DIM-3,5-Cl2 is an inverse agonist of NR4A1/NR4A2 that can downregulate pro-carcinogenic responses or gene products regulated by these receptors. It induces the production of reactive oxygen species (ROS), malondialdehyde, and lipid peroxides, while decreasing the expression of GPX4 and SLC7A11 and increasing CD71 expression. Additionally, DIM-3,5-Cl2 can induce ferroptosis and is applicable in research on triple-negative breast cancer.

DIM-3,5-Cl 2 (2.5-15 μM, 24 hours) decreases the viability of various breast cancer cells in a dose-dependent manner. It induces apoptosis in breast cancer cells by modulating the NR4A1/2 pathway and downregulates multiple oncogenes at concentrations of 7-15 μM over 24 hours. At 10-12 μM for 4-24 hours, DIM-3,5-Cl 2 significantly increases reactive oxygen species (ROS) levels, detected using H2DCFDA dye, and induces lipid peroxidation in MDA-MB-231, MDA-MB-468, and 4T1 cells using the BODIPY 581/591 C11 probe. After 16 hours at the concentration of 12 μM, there is a significant increase in MDA levels in these cells. DIM-3,5-Cl 2 reduces the expression of GPX4 and SLC7A11 proteins in the aforementioned cells between 7-15 μM for 24 hours, while CD71 expression is downregulated at lower concentrations (below 10 μM) and further decreased at higher concentrations (12-15 μM). Within 1-6 hours at 7-10 μM, it elevates CD71 protein expression and CD71 mRNA levels in MDA-MB-231 cells. It also induces CD71 protein expression in PDX-derived organoids (PDxO) at 15 μM. Additionally, between 10-12 μM for 4-24 hours, DIM-3,5-Cl 2 reduces NR4A1 and NR4A2 protein levels while enhancing Sp1 and Sp4 protein expression in MDA-MB-231 cells. At 10-12 μM for 6 hours, it activates the CD71 promoter in MDA-MB-231, MDA-MB-468, and 4T1 cells. Lastly, though DIM-3,5-Cl 2 induces luciferase activity in these cells after 24 hours at 10-12 μM, it can partially inhibit this activity at higher concentrations.