2. Vitamin D Related/Nuclear Receptor Apoptosis Metabolic Enzyme/Protease Immunology/Inflammation NF-κB
  3. Nuclear Hormone Receptor 4A/NR4A Ferroptosis Glutathione Peroxidase Reactive Oxygen Species (ROS) Transferrin Receptor Apoptosis Bcl-2 Family Caspase
  4. DIM-3,5-Cl2

DIM-3,5-Cl2 is an inverse NR4A1/NR4A2 agonist with KD 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.

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DIM-3,5-Cl2

DIM-3,5-Cl2 Chemical Structure

CAS No. : 2595179-74-9

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DIM-3,5-Cl2 Related Antibodies

Top Publications Citing Use of Products

View All Nuclear Hormone Receptor 4A/NR4A Isoform Specific Products:

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Nur77/NR4A1 Nurr1/NR4A2

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GPX1 GPX4

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Bcl-2 Bcl-xL Bcl-W Mcl-1 Bfl-1 Bcl-B Bax Bak Bim BNIP3 Bad

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Caspase 1 Caspase 2 Caspase 3 Caspase 4 Caspase 5 Caspase 6 Caspase 7 Caspase 8 Caspase 9 Caspase 10 Caspase 12 Caspase Caspase 11 Caspase-13

  • Biological Activity

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Description

DIM-3,5-Cl2 is an inverse NR4A1/NR4A2 agonist with KD 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].

IC50 & Target[2][1]

Nur77/NR4A1

7.7 μM (Kd)

Nurr1/NR4A2

12 μM (Kd)

Caspase 3

 

Bcl-2

 

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 (HY-100579), 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].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

DIM-3,5-Cl2 Related Antibodies

Western Blot Analysis[1]

Cell Line: Human MDA-MB-231, MDA-MB-468, and mouse 4T1 triple negative breast cancer (TNBC) cells
Concentration: 7, 10, 12, 15 μM (MDA-MB-231, MDA-MB-468); 10, 12, 15 μM (4T1)
Incubation Time: 24 h
Result: Induced cleaved PARP and cleaved caspase-3, and decreased BCL-2, full-length PARP, and full-length caspase-3 at 7, 10, 12 μM in MDA-MB-231 cells.
Induced cleaved PARP and cleaved caspase-3, and decreased BCL-2, full-length PARP, and full-length caspase-3 at 7, 10, 12, 15 μM in MDA-MB-468 cells.
Induced cleaved PARP and cleaved caspase-3, and decreased BCL-2, full-length PARP, and full-length caspase-3 at 10, 12, 15 μM in 4T1 cells.\nDecreased GPX4 and SLC7A11 protein levels at 7, 10, 12 μM; induced CD71 protein levels at 7, 10 μM, and decreased CD71 at 12 μM in MDA-MB-231 cells.
Decreased GPX4 and SLC7A11 protein levels at 7, 10, 12, 15 μM; induced CD71 protein levels at 7, 10 μM, and decreased CD71 at 12, 15 μM in MDA-MB-468 cells.
Decreased GPX4 and SLC7A11 protein levels at 10, 12, 15 μM; induced CD71 protein levels at 10, 12, 15 μM in 4T1 cells.

Western Blot Analysis[1]

Cell Line: Human MDA-MB-231 triple negative breast cancer (TNBC) cells
Concentration: 10, 12 μM
Incubation Time: 24 h
Result: Decreased expression of EGFR, β1-integrin, and c-Myc in MDA-MB-231 cells.

Western Blot Analysis[1]

Cell Line: Human MDA-MB-231 triple negative breast cancer (TNBC) cells
Concentration: 12 μM
Incubation Time: 24 h
Result: Ferrostatin-1 significantly inhibited compound-mediated downregulation of CD71, GPX4, and SLC7A11 in MDA-MB-231 cells.

Western Blot Analysis[1]

Cell Line: Human MDA-MB-231 triple negative breast cancer (TNBC) cells
Concentration: 10 μM
Incubation Time: 4, 6, 24 h
Result: Knockdown of Sp1 or Sp4 alone decreased CD71 protein expression in MDA-MB-231 cells.
Induced Sp1, Sp4, and CD71 protein expression in MDA-MB-231 cells at 4, 6, and 24 h, with increasing induction observed over time.

Cell Migration Assay[2]

Cell Line: Immortalized human endometriotic epithelial cells (IHEEC), Immortalized human endometriotic stromal cells (IHESC)
Concentration: 6.5 μM
Incubation Time: 24 h
Result: Significantly reduced migration of IHEEC and IHESC cells into the scratch wound area compared to vehicle controls, as measured by relative migration percentage.

Western Blot Analysis[2]

Cell Line: Immortalized human endometriotic epithelial cells (IHEEC), Immortalized human endometriotic stromal cells (IHESC)
Concentration: 6.5 μM; 13 μM
Incubation Time: 24 h
Result: Significantly decreased protein levels of EGFR, mTOR, phospho-mTOR, β1-integrin, CTGF, FN, COL1A1, TWIST1, Slug, Snail, Vimentin, N-cadherin, ZEB1, ZO-1, β-catenin, NR4A1, and NR4A2 in IHEEC cells.
Significantly increased protein levels of cleaved poly(ADP-ribose) polymerase (C-PARP) and claudin-1 in IHEEC cells.
Had no significant effect on ERβ levels in IHEEC cells.
Significantly decreased protein levels of mTOR, phospho-mTOR, β1-integrin, ERβ, CTGF, COL1A1, TWIST1, Slug, Snail, N-cadherin, ZEB1, ZO-1, β-catenin, NR4A1, and NR4A2 in IHESC cells.
Significantly increased protein levels of C-PARP and claudin-1 in IHESC cells.
Had no significant effect on EGFR, FN, or α-SMA levels in IHESC cells.

Immunofluorescence[2]

Cell Line: Immortalized human endometriotic epithelial cells (IHEEC), Immortalized human endometriotic stromal cells (IHESC)
Concentration: 13 μM
Incubation Time: 12 h
Result: Significantly reduced fluorescence intensity of COL1A1, N-cadherin, and TWIST1 compared to untreated controls in both IHEEC and IHESC cells.
Caused a substantial decrease in filamentous actin structures in treated cells as shown by phalloidin staining.
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].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Model: FVB/NJ (female, 6 weeks old, surgically induced endometriosis)[2]
Dosage: 2.5 mg/kg
Administration: i.p.; daily; 34 consecutive days
Result: Significantly reduced ectopic lesion volume compared to vehicle control.
Significantly inhibited lesion growth as measured by luciferase imaging, while vehicle-treated mice showed continuous lesion growth.
Significantly reduced Ki-67 expression in stromal cells of ectopic lesions; Ki-67 expression in epithelial cells was not significantly affected.
Significantly increased TUNEL-positive (apoptotic) cells in ectopic lesions.
Significantly reduced Nr4a1 expression in both epithelial and stromal cells of ectopic lesions; significantly reduced Nr4a2 expression in stromal cells, with no significant effect on epithelial Nr4a2 expression.
Did not cause body weight loss, and liver panel analysis showed no evidence of liver damage compared to vehicle control.
Animal Model: C57BL/6 (female, 4-6 weeks old, subcutaneous implantation of MC-38 colon cancer cells)[3]
Dosage: 2.5 mg/kg/day; 7.5 mg/kg/day
Administration: i.p.; daily; 21 days
Result: Significantly decreased tumor volumes and tumor weights relative to controls.
Showed no treatment-related toxicity, with treated mice showing a slight increase in body weight.
Decreased expression of NR4A1 and PD-L1 in tumor lysates at 2.5 mg/kg/day dose.
Significantly increased the percentage of CD8+ T-cells in tumor-infiltrating lymphocytes (TILs) at 2.5 mg/kg/day dose.
Decreased the percentage of CD8+ T-cells expressing the T-cell exhaustion markers PD-1, 2B4, and TIM3, and decreased the percentage of CD8+ T-cells co-expressing PD-1 and TIM3 in TILs at 2.5 mg/kg/day dose.
Decreased mRNA expression of NR4A1, TOX, TOX2, and NFAT, and increased mRNA expression of interferon γ, granzyme B, perforin, and T-Bet in CD8+ T-cells isolated from TILs at 2.5 mg/kg/day dose.
Significantly increased cell percentages, decreased the percentage of cells expressing PD-1, 2B4, TIM3, and PD-1/TIM3, increased the percentage of Treg cells, increased the percentage of cells expressing T-Bet and TOX/TOX2, and decreased the percentage of cells expressing NFAT1 in splenic CD8+ T-cells at 2.5 mg/kg/day dose.
Molecular Weight

391.29

Formula

C23H16Cl2N2
CAS No.
Appearance

Solid

Color

White to off-white

SMILES

ClC1=CC(Cl)=CC(C(C2=CNC3=C2C=CC=C3)C4=CNC5=C4C=CC=C5)=C1
Shipping

Room temperature in continental US; may vary elsewhere.
Storage
Powder -20°C 3 years
In solvent -80°C 6 months
-20°C 1 month
Solvent & Solubility
In Vitro: 

DMSO : 100 mg/mL (255.56 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

Preparing
Stock Solutions
Concentration Solvent Mass 1 mg 5 mg 10 mg
1 mM 2.5556 mL 12.7782 mL 25.5565 mL
5 mM 0.5111 mL 2.5556 mL 5.1113 mL
View the Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

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In Vivo:

Select the appropriate dissolution method based on your experimental animal and administration route.

For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

  • Protocol 1

    Add each solvent one by one:  10% DMSO    90% Corn Oil

    Solubility: ≥ 2.5 mg/mL (6.39 mM); Clear solution

    This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown). If the continuous dosing period exceeds half a month, please choose this protocol carefully.

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The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.
 If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
Purity & Documentation
References

Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
DMSO 1 mM 2.5556 mL 12.7782 mL 25.5565 mL 63.8912 mL
5 mM 0.5111 mL 2.5556 mL 5.1113 mL 12.7782 mL
10 mM 0.2556 mL 1.2778 mL 2.5556 mL 6.3891 mL
15 mM 0.1704 mL 0.8519 mL 1.7038 mL 4.2594 mL
20 mM 0.1278 mL 0.6389 mL 1.2778 mL 3.1946 mL
25 mM 0.1022 mL 0.5111 mL 1.0223 mL 2.5556 mL
30 mM 0.0852 mL 0.4259 mL 0.8519 mL 2.1297 mL
40 mM 0.0639 mL 0.3195 mL 0.6389 mL 1.5973 mL
50 mM 0.0511 mL 0.2556 mL 0.5111 mL 1.2778 mL
60 mM 0.0426 mL 0.2130 mL 0.4259 mL 1.0649 mL
80 mM 0.0319 mL 0.1597 mL 0.3195 mL 0.7986 mL
100 mM 0.0256 mL 0.1278 mL 0.2556 mL 0.6389 mL
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DIM-3,5-Cl2 Related Classifications

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Keywords:

DIM-3,5-Cl22595179-74-9Nuclear Hormone Receptor 4A/NR4AFerroptosisGlutathione PeroxidaseReactive Oxygen Species (ROS)Transferrin ReceptorApoptosisBcl-2 FamilyCaspaseNuclear Hormone Receptor 4ANerve Growth Factor IB-like ReceptorTfRCD71IHESC cellsferroptosisIHEEC cellsNR4A2MDA-MB-231endometriosisnuclear receptor 4A1apoptosistriple negative breast cancercolorectal cancerInhibitorinhibitorinhibit

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