Anticancer drugs as enhancers of fluconazole sensitivity in Candida albicans

Prescribing fluconazole for prophylaxis and treatment of Candida albicans infections in cancer patients is a common practice. The rational of using antiCandida drugs along with cancer drugs is debating, because of contradictory results showing either increase or decrease in antifungal sensitivity. In an effort to analyse this, effect of short term exposure to thirty anticancer agents on minimum inhibitory concentration (MIC) of fluconazole was studied in a micro plate based assay. Antitumor antibiotic, 5flurouracil was the most effective sensitizer of C. albicans, causing sixty-four fold increase in fluconazole susceptibility. Eight of the selected anticancer molecules had potential to lower fluconazole MIC by sixteen fold, so that it comes down to 0.062 μg/ml. Three of the cancer drugs caused eight fold increase in the antifungal sensitivity. Effective molecules belonged to six different classes, indicating that ability to sensitize C. albicans towards fluconazole was not confined to a specific group. Our in vitro study, for the first time reveals efficacy of the thirty anticancer drugs to act as sensitizers in C. albicans.


INTRODUCTION
Candida infections in cancer patients are associated with high morbidity and mortality rates as well as increased cost of treatment (Bensadoun et al., 2011;Davies et al., 2000;Lalla et al., 2010;Safdar and Armstrong, 2002).Antifungal drugs available for treatment of candidiasis are mainly confined to four classes of molecules that are polyenes, 5-fluoro-cytosine (5-FC), azoles and recently developed echinocandins (Raut et al., 2012).Fluconazole is a drug of choice for the treatment of C. albicans infections in cancer patients (Yu et al., 2006).In immunologically debilitated cancer patients, fluconazole need to be administered in high dosages ranging from 400 to 800 mg/day (Harnicar et al., 2009), which many times results in nephrotoxicity or infusion-related toxicity (Winston et al., 2000;Yu et al., 2006).Infections associated with drug resistant strains and biofilm forms have emerged as a major challenge to successful anti-Candida treatment (Campbell, 2012;Davies et al., 2000;Safdar et al., 2001;Shinde et al., 2012;Winston et al., 2000).In this context, better options of antifungal therapy in cancer patients need to be explored.Prescribing fluconazole for prophylaxis and treatment of Candida infections in cancer patients is a routine (Yu et al., 2006).Also, use of anti-Candida drugs along with anticancer agents is a common practice in clinics (Cornely et al., 2007;Winston et al., 2000).Interestingly, anticancer drugs alone were reported to possess antifungal properties.Cisplatin and 5-fluorouracil were shown to alter the morphology and growth of C. albicans (Kesavan et al., 2005a, b).Tamoxifen and its structural analog clomiphene were shown to possess good anti-Candida activities in vitro and in vivo in a mouse model (Dolan et al., 2009).However, combination of antifungal and antineoplastic drugs may not be always synergistic.Negative interactions among these drugs may result in emergence of antifungal drug resistance and failure of the treatment (Ghannoum et al., 1989(Ghannoum et al., , 1990)).For example, exposure to anticancer drugs was found to increase C. albicans tolerance to antifungal drugs like amphotericin B (O' Keeffe et al., 2003).Therefore, rational of using anti-Candida drugs like fluconazole or amphotericin B along with cancer drugs is a debating issue.A report by Bulatova and Darwish (2008) showed that 16 different drugs, including an anticancer agent (tamoxifen), sensitize C. albicans towards activity of fluconazole.Exposure to 27.5 µg/ml of tamoxifen was found to lower down fluconazole MIC from 5.5 to 0.01 µg/ml (Bulatova and Darwish, 2008).Recently, a comprehensive study by our group elucidated the antifungal properties of thirty commonly prescribed drugs belonging to twelve different classes of anticancer agents.It showed that many of the anticancer drugs inhibit in vitro growth of C. albicans, at concentrations which may exist in cancer patients undergoing chemotherapy (Routh et al., 2011).Effects of these drugs in combination with the most widely prescribed antifungal, fluconazole, are not well studied.Information on the effect of exposure to these drugs on fluconazole susceptibility of C. albicans may help to design the antifungal drug regimens in cancer patients infected with Candida.In this study we report alteration of fluconazole sensitivity of C. albicans exposed to selected antineoplastic drugs.

Culture and growth conditions
A standard strain of Candida albicans, ATCC 90028, was obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India, and maintained on yeast peptone dextrose (YPD) agar slants at 4°C.Activation of culture was done by inoculating a single colony from YPD agar plate (yeast extract 1%, peptone 2%, dextrose 2%, agar 2.5%) into 50 ml YPD broth in a 250 ml conical flask.The flasks were incubited at 30°C, at 100 rpm on an orbital shaking incubator for 24 h.Cells were harvested by centrifugation at 2000 × g and washed thrice with 10 mM Phosphate Buffered Saline (PBS), of pH 7.4.The cell density was determined by hemocytometer count and cells resuspended in PBS were used as inoculum for further experiments.

Anticancer and antifungal drugs
Thirty drugs from twelve different classes of anticancer agents were obtained from the local market.Details on their classification and manufacturers are mentioned in Table 1.Twelve classes of the drugs were varying in their mode of action and their cellular targets (Table 1).Fluconazole (Forcan) the standard antifungal drug was purchased from, Cipla Pharm.Ltd., India.

Anticancer drug exposure assay
Activated cells of C. albicans were exposed to various anticancer drugs in an in vitro assay as per standard methodology with modification (Bulatova and Darwish, 2008).Briefly, a concentration half the known minimum inhibitory concentration (MICs) of each anticancer drug for C. albicans was selected and this sub-inhibitory concentration was used to treat the cells.In the exposure assay, cells were incubated in ½ × MIC of each anticancer drug for 1 h, at 30°C with mild shaking and then washed with PBS to remove any traces of the drugs.Washed cells were used in growth assay to check the effect of exposure on the MIC of fluconazole.

Growth assay
Growth susceptibility study was carried out by the standard micro broth dilution methodology as per Clinical Laboratory Standards Institute (CLSI) guidelines (Routh et al., 2011).Briefly, various concentrations of the antifungal drug fluconazole were prepared in RPMI-1640 medium by double dilution in the 96 well plates.Cells obtained in the exposure assay were used as inoculums for growth assay.Each well contained 1×10 3 cells/ml in the final volume of 200 µl RPMI-1640 medium.Cells without any pre-exposure of anticancer drugs were used as inoculum for the control.Micro plates were incubated at 35°C for 48 h and absorbance was read at 620 nm using microplate reader (Multiskan EX, Thermo Electron Corp. USA).The lowest concentration of a drug which caused > 50 % reduction in the absorbance compared to that of control was considered as Minimum Inhibitory Concentration (MIC).MICs after fluconazole susceptibility in treatment group (that is with prior exposure to anticancer drugs and control group (without prior exposure) were compared.

Statistical analysis
Experiments were repeated three times and the values mentioned are mean with standard deviations.Values of the percentage growth inhibited by fluconazole after exposure to a drug and that of in the control (that is without exposure to the drug), were compared by using Student's 't' test.For each group representing individual drugs, a 'P' value < 0.05 was considered significant.

Paclitaxel and docetaxel were the most efficient sensitizers among anti-microtubule agents
Among the four antimicrotubule anticancer agents, paclitaxel was the most efficient to sensitize C. albicans, so that fluconazole susceptibility increased significantly (p < 0.05).Exposure to sub-MIC concentration of paclitaxel was found to lower MIC of fluconazole from 1 to 0.062 µg/ml.Docetaxel followed an effect similar to that of paclitaxel and decreased the fluconazole sensitivity by eight folds to obtain MIC at 0.125 µg/ml with (p < 0.05).Treatment with vinblastine resulted in lowering of fluconazole MIC by four fold, while vincristine exposure did not alter the sensitivity significantly (Table 2; Figure 1).

5-Flurouracil the most effective anticancer agent to modulate fluconazole susceptibility of C. albicans
Efficacy of anti-tumor antibiotics to potentiate susceptibility to fluconazole varied over a broad range.5-Flurouracil showed the highest that is sixty-four fold decreases in MIC of fluconazole, followed by doxorubicin and mitoxantrone with sixteen folds (p < 0.01) and daunorubicin with 8 fold increase in susceptibility to fluconazole.
Remaining of the antitumor molecules did not show significant effect on the MIC of fluconazole as indicated by p > 0.05 (Table 2; Figure 2).

Two of the alkalyting agents efficiently sensitized the C. albicans cells to fluconazole
Treatment of C. albicans cells to busulfan and cyclophosphamide resulted in 16 fold decrease in the fluconazole susceptibility with a significant p value of 0.01.Melphalan and carmustine exposure brought down the fluconazole MIC from 1 to 0.25 µg/ml (p < 0.05), while ifosamide treatment exerted no change (Table 2; Figure 3).

Methotrexate exhibited ability to sensitize C. albicans
Among the three anti-metabolites, methotrexate was the most potential one.Exposure of C. albicans cells to subinhibitory concentrations of methotrexate lowered the MIC of fluconazole by sixteen fold (p < 0.01).Two other anti-metabolites anticancer agents, gemcitabine and hydroxyurea were not very effective in this regard and no significant change in the fluconazole MIC could be observed (Table 2; Figure 4).

Exposure to platinum analogs showed their prominent effect
Prior exposure of C. albicans to three platinum analogs enhanced the susceptibility of C. albicans to fluconazole.Carboplatin exposure altered the fluconazole susceptibility, making it sixteen times more sensitive (p < 0.01).

Efficiency of other anticancer agents
Etoposide (an epipodophylotoxin anticancer drug), significantly (p < 0.01) increased the C. albicans susceptibility to fluconazole.MIC of fluconazole decreased from 1 to 0.62 µg/ml.Four of the drugs tamoxifen, aromatase inhibitor formestane, non-classic alkalyting anticancer agent, dacarbazine and leuprolide (LHRH agonist) altered the fluconazole sensitivity to make it four times efficient with a significant p < 0.05 value, while leucovorine had no effect.Interestingly, irinotecan which is an active topoisomarase I inhibitor exhibited an effect totally different from rest of the twenty nine drugs used in this study.Exposure to irinotecan increased fluconazole concentration required to inhibit C. albicans growth by four times compared to that of MIC for unexposed cells (Table 2; Figure 6).

DISCUSSION
In this study, effect of anticancer drug exposure on the fluconazole sensitivity of C. albicans was analysed.Preexposure for short time to many of the selected drugs sensitized C. albicans cells to antifungal activity of fluconazole.Out of the thirty anticancer drugs belonging to twelve different classes, antitumor antibiotic 5-flurouracil was the most effective sensitizer (p < 0.01) of fluconazole.
A known antifungal agent flucytosine, when crosses the fungal cell wall and membrane, gets converted by cytosine deaminase to 5-flurouracil.It is then incorporated into fungal RNA in place of uracil to block the protein synthesis (Polak and Scholer, 1975).The exact reason behind strong sensitization after exposure to this drug is not very clear.Eight anticancer molecules -paclitaxel, doxorubicin, mitoxantrone, cyclo-phosphamide, methotrexate, and carboplatin had potential to lower fluconazole MIC by sixteen fold; so that it comes down to 0.062 µg/ml.Whereas, three cancer drugs-docetaxel, daunorubicin and cisplatin caused eight fold increase in the antifungal susceptibility.This was found to be a significant (p < 0.05) decrease in fluconazole MIC.Effective molecules belonged to six different classes, indicating that activity was not specific to a single class.Fourteen of the selected drugs enhanced the fluconazole sensitivity of C. albicans by two to four folds, with significant p value of 0.05.Remaining of the anticancer agents exerted no activity, while irinotecan induced the negative effect to increase the MIC of fluconazole by four fold that is at 4 µg/ml.Being eukaryotes, yeast and mammalian cells exhibit similarities in basic cellular machinery and mechanisms.In many instances, Saccharomyces cerevisiae have provided invaluable insights into the actions of a diverse array of anticancer agents like topoisomerase inhibitors, microtubule aggregation and disaggregation inhibitors, immunosuppressants which block T-lymphocyte function, phosphatidylinositol kinase inhibitors, and steroid receptor antagonists (Cardenas et al., 1999;Hartwell et al., 1997;Shen et al., 1992).Challenges in the development of antifungal drugs and anticancer agents are more

Figure 1 .
Figure 1.Growth of C. albicans ATCC 90028 in presence of fluconazole, after a short term exposure to anticancer anti-microtubule agents.Note down the difference in percentage of growth compared to the unexposed cells.

Figure 3 .
Figure 3. Growth of C. albicans in fluconazole containing medium after exposure to selected alkalyting anticancer drugs.

Figure 5 .
Figure 5. Sensitization of C. albicans to fluconazole after prior exposure to different antineoplastic platinum analogs.

Figure 6 .
Figure 6.Exposure to selected anticancer drugs results in variation in fluconazole sensitivity of C. albicans ATCC 90028.

Table 1 .
List of thirty anticancer drugs used in this study.Drugs belonged to twelve different classes of anticancer agents.Details on manufacturers and their brand names are mentioned.

Table 2 .
Fluconazole MICs for growth of C. albicans ATCC 90028, after exposure to thirty drugs from twelve classes of anticancer agents.Change in fluconazole MICs of the cells exposed to various anticancer drugs was indicated in terms of fold increase in fluconazole sensitivity compared to that of unexposed cells.