Article
The nitric oxide donor JS-K induces cell death of GBM cells by nonapoptotic mechanisms in vitro
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Authors
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Jessica Günzle
- Klinik für Neurochirurgie, Universitätsklinikum Freiburg
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Nadja Osterberg
- Klinik für Neurochirurgie, Universitätsklinikum Freiburg
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Astrid Weyerbrock
- Klinik für Neurochirurgie, Universitätsklinikum Freiburg
| Published: | June 2, 2015 |
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Outline
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Objective: Glioblastoma multiforme (GBM) is the most malignant primary brain tumor showing upregulated levels of gluthatione S transferases (GSTs). The nitric oxide (NO) donor JS-K is activated by GSTs in GBM cells and induces cell death at high doses. The aim of this study was to determine whether NO-induced cell death is triggered by apoptotic or necrotic pathways.
Method: U87, LN229 and primary GBM cells were treated with JS-K (1-15μM) for 48h or 72h. Cell viability was analyzed by MTT. Apoptosis was assessed by Western Blot for caspase3 and cleaved PARP1. For discrimination of apoptosis and necrosis FACS analysis was performed with propidium iodide and targeting AnnexinV. Apoptosis and mitotic catastrophe were assessed by TUNEL assay. To analyze energy metabolism in the glioma cells, an adenosine triphosphate (ATP) assay was performed. Statistical analysis was performed by the student's t-test.
Results: JS-K decreases cell viability in the 3 GBM cell lines to <5% (15μM) in a dose-dependent manner compared to untreated controls. JS-K treatment causes cleavage of PARP1 but not activation of caspase3 indicating that cells do not undergo classical apoptosis. FACS analysis after JS-K treatment reveals a NO dose-dependent increase of necrotic cells but no significant increase in apoptosis. ATP assay after JS-K treatment shows a strong reduction in energy level indicating necrosis (p<0.001). TUNEL assay shows the impact of NO on mitotic catastrophe indicated by multifragmented nuclei as part of necrosis but no significant number of apoptotic cells.
Conclusions: For the first time we demonstrate that JS-K induces cell death via nonapoptotic mechanisms. In addition we show that PARP1 is not an exclusive marker for late apoptosis. The level of morphological changes of GBM cells indicating mitotic catastrophe correlates with the increase of necrosis after JS-K treatment. Therefore, we conclude that mitotic catastrophe is associated with necrosis rather than with apoptosis. However, in combination with other cancer therapies, JS-K can contribute to apoptotic cell death by triggering necrosis.
