Cell experiment:

The human prostate adenocarcinoma cell lines LNCaP-FGC and PC-3 are grown as adherent monolayers in 10% FBS-DMEM, supplemented with 4.0 g/L glucose and 3.7 g/L sodium bicarbonate in a humidified incubator at 37℃ and 5% CO2, and passaged at ~80% confluency. Cultures used in subsequent experiments are at less than 40 passages. Cells grown in stripped conditions are in 5% DCC-FBS-DMEM base supplemented with 4 g/L glucose, 3.7 g/L sodium bicarbonate, and 0.293 g/L L-glutamine. Before the beginning of the treatments, cells are depleted of androgen for 4-7 days in medium composed of DMEM base without phenol red and with 4 g/L glucose and 3.7 g/L sodium bicarbonate. During the depletion period, medium is changed every 48 h. Treatments are administered by the addition of 1 μL of a 1,000-fold concentrated solution of 3,3'-Diindolylmethane in Me2SO/mL of medium. Once the treatment period started, medium is changed daily to counter possible loss of readily metabolized compounds[1].

Animal experiment:

Mice[2] Four-week-old female SPF/VAF immunodeficient mice are injected subcutaneously (s.c.) into the right flank with 0.1 mL Matrigel containing 3.5×106 human gastric cancer cells (SNU-484). The mice are randomized into 2 groups 1 week after tumor implantation: i) the untreated control group (n=5, DMSO in 50 μL PBS daily) and ii) the 3,3'-Diindolylmethane-treated group (n=5, 10 mg/kg in 50 μL PBS once daily). Gastric primary tumors are excised, and the final tumor volume is measured once every 3 days using a caliper and calculated as (width)2×length/2. The experiment is terminated on day 39. Half of the tumor tissue is prepared for western blotting and the other half is snap frozen in liquid nitrogen and stored at –80℃.

3,3'-Diindolylmethane is an anticancer agent [1].

3,3'-Diindolylmethane (DIM) is a dimer of indole-3-carbinol generated in low pH environment. It inhibited cell growth with IC50 values of 17, 24 and 30 μM in MCF-7, T47D and Saos2 cell lines, respectively. DIM induced apoptosis in these cells without affecting the p53 pathway. In human nasopharyngeal carcinoma (NPC) cell line (5-8F NPC), treatment of DIM decreased the ability of cell migration and invasion dose-dependently. In rats, transplanted with NPC cells, DIM administration resulted in suppressing lymph node metastasis. Besides that, DIM was found to induce ERα target gene expression and the concomitant cell proliferation at low concentration (10μM) in the absence of E2 [1, 2 and 3].

References:
1. Ge X, Yannai S, Rennert G, et al. 3, 3′-Diindolylmethane induces apoptosis in human cancer cells. Biochemical and Biophysical Research Communications, 1996, 228(1): 153-158.
2. Wu T, Chen C, Li F, et al. 3, 3' Diindolylmethane inhibits the invasion and metastasis of nasopharyngeal carcinoma cells in vitro and in vivo by regulation of epithelial mesenchymal transition. Experimental and Therapeutic Medicine, 2014, 7(6): 1635-1638.
3. Marques M, Laflamme L, Benassou I, et al. Low levels of 3, 3 [prime]-diindolylmethane activate estrogen receptor alpha and induce proliferation of breast cancer cells in the absence of estradiol. BMC cancer, 2014, 14(1): 524.