In Vitro and in Vivo Antitumour Effects of Coconut Water Vinegar On
In Vitro and in Vivo Antitumour Effects of Coconut Water Vinegar On
In Vitro and in Vivo Antitumour Effects of Coconut Water Vinegar On
research
ORIGINAL ARTICLE
In vitro and in vivo antitumour effects of coconut water vinegar on
4T1 breast cancer cells
Nurul Elyani Mohamad1, Swee Keong Yeap2,3, Nadiah Abu1,4, Kian Lam Lim5,
Nur Rizi Zamberi1, Noraini Nordin1, Shaiful Adzni Sharifuddin6, Kamariah Long6
and Noorjahan Banu Alitheen1,3*
1
Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra
Malaysia, Serdang, Selangor, Malaysia; 2China-ASEAN College of Marine Sciences, Xiamen University Malaysia, Jalan
Sunsuria, Bandar Sunsuria, Sepang, Selangor, Malaysia; 3Institute of Bioscience, Universiti Putra Malaysia, Serdang,
Selangor, Malaysia; 4UKM Medical Centre, UKM Medical Molecular Biology Institute (UMBI), Cheras, Wilayah
Persekutuan, Malaysia; 5Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Sungai Long Campus,
Jalan Sungai Long, Bandar Sungai Long, Cheras, Kajang, Selangor, Malaysia; 6Biotechnology Research Centre, Malaysian
Agricultural Research and Development Institute (MARDI), Serdang, Selangor, Malaysia.
Abstract
Background: Coconut water and vinegars have been reported to possess potential anti-tumour and immuno-
stimulatory effects. However, the anti-tumour, anti-inflammatory and immunostimulatory effects of coconut
water vinegar have yet to be tested.
Objective: This study investigated the in vitro and in vivo anti-tumour effects of coconut water vinegar on 4T1
breast cancer cells.
Methods: The 4T1 cells were treated with freeze-dried coconut water vinegar and subjected to MTT cell viabil-
ity, BrdU, annexin V/PI apoptosis, cell cycle and wound healing assays for the in vitro analysis. For the in vivo
chemopreventive evaluation, mice challenged with 4T1 cells were treated with 0.08or 2.00 mL/kg body weight
of fresh coconut water vinegar for 28 days. Tumour weight, apoptosis of tumour cells, metastasis and immu-
nity of untreated mice and coconut water vinegar-treated 4T1 challenged mice were compared.
Results: Freeze-dried coconut water vinegar reduced the cell viability, induced apoptosis and delayed the
wound healing effect of 4T1 cells in vitro. In vivo, coconut water vinegar delayed 4T1 breast cancer progression
in mice by inducing apoptosis and delaying the metastasis. Furthermore, coconut water vinegar also promoted
immune cell cytotoxicity and production of anticancer cytokines. The results indicate that coconut water vin-
egar delays breast cancer progression by inducing apoptosis in breast cancer cells, suppressing metastasis and
activating anti-tumour immunity.
Conclusion: Coconut water vinegar is a potential health food ingredient with a chemopreventive effect.
Keywords: coconut water vinegar; breast cancer; metastasis
To access the supplementary material, please visit the article landing page
Received: 15 April 2017; Revised: 12 September 2018; Accepted: 28 November 2018; Published: 10 January 2019
Food & Nutrition Research 2019. © 2019 Nurul Elyani Mohamad et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License 1
(http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose,
even commercially, provided the original work is properly cited and states its license. Citation: Food & Nutrition Research 2019, 63: 1616 - http://dx.doi.org/10.29219/fnr.v63.1616
(page number not for citation purpose)
Nurul Elyani Mohamad et al.
C
ancer is a major public health issue and the sec- correlated the consumption of vinegar with prevention
ond leading cause of death in developing coun- of oesophageal cancer (14). In other works, vinegar from
tries. Despite intensive research and efforts, unpolished rice demonstrated in vitro cytotoxic effects on
breast cancer incidence remains the most common cancer squamous carcinoma (15) and in vivo anti-colon tumour
among women worldwide, and with the highest mortal- effects (16). Also, sugar cane vinegar was reported to
ity rates. Advances in screening and breast cancer treat- kill leukaemia cells via induction of apoptosis (17). Guo
ment are reported to reduce the incidence and mortality et al. (18) noted that vinegar prevented the formation of
of breast cancer, and strategies, including maintaining a N-nitroso compounds, which are known carcinogens.
healthy lifestyle, can help reduce the breast cancer risk These studies (15–18) helped justify the correlation of the
(1). Regarding a healthy lifestyle, evidence exists for use of vinegar with reduced cancer risk (14).
the role of a healthy diet and physical activity in pre- Vinegar can be produced from various sources of fruit
venting and delaying cancer progression. For example, and vegetables (12, 13). Although acetic acid is the main
an Asian diet that is rich in plant-based food has been component in all types of vinegar, the health benefits of
conveyed as a potential approach to reduce the risk and different types of vinegar may vary due to variations in the
delay the progression of breast cancer (2). More specif- levels of antioxidants from both the source of carbohydrate
ically, an ethnic lifestyle and ethnic food, such as high and bacterial strains used in alcohol and acetous fermenta-
consumption of soy-based products by Chinese women tion (19). Sugar-rich coconut water (6) is commonly used
in South East Asia, may contribute to their higher 5-year to produce vinegar. However, the bioactivities, particularly
overall survival compared to other ethnicities in Malaysia the antitumour effect on breast cancer, of this coconut water
and Singapore (3). Besides soy-based products, the intake vinegar have not yet been tested. Thus, this study aimed to
of many other food ingredients, such as vegetables, fruit evaluate the in vitro and in vivo antitumour effects of coconut
and fibre, which have been recommended by the National water vinegar on murine 4T1 breast cancer cells. In addition,
Cancer Institute, also helps reduce the cancer risk (4). the role of the anti-inflammatory and immunostimulatory
Coconut (Cocos nucifera L.) is an important tropi- influences of the coconut water vinegar that may indirectly
cal fruit. Coconut water that is commonly consumed as contribute to the antitumour effects was also assessed.
a refreshing beverage in the tropical regions has been
associated with various health and medicinal benefits, Materials and methods
including antibacterial, antifungal, antiviral, anti-parasitic,
anti-dermatophyte, antioxidant, hypoglycaemic and Preparation of coconut water vinegar
hepatoprotective benefits (5). These health benefits may be Coconut water vinegar was prepared according to a pre-
attributed to the presence of several bioactive compounds vious study (20). Pure and fresh coconut juice was bought
in its composition, including vitamins, amino acids, or- from the local market in Malaysia (Pasar Borong, Selan-
ganic acids, enzymes (6) and phenolic acids (7). Coconut gor). The coconut juice was first fermented using Sacchar-
water has also been linked with anti-inflammatory (8) and omyces cerevisiae 7013 INRA to produce alcohol and then
immunostimulatory effects (9). In addition, peptides iso- further fermented with Acetobacter aceti vat Europeans to
lated from coconut water have been suggested as potential give the final product, acetic acid. The sample was then left
anticancer agents (9). Given that cancer has been identified to mature at room temperature for 1 month and finally kept
as a disease of uncontrollable cell growth, associated with in a glass container at 4°C until use. For the in vitro study,
chronic inflammation and an immunosuppressive tumour coconut water vinegar was freeze-dried and stored frozen
microenvironment (10), coconut water, with its anti-inflam- at −20°C. Before cell treatment, the freeze-dried coconut
matory, immunostimulatory and cytotoxic activities (8, 9), water vinegar was diluted using RPMI-1640 media, titrated
may be beneficial in delaying cancer progression. to pH 7 and filtered through a 0.25 µm syringe filter.
However, fresh fruit and vegetables have a limited shelf
life. To overcome this limitation, fruit and vegetables can Cell culture
be fermented to prolong the shelf life or even enhance the Mouse breast cancer cell line (4T1) was purchased from the
availability of several bioactive components (11). Vin- American Type Cell Culture (ATCC) collection (ATCC,
egar is a natural food additive, which is produced from Rockville, MD, USA). The cells were preserved in RPMI
fruits or vegetable rich in glucose, by a two-step process: containing 10% foetal bovine serum (FBS) and were grown
alcohol fermentation and acetic acid fermentation. The at 37°C in a humidified incubator containing 5% CO2.
common use of vinegar as a food seasoning and thera-
peutic agent is well established (12). Vinegar has been 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium
reported as an effective anti-obesity and anti-hyperglycae- bromide (MTT) assay
mic agent, mainly due to the presence of acetic acid and Cytotoxicity of coconut water vinegar on murine 4T1 breast
phenolic compounds (12, 13). Moreover, a previous study cancer cells was measured by the MTT assay. Firstly, the cells
2
(page number not for citation purpose)
Citation: Food & Nutrition Research 2019, 63: 1616 - http://dx.doi.org/10.29219/fnr.v63.1616
Effects of coconut water vinegar on 4T1 breast cancer cells
(0.8 × 105 cells/well) were seeded in a 96-well plate overnight. 15 min in the dark and analysed using a FACSCalibur
Then, a twofold serial dilution of coconut juice vinegar was flow cytometer (Beckman Coulter, California, USA). The
pipetted to the plate and incubated for 48 h. Afterwards, experiment was repeated in triplicate.
the MTT solution was added to all the wells and the plates
were incubated for 3 h to allow the formation of formazan Wound healing assay
crystals. Then, the formazan crystals were solubilised by The 4T1 cells were seeded in a 6-well plate overnight to
DMSO (Fisher Scientific, New Hampshire, USA) and the reach full confluency. Then, a scratch was introduced in
absorbance [optical density (OD)] was measured using an the middle of the well by using a sterile yellow tip, and the
enzyme-linked immunosorbent assay (ELISA) plate reader cells were incubated with IC25 (120 µg/mL) of freeze-dried
(Bio-Tek Instruments, Vermont, USA) at 570 nm. The assay coconut water vinegar for 24 h. A well with a scratch but
was done in triplicate, and the cytotoxicity effect of coconut without treatment was used as the control. After 24 h, the
juice vinegar was analysed using the following formula: percentage of wound closure was calculated using the fol-
lowing formula:
Cell viability (%) = (OD sample/OD control) × 100% (1)
Wound closure (%) =
5-Bromo-2´-deoxyuridine (BrdU) cell proliferation assay [(width of wound at 0 h – width of wound
The BrdU proliferation assay was measured using a at 24 h)/width of wound at 0 h] × 100% (2)
BrdU cell proliferation kit (Merck, Germany). Firstly,
the 4T1 cells (8 × 104 cells/mL) were seeded in the plates and Animals
incubated overnight. The next day, the cells were treated The female BALB/c mice (n = 32; age 4–6 weeks; aver-
with two different concentrations (IC50=0.27 mg/mL; age weight 20–22 g) were purchased from the Institute
IC75=0.35 mg/mL) of coconut juice vinegar, added with 20 of Bioscience, Universiti Putra Malaysia. The mice were
μL of BrdU and incubated for another 72 h. Later, the cells acclimatised and were given distilled water and standard
were fixed and denatured using a fixing solution and stored pellet diet ad libitum. All procedures were performed ac-
at 4°C. The plates were washed and then incubated with 100 cording to the guidelines of the Animal Care and Use
μL of the detector antibody per well for 1 h. Next, 100 μL Committee, Universiti Putra Malaysia (UPM/FPV/
of goat anti-mouse immunoglobulin G-horseradish perox- PS/3.2.1.551/AUP-R168).
idase (HRP) conjugate was added for 30 min, followed by
further incubation with 100 μL of the 3,3′,5,5′-tetrameth- In vivo antitumour effect of coconut water vinegar
ylbenzidine substrate for 30 min. Finally, 100 μL of stop The study was conducted according to Yeap et al. (21),
solution was added to the plates, and the absorbance was with a slight modification. Dosages of the coconut water
measured at 450 nm, using a μQuant plate reader (Bio-Tek vinegar were derived from previous studies, where 0.08 mL/
Instruments, Vermont, USA). kg body weight (BW) was based on 1 Tbsp of vinegar in
250 mL of water, while 2 mL/kg BW has shown in vivo an-
Cell cycle analysis tioxidant and anti-inflammatory effects (20, 22). Initially,
Cells were cultured at 2.3 × 105 cells/well and treated the acclimatised female mice (n = 32) were randomly di-
with coconut juice vinegar at two different concentra- vided into four groups with eight mice per group. The mice
tions (IC50=0.27 and IC75=0.35 mg/mL) for 48 h. The cells were pretreated with either distilled water or coconut juice
were trypsinised and centrifuged at 2,000 rpm for 5 min. vinegar for 6 weeks before being induced with 4T1 cells [1
The pellet was collected and fixed with 70% ethanol for × 105 cells in 100 μL phosphate-buffered saline (PBS)] via
1 week. Lastly, the fixed cells were stained with propidium subcutaneous injection on week 7, except for the normal
iodide (PI) and analysed using fluorescence-activated cell control group (G1). The treatment continued for another
sorting (FACS) flow cytometry (Becton Dickinson, New 28 days before all mice were sacrificed (week 11).
Jersey, USA).
G1 (n = 8): Non-induced mice were given 100 µL
Annexin V analysis distilled water once per day throughout the study
The effect of coconut juice vinegar on apoptosis of the cells (normal control);
was measured using fluorescein isothiocyanate (FITC)-la- G2 (n = 8): Induced mice were given 100 µL distilled
belled annexin V antibody by flow cytometry, according to water once per day throughout the study (negative
the manual protocol of the FITC annexin V apoptosis de- control);
tection kit (BD Biosciences, California, USA). Briefly, the G3 (n = 8): Induced mice were pre-treated with coconut
cells (2.3 × 105 cells/well) were treated with coconut juice juice vinegar (100 µL of 0.08 mL/kg BW) once per day;
vinegar at 0.27 and 0.35 mg/mL, respectively, for 48 h. G4 (n = 8): Induced mice were pretreated with coconut
Then, the cells were double-stained with PI and FITC for juice vinegar (100 µL of 2 mL/kg BW) once per day.
The tumours, spleen and blood were collected from the then subjected to qRT-PCR analysis, for the intracellu-
mice. The tumours were weighed and washed in PBS be- lar adhesion molecule-1 (iCAM), c-myc, inducible nitric
fore they were divided into several parts and kept in liquid oxide synthase (iNOS), nuclear factor kappa B (NF-κB),
nitrogen until use. glyceraldehyde phosphate dehydrogenase (GAPDH),
California, β-actin and hypoxanthine phosphoribosyl-
Terminal dUTP nick end labelling (TUNEL) assay transferase (HRPT), using an iQ5 real-time PCR machine
The terminal deoxynucleotidyl transferase (TdT)-mediated (Bio-Rad, California, USA). The differential expression
dUTP nick end labelling assay was performed using a results were normalised and analysed using the house-
DeadEnd colourimetric apoptosis detection kit (Promega, keeping genes (GAPDH, β-actin and HRPT), by the iQ5
Wisconsin, USA), according to the manufacturer’s proto- Optical System software. The sequences of the primers
col. In brief, tumours were excised and embedded on a used in the study are given in Table 1.
slide. Then, the slides were deparaffinised in xylene twice
for 5 min, followed by rehydration in decreasing concen- Western blot analysis of matrix metalloproteinase
trations of ethanol (100, 95, 85, 70 and 50%). Next, the 9 (MMP9) and vascular endothelial growth factor
slides were washed in PBS. To detect apoptotic cells, the (VEGF) protein levels and quantification of nitric
slides were fixed in 4% paraformaldehyde and perme- oxide (NO) by the Griess assay
abilised using proteinase K and then fixed again in 4% The tumours were harvested, snap frozen and kept at
paraformaldehyde. The slides were then equilibrated using −80°C until use. For the extraction of protein, each tumour
the equilibration buffer and labelled using TdT. Next, the was weighed and ground in a mortar and pestle using liquid
slides were blocked in hydrogen peroxide before being nitrogen. Then, it was lysed in radioimmunoprecipitation
incubated with streptavidin HRP. The slides were then assay buffer [150 mM sodium chloride, 1.0% NP-40 or Tri-
developed using 3,3′-diaminobenzidine, mounted in glyc- ton X-100, 0.5% sodium deoxycholate, 0.1% sodium do-
erol and viewed under a bright-field inverted microscope decyl sulphate (SDS) and 50 mM Tris, pH 8.0] added with
(Nikon, Japan). The degree of DNA fragmentation was protease inhibitor cocktail (Pierce, Thermo Fisher Scien-
measured based on the presence of dark brown cells (ap- tific, New Hampshire, USA). Protein was quantified using
optotic cell indicator) against a light brown background. the Bradford assay (Bio-Rad, USA). The protein samples
were then either kept frozen at −80°C or freshly used for
Metastasis analyses using clonogenic assay western blot analysis and the Griess assay.
The metastasis of 4T1 cells was investigated using a clono- Equal amounts of protein were separated by sodium
genic assay and bone marrow test, respectively. Briefly, dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-
the mice were killed. The liver, spleen, lungs and legs were PAGE) and then transferred to a nitrocellulose membrane
removed from the body under a sterile condition, washed (Pall Corporation, Ann Arbor, MI, USA) that was sub-
with PBS and prepared according to the assay, respectively. sequently blocked with 5% non-fat milk (Bio Basic, New
The clonogenic assay was done according to a previous
study (23). In brief, the organs (liver, spleen and kidney)
were chopped into small pieces (< 1 cm3). Then, each Table 1. Primer sequences
organ was incubated in PBS and collagenase for 20–30 Target genes
min at 37°C, with thorough mixing every 5 min. Next, the
solution was passed through a 70-mm cell strainer (SPL, c-Myc Forward:5’- TGATGTGGTGTCTGTGGAGAA-3’
Korea) and spun at 2,000 rpm for 10 min. The pellet was Reverse:5’- CGTAGTTGTGCTGGTGAGTG-3’
then washed with PBS and re-suspended in 10 mL suspen- ICAM-1 Forward:5’- TGCTCAGGTATCCATCCATCC-3’
sion medium (RPMI 1640, 10% FBS, 1% penicillin-strep- Reverse:5’- ACGGTGCCACAGTTCTCAA-3’
tomycin, 60 µM 6-thioguanine). Six serial dilutions were iNOS Forward:5’-GCACCGAGATTGGAGTTC-3’
made in a 6-well plate, and the plate was incubated for Reverse:5’-GAGCACAGCCACATTGAT-3’
10 days. On day 10, the plate was fixed with methanol, NFκβ Forward:5’-CATTCTGACCTTGCCTATCT-3’
stained with crystal violet, and then the number of colo- Reverse:5’-CTGCTGTTCTGTCCATTCT-3’
nies was counted. Reference genes
GAPDH Forward:5’-TTCCAGCCTTCCTTCTTG-3’
Quantitative reverse transcription polymerase chain reaction
Reverse:5’- GGAGCCAGAGCAGTAATC-3’
(qRT-PCR) analysis
Equal amounts of total RNA were extracted using β-actin Forward:5’-GAAGGTGGTGAAGCAGGCATC-3’
4
(page number not for citation purpose)
Citation: Food & Nutrition Research 2019, 63: 1616 - http://dx.doi.org/10.29219/fnr.v63.1616
Effects of coconut water vinegar on 4T1 breast cancer cells
York, USA) overnight. Next, the membrane was washed Statistical analyses
with Tris-buffered saline (10 mM Tris, 140 mM NaCl, pH Quantitative data were expressed as mean ± standard de-
7.6) containing 0.1% Tween-20 (TBST) and further incu- viation (SD) and were analysed using one-way analysis
bated in primary antibody at 4°C for 1 h, followed by wash- of variance (ANOVA).The group means were compared
ing with TBST, before incubation with secondary antibody by Duncan’s test. Statistical Package for the Social Sci-
for another hour. It was then rewashed and incubated with ences (SPSS) version 16.0 was used for all data analysis.
HRP substrate for 10 min before it was viewed using a Che- P-values of <0.05 were considered statistically significant.
midoc imager (UVP, California, USA). Β-actin (Abcam,
USA) was used as a housekeeping control. The results ob- Results
tained were analysed using Vision Work LS Analysis soft-
ware (UVP, California, USA). Coconut water vinegar reduced viability, induced proliferation
The NO level in each tumour was quantified using the and delayed wound healing of 4T1 cells
Griess assay (Invitrogen, California, USA), according to The MTT assay showed that freeze-dried coconut water
the manufacturer’s protocol. vinegar lowered the viability of 4T1 cells in a dose-
dependent manner after treatment at IC50 (270 µg/mL) and
Immunophenotyping by flow cytometry IC75 (350 µg/mL), respectively, at 48 h (Fig. 1a). The BrdU
Briefly, the harvested spleen was washed and passed proliferation test showed that coconut water vinegar treat-
through a 70-mm cell strainer (SPL, Korea) in PBS. The ment at IC50 and IC75 reduced 13 and 49% cell prolifera-
supernatant was spun at 2,000 rpm for 15 min. The pel- tion, respectively, compared to the untreated 4T1 cells, at
let was lysed using NH4Cl buffer. Then, the splenocytes 72 h of incubation (Fig. 1b). Moreover, cell cycle profiling
were stained with four different antibodies (CD3 and showed that at IC50, coconut water vinegar significantly
CD4, CD3 and CD8, CD3 and NK1.1, F4/80) and anal- (p < 0.05) decreased the G0/G1 phase-associated increase
ysed by FACSCalibur flow cytometry (Becton Dickson, of the subG0/G1 phase at 48 and 72 h. At IC75, coconut
BD, New Jersey, USA). water vinegar further reduced the population of cells in
the S and G2/M phases, besides reducing those in the G1
Cytokine assay phase and increasing cells in the subG0/G1 phase, at 72 h
Blood was collected from each mouse using BD Micro- (Fig. 1c; Supplementary Fig. 1). Also, the annexin V/PI
tainer® tubes (Becton Dickinson, New Jersey, USA) and apoptosis assay showed that most of the 4T1 cells treated
spun at 10,000 rpm for 15 min. The resultant serum was with coconut water vinegar at both IC50 and IC75 were
kept at −20°C until use. On the allocated day, the serum under the early, rather than late, apoptosis stage at 48 h,
was diluted 10-fold using an assay diluent buffer and as- and vice versa at 72 h (Fig. 2a).
sayed by the ELISA cytokine assay; interleukin-2 (IL-2), After incubation for 24 h, the untreated 4T1 cells ex-
IL-1 beta (IL-1β), IL-10 and interferon gamma (IFNγ). All hibited an approximate 80% wound closure in the scratch
assays were performed according to the supplied mouse assay. Conversely, the coconut water vinegar treatment
cytokine kit protocols (R&D Systems, Minnesota, USA). at IC50 and IC75 had around 66 and 37% wound closure
in the scratch assay, respectively (Fig. 2b; Supplementary
Lactate dehydrogenase (LDH) cytotoxicity assay Fig. 2).
The LDH procedure was performed as described elsewhere
(21). YAC-1 cells are murine lymphoma cells that are sen- Coconut water vinegar delayed tumour formation,
sitive to cytotoxic immune cells, including CD3+CD8+ T induced apoptosis in tumour and inhibited metastasis
and NK cells. Co-culture of the splenocytes with YAC-1 of the tumour cells
cells has been commonly used to evaluate the cytotoxic- In vivo treatment with coconut water vinegar, particularly
ity of CD8 T and NK cells (24). The cytotoxicity activity at 2 mL/kg body weight (CH), exhibited delayed tumour
of coconut juice vinegar was measured using a Cytotox formation, when mice post-challenged with 4T1 cells were
96 nonradioactive cytotoxicity assay kit (Promega, Wis- compared to the untreated 4T1-challenged mice (Fig. 3a).
consin, USA). In brief, the spleen was harvested from the At 28 days, the tumour size and weight of CH-treated
untreated or coconut water vinegar-treated mice, and the mice were significantly (p < 0.05) lower than untreated
splenocytes were isolated and incubated with YAC-1 cells mice post-challenged with 4T1 cells (Fig. 3a & b). In mice
for 24 h. The ratios of splenocytes (effector spontaneous) that received 0.08 mL/kg body weight (CL) of coconut
to YAC-1 cells (target spontaneous) used in this study water vinegar, tumour formation was also later relative
were 2:1 and 5:1. The cytotoxicity of the splenocytes on to the untreated mice post-challenged with 4T1 cells. At
YAC-1 cells was tested by the Cytotox 96 nonradioactive 28 days, post-challenged CL mice were recorded with a
LDH assay (Promega, Wisconsin, USA) according to the lower but not statistically significant (p < 0.05) tumour
manufacturer’s protocol. volume and weight than untreated mice (Fig. 3a & b).
Fig. 1. (a) The percentage of 4T1 cell viability after treatment with freeze-dried coconut vinegar for 48 h. (b) The percentage of
4T1 cell proliferation quantified by BrdU ELISA test after treatment with freeze-dried coconut water vinegar (IC50 and IC75) for
72 h. (c) Cell cycle progression of untreated and freeze-dried coconut water vinegar (IC50 and IC75)-treated 4T1 cells at 48 and
72 h. Data are expressed as mean ± SEM. *p < 0.05.
From Fig. 3c and Supplementary Fig. 3, it is clear the tumour of both CL- and CH-treated mice were sig-
that mice treated with CH displayed a high number of nificantly (p < 0.05) lower than that in untreated mice
TUNEL-positive cells in the tumour immunohistochem- (Fig. 5c).
istry (IHC). Likewise, the number of TUNEL-positive
cells in the tumour of CL-treated mice was higher (though Coconut water vinegar increased serum level of anti-cancer-
not significant) compared to the untreated mice. related cytokines (IL-2 and IFNγ), spleen CD3+CD8+ and
In the CL-treated mice, metastasis of 4T1 cells was only NK1.1 cells population and cytotoxicity of splenocytes
observed in the liver but not in the lungs and spleen (single The populations of cytolytic T lymphocyte (CD3+CD8+)
event). Regarding CH treatment, no metastasis was no- and NK cells (NK1.1+) in the spleen of CL- and CH-treated
ticed in the liver, lungs and spleen (Fig. 4). mice were higher relative to the untreated 4T1-challenged
mice. The helper T cell population (CD3+CD4+) and mac-
Coconut water vinegar downregulated mRNA expression of rophages were slightly reduced in the spleen of CH-treated
iCAM, c-myc, iNOS and NF-κB, protein expression of VEGF mice (Fig. 6a).
and MMP9, and NO level in the tumour When compared with the untreated mice, the anti-
Relative to the untreated mice, CL treatment only sig- cancer-related cytokines, IL-2 and IFNγ, occurred at a
nificantly (p < 0.05) downregulated the expression of higher level in the serum of CH-treated mice, whereas the
the iCAM gene, while CH treatment downregulated the amounts were not significantly (p < 0.05) higher in the
expression of all iCAM, c-myc, iNOS and NF-κB genes CL-treated mice (Fig. 6b).
(Fig. 5a). In addition, differential protein expression of Splenocytes harvested from untreated 4T1-challenged
MMP9 and VEGF was measured using western blot anal- mice were recorded with about 50% cytotoxicity on
ysis. CH treatment significantly (p < 0.05) reduced the murine YAC-1 lymphoma. Interestingly, both CL and CH
protein expression of VEGF and MMP9 in the tumour treatments recorded higher cytotoxicity against YAC-1
of the mice compared to the untreated 4T1-challenged cells at both 5:1 and 1:2 ratios of splenocytes-to-YAC-1
mice (Fig. 5b; Supplementary Fig. 4). The NO levels in cells (Fig. 6c).
6
(page number not for citation purpose)
Citation: Food & Nutrition Research 2019, 63: 1616 - http://dx.doi.org/10.29219/fnr.v63.1616
Effects of coconut water vinegar on 4T1 breast cancer cells
Fig. 4. Representative image of the 4T1 colonies formed in the lungs, liver and spleen of untreated, 0.08 (CL) and 2 (CH) mL/kg
body weight coconut water vinegar-treated 4T1-challenged mice.
Fig. 5. (a) Differential expression of iCAM, c-myc, iNOS and NF-κB genes in the tumour of untreated, 0.08 (CL) and 2 (CH)
mL/kg body weight coconut water vinegar-treated 4T1-challenged mice quantified by qRT-PCR. Expression was normalised to
GAPDH, HRPT and β-actin. (b) Differential expression of MMP9 and VEGF proteins in the tumour of untreated and 2 (CH)
mL/kg body weight coconut water vinegar-treated 4T1-challenged mice quantified by western blot analysis. Expression was
normalised to β-actin. (c) Nitric oxide (NO) level in the tumour of untreated, 0.08 (CL) and 2 (CH) mL/kg body weight coconut
water vinegar-treated 4T1-challenged mice quantified by the Griess assay. Data are expressed as mean ± SEM. *p < 0.05.
8
(page number not for citation purpose)
Citation: Food & Nutrition Research 2019, 63: 1616 - http://dx.doi.org/10.29219/fnr.v63.1616
Effects of coconut water vinegar on 4T1 breast cancer cells
Fig. 6. (a) Population of CD3+CD4+, CD3+CD8+, NK1.1 and macrophages in the spleen of untreated, 0.08 (CL) and 2 (CH)
mL/kg body weight coconut water vinegar-treated 4T1-challenged mice quantified by flow cytometer immunophenotyping.
(b) IL-2 and IFNγ levels in the serum of untreated, 0.08 (CL) and 2 (CH) mL/kg body weight coconut water vinegar-treated
4T1-challenged mice quantified by ELISA. (c) Co-cultivation of splenocytes from untreated, 0.08 (CL) and 2 (CH) mL/kg body
weight coconut water vinegar-treated 4T1-challenged mice with YAC-1 cells at 5:1 and 2:1 ratios. Cytotoxicity was quantified by
the Cytotox 96 non-radioactive LDH assay. Data are expressed as mean ± SEM. *p < 0.05.
of bioactivities displayed by the various types of vinegar the induction of apoptosis, which further supports the
may be attributed to the different levels of active metabo- in vitro findings. In addition, the effective inhibition of
lites that are present in the raw materials used for the fer- primary tumour development by coconut water vinegar
mentation. Active ingredients that present in the coconut is possibly attributed to the suppression of c-myc gene
water vinegar may contribute to the reduction of 4T1 cells expression, as seen in the qRT-PCR assay. C-myc is an
viability via induction of apoptosis and delay the prolifer- oncogene that plays an important role in tumorigenesis
ation and closure of 4T1 cells, which need further studies. (28). Coconut water vinegar treatment had little effect
Given that coconut water vinegar illustrated in vitro in controlling primary tumour development in CL mice,
cytotoxicity in 4T1 cells, the in vivo antitumour effect which might be explained by the non-significant down-
was validated on the 4T1-challenged mice. Mice pre- regulation of c-myc oncogene by the treatment.
treated with 0.08 (CL) and 2 (CH) mL/kg body weight Breast 4T1 cells are highly metastatic, triple-negative
of coconut water vinegar, respectively, were observed breast cancer cells and have been widely used as an aggres-
with delayed tumour formation post-4T1 challenge in a sive murine breast cancer model that represents human
dose-dependent manner. The results indicated that coco- stage 4 breast cancer (29, 30). Similar to an earlier report
nut water vinegar possessed an in vivo chemopreventive (29), metastasis of 4T1 cells was observed in the lungs,
effect on breast cancer and delayed the progression of liver and spleen of the untreated mice. Some colonies of
primary tumours in a dosage-dependent manner, which 4T1 metastatic cells were still noted in the liver of the CL-
is similar to the anti-carcinogenetic impact of unpolished treated mice. Conversely, both CL and CH were effective
rice vinegar (16). The in vitro experiment revealed that in inhibiting metastasis in the lungs and spleen. The an-
induction of apoptosis in 4T1 cells was the main mode ti-metastatic effect is potentially due to the suppressed
of cell death induced by coconut water vinegar. The expression of iCAM, VEGF and MMP9 genes, as indi-
IHC TUNEL assay result showed that the decreased tu- cated by the qRT-PCR and western blot analyses. ICAM,
mour size recorded in CH-treated mice was because of VEGF and MMP9 are useful targets in treating aggressive
10
(page number not for citation purpose)
Citation: Food & Nutrition Research 2019, 63: 1616 - http://dx.doi.org/10.29219/fnr.v63.1616
Effects of coconut water vinegar on 4T1 breast cancer cells
13. Samad A, Azlan A, Ismail A. Therapeutic effects of vinegar: a 26. Gullett NP, Ruhul Amin AR, Bayraktar S, Pezzuto JM, Shin
review. Curr Opin Food Sci 2016; 8: 56–61. DM, et al. Cancer prevention with natural compounds. Semin
14. Sun X, Hu M, Henrik M, Helen SE, Dai D, Duan W, et al. Risk Oncol 2010; 37: 258–81.
factors for oesophageal cancer in Linzhou, China: a case control 27. Nanda K, Miyoshi N, Nakamura Y, Shimoji Y, Tamura Y,
study. Asian Pacific J Cancer Prev 2003; 4: 119–24. Nishikawa Y, et al. Extract of vinegar ‘Kurosu’ from unpolished
15. Baba N, Higashi Y, Kanekura T. Japanese black vinegar ‘Izumi’ rice inhibits the proliferation of human cancer cells. J Exp Clin
inhibits the proliferation of human squamous cell carcinoma Cancer Res 2004; 23: 69–75.
cells via necroptosis. Nutr Cancer 2013; 65: 1092–7. 28. Garte SJ. The c-myc oncogene in tumor progression. Crit Rev
16. Shimoji Y, Kohno H, Nanda K, Nishikawa Y, Ohigashi H, Oncog 1993; 4: 435–49.
Uenakai K, et al. Extract of Kurosu, a vinegar from unposli- 29. Heppner GH, Miller FR, Shekhar PVM. Nontransgenic models
hed rice, inhibits azoxymethane-induced colon carcinogenesis in of breast cancer. Breast Cancer Res 2000; 2: 331–4.
male F344 rats. Nutr Cancer 2004; 49: 170–3. 30. Wenzel J, Zeisig R, Fichtner I. Inhibition of metastasis in a
17. Mimura A, Suzuki Y, Toshima Y, Yazaka S, Ohtsuki T, Ui S, murine 4T1 breast cancer model by liposomes preventing tumor
et al. Induction of apoptosis in human leukemia cells by natu- cells-platelet interactions. Clin Exp Metastasis 2010; 27: 25.
rally fermented sugar cane vinegar (kibizu) of Amami Ohshima 31. Dhaheri YA, Attoub S, Arafat K, AbuQamar S, Viallet J, Saleh
Island. BioFactors 2004; 22: 93–7. A, et al. Anti-metastatic and anti-tumor growth effects of Orig-
18. Guo XK, Wang TJ, Gu JF. Effect of esophageal cancer- and anum majorana on highly metastatic human breast cancer cells:
stomach cancer-preventing vinegar on N-nitrosoproline forma- inhibition of NFkB signaling and reduction of nitric oxide pro-
tion in the human body. World J Gastroenterol 1997; 3: 269–70. duction. Plos One 2013; 8: e68808.
19. Nishidai S, Nakamura Y, Torikai K, Yamamoto M, Ishihara N, 32. Arias-Salvatierra D, Silbergeld EK, Acosta-Saavedra LC,
Mori H, et al. Kurosu, a traditional vinegar produced from un- Calderon-Aranda ES. Role of nitric oxide produced by iNOS
polished rice, suppresses lipid peroxidation in vitro and in mouse through NF-kB pathway in migration of cerebellar granule neu-
skin. Biosci Biotechnol Biochem 2000; 64:1909–14. rons induced by lipopolysaccharide. Cell Sign 2011; 23: 425–35.
20. Mohamad N., Yeap S, Lim K, Yusof H, Beh B, Tan S, et al. 33. Yerlikaya A, Okur E, Ulukaya E. The p53-independent induc-
Antioxidant effects of pineapple vinegar in reversing of paraceta- tion of apoptosis in breast cancer cells in response to protea-
mol-induced liver damage in mice. Chinese Medicine 2015; 10: 3. some inhibitor bortezomib. Tumor Biol 2012; 33: 1385–92.
21. Yeap SK, Mohd Yusof H, Mohamad NE, Beh BK, Ho WY, Ali 34. Christina YI, Ibrahim M, Rifa’I M. Polyherbal EMSA ERITIN
NM, et al. In vivo immunomodulation and lipid peroxidation blocks nuclear factor-kapppa B (NF-kB) and proinflammatory
activities contributed to chemoprevention effects of fermented cytokines in irradiated mice. Am J Immunol 2015; 11: 17–25.
mung bean against breast cancer. Evid Based Complementary 35. Mohamad NE, Yeap SK, Lim KL, Yusof HM, Beh BK, Tan
Altern Med 2013; 2013: 708464. SW, et al. Antioxidant effects of pineapple vinegar in reversing of
22. Mohamad NE, Yeap SK, Ky H, Ho WY, Boo SY, Chua J, et al. paracetamol-induced liver damage in mice. Chin Med 2015; 10: 3.
Dietary coconut water vinegar for improvement of obesity-as- 36. Kano A. Tumor cell secretion of soluble factor(s) for specific
sociated inflammation in high-fat-diet-treated mice. Food Nutr immunosuppression. Sci Rep 2015; 5: 8913.
Res 2017;61:1368322. 37. Cha E, Graham L, Manjili MH, Bear HD. IL-7+IL-15 are su-
23. Abu N, Mohamed NE, Yeap SK, Lim KL, Akhtar MN, Zulfadli perior to IL-2 for the ex vivo expansion of 4T1 mammary carci-
AJ, et al. In vivo antitumor and antimetastatic effects of fla- noma-specific T cells with greater efficacy against tumors in vivo.
vokawain B in 4T1 breast cancer cell-challenged mice. Drug Des Breast Cancer Res Treat 2010; 122: 359–69.
Dev Ther 2015; 9: 1401–17.
24. Wright SC, Bonavida B. YAC-1 variant clones selected for resis-
tance to natural killer cytotoxic factors are also resistant to nat- *Noorjahan Banu Alitheen
ural killer cell-mediated cytotoxicity. Proc Natl Acad Sci USA Department of Cell and Molecular Biology
1983; 80: 1688–92. Faculty of Biotechnology and Biomolecular Science
25. Glade MJ. Food, nutrition, and the prevention of cancer: a Universiti Putra Malaysia
global perspective. American Institute for Cancer Research/ Serdang, Selangor 43400
World Cancer Research Fund, American Institute for Cancer Malaysia
Research, 1997. Nutrition 1999; 15: 523–6. Email: [email protected]