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
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.