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Ammonia mediates mitochondrial uncoupling and promotes glycolysis via HIF-1 activation in human breast cancer MDA-MB-231 cells
Biochemical and Biophysical Research Communications 519 (2019) 153e159
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Ammonia mediates mitochondrial uncoupling and promotes glycolysis via HIF-1 activation in human breast cancer MDA-MB231 cells Yapeng Lu*, Lu Wang, Wangwang Ding, Dan Wang, Xueting Wang, Qianqian Luo, Li Zhu** Institute of Special Environmental Medicine, Nantong University, Nantong, 226019, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 August 2019 Accepted 28 August 2019 Available online 31 August 2019
It has been reported that ammonia produced by glutaminolysis activates the HIF-1 pathway in several types of cancer cells, but the underlying mechanisms remain unclear. In this study, the effects of ammonia on the activation of HIF-1 pathway and glycolysis in MDA-MB-231 breast cancer cells were investigated and the underlying mechanisms involved were elucidated. The results showed that NH4Cl concentration-dependently increased the protein level of HIF-1a and enhanced the transactivation activity of HIF-1 in MDA-MB-231 cells. In addition, NH4Cl increased the expression of GluT1 and LDHA and promoted aerobic glycolysis by activating the HIF-1 pathway. Further study revealed that NH4Cl increased the mitochondrial ROS level and decreased the cellular Fe2þ level in MDA-MB-231 cells. Activation of the HIF-1 pathway induced by NH4Cl was inhibited by addition of the antioxidant NAC or the NADPH oxidase (NOX) inhibitor apocynin, indicating the involvement of the NOX-induced ROS generation. When MDA-MB-231 cells were treated with NH4Cl, the oxygen consumption of cells increased, followed by the decreased mitochondrial membrane potential and cellular ATP level, indicating the uncoupling of mitochondria. In conclusion, NH4Cl activated the HIF-1 signaling pathway and promoted aerobic glycolysis in MDA-MB-231 cells, likely through the promotion of mitochondrial ROS release and mitochondrial uncoupling. © 2019 Elsevier Inc. All rights reserved.
Keywords: Ammonia HIF-1 pathway Mitochondrial ROS Mitochondrial uncoupling
1. Introduction Cancer is a kind of multifactorial disease and the second leading cause of human death [1]. Modern medical research shows that reprogramming of metabolism is one of the classical characteristics of cancer [2]. Especially, reprogramming of glucose metabolism from oxidative phosphorylation to glycolysis is very important for the growth and survival of cancer cells [3]. In the 1920s, Warburg and colleagues found that cancer cells are partly or mainly dependent on glycolysis to produce ATP in spite of oxygen is sufficient, a phenomenon known as aerobic glycolysis [4]. More and more evidence shows that aerobic glycolysis is an adaptive metabolic shift, which effectively supports the synthesis of biological macromolecules in the process of rapid proliferation of cancer cells [5]. Although the mechanism of aerobic glycolysis in cancer cells is
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Lu), [email protected] (L. Zhu). https://doi.org/10.1016/j.bbrc.2019.08.152 0006-291X/© 2019 Elsevier Inc. All rights reserved.
still not fully elucidated, HIF-1 has been identified as the key regulator of aerobic glycolysis and plays an important role in the proliferation of cancer cells [6]. HIF-1 is a heterodimer composed of HIF-1a and HIF-1b [7]. Under normoxic conditions, the proline and aspartate in the oxygen-dependent degradation domain of HIF-1a are hydroxylated by proline hydroxylases (PHDs), and eventually degraded by the ubiquitin-proteasome system [8,9]. Under hypoxic or some stress conditions, the activity of PHDs is inhibited and HIF-1a enters the nucleus, forming a heterodimer with HIF-1b, binding to the promoter regions of the downstream target genes [10,11]. Most of the HIF-1-targeted genes are closely associated with cancer development [12]. In particular, the genes involved in the glycolysis including PDK1, LDHA, GluT1, HK2 and microRNA-210, etc. are directly regulated by HIF-1 [13,14]. Recently, more and more evidence have shown that glutamine metabolism plays an irreplaceable role in fueling growth and proliferation of cancer cells [15,16]. Glutamine is catalyzed by glutaminase to produce ammonia and glutamate, through which a large amount of ammonia is released. It has been reported that ammonia
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produced by glutaminolysis activates the HIF-1 signaling pathway in several types of cancer cells [17,18]. Therefore, ammonia may be a key molecule linking the glutamine metabolism and the aerobic glycolysis. But to date, the underlying mechanism by which ammonia activates the HIF-1 signaling pathway remains unclear. In this study, the activation effect of ammonia on the HIF-1 signaling pathway and the aerobic glycolysis in breast cancer cell line MDAMB-231 was studied, and the possible underlying mechanisms were elucidated.
2.5. Detection of cellular oxygen consumption The cultured MDA-MB-231 cells were made into a single cell suspension (1 106/ml) and treated with 5 mM NH4Cl. Oxygen consumption in cells were assessed polarographically using a thermostatically controlled (37 C) Clark-type oxygen electrode (Hansatech instruments, UK).
2.6. Measurement of mitochondrial membrane potential 2. Materials and methods 2.1. Materials MitoSOX fluorescence probe was purchased from Invitrogen Corporation (Carlsbad, USA). HIF-1a antibody was purchased from BD Biosciences (San Diego, CA, USA). VEGFA and b-actin antibodies were purchased from Cell Signaling Technology (Beverly, USA). GluT1 and LDHA antibodies were purchased from Abcam (Cambridge, UK). Trizol was obtained from Sigma-Aldrich Corp. (St. Louis, MO, US). The ATP assay kit and JC-1 fluorescence probe were obtained from Beyotime Biotechnology Corporation (Shanghai, China). Cell culture reagents were obtained from Gibco (Carlsbad, USA). 2.2. Cell culture The human breast cancer cell lines MDA-MB-231 and MCF-7 were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). After receiving the cell lines, the frozen stocks were established immediately. Only the early passage cells (<2 months) from the initially established frozen cell lines were used in this study. Cells were cultured at 37 C in a humidified 5% CO2 incubator (Thermo Forma Electron Co., Marietta, OH, USA). 2.3. Western blot analysis The cells were homogenized in a lysis buffer containing 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 0.2 mM PMSF, and 1.0 mM DTT (pH 7.6). The protein concentration was quantified by BCA assay. The protein samples were subjected to SDSpolyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). Then the membranes were incubated with HIF-1a antibody (1:500), VEGFA antibody (1:600), GluT1 antibody (1:800) and LDHA antibody (1:800), at 4 C overnight. Finally, IRDye 800-conjugated affinity purified IgG (1:10000) was applied at room temperature for 2 h and the membranes were scanned with the ODYSSEY® Infrared Imaging System (LI-COR, Lincoln, NE, USA). 2.4. Quantitative real-time PCR (qRT-PCR) Total cellular RNA was extracted using the Trizol RNA Purification Kit. Then, the RNA was reverse transcribed into cDNA using the Omniscript RT kit. qRT-PCR amplifications were conducted in triplicate and performed on a 7300 real-time PCR system (Applied Biosystems, CA, USA). The relative mRNA levels were normalized against b-actin. The primers were as follows: HIF-1a, 50 - AAG TGT ACC CTA ACT AGC CG-30 (forward), 50 - CAC AAA TCA GCA CCA AGC30 (backward); b-actin, 50 -TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-30 (forward), 50 -CTA GAA GCA TTG CGG TCG ACG ATG GAG GG30 (backward); GluT1,50 -ATT GGC TCC GGT ATC GTC AAC -30 (forward), 50 -GCT CAG ATA GGA CAT CCA GGG TA-30 (backward); LDHA, 50 -TTG ACC TAC GTG GCT TGG AAG-30 (forward), 50 -GGT AAC GGA ATC GGG CTG AAT-30 (backward).
Mitochondrial membrane potential changes in cells were measured using a fluorescent probe JC-1. Briefly, the cells were incubated with 5 mg/ml JC-1 at 37 C for 30 min after treated with 5 mM NH4Cl for 6 h. Then, the cells were observed by a fluorescence microscope (Leica DMI 4000B, Germany). A 488 nm filter was used for the excitation of JC-1. Emission filters of 535 nm and 595 nm were used to observe JC-1 monomers and JC-1 aggregates, respectively.
2.7. Measurement of cellular ATP After the cells were treated with various concentrations of NH4Cl for 6 h, the cellular ATP content was determined using a commercial kit according to the manufacturer's instructions.
2.8. Quantification of mitochondrial ROS levels The mitochondrial ROS level was analyzed using a fluorescent probe MitoSOX. In brief, the cells were loaded with 5 mM MitoSOX for 15 min at 37 C after treated with 5 mM NH4Cl for 6 h. Then the cells were observed by a fluorescence microscope (Leica DMI 4000B, Germany). The fluorescence intensity represents the mitochondrial ROS level.
2.9. Glucose and lactate measurement After the cells were treated with 5 mM NH4Cl for 24 h, the supernatants were collected. Then the glucose consumption and lactate release were measured using a Glucose Assay Kit (Applygen Technologies, Beijing, China) and a Lactate Assay Kit (BioVision, Inc, CA, USA) according to the manufacturer’ s indication.
2.10. Luciferase assay Luciferase assay was performed according to the method we reported previously [19]. In brief, HRE-luciferase plasmid was cotransfected with an internal control vector pRL-TK (Promega, WI, USA) (100:1 ratio) into the cells by Lipofectamine 2000 (Invitrogen, CA, USA). After the cells were treated with NH4Cl for 24 h, luciferase activity was quantitated using Dual-Luciferase Reporter System (Promega, WI, US).
2.11. Statistical analysis All data are presented as mean ± S.D. of at least three independent experiments. Statistical analysis was done using GraphPad Prism 6. The statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. Differences were considered statistically significant at p < 0.05.
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3. Results 3.1. NH4Cl activated the HIF-1 signaling pathway in MDA-DB231 cells Treatment of MDA-DB-231 cells and MCF-7 cells with 0.5-5 mM NH4Cl for 24 h did not affect cell viability (data not shown). Western blot analysis showed that NH4Cl up-regulated HIF-1a protein level in a concentration-dependent manner in these two cell lines (Fig. 1A and B). The protein level of HIF-1b did not change significantly under the same conditions in MDA-DB-231 cells. The effect of NH4Cl on the transactivation activity of HIF-1 was examined by luciferase assay. The results showed that NH4Cl increased the luciferase reporter gene activity in a concentration-dependent manner in MDA-DB-231 cells (Fig. 1C). As expected, NH4Cl also promoted the expression of VEGFA, a well-known downstream target of HIF-1 (Fig. 1D and E).
3.2. NH4Cl promoted glycolysis in MDA-DB-231 cells via the HIF-1 signaling pathway The transcription level of the HIF-1-target genes that regulate glycolysis was detected by qRT-PCR after MDA-DB-231 cells were treated with 0.5e5 mM of NH4Cl for 24 h. The results clearly showed that NH4Cl increased the mRNA level of LDHA and GluT1 in a concentration-dependent manner (Fig. 2A and B). Western blot analysis displayed that 5 mM NH4Cl significantly increased the
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protein level of LDHA and GluT1 (Fig. 2C). Under the same conditions, glucose consumption and lactate release in MDA-DB231 cells was also increased significantly (Fig. 2D). When the expression of HIF-1a was down-regulated by siRNA, the protein level of LDHA and GluT1 was significantly decreased, and so was the glucose consumption and lactate secretion, suggesting the decrease in glycolytic activity in MDA-DB-231 cells (Fig. 2C and D).
3.3. NH4Cl activated the HIF-1 signaling pathway in MDA-DB231 cells via mitochondrial ROS generation The effect of NH4Cl on the mRNA level of HIF-1a in MDA-DB231 cells was examined by qRT-PCR. It was found that NH4Cl did not affect the mRNA level of HIF-1a at 0.5e5 mM (Fig. 3A). The mitochondrial ROS was labeled by MitoSOX fluorescence and the effect of NH4Cl on mitochondrial ROS level was detected. The results showed that 5 mM NH4Cl treatment significantly enhanced the fluorescence intensity of mitoSOX, indicating the increased mitochondrial ROS level (Fig. 3B). Then, the intracellular Fe2þ level was detected and the results indicated that 5 mM NH4Cl induced a decrease in intracellular Fe2þ level (Fig. 3C). When the cells were pre-treated with the antioxidant NAC or the NOX inhibitor apocinin, NH4Cl-induced mitochondrial ROS generation was reduced (Fig. 3B) and activation of the HIF-1 signaling pathway was also inhibited, as was characterized by the decreased protein level of HIF-1a and the reduced transactivation activity of HIF-1 (Fig. 3D and E).
Fig. 1. NH4Cl activated the HIF-1 signaling pathway in MDA-DB-231 cells. (A) MDA-MB-231 cells were treated with various concentrations of NH4Cl for 24 h and the protein level of HIF-1a and HIF-1b was detected by Western blotting. (B) MCF-7 cells were treated with various concentrations of NH4Cl for 24 h and the protein level of HIF-1a was detected by Western blotting. (C) MDA-MB-231 cells were transiently cotransfected with the HRE-luciferase plasmid and an internal control vector pRL-TK for 24 h, and then treated with various concentrations of NH4Cl for 24 h. Finally, the luciferase activity was quantitated. Data are expressed as mean ± S. D. (n ¼ 4). **p < 0.01 and ***p < 0.001 versus CON. (D) MDA-MB-231 cells were treated with various concentrations of NH4Cl for 24 h and the mRNA level of VEGFA was analyzed by qRT-PCR and normalized with b-actin. Data are expressed as means ± S. D. (n ¼ 3). **p < 0.01 and ***p < 0.001 versus CON. (E) MDAMB-231 cells were treated with various concentrations of NH4Cl for 24 h and the protein level of VEGFA was detected by Western blotting.
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Fig. 2. NH4Cl promoted glycolysis in MDA-DB-231 cells via the HIF-1 signaling pathway. (A) and (B) MDA-MB-231 cells were treated with various concentrations of NH4Cl for 24 h and the mRNA level of GluT1 and LDHA was analyzed by qRT-PCR and normalized with bactin. Data are expressed as means ± S. D. (n ¼ 3). *p < 0.05 and ***p < 0.001 versus CON. (C) MDA-MB-231 cells were transfected with HIF-1a siRNA for 48 h. Then the cells were treated 5 mM NH4Cl for 24 h and the protein level of GluT1 and LDHA was analyzed by Western blotting. (D) MDA-MB-231 cells were transfected with HIF-1a siRNA for 48 h. Then the cells were treated with 5 mM NH4Cl for 24 h and the glucose consumption and lactate release was detected by commercial kits according to the manufacturer’ s indication. Data are expressed as means ± S. D. (n ¼ 4). **p < 0.01 and ***p < 0.001 versus NC, #p < 0.05 and ##p < 0.01 versus NC þ NH4Cl-treated group.
3.4. NH4Cl induced mitochondrial uncoupling in MDA-DB-231 cells After MDA-DB-231 cells were treated with various concentrations of NH4Cl for 6 h, the intracellular ATP level was detected. The results showed that NH4Cl induced a concentration-dependently decrease in intracellular ATP level (Fig. 4A). Furthermore, the oxygen consumption in MDA-DB-231 cells at present of 5 mM NH4Cl was significantly increased, indicating the enhanced mitochondrial respiration (Fig. 4B and C). By JC-1 fluorescent labeling, we found that 5 mM NH4Cl treatment decreased the mitochondrial membrane potential in MDA-MB-231 cells, which was manifested by an increase in green JC-1 monomer (Fig. 4D). Taken together, the above results indicated that NH4Cl induced mitochondrial uncoupling in MDA-DB-231 cells. 4. Discussion In recent years, the role of glutamine metabolism in cancer development has received more and more attention [20]. Cancer cells consume a large amount of glutamine during rapid proliferation which makes the concentration of ammonia high in cancer cells [21]. Increasing evidence suggests that ammonia is more than
a cellular waste and serves as an intracellular signaling molecule under sub-toxic conditions [22]. For example, it was found that ammonia is an autophagy regulator in MDA-MB-231 cells and mouse myoblast C2C12 [23]. In addition, ammonia produced by glutaminolysis activates the HIF-1 signaling pathway in several types of cancer cells [17,18]. In this study, we also identified that NH4Cl at low concentrations activated the HIF-1 signaling pathway in breast cancer MDB-DA-231 cells under normoxic conditions. When the HIF-1 signaling pathway is activated, HIF-1a undergoes nuclear translocation and binds to HIF-1b to form a heterodimer, which regulates the transcription of the downstream target genes [24]. In this study, we found that NH4Cl promoted the transactivation activity of HIF-1. Furthermore, NH4Cl up-regulated the mRNA and protein level of VEGFA, a classical target of HIF-1, in a concentration-dependent manner, which further confirmed the activation of HIF-1 signaling pathway by NH4Cl. HIF-1 plays an important role in the development of cancer, including promoting cell proliferation through reprogramming of glucose metabolism [25]. HIF-1 regulates glycolysis by upregulating the expression of glycolysis-related genes such as HK2, GluT1, LDHA, and PKM2 et al., promoting glucose uptake and lactate release [26]. In the current study, we found that NH4Cl up-
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Fig. 3. NH4Cl activated the HIF-1 signaling pathway in MDA-DB-231 cells via mitochondrial ROS generation. (A) MDA-MB-231 cells were treated with various concentrations of NH4Cl for 24 h and the mRNA level of HIF-1a was analyzed by qRT-PCR and normalized with b-actin. (B) MDAMB-231 cells were treated with NAC and apocynin with or without 5 mM NH4Cl for 24 h and the mitochondrial ROS level was detected by mitoSOX labeling. (C) MDA-MB-231 cells were treated with 5 mM NH4Cl for 24 h and intracellular ferrous ion level was detected by a commercial kit according to the manufacturer’ s indication. Data are expressed as means ± S. D. (n ¼ 4). *p < 0.05 versus CON. (D) MDA-MB-231 cells were treated with NAC and apocynin with or without 5 mM NH4Cl for 24 h and the protein level of HIF-1a was detected by Western blotting. (E) MDA-MB-231 cells were transiently cotransfected with the HRE-luciferase plasmid and an internal control vector pRL-TK for 24 h. Then the cells were treated with NAC and apocynin with or without 5 mM NH4Cl for 24 h and the luciferase activity was quantitated. Data are expressed as mean ± S. D. (n ¼ 4). ***p < 0.001 versus CON, ###p < 0.001 versus NH4Cl-treated group.
regulated the mRNA and protein levels of GluT1 and LDHA and promoted glucose uptake and lactate release in MDB-DA-231 cells. As expected, the NH4Cl-induced activation of glycolysis in MDBDA-231 cells is HIF-1 dependent. PHDs are known to play a key role in regulating the HIF-1 signaling pathway via regulating the stability of HIF-1a. Hydroxylated HIF-1a by PHDs is then ubiquitinated and finally degraded by the proteasome [8,9]. In addition to hypoxia-induced inactivation of PHDs, the activities of PHDs are also inhibited by mitochondrial-driven ROS [27,28]. Mitochondria-derived ROS decreases intracellular Fe2þ levels, which is required for PHDs activity, through its oxidation into Fe3þ, thus inactivating PHDs and leading to the stabilization of HIF-1a [29,30]. In this study, we found that NH4Cl promoted mitochondrial ROS generation and decreased the intracellular Fe2þ level in MDB-DA-231 cells. When NAC was
applied to scavenge ROS, the activation of HIF-1 pathway was significantly inhibited, indicating the up-regulation of HIF-1a protein induced by NH4Cl is mitochondrial ROS-dependent. It has been reported that NH4Cl activates NOX, which leads to the production of ROS [31,32]. NOX family, including NOX1, NOX2, NOX3, NOX4, NOX5, Duox1, and Duox2, is capable of using NADPH as substrate to promote ROS production, participating in the regulation of cell inflammation, signal transduction, proliferation and autophagy [33,34]. Especially, Kelly et al. found that NOX4 is localized in the outer membrane of mitochondria and its activation promotes mitochondrial ROS production [35]. NOX4 activationinduced mitochondrial ROS inactivates PHDs by depleting intracellular Fe2þ, thereby stabilizing the HIF-1a protein [36]. Our study found that the NOX-specific inhibitor apocynin significantly inhibited NH4Cl-induced increase in mitochondrial ROS level,
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Fig. 4. NH4Cl induced mitochondrial uncoupling in MDA-DB-231 cells. (A) MDA-MB-231 cells were treated with various concentrations of NH4Cl for 6 h and the ATP level was detected by a commercial kit according to the manufacturer's indication. Data are expressed as mean ± S. D. (n ¼ 4). **p < 0.01 and ***p < 0.001 versus CON. (B) MDA-MB-231 cells were treated with 5 mM NH4Cl and the oxygen consumption was assessed at the present of 5 mM NH4Cl. (C) Quantitative analysis of the oxygen consumption described in (B). Data are expressed as mean ± S. D. (n ¼ 3). *p < 0.05 versus CON. (D) MDA-MB231 cells were treated with 5 mM NH4Cl for 6 h and mitochondrial membrane potential change in cells was measured by using a fluorescent probe JC-1.
meanwhile the protein level of HIF-1a was also significantly reduced. These results suggest that NH4Cl may activate the HIF-1 signaling pathway through activating the NOX and promoting the mitochondrial ROS generation in MDB-DA-231 cells. Interestingly, we found accidently in this study that NH4Cl induced a significant increase in oxygen consumption in MDB-DA231 cells. Meanwhile, the mitochondrial membrane potential and ATP level in MDB-DA-231 cells decreased significantly. Together, these results indicated that NH4Cl treatment induced mitochondrial uncoupling in MDB-DA-231 cells. The mitochondrial uncoupling induced by NH4Cl may lead to an imbalance in intracellular oxygen distribution and a local hypoxic state, which might involved in the activation of HIF-1 pathway. These results are in agreement with the previously shown key role of mitochondrial uncoupling in stabilization of HIF-a [37,38]. In conclusion, our study showed that NH4Cl activated the HIF-1 signaling pathway in MDB-DA-231 cells under normoxic conditions and promoted HIF-1-dependent aerobic glycolysis. In addition, NH4Cl activated the HIF-1 signaling pathway probably by inducing mitochondrial ROS generation and mitochondrial uncoupling. Therefore, ammonia may participate in the reprogramming of glucose metabolism in MDB-DA-231 cells, which provides a new idea for further investgation of the metabolic changes in glutamine-dependent cancer cells. Conflicts of interest The authors declare that there is no conflict of interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (31671206, 31500965, 81702874, and
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Ammonia mediates mitochondrial uncoupling and promotes glycolysis via HIF-1 activation in human breast cancer MDA-MB231 cells Yapeng Lu*, Lu Wang, Wangwang Ding, Dan Wang, Xueting Wang, Qianqian Luo, Li Zhu** Institute of Special Environmental Medicine, Nantong University, Nantong, 226019, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 August 2019 Accepted 28 August 2019 Available online 31 August 2019
It has been reported that ammonia produced by glutaminolysis activates the HIF-1 pathway in several types of cancer cells, but the underlying mechanisms remain unclear. In this study, the effects of ammonia on the activation of HIF-1 pathway and glycolysis in MDA-MB-231 breast cancer cells were investigated and the underlying mechanisms involved were elucidated. The results showed that NH4Cl concentration-dependently increased the protein level of HIF-1a and enhanced the transactivation activity of HIF-1 in MDA-MB-231 cells. In addition, NH4Cl increased the expression of GluT1 and LDHA and promoted aerobic glycolysis by activating the HIF-1 pathway. Further study revealed that NH4Cl increased the mitochondrial ROS level and decreased the cellular Fe2þ level in MDA-MB-231 cells. Activation of the HIF-1 pathway induced by NH4Cl was inhibited by addition of the antioxidant NAC or the NADPH oxidase (NOX) inhibitor apocynin, indicating the involvement of the NOX-induced ROS generation. When MDA-MB-231 cells were treated with NH4Cl, the oxygen consumption of cells increased, followed by the decreased mitochondrial membrane potential and cellular ATP level, indicating the uncoupling of mitochondria. In conclusion, NH4Cl activated the HIF-1 signaling pathway and promoted aerobic glycolysis in MDA-MB-231 cells, likely through the promotion of mitochondrial ROS release and mitochondrial uncoupling. © 2019 Elsevier Inc. All rights reserved.
Keywords: Ammonia HIF-1 pathway Mitochondrial ROS Mitochondrial uncoupling
1. Introduction Cancer is a kind of multifactorial disease and the second leading cause of human death [1]. Modern medical research shows that reprogramming of metabolism is one of the classical characteristics of cancer [2]. Especially, reprogramming of glucose metabolism from oxidative phosphorylation to glycolysis is very important for the growth and survival of cancer cells [3]. In the 1920s, Warburg and colleagues found that cancer cells are partly or mainly dependent on glycolysis to produce ATP in spite of oxygen is sufficient, a phenomenon known as aerobic glycolysis [4]. More and more evidence shows that aerobic glycolysis is an adaptive metabolic shift, which effectively supports the synthesis of biological macromolecules in the process of rapid proliferation of cancer cells [5]. Although the mechanism of aerobic glycolysis in cancer cells is
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Lu), [email protected] (L. Zhu). https://doi.org/10.1016/j.bbrc.2019.08.152 0006-291X/© 2019 Elsevier Inc. All rights reserved.
still not fully elucidated, HIF-1 has been identified as the key regulator of aerobic glycolysis and plays an important role in the proliferation of cancer cells [6]. HIF-1 is a heterodimer composed of HIF-1a and HIF-1b [7]. Under normoxic conditions, the proline and aspartate in the oxygen-dependent degradation domain of HIF-1a are hydroxylated by proline hydroxylases (PHDs), and eventually degraded by the ubiquitin-proteasome system [8,9]. Under hypoxic or some stress conditions, the activity of PHDs is inhibited and HIF-1a enters the nucleus, forming a heterodimer with HIF-1b, binding to the promoter regions of the downstream target genes [10,11]. Most of the HIF-1-targeted genes are closely associated with cancer development [12]. In particular, the genes involved in the glycolysis including PDK1, LDHA, GluT1, HK2 and microRNA-210, etc. are directly regulated by HIF-1 [13,14]. Recently, more and more evidence have shown that glutamine metabolism plays an irreplaceable role in fueling growth and proliferation of cancer cells [15,16]. Glutamine is catalyzed by glutaminase to produce ammonia and glutamate, through which a large amount of ammonia is released. It has been reported that ammonia
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produced by glutaminolysis activates the HIF-1 signaling pathway in several types of cancer cells [17,18]. Therefore, ammonia may be a key molecule linking the glutamine metabolism and the aerobic glycolysis. But to date, the underlying mechanism by which ammonia activates the HIF-1 signaling pathway remains unclear. In this study, the activation effect of ammonia on the HIF-1 signaling pathway and the aerobic glycolysis in breast cancer cell line MDAMB-231 was studied, and the possible underlying mechanisms were elucidated.
2.5. Detection of cellular oxygen consumption The cultured MDA-MB-231 cells were made into a single cell suspension (1 106/ml) and treated with 5 mM NH4Cl. Oxygen consumption in cells were assessed polarographically using a thermostatically controlled (37 C) Clark-type oxygen electrode (Hansatech instruments, UK).
2.6. Measurement of mitochondrial membrane potential 2. Materials and methods 2.1. Materials MitoSOX fluorescence probe was purchased from Invitrogen Corporation (Carlsbad, USA). HIF-1a antibody was purchased from BD Biosciences (San Diego, CA, USA). VEGFA and b-actin antibodies were purchased from Cell Signaling Technology (Beverly, USA). GluT1 and LDHA antibodies were purchased from Abcam (Cambridge, UK). Trizol was obtained from Sigma-Aldrich Corp. (St. Louis, MO, US). The ATP assay kit and JC-1 fluorescence probe were obtained from Beyotime Biotechnology Corporation (Shanghai, China). Cell culture reagents were obtained from Gibco (Carlsbad, USA). 2.2. Cell culture The human breast cancer cell lines MDA-MB-231 and MCF-7 were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). After receiving the cell lines, the frozen stocks were established immediately. Only the early passage cells (<2 months) from the initially established frozen cell lines were used in this study. Cells were cultured at 37 C in a humidified 5% CO2 incubator (Thermo Forma Electron Co., Marietta, OH, USA). 2.3. Western blot analysis The cells were homogenized in a lysis buffer containing 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 0.2 mM PMSF, and 1.0 mM DTT (pH 7.6). The protein concentration was quantified by BCA assay. The protein samples were subjected to SDSpolyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). Then the membranes were incubated with HIF-1a antibody (1:500), VEGFA antibody (1:600), GluT1 antibody (1:800) and LDHA antibody (1:800), at 4 C overnight. Finally, IRDye 800-conjugated affinity purified IgG (1:10000) was applied at room temperature for 2 h and the membranes were scanned with the ODYSSEY® Infrared Imaging System (LI-COR, Lincoln, NE, USA). 2.4. Quantitative real-time PCR (qRT-PCR) Total cellular RNA was extracted using the Trizol RNA Purification Kit. Then, the RNA was reverse transcribed into cDNA using the Omniscript RT kit. qRT-PCR amplifications were conducted in triplicate and performed on a 7300 real-time PCR system (Applied Biosystems, CA, USA). The relative mRNA levels were normalized against b-actin. The primers were as follows: HIF-1a, 50 - AAG TGT ACC CTA ACT AGC CG-30 (forward), 50 - CAC AAA TCA GCA CCA AGC30 (backward); b-actin, 50 -TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-30 (forward), 50 -CTA GAA GCA TTG CGG TCG ACG ATG GAG GG30 (backward); GluT1,50 -ATT GGC TCC GGT ATC GTC AAC -30 (forward), 50 -GCT CAG ATA GGA CAT CCA GGG TA-30 (backward); LDHA, 50 -TTG ACC TAC GTG GCT TGG AAG-30 (forward), 50 -GGT AAC GGA ATC GGG CTG AAT-30 (backward).
Mitochondrial membrane potential changes in cells were measured using a fluorescent probe JC-1. Briefly, the cells were incubated with 5 mg/ml JC-1 at 37 C for 30 min after treated with 5 mM NH4Cl for 6 h. Then, the cells were observed by a fluorescence microscope (Leica DMI 4000B, Germany). A 488 nm filter was used for the excitation of JC-1. Emission filters of 535 nm and 595 nm were used to observe JC-1 monomers and JC-1 aggregates, respectively.
2.7. Measurement of cellular ATP After the cells were treated with various concentrations of NH4Cl for 6 h, the cellular ATP content was determined using a commercial kit according to the manufacturer's instructions.
2.8. Quantification of mitochondrial ROS levels The mitochondrial ROS level was analyzed using a fluorescent probe MitoSOX. In brief, the cells were loaded with 5 mM MitoSOX for 15 min at 37 C after treated with 5 mM NH4Cl for 6 h. Then the cells were observed by a fluorescence microscope (Leica DMI 4000B, Germany). The fluorescence intensity represents the mitochondrial ROS level.
2.9. Glucose and lactate measurement After the cells were treated with 5 mM NH4Cl for 24 h, the supernatants were collected. Then the glucose consumption and lactate release were measured using a Glucose Assay Kit (Applygen Technologies, Beijing, China) and a Lactate Assay Kit (BioVision, Inc, CA, USA) according to the manufacturer’ s indication.
2.10. Luciferase assay Luciferase assay was performed according to the method we reported previously [19]. In brief, HRE-luciferase plasmid was cotransfected with an internal control vector pRL-TK (Promega, WI, USA) (100:1 ratio) into the cells by Lipofectamine 2000 (Invitrogen, CA, USA). After the cells were treated with NH4Cl for 24 h, luciferase activity was quantitated using Dual-Luciferase Reporter System (Promega, WI, US).
2.11. Statistical analysis All data are presented as mean ± S.D. of at least three independent experiments. Statistical analysis was done using GraphPad Prism 6. The statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. Differences were considered statistically significant at p < 0.05.
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3. Results 3.1. NH4Cl activated the HIF-1 signaling pathway in MDA-DB231 cells Treatment of MDA-DB-231 cells and MCF-7 cells with 0.5-5 mM NH4Cl for 24 h did not affect cell viability (data not shown). Western blot analysis showed that NH4Cl up-regulated HIF-1a protein level in a concentration-dependent manner in these two cell lines (Fig. 1A and B). The protein level of HIF-1b did not change significantly under the same conditions in MDA-DB-231 cells. The effect of NH4Cl on the transactivation activity of HIF-1 was examined by luciferase assay. The results showed that NH4Cl increased the luciferase reporter gene activity in a concentration-dependent manner in MDA-DB-231 cells (Fig. 1C). As expected, NH4Cl also promoted the expression of VEGFA, a well-known downstream target of HIF-1 (Fig. 1D and E).
3.2. NH4Cl promoted glycolysis in MDA-DB-231 cells via the HIF-1 signaling pathway The transcription level of the HIF-1-target genes that regulate glycolysis was detected by qRT-PCR after MDA-DB-231 cells were treated with 0.5e5 mM of NH4Cl for 24 h. The results clearly showed that NH4Cl increased the mRNA level of LDHA and GluT1 in a concentration-dependent manner (Fig. 2A and B). Western blot analysis displayed that 5 mM NH4Cl significantly increased the
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protein level of LDHA and GluT1 (Fig. 2C). Under the same conditions, glucose consumption and lactate release in MDA-DB231 cells was also increased significantly (Fig. 2D). When the expression of HIF-1a was down-regulated by siRNA, the protein level of LDHA and GluT1 was significantly decreased, and so was the glucose consumption and lactate secretion, suggesting the decrease in glycolytic activity in MDA-DB-231 cells (Fig. 2C and D).
3.3. NH4Cl activated the HIF-1 signaling pathway in MDA-DB231 cells via mitochondrial ROS generation The effect of NH4Cl on the mRNA level of HIF-1a in MDA-DB231 cells was examined by qRT-PCR. It was found that NH4Cl did not affect the mRNA level of HIF-1a at 0.5e5 mM (Fig. 3A). The mitochondrial ROS was labeled by MitoSOX fluorescence and the effect of NH4Cl on mitochondrial ROS level was detected. The results showed that 5 mM NH4Cl treatment significantly enhanced the fluorescence intensity of mitoSOX, indicating the increased mitochondrial ROS level (Fig. 3B). Then, the intracellular Fe2þ level was detected and the results indicated that 5 mM NH4Cl induced a decrease in intracellular Fe2þ level (Fig. 3C). When the cells were pre-treated with the antioxidant NAC or the NOX inhibitor apocinin, NH4Cl-induced mitochondrial ROS generation was reduced (Fig. 3B) and activation of the HIF-1 signaling pathway was also inhibited, as was characterized by the decreased protein level of HIF-1a and the reduced transactivation activity of HIF-1 (Fig. 3D and E).
Fig. 1. NH4Cl activated the HIF-1 signaling pathway in MDA-DB-231 cells. (A) MDA-MB-231 cells were treated with various concentrations of NH4Cl for 24 h and the protein level of HIF-1a and HIF-1b was detected by Western blotting. (B) MCF-7 cells were treated with various concentrations of NH4Cl for 24 h and the protein level of HIF-1a was detected by Western blotting. (C) MDA-MB-231 cells were transiently cotransfected with the HRE-luciferase plasmid and an internal control vector pRL-TK for 24 h, and then treated with various concentrations of NH4Cl for 24 h. Finally, the luciferase activity was quantitated. Data are expressed as mean ± S. D. (n ¼ 4). **p < 0.01 and ***p < 0.001 versus CON. (D) MDA-MB-231 cells were treated with various concentrations of NH4Cl for 24 h and the mRNA level of VEGFA was analyzed by qRT-PCR and normalized with b-actin. Data are expressed as means ± S. D. (n ¼ 3). **p < 0.01 and ***p < 0.001 versus CON. (E) MDAMB-231 cells were treated with various concentrations of NH4Cl for 24 h and the protein level of VEGFA was detected by Western blotting.
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Fig. 2. NH4Cl promoted glycolysis in MDA-DB-231 cells via the HIF-1 signaling pathway. (A) and (B) MDA-MB-231 cells were treated with various concentrations of NH4Cl for 24 h and the mRNA level of GluT1 and LDHA was analyzed by qRT-PCR and normalized with bactin. Data are expressed as means ± S. D. (n ¼ 3). *p < 0.05 and ***p < 0.001 versus CON. (C) MDA-MB-231 cells were transfected with HIF-1a siRNA for 48 h. Then the cells were treated 5 mM NH4Cl for 24 h and the protein level of GluT1 and LDHA was analyzed by Western blotting. (D) MDA-MB-231 cells were transfected with HIF-1a siRNA for 48 h. Then the cells were treated with 5 mM NH4Cl for 24 h and the glucose consumption and lactate release was detected by commercial kits according to the manufacturer’ s indication. Data are expressed as means ± S. D. (n ¼ 4). **p < 0.01 and ***p < 0.001 versus NC, #p < 0.05 and ##p < 0.01 versus NC þ NH4Cl-treated group.
3.4. NH4Cl induced mitochondrial uncoupling in MDA-DB-231 cells After MDA-DB-231 cells were treated with various concentrations of NH4Cl for 6 h, the intracellular ATP level was detected. The results showed that NH4Cl induced a concentration-dependently decrease in intracellular ATP level (Fig. 4A). Furthermore, the oxygen consumption in MDA-DB-231 cells at present of 5 mM NH4Cl was significantly increased, indicating the enhanced mitochondrial respiration (Fig. 4B and C). By JC-1 fluorescent labeling, we found that 5 mM NH4Cl treatment decreased the mitochondrial membrane potential in MDA-MB-231 cells, which was manifested by an increase in green JC-1 monomer (Fig. 4D). Taken together, the above results indicated that NH4Cl induced mitochondrial uncoupling in MDA-DB-231 cells. 4. Discussion In recent years, the role of glutamine metabolism in cancer development has received more and more attention [20]. Cancer cells consume a large amount of glutamine during rapid proliferation which makes the concentration of ammonia high in cancer cells [21]. Increasing evidence suggests that ammonia is more than
a cellular waste and serves as an intracellular signaling molecule under sub-toxic conditions [22]. For example, it was found that ammonia is an autophagy regulator in MDA-MB-231 cells and mouse myoblast C2C12 [23]. In addition, ammonia produced by glutaminolysis activates the HIF-1 signaling pathway in several types of cancer cells [17,18]. In this study, we also identified that NH4Cl at low concentrations activated the HIF-1 signaling pathway in breast cancer MDB-DA-231 cells under normoxic conditions. When the HIF-1 signaling pathway is activated, HIF-1a undergoes nuclear translocation and binds to HIF-1b to form a heterodimer, which regulates the transcription of the downstream target genes [24]. In this study, we found that NH4Cl promoted the transactivation activity of HIF-1. Furthermore, NH4Cl up-regulated the mRNA and protein level of VEGFA, a classical target of HIF-1, in a concentration-dependent manner, which further confirmed the activation of HIF-1 signaling pathway by NH4Cl. HIF-1 plays an important role in the development of cancer, including promoting cell proliferation through reprogramming of glucose metabolism [25]. HIF-1 regulates glycolysis by upregulating the expression of glycolysis-related genes such as HK2, GluT1, LDHA, and PKM2 et al., promoting glucose uptake and lactate release [26]. In the current study, we found that NH4Cl up-
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Fig. 3. NH4Cl activated the HIF-1 signaling pathway in MDA-DB-231 cells via mitochondrial ROS generation. (A) MDA-MB-231 cells were treated with various concentrations of NH4Cl for 24 h and the mRNA level of HIF-1a was analyzed by qRT-PCR and normalized with b-actin. (B) MDAMB-231 cells were treated with NAC and apocynin with or without 5 mM NH4Cl for 24 h and the mitochondrial ROS level was detected by mitoSOX labeling. (C) MDA-MB-231 cells were treated with 5 mM NH4Cl for 24 h and intracellular ferrous ion level was detected by a commercial kit according to the manufacturer’ s indication. Data are expressed as means ± S. D. (n ¼ 4). *p < 0.05 versus CON. (D) MDA-MB-231 cells were treated with NAC and apocynin with or without 5 mM NH4Cl for 24 h and the protein level of HIF-1a was detected by Western blotting. (E) MDA-MB-231 cells were transiently cotransfected with the HRE-luciferase plasmid and an internal control vector pRL-TK for 24 h. Then the cells were treated with NAC and apocynin with or without 5 mM NH4Cl for 24 h and the luciferase activity was quantitated. Data are expressed as mean ± S. D. (n ¼ 4). ***p < 0.001 versus CON, ###p < 0.001 versus NH4Cl-treated group.
regulated the mRNA and protein levels of GluT1 and LDHA and promoted glucose uptake and lactate release in MDB-DA-231 cells. As expected, the NH4Cl-induced activation of glycolysis in MDBDA-231 cells is HIF-1 dependent. PHDs are known to play a key role in regulating the HIF-1 signaling pathway via regulating the stability of HIF-1a. Hydroxylated HIF-1a by PHDs is then ubiquitinated and finally degraded by the proteasome [8,9]. In addition to hypoxia-induced inactivation of PHDs, the activities of PHDs are also inhibited by mitochondrial-driven ROS [27,28]. Mitochondria-derived ROS decreases intracellular Fe2þ levels, which is required for PHDs activity, through its oxidation into Fe3þ, thus inactivating PHDs and leading to the stabilization of HIF-1a [29,30]. In this study, we found that NH4Cl promoted mitochondrial ROS generation and decreased the intracellular Fe2þ level in MDB-DA-231 cells. When NAC was
applied to scavenge ROS, the activation of HIF-1 pathway was significantly inhibited, indicating the up-regulation of HIF-1a protein induced by NH4Cl is mitochondrial ROS-dependent. It has been reported that NH4Cl activates NOX, which leads to the production of ROS [31,32]. NOX family, including NOX1, NOX2, NOX3, NOX4, NOX5, Duox1, and Duox2, is capable of using NADPH as substrate to promote ROS production, participating in the regulation of cell inflammation, signal transduction, proliferation and autophagy [33,34]. Especially, Kelly et al. found that NOX4 is localized in the outer membrane of mitochondria and its activation promotes mitochondrial ROS production [35]. NOX4 activationinduced mitochondrial ROS inactivates PHDs by depleting intracellular Fe2þ, thereby stabilizing the HIF-1a protein [36]. Our study found that the NOX-specific inhibitor apocynin significantly inhibited NH4Cl-induced increase in mitochondrial ROS level,
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Fig. 4. NH4Cl induced mitochondrial uncoupling in MDA-DB-231 cells. (A) MDA-MB-231 cells were treated with various concentrations of NH4Cl for 6 h and the ATP level was detected by a commercial kit according to the manufacturer's indication. Data are expressed as mean ± S. D. (n ¼ 4). **p < 0.01 and ***p < 0.001 versus CON. (B) MDA-MB-231 cells were treated with 5 mM NH4Cl and the oxygen consumption was assessed at the present of 5 mM NH4Cl. (C) Quantitative analysis of the oxygen consumption described in (B). Data are expressed as mean ± S. D. (n ¼ 3). *p < 0.05 versus CON. (D) MDA-MB231 cells were treated with 5 mM NH4Cl for 6 h and mitochondrial membrane potential change in cells was measured by using a fluorescent probe JC-1.
meanwhile the protein level of HIF-1a was also significantly reduced. These results suggest that NH4Cl may activate the HIF-1 signaling pathway through activating the NOX and promoting the mitochondrial ROS generation in MDB-DA-231 cells. Interestingly, we found accidently in this study that NH4Cl induced a significant increase in oxygen consumption in MDB-DA231 cells. Meanwhile, the mitochondrial membrane potential and ATP level in MDB-DA-231 cells decreased significantly. Together, these results indicated that NH4Cl treatment induced mitochondrial uncoupling in MDB-DA-231 cells. The mitochondrial uncoupling induced by NH4Cl may lead to an imbalance in intracellular oxygen distribution and a local hypoxic state, which might involved in the activation of HIF-1 pathway. These results are in agreement with the previously shown key role of mitochondrial uncoupling in stabilization of HIF-a [37,38]. In conclusion, our study showed that NH4Cl activated the HIF-1 signaling pathway in MDB-DA-231 cells under normoxic conditions and promoted HIF-1-dependent aerobic glycolysis. In addition, NH4Cl activated the HIF-1 signaling pathway probably by inducing mitochondrial ROS generation and mitochondrial uncoupling. Therefore, ammonia may participate in the reprogramming of glucose metabolism in MDB-DA-231 cells, which provides a new idea for further investgation of the metabolic changes in glutamine-dependent cancer cells. Conflicts of interest The authors declare that there is no conflict of interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (31671206, 31500965, 81702874, and
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