Dehydroleucodine Induces a TP73-dependent Transcriptional Regulation of Multiple Cell Death Target Genes in Human Glioblastoma Cells

Background: Dehydroleucodine, a natural sesquiterpene lactone from Artemisia douglassiana Besser (Argentine) and Gynoxys verrucosa (Ecuador). Objective: To define the molecular mechanisms underlying the effect of dehydroleucodine on the human glioblastoma cells. Method: Various techniques (cDNA expression array, real-time quantitative PCR, chromatin immunprecipitation, luciferase reporter assay, use of phosphospecific antibodies, immunoprecipitation, immunoblotting, apoptosis and autophagy assays) were employed to define and validate multiple molecular gene targets affected in human glioblastoma cells upon dehydroleucodine exposure. Results: Dehydroleucodine exposure upregulated the total and phosphorylated (p-Y99) levels of TP73 in U87- MG glioblastoma cells. We found that TP73 silencing led to a partial rescue of U87-MG cells from the cell death induced by dehydroleucodine. Upon the dehydroleucodine exposure numerous gene targets were upregulated and downregulated through a TP73-dependent transcriptional mechanism. Some of these gene targets are known to be involved in cell cycle arrest, apoptosis, autophagy and necroptosis. Dehydroleucodine induced the TP73 binding to the specific genes promoters (CDKN1A, BAX, TP53AIP1, CYLD, RIPK1, and APG5L). Moreover, the exposure of U87-MG cells to dehydroleucodine upregulated the protein levels of CDKN1A, BAX, TP53AIP1, CYLD, RIPK1, APG5L, and downregulated the CASP8 level. The formation of RIPK1 protein complexes and phosphorylation of MLKL were induced by dehydroleucodine supporting the notion of multiple cell death mechanisms implicated in the tumor cell response to dehydroleucodine. Conclusion: This multifaceted study led to a conclusion that dehydroleucodine induces the phosphorylation of tumor protein TP73 and in turn activates numerous TP73-target genes regulating apoptosis, autophagy and necroptosis in human glioblastoma cells.