A substantial proportion of patients with melanoma brain metastases (MBM), especially those symptomatic at therapy start, continue to respond poorly to currently available systemic therapies, including immune checkpoint inhibitors. Durable systemic treatment options beyond established regimens remain limited for this patient population. Drug repurposing offers a rapid translational path for identifying novel therapeutic strategies. Thioridazine, a phenothiazine antipsychotic with known blood–brain barrier permeability, has shown anticancer effects in several malignancies but has not previously been evaluated in MBM. This study investigates the anti-tumoral potential and underlying mechanisms of thioridazine in patient-derived MBM cell lines.

Cytotoxic, anti-proliferative, and anti-migratory effects of thioridazine were assessed in four MBM cell lines (H1, H2, H3, H10) using monolayer and soft agar viability assays, live-cell imaging, and clonogenic assays. Potential off-target toxicity was evaluated using fetal rat brain organoids (FRBOs). Mechanistic effects were examined using Annexin V/PI flow cytometry, Western blotting of apoptosis- and autophagy-related proteins (cleaved PARP1, cleaved caspase-3, Bcl-2, p62, LC3A/B), and confocal microscopy to assess autophagosome formation and lysosomal dynamics.

Thioridazine reduced viability of MBM cells in a dose-dependent manner, with IC₅₀ values ranging from 8.7 to 12.0 µM. Anchorage-independent growth was similarly inhibited. FRBOs displayed no treatment-associated cytotoxicity at MBM-relevant doses in vitro. Thioridazine markedly suppressed MBM cell proliferation, migration, and clonogenic growth in a dose-dependent manner. Apoptosis induction was inconsistent across cell lines, with variable activation of cleaved PARP1, cleaved caspase-3, and Bcl-2. In contrast, autophagy-associated markers p62 and LC3A/B were consistently upregulated, accompanied by intracellular accumulation of punctate autophagosomes and altered lysosomal morphology, indicating impaired autophagic flux.

Thioridazine demonstrates robust anti-tumoral activity in MBM cells in vitro while sparing healthy brain organoid tissue. Its cytotoxic effects appear independent of classical apoptosis and instead correlate with disrupted autophagic flux and altered lysosomal dynamics. These findings highlight thioridazine as a promising candidate for therapeutic repurposing in MBM and support further investigation into its autophagy-modulating mechanisms.