Experimental Study Of Linamarase Gene-directed Enzyme Prodrug Suicide System For The Treatment Of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignances worldwide, and there is no effective treatment for it until now. Suicide gene therapy, also known as gene-directed enzyme prodrug therapy, is an important strategy for tumor treatment by improving the specifical concentrations of drugs in tumors and a unique bystander effect. The research here presents a novel suicide gene therapy strategy against HCC based on the linamarase (lis), a gene from cassava, which can hydrolyze prodrug linamarin (lin) into HCN. Lis cDNA was stably expressed in HCC cells. Studies in vitro indicated that stable expression of lis had no direct effects on the proliferation and cell cycle of hepatoma cells. In addition, the administration of lin lonely is harmless too, indicating that the system is safe. In order to enhance target gene expression in HCC cells, we reconstructed adenoviruses involving lis with CMV or AFP promoter based on ViraPower? adenovirus system respectively. In vitro studies showed that Ad-CMV-lis combined with lin had strong antitumor effect and bystander effect on a variety of tumor cells. While Ad-AFP-lis with lin only showed inhibiting effect on AFP-positive hepatoma cells, which indicated the target specificity of the adenovirus to HCC. Animal experiments showed that lis was expressed highly in the tissue of tumors specifically , and significantly inhibited tumor growth and prolonged animal survival combined with lin. All findings suggest that lis/lin system may provide a practical and promising approach for the treatment of HCC and be worthy of further study.Objective:1. To construct a new hepatoma cell line which can stably express lis cDNA and evaluate the activity of lis enzyme expressing in eukaryotic cells. To observe the effect of lis on the biological characteristics of hepatoma cells in vitro and in vivo.2. At the base of ViraPower ? adenovirus system, to reconstruct an adenoviruse vector containing lis cDNA which is regulated by CMV promoter. To Evaluate the infective efficiency of the virus and the level of gene expression in cells infected by the adenovirus. The antitumor effect and the mechanism of the adenovirus mediated lis / lin system is explored in vitro and in vivo.3. To clone AFP promoter and detect its target transcription activity. Reconstruction of an adenovirus containing lis cDNA transcripted by AFP promoter. To test the inhibitory effect of the system against the AFP positive HCC cells by in vitro and tumor-bearing nude mice. Finally, achieving the targeted transcription regulation of lis / lin system in HCC.Methods:1. Lis cDNA was cloned and amplified from cassava cDNA by PCR method. the sequence was then subcloned into pcDNA3.1+ plasmid, an eukaryotic expression vector. HepG2/lis cell which was stablely transfected with lis plasmid was built by lipofectamine and drug screening. The characteristics of HepG2/lis was observed, including cell morphology, growth curve, cell cycle, tumorigenesis in nude mice and enzyme activity of lis. The toxicity of lin itself in cells was also observed by administrated HepG2 cells using different concentrations of lin;2. Lis cDNA was subcloned into the pENTRTM2B entry vector of ViraPower? system, and then delivered to pAd/CMV/V5-DEST destination vector (containing CMV promoter) by using specific LR reaction between th entry vector and the destination vector. The newly obtained pAd/CMV/lis destination vector was linearized and then transfected into 293A cells for packaging recombinant adenovirus. The recombinant adenovirus (named Ad-CMV-lis) was amplified and purified, and the titers of the virus was also determined by TCID50 method. Hepatoma cells was infected by Ad-CMV-EGFP which contained the EGFP reporter gene to explore the best MOI of virus infection. A variety of tumor cell lines was treated by Ad-CMV-lis and lin in vitro and in vivo to observe the antitumor activity and bystander effect of the system. The inhibitory mechanism of lis/lin system was further explored by a variety of detection, including DNA fragmentation detection, Annexin-V/PI flow cytometry analysis, tumor histopathology and TUNEL staining.3. AFP promoter was cloned by RT-PCR from the genome of HepG2 cells (AFP+), then subcloned into plasmid pEGFP-1. The specific transcriptive activity of AFP promoter was detected by using EGFP reporter gene analysis. CMV promoter of pcDNA3.1+ plasmid was replaced by AFP promoter, then lis cDNA was inserted into the multiple cloning sites of the plasmid, achieving the tandem of AFP promoter with lis. By construction of adenovirus entry plasmid and the LR reaction, the AFP-lis sequence was subcloned into the pAd/PL-DEST backbone vector (no promoter). 293A cells was transfected with pAd/AFP/lis plasmid to package recombinant adenovirus Ad-AFP-lis. The recombinant virus was amplificated, purified and titered. The targeting specificity and antitumor effect of the new adenovirus with lin to AFP-positive tumor cells were detected in vitro and in vivo.Results:1. Lis was successfully introduced to stable transgenic HepG2/lis cell line. There were no significant differences in morphology, growth curve, cell cycle distribution and in vivo tumor growth curve of HepG2/lis cells compared with those of the empty plasmid transfection group or blank control cells (P> 0.05). When lin was administered to HepG2/lis cells, HCN could be detected in the metabolites and the amount of it was increaseing with the increased concentration of lin, suggesting that the protein of lis expressed in eukaryotic cells can be enzyme activity. In addition, lin itself shown no inhibitory effect on cell growth.2. Recombinant adenovirus Ad-CMV-lis was successfully constructed. The inhibitory effect treated with Ad-CMV-lis and lin in a variety of tumor cells was significant in a time- and dose-dependent manner in vitro and in vivo. There was no typical DNA ladder detected by the DNA fragment assay in the tumor cells after treatment. Annexin-V/PI flow cytometry analysis showed that apoptosis and necrosis cells were all increased, but consisted mainly of necrotic cells. Histopathological observation of tumors found many necrotic area arising in the Ad-CMV-lis/lin treatment group, and a more positive TUNEL staining cells detected.3. The 276bp sequence of AFP promoter was cloned and then reorganized. EGFP reporter gene analysis showed that the promoter had specific transcription activity in AFP-positive HCC cells. The recombinant adenovirus Ad-AFP-lis transcripted under the AFP promoter was successfully constructed. By using this adenovirus, lis could be highly and specifically expressed in AFP-positive HCC cell line. Treatment experiments in vitro and in vivo have proved that the united lin/Ad-AFP-lis treatment can inhibit the growth of AFP-positive HCC cells, and prolong animal survival time, while the AFP-negative hepatoma cell line was free of inhibitory effect.Conclusion:1. Lis cDNA of plant origin can be stably expressed in eukaryotic cells and be correctly packaged to protein at a similar size, withβ-glucosidase activity. The stable integration of lis cDNA into cells had no significant impact in the cell morphology, growth, cell cycle, tumorigenesis and other biological characteristics. The prodrug lin also had low toxicity, so lis/lin system is a safe suicide gene therapy strategy.2.Through recombinant adenovirus vector, we found that adenovirus has many advantages, including efficient infection , a high level of gene expression, easy to packaging in high-titer, not integrated into the host genome. So it can be a more efficient expression vector for lis/lin suicide gene therapy.3. Recombinant adenovirus Ad-CMV-lis shown a wide range of inhibition of tumor cell lines in vitro and in vivo, suggesting that lis/lin suicide gene system has a wider scope of application. Due to the different biology characteristics in different tumor, the potential applications of this system to other tumors must be further studied.4. The AFP promoter of 276bp can regulate the targeting transcription of lis/lin system in HCC and achieve its target of AFP-positive hepatoma cell-specific destruction. However, due to negative expression of AFP in a small number of primary liver cancer, the transformation of existing AFP promoter, enhancement its transcription level to AFP-negative hepatoma cells, is worthy of in-depth study.