Ferroptosis Induction in Glioma by Calceolarioside A via Modulation of the PI3K/Akt/Nrf2 Pathway

This protocol demonstrates the experimental workflow used to investigate the effects of Calceolarioside A (CaA) on ferroptosis induction and modulation of the Phosphatidylinositol 3-kinase (PI3K)/Protein Kinase B (Akt)/Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in glioma models. The procedures include a series of cell-based assays in U251 and U87 human glioma lines to evaluate cell viability, proliferation, and chemosensitivity following CaA treatment. Ferroptosis-associated changes are assessed by measuring reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and labile iron levels, along with expression of ferroptosis-related proteins such as glutathione peroxidase 4 (GPX4), cysteine/glutamate antiporter subunit (xCT), and ferritin via western blot. To assess pathway involvement, the protocol details qRT-PCR, western blot, and immunoprecipitation-based assays for Nrf2 expression and ubiquitination. Nrf2 overexpression experiments are included to confirm its role in ferroptosis regulation. The protocol further demonstrates the use of a mouse xenograft model, where U87 cells are implanted subcutaneously, followed by intraperitoneal CaA administration to evaluate in vivo tumor growth and toxicity. Histological analysis of major organs is performed to assess systemic safety. Additionally, molecular docking is used to predict direct binding between CaA and the PI3K p110α subunit (PIK3CA). Together, these procedures provide a reproducible framework to examine ferroptosis mechanisms and the therapeutic efficacy of natural compounds in glioma research.