RESUMO
The G12 family of G protein alpha subunits has been shown to participate in the regulation of various physiological processes. However, the role of Gα12 in bone physiology has not been well described. Here, by micro-CT analysis, we discovered that Gα12-knockout mice have an osteopetrotic phenotype. Histological examination showed lower osteoclast number in femoral tissue of Gα12-knockout mice compared to wild-type mice. Additionally, in vitro osteoclastic differentiation of precursor cells with receptor activator of nuclear factor-κB ligand (RANKL) showed that Gα12 deficiency decreased the number of osteoclast generated and the bone resorption activity. The induction of nuclear factor of activated T-cell c1 (NFATc1), the key transcription factor of osteoclastogenesis, and the activation of RhoA by RANKL was also significantly suppressed by Gα12 deficiency. We further found that the RANKL induction of NFATc1 was not dependent on RhoA signalling, while osteoclast precursor migration and bone resorption required RhoA in the Gα12-mediated regulation of osteoclasts. Therefore, Gα12 plays a role in differentiation through NFATc1 and in cell migration and resorption activity through RhoA during osteoclastogenesis.
Assuntos
Fatores de Transcrição NFATC/metabolismo , Animais , Células da Medula Óssea/metabolismo , Reabsorção Óssea/patologia , Diferenciação Celular/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Deleção de Genes , Humanos , Macrófagos/metabolismo , Masculino , Camundongos Knockout , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese , Osteopetrose/patologia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
A balance between bone formation and bone resorption is critical for the maintenance of bone mass. In many pathological conditions, including chronic inflammation, uncontrolled activation of osteoclast differentiation often causes excessive bone resorption that results in osteoporosis. In this study, we identified the osteopenia phenotype of mice lacking Usp18 (also called Ubp43), which is a deISGylating enzyme and is known as a negative regulator of type I IFN signaling. The expression of Usp18 was induced in preosteoclasts upon receptor activator of NF-κB ligand (RANKL) treatment. In an in vitro osteoclast-differentiation assay, bone marrow macrophages from Usp18-deficient mice exhibited an enhanced differentiation to multinucleated cells, elevated activation of NFATc1, and an increased expression of osteoclast marker genes upon RANKL treatment. Furthermore, in vitro quantification of bone resorption revealed a great increase in osteoclastic activities in Usp18-deficient cells. Interestingly, proinflammatory cytokine genes, such as IP-10 (CXCL10), were highly expressed in Usp18-deficient bone marrow macrophages upon RANKL treatment compared with wild-type cells. In addition, serum cytokine levels, especially IP-10, were significantly high in Usp18-knockout mice. In sum, we suggest that, although type I IFN is known to restrict osteoclast differentiation, the exaggerated activation of the type I IFN response in Usp18-knockout mice causes an osteopenia phenotype in mice.
Assuntos
Macrófagos/fisiologia , Osteoclastos/fisiologia , Osteogênese , Osteoporose/imunologia , Ubiquitina Tiolesterase/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Quimiocina CXCL10/metabolismo , Interferon Tipo I/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Osteogênese/genética , Osteogênese/imunologia , Ligante RANK/metabolismo , Ubiquitina Tiolesterase/genéticaRESUMO
Lipid raft microdomains have important roles in various cellular responses. Caveolae are a specialized type of lipid rafts that are stabilized by oligomers of caveolin proteins. Here, we show that caveolin-1 (Cav-1) plays a crucial role in the regulation of osteoclastogenesis. We found that caveolin-1 was dramatically up-regulated by receptor activator of nuclear factor κB ligand (RANKL), the osteoclast differentiation factor. Knockdown of Cav-1 reduced osteoclastogenesis and induction of NFATc1, the master transcription factor for osteoclastogenesis, by RANKL. Consistent with the in vitro results, injection of caveolin-1 siRNA onto mice calvariae showed reduction in RANKL-induced bone resorption and osteoclast formation. Moreover, Cav-1(-/-) female mice had higher bone volume and lower osteoclast number compared with wild type mice. However, Cav-1(-/-) male mice had both osteoclast and osteoblast numbers higher than wild type mice with no difference in bone volume. The sex dependence in the effect of Cav-1 deficiency was partly attributed to decreased receptor activator of nuclear factor κB and increased cFms expression in osteoclast precursors of female and male mice, respectively. Taken together, these data demonstrate that Cav-1 has a complicated but critical role for osteoclastogenesis.
Assuntos
Desenvolvimento Ósseo/genética , Caveolina 1/genética , Diferenciação Celular/genética , Osteoclastos/metabolismo , Animais , Caveolina 1/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Masculino , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Camundongos , Fatores de Transcrição NFATC/biossíntese , Osteoclastos/química , Osteogênese/genética , Ligante RANK/biossíntese , Caracteres SexuaisRESUMO
Tetraspanin family proteins regulate morphology, motility, fusion, and signaling in various cell types. We investigated the role of the tetraspanin 7 (Tspan7) isoform in the differentiation and function of osteoclasts. Tspan7 was up-regulated during osteoclastogenesis. When Tspan7 expression was reduced in primary precursor cells by siRNA-mediated gene knock-down, the generation of multinuclear osteoclasts was not affected. However, a striking cytoskeletal abnormality was observed: the formation of the podosome belt structure was inhibited and the microtubular network were disrupted by Tspan7 knock-down. Decreases in acetylated microtubules and levels of phosphorylated Src and Pyk2 in Tspan7 knock-down cells supported the involvement of Tspan7 in cytoskeletal rearrangement signaling in osteoclasts. This cytoskeletal defect interfered with sealing zone formation and subsequently the bone-resorbing activity of mature osteoclasts on dentin surfaces. Our results suggest that Tspan7 plays an important role in cytoskeletal organization required for the bone-resorbing function of osteoclasts by regulating signaling to Src, Pyk2, and microtubules.
Assuntos
Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Podossomos/metabolismo , Tetraspaninas/metabolismo , Animais , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Feminino , Camundongos , Osteogênese , Podossomos/patologiaRESUMO
Triggering receptor expressed on myeloid cells (TREM)-like transcript-1 (TLT-1) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-baring TREM family protein. In this study, we identified an alternative transcript form of TLT-1, namely TLT-1s, which has very short extracellular immunoglobulin domain consisting of only 202 amino acids. TLT-1s was mainly expressed in macrophages and osteoclast precursor cells. Upon receptor activator of nuclear factor-κB ligand stimulation, TLT-1s mRNA and protein levels were gradually decreased in BMMs. We also showed the TLT-1s is localized to the cytoplasmic membrane in osteoclast precursor cells. TLT-1s silencing strongly enhanced the formation and resorption activity of osteoclast. In addition, forced expression of TLT-1s showed reduced formation of osteoclast. Because ITIM-baring proteins inhibit immunoreceptor tyrosine-based activation motif (ITAM)-mediated receptor signaling, we tested whether TLT-1s physically interacted with TREM-2, the ITAM-associated co-stimulatory receptor essential for osteoclast differentiation. We showed that TLT-1s is associated with TREM-2 in osteoclast precursor cells. TLT-1s is also associated with tyrosine Src homology 2 domain-containing phosphatase-1 and SH2 domain-containing inositol phosphatase-1 and recruited them to the TREM2-ITAM signaling complex. In addition, knockdown of TLT-1s markedly elevated the intracellular calcium concentration and oscillation in osteoclast precursor cells. In addition, calcium-mediated induction of nuclear factor of activated T cells was also increased by TLT-1s silencing. Furthermore, TREM-2-mediated Akt activation and proliferation of osteoclast precursor cells were also enhanced in TLT-1s silenced cells. In this paper, we found the noble ITIM-baring inhibitory membrane protein; TLT-1s, which regulates ITAM-mediated signaling on osteoclastogenesis.
Assuntos
Processamento Alternativo/fisiologia , Glicoproteínas de Membrana/metabolismo , Osteoclastos/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais/fisiologia , Motivos de Aminoácidos , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Ativação Enzimática/fisiologia , Inativação Gênica , Glicoproteínas de Membrana/genética , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Osteoclastos/citologia , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Imunológicos/genética , Domínios de Homologia de srcRESUMO
Acne vulgaris is a common skin disease caused by multifactorial reasons involving excessive sebum secretion and inflammation by Cutibacterium acnes (C. acnes). Various conventional therapies are available for the treatment of acne vulgaris; however, topical photodynamic therapy (PDT) has attracted much attention because of its great potential for sebum-reducing, anti-inflammatory, and antimicrobial activities. Although 5-aminolevulinic acid (ALA) has been broadly used as a photosensitizer for topical PDT, it has several limitations such as long incubation time, pain, and post-inflammatory hyperpigmentation. Here, we report a biocompatible nanoformulation consisting of methylene blue and salicylic acid (MBSD), as a potent PDT and acne therapeutics, enclosed within oleic acid. Photoactivated MBSD showed antimicrobial activity against C. acnes along with long-term stability. When 24 patients with acne were treated with MBSD and light irradiation 5 times at 1-week intervals, MBSD-based PDT exhibited a remarkable reduction in acne lesions and sebum production. In addition, the therapeutic procedure was painless and safe, without any adverse events. Therefore, MBSD is a promising topical PDT agent for biocompatible, safe, and effective acne treatment.
Assuntos
Acne Vulgar , Anti-Infecciosos , Fotoquimioterapia , Humanos , Azul de Metileno/uso terapêutico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes , Ácido Aminolevulínico , Acne Vulgar/patologia , Resultado do Tratamento , Propionibacterium acnes , Anti-Infecciosos/uso terapêuticoRESUMO
Bone marrow-derived stem cells are self-renewing and multipotent adult stem cells that differentiate into several types of cells. Here, we investigated a unique combination of 4 differentiation-inducing factors (DIFs), including putrescine (Put), glucosamine (GlcN), nicotinamide, and BP-1-102, to develop a differentiation method for inducing mature insulin-producing cells (IPCs) and apply this method to bone marrow mononucleated cells (BMNCs) isolated from mice. BMNCs, primed with the 4 soluble DIFs, were differentiated into functional IPCs. BMNCs cultured under the defined conditions synergistically expressed multiple genes, including those for PDX1, NKX6.1, MAFA, NEUROG3, GLUT2, and insulin, related to pancreatic beta cell development and function. They produced insulin/C-peptide and PDX1, as assessed using immunofluorescence and flow cytometry. The induced cells secreted insulin in a glucose-responsive manner, similar to normal pancreatic beta cells. Grafting BMNC-derived IPCs under kidney capsules of mice with streptozotocin (STZ)-induced diabetes alleviated hyperglycemia by lowering blood glucose levels, enhancing glucose tolerance, and improving glucose-stimulated insulin secretion. Insulin- and PDX1-expressing cells were observed in the IPC-bearing graft sections of nephrectomized mice. Therefore, this study provides a simple protocol for BMNC differentiation, which can be a novel approach for cell-based therapy in diabetes mellitus.
Assuntos
Diabetes Mellitus Experimental , Células Secretoras de Insulina , Células-Tronco Mesenquimais , Camundongos , Animais , Medula Óssea , Diferenciação Celular , Glucose , Diabetes Mellitus Experimental/terapia , Insulina , Células da Medula ÓsseaRESUMO
A new type of heavy-atom-affected Pluronic (F-127) nanoparticle (FIC NP) for photodynamic therapy (PDT) is reported. FIC NPs are formulated with biocompatible constituents, and contain densely integrated iodinated aromatic molecules that form a structurally rigid core matrix and stably encapsulate photosensitizers in a monomeric form. Tiny nanoparticles (≈10 nm) are prepared by aqueous dispersion of photosensitizer-embedded aromatic nanodomains, which self-assemble by phase separation from the Pluronic melt mixture. By using spectroscopic studies and cellular experiments, the following is demonstrated: 1) enhanced singlet-oxygen generation by means of the intraparticle heavy-atom effect on the embedded photosensitizer, 2) facilitated cell uptake due to the small nanoscopic size as well as the Pluronic surface characteristics, and thereby 3) actual enhancement of PDT efficacy for a human breast-cancer cell line (MDA-MB-231), which validates a photophysically motivated nanoformulation approach toward an advanced photosensitizing nanomedicine.
Assuntos
Nanopartículas/química , Neoplasias/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , HumanosRESUMO
Indocyanine green (ICG) is a clinically approved dye that has shown great promise as a phototheranostic material with fluorescent, photoacoustic and photothermal responses in the near-infrared region. However, it has certain limitations, such as poor photostability and non-specific binding to serum proteins, subjected to rapid clearance and decreased theranostic efficacy in vivo. This study reports stable and biocompatible nanoparticles of ICG (ICG-Fe NPs) where ICG is electrostatically complexed with an endogenously abundant metal ion (Fe3+) and subsequently nanoformulated with a clinically approved polymer surfactant, Pluronic F127. Under near-infrared laser irradiation, ICG-Fe NPs were found to be more effective for photothermal temperature elevation than free ICG molecules owing to the improved photostability. In addition, ICG-Fe NPs showed the markedly enhanced tumor targeting and visualization with photoacoustic/fluorescent signaling upon intravenous injection, attributed to the stable metal complexation that prevents ICG-Fe NPs from releasing free ICG before tumor targeting. Under dual-modal imaging guidance, ICG-Fe NPs could successfully potentiate photothermal therapy of cancer by applying near-infrared laser irradiation, holding potential as a promising nanomedicine composed of all biocompatible ingredients for clinically relevant phototheranostics.
RESUMO
Hybrid nanostructures are promising for ultrasound-triggered drug delivery and treatment, called sonotheranostics. Structures based on plasmonic nanoparticles for photothermal-induced microbubble inflation for ultrasound imaging exist. However, they have limited therapeutic applications because of short microbubble lifetimes and limited contrast. Photochemistry-based sonotheranostics is an attractive alternative, but building near-infrared (NIR)-responsive echogenic nanostructures for deep tissue applications is challenging because photolysis requires high-energy (UV-visible) photons. Here, we report a photochemistry-based echogenic nanoparticle for in situ NIR-controlled ultrasound imaging and ultrasound-mediated drug delivery. Our nanoparticle has an upconversion nanoparticle core and an organic shell carrying gas generator molecules and drugs. The core converts low-energy NIR photons into ultraviolet emission for photolysis of the gas generator. Carbon dioxide gases generated in the tumor-penetrated nanoparticle inflate into microbubbles for sonotheranostics. Using different NIR laser power allows dual-modal upconversion luminescence planar imaging and cross-sectional ultrasonography. Low-frequency (10 MHz) ultrasound stimulated microbubble collapse, releasing drugs deep inside the tumor through cavitation-induced transport. We believe that the photoechogenic inflatable hierarchical nanostructure approach introduced here can have broad applications for image-guided multimodal theranostics.
Assuntos
Nanopartículas , Neoplasias , Humanos , Estudos Transversais , Microbolhas , Nanopartículas/química , Sistemas de Liberação de MedicamentosRESUMO
Osteoclasts (OCs), cells specialized for bone resorption, are generated from monocyte/macrophage precursors by a differentiation process governed by RANKL. Here, we show that DCTN1, a key component of the dynactin complex, plays important roles in OC differentiation. The expression of DCTN1 was upregulated by RANKL. The inhibition of DCTN1 expression by gene knockdown suppressed OC formation, bone resorption, and the induction of NFATc1 and c-Fos, critical transcription factors for osteoclastogenesis. More importantly, the activation of Cdc42 by RANKL was inhibited upon DCTN1 silencing. The forced expression of constitutively active Cdc42 restored the OC differentiation of precursors with DCTN1 deletion. In addition, PAK2 was found to be activated by RANKL and to function downstream of Cdc42. The DCTN1-Cdc42 axis also inhibited apoptosis and caspase-3 activation. Furthermore, the anti-osteoclastogenic effect of DCTN1 knockdown was verified in an animal model of bone erosion. Intriguingly, DCTN1 overexpression was also detrimental to OC differentiation, suggesting that DCTN1 should be regulated at the appropriate level for effective osteoclastogenesis. Collectively, our results reveal that DCTN1 participates in the activation of Cdc42/PAK2 signaling and the inhibition of apoptosis during osteoclastogenesis.
Assuntos
Complexo Dinactina/metabolismo , Osteoclastos/metabolismo , Osteogênese/fisiologia , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Apoptose/fisiologia , Reabsorção Óssea/metabolismo , Caspase 3/metabolismo , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Feminino , Macrófagos/metabolismo , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ligante RANK/metabolismo , Regulação para Cima/fisiologiaRESUMO
Fluorogenic nanoprobes capable of providing microenvironmental information have extensively been developed to improve the diagnostic accuracy for early or metastatic cancer detection. In cancer-associated microenvironment, matrix metalloproteinase-2,9 (MMP-2,9) has drawn attention as a representative enzymatic marker for diagnosis, prognosis, and prediction of various cancers, which is overexpressed in the primary site as well as metastatic regions. Here, we devised dual-emissive fluorogenic nanoprobe (DFNP) emitting both MMP-2,9-sensitive and insensitive fluorescence signals, for accurate monitoring of the MMP-2,9 activity in metastatic regions. DFNP was nanoscopically constructed by amphiphilic self-assembly between a constantly fluorescent polymer surfactant labeled with Cy7 (F127-Cy7) and an initially nonfluorescent hydrophobic peptide (Cy5.5-MMP-Q) that is fluorogenic in response to MMP-2,9. Ratiometric readout (Cy5.5/Cy7) by dual-channel imaging could normalize the enzyme-responsive sensing signal relative to the constantly emissive internal reference that reflects the probe amount, allowing for semi-quantitative analysis on the MMP-2,9-related tissue microenvironment. In addition to the dual-channel emission, the nanoconstructed colloidal structure of DFNP enabled efficient accumulation to lymph node in vivo. Because of these two colloidal characteristics, when injected intradermally to a mouse model of lymph node metastasis, DFNP could produce reliable ratiometric signals to provide information on the MMP-2,9 activity in the lymph nodes depending on metastatic progression, which corresponded well to the temporal histologic analysis. Furthermore, ratiometric lymph node imaging with DFNP after photodynamic therapy allowed for monitoring a therapeutic response to the given cancer treatment, demonstrating diagnostic and prognostic potential of the nanoconstructed colloidal sensor of tumor microenvironment in cancer treatment.
Assuntos
Diagnóstico por Imagem , Corantes Fluorescentes/química , Metástase Linfática/diagnóstico por imagem , Nanopartículas/química , Microambiente Tumoral , Animais , Carbocianinas/química , Linhagem Celular Tumoral , Fluorescência , Linfonodos/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/ultraestrutura , FototerapiaRESUMO
Transplantation of stem cell-derived insulin producing cells (IPCs) has been proposed as an alternative to islet transplantation for the treatment of diabetes mellitus. However, current IPC differentiation protocols are focused on generating functional cells from the pluripotent stem cells and tend to rely on multistep, long-term exposure to various exogenous factors. In this study, we addressed the observation that under stress, pancreatic ß-cells release essential components that direct the differentiation of the bone marrow nucleated cells (BMNCs) into IPCs. Without any supplementation with known differentiation-inducing factors, IPCs can be generated from BMNCs by in vitro priming for 6 days with conditioned media (CM) from the ß-cells. In vitro primed BMNCs expressed the ß-cell-specific transcription factors, as well as insulin, and improved hyperglycemia and glucose intolerance after transplantation into the streptozotocin-induced diabetic mice. Furthermore, we have found that components of the CM which trigger the differentiation were enclosed by or integrated into micro particles (MPs), rather than being secreted as soluble factors. Identification of these differentiation-directing factors might enable us to develop novel technologies required for the production of clinically applicable IPCs.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Animais , Biomarcadores , Glicemia , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Diabetes Mellitus Experimental , Imunofluorescência , Genes Reporter , Humanos , Insulina/biossíntese , Células Secretoras de Insulina/transplante , Células-Tronco Mesenquimais/citologia , CamundongosRESUMO
Reactive oxygen species (ROS) are an indispensable element of cellular signal transduction in various cell types, including bone cells. In particular, osteoclasts (OCs), cells specialized for bone resorption, utilize ROS as second messengers during receptor activator of NF-κB ligand (RANKL)-induced differentiation and activation. In addition, because of the high energy demands of bone-resorbing activity, OCs contain large amounts of mitochondria, the source of the majority of total ROS. In this study, we focused on the regulation of ROS generated from mitochondria during osteoclastogenesis. We observed that the level of mitochondrial superoxide dismutase 2 (SOD2), an enzyme responsible for reducing superoxide radicals in mitochondria, was increased by RANKL. siRNA-mediated knockdown (KD) of SOD2 increased ROS levels and enhanced OC differentiation. Conversely, overexpression of SOD2 reduced osteoclastogenesis by decreasing ROS levels. Moreover, we found that NAD-dependent deacetylase sirtuin 3 (Sirt3), an activator of SOD2 in mitochondria, was induced by RANKL. Sirt3-targeted siRNA decreased SOD2 activity by reducing deacetylation of lysine 68 of SOD2, leading to increased osteoclastogenesis. Furthermore, in vivo KD of SOD2 or Sirt3 in ICR mouse calvariae decreased bone volume and increased OC surface, supporting the results of in vitro experiments. Taken together, our findings demonstrate for the first time to our knowledge that the regulation of mitochondrial ROS by SOD2 and Sirt3 plays an important role in fine-tuning the OC differentiation program. © 2016 American Society for Bone and Mineral Research.
Assuntos
Osteoclastos/metabolismo , Osteogênese , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 3/metabolismo , Superóxido Dismutase/metabolismo , Acetilação , Animais , Reabsorção Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Lisina/metabolismo , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , RNA Interferente Pequeno/metabolismoRESUMO
Triggered cellular uptake of a synthetic graft copolymer carrying an anticancer drug is achieved through self-immolation of the side-chain azobenzene groups. In this concept, the conjugate is initially chemically neutral and does not possess cell-penetrating function. However, upon cleavage of the azobenzene moieties, a cascade process is initiated that ultimately reveals an ammonium cation in the vicinity of the polymer backbone. Hence, self-immolation results in the transformation of the neutral polymer chain into a polycation. This structural transformation allows the conjugate to be taken up by the cancer cells through favorable electrostatic interactions with the negatively charged phospholipid components of the cell membrane. Once inside the cells, the polymer releases covalently attached doxorubicin in a pristine form through a low pH activated release mechanism. The significance of this approach lies in the sensitivity of the azobenzene group to hypoxic conditions and to the enzyme azoreductase that is secreted by the microbial flora of the human colon and suggests a pathway to targeted drug delivery applications under these conditions.
RESUMO
Osteoclasts are responsible for the bone erosion associated with rheumatoid arthritis (RA). The upregulation of the chemokines CCL19 and CCL21 and their receptor CCR7 has been linked to RA pathogenesis. The purpose of this study was to evaluate the effects of CCL19 and CCL21 on osteoclasts and to reveal their underlying mechanisms. The expression of CCL19, CCL21 and CCR7 was higher in RA patients than in osteoarthritis patients. In differentiating osteoclasts, tumor necrosis factor-α, interleukin-1ß and lipopolysaccharide stimulated CCR7 expression. CCL19 and CCL21 promoted osteoclast migration and resorption activity. These effects were dependent on the presence of CCR7 and abolished by the inhibition of the Rho signaling pathway. CCL19 and CCL21 promoted bone resorption by osteoclasts in an in vivo mice calvarial model. These findings demonstrate for the first time that CCL19, CCL21 and CCR7 play important roles in bone destruction by increasing osteoclast migration and resorption activity. This study also suggests that the interaction of CCL19 and CCL21 with CCR7 is an effective strategic focus in developing therapeutics for alleviating inflammatory bone destruction.
Assuntos
Reabsorção Óssea/metabolismo , Movimento Celular , Quimiocina CCL19/metabolismo , Quimiocina CCL21/metabolismo , Osteoclastos/metabolismo , Receptores CCR7/metabolismo , Animais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Diferenciação Celular , Quimiocina CCL19/sangue , Quimiocina CCL21/sangue , Citocinas/metabolismo , Feminino , Humanos , Ligantes , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos ICR , Osteoartrite/metabolismo , Osteoartrite/patologia , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
PURPOSE: Minimally invasive treatments for early breast cancer have been reported. The objective of this study was to evaluate and compare two such treatments, laser ablation and photodynamic therapy (PDT), regarding their therapeutic efficacy for breast cancer. METHODS: Breast tumors were induced in 12 mice models. The treatment options were classified into four groups: control group (without any treatment, A), group treated only with laser ablation (B), group treated only with PDT (C), and group treated with the combination of laser ablation followed by PDT (D). The treatment effects were compared among these groups. RESULTS: Among the groups, the group D underwent the most effective treatment for breast cancer. Not only were the breast cancer cells necrotized by laser ablation, but the tumor margin was also managed by PDT. CONCLUSIONS: The treatment method combining laser ablation and PDT showed superior results to single treatment techniques using just one of these approaches.
Assuntos
Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Terapia a Laser/métodos , Fotoquimioterapia/métodos , Animais , Biópsia por Agulha , Terapia Combinada , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Fármacos Fotossensibilizantes/farmacologia , Projetos Piloto , Distribuição Aleatória , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Patients with inflammatory bone disease or cancer exhibit an increased risk of fractures and delayed bone healing. The S100A4 protein is a member of the calcium-binding S100 protein family, which is abundantly expressed in inflammatory diseases and cancers. We investigated the effects of extracellular S100A4 on osteoblasts, which are cells responsible for bone formation. Treating primary calvarial osteoblasts with recombinant S100A4 resulted in matrix mineralization reductions. The expression of osteoblast marker genes including osteocalcin and osterix was also suppressed. Interestingly, S100A4 stimulated the nuclear factor-kappaB (NF-κB) signaling pathway in osteoblasts. More importantly, the ex vivo organ culture of mouse calvariae with recombinant S100A4 decreased the expression levels of osteocalcin, supporting the results of our in vitro experiments. This suggests that extracellular S100A4 is important for the regulation of bone formation by activating the NF-κB signaling pathway in osteoblasts. [BMB Reports 2017; 50(2): 97-102].
Assuntos
Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Proteína A4 de Ligação a Cálcio da Família S100/farmacologia , Animais , Animais Recém-Nascidos , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Espaço Extracelular/química , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Nanocarriers (NCs) are a group of nano-sized vehicles devised to deliver drugs to targeted malignant tissues or organs that provide remarkably improved targeting efficiency and therapeutic efficacy for cancer therapy. A variety of NCs have been developed to accommodate appropriate loading and release of drugs with a wide spectrum of chemical and physical characteristics. In addition, physicochemical modifications to the surface or interior of NCs allow for modulation of pharmacokinetic features reflecting clinical demands. However, cancer-related mortality is still high and drug-mediated cancer treatment remains a challenging research field despite the remarkable advances in targeting efficiency and therapeutic efficacy resulting from NCs. In this review, we focus on typical approaches and recent trends in NC-mediated drug delivery systems and their potential for targeted cancer therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Animais , HumanosRESUMO
Current approaches in use of water-insoluble photosensitizers for photodynamic therapy (PDT) of cancer often demand a nano-delivery system. Here, we report a photosensitizer-loaded biocompatible nano-delivery formulation (PPaN-20) whose size was engineered to ca. 20nm to offer improved cell/tissue penetration and efficient generation of cytotoxic singlet oxygen. PPaN-20 was fabricated through the physical assembly of all biocompatible constituents: pyropheophorbide-a (PPa, water-insoluble photosensitizer), polycaprolactone (PCL, hydrophobic/biodegradable polymer), and Pluronic F-68 (clinically approved polymeric surfactant). Repeated microemulsification/evaporation method resulted in a fine colloidal dispersion of PPaN-20 in water, where the particulate PCL matrix containing well-dispersed PPa molecules inside was stabilized by the Pluronic corona. Compared to a control sample of large-sized nanoparticles (PPaN-200) prepared by a conventional solvent displacement method, PPaN-20 revealed optimal singlet oxygen generation and efficient cellular uptake by virtue of the suitably engineered size and constitution, leading to high in vitro phototoxicity against cancer cells. Upon administration to tumor-bearing mice by peritumoral route, PPaN-20 showed efficient tumor accumulation by the enhanced cell/tissue penetration evidenced by in vivo near-infrared fluorescence imaging. The in vivo PDT treatment with peritumorally administrated PPaN-20 showed significantly enhanced suppression of tumor growth compared to the control group, demonstrating great potential as a biocompatible photosensitizing agent for locoregional PDT treatment of cancer.