Synergistic Antifungal Effect of Fluconazole Combined with Licofelone against Resistant Candida ...

13 Pages·2017·3.32 MB·English

by  Shujuan Sun

fmicb-08-02101 November6,2017 Time:16:42 #1
ORIGINALRESEARCH
published:07November2017
doi:10.3389/fmicb.2017.02101
Synergistic Antifungal Effect of
Fluconazole Combined with
Licofelone against Resistant
Candida albicans
XinningLiu1,2,TaoLi3,DecaiWang1,YileiYang4,WenwenSun1,JianqiaoLiu5and
ShujuanSun4*
1DepartmentofClinicalPharmacy,TaishanMedicalUniversity,Taian,China,2DepartmentofMicrobialandBiochemical
Pharmacy,SchoolofMedicineandPharmacy,OceanUniversityofChina,Qingdao,China,3IntensiveCareUnit,Qianfoshan
HospitalAffiliatedtoShandongUniversity,Jinan,China,4DepartmentofPharmacy,QianfoshanHospitalAffiliatedto
ShandongUniversity,Jinan,China,5GeneralPractice,ShandongProvincialHospital,Jinan,China
Candida albicans (C. albicans) is one of the important opportunistic fungal pathogens
that is closely associated with disseminated or chronic infections. The objective
of this study is to evaluate the synergistic antifungal effect of licofelone, which is
dual microsomal prostaglandin E2 synthase/lipoxygenase (mPGES-1/LOX) inhibitor
in combination with fluconazole against C. albicans. Here our results showed that
Editedby:
JackWong, licofelone (16 µg/mL) can synergistically work with fluconazole (1 µg/mL) against
TheChineseUniversityofHongKong,
planktoniccellsoffluconazole-resistantC.albicans.Thetwo-drugcombinationinhibited
HongKong
theC.albicansbiofilmformationover12h,andreducedtheexpressionofextracellular
Reviewedby:
TaissaVila, phospholipase genes, biofilm-specific genes and RAS/cAMP/PKA pathway related
UniversityofTexasatSanAntonio, genes.Inaddition,thetwo-drugcombinationinhibitedthetransitionfromyeasttohyphal
UnitedStates
growthform,anddecreasedthesecretedaspartylproteinaseactivity,whilenotaffecting
ChamindaJayampathSeneviratne,
NationalUniversityofSingapore, the drug efflux pumps activity. Galleria mellonella model was also used to confirm
Singapore
the antifungal activity of the drug combination in vivo. This study first indicates that
*Correspondence:
the combination of fluconazole and licofelone has synergistic effect against resistant
ShujuanSun
[email protected] C.albicansandcouldbeapromisingtherapeuticstrategyfortheantifungaltreatment.
Specialtysection: Keywords:biofilms,cyclooxygenase(COX),drugresistancemechanisms,enzymeinhibitor,phospholipase
Thisarticlewassubmittedto
Antimicrobials,Resistance
andChemotherapy, INTRODUCTION
asectionofthejournal
FrontiersinMicrobiology Candida albicans is one of the important fungal pathogens that forms biofilm on the surface of
Received:04August2017 catheters and other medical devices. It is also believed to be mainly implicated in disseminated
Accepted:13October2017 or chronic fungal infections in ill and immunocompromised individuals (Carrillo-Munoz et al.,
Published:07November2017 2006). Currently, the antifungal drugs used in a clinical practice include azoles, amphotericin
Citation: B, and echinocandins. Amphotericin B is a potent antifungal agent against an array of yeast
Liu X,Li T,Wang D,Yang Y,Sun W, andfilamentousfungalpathogens,however,theapplicationislimitedbythesignificanttoxicities
Liu JandSun S(2017)Synergistic suchasrenaltoxicity,infusionreactions,andhepatotoxicity(ClementsandPeacock,1990).Both
AntifungalEffectofFluconazole
amphotericin B and echinocandin drugs also have minimal gastrointestinal absorption and are
CombinedwithLicofeloneagainst
onlyavailableasparenteralformulations,whilefluconazole,oneofthecommonlyusedfirst-line
Resistant Candidaalbicans.
drug of the azole family in clinical prevention and treatment of Candida infections, is readily
Front.Microbiol.8:2101.
doi:10.3389/fmicb.2017.02101 absorbedwithhighbioavailability(NettandAndes,2016).Thewide-spreaduseofantifungaldrugs
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Liuetal. SynergisticEffectofDrugCombination
has increased the incidence of candida-resistance, ultimately thatphosphatidylcholine-specificphospholipaseD1isimportant
leadingtorefractoryfungalinfections(Chenetal.,2012;Pfaller for yeast to hyphal transitions under certain conditions in
et al., 2015). In the United States, fluconazole-resistance has C. albicans (Hube et al., 2001). The gene expression levels
caused significant additional hospitalization costs and deaths includingRAS/cAMP/PKApathwayrelatedgenes(RAS1,CYR1,
(Sagatovaetal.,2015).Therefore,seekingnovelagentcombined TPK2), biofilm-specific related genes (EFG1, BCR1, ALS1,
withfluconazoleagainstresistantC.albicansisaurgentneed. ALS3, HWP1), and secreted aspartyl proteinase (SAP) genes
Licofelone, a dual mPGES-1/LOX inhibitor is the terminal (SAP1, SAP2, SAP3, SAP4) were assessed by RT-PCR. The
enzyme in the biosynthesis of Prostaglandin E2 (PGE ). morphogenesisofC.albicanswasalsoobservedbyfluorescence
2
Previous studies have noted that PGE , as the final product microscope. We also detected the effect of the two-drug
2
of arachidonic acid metabolic pathway can mediate several combinationonextracellularphospholipaseactivitiesbyeggyolk
biological phenomena (Harizi et al., 2008). It is also associated agar method and measured the drug efflux pumps activity by
with the ability of C. albicans to switch between yeast rhodamine6Gassay.
and hyphal growth forms. Recent studies indicated that
PGE synthesized from host and fungal can promote cell
2 MATERIALS AND METHODS
adhesion, germ tube formation and enhance fungal resistance
in C. albicans (Kalo-Klein and Witkin, 1990; Noverr et al.,
Strains and Media
2001).RAS/cAMP/PKApathwayisimportantformediatingthe
transition from yeast to hyphal growth form in C. albicans EightclinicalisolateswereobtainedfromShandongQianfoshan
and is known to regulate the expression of many biofilm- HospitalinChina.Sixresistantstrains,Candidaalbicans(CA10,
specific genes including EFG1, BCR1, ALS1, ALS3, and HWP1 CA16), Candida glabrata (CG2, CG3) and Candida parapsilosis
(Hogan and Sundstrom, 2009). Latest study shows that PGE (CP2, CP3); and two sensitive strains, Candida albicans (CA4,
2
increased cAMP level, activated PKA in C. albicans-infected CA8) were used in this study. Their susceptibilities were
macrophages, and therefore stimulated C. albicans germ tube determined according to Clinical and Laboratory Standards
formation (Yun et al., 2016). Based on this, we hypothesize Institute document M27-A3 (Wayn, 2008) with C. albicans
that the regulation of PGE influencing biofilm formation (ATCC 10231) as the quality control strain. The strains
may be associated with the2 RAS/cAMP/PKA pathway. In were maintained at −80◦C and subcultured at least twice
several studies, the synergistic effects of cyclooxygenase (COX) on the yeast–peptone–dextrose (YPD) solid medium at 35◦C.
inhibitors combined with antifungal drugs against C. albicans Licofelone and fluconazole were purchased from Shanghai
biofilmandplanktoniccellshavebeenobserved.Thesepotential Boylechem Co., Ltd., China and Dalian Meilun Biotech Co.,
antifungal activities are supposed to be associated with the Ltd., China, respectively. The stock solution was prepared
regulation of PGE production (Alem and Douglas, 2004; de accordingtothemanufacturer’sinstructions.Briefly,fluconazole
2
Quadros et al., 2011; Rusu et al., 2014; Liu et al., 2016). was dissolved in sterile deionized water at room temperature,
A dual mPGES-1/LOX inhibitor, licofelone can suppress PGE and licofelone was dissolved in dimethyl sulfoxide (DMSO)
2
formation (Koeberle et al., 2008) and has recently succeeded with 0.2% Tween 80. Each stock solution was prepared at
in reaching the required criteria in phase III clinical trials for a final concentration of 2560 µg/mL and stored at −20◦C
osteoarthritis (Payandemehr et al., 2015), but no researches until needed. RPMI1640 was used as a diluent medium
in regards with its antifungal activity and mechanism against for drugs and strains. The minimal inhibitory concentration
planktonicandbiofilmcellsofC.albicanshavebeencarriedout (MIC) was defined as the lowest concentration of the drug
sofar. that inhibited fungal growth by 80% (MIC80) compared
In this study, we first evaluated the in vitro efficacy of with that of the growth control and sessile MIC (sMIC)
licofelone alone and in combination with fluconazole against was read as the lowest concentration that produced a 50%
C. albicans by the checkerboard microdilution method, and reduction in growth compared with that of the drug-free
observed their antimicrobial effects on biofilm formation. In control. Before conducting the large number of experiments,
addition, the toxicity of C. albicans treatment was investigated the MIC and sMIC of which fluconazole and licofelone used
by Galleria mellonella model in vivo, survival analysis, and alone in the experiments was first evaluated as a preliminary
histology.Thefungalburdendeterminationwasusedtoconfirm experiment.
the antifungal effect of combined treatment during C. albicans
MIC Determination by Broth
infections. Related antifungal mechanisms were also explored
Microdilution Assays
in this study. Various virulence factors, other than biofilm
formation,havebeencontributedtoC.albicanspathogenicityin TheMICsoflicofeloneandfluconazoleagainstC.albicansstrains
thehosts,suchassecretedaspartylproteinaseandphospholipase weredeterminedbythebrothmicrodilutionmethodaccordingto
activity (Garcia-Vidal et al., 2013; Cui et al., 2015). Aspartyl theClinicalandLaboratoryStandardsInstitute(CLSI)standard
proteinase secreted from C. albicans is directly related to M27-A3 (Lockhart et al., 2011). For the checkerboard assays
virulence properties such as adhesion, tissue invasion and (Kuykendall and Lockhart, 2016), the final concentration of
immune evasion (Braga-Silva and Santos, 2011). C. albicans fluconazole and licofelone used were 0.125–64 µg/mL and
produces phospholipases, which can destroy cell membranes 2–128µg/mL, respectively, for resistant candida strains (CA10,
duringhostcellinvasion(Ghannoum,2000).Ithasbeenshown CA16, CG2, CG3, CP2, CP3); and 0.03125–16 µg/mL and
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2–128µg/mL, respectively, for sensitive candida strains (CA4, Efficacy of Fluconazole and Licofelone in
CA8). All drugs were diluted in 50 ml sterile RPMI 1640 G. mellonella Infected with C. albicans by
medium. Fluconazole and licofelone were added to the wells
Survival Assay
in the second to eleventh columns and A through G lines
of the 96-well plate, respectively. 100 µl of C. albicans cell Galleriamellonellainthefinalinstarslarvalstageofdevelopment
suspensions (0.5∼2.5 × 103 cells/mL) were then inoculated was used as previously described with some modifications
(Mylonakis et al., 2005; Amorim-Vaz et al., 2015). Larvae were
into each well of 96-well plates. Each wells were filled with
RPMI-1640 to a final volume of 200 µl except the control divided into four groups: growth control group (PBS only),
plate. Negative controls were performed in 200 µl RPMI1640 fluconazole (160 µg/mL) treated group, licofelone (80 µg/mL)
treated group, and fluconazole (160 µg/mL) with licofelone
in A12-H12 wells, and growth controls were performed in
100 µl RPMI1640 and 100 µl microorganisms in the H1 well. (80 µg/mL) treated group. Control group of larvae inoculated
The plate was incubated at 35◦C for 24 h. Both visual reading wasstudiedinparallelineveryinfectioninvestigation.Thelarvae
were stored in wood shavings in petri dishes at 35◦C in the
and optical density (OD) were performed to determine the
dark condition for 2 days before the experiments. Larvae with
MIC value (Bertout et al., 2011). OD was measured on the
dark spots or apparent melanization were excluded. Resistant
absorbance at 492 nm on a microplate reader. MIC endpoints
C. albicans isolates (CA10) were grown overnight on liquid
were defined as the lowest concentration of drugs causing 80%
Sabouraud medium, and the inocula were suspended in PBS
decreaseinviabilitycomparedtothedrug-freecontrol(MIC80).
buffer supplemented with 20 µg/mL of ampicillin to prevent
Allexperimentswereperformedintriplicate.Druginteractions
bacterialcontamination.10µlofeachlarvasuspension(5×106
wereinterpretedbythefractionalinhibitoryconcentrationindex
(FICI) model and the percentage of growth difference ((cid:49)E) cells/mL)wereinjectedofthroughthelastleftprolegusinga10-l
model.TheFICI≤0.5representssynergy,FICI0.5<FICI≤4 syringe (Gaoge, China). The infected larvae were incubated at
representsnointeraction,andFICI>4.0representsantagonism 35◦Cfor2h,andthentreatedwithdifferentgroupofantifungal
(Odds, 2003). The (cid:49)E model was defined by the following drugs.Thedeathoflarvaewasmonitoredbyvisualinspectionof
equation: (cid:49)E = E − E . The E value was theircolor(brown-darkbrown)each24hfor4days.Thesurvival
predicted measured
experiments were terminated at 4 days after infection. Each
calculated by the data obtained directly from experiments.
Statistically significant interactions of <100% were considered experiment groups contained 20 larvae (0.2–0.25 g in weight)
andtheexperimentwasrepeatedthreetimesusinglarvaefrom
weak, those from 100 to200% were considered moderate, and
thoseof>200%wereconsideredstrong,aspreviouslydescribed differentbatches.
(Katragkouetal.,2015).
Fungal Burden Determination
Fungal burden was determined by colony-forming unit (CFU)
sMIC Determination by Broth
countsfor4daysafterinfection(Krezdornetal.,2014).Larvae
Microdilution Assays weredividedintofourgroups:growthcontrolgroup(PBSonly),
Candidaalbicansbiofilmwasdevelopedusingaslightlymodified fluconazole (160 µg/mL) treated group, licofelone (80 µg/mL)
method described above (Gu et al., 2016; Sun et al., 2017). treated group, and fluconazole (160 µg/mL) with licofelone
The sMICs of fluconazole and licofelone against C. albicans (80µg/mL)treatedgroup.Eachlarvawasinfectedwith5×106
(CA10, CA16, CA4, CA8) biofilms were tested. The biofilms cells/larvaofresistantC.albicans(CA10)andincubatedat35◦C
were formed over three time intervals (8, 12, and 24 h) at for 2 h, then treated with different drug. At every 24 h, 3
35◦C by pipetting 100 ml of the standardized cell suspension larvae from each group were selected with no discrimination,
(103 cells/mL) into selected wells of 96-wellplate. After each suspended in 1 ml of PBS-ampicillin, and gently homogenized
adhesion phase (8, 12, and 24 h), the cell suspensions were forafewseconds.Themixwas10-folddilutedwithPBSbuffer
gentlywashedthreetimeswithsterilephosphate-bufferedsaline and 10 µl of these dilutions were inoculated on the sabouraud
(PBS) and the planktonic yeast were removed. Fluconazole agarplates.Theplateswereincubatedat35◦Cfor24handthe
and licofelone were then added to the corresponding wells number of CFUs was counted. The results were expressed as
of 96-well plate in serially double-diluted concentrations. The meanstandarddeviation(SD).Eachexperimentgroupcontained
final concentration of fluconazole and licofelone in wells was 20 larvae (0.2–0.25g in weight) and experiment was repeated
ranged from 1–512µg/mL and 2-128 µg/mL for each isolate, threetimesusinglarvaefromdifferentbatches.
respectively. The control wells were filled with 200 µl RPMI
Histological Study
1640 only. The plates were then incubated for another 24 h at
35◦C in an orbital shaker. A colorimetric reduction assay was ToevaluatethepresenceofresistantC.albicans(CA10)intissue
carriedoutwith2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H- of G. mellonella, three larvae from different groups (infected
tetrazolium-5-carboxanilide(XTT)accordingtotheprotocolof only; and infected and treated with licofelone, fluconazole and
Meloetal.Theabsorbancewasmeasuredwithamicrotiterplate licofelone-fluconazole)werecollectedatday3afterinfection.The
readerattheabsorbanceof492nm,andthedrugconcentration larvae were preserved in 70% ethanol and cut into 8-µm-thick
thatbroughtaboutareductioninabsorptionby50%contrasted sections using a cryostat. The samples were dried naturally at
to that in the control well was reported as the sMIC endpoint. room temperature for 2 days, stained with Periodic Acid Schiff
Eachtestwasperformedintriplicate. reagent (PAS), and dehydrated with increasing concentrations
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of ethanol and xylol (70, 80, 90, 96, and 100%). Finally, the suitableforuseasanendogenouscontrolgene(Caoetal.,2012;
samples were fixed in neutral balata and dried naturally at Buetal.,2016).PrimersusedinthisstudyareinSupplementary
roomtemperaturefor2days.Thestainedtissueswereobserved Table S1. The experiment was repeated on 3 independent
under an Olympus FSX100 fluorescence microscope with ×4.2 occasions.
objectives.Theexperimentwasrepeatedthreetimesusinglarvae
fromdifferentbatches. C. albicans Morphogenesis Analysis
Themorphogenesisanalysiswascarriedoutasdescribedbefore
Phospholipase Activity of C. albicans (Pierceetal.,2015).ThecellsofresistantC.albicans(CA10)were
Treated with Licofelone grownovernightat30◦CinRPMI1640andtreatedwithdifferent
drugs including growth control (drug-free) group, fluconazole
Phospholipase activity of resistant C. albicans (CA10) treated
(1µg/mL)group,licofelone(16µg/mL)group,andfluconazole
withdifferentdrugswasdetectedbyeggyolkagarplatemethod
(1 µg/mL) combined with licofelone (16 µg/mL) group in the
withsomemodifications(NiewerthandKorting,2001).Theegg
well plates. The treated suspensions were then examined by
yolk medium consisted of 15 g/L peptone, 3 g/L beef extract
fluorescence microscope to determine the percentage of hyphal
ointment, 5 g/L NaCl, 15 g/liter agar, 10 g/L glucose, and
10% sterile egg yolk emulsion. Each culture with 107 CFU/mL formation. The experiment was repeated on 3 independent
occasions.
were treated with different drug group including fluconazole
(1 µg/mL) group, licofelone (16 µg/mL or 32 µg/mL) group,
Rhodamine 6G Efflux Assays
fluconazole (1 µg/mL) combined with licofelone (16 µg/mL
or 32 µg/mL) group, and growth control (drug-free) group, Thedeterminationofthefunctionalactivityofdrugeffluxpumps
transferred into separate eppendorf tubes, and then incubated affected by licofelone was carried out (Sun et al., 2015). Briefly,
at 35◦C for 24 h. Then, 10 µl suspension of each tubes were resistantC.albicans(CA10)cellswereshakenovernightat35◦C
inoculatedontothemedium,theplateswereincubatedat35◦C in YPD broth and then washed three times with PBS. The
for 72 h. The diameter of the precipitation zone (a) and the cells were harvested by centrifugation and adjusted to 1 × 107
diameteroftheprecipitationzoneplusthediameterofthecolony cells/ml in PBS. In order to assess the efflux of Rh6G, we set 2
(b) were measured. Phospholipase zone (Pz) was designated groupsinthisexperiment:growthcontrolgroup(drug-free)and
as Pz = a/b, as described above. According to this definition, licofelone(16µg/mL)group.Rhodamine6G(finalconcentration
phospholipase index was scored and categorized as follows: of 10 µM) was added to each cell suspension. After incubating
negative(Pz=1);verylow(Pz=0.90to0.99);low(Pz=0.80 55 min at 35◦C, C. albicans cells were collected by centrifuging
to 0.89); high (Pz = 0.70 to 0.79), and very high (Pz ≤ 0.69), at 3,000 rpm for 5 min, washed three times with PBS, and
aspreviouslyreported(Priceetal.,1982).Eachexperimentwas resuspended with PBS containing 5% glucose. Five hundred
tested in triplicate and the phospholipase activity value was microlliterofeachsamplewerewithdrawnatevery10min.The
recordedastheaverageofthe3measurements. mean fluorescent intensity (MFI) was immediately determined
usingtheflowcytometerwithexcitationat488nmandemission
at 530 nm at each specific time intervals. The experiment was
Real-Time Quantitative PCR
repeatedon3independentoccasions.
mRNA expression of secreted aspartyl proteinase-related genes
(SAP1, SAP2, SAP3, and SAP4), RAS/cAMP/PKA pathway
Statistical Analysis
relatedgenes(RAS1,CYR1,TPK2),andbiofilmformationrelated
Data are presented as mean ± SEM. All experiments were
genes (EFG1, BCR1, HWP1, ALS1, ALS3) was measured by
performedintriplicate.Graphsproduction,datadistributionand
RT-PCR.ResistantC.albicans(CA10)cellsweregrowntomid-
log phase in RPMI-1640 medium at 35◦C after treatment with statistical analyses were performed using Graph Pad Prism 5.
fluconazole(1µg/mL)alonegroup,licofelone(16µg/mL)alone Afterensuringdataconformedtoanormaldistribution,before
group and fluconazole (1 µg/mL) combined with licofelone andafterdatatransformation,analysisofvariance(ANOVA)and
(16 µg/mL) group. Cultures without drugs were served as t-tests were used to investigate significant differences between
independent groups. The G. mellonella survival curves were
controls.TotalRNAwasextractedandisolatedfromcellsusing
analyzedbytheKaplan–Meiermethod,andfungalburdenswere
RNA pure yeast kit (DNase I) (CWBiotech, Beijing, China).
analyzedusingat-testtomeasurestatisticaldifferencesbetween
A diluted RNA was reverse transcribed to cDNA using first-
strandcDNAsynthesisSuperMixkit(CWBiotech)at42◦Cfor twoindependentgroups.P<0.05wasconsideredasstatistically
30minfollowing85◦Cfor5minaccordingtothemanufacturer’s significant.
instructions. RT-PCR reactions were performed using cDNA,
ultra SYBR mixture (with ROX) (CWBiotech), and primers
RESULTS
with an ABI ViiA 7 (Applied Biosystems) sequence detection
system (CWBiotech). An aliquot of 25 µl PCR mix was used
foreachgeneandthecyclingconditionswere95◦Cfor10min, MICs Determined by Broth Microdilution
followed by 40 cycles of 95◦C for 15 s and 60◦C for 1 min. Assays
ACT1 was found to be performed well under all experimental The susceptibilities of 8 Candida isolates (CA4, CA8, CA10,
conditions, therefore, in this study, ACT1 was chosen to be CA16, CG2, CG3, CP2, CP3) were assessed under planktonic
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FIGURE1|Druginteractionsoffluconazoleandlicofeloneareinterpretedbythe(cid:49)Emodel.Three-dimensionalplotsoffluconazolecombinedwithlicofeloneagainst
CandidaalbicanswerecreatedbyusingMATLABprogram.Theconcentrationsoffluconazoleandlicofelonearedepictedonthexaxisandyaxis,respectively,and
theE-valuesobtainedforeachcombinationisdepictedonthezaxistoconstructathree-dimensional(3D)graphic.Peaksabovethe0planerepresentsynergistic
combinations.Thecolor-codingbarontherightindicatesthattheclosertothetopofthebar,themoreeffectivethedrugcombination.(A)The(cid:49)EmodelofCandida
albicans10,(B)the(cid:49)EmodelofCandidaalbicans16.
states. For all of the strains, the MIC of licofelone used alone C. albicans (CA10, CA16) and sensitive C. albicans (CA4,
was 128 µg/mL, and the MICs of fluconazole alone were CA8), the sMIC of fluconazole and licofelone used alone was
rangedfrom1to512µg/ml(Figures1A,B).Susceptibilityassay 512µg/mLand128µg/mL,respectively.Thesusceptibilitytest
showed that the combination of licofelone and fluconazole has showed that licofelone (16 µg/mL) combined with fluconazole
strongsynergisticantifungaleffectsagainstresistantC.albicans: (1 µg/mL) had synergistic antifungal effects against C. albicans
the MIC of fluconazole alone against resistant C. albicans biofilms (FICI < 0.5) at both 8 and 12 h time point. However,
80
(CA10,CA16)were>512µg/mL,whereaswhencombinedwith after 12 h formation, the combined effect of fluconazole and
licofelone (16 µg/mL), the MICs of fluconazole were decreased licofelone on biofilm has gradually reduced. The combined
to 1 and 0.5, respectively. These effects were also illustrated by antifungal effect was barely observed on the biofilm formed
the FICI in vitro: the FICI for the resistant C. albicans (CA10, over 24 h with FICI > 1, suggesting that the two-drug
CA16)strainsis0.127and0.250,respectively(Table1),however, combination has synergistic antifungal effect at the early stage,
the FICI of sensitive C. albicans (CA4, CA8) and non-albicans but not the mature biofilm. The following experiments were
(CG2,CG3,CP2,CP3)strainswereall>0.5,indicatingthatthe performed in resistant C. albicans (CA10) isolate due to its
two-drug combination exhibited no antifungal activity against strong susceptibility to the combination of licofelone and
thesensitiveC.albicansandnon-albicans.Theseresultswerealso fluconazole.TheSMICsofcombinedantifungaldrugsareshown
interpretedby(cid:49)Emethod. inTable2.
sMIC Determined by Broth Microdilution Efficacy of in G. mellonella Infected with
Assays C. albicans
The susceptibilities of 4 C. albicans (CA4, CA8, CA10, CA16) Invivoeffectoffluconazole(160µg/mL),licofelone(80µg/mL),
isolates were assessed under biofilm states. For the resistant and their combination on infected G. mellonella treated was
TABLE1|ThecombinedantifungaleffectsoffluconazolecombinedwithlicofeloneagainstCandidaalbicans.
Drug Strains MICs(µg/mL) LAtheory BItheory
MICA CA MICB CB FICI IN (cid:54)SYN
Fluconazole+Licofelone CA10(R) >512 1 128 16 0.127 SYN 2704%
CA16(R) >512 0.5 128 32 0.250 SYN 1112%
CA4(S) 1 0.5 128 64 1.000 NI <100%
CA8(S) 2 1 128 64 1.000 NI <100%
CG2(R) 128 32 128 64 0.750 NI <100%
CG3(R) 64 16 128 64 0.750 NI <100%
CP2(R) 128 2 128 64 0.516 NI <100%
CP3(R) 128 4 128 64 0.531 NI <100%
EightisolateswithdifferentsusceptibilitiesinvolvedthreeCandidaspp.CA,Candidaalbicans;CG,Candidaglabrata;CP,Candidaparapsilosis.MICsdenotetheMIC80
ofeachdrugaloneorincombinationagainsttheisolatesandareshownasthemedianofthreeindependentexperiments;S,susceptible;R,resistant.TheFICIvalueis
themedianofthreeindependentexperiments;FICI≤0.5forsynergism,FICI>4.0forantagonismand0.5<FICI≤4.0fornointeraction.
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TABLE2|AntifungaleffectoffluconazolecombinedwithlicofeloneagainstbiofilmofresistantCandidaalbicans.
Strains Times(h) SMIC(µg/mL) LAtheory
MICA CA MICB CB FICI IN
CA10(R) 8 >512 2 >128 32 0.252 SYN
12 >512 4 >128 32 0.254 SYN
24 >512 256 >128 128 >1 NI
CA16(R) 8 >512 4 128 16 0.133 SYN
12 >512 4 128 32 0.258 SYN
24 >512 256 128 128 >1 NI
CA4(S) 8 >512 8 128 16 0.141 SYN
12 >512 16 128 16 0.156 SYN
24 >512 256 128 128 >1 NI
CA8(S) 8 >512 4 128 8 0.070 SYN
12 >512 8 128 16 0.141 SYN
24 >512 256 128 128 >1 NI
Timeindicatestheincubationperiodofbiofilmformation.SMICswerereadasthelowestconcentrationsthatproduceda50%reductioningrowthcomparedwiththatof
thedrug-freecontrol.MICA,TheMICsoffluconazoleusedalone;MICB,TheMICsoflicofeloneusedalone;CA,TheMICsoffluconazolecombinedwithlicofelone;CB,
TheMICsoflicofelonecombinedwithfluconazole;IN,interaction;SYN,synergism;NI,nointeraction.TheFICIvalueisthemedianfromthreeindependentexperiments.
FICI≤0.5forsynergism,FICI>4.0forantagonismand0.5<FICI≤4.0fornointeraction.
FIGURE2|SurvivalcurveofdifferenttreatmentonG.mellonellainfectedwithresistantC.albicans(CA10).Theconcentrationofyeastcellswas5×106cells/larva.
Thecurveswereconsistedgrowthcontrol(PBS)group,fluconazole(160µg/mL)group,licofelone(80µg/mL)groupandlicofelone(80µg/mL)combinedwith
fluconazole(160µg/mL)group.Thesefourcurveswereputinthesamecoordinatesystemtocomparethesurvivalrates.∗P<0.05comparedtothecontrol,
fluconazolealoneandlicofelonealonegroup.Theexperimentwasrepeatedon3independentoccasions(n=3).Valuesrepresentthemeansstandarddeviations
fromthreereplicates.
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thegrowthcontrolgroupby1.3and4-fold,respectively.Thedrug
combinationgroupwasalsohighest(P<0.05).Thesurvivalrate
of larvae treated with licofelone alone group was a little higher
thanthatoflarvaetreatedwithfluconazolegroupalone,thatmay
beexplainedbythefactthattheantifungalactivityoflicofelone
aloneisstrongerthanfluconazole.
Fungal Burden Determination
After 3 days of infection, the fungal burden was determined
by recovering yeast cells from the larvae infected with resistant
C.albicans(CA10).ThenumberofCFUsinlarvaewereincreased
over the time of infection (Figure 3). Infected larvae that
were treated with fluconazole (160 µg/mL) in combination
with licofelone (80 µg/mL) exhibited lower fungal burden
FIGURE3|Effectofdrugcombinationonlarvalburdensofresistant
thanfluconazolegroup.Thetwo-drugcombinationsignificantly
C.albicans(CA10).Alllarvaewereinfectedwith5×106cells/larva
C.albicans,thetreatmentconsistedofcontrol(PBSalone)group,fluconazole decreasedCFUnumberbyalmost4-foldcomparedtothecontrol
(160µg/mL)alonegroup,andfluconazole(160µg/mL)combinedwith group(P<0.05).TheCFUnumberofthecontrolgroupwasalso
licofelone(80µg/mL)group.∗P<0.05comparedtothecontrol,and slightlydecreased,whichmaybecausedbyhemocytesinlarvae.
fluconazolealonegroup.Theexperimentwasrepeatedon3independent
occasions(n=3).Valuesrepresentthemeansstandarddeviationsfromthree
replicates. Histopathology Study
Histopathologic staining of larvae infected with resistant
C. albicans (CA10) and treated with different drugs was
determined,andthemelanizationofG.mellonellainfectedwith performed at 3 day post infection. The differences in shape
resistant C. albicans (CA10) was observed. The selection of andcytoplasmicstainingweredetected.Bothyeastandfilament
drug concentration is not shown in this research. The results forms of C. albicans formed clusters in infected larvae. In the
showedthatafter2daysinfection,allofthedrug-treatedgroups treatment with antifungal drugs, the formation of a majority
showedattenuatedmelanization(Figure2).Specifically,thetwo- of yeasts was decreased in the combination of fluconazole
drug combination group significantly reduced the melanization (160µg/mL)andlicofelone(80µg/mL)group(Figures4G,H).
comparedtothefluconazolegroupandwasthehighestsurvival In addition, licofelone alone group (Figures 4E,F) decreased
rate(P<0.05).After4dayinfection,thesurvivalrateoflarvae more yeast cells than fluconazole alone group (Figures 4C,D).
treatedwithfluconazoleandcombinationgroupwashigherthan The growth control (treatment with the PBS alone) group
FIGURE4|HistopathologystudyofinfectedG.mellonellatreatedwithdifferentdrugs.After72hofinfection,larvaeinfectedwith5×106cells/larvaofresistant
C.albicans(CA10)wereprocessedforhistopathologyasdescribed.(A,B)Untreatedcontrolsgroup;(C,D)larvaetreatedwithfluconazole(160µg/mL)group;
(E,F)larvaetreatedwithlicofelone(80µg/mL)group;(G,H)larvaetreatedwiththecombinationoffluconazole(160µg/mL)andlicofelone(80µg/mL)group.The
experimentwasrepeatedon3independentoccasions.
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TABLE3|ExtracellularphospholipaseactivityofC.albicanstreatedwith
licofeloneandfluconazole.
Group(µg/mL) Precipitation Phospholipase
zone activity
Control 0.61 High
Fluconazole(1) 0.62 High
Licofelone(16) 0.76 High
Fluconazole+Licofelone(16) 0.91a Verylow
Licofelone(32) NZb
Fluconazole+Licofelone(32) NZb
Theprecipitationzonerepresentstheratioofthediameterofthecolonytothe
cloudyzonepluscolonydiameter.aP<0.05comparedtothecontrol.bNZ,no FIGURE5|RelativegeneexpressionlevelsofSAP1,SAP2,SAP3,andSAP4
inresistantC.albicans(CA10).Cellsweretreatedwithfluconazole(1µg/mL),
zoneofprecipitation.
licofelone(16µg/mL)andtheircombination.TotalRNAwasextractedand
reversetranscribedtocDNAforfurtherreal-timequantitativePCRtodetect
(Figures 4A,B) has higher levels of infection than any other geneexpressionlevels.∗P<0.05comparedtothecontrol,fluconazolealone
group. The mean size of the infected areas was smaller andlicofelonealonegroup.Theexperimentwasrepeatedon3independent
occasions(n=3).Valuesrepresentthemeansstandarddeviationsfromthree
in the combination group compared to the fluconazole
replicates.
group.
Effect of Licofelone on Extracellular
Phospholipases Activity of Resistant
C. albicans
The extracellular phospholipase activity of resistant C. albicans
(CA10) was measured (Table 3). There were no obvious
differences in average Pz between control group, fluconazole
(1 µg/mL) group, and licofelone (16 µg/mL) group. The
precipitationzoneofthecombinationoffluconazole(1µg/mL)
and licofelone (16 µg/mL) group was significantly increased
FIGURE6|RelativegeneexpressionlevelsofRAS1,CYR1,TPK2,EFG1,
to 0.91 compared to the control group (P < 0.05). No
BCR1,HWP1,ALS1andALS3inresistantC.albicans(CA10).Cellswere
precipitation zone was observed in licofelone (32 µg/mL) treatedwithfluconazole(1µg/mL),licofelone(16µg/mL)andtheir
combinedwithfluconazolegroup(P<0.05)andlicofeloneused combination.TotalRNAwasextractedandreversetranscribedtocDNAfor
alone(32µg/mL)group. furtherreal-timequantitativePCRtodetectgeneexpressionlevels.∗P<0.05
comparedtothecontrol,fluconazolealoneandlicofelonealonegroup.The
experimentwasrepeatedon3independentoccasions(n=3).Values
Effect of Fluconazole-Licofelone on SAP
representthemeansstandarddeviationsfromthreereplicates.
Gene Expression Levels
To investigate the effects of licofelone and fluconazole on
resistant C. albicans (CA10) secreted aspartyl proteinase The results showed that the combination of licofelone with
activities, the expression of 4 SAP family genes was analyzed fluconazolegroupdown-regulatedtheRAS1,CYR1,TPK2,BCR1,
by RT-PCR. The results showed that both licofelone combined HWP1, ALS1 and ALS3 expression in comparison with control
withfluconazolegroupandfluconazolealonegroupcandown- group(Figure6).Thecombinationgroupsignificantlydecreased
regulate the expression levels of SAP1, SAP2, SAP3, and SAP4 the expression level of RAS1 (3-fold), CYR1 (10-fold), TPK2
comparedtothecontrolgroup(Figure5).Thelevelofdecreased (3-fold), BCR1 (4-fold), HWP1 (3-fold), ALS1 (2-fold) and
expressioninthecombinationgroupwasmoreremarkablethan ALS3 (8-fold) compared to the fluconazole group (P < 0.05).
thefluconazolealonegroup(P <0.05).Theexpressionlevelof While the expression of RAS1, CYR1, TPK2, ALS1, ALS3,
SAP1inthefluconazolealonegroupwassignificantlydecreased and HWP1 expression on licofelone alone were higher than
comparedtothecontrolgroup(P<0.05).Whiletheexpression fluconazolealonegroup.However,thedown-regulationofEFG1
of SAP1 to SAP4 in licofelone alone group shows no obvious expression has no difference between the drug combination
differencecomparedtothecontrolgroup. group and the fluconazole group (P > 0.05). In addition,
both groups decreased EFG1 expression relative to the growth
Effect of Fluconazole-Licofelone on controlgroup.
RAS1, CYR1, TPK2, EFG1, BCR1, ALS1,
ALS3 and HWP1 Gene Expression Levels Effect of Fluconazole-Licofelone on
C. albicans Morphogenesis
Tofurtherinvestigatetheeffectsoflicofeloneandfluconazoleon
resistantC.albicans(CA10)biofilmdevelopment,theexpression Thehyphalformationwasobservedbyfluorescencemicroscope.
levels of 8 biofilm-related genes were analyzed by RT-PCR. The images of resistant C. albicans (CA10) showed that the
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FIGURE8|TheeffectoflicofeloneontheeffluxofRh6Ginresistant
C.albicans.TheeffluxofRh6Gintheabsenceandpresenceoflicofelone
(16µg/mL)weredeterminedbyaflowcytometer.MFIsrepresentthe
intracellularRh6GinC.albicans.∗P>0.05comparedtotheRh6Galone
group.Theexperimentwasrepeatedon3independentoccasions.
in the combination group. In the control group, there were a
large area of hypha gather together, while in the fluconazole or
licofelone alone group, there were still some hypha gathered,
but it was much lesser than the control group. This may be
causedthatthetwo-drugcombinationcandecreasethefilament
formation.
Effect of Licofelone on Drug Efflux
Pumps Activity of Resistant C. albicans
The rhodamine 6G assay results showed that the licofelone
group and the growth control group have the same decline
tendency (Figure 8). No significant differences between groups
wereobserved(P>0.05).
DISCUSSION
Therapies such as the use of novel compounds combined with
the azoles could be an effective solution for candida infections,
asitcanexpandantibioticspectrum,improveantifungalefficacy,
and reduce side effects (Shrestha et al., 2015; Gu et al., 2016).
Several in vitro and in vivo studies demonstrated that COX
inhibitors, for instance, ibuprofen, aspirin, and indomethacin
have certain antifungal activities by suppressing C. albicans
PGE production and biofilm formation (Alem and Douglas,
2
2005; Bink et al., 2012; Liu et al., 2014). The combination of
ibuprofen and fluconazole can enhance fluconazole susceptibly
FIGURE7|Fluorescencemicroscopeexaminedtheyeast-filamenttransition to C. albicans by decreasing the MIC of fluconazole (Costa-
ofresistantC.albicans(CA10).After24hofincubation,C.albicanstreated de-Oliveira et al., 2015). mPGES-1, as the terminal enzyme
withdifferentdrugsasdescribed.(A,B)Untreatedcontrolsgroup;(C,D)
downstreamofCOX-2cancatalyzethePGE biosynthesiswith
fluconazoletreated(1µg/mL)group;(E,F)licofelonetreated(16µg/mL) 2
group;(G,H)thecombinationoffluconazole(1µg/mL)andlicofelone fewer side effects, ideally not affecting the formation of other
(16µg/mL)treatedgroup.Theexperimentwasrepeatedon3independent housekeeping PGs (Vidal et al., 2007; Koeberle et al., 2010).
occasions. Various pharmacodynamic studies have confirmed that a novel
dual mPGES-1/LOX inhibitor, licofelone was effective in many
types of diseases and has anti-asthmatic and anticonvulsant
filamentation was reduced with the presence of licofelone effects (Rotondo et al., 2002; Kulkarni and Singh, 2007). The
combined with fluconazole (Figures 7G,H) compared to the simultaneous inhibitions of both enzymes enable licofelone
fluconazole treated group (Figures 7C,D) and control group to have a superior anti-inflammatory effect and obviate the
(Figures7A,B).Therewereentirelyyeastcellsandnofilaments gastrointestinalside-effectscomparedtotheNSAIDS.Thisbetter
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safety profile has been authenticated in healthy individuals is based on their activity of inhibiting biofilm formation by
(CharlierandMichaux,2003;Biasetal.,2004).However,weather decreasing PGE production. Here we investigated the further
2
licofelone has the similar antifungal activity as COX inhibitor antifungalmechanismsoflicofelonecombinedwithfluconazole.
has not been elucidated. In this study, we first evaluated the RAS/cAMP/PKApathwayisimportantforC.albicansvirulence
effect of fluconazole combined with licofelone against resistant by regulating hyphal growth (Rocha et al., 2001). In this
C.albicansinvitro,licofelonealonehasamoderatelygreateffect pathway, the activation of Ras1 can lead to the generation
on resistant C. albicans: the MIC of licofelone alone against of a cAMP signal that promotes PKA-mediated activation of
resistant C. albicans was 128 µg/mL, and it can synergistically the transcriptional regulator proteins EFG1 and BCR1, which
workwithfluconazole:theMIC offluconazolewasdecreased that play an essential role in regulating the morphogenesis and
80
from512to1µg/mLwhencombinedwithlicofelone(16µg/mL), expression level of cell wall-associated adhesion gene such as
while the combination has little effect on sensitive C. albicans ALS1,ALS3,andHWP1thatareassociatedwithmorphogenetic
and non-albicans strains (Table 1). Secondly, the combined response (Nobile and Mitchell, 2005). Our in vitro result
antifungal effect of fluconazole-licofelone against C. albicans showed that licofelone combined with fluconazole has great
biofilmcellswasobservedindifferenttimepoint.Thetwo-drug effects on inhibiting biofilm formation in different stages.
combinationsignificantlyreducedC.albicansbiofilmformation Regarding how the drug combination influences the biofilm
over12h,butafter24h,therewasalmostnoanti-biofilmeffect formation, the results showed that the combination has no
observed(Table2).TheC.albicansbiofilmformationhasthree influence on EFG1 expression level, but dramatically down-
phases:adherencestage(0–11h)inwhichthefungalpathogens regulated the expression of BCR1 (Figure 6), suggesting that
adheretothesurface,developmentstage(12–30h)inwhichthe thepossibilityofdrugcombinationagainstresistantC.albicans
carbohydrate-richextracellularmatrix(ECM)areproduced,and biofilm is through the regulation of the central regulator
maturationstage(31–72h)inwhichthebiofilmstructureisheld BCR1, not by EFG1. We further examined the adhesion-
togetherbyanECM.Theresistanceofbiofilmstoantimicrobial related genes HWP1, ALS1 and ALS3 expression levels in the
agentsisoftenattributedtoitsfailuretopassthebiofilmmatrix presence of the combination. We found that the expression
(Al-Fattani and Douglas, 2006). This may be the reason why level of those genes was decreased 3-fold, 2-fold, and 8-fold,
theantifungaleffectofthecombinationcanonlyinhibitbiofilm respectively, compared to the fluconazole group (P < 0.05)
over the process of formation, but has no inhibiting effect on (Figure 6). Mutational analysis indicated that strains lacking
the mature biofilm. Further, for the future experiments, we all functional HWP1, ALS1 and ALS3 alleles (hwp1(cid:49)/hwp1(cid:49)
will focus on the effect and antifungal mechanisms of resistant als1(cid:49)/als1(cid:49) als3(cid:49)/als3(cid:49)) were not able to generate adherent
C.albicans. cells in biofilm models (Nobile et al., 2008). Thus, our results
In addition, G. mellonella model recently has been used demonstrated that the fluconazole combined with licofelone
to study fungal virulence and antifungal drug activity, since may reduce the biofilm formation of C. albicans by inhibiting
it has a similar immune response to mammals (Vilcinskas, adhesion, and this inhibition is partly due to the down-
2011; Favre-Godal et al., 2014), and the correlation between regulation of biofilm cell wall adhesion-related genes ALS1,
virulence of C. albicans mutants in mice and G. mellonella ALS3 and HWP1. These results were further confirmed by
model has been confirmed (Brennan et al., 2002). It is florescence microscope analysis. The two-drug combination-
worth mentioning that, with these advantages and the treatedstrainsproducedabiofilmwithonlyyeastcellsandlittle
low cost, lack of ethical concerns and easy manipulation, filament, but the filamentation was more abundant with the
G. mellonella model has gained wide acknowledgment. presence of fluconazole group and the growth control group
As documented in our in vivo study, the combination of (Figure 7). Moreover, licofelone combined with fluconazole
fluconazole with licofelone in G. mellonella infected model decreased the expression level of RAS1, CYR1 and TPK2,
significantly improved the survival rate of larvae compared indicating that the inhibition of biofilm-specific related genes
to the fluconazole treated group (P < 0.05). The death rate is probably through RAS/cAMP/PKA pathway (Figure 6).
of the larvae is a dose dependent (Figure 2), as previously Previousstudieshavealsoshownthatsecretedaspartylproteinase
described (Scorzoni et al., 2013). To complement the studies and phospholipases were believed to be important factors
in vivo, we further performed the fungal burden determination involved in the pathogenicity and virulence of C. albicans
(Figure 3) and microscopic observation (Figure 4) in larvae (Ghannoum,2000;Fengetal.,2016).Thesehydrolyticenzymes
after infection. Our results confirmed the correlation of the were secreted by the fungus that lead to the colonization and
virulence with the degree of damage on the histological tissue infections (Nailis et al., 2010). Secreted aspartyl proteinases
ofG.mellonellawithfewerCFUswasobservedinthecombined are related to the adhesion and hydrolytic activity in mucosal
group. tissue (Ramage et al., 2012). The study by Mendes et al.
Candida albicans biofilm is a complex three-dimensional has indicated that biofilms of C. albicans secreted more Saps
architecturewithanabundantextracellularmatrix.Itisknownto than planktonic cells (Mendes et al., 2007), and Saps are
enhancethefungalresistancetomostcommonlyusedantifungal known to be intrinsically associated with the hyphal phase
agent and the ability of cells to adhere is indispensable for all of C. albicans (Chen et al., 2002; Felk et al., 2002). We also
stages of C. albicans biofilm formation (Chandra et al., 2001; examinedtheSAPgeneexpressionbyRT-PCR.Thecombination
Nobile and Johnson, 2015). Earlier evidence only indicated of fluconazole and licofelone significantly down-regulated the
that the effectiveness of COX inhibitors against C. albicans SAP1-SAP4 expression levels compared to fluconazole group
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