Authors: Sirisha Potala Rama S Verma
Publish Date: 2010/08/01
Volume: 38, Issue: 2, Pages: 1389-1397
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 toxinHN1 peptide DT/HN1 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/HN1 fusion toxin against the HNSCC cells The purified DT/HN1 fusion toxin was characterized by SDSPAGE and western blotting Refolding of purified fusion toxins was monitored by fluorescence spectra and circular dichroism spectra The activity of DT/HN1 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/HN1 demonstrated remarkably high degree of cytotoxicity specific to the HNSCC cells The IC50 of DT/HN1 fusion toxin was ~1 to 5 nM in all the three HNSCC cell lines The percentage apoptotic cells in DT/HN1 treated UMBSCC745 cells are 16 compared to 4 in untreated To further demonstrate the specific toxicity of DT/HN1 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 HN1 peptide an additional construct DTΔT/HN1 was constructed characterized and evaluated for its cytotoxic activity The fusion toxin DTΔT/HN1 deficient of the translocation domain of diphtheria toxin showed no cytotoxicity on all the cell lines clearly indicating the inability of HN1 peptide to translocate catalytic domain of the toxin into the cytosolWe thank Robert Mandic Department of Otolaryngology University of Marburg Marburg Germany for UMBSCC745 cell line We thank Reidar Grenman Department of Otorhinolaryngology University of Turku Turku Finland for providing UTSCC36 UTSCC38 cell lines We also thank A Jayakumar MD Anderson Cancer Centre Houston TX for his timely advice and Ms A Jayasree for manuscript correction
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