetate/methanol, 6 : 4)
[Rf = 0.2 (CH2Cl2/methanol/triethylamine 10 : 89 : 1)].
A similar procedure was followed for the syntheses of
In the present work, we investigate the anticancer
properties of a series of PA-1,4-NQ conjugates against
two lines of GBM cells: U87MG and GBM95. These
molecules exhibit a 1,4-NQ scaffold linked with a sper-
midine analog. Our results show that the conjugation
with PA enhanced the antitumor activity of lapachol,
lawsone and nor-lapachol against GBM cells. In addition,
the attachment to PA significantly improved the ability of
1,4-NQs in inhibiting tumor invasion in vitro. By using
molecular docking tools, we demonstrated that the con-
jugation with PA favored the interaction of 1,4-NQs with
ATP-binding site of topo2α. Collectively, our findings
suggest that PA-1,4-NQs are promising antitumor agents
against malignant brain tumors.
Fig. 1
Steps of the synthesis of PA-1,4-NQ conjugates. 1,4-NQs, 1,4-naphthoquinone; PA, polyamine.
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Antitumor action of PA-conjugated 1,4-NQs Romão et al.
3
compounds 4a and 4c, which were purified as mentioned
above using ethyl acetate/methanol 6 : 4 as eluent and
obtained as a brown reddish oil (57.9 mg, 97%) [Rf =0.2
(CH2Cl2/methanol/triethylamine 10 : 89 : 1)]. Characterization
of all the compounds by MS, FTIR and NMR was previously
described by our group [9].
described [22]. The cell survival fraction was measured at
each drug concentration as the ratio of absorbance at
560 nm, relative to vehicle-treated cells.
Tumor invasion assay
For formation of the spheroids, a single-cell suspension
was generated from trypsinized monolayers and diluted
at the desired cell density, and the suspension was mixed
in DMEM/10% FBS. Spheroids were formed using the
well-known hanging drop method (25 μl, 2000 cells per
sphere). Spherical organization of spheroids/condition
was followed regularly from 0 to 72 h on phase-contrast
images (Nikon TE 2000 inverted microscope, Nikon
Instruments Inc., Melville, New York, USA). Area mea-
surements were measured using shape descriptors of
ImageJ software (https://imagej.nih.gov/ij/ ). For spheroid
invasion assays, the 3D component of the system con-
sisted of a collagen gel matrix, prepared immediately
before the spheroid culture, using a 4 : 1 mixture of
purified bovine collagen I and DMEM with 1.0 mol/l
NaOH, as necessary for neutralization of the mixture
[23]. For each experiment, 50 µl of this neutralized
solution was pipetted into each well of a 96-well plate
and placed into a 37°C, humidified incubator for 1 h to
induce gelation. One spheroid was added to the surface
of each gel using forceps, and the medium with the
treatments was added. The spheroids were treated with
25 and 50 µmol/l of a, 3a, 3b, and 4a, and the area was
analyzed for 24, 48, and 72 h. The capacity of tumor
invasion was measured through the area of the GBM95
cell spheroids (μm2) after 24, 48 or 72 h, normalized to the
area at the initial time (0 h).
Astrocyte primary cultures
Astrocyte primary cultures were prepared from the cer-
ebral cortex of newborn mice as previously described
[20]. Briefly, single-cell suspensions were obtained by
dissociating cells from the cerebral cortex in a medium
consisting of Dulbecco’s minimum essential medium
(DMEM) and nutrient mixture F12 (DMEM/F12;
Invitrogen, Carlsbad, California, USA), enriched with
glucose (33 mmol/l), glutamine (2 mmol/l) and sodium
bicarbonate (30 mmol/l). The cells were plated in dishes
or onto coverslips treated with poly-L-ornithine (Sigma-
Aldrich Co., St. Louis, Missouri, USA) and maintained in
DMEM/F12 medium supplemented with 10% fetal
bovine serum (Invitrogen). The cultures were incubated
at 37°C in humidified 5% CO2 and 95% air atmosphere.
The medium was changed every 2 days until the cells
reached confluence. The astrocyte primary cultures were
assessed by immunocytochemistry, using an antiglial
fibrillary acidic protein antibody, and cells presented
more than 95% purity.
Maintenance of the GBM cell lines
The human GBM cell line (GBM95) was obtained
according to Faria et al. [21], following procedures
established by the Brazilian Ministry of Health Ethic
Committee (CONEP no. 2340). The GBM95 cell lines
were derived from human samples and obtained with
consent from the Brazilian Ministry of Health Ethic
Committee and with consent from the patients in the
form of written statements. The cell lines were grown
and maintained in DMEM/F12 supplemented with 10%
(v/v) fetal bovine serum. The culture flasks were main-
tained at 37°C in 5% CO2 and 95% air. Cells displaying
exponential growth were detached from the culture
flasks with 0.25% trypsin/EDTA, and they were seeded
at different densities, depending on the assay. The
U87MG cell line was obtained from the American Type
Culture Collection (Manassas, Virginia, USA).
Molecular docking
The structures of lapachol, lawsone, nor-lapachol, the
spermidine-1,4-NQs (3a, 3b, 3c, 4a, 4b, and 4c) and
nucleotides ATP, ADP and ANP were built with
GaussView 4.1 [24] and optimized with RHF/6-31G(d,p)
using GAUSSIAN 09 [25]. Using AutoDock Tools [26],
the nonpolar hydrogen atoms were merged to the heavy
atoms. Gasteiger–Marsili partial charges were assigned as
described [27], and the final structures of ligands were
written as pdbqt files for further use in docking with
AutoDock Vina [28].
The topo2α receptor structure was built as a modification
of a structure obtained from the Protein Data Bank (PDB
ID: 1ZXN). As described in the text, the topo2α receptor
structure with AMP-PNP ligand (PDB ID: 1ZXM) was
also built with the same procedure and used for structural
comparison. These structures are ATPase domains of
human DNA topo2α bound to ADP [29]. For docking
purposes, both chain A and B were considered as the
starting point. The grid box was built with 48 × 48 × 48
points and constituted a large region surrounding the
ATPase site of chain A and B. All dockings, using
AutoDock Vina, were carried out in triplicate. The
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide cytotoxicity assay
GBM95 and U87MG tumor cell lines and mouse astro-
cyte cell cultures were seeded (3 × 104 cells/well) with
10% FBS DMEM-F12 medium in 96-well culture plates
and cultured for 24 h. The cells were treated with 10, 25,
50 and 100 µmol/l of lapachol (a), lawsone (b), nor-
lapachol (c), or their PA-conjugate derivatives (3a, 3b,
3c, 4a, 4b, and 4c) for 24, 48 or 72 h. Viable cells were
quantified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide cytotoxicity assay, as previously
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4
Anti-Cancer Drugs 2018, Vol 00 No 00
interactions between the ligand and protein residues
were analyzed with AutoDock Tools and Ligplot [30].
anticancer activity for the most active molecules occurred
in a concentration-dependent manner (Fig. 2b and c). 4b
exhibited a poor specificity for tumor cells, affecting
similarly GBM95 and astrocyte cells. For the other PA-
conjugates, the gain in toxic activity fostered by PA
conjugation was selective for GBM95 cells, being not
observed on astrocytes. We can also note that the removal
of the protecting group BOC from the PA backbone has
controversial effects on the antitumor properties. For
instance, removal of the BOC group from 3c (providing
4c) negatively affected the activity of this molecule,
whereas an opposite effect was observed for PA-lapachol
conjugates. The explanation for this inconsistent effect of
the BOC group on the activities of PA-1,4-NQs is
unclear. However, in most of the cases, the presence of
the protecting group has a less significant impact on the
antitumor action of these molecules than the conjugation
with PA.
Results and Discussion
Conjugation with polyamine increases the antitumor
activity of 1,4-naphthoquinones
GBM cells are remarkably resistant to radio and che-
motherapies, and the current treatment still has poor
outcomes; thereby, the efficient inhibition of both cell
growth and tumor invasion is one of the major targets in
GBM research. In the present study, we set out to
investigate the effect of 1,4-NQs lapachol (a), lawsone (b)
and nor-lapachol (c), and their PA-conjugate derivatives
on the growth of GBM95 cell line. Figure 2a shows the
cell viability, measured by 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide assay, after incubation
for 24 h. Lapachol and lawsone at 100 µmol/l exhibited a
significant decrease in GBM95 cell viability (30–40%),
without any observable toxicity on astrocyte cells.
Nor-lapachol was ineffective at this concentration.
Interestingly, the conjugation of these molecules with a
spermidine analog significantly increased the toxicity
of all 1,4-NQs. For 4a, 3b, 4b and 3c, the GBM
cell viability is reduced in ∼ 80%. In addition, the
Next, we investigated the time dependence of the anti-
tumor action against GBM cell lines and astrocyte cells
using a lower concentration of PA-1,4-NQs (25 µmol/l).
The toxicity of lapachol, lawsone and their PA-conjugates
on GBM95 cells increased in a time-dependent manner
Fig. 2
(a)
(b)
140
120
100
80
120
100
80
GBM95
Astrocytes
60
60
a
40
40
3a
4a
20
20
0
a
3a 4a
b
3b 4b
c
3c 4c
0
10 20 30 40 50 60 70 80 90 100 110
Concentration (μM)
Compound
(c)
(d)
140
120
100
80
140
120
100
80
60
60
c
b
40
40
3c
4c
3b
4b
20
20
0
0
0
10 20 30 40 50 60 70 80 90 100 110
0
10 20 30 40 50 60 70 80 90 100 110
Concentration (μM)
Concentration (μM)
Effect of 1,4-NQs and their PA-conjugates on the viability of GBM95 and astrocyte cells. (a) Toxicity of 100 μmol/l of the compounds, measured by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, on cell viability after 24 h of incubation. Concentration-dependent effect of the
compounds a (lapachol), 3a and 4a (b), b (lawsone), 3b and 4b (c) or c (nor-lapachol), 3c and 4c (d) on the viability of GBM95 after 24 h of
treatment. Toxicity assays were performed in triplicate, and the results are expressed as arithmetic mean SEM. 1,4-NQs, 1,4-naphthoquinone;
PA, polyamine.
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Antitumor action of PA-conjugated 1,4-NQs Romão et al.
5
Fig. 3
GBM95
(b)120
(c)140
(a) 120
120
100
80
100
80
60
40
20
0
100
80
60
40
20
0
60
a
3a
4a
b
3b
4b
40
20
0
c
3c
4c
0
10 20 30 40 50 60 70 80
Time (hours)
0
10 20 30 40 50 60 70 80
Time (hours)
0
10 20 30 40 50 60 70 80
Time (hours)
U87MG
(d)120
(e) 120
(f)
120
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
b
3b
4b
c
3c
4c
a
3a
4a
0
10 20 30 40 50 60 70 80
0
10 20 30 40 50 60 70 80
0
10 20 30 40 50 60 70 80
Time (hours)
Time (hours)
Time (hours)
Astrocyte
(h) 160
(g)120
(i)
120
100
80
60
40
20
0
140
120
100
80
100
80
60
40
20
0
60
a
3a
4a
b
3b
4b
c
3c
4c
40
20
0
0
10 20 30 40 50 60 70 80
Time (hours)
0
10 20 30 40 50 60 70 80
0
10 20 30 40 50 60 70 80
Time (hours)
Time (hours)
Antitumor activity of 1,4-NQs and their PA-conjugates occurs in a time-dependent manner. The cell lines, GBM95 (a–c) and U87MG (d–f), and
astrocytes (g–i) were incubated in the presence of 25 μmol/l of each compound and the cell viability monitored over time. Toxicity assays were
performed in triplicate, and the results are expressed as arithmetic mean SEM. 1,4-NQs, 1,4-naphthoquinone; PA, polyamine.
for most of the compounds (Fig. 3a and b). The attach-
ment of PA seems to not increase the toxicity of nor-
lapachol at 25 µmol/l, even after 72 h of incubation
(Fig. 3c). The time-dependent toxicity was also eval-
uated using the U87MG, a commonly studied grade IV
GBM-astrocytoma cell line, derived from human malig-
nant glioma. In this case, U87MG seems to be slightly
more sensitive than GBM95 to the toxic effects of lapa-
chol (a), 3a and 4a, in which almost 100% of cell deaths
was observed for this cell line after 72 h of treatment with
4a (Fig. 3d). In contrast, U87MG was less susceptible to
the PA-lawsone conjugates (3b and 4b) than GBM95
(Fig. 3e). PA conjugation also increased the toxicity of
the nor-lapachol derivative, 3c, on U87MG (Fig. 3f).
Collectively, these findings point out that lapachol and
lawsone exhibit higher toxicity on GBM95 and U87MG
cell lines than nor-lapachol, which is significantly
increased by the conjugation with PA. In addition, PA
conjugates 3a, 4a and 3b display a higher specificity to
GBM cells in comparison with astrocyte cells (Fig. 3g–i).
The half maximal inhibitory concentration (IC50) values,
after 72 h of incubation, were estimated for the most
potent molecules, that is, lapachol, 3a, 4a and 3b, using
GBM95 and U87MG cell lines (Table 1). Although 3b
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6
Anti-Cancer Drugs 2018, Vol 00 No 00
potentially inhibited GBM95 cells with an IC50 value of
3.6 µmol/l, it displayed a poor inhibition on U87MG
growth (34 µmol/l). 4a was the only compound capable of
efficiently inhibiting both GBM95 and U87MG with
similar IC50 values for these cell lines (6.6 and 4.3 µmol/l,
respectively).
The capacity of GBM95 cell invasion was measured
through the relative growth of cell spheroids in a collagen
I matrix, monitored for three days after their formation, in
the presence of either DMSO or the compounds a, 3a,
4a and 3b (Fig. 4). The effect on tumor invasion was
evaluated only for the most promising compounds on the
basis of the toxicity on GBM95 cells and tumor selec-
tivity. The compound 4a was excluded, because it dis-
played high toxicity against astrocytes. We can notice that
DMSO-treated cells exhibit a remarkable increase in the
area of invasive cells measured after 72 h of incubation
(∼60-fold in relation to the initial time) (Fig. 4b).
Lapachol exhibited only a slight reduction in the tumor
invasion in comparison with DMSO. In contrast, the
Table 1 The IC50 (µmol/l) values for the most active compounds
against GBM cell lines GBM95 and U87MG (72 h of incubation)
GBM
Lapachol
3a
4a
3b
GBM95
U87MG
23.4 4.8
18.4 4.1
33 7.2
23.6 4.3
6.6 2.6
4.3 2.2
3.6 1.2
34 11
Fig. 4
(a)
0 h
72 h
(b)
50 μM
70
60
50
40
30
20
10
0
24 h
48 h
72 h
a
3a
4a
3b
DMSO
Compound
(c)
25 μM
60
50
40
30
20
10
0
24 h
48 h
72 h
a
3a
4a
3b
DMSO
Compound
100 μm
PA conjugation increases the inhibitory properties of 1,4-NQs on GBM95 cell invasion. (a) Growth of GBM95 cells as spheroids in culture at the
initial time (0 h) or after 72 h of incubation with either DMSO or 50 μmol/l of the compounds a, 3a, 4a and 3b. (b, c) show the relative growth of
GBM95 spheroids after treatment with either 50 or 25 μmol/l of the compounds, respectively. DMSO alone was used as the control. Tumor invasion
was measured through the area of the tumor spheroid (in μm2) at a specific time normalized to the area at the initial time (0 h). The assays were
performed in triplicate, and the results are expressed as arithmetic mean SEM. 1,4-NQs, 1,4-naphthoquinone; PA, polyamine.
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Antitumor action of PA-conjugated 1,4-NQs Romão et al.
7
Table 2 Binding energy (kcal/mol) of 1,4-naphthoquinones and
of PA led to a remarkable increase in the capacity of lapachol
in inhibiting topo2α. In contrast, none of the compounds
were capable of inhibiting the enzyme, DNA-topo I [19].
their polyamine-conjugates into the ATPase site of topoisomerase
II-α
Ligand
Free enzyme
Bound to ADP
ΔEa
To get further information on the mechanisms lying
behind the antitumor activity of 1,4-NQ, we apply
molecular docking to evaluate the binding site interac-
tions of PA-1,4-NQs inside the human topo2α ATPase
domain. For this purpose, we utilized the x-ray crystal-
lography structure of dimeric enzyme with cocrystal ADP
ligand as a reference molecule (PDB ID: 1ZXN). The
binding energies for the best-docked conformations of
the ligand with ATPase domain of topo2α are presented
in Table 2. Lapachol, lawsone and nor-lapachol have
binding energies with the free topo2α (without ADP) of
− 8.7, − 7.9 and − 8.7 kcal mol− 1, respectively, which are
consistently more favorable (more negative energies)
than those observed in the case of docking with the
enzyme bound to ADP. In addition, the site of interac-
tion with 1,4-NQs shifts to the neighborhood of the
ATPase site when the enzyme is complexed with ADP.
To verify whether lapachol occupies essentially the same
position of ATP in the enzyme, the critical residues
surrounding the ligands, ATP and lapachol, were inves-
tigated. When ATP is docked into the free enzyme, it
interacts exactly in the same position of ADP
(Supplementary Fig. 1SA, Supplemental digital content
1, http://links.lww.com/ACD/A253). Similar results were
verified using as receptor topo2α with the cocrystal ligand
AMP-PNP (a nonhydrolysable ATP analog) (PDB ID:
1ZXM) (Supplementary Fig. 1SB, Supplemental digital
content 1, http://links.lww.com/ACD/A253). Lapachol
docked into the free enzyme interacted with residues
involved in ATP binding, which includes hydrogen
bonds with Ser149, and Asn150 and van der Waals
interactions with Tyr34, Asn91, Asn95, Ile125, Ser148,
Gly161, Gly164, Gly166, Al3a67, Arg162 (Fig. 5a, b). The
existence of additional residues participating in the ATP
binding, that is, ATP has four more hydrogen bond than
lapachol, could explain the more favorable binding
energy observed for this ligand in comparison with lapachol
(− 10.0 kcal mol−1 for ATP against − 8.7 kcal mol−1 for
lapachol). Superposition of ATP and lapachol into
the ATPase site indicates that these ligands compete
essentially for the same binding site (Fig. 5d).
a (lapachol)
3a
4a
b (lawsone)
3b
4b
c (nor-lapachol)
3c
4c
ATP
ADP
AMP
− 8.7b
− 10.0c
− 9.0b
− 8.0c
− 8.9b
− 8.6c
− 6.8c
− 8.9c
− 8.2c
− 7.9c
− 8.2c
− 8.9c
− 9.5c
− 8.7c
− 8.9c
− 1.3
− 0.3
− 7.9b
− 10.3c
− 2.4
− 9.5b
− 1.6
− 8.7b
− 10.4c
− 9.0c
− 1.7
− 0.3
− 10.0b
− 10.0b
− 9.5b
1,4-NQs, 1,4-naphthoquinone; PA, polyamine; topo2α, topoisomerase II-α.
aChange in the binding energy caused by PA attachment (free enzyme).
bActive site.
cNeighborhood of the active site, based on GBM95 cell line.
tumor invasion was significantly reduced by the presence
of 50 μmol/l of the PA-conjugated 3a, 4a and 3b. At a low
concentration (25 μmol/l), PA conjugates and lapachol
exhibited similar inhibitory activities on tumor invasion,
which were still higher than the control (Fig. 4c).
PAs such as spermidine are present in high concentra-
tions in rapidly proliferating cells [31]. PA analogues
might work as potential vectors for the incorporation of
chemotherapeutics into tumors, which might result in an
increase of the selectivity and efficiency of the drug,
either by facilitating uptake (through the active PA
transporter), or by competition with PAs for ‘critical
binding sites’ in the cells. In this context, an improve-
ment of the activity of certain drugs such as antibiotics
and anticancer agents upon PA conjugation has been
reported by several studies [32–34]. For instance, anti-
bacterial activity of chloramphenicol is enhanced when it
is linked to PA, particularly against chloramphenicol-
resistant strains. Chloramphenicol-PA conjugates also
display an increased and more selective toxicity against
human cancer cells [15]. The enhanced antitumor activ-
ity of PA-1,4-NQs observed in our studies might be
attributed to a combination of diverse factors, which
include an increase in the affinity for the already descri-
bed cancer-related biological targets of 1,4-NQs.
Interaction of 1,4-naphthoquinones with the ATP-binding
site of topoisomerase II-α is favored by polyamine
conjugation
Afterwards, the effect of the attachment of PA to 1,4-NQs
on the interaction with ATPase site of topo2α was
addressed. For all 1,4-NQs, the linkage of PA favored the
interaction with the enzyme, which might be attributed
to the fact that the PA backbone provides additional
interactions, with residues surrounding the ATPase
binding site. The perturbation in the binding energy
(ΔE) caused by PA conjugation is shown in Table 2. ΔE
associated with the PA conjugation to b is considerably
higher than that observed with the PA conjugation to a:
− 2.4 kcal mol− 1 from b to 3b; − 1.3 kcal mol− 1 from a to
3a. Interestingly, the attachment of PA has a more
Previous biochemical experiments using the purified
ATPase domain of topo2α revealed that 1,4-NQ is capable
of inhibiting this enzyme [7]. In addition,relaxation assays
using pBR322 supercoil DNA in the presence of ATP
indicated that the, PA-1,4-NQs 3a, 3b and 3c exhibit
significant inhibition of topo2α catalytic activity, whereas PA
alone did not have any effect [19]. Importantly, lapachol was
able to only partially inhibit the enzyme even at a high
concentration, which clearly demonstrates that the linkage
Copyright r 2018 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

8
Anti-Cancer Drugs 2018, Vol 00 No 00
Fig. 5
Molecular docking indicates that PA-1,4-NQs might act as topo2α inhibitors. Best-docked conformation of lapachol, ATP and 4a into the ATPase site
of topo2α are displayed in the (a–c), respectively. Superposition of ATP with lapachol (d) and ATP with 4a (e) into the ATPase site indicates that these
ligands compete essentially for the same binding site. 1,4-NQs, 1,4-naphthoquinone; PA, polyamine; topo2α, topoisomerase II-α.
significant influence on the antitumor activity of b than
a (Fig. 3a vs. Fig. 3b). Despite 3a exhibiting more
favorable binding energy (− 10.0 kcal mol−1) than 4a, the
interaction of 3a occurred at the neighborhood of
the ATPase active site (Supplementary Fig. 2SA,
Supplemental digital content 1, http://links.lww.com/ACD/
A253). In contrast, 4a, like lapachol, interacts at the
ATPase site in the same position of ATP (Fig. 5e).
Curiously, 3a exhibits lower antitumor activity than 4a.
Similar results were obtained for lawsone derivatives. Like
3a, 3b interacts preferentially at the neighborhood ATP
site (Supplementary Fig. 2SB, Supplemental digital con-
tent 1, http://links.lww.com/ACD/A253). However, 3a and 3b
have opposite orientation to the ATPase site. The analysis
of the superposition of ATP with either 3a or 3b into the
ATP-binding site indicates that the 1,4-NQ ring of 3b
occupies a position very close to the adenine ring of ATP,
whereas for 3a the 1,4-NQ ring is far off the ATP site
(Supplementary Fig. 2SC,D, Supplemental digital content
1, http://links.lww.com/ACD/A253). This could explain the
Copyright r 2018 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

Antitumor action of PA-conjugated 1,4-NQs Romão et al.
9
distinct activities of 3a and 3b. For nor-lapachol deriva-
tives 3c and 4c, linkage to PA also favors the interaction at
the neighborhood of the active site, and neither 3c nor
4c exhibited significant antitumor action on GBM95 or
U87MG cell lines.
GBM cells. Previous biochemical assays as well as
in-silico data collectively suggest that the main target of
PA-1,4-NQs and 1,4-NQ themselves is the inhibition of
topo2α. Although the exact reason why PA improves the
antitumor action of 1,4-NQs is not totally elucidated, it
likely involves a combination of factors that includes an
enhanced selectivity and efficiency of the drug uptake
into cells, as well as an increase in the affinity of 1,4-NQs
for cancer-related biological targets such as topo2α
and/or MAO.
Topo2α is a homodimer enzyme that uses the energy of
ATP hydrolysis to resolve topological problems that
occur with the DNA during cell growth and division. A
series of coordinated conformational changes regulates
the binding and dissociation of ATP, whose hydrolysis
regulates the opening and closing of molecular ‘gates’
that allow capture and restricting passage of duplex DNA
[29]. Topo2α is a marker of cell proliferation and survival
in GBM [35]. For instance, an increase in the expression
of topo2α gene transcript have been reported in the GBM
cell line U87MG [36]. In this context, inhibition of
topo2α represents an important strategy in the design and
development of new chemotherapy for GBM. The cel-
lular consequence of such inhibition is an increase in the
number of topo2α-DNA cleavage complexes entering
into the cells, leading to apoptosis [37]. Our in-silico
findings suggest that 1,4-NQs can bind to the ATPase
site of topo2α, likely by occupying the ATP-binding site,
which is favored by the conjugation with PA. In addition,
a reasonable concordance was observed between the
increased affinity for topo2α and the antitumor activity of
PA-1,4-NQs.
Acknowledgements
This work was supported by Carlos Chagas Filho
Foundation for Research Support of the State of Rio de
Janeiro (FAPERJ) and National Counsel of Technological
and Scientific Development (CNPq).
L.R. conducted most of the experiments. C.F. conceived
the idea, analyzed the results and wrote the paper. V.P.C.
and P.A.N. conducted the molecular docking analysis.
V.M.N. participated in critically revising the manuscript.
Conflicts of interest
There are no conflicts of interest.
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