Cell cultrue, plasmid construction and transfection
Human oral squamous cell carcinoma SCC9 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in a mixture of Dulbecco’s Modified Eagle’s medium and Ham’s F12 medium (1:1) (Invitrogen, Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 400 ng/ml hydrocortisone (Sigma-Aldrich, St Louis, MO, USA) and penicillin (100 U/ml)/streptomycin (100 μg/ml) (Invitrogen). A full-length cDNA for human MT1-MMP (NM_004995.2) was amplified using RT-PCR and then ligated into the PCR2.1-TOPO vector. The constructed PCR gene product was cloned into the pEGFP-N1 vector. The final gene synthesis was verified via sequencing and amplified using DH5α competent cells. The Endo-free Plasmid Mini Kit II (OMEGA) was used for all plasmid preparations. For transfection experiments, cells were maintained in six-well plates (Corning, Lowell, MA, USA) and cultured to 80% confluence, after which the medium was changed to serum-free medium for overnight incubation. The cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. G418 (400 μg/ml; Invitrogen) was added to the media 48 h after transfection. The cells were allowed to grow in the presence of G418 for two weeks, and clones were picked for growth on plates to confluence. Thus, stably expressing empty vector--SCC9-pEGFP-N cells (SCC9-N) and a vector encoding human MT1-MMP--SCC9-pEGFP-M cells (SCC9-M) were obtained for our study.
For the experiment of addition of inhibitors, 2×105/ml SCC9-M cells were added to six-well plates (Corning). The cells were then treated with 5 nM tissue inhibitor of metalloproteinase (TIMP)-1 (Calbiochem, Darmstadt, Germany), 5 nM of TIMP2 (Calbiochem) and incubated for three days at 37°C.
Total RNA was extracted from cells using the TRIzol reagent (Invitrogen). For cDNA synthesis, mRNA was reverse-transcribed into cDNA using the 5×PrimeScript RT Master Mix (TaKaRa) at 37°C for 15 min and 85°C for 5 s according to the manufacturer’s protocol. Gene expression was quantified by real-time quantitative PCR using 2×SYBR Premix Ex Taq (TaKaRa) with a 7300 ABI Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) under the conditions of 95°C for 30 s, 95°C for 5 s, and 60°C for 31 s for 40 cycles. The relative gene expression was calculated using the 2(−ΔΔCT) method. Briefly, the resultant mRNA was normalized to its own GAPDH . The following primers were utilized for the real-time RT-PCR. GAPDH (5′-GAAGGTGAAGGTCGGAGTC-3′, 5′-GAGATGGTGATGGGATTTC -3′), MT1-MMP (5′-GGAACCCTGTAGCTTTGTGTCTGTC-3′, 5′-TGAGGGTCCTGCCTTCAAGTG-3′), E-cadherin (5′-TACACTGCCCAGGAGCCAGA-3′, 5′-TGGCACCAGTGTCCGGATTA-3′), β-catenin (5′-GCTGAAGGTGCTATCTGTCTGCTC-3′, 5′-TGAACAAGACGTTGACTTGGATCTG-3′), cytokeratin18 (5′-AGGAGTATGAGGCCCTGCTGAA-3′, 5′-TTGCATGGAGTTGCTGCTGTC-3′), vimentin (5′-TGAGTACCGGAGACAGGTGCAG-3′, 5′-TAGCAGCTTCAACGGCAAAGTTC-3′), fibronectin (5′ –TGCCTTGCACGATGATATGGA-3′, 5′-CTTGTGGGTGTGACCTGAGTGAA-3′), snail (5′-GACCACTATGCCGCGCTCTT-3′, 5′-TCGCTGTAGTTAGGCTTCCGATT-3′), slug (5′-ATGCATATTCGGACCCACACATTAC-3′, 5′-AGATTTGACCTGTCTGCAAATGCTC-3′), Twist (5′-GGAGTCCGCAGTCTTACGAG-3′, 5′-TCTGGAGGACCTGGTAGAGG-3′), ZEB1 (5′-GAAAGTGATCCAGCCAAATGGAA-3′, 5′-TTTGGGCGGTGTAGAATCAGAG-3′), ZEB2 (5′-AAATGCACAGAGTGTGGCAAGG-3′, 5′-CTGCTGATGTGCGAACTGTAGGA-3′) and CDH1 (5′-AGATGGTGTGATTACAGTCAAAAGG-3′, 5′-CAGGCGTAGACCAAGAAAT-3′).
Western blotting and shedding of the E-cadherin ectodomain
Cells were lysed using a RIPA lysis buffer (Beyotime). Total protein (30 μg) from each sample was subjected to the SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA), which were blocked for 2 h at room temperature with 5% nonfat milk in PBST. The following antibodies were used to detect bands on the protein blots: anti-β-actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MT1-MMP (1:500, Abcam, Cambridge, MA, USA), anti-E-cadherin (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-β-catenin (1:500, Santa Cruz Biotechnology), anti-cytokeratin18 (1:500, Bioworld Technology, MN, USA), anti-vimentin (1:500, Santa Cruz Biotechnology), anti-fibronectin (1:500, Santa Cruz Biotechnology), anti-Snail (1:500, Abcam), anti-Slug (1;1000, Cell Signaling Technology), anti-Twist (1:500, Abcam), anti-ZEB1 (1:300, Abcam) and anti-ZEB2 (1:500, Novus Biologicals, Littleton, USA). Immunoreactive material was visualized using the Immun-Star WesternC Kit (Bio-Rad, Hercules, CA, USA) products and bands were detected via exposure to film (Kodak, Japan). For detecting the expression of extracellular E-cadherin, the cells were cultured with serum-free medium for 24 h. Next, the conditioned medium was collected via centrifugation and concentrated 10-fold with a VirTis freeze dryer (SP Scientific, NY, USA). An immunoblot was performed as described above using an anti-E-cadherin ectodomain antibody (1:500, Santa Cruz Biotechnology). All western bolt analyses were performed at least three independent experiments.
Cells were cultured on glass coverslips, fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature, permeabilized with 1% Triton X-100 for 15 min and blocked with goat serum albumin for 30 min 37°C, followed by an overnight incubation at 4°C with antibodies specific for E-cadherin (1:100, Cell Signaling Technology) and vimentin (1:100, Santa Cruz Biotechnology), or cytokeratin 18 (1:100, Bioworld technology) and fibronectin (1:100, Santa Cruz Biotechnology). The appropriate secondary antibodies (diluted 1:50) were then used, and then nuclei were stained by 4, 6-diamidino-2-phenylindole (DAPI; 1:1000, Invitrogen) for 2 min. Immunofluorescence was visualized using a Zeiss LSM-710 laser-scanning confocal microscopy.
Adhesion, invasion and wound healing assays
The cells were plated in six-well plates (Corning) at a density of 4×105 per well and then trypsinized after 1 and 2 h, respectively. The attached cells were counted under an inverted microscope (Olympus), and the adherent rate of the three different cell populations was calculated. The cell invasion was assessed using Transwell filters with 6.5-mm diameters and 8-μM pore sizes (Costar, Lowell, MA, USA). The filters were precoated for 30 min at 37°C with 50 μL per square centimeter of growth surface with Matrigel Basement Membrane Matrix (BD Biosciences, MA, USA) diluted with serum-free medium (1:3) according to the manufacturer’s procedures. The cells (3×105) were resuspended with 100 μl serum-free medium inoculated in the upper chamber while 500 μl medium containing 10% FBS was placed in the lower chamber. The plates were placed at 37°C in 5% CO2 for 24 h. The chambers were fixed with 4% PFA and stained with 0.1% crystal violet (Beyotime) for 30 min. The non-migratory cells were removed, and the migratory cells were counted as those presenting on the lower surface of the upper chamber. Images of at least ten random fields per chamber were captured (×100 magnification). For the wound healing assay, the cells were allowed to grow to 90% confluence and then wounded by scratching with a pipette tip in the central area. Floating cells and debris were removed, and the medium was changed to serum-free. The cells were incubated for 48 h to allow cells to grow and close the wound. Photographs were taken at the same position of the wound at the indicated time points.
For flow cytometric cell-cycle analysis, the cells were synchronized with serum-free medium for 24 h, released and then cultured for three days. The cells were detached from the culture plate with trypsin, washed with PBS, and then resuspended in 75% alcohol. The prepared cells were stained with 100 mg/ml of propidium iodide (BD Pharmingen, San Jose, CA, USA) prior to analysis using flow cytometry with a BD FACS Calibur (BD Biosciences) and CellQuest Pro software (BD Biosciences). For surface marker analysis, the cells were collected and then labeled with human-fluorochrome-conjugated anti-CD24-PE (10 μl per test, Beckman Coulter, Los Angeles, CA, USA), anti-CD44-APC (20 μl per test, BD Pharmingen), anti-CD133-PE (10 μl per test, Miltenyi Biotech, Auburn, CA, USA). The corresponding mouse immunoglobulins conjugated to PE or APC (BD Pharmingen) were used as isotype controls in each experiment. For apoptosis analysis, the cells were dealed with mitomycin at concentration gradients of 16 and 128 mg/ml for 24 h. Then the prepared cells were collected and stained with PE Annexin V Apoptosis Detection Kit I (BD Pharmingen) for 15 min according to the manufacturer’s protocol. The rate of apoptosis cells was relative to each untreated group.
The cells were plated in 100-mm dishes (Corning) at a density of 1000 cells per dish and cultured at 37°C for two weeks. The dishes were fixed in 4% PFA, stained with crystal violet, and photographed. The colonies were visualized under an inverted microscope (Olympus). Aggregations of more than 50 cells were defined as a colony.
The survival rate of cells was analyzed using an MTT (Sigma) assay, which is a colorimetric assay for measuring the activity of enzymes that reduce MTT to formazan dyes, producing a purple color. The MTT assay is the preferred method used to assess the viability and proliferation of cells . The SCC9-N and SCC9-M cells were plated in 96-well plates (Corning) at an initial density of 2×103 cells per well, and then synchronized with serum-free medium for 24 h. For consecutive culturing at 0, 1, 3, 5, 7, 9 d, the cells were treated with 5 mg/ml MTT and incubated at 37°C for 4 h, and then treated by dimethylsulfoxide (Sigma). The absorbance of samples in triplicate wells was measured with an automatic enzyme-linked immunosorbent assay reader (ELx800, BioTek Instruments, Inc., USA) at a wavelength of 490 nm. Population doubling time (PDT) was calculated according to Patterson formulation. For drug resistant experiment, the SCC9-N and SCC9-M cells were plated in 96-well plates (Corning) at the same density of 5×104 cells. After serum-starvation, mitomycin at concentration gradients of 16 and 128 mg/ml was added separately to the culture medium and maintained for 24 h. The absorbance of samples in triplicate wells was measured as introduced above. The survival rate of the cells relative to each untreated group was calculated. The data were analyzed using three independent experiments.
The data were representative of three or more independent experiments as the mean ± s.d. Statistical significance was assessed using one-way analysis of variance and Student’s unpaired t test. P-value <0.05 was considered significant.