Proof-of-concept study investigating the role of S100P-RAGE in nasopharyngeal carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is an epithelial carcinoma that arises from the lining of the nasopharyngeal mucosa. The efficacy of radiation therapy is limited due to radiation resistance, particularly in the advanced stages of NPC. The S100P protein is a small isoform of the S100 protein family, which is involved in the regulation of various intracellular and extracellular processes, including proliferation, differentiation and intracellular signaling. The aim of the current study was to investigate the significance of the S100P-RAGE axis in NPC progression. The expression levels of S100P and receptor for activated glycation end-products (RAGE) in NPC specimens were determined by western blotting. In addition, the effect of the S100P-RAGE axis on NPC was evaluated in vitro by proliferation and migration assays using C666-1 cells treated with S100P or the RAGE inhibitor FPS-ZM1. The underlying mechanism was also investigated by western blotting. The expression of S100P and RAGE was detected in clinical specimens from 15 patients with NPC and 15 patients with benign nasopharyngeal inflammation, and was observed to be higher in NPC tissues compared with inflamed tissues. Furthermore, the interaction of S100P with RAGE increased the proliferation and migration potential of C666-1 cells, and activated mitogen-activated protein kinase and NF-κB signaling. These results indicate that the S100P-RAGE axis exerts a promoting effect on the progression of NPC. Therefore therapeutic strategies targeting S100P-RAGE merit further exploration for the treatment of NPC.

Keywords: C666-1 cells; RAGE; S100P; migration; proliferation.

Figure 1

Protein expression of S100P and RAGE in patients with NPC or benign nasopharyngeal inflammation. (A) The protein expression of S100P and RAGE in NPC tissues and nasopharyngeal tissues with benign inflammation by western blotting. Quantification of (B) S100P/GAPDH and (C) RAGE/GAPDH protein expression ratios. ^*P<0.05 and ^***P<0.001. RAGE, receptor for activated glycation end products; NPC, nasopharyngeal carcinoma.

Figure 2

Effects of S100P and RAGE on the proliferation and colony formation of C666-1 cells. Cell Counting Kit-8 assay results for the growth evaluation of cells treated with (A) S100P protein or (B) RAGE inhibitor FPS-ZM1 at the indicated concentrations for 6 h. The effects of S100P and FPS-ZM1 on cell viability were concentration-dependent. (C) The effects of S100P protein and FPS-ZM1 (both 1,000 ng/ml) on cell viability were also time-dependent. (D) Representative images of colony formation assay results. (E) The colony forming ability of C666-1 cells was increased in the S100P protein group and reduced in the FPS-ZM1 group. ^**P<0.01 and ^***P<0.001 vs. untreated control. RAGE, receptor for activated glycation end products; OD450, optical density at 450 nm.

Figure 3

Effects of S100P and RAGE on C666-1 cell migration. (A) Representative images of the wound healing assay (magnification, x400). (B) Measurements of the wound in the cell monolayer revealed that wound closure was induced by treatment with S100P protein and delayed by treatment with the RAGE inhibitor FPS-ZM1 compared with the untreated control. (C) The percentage of wound closure was different in the S100P protein and FPS-ZM1 groups compared with the untreated control group. (D) Representative images of the Transwell assay results. Scale bar, 200 µm. (E) The Transwell assay revealed that the migration ability of C666-1 cells was significantly increased by S100P protein and significantly reduced by the RAGE inhibitor FPS-ZM1. RAGE, receptor for activated glycation end products. ^***P<0.001.

Figure 4

S100P-RAGE activates MAPK and NF-κB signaling in C666-1 cells. (A) Representative western blots of cells treated with S100P protein or RAGE inhibitor FPS-ZM1. (B) Quantified western blotting data showing that p-ERK1/2/ERK1/2, p-p38/p38 and p-MAPK7/MAPK7 ratios and NF-κB1 and p65 expression levels were significantly increased in the S100P protein group and significantly reduced in the FPS-ZM1 group compared with those in untreated cells. ^*P<0.05 and ^***P<0.001. RAGE, receptor for activated glycation end products; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase.