RNA Profile of Cell Bodies and Exosomes Released by Tumorigenic and Non-Tumorigenic Thyroid Cells.

Tumor cells release exosomes, extracellular vesicle containing various bioactive molecules such as protein, DNA and RNA. The analysis of RNA molecules packaged in exosomes may provide new potential diagnostic or prognostic tumor biomarkers. The treatment of radioiodine-refractory aggressive thyroid cancer is still an unresolved clinical challenge, and the search for biomarkers that are detectable in early phase of the disease has become a fundamental goal for thyroid cancer research. By using transcriptome analysis, this study aimed to analyze the gene expression profiles of exosomes secreted by a non-tumorigenic thyroid cell line (Nthy-ori 3.1-exo) and a papillary thyroid cancer (TPC-1-exo) cell line, comparing them with those of cell bodies (Nthy-ori 3.1-cells and TPC-1-cells). A total of 9107 transcripts were identified as differentially expressed when comparing TPC-1-exo with TPC-1-cells and 5861 when comparing Nthy-ori 3.1-exo with Nthy-ori 3.1-cells. Among them, Sialic acid-binding immunoglobulin-like lectins 10 and 11 (SIGLEC10, SIGLEC11) and Keratin-associated protein 5 (KRTAP5-3) transcripts, genes known to be involved in cancer progression, turned out to be up-regulated only in TPC-1-exo. Gene ontology analysis revealed significantly enriched pathways, and only in TPC-1-exo were the differential expressed genes associated with an up-regulation in epigenetic processes. These findings provide a proof of concept that some mRNA species are specifically packaged in tumor-cell-derived exosomes and may constitute a starting point for the identification of new biomarkers for thyroid tumors.

Emerging roles of the cellular prion protein (PrPC) and 37/67 kDa laminin receptor (RPSA) interaction in cancer biology.

The cellular prion protein (PrPC) is well-known for its involvement, under its pathogenic protease-resistant form (PrPSc), in a group of neurodegenerative diseases, known as prion diseases. PrPC is expressed in nervous system, as well as in other peripheral organs, and has been found overexpressed in several types of solid tumors. Notwithstanding, studies in recent years have disclosed an emerging role for PrPC in various cancer associated processes. PrPC has high binding affinity for 37/67 kDa laminin receptor (RPSA), a molecule that acts as a key player in tumorigenesis, affecting cell growth, adhesion, migration, invasion and cell death processes. Recently, we have characterized at cellular level, small molecules able to antagonize the direct PrPC binding to RPSA and their intracellular trafficking. These findings are very crucial considering that the main function of RPSA is to modulate key events in the metastasis cascade. Elucidation of the role played by PrPC/RPSA interaction in regulating tumor development, progression and response to treatment, represents a very promising challenge to gain pathogenetic information and discover novel specific biomarkers and/or therapeutic targets to be exploited in clinical settings. This review attempts to convey a detailed description of the complexity surrounding these multifaceted proteins from the perspective of cancer hallmarks, but with a specific focus on the role of their interaction in the control of proliferation, migration and invasion, genome instability and mutation, as well as resistance to cell death controlled by autophagic pathway.

Expression of miR-31-5p affects growth, migration and invasiveness of papillary thyroid cancer cells.

PURPOSE: In this study, we evaluated the biological role of miRNA-31-5p in papillary thyroid cancer (PTC). METHODS: By using the real-time PCR, we measured miRNA-31-5p expression levels in 25 PTC tissues and in two human PTC cell lines (K1 and TPC-1). Then, K1 cells were transiently transfected with mirVana inhibitor or mirVana mimic to miRNA-31-5-p. Cell proliferation was determined by MTT and colony formation assays. The in vitro metastatic ability of thyroid cancer cells was evaluated by adhesion, migration and invasion assays. Epithelial mesenchymal transition (EMT) and Hippo pathway related gene and protein levels were evaluated by using the TaqMan™ Gene Expression Assays and western blot analysis, respectively. RESULTS: We found a significant increase of miR-31-5-p expression in tumor tissue and in K1 cells harboring the BRAF p.V600E mutation. Knockdown of miR-31-5p determined a reduction of cell proliferation, associated with a significant decrease in cell adhesion, migration and invasion properties. A downregulation of EMT markers and YAP/ß-catenin axis was also observed. CONCLUSIONS: Our findings suggest that miRNA-31-5p acts as oncogenic miRNA in human thyrocytes and its overexpression may be involved in the BRAF-related tumorigenesis in PTCs, providing new understanding into its pathological role in PTC progression and invasiveness.

Co-Encapsulation of Paclitaxel and JQ1 in Zein Nanoparticles as Potential Innovative Nanomedicine.

The manuscript describes the development of zein nanoparticles containing paclitaxel (PTX) and the bromo-and extra-terminal domain inhibitor (S)-tertbutyl2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno(3,2-f)(1,2,4)triazolo(4,3-a)(1,4)diazepin-6-yl)acetate (JQ1) together with their cytotoxicity on triple-negative breast cancer cells. The rationale of this association is that of exploiting different types of cancer cells as targets in order to obtain increased pharmacological activity with respect to that exerted by the single agents. Zein, a protein found in the endosperm of corn, was used as a biomaterial to obtain multidrug carriers characterized by mean sizes of ˂200 nm, a low polydispersity index (0.1-0.2) and a negative surface charge. An entrapment efficiency of ~35% of both the drugs was obtained when 0.3 mg/mL of the active compounds were used during the nanoprecipitation procedure. No adverse phenomena such as sedimentation, macro-aggregation or flocculation occurred when the nanosystems were heated to 37 °C. The multidrug nanoformulation demonstrated significant in vitro cytototoxic activity against MDA-MB-157 and MDA-MB-231 cancer cells by MTT-test and adhesion assay which was stronger than that of the compounds encapsulated as single agents. The results evidence the potential application of zein nanoparticles containing PTX and JQ1 as a novel nanomedicine.

Identification of Exosomal microRNAs and Their Targets in Papillary Thyroid Cancer Cells.

The release of molecules in exosomal cargoes is involved in tumor development and progression. We compared the profiles of exosomal microRNAs released by two thyroid cancer cell lines (TPC-1 and K1) with that of non-tumorigenic thyroid cells (Nthy-ori-3-1), and we explored the network of miRNA-target interaction. After extraction and characterization of exosomes, expression levels of microRNAs were investigated using custom TaqMan Advanced array cards, and compared with those expressed in the total cell extracts. The functional enrichment and network-based analysis of the miRNAs' targets was also performed. Five microRNAs (miR-21-5p, miR-31-5p, miR-221-3p, miR-222-3p, and let-7i-3p) were significantly deregulated in the exosomes of tumor cells vs. non-tumorigenic cells, and three of them (miR-31-5p, miR-222-3p, and let-7i-3p) in the more aggressive K1 compared to TPC-1 cells. The network analysis of the five miRNAs identified some genes as targets of more than one miRNAs. These findings permitted the identification of exosomal microRNAs secreted by aggressive PTC cells, and indicated that their main targets are regulators of the tumor microenvironment. A deeper analysis of the functional role of the targets of exosomal miRNAs will provide further information on novel targets of molecular treatments for these neoplasms.

Analysis of serum microRNA in exosomal vehicles of papillary thyroid cancer.

PURPOSE: In this study, we investigated the profile of microRNAs (miRNAs) contained in exosomes secreted in the serum of patients with papillary thyroid cancer (PTC). METHODS: Exosome were isolated by adding ExoQuick Exosome Precipitation Solution. Dynamic light scattering (DLS) and western blotting analysis were used to ensure the quality of exosomes. The expression levels of miRNAs were investigated using custom-designed TaqMan Advanced miRNA Array Cards in the screening cohort and using specific TaqMan Advanced MicroRNA Assays in the validation cohort. RESULTS: We identified miR24-3p, miR146a-5p, miR181a-5p and miR382-5p with different expression levels in two different series of 56 and 58 PTC patients as compared with healthy controls. Significant differences in the expression of three PTC exosomal miRNAs, depending on the presence of lymph node metastasis, were detected in only one PTC series. When comparing the expression levels of some PTC-specific exosomal miRNAs with those of the same miRNAs circulating free of any encapsulation, we found a significant correlation for only miR24-3p, suggesting that only select miRNAs are secreted in exosomes. CONCLUSIONS: Our findings demonstrate that four miRNAs are differently secreted in the exosomes of PTC patients, whereas no conclusive results were found to characterize PTCs with lymph node metastasis, suggesting caution in the use of circulating exosomal miRNA expression levels as lymph node metastasis biomarkers. Further investigation into the mechanisms governing miRNA secretion in tumor cells are required.

Effects of Dihydrotanshinone I on Proliferation and Invasiveness of Paclitaxel-Resistant Anaplastic Thyroid Cancer Cells.

ATC is a very rare, but extremely aggressive form of thyroid malignancy, responsible for the highest mortality rate registered for thyroid cancer. In patients without known genetic aberrations, the current treatment is still represented by palliative surgery and systemic mono- or combined chemotherapy, which is often not fully effective for the appearance of drug resistance. Comprehension of the mechanisms involved in the development of the resistance is therefore an urgent issue to suggest novel therapeutic approaches for this very aggressive malignancy. In this study, we created a model of anaplastic thyroid cancer (ATC) cells resistant to paclitaxel and investigated the characteristics of these cells by analyzing the profile of gene expression and comparing it with that of paclitaxel-sensitive original ATC cell lines. In addition, we evaluated the effects of Dihydrotanshinone I (DHT) on the viability and invasiveness of paclitaxel-resistant cells. ATC paclitaxel-resistant cells highlighted an overexpression of ABCB1 and a hyper-activation of the NF-κB compared to sensitive cells. DHT treatment resulted in a reduction of viability and clonogenic ability of resistant cells. Moreover, DHT induces a decrement of NF-κB activity in SW1736-PTX and 8505C-PTX cells. In conclusion, to the best of our knowledge, the results of the present study are the first to demonstrate the antitumor effects of DHT on ATC cells resistant to Paclitaxel in vitro.

Quercetin Protects Human Thyroid Cells against Cadmium Toxicity.

Various natural compounds have been successfully tested for preventing or counteracting the toxic effects of exposure to heavy metals. In this study, we analyzed the effects of cadmium chloride (CdCl2) on immortalized, non-tumorigenic thyroid cells Nthy-ori-3-1. We investigated the molecular mechanism underlying its toxic action as well as the potential protective effect of quercetin against CdCl2-induced damage. CdCl2 suppressed cell growth in a dose- and time-dependent manner (IC50 value ~10 µM) associated with a decrease in levels of phospho-ERK. In addition, CdCl2 elicited an increase in reactive oxygen species (ROS) production and lipid peroxidation. A significant increase in GRP78, an endoplasmic reticulum (ER) stress-related protein, was also observed. Supplementation of quercetin counteracted the growth-inhibiting action of CdCl2 by recovering ERK protein phosphorylation levels, attenuating ROS overproduction, decreasing MDA content and reducing the expression of GRP78 in cells exposed to CdCl2. Thus, in addition to revealing the molecular effects involved in cadmium-induced toxicity, the present study demonstrated, for the first time, a protective effect of quercetin against cadmium-induced damages to normal thyroid cells.

Phytochemicals in thyroid cancer: analysis of the preclinical studies.

PURPOSE: In the search for novel effective compounds to use in thyroid cancer (TC) unresponsive to current treatment, attention has recently focused on plant-derived compounds with anticancer activity. In this review, we discuss the preclinical studies demonstrating phytochemical activity against thyroid cancer cells. RESULTS/CONCLUSIONS: In particular, we describe their antiproliferative properties or ability to re-induce iodine retention, thus supporting their potential use as single agents or adjuvants in radioiodine-resistant thyroid cancer treatment.

Mediterranean Diet Nutrients to Turn the Tide against Insulin Resistance and Related Diseases.

Insulin resistance (IR), defined as an attenuated biological response to circulating insulin, is a fundamental defect in obesity and type 2 diabetes (T2D), and is also linked to a wide spectrum of pathological conditions, such as non-alcoholic fatty liver disease (NAFLD), cognitive impairment, endothelial dysfunction, chronic kidney disease (CKD), polycystic ovary syndrome (PCOS), and some endocrine tumors, including breast cancer. In obesity, the unbalanced production of pro- and anti-inflammatory adipocytokines can lead to the development of IR and its related metabolic complications, which are potentially reversible through weight-loss programs. The Mediterranean diet (MedDiet), characterized by high consumption of extra-virgin olive oil (EVOO), nuts, red wine, vegetables and other polyphenol-rich elements, has proved to be associated with greater improvement of IR in obese individuals, when compared to other nutritional interventions. Also, recent studies in either experimental animal models or in humans, have shown encouraging results for insulin-sensitizing nutritional supplements derived from MedDiet food sources in the modulation of pathognomonic traits of certain IR-related conditions, including polyunsaturated fatty acids from olive oil and seeds, anthocyanins from purple vegetables and fruits, resveratrol from grapes, and the EVOO-derived, oleacein. Although the pharmacological properties and clinical uses of these functional nutrients are still under investigation, the molecular mechanism(s) underlying the metabolic benefits appear to be compound-specific and, in some cases, point to a role in gene expression through an involvement of the nuclear high-mobility group A1 (HMGA1) protein.

Oleacein inhibits STAT3, activates the apoptotic machinery, and exerts anti-metastatic effects in the SH-SY5Y human neuroblastoma cells.

Several studies published in the last decade suggest that the beneficial role of extra-virgin olive oil (EVOO) in human health is mostly attributable to the main secoiridoid derivatives (oleuropein, oleocanthal, and oleacein). Anti-cancer properties have also been demonstrated for certain compounds present in small quantities in EVOO, including oleuropein and hydroxytyrosol, which have been extensively studied, while minor attention has been given to the most abundant secoiridoid oleacein. The aim of our research was to study the molecular mechanisms underlying the anti-proliferative and anti-metastatic capacity of oleacein in the SH-SY5Y human neuroblastoma cell line. Our results demonstrate that oleacein is able to reduce the proliferation of the SH-SY5Y cells by blocking the cell cycle in the S phase and inducing apoptotic cell death through the increase in both Bax and p53 as well as a reduction in the Bcl-2 expression and STAT3 phosphorylation. Moreover, oleacein caused reduction in the SH-SY5Y cell adhesion and migration. Overall, these findings indicate that oleacein exerts anti-cancer effects against neuroblastoma cells, suggesting a promising role as a candidate against this type of cancer.

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.

Nanoparticles Loaded with the BET Inhibitor JQ1 Block the Growth of Triple Negative Breast Cancer Cells In Vitro and In Vivo.

Inhibition of bromo-and extra-terminal domain (BET) proteins, epigenetic regulators of genes involved in cell viability, has been efficiently tested in preclinical models of triple negative breast cancer (TNBC). However, the use of the selective BET-inhibitor JQ1 on humans is limited by its very short half-life. Herein, we developed, characterized and tested a novel formulation of nanoparticles containing JQ1 (N-JQ1) against TNBC in vitro and in vivo. N-JQ1, prepared using the nanoprecipitation method of preformedpoly-lactid-co-glycolic acid in an aqueous solution containing JQ1 and poloxamer-188 as a stabilizer, presented a high physico-chemical stability. Treatment of MDA-MB 157 and MDA-MB 231 TNBC cells with N-JQ1 determined a significant decrease in cell viability, adhesion and migration. Intra-peritoneal administration (5 days/week for two weeks) of N-JQ1 in nude mice hosting a xenograft TNBC after flank injection of MDA-MB-231 cells determined a great reduction in the growth and vascularity of the neoplasm. Moreover, the treatment resulted in a minimal infiltration of nearby tissues. Finally, the encapsulation of JQ1 in nanoparticles improved the anticancer efficacy of this epigenetic compound against TNBC in vitro and in vivo, opening the way to test it in the treatment of TNBC.

Antiproliferative Effects of Cynaropicrin on Anaplastic Thyroid Cancer Cells.

BACKGROUND: The sesquiterpene lactone cynaropicrin, a major constituent of the artichoke leaves extracts, has shown several biologic activities in many preclinical experimental models, including anti-proliferative effects. OBJECTIVE: Herein we evaluated the effects of cynaropicrin on the growth of three human anaplastic thyroid carcinoma cell lines, investigating the molecular mechanism underlying its action. METHOD: MTT assay was used to evaluate the viability of CAL-62, 8505C and SW1736 cells, and flow cytometry to analyse cell cycle distribution. Western blot was performed to detect the levels of STAT3 phosphorylation and NFkB activation. Antioxidant effects were analyzed by measuring the reactive oxygen species and malonyldialdehyde dosage was used to check the presence of lipid peroxidation. RESULTS: Viability of CAL-62, 8505C and SW1736 cells was significantly reduced by cynaropicrin in a dose- and time-dependent way, with an EC50 of about 5 µM observed after 48 h of treatment with the compound. Cellular growth inhibition was accompanied both by an arrest of the cell cycle, mainly in the G2/M phase, and the presence of a significant percentage of necrotic cells. After 48 h of treatment with 10 µM of cynaropicrin, a reduced nuclear expression of NFkB and STAT3 phosphorylation were also revealed. Moreover, we observed an increase in lipid peroxidation, without any significant effect on the reactive oxygen species production. CONCLUSION: These results demonstrate that cynaropicrin reduces the viability and promotes cytotoxic effects in anaplastic thyroid cancer cells associated with reduced NFkB expression, STAT3 phosphorylation and increased lipid peroxidation. Further characterization of the properties of this natural compound may open the way for using cynaropicrin as an adjuvant in the treatment of thyroid cancer.

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.