Nitric oxide released from JS-K induces cell death by mitotic catastrophe as part of necrosis in glioblastoma multiforme

Abstract

The nitric oxide (NO) donor JS-K is specifically activated by glutathione S-transferases (GSTs) in GST-overexpressing cells. We have shown the induction of cell death in glioblastoma multiforme (GBM) cells at high JS-K doses but the mechanism remains unclear. The aim of this study was to determine whether NO-induced cell death is triggered by induction of apoptotic or necrotic pathways. For the first time, we demonstrate that NO induces cell death via mitotic catastrophe (MC) with non-apoptotic mechanisms in GBM cells. Moreover, the level of morphological changes indicating MC correlates with increased necrosis. Therefore, we conclude that MC is the main mechanism by which GBM cells undergo cell death after treatment with JS-K associated with necrosis rather than apoptosis. In addition, we show that PARP1 is not an exclusive marker for late apoptosis but is also involved in MC. Activating an alternative way of cell death can be useful for the multimodal cancer therapy of GBM known for its strong anti-apoptotic mechanisms and drug resistance.

Figure 1

Cytotoxic effect of JS-K (1–25 μM) and DMSO control (0,  48%) on U87, LN229 and primary IC cells after 48 h (a) and 72 h (b). Cell viability was determined by MTT assay at the end of the incubation time. Cytotoxicity induced by JS-K was plotted relative to viability of untreated controls set to 100% (±s.d. of three independent experiments). Asterisks (*P<0.05, **P<0.01, ***P<0.001) indicate significance between JS-K and controls

Figure 2

Western blot analysis for caspases 9 and 3, cleaved caspases 9 and 8 (cCaspase), PARP1 and cleaved PARP1 (cPARP1) in U87 (a), IC (b) and LN229 (c) cells after 48 h and 72 h exposure of JS-K (1–5 μM). 25 μg of protein lysates were separated with SDS-PAGE and probed with the different antibodies. Caspase 9 and 8 are expressed and cleaved equally in all three cell lines and both in untreated and DMSO controls. No cleavage and no decrease in protein level of caspase 3 can be shown. The figures shown are representative for three independent experiments

Figure 3

Dose-dependent increase of intracellular cGMP in U87 (a) and LN229 (b) cells mediated by JS-K after 48 h as well as in necrotic controls exposed to H₂O₂ (3 mM). cGMP level remained stable after induction of apoptosis by etoposide (10 μM). Asterisks (*P<0.05, **P<0.01, ***P<0.001) indicate significance compared to untreated controls. Western blot analysis for pAkt in LN229 (c) cells show increase of phosphorylation after exposure to JS-K for 48 h compared to control. The blot is representative for three independent experiments

Figure 4

Dose- and time-dependent decrease of intracellular ATP level in U87 (a), IC (b) and LN229 (c) cells after exposure to JS-K for 48 and 72 h. ATP level increased after treatment with etoposide (10 μM) in U87 (a) and LN229 (c) and decreased by H₂O₂ (3 mM) after 4 h exposure in all three cell lines. Mean values±s.d. of three independent experiments are plotted. Asterisks (*P<0.05, **P<0.01, ***P<0.001) indicate significance between JS-K and untreated controls

Figure 5

Flow cytometry analysis of annexin V and PI in U87 (a), IC (b) and LN229 (c) cells exposed to JS-K for 48 and 72 h and both untreated and DMSO-controls. 2 × 10⁴ cells were analysed by FACSCalibur and show significant increase of necrotic cell population. Treatment groups were normalized to total cell number and plotted relative to untreated controls±s.d. of three independent experiments. Asterisks (*P<0.05, **P<0.01, ***P<0.001) indicate significance between apoptotic and necrotic population

Figure 6

Figures show MC induced by JS-K (1–10 μM) in U87, IC and LN229 cells after 48 and 72 h. Characteristics of MC are indicated with asterisk as example for 10 μM JS-K after 48 h. Samples were analysed by microscopy (40 ×, scale bar represent 25 μm). Data are representative for three independent experiments

Figure 7

Quantitative analysis of TUNEL and MC in U87 (a), IC (b) and LN229 (c) cells after exposure to JS-K for 48 and 72 h. The induction of MC was determined by counting cells with characteristics described by Roninson et al. Induction of cell death by JS-K was plotted relative to total cell number and show a significant dose- and time-dependent increase in MC compared to apoptosis. Asterisks (*P<0.05, **P<0.01, ***P<0.001) indicate significance between apoptosis and MC with ±s.d. of three independent experiments