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. 2007 Sep 13:7:176.
doi: 10.1186/1471-2407-7-176.

Differential regulation of iron chelator-induced IL-8 synthesis via MAP kinase and NF-kappaB in immortalized and malignant oral keratinocytes

Hwa-Jeong Lee  1 , Jun Lee, Sun-Kyung Lee, Suk-Keun Lee, Eun-Cheol Kim
Affiliations

Differential regulation of iron chelator-induced IL-8 synthesis via MAP kinase and NF-kappaB in immortalized and malignant oral keratinocytes

Hwa-Jeong Lee et al. BMC Cancer. .

Abstract

Background: Interleukin-8 (IL-8) is a cytokine that plays an important role in tumor progression in a variety of cancer types; however, its regulation is not well understood in oral cancer cells. In the present study, we examined the expression and mechanism of IL-8 in which it is involved by treating immortalized (IHOK) and malignant human oral keratinocytes (HN12) cells with deferoxamine (DFO).

Methods: IL-8 production was measured by an enzyme-linked immunoabsorbent assay and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Electrophoretic mobility shift assays was used to determine NF-kappaB binding activity. Phosphorylation and degradation of the I-kappaB were analyized by Western blot.

Results: IHOK cells incubated with DFO showed increased expression of IL-8 mRNA, as well as higher release of the IL-8 protein. The up-regulation of DFO-induced IL-8 expression was higher in IHOK cells than in HN12 cells and was concentration-dependent. DFO acted additively with IL-1beta to strongly up-regulate IL-8 in IHOK cells but not in HN12 cells. Accordingly, selective p38 and ERK1/2 inhibitors for both kinases abolished DFO-induced IL-8 expression in both IHOK and HN12 cells. Furthermore, DFO induced the degradation and phosphorylation of IkappaB, and activation of NF-kappaB. The IL-8 inducing effects of DFO were mediated by a nitric oxide donor (S-nitrosoglutathione), and by pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB, as well as by wortmannin, which inhibits the phosphatidylinositol 3-kinase-dependent activation of NAD(P)H oxidase.

Conclusion: This results demonstrate that DFO-induced IL-8 acts via multiple signaling pathways in immortalized and malignant oral keratinocytes, and that the control of IL-8 may be an important target for immunotheraphy against human oral premalignant lesions.

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Figures

Figure 1
Figure 1
Effects of Iron chelator on IL-8 production in immortalized (IHOK) and malignant human oral keratinocytes (HN12). Cells were treated for 16 h with the indicated concentrations of DFO (0.2–2 mM) in IHOK and HN12 cells (A), or DFO (1.0 mM), FC (0.5 mM), MIM (1.0 mM) in IHOK and HN12 cells (B), IL-1 β (10 ng/ml), TNF-α (10 ng/ml), or combinations thereof in IHOK (C) and HN12 (D) cells. Levels of IL-8 secretion were determined by ELISA. Results are expressed as means ± SD of three independent experiments. Numbers below the gels represent the intensity of IL-8 mRNA relative to GAPDH mRNA.*: Statistically significant difference compared to control group, p < 0.05. #: Statistically significant difference compared to DFO group, p < 0.05.
Figure 2
Figure 2
Iron chelator induces IL-8 protein secretion (A, C) and IL-8 mRNA accumulation (B, D) in IHOK and HN12 cells in a time-dependent manner. Cells were incubated with DFO (1.0 mM) or IL1-β (10 ng/ml) for the indicated time periods. Levels of IL-8 protein and mRNA were determined by ELISA and semiquantitative RT-PCR, respectively. Numbers below the gels represent the intensity of IL-8 mRNA relative to GAPDH mRNA. These data are representative of three independent experiments.
Figure 3
Figure 3
Iron chelator induced phosphorylated IκB-α in IHOK and HN12 cells on time dependent. Cells were treated with DFO (1.0 mM) or IL-1α (10 ng/ml) for the indicated time periods. Levels of IκB-α, p IκB-α were determined by Western blotting. The protein fraction was extracted, electrophoresed, transferred to membrane and blotted with respective antibodies. These data are representative of three independent experiments.
Figure 4
Figure 4
DFO induced the DNA binding activities of NF-kB in IHOK and HN12 cells. The nuclear extracts from the cells treated with DFO 1.0 mM at the indicated times were incubated with [γ-32P] ATP-labeled NF-kB probe, and analyzed by EMSA. The specificity of the bands was verified by adding a 10-fold excess of a completing unlabeled NF-kB probe (cold probe) to the 4 h DFO treated unclear proteins. The results were confirmed by two independent experiments.
Figure 5
Figure 5
The effect of inhibitor for p38 and ERK MAPK on iron chelator induced IL-8 protein secretion and IL-8 mRNA in IHOK and HN12 cells. IHOK (A) & HN12 cells (C) were pretreated with ERK inhibitor PD98059 and the p38 inhibitor SB203580 for 1 h and followed by the treatment with DFO (1.0 mM) for 16 h. Levels of IL-8 secretion and IL-8 mRNA were determined by ELISA and RT-PCR in IHOK (B) & HN12 cells (D). Same procedure as described in the legend to Fig. 1 was performed. Results are expressed as means ± SD of three independent experiments. *: Statistically significant difference compared to control group: p < 0.05, #: Statistically significant difference compared to DFO group, p < 0.05.
Figure 6
Figure 6
Iron chelator stabilized IL-8 mRNA through activation of p38 and ERK1/2. IHOK and HN12 cells were treated with DFO (1.0 mM) for 16 h to allow accumulation of IL-8 mRNA. Cells were then treated with the mRNA synthesis inhibitor actinomycin D (ActD; 5 μg/ml), and either ERK pathway inhibitor (PD98059) or p38 inhibitor (SB203580). GAPDH mRNA was used as an endogenous control message. Same procedure as described in the legend to Fig. 1 was performed. Results are representative of three independent experiments.
Figure 7
Figure 7
Effect of various inhibitors on the DFO-induced IL-8 mRNA expression. Cells were incubated for 16 h in the presence or absence of DFO 1.0 mM with and without GSNO (1.0 mM), PDTC (50 μM), hemoglobin (1 mg), wortmannin (0.5 μM), and cycloheximede (10 μg/ml). Same procedure as described in the legend to Fig. 1 was performed. The results are representative of three independent experiments.
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