Ascorbic Acid Potentiates Cisplatin-Induced Antitumor Activity in HeLa Cervical Cancer Cells: Oxidative Stress, Mitochondrial Dysfunction, Apoptosis, and 3D Spheroid Responses

Cisplatin is widely used in cervical cancer chemotherapy; however, dose-limiting toxicity and the development of treatment resistance restrict its therapeutic efficacy. Ascorbic acid (AsA) may exert pro-oxidant and anticancer effects at pharmacological concentrations and has the potential to enhance the activity of conventional chemotherapeutic agents. This study aimed to investigate whether AsA enhances the anticancer activity of CDDP in HeLa cervical cancer cells and to characterize the underlying mechanisms, with particular emphasis on oxidative stress, mitochondrial dysfunction, apoptotic signaling, and the persistence of the combination effect in a three-dimensional (3D) tumor spheroid model. The effects of AsA and CDDP, alone and in combination, were evaluated in HeLa cells using cell viability and Chou–Talalay combination index (CI) analyses. Apoptosis, cell-cycle distribution, intracellular reactive oxygen species (ROS), N-acetylcysteine (NAC)-mediated antioxidant rescue, mitochondrial membrane potential (ΔΨm) using JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide), caspase-3/7 activity, and apoptosis-related gene expression were assessed. The antitumor activity of the combination was further evaluated in 3D HeLa spheroids by assessing spheroid morphology, diameter, adenosine triphosphate (ATP)-based viability, and Live/Dead fluorescence staining. AsA and CDDP exhibited 48-h half-maximal inhibitory concentration (IC50) values of 3.73 mM and 3.93 µM, respectively, and their combination produced synergistic cytotoxicity (CI = 0.72). Total apoptosis increased from 6.8% in control cells to 47.6% following combined treatment, accompanied by increased ROS generation, caspase-3/7 activity, and G2/M accumulation. AsA + CDDP markedly decreased ΔΨm and increased the BAX/BCL2 ratio and TP53 expression, supporting activation of the mitochondrial apoptotic pathway. NAC attenuated ROS accumulation and partially rescued cell viability, supporting the involvement of oxidative stress in the combination response. In the 3D spheroid model, AsA + CDDP produced the greatest reduction in spheroid diameter and ATP-based viability, accompanied by marked structural disintegration and an increased relative EthD-1 red fluorescence-signal fraction. These findings identify AsA as a potential CDDP-sensitizing strategy and support further preclinical investigation. Because the study was limited to in vitro cell and spheroid models and did not establish in vivo efficacy or safety, future studies should validate the combination in more representative cervical cancer models and assess its efficacy and safety in vivo before clinical translation.