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Extracellular vesicles (EVs) are small, membranous structures involved in intercellular communication. Here, we analyzed the effects of thyroid cancer-derived EVs on the properties of normal thyroid cells and cells contributing to the tumor microenvironment. EVs isolated from thyroid cancer cell lines (CGTH, FTC-133, 8505c, TPC-1 and BcPAP) were used for treatment of normal thyroid cells (NTHY), as well as monocytes and endothelial cells (HUVEC). EVs' size/number were analyzed by flow cytometry and confocal microscopy. Gene expression, protein level and localization were investigated by qRT-PCR, WB and ICC/IF, respectively. Proliferation, migration and tube formation were analyzed. When compared with NTHY, CGTH and BcPAP secreted significantly more EVs. Treatment of NTHY with cancer-derived EVs changed the expression of tetraspanin genes, but did not affect proliferation and migration. Cancer-derived EVs suppressed tube formation by endothelial cells and did not affect the phagocytic index of monocytes. The number of 6 µm size fraction of cancer-derived EVs correlated negatively with the CD63 and CD81 expression in NTHY cells, as well as positively with angiogenesis in vitro. Thyroid cancer-derived EVs can affect the expression of tetraspanins in normal thyroid cells. It is possible that 6 µm EVs contribute to the regulation of NTHY gene expression and angiogenesis.
Assuntos
Vesículas Extracelulares , Neoplasias da Glândula Tireoide , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Tetraspaninas/metabolismo , Neoplasias da Glândula Tireoide/patologia , Microambiente TumoralRESUMO
Transcription factor Prospero homeobox 1 (PROX1) is continuously expressed in the lymphatic endothelial cells, playing an essential role in their differentiation. Many reports have shown that PROX1 is implicated in cancer development and acts as an oncoprotein or suppressor in a tissue-dependent manner. Additionally, the PROX1 expression in many types of tumors has prognostic significance and is associated with patient outcomes. In our previous experimental studies, we showed that PROX1 is present in the thyroid cancer (THC) cells of different origins and has a high impact on follicular thyroid cancer (FTC) phenotypes, regulating migration, invasion, focal adhesion, cytoskeleton reorganization, and angiogenesis. Herein, we discuss the PROX1 transcript and protein structures, the expression pattern of PROX1 in THC specimens, and its epigenetic regulation. Next, we emphasize the biological processes and genes regulated by PROX1 in CGTH-W-1 cells, derived from squamous cell carcinoma of the thyroid gland. Finally, we discuss the interaction of PROX1 with other lymphatic factors. In our review, we aimed to highlight the importance of vascular molecules in cancer development and provide an update on the functionality of PROX1 in THC biology regulation.
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Adenocarcinoma Folicular/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Neoplasias da Glândula Tireoide/genética , Proteínas Supressoras de Tumor/genética , Adenocarcinoma Folicular/metabolismo , Adenocarcinoma Folicular/patologia , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: Podoplanin (PDPN) is a mucin-type transmembrane glycoprotein specific to the lymphatic system. PDPN expression has been found in various human tumors and is considered to be a marker of cancer. We had previously shown that PDPN expression contributes to carcinogenesis in the TPC1 papillary thyroid cancer-derived cell line by enhancing cell migration and invasiveness. The aim of this study was to determine the effect of PDPN down-regulation in another thyroid cancer-derived cell line: BcPAP. METHODS: In order to determine the effects of PDPN on malignant features of BcPAP cells (harboring the BRAFV600E mutated allele) and TPC1 cells (carrying the RET/PTC1 rearrangement), we silenced PDPN in these cells using small interfering RNA (siRNA). The efficacy of PDPN silencing was confirmed by qRT-PCR and Western blotting. Then, we tested the motility and invasiveness of these cells (using scratch test and Transwell assay), their growth capacities F(cell cycle analysis, viability, clonogenic activity) and apoptosis assays), adhesion-independent colony-formation capacities, as well as the effect of PDPN silencing on MMPs expression and activity (zymography). RESULTS: We found that PDPN-induced cell phenotype depended on the genetic background of thyroid tumor cells. PDPN down-regulation in BcPAP cells was negatively correlated with the migration and invasion, in contrast to TPC1 cells in which PDPN depletion resulted in enhanced migration and invasiveness. Moreover, our results suggest that in BcPAP cells, PDPN may be involved in the epithelial-mesenchymal transition (EMT) through regulating the expression of the ezrin, radixin and moesin (E/R/M) proteins, MMPs 9 and MMP2, remodeling of actin cytoskeleton and cellular protrusions. We also demonstrated that PDPN expression is associated with the MAPK signaling pathway. The inhibition of the MAPK pathway resulted in a decreased PDPN expression, increased E/R/M phosphorylation and reduced cell migration. Additionally, PDPN depleted BcPAP cells treated with inhibitors of MEK1/2 kinases (U0126) or of the BRAF V600E protein (PLX4720) had reduced motility, similar to that previously observed in TPC1 cells after PDPN knock-down. CONCLUSIONS: Altogether, our data suggest that PDPN may play an important role in the control of invasion and migration of papillary thyroid carcinoma cells in association with the E/R/M, MMPs and MAPK kinases.
Assuntos
Glicoproteínas de Membrana/metabolismo , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Butadienos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proteínas do Citoesqueleto/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Nitrilas/farmacologia , Fosforilação , Sulfonamidas/farmacologia , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genéticaRESUMO
It is well known that Prospero homeobox 1 (PROX1) is a crucial regulator of lymphangiogenesis, that reprograms blood endothelial cells to lymphatic phenotype. However, the role of PROX1 in tumor progression, especially in angiogenesis remains controversial. Herein, we studied the role of PROX1 in angiogenesis in cell lines derived from follicular thyroid cancer (FTC: FTC-133) and squamous cell carcinoma of the thyroid gland (SCT: CGTH-W-1) upon PROX1 knockdown. The genes involved in angiogenesis were selected by RNA-seq, and the impact of PROX1 on vascularization potential was investigated using human umbilical vein endothelial cells (HUVECs) cultured in conditioned medium collected from FTC- or SCT-derived cancer cell lines after PROX1 silencing. The angiogenic phenotype was examined in connection with the analysis of focal adhesion and correlated with fibroblast growth factor 2 (FGF2) levels. Additionally, the expression of selected genes involved in angiogenesis was detected in human FTC tissues. As a result, we demonstrated that PROX1 knockdown resulted in upregulation of factors associated with vascularization, such as metalloproteinases (MMP1 and 3), FGF2, vascular endothelial growth factors C (VEGFC), BAI1 associated protein 2 (BAIAP2), nudix hydrolase 6 (NUDT6), angiopoietin 1 (ANGPT1), and vascular endothelial growth factor receptor 2 (KDR). The observed molecular changes resulted in the enhanced formation of capillary-like structures by HUVECs and upregulated focal adhesion in FTC-133 and CGTH-W-1 cells. The signature of selected angiogenic genes' expression in a series of FTC specimens varied depending on the case. Interestingly, PROX1 and FGF2 showed opposing expression levels in FTC tissues and seven thyroid tumor-derived cell lines. In summary, our data revealed that PROX1 is involved in the spreading of thyroid cancer cells by regulation of angiogenesis.
Assuntos
Adenocarcinoma Folicular/patologia , Carcinoma de Células Escamosas/patologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Neovascularização Patológica/patologia , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Proteínas Supressoras de Tumor/metabolismo , Adenocarcinoma Folicular/irrigação sanguínea , Adenocarcinoma Folicular/metabolismo , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/metabolismo , Células Cultivadas , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Humanos , Neovascularização Patológica/metabolismo , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/irrigação sanguínea , Neoplasias da Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genéticaRESUMO
The prospero homeobox 1 (PROX1) transcription factor is a product of one of the lymphangiogenesis master genes. It has also been suggested to play a role in carcinogenesis, although its precise role in tumour development and metastasis remains unclear. The aim of this study was to gain more knowledge on the PROX1 function in thyroid tumorigenesis. Follicular thyroid cancer-derived cells-CGTH-W-1-were transfected with PROX1-siRNA (small interfering RNA) and their proliferation, cell cycle, apoptosis and motility were then analysed. The transcriptional signature of PROX1 depletion was determined using RNA-Sequencing (RNA-Seq) and the expression of relevant genes was further validated using reverse transcriptase quantitative PCR (RT-qPCR), Western blot and immunocytochemistry. PROX1 depletion resulted in a decreased cell motility, with both migratory and invasive potential being significantly reduced. The cell morphology was also affected, while the other studied cancer-related cell characteristics were not significantly altered. RNA-seq analysis revealed significant changes in the expression of transcripts encoding genes involved in both motility and cytoskeleton organization. Our transcriptional analysis of PROX1-depleted follicular thyroid carcinoma cells followed by functional and phenotypical analyses provide, for the first time, evidence that PROX1 plays an important role in the metastasis of thyroid cancer cells by regulating genes involved in focal adhesion and cytoskeleton organization in tumour cells.
Assuntos
Adenocarcinoma Folicular/genética , Proteínas de Homeodomínio/genética , Proteínas Supressoras de Tumor/genética , Adenocarcinoma Folicular/patologia , Apoptose/genética , Biomarcadores Tumorais , Biópsia , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , RNA Interferente Pequeno/genética , TranscriptomaRESUMO
BACKGROUND: Chronic rhinosinusitis (CRS) is a multifactorial disease of unknown cause characterized by sinonasal inflammation, increased mucus production, and defective mucociliary clearance. Expression of Pendrin, an epithelial anion transporter, is increased in asthma and chronic obstructive pulmonary disease. Pendrin increases mucus production and regulates mucociliary clearance. OBJECTIVES: We sought to investigate the expression of pendrin and the mucus-related protein Muc5AC in sinonasal tissues of control subjects and patients with CRS and to evaluate the regulation of pendrin expression in nasal epithelial cells (NECs) in vitro. METHODS: The expression and distribution of pendrin in sinonasal tissues was analyzed by using real-time PCR, immunoblot analysis, and immunohistochemistry. Differentiated NECs were used to study the regulation of pendrin expression. RESULTS: Increased pendrin expression was observed in nasal polyp (NP) tissue of patients with CRS. Immunohistochemistry analysis revealed that pendrin was largely restricted to the epithelial layer. Pendrin expression significantly correlated with inflammatory cell markers, suggesting that the factors made by these cells might induce pendrin expression. Furthermore, both pendrin and periostin levels (a biomarker in asthma) correlated with IL-13 levels, suggesting that pendrin can be induced by this cytokine in sinonasal tissues. Expression of the mucus component protein Muc5AC correlated weakly with pendrin expression, indicating that pendrin might modulate mucus production in NPs. In cultured NECs pendrin expression was induced by TH2 cytokines and induced synergistically when TH2 cytokines were combined with IL-17A. Interestingly, human rhinovirus had a potentiating effect on IL-13-induced pendrin expression. Dexamethasone suppressed pendrin expression, suggesting that the therapeutic benefit of dexamethasone in asthmatic patients and those with CRS might involve regulation of pendrin expression. CONCLUSIONS: TH2-mediated pendrin expression is increased in NPs of patients with CRS and might lead to increased inflammation, mucus production, and decreased mucociliary clearance.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Pólipos Nasais/metabolismo , Rinite/metabolismo , Sinusite/metabolismo , Adolescente , Adulto , Idoso , Doença Crônica , Citocinas/genética , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Mucina-5AC/genética , Mucosa Nasal/citologia , Mucosa Nasal/metabolismo , RNA Mensageiro/metabolismo , Transportadores de Sulfato , Adulto JovemRESUMO
BACKGROUND: Although elevated intraocular pressure is a major risk factor for the development of glaucoma, there is increasing evidence that the immune system may be involved in the development of normal-tension glaucoma (NTG). The aim of this study was to determine if NTG is associated with elevated levels of antibodies against human heat shock protein (HSP) 60. MATERIAL AND METHODS: The study was conducted in 139 subjects (35 subjects with NTG [Group 1], 34 subjects with primary open-angle glaucoma /POAG/ [Group 2], 24 subjects with autoimmune rheumatic diseases [Group 3], and 36 healthy controls [Group 4]). All subjects had complete ophthalmologic examination (visual acuity, slit-lamp examination, tonometry, gonioscopy; visual-field examination, and optical coherence tomography /OCT/ of the optic nerve head and the macula). Blood samples were collected for the measurements of serum levels of antibodies against human HSP60. RESULTS: The subjects with rheumatic diseases had the highest median serum level of antibodies against HSP60 - 20.49 ng/mL. The values in the subjects with NTG, POAG, and in controls were 18.79 ng/mL, 18.61 ng/mL and 17.61 ng/mL, respectively (p=0.96). CONCLUSIONS: This study does not confirm the hypothesis that normal-tension glaucoma is associated with elevated blood levels of antibodies against human heat shock protein (HSP) 60.
Assuntos
Anticorpos/sangue , Chaperonina 60/imunologia , Glaucoma/sangue , Glaucoma/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Demografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , PolôniaRESUMO
OBJECTIVE: It has been reported that patients experiencing side effects of amiodarone (AM) therapy, such as amiodarone-induced thyrotoxocosis (AIT) or amiodarone-induced hypothyroidism (AIH), have changes in serum concentrations of anti-TSH receptor (TSHR), antithyroglobulin (Tg), and antiperoxidase (TPO) autoantibodies (Abs). The purpose of our study was to identify and analyze the changes in levels of listed antibodies in patients with several thyroid disorders. METHODS: 280 patients from two centers in Poland were included. Titers of TSHR-Abs, TPO-Abs and Tg-Abs were analyzed retrospectively in the following groups of patients: A - euthyroid patients with a history of hyperthyroidism prior to re-administration of AM; B - patients with AIT who discontinued the AM therapy; C - patients with AIT chronically treated with AM; D - hypothyroid patients. RESULTS: Serum Tg-Abs were not elevated in any of the studied groups. However, there were significant differences between A and B and also D and other groups (p<0.05). TPO-Abs titers were not elevated in most cases, there were no significant differences between groups. The serum titers of TSHR-Abs were not elevated in any group. We found statistically significant differences between B and D, C and other groups (p<0.05). CONCLUSIONS: Regardless of the statistically significant differences observed for Tg-Abs and TSHR-Abs levels, this observation have a limited clinical applicability. In almost all cases we observed normal to slightly increased titers of TPO-Abs, Tg-Abs, TSHR-Abs. Discontinuation or continuation of AM therapy had no influence on autoantibody titers. Furthermore, we found it impossible to differentiate between the type I and II of AIT based on autoantibody titers.
RESUMO
Antibodies against the Na/I symporter (anti-NIS ab) have been found in adult patients with autoimmune thyroid diseases. As easily available for the immune system, NIS can play a role in the initial stage of autoimmune thyroid diseases. Children with Turner's syndrome (TS) being at high risk of autoimmune thyroid disease development seem a valuable group for the investigation of the early autoimmune process. The aim of the study was to investigate the presence of anti-NIS ab and its potential clinical significance in TS children. Fifty four girls with TS were examined (age 11.9 ± 2.46 years), and 23 healthy girls with normal thyroid function, free of autoimmune diseases. Anti-NIS antibodies were measured by the in-house ELISA method and the Western blotting. Sera considered positive for anti-NIS ab were used for the iodide uptake bioassay using COS7 cells stably transfected with hNIS. In all patients the thyroid function, antithyroid antibodies presence and thyroid ultrasonography were evaluated. In 20% of the patients a subclinical hypothyroidism was diagnosed and 70.4% had antithyroid antibodies (anti-TPO - 64.8% and Anti-Tg - 24%). Anti-NISab were present in 14.8% girls with TS and in none of the control group. Their presence was unrelated to other antithyroid antibodies titre or patients' age. A positive correlation between the anti-NIS ab presence and the hypothyroidism was found (p < 0.04). Anti-NIS ab-positive sera did not suppress iodine uptake. In conclusion, anti-NIS antibodies were present in 14.8% of children with TS and they were related to the presence of hypothyroidism.
Assuntos
Autoanticorpos/sangue , Simportadores/imunologia , Glândula Tireoide/imunologia , Síndrome de Turner/imunologia , Animais , Autoanticorpos/imunologia , Células COS , Criança , Chlorocebus aethiops , Feminino , Humanos , Hipotireoidismo/imunologia , Hipotireoidismo/fisiopatologia , Tireoidite Autoimune/imunologia , Tireoidite Autoimune/fisiopatologia , Síndrome de Turner/fisiopatologiaRESUMO
Agrin (AGRN) is a matricellular glycoprotein involved in extracellular signal transduction. AGRN is involved in tumorigenesis and cancer progression; however, the role of AGRN in thyroid cancer (TC) remains unclear. In the present study, using cell lines derived from various subtypes of TC including CGTH, FTC-133 and BcPAP and transcriptomic data from patients with TC, the role of AGRN in TC was analyzed by migration, invasion, viability and proliferation assays as well as Western blot with EMT markers. AGRN expression was significantly increased in thyroid tumors and cell lines derived from various TC subtypes. The highest AGRN expression was found in follicular and papillary thyroid carcinoma subtypes. Immunocytochemistry revealed nuclear AGRN localization in normal (NTHY) and TC cells. Silencing of AGRN decreased viability, proliferation, migration and invasion of TC cell lines by upregulating vimentin and downregulating N-cadherin and E-cadherin. Furthermore, the expression of AGRN was associated with neutrophil infiltration in thyroid tumors. In conclusion, the present results indicated that increased AGRN expression promoted tumorigenic phenotypes of TC cells, while AGRN expression was associated with immune infiltration in thyroid tumors. AGRN may represent a target for future cancer therapy and requires further evaluation.
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Introduction: Thyroid iodide transporters, Na+/I- symporter (NIS) and pendrin (PDS), are responsible for supplying this vital micronutrient for thyroid hormone synthesis by thyroid peroxidase (TPO). Both proteins were shown to be expressed, apart from the thyroid, also in other human tissues, including lactating mammary gland. NIS expression in human breast cancers has been widely studied. On the other hand, until now PDS mRNA levels in breast tumor tissue have been estimated only in high throughput analyses. Previously, we have observed that TPO is expressed in normal and cancerous human breast tissues and shows enzymatic activity. However, biochemical activity of TPO in human breast cancer cells requires iodide transport by NIS and PDS. Therefore, to extend our previous study on TPO expression and function in human breast tumors we performed analysis of NIS and PDS levels in the same group of patients. Material and methods: The study involved detection of NIS and PDS protein levels by immunohistochemistry and Western blotting, as well as mRNA levels by real-time quantitative polymerase chain reaction. Results: Here we provide direct evidence that NIS and PDS are expressed in human breast cancer tissue, with NIS levels being increased and PDS levels decreased in tumor tissue. Interestingly, PDS mRNA levels in breast cancer tissue seem to be influenced by the estrogen receptor status and age of the patients, while NIS mRNA levels were dependent on histological type of the tumor. Conclusions: This study provides valuable information important for consideration in diagnostic or therapeutic application of radioiodine in breast cancer management.
RESUMO
Oxidative stress (OS) is recognized as disturbance of cellular equilibrium between reactive oxygen species (ROS) formation and their elimination by antioxidant defense systems. One example of ROS-mediated damage is generation of potentially mutagenic DNA precursor, 8-oxodGTP. In human cells genomic 8-oxodGTP incorporation is prevented by the MutT homologue 1 (MTH1 or hMTH1 for human MTH1) protein. It is well established that malignant cells, including thyroid cancer cells, require hMTH1 for maintaining proliferation and cancerous transformation phenotype. Above observations led to the development of hMTH1 inhibitors as novel anticancer therapeutics. In the current study we present extensive analysis of oxidative stress responses determining sensitivity to hMTH1 deficiency in cultured thyroid cells. We observe here that hMTH1 depletion results in downregulation of several glutathione-dependent OS defense system factors, including GPX1 and GCLM, making some of the tested thyroid cell lines highly dependent on glutathione levels. This is evidenced by the increased ROS burden and enhanced proliferation defect after combination of hMTH1 siRNA and glutathione synthesis inhibition. Moreover, due to the lack of data on hMTH1 expression in human thyroid tumor specimens we decided to perform detailed analysis of hMTH1 expression in thyroid tumor and peri-tumoral tissues from human patients. Our results allow us to propose here that anticancer activity of hMTH1 suppression may be boosted by combination with agents modulating glutathione pool, but further studies are necessary to precisely identify backgrounds susceptible to such combination treatment.
Assuntos
Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Regulação da Expressão Gênica , Glutationa Peroxidase/metabolismo , Estresse Oxidativo/genética , Monoéster Fosfórico Hidrolases/metabolismo , Glândula Tireoide/metabolismo , Linhagem Celular Tumoral , Enzimas Reparadoras do DNA/genética , Glutationa Peroxidase/genética , Humanos , Monoéster Fosfórico Hidrolases/genética , RNA Mensageiro/genética , Glândula Tireoide/citologia , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Glutationa Peroxidase GPX1RESUMO
PURPOSE: The aim of this study was to evaluate the association between vitamin D (vitD) and changes in the titers of anti-TSH receptor (TSHR-Abs), antithyroglobulin (Tg-Abs), and antiperoxidase (TPO-Abs) autoantibodies. MATERIALS/METHODS: The study involved 269 patients with Graves' disease (GD), divided into four subgroups (1-4), i.e. 65 smokers treated with vitD(+) (1), 76 smokers not treated with vitD(-) (2), 61 non-smokers treated with vitD(+) (3) and 67 non-smokers with vitD(-) (4). All thyroid parameters were analyzed at entry and 1, 3, 6, 9 and 12 months later. RESULTS: The titer of TSHR-Abs in group 3 was significantly lower than in groups 1 and 2 across all time points. At 3, 6 and 12 months, the titers of TSHR-Abs were also lower in group 4 compared to groups 1 and 2. At 9 months, the titers in group 3 were lower than in all other groups. There was a significant inverse correlation between baseline levels of vitD and baseline titers of Tg-Abs (in group 1 only), Tg-Abs after 12 months (in group 1 only), TPO-Abs after 12 months (in groups 1 and 3), fT4 (in group 4 only), and a significant positive correlation with TPO-Abs (in group 2 only). VitD levels at 12 months were inversely correlated with Tg-Abs in group 1. CONCLUSIONS: VitD measurements in patients with GD, especially smokers with an increased TSHR-Ab titers before 131I therapy, are recommended. Immunological remission is more likely in patients with GD who receive vitD, particularly smokers.
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Autoanticorpos/sangue , Autoantígenos/imunologia , Doença de Graves/patologia , Radioisótopos do Iodo/uso terapêutico , Deficiência de Vitamina D/complicações , Vitamina D/sangue , Adulto , Autoanticorpos/imunologia , Feminino , Seguimentos , Doença de Graves/epidemiologia , Doença de Graves/imunologia , Doença de Graves/radioterapia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptores da Tireotropina/imunologia , Estudos Retrospectivos , Tireoglobulina/imunologia , Tireotropina/imunologia , Vitamina D/imunologiaRESUMO
CONTEXT: We studied two families from Galicia (northwest Spain) with Pendred syndrome (PS) and unusual thyroid phenotypes. In family A, the proposita had a large goiter and hypothyroxinemia but normal TSH and free T3 (FT3). In family B, some affected members showed deafness but not goiter. OBJECTIVE: Our objective was to identify the mutations causing PS and molecular mechanisms underlying the thyroid phenotypes. INTERVENTIONS: Interventions included extraction of DNA and of thyroid tissue. PATIENTS: Propositi and 10 members of the two families participated in the study. MAIN OUTCOME MEASURES: Main outcome measures included SLC26A4 gene analysis, deiodinase activities in thyroid tissue, and c.416-1G-->A effects on SLC26A4 splicing. In addition, a primary PS thyrocyte culture, T-PS2, was obtained from propositus B and compared with another culture of normal human thyrocytes, NT, by Western blotting, confocal microscopy, and iodine uptake kinetics. RESULTS: Proposita A was heterozygous for c.578C-->T and c.279delT, presented with goiter, and had normal TSH and FT3 but low FT4 attributable to high type 1 and type 2 iodothyronine deiodinase activities in the goiter. Propositus B bore c.279delT and a novel mutation c.416-1G-->A; some deaf relatives were homozygous for c.416-1G-->A but did not present goiter. The c.279delT mutation was associated with identical haplotype in the two families. T-PS2 showed truncated pendrin retained intracellularly and high iodine uptake with low efflux leading to iodine retention. CONCLUSIONS: c.279delT is a founder mutation in Galicia. Proposita A adapted to poor organification by increasing deiodinase activities in the goiter, avoiding hypothyroidism. Lack of goiter in subjects homozygous for c.416-1G-->A was due to incomplete penetrance allowing synthesis of some wild-type pendrin. Intracellular iodine retention, as seen in T-PS2, could play a role in thyroid alterations in PS.
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Bócio Nodular/genética , Perda Auditiva Neurossensorial/genética , Adulto , Sequência de Aminoácidos , Feminino , Bócio Nodular/enzimologia , Bócio Nodular/patologia , Haplótipos , Perda Auditiva Neurossensorial/enzimologia , Perda Auditiva Neurossensorial/patologia , Humanos , Imuno-Histoquímica , Iodeto Peroxidase/biossíntese , Iodeto Peroxidase/genética , Iodo/farmacocinética , Masculino , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Dados de Sequência Molecular , Transportadores de Ácidos Monocarboxílicos/biossíntese , Transportadores de Ácidos Monocarboxílicos/genética , Linhagem , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espanha , Transportadores de Sulfato , Simportadores , Síndrome , Testes de Função Tireóidea , Iodotironina Desiodinase Tipo IIRESUMO
Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes.
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Neoplasias da Mama/química , Mama/química , Iodeto Peroxidase/química , Western Blotting , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/patologia , Citoplasma/química , Citoplasma/metabolismo , Citoplasma/patologia , Eletroforese em Gel Bidimensional , Glicosilação , Humanos , Imuno-Histoquímica , Imunoprecipitação , Iodeto Peroxidase/metabolismo , Lactoperoxidase/química , Lactoperoxidase/metabolismo , Peso Molecular , Polissacarídeos/química , Polissacarídeos/metabolismo , Células Epiteliais da Tireoide/química , Células Epiteliais da Tireoide/metabolismoRESUMO
Cancer cells, including thyroid cancer cells, suffer from oxidative stress damaging multiple cellular targets, such as DNA and the nucleotide pool. The human MutT homologue 1 (hMTH1) controls the oxidative DNA damage load by sanitizing the nucleotide pool from the oxidized DNA precursor, 8-oxodGTP. It has previously been shown that hMTH1 is essential for cancer cell proliferation and survival, therefore hMTH1 inhibition has been proposed as a novel anticancer therapeutic strategy. Here we show that thyroid cancer cells respond to siRNA mediated hMTH1 depletion with increased DNA damage load and moderately reduced proliferation rates, but without detectable apoptosis, cell-cycle arrest or senescence. Importantly, however, hMTH1 depletion significantly reduced migration and invasion potential of the thyroid cancer cells. Accordingly, our results allow us to propose that hMTH1 may be a therapeutic target in thyroid malignancy, especially for controlling metastasis.
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Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Neoplasias da Glândula Tireoide/enzimologia , Linhagem Celular Tumoral , Movimento Celular , Humanos , Invasividade Neoplásica , Estresse Oxidativo , Neoplasias da Glândula Tireoide/patologiaRESUMO
INTRODUCTION: Apoptosis, programmed cell death is a regulating mechanism enabling the removal of superabundantly produced and unnecessary at the certain moment cells. Disturbances of the apoptosis regulation contribute to the pathogenesis of many diseases, including autoimmune thyroid disorders. The aim of this study was to estimate expression of proapoptotic Fas/FasL and caspase-8 in thyroid tissues in patients with Graves' disease (GD), non-toxic nodular goiter (NTNG) and Hashimoto's thyroiditis (HT). MATERIAL AND METHODS: Inclusion criteria of Graves' patients were: large goiter, ophthalmopathy, TRAb > 5 U/L, positive titre of anti-TPO and anti-TG antibodies and concentration of TSH < 0.45 microIU/mL for more the 2-3 months from an onset of the disease. Isolated thyrocytes were identified by indirect method: in the first stage mouse monoclonal antibodies (mAbs) anti-TPO were bound to rabbit anti-mouse antibodies IgG (Fab')2 labeled FITC. To obtained cellular suspension mAbs directed against apoptotic Fas/FasL molecules labeled with PE (Phycoerythrin) was added. All investigations were performed on Coulter EPICS XL flow cytometer. Detection of apoptotic proteins was confirmed by Western Blot and immunohistochemistry methods using mAbs in DAB chromogene visuality and marked by Mayer's haematoxylin. Evaluation of caspase-8 expression in thyroid follicular cells was performed by Western Blot test. RESULTS: The analysis of Fas and FasL expression on surface of thyroid follicular cells was higher in patients with Hashimoto's thyroiditis (38%, 26%) in comparison with patients with Graves' disease (18%, 14%). In case of patients with Hashimoto's thyroiditis significantly lower percentage of thyroid tissue infiltrating immune Fas+ (13%) and FasL+ (22%) T cells in comparison with Graves' patients (33%, 43% respectively) was observed . Identification of proapoptotic Fas and FasL molecules in the thyroid follicular cells revealed higher expression of both proteins in patients with GD (++,++) and HT (+++; +++, respectively) in comparison with NTNG patients (+/0; +/0). Caspase-8 expression was detected in band 55 kDa using Western Blot test in patients with thyroid autoimmune diseases. CONCLUSIONS: We conclude that alteration in the expression of proapoptotic proteins in thyroid follicular cells may play a role in pathogenesis of thyroid autoimmune disorders. In addition, suppression of apoptosis in Graves' disease led to increased proliferation of thyroid follicular cells which is responsible for goiter formation.
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Caspase 8/biossíntese , Proteína Ligante Fas/biossíntese , Doenças da Glândula Tireoide/metabolismo , Receptor fas/biossíntese , Adolescente , Adulto , Apoptose , Western Blotting , Criança , Feminino , Citometria de Fluxo , Expressão Gênica , Bócio Nodular/metabolismo , Bócio Nodular/fisiopatologia , Doença de Graves/metabolismo , Doença de Graves/fisiopatologia , Doença de Hashimoto/metabolismo , Doença de Hashimoto/fisiopatologia , Humanos , Imuno-Histoquímica , Masculino , Doenças da Glândula Tireoide/fisiopatologiaRESUMO
The prospero homeobox 1 (Prox1) transcription factor is a key player during embryogenesis and lymphangiogenesis. Altered Prox1 expression has been found in a variety of human cancers, including papillary thyroid carcinoma (PTC). Interestingly, Prox1 may exert tumor suppressive or tumor promoting effect, depending on the tissue context. In this study, we have analyzed Prox1 expression in normal and malignant human thyroid carcinoma cell lines. Moreover, we determined the effect of Prox1 silencing and overexpression on the cellular processes associated with the metastatic potential of tumor cells: proliferation, migration, invasion, apoptosis and anchorage-independent growth, in the follicular thyroid carcinoma (FTC) FTC-133 cell line. We found that Prox1 expression was significantly higher in FTC-derived cells than in PTC-derived cells and normal thyroid, and it was associated with the PI3K/Akt signaling pathway. In the FTC-133 cells, it was associated with cell invasive potential, motility and wound closure capacities, but not with proliferation or apoptosis. Modifying Prox1 expression also induced substantial changes in the cytoskeleton structure and cell morphology. In conclusion, we have shown that Prox1 plays an important role in the development of FTC and that its suppression prevents, whereas its overexpression promotes, the malignant behavior of thyroid follicular cancer cells.
RESUMO
BACKGROUND: Thyroid peroxidase (TPO) is essential for physiological function of the thyroid gland. The high prevalence of thyroid peroxidase antibodies (TPOAbs) in patients with breast cancer and their protective role had previously been demonstrated, indicating a link between breast cancer and thyroid autoimmunity. Recently, TPO was shown to be present in breast cancer tissue samples but its antigenicity has not been analyzed. METHODS: In this study, we investigated TPO expression levels in a series of fifty-six breast cancer samples paired with normal (peri-tumoral) tissue and its antigenic activity using a panel of well-characterized murine anti-human TPOAbs. RESULTS: We have shown that TPO transcripts were present in both normal and cancer tissue samples, although the amounts in the latter were reduced. Additionally, we observed that TPO levels are lower in more advanced cancers. TPO protein expression was confirmed in all tissue samples, both normal and cancerous. We also found that the antigenicity of the immunodominant regions (IDRs) in breast TPO resembles that of thyroid TPO, which is crucial for effective interactions with human TPOAbs. CONCLUSIONS: Expression of TPO in breast cancer together with its antigenic activity may have beneficial effects in TPOAb-positive breast cancer patients. However, further studies are needed to confirm the beneficial role of TPOAbs and to better understand the underlying mechanism.