Nachweis und Charakterisierung von RANK-Ligand und Osteoprotegerin in humanen Pankreaskarzinomzellen

Zusammenfassung: Die Modulation zellulärer Apoptoseprozesse ist nicht nur essenziell für die normale Zellphysiologie, sondern auch von fundamentaler Bedeutung in der Karzinogenese. Störungen zellulärer Kontrollmechanismen der Apoptose gelten als kritisches Ereignis in der Initiierung und Progression von Tumoren. Eine reduzierte Apoptoserate wird ferner als wichtiger Mechanismus für die Resistenz von Tumorzellen gegenüber einer Radio- und Chemotherapie angesehen. Das duktale Adenokarzinom des Pankreas gehört zu den Tumoren, die weitgehend resistent gegenüber einer konventionellen Chemotherapie sind. Basierend auf den bisherigen Befunden zur Pathogenese des Pankreas-karzinoms und den Vorarbeiten zum Thema RANKL-OPG-System war das Ziel der vorliegenden Arbeit, die Expression und Modulation der OPG-mRNA-Produktion und OPG-Proteinsekretion in humanen Pankreaskarzinomzelllinien zu untersuchen. Als Zellmodelle wurden die Adenokarzinomzelllinien PaTu 8988t, PaTu 8988s, PC-2 und Capan-2 analysiert und der Effekt von Steroidhormonen, Zytokinen und Medikamenten auf die OPG-Expression evaluiert. Die Genexpression wurde mittels Northern Blot und PCR, die Proteinsekretion mittels ELISA bestimmt. Osteoprotegerin (OPG) und Receptor activator of nuclear factor-κB ligand (RANKL) stellen spezifische Rezeptoren und Liganden der TNF-Superfamilie dar, deren Produktion bislang in verschiedenen humanen Geweben nachgewiesen werden konnte. Im Anschluss an den Nachweis von OPG-mRNA im Pankreasgewebe mittels eines kommerziellen Northern Blots konnte OPG auf mRNA- und Proteinebene in zwei der vier untersuchten Pankreaskarzinomzelllinien (PaTu 8988t und PC-2) nachgewiesen werden. Die PaTu 8988s- und Capan-2-Zellen zeigten dagegen keine Produktion von OPG. Zusätzlich exprimierten PaTu 8988t-Zellen auf mRNA-Ebene RANKL. Für Testosteron ergab sich eine dosis- und zeitabhängige Hemmung der OPG-mRNA- und -Proteinexpression in der Pankreaskarzinomzelllinie PaTu 8988t um 70 bis 77%, während 5α-DHT und 17β-Östradiol keine Effekte hatten. Das adrenale Androgen DHEA hemmte die OPG-Produktion um 83%. In den PC-2- Zellen hingegen senkte das gonadale Androgen 5α-DHT die OPG-Expression. Nach der Exposition der Zelllinie PaTu 8988t durch das Glukokortikoid Dexamethason kam es ebenfalls zu einer dosisabhängigen Hemmung der OPG-mRNA-Spiegel und der OPG-Proteinproduktion um 23%. Aufgrund der wichtigen Rolle von Zytokinen im Rahmen der Tumorimmunologie und der Modulation der Apoptose beim Pankreaskarzinom wurden unterschiedliche Zytokine (IL-1β, TNF-α und IL-8) hinsichtlich ihrer Regulation der OPG-Produktion untersucht. Im Gegensatz zu vielen anderen Zelltypen jedoch hatten diese Zytokine – IL-8 ausgenommen – keinen Einfluss auf die OPG-Expression. Die Behandlung mit IL-8 führte zu einer leichten Steigerung der OPG-mRNA-Produktion in PaTu 8988t-Zellen. Die HMG-CoA-Reduktase-Inhibitoren Lovastatin und Cerivastatin zeigten in Pankreaskarzinomzelllinien hemmende Effekte auf die OPG-mRNA- und OPG-Proteinexpression. Lovastatin als Statin der ersten Generation senkte die OPG-mRNA- und -Proteinsekretion in PaTu 8988t-Zellen um 90%. Des Weiteren wurden die OPG-mRNA- und -Proteinspiegel durch Cerivastatin in PaTu 8988t-Zellen um 27% und in PC-2-Zellen um 39% gesenkt. Diese Hemmung durch Statine war dosis- und zeitabhängig. Die Hemmung der OPG-Produktion in Pankreaskarzinomzelllinien ist ein weiterer Beleg für die pleiotropen Wirkungen der Statine, die klinisch hinsichtlich ihrer Apoptose modulierenden Effekte auf Pankreaskarzinomzellen genauer untersucht werden sollten. Zusammenfassend weisen die eigenen Ergebnisse in Zusammenschau mit den Arbeiten anderer Gruppen darauf hin, dass das Pankreas sowohl eine Produktionsstätte als auch ein potenzielles Ziel von OPG darstellt, einem wichtigen osteotropen und Apoptose modulierenden Zytokin der TNF-Rezeptorsuperfamilie. Zukünftige Studien sollten die genaue Interaktion von OPG und TRAIL sowie ihre Bedeutung für die Entstehung, Progression und Metastasierung des Pankreaskarzinoms analysieren. Diese Erkenntnisse könnten die Basis künftiger klinischer Studien sein, in denen die gezielte Modulation der Apoptose als therapeutisches Prinzip bei Patienten mit Pankreaskarzinomen eingesetzt werden kann

Glucocorticoids suppress Wnt16 expression in osteoblasts in vitro and in vivo

Glucocorticoid-induced osteoporosis is a frequent complication of systemic glucocorticoid (GC) therapy and mainly characterized by suppressed osteoblast activity. Wnt16 derived from osteogenic cells is a key determinant of bone mass. Here, we assessed whether GC suppress bone formation via inhibiting Wnt16 expression. GC treatment with dexamethasone (DEX) decreased Wnt16 mRNA levels in murine bone marrow stromal cells (mBMSCs) time- and dose-dependently. Similarly, Wnt16 expression was also suppressed after DEX treatment in calvarial organ cultures. Consistently, mice receiving GC-containing slow-release prednisolone pellets showed lower skeletal Wnt16 mRNA levels and bone mineral density than placebo-treated mice. The suppression of Wnt16 by GCs was GC-receptor-dependent as co-treatment of mBMSCs with DEX and the GR antagonist RU-486 abrogated the GC-mediated suppression of Wnt16. Likewise, DEX failed to suppress Wnt16 expression in GR knockout-mBMSCs. In addition, Wnt16 mRNA levels were unaltered in bone tissue of GC-treated GR dimerization-defective GRdim mice, suggesting that GCs suppress Wnt16 via direct DNA-binding mechanisms. Consistently, DEX treatment reduced Wnt16 promoter activity in MC3T3-E1 cells. Finally, recombinant Wnt16 restored DEX-induced suppression of bone formation in mouse calvaria. Thus, this study identifies Wnt16 as a novel target of GC action in GC-induced suppression of bone formation

Targeting Bone Metabolism in Patients with Advanced Prostate Cancer: Current Options and Controversies

Maintaining bone health remains a clinical challenge in patients with prostate cancer (PC) who are at risk of developing metastatic bone disease and increased bone loss due to hormone ablation therapy. In patients with cancer-treatment induced bone loss (CTIBL), antiresorptive agents have been shown to improve bone mineral density (BMD) and to reduce the risk of fractures. For patients with bone metastases, both zoledronic acid and denosumab delay skeletal related events (SREs) in the castration resistant stage of disease. Novel agents targeting the Wnt inhibitors dickkopf-1 and sclerostin are currently under investigation for the treatment of osteoporosis and malignant bone disease. New antineoplastic drugs such as abiraterone, enzalutamide, and Radium-223 are capable of further delaying SREs in patients with advanced PC. The benefit of antiresorptive treatment for patients with castration sensitive PC appears to be limited. Recent trials on the use of zoledronic acid for the prevention of bone metastases failed to be successful, whereas denosumab delayed the occurrence of bone metastases by a median of 4.1 months. Currently, the use of antiresorptive drugs to prevent bone metastases still remains a field of controversies and further trials are needed to identify patient subgroups that may profit from early therapy

denosumab in postmenopausal women with osteoporosis and diabetes subgroup analysis of freedom and freedom extension

Abstract Purpose Diabetes and osteoporosis occur frequently in older adults and are both associated with increased fracture risk. Denosumab treatment reduced new vertebral, nonvertebral, and hip fractures over 3 years, with continued low fracture incidence for up to 10 years in postmenopausal women with osteoporosis. However, its effects in diabetic subjects with osteoporosis have not yet been investigated. Methods Post hoc analysis of the 3-year, placebo-controlled FREEDOM study and 7-year Extension included postmenopausal women with osteoporosis and diabetes. Effects on BMD, vertebral, and nonvertebral fracture incidence were evaluated. Results Of 7808 subjects in FREEDOM, 508 with diabetes received denosumab (n = 266) or placebo (n = 242). Among those, BMD increased significantly with denosumab versus placebo in FREEDOM, and continued to increase during the Extension in long-term (continuing denosumab) and crossover (placebo to denosumab) denosumab subjects. In FREEDOM, denosumab-treated subjects with diabetes had significantly lower new vertebral fracture rates (1.6%) versus placebo (8.0%) (RR: 0.20 [95% CI 0.07–0.61]; p = .001). Nonvertebral fracture incidence was higher with denosumab (11.7%) versus placebo (5.9%) (HR: 1.94 [95% CI 1.00–3.77]; p = .046), although there were fewer hip fractures with denosumab (World Health Organization, 2017 [ 1 ]) than placebo (4; nonsignificant). During the first 3 years in FREEDOM Extension, new vertebral and nonvertebral fracture incidences were low in long-term and crossover denosumab diabetic groups (≤6%), consistent with the overall Extension population; yearly nonvertebral fracture incidence was comparable to the FREEDOM placebo group. Conclusion Denosumab significantly increased BMD and decreased vertebral fracture risk in subjects with osteoporosis and diabetes. No reduction in nonvertebral fractures was observed

Increased pore size of scaffolds improves coating efficiency with sulfated hyaluronan and mineralization capacity of osteoblasts

Background: Delayed bone regeneration of fractures in osteoporosis patients or of critical-size bone defects after tumor resection are a major medical and socio-economic challenge. Therefore, the development of more effective and osteoinductive biomaterials is crucial. Methods: We examined the osteogenic potential of macroporous scaffolds with varying pore sizes after biofunctionalization with a collagen/high-sulfated hyaluronan (sHA3) coating in vitro. The three-dimensional scaffolds were made up from a biodegradable three-armed lactic acid-based macromer (TriLA) by cross-polymerization. Templating with solid lipid particles that melt during fabrication generates a continuous pore network. Human mesenchymal stem cells (hMSC) cultivated on the functionalized scaffolds in vitro were investigated for cell viability, production of alkaline phosphatase (ALP) and bone matrix formation. Statistical analysis was performed using student's t-test or two-way ANOVA. Results: We succeeded in generating scaffolds that feature a significantly higher average pore size and a broader distribution of individual pore sizes (HiPo) by modifying composition and relative amount of lipid particles, macromer concentration and temperature for cross-polymerization during scaffold fabrication. Overall porosity was retained, while the scaffolds showed a 25% decrease in compressive modulus compared to the initial TriLA scaffolds with a lower pore size (LoPo). These HiPo scaffolds were more readily coated as shown by higher amounts of immobilized collagen (+ 44%) and sHA3 (+ 25%) compared to LoPo scaffolds. In vitro, culture of hMSCs on collagen and/or sHA3-coated HiPo scaffolds demonstrated unaltered cell viability. Furthermore, the production of ALP, an early marker of osteogenesis (+ 3-fold), and formation of new bone matrix (+ 2.5-fold) was enhanced by the functionalization with sHA3 of both scaffold types. Nevertheless, effects were more pronounced on HiPo scaffolds about 112%. Conclusion: In summary, we showed that the improvement of scaffold pore sizes enhanced the coating efficiency with collagen and sHA3, which had a significant positive effect on bone formation markers, underlining the promise of using this material approach for in vivo studies. © 2019 The Author(s)