BETULINIC ACID TRIGGERS CD95 (APO-1 FAS)-INDEPENDENT AND P53-INDEPENDENT APOPTOSIS VIA ACTIVATION OF CASPASES IN NEUROECTODERMAL TUMORS/

Betulinic acid CBA), a melanoma-specific cytotoxic agent, induced apop tosis in neuroectodermal tumors, such as neuroblastoma, medulloblastom a, and Ewing's sarcoma, representing the most common solid tumors of c hildhood. BA triggered an apoptosis pathway different from the one pre viously identified for standard chemotherapeutic drugs. BA-induced apo ptosis was independent of CD95-ligand/receptor interaction and accumul ation of wild-type p53 protein, but it critically depended on activati on of caspases (interleukin 1 beta-converting enzyme/Ced-3-like protea ses), FLICE/MACH (caspase-8), considered to be an upstream protease in the caspase cascade, and the downstream caspase CPP32/YAMA/Apopain (c aspase-3) were activated, resulting in cleavage of the prototype subst rate of caspases PARP. The broad-spectrum peptide inhibitor benzyloxyc arbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cleavage of FLIC E and PARP, also completely abrogated BA-triggered apoptosis. Cleavage of caspases was preceded by disturbance of mitochondrial membrane pot ential and by generation of reactive oxygen species. Overexpression of Bcl-2 and Bcl-x(L) conferred resistance to BA at the level of mitocho ndrial dysfunction, protease activation, and nuclear fragmentation. Th is suggested that mitochondrial alterations were involved in BA-induce d activation of caspases. Furthermore, pax and Bcl-x(s), two death-pro moting proteins of the Bcl-2 family, were up-regulated following BA tr eatment. Most importantly, neuroblastoma cells resistant to CD95- and doxorubicin-mediated apoptosis were sensitive to treatment with BA, su ggesting that BA may bypass some forms of drug resistance. Because BA exhibited significant antitumor activity on patients' derived neurobla stoma cells ex vivo, BA may be a promising new agent for the treatment of neuroectodermal tumors in vivo.