Neuropeptide Substance P Enhances Inflammation-Mediated Tumor Signaling Pathways and Migration and Proliferation of Head and Neck Cancers

Abstract

Head and neck cancers (HNC) are extremely aggressive, highly recurrent, and the sixth most common cancer worldwide. Neuropeptide substance P, along with its primary receptor, neurokinin-1 (NK-1R), is overexpressed in HNC and is a central player in inflammation and growth and metastasis of several cancers. However, the precise SP-mediated signaling that promotes HNC progression remains ill defined. Using a panel of HNC lines, in this study, we investigated the effects of SP on proliferation and migration of HNC. Tumor cells were also treated with SP and alterations in inflammatory cytokines and chemokines, and their cognate receptors were analyzed by real-time PCR. Furthermore, we investigated the role of SP in inducing epithelial-mesenchymal transition (EMT), and matrix metalloproteases that promote tumor invasion. Our results showed that SP significantly increased tumor cell proliferation and migration and induced the expression of several genes that promote tumor growth, invasion, and metastasis which was suppressed by a specific NK1R antagonist L-703606. SP also activated NFκB that was suppressed on inhibiting NK1R. Collectively, our data shows that SP-NK1R-mediated inflammatory signaling comprises an important signaling axis in promoting HNC and may prove to be effective clinical target against HNC cells that are resistant to traditional therapy.

Keywords: Cytokines; Head neck cancer; Inflammation; Neuropeptide; Tumor progression.

Fig. 1

Substance P–induced head and neck cancer (HNC) cell proliferation: After being treated with various concentrations of SP for 24 h, cellular proliferation assay of HNC cell lines (A) FaDu, (B) Detroit 562, and (C) SCC-9 was assessed by XTT assay. Values represent mean ± SE, n ≥ 3, two-way ANOVA followed by Fisher LSD for multiple comparison. Single, double, triple, and quadruple asterisks indicate p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, and p ≤ 0.0001 respectively as compared with respective control

Fig. 2

Substance P–induced expression of various cytokines and chemokines in HNC cells: HNC cells were treated with SP (100 nM) and NK1R inhibitor L-703606 (1 μM). mRNA expression of cytokines and chemokines were analyzed by PCR for (A and B) FaDu, (C and D) Detroit 562, and (E and F) SCC-9 cells. Values represent mean ± SE, n = 3, two-way ANOVA followed by Fisher LSD for multiple comparison. Single, double, triple, and quadruple asterisks or single, double, triple, and quadruple number signs indicate p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, and p ≤ 0.0001 respectively as compared with control or SP 100 nM

Fig. 3

Substance P–induced expression of various chemokine receptors in HNC cells: HNC cells were treated with SP (100 nM) and NK1R inhibitor (1 μM). mRNA expression of chemokines and chemokine receptors were analyzed by real-time PCR in (A and B) FaDu, (C and D) Detroit 562, and (E and F) SCC-9 cell lines. Values represent mean ± SE, n = 3, two-way ANOVA followed by Fisher LSD for multiple comparison. Single, double, triple, and quadruple asterisks or Single, double, triple, and quadruple number signs indicate p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, and p ≤ 0.0001 respectively as compared with control or SP 100 nM

Fig. 4

Substance P–induced expression of various MMP and EMT-inducing genes in HNC cells: HNC cells were treated with SP (100 nM) and NK1R inhibitor (1 μM). mRNA expression of various MMPs and EMT-inducing genes were analyzed by real-time PCR in (A and B) FaDu, (C and D) Detroit 562, and (E and F) SCC-9 cell lines. Values represent mean ± SE, n = 3, two-way ANOVA followed by Fisher LSD for multiple comparison. Single, double, triple, and quadruple asterisks or Single, double, triple, and quadruple number signs indicate p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, and p ≤ 0.0001 respectively as compared with control or SP 100 nM

Fig. 5

Substance P–induced NFκB nuclear translocation in HNC cells: HNC cells were treated with SP (100 nM) and with or without NK1R inhibitor for 24 h. (A) Migration of SCC-9 cells in 8-μm pores containing PTE inserts towards complete medium was assessed. Representative × 10 images were given. Graph represents number of cells migrated. (B) Rate of migration of Detroit 562 cells in the scratch wound was followed for 0 h, 2 h, 12 h, and 24 h. Representative × 4 images are shown. Graphs represent rate of migration for 12 and 24 h. (C) Immunofluorescence images of NF-kB nuclear localization in HNC cells treated with SP and its inhibitor NK1R. Fluorescent images were acquired at × 20 magnification. Bottom panel shows enlarged images of specific areas on the coverslip. The percentage of fluorescent intensity in the nucleus was calculated and plotted. Scale bar = 100 μm. Values represent mean ± SE, n ≥ 3, two-way ANOVA followed by Fisher LSD for multiple comparison. Single, double, triple, and quadruple asterisks or Single, double, triple, and quadruple number signs indicate p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, and p ≤ 0.0001 respectively as compared with control or SP 100 nM