Fig. 3

Pharmacological doses of Vit-C generate high H₂O₂ concentrations. A Schematic diagram showing the mechanism of vitamin C (Vit-C)-mediated hydrogen peroxide (H₂O₂) production via the Fenton reaction and the Haber–Weiss reaction. The transition of Vit-C between ascorbic intermediate radical (Asc^•−) and dehydroascorbic acid (DHA) can lead to H₂O₂ production in the extracellular environment, which can rapidly diffuse inside the BC cell. Following the entrance of Asc^•− via sodium-dependent Vit-C transporter (SVCT) and DHA via glucose transporter 1 (GLUT-1), they utilize the transition labile iron pool (LIP, Fe²⁺) in addition to the reduction of reduced glutathione (GSH) to glutathione disulfide (GSSG) by DHA in the presence of NADPH to produce massive amounts of intracellular hydrogen peroxide (H₂O₂), hydroxide ion (OH⁻), hydroxyls radical (^•OH), and superoxide (O₂^•−). This, in turn, will activate caspases 3 and 7, leading to cancer cell death by apoptosis. B and C H₂O₂ level measurement in MDA-MB-231 and MCF-7 spheroids after the treatment with five pharmacological doses of Vit-C (1 mM, 5 mM, 10 mM, 15 mM, and 20 mM) for 72 h, respectively. The levels of H₂O₂ production were Vit-C dose-dependent in MDA-MB-231 tumor spheroids and MCF-7 tumor spheroids, respectively (B and C). D and E Cell viability analysis of MDA-MB-231 and MCF-7 tumor spheroids treated with CAT (300 U/mL) for 2 h, then supplemented with five pharmacological doses of Vit-C (1, 5, 10, 15, and 20 mM) for 72 h. The statistical levels of significance were determined as follows: **P < 0.01, ***P < 0.001, ****P < 0.0001, and *P < 0.05. Two-tailed unpaired student's t-test was employed in both cases, and the error bars represent the mean ± SEM; the data are representation of three independent experiments (n = 3)