FOXO/TXNIP pathway is involved in the suppression of hepatocellular carcinoma growth by glutamate antagonist MK-801

BMC Cancer

Fuminori Yamaguchi¹  Yuko Hirata¹  Hossain Akram¹  Kazuyo Kamitori¹  Youyi Dong¹  Li Sui¹  Masaaki Tokuda¹ 

[1] Departments of Cell Physiology, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan

关键词: HLF;    HuH-7;    HepG2;    G1 cell cycle arrest;    p27;    TXNIP;    FOXO;    NBQX;    MK-801;   

Others  :  1079524
DOI  :  10.1186/1471-2407-13-468

 received in 2013-04-25, accepted in 2013-10-08,  发布年份 2013

【 摘 要 】

Background

Accumulating evidence has suggested the importance of glutamate signaling in cancer growth, yet the signaling pathway has not been fully elucidated. N-methyl-D-aspartic acid (NMDA) receptor activates intracellular signaling pathways such as the extracellular-signal-regulated kinase (ERK) and forkhead box, class O (FOXO). Suppression of lung carcinoma growth by NMDA receptor antagonists via the ERK pathway has been reported. However, series of evidences suggested the importance of FOXO pathways for the regulation of normal and cancer cell growth. In the liver, FOXO1 play important roles for the cell proliferation such as hepatic stellate cells as well as liver metabolism. Our aim was to investigate the involvement of the FOXO pathway and the target genes in the growth inhibitory effects of NMDA receptor antagonist MK-801 in human hepatocellular carcinoma.

Methods

Expression of NMDAR1 in cancer cell lines from different tissues was examined by Western blot. NMDA receptor subunits in HepG2, HuH-7, and HLF were examined by reverse transcriptase polymerase chain reaction (RT-PCR), and growth inhibition by MK-801 and NBQX was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of MK-801 on the cell cycle were examined by flow cytometry and Western blot analysis. Expression of thioredoxin-interacting protein (TXNIP) and p27 was determined by real-time PCR and Western blotting. Activation of the FOXO pathway and TXNIP induction were examined by Western blotting, fluorescence microscopy, Chromatin immunoprecipitation (ChIP) assay, and reporter gene assay. The effects of TXNIP on growth inhibition were examined using the gene silencing technique.

Results

NMDA receptor subunits were expressed in all cell lines examined, and MK-801, but not NBQX, inhibited cell growth of hepatocellular carcinomas. Cell cycle analysis showed that MK-801 induced G1 cell cycle arrest by down-regulating cyclin D1 and up-regulating p27. MK-801 dephosphorylated Thr24 in FOXO1 and induced its nuclear translocation, thus increasing transcription of TXNIP, a tumor suppressor gene. Knock-down of TXNIP ameliorated the growth inhibitory effects of MK-801.

Conclusions

Our results indicate that functional NMDA receptors are expressed in hepatocellular carcinomas and that the FOXO pathway is involved in the growth inhibitory effects of MK-801. This mechanism could be common in hepatocellular carcinomas examined, but other mechanisms such as ERK pathway could exist in other cancer cells as reported in lung carcinoma cells. Altered expression levels of FOXO target genes including cyclin D1 and p27 may contribute to the inhibition of G1/S cell cycle transition. Induction of the tumor suppressor gene TXNIP plays an important role in the growth inhibition by MK-801. Our report provides new evidence that FOXO-TXNIP pathway play a role in the inhibition of the hepatocellular carcinoma growth by MK-801.

【 授权许可】

   
2013 Yamaguchi et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Anwyl R: Synaptic plasticity, A molecular switch for memory. Curr Biol 1994, 4(9):854-856.
• [2]Riedel G, Platt B, Micheau J: Glutamate receptor function in learning and memory. Behav Brain Res 2003, 140(1–2):1-47.
• [3]Hinoi E, Takarada T, Ueshima T, Tsuchihashi Y, Yoneda Y: Glutamate signaling in peripheral tissues. Eur J Biochem 2004, 271(1):1-13.
• [4]Skerry TM, Genever PG: Glutamate signalling in non-neuronal tissues. Trends Pharmacol Sci 2001, 22(4):174-181.
• [5]Siegel S, Sanacora G: The roles of glutamate receptors across major neurological and psychiatric disorders. Pharmacol Biochem Behav 2012, 100(4):653-655.
• [6]Javitt DC: Glutamate as a therapeutic target in psychiatric disorders. Mol Psychiatry 2004, 979(11):984-997.
• [7]Stepulak A, Luksch H, Gebhardt C, Uckermann O, Marzahn J, Sifringer M, Rzeski W, Staufner C, Brocke KS, Turski L, et al.: Expression of glutamate receptor subunits in human cancers. Histochem Cell Biol 2009, 132(4):435-445.
• [8]Samuels Y, Prickett T: Molecular pathways: dysregulated glutamatergic signaling pathways in cancer. Clin Cancer Res 2012, 18(16):4240-4246.
• [9]Seidlitz EP, Sharma MK, Saikali Z, Ghert M, Singh G: Cancer cell lines release glutamate into the extracellular environment. Clin Exp Metastasis 2009, 26(7):781-787.
• [10]Herner A, Sauliunaite D, Michalski CW, Erkan M, De Oliveira T, Abiatari I, Kong B, Esposito I, Friess H, Kleeff J: Glutamate increases pancreatic cancer cell invasion and migration via AMPA receptor activation and Kras-MAPK signaling. Int J Cancer 2011, 129(10):2349-2359.
• [11]Hollmann M, O’Shea-Greenfield A, Rogers SW, Heinemann S: Cloning by functional expression of a member of the glutamate receptor family. Nature 1989, 342(6250):643-648.
• [12]Nakanishi N, Shneider NA, Axel R: A family of glutamate receptor genes: evidence for the formation of heteromultimeric receptors with distinct channel properties. Neuron 1990, 5(5):569-581.
• [13]Lynch DR, Guttmann RP: NMDA receptor pharmacology: perspectives from molecular biology. Curr Drug Targets 2001, 2(3):215-231.
• [14]Hardingham GE, Bading H: Synaptic versus extrasynaptic NMDA receptor signalling: implications for neurodegenerative disorders. Nat Rev Neurosci 2010, 11(10):682-696.
• [15]Sheth SS, Bodnar JS, Ghazalpour A, Thipphavong CK, Tsutsumi S, Tward AD, Demant P, Kodama T, Aburatani H, Lusis AJ: Hepatocellular carcinoma in Txnip-deficient mice. Oncogene 2006, 25(25):3528-3536.
• [16]Watanabe R, Nakamura H, Masutani H, Yodoi J: Anti-oxidative, anti-cancer and anti-inflammatory actions by thioredoxin 1 and thioredoxin-binding protein-2. Pharmacol Ther 2010, 127(3):261-270.
• [17]Papadia S, Soriano FX, Leveille F, Martel MA, Dakin KA, Hansen HH, Kaindl A, Sifringer M, Fowler J, Stefovska V, et al.: Synaptic NMDA receptor activity boosts intrinsic antioxidant defenses. Nat Neurosci 2008, 11(4):476-487.
• [18]Chen KS, DeLuca HF: Isolation and characterization of a novel cDNA from HL-60 cells treated with 1,25-dihydroxyvitamin D-3. Biochim Biophys Acta 1994, 1219(1):26-32.
• [19]Ikarashi M, Takahashi Y, Ishii Y, Nagata T, Asai S, Ishikawa K: Vitamin D3 up-regulated protein 1 (VDUP1) expression in gastrointestinal cancer and its relation to stage of disease. Anticancer Res 2002, 22(6C):4045-4048.
• [20]Escrich E, Moral R, Garcia G, Costa I, Sanchez JA, Solanas M: Identification of novel differentially expressed genes by the effect of a high-fat n-6 diet in experimental breast cancer. Mol Carcinog 2004, 40(2):73-78.
• [21]Yamaguchi F, Takata M, Kamitori K, Nonaka M, Dong Y, Sui L, Tokuda M: Rare sugar D-allose induces specific up-regulation of TXNIP and subsequent G1 cell cycle arrest in hepatocellular carcinoma cells by stabilization of p27kip1. Int J Oncol 2008, 32(2):377-385.
• [22]Aronica E, Yankaya B, Jansen GH, Leenstra S, van Veelen CW, Gorter JA, Troost D: Ionotropic and metabotropic glutamate receptor protein expression in glioneuronal tumours from patients with intractable epilepsy. Neuropathol Appl Neurobiol 2001, 27(3):223-237.
• [23]Choi SW, Park SY, Hong SP, Pai H, Choi JY, Kim SG: The expression of NMDA receptor 1 is associated with clinicopathological parameters and prognosis in the oral squamous cell carcinoma. J Oral Pathol Med 2004, 33(9):533-537.
• [24]Liu JW, Kim MS, Nagpal J, Yamashita K, Poeta L, Chang X, Lee J, Park HL, Jeronimo C, Westra WH, et al.: Quantitative hypermethylation of NMDAR2B in human gastric cancer. Int J Cancer 2007, 121(9):1994-2000.
• [25]Abdul M, Hoosein N: N-methyl-D-aspartate receptor in human prostate cancer. J Membr Biol 2005, 205(3):125-128.
• [26]Kalariti N, Pissimissis N, Koutsilieris M: The glutamatergic system outside the CNS and in cancer biology. Expert Opin Investig Drugs 2005, 14(12):1487-1496.
• [27]Rzeski W, Turski L, Ikonomidou C: Glutamate antagonists limit tumor growth. Proc Natl Acad Sci USA 2001, 98(11):6372-6377.
• [28]Stepulak A, Sifringer M, Rzeski W, Endesfelder S, Gratopp A, Pohl EE, Bittigau P, Felderhoff-Mueser U, Kaindl AM, Buhrer C, et al.: NMDA antagonist inhibits the extracellular signal-regulated kinase pathway and suppresses cancer growth. Proc Natl Acad Sci USA 2005, 102(43):15605-15610.
• [29]Greer EL, Brunet A: FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 2005, 24(50):7410-7425.
• [30]Reagan-Shaw S, Ahmad N: The role of Forkhead-box Class O (FoxO) transcription factors in cancer: a target for the management of cancer. Toxicol Appl Pharmacol 2007, 224(3):360-368.
• [31]Adachi M, Osawa Y, Uchinami H, Kitamura T, Accili D, Brenner DA: The forkhead transcription factor FoxO1 regulates proliferation and transdifferentiation of hepatic stellate cells. Gastroenterology 2007, 132(4):1434-1446.
• [32]Gross DN, van den Heuvel AP, Birnbaum MJ: The role of FoxO in the regulation of metabolism. Oncogene 2008, 27(16):2320-2336.
• [33]Gill SS, Pulido OM, Mueller RW, McGuire PF: Molecular and immunochemical characterization of the ionotropic glutamate receptors in the rat heart. Brain Res Bull 1998, 46(5):429-434.
• [34]Jeon JH, Lee KN, Hwang CY, Kwon KS, You KH, Choi I: Tumor suppressor VDUP1 increases p27(kip1) stability by inhibiting JAB1. Cancer Res 2005, 65(11):4485-4489.
• [35]Zhao X, Gan L, Pan H, Kan D, Majeski M, Adam SA, Unterman TG: Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms. Biochem J 2004, 378(Pt 3):839-849.
• [36]Ishiuchi S, Yoshida Y, Sugawara K, Aihara M, Ohtani T, Watanabe T, Saito N, Tsuzuki K, Okado H, Miwa A, et al.: Ca2 + -permeable AMPA receptors regulate growth of human glioblastoma via Akt activation. J Neurosci 2007, 27(30):7987-8001.
• [37]Lukas J, Pagano M, Staskova Z, Draetta G, Bartek J: Cyclin D1 protein oscillates and is essential for cell cycle progression in human tumour cell lines. Oncogene 1994, 9(3):707-718.
• [38]Waldman T, Kinzler KW, Vogelstein B: p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res 1995, 55(22):5187-5190.
• [39]Coats S, Flanagan WM, Nourse J, Roberts JM: Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle. Science 1996, 272(5263):877-880.
• [40]Tran H, Brunet A, Griffith EC, Greenberg ME: The many forks in FOXO’s road. Sci STKE 2003, 2003(172):RE5.
• [41]Stahl M, Dijkers PF, Kops GJ, Lens SM, Coffer PJ, Burgering BM, Medema RH: The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2. J Immunol 2002, 168(10):5024-5031.
• [42]Medema RH, Kops GJ, Bos JL, Burgering BM: AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 2000, 404(6779):782-787.
• [43]Ramaswamy S, Nakamura N, Sansal I, Bergeron L, Sellers WR: A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR. Cancer Cell 2002, 2(1):81-91.
• [44]Xuan Z, Zhang MQ: From worm to human: bioinformatics approaches to identify FOXO target genes. Mech Ageing Dev 2005, 126(1):209-215.