The human gastric cancer cell line MKN74 was obtained from the Japanese Collection of Research Bioresources cell bank (JCRB Cell Bank, Osaka, Japan) and maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA). MKN74 is a moderately differentiated adenocarcinoma cell line possessing wild-type RhoA as well as other wild type mitogen-activated protein kinase (MAPK)-related molecules, such as EGFR, FGFR2, HER2, KRAS, NRAS, and BRAF. MKN74 cells were cultured in medium supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France) and 1% penicillin/streptomycin at 37°C, in a 5% CO2 atmosphere. Cell viability and counts were evaluated with a TC20™ Automated Cell Counter (Bio-Rad Laboratories, Hercules, CA) following trypan blue staining.
Antibodies and reagents
The following antibodies were used for western blotting: monoclonal mouse anti-phospho-Vav (sc-135788) from Santa Cruz Biotechnology (Dallas, TX, USA); monoclonal rabbit anti-Vav (#4657), monoclonal rabbit anti-RhoA (#2117), monoclonal rabbit anti-ROCK1 (#4035), monoclonal rabbit anti-cofilin (#5175), monoclonal rabbit anti-phospho-cofilin (#3313), polyclonal rabbit anti-SEK/MKK4 (#9152), monoclonal rabbit anti-phosphor-SEK (#4514), monoclonal rabbit anti-p38MAPK (#4511), and polyclonal rabbit anti-phosphor-p38MAPK (#9211) from Cell Signaling Technology (Danvers, MA, USA); and the secondary antibodies peroxidase conjugated anti-GAPDH (#015-25473) from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan); and goat anti mouse IgG/horseradish peroxidase (HRP; #P0447) and goat anti-rabbit IgG/HRP (#M7106) from DAKO (Santa Clara, CA, USA). ROCK inhibitor (Y-27632) was purchased from Selleck Chemicals (Houston, TX, USA).
Vector construction and retroviral transduction
The RhoA mutants R5W, G17E, and Y42C, with or without N-terminal FLAG, were created using the InFusion® HD Cloning Kit (Takara Bio, Shiga, Japan). The DNA sequences of all these mutants were confirmed by sequencing using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, USA). The wild-type RhoA gene and RhoA mutants were inserted into the retroviral vector pDon5 Neo DNA (Takara Bio). For retroviral transduction, these vectors and an empty vector (Mock), as a control, were transfected into the Phoenix packaging cell line using PEI MAX (Polysciences, Warrington, PA, USA). The virus-containing supernatants were collected after 24, 48, and 72 h, and the MKN74 cells were infected with the retrovirus particles on RetroNectin®-coated plates (Takara Bio). Stably transfected cells were obtained after G418 (Roche, Basel, Switzerland) selection for 10 days.
Reverse transcription-quantitative PCR
Total mRNA from MKN74 cells was extracted using NucleoSpin RNAplus (Takara Bio), and cDNA was synthesized using PrimeScript RT Master Mix (TaKaRa Bio) by incubation at 37°C for 15 min and 85°C for 5 sec. Quantitative PCR was conducted using SYBER Premix Ex Taq™ II (Takara Bio) and a Mastercycler realplex2 (Eppendorf, Hamburg, Germany). The thermocycling conditions were as follows: denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec, °C for 15 sec, and 68°C for 20 sec. Relative expression level was calculated using the 2-ΔΔCq method . All data were normalized to the expression level of GAPDH mRNA and were presented as the fold-increase in expression relative to that in the control cells. The following primers were used: RhoA forward, 5′-GTGTTTTTCCATCGACAGCC-3′ and reverse, 5′-GCCTTGTGTGCTCATCATTC-3′; Vav1 forward, 5′-GCCTATGCAGCGAGTTCTCAA-3′ and reverse, 5′-TTGTCTCGCTTGACCTCGTTC-3′; E-cadherin forward, 5′-AACAAGCCCGAATTCACCCA-3′ and reverse, 5′- GCGGCATTGTAGGTGTTCAC-3′; Snail forward, 5′-ATCGGAAGCCTAACTACAGCG-3′ and reverse, 5′- TCCCAGATGAGCATTGGCAG-3′; Slug forward, 5′- ACATTAGAACTCACACGGGGG-3′ and reverse, 5′- AGAATGGGTCTGCAGATGAGC-3′; ZEB1 forward, 5′- ACCACCCTTGAAAGTGATCCA-3′ and reverse, 5′- GCATTTTCTTTTTGGGCGGTG-3′; Vimentin forward, 5′- CACTCCCTCTGGTTGATACCC-3′ and reverse, 5′- CGTGATGCTGAGAAGTTTCGT-3′; GAPDH forward, 5′-CACCACCAACTGCTTAGCACC-3′ and reverse, 5′-CAGTCTTCTGGGTGGCAGTGATG-3′.
Western blot analysis
Cells were lysed in RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology) containing phenylmethylsulfonyl fluoride, protease inhibitor cocktail, and sodium orthovanadate on ice for 30 min. The isolated protein was quantified using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and was then subjected to SDS-PAGE with NuPAGE® Bis-Tris Precast Gel (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene fluoride membrane. Membranes were incubated with primary antibodies at 4°C overnight and then with HRP-conjugated secondary antibodies. Chemiluminescence was detected using the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK). Blot imaging and analysis were performed using the ChemiDoc XRS+ System (Bio-Rad Laboratories).
Migration assay and invasion assay
Cells were dissociated with trypsin (Gibco) to form a single cell suspension using a 40-μm Corning® Cell Strainer (Corning, Inc., Corning, NY, USA) and counted with a TC20™ Automated Cell Counter (Bio-Rad Laboratories). Migration assays were performed using Falcon® Cell Culture Inserts and Companion Plates in a 24-well plate with an 8.0-µm Transparent PET Membrane (Falcon, Corning, Inc.). Cells (5 × 105) were suspended in 500 μL of serum-free medium and seeded into the upper chamber with or without 10 µM Y-27632 (Selleck Chemicals). The lower chamber was filled with medium containing 20% fetal bovine serum. After incubation for 24 h at 37℃, in an atmosphere containing 5% CO2, migrated cells were fixed in 100% methanol for 10 min and stained with Giemsa Stain Solution (FUJIFILM Wako) for 30 min. The migrated cells were counted in five random fields per sample under a microscope at 200× magnification (Olympus Corporation, Tokyo, Japan). Invasion assays were performed using the same method as that used for the migration assays, with the following exceptions: 1 × 105 cells were seeded into the upper chamber of a Matrigel-coated transwell chamber (Corning® BioCoat™ Matrigel® Invasion Chambers 24 Well, Corning, Inc.), and invaded cells were counted after incubation for 48 h.
Knockdown of Vav1 using short hairpin RNA (shRNA) and small interfering RNA (siRNA)
The Vav1 knockdown vector was constructed using the pSINsi-hU6 shRNA expression retrovirus vector (Takara Bio) modified to be hygromycin B resistant. The target sequences for Vav1 shRNA and scramble shRNA (as a negative control) were as follows: Vav1 shRNA: 5′-CGTCGAGGTCAAGCACATT-3′, scramble shRNA: 5′-TAAGCAGCGACGTATCGTC-3′ (14,15). Stably transduced cells were selected using hygromycin B (FUJIFILM Wako) for 10 days. MISSION® siRNA(#NM_005428) and MISSION® siRNA Universal Negative Control #1 (SIC001) from Sigma-Aldrich (St. Louis, MO, USA) were transfected using Lipofectamine RNAi MAX Reagent (Thermo Fisher Scientific).
Lysates prepared using NP-40 lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% NP-40, Protease/Phosphatase Inhibitor Cocktail (#5879, Cell Signaling Technology) were incubated with Anti-DDDDK-tag pAb-Agarose (PM020-8, MBL, Nagoya, Japan) for 2 h at 4°C. After centrifugation, the beads were washed with NP-40 lysis buffer and then heated to 95°C for 5 min in sample buffer (FUJIFILM Wako). Following a brief centrifugation, the supernatants were separated by SDS-PAGE, and the protein detection was performed by immunoblotting.
The cell culture supernatant was collected after 48 h of culture in serum-free medium. The supernatant was concentrated using Vivaspin® Turbo 15 (Sartorius Stedim Biotech GmbH, Goettingen, Germany). FITC-labeled precast gelatin gels and buffers for gelatin zymography were used in combination with the FITC-labeled Gelatin-zymography Kit (Cosmo Bio, Tokyo, Japan), according to the manufacturer’s instructions. Briefly, the samples (2.5 μg) on gel-plates were electrophoresed at a constant current of 15 mA; the gels were then rinsed with wash buffer for 1 h. The enzyme reaction was performed by incubating the gels with an enzyme reaction buffer at 37°C for 24 h. The zymogram was imaged using the ChemiDoc XRS + system (Bio-Rad Laboratories) at an excitation wavelength of 535 mm.
Peritoneal xenograft model
Eight-week-old male BALB/c nu/nu mice were obtained from CLEA Japan (Tokyo, Japan). Cells (2 × 106) were resuspended in 200 µL of Hank's balanced salt solution (FUJIFILM Wako) and injected into the abdominal cavity of the mice. We used a breeding room in the Division of Animal Research, Shinshu University. The rearing environment was controlled at a room temperature of 22-25°C and humidity of 40-45%. Two mice were housed in one sufficiently large cage. At 4 weeks after transplantation, the mice were sacrificed by cervical dislocation under 3% isoflurane anesthesia, and the number of disseminated nodules in the abdominal cavity was counted. All animal experiments were performed according to the national standard of the care and use of laboratory animals, and the study was approved by the Institutional Animal Care and Use Committee with Shinshu University School of Medicine.
All statistical analyses were performed using the JMP software (SAS Institute, Cary, NC, USA). Statistical significance of differences was evaluated by Student’s t-test, Tukey's test, and Dunnett's test. Continuous variables were expressed as mean ± standard deviation. A P value <0.05 was considered to indicate a statistically significant result.