Effects of let-7 microRNA on Cell Growth and Differentiation of Papillary Thyroid Cancer

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy and RET/PTC rearrangements represent key genetic events frequently associated to this cancer, enhancing proliferation and dedifferentiation by activation of the RET/PTC-RAS-BRAF-mitogen-activated protein kinase (MAPK) pathway. Recently, let-7 microRNA was found to reduce RAS levels in lung cancer, acting as a tumor suppressor gene. Here, we report that RET/PTC3 oncogenic activation in PCCL3 rat thyroid cells markedly reduces let-7f expression. Moreover, stable transfection of let-7 microRNA in TPC-1 cells, which harbor RET/PTC1 rearrangement, inhibits MAPK activation. As a result, let-7f was capable of reducing TPC-1 cell growth, and this might be explained, at least in part, by decreased messenger RNA (mRNA) expression of cell cycle stimulators such as MYC and CCND1 (cyclin D1) and increased P21 cell cycle inhibitor mRNA. In addition, let-7 enhanced transcriptional expression of molecular markers of thyroid differentiation such as TITF1 and TG. Thus, reduced expression of let-7f might be an essential molecular event in RET/PTC malignant transformation. Moreover, let-7f effects on thyroid growth and differentiation might attenuate neoplastic process of RET/PTC papillary thyroid oncogenesis through impairment of MAPK signaling pathway activation. This is the first functional demonstration of an association of let-7 with thyroid cancer cell growth and differentiation.

Recurrent sores by ill-fitting dentures and intra-oral squamous cell carcinoma in smokers

Objectives: To examine whether denture use and recurrent sores caused by ill-fitting dentures are associated with intra-oral squamous cell carcinoma (IO-SCC) in individuals exposed to tobacco. Methods: We conducted a hospital-based case-control study. The study population comprised 124 patients with IO-SCC and the same number of controls (individually paired according to gender and age) recruited from outpatient units of the same hospital. Conditional logistic regression analysis assessed the effect of denture use and recurrent oral sores by ill-fitting dentures, adjusted by covariates on the lifetime exposure to alcohol and tobacco, socioeconomic standings, and dietary patterns. Results: The use of dentures showed no association with IO-SCC [adjusted odds ratio (OR) 1.40, 95 percent confidence interval 0.51-3.87, P = 0.513] in an assessment controlled by socioeconomic position, lifetime exposure to alcohol and tobacco, and dietary patterns. However, the report of recurrent sores caused by ill-fitting dentures showed significant association with the disease (adjusted OR 4.58, 95 percent confidence interval 1.52-13.76, P = 0.007). Conclusions: The association between recurrent oral sores caused by ill-fitting dentures and squamous cell carcinoma of the mouth in smokers is in agreement with the hypothesis that the chronic physical irritation of oral mucosa contributes to the topical carcinogenic effect of tobacco, which must be taken into careful consideration in the planning of dental services for adults and the elderly.

Comparison of biological behavior between early-stage adenocarcinoma and squamous cell carcinoma of the uterine cervix

Objectives: (1) To compare the anatomopathological variables and recurrence rates in patients with early-stage adenocarcinoma (AC) and squamous cell carcinoma (SCC) of the uterine cervix; (2) to identify the independent risk factors for recurrence. Study design: This historical cohort study assessed 238 patients with carcinoma of the uterine cervix (113 and IIA), who underwent radical hysterectomy with pelvic lymph node dissection between 1980 and 1999. Comparison of category variables between the two histological types was carried out using the Pearson`s X-2 test or Fisher exact test. Disease-free survival rates for AC and SCC were calculated using the Kaplan-Meier method and the curves were compared using the log-rank test. The Cox proportional hazards model was used to identify the independent risk factors for recurrence. Results: There were 35 cases of AC (14.7%) and 203 of SCC (85.3%). AC presented lower histological grade than did SCC (grade 1: 68.6% versus 9.4%; p < 0.001), lower rate of lymphovascular space involvement (25.7% versus 53.7%; p = 0.002), lower rate of invasion into the middle or deep thirds of the uterine cervix (40.0% versus 80.8%; p < 0.001) and lower rate of lymph node metastasis (2.9% versus 16.3%; p = 0.036). Although the recurrence rate was lower for AC than for SCC (11.4% versus 15.8%), this difference was not statistically significant (p = 0.509). Multivariate analysis identified three independent risk factors for recurrence: presence of metastases in the pelvic lymph nodes, invasion of the deep third of the uterine cervix and absence of or slight inflammatory reaction in the cervix. When these variables were adjusted for the histological type and radiotherapy status, they remained in the model as independent risk factors. Conclusion: The AC group showed less aggressive histological behavior than did the SCC group, but no difference in the disease-free survival rates was noted. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

Subclinical thyroid dysfunctions are independent risk factors for mortality in a 7.5-year follow-up: the Japanese-Brazilian thyroid study

Objective: The currently available data concerning the influence of subclinical thyroid disease (STD) on morbidity and mortality are conflicting. Our objective was to investigate the relationships between STD and cardiometabolic profile and cardiovascular disease at baseline, as well as with all-cause and cardiovascular mortality in a 7.5-year follow-up. Design: Prospective, observational study. Methods: An overall of 1110 Japanese-Brazilians aged above 30 years, free of thyroid disease, and not taking thyroid medication at baseline were studied. In a cross-sectional analysis, we investigated the prevalence of STD and its relationship with cardiometabolic profile and cardiovascular disease. All-cause and cardiovascular mortality rates were assessed for participants followed for up to 7.5 years. Association between STD and mortality was drawn using multivariate analysis, adjusting for potential confounders. Results: A total of 913 (82.3%) participants had euthyroidism, 99 (8.7%) had subclinical hypothyroidism, and 69 (6.2%) had subclinical hyperthyroidism. At baseline, no association was found between STD and cardiometabolic profile or cardiovascular disease. Multivariate-adjusted hazard ratios (HRs (95% confidence interval)) for all-cause mortality were significantly higher for individuals with both subclinical hyperthyroidism (HR, 3.0 (1.5-5.9); n=14) and subclinical hypothyroidism (HR, 2.3 (1.2-4.4); n=13) than for euthyroid subjects. Cardiovascular mortality was significantly associated with subclinical hyperthyroidism (HR, 3.3 (1.4-7.5); n=8), but not with subclinical hypothyroidism (HR, 1.6 (0.6-4.2); n=5). Conclusion: In the Japanese-Brazilian population, subclinical hyperthyroidism is an independent risk factor for all-cause and cardiovascular mortality, while subclinical hypothyroidism is associated with all-cause mortality.

Paullinia cupana Mart. var. sorbilis, Guarana, Increases Survival of Ehrlich Ascites Carcinoma (EAC) Bearing Mice by Decreasing Cyclin-D1 Expression and Inducing a G0/G1 Cell Cycle Arrest in EAC Cells

The objective of this work is to report the antiproliferative effect of P. cupana treatment in Ehrlich Ascites Carcinoma (EAC)-bearing animals. Female mice were treated with three doses of powdered P. cupana (100, 1000 and 2000 mg/kg) for 7 days, injected with 10(5) EAC cells and treated up to day 21. In addition, a survival experiment was carried out with the same protocol. P. cupana decreased the ascites volume (p = 0.0120), cell number (p = 0.0004) and hemorrhage (p = 0.0054). This occurred through a G1-phase arrest (p < 0.01) induced by a decreased gene expression of Cyclin D1 in EAC cells. Furthermore, P. cupana significantly increased the survival of EAC-bearing animals (p = 0.0012). In conclusion, the P. cupana growth control effect in this model was correlated with a decreased expression of cyclin D1 and a G1 phase arrest. These results reinforce the cancer therapeutic potential of this Brazilian plant. Copyright (C) 2010 John Wiley & Sons, Ltd.

Reactive oxygen and nitrogen species balance in the determination of thyroid hormones-induced cardiac hypertrophy mediated by renin-angiotensin system

Role of reactive oxygen species (ROS)/nitric oxide (NO) balance and renin-angiotensin system in mediating cardiac hypertrophy in hyperthyroidism was evaluated in an in vivo and in vitro experimental model. Male Wistar rats were divided into four groups: control, thyroid hormone, vitamin E (or Trolox, its hydrosoluble analogue), thyroid hormone + vitamin E. Angiotensin II receptor (AT1/AT2) gene expression, immunocontent of AT1/AT2 receptors, angiotensinogen, NADPH oxidase (Nox2), and nitric oxide synthase isoforms, as well as ROS concentration (hydrogen peroxide and superoxide anion) were quantified in myocardium. Thyroid hormone increased ROS and NO metabolites, iNOS, nNOS and eNOS isoforms and it was accompanied by cardiac hypertrophy. AT1/AT2 expression and the immunocontent of angiotensinogen and Nox2 were enhanced by thyroid hormone. Antioxidants reduced ROS levels, Nox2, AT1/AT2, NOS isoforms and cardiac hypertrophy. In conclusion, ROS/NO balance may play a role in the control of thyroid hormone-induced cardiac hypertrophy mediated by renin-angiotensin system. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Thyroid hormones alter the adenine nucleotide hydrolysis in adult rat blood serum

The effects of ATP, ADP, and adenosine in the processes of platelet aggregation, vasodilatation, and coronary flow have been known for many years. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes the main system for rapid inactivation of circulating adenine nucleotides. Thyroid disorders affect a number of biological factors including adenosine levels in different fractions. Then, we intend to investigate if the soluble nucleotidases responsible for the ATP, ADP, and AMP hydrolysis are affected by variations in the thyroid hormone levels in blood serum from adult rats. Hyperthyroidism was induced by daily intraperitoneal injections of L-thyroxine (T4) (2.5 and 10.0 mu g/100 g body weight, respectively) for 7 or 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) added to their drinking water during 7 or 14 days. The treatments efficacy was confirmed by determination of hemodynamic parameters and cardiac hypertrophy evaluation. T4 treatment predominantly inhibited, and hypothyroidism (14 days after thyroidectomy) predominantly increased the ATP, ADP, and AMP hydrolysis in rat blood serum. These results suggest that both excess and deficiency of thyroid hormones can modulate the ATP diphosphohydrolase and 5`-nucleotidase activities in rat blood serum and consequently modulate the effects mediated by these enzymes and their products in vascular system. (C) 2010 International Union of Biochemistry and Molecular Biology, Inc.

Acute actions of thyroid hormone on blood vessel biochemistry and physiology

Purpose of review Description of the progress about the vascular effects promoted by thyroid hormones. Recent findings Over the past few years, a number of studies have shown that in addition to genomic effects on blood vessels, thyroid hormones exert extranuclear nongenomic effects on vascular smooth muscle cells and endothelium. These nongenomic effects occur rapidly and do not involve thyroid hormone response elements-mediated transcriptional events. In this context, the genomic and nongenomic events promoted by thyroid hormones act in concert to control the vascular hemodynamic and regulate the cardiovascular function. Summary Considering the antiatherogenic property of thyroid hormones and the rapid effects produced by this molecule as a vasodilator, including that in the coronary bed, a better understanding of the molecular mechanisms involved in its action may contribute to the development of drugs that can be clinically used to increase the known benefits promoted by thyroid hormones in cardiovascular physiology.

Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes

Aims Thyroid hormone (TH) rapidly relaxes vascular smooth muscle cells (VSMCs). However, the mechanisms involved in this effect remain unclear. We hypothesize that TH-induced rapid vascular relaxation is mediated by VSMC-derived nitric oxide (NO) production and is associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway. Methods and results NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO(2)) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001-1 mu M). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS. Conclusion Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

Pharmacological properties of purinergic receptors and their effects on proliferation and induction of neuronal differentiation of P19 embryonal carcinoma cells

We have used P19 embryonal carcinoma cells as in vitro model for early neurogenesis to study ionotropic P2X and metabotropic P2Y receptor-induced Ca2+ transients and their participation in induction of proliferation and differentiation. In embryonic P19 cells, P2Y(1), P2Y(2) and P2X(4) receptors or P2X-heteromultimers with similar P2X4 pharmacology were responsible for ATP and ATP analogue-induced Ca2+ transients. In neuronal-differentiated cells, P2Y(2), P2Y(6), P2X(2) and possibly P2X(2)/P2X(6) heteromeric receptors were the major mediators of the elevations in intracellular free calcium concentration [Ca2+](i). We have collected evidence for the involvement of metabotropic purinergic receptors in proliferation induction of undifferentiated and neural progenitor cells by using a BrdU-incorporation assay. ATP-, UTP-, ADP-, 2-MeS-ATP- and ADP-beta S-induced proliferation in P19 cells was mediated by P2Y, and P2Y2 receptors as judged from pharmacological profiles of receptor responses. ATP-provoked acceleration of neuronal differentiation, determined by analysis of nestin and neuron-specific enolase gene and protein expression, also resulted from P2Y, and P2Y2 receptor activation. Proliferation- and differentiation-induction involved the activation of inositol-trisphosphate sensitive intracellular Ca2+ stores. (C) 2008 ISDN. Published by Elsevier Ltd. All rights reserved.

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+](i)) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G alpha(q/11)-coupled M-1, M-3 and M-5 receptors and intracellular calcium stores, whereas G alpha(i/o)-protein coupled M-2 receptor activity mediated neuronal differentiation. (C) 2008 Elsevier Inc. All rights reserved.

Mechanism of acetylcholine-induced calcium signaling during neuronal differentiation of P19 embryonal carcinoma cells in vitro

Muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are involved in various physiological processes, including neuronal development. We provide evidence for expression of functional nicotinic and muscarinic receptors during differentiation of P19 carcinoma embryonic cells, as an in vitro model of early neurogenesis. We have detected expression and activity alpha(2)-alpha(7), beta(2), beta(4) nAChR and M1-M5 mAChR subtypes during neuronal differentiation. Nicotinic alpha(3) and beta(2) mRNA transcription was induced by addition of retinoic acid to P19 cells. Gene expression Of alpha(2), alpha(4)-alpha(7), beta(4) nAChR subunits decreased during initial differentiation and increased again when P19 cells underwent final maturation. Receptor response in terms of nicotinic agonist-evoked Ca2+, flux was observed in embryonic and neuronal-differentiated cells. Muscarinic receptor response, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The nAChR-induced elevation of intracellular calcium ([Ca2+](i)) response in undifferentiated cells was due to Ca2+ influx. In differentiated P19 neurons the nAChR-induced [Ca2+](i) response was reduced following pretreatment with ryanodine, while the mAChR-induced response was unaffected indicating the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses. The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initial neuronal differentiation. (C) 2007 Elsevier Ltd. All rights reserved.

Posttranscriptional regulation of sodium-iodide symporter mRNA expression in the rat thyroid gland by acute iodide administration

Serrano-Nascimento C, Calil-Silveira J, Nunes MT. Posttranscriptional regulation of sodium-iodide symporter mRNA expression in the rat thyroid gland by acute iodide administration. Am J Physiol Cell Physiol 298: C893-C899, 2010. First published January 27, 2010; doi:10.1152/ajpcell.00224.2009.-Iodide is an important regulator of thyroid activity. Its excess elicits the Wolff-Chaikoff effect, characterized by an acute suppression of thyroid hormone synthesis, which has been ascribed to serum TSH reduction or TGF-beta increase and production of iodolipids in the thyroid. These alterations take hours/days to occur, contrasting with the promptness of Wolff-Chaikoff effect. We investigated whether acute iodide administration could trigger events that precede those changes, such as reduction of sodium-iodide symporter (NIS) mRNA abundance and adenylation, and if perchlorate treatment could counteract them. Rats subjected or not to methylmercaptoimidazole treatment (0.03%) received NaI (2,000 mu g/0.5 ml saline) or saline intraperitoneally and were killed 30 min up to 24 h later. Another set of animals was treated with iodide and perchlorate, in equimolar doses. NIS mRNA content was evaluated by Northern blotting and real-time PCR, and NIS mRNA poly(A) tail length by rapid amplification of cDNA ends-poly(A) test (RACE-PAT). We observed that NIS mRNA abundance and poly(A) tail length were significantly reduced in all periods of iodide treatment. Perchlorate reversed these effects, indicating that iodide was the agent that triggered the modifications observed. Since the poly(A) tail length of mRNAs is directly associated with their stability and translation efficiency, we can assume that the rapid decay of NIS mRNA abundance observed was due to a reduction of its stability, a condition in which its translation could be impaired. Our data show for the first time that iodide regulates NIS mRNA expression at posttranscriptional level, providing a new mechanism by which iodide exerts its autoregulatory effect on thyroid.

Laminin-derived peptide AG73 regulates migration, invasion, and protease activity of human oral squamous cell carcinoma cells through syndecan-1 and beta 1 integrin

Squamous cell carcinoma is a prevalent head and neck tumor with high mortality. We studied the role played by laminin alpha 1 chain peptide AG73 on migration, invasion, and protease activity of cells (OSCC) from human oral squamous cell carcinoma. Immunohistochemistry and immunofluorescence analyzed expression of laminin alpha 1 chain and MMP9 in oral squamous cells carcinoma in vivo and in vitro. Migratory activity of AG73-treated OSCC cells was investigated by monolayer wound assays and in chemotaxis chambers. AG73-induced invasion was assessed in Boyden chambers. Invasion depends on MMPs. Conditioned media from cells grown on AG73 was subjected to zymography. We searched for AG73 receptors related to these activities in OSCC cells. Immunofluorescence analyzed AG73induced colocalization of syndecan-1 and beta 1 integrin. Cells had these receptors silenced by siRNA, followed by treatment with AG73 and analysis of migration, invasion, and protease activity. Oral squamous cell carcinoma expresses laminin alpha 1 chain and MMP9. OSCC cells treated with AG73 showed increased migration, invasion, and protease activity. AG73 induced colocalization of syndecan-1 and beta 1 integrin. Knockdown of these receptors decreased AG73-dependent migration, invasion, and protease activity. Syndecan-1 and beta 1 integrin signaling downstream of AG73 regulate migration, invasion, and MMP production by OSCC cells.

Differential gene expression analysis of iodide-treated rat thyroid follicular cell line PCCl3

The inhibitory effect of supraphysiological iodide concentrations on thyroid hormone synthesis (Wolff - Chaikoff effect) and on thyrocyte proliferation is largely known as iodine autoregulation. However, the molecular mechanisms by which iodide modulates thyroid function remain unclear. In this paper, we analyze the transcriptome profile of the rat follicular cell lineage PCCl3 under untreated and treated conditions with 10 (- 3) M sodium iodide (NaI). Serial analysis of gene expression (SAGE) revealed 84 transcripts differentially expressed in response to iodide (p <= 0.001). We also showed that iodide excess inhibits the expression of essential genes for thyroid differentiation: Tshr, Nis, Tg, and Tpo. Relative expression of 14 of 20 transcripts selected by SAGE was confirmed by real-time PCR. Considering the key role of iodide organification in thyroid physiology, we also observed that both the oxidized form of iodide and iodide per se are responsible for gene expression modulation in response to iodide excess. (c) 2008 Elsevier Inc. All rights reserved.