endothelin-1 and Bone-Neoplasms

The endothelin (ET) system includes 3 small peptide hormones and a pair of G-protein-coupled receptors. This review first outlines the ET signaling pathway and ET metabolism. Next, it summarizes the role of ET1 signaling in craniofacial development. Then, it discusses observations relating ET signaling to osteoblastic and other osteosclerotic processes in cancer. Finally, it describes recent work in our laboratory that points to endothelin signaling as an upstream mediator of WNT signaling, promoting bone matrix synthesis and mineralization. It concludes with a statement of some remaining gaps in knowledge and proposals for future research.

Topics: Bone and Bones; Bone Neoplasms; Endothelin-1; Humans; Osteogenesis; Signal Transduction

It has been long recognized that skeleton represents one of the most favored metastatic sites for common cancers like breast and prostate. During the last decade the molecular mechanisms that are responsible for the development of bone metastasis have been gradually illuminated. It appears that the bone microenvironment has a pivotal role in this process. Metastatic tumor cells interact with bone triggering a cascade of molecular events that produce osteolytic and/or osteoblastic phenomena. In this review, we summarize and discuss the most significant factors and signaling pathways implicated in bone colonization. Moreover, based on the recent literature and data, we foresee the need for designing novel agents that will efficiently disrupt these interactions among cancer cells and bone microenvironment, bringing hope for more effective treatments.

Topics: Animals; beta Catenin; Biphenyl Compounds; Bone Neoplasms; Bone Remodeling; Breast Neoplasms; Cathepsin K; Chemokine CXCL12; Diphosphonates; Endothelin-1; Female; Humans; Hypoxia; Male; Neoplastic Stem Cells; Osteoblasts; Osteoclasts; Osteolysis; Parathyroid Hormone-Related Protein; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, CXCR4; Signal Transduction; Urokinase-Type Plasminogen Activator; Wnt Proteins

Patients with prostate cancer who have progression of their disease while on androgen deprivation therapy have limited therapeutic options. Docetaxel is currently the only agent that increases overall survival in patients with metastatic, castration-resistant prostate cancer; additional agents are needed.. This review will describe the importance of endothelin-1 (ET-1) for growth of prostate cancer cells, development of bone metastases, and pain responses; the preclinical data for zibotentan, a specific inhibitor of the ET(A) receptor; and the clinical development of atrasentan, a first-generation ET receptor inhibitor, and zibotentan, a more selective inhibitor of the ET(A) receptor.. Readers will understand the importance of ET-1 as a novel pathway to target for patients with castration-resistant prostate cancer due to its association with prostate cancer growth, metastases to bone, and pain. Readers will learn about the preclinical and clinical development of zibotentan, including the promising Phase II results that have resulted in an extensive Phase III clinical trials program.. Modulating the activity of ET-1 through the ET(A) receptor is a novel target for treating patients with metastatic, castration-resistant prostate cancer. There are currently three ongoing Phase III trials with zibotentan, a selective ET(A) inhibitor, to determine the effect of this agent on overall survival in these patients.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Clinical Trials as Topic; Disease-Free Survival; Drug Evaluation, Preclinical; Endothelin A Receptor Antagonists; Endothelin-1; Humans; Male; Molecular Structure; Neoplasms, Hormone-Dependent; Orchiectomy; Prostatic Neoplasms; Pyrrolidines

Bone metastases are complications of multiple myeloma and solid tumors, including breast and prostate carcinomas. Several reports have demonstrated that the preference to metastasize to bone by tumor cells is not a casual but an addressed event, which relies on specific interactions among tumor cells, bone marrow microenvironment and bone cells. One of the features that gives tumor cells more chances to survive and proliferate into the bone tissue is osteomimicry, that is the ability to acquire a bone cell phenotype, especially osteoblast-like. As clearly demonstrated, prostate and breast cancer cells try to resemble osteoblasts by expressing bone matrix proteins, the specific marker alkaline phosphatase, and molecules regulating the osteoblast/osteoclast cross-talk. Based on this evidence it is crucial to dissect in more detail the molecular mechanisms underlying the osteomimetic properties of cancer cells and identify new therapeutic targets eventually leading to a better and prolonged life expectation for patients with bone.

Topics: Alkaline Phosphatase; beta 2-Microglobulin; Bone Matrix; Bone Morphogenetic Proteins; Bone Neoplasms; Breast Neoplasms; Core Binding Factor Alpha 1 Subunit; Endothelin-1; Female; Humans; Male; Models, Biological; Osteoblasts; Osteolysis; Phenotype; Prostatic Neoplasms; Signal Transduction; Urokinase-Type Plasminogen Activator; Wnt Proteins

Skeletal-related complications occur commonly in many solid tumors including breast, prostate and lung cancer as well as multiple myeloma. In addition, malignancies and their associated treatment may result in bone loss or osteoporosis. This review will focus solely on recent data associated with metastatic bone disease with a focus on breast cancer, prostate cancer and multiple myeloma. Bone loss or osteoporosis associated with cancer will be covered in a separate article in this issue.. Recent progress in understanding the pathophysiology of bone metastases has pointed to several novel pathways: transforming growth factor beta; receptor activator of nuclear factor beta ligand and osteoprotegerin; and Wnt signaling pathways and associated factors such as dickkopf-1 and endothelin-1.. The identification of new pathways is important in metastatic bone disease from cancer and has allowed for the development of novel therapeutics aimed at preventing the devastating complications of bone metastases. Bisphosphonates remain the predominant therapy in use for the treatment and prevention of skeletal-related adverse effects from cancer.

Topics: Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Diphosphonates; Endothelin-1; Female; Humans; Intercellular Signaling Peptides and Proteins; Male; Multiple Myeloma; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Bone metastases are highly prevalent in patients with prostate cancer, and they commonly present a therapeutic challenge. The natural history of prostatic bone metastases is characterized by skeletal morbidity, often producing distressing symptoms for individual patients and reducing patient autonomy and mobility. These bone metastases are usually radiologically osteoblastic, but there is also a strong osteolytic component as evidenced by marked increases in bone resorption markers. Malignant bone lesions can reduce the structural integrity of the skeleton, resulting in skeletal complications such as pathologic fracture, spinal cord compression, and severe bone pain, which adversely affect quality of life. Preclinical and clinical studies have provided insight into the pathophysiology of malignant bone disease from prostate cancer and suggest that bone-directed therapies, including radionuclides, endothelin-1 antagonists, and bisphosphonates, may provide both palliative and therapeutic benefits. Clinical investigations with these agents are underway in patients with prostate cancer to gain insight into the pathophysiology of bone metastases and to evaluate the role of bone-specific therapies in treating and preventing bone metastases.

Topics: Androgen Antagonists; Bone Density Conservation Agents; Bone Neoplasms; Bone Resorption; Diphosphonates; Endothelin-1; Humans; Male; Osteoblasts; Osteoclasts; Palliative Care; Prostatic Neoplasms

All bone surfaces are periodically remodeled by the coupled and balanced action of osteoclasts and osteoblasts, of which the activities are regulated by a variety of cytokines and growth factors. Patients with cancer metastatic to the skeleton often develop osteolytic bone lesions, in which the actions of osteoclasts and osteoblasts remain coupled, but become imbalanced in sites adjacent to the tumor. The result is net bone loss. Many cancers secrete osteoclast-stimulating cytokines, which increase bone resorption by osteoclasts. In turn, factors released from the bone matrix during osteolysis can stimulate tumor growth. In this so-called "vicious cycle," there are multiple sites that are targets for new bone-directed therapies. A variety of new agents for the treatment and prevention of osteolytic bone metastasis are currently being developed. These include new agents that inhibit osteoclast differentiation, bone adhesion, and osteoclast function. These new strategies have evolved from a better understanding of the interaction between tumor cells and cells in the bone marrow microenvironment. There is great promise that these new bone-targeted therapies can decrease the frequent skeletal-related events that greatly diminish quality of life of patients with bone metastases.

Topics: Angiogenesis Inhibitors; Animals; Bone Density Conservation Agents; Bone Neoplasms; Clinical Trials as Topic; Endothelin-1; Humans; Osteoclasts; Osteolysis

Alterations in bone architecture and mineral metabolism are common complications of malignancy. Cancers such as breast, prostate, and lung can affect the skeleton either indirectly through the elaboration of factors that act to disrupt normal calcium homeostasis at the level of the kidney and bone; or directly via secondary spread of tumor to bone. Although the pathophysiology of these skeletal complications is diverse, it is clear that the osteoclast and osteoblast are not just bystanders but are active participants in the development and progression of hypercalcemia and bone metastasis. Our understanding of the molecular mechanisms of metastasis leading to tumor cell escape, homing, adhesion, and secondary growth in a hospitable environment are evolving. Treatment modalities aimed at not only reducing tumor burden but altering the skeletal response to tumor have shown benefit. Newer generation bisphosphonates are quite effective in controlling hypercalcemia of malignancy and have been shown to delay progression of skeletal metastases. Clearly, cancer-associated bone morbidity remains a major public health problem. To improve therapy and prevention it is important to understand the pathophysiology of the effects of cancer on bone. This review will detail scientific advances regarding this area.

Topics: Animals; Atrasentan; Bone Neoplasms; Cell Adhesion Molecules; Cytokines; Diphosphonates; Endothelin-1; Female; Gene Expression Regulation, Neoplastic; Humans; Hypercalcemia; Immunotherapy; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, Nude; Neoplasm Proteins; Osteoblasts; Osteolysis; Pain; Pain Management; Pyrrolidines; Xenograft Model Antitumor Assays

Certain solid tumors metastasize to bone and cause an osteoblastic response. The mechanisms by which tumor cells stimulate this new bone formation are not completely understood.. The authors identified three breast cancer lines that cause osteoblastic metastases in female nude mice and provided evidence that tumor-produced endothelin-1 (ET-1) mediates the osteoblastic response.. Tumor conditioned media, as well as exogenous ET-1, stimulated osteoblast proliferation and new bone formation in cultures of mouse calvariae. These effects were blocked by antagonists of the endothelin A (ET(A)), but not ET(B), receptors. Mice inoculated with the ZR-75-1 breast cancer line and treated with a selective ET(A) receptor antagonist (ABT-627) had significantly fewer osteoblastic bone metastases and less tumor burden compared with untreated mice. In contrast, there was no effect of ABT-627 on osteolytic bone metastases caused by ET-1-negative breast cancer, MDA-MB-231. ABT-627 had no effect on growth in vitro or at the orthotopic site of ZR-75-1 or MDA-MB-231 cells.. Collectively, the data suggested that tumor-produced ET-1 mediates osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation. ET(A) receptor blockade may be useful for prevention and the treatment of osteoblastic bone metastases due to breast or prostate cancer.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Female; Mice; Mice, Nude; Neoplasms, Experimental; Osteoblasts; Receptor, Endothelin A

Certain solid tumors metastasize to bone, causing an osteoblastic response. The mechanisms by which tumor cells stimulate this new bone formation are not understood completely. We identified three breast cancer lines that cause osteoblastic metastases in female nude mice and provide evidence that tumor-produced endothelin-1 (ET-1) mediates the osteoblastic response. Tumor-conditioned media and exogenous ET-1 stimulated osteoblast proliferation and new bone formation in cultures of calvarias from mice. These effects were blocked by endothelin A (ETA) but not by ETB receptor antagonists. Mice inoculated with the ZR-75-1 breast cancer line and treated with a selective ETA receptor antagonist (ABT-627) had significantly fewer osteoblastic bone metastases and less tumor burden compared with untreated mice. In contrast, there was no effect of ABT-627 on osteolytic bone metastases caused by ET-1-negative breast cancer, MDA-MB-231. ABT-627 had no effect on cell growth in vitro or at the orthotopic site (mammary fat pad) of ZR-75-1, or MDA-MB-231 cells. Collectively, the data suggest that tumor-produced ET-1 mediates osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation. Endothelin A receptor blockade may be useful for the prevention and treatment of osteoblastic bone metastases attributable to breast or prostate cancer.

Topics: Animals; Bone Neoplasms; Bone Remodeling; Disease Models, Animal; Endothelin-1; Female; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Metastasis; Osteoblasts; Tumor Cells, Cultured

Endothelin-1 and the endothelin A (ET(A) ) receptor have been implicated in prostate cancer progression in bone. This study aimed to determine whether the specific ET(A) receptor antagonist, zibotentan, prolonged overall survival (OS) in patients with castration-resistant prostate cancer and bone metastases who were pain-free or mildly symptomatic for pain.. Patients were randomized 1:1 to zibotentan 10 mg/day or placebo, plus standard prostate cancer treatment. The primary endpoint was OS. Secondary endpoints included times to pain progression, chemotherapy use, new bone metastases, and safety. Efficacy endpoints were analyzed using a log-rank test.. A total of 594 patients were randomized (zibotentan, n = 299; placebo, n = 295). Median OS was 24.5 months in zibotentan-treated patients versus 22.5 months for placebo, but the difference did not reach statistical significance (hazard ratio, 0.87; 95.2% confidence interval, 0.69-1.10; P = .240). No statistically significant differences were observed for any secondary efficacy endpoints. Peripheral edema (44%) and headache (31%) were the most commonly reported adverse events in the zibotentan group. Cardiac failure events were higher in the zibotentan group than placebo (any grade, 5.7% and 1.7%; Common Terminology Criteria for Adverse Events grade ≥3, 3.0% and 1.0%, respectively); these were manageable and reversible.. In this large, randomized, placebo-controlled phase 3 trial, treatment with zibotentan 10 mg/day did not lead to a statistically significant improvement in OS in this patient population. Zibotentan had an acceptable safety profile.

Topics: Antineoplastic Agents; Bone Neoplasms; Disease-Free Survival; Endothelin A Receptor Antagonists; Endothelin-1; Humans; Male; Orchiectomy; Placebos; Prostatic Neoplasms; Pyrrolidines; Survival; Treatment Outcome

Bone cancer metastasis is extremely painful and decreases the quality of life of the affected patients. Available pharmacological treatments are not able to sufficiently ameliorate the pain, and as patients with cancer are living longer, new treatments for pain management are needed. Decitabine (5-aza-2'-deoxycytidine), a DNA methyltransferases inhibitor, has analgesic properties in preclinical models of postsurgical and soft-tissue oral cancer pain by inducing an upregulation of endogenous opioids. In this study, we report that daily treatment with decitabine (2 µg/g, intraperitoneally) attenuated nociceptive behavior in the 4T1-luc2 mouse model of bone cancer pain. We hypothesized that the analgesic mechanism of decitabine involved activation of the endogenous opioid system through demethylation and reexpression of the transcriptionally silenced endothelin B receptor gene, Ednrb. Indeed, Ednrb was hypermethylated and transcriptionally silenced in the mouse model of bone cancer pain. We demonstrated that expression of Ednrb in the cancer cells lead to release of β-endorphin in the cell supernatant, which reduced the number of responsive dorsal root ganglia neurons in an opioid-dependent manner. Our study supports a role of demethylating drugs, such as decitabine, as unique pharmacological agents targeting the pain in the cancer microenvironment.

Topics: Animals; Antimetabolites, Antineoplastic; beta-Endorphin; Bone Density; Bone Neoplasms; Cancer Pain; Cell Line, Tumor; Culture Media, Conditioned; Decitabine; Disease Models, Animal; Endothelin-1; Female; Ganglia, Spinal; Locomotion; Mice; Mice, Inbred BALB C; Naloxone; Neurons; Quaternary Ammonium Compounds; Receptor, Endothelin B; Weight-Bearing

We report a 46-yr-old woman with a history of breast cancer who presented with diffuse myalgias, bone pain, and osteosclerosis. She was found to have recurrent breast cancer producing endothelin-1.. Acquired osteosclerosis can be caused by various disorders. Endothelin -1 is believed to contribute to osteosclerosis caused by breast cancer.. Although the bone marrow biopsy did not reveal breast cancer, she developed skin lesions consistent with metastatic breast cancer. She ultimately died from progressive disease. At autopsy immunohistochemistry for endothelin-1 was performed on a section from the L5 vertebral body.. The section from the L5 vertebral body showed small foci of cells consistent with metastatic carcinoma and a prominent sclerotic response. Immunohistochemistry for endothelin-1 was strongly positive.. Recurrent breast cancer may present with diffuse osteosclerosis. Endothelin-1 may be a paracrine factor responsible for increased bone formation and osteosclerosis.

Topics: Adenocarcinoma; Bone Neoplasms; Breast Neoplasms; Endothelin-1; Fatal Outcome; Female; Humans; Middle Aged; Osteosclerosis; Radiography; Skin Neoplasms

Bone metastasis is a painful complication of advanced prostate cancer. Endothelin-1 is a tumor-secreted factor that plays a central role in osteoblast activation and the osteosclerotic response of prostate cancer metastatic to bone. Antagonists that block the activation of the endothelin A receptor (ETAR), located on osteoblasts, reduce osteoblastic bone lesions in animal models of bone metastasis. However, ETAR antagonists demonstrated limited efficacy in clinical trials of men with advanced prostate cancer who also received standard androgen deprivation therapy (ADT). Previous data from our group suggested that, in a mouse model, ETAR antagonists might only be efficacious when androgen signaling in the osteoblast is lowered beyond the ability of standard ADT. This notion was tested in a mouse model of prostate cancer bone metastasis. Castrated and sham-operated male athymic nude mice underwent intracardiac inoculation of the ARCaPM castration-resistant prostate cancer cell line. The mice were then treated with either the ETAR antagonist zibotentan or a vehicle control to generate four experimental groups: vehicle+sham (Veh+Sham), vehicle+castrate (Veh+Castr), zibotentan+sham (Zibo+Sham), and zibotentan+castrate (Zibo+Castr). The mice were monitored radiographically for the development of skeletal lesions. The Zibo+Castr group had significantly longer survival and a single incidental lesion. Mice in the Zibo+Sham group had the shortest survival and the largest number of skeletal lesions. Survival and skeletal lesions of the Veh+Sham and Veh+Castr groups were intermediate compared with the zibotentan-treated groups. We report a complex interaction between ETAR and androgen signaling, whereby ETAR blockade was most efficacious when combined with complete androgen deprivation.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Disease Models, Animal; Endothelin A Receptor Antagonists; Endothelin-1; Male; Mice; Orchiectomy; Osteoblasts; Prostatic Neoplasms; Pyrrolidines; Receptor, Endothelin A

We examined the influence of microenvironment stimuli on molecular events relevant to the biological functions of 1833-bone metastatic clone and the parental MDA-MB231 cells. (i) In both the cell lines, hepatocyte growth factor (HGF) and the osteoblasts' biological products down regulated nuclear Ets-1-protein level in concomitance with endogenous miR-125b accumulation. In contrast, under hypoxia nuclear Ets-1 was unchanged, notwithstanding the miR-125b increase. (ii) Also, the 1833-cell invasiveness and the expression of Endothelin-1, the target gene of Ets-1/HIF-1, showed opposite patterns under HGF and hypoxia. We clarified the molecular mechanism(s) reproducing the high miR-125b levels with the mimic in 1833 cells. Under hypoxia, the miR-125b mimic maintained a basal level and functional Ets-1 protein, as testified by the elevated cell invasiveness. However, under HGF ectopic miR-125b downregulated Ets-1 protein and cell motility, likely involving an Ets-1-dominant negative form sensible to serum conditions; Ets-1-activity inhibition by HGF implicated HIF-1α accumulation, which drugged Ets-1 in the complex bound to the Endothelin-1 promoter. Altogether, 1833-cell exposure to HGF would decrease Endothelin-1 transactivation and protein expression, with the possible impairment of Endothelin-1-dependent induction of E-cadherin, and the reversion towards an invasive phenotype: this was favoured by Ets-1 overexpression, which inhibited HIF-1α expression and HIF-1 activity. (iii) In MDA-MB231 cells, HGF strongly and rapidly decreased Ets-1, hampering invasiveness and reducing Ets-1-binding to Endothelin-1 promoter; HIF-1α did not form a complex with Ets-1 and Endothelin-1-luciferase activity was unchanged. Overall, depending on the microenvironment conditions and endogenous miR-125b levels, bone-metastatic cells might switch from Ets-1-dependent motility towards colonization/growth, regulated by the balance between Ets-1 and HIF-1.

Topics: Bone Neoplasms; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; DNA; Endothelin-1; Female; Hepatocyte Growth Factor; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; MicroRNAs; Models, Biological; Neoplasm Invasiveness; Proto-Oncogene Protein c-ets-1; Transcriptional Activation; Tumor Microenvironment

Topics: 3T3 Cells; Animals; Bone Neoplasms; CCN Intercellular Signaling Proteins; Cell Adhesion; Cell Line, Tumor; Cell Movement; Coculture Techniques; Culture Media, Conditioned; Endothelin-1; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha4beta1; Male; Mice; Neoplasm Transplantation; Osteoblasts; Osteogenesis; Prostatic Neoplasms; Proto-Oncogene Proteins; RAW 264.7 Cells; Signal Transduction; Vascular Cell Adhesion Molecule-1

Prostate cancers have a strong propensity to metastasize to bone and promote osteoblastic lesions. TMPRSS2:ERG is the most frequent gene rearrangement identified in prostate cancer, but whether it is involved in prostate cancer bone metastases is largely unknown. We exploited an intratibial metastasis model to address this issue and we found that ectopic expression of the TMPRSS2:ERG fusion enhances the ability of prostate cancer cell lines to induce osteoblastic lesions by stimulating bone formation and inhibiting the osteolytic response. In line with these in vivo results, we demonstrate that the TMPRSS2:ERG fusion protein increases the expression of osteoblastic markers, including Collagen Type I Alpha 1 Chain and Alkaline Phosphatase, as well as Endothelin-1, a protein with a documented role in osteoblastic bone lesion formation. Moreover, we determined that the TMPRSS2:ERG fusion protein is bound to the regulatory regions of these genes in prostate cancer cell lines, and we report that the expression levels of these osteoblastic markers are correlated with the expression of the TMPRSS2:ERG fusion in patient metastasis samples. Taken together, our results reveal that the TMPRSS2:ERG gene fusion is involved in osteoblastic lesion formation induced by prostate cancer cells.

Topics: Alkaline Phosphatase; Animals; Biomarkers, Tumor; Bone Neoplasms; Cell Line, Tumor; Collagen Type I, alpha 1 Chain; Endothelin-1; Gene Expression Regulation, Neoplastic; Humans; Male; Mice, SCID; Oncogene Proteins, Fusion; Osteoblasts; PC-3 Cells; Phenotype; Prostatic Neoplasms; Transplantation, Heterologous; Tumor Burden

The pathogenesis of bone metastasis is unclear, and much focus in metastatic biology and therapy relays on epigenetic alterations. Since DNA-methyltransferase blockade with 5-aza-2'-deoxycytidine (dAza) counteracts tumour growth, here we utilized dAza to clarify whether molecular events undergoing epigenetic control were critical for bone metastatization. In particular, we investigated the patterns of secreted-protein acidic and rich in cysteine (SPARC) and of Endothelin 1, affected by DNA methyltransferases in tumours, with the hypothesis that in bone metastasis a coordinate function of SPARC and Endothelin 1, if any occurs, was orchestrated by DNA methylation. To this purpose, we prepared a xenograft model with the clone 1833, derived from human-MDA-MB231 cells, and dAza administration slowed-down metastasis outgrowth. This seemed consequent to the reductions of SPARC and Endothelin 1 at invasive front and in the bone marrow, mostly due to loss of Twist. In the metastasis bulk Snail, partly reduced by dAza, might sustain Endothelin 1-SPARC cooperativity. Both SPARC and Endothelin 1 underwent post-translational control by miRNAs, a molecular mechanism that might explain the in vivo data. Ectopic miR29a reduced SPARC expression also under long-term dAza exposure, while Endothelin 1 down-regulation occurred in the presence of endogenous-miR98 expression. Notably, dAza effects differed depending on in vivo and in vitro conditions. In 1833 cells exposed to 30-days dAza, SPARC-protein level was practically unaffected, while Endothelin 1 induction depended on the 3'-UTR functionality. The blockade of methyltransferases leading to SPARC reduction in vivo, might represent a promising strategy to hamper early steps of the metastatic process affecting the osteogenic niche.

Topics: Animals; Azacitidine; Bone Neoplasms; Breast Neoplasms; DNA Methylation; DNA, Neoplasm; Endothelin-1; Female; Humans; Mice; Mice, Nude; MicroRNAs; Neoplasm Metastasis; Neoplasm Proteins; Osteonectin; RNA, Neoplasm; Tumor Microenvironment

Chondrosarcoma is a malignant tumor of mesenchymal origin predominantly composed of cartilage-producing cells. This type of bone cancer is extremely resistant to radiotherapy and chemotherapy. Surgical resection is the primary treatment, but is often difficult and not always practical for metastatic disease, so more effective treatments are needed. In particular, it would be helpful to identify molecular markers as targets for therapeutic intervention. Endothelin-1 (ET-1), a potent vasoconstrictor, has been shown to enhance chondrosarcoma angiogenesis and metastasis. We report that ET-1 promotes epithelial-mesenchymal transition (EMT) in human chondrosarcoma cells. EMT is a key pathological event in cancer progression, during which epithelial cells lose their junctions and apical-basal polarity and adopt an invasive phenotype. Our study verifies that ET-1 induces the EMT phenotype in chondrosarcoma cells via the AMP-activated protein kinase (AMPK) pathway. In addition, we show that ET-1 increases EMT by repressing miR-300, which plays an important role in EMT-enhanced tumor metastasis. We also show that miR-300 directly targets Twist, which in turn results in a negative regulation of EMT. We found a highly positive correlation between ET-1 and Twist expression levels as well as tumor stage in chondrosarcoma patient specimens. Therefore, ET-1 may represent a potential novel molecular therapeutic target in chondrosarcoma metastasis.

Topics: 3' Untranslated Regions; AMP-Activated Protein Kinases; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Chondrosarcoma; Endothelin-1; Epithelial-Mesenchymal Transition; Humans; MicroRNAs; Nuclear Proteins; Receptors, Endothelin; Twist-Related Protein 1

In order to become established in the skeleton, metastatic cells disseminating from the breast carcinoma need to acquire organ-specific traits. There are no effective predictors for who will develop bone metastasis to guide long-term predictive therapy. Our purpose was to individuate events critical for bone colonization to make a molecular classification of breast carcinoma useful for bone-metastasis outcome. In dysplasia adjacent to carcinoma and in pair-matched specimens of bone metastasis we examined SPARC expression and localization as well as Endothelin 1/ETAR signals by immunohistochemistry, and the evaluation of plasma levels of SPARC by ELISA was also performed. In patients with breast carcinoma metastasizing to bone, SPARC and Endothelin 1/ETAR axis were highly expressed from dysplasia until bone metastasis, but the SPARC plasma level was as low as that of normal women, in contrast to patients that never develop bone metastasis, suggesting that circulating SPARC was counter adhesive. Altogether, the early identification of SPARC/Endothelin 1/ETAR in dysplastic lesions would be important to devise therapies preventing metastasis engraftment, since often carcinoma cells spread to distant organs at the time or even before patients present with cancer.

Topics: Adult; Biomarkers, Tumor; Bone Neoplasms; Breast Neoplasms; Carcinoma; Case-Control Studies; Endothelin-1; Female; Humans; Middle Aged; Osteonectin; Receptor, Endothelin A

Chondrosarcoma is the second most common sarcoma in bone malignancy and is characterized by a high metastatic potential. Angiogenesis is essential for the cancer metastasis. Endothelin-1 (ET-1) has been implicated in tumor angiogenesis and metastasis. However, the relationship of ET-1 with vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma cells is mostly unknown. Here, we found that the expression of ET-1 and VEGF were correlated with tumor stage and were significantly higher than that in the normal cartilage. Exogenous ET-1 with chondrosarcoma cells promoted VEGF expression and subsequently increased migration and tube formation in endothelial progenitor cells. ET-1 increased VEGF expression and angiogenesis through ETAR, integrin-linked kinase (ILK), Akt and hypoxia-inducible factor-1α (HIF-1α) signaling cascades. Knockdown of ET-1 decreased VEGF expression and also abolished chondrosarcoma conditional medium-mediated angiogenesis in vitro as well as angiogenesis effects in the chick chorioallantoic membrane and Matrigel plug nude mice model in vivo. In addition, in the xenograft tumor angiogenesis model, knockdown of ET-1 significantly reduced tumor growth and tumor-associated angiogenesis. Taken together, these results indicate that ET-1 occurs through ETAR, ILK and Akt, which in turn activates HIF-1α, resulting in the activation of VEGF expression and contributing to the angiogenesis and tumor growth of human chondrosarcoma cells.

Topics: Animals; Bone Neoplasms; Cell Growth Processes; Cell Line, Tumor; Chick Embryo; Chondrosarcoma; Endothelin-1; Gene Knockdown Techniques; Heterografts; Humans; Male; Mice; Mice, Nude; Neovascularization, Pathologic; Signal Transduction; Vascular Endothelial Growth Factor A

Osteosarcoma (OS) is the most common childhood bone cancer. Chemoresistance is the principal reason for poor survival and disease recurrence in OS patients, and ET-1 reportedly plays an important role in the development of chemoresistance in OS cells. In the present study, we for the first time explored the association of endothelin-1 (ET-1) SNPs and haplotypes with the risk of chemoresistant pediatric OS.. We genotyped three SNPs (rs1800541, rs2070699, and rs5370) in the ET-1 gene in a case-control study, using 350 pairs of age, sex, and tumor location and stage matched pediatric patients with OS. Patients who showed <90% tumor necrosis after neochemotherapy were defined as poor responders (cases), and those who showed ≥90% tumor necrosis were defined as good responders (controls).. The G allele at rs1800541 and the G allele at rs2070699 were associated with reduced and increased risk of chemoresistant OS, respectively. The rs1800541-rs2070699 haplotypes TG and GT were respectively associated with increased (P = 0.012; adjusted OR, 1.82; 95% CI, 1.10-5.65) and reduced (P = 0.009; adjusted OR, 0.25; 95% CI, 0.14-0.84) risk of chemoresistant OS. The TG and the GT haplotypes have a gene-dosage effect on increasing and decreasing the ET-1 expression in primary OS tumor cells from chemoresistant pediatric OS subjects, respectively.. This study provides the first evidence of an association between the ET-1 gene SNPs and haplotypes and the risk of chemoresistant pediatric OS, potentially adding new insights into the pathophysiology and treatment of chemoresistant OS.

Topics: Adolescent; Antineoplastic Combined Chemotherapy Protocols; Bone Neoplasms; Case-Control Studies; Child; Child, Preschool; Drug Resistance, Neoplasm; Endothelin-1; Female; Follow-Up Studies; Genotype; Haplotypes; Humans; Male; Neoplasm Staging; Osteosarcoma; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Prognosis

The present study was undertaken to clarify the function(s) of Endothelin-1 and its receptors ETAR and ETBR in osteolytic-bone metastasis from breast cancer, and their regulation by hepatocyte and transforming growth factors (HGF, TGF-β) and hypoxia. The aim was to evaluate the adaptability of bone metastasis to microenvironmental stimuli through Endothelin-1-mediated epithelial-mesenchymal transition (EMT), or the reverse process MET, and through osteomimicry possible key features for bone colonization. We compared low (MCF-7) and high (MDA-MB231) invasive-breast carcinoma cells, and 1833-bone metastatic clone, with human pair-matched primary breast-carcinomas and bone metastases. Parental MDA-MB231 and the derived 1833-clone responded oppositely to the stimuli. In 1833 cells, TGF-β and hypoxia increased Endothelin-1 release, altogether reducing invasiveness important for engraftment, while Endothelin-1 enhanced MDA-MB231 cell invasiveness. The Endothelin-1-autocrine loop contributed to the cooperation of intracellular-signaling pathways and extracellular stimuli triggering MET in 1833 cells, and EMT in MDA-MB231 cells. Only in 1833 cells, HGF negatively influenced transactivation and release of Endothelin-1, suggesting a temporal sequence of these stimuli with an initial role of HGF-triggered Wnt/β-catenin pathway in metastatization. Then, Endothelin-1/ETAR conferred MET and osteomimetic phenotypes, with Runt-related transcription factor 2 activation and metalloproteinase 9 expression, contributing to colonization and osteolysis. Findings with human pair-matched primary ductal carcinomas and bone metastases gave a translational significance to the molecular study. Endothelin-1, ETAR and ETBR correlated with the acquisition of malignant potential, because of high expression already in the in situ carcinoma. These molecular markers might be used as predictive index of aggressive behavior and invasive/metastatic phenotype.

Topics: Bone Neoplasms; Breast Neoplasms; Endothelin-1; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Neoplasm Invasiveness; Receptor, Endothelin A; Receptor, Endothelin B; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment; Wnt Signaling Pathway

Chondrosarcoma is a type of highly malignant tumor with a potent capacity of local invasion and distant metastasis. The effect of endothelin-1 (ET-1) on migration activity in human chondrosarcoma cells is not clearly understood. Here, we found that ET-1 increased the migration and expression of cyclooxygenase (COX)-2 in human chondrosarcoma cells.. ET-1-mediated COX-2 expression was assessed by qPCR and Western blot analysis. The mechanisms of action of ET-1 in different signaling pathways were studied using Western blotting. Knockdown of proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of c-Jun to the COX-2 promoter.. Human chondrosarcoma tissues had significant expression levels of ET-1 and COX-2, which were higher than that in normal cartilage. Exogenous ET-1 increased cell migration and the expression of COX-2. In addition, COX-2 protein levels and cell migration ability were abolished by ET receptor antagonists. Activation of the mitogen-activated protein kinase (MAPK) and activator protein-1 (AP-1) pathways after ET-1 treatment was demonstrated, and ET-1-induced COX-2 expression and cell migration activity were inhibited by the specific inhibitor and mutant of MAPK and AP-1 cascades. ET-1 increased the binding of c-Jun to the AP-1 element on the COX-2 promoter. Furthermore, knockdown of ET-1 decreased cell metastasis in vitro and in vivo.. Our results indicated that ET-1 enhances the cell migration of chondrosarcoma by increasing COX-2 expression through the ET receptors, MAPK, and AP-1 signal transduction pathway.. We link high ET-1 and COX-2 expression to chondrosarcoma.

Topics: Bone Neoplasms; Cartilage; Cell Line, Tumor; Cell Movement; Chondrosarcoma; Cyclooxygenase 2; Endothelin-1; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; MAP Kinase Signaling System; Proto-Oncogene Proteins c-jun; Transcription Factor AP-1; Up-Regulation

To determine the mechanism of neuroblastoma (NB) bone invasion/metastasis, it is necessary to investigate the bone invasion/metastasis-related factors in the bone invasion/metastasis process. Some evidence has suggested that various proteins were involved in bone osteolytic response. The invasion/metastasis property and gene expression of NB, however, are still unknown.. Single-cell suspensions of SY5Y and KCNR cells were injected directly into the femur of nude mice. Radiological and histological analyses, immunohistochemistry analyses, and western blot assay were performed to characterize bone metastasis mechanism in these bone metastasis models.. SY5Y and KCNR NB cells result in osteolytic responses in bone metastasis model. Osteoprotegerin (OPG), receptor activator of NF-kappaB ligand (RANKL), parathyroid hormone-related peptide (PTHrP), endothelin 1 (ET-1), and CXCR4 were examined and compared among in vitro, in vivo, and normal bone, respectively. PTHrP, OPG, RANKL, and ET-1 except CXCR4 in SY5Y and KCNR NB cells xenografts were strikingly upregulated compared with normal bone and NB cells. However, significantly stronger expression of PTHrP and RANKL was presented than ET-1 and OPG; furthermore, the ratios of expression of PTHrP, RANKL to OPG, and ET-1 were also markedly increased in vivo versus in vitro.. Our study provided evidence that NB cell may enhance bone invasion through PTHrP, OPG, RANKL, and ET-1, especially PTHrP and RANKL which may display stronger effects. CXCR4 appeared not participating in bone invasion, but in tumor growth, and homing to bone. Targeting PTHrP, OPG, ET-1, and RANKL may provide a new insight and method for patient therapy by inhibiting NB bone metastasis and invasiveness.

Topics: Animals; Blotting, Western; Bone Neoplasms; Cell Line, Tumor; Endothelin-1; Femur; Humans; Immunohistochemistry; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neuroblastoma; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; RANK Ligand; Receptors, CXCR4

Here, we report a complex regulation of endothelin-1 (ET-1) axis driven by epigenetic reactions in 1833-bone metastatic cells, emphasizing the importance in skeletal metastasis from breast carcinoma. Inhibitors of histone deacetylases, trichostatin A (TSA), and of DNA methylases, 5'-Azacytidine (Aza), caused, respectively, reduction and increase in 1833 cell invasiveness, without affecting the basal migration of parental MDA-MB231 cells. Of note, in the two cell lines exposed to Aza the blockade of the ET-1 receptor ETAR with BQ-123 oppositely changed invasive properties. Even if in MDA-MB231 cells the ET-1 axis was scarcely influenced by epigenetic reactions, ETAR remarkably decreased after Aza. In contrast, in 1833 cells Aza exposure enhanced ET-1 coupled to ETAR wild type, being also ETAR truncated form increased, and invasiveness was stimulated. Under demethylation, the increase in ET-1 steady state protein level in 1833 clone seemed regulated at transcriptional level principally via Ets1 transcription factor. In fact, actinomycin D almost completely prevented ET-1 mRNA induction due to Aza. Only in 1833 cells, TSA exposure inactivated ET-1 axis, with reduction of the expression of ET-1 and ETAR mutated form, in agreement with Matrigel invasion decrease. This treatment favoured the ET-1 repressional control, taking place at the level of mRNA stability due to the 3'-untranslated region in the ET-1 gene, and also decreased transcription via NF-kB. Environmental conditions that alter the balance between epigenetic reactions might, therefore, affect metastasis migratory mode influencing ET-1 axis.

Topics: Azacitidine; Bone Neoplasms; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Collagen; Dactinomycin; DNA Methylation; Drug Combinations; Endothelin A Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Laminin; Mutation; Neoplasm Invasiveness; Peptides, Cyclic; Proteoglycans; Proto-Oncogene Protein c-ets-1; Receptor, Endothelin A; RNA Stability; RNA, Messenger; Transcription, Genetic

Approximately 90% of patients who die of Prostate Cancer (PCa) have bone metastases, which promote a spectrum of osteoblastic, osteolytic or mixed bone responses. Numerous secreted proteins have been reported to promote osteoblastic or osteolytic bone responses. We determined whether previously identified and/or novel proteins were associated with the osteoblastic or osteolytic response in clinical specimens of PCa bone metastases.. Gene expression was analyzed on 14 PCa metastases from 11 patients by microarray profiling and qRT-PCR, and protein expression was analyzed on 33 PCa metastases from 30 patients by immunohistochemistry on highly osteoblastic and highly osteolytic bone specimens.. Transcript and protein levels of BMP-2, BMP-7, DKK-1, ET-1, and Sclerostin were not significantly different between osteoblastic and osteolytic metastases. However, levels of OPG, PGK1, and Substance P proteins were increased in osteoblastic samples. In addition, Emu1, MMP-12, and sFRP-1 were proteins identified with a novel role of being associated with either the osteoblastic or osteolytic bone response.. This is the first detailed analysis of bone remodeling proteins in human specimens of PCa bone metastases. Three proteins not previously shown to be involved may have a role in the PCa bone response. Furthermore, our data suggests that the relative expression of numerous, rather than a single, bone remodeling proteins determine the bone response in PCa bone metastases.

Topics: Aged; Bone Morphogenetic Proteins; Bone Neoplasms; Bone Remodeling; Endothelin-1; Gene Expression; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Male; Neoplasm Proteins; Osteoblasts; Osteolysis; Phosphoglycerate Kinase; Prostatic Neoplasms; Substance P

Bone tumors strongly influence normal tissues and stimulate bone cells for the production of cytokines supporting proliferation and abnormal survival in cancer cells. We previously reported that the proteoglycan syndecan-2 controls the activity of various cytokines and growth factors and also modulates apoptosis and response to cytotoxic agents in osteosarcoma cell lines. Here, we show that syndecan-2 has a stronger tumor suppressor activity in vivo. We identify calpain-6 as a target gene downregulated by syndecan-2 in cells and in vivo. We demonstrate that calpain-6 expression in osteosarcoma cells depends on endothelin-1, a mediator of the tumor progression in bone. Syndecan-2 overexpression alters ERK1/2, PI3K/AKT and NFκB pathways that are calpain-6-promoting signals downstream of endothelin-1. Immunohistochemical analysis shows that calpain-6 is expressed in human bone tumors and metastases. A high expression of calpain-6 was specially found in recurrent osteosarcoma. Moreover, calpain-6 levels in primary tumors were inversely related to the response to chemotherapy. Consistently, calpain-6 was increased by doxorubicin and was found to be expressed at higher levels in doxorubicin-resistant U2OS osteosarcoma-derived cells as compared to responsive cells. Inhibition of calpain-6 with shRNA resulted in decreased proliferation, increased spontaneous apoptosis and increased sensitivity to doxorubicin and also methotrexate in responsive and resistant osteosarcoma cells. Taken together, our data show that syndecan-2 exerts its pro-apoptotic function through modulation of the endothelin-1/NFκB signaling and through downregulation of calpain-6, a protective factor that contributes to abnormal cell survival. Thus, this study identifies calpain-6 as a new possible therapeutic target in chemoresistant osteosarcoma.

Topics: Apoptosis; Bone Neoplasms; Calpain; Cell Line, Tumor; Drug Resistance, Neoplasm; Endothelin-1; Humans; NF-kappa B; Osteosarcoma; Signal Transduction; Syndecan-2

Tumor malignancy is associated with several cellular properties including proliferation and ability to metastasize. Endothelin-1 (ET-1) the most potent vasoconstrictor plays a crucial role in migration and metastasis of human cancer cells. We found that treatment of human chondrosarcoma (JJ012 cells) with ET-1 increased migration and expression of matrix metalloproteinase (MMP)-13. ET-1-mediated cell migration and MMP-13 expression were reduced by pretreatment with inhibitors of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR), as well as the NF-κB inhibitor and the IκB protease inhibitor. In addition, ET-1 treatment induced phosphorylation of FAK, PI3K, AKT, and mTOR, and resulted in increased NF-κB-luciferase activity that was inhibited by a specific inhibitor of PI3K, Akt, mTOR, and NF-κB cascades. Taken together, these results suggest that ET-1 activated FAK/PI3K/AKT/mTOR, which in turn activated IKKα/β and NF-κB, resulting in increased MMP-13 expression and migration in human chondrosarcoma cells.

Topics: Bone Neoplasms; Cell Line, Tumor; Cell Movement; Chondrosarcoma; Endothelin-1; Focal Adhesion Kinase 1; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; Matrix Metalloproteinase 13; NF-kappa B; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases

Osteosarcoma (OS) is the most common primary tumor in humans and dogs affecting the skeleton, and spontaneously occurring OS in dogs serves as an extremely useful model. Unacceptable toxicities using current treatment protocols prevent further dose-intensification from being a viable option to improve patient survival and thus, novel treatment strategies must be developed. Histone deacetylase inhibitors (HDACi) have recently emerged as a promising class of therapeutics demonstrating an ability to enhance the anti-tumor activity of traditional chemotherapeutics. To date, gene expression analysis of OS cell lines treated with HDACi has not been reported, and evaluation of the resultant gene expression changes may provide insight into the mechanisms that lead to success of HDACi. Canine OS cells, treated with a clinically relevant concentration of the HDACi valproic acid (VPA), were used for expression analysis on the Affymetrix canine v2.0 genechip. Differentially expressed genes were grouped into pathways based upon functional annotation; pathway analysis was performed with MetaCore and Ingenuity Pathways Analysis software. Validation of microarray results was performed by a combination of qRT-PCR and functional/biochemical assays revealing oxidative phosphorylation, cytoskeleton remodeling, cell cycle, and ubiquitin-proteasome among those pathways most affected by HDACi. The mitomycin C-bioactivating enzyme NQ01 also demonstrated upregulation following VPA treatment, leading to synergistic reductions in cell viability. These results provide a better understanding of the mechanisms by which HDACi exert their effect in OS, and have the potential to identify biomarkers that may serve as novel targets and/or predictors of response to HDACi-containing combination therapies in OS.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Line, Tumor; Dogs; Down-Regulation; Endothelin-1; Gene Expression Profiling; Histone Deacetylase Inhibitors; NAD(P)H Dehydrogenase (Quinone); Osteosarcoma; Proteasome Inhibitors; Real-Time Polymerase Chain Reaction; Systems Biology; Valproic Acid

Endothelin-1 (ET-1) participates in a wide range of cancer-relevant processes including cell proliferation, inhibition of apoptosis, matrix remodeling, bone deposition, and metastases. Although ET-1 reportedly promotes osteosarcoma (OS) cell invasion, suggesting an important role of ET-1 in OS metastasis, the role of ET-1 in OS remains unclear.. We asked whether (1) ET-1 expression is associated with the malignancy of OS, (2) ET-1 enhances the cell invasion ability of OS, and (3) ET-1 promotes OS cell survival against apoptotic stress.. We cultured primary OS specimens from 22 patients with Stages II (OS-II) and III (OS-III) in real-time quantitative RT-PCR and ELISA to compare ET-1 expression. We used Transwell(®) cell invasion assays (in triplicate) to assess the invasion ability of cells in the presence or absence of exogenous ET-1 and/or ET receptor antagonists. We compared cell apoptosis rate among the cells treated with cisplatin in the presence or absence of exogenous ET-1 and/or ET receptor antagonists. We used OS cell line MG-63 in all experiments as a reference.. Real-time quantitative RT-PCR and ELISA showed OS-III cells had greater ET-1 expression than OS-II cells at the mRNA and the secreted protein levels. Transwell(®) cell invasion assays showed OS-III cells had a greater migrated cell number than OS-II cells, which could be abrogated by ET(A) receptor antagonist BQ123 (100 pmol/L), but not ET(B) receptor antagonist BQ788 (1 μmol/L); exogenous ET-1 dose-dependently promoted OS cell migration, which could be inhibited by BQ123 (100 pmol/L). Cisplatin (10 nmol/L) induced less apoptosis in OS-III cells than in OS-II cells; exogenous ET-1 dose-dependently promoted OS cell survival against cisplatin-induced apoptosis; both effects were reversed by BQ123 (1 μmol/L), but not BQ788 (1 μmol/L).. Increased ET-1 expression appears to be associated with increased malignancy of OS. ET-1 promotes OS cell invasion and survival against cisplatin-induced apoptosis through the ET(A) receptor.. The ET-1/ET(A) pathway may represent an important target for treating OS, because blocking the ET(A) receptor with a selective antagonist can inhibit OS cell invasion and potentiate a chemotherapeutic agent's effect on OS.

Topics: Adolescent; Adult; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cell Survival; Child; Child, Preschool; Cisplatin; Drug Screening Assays, Antitumor; Endothelin A Receptor Antagonists; Endothelin-1; Female; Gene Expression; Humans; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Osteosarcoma; Receptor, Endothelin A; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Young Adult

We investigated the effects of the endothelin-1 (ET-1) receptor dual antagonist (Bosentan®) on the inflammatory cytokines and the chemoattractant molecules associated with breast cancer growth and the development of tumor infiltration in bone explants. Immunocompetent mice implanted with the murine mammary carcinoma 4T1 cells in a skin-fold chamber and treated with Bosentan® had reduced tumor growth (p < .05). ET-1 promoted the secretion of the anti-inflammatory soluble tumor necrosis factor (TNF) receptor and IL12 p40 in vitro. The Bosentan® treatment in vivo was associated with a local increase of the anti-inflammatory IL-1α cytokine concentration and decrease of the pro-inflammatory TNF-α and IL-17 cytokine concentrations (p < .05).

Topics: Animals; Antihypertensive Agents; Bone Neoplasms; Bosentan; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cytokines; Endothelin Receptor Antagonists; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Inflammation Mediators; Interleukin-17; Interleukin-1alpha; Male; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neutrophils; Receptors, Endothelin; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides; Tumor Burden; Tumor Necrosis Factor-alpha

The DLX gene family encodes for homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis. Their expression can be regulated by Endothelin1 (ET1), a peptide associated with breast cancer invasive phenotype. Deregulation of DLX gene expression was found in human solid tumors and hematologic malignancies. In particular, DLX4 overexpression represents a possible prognostic marker in ovarian cancer. We have investigated the role of DLX genes in human breast cancer progression.. MDA-MB-231 human breast carcinoma cells were grown in vitro or injected in nude mice, either subcutaneously, to mimic primary tumor growth, or intravenously, to mimic metastatic spreading. Expression of DLX2, DLX5 and DLX6 was assessed in cultured cells, either treated or not with ET1, tumors and metastases by RT-PCR. In situ hybridization was used to confirm DLX gene expression in primary tumors and in lung and bone metastases. The expression of DLX2 and DLX5 was evaluated in 408 primary human breast cancers examining the GSE1456 and GSE3494 microarray datasets. Kaplan-Meier estimates for disease-free survival were calculated for the patients grouped on the basis of DLX2/DLX5 expression.. Before injection, or after subcutaneous growth, MDA-MB-231 cells expressed DLX2 but neither DLX5 nor DLX6. Instead, in bone and lung metastases resulting from intravenous injection we detected expression of DLX5/6 but not of DLX2, suggesting that DLX5/6 are activated during metastasis formation, and that their expression is alternative to that of DLX2. The in vitro treatment of MDA-MB-231 cells with ET1, resulted in switch from DLX2 to DLX5 expression. By data mining in microarray datasets we found that expression of DLX2 occurred in 21.6% of patients, and was significantly correlated with prolonged disease-free survival and reduced incidence of relapse. Instead, DLX5 was expressed in a small subset of cases, 2.2% of total, displaying reduced disease-free survival and high incidence of relapse which was, however, non-significantly different from the other groups due to the small size of the DLX+ cohort. In all cases, we found mutually exclusive expression of DLX2 and DLX5.. Our studies indicate that DLX genes are involved in human breast cancer progression, and that DLX2 and DLX5 genes might serve as prognostic markers.

Topics: Animals; Biomarkers, Tumor; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Disease-Free Survival; Endothelin-1; Female; Gene Expression Profiling; Homeodomain Proteins; Humans; In Situ Hybridization; Kaplan-Meier Estimate; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transcription Factors

Lung cancer is commonly associated with multiple-organ metastasis, and bone is a frequent metastatic site for lung cancer. Lung cancer frequently develops osteolytic, and less frequently osteoblastic, metastasis to bone. Osteolytic metastasis models of lung cancer have been reported, but no osteoblastic metastasis model is available for lung cancer. In the present study, we established a reproducible model of human lung cancer with both osteolytic and osteoblastic changes in natural killer cell-depleted severe combined immunodeficient mice. Intravenous inoculation of ACC-LC-319/bone2 cells resulted in the development of metastatic colonies in the lung, liver, and bone of the mice. As assessed sequentially by X-ray photographs, osteolytic bone lesions were observed by day 28, and then osteoblastic lesions were detected by day 35. Histological examination revealed the presence of bony spurs, a hallmark of osteoblastic bone metastasis, where osteoclasts were hardly observed. Treatment with an anti-human vascular endothelial growth factor antibody, bevacizumab, as well as zoledronate, inhibited the number of experimental bone metastases, including osteoblastic changes produced by ACC-LC-319/bone2 cells. These results indicate that our bone metastasis model by ACC-LC319/bone2 might be useful to understand the molecular pathogenesis of osteolytic and osteoblastic metastasis, and to identify molecular targets to control bone metastasis of lung cancer.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Bone Neoplasms; Cell Line, Tumor; Diphosphonates; Disease Models, Animal; Endothelin-1; Humans; Imidazoles; Killer Cells, Natural; Lung Neoplasms; Male; Mice; Mice, Inbred ICR; Mice, SCID; Osteoblasts; Osteolysis; Vascular Endothelial Growth Factor A; Zoledronic Acid

Whilst not strictly a neuropathic injury, cancer-induced bone pain (CIBP) is a unique state with features of neuropathy and inflammation. Recent work has demonstrated that osteoclasts damage peripheral nerves (peptidergic C fibres and SNS) within trabeculated bone leading to deafferentation. In addition, glia cell activation and neuronal hyperexcitability within the dorsal horn, are all similar to a neuropathy. Gabapentin and carbamazepine (both anti-convulsants that modulate neuropathy) are effective at attenuating dorsal horn neuronal excitability and normalizing pain-like behaviours in a rat model of CIBP. However alterations in neuroreceptors in the dorsal horn do not mimic neuropathy, rather only dynorphin is upregulated, glia cells are active and hypertrophic and c-fos expression is increased post-noxious behavioural stimulus. CIBP perhaps illustrates best the complexity of cancer pains. Rarely are they purely neuropathic, inflammatory, ischaemic or visceral but rather a combination. Management is multimodal with radiotherapy, analgesics (opioids, NSAIDs), bisphosphonates, radioisotopes and tumouricidal therapies. The difficulty with opioids relates to efficacy on spontaneous pain at rest and movement-related pain. Potential adjuvants to standard analgesic therapies for CIBP are being explored in clinical trials and include inhibitors of glutamate release.

Topics: Analgesics; Anti-Inflammatory Agents, Non-Steroidal; Bone Density Conservation Agents; Bone Diseases; Bone Neoplasms; Diphosphonates; Drug Administration Routes; Endothelin-1; Humans; Lidocaine; N-Methylaspartate; Osteoprotegerin; Pain; Receptors, Vitronectin

In the present study, we have investigated the effect of (i) ET-1 (endothelin-1) and its precursor, big ET-1, on MMP (matrix metalloproteinase)-2 and MMP-9 synthesis and activity in osteosarcoma tissue, and (ii) ET-1 receptor antagonists on cell invasion. Using Western blotting, zymography, RT-PCR (reverse transcription-PCR), immunohistochemistry, immunofluorescence and Northern blotting, we have shown that ET-1 and ET-1 receptors (ET(A) and ET(B)) were expressed in these cells. Additionally, we have demonstrated that ET-1 markedly induced the synthesis and activity of MMP-2, which was significantly increased when compared with MMP-9. Furthermore, inhibition of NF-kappaB (nuclear factor kappaB) activation blocked MMP-2 production and activity, indicating the involvement of NF-kappaB, a ubiquitous transcription factor playing a central role in the differentiation, proliferation and malignant transformation. Since ET-1 acts as an autocrine mediator through gelatinase induction and because inhibition of ET(A) receptor is beneficial for reducing both basal and ET-1-induced osteosarcoma cell invasion, targeting this receptor could be an attractive therapeutic alternative for the successful treatment of osteosarcoma.

Topics: Adolescent; Adult; Antioxidants; Blotting, Northern; Blotting, Western; Bone Neoplasms; Cell Line, Tumor; Child; Endothelin-1; Enzyme Activation; Female; Humans; Immunohistochemistry; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Middle Aged; NF-kappa B; Osteosarcoma; Pyrrolidines; Reverse Transcriptase Polymerase Chain Reaction; Statistics, Nonparametric; Thiocarbamates; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Tumor Cells, Cultured

Angiogenic factors including endothelin-1 (ET-1) play a key role in the progression of breast metastases to bone. We investigated the impact of ET-1 on the development of bone metastases in an immunocompetent murine skin-fold chamber model. Murine mammary carcinoma 4T1 was injected in a skin-fold chamber implanted on CB6 mice along with bone explants. Furthermore, mice were treated with or without a dual selective antagonist of both ET-1 receptors. The progression of the vascularization within the chamber was monitored over time by intravital microscopy (IVM). The tumor growth and the development of bone metastases were assessed by cytokeratin-19 gene expression and histological studies. Results indicate that this new model associated with IVM allows for the continuous monitoring of the change in vascularization associated with the development of bone metastases. Additionally, treatment with an antagonist of both ET-1 receptors was associated with the presence of significantly less vessels near the tumor mass compared to control mice. These changes were correlated with smaller tumor masses and reduced bone invasion (P < 0.05). Thus, in an immunocompetent murine model of breast carcinoma metastases to bone, our data support the hypothesis that vascularization plays a role in tumor development and progression and that ET-1 specifically modulates the angiogenesis associated with breast metastases to the bone.

Topics: Angiogenesis Inhibitors; Animals; Bone Neoplasms; Bosentan; Breast Neoplasms; Cell Division; Endothelin-1; Mammary Neoplasms, Animal; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Models, Biological; Neoplasm Metastasis; RNA, Messenger; Skin; Sulfonamides

The success of a free flap transplantation is based on a sufficient microanastomosis which meets the following requirements: a pedicle placed without kinking or twisting, a good drainage, a well defined recipient vessel and integrity of the endothelium. The aim of this study was to determine whether operation-related ischaemia through flap transplantation and tourniquet induces an increase of Endothelin-1 plasma levels as one cause of vasospasm during microvascular procedures. We focused our attention in particular on the reperfusion period which is often limited to an irreversible perfusion failure of microcirculation due to free radicals, interleukin and Endothelin-1. Twenty-one patients with tissue injury of the lower leg were included in our study, fourteen underwent a latissimus dorsi muscle transplantation with a combined ischaemia, seven patients had a tourniquet ischaemia for tumour resection, debridement and local flap transfer. The duration of ischaemia varied due to the course of operation. The withdrawal of venous blood via central vein catheter, flap vein and wound bed followed a fixed time table pre- and post-reperfusion (T1: preoperative day via cubital vein, T2: 6th postoperative day, T3: 5 min, T4: 10 min, T5: 15 min, T6: 1 h post-declamping and after tourniquet ischaemia via central vein catheter and T7: within 5 min from the flap vein immediate after recharging the flap). The vessel anastomosis determined the withdrawal from the local wound bed. ET-1 in venous blood samples were measured with ELISA. The duration of ischaemia in the tourniquet group ranged from 22 min up to 210 min with a mean of 76.58 min and in the latissimus group from 87 min up to 203 min with a mean of 139.21 min. The mean ET-1 plasma concentration measured systemically before operation in the 21 patients was 0.51 +/- 0.08 pg/ml (Mean +/- SD). This result corresponds with data published in literature. The locally measured plasma levels of ET-1 after tourniquet and flap ischaemia were increased with 0.34 up to 3.90 pg/ml (0.95 +/- 0.79 pg/ml [Mean +/- SD]) for the tourniquet group and with 0.34 up to 14.87 pg/ml (1.85 +/- 3.64 pg/ml [Mean +/- SD]) for the latissimus group. This is an increase compared to systemically measured values as 0.75 +/- 0.06 pg/ml (Mean +/- SD) for the tourniquet group and 0.58 +/- 0.21 pg/ml (Mean +/- SD) for the latissimus group. We conclude that Endothelin-1 is increased locally in the early reperfusion period after free latissimus dorsi-t

Topics: Adult; Anastomosis, Surgical; Bone Neoplasms; Endothelin-1; Female; Follow-Up Studies; Fractures, Open; Graft Survival; Humans; Leg; Leg Injuries; Male; Microsurgery; Middle Aged; Postoperative Complications; Reference Standards; Reperfusion Injury; Risk Factors; Smoking; Soft Tissue Injuries; Soft Tissue Neoplasms; Surgical Flaps; Tourniquets

Prostate cancer frequently metastasizes to bone where it forms osteoblastic lesions through unknown mechanisms. Bone morphogenetic proteins (BMP) are mediators of skeletal formation. Prostate cancer produces a variety of BMPs, including BMP-6. We tested the hypothesis that BMP-6 contributes to prostate cancer-induced osteosclerosis at bone metastatic sites. Prostate cancer cells and clinical tissues produced BMP-6 that increased with aggressiveness of the tumor. Prostate cancer-conditioned medium induced SMAD phosphorylation in the preosteoblast MC3T3 cells, and phosphorylation was diminished by anti-BMP-6 antibody. Prostate cancer-conditioned medium induced mineralization of MC3T3 cells, which was blocked by both the BMP inhibitor noggin and anti-BMP-6. Human fetal bones were implanted in severe combined immunodeficient mice and after 4 weeks, LuCaP 23.1 prostate cancer cells were injected both s.c. and into the bone implants. Anti-BMP-6 or isotype antibody administration was then initiated. Anti-BMP-6 reduced LuCaP 23.1-induced osteoblastic activity, but had no effect on its osteolytic activity. This was associated with increased osteoblast numbers and osteoblast activity based on bone histomorphometric evaluation. As endothelin-1 has been implicated in bone metastases, we measured serum endothelin-1 levels but found they were not different among the treatment groups. In addition to decreased bone production, anti-BMP-6 reduced intraosseous, but not s.c., tumor size. We found that BMP-2, BMP-4, BMP-6, and BMP-7 had no direct effect on prostate cancer cell growth, but BMP-2 and BMP-6 increased the in vitro invasive ability of prostate cancer cell. These data show that prostate cancer promotes osteoblastic activity through BMP-6 and that, in addition to its bone effects, suggest that BMPs promote the ability of the prostate cancer cells to invade the bone microenvironment.

Topics: Animals; Bone Development; Bone Morphogenetic Protein 6; Bone Morphogenetic Proteins; Bone Neoplasms; Cell Line, Tumor; Culture Media, Conditioned; Endothelin-1; Humans; Male; Mice; Mice, SCID; Osteoblasts; Phosphorylation; Prostatic Neoplasms; Smad Proteins

(1) Atrasentan (Xinlay(R)) is an anti-cancer drug from a new class of agents called selective endothelin-A receptor antagonists. The orally administered drug is being studied in a subset of patients with advanced prostate cancer. (2) Phase II and III studies evaluating time to clinical and radiographic progression failed to demonstrate a significant benefit with atrasentan versus placebo. (3) The adverse effects, observed more frequently in those treated with atrasentan than in placebo-treated patients, were peripheral edema, rhinitis, headache, infection, dyspnea, and heart failure. (4) Atrasentan's role in the various stages of advanced prostate cancer, and relative to the chemotherapeutic agent docetaxel, has not been determined.

Topics: Bone Neoplasms; Canada; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Drug Approval; Endothelin Receptor Antagonists; Endothelin-1; Humans; Male; Neoplasm Metastasis; Prostatic Neoplasms; Pyrrolidines; Randomized Controlled Trials as Topic; Survival Rate; Treatment Outcome; United States; United States Food and Drug Administration

Tumors including sarcomas and breast, prostate, and lung carcinomas frequently grow in or metastasize to the skeleton where they can induce significant bone remodeling and cancer pain. To define products that are released from tumors that are involved in the generation and maintenance of bone cancer pain, we focus here on endothelin-1 (ET-1) and endothelin receptors as several tumors including human prostate and breast have been shown to express high levels of ETs and the application of ETs to peripheral nerves can induce pain. Here we show that in a murine osteolytic 2472 sarcoma model of bone cancer pain, the 2472 sarcoma cells express high levels of ET-1, but express low or undetectable levels of endothelin A (ETAR) or B (ETBR) receptors whereas a subpopulation of sensory neurons express the ETAR and non-myelinating Schwann cells express the ETBR. Acute (10 mg/kg, i.p.) or chronic (10 mg/kg/day, p.o.) administration of the ETAR selective antagonist ABT-627 significantly attenuated ongoing and movement-evoked bone cancer pain and chronic administration of ABT-627 reduced several neurochemical indices of peripheral and central sensitization without influencing tumor growth or bone destruction. In contrast, acute treatment (30 mg/kg, i.p.) with the ETBR selective antagonist, A-192621 increased several measures of ongoing and movement evoked pain. As tumor expression and release of ET-1 has been shown to be regulated by the local environment, location specific expression and release of ET-1 by tumor cells may provide insight into the mechanisms that underlie the heterogeneity of bone cancer pain that is frequently observed in humans with multiple skeletal metastases.

Topics: Analysis of Variance; Animals; Atrasentan; Behavior, Animal; Bone Neoplasms; Calcitonin Gene-Related Peptide; Disease Models, Animal; Dynorphins; Endothelin Receptor Antagonists; Endothelin-1; Ganglia, Spinal; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Immunohistochemistry; Male; Mice; Mice, Inbred Strains; Pain; Pain Measurement; Pyrrolidines; Receptors, Endothelin; Sarcoma; Sciatic Nerve; Time Factors

Osteoblastic bone metastases are common in prostate and breast cancer patients, but mechanisms by which tumor cells stimulate new bone formation are unclear. We identified three breast cancer cell lines that cause osteoblastic metastases in a mouse model and secrete endothelin-1. Tumor-produced endothelin-1 stimulates new bone formation in vitro and osteoblastic metastases in vivo via the endothelin A receptor. Treatment with an orally active endothelin A receptor antagonist dramatically decreased bone metastases and tumor burden in mice inoculated with ZR-75-1 cells. Tumor-produced endothelin-1 may have a major role in the establishment of osteoblastic bone metastases, and endothelin A receptor blockade represents effective treatment.

Topics: Bone Neoplasms; Bone Remodeling; Endothelin-1; Humans; Osteoblasts; Tumor Cells, Cultured

Angiogenesis is necessary for the growth of primary tumors and the formation of metastases. It is well known that vascular endothelial growth factor (VEGF) and its receptors play a major role in this process. To date, the formation of bone metastases has been poorly understood. Tumor cells must interact with the microenvironment of the bone and new blood vessels must spread. The ET/ET(A) (endothelin) receptor system seems to play an important role in this process. Specimens from metastatic bone lesions and non-malignant bone tissue were analyzed by histological and immunohistochemical staining. Sections were stained with antibodies against CD31, Flt-1, KDR, endothelin-1 (ET-1) and endothelin receptor A (ET(A)). Our studies show that there is an increased microvessel density (MVD) in metastatic bone lesions from different primary tumors in contrast to normal bone tissue. In nearly all tumor formations of the bone, ET-1 and its receptor ET(A) was found by immunohistochemistry. We also performed immunohistochemical staining for the VEGF-receptors Flt-1 and KDR. In conclusion, there is an increased vessel density in metastatic bone lesions in contrast to normal bone tissue. The ET/ET(A) system can be detected in nearly all bone specimens and is upregulated in metastatic bone lesions in contrast to healthy bone tissue.

Topics: Bone Neoplasms; Case-Control Studies; Endothelin-1; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Neoplasms; Neovascularization, Pathologic; Receptor, Endothelin A; Up-Regulation; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

To improve the therapy of advanced prostate cancer (CaP), it is critical to develop animal models that mimic CaP bone metastases. Unlike the human disease, CaP xenograft models rarely metastasize spontaneously to bone from the orthotopic site of primary tumor growth.. Single-cell suspensions of LNCaP, PC-3, LuCaP 35, and LuCaP 23.1 CaP cells were injected directly into tibia of SCID mice. Immunohistochemistry and bone histomorphometrical analyses were performed to characterize these osseous-CaP models.. LuCaP 23.1 yields an osteoblastic response, LNCaP yields mixed lesions, and LuCaP 35 and PC-3 result in osteolytic responses. We have detected osteoprotegerin, RANK ligand, parathyroid hormone-related protein, and endothelin-1, proteins associated with bone growth and remodeling, in the CaP cells grown in the bone.. These animal models can be used to study biological interactions, pathways, and potential therapeutic targets, and also to evaluate new agents for treatment and prevention of CaP bone metastasis.

Topics: Animals; Bone Density; Bone Neoplasms; Bone Remodeling; Carrier Proteins; Disease Models, Animal; Endothelin-1; Glycoproteins; Humans; Immunohistochemistry; Kinetics; Male; Membrane Glycoproteins; Neoplasm Transplantation; Orchiectomy; Osteoblasts; Osteoclasts; Osteoprotegerin; Parathyroid Hormone-Related Protein; Prostatic Neoplasms; Proteins; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tibia; Transplantation, Heterologous; Tumor Cells, Cultured

Topics: Antineoplastic Agents; Bone and Bones; Bone Neoplasms; Endothelin Receptor Antagonists; Endothelin-1; Humans; Male; Prostatic Neoplasms; Receptor, Endothelin A

Recent data demonstrate that endothelin-1 (ET-1) concentration increases in plasma of men with advanced, hormone-refractory prostate adenocarcinoma. In addition, ET-1 is involved in osteblastic remodelling and new bone formation, suggesting a role for this vasoactive peptide in the metastatic progression of prostate cancer to the bone.. We investigated the regulation of ET-1 expression in androgen-sensitive and insensitive prostate cancer cell lines by androgens and several factors involved in progression of prostate cancer (EGF) and bone remodelling (TGFbeta-1, IL1-alpha and IGF-1).. Northern analysis and radio immunoassay demonstrated that all the ET-1 pathways are tuned off in the androgen-sensitive LNCaP cell line when compared to the androgen-insensitive PC-3 and DU145. In PC-3 cells transfected with a full-length androgen receptor expression vector (PC-3-AR), treatment with androgens reduced gene expression and secretion of ET-1 without affecting the gene expression of ET-3. Collectively, these data support a role for androgens in the regulation of ET-1 production by prostate adenocarcinoma cells. In PC-3 and DU145 cells, ET-1 gene expression and secretion were up-regulated by TGFbeta-1, EGF and IL1-alpha, whereas IGF-1 was ineffective. Conversely, none of the treatments affected ECE-1 or ET-3 gene expression.. In conclusion, ET-1 production by prostate adenocarcinoma cells is down-regulated by androgens and up-regulated by factors involved in tumour progression indicating a role for this peptide in the biology of prostate cancer. In view of the role exerted by ET-1 in the process of bone metastasis, our data suggest the use of ET-1 receptor antagonists in the treatment of advanced prostate cancer.

Topics: Adenocarcinoma; Androgens; Blotting, Northern; Bone Neoplasms; Cytokines; Endothelin-1; Endothelin-3; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Male; Metalloendopeptidases; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Modulation of endothelin (ET-1)-induced [Ca(2+)](i)transients and receptor expression by parathyroid hormone (PTH) was studied in UMR-106 osteoblastic osteosarcoma cells. Ca(2+)signaling was assessed with Fura-2, and ET receptor mRNA expression was determined using ET(A)- and ET(B)-specific primers and RT-PCR amplification. ET-1 binding in UMR-106 cell membranes was also measured. PTH pretreatment for 8 h decreased the [Ca(2+)](i)transients elicited by ET-1 and by the ET(B)-selective agonist sarafotoxin 6c (S6c). When ET(B)receptors were desensitized by pretreatment with S6c or blocked with the ET(B)-selective antagonist BQ-788, the remaining ET(A)component of the signal was also decreased by PTH pretreatment. In contrast, [Ca(2+)](i)transients elicited by PGF(2alpha)and ionomycin were increased following PTH pretreatment, indicating that the effect of PTH to decrease ET-1-stimulated transients was selective. PTH pretreatment also decreased [(125)I]ET-1 binding and ET(A)and ET(B)mRNA, with maximal effects at approximately 8 h. ET-1 was not detectable in medium from either control or PTH treated UMR-106 cultures, suggesting that the decreased expression of ET receptors was not due to enhanced ET production and subsequent homologous desensitization. The downregulation of ET receptors in osteoblasts by PTH pretreatment may serve as a homeostatic mechanism in bone.

Topics: Bone Neoplasms; Calcium Signaling; Cell Membrane; Dinoprost; Down-Regulation; Endothelin Receptor Antagonists; Endothelin-1; Ionomycin; Ionophores; Oligopeptides; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Piperidines; Protein Isoforms; Receptors, Endothelin; RNA, Messenger; Tumor Cells, Cultured; Viper Venoms