Investigation into the Effect and Mechanism of Temozolomide (TMZ) Inducing Ferroptosis to Inhibit Glioma Cell Proliferation

Abstract Although temozolomide (TMZ) is a first-line chemotherapeutic agent for treating malignant gliomas, its mechanisms of action remain incompletely understood, and primary or acquired resistance is prevalent in certain patients, severely limiting its clinical efficacy. Therefore, elucidating the molecular mechanisms underlying TMZ's mode of action is crucial for improving its therapeutic efficacy. This study aimed to delineate the effects of TMZ on glioma cells and the molecular mechanisms underlying its induction of ferroptosis. Using human glioma cell lines U87 and U251 as models, we systematically evaluated the impact of TMZ on cell proliferation, cell cycle, DNA integrity, and oxidative stress status through experiments including CCK-8, flow cytometry, and immunofluorescence. Further mechanistic investigations utilizing gene silencing and small molecule inducers elucidated the pivotal role of glutathione peroxidase 4 (GPX4) in the ferroptosis process induced by TMZ. Results demonstrated that TMZ inhibited glioma cell proliferation, induced cell cycle arrest and DNA damage, while concurrently promoting reactive oxygen species (ROS) accumulation and glutathione (GSH) depletion, along with downregulation of GPX4 expression. Ferroptosis inducers Erastin or RSL3 significantly augmented TMZ's cytotoxicity, exacerbating lipid peroxidation and GSH consumption. GPX4 gene silencing also enhanced TMZ's cytotoxic effects and synergistically interacted with Erastin to promote ferroptosis. Furthermore, knockdown of the GPX4 downstream target ATP-binding cassette transporter ABCC1 increased sensitivity to TMZ. This study unveiled a novel mechanism by which TMZ induces ferroptosis via the GPX4/ABCC1 pathway, providing important insights into understanding its molecular modes of action and improving clinical therapeutic efficacy.