Synergistic Effect of 5-Fluorouracil Combined with Naringin in MDA-MB-231 Human Breast Cancer Cells
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International Research Journal of Oncology
3(3): 13-27, 2020; Article no.IRJO.59207
Synergistic Effect of 5-Fluorouracil Combined with
Naringin in MDA-MB-231 Human Breast Cancer Cells
Thangavel Muthusamy1,2*, L. Roshini Yadav2, Satish Ramalingam3,
Ilangovan Ramachandran4 and Prabakaran Nagarajan5
1
Faculty of Allied Health Sciences, Meenakshi Academic of Research and Education, MAHER
(Deemed to be a University), West K.K. Nagar, Chennai, 600078, Tamil Nadu, India.
2
Faculty of Allied Health Sciences, Chettinad Academy of Research and Education (CARE),
Kelambakkam, Chennai, 603103, Tamil Nadu, India.
3
Department of Genetic Engineering, School of Bio-engineering, SRM Institute of Science and
Technology, Kattankulathur, Kanchipuram, 603203, Tamil Nadu, India.
4
Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences,
University of Madras, Taramani Campus, Chennai, 600113, Tamil Nadu, India.
5
Department of Biological Chemistry and Pharmacology, The Ohio State University, USA.
Authors’ contributions
This work was carried out in collaboration among all authors. Author TM designed the work, wrote
Introductions, materials and methods, results and discussion of the manuscript and did 60% of the
experiments. Author LRY carried out cell culture and assist for western blot and statistical analysis.
Author SR helped out for cell migrations assay. Author IR helped for secondary antibodies for the
western blot probe. Author PN given valuable suggestions for the manuscript preparation. All authors
read and approved the final manuscript.
Article Information
Editor(s):
(1) Dr. Lomas Kumar Tomar, Galway University Hospital, National University of Ireland, Ireland.
Reviewers:
(1) Oseni Oyediran Ganiyu, Bayero University, Nigeria.
(2) Manar Montasser Mohamed Attia, Suez Canal University, Egypt.
Complete Peer review History: http://www.sdiarticle4.com/reviewhistory/59207
Received 10 May 2020
Accepted 17 July 2020
Original Research Article Published 31 July 2020
ABSTRACT
Antimetabolite established as a successful therapeutics for advancedstage breast cancers, but has
recurrently shown to exhibit major side effects, which restrict drug therapy interventions. The most
widely used chemotherapeutic agent is 5Fluorouracil, however the recent report suggests that its
antineoplastic activity is limited due to drug resistance developed by the cancer cells. The
resistance results in the decrease in efficacy of 5FU to improve either invasive diseasefree
_____________________________________________________________________________________________________
*Corresponding author: E-mail: evmthangavel@gmail.com;
Muthusamy et al.; IRJO, 3(3): 13-27, 2020; Article no.IRJO.59207
survival or overall survival. In order to enhance 5Fluorouracil mediated cytotoxicity, we have
attempted the combination of the natural compound Naringin with 5Fluorouracil. The combination
of 5Fluorouracil and Naringin inhibited the proliferation of MDAMB231 breast cancer cells
determined using MTT assay. Importantly, combined treatment showed synergistic enhancement of
breast cancer cell cytotoxicity. We next confirmed the cytotoxicity profile of the combination 5
Fluorouracil and Naringin in MDAMB231 breast cancer cells with trypan bluebased cell viability
assay and determined that the IC50 value is 80 μM. When 5Fluorouuracil and Naringin incubated
there is a 7–10fold increase in cytotoxic effect on breast cancer cells. These results demonstrates
that application of naringin as an adjuvant treatment during 5Fluorouracil administration might
enhance the chemotherapeutic efficacy of 5Fluorouracil.
Keywords: 5-fluorouracil; Naringin; cytotoxicity; MDA-MB-231 breast cancer cells.
1. INTRODUCTION is shown to disrupt the nucleotide metabolism by
blocking nucleic acid synthesis. 5FU as a
The most prevalent type of invasive cancer is chemotherapeutic agent is an active metabolite
human breast cancer (BC) and the second of capecitabine utilized highly for the cure of solid
common reason of mortality in women [13]. BC cancer such as mammary tumor [8]. The
is most frequently diagnosed malignancy in utilization of drugs such as 5Florouracil is limited
women of younger age and predominant among by acquiring drugresistance and lowering the
tumors causing mortality in this age span [4]. drug systematic efficacy [8]. Several techniques
This issue appears as a major public health practiced in attempt to overcome this
threat in developing nations where the complication such as the combined use of
prevalence of breast carcinoma is promptly antitumor metabolites [9,10]. Further, it was
increasing. It is estimated that women of younger recently indicated that joint treatment with natural
age group are the highest percentage of the agents improved the competence and inflated
overall breast tumor patient population compared the cytotoxicity levels of several anticancer
to developed nations [5]. Obesity, age, initial medications. Naringin [Fig. 1] has showed to
pregnancy at delayed age, late menopause, prevent the progression of mammary tumor cells
early menarche, using of postmenopausal MDAMB435 in vitro and Molecular docking also
hormones, like progesterone and estrogen, and showed that naringin potentially inhibits estrone
the incidence of an inherited mutation in the sulfatase and attenuates the hormonal prompt of
BRCA1 or BRCA2 breast genes are main risk breast malignant cells [11]. Research studies
factors for the cause of breast carcinoma [6]. have demonstrated that naringin mediates
Accordingly, novel methods for the treatment of antitumor action against different cancers (e.g.,
breast cancer are necessary. Plants are the bladder, breast, cervical, colon, liver, and
source to provide us new therapeutic candidate stomach cancers). It has been previously
compounds. Recognizing novel and less toxic reported that the anticancer property of naringin
phytochemical, which particularly destroys cells, might involve antiangiogenic effect, cell cycle
which are malignant, can direct to the raise of modulation, or apoptosis. This complete
potentially improved therapy for breast cancer mechanisms [Fig. 2] by which naringin prevents
patients [13]. Tumor biology studies suggest that cell growth of tumors (especially Triple Negative
breast cancer metastasis involves the Breast Cancer) is yet to be entirely elucidated
detachment of tumor cells from the primary [12,13]. In this study, using in vitro models, we
tumor, infiltration of surrounding tissues, invasion report the inhibitory effect of naringin on BC cells.
into blood vessels, and transport to distant sites, Earlier study demonstrated the experiments on
and tumor metastasis in distal organs [4,5]. estrogen receptor (+) MCF7 breast cancer cells
Studies of the mechanism of breast cancer and estrogen receptor () MDAMB231 breast
metastasis provide a theoretical basis for the malignant cells have dissolved that naringin
development of drugs that prevent metastasis [4, possesses both estrogenic (at lower dose) and
5]. The pyrimidine analog 5Fluorouracil is an antiestrogenic (at higher dose) behavior mainly
antineoplastic antimetabolite medication, which is during selectively binding with alpha and beta
the first line of anticancer chemotherapeutic (ERα and ERβ) estrogen receptors. Siegelin et
agents originated and extensively administrated al., reported that naringin (0.1 μmol /L)
in the treatment of colorectal carcinoma and considerably prevented the discharge of growth
breast carcinoma [7]. 5Fluorouracil mechanism factor of vascular endothelium from MDAMB
14Muthusamy et al.; IRJO, 3(3): 13-27, 2020; Article no.IRJO.59207
231 breast malignant cells and, compacted the survival of cervical SiHa cancer cells at IC50 of
incidence of angiogenesis [14]. Pretreatment 750 μM [11]. In 1928, naringin chemical structure
with naringin triggered death receptor, was first interpreted by Asahina and Incubuses
mitochondriaregulated apoptosis and, lower as shown in [Fig. 1].
Fig. 1. Structure of Naringin (Flavanone-7-O-glycoside)
Fig. 2. Anticancer and anti-oxidant activity of naringin
15Muthusamy et al.; IRJO, 3(3): 13-27, 2020; Article no.IRJO.59207
As illustrated earlier naringin inhibit tumor cell shows that combination of curcumin and 5
growth via promoting cell apoptosis, including Fluorouracil generated synergistic prevention of
triplenegative breast cancer cells, cervical tumor development and stimulation of effective
(SiHa) cells and bladder cancer cells. In triple apoptosis in the challenging cancer cell lines, this
negative breast cancer cells, the proapoptotic demonstrates that the combination of curcumin
action of naringin results from G1stage cell cycle and 5Fluorouracil generated synergistic
arrest. Suppression of the growth of breast prevention of proliferation and beginning of
cancer cells by naringin is regulated by inhibition apoptosis in the resistant malignant cell lines
of the βcatenin pathway, leading to a [19].
significantly increased p21 level and decreased
cell survival [15,16]. Phytotherapeutics involving A natural flavonoid for hepatocellular carcinoma
natural source for therapy of many diseases and cells in vivo and in vitro signified that oroxylin A
also widely used in combination with already raises the sensitivity in hepatocellular carcinoma
existing therapeutics such as for tumors [16]. The cells to 5Fluorouracil [20]. The inhibitory
combine action or synergistic effects, influence effects of oroxylin A or 5Fluorouracil on cell
the drug efficacy enhance the anticancer drug viability in HepG2 cells evaluated that oroxylin A
toxicity for the decreased survival rate of the and 5Fluorouracil showed inhibitory effect by a
cancer cells and also drug sensitizing agents dosedependent manner [20]. Previous study
with reducing toxic side effects. Although 5 indicated that in colon cancer SW620 cells
Fluorouracil exhibits as effective anticancer melatonin has showed synergistic effect
chemotherapeutic agent, unique remedial combined with 5Fluorouracil to restrain the
process are required to improve its anticancer increase of colon tumor by enhancing anti
effectiveness and alter its cytotoxity, in mixture proliferation, antimigration, and proapoptotic
with additional compounds through the activities [21].
expectation that this synergy strengthen the
antiproliferative agitation of 5Fluorouracil [17]. Therefore, in this study, we investigated the
Synergistic measure of 5Flurouracil and positive outcome of 5fluorouracil in combination
apigenin on Solid Ehrlich carcinoma (SEC) with naringin on cellular proliferation and
showed apigenin increases the effect of 5 apoptosis using MDAMB231 human breast
Fluorouracil on SEC thereby, decreasing drug cancer cells.
toxicity and resistance by downregulating the
expression in Myeloid cell leukemia1 (Mcl1) 2. MATERIALS AND METHODS
important in determining the cell fate by
controlling the balance between autophagy and
apoptosis, and accompanied 5Fluorouracil and 2.1 Chemicals and Reagents
apigenin exhibits better result than each of 5
Fluorouracil or apigenin unaccompanied [18]. 5Fluorourcil was obtained from Sisco Research
Combination treatment of curcumin with 5 Laboratories (SRL) (INDIA). MTTThiazolyl Blue
Fluorouracil in adjacent cells not in favor of the Tetrazolium Bromide (M5655) (Aldrich Co.,
cytological effects of 5FU although it maintains USA), Trypan Blue (TC193) and Dimethyl
its anti proliferation function towards cancer cells Sulphoxide (DMSO) (TC185) were obtained from
with effects including antioxidant property or its Himedia. Breast cancer cells MDAMB231 was
hindrance towards prosurvival directing pathway from National Centre for Cell SciencePune
in breast carcinoma [8]. They showed that (NCCS). Dulbecco’s Modified Eagles’s Medium
curcumin radically hindered 5Fluorouracil (AL007), Fetal Bovine Serum, 0.25% trypsin and
caused cytotoxicity by preserving viability of 0.03% EDTA Ethylenediaminetetraacetic acid
normal cells. As noted, adding together of (EDTA) was obtained from Himedia. Antibiotic
curcumin as an protreatment to 5Fluorouracil Antimycotic (100X) (15240096) was obtained
action in breast malignancy therapy might from Gibco Life Technologies (USA).
enhance the chemotherapeutic effectiveness
reaction by cooperative administration of high 2.2 Cell Culture
doses of 5Fluorouracil and lengthy management
period and most importantly with DPD Human breast cancer cell line MDAMB231 cells
deficiencies leading with adverse cytotoxicities were cultured in DMEM medium with FBS of
that necessitates numerous disruption, or 10% and AntibioticAntimycotic of 1% solution
0
constant execution of curative procedures [8]. 5 incubated at 37 C with 5% CO2 until cells
Fluorouracil resistance in gastric cancer cells reached 7580% confluent.
16Muthusamy et al.; IRJO, 3(3): 13-27, 2020; Article no.IRJO.59207
Cells were routinely subcultured by having clear cytoplasm (viable cells) compared to
trypsinization [(0.25% trypsin, 0.03% EDTA (nonviable cells) with blue cytoplasm. All the
(Ethylenediaminetetraacetic acid)], incubated for experiments were repeated in triplicate. By
510 minutes. Upon reaching confluence calculating the viability of cells with CELL
examined by inverted microscope for bacterial VIABILITY (%) percentage formula the % of cell
and fungal contamination prior to experiments. viability of control and 5 Fluorouracil in
Additionally, cells were determined to be free of combination with Naringin were plotted using
mycoplasma. graph.
2.3 Cell Proliferation Assay 2.5 Cell Migration Assay
The MDAMB231 cells grown in logarithmic The cells were inoculated in sixwell plate on
4
stage were seeded in a 96well plates at 5×10 reaching 70%80% confluency; cross lines were
cells per well and held overnight to adhere, made using 100μL sterile pipette tips and
following which a sequence of concentrations of washed three times with sterile PBS to eliminate
5Fluorouracil (20, 40, 80,120,160 in µM) and/or dislodged cells. Addition of 5Fluorouracil in
Naringin (40, 80, 120, 160, 200 in µM) and 5 combination with Naringin (20+160, 40+160,
Fluorouracil in combination with Naringin 80+160, 120+160, in µM) to culture medium.
[20+160, 40+160, 80+160, 120+160, 160+160 in After 0 h, and 48 h, the cultured flask was
µM] was subjected to the well for the incubation photographed. Cell migration distance = distance
period of 48 h. Control experiments were done in at 0h distance at 48 h.
MDAMB231 cells by addition of DMSO without
Naringin. Following 48 h of treatment, cell 2.6 Effect of 5-FU and Naringin Alone or
proliferation was tested with the Thiazolyl Blue in Combination Effect on the Cell
Tetrazolium Bromide MTT assay [22]. In Death Protein Expressions in MDA-
exposure of either 5Fluorouracil or Naringin and MB-231 Cells
5FU in combination with Naringin the cells were
incubated again for the next 4h at 37°C. Four Cells on ~90% confluency were seeded on 6well
hours later it was solubilized by adding DMSO to plates at 5×105 cells/plate, after 24h incubation
all the wells also resulted in purple formazan treated with Naringin (160 μM/) and 5FU (40μM)
crystals entirely and absorbance values were alone or in combination (an accompanying group
recorded at 570 nm measured as a percentage of 160µM naringin + 40μM 5FU) for 48h, as
of growth in accordance to the control. All the DMEM medium holding equal quantity of DMSO
experiments were practiced in triplicate manner. was used as control. For harvesting the culture
The concentration of the different doses of the medium was discarded and using cold PBS
drug that lead to 50% inhibition of cell buffer the cells were washed twice. Disruptions
proliferation [IC50] was considered and analyzed of pellets of cell made with RIPA buffer and were
for 5Fluorouracil, Naringin and the various assembled after centrifugation at 800 ×g at 4 °C
combinations. The IC50 value was estimated from for 5 min, and centrifugation of lysates at 9600×g
the plot. at 4 °C for 15 min for collection of the
cytoplasmic and nuclear proteins respectively.
2.4 Cell Viability Assay Phenylmethylsulfonyl fluoride method was used
for determining the protein concentrations.
The MDAMB231 cells were cultured in 6well
4
plates at 5×10 cells per well and treated with Protein samples were transferred (40μl each) on
several dose of 5Fluorouracil in combination PVDF membrane after been loaded on sodium
with Naringin (20+160, 40+160, 80+160, dodecyl sulfatepolyacrylamide gel 12% and
120+160, 160+160 in µM) for 48h. Control was separation of proteins by electrophoresis.
reviewed by adding only DMSO to MDAMB231 Blockage of the nonspecific binding with 5% milk
cells. After 48h of treatment period, assessment in TBST buffer for 1 h. Then the membranes
of cell viability using Trypan blue dye exclusion were attached with primary antibodies against
test allowing for light microscopic quantization of Caspases9 (cat# PA522252, Thermo Fisher
viable cells using hemocytometer. Derived on the Scientific), PARP (Cat# P7605, Sigma Aldrich),
principle of exclusion of certain dyes by the Bad (Cat# PA517896 Sigma Aldrich) Bax (PA5
membranes of live cells that are intact, 70418, Sigma Aldrich) or βactin (Cat# MAB1501
suspension of treated cells in PBS contained Sigma, Aldrich) [1:1000] overnight at 4°C.With
trypan blue dye to detect the percentage of cells 1× TBST the membrane was washed three
17Muthusamy et al.; IRJO, 3(3): 13-27, 2020; Article no.IRJO.59207
times, which was followed by culturing with particularly to the subgroup of flavanone and as
secondary antibodies [1:3000 dilution] for 1 h at water dissolving molecule, passive transport is
room temperature and washed again three times found to take part as major role in the biological
in 1× TBST. By means of an ECL system protein process [15]. The development of therapeutics
bands were visualized. With Quantity One against cancer and to elevate the productivity of
software [BioRad Laboratories, Hercules, CA, 5Fluorouracil, anticipation of its regulation
USA] the grayscale values of the bands were combined with other compounds might improve
analyzed. the growth inhibition action of 5FU. Naringin is
one such product derived from natural source is
2.7 Statistical Analysis used, shown to arrest prosurvival signals in
breast carcinomas [16]. The effects of 5FU or
All data analyzed with Pvalues with statistical naringin on cell growth of MDAMB231 human
significance confirmed when P < 0.05.This was breast cancer cells by Thiazolyl Blue Tetrazolium
marked by using Graph Pad Prism [Version 7.0] Bromide assay. Regardless of exposure time a
performing ttests on triplicate samples. small dosedependent antiproliferative effect
indicating an IC50 of 80 µM of 5FU [Fig. 2A].
3. RESULTS AND DISCUSSION Whereas, naringin in a dose and time dependent
method [pMuthusamy et al.; IRJO, 3(3): 13-27, 2020; Article no.IRJO.59207 slice and operate downstream effector organs, accumulation of naringin with 5FU caspases (for example caspases 3 and 7), secured distinctive cells from the cytotoxic side results in fragmentation of PARP nuclear effects of 5Fluorouracil at the same time as proteins, and carry cell apoptosis [11]. To regulating its cell death activity towards tumor additionally explore providing reduced cell cells [33]. We reported here that upon working of proliferation the induction of cell apoptosis. The various concentrations that (40, 80, 120, 160 Analysis of apoptosis using trypan blue for MDA µM) of the antimetabolite 5Fluorouracil, MB231 cells [Fig. 3]. Following combined interrupting typical actin organization and treatment 5Fluorouracil+Naringin (20+160, inactivity of migrating cells elongated process 40+160, 80+160, 120+160, 160+160 µM) for characteristic of control cells and injury 48h, significantly (p
Muthusamy et al.; IRJO, 3(3): 13-27, 2020; Article no.IRJO.59207 utilizing the MDAMB231 cells lysate and the that the combination with the same data are reported in Fig. 5. The protein bands concentrations was more effective to enhance intensities of Caspases, PARP, Bad and Bax and Caspases, PARP, Bad and Bax expression of βactin (as control) are summarized in Fig. 5. protein than 5Fluorouracil and naringin alone (P The increased expression of Caspases, PARP, < 0.05). Additionally, the combination of 5 Bad and Bax proteins was by 44.79%, 47.17%, Fluorouracil and naringin presented a 45.82% and 60.21% by 5FU (40 μmol/l), concentrationdependent increase in significance naringin (160 μmol/l), 5FU (40 μmol/l) + 160 in statistical range (P
Muthusamy et al.; IRJO, 3(3): 13-27, 2020; Article no.IRJO.59207
C
Fig. 3C. Cells were exposed to 5-Fluorouracil+Naringin (control, 20+160, 40+170, 80+160,
120+160, 160+160 in µM) and incubated for 48 h. The IC50 at 40+170 µM was determined
All data were expressed as (treated vs. control), mean±SD for three replications with *pMuthusamy et al.; IRJO, 3(3): 13-27, 2020; Article no.IRJO.59207
B
Fig. 5B. Effect of 5-FU+Naringin on cell adhesion and migration of human breast cancer MDA-
MB-231 cells control - 0 hrs
C
Fig. 5C. Effect of 5-FU+Naringin (20+160 µM) 48h on cell adhesion and migration of human
breast cancer MDA-MB-231 cells
D
Fig. 5D. Effect of 5-FU+Naringin (40+160 µM) 48h on cell adhesion and migration of human
breast cancer MDA-MB-231 cells
22Muthusamy et al.; IRJO, 3(3): 13-27, 2020; Article no.IRJO.59207
E
Fig. 5E. Effect of 5-FU+Naringin (80+160 µM) 48h on cell adhesion and migration of human
breast cancer MDA-MB-231 cells
Fig. 5F. Effect of 5-FU+Naringin (120+160 µM) 48h on cell adhesion and migration of human
breast cancer MDA-MB-231 cells
Caspase9 is engaged and rendered by naringin promoted apoptosis by regulating
mitochondriadependent death signal, whereas mitochondrionmediated pathway.
caspase2, 8, or 10 activation is induced by
death receptors, which turns on caspase3, 6 Disruption of MMP by release of cytochrome c
and 7 [34]. Then cleaving of PARP into p89 and takes place when Bax, a pro apoptotic protein is
p24, which hence, leads to fragmentation of DNA translocated to the mitochondrial outer
and developing apoptosis [36]. Mitochondrial membrane. By contrast, in order to prevent the
dependent apoptotic pathway with rising process antiapoptotic Bcl2 protecting the
expression of apoptotic genes p53, caspase8, mitochondrial integrity. Stimulating Bax level
caspase3 and Bax causing apoptosis in MCF7 SN38 resulted in apoptosis [38]. The initial Bcl2
and MDAMB231 cells with mixture of homologue to be recognized Bax inhibits the
resveratrol and raloxifene [37]. Current results antiapoptotic function of the Bcl2 protein;
show that 5FU + naringin affected the inducing apoptosis through antagonizing the
mitochondrial membrane potential. Western blot preserving effects of Bcl2 [39].
analyses indicated that there is an increase in
caspase9 and PARP activity following the Previous study demonstrated that the Bax to Bcl
combination treatment. Therefore, 5FU and 2 ratio is a key aspect of facilitating apoptosis
23Muthusamy et al.; IRJO, 3(3): 13-27, 2020; Article no.IRJO.59207
thus, the recognition of the differential Over the last decade previous findings have
expressional level of Bax and Bcl2 by shown that several regulatory proteins of
immunohistochemistry associating the radio apoptosis contain other functions distinct to
sensitizing property of gemcitabine [40]. Usage apoptosis [41]. Studies established that cyclin D1
of gemcitabine could down regulate Bcl2 protein synthesis inhibition imposed G1 phase block via
expression and up regulate Bax protein BAD in breast cancer cells which is situated in
expression to impact degrees (P < 0.05). Their the nucleus and cytoplasm of normal breast
results indicated that major impact on triggering tissue signifying a physiological part for nuclear
the apoptosis response of tumor cells was BAD [41]. Distinguishing the link in BAD/pBAD
through the combination of gemcitabine and declined expression in breast carcinoma and the
radiotherapy rather than the single functioning of involvement of enhanced BAD expression with
either radiotherapy or gemcitabine [40]. prolonged survival. As breast cancers patients
Fig. 6. Western blot analysis for Caspase-9, PARP, Bad, and Bax -induced signaling leads to
apoptotic cell death 5-FU combination with naringin induces protein expression
Fig. 7. Western blot quantification analysis for Caspase-9, PARP, Bad, and Bax -induced
signaling leads to apoptotic cell death 5-FU combination with naringin induces protein
expression
All data were expressed as (treated vs. control), mean±SD for three replications with *pMuthusamy et al.; IRJO, 3(3): 13-27, 2020; Article no.IRJO.59207
comprising high BAD protein level extended COMPETING INTERESTS
survival and which is associated directly to
metastasis, it is plausible that lessen metastatic Authors have declared that no competing
potential could be linked with an improved BAD interests exist.
expression [42]. In addition, Mary et al., (2018)
have clearly demonstrated that BAD over REFERENCES
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