Human HCT-116 cell lines were cultured in RPMI-1640 (Invitrogen Life Technologies, San Diego, CA, USA) supplemented with 10% fetal bovine serum at 37°C and 5% of CO₂/air.

3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to estimate the cell viability. HCT-116 cells were seeded at an initial density of 5,000 cells per well in a flat-bottomed 96-well cell culture plate and allowed to grow for 48 h in a humidified 5% CO₂, 95% air atmosphere in an incubator maintained at 37°C. Twenty microliters of MTT (5 mg/ml) solution (Sigma Chemical Co., St. Louis, MO, USA) were added to each well and then incubated for 4 h at 37°C. After the media were removed, 200 µl dimethyl sulfoxide was added to each well to dissolve the formazan formed. After 30 min incubation at room temperature, the plates were scanned with a microplate reader that was set at 490 nm for measuring the absorbance.

The migration of cells was assayed in Transwell cell culture chambers with 6.5-mm diameter polycarbonate membrane filters having an 8 µm pore size. Briefly, 4×10⁴ cells in 100 µl serum-free medium were added to the upper chamber of the device, and the lower chamber was filled with 600 µl culture medium with 20% fetal bovine serum. After 10 h of incubation at 37°C, the cells of the lower chamber were analyzed.

HCT-116 cells at a density of 2×10⁶ cells/well were placed in 12-well plates and incubated with inhibitors of the Wnt and Notch signaling pathways for 48 h before the cells were harvested by centrifugation. The cells were then trypsinized, washed with phosphate-buffered saline (PBS) and treated with 50 µg/ml cold propidium iodide solution for 30 min in the dark. Flow cytometric analysis was performed on a FACSCalibur instrument (Becton-Dickinson, San Jose, CA, USA). The percentage of cells in the G0/G1, S and G2/M phases was determined by flow cytometry.

Cells were cultivated on cover glass slides in 24-well culture plates. After washing with PBS four times, cells were fixed in 4% paraformaldehyde for 30 min. All of the following washing and incubation steps were carried out with PBS/Tween-20 (PBST) on a rocker: cells were washed for 5 min four times and permeabilized with 0.05% saponin for 5 min. After another three washing steps, cells were fixed with pre-chilled ice-cold 100% acetone for 2 min on ice and immediately washed three times. Blocking with 4% bovine serum albumin in PBST for 30 min was followed by overnight incubation at 4°C with the primary antibody. The next day cells were washed three times and incubated with the secondary antibody for 1 h. For double-staining, the procedure was repeated with the next antibodies. Cells were imaged with a fluorescence microscope (Axiovert 40C; Carl Zeiss Microscopy, Inc., Thornwood, NY, USA).

A total of 24, 6–8-week-old, female BALB/c mice (Center of Experimental Animal, Kunming Medical University, Kunming, China) were subcutaneously injected with 1×10⁶ HCT-116 cells/animal. All animals were housed under a controlled room humidity (50±10%), and maintained under a 12-h light/dark cycle with free access to water and food. Upon tumor formation, the animals were divided into four groups: i) control group (n=6); ii) rDDK-1 group (5 mg/kg, administered once weekly, all three times, n=6); iii) LY374973 group (4 mg/kg, administered once weekly, all three times, n=6); and iv) rDDK-1+LY374973 group (n=6). The study was approved by the ethics committee of Guangdong General Hospital (Guangzhou, China; approval number 2015268A).

qPCR was used to detect the mRNA expression of molecules in the Wnt and Notch signaling pathways. The PCR reactions were performed in a total volume of 20 µl, including 10 µl 2X Power SYBR^®-Green PCR master mix (Applied Biosystems, Warrington, UK), 2 µl cDNA (5 ng/µl) and 1 µl primer mix (10 µM each). The PCR amplification and detection were carried out using the LightCycler 480 II (Roche Applied Science, Basel, Switzerland) as follows: an initial denaturation at 95°C for 10 min; 40 cycles of 95°C for 15 sec and 60°C for 1 min. The relative gene expression was calculated using the comparative CT method (9). The gene expression of the target gene normalized to an endogenous reference (GAPDH) and relative to the calibrator was given by the formula 2^−ΔΔCq (10).ΔCq was calculated by subtracting the average GAPDH Cq from the average Cq of the gene of interest. The ratio defines the level of relative expression of the target gene to that of GAPDH.

Cells were washed with ice-cold PBS and lysed in RIPA buffer [50 mmol/l Tris (pH 7.5), 150 mmol/l NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS)] containing phenylmethylsulfonyl fluoride (PMSF; 1 mmol/l) and protease inhibitors (2 g/ml; Protease inhibitor cocktail set III, Calbiochem, Billerica, MA, USA) on ice for 30 min. The lysates were clarified by centrifugation at 13,000 × g for 30 min at 4°C. The total protein concentration was estimated using a Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel, transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), blocked and probed with antibodies against β-catenin (1:1,000; sc-65480; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), c-myc (1:1,000; sc-40; Santa Cruz Biotechnology, Inc.), Jagged (1:1,000; sc-390177; Santa Cruz Biotechnology, Inc.), Notch1 (1:1,000; sc-373891; Santa Cruz Biotechnology, Inc.), Notch2 (1:1,000; sc-5545; Santa Cruz Biotechnology, Inc.), DLL4 (1:1,000; ab7280; Abcam, Cambridge, MA, USA) and Pra-1 (1:1,000; ab76413; Abcam). Upon washing, blots were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized by super enhanced chemiluminescence detection reagent (Applygen, Beijing, China).

Statistical analyses were conducted using Student's t-tests with the statistical software SPSS 15.0 (SPSS, Inc., Chicago, IL, USA). A corresponding two-sided P-value <0.05 was considered to indicate a statistically significant difference.

To investigate the effects of inhibition of the Wnt and Notch signaling pathway on the proliferation, migration and cell cycle of HCT-116 cells, we used the specific inhibitor rDDK-1 for the Wnt signaling pathway and LY374973 for the Notch signaling pathway. We observed that rDDK-1 and LY374973 reduced the proliferation of HCT-116 cells when used separately, and synergistically inhibited the proliferation (Fig. 1A). We also revealed that rDDK-1 and LY374973 reduced the migration ability and percentage of G2/M phase cells. rDDK-1 and LY374973 could further synergistically inhibit migration and the G2/M percentage (Fig. 1B and C).

Our results revealed that rDDK-1 reduced the mRNA and protein expression of Wnt signaling pathway molecules β-catenin, Pra-1 and c-myc, and also inhibited the expression of Notch signaling pathway genes Notch2, Jagged and DLL4 (Figs. 2 and 3). LY374973 reduced the mRNA and protein expression level of Jagged and DLL4, but had no effect on Wnt signaling pathway molecules Pra-1 and c-myc (Figs. 2 and 3). With regard to the mRNA expression level, LY374973 further increased the rDDK-1 inhibition of the mRNA expression of β-catenin, Jagged and DLL4 but not Pra-1, c-myc, Notch1 and Notch2 compared with rDDK-1 alone (Fig. 2). Inconsistently, with regard to the protein expression level, LY374973 together with rDDK-1 reduced the protein expression level of all detected Wnt and Notch pathway genes (Fig. 3).

In the immunofluorescence assay, rDDK-1 was able to inhibit the expression of β-catenin and Notch1, and LY374973 increased this effect on β-catenin. However, LY374973 could only slightly inhibit the expression of Notch1 but not β-catenin (Fig. 4).

We further investigated the effects of inhibition of Wnt and Notch signaling pathway in a transplantation tumor model. The results revealed that rDDK-1 and LY374973 decreased the tumor volume and weight when used separately, and could further synergistically inhibit the tumor volume and weight (Fig. 5B and C).

Our results indicated that LY374973 could inhibit the mRNA and protein expression of Notch2, Jagged and DLL4, but had no effect on Wnt signaling pathway genes in tumor tissues (Figs. 6 and 7). With regard to the protein expression level, rDDK-1 together with LY374973 could further synergistically inhibit the expression level of all detected Wnt and Notch pathway genes compared with rDDK-1 or LY374973 alone (Fig. 7).