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Cytotoxic effect of sea anemone pore-forming toxin on K562 chronic myeloid leukemia cells.

ABSTRACT: Chronic myelogenous leukemia (CML) is one of prevalent cancer worldwide. In spite of various designed drugs, chemoresistance remains the main obstacle in cancer cure. Therefore, developing novel strategy for treatment of CML is an urgent need. Fragaceatoxin C (FraC) is novel protein toxin from a sea anemone called actinia fragacea with great impacts against cells by pore formation and disturbing cell membrane integrity. The aim of this study was evaluation of FraC toxin toxicity against K562. The bacteria cells harboring expression‌‌‌‌‌‌‌ vector of FraC were induced by IPTG and purified by Ni²⁺-NTA sepharose affinity chromatography. Then, purified toxin activity was evaluated using RBC hemolytic test. Eventually, evaluation of FraC cytotoxicity and apoptosis were performed using MTT and flow cytometery assays, respectively. Our results revealed that FraC toxin decreased K562 cells viability in a dose- and time-dependent manner with a whole destroy of cancer cells at 35.00 µg mL^-1 after 72 hr. Furthermore, flow cytometery analysis indicated that FraC toxin enhanced necrosis along with apoptosis in K562 cells in a dose dependent manner. We speculated that FraC toxin could be considered as a novel candidate for cancer cell researches and treatments provided that it should be turned into a specific agent by engineering and directing to cancer cell membrane.

SUBMITTER: Abdoli V

PROVIDER: S-EPMC9010839 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Cytotoxic effect of sea anemone pore-forming toxin on K562 chronic myeloid leukemia cells.

Abdoli Vali V Sarkhosh-Inanlou Roya R Delirezh Nowruz N Aghazadeh Safiyeh S Shaykh-Baygloo Nima N Imani Mehdi M

Veterinary research forum : an international quarterly journal 20211215 4

Chronic myelogenous leukemia (CML) is one of prevalent cancer worldwide. In spite of various designed drugs, chemoresistance remains the main obstacle in cancer cure. Therefore, developing novel strategy for treatment of CML is an urgent need. Fragaceatoxin C (FraC) is novel protein toxin from a sea anemone called <i>actinia fragacea</i> with great impacts against cells by pore formation and disturbing cell membrane integrity. The aim of this study was evaluation of FraC toxin toxicity against K ...[more]

PMID: 35529825

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Project description:BackgroundPore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17.MethodsCytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18.ResultsThe amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 μg), goat (HU50 = 13.2 ± 0.3 μg), rabbit (HU50 = 34.7 ± 0.5 μg), and human (HU50 = 25.6 ± 0.6 μg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 μg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.

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Cytotoxic effect of sea anemone pore-forming toxin on K562 chronic myeloid leukemia cells.

Publications

Cytotoxic effect of sea anemone pore-forming toxin on K562 chronic myeloid leukemia cells.

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