ARH1 (tumor angiogenesis) Mechanism of a novel tumor angiogenesis inhibitor by ARH1 protein
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1 ARH1 (tumor angiogenesis) Mechanism of a novel tumor angiogenesis inhibitor by ARH1 protein
2 ... ARH1 (tumor angiogenesis)...
3 ARH1 (tumor angiogenesis)
4 Title of Project Key Words Mechanism of a novel tumor angiogenesis inhibitor by ARH1 protein tumor angiogenesis, apoptosis, protein-protein interaction, signal pathway Project Leader Seung Bae Rho Associated Company κ κ κ κ κ κ κ Our study of the protein ARH1 has uncovered a novel mechanism that is capable of inhibiting angiogenesis. Through our study, ARH1 was determined to be a new potent antiangiogenic and antitumor molecule that targets TRADD. Here, we suggest a possible mechanism for the inhibition of angiogenesis and tumor growth by ARH1, which involves direct binding to TRADD thereby inhibiting the NF-kB-activated JNK signaling pathway. Human tumor necrosis factor 1 (TNFR1)-associated death domain protein (TRADD) plays an essential role as a multifunctional signaling molecule to the TNFR1 receptor complex located at the cell membrane. Previous data shows that TRADD-TRAF2 complex leads to the activation of JNK and NF-kB in an animal model. Here we identified human ARH1, well known as a tumor suppressor protein in ovarian carcinomas, to be the TRADD-interacting protein. Extensive protein-protein interaction studies demonstrated strong evidence that ARH1 is a component of TNFR1 signaling. In cells lacking ARH1 (RNAi), NF-kB transcriptional activation was markedly enhanced, whereas transient over-expression of ARH1 in SKOV-3 and 2774 cells repressed NF-kB-mediated transcription. Thus, ARH1 acts as a TNFR1 signaling protein that represses NF-kB transcriptional activation by the formation of a protein complex in cells. Since no X-ray and NMR structures are available for ARH1 protein, we built the three-dimensional structure of the protein using the established molecular modeling methods such as homology modeling and molecular dynamics simulations.
5 Based on the structure observation, the Lys15 and Arg16 were suggested as the potential key residues in N-terminal domain. We also show that ARH1 acts as a potent inhibitor of angiogenesis. The data indicates that ARH1 play crucial functions in maintaining cellular homeostasis by not only binding to TRADD, but also competing or preventing the protein complex formation of TRADD with TRAF2. These findings show that the inhibition of TNF signal transduction by ARH1 results in antiangiogenic and antitumor effects, suggesting that ARH1 may be valuable in anticancer therapy. k
6 ARH1 DAPI Merge Figure 2. Intracellular localization of ARH1 in ovarian cancer cells. Figure 1. Microarray analysis of ARH1 expression in ovarian cancer tissues. cdnas obtained from 11 ovarian cancer tissues were analyzed by microarray. The values are represented by fold of the mrna level in the paired normal tissues.
7 TRADD mrna GAPDH mrna TRADD TRAF2 HEK293 MRC-5 SKOV HEK293 MRC-5 SKOV TRAF2 mrna GAPDH mrna Figure 3. ARH1 inhibits endothelial cell proliferation in vitro. (A) The angiogenic effects of ARH1 on DNA synthesis in endothelial cells were followed with/without VEGF (10 ng/) treatment for 48 hours. cpm values of [ 3 H]thymidine were determined using a liquid scintillation counter. (B) Ablation of GFP-ARH1 mrna expression by sirna in HUVECs. Anti-TRADD Anti-TRAF2 Anti-beta-actin Anti-beta-actin Figure 5. Endogenous expression of TRADD and TRAF2 in normal and ovarian cancer cells. β Whole cell IP lysate Anti-TRADD Anti-IgG Anti-ARH1 Whole cell lysate IP Anti-ARH1 Anti-IgG Anti-TRADD Figure 6. Co-immunoprecipitation between the endogenous TRADD and the exogenously introduced GFP-tagged ARH1 (GFP-ARH1) display the interactions of the two proteins. κ ARH p65 CE NE HSP90 Histone H1 Figure 7. ARH1 inhibits nuclear localization of p65 Figure 4. Interaction Aanalysis between Human ARH1 and TRADD-N Terminus.
8 κ κ κ κ κ κ κ NF-κB 활성이저해되는것인지를재확인하기위하여 Ras family (H-ras, K-ras, N-ras) 로알려져있는단백질들을가지고상호결합력을측정하였다. 그결과그림 10에서보시는바와같이 TRADD 단백질은유일하게암억제단백질인 ARH1 단백질과상호결합한다는사실을알수있었다. ARH1 H-ras K-ras N-ras TRADD IP : anti-gfp WB : TRADD WB : ARH1 WB : beta-actin in WCL Figure 10. Interaction of TRADD with RAS family Figure 8. ARH1 competes with TRAF2 for TRADD binding ARH1 VEGF tubulin SKOV ES-2 SNU-8 P1 P2 P3 P4 P5 ARH1 methyl ARH1 methyl Figure 9. ARH1 inhibits VEGF expression in HUVECs Figure 11. Methylation status of candidate gene in ovarian cancer cells and clinical cancer tissues
9 k k G1 G2 (Effector) G3 G4 G5 N-terminal Extension GTP Binding Domain C-terminal Domain Figure 12. Comparison of the amino-acid sequence of ARH1, Rig, and H-Ras. 그림 13에서보시는바와같이 ARH1-N 단백질을난소암세포주에 transfection 시킨결과 early stage에서 39.15% 의난소암세포를사멸시키는효과를보여주고있다. 그리고 growth 를조사한결과 ARH1-N 과발현시켰을때난소암세포주에서성장이일어나지않았다. 동시에 TUNEL assay와 caspase-3 activity를조사한결과에서도현저히사멸하는세포가증가하는것을관찰하였다. Figure 13. ARH1-N induces apoptosis in ovarian cancer cells. A. FACS analysis B. TUNEL assays (apoptotic cells). C. Caspase-3 activity assay.
10 Figure 15. ARH1-N inhibits migration of HUVECs. Figure 14. ARH1-N inhibits endothelial cell proliferation in vitro. (A) The angiogenic effects of ARH1-N on DNA synthesis in endothelial cells were followed with/without VEGF (10 ng/) treatment for 48 hours. cpm values of [ 3 H]thymidine were determined using a liquid scintillation counter. Figure 16. ARH1-N inhibits tube formation in vitro.
11 Figure 19. ARH1-N VEGF expression in HUVECs. Figure 17. ARH1-N inhibits vessel sprouting ex vivo. κ Figure 18. ARH1-N inhibits nuclear localization of p65. κ κ κ Figure 20. ARH1-N blocks with VEGF-mediated PI3K-Akt signaling in HUVECs.
12 Figure 21. 3D structure of ARH1-N via computer modeling. Figure 22. Angiostatic activity in the chicken chorioallantoic membrane (CAM) assay.
13 Figure 23. Reporter activity test for various ARH1 promoters in human ovarian cancer cell line, SKOV-3 and Figure 24. Effect of cisplatin and etoposide with ARH1-N in ovarian cancer cells.
14 다. 바.
15 A. B. Figure 25. Adenovirus-mediated systemic gene delivery of ARH1 inhibits tumor growth and angiogenesis. (A) Xenograft mouse model. (B) Orthotopic mouse model.
16 C. D.
17 Interaction between fortilin and transforming growth factor-beta stimulated clone-22 (TSC-22) prevents apoptosis via the destabilization of TSC-22 Tissue inhibitor of metalloproteinases-3 interacts with angiotensin II type 2 receptor and additively inhibits angiogenesis Interferon regulatory factor-1 (IRF-1) regulates VEGF-induced angiogenesis in HUVEC Calpain 6 supports tumorigenesis by inhibiting apoptosis and facilitating angiogenesis Insulin-like growth factor-binding protein-5 (IGFBP-5) acts as a tumor suppressor by inhibiting angiogenesis FEBS Letters (3.263) Cardiovascular Research (6.127) Biochimica et Biophysica Acta (BBA) - Molecular Cell Research (4.374) Cancer Letters (3.398) Carcionogenesis (5.406) 2008: 271 (2), : 29 (11),
18 Cysteine-rich 61 (CYR61) inhibits cisplatin-induced apoptosis in ovarian carcinoma cells Dkk3, downregulated in cervical cancer, functions as a negative regulator of beta-catenin Leucyl-tRNA synthetase-dependent and -independent activation of a group I intron Inhibition of angiogenesis by the BTB domain of promyelocytic leukemia zinc finger protein A gene signature-based approach identifies thioridazine as an inhibitor of phosphatidylinositol-3 -kinase (PI3K)/AKT pathwa Carbonic anhydrase IX (CA 9) modulates tumor-associated cell migration and invasion Biotechnology Letters (1.595) International Journal of Cancer (4.734) Journal of Biological Chemistry (5.520) Cancer Letters (3.741) Gynecologic Oncology (3.733) Journal of Cell Science (6.144) FEBS Letters (3.541) 2009: 31 (1), : 124 (2), : 284 (39), : 294 (1), NF-kB inhibitor containing ARH1 protein or gene encoding the same
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수시과제 2015-13 경기도 지역별 여성일자리 정책 추진 전략 연구책임자 : 최 윤 선 (본원선임연구위원) : 남 승 연 (본원연구위원) 연 구 지 원 : 이 상 아 (본원위촉연구원) 연 구 기 간 : 2015. 9 ~12 2015 발 간 사 여성 일자리는 사회 내 여성과 남성간의 차이를 좁히고 개개인의 삶을 윤택하게 만드는 중요 한 부분입니다. 이에 정부는
한약치료와표적항암요법 ( 아피니토 ) 을병행하여부분관해된신세포암간전이환자 1 례 Abstract Sung-Hwan Chang 1, Ji-Hye Park 1,2, Hwa Seung Yoo,2*
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Review on Conservative Treatment of Spinal Scoliosis Moon-kyu Lee, O.M.D., Gil-jae Lee, O.M.D., Yun-kyung Song, O.M.D., Hyung-ho Lim, O.M.D. Dept. of Oriental Rehabilitation Medicine College of Oriental
216 동북아역사논총 41호 인과 경계공간은 설 자리를 잃고 배제되고 말았다. 본고에서는 근세 대마도에 대한 한국과 일본의 인식을 주로 영토와 경계인 식을 중심으로 고찰하고자 한다. 이 시기 대마도에 대한 한일 양국의 인식을 살펴볼 때는 근대 국민국가적 관점에서 탈피할
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connect educational content with entertainment content and that production of various contents inducing educational motivation is important. Key words: edutainment, virtual world, fostering simulation