中文摘要

PTEN是調控PI3K/Akt訊號傳遞、抑制細胞存活及過度增生不可或缺的重要蛋白質。已知鋅離子可影響人類支氣管上皮細胞(BEAS-2B)中的PTEN蛋白質的表現，本篇實驗進一步的探討在BEAS-2B中，鋅離子是如何對PTEN蛋白質造成影響及其生物效應。首先我們利用西方墨點法證實鋅離子確實會影響PTEN與報導中和PTEN降解有關的蛋白質表現。由於先前研究指出PTEN上K13及K289的泛素化會造成PTEN降解。因此我們將該位置被突變的PTEN送入BEAS-2B中，但結果顯示，單泛素化位置被破壞的PTEN仍會受到鋅離子的影響，鋅離子會增加IKK的活性並降低IκB的量，給予PTEN下游的Akt抑制劑並無法改變鋅離子對NFκB造成的影響。最後我們也觀察鋅對PTEN及NFκB的影響與死亡途徑是否有關，然而我們的實驗結果顯示鋅並不會誘導細胞走向凋亡(Apoptosis)或壞死(Necrosis)。近年來的文獻顯示鋅離子確實參與許多重要的訊息調控，然而，對鋅在生理及毒理方面的角色仍不清楚。本篇研究我們推測鋅會透過一種非細胞凋亡的訊號途徑影響PTEN及NFkB蛋白質的表現及分布情形，但詳細的作用機制尚未明瞭，而這些未知的生理現象可提供我們未來作更深入探討的方向。

Abstract

PTEN is a tumor suppressor protein that inhibits cell growth and viability by modulating the PI3K/AKT signaling pathway. Zinc has been reported to cause PTEN degradation in the airway epithelium. In this study, we further investigate how zinc impact on the PTEN protein and bioeffect in BEAS-2B cells. First, we showed that zinc can suppress the level of PTEN. In previous studies, Reportedly, K13 and K289 are two major monoubiquitination sites that related to PTEN stability. We expressed PTENK13/K289E mutant into BEAS-2B cells and found that BEAS-2B cell was still sensitive to Zinc treatment. Noticeably, zinc activates IKK activity and reduces IκB level in the cells. Administration of Akt inhibitor did not block the zinc-induced IKK activation. Finally, we also examined whether cell viability is associated with the zinc-induced PTEN degradation. The result shows that zinc induce BEAS-2B cell death which is not related to apoptosis, necrosis or autophagy. There is increasing evidence showing that zinc plays a key role in many signaling pathway. However, the underlying mechanisms are poorly understood. Here we assessed the effect of zinc on PTEN and NFκB through a non apoptotic cell death, and the detailed mechanisms needs further investigation.

參考文獻

Ahner A, Gong X, Frizzell RA (2013) Cystic fibrosis transmembrane conductance regulator degradation: cross-talk between the ubiquitylation and SUMOylation pathways. The FEBS journal 280: 4430-4438

Akca H, Demiray A, Tokgun O, Yokota J (2011) Invasiveness and anchorage independent growth ability augmented by PTEN inactivation through the PI3K/AKT/NFkB pathway in lung cancer cells. Lung cancer 73: 302-309

Basu K, Mukhopadhyay A, Ghosh I, Datta K (2012) Nuclear morphology and c-Jun N-terminal kinase 1 expression differentiate serum-starved oxidative stress signalling from hydrogen peroxide-induced apoptosis in retinal neuronal cell line. Cell biology international 36: 1021-1027

Bremner I (1991) Nutritional and physiologic significance of metallothionein. Methods in enzymology 205: 25-35

Chen LF, Greene WC (2004) Shaping the nuclear action of NF-kappaB. Nature reviews Molecular cell biology 5: 392-401

Chen X, Li M, Chen D, Gao W, Guan JL, Komatsu M, Yin XM (2012) Autophagy induced by calcium phosphate precipitates involves endoplasmic reticulum membranes in autophagosome biogenesis. PloS one 7: e52347

Eskelinen EL, Saftig P (2009) Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochimica et biophysica acta 1793: 664-673

Forgacs E, Biesterveld EJ, Sekido Y, Fong K, Muneer S, Wistuba, II, Milchgrub S, Brezinschek R, Virmani A, Gazdar AF, Minna JD (1998) Mutation analysis of the PTEN/MMAC1 gene in lung cancer. Oncogene 17: 1557-1565

Frederickson CJ, Koh JY, Bush AI (2005) The neurobiology of zinc in health and disease. Nat Rev Neurosci 6: 449-462

Galic S, Hauser C, Kahn BB, Haj FG, Neel BG, Tonks NK, Tiganis T (2005) Coordinated regulation of insulin signaling by the protein tyrosine phosphatases PTP1B and TCPTP. Molecular and cellular biology 25: 819-829

Georgiades SN, Mak LH, Angurell I, Rosivatz E, Firouz Mohd Mustapa M, Polychroni C, Woscholski R, Vilar R (2011) Identification of a potent activator of Akt phosphorylation from a novel series of phenolic, picolinic, pyridino, and hydroxamic zinc(II) complexes. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 16: 195-208

Gonzalez-Santamaria J, Campagna M, Ortega-Molina A, Marcos-Villar L, de la Cruz-Herrera CF, Gonzalez D, Gallego P, Lopitz-Otsoa F, Esteban M, Rodriguez MS, Serrano M, Rivas C (2012) Regulation of the tumor suppressor PTEN by SUMO. Cell death & disease 3: e393

Grandage VL, Gale RE, Linch DC, Khwaja A (2005) PI3-kinase/Akt is constitutively active in primary acute myeloid leukaemia cells and regulates survival and chemoresistance via NF-kappaB, Mapkinase and p53 pathways. Leukemia 19: 586-594

Hafsi S, Pezzino FM, Candido S, Ligresti G, Spandidos DA, Soua Z, McCubrey JA, Travali S, Libra M (2012) Gene alterations in the PI3K/PTEN/AKT pathway as a mechanism of drug-resistance (review). International journal of oncology 40: 639-644

Hamatake M, Iguchi K, Hirano K, Ishida R (2000) Zinc induces mixed types of cell death, necrosis, and apoptosis, in molt-4 cells. Journal of biochemistry 128: 933-939

Hu X, Pandolfi PP, Li Y, Koutcher JA, Rosenblum M, Holland EC (2005) mTOR promotes survival and astrocytic characteristics induced by Pten/AKT signaling in glioblastoma. Neoplasia 7: 356-368

Huang J, Yan J, Zhang J, Zhu S, Wang Y, Shi T, Zhu C, Chen C, Liu X, Cheng J, Mustelin T, Feng GS, Chen G, Yu J (2012) SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane. Nature communications 3: 911

Hussain AR, Ahmed SO, Ahmed M, Khan OS, Al Abdulmohsen S, Platanias LC, Al-Kuraya KS, Uddin S (2012) Cross-talk between NFkB and the PI3-kinase/AKT pathway can be targeted in primary effusion lymphoma (PEL) cell lines for efficient apoptosis. PloS one 7: e39945

Jiang J, Slivova V, Harvey K, Valachovicova T, Sliva D (2004) Ganoderma lucidum suppresses growth of breast cancer cells through the inhibition of Akt/NF-kappaB signaling. Nutrition and cancer 49: 209-216

Karin M, Ben-Neriah Y (2000) Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annual review of immunology 18: 621-663

Kauppinen TM, Higashi Y, Suh SW, Escartin C, Nagasawa K, Swanson RA (2008) Zinc triggers microglial activation. J Neurosci 28: 5827-5835

Keng VW, Rahrmann EP, Watson AL, Tschida BR, Moertel CL, Jessen WJ, Rizvi TA, Collins MH, Ratner N, Largaespada DA (2012) PTEN and NF1 inactivation in Schwann cells produces a severe phenotype in the peripheral nervous system that promotes the development and malignant progression of peripheral nerve sheath tumors. Cancer research 72: 3405-3413

Kim YM, Cao D, Reed W, Wu W, Jaspers I, Tal T, Bromberg PA, Samet JM (2007) Zn2+-induced NF-kappaB-dependent transcriptional activity involves site-specific p65/RelA phosphorylation. Cellular signalling 19: 538-546

Kraft C, Peter M, Hofmann K (2010) Selective autophagy: ubiquitin-mediated recognition and beyond. Nature cell biology 12: 836-841

Ku JH, Seo SY, Kwak C, Kim HH (2012) The role of survivin and Bcl-2 in zinc-induced apoptosis in prostate cancer cells. Urologic oncology 30: 562-568

Kwak YD, Wang B, Pan W, Xu H, Jiang X, Liao FF (2010) Functional interaction of phosphatase and tensin homologue (PTEN) with the E3 ligase NEDD4-1 during neuronal response to zinc. J Biol Chem 285: 9847-9857

Lee SR, Yang KS, Kwon J, Lee C, Jeong W, Rhee SG (2002) Reversible inactivation of the tumor suppressor PTEN by H2O2. The Journal of biological chemistry 277: 20336-20342

Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R (1997) PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275: 1943-1947

Li Y, Kimura T, Huyck RW, Laity JH, Andrews GK (2008) Zinc-induced formation of a coactivator complex containing the zinc-sensing transcription factor MTF-1, p300/CBP, and Sp1. Molecular and cellular biology 28: 4275-4284

Lin MC, Liu YC, Tam MF, Lu YJ, Hsieh YT, Lin LY (2012) PTEN interacts with metal-responsive transcription factor 1 and stimulates its transcriptional activity. The Biochemical journal 441: 367-377

Little PJ, Bhattacharya R, Moreyra AE, Korichneva IL (2010) Zinc and cardiovascular disease. Nutrition 26: 1050-1057

Liu C, Li G, Chen R, Yang X, Zhao X, Zhao H (2013) A novel PTEN gene promoter mutation and untypical Cowden syndrome. Chinese journal of cancer research = Chung-kuo yen cheng yen chiu 25: 306-311

Liu SY, Chen CL, Yang TT, Huang WC, Hsieh CY, Shen WJ, Tsai TT, Shieh CC, Lin CF (2012) Albumin prevents reactive oxygen species-induced mitochondrial damage, autophagy, and apoptosis during serum starvation. Apoptosis : an international journal on programmed cell death 17: 1156-1169

Ma X, Kumar M, Choudhury SN, Becker Buscaglia LE, Barker JR, Kanakamedala K, Liu MF, Li Y (2011) Loss of the miR-21 allele elevates the expression of its target genes and reduces tumorigenesis. Proceedings of the National Academy of Sciences of the United States of America 108: 10144-10149

Maehama T, Dixon JE (1998) The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. The Journal of biological chemistry 273: 13375-13378

Manning BD, Cantley LC (2007) AKT/PKB signaling: navigating downstream. Cell 129: 1261-1274

Mocchegiani E, Muzzioli M, Cipriano C, Giacconi R (1998) Zinc, T-cell pathways, aging: role of metallothioneins. Mechanisms of ageing and development 106: 183-204

Mourani PM, Garl PJ, Wenzlau JM, Carpenter TC, Stenmark KR, Weiser-Evans MC (2004) Unique, highly proliferative growth phenotype expressed by embryonic and neointimal smooth muscle cells is driven by constitutive Akt, mTOR, and p70S6K signaling and is actively repressed by PTEN. Circulation 109: 1299-1306

Myers MP, Stolarov JP, Eng C, Li J, Wang SI, Wigler MH, Parsons R, Tonks NK (1997) P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase. Proceedings of the National Academy of Sciences of the United States of America 94: 9052-9057

Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet RJ, Jr., Sledge GW, Jr. (1997) Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth. Molecular and cellular biology 17: 3629-3639

Napetschnig J, Wu H (2013) Molecular basis of NF-kappaB signaling. Annual review of biophysics 42: 443-468

Okumura K, Mendoza M, Bachoo RM, DePinho RA, Cavenee WK, Furnari FB (2006) PCAF modulates PTEN activity. The Journal of biological chemistry 281: 26562-26568

Olive V, Bennett MJ, Walker JC, Ma C, Jiang I, Cordon-Cardo C, Li QJ, Lowe SW, Hannon GJ, He L (2009) miR-19 is a key oncogenic component of mir-17-92. Genes & development 23: 2839-2849

Osaki M, Oshimura M, Ito H (2004) PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis : an international journal on programmed cell death 9: 667-676

Planchon SM, Waite KA, Eng C (2008) The nuclear affairs of PTEN. Journal of cell science 121: 249-253

Plum LM, Rink L, Haase H (2010) The essential toxin: impact of zinc on human health. Int J Environ Res Public Health 7: 1342-1365

Prasad AS (1995) Zinc: an overview. Nutrition 11: 93-99

Prasad AS, Miale A, Jr., Farid Z, Sandstead HH, Schulert AR (1963) Zinc metabolism in patients with the syndrome of iron deficiency anemia, hepatosplenomegaly, dwarfism, and hypognadism. The Journal of laboratory and clinical medicine 61: 537-549

Rhei E, Kang L, Bogomolniy F, Federici MG, Borgen PI, Boyd J (1997) Mutation analysis of the putative tumor suppressor gene PTEN/MMAC1 in primary breast carcinomas. Cancer research 57: 3657-3659

Roohani N, Hurrell R, Kelishadi R, Schulin R (2013) Zinc and its importance for human health: An integrative review. Journal of research in medical sciences : the official journal of Isfahan University of Medical Sciences 18: 144-157

Saeki Y, Kudo T, Sone T, Kikuchi Y, Yokosawa H, Toh-e A, Tanaka K (2009) Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome. The EMBO journal 28: 359-371

Seale AP, de Jesus LA, Kim SY, Choi YH, Lim HB, Hwang CS, Kim YS (2005) Development of an automated protein-tyrosine phosphatase 1B inhibition assay and the screening of putative insulin-enhancing vanadium(IV) and zinc(II) complexes. Biotechnology letters 27: 221-225

Solomons NW (1998) Mild human zinc deficiency produces an imbalance between cell-mediated and humoral immunity. Nutrition reviews 56: 27-28

Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, Ligon AH, Langford LA, Baumgard ML, Hattier T, Davis T, Frye C, Hu R, Swedlund B, Teng DH, Tavtigian SV (1997) Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nature genetics 15: 356-362

Trotman LC, Wang X, Alimonti A, Chen Z, Teruya-Feldstein J, Yang H, Pavletich NP, Carver BS, Cordon-Cardo C, Erdjument-Bromage H, Tempst P, Chi SG, Kim HJ, Misteli T, Jiang X, Pandolfi PP (2007) Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 128: 141-156

Ulukaya E, Acilan C, Yilmaz Y (2011) Apoptosis: why and how does it occur in biology? Cell biochemistry and function 29: 468-480

Vazquez F, Ramaswamy S, Nakamura N, Sellers WR (2000) Phosphorylation of the PTEN tail regulates protein stability and function. Molecular and cellular biology 20: 5010-5018

Virolle T, Adamson ED, Baron V, Birle D, Mercola D, Mustelin T, de Belle I (2001) The Egr-1 transcription factor directly activates PTEN during irradiation-induced signalling. Nature cell biology 3: 1124-1128

von Bulow V, Dubben S, Engelhardt G, Hebel S, Plumakers B, Heine H, Rink L, Haase H (2007) Zinc-dependent suppression of TNF-alpha production is mediated by protein kinase A-induced inhibition of Raf-1, I kappa B kinase beta, and NF-kappa B. J Immunol 179: 4180-4186

Walsh CT, Sandstead HH, Prasad AS, Newberne PM, Fraker PJ (1994) Zinc: health effects and research priorities for the 1990s. Environmental health perspectives 102 Suppl 2: 5-46

Wang BJ, Sheu HM, Guo YL, Lee YH, Lai CS, Pan MH, Wang YJ (2010) Hexavalent chromium induced ROS formation, Akt, NF-kappaB, and MAPK activation, and TNF-alpha and IL-1alpha production in keratinocytes. Toxicology letters 198: 216-224

Wu FY, Wu CW (1987) Zinc in DNA replication and transcription. Annual review of nutrition 7: 251-272

Wu W, Graves LM, Jaspers I, Devlin RB, Reed W, Samet JM (1999) Activation of the EGF receptor signaling pathway in human airway epithelial cells exposed to metals. The American journal of physiology 277: L924-931

Wu W, Wang X, Zhang W, Reed W, Samet JM, Whang YE, Ghio AJ (2003) Zinc-induced PTEN protein degradation through the proteasome pathway in human airway epithelial cells. The Journal of biological chemistry 278: 28258-28263

Xia D, Srinivas H, Ahn YH, Sethi G, Sheng X, Yung WK, Xia Q, Chiao PJ, Kim H, Brown PH, Wistuba, II, Aggarwal BB, Kurie JM (2007) Mitogen-activated protein kinase kinase-4 promotes cell survival by decreasing PTEN expression through an NF kappa B-dependent pathway. The Journal of biological chemistry 282: 3507-3519

Xie L, Lee SY, Andersen JN, Waters S, Shen K, Guo XL, Moller NP, Olefsky JM, Lawrence DS, Zhang ZY (2003) Cellular effects of small molecule PTP1B inhibitors on insulin signaling. Biochemistry 42: 12792-12804

Yang L, Gou Q, Mi C (2008) [Pilot study of PTEN deletion affecting the expression of Cu/Zn SOD]. Zhonghua bing li xue za zhi Chinese journal of pathology 37: 477-480

Zhang B, Egli D, Georgiev O, Schaffner W (2001) The Drosophila homolog of mammalian zinc finger factor MTF-1 activates transcription in response to heavy metals. Molecular and cellular biology 21: 4505-4514