Hippo signaling pathway components (Mst1/2, LATS1, Mob1, Sav1, YAP); Ras association domain family 1 (RASSF1) [2]
pLATS1, pMob1, pYAP[2]

Although 3,3'-Diindolylmethane (DIM) has less visible structural similarity to the endogenous AR ligand dihydrotestosterone (DHT), it is a powerful antagonist of androgen-acquisition receptor (AR) function. The primary byproduct of indole-3-carbinol digestion, which is a potentially anticancer ingredient in cruciferous products, is 3,3'-Diindolemethane. In L., 3,3'-Diindolylmethane demonstrates strong anticancer activity as well as antiandrogenic qualities. LNCaP cell proliferation is regulated by 3,3'-diindolylmethane, which also prevents DHT from stimulating DNA synthesis. Moreover, 3,3'-diindolylmethane suppresses DHT-induced stimulation in LNCaP cells and controls the equilibrium of endogenous PSA. Furthermore, the Pioneer promoter of the DHT victim controlled the expression of the reporter construct in suspension-transfected LNCaP cells by adjusting the 3,3′-diindolylmethane concentration. Co-administration of 50 μM 3,3'-diindolylmethane showed where AR was located in the cytoplasm and nucleus and partially prevented AR translocation brought on by DHT administration. Moreover, treatment with 3,3′-diindolylmethane inhibited the nucleus's ability to create AR foci. The sole source of fluorescein, which is mostly found in the cytoplasm, is 3,3'-diindolylmethane [1].

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DIM significantly inhibited the proliferation of human gastric cancer cell lines SNU-1 and SNU-484 in a dose-dependent manner, with >50% cell growth inhibition at 50 μM after 72 h treatment as assessed by MTT assay [2].
DIM (100 μM) significantly inhibited colony formation of SNU-1 and SNU-484 cells in soft agar assay after 2 weeks of culture [2].
DIM treatment (24 h) increased the proportion of cells in G1 phase in a dose-dependent manner in SNU-1 and SNU-484 cells. At 75 μM DIM, CDK2, CDK4, CDK6, and cyclin D1 protein levels were downregulated, while p53 protein level was upregulated [2].
DIM treatment (72 h, 100 μM) induced apoptosis: increased sub-G1 cell population, increased cleaved-PARP and cleaved-caspase-9 levels, and decreased pro-caspase-3 protein levels in SNU-1 and SNU-484 cells [2].
DIM (100 μM, 72 h) activated the Hippo signaling pathway: increased pLATS1, pMob1, Sav1, and pYAP protein levels, while decreased YAP protein levels in SNU-1 and SNU-484 cells. Mst1/2 levels were not altered [2].
DIM (100 μM, 72 h) increased endogenous RASSF1 protein production in SNU-1 and SNU-484 cells. Immunoprecipitation showed increased interaction between RASSF1 and the Mst1/2-LATS1-Sav1-Mob1 complex [2].

For 30 days, subjects were split into two groups at random and given subcutaneous injections of either vehicle or 3,3'-diindolylmethane (10 mg/kg). Every three days, the subjects' body weight and tumor volume were measured with calipers. Treatment with 3,3'-Diindolylmethane (DIM) markedly slowed the growth of SNU-484 xenograft tumors. Interestingly, the turning body weight 30 days post-drug irradiation did not differ appreciably from the vehicle simulator's, suggesting that 3,3'-diindolylmethane is not highly harmful to mice. When combined, our results show that 3,3'-diindolylmethane strongly suppresses SNU-484 xenografts' ability to develop in vivo through YAP inactivation [2].

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DIM (10 mg/kg, subcutaneous injection once daily for 30 days) significantly inhibited the growth of SNU-484 human gastric cancer xenografts in immunodeficient mice. Tumor volume was measured every 3 days and calculated as (width)² × length/2. DIM treatment resulted in marked inhibition of tumor growth compared to vehicle control [2].

Cell growth inhibition was determined by MTT assay. Cells were plated in 96-well plates at 1×10⁴ cells/well, incubated for 24 h, then treated with various concentrations of DIM for 72 h. Cell viability was assessed by a scanning multi-well spectrophotometer [2].
Soft agar colony formation assay: bottom layer of 1% agar and top layer of 0.7% agar were prepared in 6-well plates. SNU-1 or SNU-484 cells (5×10⁴ cells/well) were seeded in single-cell suspension. Cells were divided into control group (blank) and experimental group (DIM, 100 μM). Cells were cultured for 2 weeks, and colonies of >30 cells were counted. Experiments were repeated in triplicate [2].
Cell cycle analysis: cells were incubated in 100-mm dishes and treated with various DIM concentrations for 24 h. Cells were washed with PBS and nuclei stained with propidium iodide. Percentage of cells in different cell cycle phases was measured by flow cytometry [2].
Western blot analysis: cells were plated and allowed to attach for 24 h, then treated with DIM at indicated concentrations for 72 h (or 24 h for cell cycle proteins). Cells were harvested, lysed, and protein concentration determined. Whole lysate was resolved on SDS-PAGE and transferred to PVDF membranes. Membranes were probed with specific primary antibodies followed by peroxidase-conjugated secondary antibodies. Bands were visualized by enhanced chemiluminescence [2].
Immunoprecipitation: cells were scraped and suspended in lysis buffer. Extracts were incubated on ice for 1 h and centrifuged. Whole lysates (1.5 mg) were pre-cleared with Protein G-Sepharose, then incubated with anti-RASSF1 antibody overnight at 4°C. Antibody-antigen complexes were collected on Protein G-Sepharose, resolved by SDS-PAGE, transferred to PVDF membranes, and probed with specific antibodies [2].

In vivo xenograft study: Four-week-old female SPF/VAF immunodeficient mice were acclimated for 1 week. Mice were injected subcutaneously into the right flank with 0.1 ml Matrigel containing 3.5×10⁶ human gastric cancer cells (SNU-484). One week after tumor implantation, mice were randomized into two groups: untreated control group (n=5, DMSO in 50 μl PBS daily) and DIM-treated group (n=5, 10 mg/kg DIM in 50 μl PBS once daily, subcutaneous injection). Treatment lasted for 30 days. Tumor volume was measured once every 3 days using a caliper and calculated as (width)² × length/2. The experiment was terminated on day 39. Half of the tumor tissue was prepared for western blotting and the other half snap-frozen in liquid nitrogen and stored at -80°C [2].

Body weight of mice from both DIM-treated and vehicle control groups did not significantly differ following 30 days of drug exposure, suggesting that DIM (10 mg/kg daily subcutaneous) has no severe toxicity to the mice [2].

[1]. Plant-derived 3,3'-Diindolylmethane is a strong androgen antagonist in human prostate cancer cells. J Biol Chem. 2003 Jun 6;278(23):21136-45.

[2]. 3,3'-Diindolylmethane suppresses the growth of gastric cancer cells via activation of the Hippo signaling pathway. Oncol Rep. 2013 Nov;30(5):2419-26.

3,3'-Diindolemethane belongs to the indole class of compounds. It possesses antitumor activity and is also a P450 inhibitor. Diindolemethane has been used in research on the prevention and treatment of diseases such as systemic lupus erythematosus, prostate cancer, cervical dysplasia, stage I prostate cancer, and stage II prostate cancer. It has been reported that 3,3'-diindolemethane is present in cabbage, asparagus, and other organisms with relevant data. Diindolemethane is a phytonutrient and plant indole found in cruciferous vegetables such as broccoli, Brussels sprouts, cabbage, cauliflower, and kale, and possesses potential anti-androgenic and antitumor activities. Diindolemethane (DIM) is a dimer of indole-3-carbinol, which enhances antioxidant activity by promoting the metabolism of beneficial estrogen in both sexes by reducing the level of 16-hydroxyestrogens metabolites and increasing the production of 2-hydroxyestrogens metabolites. Although this compound can induce tumor cell apoptosis in vitro, the exact mechanism by which DIM exerts its antitumor activity in vivo remains unclear.

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DIM is a principal product converted from the bioactive phytochemical indole-3-carbinol (I3C) found abundantly in cruciferous vegetables. I3C is chemically unstable in aqueous environments and rapidly converted to DIM, which is a major condensation product in the stomach. DIM is the predominant bioactive compound in plasma [2].
The Hippo signaling pathway (also known as Salvador-Warts-Hippo pathway) controls organ size, cell proliferation, apoptosis, and tumorigenesis. YAP is a key effector protein negatively regulated by the Mst1/2-LATS1/2-Mob1 complex through direct phosphorylation. Phosphorylation of YAP (pYAP) inhibits cell proliferation [2].
RASSF1 is a tumor suppressor located on chromosome 3p21.3, frequently silenced by promoter hypermethylation in various cancers. RASSF1 interacts with Mst1/2 and promotes their phosphorylation, activating the Hippo pathway [2].

C17H14N2

246.30646

246.115

1968-05-4

3071

Off-white to pink solid powder

1.3±0.1 g/cm3

504.8±30.0 °C at 760 mmHg

167 °C

232.5±15.8 °C

0.0±1.2 mmHg at 25°C

1.765

4.05

2

0

2

19

286

0

C1(CC2=CNC3=C2C=CC=C3)=CNC4=C1C=CC=C4

VFTRKSBEFQDZKX-UHFFFAOYSA-N

InChI=1S/C17H14N2/c1-3-7-16-14(5-1)12(10-18-16)9-13-11-19-17-8-4-2-6-15(13)17/h1-8,10-11,18-19H,9H2

3-(1H-indol-3-ylmethyl)-1H-indole

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO : ≥ 100 mg/mL (~405.99 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.15 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 25.0 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: 2.5 mg/mL (10.15 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (10.15 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 25.0 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.)