RESUMO
CCL2 is an inflammatory cytokine that regulates macrophage activity and is implicated in increased mammographic density and early breast tumorigenesis. The role of CCL2 in mediating stromal interactions that contribute to breast tumorigenesis has yet to be fully elucidated. THP-1-derived macrophages and mammary fibroblasts were co-cultured for 72 h. Fibroblasts and macrophages were analysed for phenotype, expression of inflammatory and ECM-regulatory genes and collagen production. Mice overexpressing CCL2 in the mammary glands were analysed for global gene expression by RNAseq at 12 weeks of age. These mice were cross-bred with PyMT mammary tumour mice to examine the role of CCL2 in tumorigenesis. The co-culture of macrophages with fibroblasts resulted in macrophage polarization towards an M2 phenotype, and upregulated expression of CCL2 and other genes associated with inflammation and ECM remodelling. CCL2 increased the production of insoluble collagen by fibroblasts. A global gene expression analysis of CCL2 overexpressing mice revealed that CCL2 upregulates cancer-associated gene pathways and downregulates fatty acid metabolism gene pathways. In the PyMT mammary tumour model, CCL2 overexpressing mice exhibited increased macrophage infiltration and early tumorigenesis. Interactions between macrophages and fibroblasts regulated by CCL2 can promote an environment that may increase breast cancer risk, leading to enhanced early tumorigenesis.
Assuntos
Quimiocina CCL2 , Neoplasias , Camundongos , Animais , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Macrófagos/metabolismo , Colágeno/metabolismo , Neoplasias/metabolismo , Carcinogênese/metabolismoRESUMO
Osteopetrosis is a heterogeneous group of rare hereditary diseases characterized by increased bone mass of poor quality. Autosomal-dominant osteopetrosis type II (ADOII) is most often caused by mutation of the CLCN7 gene leading to impaired bone resorption. Autosomal recessive osteopetrosis (ARO) is a more severe form and is frequently accompanied by additional morbidities. We report an adult male presenting with classical clinical and radiological features of ADOII. Genetic analyses showed no amino-acid-converting mutation in CLCN7 but an apparent haploinsufficiency and suppression of CLCN7 mRNA levels in peripheral blood mononuclear cells. Next generation sequencing revealed low-frequency intronic homozygous variations in CLCN7, suggesting recessive inheritance. In silico analysis of an intronic duplication c.595-120_595-86dup revealed additional binding sites for Serine- and Arginine-rich Splicing Factors (SRSF), which is predicted to impair CLCN7 expression. Quantitative backscattered electron imaging and histomorphometric analyses revealed bone tissue and material abnormalities. Giant osteoclasts were present and additionally to lamellar bone, and abundant woven bone and mineralized cartilage were observed, together with increased frequency and thickness of cement lines. Bone mineralization density distribution (BMDD) analysis revealed markedly increased average mineral content of the dense bone (CaMean T-score + 10.1) and frequency of bone with highest mineral content (CaHigh T-score + 19.6), suggesting continued mineral accumulation and lack of bone remodelling. Osteocyte lacunae sections (OLS) characteristics were unremarkable except for an unusually circular shape. Together, our findings suggest that the reduced expression of CLCN7 mRNA in osteoclasts, and possibly also osteocytes, causes poorly remodelled bone with abnormal bone matrix with high mineral content. This together with the lack of adequate bone repair mechanisms makes the material brittle and prone to fracture. While the skeletal phenotype and medical history were suggestive of ADOII, genetic analysis revealed that this is a possible mild case of ARO due to deep intronic mutation.
Assuntos
Canais de Cloreto , Osteopetrose , Canais de Cloreto/genética , Homozigoto , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Mutação , Osteopetrose/diagnóstico , Osteopetrose/genética , Osteopetrose/metabolismo , Fenótipo , RNA MensageiroRESUMO
Mammographic density is associated with a 4-6-fold increase in breast cancer risk independent of age and BMI. High mammographic density is characterized by breast tissue with high proportions of stroma comprised of fibroblasts, collagen, and immune cells. This study sought to investigate whether stromal fibroblasts from high mammographic density breast tissue contributes to increased extracellular matrix deposition and pro-tumorigenic signaling. Mammary fibroblasts were isolated from women with high and low mammographic density and exposed to immune factors myeloperoxidase (MPO), eosinophil peroxidase (EPO), transforming growth factor beta 1 (TGFB1) and tumour necrosis factor alpha (TNFA) for 72 h and profiled for expression of cancer-associated fibroblast and extracellular matrix regulation markers. No differences in gene expression profiles or collagen production were observed between fibroblasts with high or low mammographic density, and they did not have a differential response to immune mediators. MPO and EPO significantly increased the production of collagen 1. TGFB and TNFA induced variable changes in gene expression. Fibroblasts cultured in vitro from women with high mammographic density do not appear to be inherently different to those from women with low mammographic density. The function of fibroblasts in mammographic density-associated breast cancer risk is likely to be regulated by immune signals from surrounding cells in the microenvironment.
RESUMO
Mammographic density is an important risk factor for breast cancer; women with extremely dense breasts have a four to six fold increased risk of breast cancer compared to women with mostly fatty breasts, when matched with age and body mass index. High mammographic density is characterised by high proportions of stroma, containing fibroblasts, collagen and immune cells that suggest a pro-tumour inflammatory microenvironment. However, the biological mechanisms that drive increased mammographic density and the associated increased risk of breast cancer are not yet understood. Inflammatory factors such as monocyte chemotactic protein 1, peroxidase enzymes, transforming growth factor beta, and tumour necrosis factor alpha have been implicated in breast development as well as breast cancer risk, and also influence functions of stromal fibroblasts. Here, the current knowledge and understanding of the underlying biological mechanisms that lead to high mammographic density and the associated increased risk of breast cancer are reviewed, with particular consideration to potential immune factors that may contribute to this process.
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Near-infrared photoimmunotherapy (NIR-PIT) is a promising cancer therapy based on a monoclonal antibody conjugated to a photosensitizer (IR700Dye) that is activated by near-infrared light irradiation. We previously reported on the use of NIR-PIT with a small protein mimetic, the Affibody molecule (6-7 kDa), instead of a monoclonal antibody. In this study, we investigated a combination of NIR-PIT for HER2-positive breast cancer cells (SK-BR3, MDA-MB361, and JIMT1) with HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate. HER2 Affibody and trastuzumab target different epitopes of the HER2 protein and do not compete. In vitro, the combination of NIR-PIT using both HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate induced necrotic cell death of HER2-positive breast cancer cells without damage to HER2-negative breast cancer cells (MCF7). It was more efficient than NIR-PIT using either the HER2 Affibody-IR700Dye conjugate alone or the trastuzumab-IR700Dye conjugate alone. Additionally, this combination of NIR-PIT was significantly effective against HER2 low-expressing cancer cells, trastuzumab-resistant cells (JIMT1), and brain metastatic cells of breast cancer (MDA-MB361). Furthermore, in vivo imaging exhibited the strong fluorescence intensity of both HER2 Affibody-IR700Dye conjugates and trastuzumab-Alexa488 conjugates in HER2-positive tumor, indicating that both HER2 Affibody and trastuzumab specifically bind to HER2-positive tumors without competing with each other. In conclusion, the combination of NIR-PIT using both HER2 Affibody and trastuzumab expands the targeting scope of NIR-PIT for HER2-positive breast cancer.
Assuntos
Materiais Biomiméticos/farmacologia , Neoplasias da Mama/terapia , Imunoterapia , Fototerapia , Receptor ErbB-2/antagonistas & inibidores , Trastuzumab/farmacologia , Neoplasias da Mama/metabolismo , Feminino , Corantes Fluorescentes/farmacologia , Humanos , Células MCF-7 , Receptor ErbB-2/metabolismoRESUMO
PURPOSE: Early detection of tumor treatment responses represents an unmet clinical need with no approved noninvasive methods. DAB4, or its chimeric derivative, chDAB4 (APOMAB®) is an antibody that targets the Lupus associated antigen (La/SSB). La/SSB is over-expressed in malignancy and selectively targeted by chDAB4 in cancer cells dying from DNA-damaging treatment. Therefore, chDAB4 is a unique diagnostic tool that detects dead cancer cells and thus could distinguish between treatment responsive and nonresponsive patients. PROCEDURES: In clinically relevant tumor models, mice bearing subcutaneous xenografts of human ovarian or lung cancer cell lines or intraperitoneal ovarian cancer xenografts were untreated or given chemotherapy followed 24h later by chDAB4 radiolabeled with [89Zr]ZrIV. Tumor responses were monitored using bioluminescence imaging and caliper measurements. [89Zr]Zr-chDAB4 uptake in tumor and normal tissues was measured using an Albira SI Positron-Emission Tomography (PET) imager and its biodistribution was measured using a Hidex gamma-counter. RESULTS: Tumor uptake of [89Zr]Zr-chDAB4 was detected in untreated mice, and uptake significantly increased in both human lung and ovarian tumors after chemotherapy, but not in normal tissues. CONCLUSION: Given that tumors, rather than normal tissues, were targeted after chemotherapy, these results support the clinical development of chDAB4 as a radiodiagnostic imaging agent and as a potential predictive marker of treatment response.
Assuntos
Cisplatino , Neoplasias Ovarianas , Animais , Anticorpos Monoclonais , Morte Celular , Linhagem Celular Tumoral , Elétrons , Xenoenxertos , Humanos , Pulmão/patologia , Camundongos , Neoplasias Ovarianas/diagnóstico por imagem , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos , Distribuição Tecidual , ZircônioRESUMO
Bone defects arising from fractures or disease represent a significant problem for surgeons to manage and are a substantial economic burden on the healthcare economy. Recent advances in the development of biomaterial substitutes provides an attractive alternative to the current "gold standard" autologous bone grafting. Despite on-going research, we are yet to identify cost effective biocompatible, osteo-inductive factors that stimulate controlled, accelerated bone regeneration.We have recently reported that enzymes with peroxidase activity possess previously unrecognised roles in extracellular matrix biosynthesis, angiogenesis and osteoclastogenesis, which are essential processes in bone remodelling and repair. Here, we report for the first time, that plant-derived soybean peroxidase (SBP) possesses pro-osteogenic ability by promoting collagen I biosynthesis and matrix mineralization of human osteoblasts in vitro. Mechanistically, SBP regulates osteogenic genes responsible for inflammation, extracellular matrix remodelling and ossification, which are necessary for normal bone healing. Furthermore, SBP was shown to have osteo-inductive properties, that when combined with commercially available biphasic calcium phosphate (BCP) granules can accelerate bone repair in a critical size long bone defect ovine model. Micro-CT analysis showed that SBP when combined with commercially available biphasic calcium phosphate (BCP) granules significantly increased bone formation within the defects as early as 4 weeks compared to BCP alone. Histomorphometric assessment demonstrated accelerated bone formation prominent at the defect margins and surrounding individual BCP granules, with evidence of intramembranous ossification. These results highlight the capacity of SBP to be an effective regulator of osteoblastic function and may be beneficial as a new and cost effective osteo-inductive agent to accelerate repair of large bone defects.
RESUMO
BACKGROUND: Transforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates mammary gland development and cancer progression through endocrine, paracrine and autocrine mechanisms. TGFB1 also plays roles in tumour development and progression, and its increased expression is associated with an increased breast cancer risk. Macrophages are key target cells for TGFB1 action, also playing crucial roles in tumourigenesis. However, the precise role of TGFB-regulated macrophages in the mammary gland is unclear. This study investigated the effect of attenuated TGFB signalling in macrophages on mammary gland development and mammary cancer susceptibility in mice. METHODS: A transgenic mouse model was generated, wherein a dominant negative TGFB receptor is activated in macrophages, in turn attenuating the TGFB signalling pathway specifically in the macrophage population. The mammary glands were assessed for morphological changes through wholemount and H&E analysis, and the abundance and phenotype of macrophages were analysed through immunohistochemistry. Another cohort of mice received carcinogen 7,12-dimethylbenz(a)anthracene (DMBA), and tumour development was monitored weekly. Human non-neoplastic breast tissue was also immunohistochemically assessed for latent TGFB1 and macrophage marker CD68. RESULTS: Attenuation of TGFB signalling resulted in an increase in the percentage of alveolar epithelium in the mammary gland at dioestrus and an increase in macrophage abundance. The phenotype of macrophages was also altered, with inflammatory macrophage markers iNOS and CCR7 increased by 110% and 40%, respectively. A significant decrease in DMBA-induced mammary tumour incidence and prolonged tumour-free survival in mice with attenuated TGFB signalling were observed. In human non-neoplastic breast tissue, there was a significant inverse relationship between latent TGFB1 protein and CD68-positive macrophages. CONCLUSIONS: TGFB acts on macrophage populations in the mammary gland to reduce their abundance and dampen the inflammatory phenotype. TGFB signalling in macrophages increases mammary cancer susceptibility potentially through suppression of immune surveillance activities of macrophages.
Assuntos
Macrófagos/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , 9,10-Dimetil-1,2-benzantraceno/efeitos adversos , Animais , Suscetibilidade a Doenças , Intervalo Livre de Doença , Células Epiteliais/metabolismo , Ciclo Estral , Feminino , Humanos , Inflamação , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Proteína Smad2/metabolismoRESUMO
The androgen receptor (AR) is the key oncogenic driver of prostate cancer, and despite implementation of novel AR targeting therapies, outcomes for metastatic disease remain dismal. There is an urgent need to better understand androgen-regulated cellular processes to more effectively target the AR dependence of prostate cancer cells through new therapeutic vulnerabilities. Transcriptomic studies have consistently identified lipid metabolism as a hallmark of enhanced AR signaling in prostate cancer, yet the relationship between AR and the lipidome remains undefined. Using mass spectrometry-based lipidomics, this study reveals increased fatty acyl chain length in phospholipids from prostate cancer cells and patient-derived explants as one of the most striking androgen-regulated changes to lipid metabolism. Potent and direct AR-mediated induction of ELOVL fatty acid elongase 5 (ELOVL5), an enzyme that catalyzes fatty acid elongation, was demonstrated in prostate cancer cells, xenografts, and clinical tumors. Assessment of mRNA and protein in large-scale data sets revealed ELOVL5 as the predominant ELOVL expressed and upregulated in prostate cancer compared with nonmalignant prostate. ELOVL5 depletion markedly altered mitochondrial morphology and function, leading to excess generation of reactive oxygen species and resulting in suppression of prostate cancer cell proliferation, 3D growth, and in vivo tumor growth and metastasis. Supplementation with the monounsaturated fatty acid cis-vaccenic acid, a direct product of ELOVL5 elongation, reversed the oxidative stress and associated cell proliferation and migration effects of ELOVL5 knockdown. Collectively, these results identify lipid elongation as a protumorigenic metabolic pathway in prostate cancer that is androgen-regulated, critical for metastasis, and targetable via ELOVL5. SIGNIFICANCE: This study identifies phospholipid elongation as a new metabolic target of androgen action that is critical for prostate tumor metastasis.
Assuntos
Elongases de Ácidos Graxos/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , RNA Interferente Pequeno/uso terapêutico , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular/métodos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/farmacologia , Receptores Androgênicos/fisiologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Female mice heterozygous for a genetic mutation in transcription factor forkhead box p3 (Foxp3) spontaneously develop mammary cancers; however, the underlying mechanism is not well understood. We hypothesised that increased cancer susceptibility is associated with an underlying perturbation in mammary gland development. The role of Foxp3 in mammary ductal morphogenesis was investigated in heterozygous Foxp3Sf/+ and wildtype Foxp3+/+ mice during puberty and at specific stages of the oestrous cycle. No differences in mammary ductal branching morphogenesis, terminal end bud formation or ductal elongation were observed in pubertal Foxp3Sf/+ mice compared with Foxp3+/+ mice. During adulthood, all mice underwent normal regular oestrous cycles. No differences in epithelial branching morphology were detected in mammary glands from mice at the oestrus, metoestrus, dioestrus and pro-oestrus stages of the cycle. Furthermore, abundance of Foxp3 mRNA and protein in the mammary gland and lymph nodes was not altered in Foxp3Sf/+ mice compared with Foxp3+/+ mice. These studies suggest that Foxp3 heterozygosity does not overtly affect mammary gland development during puberty or the oestrous cycle. Further studies are required to dissect the underlying mechanisms of increased mammary cancer susceptibility in Foxp3Sf/+ heterozygous mice and the function of this transcription factor in normal mammary gland development.
Assuntos
Ciclo Estral/fisiologia , Fatores de Transcrição Forkhead/genética , Heterozigoto , Glândulas Mamárias Animais/crescimento & desenvolvimento , Mutação , Maturidade Sexual/fisiologia , Animais , Feminino , Fatores de Transcrição Forkhead/fisiologia , Linfonodos/química , Glândulas Mamárias Animais/química , Neoplasias Mamárias Animais/genética , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese/fisiologia , RNA Mensageiro/análiseRESUMO
Near-infrared photoimmunotherapy (NIR-PIT) is a new and promising cancer therapy based on a monoclonal antibody conjugated to a photosensitizer which is activated by near-infrared light irradiation, causing cell death. We investigated NIR-PIT using a small protein mimetic (6-7 kDa), Affibody molecules, instead of a monoclonal antibody for HER2-overexpressing cancer. Because of its small size, the Affibody has rapid clearance, high imaging contrast, and good tumor penetration. Due to the small size of the Affibodies, which can cross the blood-brain barrier, NIR-PIT using Affibodies has the potential to extend the target cancer of NIR-PIT, including brain metastases. In vitro, NIR-PIT using HER2 Affibody-IR700Dye conjugates induced the selective destruction of HER2-overexpressing breast cancer cells without damage to control cells having low level expression of HER2. HER2-overexpressing cancer cells showed necrotic cell death and their viability maintained at low levels, even 5 days after NIR-PIT. In contrast, treatment with high concentration of HER2 Affibody-IR700Dye conjugate alone or irradiation with high dose of NIR light alone was without effect on cell viability. Affibody and IR700Dye are currently used clinically, and therefore, we would expect the current formulation to be safely and quickly transitioned into clinical trials.
Assuntos
Anticorpos Monoclonais/química , Antineoplásicos/química , Neoplasias da Mama/terapia , Imunoterapia/métodos , Fragmentos de Peptídeos/química , Fototerapia/métodos , Receptor ErbB-2/metabolismo , Anticorpos Monoclonais/imunologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Biomimética , Linhagem Celular Tumoral , Feminino , Humanos , Raios Infravermelhos , Fragmentos de Peptídeos/farmacologia , Fármacos Fotossensibilizantes/química , Ligação Proteica , Receptor ErbB-2/imunologiaRESUMO
Multiple myeloma is a fatal plasma cell malignancy that is reliant on the bone marrow microenvironment. The bone marrow is comprised of numerous cells of mesenchymal and hemopoietic origin. Of these, macrophages have been implicated to play a role in myeloma disease progression, angiogenesis, and drug resistance; however, the role of macrophages in myeloma disease establishment remains unknown. In this study, the antimyeloma efficacy of clodronate-liposome treatment, which globally and transiently depletes macrophages, was evaluated in the well-established C57BL/KaLwRijHsd murine model of myeloma. Our studies show, for the first time, that clodronate-liposome pretreatment abrogates myeloma tumor development in vivo. Clodronate-liposome administration resulted in depletion of CD169+ bone marrow-resident macrophages. Flow cytometric analysis revealed that clodronate-liposome pretreatment impaired myeloma plasma cell homing and retention within the bone marrow 24â¯hours postmyeloma plasma cell inoculation. This was attributed in part to decreased levels of macrophage-derived insulin-like growth factor 1. Moreover, a single dose of clodronate-liposome led to a significant reduction in myeloma tumor burden in KaLwRij mice with established disease. Collectively, these findings support a role for CD169-expressing bone marrow-resident macrophages in myeloma disease establishment and progression and demonstrate the potential of targeting macrophages as a therapy for myeloma patients.
Assuntos
Conservadores da Densidade Óssea/administração & dosagem , Ácido Clodrônico/administração & dosagem , Suscetibilidade a Doenças , Lipossomos , Macrófagos/imunologia , Macrófagos/metabolismo , Mieloma Múltiplo/etiologia , Mieloma Múltiplo/metabolismo , Animais , Biomarcadores , Contagem de Células , Linhagem Celular Tumoral , Movimento Celular , Modelos Animais de Doenças , Imunofenotipagem , Camundongos , Mieloma Múltiplo/patologia , Osteoblastos , Microambiente TumoralRESUMO
Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia also leads to treatment opportunities as demonstrated by the development of compounds that target regions of hypoxia within tumors. Evofosfamide is a hypoxia-activated prodrug that is created by linking the hypoxia-seeking 2-nitroimidazole moiety to the cytotoxic bromo-isophosphoramide mustard (Br-IPM). When evofosfamide is delivered to hypoxic regions of tumors, the DNA cross-linking toxin, Br-IPM, is released leading to cell death. This study assessed the anticancer efficacy of evofosfamide in combination with the Proapoptotic Receptor Agonists (PARAs) dulanermin and drozitumab against human osteosarcoma in vitro and in an intratibial murine model of osteosarcoma. Under hypoxic conditions in vitro, evofosfamide cooperated with dulanermin and drozitumab, resulting in the potentiation of cytotoxicity to osteosarcoma cells. In contrast, under the same conditions, primary human osteoblasts were resistant to treatment. Animals transplanted with osteosarcoma cells directly into their tibiae developed mixed osteosclerotic/osteolytic bone lesions and consequently developed lung metastases 3 weeks post cancer cell transplantation. Tumor burden in the bone was reduced by evofosfamide treatment alone and in combination with drozitumab and prevented osteosarcoma-induced bone destruction while also reducing the growth of pulmonary metastases. These results suggest that evofosfamide may be an attractive therapeutic agent, with strong anticancer activity alone or in combination with either drozitumab or dulanermin against osteosarcoma.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Nitroimidazóis/administração & dosagem , Osteossarcoma/tratamento farmacológico , Mostardas de Fosforamida/administração & dosagem , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/farmacologia , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Primary and secondary bone cancers are major causes of pathological bone fractures which are usually treated through implant fixation and chemotherapy. However, both approaches face many limitations. On one hand, implants may suffer from poor osseointegration, and their rejection results in repeated surgery, patient's suffering, and extensive expenses. On the other hand, there are severe systemic adverse effects of toxic chemotherapeutics which are administrated systemically. In this paper, in order to address these two problems, we present a new type of localized drug-releasing titanium implants with enhanced implants' biointegration and drug release capabilities that could provide a high concentration of anticancer drugs locally to treat bone cancers. The implants are fabricated by 3D printing of Ti alloy followed by an anodization process featuring unique micro- (particles) and nanosurface (tubular arrays) topography. We successfully demonstrate their enhanced bone osseointegration and drug loading capabilities using two types of anticancer drugs, doxorubicin (DOX) and apoptosis-inducing ligand (Apo2L/TRAIL). In vitro study showed strong anticancer efficacy against cancer cells (MDA-MB-231-TXSA), confirming that these drug-releasing implants can be used for localized chemotherapy for treatment of primary and secondary bone cancers together with fracture support.
Assuntos
Próteses e Implantes , Liberação Controlada de Fármacos , Nanotubos , Osseointegração , Propriedades de Superfície , TitânioRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Coriolus versicolor (CV) is a mushroom traditionally used for strengthening the immune system and nowadays used as immunomodulatory adjuvant in anticancer therapy. Breast cancer usually metastasizes to the skeleton, interrupts the normal bone remodeling process and causes osteolytic bone lesions. The aims of the present study were to evaluate its herb-drug interaction with metronomic zoledronate in preventing cancer propagation, metastasis and bone destruction. MATERIALS AND METHODS: Mice inoculated with human breast cancer cells tagged with a luciferase (MDA-MB-231-TXSA) in tibia were treated with CV aqueous extract, mZOL, or the combination of both for 4 weeks. Alteration of the luciferase signals in tibia, liver and lung were quantified using the IVIS imaging system. The skeletal response was evaluated using micro-computed tomography (micro-CT). In vitro experiments were carried out to confirm the in vivo findings. RESULTS: Results showed that combination of CV and mZOL diminished tumor growth without increasing the incidence of lung and liver metastasis in intratibial breast tumor model. The combination therapy also reserved the integrity of bones. In vitro studies demonstrated that combined use of CV and mZOL inhibited cancer cell proliferation and osteoclastogenesis. CONCLUSIONS: These findings suggested that combination treatment of CV and mZOL attenuated breast tumor propagation, protected against osteolytic bone lesion without significant metastases. This study provides scientific evidences on the beneficial outcome of using CV together with mZOL in the management of breast cancer and metastasis, which may lead to the development of CV as adjuvant health supplement for the control of breast cancer.
Assuntos
Agaricales , Conservadores da Densidade Óssea/administração & dosagem , Neoplasias Ósseas/tratamento farmacológico , Difosfonatos/administração & dosagem , Imidazóis/administração & dosagem , Neoplasias Mamárias Animais/tratamento farmacológico , Administração Metronômica , Agaricales/química , Animais , Conservadores da Densidade Óssea/uso terapêutico , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Difosfonatos/uso terapêutico , Feminino , Humanos , Imidazóis/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Animais/diagnóstico por imagem , Neoplasias Mamárias Animais/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Osteoclastos/efeitos dos fármacos , Tíbia/diagnóstico por imagem , Tíbia/efeitos dos fármacos , Tíbia/patologia , Ácido ZoledrônicoRESUMO
Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, well known for their antimicrobial activity, are released in high quantities by infiltrating immune cells in breast cancer. However, the functional importance of their presence within the tumour microenvironment is unclear. We have recently described a new role for peroxidases as key regulators of fibroblast and endothelial cell functionality. In the present study, we investigate for the first time, the ability of peroxidases to promote breast cancer development and progression. Using the 4T1 syngeneic murine orthotopic breast cancer model, we examined whether increased levels of peroxidases in developing mammary tumours influences primary tumour growth and metastasis. We showed that MPO and EPO stimulation increased mammary tumour growth and enhanced lung metastases, effects that were associated with reduced tumour necrosis, increased collagen deposition and neo-vascularisation within the primary tumour. In vitro, peroxidase treatment, robustly stimulated human mammary fibroblast migration and collagen type I and type VI secretion. Mechanistically, peroxidases induced the transcription of pro-tumorigenic and metastatic MMP1, MMP3 and COX-2 genes. Taken together, these findings identify peroxidases as key contributors to cancer progression by augmenting pro-tumorigenic collagen production and angiogenesis. Importantly, this identifies inflammatory peroxidases as therapeutic targets in breast cancer therapy.
Assuntos
Neoplasias da Mama/metabolismo , Peroxidase de Eosinófilo/metabolismo , Neoplasias Pulmonares/secundário , Neovascularização Patológica/metabolismo , Peroxidase/metabolismo , Proteínas Recombinantes/metabolismo , Microambiente Tumoral , Animais , Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Movimento Celular , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Ciclo-Oxigenase 2/metabolismo , Progressão da Doença , Feminino , Fibroblastos , Humanos , Neoplasias Mamárias Experimentais , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Cultura Primária de CélulasRESUMO
BACKGROUND: Macrophages play diverse roles in mammary gland development and breast cancer. CC-chemokine ligand 2 (CCL2) is an inflammatory cytokine that recruits macrophages to sites of injury. Although CCL2 has been detected in human and mouse mammary epithelium, its role in regulating mammary gland development and cancer risk has not been explored. METHODS: Transgenic mice were generated wherein CCL2 is driven by the mammary epithelial cell-specific mouse mammary tumour virus 206 (MMTV) promoter. Estrous cycles were tracked in adult transgenic and non-transgenic FVB mice, and mammary glands collected at the four different stages of the cycle. Dissected mammary glands were assessed for cyclical morphological changes, proliferation and apoptosis of epithelium, macrophage abundance and collagen deposition, and mRNA encoding matrix remodelling enzymes. Another cohort of control and transgenic mice received carcinogen 7,12-Dimethylbenz(a)anthracene (DMBA) and tumour development was monitored weekly. CCL2 protein was also quantified in paired samples of human breast tissue with high and low mammographic density. RESULTS: Overexpression of CCL2 in the mammary epithelium resulted in an increased number of macrophages, increased density of stroma and collagen and elevated mRNA encoding matrix remodelling enzymes lysyl oxidase (LOX) and tissue inhibitor of matrix metalloproteinases (TIMP)3 compared to non-transgenic controls. Transgenic mice also exhibited increased susceptibility to development of DMBA-induced mammary tumours. In a paired sample cohort of human breast tissue, abundance of epithelial-cell-associated CCL2 was higher in breast tissue of high mammographic density compared to tissue of low mammographic density. CONCLUSIONS: Constitutive expression of CCL2 by the mouse mammary epithelium induces a state of low level chronic inflammation that increases stromal density and elevates cancer risk. We propose that CCL2-driven inflammation contributes to the increased risk of breast cancer observed in women with high mammographic density.
Assuntos
Neoplasias da Mama/etiologia , Neoplasias da Mama/metabolismo , Quimiocina CCL2/metabolismo , Suscetibilidade a Doenças , Inflamação/metabolismo , Células Estromais/metabolismo , Animais , Densidade da Mama , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Quimiocina CCL2/genética , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Modelos Animais de Doenças , Epitélio/metabolismo , Ciclo Estral , Feminino , Expressão Gênica , Humanos , Inflamação/genética , Inflamação/patologia , Estimativa de Kaplan-Meier , Macrófagos/imunologia , Macrófagos/metabolismo , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Neoplasias Mamárias Experimentais , Camundongos , Camundongos Transgênicos , Prognóstico , RNA Mensageiro/genética , Células Estromais/patologiaRESUMO
Bone metastases occur in over 75% of patients with advanced breast cancer and are responsible for high levels of morbidity and mortality. In this study, ex vivo expanded cytotoxic Vγ9Vδ2 T cells isolated from human peripheral blood were tested for their anti-cancer efficacy in combination with zoledronic acid (ZOL), using a mouse model of osteolytic breast cancer. In vitro, expanded Vγ9Vδ2 T cells were cytotoxic against a panel of human breast cancer cell lines, and ZOL pre-treatment further sensitised breast cancer cells to killing by Vγ9Vδ2 T cells. Vγ9Vδ2 T cells adoptively transferred into NOD/SCID mice localised to osteolytic breast cancer lesions in the bone, and multiple infusions of Vγ9Vδ2 T cells reduced tumour growth in the bone. ZOL pre-treatment potentiated the anti-cancer efficacy of Vγ9Vδ2 T cells, with mice showing further reductions in tumour burden. Mice treated with the combination also had reduced tumour burden of secondary pulmonary metastases, and decreased bone degradation. Our data suggests that adoptive transfer of Vγ9Vδ2 T cell in combination with ZOL may prove an effective immunotherapeutic approach for the treatment of breast cancer bone metastases.
Assuntos
Antineoplásicos/farmacologia , Conservadores da Densidade Óssea/farmacologia , Neoplasias Ósseas/prevenção & controle , Neoplasias da Mama/terapia , Difosfonatos/farmacologia , Imidazóis/farmacologia , Imunoterapia Adotiva/métodos , Neoplasias Pulmonares/prevenção & controle , Osteólise/prevenção & controle , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T Citotóxicos/transplante , Animais , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/secundário , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Citotoxicidade Imunológica , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Ativação Linfocitária , Camundongos Endogâmicos NOD , Camundongos SCID , Osteólise/imunologia , Osteólise/patologia , Fenótipo , Linfócitos T Citotóxicos/imunologia , Fatores de Tempo , Ensaios Antitumorais Modelo de Xenoenxerto , Ácido ZoledrônicoRESUMO
Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, well known for their antimicrobial activity, are released in abundance by innate immune infiltrates at sites of inflammation and injury. We have discovered new and previously unrecognised roles for heme peroxidases in extracellular matrix biosynthesis, angiogenesis, and bone mineralisation, all of which play an essential role in skeletal integrity. In this study we used in vitro models of osteoclastogenesis to investigate the effects of heme peroxidase enzymes on osteoclast differentiation and bone resorbing activity, pertinent to skeletal development and remodelling. Receptor activator of nuclear factor kappa B-ligand (RANKL) stimulates the formation of tartate-resistant acid phosphatase (TRAP) positive multinucleated cells and increases bone resorption when cultured with human peripheral blood mononuclear cells (PBMCs) or the RAW264.7 murine monocytic cell line. When RANKL was added in combination with either MPO or EPO, a dose-dependent inhibition of osteoclast differentiation and bone resorption was observed. Notably, peroxidases had no effect on the bone resorbing activity of mature osteoclasts, suggesting that the inhibitory effect of the peroxidases was limited to osteoclast precursor cells. Mechanistically, we observed that osteoclast precursor cells readily internalize peroxidases, and inhibited the phosphorylation of JNK, p38 MAPK and ERK1/2, important signalling molecules central to osteoclastogenesis. Our findings suggest that peroxidase enzymes, like MPO and EPO, may play a fundamental role in inhibiting RANKL-induced osteoclast differentiation at inflammatory sites of bone fracture and injury. Therefore, peroxidase enzymes could be considered as potential therapeutic agents to treat osteolytic bone disease and aberrant bone resorption.