Cell‐penetrating and endoplasmic reticulum‐locating TAT‐IL‐24‐KDEL fusion protein induces tumor apoptosis

Abstract

Interleukin-24 (IL-24) is a unique IL-10 family cytokine that could selectively induce apoptosis in cancer cells without harming normal cells. Previous research demonstrated that intracellular IL-24 protein induces an endoplasmic reticulum (ER) stress response only in cancer cells, culminating in apoptosis. In this study, we developed a novel recombinant fusion protein to penetrate into cancer cells and locate on ER. It is composed of three distinct functional domains, IL-24, and the targeting domain of transactivator of transcription (TAT) and an ER retention four-peptide sequence KDEL (Lys-Asp-Glu-Leu) that link at its NH2 and COOH terminal, respectively. The in vitro results indicated that TAT-IL-24-KDEL inhibited growth in bladder cancer cells, as well as in non-small cell lung cancer cell line and breast cancer cell line, but the normal human lung fibroblast cell line was not affected, indicating the cancer specificity of TAT-IL-24-KDEL. Western blot analysis showed that apoptosis activation was induced by TAT-IL-24-KDEL through the ER stress-mediated cell death pathway. Treatment with TAT-IL-24-KDEL significantly inhibited the growth of human H460 xenografts in nude mice, and the tumor growth inhibition was correlated with increased hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. These findings suggest that the artificially designed recombinant fusion protein TAT-IL-24-KDEL may be highly effective in cancer therapy and worthy of further evaluation and development.

Breaking in and busting out: cell-penetrating peptides and the endosomal escape problem

“…The predominant approach for targeting CPP-bound cargo calls for the fusion of canonical signal sequences to cargo proteins, or even to the CPP tag itself (reviewed in (54)). Using this methodology, cargos have been delivered to the nucleus (55, 56), nucleolus (57), lysosome (58), peroxisome (55), the mitochondria (55, 59, 60) and the endoplasmic reticulum (55, 61), to name a few. Drawbacks to this approach include decreased uptake of tagged CPPs (61) and potential endosomal entrapment en route to the intended compartment.…”

TAT-IL-24-KDEL-induced apoptosis is inhibited by survivin but restored by the small molecular survivin inhibitor, YM155, in cancer cells

“…The levels of BiP/GRP78, phosphorylation of eIF2α, JNK, and c-Jun increased in a concentration-dependent manner (Figure 2D). These results indicated that TAT-IL-24-KDEL induced cancer cell apoptosis via the cell death pathway mediated by ER stress [26]. In addition, the activities of caspase-3 and caspase-7 were increased in a dose-dependent manner (Figure 2E).…”

“…In a previous paper, we established an efficient method for obtaining recombinant TAT-IL-24-KDEL in an Escherichia coli expression system containing a SUMO tag [26]. The DNA sequences coding TAT and KDEL were fused with the IL-24 (GenBank No.…”

“…After cleavage, the mixture was passed through the Ni-NTA affinity column and the recombinant TAT-IL-24-KDEL was recovered in the flow-through fraction. To evaluate the transfection efficiency of TAT-IL-24-KDEL, fluorescein isothiocyanate (FITC) was conjugated to the fused protein for the feasibility of localization [26]. Cells were co-cultured with FITC-labeled TAT-IL-24-KDEL for 1 h, and the green fluorescence of these samples was analyzed by flow cytometer (BD FACSCalibur TM ).…”

“…These peptides prevent the secretion of such proteins by binding with the KDEL receptors localized in the intermediate compartment and Golgi apparatus [24, 25]. In previous studies, we linked TAT and KDEL to the N-terminal and C-terminal of IL-24, respectively, and established an efficient method for obtaining recombinant TAT-IL-24-KDEL in an Escherichia coli expression system [26]. TAT-IL-24-KDEL has been shown to efficiently transfer into tumor cells and locate on ER, consequently inducing cell apoptosis to a much greater extent than IL-24 and TAT-IL-24.…”