[Expression of TRAIL(114-281) mediated by adeno-associated virus and its tumoricidal activity].
OBJECTIVE
To investigate the expression of the soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated by adeno-associated virus (AAV) and its tumoricidal activity in vitro and vivo.


METHODS
The recombinant AAV expression vector encoding the extracellular domain (114-281aa peptide, TRAIL(114-281)) of TRAIL was constructed and transfected into human embryotic kidney cells HEK293 for virus package. The human tumor cell lines of T lymphocyte leukemia Jurkat, liver cancer HepG2 and SMMC-7721, and cervical cancer HeLa were transduced by using the recombinant virus particles respectively. The recombinant virus particles were also injected into C57BL/6 mice via the hepatic portal vein or hypodermic, intramuscular, celiac and oral pathways to study the expression of TRAIL(114-281). The recombinant virus titer was determined by real-time PCR. The expression of TRAIL(114-281) was evaluated by ELISA, Western blotting and immunohistochemistry assay respectively. The tumoricidal activity and apoptosis were evaluated by MTT assay.


RESULTS
The recombinant AAV encoding for the soluble TRAIL (114 - 281aa) were constructed successfully. The titer of recombinant virus was 7.5 x 10(12) genome particles (Gps)/ml. Transduction of rAAV-TRAIL(114-281) led to high level expression of TRAIL(114-281) and the induction of apoptosis of Jurkat, Hela and SMMC-7721 cancer cells, but not HepG2 cells, in vitro. The recombinant peptide TRAIL(114-281) in trimeric active form was highly and constantly expressed in the hepatocytes and secreted into the serum up to 6 months in the of C57BL/6 mice injected with the recombinant virus particles via the hepatic portal vein. The peptide TRAIL(114-281) in the livers, but not other tissues, were also detected in the mice administrated with rAAV-TRAIL(114-281) particles via subcutaneous, intramuscular, intraceliac or oral pathway.


CONCLUSIONS
The long term, stable and liver-tropism expression of peptide TRAIL(114-281) in mice mediated by rAAV-TRAIL(114-281) provides a prospective novel strategy for tumor gene therapy of numerous cancers, especially liver cancer.
Ma
Hong
H
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union medical Sciences, Beijing 100005, China.
Liu
Yan-Xin
YX
Liu
Shi-Lian
SL
Xu
Rui-An
RA
Zheng
De-Xian
DX
D000260:Adenoviruses, Human / Q000235:genetics; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D015316:Genetic Therapy; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008113:Liver Neoplasms / Q000473:pathology; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000494:pharmacology*
D051017:Apoptosis Regulatory Proteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; C505654:Tnfsf10 protein, mouse; D014409:Tumor Necrosis Factor-alpha