Combinational Gene Transfer Of Endostatin And Antisense Hypoxia-Inducible Factor-1 Alpha To Combat Hepatocellular Carcinomas In Nude Mice.

[Background]Angiogenesis of the tumor appears to be the fundamental requirement for tumor growth, invasion, and metastasis. An avascular tumor rarely grows to a size of larger than 2-3mm3 and contains up to a few million cells. Tumor can not grow until a new vessel grow up to support it. So angiogenesis of the tumor is necessary for its grow and metastasis, anti-angiogeni cancer therapy become the focus of tumor treatment.Many researchs indicated that Angiogenesis of the tumor need many factors to take part in and it is a complicated process including (1)Tumor release vessel stimulated factors. (2)Stroma of extracellular around vessels to restructe and degradate. (3)Endothelial cell hyperplasia and translation.(4)The formation of new vessels.To prevent one of the process can inhibit the formation of new vessels., Endostatin which has been shown to inhibit the endothelial cells proliferation and migration in vitro, is a polypeptide of 184 amino acids. It is the globular domain found at the C-terminal of Type XVIII (18) collagen (a collagen found in blood vessels) cut off from the parent molecule. Endostatin, the 20-kDa C-terminal fragment of collagen XVIII, has previously been shown to inhibit growth and induce regression of different experimental tumors in rodents.In addition, it has been shown to be among the most potent inhibitors of tumor-induced angiogenesis in vivo. Systemic therapy with Endostatin protein has been shown to suppress tumor-induced angiogenesis and tumorgrowth. Systemic therapy with Endostatin protein has been shown to suppress tumor-induced angiogenesis and tumor growth. Notably, these studies required daily parenteral injections of large amounts of recombinant protein, illustrating a common difficulty in the pharmacologic use of therapeutic proteins. Gene therapy approaches may have advantageous properties as methods for delivery of systemic protein drugs.More challenges have recently emerged against anti-angiogenic therapy. The promising anti-angiogenic therapy is under pressure and suspicion due to recent disappointing clinical results. In the present study, we put forward a new insight that tumor hypoxia is also hindrance for anti-angiogenic therapy, and blocking tumor hypoxia pathway by antisense Hypoxia-inducible-factor-la could render angiogenic therapy a strong helper to eradicate established tumors.[Objective)1. To observe the effects of Endostatin gene to combat hepatocellular carcinomas in nude mice.2. To observe the effects of gene transfer of Endostatin and HIF-la to combat hepatocellular carcinomas in nude mice.[Materials arid Methods]Forty-eight nude mice, 4-5 weeks old, were used. The cell line of human primary hepatic carcinoma , namely SMMC-7721 , was cultured at 37 °C in 1640 medium(GIBCO), supplemented with 10% calf serum. Tumors were established by injection of 3 X106 tumor cells into the back of nude mice, and growth determined by measuring the long and short diameters.The mice were classified randomly into four groups, injected respectively with empty plamid PcDNA3 n Endostatin plasmid, antisense HIF-la plasmid> Endostatin plasmid+antisense HIF-la plasmid. Once the tumors reached 0.4cm in diameter, they were injected with lOOuJ gene transfer solution(100p.g expression plasmid). The transduction was mediated by cationic liposome DOTAP. For combinationaltreatment, reagents were delivered in a timed fashion, where angiostatin plasmid was injected first, followed by antisense HIF-1 a plasmid 24h later.Half of the nude mice were used to observe the tumor growth curve, the other to obtain the tumor samples. The tumor samples were prepared in the 4th day after gene transfer to be used in the examinations of the expression of Endostatin HIF-la and VEGF with immunohistochemistry and western blot analysis, of MVD in the tumor with immunohistochemistry, and of cell apoptosis with TUNEL staining.Results were expressed as mean values ± standard deviation ( X±s~). SNK test and x2 test were used for evaluating statistical significance, where a value less than 0.05(P<0.05 ) denote statistical significance. The software SPSS12.0 was used in the statistical analysis.[Results]1. In the control group, tumor grew rapidly reaching lcm in size 18-21 days following gene transfer; whereas, in the Endostatin group, tumors were suppressed for about 10 days after gene transfer, then they began to grow again . Immunohistochemistry analysis and western blot analysis of tumor tissue revealed Endostatin gene therapy resulted in overexpression of Endostatin in situ. In addition, the expression of VEGF in the Endostatin group is a little higher than in the control group. AI in the Endostatin group is higher than in the control group (P<0.01, concurrently MVD is lower (P<0.05 =.2. In the group transfer of Endostatin gene with antisense HIF-la gene, the tumors were inhibited significantly. In this group, the low expression of HIF-] a and VEGF was observed. MVD is lowest and AI is highest than the other groups.[Conclusion]1. Blocking hypoxia pathway with antisense HIF-la gene makes hepatocellular carcinomas feasible to antiangiogenic therapy.2. Antisense HIF-la therapy synergizes with Endostatin in inhibition of both tumor angiogenesis and cell apoptosis.3. The combinational gene transfer of Endostatin and aHlF-lct is a promisingapproach to treat hepatocellular carcinomas.