Specific Inhibition of the Redox Activity of Ape1/Ref-1 by E3330 Blocks Tnf-A-Induced Activation of Il-8 Production in Liver Cancer Cell Lines

APE1/Ref-1 is a main regulator of cellular response to oxidative stress via DNA-repair function and co-activating activity on the NF-κB transcription factor. APE1 is central in controlling the oxidative stress-based inflammatory processes through modulation of cytokines expression and its overexpression is responsible for the onset of chemoresistance in different tumors including hepatic cancer. We examined the functional role of APE1 overexpression during hepatic cell damage related to fatty acid accumulation and the role of the redox function of APE1 in the inflammatory process. HepG2 cells were stably transfected with functional and non-functional APE1 encoding plasmids and the protective effect of APE1 overexpression toward genotoxic compounds or FAs accumulation, was tested. JHH6 cells were stimulated with TNF-α in the presence or absence of E3330, an APE1 redox inhibitor. IL-8 promoter activity was assessed by a luciferase reporter assay, gene expression by Real-Time PCR and cytokines (IL-6, IL-8, IL-12) levels measured by ELISA. APE1 over-expression did not prevent cytotoxicity induced by lipid accumulation. E3330 treatment prevented the functional activation of NF-κB via the alteration of APE1 subcellular trafficking and reduced IL-6 and IL-8 expression induced by TNF-α and FAs accumulation through blockage of the redox-mediated activation of NF-κB. APE1 overexpression observed in hepatic cancer cells may reflect an adaptive response to cell damage and may be responsible for further cell resistance to chemotherapy and for the onset of inflammatory response. The efficacy of the inhibition of APE1 redox activity in blocking TNF-α and FAs induced inflammatory response opens new perspectives for treatment of inflammatory-based liver diseases

Impairment of enzymatic antioxidant defenses is associated with bilirubin-induced neuronal cell death in the cerebellum of Ugt1 KO mice

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration. \ua9 2015 Macmillan Publishers Limited All rights reserved

Correction: Specific Inhibition of the Redox Activity of Ape1/Ref-1 by E3330 Blocks Tnf-Α-Induced Activation of Il-8 Production in Liver Cancer Cell Lines.

APE1/Ref-1 is a main regulator of cellular response to oxidative stress via DNA-repair function and co-activating activity on the NF-\u3baB transcription factor. APE1 is central in controlling the oxidative stress-based inflammatory processes through modulation of cytokines expression and its overexpression is responsible for the onset of chemoresistance in different tumors including hepatic cancer. We examined the functional role of APE1 overexpression during hepatic cell damage related to fatty acid accumulation and the role of the redox function of APE1 in the inflammatory process. HepG2 cells were stably transfected with functional and non-functional APE1 encoding plasmids and the protective effect of APE1 overexpression toward genotoxic compounds or FAs accumulation, was tested. JHH6 cells were stimulated with TNF-\u3b1 in the presence or absence of E3330, an APE1 redox inhibitor. IL-8 promoter activity was assessed by a luciferase reporter assay, gene expression by Real-Time PCR and cytokines (IL-6, IL-8, IL-12) levels measured by ELISA. APE1 over-expression did not prevent cytotoxicity induced by lipid accumulation. E3330 treatment prevented the functional activation of NF-\u3baB via the alteration of APE1 subcellular trafficking and reduced IL-6 and IL-8 expression induced by TNF-\u3b1 and FAs accumulation through blockage of the redox-mediated activation of NF-\u3baB. APE1 overexpression observed in hepatic cancer cells may reflect an adaptive response to cell damage and may be responsible for further cell resistance to chemotherapy and for the onset of inflammatory response. The efficacy of the inhibition of APE1 redox activity in blocking TNF-\u3b1 and FAs induced inflammatory response opens new perspectives for treatment of inflammatory-based liver diseases

Modern strategies to identify new molecular targets for the treatment of liver diseases: the promising role of proteomics and redox proteomics investigations

Oxidative stress, due to an imbalance between the generation of ROS and the antioxidant defense capacity of the cell, is a major pathogenetic event occurring in several liver diseases, ranging from metabolic to proliferative. Main sources of ROS are represented by mitochondria and cytochrome P450 enzymes in the hepatocytes, K\ufcppfer cells, and neutrophils. Oxidative stress affects major cellular components including lipids, DNA, and proteins. Through modulation of protein structure/function, ROS can influence gene expression profile by affecting intracellular signal transduction pathways. While several enzymatic and nonenzymatic markers of chronic oxidative stress are well known in liver, early protein targets of oxidative injury are yet poorly defined. Identification of these biomarkers will enable early detection of liver diseases and will allow monitoring the degree of liver damage, the response to pharmacological therapies, and the development of new therapeutic approaches. In the era of molecular medicine, new proteomic methodologies promise to establish a relationship between pathological hallmarks of the disease and protein structural/functional modifications, thus allowing a better understanding and a more rational therapy on liver disorders. Purpose of this review is to critically analyze the application of proteomic and redox proteomic approaches to the study of oxidative stress-linked liver disease

Expression of ectopic APE1^WT protein confers cells protection to genotoxic damage but not to FAs-induced cytotoxicity.

<p><b>Panel A: <b><i>Western Blot analysis of total cell extracts from HepG2 stable cell clones.</i></b></b> Stably transfected clones have been obtained as described in Materials and Methods section. Twelve micrograms of protein extracts were separated by 12% SDS-PAGE and then transferred onto a NC membrane. The membrane was immunoblotted with anti-APE1 antibody. The values reported above refer to the ratios of the band intensities between ectopically-expressed and endogenous APE1, as measured by densitometry. The ectopic Flag-tagged recombinant protein both in the APE1^WT and the APE1^NΔ33 cell clones is expressed to a similar extent at different days of cell culture. a: clones after the sixth <i>in vitro</i> passage; b: clones after the tenth <i>in vitro</i> passage. <b>Panel B: </b><b><i>APE1 localization within HepG2 cell clones.</i></b> HepG2 cell clones were fixed and immunostained for Histone H3 (red) and for Flag-tagged APE1 with an α-Flag antibody (green). Merged images (yellow) show the localization of APE1^WT within cell nuclei and colocalization with Histone H3. The APE1^NΔ33 deletion mutant colocalizes with Histone H3 within cell nuclei but show also cytoplasmic positivity. <b>Panel C: </b><b><i>Growth curve of HepG2 cell clones.</i></b> HepG2 empty clone, APE1^WT clone and APE1^NΔ33 clone cells were seeded into each well of a 24-well plate and cell growth was monitored every two or three days as indicated, by trypan blue exclusion. APE1^NΔ33 cells (triangle) grew more slowly than the APE1^WT (square) and the empty clones (dot). <b>Panel D: </b><b><i>Cell growth by MTT colorimetric assay.</i></b> Thirty thousand cells of the control (empty clone), APE1^WT and APE1^NΔ33 clones were seeded in quadruplicate wells in a 96-well microculture plate. Cell viability was measured after 72 h of culture. MTT assay also revealed that APE1^NΔ33 cell clone has a lower level of proliferation than empty and APE1^WT clones. Data, expressed as the percentage of cell viability with respect to the control empty clone, are the means ± SD of three independent experiments. <b>Panel E</b><b><i>: Effect of MMS on viability of HepG2 cell clones.</i></b> HepG2 cell clones were treated for 2 h with 1.6 or 2.0 mM MMS and cell viability was estimated by the MTT colorimetric assay. When cells were treated with 2.0 mM MMS, cell viability was significantly decreased in the APE1^NΔ33 cell clone but not in the APE1^WT clone, suggesting that the ectopic expression of APE1^WT protects cells against MMS-induced citotoxicity. Data shown are the means ± SD of three independent experiments. <b>Panel F: </b><b><i>Effect of H₂O₂ on viability of HepG2 cell clones</i></b>. HepG2 cell clones were treated with 2.5 mM hydrogen peroxide for 1 h, then cell viability was determined by the MTT assay. After exposure to 2.5 mM H₂O₂ no significant decrease in cell viability was detected for APE1^WT clone compared to empty and APE1^NΔ33 cell clones. The histograms show the means ± SD of three independent experiments. <b>Panel G: </b><b><i>Quantification of γH2A.X foci in response to etoposide treatment.</i></b> The γH2A.X foci were detected using immunohistochemistry and quantified by image analysis. Cells were treated with etoposide (50 µM) for 1 h and the number of double strand DNA breaks (DSBs) was determined at different times of release (0 h, 15 h and 24 h). γH2A.X foci levels remain significantly higher than controls at 15 h and 24 h in etoposide treated APE1^NΔ33 cell clone (triangle). DNA damage was weaker for APE1^WT clone (square) than empty (dot) and APE1^NΔ33 cell clones, suggesting a protective role of APE1 overexpression in DNA repair.</p

E3330 treatment specifically inhibits TNF-α- and FAs-induced IL-8 endogenous gene expression.

<p><b>Panel A: <b><i>Effect of E3330 treatment on TNF-α-induced IL-8 mRNA expression.</i></b></b> JHH6 cells were pre-treated with 100 µM E3330 or with vehicle (DMSO) as a control, for 4 h prior to treatment with 2000 U/ml TNF-α for 2 h. IL-8 mRNA expression was determined by Real-Time PCR. The histograms show the detected levels of IL-8 mRNA normalized to control (DMSO) and normalized to two different housekeeping genes (GAPDH and HPRT). IL-8 mRNA expression was increased by TNF-α treatment when compared with control cells and pre-treatment with 100 µM E3330 decreased TNF-α-induced IL-8 mRNA. Data reported are the means ± SD of three independent experiments. <b>Panel B: </b><b><i>Effect of E3330 treatment on TNF-α-induced IL-8 protein production.</i></b> JHH6 cells were pre-treated with 100 µM E3330 or with vehicle (DMSO) as control, for 4 h prior to treatment with 2000 U/ml TNF-α for 2 h. The supernatants of the same cells analyzed for mRNA were assayed for IL-8 protein by FlowCytomix assay kit. TNF-α stimulated the secretion of IL-8 protein by JHH6 cells and the pre-treatment with 100 µM E3330 significantly suppressed TNF-α-induced IL-8 protein release. Data reported are the means ± SD of three independent experiments. <b>Panel C: </b><b><i>Effect of E3330 treatment on TNF-α-induced IL-6 mRNA expression in JHH6 cells.</i></b> JHH6 cells were pre-treated with 100 µM E3330 or with vehicle (DMSO) as a control, for 4 h prior to treatment with 2000 U/ml TNF-α for 3 h. IL-6 mRNA expression was determined by Real-Time PCR. The histograms show the detected levels of IL-6 mRNA normalized to control (DMSO) and normalized to two different housekeeping genes (GAPDH and HPRT). IL-6 mRNA expression was increased by TNF-α treatment and the pre-treatment with 100 µM E3330 significantly decreased TNF-α-induced IL-6 mRNA. Data reported are the means ± SD of three independent experiments. <b>Panel D: </b><b><i>Effect of E3330 treatment on TNF-α-induced IL-12 protein production in JHH6 cells.</i></b> The same supernatants analyzed for IL-8 protein were assayed for IL-12 protein by FlowCytomix assay kit. E3330 does not affect TNF-α-induced IL-12 activation suggesting a specific effect of E3330 on IL-8 gene expression. Data reported are the means ± SD of three independent experiments. <b>Panel E: </b><b><i>Effect of FAs overload on IL-8 gene expression.</i></b> JHH6 cells were treated for different times with 800 µM of mixture of oleate/palmitate (2∶1 ratio) and IL-8 mRNA expression was determined by Real-Time PCR. The histograms show the detected levels of IL-8 mRNA normalized to control (DMSO) and normalized to two different housekeeping genes (GAPDH and HPRT). IL-8 mRNA expression was increased by FAs treatment when compared with control cells in a time-dependent manner. Data reported are the means ± SD of three independent experiments. <b>Panel F: </b><b><i>Effect of E3330 treatment on FAs-induced IL-8 mRNA expression.</i></b> JHH6 cells were pre-treated with 100 µM E3330 or with vehicle (DMSO) as a control, for 4 h prior to treatment with 800 µM of mixture of oleate/palmitate (2∶1 ratio) for 3 h. IL-8 mRNA expression was determined by Real-Time PCR. The histograms show the detected levels of IL-8 mRNA normalized to control (DMSO) and normalized to two different housekeeping genes (GAPDH and HPRT). IL-8 mRNA expression was increased by FAs treatment and pre-treatment with 100 µM E3330 decreased FAs-induced IL-8 mRNA. Data reported are the means ± SD of three independent experiments.</p

NF-κB transcription factor regulates IL-8 promoter activity in JHH6 cells and E3330 treatment inhibits TNF-α-induced promoter activation.

<p><b>Panel A: <b><i>Western Blotting analysis of total cell extracts from human hepatocellular carcinoma cell lines.</i></b></b> A representative Western blot analysis for the evaluation of APE1 expression in Huh-7, HepG2 and JHH6 cell lines is shown in the upper panel. β-Tubulin was always measured as loading control and was used for data normalization. The lower panel shows expression levels of the protein obtained after densitometric analysis of the bands. An almost two-fold increase was observed in the content of APE1/Ref-1 in JHH6 cell line compared to Huh-7. Values were reported as histograms of the ratio between APE/Ref-1 band intensities and β-Tubulin. Data are the means ± SD of three independent experiments. <b>Panel B: </b><b><i>IL-8 mRNA expression in human hepatocellular carcinoma cell lines.</i></b> IL-8 mRNA levels were evaluated on HepG2 and JHH6 cell lines by Real-Time PCR. Total RNA was extracted and reverse-transcribed as described in Material and Methods section. The histograms show the detected levels of IL-8 mRNA normalized to two different housekeeping genes (18S and GAPDH). An almost thirty-fold increase was observed for the mRNA IL-8 expression in JHH6 cell line. Data are the means ± SD of three independent experiments. <b>Panel C: </b><b><i>Schematic representation of the luciferase-linked human IL-8 promoter constructs used in this study.</i></b> The plasmids −1498/+44 hIL-8/Luc and −162/+44 hIL-8/Luc (deleted of a 5′ promoter region) contain binding sites for AP-1, NF-IL-6 and NF-κB transcription factors. Site-directed mutation of the IL-8 NF-κB binding site in the context of the −162/+44 hIL-8/Luc plasmid abolished the binding of NF-κB on IL-8 promoter. <b>Panel D: </b><b><i>Effect of site-directed mutagenesis of the NF-κB binding site in the human IL-8 promoter sequence.</i></b> JHH6 cells transfected with −1498/+44 hIL-8/Luc or −162/+44 hIL-8/Luc ΔNF-κB constructs and then treated with 2000 U/ml of TNF-α for 3 h, were analyzed through gene reporter assay. In cells transfected with the −1498/+44 hIL-8/Luc construct, TNF-α stimulated IL-8 luciferase activity, whereas mutation of the NF-κB binding site significantly decreased both basal and TNF-α-induced IL-8 promoter driven activity in JHH6. Data reported are the means ± SD of three independent experiments. These data suggest a central role of NF-κB in IL-8 gene transcription. <b>Panel E: </b><b><i>Effect of E3330 treatment on JHH6 viability.</i></b> Levels of viability were measured with MTS assay in JHH6 cells treated for 7 h with increasing doses of E3330. Up to a concentration of 100 µM the treatment with E3330 did not affect the cellular viability. Data, expressed as the percentage of cell viability, are the means ± SD of three independent experiments. <b>Panel F: </b><b><i>Effect of E3330 on APE1 subcellular distribution.</i></b> APE1 subcellular localization was detected through confocal microscopy analysis using a specific α-APE1 monoclonal primary antibody. APE1 mainly localized within the nuclear compartment and accumulated into nucleoli. Treatment with 100 µM E3330 for 6 h induced a robust cytoplasmic enrichment of APE1. As control, cells were treated with DMSO without any effect on APE1 subcellular distribution. <b>Panel G: </b><b><i>Effect of E3330 treatment on TNF-α-induced IL-8 promoter activity.</i></b> JHH6 cells transfected with −1498/+44 hIL-8/Luc construct were pre-treated with increasing concentration of E3330, or with vehicle (DMSO) as a control, for 4 h prior to treatment with 2000 U/ml TNF-α for 3 h. TNF-α stimulated IL-8 luciferase activity and the pre-treatment with E3330 significantly decreased, in a dose-dependent manner, TNF-α-induced IL-8 promoter activity. Data reported are the means ± SD of three independent experiments.</p

Model of the effect of E3330 redox inhibitor on the Fatty Acid-TNFα-APE1-NFκB-IL8 axis.

<p>APE1 redox inhibitor E3330 prevents inflammatory cytokines production (IL-8 and IL-6) triggered by FAs accumulation and TNF-α stimulation in hepatic cancer cell lines. In this pathway, mitochondrial impairment and resulting oxidative stress condition may cause the functional activation of NF-κB transcription factor through APE1 regulatory redox function leading to IL-8 and IL-6 gene expression.</p