FIGURE 6.

EA induced colorectal cancer cell death via triggering ferroptosis. (A) The alteration of ferroptosis-related proteins caused by EA treatment (the diagram was drawn according to proteomic results and ferroptosis KEGG map). (B) The rescue effects of ferrostatin-1 (an iron death inhibitor) on the 20 μg/ml EA-induced cell inactivation were detected by the CCK8 kit. The difference between two groups was calculated by a two-way t-test, *p < 0.05, **p < 0.01. (C) EA-induced intracellular ROS was tested by H2DCFDA. The red curve represented the background without the ROS probe, the green curve represented the ROS intensity of cells without EA treatment, and the rose-pink curve represented the ROS intensity of EA-treated cells. The EA concentration in this experiment was 20 μg/ml. (D) The alteration of intracellular iron concentrations of EA-treated SW480 cells. The difference between two groups was calculated by a two-way t-test, *p < 0.05. (E) The EA-elevated LC3 was verified by cell immunofluorescence experiment in SW480 cells. LC3 was stained by green fluorescence and the nucleus was stained by DAPI with blue fluorescence. (F) The EA-repressed ferritin (FTL) was examined by IHC in xenograft tumors. The FTL was stained with brown color, and the nucleus was stained by hematoxylin (purple). (G–I) The expression of FTL, TF and CP in clinical colorectal cancer tissue and normal tissue. Data were from the online platform GEPIA (http://gepia.cancer-pku.cn/index.html). COAD: Colon adenocarcinoma; READ: Rectum adenocarcinoma, *p < 0.05.