Hormetic and Differential Apoptotic Effects of Bixin and Norbixin in Human Glioblastoma Cells (U87-MG)

Glioblastoma multiforme (GBM) is an aggressive, treatment-resistant brain tumor with limited therapeutic success. Natural apocarotenoids, such as Bixin (lipophilic) and Norbixin (hydrophilic), derived from annatto ( Bixa orellana ), have demonstrated anticancer potential, but their effects on glioblastoma remain largely unexplored. This study evaluated, under in vitro conditions and for the first time, the cytotoxic and pro-apoptotic activities of Bixin and Norbixin against the human glioblastoma cell line U87-MG. Molecular characterization was performed using FTIR and ¹ H-NMR spectroscopy, and reactivity was estimated via Frontier Molecular Orbitals (FMO) analysis. Cell viability was quantified using the tetrazolium-based colorimetric assay (MTS), and apoptosis was monitored via phase-contrast microscopy to determine IC ₅₀ values and evaluate morphological changes. Bixin exhibited significantly higher potency, displaying a distinctive biphasic dose-response pattern consistent with a hormetic effect at low concentrations. Bixin yielded IC ₅₀ values of 231.9 µM (viability) and 130.1 µM (apoptosis), indicating that apoptotic signaling precedes metabolic viability loss (AIIC ₅₀  < MTSIC ₅₀ ). Norbixin showed IC ₅₀ of 532.9 µM for viability; 400.1 µM for apoptosis and a slower apoptotic induction profile. Morphological analyses corroborated these findings, confirming classic apoptotic features (e.g., cell shrinkage and membrane blebbing), particularly under cytotoxic concentrations, and were more pronounced in Bixin-treated cells. These results reveal distinct cytotoxic and apoptotic profiles between the two apocarotenoids, driven by their structural and physicochemical differences (lipophilicity vs. hydrophilicity). Collectively, the findings support Bixin and Norbixin as promising molecular scaffolds for further investigation, rather than therapeutic agents per se, in the context of carotenoid-based strategies targeting malignant glioma cells, warranting further mechanistic and in vivo validation.