Thymoquinone Overcomes Hypoxia-Induced Carboplatin Resistance Through ROS-Independent Apoptosis but Promotes Cancer Stem Cell Enrichment: Implications on Oral Cancer Adaptation and Recurrence.

Background: Carboplatin is a first-line chemotherapy agent for patients with oral squamous cell carcinoma (OSCC), but chemoresistance significantly impacts treatment outcomes. This study evaluated the ability of thymoquinone, a natural metabolite found in food products, to modulate cytotoxicity, ROS, apoptosis, autophagy, and cancer stem cell markers in early- and late-stage OSCC cell models to identify mechanisms of chemoresistance and determine the influence of dietary metabolites on treatment outcomes. Methods: OECM-1 cells were treated with concentrations (1 mM to 1 pM) of thymoquinone, carboplatin, or their combination under normoxic and hypoxic conditions. HIF-1α levels were measured using ELISA, and cytotoxicity was assessed by the MTT assay. ROS, apoptosis, autophagy, and cell surface markers (CD44+, CD133+, CD147+) were evaluated. All experiments were repeated three times, and the data were analyzed using GraphPad Prism. Under hypoxia, HIF-1α increased 12-fold. Results: Carboplatin demonstrated reduced potency (110 μM) and efficacy (40%) compared to normoxia (82 μM, 88%), accompanied by increased apoptosis (75%) and decreased ROS (25%). Thymoquinone was more potent than carboplatin, further reducing ROS (50%), increasing apoptosis (95%), and downregulating autophagy, while the proportion of CD133+ expressing cells increased significantly (75%) in the hypoxic model. For the combined treatment across both models, thymoquinones' efficacy remained high (>90%). Between models, no further change in any parameter was observed, except for apoptosis induction, which increased to 65% (normoxia) and 50% (hypoxia). Conclusions: Thymoquinones' superior efficacy under hypoxic conditions demonstrates ROS-independent cytotoxic mechanisms; however, the enrichment of CD133+ cells raises essential questions about long-term therapeutic outcomes and the risks of tumor recurrence. Natural pharmaceutical metabolites can influence the tumor microenvironment, which is highly implicated in cancer therapeutics and cancer adaptation.