The human gastric cancer cell lines SNU-1 and SNU-484 were purchased from the Korean Cell Line Bank (Seoul National University, Korea). Cancer cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 mg/ml streptomycin, and 100 IU/ml penicillin (all from Gibco-BRL, Grand Island, NY, USA) as a monolayer in 100-mm dishes (BD Biosciences, Rockville, MD, USA) under standard conditions at 37°C in a 5% CO₂ humidified atmosphere. All experiments were performed with the cells at 60–80% confluence. DIM was purchased from LKT Laboratories (St. Paul, MN, USA). The following antibodies were obtained from the following commercial sources: cyclin D1, CDK4, CDK6, p27, cleaved-caspase-9, pro-caspase-3, cleaved-poly(ADP-ribose) polymerase (PARP), Mst1, Mst2, LATS1, pLATS1, Mob1, pMob1, Sav1, YAP, pYAP, Akt, pAkt (Cell Signaling Technology, Inc., Beverly, MA, USA); CDK2 and Ras association domain family 1 (RASSF1) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

The effect of DIM on cell proliferation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously (38). Briefly, cells were plated in 96-well plates (SPL, Seoul, Korea) at 1×10⁴ cells/well. After 24 h of incubation under standard conditions, the cells were treated with the indicated DIM concentration for 72 h. Cell viability was assessed by a scanning multi-well spectrophotometer (SpectraMAX 340; Molecular Devices Co., Sunnyvale, CA, USA).

The bottom layer of soft agar (1%) was prepared in a 6-well plate, and the top layer (0.7%) was prepared with 5×10⁴ SNU-1 or SNU-484 cells/well in a single-cell suspension. The cells were divided into 2 groups: i) the control group (blank group) and ii) the experimental group (DIM, 100 μM). The cells were cultured in an incubator at 37°C with 5% CO₂ for 2 weeks and observed for colony formation by microscopy. Colonies of >30 cells were counted, and the experiments were repeated in triplicate.

To determine the effect of DIM on the cell cycle, cells were incubated in 100-mm dishes and were treated for 24 h with various DIM concentrations. Subsequently, cells were washed with PBS and the nuclei were stained with propidium iodide (PI) (Sigma Chemical, St. Louis, MO, USA) as described previously (28,38). The percentage of cells in the different cell cycle phases was measured with a FACstar flow cytometer (Becton-Dickinson, San Jose, CA, USA) and analyzed using Becton-Dickinson software (Lysis II and CellFit).

Briefly, the human gastric cancer cell lines SNU-1 and SNU-484 were plated and allowed to attach for 24 h. DIM was added to the cell cultures at the indicated concentrations for 72 h. Cells with or without DIM were harvested and suspended in lysis buffer (Intron Biotechnology, Korea). Extracts were incubated on ice for 10 min and centrifuged at 13,200 rpm for 20 min at 4°C. After centrifugation, the supernatant was collected and the protein concentration was determined using a BSA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Whole lysate was resolved on a SDS-PAGE gel and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were probed with the specific primary antibodies and then with peroxidase-conjugated secondary antibodies. The bands were visualized with the enhanced chemiluminescence kit (Amersham, Arlington Heights, IL, USA). The following antibodies were used: antibody against cyclin D1, CDK2, CDK4, CDK6, p53, cleaved-PARP, cleaved-caspase-9, pro-caspase-3, Mst1, Mst2, pLATS1, LATS1, Mob1, pMob1, Sav1, pYAP, YAP, pAkt, Akt, RASSF1, β-catenin, pβ-catenin, β-actin and GAPDH.

Cells were scraped in PBS and suspended in lysis buffer (20 mM Tris-HCl pH 8, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40 and 2 mM EDTA). Extracts were incubated on ice for 1 h and pelleted by centrifugation at 12,000 rpm for 10 min. Protein quantification was determined using a BSA protein assay kit. Whole lysates (1.5 mg) were incubated with Protein G-Sepharose (Sigma) for 1 h at 4°C for pre-clearing and centrifuged 10,000 rpm for 5 min at 4°C. The supernatant was incubated with anti-RASSF1 at 4°C overnight. Antibody-antigen complexes were collected on Protein G-Sepharose. Immunoprecipitates were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with the specific primary antibodies followed by peroxidase-conjugated secondary antibodies. The bands were visualized with the enhanced chemiluminescence kit. The following antibodies were used: antibody against Mst1, Mst2, LATS1, Mob1, Sav1 and GAPDH.

Based on our in vitro results, we designed studies to determine the effects of DIM on xenografted human gastric tumors in nude mice. Animal experiments adhered to NIH guidelines (USA) and were performed under approval of the Institutional Animal Care and Use Committee of Chonbuk National University. Four-week-old female SPF/VAF immunodeficient mice were purchased from Orient Bio (Korea). The mice were allowed to acclimate to local conditions for 1 week prior to injection with cancer cells. Thirteen mice were 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 were 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 DIM-treated group (n=5, 10 mg/kg in 50 μl PBS once daily). Gastric primary tumors were excised, and the final 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 was snap frozen in liquid nitrogen and stored at −80°C.

In vitro experiments were repeated >3 times. The statistical significant difference between experimental groups and the control was determined by one-way ANOVA and then later compared among groups with an unpaired Student’s t-test. Results are expressed as the means ± SE. A p-value <0.05 was considered to indicate a statistically significant result.

The effects of DIM on human gastric adenocarcinoma cell growth were examined using SNU-1 and SNU-484 cells by treatment with various DIM concentrations for 72 h. MTT assay analyses revealed that DIM significantly suppressed the proliferation of the cell lines in a dose-dependent manner, showing >50% cell growth inhibition at 50 μM in the two cell lines (Fig. 1A). The effects of DIM on SNU-1 and SNU-484 gastric cancer cell colony formation were evaluated using a soft agar cloning assay. SNU-1 and SNU-484 gastric cancer cells were cultured in medium with 100 μM DIM for 2 weeks, and colony formation was observed by microscopy. As shown in Fig. 1B, DIM significantly inhibited colony formation of SNU-1 and SNU-484 cells when compared to the control. These results suggest that DIM significantly inhibits the growth of gastric cancer cells.

Fluorescence-activated cell sorting (FACS) analysis was performed to characterize whether DIM regulates cell cycle progression in human gastric cancer cells. As shown in Fig. 2A, DIM treatment resulted in a significant increase in the proportion of the cell population in the G1 phase of the cell cycle at 24 h in SNU-1 and SNU-484 human gastric cancer cells. These results suggest that DIM induced G1 phase arrest in human gastric cancer cells in a dose-dependent manner. The effects of DIM on CDK2, CDK4, CDK6 and cyclin D1 protein levels were examined to identify the regulation of G1 cell cycle regulatory proteins in human gastric cancer cells. A 75-μM DIM treatment in SNU-1 and SNU-484 cells downregulated the production of CDK2, CDK4, CDK6, cyclin D1 protein levels at 24 h (Fig. 2B). Since DNA damage checkpoints play a critical role in maintaining the integrity of the genome by arresting cell cycle progression in response to damaged or incompletely replicated DNA, and G1 checkpoint arrest in mammalian cells is mediated by the action of p53 protein, we also investigated the effects of DIM on the induction of the p53 protein level. A 75-μM DIM treatment in SNU-1 and SNU-484 cells resulted in the upregulation of the p53 protein level leading to cell cycle arrest in the G1 phase (Fig. 2B). These results indicate that DIM induced G1 phase arrest in human gastric cancer cells through changes in the levels of cell cycle regulatory proteins.

As shown in Fig. 3A, DIM treatment in SNU-1 and SNU-484 gastric cancer cells showed an increased accumulation of cells in the sub-G1 phase in a dose-dependent manner. To test whether DIM induces apoptotic cell death in gastric cancer cells, we further investigated the cleaved-caspase-9, cleaved-PARP, and pro-caspase-3 protein levels using western blot analysis. As shown in Fig. 3B, treatment with 100 μM DIM for 72 h inhibited pro-caspase-3 protein levels and increased levels of the cleaved form of PARP and cleaved-caspase-9, hallmarks of apoptosis, in the SNU-1 and SNU-484 gastric cancer cells. These results indicate that DIM induced apoptotic cell death in human gastric cancer cells. Overall, these results support the notion that the observed decline in cell viability by DIM was in part due to cell cycle arrest and induction of apoptosis.

The Hippo signaling pathway is a tumor-suppressor signaling system that inhibits cell proliferation and promotes cell apoptosis. To investigate whether DIM mediates gastric cancer cell death via the Hippo signaling pathway, we examined the protein levels of Mst1/2, LATS1, pLASTS1, Mob1, pMob1, and Sav1 after DIM treatment. As shown in Fig. 4, western blot analysis revealed that treatment with 100 μM DIM for 72 h significantly increased the production of pLATS1, pMOB1, and Sav1 in SNU-1 and SNU-484 cells. We also analyzed the production of YAP and pYAP after treatment with DIM. We found that the production of pYAP was significantly increased and YAP protein levels were decreased following treatment with 100 μM DIM for 72 h in gastric cancer cells. These results suggest that DIM may trigger the Hippo signaling pathway cascade that leads to phosphorylation, cytoplasmic retention and functional inactivation of YAP.

To address the question of how DIM activates the Hippo signaling pathway, we investigated the possible role of RASSF1 that has been identified as an upstream regulator of the Hippo signaling pathway (39,40). RASSF1 has been reported to interact with the mammalian kinases, Mst1, and Mst2 and activate them by promoting their autophosphorylation and subsequent phosphorylation of the downstream LATS1 kinase (41). In addition, several studies have revealed that Hippo signaling is tightly regulated by protein-protein interactions (14). Therefore, we tested whether RASSF1 associates with Hippo signaling pathway molecules due to DIM treatment. As shown in Fig. 5A, endogenous RASSF1 protein production was significantly increased following treatment with 100 μM DIM in the SNU-1 and SNU-484 cells. In addition, a dramatic increased interaction between the Mst1/2- LATS1-Sav1-Mob1 complex and RASSF1 was observed in the presence of the same DIM dose as analyzed by immunoprecipitation followed by immunoblotting (Fig. 5B). These results indicate that DIM may activate the Hippo signaling pathway through RASSF1 activation, which increases the coimmunoprecipitation of RASSF1 with Hippo signaling molecules.

Since we observed an inhibition of gastric cancer cell viability by DIM in vitro, we evaluated whether these observations could be translated into an animal model system in vivo. To address this issue, SPF/VAF immunodeficient mice were inoculated in the right flank with SNU-484 human gastric cancer cells. Mice were randomized into two groups and were treated daily s.c. with either vehicle or DIM (10 mg/kg) for 30 days. Tumor volume and the weight of mice were recorded once every 3 days using calipers (Fig. 6). As shown in Fig. 6A–E, DIM treatment resulted in a marked inhibition of SNU-484 xenograft tumor growth. Notably, the body weight of mice from both groups did not significantly differ from the vehicle control following 30 days of drug exposure (Fig. 6F), suggesting that DIM has no severe toxicity to the mice. Taken together, these findings demonstrate that DIM administration significantly inhibited SNU-484 xenograft growth in vivo mediated by the inactivation of YAP.