JP2019108277A - 細胞増殖抑制剤及びそれを含むがんの治療若しくは予防用医薬組成物 - Google Patents
細胞増殖抑制剤及びそれを含むがんの治療若しくは予防用医薬組成物 Download PDFInfo
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Abstract
Description
(1)GSTP1を抑制する薬物と、RAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物とを組み合わせて含む、RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんに対する細胞増殖抑制剤。
(2)RAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物と組み合わせて投与するための、GSTP1を抑制する薬物を含む、RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんに対する細胞増殖抑制剤。
(3)GSTP1を抑制する薬物と組み合わせて投与するための、RAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物を含む、RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんに対する細胞増殖抑制剤。
(4)前記RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんが、RASに活性化変異を有するがんである、(1)〜(3)のいずれかに記載の細胞増殖抑制剤。
(5)前記がんが、大腸がんである、(1)〜(4)のいずれかに記載の細胞増殖抑制剤。
(6)前記RAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物が、RASを抑制する薬物である、(1)〜(5)のいずれかに記載の細胞増殖抑制剤。
(7)前記GSTP1を抑制する薬物が、GSTP1に対するsiRNAである、(1)〜(6)のいずれかに記載の細胞増殖抑制剤。
(8)前記RAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物が、RAS/RAF/MEK/ERKシグナルカスケードの成分に対するsiRNAである、(1)〜(7)のいずれかに記載の細胞増殖抑制剤。
(9)GSTP1とCRAFの相互作用を阻害する薬物を含む、RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんに対する細胞増殖抑制剤。
(10)前記RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんが、RASに活性化変異を有するがんである、(9)に記載の細胞増殖抑制剤。
(11)前記がんが、大腸がんである、(9)又は(10)に記載の細胞増殖抑制剤。
(12)前記GSTP1とCRAFの相互作用を阻害する薬物が、CRAFデコイペプチド又はこれを発現するベクターである、(9)〜(11)のいずれかに記載の細胞増殖抑制剤。
(13)前記CRAFデコイペプチドが、
(a)配列番号9に示されるアミノ酸配列からなるポリペプチド、
(b)配列番号9に示されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなるポリペプチド、
(c)配列番号9に示されるアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなるポリペプチド、及び
(d)(a)〜(c)のいずれかに記載されるポリペプチドのN末端又はC末端に1〜50個のアミノ酸が付加されたポリペプチド
からなる群から選択される、(12)に記載の細胞増殖抑制剤。
(14)(1)〜(13)のいずれかに記載の細胞増殖抑制剤を含む、RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんの治療又は予防用医薬組成物。
(15)(1)〜(13)のいずれかに記載の細胞増殖抑制剤を含む、RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんの治療又は予防用キット。
1. GSTP1を抑制する薬物とRAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物の併用による細胞増殖抑制
本発明は、GSTP1を抑制する薬物と、RAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物とを組み合わせて含む、細胞増殖抑制剤に関する。本発明は、後述の実施例に示されるように、GSTP1を抑制する薬物と、RAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物とを併用し、GSTP1のオートクラインループと、RAS/RAF/MEK/ERKシグナルカスケードの両方を阻害することにより、それぞれの薬物単独よりも、がん細胞の増殖を効果的に抑制できるという本発明者の知見に基づく。
本発明はまた、GSTP1とCRAFの相互作用を阻害する薬物を含む、細胞増殖抑制剤に関する。本発明は、後述の実施例に示されるように、RAS/RAF/MEK/ERKシグナルカスケードとGSTP1のオートクラインループとの接合点である、GSTP1とCRAFの相互作用を阻害することにより、がん細胞の増殖が抑制されるという本発明者の知見に基づく。
(a)配列番号9に示されるアミノ酸配列からなるポリペプチド、
(b)配列番号9に示されるアミノ酸配列において1若しくは複数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなるポリペプチド、
(c)配列番号9に示されるアミノ酸配列に対して90%以上、95%以上、97%以上、98%以上又は99%以上の配列同一性を有するアミノ酸配列からなるポリペプチド、及び
(d)(a)〜(c)のいずれかに記載されるポリペプチドのN末端又はC末端に1〜50個(例えば、1〜30個、1〜20個、1〜10個又は1〜5個)のアミノ酸が付加されたポリペプチド
からなる群から選択されてもよい。
本発明は、上述の本発明に係る細胞増殖抑制剤を含む、組成物にも関する。組成物は、医薬組成物であってもよい。
本発明はまた、本発明に係る細胞増殖抑制剤若しくは組成物、又はそれに含まれる活性成分を、単独で若しくは組み合わせて含む1個又は2個以上の容器を含む、組成物の調製用、細胞増殖抑制用、又はがんの治療若しくは予防用キットにも関する。
(GSTP1と結合するCRAFのドメインの解析)
(1)細胞培養
KRAS変異陽性大腸がん細胞株M7609をRPMI培地(10%FBSを含む)中で37℃にて培養した。M7609細胞は、札幌医科大学第四内科から提供を受けた。M7609細胞はKRASに活性化変異を有するため、M7609細胞のRAS/RAF/MEK/ERKシグナルカスケードは活性化されている。
図1に、実施例1で後述のように用いたプラスミドが発現するタンパク質の構造が示される。(a)はFLAG-CRAF(1-648)を示し、CRAFに含まれるドメインのアミノ酸残基位置(61〜192位: CR1ドメイン、251〜266位: CR2ドメイン、333〜625位: CR3ドメイン)を示す。(b)はFLAG-CRAFΔN(193-648)を示す。(c)はFLAG-BRAF(1-766)を示し、BRAFに含まれるドメインのアミノ酸残基位置(2〜117位: BRSRドメイン、155〜280位: CR1ドメイン、360〜375位: CR2ドメイン、457〜717位: CR3ドメイン)を示す。(d)はFLAG-BRAFΔN(149-766)を示す。
4種のプラスミドをそれぞれ、M7609細胞にリポフェクタミン法によってトランスフェクトした。また、対照として、トランスフェクションしていない細胞(NT)を用意した。
トランスフェクション後の細胞を用いて、共免疫沈降を行った。細胞を、0.5%NP-40溶解バッファー(0.5%NP-40、20mM HEPES pH7.4、150mM NaCl、1mM MgCl2、1mM EGTA、10%グリセロール、Complete, Mini(Roche Diagnostics社)及びPhosSTOP(Roche Diagnostics社))中で氷上で30分間インキュベートして溶解した。遠心分離し、上清を細胞溶解物として得た。各サンプルから得られた、等量のタンパク質を含む細胞溶解物を、抗FLAG M2磁気ビーズ(SIGMA社)と4℃にて2時間〜一晩インキュベートし、ビーズにFLAGタグ付タンパク質を結合させた。ビーズを0.5%NP-40溶解バッファーで4回洗浄した。
共免疫沈降で得られたビーズから、ウェスタンブロット用のサンプルを調製し、SDS-PAGEに供して、タンパク質を分離した。分離したタンパク質をPVDFメンブレンに転写した。メンブレンを一次抗体溶液とインキュベートし、洗浄した。用いた一次抗体は、抗GSTP1抗体(MBL社)、抗FLAG抗体(F3165、SIGMA社)又は抗GAPDH抗体(Abcam社)とした。次いで、メンブレンを二次抗体(ウサギ又はマウスIgGに対するヤギ二次抗体)溶液とインキュベートし、洗浄した。ECL又はECL primeウェスタンブロッティング検出系(GE healthcare社)を用いてメンブレンにおいてシグナルを可視化した。
また、同様の方法で、細胞溶解物についてもウェスタンブロット解析を行った。
結果を図2に示す。GSTP1は、全長CRAFタンパク質と共沈降した(図2のレーン2)が、N末端部分が欠失したCRAFタンパク質とは共沈降しなかった(図2のレーン4)。この結果は、CRAFタンパク質のN末端部分(アミノ酸残基61〜192位のCR1ドメインを含むアミノ酸残基1〜192位)がGSTP1との結合に関与していることを示す。
(GSTP1によるCRAF活性への影響)
(1)細胞培養及びEGF処理
KRAS野生型HeLa細胞をDMEM培地中で5%CO2環境下で37℃にて培養した。HeLa細胞は、札幌医科大学第四内科から提供を受けた。HeLa細胞をまず血清飢餓条件下で16時間インキュベートし、次いで50ng/ml EGF(上皮成長因子、BD Biosciences社)溶液で10分間処理した。EGF処理によりRASが活性化される。RAS活性化はCRAFのリン酸化に必要である。
pcDNA3.1-FLAG-CRAF(札幌医科大学第四内科から提供)をHeLa細胞(EGF処理あり又はなし)に、FuGENE6 HD(Promega社)を用いてトランスフェクトした。トランスフェクションの48時間後、HeLa細胞を0.5%NP-40溶解バッファー(0.5%NP-40、20mM HEPES pH7.4、150mM NaCl、1mM MgCl2、1mM EGTA、10%グリセロール、Complete, Mini(Roche Diagnostics社)及びPhosSTOP(Roche Diagnostics社))中で氷上で30分間インキュベートして溶解した。13000×gで10分間、4℃にて遠心分離し、上清を細胞溶解物として得た。細胞溶解物を抗FLAG M2アフィニティゲル(SIGMA社)と4℃で2時間インキュベートし、抗FLAG M2アフィニティゲルにFLAG-CRAFを結合させた。インキュベート後の抗FLAG M2アフィニティゲルを0.5%NP-40溶解バッファーで4回洗浄した。FLAG-CRAFを結合させた抗FLAG M2アフィニティゲルをAssay Dilution Buffer I (Merck-Millipore社)で洗浄し、このゲルを、Assay Dilution Buffer I (Merck-Millipore社)中で、1μgのヒト胎盤GSTP1(SIGMA社)の存在下又は非存在下で、1μgの不活性MEK1(Merck-Millipore社)及びマグネシウム/ATPカクテル(Merck-Millipore社)と30℃で1時間インキュベートし、FLAG-CRAFによるMEK1のリン酸化反応を行った。また、FLAG-CRAFを結合させた抗FLAG M2アフィニティゲルの代わりに活性CRAF(OriGene社)を、Assay Dilution Buffer I中で不活性MEK1及びマグネシウム/ATPカクテルとインキュベートした対照サンプルを調製した。
インビトロキナーゼアッセイの反応後のサンプルについて、一次抗体として抗p-MEK1/2(Ser217/221)抗体(Cell signaling technology社)又は抗MEK1/2抗体(Cell signaling technology社)を用いたことを除き、実施例1の(5)に記載したように、ウェスタンブロット解析を行った。
結果を図3に示す。GSTP1によって、FLAG-CRAFによるMEK1のリン酸化が増強されたことが示された(図3のレーン3及び5を比較)。特に、GSTP1によるMEK1のリン酸化の増強は、EGF処理細胞から単離されたFLAG-CRAFを用いた場合により明らかだった(図3のレーン4及び6を比較)。これらの結果は、GSTP1がCRAFの活性を増強することを示す。
(CRAFタンパク質断片による細胞増殖への影響)
CRAFタンパク質断片発現プラスミドpcDNA-hRAF384(5807bp)を、ベクターpcDNA3.1(+)(Thermo Fisher Scientific社)のCMV(サイトメガロウイルス)プロモーター下にヒトCRAF遺伝子(配列番号6)の332〜718位の領域をクローニングして作製した。このプラスミドは、ヒトCRAFタンパク質(配列番号5)の56〜184位のCRAFタンパク質断片(配列番号9に示されるアミノ酸配列からなるポリペプチド)の発現をもたらす。
(GSTP1とKARSの二重抑制による細胞増殖への影響)
KRAS変異陽性大腸がん細胞株M7609にsiRNAのトランスフェクションを2回行った。各トランスフェクションはLipofectamine RNAiMAX (Thermo fisher scientific社)を用いて製造業者のプロトコールに従って行った。M7609細胞をRPMI-1640培地(抗生物質なし)中で37℃で培養し、20〜30%コンフルエントに達したとき、細胞を、Opti-MEM I(Thermo fisher scientific社)中のsiRNAと5時間インキュベートして、1回目のトランスフェクションを行った。トランスフェクション後、細胞をRPMI-1640培地(抗生物質なし)中で37℃で培養した。2日間培養した後、1回目と同様の方法で2回目のトランスフェクションを行った。トランスフェクション後、細胞をRPMI-1640培地(抗生物質なし)中で37℃で培養した。3日間培養した後、細胞の総数をカウントした。また、対照としてトランスフェクションしていない細胞(NT)について同様に細胞数をカウントした。実験は3回独立して行い、平均値及び標準偏差を算出した。
Claims (15)
- GSTP1を抑制する薬物と、RAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物とを組み合わせて含む、RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんに対する細胞増殖抑制剤。
- RAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物と組み合わせて投与するための、GSTP1を抑制する薬物を含む、RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんに対する細胞増殖抑制剤。
- GSTP1を抑制する薬物と組み合わせて投与するための、RAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物を含む、RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんに対する細胞増殖抑制剤。
- 前記RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんが、RASに活性化変異を有するがんである、請求項1〜3のいずれか一項に記載の細胞増殖抑制剤。
- 前記がんが、大腸がんである、請求項1〜4のいずれか一項に記載の細胞増殖抑制剤。
- 前記RAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物が、RASを抑制する薬物である、請求項1〜5のいずれか一項に記載の細胞増殖抑制剤。
- 前記GSTP1を抑制する薬物が、GSTP1に対するsiRNAである、請求項1〜6のいずれか一項に記載の細胞増殖抑制剤。
- 前記RAS/RAF/MEK/ERKシグナルカスケードを抑制する薬物が、RAS/RAF/MEK/ERKシグナルカスケードの成分に対するsiRNAである、請求項1〜7のいずれか一項に記載の細胞増殖抑制剤。
- GSTP1とCRAFの相互作用を阻害する薬物を含む、RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんに対する細胞増殖抑制剤。
- 前記RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんが、RASに活性化変異を有するがんである、請求項9に記載の細胞増殖抑制剤。
- 前記がんが、大腸がんである、請求項9又は10に記載の細胞増殖抑制剤。
- 前記GSTP1とCRAFの相互作用を阻害する薬物が、CRAFデコイペプチド又はこれを発現するベクターである、請求項9〜11のいずれか一項に記載の細胞増殖抑制剤。
- 前記CRAFデコイペプチドが、
(a)配列番号9に示されるアミノ酸配列からなるポリペプチド、
(b)配列番号9に示されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなるポリペプチド、
(c)配列番号9に示されるアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなるポリペプチド、及び
(d)(a)〜(c)のいずれかに記載されるポリペプチドのN末端又はC末端に1〜50個のアミノ酸が付加されたポリペプチド
からなる群から選択される、請求項12に記載の細胞増殖抑制剤。 - 請求項1〜13のいずれか一項に記載の細胞増殖抑制剤を含む、RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんの治療又は予防用医薬組成物。
- 請求項1〜13のいずれか一項に記載の細胞増殖抑制剤を含む、RAS/RAF/MEK/ERKシグナルカスケードが活性化されているがんの治療又は予防用キット。
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