IL-8 promotes inflammatory mediators and stimulates activation of p38 MAPK/ERK-NF-κB pathway and reduction of JNK in HNSCC

Abstract

This investigation identifies interleukin 8 (IL-8) as the main inflammatory mediator in head and neck squamous cell carcinoma (HNSCC). The expressions of chemokines of IL-8, IL-1β and IL-6 and the cytokines of tumor necrosis factor-α (TNF-α) were higher in HNSCC patient tissues than in non-cancerous matched tissues (NCMT) whereas the expression of IL-10 was lower. IL-8 is most highly expressed in the tissues of patients with HNSCC. Treatment of HNSCC cells with IL-8 increased the secretion of IL-1β, IL-6 and TNF-α and reduced IL-10 expression; the increase in the expression of IL-1β was particularly considerable. IL-8 silencing by siRNA reduced IL-1β expression in HNSCC cells, suggesting that IL-8 as a main inflammatory mediator improved IL-1β expression in HNSCC. The expressions of p-p38 mitogen-activated protein kinases (MAPK) and p-extracellular signal regulated kinase (p-ERK) were higher and that of p-c-Jun-NH2-terminal kinase (p-JNK) was lower in HNSCC patient tissues than in NCMT. IL-8 treatment induced p-p38 MAPK and p-ERK expression, but reduced p-JNK expressions in HNSCC cells. IL-8 siRNA suppressed p38 MAPK and ERK but increased JNK expression in HNSCC cells. Exposure of SCC25 cells to IL-8, increased the expressions of p-IκB-α and nuclear factor (NF)-κB, suggesting that IL-8 regulates inflammatory response by modulating the MAPK and NF-κB pathway in HNSCC cells. IL-8 promotes the migration of SCC25 cells and increases matrix metalloproteinase-2 (MMP-2) and MMP-9 expressions. These results reveal that IL-8 is the major stimulus of inflammatory mediation in HNSCC progression and migration by activating the p38 MAPK/ERK-NF-κB pathway and reducing JNK.

Keywords: HNSCC; IL-8; MAPK; inflammation.

Figure 1. Expression of inflammatory mediators in NCMT and HNSCC of human tissue

(A) A microarray analysis to evaluate expressions and the fold-change thresholds of IL-1α, IL-1β, IL-6, IL-8, IL-10 and TNF in NCMT and HNSCC of human tissue (patient no. 2). (B) IL-1α, IL-1β, IL-6, IL-8, IL-10 and TNF-α expressions in NCMT and HNSCC of human tissue (patient no. 3) were analyzed by western blotting. β-actin was used as internal control for sample loading.

Figure 2. IL-8 and inflammatory mediators in HNSCC cells

SCC25 were stimulated using IL-8 (10 and 100 ng/ml) for 72 h, and gene and protein expressions of IL-1α, IL-1β, IL-6, IL-10 and TNF-α were evaluated using RT-PCR (A) and western blotting (B). β-actin was used as internal controls for sample loading. (C) SCC25 cells were stimulated using IL-8 (10 and 100 ng/ml) for 72 h; culture media were then collected and tconcentrations of IL-1β, TNF-α and IL-6 were measured using an BD OptEIA ELISA kit. Each value is presented as mean ± SD across three experiments. *p<0.05 implies a significant difference from control cells.

Figure 3. IL-8 siRNA reduces IL-1β expression in HNSCC cells

(A) Immunohistochemically-stained IL-1β in NCMT and HNSCC, observed under an inverted fluorescent microscope (200× magnification). Knockdown of IL-8 by siRNA reduced expression of IL-1β in three types of HNSCC cell (SCC4, SCC9 and SCC25 cells). Cells were treated with control siRNA and IL-8 siRNA (10 μM) for 6 h, and amount of IL-1β present was determined using RT-PCR (B) and western blotting (C). Known amounts of β-actin were used as internal controls for sample loading.

Figure 4. Expression of MAPKs genes in NCMT and HNSCC of human tissue

(A) A microarray analysis of expressions and fold-change thresholds of p38α, p38β, JNK1, JNK2, JNK3, ERK1 and ERK2 in NCMT and HNSCC of human tissue (patient no. 2). (B) Levels of p38 MAPK, p-p38 MAPK, JNK, p-JNK, ERK, p-ERK and β-actin in NCMT and HNSCC of human tissue (patient no. 3) were analyzed by western blotting. (C) Gene expression of JNK in NCMT and HNSCC of human tissue were analyzed by RT-PCR. To identify role of JNK in HNSCC, levels of JNK and p-JNK in NCMT and HNSCC tissue (n = 6) (patients nos. 1, 4-8) were obtained using western blotting (D) and qRT-PCR (E). Knows amounts of β-actin were used as internal controls for sample loading. Each value is presented as mean ± SD across three experiments. *p<0.05 implies a significant difference from control cells. (F) Immunohistochemically-stained p-p38 MAPK, p-ERK and p-JNK in NCMT and HNSCC, observed under an inverted fluorescent microscope (200× magnification).

Figure 5. Activation of p38 MAPK/ERK-NF-κB pathway and reduction of JNK expressions in IL-8-stimulated HNSCC cells

(A) SCC25 cells were stimulated using IL-8 (10 and 100 ng/ml) for 72 h and levels of p38, JNK and ERK were obtained using RT-PCR. (B) SCC25 cells were exposed to IL-8 (10 and 100 ng/ml) for 72 h and levels of p38 MAPK, p-p38 MAPK, JNK, p-JNK, ERK and p-ERK, were obtained by western blotting. (C) To evaluate IL-8-reduced JNK expression, SCC4 and SCC9 cells were treated with IL-8 (10 and 100 ng/ml) for 72 h, and levels of JNK and p-JNK were evaluated by western blotting. (D) To confirm that the p38 MAPK-NF-κB signaling pathway is associated with IL-8, expressions of IκB-α, p-IκB-α, cytosol and nucleic NF-κB in SCC25 cells that were treated with IL-8 (10 and 100 ng/ml) for 72 h were obtained by western blotting. (E) Knockdown of IL-8 by siRNA reduced p38 and ERK expressions and increased JNK expression in three types of HNSCC cell (SCC4, SCC9 and SCC25 cells). Cells were treated with control siRNA and IL-8 siRNA (10 μM) for 24 h, and p38 MAPK, JNK and ERK were quantified using RT-PCR. Known amounts of β-actin were used as internal controls for sample loading.

Figure 6. IL-8 is associated with HNSCC migration via regulation of MMPs

(A) Effect of IL-8 (1, 10 and 100 ng/ml) on proliferation of SCC25 cells was determined using an established wound healing assay, and (B) quantitative analysis of migrating cells following IL-8 stimulation was carried out using Image J software. Each value is presented as mean ± SD across three experiments. *p<0.05 implies a significant difference from control cells. (C) To determine whether MMPs-mediated HNSCC migration is associated with IL-8 treatment, SCC25 cells were treated with IL-8 (10 and 100 ng/ml) for 72 h, and levels of MMP-2 and MMP-9 were evaluated using western blotting.

Figure 7. Scheme of IL-8 promotes inflammatory mediators in HNSCC progression and migration

Our previous report established that IL-8 promotes HNSCC progression along CXCR1/2-mediated NOD1/RIP2 signaling pathway. This study reveals that IL-8 promotes inflammatory mediators and stimulates both activation of p38 MAPK/ERK-NF-κB pathway and reduction of JNK level in HNSCC progression and migration. Two instances of IL-8-mediated mechanical signaling in HNSCC cells are schematically depicted. Results in this figure can be utilized to develop biomarkers and targets in HNSCC and to provide a basis for early diagnosis and treatment.