Abstract
BackgroundEndostatin (ES) is a well-established potent endogenous antiangiogenic factor. An ES variant, called zinc-binding protein-ES (ZBP-ES), is clinically available; however, its use is limited by rapid renal clearance and short residence time. PEGylation has been exploited to overcome these shortcomings, and mono-PEGylated ES (called M2ES) as well as mono-PEGylated ZBP-ES (MZBP-ES) are developed in our study. This study aimed to compare the biophysical properties and biological effects of M2ES and MZBP-ES to evaluate their druggability.MethodsCircular dichroism and tryptophan emission fluorescence were used to monitor the conformational changes of M2ES and MZBP-ES. Their resistance to trypsin digestion and guanidinium chloride (GdmCl)-induced unfolding was examined by Coomassie staining and tryptophan emission fluorescence, respectively. The biological effects of M2ES and MZBP-ES on endothelial cell migration were evaluated using Transwell migration and wound healing assays, and the uptake of M2ES and MZBP-ES in endothelial cells was also compared by Western blotting and immunofluorescence.ResultsStructural analyses revealed that M2ES has a more compact tertiary structure than MZBP-ES. Moreover, M2ES was more resistant to trypsin digestion and GdmCl-induced unfolding compared with MZBP-ES. In addition, although M2ES and MZBP-ES showed comparable levels of inhibiting transwell migration and wound healing of endothelial cells, M2ES displayed an increased ability to enter cells compared with MZBP-ES, possibly caused by the enhanced interaction with nucleolin.ConclusionsM2ES has a more compact tertiary structure, is more stable for trypsin digestion and GdmCl-induced unfolding, exhibits increased cellular uptake and shows equivalent inhibitory effects on cell migration relative to MZBP-ES, indicating that M2ES is a more promising candidate for anticancer drug development compared with MZBP-ES.
Highlights
Endostatin (ES) is a well-established potent endogenous antiangiogenic factor
The Circular dichroism (CD) spectra results revealed that no obvious change in the secondary structure was observed between ES and zinc-binding protein-ES (ZBP-ES), whereas slight differences were observed between M2ES and mono-PEGylated ZBP-ES (MZBP-ES) (Fig. 1c)
Quantitative results showed that the initial reaction rate was 0.026 mg/ for M2ES and 0.037 mg/(mL·min) for mono-PEGylated ZBP-ES (MZBPES). These results showed that M2ES was more resistant to trypsin digestion than MZBP-ES
Summary
An ES variant, called zinc-binding protein-ES (ZBP-ES), is clinically available; its use is limited by rapid renal clearance and short residence time. As one of the hallmarks of cancer [1], the process of angiogenesis is exquisitely regulated by a variety of activators and inhibitors [2, 3] These proangiogenic and anti-angiogenic factors constitute a tightly. Wang et al Chin J Cancer (2016) 35:14 successfully clinically available [5] This ES variant, known as the zinc-binding protein-endostatin (ZBP-ES), was approved by the China Food and Drug Administration (CFDA) to treat non-small cell lung cancer (NSCLC) patients in China in 2005 [6]. Hindered by its relatively rapid renal clearance and short circulation half-life in vivo [10], ZBP-ES was recommended to be taken on a daily basis in clinic
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