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1.
J Clin Med ; 12(3)2023 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-36769844

RESUMO

BACKGROUND: Vitamin C is one of the major extracellular nonenzymatic antioxidants involved in the biosynthesis of collagen. It promotes the growth of fibroblasts, wound healing processes, and enhances the survival and differentiation of osteoblasts. The potential effects of ascorbic acid on human dental pulp cells (DPC) and the cells of the apical papilla (CAP) used in actual regenerative endodontic procedures remain largely unknown. In this study, we investigated the possible employment of ascorbic acid in the differentiation and regenerative therapies of DPC and CAP. METHODS: Nine extracted human wisdom teeth were selected for this study. Subpopulations of stem cells within DPC and CAP were sorted with the mesenchymal stem cell marker STRO-1, followed by treatments with different concentrations (0 mM, 0.1 mM, 0.5 mM, and 1.0 mM) of ascorbic acid (AA), RT-PCR, and Western blot analysis. RESULTS: FACS analysis revealed the presence of cell subpopulations characterized by a strong expression of mesenchymal stem cell marker STRO-1 and dental stem cell markers CD105, CD44, CD146, CD90, and CD29. Treatment of the cells with defined amounts of AA revealed a markedly increased expression of proliferation marker Ki-67, especially in the concentration range between 0.1 mM and 0.5 mM. Further investigations demonstrated that treatment with AA led to significantly increased expression of common stem cell markers OCT4, Nanog, and Sox2. The most potent proliferative and expressional effects of AA were observed in the concentration of 0.1 mM. CONCLUSIONS: AA might be a novel and potent growth promoter of human dental cells. Increasing the properties of human dental pulp cells and the cells of the apical papilla using AA could be a useful factor for further clinical developments of regenerative endodontic procedures.

2.
Diseases ; 9(1)2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809272

RESUMO

INTRODUCTION: Calcification is a highly relevant process in terms of development of cardiovascular diseases, and its prevention may be the key to prevent disease progression in patients. In this study we investigated the expression of osteocalcin (OC), osteopontin (OPN) and RUNX2 in patients' leukocytes and their possible role as diagnostic markers for cardiovascular diseases. MATERIALS AND METHODS: Leucocytes from 38 patients were collected in the Department of Surgery of Martin-Luther-University Halle, including 8 patients without arteriosclerotic disease (PAD-) and 30 patients with symptomatic arteriosclerotic disease (PAD+). Patients' leucocytes, in vitro calcified human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC) were subjected to qPCR analyses with TaqMan probes, which are specific for OC, OPN and RUNX2. Additionally, the interaction between monocytes and calcified HUVEC and VSMC was investigated in adhesion assays. RESULTS: The leucocytes obtained from patients with symptomatic arteriosclerotic disease (PAD+) demonstrated decreased mRNA level expression of Osteocalcin, while OPN and RUNX2 were significantly upregulated in comparison to asymptomatic patients. The induction of calcification in HUVEC and VSMC cells led to an increased expression of OC, OPN and RUNX2. Immunocytochemistry of calcified HUVEC and VSMC revealed stronger expression of OC, OPN and RUNX2 in calcified cells. CONCLUSION: To conclude, these data demonstrate that symptomatic arteriosclerotic disease has a correlation with OC, OPN and RUNX2. The biological rationale of OC, OPN and RUNX-2 remains not yet entirely understood for atherosclerotic disease, which means it needs further investigation.

4.
Oncotarget ; 7(39): 63106-63123, 2016 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-27527858

RESUMO

Anaplastic (ATC) and certain follicular thyroid-carcinomas (FTCs) are radioresistant. The Phosphatidylinositide 3-kinase (PI3K) pathway is commonly hyperactivated in thyroid-carcinomas. PI3K can modify the PI3K-related kinases (PIKKs) in response to radiation: How PIKKs interact with PI3K and contribute to radioresistance in thyroid-carcinomas is unknown. Further uncertainties exist in how these interactions function under the radioresistant hypoxic microenvironment. Under normoxia/anoxia, ATC (8505c) and FTC (FTC-133) cells were irradiated, with PI3K-inhibition (via GDC-0941 and PTEN-reconstitution into PTEN-null FTC-133s) and effects on PIKK-activation, DNA-damage, clonogenic-survival and cell cycle, assessed. FTC-xenografts were treated with 5 × 2 Gy, ± 50 mg/kg GDC-0941 (twice-daily; orally) for 14 days and PIKK-activation and tumour-growth assessed. PIKK-expression was additionally assessed in 12 human papillary thyroid-carcinomas, 13 FTCs and 12 ATCs. GDC-0941 inhibited radiation-induced activation of Ataxia-telangiectasia mutated (ATM), ATM-and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inhibition of ATM and DNA-PKcs was PI3K-dependent, since activation was reduced in PTEN-reconstituted FTC-133s. Inhibition of PIKK-activation was greater under anoxia: Consequently, whilst DNA-damage was increased and prolonged under both normoxia and anoxia, PI3K-inhibition only reduced clonogenic-survival under anoxia. GDC-0941 abrogated radiation-induced cell cycle arrest, an effect most likely linked to the marked inhibition of ATR-activation. Importantly, GDC-0941 inhibited radiation-induced PIKK-activation in FTC-xenografts leading to a significant increase in time taken for tumours to triple in size: 26.5 ± 5 days (radiation-alone) versus 31.5 ± 5 days (dual-treatment). PIKKs were highly expressed across human thyroid-carcinoma classifications, with ATM scoring consistently lower. Interestingly, some loss of ATM and DNA-PKcs was observed. These data provide new insight into the mechanisms of hypoxia-associated radioresistance in thyroid-carcinoma.


Assuntos
Carcinoma/radioterapia , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Glândula Tireoide/radioterapia , Animais , Carcinoma/metabolismo , Carcinoma Papilar/metabolismo , Carcinoma Papilar/radioterapia , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta à Radiação , Ativação Enzimática , Feminino , Histonas/metabolismo , Humanos , Hipóxia , Indazóis/farmacologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Oxigênio/química , Tolerância a Radiação , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/metabolismo
5.
Artigo em Inglês | MEDLINE | ID: mdl-26322020

RESUMO

The relaxin-like RXFP1 ligand-receptor system has important functions in tumor growth and tissue invasion. Recently, we have identified the secreted protein, CTRP8, a member of the C1q/tumor necrosis factor-related protein (CTRP) family, as a novel ligand of the relaxin receptor, RXFP1, with functions in brain cancer. Here, we review the role of CTRP members in cancers cells with particular emphasis on CTRP8 in glioblastoma.

6.
BMC Cancer ; 15: 140, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25880801

RESUMO

BACKGROUND: Lactate dehydrogenase A (LDHA) and Pyruvate Kinase M2 (PKM2) are important enzymes of glycolysis. Both of them can be phosphorylated and therefore regulated by Fibroblast growth factor receptor 1 (FGFR1). While phosphorylation of LDHA at tyrosine10 leads to tetramerization and activation, phosphorylation of PKM2 at tyrosine105 promotes dimerization and inactivation. Dimeric PKM2 is found in the nucleus and regulates gene transcription. Up-regulation and phosphorylation of LDHA and PKM2 contribute to faster proliferation under hypoxic conditions and promote the Warburg effect. METHODS: Using western blot and SYBR Green Real time PCR we investigated 77 thyroid tissues including 19 goiter tissues, 11 follicular adenomas, 16 follicular carcinomas, 15 papillary thyroid carcinomas, and 16 undifferentiated thyroid carcinomas for total expression of PKM2, LDHA and FGFR1. Additionally, phosphorylation status of PKM2 and LDHA was analysed. Inhibition of FGFR was performed on FTC133 cells with SU-5402 and Dovitinib. RESULTS: All examined thyroid cancer subtypes overexpressed PKM2 as compared to goiter. LDHA was overexpressed in follicular and papillary thyroid cancer as compared to goiter. Elevated phosphorylation of LDHA and PKM2 was detectable in all analysed cancer subtypes. The highest relative phosphorylation levels of PKM2 and LDHA compared to overall expression were found in undifferentiated thyroid cancer. Inhibition of FGFR led to significantly decreased phosphorylation levels of PKM2 and LDHA. CONCLUSIONS: Our data shows that overexpression and increased phosphorylation of PKM2 and LHDA is a common finding in thyroid malignancies. Phospho-PKM2 and Phospho-LDHA could be valuable tumour markers for thyroglobulin negative thyroid cancer.


Assuntos
Proteínas de Transporte/metabolismo , L-Lactato Desidrogenase/metabolismo , Proteínas de Membrana/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Hormônios Tireóideos/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Biomarcadores , Proteínas de Transporte/genética , Linhagem Celular , Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/genética , Lactato Desidrogenase 5 , Proteínas de Membrana/genética , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Hormônios Tireóideos/genética , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/genética , Proteínas de Ligação a Hormônio da Tireoide
7.
Thyroid ; 25(5): 514-27, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25744544

RESUMO

BACKGROUND: Expression of the small calcium-binding protein S100A4 is associated with poor prognosis in patients with thyroid cancer (TC). The authors have previously shown that S100A4 is a target for relaxin and insulin-like peptide 3 signaling in TC cells and that S100A4 is secreted from human TC cells. Although the pro-migratory role of intracellular S100A4 in binding to non-muscle myosin is well known, this study investigated here whether extracellular S100A4 contributes to TC migration. METHODS: Human cell lines of follicular, papillary, and undifferentiated thyroid cancer, primary patient TC cells, and TC tissues were utilized to discover the presence of the receptor of advanced glycation end products (RAGE) in TC cells and TC tissues. Fluorescence imaging, protein pull-down assays, Western blot, siRNA protein silencing, small GTPase inhibitors, cell proliferation, and cell migration assays were used to investigate the interaction of extracellular S100A4 with RAGE in promoting a TC migratory response. RESULTS: It was demonstrated that RAGE served as receptor for extracellular S100A4 mediating cell migration in TC cells. The RAGE-mediated increase in cell migration was dependent on the intracellular RAGE signaling partner diaphanous-1 (Dia-1) and involved the activation of the small GTPases Cdc42 and RhoA. Although extracellular S100A4 consistently activated ERK signaling in TC cells, it was shown that ERK signaling was not mediated by RAGE and not essential for the migratory response in TC cells. CONCLUSION: The data have identified the RAGE/Dia-1 signaling system as a mediator for the pro-migratory response of extracellular S100A4 in human TC. Thus, therapeutic targeting of the RAGE/Dia-1/small GTPases signaling may successfully reduce local invasion and metastasis in TC.


Assuntos
Carcinoma Papilar/metabolismo , Movimento Celular/fisiologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteínas S100/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Adenocarcinoma Folicular , Carcinoma Papilar/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Prognóstico , RNA Interferente Pequeno , Receptor para Produtos Finais de Glicação Avançada/genética , Proteína A4 de Ligação a Cálcio da Família S100 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Neoplasias da Glândula Tireoide/patologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
8.
J Oral Sci ; 55(4): 319-27, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24351920

RESUMO

IGF-1 regulates the metabolism of hard dental tissue through binding to the IGF-1 receptor on target cells. Furthermore, IGF-binding-protein-3 promotes the accessibility of IGF-1. The aim of this study was to investigate the expression of IGF-1, IGFBP-3 and IGF-1R in STRO-1-positive dental pulp stem cells (DPSCs) and fully impacted wisdom teeth in relation to tooth development. Third molars were surgically removed from 60 patients and classified into two groups: teeth showing ongoing development (group 1) and teeth that had completed root shaping (group 2). The transcript and protein levels of IGF-1, IGFBP-3 and IGF-1R were investigated using RT-PCR and immunohistochemistry. The expression of the same proteins was also analyzed in DPSCs. The teeth from group 1 showed significantly stronger expression of IGF-1 and IGF-1R. The major sources of all of the proteins investigated immunohistochemically in sections of wisdom teeth were odontoblasts, cementoblasts and cell colonies in the pulpal mesenchyme. These colonies were identified as stem cells in view of their positivity for STRO-1, and the cells were subsequently sorted by flow cytometry. These DPSCs demonstrated high levels of pluripotency markers and IGF-1 and IGF-1R. We conclude that members of the IGF-1 family are involved in the late stage of tooth development and the process of pulpal differentiation.


Assuntos
Polpa Dentária/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Dente Serotino/metabolismo , Receptor IGF Tipo 1/metabolismo , Células-Tronco/metabolismo , Dente Impactado/metabolismo , Sequência de Bases , Western Blotting , Diferenciação Celular , Primers do DNA , Polpa Dentária/citologia , Citometria de Fluxo , Humanos , Dente Serotino/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
J Pathol ; 231(4): 466-79, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24014093

RESUMO

We report a novel ligand-receptor system composed of the leucine-rich G-protein-coupled relaxin receptor, RXFP1, and the C1q-tumour necrosis factor-related protein 8 (CTRP8) in human primary brain cancer, a tumour entity devoid of the classical RXFP1 ligands, RLN1-3. In structural homology studies and computational docking experiments we delineated the N-terminal region of the globular C1q region of CTRP8 and the leucine-rich repeat units 7 and 8 of RXFP1 to mediate this new ligand-receptor interaction. CTRP8 secreted from HEK293T cells, recombinant human (rh) CTRP8, and short synthetic peptides derived from the C1q globular domain of human CTRP8 caused the activation of RXFP1 as determined by elevated intracellular cAMP levels and the induction of a marked pro-migratory phenotype in established glioblastoma (GB) cell lines and primary cells from GB patients. Employing a small competitor peptide, we were able to disrupt the CTRP8-RXFP1-induced increased GB motility. The CTRP8-RXFP1-mediated migration in GB cells involves the activation of PI3K and specific protein kinase C pathways and the increased production/secretion of the potent lysosomal protease cathepsin B (cathB), a known prognostic marker of GB. Specific inhibition of CTRP8-induced cathB activity effectively blocked the ability of primary GB to invade laminin matrices. Finally, co-immunoprecipitation studies revealed the direct interaction of human CTRP8 with RXFP1. Our results support a therapeutic approach in GB aimed at targeting multiple steps of the CTRP8-RXFP1 signalling pathway by a combined inhibitor and peptide-based strategy to block GB dissemination within the brain.


Assuntos
Adiponectina/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Adiponectina/farmacologia , Sítios de Ligação , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/patologia , Catepsina B/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Ativação Enzimática/fisiologia , Glioblastoma/patologia , Humanos , Invasividade Neoplásica/fisiopatologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas
10.
Oncol Rep ; 29(3): 1087-93, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23258351

RESUMO

The aim of this study was to investigate the effect of short hairpin RNA (shRNA) targeting autotaxin (ATX) on the migratory and invasive capability of AGS human gastric cancer cells and the growth of xenografts in nude mice. pSUPER-ATX and pSUPER-mock (non-specific), which were constructed corresponding to the ATX-shRNA and negative control mock-shRNA synthesized based on gene sequence, were transfected with blank plasmid pSUPER-control into AGS human gastric cancer cells using Lipofectamine. At 24, 48 and 72 h post-transfection, the cells were harvested and analyzed. The endogenous ATX mRNA and protein of the different groups of AGS cells were detected by RT-PCR and western blot assays. Cell proliferation was measured by MTT assay. In vitro Transwell and Matrigel assays were used to detect the cell migratory and invasive capabilities. A tumor xenograft model was generated by subcutaneous injection of AGS cells into the dorsum of nude mice. The growth of xenograft tumors was monitored and measured; changes in tumor morphology and the organs of mice were observed by H&E staining. The expression of ATX, MMP-2 and MMP-9 protein in xenograft tumor tissues was detected by immunohistochemistry and western blotting. In vitro, both the mRNA and protein levels of ATX in the pSUPER-ATX group were significantly downregulated (P<0.01), and the cell proliferative, migratory and invasive capabilities were also significantly decreased. In vivo, no obvious damage to the organs was found. pSUPER-ATX significantly suppressed the tumor volume and weight from the 7th week after cell transplantation, compared to the pSUPER-mock, pSUPER-control and WT groups; the inhibition rates were approximately 50% (P<0.05). However, no significant differences in these parameters were found among the WT, pSUPER-mock and pSUPER-control groups. Furthermore, ATX, MMP-2 and MMP-9 were expressed at significantly lower levels in the pSUPER-ATX group compared to the levels in the other three groups (P<0.05), and a significant correlation between ATX and MMP-2 expression was found (r=0.869, P<0.01). The specific shRNA targeting ATX downregulated the endogenous ATX of AGS human gastric cancer cells, and inhibited AGS cell proliferative, migratory and invasive potentials. Moreover, shRNA targeting ATX inhibited the growth of human gastric cancer xenografts in nude mice.


Assuntos
Movimento Celular , Proliferação de Células , Diester Fosfórico Hidrolases/genética , RNA Interferente Pequeno/genética , Neoplasias Gástricas/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Diester Fosfórico Hidrolases/metabolismo , Neoplasias Gástricas/patologia , Carga Tumoral
11.
Oncol Rep ; 29(1): 149-54, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23128569

RESUMO

We report for the first time the distribution of human INSL5 and its cognate leucine rich G-protein coupled receptor RXFP4 in the large intestine and in neuroendocrine/carcinoid tissues. Immunoreactive INSL5 was uniquely expressed by enteroendocrine cells (EECs) located within the colonic mucosa, whereas colonocytes were immunopositive for RXFP4. INSL5+ and RXFP4+ cells were also detected in human neuroendocrine/carcinoid tissues. We employed a recently described Insl5 knockout mouse model and 2 mouse models of induced colitis to address the relevance of Insl5 in EEC development and in acute inflammation of the colon. We identified INSL5 as a specific marker for synaptophysin+ EECs in the mucosa of the normal human and mouse colon. Insl5 was not essential for the development of mouse synaptophysin+ EECs. The mouse models of chemically induced colitis (dextran sulfate sodium and dinitrobenzene-sulfonic acid) failed to show changes in the numbers of Insl5+ EECs at inflammatory sites during the acute phase of colitis. In conclusion, we showed that INSL5 is a novel marker of colorectal EECs and provide first evidence for the presence of a potentially autocrine/paracrine INSL5-RXFP4 signaling system in the normal human and mouse colon and in rare human neuroendocrine tumours.


Assuntos
Biomarcadores Tumorais/análise , Tumor Carcinoide/patologia , Células Enteroendócrinas/patologia , Insulina/fisiologia , Intestino Grosso/patologia , Tumores Neuroendócrinos/patologia , Proteínas/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Comunicação Autócrina , Tumor Carcinoide/metabolismo , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Enteroendócrinas/metabolismo , Feminino , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Inflamação/metabolismo , Inflamação/patologia , Intestino Grosso/metabolismo , Masculino , Camundongos , Camundongos Knockout , Estadiamento de Neoplasias , Tumores Neuroendócrinos/metabolismo , Comunicação Parácrina , Prognóstico , Células Tumorais Cultivadas
12.
Endocr Relat Cancer ; 20(1): 79-90, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23183267

RESUMO

CCL2 is a chemokine known to recruit monocytes/macrophages to sites of inflammation. CCL2 is also associated with tumor progression in several cancer types. Recently, we showed that the N-terminus of CCL2 is modified to a pyroglutamate (pE)-residue by both glutaminyl cyclases (QC (QPCT)) and its isoenzyme (isoQC (QPCTL)). The pE-residue increases stability against N-terminal degradation by aminopeptidases. Here, we report an upregulation of QPCT expression in tissues of patients with thyroid carcinomas compared with goiter tissues, whereas QPCTL was not regulated. In thyroid carcinoma cell lines, QPCT gene expression correlates with the mRNA levels of its substrate CCL2. Both QPCT and CCL2 are regulated in a NF-κB-dependent pathway shown by stimulation with TNFa and IL1b as well as by inhibition with the IKK2 inhibitor and RNAi of p50. In the culture supernatant of thyroid carcinoma cells, equal amounts of pECCL2 and total CCL2 were detected by two ELISAs discriminating between total CCL2 and pECCL2, concluding that all CCL2 is secreted as pECCL2. Activation of the CCL2/CCR2 pathway by recombinant CCL2 increased tumor cell migration of FTC238 cells in scratch assays as well as thyroid carcinoma cell-derived CCL2-induced migration of monocytic THP1 cells. Suppression of CCL2 signaling by CCR2 antagonist, IKK2 inhibitor, and QPCT RNAi reduced FTC238 cell growth measured by WST8 proliferation assays. Our results reveal new evidence for a novel role of QC in thyroid carcinomas and provide an intriguing rationale for the use of QC inhibitors as a means of blocking pECCL2 formation and preventing thyroid cancer metastasis.


Assuntos
Adenocarcinoma Folicular/metabolismo , Aminoaciltransferases/metabolismo , Bócio/metabolismo , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Adenocarcinoma Folicular/genética , Adenocarcinoma Folicular/patologia , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/genética , Apoptose , Western Blotting , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiotaxia , Ensaio de Imunoadsorção Enzimática , Bócio/genética , Bócio/patologia , Humanos , Técnicas Imunoenzimáticas , Isoenzimas , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores CCR2/genética , Receptores CCR2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia
13.
Int J Mol Med ; 30(3): 465-72, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22751750

RESUMO

Survivin is a novel apoptosis inhibitor. Its gene is related to the baculovirus gene, which is believed to play a crucial role in fetal development and in cancer. We attempted to determine the expression of survivin in both thyroid goiter and carcinoma tissues, and to evaluate its prognostic value in human thyroid disease. In the present study, we applied small interfering RNA (siRNA) directed against survivin to determine the effects of decreasing the high constitutive levels of this protein in the FTC-133 thyroid follicular cancer cell line. Using reverse transcription PCR and immunohistochemistry, we compared the expression of survivin with relevant clinical and pathological data of 90 postsurgical specimens from patients with primary thyroid carcinoma and patients with benign goiter (33 with papillary thyroid cancer, 24 with follicular thyroid cancer, 18 with undifferentiated thyroid cancer and 15 cases with goiter). For the siRNA treatment in a human follicular thyroid carcinoma cell line, fluorescein-labeled double-stranded ultrapure siRNAs were used. RT-PCR identified the survivin transcript in 67/75 (89.3%) tumor samples and in 4/15 benign goiter samples. Immunohistochemical analysis showed positive immunoreactivity in 65/75 (86.7%) carcinomas while no expression was noted in all of the 15 benign goiter tissues. Survivin mRNA and protein levels were significantly higher in cancer tissues compared to benign goiter tissues (P<0.001). Higher survivin expression was found in the tumor tissues of pT3/pT4 and in the tumors with lymph node metastasis (P<0.05). Tumors with distant metastasis demonstrated higher survivin expression compared to the tumors without distant metastasis. Additionally, the expression of survivin in undifferentiated carcinomas was higher than that in differentiated ones. There was no significant correlation between survivin expression and age, gender, histological subtype and pathological stage. Our additional studies demonstrated that siRNA directed against survivin markedly decreased the protein expression of survivin. In conclusion, we conclude that survivin expression indicates more aggressive behavior and metastatic ability in thyroid cancer cells in vivo. Survivin can be used as a diagnostic and therapeutic marker for thyroid carcinoma and an important target in the strategy of thyroid cancer therapy. Our results of siRNA silencing indicate that siRNA may have potential as a therapeutic modality in the treatment of human thyroid cancer.


Assuntos
Apoptose/genética , Carcinoma/genética , Proteínas Inibidoras de Apoptose/genética , Neoplasias da Glândula Tireoide/genética , Adulto , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Bócio/genética , Bócio/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Survivina , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia
14.
Neoplasia ; 14(5): 396-409, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22745586

RESUMO

The cytoplasmic domains of EGF-like ligands, including EGF cytoplasmic domain (EGFcyt), have important biological functions. Using specific constructs and peptides of human EGF cytoplasmic domain, we demonstrate that EGFcyt facilitates lysosomal and proteasomal protein degradation, and this coincided with growth inhibition of human thyroid and glioma carcinoma cells. EGFcyt and exon 22-23-encoded peptide (EGF22.23) enhanced procathepsin B (procathB) expression and procathB-mediated lysosomal degradation of EGFR/ErbB1 as determined by inhibitors for procathB and the lysosomal ATPase inhibitor BafA1. Presence of mbEGFctF, EGFcyt, EGF22.23, and exon 23-encoded peptides suppressed the expression of the deubiqitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1). This coincided with hyperubiquitination of total cellular proteins and ErbB1/2 and reduced proteasome activity. Upon small interfering RNA-mediated silencing of endogenously expressed UCH-L1, a similar hyperubiquitinylation phenotype, reduced ErbB1/2 content, and attenuated growth was observed. The exon 23-encoded peptide region of EGFcyt was important for these biologic actions. Structural homology modeling of human EGFcyt showed that this molecular region formed an exposed surface loop. Peptides derived from this EGFcyt loop structure may aid in the design of novel peptide therapeutics aimed at inhibiting growth of cancer cells.


Assuntos
Fator de Crescimento Epidérmico/química , Receptores ErbB/metabolismo , Lisossomos/metabolismo , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Catepsina B/metabolismo , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Éxons , Humanos , Modelos Moleculares , Neoplasias/genética , Peptídeos/química , Domínios e Motivos de Interação entre Proteínas/genética , Proteólise , Interferência de RNA , Receptor ErbB-2/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação
15.
Thyroid Res ; 4 Suppl 1: S5, 2011 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-21835052

RESUMO

RNA-binding proteins may regulate every aspect of RNA metabolism, including pre-mRNA splicing, mRNA trafficking, stability and translation of many genes. The dynamic association of these proteins with RNA defines the lifetime, cellular localization, processing and the rate at which a specific mRNA is translated. One of the pathways involved in regulating of mRNA stability is mediated by adenylate uridylate-rich element (ARE) binding proteins. These proteins are involved in processes of apoptosis, tumorigenesis and development. Out of many ARE-binding proteins, two of them AUF1 and HuR were studied most extensively and reported to regulate the mRNA stability in vivo. Our previously published data demonstrate that both proteins are involved in thyroid carcinogenesis. Several other reports postulate that mRNA binding proteins may participate in thyroid hormone actions. However, until now, exacts mechanisms and the possible role of post-transcriptional regulation and especially the role of AUF1 and HuR in those processes remain not fully understood. In this study we shortly review the possible function of both proteins in relation to development and various physiological and pathophysiological processes, including thyroid function and disorders.

16.
World J Surg Oncol ; 9: 43, 2011 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-21521534

RESUMO

BACKGROUND: The aim of this study was to clarify the clinical significance of TM4SF members CD9, CD63 and CD82 in human gastric carcinoma. METHODS: By employing RT-PCR and immunohistochemistry, we studied the expression of CD9, CD63 and CD82 in 49 paired tissue specimens of normal gastric mucosa and carcinoma. All tissues were obtained from patients who underwent curative surgery. RESULTS: All normal gastric epithelium and gastric ulcer tissues strongly expressed transcripts and proteins of CD9, CD63 and CD82 as compared with corresponding controls. We found a significant correlation between CD63 mRNA level and different pM statuses (P = 0.036). Carcinomas in M0 stage revealed a stronger expression of CD63 than carcinomas in M1 stage. Expression of CD9 protein was found significantly stronger in pN0, pM0 than in advanced pN stages (P = 0.03), pM1 (P = 0.013), respectively. We found the relationship between CD63 expression, gender (p = 0.09) and nodal status (p = 0.028), respectively. Additionally, advanced and metastasized tumor tissues revealed significantly down-regulated CD82 protein expression (p = 0.033 and p = 0, respectively), which correlated with the tumor pTNM stage (p = 0.001). CONCLUSION: The reduction of CD9, CD63 and CD82 expression are indicators for the metastatic potential of gastric carcinoma cells. Unlike their expression in other tumor types, the constitutive expression of CD63 may indicate that this factor does play a direct role in human gastric carcinogenesis.


Assuntos
Antígenos CD/genética , Proteína Kangai-1/genética , Glicoproteínas de Membrana/genética , Glicoproteínas da Membrana de Plaquetas/genética , Neoplasias Gástricas/genética , Regulação para Baixo , Expressão Gênica , Humanos , Prognóstico , Estudos Retrospectivos , Tetraspanina 29 , Tetraspanina 30
17.
Mol Cancer Res ; 9(6): 673-87, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21493587

RESUMO

In this study, we identified differential expression of immunoreactive matrix metalloproteinase 2 (MMP2)/gelatinase A, membrane-anchored MT1-MMP/MMP14, and human relaxin-2 (RLN2) in human benign and malignant thyroid tissues. MMP2 and MT1-MMP were detected in the majority of thyroid cancer tissues and colocalized with RLN2-positive cells. MMP2 was mostly absent in goiter tissues and, similar to RLN2, may serve as a marker for thyroid cancer. MMP2 and MT1-MMP were identified as novel RLN2 targets. RLN2 caused a significant downregulation of tissue inhibitor of MMP (TIMP) 3 protein levels but did not change the expression levels of MMP13, and TIMP1, TIMP2, and TIMP4 in human thyroid carcinoma cells. RLN2 failed to affect the expression of MMP1, 3, 8, and 9 in the thyroid carcinoma cells investigated. Stable RLN2 transfectants secreted enhanced levels of bioactive MMP2 which contributed to the increased collagenolytic activity and in vitro invasiveness into collagen matrix by human thyroid cancer cells. Three-dimensional reconstitution of confocal fluorescent microscopy images revealed larger-sized invadopodia, with intense MT1-MMP accumulation at the leading migrating edge in RLN2 transfectants when compared with enhanced green fluorescent protein clones. In RLN2 transfectants actin stress fibers contributed to pseudopodia formation. In conclusion, enhanced tumor cell invasion by RLN2 involves the formation of MT1-MMP-enriched invadopodia that lead to increased collagenolytic cell invasion by human thyroid cancer cells.


Assuntos
Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Relaxina/metabolismo , Neoplasias da Glândula Tireoide/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Movimento Celular , Criança , Colágeno/metabolismo , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Bócio/enzimologia , Bócio/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Pseudópodes/metabolismo , Relaxina/genética , Neoplasias da Glândula Tireoide/enzimologia
19.
Mol Cell Endocrinol ; 325(1-2): 110-7, 2010 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-20538039

RESUMO

Our previous studies demonstrated that retinoic acid (RA)-induced reduction of both, the key glycolytic enzyme ENO1 and proliferation-promoting c-Myc, resulted in decreased vitality and invasiveness of the follicular thyroid carcinoma cell lines FTC-133 and FTC-238. By employing two-dimensional electrophoresis and mass spectrometry, we identified proteins affected by RA treatment. In addition to previously reported decrease in ENO1 expression, we found that RA led to significantly reduced levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase isoenzymes M1/M2 (PKM1/M2), peptidyl-prolyl cis-trans isomerase A (PPIA), transketolase (TKT), annexin A2 (ANXA2), glutathione S-transferase P (GSTP1) and peroxiredoxin 2 (PRDX2) as compared to untreated control. The same proteins investigated on thyroid tissues were found to be significantly up-regulated in follicular, papillary and undifferentiated thyroid carcinomas when compared with goiter and adenoma tissues. These findings identify new target proteins for RA-mediated anti-tumor and re-differentiation therapies and provide novel insights into treatments for thyroid carcinoma.


Assuntos
Biomarcadores Farmacológicos/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma/tratamento farmacológico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Tretinoína/uso terapêutico , Adolescente , Adulto , Idoso , Biomarcadores Farmacológicos/análise , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/isolamento & purificação , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Metaboloma , Pessoa de Meia-Idade , Proteômica , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Tretinoína/farmacologia , Adulto Jovem
20.
Int J Oncol ; 36(6): 1401-8, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20428763

RESUMO

The aim of this study was to elucidate the role of CD97 isoforms in gastric carcinoma. Out of four gastric cancer cell lines investigated, BGC-823 cells demonstrating low CD97 protein expression were stably transfected with pcDNA3.1 vector containing CD97/EGF1,2,5 or CD97/EGF1,2,3,4,5 inserts. Behavior of transfected cells was systematically investigated by employing proliferation, motility and invasive assays. As a result, we found that over-expression of CD97/EGF1,2,5 isoform correlated with increased motile and invasive ability of the clones. Furthermore, CD97/EGF1,2,5 isoform over-expression (3.8 times higher) was followed by significant decrease of CD97/EGF1,2,3,4,5 isoform (10.3 times lower). In contrast, CD97/EGF1,2,3,4,5 clones revealed significantly reduced invasive properties as compared with corresponding controls. The changes in acetylation status were one of the possible mechanisms affecting behavior of transfected cells. We concluded from the study that CD97 is closely related with advanced stages and higher invasiveness of gastric carcinoma. The study further lightened the tumor promoting role of CD97 small isoform in cancer progression and indicated the possible suppressive properties of the full length isoform of CD97.


Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Invasividade Neoplásica/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/genética , Separação Celular , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Invasividade Neoplásica/patologia , Isoformas de Proteínas , Receptores Acoplados a Proteínas G , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/patologia , Transfecção
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