㻺㼍㼓㼛㼥㼍㻌㻯㼕㼠㼥㻌㼁㼚㼕㼢㼑㼞㼟㼕㼠㼥㻌㻭㼏㼍㼐㼑㼙㼕㼏㻌㻾㼑㼜㼛㼟㼕㼠㼛㼞㼥㻌
Ꮫ ࡢ ✀ 㢮 ༤ኈ ⸆Ꮫ ሗ ࿌ ␒ ྕ ⏥➨㸯㸲㸶㸵ྕ Ꮫ グ ␒ ྕ ➨ ྕ Ặ ྡ ㉿ 㝧 ᤵ ᖺ ᭶ ᪥ ᖹᡂ ᖺ ᭶ ᪥ Ꮫㄽᩥࡢ㢟ྡ ⱳࡢ⣽⬊⮴Ṛάᛶᡂศ㛵ࡍࡿ◊✲ ㄽᩥᑂᰝᢸᙜ⪅ ᰝ㸸 ୰ᕝ⚽ᙪ ᰝ㸸 ∾㔝᫂ᵽཱྀᜏᙪᒣᮧኖ⏨ྡྂᒇᕷ❧ᏛᏛㄽᩥ
ⱳࡢ⣽⬊⮴Ṛάᛶᡂศ㛵ࡍࡿ◊✲
ᖹᡂ26 ᖺᗘ㸦2015 ᖺ 3 ᭶㸧㉿ 㝧 ྡྂᒇᕷ❧ᏛᏛᏛㄽᩥ
ⱳࡢ⣽⬊⮴Ṛάᛶᡂศ㛵ࡍࡿ◊✲
㉿ 㝧 ྡྂᒇᕷ❧ᏛᏛ㝔⸆Ꮫ◊✲⛉ ⏕⸆Ꮫศ㔝 㸦ᣦᑟ㸸∾㔝 ᫂ ᩍᤵ㸧 Ph.D. DissertationCytotoxic constituents from the calyx of eggplants
Baiyang ZhaoDepartment of Pharmacognosy, Graduate School of Pharmaceutical Sciences,
Nagoya City University
1. ᮏㄽᩥࡣࠊ2015 ᖺ 1 ᭶ࠊྡྂᒇᕷ❧ᏛᏛ㝔⸆Ꮫ◊✲⛉࠾࠸࡚ᑂᰝࡉࢀࡓࡶࡢ ࡛࠶ࡿࠋ ᰝ㸸୰ᕝ ⚽ᙪ ᩍᤵ ᰝ㸸∾㔝 ᫂ ᩍᤵ ᰝ㸸ᵽཱྀ ᜏᙪ ᩍᤵ ᰝ㸸ᒣᮧ ኖ⏨ ᩍᤵ 2. ᮏㄽᩥࡣࠊᏛ⾡ሗ㞧ㄅ㍕ࡉࢀࡓḟࡢሗᩥࢆᇶ♏ࡍࡿࡶࡢ࡛࠶ࡿࠋ ᇶ♏࡞ࡿሗᩥ
1. Baiyang Zhao, Yohei Sakurai, Kiyosumi Shibata, Humitaka Kikkawa, Yutaka Tomoda, Hajime Mizukami
Cytotoxic keto octadecadienoic acids from eggplants Jpn. J. Food Chem, Safety, Vol, 21(1), 42-472 (2014)
2. Baiyang Zhao, Yutaka Tomoda, Hajime Mizukami, Toshiaki Makino
9-Oxo-10E, 12E-octadecadienoic acid, a cytotoxic fatty acid ketodiene isolated from eggplant calyx, induces apoptosis in human ovary cancer (HRA) cells
J. Nat. Med. (in press) doi:10.1007/s11418-015-0892-x
3㸬 ᮏㄽᩥࡢᇶ♏࡞ࡿ◊✲ࡣࠊỈୖ ඖ ྡᩍᤵࠊ∾㔝 ᫂ ᩍᤵࡢᣦᑟࡢୗࠊྡ ྂᒇᕷ❧ᏛᏛ㝔⸆Ꮫ◊✲⛉࠾࠸࡚⾜ࢃࢀࡓࠋ
┠ḟ ᗎㄽ 1 ᮏㄽ 2 ➨୍❶ ⱳࡽࡢ⣽⬊⮴Ṛάᛶᡂศࡢ༢㞳ྠᐃ 2 ➨❶ ⱳྵࡲࢀࡿ⣽㒊⮴Ṛάᛶᡂศࡢస⏝ᶵᗎ 14 ⪃ᐹ 25 ᐇ㦂᪉ἲ 27 ᘬ⏝ᩥ⊩ 32 ㅰ㎡ 34
1
ᗎ
ᗎㄽ
ࡀࢇࡣࠊ⫧‶ࠊ⢾ᒀࠊ㧗⬡⾑ࠊ㧗⾑ᅽྠᵝ⏕ά⩦័ࡢ୍ࡘ࡛࠶ࡾࠊ1981 ᖺ ௨᮶ࠊ᪥ᮏ࠾ࡅࡿṚᅉࡢ➨୍࡞ࡗ࡚࠸ࡿࠋࡇࢀᚰᝈࠊ⬻⾑⟶ᝈ࡞ື⬦◳ ᛶᝈࡀḟ࠸࡛࠸ࡿࠋࡀࢇ㝈ࡽࡎࠊẼ࡞ࡽ࡞࠸ࡼ࠺ண㜵㸦୍ḟண㜵㸧ࡍࡿࡢ ࡀ⌮࡛࠶ࡿࡀࠊࡦࡓࡧࡀࢇ⣽⬊ࡀ⏕ࡌࡓሙྜ࡛ࡶࡑࡢ≉ᛶࢆไᚚࡍࡿࡇࡼࡾࠊ ᝏᛶࢆ㜼Ṇࡋࡑࡢ㢧ᅾࢆᢚไ㸦ḟண㜵㸧࡛ࡁࡿࡶࡋࢀ࡞࠸ࠋࡶࡋࡑࡢࡼ࠺࡞ࡇ ࡀ㣗ရᡂศ࡛ྍ⬟࡛࠶ࡿ࡞ࡽࡤࠊデ᩿ࡉࢀ࡚ࡽฎ᪉ࡉࢀࡿ⸆ရẚ࡚ࠊ᪥ᖖⓗ ᦤྲྀࡍࡿ㣗ရࡣḟண㜵ࡢほⅬࡽ᭷࡛࠶ࡿ⪃࠼ࡽࢀࡿ[1]ࠋ ⌧ᅾࠊୡ⏺ࡢඛ㐍ᅜࡢ᪂⸆㛤Ⓨࡢᡭ㡰ࡣࠊᇶ♏◊✲ࠊ๓⮫ᗋヨ㦂ࠊ⮫ᗋヨ㦂ࠊᢎㄆ⏦ ㄳ࣭〇㐀㈍࡞ࡾࠊ5 ᖺࡽ 18 ᖺࡢ᭶᪥ࢆ㈝ࡸࡋࠊ⥲㈝⏝ࡣ 200 ൨ࡽ 300 ൨ ࡿ࠸ࢃࢀ࡚࠸ࡿ[2]ࠋࡑࡢ࡞࡛ࡶᇶ♏◊✲ࡣࠊ⸆┠ⓗࡼࡗ࡚ࢩ࣮ࢻ࠶ࡿ࠸ࡣࣜ ࣮ࢻྜ≀࡞ࡾ࠺ࡿྜ≀ࢆⓎぢ࣭ྜᡂࡋࠊ⣽⬊ᑐࡋ࡚ࡢẘᛶࡸ⸆ຠࡢヨ㦂ࢆᐇ ࡍࡿࡓࡵࠊປຊࡀࡿࡢࡣᐇ࡛࠶ࡿࠋࡀࢇᑐࡍࡿ⒪⸆ࡢ㛤Ⓨ࡛ࡣࠊṇᖖ⣽⬊ ࡣ↓࠸ࡀࢇ⣽⬊ࡢ≉ᛶ࡛࠶ࡿ↓㝈ቑṪᛶ㌿⛣ᛶࢆ㜼ᐖࡍࡿ⸆≀ࡀồࡵࡽࢀࠊࡇࢀ ࡽࢆ⣽⬊ᇵ㣴⣔࡛⌧ࡍࡿ⣔ࢆᵓ⠏ࡋࠊࡀࢇࡢ㢧ᅾࢆ㜵ࡂḟண㜵᭷⏝࡞ᡂศࡢ᥈ ⣴స⏝ᶵᵓࡢ⣽⬊ᕤᏛⓗゎᯒࡀࠊࡇࢀࡲ࡛⾜ࢃࢀ࡚ࡁ࡚࠸ࡿࠋ ࡑࡢࡼ࠺࡞୰ࠊᡃࠎࡢẖ᪥ࡢ㣗⏕άࡢ୰࡛ࠊᵝࠎ࡞㔝⳯ྵࡲࢀࡿᡂศࠊࡀࢇ⣽⬊ ࡢᡂ㛗࠾ࡼࡧቑṪࢆᢚไࡍࡿస⏝ࡀ࠶ࡿࡇࡀὀ┠ࡉࢀ࡚ࡁ࡚࠸ࡿࠋ࠼ࡤࠊࣈࣛࢼ ⛉ࡢ㔝⳯ྵࡲࢀࡿኳ↛ࡢࣥࢻ࣮ࣝ㢮ࡸࢯࢳ࢜ࢩࢿ࣮ࢺ㢮ࡣࠊࣄࢺ⭠ࡀࢇ⣽⬊ ᑐࡋ࡚࣏ࢺ࣮ࢩࢫ㸦ࣉࣟࢢ࣒ࣛ⣽⬊Ṛ㸧ࢆㄏᑟࡋࠊDNA ࡢኚ␗ᑐࡍࡿ㜵ᚚຊࢆ㧗 ࡵࡿຠᯝࡀ࠶ࡿ[3] ࠋ࣑ࢩ࢞ࣥᏛ⥲ྜࡀࢇࢭࣥࢱ࣮ࡢ◊✲⪅ࡽࡢ᪂ࡓ࡞◊✲ࡼࢀࡤࠊ ࣈࣟࢵࢥ࣮ࣜ⏤᮶ࡢྜ≀ࡣࠊࡀࢇᖿ⣽⬊ࢆᶆⓗࡍࡿࡇ࡛ࠊஙࡀࢇࡢண㜵ࡸ⒪ ᙺ❧ࡘྍ⬟ᛶࡀ࠶ࡿ>@ࠋ ᮏ◊✲࡛ࡣࠊྂࡃࡽ㣗ᮦࡋ࡚ୡ⏺ྛᅜ࡛⏝ࡉࢀ࡚࠸ࡿࢼࢫࢆྲྀࡾୖࡆࠊࢼࢫ ྵࡲࢀࡿ⭘⒆⣽⬊ᑐࡍࡿ⮴Ṛάᛶࢆᣢࡘ≀㉁ࡢ᥈⣴ࠊࡑࡢస⏝ᶵᗎࡢゎ᫂ࢆ┠ⓗ ࡋࡓ◊✲ࢆ⾜ࡗࡓࠋࢼࢫࡢཎ⏘ᆅࡣࣥࢻࡢᮾ㒊࡛ࠊࡑࡢᚋࣅ࣐ࣝࢆ⤒⏤ࡋ࡚୰ᅜΏ ࡾࠊ᪥ᮏࡣዉⰋ௦ዉ㡲ẚ㸦࡞ࡍࡧ㸧ࡋ࡚ఏࢃࡗࡓ⪃࠼ࡽࢀ࡚࠸ࡿࠋࢼࢫࡣ୰ ᅜ࡛ࡶ᪥ᮏ࡛ࡶ1000 ᖺ௨ୖΏࡾ᱂ᇵࡉࢀ࡚࠸ࡿࠋࡲࡓࠊࢼࢫࡣୡ⏺ࡢྛᆅ࡛⊂⮬ࡢရ ✀ࡀ⫱࡚ࡽࢀ࡚࠾ࡾࠊ᪥ᮏ࡛ࡣຍ㈡ⱳᏊ࡞ࡢ୍㒊ࡢእࡶ࠶ࡿࡶࡢࡢࠊ༡᪉㛗ᐇ ࡲࡓࡣ㛗ᐇ࡛ࠊ᪉ᑠᐇရ✀࡞ࡾࠊᮏᕞࡢ୰㛫ᆅ࡛ࡣ୰㛫ⓗ࡞୰㛗ရ✀ࡀ᱂ᇵ ࡉࢀ࡚ࡁࡓࠋ᪥ᮏ࡛᱂ᇵࡉࢀࡿ᱂ᇵရ✀ࡢࢇࡣᯝ⓶ࡀ⣸Ⰽཪࡣ㯮⣸Ⰽ࡛࠶ࡿࡀࠊ ࣮ࣚࣟࢵࣃࡸ࣓ࣜ࢝➼࡛ࡣⓑ࣭㯤⥳Ⰽ࣭᫂ࡿ࠸⣸ࠊࡉࡽ⦤ᶍᵝࡢရ✀ࡶᗈࡃ᱂ᇵࡉ ࢀ࡚࠸ࡿࠋࢼࢫࡢᯝ⫗ࡣᐦᗘࡀపࡃࢫ࣏ࣥࢪ≧࡛ࠊ࣊ࢱࡢ㒊ศࡣ㗦࠸ࢺࢤࡀ⏕࠼࡚࠸ ࡿࡀࠊ✭ࡢసᴗᛶྥୖࡸᐇയࡀࡘࡃ࠸࠺⌮⏤ࡽࢺࢤࡢ↓࠸ရ✀ࡶ㛤Ⓨࡉࢀ࡚࠸ࡿ [5]ࠋᮏ◊✲࡛ࡣࠊࢼࢫྵࡲࢀࡿ⣽⬊⮴Ṛάᛶᡂศࡢࢼࢫရ✀㛫ࡢẚ㍑ࡶ⾜ࡗࡓࠋ2
ᮏ
ᮏㄽ
➨୍❶ ⱳࡽࡢ⣽⬊⮴Ṛάᛶᡂศࡢ༢㞳ྠᐃ
᪥ᮏ࠾࠸࡚௦⾲ⓗ࡞ኟ㔝⳯ࡢࡦࡘ࡛࠶ࡿࢼࢫࡣࠊ918 ᖺⓎหࡉࢀࡓࠗᮏⲡྡ࠘ ㍕ࡉࢀ࡚࠸ࡿ⸆⏝᳜≀࡛ࡶ࠶ࡾࠊⰼࠊᯝᐇࡢࡓࠊᯝ⫗࡞ࡀ⸆⏝ࡋ࡚⏝ࡉࢀ ࡚࠸ࡿࠋࢼࢫࡣ᪥ᮏࡢẸ㛫⸆ࡋ࡚ࡣࠊᾘ⅖ࠊṆ⾑ࠊゎ⇕࡞ࡢ┠ⓗ࡛㣗࠶ࡓࡾࠊ࣎ 㸦㉕㸧ྲྀࡾࠊங⭢⅖ࠊ⭘ࢀ≀ࠊ࠶ࡂࢀ࡞ࢃࢀࡿࠋࡲࡓ⭘ࢀ≀ࠊ㟖↝ࡅࠊṑᵴ ⮋₃ࡢண㜵ࡢࡓࡵࡣࠊࢼࢫࡢ࣊ࢱࢆ᪥ᖸࡋࡋ࡚⇱ࡋࡓࡶࡢ㸦ⱳ㸧ࢆ↦ࡌࡓᾮࢆ ሬᕸࡍࡿࡇࡼࡾࡿࡉࢀࡿࠋࢼࢫࡢᯝᐇࡢ⓶ࡣࠊࣥࢺࢩࢽࣥࡢ୍✀࡛࠶ࡿ ࢼࢫࢽࣥࡸࠊ࣏ࣜࣇ࢙ࣀ࣮ࣝ⛠ࡉࢀࡿࢡࣟࣟࢤࣥ㓟ࡀྵࡲࢀࠊᢠ㓟స⏝ࡀ࠶ࡿ (Figure 1) [6]ࠋNasunin Chlorogenic acid Figure 1 Chemical structure of nasunin and chlorogenic acid
ࢼࢫ㸦≉ࡀࡃ∦㒊ࠊⱳ㸧ࡀ㉕ࡢ⒪ఏᢎⓗ⏝࠸ࡽࢀ࡚ࡁࡓࡇ╔┠ࡋࡓ ⏣㇏༤ኈ㸦ྡྂᒇᏛྡᩍᤵࠊ⏘⛉፬ே⛉Ꮫࠊ⏣ࢡࣜࢽࢵࢡ㝔㛗㸧ࡣࠊ㉕ྠ ᵝࣄࢺࣃࣆ࣮࣐ࣟ࢘ࣝࢫࢆཎᅉࡍࡿᑤᆂࢥࣥࢪ࣮࣒ࣟࡢ⒪ࠊࢼࢫࡀࡃ∦⏤᮶ ࡢᢳฟ≀ࢆ⏝࠸ࡓࡇࢁⰋዲ࡞⮫ᗋᡂ⦼ࢆᚓ࡚࠸ࡿ[7]ࠋࡋࡋ࡞ࡀࡽࠊࢼࢫࡢ᭷ຠᡂศ ᮍࡔ࡛᫂࠶ࡿࠋ ࡑࡇ࡛ࠊྛ✀ᵝ⣽⬊ᑐࡍࡿࢼࢫᢳฟ≀ࡢ⮴Ṛάᛶࢆホ౯ࡋࠊ᭷⏝ᡂศࡢ༢㞳ࠊྠᐃ ࢆ⾜ࡗࡓࠋࡲࡓࠊࡑࡢ⣽⬊⮴Ṛάᛶࡢᶵᗎࡘ࠸࡚ࡶ᳨ウࡋࡓࠋ
3
1.
ⱳࡽࡢ⣽⬊⮴Ṛάᛶᡂศࡢ༢㞳
1-1 ࢼࢫ࢚ࢱࣀ࣮ࣝᢳฟ࢚࢟ࢫࡢ⣽⬊⮴Ṛάᛶ࠾ࡅࡿྛ✀ࡀࢇ⣽⬊ࡢࢫࢡ࣮ࣜࢽࣥ ࢢ ⱳࢆຍ⇕⇱ᚋࠊ࢚ࢱࣀ࣮ࣝࢆຍ࠼࡚෭ᾐᢳฟࡋ࡚ᚓࡓ࢚࢟ࢫࡢࣄࢺཱྀ⭍㢮⾲⓶⒴ 㸦KB㸧ࠊࣄࢺ༸ᕢࡀࢇ⣽⬊㸦HRA㸧ࠊࣄࢺ⫾ඣ⏤᮶⭈⮚⣽⬊㸦HEK293㸧ࠊࣄࢺ⫢⣽⬊⒴⣽ ⬊ᰴ㸦HLE㸧ࠊࣄࢺᏊᐑ㢕⒴㸦HeLa㸧ᑐࡍࡿ⣽⬊⮴Ṛάᛶࢆ MTT ἲࡼࡾ ᐃࡋࡓࠋ ࡑࡢ⤖ᯝࠊⱳ࢚ࢱࣀ࣮࢚ࣝ࢟ࢫࡣࠊHEK293 ⣽⬊ HRA ⣽⬊ᑐࡋ࡚⃰ᗘ౫Ꮡⓗ⣽ ⬊⮴Ṛάᛶࢆ♧ࡋࡓ୍᪉࡛ࠊKB ⣽⬊ࠊHLE ⣽⬊ HeLa ⣽⬊ࡣ⣽⬊⮴Ṛάᛶࢆ♧ࡉ࡞ ࡗࡓࠋHRA ⣽⬊ᑐࡍࡿⱳ࢚ࢱࣀ࣮ࣝᢳฟ࢚࢟ࢫࡢ IC50ࡣࠊ0.5 μg/ml ࡛ࠊࡢ⣽⬊ ẚ㍑ࡋ࡚᭱ࡶᙉ࠸⣽⬊ቑṪᢚไάᛶࢆ♧ࡋࡓ㸦Figure 2㸧ࠋFigure 2 Cytotoxicity of the ethanol extracts of eggplant calyx against four different cell lines. Closed circles, HRA (human ovarian cancer); open circles, HEK293 (human embryonickidney); closed triangles, HLE, (human hepatoma); open triangles, HeLa (human cervical cancer), KB (open square). Data are expressed as mean ± S.E. (n = 6), and **P < 0.01, ***P < 0.001 vs control (0 mg/ml) group evaluated by Bonferroni / Dunnett’s multiple t-test.
༸ᕢࡣᏊᐑࡢ୧ഃ࠶ࡿぶᣦࡢ⮚ჾ࡛ࠊ⏕Ṫ⣽⬊࡛࠶ࡿ༸Ꮚࡀࡑࡇ࡛ᡂ⇍ࡋᨺฟࡉ ࢀࡿྠ࿘ᮇⓗዪᛶ࣍ࣝࣔࣥࢆศἪࡍࡿാࡁࢆࡋ࡚࠸ࡿࠋHRA ࡣࣄࢺ༸ᕢࡢᄞ⬊ ⭢ࡀࢇࡼࡿ⭡Ỉࡽᚓࡽࢀࡓ⣽⬊࡛ࠊࣄࢺ༸ᕢ⭘⒆⤌⧊ࣔࢹࣝࡋ࡚☜❧ࡉࢀࡓࠊṔ ྐⓗࡣẚ㍑ⓗ᪂ࡋ࠸⣽⬊✀࡛࠶ࡿࠋୖ⓶ᛶࡢ⭘⒆࡛࠶ࡿࡇࡽࠊ㉕ᑐࡍࡿస⏝ ࢆ᳨ウࡍࡿୖ࡛ࡶ㐺ษ࡞ࣔࢹࣝ࡞ࡾ࠺ࡿ⪃࠼ࠊ௨ୗࡢᐇ㦂ࡣHRA ࢆ⏝࠸࡚ࠊᐇ㦂ࢆ ⾜࠺ࡇࡋࡓࠋ 0 20 40 60 80 100 0 0.2 0.4 0.6 0.8 1 Cell viability (%) Concentration (mg/ml) *** *** ** *** ** ** ** ** ** *** ***
4 1-2 ࢼࢫᯝᐇྛ㒊ࡢ࢚ࢱࣀ࣮ࣝᢳฟ࢚࢟ࢫࡢࡀࢇ⣽⬊ᑐࡍࡿ⣽⬊⮴Ṛάᛶ ࢼࢫᯝᐇࡢࡀࡃ∦㒊ศࡑࢀ௨እࡢ㒊ศࡢάᛶࢆẚ㍑ࡍࡿࡓࡵࠊFigure 3ࡢࡼ࠺ࠊ ࢼࢫᯝᐇࡢࡀࡃ∦㒊ྍ㣗㒊ࢆศ㞳ࡋ࡚ࠊࡑࢀ ࡒࢀ⇱ᚋࠊ࢚ࢱࣀ࣮ࣝᢳฟ࢚࢟ࢫࢆᚓࡓࠋࡑ ࢀࡒࢀࡢ࢚࢟ࢫࡢHRA⣽⬊ᑐࡍࡿ⣽⬊⮴Ṛ άᛶࢆMTTἲࡼࡾ᳨ウࡋࡓࡇࢁࠊࡀࡃ∦ 㒊ศࡢ᪉άᛶࡀᙉࡃぢࡽࢀࡓ㸦Figure 4㸧ࠋࢼ ࢫᯝᐇྵࡲࢀࡿHRAᑐࡍࡿ⣽⬊⮴Ṛάᛶ ᡂศࡣࠊྍ㣗㒊ࡼࡾࡶࡀࡃ∦㒊ศከࡃྵࡲࢀ ࡿ⪃࠼ࡽࢀࡓࠋ ௨ୖࡢࡇࡽࠊᚋࡢᐇ㦂࡛ࡣࢼࢫࡢࡀࡃ ∦࡛࠶ࡿⱳࢆ◊✲ᮦᩱࡋ࡚⏝ࡍࡿࡇ ࡋࡓࠋ
Figure 3 Eggplants used in the present investigation. Eggplants (Senryo-nasu, A) were cut into the calyx parts (B) and the edible parts(C).
Figure 4 Cytotoxicity of the ethanol extracts prepared from the calyx parts (closed circles) and from the edible parts (open circles) of eggplants. Data are expressed as mean ± S.E. (n = 6) , and **P < 0.01, ***P < 0.001 vs control (0 mg/ml) group evaluated by Bonferroni / Dunnett’s multiple t-test.
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2. ࣄ
ࣄࢺ༸ᕢࡀࢇ⣽⬊㸦HRA㸧ᑐࡍࡿ⣽⬊⮴Ṛάᛶᡂศࡢⱳࡽࡢ༢㞳
2.1 HRAᑐࡍࡿ⣽⬊⮴Ṛάᛶࢆᣦᶆࡋࡓⱳ࢚ࢱࣀ࣮࢚ࣝ࢟ࢫࡢศ⏬ ࢼࢫᯝᐇࡣHRAᑐࡋ࡚⣽⬊⮴Ṛάᛶࡀ࠶ࡾࠊࡑࡢάᛶᡂศࡣࡀࡃ∦㒊ศࡼࡾከ ࡃྵࡲࢀ࡚࠸ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡑࡇ࡛ࠊࡑࡢάᛶࢆᣦᶆࡋ࡚ࠊⱳࡽࡢά ᛶᡂศࡢ༢㞳ࢆヨࡳࡓࠋ ᪂㩭࡞ࢼࢫ 10 kg ࡽࡀࡃ∦㒊 2.8 kg ࢆᚓ࡚ࠊຍ⇕⇱ࡋࠊ⇱≀2.6 x 102 g ࢆᚓࡓࠋ ࡇࢀࢆ 80%࢚ࢱࣀ࣮࡛ࣝ෭ᾐᢳฟࢆ3ᅇ⾜࠸ࠊࢁᾮࢆྜࢃࡏࠊ⃰⦰ᅛࡋࡓ㸦7.6 g㸧ࠋࡇ ࢀࢆ80%࢚ࢱࣀ࣮ࣝ⁐ゎࡋࠊn-࢟ࢧ࡛ࣥศ㓄ࡋࠊn-࣊࢟ࢧࣥ⏬ศ㸦0.8 g㸧ࢆᚓࡓࠋḟ 80%࢚ࢱࣀ࣮ࣝ⏬ศࢆ⃰⦰ᅛࡋࠊỈᠱ⃮ᚋࠊ㓑㓟࢚ࢳ࡛ࣝศ㓄ࡋࠊ㓑㓟࢚ࢳࣝ⏬ ศ㸦1.3 g㸧Ỉ⏬ศ㸦5.2 g㸧ࢆᚓࡓࠋࡑࡋ࡚ࠊHRA⣽⬊ᑐࡍࡿ⮴Ṛάᛶࢆẚ㍑ࡋࡓ ࡇࢁࠊάᛶࡣn-࢟ࢧࣥ⏬ศࠊỈ⏬ศࡣㄆࡵࡽࢀࡎࠊ㓑㓟࢚ࢳࣝ⏬ศࡰ㞟୰ࡋࠊ ࡑࡢ IC50 ࡣ 41 μg/m1࡛࠶ࡗࡓ㸦Figure 5㸧ࠋFigure 5 Activity-guided fractionation of eggplant calyx. IC50, concentration of the fraction required for 50% inhibition of
proliferation of HRA cells.
ࡑࡇ࡛ࠊ㓑㓟࢚ࢳࣝ⏬ศࡘ࠸࡚ࠊࢡ࣒ࣟࣟ࣍ࣝ㸭࣓ࢱࣀ࣮ࣝ㸭Ỉ㸦80㸸20㸸1㸧ࢆ⛣ ື┦ࡋࡓࢩࣜ࢝ࢤࣝࢡ࣐ࣟࢺࢢࣛࣇ࣮ࢆ⏝࠸࡚ศ㞳ࡋࠊFr. 1ࠊFr. 2ࠊFr. 3ࠊFr. 4ࢆᚓ ࡓࠋ⣽⬊⮴Ṛάᛶࢆẚ㍑ࡋࡓࡇࢁࠊFr. 2㸦0.68 g㸧ᙉ࠸άᛶࡀぢࡽࢀࡓ㸦IC50, 30 μg/ml㸧ࠋ
Fr. 2ࢆࡉࡽ࣓ࢱࣀ࣮ࣝ㸭Ỉࢆ⛣ື┦ࡋࡓODS㏫ᒙࢡ࣐ࣟࢺࢢࣛࣇ࣮ࡼࡗ࡚ศ㞳 ࢆ⾜ࡗࡓࡇࢁࠊFr. 2-1ࠊFr. 2-2Fr. 2-3ࢆᚓࡓ㸦Figure 6㸧ࠋࡑࢀࡽࡢHRAᑐࡍࡿ⣽⬊
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⮴Ṛάᛶࢆẚ㍑ࡋࡓࡇࢁࠊFr. 2-2ẚ㍑ⓗᙉ࠸άᛶࡀㄆࡵࡽࢀࡓ㸦IC50, 8.0 μg/ml㸧ࠋࡑ
ࡇ࡛ Fr. 2-2 ࢆࡉࡽศྲྀHPLC࡛ศ⏬ࡋࠊcompound 1 (< 0.5 mg)ࠊcompound 2 (< 0.5 mg)ࠊ compound 3 (9.1 mg)ࠊcompound 4 (2.2 mg)ࢆᚓࡓ㸦Figure 6㸧ࠋCompound 12ࡣࠊ㔞ࡀ ᑡ࡞ࡗࡓࡓࡵࠊࡇࢀ௨ୖࡢゎᯒࡣฟ᮶࡞ࡗࡓࠋCompound 34ࡢHRA⣽⬊ᑐࡍࡿ ⮴Ṛάᛶ㸦IC50㸧ࡣࠊࡑࢀࡒࢀ 9.7 μg/ml1.9 μg/ml ࡛࠶ࡗࡓࠋ
Figure 6 Activity-guided fractionation of the ethyl acetate fraction of eggplant calyx extract. (A) Cascade of the fractionation. IC50, concentration of the fraction required for 50% inhibition of proliferation of HRA cells. (B) RP-HPLC chromatograms of
Fr. 2-2. Column :C18-AR-ϩ(10150 mm), solvent:A (H2O) - B (CH3CN), 0 – 100 (0 - 30 min), flow speed:3 ml/min; wave
length: 280 nm.
A
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3. ༢
༢㞳ࡋࡓᡂศࡢᵓ㐀ゎᯒ
3.1 Compound 3 ࡢᵓ㐀ゎᯒ Compound 3 ࡢᵓ㐀ࢆ 1H-࠾ࡼࡧ13C-NMR EI-MS ࡛ゎᯒࡋࡓࠋNMR ࡢศᯒ⤖ᯝࢆ Table 1 ♧ࡍࠋ Compound 3 ࡢ13C-NMR ศᯒ࡛ࡣࠊ࣓ࢳࣝⅣ⣲ 1 ᮏࠊ࣓ࢳࣞࣥⅣ⣲⣙ 10 ᮏࠊ࣓ࢳࣥⅣ ⣲4 ᮏࡢࢩࢢࢼࣝࡀほ ࡉࢀࡓࠋ1H-NMR ศᯒ࡛ࡣࠊ࢜ࣞࣇࣥࢆ♧ࡍࢩࢢࢼࣝࡀ⤌ 㸦δH 6.16 ppm, 1H, d, 15.0 HzࠊδH 7.48 ppm, 1H, dd, 15.0 Hz / δH 6.11 ppm, 1H, dd, 10.7 HzࠊδH 5.90 ppm, 1H, dt, 10.7 Hz, 8.0 Hz㸧ほ ࡉࢀࡓࠋࡲࡓࠊ㸯H-㸯H-COSY ࡼࡾ⤌ࡢ࢜ࣞࣇ ࣥࡢࣉࣟࢺࣥ㛫ࡢ࢝ࢵࣉࣜࣥࢢࡀほ ࡉࢀࡓࡇࡽࠊࡇࢀࡽࡣඹᙺࡋࡓ࢜ࣞࣇࣥ ࡛࠶ࡾࠊ࢝ࢵࣉࣜࣥࢢᐃᩘࡼࡾ୍⤌ࡣࢺࣛࣥࢫࡶ࠺୍⤌ࡣࢩࢫࡢᵓ㐀࡛࠶ࡿ᥎ ࡉࢀ ࡓࠋ ࡲࡓ13C-NMR ࡛ࡣぢࡽࢀ࡞ࡗࡓࡀࠊḟඖ HMBC ศᯒ࡛ࡣࠊ㸦δH 2.34 ppm / δC 176.5 ppm㸧㸦δH 2.54 ppm / δC 200.1 ppm㸧ࡢࢩࢢࢼࣝࡀほ ࡉࢀࡓࡇࡽࠊ࢝ࣝ࣎ࢽࣝᇶ ࡀࡘᏑᅾࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋࡑࢀ௨እࡣδC 20 ~ 30 ppm ࡢࢩࢢࢼࣝ δH 1.2 ~ 1.6 ppmࠊ16H ࡢࢩࢢࢼࣝࡀほ ࡉࢀࡓࡇࡽࠊcompound 3 ࡣ┤㙐⬡⫫㓟ࡢྍ⬟ᛶࡀ㧗࠸ ⪃࠼ࡽࢀࡓࠋ ࡉࡽEI-MS ࢆ ᐃࡋࡓࡇࢁ[M+H+] m/z = 294 ࡢศᏊ࢜ࣥࣆ࣮ࢡࡀほ ࡉࢀࠊࡉ ࡽm/z = 276, 223, 171, 166, 155, 95 ࡢࣇࣛࢢ࣓ࣥࢺࣆ࣮ࢡࡀほ ࡉࢀࡓࠋࡲࡓ HI-MS ࡢ ゎᯒࡽࠊcompound 3 ࡢศᏊᘧࡣ C18H30O3ணࡉࢀࡓࠋ௨ୖࡢࡇࡽࠊcompound 3ࡣࠊ9-oxo-(10E,12Z)-octadecadienoic acid (9-E,Z-KODE)࡛࠶ࡿỴᐃࡋࡓ㸦Figure 7㸧ࠋ
Figure 7 Chemical structure of 9-oxo-(10E, 12Z)-octadecadienoic acid (9-E,Z-KODE) Upper number, 1H-NMR (ppm), lower number, 13C-NMR (ppm)
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Table 1. 1H- and 13C-NMR spectral data of compound 3.
Proton number 1H-NMR data (ppm) 13C-NMR data (ppm)
1 ̽ 176.5 2 2.34 (t, J = 7.7Hz) 33.5 3 1.62 (m) 24.6 4㹼7 1.2 - 1.4 (m) 29.0 - 31.5 8 2.54 (t, J = 7.5 Hz) 41.0 9 ̽ 200.1 10 6.16 (d, J = 15.0 Hz) 129.4 11 7.48 (dd, J = 15.0, 11.4 Hz) 137 12 6.11 (dd, J = 11.4, 10.7 Hz) 126.9 13 5.90 (dt, J = 10.7, 8.1 Hz) 143.0 14 2.30 (dt, J = 7.8 Hz) 28.5 15 1.63 (m) 24.6 16 1.2-1.4 (m) 29.0 17 1.3 (m) 22.5 18 0.89 (t, J = 6.9 Hz) 14.0 3.2 Compound 4 ࡢᵓ㐀ゎᯒ
Compound 4 ࡣ1H-ࠊ13C-NMR ࠾ࡼࡧ EI-MS ࡛ ᐃࡋࡓࠋNMR ࡢศᯒ⤖ᯝࢆ Table 2 ♧ࡍࠋ Compound 4 ࡢ13C-NMR ศᯒ࡛ࡣࠊ࣓ࢳࣝⅣ⣲ 1 ᮏࠊ࣓ࢳࣞࣥⅣ⣲⣙ 10 ᮏࠊ࣓ࢳࣥⅣ ⣲4 ᮏࡢࢩࢢࢼࣝࡀほ ࡉࢀࡓࠋ1H-NMR ศᯒ࡛ࡣࠊ࢜ࣞࣇࣥࢆ♧ࡍࢩࢢࢼࣝ㸦δ H 6.07 ppm, 1H, d, 15.4 HzࠊδH 7.12 ppm, 1H, dd, 15.4 Hz㸧ࠊ㸦δH 6.16 ~ 6.18 ppm, 2H, m㸧ࡀほ ࡉ ࢀࡓࠋࡲࡓࠊ㸯H-㸯H-COSY ࡼࡾࠊ⤌ࡢ࢜ࣞࣇࣥࡢࣉࣟࢺࣥ㛫ࡢ࢝ࢵࣉࣜࣥࢢࡀほ ࡉࢀࡓࡇࡽࠊࡇࢀࡽࡣඹᙺࡋࡓ࢜ࣞࣇ࡛ࣥࠊCompound 3 ࡼࡃ㢮ఝࡋࠊࡶ ࢺࣛࣥࢫᵓ㐀࡛࠶ࡿ᥎ ࡉࢀࡓࠋࡇࡢࡇࡽࠊcompound 4 ࡣ┤㙐⬡⫫㓟ࡢྍ⬟ᛶࡀ 㧗࠸⪃࠼ࡽࢀࡓࠋ ࡉࡽcompound 4 ࡢ EI-MS ࢆ ᐃࡋࡓࡇࢁࠊ[M+H+] m/z = 294 ࡢศᏊ࢜ࣥࣆ࣮ࢡ ࠊm/z = 223, 171, 166, 151, 95 ࡞ࡢࣇࣛࢢ࣓ࣥࢺࣆ࣮ࢡࡀほ ࡉࢀࡓࠋHI-MS ࡢศᯒ ⤖ᯝࡽࠊcompound 4 ࡢศᏊᘧࡣࠊC18H30O3ணࡉࢀࡓࠋ௨ୖࡢࡇࡽࠊcompound
9
Table 2 1H- and 13C-NMR spectral data of compound 4.
Proton number 1H-NMR data (ppm) 13C-NMR data (ppm)
1 – 177.6 2 2.36 (t, J = 7.3 Hz) 33.6 3㹼7 1.2 - 1.6 (m) 22.5 - 32.7 8 2.53 (t, J = 7.3 Hz) 40.4 9 – 201.0 10 6.07 (d, J = 15.4 Hz) 127.9 11 7.12 (dd, J = 15.4, 9.8 Hz) 143.0 12 6.15 (m) 128.8 13 6.18 (m) 145.7 14 2.17 (m) 33.1 15~17 1.2-1.6 (m) 22.5 - 32.7 18 0.89 (t, J = 7.0 Hz) 14.0
Figure 8 9-oxo-(10E, 12E)-octadecadienoic acid (9-E,Z-KODE) Upper number, 1H-NMR (ppm), lower number, 13C-NMR (ppm)
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4.
ྛ✀ࡀࢇ⣽⬊ᑐࡍࡿ9-E,E-KODE⣽⬊⮴Ṛάᛶࡢẚ㍑
9-EE-KODE࠾ࡼࡧ9-EZ-KODEࡣࠊ࠸ࡎࢀࡶ linoleic acid ࡽ lipoxygenase ࡢാࡁࡼ ࡗ࡚⏕ᡂࡉࢀࡿྜ≀࡛ࠊ୧᪉ࡶⅣ⣲㦵᱁ࡢⅣ⣲−Ⅳ⣲⤖ྜࡋ࡚㔜⤖ྜࡢࢺࣛࣥࢫ ࡀ᭷ࡍࡿඹᙺ࢜ࣞࣇࣥࡢ┤㙐⬡⫫㓟࡛࠶ࡾࠊoxylipin ⥲⛠ࡉࢀࡿྜ≀⩌ᒓࡋ࡚ ࠸ࡿࠋOxylipinࡣඹᙺࣜࣀ࣮ࣝ㓟ࡋ࡚ࡣⱄື≀ࡽぢࡘࡗࡓ㣬⬡⫫㓟࡛ࠊⱄ ື≀ࡢᾘ⟶ෆ࡛ᚤ⏕≀ࡀ⏘⏕ࡍࡿࠋOxylipinࡣ࢘ࢩࡢஙࡸங〇ရྵࡲࢀࡿ⬡㉁ࡢ 0.34 – 1.07%ࠊ∵⫗ྵࡲࢀࡿ⬡㉁ࡢ0.12 – 0.68%ࢆ༨ࡵࡿ࠸࠺ሗ࿌ࡀ࠶ࡿ[8]ࠋࡲࡓࠊ oxylipinࡣࠊ㐠ືࢆ⾜࠸㐺ษ࡞㛫ࣜࣀ࣮ࣝ㓟ࢆᦤྲྀࡍࡿ࡛ຠ⋡ⓗయ⬡⫫ࢆ⇞↝ࡉ ࡏࡿࡇࡸࠊ୰ᛶ⬡⫫ࡸᝏ⋢ࢥࣞࢫࢸ࣮ࣟࣝࢆῶࡽࡋࠊ⏕ά⩦័㸦⫧‶࣭㧗⾑ᅽ࣭⬻ ⾑⟶ᝈ࣭ᚰ⮚࣭ࡀࢇ➼㸧ࢆண㜵ࡍࡿࡇ࡞ࢆ┠ⓗࡋࡓᗣ㣗ရࡋ࡚⏝ࡉࢀ ࡚࠸ࡿࠋࡲࡓࠊoxylipin ࡣ㧗➼᳜≀࠾࠸࡚ࡣ㜵ᚚ㑇ఏᏊࡢⓎ⌧࠾ࡅࡿࢩࢢࢼࣝศᏊ ࡋ࡚ᗈࡃᏑᅾࡋ࡚࠸ࡿࠋ ᭱㏆࡛ࡣࠊ9-EE-KODEࡘ࠸࡚ࡣࠊ᪂㩭࡞ࢺ࣐ࢺྵࡲࢀࠊPPARαࡢࢦࢽࢫࢺάᛶ ࢆᣢࡘࡇ[9]ࠊࡑࡢࢦࢽࢫࢺάᛶࡣࠊࢺ࣐ࢺࢪ࣮ࣗࢫࡽ༢㞳ࡉࢀࡓ13-EZ-KODE ࡼ ࡾࡶᙅ࠸ࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ[10]ࠊ9-EE-KODEࡢ⣽⬊⮴Ṛάᛶ㛵ࡍࡿሗ࿌ࡣ࡞࠸ࠋ 9-EE-KODE9-EZ-KODEࡢHRA⣽⬊ᑐࡍࡿ⮴Ṛάᛶࡣࠊ9-EE-KODEࡢ࠺ࡀᙉࡗࡓ ࡇࡽࠊ9-EE-KODEࡢHRAᑐࡍࡿ⣽⮴Ṛάᛶࢆࡉࡽヲ⣽᳨ウࡍࡿࡇࡋࡓࠋ KBࠊHRAࠊACC-MESO-1㸦ࣄࢺᝏᛶ୰⓶⭘⣽⬊)ࠊMCF-7㸦ࣄࢺஙࡀࢇ⣽⬊㸧ࠊMia-PaCa-2
㸦ࣄࢺ⮅⮚࢞ࣥ⣽⬊㸧ࠊHT-1080㸦ࣄࢺ⧄⥔⫗⭘⣽⬊㸧ࠊ P388㸦࣐࢘ࢫⓑ⾑⣽⬊㸧 ᑐࡍࡿ9-EE-KODE ࡢ⣽⬊⮴Ṛάᛶࢆ᳨ウࡋࡓࡇࢁࠊ HRA ᑐࡍࡿ IC50ࡣ6.5 μM ẚ㍑ⓗᙉ࠸⣽⬊⮴Ṛάᛶࢆ♧ࡋࡓࡢᑐࡋ࡚ࠊࡢ⣽⬊ᰴᑐࡍࡿIC50ࡣ32㹼74 μM ࠊ⣙5㹼10 ಸ㧗࠸್ࢆ♧ࡋࠊHRA ᑐࡍࡿ≉␗ᛶࡀẚ㍑ⓗ㧗࠸ࡇࡀࢃࡗࡓࠋ ୍ ᪉ࠊ࣏ࢪࢸࣈࢥࣥࢺ࣮ࣟࣝࡋ࡚⏝࠸ࡓ adriamycin㸦ᢠᝏᛶ⭘⒆㸧ࡣࠊHRAࠊKBࠊ P388 ᑐࡋ࡚ᙉ࠸⣽⬊⮴Ṛάᛶࢆ♧ࡋࡓ㸦Table 3㸧ࠋ
11
Table 3 Cytotoxicity of 9-EE-KODE against various cancer cells.
Cell line Origin
IC50 (μM)
9-EE-KODE Adriamycin
HRA Human ovarian cancer 6.5 ± 1 0.15 ± 0.01
MIA PaCa-2 Human pancreatic cancer 32 ± 3 1.4 ± 0.1
HT-1080 Human fibrosarcoma 43 ± 2 0.72 ± 0.27
KB Human cervical carcinoma 54 ± 19 0.18 ± 0.00
ACC-MESO-1 Human malignant mesothelioma 59 ± 8 0.76 ± 0.08
MCF-7 Human breast cancer 74 ± 23 0.57 ± 0.17
P388 Mouse leukemia 48 ± 6 0.18 ± 0.00
Data are expressed as mean ± S.E. (n = 3).
5.
ྛ✀ࡢⱳᏊྵࡲࢀࡿ9-E,E-KODE㔞ࡢ ᐃ
ࢼࢫࡣࠊࢃࡀᅜ࠾࠸࡚㔝⳯ࡋ࡚⏝ࡍࡿ㛗࠸Ṕྐࡀ࠶ࡾࠊྂࡃࡽ᱂ᇵࡉࢀ࡚࠸ ࡿ㛵ಀ࡛ࠊ㠀ᖖከࡃࡢရ✀ࡀ☜❧ࡉࢀ࡚࠸ࡿࠋᚑࡗ࡚ࠊྛ✀ࢼࢫྵࡲࢀࡿ9-EE-KODE ࡢྵ㔞ࡀ␗࡞ࡿྍ⬟ᛶࡀ࠶ࡿࠋࡑࡇ࡛ࠊᕷሙ࡛㈍ࡉࢀ࡚࠸ࡿྛ✀ࢼࢫࢆ㉎ධࡋࠊᯝᐇ ࡢࡀࡃ∦㒊ࠊ㢌㒊ࠊྍ㣗㒊ࡑࢀࡒࢀࢆ⇱ᚋࠊ㓑㓟࢚ࢳ࡛ࣝᢳฟࡋࠊHPLC ࡛ 9-EE-KODE ࡢྵ㔞ࢆᐃ㔞ࡋࡓࠋࡑࡢ⤖ᯝࠊྍ㣗㒊࠾࠸࡚ࡣࠊຍⱱࢼࢫ࠾࠸࡚ࡢࢼࢫẚ㍑ࡋ ࡚ 9-EE-KODE ྵ㔞ࡀపࡃࠊࡲࡓ࠸ࡎࢀࡢရ✀࠾࠸࡚ࡶྍ㣗㒊ẚ㍑ࡋ࡚ࡀࡃ∦㒊 ࡼࡾ㧗࠸ྵ㔞ࢆྵࡴࡇࡀࢃࡗࡓࠋࡲࡓࠊ࠸ࡎࢀࡢရ✀࠾࠸࡚ࡶࠊࡀࡃ∦㒊ྵࡲ ࢀࡿ9-EE-KODE ࡢྵ㔞ࠊࡁ࡞㐪࠸ࡣㄆࡵࡽࢀ࡞ࡗࡓ㸦Figure 9㸧ࠋ12
㛗ࢼࢫ ༸ᙧࢼࢫ ࢼࢫ ຍⱱࢼࢫ ᑠࢼࢫ
Figure 9 The contents of 9-oxo-(10E, 12E)-octadecadienoic acid (9-EE-KODE) in the eggplants of various cultivars. Data are expressed as mean ± S.E. (n = 3)
ࡉ ࡽ ࠊ ྵ 㔞 ࡀ 㧗 ࠸ ࢃ ࡗ ࡓ 㛗 ࢼ ࢫ ࡢ ᳜ ≀ య ࢆ ⏝ ࠸ ࠊ ྛ 㒊 ྵ ࡲ ࢀ ࡿ 9-EE-KODE ࡢྵ㔞ࢆᐃ㔞ࡋࡓࠋࡑࡢ⤖ᯝࡣࠊࢼࢫࡢᯝᐇẚ㍑ࡋ࡚ࠊⴥࠊⱼ࠾ࡼࡧ᰿ࡢ 9-EE-KODE ྵ᭷㔞ࡀᑡ࡞ࡗࡓ㸦Fig. 10㸧ࠋ௨ୖࡢࡇࡽࠊࢼࢫࡢ᳜≀య࠾ࡅࡿ 9-EE-KODE ࡢྵ㔞ࡣࠊࡀࡃ∦㒊ࡀ୍␒ከ࠸ࡇࡀ᫂ࡽ࡞ࡗࡓࠋ
13
Figure 10 The contents of 9-E,E-KODE in the various parts of eggplants (Naga-nasu). Data are expressed as mean ± S.E. (n = 3)
14
➨
➨❶ ⱳྵࡲࢀࡿ⣽㒊⮴Ṛάᛶᡂศࡢస⏝ᶵᗎ
⣽⬊Ṛࡣࠊ⏕⌮ⓗ࡞࣏ࢺ࣮ࢩࢫࡢⓗ࡞ࢿࢡ࣮ࣟࢩࢫࡀ࠶ࡿࠋࣉࣟࢢ࣒ࣛࡉ ࢀࡓ⣽⬊Ṛࡋ࡚1972 ᖺKerr ࡽࡼࡾᥦၐࡉࢀࡓ࣏ࢺ࣮ࢩࢫࡣࠊ⣽⬊ᩘࢆ୍ᐃ ಖࡘᜏᖖᛶⓎ⏕㐣⛬࠾ࡅࡿᙧែᙧᡂࡸせ࡞⣽⬊ࢆ㝖ཤࡍࡿᶵ⬟ࢆ᭷ࡍࡿࠋᜏᖖᛶ ⥔ᣢࡢࡋ࡚ࡣࠊ60 ಶࡢ⣽⬊ࡽᵓᡂࡉࢀ࡚࠸ࡿࣄࢺࡢయ࡛ẖ᪥㉳ࡇࡿ⣙3000൨ಶ ࡢ⣽⬊Ṛࡀᣲࡆࡽࢀࡿࠋᙧែᙧᡂࡢࡋ࡚ࡣࠊಶయࡢⓎ⏕㐣⛬࠾࠸࡚೫ᖹ࡞⤖ྜ⤌ ⧊ࡢሢࡋ࡚Ꮡᅾࡍࡿࣄࢺࡢᡭࡀࠊ࣏ࢺ࣮ࢩࢫࡼࡗ࡚ᣦࡢ㛫Ꮡᅾࡋ࡚࠸ࡓỈࡁ 㒊ศࡀ㑅ᢥⓗṚࡠࡇࡼࡾࣄࢺࡢᣦࢆᙧᡂࡍࡿࡇࡀᣲࡆࡽࢀࡿࠋࡲࡓࠊ࠾ࡓࡲࡌ ࡷࡃࡋࡣࠊ࢚࢝ࣝ࡞ࡿ㝿ᑼᑿࡀせ࡞ࡿࡓࡵࠊ࣏ࢺ࣮ࢩࢫࡼࡾ㏥⦰ࡍࡿࠋ ࣏ࢺ࣮ࢩࢫ࡛ࡣࠊࡲࡎ⣽⬊ෆ⭷Ꮡᅾࡍࡿ࣍ࢫࣇࢳࢪࣝࢭࣜࣥࡢ⣽⬊እ⭷ࡢ⛣ ືࡀ㉳ࡇࡾࠊ⣽⬊ࡀจ⦰ࡋ࡚⣽⬊య✚ࡀῶᑡࡍࡿࡶࠊ᰾ෆ࡛ࡣࢡ࣐ࣟࢳࣥࡢจ⦰ ࡀ㉳ࡇࡿࠋ⥆࠸࡚᰾ࡀ᩿∦ࡉࢀࡓᚋࠊ⣽⬊⮬యࡀ᩿∦ࡋ࡚࣏ࢺ࣮ࢩࢫᑠయࡀᙧᡂ ࡉࢀࡿࠋ᭱ᚋ࣏ࢺ࣮ࢩࢫᑠయࡣ࣐ࢡࣟࣇ࣮ࢪ➼ࡢᤕ㣗⣽⬊ࡼࡗ࡚㈎㣗㝖ཤࡉࢀ ࡿ[11]ࠋ ୍᪉࡛ࠊࢿࢡ࣮ࣟࢩࢫ࡛ࡣࠊ⣽⬊ࡢ⭾࣑ࢺࢥࣥࢻࣜࡢ⭾ࡼࡾ⣽⬊ࡀỈ⭘≧ ࡞ࡗࡓᚋࠊ⣽⬊⭷ࡢᔂቯࡀ㉳ࡇࡿࠋ࣏ࢺ࣮ࢩࢫࡣ࿘ᅖࡢ⣽⬊ᙳ㡪ࢆཬࡰࡉ࡞࠸ࡢ ᑐࡋࠊࢿࢡ࣮ࣟࢩࢫࡣ⣽⬊ࡢෆᐜ≀ࡀ࿘ᅖᤥࡁᩓࡽࡉࢀࡿࡓࡵࠊ⅖ࢆక࠸࿘ࡾࡢ ⣽⬊ᙳ㡪ࢆ࠼ࡿࠋࡑࡢࡓࡵࠊࡀࢇ⣽⬊ᑐࡍࡿ࣏ࢺ࣮ࢩࢫㄏᑟ⬟ࢆᣢࡘྜ≀ࡣࠊ ࡢ⣽⬊ᙳ㡪ࢆ࠼࡞࠸ᢠࡀࢇ࡞ࡾ࠺ࡿ⪃࠼ࡽࢀࡿ㸦Figure 11㸧ࠋ15 ࣏ࢺ࣮ࢩࢫࡢ㐣⛬ࡣࠊ࣑ࢺࢥࣥࢻࣜ ෆ㒊⤒㊰ࠊFas ࣞࢭࣉࢱ࣮ࡽࡢእ㒊 ⤒㊰࠾ࡼࡧᑠ⬊యࢫࢺࣞࢫ⤒㊰ࡼࡾㄏ ᑟࡉࢀࡿ㸦Figure 12㸧ࠋ࣏ࢺ࣮ࢩࢫࡢ ࢩ ࢢ ࢼ ࣝ ࡀ ఏ 㐩 ࡉ ࢀ ࡿ ࠊcysteine dependent aspartate-directed proteinaseࠊࢩ ࢫࢸࣥ౫Ꮡᛶࢫࣃࣛࢠࣥ㓟ࡢC ᮎ ➃ഃࢆຍỈศゎࡍࡿࣉࣟࢸ࣮ࢮ࡛࠶ࡿ caspase ࡀḟࠎάᛶࡉࢀࡿࠋ࣑ࢺࢥࣥ ࢻࣜࡽࡢ⤒㊰ࡣࠊ࣏ࢺ࣮ࢩࢫࡢࢩ ࢢࢼࣝఏ㐩ࡼࡾࠊ࣑ࢺࢥࣥࢻࣜ⭷ࡢ ㏱㐣ᛶஹ㐍ࡸ⣽⬊ෆ࡛ࡢࢩࢺࢡ࣒ࣟ C ࡢᨺฟࡀᘬࡁ㉳ࡇࡉࢀࠊcaspase-9 ࢆάᛶ ࡋࠊ⥆࠸࡚caspase-3 ࢆάᛶࡋ࣏ࢺ ࣮ࢩࢫࢆㄏᑟࡍࡿࠋ ➨ ୍ ❶ ࡛ ࡣ ࠊ ࣄ ࢺ ༸ ᕢ ࡀ ࢇ ⣽ ⬊ ࡢHRA ᑐ ࡋ ࡚ ࢼ ࢫ ࡢ ᭱ ࡶ ከ ࡃ ྵ ࡲ ࢀ ࡿ 9-EE-KODEࡢ⣽⬊⮴Ṛάᛶࢆ᫂ࡽࡋࡓࠋ9-EE-KODEᏛᵓ㐀ࡀ㢮ఝࡋ࡚࠸ࡿඹᙺ ࣜࣀ࣮ࣝ㓟ࡢ୍ࡘ࡛࠶ࡿt10, c12-conjugated linoleic acidࡣࠊ࣑ࢺࢥࣥࢻࣜ⤒㊰࠶ࡿ Bcl-2ࡢⓎ⌧㔞ࢆῶᑡࡉࡏࡿࡇࡼࡾࠊ࣐࢘ࢫங⭢⭘⒆⣽⬊ࡢ࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿ ࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[12]ࠋࡑࡇ࡛ᮏ❶࡛ࡣࠊ9-EE-KODEࡢ⣽⬊⮴Ṛάᛶࡢ࣓࢝ࢽࢬ࣒ ࡋ࡚ࠊ࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿྍ⬟ᛶࡘ࠸᳨࡚ウࡋࡓࠋ
16
1.
Compound 4 (9-EE-KODE) ༢㞳ᡂศࡢᏛྜᡂ
➨୍❶࡛ᚓࡽࢀࡓ 9-EE-KODE ࡣ㠀ᖖࢃࡎ࡛࠶ࡿࡓࡵࠊࡉࡽ࡞ࡿస⏝ᶵᗎゎᯒࡢ ࡓࡵࡣ Ꮫ ྜ ᡂ ࡼ ࡾ 㔞 ౪ ⤥ ࢆ ┠ ᣦ ࡍ ࡇ ࡋ ࡓ ࠋ ࡼࡃ⇱ࡉࡏࡓ࣐ࢢࢿࢩ࣒࢘ࢸࢺࣛࣄࢻࣟࣇࣛࣥ㸦THF㸧ࢆຍ࠼ࠊethyl bromide ࢆ ධࢀ࡚ᛂࡉࡏࠊࢢࣜࢽ࣮ࣕࣝヨ⸆ࢆྜᡂࡋ[13]ࠊࡇࢀ8-bromo-1-octanol ᛂࡉࡏࡓࠋ ᛂᚋࠊ (E,E)-2,4-decadienal ࢆຍ࠼࡚ᛂࡉࡏࡓᚋࠊNH4Cl Ỉ⁐ᾮ࢚࣮ࢸ࡛ࣝศᾮࡋ ࡓࠋ࢚࣮ࢸࣝᒙࢆỈ࡛㸱ᅇศ㓄ࡋࠊ࢚࣮ࢸࣝᒙࢆMgSO4࡛⇱ࡋࡓࠋࡑࡢᚋࠊ࣊࢟ࢧࣥ 㸭㓑㓟࢚ࢳࣝ㸭࣓ࢱࣀ࣮ࣝ㸦45㸸3㸸1㸧ࢆ⛣ື┦ࡍࡿࢩࣜ࢝ࢤࣝࢡ࣐ࣟࢺࢢࣛࣇ࣮ ౪ࡋࠊ୰㛫⏘≀ྵ᭷⏬ศ㸦0.89 g㸧ࢆᚓࡓࠋࡉࡽ MeOH/Ỉ㸦1㸸1㸧ࢆ⛣ື┦ࡍࡿ ODS ࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮࡛ศ㞳ࡋࠊ୰㛫⏘≀㸦0.28 g㸧ࢆᚓࡓࠋ ୕㓟ࢡ࣒ࣟỈ⁐ᾮ⃰◲㓟ࢆຍ࠼ࡿࡇࡼࡾࠊJones ヨ⸆ࢆㄪ〇ࡋࡓ[14]ࠋ୰㛫⏘≀ 㸦0.046 g㸧 Jones ヨ⸆ࢆධࢀࠊᐊ ࡛ 30 min ᨩᢾࡋࠊ⁐ᾮࡢⰍࡣ㉥ࡽ㯤Ⰽ࡞ࡗࡓ ᚋࠊNaClࠊࢡ࣒ࣟࣟ࣍ࣝࠊࢭࢺࣥࢆຍ࠼ࠊศ㓄ࡋࡓࠋࢡ࣒ࣟࣟ࣍ࣝᒙ㸦0.037 g㸧ྵ ࡲࢀࡿ 9-EE-KODE ࢆ MeOH/Ỉ㸦4㸸1㸧ࢆ⛣ື┦ࡍࡿ ODS ㏫ᒙࢡ࣐ࣟࢺࢢࣛࣇ࣮ ࡛⢭〇ࡋࠊ0.029 g ࢆᚓࡓࠋࡑࢀࢆ HPLC ࠾ࡼࡧ NMR ࡛ྠᐃࡋࡓ㸦Figure 13㸧ࠋ17
B
Proton number ྜᡂ9-EE-KODE
1H-NMR data (ppm) ศ㞳ࡋࡓ9-EE-KODE 1H-NMR data (ppm) 1 – – 2 2.35 (t, J = 7.3 Hz) 2.36 (t, J = 7.3 Hz) 3㹼7 1.2 - 1.4 (m) 1.2 - 1.6 (m) 8 2.53 (t, J = 7.4 Hz) 2.53 (t, J = 7.3 Hz) 9 – – 10 6.07 (d, J = 15.4 Hz) 6.07 (d, J = 15.4 Hz) 11 7.11 (dd, J = 15.5Hz) 7.12 (dd, J = 15.4, 9.8 Hz) 12 6.15 (m) 6.15 (m) 13 6.19 (m) 6.18 (m) 14 2.15 (m) 2.17 (m) 15~17 1.2-1.4 (m) 1.2-1.6 (m) 18 0.89 (t, J = 7.0 Hz) 0.89 (t, J = 7.0 Hz) C
Figure 13 (A) Scheme of the synthesis of 9-EE-KODE. (B) Comparison of synthesized and isolated 9-EE-KODE by
1H-NMR (ppm). Solvent, CDCl
3. (C) Chromatograms of synthesized 9-EE-KODE. Column: Cosmosil 5C18-AR-II (10 × 150
mm, Nacalai Tesque, Kyoto), solvent: A (H2O) - B (CH3CN), 0 – 100 (0 – 30 min), flow speed: 1.0 ml/min; wave length: 280
nm. >PLQ@ ( ( ( ( ( ( ( ( ( X$8
18
2.
9-EE-KODE ࡢ HRA ᑐࡍࡿ⣽⬊⮴Ṛస⏝
➨୍❶࡛ࡣࠊ9-EE-KODEࡢHRA༸ᕢࡀࢇ⣽⬊ᑐࡍࡿ⣽⬊⮴Ṛస⏝ࢆMTTἲࡼࡾ᳨ ドࡋࡓࠋMTTἲࡣ⏕⣽⬊࠾࠸࡚MTTࢆ⣸Ⰽࡢ࣐࣍ࣝࢨࣥ㑏ඖࡍࡿ࠸࠺⣽⬊ࡢ௦ㅰ άᛶࡼࡾ⣽⬊⮴Ṛάᛶࢆ ᐃ࡛ࡁࡿࡀࠊ⏕ᡂࡋࡓ࣐࣍ࣝࢨࣥࡀ⣽⬊ẘᛶࢆ᭷ࡍࡿሙྜ ࡀ࠶ࡾࠊ⏕Ꮡ⋡ ᐃࡢ⤖ᯝㄗࡾࡀ⏕ࡌࡿྍ⬟ᛶࡀ࠶ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[15]ࠋ ࡑࡇ࡛MTTἲࡶBrdUἲࡼࡾࠊ⣽⬊⮴Ṛάᛶࢆ ᐃࡋࡓࠋ⺯ගᶆ㆑ࡋࡓᢠBrdU ᢠయࢆ⏝࠸࡚≉␗ⓗ᳨ฟࡍࡿ࠸࠺᪉ἲ࡛ࠊDNAྜᡂ㔞ࢆ ᐃࡍࡿࠋHRA⣽⬊ 9-EE-KODE ࢆྛ⃰ᗘ࡛ῧຍࡋࠊ24㛫ᚋࡉࡽ MTT ࡲࡓࡣ BrdU ࢆ ῧຍࡋ࡚ࠊ⣽⬊ࡢ௦ㅰ㔞DNAྜᡂ㔞ࢆ ᐃࡋࡓࡇࢁࠊ࠸ࡎࢀࡶ 9-EE-KODE ࡢ⃰ᗘ ౫Ꮡⓗ࡞⣽⬊⮴Ṛάᛶࡀㄆࡵࡽࢀࡓࠋࡑࢀࡒࢀࡢ᪉ἲ࡛ ᐃࡋࡓ IC50 ࡣࠊ9.2 μg/ml (31
μM) ࠾ࡼࡧ 2.6 μg/ml (8.7 μM) ࡛ࡁ࡞㐪࠸ࡣㄆࡵࡽࢀ࡞ࡗࡓ㸦Figure 14㸧ࠋ௨ୖࡢ⤖ ᯝࡼࡾࠊ9-EE-KODEࡣHRA⣽⬊ᑐࡋ࡚ࠊ⣽⬊⮴Ṛάᛶࡀ࠶ࡿࡇࡀ㏣ヨ࡛ࡁࡓࠋ
Figure 14 Cytotoxic effect of 9-EE-KODE on HRA cells evaluated by MTT (A) or BrdU assays (B) HRA cells were treated with 9-EE-KODE for 24 h, and the cell viability was evaluated. Data were expressed as mean ± S.E. (n = 3 for MTT, and n = 6 for BrdU tests). **P < 0.01, ***P < 0.001 vs control (0 μg/ml) group evaluated by Bonferroni/Dunnett’s multiple t-test.
0 20 40 60 80 100 0 5 10 15 20 0 20 40 60 80 100 0 5 10 15 20 9-EE-KODE (μg/ml)
% of control
9-EE-KODE (μg/ml)% of control
A B***
***
***
***
***
***
***
**
***
19
3.
9-EE-KODE ࡼࡿ DNA ᩿∦ࡢㄏᑟ
9-EE-KODE ࡼࡿ⣽⬊ቑṪࡢᢚไࡀ࣏ࢺ࣮ࢩࢫࡢㄏᑟࡼࡿࡶࡢ࠺ࢆ᫂ࡽ ࡍࡿࡓࡵࠊDNA ࡢ᩿∦ࡢ᭷↓ࢆほᐹࡋࡓࠋ9-EE-KODE ่࡛⃭ 24 㛫ᚋࠊHRA ⣽⬊ࡢDNA ࢆᢳฟࡋࠊ࣮࢞ࣟࢫࢤࣝ㟁ẼὋື࡛ほᐹࡋࡓࠋࡑࡢ⤖ᯝࠊ9-EE-KODE ࡢ ⃰ᗘ࡛ 1 μg/ml ࡽࠊ⣽⬊ DNA ࡢ᩿∦ࡀぢࡽࢀࡿࡼ࠺࡞ࡾࠊ⃰ᗘࡀ㧗ࡃ࡞ࡿࡑ ࡢ᩿∦ࡀ㢧ⴭほᐹࡉࢀࡿࡼ࠺࡞ࡗࡓ㸦Figure 15A㸧ࠋ ḟࠊ࣏ࢺ࣮ࢩࢫࡢ㝿⣽⬊㉁㐟㞳ࡍࡿࣄࢫࢺࣥ⤖ྜ DNA ᩿∦ࢆࠊศගගᗘィ ࡼࡾ᳨ฟࡍࡿELISA ࢟ࢵࢺࢆ⏝࠸࡚ᐃ㔞ࡋࡓࠋࡇࡢ࢟ࢵࢺ࡛ࡣࠊ⣽⬊⁐ゎヨᩱࡢ⣽⬊ ㉁ࣇࣛࢡࢩࣙࣥ୰࠾ࡅࡿࣔࣀ-࠾ࡼࡧ࢜ࣜࢦࢾࢡࣞ࢜ࢯ࣮࣒ࢆ≉␗ⓗ᳨ฟ࡛ࡁࡿࠋ 9-EE-KODE ࡛ HRA ⣽⬊ࢆ่⃭ࡋࡓᚋࡢ DNA ᩿∦ࡢࣄࢫࢺࣥ⤖ྜ DNA ᩿∦ࡢ┦ ᑐ࡞㔞ࡣࠊ⃰ᗘ౫Ꮡⓗቑຍࡍࡿഴྥࡀぢࡽࢀࡓ㸦Figure 15B㸧ࠋࡇࡢࡇࡽࠊ 9-EE-KODE ࡣ࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ
Figure 15 Effect of 9-EE-KODE on DNA fragmentation in HRA cells.
(A) HRA cells were treated with 9-EE-KODE for 24 h, and DNA in the cells was separated by electrophoresis. (B) HRA cells were treated with 9-EE-KODE for 4 h, and the amount of DNA-histone complex was evaluated. Data were expressed as mean ± S.E. (n = 3). *P < 0.05 vs control (0 μg/ml) group evaluated by Bonferroni/Dunnett’s multiple t-test.
4. 9-EE-KODEࡼࡿphosphatidylserineࡢ⣽⬊እ⭷ࡢ㟢ฟࡢㄏᑟ
࣏ࢺ࣮ࢩࢫࡢึᮇẁ㝵࡛ࡣࠊ⣽⬊⭷ࡢᵓ㐀ࢆಖࡗࡓࡲࡲ࡛⣽⬊⭷ࣜࣥ⬡㉁ࡢ㠀ᑐ⛠ ᛶࢆ႙ኻࡍࡿࠋPhosphatidylserine ࡣ㝜ᛶⲴ㟁ࡋࡓࣜࣥ⬡㉁࡛ࠊ⣽⬊⭷ࡢෆഃᒁᅾࡋ࡚ ࠸ࡿࡀࠊ࣏ࢺ࣮ࢩࢫࡢࡈࡃึᮇ⣽⬊⭷ࡢእഃ⾲ฟࡍࡿࠋࡑࢀᑐࡋࠊAnnexin V ࡣ࢝ࣝࢩ࣒࢘౫Ꮡᛶࡢࣜࣥ⬡㉁⤖ྜࢱࣥࣃࢡ࡛ࠊphosphatidylserine ࡣ㧗࠸ぶᛶ࡛⤖ ྜࡍࡿࡇࡼࡾࠊ⥳Ⰽࡢ⺯ගࢆⓎࡍࡿࠋࡲࡓࠊṇᖖ࡞⣽⬊ࡣࢿ࢟ࢩࣥ V ᰁⰍࡉࢀ ࡞࠸ࠋࡋࡓࡀࡗ࡚ࠊ࣏ࢺ࣮ࢩࢫࢆ㉳ࡇࡋࡓ⣽⬊ࡣࠊ⺯ග㢧ᚤ㙾࡛ࡑࡢ⣽⬊Ṛࡀ࣏ࢺ A B 9-EE-KODE (μg/ml) 㻌䚷0 1 5䚷 10 100 0 0.5 1 1.5 2 2.5 Control 1 5 10 100Relative enrichment factor
9-EE-KODE (μg/ml) *
20 ࣮ࢩࢫࢿࢡ࣮ࣟࢩࢫࡢุᐃࡀྍ⬟࡛࠶ࡿ[16]ࠋ
ࡑࡇ࡛⺯ගᶆ㆑ࡉࢀࡓAnnexin V ࢆ⏝࠸࡚ࠊ9-EE-KODE ่࡛⃭ࡋࡓ HRA ⣽⬊ࢆᰁⰍ ࡋࡓࡇࢁࠊ⃰ᗘ౫Ꮡⓗ⺯ගᶆ㆑ࡉࢀࡓ⣽⬊ᩘࡢቑຍࡀほᐹࡉࢀࡓ㸦Figure 16㸧ࠋ௨ୖ ࡢࡇࡽࠊ9-EE-KODE ࡣࠊHRA ⣽⬊ᑐࡋ࡚ࠊ࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿࡇࡀ᫂ࡽ ࡞ࡗࡓࠋ
B
Figure 16 Effect of 9-EE-KODE on phosphatidylserine cell in the outer membrane of HRA cells. HRA cells were treated with 9-EE-KODE for 12 h, and phosphatidylserine in outer membrane was detected by Annexin V staining. (A) Representative microscopic photographs. Bar in a photo represents 0.02 mm. (B) Positively stained cells were counted in 4 – 6 different microscopic fields of view of a single culture well, and data were exhibited as mean ± S.E. *P < 0.05, **P < 0.01, ***P < 0.001 vs control (0 μg/ml) group evaluated by Bonferroni/Dunnett’s multiple t-test.
A
1 μg/ml
5 μg/ml 10 μg/ml Control
21
5.
9-EE-KODE ࡼࡿ HRA ⣽⬊ࡢ caspase-3, -7 άᛶᑐࡍࡿᙳ㡪
࢝ࢫࣃ࣮ࢮ࢝ࢫࢣ࣮ࢻࡢάᛶࡣࠊ࣏ࢺ࣮ࢩࢫ⤒㊰ࡢ࡞࡛Ḟࡃࡇࡢ࡛ࡁ࡞࠸ ࣋ࣥࢺ࡛࠶ࡿࠋCaspase-3 ࡣ⣽⬊ࢆᩚ↛ࡋࡓᔂቯᑟࡃᩘከࡃࡢࢱࣥࣃࢡ㉁ษ᩿ࡢᘬࡁ 㔠ࢆᘬࡃ“ᇳ⾜ே”ࡢᙺࢆᣢࡘ㔜せ࡞࢝ࢫࣃ࣮ࢮ㓝⣲ࡢ 1 ࡘ࡛࠶ࡾࠊcaspase-3 άᛶࡢቑ ຍࡣ࣏ࢺ࣮ࢩࢫࡢ࣐࣮࣮࢝ࡋ࡚ỗ⏝ࡉࢀ࡚࠸ࡿ[17]ࠋ
ࡑࡇ࡛ࠊ 9-EE-KODE ࢆ HRA ⣽⬊ῧຍࡋ࡚ࡽ 24 㛫ࡢ⣽⬊࠾ࡅࡿ caspase-3㸪 Ѹ7 ࡢάᛶࢆࠊ࢟ࢵࢺࢆ⏝࠸࡚ ᐃࡋࡓࠋࡑࡢ⤖ᯝࠊ9-EE-KODE ࡣ HRA ⣽⬊ࡢ caspase-3㸪 −7 ࡢάᛶࢆ⃰ᗘ౫Ꮡⓗቑຍࡉࡏࠊ0.1 μg/ml ࡢ⃰ᗘ࡛᭷ពᕪࡀㄆࡵࡽࢀࡓ㸦Figure 17㸧ࠋ ௨ୖࡢࡇࡽࠊ9-EE-KODE ࡢ HRA ⣽⬊ᑐࡍࡿࠊ࣏ࢺ࣮ࢩࢫࡢㄏᑟࡀ࢝ࢫࣃ࣮ ࢮ࢝ࢫࢣ࣮ࢻࢆࡍࡿࡇࡀࢃࡗࡓࠋ
Figure 17 Effect of 9-EE-KODE on the activity of caspase-3/7 in HRA cells. HRA cells were treated with 9-EE-KODE for 24 h, and the activities of caspase-3/7 were measured. Data were exhibited as mean ± S.E (n = 3). **P < 0.01, ***P < 0.001 vs control (0 μg/ml) group evaluated by Bonferroni/Dunnett’s multiple t-test.
6. 9-EE-KODE ࡢ HRA ⣽⬊࣑ࢺࢥࣥࢻࣜ⭷㟁ᕪᑐࡍࡿస⏝
HRA༸ᕢࡀࢇ⣽⬊ᑐࡍࡿ9-EE-KODEࡢస⏝ࡀࠊ࣑ࢺࢥࣥࢻࣜࢆࡍࡿ࣏ࢺ࣮ࢩ ࢫㄏᑟ⤒㊰ࡼࡿྍ⬟ᛶࡘ࠸᳨࡚ウࡋࡓࠋ࣑ࢺࢥࣥࢻࣜࡢ⭷㟁ࡣࠊDNAᦆയ࡞ ࡢࢫࢺࣞࢫࡼࡾࠊ࣏ࢺ࣮ࢩࢫㄏᑟศᏊp53ࡸ࣏ࢺ࣮ࢩࢫࢆㄪ⠇ࡍࡿBcl-2ࣇ࣑ࣜ ࣮ࢱࣥࣃࢡ㉁ࢆࡋ࡚ኚࡋࠊࡑࡢ⤖ᯝࠊ࣑ࢺࢥࣥࢻࣜࡽcytochrome cࡀ₃ฟࡋࠊ ࣏ࢺ࣮ࢩࢫࡀㄏᑟࡉࢀࡿࠋ ࡑࡇ࡛ࠊ࣏ࢺ࣮ࢩࢫ㛵ಀࡍࡿ࣑ࢺࢥࣥࢻࣜෆ㒊⤒㊰࡛ぢࡽࢀࡿࠊ࣑ࢺࢥࣥࢻࣜ ⭷㟁ࡢኚࢆほᐹࡋࡓࠋ࣑ࢺࢥࣥࢻࣜࡢ⭷㟁ࡢᾘኻࡣࠊ࣏ࢺ࣮ࢩࢫࡢㄏᑟ Luminescence 䚷 (r elative to control) 9-EE-KODE (μg/ml) 0 1 2 3 4 5 6 Control 0.1 1 10 *** ** **22 ࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡿึᮇࡢ⌧㇟ࡢ୍ࡘ࡛࠶ࡿࠋ ࡇࡢኚࢆࠊJC-10 ࡤࢀࡿⰍ⣲ࢆ ⏝࠸࡚ホ౯ࡋࡓࠋ ࡇࡢⰍ⣲ࡣࠊṇᖖ⣽⬊ࡢ࣑ࢺࢥࣥࢻࣜࡢ⭷㟁ࡢࡁࡣจ㞟ࡋ࡚ Ꮡᅾࡋ㉥Ⰽࡢ⺯ගࢆⓎࡍࡿࡀࠊ࣏ࢺ࣮ࢩࢫࡋࡓ⣽⬊࡛ࡣ࣑ࢺࢥࣥࢻࣜ⭷㟁ࡀᾘኻ ࡋࠊ࣑ࢺࢥࣥࢻࣜእࣔࣀ࣐࣮≧ᣑᩓࡋ࡚ࠊ⥳Ⰽࡢ⺯ගࢆⓎࡍࡿ[18]ࠋHRA ⣽⬊ࢆ 9-EE-KODE ࡛ฎ⌮ࡋ࡚ 3 㛫ᚋ JC-10 ࢆῧຍࡋࠊ࣑ࢺࢥࣥࢻࣜ⭷㟁ࡢኚࢆ ᐃࡋࡓࡇࢁࠊ⥳Ⰽࡢ⺯ගࢆ♧ࡋࡓHRA ⣽⬊ࡢྜࡣࠊ9-EE-KODE ࡢ⃰ᗘ౫Ꮡⓗቑ ຍࡋࠊ20 μg/mL ࡢ⃰ᗘ࡛᭷ពᕪࡀㄆࡵࡽࢀࡓ㸦Figure 18㸧ࠋ ࡇࡢࡇࡽࠊ9-EE-KODEࡢῧຍࡼࡾࠊHRA⣽⬊ෆࡢ࣑ࢺࢥࣥࢻࣜࡢ⭷㟁ࡀῶ ᑡࡋ࡚࠸ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ
Figure 18 Effect of 9-EE-KODE on mitochondrial membrane potential in HRA cells.
HRA cells were treated with 9-EE-KODE for 3 h, and the mitochondrial membrane potential was evaluated by JC-10 staining. Data were exhibited as mean ± S.E. (n = 6). *P < 0.05 control (0 μg/ml) group evaluated by Bonferroni/Dunnett’s multiple t-test.
7. 9-EE-KODEࡢ
ࡢHRA⣽⬊࣑ࢺࢥࣥࢻࣜࡽࡢcytochrome cࡢᨺฟࡢ
స⏝
⣽⬊㉁ෆᨺฟࡉࢀࡓcytochrome c ࡣࠊApaf-1㸦apoptic protease activating factor 1㸧 ⤖ྜࡋ࡚Apaf-1ࡢከ㔞యࢆಁࡋࠊcaspaseࡀࣜࢡ࣮ࣝࢺࡉࢀࠊ࣏ࢺࢯ࣮࣒ࡤࢀࡿ ᕧ࡞」ྜయࡀᙧᡂࡉࢀࡿࠋ࣏ࢺࢯ࣮࣒ࡣcytochrome cࠊApaf-1࠾ࡼࡧ caspase-9 ࢆྵ ࢇࡔ」ྜయ࡛ࠊcaspase-9 ࡣࡇࡢ」ྜయ୰࡛άᛶࡍࡿࠋάᛶࡉࢀࡓ caspase-9 ࡣࠊࢹ ࢫࣞࢭࣉࢱ࣮ࢆࡍࡿ⤒㊰ྠᵝࠊcaspase-3 ࡞ࡢୗὶࡢ caspase ࢆάᛶࡋࠊ࣏ ࢺ࣮ࢩࢫࡀㄏᑟࡉࢀࡿࠋ ࣑ࢺࢥࣥࢻࣜෆࡢcytochrome c⣽⬊㉁ෆcytochrome cࡢⓎ⌧ࡢኚࡣࠊ⣽⬊࣏ࢺ ࣮ࢩࢫࡢ㘽࡞ࡿ࣋ࣥࢺ࡛࠶ࡿࠋࡑࡇ࡛ࠊ9-EE-KODEࡢῧຍࡼࡗ࡚ࠊ࣑ࢺࢥࣥࢻࣜ ෆ⣽⬊㉁ෆࡢcytochrome cࡢࢱࣥࣃࢡⓎ⌧ࢆࠊ࢙࢘ࢫࢱࣥࣈࣟࢵࢺἲࡼࡾ᳨ウࡋࡓࠋ
23 ࡑࡢ⤖ᯝࠊ9-EE-KODE ࡛ฎ⌮ࡋࡓ HRA ⣽⬊࡛ࡣࠊ⃰ᗘ౫Ꮡⓗ࣑ࢺࢥࣥࢻࣜࡢ cytochrome c ࡢⓎ⌧㔞ࡀῶᑡࡋࠊ⣽⬊㉁ࡢ cytochrome c ࡢⓎ⌧㔞ࡢቑຍࡀㄆࡵࡽࢀࡓࠋࡇ ࡢࡇࡽࠊ9-EE-KODE ࡢῧຍࡼࡾࠊcytochrome c ࡀ࣑ࢺࢥࣥࢻࣜࡽ⣽⬊㉁ࡢ ᨺฟࡀಁ㐍ࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓ㸦Figure 19㸧ࠋ A B
Figure 19 Effect of 9-EE-KODE on cytochrome c localization in mitochondrial and cytosol fractions in HRA cells.
HRA cells were treated with 9-EE-KODE for 24 h, and the cell lysate was separated into mitochondrial and cytosolic fractions. The content of cytochrome c was evaluated by western blot analysis. (A) Representative photographs of the membrane. (B) Area of the band was measured by image analyzer, and data were exhibited as mean ± S.E. (n = 3). **P < 0.01, ***P < 0.001 vs each control (0 μg/ml) group evaluated by Bonferroni/Dunnett’s multiple t-test.
24
8.
9-EE-KODE ࡢ HRA ⣽⬊࠾ࡅࡿ Bcl-2 ࣇ࣑࣮ࣜࢱࣥࣃࢡ㉁Ⓨ⌧㔞
ࡢస⏝
Bcl-2 ࣇ࣑࣮ࣜࢱࣥࣃࢡ㉁ࡣࠊ࣑ࢺࢥࣥࢻࣜࡢ㏱㐣ᛶࢆࢥࣥࢺ࣮ࣟࣝࡍࡿࡇ ࡼࡗ࡚࣏ࢺ࣮ࢩࢫࢆไᚚࡋ࡚࠸ࡿࠋᢠ࣏ࢺ࣮ࢩࢫࢱࣥࣃࢡ㉁ࡢ Bcl-2ࠊBcl-xL ࡣ࣑ ࢺࢥࣥࢻࣜእ⭷Ꮡᅾࡋࠊcytochrome c ࡢᨺฟࢆᢚไࡍࡿࠋࣉ࣏ࣟࢺ࣮ࢩࢫࢱࣥࣃ ࢡ㉁ࡢ BadࠊBidࠊBaxࠊBim ࡣ⣽⬊㉁Ꮡᅾࡍࡿࡀࠊdeath ࢩࢢࢼࣝᚑࡗ࡚࣑ࢺࢥࣥ ࢻࣜ⛣ືࡋࠊࡑࡇ࡛ࡣcytochrome c ࡢᨺฟࢆಁ㐍ࡍࡿࠋBad ࡣ࣑ࢺࢥࣥࢻࣜ ⛣ືࡋࠊBcl-xL ࣉ࣏ࣟࢺ࣮ࢩࢫ」ྜయࢆᙧᡂࡍࡿࠋࡇࡢ⛣ືࡣࠊBad ࡢࣜࣥ㓟 ࢆᘬࡁ㉳ࡇࡍ⏕ᏑᅉᏊࡼࡗ࡚ᢚไࡉࢀࠊ⣽⬊㉁ᘬࡁṆࡵࡽࢀࡿࠋ⣽⬊㉁ෆࡢ Bid ࡣ Fas ࢆࡋࡓࢩࢢࢼࣝࡋࡓࡀࡗ࡚ caspase-8 ࡼࡗ࡚ᾘࡉࢀࠊࡑࡢάᛶᆺࣇࣛࢢ࣓ࣥ ࢺ㸦tBid㸧ࡣ࣑ࢺࢥࣥࢻࣜ⛣ືࡍࡿࠋBax Bim ࡣ⏕ᏑᅉᏊࡢ㝖ཤ࡞ࡢ death ่⃭ᛂ⟅ࡋ࡚࣑ࢺࢥࣥࢻࣜ⛣ືࡍࡿࠋDNA ᦆയࡼࡗ࡚ caspase ࡀάᛶࡉࢀ ࡿࠊp53 ࡣ Bax Noxa ࡢ㌿ࢆஹ㐍ࡍࡿࠋ࣏ࢺ࣮ࢩࢫࡢ࣑ࢺࢥࣥࢻࣜ㏱㐣ᛶ cytochrome c ᨺฟࡢไᚚ࣓࢝ࢽࢬ࣒ࡣࡣศࡗ࡚࠸࡞࠸ࡀࠊBcl-xLࠊBcl-2ࠊBax ࡣ㟁౫Ꮡᛶ㝜࢜ࣥࢳࣕࢿࣝ㸦VDAC㸧ᙳ㡪ࢆ࠼ࠊࡑࢀࡀ cytochrome c ࡢᨺฟไ ᚚࡢ୍➃ࢆᢸࡗ࡚࠸ࡿ⪃࠼ࡽࢀ࡚࠸ࡿ[19] 㸦Figure 20㸧ࠋ 9-EE-KODE࡛ฎ⌮ࡋࡓHRA⣽⬊࡛ࡣࠊ࣑ࢺࢥࣥࢻࣜࡢ⭷㟁ࡣ⃰ᗘ౫Ꮡᛶ࡛పୗࡋ ࡚࠸ࡓࡢ࡛ࠊ9-EE-KODEࡣBcl-2 ࣇ࣑࣮ࣜࢱࣥࣃࢡ㉁స⏝ࡋ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋ ࡑࡇ࡛ࠊࡑࡢࡼ࠺࡞ࢱࣥࣃࢡ㉁ࡢ࠺ࡕࠊᢠ࣏ࢺ࣮ࢩࢫࢱࣥࣃࢡ㉁࡛࠶ࡿBcl-2Bcl-xLࠊ ࣉ࣏ࣟࢺ࣮ࢩࢫࢱࣥࣃࢡ㉁࡛࠶ࡿBaxࡢࢱࣥࣃࢡࡢⓎ⌧㔞ᑐࡍࡿ9-EE-KODEࡢᙳ㡪 ࢆ᳨ウࡋࡓࠋ9-EE-KODE࡛ྛ㛫ฎ⌮ࡋࡓHRA⣽⬊࡛ࡣࠊBcl-2ࡢⓎ⌧㔞ࡀ⤒ⓗῶ ᑡࠊBaxࡢⓎ⌧㔞ࡀ⤒ⓗቑຍࡋࠊ24㛫ᚋ࠾࠸࡚᭷ព࡞ኚࡀㄆࡵࡽࢀࡓࡀࠊ Bcl-xLⓎ⌧㔞ࡣኚࡀㄆࡵࡽࢀ࡞ࡗࡓ㸦Figure 21㸧ࠋࡲࡓࠊ9-EE-KODE࡛24㛫 ฎ⌮ࡋࡓHRA⣽⬊࡛ࡣࠊBcl-2ࡢⓎ⌧㔞ࡀ⃰ᗘ౫ᏑⓗῶᑡࠊBaxࡢⓎ⌧㔞ࡀቑຍࡋࠊ20 wg/mlࡢ⃰ᗘ࡛᭷ព࡞ኚࡀㄆࡵࡽࢀࡓࡀࠊBcl-xLⓎ⌧㔞ࡣኚࡀㄆࡵࡽࢀ࡞ࡗࡓ 㸦Figure 22㸧ࠋ25
A
Figure 21 Effect of 9-EE-KODE on the expression levels of Bcl-2 subfamily proteins in HRA cells. HRA cells were treated with 9-EE-KODE for 0, 6, 12 and 24 h, and the proteins levels of Bcl-2, Bcl-xL and Bax were evaluated by western blot analysis. (A) Representative photographs of the membrane. (B – D) Area of each band was measured by an image analyzer and the data were exhibited as mean ± S.E. (n = 3). *P < 0.05, **P < 0.01 vs each control (0 μg/ml) group evaluated by Bonferroni/Dunnet’s multiple t-test.
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Figure 22 Effect of 9-EE-KODE on the expression levels of Bcl-2 subfamily proteins in HRA cells.
HRA cells were treated with 9-EE-KODE for 24 h, and the proteins levels of Bcl-2, Bcl-xL and Bax were evaluated by western blot analysis. (A) Representative photographs of the membrane. (B – D) Area of each band was measured by an image analyzer and the data were exhibited as mean ± S.E. (n = 3). *P < 0.05, **P < 0.01 vs each control (0 μg/ml) group evaluated by Bonferroni/Dunnet’s multiple t-test.
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ᮏ◊✲࡛ࡣࠊࢼࢫࡀࡃ∦㒊࢚ࢱࣀ࣮ࣝᢳฟᾮࢆ⏝࠸࡚ 5 ✀㢮ࡢ⒴⣽⬊ᑐࡍࡿ⣽⬊⮴ Ṛάᛶࢆ᳨ウࡋࡓࠋࡑࡢ⤖ᯝࠊࣄࢺ༸ᕢ⒴⏤᮶HRA ⣽⬊ᑐࡋ࡚᭱ࡶ㧗࠸⮴Ṛάᛶࢆ♧ ࡋࡓࠋࢼࢫࡀࡃ∦㒊࢚ࢱࣀ࣮ࣝᢳฟ≀ࡀᑤᙧࢥࣥࢪ࣮࣐ࣟࡢ⒪᭷ຠ࡛࠶ࡗࡓࡇ ࡽ[5]ࠊࢼࢫࡀࡃ∦㒊ᢳฟᾮࡀࡢࡀࢇ⣽⬊ẚ㍑ࡋ࡚ࠊࣄࢺࡢ⏕Ṫჾᐁ⏤᮶ࡢ⒴⣽⬊ ᑐࡍࡿ᭷ຠᛶࢆ᥎ ࡋ⮴Ṛάᛶࡀ᭱ࡶ㧗ࡗࡓࠋ௨ୖࡢࡇࡽࠊࢼࢫࡀࡃ∦㒊ྵࡲ ࢀࡿ⣽⬊⮴Ṛάᛶᡂศࡣࠊᛶ࣍ࣝࣔࣥࡣ↓㛵ಀࠊఱࡽࡢHRA ⣽⬊≉᭷ࡢᶵ⬟ ᛂࡋ࡚⣽⬊Ṛࢆㄏᑟࡍࡿྍ⬟ᛶࡀ㧗࠸⪃࠼ࡽࢀࡓࠋ ࡇࡢᢳฟ≀ࡽάᛶᇶ࡙ࡃศ⏬ࢆ⾜࠺ࡇࡼࡾࠊ2 ࡘࡢ⣽⬊⮴Ṛάᛶᡂศ 9-oxo-(10E, 12Z)-octadecadienoic acid (9-EZ-KODE)࠾ࡼࡧ 9-oxo-(10E, 12E)-octadecadienoic acid (9-EE-KODE)ࢆ༢㞳࣭ྠᐃࡋࡓࠋ9-EE-KODE ࠾ࡼࡧ 9-EZ-KODE ࡢྵ㔞ࢆࢼࢫᯝᐇ ࡢ㒊ูᐃ㔞ࡋࡓࡇࢁࠊྍ㣗㒊ࡼࡾࡶࡀࡃ∦㒊ከࡃྵࡴࡇࢆㄆࡵࡓࡇࡽࠊ ࡑࢀࡽࡢྜ≀ࡣẸ㛫⒪ἲ࡛ࢼࢫࡢྍ㣗㒊࡛ࡣ࡞ࡃࢆ⏝ࡍࡿ⛉Ꮫⓗ᰿ᣐ࡞ࡿࡶࡢ ⪃࠼ࡽࢀࡿࠋࡲࡓࠊྛ✀ࢼࢫࡢᯝᐇࡀࡃ∦㒊࠾ࡅࡿࡑࢀࡽࡢྜ≀ࡢྵ㔞ࡁ࡞ ᕪࡣぢࡽࢀ࡞ࡗࡓࡇࡽࠊẸ㛫⸆ࡋ࡚ࢼࢫࡀࡃ∦㒊ࢆ⏝ࡍࡿࡁࡣ≉ࢼࢫ ࡢ✀㢮ࡣၥࢃ࡞࠸ࡇࡀ᥎ ࡉࢀࡓࠋ9-EE-KODE ࠾ࡼࡧ 9-EZ-KODE ࡣࠊlinoleic acid ࡽ lipoxygenase ࡼࡾ㓟ࡉࢀࡿࡇࡼࡗ࡚⏕ᡂࡉࢀࡿྜ≀⪃࠼ࡽࢀࡿࡀࠊ 9-EE-KODE ࡘ࠸࡚ࡣྠࡌࢼࢫ⛉ᒓࡍࡿࢺ࣐ࢺࡶྵࡲࢀࡿሗ࿌࠸࠺ࡀ࠶ࡿ[7]ࠋࡑ ࢀࡽࡢྜ≀ࡣࠊlinoleic acid ࡀ⮬↛㓟ࡉࢀࡓᚋ࡛ศゎࡉࢀࡿࡇࡼࡾ⏕ᡂࡍࡿྍ ⬟ᛶࡶ࠶ࡿࡀࠊࣜࣀ࣮ࣝ㓟ࢆࣅ࣮࣮࢝ධࢀ࡚࡞ࡋ࡛2 㐌㛫ᨺ⨨ࡋࡓᚋ࡛ HPLC ࡛ ᐃࡋ࡚ࡶࠊࡇࡢ2 ࡘྜ≀ࡣ⏕ᡂࡋ࡞ࡗࡓࠋࡇࡢࡇࡽࠊࡑࢀࡽࡢྜ≀ࡣࢼࢫ⛉ ᳜≀≉᭷ࡢ㓟㓝⣲ࡀ linoleic acid ࡽࡑࢀࡽࡢྜ≀ࢆ⏕ྜᡂࡋࡓ௦ㅰ⏘≀࡛࠶ࡿ ྍ⬟ᛶࡀ㧗࠸⪃࠼ࡽࢀࡓࠋ 9-EE-KODE ࠾ࡼࡧ 9-EZ-KODE ࡣࠊࢼࢫࡀࡃ∦ᢳฟ≀ࡑࢀࡒࢀ 0.03%࠾ࡼࡧ 0.12% ྵࡲࢀࠊࡑࢀࡒࢀࡢHRA ⣽⬊ᑐࡍࡿ IC50ࡣ1.9 ࠾ࡼࡧ 9.7 μg/ml ࡛࠶ࡗࡓࠋࢼࢫࡀࡃ ∦ᢳฟ≀ࡢ⣽⬊⮴ṚάᛶࡢIC50ࡣࠊ0.5 mg/ml ࡛࠶ࡿࡇࡽࠊࡑࢀࡒࢀࡢྜ≀ࡢྵ㔞 ẚάᛶࢆồࡵࡓࡇࢁࠊ9-EE-KODE 9-EZ-KODE ࡣࡑࢀࡒࢀ 0.08%࠾ࡼࡧ 0.1%ࡋ ㈉⊩ࡋ࡚࠸࡞ࡗࡓࠋᚑࡗ࡚ࠊࢼࢫࡀࡃ∦ᢳฟ≀ࡣࡑࡢࡢ⣽⬊⮴Ṛάᛶᡂศࢆከࡃ ྵࡴࡇࡀ᥎ ࡉࢀࡓࠋ ⣽⬊യᐖάᛶ⣽⬊㑅ᢥᛶࡣࠊࡉࡲࡊࡲࡢᅉᏊࡼࡾᙳ㡪ࢆཷࡅࡿࠋྜ≀ഃࡢᅉᏊ ࡋ࡚ࡣࠊྠ୍ศᏊ࠾ࡅࡿぶỈᛶᇶỈᛶᇶࡢඹᏑࡸࠊࢯࣉࣞࢽࣝᇶ࡞Ỉᛶ 㒊ࡢᏑᅾࠊࣁࣟࢤࣥࡢᏑᅾ࡞ࡀᣲࡆࡽࢀࡿࠋ㏆ᖺࡢ◊✲ࡼࡾࠊࢣࢺࣥᇶࡀ࠶ࡿ α,β-unsaturated ketones ࡣཱྀ⭍ෆࡀࢇᑐࡍࡿ㑅ᢥᛶࡀᙉ࠸ࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ>@ࠋࡲ ࡓࠊ⎔≧ α, β 㣬ࢣࢺࣥ㢮ࡢ 3-arylidene-1-(4-nitrophenylmethylene)-3,4-dihydro-1H-28
naphthalen-2-ones ࡣ㠀ᖖ㧗࠸⭘⒆㑅ᢥᛶࢆ♧ࡋ࡚࠸ࡿ㹙㹛ࠋ9-EE-KODE ࡣ HRA ⣽⬊
ᑐࡍࡿ⣽⬊ࡢ㑅ᢥᛶࡀ࠶ࡗࡓ⌮⏤ࡋ࡚ࠊࡑࡢᵓ㐀ෆ࠶ࡿࢣࢺࣥᇶࡢ㛵ࡀ᥎ ࡉࢀ ࡿࠋ
ᮏ◊✲࡛ࡣࠊ9-EE-KODE ࡢ HRA ⣽⬊ᑐࡍࡿ⮴Ṛάᛶࡣࠊᑡ࡞ࡃࡶ࣑ࢺࢥࣥࢻࣜ ෆ㒊⤒㊰ࢆࡍࡿ࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿࡇࢆᶵᗎࡍࡿࡇࢆ᫂ࡽࡋࡓࠋࡋ ࡋࠊ1 μg/ml ࡢ⃰ᗘ࡛ 9-EE-KODE ࢆฎ⌮ࡋࡓ HRA ⣽⬊࡛ࡣࠊ᭷ព࡞ DNA ྜᡂ㔞ࡢప ୗࠊphosphatidylserine ࡢ⣽⬊እ⭷ࡢ㟢ฟࠊcaspase-3 άᛶࡢಁ㐍ࡀㄆࡵࡽࢀࡓࡀࠊ࣑ࢺ ࢥࣥࢻࣜ⭷㟁ᕪࡢஹ㐍ࠊcytochrome c ࡢ⣽⬊㉁ࡢᨺฟࠊBcl-2 Ⓨ⌧㔞ࡢపୗ Bax Ⓨ⌧㔞ࡢቑຍࡣࠊ1 μg/ml ࡢ⃰ᗘ࡛ࡣ᭷ព࡞ኚࡣㄆࡵࡽࢀࡎࠊ10ࠥ20 μg/ml ௨ୖࡢ⃰ᗘ ࡀᚲせ࡛࠶ࡗࡓࠋ9-EE-KODE ࡣồ㟁Ꮚᛂࡋࡸࡍ࠸Ꮫᵓ㐀ࢆᣢࡘࡢ࡛ࠊ⣽⬊ෆ࠶ࡿ ࢱࣥࣃࢡ㉁ᛂࡋࡸࡍࡃࠊࡲࡓࣛࢪ࢝ࣝࢆྵࡴ௦ㅰ⏘≀ࢆᙧᡂࡋࡸࡍ࠸⪃࠼ࡽࢀࡿࠋ ࡇࡢࡇࡽࠊ9-EE-KODE ࡢ HRA ⣽⬊ᑐࡍࡿ࣏ࢺ࣮ࢩࢫࡢㄏᑟࡣ࣑ࢺࢥࣥࢻࣜ ෆ㒊⤒㊰ࡔࡅ࡛࡞ࡃࠊ9-EE-KODE ⮬㌟࠶ࡿ࠸ࡣࡢࢱࣥࣃࢡ㉁⤖ྜࡋࡓ≧ែ࡛ᑠ⬊య ࢫࢺࣞࢫ࡞ࢆㄏᑟࡋࠊࡢ⤒㊰ࢆࡍࡿࡇࡶ༑ศ᥎ ࡉࢀࡓࠋ ࢼࢫࡀࡃ∦㒊ࡢẸ㛫⒪ἲ࠾ࡅࡿ⛉Ꮫⓗ࡞࢚ࣅࢹࣥࢫࡸᢠ⒴㣗≀ࡋ࡚㛤Ⓨࡍࡿࡓࡵ ࡣࠊᮏ◊✲ࡢࡼ࠺࡞ in vitro ࡢᐇ㦂ࡔࡅ࡛ࡣ࡞ࡃ in vivo ᐇ㦂ࡶ⾜࠺ࡇࡀồࡵࡽࢀࡿࠋ ᮏ◊✲࡛ᚓࡽࢀࡓ▱ぢᇶ࡙ࡁ㸪ඹᙺࣜࣀ࣮ࣝ㓟ྜ≀ࢆ࣮ࣜࢻྜ≀ࡋࡓ᪂つ⏕≀ άᛶ≀㉁ࡢ〇㸪9-EE-KODE ࡢᶆⓗศᏊࡢゎ᫂࡞ࡀᮇᚅࡉࢀࡿࠋ
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ᐇ㦂ࡢ㒊
➨୍❶
࠙⣽⬊ࠚKB (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 1640 ᇵᆅ㸦ࢼ࢝ࣛ㸧࡛ࠊ5% CO2ୗ37Υ࡛ᇵ 㣴ࡋࡓࠋ ⥅௦ࡣࠊ0.25%ࢺࣜࣉࢩࣥ in 0.53 mM EDTA ⁐ᾮ㸦ࢼ࢝ࣛ㸧࡛⣽⬊ࢆ 3 ศ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 h ᇵ㣴ࡋࡓࠋࡑࡢᚋࠊDMSO ࡛ 10 mg/mlࠊ1 mg/mlࠊ100 μg/mlࠊ10 μg/ml ㄪ〇ࡋࡓࢧࣥࣉࣝࢆ2 μl ࡎࡘῧຍࡋ㸦᭱⤊⃰ᗘ㸸100ࠊ10ࠊ1ࠊ0.1 μg/ml㸧ࠊࡉࡽ 72 h ᇵ㣴ࡋࡓࠋࢥࣥࢺ࣮ࣟࣝࡣDMSO ࢆࠊ㝧ᛶࢥࣥࢺ࣮ࣟࣝࡋ࡚ adriamycin㸦༠Ⓨ㓝࣭ ࣇࢨ࣮ࠊᮾி㸧ࢆ㝶ᕼ㔘ࡋ࡚⏝ࡋࡓࠋMTT㸦ࢼ࢝ࣛ㸧ࢆ 3 mg/ml ࡢ⃰ᗘ࡛ 20 μl ࡎࡘῧຍࡋࠊࡉࡽ 4 h ᇵ㣴ࡋࡓࠋᇵᆅࢆ㝖ཤᚋࠊDMSO 170 μl ࢆῧຍࡋ࡚⏕ᡂࡋࡓ formazan ࢆ⁐ゎࡋࠊ570 nm ࡛ࡢ྾ගᗘࢆ ARVO MX 1420 multilabel counter (Perkin Elmerࠊ Winter Street WalthamࠊMAࠊUSA)ࠊࡲࡓࡣ iMark Microplate Reader (Bio-RadࠊPhiladelphiaࠊ PaࠊUSA) ࡼࡾ ᐃࡋࡓࠋྛ⸆≀ࡢ⃰ᗘ࠾ࡅࡿ⣽⬊ቑṪᢚไ⋡ (%)ࢆ྾ගᗘࡼࡾ⟬ ฟࡋࠊ50%ࢆྵࡴ 3 ࡘࡢ␗࡞ࡿ⃰ᗘࡢᑐᩘᑐࡍࡿ⣽⬊⏕Ꮡ⋡ࢆࣉࣟࢵࢺࡋࡓࡁࡢ㏆ ఝ┤⥺ࢆ⏝࠸ࠊ50%ࢆ♧ࡍ⃰ᗘࢆ IC50ࡋࡓࠋ ࠙ࢼࢫࡀࡃ∦࢚࢟ࢫࡢㄪ〇ศ⏬ࠚ ᐇ㦂⏝ࡋࡓࢼࢫᯝᐇࡣࠊྡྂᒇᕷෆࡢᕷሙ࡛㉎ධࡋࡓࠋ᪂㩭࡞༸ᙧࢼࢫᯝᐇ14 kg ࢆࠊࡀࡃ∦㒊ศ2.8 kg ࡑࢀ௨እࡢ㒊ศศࡅࠊࡀࡃ∦㒊ศࡣ⤖⇱ࡋࠊ⤖⇱≀ 2.6 x 102 g ࢆᚓࡓࠋࡇࢀ࢚ࢱࣀ࣮ࣝ 2 l ࢆຍ࠼୍࡚ᬌ෭ᾐᢳฟࢆ 3 ᅇ⾜࠸ࠊᢳฟᾮࢆྜ ࢃࡏ࡚⃰⦰ᅛࡍࡿࡇࡼࡾࠊᢳฟ࢚࢟ࢫ7.6 g ࢆᚓࡓࠋࡇࢀࢆ 80㸣࣓ࢱࣀ࣮ࣝ 3.6 l ᠱ⃮ࡋࠊ࣊࢟ࢧࣥ1.2 l ࡛ 3 ᅇ࡛ศ㓄ࡋࠊࡑࢀࡒࢀࡢ⏬ศࢆ⃰⦰ᅛࡋࡓࠋḟ 80%࣓
30 ࢱࣀ࣮ࣝ⏬ศࢆỈ3.6 l ᠱ⃮ࡋࠊ㓑㓟࢚ࢳࣝ 1.2 l ࡛ 3 ᅇศ㓄ࡋࠊࡉࡽỈ⏬ศࢆỈ 1.2 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 g ࢆࠊ࢚ࢱࣀ࣮ࣝ 2 l ࡛ྠᵝᢳฟࡋࠊᢳฟ࢚ ࢟ࢫ12.5 g ࢆᚓࡓࠋ ࡑࢀࡒࢀࡢࢼࢫࡀࡃ∦࢚ࢱࣀ࣮ࣝᢳฟ࢚࢟ࢫ࠾ࡼࡧࡑࡢྛ⏬ศࡣࠊ10 mg/ml ࡞ࡿࡼ ࠺DMSO ⁐ゎࡋࠊ–20Υ࡛ಖᏑࡋࡓࠋ 㓑㓟࢚ࢳࣝ⏬ศ1.3 g ࢆࠊࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࣮㸦ࢩࣜ࢝ࢤࣝ 60Nࠊ㛵 ᮾᏛࠊᮾிࠊ4 cm × 20 cm㸧౪ࡋࠊ࣓ࢱࣀ࣮ࣝ㸸ࢡ࣒ࣟࣟ࣍ࣝ㸸Ỉ㸦2 : 8 : 0.1㸧[13] ࡛⁐ฟࡉࡏࡓࠋ࣒࢝ࣛࡢ୰㢧ⴭⓗ࡞ᅄࡘࡢࣂࣥࢻࢆྲྀࡗ࡚ࠊFr. 1ࠥFr. 4 ࢆᚓࡓ㸦ࡑࢀ ࡒࢀ7.2 mgࠊ683 mgࠊ142 mgࠊ258 mg㸧ࠋFr. 2㸦683mg㸧ࢆ ODS ࢩࣜ࢝ࢤࣝࢡ࣐ࣟࢺࢢ ࣛࣇ࣮㸦ࢥࢫࣔࢩࣝ140C18-OPNࠊࢼ࢝ࣛࠊ4 cm × 20 cm㸧౪ࡋࠊ࣓ࢱࣀ࣮ࣝ㸭Ỉ 㸦1:1ࠊ4:1ࠊ1:0ࠊ300 ml ࡎࡘẁ㝵ⓗ㸧࡛⁐ฟࡉࡏࡓࠋFr. 2-1ࠥ2-3㸦ࡑࢀࡒࢀ 225.1 gࠊ48 mgࠊ 176 mg㸧ࢆᚓࡓࠋFr. 2-2㸦48 mg㸧ࢆศྲྀ HPLC㸦ࢥࢫࣔࢩࣝ C18-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 nm ࡢ UV ྾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
4 ࡢᏛᵓ㐀ࡣࠊNMR㸦Bruker ABANCE 600ࠊMAࠊUSA㸧 EI-MS㸦᪥ᮏ㟁Ꮚࠊᮾி)
ࢆ ᐃࡋࡓࠋ
Compound 3 4 ࡢࢼࢫᢳฟᾮ୰ࡢྵ㔞ࡢ ᐃࡣࠊ௨ୗࡢ᮲௳ࡢ HPLC ࡼࡾ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 g ࢆᐇ㦂ᮦᩱࡋ࡚ࠊ㓑㓟࢚ࢳࣝ 10 ml ࢆຍ࠼࡚ min ࡢࢯࢽࢣ࣮ࢩࣙࣥࡼ ࡿᢳฟࢆ3 ᅇ⾜࠸ࠊᢳฟᾮࢆྜࢃࡏ࡚⃰⦰ᅛࡍࡿࡇࡼࡾࠊྛᢳฟ࢚࢟ࢫࢆᚓࡓࠋ ࡚ࡢᢳฟ࢚࢟ࢫࢆ࣓ࢱࣀ࣮࡛ࣝ10 mg/ml ㄪ〇ࡋࡓࠋࡑࡢᚋࠊྛ⃰ᗘ㸦6.25ࠊ12.5ࠊ 25ࠊ50 μg/ml㸧ࡢ 9-EE-KODE ࢆ⏝࠸᳨࡚㔞⥺ࢆసᡂࡋࡓࠋࡑࢀࡒࢀࡢࢼࢫࡢྛ㒊ࡢᢳ ฟ࢚࢟ࢫࢆHPLC ࡛ 9-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
࠙ࢾࢡࣞ࢜ࢯ࣮࣒ࡼࡿDNA ᩿∦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 ⣽⬊ࢆ 1.5 × 104 cells/well ࡢ HRA ⣽⬊ࢆ✀ࡋࠊ6 h ᇵ 㣴ࡋࡓᚋࠊྛ⃰ᗘࡢ9-EE-KODE ࢆῧຍࡋࡓࠋ24 h ᇵ㣴ᚋࠊPBS ࡛Ὑίࡋࠊ⣽⬊ࢿ ࢟ࢩࣥV-ࣇࣝ࢜ࣞࢭࣜࣥ㸦Medical & Biological Laboratoriesࠊྡྂᒇ㸧ࢆ 10 μl ῧຍࡋࡓࠋ ᬯᡤᐊ ࡛15 min ࣮ࣥ࢟ࣗ࣋ࢺࡋࡓᚋࠊPBS ࡛Ὑίࡋࠊ⺯ග㢧ᚤ㙾࡛ほᐹࡋࡓࠋ
࠙Caspase-3/-7 ࡢ ᐃࠚ
96 ✰ࣉ࣮ࣞࢺ HRA ⣽⬊ࢆ 2 × 104 cells/well ✀ࡋࠊ24 h ᇵ㣴ᚋࠊ9-EE-KODE ࢆῧ ຍࡋ࡚24 h ᇵ㣴ࡋࡓࠋᇵᆅࢆ྾ᘬ㝖ཤᚋࠊCaspase-3/7 reagent㸦PromegaࠊMadisonࠊWIࠊ USA㸧ࢆ 100 μl ῧຍࡋ࡚ᐊ ࡛ 1 h ᇵ㣴ࡋࡓᚋࠊບ㉳Ἴ㛗 498 nmࠊ྾Ἴ㛗 521 nm ࡢ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 c ࡢ ᐃࠚ
6 ✰ࣉ࣮ࣞࢺ HRA ⣽⬊ࢆ 1.5 × 104 cells/well ✀ࡋ࡚ 24 h ᇵ㣴ࡋࠊ9-EE-KODE ࡛ 24 h ᇵ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
34
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36 ㅰ ㅰ㎡ ᮏ◊✲ࢆ㐙⾜ࡍࡿᙜࡓࡗ࡚ࠊ⤊ጞᚚᣦᑟࠊᚚ㠴᧡ࢆ㈷ࡾࡲࡋࡓ㧗▱┴❧∾㔝᳜≀ᅬ Ỉୖ ඖ ᅬ㛗㸦ྡྂᒇᕷ❧Ꮫྡᩍᤵ㸧ᚰࡼࡾ῝ㅰ࠸ࡓࡋࡲࡍࠋ ᮏㄽᩥࢆᇳ➹ࡍࡿᙜࡓࡗ࡚ࠊᚚᣦᑟࢆ㈷ࡾࡲࡋࡓྡྂᒇᕷ❧ᏛᏛ㝔⸆Ꮫ◊✲⛉⏕ ⸆Ꮫศ㔝 ∾㔝 ᫂ ᩍᤵ῝ㅰ࠸ࡓࡋࡲࡍࠋ ᮏㄽᩥࢆⓎ⾲ࡍࡿᙜࡓࡗ࡚ࠊᚚᣦᑟࢆ㈷ࡾࡲࡋࡓྡྂᒇᕷ❧ᏛᏛ㝔⸆Ꮫ◊✲⛉⏕ ⸆Ꮫศ㔝 ᑎᆏ ⚈ ㅮᖌ῝ㅰ࠸ࡓࡋࡲࡍࠋ ᮏ◊✲ࢆ㐙⾜ࡍࡿᙜࡓࡗ࡚ࠊ⤊ጞ᭷┈࡞ᚚຓゝࠊᚚᣦᑟࢆ㈷ࡾࡲࡋࡓྡྂᒇᏛྡ ᩍᤵ㸦⏘⛉፬ே⛉Ꮫ㸧ࠊ⏣ࢡࣜࢽࢵࢡ㝔㛗 ⏣ ㇏ ༤ኈࠊྡྂᒇᏛᏛ㝔་Ꮫ◊ ✲⛉ ྜྷᕝ ྐ㝯 ᩍᤵࠊᰘ⏣ Ύ ᩍᤵࠊឡ▱Ꮫ㝔Ꮫ⸆Ꮫ㒊 ⸆⏝㈨※Ꮫㅮᗙ ⏣㑓 ᏹ ᶞ ㅮᖌࠊࢺࣚࢱグᛕ㝔 ᱜ 㝧ᖹ ಟኈཌࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ ᮏ◊✲ࢆ㐙⾜ࡍࡿᙜࡓࡗ࡚ࠊᵝࠎ࡞ᚚຓゝࠊᐇ㦂ࡢ୍㒊ࢆ༠ຊࡋ࡚࠸ࡓࡔࡁࡲࡋࡓྡ ྂᒇᕷ❧ᏛᏛ㝔⸆Ꮫ◊✲⛉⏕⸆Ꮫศ㔝 Ლ⏣ ᜨ ಟኈࠊ༡ Ᏻ౫㔛 ಟኈཌࡃᚚ♩ ⏦ࡋୖࡆࡲࡍࠋ ᮏ◊✲ࢆ㐙⾜ࡍࡿᙜࡓࡗ࡚ࠊ⤊ጞ᭷┈࡞ᚚຓゝࠊᚚ༠ຊࢆࡋ࡚㡬ࡁࡲࡋࡓྡྂᒇᕷ❧ ᏛᏛ㝔⸆Ꮫ◊✲⛉⏕⸆Ꮫศ㔝ࡢⓙᵝ῝ࡃឤㅰ࠸ࡓࡋࡲࡍࠋ
