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RESEARCH ARTICLE Anti-Glioma Effect of Pseudosynanceia

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RESEARCH ARTICLE Anti-Glioma Effect of Pseudosynanceia DOI:10.31557/APJCP.2021.22.7.2295 Effect of Pseudosynanceia Melanostigma Venom on Glioblastoma Cells RESEARCH ARTICLE Editorial Process: Submission:02/14/2021 Acceptance:07/15/2021 Anti-Glioma Effect of Pseudosynanceia Melanostigma Venom on Isolated Mitochondria from Glioblastoma Cells Maral Ramezani, Fatemeh Samiei, Jalal Pourahmad* Abstract Background: Glioblastoma is the most common primary malignant tumor of the central nervous system that occurs in the spinal cord or brain. Pseudosynanceia Melanostigma is a venomous stonefish in the Persian Gulf, which our knowledge about is little. This study’s goal is to investigate the toxicity of stonefish crude venom on mitochondria isolated from U87 cells. Methods: In the first stage, we extracted venom stonefish and then isolated mitochondria have exposed to different concentrations of venom. Finally, mitochondrial toxicity parameters (Succinate dehydrogenase (SDH) activity, Reactive oxygen species (ROS), cytochrome c release, Mitochondrial Membrane Potential (MMP), and mitochondrial swelling) have evaluated. Results: To determine mitochondrial parameters, we used 115, 230, and 460 µg/ml concentrations. The results of our study show that the venom of stonefish selectively increases upstream parameters of apoptosis such as mitochondrial swelling, cytochrome c release, MMP collapse and ROS. Conclusion: This study suggests that Pseudosynanceia Melanostigma crude venom has selectively caused toxicity by increasing active mitochondrial oxygen radicals. This venom could potentially be a candidate for the treatment of glioblastoma. Keywords: Mitochondria- glioblastoma- fish venoms- cell line- tumor- toxicity-Persian Gulf Asian Pac J Cancer Prev, 22 (7), 2295-2302 Introduction freshwater or saltwater. Marine animals most commonly used for animals live in saltwater, i.e. in oceans, seas, Glioblastoma multiform is the most common group of etc. Fish make up about half of the vertebrates on Earth, brain tumors and malignant gliomas are the deadliest types and there are approximately 44,222 species of fish in 12 of adult brain tumors. Glioblastoma accounts for more than orders and 221 families. Among them, there are about 50% of all types of gliomas. The overall prevalence is 2-3 1,422 species of poisonous marine fish, which include per 100,000 and 20% of all intracranial tumors, and it is biting superficial fish, scorpionfish, zebrafish, stonefish, more common in men over 60 years (Wang et al., 2018). toadfish, fish astronomers, and species of sharks, mice, fish In cellular studies, glioblastoma multiform cells surgeons (Smith and Wheeler, 2006). Because the venom (U87) are very different in shape and size; they have of various animals, especially fish, has a wide range of high vascular proliferation and necrosis and increase the pharmacological effects on the human nerves, muscles, involvement of tumor cells in different areas of the brain. and cardiovascular system, venom proteins are a source for This type of tumor has a poor prognosis with various the emergence of drugs for the treatment of pain, cancer, clinical features such as cognitive impairment, personality infectious diseases, and autoimmune diseases (Smith changes, headache, loss of sensation (paresthesia), and and Wheeler, 2006). Apoptosis is programmed cell death gait imbalance. In the treatment of the disease, this type in which damaged and redundant cells are removed by of cancer is still considered incurable; because despite various mechanisms, thus preventing damage to adjacent the common treatments including surgical removal of the healthy cells (McConkey, 1998). Two pathways exist for tumor and subsequent radiation therapy in addition to drug apoptosis. The mitochondrial pathway of apoptosis begins treatment with Temozolomide (TMZ), people with the with any metabolic stress in the cells; the main result is a disease are usually die within 12 to 15 months (Anjum et change in the permeability of the outer membrane of the al., 2017). One of the treatments for glioma is cytotoxicity mitochondria that leads to the exit of cytochrome c. An as well as induction of apoptosis to inhibit the proliferation apoptosome formed in the cytosol and eventually leads of cancer cells (Yan et al., 2012). Therefore, the need for to the activation of the caspase cascade (Wang, 2001). new therapies or drugs seems to be necessary. Various compounds of marine animals known to use in the Aquatic animals are used for animals that live in either treatment of cancer using apoptosis such as Dolastatin 10, Department of Toxicology and Pharmacology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran. *For Correspondence: [email protected] Asian Pacific Journal of Cancer Prevention, Vol 22 2295 Maral Ramezani et al Didemnin B and Aplidine, Bryostatin 1, Ecteinascidin-743 from R & D Systems, Inc. (Minneapolis, Minn.). (ET-743) and etc.(Beesoo et al., 2014). Stonefish (Pseudosynanceia melanostigma) a native Crude Venom Preparation of the Persian Gulf that considers as one of the fish that To extract Pseudosynanceia melanostigma stonefish have poison. This fish is about 2-3 cm long and has 2-5 venom, stonefish has been caught from the coastal areas dorsal thorns, 2 thoracics, and 4-6 anal thorns that are of Bushehr beaches. Then, the thorns attached to the equipped with poison glands. This fish lives in muddy, venomous glands have cut and incubated for 24 hours in quiet, and shallow coastal areas (Vazirizadeh et al., 2014). 0.15 M sodium chloride solution at 4°C. After 24 hours, This fish’s toxin is a myotoxin that effects on skeletal, the obtained solution has been centrifuged at 12,000 g cardiac, and involuntary muscles and blocks conduction for 20 minutes at 4°C and then the supernatant has been in these tissues. It also causes the release of acetylcholine, collected (Vazirizadeh et al., 2014). The crude venom has substance P ,and cyclooxygenase (Gwee et al., 1994; been immediately freeze-dried and kept at -20°C until use Carrijo et al., 2005). Three major venom types have been for tests (Bradford, 1976). isolated from different species of stonefish, Stonustoxin (SNTX), verrucotoxin (VTX), and trachynilysin (TLY) Isolation of mitochondria from U87 cells (Vazirizadeh et al., 2014). The most important enzymes U87 cells have been purchased from Pasteur Institute in the venom include Hyaluronidase, Phospholipase, (Iran) and mitochondria have been separated by using Acetylcholinesterase and Metalloproteinase enzymes multiple centrifugation methods. Briefly, after cells have (Austin et al., 1965; Birkedal-Hansen et al., 1993; Kreil, washed with cold PBS, they have centrifuged at 450g. 1995; Dori et al., 2007). The venom can generate severe The sediment has been suspended in isolation buffer (70 pain, respiratory arrest, cardiovascular injury, muscle mM sucrose, 20 mM HEPES, 220 mM D-mannitol, and 1 paralysis and convulsions, and even death (Diaz, 2015). mM EDTA, pH = 7.2), and then the cells have been lysed Multiple studies have revealed the medicinal properties by homogenization. Then, cells have been centrifuged at of each subunit of stonefish’s venom (Carlson et al., 750g for 20 min. Supernatant has re-centrifuged at 8000g 1971; Church and Hodgson, 2000). In a study, researchers for 15 min. The sediment has been called as mitochondrial represented that this venom has catalytic effects against fraction and maintained in buffer solution (140 mM KCl, numerous cell types (Kreger, 1991). In addition, anticancer 10 mM NaCl, 2.0 mM MgCl2, 0.5 mM KH2PO4, 20 mM effects of venom are described by using in Vivo cancer HEPES ,and 0.5 mM EGTA, pH=7.2) (Salimi et al., 2017). model. They represented that the venom, induced apoptosis in Ehrlich’s associate’s carcinoma (EAC), Isolation of mitochondria from normal rat brain splenocytes, and P388 cells (Rajeshkumar et al., 2015). After anesthesia, the Male Wistar rat’s (250-300 g) This study goal is to investigate the toxicity of brain has quickly excised and used for the separation of Pseudosynanceia melanostigma stonefish crude venom on mitochondria. Brain mitochondria have been prepared mitochondria isolated from U87 cells and a comparison from rats using the differential centrifugation method. of its effect on mitochondria of normal cells. At first, the brains have been removed and torn to pieces in cold mannitol solution (mitochondria isolation Materials and Methods buffer) and then have mildly homogenized in a glass handheld homogenizer. Then, the homogenized brain In this study after determining IC50 by applied 0, 100, has centrifuged at 1,500 g for 10 min at 4°C. The 200, 500, and 1000 µg/ml crude venom on mitochondria supernatant has re-centrifuged at 12,000 g for 10 min at from U87 cells, we had six groups: 4°C. The mitochondrial sediment has been rinsed by mild 1) Control normal mitochondria (mitochondria from suspension in the buffer solution (0.25 M sucrose, 20 mM normal rat’s brain cells). KCl, 2.0 mM MgCl2, 1.0 mM Na2HPO4, 0.25 M sucrose 2) Control cancerous mitochondria (mitochondria and 0.05 mM TRIS-HCl, pH = 7.2) at 4°C and centrifuged from U87 cells). again at 12,000 × g for 10 min. Finally, mitochondrial 3) Normal mitochondria + 230 µg/ml crude venom. sediment has been suspended in Tris buffer (0.05 M 4) Cancerous mitochondria + 115 µg/ml crude venom. Tris-HCl, 0.25 M sucrose, 20 mM KCl, 2.0 mM MgCl2, 5) Cancerous mitochondria + 230 µg/ml crude venom. and 1.0 mM Na2HPO4, pH of 7.4) at 4° C and kept for 6) Cancerous mitochondria + 460 µg/ml crude venom. use in
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