Yu Fu

PI3 kinase/Akt signaling mediates epithelial-mesenchymal transition in hypoxic hepatocellular carcinoma cells.

Hypoxia activates genetic programs that facilitate cell survival; however, in cancer, it may promote invasion and metastasis. Although the exact mechanisms driving hypoxia-induced invasion and metastasis remain elusive, we hypothesized that epithelial-mesenchymal transition (EMT) may play a major role. We investigated this in vitro by treating hepatocellular carcinoma cells under 1.0% O(2). After the hypoxia treatment, the cells exhibited some morphological changes including cell elongation, cytoskeletal rearrangement, and junctional disruption. Moreover, expression of the epithelia-specific marker E-cadherin was decreased and expression of the myofibroblast-specific marker vimentin was detected in the treated cells. Cell migration and ECM gel invasion were increased. These findings were consistent with events observed during EMT. Hypoxia-induced EMT is accompanied by increased phosphorylation, activation of Akt and the downstream signaling. Hypoxia-induced EMT was blocked by PI3K inhibitor LY294002. The results suggest that the PI3K/Akt-dependent signaling pathways serve to regulate hypoxia-induced EMT of hepatocellular carcinoma cells.

Are There Any Different Effects of Bifidobacterium, Lactobacillus and Streptococcus on Intestinal Sensation, Barrier Function and Intestinal Immunity in PI-IBS Mouse Model?

Background and Aims Research has increasingly suggested that gut flora plays an important role in the development of post-infectious irritable bowel syndrome (PI-IBS). Studies of the curative effect of probiotics for IBS have usually been positive but not always. However, the differences of treatment effects and mechanisms among probiotic stains, or mixture of them, are not clear. In this study, we compared the effects of different probiotics (Befidobacterium, Lactobacillus, Streptococcus or mixture of the three) on intestinal sensation, barrier function and intestinal immunity in PI-IBS mouse model. Methods PI-IBS model was induced by Trichinella spiralis infection in mice. Different probiotics were administered to mice after 8 weeks infection. Visceral sensitivity was measured by scores of abdominal withdrawal reflex (AWR) and the threshold intensity of colorectal distention. Colonic smooth muscle contractile response was assessed by contraction of the longitudinal muscle strips. Plasma diamine oxidase (DAO) and d-lactate were determined by an enzymatic spectrophotometry. Expression of tight junction proteins and cytokines in ileum were measured by Western blotting. Results Compared to control mice, PI-IBS mice treated either alone with Befidobacterium or Lactobacillus (but not Streptococcus), or the mixture of the three exhibited not only decreased AWR score and contractile response, but also reduced plasma DAO and D-lactate. These probiotic treatments also suppressed the expression of proinflammatory cytokine IL-6 and IL-17 and promoted the expression of major tight junction proteins claudin-1 and occludin. The mixture of the three probiotic strains performed better than the individual in up-regulating these tight junction proteins and suppressing IL-17 expression. Conclusions Bifidobacterium and Lactobacillus, but not Streptococcus, alleviated visceral hypersensitivity and recovered intestinal barrier function as well as inflammation in PI-IBS mouse model, which correlated with an increase of major tight junction proteins. In addition, Mixture of three species was indicated to be superior to a single one.

Hypoxia induced HMGB1 and mitochondrial DNA interactions mediate tumor growth in hepatocellular carcinoma through Toll-like receptor 9

BACKGROUND & AIMS The mechanisms of hypoxia-induced tumor growth remain unclear. Hypoxia induces intracellular translocation and release of a variety of damage associated molecular patterns (DAMPs) such as nuclear HMGB1 and mitochondrial DNA (mtDNA). In inflammation, Toll-like receptor (TLR)-9 activation by DNA-containing immune complexes has been shown to be mediated by HMGB1. We thus hypothesize that HMGB1 binds mtDNA in the cytoplasm of hypoxic tumor cells and promotes tumor growth through activating TLR9 signaling pathways. METHODS C57BL6 mice were injected with Hepa1-6 cancer cells. TLR9 and HMGB1 were inhibited using shRNA or direct antagonists. HuH7 and Hepa1-6 cancer cells were investigated in vitro to determine how the interaction of HMGB1 and mtDNA activates TLR9 signaling pathways. RESULTS During hypoxia, HMGB1 translocates from the nucleus to the cytosol and binds to mtDNA released from damaged mitochondria. This complex subsequently activates TLR9 signaling pathways to promote tumor cell proliferation. Loss of HMGB1 or mtDNA leads to a defect in TLR9 signaling pathways in response to hypoxia, resulting in decreased tumor cell proliferation. Also, the addition of HMGB1 and mtDNA leads to the activation of TLR9 and subsequent tumor cell proliferation. Moreover, TLR9 is overexpressed in both hypoxic tumor cells in vitro and in human hepatocellular cancer (HCC) specimens; and, injection in mice to knockdown either HMGB1 or TLR9 from HCC cells suppressed tumor growth in vivo. CONCLUSIONS Our data reveals a novel mechanism by which the interactions of HMGB1 and mtDNA activate TLR9 signaling during hypoxia to induce tumor growth.

AEG-1 promotes anoikis resistance and orientation chemotaxis in hepatocellular carcinoma cells.

Metastasis contributes to the poor prognosis of hepatocellular carcinoma (HCC). Anoikis resistance and orientation chemotaxis are two important and sequential events in tumor cell metastasis. The process of tumor metastasis is known to be regulated by AEG-1, an important oncogene that plays a critical role in tumor metastasis, though the effects of this oncogene on anoikis resistance and orientation chemotaxis in HCC cells are currently unknown. To directly assess the role of AEG-1 in these processes, we up-regulated AEG-1 expression via exogenous transfection in SMMC-7721 cells, which express low endogenous levels of AEG-1; and down-regulated AEG-1 expression via siRNA-mediated knockdown in MHCC-97H and HCC-LM3 cells, which express high endogenous levels of AEG-1. Our data directly demonstrate that AEG-1 promotes cell growth as assessed by cell proliferation/viability and cell cycle analysis. Furthermore, the prevention of anoikis by AEG-1 correlates with decreased activation of caspase-3. AEG-1-dependent anoikis resistance is activated via the PI3K/Akt pathway and is characterized by the regulation of Bcl-2 and Bad. The PI3K inhibitor LY294002 reverses the AEG-1 dependent effects on Akt phosphorylation, Bcl-2 expression and anoikis resistance. AEG-1 also promotes orientation chemotaxis of suspension-cultured cells towards supernatant from Human Pulmonary Microvascular Endothelial Cells (HPMECs). Our results show that AEG-1 activates the expression of the metastasis-associated chemokine receptor CXCR4, and that its ligand, CXCL12, is secreted by HPMECs. Furthermore, the CXCR4 antoagonist AMD3100 decreases AEG-1-induced orientation chemotaxis. These results define a pathway by which AEG-1 regulates anoikis resistance and orientation chemotaxis during HCC cell metastasis.

Comparison of non-invasive biomarkers faecal BAFF, calprotectin and FOBT in discriminating IBS from IBD and evaluation of intestinal inflammation

Faecal calprotectin and faecal occult blood test (FOBT) were widely used in the diagnosis and assessment of intestinal inflammation in inflammatory bowel disease (IBD). Recently we identified an excellent new biomarker B cell-activating factor (BAFF) for IBD. Here in this study we compared the efficacy of faecal BAFF, calprotectin and FOBT to find the “best non-invasive marker”. Results showed that for discriminating IBD from IBS, BAFF ≥227.3 pg/ml yield 84% sensitivity, 100% specificity, 100% positive predictive value (PPV) and 64% negative predictive value (NPV) while calprotectin ≥50 µg/g yield 76% sensitivity, 93% specificity, 97% PPV and 53% NPV. FOBT yield 65% sensitivity, 93% specificity, 97% PPV and 43% NPV. Combining BAFF with calprotectin tests yield 94% sensitivity, 93% specificity, 98% PPV, 81% NPV. Faecal BAFF level showed the stronger correlation with endoscopic inflammatory score as compared to calprotectin not only in UC (correlation coefficient [r] = 0.69, p < 0.0001 vs. r = 0.58, p < 0.0001), but also in CD (r = 0.58, p < 0.0001 vs. r = 0.52, p = 0.0003). Our results indicating that faecal BAFF is a promising non-invasive biomarker in IBD differential diagnosis and monitoring of intestinal inflammation.

Bifidobacterium longum and VSL#3® amelioration of TNBS-induced colitis associated with reduced HMGB1 and epithelial barrier impairment

ABSTRACT Probiotics are a beneficial treatment for inflammatory bowel disease (IBD). However, studies comparing the effects of similar doses of single and mixed probiotics on IBD are scarce. High mobility group box 1 (HMGB1) is an important proinflammatory mediator involved IBD development. The present study assessed fecal HMGB1 levels in IBD patients and compared the effects of similar doses of Bifidobacterium longum (Bif) versus VSL#3® on HMGB1 levels in 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐induced murine colitis. Twenty‐four mice were divided into four treatment groups (n = 6 per group): ethanol (control), TNBS, TNBS + Bif, and TNBS + VSL#3®. Bif and VSL#3® (4 × 109 CFU/dose) were administered daily by intragastric gavage, beginning 3 d before TNBS treatment, for a total of 7 d. Fecal HMGB1 levels were higher in both active IBD patients and TNBS‐induced colitis mice versus their respective controls. Both Bif and VSL#3® improved intestinal inflammation and fecal microbiota imbalance in TNBS‐induced colitis mice. Both treatments also reduced serum and fecal HMGB1 levels as well as increased expression of zonula occludins‐1, occludin, and claudin‐1 in colon tissues. In Caco‐2 cells, HMGB1 reduced transepithelial electrical resistance, zonula occludins‐1 protein expression, and increased paracellular permeability of FITC‐dextran; the opposite was found with both probiotic treatments. These findings suggest Bif and VSL#3® have similar beneficial effects on TNBS‐induced colitis, possibly through inhibition of HMGB1 release and subsequent HMGB1‐mediated gut barrier dysfunction. The present study provides novel insights into probiotic treatment of IBD. HighlightsBifidobacterium longum and VSL#3® have similar beneficial effects on TNBS‐induced colitis.Both probiotics reduce HMGB1 levels and epithelial barrier impairment in colitis mice.Both probiotics prevent HMGB1‐induced epithelial barrier impairment in caco‐2 cells.

Mo1639 Hypoxia Induces Caspase-1 Activation and Promotes Hepatocellular Carcinoma Invasiveness

Background: The homeobox gene Barx2 was recently identified as a regulator of ovarian and breast cancer; however, the expression level of BARX2 and its significance in hepatocellular carcinoma (HCC) remain unknown. Methods: Protein and mRNA expression levels of Barx2 were examined using Western blotting and real-time PCR respectively, in paired HCC tissue and matched adjacent noncancerous tissue from 12 patients. The expression levels of epithelial-mesenchymal transition (EMT) markers were also detected in relation to BARX2 expression. Lastly, immunohistochemistry for BARX2 was also performed on a tissue microarray containing 231 HCC tissue samples. Results: We observed that BARX2 expression was lower in HCC tissues compared to matching adjacent noncancerous tissue. The low expression level of BARX2 was significantly correlated with metrics of tumor size, tumor differentiation, clinical stage, metastasis and relapse. Furthermore, the patients with low BARX2 expression had adverse survival outcomes. Importantly, multivariate cox regression analysis revealed that low BARX2 expression was an independent marker for lower overall survival (p = 0.007). Moreover, a significant negative relationship was observed between the expression of BARX2 and markers of EMT. Conclusions: These findings provide evidence that the low expression level of BARX2 in HCC is significantly correlated with tumor metastasis, and that BARX2 may be an independent prognostic biomarker for patients with HCC. Key word: BARX2, Hepatocellular carcinoma, Prognosis, EMT, Metastasis Cox multivariate analyses of prognostic factors on overall survival

Sa1692 BVES Is Critical for Crowding Induced Cancer Cell Extrusion in Liver Cancer Metastasis

Background and aim: Angiotensin 2, an important byproduct of renin angiotensin system can lead to increased extracellular matrix formation and fibrosis via enhanced TGF β-1 production in many organ systems including liver. Angiotensin converting enzyme (ACE) coding gen polymorphisms can lead to different degree of ACE expressions and hence, various angiotensin 2 tissue levels. ACE gen polymorphisms have been reported as ACE I/ D. Especially, ACE gen D homozygotic patients produce more tissue levels of ACE and this may be a potential hazard for many organ systems. We aimed to investigate if there is any role of ACE I/D gen polymorphism in the degree of liver fibrosis due to various etiologies. Methods: We enrolled 411 patients with a histopathological diagnosis of liver fibrosis. There were 240 patients with the diagnosis of mild and moderate fibrosis (Ishaks stage; 1-3) and the rest, 171 patients had advanced liver fibrosis (Ishaks stage; 4-6). Polymerase chain reaction was used to determine the type of ACE I/D gen polymorphisms. We also studied serum ACE levels with enzymatic kinetic testing method on spectrometry. Results: There were 208 male and 203 female subjects with a mean age of 51.1±13.5 years, (range; 1880 years). Within the mild and moderate liver fibrosis group, the etiologies were chronic viral hepatitis B in 131, chronic viral hepatitis C in 59, non-alcoholic steatohepatitis in 46, autoimmune hepatitis in 1 and primary biliary cirrhosis in 3 patients. Within the advanced liver fibrosis group, the etiologies were chronic viral hepatitis B in 71, chronic viral hepatitis C in 61, non-alcoholic steatohepatitis in 24, alcoholic liver disease in 11 and autoimmune hepatitis in 4 patients. On statistical analysis, there was no difference between the two groups with regard to ACE genotypes. Moreover, we could not find any significant difference between the groups regarding serum levels of angiotensin converting enzyme. Conclusion: The ACE I/D gen polymorphism do not seem to contribute as a significant risk factor for advanced liver fibrosis.