The Novel Tubulin-binding Drug BTO-956 Inhibits R3230Ac Mammary Carcinoma Growth and Angiogenesis in Fischer 344 Rats 1 Siqing Shan, A. Craig Lockhart, Wilfred Y. Saito, A. Merrill Knapp, Keith R. Laderoute, and Mark W. Dewhirst 2 Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina 27710 [S. S., A. C. L., W. Y. S., M. W. D.], and Pharmaceutical Discovery Division, SRI International, Menlo Park, California 94025 [A. M. K., K. R. L.] ABSTRACT BTO-956 [methyl-3,5-diiodo-4-(4-methoxyphenoxy)- benzoate], a novel tubulin-binding drug and thyroid hor- mone analogue, was originally found to inhibit human car- cinoma cell proliferation in vitro and to have potent growth delay activity in human breast and ovarian carcinoma xe- nografts in nude mice. Here we report that BTO-956 given to Fischer 344 rats also inhibits corneal angiogenesis and the growth and neovascularization of the R3230Ac rat mam- mary carcinoma tumor implanted in skin-fold window chambers. Hydron pellets containing recombinant human basic fibroblast growth factor (50 ng) and Sucralfate (20 g) were implanted into surgically created corneal micropockets (day 0). BTO-956 was administrated by oral gavage (500 mg/kg, twice a day for 6 days) on days 1– 6 (controls received vehicle alone). On day 7, rats received retrogade infusions of India ink via the thoracic aorta to visualize the corneal vasculature. Digitized images of slide-mounted corneas from control and treated animals were taken with a microscope. For the tumor growth and angiogenesis study, small pieces of R3230Ac tumor from a donor rat were implanted into surgically prepared window chambers (day 0). BTO-956 was given during days 5–11, and images of the tumors and their vasculature were recorded on day 12. No body weight loss was observed in either study. BTO-956 significantly inhibited corneal angiogenesis (by 50 – 80%), as assessed by measurements of limbal circumference displaying neovascu- larization, vessel length, vascularized area, and vascular area density. In the window chamber assay, tumors from treated animals were >50% smaller than tumors in control animals. In addition, vascular length densities in peripheral tumor zones were 30% less in treated compared with control animals. Together, these findings demonstrate that BTO-956 can inhibit angiogenesis induced by a growth factor in the rat cornea and in the peripheral area of implanted tumors, where tumor angiogenesis is most active. INTRODUCTION Angiogenesis, the formation of new vessels from existing vasculature, has a critical role in primary tumor growth, inva- sion, and metastasis (1). Tumor-associated angiogenesis is a multistep process that is controlled by both positive and nega- tive factors as well as by complex interactions among tumor cells, host endothelium, stromal cells, and extracellular matrix components. Moreover, because tumor vascular networks are formed within microenvironments intrinsic to the tumor mass, they have markedly different properties compared with those in normal tissues. These tissue-level differences make angiogenic tumor endothelial cells an attractive target for the development of new anticancer therapies. Current antivascular or antiangio- genic approaches include treatment with cytotoxic agents hav- ing some selectivity for proliferating vascular endothelial cells, natural angiogenesis inhibitors, modified soluble receptors that interfere with angiogenic signal transduction, and synthetic compounds capable of selectively inhibiting endothelial cell proliferation or remodeling of the extracellular matrix (2, 3). Some conventional anticancer drugs have antivascular or antiangiogenic activity in addition to their cytotoxic effects toward tumor cells (4, 5). For example, tubulin-binding agents that cause mitotic arrest can have antivascular or antiangiogenic activity. Colchicine is a tubulin-binding agent that was reported to produce hemorrhagic necrosis in experimental tumors (6). In addition, colchicine was reported to be cytotoxic toward endo- thelial cells in capillary sprouts (7). Combretastatin A4, another tubulin-binding molecule isolated from the South African tree Combretum caffrum, displays potent and selective toxicity to- ward tumor vasculature and to perturb tumor perfusion and energy status (8, 9). This latter toxicity is termed vascular targeting. BTO-956 [methyl-3,5-diiodo-4-(4'-methoxyphenoxy)ben- zoate; Fig. 1] was originally developed as a thyroid hormone analogue, but failed to show any physiological activity associ- ated with thyroid hormone metabolism (10). However, in recent studies we demonstrated that BTO-956 inhibits the proliferation of human carcinoma cells in vitro and that it has potent growth delay activity in human breast and ovarian carcinoma xenografts in nude mice (11, 12). In addition, we found that the cytotoxicity of BTO-956 toward tumor cells involves mitotic arrest associ- ated with disruption of cellular microtubule assembly that prob- ably arises from binding of the drug to the colchicine site of Received 1/2/01; revised 5/14/01; accepted 5/15/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This research was supported by the California Breast Cancer Research Program (Grant 4CB-0032 to K. R. L.) and Large Scale Biology Cor- poration, Vacaville, CA. 2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710-3455. Phone: (919) 684-4711; Fax: (919) 684-8718; E-mail: dewhirst@radonc.duke.edu. 2590 Vol. 7, 2590 –2596, August 2001 Clinical Cancer Research Research. on December 8, 2021. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from