Production and Evaluation of In-vitro and In-vivo Effects of P28-IL24, a Promising Anti-breast Cancer Fusion Protein

Abstract

Interleukin-24 (IL-24) is known to selectively induce apoptosis in cancer cells through endoplasmic reticulum (ER) stress. However, this protein alone is unable to enter cells and consequently it is not able to inhibit cell proliferation. In this study, we developed a novel recombinant chimeric protein containing IL-24 which is linked to P28, a cancer specific cell-penetrating peptide and P53 stabilizer, to target IL-24 into breast cancer cells. After design, expression and purification of the P28-IL-24 fusion protein and IL-24 as control, specific and non-specific cytolethal effects of this fusion protein were evaluated in-vitro and in-vivo. Cell proliferation assay and cell death mechanism analysis carried out by MTT and flow cytometry, respectively. Afterward, anti-tumor effects of P28-IL-24 in animal model were investigated by hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, respectively. Results showed inhibitory effects on proliferation of MCF-7 and MDA-MB-231 cancer cells by the P28-IL-24 protein. However, HUVEC cells were affected by the fusion protein just at 1.8 mu g/ml concentration. In addition, p28-IL-24 showed to induce apoptosis in the mentioned cell lines. Moreover, treatment of inbred Balb/C mice bearing syngenic 4T1 tumor cells with the p28-IL-24 reduced tumor size within the study period (p < 0.05). H&E staining and TUNEL assay, revealed that this tumor growth suppression was associated with increase in necrotic and apoptotic cells. Taken together, the findings of the current study suggest that the chimeric protein p28-IL-24 can serve as a potent candidate for further preclinical evaluations.