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Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal
1Division of Hematology/Oncology, University Children's Hospital, German Cancer Research Center, Heidelberg.
Abstract:
Betulinic acid (BA), a melanoma-specific cytotoxic agent, induced apoptosis in neuroectodermal tumors, such as neuroblastoma, medulloblastoma, and Ewing's sarcoma, representing the most common solid tumors of childhood. BA triggered an apoptosis pathway different from the one previously identified for standard chemotherapeutic drugs. BA-induced apoptosis was independent of CD95-ligand/receptor interaction and accumulation of wild-type p53 protein, but it critically depended on activation of caspases (interleukin 1beta-converting enzyme/Ced-3-like proteases). FLICE/MACH (caspase-8), considered to be an upstream protease in the caspase cascade, and the downstream caspase CPP32/YAMA/Apopain (caspase-3) were activated, resulting in cleavage of the prototype substrate of caspases PARP. The broad-spectrum peptide inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cleavage of FLICE and PARP, also completely abrogated BA-triggered apoptosis. Cleavage of caspases was preceded by disturbance of mitochondrial membrane potential and by generation of reactive oxygen species. Overexpression of Bcl-2 and Bcl-XL conferred resistance to BA at the level of mitochondrial dysfunction, protease activation, and nuclear fragmentation. This suggested that mitochondrial alterations were involved in BA-induced activation of caspases. Furthermore, Bax and Bcl-xs, two death-promoting proteins of the Bcl-2 family, were up-regulated following BA treatment. Most importantly, neuroblastoma cells resistant to CD95- and doxorubicin-mediated apoptosis were sensitive to treatment with BA, suggesting that BA may bypass some forms of drug resistance. Because BA exhibited significant antitumor activity on patients' derived neuroblastoma cells ex vivo, BA may be a promising new agent for the treatment of neuroectodermal tumors in vivo.
Insights
Betulinic acid (BA) effectively triggers apoptosis in childhood neuroectodermal tumors via a novel caspase-dependent pathway. This agent shows promise in overcoming drug resistance and treating these common childhood cancers.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Betulinic acid (BA) is a cytotoxic agent with demonstrated activity against melanoma.
- Neuroectodermal tumors, including neuroblastoma, medulloblastoma, and Ewing's sarcoma, are common childhood solid tumors.
- Standard chemotherapeutic agents often induce apoptosis through pathways involving CD95 or p53.
Purpose of the Study:
- To investigate the mechanism of apoptosis induced by Betulinic acid (BA) in neuroectodermal tumors.
- To determine if BA-induced apoptosis utilizes pathways distinct from those of conventional chemotherapeutics.
- To evaluate the potential of BA as a novel therapeutic agent for childhood neuroectodermal tumors.
Main Methods:
- Assessing apoptosis induction by BA in neuroectodermal tumor cell lines.
- Analyzing the involvement of caspases (FLICE/MACH and CPP32/YAMA) and PARP cleavage.
- Investigating the role of mitochondrial membrane potential and reactive oxygen species (ROS) generation.
- Examining the effects of Bcl-2 family proteins (Bcl-2, Bcl-XL, Bax, Bcl-xs) on BA sensitivity.
- Testing BA efficacy on drug-resistant neuroblastoma cells and patient-derived xenografts.
Main Results:
- BA induced apoptosis in neuroectodermal tumors through a pathway independent of CD95 and wild-type p53.
- BA-induced apoptosis critically depended on the activation of caspases, including FLICE/MACH (caspase-8) and CPP32/YAMA (caspase-3), leading to PARP cleavage.
- Mitochondrial membrane potential disruption and ROS generation preceded caspase activation.
- Overexpression of Bcl-2 and Bcl-XL conferred resistance, while Bax and Bcl-xs were upregulated by BA.
- Neuroblastoma cells resistant to CD95 and doxorubicin were sensitive to BA, which also showed ex vivo antitumor activity.
Conclusions:
- Betulinic acid activates a unique caspase-dependent apoptotic pathway in neuroectodermal tumors.
- Mitochondrial alterations and ROS generation are key early events in BA-induced apoptosis.
- BA demonstrates potential to overcome conventional drug resistance mechanisms in neuroblastoma.
- BA represents a promising therapeutic candidate for treating childhood neuroectodermal tumors.