Figures
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 Ki 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.
Citation: Furtado CM, Marcondes MC, Sola-Penna M, de Souza MLS, Zancan P (2012) Clotrimazole Preferentially Inhibits Human Breast Cancer Cell Proliferation, Viability and Glycolysis. PLoS ONE 7(2): e30462. https://doi.org/10.1371/journal.pone.0030462
Editor: Andrei L. Gartel, University of Illinois at Chicago, United States of America
Received: June 28, 2011; Accepted: December 20, 2011; Published: February 8, 2012
Copyright: © 2012 Furtado et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Among the physiological hallmarks of cancer, altered glucose metabolism is perhaps the most common. The Warburg effect has been observed in approximately 90% of human tumors and the biochemical origins of this phenomenon have been extensively studied [1]–[4]. Aerobic glycolysis may be required for new biomass formation [5]. In fact, proliferation of cancer cells is accompanied by activation of glycolysis [6], which occurs even at normal oxygen concentrations. Moreover, glycolysis may confer tumor cells with the ability to adapt to new microenvironments or cope with stress during tumor progression and metastasis [7].
Observations suggest that blocking glycolysis might diminish tumor progression and enhance the efficacy of chemo- and radiotherapy. However, inhibition of glycolytic enzymes is expected to have secondary effects on cell physiology, due to the additional functions of these proteins. Clotrimazole, an antifungal drug, has been successfully used to diminish the size and development of intracranial gliomas (C6 and 9L), prolonging survival in rodents [8]. Moreover, the drug also affects glycolytic enzymes decreasing hexokinase (HK) binding to the outer mitochondrial membrane [9] and detaching phosphofructokinase-1 (PFK-1) and aldolase from the cytoskeleton [10]–[12]. Indeed, clotrimazole is able to trigger apoptosis, which is directly correlated with its ability to displace HK from mitochondria [9] and PFK-1 and aldolase from the cytoskeleton [12]. The location of these glycolytic enzymes within the intracellular milieu is an important feature of glycolysis regulation [13] and thus, altering the intracellular distribution of these enzymes, clotrimazole is probably affecting the glycolytic flux.
The aim of this study was to analyze the effects of the clotrimazole on viability, growth, mobility and glycolytic profile of three human breast cell lines: MCF10A, MCF-7 and MDA-MB-231. The MCF10A human mammary epithelial cell is a normal strain, while MCF-7 and MDA-MB-231 cells are human breast-derived cell lines with tumorigenic and metastatic profiles, respectively. Here we present evidences that clotrimazole presents more pronounced effects on the tumorigenic and metastatic cells, while presenting minimal effects over the non-tumoral cell strain.
Results
Clotrimazole inhibits the migratory phenotype in breast cancer cells
To assess the effects of clotrimazole on the growth profiles of breast cell lines, cellular migration and proliferation were evaluated. The migration potential of MCF10A, MCF-7 and MDA-MB-231 cells was initially assessed using the Transwell assay (Fig. 1A). Our results confirm that there is a significant difference between the migration potential of these cells, which increases with the aggressiveness of the cell (MCF10A<MCF-7<MDA-MB-231). The presence of 50 µM clotrimazole inhibits migration of MCF-7 and MDA-MB-231 cells, but has no effect on MCF10A cells. Proportionally, the effects of clotrimazole were more pronounced on MDA-MB-231 migration (59±6% inhibition) than on MCF-7 (32±5% inhibition). Moreover, using the scratch assay, we compared cell mobility in the presence or absence of 50 µM clotrimazole (Fig. 1B and 1C). In accordance with the previous results, there is a significant difference between the migration rates of the cell lines assessed through this later technique. The evaluated migration increased approximately 1.5-fold and 2- to 3-fold for MCF-7 and MDA-MB-231 cells, respectively, when compared with control cells (black bars, Fig. 1B). Within 12 hours, the migration rate of the tumorigenic cells was significantly reduced by clotrimazole (grey bars, Fig. 1B). Clotrimazole inhibited mobility of MDA-MB-231 cells by 36±4%, of MCF-7 cells by 16±3%, but was unable to affect the MCF10A migration rate (Fig. 1B and 1C). These results suggest that clotrimazole prevents cell migration, which may contribute to the anti-metastatic potential of the drug.
(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.
To clarify that these results were not due to the effect of clotrimazole on the cell proliferation, we determined the incorporation of BrdU into DNA of synchronized proliferating breast cell lines in the absence or presence of different concentration of clotrimazole. Fig. 2A shows cell proliferation in the absence of clotrimazole after 24 h of BrdU incubation. MCF-7 and MDA-MB-231 cells display a 10- and 20-fold higher proliferation rate, respectively, when compared with the MCF10A cells. Treatment with clotrimazole (0–100 µM) inhibited the cell proliferation in all cell lines (Fig. 2B). Twenty-four hours after cell cycle reentry, which is initiated by adding fresh culture medium containing fetal serum, the percentage of proliferating cells was significantly decreased by clotrimazole. Surprisingly, 25 µM clotrimazole completely inhibited MCF10A proliferation (98±13% inhibition). However, this same concentration had no effect on tumoral cell lines. MCF-7 cells displayed a linear response to increasing clotrimazole concentrations, reaching 70% inhibition at 100 µM. Furthermore, clotrimazole also inhibited proliferation of MDA-MB-231 cells, but the maximum inhibition (50%) occurred at 50 µM and persisted even at the highest concentration tested (100 µM).
(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).
Clotrimazole diminishes glucose uptake and intracellular ATP
Proliferation in cancer cells is accompanied by activation of glycolysis [6], which occurs even in the presence of a normal oxygen concentration. This preferential use of glycolysis by cancer cells under aerobic conditions is known as the Warburg effect [14] and is characterized by high glycolytic rates in cancer cells with high rates of lactate production per glucose consumed. Our previous results have shown that breast cancer cells present a distinct glycolytic rate; the metastatic cell line (MDA-MB-231) showed lower consumption of glucose and higher production of lactate when compared to a non-tumorigenic cell line (MCF10A). Moreover, MCF-7 cells, which are tumorigenic and non-metastatic, presented an intermediate glycolytic rate [15]. In order to evaluate the metabolic profiles of these breast cell lines after treatment with clotrimazole, we incubated the cells in 96 well plates with different concentrations of clotrimazole (0–100 µM) for 24 h. Clotrimazole reduces glucose uptake by the cells in a dose-dependent fashion (Fig. 3A). The inhibition constant (Ki) for glucose uptake inhibition by clotrimazole was calculated and found to be 114.3±11.7, 77.1±7.8 and 37.8±4.2 µM for MCF10A, MCF-7 and MDA-MB-231 cells, respectively (Fig. 3B). These results clearly demonstrate that clotrimazole has more pronounced effects on cells with the highest glycolytic rates and Warburg effect.
(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 Ki 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.
We also analyzed the mitochondrial reduction activity and the cellular ATP levels after the treatment with clotrimazole. Fig. 3C shows a dose-dependent reduction of the mitochondrial activity in these cells, with the maximum inhibition being approximately 60% for MDA-MB-231 cells treated with 100 µM clotrimazole. At that concentration, both MCF10A and MCF-7 cell lines demonstrate 40% lower mitochondrial activity than corresponding cells treated with 50 µM clotrimazole. Once more, the drug was more effective in the metastatic cell line than the non-tumoral cells in the same condition (Fig. 3C). Furthermore, we evaluated the effects of clotrimazole on the cellular ATP levels. Clotrimazole reduces the cellular ATP of tumorigenic cells in a dose-dependent manner (Fig. 3D). Interestingly, MCF10A cells are completely resistant to the presence of the drug. In fact, we observed 26% and 35% higher ATP content after the treatment with 50 and 75 µM clotrimazole, respectively. However, MCF-7 and MDA-MB-231 cells display a progressive dose-dependent inhibition curve reaching 95% reduction of the ATP content with 75 µM clotrimazole.
Glycolytic enzymes from tumorigenic cells are selectively inhibited by clotrimazole
The results above show that clotrimazole alters the rates of glucose uptake, mitochondrial activity and generation of ATP in the cell lines, with more pronounced effects on the cancerous cell lines than non-tumorigenic cells. Additionally, these effects probably are not due to a specific action of clotrimazole on mitochondrial activity because all the cell lines, regardless of their phenotype, are not responsive to clotrimazole up to 100 µM (Fig. 3C). Therefore, our next objective was to investigate whether the antineoplasic effects of clotrimazole occurs by its direct action on glycolysis. Therefore, we evaluated the effects of the drug on the three enzymes that regulate the glycolytic flux: hexokinase (HK), phosphofructokinase-1 (PFK-1) and pyruvate kinase (PK).
We evaluated HK activity in all breast cells using a 24 h incubation time with different concentrations of clotrimazole. Fig. 4A shows HK dose-dependent inhibition curves for MCF10A, MCF-7 and MDA-MB-231 cells, where clotrimazole has a more pronounced effect in tumoral cell lines compared to the non-tumoral cells. HK activity in MCF10A cells was inhibited by only 46% by clotrimazole, whereas, at the same concentration (100 µM clotrimazole), HK activity in MCF-7 and MDA-MB-231 cells was inhibited by 90% and 96%, respectively. To align our results with the data in the literature, we used the 3-bromopyruvate (3-BrPA), a well-known inhibitor of energy metabolism, which has been proposed as a specific anticancer agent due to its high tumor selectivity [16], [17]. Surprisingly, 3-BrPA has different effects on these cell lines, showing a more pronounced inhibition of HK in the MCF10A cells (Table 1). This is an interesting result because, until now, both drugs (3-BrPA and clotrimazole) were thought to have the same mechanism of action in cancer cells: through the detachment of the mitochondria-associated HKII. Although this is an important mechanism to cell survival, our results strongly suggest an additional mechanism of cell death induced by the direct inhibition of HK by clotrimazole (Fig. 4A, inset).
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.
Meira and co-workers showed that clotrimazole inhibits phosphofructokinase-1 in MCF-7 cells [12]. To investigate this inhibition further, we analyzed the effects of clotrimazole on breast cell lines. Similar to what was observed for HK, clotrimazole inhibits PFK-1 activity in a dose-dependent manner (Fig. 4B). The effects of clotrimazole are dependent on the tumorigenic phenotype of the cell, as MCF10A cells are more resistant to the inhibition than MCF-7 and MDA-MB-231 cells. Once again, we observed 90–95% inhibition in tumorigenic cells and a less dramatic effect of clotrimazole on MCF10A cells.
We also evaluated the effects of clotrimazole on the last enzyme of glycolysis, pyruvate kinase (PK). Once more, clotrimazole progressively inhibited enzyme activity, with a substantial effect on MCF-7 and MDA-MB-231 cells (Fig. 4C). However, there was a more significant inhibition of PK in MCF10A cells when compared with the results for HK and PFK in this cell line. This result may be due to the effects of clotrimazole on previous steps of glycolysis or on glucose uptake in these cells.
After entering the cell, glucose is converted by HK to glucose 6-phosphate, which can be shunted to the pentose phosphate pathway (PPP). The increase in the PPP flux is directly associated with inhibition of the apoptosis. This is due to an increased generation of NADPH and, therefore, reduced glutathione, which also promotes the removal of reactive oxygen species (ROS) in cells [18]. To investigate the diversion of glucose 6-phosphate from glycolysis to PPP, we verified the activity of glucose 6-phosphate dehydrogenase (G6PDH), which is the enzyme responsible for the first irreversible reaction of the PPP. Similar to the results observed for the glycolytic enzymes, clotrimazole inhibits the PPP (Fig. 4D). The inhibitory effect of clotrimazole is more pronounced in MCF-7 and MDA-MB-231 cells than in MCF10A cells. The complete inhibition of PPP was obtained with 75 µM clotrimazole for tumorigenic cell lines and 100 µM clotrimazole for the MCF10A cells.
Metabolic effects of clotrimazole induce the loss of viability
Finally, we investigated the consequences of the metabolism inhibition induced by clotrimazole on cell viability. Published data have shown that clotrimazole reduces the viability of MCF-7 cells, CT-26 colon adenocarcinoma cells and Lewis lung carcinoma [11], [19], as well as B16 melanoma cells [10], [20]. However, as most of our results demonstrate a cellular phenotype-dependent effect of clotrimazole, our objective was to evaluate whether cell viability also responds in a similar way. The cell viability was evaluated by the activity of lactate dehydrogenase (LDH) leaked to the medium and permeability of the cells to trypan blue dye. As shown in Fig. 5A, clotrimazole is does not affect the leakage of LDH of MCF10A cells in any of the concentrations tested, even at 100 µM clotrimazole (data not shown), as compared to untreated controls. This result strongly suggests that the drug does not affect MCF-10A cells integrity. This picture changes when we analyze the effects of clotrimazole on LDH leakage from MCF-7 cells. Although 50 µM clotrimazole does not alter LDH leakage from these cells, 75 µM clotrimazole promotes a significant increase of the leaked enzyme suggesting the loss of cell integrity and viability. On the other hand, MDA-MB-231 cells viability is more susceptible to clotrimazole effects, since this cell line presents increased LDH leakage upon the treatment with 50 µM clotrimazole for 24 hours. Moreover, treatment of MDA-MB-231 cell with 75 µM clotrimazole increases 3.5 times the leaked LDH suggesting that the viability of these cells is severely compromised upon this treatment. These data are confirmed analyzing the exclusion of trypan blue dye by the different cell lines treated with clotrimazole (Fig. 5B). These results corroborate that MCF10A cells integrity is not affected up to 100 µM clotrimazole. Moreover, it is clear now that upon the treatment with clotrimazole the viability of MCF-7 and MDA-MB-231 cells decrease in a dose-responsive mode. Furthermore, MDA-MB-231 cells are more sensitive to 50 µM clotrimazole and higher concentrations, as compared to MCF-7 cells. Altogether, these data reinforce the preferential action of clotrimazole over the tumorigenic cell lines among those tested here, with an even more preferential effect over the most aggressive cell line.
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.
Discussion
The use of metabolic pathways as useful molecular targets in tumors prompted us to investigate the effects of drugs that affect cell metabolism, especially glycolysis. In this paper, we report the metabolic and anti-metastatic effects of the imidazole derivative clotrimazole in breast cell lines. Our data reveal that clotrimazole has more pronounced effects on tumorigenic cell lines that present the Warburg effect [15]. There are, at least, three major possible explanations to the preferential effects of clotrimazole on tumorigenic cell lines: (1) cancerous cells accumulate clotrimazole more readily and in an aggressiveness-dependent pattern, (2) glycolytic enzymes in cancerous cells are more sensitive to the effects of clotrimazole what could be due to an isoform expression specificity, and (3) cancerous cells rely on glycolysis more heavily and are therefore more sensitive, which is strongly supported by the Warburg effect observed in tumoral and not in non-tumoral cells. The first possibility is reasonably plausible, but we have not evaluated it and therefore we cannot discuss it. The other two possibilities seem to be supported by our results, although we cannot unambiguously discriminate between them.
In non-tumoral cells, at least 90% of ATP production is provided by mitochondrial oxidative phosphorylation, while in tumor cells, approximately 50% is dependent on cytoplasmic, aerobic glycolysis [21], [22]. Here, we demonstrate that clotrimazole inhibits glucose uptake (Fig. 3A) and mitochondrial activity (Fig. 3C) more efficiently in the tumorigenic cell lines. These effects result in alterations in ATP content that are also more pronounced in the tumorigenic cell lines. However, it seems that this later effect is more related to the inhibition of glycolysis, since mitochondrial activity is not reduced by 50 µM clotrimazole, while glucose consumption and ATP content are affected by even lower concentrations of the drug. These data highlight the importance of aerobic glycolysis for energy production in tumorigenic cells. The increased content of ATP measured in MCF-10A cell upon clotrimazole treatment might be the result of clotrimazole effects on this cell metabolism and proliferation. Comparing clotrimazole effects on cells proliferation and metabolism one can observe that the drug blocks the proliferation of MCF-10A cells (Fig. 2B), decreases its glucose consumption (Fig. 3A) and do not affect its mitochondrial activity (Fig. 3C). Similar results are observed for the tumorigenic cell lines but with lesser extent on proliferation and greater extent on metabolism. Since proliferation if the highest ATP-consuming cellular process, the resultant ATP content of MCF-10A cells increases upon clotrimazole treatment.
It is important to mention that the lack of clotrimazole effects on MCF10A cell line cannot be attributed to fact that this cell line is grown in a different medium (DMEM/F12 with 5% FHS, instead of DMEM with 10% FBS used for MCF-7 and MDA-MB-231). We have performed control experiments assaying the effects of clotrimazole on cell viability and enzymes activity of MCF-10A cells grown in DMEM/F12 but incubated with DMEM for 24 hours during the clotrimazole treatment and the results were not different from those presented here. We decided to perform all the experiments using DMEM/F12 for the MCF10A cell to assure that all the cells would be at optimal growing conditions (MCF-10A does not grow well in DMEM with 10% FBS).
Many human tumors display a high rate of aerobic glycolysis, de novo fatty acid synthesis, and nucleotide biosynthesis [23], [24]. Previous findings suggest that the increased glucose metabolism promotes lipogenesis and nucleotide biosynthesis, and enhances tumor cell growth and proliferation by providing essential synthetic and bioenergetic requirements [25]–[29]. Although the metabolic alterations might not be initiating events in oncogenesis, recent success in blocking carcinogenesis by targeting tumor metabolism suggests that aerobic glycolysis plays an important role in sustaining tumor growth [27]–[33].
After 24 hours of incubation, clotrimazole promote a phenotype-dependent inhibition of the key glycolytic enzymes: HK, PFK-1 and PK. The results always present the same pattern: MCF10A cells are less responsive to clotrimazole, whereas MCF-7 and MDA-MB-231 are sensitive to the increasing concentration of the drug (Fig. 4). More importantly, the metastatic cell line presents the highest clotrimazole-induced inhibition of glycolysis. These observations are accompanied by the dose-dependent inhibition of G6PDH, the enzyme responsible for the first irreversible reaction of the PPP (Fig. 4D). We have not observed differences between the effects of clotrimazole on enzymes activity of MCF-7 and MDA-MB-231 cell lines. The drug appears to be more effective inhibiting tumoral than non-tumoral enzymes, without distinction on the aggressiveness of the tumoral cell line. However, this can be an underestimated result, since here we measured the whole enzyme activity. These measurements were achieved under optimal substrate concentration and other reaction conditions, which can mask some fine regulatory properties of these enzymes, such as their association with cellular macromolecular structures, described as important to cancer increased glycolytic rates [34]. However, in order to assess these possibilities, a profound study of the effects of clotrimazole on glycolytic enzyme kinetics and cellular distribution should be performed, which was not the objective of the current work.
Although cancer cells are capable of carrying out oxidative phosphorylation, they undergo the glycolytic shift because aerobic glycolysis is advantageous for cell proliferation and tumorigenicity [35]. The metabolism of cancer cells, and indeed all proliferating cells, is adapted to facilitate the uptake and incorporation of nutrients into the biomass needed to produce a new cell. Supporting this idea are recent studies showing that (i) several signaling pathways implicated in cell proliferation also regulate metabolic pathways that incorporate nutrients into biomass; and that (ii) certain cancer-associated mutations enable cancer cells to acquire and metabolize nutrients in a manner conducive to proliferation rather than efficient ATP production [5]. Moreover, in metastases, the glycolytic phenotype persists due to increased production of glucose-derived acid, which potentiates invasiveness of cells into the extracellular matrix of the host tissue [36].
Our results here demonstrated that clotrimazole was able to decrease cancer cells proliferation and viability, with minimal effects on non-tumoral cells. Clotrimazole has been implicated in other mechanisms affecting tumor cells migration and invasion, such as the inhibition of the Ca2+-activated potassium channels [37], [38] and inhibition of the enzymes of the cytochrome P450 family [39]. This latter effect of clotrimazole is critically important and represents a reason for why clotrimazole is practically not used for systemic treatments due to its impact on liver CYP450 metabolism. However, clotrimazole have been used to design more selective and less toxic compounds, such as TRAM-34 [40], which, in addition to clotrimazole, was recently demonstrated to inhibit glioblastoma proliferation [41]. An interesting observation is that the effects of clotrimazole and its analogue TRAM-34, originally designed as a specific Ca2+-activated potassium channels blocker, on glioblastoma proliferation are not due to any effects on this ion channel. It is not difficult to imagine that the effects of these drugs might be related to inhibition of glioblastoma metabolism, such as described here. Interestingly, intracranial administration of clotrimazole has been used to prolong the survival of rats with glioma, but no mechanisms have been reported for this effect [8]. Clotrimazole has been shown to induce the nuclear condensation and f-actin depolymerization in MCF-7 cells [12] suggesting that clotrimazole-treated cells undergo apoptosis. In fact, decrease in glycolytic flux has been associated elsewhere to some apoptotic events [42], [43]. In addition, studies on cell metabolism during apoptosis in murine interleukin-3-dependent cell line revealed that the activation of glycolytic flux is related to cell survival [42]. However, we didn't yet explore the death mechanisms involved in the decrease of cellular viability in response to clotrimazole on tumor cells.
Our current results add to the growing evidence that aerobic glycolysis contributes to cancer cell proliferation and tumorigenicity. Although the systemic therapeutic use of clotrimazole is strongly limited due to its toxic effects, the present findings point to the development of novel treatment approaches that selectively targets glycolysis in tumor cells. These novel approaches may identify treatments that would be more selective to aggressive tumors with minimal effects over non-tumoral cells.
Materials and Methods
Cell lines and Materials
The human breast cancer cell line MCF-7 was obtained from the Cell Bank of Hospital Universitário Clementino Fraga Filho, UFRJ, Brazil. MDA-MB-231 and the non-tumorigenic epithelial MCF10A cell line were kindly supplied by Prof. Dr. Mitzi Brentani from University of São Paulo, USP, Brazil. The tumor cell lines were maintained in DMEM (Dulbecco's modified Eagle's medium; Invitrogen) supplemented with 10% (v/v) FBS (fetal bovine serum; Invitrogen) and L-glutamine. The MCF10A cell line was maintained in DMEM/F12 medium (Invitrogen) supplemented with 5% FHS (fetal horse serum; Gibco) plus 0.02 µg/ml EGF, 5 µg/ml insulin, 1.25 µg/ml hydrocortisone and 0.1 µg/ml cholera toxin (Sigma Chemical, St. Louis, MO, USA). Cells were grown at 37°C in 5% CO2 atmosphere.
Clotrimazole, 3-bromopyruvate, NAD+, NADH, ATP, hexokinase, glucose 6-phosphate dehydrogenase, fructose 6-phosphate, aldolase, lactate dehydrogenase, phosphoenolpyruvate, NADP+ and glucose 6-phosphate were obtained from Sigma. Other reagents were of the highest purity available.
Cell viability assay, glucose uptake, mitochondrial activity and ATP quantitative evaluation
To measure metabolic rates, cells were seeded in 96-well plates in the appropriate medium and grown to confluence. Then, the medium was removed, fresh medium was added, and the cells were returned to the incubator in the presence of different concentrations of clotrimazole (0–100 µM). After 24 h, the medium was removed and the amount of leaked lactate dehydrogenase (LDH) was evaluated, while mitochondrial activity and trypan blue dye exclusion was determined in the remaining cells using an MTT assay [44], [45] and counting the cells that exclude trypan blue [46], respectively.
Glucose uptake was determined by incubating cells with fresh medium containing 5 mM 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose (6-NBDG, Molecular Probes, Invitrogen, Life Technologies, Carisbad, CA, USA), a fluorescent glucose analogue. After 15, 30 and 45 min incubation, the medium was removed, the cells were washed with 6-NBDG-free medium, and 6-NBDG taken up by the cells was evaluated by fluorescence emission, according to the manufacturer's instructions. The glucose uptake rate was calculated by the linear regression of the increase in fluorescence incorporated by the cells, and these results are plotted as a percentage of control in a function of clotrimazole concentration. The parameters of the following equation were fitted to the experimental data in order to evaluate the effectiveness of clotrimazole effects by means of the concentration promoting half of the maximal glucose uptake inhibition. The equation used was:(1)where Gin is the percentage of glucose taken up by the cells at a given concentration of clotrimazole ([CTZ]) and Ki is the inhibitory constant, equivalent to the concentration of clotrimazole promoting 50% of the maximal inhibition.
LDH activity was determined spectrophotometrically by monitoring the conversion of NAD+ to NADH in a lactate-utilizing reaction [47]. Cellular ATP was assessed using a system based on firefly (Photinus pyralis) luciferase (PerkinElmer ATPLite) using a VICTOR3 multilabel reader (PerkinElmer).
Spectrophotometric assay for enzymes activity
Cells were seeded in 24-well plates and grown to confluence. Then, medium was removed and fresh medium was added, and cells were returned to the incubator in the presence of different concentrations of clotrimazole (0–100 µM) for 24 h. After this incubation, cells were removed from the plates by trypsinization and counted using a hemocytometer. Protein concentrations of cell lysates were measured [48], and the glycolytic enzyme activities were evaluated.
Phosphofructokinase-1 (PFK-1) activity was assayed using an enzyme-coupled method, where the oxidation of NADH was monitored spectrophotometrically [49]. The basic medium contain 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM fructose 6-phosphate, 1 mM ATP, 0.2 mM NADH, 2 U/ml aldolase, 4 U/ml triosephosphate isomerase and 2 U/ml α-glycerophosphate dehydrogenase.
Hexokinase (HK) activity was assessed in a basic medium containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM glucose, 1 mM ATP, 0.2 mM NAD+ and 1 U/ml glucose 6-phosphate dehydrogenase as described previously [50].
Pyruvate kinase (PK) activity was measured in a medium containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 120 mM KCl, 1 mM phosphoenolpyruvate, 1 mM ADP, 0.2 mM NADH and 20 U/ml lactate dehydrogenase as described previously [50].
Glucose-6-phosphate dehydrogenase (G6PDH) activity was assayed as previously described [51]. The basic medium contains 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 0.5 mM glucose 6-phosphate and 0.2 mM NADP+.
For all enzymatic analyses, NADH oxidation or NAD(P)+ reduction was determined by measuring the absorbance at 340 nm in a microplate reader (VICTOR 3, PerkinElmer). Reactions were initiated by the addition of an aliquot of cellular homogenate and followed online during the first-order kinetics period. Enzymes rates was determined calculating the first derivative of these curves. Blanks containing none of the coupled enzymes (or without glucose 6-phosphate in the case of G∧PDH) were performed to control for non-specific oxidation/reduction. Each curve was performed in quadruplicate.
Cell migration assays
Cell migration was determined by using the 24-well Millicell Hanging Cell Culture Insert (Millipore) chamber with 8 µm pore polyethylene terephthalate (PET) membranes. The chambers were rehydrated in serum-free medium. Complete medium with 10% FBS or FHS was used as a chemoattractant. Suspension of 5×104 cells/ml in serum-free medium was added to the inserts and incubated for 24 h in the presence or absence of 50 µM clotrimazole. Cells remaining on the upper membrane surface of the inserts were removed with a cotton swab whereas the cells on the lower surface were trypsinized and counted.
Cell migration was also assessed in scratch assays [52]. Briefly, confluent MCF10A, MCF-7 and MDA-MB-231 cells plated on tissue culture dishes were manually scratched with a 200 µl pipette tip, washed with PBS and incubated at 37°C in complete media in the presence or absence of 50 µM clotrimazole. At the indicated time points, phase contrast images at specific scratch sites were captured as previously described [53]. The images were analyzed using the software BELView (BEL Engineering, It) and data were expressed in µm×h−1.
Cell proliferation assay
Cells were seeded in 96-well culture plates and grown until confluence was reached. After 72 h serum was withdrawn to synchronize the cell cycle, cells were evaluated for proliferation in the presence of different concentrations of clotrimazole (0–100 µM) and 10 µM 5-bromo-2′-deoxyuridine (BrdU). BrdU incorporation was analyzed after 24 h of incubation using a europium-based time-resolved fluorescence (TRF) cell proliferation assay according to the manufacturer's instruction (PerkinElmer). Cells incubated without BrdU served as the negative control. The mean fluorescence for each group was determined after subtracting the mean value of the negative control from each cell.
Author Contributions
Conceived and designed the experiments: CMF MCM MSP MLSS PZ. Performed the experiments: CMF MCM MSP MLSS PZ. Analyzed the data: CMF MCM MSP MLSS PZ. Contributed reagents/materials/analysis tools: MSP PZ. Wrote the paper: MSP PZ.
References
- 1. Marin-Hernandez A, Rodriguez-Enriquez S, Vital-Gonzalez PA, Flores-Rodriguez FL, Macias-Silva M, et al. (2006) Determining and understanding the control of glycolysis in fast-growth tumor cells. Flux control by an over-expressed but strongly product-inhibited hexokinase. FEBS J 273: 1975–1988.
- 2. Meadows AL, Kong B, Berdichevsky M, Roy S, Rosiva R, et al. (2008) Metabolic and morphological differences between rapidly proliferating cancerous and normal breast epithelial cells. Biotechnol Prog 24: 334–341.
- 3. Hsu PP, Sabatini DM (2008) Cancer Cell Metabolism: Warburg and Beyond. Cell 134: 703–707.
- 4. Gillies RJ, Robey I, Gatenby RA (2008) Causes and consequences of increased glucose metabolism of cancers. J Nucl Med 49: Suppl 224S–42S.
- 5. Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324: 1029–1033.
- 6. Almeida A, Bolanos JP, Moncada S (2010) E3 ubiquitin ligase APC/C-Cdh1 accounts for the Warburg effect by linking glycolysis to cell proliferation. Proc Natl Acad Sci U S A 107: 738–741.
- 7. Lee HC, Wei YH (2009) Mitochondrial DNA instability and metabolic shift in human cancers. Int J Mol Sci 10: 674–701.
- 8. Khalid MH, Tokunaga Y, Caputy AJ, Walters E (2005) Inhibition of tumor growth and prolonged survival of rats with intracranial gliomas following administration of clotrimazole. J Neurosurg 103: 79–86.
- 9. Majewski N, Nogueira V, Bhaskar P, Coy PE, Skeen JE, et al. (2004) Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak. Mol Cell 16: 819–830.
- 10. Glass-Marmor L, Beitner R (1997) Detachment of glycolytic enzymes from cytoskeleton of melanoma cells induced by calmodulin antagonists. EurJPharmacol 328: 241–248.
- 11. Penso J, Beitner R (2002) Detachment of glycolytic enzymes from cytoskeleton of Lewis lung carcinoma and colon adenocarcinoma cells induced by clotrimazole and its correlation to cell viability and morphology. MolGenetMetab 76: 181–188.
- 12. Meira DD, Marinho-Carvalho MM, Teixeira CA, Veiga VF, Da Poian AT, et al. (2005) Clotrimazole decreases human breast cancer cells viability through alterations in cytoskeleton-associated glycolytic enzymes. MolGenetMetab 84: 354–362.
- 13. Sola-Penna M, Da Silva D, Coelho WS, Marinho-Carvalho MM, Zancan P (2010) Regulation of mammalian muscle type 6-phosphofructo-1-kinase and its implication for the control of the metabolism. IUBMB Life 62: 791–796.
- 14. Warburg O (1956) On the origin of cancer cells. Science 123: 309–314.
- 15. Zancan P, Sola-Penna M, Furtado CM, Da Silva D (2010) Differential expression of phosphofructokinase-1 isoforms correlates with the glycolytic efficiency of breast cancer cells. Mol Genet Metab 100: 372–378.
- 16. Geschwind JF, Ko YH, Torbenson MS, Magee C, Pedersen PL (2002) Novel therapy for liver cancer: direct intraarterial injection of a potent inhibitor of ATP production. Cancer Res 62: 3909–3913.
- 17. Chen Z, Zhang H, Lu W, Huang P (2009) Role of mitochondria-associated hexokinase II in cancer cell death induced by 3-bromopyruvate. Biochim Biophys Acta 1787: 553–560.
- 18. Bensaad K, Tsuruta A, Selak MA, Vidal MN, Nakano K, et al. (2006) TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell 126: 107–120.
- 19. Penso J, Beitner R (2002) Clotrimazole decreases glycolysis and the viability of lung carcinoma and colon adenocarcinoma cells. EurJPharmacol 451: 227–235.
- 20. Glass-Marmor L, Morgenstern H, Beitner R (1996) Calmodulin antagonists decrease glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, ATP and viability of melanoma cells. EurJPharmacol 313: 265–271.
- 21. Shaw RJ (2006) Glucose metabolism and cancer. Current Opinion in Cell Biology 18: 598–608.
- 22. Matoba S, Kang JG, Patino WD, Wragg A, Boehm M, et al. (2006) p53 regulates mitochondrial respiration. Science 312: 1650–1653.
- 23. Kim JW, Dang CV (2006) Cancer's molecular sweet tooth and the Warburg effect. Cancer Res 66: 8927–8930.
- 24. Tong X, Zhao F, Thompson CB (2009) The molecular determinants of de novo nucleotide biosynthesis in cancer cells. Curr Opin Genet Dev 19: 32–37.
- 25. Boros LG, Torday JS, Lim S, Bassilian S, Cascante M, et al. (2000) Transforming growth factor beta2 promotes glucose carbon incorporation into nucleic acid ribose through the nonoxidative pentose cycle in lung epithelial carcinoma cells. Cancer Res 60: 1183–1185.
- 26. Hatzivassiliou G, Zhao F, Bauer DE, Andreadis C, Shaw AN, et al. (2005) ATP citrate lyase inhibition can suppress tumor cell growth. Cancer Cell 8: 311–321.
- 27. Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A, Gerszten RE, et al. (2008) The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452: 230–233.
- 28. Liu YC, Li F, Handler J, Huang CR, Xiang Y, et al. (2008) Global regulation of nucleotide biosynthetic genes by c-Myc. PLoS ONE 3: e2722.
- 29. Mannava S, Grachtchouk V, Wheeler LJ, Im M, Zhuang D, et al. (2008) Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells. Cell Cycle 7: 2392–2400.
- 30. Ko YH, Smith BL, Wang Y, Pomper MG, Rini DA, et al. (2004) Advanced cancers: eradication in all cases using 3-bromopyruvate therapy to deplete ATP. Biochem Biophys Res Commun 324: 269–275.
- 31. Fantin VR, St-Pierre J, Leder P (2006) Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell 9: 425–434.
- 32. Telang S, Yalcin A, Clem AL, Bucala R, Lane AN, et al. (2006) Ras transformation requires metabolic control by 6-phosphofructo-2-kinase. Oncogene 25: 7225–7234.
- 33. Kim JW, Gao P, Liu YC, Semenza GL, Dang CV (2007) Hypoxia-inducible factor 1 and dysregulated c-Myc cooperatively induce vascular endothelial growth factor and metabolic switches hexokinase 2 and pyruvate dehydrogenase kinase 1. Mol Cell Biol 27: 7381–7393.
- 34. El-Bacha T, de Freitas MS, Sola-Penna M (2003) Cellular distribution of phosphofructokinase activity and implications to metabolic regulation in human breast cancer. MolGenetMetab 79: 294–299.
- 35. Tong X, Zhao F, Mancuso A, Gruber JJ, Thompson CB (2009) The glucose-responsive transcription factor ChREBP contributes to glucose-dependent anabolic synthesis and cell proliferation. Proc Natl Acad Sci U S A 106: 21660–21665.
- 36. Gatenby RA, Gillies RJ (2004) Why do cancers have high aerobic glycolysis? NatRevCancer 4: 891–899.
- 37. Ouadid-Ahidouch H, Roudbaraki M, Ahidouch A, Delcourt P, Prevarskaya N (2004) Cell-cycle-dependent expression of the large Ca2+-activated K+ channels in breast cancer cells. Biochem Biophys Res Commun 316: 244–251.
- 38. Khaitan D, Sankpal UT, Weksler B, Meister EA, Romero IA, et al. (2009) Role of KCNMA1 gene in breast cancer invasion and metastasis to brain. BMC Cancer 9: 258.
- 39. Zhang W, Ramamoorthy Y, Kilicarslan T, Nolte H, Tyndale RF, et al. (2002) Inhibition of cytochromes P450 by antifungal imidazole derivatives. Drug Metab Dispos 30: 314–318.
- 40. Wulff H, Miller MJ, Hansel W, Grissmer S, Cahalan MD, et al. (2000) Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant. Proc Natl Acad Sci U S A 97: 8151–8156.
- 41. Abdullaev IF, Rudkouskaya A, Mongin AA, Kuo YH (2010) Calcium-activated potassium channels BK and IK1 are functionally expressed in human gliomas but do not regulate cell proliferation. PLoS ONE 5: e12304.
- 42. Garland JM, Halestrap A (1997) Energy metabolism during apoptosis. Bcl-2 promotes survival in hematopoietic cells induced to apoptose by growth factor withdrawal by stabilizing a form of metabolic arrest. JBiolChem 272: 4680–4688.
- 43. Gonin-Giraud S, Mathieu AL, Diocou S, Tomkowiak M, Delorme G, et al. (2002) Decreased glycolytic metabolism contributes to but is not the inducer of apoptosis following IL-3-starvation. Cell Death Differ 9: 1147–1157.
- 44. Oddis CV, Finkel MS (1995) Cytokine-stimulated nitric oxide production inhibits mitochondrial activity in cardiac myocytes. Biochem Biophys Res Commun 213: 1002–1009.
- 45. Spitz GA, Furtado CM, Sola-Penna M, Zancan P (2009) Acetylsalicylic acid and salicylic acid decrease tumor cell viability and glucose metabolism modulating 6-phosphofructo-1-kinase structure and activity. Biochemical Pharmacology 77: 46–53.
- 46. Kennelly KP, Wallace DM, Holmes TM, Hankey DJ, Grant TS, et al. (2011) Preparation of pre-confluent retinal cells increases graft viability in vitro and in vivo: a mouse model. PLoS ONE 6: e21365.
- 47. Schwartz MK, Bodansky O (1968) Utilization of automation for studies of enzyme kinetics. Methods Biochem Anal 16: 183–218.
- 48. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265–275.
- 49. Coelho WS, Costa KC, Sola-Penna M (2007) Serotonin stimulates mouse skeletal muscle 6-phosphofructo-1-kinase through tyrosine-phosphorylation of the enzyme altering its intracellular localization. MolGenetMetab 92: 364–370.
- 50. Leite TC, Coelho RG, Silva DD, Coelho WS, Marinho-Carvalho MM, et al. (2011) Lactate downregulates the glycolytic enzymes hexokinase and phosphofructokinase in diverse tissues from mice. FEBS Letters 585: 92–98.
- 51. Sola-Penna M, Meyer-Fernandes JR (1998) Stabilization against thermal inactivation promoted by sugars on enzyme structure and function: why is trehalose more effective than other sugars? ArchBiochemBiophys 360: 10–14.
- 52. Liang C-C, Park AY, Guan J-L (2007) In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nature Protocols 2: 329–333.
- 53. Chan SW, Lim CJ, Guo K, Ng CP, Lee I, et al. (2008) A role for TAZ in migration, invasion, and tumorigenesis of breast cancer cells. Cancer Res 68: 2592–2598.