Protective Role of Gossypetin against Cyclophosphamide Toxicity in Human Lymphocyte Culture In vitro

Abstract

Gossypetin is a flavonoid which has anti-mutagenic, anti-atherosclerotic, antioxidant, as well as cytoprotective and antimicrobial effects. The objective of this study was to investigate the cytoprotective role of gossypetin (GP) against cyclophosphamide (CP) toxicity in the human lymphocyte culture. Cytotoxic, necrotic and apoptotic effects of CP (1mM), GP (25, 50 and 100 µM) and combination of them (CP+GP) were studied by using MTT assay and Flow cytometry analysis. It was detected that CP significantly decreased cell viability rate via arresting cell cycle and increasing apoptosis/necroptosis. However, GP treatment reduced negative effects of CP at different concentrations. The most effective concentration of GP against CP toxicity was 25 µM. This concentration GP increased live cell number and cell viability, in addition decreased necrotic and late apoptotic cell quantity which were treated with CP. These results suggest that GP could attenuate the cytotoxic effects of CP and protect the healthy cells when it is used during chemotherapy.

Full text

Medicinal chemistry
Ustunsoy et al., Med chem 2016, 6:2
http://dx.doi.org/10.4172/2161-0444.1000330
Research Article Open Access
Med chem
ISSN: 2161-0444 Med chem, an open access journal Volume 6(2): 088-092 (2016) - 88
*Corresponding author: Seyfettin Ustunsoy, Medical Faculty, Medical
Biochemistry Department, Fatih University, 34500, Istanbul, Turkey, Tel: +90-212-
8663300/7021; E-mail: seyfettin.ustunsoy@fatih.edu.tr
Received January 25, 2016; Accepted February 17, 2016; Published February
22, 2016
Citation: Ustunsoy S, Akal ZU, Alpsoy L (2016) Protective Role of Gossypetin
against Cyclophosphamide Toxicity in Human Lymphocyte Culture In vitro. Med
chem 6: 088-092. doi:10.4172/2161-0444.1000330
Copyright: © 2016 Ustunsoy S, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Protective Role of Gossypetin against Cyclophosphamide Toxicity in
Human Lymphocyte Culture
In vitro
Ustunsoy S1*, Akal ZU2 and Alpsoy L3
1Medical Faculty, Medical Biochemistry Department, Fatih University, 34500, Istanbul, Turkey
2Science and Art Faculty, Biology Department, Fatih University, 34500, Istanbul, Turkey
3Medical Faculty, Medical Biology Department, Fatih University, 34500, Istanbul, Turkey
Abstract
Gossypetin is a avonoid which has anti-mutagenic, anti-atherosclerotic, antioxidant, as well as cytoprotective
and antimicrobial effects. The objective of this study was to investigate the cytoprotective role of gossypetin (GP)
against cyclophosphamide (CP) toxicity in the human lymphocyte culture. Cytotoxic, necrotic and apoptotic effects
of CP (1mM), GP (25, 50 and 100 µM) and combination of them (CP+GP) were studied by using MTT assay and
Flow cytometry analysis. It was detected that CP signicantly decreased cell viability rate via arresting cell cycle and
increasing apoptosis/necroptosis. However, GP treatment reduced negative effects of CP at different concentrations.
The most effective concentration of GP against CP toxicity was 25 µM. This concentration GP increased live cell
number and cell viability, in addition decreased necrotic and late apoptotic cell quantity which were treated with CP.
These results suggest that GP could attenuate the cytotoxic effects of CP and protect the healthy cells when it is used
during chemotherapy.
Keywords: Cyclophosphamide; Gossypetin; Anticancer; Apoptosis;
Necrosis; Cytotoxicity; Cell cycle
Introduction
Flavonoids are natural ingredients of plants and their by-products
obtained by dierent methods like heating, boiling, fermenting, etc.
ey have been used as therapeutic solution to the health problems by
humans for centuries even though they do not know the exact acting
mechanism. Nowadays, there are approximately 4000 plant originated
potentially therapeutic natural avonoids in the market. ey are
classied into dierent groups according to their chemical structure
[1]. ese bioactive compounds have crucial roles at regulating
important metabolic signaling pathways during physiological or
pathophysiological cascades in human body [2,3]. Hence, there
are hundreds of studies in the literature which have revealed the
signicance of these nutrients at both providing the healthy life and
preventing/curing diseases such as atherosclerosis, diabetes, metabolic
syndrome, neurodegenerative diseases and cancer by means of anti-
inammatory or antioxidant properties. Moreover, avonoids could
increase or decrease the eect of key regulatory enzymes of metabolism
via changing their expressions at DNA levels [2,3].
Despite this huge number of avonoids, quercetin is the most
studied one due to its unique chemical and physiological properties
in addition to broad prevalence in plants. Furthermore; luteolin,
kaempferol, myricetin, catechin, rutin, setin, thymoquinone and
gossypetin are the other remarkable bioactive compounds [4,5].
Gossypetin is usually extracted from the owers of the hibiscus species.
is molecule is named in chemistry as 3,5,7,8,3',4' hexahydroxy
avone. Previous studies revealed that gossypetin has anti-mutagenic,
anti-atherosclerotic, antioxidant, cytoprotective and antimicrobial
eects likewise quercetin and luteolin because of its unique chemical
structure [6]. Ngai reported that gossypetin has antiproliferative
eects on breast cancer (MCF-7) and hepatocellular cancer (HepG-
2) cell lines. Especially between 25 and 100 µM GP is more eective
on MCF-7 and HepG-2 [7]. In addition, a newly published paper
has shown that gossypetin could protect cells against both radiation
induced oxidative stress and radiation induced cellular damage via
decreasing reactive oxygen species (ROSs) [8]. Previous studies have
shown that ROSs make vital modications on biomolecules like
lipids, proteins and DNAs which change their morphology, biological
functions, life spans and leads to chronic progressive diseases like
atherosclerosis, cancer, inammation, neurodegenerative diseases, and
aging [9]. At this point, avonoids have crucial importance because
of their antioxidant properties. ese bioactive substances can both
decrease the production of reactive oxygen species and neutralize their
toxic eects [9,10]. Moreover, Chen et al. have demonstrated another
important eect of the gossypetin which inhibits LDL cholesterol
oxidation, intracellular lipid accumulation and foam cell formation
through intra cellular signaling molecules that have signicant roles in
the development of atherosclerosis such as PPRα, PPRγ, ABCA1 and
CD36 [11]. In addition, there are recently published papers suggesting
new treatment regimens including antioxidant avonoid molecules
either for protecting the healthy cells against antineoplastic, cytotoxic,
immunosuppressive agents or as a therapeutic agent alone [12].
Cyclophosphamide (CP) is a prodrug that is commonly used in the
treatment of a broad spectrum of cancers and some sort of autoimmune
diseases. Its mechanism of action depends on the active metabolite
of 4-hyroxycyclophosphamide which is produced in the liver by
cytochrome enzyme complexes [13,14]. In summary, this molecule
adds an alkyl group to the DNA and prevents cellular replication.
As a result, CP directs the cells to the apoptosis [13,14]. Although
immunosuppression and cytotoxicity are unwanted side eects of CP,
these properties also make this drug a choice in the treatment of severe
autoimmune diseases for reducing the disease burden and improving
the patient's general health status [15].
In this study, we aimed to investigate the protective eects of GP
against cellular damage which is formed by CP. As far as we know, this

Citation: Ustunsoy S, Akal ZU, Alpsoy L (2016) Protective Role of Gossypetin against Cyclophosphamide Toxicity in Human Lymphocyte Culture In
vitro. Med chem 6: 088-092. doi:10.4172/2161-0444.1000330
Med chem
ISSN: 2161-0444 Med chem, an open access journal Volume 6(2): 088-092 (2016) - 89
is the rst paper in the literature that is revealing the protective eect of
the GP during CP chemotherapy.
Materials and Methods
All products used for cell culture studies (RPMI-1640,
phytohaemaglutinin (PHA), fetal bovine serum (FBS), phosphate
buer saline (PBS), Ficoll, L-glutamine and penicillin–streptomycin
(PS) were purchased from Biochrom AG (Mannheim, Germany) and
Biological Industries (Kibbutz Beit Haemek, Israel). Cell proliferation
assay kit (MTT) and Annexin-V CY3 Apoptosis Detection Kit Plus
(containing SYTOX Green Dye) were purchased from Biovision
(Mannheim, Germany), Ficoll-Hypaque, Propidium iodide (Sigma
Aldrich, St Louis, Missouri, USA). Gossypetin is obtained from
ChromaDex (Irvine, USA). All other chemicals used for cell culture
studies and ow cytometry analysis were of reagent grade quality
obtained from commercial sources.
Lymphocyte isolation and culture of PMBC
e peripheral blood mononuclear cells (PBMCs) were isolated
from heparinized blood samples of a healthy volunteer person by
centrifugation at 2500 rpm for 25 min at room temperature according
to the Ficoll-Hypaque density gradient method. All donors gave full
written informed consent. e cloudy layer (containing thrombocyte,
lymphocyte, monocyte, etc.) between the plasma and coll solution
was separated with the help of a pipet. Lymphocytes were washed two
times with PBS, and pellet was suspended in RPMI-1640 media. e
number of viable PMBC cells was determined with the trypan blue
method. Adjusted cell suspension density was equal to 1 × 106 cell/
mL. e PBMCs were cultured on a 96 micro-well plate at a density
of 1 × 106 cell/mL and incubated with CP (1 mM), GP (25 µM, 50 µM
and 100 µM) and CP(1mM)+GP (25, 50 and 100 µM) combinations
supplemented with 2 mM/l L-glutamine, 10% FBS and antibiotics
(penicillin and streptomycin) for 4 days.
MTT assay
e cytotoxicity was measured by MTT assay. A cell viability test
of GP was performed on cultured human lymphocytes to detect the
maximal non-toxic concentration according to the manufacturer’s
protocol. A total of 1 × 104 cells were seeded on each well of a 96-well
plate and cultured for 24 hours. e cells were treated with dierent
concentra tions of CP (1mM), GP (25, 50 and 100 µM) and CP+GP
(25, 50 and 100 µM) combinations in a serum-free medium for 24
and 48 hours. Gossypetin concentrations were selected according to
previous studies [7]. At the end of the incubation process/time, the
medium containing compound was exchanged with a fresh RPMI-
1640 medium, and 10 μL of the reconstitute MTT mixture kit reagent
were added to each well. Aer cultur ing for 4 hours, the absorbance of
the sample was measured by using a microtiter plate reader (Bio-TEK)
at 590 nm. e viability of the cells was determined as the percentage of
viable cells of the untreated control and experiments were performed
in triplicate, and data are expressed as mean of triplicate measurements
(mean ± SD) in percentage of untreated cells (100%).
Measuring apoptosis using ow cytometry
Apoptotic and necrotic cells were identied with Annexin V-Cy3
and Sytox green according to the manufacturer’s protocol, respectively.
PMBC cells (2 × 104 cells/well) were exposed to CP (1 mM), GP (25, 50
and 100 µM) and CP (1 mM) + GP (25, 50 and 100 µM) combinations
for 24 h, aer trypsinizaiton cells were collected and then washed with
PBS, and incubated with 5 µl Annexin V-Cy3 and 1 µl Sytox green for 10
min in the dark. Cells incubated in the CP and GP free growth medium
were used as the negative control. Aer staining a cell population with
Annexin V-Cy3 and Sytox Green dye in the provided binding buer,
cell counting data were acquired BD Accuri C6 soware. Apoptotic
cells show red uorescence (Cy3), dead cells show green uorescence
(sytotox green) and live cells show little or no uorescence. ese
populations can be distinguished easily by ow cytometry detecting in
the FL1 (FITC) channel (Ex. 488 nm/Em. 530 nm) for Sytox Green dye
and FL2 (PE) channel for Annexin V-Cy3 (Ex. 543 nm/Em. 570 nm).
Cell cycle proling with PI staining
Aer trypsinization, cells were collected into 5 ml propylene tubes
(1 × 106) and centrifuged at 15,000 rpm, then stained with propidium
iodide (PI) and analyzed by using BD Calibur ow cytometer, and
data were evaluated with BD Accuri C6 soware. In summary, cells
were washed with 2 ml PBS then centrifuged and supernatant was
discarded. In addition, cells were xed by adding 1 mL of 70% ice
cold EtOH slowly on the pellet while vortexing to avoid cell clumping.
Cells were incubated on ice for 30 minutes then centrifuged to get
rid of the EtOH. Cell pellet was washed with PBS and centrifuged
again. Supernatant was discarded and cell pellet was re-suspended
in 1 mL of PI staining solution. Cells were incubated at 37°C for 40
minutes in PI staining solution and centrifuged again to remove the
staining solution. Supernatant was discarded and the cell pellet was re-
suspended in 500 μl PBS then analyzed at BD Calibur ow cytometer.
All the centrifugations were carried out at 2000 g for 5 minutes.
Statistical analysis
All data are expressed as the mean ± standard deviation (SD).
Statistical dierences between experimental groups were determined
using Student’s t-test. All statistical analyses were performed using
GraphPad Prism 5 and SPSS 13.0. A value of p<0.05 was considered
statistically significant.
Results
Gossypetin attenuate CP induced cytotoxicity
In order to evaluate the cytoprotective eect of gossypetin we
performed MTT cell viability assay. Human lymphocytes were
incubated with dierent gossypetin concentrations and 1 mM CP.
Figure 1 presents MTT assay results. At 24th hour, cells that were treated
with CP showed signicantly minimal cell viability rate when compared
to the control group (p<0.05). In addition, GP100 concentration has a
slight inhibitory eect on cell viability in human lymphocytes however
GP25 concentration increased cell viability. Cell viability was 57.75% in
CP group whereas in GP25, GP50 and GP100 was 130.74%, 86.20% and
81.89% respectively (p<0.05). When CP and dierent concentrations of
GP were applied on lymphocyte culture, they decreased the cytotoxic
eects of CP. Percentage of cell viabilities were 78.87% in CP+GP25,
79.74% in CP+GP50 and 81.89% in CP+GP100 group. MTT results
showed us all GP concentrations have protective role on CP toxicity
(Figure 1).
Flow cytometric analysis of Annexin V on human lymphocytes
e Annexin V binding assay is a universal, condential, user
friendly and fast ow cytometric technique that is used to discriminate
apoptotic and necrotic cell death, the cells were stained with Annexin
V-Cy3 and Sytotox Green, and the time course study was performed
using flow cytometry (Figure 2a and 2b). More than 39.9% of CP-
treated cells showed Annexin V-negative/Sytotox green-positive
staining at 2 h, and approximately 16.6% of the cells were Annexin
V-positive/Sytotox green-negative. When the cells were treated with
GP25 most of the cells remained double negative at 2 h, moreover,

Citation: Ustunsoy S, Akal ZU, Alpsoy L (2016) Protective Role of Gossypetin against Cyclophosphamide Toxicity in Human Lymphocyte Culture In
vitro. Med chem 6: 088-092. doi:10.4172/2161-0444.1000330
Med chem
ISSN: 2161-0444 Med chem, an open access journal Volume 6(2): 088-092 (2016) - 90
GP25 increased the live cell number and decreased the number of
necrotic and late apoptotic cells, suggesting that GP25 did not induce
apoptosis at this concentration. In addition, 13.5% of CP+GP25 treated
cells showed Annexin V-negative/Sytotox green positive staining at 2
h, and approximately 9% of the cells were Annexin V-positive/Sytotox
green negative, and the percent of double negative cells were about
77%. is means that GP25+CP signicantly increased live cell number
and decreased necrotic and apoptotic cells when compared with the
CP treated group. GP50 treated cells showed 24% Annexin V-negative/
Sytotox green positive staining at 2 h, and approximately 15.2% of the
cells were Annexin V-positive/Sytotox green positive, and the rest 60%
were live cells. CP+GP50 treated group cells showed 21.9% Annexin
V-negative/Sytotox green-positive staining at 2 h, and approximately
25.4% of the cells were Annexin V-positive/Sytotox green negative. As a
result, GP50+CP combination increased apoptotic cells and decreased
necrotic cells when compared GP50 treated group. Last group GP100
treated cells showed 28.2% Annexin V-negative/Sytotox green positive
staining at 2 h, and approximately 16.4% of the cells were Annexin
V-positive/Sytotox green negative. When the cells were treated with
CP+GP100 combination, cells showed 36.9% Annexin V-negative/
Sytotox green positive staining at 2 h, and approximately 7% of the cells
were Annexin V-positive/Sytotox green negative. Our experimental
data showed that GP100+CP increased necrotic cell and decreased
apoptotic cells when compared with GP100 treated cells (Figure 3).
In summary, our ow cytometry assay results were complementary
to the MTT results; CP decreased the live cell number, on the other
hand GP25 increased the cell number near to the control group
levels. However, GP50 and GP100 decreased live cell number slightly.
Additionally, when we applied CP in combination with dierent
GP concentrations on human lymphocytes, both live cell numbers
increased and the percentages of apoptotic and necrotic cell decreased
signicantly when compared to the CP group. Also, our results
demonstrated that CP has necrotic and apoptotic eects on human
lymphocytes at 1 mM concentration. According to the data presented
in this article, the most eective concentration of GP was 25 µM against
CP toxicity (Figure 4).
Flow cytometric analysis of cell cycle proling with PI staining
Intranucleosomal DNA breaks are important markers of cellular
damage. eir presence means that apoptotic or necrotic common
Figure 1: Cell viability rate of human lymphocyte after exposure to CP, various
concentrations of GP and CP+GP combinations. ap<0.05 compare with control
group, bp<0.05 compare with CP group and cp<0.05 compare with G25 group.
Figure 2a and b: Analysis of living, apoptotic and necrotic cell numbers by
ow cytometer.

Citation: Ustunsoy S, Akal ZU, Alpsoy L (2016) Protective Role of Gossypetin against Cyclophosphamide Toxicity in Human Lymphocyte Culture In
vitro. Med chem 6: 088-092. doi:10.4172/2161-0444.1000330
Med chem
ISSN: 2161-0444 Med chem, an open access journal Volume 6(2): 088-092 (2016) - 91
cascades takes place in the cell death arising from dierent causes. Cell
cycle proling with PI staining is a familiar, reliable, fast and easy ow
cytometric method for the detection of DNA breaks, cell cycle arrest
points and for the discrimination of viable, apoptotic or necrotic cells.
To determine whether the inhibitory eect of CP on human
lymphocyte cell proliferation involved cell cycle changes, we examined
cell cycle phase distribution of treated cells by ow cytometer. Our
ow cytometric analysis of cell cycle proling with PI staining revealed
complementary data to the MTT assay results. According to this study
results CP signicantly inhibited cell cycle in G0/G1 phase. On the
other hand, all of the applied gossypetin concentrations (GP25, GP50
and GP100) signicantly induced cell viability but G25 was the most
eective concentration among them. When GPs were applied with CP,
they decreased the blocking eect of CP on cell cycle. As a result, all of
the GPs have protected the cells against CP toxicity.
Discussion
CP is an antineoplastic prodrug belongs to the nitrogen mustard
group which is converted to its active form in the liver. It is commonly
used in the treatment of autoimmune diseases and various cancer types
[16]. Although, CP takes place so oen in cancer therapy regimens, like
all other antineoplastic agents, may lead to new dysplasias which has the
possibility of emergence of new cancer types during treatment because
of its high toxic side eects. us, oncologists do not prefer to use CP
alone, instead of that they combine it with other drugs, for example
doxorubicin, to minimize the dosage for protecting normal healthy
cells from CP toxicity [17-19]. Besides this, scientists have improved
a new strategy against cytotoxic side eects of chemotherapeutic drugs
which is adding avonoids to the treatment regimens. erefore,
antineoplastic-immonosuppressive agents+avonoid combinations
are hot topic nowadays between researchers who are dealing with
cancer therapy or chronic progressive diseases. Xu et al. have worked
on ginsenoide Rg3 in combination with CP and revealed that this
drug+avonoid combination has prolonged the survival rate of mice
by eliminating the drug's toxic side eects, and inhibiting angiogenesis
via down regulating the VEGF (vascular endothelial growth factor)
expression [20]. In addition to that, Sammy et al. have combined CP
with luteolin and showed that their combination successfully decreased
the tumor volume, however, prevented the weight loss which might be
the sign of the healthy cell protection (one of the most common adverse
Figure 3: Percentages of live, necrotic and apoptotic cells in control and
experiment groups analyzed by ow cytometer.
Figure 4a and b: Flow cytometric analysis in cell cycle progression between
untreated and treated cultured human lymphocytes.

Citation: Ustunsoy S, Akal ZU, Alpsoy L (2016) Protective Role of Gossypetin against Cyclophosphamide Toxicity in Human Lymphocyte Culture In
vitro. Med chem 6: 088-092. doi:10.4172/2161-0444.1000330
Med chem
ISSN: 2161-0444 Med chem, an open access journal Volume 6(2): 088-092 (2016) - 92
eects of chemotherapy) [21]. Furthermore, resveratrol+CP mixture
was tested in the treatment of breast cancer cells and found to be very
eective at inhibiting cell proliferation through arresting cell cycle and
leading them to the apoptosis [22]. Another study, which was designed
by Alkan et al. demonstrated that curcumin+CP combination arrested
breast cancer cell cycle at the G2/M phase, and led to cell cycle jamming
at S phase. Moreover, the combination of curcumin and CP upregulated
apoptotic Bax protein expression, however, downregulated anti-
apoptotic Bcl-2 protein expression. As a result, Bax/Bcl-2 balance was
changed in favor of apoptosis and breast cancer cells were directed to
the apoptosis by this way [23]. In the light of these ndings, we thought
that gossypetin too could be a good candidate for drug+avonoid
combinations strategy in cancer treatment for preserving healthy cells
against drug caused cellular damage because of its high ROS scavenging
capacity. Moreover, in this study, we have tested dierent GP and CP
concentrations on the human lymphocytes in vitro and observed cell
survival rates along with cell cycle proliferation phases. Consistent
with other publications in the literature [24] our study conrmed
that avonoids are capable of protecting normal healthy cells against
cytotoxic eects of chemotherapeutic agents like CP. According to our
results, GP has signicantly decreased cytotoxic eects of CP on human
lymphocytes. While all applied GP concentrations were successful
against CP damage, especially, the GP25 was the most eective dose
at reducing CP toxicity. Furthermore, our ow cytometric analysis of
cell cycle proling with PI staining was consistent with cell viability
results. CP arrested cell cycle in G0/G1 phase. On the other hand,
gossypetin addition to the culture media increased cell viability close
to the control group levels. Besides, GP25 increased cell viability via
suppressing apoptotic and necrotic impacts of CP. When GPs were
applied with CP, they decreased the blocking eect of CP on cell cycle.
As a result, all of the GPs have protected the cells against CP toxicity.
It means that, gossypetin ghts against CP derived cellular damage
through scavenging ROS, decreasing oxidative stress and supporting
antioxidant mechanisms. Finally, it is important to mention that GP is a
valuable and remarkable antioxidant biomolecule that could achieve to
be a good partner in cancer chemotherapy like luteolin and quercetin.
Conclusion
In conclusion, as far as we know this is the rst study in the literature
which demonstrates that GP has cytoprotective activity against CP
toxicity. e protective eects appear to be the result of the reduction
of cytotoxicity via inhibition of ROS production and restoration of the
antioxidant system. Although, we obtained promising results on the
lymphocyte cell culture experiments, further in vitro cancer cell culture
and animal studies are necessary for conrming whether the GP+CP
complex is ecient in physiological conditions to become a potential
new antitumour drug+avanoid combination.
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