Antifungal Activity And Mechanism Of Panobinostat In Combination With Fluconazole Against Candida Albicans

BackgroundInvasive Candida infections bring huge health challenges.Candida albicans(C.albicans,CA),the most common opportunistic fungal pathogen in humans,can cause bloodstream infections with high mortality in susceptible hosts.At present,available antifungal agents used in clinical practice are limited,and most of them also have some serious adverse effects.The emergence of drug resistance because of the wide use of antifungal agents is a new limitation to successful patient therapy.Nowadays,drug combination therapy is increasingly becoming a way to enhance antifungal efficacy,reduce drug resistance and potential toxicity.Panobinostat(Pano)is a histone deacetylase(HDAC)inhibitor,which has an activity of anti-cancer.There are researches exhibited that the HDACs influence growth,virulence,drug resistance of C.albicans.No active antifungal effect has been reported between Pano and fluconazole(FLC)against Candida albicans.ObjectivesTo investigate the antifungal activity of Pano combined with FLC against resistant Candida albicans both in vitro and in vivo,and explore the antifungal mechanisms when Pano plus FLC.MethodsTo evaluate the in vitro antifungal activity,the broth microdilution method was carried out according to the Clinical and Laboratory Standards Institute standard M27-A3 document(CLSI,M27-A3),fractional inhibitory concentration index(FICI)model was used to analyze the interaction of the drug combination;XTT colorimetric method was used to determine the combined antifungal activity of Pano and FLC against Candida albicans biofilms.To evaluate the in vivo antifungal activity,the Galleria mellonella infection model was established.After the intervention of different groups of drugs,the survival rate,fungal load and tissue damage of infected larvae were measured,to evaluate the antifungal effect of Pano combined FLC in vivo.To explore the potential antifungal mechanism of drug combination,the hyphal growth assay was performed using the microscope to explore the combined activity of FLC and Pano;the impact of Pano on the extrusion and uptake of fluorescent substrates rhodamine 6G(Rh 6G)were examined to explore the effect of drug combination on drug transporters;the effect of FLC combined with Pano on the Metacaspase activation of Candida albicans was observed using fluorescent probe FITC-VAD-FMK under a fluorescence microscope,to further elucidate the potential mechanism of Pano and FLC synergistic effect against drug-resistant Candida albicans.Results(1)Pano acts synergistically against drug-resistant Candida albicans planktonic cells when combined with FLCWhen Pano combined with FLC,Pano could reduce the MIC value of FLC from≥512 μg/mL to 0.25-0.5 μg/mL,and the FICI value is 0.0024-0.0166,which is much less than 0.5,exhibiting that the combination of Pano and FLC has a strong synergistic effect on drug-resistant Candida albicans.(2)Pano acts synergistically against Candida albicans biofilms when combined with FLCFor early-formed biofilms(4h,8h),Pano reduced the sMIC value of the FLC from≥ 512 μg/mL to 0.5-2 μg/mL,FICI value was 0.0049-0.0635.While the combination of Pano and FLC had an irrelevant effect on the biofilms formed at 12 hours and 24 hours.Thus,the combination of Pano and FLC showed antifungal effects against early-stage(≤ 8 h)biofilms formed by Candida albicans.(3)Pano acts synergistically FLC against drug-resistant Candida albicans in Galleriar mellonella larvae infection modelFor the survival assay,the survival rate of the control group,Pano alone,and FLC alone group was 10%,20%,and 35%,respectively(P>0.05),however,the combination of Pano and FLC significantly enhanced the survival rate to 80%(P<0.05).For fungal burden determination,the colonies were decreased in combination group,comparing with other groups.For histopathology assay,the damage was significantly reduced in the combined drug group,comparing with other groups.(4)Pano combined with FLC could inhibit the hyphal growth of drug-resistant Candida albicansThe hyphae growth of Candida albicans was induced by RPMI-1640 medium and observed by microscope after drug intervention.Long and interlaced hyphae were formed in the control group,and there was no obvious difference when comparing with Pano-alone group or FLC-alone group.While the number of hyphae was significantly reduced in the field of vision in the combined group and the hyphal length was also reduced slightly.The results showed that the intervention of Pano combined with FLC could inhibit the hyphal growth of drug-resistant Candida albicans.(5)Pano had no effect on drug uptake and efflux of drug-resistant Candida albicansThe results of the combined group had no significant difference compared with the control group no matter the uptake or efflux of Rh 6G(P>0.05).It could be inferred that the combination of pano and FLC had no effect on the regulation of drug efflux pump.(6)Pano combined with FLC activated intracellular metacaspase and then induced the fungal cell deathMetacaspase is a major factor in apoptosis,and its activation can induce morphological changes and DNA fragmentation leading to death.FITC-VAD-FMK has an ability to penetrate the cell membrane and bind caspase to become fluorescent,it is used to monitor the caspase activation in this assay.The fluorescence microscope,showed that,on one hand,there were much fewer cells in the combined group compared with other groups.On the other hand,the combination group showed bright green fluorescence.In contrast,little fluorescence was observed in the other three groups.Furthermore,the ratio of fluorescence cells in the Pano+FLC group was much higher than in other groups.The results demonstrated that the drug combination activated intracellular metacaspases and then induced the fungal cell death.ConclusionIn summary,Pano showed synergism with FLC against both resistant planktonic C.albicans and biofilms.Moreover,Pano combined with FLC caused the higher rate for G.mellonella larvae infected with C.albicans to survive,it could also decrease fungi load and tissue damage.Mechanism studies elucidated that the synergism between Pano and FLC could inhibit the hyphal growth and activation of metacaspase.These findings might provide insights into overcoming the fungal resistance,and we hope this study could be helpful for novel antifungal drug development.