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1.
JBMR Plus ; 6(11): e10690, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36398113

RESUMO

Although changes in bone mineral density (BMD) are important indexes in osteoporosis treatment, no markers are available to predict them. Given the importance of assessing the therapeutic windows of antiresorptives, we explored potential biomarkers of bone remodeling in patients receiving treatment for osteopenia. Postmenopausal women with osteopenia (defined as a lumbar BMD T-score <-1.0 standard deviation (SD) below that of a reference population but >-2.5 SD) were administered estradiol 1 mg/d and bazedoxifene 20 mg/d. After 3 months of treatment, we evaluated their ratio of serum bone-specific tartrate-resistant acid phosphatase to bone-specific alkaline phosphatase (TRACP-5b/BAP), which is widely used for evaluating bone turnover in postmenopausal patients with osteoporosis in Japan because their minimum significant changes are smaller than other bone turnover markers such as carboxy-terminal collagen cross-links (CTX) or N-terminal propeptide of type I procollagen (P1NP) and thus, accurately reflect bone turnover. After 1 year of treatment, we assessed changes in lumbar BMD. The cut-off TRACP-5b/BAP scores for a ≤-2% decrease and ≥2% increase in lumbar spine BMD were 38.4 and 29.0, respectively. The TRACP-5b/BAP scores were associated with significantly greater areas under the curve than the other evaluated parameters. These results suggest that the TRACP-5b/BAP score after 3 months of osteopenia treatment can predict changes in lumbar BMD after 1 year of treatment. Moreover, a receiver operating characteristic curve analysis of TRACP-5b/BAP scores after 3 months of antiresorptive therapy and percent changes in BMD at 1 year revealed that the TRACP-5b/BAP score, as an index of the balance between bone resorption and formation markers, has the potential to serve as a modulator of the anabolic window reflective of bone remodeling. This study's findings also suggested a role for TRACP-5b/BAP score as a predictor of a non-response to antiresorptive therapy, thus offering health economic implications for osteoporosis treatment. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

2.
Cancer Treat Res ; 169: 251-270, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27696267

RESUMO

Bone involvement represented by osteolytic bone disease (OBD) or osteopenia is one of the pathognomonic and defining characteristics of multiple myeloma (MM). Nearly 90 % of patients with MM develop osteolytic bone lesions, frequently complicated by skeletal-related events (SRE) such as severe bone pain, pathological fractures, vertebral collapse, hypercalcemia, and spinal cord compression. All of these not only result in a negative impact on quality of life but also adversely impact overall survival. OBD is a consequence of increased osteoclast (OC) activation along with osteoblast (OB) inhibition, resulting in altered bone remodeling. OC number and activity are increased in MM via cytokine deregulation within the bone marrow (BM) milieu, whereas negative regulators of OB differentiation suppress bone formation. Inhibition of osteolysis and stimulation of OB differentiation leads to reduced tumor growth in vivo. Therefore, novel agents targeting OBD are promising therapeutic strategies not only for the treatment of MM OBD but also for the treatment of MM. Several novel agents in addition to bisphosphonates are currently under investigation for their positive effect on bone remodeling via OC inhibition or OB stimulation. Future studies will look to combine or sequence all of these agents with the goal of not only alleviating morbidity from MM OBD but also capitalizing on the resultant antitumor activity.


Assuntos
Doenças Ósseas/etiologia , Doenças Ósseas/terapia , Mieloma Múltiplo/complicações , Doenças Ósseas/patologia , Humanos
3.
J Clin Invest ; 126(4): 1300-10, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26927669

RESUMO

Regulation of STAT3 activation is critical for normal and malignant hematopoietic cell proliferation. Here, we have reported that the endogenous transmembrane protein upstream-of-mTORC2 (UT2) negatively regulates activation of STAT3. Specifically, we determined that UT2 interacts directly with GP130 and inhibits phosphorylation of STAT3 on tyrosine 705 (STAT3Y705). This reduces cytokine signaling including IL6 that is implicated in multiple myeloma and other hematopoietic malignancies. Modulation of UT2 resulted in inverse effects on animal survival in myeloma models. Samples from multiple myeloma patients also revealed a decreased copy number of UT2 and decreased expression of UT2 in genomic and transcriptomic analyses, respectively. Together, these studies identify a transmembrane protein that functions to negatively regulate cytokine signaling through GP130 and pSTAT3Y705 and is molecularly and mechanistically distinct from the suppressors of cytokine signaling (SOCS) family of genes. Moreover, this work provides evidence that perturbations of this activation-dampening molecule participate in hematologic malignancies and may serve as a key determinant of multiple myeloma pathophysiology. UT2 is a negative regulator shared across STAT3 and mTORC2 signaling cascades, functioning as a tumor suppressor in hematologic malignancies driven by those pathways.


Assuntos
Neoplasias Hematológicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mieloma Múltiplo/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Feminino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Fator de Transcrição STAT3/genética , Proteínas Supressoras de Tumor/genética , Transportadores de Ureia
4.
J Bone Miner Res ; 31(6): 1225-34, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26763740

RESUMO

Sclerostin is a potent inhibitor of osteoblastogenesis. Interestingly, newly diagnosed multiple myeloma (MM) patients have high levels of circulating sclerostin that correlate with disease stage and fractures. However, the source and impact of sclerostin in MM remains to be defined. Our goal was to determine the role of sclerostin in the biology of MM and its bone microenvironment as well as investigate the effect of targeting sclerostin with a neutralizing antibody (scl-Ab) in MM bone disease. Here we confirm increased sclerostin levels in MM compared with precursor disease states like monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM. Furthermore, we found that a humanized MM xenograft mouse model bearing human MM cells (NOD-SCID.CB17 male mice injected intravenously with 2.5 million of MM1.S-Luc-GFP cells) demonstrated significantly higher concentrations of mouse-derived sclerostin, suggesting a microenvironmental source of sclerostin. Associated with the increased sclerostin levels, activated ß-catenin expression levels were lower than normal in MM mouse bone marrow. Importantly, a high-affinity grade scl-Ab reversed osteolytic bone disease in this animal model. Because scl-Ab did not demonstrate significant in vitro anti-MM activity, we combined it with the proteasome inhibitor carfilzomib. Our data demonstrated that this combination therapy significantly inhibited tumor burden and improved bone disease in our in vivo MM mouse model. In agreement with our in vivo data, sclerostin expression was noted in marrow stromal cells and osteoblasts of MM patient bone marrow samples. Moreover, MM cells stimulated sclerostin expression in immature osteoblasts while inhibiting osteoblast differentiation in vitro. This was in part regulated by Dkk-1 secreted by MM cells and is a potential mechanism contributing to the osteoblast dysfunction noted in MM. Our data confirm the role of sclerostin as a potential therapeutic target in MM bone disease and provides the rationale for studying scl-Ab combined with proteasome inhibitors in MM. © 2016 American Society for Bone and Mineral Research.


Assuntos
Doenças Ósseas/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/metabolismo , Osteoblastos/metabolismo , Microambiente Tumoral , Proteínas Adaptadoras de Transdução de Sinal , Animais , Doenças Ósseas/genética , Doenças Ósseas/patologia , Feminino , Glicoproteínas/genética , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Osteoblastos/patologia
5.
Br J Haematol ; 169(3): 423-34, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25709080

RESUMO

Proteasome inhibition induces the accumulation of aggregated misfolded/ubiquitinated proteins in the aggresome; conversely, histone deacetylase 6 (HDAC6) inhibition blocks aggresome formation. Although this rationale has been the basis of proteasome inhibitor (PI) and HDAC6 inhibitor combination studies, the role of disruption of aggresome formation by HDAC6 inhibition has not yet been studied in multiple myeloma (MM). The present study aimed to evaluate the impact of carfilzomib (CFZ) in combination with a selective HDAC6 inhibitor (ricolinostat) in MM cells with respect to the aggresome-proteolysis pathway. We observed that combination treatment of CFZ with ricolinostat triggered synergistic anti-MM effects, even in bortezomib-resistant cells. Immunofluorescent staining showed that CFZ increased the accumulation of ubiquitinated proteins and protein aggregates in the cytoplasm, as well as the engulfment of aggregated ubiquitinated proteins by autophagosomes, which was blocked by ricolinostat. Electron microscopy imaging showed increased autophagy triggered by CFZ, which was inhibited by the addition of ACY-1215. Finally, an in vivo mouse xenograft study confirmed a decrease in tumour volume, associated with apoptosis, following treatment with CFZ in combination with ricolinostat. Our results suggest that ricolinostat inhibits aggresome formation, caused by CFZ-induced inhibition of the proteasome pathway, resulting in enhanced apoptosis in MM cells.


Assuntos
Apoptose/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Mieloma Múltiplo/metabolismo , Oligopeptídeos/farmacologia , Pirimidinas/farmacologia , Animais , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Xenoenxertos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Fagossomos/metabolismo , Inibidores de Proteassoma/farmacologia
6.
J Bone Miner Res ; 30(3): 465-70, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25407518

RESUMO

Decorin is a small, leucine-rich proteoglycan found in the extracellular matrix of various connective tissues with potential effective tumor suppressive properties. Recent data suggest low levels of decorin in multiple myeloma (MM) patients compared to healthy volunteers, as well as in patients with osteolytic bone lesions compared to non-osteolytic lesions. In the present report, we investigated the role of decorin in the MM microenvironment or niche. Our data suggests that decorin is produced by osteoblasts (OBs) but not by MM cells. Furthermore, MM cells decrease OB-induced decorin secretion and this effect is mediated by CCL3. Importantly, neutralizing CCL3 from MM cells restores decorin levels in OBs as does proteasome inhibitors such as carfilzomib. These findings indicate that decorin may indirectly act as an antagonist to MM cell survival and that the interplay between MM and decorin may be an important target to explore in manipulating the tumor niche to inhibit tumorigenesis.


Assuntos
Medula Óssea/patologia , Decorina/fisiologia , Mieloma Múltiplo/patologia , Microambiente Tumoral , Animais , Linhagem Celular Tumoral , Humanos , Camundongos
7.
Mol Cancer Ther ; 13(11): 2489-500, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25172964

RESUMO

Despite promising preclinical results with mTOR kinase inhibitors in multiple myeloma, resistance to these drugs may arise via feedback activation loops. This concern is especially true for insulin-like growth factor 1 receptor (IGF1R), because IGF1R signaling is downregulated by multiple AKT and mTOR feedback mechanisms. We have tested this hypothesis in multiple myeloma using the novel selective mTOR kinase inhibitor AZD8055. We evaluated p-mTOR S(2481) as the readout for mTORC2/Akt activity in multiple myeloma cells in the context of mTOR inhibition via AZD8055 or rapamycin. We next validated AZD8055 inhibition of mTORC1 and mTORC2 functions in multiple myeloma cells alone or in culture with bone marrow stroma cells and growth factors. Unlike rapamycin, AZD8055 resulted in apoptosis of multiple myeloma cells. AZD8055 treatment, however, induced upregulation of IGF1R phosphorylation in p-Akt S(473)-expressing multiple myeloma cell lines. Furthermore, exposure of AZD8055-treated cells to IGF1 induced p-Akt S(473) and rescued multiple myeloma cells from apoptosis despite mTOR kinase inhibition and TORC2/Akt blockage. The addition of blocking IGF1R antibody resulted in reversing this effect and increased AZD8055-induced apoptosis. Our study suggests that combination treatment with AZD8055 and IGF1R-blocking agents is a promising strategy in multiple myeloma with potential IGF1R/Akt signaling-mediated survival.


Assuntos
Morfolinas/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/enzimologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Bone ; 53(2): 487-96, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23333523

RESUMO

Over-expression of the protein Dickkopf-1 (Dkk1) has been associated with multiple myeloma bone disease. Previous reports with the use of anti-Dkk1 neutralizing Ab directed strategies have demonstrated a pro-anabolic effect with associated anti-myeloma activity in 2 in vivo mouse models. However new insights on the role of the wnt pathway in osteoclasts (OC) are emerging and the potential effect of a neutralizing Ab to Dkk1 in osteoclastogenesis remains to be elucidated. In order to better define the effect of an anti-Dkk1 neutralizing Ab on osteoclastogenesis and myeloma, we studied a novel anti-Dkk1 monoclonal Ab in our preclinical models. In vivo data confirmed the pro-anabolic and anti-MM effect. In vitro data in part confirmed the in vivo observation, suggesting an indirect anti-MM effect secondary to inhibition of osteoclastogenesis and thus the interaction between MM and bone microenvironment. However, when studies on osteoclastogenesis were extended to samples derived from MM patients, we observed a variable response to anti-Dkk1 treatment without correlation to expression of surface receptors for Dkk1 in OCs suggesting potential heterogeneity in the efficacy of such a strategy. In conclusion, Dkk1 is a promising target for the treatment of both MM and bone disease, and ongoing clinical studies will help elucidate its efficacy.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Mieloma Múltiplo/tratamento farmacológico , Animais , Linhagem Celular , Células Cultivadas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/análise , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos SCID , Mieloma Múltiplo/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Microtomografia por Raio-X
9.
Blood ; 119(11): 2579-89, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22262760

RESUMO

Histone deacetylase (HDAC) enzymatic activity has been linked to the transcription of DNA in cancers including multiple myeloma (MM). Therefore, HDAC inhibitors used alone and in combination are being actively studied as novel therapies in MM. In the present study, we investigated the preclinical activity of ACY-1215, an HDAC6-selective inhibitor, alone and in combination with bortezomib in MM. Low doses of ACY-1215 combined with bortezomib triggered synergistic anti-MM activity, resulting in protracted endoplasmic reticulum stress and apoptosis via activation of caspase-3, caspase-8, and caspase-9 and poly (ADP) ribosome polymerase. In vivo, the anti-MM activity of ACY-1215 in combination with bortezomib was confirmed using 2 different xenograft SCID mouse models: human MM injected subcutaneously (the plasmacytoma model) and luciferase-expressing human MM injected intravenously (the disseminated MM model). Tumor growth was significantly delayed and overall survival was significantly prolonged in animals treated with the combination therapy. Pharmacokinetic data showed peak plasma levels of ACY-1215 at 4 hours after treatment coincident with an increase in acetylated α-tubulin, a marker of HDAC6 inhibition, by immunohistochemistry and Western blot analysis. These studies provide preclinical rationale for acetylated α-tubulin use as a pharmacodynamic biomarker in future clinical trials.


Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/farmacocinética , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/farmacocinética , Plasmocitoma/tratamento farmacológico , Pirazinas/farmacologia , Pirimidinas/farmacologia , Pirimidinas/farmacocinética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Western Blotting , Bortezomib , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Imunofluorescência , Desacetilase 6 de Histona , Histona Desacetilases/genética , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos SCID , Plasmocitoma/metabolismo , Plasmocitoma/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Int J Oncol ; 39(5): 1327-36, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21785823

RESUMO

In vitro tumor growth in a three-dimensional (3D) architecture has been demonstrated to play an important role in biology not only for developmental organogenesis and carcinogenesis, but also for analyses on reconstitution and maintenance in a variety of biological environments surrounding the cells. In addition to providing architectural similarity to living organisms, 3D culture with a radial flow bioreactor (RFB) can also closely mimic the living hypoxic microenvironment under which specific organogenesis or carcinogenesis occurs. The findings of the present study under the RFB culture conditions show that cancer cells underwent a shift from aerobic to hypoxic energy metabolism, in addition to protein expression to maintain the 3D structure. In RFB-cultured cells, protein stability of hypoxia-inducible factor 1 (HIF1) α, a subunit of HIF1, was increased without upregulation of its mRNA. Under these conditions, PHD2, HIF-prolyl-4-hydroxy-lase 2 and a HIF1 downstream enzyme, were stabilized without affecting the mRNA levels via downregulation of FK506-binding protein 8. PHD2 accumulation, which occurred concomitant with HIF1 stabilization, may have compensated for the lack of oxygen under hypoxic conditions to regulate the HIF levels. 3D-culture-induced overexpression of carbonic anhydrase (another representative HIF downstream enzyme) was found to occur independently of cell density in RFB--cultured cells, suggesting that the RFB provided an adequately hypoxic microenvironment for the cultured cells. From these results, it was hypothesized that the key factors are regulatory molecules, which stabilize and degrade HIF molecules, thereby activating the HIF1 pathway under a hypoxic milieu.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Carcinoma de Células Escamosas/genética , Técnicas de Cultura de Células , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Nus , Esferoides Celulares , Carcinoma de Células Escamosas de Cabeça e Pescoço , Ativação Transcricional/genética , Células Tumorais Cultivadas , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Eur J Haematol ; 85(1): 68-75, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20192985

RESUMO

OBJECTIVES: Bortezomib (PS-341; Velcade), a proteasome inhibitor, is used as a therapeutic agent for multiple myeloma. Bortezomib has been shown to strongly induce osteoblast differentiation and elevate the levels of osteoblast-related differentiation markers in the serum of patients with myeloma. Bortezomib also reportedly increases the activity of the transcription factor, Runx2. However, the mechanism of action by which bortezomib-elevated Runx2 activity mediates osteoblast differentiation remains unclear. On the other hand, fibroblast growth factor 2 (FGF-2) is found at high levels in patients with multiple myeloma. We previously reported that FGF-2 reduces the levels of the transcriptional coactivator with PDZ-binding motif (TAZ). We therefore investigated the effects of bortezomib on TAZ protein levels in the presence of FGF-2. METHODS: Osteoblastic MC3T3-E1 cells were treated with different concentrations of bortezomib in the presence or absence of FGF-2 and various biologic responses were investigated by immunoblotting, RT-PCR, quantitative PCR, and alizarin red staining. RESULTS: We found that bortezomib inhibited FGF-2-induced reduction of TAZ levels through a pathway other than that used for proteasome inhibition, while maintaining TAZ function, which in turn, enhanced the expression of Runx2-transcribed osteogenic differentiation markers. Bortezomib also suppressed the antimineralization effect of FGF-2. CONCLUSIONS: These findings suggest that bortezomib inhibited FGF-2-induced reduction of TAZ and consequently stimulated osteogenic differentiation independently of proteasome inhibition. These findings may contribute to elucidate the osteolytic mechanism in multiple myeloma, and to the development of new drugs for multiple myeloma and other osteolytic diseases.


Assuntos
Ácidos Borônicos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Inibidores de Proteassoma , Pirazinas/farmacologia , Fatores de Transcrição/metabolismo , Células 3T3 , Aciltransferases , Animais , Sequência de Bases , Ácidos Borônicos/administração & dosagem , Bortezomib , Diferenciação Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Primers do DNA/genética , Humanos , Camundongos , Modelos Biológicos , Mieloma Múltiplo/complicações , Mieloma Múltiplo/tratamento farmacológico , Osteoblastos/citologia , Osteogênese/efeitos dos fármacos , Osteólise/tratamento farmacológico , Osteólise/etiologia , Inibidores de Proteases/administração & dosagem , Inibidores de Proteases/farmacologia , Pirazinas/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Transcrição/genética
12.
Int J Oncol ; 34(5): 1433-48, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19360357

RESUMO

To confirm the usefulness of the radial flow type bioreactor (RFB) for a three-dimensional (3D) culture system, which provides a tissue architecture and molecular function mimicking the in vivo environment, molecular expression in the A431 human squamous carcinoma cell line during culture were analyzed under the physically different environments of 3D culture in the RFB, 2D culture in a monolayer as well as in nude mice. Time-dependent accumulation of autocrine transforming growth factor (TGF) beta1 was found in spent culture media obtained only from 3D cultured A431 cancer cells, which grew well with a stratified-sheet morphology. Cells in the RFB overexpressed matrix metalloproteinase 7 (MMP7) and showed an increased release of soluble 80-kDa fragments of E-cadherin into the media time-dependently, resulting in the reduction of E-cadherin protein at the cell surface without down-regulation of the mRNA. beta-Catenin and its nuclear partner, LEF1, were up-regulated and Wnt protein secretion was also accelerated. Additional up-regulation of the transcriptional factors, HMGA2 and down-stream Slug, was noted. TGFbeta1-dependent, MMP7-mediated up-regulation of beta-catenin/LEF1 signaling and TGFbeta1-activated HMGA2 pathways consequently converged with Slug overexpression, due to disassembly and further repression of E-cadherin expression, which was reproducible in the epithelial mesenchymal transition process without any manipulation. Other transcriptional factors, Notch/HEY1 and NF-kappaB, were also up-regulated in 3D-cultured cells. These signals recruited molecules related to extracellular matrix-cell remodeling and angiogenesis. Expression of several representative molecules in the 3D cultured cells was parallel with that in xenotransplanted A431 tumor tissues in nude mice. 3D culture of tumor cells in the RFB is a useful tool for cancer experimental biology and evaluation of cancer therapeutic-like systems in nude mice.


Assuntos
Reatores Biológicos , Transdiferenciação Celular/genética , Metaloproteinase 7 da Matriz/fisiologia , Neoplasias/patologia , Proteínas Smad/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Técnicas de Cultura de Células/métodos , Transdiferenciação Celular/fisiologia , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Metaloproteinase 7 da Matriz/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Biochem Biophys Res Commun ; 366(2): 471-5, 2008 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-18067853

RESUMO

Transcriptional coactivator with PDZ-binding motif (TAZ) protein is a coactivator of Runx2 and corepressor of PPARgamma. It also induces differentiation of mesenchymal cells into osteoblasts. In this study, we found that FGF-2, which inhibits bone mineralization and stimulates cell proliferation, reduced the TAZ protein expression level in osteoblast-like cells, MC3T3-E1. This reduction was recovered by removing FGF-2 from the culture medium, which also restored the osteoblastic features of MC3T3-E1 cells. Furthermore, FGF-2-induced reduction of TAZ is blocked by a SAPK/JNK-specific inhibitor. These findings suggest that the expression of TAZ protein is involved in osteoblast proliferation and differentiation. This may help elucidate the discrepancies in the effect of FGF-2 and contribute to the understanding of FGF/FGFR-associated craniosynostosis syndrome etiology and treatment.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Aciltransferases , Animais , Diferenciação Celular , Regulação para Baixo , Camundongos
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