Clotrimazole Preferentially Inhibits Human Breast Cancer Cell Proliferation, Viability and Glycolysis

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

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

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

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

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

Evidenciação ambiental dos resíduos sólidos de companhias abertas no Brasil potencialmente poluidoras

Esta pesquisa tem como motivação a obrigatoriedade das empresas de apresentarem informações ambientais sobre os resíduos sólidos no ano de 2010. Parte da seguinte pergunta de pesquisa: como estão sendo evidenciadas as informações ambientais relativas aos resíduos sólidos das companhias abertas no Brasil potencialmente poluidoras no ano de 2010? Com vista a uma resolução plausível, tem-se como objetivo geral verificar a evidenciação ambiental quanto aos resíduos sólidos das companhias abertas no Brasil potencialmente poluidoras no ano de 2010. Para atingir esse objetivo, são estipulados os seguintes objetivos específicos: (i) propor um modelo para identificar itens de evidenciação ambiental dos resíduos sólidos; (ii) avaliar o nível da evidenciação ambiental e correlacioná-lo com variáveis financeiras. Para a análise dos dados é construído um modelo de análise da evidenciação ambiental dos resíduos sólidos (Waste-Ede), que compreende a junção das ideias contidas no modelo Environmental Disclosure Evaluation, na política nacional de resíduos sólidos (Lei Federal n. 12.305/2010) e nas diretrizes da Global Reporting Initiative (2006). Os resultados demonstram que a maioria das companhias não publicou o relatório de sustentabilidade. A amostra final não probabilística contemplou 86 companhias. Os resultados limitados a essa amostra revelam que as companhias estão evidenciando informações de resíduos sólidos no nível mercado (conforme modelo), com média de 12,44 pontos, e que há correlação significante em 95% do índice Waste-Ede com as variáveis financeiras: investimentos ambientais, ativo total, patrimônio líquido e receita bruta. Conclui-se que, no ano de 2010, as companhias abertas no Brasil potencialmente poluidoras que fizeram parte do estudo respeitaram, provavelmente, as pressões de seus investidores, uma vez que os reguladores não apresentaram poder de enforcement