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Cytotoxic activity of eucheuma cottonii on MCF-7 human breast cancer



Studies of seaweeds are essential as these marine macro algae are the only source that produces agar, alginate and carrageenan. Eucheuma cottonii belongs to the genus Eucheuma, in the division of Rhodophyta. It is one of the important genera of carrageenophytes. It was reported to possess anticancer effects for the prevention and management of cancers. This study investigates the cytotoxic effect of crude extracts from E. cottonii on breast cancer cell line (MCF-7) in vitro. E. cottonii collected from Sabah was used for preparation of crude extracts using absolute methanol. The cytotoxic effect of the extract was determined using MTT assay whereas cell apoptosis was detected using GeneTex Enhanced Apoptotic DNA Ladder Detection Kit. Result of MTT assay shows that the growth of MCF-7 cancer cells was suppressed by the crude extracts of E. cottonii in a dose-dependent manner of IC50 at 3.5mg/mL (24h) and 0.85mg/mL (48h). The presence of fragmented DNA of cells treated with the crude extract indicated cytotoxic effect is through apoptosis. In conclusion, E. cottonii extract that exerts cytotoxic effect on the MCF-7 cancer cell line is dose dependent and via apoptosis induction. It can be a potential source of anticancer drugs from nature.
International Journal of
Current Multidisciplinary Studies
Available Online at
Vol. 1, Issue, 2, pp.69-73, july, 2015
Lee,JW*., Wang, JH., Ng, KM., Tan, CH Rabina P and Teo, SS
Department of Applied Sciences, UCSI University, No.1 JalanMenaraGading, UCSI Heights,
56000 Cheras, Kuala Lumpur, W. P. Kuala Lumpur, Malaysia
Red Seaweeds, E.cottonii,
Anticancer, MTT Assay, Apoptosis
Article history :
Received on June 22,2015
Received in revisedform, June 31, 2015
Accepted, July 15, 2015
Published July 28, 2015
In recent years, much focus has been put on finding new anticancer drugs that has lower
side effects.Seaweed contains high level of polysaccharides especially sulphated
polysaccharides which exhibit strong biological activities such as antitumor.
Eucheumacottonii, the edible marine red algae is cultivated abundantly in Sabah of East
Malaysia mainly for its kappa-carrageenan production. Recent studies have shown that E.
cottonii was tumour-suppressive via apoptosis induction. In this in-vitro study, the
anticancer effect of E. cottonii on various cell linewere evaluated. Cancer cells were
exposed to various concentrations of E. cottonii crude extract for 24 hours. The results
have shown that E. cottonii crude extracts induced cytotoxicity in various cancer cells in
dose-dependent manner, as measured in MTT viability assay. A complete cessation in
cell proliferation was observed using highest dose of extract (20.0mg/mL) for 24 hours
incubation, showing strong cytotoxic effect of E. cottonii extract. However, the extracts
of E. cottonii did not showed any effect on fibroblast, a human normal cell line. Overall,
this study has demonstrated that E. cottoniiextracts may exhibit potential anticancer
properties against various cancer cell line yet to prevent normal human cell line to be
INTRODUCTION: Cancer is a complex group of
disease that have caused major global health
problem, with significant association with death and
disability. It arises from a series of mutations, as a
result of genetic instability and environmental
factors (Al-Hajj et al., 2003). According to World
Health Organisation, the second leading cause of
death in developed countries is cancer and it is also
the three leading causes of death for adults in
developing countries (GmbH, 2009). Cancer can be
treated by surgery, radiation, chemotherapy,
hormones and immunotherapy (American Cancer
Society, 2011).
Corresponding author:
Department of Applied Sciences, UCSI University, No.1
JalanMenaraGading, UCSI Heights, 56000 Cheras, Kuala Lumpur, W. P.
Kuala Lumpur, Malaysia
Article can be accessed online on:
Copyright © Le e,JW et al ., This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution and rep roduction in any medium, provided the original work is properly cited.
However, there is no potent medicine in the existing
cancer treatments as many of these drugs can cause
side effects in many circumstances.
Various studies have been carried out to look for
alternative treatment for cancer. Researchers have
been focusing on the discovery of new compounds
derived from the natural products which have
potential anticancer properties. Antioxidant is one of
the main compounds which act to protect against
damages by free radicals and other reactive oxygen
species. It was proven that there is a positive
correlation between the amount of antioxidants in the
food diet and the lowering of cancer mortality in an
individual (Boopathy and Kathiresan, 2010). The
possible explanation for this is that the antioxidant
can cause regression of premaglinant lesions and
thus, inhibit the development of these lesions into
Natural products produced by living organisms have
Lee,JW et al., IJCMS, 2015; Vol. 1(2): 69-73.
been widely exploited by people for the
development of various products including food,
fragrances, insecticides, pigments and therapeutic
drugs (Carte, 1996). Apart from using plant
derivatives and microbial products as a source to
produce medicine, researches on marine organisms
have been increased drastically over the years to
discover new pharmaceutical agents. It has been
found that the isolated bioactive compounds from
the marine organisms have much health beneficial
effects as their biological activities have the
potential of giving better efficacy and specificity of
drugs against certain diseases. These newly isolated
compounds from the marine environment have the
ability to withstand extreme conditions in terms of
pressure, salinity and temperature, as well as having
unique structural and functional features compared
to the terrestrial organisms (Boopathy and
Kathiresan, 2010).
Among the marine organisms, seaweed is a major
source of structurally diverse bioactive compounds.
Over the years, seaweeds have been consumed in
diet for its high nutrition content. In addition, their
richness in sulfated polysaccharide which can be
found in the cell wall also results in the diverse use
of seaweed in various fields for example, cosmetics,
pharmaceutical, microbial and biotechnology
industries (Wijesekara et al., 2011). This is because
sulfated polysaccharides in seaweeds have been
discovered to exhibit biological activities such as
anticoagulant, antiviral, antioxidative, anticancer and
immunomodulating properties. These beneficial
properties of seaweed have given us a promising
future through the development of therapeutic drugs
in the biomedical area.
Many studies have proven that E. cottonii has
potential therapeutic properties due to its antioxidant
properties. In a study carried out by Fard et al.,
(2011), E. cottonii is obtained from the coastal area
of Semponia at Sabah, Malaysia. It was found that
the antioxidant-rich E. cottonii had a significant
effect on accelerating the wound healing process.
Presence of antioxidants such as fucoxanthin,
astaxanthin, carotenoid, phenolic acid, flavanoid and
tannins in ethanolic E. cottonii may be the one
responsible for the aceelerated wound healing
properties. There was another study supported this
explanation in which flavonoids like catechol,
quercitrin and myricetin were proven to cause wound
contraction and increased in the rate of epithelisation
during the process of wound healing (Yoshie et al.,
2003). Besides, a comparison between the ethanolic
and aqueous E. cottonii extracts was also carried out
by Fard et al., (2011). It was found that ethanolicE.
cottonii extract had faster wound healing process as
compared to aqueous extract that has lower
antioxidant and polyphenol activities.
Among all types of cancer, breast cancer is the
leading cause of death among the women.
Consumption of food containing antioxidant was
proven to be effective in reducing cancer incidence
caused by oxidative damages. In a study carried out
by Namvar et al., (2012), E. cottonii samples are
obtained from Kudat, the north coast of Sabah in
Malaysia. E. cottoniipolyphenol-rich extract (ECME)
was discovered to exhibit anti-proliferative and
apoptotic effect on the oestrogen-dependent MCF-7
and oestrogen-independent MB-MDA-431 breast
cancer cell lines. Besides, ECME was found to be
more potent towards oestrogen-dependent MCF-7 as
compared to oestrogen-independent MB-MDA-431.
The mechanisms of the anti-tumour activity against
MCF-7 breast cancer are via hormone modulation
and apoptosis induction without cell cycle arrest.
During hormonal regulation, the biosynthesis of
osetrogen in the cancer cells was downregulated, thus
giving it its anti-oestrogenic effect. Most importantly,
ECME does not have any cytotoxic effect on normal
Vero (African green monkey kidney) cells. Hence,
ECME provides a better alternative to treat breast
cancer and possibly to other types of cancer cells as
In this study, E. cottonii has been used to treat
various cancer cells in vitro. In the cytotoxicity test,
E.cottonii crude extracts was evaluated using methyl
thiazol tetrazolium (MTT) assay. It is a simple,
economic and reliable method to determine the
viability of the cells after being treated with certain
drugs (Shetty et al., 1996). It involves the ability of
the living cells to reduce the MTT compound which
results in colour changes.The present study aims to
determine the anticancer and cytotoxicity potential of
the crude extracts from E. cottonii.
Lee,JW et al., IJCMS, 2015; Vol. 1(2): 69-73.
reagents Potassium chloride, potassium dihydrogen
orthophosphate, disodium hydrogen phosphate,
sodium chloride, Minimum Essential Medium
premix, sodium pyruvate, sodium bicarbonate,
penicillin/streptomycin, fetal bovine serum, trypsin,
trypan blue, methanol, dimethyl sulfoxide, MTT
Samples and preparation of crude extracts from
E.cottonii Seaweed, E. cottonii samples used was
from Sabah, East Malaysia. According to Taskin et
al., (2010), seaweed samples were washed and
grinded in liquid nitrogen. A total of 10g of grinded
samples were added into 150mL methanol and left
for 24 hours at room temperature with stirring at 200
rpm. The solvent extracts were then filtered and the
filtrate was concentrated by rotary evaporation at 45
50 °C. After the evaporation process, resulting
extracts were dissolved in dimethyl sulfoxide
(DMSO) and kept in 4 °C.
Cell Culture: HeLa (Human Cervix Adeno
carcinoma), human lung carcinoma cell line (SK-
LU-1), human colon carcinoma cell line (HCT-116),
and Fibroblastwere used. HeLa cell line was
cultured in Minimum Essential Medium (MEM)
with supplemented with 5% of FBS. SK-LU-1 and
HCT-116 cell line were cultured in Eagle's minimal
essential medium (EMEM) supplemented with 10%
of FBS. Fibroblast cell line was cultured in
Dulbecco's modification of Eagle's medium
(DMEM) supplemented with 10% of FBS. T-25
flask was placed in laminar flow and the spent
cultured medium was discarded with a sterile
Pasteur pipette. A total volume of 2mL of 1xPBS
was added to the side of the T-25 flask opposite the
cells to avoid dislodging the cells. Then, the cells
were rinsed and the rinse was discarded afterwards.
A total volume of 2mL of trypsin solution was
added to the cells, ensuring the monolayer is
completely covered. The T-25 flask was incubated
for 5-10 minutes or the flask gently tilted until the
monolayer can be seen detaching from the culture
surface. Cells then observed under microscope and
resulting cells should be rounded and floating. A
total volume of 2mL of medium and cells were
dispersed gently by repeated pipetting. Subculture
was performed in a ratio of 1:4 (cell suspension:
culture medium) which 1mL of cell suspension added
with 4mL of medium to a new T-25 flask for a total
volume of 5mL. The T-25 flask was placed into CO2
incubator and incubated at 37 °C. HeLa cells in the
T-25 flask were checked under microscope daily to
monitor the cell growth.
Cell viability test with MTT [3-(4,5-Dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide] assay Blank,
positive control, negative control, solvent control,
and a range of concentrations including 0.5,
1.0,5.0,10.0,15.0,and 20 mg/mL which a total 9 set of
triplicate test were carried out in a 96 wells plate.
Cancer cells were counted by using cell quantitation
with trypan blue exclusion assay and 10,000 cells
were plated per well with total volume of 200µL of
medium except for blank set. The 96 wells plate was
then incubated in CO2incubator overnight. The next
day, spent cultured medium was removed by pipette
it out from each well. After that, 200µL inhibitors or
crude extracts of E.cottoniiwere added to each well
accordingly. The 96 wells plate was then incubated in
CO2incubator for 24 hours. After 24 hours, 50µL of
MTT reagent was added to each well and the
aluminium foil wrapped 96 wells plate was again
incubated in CO2incubator for another 4 hours until
purple precipitate is visible. Next, all the solution in
each well was discarded and 200µL of absolute
DMSO was added into each well. Absorbance read at
570nm with a reference filter of 620nm by using
Elisa reader and absorbance reading recorded.
RESULTS AND DISCUSSION: Cytotoxicity assay
like MTT test is often used in the development of
new drugs (Langdon, 2004). The effect of a potential
cytotoxic agent on a population of targeted cells was
determined and the final percentage of cell viability
was measured. In this study, HCT-116 cells at
exponential phase were treated with
Eucheumacottonii crude extracts and the suitable
duration of exposure to this cytotoxic agent was
Based on this study, the in-vitro cytotoxicity of E.
cottonii was determined using 3-(4,5-di
methylthiasol-2-yl)-2, 4,-di phenyl tetrazolium
bromide (MTT) assay. It is a colorimetric test which
commonly used to measure cell viability based on the
ability of mitochondria in the viable cells to produce
Lee,JW et al., IJCMS, 2015; Vol. 1(2): 69-73.
dehydrogenase enzyme (Yedjou and Tchounwou,
2007). MTT is a water soluble tetrazolium salt
which turns yellowish when dissolves in solution.
When MTT was added to the cells, the
dehydrogenase enzyme reduces the soluble yellow
tetrazolium salt to insoluble purple formazan
crystals. Crystals can besolubilised using organic
solvent like dimethyl sulfoxide (DMSO) that was
used in this study and then to be quantitated
spectrometrically using ELISA reader. Thus, there is
a linear relationship between the concentrations of
purple crystals formed with the number of cells that
are metabolically active. The cytotoxicity of E.
cottonii can be studied when comparison was made
between the amount of formazan crystals produced
in cells treated with E. cottoniicrude extracts and
the untreated control cells. This enables the
effectiveness of the seaweed crude extract to be
determined using a dose-response curve (Taskin et
al., 2010).
Mitochondria are the site where intracellular
reduction of tetrazolium salts by metabolically
active dehydrogenase enzymes are carried out.
During this reaction, reducing agents such as NADH
and NADPH are also generated at the same time
(Selvi et al., 2011). However, dead cells do not have
the ability to cleave the tetrazolium ring, hence,
colour change does not occur. This is because
dehydrogenase enzyme only present in the
mitochondria of live cells, not dead cells. In this
study, untreated cells were used as negative control
whereas positive control was prepared by using 50%
DMSO to kill the cancer cells.
When MTT reagent was added into the culture
medium in 96-well plate, the appearance of the
medium was yellowish in colour. After the 4 hours
incubation, purple precipitate can be seen at the
bottom of the well, together with the yellowish
solution. After that, DMSO was used to solubilise
the purple formazan crystals that were released from
the cytosol of the cells, giving rise to the purple
In MTT assay, different concentrations of E. cottonii
crude extract were used to treat with various cancer
cells and fibroblast cells. The absorbance readings at
570nm with reference filter at 630nm for 24 hours
incubation are shown in table 1 and graph was
plotted as shown in figure 1.
In this study, MTT cell viability test shown that
various concentration of the crude extracts from E.
cottonii inhibited the growth of the various cancer
cells for 24 hours incubated with crude extracts from
E. cottonii respectively as results shown in Table 1
and Figure 1. Cells viability percentage decreased
gradually when the doses are increasing for 24 hours.
On the other hands, cell viability of fibroblast did not
decrease even though the highest dose of 20mg/mL
was applied. In Figure 1, fibroblast cells still growing
and did not shown any decreasing sign after 24 hours
treated with E. Cottonii extracts. Consequently, the
percentage of various cancer cells viability decreased
in a dose dependent manner yet to prevent fibroblast,
human normal cell lines from elimination.
When metabolic events lead to apoptosis or necrosis,
it showed reduction in cell viability in MTT
proliferation assay(Selvi et al., 2011). The linear
relationship between cell number and signal
produced is established, thus allowing an accurate
quantification of changes in the rate of cell
HeLa cells
Positive control
Negative control
Solvent control
0.5 mg/ml
1.0 mg/ml
5.0 mg/ml
10.0 mg/ml
15.0 mg/ml
20.0 mg/ml
Figure 1 Graph of cell viability percentage
Lee,JW et al., IJCMS, 2015; Vol. 1(2): 69-73.
proliferation.Triplicate test was carried out in this
MTT in the study. Standard deviation is important in
determining the dispersion of the triplicate readings
from its mean value. Therefore, outliers that were
found among the absorbance readings were removed
to ensure consistency of the results was established.
Inaccurate cell seeding and inaccurate reagent
pipetting might be the possible causes of the poor
consistency of replicates. A repeating pipettor would
be ideal to increase the accuracy of the cell seeding
and reagent pipetting.
Cancer has a scourge on the human population for
many years. Although, numerous advances have
been made in prevention, diagnosis and treatment of
the disease, it still continues to torment mankind
(Hanahanand Weinberg, 2000). There are limited
research published for the anti-cancer effect of E.
cottonii seaweed but the potential of other seaweed
species as an anti-tumor, anti-inflammatory have
been well known. In previous studies, phenol-rich
extracts of E. cottonii have suppressed and
prevented breast cancer tumour. With this
background knowledge as supportive evidence, this
study revealed the in vitro anticancer activity of the
crude extracts from E. cottonii (Namvar et al.,
2012). Thus, from the MTT analysis on the various
cancer cells line revealed the crude extracts from the
E. cottonii could effectively reduce the cancer cell
proliferation. The anticancer effect of the E. cottonii
may be due to the presence of the secondary
metabolite compounds such as phenol or antioxidant
contents which yet to be identified (Selvi et al.,
2011). Those foods rich in antioxidant have been
shown to play an essential role in the prevention of
some human diseases especially cancer.
CONCLUSION: In conclusion, this study has
demonstrated that E. cottonii red seaweed has good
antiproliferative properties and strong cytotoxicity
characteristic on HeLa cells, HCT-116 human colon
carcinoma cell line and SK-LU-1 human lung
carcinoma cell linein vitro. Although the specific
bioactive compounds which responsible for the
anticancer properties of E. cottonii have yet to be
discovered, E. cottonii has shown its potential of
contributing in the development of novel anticancer
drug based on this study. In future studies, different
assay can be carried out or the same methods can be
done on other cancer cell line followed by in vivo test
in order to prove the anticancer effect of E. cottonii
on animal model.
Acknowledgement: We wish to thankUCSI
University for the supports throughout the projects.
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For elucidation of the entity of antitumour activity of marine algae, 17 kinds of polysaccharide preparations of seaweed origin and 24 kinds of lipid fractions extracted from various seaweeds were selected as experimental material in addition to pectin. Mice implanted with Ehrlich carcinoma and Meth A fibrosarcoma were used as assay animal. Appreciable inhibition of tumour growth was found for fucoidan preparations form Undaria pinnatifida and Sargassum ringgoldianum, carrageenans and porphyran, being 51.7-54.5, 78.1, 40.1-54.0, and 45.3-58.4 %, respectively. The antitumour activities of lipid fractions against Meth A fibrosarcoma were found to be 36.1-47.1% with all the 3 fractions from S. ringgoldianum, and those of glycolipid and phospholipid fractions from L. angustata were as high as 45.9 and 58.0%, the latter of which was obtained from nearly 1/6 amount of injection. Phospholipid fraction from P. yezoensis showed very high activity such as 64.0% which was the value at the injection of nearly the same amounts as above, and it was the highest value among all the fractions tested in this work.
The edible red seaweed Eucheuma cottonii is abundantly cultivated for carrageenan production. This study investigated the effects of dietary E. cottonii polyphenol-rich extract (ECME) on breast cancer. In vitro assays showed that ECME was antiproliferative against oestrogen-dependent MCF-7 and oestrogen-independent MB-MDA-231 human breast-cancer cells (IC50 values of 20 and 42 μg/ml, respectively) but was non-toxic to normal cell lines. The ECME (150 and 300 mg/kg BW) was fed to female rats and, after 4 weeks, rat mammary tumour was induced using LA7 cells (inoculated subcutaneously). The ECME inhibited tumour development and erythrocyte lipid peroxidation in the cancer-induced rats, dose-dependently. It showed anti-oestrogenic effects on the rat estrous cycle and serum hormone levels. Electron microscopy and histopathology observations confirmed apoptosis in the rat mammary tumours. The polyphenol-rich ECME was tumour-suppressive via apoptosis induction, downregulating the endogenous oestrogen biosynthesis, and improving antioxidative status in the rats.