DIM-3,5-Cl2

DIM-3,5-Cl2 is an inverse NR4A1/NR4A2 agonist with K D values of 7.7 μM and 12.0 μM for NR4A1 and NR4A2, respectively. DIM-3,5-Cl2 acts as an inverse agonist to downregulate pro-oncogenic and proendometriotic gene products, and as an agonist to enhance NR4A1/2 / Sp1 / Sp4 -mediated CD71 transactivation. DIM-3,5-Cl2 induces ferroptosis via ROS formation, lipoperoxidation, MDA production, and reduced GPX4, SLC7A11 expression. DIM-3,5-Cl2 induces apoptosis via PARP and caspase-3 cleavage, reduced BCL-2 expression, and inhibits cancer cell viability. DIM-3,5-Cl2 can be used for the research of triple negative breast cancer, endometriosis, and colorectal cancer ^{[1] [2] [3]}.

In Vivo, DIM-3,5-Cl2 (2.5 mg/kg; i.p.; daily; 34 consecutive days) administered at 2.5 mg/kg daily for 34 days significantly reduces endometriotic lesion growth, suppresses cellular proliferation, increases apoptosis, downregulates Nr4a1 and Nr4a2 in lesions, and does not induce overt toxicity in a surgically induced Mus musculus endometriosis model^[2]. DIM-3,5-Cl2 (2.5-7.5 mg/kg/day; i.p.; daily; 21 days) significantly inhibits MC-38 colon tumor growth in syngeneic C57BL/6 mice, downregulates tumor PD-L1 expression, and reverses T-cell exhaustion in both TILs and splenic CD8+ T-cells^[3].

In Vitro, DIM-3,5-Cl2 (2.5-15 μM) potently inhibits the viability of human MDA-MB-231, MDA-MB-468, and mouse 4T1 TNBC cells^[1]. DIM-3,5-Cl2 (7-15 μM; 24 h for MDA-MB-231, MDA-MB-468; 10-15 μM; 24 h for 4T1) induces apoptosis in human MDA-MB-231, MDA-MB-468, and mouse 4T1 TNBC cells via induction of cleaved PARP and cleaved caspase-3, and downregulation of pro-survival proteins BCL-2, full-length PARP, and full-length caspase-3^[1]. DIM-3,5-Cl2 (7-15 μM; 24 h for MDA-MB-231, MDA-MB-468; 10-15 μM; 24 h for 4T1) modulates ferroptosis-related proteins in human MDA-MB-231, MDA-MB-468, and mouse 4T1 TNBC cells by decreasing GPX4 and SLC7A11 expression, and inducing CD71 expression at lower/mid concentrations (7, 10 μM) while decreasing CD71 at higher concentrations (12, 15 μM)^[1]. DIM-3,5-Cl2 (10-12 μM; 24 h) downregulates pro-oncogenic proteins EGFR, β1-integrin, and c-Myc in human MDA-MB-231 TNBC cells^[1]. DIM-3,5-Cl2 (12 μM; 16 h) potently induces ROS formation in human MDA-MB-231, MDA-MB-468, and mouse 4T1 TNBC cells^[1]. DIM-3,5-Cl2 (12 μM; 16 h) potently induces lipid peroxidation in human MDA-MB-231, MDA-MB-468, and mouse 4T1 TNBC cells^[1]. DIM-3,5-Cl2 (12 μM; 24 h) has its mediated downregulation of CD71, GPX4, and SLC7A11 in human MDA-MB-231 TNBC cells reversed by the ferroptosis inhibitor Ferrostatin-1, confirming DIM-3,5-Cl2 acts via a ferroptotic pathway^[1]. DIM-3,5-Cl2 (7-10 μM; 1, 2, 4, 6 h) time-dependently induces CD71 protein and CD71 mRNA expression in human MDA-MB-231 TNBC cells, with protein induction at 6 h and mRNA peaking at 4 h^[1]. DIM-3,5-Cl2 (15 μM; 24 h) induces CD71 protein expression in NR4A1/NR4A2-expressing TNBC PDxO models (BCM-HCI-3561, BCM-4175)^[1]. DIM-3,5-Cl2 (10 μM) has mediated induction of CD71 promoter activity in human MDA-MB-231 TNBC cells dependent on NR4A1 and NR4A2 expression, as knockdown of either receptor abrogates the induction^[1]. DIM-3,5-Cl2 (10 μM; 4-24 h) time-dependently induces Sp1, Sp4, and CD71 protein expression in human MDA-MB-231 TNBC cells, and CD71 expression is dependent on Sp1 and Sp4 expression, as knockdown of either transcription factor decreases CD71 levels^[1]. DIM-3,5-Cl2 (0.25-20 μM; 48 h) potently inhibits proliferation of IHEEC cells (IC₅₀ = 7.34 μM) and IHESC cells (IC₅₀ = 5.78 μM) after 48 h of treatment^[2]. DIM-3,5-Cl2 (6.5 μM; 24 h) significantly inhibits migration of both IHEEC and IHESC cells^[2]. DIM-3,5-Cl2 (6.5-13 μM; 24 h) modulates expression of pro-endometriotic pathway proteins in a cell-type specific manner, downregulating growth, fibrosis, and epithelial-to-mesenchymal transition (EMT) markers, upregulating apoptotic markers, and reducing NR4A1/NR4A2 levels in both IHEEC and IHESC cells^[2]. DIM-3,5-Cl2 (13 μM; 12 h) reduces expression of fibrosis and EMT markers (COL1A1, N-cadherin, TWIST1) and disrupts actin filament structures in both IHEEC and IHESC cells^[2]. DIM-3,5-Cl2 (5-10 μM; 24 h) decreases NR4A1, Sp1, and PD-L1 protein expression in SW480 human colon cancer cells^[3]. DIM-3,5-Cl2 (2.5-7.5 μM; 24 h) decreases NR4A1, Sp1, and PD-L1 protein expression in RKO human colon cancer cells^[3]. DIM-3,5-Cl2 (2.5-7.5 μM; 24 h) decreases NR4A1, Sp1, and PD-L1 protein expression in MC-38 murine colon cancer cells^[3]. DIM-3,5-Cl2 (7.5 μM; 3 h) reduces the binding of NR4A1, Sp1, and PolII to the GC-rich proximal promoter of the PD-L1 gene in MC-38 murine colon cancer cells^[3]. DIM-3,5-Cl2 (10 μM; 3 h) reduces the binding of NR4A1, Sp1, and PolII to the GC-rich proximal promoter of the PD-L1 gene in SW480 human colon cancer cells^[3]. DIM-3,5-Cl2 (7.5 μM; 3 h) reduces the binding of NR4A1, Sp1, and PolII to the GC-rich proximal promoter of the PD-L1 gene in RKO human colon cancer cells^[3].

References:
[1]. Oany AR, et al. Orphan nuclear receptor 4A1 (NR4A1) and NR4A2 are endogenous regulators of CD71 and their ligands induce ferroptosis in breast cancer. Cell Death Dis. 2025 Nov 3;16(1):776.
[2]. Tsui WNT, et al. Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy. Endocrinology. 2025;166(11):bqaf144.
[3]. Mohankumar K, et al. Bis-indole-derived NR4A1 antagonists inhibit colon tumor and splenic growth and T-cell exhaustion. Cancer Immunol Immunother. 2023;72(12):3985-3999.