Dual Pharmacological Targeting of HDACs and PDE5 Inhibits Liver Disease Progression in a Mouse Model of Biliary Inflammation and Fibrosis

Liver fibrosis, a common hallmark of chronic liver disease (CLD), is characterized by the accumulation of extracellular matrix secreted by activated hepatic fibroblasts and stellate cells (HSC). Fibrogenesis involves multiple cellular and molecular processes and is intimately linked to chronic hepatic inflammation. Importantly, it has been shown to promote the loss of liver function and liver carcinogenesis. No effective therapies for liver fibrosis are currently available. We examined the anti-fibrogenic potential of a new drug (CM414) that simultaneously inhibits histone deacetylases (HDACs), more precisely HDAC1, 2, and 3 (Class I) and HDAC6 (Class II) and stimulates the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway activity through phosphodiesterase 5 (PDE5) inhibition, two mechanisms independently involved in liver fibrosis. To this end, we treated Mdr2-KO mice, a clinically relevant model of liver inflammation and fibrosis, with our dual HDAC/PDE5 inhibitor CM414. We observed a decrease in the expression of fibrogenic markers and collagen deposition, together with a marked reduction in inflammation. No signs of hepatic or systemic toxicity were recorded. Mechanistic studies in cultured human HSC and cholangiocytes (LX2 and H69 cell lines, respectively) demonstrated that CM414 inhibited pro-fibrogenic and inflammatory responses, including those triggered by transforming growth factor β (TGFβ). Our study supports the notion that simultaneous targeting of pro-inflammatory and fibrogenic mechanisms controlled by HDACs and PDE5 with a single molecule, such as CM414, can be a new disease-modifying strateg

Epigenetic mechanisms and metabolic reprogramming in fibrogenesis: dual targeting of G9a and DNMT1 for the inhibition of liver fibrosis

OBJECTIVE: Hepatic stellate cells (HSC) transdifferentiation into myofibroblasts is central to fibrogenesis. Epigenetic mechanisms, including histone and DNA methylation, play a key role in this process. Concerted action between histone and DNA-mehyltransferases like G9a and DNMT1 is a common theme in gene expression regulation. We aimed to study the efficacy of CM272, a first-in-class dual and reversible G9a/DNMT1 inhibitor, in halting fibrogenesis. DESIGN: G9a and DNMT1 were analysed in cirrhotic human livers, mouse models of liver fibrosis and cultured mouse HSC. G9a and DNMT1 expression was knocked down or inhibited with CM272 in human HSC (hHSC), and transcriptomic responses to transforming growth factor-β1 (TGFβ1) were examined. Glycolytic metabolism and mitochondrial function were analysed with Seahorse-XF technology. Gene expression regulation was analysed by chromatin immunoprecipitation and methylation-specific PCR. Antifibrogenic activity and safety of CM272 were studied in mouse chronic CCl4 administration and bile duct ligation (BDL), and in human precision-cut liver slices (PCLSs) in a new bioreactor technology. RESULTS: G9a and DNMT1 were detected in stromal cells in areas of active fibrosis in human and mouse livers. G9a and DNMT1 expression was induced during mouse HSC activation, and TGFβ1 triggered their chromatin recruitment in hHSC. G9a/DNMT1 knockdown and CM272 inhibited TGFβ1 fibrogenic responses in hHSC. TGFβ1-mediated profibrogenic metabolic reprogramming was abrogated by CM272, which restored gluconeogenic gene expression and mitochondrial function through on-target epigenetic effects. CM272 inhibited fibrogenesis in mice and PCLSs without toxicity. CONCLUSIONS: Dual G9a/DNMT1 inhibition by compounds like CM272 may be a novel therapeutic strategy for treating liver fibrosis

Methylthioadenosine reprograms macrophage activation through adenosine receptor stimulation

Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA), which inhibits TNFα production following LPS stimulation. We found that MTA could block TNFα production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1β production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation. © 2014 Keyel et al

Novel Combination of Sorafenib and Celecoxib Provides Synergistic Anti-Proliferative and Pro-Apoptotic Effects in Human Liver Cancer Cells

Molecular targeted therapy has shown promise as a treatment for advanced hepatocellular carcinoma (HCC). Sorafenib, a multikinase inhibitor, recently received FDA approval for the treatment of advanced HCC. However, although sorafenib is well tolerated, concern for its safety has been expressed. Celecoxib (Celebrex®) is a selective cyclooxygenase-2 (COX-2) inhibitor which exhibits antitumor effects in human HCC cells. The present study examined the interaction between celecoxib and sorafenib in two human liver tumor cell lines HepG2 and Huh7. Our data showed that each inhibitor alone reduced cell growth and the combination of celecoxib with sorafenib synergistically inhibited cell growth and increased apoptosis. To better understand the molecular mechanisms underlying the synergistic antitumor activity of the combination, we investigated the expression profile of the combination-treated liver cancer cell lines using microarray analysis. Combination treatment significantly altered expression levels of 1,986 and 2,483 transcripts in HepG2 and Huh7 cells, respectively. Genes functionally involved in cell death, signal transduction and regulation of transcription were predominantly up-regulated, while genes implicated in metabolism, cell-cycle control and DNA replication and repair were mainly down-regulated upon treatment. However, combination-treated HCC cell lines displayed specificity in the expression and activity of crucial factors involved in hepatocarcinogenesis. The altered expression of some of these genes was confirmed by semi-quantitative and quantitative RT-PCR and by Western blotting. Many novel genes emerged from our transcriptomic analyses, and further functional analyses may determine whether these genes can serve as potential molecular targets for more effective anti-HCC strategies

Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis

Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liverenriched transcription factors (LETFs). TGFβ1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFβ1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH

Differential neuroprotective effects of 5'-deoxy-5'-methylthioadenosine

Background 5′-deoxy-5′-methylthioadenosine (MTA) is an endogenous compound produced through the metabolism of polyamines. The therapeutic potential of MTA has been assayed mainly in liver diseases and, more recently, in animal models of multiple sclerosis. The aim of this study was to determine the neuroprotective effect of this molecule in vitro and to assess whether MTA can cross the blood brain barrier (BBB) in order to also analyze its potential neuroprotective efficacy in vivo. Methods Neuroprotection was assessed in vitro using models of excitotoxicity in primary neurons, mixed astrocyte-neuron and primary oligodendrocyte cultures. The capacity of MTA to cross the BBB was measured in an artificial membrane assay and using an in vitro cell model. Finally, in vivo tests were performed in models of hypoxic brain damage, Parkinson's disease and epilepsy. Results MTA displays a wide array of neuroprotective activities against different insults in vitro. While the data from the two complementary approaches adopted indicate that MTA is likely to cross the BBB, the in vivo data showed that MTA may provide therapeutic benefits in specific circumstances. Whereas MTA reduced the neuronal cell death in pilocarpine-induced status epilepticus and the size of the lesion in global but not focal ischemic brain damage, it was ineffective in preserving dopaminergic neurons of the substantia nigra in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyr​idine(MPTP)-mice model. However, in this model of Parkinson's disease the combined administration of MTA and an A2A adenosine receptor antagonist did produce significant neuroprotection in this brain region. Conclusion MTA may potentially offer therapeutic neuroprotection

Dual targeting of histone methyltransferase G9a and DNA-methyltransferase 1 for the treatment of experimental hepatocellular carcinoma

Epigenetic modifications like DNA and histone methylation functionally cooperate fostering tumor growth, including that of hepatocellular carcinoma (HCC). Pharmacological targeting of these mechanisms may open new therapeutic avenues. We aimed to determine the therapeutic efficacy and potential mechanism of action of our new dual G9a histone-methyltransferase and DNA-methytransferase 1 (DNMT1) inhibitor in human HCC cells and their crosstalk with fibrogenic cells. The expression of G9a and DNMT1, along with that of their molecular adaptor ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was measured in human HCCs (n=268), peritumoral tissues (n=154) and HCC cell lines (n=32). We evaluated the effect of individual and combined inhibition of G9a and DNMT1 on HCC cells growth by pharmacological and genetic approaches. The activity of our lead compound, CM-272, was examined in HCC cells under normoxia and hypoxia, human hepatic stellate cells and LX2 cells, and xenograft tumors formed by HCC or combined HCC+LX2 cells. We found a significant and correlative overexpression of G9a, DNMT1 and UHRF1 in HCCs in association with poor prognosis. Independent G9a and DNMT1 pharmacological targeting synergistically inhibited HCC cell growth. CM-272 potently reduced HCC and LX2 cells proliferation and quelled tumor growth, particularly in HCC+LX2 xenografts. Mechanistically, CM-272 inhibited the metabolic adaptation of HCC cells to hypoxia, and induced a differentiated phenotype in HCC and fibrogenic cells. The expression of the metabolic tumor suppressor gene fructose-1,6-bisphosphatase (FBP1), epigenetically repressed in HCC, was restored by CM-272. CONCLUSION: Combined targeting of G9a/DNMT1 with compounds like CM-272 is a promising strategy for HCC treatment. Our findings also underscore the potential of differentiation therapy in HCC. This article is protected by copyright. All rights reserved