Pathways involved in the antiproliferative effect of calcium in colon cancer cells

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tubular cells. pSmad was expressed in all normal and adjacent to carcinoma tissue samples. We also found the activity of BMP signaling pathway in renal carcinoma cells what was expressed through positive staining for pSmad 1,5,8. Immunostaining was localized in citoplasm and nucleus, too.Conclusion: These results indicate that BMP signaling pathway is active in renal carcinoma cells as well as in tubular cells of normal kidney. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.489

P064 Zoledronic acid use in men with advanced prostatic cancer led to improvement in general well being and performance E. Segala,*, E. Gezb, S. Serafimovicha, S. Ish-Shaloma a Metabolic Bone Diseases Unit b Oncology, Rambam Medical Care Campus, Haifa, Israel Use of zoledronic acid(ZA)was shown to slow progression of bone involvement,to cause pain palliation in patients with bone metastases. Patients and methods:14prostate cancer patients with bone metastases,age70 ± 10 who received monthly intravenouly 4 mg of ZA during one year,calcium carbonate 600 mg,Vit D1000 IU/ d.Quality of life(QOL)assessed by:brief pain inventory form,scale from 1 to 10 for pain,from 10 to1 for pain interference with daily activities; standard analgesic score from 0 to 4;functional assessment of cancer therapy-prostate(FACT-P)questionnaire to assess functional abilities, performance status by Eastern Cooperative Oncology Group (ECOG) scale. Results: 10 patients completed the one year follow-up, 3 died; one lost to follow-up.The pain decreased during the first 6 mo, deteriorated later. In Table 1 are presented the data as changes during continuous one year process using Friedman's test.Positive correlation observed between strength of pain and sleep, contacts with people, enjoyment of life, ability to work: Pearson's test p < 0.01 at3 and 6 mo, 0.005 at 12; p = 0.01 at 3 and 6 mo 0.05 at 12;: p < 0.01 at 3 and 9 mo, 0.001 at 12, p < 0.01 at 3 and 6 mo, 0.025 at 12 mo. Improvement in mood:p < 0.001 at all time points.Despite deterioration in pain after6 mo the enjoyment from life was significantly higher in the end of study, compared to the start point. Changes in the analgesics scoreswere non-significant,due to small sample size. Conclusion: monthly treatment with infusions of ZA might improve QOL in prostate cancer patients with bone metastases. Table 1 0 Mean pain score Influence activity Influence on mood Influenceon work Influence home work Influence enjoyment

3 mo

6 mo

9 mo

12 mo

Friedman test p

3.88 ± 2.4 1.63 ± 1.6 2.13 ± 3.2 2.13 ± 2 4 ± 2.8 0.011 3.57 ± 3.3 0.86 ± 1.25 1.86 ± 3.3 3.29 ± 2.4 4 ± 2.7 0.06 2.86 ± 3.13 1.57 ± 1.5 1 ± 1.4 2.57 ± 2.2 4.14 ± 3.1 0.06 2.63 ± 2.8 4.25 ± 3.6

1 ± 1.6 1.38 ± 1.6

4.14 ± 3.6

2 ± 2.3

1.75 ± 3.4 1.38 ± 3.3 4 ± 3.5 0.047 1.88 ± 3.2 4 ± 3.1 4.38 ± 2.9 0.06 1.14 ± 1.5

2 ± 2.2 3.29 ± 1.9 0.026

Conflict of interest: S.Ish-Shalom, MSD, Lilly, Transpharma, Novartis, Aventis, Grant. Research Support, MSD, Lilly, Novartis, Transpharma, Consultant. doi:10.1016/j.bone.2009.03.490

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P065 Local management for post operative by associating calcium deficient apatite with a local anesthetic E. Verrona,*, O. Gauthiera, P. Janvierb, H. Le Guena, D. Holophernea, R. Cavagnaa, B. Bujolib, J. Boulera a INSERM U791, laboratory of osteoarticular and dental tissue engineering b CNRS UMR, LSO, NANTES, France Synthetic calcium-deficient apatites (CDA) are chemical precursor for biphasic calcium phosphates that are widely used as bone substitutes in human surgery. The purpose of the study was to define in vitro release profiles of an analgesic which has been previously loaded onto CDA using isostatic compaction and to evaluate in vivo performance on pain. CDA powder was loaded with 1%, 4% and 16% w/w of bupivacaïn using isostatic compression. We determined the profile of release by using UV spectrophotometry. Rats were implanted in femur with bupivacain-loaded CDA (N = 10). Analgesia was measured using electronic Von Frey monofilament electronic version, inflammatory response and neurological score. Bupivacaïn was totally released after 48 h of in vitro incubation. During the first post-operative day, we observed a dose dependant analgesic effect with the bupivacaïn adsorbed quantity (figure). Both inflammatory response and neurological score confirmed this result. This drug-combined device has been shown to provide a release of local anesthetic adapted to prevent short-term post operative pain. This approach could be integrated in the global management of pain needed for bone graft surgery. Although rat is considered as the validated model for evaluating pain, it seems not to be optimal to measure mid-term post op pain. Therefore additional in vivo experiments are under progress with rabbits and dogs. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.491

P066 Pathways involved in the antiproliferative effect of calcium in colon cancer cells E. Kallay⁎ Pathophysiology, Medical University of Vienna, Vienna, Austria Numerous studies have linked high calcium intake with lower colon cancer risk and identified calcium as possible cancer preventive agent. The aim of our study was to find pathways involved in the anitproliferative effect of calcium in colon cancer cells and to test if the calcium sensing receptor (CaSR) has a role in mediating this effect. Confluent Caco-2 cells, exposed to serum-free, calcium-free DMEM for 48 h were treated with 2.0 mM calcium for 1, 4, and 24 h. Effect of calcium was assessed by microarray analysis with the Human Genome 133 plus 2 Array from Affymetrix and validated with real time RT-PCR. Image data were analyzed with GCOS 1.4 using Affymetrix's default analysis settings and global scaling for normalization. Data was analysed by parametric ANOVA and pair-wise comparisons with Student's t-test. The criteria for restricting number of differentially expressed genes (n = 1571 probe sets) was an ANOVA value <10-5 and a false discovery rate <10-3. Earliest changes involved tight junctions, focal adhesion, adherens junctions, with some of the parameters being up-others downregulated. Among the canonical pathways mostly affected were: arylhydrocarbon receptor signalling, aminoacyl-tRNA biosynthesis, cell cycle: G1/S checkpoint regulation and protein ubiquitination pathway. Cell cycle was inhibited in a time-dependent manner: While after 1 h only a few parameter were changed, after 4 h there was a clear effect: 24 genes were inhibited. After 24 h, calcium reduced

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expression of 48 genes involved in cell-cycle while expression of only one gene, the cyclin-dependent kinase inhibitor p15 was enhanced. Validation experiments shoved that gadolinium, an agonist of the CaSR, had similar effects, suggesting that the receptor might mediate the antiproliferative effect of calcium. DNA replication, recombination and post-transcriptional RNA-modification were also suppressed. Almost every enzyme involved in aminoacyl-tRNA synthesis was down-regulated by calcium treatment for 24 h as well as the majority of the proteasome subunits. The gene with the highest increase in expression after 24 h of calcium treatment was sucrase-isomaltase, a specific differentiation marker of Caco-2 cells. Our data suggest that calcium supplementation inhibited proliferation and stimulated differentiation of Caco-2 cells in a manner similar to the natural process of cell maturation. This study was supported by a Marie Curie Inter-European Fellowship. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.492

P067 Development of a sclerostin elisa for clinical use G. Schetta,*, M. Henniesb a Department of Internal Medicine and Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany b Technical Support and Development, TECOmedical AG, Sissach, Switzerland Sclerostin, the product of the SOST gene is an osteocyte specific protein, which inhibits bone formation. Mutation of the SOST gene leads to loss of function of sclerostin and sclerosteosis- a disease characterized by increased bone mass. Pharmacological inhibition of sclerostin is thus considered as an attractive therapeutic for osteoporosis. As direct assessment of sclerostin expression is difficult in humans we aimed to measure sclerostin in the serum to define whether sclerostin levels differs among population groups. We thus developed a sandwich ELISA using a monoclonal antibody and a biotinylated polyclonal antibody for the quantitative determination of sclerostin in serum and plasma for routine use. The assay allows analyzing 50 μl of undiluted specimens within 4 h. With a measuring range of 0.3 – 5 ng/ml, the analytical sensitivity of the ELISA was at 0.2 ng/ml using recombinant human sclerostin for standard preparation. The mean recovery of sclerostin in serially diluted human serum samples was 110% of the expected values. For the evaluation of the assay, serum samples from 58 healthy individuals (16 to 91 years, mean 47.2 years) were tested. The mean sclerostin concentration was 0.48 ± 0.26 ng/ml. Corresponding plasma samples were 13% higher. No age dependence of sclerostin levels was found, but an influence by gender: In 19 males we found a mean sclerostin level of 0.64 ±0.22 ng/ml, in premenopausal women 0.28 ± 0.15 ng/ ml (n = 20) and in postmenopausal women 0.47 ± 0.16 ng/ml (n = 19). The differences between groups are statistically significant (ANOVA, P < 0.01). The data show that sclerostin can be reliably measured in serum and plasma of normal individuals. Serum levels of sclerostin are not dependent on age but are influenced by gender, with higher levels found in males than in females, and also between pre- and post-menopausal females. Assessment of sclerostin levels could thus emerge as a tool to define differences in expression level of sclerostin among individuals and also allow monitor to effects of anti-osteoporotic therapy on osteocyte function. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.493

P068 Multimarker approach for linking bone disorders and cardiovascular events in Chronical Kidney Disease (CKD) N. Brinskelle-Schmala, G. Hawaa,*, N. Voorzanger-Rousselotb, A. Lukasc, P. Garnerob a Research and Development, Biomedica Medizinprodukte GmbH Co Kg, Vienna, Austria b Biomarkers, CCBR-Synarc, Lyon c Bioinformatics, emergentec biodevlopment GmbH, Vienna, France Some studies suggest a link between low bone density as found in osteoporotic patients and the risk to develop and die from cardiovascular disease (Relationship between osteoporosis and cardiovascular disease in postmenopausal women, Tankó LB et al. J Bone Miner Res. 2005; 20(11):1912–20). Similar evidence has been found in patients with chronic kidney disease where disturbances in calcium/phosphate and parathyroid hormone metabolism not only lead to “uremic osteodystrophy” but also is furthermore highly associated with cardiovascular calcification (Mineral metabolism, mortality, and morbidity in maintenance hemodialysis., Block GA et al. J Am Soc Nephrol 2004;15:2208–2218). Recently (Farhleitner-Pammer et al. (JBMR 23, 11, 1850–1858, 2008) the predictive value of Bone Specific Alkaline Phosphatase (BSALP) and tartrate-resistant acid phosphatase 5b (TRACP-5b) for cardiovascular events in CKD has been demonstrated. We also chose CKD as a model for a population with a high incidence of cardiovascular and bone disorders to evaluate the predictive value of heart and bone markers and investigating the underlying biochemical pathology. Bioinformatics has been applied to propose a set of potential biomarkers. Using a context graph approach, proteins were selected and ranked that show clear biomarker profiles. These profiles are based on a pre-selection of proteins known to play a role in osteoporosis and/or cardiovascular pathophysiology. The ranking was based on graph theoretical neighbourhood measures derived from protein interaction networks, subcellular localization and cooccurrence in functional pathways. In addition to the ranked preselected protein list, further potential markers were selected as nearest neighbours in the context graph. Final selection was done from ranked lists by educated guess. Exclusion criteria were market consideration and technological issues. Therefore this approach can be understood as a systems biology driven probability filter to accumulate potential biomarkers in a certain context as the interface of osteoporosis and cardiovascular disease. Data sets of established cardiovascular markers (proANP, BigET. BNP fragment) and bone markers (OPG, sRANKL, CTX, sPINP, OC) and first results of new target molecules with a potential role in linking bone and cardiovascular disorders are presented. This work was supported by an EUROTRANS-BIO Grant, as project Nr. 2006–076. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.494

P069 Osteoprotegerin/rank ligand profile related to decreased bone turnover in cushing syndrome B. Galuscaa, B. Lindnerb, N. Germaina, G. Hawab,*, D. Frerec, B. Estoura a Endocrinology Department, CHU Saint Etienne, Saint Etienne, France b Research and Development, Biomedica Medizinprodukte GmbH Co Kg, Vienna, Austria c Nuclear Medicine Department, CHU Saint Etienne, Saint Etienne, France Context. Cushing syndrome (CS) impact on bone turnover, especially on bone resorption remains controversial. The disease is diagnosed in a large age range, inside of which the bone turnover of