Chronic alcohol consumption is a well-known risk factor for liver disease. Progression of alcohol-induced liver disease (ALD) is a multifactorial process that involves a number of genetic, nutritional and environmental factors. Experimental and clinical studies increasingly show that oxidative damage induced by ethanol contribute in many ways to the pathogenesis of alcohol hepatoxicity. Oxidative stress appears to activate AMP-activated protein kinase (AMPK) signaling system, which has emerged in recent years as a kinase that controls the redox-state and mitochondrial function. This review focuses on the most recent insights concerning the activation of AMPK by reactive oxygen species (ROS), and describes recent evidences supporting the hypothesis that AMPK signaling pathways play an important role in promoting cell viability under conditions of oxidative stress, such as during alcohol exposure. We suggest that AMPK activation by ROS can promote cell survival by inducing autophagy, mitochondrial biogenesis and expression of genes involved in antioxidant defense. Hence, increased intracellular concentrations of ROS may represent a general mechanism for enhancement of AMPK-mediated cellular adaptation, including maintenance of redox homeostasis. On the other hand, AMPK inhibition in the liver by ethanol appears to play a key role in the development of steatosis induced by chronic alcohol consumption. Although more studies are needed to assess the functions of AMPK during oxidative stress, AMPK may be a possible therapeutic target in the particular case of alcohol-induced liver disease.

Cancer metastasis is a major cause for cancer-related death and inhibiting cancer metastasis is an alternative way to treat cancer. Several lines of reported evidence suggest that NADPH oxidase 4 (NOX4) is a potential target for intervention of cancer metastasis, as the reactive oxygen species (ROS) generated by this enzyme plays important roles in TGF-β signaling, an important inducer of cancer metastasis. Here we show (1) that TGF-β induces ROS production in breast cancer 4T1 cells and enhances cell migration and that the effect of TGF-β depends on NOX4 expression, (2) that knockdown of NOX4 via RNAi significantly decreases the migration ability of 4T1 cells in the presence or absence of TGF-β and significantly attenuates distant metastasis of 4T1 cells to lung and bone, (3) that Schisandrin B (Sch B), a naturally-occurring dibenzocyclooctadiene lignan with very low toxicity, is a novel NOX inhibitor and its IC50 toward NOX4 is 9.3μM, and (4) that Sch B suppresses TGF-β-induced and NOX4-associated ROS production in 4T1 cells and inhibits TGF-β-enhanced cell migration. Similar to NOX4 knockdown observed in this study, Sch B significantly attenuated 4T1 cells distant metastasis to lung and bone in our recently-published study. In line with previous reports, the study suggests that pharmacologically targeting NOX4 may be a potential approach to disrupt cancer metastasis.

Induction of proteins involved in drug metabolism and in drug delivery has a significant impact on drug-drug interactions and on the final therapeutic effects. Two antitumor acridine derivatives selected for present studies, C-1748 (9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine) and C-1305 (5-dimethylaminopropylamino-8-hydroxy-triazoloacridinone), expressed high and low susceptibility to metabolic transformations with liver microsomes, respectively. In the current study, we examined the influence of these compounds on cytochrome P450 3A4 (CYP3A4) and 2C9 (CYP2C9) enzymatic activity and gene expression in HepG2 tumor cells. Luminescence and HPLC examination, real-time RT-PCR and western blot analyses along with transfection of pregnane X receptor (PXR) siRNA and CYP3A4 reporter gene assays were applied. We found that both compounds strongly induced CYP3A4 and CYP2C9 activity and expression as well as expression of UGT1A1 and MDR1 in a concentration- and time-dependent manner. C-1748-mediated CYP3A4 and CYP2C9 mRNA induction equal to rifampicin occurred at extremely low concentrations (0.001 and 0.01μM), whereas 10μM C-1305 induced three-times higher CYP3A4 and CYP2C9 mRNA levels than rifampicin did. CYP3A4 and CYP2C9 expressions were shown to be PXR-dependent; however, neither compound influenced PXR expression. Thus, the observed drug-mediated induction of isoenzymes occurs on a PXR-mediated regulatory level. Furthermore, C-1748 and C-1305 were demonstrated to be selective PXR agonists. These effects are hypoxia-inhibited only in the case of C-1748, which is sensitive to P450 metabolism. In summary, PXR was found to be a new target of the studied compounds. Thus, possible combinations of these compounds with other therapeutics might lead to the PXR-dependent enzyme-mediated drug-drug interactions.

Activation of Toll-like receptor 4 (TLR4) triggers both innate and adaptive immunity. We previously identified a synthetic glycolipid, CCL-34, which can induce anticancer immunity in a TLR4-dependent manner. In the present study, we demonstrated the involvement of THO complex 1 (thoc1) in the CCL-34-induced anticancer mechanism. The expression of thoc1 was suppressed in bladder cancer cells (MBT-2) co-cultured with CCL-34-activated macrophages, whereas treatment with an iNOS inhibitor could restore the expression of thoc1. Direct treatment of MBT-2 cells with an NO donor also repressed thoc1 expression. Importantly, the thoc1-overexpressing MBT-2 cells (MBT/thoc1) exhibited greater resistance than the MBT-2 cells to cytotoxicity induced by the NO donor or the CCL-34-activated macrophages. In addition, treatments with CCL-34-activated macrophages or the NO donor resulted in the suppression of thoc1 promoter activity in MBT-2 cells, and mutations in the antioxidant response element (ARE) of the thoc1 promoter abolished the repression induced by these treatments. Furthermore, NO treatment increased the expression and nuclear localization of nuclear factor E2-related factor 2 (Nrf2) in MBT-2 cells. Overexpression of Nrf2 suppressed thoc1 promoter activity in an ARE-dependent manner, and knock-down of nrf2 reversed the suppression. Notably, Bcl-2 expression was suppressed in MBT-2 cells, but not in MBT-2/thoc1 cells, treated with CCL-34-activated macrophages or the NO donor. In summary, our results demonstrate that NO-mediated thoc1 downregulation, via Nrf2, is a key step in the cancer cell apoptosis induced by CCL-34-treated macrophages and that downregulated thoc1 could lead to Bcl-2 downregulation and subsequent cancer cell apoptosis.

Ginsenoside-Rg1 (Rg1) has been identified as potent proangiogenic agent, which plays an important role in wound healing promotion or treatment of ischemic injury. We previously reported that miR-214/eNOS pathway was involved in Rg1-induced angiogenesis. Following the same microRNA microarray profiling data, we proposed miR-15b would be another microRNA candidate involved in Rg1-induced angiogenesis. Using human umbilical vein endothelial cells (HUVECs), it was showed that Rg1 could reduce miR-15b expression rapidly and steadily, leading to a temporal induction of vascular endothelial growth factor receptor-2 (VEGFR-2). The in vitro motility and tubulogenesis via VEGFR-2 in Rg1-treated HUVECs were also demonstrated. Besides, the reduction of VEGFR-2 3'-UTR reporter activity by miR-15b in the luciferase reporter gene assay clearly indicated that miR-15b could affect the VEGFR-2 transcript through targeting its 3'-UTR region. Diminishing expression of endogenous miR-15b could increase VEGFR-2 expression and HUVECs migration and tubulogenesis; while over-expression of miR-15b was found to associate with the reduction of VEGFR-2 expression as well as cellular migration and tubulogenesis. In vivo, artificial increment of miR-15b by injecting Pre-miR-15b precursor into zebrafish embryos was also found to significantly suppress the subintestinal vessels formation. In conclusion, our results further demonstrated the involvement of microRNAs in Rg1-induced angiogenesis.

Because of their favourable safety profile and beneficial anti-inflammatory properties, the CysLT1 receptor antagonists (LTRA), montelukast, zafirlukast and pranlukast are approved for the treatment of asthma and are frequently prescribed as add-on therapeutics to reduce the amount of inhaled glucocorticoids and β2-agonists. There is evidence that some of these anti-inflammatory properties might be of a secondary nature and therefore, unrelated to the CysLT1 antagonism. Here, we show that LTRA inhibit PGE2 formation in cytokine- stimulated Hela and A549 carcinoma cells and in lipopolysaccharide (LPS)-stimulated human leukocyte preparations (IC50∼ 20μM). Neither expression of enzymes involved in PGE2 synthesis nor arachidonic acid release and COX activities were inhibited by the compounds. In contrast, mPGES-1 activity was suppressed at low micromolar levels (IC50 between 2-4μM). This suppression was specific for PGE2 synthesis, since PGD2 and PGI2 levels in LPS- stimulated leukocyte preparations were not negatively affected. PGF2α levels were concomitantly inhibited, probably due to its direct synthesis from PGE2. Several major conclusions can be drawn from this study: (A) Clinical trials investigating elevated doses of the compounds are helpful to confirm suppression of PGE2 synthesis in vivo; (B) Studies investigating the role of CysLTs in cell culture or animal models of inflammation and cancer have to be reassessed carefully, if higher doses of LTRA were applied or serum levels in cell culture assays were low; (C) LTRA may serve as new scaffolds for the development of potent, selective and well tolerated mPGES-1 inhibitors.

Resveratrol has many biological effects, including anti-tumor, antiviral activites, and vascular protection. Recent studies have suggested that resveratrol exert its antitumor effects through induction of autophagy by an unknown mechanism. In this study, we investigated the involvement of autophagy in resveratrol-induced cell death and its potential molecular mechanisms in A549 human lung adnocarcinoma cells. Resveratrol-induced growth inhibition and cell death was assessed by MTT and clonogenic assays. Activation of autophagy was characterized by monodansylcadaverine, transmission electron microscopy, and expression of autophagy marker protein LC3. Western blot analysis was used to study the cell signals involved in the mechanisms of autophagic death. Intracellular free calcium was detected with Fura2-AM staining. Our results indicated that resveratrol induced A549 cell death was mediated by autophagy. 3-methyladenine, an inhibitor of autophagy, suppressed resveratrol-induced autophagic cell death, and knockdown of autophagy-related genes Atg5 and Beclin-1 with siRNAs reversed RSV-induced cell death. Intracellular free calcium accumulated immediately following resveratrol addtion, which led to the activation of phospho-AMPK and phospho-Raptor, and a reduction in the amount of phospho-p70S6K. These effects could be reversed by the AMPK inhibitor compound C, and the calcium ion-chelating agent EGTA. In conclusion, we demonstrate that resveratrol-induced A549 cell death was mediated by the process of autophagic cell death via Ca(2+)/AMPK-mTOR signaling pathway.

The genus Mycobacterium includes non-pathogenic species such as M. smegmatis, and pathogenic species such as M. tuberculosis, the causative agent of tuberculosis (TB). Treatment of TB requires a lengthy regimen of several antibiotics, whose effectiveness has been compromised by the emergence of resistant strains. New antibiotics that can shorten the treatment course and those that have not been compromised by bacterial resistance are needed. In this study, we report that thiadiazolidinones, a relatively little-studied heterocyclic class, inhibit the activity of mycobacterial alanine racemase, an essential enzyme that converts L-alanine to D-alanine for peptidoglycan synthesis. Twelve members of the thiadiazolidinone family were evaluated for inhibition of M. tuberculosis and M. smegmatis alanine racemase activity and bacterial growth. Thiadiazolidinones inhibited M. tuberculosis and M. smegmatis alanine racemases to different extents with 50% inhibitory concentrations (IC50) ranging from <0.03 to 28μM and 23 to >150μM, respectively. The compounds also inhibited the growth of these bacteria, including multidrug resistant strains of M. tuberculosis. The minimal inhibitory concentrations (MIC) for drug-susceptible M. tuberculosis and M. smegmatis ranged from 6.25μg/ml to 100μg/ml, and from 1.56 to 6.25μg/ml for drug-resistant M. tuberculosis. The in vitro activities of thiadiazolidinones suggest that this family of compounds might represent starting points for medicinal chemistry efforts aimed at developing novel antimycobacterial agents.

Two synthetic LXR agonists were recently reported to inhibit collagen-induced platelet aggregation and thrombus formation in mice. We therefore studied whether also natural LXR agonists inhibit human platelet activation and whether they can be fluorescence-labelled preserving their bioactivity for LXR-related functional imaging. The natural LXR agonist 22(R)-OH-cholesterol -but not its stereoisomer 22(S)-OH-cholesterol- inhibited collagen induced platelet shape change and aggregation similar to synthetic LXR agonists in a concentration- and time-dependent manner. First exposure to 22(S)-OH-cholesterol prevented the subsequent inhibition of platelets by 22(R)-OH-cholesterol but not vice versa. 22(R)- and 22(S)-OH-cholesterol could be fluorescence-labelled as 22(R)- and 22(S)-OH-cholesteryl-3-duodecanoic-3-BODIPY esters with high yield and purity using the Steglich acylation. Labelled 22(R)- and 22(S)-OH-cholesterol esters retained the stereo specific bioactivity of their parent compounds, were metabolically stable and not cytotoxic at LXR agonistic concentrations. Live staining with labelled 22(R)- or 22(S)-OH-cholesterol esters demonstrated stereo specific inhibition of platelet spreading and chiral handling by macrophages that reflects LXR activation. The rapid inhibition of platelet reactivity to collagen by natural and pharmacologic LXR agonists offers a mechanism that could attenuate platelet activation by denuded plaques that expose collagen and LXR agonistic oxysterols. Stable fluorescence labelled 22(R)- and 22(S)-OH-cholesterol analogues with preserved stereo specific bioactivity and staining characteristics provide valuable tools for LXR-related functional imaging in pathophysiologic studies, for binding assays and for LXR-targeted drug development.