DOI:10.31557/APJCP.2020.21.8.2423 Cytotoxicity and Oxidative Properties of Philippine Spider Venom RESEARCH ARTICLE Editorial Process: Submission:06/30/2020 Acceptance:08/20/2020 The Venom of Philippine Tarantula (Theraphosidae) Contains Peptides with Pro-Oxidative and Nitrosative-Dependent Cytotoxic Activities against Breast Cancer Cells (MCF-7) In Vitro Simon Miguel M Lopez1, Jeremey S Aguilar1, Jerene Bashia B Fernandez1, Angelic Gayle J Lao1, Mitzi Rain R Estrella1, Mark Kevin P Devanadera1,2, Anna Beatriz R Mayor2,3, Leonardo A Guevarra Jr1,2, Myla R Santiago-Bautista1,2,3*, Olga M Nuñeza4, Librado A Santiago1,2,3 Abstract Background: Breast cancer is a multifactorial disease that affects women worldwide. Its progression is likely to be executed by oxidative stress wherein elevated levels of reactive oxygen and nitrogen species drive several breast cancer pathologies. Spider venom contains various pharmacological peptides which exhibit selective activity to abnormal expression of ion channels on cancer cell surface which can confer potent anti-cancer activities against this disease. Methods: Venom was extracted from a Philippine tarantula by electrostimulation and fractionated by reverse phase-high performance liquid chromatography (RP-HPLC). Venom fractions were collected and used for in vitro analyses such as cellular toxicity, morphological assessment, and oxidative stress levels. Results: The fractionation of crude spider venom generated several peaks which were predominantly detected spectrophotometrically and colorimetrically as peptides. Treatment of MCF-7 cell line of selected spider venom peptides induced production of several endogenous radicals such as hydroxyl radicals (•OH), nitric oxide radicals (•NO), superoxide anion radicals (•O2−) and lipid peroxides via malondialdehyde (MDA) reaction, which is comparable with the scavenging effects afforded by 400 µg/mL vitamin E and L-cysteine (p<0.05). Concomitantly, the free radicals produced decrease the mitochondrial membrane potential and metabolic activity as detected by rhodamine 123 and tetrazolium dye respectively (p>0.05). This is manifested by cytotoxicity in MCF-7 cells as seen by increase in membrane blebbing, cellular detachment, caspase activity and nuclear fragmentation. Conclusion: These data suggest that the Philippine tarantula venom contains peptide constituents exhibiting pro-oxidative and nitrosative-dependent cytotoxic activities against MCF-7 cells and can indicate mechanistic insights to further explore its potential application as prooxidants in cancer therapy. Keywords: Philippine tarantula- spider venom- cytotoxicity- MCF-7- oxidative stress Asian Pac J Cancer Prev, 21 (8), 2423-2430 Introduction cell proliferation, differentiation, apoptosis and genomic stability (Dexheimer, 2013; Dumitrescu and Cotrala, 2005; Breast cancer is a heterogeneous and multifactorial Wu et al., 2008). disease that affects mainly women and ranks second Likewise, reactive oxygen/nitrogen species (ROS/ with respect to major cancer deaths worldwide (Pinkert RNS) are group of free radicals, ions, and molecules et al., 2018). Moreover, several epidemiological factors containing singular and unpaired electrons which exhibits have been associated with breast cancer especially highly combative processes in the cells. They are broadly exposure to ionizing radiation, tobacco smoking, alcohol considered into oxygen and nitrogen-free radicals and consumption, and hormonal or oral contraceptives which non-radicals that exhibit normal cellular proliferations, are well-known source of free radicals that could induce communication, and survival at physiological levels several mutations and impairment of the DNA repair (Kumari et al., 2018). However, internal and external system such as double strand breaks (DSBs) that could perturbations and insults trigger elevated levels of eventually harbor counterbalance and irregularities to ROS/RNS that confer oxidative stress (OS) which 1Department of Biochemistry, Faculty of Pharmacy, University of Santo Tomas, Manila, Philippines. 2Research Center for Natural and Applied Sciences, University of Santo Tomas, Manila Philippines. 3The Graduate School, University of Santo Tomas Manila Philippines. 4Department of Biological Sciences, College of Science and Mathematics, Mindanao State University – Iligan Institute of Technology, Iligan City, Philippines. *For Correspondence: [email protected] Asian Pacific Journal of Cancer Prevention, Vol 21 2423 Simon Miguel M. Lopez et al is one of the known pathological causes of breast The lyophilized crude venom was diluted with 195 cancer progression (Hecht et al., 2016). Interestingly, μL of 0.1% trifluoroacetic acid (TFA) in distilled water mitochondria are a prime source of ROS/RNS wherein (dH2O) and fractionated using Waters Alliance e2695 dysfunction and altered morphology can drive excessive RP-HPLC system in a 0-95% gradient elution system with ROS formation that could exacerbate OS that may endure a flow rate of 1 mL/min for 100 minutes using C18 column in a self-perpetuating cycle of breast cancer activity (Jezek as stationary phase and 0.1% TFA in dH2O and 0.1% TFA et al., 2018). in 90% acetonitrile (ACN) as the solvent system mobile Conversely, spider venom contains various forms phase. The peaks fractions monitored at 215 nm and 280 of complex active low molecular weight compounds, nm were collected to detect the presence of peptide bonds peptides and proteins which target a different receptor, ion and aromatic amino acids. channels, and enzymes (Chen et al., 2018). Consequently, The crude and fractionated venom samples were spider venom toxins exhibit several potent cytotoxic qualitatively determined for the presence of peptides properties to several cancer cells through apoptosis through bicinchoninic acid (BCA) assay. For this assay, modulation, proliferation management, inhibition of the mixture contained 500 μL of BCA and 10 μL of each enzymatic activities, and cell cycle alterations (Chen et al., sample. The samples were incubated at 60ºC for 30 2018). Also, it may exhibit selective activity to abnormal minutes and read at 562 nm (He, 2011). expression of ion channels on cancer cell surface which can confer potent anti-cancer activities (Zhao et al., 2011). Cell Seeding, Treatment and Lysis This is supported by several studies on other The breast cancer adenocarcinoma, MCF-7 cell line spiders such as Macrothele raven, Lycosa singoriensis, from the American Type Culture Collection (ATCC), Lachesanatara baevi and Psalmopoeus cambridgei passage number- P12, were seeded followed by incubation that were found to exhibit anti-tumor activity on at 37ºC in a humidified incubator with 5% CO2 for 24 erythroleukemia K562 cells, hepatocellular BEL-7402 hours. Venom fractions (50 μL of fractions 1, 3, 10, 12, 14, cells, and HeLa cells which is essential towards potential and 19) and controls (400 μg/mL vitamin E and 400 μg/mL cancer treatment (Gao et al., 2007; Liu et al., 2012; of L-cysteine) were administered in the cell culture flasks Dubovskii et al., 2015). Moreover, venom peptides containing Dulbecco’s Modified Eagle Media (DMEM) offer several advantages for targeted therapy such as and incubated for 24 hours. The medium was removed, specificity and selectivity to proteins highly expressed treated with trypsin, centrifuged at 5,000 rpm, and treated in cancer cells (Barrion-Dupo et al, 2016; Garciano with 6 mL lysis buffer to obtain the lysate. et al., 2014). However, potential anti-cancer activities have not been explored yet on venom from Philippine Detection of Cellular Stress Levels Theraphosidae as these spiders were only studied based Production of hydroxyl radical (•OH) was conducted on their morphological characteristics and features. according to the methodology of Santiago and Mayor Therefore, this study aims to evaluate the oxidative and (2014) with some modifications. In every treatment group, nitrosative activities of venom peptide constituents from 10 μL of 0.1 mM EDTA, 1 μL of 0.1 mM FeCl3, 10 μL of Philippine tarantula as well as their cytotoxic activity 2 mM H2O2, 36 μL of 3 mM deoxyribose, 33 μL of 20 mM against human breast adenocarcinoma (MCF-7) cells for phosphate pH 7.4, and 100 μL of cell lysate were mixed potential therapeutics. and incubated for 30 minutes at 37ºC. Afterwards, the samples were added with 50 μL of 5% (w/v) trichloroacetic Materials and Methods acid (TCA) and 50 μL of thiobarbituric acid (TBA) and were incubated for 30 minutes in 37ºC. The absorbance Spider Collection, Rearing, Maintenance, and of each solution was read at 532 nm. Identification The production of nitric oxide radical (NO•) was The spiders were collected from the caves of Bagacay, conducted based on the methodology of Santiago et Surigao in Mindanao, Philippines through hand-grabbing al., (2016) with some modifications. In every treatment and the use of long forceps. Spiders were maintained in group, 400 μL of 10 mM SNP, 100 μL of PBS and 100 twelve-hour daylight and darkness, fed with Pycnoscelus μL of cell lysate were combined and incubated for 25 striatus once per week and provided with water ad ºC for 150 minutes. Afterwards, an aliquot of 50 μL was libitum (Estrada-Gomez et al., 2015). Spiders were combined with 100 μL of Griess reagent containing 1% submitted at the Museum of Natural History, University (w/v) sulfanilamide, 2% (v/v) H3PO4 and 0.1% (w/v) of the Philippines Los Baños for the morphological NED-HCl. The absorbance of each solution was read at characterization