Hemolytic, Anticancer and Antigiardial Activity of Palythoa Caribaeorum Venom

Lazcano-Pérez et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2018) 24:12 https://doi.org/10.1186/s40409-018-0149-8 RESEARCH Open Access Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom Fernando Lazcano-Pérez1, Ariana Zavala-Moreno1, Yadira Rufino-González2, Martha Ponce-Macotela2, Alejandro García-Arredondo3, Miguel Cuevas-Cruz1, Saúl Gómez-Manzo4, Jaime Marcial-Quino5, Barbarín Arreguín-Lozano and Roberto Arreguín-Espinosa1* Abstract Background: Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. Methods: The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. Results: P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. Conclusion: These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer. Keywords: Cnidarian, Palythoa caribaeorum, Cytotoxin, Antitumoral effect, Giardiasis Background peptides and proteins that together cause the paralysis The phylum Cnidaria comprises approximately 11,000 and envenomation of their prey or predator [4, 5]. species classified into seven classes (Anthozoa, Scypho- Venoms isolated from almost all classes of cnidarians zoa, Cubozoa, Staurozoa, Polypodiozoa, Myxozoa and have been found to be cytotoxic in several cellular or Hydrozoa) [1]. All of them are considered to be toxic animal models [6]. Among the best known cytotoxic [2]. Moreover, some of them have been reported to be venoms are the Portuguese man-of-war hydrozoan Phy- capable of causing severe intoxication by stinging with salia physalis, the box jellyfish Chironex fleckeri, the their specialized organelles called nematocysts [3]. Ex- jellyfish Pelagia noctiluca, the fire coral Millepora com- tracts of cnidarian tissues have been found to contain a planata and many sea anemones extracts [7–14]. Due to complex mixture of low molecular weight compounds, the wide range of biological activities of these venoms, many substances isolated from them, especially those de- rived from sea anemones, have served as useful molecu- * Correspondence: [email protected] lar models and probes in biomedical research [15]. 1Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, Av. Universidad 3000, Ciudad However, the antimicrobial activity of such extracts has Universitaria, C.P. 04510. Apdo, Postal 70250 Mexico City, Mexico been little explored. A few reports can be found in the Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lazcano-Pérez et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2018) 24:12 Page 2 of 7 literature about the antiparasitic and antibacterial prop- The solution was then centrifuged twice at 70,000 g for erties of some cnidarians and even an antimicrobial pep- 15 min at 4 °C, lyophilized, and stored at − 70 °C until tide isolated from Aurelia aurita has been sequenced use. [16, 17]. Zoanthids (order Zoantharia, class Anthozoa) are or- Hemolytic activity assay ganisms commonly found in shallow zones of coral reefs. The hemolytic assay was performed as described by Rot- This group of cnidarians has not been extensively stud- tini et al. [23] with some modifications. Human erythro- ied as other cnidarians such as sea anemones or jellyfish. cyte suspension was prepared from fresh blood from a Some biochemical and toxicological research on healthy donor. Blood was collected in a flask with Als- zoanthids have proved that they possess compounds ever’s solution buffer (pH 6.4) containing dextrose (0. with biological activity. For instance, the existence of 116 M), NaCl (0.071 M), sodium citrate (0.027 M) and palytoxin, one of the most potent marine toxins known citric acid (0.002 M). The suspension was centrifuged at to man and first isolated on a zoanthid of the gender 2500 rpm for 5 min at 4 °C and the supernatant was Palythoa, later discovered to be synthesized by dinofla- decanted. This step was repeated three times and the gellates [18, 19]. Besides palytoxin, not many studies on final pellet was resuspended in Alsever’s buffer. Erythro- the biological activity of zoanthid venoms or toxins have cytes were incubated at two temperatures 37 °C and 60 ° been characterized to date. An extract of their soft tis- C for 30 min in the presence of different venom concen- sues was tested for antibacterial activity and it was found trations ranging from 1 to 10 mg/mL. Immediately after that it inhibits Escherichia coli and Staphylococcus aur- incubation, the samples were centrifuged at 2500 rpm eus in 97.7 and 100%, respectively [20]. More recently, P. for 5 min at 4 °C and the optical density of supernatant caribaeorum extracts were found to have antioxidant ef- was measured using a spectrophotometer at 415 nm. fects and cytotoxic activities [21]. The results were normalized to 100% hemolysis by dilut- According to Suput [15], an assessment of the ing the erythrocytes in deionized water and adjusting the pharmacological actions of cnidarian venoms and crude absorbance A415 to 0.9 when total lysis occurred. extracts is still missing due to the fact that several types of toxins coexist in the same venom. Therefore, it would Phospholipase A2 assay be important to know not only the effect of a particular Phospholipase A2 (PLA2) activity of the aqueous extract toxin but the total effect of the whole venom in vitro was determined using a secretory PLA2 colorimetric and in vivo. Hence, the aim of the present work is to assay kit (Cayman Chemical, USA). This assay uses the characterize some pharmacological aspects of Palythoa 1,2-dithio analogue of diheptanoyl phosphatidylcholine caribaeorum venom in terms of hemolytic, antiparasitic as substrate. Free thiols generated upon hydrolysis of the and anticancer activities in order to use this organism as thioester bond at the sn-2 position by PLA2 were de- a source of new compounds with potential use as candi- tected using DTNB [5,5′-dithio-bis-(2-nitrobenzoic acid) date drugs. ]. Color changes were monitored by a Benchmark Plus microplate spectrophotometer at 414 nm, sampling Methods every minute for 10 min. As reference for PLA2 activity, Laboratory animals 10 μL (10 μg) of bee venom PLA2 was used as control. All experiments were performed in accordance with the PLA2 activity was expressed in μmol of hydrolyzed phos- Official Standard NOM-062-ZOO-1999 for the produc- phatidylcholine per minute per mg of protein (n = 3). tion, care, and use of laboratory animals. The care and use of the animals was approved by the Bioethics Com- Isolated rat aortic ring assay mittee of the School of Medicine, UAQ. Male Wistar rats (275–325 g) were anesthetized with chloroform, sacrificed by decapitation and the descend- Venom extraction ing thoracic aorta was removed and placed in ice-cold, P. caribaeorum organisms were collected by free diving oxygenated Krebs-Henseleit solution (126.8 mM NaCl, in La Gallega coral reef in Veracruz, México. The crude 5.9 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM extract was obtained according to the method described KH2PO4, 30 mM NaHCO3, and 5 mM D-glucose, pH 7. elsewhere [22]. Briefly, the organisms were carefully sep- 4) and immediately flushed with Krebs-Henseleit solu- arated from the rocks using a chisel and a hammer. In tion to prevent intravascular clot formation. The aorta the laboratory, the material was cleaned from remaining was dissected free of adipose and connective tissue and rock and soaked in water to eliminate superficial mucus. cut into 4 to 5-mm rings. The aortic rings were mounted In order to extract nematocyst venom, the organisms between stainless steel hooks and suspended in 7-mL of were carefully squeezed in deionized water

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