Figure 1.
Expression of ectopic APE1WT protein confers cells protection to genotoxic damage but not to FAs-induced cytotoxicity.
Panel A: Western Blot analysis of total cell extracts from HepG2 stable cell clones. 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 APE1WT and the APE1NΔ33 cell clones is expressed to a similar extent at different days of cell culture. a: clones after the sixth in vitro passage; b: clones after the tenth in vitro passage. Panel B: APE1 localization within HepG2 cell clones. 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 APE1WT within cell nuclei and colocalization with Histone H3. The APE1NΔ33 deletion mutant colocalizes with Histone H3 within cell nuclei but show also cytoplasmic positivity. Panel C: Growth curve of HepG2 cell clones. HepG2 empty clone, APE1WT clone and APE1NΔ33 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. APE1NΔ33 cells (triangle) grew more slowly than the APE1WT (square) and the empty clones (dot). Panel D: Cell growth by MTT colorimetric assay. Thirty thousand cells of the control (empty clone), APE1WT and APE1NΔ33 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 APE1NΔ33 cell clone has a lower level of proliferation than empty and APE1WT clones. Data, expressed as the percentage of cell viability with respect to the control empty clone, are the means ± SD of three independent experiments. Panel E: Effect of MMS on viability of HepG2 cell clones. 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 APE1NΔ33 cell clone but not in the APE1WT clone, suggesting that the ectopic expression of APE1WT protects cells against MMS-induced citotoxicity. Data shown are the means ± SD of three independent experiments. Panel F: Effect of H2O2 on viability of HepG2 cell clones. 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 APE1WT clone compared to empty and APE1NΔ33 cell clones. The histograms show the means ± SD of three independent experiments. Panel G: Quantification of γH2A.X foci in response to etoposide treatment. 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 APE1NΔ33 cell clone (triangle). DNA damage was weaker for APE1WT clone (square) than empty (dot) and APE1NΔ33 cell clones, suggesting a protective role of APE1 overexpression in DNA repair.
Figure 2.
Overexpression of APE1 protein does not protect HepG2 cells from FAs treatment.
Panel A: Nile Red staining. Fluorescence of Nile Red-stained were measured on HepG2 cells previously incubated with 600 µM FAs mixture (oleate/palmitate 2∶1) for 24 h (b and d) or left untreated as control (a and c). Cells were fixed with 1.5% glutaraldehyde in PBS, washed with buffered saline and then were stained with Nile Red 10 µg/ml (a and b) or 100 μg/ml (c and d). As confirmed by Nile Red staining, HepG2 cell line exhibited a fat overloading profile. Panel B: Transmission electron microscopy analysis. HepG2 cells were treated with the 600 µM FAs mixture at the final ratio of 2∶1 (oleate/palmitate) for 24 h (b and d) or left untreated (a and c). Cells were then fixed and paraffin-embedded. Transmission electron microscopy confirmed fat overloading induction in HepG2 cell line. Magnification: 6300X (a and b) and 8000X (c and d). Panel C: Western Blotting analysis of total cell extracts from HepG2 stable cell clones after FAs treatment. HepG2 cell clones were treated with 600 µM of FAs mixture (2∶1 ratio of oleate/palmitate) for 24 h or left untreated as control. After FAs treatment, total cell extracts were prepared and 12 µg of protein extract was loaded onto a 12% SDS-PAGE, blotted and probed with anti-APE1 antibody. FAs treatment does not alter the endogenous levels of APE1 both in the APE1WT and the APE1NΔ33 cell clones. Panel D: Effect of FAs treatment on viability of HepG2 cell clones. HepG2 cell clones were treated with 600 µM of FAs mixture (2∶1 ratio of oleate/palmitate) for 24 h. Cytotoxicity was assessed by trypan blue exclusion test. After exposure to FAs there was a significant reduction in cell viability but no significant difference between the clones. Data, expressed as the percentage of cell viability, are the means ± SD of three independent experiments.
Figure 3.
NF-κB transcription factor regulates IL-8 promoter activity in JHH6 cells and E3330 treatment inhibits TNF-α-induced promoter activation.
Panel A: Western Blotting analysis of total cell extracts from human hepatocellular carcinoma cell lines. 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. Panel B: IL-8 mRNA expression in human hepatocellular carcinoma cell lines. 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. Panel C: Schematic representation of the luciferase-linked human IL-8 promoter constructs used in this study. 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. Panel D: Effect of site-directed mutagenesis of the NF-κB binding site in the human IL-8 promoter sequence. 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. Panel E: Effect of E3330 treatment on JHH6 viability. 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. Panel F: Effect of E3330 on APE1 subcellular distribution. 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. Panel G: Effect of E3330 treatment on TNF-α-induced IL-8 promoter activity. 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.
Figure 4.
E3330 treatment specifically inhibits TNF-α- and FAs-induced IL-8 endogenous gene expression.
Panel A: Effect of E3330 treatment on TNF-α-induced IL-8 mRNA expression. 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. Panel B: Effect of E3330 treatment on TNF-α-induced IL-8 protein production. 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. Panel C: Effect of E3330 treatment on TNF-α-induced IL-6 mRNA expression in JHH6 cells. 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. Panel D: Effect of E3330 treatment on TNF-α-induced IL-12 protein production in JHH6 cells. 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. Panel E: Effect of FAs overload on IL-8 gene expression. 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. Panel F: Effect of E3330 treatment on FAs-induced IL-8 mRNA expression. 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.
Figure 5.
Model of the effect of E3330 redox inhibitor on the Fatty Acid-TNFα-APE1-NFκB-IL8 axis.
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.