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.