Nebivolol triggers apoptosis, ferroptosis and necroptosis in triple-negative breast cancer

Background: Triple-negative breast cancer (TNBC) lacks effective targeted therapies and remains associated with poor outcomes. We are investigating nebivolol, an FDA-approved third-generation beta-blocker, as a candidate for drug repurposing in TNBC. We have previously reported that nebivolol inhibits TNBC cell growth, proliferation, clonogenic potential and disrupts autophagic flux, leading to accumulation of autophagosomes and lysosomes. RNA-seq analysis further revealed enrichment of ferroptosis (FDR = 0.076), apoptosis (FDR = 0.111), and necroptosis (FDR = 0.133) pathways following nebivolol treatment. Here, we aimed to conduct deeper mechanistic investigation of nebivolol-induced cell death pathways. Methods: Two TNBC cell lines (MDA-MB-231 and SUM159) were used for all the experiments. The effects of nebivolol (10 μM, 24h) on ferroptosis were assessed using the BODIPY C11 lipid peroxidation assay kit. Apoptosis and necroptosis were evaluated after treatment with 10 μM nebivolol for 24, 48, and 72h using FITC Annexin V Apoptosis Detection kit. Annexin V-positive cells were classified as apoptotic, whereas Annexin V-positive/PI-positive cells represented necroptotic and late-stage apoptotic populations. Nebivolol-induced apoptosis was confirmed using western blot analysis of cleaved/total PARP and caspase-3 and -7 at 24, 48, and 72h. Necroptosis-mediated membrane damage was measured by LDH release following 3-100 μM nebivolol treatment for 24h. All experiments included a minimum of three biological replicates, each with at least three technical replicates. Data were analyzed using GraphPad Prism 10.1, and statistical significance was determined by one-way ANOVA with multiple comparisons ( p < 0.05). Results: Consistent with RNA-seq results, nebivolol 10 μM significantly increased lipid peroxidation by 1.5- to 2-fold, confirming ferroptosis induction. Nebivolol 10 μM significantly increased Annexin V-positive/PI-positive but not Annexin V-positive/PI-negative populations in a time-dependent manner, suggesting its role in late-stage apoptosis and necroptosis. Nebivolol 10 μM treatment elevated cleaved PARP and caspase-7,but not caspase-3 levels, in a time-dependent manner, confirming apoptosis induction. Nebivolol 30 and 100 μM significantly increased LDH release indicating pronounced necroptosis-mediated membrane rupture. Conclusion: Overall, nebivolol elicits multimodal programmed cell death in TNBC by activating both apoptotic and non-apoptotic pathways. These findings support nebivolol as a promising repurposed drug candidate for TNBC and identify ferroptosis and necroptosis as previously unrecognized components of its antitumor mechanism.