Targeting head and neck squamous cell carcinoma using a novel fusion toxin-diphtheria toxin/HN-1 |
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Authors: | Sirisha Potala Rama S Verma |
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Institution: | (1) Stem Cell & Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, #201, Bhupat and Jyoti Mehta School of Biosciences Building, Chennai, 600036, India; |
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Abstract: | The current treatment strategies, chemotherapy and radiation therapy being used for the management of cancer are deficient
in targeted approach leading to treatment related toxicities and relapse. Contrarily, fusion toxins exhibit remarkable tumor
specificity thus emerging as an alternative therapy for the treatment of cancer. Diphtheria toxin-HN-1 peptide (DT/HN-1) is
a fusion toxin designed to target the head and neck squamous cell carcinoma (HNSCC). The aim of this study was to construct,
characterize, and evaluate the cytotoxicity and specificity of DT/HN-1 fusion toxin against the HNSCC cells. The purified
DT/HN-1 fusion toxin was characterized by SDS-PAGE and western blotting. Refolding of purified fusion toxins was monitored
by fluorescence spectra and circular dichroism spectra. The activity of DT/HN-1 fusion toxin was demonstrated on various HNSCC
cell lines by cell viability assay, cell proliferation assay, protein synthesis inhibition assay, apoptosis and cell cycle
analysis. The fusion toxin DT/HN-1 demonstrated remarkably high degree of cytotoxicity specific to the HNSCC cells. The IC50 of DT/HN-1 fusion toxin was ~1 to 5 nM in all the three HNSCC cell lines. The percentage apoptotic cells in DT/HN-1 treated
UMB-SCC-745 cells are 16% compared to 4% in untreated. To further demonstrate the specific toxicity of DT/HN-1 fusion toxin
towards the HNSCC cells we constructed, characterized and evaluated the efficacy of DT protein. The DT protein coding for
only a fragment of diphtheria toxin without its native receptor binding domain failed to exhibit any cytotoxicity on all the
cell lines used in this study thus establishing the importance of a ligand in achieving targeted toxicity. To evaluate the
translocation ability of HN-1 peptide, an additional construct DTΔT/HN-1 was constructed, characterized and evaluated for
its cytotoxic activity. The fusion toxin DTΔT/HN-1 deficient of the translocation domain of diphtheria toxin showed no cytotoxicity
on all the cell lines clearly indicating the inability of HN-1 peptide to translocate catalytic domain of the toxin into the
cytosol. |
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