Abstract
Bone is a very common metastatic site for breast cancer. In bone metastasis, there is a vicious circle wherein bone-residing metastatic cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone promote tumor growth. The contribution of tumor angiogenesis in the growth of bone metastases is, however, unknown. By using an experimental model of bone metastasis caused by MDA-MB-231/B02 breast cancer cells that quite closely mimics the conditions likely to occur in naturally arising metastatic human breast cancers, we demonstrate here that when MDA-MB-231/B02 cells were engineered to produce at the bone metastatic site an angiogenesis inhibitor, angiostatin, there was a marked inhibition in the extent of skeletal lesions. Inhibition of skeletal lesions came with a pronounced reduction in tumor burden in bone. However, although angiostatin produced by MDA-MB-231/B02 cells was effective at inhibiting in vitro endothelial cell proliferation and in vivo angiogenesis in a Matrigel implant model, we have shown that it inhibited cancer-induced bone destruction through a direct inhibition of osteoclast activity and generation. Overall, these results indicate that, besides its well known anti-angiogenic activity, angiostatin must also be considered as a very effective inhibitor of bone resorption, broadening its potential clinical use in cancer therapy.
Highlights
Bone is a very common metastatic site for human breast, prostate, lung, kidney, and thyroid cancer (1, 2)
By using an experimental model of bone metastasis caused by MDA-MB-231/B02 breast cancer cells that quite closely mimics the conditions likely to occur in naturally arising metastatic human breast cancers, we demonstrate here that when MDA-MB-231/B02 cells were engineered to produce at the bone metastatic site an angiogenesis inhibitor, angiostatin, there was a marked inhibition in the extent of skeletal lesions
Current experimental studies support the notion that there is a vicious circle at the bone metastatic site at which bone-residing metastatic cells stimulate osteoclast-mediated bone resorption and bone-derived growth factors released from resorbed bone promote tumor growth (2)
Summary
Construction of Mock- and Angiostatin-inducible Cell Lines Using the Tet-Off-Regulated Expression System—Characteristics of MDA-MB231/B02 and MDA-MB-231/B02-GFP breast cancer cell lines have been described elsewhere (9, 10). For bone metastasis and tumorigenesis experiments, animals received 5% (w/v) sucrose with or without doxycycline (1 mg/ml) in the water supply 2 days before tumor cell inoculation. Tumor cells (106) in 50% Matrigel (v/v) (BD Biosciences, Le Pont de Claix, France) were inoculated subcutaneously (100 l) into the right flank of nude mice that have been treated previously for 2 days with doxycycline. The immunostained blood vessel area/tumor area ratio (expressed as a percentage) was quantified by using a computerized image analysis system (Visiolab 2000, Biocom) and used to assess the microvessel density. The intensity of each immunoreactive band was quantified by using the computerized image analysis system ImageQuant (Amersham Biosciences), and data were expressed in ng/ml of cell culture-conditioned medium. The mononuclear cells were isolated using lymphocyte separation media (ICN Pharmaceuticals) and seeded in cell culture
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