title: 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.
authors.affiliation: Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 1411713116, Iran.
authors.forename: Ebrahim
authors.initials: E
authors.lastname: Hosseini
authors.affiliation: Bacteriology and Virology Department, School of Medicine, Shiraz University of Medical Sciences, Shiraz 71348‑14336, Iran.
authors.forename: Seyed Younes
authors.initials: SY
authors.lastname: Hosseini
authors.affiliation: Clinical Microbiology Research Center, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz 71348‑45794, Iran.
authors.forename: Tayebeh
authors.initials: T
authors.lastname: Hashempour
authors.affiliation: Gastroenterohepatology Research Center (GEHRC), Shiraz University of Medical Sciences, Shiraz 71937‑11351, Iran.
authors.forename: Mohammad-Reza
authors.initials: MR
authors.lastname: Fattahi
authors.affiliation: Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 1411713116, Iran.
authors.forename: Majid
authors.initials: M
authors.lastname: Sadeghizadeh
mesh_terms: D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D002460:Cell Line; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D056945:Hep G2 Cells; D006801:Humans; D007378:Interleukins / Q000235:genetics*; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D009842:Oligopeptides / Q000235:genetics*; D014162:Transfection / Q000379:methods; D000099180:Interleukin-24
chemical_list: D007378:Interleukins; D009842:Oligopeptides; D000099180:Interleukin-24; C047981:arginyl-glycyl-aspartic acid