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The polyphenolic compounds present in grape extracts have chemopreventive and anticancer properties. Here, we studied the ability of two grape skin extracts (GSEs), Autumn Royal and Egnatia, to influence the cell motility and membrane fluidity regulated by the enzyme Stearoyl-CoA desaturase-1 (SCD1) which increases with the cancer aggressiveness. Caco2 and SW480 human colon cancer cell lines were treated with increasing concentrations of GSEs to evaluate cell proliferation and motility. SCD1 levels were evaluated in both treated cell lines, by membrane lipidomic analysis conducted by gas chromatography. The expression levels of SCD1 and other factors involved in the reorganization of the cytoskeleton and focal adhesions were assessed by Real-time PCR, Western Blotting, and Immunofluorescence staining. High-performance liquid chromatography (HPLC) analyses were performed to determine the phenolic composition in the GSEs, finding them more expressed in Autumn Royal than in Egnatia. Both treatments reduced the levels of SCD1, phospho-Rac1/Cdc42/Rac1/Cdc42 ratio, Cofilin, Vimentin, and phospho-Paxillin especially in Caco2 compared to SW480, showing a different behavior of the two cell lines to these natural compounds. Our findings show that GSEs block the cell migration and membrane fluidity through a new mechanism of action involving structural cellular components.

Exosomes are membrane-bound extracellular vesicles (EVs) released by most cells, having a size ranging from 30 to 150 nm, and are involved in mechanisms of cell-cell communication in physiological and pathological tissues. Exosomes are engaged in the transport of biomolecules, such as lipids, proteins, messenger RNAs, and microRNA, and in signal transmission through the intercellular transfer of components. In the context of proteins and nucleic acids transported from exosomes, our interest is focused on the Frizzled proteins family and related messenger RNA. Exosomes can regenerate stem cell phenotypes and convert them into cancer stem cells by regulating the Wnt pathway receptor family, namely Frizzled proteins. In particular, for gastrointestinal cancers, the Frizzled protein involved in those mechanisms is Frizzled-10 (FZD-10). Currently, increasing attention is being devoted to the protein and lipid composition of exosomes interior and membranes, representing profound knowledge of specific exosomes composition fundamental for their application as new delivering drug tools for cancer therapy. This review intends to cover the most recent literature on the use of exosome vesicles for early diagnosis, follow-up, and the use of these physiological nanovectors as drug delivery systems for gastrointestinal cancer therapy.

The administration of a ketogenic diet (KD) has been considered therapeutic in subjects with irritable bowel syndrome (IBS). This study aimed to investigate the molecular mechanisms by which a low-carbohydrate diet, such as KD, can improve gastrointestinal symptoms and functions in an animal model of IBS by evaluating possible changes in intestinal tissue expression of endocannabinoid receptors. In rats fed a KD, we detected a significant restoration of cell damage to the intestinal crypt base, a histological feature of IBS condition, and upregulation of CB1 and CB2 receptors. The diet also affected glucose metabolism and intestinal membrane permeability, with an overexpression of the glucose transporter GLUT1 and tight junction proteins in treated rats. The present data suggest that CB receptors represent one of the molecular pathways through which the KD works and support possible cannabinoid-mediated protection at the intestinal level in the IBS rats after dietary treatment.

BACKGROUND: Cannabinoid- 2 (CB2) receptor is known for its anti-obesity effects silencing the activated immune cells that are key drivers of metabolic syndrome and inflammation. Nutritional interventions in experimental models of carcinogenesis have been demonstrated to modulate tissue inflammation state and proliferation. OBJECTIVE: Aim of this study was to test, in ApcMin/+ mice, whether a diet enriched with olive oil, omega- 3 and omega-6- PUFAs affects the adipose tissue inflammation status. METHODS: Four groups of animal were studied: ST group, receiving a standard diet; OO group, receiving the standard diet in which soybean oil (source of fats) was replaced with olive oil; OM-3 group, receiving the standard diet in which soybean oil was replaced with salmon oil; OM-6 group, receiving the standard diet in which soybean oil was replaced with oenothera oil. Gene and protein expression, in adipose tissue, were evaluated by RT-PCR and Western Blotting, respectively. Enzymatic activities were assayed by fluorescent and radiometric method, where appropriated. RESULTS: The diet enriched with olive oil significantly induced CB2 receptor expression and it was able to control inflammatory and proliferative activity of mice adipose tissue. CONCLUSIONS: The present findings open opportunities for developing novel nutritional strategies considering olive oil a key ingredient of a healthy dietary pattern.

The statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and orlistat, an inhibitor of fatty acid synthase (FAS), inhibit tumor cell growth by restricting cholesterol and fatty acid synthesis, respectively. We previously demonstrated that an omega (ω)-3 polyunsaturated fatty acid (PUFA)- or olive oil-enriched diet reduced the polyp number and volume in ApcMin/+ mice. This phenomenon was associated with a significant inhibition of FAS and HMGCoAR, as well as an increase in the estrogen receptor (ER)β/α ratio. Herein, we evaluated the effect of lovastatin and orlistat on polyp development and ER expression in ApcMin/+ mice, in order to confirm previous data obtained with ω‑3-PUFAs and olive oil. As expected, the use of lovastatin and orlistat significantly reduced HMGCoAR and FAS enzymatic activities and gene expression in colonic tissues, but did not affect the number of intestinal polyps, while there was a statistically significant reduction in polyp volume only in the mouse group treated with lovastatin. In the mice receiving orlistat, we observed a significant increase in cell proliferation in the polyp tissue, as well as enhanced expression of ERα. Moreover, the overexpression of ERα was associated with a statistically significant increase in PES1, Shh and Gli1 protein levels, considered ERα-related molecular targets.

Background/aim The expression of cannabinoid receptor-1 (CB1-R) seems to be modulated by bioactive natural components such as the flavonoid quercetin. The aim of this study was to determine in an animal model of induced-colon cancer, whether quercetin inhibits colon carcinogenesis through changes in the expression of CB1-R. Materials and methods C57BL/6J male mice were randomly assigned to standard diet or experimental diet supplemented with 0.5% quercetin. Azoxymethane (AOM) (10 mg/kg body weight) or saline solution (PBS) was intraperitoneally injected, once weekly for 6 weeks. Results The diet supplemented with quercetin induced CB1-R gene expression and protein, inhibiting the protein levels of STAT3 and p-STAT3 (both mediators of cell proliferation). Dietary quercetin also caused a significant increase in Bax/Bcl2 ratio protein expression. Conclusion The anti-proliferative and pro-apoptotic effects of quercetin in AOM-treated mice are mediated by induction of the protein and gene expression levels of CB1-R.

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) have been demonstrated to reduce tumor load in Apc(Min/+) mice, supporting a role for n-3 PUFAs in the inhibition of colon carcinogenesis and progression. The aim of the present study was to investigate whether a diet enriched with n-3 PUFAs, known already to have anti-neoplastic efficacy in Apc(Min/+) mice, would reverse the development of intestinal polyps. For this purpose, Apc(Min/+) mice were randomly divided into 3 groups of 5 animal each and fed as follows: control ST1 and ST2 groups, received a purified AIN-93M standard diet for 5 and 10 weeks, respectively; the OM-3R group received a purified AIN-93M standard diet for 5 weeks and a diet supplemented with salmon oil, rich in n-3 PUFAs, for another 5 weeks. After dietary treatment, in intestinal tissue, we evaluated the polyp number and volume, expression levels of cell proliferation- and apoptosis-related proteins, as well as the protein expression of LDL receptor and the levels of fatty acid synthase (FAS) activity. The results showed the ability of a diet enriched with n-3 PUFAs to suppress intestinal polyps in Apc(Min/+) mice, and to significantly reverse polyp development associated with the downregulation of cell proliferation markers and with the induction of estrogen receptor β and LDL receptor, which are negative modulators of cellular proliferation. This noteworthy finding is important for a translational study evaluating the therapeutic role of n-3 PUFAs in the prevention and treatment of subjects with gastrointestinal diseases.

The 3T3-L1 preadipocyte cell line is a well characterized cell model for studying the adipocyte status and the molecular mechanisms involved in differentiation of these cells. 3T3-L1 preadipocytes have the ability to synthesize and degrade endocannabinoid anandamide (AEA) and their differentiation into adipocytes increases the expression of cannabinoid (CB1) and PPAR-γ receptors. Clinically, the blocking stimulation of the endocannabinoid pathway has been one of the first approaches proposed to counteract the obesity and obesity-associated diseases (such as diabetes, metabolic syndrome and cancer). In this connection, here we studied in cultured 3T3-L1 pre-adipocytes the effects of n-3-PUFA, α-Linolenic acid (OM-3), n-6-PUFA, Linoleic acid (OM-6), and hydroxytyrosol (HT) on the expression of CB1 receptor gene and the adipogenesis-related genes PPAR-γ, Fatty Acid Synthase (FAS) and Lipoprotein Lipase (LPL). HT was able to inhibit 3T3-L1 cell differentiation by down-regulating cell proliferation and CB1 receptor gene expression. HT exhibited anti-adipogenic effects, whereas OM-3 and OM-6 exerted an inhibitory action on cell proliferation associated with an induction of the preadipocytes differentiation and CB1 receptor gene expression. Moreover, the expression of FAS and LPL genes resulted increased after treatment with both HT and OM-3 and OM-6. The present study points out that the intake of molecules such as HT, contained in extra virgin olive oil, may be considered also in view of antiobesity and antineoplastic properties by acting directly on the adipose tissue and modulating CB1 receptor gene transcription. J. Cell. Physiol. 231: 483-489, 2016. © 2015 Wiley Periodicals, Inc.

Quercetin, the major constituent of flavonoid and widely present in fruits and vegetables, is an attractive compound for cancer prevention due to its beneficial anti proliferative effects, showing a crucial role in the regulation of apoptosis and cell cycle signaling. In vitro studies have demonstrated that quercetin specifically influences colon cancer cell proliferation. Our experiments, using human colon adenocarcinoma cells, confirmed the anti proliferative effect of quercetin and gave intriguing new insight in to the knowledge of the mechanisms involved. We observed a significant increase in the expression of the endocannabinoids receptor (CB1-R) after quercetin treatment. CB1-R can be considered an estrogen responsive receptor and quercetin, having a structure similar to that of the estrogens, can interact with CB1-R leading to the regulation of cell growth. In order to clarify the contribution of the CB1-R to the quercetin action, we investigated some of the principal molecular pathways that are inhibited or activated by this natural compound. In particular we detected the inhibition of the major survival signals like the PI3K/Akt/mTOR and an induction of the pro apoptotic JNK/JUN pathways. Interestingly, the metabolism of β-catenin was modified by flavonoid both directly and through activated CB1-R. In all the experiments done, the quercetin action has proven to be reinforced by anandamide (Met-F-AEA), a CB1-R agonist, and partially counteracted by SR141716, a CB1-R antagonist. These findings open new perspectives for anticancer therapeutic strategies.

Background The lipidomic profiling of erythrocyte membranes is expected to provide a peculiar scenario at molecular level of metabolic and nutritional pathways which may influence the lipid balance and the adaptation and homeostasis of the organism. Considering that lipid accumulation in the cell is important in promoting tissue inflammation, the purpose of this study is to analyze the fatty acid profile in red blood cell membranes of patients with Non-Alcoholic Fatty Liver Disease (NAFLD), in order to identify and validate membrane profiles possibly associated with the degree of hepatic damage. Methods This work presents data obtained at baseline from 101 subjects that participated to a nutritional trial (registration number: NCT02347696) enrolling consecutive subjects with NAFLD. Diagnosis of liver steatosis was performed by using vibration-controlled elastography implemented on FibroScan. Fatty acids, extracted from phospholipids of erythrocyte membranes, were quantified by gas chromatography method. Results The subjects with severe NAFLD showed a significant decrease of the ratio of stearic acid to oleic acid (saturation index, SI) compared to controls, 1.281 ± 0.31 vs 1.5 ± 0.29, respectively. Low levels of SI in red blood cell membranes, inversely associated with degree of liver damage, suggest that an impairment of circulating cell membrane structure can reflect modifications that take place in the liver. Subjects with severe NAFLDalso showed higher levels of elongase 5 enzymatic activity, evaluated as vaccenic acid to palmitoleic acid ratio. Conclusions Starting from these evidences, our findings show the importance of lipidomic approach in the diagnosis and the staging of NAFLD.

Mediterranean diet components, such as olive oil and ω-3 polyunsaturated fatty acids (ω-3 PUFAs), can arrest cell growth and promote cell apoptosis. Recently, olive oil has been demonstrated to modulate type-1 cannabinoid (CB1) receptor gene expression in both human colon cancer cells and rat colon. The aim of this study was to investigate a possible link between olive oil and ω-3 PUFAs effects and CB1 receptor expression in both intestinal and adipose tissue of ApcMin/+ mice. To confirm the role for the CB1 receptor as a negative modulator of cell proliferation in human colon cancer, CB1 receptor gene expression was also detected in tumor tissue and in surrounding normal mucosa of patients with colorectal cancer (CRC). Dietary ω-3 PUFAs significantly inhibited intestinal polyp growth in mice, correlating with CB1 receptor gene and protein expression induction. CB1 receptor gene up-regulation was also detected in adipose tissue, suggesting a close communication between cancer cells and the surrounding environment. Tissue CB1 receptor induction was associated with a concurrent inactivation of the Wnt/β-catenin pathway. Moreover, there was a significant reduction in CB1 receptor gene expression levels in cancer tissue compared to normal surrounding mucosa of patients with CRC, confirming that in cancer the “protective” action of the CB1 receptor is lost.

Alterations of lipid metabolism have been increasingly recognized as a hallmark of cancer cells. Cancer cells esterify fatty acids predominantly to phospholipids, an essential component of cell membranes. The main pathway along which proliferating cells gain lipids for membrane synthesis is the endogenous mevalonate pathway. Increased synthesis of mevalonate and mevalonate-derived isoprenoids supports increased cell proliferation through activating growth-regulatory proteins and oncoproteins and promoting DNA synthesis. The importance of a better knowledge of metabolic changes in lipogenic enzymes pathways, as well as of the role of each biochemical pathway in carcinogenesis, provides the rationale for in-depth study of the oncogenic signaling important for the initiation and progression of tumors. The dependence of tumor cells on a dysregulated lipid metabolism suggests that the proteins involved in this process may be excellent chemotherapeutic targets for cancer treatment. Here, we confirm the vital link between lipogenesis and cell proliferation, and our recent findings suggest that nutritional intervention is an effective and safe way to reduce cell proliferation in experimental models of carcinogenesis. The olive oil diet significantly reduces the protein activities of lipogenic enzymes associated with cell growth. The use of natural dietary components could potentially assist in the management of subjects with metabolic disorders-related tumors.

Lipoprotein lipase (LPL) is the crucial enzyme for intravascular catabolism of triglyceride-rich lipoproteins. Fatty acid synthase (FAS) is a key anabolic enzyme that catalyzes the terminal steps in the novo biosynthesis of 18:2n-6. The involvement of both LPL and FAS in tumor biology has been widely demonstrated in different studies and to verify whether there are regional differences in the expression of these enzymes in visceral adipose tissue from patients with colorectal cancer might be representative of events which sustain tumor growth. The objective of this study was to evaluate LPL and FAS activity and expression of their genes in adipose tissue adjacent to neoplasia and distant from it from patients operated for colorectal cancer. LPL enzymatic activity was evaluated by a fluorescent method and FAS activity by a radiometer assay. Reverse-transcription and real-time PCR were used to detect mRNA levels of two enzymes. Our findings show a significant reduction in both LPL and FAS gene expression and activity levels in adipose tissue adjacent to tumor lesion compared to those detected in paired tissue distant from neoplasia. These results underline the influence of tumor microenvironment on lipid metabolism in adipose tissue, demonstrating a tumor-induced impairment in the formation and lipid storing capacity of adipose tissue in patients with colorectal cancer.

Fatty acid synthase is a common phenotype to various human cancers including those of prostate, colon, lung, endometrium, and stomach. Increased fatty acid synthase levels have been detected in serum from patients with breast and pancreatic cancer. In this study, serum levels of fatty acid synthase were measured in colorectal cancer patients at different stages of disease.Consecutive 67 patients with colorectal cancer were enrolled in the study. Serum levels of fatty acid synthase were examined by ELISA test. The Kruskal-Wallis test and the χ (2) method for trend have been used to analyze data.Serum fatty acid synthase levels of patients belonging to three groups of stage disease are statistically different. The patients with stage III and IV have significantly higher serum levels of fatty acid synthase than patients with stage I-II. There is a positive trend in serum fatty acid synthase levels from stage I-II to stage III and IV of disease.Fatty acid synthase levels are associated with the stage of disease in patients with colorectal cancer.

Fructosamine-3-Kinase (FN3K) is an enzyme phosphorilating fructoselysine (FL) residues on glycated proteins, resulting in the production of protein-bound FL-3-phosphate. The pathological role of the non-enzymatic modification of proteins by reducing sugars has become increasingly evident in various types of disorders, including the cancer. In this study, our aim was to study FN3K enzyme activity, as well as its mRNA in human colorectal cancer (CRC). Thirty consecutive CRC patients undergoing surgery of the colon were enrolled in the study. FN3K enzymatic activity and gene expression were analyzed using a radiometric assay and quantitative RT-PCR, respectively. FN3K is a functionally active enzyme in human colon tissue, without significant differences between normal mucosa and cancer. The mean level of FN3K mRNA was significantly lower in cancer than in the corresponding normal colorectal mucosa The colorectal tumors located on the left side showed lower levels of both enzymatic activity and mRNA FN3K than tumors located in the right side of colon. This paper is the first studying FN3K enzyme activity in human CRC, showing a significant relationship between enzymatic activity, its mRNA and tumor side.

The major soy-derived isoflavones such as genistein has been demonstrated to possess anticarcinogenic activity in animal model systems. The present study was designed to investigate the effects of isoflavone genistein exposure at concentrations ranging from 0.01 to 50 muM on the LDL receptor and HMG-CoA reductase gene expression in the estrogen receptor positive DLD-1 human colon cancer cell line. LDL receptor and HMG-CoA reductase gene expressions were evaluated by reverse transcription followed by real-time PCR. Genistein induced an increase of LDL receptor gene expression and later decrease of HMG-CoA reductase mRNA expression in DLD-1 cells. These findings provide direct evidence on the role of genistein in regulating LDL receptor and HMG-CoA reductase gene expression in colon cancer.

Gastric and colon cancers remain the leading cause of cancer mortality throughout the world. Since the gastrointestinal tract works in a constant link with the external environment, chemoprevention by dietary constituents could represent a possible approach to reduce cancer risk. Dietary vitamin K1 (VK1) has been shown to prevent the growth of many types of cancer cells. However, no data are available on possible different susceptibility to VK1 by gastric or colon neoplastic cell lines. Moreover, the exact mechanism of action of VK1 is still object of investigation, even if it has been reported that VK1 may induce cell cycle arrest and apoptosis. Therefore, molecules affecting cell growth such as the natural polyamines could be of interest in VK1 action. The aim of the present study was to investigate the effects of increasing concentrations of VK1 (from 10 to 200 µM) administered up to 72 h, on the cell proliferation and apoptosis of a gastric (HGC-27) and a colon (SW480) cancer cell line. Additionally, the polyamine biosynthesis and the MAPK pathway were also examined. VK1 treatments caused an inhibition of cell proliferation and an induction of apoptosis in both cell lines, with a concomitant significant decrease of the polyamine biosynthesis, increased phospho-ERK 1/2 expression was also observed. A different proliferative behavior and a different response to VK1 by gastric and colon cancer cells was evident, with colon cells showing a more pronounced susceptibility to VK1 action. VK1 is safe and without known toxicities in adult humans, consequently it could be effective in prevention and treatment of selected gastrointestinal neoplasms. Protocols based on the use of VK1, along with polyamine inhibitors and/or analogues, could represent a suitable alternative option for improving the efficacy of chemoprevention and treatment in future strategies for gastrointestinal cancer management.

Vitamin K1 has been demonstrated as having anticancer potentiality mainly in liver cancer cells. Beyond the reported mechanisms of cancer inhibition (cell cycle arrest and induction of apoptosis), a possible control by vitamin K1 on molecules affecting cell growth could be hypothesized. In the literature, few (if any) data are available on its antitumor effects on colon cancer cells. Therefore, the aims of the study were to investigate in three differently graded human colon cancer cell lines (Caco-2, HT-29, and SW480) the effects of increasing concentrations of vitamin K1 (from 10 μM to 200 μM) administered up to 72 h on (1) cell proliferation, (2) apoptosis with the possible involvement of the MAPK pathway, and (3) polyamine biosynthesis. Vitamin K1 treatment caused a significant antiproliferative effect and induced apoptosis in all the cell lines, with the involvement of the MAPK pathway. A concomitant and significant decrease in the polyamine biosynthesis occurred. This is the first study demonstrating a significant polyamine decrease in addition to the antiproliferative and proapoptotic effects following vitamin K1 administration to colon cancer cell lines. Therapeutically, combinations of vitamin K1 with polyamine inhibitors and/or analogues may represent a suitable option for chemoprevention and/or treatment in future strategies for colorectal cancer management.

The promotion and progression of carcinogenesis are susceptible to nutritional interventions aimed at counteracting cancer development. Lipid metabolism is essential in the onset and progression of tumors and for cancer cell survival. In this study, we tested the effects of diets enriched with natural compounds, such as olive oil and salmon oil, in mice that spontaneously develop intestinal polyps (Apc(Min/+) mice). For this purpose, we evaluated polyp number and volume, intestinal mucosa proliferation/apoptosis, estrogen receptors (ERs) expression, fatty acid synthase and 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase gene expression and enzymatic activity. Compared with the standard diet, the salmon oil-enriched diet, containing a high percentage of omega-3 polyunsaturated fatty acids, and, to a lesser extent, olive oil-enriched diet reduced polyp number and volume through a reduction of proliferation and a marked proapoptotic effect. These biological effects were mediated by an inhibition of fatty acid synthase and HMGCoA reductase gene expression and activity and an increase of ERβ/ERα ratio. Our findings suggest that a proper dietary lifestyle could contribute to primary cancer prevention.

Background Celiac disease is characterized by enhanced intestinal paracellular permeability due to alterations of function and expression of tight junction (TJ) proteins including ZO-1, Claudin-1 and Occludin. Polyamines are pivotal in the control of intestinal barrier function and are also involved in the regulation of intercellular junction proteins. Different probiotic strains may inhibit gliadin-induced toxic effects and the Lactobacillus rhamnosus GG (L.GG) is effective in the prevention and treatment of gastrointestinal diseases. Aims of the study were to establish in epithelial Caco-2 cells whether i) gliadin affects paracellular permeability and polyamine profile; ii) co-administration of viable L.GG, heat-killed L.GG (L.GG-HK) or its conditioned medium (L.GG-CM) preserves the intestinal epithelial barrier integrity. Additionally, the effects of L.GG on TJ protein expression were tested in presence or absence of polyamines. Results Administration of gliadin (1 mg/ml) to Caco-2 cells for 6 h caused a significant alteration of paracellular permeability as demonstrated by the rapid decrease in transepithelial resistance with a concomitant zonulin release. These events were followed by a significant increase in lactulose paracellular transport and a slight lowering in ZO-1 and Occludin expression without affecting Claudin-1. Besides, the single and total polyamine content increased significantly. The co-administration of viable L.GG (108 CFU/ml), L.GG-HK and L.GG-CM with gliadin significantly restored barrier function as demonstrated by transepithelial resistance, lactulose flux and zonulin release. Viable L.GG and L.GG-HK, but not L.GG-CM, led to a significant reduction in the single and total polyamine levels. Additionally, only the co-administration of viable L.GG with gliadin significantly increased ZO-1, Claudin-1 and Occludin gene expression compared to control cells. When Caco-2 cells treated with viable L.GG and gliadin were deprived in the polyamine content by α-Difluoromethylornithine, the expression of TJ protein mRNAs was not significantly different from that in controls or cells treated with gliadin alone. Conclusions Gliadin modifies the intestinal paracellular permeability and significantly increases the polyamine content in Caco-2 cells. Concomitant administration of L.GG is able to counteract these effects. Interestingly, the presence of cellular polyamines is necessary for this probiotic to exert its capability in restoring paracellular permeability by affecting the expression of different TJ proteins.

Background Lipid metabolism is altered in subjects with liver steatosis. FAS is a key enzyme in de novo lipogenesis and both FAS gene expression and enzymatic activity are primarily regulated by metabolic signals in the liver. Lipoprotein lipase (LPL), the rate-limiting enzyme for the hydrolysis of core triglycerides, plays a pivotal role in lipid metabolism. This study aims to investigate if circulating levels of FAS and LPL could be clinically associated with liver steatosis. Methods In this work, we present data obtained from a subsample of 94 subjects with liver steatosis enrolled by NUTRIEPA study, a nutritional trial in subjects with liver steatosis. Serum levels of FAS protein and LPL activity were evaluated by ELISA test and by a fluorescent method, respectively. The diagnosis and the degree of liver steatosis were based on laboratory and ecographic measurements. Statistical methods included Kruskal-Wallis analysis of variance and Wilcoxon signed-rank test, where appropriate. The χ 2 test has been performed to analyse categorical variables. Results The subjects with severe steatosis had significantly higher serum levels of FAS protein and LPL activity compared to subjects with mild and moderate liver steatosis. Moreover, a positive trend in serum levels of FAS expression from lower to higher degree of steatosis was also detected. Conclusions We describe a relationship between human liver steatosis and elevated levels of circulating lipogenic enzymes. Increased serum levels of FAS expression and LPL activity could be considered a marker of severe liver steatosis.

Background n-3 and n-6 polyunsaturated fatty acids (PUFAs) are the two major classes of PUFAs encountered in the diet, and both classes of fatty acids are required for normal human health. Moreover, PUFAs have effects on diverse pathological processes impacting chronic disease, such as cardiovascular and immune disease, neurological disease, and cancer. Aim To investigate the effects of eicosapentaenoic acid (EPA) and arachidonic acid (ARA) on the proliferation and apoptosis of human hepatoma cell line HepG2 after exposure to increasing concentrations of EPA or ARA for 48 h. Moreover, in the same cells the gene expression of Fatty Acid Synthase (FAS) and 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A Reductase (HMG-CoAR) was also investigated. Method Cell growth and apoptosis were assayed by MTT and ELISA test, respectively after cell exposure to increasing concentrations of EPA and ARA. Reverse-transcription and real-time PCR was used to detect FAS and HMG-CoAR mRNA levels in treated cells. Results Our findings show that EPA inhibits HepG2 cell growth in a dose-dependent manner, starting from 25 μM (P < 0.01, one-way ANOVA test and Dunnett's post test) and exerts a statistically significant pro-apoptotic effect already at 1 μM of EPA. Higher doses of ARA were need to obtain a statistically significant inhibition of cell proliferation and a pro-apoptotic effect in these cells (100 μM, P < 0.01, one-way ANOVA test and Dunnett's post test). Moreover, a down-regulation of FAS and HMG-CoAR gene expression was observed after EPA and ARA treatment in HepG2 cells, starting at 10 μM (P < 0.05, one-way ANOVA test and Dunnett's post test). Conclusion Our results demonstrate that EPA and ARA inhibit HepG2 cell proliferation and induce apoptosis. The down-regulation of FAS and HMG-CoAR gene expression by EPA and ARA might be one of the mechanisms for the anti-proliferative properties of PUFAs in an in vitro model of hepatocellular carcinoma.

To investigate Glyoxalase I and fructosamine-3-kinase (FN3K) activity in red blood cells from patients with colorectal adenomas and cancer.Thirty three consecutive subjects with one or more histologically confirmed colorectal adenomatous polyps, 16 colorectal cancer patients and a group of 11 control subjects with normal colonoscopy were included in the study. Glyoxalase I and FN3K activities were measured in red blood cells using a spectrophotometric and radiometric assay, respectively.A significant reduction in both Glyoxalase I and FN3K activity was detected in patients with tumors compared to patients with adenomas and the controls. Erythrocyte Glyoxalase I activity in colorectal cancer was approximately 6 times lower than that detected in patients with adenoma (0.022 ± 0.01 mmol/min per milliliter vs 0.128 ± 0.19 mmol/min per milliliter of red blood cells, P = 0.003, Tukey's test). FN3K activity in red blood cells from patients with colon cancer was approximately 2 times lower than that detected in adenoma patients (19.55 ± 6.4 pmol/min per milliliter vs 38.6 ± 31.7 pmol/min per milliliter of red blood cells, P = 0.04, Tukey's test).These findings suggest that deglycating enzymes may be involved in the malignant transformation of colon mucosa.

Oleuropein (OL) and hydroxytyrosol (HT), the main olive oil polyphenols, possess anti-proliferative effects in vitro. Fatty acid synthase, a key anabolic enzyme of biosynthesis of fatty acids, plays an important role in colon carcinoma development. Our aim was to investigate whether gene expression of FAS, as well as its enzymatic activity, is regulated by HT and OL in two human colon cancer cell lines, as HT-29 and SW620. In addition, we investigated the effects of these polyphenols on growth and apoptosis in these cells. FAS gene expression and activity in treated HT-29 and SW620 cells were evaluated by real-time PCR and radiochemical assay, respectively. Cell growth and apoptosis, after polyphenols treatment, were measured by MTT test and flow cytometry, respectively. The inhibition of proliferation, detected after HT treatment, was mediated by an inhibition of FAS expression and its enzymatic activity in SW620 cells, while the anti-proliferative effect in HT-29 cells seems to be independent from FAS. OL exerted an anti-proliferative effect only on SW620 cells with a mechanism which excluded FAS. Olive oil polyphenols used were able to induce apoptosis in both cell lines studied. The increase of apoptosis in these cells was accompanied by the block of cell cycle in the S phase. This study demonstrates that HT and OL may induce anti-proliferative and pro-apoptotic effects only in certain human colorectal cancer cell types. These effects are FAS mediated only in SW620 cells after treatment with HT.

AIM: To study, in intact male transgenic mice, the effects of three diets based on olive oil and olive oil diet supplemented with lovastatin and orlistat on hepatic lipogenic enzymes expression, considered markers of cell proliferation. METHODS: Forty ApcMin/+ mice were randomly divided into 4 groups and fed for 10 wk: olive oil (OO) group, n = 10 animals received a diet with olive oil 12%; olive oil plus lovastatin (LOVA) group, n = 10 animals received the same diet with olive oil supplemented with lovastatin 5 mg/kg; olive oil plus orlistat (OR) group, n = 10 animals fed the diet with olive oil supplemented with orlistat 50 mg/kg and SD group, n = 10 animals fed a standard diet. The activity of lipogenic enzymes and their gene expression were evaluated by radiometric and real-time reverse transcription-polymerase chain reaction assay, respectively. RESULTS: After 10 wk of dietary treatment, the body weight was no different among animal groups (21.3 ± 3.1 g for standard group, 22.1 ± 3.6 g for OO group, 22.0 ± 3.2 g for LOVA group and 20.7 ± 3.4 g for OR group, data expressed as mean ± SD), observing a generalized well-being in all animals. All the dietary managed treated groups presented significantly reduced hepatic levels of fatty acid synthase, farnesyl pyrophosphate synthase and 3-hydroxyl-3-methyl-glutaryl CoA reductase activity and gene expression when compared with the mice fed the standard diet. To evaluate cell proliferation in the liver of treated mice, the levels of cyclin E mRNA have been measured, demonstrating a significant reduction of cyclin E gene expression in all treated groups. Evidence of reduced hepatic cell proliferation was present overall in OO group mice. CONCLUSION: We confirm the role of lipogenic enzymes as markers of cell proliferation, suggesting that appropriate dietary management alone or with drugs can be a feasible approach to counteract hepatic cell proliferation in mice.

Background PUFAs are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an enzyme catalyzing the conversion of HMGCoA to mevalonate, the rate limiting step in cholesterol biosynthesis. Statins represent a class of drugs that are widely used to treat hypercholesterolemia for their ability to inhibit cholesterol biosynthesis and to up-regulate the synthesis of Low Density Lipoprotein (LDL) receptors in the liver. PUFAs mediate many, if not all, actions of statins and this could be one mechanism by which they lower cholesterol levels. The purpose of this study was to investigate whether combined treatment with Eicosapentaenoic acid (EPA) and lovastatin enhanced the regulatory effect on gene expression of HMGCoA reductase and LDL receptor in HepG2 cell line. Results The combined treatment with EPA and lovastatin enhanced the regulatory effect on gene expression of HMGCoA reductase and LDL receptor in HepG2 cell line. Moreover, we detected a synergistic effect on the inhibition of cancer cell proliferation obtained by combination of EPA and Lovastatin. Conclusions The use of EPA, in combination with low doses of Lovastatin may have potential value in treatment of neoplastic diseases.