Supplementary Methods, Table S1, and Figure S1

Supplementary Methods from KD5170, a novel mercaptoketone-based histone deacetylase inhibitor that exhibits broad spectrum antitumor activity <i>in vitro</i> and <i>in vivo</i>

Supplementary Fig. S2 from KD5170, a novel mercaptoketone-based histone deacetylase inhibitor that exhibits broad spectrum antitumor activity <i>in vitro</i> and <i>in vivo</i>

Supplementary Table S1 from KD5170, a novel mercaptoketone-based histone deacetylase inhibitor that exhibits broad spectrum antitumor activity <i>in vitro</i> and <i>in vivo</i>

Supplementary Fig. S2 from KD5170, a novel mercaptoketone-based histone deacetylase inhibitor that exhibits broad spectrum antitumor activity in vitro and in vivo

microRNA-21 (miRNA) is one of the most abundant miRNAs in chronic liver injuries including alcoholic liver injury. Previous studies have demonstrated that miR-21 plays a role in inflammation in the liver and functions in hepatic stellate cells (HSCs), which reside in the perisinusoidal space between sinusoidal endothelial cells and hepatocytes and regulate sinusoidal circulation. HSCs integrate cytokine-mediated inflammatory responses in the sinusoids and relay them to the liver parenchyma. Here, we showed that the activation of Von Hippel-Lindau (VHL) expression, by miR-21 knockout in vivo and anti-miR-21 or VHL overexpression in vitro, suppressed the production of proinflammatory cytokines, such as interleukin (IL)-6, monocyte chemoattractant protein-1, and IL-1β, in human HSCs during alcoholic liver injury. Sequence and functional analyses confirmed that miR-21 directly targeted the 3'-untranslated region of VHL. Immunofluorescence and real-time PCR analysis revealed that miR-21 depletion blocked NF-κB activation in human HSCs both in cultured HSCs as well as HSCs isolated from alcohol-related liver disease mice liver by laser capture microdissection. We also showed that conditioned medium from anti-miR-21-transfected HSCs suppressed human monocyte-derived THP-1 cell migration. Taken together, our study indicates that depletion of miR-21 may downregulate cytokine production in HSCs and macrophage chemotaxis during alcoholic liver injury and that the targeting of miR-21 may have therapeutic potential for preventing the progression of alcoholic liver diseases. NEW & NOTEWORTHY This study demonstrates that silencing microRNA-21 can inhibit cytokine production and inflammatory responses in human hepatic stellate cells during alcoholic liver injury and that the targeting of microR-21 in hepatic stellate cells may have therapeutic potential for prevention and treatment of alcoholic liver diseases.

The let-7/Lin28 axis is associated with the regulation of key cellular regulatory genes known as microRNAs in various human disorders and cancer development. This study evaluated the role of the let-7/Lin28 axis in regulating a mesenchymal phenotype of hepatic stellate cells in alcoholic liver injury. We identified that ethanol feeding significantly down-regulated several members of the let-7 family in mouse liver, including let-7a and let-7b. Similarly, the treatment of human hepatic stellate cells (HSCs) with lipopolysaccharide (LPS) and transforming growth factor-β (TGF-β) significantly decreased the expressions of let-7a and let-7b. Conversely, overexpression of let-7a and let-7b suppressed the myofibroblastic activation of cultured human HSCs induced by LPS and TGF-β, as evidenced by repressed ACTA2 (α-actin 2), COL1A1 (collagen 1A1), TIMP1 (TIMP metallopeptidase inhibitor 1), and FN1 (fibronectin 1); this supports the notion that HSC activation is controlled by let-7. A combination of bioinformatics, dual-luciferase reporter assay, and Western blot analysis revealed that Lin28B and high-mobility group AT-hook (HMGA2) were the direct targets of let-7a and let-7b. Furthermore, Lin28B deficiency increased the expression of let-7a/let-7b as well as reduced HSC activation and liver fibrosis in mice with alcoholic liver injury. This feedback regulation of let-7 by Lin28B is verified in hepatic stellate cells isolated by laser capture microdissection from the model. The identification of the let-7/Lin28 axis as an important regulator of HSC activation as well as its upstream modulators and down-stream targets will provide insights into the involvement of altered microRNA expression in contributing to the pathogenesis of alcoholic liver fibrosis and novel therapeutic approaches for human alcoholic liver diseases.

Alcoholic liver disease remains a major cause of liver-related morbidity and mortality, which ranges from alcoholic steatohepatitis to fibrosis/cirrhosis and hepatocellular carcinoma, and the related mechanisms are understood poorly. In this study, we aimed to investigate the role of miR-34a in alcohol-induced cellular senescence and liver fibrosis. We found that hepatic miR-34a expression was upregulated in ethanol-fed mice and heavy drinkers with steatohepatitis compared with respective controls. Mice treated with miR-34a Vivo-Morpholino developed less severe liver fibrosis than wild-type mice after 5 weeks of ethanol feeding. Further mechanism exploration showed that inhibition of miR-34a increased cellular senescence of hepatic stellate cells (HSCs) in ethanol-fed mice, although it decreased senescence in total liver and hepatocytes, which was verified by the changes of senescence-associated β-galactosidase and gene expression. Furthermore, enhanced cellular senescence was observed in liver tissues from steatohepatitis patients compared with healthy controls. In addition, the expression of transforming growth factor-β1, drosophila mothers against decapentaplegic protein 2 (Smad2), and Smad3 was decreased after inhibition of miR-34a in ethanol-fed mice. Our in vitro experiments showed that silencing of miR-34a partially blocked activation of HSCs by lipopolysaccharide and enhanced senescence of HSCs. Furthermore, inhibition of miR-34a decreased lipopolysaccharide-induced fibrotic gene expression in cultured hepatocytes. In conclusion, our data suggest that miR-34a functions as a profibrotic factor that promotes alcohol-induced liver fibrosis by reducing HSC senescence and increasing the senescence of hepatocytes.

IL-12.The expression of iNOS mRNA was detected in M2b monocytes with CCL1 antisense ODN but not with scrambled ODN.Furthermore, M2b monocytes co-cultured with HepG2 cells have 70% of the tumorcidal activity after treatment of CCL1 antisense ODN, while these cells incubated with scrambled ODN did not have.Conclusions: M2bMϕ treated with CCL1 antisense ODN did not show any properties of M2Mϕ.These Mϕ were converted to M1Mϕ and have an ability of tumorcidal activity after cultivation with HepG2 cells.These results indicate that the generation of M1Mϕ from M2bMϕ isolated from patients with advance stages of HCC restore the host antitumor surveillance in these patients.

A S L D A b st ra ct s BGJ398, a pan-FGFR inhibitor currently being employed in human trials, resulted in a significant increase in cellular apoptosis. Tumor tissue from mice sacrificed 10 weeks after biliary oncogene transduction of AKT and YAP also demonstrated increased expression of FGFR 1-4. BGJ398 treatment resulted in a significant reduction in tumor burden and increase in tumor cell apoptosis as assessed by the TUNEL assay in our mouse model of CCA. In Conclusion, YAP is a critical oncogene in CCA and promotes biliary carcinogenesis, in part, via upregulation of FGFR. In a murine genetic model of CCA, the FGFR specific inhibitor BGJ398 significantly reduces tumor burden by inducing apoptosis. Thus, inhibition of FGFR represents a promising therapeutic approach in YAP-driven human CCA.

Diabetes mellitus is one of the most severe endocrine metabolic disorders in the world that has serious medical consequences with substantial impacts on the quality of life. Type 2 diabetes is one of the main causes of diabetic liver diseases with the most common being non-alcoholic fatty liver disease. Several factors that may explain the mechanisms related to pathological and functional changes of diabetic liver injury include: insulin resistance, oxidative stress and endoplasmic reticulum stress. The realization that these factors are important in hepatocyte damage and lack of donor livers has led to studies concentrating on the role of stem cells (SCs) in the prevention and treatment of liver injury. Possible avenues that the application of SCs may improve liver injury include but are not limited to: the ability to differentiate into pancreatic β-cells (insulin producing cells), the contribution for hepatocyte regeneration, regulation of lipogenesis, glucogenesis and anti-inflammatory actions. Once further studies are performed to explore the underlying protective mechanisms of SCs and the advantages and disadvantages of its application, there will be a greater understand of the mechanism and therapeutic potential. In this review, we summarize the findings regarding the role of SCs in diabetic liver diseases.

BGJ398, a pan-FGFR inhibitor currently being employed in human trials, resulted in a significant increase in cellular apoptosis.Tumor tissue from mice sacrificed 10 weeks after biliary oncogene transduction of AKT and YAP also demonstrated increased expression of FGFR 1-4.BGJ398 treatment resulted in a significant reduction in tumor burden and increase in tumor cell apoptosis as assessed by the TUNEL assay in our mouse model of CCA.In Conclusion, YAP is a critical oncogene in CCA and promotes biliary carcinogenesis, in part, via upregulation of FGFR.In a murine genetic model of CCA, the FGFR specific inhibitor BGJ398 significantly reduces tumor burden by inducing apoptosis.Thus, inhibition of FGFR represents a promising therapeutic approach in YAP-driven human CCA.

GDNF suppresses expression of genes associated with hepatic steatosisReal-time PCR analyses of gene expression in liver from WT and GDNF Tg mice maintained on RD or HFD for 12 weeks.Plotted are means + SEM.*, P<0.5; **, P<0.01; ***, P<0.001, relative to WT mice maintained on the RD.†, P<0.05; ‡, P<0.01; §, P<0.001, relative to GDNF Tg mice maintained on the HFD.n > 5 mice per group.GDNF enhances liver miR-122 and miR-107 levels miR-122 (A) and miR-107 (B) levels in liver from WT

We report the identification of KD5170, a potent mercaptoketone-based Class I and II-histone deacetylase inhibitor that demonstrates broad spectrum cytotoxic activity against a range of human tumor-derived cell lines. KD5170 exhibits robust and sustained histone H3 hyperacetylation in HCT-116 xenograft tumors following single oral or i.v. dose and inhibition of tumor growth following chronic dosing.