Synergistic Effects of Betanin and Theobromine on the PI3K/AKT/mTOR Pathway in KB Cells: A Molecular Insight into ROS-Mediated Apoptosis

Background: Oral squamous cell carcinoma (OSCC) is a common cancer that has a high recurrence rate and little response to treatment. Natural substances that alter survival and apoptotic pathways, like betanin and theobromine, have promising anticancer effects. This study examines the combined cytotoxic effects of betanin and theobromine on KB oral cancer cells, with a particular emphasis on the production of reactive oxygen species (ROS) and the modulation of the PI3K/AKT/mTOR pathway. Methods: KB cells were treated with varying concentrations of betanin (1–100 µg/mL), theobromine (1–100 µg/mL), and their combinations (Combo_IC20, Combo_IC50, Combo_IC70). Cell viability was assessed using the MTT assay. Using DCFH-DA staining and fluorescence intensity, intracellular ROS levels were determined. Following a 24-hour treatment, the expression of PIK3CA, AKT1, mTOR, BAX, BCL2, and CASP3 was examined using quantitative real-time PCR (qRT-PCR). Results: Both betanin and theobromine showed dose-dependent cytotoxicity, with combination treatments—especially Combo_IC50—exhibiting significantly enhanced cell death. ROS levels increased substantially in all treated groups, with the highest levels observed in the combination groups, indicating oxidative stress-mediated apoptosis. Gene expression analysis revealed significant downregulation of PIK3CA, AKT1, and mTOR in combination groups, along with upregulation of BAX and CASP3, and downregulation of anti-apoptotic BCL2, confirming mitochondrial apoptotic pathway activation. Conclusion: Betanin and theobromine act synergistically to inhibit KB cell proliferation by inducing oxidative stress and suppressing the PI3K/AKT/mTOR pathway, leading to apoptosis. These findings support the potential application of this phytochemical combination as a safe and effective strategy for oral cancer therapy.