title: Synergistic effect of fluconazole and doxycycline against Candida albicans biofilms resulting from calcium fluctuation and downregulation of fluconazole-inducible efflux pump gene overexpression.
abstract: Candida albicans biofilms are intrinsically resistant to antimicrobial agents. Previous work demonstrated that the antifungal activity of fluconazole against C. albicans biofilms is notably enhanced by doxycycline. In order to explore the synergistic mechanism of fluconazole and doxycycline, we investigated the changes of efflux pump gene expression, intracellular calcium concentration and cell cycle distribution after drug intervention in this study. The expression levels of CDR1, CDR2 and MDR1 were determined by real-time PCR, and the results showed that fluconazole alone could stimulate the high expression of CDR1, CDR2 and MDR1, and the combination of doxycycline and fluconazole downregulated the gene overexpression induced by fluconazole. Intracellular calcium concentration was determined using Fluo-3/AM by observing the fluorescence with flow cytometry. A calcium fluctuation, which started 4 h and peaked 8 h after the treatment with fluconazole, was observed. The combined drugs also initiated a calcium fluctuation after 4 h treatment and showed a peak at 16 h, and the peak was higher than that stimulated by fluconazole alone. The cell cycle was measured using flow cytometry. Fluconazole alone and the combined drugs both induced a reduction in the percentages of S-phase cells and an elevation in the percentages of cells in the G2/M phase. The results of this research showed that the synergism of fluconazole and doxycycline against C. albicans biofilms is associated with blockade of the efflux pump genes CDR1, CDR2 and MDR1, and stimulation of high intracellular calcium concentration. The findings of this study are of great significance in the search for new antifungal mechanisms.
authors.affiliation: Department of Pharmacy, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, PR China.|School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong Province, PR China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Gao
authors.affiliation: School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong Province, PR China.
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.affiliation: School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong Province, PR China.
authors.forename: Shuyuan
authors.initials: S
authors.lastname: Liu
authors.affiliation: Department of Pharmacy, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong Province, PR China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Pharmacy, Qianfoshan Hospital Affiliated to Shandong University, Jinan, Shandong Province, PR China.
authors.forename: Shujuan
authors.initials: S
authors.lastname: Sun
mesh_terms: D000900:Anti-Bacterial Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D000935:Antifungal Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D002118:Calcium / Q000378:metabolism; D002176:Candida albicans / Q000187:drug effects* / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D004318:Doxycycline / Q000493:pharmacokinetics / Q000494:pharmacology*; D004357:Drug Synergism; D015725:Fluconazole / Q000493:pharmacokinetics / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects
chemical_list: D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D002352:Carrier Proteins; D005656:Fungal Proteins; D015725:Fluconazole; D004318:Doxycycline; D002118:Calcium