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Background: Neuroblastoma (NB) is the second most common solid childhood tumour, an aggressive disease for which new therapeutic strategies are strongly needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumour cells, but not in normal tissues and therefore represents a valuable candidate in apoptosis-inducing therapies. Caspase-8 is silenced in a subset of highly malignant NB cells, which results in full TRAIL resistance. In addition, despite constitutive caspase-8 expression, or its possible restoration by different strategies, NB cells remain weakly sensitive to TRAIL indicating a need to develop strategies to sensitise NB cells to TRAIL. Histone deacetylase inhibitors (HDACIs) are a new class of anti-cancer agent inducing apoptosis or cell cycle arrest in tumour cells with very low toxicity toward normal cells. Although HDACIs were recently shown to increase death induced by TRAIL in weakly TRAIL-sensitive tumour cells, the precise involved sensitisation mechanisms have not been fully identified., Methods: NB cell lines were treated with various doses of HDACIs and TRAIL, then cytotoxicity was analysed by MTS/PMS proliferation assays, apoptosis was measured by the Propidium staining method, caspases activity by colorimetric protease assays, and (in)activation of apoptotic proteins by immunoblotting., Results: Sub-toxic doses of HDACIs strongly sensitised caspase-8 positive NB cell lines to TRAIL induced apoptosis in a caspases dependent manner. Combined treatments increased the activation of caspases and Bid, and the inactivation of the anti-apoptotic proteins XIAP, Bcl-x, RIP, and survivin, thereby increasing the pro- to anti-apoptotic protein ratio. It also enhanced the activation of the mitochondrial pathway. Interestingly, the kinetics of caspases activation and inactivation of anti-apoptotic proteins is accelerated by combined treatment with TRAIL and HDACIs compared to TRAIL alone. In contrast, cell surface expression of TRAIL-receptors or TRAIL is not affected by sub-toxic doses of HDACIs., Conclusion: HDACIs were shown to activate the mitochondrial pathway and to sensitise NB cells to TRAIL by enhancing the amplitude of the apoptotic cascade and by restoring an apoptosis-prone ratio of pro- to anti-apoptotic proteins. Combining HDACIs and TRAIL could therefore represent a weakly toxic and promising strategy to target TRAIL-resistant tumours such as neuroblastomas.

Neuroblastoma (NB) is a childhood neoplasm which heterogeneous behavior can be explained by differential regulation of apoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces rapid apoptosis in most tumor cells and thus represents a promising anticancer agent. We have reported silencing of caspase-8 expression in highly malignant NB cells as a possible mechanism of resistance to TRAIL-induced apoptosis. To explore the particular contribution of caspase-8 in such resistance, retroviral-mediated stable caspase-8 expression was induced in the IGR-N91 cells. As a result, sensitivity to TRAIL was fully restored in the caspase-8-complemented cells. TRAIL-induced cell death could be further enhanced by cotreatment of IGR-N91-C8 and SH-EP cells with cycloheximide or subtoxic concentrations of chemotherapeutic drugs in a caspase-dependent manner. Sensitization to TRAIL involved enhanced death receptor DR5 expression, activation of Bid and the complete caspases cascade. Interestingly, combined treatments also enhanced the cleavage-mediated inactivation of antiapoptotic molecules, XIAP, Bcl-x(L) and RIP. 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. Moreover, the synergistic effect of drugs and TRAIL results from activation of the caspase cascade via a mitochondrial pathway-mediated amplification loop and from the inactivation of apoptosis inhibitors.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumor cells, a process sometimes potentiated by chemotherapeutic drugs or cycloheximide (CHX). Childhood neuroblastoma (NB) is a clinically and biologically heterogeneous neoplasm whose behavior can be explained by differential regulation of apoptosis. The non-invasive S-type NB cell lines are sensitive to TRAIL, whereas the invasive N-type NB cell lines are resistant. We have reported the silencing of caspase-8 expression in N-type cells as a possible mechanism of death receptor-mediated resistance to apoptosis in NB. The recently observed deregulation of caspase-10 in these cells prompted us to investigate the particular contribution of caspase-8 silencing in the resistance to TRAIL in N-type cells. Stable caspase-8 expression was therefore restored in the IGR-N91 cell line by retroviral infection. The IGR-N91-C8 cells became sensitive to TRAIL-mediated apoptosis, whereas the control vector-infected IGR-N91-M cells remained resistant. Interestingly, the apoptotic response to TRAIL was enhanced by co-treatment of SH-EP and IGR-N91-C8 cells with CHX or with sub-toxic concentration of doxorubicin (DOX) in a caspase-dependent manner, as cells could be protected from death by specific caspase-8 or pan-caspase inhibitors. CHX or DOX was shown to enhance TRAIL-induced caspase-8 activation and loss of mitochondrial transmembrane potential. In conclusion, restoration of active caspase-8 expression in caspase-8- and caspase-10-deficient IGR-N-91 cell line is necessary and sufficient to fully restore TRAIL-mediated cell death. Moreover, DOX and CHX were able to sensitize NB cell lines to TRAIL-induced apoptosis in a caspase-8-dependent manner by engaging death receptor and mitochondrial signaling pathways.

Neuroblastoma (NB) is a childhood neoplasm which heterogeneous behavior can be explained by differential regulation of apoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces rapid apoptosis in most tumor cells and thus represents a promising anticancer agent. We have reported silencing of caspase-8 expression in highly malignant NB cells as a possible mechanism of resistance to TRAIL-induced apoptosis. To explore the particular contribution of caspase-8 in such resistance, retroviral-mediated stable caspase-8 expression was induced in the IGR-N91 cells. As a result, sensitivity to TRAIL was fully restored in the caspase-8-complemented cells. TRAIL-induced cell death could be further enhanced by cotreatment of IGR-N91-C8 and SH-EP cells with cycloheximide or subtoxic concentrations of chemotherapeutic drugs in a caspase-dependent manner. Sensitization to TRAIL involved enhanced death receptor DR5 expression, activation of Bid and the complete caspases cascade. Interestingly, combined treatments also enhanced the cleavage-mediated inactivation of antiapoptotic molecules, XIAP, Bcl-xL and RIP.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. Moreover, the synergistic effect of drugs and TRAIL results from activation of the caspase cascade via a mitochondrial pathway-mediated amplification loop and from the inactivation of apoptosis inhibitors.Oncogene (2004) 23, 5415-5425. doi:10.1038/sj.onc.1207704 Published online 19 April 2004 [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumor cells, a process sometimes potentiated by chemotherapeutic drugs or cycloheximide (CHX). Childhood neuroblastoma (NB) is a clinically and biologically heterogeneous neoplasm whose behavior can be explained by differential regulation of apoptosis. The non-invasive S-type NB cell lines are sensitive to TRAIL, whereas the invasive N-type NB cell lines are resistant. We have reported the silencing of caspase-8 expression in N-type cells as a possible mechanism of death receptor-mediated resistance to apoptosis in NB. The recently observed deregulation of caspase-10 in these cells prompted us to investigate the particular contribution of caspase-8 silencing in the resistance to TRAIL in N-type cells. Stable caspase-8 expression was therefore restored in the IGR-N91 cell line by retroviral infection. The IGR-N91-C8 cells became sensitive to TRAIL-mediated apoptosis, whereas the control vector-infected IGR-N91-M cells remained resistant. Interestingly, the apoptotic response to TRAIL was enhanced by co-treatment of SH-EP and IGR-N91-C8 cells with CHX or with sub-toxic concentration of doxorubicin (DOX) in a caspase-dependent manner, as cells could be protected from death by specific caspase-8 or pan-caspase inhibitors. CHX or DOX was shown to enhance TRAIL-induced caspase-8 activation and loss of mitochondrial transmembrane potential. In conclusion, restoration of active caspase-8 expression in caspase-8- and caspase-10-deficient IGR-N-91 cell line is necessary and sufficient to fully restore TRAIL-mediated cell death. Moreover, DOX and CHX were able to sensitize NB cell lines to TRAIL-induced apoptosis in a caspase-8-dependent manner by engaging death receptor and mitochondrial signaling pathways. [ABSTRACT FROM AUTHOR]