Ouabain exerts both anti-tumor and senolytic effects in brain cancer stem and tumor cells

Glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumors with dismal prognoses. These tumors contain therapy-resistant cancer stem cells (CSCs) that drive tumor progression and relapse. Standard treatments such as radiation and temozolomide (TMZ) induce senescence in a subset of tumor cells, generating a persistent, treatment-refractory population. Senolytic agents that selectively eliminate senescent cells may overcome this resistance. Here, we investigated the anti-tumor and senolytic effects of the cardiac glycoside ouabain in glioblastoma stem cells (GSCs), DIPG stem cells, and glioma cell lines. Neurosphere formation and transwell assays were used to assess self-renewal and migration, and senolytic activity was examined in TMZ-treated cells using beta-galactosidase staining. RT-PCR quantified stemness-associated gene expression.Ouabain inhibited the self-renewal of GSCs and DIPG stem cells in a dose-dependent manner and reduced their migratory capacity. TMZ pre-treatment sensitized GSCs to ouabain, resulting in a more pronounced suppression of neurosphere formation and an enhanced inhibition of migration. Ouabain decreased the expression of stemness and EMT-related genes, including SOX2, YKL-40, OCT4, and NANOG.In addition, ouabain selectively eliminated TMZ-induced senescent cells in both glioma cell lines and differentiating GSCs. Together, these findings demonstrate that ouabain exerts dual anti-tumor and senolytic effects in GBM and DIPG cells by suppressing CSC stemness, migration, and eliminating senescent, stem-like populations. These combined effects highlight ouabain as a potential therapeutic strategy for targeting treatment-resistant cells in malignant gliomas.