Evaluation of IFN-γ effects on apoptosis and gene expression in neuroblastoma—Preclinical studies

Abstract

Loss of caspase-8 expression and resistance to cytotoxic agents occurs frequently in late stage neuroblastoma (NB). Interferon-gamma (IFN-gamma) induces caspase-8 in NB cells, sensitizing them to death receptor mediated apoptosis. This study characterizes the kinetics of this phenomenon and examines the effects of IFN-gamma on global gene expression to determine whether IFN-gamma responses are achievable at physiologically relevant doses and to define the biological effects of this cytokine. Here we examine the IFN-gamma responses of 16 NB cell lines. A single <5-min exposure to IFN-gamma (0.5 ng/ml) induced caspase-8 expression in all non-expressing cell lines and in 3/6 cell lines which already expressed high caspase-8. This increase in caspase-8 proteins was observed within 16 h and persisted for up to 9 days. Furthermore, IFN-gamma pretreatment of NB cells increased doxorubicin-induced apoptosis nearly 3-fold. Microarray analysis was used to identify additional genes involved in proliferation, signaling and apoptosis whose expression was modulated via IFN-gamma. Altered expression of these genes should further enhance the responsiveness of NB cells to chemotherapeutics. Thus, the use of IFN-gamma to sensitize NB cells to cytotoxic agents represents an attractive therapeutic strategy and warrants further investigation.

The Protumorigenic Role of Caspase-8 in Neuroblastoma

“…Caspase-8 has been shown to be silenced in up to 70% of advanced-stage NB patient tumors, while 30-50% maintained casp8 expression [17]. Significance of casp8 deficiency decreased chemotherapeutic drug sensitivity and enhanced metastatic cell survival by preventing integrin-mediated cell death [127,128,145,153]. In contrast, casp8 expression enhanced cell migration, adhesion, and promoted ruffle and lamellipodial formation [88,91,149].…”

“…Caspase-8 is silenced in up to 70% of advanced-stage NB patient tumors and the biological consequences of casp8 deficiency results in enhanced metastatic cell survival by preventing integrin-mediated cell death and decreased cell sensitivity to chemotherapeutic drugs in NB [127,128,145,153]. On the other hand, casp8 expression is maintained in 30-50% of NB and promotes ruffle and lamellipodial formation, enhanced cell migration, and adhesion [88,91,149].…”

“…Research suggests that the loss of casp8 in NB significantly enhanced tumor cell metastasis and survival at secondary sites by inhibiting integrin-mediated cell death (IMD) [88,145,156]. Loss of casp8 in NB decreased cell sensitivity to chemotherapeutic drugs [127,128,145,153].…”

“…The hypermethylation of casp8 showed a statistically significant correlation with relapse susceptibility, a decreased overall survival rate, and an increase in resistance against death receptor-mediated apoptosis and some chemotherapeutics [116,[123][124][125][126][127]. Studies demonstrated that by using demethylating reagents such as, gamma interferon (γ-IFN), 5'-aza-2-deoxycytidine, or retinoic acid, casp8 expression could be restored [127][128][129][130].…”

“…Human neuroblastoma, NB7 and NB1691 cell lines have been previously described [17,128]. Briefly, neuroblastoma cell lines were established by St. Jude Children's Research Hospital.…”