Yonsei Med J.  2016 May;57(3):588-598. 10.3349/ymj.2016.57.3.588.

Tolfenamic Acid Inhibits the Proliferation, Migration, and Invasion of Nasopharyngeal Carcinoma: Involvement of p38-Mediated Down-Regulation of Slug

• Jittreetat T
• Shin YS
• Hwang HS
• Lee BS
• Kim YS
• Sannikorn
• Kim CH

• KMID: 2374076
• DOI: http://doi.org/10.3349/ymj.2016.57.3.588

Abstract

PURPOSE
Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug, is known to exhibit antitumor effects in various cancers apart from nasopharyngeal cancer (NPC). NPC exhibits high invasiveness, as well as metastatic potential, and patients continue to suffer from residual, recurrent, or metastatic disease even after chemoradiation therapy. Therefore, new treatment strategies are needed for NPC. In this study, we investigated the efficacy and molecular mechanisms of TA in NPC treatment.
MATERIALS AND METHODS
TA-induced cell death was detected by cell viability assay in the NPC cell lines, HNE1 and HONE1. Wound healing assay, invasion assay, and Western blot analysis were used to evaluate the antitumor effects of TA in NPC cell lines.
RESULTS
Treatment with TA suppressed the migration and invasion of HNE1 and HONE1 cells. Hepatocyte growth factor enhanced the proliferation, migration, and invasion abilities of NPC cells. This enhancement was successfully inhibited by TA treatment. Treatment with TA increased phosphorylation of p38, and the inhibition of p38 with SB203580 reversed the cytotoxic, anti-invasive, and anti-migratory effects of TA treatment in NPC cell lines. Moreover, inhibition of p38 also reversed the decrease in expression of Slug that was induced by TA treatment.
CONCLUSION
In conclusion, the activation of p38 plays a role in mediating TA-induced cytotoxicity and inhibition of invasion and migration via down-regulation of Slug.

Keyword

Tolfenamic acid; nasopharyngeal cancer; p38 mitogen-activated protein kinase; Slug

MeSH Terms

Animals
Anti-Inflammatory Agents, Non-Steroidal/*pharmacology/therapeutic use
Cell Line, Tumor
Cell Movement/*drug effects
Cell Proliferation/*drug effects
Cell Survival/*drug effects
Down-Regulation
Gastropoda
Gene Expression Regulation, Neoplastic/drug effects
Hepatocyte Growth Factor/metabolism/*pharmacology
Humans
Imidazoles
MAP Kinase Signaling System/drug effects
Nasopharyngeal Neoplasms/*drug therapy/metabolism/pathology
Neoplasm Invasiveness/*prevention & control
Phosphorylation/drug effects
Pyridines
ortho-Aminobenzoates/*pharmacology/therapeutic use
Anti-Inflammatory Agents, Non-Steroidal
Hepatocyte Growth Factor
Imidazoles
Pyridines
ortho-Aminobenzoates

Figure

• 

  Fig. 1 Effects of tolfenamic acid on proliferation of nasopharyngeal cancer cell lines and a normal keratinocyte cell line. Cells were seeded in 96-well tissue-culture plates, at a density of 2×103 cells/well in a final volume of 100 µL of medium, and were allowed to attach for 24 h. The cells were then treated with ten different concentrations of tolfenamic acid (TA) for 24 h and 48 h. Cell proliferation was estimated by the MTT assay. (A) Nasopharyngeal cancer cell lines, (B) HaCaT cell lines. Values are represented as mean±SD from three independent experiments. *p<0.05 and ***p<0.001 compared to the control group. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

• 

  Fig. 2 Effect of tolfenamic acid (TA) on the migratory and invasive abilities of nasopharyngeal cancer cells. The migration capability after HGF/(TA) treatment was investigated using wound healing assay. (A) HNE1 and HONE1 cells were either treated with 10 ng/mL HGF along with TA (0, 10, 30, 50, 75, 100 µM) or left untreated. Confluent monolayers of HNE1 were wounded by scratching the surface as uniformly as possible with a 1-mL pipette tip. TA treatment significantly inhibited HGF-induced enhancement of cell migration in a dose-dependent manner. (B) HNE1 and HONE1 cells (2×104) in the upper chamber were pretreated with TA (0, 10, 30, 50, 75, 100 µM) and then either treated with 10 ng/mL HGF or left untreated. After 24 h, the number of invading cells was counted in four representative fields per membrane. Co-treatment with TA and HGF significantly inhibited cell invasion compared to treatment with HGF alone. The data represent the mean±SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.001. HGF, hepatocyte growth factor.

• 

  Fig. 3 Effect of tolfenamic acid (TA) on the MAPK pathway in nasopharyngeal cancer cells. (A) Immunoblot of TA-treated cells stained with antibodies against T-p38, p-p38, T-JNK, and p-JNK. Western blot analysis showed that TA-enhanced phosphorylation of p38 and JNK in HNE1 and HONE1 cells. (B) HNE1 and HONE1 cells were treated with HGF (10 ng/mL), SB203580 (0.5 µM), and TA (50 µM) alone or in combination, as indicated. Cell viability assay was performed to investigate the cytotoxic effect of TA in nasopharyngeal cancer cells in the absence or presence of SB203580. The data represent the mean±SD of three independent experiments. *p<0.05, ***p<0.001 by one-way ANOVA. MAPK, mitogen-activated protein kinase.

• 

  Fig. 4 Effect of a p38 inhibitor on anti-migratory and anti-invasive abilities of tolfenamic acid (TA) in nasopharyngeal cancer cells. HNE1 and HONE1 cells were treated with HGF (10 ng/mL), SB203580 (0.5 µM), and TA (50 µM) alone or in combination, as indicated. (A) Wound healing and (B) invasion assays were used to determine the anti-migratory and anti-invasive abilities of TA in nasopharyngeal cancer cells in the absence or presence of SB203580. The data represent the mean±SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA. DMSO, dimethyl sulfoxide; HGF, hepatocyte growth factor.

• 

  Fig. 5 Effect of p38 inhibition on Slug expression in tolfenamic acid (TA)-treated nasopharyngeal cancer cells. HNE1 and HONE1 cells were treated with TA (50 µM) and SB203580 (0.5 µM) alone or in combination, as indicated. Immunoblot of treated cells stained with antibodies against (A) p-p38 (30 min) and (B) Slug (3 h). Western blot analysis showed that TA reduced phosphorylation of p38 and Slug, while this effect was reversed by SB203580 treatment. DMSO, dimethyl sulfoxide.

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