Isolating and Screening Mangrove Microalgae for Anticancer Activity
Microalgae are valuable source of many unique biologically active compounds including anticancer compounds. In this study, sixteen microalgal strains were successfully isolated from mangrove in Xuanthuy National Park, Namdinh, Vietnam. Relative identification for each train was obtained based on morphological properties and 18 S rDNA sequence analysis. Culture extracts of these strains were tested against KB (human epidermal carcinoma) cell line. Ankistrodesmus gracilis VACC-010 and Amphiprora alata VACC-007 showed strongest inhibition with their IC50 values of 26.50 and 29.82 μg mL-1, respectively. Ankistrodesmus gracilis VACC-010 was also significantly effective against HepG2 (hepatocellular carcinoma) cell line (IC50 values of 9.64 μg mL-1), suggesting a potential source of anticancer compounds. To our knowledge, this is the first report on the anticancer activity of this strain as well as the first screening of mangrove microalgae for biologically active compounds, providing a new lead to the characterization and development of promising anticancer drugs.
Received: March 14, 2011;
Accepted: July 26, 2011;
Published: September 27, 2011
Microalgae are a highly diversified group of microorganisms, which are mostly
unicellular, colorful, photoautotrophic and constitute major oceanic as well
as freshwater primary producers (Olaizola, 2003). They
have been utilized by man for hundreds of years in various fields ranging from
human and animal nutrition, cosmetics to therapeutic purposes. They do possess
high-value compounds such as carotenoids, polyunsaturated fatty acids, vitamins
and many biologically active compounds (Spolaore et al.,
2005). Previous investigations of microalgae have shown that they are promising
sources for a wide range of novel biologically active molecules with antibacterial,
antiviral, antifungal and anticancer activities (Ghasemi
et al., 2007). As microalgae are assumed to be easier to culture
commercially than macroalgae, they have the potential to produce those compounds
which are difficult to synthesize (Borowitzka, 1995).
Approximately 60% of new drugs for cancer treatment are derived from natural
sources. Many of them have been found in cyanobacteria, which used to be considered
as blue-green algae, such as calothrixin A and B (Calothrix spp.), apratoxin
A, curacin-A (Lyngbya majuscula), largazole (Symploca sp.), borophycin
(Nostoc linckia, N. spongiaeforme var. tenue) (Boopathy
and Kathiresan, 2010; Baharum et al., 2010;
Vignesh et al., 2011). Many compounds are active
in either killing the cancer cells by blocking cancer cell growth and inducing
apoptosis or affecting the cell signaling through the activation of protein
kinase family members (Borowitzka, 1995; Khorshid
et al., 2011). They have unprecedented structures thus can be the
potential for the development of new classes of drug agents. However, other
microalgal groups have gained increasing interest and become promising sources
for exploration. Mechanism based screening for potential anticancer activity
including protein kinase C, protein tyrosine kinase and inosine monophosphate
dehydrogenase assays has found a range of potential candidates from various
microalgae such as Rhodophyta, Chlorophyta, Phaeophyta, Chrysophyta and Cryptophyta
(Gerwick et al., 1994). Noda
et al. (1996) purified and identified a glycoprotein from the culture
media of Chlorella vulgaris with a β-1, 6-D-galactopyranose backbone
and 15 amino acid sequence as DVGEAFPTVVDALVA at the NH2-terminus,
which was considered necessary for the antitumor activity. The vitamin extracts
of 7 chlorophyte strains were proved to be potential chemopreventive agents
by inducing the activity of detoxifying enzyme glutathione-S-transferase in
many tissues of tumorous mice (El-Baz et al., 2002).
Recent survey of nearly two hundred microalgal strains, resulted in ten chlorophytes
from three genera Desmococcus, Chlorella and Scenedesmus
with high antimicrobial activity and effective against some tumour cell lines
such as MCF7 (human breast adenocarcinoma), CEM (human lymphoblastoid leukaemia)
and G361 (human malignant melanoma) (Ordog et al.,
Mangroves are one of the most productive ecosystems, a rich source in biodiversity
including phytoplankton and have long been used in traditional medicine for
many diseases (Datta et al., 2011). Many mangrove
plants were reported to be good sources of anticancer drugs (Boopathy
and Kathiresan, 2010). In an attempt to search for biologically active compounds
as potential anticancer drug agents in various microalgal species from mangrove,
this research focused on the isolation of various microalgal strains from the
mangrove of Xuanthuy National Park, Namdinh, Vietnam, their identification and
laboratory culture and crude extraction thereafter for assays against several
cancer cell lines.
MATERIALS AND METHODS
Selection, isolation and identification of microalgal strains: Samples
were collected from different sites of mangrove in Xuanthuy National Park, Namdinh,
Vietnam from March to October, 2010 and cultured in 10 mL jars of f/2 medium.
Microalgal strains were isolated to a unialgal state using micropipettes and
agar plates by the procedure according to Shirai et al.
(1989). Each strain was taken picture under 400-fold OLYMPUS CX41 microscopy.
Biomass culture for anticancer activity assays was carried out in 500 mL conical
flasks and then in 4 L flat-bottom round flasks at room temperature with illumination
by neon light (Philips daylight tubes) of 3000-4000 lux on 10: 14 h light: dark
cycles. Chlorophytes were grown in BBM, C, BG11 media and the other strains
in f/2, ASW, ESM media (Kasai et al., 2009).
Total DNA was extracted and PCR amplification was performed according to the
method described by Fawley and Fawley (2004) using following
||Forward primers 2F: (2-21) 5-ATCTGGTTGATCCTGCCAGT-3
or 1315 F: 5-CGATAAGGAACGAGACCTT-3
||Reverse primer 1794R: (1794-1775) 5-GATCCTTCCGCAGGTTCACC-3
PCR products were directly sequenced in an ABM Prism 3100-Avant Sequencer. The obtained sequences were analyzed using BLASTn tool to get the relative identification of each algal species.
Preparation of microalgal extracts: Algal samples were harvested in
early stationery phase (around 11-12th day of culture) by continuous centrifugation
at 10000 rpm at 4°C in 15 min and extracted thereafter with 10 mL of methanol/chloroform
(1:1, v/v). The extracts were concentrated under vacuum to give residues and
then stored at -20°C until analyzed.
Anticancer assay: Microalgal extracts were tested against KB (human
epidermic carcinoma) and then against HepG2 (hepatocellular carcinoma), SK-LU-1
(human lung carcinoma) and MCF-7 (human breast carcinoma) cell lines from ATCC
(American Type Culture Collection) using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) assay (Scudiero et al., 1988).
Cell lines were cultured in RPMI 1640 medium supplemented with 10% Fetal Bovine
Serum (FBS) in standard condition, steriled with 5% CO2 at 37°C,
98% humidity and harvested at the log phase for assays. Two hundred microliter
volumes of cells at the concentration of 3x104 cells mL-1
were inoculated into a 96-well plate in RPMI 1640 medium. Microalgal extracts
were applied at final concentrations of 128, 32, 8, 2 and 0.5 μg mL-1
and cultures were incubated for 3 days at 37°C with 5% CO2. Then
50 μL of MTT prepared at the concentration of 1 mg mL-1 in FBS
was added to microculture wells. After 4 hours incubation, 250 μL supernatant
were removed from each well and 100 μL of DMSO was added and mixed thoroughly.
Absorbance was measured at 540 nm in a Genios TECAN spectrophotometer. IC50
value (extract concentrations resulting in a 50% inhibition of growth) was calculated
based on the percentage of growth:
RESULTS AND DISCUSSION
Isolation and identification of microalgal strains: Fifteen microalgal
strains (Fig. 1) belonging to 11 genera including 6 chlorophytes,
8 diatoms and 1 eustigmatophyte were selected and isolated to a unialgal state
according to standard literature procedures based on morphological properties
||Microscopic morphology of microalgal strains isolated from
mangrove in Xuanthuy National Park
Sequence analysis and alignment with sequences on NCBI database gave the positive
identity for almost all strains. Data were shown in Table 1
together with short description of their morphology.
Isolated microalgal strains were cultured in several media: BBM, C and BG11
for chlorophytes and f/2, ESM and ASW for the other. The growth of all strains
was promoted well in these media, especially in BBM and f/2 (data not shown).
|| Characteristics of microalgal strains isolated from Xuanthuy
|-: not determined
BBM and f/2 were used for biomass culture of chlorophytes and the other strains,
respectively for anticancer activity assays. All the glassware, pipettes and
forceps that are used in isolations and culture work are sterilized to prevent
bacteria contamination and interference to following activity assays. This is
to ensure that it is the algae which produce the activity not the associated
bacteria or fungi.
Anticancer activity of cultured microalgal strains: Screening of crude
extracts plays an important role in the procedure of finding and developing
new drugs. Cytotoxicity tests against various cancer cell lines are the most
common screening methods for anticancer compounds (Umamaheswari
and Govindan, 2007). Extracts of fifteen microalgal strains were evaluated
for cytotoxicity against KB cells using MTT based assay. The result is shown
in Table 2.
Fifteen tested strains showed various activities against KB cell growth. Some
strains of Scenedesmus sp. were reported to have antibacterial activity
and inhibition against the development of echinoderm eggs (Murakami
et al., 1988) as well as activity against some tumour cell lines
(Ordog et al., 2004) but the isolated strains
in this study had no clear activity. The activity of Chlorella strains
in et al., this study was also at low level. Chlorella vulgaris
was shown to have chemopreventive effect in induced liver cancer and breast
cancer rats and possess a glycoprotein with antitumour effects (Sulaiman2006;
Amin, 2009). The Chlorella vulgaris strain in
this study had some inhibition to KB cells but at low level. Previous report
emphasized the importance of strain selection as activity varied between strains
of the same species (Ordog et al., 2004). Ankistrodesmus
gracilis VACC-010 and Amphiprora alata VACC-007 exhibited strongest
inhibition to KB cells (26.50 and 29.82 μg mL-1, respectively)
and thus were subjected to test against HepG2, SK-LU-1 and MCF7 cancer cell
lines. Inhibitory concentration (IC50) of each strain was shown in
Amphiprora alata VACC-007 had no remarkable activity against these cell lines while Ankistrodesmus gracilis VACC-010 inhibited them all with the strongest activity against hepatic cancer cell line (HepG2) at the concentration of 9.64 μg mL-1. The result suggested that Ankistrodesmus gracilis VACC-010 contain useful biological compounds and need further characterization. As they are relatively easily grown in mass culture with BBM medium, they can provide a potential source for anticancer pharmaceutical application.
|| Activity of extracts of microalgal strains against KB cells
||Activity of extracts of microalgal strains against three cancer
In this study, fifteen microalgal strains were successfully isolated and characterized from mangrove in Xuanthuy National Park. Although, the extent of screening for anticancer activity is still modest, a potential candidate, Ankistrodesmus gracilis VACC-010 was found. It has effective inhibition against two tested cell lines. This is the first report on the anticancer activity of this strain as well as the first screening of mangrove microalgae for biologically active compounds. More diverse pharmacological evaluation of algal extracts and isolation of active compounds from Ankistrodesmus gracilis VACC-010 are underway.
This research was supported by the Ministry of Education and Training, Vietnam through Hanoi National University of Education (Project No. B2009-17-169 TD).
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