Auraptene was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Lacinartin, an oxyisopentenylated coumarin, was produced using a previously reported procedure
. Briefly, commercially available propiolic acid and pyrogallol were condensed by concentrated H2SO4 catalysis into daphnetin via a Pechmann reaction. The daphnetin was then selectively alkylated on position 7 of the coumarin ring with 3,3-dimethylallyl bromide and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). It was then methylated on position 8 with methyl iodide and triethylamine to yield lacinartin. The final yield was 62%. Stock solutions of auraptene and lacinartin were prepared in dimethyl sulfoxide (10 mg/ml) and stored at 4°C in the dark.
Effect on Porphyromonas gingivalis growth
P. gingivalis ATCC 33277 was purchased from the American Type Culture Collection (Manassas, VA, USA). Bacteria were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 20 μM hemin and 0.0001% vitamin K (THB-HK) at 37°C under anaerobic conditions (80% N2/10% H2/10% CO2) for 24 h. The effect of auraptene and lacinartin on P. gingivalis growth was assessed in two different culture media using a microplate dilution assay. THB-HK contained excess iron, while Mycoplasma broth base (MBB; BBL Microbiology Systems) supplemented with 10 μM hemin (MMB-H) contained limited iron. Briefly, 24-h cultures of P. gingivalis in THB-HK, or MBB-H were diluted in fresh broth medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal volumes (100 μl) of P. gingivalis suspension and auraptene or lacinartin (0, 12.5, 25, 50, 100 μg/ml) in THB-HK, or MBB-H were mixed in the wells of 96-well plates (Sarstedt, Newton, NC, USA). Wells with no P. gingivalis, auraptene, or lacinartin were used as controls. After a 48-h incubation at 37°C under anaerobic conditions, bacterial growth was determined by measuring the OD660 using a microplate reader.
Effect on P. gingivalis biofilm formation/desorption
P. gingivalis was grown in THB-HK supplemented or not with auraptene or lacinartin as described above. After a 48-h incubation under anaerobic conditions, spent medium and free-floating bacteria were removed by aspiration using a 26 G needle, and the wells were washed three times with 50 mM phosphate-buffered saline (PBS) pH 7.0. The biofilms were stained with 100 μl of 0.02% crystal violet for 15 min. The wells were then washed three times with PBS to remove unbound dye and were dried for 2 h at 37°C. Ethanol (100 μl, 95% (v/v)) was added to the wells, and the plate was shaken for 10 min to release the dye from the biofilms. The absorbance at 550 nm (A550) was measured to quantify biofilm formation. We also investigated the capacity of auraptene and lacinartin to promote the desorption of a P. gingivalis biofilm. Briefly, a 48-h P. gingivalis biofilm was prepared as described above and was treated for 2 h with auraptene or lacinartin at final concentrations ranging from 0 to 100 μg/ml. The biofilms were stained with crystal violet as described above. All the above assays were performed in triplicate.
Effect on P. gingivalis adherence to oral epithelial cells
P. gingivalis cells were first labeled with fluorescein isothyocyanate (FITC). Briefly, a 10-ml aliquot of a 24-h culture (THB-HK) of P. gingivalis was centrifuged at 7000 x g for 10 min, and the pellet was suspended in 12 ml of 0.5 M NaHCO3 (pH 8) containing 0.03 mg/ml FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 x g for 5 min) and were suspended in the original volume of PBS. The immortalized human oral epithelial cell line GMSM-K was kindly provided by Dr. Valerie Murrah (University of North Carolina, Chapel Hill, NC, USA). The epithelial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM L-glutamine (HyClone Laboratories, Logan, UT, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma Aldrich Corp.), and 100 μg/ml of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere until they reached confluence. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37°C and were suspended in DMEM (without FBS). Aliquots of cell suspension (100 μl, 1.5 x 106 cells/ml) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After an overnight incubation to allow the formation a confluent monolayer, spent medium was aspirated, 100 μl of formaldehyde (3.7%) was added to the wells, and the plate was incubated at room temperature for 15 min. The formaldehyde was removed by aspiration and the wells were washed three times with PBS. Filtered 1% BSA (100 μl) was added to each well, and the plate was incubated for 30 min at 37°C in a 5% CO2 atmosphere. The wells were washed once with PBS, 100 μl of auraptene or lacinartin was added to each cell (final concentrations ranging from 0 to 100 μg/ml), and the plates were incubated for 30 min. The auraptene and lacinartin were not cytotoxic at these concentrations (data not shown). The FITC-labeled P. gingivalis cells were then added (100 μl) to the wells, and the plates were incubated in the dark for a further 90 min at 37°C under anaerobic conditions. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader. Control wells without auraptene or lacinartin were used to determine 100% adherence values. Wells containing only cells and auraptene or lacinartin were also prepared to determine the autofluorescence values of the two compounds. The assays were run in triplicate.
Anti-inflammatory properties in a macrophage model
U937 human monocytes (ATCC CRL-1593.2), a monoblastic leukemia cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultivated at 37°C in a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 medium (RPMI-1640; HyClone Laboratories) supplemented with 10% heat-inactivated FBS and 100 μg/ml of penicillin G/streptomycin. The monocytes (2.5 x 105 cells/ml) were then incubated in RPMI-FBS (1%) containing 10 ng/ml of phorbol myristic acid (PMA; Sigma Aldrich Corp.) for 48 h to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 h prior to use. The macrophages were incubated with auraptene or lacinartin (6.25 to 50 μg/ml) at 37°C in a 5% CO2 atmosphere for 2 h. They were then stimulated with 1 μg/ml of Aggregatibacter actinomycetemcomitans ATCC 29522 (serotype b) lipopolysaccharide (LPS) isolated using the procedure described by Darveau and Hancock
. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture medium supernatants were collected and were stored at –20°C until used. Cells incubated in culture medium with or without auraptene or lacinartin but not stimulated with LPS were used as controls. Commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-8, TNF-α, MMP-8, and MMP-9 concentrations in the cell-free culture supernatants according to the manufacturer’s protocols. The absorbance at 450 nm was read using a microplate reader with the wavelength correction set at 550 nm.
Inhibition of MMP-9 and P. gingivalis collagenase activity
Human recombinant MMP-9 (active form) purchased from Calbiochem (San Diego, CA, USA) was diluted in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35) to a concentration of 1 μg/ml and was incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). To determine the effect of auraptene and lacinartin on P. gingivalis collagenase activity, a 48-h THB-HK culture was centrifuged at 10 000 x g for 10 min. The supernatant was then incubated for 18 h in the absence or presence of auraptene or lacinartin (0-100 μg/ml) and fluorogenic substrate (100 μg/ml). Gelatin DQTM and collagen DQTM (Molecular Probes, Eugene, OR, USA) were used to quantify MMP-9 and P. gingivalis collagenase activities, respectively. The assay mixtures were incubated for 18 h at 37°C for MMP-9 and at room temperature for P. gingivalis collagenase. The fluorescence was measured after 4 h using a microplate reader with the excitation and emission wavelengths set at 495 nm and 525 nm, respectively. Fluorescent substrates alone or with auraptene and lacinartin were used as controls. Specific inhibitors of MMP-9 (0.025 μM GM6001) and P. gingivalis collagenase (1 μM leupeptin) were tested. The assays were run in triplicate.
Results are expressed as the means ± standard deviations of three independent experiments. The data were analyzed using the Student’s t-test. A p value ≤ 0.05 was considered statistically significant.