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1 Vol pp / 1 1, /diallyltrisulfide-induced apoptosis in human melanoma cells Yoshihiro SUZUKI 1 Mayumi MURI 1,2 Shigeru NISHID 3 Miki SUZUKI-KRSKI 2 Chisei R 1 Toyoko OCHII 2 Tumor necrosis factor (TNF)-related apoptosis-inducing ligand TNF DTS DTS 1. Tumor necrosis factor TNF -related apoptosis-inducing ligand TNF death receptor DR 4/ receptor -R 1 DR5/-R2 -R3- R4 1,2 DR4 DR5 death domain -R3 - R4 3 extrinsic pathway intrin- sic pathway 1-3 DTS 4-7 DTS DTS suzuki.yoshihro@nihon-u.ac.jp 64

2 / 2. Caspase-3/7/ calcein-m/ethd-1 DTS DTS 1C annexin V/ PI 1DDR5 DR4 DTS DR5 DR4 9 DTS z-vd-fmk caspase-8 z-ietdfmkcaspase-9 z-lehd- 375 cells 258 cells 図 1 Vehicle (25ng/ml) DTS (1 M) + DTS C Melanocytes D poptotic cells % Vehicle DTS 1 M 25ng/ml +DTS 1 -C C 825 ng/ml 1 M DTS 24 calcein-m Ethd-1 calcein-m Ethd-1 Scale bar = 1 m. (D) /well V/PI FCSCalibur CellQuest V n=3-9, p<.1; p<.1. 65

3 fmk75 caspase-3/7 z-devd-fmk 5 9 m caspase-3/7 m caspase-3/7 25 ng/ml 72 DTS m caspase-3/7 9 DR4 caspase-3/7 m DR5 caspase-3/7 m DTS caspase-3/7 m DR4 DR5 caspase-3/7 m 9 caspase-3/ ng/mldr5 caspase-3/7 DTS caspase-3/7 DR5 capsase-3/7 9 DTS unfolded protein response UPR GRP78 XP1 DR5 XP-1 DTS GRP78 XP-1 2DR4 DTS 2 Tg GRP78 XP DTS caspase-12 z-td-fmk caspase-4 z- LEVD-fmk 2caspase-12 3 DTS 3 DR5 caspase-12 DTS DR4 caspase-12 DTS DTS caspase caspase-12 DR5 caspase-12 DTS DR4 DTS caspase-12 9 DTS (1 M) GRP78 XP-1 -actin poptotic cells % Inhibitor (1 M) (25 ng/ml) DTS (1 M) C3/7 C12 C4 C ll 図 2 XP-1s XP-1u 2 () / well 1 g/ml DR5/DR4 1 M DTS 3724 SDS PVDF PVDF GRP78 XP-1 PVDF -actin () M z-devd-fmk; C3/7 z-td-fmk C12z-LEVD-fmk C4 z-vd-fmk C ll 25ng/ml 1 M DTS 3772 V/PI FCSCalibur CellQuest V- n=3, p<.1. 66

4 / Caspase-12-activated cells % Caspase-12-activated cells % (1 ng/ml) - DTS (1 M) - (ng/ml) 24 h * Melanocytes 24 h 4. DTS DR 1 DTS 11 DTS DTS 4-7, M * () ng/ml Caspase-12 CaspGLOW Fluorescein Caspase-12 Staining Kit FCSCalibur CellQuest n=3, *p<.5, p<.1, p<.1 () D,E /well1 ng/ ml 1 M DTS Caspase-12 CaspGLOW Fluorescein Caspase-12 Staining Kit FCSCalibur CellQuest n= 3, *p<.5, p<.1. DTS 24 5 M 1 M DTS 24 < 2 DTS DTS 4-7 DTS 13 intrinsic pathway DTS intrinsic pathway caspase-9 caspase-3/7 DTS caspase-3/7 intrinsic pathway intrinsic pathway extrinsic pathway intrinsic pathway IRE TF6 Perk GRP78/ip UPR UPR 18,19 XP-1 DR5 2 XP-1 DTS caspase DTS 67

5 5. DTS DTS 1 Yerbes R, Palacios C, Lopez-Rivas. The therapeutic potential of receptor signaling in cancer cells. Clin Transl Oncol 211; 13: Wang S. The promise of cancer therapeutics targeting the TNF-related apoptosis-inducing ligand and receptor pathway. Oncogene 28; 27: Lelanc, HN, shkenazi. po2l/ and its death and decoy receptors. Cell Death Differ 23; 1: Das, anik NL, Ray SK. Garlic compounds generate reactive oxygen species leading to activation of stress kinases and cysteine proteases for apoptosis in human glioblastoma T98G and U87MG cells. Cancer 27; 11: Kim Y, Xiao D, Xiao H et al. Mitochondria-mediated apoptosis by diallyl trisulfide in human prostate cancer cells is associated with generation of reactive oxygen species and regulated by ax/ak. Mol Cancer Ther 27; 6: Xiao D, Choi S, Johnson DE et al. Diallyl trisulfide-induced apoptosis in human prostate cancer cells involves c-jun N-terminal kinase and extracellularsignal regulated kinase-mediated phosphorylation of cl-2. Oncogene 24; 22: usch C, Jacob C, nwar et al. Diallylpolysulfides induce growth arrest and apoptosis. Int J Oncol 21; 36: Suzuki Y, Inoue T, Murai M et al. Depolarization potentiates -induced apoptosis in human melanoma cells: Role for TP-sensitive K+ channels and endoplasmic reticulum stress. Int J Oncol 212; 41: Murai M, Inoue T, Suzuki-Karasaki M et al. Diallyl trisulfide sensitizes human melanoma cells to induced cell death by promoting endoplasmic reticulum-mediated apoptosis. Int J Oncol 212; 41: shkenazi, Pai R, Fong S et al. Safety and antitumor activity of recombinant soluble po2 ligand. J Clin Invest 1999; 14: Powlny, Singh SV. Multitargeted prevention and therapy of cancer by diallyl trisulfide and related llium vegetable-derived organosulfur compounds. Cancer Lett 28; 269: Lee C, Park H, Kim SY et al. Role of im in diallyl trisulfide-induced cytotoxicity in human cancer cells. J Cell iochem 211; 112: Shankar S, Chen Q, Ganapathy S et al. Diallyl trisulfide increases the effectiveness of and inhibits prostate cancer growth in an orthotropic model: molecular mechanisms. Mol Cancer Ther 28; 7: alch CM, uzzaid C, Soong SJ et al. Final version of the merican Joint Committee on Cancer staging system for cutaneous melanoma. J Clin Oncol 21; 19: Dimberg LY, nderson CK, Camidge R et al. On the to successful cancer therapy? Predicting and counteracting resistance against -based therapeutics. Oncogene 212; 14: Dyer MJ, MacFarlane M, Cohen GM. arriers to effective -targeted therapy of malignancy. J Clin Oncol 27; 25: Hersey P, Zhang XD. How melanoma cells evade trail-induced apoptosis. Nat Rev Cancer 21; 12: reckenridge DG, Germain M, Mathai JP et al. Regulation of apoptosis by endoplasmic reticulum pathways. Oncogene 23; 22: oyce M, Yuan J. Cellular response to endoplasmic reticulum stress: a matter of life or death. Cell Death Differ 26; 13: Liu H, Jiang CC, Lavis CJ et al. 2-Deoxy-D-glucose enhances -induced apoptosis in human melanoma cells through XP-1-mediated up-regulation of -R2. Mol Cancer 29; 8: Hitomi J, Katayama T, Eguchi, Y et al. Involvement of caspase-4 in endoplasmic retidulum stress-induced apoptosis and a beta-induced cell death. J Cell iol 24; 165: Morishima N, Nakanishi K, Takenouchi H et al. n endoplasmic reticulum stress-specific Caspase cascade in apoptosis. Cytochrome c-independent activation of Caspase-9 by Caspase-12. J iol Chem 22; 277: Nakagawa T, Zhu H, Morishima N et al. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 2; 43:

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