Integrated Molecular Analysis of Thymoquinone–Methotrexate Synergy in Breast Cancer Cells: Apoptosis, Oxidative Stress, and Pathway Modulation

<b>Background/Objectives:</b> Breast cancer remains one of the leading causes of cancer-related mortality in women worldwide, highlighting the urgent need for effective and less toxic therapeutic strategies. Thymoquinone (TQ), a bioactive phytochemical derived from Nigella sativa, possesses antioxidant and anticancer activities. Methotrexate (MTX), a widely used folate antagonist, is an established chemotherapeutic agent but is limited by toxicity and resistance. This study aimed to investigate the potential synergistic effects of TQ and MTX in estrogen receptor-positive MCF-7 breast cancer cells. <b>Methods</b>: MCF-7 cells were exposed to TQ (0-100 μM), MTX (0-10 μM), and their combinations for 24-72 h. Cell viability was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and drug interactions were evaluated using the Chou-Talalay method. Apoptosis was quantified by Annexin V/Propidium Iodide (PI) flow cytometry, and cell cycle distribution was analyzed by PI staining. Intracellular reactive oxygen species (ROS) generation was measured using a 2',7'-Dichlorofluorescin diacetate (DCFH-DA) assay, while antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT)) activities were quantified spectrophotometrically. Gene expression of Bax, Bcl-2, NF-κB, MMP-2, and MMP-9 was determined by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). <b>Results:</b> TQ and MTX each reduced cell viability in a dose- and time-dependent manner, while combination treatment significantly enhanced cytotoxicity compared with single agents (<i>p</i> < 0.01). Combination Index (CI) values < 1 confirmed a synergistic interaction, particularly at 50 μM TQ + 5 μM MTX and 100 μM TQ + 10 μM MTX. Combination therapy increased total apoptosis up to 83.6%, markedly elevated the Bax/Bcl-2 ratio, and enhanced caspase-3 activation. Cell cycle analysis revealed pronounced G2/M arrest. ROS levels increased approximately six-fold, accompanied by significant suppression of SOD and CAT activities. qRT-PCR results demonstrated upregulation of pro-apoptotic Bax and downregulation of anti-apoptotic B-cell lymphoma 2 (Bcl-2), nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-2, and MMP-9. <b>Conclusions:</b> TQ potentiates the anticancer activity of MTX in MCF-7 breast cancer cells by synergistically inducing apoptosis, oxidative stress, and cell cycle arrest while suppressing metastasis-related genes. This combination may represent a promising therapeutic strategy for breast cancer, warranting further validation in in vivo and clinical studies.