ABSTRACT
A new series of bisphosphonates bearing either the nitrogen-containing NO-donor furoxan (1,2,5-oxadiazole 2-oxide) system or the related furazan (1,2,5-oxadiazole) in lateral chain has been developed. pK(a) values and affinity for hydroxyapatite were determined for all the compounds. The products were able to inhibit osteoclastogenesis on RAW 246.7 cells at 10microM concentration. The most active compounds were further assayed on human PBMC cells and on rat microsomes. Unlike most nitrogen-containing bisphosphonates which target farnesyl pyrophosphate synthase, experimental and theoretical investigations suggest that the activity of our derivatives may be related to different mechanisms. The furoxan derivatives were also tested for their ability to relax rat aorta strips in view of their potential NO-dependent vasodilator properties.
Subject(s)
Bone Density Conservation Agents/chemical synthesis , Bone Density Conservation Agents/pharmacology , Diphosphonates/chemical synthesis , Diphosphonates/pharmacology , Nitrogen/chemistry , Animals , Aorta, Thoracic/drug effects , Bone and Bones/drug effects , Bone and Bones/metabolism , Cells, Cultured , Chromatography, Affinity , Durapatite/chemistry , Farnesyl-Diphosphate Farnesyltransferase/antagonists & inhibitors , Humans , Ibandronic Acid , In Vitro Techniques , Macrophages/drug effects , Macrophages/enzymology , Magnetic Resonance Spectroscopy , Male , Mice , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Models, Molecular , Muscle, Smooth, Vascular/drug effects , Nitric Oxide Donors/chemical synthesis , Nitric Oxide Donors/pharmacology , Osteogenesis/drug effects , Rats , Rats, Wistar , Vasodilator Agents/chemical synthesis , Vasodilator Agents/pharmacologyABSTRACT
High levels of interleukin-7 (IL-7) have been associated with bone loss due to its stimulatory osteoclastogenic activity. Osteolytic patients' peripheral blood mononuclear cells (PBMCs) differentiate into osteoclasts without adding stimulating factors. Now, we investigated the potential role of IL-7 in the spontaneous osteoclastogenesis occurring in these patients. We identified significant differences in serum IL-7 levels between patients with/without bone metastases, suggesting that IL-7 might be effective as a clinical marker of disease progression. In patients' PBMC cultures we demonstrated that IL-7 stimulates osteoclastogenesis by inducing TNF-alpha release by T and B cells. These findings add further details to the disclosure of the mechanisms controlling bone metastases in solid tumors.
Subject(s)
Gene Expression Regulation, Neoplastic , Interleukin-7/physiology , Neoplasms/metabolism , Osteoclasts/metabolism , Tumor Necrosis Factor-alpha/metabolism , B-Lymphocytes/metabolism , Case-Control Studies , Disease Progression , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Humans , Interleukin-7/biosynthesis , Interleukin-7/blood , Interleukin-7/metabolism , Leukocytes, Mononuclear/metabolism , Male , Models, Biological , Neoplasm Metastasis , Neoplasms/pathology , T-Lymphocytes/metabolismABSTRACT
BACKGROUND: Bone forming metastases are a common and disabling consequence of prostate cancer (CaP). The potential role of osteoclast activity in CaP bone metastases is not completely explained. In this study, we investigated ex vivo whether the osteolytic activity is present and how it is ruled in CaP patients with bone forming metastases. METHODOLOGY: Forty-six patients affected by newly diagnosed CaP and healthy controls were enrolled. At diagnosis, 37 patients had a primary tumour only, while 9 had primary tumour and concomitant bone forming metastases. In all patients there was no evidence of metastasis to other non-bone sites. For all patients and controls we collected blood and urinary samples. We evaluated patients' bone homeostasis; we made peripheral blood mononuclear cell (PBMC) cultures to detect in vitro osteoclastogenesis; we dosed serum expression of molecules involved in cancer induced osteoclatogenesis, such as RANKL, OPG, TNF-alpha, DKK-1 and IL-7. By Real-Time PCR, we quantified DKK-1 and IL-7 gene expression on micro-dissected tumour and healthy tissue sections. PRINCIPAL FINDINGS: CaP bone metastatic patients showed bone metabolism disruption with increased bone resorption and formation compared to non-bone metastatic patients and healthy controls. The CaP PBMC cultures showed an enhanced osteoclastogenesis in bone metastatic patients, due to an increase of RANKL/OPG ratio. We detected increased DKK-1 serum levels and tissue gene expression in patients compared to controls. IL-7 resulted high in patients' sera, but its tissue gene expression was comparable in patients and controls. CONCLUSIONS: We demonstrated ex vivo that osteoclastogenesis is an active mechanism in tumour nesting of bone forming metastatic cancer and that serum DKK-1 levels are increased in CaP patients, suggesting to deeply investigate its role as tumour marker.
Subject(s)
Bone Neoplasms/etiology , Bone Neoplasms/secondary , Carcinoma/pathology , Osteoclasts/physiology , Prostatic Neoplasms/pathology , Aged , Bone Neoplasms/blood , Bone Neoplasms/physiopathology , Bone Remodeling/genetics , Bone Remodeling/physiology , Bone Resorption/genetics , Bone Resorption/physiopathology , Carcinoma/blood , Carcinoma/genetics , Carcinoma/physiopathology , Case-Control Studies , Cell Differentiation/genetics , Cell Differentiation/physiology , Cells, Cultured , Cross-Sectional Studies , Gene Expression Regulation, Neoplastic , Humans , Intercellular Signaling Peptides and Proteins/blood , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Interleukin-7/blood , Interleukin-7/genetics , Male , Middle Aged , Osteoclasts/metabolism , Osteoprotegerin/blood , Osteoprotegerin/genetics , Prostatic Neoplasms/blood , Prostatic Neoplasms/genetics , Prostatic Neoplasms/physiopathology , RANK Ligand/blood , RANK Ligand/geneticsABSTRACT
BACKGROUND: Interleukin-7 (IL-7) is a potent regulator of lymphocyte development, which has also significant effects on bone; in fact it is a potent osteoclastogenic factor. Some human solid tumors produce high IL-7 levels, suggesting a potential IL-7 role on tumor development and progression. METHODOLOGY: We studied 50 male patients affected by solid tumors, and their blood samples were collected at tumor diagnosis. PBMCs were isolated and cultured with/without IL-7 to study its influence on osteoclastogenesis. Serum and cell culture supernatant IL-7 levels were measured by ELISA. The quantitative analysis of IL-7 expression on T and B cells was performed by Real-Time PCR. PRINCIPAL FINDINGS: Serum IL-7 levels were highest in osteolytic cancer patients, followed by cancer patients without bone lesions, and then healthy controls. We showed the IL-7 production in PBMC cultures and particularly in monocyte and B cell co-cultures. A quantitative analysis of IL-7 expression in T and B cells confirmed that B cells had a high IL-7 expression. In all cell culture conditions, IL-7 significantly increased osteoclastogenesis and an anti-IL-7 antibody inhibited it. We demonstrated that IL-7 supports OC formation by inducing the TNF-alpha production and low RANKL levels, which synergize in promoting osteoclastogenesis. CONCLUSIONS: We demonstrated the presence of high serum IL-7 levels in patients with bone metastasis, suggesting the use of serum IL-7 level as a clinical marker of disease progression and of bone involvement. Moreover, we showed the capability of IL-7 to stimulate spontaneous osteoclastogenesis of bone metastatic patients and to induce osteoclastogenesis in cancer patients without bone involvement. These findings add further details to the disclosure of the mechanisms controlling bone metastasis in solid tumors.