Synergistic inhibition of tumor growth and metastasis by combined treatment with TNP-470 and gemcitabine in a human bladder cancer KoTCC-1 model.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
These findings demonstrate that TNP-470 and gemcitabine act synergistically to inhibit tumor growth and metastasis by enhancing apoptosis and suppressing angiogenesis.
Muramaki
Mototsugu
M
Division of Urology, Department of Organ Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, Kobe, Japan.
Miyake
Hideaki
H
Hara
Isao
I
Kawabata
Gaku
G
Kamidono
Sadao
S
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D002295:Carcinoma, Transitional Cell / Q000098:blood supply / Q000473:pathology*; D002455:Cell Division / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects*; D003510:Cyclohexanes; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004357:Drug Synergism; D005260:Female; D006801:Humans; D008207:Lymphatic Metastasis / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness / Q000473:pathology*; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000473:pathology; D000077609:O-(Chloroacetylcarbamoyl)fumagillol; D012717:Sesquiterpenes / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects*; D001749:Urinary Bladder Neoplasms / Q000098:blood supply / Q000473:pathology*; D000093542:Gemcitabine
D020533:Angiogenesis Inhibitors; D003510:Cyclohexanes; D012717:Sesquiterpenes; D003841:Deoxycytidine; D000077609:O-(Chloroacetylcarbamoyl)fumagillol; D000093542:Gemcitabine