https://doi.org/10.52973/rcfcv-e34311
Received: 21/08/2023 Accepted: 27/12/2023 Published: 29/02/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34311
ABSTRACT
This study aimed to investigate the effect of propolis on pyruvate
kinase (PK) which is a key enzyme in glycolysis and superoxide
dismutase (SOD), an antioxidant enzyme on toxicity induced by DOX
in different tissues. Using molecular docking, It was looked into
how propolis affected the enzymes responsible for glycolysis and
the antioxidant system. There was no application in the rst group
(control). The second group received 100 mg·kg
-1
day of propolis by
gavage needle for 7 days, a single dose of 20 mg·kg
-1
intraperitoneal
DOX to the third group, and propolis+DOX to the fourth group. Two
days prior to DOX administration, propolis application began, and
it lasted for seven days. PK and SOD activities were determined in
liver, heart, kidney, and testis tissues, and molecular docking was
applied to ratify the activity of some propolis components (caffeic
acid phenethyl ester (CAPE) and Quercetin) on PK and SOD enzymes.
When the DOX group was compared with the control group, a decrease
in PK and SOD activities were found, and signicant difference was
found in PK and SOD activities. Administration of DOX decreased
PK and SOD activities of liver, heart, kidney, and testis tissues. In
conclusion, our study reveals that DOX disrupts glycolysis in rat
tissues. CAPE and Quercetin compounds were shown to interact
similarly with the cocrystal ligands of PK and SOD. In addition, when
the interaction types of these compounds especially on PK and the
docking scores obtained were examined, it can be said that they
show higher anity than DOX.
Key words: Doxorubicin; toxicity; pyruvate kinase; superoxide
dismutase; molecular docking
RESUMEN
El estudio tuvo como objetivo, evaluar el efecto del propóleo sobre
la piruvato quinasa (PK), que es una enzima clave en la glucólisis
y la superóxido dismutasa (SOD), una enzima antioxidante sobre
la toxicidad inducida por DOX en diferentes tejidos. Mediante el
acoplamiento molecular, analizamos cómo afectaba el propóleo a
las enzimas responsables de la glucólisis y el sistema antioxidante.
No hubo solicitud en el primer grupo (control). El segundo grupo
recibió 100 mg·kg
-1
día de propóleo por sonda gástrica durante 7
días, el tercer grupo recibió una dosis única de 20 mg·kg
-1
de DOX
intraperitoneal y el cuarto grupo propóleo+DOX. Dos días antes de la
administración de DOX, se inició la aplicación de propóleo, que duró
siete días. Se determinaron las actividades de PK y SOD en tejidos de
hígado, corazón, riñón y testículos, y se aplicó acoplamiento molecular
para raticar la actividad de algunos componentes del propóleo
(éster fenetílico del ácido cafeico (CAPE) y quercetina) sobre las
enzimas PK y SOD. Cuando se comparó el grupo DOX con el grupo de
control, se encontró una disminución en las actividades de PK y SOD,
y se encontró una diferencia signicativa en las actividades de PK
y SOD. La administración de DOX disminuyó las actividades de PK y
SOD de los tejidos del hígado, el corazón, los riñones y los testículos.
En conclusión, el presente estudio revela que DOX interrumpe la
glucólisis en tejidos de rata. Se demostró que los compuestos
CAPE y quercetina interactúan de manera similar con los ligandos
cocristalinos de PK y SOD. Además, cuando se examinaron los
tipos de interacción de estos compuestos, especialmente en PK,
y las puntuaciones de acoplamiento obtenidas, se puede decir que
muestran mayor anidad que DOX.
Palabras clave: Doxorrubicina; toxicidad; piruvato quinasa;
superóxido dismutasa; acoplamiento molecular
Effect of propolis on pyruvate kinase and superoxide dismutase activities
in doxorubicin–induced tissue damage: Molecular docking analysis
Efecto del propóleo sobre la actividad de la piruvato quinasa y la superóxido dismutasa
en el daño tisular inducido por doxorrubicina: análisis de acoplamiento molecular
Seval Yilmaz
1
* , Emre Kaya
1
, Harun Yonar
2
, Harun Uslu
3
1
Firat University, Faculty of Veterinary Medicine, Department of Biochemistry. Elazig, Türkiye.
2
Selcuk University, Faculty of Veterinary Medicine, Deparment of Biostatistics. Konya, Türkiye.
3
Firat University, Faculty of Pharmacy, Department of Pharmaceutical Professional Sciences, Department of Pharmaceutical Chemistry. Elazig, Türkiye.
*Corresponding author: sevalyilmaz@rat.edu.tr
Molecular docking analysis / Yilmaz et al. ___________________________________________________________________________________________
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INTRODUCTION
Doxorubicin (DOX), also known as Adriamycin®, is an active
chemotherapy medication used to treat a variety of cancers, including
uterine, ovarian, lung, and breast cancers. However, its clinical
ecacy is constrained by signicant toxicities, such as cardiac,
hepatic, renal, pulmonary, hematological, and testicular harm [1].
The rate–limiting enzyme of glycolysis known as pyruvate kinase
(PK), is essential for the metabolism of cancer cells [2]. Most
cancers utilize glucose substantially more than normal tissue does.
Aerobic glycolysis, also known as increased glucose consumption
and elevated lactate generation in the presence of oxygen, is more
prevalent in cancer cells (the Warburg effect)
[3, 4]
, of which PK is
considered a key regulator. An enzyme called PK catalyzes the last
step of glycolysis by phosphorylating adenosine diphosphate (ADP) to
adenosine triphosphate (ATP) and converting phosphoenolpyruvate
to pyruvate. Previous research has shown that PK is essential for
cell cycle progression, tumor growth, maintenance of the malignant
phenotype, and cell migration. The major multi–ability enzyme PK still
has unidentied activities in malignancies [2, 5].
Some of the postulated causes of DOX toxicities include oxidative
stress, inammation, endoplasmic reticulum–mediated apoptosis,
and DNA / RNA damage. Superoxide (O
2
·
–
) and hydroxyl radicals
(OH·) are produced as a result of the cytochrome P–450 enzyme's
metabolism of the DOX, which damages cellular membranes [6].
After passing through the cell membrane and being reduced by
cellular flavoenzymes, the drug DOX causes an increase in the
production of intracellular free radicals [7]. Reactive oxygen species
(ROS) are produced by disrupting complex I of the mitochondrial
electron transport chain (ETC), which is DOX–mediated redox cycling,
according to a number of methods. When reactive species are
present, macromolecules (such as lipids, proteins, DNA, etc.) undergo
oxidative changes that have harmful effects
[8, 9]. The increase in ROS
generation, particularly O
2
·
–
and OH
.
, leads to tissue damage from DOX
as well as certain non–radical substances including singlet oxygen
(
1
O
2
), hydrogen peroxide (H
2
O
2
), and others [10, 11]. The myocardium
typically causes DOX poisoning, which eventually destroys other
organs [12]. The heart's work consumes a lot of ATP, which is produced
by a variety of metabolic processes using glucose, free fatty acids,
pyruvate, and ketone bodies. The heart may also adjust to variations in
the fuel supply. DOX slows lipogenesis, which then prevents lipolysis
[12, 13]. Overall, it is clear that the ATP produced by the metabolism
of fatty acids may have been altered, forcing the cardiomyocytes to
move to alternative substrates to produce ATP, such as glycolysis [14].
Normally, intracellular enzymes including glutathione reductase,
superoxide dismutase (SOD), and catalase detoxify ROS [15]. SOD
facilitates the dismutation of O
2
·–
into either H
2
O
2
or regular molecular
oxygen (O
2
). As a result, SOD provides excellent antioxidant protection
in almost all live cells exposed to oxygen [16].
By incorporating a protective agent into DOX treatment methods,
numerous strategies have been developed to reduce these
adverse effects. Based on these beliefs, numerous antioxidants,
anti–inflammatory, and anti–apoptotic medications have been
recommended to combat produced damage sales [17, 18].
Honeybees (Apis mellifera L.) gather propolis, often known as bee
glue, from the leaf buds and bark ssures of many different types of
trees [19]. The composition and effectiveness of propolis varies widely
depending on its source, location, environment, and age. It contains
more than 150 polyphenols, including their esters, and avonoids like
phenolic acid. The avonoids in propolis are abundant and exhibit
potent free radical scavenging properties. Additionally, it includes
several vital minerals, including Ca, Zn, Mg, Cu, Mn, Fe, and Ni, as well
as vitamins E and C, vitamin B complexes, certain elements, and other
vitamins. As a result, it demonstrates a variety of biological functions,
including actions that are antibacterial, antioxidant, and capable of
scavenging free radicals [20, 21].
Propolis has recently been found
to exhibit a variety of biological activities, including those that are
antibacterial, antifungal, antiviral, immunoregulatory, antioxidative,
anticancer, hepatotropic, and antiinammatory, as well as potential use
for coronavirus 2019 (COVID–19) [22, 23]. The results indicate that CAPE–
like compounds could be employed as possible chemotherapeutic
agents against oral cancer. Additionally, oral submucosal broblast,
neck gingival carcinoma, and tongue squamous cell carcinoma cells
were revealed to be highly cytotoxic to CAPE [24]. The widely dispersed
avonoids are quercetin and rutin, which are signicant representatives
of the biologically active components in propolis and have strong
antioxidant and anticancer effects [25].
One can describe the behavior of ligands and target proteins at the
binding site and understand biochemical processes by using molecular
docking to mimic the interaction between a ligand and a protein [26].
For molecular docking investigations, CAPE and Quercetin compounds
were chosen because they have different chemical structures, have
a greater proportion of propolis components, and exhibit similar
activity to these molecules [27].
It was aimed to investigate the effect of propolis on pyruvate
kinase and superoxide dismutase activity in doxorubicin–induced
tissue damage. The molecular docking study was planned to analyze
to understand the interaction between DOX and enzymes. Due to
its broad range of biological activity, propolis has recently been
increasingly used in foods and beverages to promote health and
prevent diseases. This evaluation included the biochemical, molecular
docking chain that uses rats to investigate propolis mechanism in vivo
against tissue toxicity induced by DOX as an antioxidant, inammatory
and antiapoptotic agent.
MATERIALS AND METHODS
Experimental design
The local animal experiment ethics committee of Firat University
gave its consent to the conduct of this investigation (Protocol No:
2012/03–43). Forty–eight, three–month–old male Wistar–Albino rats
(Rattus norvegicus) weighing 250–300 g were utilized in the study;
they were procured from the Firat University Laboratory Animals
Breeding Unit. The rats were kept in air–conditioned rooms with a
xed temperature of 25 ± 2°C and 60–65% humidity, with a 12/12h light/
dark cycle, under standard conditions, and were fed on standard rat
food (pellet) and tap water ad libitum throughout the experimental
practices [28]. Experimental practices on rats were performed in
the Firat University Experimental Research Center.
In the study, rats were put into 4 groups, each including 7 rats: 1
st
group: control group, 2
nd
group: the group that received 100 mg·kg
-1
day (d) propolis by gavage for 7 d, 3
rd
group: the group that received
single dose 20 mg·kg
-1
body weight DOX (Fresenius Kabi Oncology Ltd.
19 Industrial Area, Baddi, Distt. Solan–India) intraperitoneally, and 4
th
group: the group that received propolis (100 mg·kg
-1
d by gavage, 7 d)
+ DOX (20 mg·kg
-1
body weight, intraperitoneal single dose). Propolis
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application was started 2 d before DOX administration and continued
for 7 d. The amount of DOX used in the study was determined based
on the previous studies [29, 30].
Rats were sacriced by decapitation
method 5 d after DOX administration in DOX treated group, rats in the
control and propolis groups, start of the experiment were sacriced
after 7 d, in the DOX+Propolis group, propolis pre–treatment was
performed for 2 d, then DOX administrated, were sacrificed by
decapitation method 5 d after DOX administration. The activities
of PK and SOD enzymes were determined spectrophotometrically
(Thermo Scientic, Genesys 10S UV–VIS Spectrophotometer, USA)
in the liver, heart, kidney and testis tissues.
Biochemical analysis
At the conclusion of the experiment, the rats were slaughtered, and
tissue samples of the liver, heart, kidney, and testis were collected. Until
biochemical analysis, tissue samples were kept at -80 °C in a freezer.
Physiological saline solution was used to wash the tissue samples, and
they were subsequently diluted with distilled water at a weight–to–
volume ratio of 1:10 and homogenized in a Potter–elvehjem homogenizer
(CAT R50D, Germany). Centrifugation (NUVE NF800R, Turkey) was done
on homogenates at +4 °C for 15 minutes at 3,500 rpm for SOD activity
analysis, and for 55 min at 13,500 rpm for PK activity analysis.
The PK activity were measured spectrophotometrically using the
method modied from Beutler et al.
[31], which is based on measuring
the decreasing absorbance rate of NADH at 340 nm. SOD activity
was determined according to the method modied by Sun et al.
[32]. SOD activity was measured using the method based on the
measurement of color development upon the reduction of nitroblue
tetrazolium by the O
2
·
–
produced by the xanthine–xanthine oxidase
system. The Lowryet al. [33] method was used to determine the
protein concentration in tissue homogenate.
Molecular docking
The chemical structures of the selected propolis components
were obtained from the internet in the form of SMILES (https://
pubchem.ncbi.nlm.nih.gov/). Energy minimization of ligands were
performed using ChemOce software. In silico study was performed
with Autodock4 program to validate the in vivo tests of propolis on
PK and SOD. Grid box points as different Å
3
size and 0.375 Å regular
spacing were made by centering separately for active sites of PK
and SOD enzymes previously determined or predicted. Pdb le of
enzymes were get (https://www.rcsb.org/) and were optimized using
the Maestro program (Maestro, Schrödinger, LLC, New York, NY,
2020). The 50–run Lamarckian Genetic Algorithm was used, while
standard settings were used for all ligands. The molecular docking tool
AutoDock 4.2 was used to calculate docking scores [34]. Cocrystal
was redocked on the SOD enzyme in order to validate the docking
algorithm, and the RMSD value was found to be 1.33.
Statistical analysis
All statistical analyses were conducted using IBM SPSS Statistics
(Version 22) and R 4.2.2 (R Core Team, 2021) (https://www.r-project.org/).
Numerical variables were reported as means, standard deviations,
medians, and interquartile ranges (IQR). Normality was assessed using
the Kolmogorov-Smirnov/Shapiro-Wilk tests. Differences in measured
parameters were analyzed using the Kruskal-Wallis test. Pairwise
differences were evaluated using the Mann-Whitney U test with
Bonferroni correction. The Spearman test was performed to identify
relationships between the measured parameters. A signicance level
of P<0.05 was used for all analyses.
RESULTS AND DISCUSSIONS
The TABLE I presents the PK and SOD activities in the liver, heart,
kidney and testis of the control and experimental groups. When the DOX
group was compared with the control group, a signicative decrease
in PK and SOD activities were found, and a statistically signicant
difference was found in PK and SOD activities. Compared to the DOX–
treated group, it was observed that there were signicant increases
in both PK activities and SOD activities in the DOX–administered
propolis group (P<0.05) and the values reached the control group
values (TABLE I).
Correlation between PK and SOD in the liver tissue
A positive correlation of nearly 100% was found between PK and
SOD activities in liver tissue (FIG. 1). As PK activity decreases, SOD
activity also decreases. A positive correlation of nearly 100% was
found between PK and SOD activities in the DOX+propolis group. As
PK activity increases, SOD activity also increases. In terms of SOD
activity, an inverse relationship of 97% was found between the DOX
group and the DOX+Propolis group (FIG. 2).
TABLE I
Eects of propolis on the PK and SOD activities in liver, heart, kidney and testis tissues of DOX treated rats
Activities
Control Propolis Doxorubicin Doxo.+Prop.
P
Mean ± SD Med(IQR) Mean ± SD Med(IQR) Mean ± SD Med(IQR) Mean ± SD Med(IQR)
PK
Liver (U·mg
-1
Protein) 10.80 ± 2.95 9.49 (5.03)
a
11.31 ± 2.09 11.63 (4.80)
a
6.80 ± 1.21 7.31 (2.24)
b
9.39 ± 1.47 8.98 (2.06)
a
<0.001
Kidney (U·mg
-1
Protein) 2.37 ± 0.15 2.34 (0.09)
a
2.58 ± 0.55 2.83 (1.03)
a
1.91 ± 0.20 2.04 (0.30)
b
2.22 ± 0.43 2.33 (0.87)
ab
<0.001
Heart (U·mg
-1
Protein) 5.03 ± 1.67 5.54 (3.32)
ab
5.79 ± 1.04 5.86 (2.08)
a
3.81 ± 0.86 3.78 (1.36)
b
5.05 ± 1.18 4.70 (1.47)
ab
<0.001
Testis (U·mg
-1
Protein) 6.39 ± 0.73 6.54 (1.62)
a
6.45 ± 0.22 6.43 (0.42)
a
4.13 ± 2.27 5.03 (4.61)
b
6.24 ± 0.90 6.48 (1.24)
a
<0.001
SOD
Liver (U·mg
-1
Protein) 0.19 ± 0.03 0.18 (0.06)
ac
0.204 ± 0.02 0.20 (0.02)
a
0.14 ± 0.04 0.16 (0.07)
bc
0.17 ± 0.02 0.17 (0.04)
c
<0.001
Kidney (U·mg
-1
Protein) 0.22 ± 0.04 0.22 (0.09)
a
0.216 ± 0.02 0.21 (0.03)
a
0.19 ± 0.01 0.18 (0.03)
b
0.22 ± 0.02 0.22 (0.03)
a
<0.001
Heart (U·mg
-1
Protein) 2.52 ± 0.24 2.47 (0.50)
a
2.62 ± 0.27 2.76 (0.32)
a
2.06 ± 0.48 2.28 (0.97)
b
2.52 ± 0.13 2.53 (0.25)
a
<0.001
Testis (U·mg
-1
Protein) 0.14 ± 0.06 0.13 (0.14)
a
0.13 ± 0.05 0.15 (0.11)
ab
0.09 ± 0.04 0.08 (0.06)
b
0.13 ± 0.05 0.13 (0.11)
a
<0.001
The dierent letters in the rows indicate a statistically signicant dierence between the groups and
P values are for Kruskall–Wallis test. Signicance values of
multiple comparisons have been adjusted by the Bonferroni correction
FIGURE 1. Bar graphs of PK and SOD values of each tissue in terms of groups of interest
Molecular docking analysis / Yilmaz et al. ___________________________________________________________________________________________
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Correlation between PK and SOD in the heart tissue
A statistically insignicant positive correlation was found between
PK and SOD activities in heart tissue. A very low correlation was found
between PK and SOD activities in the DOX+Propolis group (FIGS. 1, 2).
Correlation between PK and SOD in the kidney tissue
An inverse correlation of 0.50% was found between PK and SOD
activities in kidney tissue. While PK activity increased, SOD activity
decreased. The relationship between PK and SOD activities was not
signicant in the DOX+Propolis group (FIGS. 1, 2).
Correlation between PK and SOD in the testis tissue
A positive correlation of 48% was found between PK and SOD
activities in the DOX group in testicular tissue. As PK activity
decreases, SOD activity also decreases. There was a 68% negative
correlation between PK activity in the DOX group and SOD activity in the
DOX+propolis group. The relationship between PK and SOD activities
in the DOX+propolis group was not statistically signicant (FIGS. 1, 2).
The liver is the tissue most affected by DOX application in terms
of PK and SOD activities, followed by testis (FIGS. 1, 2).
Molecular docking analyses were carried out to ascertain the modes
of interaction at the active sites of these enzymes for some propolis
components and docking scores were obtained (TABLE II). It showed
higher anity for PK enzyme than DOX, with higher binding scores and
similar interaction modes of CAPE and quercetin. In another enzyme,
SOD, DOX was found to have a higher binding score than CAPE and
quercetin. The related amino acids and binding types were shown in
detail in 2D and 3D gures by the Maestro program. The interactions
of PK and SOD enzymes, whose active site was previously dened
or predicted, with CAPE, Quercetin and DOX are presented in detail
(FIGS. 3, 4, 5, 6, 7, 8, 9).
The structures of PK as determined by X–ray crystallography and
SOD main binding sites have been determined (https://www.rcsb.org/)
[35]. It has been previously represented that 2–phosphoglycolic
acid (PDB ID: PGA), which is a cocrystal in PK (PDB ID: 1A3X), bonds
hydrogen ARG49, GLY265 and THR298. It has been previously
represented covalent metal complex interaction with MN1001 for
PGA on PK (https:/www.ebi.ac.uk/pdbe/). Quercetin has been found to
form H–bonds with ARG49 and metal coordination with MN1001 in this
instance, similar to PGA. In addition, Quercetin has been detected to
make Pi cation interaction with K1002. In order to determine the likely
binding model of propolis components (CAPE, Quercetin) and DOX
into the active site of SOD, molecular docking studies were carried
out. It has been previously represented that SOD (PDB ID: 3LSU)
binding site includes HIS172, GLN179, MLY181 and ASP184 (https://goo.
su/7qZOt1). CAPE interacted with GLN179, MLY181 and ASP184 and,
Quercetin interacted with HIS172 and MLY181, these results showed
that they were compatible with the previously shared binding site
and that they interacted similarly (FIG: 3, 4, 5, 6, 7, 8 and 9) (TABLE II).
It is evident from our molecular docking observations that DOX has
a detrimental effect on PK catalytic activity. Given the decreased ATP
and accumulation of glycolytic intermediates, it is logical to hypothesize
that glycolysis may be imperfect and cause a metabolic meltdown [16].
It's feasible that a deciency in the availability of pyruvate caused by
a dysfunctional PK could prevent acetyl CoA from being available as a
substrate for the TCA cycle or the lipogenic pathway [13, 36].
The administration of propolis reduced liver, heart, kidney, and
testis damage in a manner similar to the biochemical results,
according to the results of molecular docking. Propolis pre–treatment
FIGURE 2. Spearman correlation values and scatter plots of PK and SOD enzyme activities results for the groups
of interest of each tissue
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TABLE II
Molecular docking binding scores of DOX, CAPE and Quercetin with PK and SOD enzymes binding residues
Enzymes Ligands
Visualization Results of Docking Autodock Results
H-Bond Metal Coord. Pi Cation Pi Stacking
Estimated Inhibition
Constant, Ki
Best Docking Score
PK
(1A3X)
DOX
– MN1001 – – 277.60 µM -4.85
CAPE
ASP147
MN1001 – – 194.32 µM -5.06
ASP266
Quercetin
ARG49
MN1001
K1002
– 75.85 µM -5.62
HIS5 LYS240
SOD
(3LSU)
DOX
D:ASP6
– – – 10.60 µM -6.79
D:LEU7
D:PHE11
CAPE C:GLU171 – – D:HIS30 37.46 µM -6.04
Quercetin
C:GLU171
– – D:HIS30 89.70 µM -5.52
D:MLY29*
µM: micromolar, Docking Score: Estimated Free Energy of Binding (kcal·mol
-1
), MLY: Modied residue of LYS, MN: Mn
2+
, K: K
+
for two days before to DOX results in substantial cause decorating
in all prior markers DOX group, suggesting that propolis assisted in
maintaining membrane integrity and prevented enzyme leakage.
Additionally, propolis antioxidants contain polyphenols that may
guard against oxidative cardiac, hepatic, and renal harm.
The negative effects of DOX on several organs were examined as
biochemical and molecular dockening, and it was aimed to evaluate
the possible effects of propolis, which has the potential for multiple
life when faced with DOX toxic damage. Due to its numerous stated
advantages and affordability, propolis has gained a lot of favor as a
medicinal and possible protective agent in recent years.
FIGURE 3. DOX is presented in the PK binding site with 2D FIGURE 6. Quercetin is presented in the PK binding site with 2D
FIGURE 7. CAPE is presented in the SOD binding site with 2D
FIGURE 8. Quercetin is presented in the SOD binding site with 2D
FIGURE 4. DOX is presented in the SOD binding site with 2D
FIGURE 5. CAPE is presented in the PK binding site with 2D
Molecular docking analysis / Yilmaz et al. ___________________________________________________________________________________________
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FIGURE 9. PGA (yellow), CAPE (cyan), Quercetin (purple), and DOX (black) are
presented in the PK (PGA, Mn
2+
and K
+
complex binding site) with 3D
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Since 1969, DOX, also known as Adriamycin® or Doxilthe®, has been
the most widely used and successful chemotherapeutic medication
for the treatment of a variety of malignancies, including solid and
hematogenous cancers [37].
Despite being a powerful active
anticancer medication, DOX's therapeutic use is constrained by its
side effects [38].
Glycolytic enzymes enable the switch from oxidative
phosphorylation to aerobic glycolysis [39]. Particularly PK, glycolytic
enzymes have crucial roles in the proliferation of cancer cells.
Phosphoenolpyruvate is converted into cytosolic pyruvate by PK,
which also simultaneously produces one ATP molecule [40]. Pyruvates
generated in the cytoplasm are headed for oxidative decarboxylation
to create acetyl CoA, which is then either used to power the citric acid
cycle or another metabolic route. Previous studies showed that DOX
inhibits adipogenic and lipogenic pathways [12, 13]. It was therefore
tried to nd out how DOX affected the glycolytic process. According to
gene expression studies, PK is downregulated by DOX, which results
in dysregulation of glycolysis. Glycolysis disruption may result in a
reduction in ATP synthesis and energy deprivation in cells [41].
As a model organism, Mohan et al. [16] treated Saccharomyces
cerevisiae with PK at varying concentrations (5–50 µM) to examine the
impact of DOX on the glycolytic pathway and apoptosis. As medication
concentration rose, a decline in growth rate was seen. The biphasic
character of DOX was revealed by an increase in cell density at the
highest dose (50 µM). Mohan et al. [16] reveal that Adriamycin disrupts
glycolysis in yeast cells, causing the cell to undergo apoptosis due to
oxidative stress. In the current study, the change in PK activities, an
important enzyme in the glycolytic pathway, after DOX (Adriamycin)
application between groups supports this. The maximal growth rate
was observed to be decreased by the PK concentration from 0.3 to
0.1 OD h
–1
at 20 µM. At a 50 µM concentration, it was found that more
than 80% of yeast cells were still alive [42].
After treatment, acquired drug resistance poses a signicant issue
for DOX and other chemotherapeutic drugs. miR–122 expression levels
were found to be reduced in DOX–resistant Huh7/R cells compared to
wild–type cells by Pan et al. [43], proving that miR–122 is connected to
DOX chemoresistance. High levels of miR–122 expression in Huh7/R
cells have been demonstrated to reverse DOX resistance by inhibiting
PK, causing DOX–resistant cancer cells to undergo apoptosis. As
a result, it has been shown that upregulating glucose metabolism
makes people more resistant to DOX; as a result, miR–122's restriction
of glycolysis may be a useful therapeutic approach to combat DOX
resistance in liver cancer. Pan et al. [43] revealed that dysregulated
glucose metabolism contributes to DOX resistance and inhibition of
miR–122–induced glycolysis may be a promising therapeutic strategy
to overcome doxorubicin resistance in hepatocellular carcinoma. The
decrease in PK activity observed in all tissues with DOX application
in the current study is related to this.
Cardiovascular damage attributable to dosage is one DOX drawback.
80% of the heart energy comes from lipids, with the remaining 20%
coming from other sources like glucose. Previous studies have
demonstrated that DOX inhibits cardiomyocyte fatty acid oxidation,
and that as a result, only a change in the substrate occurs [44]. Since
the medication prevents –oxidation of acids, glucose is employed
in this situation rather than fatty acids as the substrate [14]. The
amount of mitochondria in cardiomyocytes is typically 35–40% more
than that in other organs. Consequently, altering the ultrastructure
of the mitochondria can impair ATP synthesis [45].
Our study showed that propolis leads to increase activities
of PK. This shows that propolis has the ability to control glucose
metabolism, increasing PK activity. According to our findings,
administering propolis along with DOX treatment improves glucose
metabolism [46, 47].
Because the aerobic organism uses oxygen to produce energy, it
produces a variety of free radicals and other ROS. As a response, cells
have evolved an antioxidant system that can stop and reverse ROS–
mediated damage. SOD is a highly conserved enzyme that is found
in all living things. Its primary job is to turn the O
2
·–
produced during
respiration into O
2
and H
2
O
2
, which is crucial for detoxication [48].
Several avoenzymes, such as NADH dehydrogenase and NADPH
cytochrome P–450 reductase, can enzymatically convert DOX to
its semiquinone radicals [49]. Superoxide anion radical is created
when DOX radical gives up its electron to O
2
in an aerodynamic
environment [50]. O
2
·
–
then can dismutate to H
2
O
2
and/or participate
in the formation of highly toxic OH via the Haber–Weiss reaction cycle.
Specically, exposure to DOX has been shown to increase intracellular
H
2
O
2
levels. These causes this stress by receiving electrons from the
lipids in cell membranes, leading to lipid pleading toon, and oxidant–
induced cell injuries [17].
In the current study, as described by Ciaccio et al. [17], changes
in the activity of SOD, an antioxidant enzyme, after DOX application
prove that the SOD enzyme catalyzes the reaction of oxidizing
the superoxide radical to molecular oxygen and reducing another
superoxide radical to H
2
O
2
.
The availability of O
2
and NADPH, as well
as the activity of many intracellular enzymes like SOD, glutathione
peroxidase, NADPH oxidases, and thioredoxin, are all necessary for
DOX to conduct the reductive conversion. The OH
.
that is formed
secondarily has the potential to damage proteins and DNA as well as
start the process of lipid peroxidation (LPO), which has harmful effects
on tissues and cells. The cytotoxicity of DOX on malignant cells and its
detrimental effects on various organs are also a result of nucleotide
base intercalation and cell membrane lipid binding activities.
Sarvazyan et al. [7], in their study examining the effects of
doxorubicin on cardiomyocytes with reduced SOD levels, obtained
Molecular docking analysis / Yilmaz et al. ___________________________________________________________________________________________
8 of 11
results that coincide with the results in the current study and
concluded that, as in the current study, a signicant part of the
cytotoxicity of DOX can be explained by the formation of superoxide
anion and that the level of intracellular SOD activity is important for
cell protection. Researchers concluded that it should be taken into
consideration as a factor. Additionally, the enzyme topoisomerase
II, which is essential for DNA replication, is inhibited by the DOX,
which also intercalates into the cellular DNA [51]. Damage to cells is
caused by DOX because it interacts strongly with cellular nuclei and
intercalates with DNA bases to form DOX–DNA complexes [52]. By
causing ROS like H
2
O
2
and O
2
·
-
to develop, DOX destroys malignant
cells. It has been suggested that the reductive reduction of DOX is the
primary determinant of DOX cytotoxicity and the underlying process
determining drug resistance in cancer cells [53]. Although biomarkers
of DNA damage were not determined in the current study, changes in
PK and SOD activity after DOX administration suggest that there are
changes in both the glycolytic pathway and the oxidative mechanism.
In the present work, we determined changes in PK and SOD enzyme
activities after DOX application. In their study, Swamyet al. obtained
results similar to our study after DOX application. They suggested
that DOX causes this by causing oxidation of fatty acids, disturbance
in myocardial adrenergic signaling/regulation, and cellular toxicity,
which leads to depression of energy metabolism in cardiac tissue [54].
Numerous research conducted over the past few decades
have exhibit the propolis and its compounds extensive medicinal
potential. Propolis and its phytochemicals have been shown to have
antioxidant, antibacterial, antiviral, antifungal, anti–inammatory,
antiproliferative, and anticancer properties, and the list is expanding
[57, 58, 59, 60, 61].
But nothing is understood about how propolis
might impact tumor cells' glycolysis
In the present work, tissues exposed to DOX exhibit decreased
SOD enzyme activity. The obtained results recommend the function
of SOD in reducing intracellular oxidative stress and protecting rat
tissues. Some researchers have demonstrated in experimental
models that such tissue damage may be at least somewhat related
to increased oxidative stress, which is characterized by increased
free radical production or decreased endogenous antioxidant activity
[62].
Xanthine oxidase/xanthine systems, H
2
O
2
, glucose oxidase/
glucose, and isolated adult rat cardiomyocytes with normal and
reduced Cu/Zn SOD activity are similarly sensitive to extracellularly
produced oxidants, according to research by Sarvazyan et al. [7]
DOX
was administered to myocytes that had decreased SOD activity. The
formation of an O
2
·
–
inside the cell is thought to be the cause of DOX's
cardiotoxicity. It is still unclear why DOX is toxic to the kidneys, but
possible causes include an unbalanced oxidant–antioxidant system,
the production of free radicals, oxidative damage to biological
macromolecules, membrane LPO, and protein oxidation [63].
In the current study, decreased SOD activity in the hepatic, cardiac,
renal, and testis tissues of the DOX group suggested elevated
oxidative stress. Prior to DOX therapy, the liver, heart, kidneys, and
testis of animals receiving propolis had higher SOD activity. Propolis
treatment decreased the production of free radicals, which would
have been the cause of this. Due to an increase in DOX content in the
liver throughout the detoxication process, the effects of DOX toxicity
were more severe in life compared to the heart. While in the heart, the
same cause propolis therapy failed to lessen this concentrated liver.
In line with the obtained ndings, recent research on rats exposed
to DOX found that their liver, heart, kidney, and testis tissues had
dramatically reduced SOD activity [62, 63, 64, 65]. In addition, DOX–
induced hepatotoxicity in rats was reduced by hepatic SOD, as has
already been noted in a number of investigations [66, 67]. In other
respects, the PK and SOD activities in the group 4, were all discovered
to be very similar to those seen in the control group. This can be
explained by the ability of propolis to reduce free radical activity
and strengthen the antioxidant defense system. Propolis may have
a protective effect because its phenolic components maintain the
structural and functional integrity of the tissue, reducing oxidative
tissue damage. Propolis's antioxidant of these polyphenols, which
when combined with DOX, would increase the antitumor and prevent
a number of damages brought on by DOX, may be responsible for the
protective actions of propolis.
Rizk et al. [46] shown that pretreatment with propolis could
successfully reduce the toxic effects of DOX on the testes without
compromising the drug's anticancer effectiveness. These ndings
prompted speculation that propolis might function as an adjuvant
therapy, possibly shielding the testes from the oxidative and apoptotic
effects of DOX and ultimately aiding in the prevention of this severe
detrimental effect of DOX in clinical practice.
Fuliang et al. [68] demonstrated how propolis extracts boosted
SOD activity. This implies that propolis may alter the metabolism
of blood lipids, resulting in a reduction in LPO outputs and the
scavenging of free radicals. This agrees with our results, as DOX
administration reduces PK and SOD, while propolis administration
prior to DOX administration reduces oxidative stress and increases
glucose metabolism. Propolis may have a protective effect because
its phenolic components shield tissue's structural and functional
integrity and stop oxidative tissue damage.
In a study by Köse et al. [56], renal oxidative stress and excessive
free radical emission were demonstrated to cause nephrotoxicity,
which is consistent with the ndings of our investigation. Propolis'
nephroprotective activity is evaluated by Baykara et al. [66] by lowering
serum urea and enhancing kidney oxidation. According to a study
by Promsan et al. [70], pretreatment with the primary avonoid of
propolis, pinocembrin (5,7–dihydroxyavone), improved kidney function
and decreased oxidative stress and apoptotic conditions. According
to these results, pinocembrin protects against nephrotoxicity, which
may be at least in part because of its antioxidant and antiapoptotic
properties. By reducing the rise in oxidative stress and controlling
antioxidant enzymes through the Nrf2/HO–1 and NQO1 pathways, it
improves cellular function by lowering protein–related apoptosis.
CONCLUSIONS
In conclusion, propolis treatment substantially increase glucose
consumption by enhancing the activities of PK and SOD. These results
enhanced the likelihood that propolis might be used as an adjuvant
therapy to shield organs from the oxidative stress caused by DOX.
Molecular docking studies reported the interaction mode of some
propolis components (CAPE and Quercetin) and DOX with PK and
SOD including some H–bonds, metal coordination and close contact
interactions, similarly to current cocrystals, in previously represented
binding sites. Especially when the interaction types and docking
scores obtained on PK are examined, it can be said that CAPE and
Quercetin show higher anity than DOX. Studies have revealed that
CAPE and Quercein will inhibit both PK and SOD at an estimated
micromolar (µM) level. To conrm the signicance of propolis in
the curative management of DOX–induced multiple toxicities or to
develop new drug delivery techniques, more research is required.
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34311
9 of 11
Conict of interest
There are no conflicts of interest, according to the authors,
regarding this article.
Credit author statement
Seval Yilmaz: This author was involved in the design of the study,
animal applications, laboratory experiments, and evaluation of results.
Emre Kaya: This author was involved in the animal applications,
laboratory experiments, and evaluation of results.
Harun Yonar: This author supported the statistical evaluation of
the results of the study.
Harun Uslu: This author took part in the molecular docking analyzes
in the study.
Ethics approval and consent to participate
The Firat University Animal Studies Local Ethics Committee
accepted the experiments (Protocol No: 2012/03–43), which were
carried out strictly in compliance with the Experimental Animal Ethics
Committee's Guiding Principles.
Disclosure statement
The authors state that there are no interests at odds with
one another.
BIBLIOGRAPHIC REFERENCES
[1] Eleiwa NZ, Galal AA, El–Aziz A, Reda M, Hussin EM. Antioxidant
activity of Spirulina platensis alleviates doxorubicin–induced
oxidative stress and reprotoxicity in male rats. Oriental Pharm.
Exp. Med. [Internet]. 2018; 18(2):87–95. doi: https://doi.org/mjdx
[2] Yang W, Lu Z. Pyruvate kinase M2 at a glance. J. Cell Sci.
[Internet]. 2015; 128(9):1655–1660. doi: https://doi.org/f7ck3d
[3] Warburg O. On the origin of cancer cells. Sci. [Internet]. 1956;
123(3191):309–314. doi: https://doi.org/fj737q
[4] Wu SF, Le HY. Dual roles of PKM2 in cancer metabolism. Acta
Biochim. Biophys. Sin. [Internet]. 2013; 45(1):27–35. doi: https://
doi.org/f4kndk
[5] Mazurek S, Boschek CB, Hugo F, Eigenbrodt E. Pyruvate kinase
type M2 and its role in tumor growth and spreading. Semin. Cancer
Biol. [Internet]. 2005; 15(4):300–308. doi: https://doi.org/bpg6sb
[6] Abdel–Daim MM, Kilany O, Khalifa HA, Ahmed AA. Allicin ameliorates
doxorubicin–induced cardiotoxicity in rats via suppression of oxidative
stress, inflammation and apoptosis. Cancer chem. Pharmacol.
[Internet]. 2017; 80(4):745–753. doi: https://doi.org/gbzxnw
[7] Sarvazyan NA, Askari A, Huang WH. Effects of doxorubicin on
cardiomyocytes with reduced level of superoxide dismutase. Life
Sci. [Internet]. 1995; 57(10):1003–1010. doi: https://doi.org/bhgvdr
[8] Cooke MS, Evans MD, Dizdaroglu M, Lunec J. Oxidative DNA
damage: mechanisms, mutation, and disease. FASEB J.
[Internet]. 2003; 17(10):1195–1214. doi: https://doi.org/brndsj
[9] Fialkow L, Wang Y, Downey GP. Reactive oxygen and nitrogen species
as signaling molecules regulating neutrophil function. Free Rad. Biol.
Med. [Internet]. 2007; 42(2):153–164. doi: https://doi.org/fnx4d8
[10] Raj S, Franco VI, Lipshultz SE. Anthracycline–induced
cardiotoxicity: a review of pathophysiology, diagnosis, and
treatment. Curr. Treat. Opt. Cardio. Med. [Internet]. 2014;
16(6):1–14. doi: https://doi.org/mjd6
[11] Octavia Y, Tocchetti CG, Gabrielson KL, Janssens S, Crns HJ,
Moens AL. Doxorubicin–induced cardiomyopathy: from molecular
mechanisms to therapeutic strategies. J. Mol. Cell. Cardiol.
[Internet]. 2012; 52(6):1213–1225. doi: https://doi.org/f3xjqt
[12] Renu K, Sruthy KB, Parthiban S, Sugunapriyadharshini S, George
A, Tirupathi–Pichiah TB, Suman S, Abilash V G, Arunachalam S.
Elevated lipolysis in adipose tissue by doxorubicin via PPARα
activation associated with hepatic steatosis and insulin
resistance. Eur. J. Pharmacol. [Internet]. 2019; 843:162–176.
doi: https://doi.org/mjd7
[13] Arunachalam S, Tirupathi–Pichiah PB, Achiraman S. Doxorubicin
treatment inhibits PPARγ and may induce lipotoxicity by mimicking
a type 2 diabetes–like condition in rodent models. FEBS Lett.
[Internet]. 2013; 587(2):105–110. doi: https://doi.org/f4h3vh
[14] Doenst T, Nguyen TD, Abel ED. Cardiac metabolism in heart
failure: implications beyond ATP production. Circ. Res.
[Internet]. 2013; 113(6):709–724. doi: https://doi.org/f5nxvg
[15] Van Raamsdonk JM, Hekimi S. Reactive oxygen species and
aging in Caenorhabditis elegans: causal or casual relationship?
Antioxid. Redox Sig. [Internet]. 2010; 13(12):1911–1953. doi:
https://doi.org/cwg6wb
[16] Mohan UP, Kunjiappan S, Tirupathi–Pichiah PB, Arunachalam
S. Adriamycin inhibits glycolysis through downregulation of
key enzymes in Saccharomyces cerevisiae. Biotech. [Internet].
2021; 11(1):1–13. doi: https://doi.org/mjd8
[17] Ciaccio M, Valenza M, Tesoriere L, Bongiorno A, Albiero R,
Livrea MA. Vitamin A inhibits doxorubicin–induced membrane
lipid peroxidation in rat tissues in vivo. Arc. Biochem. Biophy.
[Internet]. 1993; 302(1):103–108. doi: https://doi.org/cprnh6
[18] Mohamed EK, Osman AA, Moghazy AM, Rahman A, As A. Propolis
protective effects against doxorubicin–ınduced multi–organ toxicity
via suppression of oxidative stress, inammation, apoptosis, and
histopathological alterations in female albino rats. Biointerface Res.
Appl. Chem. [Internet]. 2021; 12:1762–1777. doi: https://doi.org/mjd9
[19] Sameni HR, Ramhormozi P, Bandegi AR, Taherian AA,
Mirmohammadkhani M, Safari M. Effects of ethanol extract of
propolis on histopathological changes and anti‐oxidant defense
of kidney in a rat model for type 1 diabetes mellitus. J. Diabetes
Invest. [Internet]. 2016; 7(4):506–513. doi: https://doi.org/f9hhpz
[20] Kaya E, Yılmaz S, Ceribasi S. Protective role of propolis on low and
high dose furan–induced hepatotoxicity and oxidative stress in rats.
J. Vet. Res. [Internet]. 2019; 63(3):423–431. doi https://doi.org/mhzs
[21] Rivero–Cruz JF, Granados–Pineda J, Pedraza–Chaverri J, Pérez–Rojas
JM, Kumar–Passari A, Diaz–Ruiz G, Rivero–Cruz BE. Phytochemical
constituents, antioxidant, cytotoxic, and antimicrobial activities of
the ethanolic extract of Mexican brown propolis. Antioxid. [Internet].
2020; 9(1):70. doi: https://doi.org/gp63pm
Molecular docking analysis / Yilmaz et al. ___________________________________________________________________________________________
10 of 11
[22] Ripari N, Sartori AA, da Silva–Honorio M, Conte FL, Tasca KI, Santiago
KB, Sforcin JM. Propolis antiviral and immunomodulatory activity:
a review and perspectives for COVID–19 treatment. J. Pharm.
Pharmacol. [Internet]. 2021; 73(3):281–299. doi: https://doi.org/mjfd
[23] Yilmaz S, Kandemir FM, Kaya E, Ozkaraca M. Chemoprotective
effects of propolis on aatoxin b1–induced hepatotoxicity in
rats: Oxidative damage and hepatotoxicity by modulating TP53,
oxidative stress. Curr. Prot. [Internet]. 2020; 17(3):191–199. doi:
https://doi.org/mh2j
[24] Lee YJ, Liao PH, Chen WK, Yang CC. Preferential cytotoxicity of
caffeic acid phenethyl ester analogues on oral cancer cells. Cancer
Lett. [Internet]. 2000; 153(1–2):51–56. doi: https://doi.org/cgsk64
[25] Erlund I. Review of the avonoids quercetin, hesperetin, and
naringenin. Dietary sources, bioactivities, bioavailability, and
epidemiology. Nut. Res. [Internet]. 2004; 24(10):851–874. doi:
https://doi.org/b5twkq
[26] McConkey BJ, Sobolev V, Edelman M. The performance of current
methods in ligand–protein docking. Curr. Sci. [Internet]. 2002 [cited
20 July 2023]; 83(7):845–856. Available in: https://goo.su/MnXD
[27] Ristivojević P, Dimkić I, Guzelmeric E, Trifković J, Knežević M, Berić
T, Yesilada E, Milojković–Opsenica D, Stanković S. Proling of Turkish
propolis subtypes: Comparative evaluation of their phytochemical
compositions, antioxidant and antimicrobial activities. LWT.
[Internet]. 2018; 95:367–379. doi: https://doi.org/mjfj
[28] Yamauchi C, Fujita S, Obara T, Ueda T. Effects of room
temperature on reproduction, body and organ weights, food and
water intake, and hematology in rats. Lab. Anim. Sci. [Internet].
1981; 31(3):251–258. doi: https://doi.org/mjfk
[29] Iqbal M, Dubey K, Anwer T, Ashish A, Pillai KK. Protective effects
of telmisartan against acute doxorubicin–induced cardiotoxicity
in rats. Pharmacol. Rep. [Internet]. 2008 [cited 20 July 2023];
60(3):382–390. Available in: https://goo.su/KkQ6u. Cited in:
PubMed; PMID 18622063.
[30] Kaya E, Yılmaz S. Determination of effects of Nigella sativa oil
on doxorubicin–ınduced cardiotoxicity in rats. Firat Uni. Vet. J.
Health Sci. [Internet]. 2019 [cited 15 July; 2023]; 33(1):31–36.
Available in: https://goo.su/l5vOqN. Turkish
[31] Beutler E, Blume KG, Kaplan JC, Löhr GW, Ramot B, Valentine WN.
International Committee for Standardization in Haematology:
Recommended methods for red‐cell enzyme analysis. British J.
Haematol. [Internet]. 1977; 35(2):331–340. doi: https://doi.org/csvd2s
[32] Sun YI, Oberley LW, Li Y. A simple method for clinical assay of
superoxide dismutase. Clin. Chem. [Internet]. 1988; 34(3):497–
500. doi: https://doi.org/74fn
[33] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein
measurement with the Folin phenol reagent. J. Biol. Chem.
[Internet]. 1951; 193(1):265–275. doi: https://doi.org/ghv6nr
[34] Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell
DS, Olson AJ. AutoDock4 and AutoDockTools4: Automated
docking with selective receptor exibility. J. Comp. Chem.
[Internet]. 2009; 30(16):2785–2791. doi: https://doi.org/dvtqjs
[35] Jurica MS, Mesecar A, Heath PJ, Shi W, Nowak T, Stoddard
BL. The allosteric regulation of pyruvate kinase by fructose–1,
6–bisphosphate. Structure [Internet]. 1998; 6(2):195–210. doi:
https://doi.org/dwzzwc
[36] Renu K, Abilash VG, PB TP, Arunachalam S. Molecular mechanism of
doxorubicin–induced cardiomyopathy–An update. Eur. J. Pharmacol.
[Internet]. 2018; 818:241–253. doi: https://doi.org/gcrvs2
[37] Damiani RM, Moura DJ, Viau CM, Caceres RA, Henriques JAP,
Saffi J. Pathways of cardiac toxicity: comparison between
chemotherapeutic drugs doxorubicin and mitoxantrone. Arch.
Toxicol. [Internet]. 2016; 90(9):2063–2076. doi: https://doi.org/grcdr2
[38] Lommi J, Koskinen P, Näveri H, Härkönen M, Kupari M. Heart
failure ketosis. J. Int. Med. [Internet]. 1997; 242(3):231–238. doi:
https://doi.org/c86nj7
[39] Wilson RB, Solass W, Archid R, Weinreich FJ, Königsrainer A,
Reymond MA. Resistance to anoikis in transcoelomic shedding:
The role of glycolytic enzymes. Pleura Perit. [Internet]. 2019;
4(1): 20190003. doi: https://doi.org/ghfxw6
[40] Israelsen WJ, Vander–Heiden MG. Pyruvate kinase: Function,
regulation and role in cancer. Semin. Cell Dev. Biol. [Internet].
2015; 43:43–51. doi: https://doi.org/f7zc9s
[41] Van den Brink J, Canelas AB, Van Gulik WM, Pronk JT, Henen
JJ, De Winde JH, Daran–Lapujade P. Dynamics of glycolytic
regulation during adaptation of Saccharomyces cerevisiae to
fermentative metabolism. App. Env. Microbiol. [Internet]. 2008;
74(18):5710–5723. doi: https://doi.org/brgs4b
[42] Taymaz–Nikerel H, Karabekmez ME, Eraslan S, Kırdar B.
Doxorubicin induces an extensive transcriptional and metabolic
rewiring in yeast cells. Sci. Rep. [Internet]. 2018; 8(1):1–14. doi:
https://doi.org/gd58qk
[43] Pan C, Wang X, Shi K, Zheng Y, Li J, Chen Y, Jin L, Pan Z. MiR–122
reverses the doxorubicin–resistance in hepatocellular carcinoma
cells through regulating the tumor metabolism. PloS One
[Internet]. 2016; 11(5):e0152090. doi: https://doi.org/f9v9mn
[44] Abdel–aleem S, El–Merzabani MM, Sayed–Ahmed M, Taylor DA,
Lowe JE. Acute and chronic effects of adriamycin on fatty acid
oxidation in isolated cardiac myocytes. J. Mol. Cell. Cardiol.
[Internet]. 1997; 29(2):789–797. doi: https://doi.org/bddkkd
[45] Goffart S, von Kleist–Retzow JC, Wiesner RJ. Regulation of
mitochondrial proliferation in the heart: power–plant failure
contributes to cardiac failure in hypertrophy. Cardio. Res.
[Internet]. 2004; 64(2):198–207. doi: https://doi.org/cq5wbz
[46] Rizk SM, Zaki HF, Mina MA. Propolis attenuates doxorubicin–
induced testicular toxicity in rats. Food Chem. Toxicol. [Internet].
2014; 67:176–186. doi: https://doi.org/f53g96
[47] Orak N, Gakmuk G, Kaya S. An evaluation of damages caused
by doxorubicin in liver tissue and potential protective effect of
propolis on these damages. Konuralp Med. J. [Internet]. 2022;
14(1):104–113. doi: https://doi.org/mjfr
[48] Perry JJP, Shin DS, Getzoff ED, Tainer JA. The structural
biochemistry of the superoxide dismutases. Biochim. Biophys.
Acta – Proteins Proteom. [Internet]. 2010; 1804(2):245–262. doi:
https://doi.org/fc4wd6
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34311
11 of 11
[49] Doroshow JH, Akman S, Chu FF, Esworthy S. Role of the
glutathione–glutathione peroxidase cycle in the cytotoxicity
of the anticancer quinones. Pharmacol. Therap. [Internet].
1990; 47(3):359–370. doi: https://doi.org/fmbwrd
[50] Olson RD, Mushlin PS. Doxorubicin cardiotoxicity: analysis of
prevailing hypotheses. FASEB J. [Internet]. 1990; 4(13):3076–
3086. doi: https://doi.org/mjfs
[51] Nitiss JL. DNA topoisomerase II and its growing repertoire
of biological functions. Nature Rev. Canc. [Internet]. 2009;
9(5):327–337. doi: https://doi.org/ctfdqc
[52] Cheung KG, Cole LK, Xiang B, Chen K, Ma X, Myal Y, Hatch GM, Tong
Q, Dolinsky VW. Sirtuin–3 (SIRT3) protein attenuates doxorubicin–
induced oxidative stress and improves mitochondrial respiration
in H9c2 cardiomyocytes. J. Biol. Chem. [Internet]. 2015;
290(17):10981–10993. doi: https://doi.org/f69bnn
[53] Kostrzewa–Nowak D, Paine MJI, Wolf CR, Tarasiuk J. The role
of bioreductive activation of doxorubicin in cytotoxic activity
against leukaemia HL60–sensitive cell line and its multidrug–
resistant sublines. British J. Canc. [Internet]. 2005; 93(1):89–97.
doi: https://doi.org/c9fcdw
[54] Swamy AV, Wangikar U, Koti BC, Thippeswamy AHM, Ronad PM,
Manjula DV. Cardioprotective effect of ascorbic acid on doxorubicin–
induced myocardial toxicity in rats. Indian J. Pharmacol. [Internet].
2011; 43(5): 507–511. doi: https://doi.org/c8v99c
[55] Rohde LE, Belló–Klein A, Pereira RP, Mazzotti NG, Geib G, Weber
C, Clausell N. Superoxide dismutase activity in adriamycin–
induced cardiotoxicity in humans: a potential novel tool for risk
stratication. J. Card. Fail. [Internet]. 2005; 11(3):220–226. doi:
https://doi.org/dt5c96
[56] Köse E, Oğuz F, Vardi N, Sarihan ME, Beytur A, Yücel A, Ekı̇ncı̇
N. Therapeutic and protective effects of montelukast against
doxorubicin–induced acute kidney damage in rats. Iranian J. Basic
Med. Sci. [Internet]. 2019; 22(4):407–411. doi: https://doi.org/mjft
[57] Kuropatnicki AK, Szliszka E, Krol W. Historical aspects of propolis
research in modern times. Evidence–Based comp. Alt. Med.
[Internet]. 2013; (Spec No):964149. doi: https://doi.org/gbc5ft
[58] Cornara L, Biagi M, Xiao J, Burlando B. Therapeutic properties of
bioactive compounds from different honeybee products. Front.
Pharmacol. [Internet]. 2017; 8(412)1–20. doi: https://doi.org/mjfw
[59] Huang S, Zhang CP, Wang K, Li GQ, Hu FL. Recent advances in
the chemical composition of propolis. Molec. [Internet]. 2014;
19(12):19610–19632. doi: https://doi.org/f6vfn2
[60] Burdock GA. Review of the biological properties and toxicity of
bee propolis (propolis). Food Chem. Toxicol. [Internet]. 1998;
36(4):347–363. doi: https://doi.org/d2t9t4
[61] Sforcin JM, Bankova, V. Propolis: is there a potential for the
development of new drugs? J. Ethnopharmacol. [Internet].
2011; 133(2):253–260. doi: https://doi.org/fnqzwd
[62] Sheibani M, Nezamoleslami, S, Faghir–Ghanesefat H, Dehpour
AR. Cardioprotective effects of dapsone against doxorubicin–
induced cardiotoxicity in rats. Canc. Chem. Pharmacol.
[Internet]. 2020; 85(3):563–571. doi: https://doi.org/grb2cm
[63] Aksu EH, Kandemir FM, Yıldırım S, Küçükler S, Dörtbudak MB,
Çağlayan C, Benzer F. Palliative effect of curcumin on doxorubicin‐
induced testicular damage in male rats. J. Biochem. Mol. Toxicol.
[Internet]. 2019; 33(10):e22384. doi: https://doi.org/mjfx
[64] Liu HX, Li J, Li QX. Therapeutic effect of valsartan against
doxorubicin–induced renal toxicity in rats. Iranian J. Basic Med.
Sci. [Internet]. 2019; 22(3):251–254. doi: https://doi.org/mjfz
[65] Husna F, Arozal W, Yusuf H, Safarianti S. Protective effect of
mangiferin on doxorubicin–induced liver damage in animal
model. J. Res. Pharm. [Internet]. 2022; 26(1):44–51. doi: https://
doi.org/mjf2
[66] Nagai K, Fukuno S, Oda A, Konishi, H. Protective effects of taurine
on doxorubicin–induced acute hepatotoxicity through suppression
of oxidative stress and apoptotic responses. Anti–Cancer Drugs
[Internet]. 2016; 27(1):17–23. doi: https://doi.org/mjf3
[67] Omobowale TO, Oyagbemi AA, Ajufo UE, Adejumobi OA, Ola–
Davies OE, Adedapo AA, Yakubu MA. Ameliorative effect of
gallic acid in doxorubicin–induced hepatotoxicity in Wistar rats
through antioxidant defense system. J. Dietary Supp. [Internet].
2018; 15(2):183–196. doi: https://doi.org/mjf4
[68] Fuliang HU, Hepburn HR, Xuan H, Chen M, Daya S, Radloff SE.
Effects of propolis on blood glucose, blood lipid and free radicals
in rats with diabetes mellitus. Pharmacol. Res. [Internet]. 2005;
51(2):147–152. doi: https://doi.org/dfcqmx
[69] Baykara M, Silici S, Özçelik M, Güler O, Erdoğan N, Bilgen M. In vivo
nephroprotective ecacy of propolis against contrast–induced
nephropathy. Diag. Inter. Radiol. [Internet]. 2015; 21(4):317–321.
doi: https://doi.org/gs8p7d
[70] Promsan S, Jaikumkao K, Pongchaidecha A, Chattipakorn N,
Chatsudthipong V, Arjinajarn P, Lungkaphin A. Pinocembrin
attenuates gentamicin–induced nephrotoxicity in rats. Canadian
J. Physiol. Pharmacol. [Internet]. 2016; 94(08):808–818. doi:
https://doi.org/f8zkx6
