TRAIL induces apoptosis in cancer cells, but it does not affect normal cells (Walczak et al., 1999
). However, a significant proportion of cancer cells exhibit resistance to the cytotoxic effect of this ligand, suggesting that the use of TRAIL alone may not be enough to treat cancers (Wajant et al., 2002
). TRAIL-resistance is due to de-regulated expression of the TRAIL receptors or the intracellular components acting downstream of the receptors. Previous reports have shown that conventional chemotherapeutic agents (Gibson et al., 2000
; Singh et al., 2003
), irradiation (Shankar et al., 2004b
; Marini et al., 2005
) and HDAC (histone deacetylase) inhibitors (Singh et al., 2005
) enhance the cytotoxicity of TRAIL via up-regulation of TRAIL receptors. Up-regulation of DRs' expression by the chemotherapeutic agent is dependent on the activity of NF-κB (nuclear factor κB; Mendoza et al., 2008
) or p53 (Shankar et al., 2004a
; Seitz et al., 2010
), which transcriptionally regulate the expression of DRs by binding to sites in the promoter (Yoshida et al., 2001
; Liu et al., 2004
). Overexpression of the NF-κB p65 subunit up-regulates TRAIL-R2 expression in epithelial-derived cell lines (Shetty et al., 2002
). We have shown that etoposide treatment significantly increases the surface expression of TRAIL-R2 in the neuroblastoma cell lines IMR-32 and SK-N-MC. Our preliminary data demonstrated that etoposide treatment increased NF-κB p65 activity (data not shown), suggesting the possibility that etoposide increases TRAIL-R2 expression via an NF-κB pathway in neuroblastoma cells. This hypothesis still requires examination.
Treatment with etoposide prior to TRAIL treatment significantly enhanced cell death in SK-N-MC cells, compared with etoposide or TRAIL treatment alone. Moreover, the enhanced cell death was completely inhibited by treatment with the fusion protein DR5:Fc, which acts as a dominant-negative by competing with endogenous DR5 on the cell surface. Although etoposide treatment dramatically increased TRAIL-R2 expression and slightly increased expression of TRAIL-R1, -R3 and -R4, cell death induced by serial treatment with etoposide and TRAIL did not increase in comparison with etoposide alone in IMR-32 cells. This result may reflect the deregulation of intracellular components rather than the slight increase in DcRs.
Caspase 8 is an essential mediator of the initiation of DR-induced apoptosis (Varfolomeev et al., 1998
) and is frequently lacking in cancers, such as neuroblastoma, medulloblastoma, rhabdomyosarcoma, small cell lung cancer and melanoma (Teitz et al., 2000
; Fulda et al., 2001
; Pingoud-Meier et al., 2003
). Loss of caspase 8 expression correlates with low sensitivity to TRAIL cytotoxicity. Caspase 8 expressing cancer cells are sensitive to TRAIL cytotoxicity, whereas cells lacking caspase 8 are TRAIL-resistant. Moreover, cells lacking caspase 8 are sensitized to TRAIL cytotoxicity when caspase 8 expression, which can be induced by AzaC (5-aza-2′ deoxycytidine) or IFNγ, is restored (Hopkins-Donaldson et al., 2000
). The loss of caspase 8 expression is the result of gene silencing by aberrant methylation and can be restored by demethylating agents such as AzaC. However, the clinical use of demethylating agents has been limited by the toxic side effects of these drugs (Hopkins-Donaldson et al., 2000
; Teitz et al., 2000
; Michalowski et al., 2008
). IFNγ restores caspase 8 expression through transcriptional activation, which involves a Stat1/IRF1 pathway (Ruiz-Ruiz and Lopez-Rivas, 2002
; Fulda and Debatin, 2002
). We have shown that TRAIL treatment increases caspase 8 activity in SK-N-MC cells, but not in IMR-32 cells, because the gene was not expressed in IMR-32 cells. Treatment with IFNγ was used to restore caspase 8 expression in IMR-32 cells. Caspase 8 expression gradually increased in response to IFNγ treatment and IMR-32 cells became sensitized to TRAIL. As a result, treatment with etoposide prior to TRAIL treatment enhanced cell death. In addition, treatment with a caspase 8 inhibitor or the dominant negative DR5:Fc decreased etoposide and TRAIL-induced cell death, indicating that etoposide potentiated the TRAIL-induced cell death in caspase 8 restored IMR-32 cells.
SK-N-MC is non-MYCN amplified neuroblastoma cells, whereas IMR-32 contains 25 copies of the MYCN gene per cell (Reynolds et al., 1988
). A subset of neuroblastoma with amplification of the oncogene MYCN has a particularly poor prognosis (Hopkins-Donaldson et al., 2000
). The aberrant caspase 8 methylation has been found exclusively in neuroblastoma patient biopsies or cancer cell lines with MYCN amplification in several studies (Fulda et al., 1999
; Hopkins-Donaldson et al., 2000
). The inactivation of caspase 8 by hypermethylation has become a hallmark of defective apoptosis in advanced disease, suggesting that caspase 8 may act as a tumour suppressor gene in neuroblastoma (Fulda et al., 1999
; Gonzalez-Gomez et al., 2003
). These studies focused on the association between MYCN amplification and the methylation status of caspase 8 gene rather than caspase 8 expression. However, Fulda et al. (2001)
found no correlation between MYCN amplification and caspase 8 mRNA or protein expression. In addition, another study in neuroblastoma cell line models clearly showed that MYCN has no direct effect on caspase 8 expression (van Noesel et al., 2003
). Thus, the correlation between MYCN amplification and caspase 8 expression needs further investigation.
TRAIL-induced apoptosis has been correlated with the expression of Bcl-2 family members (Walczak et al., 2000
). Mcl-1 is an anti-apoptotic Bcl-2 family protein that can bind to BH3-only proteins such as Bid and thereby inhibits tBid (truncated Bid)-mediated cytochrome c
release from mitochondria (Clohessy et al., 2006
; Adams and Cory, 2007
). A decrease of Mcl-1 expression leads to cytochrome c
release and the activation of caspases 9 and 3, which results in cells undergoing apoptosis (Clohessy et al., 2006
). DNA damage (Arbour et al., 2008
) and activated caspase 8 (Han et al., 2004
; Weng et al., 2005
; Han et al., 2006
) can induce Mcl-1 cleavage and initiate the mitochondrial cascade. Caspases 8, 9 and 3 activation, Mcl-1 cleavage and Bid truncation increased in response to consecutive treatment with etoposide and TRAIL in SK-N-MC cells. The combined etoposide and TRAIL treatment increased caspase activation, Mcl-1 cleavage and Bid truncation in caspase 8 restored IMR-32 cells. The data suggest that etoposide-potentiated TRAIL-induced cell death is mediated by intrinsic cell death signalling pathways.
Our results indicate that etoposide treatment can enhance TRAIL cytotoxicity in neuroblastoma cells by up-regulating TRAIL-R2 expression. Furthermore, TRAIL cytotoxicity requires caspase 8 expression. Combined treatment with etoposide and TRAIL may be useful as a clinically applicable strategy for the treatment of neuroblastoma.