Biobank Education for Future Physicians: Training Medical Students Through Student Research Association Networks.

Research biobanks have become crucial collaborators in a variety of basic and clinical research projects with comprehensive biological sample collection and associated data storage. Medical students, who are the most important stakeholders of biobanks as future physicians, need to be trained in biobanking; however, there is no consensus on how to include it in formal education. This study aimed to determine and increase awareness among medical students regarding biobanks through peer training organized online by medical student research association networks. Volunteer medical or graduate students were trained by biobank professionals at the Izmir Biomedicine and Genome Center (IBG) biobank for 6-9 months. Then, a biobank event was planned by these trainees, the Ege Scientific Research Team (ESRT), and IBG-Biobank with the support of The Biobanking and BioMolecular Resources Research Infrastructure (BBMRI) Turkey. The study reached students of 46 different medical faculties. Before the event, students' level of knowledge about biobanks was identified using a pre-event questionnaire (n = 239). Following 2 days (4 main sessions) of online events, a post-event questionnaire was administered to event participants (n = 110) and 80.9% of them answered (n = 89). The pre-event survey revealed that only 34.3% of the medical students had heard of the term "Biobank" in Turkey. After the event, medical students were significantly more enthusiastic about putting effort into biobanking and using and sharing stored biobank samples of their patients compared with the pre-event (p < 0.0001). Moreover, 92% of the students stated that they would consider attending an advanced course in biobanking. In conclusion, the current study demonstrates that extracurricular courses with peer learning methods coordinated with medical student associations can be valuable in increasing future physicians' awareness and knowledge of biobanking.

c-Met activation promotes extravasation of hepatocellular carcinoma cells into 3D-cultured hepatocyte cells in lab-on-a-chip device.

Activation of c-Met signaling is associated with an aggressive phenotype and poor prognosis in hepatocellular carcinoma (HCC); however, its contribution to organ preference in metastasis remains unclear. In this study, using a Lab on a Chip device, we defined the role of aberrant c-Met activation in regulating the extravasation and homing capacity of HCC cells. Our studies showed that (i) c-Met overexpression and activation direct HCC cells preferentially towards the hepatocytes-enriched microenvironment, and (ii) blockage of c-Met phosphorylation by a small molecule inhibitor attenuated extravasation and homing capacity of HCC cells. These results, thus, demonstrate the role of c-Met signaling in regulating the colonization of HCC cells preferentially in the liver.

Toll-interacting protein may affect doxorubicin resistance in hepatocellular carcinoma cell lines.

BACKGROUND: Liver cancer is the third leading cause of cancer-related deaths worldwide, and hepatocellular carcinoma (HCC) is the most common type of liver cancer. Transarterial interventions are among the chemotherapeutic approaches used in hardly operable regions prior to transplantation, and in electrochemotherapy, where doxorubicin is used. However, the efficacy of treatment is affected by resistance mechanisms. Previously, we showed that overexpression of the CUE5 gene results in doxorubicin resistance in Saccharomyces cerevisiae (S. cerevisiae). In this study, the effect of Toll-interacting protein (TOLLIP), the human ortholog of CUE5, on doxorubicin resistance was evaluated in HCC cells to identify its possible role in increasing the efficacy of transarterial interventions. METHODS AND RESULTS: The NIH Gene Expression Omnibus (GEO) and Oncomine datasets were analyzed for HCC cell lines with relatively low and high TOLLIP expression, and SNU449 and Hep3B cell lines were chosen, respectively. TOLLIP expression was increased by plasmid transfection and decreased by TOLLIP-siRNA in both cell lines and evaluated by RT-PCR and ELISA. Cell proliferation and viability were examined using xCELLigence and MTT assays after doxorubicin treatment, and growth inhibitory 50 (GI 50) concentrations were evaluated. Doxorubicin GI 50 concentrations decreased approximately 2-folds in both cell lines upon silencing TOLLIP after 48 h of drug treatment. CONCLUSIONS: Our results showed for the first time that silencing TOLLIP in hepatocellular carcinoma cells may help sensitize these cells to doxorubicin and increase the efficacy of chemotherapeutic regimens where doxorubicin is used.

Role of Biobanks for Cancer Research and Precision Medicine in Hepatocellular Carcinoma.

INTRODUCTION: Hepatocellular carcinoma (HCC) is a highly complex and deadly cancer. There is an urgent need for new and effective treatment modalities. Since the primary goal in the management of cancer is to cure and improve survival, personalized therapy can increase survival, reduce mortality rates, and improve quality of life. Biobanks hold potential in leading to breakthroughs in biomedical research and precision medicine (PM). They serve as a biorepository, collecting, processing, storing, and supplying specimens and relevant data for basic, translational, and clinical research. OBJECTIVE: We aimed to highlight the fundamental role of biobanks, harboring high quality, sustainable collections of patient samples in adequate size and variability, for developing diagnostic, prognostic, and predictive biomarkers to develop and PM approaches in the management of HCC. METHOD: We obtained information from previously published articles and BBMRI directory. RESULTS AND CONCLUSION: Biobanking of high-quality biospecimens along with patient clinical information provides a fundamental scientific infrastructure for basic, translational, and clinical research. Biobanks that control and eliminate pre-analytical variability of biospecimens, provide a platform to identify reliable biomarkers for the application of PM. We believe, establishing HCC biobanks will empower to underpin molecular mechanisms of HCC and generate strategies for PM. Thus, first, we will review current therapy approaches in HCC care. Then, we will summarize challenges in HCC management. Lastly, we will focus on the best practices for establishing HCC biobanking to support research, translational medicine in the light of new experimental research conducted with the aim of delivering PM for HCC patients.

Evaluation of ATAD2 as a Potential Target in Hepatocellular Carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide with lack of effective systemic chemotherapy. In this study, we aimed to evaluate the value of ATPase family AAA domain-containing protein 2 (ATAD2) as a biomarker and potential therapeutic target for HCC. METHODS: The expression of ATAD2 was tested in different HCC patient cohorts by immunohistochemistry and comparative transcriptional analysis. The co-expression of ATAD2 and proliferation markers was compared during liver regeneration and malignancy with different bioinformatics tools. The cellular effects of ATAD2 inactivation in liver malignancy was tested on cell cycle, apoptosis, and colony formation ability as well as tumor formation using RNA interference. The genes affected by ATAD2 inactivation in three different HCC cell lines were identified by global gene expression profiling and bioinformatics tools. RESULTS: ATAD2 overexpression is closely correlated with HCC tumor stage. There was gradual increase from dysplasia, well-differentiated and poorly-differentiated HCC, respectively. We also observed transient upregulation of ATAD2 expression during rat liver regeneration in parallel to changes in Ki-67 expression. ATAD2 knockdown resulted in apoptosis and decreased cell survival in vitro and decreased tumor formation in some HCC cell lines. However, three other HCC cell lines tested were not affected. Similarly, gene expression response to ATAD2 inactivation in different HCC cell lines was highly heterogeneous. CONCLUSIONS: ATAD2 is a potential proliferation marker for liver regeneration and HCC. It may also serve as a therapeutic target despite heterogeneous response of malignant cells.

Increased telomerase activity in major depressive disorder with melancholic features: Possible role of pro-inflammatory cytokines and the brain-derived neurotrophic factor.

The biological mechanisms responsible for depression symptoms are not yet understood. For this reason, it is important to reveal the etiopathogenetic mechanisms in this disease. This study aims to compare the levels of pro-inflammatory cytokines, Brain-Derived Neurotrophic Factor (BDNF), and telomerase activity in patients with major depressive disorder (MDD) and healthy controls. Plasma BDNF, interleukin-6 (IL-6), IL-1beta, and Tumor Necrosis Factor-alpha (TNF-alpha) levels, and telomerase activity were measured in 39 patients with major depression and 39 healthy controls matched with patients in terms of age, gender, and education year. Plasma concentration of BDNF, IL-6 levels, and telomerase activity was significantly different between patients with MDD and healthy controls. Correlation analysis showed a positive trend between plasma BDNF levels and plasma IL-6 levels in patients with MDD with melancholic features. Furthermore, the path analysis results showed that the telomerase activity was indirectly affected by gender, IL-1ß, IL-6, BDNF, and BMI, via the severity of depression and anxiety and MDD status as the mediators. Further studies are needed to examine the molecular mechanism of the telomerase activity and the role of BDNF and pro-inflammatory cytokines in the telomerase activation in MDD.

High glucose induced c-Met activation promotes aggressive phenotype and regulates expression of glucose metabolism genes in HCC cells.

Hepatocellular carcinoma (HCC) is strongly associated with metabolic dysregulations/deregulations and hyperglycemia is a common metabolic disturbance in metabolic diseases. Hyperglycemia is defined to promote epithelial to mesenchymal transition (EMT) of cancer cells in various cancers but its molecular contribution to HCC progression and aggressiveness is relatively unclear. In this study, we analyzed the molecular mechanisms behind the hyperglycemia-induced EMT in HCC cell lines. Here, we report that high glucose promotes EMT through activating c-Met receptor tyrosine kinase via promoting its ligand-independent homodimerization. c-Met activation is critical for high glucose induced acquisition of mesenchymal phenotype, survival under high glucose stress and reprogramming of cellular metabolism by modulating glucose metabolism gene expression to promote aggressiveness in HCC cells. The crucial role of c-Met in high glucose induced EMT and aggressiveness may be the potential link between metabolic syndrome-related hepatocarcinogenesis and/or HCC progression. Considering c-Met inhibition in hyperglycemic patients would be an important complementary strategy for therapy that favors sensitization of HCC cells to therapeutics.

LGR5/R-Spo1/Wnt3a axis promotes stemness and aggressive phenotype in hepatoblast-like hepatocellular carcinoma cell lines.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly defined stem cell marker in endoderm-derived organs such as the small intestine, colon and pancreas. Recently, LGR5 was demonstrated to be an important factor in liver regeneration and stem cell maintenance. Moreover, LGR5 expression is highly up-regulated in various cancers including hepatocellular carcinoma. Herein, we demonstrate that LGR5 expression is specifically observed in certain subset of HCC cell lines with "hepatoblast-like" appearance, characterized by the expression of liver fetal/progenitor markers. Notably, the activation of the canonical Wnt pathway significantly increases the expression of LGR5 in this subset of cell lines, whereas it does not cause any induction of LGR5 expression in mesenchymal like cell lines SNU-475 and SNU-449. Furthermore, we showed that treatment of the hepatoblast-like HCC cell lines HuH-7 and Hep3B with LGR5 ligand R-Spo1 significantly amplifies the induction of LGR5 expression, the phosphorylation of LRP6 and ß-catenin resulting in enhanced TCF/LEF activity either alone or in combination with Wnt3a. Consistently, the silencing of the LGR5 gene attenuates the co-stimulatory effect of R-Spo1/Wnt3a on TCF/LEF activity while overexpression of LGR5 enhances it. On the contrary, overexpression of LGR5 does not change TCF/LEF activity induced by R-Spo1/Wnt3a in mesenchymal-like HCC line, SNU-449. Importantly, LGR5-overexpressing cells have increased expression of several Wnt target genes and stemness-related genes including EpCAM and CK19 upon R-Spo1/Wnt3a treatment. LGR5-overexpressing cells also have increased spheroid forming, migration and invasion abilities and stimulation with R-Spo1/Wnt3a augments these abilities of LGR5-overexpressing cells. In addition, ectopic overexpression of LGR5 significantly increases cell proliferation rate independent of R-Spo1/Wnt3a stimulation. Moreover, in vitro tubulogenesis assay demonstrates that treatment with R-Spo1/Wnt3a enhances the sprouting of capillary tubules in only LGR5-overexpressing cells. Finally, R-Spo1/Wnt3a significantly promotes dissemination of LGR5-overexpressing cells in vivo in a zebrafish xenograft model. Our study unravels a tumor-promoting role for LGR5 through activation of canonical Wnt/ß-catenin signaling in the hepatoblast-like HCCs. In conclusion, our results suggest that LGR5/R-Spo1/Wnt3a generates an axis that mediates the acquisition of aggressive phenotype specifically in hepatoblast-like subset of HCCs and might represent a valuable target for treatment of HCC tumors with aberrant activation of Wnt/ß-catenin pathway.

Engineered silica nanoparticles are biologically safe vehicles to deliver drugs or genes to liver cells.

Engineered silica nanoparticles (SiNP) are emerging materials for medical applications. Evaluating biological responses of specific cells treated with engineered silica nanoparticles is however essential. We synthesized and characterized the physicochemical properties of silica nanoparticles with two different sizes of 10 and 100 nm (10SiNP and 100SiNP) dispersed in cell culture medium. HuH-7, an epithelial-like human hepatoblastoma cell line and SK-HEP-1, a liver sinusoidal endothelial cell line (LSEC) are employed to evaluate their biological responses for the SiNP treatment. Primary human lymphocytes are used to assess genotoxicity recommended by OECD guidelines while erythrocytes are used to assess hemolytic activity. The engineered silica nanoparticles are not able to produce radical species, to alter the mitochondrial membrane potential, and induce any adverse effects on cell proliferation. The colony formation ability of HuH-7 hepatoblastoma cells was not affected following the SiNP treatment. Furthermore, SiNPs do not induce hemolysis of red blood cells and are not genotoxic. These findings suggest that SiNPs regardless of the size, amount, and incubation time are biologically safe vehicles to deliver drugs or genes to the liver.

Differential expression of full-length and NH2 terminally truncated FAM134B isoforms in normal physiology and cancer.

Selective autophagy of the endoplasmic reticulum (ER), namely ER-phagy, is mediated by ER-localized receptors, which are recognized and sequestered by GABARAP/LC3B-decorated phagophores and transferred to lysosomes for degradation. Being one such receptor, FAM134B plays critical roles in cellular processes such as protein quality control and neuronal survival. FAM134B has also been associated with different cancers, although its exact role remains elusive. We report here that the FAM134B gene encodes not one but at least two different protein isoforms: the full-length and the NH2 terminally truncated forms. Their relative expression shows extreme variation, both within normal tissues and among cancer types. Expression of full-length FAM134B is restricted to the brain, testis, spleen, and prostate. In contrast, NH2 terminally truncated FAM134B is dominant in the heart, skeletal muscle, kidney, pancreas, and liver. We compared wild-type and knockout mice to study the role of the Fam134b gene in starvation. NH2 terminally truncated FAM134B-2 was induced in the liver, skeletal muscle, and heart but not in the pancreas and stomach following starvation. Upon starvation, Fam134b-/- mice differed from wild-type mice by less weight loss and less hyperaminoacidemic and hypocalcemic response but increased levels of serum albumin, total serum proteins, and α-amylase. Interestingly, either NH2 terminally truncated FAM134B or both isoforms were downregulated in liver, lung, and colon cancers. In contrast, upregulation was observed in stomach and chromophobe kidney cancers.NEW & NOTEWORTHY We reported tissues expressing FAM134B-2 such as the kidney, muscle, heart, and pancreas, some of which exhibit stimulated expression upon nutrient starvation. We also demonstrated the effect of Fam134b deletion during ad libitum and starvation conditions. Resistance to weight loss and hypocalcemia, accompanied by an increase in serum albumin and α-amylase levels, indicate critical roles of Fam134b in physiology. Furthermore, the differential expression of FAM134B isoforms was shown to be significantly dysregulated in human cancers.

lncRNA HOTAIR overexpression induced downregulation of c-Met signaling promotes hybrid epithelial/mesenchymal phenotype in hepatocellular carcinoma cells.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) are both reversible processes, and regulation of phenotypical transition is very important for progression of several cancers including hepatocellular carcinoma (HCC). Recently, it is defined that cancer cells can attain a hybrid epithelial/mesenchymal (hybrid E/M) phenotype. Cells with hybrid E/M phenotype comprise mixed epithelial and mesenchymal properties, they can be more resistant to therapeutics and also more capable of initiating metastatic lesions. However, the mechanisms regulating hybrid E/M in HCC are not well described yet. In this study, we investigated the role of the potential crosstalk between lncRNA HOTAIR and c-Met receptor tyrosine kinase, which are two essential regulators of EMT and MET, in acquiring of hybrid E/M phenotype in HCC. METHODS: Expression of c-Met and lncRNA HOTAIR were defined in HCC cell lines and patient tissues through HCC progression. lncRNA HOTAIR was overexpressed in SNU-449 cells and its effects on c-Met signaling were analyzed. c-Met was overexpressed in SNU-398 cells and its effect on HOTAIR expression was analyzed. Biological significance of HOTAIR/c-Met interplay was defined in means of adhesion, proliferation, motility behavior, invasion, spheroid formation and metastatic ability. Effect of ectopic lncRNA HOTAIR expression on phenotype was defined with investigation of molecular epithelial and mesenchymal traits. RESULTS: In vitro and in vivo experiments verified the pivotal role of lncRNA HOTAIR in acquisition of hybrid E/M phenotype through modulating expression and activation of c-Met and its membrane co-localizing partner Caveolin-1, and membrane organization to cope with the rate limiting steps of metastasis such as survival in adhesion independent microenvironment, escaping from anoikis and resisting to fluidic shear stress (FSS) in HCC. CONCLUSIONS: Our work provides the first evidence suggesting a role for lncRNA HOTAIR in the modulation of c-Met to promote hybrid E/M phenotype. The balance between lncRNA HOTAIR and c-Met might be critical for cell fate decision and metastatic potential of HCC cells. Video Abstract.

The role of resistin on metabolic syndrome-induced erectile dysfunction and the possible therapeutic effect of Boldine.

BACKGROUND: Resistin is known as a potential mediator of obesity-associated insulin resistance. The high resistin level disrupts nitric oxide (NO)-mediated relaxation which is also important in erectile function. An antioxidant alkaloid, Boldine, is known as anti-diabetic and protects endothelial functions. OBJECTIVES: We aimed to investigate resistin expression in penile tissue in the presence of insulin resistance (IR) and the effect of Boldine treatment on erectile functions in the metabolic syndrome (MetS) rat model. MATERIALS AND METHODS: Wistar rats were randomly divided into three groups: Control, MetS, and boldine treated MetS group. MetS parameters were assessed by serum triglycerides (TG), uric acid (UA), glucose, insulin levels, HOMA index, and waist circumference (WC)/tibia length (TL) ratio. To evaluate erectile functions, intracavernous pressure (ICP)/mean arterial pressure (MAP) ratio was performed during cavernous nerve stimulation. Protein expressions of resistin, endothelial nitric oxide synthase (eNOS), p(S1177) eNOS, and insulin receptor-ß were evaluated by Western blotting. RESULTS: TG, glucose, insulin levels, weight, WC/TL ratio, HOMA index and resistin expression in penile tissue were significantly increased and ICP/MAP values, and p (S1177) eNOS expression in penile tissue were decreased in MetS group. Boldine treatment enhanced ICP/MAP values, insulin receptor-ß and p(S1177) eNOS expressions compared with the MetS group. DISCUSSION AND CONCLUSION: MetS caused a deterioration in erectile function accompanied by an increase in resistin expression and a reduction in eNOS enzyme activation in the rat penile tissues. Boldine treatment resulted in an improvement in erectile function, independent of resistin expression.

A Novel Function for KLF4 in Modulating the De-differentiation of EpCAM-/CD133- nonStem Cells into EpCAM+/CD133+ Liver Cancer Stem Cells in HCC Cell Line HuH7.

The complex and heterogeneous nature of hepatocellular carcinoma (HCC) hampers the identification of effective therapeutic strategies. Cancer stem cells (CSCs) represent a fraction of cells within tumors with the ability to self-renew and differentiate, and thus significantly contribute to the formation and maintenance of heterogeneous tumor mass. Increasing evidence indicates high plasticity in tumor cells, suggesting that non-CSCs could acquire stem cell properties through de-differentiation or reprogramming processes. In this paper, we reveal KLF4 as a transcription factor that can induce a CSC-like phenotype in non-CSCs through upregulating the EpCAM and E-CAD expression. Our studies indicated that KLF4 could directly bind to the promoter of EpCAM and increase the number of EpCAM+/CD133+ liver cancer stem cells (LCSCs) in the HuH7 HCC cell line. When KLF4 was overexpressed in EpCAM-/CD133- non-stem cells, the expressions of hepatic stem/progenitor cell genes such as CK19, EpCAM and LGR5 were significantly increased. KLF4 overexpressing non-stem cells exhibited greater cell viability upon sorafenib treatment, while the cell migration and invasion capabilities of these cells were suppressed. Importantly, we detected an increased membranous expression and colocalization of ß-CAT, E-CAD and EpCAM in the KLF4-overexpressing EpCAM-/CD133- non-stem cells, suggesting that this complex might be required for the cancer stem cell phenotype. Moreover, our in vivo xenograft studies demonstrated that with a KLF4 overexpression, EpCAM-/CD133- non-stem cells attained an in vivo tumor forming ability comparable to EpCAM+/CD133+ LCSCs, and the tumor specimens from KLF4-overexpressing xenografts had increased levels of both the KLF4 and EpCAM proteins. Additionally, we identified a correlation between the KLF4 and EpCAM protein expressions in human HCC tissues independent of the tumor stage and differentiation status. Collectively, our data suggest a novel function for KLF4 in modulating the de-differentiation of tumor cells and the induction of EpCAM+/CD133+ LCSCs in HuH7 HCC cells.

Pleiotropic Effects of Heparins: From Clinical Applications to Molecular Mechanisms in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a major health problem worldwide and most cases are incurable because of late presentation. It is the most common primary neoplasm of the liver and often arises in the context of a chronic liver disease that impairs coagulation. Portal vein thrombosis (PVT) is a common complication of HCC that is associated with a poor prognosis. Heparin derivatives are widely used in the management of venous thromboembolism (VTE). Among them low molecular weight heparin (LMWH) favorably influences the survival in patients with advanced cancer, including HCC. Due to their pleiotropic function, heparins affect tumorigenesis in many ways and may promote or hamper tumorigenic transformation depending on the cancer type and cancer stage along with their structural properties and concentration. Thus, their application as an antithrombotic along with the conventional therapy regime should be carefully planned to develop the best management strategies. In this review, we first will briefly review clinical applications of heparin derivatives in the management of cancer with a particular focus on HCC. We then summarize the state of knowledge whereby heparin can crosstalk with molecules playing a role in hepatocarcinogenesis. Lastly, we highlight new experimental and clinical research conducted with the aim of moving towards personalized therapy in cancer patients at risk of thromboembolism.

Thioredoxin interacting protein promotes invasion in hepatocellular carcinoma.

BACKGROUND: Considerable evidence suggests that oxidative stress plays an essential role in the progression of hepatocellular carcinoma (HCC). While acquired resistance to oxidative stress is the main driver of aggressive cell phenotype, the underlying mechanisms remain unknown. Here, we tested the hypothesis that elevated expression of Thioredoxin-interacting protein (TXNIP) is a main regulator of the aggressive phenotype in HCC. MATERIALS AND METHODS: To test this hypothesis, we measured TXNIP expression levels in 11 HCC cell lines by qPCR and western blotting. In addition, 80 pairs of HCC tissues and matched liver tissues of 73 cases, as well as 11 normal liver tissue samples were examined by immunohistochemistry. Besides, TXNIP expression levels were analyzed by Oncomine Platform in seven independent microarray datasets. Finally, the functional role of TXNIP in HCC was investigated in vitro and in vivo by silencing and overexpression studies. RESULTS: Our results show that TXNIP expression is significantly increased in HCC compared to non-tumor counterparts (p < 0.0001) as well as to normal (p < 0.0001) and cirrhotic (p < 0.0001) liver tissues. Moreover, stable overexpression of TXNIP in HCC cells (i) significantly increases ROS levels, (ii) induces EMT phenotype, (iii) increases motility, invasion and 3D branching tubulogenesis, (iv) decreases apoptosis, and (v) elevates in vivo metastasis in zebrafish embryos. Finally, we identify sinusoidal/stromal and cytoplasmic TXNIP staining patterns as risk factors for intrahepatic vascular invasion (p:0.0400). CONCLUSION: Our results strongly suggest that overexpression of TXNIP has a pivotal role in HCC progression by inducing cell survival, invasion, and metastasis.

A Comparison of the Effectiveness of Silibinin and Resveratrol in Preventing Alpha-Amanitin-Induced Hepatotoxicity.

Amanita phalloides species mushrooms containing alpha-amanitin (α-AMA) are responsible for the majority of fatal mushroom intoxications and can lead to severe poisonings resulting in hepatotoxicity and acute hepatic failure. Existing antidotes, such as silibinin, are not sufficiently effective in the prevention and/or resolution of α-AMA-induced hepatotoxicity. We investigated the effects of resveratrol on α-AMA-induced hepatotoxicity and compared with silibinin, a known antidote using in vivo and in vitro toxicity models. In the in vivo protocol, resveratrol (30 mg/kg) was given simultaneously with α-AMA (α-AMA + SR) or 12 (α-AMA + 12R) or 24 (α-AMA + 24R) hr after α-AMA administration. Silibinin (5 mg/kg) (α-AMA + Sil) and normal saline (α-AMA + NS) were given simultaneously with α-AMA. We found that liver transaminase levels in α-AMA + SR and α-AMA + 12R groups and histomorphologic injury score in the α-AMA + SR, α-AMA + 12R, α-AMA + 24R and α-AMA + Sil groups were significantly lower than that of the α-AMA + NS group. Resveratrol decreased mononuclear cell infiltration, necrosis and active caspase-3 immunopositivity in the liver. In the in vitro protocol, the effects of resveratrol and silibinin were evaluated in a reduction in cell viability induced by α-AMA in THLE-2 and THLE-3 hepatocytes. Neither resveratrol nor silibinin was found to be effective in increasing cell viability decreased by α-AMA + NS. As a conclusion, resveratrol was found to be effective in α-AMA-induced hepatotoxicity with its anti-inflammatory properties in in vivo conditions. It is a promising compound with the potential for use in the treatment of hepatotoxicity associated with Amanita phalloides type mushroom poisonings.

Role of Albumin in Growth Inhibition in Hepatocellular Carcinoma.

OBJECTIVE: Levels of serum albumin have recently emerged, together with C-reactive protein, as an important prognostic indicator for hepatocellular carcinoma (HCC). It has recently been reported that larger HCCs are associated with lower albumin levels. However, the albumin-mediated growth decrease has yet to be determined. METHODS: We examined a large HCC cohort and then by direct exposure of HCC cells in vitro, the relationship of albumin levels to HCC growth. RESULTS: We found that patients with lower albumin levels had significantly larger maximum tumor diameters, more portal vein thrombosis, more tumor multifocality, higher α-fetoprotein levels, and a lower survival than patients with higher albumin levels. Direct addition of exogenous albumin at physiological concentrations resulted in decreased growth in several HCC cell lines in vitro. We found a decrease in MAP kinase levels and in levels of Cdk2 and Cdk4, cyclinE, as well as in α-fetoprotein. CONCLUSION: These results indicate that in addition to its role as a monitor of systemic inflammation, albumin may have a direct role in HCC growth inhibition, either through modulation of α-fetoprotein or through its actions on growth-controlling kinases.

The regulatory role of heparin on c-Met signaling in hepatocellular carcinoma cells.

The role of heparin as an anticoagulant is well defined; however, its role in tumorigenesis and tumor progression is not clear yet. Some studies have shown that anticoagulant treatment in cancer patients improve overall survival, however, recent clinical trials have not shown a survival benefit in cancer patients receiving heparin treatment. In our previous studies we have shown the inhibitory effects of heparin on Hepatocyte Growth Factor (HGF)-induced invasion and migration in hepatocellular carcinoma (HCC) cells. In this study, we showed the differential effects of heparin on the behaviors of HCC cells based on the presence or absence of HGF. In the absence of HGF, heparin activated HGF/c-Met signaling and promoted motility and invasion in HCC cells. Heparin treatment led to c-Met receptor dimerization and activated c-Met signaling in an HGF independent manner. Heparin-induced c-Met activation increased migration and invasion through ERK1/2, early growth response factor 1 (EGR1) and Matrix Metalloproteinases (MMP) axis. Interestingly, heparin modestly decreased the proliferation of HCC cells by inhibiting activatory phosphorylation of Akt. The inhibition of c-Met signaling reversed heparin-induced increase in motility and invasion and, proliferation inhibition. Our study provides a new perspective into the role of heparin on c-Met signaling in HCC.

Effect of adipocyte-secreted factors on EpCAM+/CD133+ hepatic stem cell population.

Recent epidemiological studies have associated obesity with a variety of cancer types including HCC. However, the tumor initiating role of obesity in hepatocarcinogenesis is still unknown. The objective of this paper is to investigate the effect of adipocyte-secreted factors on EpCAM+/CD133+ cancer stem cells and to identify which factors play a role in modulating hepatic cancer stem cell behavior. Our results demonstrated that adipocyte-secreted factors affect motility and drug resistance of EpCAM+/CD133+ cells. When incubated with adipocyte conditioned media, EpCAM+/CD133+ cells exhibited augmented motility and reduced sorafenib-induced apoptosis. Using array-based system, we identified secretion of several cytokines such as IL6, IL8 and MCP1 by cultured adipocytes and activation of c-Met, STAT3 and ERK1/2 signaling pathways in EpCAM+/CD133+ cells incubated with adipocyte conditioned media. Treating EpCAM+/CD133+ cancer stem cells with IL6 receptor blocking antibody or c-Met inhibitor SU11274 both reduced the increase in motility; however SU11274 had greater effect on relieving protection from sorafenib-induced apoptosis. These results indicate that adipocyte-secreted factors might regulate cancer stem cell behavior through several signaling molecules including c-Met, STAT3 and ERK1/2 and inhibition of these signaling pathways offer novel strategies in targeting the effect of adipose-derived cytokines in cancer.