Thymoquinone and Metabolic Reprogramming in Breast Cancer: A New Dimension From Proteomic Analysis

Thymoquinone (TQ) has shown antitumorigenic effects in breast cancer; however, its detailed impact on cell signaling mechanisms requires further investigation. This study aims to elucidate the molecular mechanisms behind TQ's antiproliferative effects in breast cancer by analyzing proteome-level changes. MCF-7 cells were treated with 15 µM TQ, the inhibitory concentration (IC50), for 48 h. Proteins from treated and untreated (control) groups were isolated and subjected to liquid chromatography–tandem mass spectrometry (LC–MS/MS) proteomic analysis. Identified proteins were functionally annotated, with hub proteins identified using Cytoscape software, and verification conducted through Western blot analysis. Label-free quantitation identified 629 master proteins, with 104 upregulated and 477 downregulated in TQ-treated samples compared to controls. Among these, 150 proteins showed dramatic regulation, including 11 upregulated and 139 downregulated proteins, with ribosomal proteins emerging as central. The heatmap demonstrated robust clustering of replicates. Functional annotations indicated that TQ significantly impacts crucial mechanisms such as carbon metabolism, amino acid biosynthesis, protein synthesis, and the citrate cycle, essential for metabolic reprogramming. This study identifies novel molecular targets associated with metabolic reprogramming, previously underexplored in TQ's effects, highlighting their pivotal role in TQ's anticancer mechanisms in breast cancer. These findings could lay the groundwork for developing future TQ-based therapies.