Effect of RGD coupled MDA-7/IL-24 on apoptosis induction in a hepatocellular carcinoma cell line

Abstract

The melanoma differentiation-associated gene-7 (MDA-7) gene, also termed interleukin-24 (IL-24), is a tumor suppressor gene that induces apoptosis in a broad scope of malignant neoplastic cells. The apoptosis induction capacity of the MDA-7/IL-24 gene is partially associated with adhering to cognate receptors. The current study aimed to enhance the antitumor effect of IL-24. The intrinsic signal sequence of IL-24 replaced with a fused artificial signal (secrecon)-RGD4C sequence and its impact was evaluated in HepG2 cells. The modified SP.RGD.IL-24 and native IL-24 cDNA sequences were cloned into the pcDNA3.1 expression vector. Subsequently, the expression level, secretion efficacy and targeting propensity of the modified SP.RGD.IL-24 product compared with normal IL-24 by were determined by enzyme-linked immunosorbent assay. The constructs were then transfected into HepG2 and LX-2 cells as tumor and normal hepatic cell lines, respectively. The expression level of the pro-apoptotic DNA damage inducible transcript 3 (Gadd153) and BCL2 associated X apoptosis regulator (Bax) genes in the different groups were compared by reverse transcription-quantitative polymerase chain reaction. Additionally, the rate of apoptosis induction of modified and intact IL-24 sequences was compared by flow cytometry analysis of cells following their propidium iodide/annexin V staining. SP.RGD-IL-24 protein was expressed and secreted in a similar manner to native IL-24, however, the modified SP.RGD.IL-24 adhered to tumor cells more efficiently than IL-24 (P<0.05). SP.RGD.IL-24 significantly induced upregulation of Gadd153 and Bax in HepG2 cells compared with native IL-24 (P<0.05). However, neither had a significant impact on the expression level of pro-apoptotic genes in LX-2 cells. Flow cytometry analysis also indicated that modified SP.RGD.IL-24 induced apoptosis more than native IL-24 in HepG2 cells (P<0.05). In conclusion, the novel generated RGD-coupled IL-24 construct exhibited sufficient anticancer activity compared with the native IL-24. The results of the current study provide novel insights for the future of cytokine targeting and indicates its potential capacity as a valuable candidate for gene therapy methods.