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

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030462#s4" target="_blank">Methods</a> 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.</p

3-BrPA effects on HK activity from breast cell lines.

<p>3-BrPA effects on HK activity from breast cell lines.</p

Cellular viability decreases in breast cancer cells treated with clotrimazole.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030462#s4" target="_blank">Methods</a>. * 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.</p

Glycolytic enzymes activity and G6PDH activity are inhibited by clotrimazole.

<p>Cell lines were grown to confluence in the indicated media as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030462#s4" target="_blank">Methods</a>. Cell lysates were used to evaluate HK, PFK-1, PK and G6PDH activities (panel A, B, C and D respectively) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030462#s4" target="_blank">Methods</a>. 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.</p

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

<p>(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).</p