Clotrimazole preferentially inhibits human breast cancer cell proliferation, viability and glycolysis

Abstract

Background: Clotrimazole is an azole derivative with promising anti-cancer effects. This drug interferes with the activity of glycolytic enzymes altering their cellular distribution and inhibiting their activities. The aim of the present study was to analyze the effects of clotrimazole on the growth pattern of breast cancer cells correlating with their metabolic profiles.

Methodology/principal findings: Three cell lines derived from human breast tissue (MCF10A, MCF-7 and MDA-MB-231) that present increasingly aggressive profiles were used. Clotrimazole induces a dose-dependent decrease in glucose uptake in all three cell lines, with K(i) values of 114.3±11.7, 77.1±7.8 and 37.8±4.2 µM for MCF10A, MCF-7 and MDA-MB-231, respectively. Furthermore, the drug also decreases intracellular ATP content and inhibits the major glycolytic enzymes, hexokinase, phosphofructokinase-1 and pyruvate kinase, especially in the highly metastatic cell line, MDA-MB-231. In this last cell lineage, clotrimazole attenuates the robust migratory response, an effect that is progressively attenuated in MCF-7 and MCF10A, respectively. Moreover, clotrimazole reduces the viability of breast cancer cells, which is more pronounced on MDA-MB-231.

Conclusions/significance: Clotrimazole presents deleterious effects on two human breast cancer cell lines metabolism, growth and migration, where the most aggressive cell line is more affected by the drug. Moreover, clotrimazole presents little or no effect on a non-tumor human breast cell line. These results suggest, at least for these three cell lines studied, that the more aggressive the cell is the more effective clotrimazole is.

Figure 1. Clotrimazole inhibits the migration of breast cell lines.

(A) The migration of MCF10A, MCF-7 and MDA-MB-231 cells was assessed by transwell assays after 24 h incubation in the absence or presence of 50 µM clotrimazole; (B) Cell migration rate was calculated from scratch assays. The images were analyzed and data were expressed in µm×h⁻¹. (C) Scratch migration assay for control and 50 µM clotrimazole-treated cells. The filling of scratchs by migrated cells at time 0, 6 and 12 h was imaged. MCF-7 and MDA-MB-231 cells have a more motile appearance and migrate faster than MCF10A. Bars are mean ± SEM of three independent experiments. * P<0.05 compared with control condition in the absence of the drug; ^# P<0.05 compared with MCF10A cells.

Figure 2. Clotrimazole decreases cell proliferation of MCF10A, MCF-7 and MDA-MB-231 cells.

(A) Basal cell proliferation of human breast cell lines was analyzed using the BrdU incorporation assay. The graph shows the europium-based TRF cell proliferation assay. * P<0.05, in comparison with MCF10A cell line. (B) Effects of clotrimazole on cell proliferation after 24 h treatment using BrdU incorporation assay. Values are mean ± standard error (SE) of four different experiments. ^# P<0.05, in comparison with the control; * P<0.05, comparing 50, 75 and 100 µM clotrimazole with the control in the absence of the drug for both cell lines (MCF-7 and MDA-MB-231).

Figure 3. Effects of clotrimazole on glucose uptake, mitochondrial reduction activity and cellular ATP content in breast cell lines.

(A) Comparison of glucose uptake in MCF10A, MCF-7 and MDA-MB-231 cells. Glucose uptake was determined after 15, 30 and 45 min incubation through cells incubation with 5 mM 6-NBDG, a fluorescent glucose analogue. The results obtained are plotted as percentage of control in a function of clotrimazole concentration. (B) Glucose uptake experimental data was fitted in an equation as described in Methods and the K_i values were plotted. Bars are mean ± SEM of three independent experiments. * P<0.05, comparing with MCF10A cells. (C) Percent of mitochondrial reduction activity, evaluated by MTT assay. All values were normalized to that of control condition in the absence of the drug. (D) Intracellular ATP content measured by relative firefly luciferase activity (PerkinElmer ATPLite Kit). Error bars represent standard errors from five independent experiments. * P<0.05 compared to control for MCF10A cells. ^# P<0.05, compared to control for MCF-7 and MDA-MB-231 cell lines.

Figure 4. Glycolytic enzymes activity and G6PDH activity are inhibited by clotrimazole.

Cell lines were grown to confluence in the indicated media as described in Methods. Cell lysates were used to evaluate HK, PFK-1, PK and G6PDH activities (panel A, B, C and D respectively) as described in Methods. Plotted values are mean ± standard errors of five independent experiments. (A) ^# P<0.05 compared to control in the absence of clotrimazole; * P<0.05, compared to control in the absence of clotrimazole. (B) ^# P<0.05 compared to control in the absence of clotrimazole; * P<0.05, compared to control and to MCF10A in the presence of clotrimazole. (C) The differences among the results obtained with the distinct clotrimazole concentrations tested are statistically significant. * indicate differences between MCF10A and tumoral breast cell lines. (D) ^# P<0.05 compared to control in the absence of clotrimazole; * P<0.05, compared to control and to MCF10A in the presence of clotrimazole.

Figure 5. Cellular viability decreases in breast cancer cells treated with clotrimazole.

Data are presented as mean ± SE of at least five experiments. Panel A: lactate dehydrogenase (LDH) leaked to culture medium by clotrimazole-induced cellular lyse was evaluated as described in Methods. * P<0.05 compared to MCF10A cells in the same clotrimazole concentration. Panel B: the percentages of cells that exclude trypan blue dye were evaluated counting the total cells and those that were intracellularly stained with the dye. Cells were counted using a TC10 Automated Cell Counter (Bio-Rad Laboratories, CA, USA). * P<0.05 compared to MCF10A in the same clotrimazole concentration. # P<0.05 compared to MCF-7 in the same clotrimazole concentration.