The cell line HSC-3 (Japan Cancer Research Resources Bank, Tokyo, Japan), was established in 1985 from a primary oral squa-mous cell carcinoma removed from the tongue of a 64-year-old man. The HSC-3 cells were maintained in Dulbecco Modified Eagle Medium supplemented with 10% fetal bovine serum. Cells were routinely serum starved (and therefore deprived of serum HA) before adding HA.
ANTIBODIES AND REAGENTS
We used the following antibodies and reagents. Monoclonal rat anti—human CD44 antibody (clone, 020; isotype, IgG2b; CMB Technologies, Milford, Massachusetts) recognizes a common determinant of the CD44 class of glycoproteins. Polyclonal rabbit anti-phospho-MYPTl (Thr696) antibody (Millipore Corp, Chicago, Illinois) recognizes the phosphorylated myosin phosphatase regulatory subunit. Polyclonal rabbit anti-phospho-AKT1/2/3 antibody (Santa Cruz Biotechnology, Santa Cruz, California) recognizes the phosphorylated AKT; in addition, we used polyclonal goat anti-actin (I–19) (Santa Cruz Biotechnology). The Rho kinase inhibitor Y-27632 (No. 688000) and the PI-3 kinase inhibitor LY-294002 (No. 440202) were also used (Calbiochem, La Jolla, California). Healon HA polymers (roughly 500 000-Da polymers) (Healon Pharmacia, Erlangen, Germany) were prepared by gel filtration chromatography using a superfine gel column (Sephacryl S1000; GE Healthcare Biosciences, Piscataway, New Jersey). The purity of the high-molecular-mass HA polymers used in our experiments was further verified by anion-exchange high-performance liquid chromatography.
RHO KINASE ACTIVITY ASSAY
The HSC-3 cells were plated in 10-cm dishes at 0.5 × 106 cells per dish and were serum starved overnight before various treatments, including no treatment, HA treatment, blocking with Y-27632 (5 µM) followed by HA treatment (50 µg/mL), or blocking with rat anti-CD44 antibody followed by HA treatment (50 µg/mL). Ten minutes after HA treatment, cells were immediately prepared in NP-40 buffer (50mM Hepes [pH, 7.5], 150mM sodium chloride, 20mM magnesium chloride 2,1% NP-40, 1mM sodium orthovanadate, 1mM sodium fluoride, complete protease inhibitor cocktail [Roche Applied Science, Indianapolis, Indiana], 1mM phenylmethylsulfonyl fluoride, and 1 × Halt phosphatase inhibitor cocktail [Pierce Protein Research Products, Rockford, Illinois]) at 4°C and centrifuged to obtain the lysates. Equal amounts (verified by immunoblotting) of total lysates approximately 10 µg) or immunoprecipitation-purified Rho kinase obtained by preincubating lysates (approximately 100 µg) with a rabbit anti-Rho kinase antibody and agarose-conjugated antirabbit secondary antibody were assayed for Rho kinase activity using a kit (catalogue No. CY-1160; CycLex Co, Ltd, Nagano, Japan), following a protocol provided by the vendor. Briefly, samples were incubated with a kinase reaction buffer with 0.1mM adenosine triphosphate at 30°C for 45 minutes in plates precoated with a Rho kinase substrate corresponding to the C-terminal of the recombinant myosin-binding subunit of myosin phosphatase, which contains a threonine residue that can be phosphorylated, and the product was detected by a horseradish peroxidase—conjugated antibody AF20 recognizing Thr696 of the myosin-binding subunit. The horseradish peroxidase-mediated color reaction was then measured in a spectrophotometric plate reader at dual wavelengths of 450/540 nm. The absorbance data were analyzed. Control samples include a solvent control (no protein lysate) and an inhibitor control (10µM Y-27632 with protein lysate). Each assay was repeated at least 3 times.
PI-3 KINASE ACTIVITY ASSAY
The HSC-3 cells were plated in 10-cm dishes at 0.5 × 106 cells per dish and were serum starved overnight before various treatments, including no treatment, HA treatment, blocking with LY294002 (5µM) followed by HA treatment (50 µg/mL), or blocking with a rat anti-CD44 antibody followed by HA treatment (50 µg/mL). Ten minutes after HA treatment, cells were rinsed with ice-cold buffer A (20mM TRIS hydrochloride [pH, 7.4], 137mM sodium chloride, 1mM calcium chloride2, 1mM magnesium chloride2, and 0.1mM sodium orthovanadate) and immediately lysed in NP-40 buffer at 4°C to obtain the lysates. The PI-3 kinase activity was assayed using a competitive enzyme-linked immunosorbent assay kit (catalogue No. K-1000; Echelon Biosciences, Salt Lake City, Utah) because of its ability to produce PIP3 from PIP2. In brief, approximately 200 µg of lysates from each sample were incubated with an anti-PI-3 kinase and p85 antibody (catalogue No. 06-195; Millipore Corp), followed by an agarose-conjugated goat antirabbit antibody. After extensive washing with buffer A plus 1% NP-40 (3 times); with 0.1M TRIS hydrochloride (pH, 7.4), 5mM lithium chloride, and 0.1mM sodium orthovanadate (3 times); and with 10mM TRIS hydrochloride [pH, 7.4], 150mM sodium chloride, and 5mM EDTA containing 0.1mM sodium orthovanadate (2 times), the beads were incubated with PI-3 kinase reaction buffer (5mM Hepes [pH, 7], 2.5mM magnesium chloride2, and 25mM adenosine triphosphate) and 240pmol PIP2 for 2 hours at room temperature. The reaction was stopped by adding PIP3 detector protein, and the supernatant was transferred to the PIP3-coated detection plate and incubated for 1 hour at room temperature with agitation. The PIP3 detector protein binding to the plate was detected by a peroxidase-linked secondary detector, and the colorimetric readout, which was inversely proportional to the amount of PIP3 produced by PI-3 kinase, was determined at 450 nm in a plate reader. The PIP3 standards were used as references for the experimental samples, and control samples containing no enzyme and no lipid were also included. Each assay was repeated at least 3 times.
After growing in serum-free media for 24 hours, HSC-3 cells underwent one of the following procedures: incubation with or without HA, 50 µg/mL, for 10 minutes, or pretreatment with anti-CD44 antibody (1:1000), Y-27632 (5µM), orLY-294002 (5µM), followed by the addition of HA for 10 minutes. Subsequently, cells were solubilized in 50mM Hepes (pH, 7.5), 150mM sodium chloride, 20mM magnesium chloride, 1.0% NP-40, 0.2mM sodium orthovanadate, 0.2mM phenylmethylsulfonyl fluoride, 10-µg/mL leupeptin, and 5-µg/mL aprotinin. After brief centrifugation, the samples were directly solubilized in sodium dodecyl sulfate buffer, electrophoresed on 4% to 12% TRIS-glycine gels (Novex; Invitrogen, Carlsbad, California), and blotted onto nitrocellulose. After blocking nonspecific sites with 5% milk, the nitrocellulose filters were incubated with rabbit anti–phospho-AKT antibody or anti–phosphomyosin phosphatase antibody followed by incubation with horseradish peroxidase–conjugated goat antirabbit IgG. The blots were then developed by the enhanced chemiluminescence system (GE Healthcare Biosciences).
Logarithmically growing HSC-3 cells were cultured, washed, counted, and plated at 3000 cells per well in triplicate on 96-well plates and incubated in serum-free media overnight. The following day, the cells were treated with various concentrations of cisplatin without or with HA (50 µg/mL), anti-CD44 antibody, the Rho kinase inhibitor Y-27632 (5µM), or the PI-3 kinase inhibitor LY-294002 (5µM). Two days later, we performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays according to the manufacturer’s protocol (Promega, Madison, Wisconsin). The MTT assay measures cell survival based on mitochondrial conversion of MTT from a soluble tetrazolium salt into an insoluble colored formazan precipitate, which is dissolved in dimethyl sulfoxide and quantitated by spectrophotometry. The percentage of absorbance relative to controls was plotted as a linear function of drug concentration. Each assay was repeated at least 3 times. The 50% inhibitory concentrations (IC50) were identified as a concentration of drug required to achieve a 50% growth inhibition relative to the untreated control.
TUMOR CELL MIGRATION ASSAYS
A scratch wound was made by scraping the cell monolayer across the cover glass with a sterile pipette. We changed the culture medium immediately after scraping to prevent the medium from being conditioned with cell debris and factors released from detached cells. Scratched cultures were pretreated with anti-CD44 antibody, the Rho kinase inhibitor Y-27632 (5µM), or the PI-3 kinase inhibitor LY-294002 (5µM) for 1 hour before adding HA (50 µg/mL). The progress of cell migration was recorded with an inverted phase-contrast microscope at 0 hours and 24 hours from the time HA was added.
Gelatin zymography for detecting picograms of MMP-2 and MMP-9 and nanograms of other MMPs and proteases was performed. Sodium dodecyl sulfate polyacrylamide gel electropho-resis was performed in 7.5% or 10% polyacrylamide containing a 0.33-mg/mL concentration of gelatin. The gels were then rinsed twice in 0.25% surfactant (Triton X-100; Dow Chemical Company, Midland, Michigan) and incubated in the assay buffer (0.05M TRIS hydrochloride [pH, 7.5], 0.2M sodium chloride, 0.01M calcium chloride2, 1mM zinc chloride, 3mM phenylmethylsulfonyl fluoride, 0.02% sodium azide, and 0.005% polyoxy-ethylene lauryl ether [Brij 35; Caledon Laboratories Ltd, Georgetown, Ontario, Canada]) at 37°C for 18 hours. Gels were then stained with Coomassie blue 1995 R-250. Latent and active forms of gelatinases or other MMPs produce clear areas in the gel.
From the experimental results of the in vitro assays, a mean value and standard error of the mean were calculated for each experimental group. We used a t test with the resultant P value representing a 2-sided test of statistical significance. Significance was set at 95% (P = .05).