In vivo anti-tumor effect of expressing p14ARF-TAT using a FGF2-targeted cationic lipid vector.
OBJECTIVE
To develop an efficient and safe strategy to introduce a therapeutic gene into target cells in vivo for cancer therapy. The overall efficiency is based on proper selection of the delivery vector and expressed protein.


METHODS
A plasmid coding for a specific cytotoxic fusion peptide, p14ARF-TAT, was evaluated in a xenograft mouse tumor model. The expressed peptide consisted of three domains, a secretory signal, a membrane permeability segment and a cytotoxic fragment. Gene expression was verified in U87-MG cells by Western blot and cytotoxicity confirmed with CyQuant assay. To improve the delivery, a FGF2 targeting peptide, MQLPLATC, was incorporated into the vector, which was evaluated using a luciferase-expressing plasmid.


RESULTS
The luciferase activity in vitro was two-fold higher with the targeted formulations, and cytotoxicity was three-fold higher with expression of the p14ARF-TAT protein. A murine xenograph model of human glioma (U87-MG cells) tumors was used to address in vivo activity. FGF2-targeted lipoplexes demonstrated increased tumor volume reduction as compared to non-targeted formulations. RT-PCR and Western blot of tumor homogenizes indicated p14ARF-TAT expression in tumors along with other tissues.


CONCLUSIONS
p14ARF-TAT was cytotoxic and is a promising approach when combined with an efficient targeting.
Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, Florida 32610, USA.
Guoqin
G
Niu
Wouter H P
WH
Driessen
Sean M
SM
Sullivan
Jeffrey A
JA
Hughes
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002412:Cations; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002470:Cell Survival; D016222:Fibroblast Growth Factor 2 / Q000235:genetics*; D015696:Gene Products, tat / Q000235:genetics*; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D006801:Humans; D020287:In Situ Nick-End Labeling; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D015394:Molecular Structure; D009368:Neoplasm Transplantation; D010316:Particle Size; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D011092:Polyethylene Glycols / Q000737:chemistry*; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014162:Transfection; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics*
D002412:Cations; D015696:Gene Products, tat; D010714:Phosphatidylethanolamines; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins; D025803:Tumor Suppressor Protein p14ARF; C095031:pyridyldithiopropionoylamino-poly(ethylene glycol)-distearoylphosphatidylethanolamine; D016222:Fibroblast Growth Factor 2; D011092:Polyethylene Glycols; D008156:Luciferases
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