The NF-κB signaling pathway is known to play an important role in the regulation of osteoclastic bone resorption and cancer cell growth. Previous studies have shown that genetic inactivation of IκB kinase (IKK), a key component of NF-κB signaling, inhibits osteoclastogenesis, but the effects of pharmacologic IKK inhibitors on osteolytic bone metastasis are unknown. Here, we studied the effects of the IKK inhibitors celastrol, BMS-345541, parthenolide, and wedelolactone on the proliferation and migration of W256 cells in vitro and osteolytic bone destruction in vivo. All compounds tested inhibited the growth and induced apoptosis of W256 cells as evidenced by caspase-3 activation and nuclear morphology. Celastrol, BMS-345541, and parthenolide abolished IL1β and tumor necrosis factor α-induced IκB phosphorylation and prevented nuclear translocation of NF-κB and DNA binding. Celastrol and parthenolide but not BMS-345541 prevented the activation of both IKKα and IKKβ, and celastrol inhibited IKKα/β activation by preventing the phosphorylation of TAK1, a key receptor-associated factor upstream of IKK. Celastrol and parthenolide markedly reduced the mRNA expression of matrix metalloproteinase 9 and urinary plasminogen activator, and inhibited W256 migration. Administration of celastrol or parthenolide at a dose of 1 mg/kg/day suppressed trabecular bone loss and reduced the number and size of osteolytic bone lesions following W256 injection in rats. Histomorphometric analysis showed that both compounds decreased osteoclast number and inhibited bone resorption. In conclusion, pharmacologic inhibitors of IKK are effective in preventing osteolytic bone metastasis in this model and might represent a promising class of agents to the prevention and treatment of metastatic bone disease associated with breast cancer.

BackgroundAngiogenesis contributes to proliferation and metastatic dissemination of cancer cells. Anatomy of blood vessels in tumors has been characterized with 2D techniques (histology or angiography). They are not fully representative of the trajectories of vessels throughout the tissues and are not adapted to analyze changes occurring inside the bone marrow cavities.Methodology/Principal FindingsWe have characterized the vasculature of bone metastases in 3D at different times of evolution of the disease. Metastases were induced in the femur of Wistar rats by a local injection of Walker 256/B cells. Microfil®, (a silicone-based polymer) was injected at euthanasia in the aorta 12, 19 and 26 days after injection of tumor cells. Undecalcified bones (containing the radio opaque vascular casts) were analyzed by microCT, and a first 3D model was reconstructed. Bones were then decalcified and reanalyzed by microCT; a second model (comprising only the vessels) was obtained and overimposed on the former, thus providing a clear visualization of vessel trajectories in the invaded metaphysic allowing quantitative evaluation of the vascular volume and vessel diameter. Histological analysis of the marrow was possible on the decalcified specimens. Walker 256/B cells induced a marked osteolysis with cortical perforations. The metaphysis of invaded bones became progressively hypervascular. New vessels replaced the major central medullar artery coming from the diaphyseal shaft. They sprouted from the periosteum and extended into the metastatic area. The newly formed vessels were irregular in diameter, tortuous with a disorganized architecture. A quantitative analysis of vascular volume indicated that neoangiogenesis increased with the development of the tumor with the appearance of vessels with a larger diameter.ConclusionThis new method evidenced the tumor angiogenesis in 3D at different development times of the metastasis growth. Bone and the vascular bed can be identified by a double reconstruction and allowed a quantitative evaluation of angiogenesis upon time.

Beta-tricalcium phosphate (beta-TCP) is a suitable biomaterial in oral and maxillofacial surgery since it can induce a rapid proliferation of woven bone. Granules, prepared by the polyurethane foam method, were implanted in critical size defects performed in the femoral condyles of New Zealand rabbits. Animals were studied after 8 and 28 days. Ketoprofen (a non-steroidal anti-inflammatory drug (NSAID)) was given for 8 and 28 days to evaluate its effects on the healing of the graft. Before euthanasia, the rabbits received an intravenous injection of fluorescent microbeads. Bones were analyzed by microcomputed tomography. beta-TCP granules induced metaplastic bone trabeculae as early as 8 days post-surgery. At 28 days, the amount of bone was increased and the biomaterial volume decreased due to simultaneous macrophagic resorption. The amount of macrophages labeled with microbeads was less in the grafted area than in the vicinal intact marrow spaces. Ketoprofen had no effect on the amount of bone formed and on the number of labeled macrophages. The influence of small doses of NSAID, given in a short duration period, did not present deleterious effects on bone graft healing.

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Cancer has become a major problem in public health and the resulting bone metastases a worsening factor. Facing it, different strategies have been proposed and mechanisms involved in tumor angiogenesis are being studied. Enhanced permeability retention (EPR) effect is a key step in designing new anticancer drugs. We have prepared poly 2-hydroxyethyl methacrylate (pHEMA) microbeads to target human endothelial EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells. Microbeads were synthesized by emulsion precipitation method and carried positive or negative charges. EA.hy 926 cells were cultured in 24-well plates and microbeads were deposited on cells at various times. Scanning and transmission electron microscopy, flow cytometry, confocal microscopy, and three-dimensional (3D) reconstruction were used to characterize microbeads and their location outside and inside cells. Microbeads were uptaken by endothelial cells with a better internalization for negatively charged microbeads. 3D reconstruction of confocal optical sections clearly evidenced the uptake and internalization of microbeads by endothelial cells. pHEMA microbeads could represent potential drug carrier in tumor model of metastases.

Bone metastases are severe complications of cancers associated with increased morbidity, pain, risk fracture, and reduced life span for patients. Bisphosphonates emerged as a relief treatment in bone metastases. A single dose of zoledronic acid (78 microg/kg) was injected into six Copenhagen rats 4 days before receiving an intraosseous inoculation of metastatic anaplastic tumor of lymph node and lung cell (MLL) prostate cancer cells. Rat femurs were analyzed for changes by microCT and histomorphometry; trabecular volume, trabecular characteristics, osteoid parameters, osteoblastic surfaces, and osteoclast number were measured. Values were compared to a group of SHAM animals, a group of SHAM animals having received zoledronic acid and animals inoculated with MLL cells. All rats were euthanized after 1 month. MLL cells induced osteolysis in the metaphysis with extension of the tumor to soft tissues through cortical perforations. Zoledronic acid induced a marked osteosclerosis in the primary spongiosa in both SHAM and rats inoculated with MLL. Osteosclerosis was obtained in the secondary spongiosa of MLL rats. The bisphosphonate preserved cortical integrity in all animals, and no extension to soft tissues was observed in most animals. The number of osteoclasts was elevated, indicating that there was no apoptosis of osteoclasts but they became inactive. Osteosclerosis was associated with increased osteoblastic surfaces. A single zoledronic acid injection turned osteolytic metastases into osteosclerotic and preserved cortical integrity.

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