KAAD-cyclopamine sensitize malignant glioma cells to TRAIL-mediated apoptosis

Abstract

2025 Background: Malignant gliomas are the most aggressive human brain tumors without any curative treatment. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic because TRAIL induces almost selectively apoptosis in cancer cells. The main obstacle in TRAIL-based therapy is that many glioblastoma cells are resistant. In the present study we found that the Hedgehog inhibitor, KAAD-cyclopamine, potently sensitized primary glioma cells to TRAIL-mediated apoptosis. Methods: Established cell lines and isolated primary tumor glioblastoma cells were treated with achievable plasma concentrations of TRAIL, KAAD-cyclopamine, or the combination of both. Apoptosis was assessed by Annexin V/Propidiumiodide staining and subsequent flow cytometric analysis. Activation of caspases and expression of apoptosis related proteins were determined by immunoblotting. For transfection experiments primary glioma cells were electroporated with plasmids encoding the cDNA of c-FLIP and bcl-2. Results: We established a stable culture model for isolated primary human glioma cells. In contrast to cell lines, isolated primary tumor cells from all investigated glioma patients were highly TRAIL resistant. Single treatment with KAAD-cyclopamine or TRAIL does not induce cytotoxicity in malignant glioma cells. However, treatment with KAAD-cyclopamine in combination with TRAIL induces rapid apoptosis in TRAIL-resistant glioma cells. Notably, normal human astrocytes were not affected by the combination treatment consisting of KAAD-cyclopamine and TRAIL. KAAD-cyclopamine led to an upregulation of death receptor 4 and 5 and down-regulation of bcl-2 and c-FLIP. Furthermore, over-expression of both bcl-2 and c-FLIP attenuated KAAD-cyclopamine facilitated TRAIL-mediated apoptosis. Conclusion: The widespread TRAIL resistance in primary glioma cells described here questions the therapeutic clinical benefit of TRAIL as a monotherapeutic agent. Overcoming TRAIL resistance by KAAD-cyclopamine cotreatment might, however, provide a powerful therapeutic option for glioma patients. No significant financial relationships to disclose.