Ceramide induces apoptosis in neuroblastoma cell cultures resistant to CD95 (Fas/APO-1)-mediated apoptosis

Background/Purpose: Spontaneous tumor regression is a well-known characteristic in neuroblastomas. Because preliminary reports have shown that regression may be caused by apoptosis (a lethal cascade mediated by the CD95 (APO-1/ Fas)-receptor), we analyzed the expression of CD95-receptors in 5 human neuroblastoma cell lines, Ceramides (known stimuli of apoptosis downstream from the CD95-receptor complex) also were used to test whether apoptosis would be induced in neuroblastoma cell cultures resistant to CD95-mediated programmed cell death. Methods: The expression of the CD95-receptor was assessed by flow cytometry after incubation with either fluorisothiocyanate-conjugated (FITC) anti-CD95-antibody (UB2) or CD95-ligand for 16 hours. Apoptotic cell death was detected via microscopy, cell viability testing (MTT, 3-[4,5 dimehylthiazole-2-yl]-2,5 diphenyltetrazoliumbromide), and flow cytometric analysis after propidium iodide staining of the DNA. Results: CD95-receptor expression was found on all neuroblastoma cell lines. Stimulation of the CD95-receptor of the malignant glioblastoma cell line LN229 (positive control) with either anti-CD95-antibody or CD95-ligand induced apoptosis. Apoptosis was not seen, however, in any of the neuroblastoma cell lines when the CD95-receptor was stimulated with anti-CD95-antibody or the CD95-ligand. Significant apoptosis was detected in all neuroblastoma cell lines after the addition of 25 mu mol/L C2- and C6-ceramide. Conclusions: CD95-receptors are present on neuroblastoma cell lines, and these cells are resistant to apoptosis stimulated by anti-CD95-antibody or CD95-ligand. Apoptosis is induced, however, when these cells are treated with ceramide. A signal blockage downstream from the CD95-receptor complex and upstream of ceramide may account for this finding, and the "cellular FLICE inhibitory protein" (cFLIP) may be primarily responsible. Copyright (C) 2000 by W.B. Saunders Company.