Synergistic Effect of Fluconazole and Calcium Channel Blockers against Resistant Candida albicans

Candidiasis has increased significantly recently that threatens patients with low immunity. However, the number of antifungal drugs on the market is limited in comparison to the number of available antibacterial drugs. This fact, coupled with the increased frequency of fungal resistance, makes it necessary to develop new therapeutic strategies. Combination drug therapy is one of the most widely used and effective strategy to alleviate this problem. In this paper, we were aimed to evaluate the combined antifungal effects of four CCBs (calcium channel blockers), amlodipine (AML), nifedipine (NIF), benidipine (BEN) and flunarizine (FNZ) with fluconazole against C. albicans by checkerboard and time-killing method. In addition, we determined gene (CCH1, MID1, CNA1, CNB1, YVC1, CDR1, CDR2 and MDR1) expression by quantitative PCR and investigated the efflux pump activity of resistant candida albicans by rhodamine 6G assay to reveal the potential mechanisms. Finally, we concluded that there was a synergy when fluconazole combined with the four tested CCBs against resistant strains, with fractional inhibitory concentration index (FICI) <0.5, but no interaction against sensitive strains (FICI = 0.56 ~ 2). The mechanism studies revealed that fluconazole plus amlodipine caused down-regulating of CNA1, CNB1 (encoding calcineurin) and YVC1 (encoding calcium channel protein in vacuole membrane).


Introduction
Candidiasis is one of the most important human opportunistic fungal infections, which threatens peoples living with immune system compromise, including solid organ transplant, AIDS or cancer patients, and hematopoietic stem cell transplant recipients. Candida albicans remains the most common etiological agent of candidiasis [1,2]. Fluconazole (FLC) which belongs to azole antifungals, is the mainstay in prevention and treatment of Candida albicans infections due to the great efficacy, reduced toxicity and inexpensive [3]. However, with the wide-spread use of FLC, the incidence of FLC-resistance or cross-resistance to multiple azole derivatives emerged [4][5][6]. Despite the availability of several other classes of antifungal drugs (Polyenes, Pyrimidines, Echinocandins), inherent toxicity, cost of care, and emergence of resistance remain major problems in clinic, and the available antifungal agents are still limited [7]. These problems point to an urgent and unmet need for the development of new antifungal agents or searching for new antifungal approaches. Unfortunately, the pace of developing new antifungal drugs has been extremely slow. Therefore, more and more attentions have been paid to drug combination, of which, the combination of antifungals with non-antifungals was focused on [8,9].
Calcium channel blockers (CCBs), which are commonly used drugs in treatment of cardiovascular disease, were demonstrated to have antifungal activity against Candida albicans or Aspergillus fumigates [10][11][12]. The synergistic antifungal properties in combination with some known antifungal agents (such as fluconazole, ketoconazole, itraconazole) have also been reported [13][14][15][16]. In addition to inhibition of fungal tube or biofilm formation, the established antifungal mechanisms were identified as targeting calcium channel protein Cch1-Mid1 in membrane and disturbing calcium homeostasis. Recently, more and more reports documented that calcium and some components in calcium-signaling pathway were related to fungal resistance and virulence [17,18]. For example, calcineurin is involved in hyphal growth, drug tolerance, virulence and stress responses in serum of some candidas [19][20][21][22], vacuolar acidification contributed to the ability of Candida albicans to form hyphae and establish infection [23], and that Yvc1p, the putative vacuolar calcium channel, plays an important role in Candida albicans infection and survival in host tissues as it is associated with fungal stress response, morphogenesis, and polarized growth [24]. Thus, some non-antifungal agents with action of disturbing calcium homeostasis may become the potential candidates to combine with FLC to fight against fungal infections [11,12,15,25,26].
In this study, we first evaluated the in Vitro activity of four clinic-commonly used CCBs, amlodipne (AML), nifedipien (NIF), benidipine (BEN), flunarizine (FNZ) combined with FLC against resistant Candida albicans (CA10, CA16) and sensitive Candida albicans (CA14, CA129). In addition, we performed time-killing curves to investigate the antifungal effects of the four CCBs combined with FLC against resistant Candida albicans (CA10) in different time points dynamically. Since some CCBs could disturb the calcium homeostasis by interfering with Cch1-Mid1 protein, which is a homologue of mammalian voltage-gated Ca 2+ channels (VGCCs) [27], we determined the gene expression of CCH1, MID1, CNA1, CNB1 and YVC1, which are closely related to the regulation of calcium concentration by real-time quantitative PCR (RT-PCR). In order to identify whether the combinations could also reverse one of the most common resistant mechanism of increased drug efflux, we conducted rhodamine 6G assays to investigate activity of efflux pump protein and RT-PCR to determine gene expression of CDR1, CDR2 and MDR1 [28].

Strains and media
The following Candida albicans strains were used in this study: the FLC resistant isolates (CA10, CA16) and FLC susceptible isolates (CA14, CA129) and the quality control strain (ATCC10231). The strains were refreshed from the frozen storage at -80°C and subcultured on the yeast-peptone-dextrose (YPD) solid medium (1% yeast extract, 2% peptone, 2% glucose and 2% agar) at least twice at 35°C before each experiment to ensure viability and purity. RPMI1640 was used as liquid medium to dilute drugs and strains. FLC was provided by Cheng Chuang Pharmaceutical and a stock solution of 2560 μg ml -1 was prepared in distilled water. AML, NIF, BEN and FNZ were purchased from Chinese medicine institute and stock solutions of 2560 μg ml -1 were prepared in dimethyl sulphoxide (DMSO). All stock solutions were stored at -20°C.

MIC testing in broth microdilution assays
The determination of FLC MIC and the investigation of the interaction of tested CCBs with FLC by way of checkerboard tests on 96-well plates were performed according to the approved CLSI standard reference method for broth dilution antifungal susceptibility testing of yeasts M27-A3 [29]. For the checkerboard assays [30], 50 μl of RPMI-1640 medium containing FLC with concentration ranging from 0.125~64 μg ml -1 , was added to the wells in the second to eleventh columns of the microtitre plate, and 50 μl of RPMI-1640 medium containing AML (or NIF, BEN and FNZ) with concentration ranging from 32~0.5 μg ml -1 was added to the wells in A to G line of the 96-well plate. Then, 100 μl of Candida albicans cell suspensions (0.5~2.5×10 3 cells/ml) was added to each well mentioned above. All the wells on the plate were filled with RPMI-1640 to a final volume of 200 μl. Thus, the well H1 (the intersection of row H and column 1) was drug free that served as the growth control and wells of the twelfth column filled with only RPMI-1640 medium served as the negative control. The plate was covered with its lid, sealed with parafilm and incubated at 35°C for 24 h [31]. Readings were performed by both visual reading and optical density (OD) by determining the absorbance at 490 nm on a microplate reader. MIC endpoints were defined as the lowest concentration of drugs causing 80% decrease in viability compared to the drug-free control (MIC 80 ). All experiments were performed in triplicate. Drug interactions were interpreted by fractional inhibitory concentration index (FICI) model and the percentage of growth difference (ΔE) model, respectively. The FICI was calculated as the sum of the FICs of either drug (MIC FLC + CCB /MIC FLC + MIC CCB + FLC /MIC CCB ). The FICI 0.5 represents synergy, an FICI > 4 antagonism, and a 0.5<FICI 4 no interaction [32]. The ΔE model was defined by the following equation: ΔE = E predicted -E measured , with E measured obtained directly from the experimental data. The following equation was derived: E predicted = E A ×E B , where E A and E B are the experimental percentages of fungal growth of each drug action alone, respectively. Statistically significant interactions of <100% were considered weak, those of 100-200% were considered moderate, and those of >200% were considered strong, as described previously [33].

Time-killing curves
Inoculum of (0.5~2.5) × 10 3 cells/ml of Candida albicans (CA10) was used in this experiment. The final concentration was 1μg ml -1 for FLC when combined with AML (8 μg ml -1 ) and NIF (8 μg ml -1 ), and 2μg ml -1 for FLC when combined with BEN (16 μg ml -1 ) and FNZ (16 μg ml -1 ). A drug-free sample served as a growth control. The XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] test was performed to detect the cell viability after different treatments according to the method described previously [30,34]. Briefly, at predetermined time points (0, 6, 12, 24, and 48 h after incubation at 35°C), 100 μl aliquot from each treatment mixture was transferred to a well of new 96-well microtitre plate, and then a 100 μl aliquot of XTT-menadione solution was added (Prior to each assay, XTT purchased from Sigma was dissolved in a saturated solution at 0.5 μg ml -1 in Ringer's lactate. The solution was filter sterilized through a 0.22 μm filter, and then 100 mM menadione in acetone was added to a final concentration of 10 μM). The plate was then incubated in the dark for up to 2 h at 35°C. After that, XTT reduction was assessed by determining the absorbance at 490 nm on a microplate reader (SPEC TRA MAX190, Thermo lab systems, USA). All experiments were conducted in triplicate, and the results were reported as mean values. Thus, growth-and metabolism-inhibitory effects of the drugs alone and in combination were observed based on the results of spectrophotometric methods.

Real-time quantitative PCR
For determining the expression levels of calcium-regulation related genes CCH1, MID1, CNA1, CNB1, YVC1, and genes encoding membrane transport protein (CDR1, CDR2 and MDR1), Candida albicans (CA10) cells were grown to mid-log phase in RPMI-1640 medium at 35°C after treatment with drugs alone or in combination at the following final concentrations: FLC at 1 μg ml -1 ; AML at 8 μg ml -1 . Cultures without drugs served as the control. Cells were then harvested for RNA extraction. Cell total RNA was isolated using the hot phenol method described as previous [35]. Then, diluted RNA was treated with First-Stand cDNA Synthesis-SuperMix kit (TransGen Biotech) and was reversely transcribed at 42°C for 30 min and 85°C for 5 min according to the manufacturer's instructions. RT-PCR reactions were mixed with RNA, Ultro SYBR Mixture (with ROX) (Transgene, Beijing, China) and gene primers (The sequences of the primers are listed in Table 1) in triplicate. The ACT gene was used as the endogenous control. The RT-PCR was carried out with an ABI ViiA 7 (Applied Biosystems) sequence detection system using SYBR Green I (Cwbiotech) in duplicate for three separate experiments. An aliquot of 25 ml PCR mix was used for each gene and the cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.

Rhodamine 6G efflux assay
To identify whether efflux pump function could be affected by these four CCBs, the functional activity of drug efflux pumps was tested. Briefly [36], cells were shaken overnight at 35°C in YPD broth, transferred to fresh YPD broth and incubated at 35°C for 4 h. The cells were harvested by centrifuging and adjusted to 1× 10 7 Cells/ml in YPD broth. A final concentration of 10 μmol Rh6G was added to the cell suspension. Candida albicans (CA10) cells grown to midlog phase in RPMI-1640 medium at 35°C were collected by centrifuging, washed and resuspended in PBS. The cells were then de-energized for 2 h in PBS (without glucose). The de-energized cells were pelleted, washed three times with PBS and resuspended with PBS containing 5% glucose. Energy dependent efflux was measured after the addition of tested CCBs to the cells. Samples, 500 μl each, were withdrawn at specific time intervals (5, 10, 15, 20, 25, 30 min) Table 1. Gene-specific primers used for RealTime-PCR.

Gene
Primer sequences ( and the fluorescence of the reaction mixture was recorded using a BD FACScalibur flow cytometer (Becton Dickinson) with excitation at 485 nm and emission at 538 nm, respectively. Rh6G-free and Rh6G-alone groups as controls were included in all the experiments.

Statistical analysis
Data were analyzed using SPSS Statistics software with Student's t-test. Values presented as means ± standard deviation of three replicates, Ã P <0.01 indicated the levels of significant difference (at least 2.0-fold compared to the control group).

Calcium channel blockers tested significantly increased the sensitivity of fluconazole to resistant Candida albicans
Using the CLSI standard reference method (M27-A3) for broth dilution antifungal susceptibility testing of yeasts [29], the MIC 80 for fluconazole of sensitive strains CA14, CA129 were 0.5 μg ml -1 and 2 μg ml -1 , of resistant strains (CA10, CA16) were all 512 μg ml -1 . Whereas the MIC for CCBs (AML, NIF, BEN, FNZ) of all the tested strains were >512 μg ml -1 , exhibiting no antifungal activity. However, the MIC 80 of FLC for resistant strains were decreased to 1~2 μg ml -1 in the presence of AML (8 μg ml -1 ), NIF (8 μg ml -1 ), BEN (16 μg ml -1 ), FNZ (16 μg ml -1 ). Calculation of the fractional inhibitory concentration index (see Materials and Methods) produced values of 0.017~0.035, indicating significant synergism (Table 2 and S1 Table). The results interpreted by ΔE method also demonstrated to be strong synergistic with very high percentages of interactions ranging from 964.71% to 1866.19% (Fig 1). For sensitive strains, the MIC 80 of FLC showed almost no change when combined with these four CCBs compared with FLC alone with FICI ranging from 0.56 to 2 and the ΔE method showed very low percentages of synergistic interaction, indicating no interaction between fluconazole and AML (NIF, BEN, FNZ) (data not shown).These observations indicate that tolerance to fluconazole of resistant Candida albicans is lost in the presence of the four CCBs and that AML proved to be the best candidate for combination with FLC against resistant Candida albicans.

Time-kill curves
The dynamic combined antifungal effects of FLC with CCBs (AML, NIF, BEN, FNZ) against resistant Candida albicans (CA10) were confirmed by time-kill studies. The intensity and nature of interactions between FLC and the four CCBs were depicted by time-kill curves with OD value obtained from the XTT reduction assay as Y-axis and time as X-axis (Fig 2 and S1  Fig). The results showed little difference between groups containing drugs and control group in the first 6 h. While, a growth delay exhibited in groups containing FLC from the 6 th hours, and that drug combination groups showed more obvious growth delay. At 24 th and 48 th , the OD value was reduced more than two-fold in the groups of FLC combined with AML, NIF and BEN compared with FLC alone group, indicating a synergism [37]. Although the OD values were not significant different between FLC/FNZ combination group and FLC alone group, the results showed an apparent growth delay in FLC/FNZ combination group compare with FLC alone group. Among these four combinations, FLC/AML group showed the best antifungal effects against CA10 as times goes on, and FLC/NIF group showed weaker antifungal effects compared with FLC/AML. The results demonstrated that AML, NIF, BEN and FNZ could eliminate FLC tolerance against resistant Candida albicans, which was consistent with those from the checkerboard microdilution assays.

Fluconazole combined with amlodipine down-regulated the expression of CNA1, CNB1 and YVC1
The results of RT-PCR assays showed that AML alone caused almost three-fold up-regulation in the level of CNA1 and CNB1 transcription and the expression of CNB1 was increase on FLC challenge by two-fold compared with the control group (P <0.01). In contrast, the combination of FLC and AML significantly down-regulated the CNA1 and CNB1 compared with AML challenge alone by a greater than almost ten-fold and three-fold, respectively (P < 0.01). The expression of CNB1 was up-regulated on FLC challenge compared with the control group by two-fold (P < 0.01). For the expression level of YVC1, it was increased almost two-fold (P < 0.01) after the treatment of FLC or AML alone, while the drug combination two-fold down-regulated its expression compared with the two drugs alone (P <0.01) (Fig 3 and S2  Fig). What was contrary to the nature mechanisms of CCBs, there was no significant difference in the expression level of CCH1 and MID1 among drugs alone group, drugs combination group and control group (data not shown). As calcineurin is an important enzyme in regulating calcium homeostasis in fungal cells [38,39], and YVC1p mainly transports excess calcium in cytosol to vacuole [24,40], we speculated that: ①the treatment of FLC combined with AML disrupted calcium concentration in CA10. The results may cause an increase of calcium The resistance of Candida albicans to FLC has been identified as different mechanisms, such as mutations in ERG11 (encoding the drug target protein lanosterol 14α-demethylase) and overexpression of multidrug transporters and efflux pump-encoding genes CDR1, CDR2, and MDR1 [5,41]. In the purpose of testing whether the presence of tested CCBs could also reverse the increase of drug efflux, we investigated the activity of efflux pumps by Rhodamine 6G assay and determined the expression changes of drug resistant genes CDR1, CDR2 and MDR1 by RT-PCR. The results demonstrated a stress increase in the expression level of CDR1, CDR2 and MDR1 after treatment of FLC alone compared with control group. The combined group downregulated the expression level of CDR1 and CDR2 compared with drug alone groups, but there was no significant differences ( Fig 4A and S3A Fig). For the expression level of MDR1, there was almost no difference between the drugs alone group and combined group. The results of Rhodamine 6G efflux assay indicated that when cells were resuspended in PBS containing 5% glucose could induce an ATP-driven drug efflux, FLC-resistant Candida albicans pumped out Rh6G. However, the addition of tested CCBs did not inhibit the efflux of Rh6G (Fig 4B and  S3B Fig). Taken together, the results of Rhodamine 6G assay and RT-PCR method demonstrated that the synergistic antifungal effects of FLC combined with tested CCBs was irrelevant to reverse the mechanism of drug efflux. Supporting Information