Abstract
BackgroundDiphtheria toxin (DT) has been utilized as a prospective anti-cancer agent for the targeted delivery of cytotoxic therapy to otherwise untreatable neoplasia. DT is an extremely potent toxin for which the entry of a single molecule into a cell can be lethal. DT has been targeted to cancer cells by deleting the cell receptor-binding domain and combining the remaining catalytic portion with targeting proteins that selectively bind to the surface of cancer cells. It has been assumed that “receptorless” DT cannot bind to and kill cells. In the present study, we report that “receptorless” recombinant DT385 is in fact cytotoxic to a variety of cancer cell lines.Methods In vitro cytotoxicity of DT385 was measured by cell proliferation, cell staining and apoptosis assays. For in vivo studies, the chick chorioallantoic membrane (CAM) system was used to evaluate the effect of DT385 on angiogenesis. The CAM and mouse model system was used to evaluate the effect of DT385 on HEp3 and Lewis lung carcinoma (LLC) tumor growth, respectively.ResultsOf 18 human cancer cell lines tested, 15 were affected by DT385 with IC50 ranging from 0.12–2.8 µM. Furthermore, high concentrations of DT385 failed to affect growth arrested cells. The cellular toxicity of DT385 was due to the inhibition of protein synthesis and induction of apoptosis. In vivo, DT385 diminished angiogenesis and decreased tumor growth in the CAM system, and inhibited the subcutaneous growth of LLC tumors in mice.ConclusionDT385 possesses anti-angiogenic and anti-tumor activity and may have potential as a therapeutic agent.
Highlights
Diphtheria toxin (DT) is synthesized in Corynebacterium diphtheriae as a single-chain enzyme of 535 amino acids with a molecular weight of 63,000 [1,2]
We expressed a fusion protein in E. coli in which p22 was substituted for the receptorbinding domain of DT (DT385-p22)
The activity of this fusion protein was compared with recombinant DT385 and recombinant p22
Summary
Diphtheria toxin (DT) is synthesized in Corynebacterium diphtheriae as a single-chain enzyme of 535 amino acids with a molecular weight of 63,000 [1,2]. DT has been shown to enter toxinsensitive mammalian cells by receptor-mediated endocytosis which involves the interaction of the receptor-binding domain of the protein with a transmembrane cell surface precursor of the heparin-binding epidermal growth factor-like growth factor [3,4]. After binding to this cell-surface receptor, DT is endocytosed and trafficked to an acidic vesicular compartment, where it undergoes a pH-dependent conformational change, cleavage and release of the catalytic domain. We report that ‘‘receptorless’’ recombinant DT385 is cytotoxic to a variety of cancer cell lines
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