Antitumor Activity Of An Adenovirus Harboring Two Therapeutic Genes, Anti-VEGF Ribozyme And Human IL-24, In Colon Cancer

Background and Objective: The morbidity of colon cancer is increasing, but the current treatment for colon cancer is still unsatisfactory, especially for relapsed and metastatic patients. VEGF plays a key role in tumor angiogenesis. In general, VEGF is commonly upregulated in colon cancer, and increased VEGF expression and tumor vascularization are correlated with tumor progression and a poor clinical prognosis in colon cancer. The hairpin ribozyme is a small RNA molecule with endoribonuclease activity that exhibits catalytic sequence-specific cleavage of the target RNA and down-regulates the expression of target gene.Mda-7/IL-24 (melanoma differentiation associated gene-7) is a member of the IL-10 subfamily .When expressed at high levels, Mda-7/IL-24 suppresses growth and induces programmed cell death (apoptosis) in a broad spectrum of human cancers, including colon cancer. In contrast, Mda-7/IL-24 does not induce apoptosis in, and has a negligible effect on the growth of, normal cells. In this study, we constructed an adenovirus that simultaneously expresses anti-VEGF hairpin ribozyme and human IL-24 and examined its VEGF expression inhibitory effect and anti-tumor efficacy against human colon cancer HT-29 cells in vitro and in vivo.Methods: Designing and synthesizing the double strands DNA which encodes the anti-VEGF hairpin ribozyme according to declared VEGF165 gene sequence from GenBank. These oligonucleotide sequences, which include Bgl II and Sal I linker sites at each end, were inserted into the multiple cloning site (MCS) of the shuttle plasmid pTrack-CMV which harbored an IRES between Sal I and Not I digestion sites and human IL-24 cDNA between Xho I and Xba I sites (previously constructed by the Cell and Molecular Biology Institute of Soochow University). The resultant shuttle vector (pAdTrack-CMV-Rz/IL-24) was linearized by the restriction endonuclease PmeI and subsequently cotransformed into BJ5183 bacterial cells with the adenoviral backbone plasmid (pAdEasy-1). After PacI digestion, the recombinant pAd-Rz/IL-24 plasmid was transfected into human embryonic kidney 293 (QBI-293A) cells with lipofectamine (Sigma; CA) to package the viruses. The constructed adenovirus, Ad-Rz/IL24 ,were amplified in QBI-293A cells. The expression of anti-VEGF hairpin ribozyme and IL-24 mRNA in HT-29 cells were detected by RT-PCR and the inhibitory effect of VEGF expression were tested by Real-time PCR and Elisa. The effect of growth inhibition and apoptosis-inducing of Ad-Rz/IL24 on HT-29 cells were detected by MTT and FCAS. The effect and mechanism of Ad-Rz/IL24 treatment on colon carcinoma in vivo were observed and studied through the HT-29 human colon cancer subcutaneous model in nu/nu mice. The growth inhibition effect of Ad-Rz/IL24 on xenografts was teseted and IL-24, GADD, and VEGF expression in xenograft tumor tissue were detected by Immunohistochemical analysis. The microvessle density (MVD) of the xenograft tumor was determined by CD34 staining.Results: The anti-VEGF ribozyme and IL-24 were successfully inserted into the adenoviral vector. The adenovirus of Ad-Rz/IL24 can effectively infect the colon cancer HT-29 cells. Anti-VEGF ribozyme and IL-24 could effectively express in HT-29 cells after treated by Ad-Rz/IL24. Ad-Rz/IL24 could inhibit the expression of VEGF mRNA in HT-29 cells .The relative expression of VEGF mRNA in HT-29 cell treated with Ad-Rz/IL24, Ad-Rz (adenovirue carrying anti-VEGF ribozyme ) or Ad-IL-24 (adenovirue carrying IL-24 )decreased to about (39.0±1.0)%, (45.0±1.0)%, or (91.0±1.0)% of that of PBS control group in HT-29 cells respectively(P<0.05) and the VEGF expression in cells treated with Ad-GFP (vacant vector) was about (122.0±2.0)%. The amount of VEGF protein in the supernatant of Ad-Rz/IL24, Ad-Rz ,Ad-IL-24 ,Ad-GFP or PBS treated cells were 0.37±0.28/million cells, 0.46±0.35/ million cells, 0.56±0.30/ million cells ,0.74±0.13/ million cells ,and 0.81±0.16/ million cells, respectively (P<0.05). MTT assay showed significant growth inhibition for colon cancer cell lines by Ad-Rz/IL-24 or Ad-IL-24 (p<0.05, compared to PBS), while cells treated with Ad-Rz did not show significant growth inhibition (p > 0.05). Ad-Rz/IL-24 treatment induced apoptosis in about (11.0±0.8) % of the cells, which was about twice as much as Ad-IL-24 treatment (The apoptosis induced by Ad-IL-24 is about (5.6±0.3) %). Ad-Rz treatment induced apoptosis in only about (1.8±0.2) % of the cells, but it was still significantly different from both the control and Ad-GFP-treated cells (p <0.05). The growth of xenograft tumors were inhibited to some extent by Ad-Rz/IL24 treatment, but the significance was under borderline (P=0.0619, CMH test, compared to PBS). IL-24 and GADD protein was detected only in the Ad-Rz/IL-24 and Ad-IL-24 treated groups. Ad-Rz/IL24 can significantly inhibit the VEGF expression in xenograft tumor tissue and the MVD of xenograft tumors treated with Ad-Rz/IL24, Ad-Rz ,Ad-IL-24 ,Ad-GFP or PBS were 3.5±0.6, 4.3±0.5, 6.0±1.0, 11.5±1.0 and 12.8±11.1, respectively(P<0.05).Conclusion: Dual-gene, anti-VEGF hairpin ribozyme and interleukin-24, mediated by adenovirus(Ad-Rz/IL24) can significantly down-regulate the expression of VEGF in HT-29 cells and inhibit tumor growth of colon cancer HT-29 cells. Forthermore, Ad-Rz/IL24 can significantly inhibit the tumor angiogenesis and inhibit the xenograft tumor growth to some extent in vivo.