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To assess the effect of sequential treatment with Vitamin C (VC) and Quercetin (Q) on Nrf2-related oxidative stress in PC3 and DU145 cells, viability was measured by MTT assay. Intracellular ROS levels were determined, using 2'-7'-dichlorodihydrofluorescein diacetate fluorescent as a probe. Nrf2 gene expression was investigated by quantitative reverse transcription polymerase chain reaction, and Nrf2 protein levels were defined by western blot analysis. The activity of glutathione peroxidase (GPx), glutathione reductase (GR), nicotinamide adenine dinucleotide phosphate dehydrogenase quinone 1 (NQO1) and hemeoxygenase 1 (HO-1) enzymes were measured. The IC50 values for VC + Q were 263.03–372.1 µM and 144.2–194.1 µM respectively and 200 µM VC + 50 µM Q (dose no.1) and 100 µM VC + 75 µM Q (dose no.2) were selected. Sequential treatment of PC3 cells led to a significant reduction of Nrf2 mRNA expression and protein levels in addition to a significant reduction of GPx, GR and NQO1 enzymatic activity. Although the data was slightly different for DU145 cells after the treatments, in terms of Nrf2 gene expression, we obtained the same results. Our study revealed the significant effects of sequential treatment with VC + Q on Nrf2 suppression in prostate cancer cells. [ABSTRACT FROM AUTHOR]