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.

中文翻译：

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新型融合蛋白NGR-sIL-24可通过凋亡靶向抑制癌细胞的生长。

促凋亡肽由于其高活性而作为有希望的抗癌剂引起了广泛的关注。然而，肽对细胞的不良吸收通常与有限的治疗应用有关。发现细胞穿透性归巢肽（CPHP）可以增加细胞内化以及肽缀合物的抗癌功效。在这项研究中，我们开发了一种新型重组融合蛋白，该蛋白由sIL-24肽作为促凋亡部分，而天冬酰胺-甘氨酸-精氨酸（NGR）主题作为CD13靶向CPHP组件。在计算机分析中表明，灵活的GGGGS接头为我们设计的融合蛋白提供了最佳的结构和稳定性。细胞粘附实验表明，它对高表达CD13的细胞（U937和A549）具有NGR-sIL-24的显着结合亲和力。此外，共聚焦显微镜显示，NGR强烈促进了U937和A549癌细胞中sIL-24的结合和细胞摄取。NGR-sIL-24处理以剂量和时间依赖性方式显着抑制U937和A549癌细胞的生长，而不会影响正常细胞系MRC-5。流式细胞仪分析和Hoechst 33342染色在用NGR-sIL-24处理的U937和A549细胞中显示出有效的凋亡诱导作用。进一步的机制揭示发现，NGR-sIL-24促进凋亡的死亡归因于BiP / GRP78，Bax / Bcl-2，GADD34，细胞色素c的释放和caspase-3的裂解，这表明NGR-sIL-24渗透进入主要通过内质网（ER）应激依赖性和线粒体依赖性信号传导途径诱导癌细胞和随后的凋亡诱导。