RESUMO
Adrenocortical carcinomas (ACCs) overexpress insulin-like growth factor 2 (IGF2), that drives a proliferative autocrine loop by binding to IGF1R and IR, but IGF1R/IR-targeted therapies failed in ACC patients. The cytoskeleton actin-binding protein filamin A (FLNA) impairs IR signalling in melanoma cells. Aims of this study were to test FLNA involvement in regulating IGF1R and IR responsiveness to both IGF2 and inhibitors in ACC. In ACC cells H295R and SW13 and primary cultures (1ACC, 4 adenomas) we found that IGF1R and IR interacted with FLNA, and FLNA silencing increased IGF1R and reduced IR expression, with a downstream effect of increased cell proliferation and ERK phosphorylation. In addition, FLNA knockdown potentiated antiproliferative effects of IGF1R/IR inhibitor Linsitinib and IGF1R inhibitor NVP-ADW742 in H295R. Finally, Western blot showed lower FLNA expression in ACCs (n = 10) than in ACAs (n = 10) and an inverse correlation of FLNA/IGF1R ratio with ERK phosphorylation in ACCs only. In conclusion, we demonstrated that low FLNA levels enhance both IGF2 proliferative effects and IGF1R/IR inhibitors efficacy in ACC cells, suggesting FLNA as a new factor influencing tumor clinical behavior and the response to the therapy with IGF1R/IR-targeted drugs.
Assuntos
Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/patologia , Biomarcadores Tumorais/metabolismo , Filaminas/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Citoesqueleto de Actina/metabolismo , Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Neoplasias do Córtex Suprarrenal/metabolismo , Carcinoma Adrenocortical/tratamento farmacológico , Carcinoma Adrenocortical/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Filaminas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Imidazóis/farmacologia , Fator de Crescimento Insulin-Like II/genética , Mitógenos/farmacologia , Pirazinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Medullary thyroid carcinoma (MTC) is a rare neuroendocrine neoplasm of the parafollicular thyroid C cells. Although somatostatin receptors are expressed by MTCs, treatment with octreotide has shown poor efficacy, whereas recently pasireotide has demonstrated antiproliferative effects in persistent postoperative MTCs. Aim of this study was to test the effects of octreotide and pasireotide on MTC cells proliferation, cell cycle proteins expression, MAPK activation, apoptosis, calcitonin secretion, migration and invasion in TT cell line as well as in primary MTC cultured cells. Our results showed that both octreotide and pasireotide reduced TT cell proliferation (-35.2 ± 12.1%, p < 0.001, and -25.3 ± 24.8%, p < 0.05, at 10-8 M, respectively), with concomitant inhibition of ERK phosphorylation and cyclin D1 expression. This cytostatic effect was accompanied by a proapoptotic action, with an increase of caspase3/7 activity of 1.5-fold. Moreover, both octreotide and pasireotide inhibited cell migration (-50.9 ± 11.3%, p < 0.01, and -40.5 ± 17%, p < 0.05, respectively) and invasion (-61.3 ± 35.1%, p < 0.05, and -49.7 ± 18%, p < 0.01, respectively). No effect was observed on calcitonin secretion. We then tried to extend these observations to primary cultures (n = 5). Octreotide and/or pasireotide were effective in reducing cells proliferation in 3 out of 5 tumors, and to induce cell apoptosis in 1 out of 3 MTCs. Both octreotide and pasireotide were able to reduce cell migration in all MTC tested. SST2, SST3 and SST5 were expressed in all MTC, with a tendency to increased expression of SST2 in RET mutated vs wild type MTCs. In agreement, inhibition of mutated RET in TT cells reduced SST2 expression. In conclusion, we demonstrated that octreotide and pasireotide inhibited cell proliferation and invasiveness in a subset of MTC, supporting their potential use in the control of tumor growth.
Assuntos
Carcinoma Neuroendócrino/patologia , Octreotida/farmacologia , Somatostatina/análogos & derivados , Neoplasias da Glândula Tireoide/patologia , Apoptose/efeitos dos fármacos , Calcitonina/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Mutação/genética , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-ret/genética , Somatostatina/metabolismo , Somatostatina/farmacologia , Células Tumorais CultivadasRESUMO
The antidiabetic drug metformin displays anticancer properties in several neoplasms. In pituitary NETs, aryl hydrocarbon receptor-interacting protein (AIP) is up-regulated by the somatostatin analog octreotide. Metformin inhibited QGP-1 cell proliferation in a dose- and time-dependent manner, at concentrations similar to those achievable in treated patients (-31 ± 12%, p < 0.05 vs basal at 100 µM). Moreover, metformin decreased pancreatic neuroendocrine tumors (PAN-NETs) cell proliferation (-62 ± 15%, p < 0.0001 vs basal at 10 mM), without any additive effect when combined with octreotide. Both octreotide and metformin induced AIP up-regulation. AIP silencing abolished the reduction of mTOR phosphorylation induced by metformin and octreotide. Moreover, metformin decreased HSP70, increased Zac1 and AhR expression; these effects were abolished in AIP silenced QGP-1 cells. In conclusion, metformin acts as an anticancer agent in PAN-NET cells, its activity is mediated by AIP and its interacting proteins. These findings provide a novel insight into the antitumorigenic mechanism of metformin.
Assuntos
Antineoplásicos/uso terapêutico , Metformina/uso terapêutico , Tumores Neuroendócrinos/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Inativação Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Metformina/farmacologia , Pessoa de Meia-Idade , Modelos Biológicos , Tumores Neuroendócrinos/patologia , Octreotida/farmacologia , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Ensaio Tumoral de Célula-Tronco , Proteínas Supressoras de Tumor/metabolismoRESUMO
Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular thyroid C cells and accounts for 5% of thyroid cancers. In inherited cases of MTC, and in about 40% of sporadic cases, activating mutations of the receptor tyrosine kinase proto-oncogene RET are found. Constitutively active RET triggers signaling pathways involved in cell proliferation, survival and motility, but the mechanisms underlying malignant transformation of C-cells have been only partially elucidated. Cofilin is a key regulator of actin cytoskeleton dynamics. A crucial role of cofilin in tumor development, progression, invasion and metastasis has been demonstrated in different human cancers, but no data are available in MTC. Interestingly, RET activation upregulates cofilin gene expression. The aim of this study was to investigate cofilin contribution in invasiveness and growth of MTC cells, and its relevance in the context of mutant RET signaling. We found that cofilin transfection in human MTC cell line TT significantly increased migration (178⯱â¯44%, pâ¯<â¯0.001), invasion (165⯱â¯28%, pâ¯<â¯0.01) and proliferation (146⯱â¯18%, pâ¯<â¯0.001), accompanied by an increase of ERK1/2 phosphorylation (2.23-fold) and cyclin D1 levels (1.43-fold). Accordingly, all these responses were significantly reduced after genetic silencing of cofilin (-55⯱â¯10% migration, pâ¯<â¯0.001, -41⯱â¯8% invasion, pâ¯<â¯0.001, -17⯱â¯3% proliferation, pâ¯<â¯0.001). These results have been confirmed in primary cells cultures obtained from human MTCs. The inhibition of constitutively active RET in TT cells by both the RET pharmacological inhibitor RPI-1 and the transfection of dominant negative RET mutant (RETΔTK) resulted in a reduction of cofilin expression (-37⯱â¯8%, pâ¯<â¯0.001 and -31⯱â¯16%, pâ¯<â¯0.01, respectively). Furthermore, RPI-1 inhibitory effects on TT cell migration (-57⯱â¯13%, pâ¯<â¯0.01), but not on cell proliferation, were completely abolished in cells transfected with cofilin. In conclusion, these data indicate that an unbalanced cofilin expression, induced by oncogenic RET, contributes to promote MTC invasiveness and growth, suggesting the possibility of targeting cofilin pathway for more effective treatment of MTC.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Carcinoma Neuroendócrino/metabolismo , Carcinoma Neuroendócrino/patologia , Movimento Celular , Proteínas Proto-Oncogênicas c-ret/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Mutação/genética , Invasividade Neoplásica , Inibidores de Proteínas Quinases/farmacologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidoresRESUMO
Although generally benign, pituitary tumors are frequently locally invasive, with reduced success of neurosurgery and unresponsive to pharmacological treatment with somatostatin or dopamine analogues. The molecular basis of the different biological behavior of pituitary tumors are still poorly identified, but a body of work now suggests that the activity of specific cytoskeleton proteins is a key factor regulating both the invasiveness and drug resistance of these tumors. This review recapitulates the experimental evidence supporting a role for the actin-binding protein filamin A (FLNA) in the regulation of somatostatin and dopamine receptors expression and signaling in pituitary tumors, thus in determining the responsiveness to currently used drugs, somatostatin analogues and dopamine receptor type 2 agonists. Regarding the regulation of invasive behavior of pituitary tumoral cells, we bring evidence to the role of the actin-severing protein cofilin, whose activation status may be modulated by dopaminergic and somatostatinergic drugs, through FLNA involvement. Molecular mechanisms involved in the regulation of FLNA expression and function in pituitary tumors will also be discussed.
Assuntos
Filaminas/metabolismo , Neoplasias Hipofisárias/metabolismo , Animais , Citoesqueleto/metabolismo , Humanos , Neoplasias Hipofisárias/tratamento farmacológico , Neoplasias Hipofisárias/patologiaRESUMO
An efficient intracellular response to somatostatin analogs (SSA) in pituitary tumors requires filamin A (FLNA). Since cAMP pathway plays an important role in GH-secreting pituitary tumors pathogenesis and FLNA is phosphorylated by PKA on S2152, aim of this study was to investigate in tumoral somatotrophs the impact of cAMP pathway activation and SSA stimulation on FLNA phosphorylation and the consequences on SST2 function. We found a PKA-mediated increase (2-fold) and SST2 agonist-induced decrease (-50%) of FLNA phosphorylation in GH3, GH4C1 and primary somatotroph tumor cells. This modification regulates FLNA function. Indeed, phosphomimetic S2152D FLNA mutant, but not phosphodeficient S2152A, abolished the known SSA antitumoral effects, namely: 1) inhibition of cell proliferation, reduction of cyclin D3 and increase of p27; 2) increase of cell apoptosis; 3) inhibition of cell migration via RhoA activation and cofilin phosphorylation. Coimmunoprecipitation and immunofluorescence assays showed that S2152A FLNA was recruited to activated SST2, whereas S2152D FLNA constitutively bound SST2 on the plasma membrane, but prevented Gαi proteins recruitment to SST2. In conclusion, we demonstrated that FLNA phosphorylation, promoted by cAMP pathway activation and inhibited by SSA, prevented SST2 signaling in GH-secreting tumoral pituitary cells.
Assuntos
AMP Cíclico/metabolismo , Filaminas/metabolismo , Adenoma Hipofisário Secretor de Hormônio do Crescimento/metabolismo , Neoplasias Hipofisárias/metabolismo , Proteínas Quinases/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Adenoma Hipofisário Secretor de Hormônio do Crescimento/genética , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Hormônios/farmacologia , Humanos , Fosforilação/efeitos dos fármacos , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Somatostatina/farmacologia , Somatotrofos/efeitos dos fármacos , Somatotrofos/metabolismo , Células Tumorais CultivadasRESUMO
OBJECTIVE: To evaluate if the parameters of hypothalamic-pituitary-adrenal (HPA) axis activity could predict the occurrence and duration of post-surgical hypocortisolism (PSH) in patients with Cushing's syndrome (CS) and with adrenal incidentaloma (AI). METHODS: We studied 80 patients (54 females, age 53.3 ± 11 years), who underwent adrenalectomy for CS (17 patients) or for AI (53 patients). Before surgery, we measured adrenocorticotroph hormone (ACTH), urinary free cortisol (UFC) and serum cortisol after 1 mg dexamethasone suppression test (1 mg-DST) levels. After surgery, all patients were given a steroid replacement therapy, and PSH was searched after 2 months by a low-dose (1 µg, iv) corticotropin stimulation test, that was repeated every 6 months in PSH patients for at least 4 years. RESULTS: The PSH occurred in 82.4 and 46% of CS and AI patients, respectively. In the whole sample and in AI patients separately considered, the PSH was independently predicted by the preoperative cortisol levels after 1 mg-DST, however, with a low (< 70%) accuracy. In AI patients the PSH occurrence was not ruled out even by the cortisol levels after 1 mg-DST lower than 1.8 µg/dL (50 nmol/L). In the 50% of CS patients and in 31% of AI patients the PSH lasted more than 18 months and in 35.7% of CS patients it persisted for more than 36 months. In AI patients, the PSH duration was not predictable by any parameter. However, a PSH duration of at least 12 months was significantly predicted before adrenalectomy (sensitivity 91.7%, specificity 41.2%, positive predictive value 52.4%, negative predictive value 87.5%, p = 0.05) by the presence of at least 2 out of low ACTH levels, increased UFC levels and cortisol levels after 1 mg-DST ≥ 3.0 µg/dL (83 nmol/L). CONCLUSION: The PSH occurrence and its duration are hardly predictable before surgery. All patients undergoing unilateral adrenalectomy should receive a steroid substitutive therapy.
Assuntos
Doença de Addison/diagnóstico , Doença de Addison/epidemiologia , Neoplasias das Glândulas Suprarrenais/cirurgia , Adrenalectomia/efeitos adversos , Hormônio Adrenocorticotrópico/sangue , Síndrome de Cushing/cirurgia , Complicações Pós-Operatórias , Doença de Addison/sangue , Doença de Addison/etiologia , Adulto , Idoso , Feminino , Humanos , Sistema Hipotálamo-Hipofisário , Incidência , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Sistema Hipófise-SuprarrenalRESUMO
The pharmacological therapy of GH-secreting pituitary tumors is based on somatostatin (SS) analogs that reduce GH secretion and cell proliferation by binding mainly SS receptors type 2 (SST2). Antimigratory effects of SS have been demonstrated in different cell models, but no data on pituitary tumors are available. Aims of our study were to evaluate SST2 effects on migration and invasion of human and rat tumoral somatotrophs, and to elucidate the molecular mechanism involved focusing on the role of cofilin and filamin A (FLNA). Our data revealed that SST2 agonist BIM23120 significantly reduced GH3 cells migration (-22% ± 3.6%, p < 0.001) and invasion on collagen IV (-31.3% ± 12.2%, p < 0.01), both these effects being reproduced by octreotide and pasireotide. Similar results were obtained in primary cultured cells from human GH-secreting tumors. These inhibitory actions were accompanied by a marked increase in RhoA/ROCK-dependent cofilin phosphorylation (about 2.7-fold in GH3 and 2.1-fold in human primary cells). Accordingly, the anti-invasive effect of the SS analog was mimicked by the overexpression in GH3 cells of the S3D phosphomimetic cofilin mutant, and abolished by both phosphodeficient S3A cofilin and a specific ROCK inhibitor that prevented cofilin phosphorylation. Moreover, FLNA silencing and FLNA dominant-negative mutants FLNA19-20 and FLNA21-24 transfection demonstrated that FLNA plays a scaffold function for SST2-mediated cofilin phosphorylation. Accordingly, cofilin recruitment to agonist-activated SST2 was completely lost in FLNA silenced cells. In conclusion, we demonstrated that SST2 inhibits rat and human tumoral somatotrophs migration and invasion through a molecular mechanism that involves FLNA-dependent cofilin recruitment and phosphorylation.
Assuntos
Citoesqueleto/metabolismo , Neoplasias Hipofisárias/tratamento farmacológico , Neoplasias Hipofisárias/metabolismo , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Fatores de Despolimerização de Actina/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Filaminas/metabolismo , Humanos , Invasividade Neoplásica , Fosforilação , Neoplasias Hipofisárias/patologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Somatostatina/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
cAMP pathway plays a major role in the pathogenesis of cortisol-producing adrenocortical adenomas (CPA). cAMP-induced steroidogenesis is preceded by actin cytoskeleton reorganization, a process regulated by cofilin activity. In this study we investigated cofilin role in mediating cAMP effects on cell morphology and steroidogenesis in adrenocortical tumor cells. We demonstrated that forskolin induced cell rounding and strongly reduced phosphorylated (P)-cofilin/total cofilin ratio in Y1 (-52 ± 16%, p < 0.001) and human CPA cells (-53 ± 18%, p < 0.05). Cofilin silencing significantly reduced both forskolin-induced morphological changes and progesterone production (1.3-fold vs 1.8-fold in controls, p < 0.05), whereas transfection of wild-type or S3A (active), but not S3D (inactive) cofilin, potentiated forskolin effects on cell rounding and increased 3-fold progesterone synthesis with respect to control (p < 0.05). Furthermore, cofilin dephosphorylation by a ROCK inhibitor potentiated forskolin-induced cell rounding and steroidogenesis (2-fold increase vs forskolin alone). Finally, we found a reduced P-cofilin/total cofilin ratio and increased cofilin expression in CPA vs endocrine inactive adenomas by western blot and immunohistochemistry. Overall, these results identified cofilin as a mediator of cAMP effects on both morphological changes and steroidogenesis in mouse and human adrenocortical tumor cells.
Assuntos
Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/metabolismo , AMP Cíclico/farmacologia , Esteroides/biossíntese , Fatores de Despolimerização de Actina/antagonistas & inibidores , Fatores de Despolimerização de Actina/genética , Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Neoplasias do Córtex Suprarrenal/patologia , Adenoma Adrenocortical/tratamento farmacológico , Adenoma Adrenocortical/patologia , Animais , Colforsina/farmacologia , Humanos , Hidrocortisona/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas , Vasodilatadores/farmacologiaRESUMO
The role of progenitor/stem cells in pituitary tumorigenesis, resistance to pharmacological treatments and tumor recurrence is still unclear. This study investigated the presence of progenitor/stem cells in non-functioning pituitary tumors (NFPTs) and tested the efficacy of dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists to inhibit in vitro proliferation. They found that 70% of 46 NFPTs formed spheres co-expressing stem cell markers, transcription factors (DAX1, SF1, ERG1) and gonadotropins. Analysis of tumor behavior showed that spheres formation was associated with tumor invasiveness (OR = 3,96; IC: 1.05-14.88, p = 0.036). The in vitro reduction of cell proliferation by DRD2 and SSTR2 agonists (31 ± 17% and 35 ± 13% inhibition, respectively, p < 0.01 vs. basal) occurring in about a half of NFPTs cells was conserved in the corresponding spheres. Accordingly, these drugs increased cyclin-dependent kinase inhibitor p27 and decreased cyclin D3 expression in spheres. In conclusion, they provided further evidence for the existence of cells with a progenitor/stem cells-like phenotype in the majority of NFPTs, particularly in those with invasive behavior, and demonstrated that the antiproliferative effects of dopaminergic and somatostatinergic drugs were maintained in progenitor/stem-like cells.
Assuntos
Carcinogênese/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias Hipofisárias/tratamento farmacológico , Receptores de Dopamina D2/genética , Receptores de Somatostatina/genética , Adulto , Carcinogênese/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D3/biossíntese , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Receptor Nuclear Órfão DAX-1/biossíntese , Dopaminérgicos/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/genética , Canal de Potássio ERG1/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Gonadotropinas/biossíntese , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Fatores de Processamento de RNA/biossíntese , Receptores de Dopamina D2/agonistas , Receptores de Somatostatina/agonistas , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologiaRESUMO
All tissues and organs derive from stem cells, which are undifferentiated cells able to differentiate into specialized cells and self-renewal. In mammals, there are embryonic stem cells that generate germ layers, and adult stem cells, which act as a repair system for the body and maintain the normal turnover of regenerative organs. Mesenchymal stem cells (MSCs) are nonhematopoietic adult multipotent cells, which reside in virtually all postnatal organs and tissues, and, under appropriate in vitro conditions, are capable to differentiate into osteogenic, adipogenic, chondrogenic, myogenic, and neurogenic lineages. Their commitment and differentiation depend on several interacting signaling pathways and transcription factors. Most GNAS-based disorders have the common feature of episodic de novo formation of islands of extraskeletal, qualitatively normal, bone in skin and subcutaneous fat. The tissue distribution of these lesions suggests that pathogenesis involves abnormal differentiation of MSCs and/or more committed precursor cells that are present in subcutaneous tissues. Data coming from transgenic mice support the concept that GNAS is a key factor in the regulation of lineage switching between osteoblast and adipocyte fates, and that its role may be to prevent bone formation in tissues where bone should not form. Despite the growing knowledge about the process of heterotopic ossification in rare genetic disorders, the pathophysiological mechanisms by which alterations of cAMP signaling lead to ectopic bone formation in the context of mesenchymal tissues is not fully understood.
Assuntos
Células-Tronco Adultas/metabolismo , Diferenciação Celular , Cromograninas/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Impressão Genômica , Células-Tronco Mesenquimais/metabolismo , Sistemas do Segundo Mensageiro , Células-Tronco Adultas/patologia , Animais , Cromograninas/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Células-Tronco Mesenquimais/patologiaRESUMO
Non-functioning pituitary tumors (NFPTs) frequently present local invasiveness. Dopamine receptor 2 (DRD2) agonists are the only medical therapy that induces tumor shrinkage in some patients. Invasion requires cytoskeleton rearrangements that are tightly regulated by cofilin pathway, whose alterations correlate with invasion in different tumors. We investigated the effect of DR2D agonist on NFPT cells migration/invasion and the molecular mechanisms involved. We demonstrated that DRD2 agonist reduced migration (-44 ± 25%, p < 0.01) and invasion (-34 ± 6%, p < 0.001) and increased about 4-fold Ser3-phosphorylated inactive cofilin (P-cofilin) in NFPT cells. These effects were abolished by inhibiting ROCK, a kinase that phosphorylates cofilin. The overexpression of wild-type or phosphodeficient S3A-cofilin increased HP75 cells migration (+49 ± 6% and +57 ± 9% vs empty vector, respectively, p < 0.05), while phosphomimetic mutant had no effect. Interestingly, P-cofilin levels were lower in invasive vs non-invasive tumors by both western blot (mean P-cofilin/total cofilin ratio 0.77 and 1.93, respectively, p < 0.05) and immunohistochemistry (mean percentage of P-cofilin positive cells 17.6 and 45.7, respectively, p < 0.05). In conclusion, we showed that the invasiveness of pituitary tumors is promoted by the activation of cofilin, which can be regulated by DRD2 and might represent a novel biomarker for pituitary tumors' invasive behavior.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Movimento Celular , Neoplasias Hipofisárias/enzimologia , Receptores de Dopamina D2/metabolismo , Quinases Associadas a rho/metabolismo , Fatores de Despolimerização de Actina/genética , Adulto , Idoso , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Fosforilação , Neoplasias Hipofisárias/tratamento farmacológico , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Mutação Puntual , Inibidores de Proteínas Quinases/farmacologia , Receptores de Dopamina D2/agonistas , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
BACKGROUND: The optimal duration of cabergoline (CAB) treatment of prolactinomas that minimizes recurrences is not well established. 2011 Endocrine Society Guidelines suggested that withdrawal may be safely undertaken after 2 years in patients achieving normoprolactinemia and tumor reduction. MATERIALS: We analyzed 74 patients (mean age = 46.9 ± 14.4, M/F = 19/55, macro/micro = 18/56) bearing a prolactinoma divided in 3 groups: group A (23) treated for 3 years, group B (23) for a period between 3 and 5 years, and group C (28) for a period >5 years. CAB therapy was interrupted according to Endocrine Society Guidelines. Prolactin (PRL) levels were measured 3, 6, 12 and 24 months after withdrawal. Recurrence was defined with PRL levels ≥30 ng/ml. RESULTS: Groups did not differ in pretreatment PRL levels (123.2 ± 112.1, 120.9 ± 123.8, 176.6 ± 154.0), pituitary deficit (4, 17, 17 %), mean CAB weekly dose (0.7 ± 0.4, 0.6 ± 0.3, 0.7 ± 0.4) and PRL levels before withdrawal (17.1 ± 19.6, 11.4 ± 8.8, 13.8 ± 13.5). Recurrence occurred within 12 months in 34 patients (45.9 %), without significant differences among groups. Neuroradiological evaluation showed a significantly higher presence of macroadenoma in group C (13, 17 and 39 %, respectively). Recurrence rate of hyperprolactinemia did not depend on sex, tumor size or CAB dose but it was significantly correlated with PRL levels at diagnosis and before withdrawal (p = 0.03). Finally, patients with pituitary deficit at diagnosis showed a significantly higher recurrence rate (p = 0.03). CONCLUSIONS: The study provides additional evidence that prolonging therapy for more than 3 years does not reduce recurrence rate. In particular, recurrence risk was similar in micro- and macroadenomas, and higher in patients with pituitary deficits at diagnosis.
Assuntos
Agonistas de Dopamina/uso terapêutico , Ergolinas/uso terapêutico , Hiperprolactinemia/etiologia , Recidiva Local de Neoplasia/etiologia , Neoplasias Hipofisárias/tratamento farmacológico , Prolactinoma/tratamento farmacológico , Adulto , Idoso , Biomarcadores/sangue , Cabergolina , Feminino , Humanos , Hiperprolactinemia/diagnóstico , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/diagnóstico , Neoplasias Hipofisárias/complicações , Neoplasias Hipofisárias/patologia , Prognóstico , Prolactinoma/complicações , Prolactinoma/patologia , Tomografia Computadorizada por Raios X/métodos , Suspensão de TratamentoRESUMO
cAMP effects have been initially attributed to protein kinase A (PKA) activation. Subsequently, two exchange proteins directly activated by cAMP (Epac1/2) have been identified as cAMP targets. Aim of this study was to investigate cAMP effects in pancreatic-NET (P-NET) and bronchial carcinoids and in corresponding cell lines (QGP-1 and H727) on cell proliferation and adhesion and to determine PKA and Epac role in mediating these effects. We found that cAMP increased cyclin D1 expression in P-NET and QGP-1 cells, whereas it had opposite effects on bronchial carcinoids and H727 cells and it promoted cell adhesion in QGP-1 and H727 cells. These effects are mimicked by Epac and PKA specific analogs, activating the small GTPase Rap1. In conclusion, we demonstrated that cAMP exerted divergent effects on proliferation and promoted cell adhesion of different neuroendocrine cell types, these effects being mediated by both Epac and PKA and involving the same effector GTPase Rap1.
Assuntos
Proliferação de Células , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Tumores Neuroendócrinos/metabolismo , Adesão Celular , Humanos , Tumores Neuroendócrinos/patologia , Células Tumorais CultivadasRESUMO
Emricasan, formerly IDN-6556, is a small molecule currently being evaluated in clinical trials to reduce hepatic injury and liver fibrosis. Since emricasan is an irreversible pan-caspase inhibitor that potently inhibits caspase-mediated apoptosis and inflammation, its carcinogenic potential was evaluated in a humanized mouse model. Tg.rasH2 mice received LabDiet formulated with 0, 10, 25, and 75mg/kg/day of emricasan, for 26weeks. At terminal sacrifice, blood was collected for clinical pathology analysis and tissues were collected, processed, and evaluated microscopically. There were no treatment related deaths or overt signs of toxicity for the duration of the study. There was no evidence of a carcinogenic effect in the peripheral blood leukocyte counts. Liver microgranulomas, which are background lesions, were slightly increased, especially in males. Increases in the incidence of the activated germinal centers were seen in the spleens and mesenteric lymph nodes of males and females, and in the mandibular lymph nodes of male mice. Atrophy of ovaries and testicular degeneration were also seen in emricasan treated animals. Although several non-neoplastic lesions were observed, there was no evidence of emricasan-related tumor formation in any tissue. In addition, the non-neoplastic lesions were not considered pre-neoplastic. Thus, emricasan is not considered carcinogenic.
Assuntos
Inibidores de Caspase/toxicidade , Ácidos Pentanoicos/toxicidade , Animais , Testes de Carcinogenicidade , Inibidores de Caspase/sangue , Inibidores de Caspase/farmacocinética , Feminino , Genes ras , Granuloma/induzido quimicamente , Fígado/efeitos dos fármacos , Fígado/patologia , Pulmão/anatomia & histologia , Pulmão/efeitos dos fármacos , Masculino , Camundongos Transgênicos , Ovário/efeitos dos fármacos , Ovário/patologia , Ácidos Pentanoicos/sangue , Ácidos Pentanoicos/farmacocinética , Baço/anatomia & histologia , Baço/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/patologiaRESUMO
CONTEXT: Pituitary incidentalomas (PIs) are commonly encountered in clinical practice. The management of these asymptomatic pituitary lesions is still controversial. Systematic screening for subclinical or mild ACTH-dependent hypercortisolism (AH) is not presently recommended, due to the limited data available thus far on the epidemiological and clinical relevance of this condition in patients with PIs. As subclinical hypercortisolism (SH) was considered to be associated with chronic complications of overt cortisol excess, such as hypertension, diabetes, and osteoporosis, this disorder should be diagnosed at the early stage. OBJECTIVE: The objective of this study was to evaluate the prevalence of hypercortisolism in a population of subjects with PIs. DESIGN, SUBJECTS, AND METHODS: A total of 68 consecutive patients (48 females and 20 males, aged 18-82 years) without clinically overt hypercortisolism, who were referred for evaluation of PIs between January 2010 and March 2013, were prospectively investigated for AH. Pituitary hypercortisolism was diagnosed in the presence of cortisol >50ânmol/l after 1âmg dexamethasone suppression test, non-suppressed ACTH, and the additional finding of one of the following: urinary free cortisol (UFC) >193ânmol/24âh, and midnight serum and salivary cortisol levels >207 and 2.8ânmol/l respectively. RESULTS: Among patients with PIs, we found a 7.3% rate of pituitary hypercortisolism diagnosed with biochemical criteria and a 4.4% rate of histologically confirmed AH. CONCLUSIONS: Subclinical or mild hypercortisolism may be more common than generally perceived in patients with PIs.
Assuntos
Adenoma Hipofisário Secretor de ACT/epidemiologia , Adenoma/epidemiologia , Achados Incidentais , Hipersecreção Hipofisária de ACTH/diagnóstico , Hipersecreção Hipofisária de ACTH/epidemiologia , Adenoma Hipofisário Secretor de ACT/complicações , Adenoma Hipofisário Secretor de ACT/diagnóstico , Adenoma/complicações , Adenoma/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Subclinical hypercortisolism (SH) has been associated with metabolic complications such as type 2 diabetes mellitus, obesity and dyslipidemia. Scarce data are available regarding the lipid pattern abnormalities in SH, in relation to insulin resistance and impaired glucose metabolism (IGM). We aimed to evaluate the possible influence of SH on lipid pattern in relation to the presence/absence of impaired glucose metabolism. METHODS: In 338 patients with adrenal incidentaloma, the presence of SH, hypertension, dyslipidemia and IGM was evaluated. According to the presence of SH and IGM the patients were divided into 4 groups (IGM+SH+, IGM+SH-, IGM-SH+, IGM-SH-). We recruited 98 subjects without IGM (IGM-) and 100 with IGM (IGM+) as control groups. RESULTS: The prevalence of dyslipidemia was comparable among Group IGM+SH+, Group IGM+SH- and IGM+ controls (57.9, 58.4, 56%, P = NS). No difference in dyslipidemia prevalence among IGM- patients and IGM- controls was observed. The IGM+SH+ patients had a higher prevalence of dyslipidemia (57.9%) than IGM-SH+ ones (29.1%, P < 0.01). The IGM+SH- patients showed an increased prevalence of hypertension (76.6 vs 54.8%, P < 0.01) and dyslipidemia (58.4 vs 23.8%, P < 0.0001) as compared with IGM-SH- patients. Logistic regression analysis showed that only IGM was associated to dyslipidemia (OR 4.31, 95% CI 2.61-7.12, P = 0.0001) regardless of age, SH and gender. CONCLUSIONS: In the absence of alterations of glucose metabolism the presence of a subtle cortisol excess has no effect on lipid pattern. IGM seems to influence the lipid metabolism regardless of the presence of SH.
Assuntos
Neoplasias das Glândulas Suprarrenais/epidemiologia , Síndrome de Cushing/epidemiologia , Dislipidemias/epidemiologia , Intolerância à Glucose/epidemiologia , Lipídeos/sangue , Neoplasias das Glândulas Suprarrenais/sangue , Neoplasias das Glândulas Suprarrenais/patologia , Idoso , Comorbidade , Síndrome de Cushing/sangue , Síndrome de Cushing/patologia , Dislipidemias/sangue , Dislipidemias/patologia , Feminino , Intolerância à Glucose/sangue , Intolerância à Glucose/patologia , Humanos , Masculino , Pessoa de Meia-Idade , PrevalênciaRESUMO
PURPOSE: Adult patients operated for craniopharyngioma develop more frequently GH deficiency (GHD) than patients operated for non-functioning pituitary adenoma (NFPA). The aim of the study was to compare both short- (1 year) and long-term (5 years) effects of rhGH in 38 GHD adult patients (19 operated for Craniopharyngioma (CP) and 19 for NFPA). METHODS: IGF-I levels, body composition (BF%), BMI, lipid profile and glucose homeostasis were evaluated in all patients. Pituitary MRI was performed at baseline and during follow-up, as needed. RESULTS: At baseline no difference between the two groups was observed, apart from a higher prevalence of diabetes insipidus in CP patients (79 vs 21%). After 12 months, IGF-I SDS normalized and BF% significantly decreased only in the NFPA group. During long-term treatment, decrease in BF% and improvement in lipid profile shown by reduction in total- and LDL-cholesterol were present in NFPA group only, while increase in insulin levels and HbA1c and decrease of QUICKI were observed in CP patients only. Accordingly, after long-term therapy, the prevalence of metabolic syndrome (MS) was significantly higher in CP than in NFPA group (37% in CP and in 5% in NFPA group; p < 0.05). CONCLUSION: The present data suggest that CP patients are less sensitive to the positive rhGH effects on lipid profile and BF% and more prone to insulin sensitivity worsening than NFPA patients, resulting in increased prevalence of MS in CP only.
Assuntos
Adenoma/sangue , Adenoma/metabolismo , Craniofaringioma/sangue , Terapia de Reposição Hormonal , Hormônio do Crescimento Humano/farmacologia , Síndrome Metabólica/sangue , Neoplasias Hipofisárias/sangue , Adenoma/tratamento farmacológico , Adenoma/cirurgia , Adulto , Craniofaringioma/tratamento farmacológico , Craniofaringioma/cirurgia , Feminino , Hormônio do Crescimento Humano/administração & dosagem , Hormônio do Crescimento Humano/deficiência , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Hipofisárias/tratamento farmacológico , Neoplasias Hipofisárias/cirurgia , Adulto JovemRESUMO
Components of the tumour microenvironment initiate and promote cancer development. In this study, we investigated the stromal component of parathyroid neoplasia. Immunohistochemistry for alpha-smooth muscle actin (α-SMA) showed an abundant periacinar distribution of α-SMA(+) cells in normal parathyroid glands (n=3). This pattern was progressively lost in parathyroid adenomas (PAds; n=6) where α-SMA(+)cells were found to surround new microvessels, as observed in foetal parathyroid glands (n=2). Moreover, in atypical adenomas (n=5) and carcinomas (n=4), α-SMA(+) cells disappeared from the parenchyma and accumulated in the capsula and fibrous bands. At variance with normal glands, parathyroid tumours (n=37) expressed high levels of fibroblast-activation protein (FAP) transcripts, a marker of tumour-associated fibroblasts. We analysed the ability of PAd-derived cells to activate fibroblasts using human bone-marrow mesenchymal stem cells (hBM-MSCs). PAd-derived cells induced a significant increase in FAP and vascular endothelial growth factor A (VEGFA) mRNA levels in co-cultured hBM-MSCs. Furthermore, the role of the calcium-sensing receptor (CASR) and of the CXCL12/CXCR4 pathway in the PAd-induced activation of hBM-MSCs was investigated. Treatment of co-cultures of hBM-MSCs and PAd-derived cells with the CXCR4 inhibitor AMD3100 reduced the stimulated VEGFA levels, while CASR activation by the R568 agonist was ineffective. PAd-derived cells co-expressing parathyroid hormone (PTH)/CXCR4 and PTH/CXCL12 were identified by FACS, suggesting a paracrine/autocrine signalling. Finally, CXCR4 blockade by AMD3100 reduced PTH gene expression levels in PAd-derived cells. In conclusion, i) PAd-derived cells activated cells of mesenchymal origin; ii) PAd-associated fibroblasts were involved in tumuor neoangiogenesis and iii) CXCL12/CXCR4 pathway was expressed and active in PAd cells, likely contributing to parathyroid tumour neoangiogenesis and PTH synthesis modulation.