All reagents were purchased from Sigma Aldrich (Sigma-Aldrich, St. Louis, USA) unless stated otherwise. Nutritional grade glucosamine sulfate powder (2(C6H13NO5).H2SO4) was purchased from Vita Natura (Bonn, Germany). A glucosamine sulfate solution was prepared to a final concentration of 100 μg/ml in cell culture medium (detailed below). The solution was sterile filtered, aliquoted, and stored at −20 °C until further use.
MG-63 osteosarcoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, USA). SaOS-2 cells were purchased from Deutsche Sammlung für Mikroorganismen (DMSZ, Braunschweig, Germany). Both cell lines were cultured as a monolayer in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1 % MEM-Vitamine, 1 % glutamine, 10 % fetal bovine serum (FBS), 1 U/mL penicillin/streptomycin and 2 % HEPES buffer (all obtained from Biochrom, Berlin, Germany) at 37 °C and 5 % CO2. Cells were passaged at 80–90 % confluency and culture medium was replaced every second day.
Glucosamine sulfate treatment
Treatments were performed in a 6 well plates. MG-63 (2 × 106) and SaOS-2 (4 × 106) were seeded in 3 ml of medium and cultured for 24 h. Different seeding densities were used to compensate for the higher proliferation rate of MG-63 cells. The following day, the medium was replaced with fresh medium containing 2 % FBS and a final concentration of 10, 50 or 100 μg/ml glucosamine sulfate (achieved with a 1:9, 1:1 and 1:0 dilution of the glucosamine stock solution) and cells were incubated for 48 h. Following the incubation period, cells were dissociated with trypsin, counted and collected by centrifugation. RNA was then isolated from the cell pellets as described below.
To detect potential toxicity associated with glucosamine sulfate treatment and exclude a downregulation of mRNA and protein expression due to cytotoxic cell death, a WST-1 cytotoxicity assay (Roche, Mannheim, Germany) was performed. Briefly, 2 × 105 MG-63 and 4 × 105 SaOS-2 were seeded in 200 μL medium in 48 well plates. Cells were then stimulated with different concentrations of glucosamine sulfate for 48 h (described above) prior to addition of 20 μl of WST reagent were added. Plates were incubated for 2 h before absorption was measured at 450 nm and a reference wavelength of 690 nm.
Quantitative gene expression analysis
RNA was isolated from cells using the RNeasy Tissue Kit® (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. In brief, MG-63 or SaOS-2 cells were resuspended in RLT buffer, transferred to a Qiashredder® and then lysed. RNA from lysates was immobilized on a silica matrix and eluted with distilled water. Harvested RNA was quantified by photometry.
Isolated RNA was transcribed to cDNA using QuantiTect® Reverse Transcriptions Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The relative gene expression of MMP-2, -3 and -9 was quantified by real-time polymerase chain reaction (PCR) using a thermo cycler with TaqMan® Array and specific primers (MMP-2: Hs01548727_m1; MMP-3: Hs01548727_m1; MMP-9: Hs01548727_m1; glyceraldehyde 3-phosphate dehydrogenase (GAPDH): NM_002046.3) (Applied Biosystems, Grand Island, USA). GAPDH was used as internal control. The thermal cycler conditions were: 40 cycles consisting of a 15 s denaturation phase at 95 °C and a combined 1 min annealing and extension phase at 60 °C.
Enzyme-linked immunosorbent assay (ELISA)
Protein analysis was performed from cells stimulated with 10 μg/ml glucosamine sulfate. After washing with PBS, a precipitation step with acetone and resuspension with cell extraction buffer (Invitrogen™, Frederick, USA) was performed.
Sandwich ELISA Kits for human MMP-3 and MMP-9 (Invitrogen™, Frederick, USA) were used for quantitative analysis according to the manufacturer’s instructions. In brief, 50 μl of diluted cell lysate was transferred to each microplate well previously coated with monoclonal antibody. After incubation with MMP-3 and MMP-9 specific detection antibodies as well as substrate solution the enzyme concentration was quantified spectrophotometrically in an ELISA Reader at 450 nm.
Quantitative PCR and ELISA data were compared using SPSS software (IBM, Armonk, USA) and the t-test for unpaired samples. The results are shown as mean ± standard deviation. A p-value of ≤0,05 was considered statistically significant.