Fig. 4.

IRP2 inhibitors induce the ubiquitin dependent degradation of IRP2 which has anti-tumor activity. (A) Cytotoxicity of KS-20073 and KS-20226 in CRC cells and divergent normal cells. CCD18-Co; Human normal colon cell line, VERO; African green monkey kidney cell line, HFL-1; Human embryonic lung cell line, L929 (NCTC clone 929); Mouse fibroblast cell line, NIH 3T3; Mouse embryonic fibroblast cell line, CHO-K1; Chinese hamster ovary cell line. (B) 3D Spheroid toxicity of KS-20073 (30 µM) and KS-20226 (20 µM). For spheroid formation, cells were grown in U-bottom plates coated with poly-HEMA to maintain low adherent condition. Cells were treated with IRP2 inhibitors at days 3, 5, and 7 days, stained with Calcein AM (AM), and Ethidium homodimer-1 (EthD-1), and observed using confocal microscopy. The region area and fluorescence intensity of EthD-1 was assessed and quantified using the Harmony software. Data are represented as the mean ± SEM (n = 3). ***p < 0.001. (C) IRP1, IRP2, TFRC, and FTH1 protein expression levels were assessed via the immunoblotting of SW480 and LOVO cells treated with KS-20073 and KS-20226 (3 and 10 µM) for 72 h. β-actin was used as the protein-loading control. Relative protein levels were calculated using the Image J software. (D) HEK-293T cells were transfected with the HA-Ub and Myc-IRP2 plasmids for the ubiquitination assay. After 36 h of transfection, the cells were treated with KS-20073 (10 µM), KS-20226 (10 µM) and MG-132 (5 µM) for 24 h and subjected to immunoprecipitation. (E) Labile iron pool (LIP) was determined using Calcein AM (green). SW480 and LOVO cells were incubated with KS-20073 (30 µM) and KS-20226 (20 µM) for 24 h and then, quantified using the Harmony software. Data are represented as the mean ± SEM (n = 3). **p < 0.01, ***p < 0.001