Nonsteroidal anti-inflammatory medications are cytostatic against human vestibular schwannomas

Abstract

Vestibular schwannomas (VSs) are the most common tumors of the cerebellopontine angle. Significant clinical need exists for pharmacotherapies against VSs. Motivated by previous findings that immunohistochemical expression of cyclooxygenase 2 (COX-2) correlates with VS growth rate, we investigated the role of COX-2 in VSs and tested COX-2 inhibiting salicylates against VSs. COX-2 was found to be aberrantly expressed in human VS and primary human VS cells in comparison with control human nerve specimens and primary Schwann cells (SCs), respectively. Furthermore, levels of prostaglandin E2, the downstream enzymatic product of COX-2, were correlated with primary VS culture proliferation rate. Because COX-2 inhibiting salicylates such as aspirin are well tolerated and frequently clinically used, we assessed their repurposing for VS. Changes in proliferation, cell death, and cell viability were analyzed in primary VS cultures treated with aspirin, sodium salicylate, or 5-aminosalicylic acid. These drugs neither increased VS cell death nor affected healthy SCs. The cytostatic effect of aspirin in vitro was in concurrence with our previous clinical finding that patients with VS taking aspirin demonstrate reduced tumor growth. Overall, this work suggests that COX-2 is a key modulator in VS cell proliferation and survival and highlights salicylates as promising pharmacotherapies against VS.

Figure 1

COX-2 is aberrantly upregulated in VS and derived primary cultures. A. PTGS2 gene expression in human VSs (n=9 different tumors) versus great auricular nerves (GAN, n=8 different nerves) as measured through qPCR. Error bars represent range. B. Representative images of COX-2 expression (green), as visualized through immunohistochemistry, in (a) GAN (n=5 different nerves) and (b) VS (n=6 different VSs). Schwann or schwannoma cells are labeled with S100 (red) and nuclei are labeled with DAPI (blue). C. Prostaglandin (PTG) levels in tissue lysates of VS (n=5 different tumors) and GAN (n=3 different nerves). Error bars represent SEM. D. COX-2 expression in cultured human VS (n=6 different cultures) normalized to expression in SC cultures (n=6 different cultures) as quantified through western blot analysis. Error bars represent SD. E. COX-2 expression in tissue specimens of VS (n=3 different tumors) and GAN (n=3 different nerves) assessed by western blot analysis. Error bars represent SD. F. Correlation of PTG concentrations secreted in VS culture media with VS proliferation rate (% BrdU-positive cells) in vitro. R represents Spearman's correlation coefficient (n=6 different cultures). *p<0.05. re = in comparison to.

Figure 2

Salicylates decrease proliferation of VS cells. A. Representative VS culture proliferation images are shown after treatment for (a) no treatment control (NT), (b) 5 mM aspirin, and (c) 1 mM NaSal. S100 marks schwannoma cells, BrdU in nuclei marks proliferating cells. Nuclei are labeled with DAPI. Scale bar = 100 μm for all images. B. Quantification of proliferation changes after treatment with aspirin, NaSal and 5-ASA in primary VS cells normalized to proliferation in NT cells (n=3-7 different cultures). Error bars represent SEM. C. Representative VS culture cell death images are shown after treatment for (a) NT, (b) 5 mM aspirin, and (c) 5 mM NaSal. TUNEL (green) marks dying cells. Nuclei are labeled with DAPI, Scale bar = 100 μm for all images; D. Quantification of cell death rate changes after treatment with aspirin, NaSal and 5-ASA in primary VS cells (n=5-6 different cultures for each). Error bars represent SEM. *p<0.05. **p<0.01. ***p<0.001. re = in comparison to.

Figure 3

Salicylates and a specific COX-2 inhibitor II decrease VS cell viability but do not affect proliferation of normal Schwann cells. A. Quantification of proliferation changes after treatment with COX-2 inhibitor II (n=5 different cultures). Error bars represent SD. B. MTT assays of cell viability after treatment of GAN cells and VS cells with 2 mM aspirin, 2 mM NaSal, 2 mM 5-ASA, and 10 μM COX-2 inhibitor II (5-6 different wells from 3 different samples for each treatment). Error bar represent standard deviation. There is no error bar associated with NT because it was set to 100% for every comparison of non-treated VS and GAN cells. C. Secreted PTG levels in VS culture media after treatment for NT, 1 mM and 5 mM aspirin, and 5 mM NaSal (n=3-4 different cultures). Error bars represent SD. D. Quantification of proliferation changes after treatment with aspirin, NaSal and 5-ASA in primary SCs normalized to proliferation in NT cells (n=3-4 different cultures). Error bars represent SEM. *p<0.05. **p<0.01. ***p<0.001. re = in comparison to.