Abstract: 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.
INTRODUCTION
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., 2004).
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 primers:
| • | 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 (Shirora, 1966).
| Fig. 1: | 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).
| Table 1: | Characteristics of microalgal strains isolated from Xuanthuy National Park |
| -: 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 Table 3.
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.
| Table 2: | Activity of extracts of microalgal strains against KB cells |
| Table 3: | Activity of extracts of microalgal strains against three cancer cell lines |
CONCLUSION
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.
ACKNOWLEDGMENTS
This research was supported by the Ministry of Education and Training, Vietnam through Hanoi National University of Education (Project No. B2009-17-169 TD).