Title Warburg効果とTRAIL誘導がん細胞死( 本文(Fulltext) ) Author(s) 熊崎, 実南 Report No.(Doctoral Degree) 博士(薬科学) 連創博甲第36号 Issue Date 2017-03-25 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/56206 ※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。
Warburg ຠᯝ TRAIL ㄏᑟࡀࢇ⣽⬊Ṛ
Perturbation of the Warburg effect increases the sensitivity of cancer
cells to TRAIL-induced cell death
2017
┠ḟ ➨1 ❶ ⥴ゝ 1 ➨2 ❶ ◊✲ࡢ⫼ᬒ┠ⓗ ➨㸯⠇ TRAIL ࢩࢢࢼࣝ 4 ➨2 ⠇ TRAIL ࢩࢢࢼࣝࢆᶆⓗࡋࡓ⒪ࡢኚ㑄ၥ㢟Ⅼ 8 ➨3 ⠇ TRAIL ⪏ᛶᶵᵓ 11 ➨4 ⠇ ࡀࢇ⣽⬊≉␗ⓗ࢚ࢿࣝࢠ࣮௦ㅰไᚚᶵᵓ㸸Warburg ຠᯝ 14 ➨5 ⠇ CCN1 ࡢⓎ⌧ TRAIL ㄏᑟ⣽⬊Ṛࡢ㛵 17 ➨3 ❶ Warburg ຠᯝ㛵㐃 PTBP1 TRAIL ㄏᑟ࣏ࢺ࣮ࢩࢫ ➨1 ⠇ ᗎ 20 ➨2 ⠇ PTBP1 ࡢⓎ⌧ TRAIL ㄏᑟࡀࢇ⣽⬊Ṛࡢ㛵 21 ➨3 ⠇ TRAIL ⪏ᛶᶵᵓ࠾ࡅࡿ PTBP1 ࡢᙺ 29 ➨4 ⠇ άᛶ㓟⣲㸦ROS㸧ࡢⓎ⏕ TRAIL ⪏ᛶゎ㝖ᶵᵓࡢ㛵 31 ➨5 ⠇ CCN1 ࡢⓎ⌧ TRAIL ㄏᑟ⣽⬊Ṛࡢ㛵 33 ➨4 ❶ ⥲ᣓ 37 ㅰ㎡ 41 ᐇ㦂ࡢ㒊 42 ཧ⪃ᩥ⊩ 51 ◊✲ᴗ⦼┠㘓 57
1
➨1 ❶ ⥴ゝ
⭘⒆ቯṚᅉᏊ㸦TNF: tumor necrosis factor㸧ࡣࠊ1975 ᖺ Sloan-Kettering ⒴
◊✲ᡤࡢCarswell ࡽࡼࡗ࡚࣐࢘ࢫ⛣᳜ࡋࡓ Meth A ⫗⭘ฟ⾑ᛶࡢቯṚࢆ
ᘬࡁ㉳ࡇࡍᅉᏊࡋ࡚Ⓨぢࡉࢀࡓࢧࢺ࡛࢝ࣥ࠶ࡿ1ࠋࡑࢀ௨ᚋࠊTNF ࣇ࣑
࣮ࣜᒓࡍࢧࢺ࢝ࣥࡋ࡚࣏ࢺ࣮ࢩࢫ㛵ࡍࡿ TNF-DࠊFasLࠊTRAIL
㸦Tumor necrosis-factor related apoptosis-inducing ligand㸧࡞ 20 ✀㢮௨ୖࡀ
ྠᐃࡉࢀ࡚࠸ࡿ2, 3ࠋTRAIL ࡣࠊ⣽⬊⭷Ꮡᅾࡍࡿ≉␗ⓗཷᐜయࡢ⤖ྜࢆࡋ
࡚࣏ࢺ࣮ࢩࢫㄏᑟࢩࢢࢼࣝࢆ⣽⬊ෆఏ㐩ࡍࡿᢠ⭘⒆ᛶࡢࢧࢺ࡛࢝ࣥ࠶ ࡿࠋTRAIL ࡣ Death receptor㸦DR㸧4/5 ⤖ྜࡋ࡚ࡀࢇ⣽⬊ᑐࡋ࡚≉␗ⓗ ࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿࡇࡽࠊࡀࢇ⒪ཬࡧࡀࢇண㜵࠾࠸࡚ᴟࡵ࡚㔜せ ࡞ാࡁࢆᢸ࠺ࡇࡀᮇᚅࡉࢀ࡚࠾ࡾୡ⏺୰࡛◊✲ࡀ㐍ࡵࡽࢀ࡚࠸ࡿ4ࠋ⌧ᅾࡲ࡛ ࣄࢺ⤌ࡳ࠼ᆺTRAIL ࡸ DR4/5 ࢆᶆⓗࡋࡓᢠయ་⸆ࡀ㛤Ⓨࡉࢀᵝࠎ࡞ࡀࢇ ࠾࠸࡚⮫ᗋヨ㦂ࡀ⾜ࢃࢀ࡚࠸ࡿࡀTRAIL ⪏ᛶࡢ⋓ᚓࠊస⏝ࡢၥ㢟ࡽ୰Ṇ ࡞ࡗࡓ5, 6ࠋࡇࡢࡼ࠺࡞⫼ᬒࡽTRAIL ⪏ᛶᶵᵓࡢゎ᫂ࠊ⪏ᛶゎ㝖㛵ࢃࡿ ศᏊࢆྠᐃࡍࡿࡇࡀᚲ㡲ㄢ㢟࡛࠶ࡿゝ࠼ࡿࠋ ᮏ◊✲࡛ࡣࠊTRAIL ⪏ᛶゎ㝖᭷ຠ࡞ᶆⓗศᏊࡋ࡚ࠊࡀࢇ⣽⬊≉␗ⓗ࡞࢚ࢿ ࣝ ࢠ ࣮ ௦ ㅰ ไ ᚚ ᶵ ᵓ 㸦Warburg ຠ ᯝ 㸧 ࡢ ᡂ ❧ ᚲ 㡲 ࡞ 㑇 ఏ Ꮚ PTBP1 㸦Polypyrimidine tract-binding protein㸧╔┠ࡋࡓࠋWarburg ຠᯝࡣࠊࡀࢇ⣽
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⬊ࡀ㓟⣲ࡢ᭷↓㛵ࢃࡽࡎゎ⢾⣔౫Ꮡࡋࡓ࢚ࢿࣝࢠ࣮௦ㅰࢆ⾜࠺⌧㇟࡛࠶ࡿ
7ࠋPTBP1 ࡣ PKM㸦Pyruvate kinase muscle㸧1/2 㑇ఏᏊࡢࢫࣉࣛࢧ࣮ࡋ࡚
ᶵ⬟ࡋࠊࡀࢇ⣽⬊࠾࠸࡚ࡣ㑅ᢥⓗࢫࣉࣛࢩࣥࢢࡼࡾ PKM2 ࡢⓎ⌧ࡀඃ ࡞ࡗ࡚࠸ࡿ 8ࠋᡃࠎࡢ◊✲ࢢ࣮ࣝࣉࡣࡇࢀࡲ࡛⭠ࡀࢇࡢ⮫ᗋ᳨యࡢ 90㸣 ௨ୖ࡛ PTBP1 ࡀ㧗Ⓨ⌧ࡋ࡚࠸ࡿࡇࢆሗ࿌ࡋ࡚࠾ࡾࠊPTBP1 ࡀࡀࢇ࠾ࡅࡿ Warburg ຠᯝࡢᡂ❧ᚲ㡲࡞㑇ఏᏊ࡛࠶ࡿࡇࢆ♧ࡋࡓ9ࠋ ᮏ◊✲࡛ࡣࠊWarburg ຠᯝࡢᡂ❧ᚲ㡲࡞㑇ఏᏊ PTBP1 ࡢⓎ⌧ TRAIL ㄏᑟ ࡀࢇ⣽⬊Ṛࡢ㛵㸦➨3 ❶-2 ⠇㸧 Warburg ຠᯝࡢ⬺ไᚚࢆࡋࡓάᛶ㓟⣲ ࡢⓎ⏕ࡀTRAIL ⪏ᛶゎ㝖ᶵᵓ㛵ࡍࡿᶵᵓ㸦➨ 3 ❶-3ࠊ4 ⠇㸧ࢆ᪂ࡓ᫂ࡽ ࡋࡓࠋࡉࡽࠊCCN1 TRAIL ㄏᑟ⣽⬊Ṛࡢ㛵㸦➨ 3 ❶-5 ⠇㸧ࡘ࠸ ࡚ሗ࿌ࡍࡿࠋ ᮏ◊✲࡛ᚓࡽࢀࡓ▱ぢࡽࠊWarburg ຠᯝࡢᡂ❧ᚲ㡲࡞㑇ఏᏊ࡛࠶ࡿ PTBP1 ࡀTRAIL ⪏ᛶゎ㝖᭷ຠ࡞ᶆⓗศᏊ࡞ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋᚋࠊPTBP1 ࢆไᚚࡍࡿྜ≀ࡸsiRNA ࡀࡀࢇࡢ࢚ࢿࣝࢠ࣮௦ㅰࡢ◚⥢ࡢࡳ࡞ࡽࡎ TRAIL ㄏ ᑟ⣽⬊Ṛࢆ⏝ࡋࡓࡀࢇࡢ་⸆ࢩ࣮ࢬࡋ࡚ᮇᚅࡉࢀࡿࠋ
3
ᮏᏛㄽᩥࡣࠊୗグࡢཎⴭㄽᩥࢆࡶసᡂࡋᒱ㜧ᏛᏛ㝔㐃ྜ⸆་⒪ ሗ◊✲⛉ᥦฟࡋࡓࡶࡢ࡛࠶ࡿࠋ
(1) Perturbation of the Warburg effect increases the sensitivity of cancer cells to TRAIL-induced cell death.
Minami Kumazaki, Haruka Shinohara, Kohei Taniguchi, Tomoaki Takai, Yuki Kuranaga, Nobuhiko Sugito, Yukihiro Akao.
Experimental Cell Research; 347(1): 133-142 (2016)
(2) Understanding of tolerance in TRAIL-induced apoptosis and cancelation of its machinery by D-mangostin, a xanthone derivative.
Minami Kumazaki, Haruka Shinohara, Kohei Taniguchi, Hiroshi Ueda, Mayu ko Nishi, Akihide Ryo, Yukihiro Akao
4 ➨2 ❶ ◊✲ࡢ⫼ᬒ┠ⓗ ➨ ➨1 ⠇ TRAIL ࢩࢢࢼࣝ TRAIL ࡣࠊ1995 ᖺ Wiley ࡽࡢ◊✲ࢢ࣮ࣝࣉࡼࡗ࡚༢㞳ࡉࢀ10ࠊάᛶT ⣽⬊ࡢࠊNK ⣽⬊ࠊ༢⌫ࠊᶞ≧⣽⬊ࠊዲ୰⌫࡞ࡢච⣽⬊࡛ࡑࡢⓎ⌧ࡀㄆ ࡵࡽࢀ࡚࠾ࡾࠊ⣽⬊⭷Ꮡᅾࡍࡿ≉␗ⓗཷᐜయࡢ⤖ྜࢆࡋ࡚࣏ࢺ࣮ࢩࢫ
ㄏᑟࢩࢢࢼࣝࢆ⣽⬊ෆఏ㐩ࡍࡿ 11ࠋTRAIL ཷᐜయࡣ TRAIL-R1 㸦Death
receptor 4: DR4㸧ࠊTRAIL-R2 㸦Death receptor 5: DR5㸧ࠊTRAIL-R3 㸦Decoy receptor 1: DcR1㸧ࠊTRAIL-R4 㸦Decoy receptor 2: DcR2㸧ࠊOsteoprotegerin
㸦OPG㸧ࡢ 5 ✀㢮ࡀᏑᅾࡍࡿ㸦Fig-1㸧12ࠋDR4/5 ࡣࠊࢩࢫࢸࣥṧᇶᐩࡴࢻ ࣓ࣥࡢ⧞ࡾ㏉ࡋᵓ㐀ࢆ≉ᚩࡋࡓ⣽⬊⭷ࣞࢭࣉࢱ࣮࡛࠶ࡾࠊ⣽⬊ෆ㡿ᇦࡣ DD 㸦Death domain㸧ࡤࢀࡿ࣏ࢺ࣮ࢩࢫࡢㄏᑟᚲ㡲ࡢᵓ㐀ࢆ᭷ࡋ࡚࠸ ࡿࠋࡑࡢ3 ࡘࡢࣞࢭࣉࢱ࣮ࡣࠊTRAIL ㄏᑟ࣏ࢺ࣮ࢩࢫࢆ㜼ᐖࡍࡿࠊ‛Decoy 㸦࠾ ࡾ㸧‘ࡋ࡚ᶵ⬟ࡋ࡚࠸ࡿ⪃࠼ࡽࢀ࡚࠸ࡿ13ࠋDcR1/2 ࡣࠊDR4/5 ⣽⬊እ㡿 ᇦࡢ┦ྠᛶࡣ㧗࠸ࡀࠊ⣽⬊ෆ㡿ᇦࡢDD ࡢḞኻཬࡧᶵ⬟ࡽ TRAIL ࡀ⤖ྜ ࡋ࡚ࡶ࣏ࢺ࣮ࢩࢫࢆㄏᑟࡋ࡞࠸4, 13ࠋOPG ࡣ TRAIL ࡢ⤖ྜぶᛶࡀ㠀ᖖ ప࠸ࡓࡵTRAIL ࣞࢭࣉࢱ࣮ࡋ࡚ࡢ⏕≀Ꮫⓗព⩏ࡘ࠸࡚ࡣ᫂ࡽࡉࢀ࡚࠸ ࡞࠸ࠋ
5
Figure-1 Characteristics of TRAIL-R in humans
TRAIL ࡀ DR4/5 ⤖ྜࡍࡿࠊ⭘⒆⣽⬊ࡸᙧ㉁㌿ࡋࡓ⣽⬊ᑐࡋ࡚≉␗ⓗ ࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿࠋX ⥺⤖ᬗᵓ㐀ゎᯒࡢ⤖ᯝࠊTRAIL ࡀ⤖ྜࡍࡿ DR
ࡣ࣍ࣔ୕㔞యࡋࠊ⣽⬊ෆࡢDD ࡀࢲࣉࢱ࣮ศᏊ࡛࠶ࡿ FADD㸦Fas-associated
death domain㸧ࢆࣜࢡ࣮ࣝࢺࡉࡏࠊFADD ࢆࡋ࡚࢝ࢫࣃ࣮ࢮ 8 ࡀ⤖ྜࡍࡿࡇ
ࡼࡾDISC㸦Death-inducing signaling complex㸧ࡤࢀࡿ」ྜయࢆᙧᡂࡍ
ࡿ 14, 15ࠋࡑࡢᚋࠊ࢝ࢫࣃ࣮ࢮ 8 ࡣ⮬ᕫศゎࡼࡗ࡚άᛶࡉࢀࠊ2 ࡘࡢ⤒㊰ࢆ
ࡋ࡚࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿࠋ1 ࡘࡣᐇ⾜࢝ࢫࣃ࣮ࢮ࡛࠶ࡿ࢝ࢫࣃ࣮ࢮ 3 ࡢά
6 ࡢᾘኻࠊࢩࢺࢡ࣒ࣟc ࡢᨺฟࠊ࢝ࢫࣃ࣮ࢮ 9 ࡢάᛶࠊ࢝ࢫࣃ࣮ࢮ 3 ࡢάᛶ 㐍ࡴෆᅉᛶ⤒㊰࡛࠶ࡿ㸦Fig-2㸧12, 14, 16ࠋ TRAIL ࡢ⏕≀Ꮫⓗព⩏ࡘ࠸࡚ࡣࠊචᏛⓗ┘どᶵᵓ⭘⒆ᢚไ࡛࠶ࡿࡇ ࡀከᩘሗ࿌ࡉࢀ࡚࠸ࡿࠋTRAIL ࡣච⣽⬊ࡢࡳⓎ⌧ࡋ࡚࠾ࡾࠊ࣏ࣜከ⢾య 㸦lipopolysacharides㸧ࡸࣥࢱ࣮ࣇ࢙ࣟࣥ࡞ࡢࢧࢺ࢝ࣥࡼࡗ࡚ㄏᑟࡉ ࢀࡿࡇࡽࡶචᏛⓗ┘どᶵᵓࢆᢸࡗ࡚࠸ࡿ⪃࠼ࡽࢀ࡚࠸ࡿ 17, 18ࠋࡲࡓࠊ Zheng ࡽࡢ◊✲ࢢ࣮ࣝࣉࡼࡾ TRAIL ࣀࢵࢡ࢘ࢺ࣐࢘ࢫ࠾࠸࡚ࠊࣜࣥࣃ⌫ ᩘཬࡧ㦵㧊⣽⬊Ṛࡢῶᑡࠊࣜࢫࢸࣜ⳦ࡢឤᰁᑐࡋ࡚పឤཷᛶࢆ♧ࡍࡇࡀ ሗ࿌ࡉࢀ࡚࠸ࡿ19ࠋ୍᪉ࠊ⭘⒆ᢚไ࠾ࡅࡿTRAIL ࡢാࡁࡘ࠸࡚ࡣࠊWalczack ࡽࡢ◊✲ࢢ࣮ࣝࣉࡼࡾࣄࢺࡢஙࡀࢇ⣽⬊ࢆ⛣᳜ࡋࡓ࣐࢘ࢫ recombinant TRAIL ࢆᢞࡋࡓ⤖ᯝࠊ⭘⒆ࢧࢬࡢ⦰ᑠຠᯝࡀㄆࡵࡽࢀࡓࡇࡽึࡵ࡚᫂ ࡽ࡞ࡗࡓ 20ࠋTRAIL ࢩࢢࢼࣝࡢ⭘⒆ᢚไ࠾ࡅࡿ⏕≀Ꮫⓗព⩏ࡑࡢࡀࢇ ⣽⬊ᑐࡍࡿ≉␗ᛶࡣ㠀ᖖ⯆῝࠸Ⅼ࡛࠶ࡿࠋTRAIL ࢩࢢࢼࣝࡣࠊࡀࢇ⣽⬊ ≉␗ⓗㄏᑟࡉࢀࡿࡇࡽࠊࡀࢇᑐࡍࡿ᭱ึࡢ㜵ᚚᶵᵓ࡛࠶ࡾࠊࡀࢇ⣽⬊ ṇᖖ⣽⬊࠾ࡅࡿDR4/5 ཬࡧ DcR1/2 ࡢⓎ⌧ࣞ࣋ࣝࡢᕪࡀࡑࡢ≉␗ᛶࢆࡶࡓ ࡽࡋ࡚࠸ࡿ⪃࠼ࡽࢀ࡚࠸ࡿࡀヲ⣽࡞ᶵᵓࡣᮍࡔ᫂ࡽࡉࢀ࡚࠸࡞࠸ࠋ
7 Figure-2 TRAIL-induced apoptosis pathways
8 ➨ ➨2 ⠇ TRAIL ࢩࢢࢼࣝࢆᶆⓗࡋࡓ⒪ࡢኚ㑄ࡑࡢၥ㢟Ⅼ TRAIL ㄏᑟ⣽⬊Ṛࡣࡀࢇ⣽⬊ᑐࡋ࡚㧗࠸≉␗ᛶࢆ᭷ࡍࡿࡇࡽࠊࡀࢇ ⒪ཬࡧࡀࢇண㜵࠾࠸࡚ᴟࡵ࡚㔜せ࡞ാࡁࢆᢸ࠺ࡇࡀᮇᚅࡉࢀ࡚࠾ࡾୡ⏺୰ ࡛◊✲ࡀ㐍ࡵࡽࢀ࡚࠸ࡿࠋ1999 ᖺࡣࣄࢺ⤌ࡳ࠼ᆺ TRAIL ࢆ⏝࠸ࡓ๓⮫ᗋヨ 㦂ࡀ⾜ࢃࢀࠊᙉຊ࡞⭘⒆㏥⦰ຠᯝࢆ♧ࡋࡓࡇࡽࠊTRAIL ⤒㊰ࢆࡍࡿ⣽⬊ Ṛὀ┠ࡀ㧗ࡲࡗࡓ21ࠋDulanermin ࡣࠊDR4/5 ࢆⓎ⌧ࡋ࡚࠸ࡿࡀࢇ⣽⬊ᑐࡋ ࡚࢝ࢫࣃ࣮ࢮࡢάᛶ p53 ౫Ꮡᛶࡢ࣏ࢺ࣮ࢩࢫࡢㄏᑟࢆᮇᚅࡋ࡚㛤Ⓨࡉࢀ ࡓࣄࢺ⤌ࡳ࠼ᆺTRAIL ࡛࠶ࡿࠋࣇࣛࣥࢫࡢ Soria ࡽࡣࠊ213 ྡࡢ㐍⾜ᛶ/Ⓨ ᛶ㠀ᑠ⣽⬊⫵ࡀࢇᝈ⪅ࢆᑐ㇟ࡋ࡚ࠊDulanermin ࡢ paclitaxel࣭carboplatin 㸦PC㸧 / paclitaxel࣭carboplatin࣭bevacizumab 㸦PCB㸧ࡢే⏝ຠᯝཬࡧᏳᛶࢆホ ౯ࡍࡿ➨ II ┦ヨ㦂ࢆ⾜ࡗࡓ 5 㸦Table-1A㸧ࠋDulanermin ࡣࠊᝈ⪅ᑐࡋ࡚ẘᛶ ࡸస⏝ࡣ࡞ࡃⰋዲ࡞᭷⏝ᛶࢆ♧ࡋࡓࡀࠊPC/PCB ࡢ㏣ຍࡼࡾ┦ⓗ࡞ᢠ⭘ ⒆ຠᯝࢆ♧ࡉ࡞ࡗࡓࠋࡑࡢၥ㢟Ⅼࡋ࡚ࠊࣄࢺ⤌ࡳ࠼ᆺTRAIL ࡣ⣽⬊Ṛࢆ ㄏᑟࡋ࡞࠸DcR1/2 ⤖ྜࡍࡿࡇࠊ༙ῶᮇࡀ㠀ᖖ▷࠸ࡇࡀᣲࡆࡽࢀࡓ22ࠋ ㏆ᖺ࡛ࡣࠊTRAIL ཷᐜయࢆάᛶࡍࡿࣄࢺᆺࣔࣀࢡࣟࢼ࣮ࣝᢠయࡀ㛤Ⓨࡉ ࢀᵝࠎ࡞ࡀࢇᑐࡋ࡚⮫ᗋヨ㦂ࡀ⾜ࢃࢀࠊᢠయ་⸆࡛ࡢ◊✲㛤ⓎࡣᩘከࡃᏑ ᅾࡍࡿࠋ⭠ࡀࢇཬࡧஙࡀࢇ⣽⬊ࡣ࣏ࢺ࣮ࢩࢫࡢㄏᑟ DR5 ࢆ⏝ࡋࠊ ᝏᛶࣜࣥࣃ⭘ࡸ⮅⮚ࡀࢇ⣽⬊ࡣDR4 ࢆ⏝ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾࠊࡀࢇ✀
9
ࡼࡾᶆⓗ࡞ࡿࣞࢭࣉࢱ࣮ࡀ␗࡞ࡿࡇࡽDR4 㸦mapatumumab㸧ࡶࡋࡃ
ࡣDR5 㸦drozitumumab, conatumumab, lexatumumab, tigatuzumab, LY-135㸧
ࢆࡑࢀࡒࢀᶆⓗࡋࡓᢠయ་⸆ࡀ㛤Ⓨࡉࢀ࡚࠸ࡿ6, 23-26ࠋᢠయ་⸆ࢆ⏝࠸ࡓ⮫ᗋ ヨ㦂ࡣࠊ࠸ࡃࡘࡢ࡛࣏ࢪࢸࣈ࡞ഴྥࡀᚓࡽࢀࡓࡶࡢࡢᶆ‽ⓗᏛ⒪ἲ ࡢే⏝࡛㢧ⴭ࡞ຠᯝࡣᚓࡽࢀ࡞ࡗࡓ㸦Table-1B㸧ࠋ (A) (B)
Table-1 Results of Dulanermin and Conatumumab in clinical trials
(A and B) Abbreviations: BV, bevacizumab; Chemo, chemotherapy; CR, complete response; PR, partial response; n, number of patients; RCT, randomized-controlled trials
Anticancer activity was considered when the addition of the TRAIL-receptor aganist demonstrated statistically significant activity compared with the standard therapy
10 TRAIL ཷᐜయࢆᶆⓗࡋࡓᢠయ་⸆ࡢၥ㢟Ⅼࡋ࡚ࠊTRAIL ⪏ᛶࡢ⋓ᚓࡀᣲࡆ ࡽࢀࡓ27ࠋᐇ㝿ࠊ㌿⛣ࡋࡓ⭘⒆ࡢ㒊ศࡣTRAIL ⪏ᛶࢆ♧ࡋࠊTRAIL ⪏ᛶ ࡣࡀࢇ⒪ࡢ㐣⛬࡛ᘬࡁ㉳ࡇࡉࢀ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀTRAIL ࢩࢢࢼࣝࢆᶆ ⓗࡋࡓ⒪ࡢࡁ࡞㞀ቨ࡞ࡗ࡚࠸ࡿ 28ࠋTRAIL ⪏ᛶࢆ⋓ᚓࡋࡓ⣽⬊࡛ࡣࠊ ࣏ࢺ࣮ࢩࢫㄏᑟᛶࡢDR4/5 ࡢⓎ⌧పୗࡸࠊ࣏ࢺ࣮ࢩࢫ㜼ᐖᅉᏊࡢⓎ⌧␗ᖖ ࡢ㛵ࡀ♧၀ࡉࢀ࡚࠸ࡿ29, 30ࠋ௨ୖࡢࡇࡽࡶTRAIL ⪏ᛶᶵᵓࡢゎ᫂ࠊ⪏ ᛶゎ㝖㛵ࢃࡿศᏊࢆྠᐃࡍࡿࡇࡀᚲ㡲ㄢ㢟࡛࠶ࡿゝ࠼ࡿࠋ
11 ➨ ➨3 ⠇ TRAIL ⪏ᛶᶵᵓ ௨๓ࡼࡾࠊTRAIL ⪏ᛶ࣓࢝ࢽࢬ࣒ࡣ࣏ࢺ࣮ࢩࢫࢆㄏᑟࡋ࡞࠸ DcR1/2 ࡼࡗ ࡚ㄪ⠇ࡉࢀ࡚࠸ࡿ⪃࠼ࡽࢀ࡚࠸ࡓࠋᐇ㝿ࠊDcR1/2 ࡢ㧗Ⓨ⌧ࡣึᮇࡢ⫶ࡀࢇ ࡸஙࡀࢇ࡛㧗㢖ᗘㄆࡵࡽࢀ࡚࠾ࡾணᚋⰋ┦㛵ࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚ ࠸ࡿࡀࠊTRAIL ᑐࡍࡿឤཷᛶࡢ┦㛵㛵ಀࡣᮍࡔゎ᫂ࡉࢀ࡚࠸࡞࠸ࠋ㏆ᖺࠊ TRAIL ⪏ᛶࢆ⋓ᚓࡋࡓ⣽⬊࡛ࡣࠊ࣏ࢺ࣮ࢩࢫㄏᑟᛶࡢ DR4/5 ࡢⓎ⌧పୗࡸ FLIP㸦FLICE-inhibitory protein㸧ࠊBCL-2㸦B-cell lymphoma 2㸧ࣇ࣑࣮ࣜࠊIAP 㸦Inhibitor of apoptosis protein㸧ࣇ࣑࣮ࣜ࠸ࡗࡓ࣏ࢺ࣮ࢩࢫ㜼ᐖᅉᏊࡢⓎ
⌧␗ᖖࡢ㛵ࡀ♧၀ࡉࢀ࡚࠸ࡿ29, 30ࠋ ࣏ࢺ࣮ࢩࢫㄏᑟᛶࡢDR4/5 ࡢⓎ⌧పୗ ࡀࢇ࠾ࡅࡿ㧗࣓ࢳࣝࡸⅬ✺↛ኚ␗࡞ࡢ࢚ࣆࢪ࢙ࢿࢸࢵࢡ࡞ኚࡼࡿ ࣏ࢺ࣮ࢩࢫㄏᑟᛶࡢDR4/5 ࡢⓎ⌧పୗࡀ TRAIL ⪏ᛶ㛵ࡋ࡚࠸ࡿࡇࡀሗ࿌ ࡉࢀ࡚࠸ࡿ30-32ࠋᚑࡗ࡚ࠊᑡ࡞ࡃࡶᶵ⬟ⓗ࡞DR4/5 ࡢⓎ⌧ࡣ TRAIL ㄏᑟ⣽⬊ Ṛࡢάᛶᚲせ࡛࠶ࡿ⪃࠼ࡽࢀ࡚࠸ࡿࠋᡃࠎࡣࡇࢀࡲ࡛ࣄࢺ⭠⒴⣽⬊ᰴ DLD-1 ᑐࡍࡿ TRAIL ⪏ᛶᰴ㸦DLD-1/TRAIL㸧ࢆస〇ࡍࡿࡇࡼࡾ TRAIL ⪏
ᛶᶵᵓࡋ࡚ DR5 ࡢⓎ⌧పୗ DR5 ࡢ⣽⬊⾲㠃ୖࡢࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢ
Ⰻࢆ᫂ࡽࡋࡓ㸦Fig-3A and B㸧ࠋ FLIP ࡢ㐣Ⓨ⌧
12
FLIP ࡣࠊ࢝ࢫࣃ࣮ࢮ 8 ᵓ㐀ⓗ㢮ఝࡋ࡚࠾ࡾ FLIPS FLIPRࠊFLIPLࡢ 3 ࡘ
ࡢࢯࣇ࢛࣮࣒ࡀᏑᅾࡍࡿ33ࠋFLIPSFLIPRࡣ࢝ࢫࣃ࣮ࢮ8 ➇ྜࡋ DISC
ᙧᡂࢆ㜼ᐖࡍࡿ34, 35ࠋ୍᪉ࠊFLIPLࡢ࣏ࢺ࣮ࢩࢫ࠾ࡅࡿാࡁࡣ」㞧࡛ࡑࡢⓎ ⌧ࣞ࣋ࣝ౫Ꮡࡋ࡚࠸ࡿࠋ㐣Ⓨ⌧ࡋࡓ FLIPLࡣ࢝ࢫࣃ࣮ࢮ 8 ࣊ࢸࣟ㔞య ࢆᙧᡂࡍࡿࡇ࡛࢝ࢫࣃ࣮ࢮࡢάᛶࢆ㜼ᐖࡍࡿ36ࠋFLIP ࡢ㐣Ⓨ⌧ࡣከࡃࡢ ࡀࢇ࡛ㄆࡵࡽࢀ࡚࠾ࡾࠊTRAIL ࢩࢢࢼࣝࢆ⏝ࡋࡓࡀࢇ⒪ࡢᶆⓗࡋ࡚ᮇᚅ ࡉࢀ࡚࠸ࡿࠋ BCL-2 ࣇ࣑࣮ࣜࢱࣥࣃࢡ㉁ࡢⓎ⌧␗ᖖ BCL-2 ࣇ࣑࣮ࣜࢱࣥࣃࢡ㉁ࡣࠊ࣑ࢺࢥࣥࢻࣜࡢ⭷㏱㐣ᛶࢆไᚚࡋ࣑ࢺࢥࣥ ࢻࣜࢆࡋࡓ࣏ࢺ࣮ࢩࢫࡢಁ㐍ࡸ㜼ᐖ㛵ࡋ࡚࠸ࡿࠋࡇࢀࡲ࡛ࡢ◊✲ ࡽ࣏ࢺ࣮ࢩࢫಁ㐍ᛶࡢBAX/BAK ࡢⓎ⌧పୗྠᵝࠊ࣏ࢺ࣮ࢩࢫᢚไᛶࡢ BCL-2ࠊ BCL-XLࠊMCL-1 ࡢ㐣Ⓨ⌧ࡣࠊࡀࢇ࠾ࡅࡿᏛ⒪ἲࡸᨺᑕ⥺⒪ἲ ᢠᛶࢆ♧ࡍࡇࡀ᫂ࡽ࡞ࡗ࡚࠸ࡿ 37-39ࠋTRAIL ࢩࢢࢼࣝ㛵ࡋ࡚ࡶ BCL-2 ࣇ࣑࣮ࣜࢱࣥࣃࢡ㉁ࡢⓎ⌧␗ᖖࡀ TRAIL ⪏ᛶ㛵ࡋ࡚࠸ࡿྍ⬟ᛶࡀ ♧၀ࡉࢀ࡚࠸ࡿࠋ IAP ࣇ࣑࣮ࣜࢱࣥࣃࢡ㉁ࡢ㐣Ⓨ⌧ IAP ࣇ࣑࣮ࣜࢱࣥࣃࢡ㉁ࡣ࢝ࢫࣃ࣮ࢮࢆ┤᥋ⓗ㜼ᐖࡍࡿࡇࡼࡗ࡚ෆᅉ ᛶࠊእᅉᛶ࠸ࡎࢀࡶࡢ࣏ࢺ࣮ࢩࢫࢆ㜼ᐖࡍࡿࠋࡀࢇ࠾࠸࡚IAP ࡸ XIAP ࡢ㐣
13 Ⓨ⌧ࡀㄆࡵࡽࢀ࡚࠸ࡿࠋXu ࡽࡢ◊✲ࢢ࣮ࣝࣉࡣ IAP ࡢⓎ⌧ᢚไ㛵ࡍࡿࢩ ࢫࣉࣛࢳࣥࢆᢞࡍࡿࡇࡼࡾTRAIL ࡢឤཷᛶࡀᅇࡍࡿࡇࢆሗ࿌ࡋ࡚࠾ ࡾIAP ࣇ࣑࣮ࣜࢱࣥࣃࢡ㉁ࡢ㐣Ⓨ⌧ TRAIL ⪏ᛶࡢ㛵ࡀ♧၀ࡉࢀ࡚࠸ࡿࠋ ᚋ TRAIL ࣞࢭࣉࢱ࣮࡛࠶ࡿ DR4/DR5 ࡢⓎ⌧ࢆࡉࡽቑᙉࡍࡿపศᏊྜ≀ TRAIL ࡢే⏝ࡶࡋࡃࡣࠊ࣏ࢺ࣮ࢩࢫ㜼ᐖᅉᏊࡢⓎ⌧ᢚไ TRAIL ࡢే⏝ ࡼࡾእᅉᛶ⤒㊰ࡢ࣏ࢺ࣮ࢩࢫࢆቑᙉࡉࡏࡿ࡞ࠊTRAIL ⪏ᛶᶵᵓྜࢃࡏ ࡓඞ᭹ἲࡀᮇᚅࡉࢀࡿࠋ (A) (B) 㸦Kumazaki et al., 2015 ࡼࡾᢤ⢋) Figure-3
(A) Western blot analysis was performed to determine steady-state expression of DR5, DR4, and adaptor molecule FADD. E-actin was used as an internal control. Also shown are the steady-state expression levels of DR5 mRNA as relative ratios with respect to the GAPDH expression level. The expression level of mRNA was calculated by the
''Ct method. Means S.D. indicated by error bars are shown. (B)The photomicrograph shows the results of immunofluorescence staining for DR5 (anti-DR5) on the cell surface and in the cytosol of DLD-1 and DLD-1/TRAIL cells. Nuclei were counterstained in blue with Hoechst33342.
14 ➨ ➨4 ⠇ ࡀࢇ⣽⬊≉␗ⓗ࢚ࢿࣝࢠ࣮௦ㅰไᚚᶵᵓ㸸Warburg ຠᯝ ㏆ᖺࠊࡀࢇᑐࡍࡿ᪂ࡓ࡞⸆ࡢࢱ࣮ࢤࢵࢺࡋ࡚࢚ࢿࣝࢠ࣮௦ㅰࡀὀ┠ࡉࢀ ࡚࠸ࡿࠋࡀࢇ⣽⬊ࡣప㓟⣲࣭పᰤ㣴≧ែ࠾࠸࡚㝈ࡽࢀࡓ࢚ࢿࣝࢠ࣮※ࢆ᭷ຠ ⏝ ࡍ ࡿ ࡓ ࡵ ௦ ㅰ ᶵ ᵓ ࡢ ࣜ ࣉ ࣟ ࢢ ࣛ ࣑ ࣥ ࢢ ࢆ ⾜ ࡗ ࡚ ࠸ ࡿ ࠋ ࡑ ࡢ ୍ ࡘ ࡀ ࠕWarburg ຠᯝࠖ࠸࠺⌧㇟࡛࠶ࡿࠋWarburg ຠᯝࡣࠊࠕࡀࢇ⣽⬊ࡣዲẼⓗ᮲௳ ୗ࠾࠸࡚ࡶTCA ࢧࢡࣝࢆά⏝ࡏࡎࠊᖖ᎘Ẽⓗゎ⢾ࢆ⏝ࡋ࡚ࢢࣝࢥ࣮ࢫ ࢆ௦ㅰࡋࠊ㔞ࡢங㓟ࢆศἪࡍࡿࠖ࠸࠺⌧㇟ࢆOtto Warburg ࡀᥦၐࡋࡓࡶࡢ ࡛࠶ࡿ 7, 40ࠋࡀࢇ⣽⬊ࡀ Warburg ຠᯝࢆ⋓ᚓࡍࡿࡢ㔜せ࡞ᙺࢆᯝࡓࡋ࡚࠸ ࡿࡢࡀࠊPKM ࡛࠶ࡿࠋWarburg ຠᯝࡢ⋓ᚓᶵᵓࢆ Figure-4 ♧ࡋࡓࠋPKM ࡣ ゎ⢾⣔ࡢ᭱⤊ࢫࢸࢵࣉ࡛࠶ࡿ࣍ࢫ࢚࣍ࣀ࣮ࣝࣆࣝࣅࣥ㓟㸦PEP㸧ࡽࣆࣝࣅࣥ 㓟ࡢᛂࢆゐ፹ࡍࡿ㓝⣲࡛࠶ࡾࠊゎ⢾⣔ࡢᚊ㏿㓝⣲ࡋ࡚ാࡃ41ࠋPKM ࡣ PKM1 PKM2 ࡢ 2 ࡘࡢࢯࣇ࢛࣮࣒ࡀᏑᅾࡋࠊࢫࣉࣛࢩࣥࢢ㐣⛬࠾࠸ ࡚࢚࢟ࢯࣥ 8ࠊ9ࠊ11 ࢆྲྀࡾ㎸ࡴ PKM1ࠊ࢚࢟ࢯࣥ 8ࠊ10ࠊ11 ࢆྲྀࡾ㎸ࡴ PKM2 ࡀసࡽࢀࡿ 42ࠋࡀࢇ⣽⬊࠾࠸࡚ࡣࢫࣉࣛࢧ࣮࡛࠶ࡿ hnRNPA1ࠊ hnRNPA2ࠊPTBP1 ࡀ㧗Ⓨ⌧ࡍࡿࡇ࡛࢚࢟ࢯࣥ 9 ࡢྲྀࡾ㎸ࡳࡀ㜼ᐖࡉࢀࠊ࢚࢟ ࢯࣥ10 ࢆྲྀࡾ㎸ࢇࡔ PKM2 ࡀ㧗Ⓨ⌧ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ43-45ࠋⴭ⪅ࡽࡣࠊ ⭠⒴ࡢ⮫ᗋ᳨యࡢ90%௨ୖ࡛ PTBP1 ࡀ㧗Ⓨ⌧ࡋ࡚࠾ࡾࠊࡰࡍ࡚ࡢࡀࢇ✀ ࠾࠸࡚PKM2 ࡀ㧗Ⓨ⌧ࡋ࡚࠸ࡿࡇࢆ☜ㄆࡋ࡚࠸ࡿ46ࠋ
15 TCA ࢧࢡࣝࢆࡋࡓࢢࣝࢥ࣮ࢫ௦ㅰ࡛ࡣࢢࣝࢥ࣮ࢫ 1 ࣔࣝᙜࡓࡾ 36ATP ࡀ⏘ ⏕ࡉࢀࡿࡢᑐࡋࠊゎ⢾⣔࡛ࡣ2ATP ࡋ⏘⏕ࡍࡿࡇࡀ࡛ࡁ࡞࠸ࡓࡵ ATP ࡢ ⏘⏕ຠ⋡ࡀప࠸ࠋࡋࡋ࡞ࡀࡽࠊゎ⢾⣔ࡣTCA ࢧࢡࣝẚ㍑ࡋ࡚ᛂࢫࢸࢵ ࣉࡀ༢⣧࡛࠶ࡿࡓࡵATP ࡢ⏘⏕㏿ᗘࡀ㏿ࡃࠊࡀࢇ⣽⬊ࡣࢢࣝࢥ࣮ࢫࡢྲྀࡾ㎸ࡳ ࢆஹ㐍ࡉࡏࡿࡇ࡛㔞ࡢATP ⏘⏕ࢆྍ⬟ࡋ࡚࠸ࡿࠋࡲࡓࠊゎ⢾⣔ࡢ⏝ ࡣ 2 ࡘࡢⅬࡀᣲࡆࡽࢀࠊࡘࡲࡾ TCA ࢧࢡࣝࡢ㐣⛬࡛Ⓨ⏕ࡍࡿάᛶ㓟⣲
㸦reactive oxygen species; ROS㸧ࡢⓎ⏕ࢆᢚไ࡛ࡁࡿࡇ࡛࠶ࡿࠋ⣽⬊ෆ࡛㐣
⏘⏕ࡉࢀࡓROS ࡣ࣏ࢺ࣮ࢩࢫࡸ࣮࢜ࢺࣇࢪ࣮➼ࡢࣉࣟࢢ࣒ࣛ⣽⬊Ṛࡢཎ ᅉ࡞ࡿࡓࡵࠊゎ⢾⣔ࢆ⏝ࡍࡿࡇ࡛ROS ࡢⓎ⏕ࢆᢚไࡋ࡚࠸ࡿ⪃࠼ࡽࢀ ࡚࠸ࡿࠋࡶ࠺୍ࡘࡢⅬࡣࠊゎ⢾⣔ࡢ୰㛫௦ㅰ⏘≀ࢆ⏝ࡋࡓ࣌ࣥࢺ࣮ࢫࣜࣥ 㓟ᅇ㊰ࡼࡿ᰾㓟ྜᡂࡢಁ㐍࡛࠶ࡿࠋPKM1 ࡀᖖάᛶᆺࡢᅄ㔞య࡛Ꮡᅾࡍࡿ ࡢᑐࡋ࡚ࠊPKM2 ࡣẚ㍑ⓗάᛶࡢప࠸༢㔞యࡶࡋࡃࡣ㔞య࡛Ꮡᅾࡍࡿࠋࡇ ࡢከ㔞యࡢไᚚᶵᵓࡣ PKM2 ࡢࢳࣟࢩࣥࣜࣥ㓟ࡀ㛵ࡋ࡚࠾ࡾࠊࣜࣥ㓟 ࢆཷࡅࡓ PKM2 ࡣᅄ㔞యࡢᙧᡂࡀᅔ㞴࡞ࡿࠋPKM2 ࡢ࢟ࢼ࣮ࢮάᛶࡀప࠸ ≧ែಖࡓࢀࡿࡇ࡛ࠊ⣽⬊ෆ࡛ࡣゎ⢾⣔ࡢ୰㛫௦ㅰ⏘≀ࡀ✚ࡋࡸࡍࡃ࡞ࡿࠋ ⣽⬊ࡣࡇࡢ୰㛫௦ㅰ⏘≀ࢆ࣌ࣥࢺ࣮ࢫࣜࣥ㓟⤒㊰㸦PPP㸧ືဨࡋࠊ⣽⬊ศ ᚲせ࡞᰾㓟ྜᡂ⏝ࡋ࡚࠸ࡿࠋ ௨ୖࡢ㏻ࡾࠊWarburg ຠᯝࡣࡀࢇ⣽⬊ࡢቑṪࠊ⏕Ꮡࠊ࢚ࢿࣝࢠ࣮ࡢ⋓ᚓᴟࡵ
16
࡚㔜せ࡞ᶵᵓ࡛࠶ࡿࠋ
17 ➨ ➨5 ⠇ CCN1 ࡢⓎ⌧ TRAIL ㄏᑟ⣽⬊Ṛࡢ㛵 CCN1㸦CYR61㸧ࡣ CCN ࣇ࣑࣮ࣜᒓࡋࠊቑṪᅉᏊࠊࢧࢺ࢝ࣥࠊప㓟 ⣲≧ែࡼࡿ⣽⬊ࢫࢺࣞࢫ࡞ᵝࠎ࡞่⃭ࡼࡗ࡚ㄏᑟࡉࢀࡿ⣽⬊እ࣐ࢺࣜࢵ ࢡࢫࢱࣥࣃࢡ㉁࡛࠶ࡿ 47ࠋCCN1 ࡣ 4 ࡘࡢࢻ࣓ࣥࡽᵓᡂࡉࢀ࡚࠾ࡾࡑࢀࡒ ࢀࡀ⤖ྜࡍࡿࣥࢸࢢࣜࣥࣞࢭࣉࢱ࣮ࡀ␗࡞ࡿࡇࡽ⣽⬊ࡢ᥋╔ࠊ㐟㉮ࠊቑ Ṫࠊ⏕Ꮡ࡞ࡢᵝࠎ࡞⣽⬊άᛶࡢไᚚ㛵ࡋ࡚࠸ࡿ 48ࠋࣥࢸࢢࣜࣥࡣ 2 㔞 యࢱࣥࣃࢡ㉁࡛࠶ࡾࠊȘșࡢࢧࣈࣘࢽࢵࢺࡀ㠀ඹ᭷⤖ྜ࡛ྜࡋάᛶࡢ࠶ࡿ2 㔞యࢆᙧᡂࡋ࡚࠸ࡿ49, 50ࠋȘșࡢࢧࣈࣘࢽࢵࢺࡣᩘከࡃᏑᅾࡋࠊ20 ✀㢮௨ୖ ࡢȘșࡢࣥࢸࢢࣜࣥࡀྠᐃࡉࢀ࡚࠾ࡾࠊࡑࡢ⤌ࡳྜࢃࡏࡼࡾ⤖ྜࡍࡿศᏊ ࡀ␗࡞ࡿ 50, 51ࠋCCN1 ࡣ⣽⬊ࡢቑṪࡸ⏕Ꮡ㛵ࡍࡿ୍᪉ࠊ⥺⥔ⱆ⣽⬊࠾࠸ ࡚CCN ࢱࣥࣃࢡ㉁ࡣࠊTNF-Dࡶࡋࡃࡣ FasL Ꮡᅾୗ࡛࣏ࢺ࣮ࢩࢫࢆቑᙉࡍࡿ ࡇࡀ in vitro ཬࡧ in vivo ࡢ⣔࡛᫂ࡽ࡞ࡗ࡚࠸ࡿ 52ࠋࡑࡢ࣓࢝ࢽࢬ࣒ࢆ Figure-5 ♧ࡍࠋCCN1 ࡣࣥࢸࢢࣜࣥD6E1ࢩࣥࢹ࢝ࣥ 4㸦Syndecan-4㸧 ࡢ⤖ྜࢆࡋ࡚5-࣏ࣜ࢟ࢩࢤࢼ࣮ࢮ㸦5-Lox㸧ࡢάᛶࢆྵࡴᵝࠎ࡞࣓࢝ࢽࢬ࣒ ࡼࡾ㔞ࡢROS ࢆⓎ⏕ࡉࡏࡿࠋ㏻ᖖ TNF-D༢⊂࡛ࡣࠊNF-NEࡀ࢝ࢫࣃ࣮ࢮࡢ
άᛶࢆ㜼ᐖࡍࡿc-FLIP ࡢㄏᑟࡸ ROS ࢆᢚไࡍࡿࡇ࡛ JNK㸦c-Jun N-terminal
kinase㸧ࡢάᛶࢆ㜼ᐖࡍࡿࣇ࢛ࢫࣇࢱ࣮ࢮࢆㄏᑟࡍࡿࡇࡼࡾ TNF-Dㄏ ᑟᛶ࣏ࢺ࣮ࢩࢫࢆ㜼ᐖࡋ࡚࠸ࡿࠋࡋࡋ࡞ࡀࡽࠊCCN1 ࡼࡗ࡚⏘⏕ࡉࢀࡓ
18 ROS ࡣ JNK ࡢάᛶࢆಁ㐍⥔ᣢࡋࠊࡑࡢ⤖ᯝ FLIP ࡢാࡁࢆ㜼ᐖࡍࡿࡇ࡛ TNF-Dㄏᑟᛶࡢ࣏ࢺ࣮ࢩࢫࢆಁ㐍ࡍࡿࠋࡉࡽࠊROS ࡢⓎ⏕ࡣ p38/MAPK ࢆ άᛶࡉࡏࠊBAX ࡢ࣑ࢺࢥࣥࢻࣜࡢᒁᅾཬࡧࢩࢺࢡ࣒ࣟ c ࡢᨺฟࢆㄏᑟࡋࠊ FasL ࢆࡋࡓ࣏ࢺ࣮ࢩࢫࢆಁ㐍ࡍࡿࠋࡀࢇ⣽⬊࠾ࡅࡿ CCN1 ࡢാࡁࡘ࠸ ࡚ࡶ⥺⥔ⱆ⣽⬊㢮ఝࡋ࡚࠸ࡿࠋࡀࢇࡢ㛫㉁࡛ࡣ⥺⥔ⱆ⣽⬊ࡀάᛶࡉࢀ࡚࠾ ࡾࠊࡀࢇ࠾ࡅࡿ CCN1 ࡢ㐣Ⓨ⌧ࡣࠊAKT ࡸ ERK ࢩࢢࢼࣝࢆάᛶࡉࡏࡿ ࡇ࡛⭘⒆ቑṪࡸ⾑⟶ᐦᗘࢆୖ᪼ࡉࡏࡿࡇࡀච࣐࢘ࢫࢆ⏝࠸ࡓᐇ㦂ࡼ ࡾ᫂ࡽ࡞ࡗ࡚࠸ࡿ51ࠋ㏆ᖺࠊ๓❧⭢ࡀࢇ࠾࠸࡚ࡶTRAIL Ꮡᅾୗ࡛ࡣࣥ ࢸࢢࣜࣥࣞࢭࣉࢱ࣮DvE3ࠊD6E4ࡢ⤖ྜࢆࡋ࡚ TRAIL ㄏᑟ⣽⬊Ṛࢆቑᙉࡍࡿ ࡇࡀ᫂ࡽ࡞ࡗࡓ52ࠋ๓❧⭢ࡀࢇ⣽⬊PC3 ࠾ࡅࡿ TRAIL ࡢ┦ຠᯝ
ࡣROS ࡢⓎ⏕ࡣ㛵ࡋ࡚࠾ࡽࡎ PKC 㸦Protein kinase C㸧Dࡢάᛶ౫Ꮡⓗ
ㄏᑟࡉࢀࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀヲ⣽࡞ᶵᵓࡣ᫂ࡽࡉࢀ࡚࠸࡞࠸ 52ࠋ
CCN1 ࡢ࣏ࢺ࣮ࢩࢫㄏᑟᶵᵓࡣ CCN1 ࡢࣥࢸࢢࣜࣥࣞࢭࣉࢱ࣮௨እࡢ⤖
ྜࡶࡋࡃࡣ TRAIL ㄏᑟ⣽⬊Ṛ㛵ࡍࡿࣞࢭࣉࢱ࣮㸦DR4/5㸧ࡢྜ㛵ࡍ
19
20 ➨3 ❶ Warburg ຠᯝ㛵㐃 PTBP1 TRAIL ㄏᑟ࣏ࢺ࣮ࢩࢫ ➨1 ⠇ ᗎ TRAIL ࢆࡋࡓࡀࢇ⣽⬊≉␗ⓗ࡞⣽⬊Ṛࡣᢠ⭘⒆⸆ࡢᶆⓗࡋ࡚ᮇᚅࡉࢀ࡚ ࠸ࡿࠋࣄࢺ⤌ࡳ࠼ᆺTRAIL ࡣࣄࢺࡢ⏕యෆ࠾ࡅࡿ༙ῶᮇࡀ㠀ᖖ▷ࡃࠊ㔜 ⠜࡞⫢ᶵ⬟㞀ᐖࡢᠱᛕࡀ࠶ࡿࡇࡽࠊ⌧ᅾࡣDR4/5 ࢆᶆⓗࡋࡓᢠయ་⸆ࡢ 㛤Ⓨࡀ⾜ࢃࢀ࡚࠸ࡿࠋࡋࡋ࡞ࡀࡽࠊTRAIL ⪏ᛶࡢ⋓ᚓࡀࡁ࡞㞀ቨ࡞ࡗ࡚ ࠾ࡾᮍࡔ᭷ຠ࡞⒪ἲࡣ☜❧ࡉࢀ࡚࠸࡞࠸ࠋࡑࡇ࡛ᮏ❶࡛ࡣࠊTRAIL ㄏᑟ⣽⬊ Ṛࢆ⏝ࡋࡓࡀࢇࡢ⒪⸆ࡢ㛤ⓎྥࡅTRAIL ⪏ᛶゎ㝖㛵ࢃࡿศᏊࡢྠᐃཬ ࡧࡑࡢ⪏ᛶゎ㝖ᶵᵓࡢゎ᫂ࢆヨࡳࡓࠋ
21 ➨2 ⠇ PTBP1 ࡢⓎ⌧ TRAIL ㄏᑟࡀࢇ⣽⬊Ṛࡢ㛵 ࡀࢇ⣽⬊≉␗ⓗ࡞࢚ࢿࣝࢠ࣮௦ㅰᶵᵓ࡛࠶ࡿWarburg ຠᯝࡣ PTBP1/PKM ࢝ࢫ ࢣ࣮ࢻࡼࡗ࡚ᡂ❧ࡋ࡚࠾ࡾࠊࡇࢀࡲ࡛⭠ࡀࢇࡢ⮫ᗋ᳨యࡢ 90%௨ୖ࡛ PTBP1 ࡀ㧗Ⓨ⌧ࡋ࡚࠸ࡿࡇࢆሗ࿌ࡋ࡚࠸ࡿࠋ2 ✀⣽⬊ᰴࢆ⏝࠸ࣞゎᯒ㸦㑇 ఏᏊⓎ⌧ࣉࣟࣇࣝ㸧ࢆ⾜ࡗࡓࡇࢁࠊDLD-1 ẚ㍑ࡋ࡚ DLD-1/TRAIL ࠾ ࠸࡚PTBP1 ࡢ㧗Ⓨ⌧ࡀㄆࡵࡽࢀࡓࠋᐇ㝿ᐃᖖ≧ែ࠾ࡅࡿ PTBP1 ࡢࢱࣥࣃ ࢡ㉁࡛ࣞ࣋ࣝࡢⓎ⌧ࢆ࢚࢘ࢫࢱࣥࣈࣟࢵࢺἲࡼࡾ᳨ドࡋࡓࡇࢁࠊࣞゎ ᯒ ࡢ ⤖ ᯝ ྠ ᵝ DLD-1/TRAIL ࠾࠸࡚ PTBP1 ࡢ㧗Ⓨ⌧ࡀ☜ㄆࡉࢀࡓ 㸦Fig-6A㸧ࠋ (A) Figure-6
(A) Western blot analysis was performed to determine the steady-state expression of PTBP1 in TRAIL-sensitive DLD-1 and –resistant DLD-1 cells. E-actin was used as an internal control.
22 ࡑࡇ࡛ࠊsiRNA ࢆ⏝࠸࡚ PTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓࡢ TRAIL ᑐࡍ ࡿឤཷᛶࡢᙳ㡪ࢆホ౯ࡋࡓࠋ࠸ࡎࢀࡢ⣽⬊ᰴ࠾࠸࡚ࡶPTBP1 ࢆࣀࢵࢡࢲ࢘ ࣥ ࡋ ࡓ ⣽ ⬊ ࡛ ࡣ ࠊ ⣙ 20 㸣 ࡢ ⏕ ⣽ ⬊ ᩘ ࡢ ῶ ᑡ ࡀ ㄆ ࡵ ࡽ ࢀ ࡓ 㸦 Fig-6B 㸧ࠋ DLD-1/TRAIL ࠾࠸࡚ቑṪᢚไຠᯝࢆ♧ࡉ࡞࠸ TRAIL 5 ng/ml ࠾࠸࡚ࡶ PTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ࡛ TRAIL ࡼࡿ⣽⬊Ṛࡀ┦ⓗㄏᑟࡉࢀࡿࡇ ࡀศࡗࡓ㸦Fig-6B㸧ࠋFigure-6C ࡛♧ࡍࡼ࠺ PTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ⣽ ⬊࡛ࡣTRAIL ㄏᑟ࣏ࢺ࣮ࢩࢫࡢᣦᶆ࡛࠶ࡿ࢝ࢫࣃ࣮ࢮ 8 ࡢ㢧ⴭ࡞άᛶࡀㄆ ࡵࡽࢀࡓࠋ (B)
23
(C)
Figure-6
(B) TRAIL-sensitive and -resistant DLD-1 cells were transfected with siR-PTBP1 (2 nM) for 48 h and then treated with rTRAIL (5, 10 ng/ml) for 24 h. The cell viability was estimated at 72 h after the transfection. The cell viability of the control (0; PBS alone) is indicated as 100 %. The growth inhibition effect by TRAIL with and without the transfection with siR-PTBP1 was assessed by the average value of the growth inhibition ratios (GI) at each TRAIL concentrations (5, 10 ng/ml). We defined the synergistic effect (Syn) by introduction of siR-PTBP1 as the ratios of siR-PTBP1 to control siRNA GI value. (C) Western blot analysis was performed to determine the level of the active form of caspase-8. E-actin was used as an internal control.
ࡉࡽࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ⣽⬊࠾ࡅࡿ࢚ࢿࣝࢠ࣮௦ㅰࡢᙳ㡪ࢆ᳨ ドࡍࡿPKM2 ࡽ PKM1 ࡢࢫࢵࢳࡀほᐹࡉࢀࡓ(Fig-6D)ࠋPKM1 ࡢⓎ⌧ ࡀ᭷ព࡞ࡿࡇ࡛ゎ⢾⣔ࡽ㓟ⓗࣜࣥ㓟ࡢࢩࣇࢺࡀண ࡉࢀࠊ⣽⬊ෆ ࡢATP ࣞ࣋ࣝゎ⢾⣔ࡢ᭱⤊⏘≀࡛࠶ࡿங㓟ࣞ࣋ࣝࡢ ᐃࢆ⾜ࡗࡓࠋPTBP1 ࢆ ࣀࢵࢡࢲ࢘ࣥࡋࡓ⣽⬊࡛ࡣࠊ⣽⬊ෆࡢATP ࣞ࣋ࣝࡢቑຍங㓟ࣞ࣋ࣝࡢῶᑡࡀ ㄆࡵࡽࢀࡓ㸦Fig-6E㸧ࠋࡇࡢ⤖ᯝࡽࠊPTBP1 ࡢⓎ⌧పୗࡼࡾ⣽⬊ෆࡢ࢚ࢿࣝ ࢠ࣮௦ㅰࡀ୍㒊ゎ⢾⣔ࡽ㓟ⓗࣜࣥ㓟ࢩࣇࢺࡋ࡚࠸ࡿࡇࡀศࡗࡓࠋ
24
(D) (E)
Figure-6
(D)TRAIL-sensitive and -resistant DLD-1 cells were transfected with control or PTBP1 siRNA (siR-PTBP1; 2 nM) for 48 h. Western blot analysis was performed to determine the expression of Warburg effect-related genes. E-actin was used as an internal control. (E) TRAIL-sensitive and -resistant DLD-1 cells was transfected with control or siR-PTBP1 (2, 5 nM) for 72 h. The ATP and lactate production were normalized to cell numbers, and that of the Control (0) is indicated as “1”.
ᐇ㝿 PTBP1 ࡢࣀࢵࢡࢲ࢘ࣥࡼࡿ Warburg ຠᯝࡢ⬺ไᚚ㸦ゎ⢾⣔ࡽ㓟
ⓗࣜࣥ㓟ࡢࢫࢵࢳ㸧ࡀTRAIL ᑐࡍࡿឤཷᛶࡢᅇ㛵ࡋ࡚࠸ࡿࡢ
ࢆゎ⢾⣔ࡢ㜼ᐖ࡛࠶ࡿ 2-ࢹ࢜࢟ࢩࢢࣝࢥ࣮ࢫ㸦2-DG㸧ࢆ⏝࠸᳨࡚ドࡋࡓࠋ
DLD-1/TRAIL 2-DG ࢆ 48 㛫స⏝ࡉࡏࡓᚋ TRAIL ࢆᢞࡋࡓ⣽⬊࡛ࡣ⏕⣽
25 ⤖ᯝࡽࠊWarburg ຠᯝࡢᡂ❧ᚲ㡲࡞㑇ఏᏊ PTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ ࡼࡿWarburg ຠᯝࡢ⬺ไᚚࢆࡋ࡚ TRAIL ᑐࡍࡿឤཷᛶࡀᅇࡍࡿࡇࡀ ᫂ࡽ࡞ࡗࡓࠋ (F) Figure-6
(F) TRAIL-resistant DLD-1 cells were pretreated with 2-DG (5 mM) for 24 h and then treated with rTRAIL (5, 10, 25 ng/ml) for 24 h. The cell viability was estimated at 48 h after the start of treatment. The cell viability of the control (0; DMSO alone) is indicated as 100 %. Western blot analysis was performed to determine the level of the active form of caspase-8. E-actin was used as an internal control.
26 ࡉࡽࠊPTBP1 ࡢⓎ⌧ TRAIL ㄏᑟ⣽⬊Ṛࡢ㛵ಀࢆ᫂ࡽࡍࡿࡓࡵࠊࢺ ࣜࣃࣥࣈ࣮ࣝⰍ⣲㝖ヨ㦂ἲࡼࡾTRAIL ࡢ IC50್ࡀ20 ng/ml ௨ୖ࡛࠶ࡗࡓࣄ ࢺ⭠⒴⣽⬊ᰴSW480 ࠊṇᖖࣄࢺங⭢ୖ⓶⣽⬊ᰴ MCF10A ࢆ⏝࠸࡚ྠᵝࡢ ᐇ㦂ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝ2 ✀⣽⬊ᰴࡢ TRAIL ࡢ IC50್ࡣࠊࡑࢀࡒࢀ20.2 ng/mlࠊ 102.1 ng/ml ࡞ࡗࡓࠋึࡵࠊᐃᖖ≧ែ࠾ࡅࡿ PTBP1 ࡢⓎ⌧ࢆ࢚࢘ࢫࢱࣥ ࣈࣟࢵࢺἲࡼࡾ᳨ドࡋࡓࠋ࠸ࡎࢀࡢ⣽⬊ᰴ࠾࠸࡚ࡶDLD-1/TRAIL ྠᵝࠊ PTBP1 ࡢ㧗Ⓨ⌧ࡀㄆࡵࡽࢀࠊMCF10A ࡛ࡑࡢⓎ⌧ࣞ࣋ࣝࡀ᭱ࡶ㧗࠸ࡇࡀศ ࡗࡓ㸦Fig-6G㸧ࠋḟࠊPTBP1 ࡢⓎ⌧ࢆ siRNA ࢆ⏝࠸࡚ࣀࢵࢡࢲ࢘ࣥࡋࡓࡢ TRAIL ᑐࡍࡿឤཷᛶࡢᙳ㡪ࢆ᳨ドࡋࡓࠋ࠸ࡎࢀࡢ⣽⬊ᰴ࠾࠸࡚ࡶ TRAIL 5 ng/ml ࠾࠸࡚ PTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ࡛ TRAIL ࡼࡿ⣽⬊Ṛࡀ┦ⓗ ㄏᑟࡉࢀࡿࡇࡀศࡗࡓ㸦Fig-6H㸧ࠋ୰࡛ࡶ TRAIL ࡢ IC50 ್ࡀ᭱ࡶ㧗࠸
MCF10A ࡛ TRAIL ࡢឤཷᛶࡢᅇຠᯝࡀᙉࡃㄆࡵࡽࢀࡓ㸦Syn:2.94㸧ࠋFigure-6G
♧ࡍࡼ࠺ࠊDLD-1/TRAIL ྠᵝ PTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ
ࡼࡾPKM2 ࡽ PKM1 ࡢࢫࢵࢳࡀㄆࡵࡽࢀࡓ㸦Fig-6G㸧ࠋ௨ୖࡢ⤖ᯝࡽࠊ
PTBP1 ࡢ㧗Ⓨ⌧ࡣ TRAIL ⪏ᛶ㛵㐃ࡋ࡚࠾ࡾࠊTRAIL ⪏ᛶࡀᙉ࠸⣽⬊ᰴ PTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇࡼࡾ TRAIL ᑐࡍࡿឤཷᛶࡢᅇຠᯝ ࡀᙉ࠸ࡇࡀศࡗࡓࠋ
27
(G)
(H)
Figure-6
(G) TRAIL-sensitive DLD-1, TRAIL-resistant SW480 and MCF10A cells were transfected with control or siR-PTBP1 (2 nM) for 48 h. Western blot analysis was
28
performed to determine the expression of Warburg effect-related genes. E-actin was used as an internal control. (H) TRAIL-resistant SW480 and MCF10A cells were transfected with siR-PTBP1 (2 nM) for 48 h and then treated with rTRAIL (5, 10 ng/ml) for 24 h. The cell viability was estimated at 72 h after the transfection. The cell viability of control (0; PBS alone) is indicated as 100 %. The growth inhibition effect by TRAIL with and without the transfection with siR-PTBP1 was assessed by the average value of the growth inhibition ratios (GI) at each TRAIL concentrations (5, 10 ng/ml). We defined the synergistic effect (Syn) by introduction of siR-PTBP1 as the ratios of siR-PTBP1 to control siRNA GI value. Data are expressed as means r SD of 3 different experiments.
29 ➨ ➨3 ⠇ TRAIL ⪏ᛶᶵᵓ࠾ࡅࡿ PTBP1 ࡢᙺ ࡇࢀࡲ࡛ࡢ◊✲ࡽ DR5 ࡢⓎ⌧పୗ⣽⬊⾲㠃ୖࡢࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢ ⰋࡀTRAIL ⪏ᛶࡢせᅉ࡛࠶ࡿࡇࢆ᫂ࡽࡋࡓ53ࠋᮏ⠇࡛ࡣࠊPTBP1 ࢆࣀࢵ ࢡࢲ࢘ࣥࡋࡓ㝿2 ࡘࡢ TRAIL ⪏ᛶᶵᵓࡀゎ㝖ࡉࢀࡿ࠺ࢆ᳨ドࡋࡓࠋึ ࡵࠊsiR-PTBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋ 48 㛫࡛ࡢ DR5 ࡢⓎ⌧ࣞ࣋ࣝࢆ ࢚࢘ࢫࢱࣥࣈࣟࢵࢺἲࡼࡾ᳨ドࡋࡓࠋࡑࡢ⤖ᯝࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ ⣽⬊࠾࠸࡚ DR5 ࡢⓎ⌧ࡀ㢧ⴭቑຍࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓ㸦Fig-7A㸧ࠋ ࡑࡇ࡛ࠊPTBP1 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆస〇ࡋࠊPTBP1 ࡀ DR5 ࡢ㌿࠼ࡿᙳ㡪 ࢆ᳨ドࡋࡓࠋDLD-1 PTBP1 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋ 24 㛫ᚋ࠾ࡅࡿDR5 ࡢⓎ⌧ࣞ࣋ࣝࢆ࢚࢘ࢫࢱࣥࣈࣟࢵࢺἲࡼࡾホ౯ࡋࡓࡇࢁࠊ DR5 ࡢⓎ⌧పୗࡀㄆࡵࡽࢀࡓࡀ㸦Fig-7B㸧ࠊࢡ࣐ࣟࢳࣥචỿ㝆ἲ࡛ࡣ PTBP1 DR5 ࡢ┤᥋⤖ྜࡀㄆࡵࡽࢀࡎ PTBP1 ࡀ DR5 ࡢ㌿㛵ࡋ࡚࠸࡞࠸ࡇࡀ ᫂ࡽ࡞ࡗࡓࠋḟDR5 ࡢ⣽⬊⾲㠃ୖࡢࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢⰋࡢᙳ㡪 ࢆ᳨ドࡋࡓࠋ2 ✀⣽⬊ᰴ siR-PTBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋ 48 㛫࡛ࡢ DR5 ࡢ⣽⬊ෆᒁᅾࢆචᰁⰍἲࡼࡾホ౯ࡋࡓ⤖ᯝࠊ࠸ࡎࢀࡢ⣽⬊ᰴ࠾࠸࡚ ࡶPTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇࡼࡾ⣽⬊⾲㠃ୖࡢ DR5 ࡢⓎ⌧ቑ ຍࡀほᐹࡉࢀࡓ㸦Fig-7C㸧ࠋ௨ୖࡢ⤖ᯝࡽ PTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿ ࡇ࡛DR5 ࡢⓎ⌧⣽⬊⾲㠃ୖࡢࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡀㄏᑟࡉࢀ TRAIL ⪏ᛶࡀ
30
ゎ㝖ࡉࢀࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ
(A) (B)
(C)
Figure-7
(A) TRAIL-sensitive and -resistant DLD-1 cells were transfected with control or siR-PTBP1 (2, 5 nM) for 48 h. Western blot analysis was performed to determine the expression levels of PTBP1 and DR5. (B) TRAIL-resistant DLD-1 cells were transfected with control and PTBP1 expression plasmid vectors (0.2 Pg/ml) for 24 h. Western blot analysis was performed to determine the levels of PTBP1 and DR5. (C) TRAIL-sensitive and -resistant DLD-1 cells were transfected with siR-PTBP1 (2, 5 nM) for 48 h. The results of immunofluorescence staining for anti-DR5 antibody binding on the cell surface and in the cytosol of untreated (0: Control siRNA) or siR-PTBP1 transfected cells are shown. Nuclei were counterstained in blue with Hoechst33342. Anti-DR5 antibody bound to the cell surface, which is indicated by the white arrows.
31 ➨ ➨4 ⠇ άᛶ㓟⣲㸦ROS㸧ࡢⓎ⏕ TRAIL ⪏ᛶゎ㝖ᶵᵓࡢ㛵 ๓⠇࠾࠸࡚ࠊPTBP1 ࡣ DR5 ࡢ㌿┤᥋㛵ࡋ࡚࠸࡞࠸ࡇࡽࠊ⣽⬊ෆ ࡢ࢚ࢿࣝࢠ࣮௦ㅰࡢኚ㸦Warburg ຠᯝࡢ⬺ไᚚ㸧ࡀ DR5 ࡢⓎ⌧ቑຍ㛵ࡋ ࡚࠸ࡿࢆ᳨ウࡋࡓࠋ➨2 ⠇࠾࠸࡚ PTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇ ࡼࡾPKM2 ࡽ PKM1 ࡢࢫࢵࢳࡀㄏᑟࡉࢀࠊ⣽⬊ෆࡢ࢚ࢿࣝࢠ࣮௦ㅰࡀ୍ 㒊ゎ⢾⣔ࡽ࣑ࢺࢥࣥࢻࣜ࠾ࡅࡿTCA ࢧࢡࣝࡼࡿ㓟ⓗࣜࣥ㓟ࢩ ࣇࢺࡍࡿࡇࢆ᫂ࡽࡋࠊ⣽⬊ෆ࡛ࡣάᛶ㓟⣲㸦ROS㸧ࡀⓎ⏕ࡋ࡚࠸ࡿྍ⬟ ᛶࡀண ࡉࢀࡓࠋᡃࠎࡢ◊✲ࢢ࣮ࣝࣉࡣࡍ࡛ࠊPTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥ ࡍࡿࡇࡼࡾ⣽⬊ෆ࡛ROS ࡢⓎ⏕ࡀቑຍࡍࡿࡇࢆሗ࿌ࡋ࡚࠸ࡿ9ࠋࡑࡇ࡛ࠊ ⣽⬊ෆ࡛Ⓨ⏕ࡋࡓ ROS TRAIL ⪏ᛶゎ㝖ᶵᵓࡢ㛵ࢆ᳨ドࡍࡿࡓࡵࠊᢠ 㓟࡛࠶ࡿN-ࢭࢳࣝࢩࢫࢸࣥ㸦NAC㸧ࢆ⏝࠸࡚ᐇ㦂ࢆ⾜ࡗࡓࠋNAC ࢆ 6 㛫స⏝ࡋࡓᚋTRAIL ᑐࡍࡿឤཷᛶࡢᙳ㡪ࢆࢺࣜࣃࣥࣈ࣮ࣝⰍ⣲㝖ヨ㦂 ἲࡼࡾ ᐃࡋࡓࠋࡑࡢ⤖ᯝࠊNAC ࢆస⏝ࡉࡏࡓ⣽⬊࠾࠸࡚ࠊPTBP1 ࡢࣀࢵ ࢡࢲ࢘ࣥࡼࡿ TRAIL ㄏᑟ⣽⬊Ṛࡀ୍㒊࢟ࣕࣥࢭࣝࡉࢀࡿࡇࡀศࡗࡓ 㸦Fig-8A㸧ࠋ㠀ᖖ⯆῝࠸ࡇࠊNAC ࢆస⏝ࡉࡏࡓ⣽⬊࡛ࡣ PTBP1 ࡢࣀࢵ ࢡࢲ࢘ࣥࡼࡾゎ㝖ࡉࢀ࡚࠸ࡓDR5 ࡢⓎ⌧ࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢㄏᑟࡶ୍㒊࢟ ࣕࣥࢭࣝࡉࢀࡿࡇࡀ᫂ࡽ࡞ࡗࡓ㸦Fig-8B and C㸧ࠋࡇࢀࡽࡢ⤖ᯝࡽࠊ PTBP1 ࡢࣀࢵࢡࢲ࢘ࣥࡼࡿ DR5 ࡢⓎ⌧ࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢㄏᑟࡣ
32
Warburg ຠᯝࡢ⬺ไᚚࡼࡿ ROS ࡢⓎ⏕ࡀ㛵ࡋ࡚࠸ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ (A) (B)
(C)
Figure-8
(A)TRAIL-sensitive DLD-1 cells were pretreated with NAC (1 mM) for 6 h and then incubated with siR-PTBP1 (5 nM) and/or rTRAIL (5, 10 ng/ml) for 24 h. The cell viability was estimated at 72 h after the treatment. Data were obtained from 3 independent experiments. The cell viability of the control (Control; Control-siRNA alone) is indicated as 100 %. (B) TRAIL-sensitive DLD-1 cells were pre-treated with NAC (1 mM) before the transfection with siR-PTBP1. Western blot analysis was performed to determine the expression level of DR5 protein. E-actin was used as an internal control. (C) TRAIL-sensitive DLD-1 cells treated with NAC (1 mM) and/or siR-PTBP1 (5 nM). The results of immunofluorescence staining for anti-DR5 binding on the cell surface and in the cytosol of untreated (0: Control-siRNA) or siR-PTBP1 transfected cells are shown. Nuclei were counterstained in blue with Hoechst33342. Anti-DR5 antibody bound to the cell surface, which is indicated by the white arrows. *p<0.05, as indicated by the brackets (Student’s t test).
33 ➨ ➨5 ⠇ CCN1 TRAIL ㄏᑟ⣽⬊Ṛࡢ㛵 ᮏ⠇࡛ࡣࠊDLD-1/TRAIL ࠾࠸࡚ Control ⣽⬊ PTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ ⣽⬊ࢆ⏝࠸࡚ࣞゎᯒࢆ⾜࠸ PTBP1 ࡢࣀࢵࢡࢲ࢘ࣥࡼࡿࡑࡢࡢ TRAIL ⪏ᛶゎ㝖࣓࢝ࢽࢬ࣒ࡢゎ᫂ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ⣽ ⬊࠾࠸࡚ CCN1 㑇ఏᏊࡢ㢧ⴭ࡞Ⓨ⌧ஹ㐍ࡀㄆࡵࡽࢀࡓࠋCCN1 ࡣࠊࣥࢸࢢ ࣜࣥ⤖ྜࢱࣥࣃࢡ㉁࡛࠶ࡾࣥࢸࢢࣜࣥࣞࢭࣉࢱ࣮⤖ྜࡍࡿࡇ࡛⣽⬊᥋╔ࠊ 㐟㉮ࠊ⏕Ꮡ࡞ᵝࠎ࡞⣽⬊άᛶไᚚ㛵ࡋ࡚࠸ࡿ 48ࠋ୍᪉ࠊCCN1 ࡣ๓❧⭢ ࡀࢇ⣽⬊࠾࠸࡚TRAIL ㄏᑟ⣽⬊Ṛࢆቑᙉࡍࡿࡇࡀሗ࿌ࡉࢀ࠾ࡾࠊ࣏ࢺ࣮ ࢩࢫࡢㄏᑟ㛵ࡍࡿࡇࡀ♧၀ࡉࢀ࡚࠸ࡿ52ࠋ
DLD-1/TRAIL siR-PTBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ㝿ࡢ CCN1 ࡢ mRNAࠊ ࢱࣥࣃࢡ㉁Ⓨ⌧ࣞ࣋ࣝࢆ᳨ドࡋࡓࠋᐃᖖ≧ែ࠾࠸࡚ࡑࡢⓎ⌧ࡀㄆࡵࡽࢀ࡞࠸ ࡢᑐࡋ࡚ࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ⣽⬊࡛ࡣ㢧ⴭ mRNAࠊࢱࣥࣃࢡ㉁ࣞ ࡛࣋ࣝⓎ⌧ࡀቑຍࡋ࡚࠸ࡿࡇࡀศࡗࡓ㸦Fig-9A㸧ࠋࡑࡇ࡛ PTBP1 ࡢࣀࢵࢡ ࢲ࢘ࣥࡼࡾㄏᑟࡉࢀࡓCCN1 ࡢⓎ⌧ࡀ TRAIL ⣽⬊Ṛࡢㄏᑟಁ㐍ࡢࡼ࠺ 㛵 ࡋ ࡚ ࠸ ࡿ ࡢ ᳨ ド ࡋ ࡓ ࠋ ึ ࡵ ࠊCCN1 ࡢ Ⓨ ⌧ ࣋ ࢡ ࢱ ࣮ ࢆ స 〇 ࡋ DLD-1/TRAIL CCN1 ࢆ㐣Ⓨ⌧ࡋࡓࡢ TRAIL ᑐࡍࡿឤཷᛶࢆホ౯ࡋࡓࠋ CCN1 ࣋ࢡࢱ࣮ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋ 24 㛫࠾࠸࡚ࡑࡢⓎ⌧ஹ㐍ࡀㄆࡵ ࡽࢀࡓࡓࡵᮏᐇ㦂࡛ࡣࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥᚋ24 㛫ࡢ⣽⬊ࢆ CCN1 㐣Ⓨ⌧
34 ⣽⬊ࡋ࡚⏝ࡋࡓ㸦Fig-9B㸧ࠋ⯆῝࠸ࡇࠊCCN1 ࢆ㐣Ⓨ⌧ࡋࡓ⣽⬊࡛ ࡣࠊࢥࣥࢺ࣮ࣟࣝ⣽⬊ẚ㍑ࡋ࡚࢝ࢫࣃ࣮ࢮ8 ࡢάᛶࢆకࡗࡓ TRAIL ㄏᑟ⣽ ⬊Ṛࡀቑᙉࡉࢀࡿࡇࡀศࡗࡓ㸦Fig-9C㸧ࠋࡲࡓࠊPTBP1 ࡢ CCN1 ᑐࡍࡿ ㌿ࡢᙳ㡪ࢆࢡ࣐ࣟࢳࣥචỿ㝆ἲࡼࡾホ౯ࡋࡓ⤖ᯝࠊPTBP1 ࢱࣥࣃࢡࡀ CCN1 ࡢ mRNA ┤᥋⤖ྜࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧ࡉࢀࡓ㸦Fig-9D㸧ࠋࡉࡽࠊPTBP1 ࡢⓎ⌧࣋ࢡࢱ࣮ࢆ⏝࠸PTBP1 ࢆ㐣Ⓨ⌧ࡋࡓ⣽⬊࠾࠸࡚ CCN1 ࡢⓎ⌧ቑຍࡀ ୍㒊࢟ࣕࣥࢭࣝࡉࢀࡓ㸦Fig-9E㸧ࠋ௨ୖࡢ⤖ᯝࡽࠊPTBP1 ࡣ TRAIL ㄏᑟ⣽⬊ Ṛ㛵ࡍࡿCCN1 ࡢࣜࣉࣞࢵࢧ࣮ࡋ࡚ᶵ⬟ࡋ࡚࠾ࡾࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ ࣥࡍࡿࡇࡼࡿCCN1 ࡢⓎ⌧ቑຍࢆࡋ࡚ TRAIL ㄏᑟ⣽⬊Ṛࡀቑᙉࡉࢀࡿྍ ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ (A)
35
(B) (C)
(D) (E)
Figure-9
(A) DLD-1/TRAIL cells were transfected with siR-PTBP1 (2, 5 nM) for 48 h. The expression levels of CCN1 mRNA as a relative ratio with respect to the GAPDH expression level was evaluated by RT-qPCR. Also shown is the expression level of CCN1 protein determined by performing Western blot analysis. Means (S.D) indicated by error bars are shown. (B) Control and CCN1-expression plasmid vectors (0.2 Pg/ml) were used to transfect DLD-1/TRAIL cells for 24 or 48 h. Western blot analysis was
36
performed to determine the expression of CCN1 with E-actin used as the internal control. (C) Control and CCN1-expression plasmid vectors (0.2 Pg/ml) were used to transfect DLD-1/TRAIL cells for 24 h, and the cells were then exposed to rTRAIL (10, 25, 50 ng/ml) for 24 h. The cell viability was estimated at 48 h after the treatment. The cell viability of the control (0; PBS alone) is indicated as 100 %. Western blot analysis was performed to determine the expression of activation of caspase-8 with E-actin used as the internal control. (D) MCF10A cells transfected with control or siR-PTBP1 (2 nM) for 48 h. The expression levels of CCN1 mRNA as relative ratios with respect to the GAPDH expression level was evaluated by RT-qPCR. The expression level of input DNA is indicated as “1”. (E) DLD-1/TRAIL cells were transfected with control or PTBP1-expression plasmid vectors (0.2 Pg/ml) for 24 h. Western blot analysis was performed to determine the expression of PTBP1 and CCN1 proteins, with E-actin used as the internal control. *p<0.05, as indicated by the brackets (Student’s t test).
37 ➨4 ❶ ⥲ᣓ ࡀࢇᑐࡍࡿTRAIL ㄏᑟ࣏ࢺ࣮ࢩࢫࢆᶆⓗࡋࡓከࡃࡢ⮫ᗋヨ㦂ࡢ⤖ᯝࡣࠊ TRAIL ⪏ᛶࡢ⋓ᚓࠊࡉࡽࡣ㔜⠜࡞⫢ᶵ⬟㞀ᐖ➼ࡢస⏝ࡢࡓࡵኻᩋ⤊ࢃࡗ ࡓࠋࡇࡢࡼ࠺࡞⫼ᬒࡽTRAIL ⪏ᛶᶵᵓࡢゎ᫂ࠊ⪏ᛶゎ㝖᭷ຠ࡞ᶆⓗศᏊ ࢆྠᐃࡍࡿࡇࡀࡀࢇ⣽⬊≉␗ⓗ࡞TRAIL ㄏᑟ⣽⬊Ṛࢆᶆⓗࡍࡿ᪂つ⒪⸆ ࡢ㛤Ⓨồࡵࡽࢀ࡚࠸ࡿࠋ ᮏ◊✲࡛ࡣࠊWarburg ຠᯝࡋ࡚▱ࡽࢀࡿࡀࢇ⣽⬊≉␗ⓗ࡞࢚ࢿࣝࢠ࣮௦ㅰ ᶵᵓࡢᡂ❧ᚲ㡲࡞㑇ఏᏊPTBP1 ╔┠ࡋࡓࠋPyruvate kinase 㸦PK㸧ࡣࠊ4 ࡘࡢࢯࣇ࢛࣮࣒ࡀᏑᅾࡍࡿゎ⢾⣔ࡢᚊ㏿㓝⣲࡛࠶ࡿࠋᐇ㝿 PKM1/2 ࡢⓎ ⌧ࣞ࣋ࣝࡣࠊࢫࣉࣛࢧ࣮ࢱࣥࣃࢡ㉁࡛࠶ࡿ PTBP1 ࡼࡗ࡚ไᚚࡉࢀ࡚࠸ࡿࠋ ࡀࢇ⣽⬊࠾࠸࡚ࡣࠊPTBP1 ࡀ㧗Ⓨ⌧ࡋ࡚࠾ࡾ㑅ᢥⓗࢫࣉࣛࢩࣥࢢࡼࡾ PKM2 ࡢⓎ⌧ࡀ᭷ព࡞ࡿࡇ࡛ Warburg ຠᯝࢆᡂ❧ࡉࡏ࡚࠸ࡿࠋᮏ◊✲࡛ࡣ TRAIL ⪏ᛶᰴ࠾࠸࡚ PTBP1 ࡢ㧗Ⓨ⌧ࢆㄆࡵࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࠊ TRAIL ࡢឤཷᛶࡀᅇࡍࡿࡇࡀศࡗࡓ㸦➨ 3 ❶-2 ⠇㸧ࠋࡑࡢຠᯝࡣ TRAIL ᑐࡋ࡚᭱ࡶᙉ࠸⪏ᛶࢆ♧ࡋࡓṇᖖࣄࢺங⭢ୖ⓶⣽⬊ᰴMCF10A ࠾࠸࡚㢧ⴭ ㄆࡵࡽࢀࡓࠋࡇࢀࡽࡢ⤖ᯝࡽࠊPTBP1 ࡢⓎ⌧ TRAIL ⪏ᛶࡣṇࡢ┦㛵ࡀ ࠶ࡾࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿ TRAIL ឤཷᛶࡀᅇࡍࡿࡇࡀศࡗࡓࠋ ᡃࠎࡣࡇࢀࡲ࡛ࠊ࣏ࢺ࣮ࢩࢫㄏᑟᛶࡢ DR5 ࡢⓎ⌧పୗຍ࠼࡚ DR5 ࡢ⣽
38 ⬊⾲㠃ୖࡢࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢⰋࡀTRAIL ⪏ᛶࡢせᅉ࡛࠶ࡿࡇࢆ᫂ࡽ ࡋࡓ53ࠋPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿࡇࡼࡾࠊDR5 ࡢⓎ⌧పୗࣜࢡ࣮ࣝ ࢺ࣓ࣥࢺࡢⰋࡀゎ㝖ࡉࢀࡿࡇࡀศࡗࡓ㸦➨ 3 ❶-3 ⠇㸧ࠋࡋࡋ࡞ࡀࡽࠊ PTBP1 ࡣ DR5 ࡢ㌿┤᥋ⓗ㛵ࡋ࡚࠸࡞࠸ࡇࡽ⣽⬊ෆ࡛ࡢ࢚ࢿࣝࢠ ࣮௦ㅰࡢኚ╔┠ࡋTRAIL ⪏ᛶゎ㝖ᶵᵓࡢゎ᫂ࢆヨࡳࡓࠋPTBP1 ࢆࣀࢵࢡࢲ ࢘ࣥࡋࡓ⣽⬊࡛ࡣࠊPKM2/PKM1 ࡢẚࡀᐃᖖ≧ែẚ㍑ࡋ࡚㢧ⴭῶᑡࡋ࡚࠾ ࡾࠊࡉࡽࡣ⣽⬊ෆࡢATP ࣞ࣋ࣝࡢቑຍࠊゎ⢾⣔ࡢ᭱⤊⏘≀࡛࠶ࡿங㓟㔞ࡢ ῶᑡࡀㄆࡵࡽࢀࡓࡇࡽ⣽⬊ෆࡢ࢚ࢿࣝࢠ࣮௦ㅰࡀ୍㒊ゎ⢾⣔ࡽ࣑ࢺࢥࣥ ࢻࣜ࠾ࡅࡿ㓟ⓗࣜࣥ㓟ࢩࣇࢺࡋ࡚࠸ࡿࡇࡀศࡗࡓࠋ2006 ᖺࠊ ࢘ࢥࣥࡢせ࡞ᵓᡂᡂศ࡛࠶ࡿࢡࣝࢡ࣑ࣥࡀROS ࡢⓎ⏕ࢆࡋ࡚ DR5 ࡢⓎ⌧ ࡀቑຍࡍࡿࡇࡼࡾTRAIL ࡢឤཷᛶࢆᅇࡉࡏࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ54ࠋ ᡃࠎࡣࠊPTBP1 ࡢⓎ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿ PKM2 ࡽ PKM1 ࡢࢫࢵࢳ ࡼࡾROS ࡢ⏘⏕㔞ࡀୖ᪼ࡍࡿࡇࢆ᫂ࡽࡋ࡚࠸ࡿ46ࠋᐇ㝿ࠊᢠ㓟࡛ ࠶ࡿNAC ࢆస⏝ࡉࡏࡿࡇ࡛ PTBP1 ࡢࣀࢵࢡࢲ࢘ࣥࡼࡿ TRAIL ㄏᑟ⣽⬊Ṛ ࡀ୍㒊࢟ࣕࣥࢭࣝࡉࢀࡿࡇࡀ♧ࡉࢀࡓࠋ⯆῝࠸ࡇࠊDR5 ࡢⓎ⌧ࣜࢡ ࣮ࣝࢺ࣓ࣥࢺࡢㄏᑟࡶ࢟ࣕࣥࢭࣝࡉࢀࡿࡇࡀศࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡽࠊ Warburg ຠᯝࡢ⬺ไᚚࡼࡿ ROS ࡢⓎ⏕ࡀ DR5 ࡢⓎ⌧ࣜࢡ࣮ࣝࢺ࣓ࣥࢺࡢ ㄏᑟ㛵ࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓ㸦➨ 3 ❶-4 ⠇㸧ࠋࡋࡋࠊROS ࡢⓎ⏕
39 ࡀDR5 ࡢⓎ⌧ࢆቑຍࡉࡏࡿ࣓࢝ࢽࢬ࣒ࡘ࠸࡚ࡣ᫂ࡽࡉࢀ࡚࠸࡞࠸ࠋࡉࡽ ᮏ◊✲࡛ࡣࠊPTBP1 ࡢࣀࢵࢡࢲ࢘ࣥࡼࡿࡑࡢࡢ TRAIL ⪏ᛶゎ㝖࣓࢝ࢽࢬ ࣒ࡢゎ᫂ࢆ⾜ࡗࡓࠋࣞゎᯒࡢ⤖ᯝࠊPTBP1 ࢆࣀࢵࢡࢲ࢘ࣥࡋࡓ⣽⬊࠾࠸ ࡚Ⓨ⌧ஹ㐍ࡀㄆࡵࡽࢀࡓCCN1 㑇ఏᏊ TRAIL ㄏᑟ⣽⬊Ṛࡢ㛵㐃ࡘ࠸࡚ࡶ ᳨ドࢆ⾜ࡗࡓࠋChen ࡽࡣࠊCCN1 ࡀࣥࢸࢢࣜࣥࣞࢭࣉࢱ࣮ཬࡧࢩࣥࢹ࢝ࣥ 4 ⤖ྜࡍࡿࡇ࡛㔞ࡢROS ࢆⓎ⏕ࡉࡏ JNK ࡢάᛶࢆࡋ࡚ TRAIL ㄏᑟ⣽ ⬊Ṛࢆಁ㐍ࡍࡿࡇࢆሗ࿌ࡋ࡚࠸ࡿ 52ࠋᐇ㝿ࠊCCN1 ࢆ㐣Ⓨ⌧ࡉࡏࡓ⣽⬊ ࠾࠸࡚ TRAIL ㄏᑟࡼࡿ⣽⬊ṚࡀቑᙉࡉࢀࡿࡇࡀศࡗࡓࠋPTBP1 ࡣࠊ Pre-miRNA ࡸᵝࠎ࡞㑇ఏᏊࡢࢫࣉࣛࢩࣥࢢࡢไᚚ㛵ࡍࡿࡇࡀሗ࿌ࡉࢀ ࡚࠸ࡿ55ࠋᡃࠎࡣࠊࢡ࣐ࣟࢳࣥචỿ㝆ἲࡼࡾPTBP1 ࡀ CCN1 ࡢ㌿࠾࠸ ࡚ࣜࣉࣞࢵࢧ࣮ࡋ࡚ᶵ⬟ࡋ࡚࠸ࡿࡇࢆ᪂ࡓ᫂ࡽࡋࡓ㸦➨ 3 ❶-5 ⠇㸧ࠋ ࡇࢀࡽࡢ⤖ᯝࡽࠊDR5 ࡢⓎ⌧ቑຍࡣ Warburg ຠᯝࡢ⬺ไᚚࡢࡳ࡞ࡽࡎࠊ CCN1 ࡢⓎ⌧ቑຍࡼࡿ ROS ࡢ㛵ࡀ♧ࡉࢀࡓࠋ ᮏ◊✲ࡼࡾࠊWarburg ຠᯝ TRAIL ㄏᑟ⣽⬊Ṛࡢ㛵㐃ࡀ᫂ࡽ࡞ࡾࠊPTBP1 ࡣTRAIL ⪏ᛶゎ㝖᭷ຠ࡞ᶆⓗศᏊ࡞ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋᚋࠊPTBP1 ࢆไᚚࡍࡿྜ≀ࡸsiRNA ࡀࡀࢇࡢ࢚ࢿࣝࢠ࣮௦ㅰࡢ◚⥢ࡢࡳ࡞ࡽࡎ TRAIL ㄏ ᑟ⣽⬊Ṛࢆ⏝ࡋࡓࡀࢇࡢ་⸆ࢩ࣮ࢬࡋ࡚ᮇᚅࡉࢀࡿࠋ
40
Figure-10 Schematic diagram of the mechanism and machinery involved in the TRAIL-induced apoptosis by silencing PTBP1
41
ㅰ㎡
ᮏ◊✲ࡢ㐙⾜࠾ࡼࡧㄽᩥࡢసᡂ࠶ࡓࡾࠊ⤊ጞࡈ᠓⠜࡞ࡿࡈᣦᑟࠊࡈ㠴᧡ࢆ㈷ ࡾࡲࡋࡓᒱ㜧ᏛᏛ㝔㐃ྜ⸆་⒪ሗ◊✲⛉ ㉥ᑿ ᖾ༤ ᩍᤵᚰࡼࡾ ឤㅰࡢពࢆ⾲ࡋࡲࡍࠋ ࡲࡓࠊᮏㄽᩥࡢసᡂ㝿ࡋࠊࡈຓゝ࠾ࡼࡧࡈᰯ㜀ࢆ㈷ࡾࡲࡋࡓᒱ㜧ᏛᏛ㝔 㐃ྜ⸆་⒪ሗ◊✲⛉ Ᏹ㔝 ᩥ ᩍᤵࠊ ୖ⏣ ᾈ ᩍᤵࠊ⏣୰ 㤶࠾ 㔛 ᩍᤵ῝ࡃឤㅰ࠸ࡓࡋࡲࡍࠋ ᮏ◊✲ࡢ㐙⾜࠶ࡓࡾࠊ◊✲άືࢆඹࡋ࡚ࡁࡓᒱ㜧ᏛᏛ㝔㐃ྜ⸆་⒪ ሗ◊✲⛉㉥ᑿ◊✲ᐊࡢⓙᵝᚰࡼࡾ࠾♩⏦ࡋୖࡆࡲࡍࠋ42
ᐇ㦂ࡢ㒊 1. ヨ⸆
Recombinant Human TRAIL-Apo2L Ligand (#4354-10; BioVision, CA, USA)ࠊ2-ࢹ࢜࢟ࢩ-D-ࢢࣝࢥ࣮ࢫ(Sigma-Aldrich, St. Louis, MO,USA)ࠊN-ࢭࢳࣝ-L-ࢩࢫ ࢸࣥ(Sigma-Aldrich, St. Louis, MO, USA)ࢆ⏝ࡋࡓࠋ
2. ⣽⬊ᇵ㣴
ࣄ ࢺ ⭠ ⒴ ⣽ ⬊ ᰴ DLD-1 ࠊ SW480 ࡣ ࠊ Japanese Collection Research Bioresources Cell Bank (Osaka, Japan)ࡼࡾ㉎ධࡋࡓࠋMCF10A ࡣࠊAmerican Type Culture Collection (ATCC Manassas, VA, USA)ࡼࡾ㉎ධࡋࡓࠋ⣽⬊ࡣ㉎ධ ᚋ6 ᭶௨ෆࡶࡋࡃࡣ MycoAlert Mycoplasma Detection Kit (LT07-118; Lonza, Rockland, ME, USA)࡛⟶⌮ࡋࡓࡶࡢࢆ⏝ࡋࡓࠋDLD-1ࠊDLD-1/TRAIL ཬࡧ SW480 ࡣ 10 %FBS ྵ᭷ RPMI-1640(189-02025; Invitrogen, Carlsbad, CA, USA)
୰࡛5 % CO2ࠊ37Υࡢ᮲௳ୗ࡛ᇵ㣴ࡋࡓࠋMCF10A ࡣ MEBM(CC3150; Lonza,
Tokyo, Japan)୰࡛ 5 % CO2ࠊ37Υࡢ᮲௳ୗ࡛ᇵ㣴ࡋࡓࠋ
⏕⣽⬊ᩘࡣࠊࢺࣜࣃࣥࣈ࣮ࣝⰍ⣲㝖ヨ㦂ἲࡼࡾホ౯ࡋࡓࠋᇵ㣴ᚋࡢ⣽⬊⁐ ᾮࢆࢺࣜࣃࣥࣈ࣮ࣝ➼㔞ΰྜࡋࠊ⾑⌫ィ⟬┙࡚⏕⣽⬊ᩘࢆィᩘࡋࡓࠋࢥࣥ
43
ࢺ࣮ࣟࣝ⣽⬊ᑐࡍࡿ⏕⣽⬊ᩘ(%)ࢆ⣽⬊⏕Ꮡ⋡ࡋࡓࠋ
3. ࢚࢘ࢫࢱࣥࣈࣟࢵࢺゎᯒ 3-1 ࢱࣥࣃࢡ㉁ᢳฟ
ࢱࣥࣃࢡ㉁ᢳฟᾮࡣࠊProtein lysis buffer (10 nM Tris-HCLࠊ0.1% SDSࠊ1% NP-40ࠊ0.1% ࢹ࢜࢟ࢩࢥ࣮ࣝ㓟ࢼࢺ࣒ࣜ࢘ࠊ150 mM NaClࠊ1 mM EDTA) 1% Protease inhibitor cocktailࠊPhosphatase inhibitor cocktail II ཬࡧ III ࢆΰྜࡋ࡚ ⏝࠸ࡓࠋࢱࣥࣃࢡ㉁ᢳฟᾮᅇࡋࡓ⣽⬊ࢆᠱ⃮ࡉࡏࠊ20 ศ㛫ị୰㟼⨨ࡉࡏ ࡓࠋࡑࡢᚋࠊ13,000rpmࠊ4Υࠊ20 ศ㛫㐲ᚰศ㞳ࡋࡓࠋ㐲ᚰศ㞳ࡋࡓୖΎࢆᅇ
ࡋࠊࢱࣥࣃࢡ㉁ࢧࣥࣉࣝࡋࡓࠋࢱࣥࣃࢡ㉁ᐃ㔞ࡣࠊDC Protein assay kit (Biorad,
Hercules, CA, USA)ࢆ⏝࠸࡚⾜ࡗࡓࠋᐃ㔞ࡋࡓࢱࣥࣃࢡ㉁ࢆ SDS sample buffer (62.5 mM Tris-HCLࠊ2% SDSࠊ10% ࢢࣜࢭ࣮ࣟࣝࠊ50 mM DTTࠊ0.01%ࣈࣟࣔ
ࣇ࢙ࣀ࣮ࣝࣈ࣮ࣝ)ΰࡋ࡚ 50 Pg/μL ㄪᩚࡋࠊ98Υ࡛ 5 ศ㛫Ἓ㦐ฎ⌮ࡋࡓ
ᚋࠊịୖ࡛5 ศ㛫㟼⨨ࡋࡓࠋ
3-2 㟁ẼὋື࠾ࡼࡧ㌿
44
ࡓࠋὋືᚋࠊࢤࣝࢆblotting buffer (25 mM Trisࠊ0.2 M ࢢࣜࢩࣥࠊ20%࣓ࢱࣀ࣮ ࣝ) 5 ศ㛫ᾐࡋࡓࠋPVDF ࣓ࣥࣈࣞࣥ(PerkinElmer Life Sciences, Boston, MA, USA)ࡣ࣓ࢱࣀ࣮ࣝ 3 ศ㛫ᾐࡋࠊ㉸⣧Ỉ 5 ศ㛫ᾐࡋࡓࠋࡑࡢᚋࠊblotting buffer
5 ศ㛫ᾐࡋࡓࠋ㝧ᴟഃࡽࠊblotting buffer ᾐࡋࡓࢁ⣬ࠊPVDF ࣓ࣥࣈࣞࣥࠊ
ࢤࣝࠊࢁ⣬ࡢ㡰㔜ࡡࠊ15 Vࠊ370 mA ࡛ 40 ศ㛫㌿ࡋࡓࠋ
3-3 ࢚࢘ࢫࢱࣥࣈࣟࢵࢸࣥࢢ
㌿ᚋࠊ0.1% Tween 20 ྵ᭷ 50 mM Tris-HCL buffer (TBST)࡛Ὑίࡋࠊ5%ࢫ࢟
࣒࣑ࣝࢡ⁐ᾮᾐࡋ࡚ 1 㛫ࣈࣟࢵ࢟ࣥࢢࡋࡓࠋTBST ࡛Ὑίࡋࠊᢠయᕼ㔘ᾮ
(2%BSAࠊ0.01%ࢪࢼࢺ࣒ࣜ࢘ࠊTBST)࡛ᕼ㔘ࡋࡓ୍ᢠయᾐࡋ࡚ 4Υ࡛
୍ᬌᛂࡉࡏࡓࠋTBST ࡛Ὑίࡋࡓᚋࠊ5%ࢫ࣒࣑࢟ࣝࢡ⁐ᾮ࡛ᕼ㔘ࡋࠊLuminate
Forte Western HRP Substrate (WBLUF0500; Millipore)࡛Ⓨගࡉࡏࡓᚋࠊ Luminescent image analyzer LAS-4000 UV mini (Fujifilm, Tokyo, Japan)ࢆ⏝࠸ ᳨࡚ฟࡋࡓࠋ୍ḟᢠయࡣPTBP1 (#8776)ࠊDR5 (#8074)ࠊ Caspase-8 (#9496)ࠊ CCN1 (#14479)ࡣ Santa Cruz Biotechnology (Santa Cruz, CA, USA)ࡼࡾࠊFADD (M035-3)ࡣ MBL (MEDICAL & BIOLOGICAL LABORATORIES CO, LTD, Nagoya, Japan) ࡼ ࡾ ࠊ PKM1 (NBP2-14833SS) ࠊ PKM2 (NBP1-48308SS) ࡣ Novus Biologicals (Littleton, CO, USA)ࢆ⏝ࡋࡓࠋࢥࣥࢺ࣮ࣟࣝࡣ anti-E-actin
45
antibody (A5316; Sigma-Aldrich)ࢆ⏝ࡋࡓࠋ
4. Real-time PCR 4-1 RNA ᢳฟ
⣽⬊ࡢRNA ࡣ NucleaseSpin miRNA kit (TaKaRa, Osaka, Japan)ࢆ⏝ࡋᢳฟࡋ ࡓࠋRNA 㔞ࡣ UV spectrophotometry ࡚ᐃ㔞ࡋࡓࠋ
4-2 mRNA ࡢᐃ㔞
PrimeScript RT reagent kit (TaKaRa)ࢆ⏝ࡋࠊ37Υ15 ศࠊ85Υ5 ⛊ࠊ4Υ࡛ RNA
ࢧࣥࣉࣝࡢ㏫㌿ᛂࢆ⾜࠸ࠊ㗪ᆺ cDNA ࢆྜᡂࡋࡓࠋQuantitative reverse
transcription-PCR (qRT-PCR) ᛂ ࡣ Universal SYBR select Master Mix (Applied Biosystems, Forester City, CA)ཬࡧ Tabele-2 ࡢࣉ࣐࣮ࣛࢭࢵࢺࢆ
⏝ࡋࡓࠋGAPDH ࡢ mRNA 㔞ࢆෆ㒊ࢥࣥࢺ࣮ࣟࣝࡋࡓࠋ95Υ30 ⛊࡛ึᮇኚᛶ
ࢆ⾜ࡗࡓᚋࠊ95Υ5 ⛊ࡢኚᛶᛂཬࡧ 60Υ60 ⛊ࡢࢽ࣮ࣜࣥࢢ࣭ఙ㛗ᛂࢆ 40 ࢧࢡࣝ⾜࠸ࠊ95Υ15 ⛊ࠊ60Υ30 ⛊ࠊ95Υ15 ⛊ࡢࢫࢸࢵࣉ࡛⼥ゎ᭤⥺ࢆศ
46
Primers Sequences
DR5 forward 5’-GAGAGACTATAAGAGCGT-3’ DR5 reverse 5’-CTTCCTGAAGAGAACCACAC-3’
GAPDH forward 5’-TCTAGACGGCAGGTCAGGTCCACC-3’ GAPDH reverse 5’-CCACCCATGGCAAATTCCATGGCA-3’
Table-2 Sequences of primers using in this study
4-3 miRNA ࡢᐃ㔞
miRNA ࡢᐃ㔞ࡣ TaqMan MicroRNA Assay (Applied Biosystems)ࢆ⏝ࡋࡓࠋ ࡲ ࡎ TawMan MicroRNA Reverse Transcription Kit (Applied Biosystems) ࠊ stem-loop RT primer (Applied Biosystems)ཬࡧ RNA ࢧࣥࣉࣝࢆΰྜࡋࠊ16Υ࡛ 30 ศࠊ42Υ࡛ 30 ศࠊ85Υ࡛ 5 ศࠊ4Υ࡛ 10 ศࡢ㡰࡛ RT ᛂࢆ⾜ࡗࡓࠋqRT-PCR ࡣ TapMan MicroRNA Assay ෆࡢ PCR primer (Applied Biosystems)ཬࡧ THUNDERBIRD Probe qPCR Mix (TOYOBO, Osaka, Japan)ࢆ⏝ࡋࡓࠋ95Υ 30 ⛊࡛ኚᛶࡉࡏࡓᚋࠊ95Υ5 ⛊ࠊ60Υ60 ⛊ࡢᛂࢆ 40 ࢧࢡࣝ⾜࠸''Ct ἲ
࡚miRNA 㔞ࢆィ⟬ࡋࡓࠋRNU6B ࢆෆ㒊ࢥࣥࢺ࣮ࣟࣝࡋࠊྛࢧࣥࣉࣝࡢᛂ
47
5. 㑇ఏᏊᑟධᐇ㦂
ྛ⣽⬊ࡣ0.5×105 cells/mL ࡢ⣽⬊ᐦᗘ࡛ 6 ࢙࢘ࣝࣉ࣮ࣞࢺ✀ࡋࡓࠋࢺࣛࣥ
ࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜࠺24 㛫๓✀ࡋࠊࣉ࣮ࣞࢺ᥋╔ࡉࡏࡓࠋᮏ◊✲
⏝ࡋࡓ siRNA (Invitrogen)ࡢ㓄ิࡣ Tabel-3 ♧ࡋࡓࠋControl ࡞ࡿ㠀≉␗ⓗ
RNA ࡣ Hokkaido System Sciences (Sapporo, Japan)ࡼࡾ㉎ධࡋࡓࠋsiRNA ࡣ Lipofectamine RNAiMAX (Invitrogen)ࢆ⏝ࡋ࡚࢝ࢳ࢜ࣥᛶ࣏ࣜࢯ࣮࣒ࢆᙧᡂࡉ
ࡏࠊ⣽⬊ෆᑟධࡋࡓࠋ㑇ఏᏊᑟධࢆ⾜ࡗࡓ48 㛫ᚋᅇࡋࠊྛࢵࢭ
⏝ࡋࡓࠋ
Gene Sequences
PTBP1 5’-AUCUCUGGUCUGCUAAGGUCACUUC-3’ (siR-PTBP1) Control 5’-GGCCUUUCACUACUCCUCA-3’
Table-3 Sequences of siRNAs using in this study
6. ChIP ࢵࢭ
DLD-1/TRAIL siR-PTBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࠊ5%CO2ࠊ37Υ࡛ 48
48
Carlsbad, CA, USA)ཬࡧ DR5 antibody (Cell Signaling Technology)ࢆ⏝࠸ࡓࠋࡲ ࡓࠊ࣏ࢪࢸࣈࢥࣥࢺ࣮ࣟࣝࡋ࡚Antibody against Suz12 (39357; Active Motif)ࠊ
ࢿ࢞ࢸࣈࢥࣥࢺ࣮ࣝࡋ࡚rabbit IgG (53025; Active Motif)ࢆ⏝ࡋࡓࠋච
ỿ㝆ᛂ࡛ᅇࡋࡓDNA ࡣ Real-time PCR ࡚ቑᖜࡋࠊInput DNA ࡢయ㔞
ᑐࡍࡿቑᖜ⋡ࢆồࡵࡓࠋ
7. L-lactate ࢵࢭ
DLD-1 ཬࡧ DLD-1/TRAIL ᑐࡋࠊsiR-PTBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࠊ 5%CO2ࠊ37Υ࡛ 48 㛫ᇵ㣴ࡋࡓࠋ⣽⬊ࢆᅇࡋࠊL-Lactate Assay Kit (700510;
Cayman Chemical Company, Ann Arbor, MI, USA)ࢆ⏝࠸࡚⣽⬊ෆࡢ L-lactate ࢆ ᢳฟཬࡧ ᐃࢆ⾜ࡗࡓࠋ ᐃ್ࡣྛࢧࣥࣉࣝࡢ⏕⣽⬊ᩘ࡛⿵ṇࡋࠊ⣽⬊ෆங㓟 ⏘⏕㔞ࡋࡓࠋ
8. ATP ࢵࢭ
DLD-1 ཬࡧ DLD-1/TRAIL siR-PTBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࠊ5%CO2ࠊ
49
Invitrogen)࡚ ATP ࣞ࣋ࣝࢆ ᐃࡋࡓࠋ ᐃ್ࡣྛࢧࣥࣉࣝࡢ⏕⣽⬊ᩘ࡛⿵ṇ ࡋࡓࠋ
9. ච⺯ගᰁⰍ
ච⺯ගᰁⰍࡣCell Signaling Technology ࡢࣉࣟࢺࢥ࣮ࣝᚑࡗࡓࠋDLD-1 ཬ
ࡧDLD-1/TRAIL siR-PRBP1 ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࠊ48 㛫ᚋࡢ⣽⬊ࢆ
ᅇࡋࡓࠋSmear Gell (SG-01; GenoStaff, Tokyo, Japan)ࢆ⏝࠸࡚ࢫࣛࢻ࢞ࣛ ࢫୖ⣽⬊ࢆ╔ࡉࡏࠊ4%࣒࣍ࣝࣝࢹࣄࢻᾐࡋ࡚ᐊ ࡛ 15 ศ㛫⣽⬊ࢆᅛ ᐃࡋࡓࠋPBS ࡚⣽⬊ࢆὙίࡋࣈࣟࢵ࢟ࣥࢢࣂࢵࣇ࣮(1×PBSࠊ5%ṇᖖࣖࢠ
⾑Ύࠊ0.3% TritonTMX100)࡛ 60 ศ㛫ࣈࣟࢵ࢟ࣥࢢࡋࡓࠋࡑࡢᚋࠊࣈࣟࢵ࢟ࣥࢢ
ࣂࢵࣇ࣮ࢆ྾ᘬ㝖ཤࡋࠊ୍ᢠయࢆࣉࣛࡋ࡚4Υ୍࡛ᬌ࣮ࣥ࢟ࣗ࣋ࢺࡋ
ࡓࠋPBS ࡛Ὑίࡋࡓᚋࠊ⺯ගᶆ㆑ḟᢠయࢆຍ࠼࡚ᬯᡤࠊᐊ ࡛ 2 㛫ࣥ࢟ ࣮ࣗ࣋ࢺࡋࡓࠋḟᢠయࡣࠊAnti-Rabbit IgG (H+L), F (ab’)2 Fragment Alexa
Fluor 488 (#4412; Cell Signaling Technology)ࢆ⏝ࡋࡓࠋࡲࡓࠊ⣽⬊᰾ᰁⰍ Hoechst33342ࠊ⣽⬊㦵᱁ᰁⰍ fluorescent F-actin probe Rhodamine Phalloidin (Cytoskeleton, Denver, Co, USA)ࢆ⏝ࡋࠊḟᢠయྠ࣮ࣥ࢟ࣗ࣋ࢺࡋ ࡓ ࠋ ᗘ Ὑ ί ࢆ ⾜ ࠸ ࠊ ᑒ ධ ࢆ ῧ ຍ ࡋ ࡓ ࡶ ࡢ ࢆ BIOREVO fluorescence
50
microscope (Keyence, Osaka, Japan)࡚ほᐹࡋࡓࠋ
10. ⤫ィᏛⓗゎᯒ
ྛᐇ㦂ࡣ3 ᅇࡎࡘ⾜ࡗࡓࠋᐇ㦂ᡂ⦼ࡣᖹᆒ್±ᶆ‽೫ᕪ࡛♧ࡋࠊ⤫ィᏛⓗ࡞ẚ㍑
ࡣStudent’s t test ࡼࡾ⾜ࡗࡓࠋ༴㝤⋡ 5%ᮍ‶ࢆ᭷ពᕪ࠶ࡾࡋࡓࠋྛࢢࣛࣇ
51
ཧ⪃ᩥ⊩
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