Recombinant sclerostin antagonizes effects of ex vivo mechanical loading in trabecular bone and increases osteocyte lacunar size

Sclerostin has emerged as an important regulator of bone mass. We have shown that sclerostin can act by targeting late osteoblasts/osteocytes to inhibit bone mineralization and to upregulate osteocyte expression of catabolic factors, resulting in osteocytic osteolysis. Here we sought to examine the effect of exogenous sclerostin on osteocytes in trabecular bone mechanically loaded ex vivo. Bovine trabecular bone cores, with bone marrow removed, were inserted into individual chambers and subjected to daily episodes of dynamic loading. Cores were perfused with either osteogenic media alone or media containing human recombinant sclerostin (rhSCL) (50 ng/ml). Loaded control bone increased in apparent stiffness over time compared with unloaded bone, and this was abrogated in the presence of rhSCL. Loaded bone showed an increase in calcein uptake as a surrogate of mineral accretion, compared with unloaded bone, in which this was substantially inhibited by rhSCL treatment. Sclerostin treatment induced a significant increase in the ionized calcium concentration in the perfusate and the release of -CTX at several time points, an increased mean osteocyte lacunar size, indicative of osteocytic osteolysis, and the expression of catabolism-related genes. Human primary osteocyte-like cultures treated with rhSCL also released -CTX from their matrix. These results suggest that osteocytes contribute directly to bone mineral accretion, and to the mechanical properties of bone. Moreover, it appears that sclerostin, acting on osteocytes, can negate this effect by modulating the dimensions of the lacunocanalicular porosity and the composition of the periosteocyte matrix

The Effects of Vitamin E Analogues α-Tocopherol and γ-Tocotrienol on the Human Osteocyte Response to Ultra-High Molecular Weight Polyethylene Wear Particles

Abstract: Polyethylene (PE) liners are a common bearing surface of orthopaedic prostheses. Wear particles of ultra-high molecular weight PE (UHMWPE) contribute to periprosthetic osteolysis, a major cause of aseptic loosening. Vitamin E is added to some PE liners to prevent oxidative degradation. Osteocytes, an important cell type for controlling both bone mineralisation and bone resorption, have been shown to respond UHMWPE particles by upregulating pro-osteoclastogenic and osteocytic osteolysis. Here, we examined the effects of the vitamin E analogues α-tocopherol and γ-tocotrienol alone or in the context of UHMWPE particles on human osteocyte gene expression and mineralisation behaviour. Human osteoblasts differentiated to an osteocyte-like stage were exposed to UHMWPE wear particles in the presence or absence of either α-Tocopherol or γ-Tocotrienol. Both α-Tocopherol and γ-Tocotrienol induced antioxidant-related gene expression. UHMWPE particles independently upregulated antioxidant gene expression, suggesting an effect of wear particles on oxidative stress. Both vitamin E analogues strongly induced OPG mRNA expression and γTocotrienol also inhibited RANKL mRNA expression, resulting in a significantly reduced RANKL:OPG mRNA ratio (p < 0.01) overall. UHMWPE particles reversed the suppressive effect of α-Tocopherol but not of γ-Tocotrienol on this pro-osteoclastogenic index. UHMWPE particles also upregulated osteocytic-osteolysis related gene expression. Vitamin E analogues alone or in combination with UHMWPE particles also resulted in upregulation of these genes. Consistent with this, both vitamin E analogues promoted calcium release from mineralised cultures of osteocyte-like cells. Our findings suggest that while vitamin E may suppress osteocyte support of osteoclastogenesis in the presence of UHMWPE particles, the antioxidant effect may induce osteocytic osteolysis, which could promote periprosthetic osteolysis. It will be important to conduct further studies of vitamin E to determine the long-term effects of its inclusion in prosthetic materials.Renee T. Ormsby, Kunihiro Hosaka, Andreas Evdokiou, Andreani Odysseos, David M. Findlay, Lucian B. Solomon, and Gerald J. Atkin

Immune Regulation of Mammary Fibroblasts and the Impact of Mammographic Density

Mammographic density is associated with a 4–6-fold increase in breast cancer risk independent of age and BMI. High mammographic density is characterized by breast tissue with high proportions of stroma comprised of fibroblasts, collagen, and immune cells. This study sought to investigate whether stromal fibroblasts from high mammographic density breast tissue contributes to increased extracellular matrix deposition and pro-tumorigenic signaling. Mammary fibroblasts were isolated from women with high and low mammographic density and exposed to immune factors myeloperoxidase (MPO), eosinophil peroxidase (EPO), transforming growth factor beta 1 (TGFB1) and tumour necrosis factor alpha (TNFA) for 72 h and profiled for expression of cancer-associated fibroblast and extracellular matrix regulation markers. No differences in gene expression profiles or collagen production were observed between fibroblasts with high or low mammographic density, and they did not have a differential response to immune mediators. MPO and EPO significantly increased the production of collagen 1. TGFB and TNFA induced variable changes in gene expression. Fibroblasts cultured in vitro from women with high mammographic density do not appear to be inherently different to those from women with low mammographic density. The function of fibroblasts in mammographic density-associated breast cancer risk is likely to be regulated by immune signals from surrounding cells in the microenvironment.Maddison Archer, Pallave Dasari, David Walsh, Kara L. Britt, Andreas Evdokiou, and Wendy V. Ingma

Elevated expression of caspase-3 inhibitors, survivin and xIAP correlates with low levels of apoptosis in active rheumatoid synovium

Introduction: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a tumour necrosis factor (TNF) family member capable of inducing apoptosis in many cell types. Methods: Using immunohistochemistry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and real-time PCR we investigated the expression of TRAIL, TRAIL receptors and several key molecules of the intracellular apoptotic pathway in human synovial tissues from various types of arthritis and normal controls. Synovial tissues from patients with active rheumatoid arthritis (RA), inactive RA, osteoarthritis (OA) or spondyloarthritis (SpA) and normal individuals were studied. Results Significantly higher levels of TRAIL, TRAIL R1, TRAIL R2 and TRAIL R4 were observed in synovial tissues from patients with active RA compared with normal controls (p < 0.05). TRAIL, TRAIL R1 and TRAIL R4 were expressed by many of the cells expressing CD68 (macrophages). Lower levels of TUNEL but higher levels of cleaved caspase-3 staining were detected in tissue from active RA compared with inactive RA patients (p < 0.05). Higher levels of survivin and x-linked inhibitor of apoptosis protein (xIAP) were expressed in active RA synovial tissues compared with inactive RA observed at both the protein and mRNA levels. Conclusions: This study indicates that the induction of apoptosis in active RA synovial tissues is inhibited despite stimulation of the intracellular pathway(s) that lead to apoptosis. This inhibition of apoptosis was observed downstream of caspase-3 and may involve the caspase-3 inhibitors, survivin and xIAP.Anak ASSK Dharmapatni, Malcolm D Smith, David M Findlay, Christopher A Holding, Andreas Evdokiou, Michael J Ahern, Helen Weedon, Paul Chen, Gavin Screaton, Xiao N Xu and David R Hayne

Pharmacological blockade of aquaporin-1 water channel by AqB013 restricts migration and invasiveness of colon cancer cells and prevents endothelial tube formation in vitro

BACKGROUND Aquaporins (AQP) are water channel proteins that enable fluid fluxes across cell membranes, important for homeostasis of the tissue environment and for cell migration. AQP1 knockout mouse models of human cancers showed marked inhibition of tumor-induced angiogenesis, and in pre-clinical studies of colon adenocarcinomas, forced over-expression of AQP1 was shown to increase angiogenesis, invasion and metastasis. We have synthesized small molecule antagonists of AQP1. Our hypothesis is that inhibition of AQP1 will reduce migration and invasiveness of colon cancer cells, and the migration and tube-forming capacity of endothelial cells in vitro. METHODS Expression of AQP1 in cell lines was assessed by quantitative (q) PCR, western blot and immunofluorescence, while expression of AQP1 in human colon tumour tissue was assessed by immunohistochemistry. The effect of varying concentrations of the AQP1 inhibitor AqB013 was tested on human colon cancer cell lines expressing high versus low levels of AQP1, using wound closure (migration) assays, matrigel invasion assays, and proliferation assays. The effect of AqB013 on angiogenesis was tested using an endothelial cell tube-formation assay. RESULTS HT29 colon cancer cells with high AQP1 levels showed significant inhibition of migration compared to vehicle control of 27.9 % ± 2.6 % (p < 0.0001) and 41.2 % ± 2.7 (p <0.0001) treated with 160 or 320 μM AqB013 respectively, whereas there was no effect on migration of HCT-116 cells with low AQP1 expression. In an invasion assay, HT29 cells treated with 160 μM of AqB013, showed a 60.3 % ± 8.5 % decrease in invasion at 144 hours (p < 0.0001) and significantly decreased rate of invasion compared with the vehicle control (F-test, p = 0.001). Almost complete inhibition of endothelial tube formation (angiogenesis assay) was achieved at 80 μM AqB013 compared to vehicle control (p < 0.0001). CONCLUSION These data provide good evidence for further testing of the inhibitor as a therapeutic agent in colon cancer.Hilary S. Dorward, Alice Du, Maressa A. Bruhn, Joseph Wrin, Jinxin V. Pei, Andreas Evdokiou, Timothy J. Price, Andrea J. Yool, and Jennifer E. Hardingha

Intracellular zinc depletion induces caspase activation and p21Waf1/Cip1 cleavage in human epithelial cell lines

To better understand the mechanisms by which zinc deficiency induces epithelial cell death, studies were done of the effects of intracellular zinc depletion induced by the zinc chelator TPEN on apoptosis-related events in human malignant epithelial cell lines LIM1215 (colonic), NCI-H292 (bronchial), and A549 (alveolar type II). In TPEN-treated cells, depletion of zinc was followed by activation of caspase-3 (as demonstrated by enzymatic assay and Western blotting), DNA fragmentation, and morphologic changes. Increase in caspase-3 activity began 1–2 h after addition of TPEN, suggesting that zinc may suppress a step just before the activation of this caspase. Caspase-6, a mediator of caspase-3 processing, also increased, but later than caspase-3. Effects of TPEN on apoptosis were completely prevented by exogenous ZnSO4 and partially prevented by peptide caspase inhibitors. A critical substrate of caspase-3 may be the cell cycle regulator p21Waf1/Cip1, which was rapidly cleaved in TPEN-treated cells to a 15-kDa fragment before further degradation.F. Chai, A. Q. Truong-Tran, A. Evdokiou, G. P. Young and P. D. Zalewsk

Plant-derived soybean peroxidase stimulates osteoblast collagen biosynthesis, matrix mineralization, and accelerates bone regeneration in a sheep model

Bone defects arising from fractures or disease represent a significant problem for surgeons to manage and are a substantial economic burden on the healthcare economy. Recent advances in the development of biomaterial substitutes provides an attractive alternative to the current “gold standard” autologous bone grafting. Despite on-going research, we are yet to identify cost effective biocompatible, osteo-inductive factors that stimulate controlled, accelerated bone regeneration.We have recently reported that enzymes with peroxidase activity possess previously unrecognised roles in extracellular matrix biosynthesis, angiogenesis and osteoclastogenesis, which are essential processes in bone remodelling and repair. Here, we report for the first time, that plant-derived soybean peroxidase (SBP) possesses pro-osteogenic ability by promoting collagen I biosynthesis and matrix mineralization of human osteoblasts in vitro. Mechanistically, SBP regulates osteogenic genes responsible for inflammation, extracellular matrix remodelling and ossification, which are necessary for normal bone healing. Furthermore, SBP was shown to have osteo-inductive properties, that when combined with commercially available biphasic calcium phosphate (BCP) granules can accelerate bone repair in a critical size long bone defect ovine model. Micro-CT analysis showed that SBP when combined with commercially available biphasic calcium phosphate (BCP) granules significantly increased bone formation within the defects as early as 4 weeks compared to BCP alone. Histomorphometric assessment demonstrated accelerated bone formation prominent at the defect margins and surrounding individual BCP granules, with evidence of intramembranous ossification. These results highlight the capacity of SBP to be an effective regulator of osteoblastic function and may be beneficial as a new and cost effective osteo-inductive agent to accelerate repair of large bone defects.Alexandra J. Barker, Agnes Arthur, Mark O. DeNichilo, Romana Panagopoulos, Stan Gronthos, Peter J. Anderson, Andrew C.W. Zannettino, Andreas Evdokiou, Vasilios Panagopoulo

Progressive resistance of BTK-143 osteosarcoma cells to Apo2L/TRAIL-induced apoptosis is mediated by acquisition of DcR2/TRAIL-R4 expression: resensitisation with chemotherapy

© 2003 Cancer Research UKApo2 ligand (Apo2L, also known as TRAIL) is a member of the tumour necrosis factor (TNF) family of cytokines that selectively induces the death of cancer cells, but not of normal cells. We observed that recombinant Apo2L/TRAIL was proapoptotic in early-passage BTK-143 osteogenic sarcoma cells, inducing 80% cell death during a 24 h treatment period. Apo2L/TRAIL-induced apoptosis was blocked by caspase inhibition. With increasing passage in culture, BTK-143 cells became progressively resistant to the apoptotic effects of Apo2L/TRAIL . RNA and flow cytometric analysis demonstrated that resistance to Apo2L/TRAIL was paralleled by progressive acquisition of the decoy receptor, DcR2. Blocking of DcR2 function with a specific anti-DcR2 antibody restored sensitivity to Apo2L/TRAIL in a dose-dependent manner. Importantly, treatment of resistant cells with the chemotherapeutic agents doxorubicin, cisplatin and etoposide reversed the resistance to Apo2L/TRAIL, which was associated with drug-induced upregulation of mRNA encoding the death receptors DR4 and DR5. BTK-143 cells thus represent a useful model system to investigate both the mechanisms of acquisition of resistance of tumour cells to Apo2L/TRAIL and the use of conventional drugs and novel agents to overcome resistance to Apo2L/TRAIL.S Bouralexis, D M Findlay, G J Atkins, A Labrinidis, S Hay & A Evdokio

DNA Methylation of the ABO Promoter Underlies Loss of ABO Allelic Expression in a Significant Proportion of Leukemic Patients

Background: Loss of A, B and H antigens from the red blood cells of patients with myeloid malignancies is a frequent occurrence. Previously, we have reported alterations in ABH antigens on the red blood cells of 55% of patients with myeloid malignancies. Methodology/Principal Findings: To determine the underlying molecular mechanisms of this loss, we assessed ABO allelic expression in 21 patients with ABH antigen loss previously identified by flow cytometric analysis as well as an additional 7 patients detected with ABH antigen changes by serology. When assessing ABO mRNA allelic expression, 6/12 (50%) patients with ABH antigen loss detected by flow cytometry and 5/7 (71%) of the patients with ABH antigen loss detected by serology had a corresponding ABO mRNA allelic loss of expression. We examined the ABO locus for copy number and DNA methylation alterations in 21 patients, 11 with loss of expression of one or both ABO alleles, and 10 patients with no detectable allelic loss of ABO mRNA expression. No loss of heterozygosity (LOH) at the ABO locus was observed in these patients. However in 8/11 (73%) patients with loss of ABO allelic expression, the ABO promoter was methylated compared with 2/10 (20%) of patients with no ABO allelic expression loss (P = 0.03). Conclusions/Significance: We have found that loss of ABH antigens in patients with hematological malignancies is associated with a corresponding loss of ABO allelic expression in a significant proportion of patients. Loss of ABO allelic expression was strongly associated with DNA methylation of the ABO promoter.Tina Bianco-Miotto, Damian J. Hussey, Tanya K. Day, Denise S. O'Keefe and Alexander Dobrovi

Anticancer efficacy of the hypoxia-activated prodrug evofosfamide (TH-302) in osteolytic breast cancer murine models

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia offers treatment opportunities, exemplified by the development of compounds that target hypoxic regions within tumors. Evofosfamide (TH-302) is a prodrug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide mustard (Br-IPM). When evofosfamide is delivered to hypoxic regions, the DNA cross-linking effector, Br-IPM, is released. This study assessed the cytotoxic activity of evofosfamide in vitro and its antitumor activity against osteolytic breast cancer either alone or in combination with paclitaxel in vivo. A panel of human breast cancer cell lines were treated with evofosfamide under hypoxia and assessed for cell viability. Osteolytic MDA-MB-231-TXSA cells were transplanted into the mammary fat pad, or into tibiae of mice, allowed to establish and treated with evofosfamide, paclitaxel, or both. Tumor burden was monitored using bioluminescence, and cancer-induced bone destruction was measured using micro-CT. In vitro, evofosfamide was selectively cytotoxic under hypoxic conditions. In vivo evofosfamide was tumor suppressive as a single agent and cooperated with paclitaxel to reduce mammary tumor growth. Breast cancer cells transplanted into the tibiae of mice developed osteolytic lesions. In contrast, treatment with evofosfamide or paclitaxel resulted in a significant delay in tumor growth and an overall reduction in tumor burden in bone, whereas combined treatment resulted in a significantly greater reduction in tumor burden in the tibia of mice. Evofosfamide cooperates with paclitaxel and exhibits potent tumor suppressive activity against breast cancer growth in the mammary gland and in bone.Vasilios Liapis, Irene Zinonos, Agatha Labrinidis, Shelley Hay, Vladimir Ponomarev, Vasilios Panagopoulos, Aneta Zysk, Mark DeNichilo, Wendy Ingman, Gerald J. Atkins, David M. Findlay, Andrew C. W. Zannettino, Andreas Evdokio