Title 慢性骨髄性白血病細胞に対する中鎖脂肪酸誘導体の抗がん作用とエネルギー代謝( 本文(Fulltext) ) Author(s) 篠原, 悠 Report No.(Doctoral Degree) 博士(薬科学) 連創博甲第30号 Issue Date 2016-03-25 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/54543 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
៏ᛶ㦵㧊ᛶⓑ⾑⣽⬊ᑐࡍࡿ
୰㙐⬡⫫㓟ㄏᑟయࡢᢠࡀࢇస⏝࢚ࢿࣝࢠ࣮௦ㅰ
Anti-cancer effects of medium-chain fatty-acid derivative through perturbation of energy metabolism in chronic myeloid leukemia
2016
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➨ 1 ❶ ⥴ゝ 1 ➨ 2 ❶ ◊✲ࡢ⫼ᬒ┠ⓗ ➨ 1 ⠇ ៏ᛶ㦵㧊ᛶⓑ⾑ࡢⓎᶵᵓ 4 ➨ 2 ⠇ ៏ᛶ㦵㧊ᛶⓑ⾑⒪ࡢኚ㑄ၥ㢟Ⅼ 6 ➨ 3 ⠇ ࡀࢇ⣽⬊≉␗ⓗ࢚ࢿࣝࢠ࣮௦ㅰᶵᵓ: Warburg ຠᯝ 10 ➨ 4 ⠇ ࡀࢇ⣽⬊࠾ࡅࡿ⬡⫫㓟㓟ࡢᙺ 13 ➨ 5 ⠇ ୰㙐⬡⫫㓟ࡢ⏕⌮άᛶ 15 ➨ 3 ❶ ៏ᛶ㦵㧊ᛶⓑ⾑⣽⬊ᑐࡍࡿ୰㙐⬡⫫㓟ㄏᑟయࡢቑṪᢚไస⏝ ➨ 1 ⠇ ᗎ 17 ➨ 2 ⠇ ៏ᛶ㦵㧊ᛶⓑ⾑⣽⬊ᰴᑐࡍࡿ୰㙐⬡⫫㓟ㄏᑟయࡢ 18 ቑṪᢚไຠᯝ ➨ 3 ⠇ ୰㙐⬡⫫㓟ㄏᑟయࡼࡿ࣮࢜ࢺࣇࢪ࣮⣽⬊Ṛࡢㄏᑟ 19 ➨ 4 ⠇ BCR-ABL ࡢⓎ⌧ᑐࡍࡿస⏝ 23 ➨ 5 ⠇ ࡀࢇ⣽⬊ࡢ࢚ࢿࣝࢠ࣮௦ㅰᑐࡍࡿస⏝ 25 ➨ 6 ⠇ ୰㙐⬡⫫㓟ㄏᑟయࡢᶆⓗศᏊࡢ᥈⣴ 29 ➨ 7 ⠇ ᑠᣓ 31➨ 4 ❶ ࡀࢇ⣽⬊ࡢ࢚ࢿࣝࢠ࣮௦ㅰᑐࡍࡿ୰㙐⬡⫫㓟ㄏᑟయ࣐ࢳࢽࣈ ࡢẚ㍑ ➨ 1 ⠇ ᗎ 33 ➨ 2 ⠇ ୰㙐⬡⫫㓟ㄏᑟయ࠾ࡼࡧ࣐ࢳࢽࣈࡼࡿ⣽⬊Ṛࡢẚ㍑ 33 ➨ 3 ⠇ BCR-ABL ゎ⢾⣔ᑐࡍࡿస⏝ 36 ➨ 4 ⠇ ⬡⫫㓟㓟ᑐࡍࡿస⏝ 38 ➨ 5 ⠇ ⓑ⾑ᖿ⣽⬊࠾ࡅࡿ AIC-47 ࡢ᭷ຠᛶ 42 ➨ 6 ⠇ ᑠᣓ 44 ➨ 5 ❶ miR-124/PTBP1 ࢝ࢫࢣ࣮ࢻࡼࡿࡀࢇ⣽⬊ࡢ࢚ࢿࣝࢠ࣮௦ㅰไᚚ ➨ 1 ⠇ ᗎ 47 ➨ 2 ⠇ miR-124 ࡢ⤌⧊ศᕸ 47 ➨ 3 ⠇ ୰㙐⬡⫫㓟ㄏᑟయ࠾ࡼࡧ࣐ࢳࢽࣈࡼࡿ miR-124 ࡢ Ⓨ⌧ኚ 48 ➨ 4 ⠇ ࡀࢇࡢⓎ㐣⛬࠾ࡅࡿ miR-124 ࡢⓎ⌧ኚ 49 ➨ 5 ⠇ miR-124 ࡼࡿࡀࢇ⣽⬊ࡢ࢚ࢿࣝࢠ࣮௦ㅰไᚚ 51 ➨ 6 ⠇ ᑠᣓ 54 ➨ 6 ❶ ⥲ᣓ 56 ⤖ㄒ 60 ㅰ㎡ 61
ᐇ㦂ࡢ㒊 62 ➨ 3 ❶㛵ࡍࡿᐇ㦂 69 ➨ 4 ❶㛵ࡍࡿᐇ㦂 72 ➨ 5 ❶㛵ࡍࡿᐇ㦂 76 ཧ⪃ᩥ⊩ 78 ◊✲ᴗ⦼┠㘓 86
1
➨ 1 ❶ ⥴ゝ
ࣄࢺࢤࣀ࣒ィ⏬⤊ᚋࠊᵝࠎ࡞ᝈࡢཎᅉ࡞ࡿ㑇ఏᏊࡢሷᇶ㓄ิࡀ᫂ࡽ ࡞ࡾࠊࡀࢇᑐࡍࡿ⸆ࡣⓎࡀࢇ㛵ࢃࡿࢻࣛࣂ࣮㑇ఏᏊ⏘≀ᑐࡍࡿศ Ꮚᶆⓗ⒪⸆ࡀࢺࣞࣥࢻ࡞ࡗࡓࠋ᭱ࡶᡂຌࡋࡓศᏊᶆⓗ⒪⸆ࡢ 1 ࡘࡀ៏ᛶ 㦵㧊ᛶⓑ⾑ (chronic myeloid leukemia; CML) ᑐࡍࡿ࣐ࢳࢽࣈ࡛࠶ࡿࠋ CML ࡣ t(9; 22) ㌿ᗙ⏤᮶ࡢ࣓࢟ࣛ㑇ఏᏊ BCR-ABL ࢆཎᅉ㑇ఏᏊࡍࡿ㐀⾑ჾ⭘
⒆࡛࠶ࡾࠊBCR-ABL ࡢ⩻ヂࢱࣥࣃࢡ㉁ࡀ♧ࡍᜏᖖⓗ࡞ࢳࣟࢩࣥ࢟ࢼ࣮ࢮάᛶ ࡼࡾࠊࡀࢇࡋ࡚ࡢᙧ㉁ࢆ⋓ᚓࡍࡿࠋ࣐ࢳࢽࣈࢆጞࡵࡋࡓࢳࣟࢩࣥ࢟ࢼ࣮ ࢮ㜼ᐖ (TKI) ࡣ BCR-ABL ࡢ ATP ⤖ྜ㒊ࢆ㑅ᢥⓗ㜼ᐖࡍࡿࡇ࡛ୗὶࡢ ࢩࢢࢼࣝࢆάࡋඃࢀࡓቑṪᢚไస⏝ࢆ♧ࡍࠋTKI ࡢᑟධࡼࡗ࡚ CML ࡢ ⒪ᡂ⦼ࡣ㣕㌍ⓗྥୖࡋࡓࡀࠊTKI ᑐࡍࡿ⪏ᛶࡢ⋓ᚓࡸṧᏑࡋࡓⓑ⾑ᖿ⣽⬊ ࡼࡿⓎࡀၥ㢟࡞ࡗ࡚࠾ࡾࠊTKI ༢ࡼࡿ CML ࡢ᰿ࡣ㞴ࡋ࠸ࠋࡑࡢࡓ ࡵࠊTKI 㠀ឤཷᛶ⣽⬊ᑐࡋ࡚ࡶ᭷ຠ࡞⒪⸆ࡢ㛤Ⓨࡀᮃࡲࢀ࡚࠸ࡿࠋ ศᏊᶆⓗ⒪⸆ࡣࢻࣛࣂ࣮㑇ఏᏊ⏘≀ࡢࢱࣥࣃࢡ㉁❧యᵓ㐀౫Ꮡࡋ࡚స ⏝ࡍࡿࡇࡽࠊኚ␗ࡼࡿ⪏ᛶࡢ⋓ᚓࡀ㧗㢖ᗘ࡛⏕ࡌࡿࠋࡲࡓࠊⓎࡢཎᅉ ࡞ࡿᖿ⣽⬊ࡣࠊ࣊ࢸࣟ࡞㑇ఏᏊⓎ⌧ࢆ᭷ࡋࢻࣛࣂ࣮㑇ఏᏊ㠀౫Ꮡⓗ⏕Ꮡ ࡋ࡚࠸ࡿࡓࡵศᏊᶆⓗ⒪⸆ᑐࡍࡿឤཷᛶࡀప࠸ࠋࡇࢀࡽࡢࡇࡽࠊTKI 㠀ឤཷᛶ⣽⬊ࡢ⒪ࡣᚑ᮶ࡢᢠࡀࢇࡣ␗࡞ࡿస⏝Ⅼࢆᣢࡘ⸆ࡀᚲせ࡛ ࠶ࡿ⪃࠼ࡽࢀࡿࠋ㏆ᖺࠊ᪂ࡓ࡞⸆ࡢࢱ࣮ࢤࢵࢺࡋ࡚ὀ┠ࡉࢀ࡚࠸ࡿࡢࡀࠊ ࡀࢇ⣽⬊ࡢ࢚ࢿࣝࢠ࣮௦ㅰ࡛࠶ࡿࠋࡀࢇ⣽⬊ࡣప㓟⣲࣭పᰤ㣴⎔ቃ࡛⏕Ꮡࡍࡿ ࡓࡵ௦ㅰࡢࣜࣉࣟࢢ࣑ࣛࣥࢢࢆ⾜ࡗ࡚࠸ࡿࠋࡑࡢ௦ㅰᶵᵓࢆࢱ࣮ࢤࢵࢺࡍ ࡿ⒪ἲࡀⓑ⾑⣽⬊ࡢࡳ࡞ࡽࡎⓑ⾑ᖿ⣽⬊ࡶ᭷ຠ࡛࠶ࡿࡇࡀ᫂ࡽ ࡉࢀࡘࡘ࠶ࡿࠋ ᮏ◊✲࡛ࡣࠊCML ᑐࡍࡿ᪂つ⒪⸆ࡢ⸆ࢩ࣮ࢬࡋ࡚୰㙐⬡⫫㓟ㄏᑟయ
2 ࡢ᭷⏝ᛶࢆホ౯ࡋࡓࠋࡇࢀࡲ࡛ࡢ◊✲࡛ࡣ㛗㙐࠾ࡼࡧ▷㙐⬡⫫㓟↔Ⅼࡀᙜ࡚ ࡽࢀ࡚࠾ࡾࠊࡇࢀࡽࡢ⬡⫫㓟㛵ࡋ࡚ࡣᢠࡀࢇάᛶࢆጞࡵࡋ࡚ᵝࠎ࡞▱ぢࡀ ᚓࡽࢀ࡚࠸ࡿࠋࡋࡋ࡞ࡀࡽࠊࣄࢺ࠾ࡅࡿ୰㙐⬡⫫㓟ࡢ⏕⌮άᛶࡣࡇࢀࡲ࡛ ࢇሗ࿌ࡉࢀ࡚࠾ࡽࡎࠊᢠࡀࢇࡢࢩ࣮ࢬࡋ࡚ࡶὀ┠ࡉࢀ࡚ࡇ࡞ࡗࡓࠋ ᮏ◊✲࡛ࡣࠊ୰㙐⬡⫫㓟ㄏᑟయࡢᢠࡀࢇάᛶࢆホ౯ࡋࠊࡑࡢస⏝࣓࢝ࢽࢬ࣒ࢆ ࢚ࢿࣝࢠ࣮௦ㅰ࠸࠺ほⅬࡽゎ᫂ࡋࡓ (➨ 3 ❶)ࠋ⥆࠸࡚࣐ࢳࢽࣈ୰㙐⬡ ⫫㓟ㄏᑟయࡢస⏝Ⅼࡢ㐪࠸ࢆ᳨ドࡋࠊ୰㙐⬡⫫㓟ㄏᑟయࡢࢻࣛࣂ࣮㑇ఏᏊ ⏘≀㠀౫Ꮡⓗ࡞స⏝Ⅼࢆ᫂ࡽࡋࡓ (➨ 4 ❶)ࠋࡉࡽࠊࡀࢇ⣽⬊≉␗ⓗ࡞࢚ ࢿࣝࢠ࣮௦ㅰᶵᵓࡢ⋓ᚓࡀࢇࡢ㐣⛬ࡢ㛵㐃ᛶࡘ࠸᳨࡚ウࢆຍ࠼ࠊ࢚ࢿ ࣝࢠ࣮௦ㅰࡀࡀࢇ⒪ࡢ᭷⏝࡞ࢱ࣮ࢤࢵࢺ࡞ࡿࡇࢆ♧ࡋࡓ (➨ 5 ❶)ࠋ
3
ᮏᏛㄽᩥࡣࠊୗグࡢཎⴭㄽᩥࢆࡶసᡂࡋᒱ㜧ᏛᏛ㝔㐃ྜ⸆་⒪ ሗ◊✲⛉ᥦฟࡋࡓࡶࡢ࡛࠶ࡿࠋ
(1) Perturbation of energy metabolism by fatty-acid derivative AIC-47 and imatinib in BCR-ABL-harboring leukemic cells.
Haruka Shinohara, Minami Kumazaki, Yosuke Minami, Yuko Ito, Nobuhiko Sugito, Yuki Kuranaga, Kohei Taniguchi, Nami Yamada, Yoshinori Otsuki, Tomoki Naoe, Yukihiro Akao.
Cancer Letters; 371 (1): 1-11 (2016).
(2) Anti-cancer fatty-acid derivative induces autophagic cell death through modulation of PKM isoform expression profile mediated by bcr-abl in chronic myeloid leukemia.
Haruka Shinohara, Kohei Taniguchi, Minami Kumazaki, Nami Yamada, Yuko Ito, Yoshinori Otsuki, Bunji Uno, Fumihiko Hayakawa, Yosuke Minami, Tomoki Naoe, Yukihiro Akao.
Cancer Letters; 360 (1): 28-38 (2015)
(3) MicroRNA-124 inhibits cancer cell growth through PTB1/PKM1/PKM2 feedback cascade in colorectal cancer.
Kohei Taniguchi, Nobuhiko Sugito, Minami Kumazaki, Haruka Shinohara, Nami Yamada, Yoshihito Nakagawa, Yuko Ito, Yoshinori Otsuki, Bunji Uno, Kazuhisa Uchiyama, Yukihiro Akao.
4 ➨ 2 ❶ ◊✲ࡢ⫼ᬒ┠ⓗ ➨ 1 ⠇ ៏ᛶ㦵㧊ᛶⓑ⾑ࡢⓎᶵᵓ CML ࡣከ⬟ᛶ㐀⾑ᖿ⣽⬊ࡢ⭘⒆ࡼࡗ࡚Ⓨࡍࡿᝈ࡛࠶ࡾࠊt(9; 22) ㌿ᗙ ࡼࡾᙧᡂࡉࢀࡿࣇࣛࢹࣝࣇ (Philadelphia; Ph) ᰁⰍయ ࢆ≉ᚩࡍࡿ1,2)ࠋ ࡇࡢ㌿ᗙᰁⰍయୖᙧᡂࡉࢀࡿ࣓࢟ࣛ㑇ఏᏊ BCR-ABL ࡣࠊCML ࡢࢻࣛࣂ࣮㑇 ఏᏊࡋ࡚ᶵ⬟ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ (Fig. 1A)3)ࠋࡲࡓࠊከࡃࡢᅛᙧ⭘⒆ࡣ 」ᩘࡢ㑇ఏᏊከẁ㝵ⓗኚ␗ࡀ⏕ࡌࡿࡇ࡛ⓎࡍࡿࡢᑐࡋࠊCML ࡣ 1 ࡘ ࡢ㑇ఏᏊኚ␗ BCR-ABL ࡢࡳ࡛Ⓨࡍࡿࡇࡀ᫂ࡽࡉࢀ࡚࠸ࡿ4)ࠋBCR-ABL ࡢ㌿⏘≀࠾ࡼࡧ⩻ヂࢱࣥࣃࢡ㉁ࡣࠊABL 㑇ఏᏊࡢࡶࡢࡣࡃ␗࡞ࡿᛶ㉁ࢆ ♧ࡍ5)ࠋABL ࡀ⮬ᕫㄪ⠇ᛶࡢࣜࣥ㓟⬟ࢆ᭷ࡍࡿࡢᑐࡋࠊBCR-ABL ࡣ BCR ࡢࡶࡘ㔜ྜ⬟ࡼࡾ㔞యࡶࡋࡃࡣᅄ㔞యࢆᙧᡂࡋ࡚ศᏊ㛫࡛ࡢࣜࣥ㓟ࡀྍ ⬟࡞ࡿࡓࡵࠊᜏᖖⓗ࡞ࢳࣟࢩࣥ࢟ࢼ࣮ࢮάᛶࢆ♧ࡍ6)ࠋࡇࡢ࢟ࢼ࣮ࢮάᛶࡼ ࡾ⣽⬊ቑṪࠊ⣽⬊࿘ᮇࡢஹ㐍ࠊࣉࣟࢢ࣒ࣛ⣽⬊Ṛࡢ⪏ᛶ㛵ࢃࡿᵝࠎ࡞ࢩࢢ ࢼࣝࡀάᛶࡉࢀࠊⓑ⾑⣽⬊ࡢ␗ᖖ࡞ቑṪࡀྍ⬟࡞ࡿ (Fig. 1B)1,7,8)ࠋ
5
Fig. 1 Pathogenic mechanism of chronic myeloid leukemia (CML)
6
➨ 2 ⠇ ៏ᛶ㦵㧊ᛶⓑ⾑⒪ࡢኚ㑄ၥ㢟Ⅼ
CML ࡣ WHO ศ㢮ࡢᇶ‽ࡼࡾᐃ⩏ࡉࢀࡓ 3 ࡘࡢᮇࠊࡍ࡞ࢃࡕⓑ⾑ࡸ⾑
ᑠᯈࡢቑຍࢆㄆࡵࡿࡀ⮬ぬ≧ࡢஈࡋ࠸៏ᛶᮇ (chronic phase: CP)ࠊ㢛⢏⌫ࡢศ ␗ᖖࡀ㐍⾜ࡍࡿ⛣⾜ᮇ (accelerated phase: AP)ࠊᮍศ࡞ⱆ⌫ࡀቑຍࡍࡿᛴᛶ ㌿ᮇ (blast crisis phase: BP) ࢆ⤒࡚㐍⾜ࡍࡿ9)ࠋ⣙ 85%ࡢᝈ⪅ࡀ CP ᮇ࡛デ᩿ࡉ
ࢀࡿࡇࡽࠊCML ⒪ࡣ Ph+⣽⬊ࡢࢥࣥࢺ࣮ࣟࣝᮇ㐍⾜ࡢᅇ㑊ࢆ┠ᶆ ࡋ࡚⾜ࢃࢀࡿ10)ࠋࡇࢀࡲ࡛ࡢ CML ⒪࡛ࡣࠊࣈࢫࣝࣇࣥࡸࣁࢻࣟ࢟ࢩ࢘ࣞ ࡼࡿᏛ⒪ἲࠊࣥࢱ࣮ࣇ࢙ࣟࣥĮ ,)1Į ⒪ἲࡀ⾜ࢃࢀ࡚ࡁࡓࠋᏛ⒪ἲ ࡛ࡣ Ph+⣽⬊ᩘࡢࢥࣥࢺ࣮ࣟࣝࡣྍ⬟࡛࠶ࡗࡓࡀࠊ5 ᖺ⏕Ꮡ⋡ࡣ 38%ࠊ10 ᖺ⏕Ꮡ ⋡ࡣ 17%࡛࠶ࡾ⏕Ꮡᮇ㛫ࡢᘏ㛗ࡣᅔ㞴࡛࠶ࡗࡓࠋ⥆࠸࡚Ⓩሙࡋࡓ IFNĮ ⒪ἲ࡛ࡣ 5 ᖺ⏕Ꮡ⋡ࡀ 63%⏕Ꮡᮇ㛫ࡢᘏ㛗ࡀㄆࡵࡽࢀࡓࡀࠊ10 ᖺ⏕Ꮡ⋡ࡣ 29%࡛࠶ࡾ 㛗ᮇᡂ⦼ࡢᨵၿࡣ⮳ࡽ࡞ࡗࡓ (Fig. 2)11)ࠋ
Fig. 2 Change of the treatment and survival of patients with CML
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ࣄࢺࢤࣀ࣒ィ⏬⤊ᚋࠊࡀࢇ⒪ࢆ┠ᶆࡋࡓ⸆ࡣࢻࣛࣂ࣮㑇ఏᏊ⏘≀ ᑐࡍࡿศᏊᶆⓗ⒪⸆ࡀࢺࣞࣥࢻ࡞ࡗࡓࠋCML ᑐࡍࡿ BCR-ABL 㑅ᢥⓗ ࢳࣟࢩࣥ࢟ࢼ࣮ࢮ㜼ᐖ (tyrosine kinase inhibitor; TKI) ࡣࡑࡢ௦⾲ⓗ࡞⸆ࡢ 1 ࡘ࡛࠶ࡿࠋ᭱ึ㛤Ⓨࡉࢀࡓ TKI ࡛࠶ࡿ࣐ࢳࢽࣈࡣࠊ5 ᖺ⏕Ꮡ⋡ 93%ࠊ8 ᖺ⏕ Ꮡ⋡ 85%ඃࢀࡓ㛗ᮇᡂ⦼ࢆ♧ࡋࡓࡇࡽࠊ,)1Į ᭰ࢃࡗ࡚ CP ᮇࡢ CML
ᑐࡍࡿ➨୍㑅ᢥ⸆࡞ࡗࡓ (Fig. 2)12)ࠋ⌧ᅾࡣࠊ➨୍ୡ௦ࡢ࣐ࢳࢽࣈࠊ➨ୡ
௦ࡢࢽࣟࢳࢽࣈࠊࢲࢧࢳࢽࣈࠊ࣎ࢫࢳࢽࣈࠊ➨୕ୡ௦ࡢ࣏ࢼࢳࢽࣈࡀ㛤Ⓨࡉࢀ ࡚࠾ࡾࠊࢽࣟࢳࢽࣈ࠾ࡼࡧࢲࢧࢳࢽࣈࡶ➨୍㑅ᢥ⸆ࡋ࡚ᢎㄆࡉࢀ࡚࠸ࡿࠋ
BCR-ABL ࢳࣟࢩࣥ࢟ࢼ࣮ࢮࡣ ATP binding site ATP ࢆ⤖ྜࡋࠊࡑࡢࣜࣥ㓟
ᇶࡼࡗ࡚ᇶ㉁ࡢࢳࣟࢩࣥṧᇶࢆࣜࣥ㓟ࡍࡿࠋ࣐ࢳࢽࣈࢆጞࡵࡍࡿ TKI ࡣࠊATP binding site ⤖ྜࡍࡿࡇ࡛ ATP ࡢ⤖ྜࢆ➇ྜⓗ㜼ᐖࡋࠊᇶ㉁ࡢࣜ
ࣥ㓟ࡑࢀ⥆ࡃୗὶࢩࢢࢼࣝࡢάᛶࢆᢚไࡍࡿ (Fig. 3A)13)ࠋࡑࡢ⤖ᯝࠊ
Ph+⣽⬊㑅ᢥⓗቑṪᢚไ࣏ࢺ࣮ࢩࢫࡀㄏᑟࡉࢀࡿ14)ࠋ୍㒊ࡢᛴᛶࣜࣥࣃᛶ
ⓑ⾑ (acute lymphoblastic leukemia; ALL) ࠾࠸࡚ࡶ Ph ᰁⰍయࡀほᐹࡉࢀࠊྠ
ᵝࡢస⏝ᶵᗎ࡛ TKI ࡀ᭷ຠᛶࢆ♧ࡍࡇࡽࠊPh+ ALL ᑐࡋ࡚ࡶ TKI ࡀ⏝ ࡉࢀ࡚࠸ࡿ15)ࠋ TKI ࡢᑟධࡼࡾ CML ࡢ⒪ᡂ⦼ࡣ㣕㌍ⓗྥୖࡋࡓࡀࠊTKI ᑐࡍࡿ⪏ᛶ ࡢ⋓ᚓࡸ᩿⸆ᚋࡢⓎࡀࡁ࡞ၥ㢟࡞ࡗ࡚࠸ࡿࠋ࣐ࢳࢽࣈᑐࡍࡿ⪏ᛶࡢ ⋓ᚓᶵᵓࡋ࡚ࡣࠊBCR-ABL ౫Ꮡᛶ㠀౫Ꮡᛶࡢࡶࡢࡀ⪃࠼ࡽࢀ࡚࠸ࡿࠋࡇࢀ ࡲ࡛ሗ࿌ࡉࢀ࡚࠸ࡿ࡞ᶵᵓࢆ Table 1 ࡲࡵࡓࠋ
8
Mechanism of imatinib-resistance References
BCR-ABL-dependent
࢟ࢼ࣮ࢮࢻ࣓ࣥࡢⅬ✺↛ኚ␗ (Point mutation) 16,17)
BCR-ABL 㑇ఏᏊࡢቑᖜ (Amplification) 18)
BCR-ABL 㑇ఏᏊࡢ㑅ᢥⓗࢫࣉࣛࢩࣥࢢ (Alternative splicing) 19)
BCR-ABL-independent ⓑ⾑ᖿ⣽⬊ (Stem cell) 20,21) ࣐ࢳࢽࣈ⤖ྜࢱࣥࣃࢡĮ-1-acid glycoprotein ࡢቑຍ 22,23) multi-drug resistance (MDR) ࡼࡿ⸆ࡢฟஹ㐍 24) ࣏ࢺ࣮ࢩࢫㄏᑟᅉᏊ Bim ࡢ㑇ఏᏊከᆺ 25) ௦᭰ࢩࢢࢼࣝࡢάᛶஹ㐍 24,26)
Table 1 Mechanism of resistance to imatinib in CML
ࡇࢀࡽࡢᶵᵓࡢ୰࡛ࠊBCR-ABL ࢟ࢼ࣮ࢮࢻ࣓ࣥࡢⅬ✺↛ኚ␗ࡀ᭱ࡶ㧗㢖ᗘ࡛ ࠶ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ16)ࠋⅬ✺↛ኚ␗ࡀ⏕ࡌࡿ࢟ࢼ࣮ࢮࢻ࣓ࣥᏑᅾ ࡍࡿ࣑ࣀ㓟ࡢ 1 ࡘࡀูࡢ࣑ࣀ㓟⨨ࡉࢀࠊࢱࣥࣃࢡࡢ❧యᵓ㐀ࡀኚࡍ ࡿࠋࡑࡢ⤖ᯝࠊBCR-ABL ࣐ࢳࢽࣈࡢ⤖ྜぶᛶࡀపୗࡋࠊࢳࣟࢩࣥ࢟ࢼ ࣮ࢮάᛶࡢ㜼ᐖࡑࢀ⥆ࡃቑṪᢚไ࣏ࢺ࣮ࢩࢫࡀㄏᑟࡉࢀࡃࡃ࡞ࡿ (Fig. 3B)ࠋ⪏ᛶࡢ⋓ᚓࡣ≉ AP ᮇࠊBP ᮇࡢᝈ⪅ከࡃࠊணᚋࢆᝏࡉࡏࡿࡇ ࡀሗ࿌ࡉࢀ࡚࠸ࡿ27)ࠋ ࡲࡓࠊCML ࡢⓎࡣⓑ⾑ᖿ⣽⬊ࡢṧᏑࡀ㛵ࡋ࡚࠸ࡿࡇࡀ▱ࡽࢀ࡚࠸ ࡿࠋⓑ⾑ᖿ⣽⬊ࡣ⣽⬊࿘ᮇࡽ⬺ࡋࠊ㟼Ṇᮇ (G0ᮇ) Ṇࡲࡗ࡚࠸ࡿࡓࡵ TKI ᑐࡍࡿឤཷᛶࡀప࠸ࡇࡀ᫂ࡽࡉࢀ࡚࠸ࡿ28)ࠋⓑ⾑ᖿ⣽⬊࡛ࡣ BCR-ABL ࡢ࢟ࢼ࣮ࢮάᛶ㠀౫Ꮡⓗ⣽⬊ࡢ⏕Ꮡࡀ⥔ᣢࡉࢀࡿࡇࡽࠊTKI ༢
9 ࡛ࡢ⒪ࡣᅔ㞴࡛࠶ࡿ20)ࠋࡉࡽࠊTKI ࡢ᭹⸆ࢆ୰᩿ࡍࡿ 2 ᖺ௨ෆ⣙ 60% ࡢᝈ⪅࡛Ⓨࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾ29)ࠊ⌧ᅾࡢࡇࢁ TKI ࢆ୰Ṇ࡛ࡁࡿᇶ ‽ࡣ࡞ࡃࠊศᏊ㑇ఏᏛⓗዌຌࡀᚓࡽࢀ࡚ࡶ⒪ࢆ⥆ࡅࡿࡇࡀ່ࡵࡽࢀ࡚ ࠸ࡿࠋ୍⯡ศᏊᶆⓗ⒪⸆ࡣ⸆㈝ࡀ㧗㢠࡛࠶ࡾࠊ⤒῭ⓗ࡞㈇ᢸࡀࡁ࠸ࡇ ࡽ⒪ࡢ⥆⾜ࡀ㞴ࡋࡃ࡞ࡗ࡚ࡋࡲ࠺ࢣ࣮ࢫࡀᑡ࡞ࡃ࡞࠸30)ࠋࡑࡢࡓࡵࠊẚ ㍑ⓗᏳ౯ᥦ౪ࡍࡿࡇࡀ࡛ࡁࠊⓎࡢཎᅉ࡞ࡿⓑ⾑ᖿ⣽⬊ࡶ᭷ຠ࡞᪂ つ⒪⸆ࡢ㛤Ⓨࡀᮃࡲࢀ࡚࠸ࡿࠋⓑ⾑ᖿ⣽⬊ࢆ᰿⤯ࡉࡏࡿࡓࡵࡢ⒪ἲࡢ◊ ✲ࡣࠊ㏆ᖺ┒ࢇ⾜ࢃࢀ࡚࠸ࡿࠋBCR-ABL ࡢୗὶศᏊ mTOR ࡢ㜼ᐖ TKI ࡢే⏝31,32)ࡸࠊⓑ⾑ᖿ⣽⬊ࡢ⣽⬊࿘ᮇࢆ㟼Ṇᮇࡽ㏻ᖖࡢ࿘ᮇ㏣࠸ฟࡍ ࡼ࠺࡞⒪ἲ33)ࡀᥦࡉࢀ࡚࠸ࡿࠋࡲࡓࠊⓑ⾑ᖿ⣽⬊ࡀ≉␗ⓗ⏝ࡋ࡚࠸ ࡿࢪ࣌ࣉࢳࢻࡢྲྀࡾ㎸ࡳࢆ㜼ᐖࡍࡿ⸆ TKI ࡢే⏝ࡀ᭷ຠ࡛࠶ࡿࡇࡶሗ ࿌ࡉࢀ࡚࠾ࡾ34)ࠊCML ࡢ᰿࢚ࢿࣝࢠ࣮௦ㅰࡢไᚚࡀ᭷⏝࡛࠶ࡿྍ⬟ᛶࡀ♧ ၀ࡉࢀ࡚࠸ࡿࠋ
Fig. 3 Schematic mechanism of anti-cancer effects of imatinib and acquisition of TKI resistance
10 ➨ 3 ⠇ ࡀࢇ⣽⬊≉␗ⓗ࢚ࢿࣝࢠ࣮௦ㅰᶵᵓ: Warburg ຠᯝ ㏆ᖺࠊࡀࢇᑐࡍࡿ᪂ࡓ࡞⸆ࡢࢱ࣮ࢤࢵࢺࡋ࡚࢚ࢿࣝࢠ࣮௦ㅰࡀὀ┠ࡉ ࢀ࡚࠸ࡿ35)ࠋࡀࢇ⣽⬊ࡣప㓟⣲࣭పᰤ㣴≧ែ࠾࠸࡚㝈ࡽࢀࡓ࢚ࢿࣝࢠ࣮※ࢆ ᭷ຠ⏝ࡍࡿࡓࡵ௦ㅰᶵᵓࡢࣜࣉࣟࢢ࣑ࣛࣥࢢࢆ⾜ࡗ࡚࠸ࡿ36)ࠋࡑࡢ 1 ࡘࡀ ࠕWarburg ຠᯝࠖ࠸࠺⌧㇟࡛࠶ࡿࠋWarburg ຠᯝࡣࠊࠕࡀࢇ⣽⬊ࡣዲẼⓗ᮲௳ୗ ࠾࠸࡚ࡶ TCA ࢧࢡࣝࢆά⏝ࡏࡎࠊᖖ᎘Ẽⓗゎ⢾ࢆ⏝ࡋ࡚ࢢࣝࢥ࣮ࢫࢆ ௦ㅰࡋࠊ㔞ࡢங㓟ࢆศἪࡍࡿࠖ࠸࠺⌧㇟ࢆ OttoWarburg ࡀᥦၐࡋࡓࡶࡢ࡛࠶ ࡿ37,38)ࠋࡀࢇ⣽⬊ࡀ Warburg ຠᯝࢆ⋓ᚓࡍࡿࡢ㔜せ࡞ᙺࢆᯝࡓࡋ࡚࠸ࡿศᏊ ࡀࠊpyruvate kinase muscle (PKM) ࡛࠶ࡿ39,40)ࠋWarburg ຠᯝࡢ⋓ᚓᶵᵓࢆ Fig. 4 ♧ࡋࡓࠋ
Fig. 4 Cancer-specific energy metabolism “Warburg effect”
PKM, pyruvate kinase muscle; PTBP1, polypyrimidine tract-binding protein 1; PPP, Pentose phosphate pathway; PEP, phosphoenolpyruvate
11 PKM ࡣゎ⢾⣔ࡢ᭱⤊ࢫࢸࢵࣉ࡛࠶ࡿ࣍ࢫ࢚࣍ࣀ࣮ࣝࣆࣝࣅࣥ㓟 (PEP) ࡽ ࣆࣝࣅࣥ㓟ࡢᛂࢆゐ፹ࡍࡿ㓝⣲࡛࠶ࡾࠊゎ⢾⣔ࡢᚊ㏿㓝⣲ࡋ࡚ാࡃ41)ࠋ PKM ࡣ PKM1 PKM2 ࡢ 2 ࡘࡢࢯࢨ࣒ࡀᏑᅾࡋࠊࢫࣉࣛࢩࣥࢢ㐣⛬ ࠾࠸࡚࢚࢟ࢯࣥ 8ࠊ9ࠊ11 ࢆྲྀࡾ㎸ࡴ PKM1ࠊ࢚࢟ࢯࣥ 8ࠊ10ࠊ11 ࢆྲྀࡾ㎸ ࡴ PKM2 ࡀసࡽࢀࡿ39,42)ࠋࡀࢇ⣽⬊࠾࠸࡚ࡣࢫࣉࣛࢧ࣮࡛࠶ࡿ hnRNP A1ࠊ
hnRNP A2ࠊpolypyrimidine tract-binding protein 1 (PTBP1) ࡀ㧗Ⓨ⌧ࡍࡿࡇ࡛࢚
࢟ࢯࣥ 9 ࡢྲྀࡾ㎸ࡳࡀ㜼ᐖࡉࢀࠊ࢚࢟ࢯࣥ 10 ࢆྲྀࡾ㎸ࢇࡔ PKM2 ࡀ㧗Ⓨ⌧ࡍࡿ ࡇࡀ▱ࡽࢀ࡚࠸ࡿ43,44)ࠋ➹⪅ࡽࡣࡰࡍ࡚ࡢࡀࢇ✀࠾࠸࡚ PKM2 ࡀ㧗Ⓨ ⌧ࡋ࡚࠸ࡿࡇࢆ☜ㄆࡋ࡚࠸ࡿ45)ࠋ TCA ࢧࢡࣝࢆࡋࡓࢢࣝࢥ࣮ࢫ௦ㅰ࡛ࡣࢢࣝࢥ࣮ࢫ 1 ࣔࣝᙜࡓࡾ 36 ATP ࡀ ⏘⏕ࡉࢀࡿࡢᑐࡋࠊゎ⢾⣔࡛ࡣ 2 ATP ࡋ⏘⏕ࡍࡿࡇࡀ࡛ࡁ࡞࠸ࡓࡵ ATP ࡢ⏘⏕ຠ⋡ࡀప࠸ࠋࡋࡋ࡞ࡀࡽࠊゎ⢾⣔ࡣ TCA ࢧࢡࣝẚ㍑ࡋ࡚ᛂࢫࢸ ࢵࣉࡀ༢⣧࡛࠶ࡿࡓࡵ ATP ࡢ⏘⏕㏿ᗘࡀ᪩ࡃࠊࡀࢇ⣽⬊ࡣࢢࣝࢥ࣮ࢫࡢྲྀࡾ㎸ ࡳࢆஹ㐍ࡉࡏࡿࡇ࡛㔞ࡢ ATP ⏘⏕ࢆྍ⬟ࡋ࡚࠸ࡿ46,47)ࠋࡲࡓࠊゎ⢾⣔ࡢ ⏝ࡣ 2 ࡘࡢⅬࡀᣲࡆࡽࢀࡿ (Fig. 4)ࠋ1 ࡘ┠ࡣ TCA ࢧࢡࣝࡢ㐣⛬࡛Ⓨ⏕ ࡍࡿάᛶ㓟⣲✀ (reactive oxygen species; ROS) ࡢⓎ⏕ࢆᢚไ࡛ࡁࡿࡇ࡛࠶ࡿ 38,48)ࠋ⣽⬊ෆ࡛㐣⏘⏕ࡉࢀࡓ ROS ࡣ࣏ࢺ࣮ࢩࢫࡸ࣮࢜ࢺࣇࢪ࣮࡞ࡢࣉ ࣟࢢ࣒ࣛ⣽⬊Ṛࡢཎᅉ࡞ࡿࡓࡵ49)ࠊゎ⢾⣔ࢆ⏝ࡍࡿࡇ࡛ ROS ࡢⓎ⏕ࢆᢚ ไࡋ࡚࠸ࡿ⪃࠼ࡽࢀ࡚࠸ࡿࠋ2 ࡘ┠ࡣࠊゎ⢾⣔ࡢ୰㛫௦ㅰ⏘≀ࢆ⏝ࡋࡓ᰾㓟 ྜᡂࡀྍ⬟࡞ࡿࡇ࡛࠶ࡿ38,39)ࠋPKM1 ࡀᖖάᛶᆺࡢᅄ㔞య࡛Ꮡᅾࡍࡿࡢ ᑐࡋࠊPKM2 ࡣẚ㍑ⓗάᛶࡢప࠸༢㔞యࡶࡋࡃࡣ㔞య࡛Ꮡᅾࡍࡿࠋࡇࡢከ㔞య ࡢไᚚᶵᵓࡣ PKM2 ࡢࢳࣟࢩࣥࣜࣥ㓟ࡀ㛵ࡋ࡚࠾ࡾࠊࣜࣥ㓟ࢆཷࡅ ࡓ PKM2 ࡣᅄ㔞యࡢᙧᡂࡀᅔ㞴࡞ࡿ50)ࠋPKM2 ࢆࣜࣥ㓟ࡍࡿୖὶศᏊࡋ ࡚ࡣࠊFGFRࠊJAK2 ࡢ BCR-ABL ࡀሗ࿌ࡉࢀ࡚࠸ࡿ50)ࠋPKM2 ࡢ࢟ࢼ࣮ࢮ
12 άᛶࡀప࠸≧ែಖࡓࢀࡿࡇ࡛ࠊ⣽⬊ෆ࡛ࡣゎ⢾⣔ࡢ୰㛫௦ㅰ⏘≀ࡀ✚ࡋ ࡸࡍࡃ࡞ࡿࠋ⣽⬊ࡣࡇࡢ୰㛫௦ㅰ⏘≀ࢆ࣌ࣥࢺ࣮ࢫࣜࣥ㓟⤒㊰ (PPP) ືဨࡋࠊ ⣽⬊ศᚲせ࡞᰾㓟ࡢྜᡂ⏝ࡋ࡚࠸ࡿ39)ࠋ PKM 㑇ఏᏊࡢࢫࣉࣛࢧ࣮࡛࠶ࡿ PTBP1 ࡢⓎ⌧ไᚚࡣࠊc-Myc44)ࠊཷᐜయ ᆺࢳࣟࢩࣥ࢟ࢼ࣮ࢮ51)࡞ࡢ㛵ࡀ▱ࡽࢀ࡚࠾ࡾࠊ➹⪅ࡽࡶ PTBP1 ࢆࢱ࣮ࢤࢵ
ࢺࡍࡿ microRNA (miRNA, miR) ࡼࡿⓎ⌧ㄪ⠇ࢆሗ࿌ࡋ࡚࠸ࡿ45)
(Fig. 4)ࠋ
miRNA ࡣ 18㹼25 ሷᇶࡽᡂࡿ୍ᮏ㙐ࡢ non-cording RNA ࡛࠶ࡾࠊᶆⓗࡍࡿ
mRNA ࡢ 3’㠀⩻ヂ㡿ᇦ (UTR) 㡿ᇦࡢ┦⿵ⓗ㓄ิ㒊⤖ྜࡍࡿࡇ࡛ࡑࡢ⩻
ヂࢆ㈇ไᚚࡍࡿ52)ࠋmiRNA ࡣ㑇ఏᏊࡢ㌿ᚋⓎ⌧ㄪ⠇ࢆᢸ࠺ࡇ࡛ࠊ⏕≀ࡢ
Ⓨ⏕53)ࠊ⣽⬊Ṛ54)ࠊศ55)ࠊ⣽⬊ቑṪ56)࠸ࡗࡓ⏕⌧㇟ࢆไᚚࡍࡿࡔࡅ࡛ࡣ
࡞ࡃࡀࢇࡢⓎࡸ㐍⾜ࡢ㛵㐃ᛶ52,57)ࡶ♧၀ࡉࢀ࡚࠸ࡿࠋmiRNA 㛵ࡍࡿࢹ࣮
ࢱ࣮࣋ࢫࠊTarget Scan 6.2 database (http://www.targetscan.org/) ࠾ࡼࡧ miRBase (http://www.mirbase.org/) ࡛ࡣࠊPTBP1 ࢆᶆⓗࡍࡿ miRNA ࡣ miR-1ࠊmiR-9ࠊ
miR-124ࠊmiR-133ࠊmiR-137 ࡢ 5 ࡘࡀ♧ࡉࢀ࡚࠾ࡾࠊࡇࡢ࠺ࡕ miR-124 ࠾ࡼࡧ miR-133 ࡣ PTBP1 ࡢ 3’UTR 㡿ᇦෆ 2 ᡤࡢ┦⿵ⓗ㓄ิ㒊ࢆ᭷ࡋ࡚࠸ࡿࠋ➹ ⪅ࡽࡣ⭠ࡀࢇᝈ⪅ࡢࡀࢇ㒊࠾࠸࡚ miR-124 ࠾ࡼࡧ miR-133 ࡢⓎ⌧ࡀపୗࡋ ࡚࠾ࡾࠊࡀࢇ㒊࠾ࡅࡿ PKM2 ࡢ㧗Ⓨ⌧ᐤࡋ࡚࠸ࡿࡇࢆሗ࿌ࡋ࡚ࡁࡓ45)ࠋ ࡋࡋ࡞ࡀࡽࠊࡀࢇࡢⓎ㐣⛬࠾ࡅࡿࡇࢀࡽࡢ miRNA ࡢⓎ⌧ኚࡣࡲ࡛᫂ ࡽࡉࢀ࡚࠸࡞࠸ࠋ ௨ୖࡢ㏻ࡾࠊWarburg ຠᯝࡣࡀࢇ⣽⬊ࡢቑṪࠊ⏕Ꮡࠊ࢚ࢿࣝࢠ࣮ࡢ⋓ᚓࢻ ࣂࣥࢸ࣮ࢪࢆ࠼ࡿᴟࡵ࡚㔜せ࡞ᶵᵓ࡛࠶ࡿࠋⓑ⾑ࠊ⭠ࡀࢇࢆጞࡵࡋࡓ ᵝࠎ࡞ࡀࢇ✀࠾࠸࡚ࠊゎ⢾㜼ᐖࡢే⏝ࡀᢠࡀࢇࡢឤཷᛶࢆ㧗ࡵࡿࡇࡀ ሗ࿌ࡉࢀ࡚࠾ࡾ35)ࠊWarburg ຠᯝࢆ◚⥢ࡉࡏࡿ⒪ἲࡀ⒪ᡂ⦼ࡢྥୖࡸ⸆⪏ ᛶࡢඞ᭹ᮇᚅࡉࢀ࡚࠸ࡿࠋ
13
➨ 4 ⠇ ࡀࢇ⣽⬊࠾ࡅࡿ⬡⫫㓟㓟ࡢᙺ
๓㏙ࡢ㏻ࡾࠊࡀࢇ⣽⬊࠾࠸࡚ࡣ Warburg ຠᯝࡼࡿࢢࣝࢥ࣮ࢫࢆ୰ᚰࡋ ࡓ࢚ࢿࣝࢠ࣮௦ㅰ࡛ ATP ࡀ⏘⏕ࡉࢀࡿࠋࡋࡋ࡞ࡀࡽࠊ⣽⬊ෆࡢ ATP 㟂せࡀ㐣 ࡞ࡿࢢࣝࢥ࣮ࢫࡢࡳ࡞ࡽࡎ⬡⫫㓟ࢆ࢚ࢿࣝࢠ࣮※ࡋ࡚ ATP ࢆ⏘⏕ࡍࡿ ࡇࡀ▱ࡽࢀ࡚࠸ࡿ58,59)ࠋ⬡⫫㓟㓟 (fatty-acid oxidation; FAO) ࡣ Fig. 5 ♧ࡍ ࡼ࠺࡞ࢫࢸࢵࣉࢆ⤒࡚㐍⾜ࡍࡿࠋࡲࡎࠊ⬡⫫㓟ࡣ⣽⬊㉁ෆ࡛⬡⫫㓟ࢩࣝ-CoA ኚࡉࢀࠊ࢝ࣝࢽࢳࣥ⤖ྜࡋࡓᚋ࣑ࢺࢥࣥࢻࣜෆྲྀࡾ㎸ࡲࢀࡿࠋࡇ ࡢ࢝ࣝࢽࢳࣥࡢ⤖ྜࢆゐ፹ࡍࡿ㓝⣲ carnitine palmitoyltransferase 1 (CPT1) ࡀ ᛂయࡢᚊ㏿㓝⣲࡛࠶ࡿ60)ࠋ࣑ࢺࢥࣥࢻࣜࡢ࣐ࢺࣜࢵࢡࢫෆྲྀࡾ㎸ࡲࢀ ࡓ⬡⫫㓟ࡣࡧ⬡⫫㓟ࢩࣝ-CoA ⏕ࡉࢀࠊࢺࣛࣥࢫ-ǻ2 -࢚ࣀࣝࠊỈࠊ ⬺Ỉ⣲ࠊࢳ࢜ࣜࢩࢫࡢ 4 ࡘࡢࢫࢸࢵࣉࢆ⤒࡚ ȕ ࡽ㡰ษ᩿ࡉࢀࠊ᭱⤊ⓗ ࢭࢳࣝ-CoA ࡞ࡿࠋࢭࢳࣝ-CoA ࡣ TCA ࢧࢡࣝࢆࡋࡓ㟁Ꮚఏ㐩⣔
ࡼࡾ ATP ኚࡉࢀࡿࠋࡲࡓࠊᛂ㐣⛬࡛⏕ࡌࡿ FADH2࠾ࡼࡧ NADH2ࡶ㟁Ꮚ
ఏ㐩⣔ࢆࡋ࡚ ATP ྜᡂ⏝ࡉࢀࡿࠋ ⬡⫫㓟㓟ࡢᚊ㏿㓝⣲࡛࠶ࡿ CPT1 ࡣ 3 ࡘࡢࢯࣇ࢛࣮࣒ࠊCPT1A (⫢⮚ ᆺ)ࠊCPT1B (➽⫗ᆺ)ࠊCPT1C (⬻ᆺ) ࡀᏑᅾࡍࡿࡀࠊࡀࢇ⣽⬊࠾ࡅࡿ⬡⫫㓟㓟 ࡢάᛶࡣ CPT1C ࡢⓎ⌧ஹ㐍ࡀ㛵ࡋ࡚࠸ࡿ60,61)ࠋBCR-ABL+⣽⬊࠾࠸ ࡚ S6K1 ࢩࢢࢼࣝࡢ㜼ᐖࡼࡾゎ⢾⣔ࢆᢚไࡋ࡚ࡶ⣽⬊ṚࡣㄏᑟࡉࢀࡎࠊCPT1C ࡢⓎ⌧ቑຍࢆࡋ࡚⬡⫫㓟㓟ࡀάᛶࡉࢀࠊ⣽⬊ࡢ࢚ࢿࣝࢠ࣮⏘⏕࠾ࡼࡧ⏕ Ꮡࡀ⥔ᣢࡉࢀࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ62)ࠋࡲࡓࠊ⬡⫫㓟㓟ࡢάᛶࡣⓑ⾑ ⣽⬊ᑐࡍࡿ⸆ឤཷᛶࡢపୗࢆㄏᑟࡍࡿࡇࡶ▱ࡽࢀ࡚࠸ࡿ20)ࠋࡀࢇ⣽⬊ ࠾ࡅࡿ࢚ࢿࣝࢠ࣮௦ㅰࡢ◊✲ࡣࠊࢢࣝࢥ࣮ࢫ௦ㅰࢆ୰ᚰ㐍ࡵࡽࢀ࡚ࡁࡓࡇ ࡽࠊ⬡⫫㓟㓟㛵ࡋ࡚ࡣ㆟ㄽࡀᚅࡓࢀ࡚࠸ࡿ㒊ศࡶከ࠸ࠋ⬡⫫㓟㓟ࡣゎ
14
⢾⣔ࡢ௦ൾᛶᶵᵓࡋ࡚ᶵ⬟ࡍࡿࡇࡀ᪤ሗ࿌ࡉࢀ࡚࠸ࡿࡀࠊWarburg ຠᯝ ࡢ┤᥋ⓗ࡞㛵㐃ᛶ㛵ࡋ࡚ࡣ᫂ࡽࡉࢀ࡚࠸࡞࠸ࠋ
15 ➨ 5 ⠇ ୰㙐⬡⫫㓟ࡢ⏕⌮άᛶ ⬡⫫㓟ࡣࠊ㙐ࢆᵓᡂࡍࡿⅣ⣲ᩘࡼࡾ▷㙐⬡⫫㓟 (C6 ௨ୗ)ࠊ୰㙐⬡⫫㓟 (C8㹼14)ࠊ㛗㙐⬡⫫㓟 (C16 ௨ୖ) ศ㢮ࡉࢀࡿ63)ࠋ㛗㙐⬡⫫㓟ࡣ᳜≀⏤᮶ࡢ ࢜ࣞࣥ㓟ࠊࣜࣀ࣮ࣝ㓟ࠊ㨶Ἔ⏤᮶ࡢ࢚ࢥࢧ࣌ࣥࢱ࢚ࣥ㓟 (EPA)ࠊࢻࢥࢧ࣊ ࢟ࢧ࢚ࣥ㓟 (DHA) ࡞ࡀศ㢮ࡉࢀࡿࠋࡇࢀࡽࡢ㛗㙐⬡⫫㓟㛵ࡋ࡚ࡣࡇࢀࡲ࡛ ✚ᴟⓗ࡞◊✲ࡀ㐍ࡵࡽࢀࠊᰤ㣴Ꮫⓗ࠾ࡼࡧ་⸆Ꮫⓗ࡞㠃࡛ࡢ▱ぢࡀከᩘᚓࡽ ࢀ࡚ࡁࡓ64-66)ࠋ▷㙐⬡⫫㓟㛵ࡋ࡚ࡶࣈࢳࣝ㓟 (C4) ࠾ࡼࡧࡑࡢㄏᑟయࡼࡿᢠ ࡀࢇάᛶࠊචᢚไస⏝࡞ࡀሗ࿌ࡉࢀ࡚࠾ࡾࠊࡑࡢ⏕⌮ᶵ⬟ࡀ᫂ࡽࡉࢀ ࡘࡘ࠶ࡿ67,68)ࠋࡋࡋ࡞ࡀࡽࠊ୰㙐⬡⫫㓟ࣄࢺࡢ㛵ࢃࡾࡘ࠸࡚ࡢ◊✲ࡣ ᴟࡵ࡚ᑡᩘ␃ࡲࡗ࡚࠸ࡿࠋࣄࢺࡢ⏕యෆ࠾࠸࡚ࡣ⬡⫫㓟㓟ࡼࡾ୍ⓗ ୰㙐⬡⫫㓟ࡀᏑᅾࡍࡿࡀࠊ୰㛫௦ㅰ≀ࡋ࡚Ṇࡲࡿࡇ࡞ࡃࢭࢳࣝ-CoA ࡲ ࡛௦ㅰࡉࢀࡿ (Fig. 5)ࠋ୍᪉ࠊ᳜≀⏺࡛ࡣࢧࣥࢩࣙ࢘ࡢ㎞ᡂศ࡞ࡀ63,69)ࠊື ≀⏺࡛ࡣ᪻ࡢࣇ࢙ࣟࣔࣥ㢮ࡀ୰㙐⬡⫫㓟࡛ᵓᡂࡉࢀ࡚࠾ࡾ70)ࠊ᳜≀ࡸ᪻㢮 ࡣ୰㙐⬡⫫㓟ࢆ⏕యෆṆࡵ࡚⏝ࡋࠊ✀ࡢಖᏑᙺ❧࡚࡚࠸ࡿࠋ ࡑࡇ࡛ࠊ➹⪅ࡽࡣࣄࢺ࠾ࡅࡿ୰㙐⬡⫫㓟࠾ࡼࡧࡑࡢㄏᑟయࡢ⏕⌮άᛶࡢ᥈ ⣴ࢆ┠ⓗࡋ࡚⣙ 800 ✀ࡢ୰㙐⬡⫫㓟ㄏᑟయࢆྜᡂࡋࠊ୰㙐⬡⫫㓟ㄏᑟయࣛ ࣈ࣮ࣛࣜࢆస〇ࡋࡓࠋ➹⪅ࡣࡇࡢ࠺ࡕ 102 ✀ࡢㄏᑟయ㛵ࡋ࡚ᢠࡀࢇάᛶࡢࢫ ࢡ࣮ࣜࢽࣥࢢࢆ⾜࠸ࠊ3-ࢹࢭࣥ㓟ㄏᑟయᙉ࠸άᛶࢆぢฟࡋࡓ71) (Fig. 6)ࠋࢹࢭ ࣥ㓟 (10-hydroxy-2-decenoic acid) ࡣ࣑ࢶࣂࢳࡀసࡿ࣮ࣟࣖࣝࢮ࣮ࣜ୰ྵࡲࢀ ࡿ୰㙐⬡⫫㓟࡛࠶ࡾࠊᢠࡀࢇάᛶࢆ᭷ࡍࡿࡇࡀ᪤ሗ࿌ࡉࢀ࡚࠸ࡿࡀ72)ࠊά ᛶࡀᙅࡃࠊస⏝࣓࢝ࢽࢬ࣒ࡀ࡛᫂࠶ࡗࡓࡇࡽࡑࡢ⏝㏵ࡣᗣ㣗ရࡢࡳ ␃ࡲࡗ࡚࠸ࡓࠋࣄࢺ CML ⣽⬊ᰴ K562 ⣽⬊ࢆ⏝࠸ࡓᵓ㐀άᛶ┦㛵ࡢ᳨ウࡼࡾࠊ 3-ࢹࢭࣥ㓟ࡀᢠࡀࢇάᛶࡢ᰾࡞ࡿᵓ㐀࡛࠶ࡾࠊ࣑ࢻࡲࡓࡣ࢚ࢫࢸࣝㄏᑟయ ኚࡍࡿࡇ࡛ࡑࡢάᛶࡀ㢧ⴭୖ᪼ࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ≉ࠊ࣑
16
ࢻㄏᑟయ࡛࠶ࡿ (E)-1-(azocan-1-yl) dec-3-en-1-one (AIC-47) ࡣ K562 ᑐࡋࠊ᪤Ꮡ
ࡢᢠࡀࢇ࡛࠶ࡿ࢚ࢺ࣏ࢩࢻࡰྠ➼ࡢᢠࡀࢇస⏝ࢆ♧ࡋ71)
(Fig. 6)ࠊCML
ᑐࡍࡿ᪂つ⒪⸆ࡢ⸆ࢩ࣮ࢬ࡞ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ
Fig.6 Anti-cancer effects of medium-chain fatty-acid derivatives on K562 cells Left panel shows growth inhibition at 72 h after treatment of medium-chain fatty-acid derivatives (AIC-8, -18, -47, -49, -82), 3-decenoic acid, Cytarabine or Etoposide in human CML K562 cells. Data are
expressed as the means ± SD of 3 different experiments. Right panel shows chemical structures of AIC-8, -18, -47, -49, -82. (Shinohara et al., 2013 ࡼࡾᢤ⢋) ࡑࡇ࡛ᮏ◊✲࡛ࡣࠊCML ᑐࡍࡿ᪂つ⒪⸆ࡋ࡚ࡢ᭷ຠᛶࡀ♧၀ࡉࢀࡓ୰ 㙐⬡⫫㓟ㄏᑟయ AIC-47 ࡘ࠸࡚ 1) ࡇࢀࡲ࡛࡛᫂࠶ࡗࡓ୰㙐⬡⫫㓟ࡢᢠࡀࢇάᛶࡢస⏝ᶵᗎࢆゎ᫂ࡍࡿࡇ 2) ➨୍㑅ᢥ⸆ࡋ࡚⏝ࡉࢀ࡚࠸ࡿ࣐ࢳࢽࣈࡢస⏝ࡢ㐪࠸ࢆ᫂ࡽࡋࠊ ࣐ࢳࢽࣈᢠᛶࢆ♧ࡍⓑ⾑ᖿ⣽⬊ᑐࡍࡿ᭷ຠᛶࢆホ౯ࡍࡿࡇ ࢆ┠ⓗࡋࠊࡀࢇ⣽⬊ࡢ࢚ࢿࣝࢠ࣮௦ㅰ࠸࠺ほⅬࡽ◊✲ࢆᒎ㛤ࡋࡓࠋ
17 ➨ 3 ❶ ៏ᛶ㦵㧊ᛶⓑ⾑⣽⬊ᑐࡍࡿ୰㙐⬡⫫㓟ㄏᑟయࡢቑṪᢚไస⏝ ➨ 1 ⠇ ᗎ ⬡⫫㓟ࡢ⏕⌮άᛶ㛵ࡍࡿࡇࢀࡲ࡛ࡢ◊✲ࡣࠊ㛗㙐࠾ࡼࡧ▷㙐⬡⫫㓟↔ Ⅼࢆᙜ࡚ࡓࡶࡢ࡛࠶ࡾࠊࣄࢺ࠾ࡅࡿ୰㙐⬡⫫㓟ࡢ⏕⌮άᛶࡣࢇ᫂ࡽ ࡉࢀ࡚࠸࡞࠸ࠋࢹࢭࣥ㓟ࡣ࣮ࣟࣖࣝࢮ࣮ࣜྵࡲࢀࡿ୰㙐⬡⫫㓟ࡢ୍✀ ࡛࠶ࡾࠊⓑ⾑ࣔࢹ࣐ࣝ࢘ࢫ࠾ࡅࡿᢠࡀࢇάᛶࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ72)ࠊࡑ ࡢヲ⣽࡞స⏝࣓࢝ࢽࢬ࣒ࡣ࠸ࡲࡔ࡛᫂࠶ࡿࠋࡑࡇ࡛ᮏ❶࡛ࡣࠊ୰㙐⬡⫫㓟 ㄏᑟయࣛࣈ࣮ࣛࣜࡼࡾぢฟࡋࡓ (E)-1-(azocan-1-yl) dec-3-en-1-one (AIC-47) ࡘ࠸࡚ࠊࣄࢺ CML ⣽⬊ᰴᑐࡍࡿቑṪᢚไຠᯝࢆ᳨ウࡋࠊቑṪᢚไ࣓࢝ࢽ ࢬ࣒ࡢゎ᫂ࢆヨࡳࡓࠋ
18 ➨ 2 ⠇ ៏ᛶ㦵㧊ᛶⓑ⾑⣽⬊ᰴᑐࡍࡿ୰㙐⬡⫫㓟ㄏᑟయࡢቑṪᢚไຠᯝ 2 ✀ࡢࣄࢺ CML ⣽⬊ᰴ K562 ࠾ࡼࡧ KCL-22 AIC-47 ࢆῧຍࡋࠊ72 㛫ᚋ ࡢ⣽⬊⏕Ꮡ⋡ࢆࢺࣜࣃࣥࣈ࣮ࣝⰍ⣲㝖ヨ㦂ἲࡼࡾ ᐃࡋࡓࠋࡑࡢ⤖ᯝࠊ AIC-47 ࡣ ȝ0 ௨ୖࡢ⃰ᗘ࡛᭷ព࡞ቑṪᢚไస⏝ࢆ♧ࡋࡓ (Fig. 7)ࠋḟࠊṇ ᖖ⣽⬊ᑐࡍࡿ AIC-47 ࡢቑṪᢚไࢆ᳨ウࡍࡿࡓࡵࠊṇᖖࣄࢺᮎᲈ⾑ࣜࣥࣃ⌫ ᑐࡍࡿຠᯝࢆ᳨ウࡋࡓࠋศಁ㐍 Concanavalin A ่࡛⃭ᚋࡢࣄࢺᮎᲈ⾑ࣜ ࣥࣃ⌫ AIC-47 ࢆస⏝ࡉࡏࡓࡇࢁࠊȝ0 ௨ୗࡢ⃰ᗘ࡛ࡣ⣽⬊⏕Ꮡ⋡ࡢప ୗࡣㄆࡵࡽࢀ࡞ࡗࡓ (Fig. 7)ࠋ௨ୖࡢ⤖ᯝࡽࠊAIC-47 ࡣࡀࢇ⣽⬊≉␗ⓗ ቑṪᢚไస⏝ࢆ♧ࡍࡇࡀ♧၀ࡉࢀࡓࠋ
Fig. 7 Anti-cancer effects of medium-chain fatty-acid derivative AIC-47 in human CML cells and normal lymphocytes
Effects of AIC-47 on cell growth of K562 and KCL-22 cells, and mitogen-stimulated human blood lymphocytes. Cells were treated with DMSO (Control) or AIC-47 for 72 h. Human peripheral blood lymphocytes were stimulated with Concanavalin A for 48 h and treated with DMSO or AIC-47.
19 ➨ 3 ⠇ ୰㙐⬡⫫㓟ㄏᑟయࡼࡿ࣮࢜ࢺࣇࢪ࣮⣽⬊Ṛࡢㄏᑟ AIC-47 ࡼࡿቑṪᢚไస⏝ࢆゎ᫂ࡍࡿࡓࡵࠊ⣽⬊ࡢ⾲⌧ᆺࡍ࡞ࢃࡕ⣽⬊Ṛ ࡢᙧែࢆ᳨ドࡋࡓࠋ⣽⬊Ṛࡣࡁࡃศ㢮ࡋ࡚ࣉࣟࢢ࣒ࣛ⣽⬊Ṛ㠀ࣉࣟࢢ࣒ࣛ ⣽⬊Ṛศࡅࡽࢀࡿࠋࡉࡽࣉࣟࢢ࣒ࣛ⣽⬊ṚࡣᙧែᏛୖࡢ㐪࠸ࡽࠊ࣏ࢺ ࣮ࢩࢫࠊ࣮࢜ࢺࣇࢪ࣮⣽⬊Ṛࠊࢿࢡࣟࣉࢺ࣮ࢩࢫࡢ 3 ࡘศ㢮ࡍࡿࡇࡀ࡛ ࡁࡿ73)ࠋ⬡⫫㓟ࡼࡿ⣽⬊ቑṪᢚไస⏝ࡢ࣓࢝ࢽࢬ࣒ࡋ࡚ࡣࠊ࣏ࢺ࣮ࢩ ࢫ64,67)ࠊ࣮࢜ࢺࣇࢪ࣮ࡢㄏᑟ74)ࡼࡿࡶࡢࡀ▱ࡽࢀ࡚࠾ࡾࠊ➹⪅ࡣ AIC-47 ࡀ࣏ࢺ࣮ࢩࢫࢆㄏᑟࡋ࡞࠸ࡇࢆ᪤ሗ࿌ࡋ࡚࠸ࡿ71)ࠋࡑࡇ࡛ࠊ࣮࢜ࢺࣇ ࢪ࣮㛵㐃ࢩࢢࢼࣝ (Fig. 8) ࡘ࠸࡚ゎᯒࢆ⾜ࡗࡓࠋ ࣮࢜ࢺࣇࢪ࣮ࡣࠊ୍⯡ᰤ㣴㣚㣹࡞ᵝࠎ࡞ࢫࢺࣞࢫ᮲௳ୗ࠾࠸࡚άᛶ ࡉࢀࠊ⣽⬊ࡢ⏕Ꮡಁ㐍ᶵᵓࡋ࡚ാࡃࠋࡲࡓࠊ࣮࢜ࢺࣇࢪ࣮ࡣ⣽⬊ෆࡢ␗ ᖖࢱࣥࣃࢡ㉁ࡸᦆയࢆཷࡅࡓ࢜ࣝ࢞ࢿࣛࢆศゎࡋࠊ⣽⬊ࡢᜏᖖᛶࢆ⥔ᣢࡍࡿࡓ ࡵࡢ⏕⌮ⓗ࡞⣽⬊ᶵ⬟࡛ࡶ࠶ࡿࠋ୍᪉ࠊ⣽⬊ᙉ࠸ࢫࢺࣞࢫࡀ㛗ᮇ㛫ຍࢃࡗࡓ 㝿ࡣ࣮࢜ࢺࣇࢪ࣮ࡀ↓⛛ᗎஹ㐍ࡋࠊ⣽⬊Ṛㄏᑟᶵᵓࡋ࡚స⏝ࡍࡿ75)ࠋ ࣮࢜ࢺࣇࢪ࣮ࢩࢢࢼࣝࡣ Fig. 8 ♧ࡍఏ㐩⤒㊰ࡼࡾάᛶࡉࢀࡿࠋቑṪ࣭ ⏕Ꮡࢩࢢࢼࣝࡢάࡼࡾ࣮࢜ࢺࣇࢪ࣮ࢩࢢࢼࣝࡀㄏᑟࡉࢀࡿࠊ Beclin-1 ࢆྵࡴ」ྜయࡀᙧᡂࡉࢀࡿࠋࡇࡢ」ྜయᙧᡂࢆࡋ࡚⣽⬊ෆ㝸㞳⭷
ࡀ⏕ࡌࠊ⭷ୖ Atg5 」ྜయࡀ⤖ྜࡍࡿࡇ࡛⭷ࡀఙ㛗ࡍࡿࠋAtg3 ࠾ࡼࡧ Atg7 ࡼࡗ࡚ษ᩿ࡉࢀࡓ LC3 I ࡀ LC3 II ࡞ࡾࠊ⭷ୖ⤖ྜࡍࡿࡇ࡛ࡉࡽ⭷ ࡢఙ㛗ࡀ㐍ࡳࠊ⭷ෆ p62 ࢆ㊊ሙࡋ࡚ࢱࣥࣃࢡ㉁ࡸ࢜ࣝ࢞ࢿࣛ࡞ࡀྲྀࡾ㎸ ࡲࢀ࡚࣮࢜ࢺࣇࢦࢯ࣮࣒ࡀᙧᡂࡉࢀࡿࠋ࣮࢜ࢺࣇࢦࢯ࣮࣒ࡢᙧᡂ LC3 II ࡀᚲ㡲࡛࠶ࡿࡇࡽࠊLC3 I ࡽ LC3 II ࡢ⛣⾜ࡣ࣮࢜ࢺࣇࢦࢯ࣮࣒࣐࣮ ࣮࢝ࡋ࡚⏝ࡉࢀ࡚࠸ࡿ76)ࠋ࣮࢜ࢺࣇࢦࢯ࣮࣒ࡣࣜࢯࢯ࣮࣒⼥ྜࡋࠊ ࣮࢜ࢺࣜࢯࢯ࣮࣒࡞ࡿࠋ࣮࢜ࢺࣇࢦࢯ࣮࣒ෆࡢࢱࣥࣃࢡ㉁ࡸ࢜ࣝ࢞ࢿࣛࡣ
20 ࣜࢯࢯ࣮࣒ෆࡢຍỈศゎ㓝⣲࡛ศゎࡉࢀࠊ࣑ࣀ㓟࡞ࡗ࡚ࢫࢺࣞࢫᛂ⟅ᚲ せ࡞ࢱࣥࣃࢡ㉁ࡢྜᡂ⏝ࡉࢀࡿ77)ࠋࡲࡓࠊ࣮࢜ࢺࣇࢦࢯ࣮࣒ࡢ⭷ෆ ⤖ྜࡋࡓ LC3 II ࠾ࡼࡧ p62 ࡶྠศゎࡉࢀࡿࡇࡽࠊp62 ࡶ࣮࢜ࢺࣇ ࢪ࣮≉␗ⓗศᏊ࣐࣮࣮࢝࡞ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ76)ࠋ
21
ࡲࡎࠊ࣮࢜ࢺࣇࢪ࣮ࢩࢢࢼࣝࢆ࢙࢘ࢫࢱࣥࣈࣟࢵࢺࡼࡾ᳨ドࡋࡓࠋ AIC-47 ῧຍ⣽⬊࡛ࡣࠊ࣮࢜ࢺࣇࢦࢯ࣮࣒ࡢᙧᡂࢆ♧၀ࡍࡿ LC3B I ࡽ II
ࡢ⛣⾜ࠊୖὶࢩࢢࢼ࡛ࣝ࠶ࡿ Bcl-2 ࡢⓎ⌧పୗࡑࢀక࠺ Beclin-1 ࡢⓎ⌧ ቑຍࠊ࠾ࡼࡧቑṪࢩࢢࢼ࡛ࣝ࠶ࡿ PI3K/Akt/mTOR ࢩࢢࢼࣝࡢάࡀほᐹࡉ ࢀࡓ (Fig. 9A)ࠋࡉࡽࠊ㟁Ꮚ㢧ᚤ㙾ゎᯒࡼࡾ AIC-47 ῧຍᚋࡢ K562 ⣽⬊ࡢ ᙧែᏛⓗኚࢆ᳨ドࡋࡓࡇࢁࠊ⣽⬊ෆ࣮࢜ࢺࣇࢦࢯ࣮࣒ࡢᙧᡂࡀほᐹࡉ ࢀࡓࠋࡉࡽ࣮࢜ࢺࣇࢦࢯ࣮࣒ෆࡣ⬡⫫ (㯮Ⰽ▮༳) ࡢ✚ࡀㄆࡵࡽࢀࠊ AIC-47 ࡀ࣮࢜ࢺࣇࢪ࣮ࡢ୍✀࡛࠶ࡿ lipophagy ࢆㄏᑟࡍࡿࡇࡀ᫂ࡽ࡞ ࡗࡓ (Fig. 9B)ࠋ ⥆࠸࡚ࠊAIC-47 ࡼࡿ࣮࢜ࢺࣇࢪ࣮ࡀ⣽⬊ࡢ⏕Ꮡಁ㐍ࡶࡋࡃࡣ⣽⬊Ṛࡢ ࠸ࡎࢀࡢᶵᵓࡢࡶࡢ࡛࠶ࡿࢆ᳨ドࡍࡿࡓࡵࠊ࣮࢜ࢺࣇࢪ࣮㜼ᐖ AIC-47 ࡢే⏝ᐇ㦂ࢆ⾜ࡗࡓࠋྛ㜼ᐖࡢస⏝Ⅼࡣ Fig. 8 ♧ࡋࡓࠋ㜼ᐖ
ࡢຠᯝุᐃࡣ p62 ࡢศゎᢚไࢆᣦᶆࡋࡓࠋPI3K Class III ࡢ㜼ᐖ࡛࠶ࡿ 3-Methyladenine (3-MA) ࢆే⏝ࡍࡿࠊAIC-47 ࡼࡿ⣽⬊⏕Ꮡ⋡ࡢపୗ
LC3B I ࡽ II ࡢ⛣⾜ࡀ୍㒊ᢚไࡉࢀࡓ (Fig. 9C)ࠋ࣮࢜ࢺࣇࢦࢯ࣮࣒ࣜ
ࢯࢯ࣮࣒ࡢ⼥ྜࢆ㜼ᐖࡍࡿ Bafilomycin A1 ࢆే⏝ࡋࡓሙྜࡶ⣽⬊⏕Ꮡ⋡ࡢ ᅇࡀㄆࡵࡽࢀࠊLC3B II ࡢ✚ࡀほᐹࡉࢀࡓ (Fig. 9C)ࠋ௨ୖࡢ⤖ᯝࡽࠊ AIC-47 ࡀ࣮࢜ࢺࣇࢪ࣮⣽⬊Ṛࢆㄏᑟࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ
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Fig. 9 Medium-chain fatty-acid derivative AIC-47 induced autophagic cell death in K562 and KCL-22 cells.
(A) Effect of AIC-47 on autophagy flux and cell growth signaling. Proteins related with autophagy flux in K562 and KCL-22 cells treated with AIC-47 for 72 h were examined by Western blot analysis. (B) Morphological evidence of AIC-47-induced autophagosome formation in K562 cells. K562 cells were treated with AIC-47 for 72 h and then examined by TEM. Scale bar, 1 or 2 ȝP The arrows indicate lipid droplets. (C) Inhibition of autophagy by 3-methyladenine (3-MA) and Bafilomycin A1. Both K562 and KCL-22 cells were pre-incubated or not ZLWKȝ0-MA or 5 nM Bafilomycin for 8 h and then treated or not with AIC-47 for 72 h, after which the cell viability was estimated. Expression of LC3B and p62 were examined by Western blot analysis. The numbers below LC3B indicate each band density relative to that of the Control (taken as 100), which values were determined by densitometry.
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➨ 4 ⠇ BCR-ABL ࡢⓎ⌧ᑐࡍࡿస⏝
CML ࡢࢻࣛࣂ࣮㑇ఏᏊ࡛࠶ࡿ BCR-ABL ࡣ࣮࢜ࢺࣇࢪ࣮ᢚไᅉᏊࡋ࡚ ᶵ⬟ࡍࡿࠋࡑࡇ࡛ࠊAIC-47 ࡼࡿ BCR-ABL ࡢⓎ⌧ኚࢆ Real-time PCR ࠾ࡼࡧ ࢙࢘ࢫࢱࣥࣈࣟࢵࢺ᳨࡚ドࡋࡓࠋAIC-47 ῧຍ⣽⬊࡛ࡣ mRNA ࠾ࡼࡧࢱࣥࣃࢡ ㉁࡛ࣞ࣋ࣝ BCR-ABL ࡢⓎ⌧ࡀ㢧ⴭపୗࡋ࡚࠾ࡾࠊࡑࢀ┦㛵ࡋ࡚ c-Myc ࡢ Ⓨ⌧పୗࡶほᐹࡉࢀࡓ (Fig. 10A)ࠋBCR-ABL ࡢࣜࣥ㓟ࢱ࣮ࢤࢵࢺ࡛࠶ࡿ CrkL ࡢࣜࣥ㓟ࣞ࣋ࣝࡀపୗࡋ࡚࠸ࡓࡇࡽࠊAIC-47 ࡼࡿ BCR-ABL ࡢᶵ⬟ప ୗࡀ☜ㄆࡉࢀࡓ (Fig. 10A)ࠋBCR-ABL ࡢⓎ⌧పୗࡀ mRNA ࡛ࣞ࣋ࣝㄏᑟࡉࢀ࡚ ࠸ࡓࡇࡽࠊAIC-47 ࡀ BCR-ABL ࡢ㌿ࢆ㜼ᐖࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ GeneCards (http://www.genecards.org/) ࢹ࣮ࢱ࣮࣋ࢫୖࠊBCR-ABL ࡢࣉ࣮ࣟࣔࢱ࣮
㡿ᇦ⤖ྜࡍࡿྍ⬟ᛶࡢ࠶ࡿ㑇ఏᏊࢆ siRNA ࡚ࣀࢵࢡࢲ࢘ࣥࡍࡿᐇ㦂ࢆ⾜ࡗ ࡓࠋࡑࡢ⤖ᯝࠊc-Myc ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓሙྜࡢࡳ BCR-ABL ࡢⓎ⌧పୗࡀㄆ ࡵࡽࢀࡓ (Fig. 10B)ࠋࡑࡇ࡛ࠊc-Myc BCR-ABL 㑇ఏᏊࡢ⤖ྜᛶࢆ᳨ドࡍࡿ ࡓࡵ ChIP ࢵࢭࢆ⾜ࡗࡓࠋBCR-ABL ࡢࣉ࣮ࣟࣔࢱ࣮㡿ᇦ c-Myc ࡀ⤖ྜࡋࠊ AIC-47 ࢆῧຍࡍࡿࡇ࡛ࡑࡢ⤖ྜࡀゎ㝖ࡉࢀࡿࡇࡀ᫂ࡽ࡞ࡗࡓ (Fig.
10C)ࠋ௨ୖࡢ⤖ᯝࡽࠊc-Myc ࡀ BCR-ABL ࡢ㌿ᅉᏊࡋ࡚స⏝ࡋ࡚࠾ࡾࠊ
AIC-47 ࡣ c-Myc ࡢⓎ⌧పୗࢆࡋ࡚ BCR-ABL ࡢⓎ⌧ࢆᢚไࡍࡿࡇࡀ♧၀ࡉ
24
Fig. 10 Transcriptional repression of BCR-ABL by AIC-47 was induced through down-regulation of c-Myc.
(A, B) Expression levels of BCR-ABL mRNA (bar graphs) and protein (Western blots) after treatment with AIC-47 (A) or transfection with siR-c-Myc (B) for 72 h. (C) Real-time PCR analysis of ChIP DNA. The data are plotted as the ratio of immunoprecipitated DNA vs. total input DNA.
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➨ 5 ⠇ ࡀࢇ⣽⬊ࡢ࢚ࢿࣝࢠ࣮௦ㅰᑐࡍࡿస⏝
ࡀࢇ⣽⬊≉␗ⓗ࡞࢚ࢿࣝࢠ࣮௦ㅰᶵᵓ࡛࠶ࡿ Warburg ຠᯝࡣ PTBP1/PKM ࢝ࢫ
ࢣ࣮ࢻࡼࡗ࡚ᡂ❧ࡋ࡚࠾ࡾࠊPTBP1 ࡢⓎ⌧ㄪ⠇ࡣ c-Myc ࡀ㛵ࡋ࡚࠸ࡿ44)ࠋ
➨ 3 ⠇࡛ AIC-47 ࡼࡿ c-Myc ࡢⓎ⌧పୗࡀㄆࡵࡽࢀࡓࡇࡽࠊAIC-47 Warburg ຠᯝࡢ㛵㐃ᛶࢆ᳨ドࡋࡓࠋWarburg ຠᯝࡢᡂ❧ᐤࡍࡿ PTBP1ࠊ
PKM1 ࠾ࡼࡧ PKM2 ࡢⓎ⌧ࢆ Real-time PCR ࠾ࡼࡧ࢙࢘ࢫࢱࣥࣈࣟࢵࢺࡼࡾ᳨
ドࡋࡓ⤖ᯝࠊPTBP1 ࠾ࡼࡧ PKM2 ࡢⓎ⌧పୗ PKM1 ࡢⓎ⌧ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 11A, B)ࠋc-MycࠊPTBP1 ࡢⓎ⌧ࢆࡑࢀࡒࢀ siRNA ࢆ⏝࠸࡚ࣀࢵࢡࢲ࢘ࣥࡋ
ࡓࡇࢁࠊPKM2 ࡽ PKM1 ࡢⓎ⌧ࢫࢵࢳࡀほᐹࡉࢀࠊ࣮࢜ࢺࣇࢪ࣮ࡢ ㄏᑟࢆ♧၀ࡍࡿ LC3B I ࡽ II ࡢ⛣⾜ࡶ☜ㄆࡉࢀࡓ (Fig. 11C, D)ࠋ ཷᐜయᆺࢳࣟࢩࣥ࢟ࢼ࣮ࢮࡣࡑࡢୗὶ࡛ PTBP1/PKM ࢝ࢫࢣ࣮ࢻࢆάᛶࡉ ࡏࠊWarburg ຠᯝࢆஹ㐍ࡉࡏ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ51)ࠋࡑࡇ࡛ࠊ㠀ཷᐜయ ᆺࢳࣟࢩࣥ࢟ࢼ࣮ࢮ࡛࠶ࡿ BCR-ABL ࡀ Warburg ຠᯝ㛵㐃ࡋ࡚࠸ࡿ࠺ࢆ ᳨ドࡍࡿࡓࡵࠊsiRNA ࢆ⏝࠸࡚ BCR-ABL ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓࡇࢁࠊ PTBP1 ࠾ࡼࡧ PKM2 ࡢⓎ⌧పୗ PKM1 ࡢⓎ⌧ቑຍࠊLC3B ࡢ I ࡽ II ࡢ⛣ ⾜ࡀほᐹࡉࢀࡓ (Fig. 11E)ࠋ PKM2 ࡽ PKM1 ࢫࢵࢳࡍࡿࡇ࡛ࠊ᎘Ẽⓗゎ⢾ඃ࡞≧ែࡽ TCA ࢧ ࢡࣝࢆࡋࡓ㓟ⓗࣜࣥ㓟ࡀඃ࡞≧ែࢫࢵࢳࡍࡿࡇࡀணࡉࢀࡓࠋ ࡑࡇ࡛᎘Ẽⓗゎ⢾ࡢ᭱⤊⏘≀࡛࠶ࡿ L-lactate ࢆᐃ㔞ࡋࡓࡇࢁࠊAIC-47ࠊ siR-BCR-ABLࠊsiR-c-MycࠊsiR-PTBP1 ࡢ࠸ࡎࢀࢆస⏝ࡉࡏࡓ⣽⬊࠾࠸࡚ࡶ⣽⬊
ෆ lactate ⏘⏕㔞ࡢపୗࡀㄆࡵࡽࢀࡓ (Fig. 11F)ࠋࡉࡽࠊTCA ࢧࢡࣝࡼࡿ ROS ࡢⓎ⏕ࢆ㟁Ꮚࢫࣆࣥඹ㬆ἲ (Electron Spin Resonance; ESR) ࢆ⏝࠸࡚ ᐃ
ࡋࡓࠋROS ࡢ୍✀࡛࠶ࡿࢫ࣮ࣃ࣮࢜࢟ࢩࢻࢽ࢜ࣥ (O2-) ࠾ࡼࡧࡑࡢࡢ㓟⣲
26
࣮ࣜࣛࢪ࢝ࣝࡢᤕᤊ࡛࠶ࡿ N-acetylcysteine (NAC) AIC-47 ࢆే⏝ࡍࡿࠊ AIC-47 ࡼࡿቑṪᢚไస⏝࠾ࡼࡧ LC3B ࡢ⛣⾜ࡀ୍㒊ᢚไࡉࢀࡓ (Fig. 11H)ࠋ
௨ୖࡢ⤖ᯝࡽࠊAIC-47 ࡣ PKM2 ࡽ PKM1 Ⓨ⌧ࢫࢵࢳࢆㄏᑟࡍࡿࡇ࡛ Warburg ຠᯝࢆ◚⥢ࡉࡏࠊࢢࣝࢥ࣮ࢫ௦ㅰ⤒㊰ࢆ᎘Ẽⓗゎ⢾ࡽ TCA ࢧࢡࣝ
ࢫࢵࢳࡉࡏࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡲࡓࠊ㠀ཷᐜయᆺࢳࣟࢩࣥ࢟ࢼ࣮ࢮ ࡛࠶ࡿ BCR-ABL ࡶ Warburg ຠᯝࢆஹ㐍ࡉࡏࡿࡇࢆぢฟࡋࡓࠋAIC-47 ࡼࡿ ࣮࢜ࢺࣇࢪ࣮⣽⬊ṚࡢㄏᑟࡣࠊTCA ࢧࢡࣝࡢᅇ㌿ࡼࡿ ROS ࢆⓎ⏕ࡀཎᅉ ࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋ
27
Fig. 11 Dys-regulation of PTBP/PKM axis through c-Myc and BCR-ABL down-regulation induced autophagy.
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(A, B) Expression levels of mRNAs (A) and proteins (B) after treatment of K562 and KCL-22 cells with AIC-47 for 72 h. (B) The numbers below PKM1 and PKM2 indicate each band density relative to that of the Control (taken as 100), which values were determined by densitometry. (C, D, E) Effects of silencing
c-Myc (C), PTBP1 (D) or BCR-ABL (E) on autophagy flux in K562 and KCL-22 cells at 72 h. (F) Lactate
production in K562 and KCL-22 cells after treatment of them with AIC-47 ȝ0 RUtransfection of them with siR-BCR-ABL, siR-c-Myc, or siR-PTBP1 (5 nM) for 72 h. (G) The production of free-radicals was measured by electron spin resonance spectroscopy (ESR). K562 cells were treated or not with AIC-47 (5 ȝ0 IRU K 7he representative ESR spectrum for the Control and AIC-47 are depicted in the upper panels. The intensities of the spin adducts are shown in the lower panel. (H) K562 and KCL-22 cells were pre-incubated or not with 1 mM N-acetylcysteine (NAC) for 4 h and then treated or not with AIC-47 (5 ȝ0 IRUK7KHUeafter, the cell viability was estimated; and the conversion of LC3B was examined by Western blot analysis.
29
➨ 6 ⠇ ୰㙐⬡⫫㓟ㄏᑟయࡢᶆⓗศᏊࡢ᥈⣴
ᮏ❶➨ 2 ⠇ࠊ➨ 3 ⠇♧ࡋࡓ᳨ウ⤖ᯝࡽࠊAIC-47 ࡼࡿቑṪᢚไ࣓࢝ࢽࢬ ࣒ࡢ୰ᚰศᏊࡀ c-Myc ࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋࡑࡇ࡛ࠊAIC-47 ࡀ┤᥋ⓗᶆ ⓗࡋ࡚࠸ࡿศᏊࢆ᥈⣴ࡋࠊc-Myc ࡢⓎ⌧పୗ (ᮏ❶➨ 2 ⠇ Fig. 10A) ࡢ㛵㐃 ᛶࢆ᳨ウࡋࡓࠋపศᏊྜ≀ࡢᶆⓗศᏊࡢ᥈⣴ࡣࣅ࣮ࢬᢸయྜ≀ࢆ⤖ྜ ࡉࡏ࡚ᅛᐃࡋࠊᶆⓗࢱࣥࣃࢡ㉁ࢆࢣ࣑࢝ࣝࣉࣝࢲ࢘ࣥࡍࡿ᪉ἲࢆ⏝࠸ࡿࡇ ࡀ࡛ࡁࡿࡀࠊAIC-47 ࡣྜ≀ࡢᵓ㐀≉ᛶୖࣅ࣮ࢬᢸయ⤖ྜࡉࡏࡿࡇࡀᅔ㞴 ࡛࠶ࡗࡓࠋࡑࡢࡓࡵࠊࢻࢵ࢟ࣥࢢ࣭ࢩ࣑࣮ࣗࣞࢩࣙࣥࡼࡿ in silico ゎᯒࢆ⾜ࡗ ࡓࠋ ࣃ࣑ࣝࢳࣥ㓟 (C16 ⬡⫫㓟) ࡣࠊSTAT3 ⤖ྜࡍࡿࡇ࡛࣮࢜ࢺࣇࢪ࣮ࢆㄏ ᑟࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ78)ࠋSTAT3 AIC-47 ࡢ⤖ྜぶᛶࢆ☜ㄆࡋࡓࠋ ࡑࡢ⤖ᯝࠊAIC-47 STAT3 ࡢ⤖ྜぶᛶࡣపࡃࠊ⤖ྜ࢚ࢿࣝࢠ࣮ࡣ-5.21 kcal/mol ࡛࠶ࡗࡓࠋࡇࡢࡇࡽࠊAIC-47 ࡢᶆⓗศᏊࡣࣃ࣑ࣝࢳࣥ㓟ࡢࡶࡢ ␗࡞ࡗ࡚࠸ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡑࡇ࡛ࠊ⬡⫫㓟ࢆࣜ࢞ࣥࢻࡍࡿཷᐜయ ࡢ⤖ྜぶᛶࢆ᳨ドࡋࡓࠋPeroxisome proliferator-DFWLYDWHGUHFHSWRUȖ 33$5Ȗ ࡣ ࢢࣝࢥ࣮ࢫࠊ⬡㉁࡞ࡢ࢚ࢿࣝࢠ࣮௦ㅰࡢㄪ⠇ᅉᏊࡋ࡚▱ࡽࢀ࡚࠸ࡿ79)ࠋࡲ ࡓࠊ33$5Ȗ ࡢࢦࢽࢫࢺࡋ࡚㛤Ⓨࡉࢀࡓࣆ࢜ࢢࣜࢱࢰࣥࡣ⢾ᒀ⒪⸆ࡋ ࡚㛗ᖺ⏝࠸ࡽࢀ࡚ࡁࡓࡀࠊCML ᖿ⣽⬊ࡢ⒪ࡶ᭷ຠ࡛࠶ࡿࡇࡀሗ࿌ࡉࢀ࡚ ࠸ࡿ80)ࠋAIC-47 33$5Ȗ ࡢ⤖ྜ࢚ࢿࣝࢠ࣮ࡣ-7.60 kcal/mol ࡛࠶ࡾࠊ᳨ウࢆ⾜ ࡗࡓᶆⓗศᏊࡢ୰࡛᭱ࡶ㧗࠸⤖ྜぶᛶࢆ♧ࡋࡓࠋAIC-47 33$5Ȗ ࡢ⤖ྜࣔࢹ ࣝࢆ Fig. 12A ♧ࡋࡓࠋࡀࢇ⣽⬊࠾࠸࡚ࡣࠊάᛶࡉࢀࡓ 33$5Ȗ ࡼࡗ࡚ ȕ-catenin ࡀ⣽⬊㉁ෆ࡛ศゎࡉࢀࠊȕ-catenin ࡼࡿ㌿ࡀᢚไࡉࢀࡿࡇࡀ▱ࡽ ࢀ࡚࠸ࡿ81)ࠋc-Myc ࡣ ȕ-catenin ࡢ㌿ᶆⓗ࡛࠶ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ82)ࠋࡑ ࡇ࡛ࠊAIC-47 ῧຍ⣽⬊ࡢ᰾ෆ࠾ࡼࡧ⣽⬊ෆࡢࢱࣥࣃࢡ㉁ࢆศ⏬ᢳฟࡋ ȕ-catenin
30
ࡢᒁᅾࢆ᳨ウࡋࡓࡇࢁࠊ⣽⬊㉁ෆ࡛ࡢศゎࡀஹ㐍ࡋࠊ᰾ෆࡢ⛣⾜ࡀᢚไࡉ ࢀ࡚࠸ࡓ (Fig. 12B)ࠋࡉࡽࠊsiRNA ࢆ⏝࠸࡚ ȕ-catenin ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥ ࡍࡿࠊc-Myc ࡢⓎ⌧పୗࡀㄆࡵࡽࢀࡓ (Fig. 12C)ࠋ௨ୖࡢ⤖ᯝࡽࠊAIC-47
ࡣ33$5Ȗ ࢆάᛶࡉࡏࠊȕ-catenin ࡢ᰾ෆ⛣⾜ࢆᢚไࡍࡿࡇ࡛ c-Myc ࡢⓎ⌧ప
ୗࢆㄏᑟࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋࡋࡋ࡞ࡀࡽࠊᮏ⠇࡛ࡢ᳨ウࡣ㛫᥋ⓗ࡞ᐇ㦂 ⣔ࡼࡿࡶࡢ࡛࠶ࡿࡓࡵࠊࡉࡽヲ⣽࡞᳨ウࡀᚲせ࡛࠶ࡿࠋ
Fig. 12 DHJUDGDWLRQRIȕ-catenin after AIC-ERXQGWR33$5ȖLQGXFHGWKH down-regulation of c-Myc.
(A) 0ROHFXODUPRGHORI33$5ȖELQGLQJZLWK$,&-33$5ȖLVVKRZQE\a ribbon diagram. The top-scored docking model was visualized. (B) Effect of AIC-RQVXEFHOOXODUGLVWULEXWLRQRIȕ-catenin. The expression OHYHOVRIF\WRSODVPLFDQGQXFOHDUȕ-catenin at 72 h after the treatment of K562 and KCL-22 cells with AIC-47 were examined by Western blot analysis. Cell lysates were collected and fractionated into cytoplasmic and nuclear extracts. The efficiency of fractionation was verified by staining for Histone H3 as a nuclear marker and for ȕ-actin as a cytoplasmic marker and also as an indicator for contamination of the nuclear fraction with cytoplasmic protein. (C) Effect of silencing ȕ-catenin with siR-ȕ-catenin on the expression of c-Myc at 72 h after transfection. Effects of the silencing with siR-ȕ-catenin on the expression of ȕ-catenin and c-Myc were examined by Western blot analysis. Numbers below blots in “B” and “C” indicate each band density relative to that of the Control (taken as 100), which values were determined by densitometry.
31 ➨ 7 ⠇ ᑠᣓ ᮏ❶࡛ࡣࠊࡇࢀࡲ࡛᫂ࡽࡉࢀ࡚࠸࡞ࡗࡓ୰㙐⬡⫫㓟ࡢ⏕⌮άᛶࡢ 1 ࡘ ࡋ࡚ࠊ୰㙐⬡⫫㓟ㄏᑟయ AIC-47 ࡀ࣮࢜ࢺࣇࢪ࣮⣽⬊Ṛࢆㄏᑟࡍࡿࡇ࡛ᢠ ࡀࢇస⏝ࢆ♧ࡍࡇࢆ᫂ࡽࡋࡓࠋ⭠ࡀࢇ⣽⬊ᑐࡍࡿࣈࢳࣝ㓟ㄏᑟయ (C4) ࡢᢠࡀࢇάᛶࡣ 2 mM ௨ୖ67)ࠊ⫢⮚ࡀࢇ⣽⬊ᑐࡍࡿࣃ࣑ࣝࢳࣥ㓟 (C16) ࠾ࡼࡧࢫࢸࣜࣥ㓟 (C18) ࡢᢠࡀࢇάᛶࡣ ȝ0 ௨ୖ83)࡛ㄆࡵࡽࢀࡿࡇࡀ ሗ࿌ࡉࢀ࡚࠾ࡾࠊࡀࢇ✀ࡣ␗࡞ࡿࡶࡢࡢࠊ▷㙐࣭㛗㙐⬡⫫㓟ẚ㍑ࡋ࡚୰㙐⬡ ⫫㓟ㄏᑟయࡀࡼࡾᙉ࠸ᢠࡀࢇάᛶࢆ♧ࡍࡇࡀ᫂ࡽ࡞ࡗࡓࠋ ࡲࡓࠊCML ࡢࢻࣛࣂ࣮㑇ఏᏊ࡛࠶ࡿ BCR-ABL ࡀࡀࢇ⣽⬊≉␗ⓗ࢚ࢿࣝࢠ ࣮௦ㅰᶵᵓ (Warburg ຠᯝ) ࡢஹ㐍ᐤࡋ࡚࠸ࡿ࠸࠺᪂ࡓ࡞▱ぢࢆᚓࡓࠋᮏ ❶ࡢ᳨ウ⤖ᯝࡽ⪃࠼ࡽࢀࡓ AIC-47 ࡢቑṪᢚไ࣓࢝ࢽࢬ࣒ࡣ Fig. 13 ♧ࡋࡓ ㏻ࡾ࡛࠶ࡿࠋࡲࡎࠊAIC-47 ࡣ P3$5Ȗ ࢆάᛶࡉࡏࠊȕ-catenin ࢆࡋ࡚ c-Myc ࡢ Ⓨ⌧ࢆᢚไࡍࡿࠋ⥆࠸࡚ c-Myc ࡢⓎ⌧పୗࡀ㌿ᶆⓗ࡛࠶ࡿ BCR-ABL ࡢⓎ⌧ ࢆῶᑡࡉࡏࡿࠋc-Myc ࠾ࡼࡧ BCR-ABL ࡢⓎ⌧పୗࡣඹ PTBP1/PKM ࢝ࢫࢣ࣮ ࢻࡢ⬺ไᚚࢆㄏᑟࡋࠊࡀࢇ⣽⬊ࡢࢢࣝࢥ࣮ࢫ௦ㅰࢆゎ⢾⣔ࡽ TCA ࢧࢡࣝ ࢫࢵࢳࡉࡏࡿࠋ᭱⤊ⓗ TCA ࢧࢡࣝࡽⓎ⏕ࡍࡿ ROS ࡀཎᅉ࡞ࡾࠊ࢜ ࣮ࢺࣇࢪ࣮⣽⬊Ṛࡀㄏᑟࡉࢀࡿ⪃࠼ࡽࢀࡓࠋ ୰㙐⬡⫫㓟ㄏᑟయࡣ BCR-ABL ࠾ࡼࡧ Warburg ຠᯝ࠸࠺ࡀࢇ⣽⬊≉␗ⓗⓎ ⌧ࡋ࡚࠸ࡿᶵᵓࢆస⏝Ⅼࡍࡿࡇ࡛ࠊࡀࢇ⣽⬊ࡢࡳࢆ㑅ᢥⓗയᐖࡋ࡚࠾ࡾࠊ ẚ㍑ⓗᏳᛶࡢ㧗࠸⸆ࢩ࣮ࢬ࡞ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ
32
Fig. 13 Schematic diagram of AIC-47-induced autophagic cell death in BCR-ABL CML cells.
33 ➨ 4 ❶ ࡀࢇ⣽⬊ࡢ࢚ࢿࣝࢠ࣮௦ㅰᑐࡍࡿ୰㙐⬡⫫㓟ㄏᑟయ࣐ࢳࢽࣈࡢ ẚ㍑ ➨ 1 ⠇ ᗎ CML ࡢ➨୍㑅ᢥ⸆ࡋ࡚⏝ࡉࢀࡿ࣐ࢳࢽࣈࡣࠊBCR-ABL ࡢ࢟ࢼ࣮ࢮ άᛶࢆᶆⓗࡋࡓ⒪⸆࡛࠶ࡾࠊ㠀ᖖඃࢀࡓ⒪ᡂ⦼ࢆ♧ࡋ࡚࠸ࡿࠋࡑࡢ ୍᪉࡛ࠊⓎࡢཎᅉ࡞ࡿⓑ⾑ᖿ⣽⬊ᑐࡋ࡚ࡣ⒪ຠᯝࡀపࡃࠊⓑ⾑ ᖿ⣽⬊ࡶ᭷ຠ࡞⒪⸆ࡢ㛤Ⓨࡀᮃࡲࢀ࡚࠸ࡿࠋ㏆ᖺࠊⓑ⾑ᖿ⣽⬊ᑐࡍ ࡿ⒪ἲࡢ◊✲ࡀ┒ࢇ⾜ࢃࢀ࡚࠾ࡾࠊᵝࠎ࡞ే⏝⸆ࡢ᭷ຠᛶࡀሗ࿌ࡉࢀ࡚ ࠸ࡿࠋࡑࡢ୰࡛ࠊ࢚ࢿࣝࢠ࣮௦ㅰࢆไᚚࡍࡿࡼ࠺࡞⒪ἲࡶᥦࡉࢀ࡚ࡁࡓࠋ ࡑࡇ࡛ࠊᮏ❶࡛ࡣࡲࡎ୰㙐⬡⫫㓟ㄏᑟయ AIC-47 ࣐ࢳࢽࣈࡢస⏝Ⅼࡢ㐪࠸ ࢆ࢚ࢿࣝࢠ࣮௦ㅰ࠸࠺ほⅬࡽ㠀ᖿ⣽⬊ᛶࡢ CML ⣽⬊࡛ẚ㍑᳨ウࡋࠊࡉࡽ ⓑ⾑ᖿ⣽⬊࠾ࡅࡿ AIC-47 ࡢ᭷ຠᛶࢆ᳨ドࡋࡓࠋ ➨ 2 ⠇ ୰㙐⬡⫫㓟ㄏᑟయ࠾ࡼࡧ࣐ࢳࢽࣈࡼࡿ⣽⬊Ṛࡢẚ㍑ K562 ࠾ࡼࡧ KCL-22 AIC-47 ࡲࡓࡣ࣐ࢳࢽࣈ (IM) ࢆῧຍࡋࠊ48 㛫 ᚋࡢ⣽⬊⏕Ꮡ⋡ࢆࢺࣜࣃࣥࣈ࣮ࣝⰍ⣲㝖ヨ㦂ἲࡼࡾ ᐃࡋࡓࠋAIC-47 ࠾ ࡼࡧ࣐ࢳࢽࣈࡣࡑࢀࡒࢀ༢࡛ࡶ᭷ព࡞ቑṪᢚไࢆ♧ࡋࡓࡀࠊ2 ࢆే⏝ࡍ ࡿࡇࡼࡾቑṪᢚไస⏝ࡢቑᙉࡀㄆࡵࡽࢀࡓ (Fig. 14A)ࠋࡑࡇ࡛ࠊే⏝ຠᯝ ࡢᐃ㔞ⓗᣦᶆ࡞ࡿే⏝ಀᩘ (Combination Index; CI) ࢆࣉࣟࢵࢺࡍࡿ CI < 1 ࡛࠶ࡗࡓ (Fig. 14B)ࠋ௨ୖࡢ⤖ᯝࡽࠊAIC-47 ࣐ࢳࢽࣈࡢే⏝ࡣ┦ⓗ
࡞ቑṪᢚไస⏝ࢆ♧ࡍࡇࡀ᫂ࡽ࡞ࡗࡓࠋ
⥆࠸࡚ࠊྛ⸆ࡼࡿ⣽⬊Ṛࡢᙧែࢆ᳨ドࡋࡓࠋ࣏ࢺ࣮ࢩࢫࡢᣦᶆࡋ࡚ PARP-1 ࢆࠊ࣮࢜ࢺࣇࢪ࣮ࡢᣦᶆࡋ࡚ p62 ࡢⓎ⌧ࢆࡑࢀࡒࢀ࢙࢘ࢫࢱࣥࣈ
34 ㄆࡉࢀࡎࠊp62 ࡢศゎࡢࡳࡀほᐹࡉࢀࡓࡀࠊ࣐ࢳࢽࣈࢆῧຍࡋࡓ⣽⬊࡛ࡣ PARP-1 ࡢษ᩿ p62 ࡢศゎࡀㄆࡵࡽࢀࡓ (Fig. 14C)ࠋࡉࡽࠊ㟁Ꮚ㢧ᚤ㙾ゎ ᯒࡼࡾྛ⸆ῧຍᚋࡢ K562 ⣽⬊ࡢᙧែᏛⓗኚࢆ᳨ドࡋࡓࠋAIC-47 ῧຍ ⣽⬊࡛ࡣࠊ➨ 3 ❶➨ 3 ⠇࡛ࡢ᳨ウ⤖ᯝྠᵝ࣮࢜ࢺࣇࢦࢯ࣮࣒ (AP) ࡢᙧ ᡂ⬡⫫ࡢ✚ࡀ☜ㄆࡉࢀࡓ (Fig. 14D)ࠋ࣐ࢳࢽࣈῧຍ⣽⬊࡛ࡣ᰾ࡢ᩿∦ ࡀㄆࡵࡽࢀࠊ࣏ࢺ࣮ࢩࢫࡢㄏᑟࡀ♧၀ࡉࢀࡓ (Fig. 14D)ࠋࡲࡓࠊ2 ࡢే ⏝ࡼࡾ AIC-47 ࡢ≉ᚩ࡛࠶ࡿ lipophagy ࣐ࢳࢽࣈࡢ≉ᚩ࡛࠶ࡿ᰾ࡢ᩿∦ ࡀྠ୍⣽⬊ෆほᐹࡉࢀࡓ (Fig. 14D)ࠋḟࠊ࣐ࢳࢽࣈࡼࡿ࣮࢜ࢺࣇ ࢪ࣮ࡀ⣽⬊ࡢ⏕Ꮡಁ㐍ࡶࡋࡃࡣ⣽⬊Ṛࡢ࠸ࡎࢀࡢᶵᵓᐤࡋ࡚࠸ࡿࢆ᳨ ドࡍࡿࡓࡵࠊ࣮࢜ࢺࣇࢪ࣮㜼ᐖ࡛࠶ࡿࢡࣟࣟ࢟ࣥࡢే⏝ᐇ㦂ࢆ⾜ࡗࡓࠋ ࢡࣟࣟ࢟ࣥࡢຠᯝุᐃࡣ p62 ࡢศゎᢚไࢆᣦᶆࡋࡓࠋࢡࣟࣟ࢟ࣥࡢే⏝ࡼ ࡾ AIC-47 ῧຍ⣽⬊࡛ࡣ⣽⬊⏕Ꮡ⋡ࡀᅇࡋ (Fig. 14E)ࠊ➨ 3 ❶➨ 3 ⠇ࡢ⤖ᯝ ୍⮴ࡋࡓࠋ୍᪉ࠊ࣐ࢳࢽࣈῧຍ⣽⬊࡛ࡣ࣮࢜ࢺࣇࢪ࣮ࡢ㜼ᐖࡼࡾ⣽⬊ ⏕Ꮡ⋡ࡀࡉࡽపୗࡋࡓ (Fig. 14E)ࠋࡇࡢ⤖ᯝࡽࠊ࣐ࢳࢽࣈῧຍ⣽⬊࡛ࡣ ⏕Ꮡ⥔ᣢᶵᵓࡋ࡚ࡢ࣮࢜ࢺࣇࢪ࣮ࡀㄏᑟࡉࢀ࡚࠸ࡿࡇࡀ♧၀ࡉࢀࠊࡇࢀ ࡲ࡛ࡢሗ࿌୍⮴ࡋࡓࠋ௨ୖࡢ⤖ᯝࡽࠊAIC-47 ࡣ࣮࢜ࢺࣇࢪ࣮⣽⬊Ṛࠊ ࣐ࢳࢽࣈࡣ࣮࢜ࢺࣇࢪ࣮ࢆక࠺࣏ࢺ࣮ࢩࢫ࡛ቑṪᢚไస⏝ࢆ♧ࡋ࡚࠾ ࡾࠊྛ⸆ࡼࡿ⣽⬊Ṛࡢᙧែࡀ␗࡞ࡗ࡚࠸ࡿࡇࡀ☜ㄆࡉࢀࡓࠋ
35
Fig. 14 Morphological features of cells treated with AIC-47 and/or imatinib Cells were treated with DMSO (Control; C), AIC-47 (47; ȝ0 LPDWLQLE IM; 0.2ȝ0IRU. ȝ0IRU.&/-22) or their combination (47+IM) for 48 h. (A) Viability of K562 and KCL-22 cells treated with AIC-47 and/or imatinib for 48 h. (B) Synergistic effects of AIC-47 and imatinib. Combination Index (CI) was calculated by Chou-Talalay’s method. The dashed line indicates the zero interaction of the isobole. (C) Effects of AIC-47, imatinib or their combination on apoptosis and autophagy. (D) Morphology of the AIC-47 and/or imatinib (IM)-treated K562 cells at 48 h was determined by TEM. RepresenWDWLYHLPDJHV VFDOHEDUȝP DUHVKRZQ N: nucleus, AP: autophagosome, AV: autophagic vacuole. (E) Effects of inhibition of autophagy by chloroquine on cytotoxicity of AIC-47, imatinib or their combination. Expression of p62 was examined by Western blot analysis.
36
➨ 3 ⠇ BCR-ABL ゎ⢾⣔ᑐࡍࡿᙳ㡪ࡢẚ㍑
➨ 3 ❶➨ 4 ⠇࡛ AIC-47 ࡀ BCR-ABL ࡢ㌿ᢚไࢆㄏᑟࡋࠊࡀࢇ⣽⬊ࡢゎ⢾ ⣔ (Warburg ຠᯝ) ࢆ◚⥢ࡉࡏࡿࡇࢆ᫂ࡽࡋࡓࠋࡑࡇ࡛࣐ࢳࢽࣈࡼ ࡿ BCR-ABL ࠾ࡼࡧ Warburg ຠᯝࡢᙳ㡪ࢆ᳨ドࡋࡓࠋࡲࡎࠊBCR-ABL ࡢⓎ ⌧࠾ࡼࡧࣜࣥ㓟ࣞ࣋ࣝࡢኚࢆ࢙࢘ࢫࢱࣥࣈࣟࢵࢺࡼࡾゎᯒࡍࡿࠊ AIC-47 ࡣ BCR-ABL ࡢⓎ⌧ࣜࣥ㓟ࣞ࣋ࣝࡢ୧᪉ࢆᢚไࡍࡿࡢᑐࡋࠊ
࣐ࢳࢽࣈࡣࣜࣥ㓟ࡢࡳࢆᢚไࡋࠊBCR-ABL ࡢⓎ⌧ࡣኚࡉࡏ࡞࠸ࡇࡀ☜ ㄆࡉࢀࡓ (Fig. 15A)ࠋࡲࡓࠊ2 ࡢే⏝࡛ࡣ BCR-ABL ࡢⓎ⌧ࠊࣜࣥ㓟ࣞ࣋ ࣝࡶ㢧ⴭᢚไࡉࢀ࡚࠸ࡓ (Fig. 15A)ࠋ⥆࠸࡚ࠊྛ⸆ࡢ Warburg ຠᯝ ᑐࡍࡿᙳ㡪ࢆ᳨ドࡋࡓࠋ࣐ࢳࢽࣈࡣࠊࢢࣝࢥ࣮ࢫࢺࣛࣥࢫ࣏࣮ࢱ࣮GLUT-1 ࡢⓎ⌧ࢆῶᑡࡉࡏࡿࡇ࡛⣽⬊ෆࡢࢢࣝࢥ࣮ࢫࡢྲྀࡾ㎸ࡳࢆᢚไࡍࡿࡇ
ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ84,85)ࠊPTBP1/PKM ࢝ࢫࢣ࣮ࢻᑐࡍࡿሗ࿌ࡣ࡞࠸ࠋࡑࡇ
࡛࢙࢘ࢫࢱࣥࣈࣟࢵࢺࡼࡿゎᯒࢆ⾜ࡗࡓࡇࢁࠊAIC-47 ྠᵝ࣐ࢳࢽ ࣈࡶ PTBP1 ࠾ࡼࡧ PKM2 ࡢⓎ⌧ࢆపୗࡉࡏࠊPKM1 ࡢⓎ⌧ࢆቑຍࡉࡏࡿࡇ ࡀ᫂ࡽ࡞ࡗࡓ (Fig. 15A)ࠋࡇࡢ⌧㇟ࡣ AIC-47 ࡼࡾࡶ࣐ࢳࢽࣈ࡛ᙉࡃㄆ ࡵࡽࢀࡓࠋ⣽⬊ෆࡢ PKM1 ࠾ࡼࡧ PKM2 ࢆචᰁⰍࡋࡓࡇࢁࠊPKM2 ࡽ PKM1 ࡢⓎ⌧ࢫࢵࢳࡀ single-cell ࡛ࣞ࣋ࣝㄏᑟࡉࢀ࡚࠸ࡿࡇࡀ᫂ࡽ ࡞ࡗࡓ (Fig. 15B)ࠋ᎘Ẽⓗゎ⢾ࡢ᭱⤊⏘≀࡛࠶ࡿ L-lactate ⏘⏕㔞ࡶྛ⸆ῧຍ ᚋ᭷ពῶᑡࡋ࡚࠸ࡓ (Fig. 15C)ࠋ௨ୖࡢ⤖ᯝࡽࠊAIC-47 ࠾ࡼࡧ࣐ࢳ ࢽࣈࡣࡶ Warburg ຠᯝࡢ◚⥢ࢆㄏᑟࡍࡿࡇࡀ᫂ࡽ࡞ࡾࠊBCR-ABL ࡼࡿ Warburg ຠᯝࡢஹ㐍ࡣࢳࣟࢩࣥࣜࣥ㓟ࡀ㔜せ࡛࠶ࡿࡇࡀ♧၀ࡉ ࢀࡓࠋ
37
Fig. 15 Effects of AIC-47 and/or imatinib on BCR-ABL and Warburg effect. Cells were treated with DMSO (Control; C), AIC-47 (47; ȝ0 LPDWLQLE IM; 0.2ȝ0IRU. ȝ0IRU.&/-22) or their combination (47+IM) for 48 h. (A) Effects of AIC-47, imatinib or their
combination on expression of BCR-ABL and Warburg effect-related proteins. The numbers below PTBP1, PKM1, and PKM2 indicate each band density relative to that of the Control (taken as “1”). (B)
Immunofluorescence of PKM1 and PKM2 in K562 cells. Representative images (scale bar, 5ȝP are shown. (C) Lactate production in K562 and KCL-22 cells. The lactate production was normalized to cell numbers.
38 ➨ 4 ⠇ ⬡⫫㓟㓟ᑐࡍࡿస⏝ ⣽⬊ࡢせ࡞࢚ࢿࣝࢠ࣮※ࡣ⢾ (ࢢࣝࢥ࣮ࢫ) ࡛࠶ࡿࡀࠊ⢾௦ㅰࡼࡿ ATP ࡢ౪⤥ࡀ㊊ࡍࡿ⬡⫫㓟ࡸ࣑ࣀ㓟ࢆ⏝ࡋ࡚ ATP ࢆ⏘⏕ࡍࡿࡇࡀ▱ࡽ ࢀ࡚࠸ࡿࠋⓑ⾑⣽⬊࠾࠸࡚ࡶゎ⢾⣔ࡢୖὶศᏊࢆ㜼ᐖࡍࡿࡇ࡛ CPT1C ࡢⓎ⌧ࡀቑຍࡋࠊ⬡⫫㓟㓟ࡀάᛶࡉࢀࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ62)ࠋࡑࡇ ࡛ࠊCPT1C ࡢⓎ⌧㔞ࢆ࢙࢘ࢫࢱࣥࣈࣟࢵࢺࡼࡾゎᯒࡍࡿࠊ࣐ࢳࢽࣈࢆ ༢࡛ῧຍࡋࡓሙྜࡢࡳ CPT1C ࡢⓎ⌧ஹ㐍ࡀㄆࡵࡽࢀࠊAIC-47 ࢆῧຍࡋࡓ ⣽⬊࡛ࡣࡑࡢⓎ⌧ࡀᢚไࡉࢀࡿࡇࡀ᫂ࡽ࡞ࡗࡓ (Fig. 16A)ࠋⓑ⾑⣽⬊ ࠾ࡅࡿ⬡⫫㓟㓟ࡢάᛶࡀᏛ⒪ἲᑐࡍࡿឤཷᛶࡢపୗ㛵ࢃࡗ࡚࠸ ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ86)ࠋCPT1 ࡢ㜼ᐖ࡛࠶ࡿ Etomoxir ࢆ⏝࠸ࠊ⬡⫫㓟㓟 ࢆ㜼ᐖࡍࡿྛ⸆ࡼࡿ⣽⬊ቑṪᢚไస⏝ࡢቑᙉࡀㄆࡵࡽࢀࠊࡑࡢస⏝ࡣ ≉࣐ࢳࢽࣈ࡛ᙉ࠸ࡇࡀ᫂ࡽ࡞ࡗࡓ (Fig. 16B)ࠋ௨ୖࡢ⤖ᯝࡽࠊ ࣐ࢳࢽࣈࡣ CPT1C ࡢⓎ⌧ቑຍࢆࡋ࡚⬡⫫㓟㓟ࢆάᛶࡍࡿࡢᑐࡋࠊ AIC-47 ࡣ⬡⫫㓟㓟ࢆᢚไࡋ࡚࠸ࡿࡇࡀ♧၀ࡉࢀࡓࠋࡑࡇ࡛ᇵᆅࡢ⤌ᡂࢆ ኚ᭦ࡋࠊྛ⸆ࢆῧຍࡋࡓ⣽⬊ࡢ ATP ࣞ࣋ࣝࢆ ᐃࡋࡓࠋࢢࣝࢥ࣮ࢫ⬡⫫ 㓟ࡀᏑᅾࡍࡿ㏻ᖖࡢᇵᆅ (Glucose +, Fatty-acid +) ࡛ࡣࠊ࠸ࡎࢀࡢ⸆ࢆῧຍ ࡋࡓሙྜࡶ⣽⬊ෆ ATP ࣞ࣋ࣝࡣపୗࡋࡓ (Fig. 16C)ࠋࢢࣝࢥ࣮ࢫࡢࡳࢆ㝖 ࠸ࡓᇵᆅ୰ (Glucose í, Fatty-acid +) ࡛ࡣ࣐ࢳࢽࣈῧຍ⣽⬊ࡢ ATP ࣞ࣋ࣝࡀ 㢧ⴭቑຍࡋࠊࡉࡽ⬡⫫㓟ࢆඹ㝖ࡃ (Glucose í, Fatty-acid í) ࣐ࢳࢽ ࣈῧຍ⣽⬊࡛ࡢ ATP ࣞ࣋ࣝࡢቑຍࡀᢚไࡉࢀࡓ (Fig. 16C)ࠋ௨ୖࡢ⤖ᯝࡽࠊ ࣐ࢳࢽࣈῧຍ⣽⬊ࡣ⬡⫫㓟ࢆ⏝ࡋ⣽⬊ෆࡢ ATP ࣞ࣋ࣝࢆ⥔ᣢࡋ࡚࠸ࡿࡇ ࡀ♧၀ࡉࢀࡓࠋ ᮏ❶➨ 3 ⠇࡛࣐ࢳࢽࣈࡀ Warburg ຠᯝࡢ◚⥢ࢆㄏᑟࡍࡿࡇࡀ♧ࡉࢀࡓࡇ ࡽࠊWarburg ຠᯝࡢ◚⥢⬡⫫㓟㓟ࡢάᛶࡢ㛵㐃ᛶࢆ᳨ドࡋࡓࠋ
39 BCR-ABL ࠾ࡼࡧ PTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓࡇࢁࠊCPT1C ࡢⓎ⌧ஹ㐍 ࡀㄆࡵࡽࢀࡓ (Fig. 16D, E)ࠋࡇࡢ⤖ᯝࡽࠊBCR-ABL/PTBP1 ࢝ࢫࢣ࣮ࢻࢆ ࡋࡓ Warburg ຠᯝࡢ◚⥢ࡀ௦ൾⓗ࡞⬡⫫㓟㓟ࡢάᛶᐤࡋ࡚࠸ࡿࡇ ࡀ♧ࡉࢀࡓࠋࡲࡓࠊAIC-47 ࡣྠࡌ࢝ࢫࢣ࣮ࢻࢆࡋ࡚ Warburg ຠᯝࢆ◚⥢ࡉ ࡏࡿࡶ㛵ࢃࡽࡎ CPT1C ࡢⓎ⌧ࢆቑຍࡉࡏ࡞࠸ࡓࡵࠊWarburg ຠᯝࡣ⊂❧ ࡋࡓ⤒㊰࡛⬡⫫㓟㓟ࡢάᛶࢆᢚไࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡓࠋ ࡑࡇ࡛ࠊ࢞ࢫࢡ࣐ࣟࢺࢢࣛࣇ࣮ࡼࡾ K562 ⣽⬊ෆࡢ⬡⫫㓟⤌ᡂࢆ᳨ドࡋ ࡓࠋAIC-47 ῧຍᚋࡢ⣽⬊ෆ࡛ࡣ㙐ࡢⅣ⣲ᩘࡀ 14 (C14) ࡢ⬡⫫㓟ࠊࡍ࡞ࢃࡕ ࣑ࣜࢫࢳࣥ㓟࠾ࡼࡧ࣑ࣜࢫࢺࣞࣥ㓟ࡢྵ᭷㔞ࡀቑຍࡍࡿഴྥࡀㄆࡵࡽࢀࡓ (Fig. 16F)ࠋ⬡⫫㓟㓟ࡢ㐣⛬࠾࠸࡚ C14 ⬡⫫㓟ࡣ࣑ࢺࢥࣥࢻࣜෆྲྀࡾ
㎸ࡲࢀࡓᚋࠊlong-chain acyl-CoA dehydrogenase (LCAD) ࡼࡾ௦ㅰࡉࢀࡿ (➨ 2 ❶➨ 4 ⠇ Fig. 5)ࠋAIC-47 ࡀ C14 ⬡⫫㓟ࡢ௦ㅰᙳ㡪ࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼
ࡽࢀࡓࡇࡽࠊ↓⣽⬊⣔࡛ LCAD ࡢ㓝⣲άᛶࢆ ᐃࡋࡓࠋAIC-47 ඹᏑୗ࡛ ࡣ LCAD ࡼࡿ C14 ⬡⫫㓟ࡢ௦ㅰࡀ➇ྜⓗ㜼ᐖࡉࢀࡓ (Fig. 16G)ࠋ௨ୖࡢ ⤖ᯝࡽࠊAIC-47 ࡣ LCAD ࡢ㜼ᐖࢆࡋ࡚ C14 ⬡⫫㓟ࡢ௦ㅰࢆጉࡆࠊWarburg ຠᯝ㠀౫Ꮡⓗ⬡⫫㓟㓟ࢆ㜼ᐖࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋ
40
Fig. 16 Compensatory activation of fatty-acid oxidation after treatment with imatinib and its suppression by AIC-47
K562 and KCL-22 cells were treated with DMSO (C; Control), AIC-47 (47; ȝ0 LPDWLQLE ,0; 0.25 ȝ0IRU.ȝ0IRU.&/-22) or their combination (47+IM) for 48 h. (A) Expression of CPT1C. The numbers below CPT1C indicate each band density relative to that of the Control (taken as “1”). (B) Inhibition of FAO by Etomoxir. K562 and KCL-22 cells were co-incubated oUQRWZLWKȝ0(WRPRxir and each agent for 48 h. (C) ATP production in K562 and KCL-22 cells after treatment with each agent for 6 h. The cells were cultured in medium as described below the graph. The ATP production was
41
normalized to cell numbers. (D, E) Expression of CPT1C in K562 and KCL-22 cells after transfection with siR-BCR-ABL (D) or PTBP1 (E) for 72 h. (F) Fatty-acid composition analyzed by gas
chromatography. (G) Enzyme activity of long-chain acyl-CoA dehydrogenase (LCAD) with or without AIC-47 (10 nM). The dimensions of apparent Vmax, Km, and Ki values were nmol min-1(mg of protein)-1, ȝ0DQGȝ0UHVSHFWLYHO\
42 ➨ 5 ⠇ ⓑ⾑ᖿ⣽⬊࠾ࡅࡿ AIC-47 ࡢ᭷ຠᛶ ⓑ⾑⣽⬊࠾ࡅࡿᖿ⣽⬊࣐࣮࣮࢝ࡣ CD34 ࡀ⏝࠸ࡽࢀ࡚࠸ࡿࠋⓑ⾑ᖿ ⣽⬊࠾ࡅࡿ AIC-47 ࡢ᭷ຠᛶࢆ᳨ドࡍࡿࡓࡵࠊCML ᝈ⪅ࡢ⾑⌫⣽⬊ࡽ CD34+ศ⏬ࡢ᥇ྲྀࢆヨࡳࡓࠋࡋࡋ࡞ࡀࡽ CML ࡢᖿ⣽⬊ࡣࡢⓑ⾑ᖿ⣽⬊ ẚ㍑ࡋ࡚㠀ᖖቯࢀࡸࡍࡃࠊ⸆ຠᯝࡢ᳨ウࢆ⾜࠺ࡇࡀᅔ㞴࡛࠶ࡗࡓࠋ ࡑࡇ࡛ࠊPh+ ALL ࡢࣔࢹ࣐ࣝ࢘ࢫࡼࡾ᥇ྲྀࡋࡓ CD34+ศ⏬ࢆᐇ㦂⏝ࡋࡓࠋ ࡲࡎࡇࡢᖿ⣽⬊ᵝศ⏬ᑐࡋ AIC-47 ࠾ࡼࡧ࣐ࢳࢽࣈࢆస⏝ࡉࡏࡓࡇࢁࠊ AIC-47 ࡣ༢⊂࡛᭷ព࡞⣽⬊⏕Ꮡ⋡ࡢపୗࡀㄆࡵࡽࢀࡓࡀࠊ0.5 ȝ0 ࡢ࣐ࢳࢽ ࣈ࡛ࡣ⣽⬊⏕Ꮡ⋡ኚࡣㄆࡵࡽࢀ࡞ࡗࡓ (Fig. 17A)ࠋ㠀ᖿ⣽⬊ᛶࡢ CML ᑐࡋ࡚࣐ࢳࢽࣈࡣ 0.1 ȝ0 ࡛᭷ព࡞ቑṪᢚไࢆ♧ࡋ࡚࠸ࡿ (ᮏ❶➨ 2 ⠇ Fig. 14A) ࡇࡽࠊCD34+ศ⏬࡛ࡣ࣐ࢳࢽࣈᑐࡍࡿឤཷᛶࡀపୗࡋ࡚࠸ࡿࡇ ࡀ᫂ࡽ࡞ࡗࡓࠋ⥆࠸࡚ࠊ⣽⬊ෆࢩࢢࢼࣝࢆ࢙࢘ࢫࢱࣥࣈࣟࢵࢺࡼࡾ ゎᯒࡋࡓࠋࡑࡢ⤖ᯝࠊ࠸ࡎࢀࡢ⸆ࢆῧຍࡋࡓ⣽⬊࠾࠸࡚ࡶ Warburg ຠᯝ 㛵㐃ศᏊࡢ⬺ไᚚࠊࡍ࡞ࢃࡕ PTBP1 ࡢⓎ⌧పୗ PKM2 ࡽ PKM1 ࡢⓎ⌧ࢫ ࢵࢳࡣඹ㏻ࡢ⌧㇟ࡋ࡚ほᐹࡉࢀࡓ (Fig. 17B)ࠋ୍᪉ࠊ࣐ࢳࢽࣈࢆ༢࡛ ῧຍࡋࡓ⣽⬊ࡢࡳ࡛ CPT1C ࡢⓎ⌧ஹ㐍ࡀㄆࡵࡽࢀࡓ (Fig. 17B)ࠋࡑࡇ࡛ࠊ Etomoxir ࢆ⏝࠸⬡⫫㓟㓟ࢆ㜼ᐖࡋࡓࡇࢁࠊ༢࡛ࡣ⣽⬊ࡢ⏕Ꮡ⋡ࢆኚ ࡉࡏ࡞ࡗࡓ࣐ࢳࢽࣈ࠾࠸࡚ࡶ᭷ព࡞⣽⬊⏕Ꮡ⋡ࡢపୗࡀㄆࡵࡽࢀࡓ (Fig. 17C)ࠋ௨ୖࡢ⤖ᯝࡽࠊᖿ⣽⬊࠾ࡅࡿ࣐ࢳࢽࣈ㠀ឤཷᛶࡢ⋓ᚓࡣ ⬡⫫㓟㓟ࡢάᛶࡀᐤࡋ࡚࠾ࡾࠊ⬡⫫㓟㓟ࢆ㜼ᐖࡍࡿ AIC-47 ࡀⓑ⾑ ᖿ⣽⬊ᑐࡋ࡚ࡶ᭷ຠ࡞⒪⸆࡞ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ
43
Fig. 17 Ex vivo effects of AIC-47 on the CD34+fraction of Ph-positive ALL cells (A) Viability of Ph-positive CD34+murine ALL stem cells treated with DMSO (Control), AIC-47, imatinib (IM) or their combination for 48 h. (B) Effects of AIC-47, imatinib or their combination on apoptosis, autophagy, Warburg effect-related proteins, and CPT1C. Cells were treated with DMSO (Control; C), AIC-47 (47; 5 ȝ0 LPDWLQLE IM; 1 ȝ0 RUtheir combination (47+IM) for 48 h. The numbers below PTBP1, PKM1, PKM2, and CPT1C indicate each band density relative to that of the Control (taken as “1”). (C) Inhibition of FAO by Etomoxir. Cells were co-incubated oUQRWZLWKȝ0 Etomoxir and AIC-47 (47; ȝ0 or imatinib (IM; ȝ0 IRUK.
44 ➨ 6 ⠇ ᑠᣓ ᮏ❶࡛ࡣࠊ୰㙐⬡⫫㓟ㄏᑟయ AIC-47 ᪤Ꮡ⸆࣐ࢳࢽࣈࡢస⏝Ⅼࡢ㐪࠸ࢆ ࢚ࢿࣝࢠ࣮௦ㅰ࠸࠺ほⅬࡽẚ㍑᳨ウࡋࡓࠋ᳨ウ⤖ᯝࢆ Fig. 18 ࠾ࡼࡧ Table 2 ࡲࡵࡓࠋAIC-47 ࣐ࢳࢽࣈࡣ␗࡞ࡗࡓ⣽⬊ṚࢆㄏᑟࡋࠊBCR-ABL ࡢ Ⓨ⌧ᑐࡍࡿస⏝ࡶ␗࡞ࡗ࡚࠸ࡿࡇࢆ☜ㄆࡋࡓࠋࡲࡓࠊ࣐ࢳࢽࣈࡀ Warburg ຠᯝࢆᙉຊ◚⥢ࡉࡏࡿࡇࢆぢฟࡋࡓࠋࡇࢀࡲ࡛ࡢ◊✲࡛ࡣ࣐ࢳ ࢽࣈ࢚ࢿࣝࢠ࣮௦ㅰࡢ㛵㐃ᛶࡣࢇ᫂ࡽࡉࢀ࡚࠾ࡽࡎࠊࢢࣝࢥ ࣮ࢫࡢྲྀࡾ㎸ࡳࢆไᚚࡍࡿ GLUT-1 ࡢⓎ⌧ࢆኚࡉࡏࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ ࡿࡢࡳ࡛࠶ࡗࡓ84,85)ࠋ࣐ࢳࢽࣈࡼࡿඃࢀࡓẅ⣽⬊ຠᯝࡣࠊBCR-ABL ࡢୗ ὶᏑᅾࡍࡿ⏕Ꮡ࣭ቑṪ㛵㐃ࢩࢢࢼࣝࡢάࡼࡿࡶࡢࡔࡅ࡛ࡣ࡞ࡃࠊࡀ ࢇ⣽⬊ࡢせ࡞࢚ࢿࣝࢠ࣮⋓ᚓᶵᵓ࡛࠶ࡿゎ⢾⣔ࢆ㜼ᐖࡍࡿࡇࡶ㉳ᅉࡋ ࡚࠸ࡿ⪃࠼ࡽࢀࡓࠋࡋࡋ࡞ࡀࡽࠊWarburg ຠᯝࡢ◚⥢ࡣ௦ൾⓗ࡞⬡⫫㓟㓟 ࡢάᛶࢆㄏᑟࡍࡿࡇࡀ᫂ࡽ࡞ࡾࠊ࣐ࢳࢽࣈࡣ⬡⫫㓟㓟ࢆάᛶ ࡉࡏࡿࡇࡀ♧ࡉࢀࡓࠋ୍᪉ࠊAIC-47 ࡣ Warburg ຠᯝ⊂❧ࡋࡓᶵᵓ࡛⬡ ⫫㓟㓟ࡢάᛶࢆᢚไࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋ ࡲࡓࠊᖿ⣽⬊ᵝࡢᛶ㉁ࢆ᭷ࡍࡿ CD34+ศ⏬ᑐࡋࠊAIC-47 ࡣ༢࡛ࡶ⣽⬊ ⏕Ꮡ⋡ࡢపୗࢆㄆࡵࡓࠋⓑ⾑ᖿ⣽⬊ࡢ TKI 㠀ឤཷᛶࡢ⋓ᚓࡣ⣽⬊࿘ᮇࡢ㟼 Ṇࡼࡿࡶࡢ࡛࠶ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࡀ28)ࠊᮏ❶࡛ࡢ᳨ウࡼࡾ௦ൾⓗ࡞ ⬡⫫㓟㓟ࡢάᛶࡶ୍ᅉ࡞ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ༢࡛ࡣ᭷ຠᛶࡢ ప࠸࣐ࢳࢽࣈࡶ⬡⫫㓟㓟ࢆ㜼ᐖࡍࡿࡇ࡛ᖿ⣽⬊ࡢ⣽⬊⏕Ꮡ⋡ࢆపୗࡉ ࡏࡿࡇࡽࠊⓑ⾑ᖿ⣽⬊ࡣ⬡⫫㓟㓟ࢆᕦࡳ⏝ࡍࡿࡇ࡛⸆⪏ᛶ ࢆ⋓ᚓࡋ࡚࠸ࡿ⪃࠼ࡽࢀࠊゎ⢾⣔⬡⫫㓟㓟ࢆඹ㜼ᐖࡍࡿ⒪ἲࡀ᭷ ຠ࡛࠶ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ
45 ⬡⫫㓟㓟ࡣ࢚ࢿࣝࢠ࣮⏘⏕ࡢࡳ࡞ࡽࡎ NADPH ࡢ౪⤥※ࡋ࡚ᶵ⬟ࡍࡿ ࡇࡀ▱ࡽࢀ࡚࠸ࡿ87)ࠋⓑ⾑࠾ࡼࡧⓑ⾑ᖿ⣽⬊࠾࠸࡚⬡⫫㓟㓟ࡽ ⏕ࡌࡓ NADPH ࡀ⣽⬊ෆࡢ ROS ࢆᤕᤊࡋࠊ⣽⬊ࡢ⏕Ꮡ⥔ᣢാࡃࡇࡀሗ࿌ ࡉࢀ࡚࠸ࡿ86,88)ࠋⓑ⾑ᖿ⣽⬊ࡣ ROS ᑐࡍࡿឤཷᛶࡀ㧗࠸ࡓࡵ89,90)ࠊ㠀ᖿ ⣽⬊ᛶࡢⓑ⾑⣽⬊ࡼࡾࡶࡉࡽ⬡⫫㓟㓟ࢆάᛶࡉࡏ࡚࠾ࡾࠊࡑࡢ⤖ᯝ⸆ ឤཷᛶࡀపୗࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡓࠋ ௨ୖࡢࡇࡽࠊAIC-47 ࡣ Warburg ຠᯝ⬡⫫㓟㓟ࡢ୧᪉ࢆ◚⥢ࡉࡏࠊ ⣽⬊ෆࡢ࢚ࢿࣝࢠ࣮ࢆᯤῬࡉࡏࡿࡶ ROS ࡢᤕᤊ㛵ࢃࡿ NADPH ࡢⓎ ⏕ࢆᢚไࡍࡿࡇ࡛ẅ⣽⬊ຠᯝࢆ♧ࡋ࡚࠸ࡿ⪃࠼ࡽࢀࠊ᪤Ꮡ⸆ࡣ␗࡞ࡿ᪂ ࡓ࡞స⏝Ⅼࢆᣢࡘࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡲࡓࠊAIC-47 ࣐ࢳࢽࣈࡣ┦ ຠᯝࢆ♧ࡋࡓࡇࡽే⏝⸆ࡋ࡚ࡢ᭷ຠᛶࡶᮇᚅ࡛ࡁࠊ⸆ࢩ࣮ࢬࡋ࡚᭷ ⏝࡛࠶ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ
46
Fig. 18 Schematic diagram of the effects of AIC-47 and imatinib on cancer metabolism in BCR-ABL-transformed leukemia cells
AIC-47 Imatinib
Cell death Autophagic cell death Apoptosis
BCR-ABL Expression suppression Inhibitory phosphorylation
Glycolysis Dys-regulation Dys-regulation
Fatty-acid oxidation Inhibition Activation
47 ➨ 5 ❶ miR-124/PTBP1 ࢝ࢫࢣ࣮ࢻࡼࡿࡀࢇ⣽⬊ࡢ࢚ࢿࣝࢠ࣮௦ㅰไᚚ ➨ 1 ⠇ ᗎ Warburg ຠᯝࡢᡂ❧㛵ࡍࡿ PTBP1 ࡣ c-Myc44)ࠊཷᐜయᆺࢳࣟࢩࣥ࢟ࢼ࣮ ࢮ51)ࡢࠊ」ᩘࡢ miRNA91)ࡼࡾⓎ⌧ࢆไᚚࡉࢀ࡚࠸ࡿࠋ➹⪅ࡽࡶࡇࢀࡲ ࡛ miR-124 ࠾ࡼࡧ miR-133 ࡢⓎ⌧ࡀࡀࢇ࡛పୗࡋ࡚࠾ࡾࠊࡑࢀ┦㛵ࡋ࡚ PKM2 ࡢⓎ⌧ࡀஹ㐍ࡋ࡚࠸ࡿࡇࢆሗ࿌ࡋ࡚ࡁࡓ45)ࠋࡋࡋ࡞ࡀࡽࠊࡀࢇࡢ Ⓨ㐣⛬࠾ࡅࡿࡇࢀࡽࡢ miRNA ࡢⓎ⌧ኚࡸ PTBP1/PKM ࢝ࢫࢣ࣮ࢻࡢไ ᚚࡣࡲ࡛᫂ࡽࡉࢀ࡚࠸࡞࠸ࠋ➨ 2 ❶ࡢ᳨ウ⤖ᯝࡽࠊAIC-47 ࡣ c-Myc ࠾ࡼࡧ㠀ཷᐜయᆺࢳࣟࢩࣥ࢟ࢼ࣮ࢮ BCR-ABL ࢆࡋ࡚ Warburg ຠᯝࡢ◚⥢ࢆ ㄏᑟࡍࡿࡇࡀ♧ࡉࢀࡓࠋࡑࡇ࡛ᮏ❶࡛ࡣࡲࡎ AIC-47 ࠾ࡼࡧ࣐ࢳࢽࣈࡼ ࡿ miR-124 ࡢⓎ⌧ኚࢆ᳨ドࡋࡓࠋࡉࡽࠊࡀࢇࡢⓎ㐣⛬࠾ࡅࡿ miR-124 ࡢⓎ⌧ኚ miR-124 ࡼࡿ PTBP1/PKM ࢝ࢫࢣ࣮ࢻࡢไᚚࢆ᳨ドࡋࡓࠋ ➨ 2 ⠇ miR-124 ࡢ⤌⧊ศᕸ miR-124 ࡣ⬻≉␗ⓗⓎ⌧ࡋ࡚࠸ࡿ miRNA ࡛࠶ࡾࠊ⚄⤒ࡢศࡸⓎ㐩ࢆ ㄪ⠇ࡋ࡚࠸ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ55)ࠋࣄࢺࡢṇᖖ⤌⧊࠾ࡅࡿ miR-124 ࡢ⤌ ⧊ศᕸࢆ᳨ドࡋࡓ⤖ᯝࠊmiR-124 ࡣ⬻࡛᭱ࡶ㧗Ⓨ⌧ࡋ࡚࠾ࡾࠊḟ࠸࡛⭁⮚ࡸ㦵 㧊ࡢࡼ࠺࡞㐀⾑ჾ࡛Ⓨ⌧ࡀ㧗࠸ࡇࡀ᫂ࡽ࡞ࡗࡓ (Fig. 19)ࠋ
48 ➨ 3 ⠇ ୰㙐⬡⫫㓟ㄏᑟయ࠾ࡼࡧ࣐ࢳࢽࣈࡼࡿ miR-124 ࡢⓎ⌧ኚ AIC-47 ࠾ࡼࡧ࣐ࢳࢽࣈࢆῧຍࡋࡓ CML ⣽⬊ෆࡢ miR-124 ࡢⓎ⌧ࢆ Real-time PCR ᳨࡚ドࡋࡓࠋࡑࡢ⤖ᯝࠊ࣐ࢳࢽࣈࡣ࠸ࡎࢀࡢ⣽⬊ᰴ࠾࠸ ࡚ࡶ᭷ព࡞ miR-124 ࡢⓎ⌧ቑຍࢆ♧ࡋࡓࠋAIC-47 ࡶቑຍഴྥࡣㄆࡵࡽࢀࡓࡀࠊ KCL-22 ⣽⬊ᑐࡋ࡚ࡣ᭷ពᕪࡀㄆࡵࡽࢀ࡞ࡗࡓ (Fig. 20)ࠋ௨ୖࡢ⤖ᯝࡽࠊ ࣐ࢳࢽࣈࡼࡿ Warburg ຠᯝࡢ◚⥢ࡣ miR-124 ࡢⓎ⌧ቑຍࡀ㛵ࡋ࡚࠸ ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊAIC-47 ࡼࡿ Warburg ຠᯝࡢ◚⥢࠾࠸࡚ࡣ miR-124 ࡢⓎ⌧ኚࡢᐤᗘࡀᑠࡉ࠸⪃࠼ࡽࢀࡓࠋ
Fig. 20 Effects of AIC-47 and/or imatinib on the expression of miRNA-124 targeting PTBP1.
Expression level of miR-124 in K562 and KCL-22 cells after treatment with DMSO (C; Control), AIC-47 (47; ȝ0 LPDWLQLE IM; ȝ0IRU.ȝ0IRU.&/-22) or their combination (47+IM) for 48 h.
49 ➨ 4 ⠇ ࡀࢇࡢⓎ㐣⛬࠾ࡅࡿ miR-124 ࡢⓎ⌧ኚ ࡀࢇࡢⓎ⏕ࡣࠊinitiationࠊpromotionࠊprogression ࠸࠺ 3 ࡘࡢẁ㝵ࡀᏑᅾ ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋmiR-124 ࡣ STAT3 ࡞⣽⬊ቑṪ㛵ࡍࡿ㑇ఏᏊࢆ ᶆⓗࡋࡀࢇᢚไⓗᶵ⬟ࡍࡿ miRNA ࡛࠶ࡾ92)ࠊᵝࠎ࡞ࡀࢇ✀࡛Ⓨ⌧ࡀపୗ ࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ92,93)ࠋࡋࡋ࡞ࡀࡽࠊࡀࢇࡢⓎ㐣⛬࠾࠸࡚ miR-124 ࡢⓎ⌧పୗࡀࡢẁ㝵⨨ࡍࡿࡣ᫂ࡽ࡛࡞࠸ࠋࡑࡇ࡛ࡀࢇࡢⓎ 㐣⛬࠾ࡅࡿ miR-124 ࡢⓎ⌧ኚࢆ᳨ドࡍࡿࡇࡋࡓࠋCML ࡣ 1 ࡘࡢ㑇 ఏᏊኚ␗ BCR-ABL ࡛Ⓨࡍࡿࡓࡵࠊẁ㝵ⓗ࡞Ⓨࡀࢇࡢ㐣⛬ࢆࡽ࡞࠸ࠋࡲࡓࠊ 㐀⾑ᖿ⣽⬊ࡢ⭘⒆࡛࠶ࡿࡓࡵྠ୍ᝈ⪅ࡽṇᖖ㦵㧊⣽⬊ࢆᚓࡿࡇࡀ࡛ࡁ࡞ ࠸ࠋࡑࡇ࡛ࠊadenoma-carcinoma sequence ࡢ㑇ఏᏊኚ␗ࡢ✚ࡼࡾẁ㝵ⓗ ⭘⒆ࡋࠊྠ୍ᝈ⪅ࡽࡢṇᖖ⤌⧊࠾ࡼࡧࡀࢇ⤌⧊ࡢ᥇ྲྀࡀྍ⬟࡞⭠ࡀࢇᝈ ⪅ࡢ⮫ᗋ᳨యࢆ⏝࠸ࡓࠋReal-time PCR ࡼࡾྛ⤌⧊୰ࡢ miR-124 ࡢⓎ⌧㔞ࢆ ᐃ㔞ࡋࠊṇᖖ⤌⧊ࡀࢇ⤌⧊࡛Ⓨ⌧ࢆẚ㍑ࡋࡓࠋྛ⮫ᗋࣃ࣓࣮ࣛࢱ࡛ศ㢮ࡋࡓ ᩘ (n) ࠊࡀࢇ⤌⧊࡛ miR-124 ࡢⓎ⌧ࡀపୗࡋ࡚࠸ࡓᩘ࠾ࡼࡧࡑࡢ ྜ (%) ࡣ Table 3 ♧ࡍ㏻ࡾ࡞ࡗࡓࠋmiR-124 ࡢⓎ⌧పୗࡣ 81.8%ࡢ adenoma ࡛☜ㄆࡉࢀࠊmiR-124 ࡣⰋᛶ⭘⒆ࡢẁ㝵ࡽࡍ࡛Ⓨ⌧ࡀపୗࡋ ࡚࠸ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡑࡢࡢ⮫ᗋࣃ࣓࣮ࣛࢱࡢ᭷ព࡞┦㛵ࡣㄆࡵ ࡽࢀ࡞ࡗࡓࡇࡽࠊmiR-124 ࡣࡀࢇࡀⓎ⏕ࡍࡿ๓ẁ㝵ࠊࡍ࡞ࢃࡕ initiation ࡢẁ㝵࠾࠸࡚Ⓨ⌧ࡀపୗࡋࠊࡀࢇᢚไ miRNA ࡋ࡚ࡢᶵ⬟ࢆኻ࠺⪃࠼ࡽ ࢀࡓࠋ
50
Characteristic n miR- Ļ) Case, (%)
Sex Male 36 25 (69.4) Female 19 12 (63.1) Tumor Cancer 33 19 (57.6) Adenoma 22 18 (81.8) Location Right colon 15 9 (60) Left colon 40 28 (70) Depth in cancer Mucosa (M) 5 2 (40) Submucosa (SM) 5 3 (60) Mucosa propria (MP) 5 5 (100) Subserosa (SS) 9 4 (44.4)
Serosa exposure, Serosa onvasion (SE, SI) 9 5 (55.6) Tumor diameter in cancer (mm)
< 45 16 10 (62.5)
> 45 17 9 (52.9)
Dukes classification system
A 18 11 (61.1)
B 1 1 (100.0)
C 14 7 (50)
Tumor diameter in adenoma (mm)
< 10 10 8 (80.0)
> 10 12 10 (83.3)
Grade in adenoma
Low-grade dysplasia 11 10 (90.9)
High-grade dysplasia 11 8 (72.7)
Table 3. Characteristics of study population and expression of miR-124 in colorectal tumors
