Isocitrate dehydrogenase 1 mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to ferroptosis

Abstract

The present study intends to clarify the hypothesis that isocitrate dehydrogenase 1 (IDH1) mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to ferroptosis through the in vitro and in vivo experiments. Cholangiocarcinoma RBE cell line was transfected with IDH1 R132C mutation plasmids and treated with erastin to induce ferroptosis, which were then microscopically photographed. Cell viability rate was calculated by trypan blue staining. The lipid ROS level was determined by using flow cytometer. The BALB/c nude mice were injected subcutaneously with IDH1 knockout (KO), WT, or R132C mutation cell line, followed by injecting erastin intraperitoneally. The tumor tissue was surgically separated for the measurement of tumor volume and weight. The results showed that IDH1 mutant RBE cell line are sensitive to erastin-induced ferroptosis, evidenced by the increased number of propidium iodide-positive cells, the decreased cell viability, and increased lipid ROS level. However, current targeted inhibitors of IDH1 mutation (AG120 and IDH305) reversed these effects caused by IDH1 mutation. The in vivo experiment showed that IDH1 mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to erastin-induced ferroptosis. This study indicated that IDH1 mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to erastin-induced ferroptosis.

Keywords: IDH1; cholangiocarcinoma; ferroptosis; mutation.

Figure 1

IDH1 mutation promotes erastin-induced ferroptosis in cholangiocarcinoma RBE cell line. The IDH1 KO RBE cell line was constructed using CRISPR-Cas9, and then the IDH1 KO cell line was, respectively, transfected with Vector, IDHI WT, or IDHI R132C mutation plasmid to construct the IDH1 WT or mutation cell lines, followed by erastin (5 μM) treatment for 12 h, and (a) cells were microscopically photographed. (b) The number of PI-positive cells was statistically quantified. (c) The trypan blue staining assay was used to determine the cell viability. (d and e) The lipid ROS levels were determined by using flow cytometer. *p < 0.05 vs DMSO treatment group, ^# p < 0.05, ^### p < 0.001 vs IDH1 WT or IDH1 R132C group.

Figure 2

Inhibitors of IDH1 mutation reversed its effects on erastin-induced ferroptosis in cholangiocarcinoma RBE cell line. Current targeted inhibitors of IDH1 (AG120 and IDH305) were administrated into IDH1 R132C mutation cell line with erastin and DMSO treatment, and (a) cells were microscopically photographed. (b) The number of PI-positive cells was statistically quantified. (c) The trypan blue staining assay was used to determine the effects of AG120 or IDH305 on the cell viability. (d) The effects of AG120 or IDH305 on the lipid ROS levels were determined by using flow cytometer, and (d and e) statistically quantified. ***p < 0.001 vs DMSO treatment group.

Figure 3

IDH1 mutation promotes erastin-induced tumor growth inhibition in cholangiocarcinoma. Male BALB/c nude mice were injected subcutaneously with IDH1 KO, WT, or mutation cell line. One week later, mice were injected intraperitoneally with erastin or DMSO at a dose of 15 mg/kg. (a) After administration, the diameter and volume of the tumor tissue were measured every 3 days until the end of the administration. The animals were executed and the tumor tissue was surgically removed for photography (b), and for the measurement of (c) tumor weight. *p < 0.05 vs DMSO treatment group, #p < 0.05 vs IDH1 WT group.