These data indicate that Fas-mediated apoptosis in neuroblastoma cells is mitochondria-dependent and inhibited both at the mitochondrial level and at the level of caspase 8 activation.
We conclude that caspase-8 acts as a tumor suppressor gene in neuroblastomas, that its silencing provides a permissive environment for MYCN gene amplification once the tumors are treated with chemotherapeutic drugs/irradiation, and that expression of this gene in these tumor cells may be of clinical benefit.
A missense mutation was detected at codon 96, GCT (Alanine) to GTT (Valine), of the caspase 8 gene in one of the NB cell lines lacking caspase 8 expression.
As frequent methylation of the CASP8 gene has also been reported in neuroblastoma, we investigated whether RASSF1A and CASP8 methylation were independent or related events.
However, approximately 20% of the neuroblastoma cell lines with methylated CASP8 alleles are also highly resistant to staurosporine (STS)- and radiation-induced cell death, presumably because cytochrome c is not released from mitochondria.
Caspase-8 silenced N-type invasive NB cell lines LAN-1 and IMR-32 were investigated for their sensitivity to dox, and compared to S-type noninvasive SH-EP NB cells expressing caspase-8.
These results indicate that the profile of caspase 8 expression is an important determinant of the response of neuroblastoma cells to Fas-mediated cell death.
The retinoic acid analogue 4HPR, IFN-gamma, and the demethylating agent 5-aza-cytidine activate this promoter in NB cells that lack endogenous caspase-8, indicating that this element may regulate both constitutive and inducible CASP8 expression.
The authors found that interferon-gamma induces caspase-8 expression in neuroblastoma cells irrespective of the gene silenced by hypermethylation of caspase-8 promoter.
Our results show that restoration of active caspase-8 expression in a caspase-8-deficient NB cell line is necessary and sufficient to fully restore TRAIL sensitivity.
Furthermore, gemcitabine-induced apoptosis was observed irrespective of the caspase-8 status of neuroblastoma cells, which indicates that apoptosis depends on the mitochondrial pathway.
Furthermore, the methylation pattern of 14.3.3sigma, RASSF1A and of an intragenic segment of CASP8 was significantly different between MYCN amplified and single copy neuroblastoma suggesting a specific role of epigenetic alterations in aggressive neuroblastoma.
Thus, by demonstrating that 5-dAzaC and IFN-gamma at relatively low individual concentrations cooperate to restore caspase-8 expression and sensitize resistant neuroblastoma and medulloblastoma cells to TRAIL-induced apoptosis, our findings have important implications for novel strategies targeting defective apoptosis pathways in neuroectodermal tumors.
We investigated whether the levels of survivin and caspase 8 and the ratio between these 2 apoptotic factors correlate with tumor biology and predicts outcome in patients with neuroblastoma.
We sought to determine whether caspase 8l is present in neuroblastoma and whether over-expression of this protein could inhibit caspase 8-dependent apoptosis.
Thus, by demonstrating that 5-dAzaC and IFN-gamma at relatively low individual concentrations cooperate to restore caspase-8 expression and sensitize resistant neuroblastoma and medulloblastoma cells to TRAIL-induced apoptosis, our findings have important implications for novel strategies targeting defective apoptosis pathways in neuroectodermal tumors.
These findings define caspase-8 as a metastasis suppressor gene that, together with integrins, regulates the survival and invasive capacity of neuroblastoma cells.