Sensitization of TNF alpha and Agonistic FAS/CD95 Antibody-Induced Apoptosis by INF gamma on Neuroblastoma Cells

ABSTRACT

PURPOSE: IFN gamma sentitizes many tumor cells to TNF alpha and FASL-mediated apoptosis by enhancing the expression of TNF or FAS/CD95 receptor and modulating the activation of caspase and Bcl-2 family. It has been reported that IFN gamma and TNF alpha synergistically caused differentiation and growth inhibition of neuroblastoma cells. Even though some neuroblastoma cell express FASR/FASL on the cell surface, they could not induce apoptosis by ligation of the FAS/CD95 receptor. But the treatment of IFN gamma is reported to induce apoptosis in some neuroblastoma cell lines through the CD95/ CD95L autocrine circuit. In this study, we examined whether IFN gamma could affect TNF alpha and agonistic FAS/CD95 antibody(CH-11)-induced apoptosis against neuroblastoma cell lines that had shown diverse drug sensitivity and resistance. METHODS: CHLA-15, CHLA-90 and LA-N-2 neuroblastoma cells were cultured in IMDM and treated with recombinant IFN gamma TNF alpha and CH-11 antibody. Cell viability was measured by DIMSCAN with a fluorescent calcein-AM. Apoptosis was analyzed through flow cytometry using Annexin V- PE and 7-ADD staining and confirmed by pancaspase and caspase-8 blocking experiments. The expression of TNF RI and FAS/CD95 receptor was evaluated by flow cytometry using the corresponding antibody and PE-conjugated secondary antibody. RESULTS: IFN gamma or TNF alpha alone had no demonstrable cytotoxic effects, whereas both cytokines in combination induced apoptosis synergistically in CHLA-15 and CHLA-90 cells. Although there was no cytotoxicity with the ligation of CH-11 alone in CHLA-90 cells, pretreatment of IFNgammaincreased the sensitivity of CH-11-mediated apoptosis. The expression of TNFRI and FAS/CD95R were nonspecifically enhanced after treatment of IFN gamma without relation to sensitivity to TNF alpha and CH-11. This finding suggest up-regulation of both receptors may contribute to sensitization of TNFalphaand CH-11-mediated apoptosis by IFN gamma in only sensitive cell lines. CONCLUSION: IFN gamma induced sensitization of TNF alpha and agonistic FAS/CD95 antibody-mediated apoptosis on some neuroblastoma cells through up-regulation of TNFRI and FAS/CD95 receptor.