Medulloblastoma, a common malignant pediatric
brain tumor, is highly resistant to
death receptor-mediated apoptosis despite
death receptor expression by
tumor cells. Developing new strategies to overcome this resistance to
death receptor activation could positively impact therapeutic outcomes. We explored the modulation of
death receptor-induced
medulloblastoma cell death by the
topoisomerase I inhibitor camptothecin (
CPT).
CPT significantly increased the human
medulloblastoma DAOY cell death response to agonistic anti-Fas antibody (CH-11). Cell death after
CPT, CH-11, and CPT+CH-11 treatment was 9, 7, and 33%, respectively. Isobologram analysis showed that CH-11 and
CPT act synergistically to induce cell death in DAOY cells. A similar pattern of synergism between
CPT and CH-11 was found in ONS-76
medulloblastoma cells. Synergistic cell death was found to be predominantly apoptotic involving both extrinsic and intrinsic pathways as evidenced by
annexin V staining, cleavage of
caspases (3, 8, and 9), Bid and PARP, and cytoprotection by
caspase inhibitors. Flow cytometric analyses showed that expression of cell surface Fas or
Fas ligand did not change with drug treatment. Western blot analyses showed that the combination of CH-11+CPT induced a significant decrease in XIAP levels. Furthermore,
reactive oxygen species, especially O2, were elevated after
CPT treatment, and even more so by the CH-11+CPT treatment. The
antioxidants glutathione and N-acetyl-
cysteine prevented cell death induced by CPT+CH-11. Moreover, the mitochondrial respiratory chain complex I inhibitor
rotenone potentiated CH-11-induced apoptosis in DAOY cells. Taken together, these findings show that
CPT synergizes with Fas activation to induce
medulloblastoma apoptosis through a mechanism involving
reactive oxygen species and oxidative stress pathways.