Nur77 (IC50 = 13.0 μM); Nur77 (IC50 = 13.6 μM)
DIM-C-pPhOH (7.5-20 μM; 24 hours; ACHN and 786-O cells) treatment significantly decreases cell proliferation[1].
DIM-C-pPhOH (20 μM; 24 hours; ACHN and 786-O cells) treatment results in Annexin V staining in ACHN and 786-O cells, confirming that DIM-C-pPhOH induces apoptosis and also induces caspase 7 and 8 cleavage[1].
DIM-C-pPhOH (15-20 μM; 24 hours; ACHN and 786-O cells) treatment inhibits NR4A1-regulated expression of survivin, bcl-2 and EGFR in ACHN and 786-O cells. Additionally, it activates AMPK, stimulates sestrin 2, and inhibits the activation of mTOR and downstream kinases[1].
DIM-C-pPhOH reduces expression of β1-integrin protein and mRNA as well as β1-integrin-dependent responses in MCF7, MDA-MB-231, and SKBR3. The latter two cell lines are also prevented from migrating[2].

DIM-C-pPhOH (7.5-20 μM; 24 hours; ACHN and 786-O cells) treatment significantly decreases cell proliferation[1].
DIM-C-pPhOH (20 μM; 24 hours; ACHN and 786-O cells) treatment results in Annexin V staining in ACHN and 786-O cells, confirming that DIM-C-pPhOH induces apoptosis and also induces caspase 7 and 8 cleavage[1].
DIM-C-pPhOH (15-20 μM; 24 hours; ACHN and 786-O cells) treatment inhibits NR4A1-regulated expression of survivin, bcl-2 and EGFR in ACHN and 786-O cells. Additionally, it activates AMPK, stimulates sestrin 2, and inhibits the activation of mTOR and downstream kinases[1].
DIM-C-pPhOH reduces expression of β1-integrin protein and mRNA as well as β1-integrin-dependent responses in MCF7, MDA-MB-231, and SKBR3. The latter two cell lines are also prevented from migrating[2].

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Treatment of human renal cell carcinoma (RCC) cell lines ACHN (p53-positive) and 786-O (p53-mutant) with DIM-C-pPhOH significantly decreased cell proliferation in a concentration-dependent manner, with an IC50 of 13.6 μM in ACHN cells and 13.0 μM in 786-O cells.
DIM-C-pPhOH (20 μM) induced apoptosis in both ACHN and 786-O cells, as evidenced by increased Annexin V staining and cleavage of caspases 3, 7, 8 and PARP.
Treatment with DIM-C-pPhOH (15-20 μM) decreased protein expression of the Sp1-regulated pro-survival/growth genes survivin, bcl-2, and EGFR in ACHN and 786-O cells.
DIM-C-pPhOH (15-20 μM) treatment decreased protein expression of TXNDC5 and IDH1, induced markers of endoplasmic reticulum (ER) stress (CHOP, ATF4, p-PERK), and increased reactive oxygen species (ROS) in both RCC cell lines.
In ACHN cells (wild-type p53), DIM-C-pPhOH (15-20 μM) induced sestrin 2 expression, activated AMPKα (increased p-AMPKα), and inhibited mTOR signaling (decreased p-mTOR, p-p70S6K, p-S6RP, p-4EBP1).
In 786-O cells (mutant p53), DIM-C-pPhOH (15-20 μM) also induced sestrin 2 and inhibited mTOR signaling; this induction was attenuated by co-treatment with the antioxidant glutathione (GSH), suggesting ROS-mediated induction.
Immunofluorescence analysis in ACHN and 786-O cells treated with DIM-C-pPhOH (20 μM) showed that NR4A1 protein remained localized in the nucleus, indicating the antagonist acts on nuclear NR4A1.
In a transactivation assay, ACHN cells co-transfected with an NBRE3-luc reporter and FLAG-NR4A1 showed that DIM-C-pPhOH (20 μM) significantly decreased NR4A1-dependent luciferase activity. [1]

DIM-C-pPhOH (30 mg/kg; oral gavage; daily; for 50 days; male athymic nude mice) treatment results in a significant inhibition of tumor growth[1].

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In an orthotopic xenograft model, male athymic nude mice bearing ACHN cell tumors were treated orally with DIM-C-pPhOH (30 mg/kg/day, in corn oil) for 50 days. This treatment significantly inhibited tumor growth compared to vehicle-treated controls.
Western blot analysis of tumor lysates from DIM-C-pPhOH-treated mice showed decreased protein levels of survivin, bcl-2, EGFR, TXNDC5, and IDH1, and increased expression of the ER stress marker CHOP, compared to control tumors. [1]

For proliferation assays, ACHN and 786-O kidney cancer cells were plated at a density of 1.0 x 10^5 cells per well in 12-well plates and allowed to attach for 24 hours.
The medium was then changed, and cells were treated with varying concentrations (0-20 μM) of DIM-C-pPhOH for 24 hours.
Cell numbers were subsequently determined.
For apoptosis analysis via Annexin V staining, cells were seeded similarly, treated with DIM-C-pPhOH (20 μM) for 24 hours in medium containing 2.5% charcoal-stripped fetal bovine serum, and then analyzed for Annexin V staining according to standard protocols.
For western blot analysis, cells were seeded, treated with DIM-C-pPhOH (15 or 20 μM) for 24 hours, and whole cell lysates were prepared.
Lysates were analyzed using specific antibodies against proteins of interest (e.g., survivin, bcl-2, EGFR, cleaved caspases, PARP, TXNDC5, IDH1, stress markers, mTOR pathway components).
β-actin was used as a loading control.
For immunofluorescence to assess NR4A1 localization, cells were treated with DIM-C-pPhOH (20 μM) for 24 hours, fixed, and immunostained with an antibody against NR4A1 and counterstained with DAPI.
Images were captured using a fluorescence microscope.
For ROS measurement, cells were treated with DIM-C-pPhOH, and cellular ROS levels were determined using the cell-permeant fluorescent probe CM-H2DCFDA.
For the NR4A1 transactivation assay, ACHN cells were plated and co-transfected with an NBRE3-luciferase reporter plasmid (400 ng) and a FLAG-NR4A1 expression plasmid (40 ng) using a transfection reagent.
After 6 hours, cells were treated with DIM-C-pPhOH (20 μM) or vehicle (DMSO) in medium containing 2.5% charcoal-stripped serum for 18 hours, after which luciferase activity was measured. [1]

Male athymic nude mice (Foxn1nu, aged 6-7 weeks) were used.
ACHN renal carcinoma cells (10^7 cells in 150 μL Matrigel) were injected subcutaneously into the flanks of the mice.
After 7 days, when tumors were palpable, mice were randomized into treatment groups (n=6 per group).
DIM-C-pPhOH was dissolved in corn oil.
Mice were dosed daily via oral gavage with DIM-C-pPhOH at 30 mg/kg body weight per day, or with corn oil vehicle alone, for 50 days.
Mice were weighed, and tumor dimensions (length and width) were measured with calipers twice a week to calculate tumor volume using the formula: V = (L x W^2) / 2.
At the end of the study, mice were euthanized by CO2 asphyxiation, and tumors were collected for lysate preparation and subsequent western blot analysis. [1]

[1]. Nuclear Receptor 4A1 (NR4A1) as a Drug Target for Renal Cell Adenocarcinoma. PLoS One. 2015 Jun 2;10(6):e0128308.

[2]. NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration. Mol Cell Biol. 2016 Apr 15;36(9):1383-94.

DIM-C-pPhOH is a 1,1-bis(3'-indolyl)-1-(para-substituted phenyl)methane (C-DIM) compound, specifically a para-hydroxyphenyl analogue. It functions as an antagonist of the orphan nuclear receptor NR4A1, acting on the nuclear form of the receptor without inducing its nuclear export. This study proposes that NR4A1 is a tumor-promoting factor in renal cell adenocarcinoma (RCC), and that DIM-C-pPhOH exerts its anti-cancer effect by antagonizing NR4A1, thereby inhibiting multiple downstream pathways. These pathways include: (1) co-activation of Sp1-regulated pro-survival/growth genes (e.g., survivin, bcl-2, EGFR); (2) maintaining low cellular stress by regulating TXNDC5 and IDH1; and (3) regulating the mTOR signaling pathway through a p53-dependent and ROS-mediated mechanism. The results indicate that DIM-C-pPhOH is a potential novel chemotherapy drug for renal cell carcinoma, particularly suitable for tumors overexpressing NR4A1. [1]

C23H18N2O

338.41

338.141

C, 81.63; H, 5.36; N, 8.28; O, 4.73

151358-47-3

2940609

Off-white to pink solid powder

1.3±0.1 g/cm3

610.2±50.0 °C at 760 mmHg

322.9±30.1 °C

0.0±1.8 mmHg at 25°C

1.770

4.8

3

1

3

26

443

0

O([H])C1C([H])=C([H])C(=C([H])C=1[H])C([H])(C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12)C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12

QCPDFNWJBQMXLI-UHFFFAOYSA-N

InChI=1S/C23H18N2O/c26-16-11-9-15(10-12-16)23(19-13-24-21-7-3-1-5-17(19)21)20-14-25-22-8-4-2-6-18(20)22/h1-14,23-26H

4-[bis(1H-indol-3-yl)methyl]phenol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 68~125 mg/mL(201.0~369.4 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.15 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)