RESUMO
To test the hypothesis that genetic and pharmacological modulation of the classical cannabinoid type 1 (CB1) and 2 (CB2) receptors attenuate cancer-induced bone pain, we searched Medline, Web of Science and Scopus for relevant skeletal and non-skeletal cancer studies from inception to July 28, 2022. We identified 29 animal and 35 human studies. In mice, a meta-analysis of pooled studies showed that treatment of osteolysis-bearing males with the endocannabinoids AEA and 2-AG (mean difference [MD] - 24.83, 95% confidence interval [95%CI] - 34.89, - 14.76, p < 0.00001) or the synthetic cannabinoid (CB) agonists ACPA, WIN55,212-2, CP55,940 (CB1/2-non-selective) and AM1241 (CB2-selective) (MD - 28.73, 95%CI - 45.43, - 12.02, p = 0.0008) are associated with significant reduction in paw withdrawal frequency. Consistently, the synthetic agonists AM1241 and JWH015 (CB2-selective) increased paw withdrawal threshold (MD 0.89, 95%CI 0.79, 0.99, p < 0.00001), and ACEA (CB1-selective), AM1241 and JWH015 (CB2-selective) reduced spontaneous flinches (MD - 4.85, 95%CI - 6.74, - 2.96, p < 0. 00001) in osteolysis-bearing male mice. In rats, significant increase in paw withdrawal threshold is associated with the administration of ACEA and WIN55,212-2 (CB1/2-non-selective), JWH015 and AM1241 (CB2-selective) in osteolysis-bearing females (MD 8.18, 95%CI 6.14, 10.21, p < 0.00001), and treatment with AM1241 (CB2-selective) increased paw withdrawal thermal latency in males (mean difference [MD]: 3.94, 95%CI 2.13, 5.75, p < 0.0001), confirming the analgesic capabilities of CB1/2 ligands in rodents. In human, treatment of cancer patients with medical cannabis (standardized MD - 0.19, 95%CI - 0.35, - 0.02, p = 0.03) and the plant-derived delta-9-THC (20 mg) (MD 3.29, CI 2.24, 4.33, p < 0.00001) or its synthetic derivative NIB (4 mg) (MD 2.55, 95%CI 1.58, 3.51, p < 0.00001) are associated with reduction in pain intensity. Bioinformatics validation of KEGG, GO and MPO pathway, function and process enrichment analysis of mouse, rat and human data revealed that CB1 and CB2 receptors are enriched in a cocktail of nociceptive and sensory perception, inflammatory, immune-modulatory, and cancer pathways. Thus, we cautiously conclude that pharmacological modulators of CB1/2 receptors show promise in the treatment of cancer-induced bone pain, however further assessment of their effects on bone pain in genetically engineered animal models and cancer patients is warranted.
Assuntos
Dor do Câncer , Canabinoides , Neoplasias , Osteólise , Masculino , Ratos , Humanos , Camundongos , Animais , Receptores de Canabinoides , Osteólise/tratamento farmacológico , Canabinoides/farmacologia , Canabinoides/uso terapêutico , Agonistas de Receptores de Canabinoides , Dor do Câncer/tratamento farmacológico , Dor do Câncer/etiologia , Neoplasias/tratamento farmacológico , Receptor CB2 de Canabinoide , Receptor CB1 de CanabinoideRESUMO
TRAF6 has emerged as a key regulator of breast cancer (BCa). However, the TRAF family constitutes of seven members that exhibit distinct and overlapping functions. To explore which TRAF represents a potential druggable target for BCa treatment, we searched Medline, Web of Science and Scopus for relevant studies from inception to June 27, 2021. We identified 14 in vitro, 11 in vivo and 4 human articles. A meta-analysis of pharmacological studies showed that in vitro inhibition of TRAF2/4 (mean difference (MD): - 57.49, 95% CI: - 66.95, - 48.02, P < 0.00001) or TRAF6 (standard(Std.)MD: - 4.01, 95% CI: - 5.75, - 2.27, P < 0.00001) is associated with reduction in BCa cell migration. Consistently, inhibition of TRAF2/4 (MD: - 51.08, 95% CI: - 64.23, - 37.94, P < 0.00001) and TRAF6 (Std.MD: - 2.80, 95% CI: - 4.26, - 1.34, P = 0.0002) is associated with reduced BCa cell invasion, whereas TRAF2/4 inhibition (MD: - 40.54, 95% CI: - 52.83, - 28.26, P < 0.00001) is associated with reduced BCa cell adhesion. Interestingly, only inhibition of TRAF6 (MD: - 21.46, 95% CI: - 30.40, - 12.51, P < 0.00001) is associated with reduced cell growth. In animal models of BCa, administration of pharmacological inhibitors of TRAF2/4 (Std.MD: - 3.36, 95% CI: - 4.53, - 2.18, P < 0.00001) or TRAF6 (Std.MD: - 4.15, 95% CI: - 6.06, - 2.24, P < 0.0001) in mice is associated with reduction in tumour burden. In contrast, TRAF6 inhibitors (MD: - 2.42, 95% CI: - 3.70, - 1.14, P = 0.0002) reduced BCa metastasis. In BCa patients, high expression of TRAF6 (Hazard Ratio: 1.01, CI: 1.01, 1.01, P < 0.00001) is associated with poor survival rate. Bioinformatics validation of clinical and pathway and process enrichment analysis in BCa patients confirmed that gain/amplification of TRAF6 is associated with secondary BCa in bone (P = 0.0079), and poor survival rate (P < 0.05). Overall, TRAF6 inhibitors show promise in the treatment of metastatic BCa. However, low study number and scarcity of evidence from animal and human studies may limit the translation of present findings into clinical practice.
Assuntos
Neoplasias da Mama , Fator 6 Associado a Receptor de TNF , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/genéticaRESUMO
Zoledronic Acid (ZA) has been shown to inhibit Osteosarcoma (OSA) progression in preclinical studies. However, the use of ZA as an intervention for OSA treatment and management remains controversial. A systematic review and meta-analysis of randomized-controlled trials comparing the use of ZA with standard treatment vs. standard treatment alone for OSA patients after resection was conducted. Primary outcomes assessed event-free survival (EFS) and overall survival (OS) rates, while secondary outcomes assessed impact of ZA on metastatic spread, histological response and adverse events occurrence. A literature search was conducted using EMBASE, MEDLINE, and the Cochrane Central Register of Controlled Trials. The Cochrane risk of bias tool (version 2) was used to assess trial quality. RevMan v5.4 was used for the meta-analysis. The between-trial heterogeneity was assessed using the Chi2 test and the I2 statistic and the GRADE methodology was utilized to assess certainty of evidence. Two studies were considered eligible for qualitative synthesis and meta-analysis. ZA had no benefit on EFS (HR, 0.95; 95% CI, 0.48-1.88; p-value 0.88), however, when compared to standard treatment it reduced OS (HR, 1.98; 95% CI, 1.49-2.64; p-value < 0.00001). ZA did not deter lung metastasis (RR, 2.56; 95% CI, 0.35-18.60; p-value 0.35), and neither did it increase good histological response (RR, 0.97; 95% CI, 0.90-1.05; p-value 0.48). ZA treatment was associated with higher risk of adverse events. Based on existing data, the use of ZA as adjuvant therapy is not recommended for the treatment of OSA patients.
Assuntos
Osteossarcoma , Humanos , Ácido Zoledrônico/uso terapêutico , Terapia Combinada , Osteossarcoma/tratamento farmacológicoRESUMO
NFκB plays a key role in inflammation and skeletal disorders. Previously, we reported that pharmacological inhibition of NFκB at the level of TRAF6 suppressed RANKL, CD40L and IL1ß-induced osteoclastogenesis and attenuated cancer-induced bone disease. TNFα is also known to regulate TRAF6/NFκB signalling, however the anti-inflammatory and osteoprotective effects associated with inhibition of the TNFα/TRAF6/NFκB axis have not been investigated. Here, we show that in vitro and ex vivo exposure to the verified small-molecule inhibitor of TRAF6, 6877002 prevented TNFα-induced NFκB activation, osteoclastogenesis and calvarial osteolysis, but it had no effects on TNFα-induced apoptosis or growth inhibition in osteoblasts. Additionally, 6877002 disrupted T-cells support for osteoclast formation and synoviocyte motility, without affecting the viability of osteoblasts in the presence of T-cells derived factors. Using the collagen-induced arthritis model, we show that oral and intraperitoneal administration of 6877002 in mice reduced joint inflammation and arthritis score. Unexpectedly, no difference in trabecular and cortical bone parameters were detected between vehicle and 6877002 treated mice, indicating lack of osteoprotection by 6877002 in the arthritis model described. Using two independent rodent models of osteolysis, we confirmed that 6877002 had no effect on trabecular and cortical bone loss in both osteoporotic rats or RANKL- treated mice. In contrast, the classic anti-osteolytic alendronate offered complete osteoprotection in RANKL- treated mice. In conclusion, TRAF6 inhibitors may be of value in the management of the inflammatory component of bone disorders, but may not offer protection against local or systemic bone loss, unless combined with anti-resorptive therapy such as bisphosphonates.
Assuntos
Anti-Inflamatórios/farmacologia , Antígenos CD40/antagonistas & inibidores , Osteólise/prevenção & controle , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Animais , Anti-Inflamatórios/química , Artrite Experimental/metabolismo , Artrite Experimental/prevenção & controle , Antígenos CD40/metabolismo , Linhagem Celular Tumoral , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos DBA , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteólise/metabolismo , Células RAW 264.7 , Roedores/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Multiple myeloma (MM) patients develop osteolysis characterised by excessive osteoclastic bone destruction and lack of osteoblast bone formation. Pharmacological manipulation of monoacylglycerol lipase (MAGL), an enzyme responsible for the degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG), reduced skeletal tumour burden and osteolysis associated with osteosarcoma and advanced breast and prostate cancers. MM and hematopoietic, immune and bone marrow cells express high levels of type 2 cannabinoid receptor and osteoblasts secrete 2-AG. However, the effects of MAGL manipulation on MM have not been investigated. Here, we report that treatment of pre-osteoclasts with non-cytotoxic concentrations of JZL184, a verified MAGL inhibitor, enhanced MM- and RANKL-induced osteoclast formation and size in vitro. Exposure of osteoblasts to JZL184 in the presence of MM cell-derived factors reduced osteoblast growth but had no effect on the ability of these cells to mature or form bone nodules. In vivo, administration of JZL184 induced a modest, yet significant, bone loss at both trabecular and cortical compartments of long bones of immunocompetent mice inoculated with the syngeneic 5TGM1-GFP MM cells. Notably, JZL184 failed to inhibit the in vitro growth of a panel of mouse and human MM cell lines, or reduce tumour burden in mice. Thus, MAGL inhibitors such as JZL184 can exacerbate MM-induced bone loss.
Assuntos
Benzodioxóis/efeitos adversos , Reabsorção Óssea/induzido quimicamente , Monoacilglicerol Lipases/antagonistas & inibidores , Mieloma Múltiplo , Piperidinas/efeitos adversos , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Células RAW 264.7RESUMO
E11/Podoplanin (Pdpn) is implicated in early osteocytogenesis and the formation of osteocyte dendrites. This dendritic network is critical for bone modelling/remodelling, through the production of receptor activator of nuclear factor κ B (RANK)-ligand (RANKL). Despite this, the role of Pdpn in the control of bone remodelling is yet to be established in vivo. Here we utilised bone-specific Pdpn conditional knockout mice (cKO) to examine the role of Pdpn in the bone loss associated with ovariectomy (OVX). MicroCT revealed that Pdpn deletion had no significant effect on OVX-induced changes in trabecular microarchitecture. Significant differences between genotypes were observed in the trabecular pattern factor (P<0.01) and structure model index (P<0.01). Phalloidin staining of F-actin revealed OVX to induce alterations in osteocyte morphology in both wild-type (WT) and cKO mice. Histological analysis revealed an expected significant increase in osteoclast number in WT mice (P<0.01, compared with sham). However, cKO mice were protected against such increases in osteoclast number. Consistent with this, serum levels of the bone resorption marker Ctx were significantly increased in WT mice following OVX (P<0.05), but were unmodified by OVX in cKO mice. Gene expression of the bone remodelling markers Rank, Rankl, Opg and Sost were unaffected by Pdpn deletion. Together, our data suggest that an intact osteocyte dendritic network is required for sustaining osteoclast formation and activity in the oestrogen-depleted state, through mechanisms potentially independent of RANKL expression. This work will enable a greater understanding of the role of osteocytes in bone loss induced by oestrogen deprivation.
Assuntos
Remodelação Óssea , Fêmur/metabolismo , Glicoproteínas de Membrana/deficiência , Osteoclastos/metabolismo , Osteogênese , Osteoporose Pós-Menopausa/prevenção & controle , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Feminino , Fêmur/patologia , Humanos , Glicoproteínas de Membrana/genética , Camundongos Knockout , Osteoclastos/patologia , Osteoporose Pós-Menopausa/genética , Osteoporose Pós-Menopausa/metabolismo , Osteoporose Pós-Menopausa/patologia , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ovariectomia , Peptídeos/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismoRESUMO
BACKGROUND: Cancer-associated bone disease is a serious complication in bone sarcomas and metastatic carcinomas of breast and prostate origin. Monoacylglycerol lipase (MAGL) is an enzyme of the endocannabinoid system, and is responsible for the degradation of the most abundant endocannabinoid in bone, 2-arachidonoyl glycerol (2AG). METHODS: The effects of the verified MAGL inhibitor on bone remodelling were assessed in healthy mice and in mouse models of bone disease caused by prostate and breast cancers and osteosarcoma. FINDINGS: JZL184 reduced osteolytic bone metastasis in mouse models of breast and prostate cancers, and inhibited skeletal tumour growth, metastasis and the formation of ectopic bone in models of osteosarcoma. Additionally, JZL184 suppressed cachexia and prolonged survival in mice injected with metastatic osteosarcoma and osteotropic cancer cells. Functional and histological analysis revealed that the osteoprotective action of JZL184 in cancer models is predominately due to inhibition of tumour growth and metastasis. In the absence of cancer, however, exposure to JZL184 exerts a paradoxical reduction of bone volume via an effect that is mediated by both Cnr1 and Cnr2 cannabinoid receptors. INTERPRETATION: MAGL inhibitors such as JZL184, or its novel analogues, may be of value in the treatment of bone disease caused by primary bone cancer and bone metastasis, however, activation of the skeletal endocannabinoid system may limit their usefulness as osteoprotective agents.
Assuntos
Benzodioxóis/farmacologia , Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Inibidores Enzimáticos/farmacologia , Monoacilglicerol Lipases/antagonistas & inibidores , Piperidinas/farmacologia , Animais , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Reabsorção Óssea/diagnóstico por imagem , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Comunicação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Camundongos , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteólise/tratamento farmacológico , Osteólise/etiologia , Osteólise/metabolismo , Osteólise/patologia , Receptores de Canabinoides/metabolismoRESUMO
Pharmacological therapy of osteoporosis reduces bone loss and risk of fracture in patients. Modulation of bone mineral density cannot explain all effects. Other aspects of bone quality affecting fragility and ways to monitor them need to be better understood. Keratinous tissue acts as surrogate marker for bone protein deterioration caused by oestrogen deficiency in rats. Ovariectomised rats were treated with alendronate (ALN), parathyroid hormone (PTH) or estrogen (E2). MicroCT assessed macro structural changes. Raman spectroscopy assessed biochemical changes. Micro CT confirmed that all treatments prevented ovariectomy-induced macro structural bone loss in rats. PTH induced macro structural changes unrelated to ovariectomy. Raman analysis revealed ALN and PTH partially protect against molecular level changes to bone collagen (80% protection) and mineral (50% protection) phases. E2 failed to prevent biochemical change. The treatments induced alterations unassociated with the ovariectomy; increased beta sheet with E2, globular alpha helices with PTH and fibrous alpha helices with both ALN and PTH. ALN is closest to maintaining physiological status of the animals, while PTH (comparable protective effect) induces side effects. E2 is unable to prevent molecular level changes associated with ovariectomy. Raman spectroscopy can act as predictive tool for monitoring pharmacological therapy of osteoporosis in rodents. Keratinous tissue is a useful surrogate marker for the protein related impact of these therapies.The results demonstrate utility of surrogates where a clear systemic causation connects the surrogate to the target tissue. It demonstrates the need to assess broader biomolecular impact of interventions to examine side effects.
Assuntos
Osteoporose Pós-Menopausa/diagnóstico , Osteoporose Pós-Menopausa/terapia , Análise Espectral Raman , Alendronato/farmacologia , Animais , Peso Corporal , Densidade Óssea , Conservadores da Densidade Óssea/farmacologia , Modelos Animais de Doenças , Estrogênios/metabolismo , Feminino , Humanos , Queratinas/química , Hormônio Paratireóideo/farmacologia , Ratos , Ratos Sprague-Dawley , Microtomografia por Raio-XRESUMO
Transcription is a process by which the rate of RNA synthesis is regulated. Here we describe the techniques for carrying out promoter-reporter assays, electrophoretic mobility shift assays, chromosome conformation capture (3C) assays, chromatin immunoprecipitation assays, and CRISPR-Cas9 assay, five commonly used methods for studying and altering gene transcription.
Assuntos
Osso e Ossos/citologia , Imunoprecipitação da Cromatina/métodos , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Regulação da Expressão Gênica , Transcrição Gênica , Sistemas CRISPR-Cas/genética , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Separação Celular/instrumentação , Separação Celular/métodos , Imunoprecipitação da Cromatina/instrumentação , Ensaio de Desvio de Mobilidade Eletroforética/instrumentação , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Edição de Genes/instrumentação , Edição de Genes/métodos , Genes Reporter/genética , Humanos , Regiões Promotoras Genéticas/genéticaRESUMO
This chapter describes the surgical procedures for ovariectomy and orchiectomy in mice and rats. In addition to providing technical details of the surgical techniques, details of anesthesia options and pre-, peri-, and postoperative care are also included.
Assuntos
Reabsorção Óssea/patologia , Modelos Animais de Doenças , Animais , Reabsorção Óssea/metabolismo , Estrogênios/metabolismo , Feminino , Humanos , Masculino , Camundongos , Orquiectomia/instrumentação , Orquiectomia/métodos , Ovariectomia/instrumentação , Ovariectomia/métodos , Ovário/metabolismo , Ovário/cirurgia , Ratos , Testículo/metabolismo , Testículo/cirurgiaRESUMO
NFκB is implicated in breast cancer bone metastasis and skeletal remodelling. However, the role of IKKß, a key component of the canonical NFκB pathway, in the regulation of breast cancer osteolytic metastasis has not been investigated. Here, we describe the cancer-specific contribution of IKKß to bone metastasis, skeletal tumour growth and osteolysis associated with breast cancer. IKKß is highly expressed in invasive breast tumours and its level of expression was higher in patients with bone metastasis. IKKß overexpression in parental MDA-MD-231 breast cancer cells, promoted mammary tumour growth but failed to convey osteolytic potential to these cells in mice. In contrast, IKKß overexpression in osteotropic sub-clones of MDA-MB-231 cells with differing osteolytic phenotypes increased incidence of bone metastasis, exacerbated osteolysis and enhanced skeletal tumour growth, whereas its knockdown was inhibitory. Functional and mechanistic studies revealed that IKKß enhanced the ability of osteotropic MDA-MB-231 cells to migrate, increase osteoclastogenesis, and to inhibit osteoblast differentiation via a mechanism mediated, at least in part, by cytoplasmic sequestering of FoxO3a and VEGFA production. Thus, tumour-selective manipulation of IKKß and its interaction with FoxO3a may represent a novel strategy to reduce the development of secondary breast cancer in the skeleton.
RESUMO
Osteoporosis is a common disease characterised by reduced bone mass and an increased risk of fragility fractures. Low bone mineral density is known to significantly increase the risk of osteoporotic fractures, however, the majority of non-traumatic fractures occur in individuals with a bone mineral density too high to be classified as osteoporotic. Therefore, there is an urgent need to investigate aspects of bone health, other than bone mass, that can predict the risk of fracture. Here, we successfully predicted association between bone collagen and nail keratin in relation to bone loss due to oestrogen deficiency using Raman spectroscopy. Raman signal signature successfully discriminated between ovariectomised rats and their sham controls with a high degree of accuracy for the bone (sensitivity 89%, specificity 91%) and claw tissue (sensitivity 89%, specificity 82%). When tested in an independent set of claw samples the classifier gave 92% sensitivity and 85% specificity. Comparison of the spectral changes occurring in the bone tissue with the changes occurring in the keratin showed a number of common features that could be attributed to common changes in the structure of bone collagen and claw keratin. This study established that systemic oestrogen deficiency mediates parallel structural changes in both the claw (primarily keratin) and bone proteins (primarily collagen). This strengthens the hypothesis that nail keratin can act as a surrogate marker of bone protein status where systemic processes induce changes.
Assuntos
Osso e Ossos/patologia , Colágeno/química , Estrogênios/deficiência , Casco e Garras/patologia , Queratinas/química , Análise Espectral Raman , Animais , Densidade Óssea , Osso e Ossos/metabolismo , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Estrogênios/metabolismo , Feminino , Casco e Garras/metabolismo , Osteoporose/metabolismo , Osteoporose/patologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Microtomografia por Raio-XRESUMO
The endocannabinoid system plays a role in regulating bone mass and bone cell activity and inactivation of the type 1 (Cnr1) or type 2 (Cnr2) cannabinoid receptors influences peak bone mass and age-related bone loss. As the Cnr1 and Cnr2 receptors have limited homology and are activated by different ligands, we have evaluated the effects of combined deficiency of Cnr1 and 2 receptors (Cnr1/2-/- ) on bone development from birth to old age and studied ovariectomy induced bone loss in female mice. The Cnr1/2-/- mice had accelerated bone accrual at birth when compared with wild type littermates, and by 3 months of age, they had higher trabecular bone mass. They were also significantly protected against ovariectomy-induced bone loss due to a reduction in osteoclast number. The Cnr1/2-/- mice had reduced age-related bone loss when compared with wild-type due to a reduction in osteoclast number. Although bone formation was reduced and bone marrow adiposity increased in Cnr1/2-/- mice, the osteoclast defect outweighed the reduction in bone formation causing preservation of bone mass with aging. This contrasts with the situation previously reported in mice with inactivation of the Cnr1 or Cnr2 receptors individually where aged-related bone loss was greater than in wild-type. We conclude that the Cnr1 and Cnr2 receptors have overlapping but nonredundant roles in regulating osteoclast and osteoblast activities. These observations indicate that combined inhibition of Cnr1 and Cnr2 receptors may be beneficial in preventing age-related bone loss, whereas blockade of individual receptors may be detrimental.
Assuntos
Densidade Óssea/genética , Osteoclastos/metabolismo , Osteoporose/genética , Receptor CB1 de Canabinoide/genética , Receptor CB2 de Canabinoide/genética , Animais , Contagem de Células , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Fêmur/metabolismo , Fêmur/patologia , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/patologia , Osteogênese/genética , Osteoporose/etiologia , Osteoporose/patologia , Osteoporose/prevenção & controle , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ovariectomia/efeitos adversos , Cultura Primária de Células , Ligante RANK/genética , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptor CB1 de Canabinoide/deficiência , Receptor CB2 de Canabinoide/deficiência , Transdução de Sinais , Tíbia/metabolismo , Tíbia/patologiaRESUMO
PURPOSE: To investigate possible associations between recreational cannabis use and bone health in humans. METHODS: Cross-sectional study of individuals recruited from primary care in the UK between 2011 and 2013. Cases were regular smokers of cannabis divided into moderate (n = 56) and heavy user (n = 144) subgroups depending on whether they reported fewer or more than 5000 cannabis smoking episodes during their lifetime. Controls comprised 114 cigarette smokers. RESULTS: Heavy cannabis users had lower total hip bone mineral density (mean ± SD Z-score: -0.20 ± 0.9 vs +0.2 ± 0.9, P < .0005), lower spine bone mineral density (-0.5 ± 1.2 vs 0.0 ± 1.2, P < .0005), and lower body mass index (BMI; 26.5 ± 6.0 vs 29.0 ± 7.0, P = .01) than controls. Fracture rate was also increased in heavy users (rate ratio = 2.17; 95% confidence interval, 1.59-2.95; P < .001). When compared with controls, serum cross-linked C-telopeptide of type 1 collagen (CTX) concentrations were raised in heavy cannabis users (0.3 ± 0.1 vs 0.2 ± 0.1 pg/mL, P = .045), as were serum N-terminal propeptide of type 1 procollagen (P1NP) concentrations (47.1 ± 19.2 vs 41.2 ± 17.8 pg/mL, P = .01). Serum total 25-hydroxyvitamin D concentrations were reduced in heavy users compared with controls (25.3 ± 16.8 vs 36.9 ± 26.7 nmol/L, P = .002). Multiple regression analysis revealed that heavy cannabis use was an independent predictor of spine bone mineral density, accounting for 5.4% of the variance (P = .035), and total hip bone mineral density, accounting for 5.8% of the variance (P = .001), but mediation analysis suggested that the effect on spine bone mineral density was indirect and mediated through low body mass index. CONCLUSIONS: Heavy cannabis use is associated with low bone mineral density, low BMI, high bone turnover, and an increased risk of fracture. Heavy cannabis use negatively impacts on bone health both directly and indirectly through an effect on BMI.
Assuntos
Densidade Óssea/efeitos dos fármacos , Fraturas Ósseas/etiologia , Abuso de Maconha/complicações , Adulto , Índice de Massa Corporal , Estudos de Casos e Controles , Colágeno Tipo I/sangue , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/sangue , Peptídeos/sangue , Pró-Colágeno/sangue , Fatores de Risco , Vitamina D/análogos & derivados , Vitamina D/sangueRESUMO
The cannabinoid type 2 receptor (CB2) has previously been implicated as a regulator of tumor growth, bone remodeling, and bone pain. However, very little is known about the role of the skeletal CB2 receptor in the regulation of osteoblasts and osteoclasts changes associated with breast cancer. Here we found that the CB2-selective agonists HU308 and JWH133 reduced the viability of a variety of parental and bone-tropic human and mouse breast cancer cells at high micromolar concentrations. Under conditions in which these ligands are used at the nanomolar range, HU308 and JWH133 enhanced human and mouse breast cancer cell-induced osteoclastogenesis and exacerbated osteolysis, and these effects were attenuated in cultures obtained from CB2-deficient mice or in the presence of a CB2 receptor blocker. HU308 and JWH133 had no effects on osteoblast growth or differentiation in the presence of conditioned medium from breast cancer cells, but under these circumstances both agents enhanced parathyroid hormone-induced osteoblast differentiation and the ability to support osteoclast formation. Mechanistic studies in osteoclast precursors and osteoblasts showed that JWH133 and HU308 induced PI3K/AKT activity in a CB2-dependent manner, and these effects were enhanced in the presence of osteolytic and osteoblastic factors such as RANKL (receptor activator of NFκB ligand) and parathyroid hormone. When combined with published work, these findings suggest that breast cancer and bone cells exhibit differential responses to treatment with CB2 ligands depending upon cell type and concentration used. We, therefore, conclude that both CB2-selective activation and antagonism have potential efficacy in cancer-associated bone disease, but further studies are warranted and ongoing.
Assuntos
Células da Medula Óssea/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Animais , Western Blotting , Células da Medula Óssea/citologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Canabinoides/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Humanos , Células MCF-7 , Camundongos Knockout , Osteoblastos/citologia , Osteoclastos/citologia , Osteogênese/efeitos dos fármacos , Osteólise , Hormônio Paratireóideo/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK/farmacologia , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Micro-computed tomography (micro-CT) is a high-resolution imaging modality that is capable of analysing bone structure with a voxel size on the order of 10 µm. With the development of in vivo micro-CT, where disease progression and treatment can be monitored in a living animal over a period of time, this modality has become a standard tool for preclinical assessment of bone architecture during disease progression and treatment. For meaningful comparison between micro-CT studies, it is essential that the same parameters for data acquisition and analysis methods be used. This protocol outlines the common procedures that are currently used for sample preparation, scanning, reconstruction and analysis in micro-CT studies. Scan and analysis methods for trabecular and cortical bone are covered for the femur, tibia, vertebra and the full neonate body of small rodents. The analysis procedures using the software provided by ScancoMedical and Bruker are discussed, and the routinely used bone architectural parameters are outlined. This protocol also provides a section dedicated to in vivo scanning and analysis, which covers the topics of anaesthesia, radiation dose and image registration. Because of the expanding research using micro-CT to study other skeletal sites, as well as soft tissues, we also provide a review of current techniques to examine the skull and mandible, adipose tissue, vasculature, tumour severity and cartilage. Lists of recommended further reading and literature references are included to provide the reader with more detail on the methods described.
RESUMO
Insulin-like growth factor 1 (IGF-1) plays an important role in both bone metabolism and breast cancer. In this study, we investigated the effects of the novel IGF-1 receptor tyrosine kinase inhibitor cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP) on osteolytic bone disease associated with breast cancer. Human MDA-MB-231 and mouse 4T1 breast cancer cells enhanced osteoclast formation in receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) stimulated bone marrow cultures, and these effects were significantly inhibited by PQIP. Functional studies in osteoclasts showed that PQIP inhibited both IGF-1 and conditioned medium-induced osteoclast formation by preventing phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) activation without interfering with RANKL or M-CSF signaling. Treatment of osteoblasts with PQIP significantly inhibited the increase in RANKL/osteoprotegerin (OPG) ratio by IGF-1 and conditioned medium and totally prevented conditioned medium-induced osteoclast formation in osteoblast-bone marrow (BM) cell cocultures, thereby suggesting an inhibitory effect on osteoblast-osteoclast coupling. PQIP also inhibited IGF-1-induced osteoblast differentiation, spreading, migration, and bone nodule formation. Treatment with PQIP significantly reduced MDA-MB-231 conditioned medium-induced osteolytic bone loss in a mouse calvarial organ culture system ex vivo and in adult mice in vivo. Moreover, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following 4T1 intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in bone resorption and formation indices, indicative of a reduced rate of cancer-associated bone turnover. We conclude that inhibition of IGF-1 receptor tyrosine kinase activity by PQIP suppresses breast cancer-induced bone turnover and osteolysis. Therefore, PQIP, and its novel derivatives that are currently in advanced clinical development for the treatment of a number of solid tumors, may be of value in the treatment of osteolytic bone disease associated with breast cancer.
Assuntos
Remodelação Óssea , Neoplasias da Mama/patologia , Osteólise , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptor IGF Tipo 1/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imidazóis/farmacologia , Camundongos , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Pirazinas/farmacologiaRESUMO
The endocannabinoid system has recently been shown to play a role in the regulation of bone metabolism. The type 2 cannabinoid receptor (CB2) has been reported to regulate bone mass, but conflicting results have been reported with regard to its effects on bone resorption and osteoclast function. Here we investigated the role that CB2 plays in regulating bone mass and osteoclast function using a combination of pharmacological and genetic approaches. The CB2-selective antagonist/inverse agonist AM630 inhibited osteoclast formation and activity in vitro, whereas the CB2-selective agonists JWH133 and HU308 stimulated osteoclast formation. Osteoclasts generated from CB2 knockout mice (CB2-/-) were resistant to the inhibitory effects of AM630 in vitro, consistent with a CB2-mediated effect. There was no significant difference in peak bone mass between CB2-/- mice and wild-type littermates, but after ovariectomy, bone was lost to a greater extent in wild-type compared with CB2-/- mice. Furthermore, AM630 protected against bone loss in wild-type mice, but the effect was blunted in CB2-/- mice. We conclude that CB2 regulates osteoclast formation and bone resorption in vitro and that under conditions of increased bone turnover, such as after ovariectomy, CB2 regulates bone loss. These observations indicate that CB2 regulates osteoclast formation and contributes to ovariectomy-induced bone loss and demonstrate that cannabinoid receptor antagonists/inverse agonists may be of value in the treatment of bone diseases characterized by increased osteoclast activity.