Human Soluble TRAIL Gene Therapy Of Liver Cancer Mediated By Recombinant Adenovirus Vector

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is one of several members of the TNF gene superfamily that induce apoptosis through engagement of death receptors. TRAIL is unusual as compared to any other cytokine as it interacts with a complex system of receptors: two pro-apoptotic death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) and two anti-apoptotic decoys (TRAIL-R3/DcRl and TRAIL-R4/DcR2). It selectively induces apoptosis in many transformed cells but not in normal cells. Because of this feature, TRAIL has generated tremendous excitement as a potential tumor-specific cancer therapeutic.To investigate the antitumor effect of TRAIL mediated by recombinant adenovirus vector, The human soluble TRAIL gene was cloned from human peripheral mononuclear cells by RT-PCR and nucleic acid sequence is as same as Genebank described. Recombinant adenovirus vector carrying TRAIL was constructed by using Cre/loxP system, which is also called site specific recombinant system. TRAIL was cloned to adenovirus shuttle vector pDC315. After cotransformation 293 cell with pBGHloxPAE!E3 by Lipofectamine then recombinat generated and amplified. The lyses supernatant of 293 waspurified by gradient ultraspeed centrifuge through three times of "infection-collection-freeza/thaw". Titer of Ad-TRAIL was 7X 10lopnVml.In vitro infection of human liver cancer cell line with Ad-TRAIL at MOI 0.1 can lead to 5 % > 58 % and 95 % infection rate respectively after 3 ^ 7^ 10 days measured by EGFP expression. The mRNA of TRAIL was detected in the infected liver cancer cell by RT-PCR. The TRAIL protein was 32.5 KD detected by western blot, and protein was 10ng/106/48h in the infected cell culture supernant by ELISA. All date suggested Ad-TRAIL can infect liver cancer cell efficiently and mediate the expression of TRAIL in vitro. MTT assay showed the ability of Ad-TRAIL is different in different liver cancer cells. Hep3B and PLC/PR/F5 were more sensitive to Ad-TRAIL while SMMC7721 and HepG2 were not sensitive. In control group Ad-EGFP can't effect the activity of cells unless at high MOI. This implied Ad-TRAIL can inhibit growth of liver cancer cell. SMMC7721 is more sensitive to Ad-TRAIL when caspase 3 gene was transformed.Intratumor injection of 1 X 109pfii Ad-TRAIL had significant therapeutic effect. Establish human liver cancer model by subcutaneous inoculation 1 X 108 Hep3B on right back flank of BALB/c mice. After five introtumor injections of 2 X 108 pfu Ad-TRAIL * Ad-EGFP and PBS, the tumor mass was observed. In contrast with control group, Ad-TRAIL can significantly inhibit tumor growth of mice 28 days after treatment and tumor mass were 62.17 +6.84 106.12 + 23.5x 124.86 + 28.77 mm3 respectively(p<0.05). Pathological staining (TUNEL) and electric microscopy showed that there were significantly more intratumor apoptosis than control group. In a word, Ad-TRAIL can inhibit the tumor of HepSB efficiently in vivo. The research paved a way for further gene therapy research of liver cancer base on human soluble TRAIL gene.