SYNERGISTIC INHIBITION OF TUMOR GROWTH AND METASTASIS BY COMBINED TREATMENT WITH TNP-470 AND GEMCITABINE IN A HUMAN BLADDER CANCER KOTCC-1 MODEL

Abstract

TNP-470 (O-(chloroacetylcarbamoyl)fumagillol) is a potent inhibitor of angiogenesis that was reported to enhance synergistically the antitumor effects of cytotoxic agents. We evaluated the effectiveness of combined treatment with TNP-470 and gemcitabine in vitro and in vivo using highly metastatic human bladder cancer KoTCC-1 cells. The in vitro growth inhibitory and apoptotic effects of gemcitabine and/or TNP-470 on KoTCC-1 cells were assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and TUNEL staining, respectively. We then evaluated the combined effect of gemcitabine and TNP-470 therapy after subcutaneous and orthotopic injection of KoTCC-1 cells into athymic nude mice. Established tumors were analyzed by TUNEL staining and immunohistochemical staining of CD31 to quantify microvessel density. In vitro TNP-470 enhanced the cytotoxic effect of gemcitabine on KoTCC-1 cells, decreasing its IC50 by more than 50%. This combined treatment significantly induced apoptosis compared with treatment with either agent alone. In vivo combined treatment with TNP-470 and gemcitabine significantly decreased tumor growth after subcutaneous and orthotopic injection into athymic nude mice and significantly decreased the incidence of lymph node metastasis after arthotopic injection compared with either agent alone. Immunohistochemical analysis of the subcutaneous tumor after each treatment demonstrated that gemcitabine administration significantly induced apoptotic cell death. In contrast, a significant decrease in microvessel density was observed after TNP-470 treatment. These findings demonstrate that TNP-470 and gemcitabine act synergistically to inhibit tumor growth and metastasis by enhancing apoptosis and suppressing angiogenesis.