➨୍❶
࠙⣽⬊ࠚ
KB (Human cervical carcinoma)ࠊACC-MESO-1 (Human malignant mesothelioma)ࠊMCF-7 (Human breast cancer)ࠊMia-PaCa-2 (Human pancreatic cancer)ࠊHT-1080 (Human fibrosarcoma) ࡣࠊឡ▱Ꮫ㝔Ꮫ⸆Ꮫ㒊⏕య᭷ᶵᏛㅮᗙ ⏣୰ ᇶḧ ᩍᤵࡼࡾࠊ HRA (Human ovarian
cancer) ࡣࠊྡྂᒇᏛᏛ㝔་Ꮫ◊✲⛉⏘፬ே⛉Ꮫศ㔝 ᰘ⏣ Ύ ඛ⏕ࡼࡾศࡉࢀ
ࡓࡶࡢࢆ⏝ࡋࡓࠋࡑࢀࡽࡢ⣽⬊ࡣࠊ 10%ศࡢ࢘ࢩ⫾ඣ⾑Ύ㸦FBSࠊSigma Aldrichࠊ St. LouisࠊMOࠊUSA㸧ࠊ࣌ࢽࢩࣜࣥ㸦100 unit/ml㸧ࠊࢫࢺࣞࣉࢺ࣐ࢩࣥ㸦100 mg/ml㸧㸦ࢼ
࢝ࣛࢸࢫࢡࠊி㒔㸧ࢆῧຍࡋࡓRPMI 1640ᇵᆅ㸦ࢼ࢝ࣛ㸧࡛ࠊ5% CO2ୗ37Υ࡛ᇵ 㣴ࡋࡓࠋ
⥅௦ࡣࠊ0.25%ࢺࣜࣉࢩࣥ in 0.53 mM EDTA⁐ᾮ㸦ࢼ࢝ࣛ㸧࡛⣽⬊ࢆ3ศ㛫ฎ⌮
ࡍࡿࡇ࡛㞳ࡋࡍࡿࡇࡼࡾ⾜ࡗࡓࠋ⣽⬊ᩘࡢ ᐃࡣࠊ⾑⌫ィ⟬ᯈࢆ⏝࠸࡚0.5㸣ࢺ
ࣜࣃࣥࣈ࣮ࣝᰁⰍᾮ㸦ࢼ࢝ࣛ㸧࡛ᰁⰍࡍࡿࡇࡼࡾ⾜ࡗࡓࠋ
࠙⣽⬊⮴Ṛάᛶࡢ ᐃࠚ
ྛ✀⣽⬊ࢆ96✰ᖹᗏࣉ࣮ࣞࢺ㸦᪥ᮏࢪ࢙ࢿࢸࢡࢫ㸧5 × 10㸲cell / 198 μl / well ࡎ ࡘ✀ࡋࠊ24 hᇵ㣴ࡋࡓࠋࡑࡢᚋࠊDMSO࡛10 mg/mlࠊ1 mg/mlࠊ100 μg/mlࠊ10 μg/ml
ㄪ〇ࡋࡓࢧࣥࣉࣝࢆ2 μlࡎࡘῧຍࡋ㸦᭱⤊⃰ᗘ㸸100ࠊ10ࠊ1ࠊ0.1 μg/ml㸧ࠊࡉࡽ72 h ᇵ㣴ࡋࡓࠋࢥࣥࢺ࣮ࣟࣝࡣDMSOࢆࠊ㝧ᛶࢥࣥࢺ࣮ࣟࣝࡋ࡚adriamycin㸦༠Ⓨ㓝࣭
ࣇࢨ࣮ࠊᮾி㸧ࢆ㝶ᕼ㔘ࡋ࡚⏝ࡋࡓࠋMTT㸦ࢼ࢝ࣛ㸧ࢆ3 mg/mlࡢ⃰ᗘ࡛20 μlࡎࡘῧຍࡋࠊࡉࡽ4 hᇵ㣴ࡋࡓࠋᇵᆅࢆ㝖ཤᚋࠊDMSO 170 μlࢆῧຍࡋ࡚⏕ᡂࡋࡓ formazanࢆ⁐ゎࡋࠊ570 nm࡛ࡢ྾ගᗘࢆARVO MX 1420 multilabel counter (Perkin Elmerࠊ Winter Street WalthamࠊMAࠊUSA)ࠊࡲࡓࡣiMark Microplate Reader (Bio-RadࠊPhiladelphiaࠊ
PaࠊUSA) ࡼࡾ ᐃࡋࡓࠋྛ⸆≀ࡢ⃰ᗘ࠾ࡅࡿ⣽⬊ቑṪᢚไ⋡ (%)ࢆ྾ගᗘࡼࡾ⟬
ฟࡋࠊ50%ࢆྵࡴ 3 ࡘࡢ␗࡞ࡿ⃰ᗘࡢᑐᩘᑐࡍࡿ⣽⬊⏕Ꮡ⋡ࢆࣉࣟࢵࢺࡋࡓࡁࡢ㏆
ఝ┤⥺ࢆ⏝࠸ࠊ50%ࢆ♧ࡍ⃰ᗘࢆIC50ࡋࡓࠋ
࠙ࢼࢫࡀࡃ∦࢚࢟ࢫࡢㄪ〇ศ⏬ࠚ
ᐇ㦂⏝ࡋࡓࢼࢫᯝᐇࡣࠊྡྂᒇᕷෆࡢᕷሙ࡛㉎ධࡋࡓࠋ᪂㩭࡞༸ᙧࢼࢫᯝᐇ14 kg
ࢆࠊࡀࡃ∦㒊ศ2.8 kgࡑࢀ௨እࡢ㒊ศศࡅࠊࡀࡃ∦㒊ศࡣ⤖⇱ࡋࠊ⤖⇱≀
2.6 x 102 gࢆᚓࡓࠋࡇࢀ࢚ࢱࣀ࣮ࣝ2 lࢆຍ࠼୍࡚ᬌ෭ᾐᢳฟࢆ3ᅇ⾜࠸ࠊᢳฟᾮࢆྜ
ࢃࡏ࡚⃰⦰ᅛࡍࡿࡇࡼࡾࠊᢳฟ࢚࢟ࢫ7.6 gࢆᚓࡓࠋࡇࢀࢆ80㸣࣓ࢱࣀ࣮ࣝ3.6 l
ᠱ⃮ࡋࠊ࣊࢟ࢧࣥ1.2 l࡛3ᅇ࡛ศ㓄ࡋࠊࡑࢀࡒࢀࡢ⏬ศࢆ⃰⦰ᅛࡋࡓࠋḟ80%࣓
30
ࢱࣀ࣮ࣝ⏬ศࢆỈ3.6 lᠱ⃮ࡋࠊ㓑㓟࢚ࢳࣝ1.2 l࡛3ᅇศ㓄ࡋࠊࡉࡽỈ⏬ศࢆỈ1.2 l
࡛3 ᅇศ㓄ࡋࠊࡑࢀࡒࢀࡢ⏬ศࢆ⃰⦰ᅛࡋࡓࠋᚓࡽࢀࡓྛ⏬ศࡢ㔜㔞ࡣࠊ࣊࢟ࢧࣥ⏬
ศ㸦0.8 g㸧ࠊ㓑㓟࢚ࢳࣝ⏬ศ㸦1.3 g㸧ࠊỈ⏬ศ㸦5.2 g㸧࡛࠶ࡗࡓࠋ
ูࢼࢫᯝᐇࡢࡀࡃ∦ศ௨እࡢ㒊ศ200 gࢆࠊ࢚ࢱࣀ࣮ࣝ2 l࡛ྠᵝᢳฟࡋࠊᢳฟ࢚
࢟ࢫ12.5 gࢆᚓࡓࠋ
ࡑࢀࡒࢀࡢࢼࢫࡀࡃ∦࢚ࢱࣀ࣮ࣝᢳฟ࢚࢟ࢫ࠾ࡼࡧࡑࡢྛ⏬ศࡣࠊ10 mg/ml࡞ࡿࡼ
࠺DMSO⁐ゎࡋࠊ–20Υ࡛ಖᏑࡋࡓࠋ
㓑㓟࢚ࢳࣝ⏬ศ1.3 gࢆࠊࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮㸦ࢩࣜ࢝ࢤࣝ60Nࠊ㛵 ᮾᏛࠊᮾிࠊ4 cm × 20 cm㸧౪ࡋࠊ࣓ࢱࣀ࣮ࣝ㸸ࢡ࣒ࣟࣟ࣍ࣝ㸸Ỉ㸦2 : 8 : 0.1㸧[13]
࡛⁐ฟࡉࡏࡓࠋ࣒࢝ࣛࡢ୰㢧ⴭⓗ࡞ᅄࡘࡢࣂࣥࢻࢆྲྀࡗ࡚ࠊFr. 1ࠥFr. 4ࢆᚓࡓ㸦ࡑࢀ
ࡒࢀ7.2 mgࠊ683 mgࠊ142 mgࠊ258 mg㸧ࠋFr. 2㸦683mg㸧ࢆODSࢩࣜ࢝ࢤࣝࢡ࣐ࣟࢺࢢ
ࣛࣇ࣮㸦ࢥࢫࣔࢩࣝ140C18-OPNࠊࢼ࢝ࣛࠊ4 cm × 20 cm㸧౪ࡋࠊ࣓ࢱࣀ࣮ࣝ㸭Ỉ 㸦1:1ࠊ4:1ࠊ1:0ࠊ300 mlࡎࡘẁ㝵ⓗ㸧࡛⁐ฟࡉࡏࡓࠋFr. 2-1ࠥ2-3㸦ࡑࢀࡒࢀ225.1 gࠊ48 mgࠊ 176 mg㸧ࢆᚓࡓࠋFr. 2-2㸦48 mg㸧ࢆศྲྀHPLC㸦ࢥࢫࣔࢩࣝC18-AR-ϩcolumn 10 × 150 mmࠊ ࢼ࢝ࣛ㸧౪ࡋࠊὶ㏿3 ml/minࠊỈ㸭ࢭࢺࢽࢺࣜࣝ㸦50:50ࠊ0 min㸹30:70ࠊ30 min㸹 0㸸100ࠊ32 min㸧࡛⁐ฟࡉࡏࠊ280 nmࡢUV྾ࡽࠊCompound 1㸦19 minࠊ0.13 mg㸧ࠊ
2㸦20 minࠊ0.25 mg㸧ࠊ3㸦24 minࠊ9.1 mg㸧ࠊ4㸦26minࠊ2.2 mg㸧ࢆᚓࡓࠋCompound 3
4ࡢᏛᵓ㐀ࡣࠊNMR㸦Bruker ABANCE 600ࠊMAࠊUSA㸧EI-MS㸦᪥ᮏ㟁Ꮚࠊᮾி)
ࢆ ᐃࡋࡓࠋ
Compound 34ࡢࢼࢫᢳฟᾮ୰ࡢྵ㔞ࡢ ᐃࡣࠊ௨ୗࡢ᮲௳ࡢHPLCࡼࡾ⾜ࡗࡓ㸸
࣒࢝ࣛࠊࢥࢫࣔࢩࣝ5C18-AR-ϩcolumn 4.6 × 150 mm㸦ࢼࢼ࢝ࣛ㸧㸹⛣ື┦ࠊỈ㸭ࢭࢺࢽ ࢺࣜࣝ㸦50:50ࠊ0 min㸹30:70ࠊ20 min㸹0㸸100ࠊ22 min㸧㸹ὶ㏿ࠊ1 ml/min㸹᳨ฟࠊ UV 280 nmࠊ⁐ฟ㛫ࠊcompound 3㸦19 min㸧ࠊcompound 4㸦20 min㸧ࠋ
࠙ྛ✀ࢼࢫࡀࡃ∦㒊ࠊ㢌㒊ࠊᯝᐇ࢚࢟ࢫࡢㄪ〇ࠚ
ᐇ㦂⏝ࡋࡓྛ✀ࡢࢼࢫᯝᐇࡣࠊ᪥ᮏᕷሙရ࡛㉎ධࡋࡓࠋ
ྛ✀᪂㩭࡞ࢼࢫᯝᐇࢆࡀࡃ∦㒊ࠊ㢌㒊ཬࡧᯝᐇࡑࢀࡒࢀศࡅ࡚ࠊ୍ᬌຍ⇕⇱ࡋࠊࡑ
ࢀࡒࢀ1 gࢆᐇ㦂ᮦᩱࡋ࡚ࠊ㓑㓟࢚ࢳࣝ10 mlࢆຍ࠼࡚minࡢࢯࢽࢣ࣮ࢩࣙࣥࡼ
ࡿᢳฟࢆ3ᅇ⾜࠸ࠊᢳฟᾮࢆྜࢃࡏ࡚⃰⦰ᅛࡍࡿࡇࡼࡾࠊྛᢳฟ࢚࢟ࢫࢆᚓࡓࠋ
࡚ࡢᢳฟ࢚࢟ࢫࢆ࣓ࢱࣀ࣮࡛ࣝ10 mg/mlㄪ〇ࡋࡓࠋࡑࡢᚋࠊྛ⃰ᗘ㸦6.25ࠊ12.5ࠊ
25ࠊ50 μg/ml㸧ࡢ9-EE-KODEࢆ⏝࠸᳨࡚㔞⥺ࢆసᡂࡋࡓࠋࡑࢀࡒࢀࡢࢼࢫࡢྛ㒊ࡢᢳ
ฟ࢚࢟ࢫࢆHPLC࡛9-EE-KODE ࡢྵ᭷㔞ࢆ ᐃࡋࡓࠋ
➨❶
࠙9-EE-KODEࡢྜᡂࠚ
31
ྜᡂ࠾ࡼࡧ☜ㄆ᪉ἲࡘ࠸࡚ࡣࠊᮏᩥグ㍕ࡋࡓࠋ
࠙MTTἲࡼࡿ9-EE-KODEࡢ⣽⬊⮴Ṛస⏝ࡢ ᐃࠚ
9-EE-KODEῧຍᚋࠊ24 hᚋMTT⁐ᾮࢆῧຍࡋࡓࢆ㝖ࡁࠊ➨୍❶‽ࡌ࡚⾜ࡗࡓࠋ
࠙BrdUἲࡼࡿ9-EE-KODEࡢ⣽⬊⮴Ṛస⏝ࡢ ᐃࠚ
5-Bromo-2'-deoxyuridine Labeling & Detection Kit Ϫ㸦Roche Applied,ScienceࠊMannheimࠊ Germany㸧ࢆ⏝ࡋࡓࠋMTTἲྠᵝ᧯సࡋࠊMTT⁐ᾮࡢ௦ࢃࡾ10 μlࡢBrdUᶆ㆑⁐
ᾮࢆῧຍࡋࠊ37Υ࡛ 2 h ࣮ࣥ࢟ࣗ࣋ࢺࡋ࡚ࠊ᪂ࡓྜᡂࡉࢀࡿDNAྲྀࡾ㎸ࡲࡏࡓࠋ ᇵᆅࢆ྾ᘬ㝖ཤᚋࠊ⣽⬊200 μl/well ࡢ FixDenat ࢆῧຍࡋ࡚30 minࠊ25°C࡛ฎ⌮ࡍࡿ
ࡇࡼࡾᅛᐃࡋࡓࠋࡑࡢᚋࠊ100 μl/well ࡢPODᶆ㆑ᢠBrdU ᢠయᛂᾮࢆῧຍࡋ࡚ࠊ
15°C ࠊ90 minฎ⌮ࡋࡓࠋὙίᾮ࡛Ὑίᚋࠊ࣌ࣝ࢜࢟ࢩࢲ࣮ࢮࡢᇶ㉁ࢆຍ࠼ࠊ25°C࡛3 min
࣮ࣥ࢟ࣗ࣋ࢺᚋࠊ405/490nmࡢ྾ගᗘࢆ࣐ࢡࣟࣉ࣮ࣞࢺ࣮ࣜࢲ࣮ࢆ⏝࠸࡚ ᐃࡋࡓ
࣮࠙࢞ࣟࢫࢤࣝ㟁ẼὋືࡼࡿ࣏ࢺ࣮ࢩࢫࡢ᳨ฟࠚ
ᇵ㣴ࣇࣛࢫࢥ㸦50 mlࠊ᪥ᮏࢪ࢙ࢿࢸࢡࢫࠊ᪥ᮏ㸧2 × 106 ಶࡢHRA⣽⬊ࢆ✀ࡋࠊ 24 h ᇵ㣴ᚋࠊᇵᆅࢆDMSO㸦Control㸧ࡲࡓࡣ9-EE-KODEࢆ1ࠊ5ࠊ10ࠊ100 μg/ml ྵࡴᇵ ᆅࡋࠊࡉࡽ24 hᇵ㣴ࡋࡓࠋ⣽⬊ࢆᅇࡋࠊ⁐ゎࣂࢵࣇ࣮㸦100 mM Tris-HClࠊ pH 8.5㸹5 mM EDTAࠊ0.2 M NaClࠊ0.2% SDSࠊ0.2 mg/ml proteinase K㸧ࢆ330 μlຍ࠼࡚37Υ
୍࡛ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡍࡿࡇࡼࡾࠊ⣽⬊ࢆ⁐ゎࡋࡓࠋ㐲ᚰศ㞳㸦1.5 x 104 rpmࠊ10 min㸧 ᚋࠊỿẊ5 M NaCl 141 μlࢆຍ࠼ࠊ㍍ࡃᨩᢾࡋࡓᚋࠊࡉࡽ㐲ᚰศ㞳ࡋࡓ㸦1.5 x 104 rpmࠊ 20 min㸧ࠋୖΎࢆ᪂ࡓ࡞ࢳ࣮ࣗࣈ⛣ࡋ࡚ࠊ࢚ࢱࣀ࣮ࣝ470 μlࢆຍ࠼ࠊ㍍ࡃᨩᢾࡋࡓᚋࠊ 㐲ᚰศ㞳ࡋࡓ㸦1.5 x 104 rpmࠊ20 min㸧ࠋୖΎࢆ྾ᘬ㝖ཤࡋࠊỿẊ70%࢚ࢱࣀ࣮ࣝ 400 μl
ࢆຍ࠼ࡓᚋࠊ㍍ࡃᨩᢾࡋ࡚ࠊ㐲ᚰศ㞳ࡋࡓ㸦1.5 x 104 rpmࠊ20 min㸧ࠋୖΎࢆ྾ᘬ㝖ཤࡋࠊ ỿẊỈࢆ15 μlῧຍࡋ࡚⁐ゎࡋࠊRNase A (10 mg/mL in Tris-EDTA buffer) ࢆ1 μlῧຍࡋࠊ 37ΥỈᾎ୍࡛ᬌ࡛࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋࡑࡢᚋࠊ㐲ᚰ㸦3 x 103 rpmࠊ20 min㸧ࡉࡏ࡚ࠊୖ
Ύࢆ㝖ཤࡋࠊỿẊࢆTris-EDTA buffer 20 μl࡛⁐ゎࡋࠊ2%࣮࢞ࣟࢫࢤࣝࢆ⏝࠸࡚㟁ẼὋ
ືࡋࡓࠋศ㞳ࡋࡓDNA ࡣࠊ࢚ࢳࢪ࣒࢘ࣈ࣑ࣟࢻ࡛ࢤࣝࢆᰁⰍࡋࡓᚋࠊUV ࢺࣛࣥࢫࣝ
࣑ࢿ࣮ࢱ࣮ࡼࡾྍどࡋࡓࠋ
࠙ࢾࢡࣞ࢜ࢯ࣮࣒ࡼࡿDNA᩿∦ࡢ᳨ฟࠚ
Cell death detection ELISAPLUS㸦Roche Applied ScienceࠊPenzbergࠊUpper BavariaࠊGermany㸧
ࢆ⏝࠸࡚ ᐃࡋࡓࠋ96✰ࣉ࣮ࣞࢺHRA⣽⬊ࢆ1.5 × 104 cells/well✀ࡋࠊ24 hᇵ㣴ᚋࠊ
9-EE-KODEࢆῧຍࡋ࡚ࡉࡽ24 hࢆᇵ㣴ࡋࡓࠋࡑࡢᚋࠊ࢟ࢵࢺࡢㄝ᫂᭩ᚑࡗ࡚ ᐃࡋ
ࡓࠋ⤖ᯝࡣࠊᮍฎ⌮㸦ᑐ↷㸧⣽⬊9-EE-KODE࡛ฎ⌮ࡋࡓ⣽⬊ࡽࡢ྾ගᗘࡢẚࡽࠊ ௨ୗࡢᘧࡼࡾィ⟬ࡋࡓenrichment factorࡋ࡚⾲ࡋࡓࠋ
32
࠙ࢿ࢟ࢩࣥV ᰁⰍࠚ
ᇵ㣴ࣇࣛࢫࢥ㸦50 ml㸧HRA⣽⬊ࢆ1.5 × 104 cells/wellࡢHRA⣽⬊ࢆ✀ࡋࠊ6 hᇵ 㣴ࡋࡓᚋࠊྛ⃰ᗘࡢ9-EE-KODEࢆῧຍࡋࡓࠋ24 hᇵ㣴ᚋࠊPBS࡛Ὑίࡋࠊ⣽⬊ࢿ
࢟ࢩࣥV-ࣇࣝ࢜ࣞࢭࣜࣥ㸦Medical & Biological Laboratoriesࠊྡྂᒇ㸧ࢆ10 μlῧຍࡋࡓࠋ ᬯᡤᐊ ࡛15 min࣮ࣥ࢟ࣗ࣋ࢺࡋࡓᚋࠊPBS ࡛Ὑίࡋࠊ⺯ග㢧ᚤ㙾࡛ほᐹࡋࡓࠋ
࠙Caspase-3/-7ࡢ ᐃࠚ
96✰ࣉ࣮ࣞࢺHRA⣽⬊ࢆ2 × 104 cells/well✀ࡋࠊ24 hᇵ㣴ᚋࠊ9-EE-KODEࢆῧ ຍࡋ࡚24 hᇵ㣴ࡋࡓࠋᇵᆅࢆ྾ᘬ㝖ཤᚋࠊCaspase-3/7 reagent㸦PromegaࠊMadisonࠊWIࠊ USA㸧ࢆ100 μlῧຍࡋ࡚ᐊ ࡛1 hᇵ㣴ࡋࡓᚋࠊບ㉳Ἴ㛗498 nmࠊ྾Ἴ㛗521 nmࡢ⺯
ගࢆ࣑ࣝࣀ࣓࣮ࢱ࣮㸦Perkin ElmerࠊWinter Street WalthamࠊMAࠊUSA㸧࡛ ᐃࡋࡓࠋ
࣑࠙ࢺࢥࣥࢻࣜ⭷㟁ࡢ ᐃࠚ
96✰㯮Ⰽࣉ࣮ࣞࢺHRA⣽⬊ࢆ1.5 × 104 cells/well✀ࡋࠊ24 hᇵ㣴ᚋࠊ9-EE-KODE ࢆ ῧຍࡋ࡚12 hᇵ㣴ࡋࡓࠋࡑࡢᚋࠊCell MeterTM JC-10 Mitochondrial Membrane Potential࢟ࢵ
ࢺ㸦BiomolࠊHamburgࠊGermany㸧ࢆ⏝ࡋ࡚ࠊບ㉳Ἴ㛗490 nmࠊ྾Ἴ㛗535(⥳)/590(㉥) ࡢ⺯ගࢆ ᐃࡋࡓࠋ
࣑࠙ࢺࢥࣥࢻࣜࡢBcl-2 ࢧࣈࣇ࣑࣮ࣜࢱࣥࣃࢡ㉁ࡢ᳨ฟࠚ
6✰ࣉ࣮ࣞࢺHRA⣽⬊ࢆ1.5 × 104 cells/well✀ࡋࠊ9-EE-KODEࢆῧຍࡋ࡚ࠊ24 hᇵ㣴 ࡋࡓࠋ⣽⬊ࢆࢺࣜࣉࢩࣥฎ⌮ࡼࡾ᥇ྲྀࡋࠊ㐲ᚰศ㞳㸦2 x 103 rpmࠊ20 min㸧ᚋࠊỿẊ
lysis buffer㸦50 M Tris-HCl, pH8.0ࠊ0.15 M NaClࠊ0.5% sodium deoxycholateࠊ0.1% SDS, 1.0 %
NP-40 substitute㸧ࢆ100 μlῧຍࡋࠊ㉸㡢Ἴ࡛⣽⬊ࢆ◚○ࡋࡓࠋࢱࣥࣃࢡ㉁⃰ᗘࡣࠊBCA ࣉ
ࣟࢸࣥࢵࢭ࢟ࢵࢺ㸦Thermo ScientificࠊRockfordࠊILࠊUSA㸧࡛ ᐃࡋࡓࠋ25 μgࡢ
ྛࢧࣥࣉࣝࢆ12%ࢡ࣑ࣜࣝࢻࢤࣝࢆ⏝ࡋࡓSDS-PAGE౪ࡋࡓࠋศᏊ㔞࣐࣮࣮࢝
ࡋ࡚ࠊPrecision Plus ProteinTM Standards Dual Color㸦Bio-Rad㸧 ࢆ⏝࠸ࡓࠋ㟁ẼὋືᚋࡢࢤ
ࣝࡣࠊࢭ࣑ࢻࣛᘧࣈࣟࢵࢸࣥࢢ⨨㸦ATTO AE-6687 HorizBLOT 2MࠊATTOࠊᮾி㸧
ࢆ⏝࠸࡚ࠊImmobilonTM-P Transfer Membrane㸦MilliporeࠊBillericaࠊMAࠊUSA㸧㌿ࡋ ࡓࠋ⭷ࢆὙίᾮ㸦20 mM Tris-HClࠊpH 7.5ࠊ170 mM NaClࠊ0.05% Tween20㸧࡚Ὑίࡋࠊ
10%ࣈࣟࢵࢡ࢚࣮ࢫ㸦DS ࣇ࣮࣐ࣂ࣓࢜ࢹ࢝ࣝࠊ㜰㸧୰࡛ᐊ ࡛2 hࣥ࢟ࣗ࣋
࣮ࢺࡋࡓࠋḟ10%ࣈࣟࢵࢡ࢚࣮ࢫ࡛ᕼ㔘ࡋࡓ௨ୗࡢ୍ḟᢠయᾮ࡛࣓ࣥࣈࣞࣥࢆ4°C୍࡛
33
ᬌᛂࡉࡏࠊὙίᾮ࡛3ᅇὙίᚋࠊ10%ࣈࣟࢵࢡ࢚࣮ࢫ࡛ᕼ㔘ࡋࡓ࣌ࣝ࢜࢟ࢩࢲ࣮ࢮᶆ㆑
ᢠ࣐࢘ࢫIgG㸦1:1 x 104ࠊJackson Immuno ResearchࠊRestonࠊVAࠊUSA㸧ࢆ⏝࠸࡚ᐊ ࡛1 hᛂࡉࡏࡓࠋὙίᾮ࡛3ᅇὙίࡋࠊLuminateTM Forte Western HRP Substrate (Merck KGaA, Darmstadt, Germany㸧ᾮ5 minᾐࡋࠊᏛⓎග᳨ฟჾ㸦LAS3000ࠊᐩኈࣇ࣒ࣝࠊᮾி㸧
ࢆ⏝ࡋ࡚Ⓨගࢆ᳨ฟࡋࡓࠋⓎග⏬ീࡢゎᯒࡣIMAGE J1.46㸦ᐩኈࣇ࣒ࣝ㸧ࢆ⏝࠸ࡓࠋ
ᙳᚋࡢ࣓ࣥࣈࣞࣥࡣࠊWB-stripping solution㸦ࢼࢼ࢝ࣛ㸧࡛37°Cࠊ9ศ㛫Ὑίࡋࠊࡧࣈ
ࣟࢵ࢟ࣥࢢࢆ⾜࠺ࡇู࡛ࡢࢱࣥࣃࢡ㉁ࡢ᳨ฟ⏝࠸ࡓࠋ
࣑࠙ࢺࢥࣥࢻࣜෆ⣽⬊㉁ෆࡢCytochrome cࡢ ᐃࠚ
6✰ࣉ࣮ࣞࢺHRA⣽⬊ࢆ1.5 × 104 cells/well✀ࡋ࡚24 hᇵ㣴ࡋࠊ9-EE-KODE࡛ 24 hᇵ㣴ࡋࡓࠋ⣽⬊ࢆࢺࣜࣉࢩࣥฎ⌮ࡼࡾ᥇ྲྀࡋࠊMitochondria/Cytosol Fractionation Kit 㸦AbcamࠊCambridgeࠊUK㸧࡛࣑ࢺࢥࣥࢻࣜ⣽⬊㉁ࢆศ㞳ࡋࠊ๓⠇ྠᵝࡢ᪉ἲ࡛
᳨ฟࡋࡓࠋ
࠙⏝ࡋࡓᢠయࠚ
ศᏊ✀ ࣓࣮࣮࢝ ᕼ㔘ẚ⋡ kDa
Bcl-2 Cell Signaling㸦DanversࠊMAࠊUSA㸧 1:1000 26
Bcl-xL Cell Signaling 1:1000 30
Bax Cell Signaling 1:1000 20
Cytochrome c Abcam 1:500 12
β-actin Santa Cruz Biotechnology㸦Santa CruzࠊCAࠊUSA㸧 1:500 43
34
ཧ
ཧ⪃ᩥ⊩
1. Yagasaki K., Miura Y.: Food components with potentialities to suppress proliferation and invasion of cancer cells. Anim. Cell Technol. Basic Appl. Asp. 10: 107–111 (1999).
2. DiMasi J., Hansen R., Grabowski H.: The price of innovation: new estimates of drug development costs. J. Health Eco. 22: 151–185 (2003).
3. Bonnesen C., Eggleston I., Hayes J.䠖Dietary indoles and isothiocyanates that are generated from cruciferous vegetables can both stimulate apoptosis and confer protection against DNA damage in human colon cell lines. Cancer Res. 61: 6120–6130 (2001).
4. Yanyan L., Tao Z., Hasan K., et al.:Sulforaphane, a dietary component of broccoli/broccoli sprouts, inhibits breast cancer stem cells.Clinical Cancer Res. 16: 2580–2590 (2010).
5. ࠕ࡞ࡍࡢ᱂ᇵ⫱࡚᪉ࠖ http://www.ktf-ririka-2.net/11-nasu.html.html (2015/01/19ࢡࢭ ࢫ)
6. Kiminori M., Takao K., Tsuyoshi M., Masaharu M.: Antiangiogenic activity of nasunin, an antioxidant anthocyanin, in eggplant peels. J. Agric. Food Chem. 53: 6272–6275 (2005).
7. Tomoda, Y., Tomoda, K., Horio, H., Mano, N., Mano, S., Morikawa, Y., Ito, T., Sugiyama, M.: Treatment results of the eggplant extract against condyloma acuminatum. 18th Annual Meeting of Japanese Society of Sexually Transmitted Infections, Kokura, Japan (2007).
8. Dhiman TR, Nam SH, Ure AL.: Factors affecting conjugated linoleic acid content in milk and meat. Crit. Rev. Food Sci. Nutr. 45: 463–482 (2005).
9. Kim, Y., Hirai S., Takahashi, H., Goto T., Ohyane C., Tsugane T., Konishi C., Fujii T., Imai S., Iijima Y., Aoki K., Shibata D., Takahashi N., Kawada T.: 9-Oxo-10(E),12(E)-
octadecadienoic acid derived from tomato is a potent PPARα agonist to decrease
triglyceride accumulation in mouse primary hepatocytes. Mol. Nutr. Food Res. 55: 585–593 (2011).
10. Kim Y., Hirai S., Goto T., Ohyane C., Takahashi H., Tsugane T., Konishi C., Fujii T., Inai S., Iijima Y., Aoki K., Shibata D., Takahashi N., Kawada T.: Potent PPARα activator derived from tomato juice, 13-oxo-9,11-octadecadienoic acid, decreases plasma and hepatic triglyceride in obese diabetic mice. PLoS One. 7: e31317 (2012).
11. Degterev A., Yuan J.: Expansion and evolution of cell death programmes. Nat. Rev. Mol.
Cell Biol. 9. 378–390 (2008).
12. Ou L., Ip C., Lisafeld B., Ip M.: Conjugated linoleic acid induces apoptosis of murine mammary tumor cells via Bcl-2 loss. Biochem. Biophys. Res. Commun. 356: 1044–1049 (2007).
13. Harding K., May L., Dick K.: Selective oxidation of allylic alcohols with chromic acid. J.
35 Org. Chem. 40: 1664–1665 (1975).
14. Tsuboi S., Maeda S., Takeda A.: A convenient synthesis of ±-dimorphecolic acid and its analogs. Bull. Chem. Soc. Jpn. 59: 2050–2052 (1986).
15. Cory A., Owen T., Barltrop J., Cory J.: Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun. 3: 207–212 (1991).
16. Koopman G., Reutelingsperger CP., Kuijten G.A., Keehnen R.M., Pals S.T., van Oers M.H.:
Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood. 84: 1415–1520 (1994).
17. Mazumder S., Plesca D., Almasan A.: Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis. Methods Mol Biol. 414: 13–21 (2008).
18. Reers M., Smiley S.T., Mottola-Hartshorn C., Chen A., Lin M., Chen L.B.: Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol. 260: 406–1417 (1995).
19. Tsujimoto Y.: Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria.
Genes Cells. 3: 697–707 (1998).
20. Bogner C., Leber B., Andrews D.W.: Apoptosis: embedded in membranes. Curr. Opin. Cell Biol. 22: 845-851 (2010).
21. Nakayachi T., Yasumoto E., Nakano K., Morshed S.R., Hashimoto K., Kikuchi H.,
Nishikawa H., Kawase M., Sakagami H.:Structure-activity relationships of α,β-unsaturated ketones as assessed by their cytotoxicity against oral tumor cells. Anticancer Rec.
24:737-742 (2004).
22. Dimmock JR., Das U., Gul H.I., Kawase M., Sakagami H., Baráth Z., Ocsovsky I., Molnár J..:
3-Arylidene-1-(4-nitrophenylmethylene)-3,4-dihydro-1H-naphthalen-2-ones and related compounds displaying selective toxicity and reversal of multidrug resistance in neoplastic cells. Bioorg Med Chem Lett. 15:1633-1636 (2005).