Abstract
BackgroundSnake 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.MethodsThe crude venom of B. fasciatus was separated by gel-filtration on Superdex HR 75 column and reversed phase HPLC on C18 column. The fractions obtained were screened for cytotoxic effect against MCF7, A549, and HK2 cell lines 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 established by ultra high resolution LC-MS/MS. The molecular mechanism of the isolated protein action on MCF7 cells was elucidated by flow cytometry.ResultsMTT cell viability assays of cancer cells incubated with fractions isolated from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa possessing significant cytotoxicity. This protein manifested the dose and time dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on human normal kidney HK2 cells. In MCF7, flow cytometry analysis revealed a decrease in the proportion of Ki-67 positive cells. As Ki-67 protein is a cellular marker for proliferation, its decline indicates the reduction in the proliferation of MCF7 cells treated with the protein. Flow cytometry analysis of MCF7 cells stained with propidium iodide and Annexin V conjugated with allophycocyanin showed that a probable mechanism of cell death is apoptosis. Mass spectrometric studies 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.
Highlights
Cancer is the second leading cause of death in the world
Cytotoxicity studies We have recently shown that krait B. fasciatus venom from Vinh Phuc province (Vietnam) possessed the capacity to inhibit proliferation of the human breast cancer cell line MCF7 and the human lung cancer cell A549 (Tran et al, 2019)
We have studied the effects of the whole B. fasciatus venom on the human breast cancer cells MCF7 and lung cancer cells A549 and observed cytotoxic effects against both cell lines (Tran et al, 2019)
Summary
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. 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. Results: MTT cell viability assays of cancer cells incubated with fractions isolated from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa possessing significant cytotoxicity. This protein manifested the dose and time dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on human normal kidney HK2 cells.
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