Abstract
Neuroblastoma resistance to apoptosis may contribute to the aggressive behavior of this tumor. Therefore, it would be relevant to activate endogenous cellular death mechanisms as a way to improve neuroblastoma therapy. We used the neuroblastoma SH-SY5Y cell line as a model to study the mechanisms involved in acetaminophen (AAP)-mediated toxicity by measuring CYP2E1 enzymatic activity, NFkB p65 subunit activation and translocation to the nucleus, Bax accumulation into the mitochondria, cytochrome c release and caspase activation. AAP activates the intrinsic death pathway in the SH-SY5Y human neuroblastoma cell line. AAP metabolism is partially responsible for this activation, because blockade of the cytochrome CYP2E1 significantly reduced but did not totally prevent, AAP-induced SH-SY5Y cell death. AAP also induced NFkB p65 activation by phosphorylation and its translocation to the nucleus, where NFkB p65 increased IL-1β production. This increase contributed to neuroblastoma cell death through a mechanism involving Bax accumulation into the mitochondria, cytochrome c release and caspase3 activation. Blockade of NFkB translocation to the nucleus by the peptide SN50 prevented AAP-mediated cell death and IL-1β production. Moreover, overexpression of the antiapoptotic protein Bcl-xL did not decrease AAP-mediated IL-1β production, but prevented both AAP and IL-1β-mediated cell death. We also confirmed the AAP toxic actions on SK-N-MC neuroepithelioma and U87MG glioblastoma cell lines. The results presented here suggest that AAP activates the intrinsic death pathway in neuroblastoma cells through a mechanism involving NFkB and IL-1β.
Highlights
Neuroblastoma is the most common tumor in infants younger than one year of age
Effect of AAP on SH-SY5Y viability In order to evaluate the effect of AAP on SH-SY5Y human neuroblastoma viability, cells were treated with various concentrations of AAP for 24, 48 and 72 h and the percentage of MTT transformed as well as the percentage of LDH activity released to the culture medium (% LDH released) were measured as indices of cellular death
LDH activity has been considered an index of necrosis or of secondary necrotic cell death after apoptosis occurring in cultures in which the phagocytic component is absent and apoptotic bodies cannot be removed [20]
Summary
Neuroblastoma is the most common tumor in infants younger than one year of age. Neuroblastoma accounts for 7–10% of childhood cancers with an annual incidence of 8 per million children under the age of 15 [1,2]. It has been proposed that resistance to extrinsic apoptosis pathway activation is one of the mechanisms that contributes to the aggressive behavior of advanced-stage neuroblastoma, in older children [5,6]. For this reason, in recent years one of the goals of research on drug treatments for neuroblastoma has been to study the activation of endogenous cellular death mechanisms in neuroblastoma to improve therapy. Apoptosis pathways may be initiated through various entry sites including death receptors (extrinsic receptor-mediated pathway) and mitochondria (intrinsic mitochondrial pathway), with the latter playing a crucial role in drug-induced apoptosis [8,9]
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