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. 2007 Mar;150(5):577-85.
doi: 10.1038/sj.bjp.0706993. Epub 2007 Jan 22.

Acetaminophen potentiates staurosporine-induced death in a human neuroblastoma cell line

I Posadas  1 , V Vellecco, P Santos, J Prieto-Lloret, V Ceña
Affiliations

Acetaminophen potentiates staurosporine-induced death in a human neuroblastoma cell line

I Posadas et al. Br J Pharmacol. 2007 Mar.

Abstract

Background and purpose: Neuroblastoma is the most common solid tumour in infants characterized by a high resistance to apoptosis. Recently, the cyclo-oxygenase pathway has been considered a potential target in the treatment of different kinds of tumours. The aim of the present work was to investigate a possible relationship between cyclo-oxygenase pathway and stauroporine-induced apoptosis in the neuroblastoma cell line SH-SY5Y.

Experimental approach: Cellular viability was measured by release of LDH. DNA fragmentation was visualized by electrophoresis on agarose gel containing ethidium bromide. Cyclo-oxygenase activity was measured in microsomal fractions obtained from cells by quantification of its final product PGE2 by RIA. Caspase-3 activity was measured fluorimetrically and Western blot analysis was performed to assess cytochrome c expression.

Key results: We have found that staurosporine (500 nM) induced cellular death in a time-dependent manner in SH-SY5Y human neuroblastoma cells. Cyclo-oxygenase enzymatic activity was present in SH-SY5Y human neuroblastoma cells under basal conditions and pharmacological experiments using COX inhibitors indicate that cyclo-oxygenase-1 and cyclo-oxygenase-3 are the active isoforms in these cells. Co-incubation of SH-SY5Y cells with staurosporine (500 nM) and acetaminophen for 24 h potentiated staurosporine-mediated cellular death in a concentration-dependent manner. This process is mediated by an increase in cytochrome c release and caspase 3 activation and is prevented by N-acetylcysteine or the superoxide dismutase mimetic, MnTBAP.

Conclusions and implications: Acetaminophen potentiates staurosporine-mediated neuroblastoma cell death. The mechanism of action of acetaminophen seems to be related to production of reactive oxygen species and decreased intracellular glutathione levels.

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Figures

Figure 1
Figure 1
Effect of staurosporine (500 nM) on SH-SY5Y neuroblastoma cell line. (a) Time course of staurosporine-induced cell death. Release of LDH is expressed as percentage of total LDH content in the cell at the beginning of the experiment. Data represent mean+s.e.m. of 12 experiments. **P<0.01 as compared to vehicle-treated cells (v). (b) Time course of staurosporine-induced caspase 3 activity in total lysates. Caspase 3 activity was measured as indicated in Methods. Data are expressed as mean±s.e.m. of 12 experiments. **P<0.01 ***P<0.001, compared to vehicle-treated cells. (c) DNA fragmentation in SH-SY5Y cells treated with vehicle (v) or staurosporine (St) 500 nM for 24 h. Nucleosomal fragmentation was visualized by agarose gel electrophoresis. M indicates DNA size markers. Data shown are representative of three different experiments. (d) Densitometric analysis of low-molecular weight DNA fragments extracted from vehicle- or staurosporine-treated cells. Optical density is expressed in arbitrary units. Data are expressed as mean±s.e.m. of three experiments. ***P<0.001 compared to vehicle-treated cells.
Figure 2
Figure 2
COX activity in SH-SY5Y neuroblastoma cell line. (a) Left panel, COX activity is expressed as PGE2 generated by the microsomal fraction obtained from SH-SY5Y cells treated with vehicle (v) or staurosporine (st; 500 nM; 24 h). (a) Right panel, the PGE2 levels released into supernatants obtained from vehicle-(v) or staurosporine (St; 500 nM)-treated SH-SY5Y cells for 24 h. Data represent mean+s.e.m. of 12 experiments. ***P<0.001 as compared with vehicle. (b) Effect of indomethacin, NS-398 and acetaminophen on COX activity measured in microsomal fraction obtained from vehicle-treated SH-SY5Y cells. Data represent mean+s.e.m. of 12 experiments. ***P<0.001 **P<0.01, compared to vehicle-treated cells. (c) Effect of indomethacin, NS-398 and acetaminophen on COX activity measured in microsomal fraction obtained from staurosporine (500 nM)-treated SH-SY5Y cells for 24 h. Data represent mean±s.e.m. of 12 experiments. ***P<0.001; *P<0.05 in comparison to staurosporine-treated cells.
Figure 3
Figure 3
Caspase 3 activity in SH-SY5Y neuroblastoma cell line. Caspase 3 activity measured in total lysates obtained from SH-SY5Y cells treated with vehicle, staurosporine 500 nM or staurosporine 500 nM+different acetaminophen concentrations (in mM) for 18 h. Data are expressed as mean±s.e.m. (n=12). ***P<0.001 compared to staurosporine-treated cells.
Figure 4
Figure 4
Effect of staurosporine (St) and staurosporine+acetaminophen (2 mM; St+AAP) on cytochrome c (Cyt C) released from mitochondria 24 h after treatment. (a) Cyt C levels in mitochondrial fractions; OxPhos Complex IV subunit IV (COX-IV) protein levels were used as mitochondrial protein loading controls. (b) Cyt C levels in cytosolic fractions; α-tubulin protein levels were used as cytosolic protein loading controls. Figures are representative of three separate experiments.
Figure 5
Figure 5
Effect of BA, NAC and MnTBAP on SH-SY5Y cell viability. (a) Percentage of LDH released from cells treated with vehicle or staurosporine 500 nM alone or in the presence of MnTBAP 1 μM, NAC 100 μM or BA 2 μM for 24 h. Data represent mean+s.e.m. of 12 experiments. ***P<0.001 as compared to staurosporine-treated cells. (b) Percentage of LDH released from cells treated with vehicle, staurosporine 500 nM+acetaminophen 2 mM or staurosporine 500 nM+acetaminophen 2 mM in the presence of MnTBAP 1 μM, NAC 100 μM or BA 2 μM for 24 h. Data represent mean±s.e.m. of 12 experiments. ***P<0.001 as compared to staurosporine+acetaminophen-treated cells.
Figure 6
Figure 6
Effect of staurosporine (St) and staurosporine+acetaminophen (St+AAP) on GSt content 18 h after treatment. (a) GSt was measured in total lysates obtained from SH-SY5Y cells treated with vehicle, staurosporine 500 nM or staurosporine 500 nM+different acetaminophen concentrations (in mM) for 18 h. Data are expressed as mean±s.e.m (n=12). **P<0.01; ***P<0.001 compared to staurosporine-treated cells. (b) Percentage of LDH released from cells treated with vehicle, staurosporine 500 nM, staurosporine 500 nM+acetaminophen 2 mM in the presence of disulfiram 100 nM for 24 h. Data are expressed as mean±s.e.m. (n=12). ***P<0.001 compared to staurosporine+acetaminophen-treated cells.
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