Fig. 4: HO-1 modulation and iron-bound transferrin are the key players in FMD-dependent sensitization to Vitamin C.

a, b Western blotting detection of HO-1 expression level in KRAS-mutant HCT116 (n = 4), CT26 (n = 4) and HCT116-derived tumor masses (n = 5 in Vit C and FMD + Vit C, n = 7 in Ad libitum and FMD) and KRAS-wild-type SW48 cancer cells (n = 4). VINCULIN as loading control. Representative blots and quantifications are shown. P values were determined by two-sided unpaired t-test. CT26: exact P values = 0.00002 (CTR vs STS), 0.00009 (CTR vs STS + Vit C). c Viability of DLD1, HCT116, and CT26 cells treated with STS with or without vitamin C, hemin (n = 3) or d zinc protoporphyrin (ZnPP; HCT116, n = 3; DLD1 and CT26, n = 4). P values were determined by two-sided unpaired t-test. HCT116 in (c): exact P value = 0.00005 (STS + Vit C vs STS + Hemin + Vit C), CT26 in (c): exact P value= 0.00001 (STS + Vit C vs STS + Hemin + Vit C). e Western blot (left) and viability (right) of HCT116 cells transfected with control siRNAs (siCTR) or anti-HO-1 siRNAs (siHO-1), n = 5. P values were determined by two-sided unpaired t-test. f Viability of HCT116 cells grown in STS medium with apo-transferrin (ApoTrf) or holo-transferrin (HoloTrf) with or without vitamin C (n = 4 in STS, n = 3 in ApoTrf and n = 5 in HoloTrf). P values were determined by two-sided unpaired t-test. Exact P value = 0.00000003 (STS + Vit C vs STS + HoloTrf + Vit C). g Quantification of transferrin bound iron in mouse blood serum (right) of HCT116-engrafted NSG mice fed ad libitum or subjected to FMD cycles, and treated with or without vitamin C (tumor growth, left). Black arrows indicate blood serum collection: upon the last FMD cycle and 24 h after refeeding (RF). P values were determined by two-sided unpaired t-test (n = 8 mice in Ad libitum, n = 7 in FMD, FMD + Vit C, Vit C, n = 3 in FMD RF, FMD + Vit C RF). All data are represented as mean ± SEM, n = independent experiments.