ABSTRACT
Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Subject(s)
Cell-Penetrating Peptides/pharmacology , Drug Carriers/pharmacology , Heparin-binding EGF-like Growth Factor/pharmacology , Recombinant Fusion Proteins/pharmacology , Ribosome Inactivating Proteins, Type 2/pharmacology , Amino Acid Sequence , Apoptosis/drug effects , Cell Line, Tumor , Cell-Penetrating Peptides/biosynthesis , Cell-Penetrating Peptides/genetics , Cloning, Molecular , Dose-Response Relationship, Drug , Drug Carriers/chemistry , Drug Carriers/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , HeLa Cells , Heparin/chemistry , Heparin/metabolism , Heparin-binding EGF-like Growth Factor/biosynthesis , Heparin-binding EGF-like Growth Factor/genetics , Humans , Momordica charantia/chemistry , Protein Binding , Protein Domains , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Ribosome Inactivating Proteins, Type 2/biosynthesis , Ribosome Inactivating Proteins, Type 2/genetics , S Phase/drug effects , Signal TransductionABSTRACT
Current databases of Phyllostachys pubescens full-length cDNAs (FL-cDNAs) provide a rich source of sequences for the development of potential FL-cDNA simple sequence repeat (SSR) markers. We screened 10,608 P. pubescens cDNAs, discovering 1614 SSRs in 1382 SSR-containing FL-cDNAs. The SSRs were more abundant within transposable elements (TEs) than expressed sequence tags (ESTs) and genome survey sequences (GSSs), and specific dinucleotide repeats tended to associate with particular TE families: (TA)n with En/Spm and (CT)n with Mutator. A selected panel of 100 FL-cDNAs containing type I SSRs yielded 68 functional SSR markers with an average polymorphism information content (PIC) value of 0.12, among which 22 loci contained polymorphisms. These markers became less transferrable (83.1% â 69.9% â 49.3%) but more polymorphic (79.4% â 92.3% â 92.8%) with increasing phylogenetic distance (intra-genus â intra-subtribe â intra-family). Transferability and polymorphism also depended on the location of the marker, with those located in the coding region being more transferrable (69.1%) and less polymorphic (89.4%) than those in the 5'-UTR (63.4% transferable, 90.7% polymorphic) and the 3'-UTR (61.8% transferable, 91.4% polymorphic). As proof of principle, we were able to use our FL-cDNA SSR markers to identify the parental stocks in interspecific hybrids of bamboo within and beyond P. pubescens, and estimate the outcrossing rate for P. pubescens. Our research should facilitate molecular breeding in bamboo species where original genetic markers are scarce.
