A major concern in cancer therapy is resistance of tumors such as glioblastoma to current treatment protocols. Here, we report that transfer of the gene encoding second mitochondria-derived activator of caspase (Smac) or Smac peptides sensitized various tumor cells in vitro and malignant glioma cells in vivo for apoptosis induced by death-receptor ligation or cytotoxic drugs. Expression of a cytosolic active form of Smac or cell-permeable Smac peptides bypassed the Bcl-2 block, which prevented the release of Smac from mitochondria, and also sensitized resistant neuroblastoma or melanoma cells and patient-derived primary neuroblastoma cells ex vivo. Most importantly, Smac peptides strongly enhanced the antitumor activity of Apo-2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in an intracranial malignant glioma xenograft model in vivo. Complete eradication of established tumors and survival of mice was only achieved upon combined treatment with Smac peptides and Apo2L/TRAIL without detectable toxicity to normal brain tissue. Thus, Smac agonists are promising candidates for cancer therapy by potentiating cytotoxic therapies.
The cytotoxic effect of anticancer drugs has been shown to involve induction of apoptosis. We report here that tumor cells resistant to CD95 (APO-1/Fas) -mediated apoptosis were cross-resistant to apoptosis-induced by anticancer drugs. Apoptosis induced in tumor cells by cytarabine, doxorubicin, and methotrexate required the activation of ICE/Ced-3 proteases (caspases), similarly to the CD95 system. After drug treatment, a strong increase of caspase activity was found that preceded cell death. Drug-induced activation of caspases was also found in ex vivo-derived T-cell leukemia cells. Resistance to cell death was conferred by a peptide caspase inhibitor and CrmA, a poxvirus-derived serpin. The peptide inhibitor was effective even if added several hours after drug treatment, indicating a direct involvement of caspases in the execution and not in the trigger phase of drug action. Drug-induced apoptosis was also strongly inhibited by antisense approaches targeting caspase-1 and -3, indicating that several members of this protease family were involved. CD95-resistant cell lines that failed to activate caspases upon CD95 triggering were cross-resistant to drug-mediated apoptosis. Our data strongly support the concept that sensitivity for drug-induced cell death depends on intact apoptosis pathways leading to activation of caspases. The identification of defects in caspase activation may provide molecular targets to overcome drug resistance in tumor cells.
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