Dulbecco's modified eagle medium was obtained from GIBCO BRL (Grand Island, NY, USA). Indole-3-Carbinol, Genistein and TRAIL were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Enzo Life Sciences (Farmingdale, NY, USA), respectively. The human endometrial cancer cell line Ishikawa was obtained from European collection of cell cultures (ECACC, Salisbury, UK). The cells were cultured and incubated at 37℃ in 5% CO₂.

The number of viable cell exposed to I3C, Genistein and TRAIL were evaluated by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Initially, cells were seeded in 96-well plate, and then cultured for 24 hr to allow their adhesion to the plate. After pre-incubation, the culture medium was changed to experimental medium supplemented with DMSO (0.1%) control, I3C (50 µM), Genistein (20 µM) and TRAIL (10, 20, and 50 ng/mL) alone or in combination for 24 hr. The intensity of the purple color formed by this assay is proportional to the number of viable cells. MTT reagent was added and incubated for an additional 4 hr at 37℃. The optical density (OD) was measured at 540 nm. The mean values and their standard deviations were calculated from triplicate experiments.

To determine the distribution of cells in the different phases of cell cycle profile, flow cytometric analysis was performed. Cells were treated with DMSO (control) or I3C (50 µM), Genistein (20 µM) and TRAIL (10, 20, and 50 ng/mL) alone or in combination. After treatment, cells were harvested by trypsinization and centrifugation, washed with ice-cold phosphate buffered saline (PBS), and was fixed in ice-cold 70% ethanol at 4℃ for 24 hr. Ethanol fixed cells were washed and treated with RNase A for 30 min at 37℃ and were stained with propidium iodide. DNA fluorescence was measured by flow cytometer using a FACS Calibur cell sorter (Becton Dickinson, San Jose, CA, USA). The staining of apoptotic cells was performed by using annexin VFITC/PI apoptosis detection kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer's instructions. The determination of apoptotic cells was calculated using Cellquest software (Becton Dickinson).

Endometrial cancer cells were treated with DMSO (control) or I3C (50 µM), Genistein (20 µM) and TRAIL (10, 20, and 50 ng/mL) alone or in combination for 24 hr. After drug treatment, cells were harvested with lysis buffer. DTT Mix and 2 × Reaction buffer was added in supernatants and incubated on ice for 30 min. Caspase-3 substrate (DEVD-pNA; 50 µM final concentration) was added and incubated at 37℃ for 1 hr in water bath. Caspase-3 activity was measured by microplate reader (Tecan, Durham, NC, USA) at 405 nm followed by ApoAlert Caspase Colorimetric Assay kits User Manual (Clontech, Mountain View, CA, USA).

Cells were harvested, centrifuged and washed once in PBS. Cell pellets were lysed in DNA isolation buffer and centrifuged at 4℃ for 20 min. After treated with Tris saturated phenol (pH 8.0), the extraction was further treated with equal volume of phenol/chloroform/isoamyl alcohol (25:24:1 v/v) and chloroform/isoamyl alcohol (24:1 v/v) separately. The DNA was precipitated by addition 0.1 volume of 3 M sodium acetate and two volume of ice-cold ethanol and incubated at -70℃ for at least 2 hr. The samples were then loaded into a 1.8% agarose gel and electrophoresed for 40 min at constant 100V. DNA was visualized by UV illumination.

Fragmented nucleosomal DNA was quantified by Cell Death Detection ELISA Plus kit (Roche Diagnostics, Mannheim, Germany) as described in the manufacture's manual. The absorbance was measured at 405 nm using microplate reader (Tecan).

Cell (4 × 10⁵/mL) extracts were prepared in lysis buffer (10 mM Tris [pH 7.4], 5 mM EDTA, 130 mM NaCl, 1% Triton X-100, serine protease inhibitor phenylmethylsulphonyl fluoride [10 µg/mL], leupeptin [10 µg/mL], aprotinin [10 µg/mL], 5 mM phenanthroline and 28 mM benzamidine-HCl). Protein concentrations were measured using Bio-Rad Protein Assay Reagent (Bio-Rad, Hercules, CA, USA) following the manufacturer's protocol. Aliquots of protein were separated by 8%-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidine difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked with Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (Becton Dickinson). After washing, the membranes were incubated with primary antibodies of DR4 (Santa Cruz Biotech, Santa Cruz, CA, USA), DR5 (Koma Biotech, Seoul, Korea), cleaved caspase-3, cleaved caspase-8 and PARP (Cell Signaling, Danvers, MA, USA), Flip (Enzo Life Science) and β-actin (Santa Cruz Biotech). After reaction with horseradish peroxidase conjugated secondary antibodies (Santa Cruz Biotech) bands on the membranes were visualized by an enhanced chemiluminescence system (Thermo Scientific, Rockford, IL, USA) following the manufacturer's suggested procedure. The density of respective bands was analyzed by the chemi-Doc XRS imaging system (Bio-Rad). The data were presented as % of controls.

The data are presented as mean ± SD. Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test for a post hoc comparison by using the Statistical Package for Social Science (SPSS 17.0) statistical software. Statistical significance was set at P < 0.05.

To determine whether I3C and Genistein can sensitize endometrial cancer, Ishikawa and HEC1A cells were grown in presence of I3C, Genistein and TRAIL for 24 hr. I3C and Genistein did not show significant cell death alone or in combination. TRAIL only also could not affect in cell viability. However, three-combination (I3C and Genistein with TRAIL) treatment induced significant suppression of cell proliferation in Ishikawa and HEC1A (data not shown). The finding indicated the potency of three-combination over the endometrial cancer and only Ishikawa cells were selected for the further experiments (Fig. 1).

Flow cytometric analysis of cell cycle was performed to examine the mechanism of inhibition of cell growth following I3C, Genistein and TRAIL treatment. There was no significant alternation in the cell cycle population in individual or two-combination treatment. However, remarkable increase in sub-G1 arrest was observed in the combination of three drugs. The treatment increased the ratio of sub-G1 fraction from 5.25% (DMSO control) to 44% (I3C Genistein and TRAIL). Consequently, the G0/G1phase decreased from 55.5% (DMSO control) to 19.35% (I3C, Genistein and TRAIL) (Fig. 2A). To observe the apoptosis, we examined Ishikawa cells for annexin V-FITC staining. Induction of apoptosis was substantiated by examining the flow cytometry pattern of annexin V-FITC stained. Apoptotic cells accounted for 3.6% (TRAIL), 4.8% (I3C and Genistein), and 13.3% (I3C, Genistein and TRAIL) (Fig. 2B).

In order to study the apoptotic response, we examined the caspase-3 activity with the treatment of I3C, Genistein and TRAIL. The TRAIL alone and combination of I3C and Genistein treatment did not show significant changes. The caspase-3 activity was increased in dose dependent manner when TRAIL was added with I3C and Genistein (Fig. 3A). ELISA analysis showed increase of histone-associated DNA fragments (Fig. 3B). The presence of distinct bands after electrophoresis of genomic DNA of cells has become a hallmark of apoptosis (Fig. 3C).

To show whether potential reduction of cell viability and cell arrest was due to apoptosis, the apoptosis inducing genes were examined. The up-regulated expression of DR4, DR5, cleaved caspase-8 and degradation of Flip was observed in three-combination treatment. In addition, the increased expression of cleavage of caspase-3 and PARP confirmed the caspase cascade in apoptotic signaling pathway (Fig. 4).