RESUMO
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.
Assuntos
Azacitidina/análogos & derivados , Caspases/biossíntese , Glicoproteínas de Membrana/farmacologia , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Fator de Necrose Tumoral alfa/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Azacitidina/farmacologia , Caspase 10 , Caspase 8 , Caspase 9 , Inibidores de Caspase , Caspases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Decitabina , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Proteínas Ligadas por GPI , Humanos , Células Jurkat/citologia , Células Jurkat/efeitos dos fármacos , Glicoproteínas de Membrana/biossíntese , Metilação , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/biossíntese , Membro 10c de Receptores do Fator de Necrose Tumoral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF , Receptores Chamariz do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa/biossínteseRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/fisiologia , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/farmacologia , Proteínas de Neoplasias/fisiologia , Tumores Neuroectodérmicos Primitivos/patologia , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Antimetabólitos Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/fisiologia , Caspase 8 , Caspase 9 , Caspases/biossíntese , Caspases/deficiência , Caspases/genética , Criança , Cicloeximida/farmacologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Metilação de DNA/efeitos dos fármacos , Decitabina , Resistência a Medicamentos , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Tumores Neuroectodérmicos Primitivos/enzimologia , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Receptores do Fator de Necrose Tumoral/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF , Transcrição Gênica , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologiaRESUMO
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.