gms | German Medical Science

Objective: Glioblastoma multiforme (GBM) is characterized by a high treatment resistance, an infiltrative growth pattern and a short overall survival. Experimental strategies against malignant gliomas include cyclooxygenase (COX) inhibition and nitric oxide-based (NO) 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 (GST). The aim of this study is to analyze the impact of NO on COX expression and metabolism and to determine potential synergistic antitumor effects of COX inhibition by acetyl salicylic acid (ASA) and JS-K in glioma cells.

Methods: U87, LN229 and primary glioblastoma cells (IC) were treated with JS-K (1-15 µM) for 6h. Cell viability was analyzed by MTT assay after therapy with JS-K and/or ASA (10 mM). COX-2 expression was determined by qRT-PCR and regulation of COX-2 activity by ELISA. To differentiate between apoptosis or necrosis as distinct forms of cell death, Western Blot for caspase-3 and FACS analysis for AnnexinV and propidium iodide were performed. Intracellular ATP and calcium levels were measured by ELISA to assess cellular energy metabolism. Phosphorylation and activation of Akt was determined by Western Blot. Statistical analysis was performed by Student´s t-test.

Results: JS-K significantly increased COX-2 expression and COX activity (LN229, 200±11 %, p=0.0001) time- and dose-dependently. Cell viability was significantly reduced by JS-K to 64±10 % (LN229, p=0.003), 57±4 % (U87, p<0.0001) and 37±8 % (IC, p=0.0002) after 6 h. Combination therapy with ASA reduced cell viability further to 26±3 % (LN229, p<=0.0001), 25±3 % (U87, p<0.0001) and 21±3 % (IC, p<0.0001). Both treatments led to a dose-dependent increase in necrotic cell death (LN229, 80±20 %, p= 0.004). The lack of caspase-3 activation and the persistent small apoptotic population in FACS analysis exclude apoptosis as the major cell death mechanism. Both mono- and combination therapy induced activation of Akt signaling which is associated with antiapoptotic pathways. JS-K decreased intracellular ATP which is indicative of a necrotic process. While ASA did not alter intracellular calcium concentration alone (LN229, 118±12 %, p=0.05), it significantly enhanced the rise in intracellular calcium induced by JS-K (289±10 %, p<=0.0001).

Conclusion: COX inhibition by ASA enhances the cytotoxic effect of NO released from JS-K at doses which are not cytotoxic and do not cause changes in energy metabolism. Glioma cells predominantly die by necrotic cell death as shown by reduced energy metabolism and activation of anti-apoptotic mechanisms. Combining COX inhibition by ASA and NO-based antitumor strategies provides the opportunity to increase treatment efficacy with a well-established remedy.