β-Lapachone enhances the antifungal activity of fluconazole against a Pdr5p-mediated resistant Saccharomyces cerevisiae strain

Abstract

Objectives: The aim of this study was to evaluate the ability of lapachones in disrupting the fungal multidrug resistance (MDR) phenotype, using a model of study which an azole-resistant Saccharomyces cerevisiae mutant strain that overexpresses the ATP-binding cassette (ABC) transporter Pdr5p.

Methods: The evaluation of the antifungal activity of lapachones and their possible synergism with fluconazole against the mutant S. cerevisiae strain was performed through broth microdilution and spot assays. Reactive oxygen species (ROS) and efflux pump activity were assessed by fluorometry. ATPase activity was evaluated by the Fiske and Subbarow method. The effect of β-lapachone on PDR5 mRNA expression was assessed by RT-PCR. The release of hemoglobin was measured to evaluate the hemolytic activity of β-lapachone.

Results: α-nor-Lapachone and β-lapachone inhibited S. cerevisiae growth at 100 μg/ml. Only β-lapachone enhanced the antifungal activity of fluconazole, and this combined action was inhibited by ascorbic acid. β-Lapachone induced the production of ROS, inhibited Pdr5p-mediated efflux, and impaired Pdr5p ATPase activity. Also, β-lapachone neither affected the expression of PDR5 nor exerted hemolytic activity.

Conclusions: Data obtained indicate that β-lapachone is able to inhibit the S. cerevisiae efflux pump Pdr5p. Since this transporter is homologous to fungal ABC transporters, further studies employing clinical isolates that overexpress these proteins will be conducted to evaluate the effect of β-lapachone on pathogenic fungi.

Keywords: Fluconazole; Lapachone; Multidrug resistance; Yeast.

Fig. 1

Structure of the compounds tested in this study: α-lapachone; α-nor-lapachone; β-lapachone; β-nor-lapachone

Fig. 2

Evaluation of the growth of Pdr5p+ cells in the presence of the tested compounds. Cells were grown in the presence of α-lapachone, α-nor-lapachone, β-lapachone, and β-nor-lapachone at 6.25–100 μg/ml at 37 °C for 48 h. Cell growth was measured spectrophotometrically (600 nm). Data represent mean ± standard error of three independent experiments. Black circle for α-lapachone. Whit circle for α-nor-lapachone. Inverted black triangle for β-lapachone. White triangle for β-nor-lapachone. *p < 0.05

Fig. 3

Combined effect of β-lapachone and fluconazole on the growth of Pdr5p+ cells in solid media through spot assay. Serial 5-fold dilution cells were spotted onto YPD agar in the presence or absence of 125 μg/ml fluconazole, 0.125% DMSO, and 12.5 μg/ml β-lapachone. Combining fluconazole and β-lapachone resulted in the complete inhibition of Pdr5p+ cell growth

Fig. 4

Effect of β-lapachone and fluconazole alone and in combination on ROS production by Pdr5p+ cells. a ROS production after treatment with 125 μg/ml fluconazole was comparable with control. Treatment with 100 μg/ml β-lapachone increased ROS production. Combining β-lap with fluconazole did not enhance ROS production in comparison with treatment with β-lapachone alone. Ascorbic acid significantly diminished ROS production, being used as a negative control. *p < 0.05 in comparison with untreated system. b Combined effect of ascorbic acid, β-lapachone, and fluconazole on the growth of Pdr5p+ cells in solid media through spot assay. Serial 5-fold dilution cells were spotted onto YPD agar in the presence or absence of 125 μg/ml fluconazole, 25 mM ascorbic acid, 0.125% DMSO, and 12.5 μg/ml β-lapachone. Combining 100 μg/ml β-lapachone, 125 μg/ml fluconazole, and 25 mM ascorbic acid promoted cell growth comparable to the positive control

Fig. 5

Assessment of Pdr5p activity by Nile red accumulation assay. Pdr5p+ cells were incubated in the presence or absence of 100 μg/ml β-lapachone for 60 min at 30 °C. Then, cells were loaded with Nile red, and the transporter was activated by adding 0.2% glucose. Treatment of Pdr5p+ cells with β-lapachone inhibited Nile red efflux by 79.4%. *p < 0.05

Fig. 6

The effect of β-lapachone on the ATPase activity of Pdr5p was evaluated by incubating membranes containing Pdr5p in the presence of different concentrations of the compound (100–6.25 μg/ml). β-Lapachone inhibited Pdr5p ATPase activity by 72.6–82.5%. The Lineweaver-Burk plot shows that the effect of β-lap on Pdr5p ATPase activity occurred due to a mixed inhibition (inset). *p < 0.05

Fig. 7

Mitochondrial membrane potential in the presence of β-lapachone was assessed by JC-1 labeling. β-Lapachone at 100 μg/ml reduced MMP by 25%, while sodium azide decreased MMP by 37%. *p < 0.05

Fig. 8

The influence of β-lapachone alone or in combination with fluconazole on PDR5 mRNA expression of Pdr5p+ cells was assessed by RT-PCR. TFC1 was used as a housekeeping gene and the untreated cells as a reference sample. The relative expression level of mRNA was calculated using the ΔΔCT method. Values represent mean + SD of three independent experiments. One hundred micrograms per milliliter of β-lapachone, 125 μg/ml fluconazole, and 100 μg/ml β-lapachone + 125 μg/ml fluconazole did not exert any significant effect on PDR5 gene expression

Fig. 9

Evaluation of β-lapachone toxicity against human erythrocytes. A human erythrocyte suspension (0.5% v/v) was incubated in the presence of serial concentrations of β-lapachone (128–0.5 μg/ml) for 60 min, and the absorbance of released hemoglobin was measured at 540 nm. Controls of 100% and 0% hemolysis were performed by incubating the cells in PBS in the presence or absence of 1% Triton X-100, respectively. DMSO (1.28%) was also used as control. β-Lapachone showed toxicity comparable with PBS and DMSO. *p < 0.05