| Invasive fungal infections have become frequent in severely immunocompromised individuals, such as transplant, cancer chemotherapy,and HIV-infected patients. Candida spp. especially C. albicans account for a large proportion in clinical isolated strains from fungal infection patients. Azoles, especially fluconazole, have frequently been used in clinical practice due to their great efficacy in the prevention and treatment of Candida albicans infections and their reduced toxicity, but their use results in the emergence of drug resistance, which brings great challenges to clinical therapy.The development of brand-new antifungal drugs is time consuming, costly and requires excellent research team. Therefore, drug combination becomes one of the great strategies against fungal resistance. Recent years, many researchers found that some non-antifungal compounds exhibit no antifungal activities by themselves, but enhance the sensitivity of fluconazole to resistant fungal strains. These findings seem to provide strategy to cope with fungal resistance and provide clues to antifungal drugs development. Azoles is extensively used antifungal drugs in clinical. We can screen non-antifungal compounds which could enhance the sensitivity of fluconazole through drugs combination. There will be great significance in increasing the clinical safety and quality of azole antifungal drugs.Calcium homeostasis is the growth foundation of C. albicans. Calcium involves in cell basal metabolism as well as maintains the physiological function of some cell organoids such as endoplasmic reticulum, Golgi complex. Moreover, as a universal message vehicle, calcium also participates various metabolic activity of C. albicans such as morphogenesis, environmental responses and pathogenicity. Calcium channel blockers are clinical safe and common used in drugs in cardiovascular disease.Recently, many reports demonstrated that some calcium channel blockers exhibit stricken antifungal activity against C. albicans by themselves or by combining with fluconazole. Nifedipine and verapamil could inhibit the formation of C. albicans germ tube when used alone. Nifedipine and Nimodipine could enhance the antifungal activity of fluconazole when used in combined. Besides, verapamil in combination with fluconazole could resist C. albicans biofilms. All these researches demonstrated that there seem to be a broad applying prospect of calcium channel blockers in antifungal filed. If we make a fully exploration in the antifungal mechanisms of calcium channel blockers further, it may provide significant clues in seeking for new antifungal approaches. In addition, we found that although there have been many antifungal researches on calcium channel blockers, there are few studies on the combined antifungal effects of amlodipine, nifedipine benidipine and flunarizine in combination with fluconazole. Thus, we select these calcium channel blockers as non-antifungal agents to combine with fluconazole. In this study, we evaluate the antifungal effects of thesed drugs combination against C. albicans, and found significant synergism. We further study the mechanisms of drug combination. If we make a fully exploration on the combined mechanisms, it will not only expand the clinical application of calcium channel blockers, but also will make a great sense in the field of antifungal research.1. The combined antifungal effects of fluconazole and calcium channel blockersPrepare series of concentrations of fluconazole and calcium channel blockers (amlodipine, nifedipine, benidipine, and flunarizine) according to the checkerboard method described in CLSI M27-A3. The concentration range of fluconazole is 0.125～64μg/ml, and the concentration range of amlodipine, nifedipine, benidipine, and flunarizine is 0.5～32μg/ml respectively. Both FLC-resistant strains and susceptive strains were tested. The final results demonstrated that these four calcium channel blockers showed no antifungal effect when used alone. While the combination of FLC with these four CCBs showed synergistic effects on resistant strains.A time-killing method was also performed to evaluate the dynamic antifungal fungal activity of the studied calcium channel blockers on FLC resistant C. albicans (CA10). At determined time point (0、6、12、24、48h), XTT assay were used to detect the the number of live cells. The results reflected that the combinations of FLC with amloipine, nifedipine, benidipine and flunarizine had a better antifungal effect than FLC used alone, and the combination of fluconazole with amlodipine showed the most synergistic antifungal effects.2. The relationship of calcium concentration with drugs combinationFluo-3/AM was used to reflect the concentration of intracellular calcium as it could bind to calcium easily and flexible and importantly, it is easy to be detected. CA10 was incubated in YPD liquid medium until log phase growth, then washed by D-Hanks buffer three times and re-suspended with a concentration of 107 CFU/ml. Cells were loaded with Fluo-3/AM at 37℃ and 120rpm for 50 min to a final concentration of 6μM. Then, flow cytometry was introduced to test the effect of drugs combinations on intracellular calcium of CA10. Results showed that the sensitization of amlodipine to fluconazole may due to the release of calcium from calcium store, and extremely raised intracellular calcium.3. Influence on calcium signaling pathway related genes expression of drugs combinationThe expression of calcium signaling pathway related genes CCH1, MID1, CNA1, CNB1 and YVC1 of resistant C. albicans were measured by reverse transcription PCR and real-time fluorescence quantitative PCR, and the result of real-time fluorescence quantitative PCR was analysed by 2-ΔΔCt method. We found that the expression of calcium channel genes in cell membrane of C. albicans (CCH1, MID1) were similar between drugs alone groups and combined group compared with control group. For CNA1, CNB1 and YVC1 genes, there is no significant differences of control group and drugs alone group were observed. However, the expressions were significantly reduced by combination of fluconazole and amlodipine. It can be speculated that the synergism is associated with blocking the expression of calcineurin genes (CNA1, CNB1) and calcium channel gene in cell vacuole (YVC1). Then, the drugs combination inhibits the activity of calcineurin and calcium channel in cell vacuole, so as to disrupt the calcium homeostasis.4. Influence on resistant genes expression and proteins of drugs combiantionThe expression of resistance genes CDR1, CDR2, and MDR1 of C. albicans were determined by general PCR and real-time fluorescence quantitative PCR, and tehn the results of real-time fluorescence quantitative PCR were analysed by 2-ΔΔCt method. We found that the expression of resistance genes CDR1, CDR2, and MDR1 of C. albicans were similar. And, there is no significant differences of control group and drugs alone group, as well as drugs alone group and drugs combined group were observed.The effects of calcium channel blockers on fluconazole efflux were evaluated using rhodamine 6G (Rh6G) as substrate. The decrease of drug accumulation in cells attributed to low uptake and high efflux was one mechanism of fungal resistance. Our results showed that the uptake (within 50 min) of Rh6G was similar in resistant strains (CA10) when they were incubated in glucose free conditions. When glucose was added (after 50 min), intracellular concentrations of Rh6G were sharply decreased. Addition of calcium channel blockers did not increase the Rh6G uptake and decrease the efflux in flluconazole resistant strains. |
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