ORIGINAL ARTICLE
Impact of the Potential Antitumor Agent
2-(4-Hydroxyphenyl) Amino-1,4-Naphthoquinone (Q7)
on Vasomotion Is Mediated by the Vascular Endothelium,
But Not Vascular Smooth Muscle Cell Metabolism
Javier Palacios, PhD,* Julio Benites, PhD,* Gareth I. Owen, PhD,†‡ Pablo Morales, PhD,‡
Downloaded from http://journals.lww.com/cardiovascularpharm by h6piS3blbHVTkO2SQJCLI3k9G8qtB5nOZ3cOO6aAaIb5eyHa+UCvNbTcD6VJx5X5bWnISE3kF49BvTOWvhdLxi9Ecdu+tqQ8KEZhJnmKBX4= on 02/04/2021
Mario Chiong, PhD,‡ Chukwuemeka R. Nwokocha, PhD,§ Adrián Paredes, PhD,¶
and Fredi Cifuentes, PhD║
Abstract: Vasomotion is defined as rhythmic oscillations in
arterial diameter that regulate the blood flow and blood pressure.
Because antitumor treatment may impair vascular functions
and increase the blood pressure, we sought to evaluate whether
a new naphthoquinone derivative, postulated as an antitumor
agent, manifests adverse effects on vascular function. In this
article, we evaluated the toxicity of 2-(4-hydroxyphenyl)
amino-1,4-naphthoquinone (Q7) and its effects on vascular
Received for publication August 1, 2020; accepted October 13, 2020.
From the *Departamento de Química y Farmacia, Facultad Ciencias de la
Salud, Universidad Arturo Prat, Iquique, Chile; †Departamento de
Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad
Católica de Chile, Santiago, Chile; ‡Advanced Center for Chronic
Diseases, ACCDiS, CEMC, Department of Biochemistry and Molecular
Biology, Faculty of Chemical and Pharmaceutical Sciences, University of
Chile, Santiago, Chile; §Department of Basic Medical Sciences Physiology
Section, Faculty of Medical Sciences, The University of the West Indies,
Mona, Kingston, Jamaica; ¶Departamento de Química, Facultad de
Ciencias Básicas, Universidad de Antofagasta, Antofagasta, Chile; and
║Laboratorio de Fisiología Experimental, Instituto Antofagasta,
Universidad de Antofagasta, Antofagasta, Chile.
Financial support was provided by FONDECYT 1200610 (J.P.), 1180241
(G.I.O.), Vicerrectoría para Investigación, Innovación y Postgrado de la
Universidad Arturo Prat (VRIIP0002-20, VRIIP0006-17, and
VRIIP0198-14), and the Network for Extreme Environments Research
project to F. Cifuentes and A. Paredes (NEXER, Project ANT1756,
Universidad de Antofagasta, Chile). CONICYT FONDAP-15130011
(M.G. and G. I. Owen) and Millennium Institute on Immunology and
Immunotherapy IMII P09/016-F (G. I. Owen).
The authors report no conflicts of interest.
F. Cifuentes, A. Paredes, and J. Palacios conceived and designed the research
study, and also carried out the vascular reactivity experiments; cytotox-
icity and angiogenesis assays were performed in the laboratory of G. I.
Owen; P. Morales, J. Palacios, and M. Chiong performed the ATP content
and glucose uptake; J. Benites synthetized Q7; and C. R. Nwokocha, G. I.
Owen, and J. Palacios edited and revised the manuscript.
Ethics approval: The trials were conducted in accordance with the Guide for
the Care and Use of Laboratory Animals published by the National
Institutes of Health of the United States (NIH, publication revised in
2013) and the Ethics Committee of Arturo Prat University (CEC-17).
Availability of data: The data sets generated during and/or analyzed during
the current study are available from the corresponding author on
reasonable request.
Reprints: Javier Palacios, PhD, Laboratorio de Bioquímica Aplicada,
Facultad de Ciencias de la Salud, Universidad Arturo Prat, Av. Arturo
Prat Chacón, 2120, 1110939 Iquique, Chile (e-mail: clpalaci@unap.cl).
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J Cardiovasc Pharmacol   Volume 77, Number 2, February 2021
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vasomotion in 3 models of vascular structure: endothelial cells,
aortic ring, and smooth muscle cells. Although showing nontoxic
effects, Q7 inhibited the formation of capillary-like structures of
the EA.hy926 endothelial cell line grown on Matrigel. In exvivo
experiments with aortic rings precontracted with phenylephrine
(PE, 1026 M), Q7 (1025 M) significantly (P , 0.05) reduced
vascular rhythmic contractions induced by the acetylcholine
(ACh; 1027-1025 M), whereas sodium nitroprusside (a nitric
oxide donor; 1028 M) recovered the vasomotion. Furthermore,
Q7 (1025 M) did not decrease KCl-induced vascular rhythmic
contractions in the aortic rings precontracted with BaCl2 (a non-
selective K+ channel blocker; 1023 M). Vascular smooth muscle
cells (A7r5) preincubated with Q7 (1025 M) for 3 hours also
demonstrated a reduced glucose uptake. However, the
Adenosine Triphosphate content was unaffected, suggesting that
the rapid reduction in vasomotion observed in vascular reactivity
experiments did not involve cellular metabolism but may be due
to faster mechanisms involving endothelial nitric oxide and K+
channels leading to oscillations in intracellular Ca2+. In summary,
the naphthoquinone derivative Q7 presents low cytotoxicity yet
may alter the endothelial cell response and vasomotion in the
absence of changes in smooth muscle cell metabolism.
Key Words: naphthoquinone, vasomotion, endothelium, nitric
oxide, ATP, aorta
(J Cardiovasc Pharmacol
2021;77:245–252)
INTRODUCTION
Vasomotion is the spontaneous rhythmic changes in
the diameter and tone of arteries and arterioles, essential in
the local regulation of blood flow.1 In vascular smooth
muscle cells, vasomotion is associated with oscillations
in intracellular Ca 2+ and with changes in membrane poten-
tial,2 whereas alterations in microvascular beds are impli-
cated in diseases, such as hypertension and diabetes.3 The
vasomotion mechanisms consist of oscillations in the
membrane potential of smooth muscle cells, followed by
rhythmic voltage-dependent Ca2+ entry. This, in turn,
allows for an intermittent release of Ca 2+ from intracellu-
lar stores.2 In addition, endothelial nitric oxide (NO) is
necessary for vasomotion regulation through K+ channels
and cytosolic Ca2+.4
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Palacios et al
Adenosine Triphosphate (ATP) content and glucose
metabolism play an important role in modulating spontaneous
rhythmic contractions in blood vessels, specifically on the
plasma membrane ion channel activity and membrane
potential.5 It is postulated that mitochondrial metabolism
stimulates vasomotion through changes in the mitochondrial
membrane potential and Ca2+ oscillations, allowing for an
intracellular communication between vascular endothelial
cells and smooth muscle cells.6
Numerous quinone-bearing molecules, such as the 1,4-
naphthoquinone pharmacophore group, are used as therapeutic
drugs. Juglone (5-hydroxy-1,4-naphthoquinone), a recognized
naphthoquinone derivative from walnut, is used in the treatment
of cancer.7,8 Naphthoquinone derivatives such as 2-(4-
hydroxyphenyl) amino-1,4-naphthoquinone (Q7) are known to
possess potent anticancer activities; however, their clinical appli-
cation is hampered by adverse side effects. Previous studies have
shown that treatment with quinone-related compounds can
impair vascular functions and increase blood pressure.9–11
The EA.hy926 cell line is a hybridized cell line derived
from the A549/8 human lung carcinoma cell line and human
umbilical vein endothelial cells. EA.hy926 is regarded as an
immortalized endothelial cell line because this hybrid kept the
human umbilical vein endothelial cell–derived endothelial phe-
notype and behavior while possessing the immortality of the
cancer cell patronage. This cell line is now an established in vitro
approximation of angiogenesis because the endothelial cells form
capillary-like structures when grown in on Matrigel (a substitute
for the 3D structure of the extracellular matrix).12 The formation
of these structures is promoted in the presence of vascular endo-
thelial growth factor and is inhibited by the presence of throm-
bin.13,14 The A7r5 cell line, which is derived from the thoracic
aorta of an embryonic rat, shares similar neural stem cell markers
with both multipotent vascular stem cells and adventitial progen-
itors and thus are widely used as an in vitro model of nondiffer-
entiated, neonatal and neointimal vascular smooth muscle cells.15
Concentration-response curves in the aortic rings of rat are used
as a tool for the evaluation of vasoactive substances in the vas-
cular endothelium and smooth muscle.16 To assess vasomotion or
vascular relaxation, the aortic rings are precontracted with an
agonist, and after the aortic rings reach a plateau of vascular tone,
the vasoactive substance is added.17
In this study, we sought to evaluate the vascular function
of the new naphthoquinone derivative compound Q7. To this
end, we assessed effects of Q7 on the several players in the
regulation of vascular function, namely the modulation of
vascular endothelium by NO, and K+ channels, ATP levels,
and glucose uptake on vascular smooth muscle cells.
METHODS AND MATERIALS
Animals
Ten 4 weeks old (150–190 g) Wistar male rats were
provided by the Height Institute of the Arturo Prat University
of Iquique and randomly distributed in cages and maintained
at a suitable temperature (228C–248C) and humidity (45%–
50%). Animals had ad libitum access to water and food
(Champion, Santiago).
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J Cardiovasc Pharmacol   Volume 77, Number 2, February 2021
Q7 was synthesized by the laboratory of Medicinal
Chemistry of the Faculty of Health Sciences, Arturo Prat
University, and doses of use selected according to a previous
study.18 The trials were conducted in accordance with the
Guide for the Care and Use of Laboratory Animals published
by the National Institutes of Health of the United States (NIH,
publication revised in 2013) and the Ethics Committee of
Arturo Prat University (CEC-17).
Isolation of Aortic Rings
After the animal was killed by cervical dislocation, the
aorta was separated and transferred to a Krebs–Ringer
bicarbonate buffer solution (48C) (in mM): 4.2 KCl, 1.19
KH2PO4, 120 NaCl, 25 NaHCO3, 1.2 MgSO4, 1.3 CaCl2, and
5 D-glucose (pH7.4). Aortic rings of 2–3 mm were prepared
and cleaned of connective tissue, taking special care to avoid
endothelial damage.
Vascular Reactivity Experiments
Aortic rings from the same animal were concurrently
studied in different organ baths19 for comparable abilities and
reactivity function. After a 30 minutes period of equilibration,
the aortic rings were stabilized by 3 successive, near-
maximum contractions with 60 mM KCl for 10 minutes.
On the aorta a passive tension of 1.0 g was maintained, a
value previously determined by our laboratory as the optimal
resting tension for obtaining maximum active tension.20
Vasodilation to 1025 M acetylcholine (ACh, muscarinic ago-
nist) in aortic rings precontracted with 1026 M phenylephrine
(PE) was used as a method to assess endothelial function.
Functionality was confirmed with a vasodilation of 70%–
80%.16
In Vitro Vascular Network Formation Assay
The vascular network formation assay was performed
as previously described.12 EA.hy926 cells were cultured on
top of growth factor–reduced Matrigel-coated plates.
Thrombin (Throm; 1 IU/mL) was used as a negative
control.14
MTS Reduction Assay
Cytotoxicity on the EA.hy926 cell line was determined
using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy
methoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduc-
tion assay as previously described by Palacios et al.11
Cytotoxicity of the naphthoquinone derivative (Q7) was as-
sessed at varying concentrations in relation to a vehicle-
treated control. Cyclophosphamide (Cyclo; 1024 M) that is
inactive in cell culture was used as a negative control (cyclo-
phosphamide is active in the human body only after metabo-
lism in the liver). The chemotherapy drug epirubicin (Epirub;
1025 M) was used as a positive control for cell death.
Epirubicin is an anthracycline chemotherapy drug that exerts
its effect by intercalating DNA strands and thus inhibiting
DNA and RNA synthesis. The cytotoxicity was calculated
in accordance with the formula: cytotoxicity (%) =
[12(absorbance sample/absorbance control)] · 100. The
absorbance was measured with a microplate reader (Infinite
200 PRO; Tecan, Switzerland) at 490 nm.
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J Cardiovasc Pharmacol   Volume 77, Number 2, February 2021
ATP Measurement
The vascular smooth muscle cell line A7r5 (ATCC
CRL-1444) was cultured as previously described.21
Intracellular ATP levels were determined with a luciferin/
luciferase-based ATP detection kit, the CellTiter-Glo
Luminescent Cell Viability Assay (Promega, Madison, WI).
Sample luminescence was quantified in a TopCount NXT
microplate luminescence counter (PerkinElmer, Waltham,
MA). Data were normalized as fold over control.
Glucose Uptake
Glucose uptake was measured using 1025
M [3H]2-
deoxyglucose solution for 10 minutes at 378C, as previously
reported.22 Radioactivity was determined by liquid scintilla-
tion counting.
Drugs
The following drugs were used in this study: L-phen-
ylephrine hydrochloride (PE; Sigma-Aldrich, St Louis,
MO), acetylcholine chloride (ACh; Sigma-Aldrich, St
Louis, MO), sodium nitroprusside (SNP; Merck,
Darmstadt, Germany), and barium chloride dehydrate
(BaCl2; Sigma-Aldrich, St Louis, MO). The 2-[(4-
hydroxyphenyl) amino]-1,4-naphthoquinone (Q7; 265.07
g/mol) was synthesized by amination of 1,4-
naphthoquinone with 4-hydroxyphenylamine, under aero-
bic conditions, using CeCl3$7H2O as the Lewis acid cata-
lyst as previously reported.23 The solutions used were
freshly prepared before each experiment.
Statistical Analysis
Values are expressed as the mean 6 SEM; n represents
the number of animals studied. For the statistical analysis of the
groups, a one-way analysis of variance was used, as appropri-
ate, followed by a Bonferroni post hoc test. A P value , 0.05
is considered statistically significant. We used GraphPad Prism
version 6.0 software (GraphPad Software, CA).
RESULTS
Chemical Composition
Figure 1 shows the chemical structure of 2-[(4-
hydroxyphenyl) amino]-1,4-naphthoquinone (Q7), synthesized
under aerobic conditions by amination of 1,4-naphthoquinone
with 4-hydroxyphenylamine, using CeCl3$7H2O as the Lewis
acid catalyst as previously reported.23 The structure of Q7 was
established on the basis of their spectral properties (infrared
radiation, 1H Nuclear Magnetic Resonance, and 13C Nuclear
Magnetic Resonance) and micro analytical data according to a
previous study.23
Effect of Q7 on Spontaneous Rhythmic
Contractions in Intact Aortic Rings of Rat
In the next experiments, we evaluated whether Q7
impaired vascular function, that is, vascular rhythmic
contractions (Fig. 2A). In the rat aorta previously con-
tracted with 1026 M PE, we found a significant reduction
in the amplitude of ACh-induced rhythmic contractions
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Impact of the Potential Antitumor Agent
FIGURE 1. Chemical structure of 2-[(4-hydroxyphenyl)
amino]-1,4-naphthoquinone (Q7; 265.07 g/mol).
by preincubation with 10 25 M Q7 (24 6 3% control vs.
12 6 2 with 1026 M ACh; P , 0.05 and 29 6 3% control
vs. 13 6 6% with 10 25 ACh; P , 0.05; Fig. 2B). In the
same tissues, Q7 significantly decreased ACh-induced
relaxation, as shown by reducing the maximal concentra-
tion (Emax) to ACh (1025 M) but not the sensitivity
(log EC 50 ) to ACh (Table 1). The preincubation of aortic
rings with Q7 did not cause any change in either the Emax
and log EC 50 of contractile response to 10 26 M PE
(Table 1).
The vasomotion described above was confirmed as
endothelium dependent. ACh is known to induce the
generation of NO from the vascular endothelium, and SNP
is a recognized NO donor. Interestingly, SNP recovered the
rhythmic contractions in the presence of Q7 (Fig. 2C),
increasing amplitude to a similar value to that of the ACh
control (Fig. 2B).
To study the role of depolarization of the plasma
membrane, a nonselective blocker of inward-rectifier K+
channels and a low concentration of KCl were used. The
preincubation of aortic rings with Q7 significantly increased
the contractile response to 1026 M PE (134 6 6% control vs.
192 6 12 with 1025 M Q7; Figs. 3A, B). However, we found
no change by Q7 in the amplitude of KCl-induced vascular
rhythmic contractions in the aortic rings precontracted with
1 mM BaCl2 (Fig. 3C).
Potential Antiangiogenic Effect of Q7 on the
EA.hy926 Endothelial Cell Line
Antiangiogenic therapy is used clinically to arrest
cancer cell growth.24 The potential proangiogenic or antian-
giogenic activity of the Q7 compound was evaluated by its
ability to alter the formation of capillary-like (tubular) struc-
tures of the EA.hy926 endothelial cell line grown on
Matrigel.25 The results demonstrated that the preincubation
with Q7 (1027 to 1025 M) of EA.hy926 cell line significantly
decreased the formation of tubular structures (Figs. 4A, B).
Therefore, the inhibition of tubular networks in this endothe-
lial cell line may suggest an inhibition of vascular endothelial
function and a potential antiangiogenic effect of Q7.
Cytotoxicity of Naphthoquinone Derivative
(Q7) on the EA.hy926 Endothelial Cell Line
Next, we assessed the cytotoxicity activity of 2-(4-
hydroxyphenyl) amino-1,4-naphthoquinone (Q7) on
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Palacios et al J Cardiovasc Pharmacol   Volume 77, Number 2, February 2021

FIGURE 2. Preincubation with Q7 decreased rhythmic contractions in aortic rings of rat. Representative traces showing a decrease
of ACh-induced (1026 to 1025 M) but not SNP-induced (1028 M) maximal amplitude in the rat aorta precontracted with 1026 M
phenylephrine in absence (control) or presence of 1025 M Q7 (A). Summary of ACh-induced (B) and SNP-induced (C) rhythmic
contraction data. The maximum amplitude at ACh or SNP was calculated as a percentage of the third contractile response to KCl
(60 mM). Data represent the SEM of 5 independent experiments. *P , 0.05 versus control.

EA.hy926 cells. No significant cytotoxicity was found in the , 0.001). Although the naphthoquinone derivative Q7
EA.hy926 cell line after 48 hours of incubation with the showed no cytotoxic activity, we assessed if it could cause
naphthoquinone derivate Q7 (1027 to 1025 M) or in the vascular dysfunction.
negative control (cyclophosphamide, 1024 M) (Fig. 5). The
positive control for cell death, epirubicin (1025 M), caused a Effect of Q7 on Metabolic ATP and Glucose
significant cytotoxicity in the EA.hy926 cell line (64 6 1%; P Uptake in the Vascular Smooth Muscle Cell
Line (A7r5)
TABLE 1. Effect of Q7 on the Contractile Response to PE and The A7r5 cell line is derived from the thoracic aorta of
Relaxation to ACh in the Rat Aorta an embryonic rat. These cells share similar neural stem cell
markers with both multipotent vascular stem cells and
Drugs Emax (%) Log EC50 adventitial progenitors and thus are widely used as an
PE in vitro model of nondifferentiated, neonatal and neointimal
Control 174 6 12 8.06 6 0.12 vascular smooth muscle cells.15 To determine whether Q7
Q7 174 6 13 8.15 6 0.13 may have metabolic implications, we measured ATP levels
ACh in a rat’s aortic vascular smooth muscle cell line (A7r5). We
Control 105 6 5 7.21 6 0.13 found only a 13% decrease of ATP levels in A7r5 cells pre-
Q7 79 6 5* 7.14 6 0.22 incubated with 10–5 M Q7 for 3 hours versus control (0.88 6
Emax represents the effect at maximal concentration to PE (1026 M) and ACh 0.01% ATP content with 10–5 M Q7; Fig. 6). Preincubation
(1025 M), and log EC50 represents the logarithm of the half maximal effective concen- with 1.2 mM oligomycin for 30 minutes decreased ATP levels
tration. The values in mean 6 SEM represent the mean of 5 independent experiments.
Statistically significant difference *P , 0.05 versus control. in A7r5 cells by 39% (0.61 6 0.07 relative units; P , 0.001)
versus control.

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J Cardiovasc Pharmacol   Volume 77, Number 2, February 2021 Impact of the Potential Antitumor Agent

FIGURE 3. Role of K+ on vascular rhythmic contractions in aortic rings. The effect of 1 mM BaCl2 (a nonselective blocker of inward
rectifier K+ channels) and 10 mM KCl in the absence or presence of 1025 M Q7. The tracings were amplified from the regular part
of the recording, 10 minutes after the addition of KCl (10 mM) (A). Vascular contraction (B) and the maximum amplitude to BaCl2
(C) were calculated as a percentage of the third contractile response to KCl 60 mM. Data represent the SEM of 5 independent
experiments. *P ,0.05 versus control.

To further understand the mechanism of ATP in the DISCUSSION

presence of Q7, we examined the role of glucose uptake in
A7r5 vascular smooth muscle cells. Preincubation with Q7
(1025 M) for 3 hours significantly decreased the glucose
uptake compared with the control condition (22 6 2 cpm/
mg protein control vs. 15 6 1 cpm/mg protein with 1025 M
Q7; P , 0.05; Fig. 7), whereas no significant change was
observed at 30 minutes.
Vasomotion, defined as rhythmic oscillations in arterial
diameter, is essential in the local regulation of blood flow and
thus vascular function.1 We assessed the effects of potential anti-
tumor agents and naphthoquinone derivative Q7 on the several
players in the regulation of vascular function, such as the mod-
ulation of vascular endothelium, K+ channels, ATP levels, and
glucose uptake.

FIGURE 4. Q7 reduces vascular mor-

phogenesis in vitro. Representative
images are shown for the EA.hy926
cell line (A). Preincubation with Q7 of
the EA.hy926 cell line decreases the
formation of tubular structures (B).
Thrombin (Throm; 1 IU/mL) was used
as a negative control. Data represent
the SEM of 4 independent experi-
ments. **P , 0.01 and ***P , 0.001
versus control. Scale bar = 100 mm.

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Palacios et al
FIGURE 5. Determination of cytotoxicity of the naph-
thoquinone derivative (Q7) on the EA.hy926 endothelial cell
line. Cyclophosphamide (Cyclo; 1024 M) was used as a neg-
ative control and epirubicin (Epiru; 1026 M) as a positive
control. Data represent the mean 6 SEM. ***P , 0.001 versus
control; n = 4.
The vasomotion phenomenon consists of 2 compo-
nents: tonic and phasic vascular response.26 For vasomotion
to occur, a certain vascular tone is required in response to
vasoactive substances.27 As illustrated in Figure 8, the tonic
response is Ca2+ dependent, and the phasic response is K+
dependent.
On the one hand, our results confirmed that ACh,
following PE, produced vascular rhythmic contractions. The
release of endothelial NO mediated by ACh produces artery
relaxation28 and vascular rhythmic contractions in the rat
FIGURE 6. Q7 does not reduce the production of ATP (after 3
hours) in the A7r5 vascular smooth muscle cell line. A7r5 cells
were stimulated with 1026 M Q7 (gray bar) or 1025 M Q7
(black bar) for different times (30 minutes, 1 hour, or 3 hours).
Mitochondrial ATP production was determined using a
luciferin/luciferase-based kit. Oligomycin (Oligo, 1.2 mM), an
ATP synthase inhibitor, was used as a negative control. Data
represent the SEM of 5 independent experiments. **P , 0.01
versus control.
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J Cardiovasc Pharmacol   Volume 77, Number 2, February 2021
FIGURE 7. Q7 reduces the glucose uptake in the A7r5 vascular
smooth muscle cell line. A7r5 cells were stimulated with Q7
(1026 M and 1025 M) for 30 minutes or 3 hours. A7r5 cells
were preincubated for 30 minutes with insulin (Ins, 1027 M) or
cytochalasin-B (Cit B; 1025 M), an inhibitor of glucose trans-
porters, as a negative control (C). Glucose uptake was deter-
mined using [3H]-2-deoxyglucose. The data represent the SEM
of 4 independent experiments. *P , 0.05 and ***P , 0.001
versus control.
aorta.17 Endothelial NO modulates rhythmic contractions
through Ca2+-activated K+ channels and Ca2+ oscillations of
vascular tissue.29,30 On the other hand, we found that Q7
decreased the ACh-induced relaxation and the maximal
amplitude of ACh-induced vasomotion in intact aortic rings
but not SNP-induced vasomotion. Interestingly, applied SNP
recovered the rhythmic contractions in the presence of Q7,
increasing amplitude to similar values to that of the ACh
control. This implies the participation of NO in vasomotion.
Because Q7-generated reactive oxygen species decrease the
bioavailability of NO induced by ACh in the rat aorta11 and
vasomotion depends on NO,5,31 we hypothesize that Q7
reduces the amplitude of vasomotion by decreasing NO bio-
availability. However, it did not happen in the presence of
SNP, probably because the release of NO by SNP saturates
the ability of the Q7 scavenger. These rhythmic oscillations in
vascular tone and vasomotion modulate the hemodynamic of
flow and, as such, could be regarded as a biomechanism that
occurs through vascular endothelial functions.32,33 It is also
important to consider that the animals were 1-month-old, and
in previous experiments, we found that vasomotion decreased
in the rat aorta from 5 to 6-month-old (data not shown), which
is associated with aging.34 Stiffness is known to increase with
aging; therefore, another physiological significance of vaso-
motion in the rat aorta is a measure of the elastic property of
conduit vessels.35
Q7 inhibited the formation of tubular networks (an
in vitro approximation of angiogenesis) in the EA.hy926
vascular endothelial cell line, without causing any cell
cytotoxicity. As endothelial NO is a mediator of angiogen-
esis,36 this potential inhibition of angiogenesis by Q7 sup-
ports the impairment of vascular endothelium and lower
generation or bioavailability of NO. The potential antiangio-
genic effect of Q7 further supports the clinical attractiveness
of this compound as an antitumor agent. The assessment of
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J Cardiovasc Pharmacol   Volume 77, Number 2, February 2021
FIGURE 8. Putative negative effect of 1,4-naph-
thoquinone derivative (Q7) on the vasomotion
phenomenon in the rat aorta. Vasomotion,
defined as rhythmic contractions of blood vessels,
consists of 2 components: tonic and phasic vas-
cular response. The tonic response is Ca2+
dependent, and the phasic response is K+
dependent. eNOS, endothelial nitric oxide syn-
thase; IP3, inositol trisphosphate.
this parameter should be incorporated into future protocols
evaluating the in vivo antitumor effects of this compound.
KCl-mediated relaxation of aortic rings precontracted
with BaCl2 generated an important vasomotion. First, BaCl2
blocks Ca2+-activated K+ channels at millimolar concentra-
tions, depolarizing the cell membrane leading to an increased
Ca2+ influx and vascular contraction.37 Second, the increase
of extracellular KCl up to 20 mM causes hyperpolarization of
the plasma membrane and relaxation in vascular smooth mus-
cle.38 The contractile response to BaCl2 increased with Q7
preincubation while KCl-induced vascular rhythmic contrac-
tions did not decrease. Rhythmic contractions require reach-
ing an intermediate vascular tone or threshold, which depends
on the concentration of cytosolic calcium. Intracellular cal-
cium levels are regulated by vasoactive substances2 and by
Ca2+-activated K+ channels.30 Our results suggest that the
increase of barium-induced vascular contraction enhanced
the vasomotion caused by the repolarization with KCl and
likely masking Q7-impaired vasomotion.
The hypothesis that Q7 (1,4-naphthoquinone deriva-
tive) may induce the release of cyclooxygenase (COX)-
derived metabolites was analyzed in the literature. Several
studies demonstrated that 1,4-naphthoquinone derivatives
inhibit the cyclooxygenase synthesis. In vivo and in vitro,
1,4-naphthoquinone derivative exhibits antiplatelet activity
by the inhibition of thromboxane synthesis39; in vitro, 1,4-
naphthoquinone derivatives inhibit COX-1 and COX-2 by a
redox-cycling mechanism,40 and menadione (2-methyl-1,4-
naphthoquinone) inhibits prostaglandin synthesis in cultured
porcine endothelial cells.41 Therefore, to understand whether
Q7 can induce the release of COX-derived metabolites (eg,
thromboxane A4) and cause a reduction in vasomotion in Q7,
further studies are required.
Cellular ATP content plays an important role in the
energy metabolism and regulation of vasomotion.4
Preincubation for 3 hours with Q7 did not significantly
decrease ATP levels in the A7r5 cell line. However, previous
reports in the bladder cancer cell line (T24), observed that a
drop in the intracellular content of ATP was found after pre-
incubation with Q7 for 6 hours.23 On the other hand, in vas-
cular tissue, glucose is metabolized to lactate when the glucose
concentration in blood is at a physiological level, whereas the
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Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Impact of the Potential Antitumor Agent
oxidation of glucose through the Krebs cycle occurs with a
lower level of glycemia.42,43 In this article, we show that Q7
significantly decreased glucose uptake in vascular smooth mus-
cle cells after 3 hours. This result is in agreement with a pre-
vious study, which showed that preincubation with Q7 or
juglone (5 hydroxy-1,4-naphthoquinone) for 1 hour decreased
glucose uptake in ex vivo in Ehrlich tumor cells from mice.44
Therefore, considering that the preincubation time with Q7 was
20 minutes in the vascular reactivity experiments of rat aorta,
this effect may not be the consequence of a decrease in glucose
uptake or a reduction in the total ATP content, which would be
slower mechanisms of action.
Previous studies reported a cytotoxic effect of Q7 (1025
M) on human bladder carcinoma T24 cells (40%)45 and breast
cancer MCF-7 cells (20%),18 whereas in Ehrlich carcinoma in
mice (20%) the Q7 doses (intraperitoneal injection) were 1
mg/Kg of body weight for 9 days.44 Naphthoquinone deriv-
atives possess potent antitumor activities because of their
ability to generate oxide-reduction processes.23 These deriv-
atives induce reactive oxygen species, such as superoxide
anion (O2-), hydrogen peroxide (H2O2), and the hydroxyl
radical (OH) that can cause cell death.46
In conclusion, vasomotion contributes to the evaluation
of the vascular function in the presence of a new naphthoqui-
none derivative with low cytotoxicity. We demonstrate that the
rapid impairment of vasomotion caused by a naphthoquinone
derivative may be mediated by a fast-acting mechanism
involving NO from vascular endothelium or K+ channels in
vascular smooth muscle and not by slower cellular metabolism
mechanisms, such as reduced glucose uptake or ATP deple-
tion. Although these results are promising, future in vivo exper-
iments will confirm if these 1,4-naphthoquinones have the
clinical potential as antitumor agents.
ACKNOWLEDGMENTS
The authors wish to express their gratitude to the
Vicerrectoría de Investigación, Inovación y Postgrado de la
Universidad de Arturo Prat and the Rectoria y Vicerrectoria de
Investigacion, Innovacion y Postgrado Universidad de
Antofagasta for their financial and technical support. Also a
www.jcvp.org | 251
Palacios et al
special thanks to Pilar Díaz Toro from the laboratory of
Medicinal Chemistry of the Faculty of Health Sciences of the
Arturo Prat University, Iquique, for synthesizing the naphthoqui-
none derivative [2-(4-hydroxyphenyl)amino-1,4-naphthoquinone]
(Q7).
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