Impact of RGD Peptide Tethering to IL24/mda-7 (Melanoma Differentiation Associated Gene-7) on Apoptosis Induction in Hepatocellular Carcinoma Cells


Background: Melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24), a unique tumorsuppressor gene, has killing activity in a broad spectrum of cancer cells. Herein, plasmids producing mda-7proteins fused to different RGD peptides (full RGD4C and shortened RGD, tRGD) were evaluated for apoptosisinduction with a hepatocellular carcinoma cell line, Hep-G2. The study aim was to improve the apoptosis potencyof mda-7 by tethering to RGD peptides. Materials and
Methods: Three plasmids including mda-7, mda-7-RGDand 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 cytometryusing PI/annexin staining. To detect early events in apoptosis, 18 hours after transfection, expression of theBAX gene was quantified by real time PCR. Modeling of proteins was also performed to extrapolate possibleconsequences of RGD modification on their structures and subsequent attachment to receptors. Results and
Conclusions: In MTT assays, while all mda-7 forms showed measurable inhibition of proliferation, unmodifiedmda-7 protein exhibited most significant effect compared to control plasmid (P<0.001). Again, flow cytometryanalysis showed a significant apoptosis induction by simple mda-7 gene but not for those RGD-fused mda-7proteins. These findings were also supported by expression analysis of BAX gene (P<0.001). Protein modellinganalysis 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