Repurposing Napabucasin as an Antimicrobial Agent against Oral Streptococcal Biofilms

Objectives Disruption of microbial biofilms is an effective way to control dental caries. Drug resistance and side effects of the existing antimicrobials necessitate the development of novel antibacterial agents. The current study was aimed at investigating the antibacterial activities of the repurposed natural compound napabucasin against oral streptococci. Methods The minimum inhibitory concentration, minimum bactericidal concentration, minimum biofilm inhibition concentration, and minimum biofilm reduction concentration of Streptococcus mutans, Streptococcus gordonii, and Streptococcus sanguinis were examined by a microdilution method. Cytotoxicity of napabucasin against human oral keratinocytes, human gingival epithelia, and macrophage RAW264.7 was evaluated by CCK8 assays. The dead/live bacterium and exopolysaccharide in the napabucasin-treated multispecies biofilms were evaluated by confocal laser scanning microscopy. Microbial composition within the napabucasin-treated biofilms was further visualized by fluorescent in situ hybridization and qPCR. And the cariogenicity of napabucasin-treated biofilms was evaluated by transverse microradiography. Results Napabucasin exhibited good antimicrobial activity against oral streptococcal planktonic cultures and biofilms but with lessened cytotoxicity as compared to chlorhexidine. Napabucasin reduced the cariogenic S. mutans and increased the proportion of the commensal S. gordonii in the multispecies biofilms. More importantly, napabucasin significantly reduced the demineralization capability of biofilms on tooth enamels. Conclusion Napabucasin shows lessened cytotoxicity and comparable antimicrobial effects to chlorhexidine. Repurposing napabucasin may represent a promising adjuvant for the management of dental caries.


Introduction
Dental caries is one of the most prevalent diseases incurring large expenditures worldwide [1,2]. It is a slowly progressive chronic disease initiated by oral biofilms and associated with multiple risk factors [3]. Cariogenic bacteria such as Streptococcus mutans dynamically compete with commensal bacteria including Streptococcus sanguinis and Streptococcus gordonii within the oral biofilm. Given the disequilibrium of the microbial ecology, the microbial metabolism of carbohydrates can lead to continuous decline of pH at the biofilm and tooth hard tissue interface, consequently causing demineralization of tooth hard tissue, and dental caries gradually occurs [3][4][5][6][7].
Mechanical plaque control is the mainstay for the control of oral biofilms and dental caries, but it heavily relies on individuals' compliance. To supplement mechanical plaque control, mouth rinses with antiplaque properties are well recommended [8]. Chlorhexidine (CHX) is one of the most common antimicrobial agents used as mouth rinse [9][10][11]. However, longtime usage of CHX could cause drug resistance and side effects such as taste confusions or tooth staining [11]. Therefore, alternative antimicrobial agents with comparable effectiveness but lessened side effects are needed for the better control of the oral biofilm [12].
Drug repurposing has garnered increasing attention as an alternative strategy to identify new antimicrobial agents for its efficiency in reducing time, cost, and risks associated with the development of novel antibiotics [13,14]. In an effort to repurpose existing drugs as antibacterial agents, we have recently screened from a library of bioactive molecules against Streptococcus mutans and identified the natural compound napabucasin (NAP) (Figure 1(a)), namely, 2-acetylfuro-1,4naphathoquinone. 2-Acetylfuro-1,4-naphathoquinone is one of the chemical constituents first isolated from Newbouldia laevis [15]. N. laevis is widely used in the African folk medicine and has been reported to reduce dental caries and other diseases [16]. Previous studies reported the antibacterial activity of 2-acetylfuro-1,4-naphathoquinone against Escherichia coli, Streptococcus faecalis, and Staphylococcus aureus [15,17]. A recent study also showed its antimycobacterial activity for the treatment of tuberculosis [18]. In addition, the NAP is in phase III clinical trials for the treatment of cancers (i.e., gastric cancer, pancreatic cancer, and colorectal cancer) [19][20][21][22][23]. However, there is no data to support its activity against oral pathogens.
The purpose of this study is to investigate the antimicrobial activity of NAP against oral streptococci.

Materials and Methods
2.1. Test Bacteria and Chemicals. Streptococcus mutans UA159, Streptococcus gordonii DL1, and Streptococcus sanguinis ATCC 10556 were kindly provided by the State Key Laboratory of Oral Diseases (Sichuan University, Chengdu, China). S. mutans, S. gordonii, and S. sanguinis were routinely grown at 37°C under aerobic condition (5% CO 2 ) in brain heart infusion broth (BHI; Difco, Sparks, MD). Inoculum for the experiment was adjusted to 1 × 10 8 CFU/mL for S. mutans, S. gordonii, and S. sanguinis based on the OD 600 nm versus CFU/mL graph of each bacterium and further 1 : 100 diluted in the growth culture. When needed, medium was supplemented with 1% sucrose (designated BHIS).
Napabucasin was purchased from Bide Pharmatech Ltd. and prepared in DMSO at a stocking concentration of 100 mg/mL.

Susceptibility Tests
2.2.1. Bacterial Susceptibility Test. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of NAP against S. mutans, S. gordonii, and S. sanguinis were determined by a microdilution method in BHI, as described previously [24,25]. The concentrations of NAP ranged from 0.97 to 1000 μg/mL (twofold dilutions). BHI broth containing equivalent DMSO (1% to 0.001%, v / v) was used as a solvent control and ran simultaneously to control for the possible growth inhibition caused by the added DMSO. CHX was used as a positive control, cell control (test bacteria and BHI broth) was used as a negative control, and BHI broth was used as a blank control.

Biofilm Susceptibility Test.
The minimum biofilm inhibition concentration (MBIC) was used to evaluate the effect of NAP on biofilm formation [26]. S. mutans, S. gordonii, or S. sanguinis (1 × 10 7 CFU/mL, 10 μL/well) were grown in BHIS with twofold serial dilution of NAP (200 μL/well) ranging from 0.12 to 125 μg/mL at 37°C for 24 h. A parallel study was also performed with BHIS as a negative control. Then, the supernatants from the wells were decanted, and the adherent biofilm was washed three times with PBS to remove the planktonic cells. Fixed with methanol for 15 min and airdried at room temperature, the biofilm was stained with 0.1% (wt/vol) crystal violet (Sigma) for 5 min, rinsed with deionized water until blank control wells were colorless, and added 200 μL of 95% ethanol to each crystal violet-stained well. Subsequently, the plate was rocked 30 min at room temperature, and the absorbance at 595 nm was recorded. The percentage of inhibition was calculated using the equation: ðA 595 of negative control − A 595 of the test groupÞ/A 595 of negative control × 100%. The MBIC was defined as the lowest agent concentration that showed 90% or more inhibition of biofilm formation.
The effect of NAP on the 1-day-developed biofilm was examined by the minimum biofilm reduction concentration (MBRC). S. mutans, S. gordonii, or S. sanguinis (1 × 10 7 CFU/mL, 200 μL/well) in BHIS was added to a 96-well polystyrene tissue culture plate. After anaerobic incubation at 37°C for 24 h, the supernatants were removed and washed with PBS three times without disrupting the integrity of biofilms. Fresh BHIS containing NAP ranging from 0.12 to 125 μg/mL were added to each well and incubated at 37°C for 24 h. The negative control was biofilms in BHIS without NAP. The biofilm was fixed, air-dried, stained, and quantified as described above. The MBRC was defined as the lowest agent concentration that showed 90% or more reduction of the biofilm.
2.3. In Vitro Cytotoxicity/Viability Assay. Cell viability was evaluated by using the Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) assay as described by Park et al. [27]. Test cells were human oral keratinocytes (HOK), human gingival epithelia (HGE), and macrophage RAW264.7 (RAW264.7). Cells were plated in 96-well plates at 10,000 cells/well in minimum essential medium with the Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic. The cells were grown in a humidified environment with 5% CO 2 at 37°C for 24 hours. Considering the short exposure duration of oral cells to mouth rinses, cells were treated with medium containing NAP (0.12 to 62.5 μg/mL) and positive control CHX for 5 min [27][28][29]. Then, the cells were washed with PBS twice and were added with the fresh medium (200 μL/well). Each well was added with 10 μL of CCK-8, and after incubation in the CO 2 incubator for 1 h to 1.5 h, absorbance was measured at the wavelength of 450 nm. The cell viability was calculated according to the following formula: ð%Þ = ðA 450 of test group − A 450 of blank controlÞ /ðA 450 of negative control − A 450 of blank controlÞ × 100%.

Multispecies Biofilm
Imaging. The multispecies biofilms were cultivated in accordance with a previous study [30].
The chemotaxis chamber μ-Slide, which has extremely low values of birefringence and autofluorescence, was used for bacterial culture and confocal microscopy [31]. Bacterial suspensions (1 × 10 5 CFU/mL for each strain) were mixed in 300 μL BHI containing 1% sucrose (BHIS) in the μ-Slide (8 wells, 80826, Ibidi) at 37°C for 24 h. Then, biofilms were exposed to PBS, 62.5 μg/mL NAP, and 0.2% CHX for 3 days (5 min, three times per day). This short-term repeated treatment was to simulate the daily exposure to the mouth rinses [32]. For dead/live imaging, biofilms were stained with fluorescent LIVE/DEAD BacLight Bacterial Viability stain (Molecular Probes, Invitrogen) containing SYTO 9 and propidium iodide according to the manufacturer's instructions. The labeled biofilms were imaged with a DMIRE2 confocal laser scanning microscope (Leica, Wetzlar, Germany) equipped with a 60x oil immersion objective lens.
For extracellular polysaccharide (EPS) staining, the bacterial cell and the EPS were stained with SYTO 9 (Molecular Probes) and Alexa Fluor 647-labeled dextran conjugate (Molecular Probes) as described previously [33]. The biofilms were captured with a Leica DMIRE2 confocal laser scanning microscope as in live/dead imaging.
For fluorescent in situ hybridization imaging, biofilms were fixed in 4% paraformaldehyde overnight and investigated by species-specific probes [34]. The multispecies biofilms were imaged with a confocal laser scanning microscope (FV1000, Olympus, Tokyo, Japan).
All three-dimensional reconstructions of the biofilms were performed with Imaris 7.0.0 (Bitplane, Zürich, Switzerland). The quantification of the dead/live and EPS/bacteria 3 BioMed Research International volume ratio was, respectively, performed with Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA) and COMSTAT (http://www.image-analysis.dk) [33].

DNA Isolation and Real-Time PCR.
Total DNA of biofilms were isolated and purified using a TIANamp Bacteria DNA kit (TIANGEN, Beijing, China). The purity and concentration of DNA were detected with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The extracts were stored at -20°C until use. TaqMan real-time polymerase chain reaction (Life Technologies, Carlsbad, CA, USA) was used to quantify the absolute number of S. mutans, S. gordonii, and S. sanguinis as described by the manufacturer (Takara, Dalian, China).
2.6. Transverse Microradiography. Human teeth free of white spots, cracks, and other defects that had been extracted for periodontal or impacted reasons were collected under a protocol approved by the Ethics Committee of West China Hospital of Stomatology, Sichuan University (WCHSIRB-D-2018-107). Crowns were separated from roots and cut into four sections measuring 5 mm × 5 mm × 2 mm by using a diamond-coated saw (Struers Minitom; Struers, Copenhagen, Denmark) under continuous water cooling. The enamel slabs were embedded in polymethylmethacrylate and painted with two layers of acid-resistant nail varnish, leaving a 4 mm × 4 mm window of the exposed enamel surface. Then, these surfaces were polished progressively with waterproof silicon carbide abrasive papers (800-4000 grit; Struers, Copenhagen, Denmark) and were ultrasonically cleaned in a deionized water for 5 min to remove the residual abrasives. All slabs were sterilized with ethylene oxide [35]. The 30 specimens were randomly divided into 3 groups: PBS, 62.5 μg/mL NAP, and 0.2% CHX. Enamel slabs obtained from the same tooth were evenly distributed to each test group.
Overnight cultures of S. mutans, S. gordonii, and S. sanguinis (1 × 10 7 CFU/mL) were simultaneously inoculated on enamel slabs in a 24-well cell culture plate with BHIS at 37°C. Slabs with biofilms were then exposed to PBS, 62.5 μg/mL NAP, and 0.2% CHX for 5 days (5 min, three times per day). The 5-day demineralization duration was in accordance with previous studies [36,37]. All specimens were then washed in PBS and refreshed with BHIS after every exposure. After 5 days of treatment, specimens were taken out and rinsed with PBS to remove the biofilms. Then, specimens were cut again and polished with a hand plane-parallel from both sides with waterproof silicon carbide abrasive papers (800-4000 grit; Struers, Copenhagen, Denmark) to thickness ranging around 150 nm [38]. X-ray films of experimental lesions were acquired with an X-ray generator (Softex, Japan) equipped with a microradiography camera and then were further examined using a Zeiss AXIO Imager A2 microscope (Carl Zeiss, Germany). Quantitative data was acquired by a calibrated analysis system TMR2006 (Inspektor Research Systems BV, Netherlands) [39]. Data are obtained as the mean of 10 separate samples.

Statistical Analysis.
All experiments were repeated at least three times independently. One-way analyses of vari-ance (ANOVAs) and the Student-Newman-Keuls test were used to compare differences. Differences were considered significant when P < 0:05. Statistical analyses were performed with the SPSS software, version 16.0 (SPSS Inc., Chicago, IL, USA).

NAP Shows Lessened Cytotoxicity against Human Oral
Cells Relative to Chlorhexidine. The cytotoxicity of NAP against human oral keratinocytes (HOK), human gingival epithelia (HGE), and macrophage RAW264.7 (RAW264.7) was evaluated by measuring the cell viability after drug exposure duration of 5 min. NAP showed lessened cytotoxicity against HOK, HGE, and RAW264.7 compared with CHX. More importantly, the IC50 of NAP on HOK, HGE, and RAW264.7 (IC50 > 62:5 μg/mL) were higher than its minimal biofilm reduction concentrations against an oral streptococcal biofilm (MBRC ranging from 3.91 μg/mL to 62.5 μg/mL), suggesting that NAP is safe for use as an antimicrobial agent at the exposure duration (Figures 1(b)-1(d)).

NAP Inhibits the Development of Multispecies Biofilms.
The antimicrobial effects of NAP were further evaluated with multispecies biofilms. Both NAP and CHX treatment disrupted the structural integrity of multispecies biofilms significantly (Figure 2(a)). NAP treatment further reduced  BioMed Research International the bacteria within the oral streptococcal biofilms as compared to CHX (Figure 2(b)). In addition, the biofilms treated with NAP showed an equivalent dead/live cell ratio as well as an EPS/bacteria ratio as compared to the CHX-treated ones (Figures 2(c)-2(e)). Fluorescent in situ hybridization imaging and qPCR were conducted to evaluate the effect of NAP on multispecies biofilm composition. As shown in Figures 3(a) and 3(b), both NAP and CHX treatments significantly reduced the total amount of streptococci. NAP inhibited S. mutans and S. sanguinis but increased the proportion of the commensal S. gordonii within the multispecies biofilms.

NAP Reduces the Demineralization Capability of
Streptococcal Biofilms on Enamel. We further evaluated the inhibitory effects of NAP on the cariogenicity of oral biofilms by quantifying biofilm-induced demineralization on human enamel slabs. As shown by the transverse microradiography data, the depth of a biofilm-induced lesion and the mineral loss of enamel were significantly reduced when treated with CHX and NAP compared to the negative control. NAP and CHX showed a comparable inhibitory effect on the biofilminduced demineralization (Figure 4).

Discussion
As a biofilm-associated chronic disease, effective biofilm control is critical for dental caries management. Antimicrobial agents, as supplements to insufficient mechanical removal, have been used to control oral biofilms for years. Clinical trials have shown that long-time usage of antimicrobial mouth rinses can significantly reduce S. mutans in saliva [40,41] and reduce the incidence of caries among people at high caries risk [42,43]. However, CHX had only a superficial bactericidal effect on dental plaque and exhibited noticeable cytotoxicity as dose increases [44,45]. Long-term repeated exposure to CHX could induce drug resistance in oral microbes such as S. gordonii, Enterococcus faecalis, Fusobacterium nucleatum, and Porphyromonas gingivalis [46,47]. In addition, CHX can cause tooth or tongue staining and

5
BioMed Research International taste confusions in patients [48]. The adversary effects of CHX necessitate the development of novel agents to control oral biofilms. Here, we demonstrated that the natural com-pound NAP possessed comparable antimicrobial activity with CHX against oral streptococcal biofilms but with relatively lower cytotoxicity, representing a promising novel  BioMed Research International agent in the control of dental caries. NAP is also an anticancer drug that is in phase III clinical trials for cancer treatment. Drug repurposing is an effective drug development strategy [49]. The repurposed use of the anticancer drug toremifene showed good antimicrobial activity against oral pathogens P. gingivalis and S. mutans by damaging the bacterial membrane [50]. The antiasthma drug zafirlukast also showed potent antimicrobial activity against P. gingivalis and S. mutans [51]. The repurposed use of NAP in the current study also showed potent antimicrobial activity against oral streptococci in either planktonic culture or biofilm. Oral biofilms are microbes embedded within an EPS matrix that functions as a "glue" to form a cohesive and adherent ecosystem. EPS is well recognized as a critical cariogenic factor of a streptococcal biofilm [5,33,[52][53][54]. Disruption of EPS can disperse the biofilm and increase the sensitivity of bacteria to antibiotics [54,55]. Previous studies verified that proanthocyanidins (PACs) in cranberry could reduce the amount of EPS, break down microarchitecture of the cariogenic biofilm, and reduce the incidence of smooth-surface caries in rats [56,57]. The current study found that NAP could significantly inhibit EPS production in the biofilm and thus disrupted the integrity of the streptococcal biofilm, further supporting its potential use as a plaque control measure that could supplement the management of dental caries.
The cariogenicity of the oral biofilm is closely associated with microbial interactions between cariogenic S. mutans and commensal streptococci such as S. sanguinis and S. gordonii. Disequilibrium within the plaque biofilms is the initiating event that mediates the transition from health to disease [58]. Changes in host diet such as excessive carbohydrate consumption promote accumulation of acid-producing organisms that trigger ecological alteration towards cariogenic microbiota [5]. The most typical acid-producing organism, S. mutans, can drive the dysbiosis of the oral microbial ecology and ultimately lead to the occurrence of dental caries [59,60]. Inhibiting S. mutans with increasing relative abundance of commensal streptococci is believed to be "ecologically safe" for the control of oral biofilms [6,24,53,61]. Our previous study showed that the combinatory use of arginine and NaF could inhibit S. mutans but enrich the commensal S. sanguinis in the multispecies biofilms, representing an ecological approach to the management of dental caries [6]. In this study, multispecies biofilms consisting of S. mutans, S. gordonii, and S. sanguinis were established and we found that NAP could suppress cariogenic S. mutans but increase the proportion of S. gordonii within the multispecies biofilms. More importantly, NAP treatment could significantly halt the biofilm-induced demineralization of tooth enamel, suggesting that NAP could be a good candidate for daily use mouth rinse.
Biocompatibility is a critical factor for daily use mouth rinse. Based on the clinical data reported in the previous phase I and II study, patients with advanced malignancies received napabucasin orally at a dose of 240 mg twice a day. Adverse events were generally mild and predominantly included diarrhea, abdominal pain, nausea, and fatigue [22,62]. The current study further evaluated the cytotoxicity of NAP on human oral cells such as HOK, HGE, and macrophage RAW264.7. Lower cytotoxicity of NAP against all these cells was observed as compared to that of CHX. These data further support repurposing NAP as an antimicrobial compound that can be tropically used for the control of oral biofilms.

Conclusion
In summary, this study for the first time demonstrated that NAP exhibited good antibacterial capability against oral streptococcal biofilms with lower cytotoxicity. NAP can disperse the multispecies biofilms and reduce the biofilm-induced demineralization of tooth hard tissue with decreasing relative abundance of S. mutans in the biofilms. Further in vivo studies are still needed to translate this promising repurposed natural compound to the management of dental caries.

Data Availability
All data used during the study are available in the article and can be solicited from the corresponding author.