Low Doses of Methylmercury Induce the Proliferation of Thyroid Cells In Vitro Through Modulation of ERK Pathway.

Exposure to environmental endocrine disruptors has been associated with an increased frequency of thyroid pathology. In this study, we evaluated the effects of various concentrations of methylmercury (MeHg) on immortalized, non-tumorigenic thyroid cells (Nthy-ori-3-1). Exposure to MeHg at 2.5 and 5 µM for 24 h caused a reduction in cell viability with a decrease of the cell population in sub-G0 phase, as detected by MTT and flow cytometry. Conversely, MeHg at the lower concentration of 0.1 µM increased the cell viability with a rise of G2/M phase. An immunoblot analysis showed higher expression levels of phospho-ERK and not of phospho-Akt. Further enhancement of the cell growth rate was observed after a prolonged exposure of the cells up to 18 days to MeHg 0.1 µM. The present findings demonstrate the toxicity of high concentrations of MeHg on thyroid cells, while showing that treatment with lower doses of Hg, as may occur after prolonged exposure to this environmental contaminant, exerts a promoting effect on thyroid cell proliferation, by acting on the ERK-mediated pro-oncogenic signal transduction pathway.

Oleacein Prevents High Fat Diet-Induced Adiposity and Ameliorates Some Biochemical Parameters of Insulin Sensitivity in Mice.

Oleacein is one of the most abundant polyphenolic compounds of olive oil, which has been shown to play a protective role against several metabolic abnormalities, including dyslipidemia, insulin resistance, and glucose intolerance. Herein, we investigated the effects of oleacein on certain markers of adipogenesis and insulin-resistance in vitro, in 3T3-L1 adipocytes, and in vivo in high-fat diet (HFD)-fed mice. During the differentiation process of 3T3-L1 preadipocytes into adipocytes, oleacein strongly inhibited lipid accumulation, and decreased protein levels of peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid synthase (FAS), while increasing Adiponectin levels. In vivo, treatment with oleacein of C57BL/6JOlaHsd mice fed with HFD for 5 and 13 weeks prevented the increase in adipocyte size and reduced the inflammatory infiltration of macrophages and lymphocytes in adipose tissue. These effects were accompanied by changes in the expression of adipose tissue-specific regulatory elements such as PPARγ, FAS, sterol regulatory element-binding transcription factor-1 (SREBP-1), and Adiponectin, while the expression of insulin-sensitive muscle/fat glucose transporter Glut-4 was restored in HFD-fed mice treated with oleacein. Collectively, our findings indicate that protection against HFD-induced adiposity by oleacein in mice is mediated by the modulation of regulators of adipogenesis. Protection against HFD-induced obesity is effective in improving peripheral insulin sensitivity.

Expression of Leptin Receptor and Effects of Leptin on Papillary Thyroid Carcinoma Cells.

BACKGROUND: Obesity has been hypothesized to contribute to the aggressiveness of thyroid cancer through the production of abnormal levels of serum adipokines. Leptin receptor (OB-R) expression has also been documented in papillary thyroid cancer (PTC). AIM: In this translational study, we analyzed in vitro the effects of leptin on the growth and migration of thyroid cancer cells (TPC-1 and K1), the molecular mechanisms underlying leptin's action, and the influence of prolonged leptin exposure on cell response to a protein kinase inhibitor lenvatinib. The expression levels of OB-R mRNA and protein were also investigated in vivo in a series of aggressive PTCs divided into two groups based on the presence of the BRAF mutation. RESULTS: In TPC-1 and K1 cells, prolonged treatment with leptin (500 ng/ml for 96 h) resulted in a mild increase in the proliferation (about 20% over control only in K1 cells, p < 0.05) and in the migration of both cancer cell lines. Immunoblot analysis revealed a slight increase in the phosphorylation of AKT, but no effect on ß-catenin and phospho-ERK expressions. The inhibitory effects of lenvatinib on the viability of both cell lines were not influenced by the leptin treatment. OB-R transcript (in fresh tissues) and proteins (in formalin-fixed and paraffin-embedded specimens) were expressed in all PTC tissues examined, with no significant differences between BRAF-mutated and BRAF-wild-type tumors. CONCLUSIONS: These results demonstrate leptin's role in mildly increasing the aggressive phenotype of PTC cells but without influencing the action of lenvatinib. Further studies will clarify whether it is possible to target OB-R, expressed in all aggressive PTCs, as an adjuvant treatment approach for these malignancies.

Secoiridoids of olive and derivatives as potential coadjuvant drugs in cancer: A critical analysis of experimental studies.

Phenolic secoiridoids from olive, including oleocanthal, oleuropein and related derivatives, are bioactive natural products with documented anticancer activities, that have mainly been attributed to their antioxidant, anti-inflammatory and antiproliferative effects. This review summarizes the results of the preclinical studies on the natural secoiridoids of olive used as single agents or in combination with other chemotherapeutics against cancer cells. The molecular targets of their action are described. A critical analysis of the importance of the experimental studies in view of the possible use in humans is also discussed.

Human telomerase reverse transcriptase in papillary thyroid cancer: gene expression, effects of silencing and regulation by BET inhibitors in thyroid cancer cells.

PURPOSE: Mutations in TERT promoter have been detected in the more aggressive papillary thyroid cancers (PTCs). To elucidate the role of TERT as an eligible molecular target in these tumors, the expression of hTERT was analyzed in a series of PTCs and the effects of both pharmacological and RNA-interference-induced hTERT silencing were investigated in two human PTC cell lines (K1 and BCPAP). METHODS: The expression levels of hTERT mRNA and protein were evaluated by real-time PCR and western blot assays, respectively. Effects of hTERT silencing on PTC cell lines were analyzed by MTT, migration and western blot assays. Pharmacological inhibition of hTERT was performed using two bromodomain and extra-terminal (BET) inhibitors, JQ1 and I-BET762. RESULTS: hTERT expression results increased in 20 out of 48 PTCs, including tumors either positive or negative for the presence of hTERT promoter and/or BRAF mutations. In K1 and BCPAP cells, hTERT silencing determined a reduction in cell viability (~50% for K1 and ~70%, for BCPAP, vs control) and migration properties that were associated with a decrease of AKT phosphorylation and ß-Catenin expression. Moreover, hTERT mRNA levels were down-regulated by two BET inhibitors, JQ1 and I-BET762, which at the same dosage (0.5 and 5 µM) reduced the growth of these thyroid cancer cells. CONCLUSIONS: These findings demonstrate that hTERT may represent an excellent therapeutic target in subgroups of aggressive PTCs.

Effects of Oleacein on High-Fat Diet-Dependent Steatosis, Weight Gain, and Insulin Resistance in Mice.

Many reports indicate that the protective action of nutraceuticals in the Mediterranean diet, against metabolic and cardiovascular diseases, can be attributed to the action of polyphenolic components of extra-virgin olive oil (EVOO). Here, we evaluated the protective effects of oleacein, one of the most abundant secoiridoids in EVOO, on the damages/metabolic alterations caused by high-fat diet (HFD) in male C57BL/6JolaHsd mice. After 5 weeks of treatment with 20 mg/kg of oleacein, body weight, glycemia, insulinemia, serum lipids, and histologic examination of liver tissue indicated a protective action of oleacein against abdominal fat accumulation, weight gain, and liver steatosis, with improvement of insulin-dependent glucose and lipid metabolism. Both serum parameters and hepatic histologic examination were altered in mice fed with HFD. By contrast, in the animals that received oleacein, plasma glucose, cholesterol and triglyceride serum levels, and liver histology were similar to controls fed with normocaloric diet. In addition, protein levels of FAS, SREBP-1, and phospho-ERK in liver were positively modulated by oleacein, indicating an improvement in liver insulin sensitivity. In a group of obese mice, treatment with oleacein determined a light, but still significant reduction of the increase in body weight, mainly due to lesser liver steatosis enlargement, associated with reduced levels of SREBP-1 and phospho-ERK and lower levels of total serum cholesterol; in these animals, altered plasma glucose and triglyceride serum levels were not reverted by oleacein. These results indicate that HFD-related hepatic insulin resistance may be partially prevented by oral administration of oleacein, suggesting a protective role of this nutraceutical against diet-dependent metabolic alterations. Additional studies are necessary to check whether oleacein can be used as an adjuvant to improve insulin sensitivity in humans.

Silencing of hTERT blocks growth and migration of anaplastic thyroid cancer cells.

Mutations in the hTERT promoter responsible for constitutive telomerase activity are the most frequent genetic alteration detected in anaplastic thyroid cancer (ATC), and proposed as diagnostic and prognostic biomarker in these tumours. In this study we analyzed hTERT expression in a series of human ATCs and investigated the effects of small-interfering RNA-mediated silencing of hTERT on viability and migration and invasive properties of three human ATC cell lines. Expression of hTERT mRNA resulted increased in 8/10 ATCs compared to normal thyroid tissues. Silencing of hTERT in CAL-62, 8505C and SW1736 cells did not modify telomere length but determined a significant decrease (about 50%) of cell proliferation in all cell lines and a great reduction (about 50%) of migration and invasion capacity. These finding demonstrate that hTERT may be considered as a molecular target for ATC treatment.

PNPLA3 overexpression results in reduction of proteins predisposing to fibrosis.

Liver fibrosis is a pathological scarring response to chronic hepatocellular injury and hepatic stellate cells (HSCs) are key players in this process. PNPLA3 I148M is a common variant robustly associated with liver fibrosis but the mechanisms underlying this association are unknown. We aimed to examine a) the effect of fibrogenic and proliferative stimuli on PNPLA3 levels in HSCs and b) the role of wild type and mutant PNPLA3 overexpression on markers of HSC activation and fibrosis.Here, we show that PNPLA3 is upregulated by the fibrogenic cytokine transforming growth factor-beta (TGF-ß), but not by platelet-derived growth factor (PDGF), and is involved in the TGF-ß-induced reduction in lipid droplets in primary human HSCs. Furthermore, we show that retinol release from human HSCs ex vivo is lower in cells with the loss-of-function PNPLA3 148M compared with 148I wild type protein. Stable overexpression of PNPLA3 148I wild type, but not 148M mutant, in human HSCs (LX-2 cells) induces a reduction in the secretion of matrix metallopeptidase 2 (MMP2), tissue inhibitor of metalloproteinase 1 and 2 (TIMP1 and TIMP2), which is mediated by retinoid metabolism. In conclusion, we show a role for PNPLA3 in HSC activation in response to fibrogenic stimuli. Moreover, we provide evidence to indicate that PNPLA3-mediated retinol release may protect against liver fibrosis by inducing a specific signature of proteins involved in extracellular matrix remodelling.

Identification and characterization of two novel mutations in the LPL gene causing type I hyperlipoproteinemia.

BACKGROUND: Type 1 hyperlipoproteinemia is a rare autosomal recessive disorder most often caused by mutations in the lipoprotein lipase (LPL) gene resulting in severe hypertriglyceridemia and pancreatitis. OBJECTIVES: The aim of this study was to identify novel mutations in the LPL gene causing type 1 hyperlipoproteinemia and to understand the molecular mechanisms underlying the severe hypertriglyceridemia. METHODS: Three patients presenting classical features of type 1 hyperlipoproteinemia were recruited for DNA sequencing of the LPL gene. Pre-heparin and post-heparin plasma of patients were used for protein detection analysis and functional test. Furthermore, in vitro experiments were performed in HEK293 cells. Protein synthesis and secretion were analyzed in lysate and medium fraction, respectively, whereas medium fraction was used for functional assay. RESULTS: We identified two novel mutations in the LPL gene causing type 1 hyperlipoproteinemia: a two base pair deletion (c.765_766delAG) resulting in a frameshift at position 256 of the protein (p.G256TfsX26) and a nucleotide substitution (c.1211 T > G) resulting in a methionine to arginine substitution (p.M404 R). LPL protein and activity were not detected in pre-heparin or post-heparin plasma of the patient with p.G256TfsX26 mutation or in the medium of HEK293 cells over-expressing recombinant p.G256TfsX26 LPL. A relatively small amount of LPL p.M404 R was detected in both pre-heparin and post-heparin plasma and in the medium of the cells, whereas no LPL activity was detected. CONCLUSIONS: We conclude that these two novel mutations cause type 1 hyperlipoproteinemia by inducing a loss or reduction in LPL secretion accompanied by a loss of LPL enzymatic activity.

Normocaloric Diet Restores Weight Gain and Insulin Sensitivity in Obese Mice.

An increased incidence of obesity is registered worldwide, and its association with insulin resistance and type 2 diabetes is closely related with increased morbidity and mortality for cardiovascular diseases. A major clinical problem in the management of obesity is the non-adherence or low adherence of patients to a hypocaloric dietetic restriction. In this study, we evaluated in obese mice the effects of shifting from high-calorie foods to normal diet on insulin sensitivity. Male C57BL/6JOlaHsd mice (n = 20) were fed with high fat diet (HFD) for a 24-week period. Afterward, body weight, energy, and food intake were measured in all animals, together with parameters of insulin sensitivity by homeostatic model assessment of insulin resistance and plasma glucose levels in response to insulin administration. Moreover, in half of these mice, Glut4 mRNA levels were measured in muscle at the end of the high fat treatment, whereas the rest of the animals (n = 10) were shifted to normocaloric diet (NCD) for 10 weeks, after which the same analyses were carried out. A significant reduction of body weight was found after the transition from high to normal fat diet, and this decrease correlated well with an improvement in insulin sensitivity. In fact, we found a reduction in serum insulin levels and the recovery of insulin responsiveness in terms of glucose disposal measured by insulin tolerance test and Glut4 mRNA and protein expression. These results indicate that obesity-related insulin resistance may be rescued by shifting from HFD to NCD.

DEPDC5 variants increase fibrosis progression in Europeans with chronic hepatitis C virus infection.

UNLABELLED: Chronic hepatitis C virus (HCV) infection may progress to cirrhosis and hepatocellular carcinoma (HCC). Recently, two genetic variants, DEPDC5 rs1012068 and MICA rs2596542, were associated with the onset of HCC in Asian subjects with chronic HCV infection. The aim of the present study was to analyze whether DEPDC5 and MICA genetic variants were associated with liver disease progression in European subjects with chronic HCV infection. In a Northern Italian discovery cohort (n = 477), neither DEPDC5 rs1012068 nor MICA rs2596542 were associated with HCC (n = 150). However, DEPDC5 rs1012068 was independently associated with cirrhosis (n = 300; P = 0.049). The association of rs1012068 with moderate to severe fibrosis was confirmed in an independent cross-sectional German cohort (n = 415; P = 0.006). Furthermore, DEPDC5 rs1012068 predicted faster fibrosis progression in a prospective cohort (n = 247; P = 0.027). Next, we examined the distribution of nonsynonymous DEPDC5 variants in the overall cross-sectional cohort (n = 912). The presence of at least one variant increased the risk of moderate/severe fibrosis by 54% (P = 0.040). To understand the molecular mechanism underlying the genetic association of DEPDC5 variants with fibrosis progression, we performed in vitro studies on immortalized hepatic stellate cells (LX-2). In these cells, down-regulation of DEPDC5 resulted in increased expression of ß-catenin and production of its target matrix metallopeptidase 2 (MMP2), a secreted enzyme involved in fibrosis progression. CONCLUSION: DEPDC5 variants increase fibrosis progression in European subjects with chronic HCV infection. Our findings suggest that DEPDC5 down-regulation may contribute to HCV-related fibrosis by increasing MMP2 synthesis through the ß-catenin pathway.

Oral Administration of Oleuropein and Its Semisynthetic Peracetylated Derivative Prevents Hepatic Steatosis, Hyperinsulinemia, and Weight Gain in Mice Fed with High Fat Cafeteria Diet.

The high consumption of olive tree products in the Mediterranean diet has been associated with a lower incidence of metabolic disorders and cardiovascular diseases. In particular, the protective effects of olive oil have been attributed to the presence of polyphenols such as oleuropein (Ole) and its derivatives. We have synthesized a peracetylated derivative of Ole (Ac-Ole) which has shown in vitro antioxidant and growth-inhibitory activity higher than the natural molecule. In this study, male C57BL/6JOlaHsd mice were fed with a standard (std), cafeteria (caf) diet, and caf diet supplemented with Ole (0.037 mmol/kg/day) and Ac-Ole (0.025 mmol/kg/day) for 15 weeks. We observed a significant reduction in the caf diet-induced body weight gain and increase of abdominal adipose tissue. Also, Ole and Ac-Ole prevented the development of hepatic steatosis. Finally, Ole and Ac-Ole determined a lower increase of HDL and LDL-cholesterol levels and corrected caf diet-induced elevation of plasma glucose concentrations by improving insulin sensitivity. The observed beneficial properties of Ole and Ac-Ole make these compounds and in particular Ac-Ole promising candidates for a potential pharmaceutic use in metabolic disorders.

Beneficial effects of the olive oil phenolic components oleuropein and hydroxytyrosol: focus on protection against cardiovascular and metabolic diseases.

The overall health beneficial action of olive oil phenolic components is well established. Recent studies have elucidated the biological effects of two isolated compounds, namely oleuropein and hydroxytyrosol, with particular attention on their antioxidant activity. Thus, a protective action has been demonstrated in preclinical studies against several diseases, especially cardiovascular and metabolic disorders. The present review will describe the biological effects of oleuropein and hydroxytyrosol, with particular attention on the molecular mechanism underlying the protective action on cardiovascular and metabolic alterations, as demonstrated by in vitro and in vivo experimental studies performed with the isolated compounds.