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Cyclooxygenase (COX) Inhibition by Acetyl Salicylic Acid (ASA) Enhances Antitumor Effects of Nitric Oxide in Glioblastoma In Vitro

  • Jessica Guenzle
  • Nicklas W. C. Garrelfs
  • Jonathan M. Goeldner
  • Astrid WeyerbrockEmail author
Article
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Abstract

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with a high recurrence rate and a median survival of about 16 months even with multimodal therapy. Novel experimental strategies against malignant gliomas include cyclooxygenase (COX) inhibition and nitric oxide (NO)–based therapies. Therapeutic doses of NO can be delivered to tumor cells by NO donors such as JS-K (O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) which releases NO upon enzymatic activation by glutathione S-transferase. COX-2 is frequently overexpressed in tumors and increases tumor invasiveness and angiogenesis. In this study, we show that pretreatment with acetyl salicylic acid (ASA) enhanced the cytotoxic antitumor effect of NO in vitro. Combination of low doses of JS-K and ASA revealed a dose-dependent synergistic increase of necrotic cell death under circumvention of classical apoptosis and alteration of the metabolic calcium level. These findings provide an opportunity to improve currently used therapeutic strategies in the treatment of gliomas with a well-established remedy.

Keywords

Nitric oxide Glioma JS-K Acetyl salicylic acid (ASA) 

Abbreviations

ASA

Acetyl salicylic acid

ATF3

Activating transcription factor 3

ATCC

American Type Culture

CO2

Carbon dioxide

DMEM

Dulbecco’s modified Eagle medium

DMSO

Dimethyl sulfoxide

ECL

Enhanced chemiluminescence

ECM

Extracellular matrix

eNOS

Endothelial nitric oxide synthase

ETO

Etoposide

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

GBM

Glioblastoma multiforme

GST

Glutathione S-transferase

H2O2

Hydrogen peroxide

iNOS

Inducible nitric oxide synthase

JS-K

(O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate)

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

nNOS

Neuronal nitric oxide synthase

NO

Nitric oxide

PBS

Phosphate-buffered saline

PI

Propidium iodide

PVDF

Polyvinylidene fluoride

SD

Standard deviation

SDS

Sodium dodecyl sulfate

TMZ

Temozolomide

Notes

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

12035_2019_1513_Fig7_ESM.png (522 kb)
Supplementary 1

The dose-dependent cytotoxic effect of ASA (0.5–25 mM) was assessed by MTT assay over 1 h (A) and 48 h (B) in primary IC cells, LN229 and U87. Viability was plotted relative to untreated controls set to 100% (± S.D. of three independent experiments). Asterisks (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001) indicate significance compared to the control. (PNG 521 kb)

12035_2019_1513_MOESM1_ESM.tif (16.2 mb)
High resolution image (TIF 16631 kb)
12035_2019_1513_Fig8_ESM.png (556 kb)
Supplementary 2

The time- and dose-dependent effect of JS-K on cell viability of primary IC cells (A), LN229 (B), and U87 (C) was assessed over 24 h for concentrations between 1 and 25 μM. Concentration of DMSO control is 0.48% according to the DMSO concentration in 25 μM JS-K. The synergistic cytotoxic effect of the combined therapy of ASA (24 h, 10 mM) and JS-K (1 h (D-F) and 24 h (G-I), 1–15 μM) compared to JS-K alone was determined by MTT viability assay for primary IC cells (D,G), LN229 (E,H) and U87 (F,I). Concentration of DMSO control is 0.29% according to the DMSO concentration in 15 μM JS-K. Viability was plotted relative to untreated controls set to 100%. Asterisks (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001) indicate significance to control. To demonstrate the difference to JS-K, cell viability of combined treatment was set to single treatment for each concentration over 1–24 h for IC cells (J), LN229 (K) and U87 (L). Asterisks (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001) indicate significance compared to JS-K monotherapy. Experiments were performed in triplicates (± S.D.). (PNG 555 kb)

12035_2019_1513_MOESM2_ESM.tif (12.9 mb)
High resolution image (TIF 13259 kb)
12035_2019_1513_Fig9_ESM.png (530 kb)
Supplementary 3

Western Blot analysis for caspase 3 (35 kDa) in primary IC cells (A), LN229 (B) and U87 (C) after 6 h exposure of JS-K (1–10 μM) and combination with ASA (24 h, 10 mM). No cleavage of caspase 3 could be observed in all three cell lines, DMSO controls and cells treated with ASA alone. The figures are representative for three independent experiments. GAPDH was used as loading control. (PNG 529 kb)

12035_2019_1513_MOESM3_ESM.tif (5.4 mb)
High resolution image (TIF 5487 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jessica Guenzle
    • 1
  • Nicklas W. C. Garrelfs
    • 1
  • Jonathan M. Goeldner
    • 1
  • Astrid Weyerbrock
    • 1
    • 2
    Email author
  1. 1.Department of Neurosurgery, Medical CenterUniversity of FreiburgFreiburgGermany
  2. 2.Department of NeurosurgeryCantonal Hospital St. GallenSt. GallenSwitzerland

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