- Email: [email protected]
c-Myc Overexpression Promotes Oral Cancer Cell Proliferation and Migration by Enhancing Glutaminase and Glutamine Synthetase Activity
BASIC INVESTIGATION
c-Myc Overexpression Promotes Oral Cancer Cell Proliferation and Migration by Enhancing Glutaminase and Glutamine Synthetase Activity Tao Wang, PhD1,3, Bolei Cai, PhD1, Mingchao Ding, PhD1, Zhongping Su, PhD1, Yanpu Liu, PhD1 and Liangliang Shen, PhD2 1
State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology and 2 State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Shaanxi Province, China; 3 Department of Stomatology, Shaanxi Provincial People’s Hospital, Shaanxi Province, China
ABSTRACT Background: This study aimed to investigate whether glutaminase (GLS) and glutamine synthetase (GS) are involved in c-Myc-mediated tumor development in oral cancer. Methods: The correlation between the expressions of c-Myc, GLS, and GS in clinical samples and the clinicopathologic features of oral cancer were examined using immunohistochemistry and quantitative real-time polymerase chain reaction. After overexpressing the c-Myc gene and using an inhibitor of GLS or GS, functional experiments were performed to confirm the effects of c-Myc, GLS and GS on proliferation, cell cycle and migration in KB oral cancer cells. The expressions of E-cadherin and N-cadherin were determined by immunofluorescence assays in KB cells overexpressing c-Myc in the presence of GLS or GS inhibitors. Results: The protein expression of GS was correlated with the Tumor, Lymph Node, and Metastasis (TNM) stage. In addition, c-Myc mRNA levels were positively correlated with GS mRNA levels. Overexpression of c-Myc increased the colonies derived from oral cancer cells and caused more cells to be in S phase compared with the mock-vehicle group. The migratory speed of KB cells was promoted by overexpression of c-Myc compared to the mock-vehicle group. However, these effects were effectively reversed in the presence of GLS or GS inhibitor. Furthermore, c-Myc could inhibit E-cadherin protein expression while promoting N-cadherin expression by enhancing the activity of GLS and GS. Conclusions: c-Myc overexpression promotes oral cancer cell proliferation and migration by enhancing GLS and GS activity. Our findings are beneficial for the identification of novel molecular targets for the prevention and treatment of oral cancer. Key Indexing Terms: Oral cancer; c-Myc; Glutaminase; Glutamine synthetase; Metabolic reprogramming. [Am J Med Sci 2019;358(3):235–242.]
INTRODUCTION
O
ral cancer is a serious dental problem with a poor 5-year survival rate and limited traditional treatment methods.1 Normal cells produce energy by mitochondrial oxidative phosphorylation, while cancer cells rely on different modes of energy metabolism, such as glycolysis, enhanced lipid synthesis and glutamine addiction. Due to differences in energy sources, cancer cells grow much faster than normal cells.2 Therefore, preventing cancer cell growth by restricting their energy supply is of interest. Glutamine addiction is a hallmark of tumor metabolism.3 Many cancer cells rely on glutamine to replenish the tricarboxylic acid (TCA) cycle to meet cellular energy requirements.4 Glutamine not only provides a carbon
source for the TCA cycle, but also provides a nitrogen source for the synthesis of biological macromolecules such as proteins, aminohexoses and nucleotides, which is critical in maintaining oxidation-reduction homeostasis against oxidative stress.5 Glutaminase (GLS) catalyzes the deamination of glutamine to glutamate, and is a key enzyme for glutamine glycolysis in tumor cells.6 Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamic acid and ammonia.7 During tumor metabolism, a large amount of glutamine is transported to cancer cells to promote cancer cell proliferation and malignant transformation.6 However, the metabolic regulation mechanisms of glutamine remain unknown. The proto-oncogene c-Myc is significantly up-regulated in oral cancer patients, and its expression is
Copyright © 2019 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved. www.amjmedsci.com www.ssciweb.org
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correlated with clinicopathologic grade and stage in oral cancer.8 Sawant et al reported that c-Myc is a key contributor to oral carcinogenesis in immunodeficient mice.8 Mechanistically, c-Myc mediates activation of transforming growth factor-b1,9 matrix metalloproteinase-9,10 insulin receptor11 and insulin-like growth factor 1 receptor,11 which are critical for the proliferation, anti-apoptosis and invasion of oral cancer cells. Recently, Dejure et al reported that c-Myc is a key transcription factor promoting glutamine metabolism in HCT116 colon cancer cells.12 Additionally, c-Myc enhanced mitochondrial GLS expression and glutamine metabolism in B lymphoma cells, human prostate cancer cells and lymphoblastoid cells.13 However, whether the activity of GLS and GS in oral cancer cells is also necessary for c-Myc-mediated tumor development remains unclear. This study aimed to examine the role of GLS and GS in c-Myc-mediated proliferation and migration of oral cancer cells. This study may foster the identification of novel molecular targets for oral cancer therapy.
MATERIALS AND METHODS Cell Culture and Transfection Oral cancer KB cells were purchased from SHBIO (Shanghai, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM, #12634-010, Thermo Scientific) supplemented with 5% fetal bovine serum (no. SH30070.02, HyClone) at 37°C with 5% CO2 in an incubator. pEGFP-C1-c-Myc plasmid and mock-vehicle
were obtained from our previous study and transfected using Lipofectamine 2000 Transfection Reagent (#11668027, thermo scientific) according to manufacturer instructions. The transfection efficiency in KB cells was measured by quantitative real-time polymerase chain reaction (qRT-PCR).
Immunohistochemistry and Immunofluorescence Protein expression levels in oral cancer tissues and KB cells were evaluated by immunohistochemistry (IHC) and immunofluorescence (IF). For IHC, 23 oral cancer tissue samples collected from patients in Shaanxi Provincial People’s Hospital (Table 1) were fixed with 4% paraformaldehyde for 12 hours and embedded in paraffin. Paraffin sections were incubated with anti-c-Myc antibody (ab139688, abcam), anti-GS1 antibody (ab176562, abcam) and anti-GLS antibody (ab156876, abcam). Paraffin sections were stained using an UltraSensitiveTM SP (Mouse/Rabbit) IHC Kit (KIT-9720, MXB) according to manufacturer instructions. Subsequently, the DAB reaction was performed using a DAB kit (DAB-0031/1031, MXB). For IF, after treatment with 1 mm/mL GLS inhibitor (LDN27219 [M01840, guidechem]) or 25 mm/mL GS inhibitor (Methylthio acetaldoxime [10533-67-2, guidechem]), KB cells transfected with the c-Myc overexpression plasmid or mock-vehicle were fixed with 4% paraformaldehyde for 20 minutes and washed with a blocking reagent. Cells were then incubated overnight at 4°C using
TABLE 1. The clinicopathologic features of patients. ID 19610849 19580523 19642618 19598364 19587011 19639354 19177810 19397966 19611957 19572178 19645781 19347388 19460500 19552811 19623050 19206580 19541452 19608976 F135060202 19397719 19364447 19412617 29662316
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Sex
Age
TNM
Clinical diagnosis
Female Male Male Male Male Male Male Female Female Female Female Female Female Male Male Male Male Male Female Male Female Male Male
52 50 58 77 62 64 63 60 76 51 65 61 65 46 48 59 66 49 86 33 22 67 61
T4N0M0 T2N0M0 T4aN0M0 PT1MONO T2N0M0 PT2N0M0 PT4NIMO T2N0M0 T4aN2bm0 PT3N0M0 PT4N0M0 T4N1M0 T2N2bM0 PT2N0M0 PT2N0M0 T4N0M0 PT3N2M0 PT4N0M0 PT4Nx T3N0M0 T4aN0M0 T4aN0M0 T4aN0M0
Right lingual squamous cell carcinoma. Highly differentiated squamous cell carcinoma of the left cheek mucosa Highly differentiated squamous cell carcinoma of the right lower jaw. Highly differentiated squamous cell carcinoma of the right lower jaw. Highly differentiated squamous cell carcinoma of the tongue. Highly differentiated tongue cancer on the right. Highly differentiated squamous cell carcinoma of the left lower jaw. Highly differentiated squamous cell carcinoma of the left cheek mucosa. Highly differentiated squamous cell carcinoma of the left lower jaw. Highly differentiated squamous cell carcinoma of the right tongue. Highly differentiated squamous cell carcinoma of the left lower jaw. Recurrence of neck after squamous cell carcinoma surgery. Cervical lymph node metastasis after lip cancer surgery. Highly differentiated squamous cell carcinoma of the left cheek mucosa. Highly differentiated squamous cell carcinoma of the left tongue. Highly differentiated squamous cell carcinoma of the left lower jaw. Highly differentiated squamous cell carcinoma of the left cheek mucosa. Highly differentiated squamous cell carcinoma of the right oral bottom. Highly differentiated maxillary gingival carcinoma with cervical lymph node metastasis. Highly differentiated squamous cell carcinoma of the left tongue. Highly differentiated squamous cell carcinoma of the right lower jaw. Highly differentiated squamous cell carcinoma of the left oral bottom. Highly differentiated squamous cell carcinoma of the left upper jaw.
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anti-c-Myc antibody (ab139688, abcam), anti-GS1 antibody (ab176562, abcam), anti-GLS antibody (ab156876, abcam), anti-E-cadherin antibody (ab1416, abcam) and anti-N-cadherin antibody (ab98952, abcam). Fluorescent secondary antibodies (#8887 and #8885, cell signaling technology) were used for detection.
qRT-PCR Total RNA was extracted with TRIzol LS Reagent (10296010, ThermoFisher). cDNA was synthesized from RNA using Advantage RT-for-PCR Kit (639505, Takara). qRT-PCR was performed by Terra qPCR Direct SYBR Premix (638319, Takara) using a Thermal Cycler Dice Real Time System II (TP900, Takara).
Colony Formation KB cells transfected with plasmids overexpressing cMyc or mock-vehicle were seeded in 6-well plates for colony formation assays. Cells were treated with GLS inhibitor (1 mm/mL) or GS inhibitor (25 mm/mL). After 7 days, colonies were fixed with 95% ethanol and stained with 0.1% crystal violet for 15 minutes and cell colonies were imaged.
Cell Cycle Assay After treatment with GLS inhibitor (1 mm/mL) or GS inhibitor (25 mm/mL), KB cells were transfected with plasmids overexpressing c-Myc or mock vehicle plasmids, then were collected and washed with cold PBS. Next, cells were fixed in absolute ethyl alcohol at 4°C for 24 hours and stained with a Cell Cycle and Apoptosis Analysis Kit (C1052, Beyotime). The cell cycle was analyzed using flow cytometry (CytoFLEX).
Wound Healing Assay Wound healing assays were performed to investigate cell migration. A straight line was scratched on the cell layer of KB cells transfected with plasmids overexpressing c-Myc or mock vehicle, then cells were treated with GLS inhibitor (1 mm/mL) or GS inhibitor (25 mm/mL). Cells were imaged from the same point of view at 0 and 24 hours. The wound closure rate was measured by ImageJ.
Statistical Analysis All statistical analyses were carried out using IBM SPSS Statistics 21. The differences between 2 groups were analyzed by 1-way analysis of variance. The correlation of related gene and protein expression with clinicopathologic features was analyzed using Spearman correlation analysis. P values less than 0.05 were considered to be statistically significant.
RESULTS Expression Profiles of c-Myc, GLS and GS in Oral Cancer Expression levels of c-Myc, GLS and GS were detected in oral carcinoma tissues. As shown in Figure 1A, IHC results indicated strong expressions of c-Myc, GLS and GS in tumor tissues. Next, the relationship of c-Myc, GLS and GS expression levels with clinicopathologic features was analyzed in oral cancer tissue samples. The protein expression of GS was significantly associated with clinical stage (Figure 1B and C). As shown in Figure 1D and E, c-Myc positively correlated with GS expression at the mRNA level. However, the other results were not statistically different, possibly due to the small sample size in this study (Figure 1). GLS and GS are Necessary for c-Myc-mediated Oral Cancer Cell Proliferation To examine the interaction of c-Myc, GLS and GS in oral cancer cells, a complementary DNA fragment encoding human c-Myc was cloned into the pEGFP-C1 vector, and the protein expression efficiency in KB cells is presented in Figure 2. Clone formation assays revealed that colonies derived from KB cells overexpressing c-Myc were increased compared to the mock-vehicle group. When KB cells were treated with GLS inhibitor, colony formation was significantly inhibited (P < 0.01). Similarly, GS inhibitor decreased c-Myc overexpression in both KB cells and control cells. These data demonstrated that GLS or GS inhibitor can suppress the c-Myc-mediated proliferation of KB cells (Figure 3A). Moreover, we found that up-regulation of c-Myc caused more cells to be in the S phase of the cell cycle compared with the mock vehicle group, indicating the high proliferative activity of KB cells after c-Myc overexpression. However, treatment with GLS or GS inhibitor decreased the percentage of KB cells overexpressing c-Myc at S phase. These results suggest that GLS and GS inhibitors exert their antiproliferative effects by inducing cell cycle arrest at G0/G1 phase (Figure 3B). In summary, GLS and GS are necessary for c-Myc-mediated oral cancer cell proliferation. GLS and GS are Necessary for c-Myc-mediated Oral Cancer Cell Migration Cell migration drives oral cancer progression and seriously affects patient survival. As shown in Figure 4, overexpression of c-Myc increased the migratory speed in KB cells compared to the mock vehicle group, and this effect was reversed in the presence of GLS or GS inhibitor, suggesting that GLS and GS also play pivotal roles in c-Myc-mediated cell migration in oral cancer. c-Myc Regulates Epithelial-mesenchymal Transition by Up-regulating GLS and GS Activity As shown in Figure 5A-D, IF results revealed that upregulation of c-Myc enhanced the expressions of GLS
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FIGURE 1. Expressions of c-Myc, glutaminase (GLS) and glutamine synthetase (GS) at the mRNA and protein levels in oral cancer tissues. (A) The expressions of c-Myc, GS and GLS in a patient diagnosed with oral cancer at T2N0M0 stage, as determined by immunohistochemistry (IHC) analysis. (B) Relative protein expressions of c-Myc, GLS and GS in oral cancer specimens based on IHC images. (C) Correlations between clinical Tumor, Lymph Node, and Metastasis (TNM) stages and c-Myc, GS and GLS protein expressions in 23 oral cancer patients. (D) Relative mRNA levels of c-Myc, GLS and GS in oral cancer specimens. (E) Correlations between clinical TNM stages and c-Myc, GS and GLS mRNA levels in 21 oral cancer patients.
and GS. Epithelial-mesenchymal transition (EMT) is the biological process by which epithelial cells become mesenchymal phenotypic cells. E-cadherin and N-cadherin are 2 markers for EMT. Overexpression of c-Myc inhibited E-cadherin protein expression but promoted N-cadherin expression compared with the mock vehicle group. Although the GLS and GS inhibitors did not change GLS and GS expressions, the E-cadherin level increased and the N-cadherin level decreased in KB cells in the presence of GLS or GS inhibitors.
DISCUSSION Although the molecular mechanisms of tumor development have not been completely elucidated, the biochemical characteristics of cancer cell energy metabolism provide opportunities for targeted therapies. Some researchers have suggested that cutting off the energy supply of cancer cells may be a way to treat cancer.14,15 Two hallmarks of classical tumor metabolic 238
reprogramming are aerobic glycolysis and dependence on glutamine to replenish the TCA cycle.16 In the TCA cycle, oxaloacetate condenses with acetyl-CoA to generate citric acid, and glutamine is an important source of oxaloacetate. By promoting citric acid synthesis, glutamine helps maintain lipid synthesis. In addition, glutamine can be deaminated to glutamate, which is a precursor for glutathione synthesis. In this way, glutamine is involved in redox balance regulation in tumor cells. Hence, glutamine is an essential amino acid for tumor growth and is preferentially consumed by tumor cells.17 GLS is an enzyme that catalyzes the hydrolysis of glutamine into glutamate and ammonia and GS is an enzyme that catalyzes the synthesis of glutamine from glutamate and ammonium ions.18 Both GLS and GS are highly expressed in tumor cells.19 Studies investigating GLS and GS regulatory mechanisms may identify new possibilities for oral cancer treatment. The expression level of the GLS protein has been shown to be up-regulated in liver cancer,20 nonsmall cell lung cancer,21 THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES VOLUME 358 NUMBER 3 SEPTEMBER 2019
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FIGURE 2. pEGFP-C1-c-Myc mediates c-Myc overexpression in KB cells. The overexpression efficacy of c-Myc was measured by green fluorescent protein (GFP) expression (A) and quantitative real-time polymerase chain reaction (qRT-PCR) (B) in KB cells. Data are represented with GraphPad Prism 5.0. ***P < 0.001.
FIGURE 3. Glutaminase (GLS) or glutamine synthetase (GS) inhibitor suppresses proliferation and induces cell cycle arrest at G0/G1 phase in KB cells through the up-regulation of c-Myc. Colony formation was performed to determine cell proliferation (A), and flow cytometry was performed to determine cell cycle phase (B) in KB cells transfected with mock vehicle and c-Myc overexpression vector, respectively. GLS inhibitor (GLSi) 1.0 mM; GS inhibitor (GSi) 25 mM. Data are represented with GraphPad Prism 5.0. *P < 0.05; **P < 0.01.
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FIGURE 4. Glutaminase (GLS) and glutamine synthetase (GS) are necessary for c-Myc-mediated oral cancer cell migration. (A) Cell migration was detected by wound healing assay in KB cells transfected with mock vehicle or c-Myc overexpression vector. (B) Data are represented with GraphPad Prism 5.0. **P < 0.01. GLS inhibitor (GLSi) 1.0 mM; GS inhibitor (GSi) 25 mM.
colorectal cancer22 and melanoma.23 Okamura et al analyzed gene expression changes in breast cancer cells after knocking out GLS1 and found that 4 genes were significantly downregulated. The 4 targeted genes, high-mobility group Hmga2, formin-like protein 3, Nedd-4 ubiquitin-protein ligase and ubiquitin carboxylterminal hydrolase 15, have relevant functions in tumor n et al cell proliferation and progression.24 Martín-Rufia reported that GLS1 silencing and oxidative stress
synergistically inhibit the proliferation of glioma cells.25 These studies demonstrated that GLS1 is required for the survival of many tumor cells. However, the relationships between glutamine metabolism and oral cancer have not been reported. In this study, we found that GLS and GS were highly expressed in oral cancer samples (Figure 1). c-Myc is known to bind to chromosomes and DNA. It is mainly activated by amplification and cleavage of
FIGURE 5. c-Myc regulates epithelial-mesenchymal transformation (EMT) by up-regulating glutaminase (GLS) and glutamine synthetase (GS) expressions. (A) Representative immunofluorescence (IF) images showing the location and intensity of c-Myc, GLS, GS, N-cadherin, and E-cadherin. (B-F) Relative protein expression levels of c-Myc, GLS, GS, N-cadherin and E-cadherin based on IF. Data are represented with GraphPad Prism 5.0. *P < 0.05; **P < 0.01. GLS inhibitor (GLSi) 1.0 mM; GS inhibitor (GSi) 25 mM.
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chromosomal translocations, and it plays a pivotal role in regulating cell growth, differentiation and malignant transformation.26 As shown in Figure 1, we found that cMyc, GLS and GS were highly expressed in oral cancer samples, and that GS expression levels were associated with TNM stage. Hence, we suspect a relationship between c-Myc and GLS and GS in oral cancer cells. Recent studies have shown that c-Myc promoted glutamine uptake and catabolism by activating the expression of genes required for glutamine metabolism. Glutamine deficiency restrained cell growth and proliferation and induces apoptosis in cells overexpressing c-Myc, while supplementation with TCA cycle intermediates reversed these effects.27 Wise et al reported that glutamine deprivation induced c-Myc-dependent apoptosis and that the reliance of c-Myc on glutamine was dependent on cell type.28 Furthermore, c-Myc was shown to directly bind the promoter of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, and upregulate its protein expression.28 In addition, c-Myc also indirectly enhanced GLS1 activity by inhibiting miR-23a/b.13 Similarly, our data showed that GLS and GS were necessary for c-Myc-mediated oral cancer cell proliferation and migration, indicating that c-Myc acts as an upstream regulator of GLS and GS in oral cancer cells (Figure 3 and Figure 4). EMT induces migration, invasion and tumor progression of tumor cells.13 Varaperez et al demonstrated that Bcl2 interacting protein 3 mediated mitochondrial quality control and promoted the glutamine metabolism-driven EMT-like transition of melanoma cells.29 Similarly, our study indicated that c-Myc inhibits E-cadherin protein expression while promoting N-cadherin expression by enhancing GLS and GS activity, which might play a significant role in the migration and progression of oral cancer cells. In summary, our findings revealed that c-Myc overexpression promotes oral cancer cell proliferation and migration by enhancing GLS and GS activity. This study is beneficial for exploring novel biochemical targets for the prevention and treatment of oral cancer.
AUTHOR CONTRIBUTIONS Y.P.L. and L.L.S. conceived of the presented idea. T. W. and B.L.C. performed the experiment and collected the data. M.C.D. and Z.P.S were responsible for data analysis and visualization. Y.P.L. and L.L.S. supervised the findings of this work. All authors discussed the results and contributed to the final manuscript.
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4. Sreedhar A, Zhao Y. Dysregulated metabolic enzymes and metabolic reprogramming in cancer cells. Biomed Rep. 2018;8:3–10. 5. Novak F, Heyland DK, Avenell A, Drover JW, et al. Glutamine supplementation in serious illness: a systematic review of the evidence. Crit Care Med. 2002;30:2022. 6. Wang JB, Erickson JW, Fuji R, et al. Targeting mitochondrial glutaminase activity inhibits oncogenic transformation. Cancer Cell. 2010;18: 207–219. 7. Norenberg MD, Martinezhernandez A. Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 2015;161: 303–310. 8. Sawant S, Gokulan R, Dongre H, et al. Prognostic role of Oct4, CD44 and c-Myc in radio−chemo-resistant oral cancer patients and their tumourigenic potential in immunodeficient mice. Clin Oral Invest. 2016;20:43. 9. Hu L, Liu J, Zhi L, et al. Transforming growth factor-b1 activates DNp63/ c-Myc to promote oral squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol. 2016;122:460–482. 10. Pramanik KK, Nagini S, Singh AK, et al. Glycogen synthase kinase-3b mediated regulation of matrix metalloproteinase-9 and its involvement in oral squamous cell carcinoma progression and invasion. Cell Oncol. 2018;41:47–60. 11. Sun J, Lu Z, Yun D, et al. Up-regulation of INSR/IGF1R by C-myc promotes TSCC tumorigenesis and metastasis through the NF-kB pathway. Biochim Biophys Acta Mol Basis Dis. 2018;1864: 1873–1882. 12. Dejure FR, Royla N, Herold S, et al. The mRNA 30 -UTR couples RNA polymerase II function to glutamine and ribonucleotide levels. EMBO J. 2017;36:1854–1868. 13. Gao P, Tchernyshyov I, Chang TC, et al. c-Myc suppression of miR23a/b enhances mitochondrial glutaminase expression and glutamine metabolism. Nature. 2009;458:762–765. 14. Granchi C, Qian Y, Lee HY, et al. Salicylketoximes that target glucose transporter 1 restrict energy supply to lung cancer cells. ChemMedChem. 2015;10:1892–1900. nchez R, Marín-Herna ndez A, Saavedra E, et al. Who 15. Moreno-Sa controls the ATP supply in cancer cells? Biochemistry lessons to understand cancer energy metabolism. Int J Biochem Cell Biol. 2014;50:10–23. 16. Fiaschi T, Chiarugi P. Oxidative stress, tumor microenvironment, and metabolic reprogramming: a diabolic liaison. Int J Cell Biol. 2012;2012: 762825. 17. Wise DR, Thompson CB. Glutamine addiction: a new therapeutic target in cancer. Trends Biochem Sci. 2010;35:427–433. 18. Labow BI, Souba WW, Abcouwer SF. Mechanisms governing the expression of the enzymes of glutamine metabolism−glutaminase and glutamine synthetase. J Nutr. 2001;131:2467S–2474S. 19. Matsuno T, Goto I. Glutaminase and glutamine synthetase activities in human cirrhotic liver and hepatocellular carcinoma. Cancer Res. 1992; 52:1192–1194. 20. Wang D, Meng G, Zheng M, et al. The glutaminase-1 inhibitor 968 enhances dihydroartemisinin-mediated antitumor efficacy in hepatocellular carcinoma cells. PLoS One. 2016;11: e0166423. 21. Owonikoko TK. Glutaminase enzyme (GLS1) in small cell lung cancer. Emory Univ. 2014. 22. Lu WQ, Hu YY, Lin XP, et al. Knockdown of PKM2 and GLS1 expression can significantly reverse oxaliplatin-resistance in colorectal cancer cells. Oncotarget. 2017;8:44171–44185. 23. He YL. Effect of granulysin (GLS) and IL-12 genes' expression products on proliferation and apoptosis of melanoma B16 cell. J Chongqing Med Univ. 2010. 24. Okamura S, Ng CC, Koyama K, et al. Identification of seven genes regulated by wild-type p53 in a colon cancer cell line carrying a wellcontrolled wild-type p53 expression system. Oncol Res. 1999;11:281–285. n M, Nascimento-Gomes R, Higuero A, et al. Both 25. Martín-Rufia GLS silencing and GLS2 overexpression synergize with oxidative stress against proliferation of glioma cells. J Mol Med. 2014;92: 277–290.
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26. David CJ, Chen M, Assanah M, et al. HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer. Nature. 2010;463:364–368. 27. Yuneva M, Zamboni N, Oefner P, et al. Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells. J Cell Biol. 2007;178:93–105. 28. Wise DR, DeBerardinis RJ, Mancuso A, et al. Myc Regulates a Transcriptional Program That Stimulates Mitochondrial Glutaminolysis and Leads to Glutamine Addiction. Proc Natl Acad Sci U S A. 2008;105: 18782–18787. 29. Varaperez M, Maes H, Van SD, et al. BNIP3 contributes to the glutamine-driven aggressive behavior of melanoma cells. Biol Chem. 2019; 400:187–193.
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Submitted November 22, 2018; accepted May 31, 2019. Funding: This study was supported by National Natural Science Foundation of China (Grant No.: 31401161). Conflict of Interest: The authors declare that they have no conflict of interest. Correspondence: Liangliang Shen, State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, 169 West Changle Road, Xi'an 710032, Shaanxi Province, China. (E-mail: [email protected], [email protected]). Yanpu Liu, State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University, 169 West Changle Road, Xi'an 710032, Shaanxi Province, China. (E-mail: [email protected]).
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES VOLUME 358 NUMBER 3 SEPTEMBER 2019
c-Myc Overexpression Promotes Oral Cancer Cell Proliferation and Migration by Enhancing Glutaminase and Glutamine Synthetase Activity Tao Wang, PhD1,3, Bolei Cai, PhD1, Mingchao Ding, PhD1, Zhongping Su, PhD1, Yanpu Liu, PhD1 and Liangliang Shen, PhD2 1
State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology and 2 State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Shaanxi Province, China; 3 Department of Stomatology, Shaanxi Provincial People’s Hospital, Shaanxi Province, China
ABSTRACT Background: This study aimed to investigate whether glutaminase (GLS) and glutamine synthetase (GS) are involved in c-Myc-mediated tumor development in oral cancer. Methods: The correlation between the expressions of c-Myc, GLS, and GS in clinical samples and the clinicopathologic features of oral cancer were examined using immunohistochemistry and quantitative real-time polymerase chain reaction. After overexpressing the c-Myc gene and using an inhibitor of GLS or GS, functional experiments were performed to confirm the effects of c-Myc, GLS and GS on proliferation, cell cycle and migration in KB oral cancer cells. The expressions of E-cadherin and N-cadherin were determined by immunofluorescence assays in KB cells overexpressing c-Myc in the presence of GLS or GS inhibitors. Results: The protein expression of GS was correlated with the Tumor, Lymph Node, and Metastasis (TNM) stage. In addition, c-Myc mRNA levels were positively correlated with GS mRNA levels. Overexpression of c-Myc increased the colonies derived from oral cancer cells and caused more cells to be in S phase compared with the mock-vehicle group. The migratory speed of KB cells was promoted by overexpression of c-Myc compared to the mock-vehicle group. However, these effects were effectively reversed in the presence of GLS or GS inhibitor. Furthermore, c-Myc could inhibit E-cadherin protein expression while promoting N-cadherin expression by enhancing the activity of GLS and GS. Conclusions: c-Myc overexpression promotes oral cancer cell proliferation and migration by enhancing GLS and GS activity. Our findings are beneficial for the identification of novel molecular targets for the prevention and treatment of oral cancer. Key Indexing Terms: Oral cancer; c-Myc; Glutaminase; Glutamine synthetase; Metabolic reprogramming. [Am J Med Sci 2019;358(3):235–242.]
INTRODUCTION
O
ral cancer is a serious dental problem with a poor 5-year survival rate and limited traditional treatment methods.1 Normal cells produce energy by mitochondrial oxidative phosphorylation, while cancer cells rely on different modes of energy metabolism, such as glycolysis, enhanced lipid synthesis and glutamine addiction. Due to differences in energy sources, cancer cells grow much faster than normal cells.2 Therefore, preventing cancer cell growth by restricting their energy supply is of interest. Glutamine addiction is a hallmark of tumor metabolism.3 Many cancer cells rely on glutamine to replenish the tricarboxylic acid (TCA) cycle to meet cellular energy requirements.4 Glutamine not only provides a carbon
source for the TCA cycle, but also provides a nitrogen source for the synthesis of biological macromolecules such as proteins, aminohexoses and nucleotides, which is critical in maintaining oxidation-reduction homeostasis against oxidative stress.5 Glutaminase (GLS) catalyzes the deamination of glutamine to glutamate, and is a key enzyme for glutamine glycolysis in tumor cells.6 Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamic acid and ammonia.7 During tumor metabolism, a large amount of glutamine is transported to cancer cells to promote cancer cell proliferation and malignant transformation.6 However, the metabolic regulation mechanisms of glutamine remain unknown. The proto-oncogene c-Myc is significantly up-regulated in oral cancer patients, and its expression is
Copyright © 2019 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved. www.amjmedsci.com www.ssciweb.org
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correlated with clinicopathologic grade and stage in oral cancer.8 Sawant et al reported that c-Myc is a key contributor to oral carcinogenesis in immunodeficient mice.8 Mechanistically, c-Myc mediates activation of transforming growth factor-b1,9 matrix metalloproteinase-9,10 insulin receptor11 and insulin-like growth factor 1 receptor,11 which are critical for the proliferation, anti-apoptosis and invasion of oral cancer cells. Recently, Dejure et al reported that c-Myc is a key transcription factor promoting glutamine metabolism in HCT116 colon cancer cells.12 Additionally, c-Myc enhanced mitochondrial GLS expression and glutamine metabolism in B lymphoma cells, human prostate cancer cells and lymphoblastoid cells.13 However, whether the activity of GLS and GS in oral cancer cells is also necessary for c-Myc-mediated tumor development remains unclear. This study aimed to examine the role of GLS and GS in c-Myc-mediated proliferation and migration of oral cancer cells. This study may foster the identification of novel molecular targets for oral cancer therapy.
MATERIALS AND METHODS Cell Culture and Transfection Oral cancer KB cells were purchased from SHBIO (Shanghai, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM, #12634-010, Thermo Scientific) supplemented with 5% fetal bovine serum (no. SH30070.02, HyClone) at 37°C with 5% CO2 in an incubator. pEGFP-C1-c-Myc plasmid and mock-vehicle
were obtained from our previous study and transfected using Lipofectamine 2000 Transfection Reagent (#11668027, thermo scientific) according to manufacturer instructions. The transfection efficiency in KB cells was measured by quantitative real-time polymerase chain reaction (qRT-PCR).
Immunohistochemistry and Immunofluorescence Protein expression levels in oral cancer tissues and KB cells were evaluated by immunohistochemistry (IHC) and immunofluorescence (IF). For IHC, 23 oral cancer tissue samples collected from patients in Shaanxi Provincial People’s Hospital (Table 1) were fixed with 4% paraformaldehyde for 12 hours and embedded in paraffin. Paraffin sections were incubated with anti-c-Myc antibody (ab139688, abcam), anti-GS1 antibody (ab176562, abcam) and anti-GLS antibody (ab156876, abcam). Paraffin sections were stained using an UltraSensitiveTM SP (Mouse/Rabbit) IHC Kit (KIT-9720, MXB) according to manufacturer instructions. Subsequently, the DAB reaction was performed using a DAB kit (DAB-0031/1031, MXB). For IF, after treatment with 1 mm/mL GLS inhibitor (LDN27219 [M01840, guidechem]) or 25 mm/mL GS inhibitor (Methylthio acetaldoxime [10533-67-2, guidechem]), KB cells transfected with the c-Myc overexpression plasmid or mock-vehicle were fixed with 4% paraformaldehyde for 20 minutes and washed with a blocking reagent. Cells were then incubated overnight at 4°C using
TABLE 1. The clinicopathologic features of patients. ID 19610849 19580523 19642618 19598364 19587011 19639354 19177810 19397966 19611957 19572178 19645781 19347388 19460500 19552811 19623050 19206580 19541452 19608976 F135060202 19397719 19364447 19412617 29662316
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Sex
Age
TNM
Clinical diagnosis
Female Male Male Male Male Male Male Female Female Female Female Female Female Male Male Male Male Male Female Male Female Male Male
52 50 58 77 62 64 63 60 76 51 65 61 65 46 48 59 66 49 86 33 22 67 61
T4N0M0 T2N0M0 T4aN0M0 PT1MONO T2N0M0 PT2N0M0 PT4NIMO T2N0M0 T4aN2bm0 PT3N0M0 PT4N0M0 T4N1M0 T2N2bM0 PT2N0M0 PT2N0M0 T4N0M0 PT3N2M0 PT4N0M0 PT4Nx T3N0M0 T4aN0M0 T4aN0M0 T4aN0M0
Right lingual squamous cell carcinoma. Highly differentiated squamous cell carcinoma of the left cheek mucosa Highly differentiated squamous cell carcinoma of the right lower jaw. Highly differentiated squamous cell carcinoma of the right lower jaw. Highly differentiated squamous cell carcinoma of the tongue. Highly differentiated tongue cancer on the right. Highly differentiated squamous cell carcinoma of the left lower jaw. Highly differentiated squamous cell carcinoma of the left cheek mucosa. Highly differentiated squamous cell carcinoma of the left lower jaw. Highly differentiated squamous cell carcinoma of the right tongue. Highly differentiated squamous cell carcinoma of the left lower jaw. Recurrence of neck after squamous cell carcinoma surgery. Cervical lymph node metastasis after lip cancer surgery. Highly differentiated squamous cell carcinoma of the left cheek mucosa. Highly differentiated squamous cell carcinoma of the left tongue. Highly differentiated squamous cell carcinoma of the left lower jaw. Highly differentiated squamous cell carcinoma of the left cheek mucosa. Highly differentiated squamous cell carcinoma of the right oral bottom. Highly differentiated maxillary gingival carcinoma with cervical lymph node metastasis. Highly differentiated squamous cell carcinoma of the left tongue. Highly differentiated squamous cell carcinoma of the right lower jaw. Highly differentiated squamous cell carcinoma of the left oral bottom. Highly differentiated squamous cell carcinoma of the left upper jaw.
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anti-c-Myc antibody (ab139688, abcam), anti-GS1 antibody (ab176562, abcam), anti-GLS antibody (ab156876, abcam), anti-E-cadherin antibody (ab1416, abcam) and anti-N-cadherin antibody (ab98952, abcam). Fluorescent secondary antibodies (#8887 and #8885, cell signaling technology) were used for detection.
qRT-PCR Total RNA was extracted with TRIzol LS Reagent (10296010, ThermoFisher). cDNA was synthesized from RNA using Advantage RT-for-PCR Kit (639505, Takara). qRT-PCR was performed by Terra qPCR Direct SYBR Premix (638319, Takara) using a Thermal Cycler Dice Real Time System II (TP900, Takara).
Colony Formation KB cells transfected with plasmids overexpressing cMyc or mock-vehicle were seeded in 6-well plates for colony formation assays. Cells were treated with GLS inhibitor (1 mm/mL) or GS inhibitor (25 mm/mL). After 7 days, colonies were fixed with 95% ethanol and stained with 0.1% crystal violet for 15 minutes and cell colonies were imaged.
Cell Cycle Assay After treatment with GLS inhibitor (1 mm/mL) or GS inhibitor (25 mm/mL), KB cells were transfected with plasmids overexpressing c-Myc or mock vehicle plasmids, then were collected and washed with cold PBS. Next, cells were fixed in absolute ethyl alcohol at 4°C for 24 hours and stained with a Cell Cycle and Apoptosis Analysis Kit (C1052, Beyotime). The cell cycle was analyzed using flow cytometry (CytoFLEX).
Wound Healing Assay Wound healing assays were performed to investigate cell migration. A straight line was scratched on the cell layer of KB cells transfected with plasmids overexpressing c-Myc or mock vehicle, then cells were treated with GLS inhibitor (1 mm/mL) or GS inhibitor (25 mm/mL). Cells were imaged from the same point of view at 0 and 24 hours. The wound closure rate was measured by ImageJ.
Statistical Analysis All statistical analyses were carried out using IBM SPSS Statistics 21. The differences between 2 groups were analyzed by 1-way analysis of variance. The correlation of related gene and protein expression with clinicopathologic features was analyzed using Spearman correlation analysis. P values less than 0.05 were considered to be statistically significant.
RESULTS Expression Profiles of c-Myc, GLS and GS in Oral Cancer Expression levels of c-Myc, GLS and GS were detected in oral carcinoma tissues. As shown in Figure 1A, IHC results indicated strong expressions of c-Myc, GLS and GS in tumor tissues. Next, the relationship of c-Myc, GLS and GS expression levels with clinicopathologic features was analyzed in oral cancer tissue samples. The protein expression of GS was significantly associated with clinical stage (Figure 1B and C). As shown in Figure 1D and E, c-Myc positively correlated with GS expression at the mRNA level. However, the other results were not statistically different, possibly due to the small sample size in this study (Figure 1). GLS and GS are Necessary for c-Myc-mediated Oral Cancer Cell Proliferation To examine the interaction of c-Myc, GLS and GS in oral cancer cells, a complementary DNA fragment encoding human c-Myc was cloned into the pEGFP-C1 vector, and the protein expression efficiency in KB cells is presented in Figure 2. Clone formation assays revealed that colonies derived from KB cells overexpressing c-Myc were increased compared to the mock-vehicle group. When KB cells were treated with GLS inhibitor, colony formation was significantly inhibited (P < 0.01). Similarly, GS inhibitor decreased c-Myc overexpression in both KB cells and control cells. These data demonstrated that GLS or GS inhibitor can suppress the c-Myc-mediated proliferation of KB cells (Figure 3A). Moreover, we found that up-regulation of c-Myc caused more cells to be in the S phase of the cell cycle compared with the mock vehicle group, indicating the high proliferative activity of KB cells after c-Myc overexpression. However, treatment with GLS or GS inhibitor decreased the percentage of KB cells overexpressing c-Myc at S phase. These results suggest that GLS and GS inhibitors exert their antiproliferative effects by inducing cell cycle arrest at G0/G1 phase (Figure 3B). In summary, GLS and GS are necessary for c-Myc-mediated oral cancer cell proliferation. GLS and GS are Necessary for c-Myc-mediated Oral Cancer Cell Migration Cell migration drives oral cancer progression and seriously affects patient survival. As shown in Figure 4, overexpression of c-Myc increased the migratory speed in KB cells compared to the mock vehicle group, and this effect was reversed in the presence of GLS or GS inhibitor, suggesting that GLS and GS also play pivotal roles in c-Myc-mediated cell migration in oral cancer. c-Myc Regulates Epithelial-mesenchymal Transition by Up-regulating GLS and GS Activity As shown in Figure 5A-D, IF results revealed that upregulation of c-Myc enhanced the expressions of GLS
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FIGURE 1. Expressions of c-Myc, glutaminase (GLS) and glutamine synthetase (GS) at the mRNA and protein levels in oral cancer tissues. (A) The expressions of c-Myc, GS and GLS in a patient diagnosed with oral cancer at T2N0M0 stage, as determined by immunohistochemistry (IHC) analysis. (B) Relative protein expressions of c-Myc, GLS and GS in oral cancer specimens based on IHC images. (C) Correlations between clinical Tumor, Lymph Node, and Metastasis (TNM) stages and c-Myc, GS and GLS protein expressions in 23 oral cancer patients. (D) Relative mRNA levels of c-Myc, GLS and GS in oral cancer specimens. (E) Correlations between clinical TNM stages and c-Myc, GS and GLS mRNA levels in 21 oral cancer patients.
and GS. Epithelial-mesenchymal transition (EMT) is the biological process by which epithelial cells become mesenchymal phenotypic cells. E-cadherin and N-cadherin are 2 markers for EMT. Overexpression of c-Myc inhibited E-cadherin protein expression but promoted N-cadherin expression compared with the mock vehicle group. Although the GLS and GS inhibitors did not change GLS and GS expressions, the E-cadherin level increased and the N-cadherin level decreased in KB cells in the presence of GLS or GS inhibitors.
DISCUSSION Although the molecular mechanisms of tumor development have not been completely elucidated, the biochemical characteristics of cancer cell energy metabolism provide opportunities for targeted therapies. Some researchers have suggested that cutting off the energy supply of cancer cells may be a way to treat cancer.14,15 Two hallmarks of classical tumor metabolic 238
reprogramming are aerobic glycolysis and dependence on glutamine to replenish the TCA cycle.16 In the TCA cycle, oxaloacetate condenses with acetyl-CoA to generate citric acid, and glutamine is an important source of oxaloacetate. By promoting citric acid synthesis, glutamine helps maintain lipid synthesis. In addition, glutamine can be deaminated to glutamate, which is a precursor for glutathione synthesis. In this way, glutamine is involved in redox balance regulation in tumor cells. Hence, glutamine is an essential amino acid for tumor growth and is preferentially consumed by tumor cells.17 GLS is an enzyme that catalyzes the hydrolysis of glutamine into glutamate and ammonia and GS is an enzyme that catalyzes the synthesis of glutamine from glutamate and ammonium ions.18 Both GLS and GS are highly expressed in tumor cells.19 Studies investigating GLS and GS regulatory mechanisms may identify new possibilities for oral cancer treatment. The expression level of the GLS protein has been shown to be up-regulated in liver cancer,20 nonsmall cell lung cancer,21 THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES VOLUME 358 NUMBER 3 SEPTEMBER 2019
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FIGURE 2. pEGFP-C1-c-Myc mediates c-Myc overexpression in KB cells. The overexpression efficacy of c-Myc was measured by green fluorescent protein (GFP) expression (A) and quantitative real-time polymerase chain reaction (qRT-PCR) (B) in KB cells. Data are represented with GraphPad Prism 5.0. ***P < 0.001.
FIGURE 3. Glutaminase (GLS) or glutamine synthetase (GS) inhibitor suppresses proliferation and induces cell cycle arrest at G0/G1 phase in KB cells through the up-regulation of c-Myc. Colony formation was performed to determine cell proliferation (A), and flow cytometry was performed to determine cell cycle phase (B) in KB cells transfected with mock vehicle and c-Myc overexpression vector, respectively. GLS inhibitor (GLSi) 1.0 mM; GS inhibitor (GSi) 25 mM. Data are represented with GraphPad Prism 5.0. *P < 0.05; **P < 0.01.
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FIGURE 4. Glutaminase (GLS) and glutamine synthetase (GS) are necessary for c-Myc-mediated oral cancer cell migration. (A) Cell migration was detected by wound healing assay in KB cells transfected with mock vehicle or c-Myc overexpression vector. (B) Data are represented with GraphPad Prism 5.0. **P < 0.01. GLS inhibitor (GLSi) 1.0 mM; GS inhibitor (GSi) 25 mM.
colorectal cancer22 and melanoma.23 Okamura et al analyzed gene expression changes in breast cancer cells after knocking out GLS1 and found that 4 genes were significantly downregulated. The 4 targeted genes, high-mobility group Hmga2, formin-like protein 3, Nedd-4 ubiquitin-protein ligase and ubiquitin carboxylterminal hydrolase 15, have relevant functions in tumor n et al cell proliferation and progression.24 Martín-Rufia reported that GLS1 silencing and oxidative stress
synergistically inhibit the proliferation of glioma cells.25 These studies demonstrated that GLS1 is required for the survival of many tumor cells. However, the relationships between glutamine metabolism and oral cancer have not been reported. In this study, we found that GLS and GS were highly expressed in oral cancer samples (Figure 1). c-Myc is known to bind to chromosomes and DNA. It is mainly activated by amplification and cleavage of
FIGURE 5. c-Myc regulates epithelial-mesenchymal transformation (EMT) by up-regulating glutaminase (GLS) and glutamine synthetase (GS) expressions. (A) Representative immunofluorescence (IF) images showing the location and intensity of c-Myc, GLS, GS, N-cadherin, and E-cadherin. (B-F) Relative protein expression levels of c-Myc, GLS, GS, N-cadherin and E-cadherin based on IF. Data are represented with GraphPad Prism 5.0. *P < 0.05; **P < 0.01. GLS inhibitor (GLSi) 1.0 mM; GS inhibitor (GSi) 25 mM.
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chromosomal translocations, and it plays a pivotal role in regulating cell growth, differentiation and malignant transformation.26 As shown in Figure 1, we found that cMyc, GLS and GS were highly expressed in oral cancer samples, and that GS expression levels were associated with TNM stage. Hence, we suspect a relationship between c-Myc and GLS and GS in oral cancer cells. Recent studies have shown that c-Myc promoted glutamine uptake and catabolism by activating the expression of genes required for glutamine metabolism. Glutamine deficiency restrained cell growth and proliferation and induces apoptosis in cells overexpressing c-Myc, while supplementation with TCA cycle intermediates reversed these effects.27 Wise et al reported that glutamine deprivation induced c-Myc-dependent apoptosis and that the reliance of c-Myc on glutamine was dependent on cell type.28 Furthermore, c-Myc was shown to directly bind the promoter of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, and upregulate its protein expression.28 In addition, c-Myc also indirectly enhanced GLS1 activity by inhibiting miR-23a/b.13 Similarly, our data showed that GLS and GS were necessary for c-Myc-mediated oral cancer cell proliferation and migration, indicating that c-Myc acts as an upstream regulator of GLS and GS in oral cancer cells (Figure 3 and Figure 4). EMT induces migration, invasion and tumor progression of tumor cells.13 Varaperez et al demonstrated that Bcl2 interacting protein 3 mediated mitochondrial quality control and promoted the glutamine metabolism-driven EMT-like transition of melanoma cells.29 Similarly, our study indicated that c-Myc inhibits E-cadherin protein expression while promoting N-cadherin expression by enhancing GLS and GS activity, which might play a significant role in the migration and progression of oral cancer cells. In summary, our findings revealed that c-Myc overexpression promotes oral cancer cell proliferation and migration by enhancing GLS and GS activity. This study is beneficial for exploring novel biochemical targets for the prevention and treatment of oral cancer.
AUTHOR CONTRIBUTIONS Y.P.L. and L.L.S. conceived of the presented idea. T. W. and B.L.C. performed the experiment and collected the data. M.C.D. and Z.P.S were responsible for data analysis and visualization. Y.P.L. and L.L.S. supervised the findings of this work. All authors discussed the results and contributed to the final manuscript.
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Submitted November 22, 2018; accepted May 31, 2019. Funding: This study was supported by National Natural Science Foundation of China (Grant No.: 31401161). Conflict of Interest: The authors declare that they have no conflict of interest. Correspondence: Liangliang Shen, State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, 169 West Changle Road, Xi'an 710032, Shaanxi Province, China. (E-mail: [email protected], [email protected]). Yanpu Liu, State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University, 169 West Changle Road, Xi'an 710032, Shaanxi Province, China. (E-mail: [email protected]).
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