RESUMO
Cerebral palsy (CP) is the most common childhood disability worldwide, yet biomarkers for predicting CP are lacking. By subjecting peripheral blood samples from 62 CP patients and 30 healthy controls to Affymetrix GeneChip® PrimeView™ HumanGene Expression Microarray analysis, we identified the novel biomarker B-cell lymphoma 6 (BCL6) as the most upregulated gene in the CP samples. Gastrodin is a traditional Chinese medicine and bioactive compound that promotes adductor angle release, as well as gross and fine motor performance by increasing Gross Motor Function Measure-66 and Fine Motor Function Measure-45 scores. Gastrodin upregulates the mRNA expression of Mgl2 and Mrc1, M2 macrophage markers, and arginase activity, an M2 polarization indicator, in murine RAW264.7 macrophages. Moreover, these effects were blocked by BCL6 siRNA, which also abrogated the protective effects of Gastrodin against hydrogen peroxide-induced apoptosis and death in RAW264.7 cells. Our work identified BCL6 as a novel biomarker for early prediction of CP. Moreover, we demonstrated that Gastrodin not only stimulated polarization toward M2-like macrophages, which promote tissue repair, but also rescued macrophages from oxidative stress, apoptosis and death by inducing BCL6 expression. BCL6-targeted therapeutic strategies have promise for improving motor performance in CP patients.
Assuntos
Antioxidantes/uso terapêutico , Álcoois Benzílicos/uso terapêutico , Paralisia Cerebral/diagnóstico , Paralisia Cerebral/tratamento farmacológico , Glucosídeos/uso terapêutico , Macrófagos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-6/genética , Animais , Apoptose/efeitos dos fármacos , Arginase/genética , Arginase/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Caspase 3/genética , Caspase 3/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Paralisia Cerebral/genética , Paralisia Cerebral/patologia , Pré-Escolar , Feminino , Regulação da Expressão Gênica , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Lactente , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Desempenho Psicomotor/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos , Transdução de SinaisRESUMO
Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to be successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Clatrina/imunologia , Dinaminas/imunologia , Células Endoteliais/imunologia , Proteínas de Neoplasias/imunologia , Frações Subcelulares/imunologia , Células Cultivadas , Células Endoteliais/ultraestrutura , HumanosRESUMO
Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane glycoprotein that regulates cell motility and proliferation. TM4SF1 is an attractive cancer target because of its high expression in both tumor cells and on the vascular endothelial cells lining tumor blood vessels. We generated mouse monoclonal antibodies against human TM4SF1 in order to evaluate their therapeutic potential; 13 of the antibodies we generated reacted with extracellular loop-2 (EL2), TM4SF1's larger extracellular, lumen-facing domain. However, none of these antibodies reacted with mouse TM4SF1, likely because the EL2 of mouse TM4SF1 differs significantly from that of its human counterpart. Therefore, to test our antibodies in vivo, we employed an established model of engineered human vessels in which human endothelial colony-forming cells (ECFC) and human mesenchymal stem cells (MSC) are incorporated into Matrigel plugs that are implanted subcutaneously in immunodeficient nude mice. We modified the original protocol by (1) preculturing human ECFC on laminin, fibronectin, and collagen-coated plates, and (2) increasing the ECFC/MSC ratio. These modifications significantly increased the human vascular network in Matrigel implants. Two injections of one of our anti-TM4SF1 EL2 monoclonal antibodies, 8G4, effectively eliminated the human vascular component present in these plugs; they also abrogated human PC3 prostate cancer cells that were incorporated into the ECFC/MSC Matrigel mix. Together, these studies provide a mouse model for assessing tumor xenografts that are supplied by a human vascular network and demonstrate that anti-TM4SF1 antibodies such as 8G4 hold promise for cancer therapy.
Assuntos
Antígenos de Superfície/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Células-Tronco Mesenquimais , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Transplante de Neoplasias , Neovascularização Patológica , Engenharia Tecidual/métodos , Transcrição GênicaRESUMO
Transmembrane-4 L-six family member-1 (TM4SF1) is an atypical tetraspanin that is highly and selectively expressed in proliferating endothelial cells and plays an essential role in blood vessel development. TM4SF1 forms clusters on the cell surface called TMED (TM4SF1-enriched microdomains) and recruits other proteins that internalize along with TM4SF1 via microtubules to intracellular locations including the nucleus. We report here that tumor growth and wound healing are inhibited in Tm4sf1-heterozygous mice. Investigating the mechanisms of TM4SF1 activity, we show that 12 out of 18 signaling molecules examined are recruited to TMED on the surface of cultured human umbilical vein endothelial cells (HUVEC) and internalize along with TMED; notable among them are PLCγ and HDAC6. When TM4SF1 is knocked down in HUVEC, microtubules are heavily acetylated despite normal levels of HDAC6 protein, and, despite normal levels of VEGFR2, are unable to proliferate. Together, our studies indicate that pathological angiogenesis is inhibited when levels of TM4SF1 are reduced as in Tm4sf1-heterozygous mice; a likely mechanism is that TM4SF1 regulates the intracellular distribution of signaling molecules necessary for endothelial cell proliferation and migration.
RESUMO
BACKGROUND: Novel therapies are needed for the treatment of invasive thyroid cancers. Aberrant activation of tyrosine kinases plays an important role in thyroid oncogenesis. Because current targeted therapies are biased toward a small subset of tyrosine kinases, we conducted a study to reveal novel therapeutic targets for thyroid cancer using a bead-based, high-throughput system. METHODS: Thyroid tumors and matched normal tissues were harvested from twenty-six patients in the operating room. Protein lysates were analyzed using the Luminex immunosandwich, a bead-based kinase phosphorylation assay. Data was analyzed using GenePattern 3.0 software and clustered according to histology, demographic factors, and tumor status regarding capsular invasion, size, lymphovascular invasion, and extrathyroidal extension. Survival and invasion assays were performed to determine the effect of Src inhibition in papillary thyroid cancer (PTC) cells. RESULTS: Tyrosine kinome profiling demonstrated upregulation of nine tyrosine kinases in tumors relative to matched normal thyroid tissue: EGFR, PTK6, BTK, HCK, ABL1, TNK1, GRB2, ERK, and SRC. Supervised clustering of well-differentiated tumors by histology, gender, age, or size did not reveal significant differences in tyrosine kinase activity. However, supervised clustering by the presence of invasive disease showed increased Src activity in invasive tumors relative to non-invasive tumors (60% v. 0%, p<0.05). In vitro, we found that Src inhibition in PTC cells decreased cell invasion and proliferation. CONCLUSION: Global kinome analysis enables the discovery of novel targets for thyroid cancer therapy. Further investigation of Src targeted therapy for advanced thyroid cancer is warranted.
Assuntos
Adenocarcinoma Papilar/enzimologia , Adenocarcinoma Papilar/patologia , Neoplasias da Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/patologia , Tirosina/metabolismo , Quinases da Família src/metabolismo , Adenocarcinoma Papilar/tratamento farmacológico , Proliferação de Células , Dasatinibe , Ensaios de Triagem em Larga Escala , Humanos , Metaboloma , Terapia de Alvo Molecular , Mutação , Invasividade Neoplásica , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Células Tumorais Cultivadas , Regulação para Cima , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
A subset of patients with papillary thyroid cancer (PTC) present with aggressive disease that is refractory to conventional treatment. Novel therapies are needed to treat this group of patients. Galectin-3 (Gal-3) is a beta-galactoside-binding protein with anti-apoptotic activity. Over 30 studies in the last 3 years have reported that Gal-3 is highly expressed in PTC relative to normal thyrocytes. In this study, we show that Gal-3 silencing with RNA interference stimulates apoptosis, while Gal-3 overexpression protects against both TRAIL- and doxorubicin-induced apoptosis in PTC cells. The anti-apoptotic activity and chemoresistance related to Gal-3 function can be partially reversed through the inhibition of the PI3K-Akt pathway, suggesting that Gal-3 acts, at least in part, on the PI3K-Akt axis. These observations support further evaluation of Gal-3 as a potential therapeutic target in patients with aggressive PTC.
Assuntos
Adenocarcinoma Papilar/metabolismo , Antibióticos Antineoplásicos/farmacologia , Apoptose/genética , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Galectina 3/fisiologia , Neoplasias da Glândula Tireoide/metabolismo , Adenocarcinoma Papilar/patologia , Linhagem Celular Tumoral , Galectina 3/genética , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Neoplasias da Glândula Tireoide/patologiaRESUMO
Lysophosphatidic acid (LPA) is a lipid bioactive mediator which binds to G-protein-coupled receptors and activates a variety of cellular functions. LPA modulates multiple behaviors in endothelial cells, including cell proliferation and migration, capillary-like tube formation in vitro, activation of proteases, interactions with leukocytes, and expressions of inflammation-related genes, thereby regulating vessel formation. LPA has been reported to modulate the angiogenesis process. However, the role of LPA in the lymphangiogenesis process has not been studied. In this study, we showed that LPA upregulated vascular endothelial growth factor-C (VEGF-C) mRNA expression in human umbilical vein endothelial cells (HUVECs) and subsequent endothelial cell tube formation in vitro and in vivo. These enhancement effects were LPA(1)- and LPA(3)-dependent and required cyclooxygenase-2 (COX-2), endothelial growth factor receptor (EGFR) transactivation and activation of nuclear factor kappaB (NF-kappaB). Moreover, LPA induced the protein expressions of the lymphatic markers, Prox-1, LYVE-1, and podoplanin, in HUVECs, and these enhancement effects were dependent on LPA(1) and LPA(3) activation and EGFR transactivation. Our results demonstrated that LPA might regulate VEGF-C and lymphatic marker expression in endothelial cells, which contributes to endothelial cell tube formation in vitro and in vivo, thus facilitating endothelial cell participation in the lymphangiogenesis process. This study clarifies the signaling mechanism of LPA-regulated VEGF-C expression and lymphatic marker expressions in endothelial cells, which suggest that LPA may be a suitable target for generating therapeutics against lymphangiogenesis and tumor metastasis.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Células Endoteliais/enzimologia , Lisofosfolipídeos/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Receptores de Ácidos Lisofosfatídicos/metabolismo , Regulação para Cima/efeitos dos fármacos , Fator C de Crescimento do Endotélio Vascular/genética , Biomarcadores/metabolismo , Células Cultivadas , Inibidores de Ciclo-Oxigenase/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Humanos , Modelos Biológicos , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Ativação Transcricional/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Thrombomodulin (TM) is an anticoagulant glycoprotein highly expressed on endothelial cell surfaces. Increased levels of soluble TM in circulation have been widely accepted as an indicator of endothelial damage or dysfunction. Previous studies indicated that various proinflammatory factors stimulate TM shedding in various cell types such as smooth muscle cells and epithelial cells. Lysophosphatidic acid (LPA) is a bioactive lipid mediator present in biological fluids during endothelial damage or injury. In the present study, we first observed that LPA triggered TM shedding in human umbilical vein endothelial cells (HUVECs). By Cyflow analysis, we showed that the LPA-induced accessibility of antibodies to the endothelial growth factor (EGF)-like domain of TM is independent of matrix metalloproteinases (MMPs), while LPA-induced TM lectin-like domain shedding is MMP-dependent. Furthermore, a stable cell line expressing TM without its lectin-like domain exhibited a higher cell proliferation rate than a stable cell line expressing full-length TM. These results imply that LPA induces TM lectin-like domain shedding, which might contribute to the exposure of its EGF-like domain for EGF receptor (EGFR) binding, thereby stimulating subsequent cell proliferation. Based on our findings, we propose a novel mechanism for the exposure of TM EGF-like domain, which possibly mediates LPA-induced EGFR transactivation.
Assuntos
Células Endoteliais/efeitos dos fármacos , Lectinas/metabolismo , Lisofosfolipídeos/farmacologia , Trombomodulina/metabolismo , Sítios de Ligação , Linhagem Celular , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Receptores ErbB/metabolismo , Citometria de Fluxo , Humanos , Metaloproteinases da Matriz/metabolismoRESUMO
Dioxins comprise a group of compounds which contain a double aromatic ring-like structure. They are among the most prevalent and toxic environmental pollutants. Accumulation of dioxins in human tissues poses a potential threat to human health. Currently, analytical chemical procedures dominate dioxin-detection protocols. In this study, we established a fluorescence resonance energy transfer (FRET)-based dioxin-detection bioassay. Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) fused-cyan fluorescent protein (CFP) and -yellow fluorescent protein (YFP) constructed were transiently co-transfected into rat hepatoma cell line, H4IIEC3 cells. Our results showed that no FRET signals were detected in AHR-CFP- and ARNT-YFP-transfected H4IIEC3 cells. However, dioxin treatments upregulated FRET signals in these transfected cells in a dose-dependent manner. This work highlighted the potential of FRET technique in the detection of dioxin-like compounds.
Assuntos
Bioensaio/métodos , Dioxinas/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Fluoresceínas/análise , Dibenzodioxinas Policloradas/análise , Dibenzodioxinas Policloradas/toxicidade , Ratos , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Teratogênicos/análise , Teratogênicos/toxicidadeRESUMO
Lysophosphatidic acid (LPA) is a low-molecular-weight lysophospholipid (LPL), which regulates endothelial cells participating in inflammation processes via interactions with endothelial differentiation gene (Edg) family G protein-coupled receptors. In this study, we attempted to determine which LPA receptors mediate the inflammatory response in human endothelial cells. Introduction of siRNA against LPA1 significantly suppressed LPA-induced ICAM-1 mRNA, total protein, and cell surface expressions, and subsequent U937 monocyte adhesion to LPA-treated human umbilical endothelial cells (HUVECs). By knock down of LPA1 and LPA3 in HUVECs, LPA-enhanced IL-1beta mRNA expression was significantly attenuated. Moreover, LPA1 and LPA3 siRNA also inhibited LPA-enhanced IL-1-dependent long-term IL-8 and MCP-1 mRNA expression, and subsequent THP-1 cell chemotaxis toward LPA-treated HUVEC-conditioned media. These results suggest that the expression of LPA-induced inflammatory response genes is mediated by LPA1 and LPA3. Our findings suggest the possible utilization of LPA1 or LPA3 as drug targets to treat severe inflammation.
Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Lisofosfolipídeos/farmacologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Adesão Celular , Células Cultivadas , Quimiocina CCL2/genética , Células Endoteliais/citologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1/genética , Interleucina-8/genética , Fragmentos de Peptídeos/genética , RNA Interferente Pequeno/genéticaRESUMO
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both low-molecular-weight lysophospholipids, which promote cell proliferation, migration, and invasion via interaction with a family of specific G protein-coupled receptors. Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes, which are involved in degradation of the extracellular matrix and play critical roles in endothelial cell migration and matrix remodeling during angiogenesis. Among these MMPs, MMP-2 is known to trigger cell migration. In our present study, we examined the effects of LPA and S1P on MMP-2 expression in human endothelial cells. We showed that LPA and S1P enhanced MMP-2 expression in mRNA, protein levels, and also enzymatic activity of cells of the EAhy926 human endothelial cell line. The enhancement effects occurred in concentration- and time-dependent manners. Results from real-time PCR, Western blots, and substrate gels indicated that these enhancement effects were mediated through MAPK kinase/ERK-, nuclear factor-kappaB-, and calcium influx-dependent pathways. Furthermore, we show that endothelial cell invasion of the gel was enhanced by lysophospholipids, and the induction could be prevented by an MMP inhibitor, GM6001. These observations suggest that LPA and S1P may play important roles in endothelial cell invasion by regulating the expression of MMP-2.
Assuntos
Endotélio Vascular/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Metaloproteinase 2 da Matriz/genética , Esfingosina/análogos & derivados , Linhagem Celular , Humanos , RNA Mensageiro/genética , Esfingosina/farmacologiaRESUMO
Antibody-drug conjugates (ADC) represent a promising therapeutic modality for managing cancer. Here, we report a novel humanized ADC that targets the tetraspanin-like protein TM4SF1. TM4SF1 is highly expressed on the plasma membranes of many human cancer cells and also on the endothelial cells lining tumor blood vessels. TM4SF1 is internalized upon interaction with antibodies. We hypothesized that an ADC against TM4SF1 would inhibit cancer growth directly by killing cancer cells and indirectly by attacking the tumor vasculature. We generated a humanized anti-human TM4SF1 monoclonal antibody, v1.10, and armed it with an auristatin cytotoxic agent LP2 (chemical name mc-3377). v1.10-LP2 selectively killed cultured human tumor cell lines and human endothelial cells that express TM4SF1. Acting as a single agent, v1.10-LP2 induced complete regression of several TM4SF1-expressing tumor xenografts in nude mice, including non-small cell lung cancer and pancreas, prostate, and colon cancers. As v1.10 did not react with mouse TM4SF1, it could not target the mouse tumor vasculature. Therefore, we generated a surrogate anti-mouse TM4SF1 antibody, 2A7A, and conjugated it to LP2. At 3 mpk, 2A7A-LP2 regressed several tumor xenografts without noticeable toxicity. Combination therapy with v1.10-LP2 and 2A7A-LP2 together was more effective than either ADC alone. These data provide proof-of-concept that TM4SF1-targeting ADCs have potential as anticancer agents with dual action against tumor cells and the tumor vasculature. Such agents could offer exceptional therapeutic value and warrant further investigation. Mol Cancer Ther; 14(8); 1868-76. ©2015 AACR.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores da Angiogênese/toxicidade , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Expressão Gênica , Humanos , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica , Coelhos , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 µm diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per µm and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 µm from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 °C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.
Assuntos
Comunicação Celular/fisiologia , Movimento Celular/fisiologia , Técnicas Citológicas/métodos , Pseudópodes/fisiologia , Antígenos de Superfície/análise , Linhagem Celular Tumoral , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas de Neoplasias/análise , Octoxinol/químicaRESUMO
BACKGROUND: We hypothesized that autophagy inhibition would enhance the anticancer efficacy of ret protooncogene-targeted therapy in medullary thyroid cancer. METHODS: Medullary thyroid cancer-1.1 and TT cells were treated with sunitinib or sorafenib in the presence or absence of everolimus, trehalose, or small interfering RNA directed against autophagy protein 5. RESULTS: Sunitinib and sorafenib each robustly induced light chain 3-II expression, indicating autophagy activation. Autophagy protein 5 silencing diminished the antiproliferative effects of sunitinib and sorafenib by 44% (P < .05) and 41% (P < .05), respectively, in medullary thyroid cancer-1.1 cells and by 43% (P < .01) and 39% (P < .05), respectively, in TT cells. In contrast, everolimus increased the antiproliferative effects of sunitinib and sorafenib by 24% (P < .01) and 27% (P < .01), respectively, in medullary thyroid cancer-1.1 cells and by 20% (P < .05) and 23% (P < .05), respectively, in TT cells. Trehalose increased the antiproliferative effects of sunitinib and sorafenib by 26% (P < .01) and 27% (P < .01), respectively, in medullary thyroid cancer-1.1 cells and by 28% (P < .05) and 29% (P < .05), respectively, in TT cells. Autophagy protein 5 silencing abrogated both everolimus- and trehalose-induced increases in tyrosine kinase inhibitor efficacy. CONCLUSION: Loss (gain) of autophagy diminishes (improves) the efficacy of sunitinib and sorafenib. Our findings suggest that autophagic activation should be combined with targeted ret protooncogene therapy for patients with advanced medullary thyroid cancer.
Assuntos
Antineoplásicos/uso terapêutico , Autofagia , Benzenossulfonatos/uso terapêutico , Proliferação de Células , Indóis/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/uso terapêutico , Pirróis/uso terapêutico , Neoplasias da Glândula Tireoide/patologia , Autofagia/efeitos dos fármacos , Benzimidazóis/farmacologia , Carcinoma Neuroendócrino , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Everolimo , Humanos , Imunossupressores/farmacologia , Niacinamida/análogos & derivados , Compostos de Fenilureia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Sorafenibe , Sunitinibe , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/fisiopatologiaRESUMO
BACKGROUND: Coactivation of tyrosine kinase limits the efficacy of tyrosine kinase inhibitors. We hypothesized that a strategic combination therapy could overcome tyrosine kinase coactivation and compensatory oncogenic signaling in patients with adrenocortical carcinoma (ACC). METHODS: We profiled 88 tyrosine kinases before and after treatment with sunitinib in H295R and SW13 ACC cells. The effects of monotherapy and strategic combination regimens were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (ie, MTS) assay. RESULTS: The minimum inhibitory concentrations (IC(min)) of sunitinib quenched its primary targets: FLT-3, VEGFR-2, and RET. In contrast, ERK, HCK, Chk2, YES, CREB, MEK, MSK, p38, FGR, and AXL were hyperactivated. Monotherapy with sunitinib or PD98059 at their IC(min) reduced proliferation by 23% and 19%, respectively, in H295R cells and by 25% and 24%, respectively, in SW13 cells. Sunitinib and PD98059 in combination decreased proliferation by 68% and 64% in H295R and in SW13 cells, respectively (P < .05 versus monotherapy). The effects of combination treatment exceeded the sum of the effects observed with each individual agent alone. CONCLUSION: We describe the first preclinical model to develop strategic combination therapy to overcome tyrosine kinase coactivation in ACC. Because many tyrosine kinase inhibitors are readily available, this model can be immediately tested in clinical trials for patients with advanced ACC.
Assuntos
Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Carcinoma Adrenocortical/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas Quinases Dependentes de Cálcio-Calmodulina/administração & dosagem , Flavonoides/administração & dosagem , Indóis/administração & dosagem , Proteínas Tirosina Quinases/metabolismo , Pirróis/administração & dosagem , Neoplasias do Córtex Suprarrenal/enzimologia , Carcinoma Adrenocortical/enzimologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , SunitinibeRESUMO
CONTEXT: Low tumoral expression of mitogen-inducible gene-6 (Mig-6) is associated with papillary thyroid cancer (PTC) recurrence after thyroidectomy. OBJECTIVE: We hypothesize that Mig-6 behaves as a tumor suppressor in PTC. DESIGN: Mig-6 expression and promoter methylation status were compared in 31 PTC specimens with matched normal thyroid tissue from the same patient. The impact of Mig-6 loss and gain of function on nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation, global tyrosine kinase phosphorylation, and cellular invasion was determined in vitro. RESULTS: Mig-6 protein was abundant in all normal thyroid specimens, whereas 77% of PTC had low Mig-6 expression. Mig-6 promoter methylation was found in 79% of PTC with low Mig-6 expression. Low Mig-6 expression in PTC specimens was associated with low NF-κB activity but high levels of epidermal growth factor receptor (EGFR) and ERK phosphorylation. Mig-6 expression inversely correlated with PTC size but had no association with other clinicopathological variables including age, extrathyroidal extension, lymphovascular invasion, or histological subtype. Mig-6 knockdown in thyroid cancer cell lines resulted in EGFR phosphorylation and diminished NF-κB activity, whereas Mig-6 overexpression had the opposite effects. Mig-6 knockdown activated ErbB2, Met, and Src phosphorylation. Furthermore, Mig-6 regulated ERK phosphorylation independent from its effects on EGFR. Mig-6 knockdown promoted cellular proliferation, as determined by clonogenic survival. Lastly, Mig-6 knockdown increased matrix metalloproteinase-2 and -9 activities and increased cellular invasion. CONCLUSIONS: Mig-6 has tumor suppressor-like activity in PTC. In vivo studies are required to confirm that Mig-6 is a putative tumor suppressor in PTC, and future studies should investigate the utility of Mig-6 as a diagnostic marker.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Papilar/genética , Genes Supressores de Tumor/fisiologia , Proteínas Supressoras de Tumor/genética , Western Blotting , Carcinoma , Carcinoma Papilar/patologia , Linhagem Celular Tumoral , Núcleo Celular/química , Células Cultivadas , Citosol/química , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Cultura em Câmaras de Difusão , Regulação para Baixo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , NF-kappa B/genética , Invasividade Neoplásica/genética , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Ensaio Tumoral de Célula-TroncoRESUMO
BACKGROUND: Necroptosis is a recently described mechanism of programmed cellular death. We hypothesize that necroptosis plays an important role in radiation-induced cell death in endocrine cancers. METHODS: Thyroid and adrenocortical carcinoma cell lines were exposed to increasing doses of radiation in the presence of necroptosis inhibitor Nec-1 and/or apoptosis-inhibitor zVAD. H295R cells deficient in receptor interacting protein 1 (RIP1), an essential kinase for necroptosis, were used as controls. Survival curves were generated at increasing doses of radiation. RESULTS: Nec-1 and zVAD increased cellular survival with increasing doses of radiotherapy in 8505c, TPC-1, and SW13. Both inhibitors used together had an additive effect. At 6 Gy, 8505c, TPC-1, and SW13 cell survival was significantly increased compared to controls by 40%, 33%, and 31% with Nec-1 treatment, by 53%, 47%, and 44% with zVAD treatment, and by 80%, 70%, and 65% with both compounds, respectively (P < .05). H295R showed no change in survival with Nec-1 treatment. The radiobiologic parameter quasithreshold dose was significantly increased in 8505c, TPC-1, and SW13 cells when both Nec-1 and zVAD were used in combination to inhibit necroptosis and apoptosis together, revealing resistance to standard doses of fractionated therapeutic radiation. CONCLUSION: Necroptosis contributes to radiation-induced cell death. Future studies should investigate ways to promote the activation of necroptosis to improve radiosensitivity.
Assuntos
Neoplasias do Córtex Suprarrenal/radioterapia , Morte Celular/efeitos da radiação , Neoplasias da Glândula Tireoide/radioterapia , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/fisiopatologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Carcinoma , Carcinoma Papilar , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Oligopeptídeos/farmacologia , Proteínas de Ligação a RNA/metabolismo , Câncer Papilífero da Tireoide , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/fisiopatologiaRESUMO
BACKGROUND: Mitogen-inducible gene 6 (Mig-6) is a putative tumor suppressor gene and prognostic biomarker in papillary thyroid cancer. We hypothesized that Mig-6 knockout would activate pro-oncogenic signaling in mouse thyrocytes. METHODS: We performed a thyroid-specific knockout using the Cre/loxP recombinase system. RESULTS: Four knockout and 4 control mouse thyroids were harvested at 2 months of age. Immunoblotting confirmed Mig-6 ablation in knockout mice thyrocytes. Epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) phosphorylation levels were increased in Mig-6 knockout compared to wild-type mice. Total EGFR levels were similar in knockout and wild-type mice. However, EGFR was absent in the caveolae-containing membrane fraction of knockout mice, indicating that Mig-6 depletion is associated with a change in the membrane distribution of EGFR. Although p65 localized to the nucleus in wild-type mice, it was distributed in both cytoplasm and nucleus in knockouts, suggesting that Mig-6 loss decreases p65 activity. CONCLUSION: Our results confirm the feasibility of targeted, thyroid-specific gene knockout as a strategy for studying the relevance of specific genes in thyroid oncogenesis. We suggest that the loss of Mig-6 alters the membrane distribution of EGFR, which may limit receptor degradation and activate this oncogenic signaling pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fator de Crescimento Epidérmico/metabolismo , Genes Supressores de Tumor/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais/genética , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biomarcadores/metabolismo , Carcinoma , Carcinoma Papilar , Técnicas de Inativação de Genes , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , NF-kappa B/antagonistas & inibidores , Transdução de Sinais/fisiologia , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
BACKGROUND: Activation of the Wnt/beta-catenin signaling pathway is implicated in thyroid tumorigenesis, and up to 90% of papillary thyroid cancer (PTC) demonstrate aberrant expression of beta-catenin. Nonsteroidal antiinflammatory drugs reverse aberrant beta-catenin expression and localization in colon cancer. In this study, we tested the hypothesis that the nonsteroidal antiinflammatory drug sulindac would reverse aberrant beta-catenin activity in thyroid cancer cells. METHODS: beta-catenin protein levels were determined in thyroidectomy specimens from six consecutive patients and in three different thyroid cancer cells lines (8505-C, KTC-1, and TPC-1) by immunoblotting. Cells of 8505-C and KTC-1 harbor the BRAF(V600E) mutation, and TPC-1 has the RET/PTC rearrangement. All cell lines were treated with sulindac (100 microM for up to 72 hours). Protein levels of c-myc and cyclin D1 were detected by immunoblotting, and beta-catenin localization was determined by immunocytochemistry in the PTC cell lines. PCCL3 rat thyroid cells that conditionally overexpress either BRAF(V600E) or RET/PTC were also treated with sulindac. RESULTS: All PTC specimens and cell lines expressed high levels of beta-catenin protein and displayed aberrant nuclear and cytoplasmic localization of beta-catenin. Exposure to sulindac for 48 hours reduced beta-catenin expression in 8505-C and KTC-1 cells, but not in TPC-1 cells. Further, sulindac treatment reduced c-myc and cyclin D1 levels in 8505-C and KTC-1 cells, but had no effect in TPC-1 cells. Immunocytochemistry demonstrated that sulindac treatment redistributed beta-catenin from the nucleus to the membrane in 8505-C and KTC-1 cells. However, sulindac did not affect beta-catenin localization in TPC-1 cells. Finally, sulindac was effective in decreasing beta-catenin expression and cellular proliferation in BRAF(V600E)-overexpressing cells, but not in RET/PTC3-overexpressing cells. CONCLUSIONS: Taken together, our findings demonstrate that sulindac treatment reverses beta-catenin activity in 8505-C and KTC-1 cell lines with the BRAF(V600E), but not in TPC-1 cells with the RET/PTC mutation. Future studies should investigate the potential for beta-catenin-directed therapy for patients with advanced thyroid cancers.
Assuntos
Sulindaco/farmacologia , Neoplasias da Glândula Tireoide/metabolismo , beta Catenina/genética , Adenocarcinoma Papilar/genética , Adenocarcinoma Papilar/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Ratos , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , beta Catenina/biossínteseRESUMO
Although autophagy is generally considered a prosurvival mechanism that preserves viability, there is evidence that it could drive an alternative programmed cell death pathway in cells with defects in apoptosis. Because the inhibition of autophagic activity promotes resistance to both chemotherapy and external beam radiation in papillary thyroid cancer (PTC), we determined if RAD001, a potent activator of autophagy, improves the efficacy of either therapy. We found that RAD001 increased the expression level of light chain 3-II, a marker for autophagy, as well as autophagosome formation in cell lines and in human PTC ex vivo. RAD001 sensitized PTC to doxorubicin and external beam radiation in a synergistic fashion, suggesting that combination therapy could improve therapeutic response at less toxic concentrations. The effects of RAD001 were abrogated by RNAi knockdown of the autophagy-related gene 5, suggesting that RAD001 acts, in part, by enhancing autophagy. Because the synergistic activity of RAD001 with doxorubicin and external radiation suggests distinct and complementary mechanisms of action, we characterized how autophagy modulates signaling pathways in PTC. To do so, we performed kinome profiling and discovered that autophagic activation resulted in Src phosphorylation and Met dephosphorylation. Src inhibition did not reverse the effects of RAD001, whereas Met inhibition reversed the effects of autophagy blockade on chemosensitivity. These results suggest that the anticancer effects of autophagic activation are mediated largely through Met. We conclude that RAD001 induces autophagy, which enhances the therapeutic response to cytotoxic chemotherapy and external beam radiation in PTC.