Cytotoxic Activity of Sponge Extract and Cancer Cell Lines from Selected Sponges  

K. Chairman , A.J.A. Ranjit Singh
Department of Advanced Zoology and Biotechnology, Sri Paramakalyani College, Manonmaniam Sundaranar University, Alwarkurichi, Tirunelveli, Tamilnadu, India-627 412.
Sri Paramakalyani College, Alwarkurichi, Tirunelveli, Tamilnadu, India - 627 412
Author    Correspondence author
Biomaterial and Biomedicine, 2013, Vol. 2, No. 1   doi: 10.5376/bb.2013.02.0001
Received: 21 Dec., 2012    Accepted: 09 Jan., 2013    Published: 19 Feb., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Chairman and Ranjit Singh, 2013, Cytotoxic Activity of Sponge Extract and Cancer Cell Lines from Selected Sponges, Biomaterial and Biomedicine, Vol 2, No.1 1-5 (doi: 10.5376/bb.2013.02.0001)


In this study, the ancancer activity exhibited by sponge fraction extracts from Aurora globostellata from Turicorin coasts. Antitumoural activities were determined by a cytotoxic assay with three different tumoural cells lines HeLa (Human cervical cancer), Raw 264.7 (Mouse leukaemic monocyte macrophage cell line) and HEK-293 (Human kidney cell line). Among the species under study, Aurora globostellata, displayed a high anticancer activity. Further to the fractionation of these crude extracts, a significant correlation was found in most fractions between the high anticancer activity and the high phenolic content. Aurora globostellata marine sponge exhibited strong cytotoxic activities against all tumoural cells.

Marine sponge; Phenolic compound; Antitumour activity; MTT assay

1. Introduction
In opposition to terrestrial natural products knowledge, the studies with marine natural products are quite young. However, despite the short time, this field has been conquering an important status among chemists and pharmacologists. Studies with marine natural products showed a variety of organic compounds derived from marine species with known and with novel biological activities. Sponges are among the most studied zoological groups by marine chemists and pharmacologists, while showing the highest rates of cytotoxic molecules. Several studies also describe antitumor activity (Osinga et al., 1998, Faulkner, 2000; Prado et al., 2004).

Natural products and their analogs or molecules derived there of comprise approximately 50% of the drugs presently used for clinical purposes. Regarding anticancer drugs, 63% of them fall into this category (Cragg and Newman, 2009; Newman and Cragg, 2007). However, and despite the high number of available drugs, there is a growing need to develop more specific agents to treat cancers, particularly chemo-resistant tumors (Lima et al., 2007). Bio-discovery is the extraction and testing of molecules for biological activity, identification of compounds with promise for further development, and research on the molecular basis for the biological activity.

Today, more than 60 % of the anticancer drugs commercially available are of natural origin (Cragg et al., 1997). The relevance of the sea as a tool to discover novel anticancer compounds was validated by the discovery, development and marketing approval of 1-beta-D-arabinofuranosylcytosine (ARA-C) (Bergmann et al., 1950). ARA-C is a basic component in the curative setting of acute myeloid leukaemia (Wolf et al., 1985). The available results clearly anticipated the potential of the marine ecosystem in cancer therapy. During the last decade about 2500 new metabolites with antiproliferative activity have been reported; about 68 of which are new marine derived anticancer chemical entities, most of them with undetermined modes of action (Mayer et al., 2003).

Ecteinascidin-743 (ET-743) (Trabectedin, Yondelis) is a tetrahydroisoquinoline alkaloid derived from the colonial tunicate Ecteinascidia turbinata (Rinehart et al., 1999), a tunicate that lives in clusters in the Caribbean and Mediterranean seas. The compound demonstrated very potent activity against a broad spectrum of tumour types in animal models (Rinehart et al., 1999). Early trial results have shown promising activity of ET-743 in the treatment of advanced soft tissue sarcoma (STS), osteosarcoma and metastatic breast cancers. It was found that ET-743 prevents the formation of P-glycoprotein, a protein associated with multidrug-resistant tumours (Zewail-Foote et al., 1999).

Kahalalide F is a depsipeptide discovered in Elysia rufescens, a marine mollusc found in Hawaii. It caused a disruption of lysosomal membranes and consequently the formation of large vacuoles. This mechanism is unique among anticancer agents and may cause increasing acidification of the intra-cellular space, a stimulatory event that initiates a pathway for apoptosis (Hamann et al., 1993). In addition, kahalaide F leads to an inhibition of erb 2 transmembrane tyrosine kinase activity and inhibits TGF–a gene expression.

Discodermolide is a polyhydroxylated lactone, isolated from the deep-sea sponge Discodermia sp. It is an immunosuppresive and cytotoxic agent (Gunasekera et al., 1990). The study of its mechanism has revealed that discodermolide was able to stabilise microtubules. In 1998, Novartis Pharma AG licensed this compound for development as a candidate agent for treatment of cancers.

Bryostatin 1 is a macrocyclic lactone isolated from the marine bryozoan Bugula neritina (Bugulidae). It has many properties including activation of T-cells, immunomodulation and stimulation of haematopoietic progenitor cells. Bryostatin 1 was found to bind to protein kinase C with high affinity, which may be the mechanistic basis for both observed anticancer and immunostimulating activities (De Vries et al., 1995).

Didemnin B is a depsipeptide isolated from the Caribbean tunicate Trididemnum solidum. It was found to display antineoplastic, antiviral and subsequently immunosuppresive activities (Rinehart et al., 1999). Mechanistically, didemnin B acts at the GTP binding protein elongation factor.

Ziconotide (Prialt) is a 25 amino acid peptide from the venom of the predatory snail Conus magnus. It acts by binding to and inhibiting presynaptic calcium channels, thereby preventing neurotransmitter release (Oliveira et al., 1985). It is licensed by Elan Pharmaceuticals as Prialt. Prialt blocks nerve impulses in a key region of the spinal cord, where pain fibres from the body connect with the nerve cells that send pain to the brain. This is why Prialt is 50 times more potent than morphine.

In the present study an attempt has been made to screen the cytotoxicity of marine sponge extracts of a Aurora globostellata.

2. Material and Methods
2.1 Sponge collection
Samples of Aurora globostellata were collected by Scuba-divers at depths of 15 m (first collection) and 7 m (second collection), on Tuticorin Gulf of Mannar, in the state of Tamilnadu. The sponge collection was made in March - 2010 and these samples were directly immersed in ethyl acetate. Samples from the second collection, in May of 2011, were immediately frozen under dry ice (-20℃). Both collections were sent to Dept. of Zoology, Sri Paramakalyani College, and Alwarkurichi. All samples were maintained at -20 ℃, and subsequently identified.

2.2. Extract preparation
The sponge was extracted in ethyl acetate four times by maceration in ethyl acetate (0.3 g/mL), over 4 days. The ethyl acetate solution was reserved each of these days. After the fourth day, the solutions were blended, filtered, concentrated in a rotary evaporator and dried in a Speed Vaccum evaporator. A portion of the solution extracted from samples collected in 2011 was partitioned against hexane (1:1, v/v) (100%), the lipid portion was removed, and then the polar fraction was dried in a SpeedVaccum evaporator. The sixteenth fraction was tested for other activities.

2.3.Cell culture and cytotoxicity evaluation
The cell lines HeLa (Human cervical cancer), Raw 264.7 (Mouse leukaemic monocyte macrophage cell line) and HEK-293 (Human kidney cell line) were obtained from American type culture collection Manassas, VA, and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine and 100 µg/mL Penicillin-Streptomycin incubated at 37℃, humidified atmosphere with 5% CO2. Cytotoxity of the crude extract II, III, A, B, C and D were measured using MTT {(3-(4, 5-dimethylthiazol-2-yl)-2, 5-dipheny- ltetrazolium bromide (Sigma, St. Louis, USA)} assay. For this, the cells were grown at a concentration of 5× 103 cells/well in 96 well plates.

After 24 h, cells were washed with fresh medium and were treated with different concentrations of crude extract (12.5 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL). The cells without the addition of crude extract were taken as control. After 24 h incubation, 100 µL of MTT (1 mg/mL) solution was added and further incubated for 4 h at 37℃. Finally 100 µL DMSO was added to solubilize the formazan salt formed and the amount of formazan salt was determined by measuring the OD at 540 nm using a GENios ® microplate reader (Tecan Austria GmbH, Austria). The relative cell viability was determined by the amount of MTT converted into formazan salt. Viability of cells was quantified as a percentage compared to that of control. The experiment was carried out in triplicate and the data were expressed as mean from these three sets of experiments.

3. Results and Discussion
In the present study, the ability of the compounds in the sixteenth fraction of the extracts of the sponge Aurora globostellata to inhibit the proliferation of human HeLa, Raw 264.7 and HEK-293 cell line was examined. Results from cell counting showed that the compound, at all the doses tested, inhibited the proliferation of cells. In addition, the HeLa, Raw 264.7 and HEK-293 cell line viability was estimated by MTT assay, and the results showed that the compounds in Aurora globostellata.

These results were expressed as percent viability and as total number of viable cells As shown in Figure 1, the extract was cytotoxic at concentrations of 12.5 g/mL and 100 g/mL. Viability decreased following a dose/time response curve. The anticancer property of cell free extracts from sponge isolates might be due to the presence of the active secondary metabolites such as alkaloids and quninine (Gordaliza, 2010). Alkaloids are microtubule interfering agents which can bind with beta tublin, thus preventing the cell from making the mitotic spindle fibres necessary to move the chromosome around as the cell divides(Solanki et al., 2008), inhibiting topoisomerase (Facompre et al., 2003), mitochondrial damage and inducing the release of cytochrome C and apoptosis inducing factor. Moreover, quinine derivatives viz., driamycin, dauno- rubicin, mitomycin C, streptonigrin and lapachol, can interfere the DNA and RNA replication and mitochondrial oxidative pathways or the formation of super oxide, peroxide and hydroxide radicals as toxic products in the cell line. Several compounds of anthro-quinone families (parimycin, trioxacarcins and gutingimycin) showed antitumor activities (Anibou et al., 2008; Gulecha and Sivakuma, 2011; Kumar et al., 2011).

Figure 1 Anti cancer activity of Aurora globostellata marine sponges

The results obtained in the present study indicated that extracts of Aurora globostellata showed good cytotoxic potential agent tumour cell lines (Figure 2). The study suggested that marine sponge extracts can be used to develop a good anticancer-agent.

Figure 2 The extracts of control and Aurora globostellata against Hela cell line

4. Conclusion
The cytotoxic nature of the compound inhibited the proliferation of Tumour cells tested. In the present study experiments were carried using HeLa, Raw-264.7 and HEK-293 cell lines. The extract of the sponge was able to inhibit the cell proliferation significantly. The present study it is concluded that the 16th and 18th fractions of the sponges Aurora globostellata and Spirastrella inconstans var. moeandrina Dendy respectively has rich bioactive compounds and that compound must be identified exactly. This will be a panacea for many pandemic problems.

Authors are thankful to Dr. A.J.A.Ranjit Singh, Principal, Sri Paramakalyani College, Alwarkurichi, Tirunelveli, Tamilnadu, lab facilities and encouragement. We thankful for supporting agency of Department of Science and Technology, Govt. of India, New Delhi.

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