Pharmacologic inhibitors of IκB kinase suppress growth and migration of mammary carcinosarcoma cells in vitro and prevent osteolytic bone metastasis in vivo

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

Three-Dimensional Characterization of the Vascular Bed in Bone Metastasis of the Rat by Microcomputed Tomography (MicroCT)

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 Findings We 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. Conclusion This 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

Synthesis and use of pHEMA microbeads with human EA.hy 926 endothelial cells

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

Biomatériaux et angiogenèse : Utilisation dans les métastases et la reconstruction osseuses

In animal models, development and vascularization of bone metastases were studied as well as biomaterials for bone filling or that can target angiogenesis. pHEMA a polymer well-known for its biocompatibility, was used to synthesize fluorescent microbeads bearing anionic and cationic charges, at their surface. pHEMA microbeads were incubated with an endothelial cell line that preferentially endocytose anionic microbeads. A radiopaque silicone was used to characterize in 3D, the vasculature in bone metastases of rat and at different stages of tumor evolution. Blood vessels in tumoral bone were hypervascular and their architecture disorganized. The vasculature was irregular in size and tortuous. In another study, zoledronic acid a potent bisphosphonate with also an inhibitor effect on angiogenesis was used preventively in a model of bone metastases in rat. Bone metastases cell model were MatLyLu carcinomas. Zoledronic acid converted metastases initially osteolytic to osteosclerosis forms, with limited cortical perforations. In rabbit, we worked on a bone filling model with beta tricalcium phosphate (β-TCP). We reported that a non steroidal anti inflammatory drugs, used in bone surgery do not delay β-TCP bone graft healing. The vascularization plays a key role in normal bone remodeling, but also in benign and malignant bone diseases. The vascularization is also important in osteointegration of biomaterials used for substitution of bone loss.Dans des études animales, nous avons étudié le développement des métastases osseuses, leur vascularisation ainsi que des biomatériaux de comblement ou pouvant cibler l'angiogenèse. Nous avons utilisé le poly 2-hydroxyéthyl méthacrylate (pHEMA) connu pour sa biocompatibilité, pour fabriquer des microbilles fluorescentes portant à leur surface des charges anioniques ou cationiques. Les microbilles ont été incubées avec une lignée de cellules endothéliales qui internalisent de préférence les microbilles portant une charge anionique. Un silicone radio-opaque a été utilisé pour caractériser l'architecture en 3D des vaisseaux sanguins à différents stades d'évolution des métastases osseuses chez le rat. Nous avons montré au niveau des os tumoraux une hypervascularisation et une désorganisation importante de l'architecture vasculaire avec un réseau très dense de micro vaisseaux à l'aspect tortueux s'étendant dans toute la zone trabéculaire, envahissant la corticale et la diaphyse en sens rétrograde. Dans une autre étude, l'acide zolédronique, un puissant bisphosphonate ayant aussi un effet anti-angiogénique, a été utilisé de façon préventive dans un modèle de métastase osseuse chez le rat. Il permet la conversion des métastases du carcinome MatLyLu, initialement ostéolytiques, en formes ostéocondensantes en limitant les perforations corticales. Enfin, chez le lapin, dans un modèle de comblement osseux par le bêta tricalcium phosphate (β−TCP), nous avons montré que des anti-inflammatoires non stéroïdiens, utilisés en chirurgie osseuse, ne retardent pas l'ostéogenèse au contact du biomatériau. La vascularisation joue un rôle important dans le remodelage osseux normal mais aussi dans les ostéopathies bénignes et malignes ainsi que dans l'ostéointégration des biomatériaux utilisés pour le traitement des pertes de substance osseuse

Biomatériaux et angiogenèse (utilisation dans les métastases et la reconstruction osseuses)

Dans des études animales, nous avons étudié le développement des métastases osseuses, leur vascularisation ainsi que des biomatériaux de comblement ou pouvant cibler l'angiogenèse. Nous avons utilisé le poly 2-hydroxyéthyl méthacrylate (pHEMA) connu pour sa biocompatibilité, pour fabriquer des microbilles fluorescentes portant à leur surface des charges anioniques ou cationiques. Les microbilles ont été incubées avec une lignée de cellules endothéliales qui internalisent de préférence les microbilles portant une charge anionique. Un silicone radio-opaque a été utilisé pour caractériser l'architecture en 3D des vaisseaux sanguins à différents stades d'évolution des métastases osseuses chez le rat. Nous avons montré au niveau des os tumoraux une hypervascularisation et une désorganisation importante de l'architecture vasculaire avec un réseau très dense de micro vaisseaux à l'aspect tortueux s'étendant dans toute la zone trabéculaire, envahissant la corticale et la diaphyse en sens rétrograde. Dans une autre étude, l'acide zolédronique, un puissant bisphosphonate ayant aussi un effet anti-angiogénique, a été utilisé de façon préventive dans un modèle de métastase osseuse chez le rat. Il permet la conversion des métastases du carcinome MatLyLu, initialement ostéolytiques, en formes ostéocondensantes en limitant les perforations corticales. Enfin, chez le lapin, dans un modèle de comblement osseux par le bêta tricalcium phosphate (b TCP), nous avons montré que des anti-inflammatoires non stéroïdiens, utilisés en chirurgie osseuse, ne retardent pas l'ostéogenèse au contact du biomatériau. La vascularisation joue un rôle important dans le remodelage osseux normal mais aussi dans les ostéopathies bénignes et malignes ainsi que dans l'ostéointégration des biomatériaux utilisés pour le traitement des pertes de substance osseuse.In animal models, development and vascularization of bone metastases were studied as well as biomaterials for bone filling or that can target angiogenesis. pHEMA a polymer well-known for its biocompatibility, was used to synthesize fluorescent microbeads bearing anionic and cationic charges, at their surface. pHEMA microbeads were incubated with an endothelial cell line that preferentially endocytose anionic microbeads. A radiopaque silicone was used to characterize in 3D, the vasculature in bone metastases of rat and at different stages of tumor evolution. Blood vessels in tumoral bone were hypervascular and their architecture disorganized. The vasculature was irregular in size and tortuous. In another study, zoledronic acid a potent bisphosphonate with also an inhibitor effect on angiogenesis was used preventively in a model of bone metastases in rat. Bone metastases cell model were MatLyLu carcinomas. Zoledronic acid converted metastases initially osteolytic to osteosclerosis forms, with limited cortical perforations. In rabbit, we worked on a bone filling model with beta tricalcium phosphate (b-TCP). We reported that a non steroidal anti inflammatory drugs, used in bone surgery do not delay b-TCP bone graft healing. The vascularization plays a key role in normal bone remodeling, but also in benign and malignant bone diseases. The vascularization is also important in osteointegration of biomaterials used for substitution of bone loss.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Histological aspect of bones after Microfil® injection and decalcification, Microfil® appears shrunk with a deep gray-black tint.

<p>Hematoxylin-phloxin staining. A) Control femur showing two medullar arteries running in parallel cf <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017336#pone-0017336-g002" target="_blank">Fig 2B</a>. B) Control femur showing the silicone rubber in a dilated vascular sinus (right arrow) and in a vascular canal in the cortice (left arrow). C) Metastatic femur at 12 days with enlarged vascular channels in the area of tumors cells, arrow points to an intracortical vessel. D) Metastatic femur at 26 days (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017336#pone-0017336-g002" target="_blank">Fig 2G</a>) with cortical perforation, large vessels coming from the periosteum (p) and extending in the tumor area having a fibrous stroma and foci of metaplastic bone. E) A large vessel running between metaplastic trabeculae (m) and tumor cells. F) Tumor cells with a spindle-shape or enlarged cytoplasm, thin metaplastic trabeculae and a vessel containing Microfil®.</p

Frequency distribution of the vascular diameter on the control side (in blue) and the metastatic side (in red).

<p>In controls, the arteries appeared to be centered on a peak at 100–120 µm whatever the time of analysis. In the metastatic side, larger arteries (up to 600 µm in diameter) were observed.</p

Time evolution of vascular volume in the control and metastatic femurs.

<p>Time evolution of vascular volume in the control and metastatic femurs.</p

Principle of over imposition of two 3D models.

<p>A) 3D model obtained on the undecalcified bone; bone and vascular bed are identified after a single threshold. The arrow points on the central medullar artery. B) 3D model of the vascular bed obtained after decalcification of the same femur. Periosteal arteries surrounding the bone shaft and epiphysis are evidenced (→). C) Overimposition of the two 3D models. The vascular bed appears with a different pseudo-color than bone.</p