Argininosuccinate synthetase 1 suppression and arginine restriction inhibit cell migration in gastric cancer cell lines

Gastric cancer metastasis remains a major cause of cancer-related deaths. There is an urgent need to develop new therapeutic approaches targeting metastatic gastric cancer. Argininosuccinate synthetase 1 (ASS1) expression is increased in gastric cancer. We detected the protein expression of ASS1 in human gastric cancer cell lines (AGS, NCI-N87, and MKN45) and in murine gastric cancer cell lines (3I and 3IB2). We used vector-mediated short hairpin RNA (shRNA) expression to silence ASS1 expression in the MKN45 and 3IB2 cell lines, and analyzed the effects of this protein on cell migration and metastasis. We demonstrated that ASS1 silencing suppressed cell migration in the MKN45 and 3IB2 cell lines. ASS1 knockdown significantly reduced liver metastasis in mice after the intrasplenic implantation of 3IB2 cancer cell clones. To determine whether arginine restriction may represent a therapeutic approach to treat gastric cancer, the sensitivity of tumor cells to arginine depletion was determined in gastric cancer cells. Arginine depletion significantly inhibited cell migration in the gastric cancer cell line. The silencing of ASS1 expression in MKN45 and 3IB2 gastric cancer cells markedly decreased STAT3 protein expression. In conclusion, our results indicate that the ASS1 protein is required for cell migration in gastric cancer cell lines.


Western blot analysis
Total cell lysates were prepared and analyzed by SDS-PAGE as previously described 1 .
Tumor cells were washed twice with PBS and lysed with ice-cold RIPA buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% NP-40 and 1% SDS) containing one protease inhibitor tablet (cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, Roche; Mannheim, Germany). The tumor cells were harvested using a cell scraper and collected in an Eppendorf tube for 20 min at 4°C. The sample was centrifuged for 10 min at 13000 RPM at 4°C, and the supernatant was transferred to an Eppendorf tube. The total protein concentrations in the cell lysates were quantitatively assessed using the Bio-Rad Bradford assay (Mississauga, ON). Immunodetection was performed using an HRP-based SuperSignal Chemiluminescent Substrate (Pierce, Rockford, IL, USA). For quantification, the bands were measured using an AlphaImager 2200 system (Alpha Innotech, San Leandro, CA, USA) and normalized to the band density of β-actin. Argininosuccinate synthetase 1 (ASS1) expression was quantified and expressed as the ASS1 to β-actin ratio. These experiments were repeated using three independent batches of cell clones or cell lysates. The quantitative data are presented as values relative to those in the control cells.
After incubation for 1 h, the absorbance at 490 nm was recorded using an ELISA plate reader.

Cell cycle analysis by flow cytometry
Tumor cells were analyzed for changes in cell cycle status via propidium iodide analysis as previously described 1 . Tumor cells (1×10 6 cells/well) were cultured in 6-well plates for 48 h.
Analysis was performed using a FACScan instrument (BD Biosciences, San Jose, CA, USA).

Experimental metastatic model of gastric carcinoma
The metastatic abilities of MKN45 and 3IB2 cell clones in vivo were evaluated using a hepatic metastasis model, in which 1 × 10 6 tumor cells in 0.05 mL of PBS were intrasplenically injected as previously described 1 . Fourteen days after MKN45 cell clone injection, the NOD/SCID mice were sacrificed, and liver metastases were examined macroscopically and histologically. Eleven days after 3IB2 cell clone injection, the ICR mice were sacrificed, and liver metastases were examined macroscopically and histologically.
Formalin-fixed paraffin-embedded sections of the liver and spleen were used for hematoxylin and eosin (H&E) staining. Each animal experiment was performed at least twice.
AGS cells were transfected with an empty vector or ASS1 pCMV6 using the transfection reagent Lipofectamine 2000. The stable transfectants were selected using G418 (neomycin sulfate, 300 μg/mL). After limiting dilution analysis, cell clones were selected and maintained for further studies using G418. We established vector control (VC) and ASS1-overexpressing (ASS1-myc) clones. The cell lines were maintained in complete medium containing G418. To monitor the efficacy of ASS1 overexpression, the expression of this protein in the stable transfectants was analyzed by Western blotting.

Cell migration assay
Cell migration was evaluated by culturing 3IB2 cell clones in modified Boyden chambers (NeuroProbe, Inc., Gaithersburg, MD, USA) for 8 h as previously described 1 . To determine cell migration, 2.5 × 10 4 cells in serum-free medium were added to the upper chamber, and either 10% FCS-supplemented or serum-free medium was added to the bottom chamber as the chemoattractant. The stained cells were counted in 6 randomly selected fields using a microscope at 100x magnification. Each experiment was performed in triplicate and was repeated three times. The results were obtained from three independent experiments. LV: lentivirus; P: parental cells; VC: vector control; RNAi-1 and RNAi-2: ASS1-specific shRNAs 1 and 2. NS, not significant, *P < 0.05, **P < 0.001, ***P < 0.0001. Figure S2. Cell lysates were immunoblotted using anti-ASS1, anti-STAT3 and anti-β-actin antibodies. These cropped blots are shown in Fig. 1a, Fig. 1c, Fig. 4a, Fig. 4b and Fig. 6a.