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American Journal of Cancer Research logoLink to American Journal of Cancer Research
. 2015 Aug 15;5(9):2673–2685.

3-bromopyruvate enhanced daunorubicin-induced cytotoxicity involved in monocarboxylate transporter 1 in breast cancer cells

Zhe Liu 1,*, Yiming Sun 1,*, Haiyu Hong 2,*, Surong Zhao 1, Xue Zou 1, Renqiang Ma 2, Chenchen Jiang 3, Zhiwei Wang 4,5, Huabin Li 2, Hao Liu 1
PMCID: PMC4633897  PMID: 26609475

Abstract

Increasing evidence demonstrates that the hexokinase inhibitor 3-bromopyruvate (3-BrPA) induces the cell apoptotic death by inhibiting ATP generation in human cancer cells. Interestingly, some tumor cell lines are less sensitive to 3-BrPA-induced apoptosis than others. Moreover, the molecular mechanism of 3-BrPA-trigged apoptosis is unclear. In the present study, we examined the effects of 3-BrPA on the viability of the breast cancer cell lines MDA-MB-231 and MCF-7. We further investigated the potential roles of monocarboxylate transporter 1 (MCT1) in drug accumulation and efflux of breast cancer cells. Finally, we explored whether 3-BrPA enhanced daunorubicin (DNR)-induced cytotoxicity through regulation of MCT1 in breast cancer cells. MTT and colony formation assays were used to measure cell viability. Western blot analysis, flow cytometric analysis and fluorescent microscopy were used to determine the molecular mechanism of actions of MCT1 in different breast cancer cell lines. Whole-body bioluminescence imaging was used to investigate the effect of 3-BrPA in vivo. We found that 3-BrPA significantly inhibited cell growth and induced apoptosis in MCF-7 cell line, but not in MDA-MB-231 cells. Moreover, we observed that 3-BrPA efficiently enhanced DNR-induced cytotoxicity in MCF-7 cells by inhibiting the activity of ATP-dependent efflux pumps. We also found that MCT1 overexpression increased the efficacy of 3-BrPA in MDA-MB-231 cells. 3-BrPA markedly suppressed subcutaneous tumor growth in combination with DNR in nude mice implanted with MCF-7 cells. Lastly, our whole-body bioluminescence imaging data indicated that 3-BrPA promoted DNR accumulation in tumors. These findings collectively suggest that 3-BrPA enhanced DNR antitumor activity in breast cancer cells involved MCT-1, suggesting that inhibition of glycolysis could be an effective therapeutic approach for breast cancer treatment.

Keywords: Breast cancer, DNR, 3-BrPA, MCT1

Introduction

Cancer cells have different metabolic requirements compared to most normal differentiated cells. Unlike normal cells, cancer cells primarily produce energy via glycolysis even in the presence of oxygen [1,2]. This phenomenon, called the Warburg effect, has received increasing attention in recent years [3]. Regardless of the underlying mechanism such as mitochondrial dysfunction, hypoxia, tumor cell signaling, or metabolic enzyme expression, its final result is the same: glycolysis-dependent production of ATP by tumor cells [4]. Because glycolysis produces less ATP than oxidative phosphorylation, tumor cells must metabolize more glucose to maintain their energy supply than normal cells [5]. Therefore, high glycolytic activity is essential for the survival and growth of tumor cells, and inhibition of glycolysis may selectively kill them via ATP deprivation.

Multidrug resistance (MDR) is a unique broad-spectrum resistance phenomenon [6]. Despite continual introduction of new chemotherapeutic drugs and improvements in clinical chemotherapy, overcoming MDR is still one of the greatest challenges in cancer chemotherapy [7,8]. MDR is produced by mutation (e.g., lactamase in bacteria) or overexpression (e.g., the ABC transporters in tumor cells) of proteins that antagonize drug activity [9,10]. The ABC transporters including P-glycoprotein, are located in the cell membrane, and are highly dependent on ATP for activity [11,12]. Inhibition of glycolysis and consequent inactivation of the ABC transporters promote intracellular retention of anti-cancer agents, thus accentuating their cytotoxic effects on malignant cells [13,14].

3-bromopyruvate (3-BrPA) and 2-deoxyglucose (2-DG) are analogs of pyruvate and glucose [15], respectively, that competitively inhibit the activity of hexokinase (HK), the enzyme that converts glucose to glucose-6-phosphate in the glycolytic pathway [16]. 3-BrPA is currently believed to have more anti-cancer activity than 2-DG: it inhibits glycolysis at a significantly lower Ki than 2-DG and also inhibits oxidative phosphorylation, leading to metabolic oxidative stress [17,18]. Moreover, 3-BrPA is a strong alkylating agent with similar chemical properties and structure as lactic acid, the final product of glycolysis [19,20]. It uses lactic acid carriers to enter cells, and upon entry, is immediately pharmacologically active [21]. Additionally, 3-BrPA may be cytotoxic for tumor cells with different aerobic glycolytic phenotypes and has extensive application prospects in combination therapy [22]. However, sensitivity to 3-BrPA differs in different tumor cell lines, and drug combination problems are also worthy of attention.

In the current study, we explored the effects of 3-BrPA on the viability of the breast cancer cell lines. We also determined whether 3-BrPA enhanced daunorubicin (DNR)-induced cytotoxicity. Furthermore, we investigated the potential roles of monocarboxylate transporter 1 (MCT1) in drug accumulation and efflux of breast cancer cells. Finally, we dissect the molecular mechanism of 3-BrPA-induced anti-tumor activity in breast cancer cells.

Material and methods

Reagents and antibodies

3-BrPA, daunorubicin (DNR), and propidium iodide were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Anti-MCT1 antibody and rabbit anti-β-actin were obtained from Millipore (Temecula, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively.

Cell lines and cell culture

Human breast cancer cell lines MDA-MB-231 and MCF-7 were obtained from American Type Culture Collection (Rockville, MD, USA). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, penicillin (10 U/ml), streptomycin (100 U/ml), and HEPES (25 mM). Cells were maintained at 37°C in a 5% CO2 humidified atmosphere.

Cell viability assay

Cells in 96-well plates received different concentrations of 3-BrPA or DNR 24 h after seeding at 1 × 104 cells/well. They were refed with phosphate-buffered saline (PBS) containing 5 mg/ml MTT 24 h after addition of compounds. After 4-h incubation at 37°C, the MTT solution was removed and replaced with 150 ul of dimethylsulfoxide/well. Absorbance was measured in a plate reader at 490 nm.

Colony formation assays

Cells in 12-well plates received different concentrations of 3-BrPA or DNR 24 h after seeding at 5 × 103 cells/well. Approximately 1 week later, colonies were visualized under a microscope, and then stained with crystal violet and counted the cell numbers.

Propidium iodide staining cells in 6-well plates (2 × 106/well) were cultured for 24 h to reach exponential growth. Cells received different concentrations of 3-BrPA, DNR, or both for 24 h and were stained with 600 ul propidium iodide/well for 2 h and evaluated via flow cytometry.

Intracellular ATP measurement

Cells were seeded at 2 × 105 cells/well in a 6-well plate for 24 h. Cellular ATP levels were determined using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Luminescence levels were measured using a microplate reader.

Drug accumulation and efflux assay

Cells were cultured in the presence or absence of 3-BrPA for 1 h followed by treatment with 6 umol/L DNR for 1 h. After washing, intracellular fluorescence levels were determined via flow cytometry [accumulation phase (AP)]. For determining drug efflux levels, the cells were further incubated in the presence or absence of 3-BrPA for 1 h before determination of intracellular fluorescence levels [efflux phase (EP)]. The difference in mean fluorescence intensity (MFI) between the AP and background represented the intracellular drug concentration during the AP. The difference in MFI between the EP and background represented intracellular drug concentration during EP.

Western blot analysis

Cells were rinsed with ice-cold PBS and lysed in radioimmune precipitation buffer for 30 min on ice. Cell lysates were centrifuged at 12,000 × g for 30 min at 4°C. Proteins in the supernatant were separated on 15% SDS-polyacrylamide gels and subsequently transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibody overnight at 4°C and subsequently with the corresponding secondary antibody. β-actin was used as the loading control.

Plasmid transfection

The pCMV-HA-MCT1 plasmid and control plasmid were obtained from GenePharma. Plasmids were transfected into MDA-MB-231 cells using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were harvested 24 h after transfection for western blot analysis.

Mitochondrial membrane potential

Cells in 12-well plates (2 × 105) cells/well were cultured for 24 h to reach exponential growth before treatment. Changes in mitochondrial membrane potential were evaluated using a mitochondrial membrane potential assay kit (Keygem Biotech, Nanjing, China) according to the manufacturers’ instructions. Stained cells were visualized using a microscope.

Live cell imaging system

Cells in 6-well plates (2 × 105 cells/well) were cultured for 24 h to reach exponential growth. Cells received 3-BrPA for 1 h, followed by 6 umol/L DNR for 1 hour. Drug fluorescence in cells was visualized using a live cell imaging system.

In vivo experiments

Five-week-old male nude mice were subcutaneously inoculated 1 × 107 MCF-7 cells. We allowed 2 weeks for palpable subcutaneous tumors to form before administering 3-BrPA and DNR. When tumors were > 100 mm3 in size, mice were intraperitoneally injected every day with 0.2 ml of PBS, DNR (0.5 mg kg-1), 3-BrPA (8 mg kg-1), or DNR plus 3-BrPA. Tumor volume and mouse body weight were measured before each injection. Tumor volume was calculated as length × width × height/2. All animal studies and procedures were approved by the BBMC Institutional Animal Care and Use Committee.

Whole-body bioluminescence imaging

Whole-body bioluminescence imaging was performed in mouse studies to monitor the distribution of chemotherapy drugs in breast tumors. After tumors were > 500 mm3 in size, the mice were injected with 0.2 ml of PBS, DNR (0.5 mg kg-1) or DNR plus 3-BrPA. Mice were anesthetized with 2% isoflurane and imaged using the Caliper IVIS Lumina II system (Caliper, Hopkinton, MA, USA) 30 minutes after drug injection.

Statistical analysis

Statistical analyses were carried out using by Student’s test. The symbol ‘*’ indicates that the values are significantly different from those of the control (*P < 0.05).

Results

Differential 3-BrPA-mediated cytotoxicity in breast cancer cells

To investigate the effects of 3-BrPA on cytotoxicity, MCF-7 and MDA-MB-231 cells were treated with different concentrations of 3-BrPA for 24 h. As determined by MTT assays, 50 uM and 100 uM 3-BrPA inhibited cell proliferation and induced apoptosis in MCF-7 cells, but not in MDA-MB-231 cells (Figure 1A and 1B). Specifically, IC50 for 3-BrPA treatment in MCF-7 and MDA-MB-231 cells are about 100 and 240 uM, respectively. Moreover, 200 uM 3-BrPA exhibited anti-tumor activity to a greater extent in MCF-7 cells compared with MDA-MB-231 cells (Figure 1A and 1B). These results show that 3-BrPA is more cytotoxic for MCF-7 cells than MDA-MB-231 cells.

Figure 1.

Figure 1

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Differential 3-BrPA-mediated cytotoxicity in breast cancer cells. A. MCF-7 and MDA-MB-231 cells were treated with 25, 50, 100, or 200 umol/L 3-BrPA for 24 h. Then, cell viability was analyzed by the MTT assay. Data represent mean ± SEM of three independent experiments. B. MCF-7 and MDA-MB-231 cells were treated with various concentrations 3-BrPA (25, 50, or 100 umol/L) for 24 h before being analyzed using flow cytometry. C. Cells were treated with DNR (2, 4, 6, or 8 umol/L) or co-treated cells with DNR (2, 4, 6, or 8 umol/L) and 3-BrPA (25 umol/L) for 24 h. Cell viability was analyzed by the MTT assay. Data represent mean ± SEM of three independent experiments. D. MCF-7 and MDA-MB-231 cells were treated with 3-BrPA (25 umol/L), DNR (8 umol/L), or 3-BrPA+DNR for 24 h before being analyzed using flow cytometry.

3-BrPA sensitized MCF-7 cells to DNR-induced cell growth inhibition and apoptosis

To determine whether 3-BrPA sensitized breast cancer cells to chemotherapeutic agents, we co-treated cells with DNR and 3-BrPA. Co-treated MCF-7 cells were less viable than cells receiving only DNR (Figure 1C). IC50 for DNR treatment was reduced from about 13,8 uM in MCF-7 cells to 7 uM in MCF-7 cells treated with 3-BrPA (Figure 1C). Co-treatment, however, did not decrease the viability of MDA-MB-231 cells (Figure 1C). IC50 of DNR treatment was about 8 uM in MDA-MB-231 cells to 9.5 uM in 3-BrPA treated MDA-MB-231 cells (Figure 1C). Furthermore, propidium iodide staining showed that 3-BrPA in combination with DNR caused to a great induction on apoptosis in MCF-7 cells compared with MDA-MB-231 cells (Figure 1D). These results suggest that 3-BrPA sensitized MCF-7 cells but not MDA-MB-231 cells to DNR-induced cell growth inhibition and significant apoptosis.

3-BrPA damaged mitochondrial membrane potential and inhibited ATP generation in MCF-7 cells

The loss of mitochondrial membrane potential and consequent production of reactive oxygen species (ROS) are the common landmark events in early apoptosis [23]. ROS closely contact mitochondrial antioxidants and induce apoptosis by releasing cytochrome c from mitochondria. ROS also inhibit the production of ATP, which in turn further increases ROS generation and apoptosis. Thus, a vicious cycle ensues. Therefore, we detected whether 3-BrPA inhibited ATP levels in breast cancer cells. We found that 3-BrPA decreased ATP levels in MCF-7 cells, but not in MDA-MB-231 cells (Figure 2A). To further assess changes in mitochondrial membrane potential, we used JC-1 as a fluorescent marker. When mitochondrial membrane potential is intact, cells fluoresce red. In contrast, when it is destroyed, cells fluoresce green. Consistently, MCF-7 cells treated with 3-BrPA showed clear damage to the mitochondrial membrane potential, appearing green after JC-1 staining (Figure 2B). Taken together, 3-BrPA damaged mitochondrial membrane potential and decreased ATP generation in MCF-7 cells.

Figure 2.

Figure 2

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3-BrPA damages mitochondrial membrane potential and inhibits ATP generation in MCF-7 cells. A. Intracellular ATP levels were measured in MCF-7 and MDA-MB-231 cells treated with 25, 50, or 100 umol/L 3-BrPA for 5 h. Data represent mean ± SEM of three independent experiments. *P < 0.05 vs. control; **P < 0.01 vs. control. B. MCF-7 cells were treated with indicated concentrations of 3-BrPA (25, 50, or 100 umol/L) for 24 h, and the mitochondrial membrane potential were assessed by JC-1 staining and fluorescence microscopy. C. MCF-7 and MDA-MB-231 cells were cultured in the presence or absence of 3-BrPA (50 umol/L), and 30 minutes later, 8 umol/L DNR was added and intracellular fluorescence levels were analyzed by flow cytometry after incubating for 30 minutes. Then cells were further incubated in the presence or absence of 3-BrPA (50 umol/L) for 1 h, and intracellular fluorescence levels were analyzed by flow cytometry. Data represent mean ± SEM of three independent experiments. AP (accumulation phase); EP (efflux phase); MFI (mean fluorescence intensity). D. MCF-7 cells were treated with 3-BrPA (50 umol/L) or 3-BrPA+DNR for 24 h, and cell fluorescence was detected by live cell imaging system.

3-BrPA enhances DNR accumulation and efflux in MCF-7 breast cancer cells

The anticancer activity of 3-BrPA requires depletion of the cellular ATP pool, as does ablation of MDR because of the ATP dependence of the ABC transporters [24]. To assess whether 3-BrPA could enhance DNR accumulation due to reduced ATP production, the effects of glycolysis inhibition on DNR accumulation and efflux were conducted in breast cancer cells. As determined by monitoring MFI via flow cytometry, 3-BrPA markedly increased DNR levels in MCF-7 cells (from 90.41 to 149.57), but not in MDA-MB-231 cells (Figure 2C). A similar result was obtained using a live imaging system. 3-BrPA increased DNR levels in MCF-7 cells (Figure 2D), but not in MDA-MB-231 cells (Figure 4B). This pattern is consistent with that seen in the viability (Figure 1C). Our results suggest that 3-BrPA enhanced DNR accumulation and efflux partly through reducing ATP levels in MCF-7 cells.

Figure 4.

Figure 4

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Overexpression of MCT1 enhances DNR accumulation and efflux in MDA-MB-231 cells. A. MDA-MB-231 cells were transfected with the control or MCT1 cDNA. Cells were cultured in the presence or absence of 3-BrPA (50 umol/L), and 30 minutes later, 8 umol/L DNR was added and intracellular fluorescence levels were analyzed by flow cytometry after incubating for 30 minutes. Then cells were further incubated in the presence or absence of 3-BrPA (50 umol/L) for 1 h, and intracellular fluorescence levels were analyzed by flow cytometry. Data represent mean ± SEM of three independent experiments. B. MDA-MB-231 cells were transfected with the control or MCT1 cDNA. After 24 h, cells were treated with 3-BrPA (50 umol/L) or 3-BrPA+DNR for 24 h, and cell fluorescence was detected by live cell imaging system.

Overexpression of MCT1 enhanced 3-BrPA-induced cell growth inhibition and apoptosis

It has been known that monocarboxylate transporter 1 (MCT1) is a potential marker for 3-BrPA sensitivity in human cancer cells [25]. To identify whether MCT-1 is involved in 3-BrPA-induced cell growth inhibition in breast cancer cells, we examined the expression of MCT1 in the two breast cancer cell lines. We observed high expression of MCT1 in MCF-7 cells and undectable level of MCT1 in MDA-MB-231 cells (Figure 3A). To further explore the function of MCT1 in 3-BrPA sensitivity, MDA-MB-231 cells were transfected with MCT1 cDNA. Overexpression efficiency was confirmed by western blotting and we found higher expression of MCT1 in transfected cells (Figure 2B). Moreover, our MTT assay results showed that overexpression of MCT1 sensitized MDA-MB-231 cells to 3-BrPA, leading to cell growth inhibition (Figure 3C), suggesting that 3-BrPA inhibited cell growth partly involved in DNR level. In line with this, our colony formation assay revealed overexpression of MCT1 caused inhibition of cell growth (Figure 3D). Notably, MCT1 overexpressing cells were significantly more sensitive to 3-BrPA-induced apoptosis (Figure 3E), compared with control cells (Figure 1B).

Figure 3.

Figure 3

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Overexpression of MCT1 enhances 3-BrPA-induced apoptosis in MDA-MB-231 cells. A. MDA-MB-231 and MCF-7 cells were exposed to 100 umol/L 3-BrPA for 24 h. At the end of the treatment, the total cell lysates were prepared, and an equal amount of protein was separated by SDS-PAGE, followed by western blotting. The membranes were probed with MCT1. B. MDA-MB-231 cells were transfected with the control or MCT1 cDNA. After 24 h, the whole-cell lysates were subjected to western blot analysis. C-E. MDA-MB-231 cells were transfected with the control or MCT1 cDNA. After 24 h, the cells were cultured with 3-BrPA (25, 50, 100, 200 umol/L) for 24 h. Then cell viability, colony formation and apoptosis were investigated. Data represent mean ± SEM of three independent experiments.

Overexpression of MCT1 enhanced DNR accumulation and efflux

Next, we determined whether up-regulation of MCT1 affected DNR accumulation and efflux in MDA-MB-231 cells. Our flow cytometry result showed that overexpression of MCT1 significantly increased DNR levels from MFI 59 to MFI 155 in MDA-MB-231 cells (Figure 4A), compared with control cells (Figure 2C). Consistently, over-expression of MCT1 led to DNR accumulation by a live imaging system (Figure 4B). More importantly, 3-BrPA increased DNR levels in MCT1 over-expressing MDA-MB-231 cells, but not in MDA-MB-231 cells (Figure 4B). Taken together, overexpression of MCT1 could increase DNR accumulation and efflux, leading to 3-BrPA-induced cell growth inhibition and apoptosis.

Depletion of MCT1 inhibited 3-BrPA-induced cell growth

To further define the role of MCT1 in 3-BrPA-induced cell proliferation, MCF-7 cells were transfected with MCT1 siRNA. Efficiency of MCT1 knockdown was validated by western blotting and we observed that MCT1 siRNA2 significantly inhibited the expression of MCT1 in MCF-7 cells (Figure 5A, 5B). We also detected the cell growth in MCT1 siRNA transfected MCF-7 cells treated with 3-BrPA. Our MTT assay result demonstrated that depletion of MCT1 rescued cell growth inhibition by 3-BrPA treatment in MCF-7 cells (Figure 5C), suggesting that 3-BrPA-mediated cell growth inhibition could be in part due to increased MCT levels.

Figure 5.

Figure 5

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Depletion of MCT1 reduces 3-BrPA sensitivity to MCF-7 cells. A. MCF-7 cells were transfected with the control or MCT1 siRNA. After 24 h, the whole-cell lysates were subjected to western blot analysis. B. Quantitative results are illustrated for panel A. C. MCF-7 cells were transfected with the control or MCT1 cDNA. After 24 h, the cells were cultured with 3-BrPA (25, 50, 100, 200 umol/L) for 24 h. Then cell viability was investigated by MTT assay. Data represent mean ± SEM of three independent experiments.

3-BrPA enhances the antitumor effects of DNR in vivo

To assess the effects of 3-BrPA in combination of DNR in vivo, nude mice were injected subcutaneously with MCF-7 cells. Daily administration of both DNR and 3-BrPA clearly suppressed subcutaneous tumor growth compared with DNR or 3-BrPA alone (Figure 6A). These results indicate that 3-BrPA sensitizes preformed tumors to DNR in vivo. We also investigated the distribution of DNR in nude mice receiving DNR alone or DNR plus 3-BrPA. The red fluorescence indicates the location of DNR in Figure 6B. It is clearly shown that DNR accumulated in tumor (Figure 6B). The results suggest that 3-BrPA could promote the accumulation of DNR in tumors.

Figure 6.

Figure 6

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3-BrPA enhances the antitumor effects of DNR in vivo. A. MCF-7 cells were subcutaneously injected into the right flank of nude mice receiving vehicle and mice were treated with 3-BrPA (8 mg kg-1), DNR (0.5 mg kg-1) or DNR plus 3-BrPA every 2 days. Mice were sacrificed after 28 days. B. MCF-7 cells were subcutaneously injected into the right flank of nude mice. After tumors were > 500 mm3 in size, the mice were injected with 0.2 ml of PBS, DNR (0.5 mg kg-1) or DNR plus 8 mg kg-1 3-BrPA. Mice were anesthetized with 2% isoflurane and imaged using the Caliper IVIS Lumina II system 30 minutes after drug injection.

Discussion

Treatment of tumors is a major concern of the medical profession, and low sensitivity or resistance of tumor cells to chemotherapeutic drugs is an ongoing problem [26,27]. Tumor cells proliferate rapidly and require the production of large amounts of proteins, nucleic acids, lipids, and ATP [28]. Glycolysis rapidly supplies the necessary energy for these processes, as well as key precursors [29]. Cancer cells are more dependent on the glycolytic pathway for ATP generation than normal cells and eventually acquire drug resistance often because of aberrant expression of the drug-expelling ABC transporters [30]. Due to that transporters require ATP for activity [31,32], inhibition of glycolysis may increase the concentration of chemotherapeutic agents in cancer cells [33,34].

3-BrPA is an analog of the cellular metabolites pyruvic acid and lactic acid. As such, it effectively inhibits glycolysis and consequent ATP generation [35,36]. Many new targets of 3-BrPA have been discovered in recent years, such as HK II, glyceraldehyde 3-phosphate dehydrogenase, and pyruvate dehydrogenase [37]. Tumor cells rely on glycolysis for ATP production and the ABC transporters require ATP for activity [38,39]. Thus, 3-BrPA may effectively overcome MDR in tumors via glycolysis inhibition [40]. Consistently, our data show that 3-BrPA decreased ATP levels and increased DNR levels in MCF-7 cells. These observations suggest that 3-BrPA enhanced DNR accumulation partly via ATP deprivation, which reduced ABC transporter activity and consequent drug efflux from MCF-7 cells.

The present study shows that MCF-7 cells are more sensitive to 3-BrPA-induced anti-tumor activity than MDA-MB-231 cells, suggesting that 3-BrPA inhibits proliferation and induces apoptosis in some but not all tumor cell lines. Recently, a novel strategy for cancer treatment has been proposed [25]. It is based on data showing that deadly toxic substances can be transported into malignant cells via specific proteins highly expressed on the surface of cancer cells (e.g., MCT1). Consequently, cancer cells can absorb toxic substances that normal cells cannot, and this distinction can be exploited to selectively kill cancer cells. MCT1 is necessary and sufficient for 3-BrPA transport into cells, and tumor cell levels of MCT1 can be used to predict tumor cell sensitivity to 3-BrPA [25,41]. In line with these, our results demonstrated that 3-BrPA enhanced the sensitivity of MCF-7 cells to DNR through MCT1. To support this concept, we found that MCT1 was more highly expressed in MCF-7 cells compared with MDA-MB-231 cells, in which expression was barely detectable. Because cancer cells express more MCT1 than normal cells, tumor cells are more sensitive to 3-BrPA. Via identification of surface transporters, we should be able to determine how drugs enter cells and induce toxicity. Doing so would allow the selective treatment of tumor as compared with normal cells.

In conclusion, our results show that 3-BrPA induced cell growth inhibition and apoptosis, decreased ATP generation, and enhanced DNR accumulation in the MCF-7 breast cancer cell line. Our data further reveal that the different sensitivities of MCF-7 and MDA-MB-231 cells to 3-BrPA reflect differences in MCT1 expression levels. Therefore, 3-BrPA may be clinically useful for overcoming the resistance of tumors to chemotherapeutic agents and relevant to anti-cancer strategies attempting to target cancer metabolism. However, further investigation for molecular mechanism of 3-BrPA-trigged anti-tumor activities is warranted.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81372899, 81172087).

Disclosure of conflict of interest

None.

References

  • 1.Ponisovskiy MR. Warburg effect mechanism as the target for theoretical substantiation of a new potential cancer treatment. Crit Rev Eukaryot Gene Expr. 2011;21:13–28. doi: 10.1615/critreveukargeneexpr.v21.i1.20. [DOI] [PubMed] [Google Scholar]
  • 2.Bayley JP, Devilee P. The Warburg effect in 2012. Curr Opin Oncol. 2012;24:62–67. doi: 10.1097/CCO.0b013e32834deb9e. [DOI] [PubMed] [Google Scholar]
  • 3.Koppenol WH, Bounds PL, Dang CV. Otto Warburg’s contributions to current concepts of cancer metabolism. Nat Rev Cancer. 2011;11:325–337. doi: 10.1038/nrc3038. [DOI] [PubMed] [Google Scholar]
  • 4.Sotgia F, Martinez-Outschoorn UE, Lisanti MP. Genetic induction of the Warburg effect inhibits tumor growth. Oncotarget. 2012;3:1266–1267. doi: 10.18632/oncotarget.739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Lai IL, Chou CC, Lai PT, Fang CS, Shirley LA, Yan R, Mo X, Bloomston M, Kulp SK, Bekaii-Saab T, Chen CS. Targeting the Warburg effect with a novel glucose transporter inhibitor to overcome gemcitabine resistance in pancreatic cancer cells. Carcinogenesis. 2014;35:2203–2213. doi: 10.1093/carcin/bgu124. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 6.Joyce H, McCann A, Clynes M, Larkin A. Influence of multidrug resistance and drug transport proteins on chemotherapy drug metabolism. Expert Opin Drug Metab Toxicol. 2015;11:795–809. doi: 10.1517/17425255.2015.1028356. [DOI] [PubMed] [Google Scholar]
  • 7.Kathawala RJ, Gupta P, Ashby CR Jr, Chen ZS. The modulation of ABC transporter-mediated multidrug resistance in cancer: a review of the past decade. Drug Resist Updat. 2015;18:1–17. doi: 10.1016/j.drup.2014.11.002. [DOI] [PubMed] [Google Scholar]
  • 8.Pavan B, Paganetto G, Rossi D, Dalpiaz A. Multidrug resistance in cancer or inefficacy of neuroactive agents: innovative strategies to inhibit or circumvent the active efflux transporters selectively. Drug Discov Today. 2014;19:1563–1571. doi: 10.1016/j.drudis.2014.06.004. [DOI] [PubMed] [Google Scholar]
  • 9.Zhang YK, Wang YJ, Gupta P, Chen ZS. Multidrug Resistance Proteins (MRPs) and Cancer Therapy. AAPS J. 2015;17:802–812. doi: 10.1208/s12248-015-9757-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Choi YH, Yu AM. ABC transporters in multidrug resistance and pharmacokinetics, and strategies for drug development. Curr Pharm Des. 2014;20:793–807. doi: 10.2174/138161282005140214165212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Meng X, Liao S, Wang X, Wang S, Zhao X, Jia P, Pei W, Zheng X. Reversing P-glycoproteinmediated multidrug resistance in vitro by alpha-asarone and beta-asarone, bioactive cistrans isomers from Acorus tatarinowii. Biotechnol Lett. 2014;36:685–691. doi: 10.1007/s10529-013-1419-8. [DOI] [PubMed] [Google Scholar]
  • 12.Piecuch A, Oblak E. Yeast ABC proteins involved in multidrug resistance. Cell Mol Biol Lett. 2014;19:1–22. doi: 10.2478/s11658-013-0111-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Hulleman E, Kazemier KM, Holleman A, VanderWeele DJ, Rudin CM, Broekhuis MJ, Evans WE, Pieters R, Den Boer ML. Inhibition of glycolysis modulates prednisolone resistance in acute lymphoblastic leukemia cells. Blood. 2009;113:2014–2021. doi: 10.1182/blood-2008-05-157842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Wu AM, Dalvi P, Lu X, Yang M, Riddick DS, Matthews J, Clevenger CV, Ross DD, Harper PA, Ito S. Induction of multidrug resistance transporter ABCG2 by prolactin in human breast cancer cells. Mol Pharmacol. 2013;83:377–388. doi: 10.1124/mol.112.082362. [DOI] [PubMed] [Google Scholar]
  • 15.Xiao H, Li S, Zhang D, Liu T, Yu M, Wang F. Separate and concurrent use of 2-deoxy-Dglucose and 3-bromopyruvate in pancreatic cancer cells. Oncol Rep. 2013;29:329–334. doi: 10.3892/or.2012.2085. [DOI] [PubMed] [Google Scholar]
  • 16.Gonzalez CD, Alvarez S, Ropolo A, Rosenzvit C, Bagnes MF, Vaccaro MI. Autophagy, Warburg, and Warburg reverse effects in human cancer. Biomed Res Int. 2014;2014:926729. doi: 10.1155/2014/926729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Dylag M, Lis P, Niedzwiecka K, Ko YH, Pedersen PL, Goffeau A, Ulaszewski S. 3-Bromopyruvate: a novel antifungal agent against the human pathogen Cryptococcus neoformans. Biochem Biophys Res Commun. 2013;434:322–327. doi: 10.1016/j.bbrc.2013.02.125. [DOI] [PubMed] [Google Scholar]
  • 18.Wolf A, Agnihotri S, Micallef J, Mukherjee J, Sabha N, Cairns R, Hawkins C, Guha A. Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme. J Exp Med. 2011;208:313–326. doi: 10.1084/jem.20101470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Lis P, Zarzycki M, Ko YH, Casal M, Pedersen PL, Goffeau A, Ulaszewski S. Transport and cytotoxicity of the anticancer drug 3-bromopyruvate in the yeast Saccharomyces cerevisiae. J Bioenerg Biomembr. 2012;44:155–161. doi: 10.1007/s10863-012-9421-8. [DOI] [PubMed] [Google Scholar]
  • 20.Pedersen PL. 3-Bromopyruvate (3BP) a fast acting, promising, powerful, specific, and effective “small molecule” anti-cancer agent taken from labside to bedside: introduction to a special issue. J Bioenerg Biomembr. 2012;44:1–6. doi: 10.1007/s10863-012-9425-4. [DOI] [PubMed] [Google Scholar]
  • 21.Shoshan MC. 3-Bromopyruvate: targets and outcomes. J Bioenerg Biomembr. 2012;44:7–15. doi: 10.1007/s10863-012-9419-2. [DOI] [PubMed] [Google Scholar]
  • 22.Ganapathy-Kanniappan S, Kunjithapatham R, Geschwind JF. Anticancer efficacy of the metabolic blocker 3-bromopyruvate: specific molecular targeting. Anticancer Res. 2013;33:13–20. [PubMed] [Google Scholar]
  • 23.El Sayed SM, Mahmoud AA, El Sawy SA, Abdelaal EA, Fouad AM, Yousif RS, Hashim MS, Hemdan SB, Kadry ZM, Abdelmoaty MA, Gabr AG, Omran FM, Nabo MM, Ahmed NS. Warburg effect increases steady-state ROS condition in cancer cells through decreasing their antioxidant capacities (anticancer effects of 3-bromopyruvate through antagonizing Warburg effect) Med Hypotheses. 2013;81:866–870. doi: 10.1016/j.mehy.2013.08.024. [DOI] [PubMed] [Google Scholar]
  • 24.Falasca M, Linton KJ. Investigational ABC transporter inhibitors. Expert Opin Investig Drugs. 2012;21:657–666. doi: 10.1517/13543784.2012.679339. [DOI] [PubMed] [Google Scholar]
  • 25.Birsoy K, Wang T, Possemato R, Yilmaz OH, Koch CE, Chen WW, Hutchins AW, Gultekin Y, Peterson TR, Carette JE, Brummelkamp TR, Clish CB, Sabatini DM. MCT1-mediated transport of a toxic molecule is an effective strategy for targeting glycolytic tumors. Nat Genet. 2013;45:104–108. doi: 10.1038/ng.2471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kartal-Yandim M, Adan-Gokbulut A, Baran Y. Molecular mechanisms of drug resistance and its reversal in cancer. Crit Rev Biotechnol. 2015:1–11. doi: 10.3109/07388551.2015.1015957. [DOI] [PubMed] [Google Scholar]
  • 27.Tomasetti C. Drug resistance. Adv Exp Med Biol. 2014;844:303–316. doi: 10.1007/978-1-4939-2095-2_15. [DOI] [PubMed] [Google Scholar]
  • 28.Granchi C, Minutolo F. Anticancer agents that counteract tumor glycolysis. ChemMedChem. 2012;7:1318–1350. doi: 10.1002/cmdc.201200176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Lee ZW, Teo XY, Tay EY, Tan CH, Hagen T, Moore PK, Deng LW. Utilizing hydrogen sulfide as a novel anti-cancer agent by targeting cancer glycolysis and pH imbalance. Br J Pharmacol. 2014;171:4322–4336. doi: 10.1111/bph.12773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Oshikata A, Matsushita T, Ueoka R. Enhancement of drug efflux activity via MDR1 protein by spheroid culture of human hepatic cancer cells. J Biosci Bioeng. 2011;111:590–593. doi: 10.1016/j.jbiosc.2011.01.006. [DOI] [PubMed] [Google Scholar]
  • 31.Park S, Shimizu C, Shimoyama T, Takeda M, Ando M, Kohno T, Katsumata N, Kang YK, Nishio K, Fujiwara Y. Gene expression profiling of ATP-binding cassette (ABC) transporters as a predictor of the pathologic response to neoadjuvant chemotherapy in breast cancer patients. Breast Cancer Res Treat. 2006;99:9–17. doi: 10.1007/s10549-006-9175-2. [DOI] [PubMed] [Google Scholar]
  • 32.Kovalev AA, Tsvetaeva DA, Grudinskaja TV. Role of ABC-cassette transporters (MDR1, MRP1, BCRP) in the development of primary and acquired multiple drug resistance in patients with early and metastatic breast cancer. Exp Oncol. 2013;35:287–290. [PubMed] [Google Scholar]
  • 33.Hou X, Huang F, Carboni JM, Flatten K, Asmann YW, Ten Eyck C, Nakanishi T, Tibodeau JD, Ross DD, Gottardis MM, Erlichman C, Kaufmann SH, Haluska P. Drug efflux by breast cancer resistance protein is a mechanism of resistance to the benzimidazole insulin-like growth factor receptor/insulin receptor inhibitor, BMS-536924. Mol Cancer Ther. 2011;10:117–125. doi: 10.1158/1535-7163.MCT-10-0438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Nambaru PK, Hubner T, Kock K, Mews S, Grube M, Payen L, Guitton J, Sendler M, Jedlitschky G, Rimmbach C, Rosskopf D, Kowalczyk DW, Kroemer HK, Weiss FU, Mayerle J, Lerch MM, Ritter CA. Drug efflux transporter multidrug resistance-associated protein 5 affects sensitivity of pancreatic cancer cell lines to the nucleoside anticancer drug 5-fluorouracil. Drug Metab Dispos. 2011;39:132–139. doi: 10.1124/dmd.110.033613. [DOI] [PubMed] [Google Scholar]
  • 35.Pelicano H, Martin DS, Xu RH, Huang P. Glycolysis inhibition for anticancer treatment. Oncogene. 2006;25:4633–4646. doi: 10.1038/sj.onc.1209597. [DOI] [PubMed] [Google Scholar]
  • 36.Pereira da Silva AP, El-Bacha T, Kyaw N, dos Santos RS, da-Silva WS, Almeida FC, Da Poian AT, Galina A. Inhibition of energy-producing pathways of HepG2 cells by 3-bromopyruvate. Biochem J. 2009;417:717–726. doi: 10.1042/BJ20080805. [DOI] [PubMed] [Google Scholar]
  • 37.Cardaci S, Desideri E, Ciriolo MR. Targeting aerobic glycolysis: 3-bromopyruvate as a promising anticancer drug. J Bioenerg Biomembr. 2012;44:17–29. doi: 10.1007/s10863-012-9422-7. [DOI] [PubMed] [Google Scholar]
  • 38.Modok S, Mellor HR, Callaghan R. Modulation of multidrug resistance efflux pump activity to overcome chemoresistance in cancer. Curr Opin Pharmacol. 2006;6:350–354. doi: 10.1016/j.coph.2006.01.009. [DOI] [PubMed] [Google Scholar]
  • 39.Holland KA, Holland IB. Adventures with ABC-proteins: highly conserved ATP-dependent transporters. Acta Microbiol Immunol Hung. 2005;52:309–322. doi: 10.1556/AMicr.52.2005.3-4.4. [DOI] [PubMed] [Google Scholar]
  • 40.Xu RH, Pelicano H, Zhou Y, Carew JS, Feng L, Bhalla KN, Keating MJ, Huang P. Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia. Cancer Res. 2005;65:613–621. [PubMed] [Google Scholar]
  • 41.Le Floch R, Chiche J, Marchiq I, Naiken T, Ilc K, Murray CM, Critchlow SE, Roux D, Simon MP, Pouyssegur J. CD147 subunit of lactate/H+ symporters MCT1 and hypoxia-inducible MCT4 is critical for energetics and growth of glycolytic tumors. Proc Natl Acad Sci U S A. 2011;108:16663–16668. doi: 10.1073/pnas.1106123108. [DOI] [PMC free article] [PubMed] [Google Scholar]

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