Abstract
BackgroundHydra actinoporin like toxin -1 (HALT-1), is a small 18.5 kDa pore forming toxin derived from Hydra magnipapillata which has been shown to elicit strong haemolytic and cytolytic activity when in contact with cell membranes. Due to its cytotoxic potency, HALT-1 was further investigated for its potential as a toxin moiety candidate in immunotoxin developmental efforts, ideally as a form of targeted therapy against cancer.MethodsIn this study, wtHALT-1 (wild type) and its Y110A mutated binding domain counterpart (mHALT-1) were produced and evaluated for their cytotoxic and apoptotic effects on various cancer cell lines. A total of seven different tumour and non-tumour cell lines including HeLa, HepG2, SW-620, MCF-7, CCD841CoN, NHDF and HCT116 were used. Immunofluorescence assays were used to observe membrane binding and localization changes between both HALT-1 recombinant proteins based on 6xHis-tag detection.ResultBased on MTT data, mHALT-1 demonstrated a significant reduction of 82% ± 12.21% in cytotoxic activity across all cell lines after the membrane recognition domain had been mutated in comparison to the wtHALT-1. Annexin V FITC/PI assay data also indicated that HeLa, HepG2 and MCF-7 demonstrated an apoptosis-mediated cell death after being treated with wtHALT-1. Additionally, a notable difference between wtHALT-1 and mHALT-1 binding affinity was clearly observed where emission of green fluorescence along the cell membrane was observed only in wtHALT-1 treated cells.DiscussionThese results suggest that mHALT-1 (Y110A) can be potentially developed as a toxin-moiety candidate for the development of future immunotoxins against various human cell-based diseases.
Highlights
Most protein-based toxins have the potential to be developed as a toxin moiety in immunotoxin-based drugs (Aruna, 2006; Becker & Benhar, 2012; Mazor et al, 2016; Mazor, King & Oastan, 2018; Schmohl et al, 2018; Shan, Liu & Wang, 2013; Mathew & Verma, 2009)
Experimental results indicated that wtHALT-1 induced potent cytotoxic effects (IC50 range of 0.30–1.62 μM) against human cancer cell lines, including MCF-7, HeLa, HepG2, HCT116 and SW-620 (Table 1)
The cytotoxic effect of wtHALT-1 was shown to be 50% stronger than mHALT-1 (p = 0.022) on MCF-7 cells (IC50 = 0.30 μM)
Summary
Most protein-based toxins have the potential to be developed as a toxin moiety in immunotoxin-based drugs (Aruna, 2006; Becker & Benhar, 2012; Mazor et al, 2016; Mazor, King & Oastan, 2018; Schmohl et al, 2018; Shan, Liu & Wang, 2013; Mathew & Verma, 2009). These toxin moieties generally need to internalize and translocate to the cytosol in order to achieve its cytotoxic effects, most of which function by enzymatically inhibiting protein synthesis (Tejuca et al, 2009; Virgilio et al, 2010). Discussion.These results suggest that mHALT-1 (Y110A) can be potentially developed as a toxin-moiety candidate for the development of future immunotoxins against various human cell-based diseases
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