Chemoresistant tumors of the nervous system exact a significant toll among patients with cancer. The American Cancer Society estimates that 20,500 malignant tumors of the brain or spinal cord will be diagnosed during 2007 in the U.S. Approximately 12,740 people will die from these malignant tumors. CNS cancer accounts for approximately 1.3% of all cancers and 2.2% of all cancer-related deaths.[20
] Brain is the most common site for solid tumors of childhood; the long-term survival rate for children who received radiation therapy for brain tumors of any type is, at best, in the neighborhood of 40%.[21
] Neuroblastoma is the single most common solid tumor of childhood; 65% of children with neuroblastoma have metastatic disease at the time of initial diagnosis, and these children have only a 5–20% 5-year survival rate.[22
] These statistics do not even include the enormous morbidity associated with these diseases.
Therapeutic failures in this arena are at least in part the result of altered expression of proteins that function to regulate apoptosis induction and/or enactment, including those in the Bcl-2 protein family. For example, altered levels of Bcl-2 and the related protein Bcl-XL
contribute to the resistance of neuroblastomas to chemotherapy.[24
] Similarly, medulloblastomas and glioblastomas have all been shown to overexpress the bcl-2 and or bcl-XL
gene, contributing to resistance to chemotherapeutic agent-induced apoptosis.[24
] The discovery and development of strategies that overcome such drug resistance and that enhance the ability to predict responsiveness of particular tumors to particular chemotherapeutic agents would contribute greatly to the conquest of these common and deadly diseases.
Studies in our laboratory and those of others demonstrate that Bcl-2 exerts its effects at least in part by a mechanism that includes a shift in the cellular capacity for glutathione turnover.[9
] Not surprisingly, other studies link both the initiating and subsequent steps involved in the apoptotic process to exposure of the cell to reactive oxygen species [39
] and demonstrate release of cytochrome c from the mitochondrion with a resultant breakdown in electron transport and increased formation of endogenous reactive oxygen species.[ 39
] The caspases, enzymes critical for the enactment of apoptosis, are accordingly cysteine-rich and redox-active.[43
We have pursued a strategy for overcoming Bcl-2-mediated chemoresistance that takes advantage of the increase in cellular free sulfhydryl content by using chemotherapeutic agents that require reduction by glutathione for their activity. Our initial studies focused on the efficacy of a member of one such class of agents, the enediynes, in tumor cells that were genetically engineered to overexpress bcl-2.[8
NCS is an enediyne DNA-cleaving natural product that induces apoptosis in neural crest tumor cells in culture.[14
] Like many other naturally-occurring enediynes, NCS is actually a prodrug that requires sulfhydryl activation for efficacy. As such, the cytotoxicity of NCS has been demonstrated, within the range of physiological manipulations performed, to vary directly with the glutathione content of the cell.[45
] This information led to our prediction that, contrary to the case for other chemotherapeutic agents studied, overexpression of bcl-2 and the resulting shift in glutathione handling in the cell would potentiate the induction of apoptosis by NCS.
We initially showed that, in PC12 rat pheochromocytoma cells that have been bcl-2-or mock-transfected, bcl-2 overexpression does indeed potentiate the apoptosis-inducing activity of NCS.[8
] In this same system, bcl-2 overexpression decreases apoptosis induction by the non-enediyne, cisplatin, and synthetic and natural enediynes that do not require reductive activation by glutathione, indicating that glutathione is important in the potentiation of NCS-induced apoptosis by Bcl-2. In addition, inhibition of glutathione synthesis completely abolishes the difference in NCS sensitivity between cells that do and do not overexpress bcl-2. The glutathione-dependent enediyne prodrugs are therefore the only class of drug that has been demonstrated to work best in those tumor cells that have become resistant to other known chemotherapeutic agents.
Potentiation of NCS-induced apoptosis by Bcl-2 overproduction is dependent, not only on altered metabolism of glutathione, but also on caspase-3-mediated cleavage of Bcl-2.[9
] The resulting cleavage product of this anti-apoptotic protein is, in fact, pro-apoptotic. Caspase-3 activation, like NCS activation, has been demonstrated to be sulfhydryl reduction-dependent. [43
NCS and related enediynes have previously been proposed for clinical use in a variety of human cancers. Initial clinical trials of NCS were hampered by anaphylactic responses to its non-covalently bound protein component, ironically a portion of the NCS structure with no relationship to its efficacy. Our studies have taken specific advantage of the need for sulfhydryl activation of these compounds or have proposed identification of particular subgroups of patients and/or tumors for which these compounds might present an improved therapeutic index. We have found that potentiation of NCS-induced apoptosis by bcl-2 overexpression depends critically upon cellular expression of caspase-3, Bcl-2-induced enhancement of glutathione recycling capacity, and cleavability of Bcl-2 by caspase-3,[9
] potentially allowing us to predict for which tumors NCS might be most efficacious.
The present study presents proof of principle for the prediction of efficacy of NCS by determination of the product of caspase-3 content and Bcl-2 content. It further suggests that, as a function of their relatively high Bcl-2 and caspase-3 content, cancers of the nervous system are particularly sensitive to NCS and perhaps other sulfhydryl-dependent enediynes. Verification of these results with larger numbers of cell lines and extension of these conclusions to primary tumors will require additional studies.