ABSTRACT
Disruption of apoptotic pathways may be involved in tumor formation, regression, and treatment resistance of neuroblastoma (NB). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in cancer cell lines, whereas normal cells are not sensitive to TRAIL-mediated apoptosis. In this study we analyzed the expression and function of TRAIL and its agonistic and antagonistic receptors as well as expression of cellular FLICE-like inhibitory protein and caspase-2, -3, -8, -9, and -10 in 18 NB cell lines. Semiquantitative RT-PCR revealed that TRAIL-R2 and TRAIL-R3 are the main TRAIL-receptors used by NB cells. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cellular FLICE-like inhibitory protein. Surprisingly, caspase-8 and caspase-10 mRNA expression was detected in only 5 of 18 NB cell lines. Interestingly, only these five NB cell lines were susceptible to TRAIL-induced apoptosis in a time- and dose-dependent manner. Treatment with 5-aza-2'-deoxycytidine restored mRNA and protein expression of caspase-8 and TRAIL sensitivity of resistant cell lines, suggesting that gene methylation is involved in caspase inactivation. The TRAIL system seems to be functional in NB cells expressing caspase-8 and/or caspase-10. Because many cytotoxic drugs induce caspase-dependent apoptosis, failure to express caspase-8 and/or caspase-10 might be an important mechanism of resistance to chemotherapy in NB.
Subject(s)
Azacitidine/analogs & derivatives , Caspases/biosynthesis , Membrane Glycoproteins/pharmacology , Neuroblastoma/enzymology , Neuroblastoma/pathology , Tumor Necrosis Factor-alpha/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Apoptosis Regulatory Proteins , Azacitidine/pharmacology , Caspase 10 , Caspase 8 , Caspase 9 , Caspase Inhibitors , Caspases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Decitabine , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , GPI-Linked Proteins , Humans , Jurkat Cells/cytology , Jurkat Cells/drug effects , Membrane Glycoproteins/biosynthesis , Methylation , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/biosynthesis , Receptors, Tumor Necrosis Factor, Member 10c , Reverse Transcriptase Polymerase Chain Reaction , TNF-Related Apoptosis-Inducing Ligand , Tumor Necrosis Factor Decoy Receptors , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
BACKGROUND: Disruption of apoptotic pathways may be involved in tumor formation, regression, and treatment resistance of neuroblastoma (NB). TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in cancer cell lines. PROCEDURE: In this study we analyzed the expression and function of TRAIL, its agonistic and antagonistic receptors, and important intracellular signaling elements in 18 NB cell lines. RESULTS: Semiquantitative RT-PCR revealed that TRAIL-R2 and TRAIL-R3 are the main TRAIL-receptors used by NB cells. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cFLIP. Surprisingly, caspase-8 and caspase-10 mRNA was detected in only 5 of 18 NB cell lines. Interestingly, only these five NB cell lines were susceptible to TRAIL-induced apoptosis in a time- and dose-dependent manner. CONCLUSIONS: Treatment with 5-aza-2'-deoxycytidine restored mRNA expression of caspase-8 and -10 and TRAIL sensitivity of resistant cell lines, suggesting that gene methylation is involved in caspase inactivation. Since many cytotoxic drugs induce caspase-dependent apoptosis, failure to express caspase-8 and/or caspase-10 might be an important mechanism of resistance to chemotherapy in NB.
Subject(s)
Apoptosis/genetics , Caspases/genetics , Gene Expression Regulation, Neoplastic , Membrane Glycoproteins/genetics , Neuroblastoma/genetics , Tumor Necrosis Factor-alpha/genetics , Apoptosis Regulatory Proteins , Caspase 8 , Caspase 9 , Humans , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/genetics , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, CulturedABSTRACT
TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in adult malignant glioma and various other human solid tumor models but not in normal tissues. To characterize the TRAIL death pathway in childhood primitive neuroectodermal brain tumor (PNET), 8 human PNET cell lines were tested for TRAIL-induced apoptosis. TRAIL-sensitivity of the PNET cell lines was correlated with mRNA expression levels of TRAIL, its agonistic (TRAIL-R1, TRAIL-R2) and antagonistic (TRAIL-R3, TRAIL-R4) receptors, cellular FLICE-like inhibitory protein (cFLIP), caspase-3 and caspase-8. Three of 8 PNET cell lines tested were susceptible to TRAIL-induced apoptosis. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cFLIP. However, all TRAIL-sensitive PNET cell lines expressed caspase-8 mRNA and protein, while none of the five TRAIL-resistant PNET cell lines expressed caspase-8 protein. Treatment with the methyltransferase inhibitor 5-aza-2'-deoxycytidine restored mRNA expression of caspase-8 and TRAIL-sensitivity in formerly TRAIL-resistant PNET cells, suggesting that gene methylation inhibits caspase-8 transcription in these cells. We conclude, that loss of caspase-8 mRNA is an important mechanism of TRAIL-resistance in PNET cells. Treatment with recombinant soluble TRAIL, possibly in combination with methyltransferase inhibitors, represents a promising therapeutic approach for PNET that deserves further investigation.
