細胞壞死經常被認為是一種不受調控且無法復原的細胞死亡型態，然而愈來愈多的研究證實，細胞壞死是受到訊息調控的程序性細胞死亡。先前的研究發現，Necrostatin-1 (Nec-1) 可以有效抑制藉由死亡受器路徑引發的細胞壞死，而非TNF-α引起的細胞凋亡。Nec-1主要是透過與ATP競爭結合到RIP1上，抑制RIP1的活性，但是Nec-1 並不影響TNFα刺激下活化的NF-κB活性。實驗室先前的研究利用鎘處理中國倉鼠卵巢細胞CHO K1，誘導細胞走向壞死，建立清晰的細胞壞死調控路徑，而Nec-1可抑制鎘所誘導的細胞壞死。本篇研究中，我們利用不同的細胞壞死誘導藥物促使不同細胞走向細胞壞死，套用鎘誘發細胞壞死的訊息調控路徑，探討Nec-1抑制細胞壞死在細胞內的專一性調控位點。Nec-1可以抑制鎘造成CHO K1細胞或ethacrynic acid (EA) 及死亡受器所誘導U937細胞的細胞壞死。然而Nec-1無法抑制EA處理造成DLD-1細胞產生的細胞壞死。一系列分析的結果顯示，Nec-1沒有調控細胞內鈣離子的能力，也不影響鈣離子依賴性蛋白酶calpain活性的變化。然而，Nec-1可以有效抑制CHO K1細胞在鎘處理下以及EA或 z-VAD-fmk/TNF-α處理U937細胞導致的粒線體膜電位下降。另一方面，DLD-1細胞在EA處理下可見粒線體膜電位上升，在共同處理Nec-1會更進一步增加粒線體膜電位。利用Hsp90抑制劑GA阻斷RIP1活性可以有效抑制z-VAD-fmk/TNF-α處理所造成的細胞壞死，卻無法抑制鎘或EA處理下造成的細胞壞死，顯示Nec-1可以透過RIP1非相關路徑調抑制細胞壞死。細胞壞死誘導藥物也會誘使細胞內氧化性物質 (ROS) 上升，並抑制NF-κB活性。Nec-1不會抑制細胞內ROS的生成。然而，Nec-1可以恢復細胞壞死誘導藥物所抑制的NF-κB活性。有趣的是，單獨Nec-1可以激活細胞內NF-κB。隨著處理時間或劑量上升，NF-κB活性會逐漸的增加。Nec-1會造成IκB的降解，處理26S蛋白酶體抑制劑MG132可以降低Nec-1誘導的IκB降解，進而抑制NF-κB的活化。進一步合併IKK抑制劑PS-1145也可以顯著的抑制Nec-1處理下誘導的NF-κB活化。這樣的結果顯示Nec-1可能是透過活化IKK complex進而促使IκB的降解。綜合本研究的結果可知，Nec-1可以透過RIP1相關或非相關路徑抑制粒線體膜電位下降及活化NF-κB。一方面，Nec-1透過抑制粒線體膜電位下降抑制細胞壞死。另一方面，Nec-1也會透過IKK-IκB路徑活化存活訊號NF-κB，而增加細胞的存活。

Accumulated evidence has indicated that necrotic cell death is not an incidental event but proceeded in a programmed manner. Recently, necrostatin-1 (Nec-1) is developed to protect cells from necrotic death that is mediated through death receptor (DR) signaling. Nec-1 acts on RIP1 kinase activity as the first-in-class inhibitors of RIP1 that is associated with necrosis but irreverent to NF-κB activation. Our previous study has demonstrated that cadmium (Cd) induces necrosis in CHO K1 cells and the signaling pathway has been established. Notably, the Cd-induced necrotic cell death can be attenuated by Nec-1. In this study, we used this established necrotic pathway as a template for us to examine the target sites for Nec-1 action. Two treatments causing necrosis were also studied in parallel for comparisons. Treating U937 cells with TNF-α/z-VAD-fmk or ethacrynic acid (EA) caused necrotic death which could be rescue by Nec-1. However, Nec-1 didn’t protect EA-treated DLD-1 cells from necrosis. Notably, RIP1 activity abolished by GA did not inhibited Cd- or EA-induced necrosis. Systematic analyses revealed that Nec-1 was ineffective to modulate intracellular calcium contents. There was also lack of consistent and conclusive result for Nec-1 in regulating the activity of calpain, a downstream protease activated by calcium. The most significant effect of Nec-1 was on cells that mitochondrial membrane potential (MMP) reduced after giving necrosis inducing factors. The MMP can be restored by Nec-1 and cells rescued. Addition of RIP1 activity inhibitor, geldanamycin (GA), did not rescue cells from Cd-induced necrosis, indicating that Cd did not cause necrosis through RIP1 pathway. Either Cd, EA or death ligand elevated reactive oxygen species (ROS). However, Nec-1 had no effect on altering the ROS level. Nec-1 by itself stimulated NF-κB activity in a dose and time-dependent manner. Additionally, Nec-1 was able to restore the activity that was inhibited by necrosis-inducing agents. Analysis of IκB level showed an acceleration of IκB degradation by Nec-1 treatment. Using IKK specific inhibitor, PS-1145, to block its activity abolished the Nec-1-induced IκB degradation and NF-κB activation. These results suggest that Nec-1 can act as an NF-κB activator by enhancing the IKK activity. Activation of NF-κB may participate partly in protecting cells from necrosis.

Aizawa S, Ookawa K, Kudo T, Asano J, Hayakari M, Tsuchida S (2003) Characterization of cell death induced by ethacrynic acid in a human colon cancer cell line DLD-1 and suppression by N-acetyl-L-cysteine. Cancer Sci 94(10): 886-893

Bano D, Young KW, Guerin CJ, Lefeuvre R, Rothwell NJ, Naldini L, Rizzuto R, Carafoli E, Nicotera P (2005) Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity. Cell 120(2): 275-285

Ben-Neriah Y (2002) Regulatory functions of ubiquitination in the immune system. Nat Immunol 3(1): 20-26

Bezabeh T, Mowat MR, Jarolim L, Greenberg AH, Smith IC (2001) Detection of drug-induced apoptosis and necrosis in human cervical carcinoma cells using 1H NMR spectroscopy. Cell Death Differ 8(3): 219-224

Chaturvedi MM, Mukhopadhyay A, Aggarwal BB (2000) Assay for redox-sensitive transcription factor. Methods Enzymol 319: 585-602

Chen G, Cao P, Goeddel DV (2002) TNF-induced recruitment and activation of the IKK complex require Cdc37 and Hsp90. Mol Cell 9(2): 401-410

Chen ZJ, Bhoj V, Seth RB (2006) Ubiquitin, TAK1 and IKK: is there a connection? Cell Death Differ 13(5): 687-692

Clarke PG (1990) Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol (Berl) 181(3): 195-213

Crompton M (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem J 341 ( Pt 2): 233-249

Degterev A, Hitomi J, Germscheid M, Ch'en IL, Korkina O, Teng X, Abbott D, Cuny GD, Yuan C, Wagner G, Hedrick SM, Gerber SA, Lugovskoy A, Yuan J (2008) Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol 4(5): 313-321

Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J (2005) Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 1(2): 112-119

Deng Y, Ren X, Yang L, Lin Y, Wu X (2003) A JNK-dependent pathway is required for TNFalpha-induced apoptosis. Cell 115(1): 61-70

Ding WX, Ni HM, Yin XM (2007) Absence of Bax switched MG132-induced apoptosis to non-apoptotic cell death that could be suppressed by transcriptional or translational inhibition. Apoptosis 12(12): 2233-2244

Earnshaw WC, Martins LM, Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68: 383-424

Espert L, Denizot M, Grimaldi M, Robert-Hebmann V, Gay B, Varbanov M, Codogno P, Biard-Piechaczyk M (2006) Autophagy is involved in T cell death after binding of HIV-1 envelope proteins to CXCR4. J Clin Invest 116(8): 2161-2172

Festjens N, Kalai M, Smet J, Meeus A, Van Coster R, Saelens X, Vandenabeele P (2006a) Butylated hydroxyanisole is more than a reactive oxygen species scavenger. Cell Death Differ 13(1): 166-169

Festjens N, Vanden Berghe T, Vandenabeele P (2006b) Necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response. Biochim Biophys Acta 1757(9-10): 1371-1387

Ghosh S, Karin M (2002) Missing pieces in the NF-kappaB puzzle. Cell 109 Suppl: S81-96

Ghosh S, May MJ, Kopp EB (1998) NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16: 225-260

Han W, Li L, Qiu S, Lu Q, Pan Q, Gu Y, Luo J, Hu X (2007) Shikonin circumvents cancer drug resistance by induction of a necroptotic death. Mol Cancer Ther 6(5): 1641-1649

Han Y, Englert JA, Delude RL, Fink MP (2005) Ethacrynic acid inhibits multiple steps in the NF-kappaB signaling pathway. Shock 23(1): 45-53

Hayden MS, Ghosh S (2004) Signaling to NF-kappaB. Genes Dev 18(18): 2195-2224
Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, Bodmer JL, Schneider P, Seed B, Tschopp J (2000) Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 1(6): 489-495

Hu X, Han W, Li L (2007) Targeting the weak point of cancer by induction of necroptosis. Autophagy 3(5): 490-492

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

Kawahara A, Ohsawa Y, Matsumura H, Uchiyama Y, Nagata S (1998) Caspase-independent cell killing by Fas-associated protein with death domain. J Cell Biol 143(5): 1353-1360

Kelliher MA, Grimm S, Ishida Y, Kuo F, Stanger BZ, Leder P (1998) The death domain kinase RIP mediates the TNF-induced NF-kappaB signal. Immunity 8(3): 297-303

Khil MS, Kim SH, Pinto JT, Kim JH (1996) Ethacrynic acid: a novel radiation enhancer in human carcinoma cells. Int J Radiat Oncol Biol Phys 34(2): 375-380

Kim YS, Morgan MJ, Choksi S, Liu ZG (2007) TNF-induced activation of the Nox1 NADPH oxidase and its role in the induction of necrotic cell death. Mol Cell 26(5): 675-687

Koechel DA, Cafruny EJ (1973) Synthesis and structure-activity relationship of some thiol adducts of ethacrynic acid. J Med Chem 16(10): 1147-1152

Kroemer G, El-Deiry WS, Golstein P, Peter ME, Vaux D, Vandenabeele P, Zhivotovsky B, Blagosklonny MV, Malorni W, Knight RA, Piacentini M, Nagata S, Melino G (2005) Classification of cell death: recommendations of the Nomenclature Committee on Cell Death. Cell Death Differ 12 Suppl 2: 1463-1467

Kroemer G, Reed JC (2000) Mitochondrial control of cell death. Nat Med 6(5): 513-519

Lee HH, Dadgostar H, Cheng Q, Shu J, Cheng G (1999) NF-kappaB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc Natl Acad Sci U S A 96(16): 9136-9141

Lee TH, Shank J, Cusson N, Kelliher MA (2004) The kinase activity of Rip1 is not required for tumor necrosis factor-alpha-induced IkappaB kinase or p38 MAP kinase activation or for the ubiquitination of Rip1 by Traf2. J Biol Chem 279(32): 33185-33191

Levine B, Yuan J (2005) Autophagy in cell death: an innocent convict? J Clin Invest 115(10): 2679-2688

Lim SY, Davidson SM, Mocanu MM, Yellon DM, Smith CC (2007) The cardioprotective effect of necrostatin requires the cyclophilin-D component of the mitochondrial permeability transition pore. Cardiovasc Drugs Ther 21(6): 467-469

Liu X, Luo X, Shi Y, Zhu GD, Penning T, Giranda VL, Luo Y (2008) Poly (ADP-ribose) polymerase activity regulates apoptosis in HeLa cells after alkylating DNA damage. Cancer Biol Ther 7(6)

Maiuri MC, Zalckvar E, Kimchi A, Kroemer G (2007) Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8(9): 741-752

Maundrell K, Antonsson B, Magnenat E, Camps M, Muda M, Chabert C, Gillieron C, Boschert U, Vial-Knecht E, Martinou JC, Arkinstall S (1997) Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1. J Biol Chem 272(40): 25238-25242

McCaughan FM, Brown AL, Harrison DJ (1994) The effect of inhibition of glutathione S-transferase P on the growth of the Jurkat human T cell line. J Pathol 172(4): 357-362

McConkey DJ (1998) Biochemical determinants of apoptosis and necrosis. Toxicol Lett 99(3): 157-168

McConkey DJ, Orrenius S (1996) The role of calcium in the regulation of apoptosis. J Leukoc Biol 59(6): 775-783

Muzio M, Stockwell BR, Stennicke HR, Salvesen GS, Dixit VM (1998) An induced proximity model for caspase-8 activation. J Biol Chem 273(5): 2926-2930

Pattingre S, Espert L, Biard-Piechaczyk M, Codogno P (2008) Regulation of macroautophagy by mTOR and Beclin 1 complexes. Biochimie 90(2): 313-323

Ploemen JH, van Ommen B, van Bladeren PJ (1990) Inhibition of rat and human glutathione S-transferase isoenzymes by ethacrynic acid and its glutathione conjugate. Biochem Pharmacol 40(7): 1631-1635

Rashmi R, Pillai SG, Vijayalingam S, Ryerse J, Chinnadurai G (2008) BH3-only protein BIK induces caspase-independent cell death with autophagic features in Bcl-2 null cells. Oncogene 27(10): 1366-1375

Ravagnan L, Roumier T, Kroemer G (2002) Mitochondria, the killer organelles and their weapons. J Cell Physiol 192(2): 131-137

Rhodes T, Twentyman PR (1992) A study of ethacrynic acid as a potential modifier of melphalan and cisplatin sensitivity in human lung cancer parental and drug-resistant cell lines. Br J Cancer 65(5): 684-690

Sato S, Fujita N, Tsuruo T (2000) Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci U S A 97(20): 10832-10837

Singh S, Aggarwal BB (1995) Activation of transcription factor NF-kappa B is suppressed by curcumin (diferuloylmethane) [corrected]. J Biol Chem 270(42): 24995-25000

Smith CC, Davidson SM, Lim SY, Simpkin JC, Hothersall JS, Yellon DM (2007) Necrostatin: a potentially novel cardioprotective agent? Cardiovasc Drugs Ther 21(4): 227-233

Suzuki S, Suzuki N, Mirtsos C, Horacek T, Lye E, Noh SK, Ho A, Bouchard D, Mak TW, Yeh WC (2003) Nur77 as a survival factor in tumor necrosis factor signaling. Proc Natl Acad Sci U S A 100(14): 8276-8280

Tartaglia LA, Ayres TM, Wong GH, Goeddel DV (1993) A novel domain within the 55 kd TNF receptor signals cell death. Cell 74(5): 845-853

Temkin V, Huang Q, Liu H, Osada H, Pope RM (2006) Inhibition of ADP/ATP exchange in receptor-interacting protein-mediated necrosis. Mol Cell Biol 26(6): 2215-2225

Thon L, Mathieu S, Kabelitz D, Adam D (2006) The murine TRAIL receptor signals caspase-independent cell death through ceramide. Exp Cell Res 312(19): 3808-3821

Thon L, Mohlig H, Mathieu S, Lange A, Bulanova E, Winoto-Morbach S, Schutze S, Bulfone-Paus S, Adam D (2005) Ceramide mediates caspase-independent programmed cell death. FASEB J 19(14): 1945-1956

Thornberry NA (1998) Caspases: key mediators of apoptosis. Chem Biol 5(5): R97-103

Tracy K, Macleod KF (2007) Regulation of mitochondrial integrity, autophagy and cell survival by BNIP3. Autophagy 3(6): 616-619

Ullman E, Fan Y, Stawowczyk M, Chen HM, Yue Z, Zong WX (2008) Autophagy promotes necrosis in apoptosis-deficient cells in response to ER stress. Cell Death Differ 15(2): 422-425

Wajant H, Pfizenmaier K, Scheurich P (2003) Tumor necrosis factor signaling. Cell Death Differ 10(1): 45-65

Wang KK (2000) Calpain and caspase: can you tell the difference? Trends Neurosci 23(1): 20-26

Wertz IE, O'Rourke KM, Zhou H, Eby M, Aravind L, Seshagiri S, Wu P, Wiesmann C, Baker R, Boone DL, Ma A, Koonin EV, Dixit VM (2004) De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling. Nature 430(7000): 694-699

Wu CJ, Conze DB, Li T, Srinivasula SM, Ashwell JD (2006) Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected]. Nat Cell Biol 8(4): 398-406

Xu X, Chua CC, Kong J, Kostrzewa RM, Kumaraguru U, Hamdy RC, Chua BH (2007) Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells. J Neurochem 103(5): 2004-2014

Xu Y, Huang S, Liu ZG, Han J (2006) Poly(ADP-ribose) polymerase-1 signaling to mitochondria in necrotic cell death requires RIP1/TRAF2-mediated JNK1 activation. J Biol Chem 281(13): 8788-8795

Yamashima T (2004) Ca2+-dependent proteases in ischemic neuronal death: a conserved 'calpain-cathepsin cascade' from nematodes to primates. Cell Calcium 36(3-4): 285-293

Yang PM, Chen HC, Tsai JS, Lin LY (2007) Cadmium induces Ca2+-dependent necrotic cell death through calpain-triggered mitochondrial depolarization and reactive oxygen species-mediated inhibition of nuclear factor-kappaB activity. Chem Res Toxicol 20(3): 406-415

You Z, Savitz SI, Yang J, Degterev A, Yuan J, Cuny GD, Moskowitz MA, Whalen MJ (2008) Necrostatin-1 reduces histopathology and improves functional outcome after controlled cortical impact in mice. J Cereb Blood Flow Metab

Yuan J (2008) Inducing autophagy harmlessly. Autophagy 4(2): 249-250

Zheng L, Bidere N, Staudt D, Cubre A, Orenstein J, Chan FK, Lenardo M (2006) Competitive control of independent programs of tumor necrosis factor receptor-induced cell death by TRADD and RIP1. Mol Cell Biol 26(9): 3505-3513

Zong WX, Thompson CB (2006) Necrotic death as a cell fate. Genes Dev 20(1): 1-15

楊培銘 (2006) 鎘誘發發細胞壞死之機制及其防護作用的研究