Construction of expressing vectors including melanoma differentiation-associated gene-7 (mda-7) fused with the RGD sequences for better tumor targeting

Document Type: Original Article


1 Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

2 Cancer Immunology Research Group, Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

3 Sciences Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

4 Department Of Biochemistry, Payame Noor University, Tehran Shargh Branch, Tehran, Iran


Objective(s): Up to now, many researches have been performed to improve the antitumoral effect of melanoma differentiation-associated gene-7 (mda-7) protein. The purpose of our research was to construct 3 expression vectors producing mda-7 in fusion with RGD (Arginine-Glycine-Aspartic acid) peptide and evaluate their expression.    
Materials and Methods: mda-7 gene with two different RGD sequences was amplified by PCR then was cloned by TA–cloning system. The colonies including these genes were selected by blue–white screening, colony PCR, and sequencing, respectively. Afterward, the genes were sub-cloned into the expression vector following confirmation by colony PCR and sequencing. In addition, these constructs were transfected into 293 and Huh-7 cells for further expression analysis. The mda-7 gene expression was evaluated by RT-PCR and IF (immunofluorescence assay). DNA laddering test and trypan blue exclusion assays were performed to screen cytotoxicity of prepared plasmids.
Results: Three different mda-7 genes with terminal RGD peptide were cloned correctly into the expression vectors and their expression was confirmed to be suitable by RT-PCR and IF assay. It was shown that expressions were limited to those transfected, GFP shining cells. No significant cytotoxicity was observed by simple assays in all plasmid treated cells. In expressing cells, all forms of mda-7 protein were localized mainly around ER prenuclear compartment while GFP protein was distributed evenly among them.
Conclusion: Theoretically RGD tagged mda-7 would be able to induce apoptosis with more specificity and stronger than the standard one, therefore, these new constructs may have the potential for further researches.


1. Fisher P.B. Is mda-7/IL-24 a magic bullet for cancer? Cancer Res. 2005. 65:10128–10138.

2. Xue XB, Xiao Ch Wen, Zhang H,  Lu AG, Gao W, Zhou ZhQ, Guo XL. Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines. World J Gastroenterol 2010; 16:4677-4684.

3. Jiang H, Su ZZ, Lin JJ, Goldstein NI, Young CS, Fisher PB. The melanoma differentiation associated gene mda-7 suppresses cancer cell growth. Proc Nat Acad Sci 1996; 93:9160-9165.

4. Sarkar D, Su Z-Z, Lebedeva IV, Sauane M, Gopalkrishnan RV, Valerie K, et al. mda-7 (IL-24) mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK. Proc Nat Acad Sci 2002; 99:10054-10059.

5. Mhashilkar AM, Schrock RD, Hindi M, Liao J, Sieger K, Kourouma F, et al. Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy. Mol Med 2001; 7:12.

6. Lebedeva IV, Su ZZ, Chang Y, Kitada S, Reed JC, Fisher PB. The cancer growth suppressing gene mda-7 induces apoptosis selectively in human melanoma cells. Oncogene 2002; 21:708-718.

7. Saeki T, Mhashilkar A, Chada S, Branch C, Roth JA, Ramesh R. Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro. Gene Ther 2000; 7.

8. Cao XX I, Mohuiddin S, Chada AM, Mhashilkar MK, Ozvaran DJ, McConkey SD, et al. Adenoviral transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL. Mol Med 2002; 8:869-876.

9. Saeki T, Mhashilkar A, Swanson X, Zou-Yang XH, Sieger K, Kawabe S, et al. Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo. Oncogene 2002; 21:4558-4566.

10. Weber WA, Haubner R, Vabuliene E, Kuhnast B, Wester HJ,Schwaiger M. Tumor angiogenesis targeting using imaging agents. Q J Nucl Med 2001; 45:179-182.

11. Wang M, Tan Z, Zhang R, Kotenko SV, Liang P. Interleukin 24 (MDA-7/MOB-5) Signals through Two Heterodimeric Receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2. J Biol Chem 2002; 277:7341-7347.

12. Sauane M, Su Z-z, Gupta P, Lebedeva IV, Dent P, Sarkar D, et al. Autocrine regulation of mda-7/IL-24 mediates cancer-specific apoptosis. Proc Nat Acad Sci 2008; 105:9763-9768.

13. Cunningham CC, Chada S, Merritt JA, Tong A, Senzer N, Zhang Y, et al. Clinical and local biological effects of an intratumoral injection of mda-7 (IL24; INGN 241) in patients with advanced carcinoma: a phase I study. Mol Ther 2005; 11:149-159.

14. Liu JJ, Zhang BF, Yin XX, Pei DS, Yang ZX, Di JH, et al. Expression, purification, and characterization of RGD-mda-7, a His-tagged mda-7/IL 24 mutant protein. J Immunoassay Immunochem 2012; 33:352-368.

15. Jin H, Varner J. Integrins: roles in cancer development and as treatment targets. Br J Cancer 2004; 90:561-565.

16. Zitzmann S, Ehemann V, Schwab M. Arginine-glycine-aspartic acid (RGD)-peptide binds to both tumor and tumor-endothelial cells in vivo. Cancer Res 2002; 62:5139-5143.

17. Kumar  cc. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets 2003; 4:9.?

18. Takagi J, Strokovich K, Springer TA, Walz T. Structure of integrin [alpha]5[beta]1 in complex with fibronectin. EMBO J 2003; 22:4607-4615.

19. Xiao B, Li W, Yang J, Guo G, Mao X-H, Zou Q-M. RGD-IL-24, a novel tumor-targeted fusion cytokine: expression, purification and functional evaluation. Mol Biotechnol 2009; 41:138-144.

20. Pei D-S, Yang Z-X, Zhang B-F, Yin X-X, Li L-T, Li H-Z,   et al. Enhanced apoptosis-inducing function of MDA-7/IL-24 RGD mutant via the increased adhesion to tumor cells. J Interferon Cytokine Res 2012; 32:66-73.

21. Craig R, Cutrera J, Zhu S, Xia X, Lee Y-H, Li S. Administering Plasmid DNA Encoding Tumor vessel–anchored IFN-α for Localizing Gene Product Within or Into Tumors. Mol Ther 2008; 16:901-906.

22. Wang X, Bai C, Zhang J, Sun A, Wang X, Wei D. Improving the mda-7/IL-24 refolding and purification process using optimized culture conditions based on the structure characteristics of inclusion bodies. Bioresources and Bioprocessing 2014, 1:21.

23. Kreis S, Philippidou D, Margue C, Rolvering C, Haan C, Dumoutier L, et al. Recombinant interleukin-24 lacks apoptosis-inducing properties in melanoma cells. PloS One 2007; 2:e1300.

24. Temming K, Schiffelers RM, Molema G, Kok RJ. RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature. Drug Resist Update 2005; 8:381-402.

25. Momtazi-borojeni Aa, Behbahani M, Sadeghi-aliabadi H. Antiproliferative activity and apoptosis induction of crude extract and fractions of avicennia marina. Iran J Basic Med Sci 2013; 16:1204-1208.

26. Sauane M, Gopalkrishnan RV, Lebedeva I, Mei MX, Sarkar D, Su ZZ, et al. Mda-7/IL-24 induces apoptosis of diverse cancer cell lines through JAK/STAT-independent pathways. J Cell Physiol 2003; 196:334-345.

27. Wang CJ, Xue XB,  Yi JL, Chen K, Zheng JW, Wang J, et al. Melanoma differentiation-associated gene-7, MDA-7/IL-24, selectively induces growth suppression, apoptosis in human hepatocellular carcinoma cell line HepG2 by replication-incompetent adenovirus vector. World J Gastroenterol  2006; 12:1774-1779.

28. Chen WY, Cheng YT, Lei HY, Chang CP, Wang CW, Chang MS. IL-24 inhibits the growth of hepatoma cells in vivo. Genes Immun 2005; 6:493-499.

29. Hynes RO. Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 1992; 69:11-25.

30. Jin ZH, Furukawa T, Claron M, Boturyn D, Coll JL, Fukumura T, et al. Positron emission tomography imaging of tumor angiogenesis and monitoring of antiangiogenic efficacy using the novel tetrameric peptide probe 64Cu-cyclam-RAFT-c(-RGDfK-)4. Angiogenesis 2012; 15:569-580.