Pre-exposure of Candida species to cytarabine and daunorubicin does not affect their in vitro antifungal susceptibility and virulence in flies

Keywords: :

• cytarabine
• daunorubicin
• Candida
• susceptibility
• virulence
• Drosophila

We found that the in vitro susceptibility of C. albicans and C. tropicalis (three isolates each) to antifungals (amphotericin B, caspofungin, and fluconazole) and their virulence in a Toll-deficient fly model of invasive candidiasis were not altered by the previous in vitro exposure to cytarabine, daunorubicin or their combination.

The year of 2013 marks the 40th anniversary for the highly successful 7 + 3 remission-induction regimen (7 d of continuous cytarabine [Ara-C] and the first 3 d of intravenous daunorubicin [DNR]) in acute myeloid leukemia (AML).¹ However, infections, including candidiasis, are well known toxicities of that regimen.² The treatment of candidiasis in cancer and/or transplant patients is difficult, and frequently is a failure. Although Candida resistance, an emerging problem in the AML population,^3,4 has been attributed to antifungal selection pressure,⁵ an intriguing hypothesis is that antineoplastic themselves could contribute to resistance-encoding mutations, through their DNA damaging action.^6-9 Doxorubicin, for example, has been reported to induce efflux pump gene expression in Candida albicans,⁸ and the antineoplastic methotrexate is likely to contribute to reduce amphotericin B activity by increasing the yeast catalase activity.⁷ In addition, such subtle mutagenic effects could affect fitness and virulence of the Candida species.

To that end, we tested whether pre-exposure of C. albicans and C. tropicalis clinical strains (three strains each) to Ara-C, DNR or their combination, affects: (1) subsequent in vitro susceptibility to antifungals and (2) their virulence in a Toll-deficient fly model of invasive candidiasis. The Candida species used in our study were isolated from cancer patients with invasive candidiasis at the University of Texas MD Anderson Cancer Center. The strains were grown overnight in the dark at 30°C on yeast nitrogen base (YNB) liquid medium (control), YNB and Ara-C (cytosine β-d-arabinofuranoside hydrochloride, Sigma; 0.5 μg/ml), YNB and DNR (daunorubicin hydrochloride, Sigma; 100 μg/ml), and YNB with Ara-C (0.5 μg/ml) plus DNR (100 μg/ml). The overnight cultures were inoculated onto fresh YNB with or without the drugs at the above concentrations, which were based on pharmacokinetic studies and simulate the therapeutic plasma concentrations of these drugs.^7,9 Yeast cells were collected by centrifugation, washed three times in sterile saline and counted with a hemacytometer.

The susceptibility of the C. albicans and C. tropicalis strains to amphotericin B, caspofungin and fluconazole was evaluated according to the Clinical and Laboratory Standards Institute-approved document M27-A23 protocol.¹⁰ We also assessed differences in growth curves in the presence or not of Ara-C, DNR and Ara-C plus DNR for Candida blastospores growing in liquid medium for 24 h with the use of a plate reader (PowerWave HT; BioTek) at 30°C.¹¹ For the virulence experiments, we used female Oregon^R Toll-deficient flies and followed standard procedures for feeding, housing and manipulation of flies.¹² The flies were exposed to a 12-h light/dark cycle. The injection method used 5 × 10⁵ Candida yeast cells/mL (~1 × 10² yeast cells per fly) accordingly to previously described procedures.¹² Survival curves were plotted using Kaplan-Meier analysis and differences in survival rates were analyzed using the log-rank test in the GraphPad Prism software (version 5.0; GraphPad Software, Inc.). A p value of ≤ 0.05 was considered statistically significant. All experiments were performed in triplicate on different days.

The growth rates of the C. albicans and C. tropicalis strains grown in the presence of Ara-C, DNR, and their combination were similar (Fig. 1A and B). Likewise, the in vitro susceptibility to amphotericin B, caspofungin, and fluconazole of the strains pre-grown in the presence of Ara-C, DNR and both drugs in combination was not different to control (Fig. 1C). Finally, the infection of Toll-deficient flies resulted in similar mortality rates among the antineoplastic pre-exposed C. albicans and C. tropicalis compared with control (p = 0.58 by the log-rank test; Fig. 2). In this preliminary study, we found that the pre-exposure of C. albicans and C. tropicalis strains to Ara-C, DNR, or their combination had no apparent impact on their antifungal susceptibility or fitness. Further studies should focus on the effect of combined antineoplastic and antifungal/antibacterials to different Candida phenotypic traits, ideally in the context of complex microbial communities such is the gut microbiome.

Figure 1. Growth rates of one representative isolate of C. albicans (A) and C. tropicalis (B) previously grown in liquid medium in the presence or not of the antineoplastic drugs cytarabine (Ara-C) and daunorubicin (DNR). Drugs were washed 3× in PBS and 1 × 10⁴ yeast cells were resuspended in YNB broth. Optical density curves were measured in triplicate in a plate reader. (C) Minimum inhibitory concentrations (MIC) of the C. albicans and C. tropicalis strains (three strains each) pre-exposure or not to the combination of Ara-C and DNR.

Figure 2. Survival rates of Toll-deficient flies infected with one representative isolate of C. albicans (A) and C. tropicalis (B) previously grown in liquid medium in the presence or not of the antineoplastic drugs cytarabine (Ara-C) and daunorubicin (DNR). Data shown are the means of three independent experiments (n = 23 flies/group).

Acknowledgments

D.P.K. acknowledges the MD Anderson Cancer Center core grant (CA 16672) and The Frances King Black Endowment. We thank Nathaniel D. Albert for invaluable technical support. This study was supported in part by the National Council of Scientific and Technological Development (CNPq, Brazil).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.