RESUMO
Müllerian inhibiting substance (MIS/AMH), produced by granulosa cells of growing follicles, is an important regulator of folliculogenesis and follicle development. Treatment with exogenous MIS in mice suppresses follicle development and prevents ovulation. To investigate the mechanisms by which MIS inhibits follicle development, we performed single-cell RNA sequencing of whole neonatal ovaries treated with MIS at birth and analyzed at postnatal day 6, coinciding with the first wave of follicle growth. We identified distinct transcriptional signatures associated with MIS responses in the ovarian cell types. MIS treatment inhibited proliferation in granulosa, surface epithelial, and stromal cell types of the ovary and elicited a unique signature of quiescence in granulosa cells. In addition to decreasing the number of growing preantral follicles, we found that MIS treatment uncoupled the maturation of germ cells and granulosa cells. In conclusion, MIS suppressed neonatal follicle development by inhibiting proliferation, imposing a quiescent cell state, and preventing granulosa cell differentiation.
Assuntos
Hormônio Antimülleriano/farmacologia , Ovário/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Diferenciação Celular/efeitos dos fármacos , Feminino , Inibinas/análise , Camundongos , Camundongos Endogâmicos C57BL , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Ovário/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Peptídeos/análise , Receptores de Fatores de Crescimento Transformadores beta/análise , Análise de Sequência de RNA , Análise de Célula Única , Transcrição Gênica/efeitos dos fármacosRESUMO
Anti-Müllerian hormone (AMH) is a glycoprotein produced by granulosa cells of preantral and small antral follicles that has multiple important roles in the ovaries. Recent studies have revealed extragonadal AMH regulation of gonadotrophin secretion from bovine gonadotrophs. In this study we investigated whether the primary receptor for AMH, AMH receptor type 2 (AMHR2), is expressed in bovine oviducts and endometria. Reverse transcription-polymerase chain reaction detected expression of AMHR2 mRNA in oviductal and endometrial specimens. Western blotting and immunohistochemistry were performed to analyse AMHR2 protein expression using anti-bovine AMHR2 antibody. Immunohistochemistry revealed robust AMHR2 expression in the tunica mucosa of the ampulla and isthmus, as well as in the glandular and luminal epithelium of the endometrium. AMHR2 mRNA (measured by real-time polymerase chain reaction) and AMHR2 protein expression in these layers did not significantly differ among oestrous phases in adult Wagyu cows (P>0.1). In addition, AMHR2 mRNA and protein expression in these layers did not differ among old Holsteins (mean (±s.e.m.) age 91.9±6.4 months) and young (26.6±0.8 months) and old (98.8±10.2 months) Wagyu cows. Therefore, AMHR2 is expressed in bovine oviducts and endometria.
Assuntos
Envelhecimento/metabolismo , Bovinos/metabolismo , Endométrio/química , Tubas Uterinas/química , RNA Mensageiro/análise , Receptores de Peptídeos/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Animais , Hormônio Antimülleriano/sangue , Bovinos/genética , Ciclo Estral/fisiologia , Feminino , Expressão Gênica , Receptores de Peptídeos/análise , Receptores de Fatores de Crescimento Transformadores beta/análise , Especificidade da EspécieRESUMO
Angiosarcoma is a rare malignant tumor derived from endothelial cells, and its prognosis is poor because advanced angiosarcoma is often resistant to taxane therapy. Endoglin (CD105) acts as a coreceptor for TGF-ß signaling and is overexpressed in tumor-associated endothelial cells and enhances tumor angiogenesis. Numerous clinical trials are testing the effectiveness of anti-endoglin antibodies in various types of malignancies. Here, we investigated the role of endoglin in the pathogenesis of angiosarcoma and whether endoglin inhibition results in antitumor activity. Endoglin was overexpressed in angiosarcoma, and its inhibition was effective in promoting apoptosis and the suppression of migration, invasion, tube formation, and Warburg effect in angiosarcoma cells. Knockdown of endoglin activated caspase 3/7 that is essential for apoptosis, reduced survivin levels, and decreased paxillin and vascular endothelial cadherin phosphorylation and matrix metalloproteinase 2 and matrix metalloproteinase 9 activities in angiosarcoma cells. Although endoglin is a coreceptor that regulates TGF-ß signaling, the antitumor effect of endoglin in angiosarcoma was not based on Smad signaling regulation but on non-Smad TGF-ß signaling. Taken together, these results indicated that endoglin could be a novel therapeutic target for angiosarcoma.
Assuntos
Endoglina/fisiologia , Hemangiossarcoma/etiologia , Fator de Crescimento Transformador beta/fisiologia , Linhagem Celular Tumoral , Endoglina/antagonistas & inibidores , Hemangiossarcoma/tratamento farmacológico , Hemangiossarcoma/patologia , Humanos , Metaloproteinases da Matriz/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/análise , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The dynamics and stoichiometry of receptors newly delivered on the plasma membrane play a vital role in cell signal transduction, yet knowledge of this process is limited because of the lack of suitable analytical methods. Here we developed a new strategy that combines single-molecule imaging (SMI) and fluorescence recovery after photobleaching (FRAP), named FRAP-SMI, to monitor and quantify individual newly delivered and inserted transmembrane receptors on plasma membranes of living cells. Transforming-growth-factor-ß type II receptor (TßRII), a typical serine/threoninekinase receptor, was studied with this method. We first eliminated the fluorescence signals from the pre-existing EGFP-labeled TßRII molecules on the plasma membrane, and then we recorded the individual newly appeared TßRII-GFP by total-internal-reflection fluorescence imaging. The fluorescence-intensity distributions, photobleaching steps, and diffusion rates of the single TßRII-GFP molecules were analyzed. We reported, for the first time, that TßRII was transported to the plasma membrane mainly in the monomeric form in both resting and TGF-ß1stimulated cells. This strongly supported our former discovery that TßRII could exist as a monomer on the cell membrane. We also found that ligand stimulation resulted in enhanced delivery rates and prolonged membrane-association times for the TßRII molecules. On the basis of these observations, we proposed a mechanism of TGF-ß1-induced TßRII dimerization for receptor activation. Our method provides a useful tool for the real-time quantification of the spatial arrangement, mobility, and oligomerization of cell-surface proteins in living cells, thus providing a better understanding of cell signaling.
Assuntos
Membrana Celular/metabolismo , Imagem Óptica , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Imagem Individual de Molécula , Membrana Celular/química , Células HeLa , Humanos , Receptores de Fatores de Crescimento Transformadores beta/análise , Células Tumorais CultivadasRESUMO
Oral squamous cell carcinoma (OSCC) is the most common type of malignant cancer affecting the oral cavity. Tumor associated macrophages (TAMs) play a vital role in the initiation, progression and metastasis of OSCC. In this study, we investigated the correlation between macrophages and several clinical and pathological indicators, and we also explored how transforming growth factor-ß1 (TGF-ß1) effect on VEGF expression in TAMs. Seventy-two paraffin-embedded OSCC samples were collected. Association between macrophages density, micro vascular density (MVD) and clinical-pathological feature were explored by immunohistochemical staining. Western blot, ELISA and qRT-PCR were conducted to assess the VEGF expression in TAMs treated with or without neutralizing TGF-ß1, TßRII and smad3 antibodies. Results showed that CD68+ macrophages were absent in normal tissues. Macrophages density was directly correlated to low pathological differentiation, late clinical staging and poor survival rate. MVD showed positive correlation with clinical staging and macrophages density. Furthermore, OSCC-associated macrophages expressed more VEGF than macrophages in healthy lymph nodes. However, when TGF-ß1 or TßRII were neutralized or the Smad3 was inhibited, VEGF expression was down regulated as well. It is concluded that TGF-ß1 could promote OSCC-associated macrophages to secrete more VEGF via TßRII/Smad3 signaling pathway. This result might explain the correlation between macrophages density and worse clinical-pathological condition.
Assuntos
Carcinoma de Células Escamosas/patologia , Macrófagos/patologia , Neoplasias Bucais/patologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismo , Humanos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Prognóstico , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/análise , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad3/análise , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
Androgens are required for normal male sex differentiation and development of male secondary sexual characteristics. Mutations in AR gene are known to cause defects in male sexual differentiation. In current study, we enrolled a 46,XY phenotypically female patient bearing testes in inguinal canal. DNA sequencing of the AR gene detected a missense mutation C.1715A > G (p. Y572C) in exon 2 which is already known to cause complete androgen insensitivity syndrome (CAIS). We focused on the effects of this mutation on the testicular histopathology of the patient. Surface spreading of testicular tissues showed an absence of spermatocytes while H&E staining showed that seminiferous tubules predominantly have only Sertoli cells. This meiotic failure is likely due to the effect of the AR mutation which ultimately leads to Sertoli cell only syndrome. Tubules were stained with SOX9 and AMH which revealed Sertoli cells maturation arrest. Western blot and realtime PCR data showed that patient had higher levels of AMH, SOX9 and inhibin-B in the testis. Therefore, we suggest that the dysfunctioning of AR by mutation enhances AMH expression which ultimately leads to the failure in maturation of Sertoli cells.
Assuntos
Síndrome de Resistência a Andrógenos/diagnóstico , Receptores Androgênicos/genética , Receptores de Peptídeos/análise , Receptores de Fatores de Crescimento Transformadores beta/análise , Testículo/química , Testículo/patologia , Adulto , Síndrome de Resistência a Andrógenos/genética , Estudos de Casos e Controles , Feminino , Disgenesia Gonadal 46 XY , Humanos , Inibinas/análise , Masculino , Mutação de Sentido Incorreto , Fatores de Transcrição SOX9/análise , Túbulos Seminíferos/patologia , Células de Sertoli , EspermatócitosRESUMO
The transforming growth factor-ß (TGFß) plays a dual role in breast cancer, acting as a tumor suppressor in early carcinomas while promoting tumor metastasis in more advanced breast carcinoma. As a result, the prognostic role of TGFß and its signaling components in breast cancer remains unclear. Here we evaluated the expression levels of TGFß signaling receptors TßRII and TßRI using human breast cancer tissue microarrays and a large publicly available gene profiling database in relation to various clinicopathological parameters. Our results indicate that breast cancer tissues express lower TßRII and TßRI protein levels compared with normal breast tissue. In contrast to TßRI expression, TßRII mRNA expression levels were also significantly downregulated in invasive breast cancer compared with normal breast tissue (4.18-fold downregulation, P=9.3×10-115). Interestingly, within the cancer cases analyzed, our results revealed a direct correlation between high TßRII and TßRI expression levels and classic poor prognostic clinicopathological parameters, including larger tumor size, advanced tumor stage, and poorly differentiated tumors. Next, we examined TGFß receptors' expression in relation to breast cancer molecular subtypes. Importantly, our results revealed that whereas expression of TGFß receptors in luminal A and triple-negative breast cancer showed no correlation with patient outcome, their expression in luminal B and HER2 subtypes showed significant association with favorable patient outcome. Together, these results indicate that although TGFß receptors are downregulated in breast cancer, their expression in tumors is an indicator of aggressive breast cancer phenotype. Moreover, the relation between TGFß pathway and patient outcome is breast cancer subtype dependent.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/química , Proteínas Serina-Treonina Quinases/análise , Receptores de Fatores de Crescimento Transformadores beta/análise , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Diferenciação Celular , Linhagem Celular Tumoral , Bases de Dados Genéticas , Intervalo Livre de Doença , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estadiamento de Neoplasias , Fenótipo , Valor Preditivo dos Testes , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Fatores de Tempo , Análise Serial de Tecidos , Carga TumoralRESUMO
BACKGROUND: Transforming growth factor beta (TGFß) signalling is involved in both tumour suppression and tumour progression. The mRNA expression levels of the TGFß isoforms and receptors in breast tumours may have prognostic value and clinical implications. METHODS: The mRNA levels of TGFB1, TGFB2, TGFB3, TGFBR1 and TGFBR2 were analysed in primary breast tumours and adjacent normal breast tissues, and the associations with tumour characteristics and patients' overall and relapse-free survival were evaluated, using the public gene expression microarray data from The Cancer Genome Atlas (n = 520) and the Gene Expression Omnibus (four datasets) and our quantitative real-time PCR validation data (n = 71). RESULTS: Significantly higher TGFB1 and TGFB3 mRNA levels and lower TGFBR2 mRNA levels were observed in primary tumours compared with their paired normal tissues. TGFB1 mRNA expression was seemly lower in triple-negative tumours and in tumours from lymph node-negative patients. TGFB3 mRNA expression was significantly lower in estrogen receptor-negative/progesterone receptor-negative/Basal-like/Grade 3 tumours. High TGFB2, TGFB3 and TGFBR2 mRNA levels in tumours were generally associated with better prognosis for patients, especially those diagnosed with lymph node-negative diseases. High TGFBR1 mRNA levels in tumours were associated with poorer clinical outcomes for patients diagnosed with small (diameter ≤ 2 cm) tumours. CONCLUSIONS: The results indicate a reduced responsiveness of tumour cells to TGFß, a preferential up-regulation of TGFB1 in malignant tumours and a preferential up-regulation of TGFB3 in premalignant tumours. The results may not only provide prognostic value for patients but also assist in classifying tumours according to their potential responses to TGFß and selecting patients for TGFß signalling pathway targeted therapies.
Assuntos
Neoplasias da Mama/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Fator de Crescimento Transformador beta/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Prognóstico , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/análise , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/metabolismoRESUMO
Appropriate cellular signaling is essential to control cell proliferation, differentiation, and cell death. Aberrant signaling can have devastating consequences and lead to disease states, including cancer. The transforming growth factor-ß (TGF-ß) signaling pathway is a prominent signaling pathway that has been tightly regulated in normal cells, whereas its deregulation strongly correlates with the progression of human cancers. The regulation of the TGF-ß signaling pathway involves a variety of physiological regulators. Many of these molecules act to alter the activity of Smad proteins. In contrast, the number of molecules known to affect the TGF-ß signaling pathway at the receptor level is relatively low, and there are no known direct modulators for the TGF-ß type II receptor (TßRII). Here we identify SPSB1 (a Spry domain-containing Socs box protein) as a novel regulator of the TGF-ß signaling pathway. SPSB1 negatively regulates the TGF-ß signaling pathway through its interaction with both endogenous and overexpressed TßRII (and not TßRI) via its Spry domain. As such, TßRII and SPSB1 co-localize on the cell membrane. SPSB1 maintains TßRII at a low level by enhancing the ubiquitination levels and degradation rates of TßRII through its Socs box. More importantly, silencing SPSB1 by siRNA results in enhanced TGF-ß signaling and migration and invasion of tumor cells.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Inativação Gênica , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/análise , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/análise , Proteínas Supressoras da Sinalização de Citocina/análise , Proteínas Supressoras da Sinalização de Citocina/genética , Ativação Transcricional , UbiquitinaçãoRESUMO
INTRODUCTION: Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-ß superfamily, playing a role in sexual differentiation and recruitment. Since a correlation exists between AMH serum levels in cord blood and fetal sex, the present study aimed to identify mRNA and protein expression of AMH and AMHRII in placenta and fetal membranes according to fetal sex. METHODS: Placenta and fetal membranes samples (n = 40) were collected from women with singleton uncomplicated pregnancies at term. Identification of AMH protein in placenta and fetal membranes was carried out by immunohistochemistry and AMH and AMHRII protein localization by immunofluorescence, while mRNA expression was assessed by quantitative real-time PCR. RESULT: AMH and AMHRII mRNAs were expressed by placenta and fetal membranes at term, without any significant difference between males and females. Placental immunostaining showed a syncytial localization of AMH without sex-related differences; while fetal membranes immunostaining was significantly more intense in male than in female fetuses (p < 0,01). Immunofluorescence showed an intense co-localization of AMH and AMHRII in placenta and fetal membranes. DISCUSSION: The present study for the first time demonstrated that human placenta and fetal membranes expresses and co-localizes AMH and AMHRII. Although no sex-related difference was found for the mRNA expression both in placenta and fetal membranes, a most intense staining for AMH in male fetal membranes supports AMH as a gender specific hormone.
Assuntos
Hormônio Antimülleriano/genética , Membranas Extraembrionárias/metabolismo , Placenta/metabolismo , Receptores de Peptídeos/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Caracteres Sexuais , Hormônio Antimülleriano/análise , Membranas Extraembrionárias/química , Feminino , Imunofluorescência , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Placenta/química , Gravidez , RNA Mensageiro/análise , Receptores de Peptídeos/análise , Receptores de Fatores de Crescimento Transformadores beta/análiseRESUMO
Coronary artery aneurysms (CAA) remain an important complication of Kawasaki disease (KD), the most common form of pediatric acquired heart disease in developed countries. Potentially life-threatening CAA develop in 25% of untreated children and 5% of children treated with high-dose intravenous immunoglobulin during the acute phase of the self-limited vasculitis. Noncoronary artery aneurysms (NCAA) in extraparenchymal, muscular arteries occur in a minority of patients with KD who also have CAA, yet little is understood about their formation and remodeling. We postulated that activation of the transforming growth factor-ß (TGF-ß) pathway in KD may influence formation and remodeling of aneurysms in iliac, femoral, and axillary arteries, the most common sites for NCAA. We studied a resected axillary artery from one adult and endarterectomy tissue from the femoral artery from a second adult, both with a history of CAA and NCAA following KD in infancy. Histology of the axillary artery aneurysm revealed destruction of the internal elastic lamina and recanalization of organized thrombus, while the endarterectomy specimen showed dense calcification and luminal myofibroblastic proliferation. Immunohistochemistry for molecules in the TGF-ß signaling pathway revealed increased expression of TGF-ß2, TGF-ß receptor 2, and phosphorylated SMAD3. These findings suggest ongoing tissue remodeling of the aneurysms decades after the acute injury and demonstrate the importance of the TGF-ß signaling pathway in this process.
Assuntos
Aneurisma/diagnóstico , Aneurisma/etiologia , Artéria Axilar/química , Artéria Femoral/química , Síndrome de Linfonodos Mucocutâneos/complicações , Transdução de Sinais , Fator de Crescimento Transformador beta2/análise , Adulto , Aneurisma/metabolismo , Aneurisma/cirurgia , Artéria Axilar/patologia , Artéria Axilar/cirurgia , Endarterectomia , Feminino , Artéria Femoral/patologia , Artéria Femoral/cirurgia , Humanos , Imuno-Histoquímica , Masculino , Síndrome de Linfonodos Mucocutâneos/diagnóstico , Síndrome de Linfonodos Mucocutâneos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/análise , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/análise , Proteína Smad3/análise , Remodelação Vascular , Adulto JovemRESUMO
BACKGROUND: TGF-ß plays an important role in growth and development but is also involved in scarring and fibrosis. Differences for this growth factor are known between scarless fetal wound healing and adult wound healing. Nonetheless, most of the data in this area are from animal studies or in vitro studies and, thus, information about the human situation is incomplete and scarce. OBJECTIVE: The aim of this study was to compare the canonical TGF-ß signaling in unwounded human fetal and adult skin. METHODS: Q-PCR, immunohistochemistry, Western Blot and Luminex assays were used to determine gene expression, protein levels and protein localization of components of this pathway in healthy skin. RESULTS: All components of the canonical TGF-ß pathway were present in unwounded fetal skin. Compared to adult skin, fetal skin had differential concentrations of the TGF-ß isoforms, had high levels of phosphorylated receptor-Smads, especially in the epidermis, and had low expression of several fibrosis-associated target genes. Further, the results indicated that the processes of receptor endocytosis might also differ between fetal and adult skin. CONCLUSION: This descriptive study showed that there are differences in gene expression, protein concentrations and protein localization for most components of the canonical TGF-ß pathway between fetal and adult skin. The findings of this study can be a starting point for further research into the role of TGF-ß signaling in scarless healing.
Assuntos
Expressão Gênica , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Pele/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adolescente , Adulto , Caveolinas/análise , Caveolinas/genética , Clatrina/análise , Clatrina/genética , Colágeno Tipo III/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Decorina/genética , Endocitose/fisiologia , Feto , Idade Gestacional , Humanos , Proteínas de Ligação a TGF-beta Latente/genética , Proteínas de Ligação a TGF-beta Latente/metabolismo , Pessoa de Meia-Idade , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Serina-Treonina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/análise , Receptores de Fatores de Crescimento Transformadores beta/genética , Pele/química , Proteínas Smad/análise , Proteínas Smad/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta2/análise , Fator de Crescimento Transformador beta3/análise , Adulto JovemRESUMO
BACKGROUND: A new diagnostic and prognostic biomarker may be of value in cancer diseases. Our study aimed to evaluate the CDKN1A/p21 and TGFBR2 level measurable in a cohort of patients with breast cancer after mastectomy, and to confirm their suitability to serve as prognostic biomarkers of the cancer. METHODS: The expression levels of CDKN1A/p21 and TGFBR2 were detected by reverse transcription-PCR (RT-PCR), western blot assay and immunohistochemical staining for 65 primary tumor samples and paired adjacent noncancerous breast tissues. Their relations to clinicopathologic parameters and to the prognosis of patients with breast cancer were analyzed. RESULTS: We found the mRNA and protein expression levels of CDKN1A/p21 were significantly upregulated in breast cancer tissues compared with adjacent nontumorous breast tissues. Increased CDKN1A/p21 expression showed a significant correlation with larger tumor size (P=0.014), higher tumor dedifferentiation grade (P=0.021), lymph node metastasis (P=0.019) and a shorter disease-free survival (P=0.044). Contrarily, the expression levels of TGFBR2 mRNA and protein were significantly decreased in breast cancer tissues compared with adjacent nontumorous breast tissues. Underexpression of TGFBR2 in breast cancer was correlated with larger tumor size (P=0.034), lymph node metastasis (P=0.039) and a shorter disease-free survival (P=0.035). Statistical analysis suggested that there was no significant association between CDKN1A/p21 and TGFBR2 expression. CONCLUSIONS: in summary, our results suggested that high CDKN1A/p21 and low TGFBR2 expression was closely correlated with adverse pathological parameters and poor prognosis in breast cancer. Both CDKN1A/p21 and TGFBR2 are presented as possible candidates for breast cancer biomarkers.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Adulto , Idoso , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Inibidor de Quinase Dependente de Ciclina p21/análise , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Serina-Treonina Quinases/análise , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Increased colorectal epithelial cell proliferation is an early, common event in colorectal carcinogenesis. We conducted a pilot, colonoscopy-based case-control study (n = 49 cases, 154 controls) of incident, sporadic colorectal adenoma to investigate endogenous cell growth factors and receptor, as well as the balance of growth factors, as potential modifiable pre-neoplastic biomarkers of risk for colorectal neoplasms. We measured transforming growth factor alpha (TGFα), TGFß(1), and TGFß receptor II (TGFßRII) expression in normal-appearing mucosa from the rectum, sigmoid colon, and ascending colon using automated immunohistochemistry and quantitative image analysis. Diet and lifestyle were assessed via questionnaires. The mean ratio of rectal TGFα to TGFß(1) expression and mean rectal TGFα expression were, respectively, 110% (P = 0.02) and 49% (P = 0.04) higher in cases than in controls, and associated with a more than two-fold (OR 2.42, 95% CI 0.85-6.87) and a 62% (OR 1.62, 95% CI 0.63-4.19) higher risk of colorectal adenoma. TGFß(1) and TGFßRII expression were 6.7% (P = 0.75) and 7.2% (P = 0.49), respectively, lower in cases than in controls. The TGFα/TGFß(1) expression ratio was 105% higher among smokers than among non-smokers (P = 0.03). These preliminary data suggest that the balance of TGFα and TGFß(1) expression, and to a lesser extend TGFα alone, in the normal-appearing rectal mucosa may be directly associated with risk for incident, sporadic colorectal neoplasms, as well as with modifiable risk factors for colorectal neoplasms.
Assuntos
Colo/patologia , Neoplasias Colorretais/patologia , Receptores de Fatores de Crescimento Transformadores beta/análise , Reto/patologia , Fator de Crescimento Transformador alfa/análise , Fator de Crescimento Transformador beta1/análise , Adulto , Idoso , Biomarcadores Tumorais/análise , Estudos de Casos e Controles , Neoplasias Colorretais/epidemiologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Our aim was to evaluate serum levels of anti-Müllerian hormone (AMH) and also immunohistochemical (IHC) staining properties of AMH receptor type II (AMHRII) in patients with endometrial cancer (EC) and a control group. Preoperatively, serum levels of AMH were assessed and AMHRII expression was evaluated by immunohistochemistry in a benign and malignant group. AMH serum levels of the control group and EC patients were comparable. For EC patients, there was no difference with respect to the AMH levels and tumour stage; grade; histological type; deep myometrial invasion; lymphovascular space invasion or lymph node involvement. However, AMH levels in patients with extrauterine involvement were higher than patients with disease confined to the uterus. EC samples were more likely to be stained positive for AMHRII than benign lesions. Also, as the stage of the lesion worsens, the rate of IHC staining of AMHRII decreases. In conclusion, AMHRII is expressed in normal endometrial cells as well as endometrial cancer cells. AMH levels increase in EC, with extrauterine involvement at least in locally advanced disease. Also AMH expression decreases as the disease is staged-up.
Assuntos
Hormônio Antimülleriano/sangue , Carcinoma Endometrioide/sangue , Carcinoma Papilar/sangue , Carcinossarcoma/sangue , Neoplasias do Endométrio/sangue , Neoplasias do Endométrio/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Endometrioide/secundário , Carcinoma Papilar/secundário , Carcinossarcoma/secundário , Estudos de Casos e Controles , Neoplasias do Endométrio/química , Endométrio/química , Feminino , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Estudos Prospectivos , Receptores de Peptídeos/análise , Receptores de Fatores de Crescimento Transformadores beta/análiseRESUMO
BACKGROUND: The pathogenesis of idiopathic pulmonary fibrosis (IPF) in dogs is poorly understood. In human, transforming growth factor ß1 (TGF-ß1) is considered central in the pathogenesis. OBJECTIVES: To investigate TGF-ß1 pathway in IPF. ANIMALS: Lung tissues from 12 affected and 11 control dogs. Serum from 16 affected West Highland white Terriers (WHWTs) and healthy dogs from predisposed (13 WHWTs, 12 Scottish Terriers and 13 Bichons Frise) and nonpredisposed breeds (10 Whippets, 10 Belgian shepherds, 8 Labradors). METHODS: In this prospective study, immunohistochemistry was used to evaluate expression and localization of TGF-ß1 protein and proteins involved in TGF-ß1 signaling (TGF-ß receptor type I and phospho-Smad2/3). Pulmonary expression of TGF-ß1 and molecules involved in its storage (latent TGF-ß binding proteins [LTBP] 1, 2, and 4), activation (ανß6 and ανß8 integrins, thrombospondin-1) and signal inhibition (Smad 7) was analyzed by quantitative reverse transcriptase PCR. Circulating TGF-ß1 concentration was measured by ELISA. RESULTS: In IPF, high level of TGF-ß1 protein was found in areas of fibrosis, epithelial cells had strong expression of TGF-ß receptor type 1 and phospho-Smad2/3, gene expression was decreased for LTBP 4 (P = .009) and ß8 integrin (P < .001) and increased for thrombospondin-1 (P = .016); no difference was seen for Smad7, LTBP1 and 2. Serum TGF-ß1 concentration was higher in predisposed compared with nonpredisposed breeds (P < .0001). CONCLUSIONS AND CLINICAL IMPORTANCE: This study identified an enhanced TGF-ß1 signaling activity in IPF. TGF-ß1 storage and activation proteins with altered expression represent potential therapeutic targets. Higher circulating TGF-ß1 concentration in predisposed breeds might partly explain their susceptibility for IPF.
Assuntos
Doenças do Cão/etiologia , Fibrose Pulmonar Idiopática/veterinária , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Animais , Estudos de Casos e Controles , Doenças do Cão/fisiopatologia , Cães , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/fisiopatologia , Pulmão/química , Pulmão/metabolismo , Pulmão/fisiopatologia , Receptores de Fatores de Crescimento Transformadores beta/análise , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/sangue , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Surface-enhanced Raman scattering (SERS) technique is becoming highly popular for multiplex biosensing due to the 'fingerprint' Raman spectra from every molecule. As a proof-of-concept, we demonstrated the actively targeted multiplex in vitro and in vivo detection of three intrinsic cancer biomarkers - EGFR, CD44 and TGFßRII in a breast cancer model using three multiplexing capable, biocompatible SERS nanoparticles/nanotags. Intra-tumorally injected antibody conjugated nanotags specifically targeting the three biomarkers exhibited maximum signal at 6 hours and no detectable signal at 72 hours. However, nanotags without antibodies showed no detectable signal after 6 hours. This difference could be due to the specific binding of the bioconjugated nanotags to the receptors on the cell surface. Thus, this study establishes SERS nanotags as an ultrasensitive nanoprobe for the multiplex detection of biomarkers and opens up its potential application in monitoring tumor progression and therapy and development into a theranostic probe.
Assuntos
Materiais Biocompatíveis/metabolismo , Biomarcadores Tumorais/análise , Nanopartículas/química , Análise Espectral Raman , Animais , Anticorpos Imobilizados/imunologia , Materiais Biocompatíveis/química , Biomarcadores Tumorais/imunologia , Linhagem Celular Tumoral , Receptores ErbB/análise , Receptores ErbB/imunologia , Feminino , Humanos , Receptores de Hialuronatos/análise , Receptores de Hialuronatos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/diagnóstico , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/análise , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Transplante HeterólogoRESUMO
Although glycoproteomics is greatly developed in recent years, our knowledge about N-glycoproteome of human tissues is still very limited. In this study, we comprehensively mapped the N-glycosylation sites of human liver by combining click maltose-hydrophilic interaction chromatography (HILIC) and the improved hydrazide chemistry. The specificity could be as high as 90% for hydrazide chemistry and 80% for HILIC. Altogether, we identified 14,480 N-glycopeptides matched with N-!P-[S|T|C] sequence motif from human liver, corresponding to 2210 N-glycoproteins and 4783 N-glycosylation sites. These N-glycoproteins are widely involved into different types of biological processes, such as hepatic stellate cell activation and acute phase response of human liver, which all highly associate with the progression of liver diseases. Moreover, the exact N-glycosylation sites of some key-regulating proteins within different human liver physiological processes were also obtained, such as E-cadherin, transforming growth factor beta receptor and 29 members of G protein coupled receptors family.
Assuntos
Glicoproteínas/análise , Hidrazinas/química , Fígado/química , Mapeamento de Peptídeos/métodos , Motivos de Aminoácidos , Caderinas/análise , Caderinas/química , Cromatografia/métodos , Feminino , Glicoproteínas/química , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fígado/metabolismo , Masculino , Dados de Sequência Molecular , Proteômica , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/química , Receptores de Fatores de Crescimento Transformadores beta/análise , Receptores de Fatores de Crescimento Transformadores beta/químicaRESUMO
The widespread use of sulfur mustard (SM) as a chemical warfare agent in the past century has proved its long-lasting toxic effects. Despite a lot of research over the past decades on Iranian veterans, there are still major gaps in the SM literature. Transforming growth factor (TGF-ß), a cytokine that affects many different cell processes, has an important role in the lungs of patients with some of chronic airway diseases, especially with respect to airway remodeling in mustard lung. Primary airway fibroblasts from epibronchial biopsies were cultured, and gene expression of TGF-ß1, TGF-ß2, TbR-I and TbR-II in fibroblasts of SM injured patients and controls were investigated. Expression of TGF-ßs and receptors was measured by RT-PCR. Protein level of TGF-ß1 was surveyed by western blot. Our findings revealed that expression levels of TGF-ß1, TGF-ß2, TbR-I and TbR-II were upregulated in the airway fibroblasts of SM exposed patients in comparison with control samples. TGF-ß1 expression was shown to be markedly increased in primary lung fibroblasts of chemically injured patients. Our novel data, suggested that over-expression of TGF-ß molecule and receptors in primary airway fibroblasts of mustard gas injured patients may be involved in progression of airway remodeling of these patients.
Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Substâncias para a Guerra Química/efeitos adversos , Fibroblastos/efeitos dos fármacos , Gás de Mostarda/efeitos adversos , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Adulto , Western Blotting , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Broncoscopia , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Inalação , Pessoa de Meia-Idade , Receptores de Fatores de Crescimento Transformadores beta/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/análiseRESUMO
BACKGROUND: Bronchopulmonary dysplasia (BPD) is one of the most common complications after preterm birth and is associated with intrauterine exposure to bacteria. Transforming growth factor-ß (TGFß) is implicated in the development of BPD. OBJECTIVES: We hypothesized that different and/or multiple bacterial signals could elicit divergent TGFß signaling responses in the developing lung. METHODS: Time-mated pregnant Merino ewes received an intra-amniotic injection of lipopolysaccharide (LPS) and/or Ureaplasma parvum serovar 3 (UP) at 117 days' and/or 121/122 days' gestational age (GA). Controls received an equivalent injection of saline and or media. Lambs were euthanized at 124 days' GA (term = 150 days' GA). TGFß1, TGFß2, TGFß3, TGFß receptor (R)1 and TGFßR2 protein levels, Smad2 phosphorylation and elastin deposition were evaluated in lung tissue. RESULTS: Total TGFß1 and TGFß2 decreased by 24 and 51% after combined UP+LPS exposure, whereas total TGFß1 increased by 31% after 7 days' LPS exposure but not after double exposures. Alveolar expression of TGFßR2 decreased 75% after UP, but remained unaltered after double exposures. Decreased focal elastin deposition after single LPS exposure was prevented by double exposures. CONCLUSIONS: TGFß signaling components and elastin responded differently to intrauterine LPS and UP exposure. Multiple bacterial exposures attenuated TGFß signaling and normalized elastin deposition.