Original Research Article 2019;2(1):e5 Selective Toxicity of Standardized Extracts of Persian Gulf Sponge (Irciniamutans) on Skin Cells and Mitochondria isolated from Melanoma induced mouse a b c d e Yalda Arast , Nina SeyedRazi , Melika Nazemi , Enayatollah Seydi , Jalal Pourahmad * a. Department of Occupational Health Engineering, Faculty of Health, Qom University of Medical Sciences, Qom, Iran. b. Pharmaceutical Sciences Research Center, ShahidBeheshti University of Medical Sciences, Tehran, Iran. c. Persian Gulf and Oman Sea Ecological Center, Iranian Fisheries Science Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Bandar Abbas, Iran. d. Research Center for Health, Safety and Environment (RCHSE), Department of Occupational Health Engineering, Alborz University of Medical Sciences, Karaj, Iran. e. Department of Pharmacology and Toxicology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Article Info: ABSTRACT: Received: March 2019 Melanoma is an aggressive and highly lethal cancer with poor prognosis and Accepted: June 2019 resistance to current treatments. Apoptosis signaling is believed to be suppressed in Published online: melanoma. Evidence suggests that compounds isolated from marine sponges have June 2019 anti-cancer properties. This study was designed to evaluate the apoptotic effect of methanolic, diethyl ether, and n-hexane extracts of Irciniamutans (I.mutans) on skin mitochondria isolated from mice animal models of melanoma. Mitochondria were * Corresponding Author: isolated by differential centrifugation. According to our results, methanolic, diethyl Jalal Pourahmad ether, and n-hexane extracts of I.mutans raised the reactive oxygen species (ROS) Email: level only in the cancerous skin mitochondria group. Furthermore, methanolic, [email protected] diethyl ether, and n-hexane extracts induced swelling in the mitochondria, decreased [email protected] the mitochondrial membrane potential (MMP) and the release of cytochrome c from the mitochondria. Based on these results, the potentially bioactive compounds found in I.mutans render the sea sponge as a strong candidate for further researches in molecular identification and confirmatory in-vivo researches. Keywords: Irciniamutans; Melanoma; Apoptosis; Mitochondria; Reactive Oxygen Species. Please Cite this article as: Arast Y, SeyedRazi N, Nazemi M, Seydi E, Pourahmad J. Selective Toxicity of Standardized Extracts of Persian Gulf Sponge (Irciniamutans) on Skin Cells and Mitochondria isolated from Melanoma induced mouse. Int. Pharm. Acta. 2019;2(1):e5 DOI: https://doi.org/10.22037/ipa.v2i1.25103 1. Introduction and Australia are rising sharply. According to the World Health Organization (WHO), nearly 132,000 new cases Melanoma is as a solid malignant tumor originated from are detected each year worldwide [5-8]. Melanoma is melanocytic cells; it is considered as one of the most the fourth most common cancer in men and the sixth in important aggressive and malignant skin cancers [1-4]. women [9, 10]. Furthermore, melanoma prognosis is To date, the incidence of melanoma is rising, especially very poor. [2, 11]. within the Caucasian population. Studies have In recent years, despite the availability of treatment demonstrated that incident rates in Europe, America, methods and strategies, such as chemotherapy, This open-access article is distributed under the terms of the Creative Commons Attribution Non Commercial 4.0 License (CC BY-NC 4.0). 2 Y. Arast et al., International Pharmacy Acta, 2019;2(1):e5 radiotherapy, surgery and immunotherapy, the average using the method described by Mamelona et al., [18]. survival rate is very low [10]. Various studies have The (I.mutans) samples were cut into small parts and shown that melanoma treatment is a major challenge for homogenized using a blender. After filtration and oncology researchers [11, 12]. Resistance to centrifugation at 30,000 ×g for 15 min in 4º C,the chemotherapyis one of the main reasons of melanoma powdered samples were kept in n-hexane about 24 treatments failures [2, 5, 6, 10]. It is well documented hours to produce non-polar and polar extractions, then that a number of chemotherapy drugs via the intrinsic or the solution was filtered and n-hexane was evaporated mitochondrial pathway induce apoptosis signaling in the to dryness, at low pressure at 35- 40ºC by using Rota- tumor cells. Studies have shown that, cells in melanoma vapors. For diethyl ether extract the sample was put in lesions show an inherently low level of spontaneous cell diethyl ether about 48 hours, the solution was filtered death, and resistance to apoptosis through activation of and diethyl ether was evaporated to dryness, at low proliferation and survival pathways. Therefore, the pressure at 35- 40ºC by using Rota-vapors. Finally the activation of apoptotic programs in melanoma cells sample wassoaked in methanol for 72 hours, after filter provide a potential key requisite to overcome drug and dryness the polar extrac was produced. Finally, the resistance and ameliorate clinical outcomes [2, 10, 13, powdered extracts of (I.mutans) werestored at -20 ºC. 14]. Most anticancer medicines have a natural origin. Standardization of extracts by GC-MS analysis: Currently, natural compounds or lead compounds from The n-hexane, diethyl ether and methanolic extracts of various sources such as, marine organisms, plants, and the sea cucumber (I.mutans) were analyzed by GC-MS microorganisms are used in clinic for the treatment of (Agilent7000 series Triple Quad GC/MS Main Frame). many chronic diseases, including cancer. Many of these The GC column dimension was: 30 mm, 0.25 mm, 0.5 natural compounds are capable of inducing apoptosis in mm AB-35MS fused silica capillary column. The GC melanoma cells [9, 15]. Marine sponges are rich sources conditions were as follows: injector temperature 250°C of anti-cancer compounds. These natural compounds column temp isothermal at 100°C then programmed to have different pharmacological activities [16]. rise up to 250°C at 6°C/min and held at this temperature Anticancer treatments (such as, alkylating agents, for 10 minutes. The ion source temperature was 200°C ionizing radiation, platinum compounds and DNA and the interface temperature was 250°C. Helium gas topoisomerase inhibitor) are used as targeted therapies was engaged for carrier gas at the rate of 1ml/min. (such as, apoptosis-inducers) to identify specific targets Spectra was obtained in the EI mode with 70eV for tumor cells to increase treatment efficacy [17]. ionization energy. The compounds were identified by Our previous studies demonstrate that marine organisms comparison with the standards. GC-MS Analysis of the (such as sea sponges, sea cucumbers and sea squirts) n-hexane, diethyl ether, and methanolic extracts of have cytotoxic effects on cancer cells (including, (I.mutans) showed the fallowing information (table 1). hepatocellular carcinoma, leukemia and melanoma) [18- 21]. Research has shown that certain changes in Animals mitochondria occur during melanoma, and these changes consequently affect the treatment [22, 23]. In The animal model for melanoma was acquired from this study, we investigate the cytotoxicity effect of Pasteur Institute. All experimentations were conducted according to the ethical standards and protocols (I.mutans) (sea sponge found in the Persian Gulf) extracts on skin mitochondria isolated from a typical approved by the Committee of Animal Experimentation animal model of melanoma. of Shahid Beheshti University of Medical Sciences, Tehran, Iran. Isolation of Mitochondria From mouse melanoma 2. Materials & Methods cells Sampling and extraction of Sponge Samples: The preparation of isolated mouse skin cells was Sponge samples (I.mutans) were collected from the completed using the two-step collagenase skin perfusion Bandar-e-lengehcoast, south of Iran. The (I.mutans) technique. To assess cellular integrity (or viability), the samples were transported to the laboratory via iced trypan blue exclusion test was performed [24, 25]. boxes. The samples were washed with cold water, Mitochondria were prepared from cancerous cells in weighed, and evaluated. Bioactive agents were extracted accordance with our previous published work. This open-access article is distributed under the terms of the Creative Commons Attribution Non Commercial 4.0 License (CC BY-NC 4.0). Selective Toxicity of Standardized Extracts of Persian Gulf Sponge (Irciniamutans) on Skin Cells and …. 3 Determination of succinate dehydrogenase (SDH) spectrophotometer (Ex = 490 nm, and λ Em= 535nm) activity [18, 19]. The SDH activity was measured using a MTT dye. At Mitochondrial Swelling assay first, 100 μl of mitochondrial suspension from both groups were incubated with various concentrations Isolated mitochondria from both groups were suspended (125, 250, 500, 1000, and 1500 µg/ml) of aqueous, in swelling buffer and incubated at 30°C with several methanol, diethyl ether, and n-hexane extracts of concentrations (250, 500, and 1000 µg/ml) of three I.mutansat 37°C for 1 hour. Furthermore, 0.4% of MTT extracts (methanol, diethyl ether, and n-hexane) of (MTT dye plus succinate dehydrogenase) was added to I.mutans. The absorbance was then measured at 549 nm the medium and then incubated at 37°C for 30 minutes. using an ELISA reader (Tecan, Rainbow Thermo, Finally, the product of formazancrystals was dissolved Austria). It should be noted that