Phospholipase A2 from Krait Bungarus Fasciatus Venom Induces Human Cancer Cell Death in Vitro
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Phospholipase A2 from krait Bungarus fasciatus venom induces human cancer cell death in vitro Thien V. Tran1,2, Andrei E. Siniavin3, Anh N. Hoang2,4, My T.T. Le5, Chuong D. Pham5, Trung V. Phung6, Khoa C. Nguyen2,4, Rustam H. Ziganshin7, Victor I. Tsetlin8, Ching-Feng Weng9 and Yuri N. Utkin3 1 Tra Vinh University, Tra Vinh City, Vietnam 2 Graduate University of Science and Technology VAST, Hanoi, Vietnam 3 Laboratory of Molecular Toxinology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russian Federation 4 Institute of Applied Materials Science VAST, Ho Chi Minh City, Vietnam 5 Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, Vietnam 6 Center for Research and Technology Transfer VAST, Ho Chi Minh City, Vietnam 7 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russian Federation 8 Department of Molecular Neuroimmune Signalling, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russian Federation 9 Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Shoufeng, Hualien, Taiwan ABSTRACT Background: Snake venoms are the complex mixtures of different compounds manifesting a wide array of biological activities. The venoms of kraits (genus Bungarus, family Elapidae) induce mainly neurological symptoms; however, these venoms show a cytotoxicity against cancer cells as well. This study was conducted to identify in Bungarus fasciatus venom an active compound(s) exerting cytotoxic effects toward MCF7 human breast cancer cells and A549 human lung cancer cells. Methods: The crude venom of B. fasciatus was separated by gel-filtration on Superdex HR 75 column and reversed phase HPLC on C18 column. The fractions 12 April 2019 Submitted obtained were screened for cytotoxic effect against MCF7, A549, and HK2 cell lines Accepted 17 October 2019 Published 3 December 2019 using colorimetric assay with the tetrazolium dye MTT- 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide. The primary structure of active protein was Corresponding author Yuri N. Utkin, [email protected] established by ultra high resolution LC-MS/MS. The molecular mechanism of the fl Academic editor isolated protein action on MCF7 cells was elucidated by ow cytometry. Bruno Lomonte Results: MTT cell viability assays of cancer cells incubated with fractions isolated Additional Information and from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa Declarations can be found on possessing significant cytotoxicity. This protein manifested the dose and time page 18 dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on DOI 10.7717/peerj.8055 human normal kidney HK2 cells. In MCF7, flow cytometry analysis revealed a Copyright decrease in the proportion of Ki-67 positive cells. As Ki-67 protein is a cellular 2019 Tran et al. marker for proliferation, its decline indicates the reduction in the proliferation of Distributed under MCF7 cells treated with the protein. Flow cytometry analysis of MCF7 cells stained Creative Commons CC-BY 4.0 with propidium iodide and Annexin V conjugated with allophycocyanin showed that a probable mechanism of cell death is apoptosis. Mass spectrometric studies How to cite this article Tran TV, Siniavin AE, Hoang AN, Le MTT, Pham CD, Phung TV, Nguyen KC, Ziganshin RH, Tsetlin VI, Weng C-F, Utkin YN. 2019. Phospholipase A2 from krait Bungarus fasciatus venom induces human cancer cell death in vitro. PeerJ 7:e8055 DOI 10.7717/peerj.8055 showed that the cytotoxic protein was phospholipase A2. The amino acid sequence of this enzyme earlier was deduced from cloned cDNA, and in this work it was isolated from the venom as a protein for the first time. It is also the first krait phospholipase A2 manifesting the cytotoxicity for cancer cells. Subjects Biochemistry, Toxicology Keywords Phospholipase A2, Venom, Krait, Cytotoxicity, Human cancer cells, Breast cancer, Lung cancer, Mass spectrometry INTRODUCTION Cancer is the second leading cause of death in the world. Despite advances in the development of new drugs, the search for new effective medicines remains a challenging task. The main problem is a high toxicity of the existing drugs to the normal cells. Snake venoms contain many bioactive proteins manifesting diverse biological activities. Some of them were shown to possess cytotoxic activity against tumor cells. Anti-cancer activity was demonstrated for venoms of snakes from different genera and species (Li, Huang & Lin, 2018). There are numerous communications on cytotoxic effects of venoms from cobras, vipers and pit-vipers on tumor cells (Zainal Abidin et al., 2019; Nalbantsoy et al., 2017; Ghazaryan et al., 2015). Several proteins, including cytotoxins (Dubovskii & Utkin, 2015), L-amino acid oxidases (Salama et al., 2018), phospholipases A2 (Sobrinho et al., 2016), disintegrins (Arruda Macêdo, Fox & De Souza Castro, 2015) and others manifesting anti-proliferative activity were isolated from these venoms. These proteins themselves, due to their inherent adverse properties (high molecular mass, high toxicity, etc.), can hardly be used as medicines. However, their active fragments may well be used for this purpose. The adverse effects of the venom proteins can be greatly diminished by use of the targeted drug delivery systems. For example, liposomal delivery of disintegrins substantially increased their therapeutic potential (David et al., 2018). The anti-cancer activity of kraits’ venoms is not so well studied as that of other venoms. Thus, it was found that β-bungarotoxin from krait Bungarus multicinctus venom showed the concentration- and time-dependent cytotoxicity against human neuroblastoma SK-N-SH cells (Cheng, Wang & Chang, 2008). Moreover, the cytotoxic effect was localized on B-subunit of β-bungarotoxin. L-Amino acid oxidases isolated from B. fasciatus (Wei et al., 2009) and B. multicinctus (Lu et al., 2018) venoms manifested strong cytotoxicity against different cancer cell lines. A protease inhibitor like protein-1 (PILP-1) from B. multicinctus venom was found to induce apoptotic death of human leukemia U937 cells (Liu & Chang, 2010). The more detailed studies showed that PILP-1-induced down-regulation of a disintegrin and metalloprotease 17 (ADAM17) which resulted in inactivation of Lyn/Akt pathways. The mitochondrion-mediated apoptosis of U937 cells was thus activated. From B. fasciatus krait venom, a protein BF-CT1 possessing capacity to induce Ehrlich ascites carcinoma (EAC) and U937 leukemic cell death was isolated (Bhattacharya et al., 2013). BF-CT1 had molecular mass of 13 kDa and induced apoptosis in EAC in vivo and in U937 cell line in vitro. Tran et al. (2019), PeerJ, DOI 10.7717/peerj.8055 2/22 The above studies indicated that krait venoms have some anti-cancer potential. In this work we present the data on activity-guided isolation from Vietnamese krait B. fasciatus venom and characterization of a phospholipase A2 manifesting cytotoxic activity against human MCF7 and A549 cell lines. MATERIALS AND METHODS Materials Snake venom Crude krait B. fasciatus venom (Vinh Son, Vinh Tuong, Vinh Phuc Province, Vietnam) was obtained as previously described (Ziganshin et al., 2015). The venom was collected from several tens of snake specimens at the farm owned by professional snake breeder Mr. Ha Van Tien by farm team members. It was lyophilized and stored at −20 C until use. Cell lines The human breast cancer cell line MCF7 (Catalog number: HTB-22), the human breast cancer cells BT-474 (Catalog number: HTB-20), the human breast cancer cells SK-BR-3 (Catalog number: HTB-30), the human prostate cancer cells PC-3 (Catalog number: CRL-1435), the human prostate cancer cells LNCaP (Catalog number: CRL-1740), the human lung cancer cells A549 (Catalog number: CCL-185EMT), and renal tubular epithelial HK-2 cells (a proximal tubular cell line derived from normal kidney; Catalog number: CRL-2190) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and stored in liquid nitrogen until use. Methods Fractionation of krait B. fasciatus venom Crude krait B. fasciatus venom was separated by gel-filtration on the SuperdexÒ 75 10/300 GL column (1 × 30 cm; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) equilibrated with the 0.1 M ammonium acetate buffer (pH 6.2). The column was eluted at flow rate of 0.5 ml/min and the eluate was monitored by measuring optical density at 226 nm (Fig. 1A). The fractions obtained were freeze-dried and used for activity measurements. Fraction 3 (Fig. 1A) was further separated by reversed-phase high-performance liquid chromatography on the Jupiter C18 column (10 × 250 mm) in a gradient of 25–40% acetonitrile in 75 min in the presence of 0.1% trifluoroacetic acid, at a flow rate of 2.0 ml/min. The eluate was monitored by measuring optical density at 275 nm (Fig. 1B). The obtained fractions were freeze-dried and used for activity measurements. Cell culture Both MCF7 and A549 cells were cultured in DMEM supplemented with 10% FBS, 1% antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin). HK2 cells were grown in DMEM/F12 supplemented with 10% FBS, 1% antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin), and 5 ng/mL of human recombinant epidermal growth factor (EGF, Gibco, Waltham, MA, USA). All of cell lines were kept at 37 Cina fi humidi ed atmosphere of 5% CO2 incubator. Tran et al. (2019), PeerJ, DOI 10.7717/peerj.8055 3/22 Figure 1 Fractionation of B. fasciatus venom. (A) Gel filtration of crude venom on the SuperdexÒ 75 10/300 GL column (1 × 30 cm) equilibrated with the 0.1 M ammonium acetate buffer (pH 6.2). Flow rate 0.5 ml/min. The eluate was monitored by spectrophotometry (OD = 226 nm). Horizontal bars indicate the collected fractions. (B) Reversed phase chromatography of Fraction 3 (From A) on the Jupiter C18 column (10 × 250 mm) in a gradient of 25–40% acetonitrile in 75 min in the presence of 0.1% tri- fluoroacetic acid, at a flow rate of 2.0 ml/min.