Synergistic anticancer effects of thymoquinone and curcumin via ROS-mediated mitochondrial apoptosis in HeLa cells.

BACKGROUND: Cervical cancer continues to pose a significant global health issue, often exacerbated by challenges such as chemoresistance and toxicity. Phytochemicals such as thymoquinone and curcumin exhibit promising multi-targeted anticancer properties. PURPOSE: This study investigated the synergistic cytotoxic and pro-apoptotic effects of Thymoquinone (TQ) and Curcumin (CUR) on HeLa cells and elucidated the underlying mechanisms. METHODS: HeLa cells were treated with TQ (0–100 µM), CUR at 30%, 50%, and 70% inhibitory concentrations (IC₃₀, IC₅₀, IC₇₀), and fixed-ratio combinations for 24, 48, 72, and 96. Cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and synergy was assessed using the Chou–Talalay method. Morphological alterations and apoptotic features were examined by phase-contrast microscopy and acridine orange/ethidium bromide (AO/EtBr) dual staining. Apoptotic signalling was evaluated through reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm), and expression of caspase-9 and caspase-3. RESULTS: TQ and CUR reduced HeLa cell viability dose-dependently. Combination treatment showed time-dependent interaction, with additive effects at 24 h, antagonist-to-additive effects at 48 h, and synergistic cytotoxicity at longer exposures. Synergy emerged at 72 h and peaked at 96 h, particularly at lower inhibitory concentrations (CI₃₀ = 0.12). The combination induced apoptotic features, including nuclear condensation, membrane blebbing, mitochondrial depolarization, elevated ROS levels, and caspase-9 and caspase-3 activation, indicating intrinsic, caspase-dependent apoptosis. CONCLUSION: TQ and CUR synergistically induce apoptosis in HeLa cells through ROS-mediated mitochondrial dysfunction and caspase activation, highlighting their potential as a phytochemical-based adjunct strategy for cervical cancer management. Hence, further in vivo studies are warranted.