Objective To investigate the effects of isorhamnetin (ISO) on the viability and apoptosis of breast cancer cells and the underlying mechanisms.

Methods The CCK-8 assay was used to evaluate the effect of ISO on the proliferative activity of MDA-MB-231 cells and to calculate the half-maximal inhibitory concentration (IC₅₀). Plate colony formation assay and flow cytometry were performed to detect cell proliferation and apoptosis. Microarray sequencing and bioinformatics analysis were conducted to identify differentially expressed genes and related biological functions after ISO treatment. Intracellular reactive oxygen species (ROS) levels were measured using the DCFH-DA fluorescent probe, and the contents of malondialdehyde (MDA) and glutathione (GSH) were determined. The expression of proteins in the Keap1-Nrf2 signaling pathway was assessed by qRT-PCR and Western Blot. An Nrf2-knockdown MDA-MB-231 cell model was established using siRNA, and the above indicators were re-evaluated following ISO treatment.

Results ISO inhibited the viability of MDA-MB-231 cells in a concentration-dependent manner (IC₅₀ = 23.38 μmol/L), reduced cell proliferation, and induced apoptosis. Sequencing analysis revealed that the biological functions affected by ISO were closely associated with the Keap1-Nrf2 signaling pathway. ISO treatment significantly increased intracellular ROS and MDA levels and decreased GSH content in MDA-MB-231 cells. It downregulated Keap1 mRNA and protein expression while upregulating the mRNA and protein expression of Nrf2, HO-1, GCLM, and NQO1. Knockdown of Nrf2 combined with ISO treatment further enhanced ROS accumulation, inhibited cell proliferation, promoted apoptosis, and weakened the activation of the Keap1-Nrf2 signaling pathway by ISO.

Conclusion ISO inhibits the malignant progression of breast cancer cells by inducing ROS accumulation, but simultaneously activates the Keap1-Nrf2 signaling pathway to attenuate its cytotoxicity. Targeted inhibition of Nrf2 effectively dismantles this defense mechanism, synergistically enhancing the antitumor effect of ISO by promoting ROS accumulation.

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