The colon cancer cell line SW1116 (ATCC no. CCL-233) was grown in RPMI-1640 medium (Gibco-BRL, Life Technologies Inc., Gaithersburg, MD, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Summit Biotechnology, Fort Collins, CO, USA). The colon cancer cell line HT-29 (ATCC no. HTB-38) was cultured in McCoy’s 5a medium (Gibco-BRL) supplemented with 10% FBS. Human liver sinusoidal endothelial cells (HLSECs) were purchased from Cell Systems (Kirkland, WA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL) supplemented with 10% FBS. Human umbilical vein endothelial cells (HUVECs) (ATCC no. PCS-100-013) were obtained from ATCC and maintained in F12K supplemented with endothelial cell growth supplement (ECGS) (0.05 mg/ml) (BD Biosciences, Bedford, MA, USA) and heparin (Sigma-Aldrich, Victoria, Australia) (0.1 mg/ml). Cells were maintained in a humidified incubator at 37°C with 5% CO₂, fed every 3 days with complete medium and subcultured when confluence was reached.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Synthesis of cDNA from 1 μg RNA was performed with a reverse transcription system kit (Promega, Madison, WI, USA). PCR reactions were carried out in a thermal cycler (model PTC-225; M J Research, Watertown, MA, USA) using HotStarTaq DNA polymerase (Qiagen GmbH, Hilden, Germany) as follows: 95°C for 15 min, 34 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec, and finally at 72°C for 5 min. The expression of GAPDH mRNA was taken as an internal loading control. The PCR products were separated on 1.5% agarose gels and stained with ethidium bromide. Table I provides the sequences of the primers used in the study.

Total RNA was extracted from HT29 cells, and the full-length cDNA of γ-synuclein was amplified using RT-PCR. The digested fragment of cDNA was inserted between the XhoI and BamHI sites of the pIRES2-EGFP plasmid to generate the pIRES2-γ-synuclein construct. The sequences (as shown in Table I) unique to the coding region of γ-synuclein were chemically synthesized and inserted between the BamHI and HindIII sites of the pGCsi-U6/neo/GFP plasmid to generate the pGCsi-γ-synuclein construct. The positive clones of pIRES2-γ-synuclein and pGCsi-γ-synuclein were confirmed by sequencing.

One day before transfection, SW1116 cells were plated in a 6-well plate at 1×10⁵ cells/well using RPMI-1640 medium without antibiotics. The cells were transfected with 3.0 μg/well of the recombinant plasmids and empty plasmids as control, respectively, using Lipofectamine (Invitrogen). Fresh growth medium was replaced after 4 h of transfection. Cells were passaged at a 1:10 dilution at 24 h after transfection and cultured in medium supplemented with G418 (Promega) at 1,000 μg/ml for 4 weeks. Stably transfected polyclones were selected from several cell islands with green fluorescent protein (GFP or EGFP) and were maintained in medium containing 400 μg/ml G418 for further study. Meanwhile, GFP or EGFP fluorescence was detected by flow cytometry to confirm that the purity of transfectants was >95%.

Cells were harvested and lysed with mammalian protein extraction reagent (Pierce, Rockford, IL, USA). The lysated proteins were quantified by bicinchoninic acid (BCA) protein assay kit (Pierce). Total protein (50 μg) was subjected to 15% SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked overnight with 5% skimmed milk in TBS buffer containing 0.05% Tween-20 at 4°C, and incubated with a 1:500 dilution of mouse anti-γ-synuclein monoclonal antibody (sc-65979; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and goat anti-mouse IgG-AP antibody (Santa Cruz Biotechnology) (1:5,000), respectively. Membranes incubated with ECL (Pierce) for 1 min were exposed to the film for 1–5 min. GAPDH served as a loading control.

Cells were plated in five copies onto 96-well plates at a density of 2×10³ cells/well in 100 μl RPMI-1640 medium containing 10% FBS at 37°C with 5% CO₂. The number of viable cells was determined daily with the WST-8 cytotoxicity assay using the Cell Counting Kit-8 (Dojindo, Japan). Briefly, 10 μl of the CCK-8 solution was added to each well of the microplate, and the absorbance at 490 nm was measured by a microplate reader (μQuant; Bio-Tek, Winooski, VT, USA) after a 4-h incubation.

Cells (1×10³) were trypsinized to a single-cell suspension and then plated in triplicate onto 6-well plates in complete culture medium containing 0.3% agar on top of 0.6% agar in the same medium. Cultures were maintained at 37°C with 5% CO₂ incubator for 14 days. The colonies were fixed with 70% ethanol and stained with 0.2% crystal violet. The colonies containing at least 50 cells were counted. Colony formation rates were calculated as the number of colonies relative to that of cells initially plated in a well (1×10³), and were expressed as mean ± SD.

Cells were plated in 35-mm dishes to form a monolayer one day before assay. After making a straight scratch with a pipette tip, cells were incubated in RPMI-1640 medium containing 10% FBS at 37°C with 5% CO₂ for 36 h. The motile cells were photographed under light microscopy to detect the speed of wound closure at various intervals. Assays were repeated three times.

Boyden chambers with 8-μm polycarbonate membranes in 24-well dishes (Nucleopore, Pleasanton, CA, USA) were coated with 4 mg/ml growth factor-reduced Matrigel (50 μg; Collaborative Biomedical, Becton Dickinson Labware, Bedford, MA, USA). Cells (1×10⁵) were resuspended in serum-free RPMI-1640 and added to the upper chamber in triplicate. Consecutively, RPMI-1640 with 10% FBS was added to the lower chamber. Chambers were incubated at 37°C with 5% CO₂ incubator. After a 48-h incubation, the chambers were fixed with 70% ethanol, and cells were stained with 0.2% crystal violet. Cells on the surface of the upper chamber were removed by swiping with cotton swabs. The number of invasive cells in the lower chamber was determined under light microscopy. The data are expressed as means ± SD of cell counts in 10 random fields of vision.

For the adhesion assay with HLSECs or HUVECs, a 96-well plate, coated by 2% gelatin, was seeded with 1×10⁴ endothelial cells/well and cultured until confluent. SW1116 cells (5×10⁴) were then transferred to each well in triplicate and left to adhere to the endothelial layer for 1 h at 37°C. The endothelial layer was washed with PBS to remove the non-adherent cells. Since γ-synuclein expression plasmids contained green fluorescence protein (GFP or EGFP), the attached cells were quantified by measuring the fluorescence intensity using a microplate reader (model Safire 2; Tecan, Männedorf, Switzerland).

Six-week-old male BALB/c nu/nu nude mice were purchased from the Experimental Animal Centre of Shanghai Institutes for Biological Sciences (SIBS, Shanghai, China) and housed in a specific pathogen-free environment. The pIRES2-EGFP, pIRES2-γ-synuclein, pGCsi-U6/neo/GFP, and pGCsi-γ-synuclein cells were harvested, washed and re-suspended in PBS, respectively. For the tumorigenicity assay, 1×10⁶ cells suspended in 100 μl PBS were injected subcutaneously into the right flank regions using a 27-gauge needle. Mice were checked every 3 days for tumor appearance, and tumor size was determined by measuring two diameters with a caliper. Tumor volume (V) was estimated by using the equation: V = 4/3π × L/2 × (W/2)², where L is the mid-axis length and W is the mid-axis width. For liver metastasis assay, mice were anesthetized intraperitoneally with 2.5% Avertin (Sigma). The spleen was exteriorized through a left lateral flank incision, and 1×10⁶ cells in 50 μl of PBS were injected into the distal tip of spleen parenchyma using a 27-gauge needle. The injection site on the spleen was pressed with a cotton stick wet in iodine-polividone solution in order to destroy extravasated cells and ensure hemostasis. The peritoneum and skin were closed in a single layer with surgical thread. Each group for the tumorigenicity assay included 10 mice and each group for the liver metastasis assay included 7 mice. All animals were sacrificed on day 30 after tumor cell implantation. Tumor specimens (in the right flank, liver and spleen) were collected, fixed in 10% neutral buffered formalin, embedded in paraffin and subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). The Ethics Committee of the Fujian Medical University approved the animal usage in the research, and all surgical procedures and care administered to the animals were in accordance with institutional guidelines.

Unstained 4-mm sections were cut from tissue blocks and stained with H&E and subjected to IHC analysis. Sections subjected to IHC were cleared with xylene and rehydrated with ethanol, and the slides were bathed in 0.01 mol/l sodium citrate and heated in a microwave oven for 12 min. The sections were incubated with anti-γ-synuclein antibody (sc-65979; Santa Cruz Biotechnology) and kept at 4°C overnight. Negative control slides were treated only with non-immunized mouse immunoglobulin fraction under equivalent conditions. For the secondary developing reagents, a labeled streptavidin-biotin kit (Dako, Carpinteria, CA, USA) was used. Slides were developed with diaminobenzaminidine and counterstained with hematoxylin.

The results are presented as means ± SD. Differences between groups were compared by two-way ANOVA, and were considered significant at P<0.05. Data analysis was carried out using SSPS 13.0 software.

After identification by sequence analysis, γ-synuclein gene eukaryotic expression plasmids and siRNA plasmids were successfully constructed. The recombinant plasmids and empty plasmids were transfected into SW1116 cells. After 4 weeks of selection with G418, stable transfected clones were successfully established, and the purity of the transfectants was >95% (Fig. 1A). To confirm the γ-synuclein expression in the stable transfectants, we examined the expression of γ-synuclein by RT-PCR and western blot analysis. The expression of γ-synuclein mRNA was upregulated in pooled pIRES2-γ-synuclein cells and downregulated in pooled pGCsi-γ-synuclein cells (Fig. 1B); these finding paralleled those of the western blot analysis (Fig. 1C).

In vitro cell proliferation assay showed that γ-synuclein knockdown significantly suppressed cell growth, and the number of pGCsi-γ-synuclein cells was significantly reduced by 48, 72, 96 and 120 h after plating, respectively, compared with the corresponding empty plasmid cells and parental (control) cells (Fig. 2A; P<0.05). The number of pIRES2-γ-synuclein cells was increased, but not significantly (Fig. 2A; P>0.05), compared with the control and pIRES2-EGFP cells, as well as the pGCsi-U6/neo/GFP and pGCsi-γ-synuclein cells, which in general presented the effects in a γ-synuclein expression quantity-dependent manner. Subsequent soft agar colony formation assay was conducted to evaluate the tumorigenicity of cells in vitro. Colony formation rates were 32.6±.1, 42.2±7.5, 30.8±5.9, 34.2±6.2 and 12.8±4.6% in the pIRES2-EGFP, pIRES2-γ-synuclein, control, pGCsi-U6/neo/GFP and pGCsi-γ-synuclein cells. The colony formation rate of the pGCsi-γ-synuclein cells was significantly reduced, compared with the empty vector and control cells (Fig. 2B; P<0.05). The size of colonies formed by the pGCsi-γ-synuclein cells was smaller than that of the two control cells. Whereas in parallel with the results of the proliferation assay, overexpression of γ-synuclein moderately enhanced colony formation of the SW1116 cells (Fig. 2B; P>0.05), which also exhibited a γ-synuclein expression quantity-dependent trend.

The results of the wound healing assay demonstrated that γ-synuclein is closely involved in cell motility. SW1116 cells overexpressing γ-synuclein moved more rapidly towards the gap, compared with the empty vector and control cells, and γ-synuclein knockdown decreased the healing rate of the SW1116 cell injury (Fig. 3A). A subsequent reconstituted basement membrane (Matrigel) invasion assay was carried out. As expected, γ-synuclein facilitated SW1116 cell invasion through Matrigel and a filter membrane, and the number of invasive pIRES2-γ-synuclein cells was increased by 93.1 and 81.5%, respectively; a higher number than that of the control and empty vector cells (Fig. 3B; P<0.05). However, γ-synuclein knockdown did not significantly attenuate invasion of SW1116 cells, compared with the two corresponding control cells (Fig. 3B; P>0.05). Generally speaking, γ-synuclein promoted SW1116 cell migration and invasion in a γ-synuclein expression quantity-dependent trend, which was observed in the following in vivo assays.

We carried out adhesion assays to investigate whether γ-synuclein influences the adhesion of SW1116 cells to endothelial cells. In the HLSEC adhesion assay, the average fluorescence intensity of EGFP was 1.07±0.25 in the pIRES2-EGFP cells and 1.73±0.32 in the pIRES2-γ-synuclein cells; γ-synuclein significantly facilitated SW1116 cell adhesion to HLSECs (Fig. 3C; P<0.05). There was no difference in fluorescence intensity of GFP between the pGCsi-U6/neo/GFP (0.94±0.26) and pGCsi-γ-synuclein (0.78±0.15) cells (Fig. 3C; P>0.05). Meanwhile, the results of the HUVEC adhesion assay revealed no effect of γ-synuclein on the adhesive ability of SW1116 cells to HUVECs (Fig. 3D; P>0.05), which suggests that SW1116 cells overexpressing γ-synuclein interact specifically with HLSECs.

We examined the in vivo tumorigenicity of SW1116 cells by injecting pIRES2-EGFP, pIRES2-γ-synuclein, pGCsi-U6/neo/GFP and pGCsi-γ-synuclein cells subcutaneously into the right flank regions of nude mice. The xenograft tumor growth rates in the different groups were compared. Xenograft tumors appeared in all mice of the three groups except for the pGCsi-γ-synuclein group on day 6, and pIRES2-γ-synuclein group mice exhibited a faster tumor growth rate from day 27 to the end of the expreriment, compared to the other three groups (Fig. 4A; P<0.05). However, only 6 of the 10 mice injected with pGCsi-γ-synuclein cells presented with tumors on day 6, and the other 4 mice remained with no tumor formation until day 9. All pGCsi-γ-synuclein cell tumors maintained a slow rate of growth, compared to the other three groups, within the time remaining until sacrifice (Fig. 4A; P<0.05). All animals were sacrificed on day 30 and tumor tissue specimens were collected as shown in Fig. 4B. The expression of γ-synuclein in tumor tissues was detected by IHC staining. The results showed that γ-synuclein protein was upregulated in tumors derived from pIRES2-γ-synuclein transfectants and downregulated in tumors derived from pGCsi-γ-synuclein transfectant, when compared with the two empty vector transfectants. (Fig. 4C).

We adopted an experimental model that entailed the injection of tumor cells into the spleen of nude mice, followed by assessment of their ability for tumorigenesis in the spleens and invasion via the portal vein into the liver and potential intraperitoneal dissemination. All animals were sacrificed by laparotomy on day 30, and tumor progression was observed macroscopically. All mice developed primary tumors in the spleen, and all mice injected with pIRES2-γ-synuclein cells developed metastases on the liver surface. Meanwhile, mice infected with pIRES2-γ-synuclein developed multiple intraperitoneal dissemination nodules in the mesentery, gastrointestinal serosa and peritoneum, whease less tumor nodules were found in the peritoneal cavity of mice in the other three groups (Table II). Liver and spleen specimens were collected for further evaluation. Liver replacement by metastases was too extensive in the liver of 71.4% (n=5 of 7) of pIRES2-γ-synuclein mice, which displayed enlarged livers covered with numerous tumor nodules throughout and with a whitish and irregular surface caused by extensive tumor growth, whereas the livers of the other groups were smaller in size, displaying a macroscopically normal appearance with a few surface nodules, and no liver surface metastases were found in 2 of 7 pIRES2-EGFP, 3 of 7 pGCsi-U6/neo/GFP and 3 of 7 pGCsi-γ-synuclein mice (Fig. 5A). The results paralleled the weight of the livers (Fig. 5B), which also exhibited a γ-synuclein expression quantity-dependent trend; the more γ-synuclein protein expressed, the more obvious the liver metastasis. As shown in Fig. 5C, the growth of these cells in the spleens was almost identical in the pIRES2-γ-synuclein and two empty vector groups, and was significantly decreased in the pGCsi-γ-synuclein group (P<0.05). Liver specimens were subsequently subjected to histologic examination by H&E staining (Fig. 5D). The results showed that liver metastases were found microscopically in all mice including micrometastases in mice without macroscopic nodules. Large regions of liver tissues were replaced by metastases with formation of neovessels, which was reported to be required for tumors to grow beyond a critical size, whereas livers containing pGCsi-γ-synuclein cells were mainly occupied by small-sized metastases with central necrosis.