Human choriocarcinoma cells, JEG-3 and BeWo (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% heat-inactivated FBS, 2 mM glutamine, penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C with 5% CO₂.

Genomic DNA was extracted and purified from cells using DNAzol (Invitrogen Life Technologies; Carlsbad, CA, USA) according to the manufacturer’s protocol. The extracted DNA was modified by treatment with sodium bisulfite using the EpiTect Bisulfite kit (Qiagen, Hilden, Germany). The methylation status of the DKK1 gene was assessed using both methylation specific PCR (MSP) and bisulfate sequencing. The modified DNA was used as a template both for MSP and unmethylated-specific PCR (USP). The primer sequences for the methylated DKK1 gene were 5′-TTAAGGGGTCGGAATGTTTC-3′ (sense) and 5′-CACGAAACCGTACCGATTC-3′ (antisense) and for the unmethylated allele were 5′-TTTTAAGGGGTTGGAATGTTTT-3′ (sense) and 5′-CCACAAAACCATACCAATTCAAC-3′ (antisense). The PCR products were separated in a 2% agarose gel with ethidium bromide and visualized under UV illumination. To examine the effect of demethylation, cells were incubated with medium containing 10 μM 5-aza-2′-deoxycytidine (5-aza-dC, Sigma-Aldrich, Carlsbad, CA, USA) for 36 h. Then DNA was isolated and MSP carried out as described above.

Cells were washed with PBS, fixed in 4% paraformaldehyde, permeabilized in 1% Triton X-100 for 15 min at room temperature and blocked with 5% bovine serum albumin in PBS for 1 h. DKK1 was detected using anti-DKK1 (Santa Cruz, sc-25516) antibody for 1 h at room temperature. Cells were washed with PBS and incubated with the appropriate fluorophore-conjugated secondary antibody, Alexa Fluor^® 594 Donkey anti-rabbit IgG (H+L), for 1 h at room temperature, washed with PBS, and mounted using Prolong anti-fade (Sigma).

Cells were seeded at 200 cells per well in 24-well tissue culture plates and incubated under normal culture conditions. After 24 h, cells were treated with various concentrations of rhDKK1 protein (e.g., 0, 10, 50, 100 and 200 ng/ml for each). Plates were incubated for 3 weeks in a humidified incubator at 37°C. Two weeks after seeding, colonies were stained with 0.05% crystal violet containing 50% methanol and counted. The colonies were counted in 4–5 random fields for each of the duplicate samples by using a microscope at ×100 magnification.

Cells were trypsinized, washed twice with cold PBS, and resuspended in 200 μl binding buffer. Annexin V-FITC was added to a final concentration of 0.5 μg/ml, according to the manufacturer’s instructions (KeyGen, Nanjing, China). After 20-min incubation at room temperature in the dark, 400 μl of binding buffer containing propidium iodide (PI, 50 μg/μl) was added, and samples were immediately analyzed on a FACSCalibur flow cytometer (Becton-Dickinson Medical Devices, Shanghai, China).

The changes in the mitochondrial potential were detected by using 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolyl-carbocyanine iodide/chloride (JC-1, KeyGen), a cationic dye that exhibits potential-dependent accumulation in mitochondria. These changes were indicated by fluorescence emission shift from red (590 nm) to green (525 nm) and analyzed on a flow cytometer (BD).

Transwell chamber (8-μM pore size polycarbonate membrane, Cell Biolabs, San Diego, CA, USA) Matrigel-invasion assays were performed to detect the invasion of cells. Cells (30,000) were placed in the upper chamber and allowed to invade for 24 h. Ten fields of cells were acquired at ×10 magnification and quantified. Relative invasion was expressed as a ratio of control cells.

All animal studies were conducted by the Animal Care and Use Committee of China Medical University. Fifty NOD SCID mice (Institute of Animal Center, Chinese Academy of Sciences, Shanghai, China), male, aged 4–6 weeks, were used in the in vivo experiment for observing antitumor efficacy of DKK1. Tumor xenografts were established by subcutaneous inoculation of 3×10⁷ JEG-3 cells into the back of mice. When tumors reached 5–7 mm in diameter, the mice were randomly divided into five groups (untreated, PBS, DKK1, DKK1+si-LGR6 #1 and DKK1+si-LGR6 #2). Mice were then intratumorally injected with 100 μl of PBS, DKK1, DKK1+si-LGR6 #1 or DKK1+si-LGR6 #2. Synthetic siRNA (100 nM) (si-LGR6) specifically targeting LGR6 (Invitrogen) were used to transfect cells using Oligofectamine (Invitrogen). The injections were repeated every two days. Tumor growth was monitored by periodic measurements with calipers. All animals were sacrificed five weeks post-treatment and tumors were dissected for pathological examination. Another 80 NOD SCID nude mice treated in the same way as described above and used to observe the survival time. The survival status of the mice was observed until the experiments were terminated for unacceptable tumor burden.

To measure the serum DKK1 concentration from mouse, a sandwich enzyme-linked immunosorbent assay (ELISA) was performed using DKK1 ELISA kit (R&D Systems) according to its protocol.

Cells and tissues were harvested, washed twice with PBS, lysed on ice for 30 min in 100 μl lysis buffer [120 mM NaCl, 40 mM Tris (pH 8.0), 0.1% NP-40] and then centrifuged at 13,000 g for 15 min. The supernatants were collected from the lysates and the protein concentration was determined. Aliquots of the lysates (50 μg of protein) were boiled for 5 min and electrophoresed using a 10% sodium dodecysulfate-polyacrylamide gel. The blots in the gels were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were then incubated with primary antibodies. Axin2 (2151, 1:500) was purchased from Cell Signaling Technology (Beverly, MA, USA). LRP6 (sc-25317, 1:500), glycogen synthase kinase (GSK-3β, sc-377213, 1:200), β-catenin (sc-7963, 1:200), Bax (sc-7480, 1:500), Bcl-xL (sc-8392, 1:200), Bcl-2 (sc-783, 1:500), phospho-Bcl-2 (Ser 87) (sc-16323, 1:200), β-tubulin (sc-5274, 1:500) and β-actin (sc-103656, 1:1,000) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The nitrocellulose membranes were further incubated with secondary immunoglobulin-G-horseradish peroxidase conjugates. Immunostaining was detected using an enhanced chemiluminescence (ECL) system (Amersham Biosciences, Westborough, MA, USA).

The interaction between DKK1 and LRP6 protein was analyzed by SYBYL-X 1.3 (Tripos International, St. Louis, MO, USA). The structure of DKK1 (PDB code: 3S8V) and LRP6 (PDB code: 3S8Z) protein were retrieved from the Protein Data Bank (http://www.rcsb.org).

FRET measurements were performed on the confocal laser scanning microscope (Bio-Rad) as previously described (15). Donor signal I1 (excitation = 488 nm, emission = 505–530 nm), FRET signal I2 (excitation = 488 nm, emission > 585 nm), and acceptor signal I3 (excitation = 543 nm, emission > 585 nm) were collected.

Statistical analyses were carried out using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). Data were expressed as mean ± standard deviation of three independent experiments performed in triplicate. Data comparisons in relation to control were performed by one-way ANOVA. The Kaplan-Meier method was used to estimate the probability of mouse survival. Differences were considered statistically significant at P-value <0.05.

A correlation was observed between DKK1 promoter methylation and decreased DKK1 mRNA levels in both JEG3 and BeWo cells (Fig. 1A). After the cells were treated with 5-aza-dC, we found that the DKK1 mRNA and protein expression was restored (Fig. 1A).

Colony formation assay was used to detect cell viability. As shown in Fig. 1B, the proliferation rate of both JEG3 and BeWo cells were inhibited by rhDKK1 protein in a dose-dependent manner (P<0.05). The IC₅₀ values for JEG3 and BeWo cells were 83.4 and 97.3 ng/ml, respectively. The results of Annexin V-FITC and PI double staining showed that the apoptotic ratio of the cells after DKK1 treatment was increased significantly in these cells (Fig. 1C, P<0.05). Mitochondrial damage was indicated by the fluorescence emission shift from red to green. JEG3 and BeWo cells showed mitochondrial damage following DKK1 treatment (Fig. 1D). In addition, we found that significantly less cells migrated to the lower membrane compared with untreated ones (Fig. 1E, P<0.05).

Furthermore, we carried out western blot analysis to identify the mechanism of apoptosis induced by DKK1. We found that LRP6 and β-catenin protein levels were lower in treated cells than untreated ones, while Axin2 and GSK-3 protein levels were increased in the treated ones (Fig. 2A). Compared with untreated control, we found a decrease in Bcl-2 and Bcl-xL protein, and an increase in p-Bcl-2 and Bax protein levels in DKK1-treated cells (Fig. 2B). Active caspase-3 and -9 were also increased in DKK1-treated cells (Fig. 2B).

We next determined the antitumor properties of DKK1 in established xenograft tumor models. As shown in Fig. 3B, the tumor volume of DKK1-treated JEG-3 mice was less than untreated or PBS treated JEG-3 mice (P<0.05). In addition, the survival rate of mice with tumors treated with DKK1 was significantly improved (Fig. 3C, P<0.05). The serum levels of DKK1 in the mice treated with DKK1 were higher than that in untreated or PBS treated ones (Fig. 3D, P<0.05). Interestingly, DKK1 showed no effects on LRP6 knockdown JEG-3 cells (Fig. 3, P<0.05). We knocked down LRP6 expression in JEG-3 cells by using synthetic siRNA (si-LGR6) and noticed that the effect of DKK1 on Wnt signaling pathway and mitochondrial apoptosis pathway was offset by si-LGR6 (Fig. 4). These results indicated that DKK1 may cause its antitumor effect by LGR6 in JEG-3 cells.

We found that DKK1 was ab;e to bind to LGR6 by using the modeling software SYBYL-X 1.3 (Fig. 5B). Furthermore, we carried out FRET to identify the interaction between DKK1 and LGR6. To permit spectroscopic analyses of receptor assembly and function in the living cell, we tagged DKK1 protein with RFP and labeled LGR6 with GFP. The results of FRET confirmed an almost linear increased donor (DKK1) and acceptor (LGR6) dimer (Fig. 5C). Furthermore, we carried out multiple sequence alignment and phylogenetic analysis. Additionally, we confirmed both the DKK1 and LGR6 as highly conserved proteins (Fig. 6), indicating that our results can be applied to other species.