SRT1720 Alleviates ANIT-Induced Cholestasis in a Mouse Model.

Intrahepatic cholestasis is a kind of clinical syndrome along with hepatotoxicity which caused by intrahepatic and systemic accumulations of bile acid. There are several crucial generating factors of the pathogenesis of cholestasis, such as inflammation, dysregulation of bile acid transporters and oxidative stress. SIRT1 is regarded as a class III histone deacetylase (HDAC). According to a set of researches, SIRT1 is one of the most important factors which can regulate the hepatic bile acid metabolism. SRT1720 is a kind of activator of SIRT1 which is 1000 times more potent than resveratrol, and this paper is aimed to study its protective influence on hepatotoxicity and cholestasis induced by alpha-naphthylisothiocyanate (ANIT) in mice. The findings revealed that SRT1720 treatment increased FXR and Nrf2 gene expressions to shield against hepatotoxicity and cholestasis induced by ANIT. The mRNA levels of hepatic bile acid transporters were also altered by SRT1720. Furthermore, SRT1720 enhanced the antioxidative system by increasing Nrf2, SOD, GCLc, GCLm, Nqo1, and HO-1 gene expressions. In conclusion, a protective influence could be provided by SRT1720 to cure ANIT-induced hepatotoxicity and cholestasis, which was partly through FXR and Nrf2 activations. These results indicated that SIRT1 could be regarded as a therapeutic target to cure the cholestasis.

Role of AMP-activated protein kinase α1 in 17α-ethinylestradiol-induced cholestasis in rats.

Estrogen-induced cholestasis occurs in many women who are susceptible due to pregnancy or hormone replacement therapy for postmenopausal syndrome. 17α-Ethinylestradiol (EE), as a synthetic estrogen, has been widely used to study the underlying mechanisms of estrogen-induced cholestasis. Recent studies have also reported that liver kinase B1 (LKB1)-mediated activation of AMP-activated protein kinase (AMPK) plays a critical role in the regulation of canalicular network formation. However, the role of AMPK in EE-induced cholestasis remains to be determined. In this study, the effects of EE (1-100 µM) on AMPK activation and the expression of farnesoid X receptor (FXR) and hepatic bile acid transporters were examined in in vitro using 3D-cultured rat primary hepatocytes and in in vivo using rat cholestasis models. We also used specific chemical agonist and antagonist of AMPK, AMPK subunit-specific antibodies and lentiviral shRNAs for AMPKα1 and AMPKα2 to delineate the role of AMPK in EE-induced cholestasis and potential cellular mechanisms. We found that EE-induced phosphorylation of AMPKα1 via extracellular signal-regulated kinases-LKB1-mediated signaling pathways and subsequent nuclear translocation accounted for the down-regulation of FXR and bile acid transporters and disruption of bile acid homeostasis. Inhibition of AMPK activation using an AMPK antagonist Compound C (2 µM) or down-regulation of AMPKα1 using gene-specific shRNA attenuated EE-induced cholestasis both in in vitro and in in vivo. In conclusion, these results revealed that activation of cAMP-ERK-LKB1-AMPKα1 signaling pathway plays a critical role in EE-mediated dysregulation of the expression of FXR and bile acid transporters. AMPKα1 may represent an important therapeutic target for estrogen-induced cholestasis.

Protective effects of SRT1720 via the HNF1α/FXR signalling pathway and anti-inflammatory mechanisms in mice with estrogen-induced cholestatic liver injury.

Sirtuin 1 (SIRT1) is the most conserved mammalian NAD+-dependent protein deacetylase and is a member of the silent information regulator 2 (Sir2) families of proteins (also known as Sirtuins). In the liver, hepatic SIRT1 modulates bile acid metabolism through the regulation of farnesoid X receptor (FXR) expression. FXR is one of the most important nuclear receptors involved in the regulation of bile acid metabolism. SIRT1 modulates the FXR expression at multiple levels, including direct deacetylation of this transcription factor and transcriptional regulation through hepatocyte nuclear factor 1α (HNF1α). Therefore, hepatic SIRT1 is a vital regulator of the HNF1α/FXR signalling pathway and hepatic bile acid metabolism. However, whether SIRT1 is a suitable therapeutic target for the treatment of cholestasis is unknown. In the present study, we examined the protective effect of SRT1720, which is a specific activator of SIRT1, against 17α-ethinylestradiol (EE)-induced cholestasis in mice. Our data demonstrated that SRT1720 significantly prevented EE-induced changes in the serum levels of total bile acids (TBA), total bilirubin (TBIL), γ-glutamyltranspeptidase (γ-GGT) and alkaline phosphatase (ALP). SRT1720 also relieved EE-induced liver pathological injuries as indicated by haematoxylin and eosin (H&E) staining. SRT1720 treatment protected against EE-induced liver injury through the HNF1α/FXR signalling pathway, which up-regulated the expression of hepatic efflux transporter (Bsep and Mrp2) and hepatic uptake transporters (Ntcp and Oatp1b2). Moreover, SRT1720 significantly inhibited the TNF-α and IL-6 levels induced by EE. These findings indicate that SRT1720 exerts a dose-dependent protective effect on EE-induced cholestatic liver injury in mice and that the mechanism underlying this activity is related to the activation of the HNF1α/FXR signalling pathway and anti-inflammatory mechanisms.

Alpha-naphthylisothiocyanate impairs bile acid homeostasis through AMPK-FXR pathways in rat primary hepatocytes.

Alpha-naphthylisothiocyanate (ANIT) is widely used to induce cholestasis in basic researches. Although direct damage induced by ANIT to bile duct epithelial cells has been documented in previous studies, few works investigated ANIT-induced effects on hepatocytes. Our previous study indicated that activated AMP-activated protein kinase (AMPK) inhibited farnesoid X receptor (FXR) expression and further participated in the pathogenesis of estrogen-induced cholestasis. However, whether ANIT has effects on bile acid homeostasis in hepatocytes, and the role of AMPK-FXR pathway played in these effects remain unclear. In this study, our results showed that ANIT induced intracellular bile acid accumulation without obvious cellular toxicity in sandwich cultured rat primary hepatocytes (SCRHs), accompanied with significant decreased expression of FXR and bile acid transporters. AMPK activation via ERK1/2-LKB1 pathway was critical for ANIT-induced effects on hepatocytes. Compound C, specific AMPK inhibitor, blocked ANIT-regulated gene expression, decreased bile acid accumulation and recovered bile canalicular structure both in vitro and in vivo. Furthermore, the expression of A1 adenosine receptor (A1AR), a potential cholestatic target, was relatively low in hepatocytes compared with expression in rat whole livers. Consistent with these findings, DPCPX, a classic antagonist of A1AR, had no effect on ANIT-induced hepatocytes injury. In summary, our results indicate that AMPK-FXR signaling is critical for ANIT-induced toxic effects on hepatocytes, provide new insights into the pathogenesis of ANIT-induced cholestasis, and suggest AMPK-FXR pathway as a potential therapeutic target for cholestasis.

Optimization of 2-(3-(arylalkyl amino carbonyl) phenyl)-3-(2-methoxyphenyl)-4-thiazolidinone derivatives as potent antitumor growth and metastasis agents.

A series of 2,3-diaryl-4-thiazolidinone derivatives were synthesized and evaluated for their antiproliferative properties against two well-known cancer cell lines (A549 as human lung cancer and MDA-MB-231 as human breast cancer). Structure activity relationship (SAR) analysis resulted in the discovery of 2-(3-(arylalkyl amino carbonyl)phenyl)-3-(2-methoxy-phenyl)-4-thiazolidinone derivatives with high potent inhibitory effects on the proliferation of both cancer cell lines. Furthermore, several compounds with potent antiproliferative activities displayed excellent inhibitory activities on migration with an IC50 of about 0.05 µM on MDA-MB-231 cells in two different migration assays. In particular, compound 39 was indicated to suppress tumor growth and metastasis as well as promote survival rate. Intriguingly, this series of analogs have been indicated to inhibit tumor cell proliferation through inducing cell cycle arrest. These results suggested that the new series of 2-(3-(arylalkyl amino carbonyl)phenyl)-3-(2-methoxyphenyl)-4-thiazolidinone derivatives could be regarded and developed as novel highly potential anticancer agents in the future.

Inhibition of breast cancer metastases by a novel inhibitor of TGFß receptor 1.

BACKGROUND: Transforming growth factor beta (TGFß), which is implicated in metastasis to various organs in breast cancer, is a potential target for new antitumor metastasis drugs. METHODS: To identify specific inhibitors of TGFß receptor 1 (TGFßR1) in breast cancer metastasis, a virtual library of more than 400000 different compounds was screened by molecular docking modeling and confirmed with Smad-binding element luciferase and TGFßR1 kinase assays. Affymetrix GeneChip expression analysis of mRNA levels and real-time polymerase chain reaction were performed to determine expression changes of TGFß-mediated, metastasis-associated genes in breast cancer cells after treatment with the small-molecule inhibitor YR-290. YR-290 was also examined for its effects on breast cancer migration, invasion, and metastasis using transwell and epithelial-to-mesenchymal transition (EMT) assays in vitro and three different mouse (BALB/c and NU/NU nude) models (n = 10 per group). Kaplan-Meier analyses were used to assess survival. All statistical tests were two-sided. RESULTS: YR-290 interacted with the kinase domain of TGFßR1, abrogated kinase activity (half maximal inhibitory concentration = 137nM, 95% confidence interval = 126.4 to 147.6nM) and inhibited the TGFß-mediated downstream signaling pathway and metastasis-associated genes in breast cancer cells. YR-290 inhibited TGFß-modulated breast cancer cell migration and invasion. In tumor metastasis mouse models, YR-290 almost completely blocked cancer metastasis. Numbers of lung tumor nodules of mice treated with 1mg/kg and 5mg/kg YR-290 were reduced by 74.93% (95% confidence interval = 61.45% to 88.41%) and 94.93% (95% confidence interval = 82.13% to 100%), respectively, compared with control mice. Treatment with YR-290 also statistically significantly prolonged the survival of tumor-bearing mice. CONCLUSIONS: YR-290 is a novel inhibitor of tumor metastasis that works by blocking TGFß signaling pathways.