Pro-apoptotic peptides have attracted much attention as promising anticancer agents due to their high activity. However, poor cellular uptake of the peptides is often associated with limited therapeutic application. Cell-penetrating homing peptides (CPHPs) were found to increase cell internalization as well as anticancer efficacy of the peptide conjugates. In this study, we developed a novel recombinant fusion protein composed of sIL-24 peptide as a pro-apoptotic moiety and asparagine-glycine-arginine (NGR) motif as a CD13-targeting CPHP component. In silico analysis demonstrated that flexible GGGGS linker provided the best structure and stability for our designed fusion protein. Cell adhesion experiments showe... More
Pro-apoptotic peptides have attracted much attention as promising anticancer agents due to their high activity. However, poor cellular uptake of the peptides is often associated with limited therapeutic application. Cell-penetrating homing peptides (CPHPs) were found to increase cell internalization as well as anticancer efficacy of the peptide conjugates. In this study, we developed a novel recombinant fusion protein composed of sIL-24 peptide as a pro-apoptotic moiety and asparagine-glycine-arginine (NGR) motif as a CD13-targeting CPHP component. In silico analysis demonstrated that flexible GGGGS linker provided the best structure and stability for our designed fusion protein. Cell adhesion experiments showed a significant binding affinity toward high CD13-expressing cells (U937 and A549) for NGR-sIL-24. Moreover, confocal microscopy revealed that NGR strongly facilitated the binding and cellular uptake of sIL-24 in U937 and A549 cancer cells. NGR-sIL-24 treatment markedly inhibited the growth of U937 and A549 cancer cells in a dose and time-dependent manner, without affecting the normal cell line MRC-5. Flow cytometric analysis and Hoechst 33342 staining exhibited potent apoptosis induction in U937 and A549 cells treated with NGR-sIL-24. Further mechanism elucidation uncovered that apoptotic death promoted by NGR-sIL-24 was attributed to upregulation of BiP/GRP78, Bax/Bcl-2, GADD34, cytochrome c release, and cleavage of caspase-3, suggesting NGR-sIL-24 penetration into cancerous cells and subsequent apoptosis induction, mainly through endoplasmic reticulum (ER) stress-dependent and mitochondria-dependent signaling pathways. Our results indicate that the designed recombinant fusion protein NGR-sIL-24 may serve as a potential targeted therapy agent for cancers with high expression of CD13.
