Fig 2: AhR activation status does not affect Nrf2 activation or transcriptional activity. RPTECs were cultured under ctrl conditions or subjected to Reox with or without the AhR inhibitor CH223191. (A) Representative western blots of Nrf2 levels (corresponding to its activation status) and the expression of the Nrf2 transcriptional targets xCT (SLC7A11) and SOD-3. (B-D) Statistical analysis of the western blots. Neither Reox nor CH223191 affects Nrf2 activity, or the expression of xCT and SOD-3. Data are presented as the mean ± SEM of six independent experiments. AhR, arylhydrocarbon receptor; Nrf2, nuclear factor erythroid 2-related factor 2; xCT, cystine-glutamate antiporter; SOD-3, superoxide dismutase; RPTEC, renal proximal tubular epithelial cell; ctrl, control; Reox, reoxygenation.

Fig 3: Quantitative results of ferroptosis-related markers. Representative images of Western blots and densitometry of ferroptosis-related markers. Caki-2 cells showed significantly increased levels of GPX4, xCT, TfRC, and FTH1, whereas Caki-1 cells showed decreased levels of GPX4, xCT, and TfRC compared to HK-2 cells. SNU-333 cells showed significantly increased levels of FTH1, while the other markers were comparable to those of HK-2 cells. *P < .05, **P < .01, ***P < .001 versus HK-2 cells.

Fig 4: Schematic representation of cell death pathways in colorectal cancer (CRC) cells following fenbendazole treatment.Fenbendazole induces G2/M arrest and apoptosis in both (A) 5-FU-sensitive SNU-C5 and (B) 5-FU-resistant SNU-C5 (SNU-C5/5-FUR) CRC cells. In SNU-C5 cells, fenbendazole is presumed to activate p53-mediated apoptosis by increasing p53 expression. In SNU-C5/5-FUR cells, fenbendazole triggers apoptosis without affecting p53 expression, whereas fenbendazole enhances ferroptosis by inhibiting the expression of GPX4 and SLC7A11. Therefore, although fenbendazole has anti-cancer effects on both 5-FU-sensitive and resistant CRC cells, the mechanism of action appears to be different. That is, fenbendazole promotes cell death by activating p53-mediated apoptosis in SNU-C5 cells, whereas by both enhancing p53-independent apoptosis and ferroptosis-augmented apoptosis in SNU-C5/5-FUR cells. PARP, poly (ADP-ribose) polymerase; HMGB1, high mobility group box 1; GPX4, glutathione peroxidase 4; LC3, light chain 3.