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A novel drug delivery system preventing Glioblastoma multiforme (GBM) recurrence after resection surgery is imperatively required to overcome the mechanical limitation of the current local drug delivery system and to offer personalised treatment options for GBM patients. In this study, 3D printed biodegradable flexible porous scaffolds were developed via Fused Deposition Modelling (FDM) three-dimensional (3D) printing technology for the local delivery of curcumin. The flexible porous scaffolds were 3D printed with various geometries containing 1, 3, 5, and 7% (w/w) of curcumin, respectively, using curcumin-loaded polycaprolactone (PCL) filaments. The scaffolds were characterised by a series of characterisation studies and in vitro studies were also performed including drug release study, scaffold degradation study, and cytotoxicity study. The curcumin-loaded PCL scaffolds displayed versatile spatiotemporal characteristics. The polymeric scaffolds obtained great mechanical flexibility with a low tensile modulus of less than 2 MPa, and 4 to 7-fold ultimate tensile strain, which can avoid the mechanical mismatch problem of commercially available GLIADEL wafer with a further improvement in surgical margin coverage. In vitro release profiles have demonstrated the sustained release patterns of curcumin with adjustable release amounts and durations up to 77 h. MTT study has demonstrated the great cytotoxic effect of curcumin-loaded scaffolds against the U87 human GBM cell line. Therefore, 3D printed curcumin-loaded scaffold has great promise to provide better GBM treatment options with its mechanical flexibility and customisability to match individual needs, preventing post-surgery GBM recurrence and eventually prolonging the life expectancy of GBM patients.
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Growth plate cartilage injuries often result in bony repair at the injury site and premature mineralisation at the uninjured region causing bone growth defects, for which underlying mechanisms are unclear. With the prior microarray study showing upregulated bone morphogenetic protein (BMP) signalling during the injury site bony repair and with the known roles of BMP signalling in bone healing and growth plate endochondral ossification, this study used a rat tibial growth plate drill-hole injury model with or without systemic infusion of BMP antagonist noggin to investigate roles of BMP signalling in injury repair responses within the injury site and in the adjacent "uninjured" cartilage. At days 8, 14 and 35 post-injury, increased expression of BMP members and receptors and enhanced BMP signalling (increased levels of phosphorylated (p)-Smad1/5/8) were found during injury site bony repair. After noggin treatment, injury site bony repair at days 8 and 14 was reduced as shown by micro-CT and histological analyses and lower mRNA expression of osteogenesis-related genes Runx2 and osteocalcin (by RT-PCR). At the adjacent uninjured cartilage, the injury caused increases in the hypertrophic zone/proliferative zone height ratio and in mRNA expression of hypertrophy marker collagen-10, but a decrease in chondrogenesis marker Sox9 at days 14 and/or 35, which were accompanied by increased BMP signalling (increased levels of pSmad1/5/8 protein and BMP7, BMPR1a and target gene Dlx5 mRNA). Noggin treatment reduced the hypertrophic zone/proliferative zone height ratio and collagen-10 mRNA expression, but increased collagen-2 mRNA levels at the adjacent growth plate. This study has identified critical roles of BMP signalling in the injury site bony repair and in the hypertrophic degeneration of the adjacent growth plate in a growth plate drill-hole repair model. Moreover, suppressing BMP signalling can potentially attenuate the undesirable bony repair at injury site and suppress the premature hypertrophy but potentially rescue chondrogenesis at the adjacent growth plate.
Assuntos
Lâmina de Crescimento , Fraturas Salter-Harris , Animais , Cartilagem , Osteogênese , Ratos , Ratos Sprague-DawleyRESUMO
Delta inulin, also known as microparticulate inulin (MPI), was modified by covalently attaching doxorubicin to its nanostructured surface for use as a targeted drug delivery vehicle. MPI is readily endocytosed by monocytes, macrophages, and dendritic cells and in this study, we sought to utilize this property to develop a system to target anti-cancer drugs to lymphoid organs. We investigated, therefore, whether MPI could be used as a vehicle to deliver doxorubicin selectively, thereby reducing the toxicity of this antibiotic anthracycline drug. Doxorubicin was covalently attached to the surface of MPI using an acid-labile linkage to enable pH-controlled release. The MPI-doxorubicin conjugate was characterized using FTIR and SEM, confirming covalent attachment and indicating doxorubicin coupling had no obvious impact on the physical nanostructure, integrity, and cellular uptake of the MPI particles. To simulate the stability of the MPI-doxorubicin in vivo, it was stored in artificial lysosomal fluid (ALF, pH 4.5). Although the MPI-doxorubicin particles were still visible after 165 days in ALF, 53% of glycosidic bonds in the inulin particles were hydrolyzed within 12 days in ALF, reflected by the release of free glucose into solution. By contrast, the fructosidic bonds were much more stable. Drug release studies of the MPI-doxorubicin in vitro, demonstrated a successful pH-dependent controlled release effect. Confocal laser scanning microscopy studies and flow cytometric analysis confirmed that when incubated with live cells, MPI-doxorubicin was efficiently internalized by immune cells. An assay of cell metabolic activity demonstrated that the MPI carrier alone had no toxic effects on RAW 264.7 murine monocyte/macrophage-like cells, but exhibited anti-cancer effects against HCT116 human colon cancer cells. MPI-doxorubicin had a greater anti-cancer cell effect than free doxorubicin, particularly when at lower concentrations, suggesting a drug-sparing effect. This study establishes that MPI can be successfully modified with doxorubicin for chemotherapeutic drug delivery.
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The use of particles for monocyte-mediated delivery could be a more efficient strategy and approach to achieve intracellular targeting and delivery of antitubercular drugs to host macrophages. In this study, the potential of inulin microparticles to serve as a drug vehicle in the treatment of chronic tuberculosis using a monocytes-mediated drug targeting approach was evaluated. Isoniazid (INH) was conjugated to inulin via hydrazone linkage in order to obtain a pH-sensitive inulin-INH conjugate. The conjugate was then characterized using proton nuclear magnetic resonance (1HNMR), Fourier transform infrared spectroscopy (FTIR) as well as in vitro, cellular uptake and intracellular Mycobacterium tuberculosis (Mtb) antibacterial efficacy. The acid-labile hydrazone linkage conferred pH sensitivity to the inulin-INH conjugate with ~95, 77 and 65% of the drug released after 5 h at pH 4.5, 5.2, and 6.0 respectively. Cellular uptake studies confirm that RAW 264.7 monocytic cells efficiently internalized the inulin conjugates into endocytic compartments through endocytosis. The intracellular efficacy studies demonstrate that the inulin conjugates possess a dose-dependent targeting effect against Mtb-infected monocytes. This was through efficient internalization and cleavage of the hydrazone bond by the acidic environment of the lysosome, which subsequently released the isoniazid intracellularly to the Mtb reservoir. These results clearly suggest that inulin conjugates can serve as a pH-sensitive intracellular drug delivery system for TB treatment.
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The propensity of monocytes to migrate into sites of mycobacterium tuberculosis (TB) infection and then become infected themselves makes them potential targets for delivery of drugs intracellularly to the tubercle bacilli reservoir. Conventional TB drugs are less effective because of poor intracellular delivery to this bacterial sanctuary. This study highlights the potential of using semicrystalline delta inulin particles that are readily internalised by monocytes for a monocyte-based drug delivery system. Pyrazinoic acid was successfully attached covalently to the delta inulin particles via a labile linker. The formation of new conjugate and amide bond was confirmed using zeta potential, Proton Nuclear Magnetic Resonance (1HNMR) and Fourier transform infrared spectroscopy (FTIR). Scanning electron microscopy (SEM) confirmed that no significant change in size after conjugation which is an important parameter for monocyte targeting. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were used to establish the change in thermal properties. The analysis of in-vitro release demonstrated pH-triggered drug cleavage off the delta inulin particles that followed a first-order kinetic process. The efficient targeting ability of the conjugate for RAW 264.7 monocytic cells was supported by cellular uptake studies. Overall, our finding confirmed that semicrystalline delta inulin particles (MPI) can be modified covalently with drugs and such conjugates allow intracellular drug delivery and uptake into monocytes, making this system potentially useful for the treatment of TB.
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Dexamethasone (Dex) is known to cause significant bone growth impairment in childhood. Although previous studies have suggested roles of osteocyte apoptosis in the enhanced osteoclastic recruitment and local bone loss, whether it is so in the growing bone following Dex treatment requires to be established. The current study addressed the potential roles of chemokine CXCL12 in chondroclast/osteoclast recruitment and bone defects following Dex treatment. Significant apoptosis was observed in cultured mature ATDC5 chondrocytes and IDG-SW3 osteocytes after 48 hours of 10-6 M Dex treatment, and CXCL12 was identified to exhibit the most prominent induction in Dex-treated cells. Conditioned medium from the treated chondrocytes/osteocytes enhanced migration of RAW264.7 osteoclast precursor cells, which was significantly inhibited by the presence of the anti-CXCL12 neutralizing antibody. To investigate the roles of the induced CXCL12 in bone defects caused by Dex treatment, young rats were orally gavaged daily with saline or Dex at 1 mg/kg/day for 2 weeks, and received an intraperitoneal injection of anti-CXCL12 antibody or control IgG (1 mg/kg, three times per week). Aside from oxidative stress induction systemically, Dex treatment caused reductions in growth plate thickness, primary spongiosa height, and metaphysis trabecular bone volume, which are associated with induced chondrocyte/osteocyte apoptosis and enhanced chondroclast/osteoclast recruitment and osteoclastogenic differentiation potential. CXCL12 was induced in apoptotic growth plate chondrocytes and metaphyseal bone osteocytes. Anti-CXCL12 antibody supplementation considerably attenuated Dex-induced chondroclast/osteoclast recruitment and loss of growth plate cartilage and trabecular bone. CXCL12 neutralization did not affect bone marrow osteogenic potential, adiposity, and microvasculature. Thus, CXCL12 was identified as a potential molecular linker between Dex-induced skeletal cell apoptosis and chondroclastic/osteoclastic recruitment, as well as growth plate cartilage/bone loss, revealing a therapeutic potential of CXCL12 functional blockade in preventing bone growth defects during/after Dex treatment. © 2018 American Society for Bone and Mineral Research.
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Apoptose/efeitos dos fármacos , Osso Esponjoso , Quimiocina CXCL12/metabolismo , Dexametasona/efeitos adversos , Lâmina de Crescimento , Músculo Esquelético/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Osso Esponjoso/crescimento & desenvolvimento , Osso Esponjoso/patologia , Linhagem Celular , Quimiocina CXCL12/antagonistas & inibidores , Dexametasona/farmacologia , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/patologia , Masculino , Camundongos , Músculo Esquelético/patologia , Células RAW 264.7 , Ratos , Ratos Sprague-DawleyRESUMO
Cancer treatments with cytotoxic drugs have been shown to cause bone loss. However, effects on bone are less clear for ErbB-targeting tyrosine kinase inhibitors or their combination use with cytotoxic drugs. This study examined the effects of individual or combination treatments with breast cancer drugs lapatinib (a dual ErbB1/ErbB2 inhibitor) and paclitaxel (a microtubule-stabilizing cytotoxic agent) on bone and bone marrow of rats. Wistar rats received lapatinib (240 mg/kg) daily, paclitaxel (12 mg/kg) weekly, or their combination for 4 weeks, and effects on bone/bone marrow were examined at the end of week 4. Microcomputed tomographical structural analyses showed a reduction in trabecular bone volume in tibia following the lapatinib, paclitaxel or their combination treatments ( P < 0.05). Histomorphometry analyses revealed marked increases in bone marrow adipocyte contents in all treatment groups. Reverse transcription polymerase chain reaction gene expression studies with bone samples and cell culture studies with isolated bone marrow stromal cells showed that the all treatment groups displayed significantly reduced levels of osterix expression and osteogenic differentiation potential but increased expression levels of adipogenesis transcription factor peroxisome proliferator-activated receptor γ. In addition, these treatments suppressed the expression of Wnt10b and/or increased expression of Wnt antagonists (secreted frizzled-related protein 1, Dickkopf-related protein 1 and/or sclerostin). Furthermore, all treatment groups showed increased numbers of bone-resorbing osteoclasts on trabecular bone surfaces, although only the lapatinib group displayed increased levels of osteoclastogenic signal (receptor activator of nuclear factor κΒ ligand/osteoclastogenesis inhibitor osteoprotegrin expression ratio) in the bones. Thus, inhibiting ErbB1 and ErbB2 by lapatinib or blocking cell division by paclitaxel or their combination causes significant trabecular bone loss and bone marrow adiposity involving a switch in osteogenesis/adipogenesis potential, altered expression of some major molecules of the Wnt/ß-catenin signalling pathway, and increased recruitment of bone-resorbing osteoclasts.
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Adiposidade/efeitos dos fármacos , Medula Óssea/metabolismo , Reabsorção Óssea/induzido quimicamente , Lapatinib/farmacologia , Paclitaxel/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Moduladores de Tubulina/farmacologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Quimioterapia Combinada , Expressão Gênica/efeitos dos fármacos , Marcadores Genéticos/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Lapatinib/administração & dosagem , Lapatinib/efeitos adversos , Proteínas de Membrana/genética , PPAR gama/genética , Paclitaxel/administração & dosagem , Paclitaxel/efeitos adversos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Ratos , Ratos Wistar , Survivina/genética , Fatores de Transcrição/genética , Moduladores de Tubulina/administração & dosagem , Moduladores de Tubulina/efeitos adversos , Proteínas Wnt/genética , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Faulty bony repair causes dysrepair of injured growth plate cartilage and bone growth defects in children; however, the underlying mechanisms are unclear. Recently, we observed the prominent induction of neurotrophin3 (NT-3) and its important roles as an osteogenic and angiogenic factor promoting the bony repair. The current study investigated its roles in regulating injury site remodelling. In a rat tibial growth plate drill-hole injury repair model, NT-3 was expressed prominently in osteoblasts at the injury site. Recombinant NT-3 (rhNT-3) systemic treatment enhanced, but NT-3 immunoneutralization attenuated, expression of cartilage-removal proteases (MMP-9 and MMP-13), presence of bone-resorbing osteoclasts and expression of osteoclast protease cathepsin K, and remodelling at the injury site. NT-3 was also highly induced in cultured mineralizing rat bone marrow stromal cells, and the conditioned medium augmented osteoclast formation and resorptive activity, an ability that was blocked by presence of anti-NT-3 antibody. Moreover, NT-3 and receptor TrkC were induced during osteoclastogenesis, and rhNT-3 treatment activated TrkC downstream kinase Erk1/2 in differentiating osteoclasts although rhNT-3 alone did not affect activation of osteoclastogenic transcription factors NF-κB or NFAT in RAW264.7 osteoclast precursor cells. Furthermore, rhNT-3 treatment increased, but NT-3 neutralization reduced, expression of osteoclastogenic cytokines (RANKL, TNF-α, and IL-1) in mineralizing osteoblasts and in growth plate injury site, and rhNT-3 augmented the induction of these cytokines caused by RANKL treatment in RAW264.7 cells. Thus, injury site osteoblast-derived NT-3 is important in promoting growth plate injury site remodelling, as it induces cartilage proteases for cartilage removal and augments osteoclastogenesis and resorption both directly (involving activing Erk1/2 and substantiating RANKL-induced increased expression of osteoclastogenic signals in differentiating osteoclasts) and indirectly (inducing osteoclastogenic signals in osteoblasts).
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Cartilagem Articular/patologia , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Neurotrofina 3/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Calo Ósseo/metabolismo , Calo Ósseo/patologia , Citocinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Lâmina de Crescimento/efeitos dos fármacos , Humanos , Masculino , Camundongos , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Receptor trkC/metabolismoRESUMO
Injured growth plate is often repaired by bony tissue causing bone growth defects, for which the mechanisms remain unclear. Because neurotrophins have been implicated in bone fracture repair, here we investigated their potential roles in growth plate bony repair in rats. After a drill-hole injury was made in the tibial growth plate and bone, increased injury site mRNA expression was observed for neurotrophins NGF, BDNF, NT-3, and NT-4 and their Trk receptors. NT-3 and its receptor TrkC showed the highest induction. NT-3 was localized to repairing cells, whereas TrkC was observed in stromal cells, osteoblasts, and blood vessel cells at the injury site. Moreover, systemic NT-3 immunoneutralization reduced bone volume at injury sites and also reduced vascularization at the injured growth plate, whereas recombinant NT-3 treatment promoted bony repair with elevated levels of mRNA for osteogenic markers and bone morphogenetic protein (BMP-2) and increased vascularization and mRNA for vascular endothelial growth factor (VEGF) and endothelial cell marker CD31 at the injured growth plate. When examined in vitro, NT-3 promoted osteogenesis in rat bone marrow stromal cells, induced Erk1/2 and Akt phosphorylation, and enhanced expression of BMPs (particularly BMP-2) and VEGF in the mineralizing cells. It also induced CD31 and VEGF mRNA in rat primary endothelial cell culture. BMP activity appears critical for NT-3 osteogenic effect in vitro because it can be almost completely abrogated by co-addition of the BMP inhibitor noggin. Consistent with its angiogenic effect in vivo, NT-3 promoted angiogenesis in metatarsal bone explants, an effect abolished by co-treatment with anti-VEGF. This study suggests that NT-3 may be an osteogenic and angiogenic factor upstream of BMP-2 and VEGF in bony repair, and further studies are required to investigate whether NT-3 may be a potential target for preventing growth plate faulty bony repair or for promoting bone fracture healing. © 2016 American Society for Bone and Mineral Research.
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Proteína Morfogenética Óssea 2/metabolismo , Regeneração Óssea/fisiologia , Cartilagem/metabolismo , Lâmina de Crescimento/metabolismo , Neurotrofina 3/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Osteogênese/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Angiogenesis plays a pivotal role in bone formation, remodeling, and fracture healing. The regulation of angiogenesis in the bone microenvironment is highly complex and orchestrated by intercellular communication between bone cells and endothelial cells. Here, we report that EGF-like domain 7 (EGFL7), a member of the epidermal growth factor (EGF) repeat protein superfamily is expressed in both the osteoclast and osteoblast lineages, and promotes endothelial cell activities. Addition of exogenous recombinant EGFL7 potentiates SVEC (simian virus 40-transformed mouse microvascular endothelial cell line) cell migration and tube-like structure formation in vitro. Moreover, recombinant EGFL7 promotes angiogenesis featuring web-like structures in ex vivo fetal mouse metatarsal angiogenesis assay. We show that recombinant EGFL7 induces phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription 3 (STAT3), and focal adhesion kinase (FAK) in SVEC cells. Inhibition of ERK1/2 and STAT3 signaling impairs EGFL7-induced endothelial cell migration, and angiogenesis in fetal mouse metatarsal explants. Bioinformatic analyses indicate that EGFL7 contains a conserved RGD/QGD motif and EGFL7-induced endothelial cell migration is significantly reduced in the presence of RGD peptides. Moreover, EGFL7 gene expression is significantly upregulated during growth plate injury repair. Together, these results demonstrate that EGFL7 expressed by bone cells regulates endothelial cell activities through integrin-mediated signaling. This study highlights the important role that EGFL7, like EGFL6, expressed in bone microenvironment plays in the regulation of angiogenesis in bone.
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Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Integrinas/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Osso e Ossos/irrigação sanguínea , Osso e Ossos/citologia , Proteínas de Ligação ao Cálcio , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Família de Proteínas EGF , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Consolidação da Fratura/fisiologia , Lâmina de Crescimento/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteocalcina/biossíntese , Osteoclastos/metabolismo , Osteogênese/fisiologia , Fosforilação/efeitos dos fármacos , Proteínas/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Fraturas Salter-Harris , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Injuries to the growth plate cartilage often lead to bony repair, resulting in bone growth defects such as limb length discrepancy and angulation deformity in children. Currently utilised corrective surgeries are highly invasive and limited in their effectiveness, and there are no known biological therapies to induce cartilage regeneration and prevent the undesirable bony repair. In the last 2 decades, studies have investigated the cellular and molecular events that lead to bony repair at the injured growth plate including the identification of the four phases of injury repair responses (inflammatory, fibrogenic, osteogenic and remodelling), the important role of inflammatory cytokine tumour necrosis factor alpha in regulating downstream repair responses, the role of chemotactic and mitogenic platelet-derived growth factor in the fibrogenic response, the involvement and roles of bone morphogenic protein and Wnt/B-catenin signalling pathways, as well as vascular endothelial growth factor-based angiogenesis during the osteogenic response. These new findings could potentially lead to identification of new targets for developing a future biological therapy. In addition, recent advances in cartilage tissue engineering highlight the promising potential for utilising multipotent mesenchymal stem cells (MSCs) for inducing regeneration of injured growth plate cartilage. This review aims to summarise current understanding of the mechanisms for growth plate injury repair and discuss some progress, potential and challenges of MSC-based therapies to induce growth plate cartilage regeneration in combination with chemotactic and chondrogenic growth factors and supporting scaffolds.
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Fraturas Salter-Harris , Animais , Desenvolvimento Ósseo/fisiologia , Regeneração Óssea/fisiologia , Remodelação Óssea/fisiologia , Terapia Baseada em Transplante de Células e Tecidos , Condrócitos/transplante , Fibrose , Lâmina de Crescimento/patologia , Lâmina de Crescimento/fisiopatologia , Substâncias de Crescimento/fisiologia , Humanos , Inflamação/patologia , Inflamação/fisiopatologia , Transplante de Células-Tronco Mesenquimais , Osteogênese/fisiologia , Transdução de Sinais , Engenharia TecidualRESUMO
Dysregulated cyclin-dependent kinases (CDKs) are considered a potential target for cancer therapy. Flavopiridol is a potent CDK inhibitor. In this study, the antiproliferative effect of the flavonoid compound flavopiridol and its mechanism in human uterine leiomyoma cells were investigated. The present study focused on the effect of flavopiridol in cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells. Cell viability and cell proliferation assays were conducted. Flow cytometry was performed to determine the effect of flavopiridol on cell cycle. The expression of cell cycle regulatory-related proteins was evaluated by Western blotting. Cell viability and proliferation of uterine leiomyoma cells were significantly reduced by flavopiridol treatment in a dose-dependent manner. Flow cytometry results showed that flavopiridol induced G1 phase arrest. Flavopiridol-induced growth inhibition in uterine leiomyoma cells was associated with increased expression of p21(cip/wafl) and p27(kip1) in a dose-dependent manner. Downregulation of CDK2/4 and Cyclin A with a concomitant increase in dephosphorylation of retinoblastoma was observed. This study demonstrates that flavopiridol inhibits cell proliferation by initiating G1 cell cycle arrest in human uterine leiomyoma. We also found that flavopiridol is effective in inhibiting xenografted human uterine leiomyoma growth. These results indicate that flavopiridol could prove to be a promising chemopreventive and therapeutic agent for human uterine leiomyoma.
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Antineoplásicos/uso terapêutico , Flavonoides/uso terapêutico , Leiomioma/tratamento farmacológico , Piperidinas/uso terapêutico , Neoplasias Uterinas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto , Animais , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Feminino , Flavonoides/farmacologia , Humanos , Leiomioma/patologia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Piperidinas/farmacologia , Resultado do Tratamento , Células Tumorais Cultivadas , Neoplasias Uterinas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Growth plate injuries often result in undesirable bony repair causing bone growth defects, for which the underlying mechanisms are unclear. Whilst the key importance of pro-angiogenic vascular endothelial growth factor (VEGF) is well-known in bone development and fracture repair, its role during growth plate bony repair remains unexplored. Using a rat tibial growth plate injury repair model with anti-VEGF antibody, Bevacizumab, as a single i.p. injection (2.5âmg/kg) after injury, this study examined the roles of VEGF-driven angiogenesis during growth plate bony repair. Histology analyses observed isolectin-B4-positive endothelial cells and blood vessel-like structures within the injury site on days 6 and 14, with anti-VEGF treatment significantly decreasing blood-vessel-like structures within the injury site (P<0.05). Compared with untreated controls, anti-VEGF treatment resulted in an increase in undifferentiated mesenchymal repair tissue, but decreased bony tissue at the injury site at day 14 (P<0.01). Consistently, microcomputed tomography analysis of the injury site showed significantly decreased bony repair tissue after treatment (P<0.01). RT-PCR analyses revealed a significant decrease in osteocalcin (P<0.01) and a decreasing trend in Runx2 expression at the injury site following treatment. Furthermore, growth plate injury-induced reduced tibial lengthening was more pronounced in anti-VEGF-treated injured rats on day 60, consistent with the observation of a significantly increased height of the hypertrophic zone adjacent to the growth plate injury site (P<0.05). These results indicate that VEGF is important for angiogenesis and formation of bony repair tissue at the growth plate injury site as well as for endochondral bone lengthening function of the uninjured growth plate.
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Cartilagem/irrigação sanguínea , Cartilagem/metabolismo , Lâmina de Crescimento/irrigação sanguínea , Lâmina de Crescimento/metabolismo , Tíbia/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Cartilagem/lesões , Cartilagem/fisiopatologia , Lâmina de Crescimento/fisiopatologia , Masculino , Neovascularização Patológica , Ratos , Ratos Sprague-Dawley , Fraturas Salter-Harris , Tíbia/irrigação sanguínea , Tíbia/lesões , Tíbia/metabolismo , CicatrizaçãoRESUMO
Cancer chemotherapy has been shown to induce long-term skeletal side effects such as osteoporosis and fractures; however, there are no preventative treatments. This study investigated the damaging effects of anti-metabolite methotrexate (MTX) subcutaneous injections (0.75 mg/kg BW) for five days and the potential protective benefits of daily oral gavage of fish oil at 0.5 mL/100 g BW (containing 375 mg of n-3 PUFA/100 g BW), genistein (2 mg/100 g BW), or their combination in young adult rats. MTX treatment alone significantly reduced primary spongiosa height and secondary spongiosa trabecular bone volume. Bone marrow stromal cells from the treated rats showed a significant reduction in osteogenic differentiation but an increase in adipogenesis ex vivo. Consistently, stromal cells had significantly higher mRNA levels of adipogenesis-related proliferator activator activated receptor-γ (PPAR-γ) and fatty acid binding protein (FABP4). MTX significantly increased the numbers of bone-resorbing osteoclasts and marrow osteoclast precursor cell pool while significantly enhancing the mRNA expression of receptor activator for nuclear factor kappa B ligand (RANKL), the RANKL/osteoprotegerin (OPG) ratio, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the bone. Supplementary treatment with fish oil and/or genistein significantly preserved trabecular bone volume and osteogenesis but suppressed MTX-induced adipogenesis and increases in osteoclast numbers and pro-osteoclastogenic cytokine expression. Thus, Fish oil and/or genistein supplementation during MTX treatment enabled not only preservation of osteogenic differentiation, osteoblast number and bone volume, but also prevention of MTX treatment-induced increases in bone marrow adiposity, osteoclastogenic cytokine expression and osteoclast formation, and thus bone loss.
Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Suplementos Nutricionais , Óleos de Peixe/farmacologia , Genisteína/farmacologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Osso e Ossos/anatomia & histologia , Osso e Ossos/citologia , Diferenciação Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Óleos de Peixe/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Genisteína/administração & dosagem , Mediadores da Inflamação/metabolismo , Masculino , Metotrexato/administração & dosagem , Metotrexato/efeitos adversos , Tamanho do Órgão , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Ratos , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
BACKGROUND: Chemo-resistance to cisplatin-centered cancer therapy is a major obstacle to effective disease treatment. Recently, salinomycin was proven to be highly-effective for the elimination of cancer stem cells both in vitro and in vivo. The objective of the present study was to evaluate the anticancer properties of salinomycin in cisplatin-resistant ovarian cancer cells (A2780cis). MATERIALS AND METHODS: The tetrazolium dye (MTT) assay was used to determine cell viability. Flow cytometric analysis was performed to analyze the effect on cell cycle and apoptosis. The expression of apoptosis-related proteins was evaluated by western blot analysis. RESULTS: Cell viability was significantly reduced by salinomycin treatment in a dose-dependent manner. Flow cytometry showed an increase in sub-G1 phase cells. Salinomycin increased the expression of death receptor-5 (DR5), caspase-8 and Fas-associated protein with death domain (FADD). A decline in the expression of FLICE-like inhibitory protein (FLIP), activation of caspase-3 and increased poly ADP-ribose polymerase (PARP) cleavage, triggered apoptosis. Furthermore, annexin-V staining also revealed the apoptotic induction. CONCLUSION: These findings provide important insights regarding the activation of caspase-8 and DR5, to our knowledge, for the first time in salinomycin-treated cisplatin-resistant ovarian cancer and demonstrate that salinomycin could be a prominent anticancer agent.
Assuntos
Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Ovarianas/patologia , Piranos/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Anexina A5/metabolismo , Antineoplásicos/farmacologia , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Feminino , Humanos , Immunoblotting , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
BACKGROUND: Self-expandable metallic stent placement is widely used to manage malignant gastroduodenal obstructions. However, there are difficulties in negotiating a guidewire (GW) and a stent delivery system (SDS). PURPOSE: To investigate feasibility, usefulness, and safety of a guiding sheath for fluoroscopic stent placement in patients with malignant gastroduodenal obstructions. MATERIAL AND METHODS: In July 2001 to August 2011, 726 patients with malignant gastroduodenal obstructions underwent stent placement. Guiding sheath was used in patients in whom a GW could not be passed through the obstruction and a SDS failed to reach the obstruction. Sheath usefulness was evaluated based on the ability of the sheath to successfully assist. The technical success rate and the most frequent reasons for the use of a sheath were evaluated. RESULTS: The guiding sheath was needed in 148 of 726 patients (20%). The overall technical success rate was 98% with the guiding sheath. In two of 148 patients, stent placement failed because, the GW could not be passed through the obstruction, in the other, the SDS could not be passed. A minority of patients reported mild discomfort. Patients with pancreatic cancer and duodenal obstruction were significantly more likely to require the use of guiding sheaths (P = 0.002, P < 0.001, respectively). CONCLUSION: Using a guiding sheath for fluoroscopic stent placement appears to be feasible, useful and safe in patients with malignant gastroduodenal obstructions.
Assuntos
Obstrução Duodenal/patologia , Obstrução Duodenal/cirurgia , Obstrução da Saída Gástrica/patologia , Obstrução da Saída Gástrica/cirurgia , Radiografia Intervencionista/instrumentação , Stents , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Desenho de Equipamento , Feminino , Fluoroscopia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do TratamentoRESUMO
INTRODUCTION: Injured growth plate cartilage is often repaired by bony tissue, causing bone growth defects in children. Currently, mechanisms for the undesirable repair remain unclear and there are no biological treatments available to prevent the associated bone growth defects. Osterix is known as a vital transcription factor for osteoblast differentiation which is critical for normal bone formation and bone repair, and osterix is known to be regulated by protein kinase-D; however it is unknown whether protein kinase-D-osterix signalling plays any roles in the bony repair of injured growth plate. METHODS: Using a rat model, this study investigated potential roles of protein kinase-D (PKD) in regulating expression of osteogenic transcription factor osterix and the growth plate bony repair. 4 days post injury at the proximal tibial growth plate, rats received four once-daily injections of vehicle or 2.35 mg/kg gö6976 (a PKD inhibitor), and growth plate tissues collected at day 10 were examined histologically and molecularly. In addition, effects of PKD inhibition on osteogenic and chondrogenic differentiation were examined in vitro using rat bone marrow mesenchymal stromal cells. RESULTS: Compared to vehicle control, PKD inhibition caused a decrease in bone volume (p<0.05), an increase in % of mesenchymal tissue (p<0.01), and an increase in cartilaginous tissue within the injury site. Consistently, gö6976 treatment tended to decrease expression of bone-related genes (osterix, osteocalcin) and increase levels of cartilage-related genes (Sox9, collagen-2a, collagen-10a1). In support, in vitro experiments showed that gö6976 presence in the primary rat marrow stromal cell culture resulted in a decrease of alkaline phosphatase(+) CFU-f colonies formed (p<0.05) and an increase in collagen-2a expression in chondrogenic pellet culture (p<0.05). CONCLUSION: These studies suggest that PKD is important for growth plate bony repair and its inhibition after growth plate injury may result in less bone formation and potentially more cartilage repair.
Assuntos
Cartilagem/lesões , Cartilagem/metabolismo , Lâmina de Crescimento/enzimologia , Proteína Quinase C/biossíntese , Fraturas Salter-Harris , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Cartilagem/patologia , Modelos Animais de Doenças , Expressão Gênica , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/patologia , Masculino , Osteoblastos , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/metabolismoRESUMO
Growth plate cartilage is responsible for longitudinal growth of the long bone in children, and its injury is often repaired by bony tissue, which can cause limb length discrepancy and/or bone angulation deformities. Whilst earlier studies with a rat growth plate injury repair model have identified inflammatory, mesenchymal infiltration, osteogenesis and remodeling responses, the molecular mechanisms involved in the bony repair remain unknown. Since our recent microarray study has strongly suggested involvement of Wnt-ß-catenin signalling pathway in regulating the growth plate repair and the pathway is known to play a crucial role in the osteogenic differentiation of mesenchymal progenitor cells, the current study investigated the potential roles of Wnt-ß-catenin signalling pathway in the bony repair of injured tibial growth plate in rats. Immunohistochemical analysis of the growth plate injury site revealed ß-catenin immunopositive cells within the growth plate injury site. Treatment of the injured rats with the ß-catenin inhibitor ICG-001 (oral gavage at 200mg/kg/day for 8days, commenced at day 2 post injury) enhanced COL2A1 gene expression (by qRT-PCR) and increased proportion of cartilage tissue (by histological analysis), but decreased level of osterix expression and amount of bone tissue, at the injury site by day 10 post-injury (n=8, P<0.01 compared to vehicle controls). Consistently, in vitro studies with bone marrow stromal cells from normal rats showed that ß-catenin inhibitor ICG-001 dose dependently inhibited expression of Wnt target genes Cyclin D1 and survivin (P<0.01). At 25mM, ICG-001 suppressed osteogenic (by CFU-f-ALP assay) but enhanced chondrogenic (by pellet culture) differentiation. These results suggest that Wnt/ß-catenin signalling pathway is involved in regulating growth plate injury repair by promoting osteoblastogenesis, and that intervention of this signalling could represent a potential approach in enhancing cartilage repair after growth plate injury.
Assuntos
Envelhecimento/patologia , Cartilagem/lesões , Cartilagem/metabolismo , Lâmina de Crescimento/metabolismo , Fraturas Salter-Harris , Via de Sinalização Wnt , Cicatrização , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/patologia , Imuno-Histoquímica , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Pirimidinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , Cicatrização/efeitos dos fármacos , Cicatrização/genética , beta Catenina/metabolismoRESUMO
In the last two decades, there has been a strong interest in searching for biological treatments for regeneration of injured growth plate cartilage and prevention of its bony repair. Various means have been tried, including implantation of chondrocytes, mesenchymal stem cell (MSC), together with exogenous growth factor and scaffolds, and gene therapy. However, with the lack of success with chondrocytes, more research has focussed on MSC-based treatments. In addition to circumvent limitations with MSC-based treatments (including cell harvest-associated morbidity, difficulties/time/cost involved in MSC isolation and ex vivo expansion, and potential disease transmission), mobilising endogenous MSCs to the growth plate injury site and enhancing in situ regeneration mechanisms would represent an alternative attractive approach. Further studies are required to investigate the potential particularly in large animal models or clinical setting of the ex vivo MSC approach and the feasibility of the endogenous MSC in situ approach in growth plate regeneration.