Understanding nutritional interventions and physical exercise in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in adults and its prevalence is rising around the world. This pathology is characterized by accumulation of liver fat, which exceeds 5% of liver weight in absence of alcohol consumption, viral infection or other hepatic etiology. Since NAFLD has been associated with obesity, insulin resistance, diabetes or alteration of lipid profiles, it is considered as the liver manifestation of metabolic syndrome. Pathogenic mechanisms of NAFLD have not been clearly elucidated, but different events such as lipid accumulation, insulin resistance, oxidative and endoplasmic reticulum stress, mitochondrial dysfunction and inflammation are involved. Modifications in lifestyle constitute the first line for the management of NAFLD. Nutritional interventions include low fat and carbohydrate diet with higher polyunsaturated fatty acids ingestion. Moreover, supplementation with antioxidant and cytoprotective agents could be useful to decrease oxidative stress, inflammation and fibrosis. Physical activity enables to reduce the expression of lipogenic genes, fat accumulation, or insulin resistance and improves cardiorespiratory fitness. Benefits have been found following both aerobic exercise and resistance training, and remain even after exercise cessation. However, more studies are required to analyze the molecular and cellular mechanisms involved in nutritional and physical intervention, and to define the volume of activity required and its association with weight loss. In this paper, we offer an updated overview of the mechanisms implicated in the progression of NAFLD, and analyze the beneficial effects of nutritional interventions and physical exercise in the prevention and treatment of this condition.

On-line surface plasmon resonance biosensing of vascular endothelial growth factor signaling in intact-human hepatoma cell lines.

Surface plasmon resonance (SPR) monitoring of biorecognition events at intracellular levels is a valuable tool for studying the angiogenic response of carcinoma living cells during tumor growth and proliferation. We report here a comparative study of two different strategies to detect human hepatoma cell interactions between transmembrane vascular endothelial growth factor receptor (VEGFR2) and vascular endothelial growth factor (VEGF). To monitor VEGFR2 activation after VEGF stimulation, intact hepatocellular carcinoma HepG2 or Huh7 cells (2 × 10(5) cells per mL) were directly immobilized on the sensor chip. Distinguishable SPR sensorgrams were obtained for each cell line depending on the time required for VEGFR2 activation. SPR signals for VEGF-VEGFR2 binding were inhibited by the VEGFR inhibitor, CBO-P11. The SPR response after VEGF stimulation/inhibition was in good agreement with the results observed by immunoblotting analysis. In a second approach we used intact cell lines as analytes. SPR analysis was done by injecting HepG2 and HuH7 cell suspensions (2-4 × 10(4) cells per mL) onto a sensor surface previously immobilized with VEGF via a thiol self-assembled monolayer (SAM). Specificity and reproducibility were evaluated reusing the same chip surface over more than 60 complete regeneration cycles. Comparison between both methods yielded differences in terms of reliability, making the latter strategy more effective for the analysis of real samples. The investigation of VEGF signaling in intact human hepatoma living cells by SPR monitoring comprises a novel and promising design for the study of tumor angiogenesis via downregulation of VEGF and VEGFR2 pathways. Further investigation on VEGFR activation and vascular function could contribute to establish a robust and meaningful tool for early cancer diagnostics.

FoxO proteins: regulation and molecular targets in liver cancer.

Human forkhead box class O (FoxO) transcription factors, activated in response to a wide range of external stimuli, like growth factors, insulin, nutrient levels and oxidative stress, are able to control several specific geneexpression programs. Besides their clear implication in metabolic processes, they appear to play a relevant role in tumour suppression by upregulation of genes involved in cell cycle arrest or apoptosis. Recent research efforts provide new insights into the molecular modulation of FoxO in liver cancer and disclose potential opportunities for developing new antitumor drugs. Through an intricate regulatory model, achieved via several post-translational modifications, including phosphorylation, acetylation, and ubiquitination, which control their subcellular localization and DNA binding activity, FoxO factors act as tumour suppressors. Low levels of FoxOs are associated with poor prognosis in cancer patients, and seem to confer chemotherapy resistance. Within FoxO members, FoxO3a appears to present anti-tumour properties in hepatocellular carcinoma, inducing the expression of pro-apoptotic genes, or interfering with signaling cascades commonly altered in this disease such as Wnt/ß-catenin, PI3K/AKT/mTOR or MAPKs pathways. Here, we describe the main mechanisms of FoxO proteins regulation, and their cross-link with altered pathways in liver cancer. Moreover, based on the current knowledge of FoxO modulation, emphasis is placed on the development of novel agents which specifically activate FoxO family members and could be useful in the treatment of hepatocarcinoma.

Inhibition of VEGF expression through blockade of Hif1α and STAT3 signalling mediates the anti-angiogenic effect of melatonin in HepG2 liver cancer cells.

BACKGROUND: Hepatocellular carcinoma (HCC) growth relies on angiogenesis via vascular endothelial growth factor (VEGF) release. Hypoxia within tumour environment leads to intracellular stabilisation of hypoxia inducible factor 1 alpha (Hif1α) and signal transducer and activator of transcription (STAT3). Melatonin induces apoptosis in HCC, and shows anti-angiogenic features in several tumours. In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate the anti-angiogenic effects of melatonin. METHODS: HepG2 cells were treated with melatonin under normoxic or CoCl2-induced hypoxia. Gene expression was analysed by RT-qPCR and western blot. Melatonin-induced anti-angiogenic activity was confirmed by in vivo human umbilical vein endothelial cells (HUVECs) tube formation assay. Secreted VEGF was measured by ELISA. Immunofluorescence was performed to analyse Hif1α cellular localisation. Physical interaction between Hif1α and its co-activators was analysed by immunoprecipitation and chromatin immunoprecipitation (ChIP). RESULTS: Melatonin at a pharmacological concentration (1 mM) decreases cellular and secreted VEGF levels, and prevents HUVECs tube formation under hypoxia, associated with a reduction in Hif1α protein expression, nuclear localisation, and transcriptional activity. While hypoxia increases phospho-STAT3, Hif1α, and CBP/p300 recruitment as a transcriptional complex within the VEGF promoter, melatonin 1 mM decreases their physical interaction. Melatonin and the selective STAT3 inhibitor Stattic show a synergic effect on Hif1α, STAT3, and VEGF expression. CONCLUSION: Melatonin exerts an anti-angiogenic activity in HepG2 cells by interfering with the transcriptional activation of VEGF, via Hif1α and STAT3. Our results provide evidence to consider this indole as a powerful anti-angiogenic agent for HCC treatment.

Melatonin induces transcriptional regulation of Bim by FoxO3a in HepG2 cells.

BACKGROUND: Melatonin induces apoptosis in many different cancer cell lines, including hepatocellular carcinoma cells. However, the responsible pathways have not been clearly elucidated. A member of the forkhead transcription factors' family, FoxO3a, has been implicated in the expression of the proapoptotic protein Bim (a Bcl-2-interacting mediator of cell death). In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate whether melatonin treatment induces Bim through regulation by the transcription factor FoxO3a. METHODS: Cytotoxicity of melatonin was compared in HepG2 hepatoblastoma cells and primary human hepatocytes. Proapoptotic Bim expression was analysed by reverse transcriptase-polymerase chain reaction and western blot. Reporter gene assays and chromatin immunoprecipitation assays were performed to analyse whether FoxO3a transactivates the Bim promoter. Small interfering RNA (siRNA) was used to study the role of FoxO3a in Bim expression. Immunofluorescence was performed to analyse FoxO3a localisation in HepG2 cells. RESULTS: Melatonin treatment induces apoptosis in HepG2 cells, but not in primary human hepatocytes. The proapoptotic effect was mediated by increased expression of the BH3-only protein Bim. During melatonin treatment, we observed increased transcriptional activity of the forkhead-responsive element and could demonstrate that FoxO3a binds to a specific sequence within the Bim promoter. Furthermore, melatonin reduced phosphorylation of FoxO3a at Thr(32) and Ser(253), and induced its increased nuclear localisation. Moreover, silencing experiments with FoxO3a siRNA prevented Bim upregulation. CONCLUSION: This study shows that melatonin can induce apoptosis in HepG2 hepatocarcinoma cells through the upregulation of proapoptotic Bim mediated by nuclear translocation and activation of the transcription factor FoxO3a.