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Proteomic Analysis of Novel Targets Associated with the Enhancement of Trka-Induced SK-N-MC Cancer Cell Deathcausedbyngf

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Proteomic Analysis of Novel Targets Associated with the Enhancement of Trka-Induced SK-N-MC Cancer Cell Deathcausedbyngf OPEN Experimental & Molecular Medicine (2016) 48, e235; doi:10.1038/emm.2016.33 & 2016 KSBMB. All rights reserved 2092-6413/16 www.nature.com/emm ORIGINAL ARTICLE Proteomic analysis of novel targets associated with the enhancement of TrkA-induced SK-N-MC cancer cell deathcausedbyNGF Eun Joo Jung1,2, Ky Hyun Chung2,3, Dong-Won Bae4 and Choong Won Kim1,2 Nerve growth factor (NGF) is known to regulate both cancer cell survival and death signaling, depending on the cellular circumstances, in various cell types. In this study, we showed that NGF strongly upregulated the protein level of tropomyosin-related kinase A (TrkA) in TrkA-inducible SK-N-MC cancer cells, resulting in increases in various TrkA-dependent cellular processes, including the phosphorylation of c-Jun N-terminal kinase (JNK) and caspase-8 cleavage. In addition, NGF enhanced TrkA-induced morphological changes and cell death, and this effect was significantly suppressed by the JNK inhibitor SP600125, but not by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. To investigate novel targets associated with the enhancement of TrkA-induced SK-N-MC cell death caused by NGF, we performed Coomassie Brilliant Blue staining and two-dimensional (2D) proteomic analysis in TrkA-inducible SK-N-MC cells. We identified 31 protein spots that were either greatly upregulated or downregulated by TrkA during NGF treatment using matrix-associated laser desorption/ionization time of flight/time of flight mass spectrometry, and we analyzed the effects of SP600125 and wortmannin on the spots. Interestingly, 11 protein spots, including heterogeneous nuclear ribonucleoprotein K (hnRNP K), lamin B1 and TAR DNA-binding protein (TDP43), were significantly influenced by SP600125, but not by wortmannin. Moreover, the NGF/TrkA-dependent inhibition of cell viability was significantly enhanced by knockdown of hnRNP K using small interfering RNA, demonstrating that hnRNP K is a novel target associated with the regulation of TrkA-dependent SK-N-MC cancer cell death enhanced by NGF. Experimental & Molecular Medicine (2016) 48, e235; doi:10.1038/emm.2016.33; published online 27 May 2016 INTRODUCTION translation regulators may have an important role in Nerve growth factor (NGF) is known to regulate both cancer NGF-induced morphological changes. Moreover, NGF induced cell survival and death through its receptors tropomyosin- c-Jun N-terminal kinase (JNK) activation to promote cell death related kinase A (TrkA) and p75 neurotrophin receptor and neurite outgrowth in PC12 cells, and NGF-induced JNK (p75NTR), depending on the tumor origin: NGF activates phosphorylation was significantly suppressed by specific survival signaling through TrkA and p75NTR in breast cancer, inhibitors of the ERK1/2, p38 MAPK, JAK3, Src and Gi whereas NGF activates cell death signaling through p75NTR proteins, but not by the PI3K inhibitor wortmannin,4 suggest- in prostate cancer and through TrkA in glioblastoma and ing that various signaling pathways, except for PI3K-Akt medulloblastoma.1 β-tubulin and a 43-kDa chromogranin signaling, are involved in NGF-induced cell death and neuronal B-derived fragment were identified as the major differentiation. Interestingly, JNK-associated neuronal death NGF-regulated proteins during neuronal differentiation of required nuclear but not cytosolic JNK activity, indicating that PC12 cells using a 2D proteomic analysis.2 Various the nuclear localization of JNK is critical for neuronal death.5 NGF-regulated proteins, including 40S ribosomal protein Although it remains unclear why NGF functions as both an S27a, 60S acidic ribosomal protein P1 and eukaryotic inducer and an inhibitor of cell death, NGF seems to promote translation initiation factor 5A, were identified using a cell death signaling rather than survival in situations of high multiplex iTRAQ proteomic analysis of neurite outgrowth ectopic TrkA expression. NGF is known to induce cell death in regulation of dorsal root ganglia (DRG) cells,3 suggesting that ectopic TrkA-expressing PC12 cells and medulloblastoma 1Department of Biochemistry, Gyeongsang National University School of Medicine, Jinju, Republic of Korea; 2Institute of Health Sciences, Gyeongsang National University, Jinju, Republic of Korea; 3Department of Urology, Gyeongsang National University School of Medicine and Gyeongsang National University Hospital, Jinju, Republic of Korea and 4Central Instrument Facility, Gyeongsang National University, Jinju, Republic of Korea Correspondence: Professor CW Kim, Department of Biochemistry, Gyeongsang National University School of Medicine, 15 Jinju-daero 816Beon-gil, Jinju 52727, Republic of Korea. E-mail: [email protected] Received 17 August 2015; revised 29 December 2015; accepted 8 January 2016 Novel targets in NGF/TrkA-dependent cell death EJ Jung et al 2 cells.6,7 In addition, ectopic TrkA causes neuronal death in ribonucleoprotein K (hnRNP K; D-6, Santa Cruz), TDP43 (E-10, mouse embryonic stem (ES) cells and inhibits angiogenesis and Santa Cruz), GAPDH (FL-335, Santa Cruz), goat anti-rabbit horse- tumor growth in SY5Y neuroblastoma cells by downregulation radish peroxidase conjugate (Bio-Rad) and goat anti-mouse horse- of angiogenic factors.8,9 The ACTB protein, tropomyosin 3, radish peroxidase conjugate (Thermo Scientific, Rockford, IL, USA). dynein intermediate chain protein, vimentin, lamin A/C isoform 1 precursor, lamin B2, hnRNPKa and heat shock Cell culture 70-kDa protein 5 have been identified as NGF-mediated SK-N-MC-TrkA cells, which can induce the expression of ectopic TrkA via the Tet-On system, were previously established from the TrkA-dependent effector proteins using a DIGE (fluorescence human neuroblastoma SK-N-MC cell line (Korean Cell Line Bank No. two-dimensional difference gel electrophoresis) proteomic 30010).12 The cells were maintained in medium A (10% Tet-screened analysis in SY5Y neuroblastoma cells stably expressing ectopic FBS, 1% penicillin/streptomycin, DMEM) containing 1.25 μgml− 1 10 − 1 TrkA. However, little is known about the proteomic factors blasticidin and 25 μgml zeocin in a humidified 5% CO2 incubator associated with the enhancement of TrkA-dependent cancer at 37 °C. The ectopic expression of TrkA was induced by adding cell death caused by NGF. 2 μgml− 1 Tet to the medium A. We recently reported that, in the absence of NGF treatment, SP600125 strongly suppressed TrkA-dependent cellular Western blot analysis processes, including the phosphorylation of JNK and c-Jun, Whole cells were extracted with sodium dodecyl sulfate (SDS) sample in TrkA-inducible SK-N-MC cancer cells, indicating that JNK buffer and were boiled for 5 min at 95 °C. The resultant proteins were is involved in TrkA-dependent signaling pathways.11 In the separated using 10% SDS polyacrylamide gel electrophoresis present study, we investigated the roles of NGF in the (SDS-PAGE) and transferred to an NC membrane. The blot was blocked for 1 h at room temperature in blocking buffer (3% skim regulation of various TrkA-dependent cellular processes, milk, 0.1% Tween-20, phosphate-buffered saline (PBS)) and then morphological changes and cell death. In addition, we incubated with primary antibody at 4 °C overnight. The blot was fi identi ed novel targets associated with the enhancement of then washed with PBST (0.1% Tween-20, PBS) three times for 10 min TrkA-induced SK-N-MC cancer cell death caused by NGF and incubated with an horseradish peroxidase-conjugated secondary through a 2D proteomic analysis, and we investigated the antibody in blocking buffer for 2 h. After being washed with PBST, the effects of small interfering RNA (siRNA) knockdown of these blot was analyzed with the SuperSignal ECL detection system. targets on the regulation of NGF/TrkA-dependent cell death. Cell morphology analysis MATERIALS AND METHODS SK-N-MC-TrkA cells were split in a six-well dish (3 × 105 cells) in the Reagents absence or presence of Tet for 12 h and were then left untreated or − 1 μ μ − 1 Tetracycline (Tet) was obtained from Duchefa Biochemie were treated with 50 ng ml NGF, 1 M wortmannin and 2 gml (Haarlem, the Netherlands). DMEM, Tet-screened FBS and SP600125 for 24 h. The cell morphology was analyzed using penicillin/streptomycin were from Life Technologies (Carlsbad, CA, phase-contrast light microscopy (EVOS XL Core, Life Technologies) fi USA). Blasticidin and zeocin were from Invitrogen (Carlsbad, CA, with × 150 ampli cation. USA). Human recombinant NGF-β, 0.4% trypan blue solution, Coomassie Brilliant Blue (CBB) G, octyl β-D-thioglucopyranoside Detection of dead cells and α-cyano-4-hydroxycinnamic acid were from Sigma-Aldrich SK-N-MC-TrkA cells were split in a six-well dish (3 × 105 cells) in the − (St Louis, MO, USA). SP600125 was from Calbiochem (Billerica, absence or presence of 2 μgml 1 Tet for 12 h and then were left − 1 MA, USA). Wortmannin was from Enzo Life Sciences (Farmingdale, untreated or were treated with 50 ng ml NGF, 1 μM wortmannin − NY, USA). Cell counting kit-8 (CCK-8) was from Dojindo (Kuma- and 2 μgml 1 SP600125 for 24 h, as indicated. Without removing the moto, Japan). Acrylamide/bis-acrylamide 37.5:1 solution (40%), urea, medium, the cells were stained with a 0.03% trypan blue staining Tween-20, ammonium sulfate and DMSO were from Amresco (Solon, solution for 20 min. Dead cells, which were stained a dark blue color, OH, USA). IPG strip (pH 4-7) 17 cm was from Bio-Rad (Quarry Bay, were visualized using bright-field phase-contrast light microscopy Hong Kong). IPG buffer (pH 4–7) was from Amersham Biosciences (EVOS XL Core, Life Technologies) with × 150 amplification. The (Piscataway, NJ, USA). Recombinant DNase I (RNase-free) was from color contrast of the images was adjusted with a Photoshop program. Takara (Shiga, Japan). Sequencing-grade modified trypsin was from Promega (Madison, WI, USA). SuperSignal West Pico stable peroxide Cell viability analysis solution was from Pierce (Rockford, IL, USA). G-fectin transfection SK-N-MC-TrkA cells were split in a 24-well dish (5 × 104 cells) in the − reagent was from Genolution Inc.
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