Preclinical report 447
3-Bromopyruvate induces apoptosis in breast cancer cells by downregulating Mcl-1 through the PI3K/Akt signaling pathway Zhe Liua,*, Yuan-Yuan Zhanga,*, Qian-Wen Zhanga, Su-Rong Zhaoa, Cheng-Zhu Wua, Xiu Chenga, Chen-Chen Jiangb, Zhi-Wen Jianga and Hao Liua The hexokinase inhibitor 3-bromopyruvate (3-BrPA) can inhibit glycolysis in tumor cells to reduce ATP production, resulting in apoptosis. However, as 3-BrPA is an alkylating agent, its cytotoxic action may be induced by other molecular mechanisms. The results presented here reveal that 3-BrPA-induced apoptosis is caspase independent. Further, 3-BrPA induces the generation of reactive oxygen species in MDA-MB-231 cells, leading to mitochondriamediated apoptosis. These results suggest that caspaseindependent apoptosis may be induced by the generation of reactive oxygen species. In this study, we also demonstrated that 3-BrPA induces apoptosis through the downregulation of myeloid cell leukemia-1 (Mcl-1) in MDA-MB-231 breast cancer cells. The results of Mcl-1 knockdown indicate that Mcl-1 plays an important role in 3-BrPA-induced apoptosis. Further, the upregulation of Mcl-1 expression in 3-BrPA-treated MDA-MB-231 cells significantly increases cell viability. In addition, 3-BrPA treatment resulted in the downregulation of p-Akt, suggesting that 3-BrPA may downregulate Mcl-1
through the phosphoinositide-3-kinase/Akt pathway. These findings indicate that 3-BrPA induces apoptosis in breast cancer cells by downregulating Mcl-1 through the phosphoinositide-3-kinase/Akt signaling c 2014 Wolters pathway. Anti-Cancer Drugs 25:447–455 Kluwer Health | Lippincott Williams & Wilkins.
Introduction
of caspase-8, leading to a signaling cascade that culminates in the death of the cell. The intrinsic pathway can be initiated by various intracellular signals, such as a variety of stressors or DNA damage, which lead to the activation of Bax. Bax induces the release of cytochrome c from the mitochondria. Subsequently, cytochrome c binds to Apaf-1, which facilitates the formation of the apoptosome, leading to the activation of caspase-9 and eventual cell death. Bid, which is the specific substrate of caspase-8, plays a key role in these two pathways. Activated caspase-8 cleaves Bid and induces the release of cytochrome c [8,9].
Cancer cells exhibit and rely on increased glycolysis for ATP production [1,2]. Hypoxia caused by the rapid growth of cancer cells limits the traditional synthesis of ATP [3,4]. Therefore, cancer cells require high glucose uptake and accelerated glycolysis rates to survive. Hexokinase (HK) is the first rate-limiting enzyme of glycolysis [5,6], and it catalyzes the phosphorylation of glucose to generate glucose-6-phosphate. Glucose-6phosphate is important particularly for maintaining high levels of glycolysis. Theoretically, HK inhibitors can effectively inhibit cancer cell glycolysis, thereby reducing the ATP supply and inducing apoptosis. Further, HK inhibitors can initiate the mitochondrial apoptosis cascade, inducing apoptosis of cancer cells. Apoptosis is a consequence of a vast array of complex cellular processes and is responsible for maintaining homeostasis and ensuring normal physiological functions within an organism. Apoptosis can occur through two pathways: extrinsic and intrinsic [7]. The extrinsic pathway primarily utilizes extracellular ligands to activate cell surface death receptors, such as the Fas receptor or tumor necrosis factor receptor. The activation of any of the death receptors results in the cleavage and activation c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4973
Anti-Cancer Drugs 2014, 25:447–455 Keywords: apoptosis, breast cancer, 3-bromopyruvate, caspases, myeloid cell leukemia-1, reactive oxygen species a Faculty of Pharmacy, Bengbu Medical College, Bengbu, Anhui, People’s Republic of China and bSchool of Medicine and Public Health, University of Newcastle, Newcastle, New South Wales, Australia
Correspondence to Hao Liu, Faculty of Pharmacy, Bengbu Medical College, Bengbu 233030, Anhui, People’s Republic of China Tel: + 86 552 317 5230; fax: + 86 552 317 5228; e-mail: [email protected] *Zhe Liu and Yuan-Yuan Zhang contributed equally to the writing of this article. Received 18 October 2013 Revised form accepted 25 December 2013
3-Bromopyruvate (3-BrPA) treatment limits HK levels during glycolysis, preventing the production of ATP [10]. In addition, 3-BrPA inhibits glycolysis, leading to the dephosphorylation of Bad, which is linked to glycolysis and apoptosis. Members of the Bcl-2 antiapoptotic family such as Bcl-2 and Bcl-xL can be combined with Bad or Bax to prevent apoptosis. The inhibition of glycolysis induces the dephosphorylation of Bad, migration of Bax to the mitochondria, and release of cytochrome c, leading to the activation of the executioner caspase, caspase3 [11]. 3-BrPA can also induce an increase in superoxide anion levels, which is a key contributor to oxidative DOI: 10.1097/CAD.0000000000000081
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448 Anti-Cancer Drugs 2014, Vol 25 No 4
stress [12]. To determine whether 3-BrPA causes apoptosis by affecting Bcl-2 family proteins, we analyzed the changes in myeloid cell leukemia-1 (Mcl-1) expression in two cancer cell lines after the administration of 3-BrPA. We observed that Mcl-1 was downregulated in MDA-MB-231 cells but not in MDA-MB-435 cells. Moreover, we report here that the small interfering (si) RNA-mediated downregulation of Mcl-1 induced an increase in MDA-MB-435 apoptosis after the administration of 3-BrPA. Therefore, we conclude that Mcl-1 plays an important role in 3-BrPA-induced apoptosis.
Materials and methods Reagents and antibodies
3-BrPA and 2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one (LY294002) were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA). An Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit was purchased from Keygen Biotech (Nanjing, China). Anti-caspase-3 monoclonal antibodies were obtained from Abcam (Cambridge, UK). Mouse monoclonal antibodies against caspase-8 were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). The antibodies for Akt and p-Akt were from Assay Biotech (Los Angeles, California, USA). Rabbit anti-b-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, California, USA). Cell lines and cell culture
Human breast cancer cell lines MDA-MB-231 and MDA-MB-435 were obtained from Shanghai Cell Bank (Shanghai, China). The 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 mmol/l). The cells were maintained at 371C in a 5% CO2 humidified atmosphere. MTT assay
The breast cancer cells were seeded at 1 104 cells/well in a 96-well plate for 24 h and then treated with different concentrations of 3-BrPA. At 24, 48 and 72 h, the cells were incubated with MTT (5 mg/ml in PBS) for 4 h at 371C. After 4 h, the MTT solution was removed and replaced with 150 ml of DMSO/well. The absorbance was measured in a plate reader at a test wavelength of 490 nm. Propidium iodide staining
Before 3-BrPA treatment, cells (2 106) were plated in each well of a six-well plate and allowed to reach exponential growth (24 h). The cells were treated with different concentrations of 3-BrPA (40, 80, and 160 mmol/l) for 24 h, subjected to PI staining, and then evaluated using flow cytometry. Detection of superoxide anion levels
Breast cancer cells were seeded at 2 105 cells/well in a six-well plate. The cells were labeled with 5 mmol/l
dihydroethidium (Beyotime, Jiangsu, China), which was immediately followed by the addition of 3-BrPA (160 mmol/l). After the treatment for the indicated time periods, the cells were harvested, washed, and analyzed by fluorescence-activated cell sorting. Annexin V-FITC/PI apoptosis assay
Cellular apoptosis analysis was carried out using the Annexin V-FITC/PI apoptosis detection kit (Keygen Biotech). Briefly, MDA-MB-231 cells (2 106) were seeded in each well of a six-well plate and treated with 3-BrPA for 24 h. The cells were harvested and collected by centrifugation and resuspended in 200 ml of ice-cold binding buffer. Thereafter, the cells were incubated with 5 ml of Annexin V FITC and 1 ml of PI for 15 min at room temperature in the dark and subsequently analyzed using a flow cytometer. Western blot analysis
The cells were rinsed with ice-cold PBS and lysed in RIPA buffer for 30 min on ice. The cell lysates were centrifuged at 12 000g for 30 min at 41C. The proteins were separated on a 15% SDS-PAGE gel and were subsequently transferred onto a nitrocellulose membrane (Bio-Rad). The membranes were incubated with the appropriate primary antibody overnight at 41C and further incubated with the corresponding secondary antibody. b-Actin was used as a loading control. siRNA
Mcl-1 siRNA (50 -GGGACUGGCUAGUUAAACATT-30 ) and nonsilencing siRNA (50 -UUCUCCGAACGUGUCACG UTT-30 ) were synthesized by Shanghai GenePharma (Shanghai, China). MDA-MB-435 cells were plated at 5 105 cells/well in a six-well plate and transfected with 20 nmol/l siRNA in Opti-MEM medium (Invitrogen, Carlsbad, California, USA) using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. To confirm the knockdown efficiency, cell extracts were prepared and subjected to SDS-PAGE, followed by immunoblotting. Plasmid transfection
The pCMV-HA-Mcl-1 plasmid and control plasmid were obtained from GenePharma. The plasmids were transfected into MDA-MB-231 cells using lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s transfection protocol. After 24 h of incubation, total cell lysates were prepared for immunoblot analysis. Statistical analysis
Statistical analyses were carried out using one-way analysis of variance. The symbol ‘*’ indicates that the values are significantly different from those of the control (*P < 0.05; **P < 0.01; ***P < 0.001).
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Antitumor effects of 3-bromopyruvate Liu et al. 449
Results Differential 3-BrPA-mediated cytotoxicity in breast cancer cells
To study the effect of 3-BrPA on cell proliferation, two breast cancer cell lines were treated with different concentrations of 3-BrPA for 24, 48, and 72 h. We established that 3-BrPA induced MDA-MB-231 cell apoptosis and inhibited MDA-MB-231 cell proliferation (Fig. 1a and b). The results demonstrated that the rate of cell proliferation had an inverse relationship with both the time of 3-BrPA exposure and concentration of 3-BrPA (Fig. 1a). However, 3-BrPA did not induce cytotoxicity in the MDA-MB-435 cell line (Fig. 2a and b). Annexin V and PI were used to determine the type of cell death induced by 3-BrPA in MDA-MB-231 cells. After 24 h of 3-BrPA treatment, the percentage of the apoptotic cells was increased significantly from 5.5 to 22.2%, and the percentage of necrotic cells increased from 0.9 to 10.7% (Fig. 1d). Taken together, these data suggest that 3-BrPA induces apoptosis in MDA-MB-231 cells and that MDA-MB-435 cells are not sensitive to 3-BrPA. 3-BrPA-induced apoptosis is independent of caspase activity
To verify whether 3-BrPA-induced cell death observed in MDA-MB-231 cells was a result of apoptosis, we performed immunoblotting to detect caspase activation. The cleaved products of caspase-8 and caspase-3 were detected in the MDA-MB-231 cells, indicating that 3-BrPA-induced cell death was apoptotic (Fig. 2a). To determine whether caspases play a role in cell death induced by 3-BrPA, we used the pan-caspase inhibitor z-VAD-FMK (Sigma Chemical Co.) to treat the 3-BrPAsensitive MDA-MB-231 cells. z-VAD treatment did not inhibit 3-BrPA-induced cell death (Fig. 2d), indicating a caspase-independent killing mechanism [13]. During cellular aerobic respiration and mitochondrial electron transport, a small portion of the oxygen necessary for these processes cannot be reduced completely, resulting in the generation of reactive oxygen species (ROS) with a strong oxidizing potential, such as the superoxide anion. To maintain a relatively stable level of intracellular ROS, intracellular antioxidant enzymes remove ROS that is produced continuously through metabolic processes. However, under certain conditions, the homeostasis between ROS production and antioxidant activity, which initiates a series of reactions, leads to apoptosis [14,15]. In this study, 3-BrPA treatment increased the generation of ROS (Fig. 2b and c). These results indicate that 3-BrPA-induced caspase-independent apoptosis might occur as a result of increased ROS production. 3-BrPA decreases the expression of Mcl-1 in MDA-MB-231 cells through the PI3K pathway
Mcl-1 and Bcl-2 family proteins are similar in structure and function, and they are involved primarily in the
maintenance of mitochondrial membrane stability and in the inhibition of the release of cytochrome c through the phosphoinositide-3-kinase (PI3K) pathway, thus protecting cells from apoptosis. 3-BrPA treatment downregulated the expression of Mcl-1 (Fig. 3a), and the PI3K/Akt inhibitor LY294002 also decreased the expression of Mcl-1 (Fig. 3b). To confirm whether 3-BrPA downregulated the expression of Mcl-1 through the PI3K/Akt pathway, we examined the expression of p-Akt in 3-BrPA-treated MDA-MB-231 cells. Our data revealed that 3-BrPA downregulated p-Akt (Fig. 3b). On the basis of these results, we hypothesize that 3-BrPA decreases the expression of Mcl-1 through the PI3K/Akt pathway. Downregulation of Mcl-1 induces apoptosis in MDA-MB-435 cells
To examine the functional significance of Mcl-1 expression in the sensitization of breast cancer cells to 3-BrPA, siRNA-mediated silencing of Mcl-1 was performed, and the role of Mcl-1 expression in the sensitivity of MDAMB-435 cells to 3-BrPA-induced apoptosis was assessed. Western blot analysis confirmed that Mcl-1 expression was reduced substantially by the Mcl-1-specific siRNA (Fig. 4a). The downregulation of Mcl-1 expression in the MDA-MB-435 cells resulted in increased sensitivity to 3-BrPA-induced antiproliferation (Fig. 4c) and activation of caspase-3 expression (Fig. 4b). These results demonstrate that the downregulation of Mcl-1 expression is critical to 3-BrPA-induced apoptosis in breast cancer cells. Upregulation of Mcl-1 protects MDA-MB-231 cells from apoptosis
To further confirm the role of Mcl-1 in 3-BrPA-induced apoptosis, we transfected complementary DNA encoding Mcl-1 into MDA-MB-231 cells (Fig. 5a). The overexpression of Mcl-1 significantly increased cell viability after the administration of 3-BrPA, indicating that the cells were protected from apoptosis (Fig. 5b). The cleaved products of caspase-3 were nearly undetectable in the Mcl-1-overexpressing MDA-MB-231 cells (Fig. 5c). Taken together, these results further confirm that Mcl-1 plays an important role in 3-BrPA-induced apoptosis.
Discussion The Bcl-2 protein family, which includes antiapoptotic and proapoptotic proteins, is the primary regulator of apoptosis, and the balance between antiapoptotic and proapoptotic proteins is the key to cell survival [16]. Mcl-1 is an antiapoptotic member of the Bcl-2 family of proteins and plays an integral role in cell survival and apoptosis [17,18]. Mcl-1 is overexpressed in a variety of cancers and contributes to cancer cell survival and apoptosis resistance [19]. Although the overexpression of Mcl-1 does not lead to increased cell proliferation, the ability of Mcl-1 to suppress apoptosis is an important contributor to the transformed state [20]. Generally, Mcl-1 expression can be induced by a variety of signaling pathways, such as the
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Fig. 1
(a)
MDA-MB-231
MDA-MB-435
Cell viability (%)
100
Cell viability (%)
24 h 48 h 72 h
120
80 60 40
100 80 60 40
20
20
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0 0 10 20 40 80 160 Concentrations of 3-BrPA (μmol/l)
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0 10 20 40 80 160 Concentrations of 3-BrPA (μmol/l)
40 μmol/l
80 μmol/l
160 μmol/l
M1 9.8%
M1 14.8%
M1 54.7%
Relative cell number
MDA-MB-231 M1 2.3%
MDA-MB-435 M1 2.8%
M1 4.7%
M1 5.3%
100 Cell apoptosis (%)
(c)
80 ∗
60
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40 μmol/l
80 μmol/l
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40 20 0
(d)
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Control Q1-UR 0.9%
Q1-UL 0.2%
MDA-MB-435 80 μmol/l
Gate: P7
Gate: P7 Q1-UL 0.6%
M1 5.2%
160 μmol/l
Gate: P7 Q1-UR 1.2%
Q1-UL 0.4%
Gate: P7 Q1-UR 12.7%
Q1-UL 1.8%
Q1-UR 10.7%
MDA-MB-231 Q1-LL 93.0%
Q1-LR 5.5%
Q1-LL 89.8%
Q1-LR 8.8%
Q1-LL 74.7%
Q1-LR 12.2%
Q1-LL 65.3%
Q1-LR 22.2%
Concentrations of 3-BrPA (μmol/l)
Differential 3-BrPA-mediated cytotoxicity of breast cancer cells. (a) MDA-MB-231 and MDA-MB-435 cells were cultured with various concentrations of 3-BrPA (20, 40, 80, and 160 mmol/l) for 24, 48, and 72 h, and the cell viability was analyzed using an MTT assay. (b) MDA-MB-231 and MDA-MB435 cells were treated with various concentrations of 3-BrPA (20, 40, 80, and 160 mmol/l) for 24 h before being analyzed using flow cytometry. (c) Bar chart presentation of the distribution of apoptotic cells in a population. This figure represents three independent experiments. (d) Distribution of viable, apoptotic, and necrotic MDA-MB-231 cells in the presence of various concentrations of 3-BrPA (20, 40, 80, and 160 mmol/l) for 24 h, as measured by Annexin V/PI staining. Apoptosis was measured by the propidium iodide method using flow cytometry. *P < 0.05. 3-BrPA, 3-bromopyruvate.
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Antitumor effects of 3-bromopyruvate Liu et al. 451
Fig. 2
MDA-MB-231
(a)
Time (h)
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24 -Pro-caspase-8 -Cleaved caspase-8 -Pro-caspase-3 -Cleaved caspase-3
-β-Actin (b)
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0 Control
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3-BrPA-induced apoptosis is independent of caspase activity. (a) Whole-cell lysates from MDA-MB-231 cells treated with 3-BrPA (160 mmol/l) for 6, 16, and 24 h were subjected to western blot analysis. Data are representative of three independent experiments. (b) MDA-MB-231 cells treated with 3BrPA (160 mmol/l) for 1, 3, and 6 h were subjected to ROS measurement by DHE staining and viewed using fluorescence microscopy. (c) After treatment with 3-BrPA (160 mmol/l) for 1, 3, and 6 h, MDA-MB-231 cells were subjected to ROS measurement by DHE staining and viewed using flow cytometry. (d) MDA-MB-231 cells treated with 3-BrPA (160 mmol/l) or the combination of 3-BrPA (160 mmol/l) and z-VAD-FMK (20 mmol/l) for 24 h were analyzed to determine cell viability using an MTT assay. *P < 0.05. 3-BrPA, 3-bromopyruvate; DHE, dihydroethidium; ROS, reactive oxygen species.
PI3K/Akt, Stat-3, and p38/MAPK pathways [21]. Splicing variations during transcription generate two Mcl-1 mRNA products: Mcl-1L and Mcl-1S [22]. Mcl-1L contains three exons, whereas Mcl-1S only contains exons 1 and 3. Although Mcl-1L and Mcl-1S are encoded by the same gene, they possess opposite functions: Mcl-1L inhibits apoptosis, whereas Mcl-1S promotes apoptosis. Therefore, the proportion of Mcl-1L or Mcl-1S in a cell plays an important role in cell apoptosis. Mcl-1L is localized primarily in the outer mitochondrial membrane and inhibits the release of cytochrome c through the formation of heterodimers or by neutralizing the proapoptotic proteins Bim and Bak. The cleavage product of Mcl-1 can promote cell apoptosis, resulting in more caspase activation [23].
3-BrPA, a small-molecule survivin inhibitor, demonstrates potent antitumor activity in a wide range of human cancer cell lines. Although many cancer cells are sensitive to 3-BrPA-induced apoptosis, others, particularly some breast cancer cells, are resistant to 3-BrPA. In the present study, we demonstrate that MDA-MB-231 cells are sensitive to 3-BrPA-induced apoptosis, whereas MDA-MB-435 cells exhibit resistance to 3-BrPA-induced apoptosis. 3-BrPA is a HK inhibitor that competitively inhibits the uptake of glucose by cancer cells and inhibits both HK and mitochondrial oxidative phosphorylation, leading to decreased ATP production, which in turn leads to cell death [24,25]. Our results demonstrate that 3-BrPA has a significant impact on ROS generation. Mitochondria-associated HK activity
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452 Anti-Cancer Drugs 2014, Vol 25 No 4
Fig. 3
MDA-MB-231
(a) Time (h)
0
6
MDA-MB-435
16
24
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24 -Mcl-1 -β-Actin
Relative expression
Relative expression
1.5
Mcl-1
1.5 ∗
1
∗∗ 0.5
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-p-Akt
-Mcl-1
-Akt
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Relative expression
∗
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∗ 1
(c)
MDA-MB-231
Time (h) 0
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∗
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∗∗
0 0h
6h
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LY294002 0h
6h
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24 h
3-BrPA decreases the expression of Mcl-1 in MDA-MB-231 cells through the PI3K pathway. MDA-MB-231 and MDA-MB-435 cells were exposed to 160 mmol/l of 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 (a) Mcl-1, Akt, and (b) p-Akt. (c) MDA-MB-231 cells were treated with 10 mmol/l of LY294002 for 24 h. The total cell lysates were prepared, and an equal amount of protein was separated using SDS-PAGE, followed by western blot analysis. The membranes were probed for Mcl-1 expression. *P < 0.05, **P < 0.01. 3-BrPA, 3-bromopyruvate; LY294002, 2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one; Mcl-1, myeloid cell leukemia-1; PI3K, phosphoinositide-3-kinase.
plays an important role in preventing mitochondrial ROS generation [26]. In our study, we observed that 3-BrPA induces ROS generation. Therefore, our results imply that 3-BrPA plays a dual role in cell death: inhibition of HK and induction of ROS production [27]. The results of the present work demonstrate that 3-BrPA downregulates the expression of Mcl-1in MDA-MB-231 cells. Generally, Mcl-1 expression can be induced by a variety of signaling pathways, such as the PI3K/Akt, Stat-3, and p38/MAPK pathways. Our results indicate that 3-BrPA downregulates the expression of p-Akt. This observation is consistent with the conclusion that 3-BrPA downregulates the expression of Mcl-1 through the
PI3K/Akt pathway. The PI3K/Akt pathway is an important regulator of the malignant transformation of cells because the pathway can promote cellular aerobic glycolysis. PI3K and Akt are activated through the insulin receptor. This activation stimulates the uptake of glucose and subsequent glycolysis [28]. The release of cytochrome c from the mitochondria to the cytosol is critical for inducing the caspase cascade leading to apoptosis [29,30]. This induction of the caspase cascade was evidenced by the increased activation of caspase-8 and caspase-3. However, z-VAD did not inhibit 3-BrPA-induced apoptosis in the 3-BrPA-sensitive cell line MDA-MB-231, suggesting that 3-BrPA-induced apoptosis is a caspase-independent process. Further, we established that 3-BrPA increases the
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Antitumor effects of 3-bromopyruvate Liu et al. 453
Fig. 4
MDA-MB-435
(a) Control siRNA Mcl-1 siRNA991 Mcl-1 siRNA1114 Mcl-1 siRNA1235
− − − −
+ − − −
− + − −
− − + −
− − − +
Relative expression
1.5
1
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(b) Time (h)
siRNA991 siRNA1114 siRNA1235
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- Cleaved caspase-3 - β-Actin
(c)
120 No 3-BrPA
Cell viability (%)
100 80
3-BrPA
∗ ∗∗
60
∗
40 20 0 Control
NC
SiRNA991
SiRNA1114
SiRNA1235
Downregulation of Mcl-1 induces apoptosis in MDA-MB-435 cells. (a) MDA-MB-435 cells were transfected with the control or Mcl-1 siRNA. After 24 h, the whole-cell lysates were subjected to western blot analysis. (b) MDA-MB-435 cells were transfected with the control or Mcl-1 siRNA. After 24 h, the total cell lysates were prepared, and an equal amount of protein was separated using SDS-PAGE, followed by western blot analysis. The membranes were probed for the activation of caspase-3. The data are representative of three independent experiments. (c) MDA-MB-435 cells were transfected with the control or Mcl-1 siRNA. The cells were incubated for 24 h and then treated with 3-BrPA (160 mmol/l) for 24 h. Cell viability was measured using an MTT assay. **P < 0.01. 3-BrPA, 3-bromopyruvate; Mcl-1, myeloid cell leukemia-1; siRNA, small interfering RNA.
generation of ROS. These results indicated that 3-BrPAinduced caspase-independent apoptosis might be the result of increased ROS production [31].
This study demonstrates that the reduction of Mcl-1 expression alone is not enough to significantly reduce MDA-MB-435 cell survival. Therefore, we combined
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Fig. 5
(a) Relative expression
2.5 MDA-MB-231 − −
Vector alone Mcl-1 cDNA
+ −
− +
∗∗ 2
1 0.5 0
(b)
∗
1.5
Control
Vector alone
Mcl-1 cDNA
3-BrPA 120
3-BrPA+Mcl-1 cDNA ∗
100 Cell viability (%)
∗∗ 80 60 40 20 0 Control
(c) Time (h)
10 20 40 80 3-BrPA concentration (μmol/l)
160
MDA-MB-231 0
6
16
24 -Pro-caspase-3
-Cleaved caspase-3
-β-Actin Upregulation of Mcl-1 protects MDA-MB-231 cells from apoptosis. (a) MDA-MB-231 cells were transfected with the control or Mcl-1 cDNA. After 24 h, the whole-cell lysates were subjected to western blot analysis. (b) MDA-MB-231 cells were transfected with the control or Mcl-1 cDNA. After 24 h, the cells were cultured with 3-BrPA (160 mmol/l) for 24 h, and the cell viability was analyzed using an MTT assay. (c) MDA-MB-231 cells were transfected with the control or Mcl-1 cDNA. After 24 h, the total cell lysates were prepared, and an equal amount of protein was separated using SDS-PAGE, followed by western blotting. The membranes were probed with caspase-3. The data are representative of three independent experiments. *P < 0.05, **P < 0.01. 3-BrPA 3-bromopyruvate; c DNA. complementary DNA; Mcl-1, myeloid cell leukemia-1.
3-BrPA treatment with siRNA-mediated Mcl-1 knockdown, which enhanced the effect of 3-BrPA in the MDA-MB-435 cell line, resulting in a significant decrease in overall cell viability and a significant decrease in the activation of caspase-3. The binding of Mcl-1 to Bak maintains the mitochondrial membrane surface activity and inhibits the release of cytochrome c from the mitochondria, leading to apoptosis. Further, the upregulation of Mcl-1 in the MDAMB-231 cell line demonstrates the important role of Mcl-1 in the response of different breast cancer cells to 3-BrPA – that is, the upregulation of Mcl-1 significantly increases the
cell survival rate. The results obtained using Mcl-1 siRNA, in conjunction with other data, indicate that 3-BrPA induces apoptosis by downregulating the expression of Mcl-1. Conclusion
The results obtained from this study confirm that 3-BrPA induces ROS generation, downregulates Mcl-1, and induces apoptosis in the breast cancer cell line MDAMB-231. Further, 3-BrPA induces apoptosis by downregulating Mcl-1 expression and generating ROS.
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Antitumor effects of 3-bromopyruvate Liu et al. 455
Therefore, 3-BrPA might be clinically useful to overcome the resistance of tumors to cell death; however, further investigation is warranted.
14
Acknowledgements
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This work was supported by the National Natural Science Foundation of China (81000992 and 81072207) and the Natural Science Foundation of Anhui Province (090413135), Graduate Scientific Research and Innovation Projects of Bengbu Medical College of Anhui Province (Byycx1328).
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Conflicts of interest
There are no conflicts of interest. 20
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Shaw RJ. Glucose metabolism and cancer. Curr Opin Cell Biol 2006; 18:598–608. Matoba S, Kang JG, Patino WD, Wragg A, Boehm M, Gavrilova O, et al. p53 regulates mitochondrial respiration. Science 2006; 312:1650–1653. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Rev Cancer 2004; 4:891–899. Gambhir SS. Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer 2002; 2:683–693. Mathupala SP, Ko YH, Pedersen PL. Hexokinase II: cancer’s double-edged sword acting as both facilitator and gatekeeper of malignancy when bound to mitochondria. Oncogene 2006; 25:4777–4786. Mathupala SP, Rempel A, Pedersen PL. Glucose catabolism in cancer cells. Isolation, sequence, and activity of the promoter for type II hexokinase. J Biol Chem 1995; 270:16918–16925. Mandelin AM 2nd, Pope RM. Myeloid cell leukemia-1 as a therapeutic target. Expert Opin Ther Targets 2007; 11:363–373. Scatena R, Bottoni P, Botta G, Martorana GE, Giardina B. The role of mitochondria in pharmacotoxicology: a reevaluation of an old, newly emerging topic. Am J Physiol Cell Physiol 2007; 293:C12–C21. Hacker G, Paschen SA. Therapeutic targets in the mitochondrial apoptotic pathway. Expert Opin Ther Targets 2007; 11:515–526. Ihrlund LS, Hernlund E, Khan O, Shoshan MC. 3-Bromopyruvate as inhibitor of tumour cell energy metabolism and chemopotentiator of platinum drugs. Mol Oncol 2008; 2:94–101. Xu RH, Pelicano H, Zhou Y, Carew JS, Feng L, Bhalla KN, et al. 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. Qin JZ, Xin H, Nickoloff BJ. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines. Biochem Biophys Res Commun 2010; 396:495–500. Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D, Reed JC. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA 1998; 95:4997–5002.
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3-Bromopyruvate induces apoptosis in breast cancer cells by downregulating Mcl-1 through the PI3K/Akt signaling pathway Zhe Liua,*, Yuan-Yuan Zhanga,*, Qian-Wen Zhanga, Su-Rong Zhaoa, Cheng-Zhu Wua, Xiu Chenga, Chen-Chen Jiangb, Zhi-Wen Jianga and Hao Liua The hexokinase inhibitor 3-bromopyruvate (3-BrPA) can inhibit glycolysis in tumor cells to reduce ATP production, resulting in apoptosis. However, as 3-BrPA is an alkylating agent, its cytotoxic action may be induced by other molecular mechanisms. The results presented here reveal that 3-BrPA-induced apoptosis is caspase independent. Further, 3-BrPA induces the generation of reactive oxygen species in MDA-MB-231 cells, leading to mitochondriamediated apoptosis. These results suggest that caspaseindependent apoptosis may be induced by the generation of reactive oxygen species. In this study, we also demonstrated that 3-BrPA induces apoptosis through the downregulation of myeloid cell leukemia-1 (Mcl-1) in MDA-MB-231 breast cancer cells. The results of Mcl-1 knockdown indicate that Mcl-1 plays an important role in 3-BrPA-induced apoptosis. Further, the upregulation of Mcl-1 expression in 3-BrPA-treated MDA-MB-231 cells significantly increases cell viability. In addition, 3-BrPA treatment resulted in the downregulation of p-Akt, suggesting that 3-BrPA may downregulate Mcl-1
through the phosphoinositide-3-kinase/Akt pathway. These findings indicate that 3-BrPA induces apoptosis in breast cancer cells by downregulating Mcl-1 through the phosphoinositide-3-kinase/Akt signaling c 2014 Wolters pathway. Anti-Cancer Drugs 25:447–455 Kluwer Health | Lippincott Williams & Wilkins.
Introduction
of caspase-8, leading to a signaling cascade that culminates in the death of the cell. The intrinsic pathway can be initiated by various intracellular signals, such as a variety of stressors or DNA damage, which lead to the activation of Bax. Bax induces the release of cytochrome c from the mitochondria. Subsequently, cytochrome c binds to Apaf-1, which facilitates the formation of the apoptosome, leading to the activation of caspase-9 and eventual cell death. Bid, which is the specific substrate of caspase-8, plays a key role in these two pathways. Activated caspase-8 cleaves Bid and induces the release of cytochrome c [8,9].
Cancer cells exhibit and rely on increased glycolysis for ATP production [1,2]. Hypoxia caused by the rapid growth of cancer cells limits the traditional synthesis of ATP [3,4]. Therefore, cancer cells require high glucose uptake and accelerated glycolysis rates to survive. Hexokinase (HK) is the first rate-limiting enzyme of glycolysis [5,6], and it catalyzes the phosphorylation of glucose to generate glucose-6-phosphate. Glucose-6phosphate is important particularly for maintaining high levels of glycolysis. Theoretically, HK inhibitors can effectively inhibit cancer cell glycolysis, thereby reducing the ATP supply and inducing apoptosis. Further, HK inhibitors can initiate the mitochondrial apoptosis cascade, inducing apoptosis of cancer cells. Apoptosis is a consequence of a vast array of complex cellular processes and is responsible for maintaining homeostasis and ensuring normal physiological functions within an organism. Apoptosis can occur through two pathways: extrinsic and intrinsic [7]. The extrinsic pathway primarily utilizes extracellular ligands to activate cell surface death receptors, such as the Fas receptor or tumor necrosis factor receptor. The activation of any of the death receptors results in the cleavage and activation c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4973
Anti-Cancer Drugs 2014, 25:447–455 Keywords: apoptosis, breast cancer, 3-bromopyruvate, caspases, myeloid cell leukemia-1, reactive oxygen species a Faculty of Pharmacy, Bengbu Medical College, Bengbu, Anhui, People’s Republic of China and bSchool of Medicine and Public Health, University of Newcastle, Newcastle, New South Wales, Australia
Correspondence to Hao Liu, Faculty of Pharmacy, Bengbu Medical College, Bengbu 233030, Anhui, People’s Republic of China Tel: + 86 552 317 5230; fax: + 86 552 317 5228; e-mail: [email protected] *Zhe Liu and Yuan-Yuan Zhang contributed equally to the writing of this article. Received 18 October 2013 Revised form accepted 25 December 2013
3-Bromopyruvate (3-BrPA) treatment limits HK levels during glycolysis, preventing the production of ATP [10]. In addition, 3-BrPA inhibits glycolysis, leading to the dephosphorylation of Bad, which is linked to glycolysis and apoptosis. Members of the Bcl-2 antiapoptotic family such as Bcl-2 and Bcl-xL can be combined with Bad or Bax to prevent apoptosis. The inhibition of glycolysis induces the dephosphorylation of Bad, migration of Bax to the mitochondria, and release of cytochrome c, leading to the activation of the executioner caspase, caspase3 [11]. 3-BrPA can also induce an increase in superoxide anion levels, which is a key contributor to oxidative DOI: 10.1097/CAD.0000000000000081
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448 Anti-Cancer Drugs 2014, Vol 25 No 4
stress [12]. To determine whether 3-BrPA causes apoptosis by affecting Bcl-2 family proteins, we analyzed the changes in myeloid cell leukemia-1 (Mcl-1) expression in two cancer cell lines after the administration of 3-BrPA. We observed that Mcl-1 was downregulated in MDA-MB-231 cells but not in MDA-MB-435 cells. Moreover, we report here that the small interfering (si) RNA-mediated downregulation of Mcl-1 induced an increase in MDA-MB-435 apoptosis after the administration of 3-BrPA. Therefore, we conclude that Mcl-1 plays an important role in 3-BrPA-induced apoptosis.
Materials and methods Reagents and antibodies
3-BrPA and 2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one (LY294002) were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA). An Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit was purchased from Keygen Biotech (Nanjing, China). Anti-caspase-3 monoclonal antibodies were obtained from Abcam (Cambridge, UK). Mouse monoclonal antibodies against caspase-8 were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). The antibodies for Akt and p-Akt were from Assay Biotech (Los Angeles, California, USA). Rabbit anti-b-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, California, USA). Cell lines and cell culture
Human breast cancer cell lines MDA-MB-231 and MDA-MB-435 were obtained from Shanghai Cell Bank (Shanghai, China). The 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 mmol/l). The cells were maintained at 371C in a 5% CO2 humidified atmosphere. MTT assay
The breast cancer cells were seeded at 1 104 cells/well in a 96-well plate for 24 h and then treated with different concentrations of 3-BrPA. At 24, 48 and 72 h, the cells were incubated with MTT (5 mg/ml in PBS) for 4 h at 371C. After 4 h, the MTT solution was removed and replaced with 150 ml of DMSO/well. The absorbance was measured in a plate reader at a test wavelength of 490 nm. Propidium iodide staining
Before 3-BrPA treatment, cells (2 106) were plated in each well of a six-well plate and allowed to reach exponential growth (24 h). The cells were treated with different concentrations of 3-BrPA (40, 80, and 160 mmol/l) for 24 h, subjected to PI staining, and then evaluated using flow cytometry. Detection of superoxide anion levels
Breast cancer cells were seeded at 2 105 cells/well in a six-well plate. The cells were labeled with 5 mmol/l
dihydroethidium (Beyotime, Jiangsu, China), which was immediately followed by the addition of 3-BrPA (160 mmol/l). After the treatment for the indicated time periods, the cells were harvested, washed, and analyzed by fluorescence-activated cell sorting. Annexin V-FITC/PI apoptosis assay
Cellular apoptosis analysis was carried out using the Annexin V-FITC/PI apoptosis detection kit (Keygen Biotech). Briefly, MDA-MB-231 cells (2 106) were seeded in each well of a six-well plate and treated with 3-BrPA for 24 h. The cells were harvested and collected by centrifugation and resuspended in 200 ml of ice-cold binding buffer. Thereafter, the cells were incubated with 5 ml of Annexin V FITC and 1 ml of PI for 15 min at room temperature in the dark and subsequently analyzed using a flow cytometer. Western blot analysis
The cells were rinsed with ice-cold PBS and lysed in RIPA buffer for 30 min on ice. The cell lysates were centrifuged at 12 000g for 30 min at 41C. The proteins were separated on a 15% SDS-PAGE gel and were subsequently transferred onto a nitrocellulose membrane (Bio-Rad). The membranes were incubated with the appropriate primary antibody overnight at 41C and further incubated with the corresponding secondary antibody. b-Actin was used as a loading control. siRNA
Mcl-1 siRNA (50 -GGGACUGGCUAGUUAAACATT-30 ) and nonsilencing siRNA (50 -UUCUCCGAACGUGUCACG UTT-30 ) were synthesized by Shanghai GenePharma (Shanghai, China). MDA-MB-435 cells were plated at 5 105 cells/well in a six-well plate and transfected with 20 nmol/l siRNA in Opti-MEM medium (Invitrogen, Carlsbad, California, USA) using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. To confirm the knockdown efficiency, cell extracts were prepared and subjected to SDS-PAGE, followed by immunoblotting. Plasmid transfection
The pCMV-HA-Mcl-1 plasmid and control plasmid were obtained from GenePharma. The plasmids were transfected into MDA-MB-231 cells using lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s transfection protocol. After 24 h of incubation, total cell lysates were prepared for immunoblot analysis. Statistical analysis
Statistical analyses were carried out using one-way analysis of variance. The symbol ‘*’ indicates that the values are significantly different from those of the control (*P < 0.05; **P < 0.01; ***P < 0.001).
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Antitumor effects of 3-bromopyruvate Liu et al. 449
Results Differential 3-BrPA-mediated cytotoxicity in breast cancer cells
To study the effect of 3-BrPA on cell proliferation, two breast cancer cell lines were treated with different concentrations of 3-BrPA for 24, 48, and 72 h. We established that 3-BrPA induced MDA-MB-231 cell apoptosis and inhibited MDA-MB-231 cell proliferation (Fig. 1a and b). The results demonstrated that the rate of cell proliferation had an inverse relationship with both the time of 3-BrPA exposure and concentration of 3-BrPA (Fig. 1a). However, 3-BrPA did not induce cytotoxicity in the MDA-MB-435 cell line (Fig. 2a and b). Annexin V and PI were used to determine the type of cell death induced by 3-BrPA in MDA-MB-231 cells. After 24 h of 3-BrPA treatment, the percentage of the apoptotic cells was increased significantly from 5.5 to 22.2%, and the percentage of necrotic cells increased from 0.9 to 10.7% (Fig. 1d). Taken together, these data suggest that 3-BrPA induces apoptosis in MDA-MB-231 cells and that MDA-MB-435 cells are not sensitive to 3-BrPA. 3-BrPA-induced apoptosis is independent of caspase activity
To verify whether 3-BrPA-induced cell death observed in MDA-MB-231 cells was a result of apoptosis, we performed immunoblotting to detect caspase activation. The cleaved products of caspase-8 and caspase-3 were detected in the MDA-MB-231 cells, indicating that 3-BrPA-induced cell death was apoptotic (Fig. 2a). To determine whether caspases play a role in cell death induced by 3-BrPA, we used the pan-caspase inhibitor z-VAD-FMK (Sigma Chemical Co.) to treat the 3-BrPAsensitive MDA-MB-231 cells. z-VAD treatment did not inhibit 3-BrPA-induced cell death (Fig. 2d), indicating a caspase-independent killing mechanism [13]. During cellular aerobic respiration and mitochondrial electron transport, a small portion of the oxygen necessary for these processes cannot be reduced completely, resulting in the generation of reactive oxygen species (ROS) with a strong oxidizing potential, such as the superoxide anion. To maintain a relatively stable level of intracellular ROS, intracellular antioxidant enzymes remove ROS that is produced continuously through metabolic processes. However, under certain conditions, the homeostasis between ROS production and antioxidant activity, which initiates a series of reactions, leads to apoptosis [14,15]. In this study, 3-BrPA treatment increased the generation of ROS (Fig. 2b and c). These results indicate that 3-BrPA-induced caspase-independent apoptosis might occur as a result of increased ROS production. 3-BrPA decreases the expression of Mcl-1 in MDA-MB-231 cells through the PI3K pathway
Mcl-1 and Bcl-2 family proteins are similar in structure and function, and they are involved primarily in the
maintenance of mitochondrial membrane stability and in the inhibition of the release of cytochrome c through the phosphoinositide-3-kinase (PI3K) pathway, thus protecting cells from apoptosis. 3-BrPA treatment downregulated the expression of Mcl-1 (Fig. 3a), and the PI3K/Akt inhibitor LY294002 also decreased the expression of Mcl-1 (Fig. 3b). To confirm whether 3-BrPA downregulated the expression of Mcl-1 through the PI3K/Akt pathway, we examined the expression of p-Akt in 3-BrPA-treated MDA-MB-231 cells. Our data revealed that 3-BrPA downregulated p-Akt (Fig. 3b). On the basis of these results, we hypothesize that 3-BrPA decreases the expression of Mcl-1 through the PI3K/Akt pathway. Downregulation of Mcl-1 induces apoptosis in MDA-MB-435 cells
To examine the functional significance of Mcl-1 expression in the sensitization of breast cancer cells to 3-BrPA, siRNA-mediated silencing of Mcl-1 was performed, and the role of Mcl-1 expression in the sensitivity of MDAMB-435 cells to 3-BrPA-induced apoptosis was assessed. Western blot analysis confirmed that Mcl-1 expression was reduced substantially by the Mcl-1-specific siRNA (Fig. 4a). The downregulation of Mcl-1 expression in the MDA-MB-435 cells resulted in increased sensitivity to 3-BrPA-induced antiproliferation (Fig. 4c) and activation of caspase-3 expression (Fig. 4b). These results demonstrate that the downregulation of Mcl-1 expression is critical to 3-BrPA-induced apoptosis in breast cancer cells. Upregulation of Mcl-1 protects MDA-MB-231 cells from apoptosis
To further confirm the role of Mcl-1 in 3-BrPA-induced apoptosis, we transfected complementary DNA encoding Mcl-1 into MDA-MB-231 cells (Fig. 5a). The overexpression of Mcl-1 significantly increased cell viability after the administration of 3-BrPA, indicating that the cells were protected from apoptosis (Fig. 5b). The cleaved products of caspase-3 were nearly undetectable in the Mcl-1-overexpressing MDA-MB-231 cells (Fig. 5c). Taken together, these results further confirm that Mcl-1 plays an important role in 3-BrPA-induced apoptosis.
Discussion The Bcl-2 protein family, which includes antiapoptotic and proapoptotic proteins, is the primary regulator of apoptosis, and the balance between antiapoptotic and proapoptotic proteins is the key to cell survival [16]. Mcl-1 is an antiapoptotic member of the Bcl-2 family of proteins and plays an integral role in cell survival and apoptosis [17,18]. Mcl-1 is overexpressed in a variety of cancers and contributes to cancer cell survival and apoptosis resistance [19]. Although the overexpression of Mcl-1 does not lead to increased cell proliferation, the ability of Mcl-1 to suppress apoptosis is an important contributor to the transformed state [20]. Generally, Mcl-1 expression can be induced by a variety of signaling pathways, such as the
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Fig. 1
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Concentrations of 3-BrPA (μmol/l)
Differential 3-BrPA-mediated cytotoxicity of breast cancer cells. (a) MDA-MB-231 and MDA-MB-435 cells were cultured with various concentrations of 3-BrPA (20, 40, 80, and 160 mmol/l) for 24, 48, and 72 h, and the cell viability was analyzed using an MTT assay. (b) MDA-MB-231 and MDA-MB435 cells were treated with various concentrations of 3-BrPA (20, 40, 80, and 160 mmol/l) for 24 h before being analyzed using flow cytometry. (c) Bar chart presentation of the distribution of apoptotic cells in a population. This figure represents three independent experiments. (d) Distribution of viable, apoptotic, and necrotic MDA-MB-231 cells in the presence of various concentrations of 3-BrPA (20, 40, 80, and 160 mmol/l) for 24 h, as measured by Annexin V/PI staining. Apoptosis was measured by the propidium iodide method using flow cytometry. *P < 0.05. 3-BrPA, 3-bromopyruvate.
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Antitumor effects of 3-bromopyruvate Liu et al. 451
Fig. 2
MDA-MB-231
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3-BrPA-induced apoptosis is independent of caspase activity. (a) Whole-cell lysates from MDA-MB-231 cells treated with 3-BrPA (160 mmol/l) for 6, 16, and 24 h were subjected to western blot analysis. Data are representative of three independent experiments. (b) MDA-MB-231 cells treated with 3BrPA (160 mmol/l) for 1, 3, and 6 h were subjected to ROS measurement by DHE staining and viewed using fluorescence microscopy. (c) After treatment with 3-BrPA (160 mmol/l) for 1, 3, and 6 h, MDA-MB-231 cells were subjected to ROS measurement by DHE staining and viewed using flow cytometry. (d) MDA-MB-231 cells treated with 3-BrPA (160 mmol/l) or the combination of 3-BrPA (160 mmol/l) and z-VAD-FMK (20 mmol/l) for 24 h were analyzed to determine cell viability using an MTT assay. *P < 0.05. 3-BrPA, 3-bromopyruvate; DHE, dihydroethidium; ROS, reactive oxygen species.
PI3K/Akt, Stat-3, and p38/MAPK pathways [21]. Splicing variations during transcription generate two Mcl-1 mRNA products: Mcl-1L and Mcl-1S [22]. Mcl-1L contains three exons, whereas Mcl-1S only contains exons 1 and 3. Although Mcl-1L and Mcl-1S are encoded by the same gene, they possess opposite functions: Mcl-1L inhibits apoptosis, whereas Mcl-1S promotes apoptosis. Therefore, the proportion of Mcl-1L or Mcl-1S in a cell plays an important role in cell apoptosis. Mcl-1L is localized primarily in the outer mitochondrial membrane and inhibits the release of cytochrome c through the formation of heterodimers or by neutralizing the proapoptotic proteins Bim and Bak. The cleavage product of Mcl-1 can promote cell apoptosis, resulting in more caspase activation [23].
3-BrPA, a small-molecule survivin inhibitor, demonstrates potent antitumor activity in a wide range of human cancer cell lines. Although many cancer cells are sensitive to 3-BrPA-induced apoptosis, others, particularly some breast cancer cells, are resistant to 3-BrPA. In the present study, we demonstrate that MDA-MB-231 cells are sensitive to 3-BrPA-induced apoptosis, whereas MDA-MB-435 cells exhibit resistance to 3-BrPA-induced apoptosis. 3-BrPA is a HK inhibitor that competitively inhibits the uptake of glucose by cancer cells and inhibits both HK and mitochondrial oxidative phosphorylation, leading to decreased ATP production, which in turn leads to cell death [24,25]. Our results demonstrate that 3-BrPA has a significant impact on ROS generation. Mitochondria-associated HK activity
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452 Anti-Cancer Drugs 2014, Vol 25 No 4
Fig. 3
MDA-MB-231
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3-BrPA decreases the expression of Mcl-1 in MDA-MB-231 cells through the PI3K pathway. MDA-MB-231 and MDA-MB-435 cells were exposed to 160 mmol/l of 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 (a) Mcl-1, Akt, and (b) p-Akt. (c) MDA-MB-231 cells were treated with 10 mmol/l of LY294002 for 24 h. The total cell lysates were prepared, and an equal amount of protein was separated using SDS-PAGE, followed by western blot analysis. The membranes were probed for Mcl-1 expression. *P < 0.05, **P < 0.01. 3-BrPA, 3-bromopyruvate; LY294002, 2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one; Mcl-1, myeloid cell leukemia-1; PI3K, phosphoinositide-3-kinase.
plays an important role in preventing mitochondrial ROS generation [26]. In our study, we observed that 3-BrPA induces ROS generation. Therefore, our results imply that 3-BrPA plays a dual role in cell death: inhibition of HK and induction of ROS production [27]. The results of the present work demonstrate that 3-BrPA downregulates the expression of Mcl-1in MDA-MB-231 cells. Generally, Mcl-1 expression can be induced by a variety of signaling pathways, such as the PI3K/Akt, Stat-3, and p38/MAPK pathways. Our results indicate that 3-BrPA downregulates the expression of p-Akt. This observation is consistent with the conclusion that 3-BrPA downregulates the expression of Mcl-1 through the
PI3K/Akt pathway. The PI3K/Akt pathway is an important regulator of the malignant transformation of cells because the pathway can promote cellular aerobic glycolysis. PI3K and Akt are activated through the insulin receptor. This activation stimulates the uptake of glucose and subsequent glycolysis [28]. The release of cytochrome c from the mitochondria to the cytosol is critical for inducing the caspase cascade leading to apoptosis [29,30]. This induction of the caspase cascade was evidenced by the increased activation of caspase-8 and caspase-3. However, z-VAD did not inhibit 3-BrPA-induced apoptosis in the 3-BrPA-sensitive cell line MDA-MB-231, suggesting that 3-BrPA-induced apoptosis is a caspase-independent process. Further, we established that 3-BrPA increases the
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Antitumor effects of 3-bromopyruvate Liu et al. 453
Fig. 4
MDA-MB-435
(a) Control siRNA Mcl-1 siRNA991 Mcl-1 siRNA1114 Mcl-1 siRNA1235
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(c)
120 No 3-BrPA
Cell viability (%)
100 80
3-BrPA
∗ ∗∗
60
∗
40 20 0 Control
NC
SiRNA991
SiRNA1114
SiRNA1235
Downregulation of Mcl-1 induces apoptosis in MDA-MB-435 cells. (a) MDA-MB-435 cells were transfected with the control or Mcl-1 siRNA. After 24 h, the whole-cell lysates were subjected to western blot analysis. (b) MDA-MB-435 cells were transfected with the control or Mcl-1 siRNA. After 24 h, the total cell lysates were prepared, and an equal amount of protein was separated using SDS-PAGE, followed by western blot analysis. The membranes were probed for the activation of caspase-3. The data are representative of three independent experiments. (c) MDA-MB-435 cells were transfected with the control or Mcl-1 siRNA. The cells were incubated for 24 h and then treated with 3-BrPA (160 mmol/l) for 24 h. Cell viability was measured using an MTT assay. **P < 0.01. 3-BrPA, 3-bromopyruvate; Mcl-1, myeloid cell leukemia-1; siRNA, small interfering RNA.
generation of ROS. These results indicated that 3-BrPAinduced caspase-independent apoptosis might be the result of increased ROS production [31].
This study demonstrates that the reduction of Mcl-1 expression alone is not enough to significantly reduce MDA-MB-435 cell survival. Therefore, we combined
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Anti-Cancer Drugs 2014, Vol 25 No 4
Fig. 5
(a) Relative expression
2.5 MDA-MB-231 − −
Vector alone Mcl-1 cDNA
+ −
− +
∗∗ 2
1 0.5 0
(b)
∗
1.5
Control
Vector alone
Mcl-1 cDNA
3-BrPA 120
3-BrPA+Mcl-1 cDNA ∗
100 Cell viability (%)
∗∗ 80 60 40 20 0 Control
(c) Time (h)
10 20 40 80 3-BrPA concentration (μmol/l)
160
MDA-MB-231 0
6
16
24 -Pro-caspase-3
-Cleaved caspase-3
-β-Actin Upregulation of Mcl-1 protects MDA-MB-231 cells from apoptosis. (a) MDA-MB-231 cells were transfected with the control or Mcl-1 cDNA. After 24 h, the whole-cell lysates were subjected to western blot analysis. (b) MDA-MB-231 cells were transfected with the control or Mcl-1 cDNA. After 24 h, the cells were cultured with 3-BrPA (160 mmol/l) for 24 h, and the cell viability was analyzed using an MTT assay. (c) MDA-MB-231 cells were transfected with the control or Mcl-1 cDNA. After 24 h, the total cell lysates were prepared, and an equal amount of protein was separated using SDS-PAGE, followed by western blotting. The membranes were probed with caspase-3. The data are representative of three independent experiments. *P < 0.05, **P < 0.01. 3-BrPA 3-bromopyruvate; c DNA. complementary DNA; Mcl-1, myeloid cell leukemia-1.
3-BrPA treatment with siRNA-mediated Mcl-1 knockdown, which enhanced the effect of 3-BrPA in the MDA-MB-435 cell line, resulting in a significant decrease in overall cell viability and a significant decrease in the activation of caspase-3. The binding of Mcl-1 to Bak maintains the mitochondrial membrane surface activity and inhibits the release of cytochrome c from the mitochondria, leading to apoptosis. Further, the upregulation of Mcl-1 in the MDAMB-231 cell line demonstrates the important role of Mcl-1 in the response of different breast cancer cells to 3-BrPA – that is, the upregulation of Mcl-1 significantly increases the
cell survival rate. The results obtained using Mcl-1 siRNA, in conjunction with other data, indicate that 3-BrPA induces apoptosis by downregulating the expression of Mcl-1. Conclusion
The results obtained from this study confirm that 3-BrPA induces ROS generation, downregulates Mcl-1, and induces apoptosis in the breast cancer cell line MDAMB-231. Further, 3-BrPA induces apoptosis by downregulating Mcl-1 expression and generating ROS.
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Antitumor effects of 3-bromopyruvate Liu et al. 455
Therefore, 3-BrPA might be clinically useful to overcome the resistance of tumors to cell death; however, further investigation is warranted.
14
Acknowledgements
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This work was supported by the National Natural Science Foundation of China (81000992 and 81072207) and the Natural Science Foundation of Anhui Province (090413135), Graduate Scientific Research and Innovation Projects of Bengbu Medical College of Anhui Province (Byycx1328).
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Conflicts of interest
There are no conflicts of interest. 20
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