Characterisation of the effect of novel anti-cancer components of Pseudechis australis and Crotalus vegrandis snake venom on acute lymphoblastic leukaemia cells.

James Boncan, Sara Dobbin, Diego Cobice, Stephen McClean, Karen McCloskey, Ken Mills

Research output: Chapter in Book/Report/Conference proceedingConference contributionpeer-review

Abstract

Introduction: Acute lymphoblastic leukaemia (ALL) is a haematological malignancy affecting lymphoid lineage cells accounting for approximately 230 UK deaths annually. Standard of care-therapies for ALL are poorly tolerated and have poor clinical responses. Snake venom represents a natural source of bioactive chemicals with numerous in vitro studies identifying several venom components with anti-cancer activity [1]. Here, we aimed to identify active components within crude snake venom and characterise their mechanisms in ALL cells. Methods: Reh and NALM6, B cell precursor ALL cell lines were used. Crude snake venom from Pseudechis australis (PA) and Crotalus vegrandis (CV) was fractionated using gel-filtration chromatography with active fractions subject to MALDI-ToF mass spectrometry [2]. Fractions were numbered sequentially. Cell viability and cytotoxicity was assessed using CellTiter-Glo luminescence and CellTox-Green assays, respectively. Lactate dehydrogenase (LDH) release was measured with LDH-Glo cytotoxicity assay kits and luminescent caspase 3/7 assays were used to study apoptosis. In functional assays, at least N=3 were performed; occasionally only N=2 was achievable due to limited snake venom fraction volume. Data sets were compared with ANOVA with p<0.05 considered as significant. Results: In viability assays using Reh cells, PA venom fractions, PA60-63 and PA68-71 reduced viability after 24, 48 and 72 hours (N=3, p<0.05). MALDI-ToF analysis of these fractions revealed distinct peaks of 13kDa, indicative of the phospholipase A2 family of enzymes. Reh cells treated with a commercially bought phospholipase A2 isolated from Crotalus adamanteus snake venom also displayed reduced cell viability at 24, 48 and 72 hours (IC50 63.5µg/mL, 58.7µg/mL and 57.9µg/mL respectively, N=3, p<0.05). In NALM6 cells, CV venom fractions, CV110-115, CV125, CV131-137 and CV139-149 reduced cell viability (N=3; p<0.05). MALDI signals were observed for CV110–115, CV125 and CV131–133 venom fractions, at 9kDa and 11kDa, slightly below the phospholipase A2 range (13kDa). Reh cell cytotoxicity was increased by active PA fractions (N=3, p<0.05). CV112, CV114, CV132 and CV134 evoked cytotoxicity and loss of NALM6 membrane integrity (N=2). LDH release was not evoked in PA venom-treated Reh cells (N=3, p<0.05). Finally, neither PA-treated Reh nor CV-treated NALM6 exhibited increased caspase 3/7 activity (N=2). Conclusions: PA and CV-derived venom fractions reduced ALL cell viability. MALDI-ToF analysis identified phospholipase A2 family as an anticancer component of interest within PA snake venom. Functional assays highlight two potential mechanisms for reduced viability in PA-treated Reh and CV-treated NALM6; both appear to be apoptosis- and necrosis-independent, indicating suppression of ALL cell proliferation. References: [1] Urra, F. and Araya-Maturana, R., 2020. Putting the brakes on tumorigenesis with snake venom toxins: New molecular insights for cancer drug discovery. Seminars in Cancer Biology. [2] Moore, S., Bhat, V., Flatt, P., Gault, V. and McClean, S., 2015. Isolation and characterisation of insulin-releasing compounds from Crotalus adamanteus, Crotalus vegrandis and Bitis nasicornis venom. Toxicon, 101, pp.48-54
Original languageEnglish
Title of host publicationBritish Journal of Pharmacology
Subtitle of host publicationBr J Pharmacol
Pages468-469
Number of pages2
Volume178
Edition2
DOIs
Publication statusPublished - 8 Dec 2020
EventPharmacology 2020 - Online (Virtually) - Online
Duration: 14 Dec 202018 Dec 2020
https://live.bethereglobal.com/s/pharmacology-2020

Conference

ConferencePharmacology 2020 - Online (Virtually)
Period14/12/2018/12/20
Internet address

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