Inhibitory efficacy of local angiostatin expression in treatment of human hepatic carcinoma cells

Abstract

OBJECTIVE: To study the efficacy of local therapy using overexpression of the mouse angiostatin gene to treat human hepatic carcinoma. To investigate the mechanism of any therapeutic effect. METHODS: Full-length mouse angiostatin cDNA was directly cloned into the eukaryotic expression plasmid pcDNA3.1(+). The recombinant plasmid was used to transfect HCC7721 human hepatic carcinoma cells (experimental group). The vector control group cells were transfected with pcDNA3.1(+), and the mock control group was not exposed to the plasmid. Angiostatin expression at the mRNA and protein levels was detected by using RNA dot blots and fluorescence-activated cell sorter (FACS), respectively. Cell growth in vitro was assayed with thiazoyl blue tetrazolium bromide and cell cycle distributions were analyzed with FACS. Angiostatin-expressing cells and the vector control cells were transplanted subcutaneously into nude BALB/c mice and tumor volumes were measured after 30 days. Microvessel densities (MVD) and angiostatin expression in the tumor tissues were detected by using immunohistochemical staining with the primary anti-vWF and anti-HA antibodies, respectively. RESULTS: The pcDNA3.1(+)-angio recombinant eukaryotic expression plasmid was successfully constructed. No significant differences existed in cell morphology, cell growth curve and cell cycle distributions among the three study groups in vitro. However, markedly smaller tumor volumes, lower microvessel densities in tumor tissues, and stronger angiostatin-positive staining were observed in the experimental group relative to the control group, indicating reduced tumorigenesis associated with angiostatin overexpression in vivo. CONCLUSIONS: Mouse angiostatin did not directly inhibit human hepatic carcinoma cell growth and proliferation in vitro. However, local production of angio­statin effectively inhibited tumorigenesis of HCC7721 in vivo, probably because of inhibition of tumor angio­genesis via a paracrine pathway. Transfer of the angiostatin gene may be an effective new approach to the treatment of hepatic carcinoma.