2
F. Ourique et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e7
Q9 (2-(4-methoxyanilino)-1,4-naphthoquinone) as well as other 3-
acyl-2-phenylamino-1,4-naphthoquines have been synthesized
and screened for antitumor activities with or without ascorbate.
Initially, a series of in vitro assays were performed. These in-
vestigations have shown inhibition of cell proliferation and
migration by oxidative stress from ascorbate-driven juglone redox
cycling in human bladder-derived T24 cells in culture [7]. In addi-
tion, it was previously shown that the antiproliferative effects of Q7
and Q9 are potentiated by ascorbate and associated with the
appearance of a senescent phenotype in T24 cells [8]. The formu-
lations prepared with juglone, Q7 and Q9 in combination with
ascorbate were tested later in vivo using Ehrlich ascites tumor-
bearing mice. These screening assays showed that the potenti-
ating effect of ascorbate was reproduced in vivo in the cases of
juglone and Q7. Q7 plus ascorbate caused up to 60% of inhibition of
tumor and the largest extension of survival of mice. Therefore,
taking into consideration some previous studies from our labora-
tory, a series of 1,4-naphthoquinones were left behind, whereas
juglone and Q7 plus ascorbate were kept in the study because they
caused the most promising antitumor activity, causing DNA dam-
age and inhibition on pAkt in Ehrlich ascites tumor in mice [9].
These previous data provided the basis for maintaining the in-
vestments on further studies in order to understand better the ef-
fects caused in vivo by juglone and Q7 in combination with
ascorbate. In the current phase, our approach took into account
some potential actions on an important part of the antioxidant
system, as well as the effects on the glycolytic metabolism and
and 100 mg/kg, respectively). Ascorbate was administered at doses
100-fold higher than the doses of juglone or Q7 [9]. Two hours after
the last dose, samples of ascitic fluid were collected for analysis.
2.4. Biomarkers of oxidative stress in Ehrlich tumor tissue
Samples of ascitic fluid were centrifuged (5000 g for 5 min).
Supernatants were diluted (1:5 v/v) in buffer containing 100 mM
sodium phosphate,150 mM NaCl and 0.1% Triton X-100 (pH 7.4) and
acidified in trichloroacetic acid (TCA 12%, 1:5 v/v) for the mea-
surement of reduced glutathione (GSH). Oxidative damage to pro-
teins was quantified as carbonyl proteins as described by Levine
et al. [11]. Carbonyl groups of proteins react covalently with 2,4-
dinitrophenylhydrazine, which triggers the formation of a 2,4
dinitrophenylhydrazone product. The reaction was followed by
spectrophotometric quantification of the acid hydrazones at
370 nm. The content of GSH was estimated as described by Beutler
et al. [12] using the reaction of small thiols in the sample with
dithiobisnitrobenzoic acid. The resulting yellow thiolate formation,
proportional to the content of GSH, can be quantified spectropho-
tometrically at 412 nm. Catalase activity was determined through
the method described by Aebi [13] based on the decomposition of
2 2
H O and the decrease in absorbance at 240 nm. Superoxide dis-
mutase (SOD) activity was measured monitoring the oxidation of
adrenaline to adrenochrome as described by Misra and Fridovich
[14]. Glutathione peroxidase (GPx) activity was measured using the
method described by Floh eꢀ and Günzler [15]. Tert-butyl hydro-
tumor survival under hypoxia by assessing HIF-1
a
, glucose trans-
peroxide was used as substrate to be reduced by GPx in a reaction
that uses GSH as a reducing agent forming GSSG. By using gluta-
thione reductase (GR), GSH can be regenerated from GSSG in a
reaction that uses NADPH as a reducing agent. The rate of NADPH
oxidation is proportional to the activity of GPx that was quantified
at 340 nm. This principle was followed as well to estimate GR ac-
tivity using the method described by Calberg and Mannervick [16].
All results were normalized to the protein content using the
method of Lowry et al. [17].
porters GLUT1 and glucose uptake in tumor tissue of Ehrlich tumor-
bearing mice. Therefore, the aim of this work was to obtain further
pharmacological data of the antitumor effects and mechanisms
triggered by juglone and Q7 with and without ascorbate in vivo.
This manuscript reports data showing that these treatments
induced oxidative stress associated with cell cycle arrest and tumor
cell death in vivo triggering changes in terms of proteins involved in
cell cycle control and also in apoptosis.
2
. Methods
2.5. Immunoblotting assays
2.1. Drugs
Samples of ascitic fluid (250
mL) were centrifuged (5.000 g for
5
min). The supernatants were discarded and the pellet washed in
Juglone 97% (Cat. H47003) and sodium ascorbate ꢀ98% (Cat.
phosphate buffered-saline (PBS) and centrifuged again. Whole cell
lysates were prepared in RIPA lysis buffer (50 mM Tris-Cl pH 7.4,
150 mM NaCl, 1% NP40, 0.25% Na-deoxycholate, 1 mM phenyl-
methylsulfonyl fluoride) supplemented with 1% protease inhibitor
and 3% phosphatase inhibitor cocktails. After further denaturation
A7631) were purchased from Sigma-Aldrich. Q7 was synthesized
by amination of 1,4 -naphthoquinone with 4-aminophenol, under
aerobic conditions using CeCl
3 2
$7H O as the Lewis acid catalyst, as
previously described by Benites et al. [10].
in Laemmli buffer (60 mM Tris-Cl pH 6.8, 2% SDS,10% glycerol, 5%
mercaptoethanol, 0.01% bromophenol blue), equal amounts of
protein (30 g) were subjected to SDS-PAGE, followed by electro-
b-
2.2. Animals
m
Male BALB/c inbred mice (20e22 g) were housed under
blotting to PVDF membranes. After blocking and washing, the
membranes were incubated overnight with the primary antibodies,
washed again and further incubated with the secondary antibodies
(1 h). Immunodetection was performed using the enhanced
chemiluminescence (ECL) detection kit (Millipore, USA) for HRP-
coupled secondary antibodies. Вeta-actin served as a loading con-
controlled conditions, receiving water and food ad libitum. This
research was conducted in accordance with internationally
accepted principles for laboratory animal use and care (NIH pub-
lication # 85e23, revised in 1985). This experimental protocol was
approved by the local ethics committee of Universidade Federal de
Santa Catarina, Brazil (CEUA-PP00784).
trol. The primary antibodies were: Rabbit polyclonal antibodies
®
from Santa Cruz raised against HIF1
a
(Cat. sc-10790), GLUT1 (Cat.
2
.3. Tumor induction and treatment
sc-7903), cyclin A (Cat. sc-596), PARP (Cat. sc-7150), p53 (Cat. sc-
243), actin (Cat. sc-7210). Mouse monoclonal antibodies for
6
6
On day zero, Ehrlich carcinoma cells (5 ꢁ 10 ) were inoculated
detection of protein Bax (Cat. sc-7480) and Bcl-XL (Cat. Sc-8392).
Rabbit polyclonal antibody for detection of p16 (Cat. 4824) from
into the abdomen of mice (n ¼ 12). Twenty-four hours later, the
treatments got started. The treatments were done via intraperito-
neal injections every 24 h for 9 days. The control group was treated
only with excipient (saline - DMSO 0.1%). Previous tests were
conducted to select the doses of juglone and Q7 plus ascorbate (1
®
Cell Signaling . The secondary antibodies: Polyclonal goat anti-
rabbit IgG antibody (Cat. AP132P) and polyclonal goat anti-mouse
IgG antibody (Cat. AP181P), both peroxidase conjugated from
Merck Millipore. Western blots were quantified by densitometry
Please cite this article in press as: F. Ourique, et al., In vivo inhibition of tumor progression by 5 hydroxy-1,4-naphthoquinone (juglone) and 2-(4-
hydroxyanilino)-1,4-naphthoquinone (Q7) in combination with ascorbate, Biochemical and Biophysical Research Communications (2016),
Ourique, Fabiana
