Recombinant Adeno-associated Virus-mediated Expression Of Human Endostatin Inhibits Growth And Metastasis Of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC), a highly malignant tumor that can easily metastasize, is highly prevalent in Southeast Asia, particularly in southern China, Hong Kong, Singapore and Malaysia. Radiotherapy is the most common treatment for NPC patients, as NPC is a fast growing tumor that is highly sensitive to radiotherapy. However, the 5-year survival rate is less than 50%. Most deaths are associated with distant metastases and/or local recurrences. There is no effective treatment for NPC during the metastatic stages.Nasopharyngeal carcinom, as well as other solid tumors, depend on angiogenesis for sustained growth and hematogenous metastatic spread. Increasing evidence demonstrates the importance of angiogenesis in solid tumor growth and metastasis. Consequently, the concept of targeting the tumor vasculature with anti-angiogenic agents has emerged as an attractive new strategy in the treatment of cancer. The process of angiogenesis consists of multiple, sequential and interdependent steps. It is controlled by the balance between positive and negative regulators. In tumors the most important pro-angiogenic growth factors are believed to be vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Many antiangiogenic factors, including thrombospondin, interferon a, platelet factor 4, angiostatin and endostatin, have been isolated and implicated in the antiangiogenic therapy of cancer. Among these, endostatin, a 20 kDa C-terminal proteolytic fragmentof collagen XVIII, has received the greatest attention. In vitro studies have show that endostatin can inhibit endothelial cell proliferation and induce endothelial cell apoptosis. In vivo, the systemic administration of recombinant endostatin resulted in growth inhibition or regression in a number of different tumor models. It may be the most potent inhibitor of tumor angiogenesis.Most therapeutic investigation of endostatin have utilized the purified protein. Endostatin, however, as a protein drug, has a short half-life in vivo. The requirement for high dosages of expensive recombinant endostatin protein, difficulties in protein production and the cumbersome daily administration may hinder any future clinical applications. Gene therapy, which can deliver gene cassettes encoding for endostatin protein, may provide a solution to these problems.The key for gene therapy is vector capable of sustained, long-term expression without vector-associated toxicity or immunity. Recombinant adeno-associated virus (rAAV) vector is a good candidate for cancer gene therapy because of its non-pathogenicity, non-immunogenicity and replication-defective characteristics. rAAV is able to integrate into the host genome, to mediate efficient and long-term gene transduction across a board range of dividing or non-dividing mammalian cells.According to the background above, this study was designed to explore the effect of endostatin mediated by recombinant adeno-associated virus on tumor growth and metastasis of NPC in an animal model. This work was supported by: the Guangdong Natural Science Fund, (04020406). Recombinant adeno-associated viral vectors carrying human endostatin gene (rAAV-hEndo) or enhanced green fluorescent protein gene (rAAV-EGFP) were kindly provided by Dr Peng Ymg from the university of Hong Kong.ObjectiveTo detect the expression of human endostatin mediated by recombinant adeno-associated viral vector, observe its biological activity in vitro and investigate the inhibitory effect on growth and metastasis of human nasopharyngeal carcinoma in nude mice by adeno-associated virus-mediated gene transfer of human endostatin.MethodsThe infectious efficiency of rAAV in vitro was detected. The endostatin expressed in transfected human umbilical vein endothelial cell ECV304 was detected by immunofluorescence staining. The effects of endostatin on ECV340 cell were evaluated by MTT-cell proliferation assay. Cell Cycle Analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) technique. Furthermore, in order to evaluate the antiangiogenic and antitumor effects of endostatin in vivo, we establish a heterotopic implantation model of human nasopharyngeal carcinoma in nude mice using human nasopharyngeal carcinoma cell line C666-1. Recombinant adeno-associated viral vectors carrying human endostatin gene (rAAV-hEndo) or enhanced green fluorescent protein gene (rAAV-EGFP) or PBS were injected into the tail vein of tumor bearing mice respectively. Three weeks after implantation, the volumes of tumor, inhibition rate, the percentage of lung metastases, microvessel density (MVD) and apoptotic index (AI) were evaluated respectively. Data were analyzed with SPSS 10.0.ResultsThe infectious efficiency of rAAV was 98% in vitro. Immunofluorescence staining showed the humam endostatin protein was expressed mainly in cytoplasm. ECV304 cell proliferation was obviously inhibited by the infecting supernatant of rAAV-hEndo. The inhibitory rate was 67.3% when the supernatant was used 72h later. ECV304 cells infected with rAAV-hEndo were arrested in Gi phase significantly. Gj cell percentage of treatment group(72.5±4.0)% were higher than that of control group(52.1±2.1)%(P<0.01). ECV304 cells infected with rAAV-hEndo demonstrated markedly enhanced apoptosis. The apoptotic index of ECV304 cells infected with rAAV-hEndo and the control cells were(32.6±3.2)%and (4.2 ±1.9)% respectively (P<0.01). The average tumor volume in hEndo group. EGFP group and PBS group was (0.4809+0.2069) cm3, (1.3109±0.4368) cm3 and (1.6442±0.5379) cm3 respectively. Compared with PBS group, the restrained percentage of tumor in hEndo group was 70.7%. The percentage of lung metastases in hEndo group, EGFP group and PBS group was 0.0%, 50.0% and 66.7% respectively. The average MVD ofhEndo group (3.67± 1.63) was significantly lower than that of EGFP group (19.67± 2.16) and PBS group (22.50 + 3.02) (P<0.01). The apoptotic index increased significantly in hEndo group(28.83 + 5.27)% versus EGFP group (6.17+2.79) % and PBS group (4.50+2.17) % (P<0.01). The survival time of tumor bearing mice in hEndo group(36.50+8.46)d was significantly longer than EGFP group(24.00+5.66)d and PBS group(21.17 + 3.92)d (P<0.01).ConclusionsThese studies show that the recombinant adeno-associated virus rAAV-hEndo can express biologically active human endostatin effectively in vitro and the gene transfer of human endostatin mediated by adeno-associate virus can significantly inhibit the growth and metastasis of human nasopharyngeal carcinoma in nude mice. The mechanism may be due to the effect of antiangiogensis and inducement of tumor cell apoptosis.