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

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

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

Discrete Fourier Transform-Based Multivariate Image Analysis: Application to Modeling of Aromatase Inhibitory Activity

We recently generalized the formerly alignment-dependent multivariate image analysis applied to quantitative structure–activity relationships (MIA-QSAR) method through the application of the discrete Fourier transform (DFT), allowing for its application to noncongruent and structurally diverse chemical compound data sets. Here we report the first practical application of this method in the screening of molecular entities of therapeutic interest, with human aromatase inhibitory activity as the case study. We developed an ensemble classification model based on the two-dimensional (2D) DFT MIA-QSAR descriptors, with which we screened the NCI Diversity Set V (1593 compounds) and obtained 34 chemical compounds with possible aromatase inhibitory activity. These compounds were docked into the aromatase active site, and the 10 most promising compounds were selected for in vitro experimental validation. Of these compounds, 7419 (nonsteroidal) and 89 201 (steroidal) demonstrated satisfactory antiproliferative and aromatase inhibitory activities. The obtained results suggest that the 2D-DFT MIA-QSAR method may be useful in ligand-based virtual screening of new molecular entities of therapeutic utility

Additional file 1: Figure S1. of Unique PFK regulatory property from some mosquito vectors of disease, and from Drosophila melanogaster

Alignment of the amino acid sequences of several insects and mammalian PFKs. The putative residues assigned to the binding of effectors/substrates are indicated. α - F6P; β - ATP substrate; χ - ATP inhibitor; δ - citrate; ε - F2, 6BP ϕ - AMP. The sequences shown are from: Oryctolagus cuniculus (ORYCU), Mus musculus (MUSMU), Homo sapiens (HOMSA), Aedes aegypti, (AEDAE), Culex quinquefasciatus (CULPI), Anopheles gambiae, ANOGA), Drosophila melanogaser (DROME), Drosophila virilis (DROVI), Drosophila pseudoobscura (DROPE), Acyrthosiphon pisum, (ACYPI), Tribolium castaneum, (TRICA), Pediculus humanus corporis (PEDHU), Bombus terretris (BOMTE), Rhodnius prolixus (RPRC). (TIF 9895 kb

Transmission electron microscopy of mitochondria of MCF-7 cells treated with nanomicellar CTZ.

<p>The experimental procedures are described in Materials and Methods. Panel A: non-treated control cells. Panel B: MCF-7 cells treated with 50 μM _nCTZ. Panel C: MCF-7 cells treated with 100 μM _nCTZ. Panel D: MCF-7 cells treated with nanomicelles prepared in the absence of CTZ. Bar = 5 μm. Images are representative of a series of at least four experiments.</p

Effects of nanomicellar CTZ on glycolytic parameters.

<p>The activities of the glycolytic enzymes HK (panel A), PFK (panel B) and PK (panel C) and lactate production (panel D) were assessed as described in Materials and Methods in the absence and in the presence of 50 or 100 μM of DMSO-solubilized or a nanomicellar preparation of CTZ. In the absence of CTZ the appropriate amount of DMSO (1% vol/vol) or CTZ-free nanomicellar preparation was added as controls. The results for these controls were not different from the control with no addition. Values are the means ± standard error of the mean, for at least 4 independent experiments (n = 4). * P < 0.05 compared to control and # P < 0.05 compared to DMSO-solubilized CTZ (two-tailed ANOVA, Bonferroni’s port-hoc test).</p

Effects of nanomicellar CTZ on mitochondrial parameters and on intracellular ATP levels.

<p>SDH activity (panel A), mitochondrial transmembrane potential (panel B) and intracellular ATP content (panel C) were assessed as described in Materials and Methods in the absence and in the presence of 50 or 100 μM of DMSO-solubilized or a nanomicellar preparation of CTZ. In the absence of CTZ the appropriate amount of DMSO (1% vol/vol) or CTZ-free nanomicellar preparation was added as controls. The results for these controls were not different from the control with no addition. Malonate (12 mM) was used as a control for SDH inhibition. Values are the means ± standard error of the mean, for at least 4 independent experiments (n = 4). * P < 0.05 compared to control and # P < 0.05 compared to DMSO-solubilized CTZ (two-tailed ANOVA, Bonferroni’s port-hoc test).</p