Repurposing Toremifene for Treatment of Oral Bacterial Infections

Bacterial strains and chemicals. P. gingivalis ATCC 33277 was routinely grown on 5% horse blood agar supplemented with hemin (5 μg/ml) and menadione (1 μg/ml) at 37°C under anaerobic conditions (90% N₂, 5% H₂, and 5% CO₂) using an Anoxomat AN2OP system (Mart Microbiology, Drachten, the Netherlands). S. mutans ATCC 25175 was routinely grown on solid Trypticase soy broth (TSB) agar (Becton Dickinson Benelux) containing 1.5% agar at 37°C. Liquid cultures of all the strains were grown in TSB.

Toremifene was purchased from TCI Europe N.V., and stock solutions of 20 mM were prepared in DMSO.

Antibacterial assays.The MIC of toremifene was evaluated in TSB as described previously (32). To determine the MBC, 10-μl aliquots were taken from the wells of the MIC assay that did not show bacterial growth and were plated onto agar plates. After incubation of the plates, the MBC was determined as the lowest concentration of toremifene for which no CFU were observed.

Antibiofilm assays.The MBIC values of toremifene were determined using crystal violet staining. P. gingivalis biofilms were grown anaerobically on the polystyrene pegs of Nunc Immuno-TSP (VWR International) lids, as described previously with minor modifications (33). Overnight cultures of P. gingivalis were diluted 1/10 in TSB. Next, 2-fold serial dilutions of toremifene in cell suspension (0 to 200 μM) were prepared at a volume of 150 μl in the polystyrene microtiter plates of the Nunc device. Subsequently, the plates were covered with a lid containing the pegs, and biofilms were allowed to grow on the pegs for 72 h at 37°C without shaking. After incubation, the pegs were washed once with phosphate-buffered saline (PBS), stained with 200 μl 0.1% (wt/vol) crystal violet in an isopropanol-methanol-PBS solution (1/1/18 [vol/vol]) for 1 h, washed with water to remove excess stain, and air dried (0.5 h). Next, the remaining crystal violet stain was removed from the pegs in 200 μl acetic acid (30%), and the intensity was measured by determining the optical density at 570 nm (OD₅₇₀), using a Synergy MX multimode reader (Biotek, Winooski, VT).

S. mutans biofilms were grown on the bottoms of the wells of polystyrene microtiter plates, as they failed to grow on pegs. To this end, overnight cultures were diluted 1/200 in brain heart infusion (BHI) medium (Becton Dickinson Benelux) supplemented with 3% sucrose, and 2-fold serial dilutions (150 μl) of toremifene in the cell suspensions (0 to 200 μM) were prepared in the microtiter plate. After 24 h of biofilm formation at 37°C, the biofilm formation was assessed by crystal violet staining as described above. The lowest concentration of toremifene required to inhibit biofilm formation was defined as the MBIC.

In addition, the biofilm-inhibitory effects of toremifene against S. mutans and P. gingivalis were tested under shaking conditions. Biofilms were grown and quantified as described above, with the difference that the biofilms were grown in a shaking incubator.

To determine the effect of toremifene on preformed biofilms, 72-h-old (P. gingivalis) or 24-h-old (S. mutans) biofilms were grown on polystyrene surfaces as described above. Subsequently, the biofilms were treated with 150 μl growth medium containing toremifene (0 to 200 μM) and incubated at 37°C for 24 h. Next, the biofilms were washed with PBS and quantified with cell titer blue (CTB; Promega Benelux) by adding 200 μl of CTB diluted 1/100 in PBS to each well. After 24 h of incubation in the dark at 37°C, fluorescence was measured (excitation wavelength [λ_ex] = 535 nm; excitation wavelength [λ_ex] = 590 nm) using the Synergy MX multimode reader (Biotek, Winooski, VT). The MBRC was defined as the lowest concentration of toremifene able to eradicate the preformed biofilm.

Inhibition of biofilm formation on titanium disks.To evaluate the biofilm-inhibitory activity of toremifene against P. gingivalis and S. mutans biofilms grown on titanium, round titanium disks (commercially pure titanium, grade 2; height, 2 mm; width, 0.5 cm) were used. Tests with P. gingivalis were performed under anaerobic conditions. First, bacterial suspensions were prepared by diluting overnight cultures of P. gingivalis 1/10 in TSB and those of S. mutans 1/200 in BHI medium supplemented with 3% sucrose. Next, the titanium disks were placed at the bottoms of the wells of a 96-well plate and challenged with 200 μl of a bacterial suspension containing 0 to 50 μM toremifene. After 72 h (P. gingivalis) or 24 h (S. mutans) of incubation at 37°C under static conditions, the disks were removed from the wells and subsequently washed with PBS to remove nonadherent bacteria and placed in centrifuge tubes containing 1 ml PBS. Adherent bacteria were removed from the disks by sonication (45,000 Hz in a water bath sonicator [VWR USC 300-T] for 10 min), followed by vortexing (1 min). Bacterial viability was quantified by serial dilution plating (CFU counts).

In addition, the BacLight LIVE/DEAD bacterial-viability staining kit (Molecular Probes, Invitrogen) was used to microscopically evaluate the viability of the biofilms formed on titanium disks. After incubation, the disks were washed with 1× PBS and transferred to a LIVE/DEAD staining solution containing SYTO 9 and PI (prepared according to the manufacturer's instructions). After 10 min of incubation at room temperature in the dark, the disks were washed again in 1× PBS and mounted on a coverslip for imaging. The stained biofilm cells were visualized under a Zeiss Axio imager Z1 fluorescence microscope equipped with an EC Plan-Neofluar 20× objective using the SYTO 9 (λ_ex = 483 nm; λ_em = 500 nm) and PI (λ_ex = 305 nm; λ_em = 617 nm) channels.

Time-kill assay.Exponential-phase cells of S. mutans were incubated with 1× and 4× the MIC of toremifene or chlorhexidine at 37°C under shaking conditions (see Table S1 in the supplemental material). At periodic intervals, aliquots taken from the samples were serially diluted in MgSO₄ and subsequently plated on TSB agar. After incubation for 2 days as 37°C, cell viability was determined by CFU counting.

Single-step resistance selection.The frequency at which mutants of P. gingivalis and S. mutans that are resistant to antibacterial agents emerge was determined as described previously (34). Briefly, 500 μl of an overnight culture of P. gingivalis or S. mutans was plated on agar plates containing antibacterial agents at 5× the MIC (see Table S1 in the supplemental material). In parallel, the overnight cultures were serially diluted and plated on nonselective agar. After incubation of the plates for 7 (P. gingivalis) or 2 (S. mutans) days, the MICs of the antibacterial agents for the surviving colonies on selective agar were determined to verify resistance. The spontaneous-mutation frequency was calculated by dividing the number of surviving colonies on selective plates by the total number of colonies on nonselective plates after incubation.

Macromolecular synthesis assay.The effects of toremifene on the macromolecular synthesis pathways in S. mutans were determined by monitoring the incorporation of radiolabeled precursors of macromolecules. Briefly, S. mutans exponential-phase cells (OD₅₉₅, 0.2 to 0.3) were incubated with radiolabeled precursors for DNA ([³H]thymidine [1 μCi]), RNA ([³H]uridine [2.5 μCi]), and proteins ([³H]leucine [2.5 μCi]). Next, the cells were treated with 4× the MIC of toremifene or control antibacterials (ciprofloxacin, rifampin, tetracycline, and triclosan) (see Table S1 in the supplemental material). After 10 min of incubation at 37°C, 100 μl was taken from the samples and resuspended in 3 ml ice-cold 10% trichloroacetic acid to stop the reactions and to release free radiolabeled precursors from the cells. Next, the samples were filtered through Whatman 25-mm GF/C glass microfiber filters and washed three times with 3 ml ice-cold water. Subsequently, the dried filters were transferred to scintillation vials containing 3.5 ml scintillation fluid. Each vial was counted in a liquid scintillation counter (Hidex 300 SL) for 2 min. The results are expressed as the percentage of incorporation compared to the untreated control.

Membrane permeabilization assays.The ability of toremifene to permeabilize the outer membrane of P. gingivalis was determined using the fluorescent dye NPN, as previously described (34) with some modifications. Briefly, exponential-phase cells were washed and resuspended to an OD₅₉₅ of 0.1 in buffer (5 mM HEPES, pH 7.4). Next, toremifene (0 to 25 μM) and NPN (10 μM) were added, and changes in fluorescence were recorded after incubation for 5 min using a Synergy MX multimode reader (Biotek, Winooski, VT; λ_ex = 350 nm, and λ_em = 420 nm). Triclosan at 1× the MIC was used as a positive control because of its strong outer-membrane-permeabilizing properties. Ciprofloxacin at 1× the MIC was used as a negative control (see Table S1 in the supplemental material).

The ability of toremifene to permeabilize the inner membrane of P. gingivalis and the membrane of S. mutans was determined using the fluorescent dye Sytox green (Invitrogen, USA), as previously described (34). Briefly, exponential-phase cells were washed and resuspended to an OD₅₉₅ of 0.5 in PBS. Next, the cells were incubated with toremifene (0 to 25 μM) and Sytox green (1 μM) at 37°C for 15 min. Thereafter, the increase in fluorescence was measured using a Synergy MX multimode reader (Biotek, Winooski, VT; λ_ex = 504 nm, and λ_em = 523 nm). Melittin (10 μg/ml for P. gingivalis; 2.5 μg/ml for S. mutans) was used as a positive control because of its strong inner-membrane-permeabilizing properties. Ciprofloxacin at 1× the MIC was used as a negative control (see Table S1 in the supplemental material).

The fluorescence values for each condition were divided by the respective OD₅₉₅ values to correct for the cell density of the culture. In addition, the ratio was corrected for background fluorescence by subtracting the fluorescence values of untreated cells.

Bodipy TR cadaverine displacement assay.To determine the ability of toremifene to bind with the lipid A part of LPS, the fluorescent probe BC (Thermo Fisher Scientific, USA) was used. In this study, exponential-phase cells of P. gingivalis were washed and resuspended to an OD₅₉₅ of 0.3 in PBS. Next, the cell suspensions were transferred to the wells of a black 96-well microtiter plate and mixed with 2.5 μM Bodipy TR cadaverine. After 2 h of incubation at 37°C, a 2-fold serial dilution of toremifene (0 to 50 μM) was added to the wells. Then, fluorescence was assessed for 30 min using a Synergy MX multimode reader (Biotek, Winooski, VT; λ_ex = 580 nm, and λ_em = 620 nm). Cells treated with 1× the MIC of ciprofloxacin were used as a negative control (see Table S1 in the supplemental material). Cells treated with 1× and 4× the MIC of chlorhexidine were used as a positive control (see Table S1). BC displacement from LPS was calculated using the following formula: [(F − F₀)/(F_max − F₀)] × 100, where F_max is the fluorescence intensity of BC without cells, F₀ is the intensity in the presence of cells alone, and F is the intensity of the mixture of cells and BC at various concentrations of toremifene, chlorhexidine, or ciprofloxacin.

Fluorescence microscopy.Exponential-phase cells of P. gingivalis and S. mutans were treated with 4× the MIC of toremifene or control antibacterials (ciprofloxacin, rifampin, tetracycline, and triclosan) (see Table S1 in the supplemental material). After 30 min of incubation at 37°C, the cells were centrifuged and stained with 10 μg/ml FM 4-64 (Molecular Probes) for 10 min at room temperature before being imaged. The cells were visualized using a Zeiss Axio imager Z1 fluorescence microscope equipped with an EC Plan-Neofluar 100× objective, using the FM 4-64 channel (λ_ex = 506 nm; λ_em = 751 nm).

Hemolysis assay.The hemolysis assay was performed as described previously, with some modifications (35). Briefly, fresh horse RBCs were rinsed three times with PBS by centrifugation for 10 min at 800 × g and diluted in PBS to achieve a final RBC concentration of 4%. The resulting suspension was incubated at 37°C for 10 min under shaking conditions. Subsequently, 200 μl of the suspensions was transferred to the wells of a microtiter plate, and the assay was initiated by addition of different concentrations of toremifene to the suspensions. Controls included RBC suspensions treated with PBS and with Triton X-100 (1%) to provide references for 0% and 100% hemolysis, respectively. The resulting suspensions were incubated for 60 min at 37°C. Following centrifugation for 10 min at 800 × g, hemolysis was assessed by measuring the absorbance of the supernatant at 540 nm. The percentage of hemolysis was calculated relative to 100% hemolysis with Triton X-100.

Cytotoxicity assay.A cytotoxicity test of toremifene was performed on a cell type relevant to the oral cavity homeostasis, with the aim of screening for concentrations that did not inhibit cell growth or induce cell death. HOC18 cells, an immortalized human oral gingival epithelial cell line, were used (36). Cells were plated in 96-well plates at 15,000 cells/well in minimum essential medium with the Eagle-alpha modification (αMEM; Sigma, Bornem, Belgium) with 0.292 g/liter l-glutamine (G7513; Sigma, Bornem, Belgium) supplemented with 10% fetal bovine serum (PAA Laboratories GmbH, Pasching, Austria) and 1% antibiotic-antimycotic (Gibco 15240; Life Technologies SAS, Saint Aubin, France). The cells were maintained overnight at 37°C in a humidified environment with 5% CO₂.

At day 1 postseeding, the cells were incubated with toremifene by adding the compound to the culture medium. A 2-fold serial dilution assay of toremifene was used, starting from 50 μM. Suspensions of the same cell line under the same conditions exposed to Triton X-100 (5%) or cultured without chemicals were used as controls. The proliferation of the HOC18 cells in the presence or absence of chemicals was investigated after 1 day of compound addition. Cell viability was monitored using the XTT assay according to the manufacturer's instructions (XTT cell proliferation kit II; Roche Diagnostics GmbH, Roche Applied Science, Penzberg, Germany). Briefly, this is a colorimetric assay, performed by adding XTT solution 4 h prior to the end of toremifene (or Triton X-100) exposure. In the assay, metabolically active cells cleave the yellow tetrazolium salt to form the orange formazan dye, whose absorbance is recorded at 450 nm and 650 nm (reference wavelength) using a spectrophotometer (Multiskan Ascent 96/384; Thermo Scientific, Waltham, MA, USA) associated with Ascent software version 2.6 (FIN-01621; Thermo Electron Corporation, Vantaa, Finland). The percentage of cytotoxicity was calculated relative to 100% cytotoxicity with Triton X-100.

Statistical analysis and reproducibility of results.Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by Dunnett's multiple-comparison test. P values of <0.001, <0.01, and <0.05 were considered to be statistically significant. All the experiments were repeated at least three times.