Antifungal activity of tamoxifen: in vitro and in vivo activities and mechanistic characterization

Abstract

Tamoxifen (TAM), an estrogen receptor antagonist used primarily to treat breast cancer, has well-recognized antifungal properties, but the activity of TAM has not been fully characterized using standardized (i.e., CLSI) in vitro susceptibility testing, nor has it been demonstrated in an in vivo model of fungal infection. In addition, its mechanism of action remains to be clearly defined at the molecular level. Here, we report that TAM displays in vitro activity (MIC, 8 to 64 microg/ml) against pathogenic yeasts (Candida albicans, other Candida spp., and Cryptococcus neoformans). In vivo, 200 mg/kg of body weight per day TAM reduced kidney fungal burden (-1.5 log(10) CFU per g tissue; P = 0.008) in a murine model of disseminated candidiasis. TAM is a known inhibitor of mammalian calmodulin, and TAM-treated yeast show phenotypes consistent with decreased calmodulin function, including lysis, decreased new bud formation, disrupted actin polarization, and decreased germ tube formation. The overexpression of calmodulin suppresses TAM toxicity, hypofunctional calmodulin mutants are hypersensitive to TAM, and TAM interferes with the interaction between Myo2p and calmodulin, suggesting that TAM targets calmodulin as part of its mechanism of action. Taken together, these experiments indicate that the further study of compounds related to TAM as antifungal agents is warranted.

Figures

FIG. 1.

Tamoxifen and clomiphene disrupt yeast cell integrity. (A) Chemical structure of TAM and CLM. (B) TAM and CLM causes the release of AK into culture medium in C. albicans (SC5314) and C. neoformans (NYS-2). Yeast cells were treated with TAM (SC5314, 32 μg/ml; NYS-2, 64 μg/ml) or CLM (SC5314, 32 μg/ml; NYS-2, 64 μg/ml) for 5 h and processed for extracellular AK activity as described in Materials and Methods. Mean relative light units (R.L.U.) and standard deviations (error bars) from three independent experiments performed in triplicate are shown. (C) TAM and CLM treatment of SC5314 and NYS-2 cells causes cell death by propidium iodide staining. SC5314 and NYS-2 cells were treated with TAM and CLM as described for panel B and processed for propidium iodide staining as previously described (24). Three independent experiments (100 cells per experiment) were conducted for each treatment. The percentage of cells stained by propidium iodide was calculated for each experiment. Bars indicate the means, and error bars indicate standard deviations.

FIG. 2.

TAM treatment decreases kidney fungal burden on day 2 postinfection in a murine model of disseminated candidiasis. Mice were treated with TAM (200 mg/kg; n = 5) or vehicle (peanut oil; n = 5) by oral gavage for 7 days, injected with C. albicans SC5314 (1.5 × 104 CFU/g) by the lateral tail vein, and treated for 2 days before sacrifice. The kidney fungal burden was determined as described in Materials and Methods and expressed as log10 CFU per gram of tissue. The points of the graph represent individual animals in each group, and the horizontal line indicates the means for the group as a whole. Differences between groups were analyzed by Mann-Whitney U test (*, P = 0.008).

FIG. 3.

Calmodulin modulates TAM toxicity in S. cerevisiae. (A and B) A 10-fold dilution series of BY4741 cells transformed with a multicopy plasmid (2μ) expressing PKC1 (A), CMD1 (B), or empty vector were spotted on YPD plates containing either 1% DMSO or TAM at the indicated concentrations, incubated at 30°C for 3 days, and photographed. (C) Functional classes of the calmodulin separation of function mutants (31). (D) The indicated calmodulin mutants and wild-type cells (WT; S288c genetic background) were spotted on TAM containing plates as described for panels A and B. For each phenotype, independent isolates of each strain were tested in duplicate, and a representative experiment is shown.

FIG. 4.

TAM affects calcium dependent processes and polarized growth. (A) Extracellular calcium, but not sorbitol, suppresses TAM toxicity. Cultures (25 ml; 0.1 OD600 unit) of S. cerevisiae (BY4741) in YPD supplemented with either 30 mM CaCl2 or 1 M sorbitol (SYPD) were treated with TAM (8 μg/ml) or 1% DMSO solvent and incubated for 5 h at 30°C, and growth was assessed according to the OD600. Means and standard deviations (error bars) from three separate experiments are shown. (B) TAM inhibits bud formation in S. cerevisiae. Logarithmic-phase BY4741 cells in YPD (0.1 OD600 unit) were treated with the indicated concentrations of TAM, incubated for 5 h at 30°C, and fixed with formalin, and the percentage of budded cells was determined by light microscopy. Mean percentages of budded cells from three separate experiments (100 cells/experiment) are presented. Error bars indicate standard deviations. (C) TAM causes actin depolarization in S. cerevisiae. BY4741 cells were treated with the indicated concentrations of TAM or 1% DMSO, fixed, and stained with Alexa Fluor 488-phalloidin to visualize actin. (D) TAM inhibits germ tube (GT) formation. Germ tubes were induced in C. albicans (SC5314) cells with and without TAM (2 h of incubation). The percentage of germ tubes and yeast was determined by light microscopy. The means of three independent experiments of 100 cells are shown, with error bars indicating standard deviations (*, P < 0.005 by Student's t test). Propidium iodide staining indicated that >95% of cells were viable at the time of germ tube assessment.

FIG. 5.

TAM interferes with the in vitro binding of Myo2p to calmodulin and the cellular localization of Myo2p. (A) Cell lysates of S. cerevisiae BY4741 containing Myo2-GFP were immunoprecipitated (IP) with calmodulin-Sepharose beads in the presence of 1% DMSO carrier, TAM (50 μg/ml), or the bona fide calmodulin inhibitor prochlorperazine (PRO; 50 μg/ml). Immunoprecipitates were eluted from beads with SDS-PAGE loading buffer and analyzed by Western blotting (WB) with anti-GFP antibodies. (B) Semiquantitative analysis of the effect of TAM and PRO on Myo2-GFP immunoprecipitation by calmodulin-Sepharose. The amount of Myo2-GFP precipitated by calmodulin-Sepharose was estimated by the densitometry of the bands visualized by Western blotting, of which the blot in panel A is a representative example. The amount precipitated in the presence of 1% DMSO was set at 100%. Bars represent the means of three independent experiments, and error bars indicate standard errors of the means. (C) S. cerevisiae BY4741 cells containing Myo2-GFP were treated with 1% DMSO or 12 μg/ml TAM for 4 h and analyzed by fluorescence microscopy.