title: Impact of RGD Peptide Tethering to IL24/mda-7 (Melanoma Differentiation Associated Gene-7) on Apoptosis Induction in Hepatocellular Carcinoma Cells.
abstract: BACKGROUND
  Melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24), a unique tumor suppressor gene, has killing activity in a broad spectrum of cancer cells. Herein, plasmids producing mda-7 proteins fused to different RGD peptides (full RGD4C and shortened RGD, tRGD) were evaluated for apoptosis induction with a hepatocellular carcinoma cell line, Hep-G2. The study aim was to improve the apoptosis potency of mda-7 by tethering to RGD peptides.
  
  
  METHODS
  Three plasmids including mda-7, mda-7-RGD and mda-7-tRGD genes beside a control vector were transfected into Hep-G2 cells. After 72 hours incubation, cell viability was evaluated by MTT assay. In addition, the rate of apoptosis was analyzed by flow cytometry using PI/annexin staining. To detect early events in apoptosis, 18 hours after transfection, expression of the BAX gene was quantified by real time PCR. Modeling of proteins was also performed to extrapolate possible consequences of RGD modification on their structures and subsequent attachment to receptors.
  
  
  CONCLUSIONS
  In MTT assays, while all mda-7 forms showed measurable inhibition of proliferation, unmodified mda-7 protein exhibited most significant effect compared to control plasmid (P<0.001). Again, flow cytometry analysis showed a significant apoptosis induction by simple mda-7 gene but not for those RGD-fused mda-7 proteins. These findings were also supported by expression analysis of BAX gene (P<0.001). Protein modelling analysis revealed that tethering RGD at the end of IL-24/Mda7 disrupt attachment to cognate receptor, IL-20R1/ IL-20R2. In conclusion, fusion of RGD4C and shortened RGD peptides to carboxyl terminal of mda7, not only reduce apoptosis property in vitro but also disrupt receptor attachment as demonstrated by protein modelling.
authors: Gastroenterohepatology Research Center (GEHRC), Shiraz University of Medical Sciences, Shiraz, Iran E-mail : Erfanin@sums.ac.ir, Hosseinisy22@yahoo.com.
authors: Samaneh
authors: S
authors: Bina
authors: Fatemeh
authors: F
authors: Shenavar
authors: Mahboobeh
authors: M
authors: Khodadad
authors: Mohammad Reza
authors: MR
authors: Haghshenas
authors: Mojtaba
authors: M
authors: Mortazavi
authors: Mohammad-Reza
authors: MR
authors: Fattahi
authors: Nasrollah
authors: N
authors: Erfani
authors: Seyed Younes
authors: SY
authors: Hosseini
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology* / Q000628:therapy*; D049109:Cell Proliferation; D005434:Flow Cytometry; D015316:Genetic Therapy; D006801:Humans; D007378:Interleukins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D009842:Oligopeptides / Q000378:metabolism*; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D000099180:Interleukin-24
chemical_list: D007378:Interleukins; D009842:Oligopeptides; D012333:RNA, Messenger; D000099180:Interleukin-24; C047981:arginyl-glycyl-aspartic acid