Commagene Journal of Biology

Yüksel & Deveci Özkan (2021) Comm. J. Biol. 5(1), 84-89. e-ISSN 2602-456X
DOI: 10.31594/commagene.895415
Research Article / Araştırma Makalesi

The Role of Citrus Nobiletin on Oxidative Stress Levels and Superoxide

Dismutase Activities in Metastatic Castration-Resistant Prostate Cancer
Burcu YUKSEL1*, Asuman DEVECI OZKAN2
1Kocaeli
University, Vocational School of Kocaeli Health Sciences, Medical Lab. Tech., Kocaeli, Turkey
2Sakarya
University, Faculty of Medicine, Department of Medical Biology, Sakarya, Turkey
ORCID ID: Burcu YUKSEL: https://orcid.org/0000-0001-8691-6447; Asuman DEVECI OZKAN: https://orcid.org/0000-0002-3248-4279

Received: 11.03.2021 Accepted: 15.06.2021 Published online: 24.06.2021 Issue published: 30.06.2021

Abstract: Nobiletin (NOB) is a polymethoxylated flavone. It has multiple biologic activities that can modulate oxidative stress
in many cancer types. However, there is no study in the literature that has examined the effects of NOB on oxidative stress
levels in Metastatic Castration-Resistant Prostate Cancer (MCRPC) yet. Motivated from this gap, we investigated the impact
of NOB on oxidative stress and superoxide dismutase (SOD) enzyme activities in MCRPC as a preliminary study. For this
purpose, PC-3 and HUVEC cells were used to determine the effects of NOB on the amount of Malondialdehyde (MDA),
hydrogen peroxide (H2O2), and proline as well as SOD enzyme activities. NOB potentially induced SOD enzyme activities but
the level of MDA, H2O2, and proline decreased after incubation with NOB in PC-3 cells (p<.05 and p<.001 were considered
statistically significant). Our results confirmed that NOB acted as a protective agent for cancer cells and could selectively
regulate oxidant status in MCRPC cells. Consequently, these preliminary findings provide better insight into the role of citrus
NOB on oxidative stress levels and antioxidant enzyme activities in MCRPC. Additionally, there is a need to elucidate the
molecular mechanisms of this cytoprotective effect of NOB as a potential chemotherapeutic agent.
Keywords: Antioxidant effect, cancer, flavanoid, malondialdehyde, proline.

Metastatik Kastrasyona Dirençli Prostat Kanserinde Narenciye Nobiletin'in Oksidatif Stres

Düzeyleri ve Süperoksit Dismutaz Aktiviteleri Üzerindeki Rolü

Öz: Nobiletin (NOB) polimetoksile bir flavondur ve birçok kanser türünde oksidatif stresi modüle edebilen çok sayıda
biyolojik aktiviteye sahiptir. Bununla birlikte, literatürde, MCRPC'de Metastatik Kastrasyona Dirençli Prostat Kanserinde
(MCRPC) NOB'nin oksidatif stres seviyeleri üzerindeki etkilerine dair henüz bir çalışma bulunmamaktadır. Bu nedenle, ön
çalışma olarak MCRPC'de NOB'nin oksidatif stres ve süperoksit dismutaz (SOD) enzim aktiviteleri üzerindeki etkilerini
belirlemeyi amaçladık. Bu amaçla, çalışmada NOB'nin Malondialdehit (MDA), hidrojen peroksit (H2O2) ve prolin miktarı ile
SOD enzim aktiviteleri üzerindeki etkilerini belirlemek için PC-3 ve HUVEC hücreleri kullanıldı. NOB’un potansiyel olarak
SOD enzim aktivitelerini indüklediği, ancak PC-3 hücrelerinde NOB ile inkübasyondan sonra MDA, H2O2 ve prolin
seviyesinin azaldığı tespit edildi (p<.05 ve p <.001 istatistiksel olarak anlamlı kabul edildi). Elde edilen veriler, NOB'nin kanser
hücreleri için koruyucu bir ajan olarak hareket ettiğini ve MCRPC hücrelerinde oksidan durumunu seçici olarak
düzenleyebildiğini doğruladı. Sonuç olarak, bu ön bulgular, MCRPC'de turunçgil NOB'nin oksidatif stres seviyeleri ve
antioksidan enzim aktiviteleri üzerindeki rolü hakkında daha iyi fikir vermektedir. Ek olarak, potansiyel bir kemoterapötik
ajan olarak NOB'nin bu sitoprotektif etkisinin moleküler mekanizmalarının aydınlatılmasına ihtiyaç vardır.
Anahtar kelimeler: Antioksidan etki, flavonoid, kanser, malondialdehyde, proline.

1. Introduction and result in DNA damage, apoptosis, and genetic

alterations of essential cellular proteins that regulate the
In metastatic castration-resistant prostate cancer
cell cycle (Ma, 2010). To balance ROS-induced oxidative
(MCRPC), therapies such as androgen ablation cannot be
damage, cells have developed various antioxidant
successful and oxidative stress has been regarded as one of
processes to sustain their genomic constancy such as
the qualities of the aggressive disease phenotype (Shen &
enzymatic scavengers including superoxide dismutase
Abate-Shen, 2010). In particular, oxidative stress is related
(SOD), catalase as well as glutathione peroxidase,
to MCRPC development, advancement, and response to
antioxidant enzymes involved in cell defence, DNA repair
therapy. Reactive oxygen species (ROS) are produced
enzymes, and cellular mechanisms of genomic control.
directly in damaged tissue in response to pro-
While SOD superoxide anions catalyze H2O2, catalase and
inflammatory cytokines, growth factors, exposure to
glutathione peroxidase convert H2O2 into the water and
chemicals, and other stressors. Oxidative signals can
the accumulation of peroxides is prevented (Acharya et al.,
regulate the expression of various cytokines and
2010). In this context, current treatment options in MCRPC
chemokines (TNFα, IL-1β, IL-6, IL-8). In inflammatory
are low and oxidative stress plays a vital role in MCRPC
microenvironment conditions, the subsequent stimulation
development (Amaral et al., 2012). Therefore, it is essential
of ROS production causes DNA damage and increases
to investigate the effects of alternative treatment strategies
mutation rate when it continues for a long time. This may
on oxidative stress markers in MCRPC.
induce oncogenic transformation in cancer-related genes

*Corresponding author: [email protected]

 Yüksel & Deveci Özkan (2021) Comm. J. Biol. 5(1), 84–89.

Nobiletin (NOB) is a bioactive polymethoxylated 5% CO2. The optimal treatment concentration and times
flavone (5,6,7,8,3’,4’‐hexamethoxyflavone) which was for NOB in PC-3 and HUVEC cell lines were determined
found in citrus peels and had multiple biologic activities in our previous studies (Deveci Ozkan et al., 2020).
(Bernini et al., 2011; Zheng et al., 2019; Dusabimana et al., Therefore, all cells were treated with NOB (80 µM for PC-
2019). Flavonoids exhibit anti-carcinogenic characteristics 3 and HUVEC, respectively) for 48 h in all experiments.
in vitro and reduce cancer risk by protecting cells from Additionally, cells were grown in a culture medium
oxidation or inflammation by changing the level of sex without NOB used as a negative control.
hormones, reducing angiogenesis or cell proliferation, and
2.2. Antioxidant enzyme activity and oxidative stress
regulating apoptosis (Gates et al., 2009). NOB is anti-
assays
apoptotic, anti-inflammatory, anti-tumor, and antioxidant
and an effective inhibitor against human prostate cancer 2.2.1. Preparation of cell lysate
cells and melanoma cells (Kunimasa et al., 2010; Lee et al.,
To determine the effects of NOB on antioxidant enzyme
2013; Xiao et al., 2016; Huang et al., 2016; Surichana et al.,
activity and oxidative stress level, the cells (5x10 5) were
2018). Additionally, studies have shown that NOB has an
seeded six-well plates and treated with indicated
anti-inflammatory effect and exhibit an inhibiting effect on
concentrations (80 µM for PC-3 and HUVEC, respectively)
tumor invasion, proliferation, and metastasis and inhibit
of NOB for 48h. After incubation, the cells were treated
the creation of reactive oxygen species (ROS) by increasing
with RIPA lysis and extraction buffer (Sigma Aldrich,
the superoxide dismutase (SOD) and glutathione (GSH)
USA), centrifuged for 10 min at 10.000 rpm at 4°C. After
activity and reduction in the making of malondialdehyde
centrifugation, obtained supernatant was used for the
(MDA) and H2O2‐induced PC12 cells (Luo et al., 2008; Lu
analysis of antioxidant enzyme activity and oxidative
et al., 2010; Güney Eskiler et al.; 2018; Liu et al., 2019;
stress.
Deveci Ozkan et al., 2020). In addition, researchers
reported that nobiletin impeded cell proliferation both 2.2.2. Determination of Superoxide Dismutase (SOD)
depending on the dose and duration and prevented cell activity
cycle progression in G1 (Morley et al., 2007). Nobiletin can
SOD activity was established phytochemically according
induce DNA damage that contributes to apoptosis by
to the proposed method of Beauchamp and Fridovich
regulating polymerase activity in cancer cells (Zhang et al.,
(1971). SOD accelerates superoxide radicals' degradation
2020). It has been suggested that NOB may be a pathway
(O2-), H2O2, and molecular oxygen formed during
that triggers pyroptosis associated with apoptosis by
oxidative energy production. SOD activity is determined
inducing IL-1β expression (He et al., 2015). NOB is
by the inhibition of NBT (nitrobluetetrazolium) and
hypothesized to inhibit metastasis by impeding the
formazan (blue crystals) formation. The blue-purple color
activity of activator protein-1, a dimeric protein,
formation from NBT with the effect of light is inversely
preventing DNA binding (Kawabata et al., 2005; Goh et al.,
proportional to the activity of the SOD enzyme. One unit
2019). Another suggestion proposes that NOB functions
of SOD activity was determined as the quantity of enzyme
through the Nuclear Factor-kappa B route and
needed to reduce 50% of the NBT kept under light at 560
differentiates gene expression via regulating its promoter
nm. Therefore, SOD enzyme activity was calculated
regions (Xiong et al., 2015; Park et al., 2016). As a food
according to the formula below.
ingredient, nobiletin may be a favorable new anti-prostate
cancer functional food component that can generate ROS- %Inhibition = [(Blank OD – Sample OD)/Blank OD] x 100
mediated apoptosis in prostate cancer cells.
2.2.3. Determination of Malondialdehyde (MDA) level
No study has been conducted in the literature that
MDA level was verified by the proposed method of
shows the effects of NOB on oxidative stress levels and
Ohkawa et al. (1979). According to this method, the
antioxidant enzyme activities in MCRPC. Therefore, in this
thiocarboxylic acid (TBA) test, which accepts MDA as the
study, we aimed to determine the effects of NOB on
final product of lipid peroxidation, was used. The
oxidative stress in MCRPC (PC-3) and control cells
formation of MDA content resulting from the TBA reaction
(HUVEC). To clarify if NOB affects oxidative stress, we
is accepted as a lipid peroxidation measure. The amount of
measured the amount of Malondialdehyde (MDA),
MDA in 1 ml of solution was calculated according to the
hydrogen peroxide (H2O2), and proline as well as SOD
formula below and the results were given as MDA
(superoxide dismutase) enzyme activities. Thus, it is
(nmol/gram tissue) (Ananieva et al., 2002).
aimed to obtain information that will contribute to the
potential of NOB as a functionally unique and possible MDA (nmol/g): [(A532-A600)/155000] x 106
chemopreventive agent in inflammation-related tumor
2.2.4. Determination of Proline Level
formation such as MCRPC.
Proline level was determined by the proposed method of
2. Material and Methods
Myara et al. (1982). According to this method, the proline
2.1. Cell culture and NOB treatment level was determined by measuring absorbance in a
spectrophotometer. Absorbance values were obtained
In this study, the PC-3 (prostate cancer) cell line was used
from an ultraviolet spectrophotometer (Shimadzu UV
as MCRPC cells and human umbilical vein endothelial
mini-1240 spectrophotometer) at 520 nm wavelength.
cells (HUVEC) were evaluated as a control cell line. All
Proline concentration was calculated as μmol/g according
cells were bought from American Type Culture Collection
to the proline standard curve.
(ATCC). PC-3 was cultured in RPMI-1640 (Thermo Fisher,
USA) and HUVEC cells were grown in DMEM medium 2.2.5. Determination of Hydrogen Peroxide (H2O2) level
(Thermo Fisher, USA) supplemented with 0.1% penicillin
H2O2 level was determined by the proposed method of
and streptomycin, 10% FBS, and incubated at 37°C with

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 Yüksel & Deveci Özkan (2021) Comm. J. Biol. 5(1), 84–89.

Jana and Choudhuri (1981). According to this method, the a similar significant increase was observed in HUVEC cells
H2O2 level was determined by measuring absorbance in a treated with NOB compared to the control group level in
spectrophotometer. Absorbance values were obtained both treatment times (*p<.05, **p<.001, Table 1 and Fig. 1).
from an ultraviolet spectrophotometer (Shimadzu UV As we expect that a significant decrease was observed in
mini-1240 spectrophotometer) at 410 nm wavelength. MDA levels of the PC-3 cells treated with NOB (0.19 ± 0.03)
H2O2 concentration was calculated according to the compared to the control (0.37 ± 0.06) for 48 h but a
proline standard curve. significant increase was determined in HUVEC cells
treated with NOB (0.69 ± 0.28) compared to the control
2.3. Statistical analysis
(0.60 ± 0.47) for 48h (*p<.05, **p<.001, Table 1 and Fig. 2).
Statistical analyses were carried out via Graph pad Prism Additionally, a similar significant decrease was observed
v9.0 (Software, CA) and showed the mean ± standard for in H2O2 concentration of the PC-3 cells treated with
deviation of three independent experiments. One-way NOB for 24 and 48h (0.21 ± 0.08 and 0.34 ± 0.01,
analysis of variance (ANOVA) and Tukey's test was respectively) compared to the control (1.49 ± 0.05 and 0.12
utilized to obtain multiple comparisons (p<.05 and p<.001 ± 0.05, respectively) but a significant increase was
were taken as statistically significant). determined in HUVEC cells treated with NOB compared
to the control for both 24 and 48h (*p<.05, **p<.001, Table 1
3. Results
and Fig. 3). Besides, the proline level of the cells incubated
Our results showed that the SOD activities of the cells with NOB showed a statistically non-significant decrease
incubated with NOB for 24 and 48 h showed a significant in PC-3 and HUVEC cells compared to the control cells for
increase in PC-3 cells (9.71 ± 2.04 and 9.36 ± 2.32, 48h (Table 1 and Fig. 4). Therefore, according to our
respectively) compared to the control cells not treated with results, MDA and H2O2 levels were significantly lower
NOB (7.98 ± 0.41 and 6.87 ± 0.13, respectively). Moreover, whereas SOD activity was significantly higher in the
MCRPC cells treated with NOB.
Table 1. Comparison of the oxidative stress levels and antioxidant enzyme activities according to the control group and NOB treated
group in PC-3 and HUVEC for 24 and 48h.
PC-3-Control PC-3-NOB HUVEC-Control HUVEC-NOB
Parameters
Mean ± SD (n=3) Mean ± SD (n=3) Mean ± SD (n=3) Mean ± SD (n=3)
24h 7.98 ± 0.41 9.71 ± 2.04* 8.36 ± 0.38 14.21 ± 1.29**
SOD (unit/mg)
48h 6.87 ± 0.13 9.36 ± 2.32* 3.38 ± 0.78 11.06 ± 0.86**
24h 0.53 ± 0.21 0.56 ± 0.15 0.42 ± 0.21 0.47 ± 0.08*
MDA (µM)
48h 0.37 ± 0.06 0.19 ± 0.03* 0.60 ± 0.47 0.69 ± 0.28**
24h 1.49 ± 0.05 0.21 ± 0.08** 0.16 ± 0.05 0.21 ± 0.01**
H2O2 (µM)
48h 0.12 ± 0.05 0.34 ± 0.01** 0.06 ± 0.01 0.10 ± 0.07**
24h 9.46 ± 1.83 6.68 ± 0.83 4.27 ± 0.57 6.85 ± 1.18
Prolin (µM)
48h 11.22 ± 1.22 7.63 ± 1.46 7.98 ± 0.08 7.27 ±1.07**

The results are expressed as mean ± SD of eight experiments. *p< .05 and **p< .001 significantly different compared to the controls.

Figure 1. Relative enzyme activity level of SOD in PC-3 and Figure 2. Relative level of MDA in PC-3 and HUVEC cells. To
HUVEC cells. To determine the enzyme activity level of SOD in determine the level of MDA in PC-3 and HUVEC cells were
PC-3 and HUVEC cells were treated with 80 mM NOB, for 24 and treated with 80 mM NOB, for 24 and 48 h. (*p<.05, **p<.001, NOB:
48 h. (*p<.05, **p<.001, NOB: Nobiletin). Nobiletin).

4. Discussion diseases such as prostate cancer. Besides, if this oxidative

stress that can cause damage to the cells is not repaired, the
In this study, the role of citrus nobiletin on oxidative stress development of carcinogenesis will be inevitable over the
levels and antioxidant enzyme activities was investigated years (Cooke et al., 2000; DeWeese et al., 2001; Cooke et al.,
in metastatic castration-resistant prostate cancer cells for 2003). Moreover, antioxidant enzymes' activities decrease
the first time. Our preliminary findings demonstrated that when the balance of ROS-antioxidant is disturbed in
NOB could modulate oxidative stress in PC-3 cells. prostate cancer cells (Zhou et al., 2006). Our study found
Oxidative stress is one of the inevitable consequences significantly higher SOD (antioxidant enzyme) activity
of aerobic life and increasing evidence indicates that the levels in NOB-treated PC-3 cells. This finding has
accumulation and formation of ROS and reactive nitrogen supported that NOB has a potential protective role on the
species play a significant role in various age-related cells from oxidative stress by increasing the level of
antioxidant enzymes.

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data, nobiletin reduced the cytotoxicity induced by H2O2,

scavenging ROS, reducing MDA, especially in PC-3 cells,
and restoring the activities of antioxidants (Lu et al., 2010;
Malik et al., 2015). In this study, it can be assumed that the
protection provided by nobiletin may be due to its
antioxidant effect.
Extracellular matrix (ECM) proteins are a large
source of amino acids that are likely to be discharged into
the tumor microenvironment through the activity of
matrix metalloproteinases and/or collagenases emitted by
cancer cells, thereby affecting the metabolism of cancer
cells. There are specifically Glycine and Proline, as a non-
Figure 3. Relative concentration of H2O2 in PC-3 and HUVEC cells. essential amino acid, among the ECM proteins.
To determine the concentration of H2O2 in PC-3 and HUVEC cells Additionally, many studies have demonstrated that
were treated with 80 mM NOB, for 24 and 48 h. (*p<.05, **p<.001, proline metabolism impacted many pathways in the
NOB: Nobiletin). control of cancer cell plasticity and had the potential to be
a prognostic marker and potential therapeutic target
(Phang & Liu, 2012; Phang et al., 2015). Many cancer cells
can use proline to produce ATP and ROS and proline
oxidation had an essential role in cancer cells' survival
(Phang, 2019; Huynh et al., 2020). Thus, our results showed
that NOB treatment decreased the proline level in PC-3
cells but increased in HUVEC cells compared to those not
treated with NOB. These findings suggested that NOB can
modulate oxidant status through ECM and selectively
regulate oxidant status in MCRPC cells, consistent with the
literature. Nobiletin may be an efficient cytostatic anti-
cancer agent. Inhibition of cell spread without stimulating
cell mortality might be valuable in the treatment of tumors
in a way that is less likely to generate cytotoxicity and
Figure 4. Relative level of proline in PC-3 and HUVEC cells. To mortality in non-tumor tissues.
determine the level of proline in PC-3 and HUVEC cells were
treated with 80 mM NOB, for 24 and 48 h (*p<.05, **p<.001, NOB: 5. Conclusion
Nobiletin).
The imbalance between antioxidants and oxidants occurs
Oxidation of lipid peroxidation is one of the most by decreasing the number of antioxidants or increasing the
widely reported markers of oxidative stress as a number of oxidants in the cell and can induce positive
contributor to cancer and MDA is the most frequently responses, including cellular proliferation or activation
studied of these markers. In one study, Meng et al. (2017) and negative responses as growth suppression and cell
showed that Cu2+ -induced intracellular ROS mortality. Thus, stress development studies have proven
accumulation and increased MDA levels with apoptosis to be instructive for the development of cancer treatments
reduced by astaxanthin treatment and this might have a and are useful in developing anti-cancer strategies.
protective effect against Cu2+ -induced oxidative damage Therefore, these preliminary findings provide better
by reducing ROS and MDA levels. Similarly, our results insight into citrus nobiletin's role, as a cytoprotective and
revealed that NOB treatment decreased the MDA level in potential chemotherapeutic agent, on oxidative stress
PC-3 cells compared to the control cells not treated with levels and antioxidant enzyme activities in MCRPC
NOB. through by antioxidant properties of NOB.
Acute and chronic inflammatory cells such as Acknowledgements: We would like to thank Dr. Ozlem AKSOY
prostate cancer produce superoxide, H2O2, and other ROS (Kocaeli University) for providing the lab facilities
and recently it has been shown that oxidative stress is
Ethics committee approval: Ethics committee approval is not
higher in prostate cancer cells compared to normal cells. In
required for this study.
a study, the effect of doxorubicin, a chemotherapeutic
drug, on H2O2 production in PC-3 cells was examined and Conflict of interest: The authors declare that there is no conflict
an increase in intracellular H2O2 was observed as of interest.
doxorubicin concentration increased (Wagner+ et al.,
2005). However, according to the results of our study, as a References
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