Journal: Free radical biology & medicine
Article Title: Antioxidants tiron and N-acetyl-L-cysteine differentially mediate apoptosis in melanoma cells via a reactive oxygen species-independent NF-?B pathway
Figure Lengend Snippet: Regulation of tiron or NAC on NF-κB pathway. A, NF-κB reporter luciferase activity and CXCL1 secretion are induced by tiron and suppressed by NAC. SK-Mel-5 cells were co-transfected with an NF-κB transcriptional luciferase reporter vector and a pRSV β-galactosidase reporter vector for 24 h followed by treatment with increasing concentrations of tiron or NAC (left panel) for an additional 24 h. Subsequently, luciferase activity was determined as described in “Materials and Methods.” For assessment of NF-κB target gene expression, release of the CXCL1 chemokine into the medium of cultured SK-MEL-5 cells exposed to tiron or NAC was measured using ELISA assay (right panel). Both luciferase activity and CXCL1 production are expressed as mean ± S.D. and the experiments were repeated three times with consistent results. B, IKK activity is down-regulated by NAC, but not tiron. SK-MEL-5 cells were treated with the indicated concentrations of either tiron or NAC for 24 h. The cellular IKKα/β proteins were immunoprecipitated and subjected to IKK activity assay and immunoblotting for IKKα/β proteins (upper panel) as described in “Materials and Methods.” The mean ± S.D. of IKK activity from three independent experiments is shown in the lower panel. C, tiron increased cellular NF-κB DNA binding in vivo . Nuclear extracts (NE) were prepared from the SK-MEL-5 cells treated 24 h with the indicated concentrations of tiron (lanes 1–4). Nuclear NF-κB proteins binding to labeled NF-κB oligonucleotide probe was assessed by EMSA as described in “Materials and Methods.” The specificity of the protein/DNA complex was identified by competition with NF-κB oligonucleotides and by super-shift analysis using specific NF-κB antibodies (p50 or/and p65) (lanes 5–10). (s), specific NF-κB band. (a, b), shifted bands. D, tiron directly increased the binding affinity between NF-κB complex and DNA. Five microgram of SK-MEL-5 nuclear extract were incubated with γ- 32 P labeled NF-κB oligonucleotides. The indicated concentrations of tiron were added to the “cell-free” mixture and NF-κB/DNA binding activity was assessed by EMSA. The binding of nuclear protein and OCT-1 oligo with the addition of different concentrations of tiron serves as loading control. Data shown here are representative of three independent experiments. E, the AP-1 activity profile in melanoma cells treated with either tiron or NAC. SK-MEL-5 cells were co-transfected with AP-1 luciferase reporter and β-galactosidase reporter. After 24 h transfection, cells were subsequently treated with different concentrations of either tiron or NAC for an additional 24 h. Cellular luciferase activity was determined as described in “Materials and Methods.” Luciferase activity in the treated cells was normalized to that of control cells. Mean ± S.D. of luciferase activity from three experiments is shown.
Article Snippet: Double-stranded NF-κB consensus oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′, Promega, Madison, WI) was labeled with γ-32 P (Amersham Pharmacia Biotech), and 105 cpm of labeled NF-κB oligonucleotides were used in binding reactions with 5 μg nuclear extract at room temperature for 30 min as per the manufacturer’s instructions (Promega).
Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Binding Assay, In Vivo, Labeling, Incubation