Characterization, purification and phylogenetic analysis of a cytolysin from the sea anemone Heteractis magnifica of the Indian Ocean

Karthikayalu, S; Rama,; Kirubagaran, Ramalingam; Venkatesan, R.

doi:10.1590/s1678-91992010000200006

Cited by 6 publications

(7 citation statements)

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“…The sea anemone mouth is surrounded by a ring of tentacles that are armed with stinging cells (nematocysts), which are used both in defense and in capturing prey. This cytolysin from the sea anemone H. magnifica shares close homology with reported poreforming cytolysins from other sea anemones such as sticholysin I and II from Stichodactyla helianthus, actinoporin (RTX-A) from Heteractis crispa, actinoporin Or-A from Oulactis orientalis and equinatoxin II, IV and V from Actina equina (16).…”

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Purification of a 19-kDa pore-forming cytolysin from the sea anemone Heteractis magnifica

Karthikayalu¹,

Rama²,

Venkatesan³

2010

J. Venom. Anim. Toxins incl. Trop. Dis

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Pore-forming cytolysins of 19 kDa from sea anemones present a remarkable cytolytic property. In the present work, a purified 19-kDa cytolysin was obtained from the sea anemone Heteractis magnifica. The purification steps involved ammonium sulfate precipitation and subsequently desalting by dialysis against 10 mM sodium phosphate buffer (pH 7.4), followed by anion exchange chromatography in DEAESepharose® column (GE Healthcare, Sweden) and gel filtration chromatography using Sephadex® G-50 matrix (GE Healthcare, Sweden). The active fractions from the gel filtration chromatography were pooled and rechromatographed in the same column. The final active fraction showed a prominent protein band of molecular mass of 19 kDa when analyzed by SDS-PAGE. Key words:Heteractis magnifica, cytolysin, hemolysin, pore-forming toxin. Dr. Daphne Fautin (17)] was collected from Andaman Islands, India, at a depth of 5 m by scuba diving. Samples were deposited in the National Marine Repository at the National Institute of Oceanography, Goa, India. In the laboratory, the live animal was induced by osmotic thermal stress to eject the epithelial mucus that contains the toxins (6). Cnidarian toxins are stored in the cnidocytes, which are intracellular organelles that fire under pressure or osmotic variations and deeply injects the venom. Approximately 2 kg of anemone was placed in 1 L of warm distilled water (40 to 45°C) for 15 minutes with intermittent stirring to minimize tissue damage. After 15 minutes, the anemone was removed and the solution was filtered. The filtrate was stored as 100 mL aliquots in liquid nitrogen during transportation. The extract was removed from liquid nitrogen after transportation and stored at -70°C until analysis. When required, the aliquots were thawed and concentrated by lyophilization and reconstituted in phosphate buffered saline at pH 7.4. Short CommuniCationConcentration of the protein was determined by the method described by Lowry et al. (18) using bovine serum albumin (BSA) as the standard.Hemolytic activity of the toxin was measured quantitatively in terms of attenuance on human red blood cells at room temperature using Spectramax Microtiter® plate reader (Molecular devices, USA). Freshly collected human blood with heparin was centrifuged to remove the buffy coat, and the erythrocytes (RBC) obtained were washed three times in 0.85% saline and stored at 4°C. Toxins at desired concentrations were added in the first well to erythrocyte buffer (140 mM NaCl, 10 mM Tris-HCl, pH 7.4), and then serially diluted two-fold. RBCs (100 μL; D 630 = 0.5) in erythrocyte buffer were added to the toxins, and hemolysis was monitored by measuring attenuance at 630 nm for 20 minutes at room temperature. The final volume was 200 μL per well. The percentage of hemolysis was determined at the end of the assay using the following equation of Malovrh et al. (19):In which D obs was the measured attenuance in the well after 20 minutes and D max is the maximal attenuance by distilled water and D min is the minimal a...

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confidence: 65%

“…A highly active 19-kDa pore-forming cytolysin has been isolated from the mucus secretion and body homogenate of the sea anemone Heteractis magnifica (13)(14)(15)(16). The sea anemone mouth is surrounded by a ring of tentacles that are armed with stinging cells (nematocysts), which are used both in defense and in capturing prey.…”

mentioning

confidence: 99%

Purification of a 19-kDa pore-forming cytolysin from the sea anemone Heteractis magnifica

Karthikayalu¹,

Rama²,

Venkatesan³

2010

J. Venom. Anim. Toxins incl. Trop. Dis

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“…For example, a 19 kDa cytolysin was purified from the sea anemone H. magnifica using anion exchange chromatography and gel filtration (Karthikayalu et al 2010). Three lethal and hemolytic toxins were isolated from sea anemone Actinia equine.…”

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confidence: 99%

The effect of sea anemone (H. magnifica) venom on two human breast cancer lines: death by apoptosis

2013

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Venom from the sea anemone, Heteractis magnifica, has multiple biological effects including, cytotoxic, cytolytic and hemolytic activities. In this study, cytotoxicity induced by H. magnifica venom was investigated using the crystal violet assay on human breast cancer T47D and MCF7 cell lines and normal human breast 184B5 cell line. Apoptosis was also assayed via Annexin V-flourescein isothiocyanate and propidium iodide (PI) staining followed by flow cytometric analysis. Cell cycle progression and mitochondria membrane potential were studied via flow cytometry following PI and JC-1 staining respectively. H. magnifica venom induced significant reductions in viable cell numbers and increases in apoptosis in T47D and MCF7 in dose-dependent manners. A significant apoptosisrelated increase in the sub G1 peak of the cell cycle in both breast cancer cell lines was also observed. Moreover, treatment by venom cleaved caspase-8, caspase-9, and activated caspase-3. Overall, H. magnifica venom was highly cytotoxic to T47D and MCF7 human breast cancer cells, and the phenomenon could be the killing phenomenon via the death receptor-mediated and the mitochondria-mediated apoptotic pathways. Consequently, H. magnifica venom has potential for the development of a breast cancer therapeutic.

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“…Sea anemones are marine-dwelling, solitary invertebrates belonging to the phylum Cnidaria and the class Anthozoa (1). Sea anemones were named after a terrestrial flower and have a central mouth surrounded by tentacles in a symmetrical structure (2).…”

Section: Introductionmentioning

confidence: 99%

Differential susceptibilities of human lung, breast and skin cancer cell lines to killing by five sea anemone venoms

Ramezanpour¹,

Silva²,

Sanderson³

2012

J. Venom. Anim. Toxins incl. Trop. Dis

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Although sea anemones are well known for being rich sources of toxins, including cytolysins and neurotoxins, their venoms and toxins have been poorly studied. In the present study, the venoms from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor) were obtained by the milking technique, and the potential of these venoms to kill cancer cells was tested on three cell lines (A549 lung cancer, T47D breast cancer and A431 skin cancer). The total protein level in the crude extract was determined by the bicinchoninic acid (BCA) protein assay. The cytotoxicity on different cell lines was assayed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay which measures survival based on the detection of mitochondrial activity and by the crystal violet assay, which measures survival based on the ability of cells to remain adherent to microplates. The results indicate that the sea anemone venom is cytotoxic to human cancer cells. The A549 cell line was the most sensitive of the cell lines tested with a significant reduction in viability observed at 40 µg/mL. H. malu, C. adhaesivum and E. quadricolor had a significant inhibitory effect on A431 cells. Furthermore, H. malu and C. adhaesivum had a significant inhibitory effect on T47D cell line at 40 µg/mL. In conclusion, the sea anemone venoms tested have the potential to be developed as anticancer agents.

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Characterization, purification and phylogenetic analysis of a cytolysin from the sea anemone Heteractis magnifica of the Indian Ocean

Cited by 6 publications

References 24 publications

Purification of a 19-kDa pore-forming cytolysin from the sea anemone Heteractis magnifica

Purification of a 19-kDa pore-forming cytolysin from the sea anemone Heteractis magnifica

The effect of sea anemone (H. magnifica) venom on two human breast cancer lines: death by apoptosis

Differential susceptibilities of human lung, breast and skin cancer cell lines to killing by five sea anemone venoms

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