Human colon cancer cell lines, HCT-116 and SW480, were purchased from the The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 g/ml streptomycin (HyClone™; Cytiva) at 37°C in a 5% CO₂ incubator. UA (Selleck Chemicals) was dissolved in dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) and stored at −20°C. LY294002, rapamycin and MG-132 were purchased from Selleck Chemicals.

HCT-116 and SW480 cells were seeded in 96-well plates at a density of 1×10⁴ cells/well and incubated overnight at 37°C. The cells were treated with various concentrations of UA (0, 5, 10, 15, 20, 25, 30 and 40 µmol/l) for 24 or 48 h. Subsequently, serum-free medium containing 10% Cell Counting Kit-8 (Dojindo Laboratories, Inc.) reagent was added and incubated at 37°C for 2 h. The absorbance was measured at 450 nm with a microplate reader.

For the migration analysis, HCT-116 and SW480 cells (5×10⁴ cells in RPMI-1640 serum-free medium) pretreated with different concentrations of UA were evenly seeded in the Transwell upper chamber. A total of 500 µl of medium containing 10% FBS was added to the lower chamber. After 36 h of incubation at 37°C, the migrating cells were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with crystal violet solution at room temperature for 10 min. The number of cells in three random fields was recorded using a light microscope (Leica Microsystems GmbH). Regarding the invasion analysis, 100 µl Matrigel (BD Biosciences) were placed on the bottom of the Transwell upper chamber in advance and incubated at 37°C for 6 h. The remaining steps were the same as those for the migration analysis.

Human tissue samples were obtained from a tissue microarray (cat. no. HCol-Ade060CS-01) provided by Shanghai Xinchao Biological Technology Co., Ltd., including 40 colon cancer tissue samples and 40 adjacent normal tissues. The present study was approved (approval no. PTEC-A-2021-29-1) by the Ethics Committee of Putuo Hospital affiliated to Shanghai University of Traditional Chinese Medicine (Shanghai, China).

The mouse lung tissue was fixed in 4% paraformaldehyde (Ruibaohe Biotechnology, Inc.) at room temperature for 24 h, and then was dehydrated in an automatic dehydrator. After being embedded in paraffin, the tissue was sliced into 5-µm-thick slices. Subsequently, immunohistochemical staining was performed according to the manufacturer's instructions. The slides were placed in 100% xylene for dewaxing and were then rehydrated in the graded ethanol series (70, 80, 90, 95, and 100%; Shanghai Lingfeng Chemical Reagent Co., Ltd.). For heat-mediated antigen retrieval, the slides were heated at 100°C for 15 min and 50°C for 6 min in citrate buffer (0.01 mmol/l, pH 6.0). Tissue sections were incubated with endogenous peroxygenase inhibitor (cat. no. PV-9001; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) for 10 min at room temperature and subsequently with 5% BSA (cat. no. ST025; Beyotime Institute of Biotechnology) at 37°C for 30 min. The sections were incubated with primary antibody against ARL4C (1:200; cat. no. ab122025, Abcam) at 4°C overnight and then incubated with a secondary, horseradish peroxidase (HRP)-labelled antibody at 37°C for 30 min. DAB staining solution was added (Fuzhou Maixin Biotechnology, Inc.) and the sections were incubated at room temperature for 3 min. Finally, the slides were counterstained with hematoxylin at room temperature for 10 sec, washed with running water, soaked in deionized water for 30 min and dried overnight at room temperature. The slides were observed and images were captured under a light microscope (BX43; Olympus Corporation). Analysis of the pathological changes was conducted using Image-Pro Plus software (v 6.0; Media Cybernetics, Inc.).

Cells were lysed with the use of RIPA buffer (Beyotime Institute of Biotechnology) containing a protease inhibitor on ice for 15 min. Cell lysates were subjected to centrifugation at 8,051 × g for 15 min at 4°C and the supernatant was drawn. The total protein was quantified using a BCA kit (Beyotime Institute of Biotechnology). A total of 20 µg protein was separated using 10% SDS-PAGE and transferred onto PVDF membranes (MilliporeSigma). The membranes were blocked with 5% non-fat milk at room temperature for 2 h and then incubated with primary antibodies against β-actin (1:5,000; cat. no. ab6276, Abcam), ARL4C (1:1,000; cat. no. ab122025, Abcam) and matrix metallopeptidase 2 (MMP2) (1:1,000; cat. no. ab92536, Abcam), p-AKT (1:1,000; cat. no. 4060S, Cell Signaling Technology, Inc.) and p-mTOR (1:1,000; cat. no. 5536, Cell Signaling Technology, Inc.) overnight at 4°C. The membranes were washed three times with TBST buffer containing 0.05% Tween-20 (cat. no. ST1726, Beyotime Institute of Biotechnology) and incubated with anti-rabbit IgG, HRP-conjugated secondary antibody (1:1,000; cat. no. 7074, Cell Signaling Technology, Inc.) for 2 h at room temperature. Finally, an enhanced chemiluminescence reagent (Bio-Rad Laboratories, Inc.) was added to visualize the immunoreactive bands. The band intensity was analysed with ImageJ software (version 1.51J8; National Institutes of Health) using β-actin as the loading control.

To generate the ARL4C-overexpressing cell lines (ARL4C-OE), the 2nd generational system was used in the lentivirus transfection experiment. Briefly, the ARL4C coding sequence was cloned into the GV358 (11.6 kb) lentiviral vector (Shanghai GeneChem Co., Ltd.) to form recombinant plasmids. Subsequently, the recombinant GV358, Helper 1.0 and Helper 2.0 plasmids were co-transfected into 293T cells at 37°C. The quantity of plasmids, Helper 1.0 (packaging) and Helper 2.0 (envelope) were 20, 15 and 10 µg, respectively. After 72 h, the culture supernatant was harvested, concentrated and purified (82,700 × g for 2 h at 4°C). The concentration of lentivirus was 1.5×10⁹ TU/ml. The HCT-116 and SW480 cells (5×10⁴) were then seeded on a 12-well plate wherein cells were transfected with lentivirus containing human ARL4C-Flag cDNA or lentiviral vectors (Ubi-MCS-SV40-puromycin; cat. no. con254; Shanghai GeneChem Co., Ltd.) for 24 h at 37°C (multiplicity of infection=10). Subsequently, the transfection solution was replaced with complete medium, and the stable strain selection experiment was carried out after culturing for 96 h with puromycin at 37°C. The concentration of puromycin for induction was 5 µg/ml determined in the preliminary experiment and that for maintenance was 2 µg/ml. As the lentiviral vector contains Flag elements instead of enhanced green fluorescent protein (EGFP), western blot analysis detecting Flag antibody and PCR detecting ARL4C gene expression were conducted for determining the success of the transfection. The time interval between transduction and subsequent experimentation was ~2 weeks. The stable strain expressing ARL4C, and the negative control were named ARL4C-OE and ARL4C-NC respectively.

According to the manufacturer's instructions, the secretion of MMP2 in cell supernatant was detected using an ELISA kit (cat. no. orb1494431, Biorbyt, Ltd.).

The male BALB/c-nude mice (5 weeks old; weighing 15–18 g; n=18) used in the present study were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd. The mice were randomly divided into three groups with 6 mice in each group as follows: Control, UA (10 mg/kg), UA (20 mg/kg). According to institutional guidelines, the mice were raised in a specific pathogen-free level barrier system [22°C; 50% humidity; 12:12 light/dark cycle; food and water were freely available (ad libitum)]. HCT-116 cells (5×10⁶) were injected into the lateral tail vein of male nude mice to establish the lung metastatic model of colon cancer. After 1 week, the mice were injected intraperitoneally with different concentrations of UA (10 and 20 mg/kg) every other day for 4 weeks. After 6 weeks, all of the mice were sacrificed by cervical dislocation and the lung tissues were fully excised. In addition, the number of lung metastases was measured. The lung samples were processed into paraffin-embedded sections and stained with haematoxylin and eosin (H&E) to visualize the presence of metastasis. All animal experimental protocols were reviewed and approved (approval no. TJ-HB-LAC-2020-92) by the Animal Experimental Ethics Committee of Shanghai Tongfeng Huiji Biological Medicine Technology Co., Ltd. (Shanghai, China).

H&E staining was performed using an H&E Staining Kit (cat. no. G1120, Beijing Solarbio Science & Technology Co., Ltd.). Briefly, the steps of tissue fixation and slicing were same as those described for immunohistochemistry. After dewaxing and rehydrating, the sections were stained with hematoxylin for 2 min and with differentiation solution for 10 sec at room temperature. Subsequently, eosin staining was carried out for 30 sec. Finally, the sections were treated with dehydration, transparency and sealing. Images were captured using a light microscope at the magnification of ×40 and ×100.

TCGA data analysis was performed using the publicly accessible UALCAN database (https://ualcan.path.uab.edu/index.html). The mRNA values of ARL4C were collected from healthy (normal) individuals and colon cancer patients, and the mRNA levels of ARL4C related to lymph node metastasis and tumor staging in patients with colon cancer were analyzed based on the TCGA dataset.

HCT-116 and SW480 cells with ARL4C-OE and ARL4C-NC were treated with LY294002 (15 µmol/l) or UA (20 µmol/l) for 8 h at 37°C. The cells were lysed with 200 µl Pierce™ IP Lysis Buffer (cat. no. 87787, Thermo Fisher Scientific, Inc.) on ice for 15 min. Subsequently, the samples were immunoprecipitated with anti-Flag magnetic beads (cat. no. B26101, Selleck Chemicals) overnight at 4°C to pull down ARL4C protein. The immunoprecipitated beads were then washed three times with PBST containing 0.5% Tween-20 for 5 min at room temperature. The beads with 50 µl SDS-PAGE sample loading buffer (cat. no. P0015A, Beyotime Institute of Biotechnology) were then boiled for 5 min at 100°C. The resulting complexes were subjected to immunoblotting with anti-ubiquitin antibody (1:100; cat. no. ab134953; Abcam).

SPSS 21.0 software (IBM Corp.) was used for statistical analysis. All data were obtained from three independently replicated experiments and are expressed as the mean ± standard deviation. Comparisons between two groups were performed using an unpaired Student's t-test or the Mann-Whitney U test, while comparisons among ≥3 groups were performed using either one- or two-way ANOVA followed by the Fisher's least significant difference (LSD) test. P<0.05 was considered to indicate a statistically significant difference.

First, the present study investigated the effect of UA on the proliferation of HCT-116 and SW480 cells. By increasing the UA concentration, the proliferation of HCT-116 and SW480 cells gradually decreased at 24 or 48 h (Fig. 1A and B) and the pharmacological effect of the drug at 48 h was improved compared with that at 24 h. The IC₅₀ value of UA for the HCT-116 cell line at 48 h was 20.47 µM while for the SW480 cell line it was 25.18 µM. Thus, three concentrations lower than this dose were selected in the subsequent experiment. Subsequently, Transwell assays were performed to verify the inhibitory effect of UA on the migration and invasion of colon cancer cells. The results revealed that UA inhibited the migration and invasion of HCT-116 and SW480 cells in a dose-dependent manner (Fig. 1C and D). These findings indicated that UA significantly inhibited the proliferation, migration and invasion of HCT-116 and SW480 cells.

The present study aimed to evaluate the effect of UA on colon cancer metastasis in vivo. To achieve this, a lung metastatic model of colon cancer was established by injecting HCT-116 cells into the tail vein of male BALB/c-nude mice. The organs of the lung were excised after 6 weeks and the number of lung metastatic tumours was measured. The findings indicated that UA could relieve the weight loss caused by colon cancer on day 42 (Fig. 2A). A significant decrease in the number of lung metastases was observed after administering low (10 mg/kg) and high (20 mg/kg) doses of UA, particularly at 20 mg/kg (Fig. 2B and C). Similarly, the presence of lung metastases from colon cancer was confirmed using H&E staining and a significant improvement in lung metastases was observed after UA treatment (Fig. 2D). Taken together, these results demonstrated that UA could inhibit the lung metastasis of colon cancer in vivo.

A previous study reported that high expression of ARL4C is associated with poor prognosis, depth of tumour invasion and peritoneal metastasis (10). Therefore, UA may inhibit metastasis by targeting ARL4C. First, the present study analyzed the expression of ARL4C in normal samples and samples from patients with colon cancer by employing the UALCAN database from The Cancer Genome Atlas (TCGA). In Fig. 3A, the expression of ARL4C was revealed to be increased in colon cancer tissues compared with normal tissues. Subsequently, the association between ARL4C expression and nodal metastatic status or individual cancer stages was analysed using data from TCGA database. The data indicated that the expression level of ARL4C was positively associated with the number of lymph node metastases (Fig. 3B) and cancer stage (Fig. 3C). To further determine the role of ARL4C in human colon cancer tissue, the protein expression of ARL4C in 40 paired human colon cancer specimens was detected through immunohistochemistry. The results revealed that ARL4C was highly expressed in colon cancer tissues compared with the adjacent tissues (Fig. 3D and E). The effects of UA on the expression of ARL4C in HCT-116 and SW480 cells were then evaluated. Western blot analyses demonstrated that the expression level of ARL4C was significantly decreased after UA treatment (Fig. 3F-H). In addition, the protein expression of MMP2 was also decreased (Fig. 3F-H). Similarly, the effect of UA on the expression of ARL4C in mouse lung metastases was examined. The immunohistochemical results (Fig. 3I and J) revealed that the expression of ARL4C was higher in lung tissues not treated with UA while it was significantly decreased after treatment with high-dose UA. These findings indicated that ARL4C was highly expressed in human colon cancer tissue and was associated with local lymph node metastasis and tumour stage. UA effectively inhibited colon cancer metastasis in vivo and in vitro and the mechanism may be associated with the inhibition of ARL4C expression.

To explore whether the UA-inhibited migration and invasion was dependent on the ARL4C protein, ARL4C-overexpressing HCT-116 and SW480 cell lines were constructed using lentivirus transfection. The transfection efficiency was verified using western blot analysis (Fig. 4A-C). Subsequently, Transwell assays revealed that overexpression of ARL4C reversed the inhibitory effect of UA on cell migration and invasion, compared with cells only treated with UA (Fig. 4D and E). To rule out any toxic effect of UA in determining the decrease in cell migration and invasion, the assays were also performed at the 90% viability (IC₁₀) concentration of UA. The results in Fig. S1 showed that low concentrations of UA inhibited cell migration and invasion, while overexpression of ARL4C reversed the inhibitory effect of UA. Western blot analysis revealed that the protein expression of ARL4C and MMP2 in the UA group was lower than that in the ARL4C overexpression group combined with the UA treatment (Fig. 4F and H). As MPP2 is a type of secreted protein (32), the secreted MPP2 was detected using ELISA with the cell supernatant. As expected, UA treatment decreased the secretion of MPP2, which was reversed by ARL4C overexpression (Fig. 4G and I). These findings indicated that the pharmacological effect of UA on colon cancer invasion and migration was mediated through the downregulation of ARL4C protein expression.

It was reported that the AKT/mTOR pathway regulated the expression of ARL4C in glioblastoma and lung cancer (33,34). Therefore, the present study further investigated whether the inhibitory effect of ARL4C by UA is associated with the inhibition of AKT signalling activation. First, ARL4C protein expression was decreased in the presence of the AKT inhibitor, LY294002 (Fig. 5A-C), or the mTOR inhibitor, rapamycin (Fig. 5D-F). In addition, UA significantly inhibited the phosphorylation of AKT and mTOR in HCT-116 and SW480 cells (Fig. 5G-J).

However, the mechanism by which AKT regulates ARL4C expression still requires investigation. As the stability of ARL4C is controlled by ubiquitination in hippocampal morphogenesis (35), MG-132, a proteasome inhibitor, was utilized. The results demonstrated that MG-132 reversed the decreased expression of the ARL4C protein caused by UA or LY294002 (Fig. 6A-F). Finally, using an immunoprecipitation experiment, it was observed that the ubiquitination level of ARL4C was increased after the administration of UA or LY294002 in HCT-116 and SW480 cells (Fig. 6G). Overall, these findings indicated that UA increased the ubiquitination of ARL4C by inhibiting the AKT pathway.