JP5469282B1 - Anti-pancreatic cancer peptide - Google Patents
Anti-pancreatic cancer peptide Download PDFInfo
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- JP5469282B1 JP5469282B1 JP2013539059A JP2013539059A JP5469282B1 JP 5469282 B1 JP5469282 B1 JP 5469282B1 JP 2013539059 A JP2013539059 A JP 2013539059A JP 2013539059 A JP2013539059 A JP 2013539059A JP 5469282 B1 JP5469282 B1 JP 5469282B1
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- peptide
- pancreatic cancer
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- aspc1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Life Sciences & Earth Sciences (AREA)
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Abstract
本発明により、Ｐａｔｃｈｅｄ−１蛋白質と相互作用することにより膵臓癌細胞のヘッジホッグシグナル伝達経路の活性を抑制し、膵臓癌細胞の増殖を抑制する膵臓癌の治療に有効な新規抗膵臓癌ペプチド、該ペプチドを含む膵臓癌の抗癌剤および該抗がん剤を投与する工程を含む膵臓癌の治療方法を提供する。 According to the present invention, a novel anti-pancreatic cancer peptide effective in treating pancreatic cancer that inhibits the activity of the hedgehog signaling pathway of pancreatic cancer cells by interacting with Patched-1 protein and suppresses the proliferation of pancreatic cancer cells, An anticancer agent for pancreatic cancer comprising the peptide and a method for treating pancreatic cancer comprising the step of administering the anticancer agent are provided.
Description
本発明は、Ｐａｔｃｈｅｄ−１（以下「Ｐａｔｃｈ１」ともいう。）蛋白質と相互作用して膵臓癌細胞のヘッジホッグシグナル伝達経路の活性を抑制し、膵臓癌細胞の増殖を抑制する膵臓癌の治療に有効な新規抗膵臓癌ペプチド、該ペプチドを含む膵臓癌の抗癌剤および該ペプチドによる治療方法に関する。 The present invention relates to treatment of pancreatic cancer that interacts with Patched-1 (hereinafter also referred to as “Patch1”) protein to suppress the activity of the hedgehog signaling pathway of pancreatic cancer cells and suppress the proliferation of pancreatic cancer cells. The present invention relates to an effective novel anti-pancreatic cancer peptide, an anticancer agent for pancreatic cancer containing the peptide, and a method for treating with the peptide.
膵臓癌では、ヘッジホッグシグナル伝達経路がそのリガンドであるソニックヘッジホッグ（Ｓｏｎｉｃ Ｈｅｄｇｅｈｏｇ。以下、「Ｓｈｈ」ともいう。）により異常に活性化されていることが報告されており、該活性化されたヘッジホッグシグナル伝達経路のコントロールにより膵臓癌の成長が抑制されるとの報告がされている。 In pancreatic cancer, it has been reported that the hedgehog signaling pathway is abnormally activated by its ligand, sonic hedgehog (hereinafter also referred to as “Shh”), and the activation is reported. It has been reported that the growth of pancreatic cancer is suppressed by controlling the hedgehog signaling pathway.
上記の報告は、いずれも実験的研究のレベルのものであり、ヘッジホッグシグナル伝達経路をコントロールし、且つ癌の成長を抑制する臨床レベルで使用可能な薬は未だ開発されていないため、新規治療薬の開発が切実に要望されている。本発明者らは、ソニックヘッジホッグの結合部位に対して産生した抗Ｐｔｃｈ１ポリクロナール抗体がヘッジホッグシグナル伝達活性と癌細胞の成長の双方を抑制することを既に報告しているが、該ポリクロナール抗体は、未だ臨床的に使用可能な段階に至っておらす、またハイブリッド抗体の産生には高コストを要するという問題がある。そこで、本発明は、Ｐａｔｃｈｅｄ−１蛋白質と相互作用することにより膵臓癌細胞のヘッジホッグシグナル伝達経路の活性を抑制し、膵臓癌細胞の増殖を抑制する膵臓癌の臨床治療に有効な新規抗膵臓癌ペプチド、該ペプチドを含む膵臓癌の抗癌剤および該ペプチドによる膵臓癌の治療方法を提供することを目的とする。 All of the above reports are at the level of experimental research, and no drugs that can be used at the clinical level that control the hedgehog signaling pathway and suppress cancer growth have been developed. There is an urgent need for drug development. The present inventors have already reported that the anti-Ptch1 polyclonal antibody produced against the binding site of sonic hedgehog suppresses both hedgehog signaling activity and cancer cell growth. However, there is still a problem that it has reached a stage where it can be clinically used, and production of hybrid antibodies requires high costs. Thus, the present invention provides a novel anti-pancreas effective for clinical treatment of pancreatic cancer that inhibits the activity of the hedgehog signaling pathway of pancreatic cancer cells by interacting with Patched-1 protein and suppresses the proliferation of pancreatic cancer cells. An object of the present invention is to provide a cancer peptide, an anticancer agent for pancreatic cancer containing the peptide, and a method for treating pancreatic cancer using the peptide.
上記課題解決のため、本発明は、配列表の配列番号３に記載のアミノ酸配列からなるペプチドを提供する。該ペプチドは、そのＮ末端のアミノ基をアセチル化してもよい。また、該ペプチドのＣ末端のカルボキシル基をアミド化してもよい。
本発明は、また、配列表の配列番号３に記載のアミノ酸配列からなるペプチド、そのＮ末端のアミノ基をアセチル化したもの、そのＣ末端のカルボキシル基をアミド化したもののいずれか、又はそれらのペプチドの薬学的に許容される塩を含有する膵臓癌に対する抗癌剤を提供する。
本発明は、さらに、上記抗癌剤を投与する工程を含む膵臓癌の治療方法を提供する。In order to solve the above problems, the present invention provides a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing. The peptide may be acetylated at its N-terminal amino group. Further, the C-terminal carboxyl group of the peptide may be amidated.
The present invention also includes a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing, an acetylated amino group at the N-terminal thereof, an amidated carboxyl group at the C-terminal, or a An anticancer agent for pancreatic cancer containing a pharmaceutically acceptable salt of a peptide is provided.
The present invention further provides a method for treating pancreatic cancer, comprising the step of administering the anticancer agent.
本発明により、臨床上、膵臓癌に対して有効性が優れ且つより低コストで生産し得る新規なペプチドが提供される。 The present invention provides a novel peptide that is clinically effective for pancreatic cancer and can be produced at a lower cost.
図１は、膵臓癌細胞ＡｓＰＣ１に対する相互作用ペプチドの３−（４，５−ジメチルチアゾール−２−イル）−２，５−ジフェニルテトラゾリウムブロミドを用いるアッセイ（以下「ＭＴＴアッセイ」という。）による吸光度を指標とした増殖抑制効果を示す。横軸は、ＡｓＰＣ１細胞と共に培養される相互作用ペプチド（図中、ペプチドＡ、Ｂ、Ｃ、Ｄ、Ｆ、ＧおよびＨ）の濃度（μｇ／ｍｌ（ＲＰＭＩ＋５％ＦＣＳ））を示し、縦軸は、ＭＴＴアッセイによる吸光度（Ａ５７０）を示す。また、ＮＣは、正常対照（緩衝液単独（ＲＰＭＩ＋５％ＦＣＳ））を示し（以下同じ。）、吸光度については、その平均値±標準偏差（ＳＤ）を示す（以下、図２において同じ。）。アステリスク（図中＊）は、危険率が５％未満であること（Ｐ＜０．０５）を示す（以下同じ。）。
図２は、膵臓癌細胞系ＳＵＩＴ２に対する相互作用ペプチドのＭＴＴアッセイによる吸光度を指標とした増殖抑制効果を示す。横軸は、ＳＵＩＴ２細胞と共に培養される相互作用ペプチド（図中、ペプチドＡ、Ｂ、Ｃ、Ｄ、Ｆ、ＧおよびＨ）の濃度（μｇ／ｍｌ（ＲＰＭＩ＋５％ＦＣＳ））を示し、縦軸は、ＭＴＴアッセイによる吸光度（Ａ５７０）を示す。
図３Ａは、膵臓癌細胞ＡｓＰＣ１に対する相互作用ペプチドの細胞数を指標とした増殖抑制効果を示す。縦軸は、膵臓癌細胞ＡｓＰＣ１のコールター計数による細胞数を示し、横軸は、膵臓癌細胞ＡｓＰＣ１と共に異なる培養時間（図中０、４８、７２時間）で培養された相互作用ペプチドを示す。ＮＣ、ペプチドＢ、ペプチドＣおよびペプチドＧについては、更に細胞数の平均値±標準偏差（ＳＤ）を示す（以下、図３Ｂにおいて同じ。）。また、Ａ、Ｂ、Ｃ、Ｄ、Ｆ、Ｇ、Ｈは、それぞれペプチドＡ、ペプチドＢ、ペプチドＣ、ペプチドＤ、ペプチドＦ、ペプチドＧ、ペプチドＨを示す（以下同じ）。
図３Ｂは、膵臓癌細胞ＳＵＩＴ２に対する相互作用ペプチドの細胞数を指標とした増殖抑制効果を示す。縦軸は、膵臓癌細胞ＳＵＩＴ２のコールター計数による細胞数を示し、横軸は、膵臓癌細胞ＳＵＩＴ２と共に異なる培養時間（図中０、４８、７２時間）で培養された相互作用ペプチドを示す。
図４Ａは、ＡｓＰＣ１に関するＧｌｉ１のｍＲＮＡ量に対する相互作用ペプチドの影響を示す。縦軸は、ヘッジホッグシグナル伝達経路の活性のマーカーであるＧｌｉ１の相対的ｍＲＮＡ量を示し、横軸は、ＡｓＰＣ１と７２時間培養された相互作用ペプチドを示す。また、相対ｍＲＮＡ量についてはその平均値±標準偏差（ＳＤ）を示す（以下、図４Ｂ、図６Ｃおよび図６Ｄにおいて同じ。）。
図４Ｂは、ＳＵＩＴ２に関するＧｌｉ１の相対的ｍＲＮＡ量に対する相互作用ペプチドの影響を示す。縦軸は、ヘッジホッグシグナル伝達経路の活性のマーカーであるＧｌｉ１の相対的ｍＲＮＡ量を示し、横軸は、ＳＵＩＴ２と７２時間培養された相互作用ペプチドを示す。
図５は、ペプチドＡ、ペプチドＢ、ペプチドＣおよびペプチドＧと共に培養されたＡｓＰＣ１細胞に関するソニックヘッジホッグの染色レベル（上部の写真）およびそれらの位相差画像（下部の写真）を示す。また、横棒は５０μｍを示す。
図６Ａは、 移植したＡｓＰＣ１膵臓癌細胞に対するペプチドＡおよびペプチドＣの増殖抑制効果を示す。縦軸は、膵臓癌細胞の容積を示し、横軸は、ペプチドＡおよびペプチドＣの膵臓癌細胞移植後の日数を示す。
図６Ｂは、膵臓癌細胞を移植されたマウスであって、それぞれペプチドＡを注入されたもの（写真左）およびペプチドＣを注入されたもの（写真右）を示し、矢印は移植腫瘍を示す。
図６Ｃは、ＡｓＰＣ１に関するＧｌｉ１の相対的ｍＲＮＡ量に対するペプチドＡ（左図）およびペプチドＣ（右図）の影響を示す。縦軸は、ヘッジホッグシグナル伝達経路の活性のマーカーであるＧｌｉ１の相対的ｍＲＮＡ量を示し、横軸は、投与されたペプチドを示す。
図６Ｄは、ＳＵＩＴ２に関するＧｌｉ１の相対的ｍＲＮＡ量に対するペプチドＡ（左図）およびペプチドＧ（右図）の影響を示す。縦軸は、ヘッジホッグシグナル伝達経路の活性のマーカーであるＧｌｉ１の相対的ｍＲＮＡ量を示し、横軸は、投与されたペプチドを示す。
図６Ｅは、相互作用ペプチドがＰｔｃｈ１とヘッジホッグシグナル伝達経路の間の相互作用を妨げる推論上のメカニズムを示す。ＮおよびＣはそれぞれ、Ｎアミノ末端およびＣカルボキシ末端を示す。FIG. 1 shows the absorbance by an assay using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide as an interacting peptide for pancreatic cancer cell AsPC1 (hereinafter referred to as “MTT assay”). The growth inhibitory effect as an index is shown. The horizontal axis shows the concentration (μg / ml (RPMI + 5% FCS)) of interacting peptides (in the figure, peptides A, B, C, D, F, G and H) cultured with AsPC1 cells, and the vertical axis , Shows absorbance (A570) by MTT assay. NC represents a normal control (buffer alone (RPMI + 5% FCS)) (hereinafter the same), and the absorbance represents an average value ± standard deviation (SD) (hereinafter the same in FIG. 2). An asterisk (* in the figure) indicates that the risk rate is less than 5% (P <0.05) (the same applies hereinafter).
FIG. 2 shows the growth inhibitory effect of the interacting peptide on pancreatic cancer cell line SUIT2 as an index of absorbance by MTT assay. The horizontal axis indicates the concentration (μg / ml (RPMI + 5% FCS)) of interacting peptides (in the figure, peptides A, B, C, D, F, G and H) cultured with SUIT2 cells, and the vertical axis , Shows absorbance (A570) by MTT assay.
FIG. 3A shows the growth inhibitory effect using the number of interacting peptide cells for pancreatic cancer cell AsPC1 as an index. The vertical axis shows the number of cells obtained by Coulter counting of pancreatic cancer cells AsPC1, and the horizontal axis shows interacting peptides cultured with different culture times (0, 48, 72 hours in the figure) with pancreatic cancer cells AsPC1. For NC, Peptide B, Peptide C and Peptide G, the mean number of cells ± standard deviation (SD) is also shown (hereinafter the same in FIG. 3B). A, B, C, D, F, G, and H represent peptide A, peptide B, peptide C, peptide D, peptide F, peptide G, and peptide H, respectively (the same applies hereinafter).
FIG. 3B shows the growth-suppressing effect using the number of interacting peptides on pancreatic cancer cell SUIT2 as an index. The vertical axis shows the number of cells by Coulter counting of pancreatic cancer cells SUIT2, and the horizontal axis shows interacting peptides cultured with different culture times (0, 48, 72 hours in the figure) with pancreatic cancer cells SUIT2.
FIG. 4A shows the effect of interacting peptides on the amount of Gli1 mRNA for AsPC1. The vertical axis represents the relative mRNA level of Gli1, which is a marker for the activity of the hedgehog signal transduction pathway, and the horizontal axis represents an interacting peptide cultured with AsPC1 for 72 hours. In addition, the average mRNA standard deviation (SD) is shown for the relative mRNA amount (hereinafter the same in FIG. 4B, FIG. 6C and FIG. 6D).
FIG. 4B shows the effect of interacting peptides on the relative mRNA level of Gli1 for SUIT2. The vertical axis represents the relative mRNA level of Gli1, which is a marker for the activity of the hedgehog signaling pathway, and the horizontal axis represents an interacting peptide cultured with SUIT2 for 72 hours.
FIG. 5 shows Sonic hedgehog staining levels (top photo) and phase contrast images (bottom photo) for AsPC1 cells cultured with peptide A, peptide B, peptide C and peptide G. Moreover, a horizontal bar shows 50 micrometers.
FIG. 6A shows the growth inhibitory effect of peptide A and peptide C on transplanted AsPC1 pancreatic cancer cells. The vertical axis shows the volume of pancreatic cancer cells, and the horizontal axis shows the number of days after transplantation of pancreatic cancer cells of peptide A and peptide C.
FIG. 6B shows mice transplanted with pancreatic cancer cells, each injected with peptide A (left photo) and injected with peptide C (right photo), and arrows indicate transplanted tumors.
FIG. 6C shows the effect of peptide A (left panel) and peptide C (right panel) on the relative mRNA level of Gli1 with respect to AsPC1. The vertical axis shows the relative mRNA level of Gli1, which is a marker of the activity of the hedgehog signaling pathway, and the horizontal axis shows the administered peptide.
FIG. 6D shows the effect of peptide A (left panel) and peptide G (right panel) on the relative mRNA level of Gli1 for SUIT2. The vertical axis shows the relative mRNA level of Gli1, which is a marker of the activity of the hedgehog signaling pathway, and the horizontal axis shows the administered peptide.
FIG. 6E shows a speculative mechanism by which interacting peptides prevent the interaction between Ptch1 and the hedgehog signaling pathway. N and C represent the N amino terminus and the C carboxy terminus, respectively.
以下、本発明を実施するための好適な形態について図面を参照しながら説明する。なお、以下に説明する実施形態は、本発明の代表的な実施形態の一例を示したものであり、これにより本発明の範囲が狭く解釈されることはない。
（Ａ）新規ペプチド
本発明に係る新規ペプチドは、配列表の配列番号３に記載のアミノ酸配列で表される。以下該ペプチド（実施例におけるペプチドＣを参照）について説明する。
該ペプチドは、図６Ｅに示す推論上のメカニズムに従いＰａｔｃｈｅｄ−１蛋白質と相互作用することが予想されるペプチドをソフトウェアおよび独自のノウハウを用いて設計したもののうち、該相互作用の可能性が高い７種のペプチド（以下「相互作用ペプチド」という。）について合成を行ない（実施例１参照）、Ｐａｔｃｈｅｄ−１蛋白質との相互作用を検証した結果、試験管内（ｉｎ ｖｉｔｒｏ）および生体内（ｉｎ ｖｉｖｏ）においてヒト膵臓癌細胞系（ＡｓＰＣ１およびＳＵＩＴ２）に対する特に優れた増殖抑制効果を有することが見出されたものである。該ペプチドは、１６個のアミノ酸（Ａｓｐ Ｔｈｒ Ｌｅｕ Ｓｅｒ Ｃｙｓ Ｇｌｎ Ｓｅｒ Ｐｒｏ Ｇｌｕ Ｓｅｒ Ｔｈｒ Ｓｅｒ Ｓｅｒ Ｔｈｒ Ａｒｇ Ａｓｐ）により構成され、その構成アミノ酸Ａｓｐ、Ｔｈｒ、Ｌｅｕ、Ｓｅｒ、Ｃｙｓ、Ｇｌｎ、Ｐｒｏ、Ｇｌｕ、Ａｒｇは、それぞれ、天然型アミノ酸のアスパラギン酸、スレオニン、ロイシン、セリン、システイン、グルタミン、プロリン、グルタミン酸、アルギニンを示す。
本発明の新規ペプチドのＮ末端のアミノ基はアセチル化してもよいが、該アセチル化は、自体公知の方法（例えば、Ａｔｈｅｒｔｏｎ，Ｅ．ａｎｄ Ｓｈｅｐｐａｒｄ，Ｒ．Ｃ．（１９８９）Ｓｏｌｉｄ ｐｈａｓｅ ｐｅｐｔｉｄｅ ｓｙｎｔｈｅｓｉｓ；ａ ｐｒａｃｔｉｃａｌ ａｐｐｒｏａｃｈ．ＩＲＬ Ｐｒｅｓｓ，Ｏｘｆｏｒｄ．および生化学実験講座 蛋白質の化学 ＩＶ ｐ４４９、日本生化学会編、東京化学同人（１９７７））により、もしくはこれらに準じた方法により、行なうことができる。
本発明のペプチドのＣ末端のカルボキシル基はアミド化してもよいが、該アミド化は、自体公知の方法（例えば、Ｎｅｉｓｅｓ，Ｂ．；Ｓｔｅｇｌｉｃｈ，Ｗ．Ａｎｇｅｗ．Ｃｈｅｍ．Ｉｎｔ．Ｅｄ．１９７８，１７，５２２．）により、もしくはこれに準じた方法により、行なうことができる。
該Ｃ末端のカルボキシル基のアミド化または前記のＮ末端のアミノ基のアセチル化により、本発明に係る新規ペプチドの安定化を図ることができる。
（Ｂ）膵臓癌に対する抗癌剤
本発明に係る膵臓癌に対する抗癌剤、すなわち、前記の新規ペプチド（配列表の配列番号３に記載のアミノ酸配列からなるペプチド、そのＮ末端のアミノ基をアセチル化したもの、そのＣ末端のカルボキシル基をアミド化したもの）のいずれか、又はそれらのペプチドの薬学的に許容される塩を含有する膵臓癌の抗癌剤について、以下に説明する。
本発明のペプチドの薬学的に許容される塩とは、塩酸塩、硫酸塩、臭化水素酸塩等の無機酸との塩、酢酸塩、クエン酸塩、メタンスルホン酸塩などの有機酸との塩、ナトリウム塩、カリウム塩等の無機塩基との塩、イソプロピルアミン、２−エチルアミノエタノール等の有機塩基との塩をいう。
本発明の抗癌剤は、有効成分として本発明のペプチドを含有し、経直腸、経鼻、経肺、経膣、外用（局所）、経口または非経口（皮下、植込み、静脈内および筋肉内を含む）投与に適した固体、半固体または液体（錠剤、ペレット剤、トローチ剤、カプセル剤、坐剤、クリーム剤、軟膏剤、エアゾール剤、散剤、液剤、乳剤、懸濁剤、シロップ剤、注射液など）等の医薬製剤の形態で使用できる。
本発明の抗癌剤は、製薬目的で慣用されている種々の有機または無機担体、例えば賦形剤（スクロース、デンプン、マンニトール、ソルビトール、ラクトース、グルコース、セルロース、タルク、リン酸カルシウム、炭酸カルシウムなど）、縮合剤（セルロース、メチルセルロース、ヒドロキシプロピルセルロース、ポリプロピルピロリドン、ゼラチン、アラビアゴム、ポリエチレングリコール、スクロース、デンプンなど）、崩壊剤（デンプン、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、ヒドロキシプロピルデンプン、炭酸水素ナトリウム、リン酸カルシウム、クエン酸カルシウムなど）、滑沢剤（ステアリン酸マグネシウム、エアロシル、タルク、ラウリル硫酸ナトリウムなど）、矯味剤（クエン酸、メントール、グリシン、オレンジ末など）、保存剤（安息香酸ナトリウム、亜硫酸水素ナトリウム、メチルパラベン、プロピルパラベンなど）、安定化剤（クエン酸、クエン酸ナトリウム、酢酸など）、懸濁剤（メチルセルロース、ポリビニルピロリドン、ステアリン酸アルミニウムなど）、分散剤（ヒドロキシプロピルメチルセルロースなど）、希釈剤（水など）、基材ワックス（カカオバター、ポリエチレングリコール、白色ワセリンなど）を用いる常法によっても製造することができる。
（Ｃ）膵臓癌の治療方法
本発明に係る膵臓癌の治療方法、すなわち、前記の抗癌剤を投与する工程を含む膵臓癌の治療方法において、本発明の抗癌剤は、ヒトを含む哺乳動物に上記慣用の医薬製剤の形で、特に限定なく投与することができる。その中でも静脈内、筋肉内または経口投与するのが好ましい。なお、相互作用ペプチドのうち、配列表の配列番号２、配列番号５および配列番号６に記載のアミノ酸配列で表されるペプチド（それぞれ実施例におけるペプチドＢ、ＦおよびＧを参照）についてもヒト膵臓癌細胞系に対して優れた増殖抑制効果を有しているため、これらのペプチド（そのＮ末端のアミノ基をアセチル化したものおよびそのＣ末端のカルボキシル基をアミド化したものを含む。）は、本発明に係るペプチドと同様に、それぞれのペプチド又はそれらのペプチドの薬学的に許容される塩を含有する膵臓癌の抗癌剤において用いることができ、また該抗癌剤を投与する工程を含む膵臓癌の治療方法においても用いることができる。DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.
(A) Novel peptide The novel peptide according to the present invention is represented by the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing. The peptide (see peptide C in the examples) will be described below.
Among the peptides that are designed using software and unique know-how, peptides that are expected to interact with the Patched-1 protein according to the inference mechanism shown in FIG. As a result of synthesizing a kind of peptide (hereinafter referred to as “interactive peptide”) (see Example 1) and verifying its interaction with Patched-1 protein, in vitro and in vivo Has been found to have a particularly excellent growth inhibitory effect on human pancreatic cancer cell lines (AsPC1 and SUIT2). The peptide is composed of 16 amino acids (Asp Thr Leu Ser Cys Gln Ser Pro Glu Ser Thr Ser Ser Thr Arg Asp), and its constituent amino acids Asp, Thr, Leu, Ser, Cys, Gln, Pro, Glu, Arg Respectively represent the natural amino acids aspartic acid, threonine, leucine, serine, cysteine, glutamine, proline, glutamic acid, and arginine.
Although the N-terminal amino group of the novel peptide of the present invention may be acetylated, the acetylation may be performed by a method known per se (for example, Atherton, E. and Sheppard, RC (1989) Solid phase peptide synthesis; a practical appl.IRL Press, Oxford, and Biochemistry Experiment Course Protein Chemistry IV p449, edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin (1977)), or a method based thereon.
Although the carboxyl group at the C-terminal of the peptide of the present invention may be amidated, the amidation is carried out by a method known per se (for example, Neises, B .; Steglich, W. Angew. Chem. Int. Ed. 1978, 17). , 522.) or a method according thereto.
The novel peptide according to the present invention can be stabilized by amidation of the C-terminal carboxyl group or acetylation of the N-terminal amino group.
(B) Anticancer agent against pancreatic cancer Anticancer agent against pancreatic cancer according to the present invention, that is, the above-mentioned novel peptide (peptide comprising the amino acid sequence described in SEQ ID NO: 3 in the sequence listing, acetylated N-terminal amino group, An anticancer agent for pancreatic cancer containing any one of the C-terminal carboxyl groups) or a pharmaceutically acceptable salt of the peptide thereof will be described below.
The pharmaceutically acceptable salts of the peptides of the present invention include salts with inorganic acids such as hydrochloride, sulfate, hydrobromide, organic acids such as acetate, citrate, methanesulfonate, and the like. Salts with inorganic bases such as sodium salts, sodium salts and potassium salts, and salts with organic bases such as isopropylamine and 2-ethylaminoethanol.
The anticancer agent of the present invention contains the peptide of the present invention as an active ingredient and includes transrectal, nasal, pulmonary, vaginal, topical (topical), oral or parenteral (subcutaneous, implantation, intravenous and intramuscular) ) Solid, semi-solid or liquid suitable for administration (tablets, pellets, troches, capsules, suppositories, creams, ointments, aerosols, powders, solutions, emulsions, suspensions, syrups, injections) Etc.) and the like.
The anticancer agent of the present invention includes various organic or inorganic carriers conventionally used for pharmaceutical purposes, such as excipients (sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, etc.), condensing agents (Cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, etc.), disintegrant (starch, carboxymethylcellulose, carboxymethylcellulose calcium, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, citrate Acid), lubricants (magnesium stearate, aerosil, talc, sodium lauryl sulfate, etc.), flavoring agents (citric acid, men , Glycine, orange powder, etc.), preservatives (sodium benzoate, sodium bisulfite, methylparaben, propylparaben, etc.), stabilizers (citric acid, sodium citrate, acetic acid, etc.), suspending agents (methylcellulose, polyvinyl) It can also be produced by a conventional method using pyrrolidone, aluminum stearate, etc.), a dispersant (hydroxypropylmethylcellulose, etc.), a diluent (water, etc.), and a base wax (cocoa butter, polyethylene glycol, white petrolatum, etc.).
(C) Method for treating pancreatic cancer In the method for treating pancreatic cancer according to the present invention, that is, the method for treating pancreatic cancer comprising the step of administering the anticancer agent, the anticancer agent of the present invention is commonly used for mammals including humans. And can be administered without any particular limitation. Of these, intravenous, intramuscular or oral administration is preferred. Of the interacting peptides, peptides represented by the amino acid sequences set forth in SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 6 (see peptides B, F and G in the examples, respectively) also represent human pancreas. These peptides (including those obtained by acetylating the N-terminal amino group and those obtained by amidating the C-terminal carboxyl group) have an excellent growth inhibitory effect on cancer cell lines. The pancreatic cancer can be used in an anticancer agent for pancreatic cancer containing each peptide or a pharmaceutically acceptable salt of the peptide as well as the peptide according to the present invention, and the method further comprises a step of administering the anticancer agent. It can also be used in therapeutic methods.
癌細胞の生育に対するペプチドの生物学的効果が ヘッジホッグシグナル伝達経路の（転写）活性の減衰により生じたことを確認するために、該経路に対する合成ペプチドである相互作用ペプチド（ペプチドＡ、Ｂ、Ｃ、Ｄ、Ｆ、Ｇ、Ｈ）の影響を検討した結果を実施例に示す。以下に実施例を示して本発明をより具体的に説明するが、本発明はこれらの実施例に限定されるものではない。
実施例１
（Ｐｔｃｈ１に対する相互作用ペプチドの設計および合成）
Ｐｔｃｈ１の標的配列に対する相互作用ペプチドは、アミノ酸をランダムに変更することで標的に対する親和性が高いと予想されるペプチド配列５０００種を設計する遺伝的アルゴリズムを採用し、且つ設計した各ペプチド配列について、疎水性親水性指標の相補性最適化、平均的構造類似性最適化、側鎖の干渉の最小化および骨格の配列を含む幾つかの物理化学的パラメータに基づき点数が割り当てられるＭＩＭＥＴＩＣプログラムと称するソフトウェア（Ｃａｍｐｂｅｌｌ Ｗ，Ｋｌｅｉｍａｎ Ｌ，Ｂａｒａｎｙ Ｌ，Ｌｉ Ｚ，Ｋｈｏｒｃｈｉｄ Ａ，Ｆｕｊｉｔａ Ｅ，ｅｔ ａｌ．Ａ ｎｏｖｅｌ ｇｅｎｅｔｉｃ ａｌｇｏｒｉｔｈｍ ｆｏｒ ｄｅｓｉｇｎｉｎｇ ｍｉｍｅｔｉｃ ｐｅｐｔｉｄｅｓ ｔｈａｔ ｉｎｔｅｒｆｅｒｅ ｗｉｔｈ ｔｈｅ ｆｕｎｃｔｉｏｎ ｏｆ ａ ｔａｒｇｅｔ ｍｏｌｅｃｕｌｅ．Ｍｉｃｒｏｂｉｏｌ Ｉｍｍｕｎｏｌ．２００２；４６：２１１−５．およびＢａｒａｎｙｉ Ｌ，Ｃａｍｐｂｅｌｌ Ｗ，Ｏｈｓｈｉｍａ Ｋ，Ｆｕｊｉｍｏｔｏ Ｓ，Ｂｏｒｏｓ Ｍ，Ｏｋａｄａ Ｈ．Ｔｈｅ ａｎｔｉｓｅｎｓｅ ｈｏｍｏｌｏｇｙ ｂｏｘ：ａ ｎｅｗ ｍｏｔｉｆ ｗｉｔｈｉｎ ｐｒｏｔｅｉｎｓ ｔｈａｔ ｅｎｃｏｄｅｓ ｂｉｏｌｏｇｉｃａｌｌｙ ａｃｔｉｖｅ ｐｅｐｔｉｄｅｓ．Ｎａｔ Ｍｅｄ．１９９５；１：８９４−９０１．を参照）により設計され、下記の表１に示す高い点数の７個のペプチドが選択された。これら７個のペプチドは、固相合成法（Ｇｅｒａｌｄ Ｆ．Ｓｉｇｌｅｒ，Ａｎｎｅ Ｋ．Ｓｏｕｔａｒ，Ｌｏｕｉｓ Ｃ．Ｓｍｉｔｈ，Ａｎｔｏｎｉｏ Ｍ．Ｇｏｔｔｏ，Ｊｒ．，Ｊａｍｅｓ Ｔ．Ｓｐａｒｒｏｗ．Ｔｈｅ ｓｏｌｉｄ ｐｈａｓｅ ｓｙｎｔｈｅｓｉｓ ｏｆ ａ ｐｒｏｔｅｉｎ ａｃｔｉｖａｔｏｒ ｆｏｒ ｌｅｃｉｔｈｉｎ−ｃｈｏｌｅｓｔｅｒｏｌ ａｃｙｌｔｒａｎｓｆｅｒａｓｅ ｃｏｒｒｅｓｐｏｎｄｉｎｇ ｔｏ ｈｕｍａｎ ｐｌａｓｍａ ａｐｏＣ−Ｉ．Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，Ｖｏｌ．７３，Ｎｏ．５，ｐｐ．１４２２−１４２６，Ｍａｙ １９７６，Ｂｉｏｃｈｅｍｉｓｔｒｙを参照）により製造した。
（ヒト膵臓癌細胞系（ＡｓＰＣ１およびＳＵＩＴ２）の増殖アッセイ）
ヒト膵臓癌細胞系ＡｓＰＣ１（アメリカンタイプカルチャーコレクション、Ｒｏｃｋ ｖｉｌｌｅ、Ｍｅｄ．、ＵＳＡより入手）およびＳＵＩＴ２（ＪＣＲＢ細胞バンクより入手）の増殖アッセイは、次の細胞培養条件による培養細胞について、分光光度的に測定可能なブルーホルマザンに対する、代謝的活性細胞のミトコンドリアデヒドロゲナーゼによる黄色３−（４，５−ジメチルチアゾール−２−イル）−２，５−ジフェニルテトラゾリウムブロミド（ＭＴＴ）（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．セントルイス、ＭＯ）の還元に基づくＭＴＴアッセイ法およびコールターカウンター（Ｂｅｃｋｍａｎ Ｃｏｕｌｔｅｒ，Ｆｕｌｌｅｒｔｏｎ，ＣＡ，ＵＳＡ）による細胞数計測法（Ｋｕｂｏ Ｍ，Ｎａｋａｍｕｒａ Ｍ，Ｔａｓａｋｉ Ａ，Ｙａｍａｎａｋａ Ｎ，Ｎａｋａｓｈｉｍａ Ｈ，Ｎｏｍｕｒａ Ｍ，ｅｔ ａｌ．Ｈｅｄｇｅｈｏｇ ｓｉｇｎａｌｉｎｇ ｐａｔｈｗａｙ ｉｓ ａ ｎｅｗ ｔｈｅｒａｐｅｕｔｉｃ ｔａｒｇｅｔ ｆｏｒ ｐａｔｉｅｎｔｓ ｗｉｔｈ ｂｒｅａｓｔ ｃａｎｃｅｒ．Ｃａｎｃｅｒ Ｒｅｓ．２００４；６４：６０７１−４．を参照）により行なった。
（細胞培養条件）
ヒト膵臓癌細胞系ＡｓＰＣ１およびＳＵＩＴ２は、ウシ胎児血清（ＦＢＳ；Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）と抗生物質（１００ｕｎｉｔｓ／ｍｌのペニシリンおよび１００μｇ／ｍｌのストレプトマイシン、明治製菓株式会社製）を補ったＲＰＭＩ１６４０完全培地（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｇｒａｎｄ Ｉｓｌａｎｄ，ＮＹ，ＵＳＡ製）中で、加湿した二酸化炭素５％および空気９５％下３７℃で培養した（Ｎａｋａｍｕｒａ Ｍ，Ｍａｓｕｄａ Ｈ，Ｈｏｒｉｉ Ｊ，Ｋｕｍａ Ｋ，Ｙｏｋｏｙａｍａ Ｎ，Ｏｈｂａ Ｔ，ｅｔ ａｌ．Ｗｈｅｎ ｏｖｅｒｅｘｐｒｅｓｓｅｄ，ａ ｎｏｖｅｌ ｃｅｎｔｒｏｓｏｍａｌ ｐｒｏｔｅｉｎ，ＲａｎＢＰＭ，ｃａｕｓｅｓ ｅｃｔｏｐｉｃ ｍｉｃｒｏｔｕｂｕｌｅ ｎｕｃｌｅａｔｉｏｎ ｓｉｍｉｌａｒ ｔｏ ｇａｍｍａ−ｔｕｂｕｌｉｎ．Ｊ Ｃｅｌｌ Ｂｉｏｌ．１９９８；１４３：１０４１−５２．およびＫａｍｅｄａ Ｃ，Ｔａｎａｋａ Ｈ，Ｙａｍａｓａｋｉ Ａ，Ｎａｋａｍｕｒａ Ｍ，Ｋｏｇａ Ｋ，Ｓａｔｏ Ｎ，ｅｔ ａｌ．Ｔｈｅ Ｈｅｄｇｅｈｏｇ ｐａｔｈｗａｙ ｉｓ ａ ｐｏｓｓｉｂｌｅ ｔｈｅｒａｐｅｕｔｉｃ ｔａｒｇｅｔ ｆｏｒ ｐａｔｉｅｎｔｓ ｗｉｔｈ ｅｓｔｒｏｇｅｎ ｒｅｃｅｐｔｏｒ−ｎｅｇａｔｉｖｅ ｂｒｅａｓｔ ｃａｎｃｅｒ．Ａｎｔｉｃａｎｃｅｒ Ｒｅｓ．２００９；２９：８７１−９．を参照）。
（ＭＴＴアッセイ法による増殖アッセイ）
ヒト膵臓癌細胞系ＡｓＰＣ１およびＳＵＩＴ２は、３ｘ１０３細胞／ウェルの濃度で９６ウェルプレートに接種され、上記の細胞培養条件に従い、一夜培養した後、それぞれ１．０μｇ／ｍｌおよび１０．０μｇ／ｍｌの７種の各相互作用ペプチド（ペプチドＡ、Ｂ、Ｃ、Ｄ、Ｆ、Ｇ、Ｈ）を含む新たな培地に変えられ７２時間培養された。その後、細胞はトリプシンを用いて収集し、生存細胞はＭＴＴアッセイに付された。正常対照（ＮＣ）としては緩衝液単独（ＲＰＭＩ＋５％ＦＣＳ）を用い、各相互作用ペプチドは当該緩衝液（ＲＰＭＩ＋５％ＦＣＳ）に溶解して使用した（以下同じ。）。
結果を図１および図２に示す。ペプチドＢ、ＣおよびＧは、ヒト膵臓癌細胞系ＡｓＰＣ１細胞の成長に対して、いずれも１．０μｇ／ｍｌの濃度および１０．０μｇ／ｍｌの濃度において用量依存的に、それぞれ抑制傾向および有意な抑制効果を示した（図１参照）。また、ペプチドＢ、Ｃ、ＦおよびＧは、ヒト膵臓癌細胞系ＳＵＩＴ２細胞の成長に対して、いずれも１．０μｇ／ｍｌの濃度および１０．０μｇ／ｍｌの濃度において用量依存的に、それぞれ抑制傾向および有意な抑制効果を示した（図２参照）。ペプチドＦの例外はあるものの、前記ＡｓＰＣ１の場合と類似していた。なお、ペプチドＡ、ＤおよびＨは、いずれもヒト膵臓癌細胞系ＡｓＰＣ１およびＳＵＩＴ２細胞の成長に対して抑制効果を示さなかった（図１および図２参照）。
（コールターカウンターによる細胞数計測法による増殖アッセイ）
ＭＴＴアッセイにより検出された相互作用ペプチドの生物学的効果を更に確認するため、相互作用ペプチドと共に培養されたヒト膵臓癌細胞系ＡｓＰＣ１細胞およびＳＵＩＴ２細胞について以下に示すコールター計測を行なった。ヒト膵臓癌細胞系ＡｓＰＣ１およびＳＵＩＴ２は、１ｘ１０４細胞／ウェルの濃度で２４ウェルプレートに接種され、上記の細胞培養条件に従い、一夜培養した後、それぞれ１０μｇ／ｍｌの７種の各相互作用ペプチド（ペプチドＡ、Ｂ、Ｃ、Ｄ、Ｆ、Ｇ、Ｈ）を含む新たな培地に変えられ４８時間および７２時間培養された。その後、細胞はトリプシンを用いて収集し、生存細胞はコールターカウンターを用いる細胞数計測に付された。
結果を図３Ａ、３Ｂに示す。ＭＴＴアッセイの結果と矛盾すること無く、同様の結果が得られた。すなわち、ペプチドＢ、ＣおよびＧは、ヒト膵臓癌細胞系ＡｓＰＣ１細胞の増殖に対して、いずれも４８時間および７２時間の培養時間において、それぞれ抑制傾向および有意な抑制効果を示した（図３Ａ）。ペプチドＢ、Ｃ、ＦおよびＧは、ヒト膵臓癌細胞系ＳＵＩＴ２細胞の増殖に対して、いずれも４８時間および７２時間の培養時間において、それぞれ抑制傾向および有意な抑制効果を示した（図３Ｂ）。ペプチドＦは、ＳＵＩＴ２についてのみ効果を示した。
なお、ペプチドＡ、ＤおよびＨは、いずれもヒト膵臓癌細胞系ＡｓＰＣ１およびＳＵＩＴ２細胞の増殖に対して抑制効果を示さなかった（図１および図２参照）。
実施例３
（Ｇｌｉ１のｍＲＮＡの発現量に対するＰｔｃｈ１相互作用ペプチドの影響）
Ｇｌｉ１はヘッジホッグシグナル伝達経路の転写因子であるだけでなくヘッジホッグ経路の標的遺伝子でもあるため、Ｇｌｉ１のｍＲＮＡの発現量をヘッジホッグ経路の活性のマーカーとして用いた。実施例２においてＡｓＰＣ１の増殖を抑制することが確認された、３個のペプチド、即ちペプチドＢ、ＣおよびＧは、ＡｓＰＣ１と共に７２時間培養され、陰性対照としてペプチドＡもＡｓＰＣ１と共に同時間培養された。培養後、ｍＲＮＡは培養細胞から調製され、次に述べるリアルタイムＲＴ−ＰＣＲによる定量分析に付された。また、実施例２においてＳＵＩＴ２の増殖を抑制することが確認された、４個の相互作用ペプチド、即ちペプチドＢ、Ｃ、ＦおよびＧについても、ＡｓＰＣ１の場合と同様に、ＳＵＩＴ２の増殖に対して抑制効果を示したので、これら４個の相互作用ペプチドについてもＳＵＩＴ２と共に７２時間培養され、陰性対照としてペプチドＡもＳＵＩＴ２と共に同時間培養された。培養後、ｍＲＮＡは培養細胞から調製され、次に述べるリアルタイムＲＴ−ＰＣＲによる定量分析に付された。
（ＲＴ−ＰＣＲを用いたＧｌｉ１のｍＲＮＡの発現量の定量）
Ｇｌｉ１のｍＲＮＡの発現量の定量は、リアルタイム逆転写ポリメラーゼ連鎖反応（ＲＴ−ＰＣＲ）を用いて以下のように行なった。即ち、総ＲＮＡは、ＲＮｅａｓｙミニキット（Ｑｉａｇｅｎ，Ｖａｌｅｎｃｉａ，ＣＡ，ＵＳＡ）を用いて抽出され、分光光度法（Ｕｌｔｒｏｓｐｅｃ ２１００ Ｐｒｏ；Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ，Ｃａｍｂｒｉｄｇｅ，ＵＫ）により定量された。ＲＮＡ（７００ｎｇ）は、ＤＮａｓｅで処理され、Ｑｕａｎｔｉｔｅｃｔ Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔｉｏｎ Ｋｉｔ（Ｑｉａｇｅｎ）を用いてそのプロトコールに従いｃＤＮＡに逆転写された。反応は、ＳＹＢＲ Ｐｒｅｍｉｘ ＥｘＴａｑ（タカラバイオ株式会社製）を用い、ＤＮＡ Ｅｎｇｉｎｅ Ｏｐｔｉｃｏｎ ２ Ｓｙｓｔｅｍ（ＭＪ Ｒｅｓｅａｒｃｈ，Ｗａｌｔｈａｍ，ＭＡ，ＵＳＡ）により実施された。ｐＧｌｉ１−ＧＦＰ（緑色蛍光蛋白質）は、連続的に１０倍単位で希釈され、Ｇｌｉ１の標準曲線を作成するためにプライマー対を用いて増幅された。各サンプルは３回実施された。全てのプライマーの組は２００塩基対の長さの断片を増幅した。使用されたプライマーの配列は次の通りである。ｂｅｔａ−ａｃｔｉｎ、順方向、５０−ＴＴＧ ＣＣＧ ＡＣＡ ＧＧＡ ＴＧＣ ＡＧＡ ＡＧＧ Ａ−３０、逆方向、５０−ＡＧＧ ＴＧＧ ＡＣＡ ＧＣＧ ＡＧＧ ＣＣＡ ＧＧＡ Ｔ−３０およびＧｌｉ１、順方向、５０−ＧＧＴ ＴＣＡ ＡＧＡ ＧＣＣ ＴＧＧ ＧＣＴ ＧＴＧ Ｔ−３０、逆方向、５０−ＧＧＣ ＡＧＣ ＡＴＴ ＣＴＣ ＡＧＴ ＧＡＴ ＧＣＴ Ｇ−３０。あるサンプル中の遺伝子の量は、そのサンプル中のβ−ａｃｔｉｎ量に対して規準化された（Ｋａｍｅｄａ Ｃ，Ｎａｋａｍｕｒａ Ｍ，Ｔａｎａｋａ Ｈ，Ｙａｍａｓａｋｉ Ａ，Ｋｕｂｏ Ｍ，Ｔａｎａｋａ Ｍ，ｅｔ ａｌ．Ｏｅｓｔｒｏｇｅｎ ｒｅｃｅｐｔｏｒ−ａｌｐｈａ ｃｏｎｔｒｉｂｕｔｅｓ ｔｏ ｔｈｅ ｒｅｇｕｌａｔｉｏｎ ｏｆ ｔｈｅ ｈｅｄｇｅｈｏｇ ｓｉｇｎａｌｌｉｎｇ ｐａｔｈｗａｙ ｉｎ ＥＲ ａｌｐｈａｐｏｓｉｔｉｖｅ ｇａｓｔｒｉｃ ｃａｎｃｅｒ．Ｂｒ Ｊ Ｃａｎｃｅｒ．２０１０；１０２：７３８−４７．を参照）。
結果を図４Ａおよび図４Ｂに示す。Ｇｌｉ１の相対ｍＲＮＡ量は、ペプチドＡと共に若しくは相互作用ペプチド無しで培養されたＡｓＰＣ１細胞（ＮＣ参照）に比して、ペプチドＢ、ＣおよびＧと共に培養されたＡｓＰＣ１細胞においてより有意に抑制されている（図４Ａ参照）。また、ペプチドＢ、Ｃ、ＦおよびＧと共に培養されたＳＵＩＴ２細胞のｍＲＮＡ量が、ペプチドＡと共にまたは相互作用ペプチド無しに培養されたＳＵＩＴ２細胞のｍＲＮＡ量と比較して有意に抑制されることを示している（図４Ｂ参照）。この結果は、Ｐｔｃｈ１に対する相互作用ペプチドの癌細胞の生育に対する抑制効果がヘッジホッグシグナル伝達経路の（転写）活性を減衰させることにより誘起されることを強く示唆している。
実施例４
（培養細胞のソニックヘッジホッグ（Ｓｈｈ）の免疫染色）
Ｐｔｃｈ１に対する相互作用ペプチドは、小さ過ぎて抗体により染色されないため、相互作用ペプチドのＰｔｃｈ１タンパクに対する特異性を、ペプチドが競合的にＰｔｃｈ１とＳｈｈとの間の相互作用を阻害する効果により確証した。ＡｓＰＣ１細胞（２ｘ１０４／ｗｅｌｌ）は、２４ーウェルプレートのカバーグラス（ＡＧＣテクノグラス株式会社製）の上で実施例２の細胞培養条件に従い一夜培養された。次にその培地は、Ｐｔｃｈ１に対する１０μｇ／ｍｌの相互作用ペプチド（ペプチドＡ、ペプチドＢ、ペプチドＣ，ペプチドＧ）を含む新しい培地にかえられた。２４時間培養後、スライドは、空気乾燥され８％フォルムアルデヒドに３０分間浸漬された。一次抗体は、４℃で一夜浸漬された。使用された一次抗体は、１：２５０の濃度のＳｈｈ（Ｎ−１９；Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，Ｓａｎｔａ Ｃｒｕｚ，ＣＡ，ＵＳＡ社製）であった。二次抗体（ウサギ抗ヤギ免疫グロプリン；株式会社ニチレイ製）は室温下で１時間反応した。ベクタシールド マウンティング メディウム［Ｖｅｃｔａｓｈｉｅｌｄ ｍｏｕｎｔｉｎｇ ｍｅｄｉｕｍ、ベクターラボ（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｂｕｒｌｉｎｇａｍｅ，Ｃａｌｉｆｏｒｎｉａ，ＵＳＡ）社製］に載せた後、サンプルは、レーザー走査型共焦点蛍光顕微鏡（ＬＳＭ−ＧＢ２００ Ｓｙｓｔｅｍ；オリンパス光学工業株式会社製）下で視覚化した。
結果を図５に示す。図５の上部パネルは、Ｐａｔｃｈｅｄ−１（Ｐｔｃｈ１）に対する相互作用ペプチド（ペプチドＢ、ＣおよびＧ）を伴うＡｓＰＣ１細胞の周囲にあるＳｈｈの染色レベルは、ペプチドＡ（対照ペプチド）を伴う細胞の染色レベルより低いことを示している。これは、ペプチドＢ、ＣおよびＧがＳｈｈとの所定の結合部位において特異的にＰｔｃｈ１と相互作用をしていることを支持している。なお、下部のパネルは、ペプチドＡ、ＢおよびＧ（左から右へ）の位相差による画像（位相）であり、細胞の外形が示されている。ペプチドＡに比較して、ペプチドＢ、Ｃ、Ｄを加えた細胞では、位相差画像で認められる細胞外形に従った染色像が認められない。
実施例５
（動物モデルおよびＡｓＰＣ１の臀部皮下移植
動物実験のために遵守する手順は、九州大学の実験動物の管理と使用に関するガイドライおよび文部科学省の研究機関等における動物実験等の実施に関する基本指針に従った。４〜６週令の雌のＮＯＤ―ＳＣＩＤ（ＮＯＤ／ｓｅｖｅｒｅ ｃｏｍｂｉｎｅｄ ｉｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙ）マウスは、日本エスエルシー株式会社より購入した。マウスは、九州大学で承認された施設の特定病原体除去の状態下にある水平層流キャビネットに収容された。ＡｓＰＣ１またはＳＵＩＴ２細胞は、無血清のＲＰＭＩ／Ｍａｔｒｉｇｅｌ（日本ベクトン・ディッキンソン株式会社製）混合物（１：１容量、合計１×１０５ｃｅｌｌｓ／０．４ｍｌ）に懸濁し、次にマウスの臀部に２７番径の針を用いて注入した。腫瘍形成後、２００μｇの相互作用ペプチド（ＡｓＰＣ１細胞については、ペプチドＡ、ペプチドＣ、ＳＵＩＴ２細胞については、ペプチドＡ，ペプチドＧ）が腫瘍部位に２７番径の針を用いて、一日１回で５日間注入された。腫瘍形成、腫瘍サイズおよびマウスの体重は、２日置きに４６日間測定された（Ａｋｉｙｏｓｈｉ Ｔ，Ｎａｋａｍｕｒａ Ｍ，Ｙａｎａｉ Ｋ，Ｎａｇａｉ Ｓ，Ｗａｄａ ｊ，Ｋｏｇａ Ｋ，ｅｔ ａｌ．Ｇａｍｍａ−ｓｅｃｒｅｔａｓｅ ｉｎｈｉｂｉｔｏｒｓ ｅｎｈａｎｃｅ ｔａｘａｎｅ−ｉｎｄｕｃｅｄ ｍｉｔｏｔｉｃ ａｒｒｅｓｔ ａｎｄ ａｐｏｐｔｏｓｉｓ ｉｎ ｃｏｌｏｎ ｃａｎｃｅｒ ｃｅｌｌｓ．Ｇａｓｔｒｏｅｎｔｅｒｏｌｏｇｙ．２００８；１３４：１３１−４４．に記載の方法に順じて行なった）。
結果を図６Ａ、図６Ｂに示す。図６Ａ、図６Ｂ共に、ペプチドＡと比較してペプチドＣがＡｓＰＣ１細胞の異種移植による膵臓腫瘍の生体内での生育を抑制する傾向を示している。
（ＲＴ−ＰＣＲを用いたＧｌｉ１のｍＲＮＡの発現量の定量）
前記臀部皮下移植後の最終日（４６日目）に腫瘍を切除し回収した当該腫瘍組織を用い実施例３に記載のリアルタイムＰＣＲにより、そのＧｌｉ１のｍＲＮＡの発現量の定量を行なった。
結果を図６Ｃに示す。図６Ｃから明らかなように、ペプチドＣが注入されたＡｓＰＣ１腫瘍細胞のＧｌｉ１発現量は、ペプチドＡが注入されたＡｓＰＣ１腫瘍細胞に比して有意に減少した。また、図６Ｄに示すように、ペプチドＧが注入されたＳＵＩＴ２腫瘍細胞のＧｌｉ１発現量は、ペプチドＡが注入された腫瘍細胞に比して有意に減少している。この結果は、Ｐｔｃｈ１に対する相互作用ペプチド（ペプチドＣおよびペプチドＧ）の癌細胞の生育に対する抑制効果がヘッジホッグシグナル伝達経路の（転写）活性を減衰させることにより誘起されることを強く示唆している。
（統計解析）
スチューデントｔ検定が統計解析に用いられた。全ての計算は、スタットビュー５．０Ｊソフトウェア（ＳｔａｔＶｉｅｗ ５．０Ｊ ｓｏｆｔｗａｒｅ、Ａｂａｃｕｓ Ｃｏｎｃｅｐｔｓ，Ｂｅｒｋｅｌｅｙ，ＣＡ，ＵＳＡ）を用いて行なわれた。０．０５未満のＰ値は有意と看做された（Ｋａｍｅｄａ Ｃ，Ｎａｋａｍｕｒａ Ｍ，Ｔａｎａｋａ Ｈ，Ｙａｍａｓａｋｉ Ａ，Ｋｕｂｏ Ｍ，Ｔａｎａｋａ Ｍ，ｅｔ ａｌ．Ｏｅｓｔｒｏｇｅｎ ｒｅｃｅｐｔｏｒ−ａｌｐｈａ ｃｏｎｔｒｉｂｕｔｅｓ ｔｏ ｔｈｅ ｒｅｇｕｌａｔｉｏｎ ｏｆ ｔｈｅ ｈｅｄｇｅｈｏｇ ｓｉｇｎａｌｌｉｎｇ ｐａｔｈｗａｙ ｉｎ ＥＲ ａｌｐｈａｐｏｓｉｔｉｖｅ ｇａｓｔｒｉｃ ｃａｎｃｅｒ．Ｂｒ Ｊ Ｃａｎｃｅｒ．２０１０；１０２：７３８−４７．を参照）。In order to confirm that the biological effect of the peptide on the growth of cancer cells was caused by the attenuation of the (transcriptional) activity of the hedgehog signaling pathway, interacting peptides (peptides A, B, The results of studying the influence of C, D, F, G, and H) are shown in Examples. EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
Example 1
(Design and synthesis of interacting peptides for Ptch1)
The interaction peptide for the target sequence of Ptch1 employs a genetic algorithm for designing 5000 kinds of peptide sequences that are expected to have high affinity for the target by randomly changing amino acids, and for each designed peptide sequence, Software called MIMETIC program in which scores are assigned based on several physicochemical parameters including optimization of complementation of hydrophobic hydrophilicity index, optimization of average structural similarity, minimization of side chain interference and backbone sequence (Campbell W, Kleiman L, Barany L, Li Z, Khorchid A, Fujita E, et al. A novel genetic algorithm for designing peptides interstituting. e function of a target molecule.Microbiol Immunol.2002; 46:. 211-5 and Baranyi L, Campbell W, Ohshima K, Fujimoto S, Boros M, Okada H.The antisense homology box: a new motif within proteins that encodes biologically active peptides.Nat Med.1995; 1: 894-901.) and the high score 7 peptides shown in Table 1 below were selected. These seven peptides were synthesized by the solid phase synthesis method (Gerald F. Sigler, Anne K. Soutar, Louis C. Smith, Antonio M. Gotto, Jr., James T. Sparrow. The solid phase of the synthetic phase. -Manufactured by cholesterol acyltransferase corresponding to human plasma apo C-I. Proc. Natl. Acad. Sci. USA, Vol. 73, No. 5, pp. 1422-1426, May 1976, Biochemistry.
(Proliferation assay of human pancreatic cancer cell lines (AsPC1 and SUIT2))
Proliferation assays of the human pancreatic cancer cell lines AsPC1 (obtained from American Type Culture Collection, Rockville, Med., USA) and SUIT2 (obtained from JCRB cell bank) were performed spectrophotometrically for cultured cells under the following cell culture conditions: Yellow 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehydrogenase of metabolically active cells against measurable blue formazan (Sigma Chemical Co. St. Louis, MO) MTT assay based on reduction of cells and cell counting (Kubo M, Nakamura M, Tasaki A, Beckman Coulter, Fullerton, CA, USA) Yamanaka N, Nakashima H, Nomura M, et al., Hedgehog signaling pathway is a new therapeutic target for patent with breast.
(Cell culture conditions)
Human pancreatic cancer cell lines AsPC1 and SUIT2 were prepared from RPMI 1640 complete medium (Life Technologies) supplemented with fetal bovine serum (FBS; Life Technologies) and antibiotics (100 units / ml penicillin and 100 μg / ml streptomycin, manufactured by Meiji Seika Co., Ltd.). , Grand Island, NY, USA) at 37 ° C. under humidified carbon dioxide 5% and air 95% (Nakamura M, Masuda H, Horii J, Kuma K, Yokoyama N, Ohba T, et al. When overexpressed, a novel centrosomal protein, RanBPM, casuals electrical microtubule nucleation similar to gamma-tubulin.J Cell Biol.1998; 143: 1041-52. and Kameda C, Tanaka H, Yamazaki A, Nakamura M, Koga K, Sato N, et al. The Hedgehep. with estrogen receptor-negative breast cancer. Anticancer Res. 2009; 29: 871-9.).
(Proliferation assay by MTT assay)
Human pancreatic cancer cell lines AsPC1 and SUIT2 were inoculated into 96-well plates at a concentration of 3 × 10 3 cells / well and cultured overnight according to the cell culture conditions described above, then 1.0 μg / ml and 10.0 μg / ml, respectively. The medium was changed to a new medium containing 7 kinds of interacting peptides (peptides A, B, C, D, F, G, H) and cultured for 72 hours. Cells were then harvested using trypsin and viable cells were subjected to MTT assay. As a normal control (NC), buffer alone (RPMI + 5% FCS) was used, and each interacting peptide was dissolved in the buffer (RPMI + 5% FCS) and used (the same applies hereinafter).
The results are shown in FIG. 1 and FIG. Peptides B, C and G both inhibited and significantly inhibited the growth of human pancreatic cancer cell line AsPC1 cells in a dose-dependent manner at concentrations of 1.0 μg / ml and 10.0 μg / ml, respectively. The suppression effect was shown (refer FIG. 1). In addition, peptides B, C, F and G each inhibited the growth of human pancreatic cancer cell line SUIT2 cells in a dose-dependent manner at a concentration of 1.0 μg / ml and a concentration of 10.0 μg / ml, respectively. The tendency and the significant inhibitory effect were shown (refer FIG. 2). Although there was an exception for peptide F, it was similar to that for AsPC1. Peptides A, D and H did not show an inhibitory effect on the growth of human pancreatic cancer cell lines AsPC1 and SUIT2 cells (see FIGS. 1 and 2).
(Proliferation assay by cell counting with Coulter counter)
In order to further confirm the biological effect of the interacting peptide detected by MTT assay, the following Coulter measurements were performed on human pancreatic cancer cell line AsPC1 cells and SUIT2 cells cultured with interacting peptides. Human pancreatic cancer cell lines AsPC1 and SUIT2 were inoculated into a 24-well plate at a concentration of 1 × 10 4 cells / well, cultured overnight according to the above cell culture conditions, and then each of the 7 interacting peptides (10 μg / ml each) ( The medium was changed to a fresh medium containing peptides A, B, C, D, F, G, and H) and cultured for 48 hours and 72 hours. Thereafter, cells were collected using trypsin, and viable cells were subjected to cell count using a Coulter counter.
The results are shown in FIGS. 3A and 3B. Similar results were obtained consistent with the results of the MTT assay. That is, peptides B, C, and G showed a tendency to suppress and a significant inhibitory effect on the proliferation of human pancreatic cancer cell line AsPC1 cells, respectively, at a culture time of 48 hours and 72 hours, respectively (FIG. 3A). . Peptides B, C, F and G both showed a tendency to suppress and a significant inhibitory effect on the growth of human pancreatic cancer cell line SUIT2 cells at 48 hours and 72 hours of culture, respectively (FIG. 3B). . Peptide F was only effective for SUIT2.
Peptides A, D and H did not show an inhibitory effect on the growth of human pancreatic cancer cell lines AsPC1 and SUIT2 cells (see FIGS. 1 and 2).
Example 3
(Effect of Ptch1-interacting peptide on Gli1 mRNA expression level)
Since Gli1 is not only a transcription factor of the hedgehog signaling pathway but also a target gene of the hedgehog pathway, the expression level of Gli1 mRNA was used as a marker for hedgehog pathway activity. Three peptides confirmed to inhibit AsPC1 growth in Example 2, namely peptides B, C and G, were cultured with AsPC1 for 72 hours, and peptide A was also incubated with AsPC1 for the same time as a negative control. . After culture, mRNA was prepared from cultured cells and subjected to quantitative analysis by real-time RT-PCR described below. In addition, the four interacting peptides that were confirmed to suppress the proliferation of SUIT2 in Example 2, ie, peptides B, C, F, and G, also against the proliferation of SUIT2 as in the case of AsPC1. Since they showed an inhibitory effect, these four interacting peptides were also cultured with SUIT2 for 72 hours, and peptide A was also cultured with SUIT2 for the same time as a negative control. After culture, mRNA was prepared from cultured cells and subjected to quantitative analysis by real-time RT-PCR described below.
(Quantification of expression level of Gli1 mRNA using RT-PCR)
The expression level of Gli1 mRNA was quantified using real-time reverse transcription polymerase chain reaction (RT-PCR) as follows. That is, total RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA, USA) and quantified by spectrophotometry (Ultratroc 2100 Pro; Amersham Pharmacia Biotech, Cambridge, UK). RNA (700 ng) was treated with DNase and reverse transcribed into cDNA according to the protocol using Quantitative Reverse Transcription Kit (Qiagen). The reaction was carried out using DNA Engineer Opticon 2 System (MJ Research, Waltham, MA, USA) using SYBR Premix ExTaq (manufactured by Takara Bio Inc.). pGli1-GFP (green fluorescent protein) was serially diluted 10-fold and amplified using primer pairs to create a standard curve for Gli1. Each sample was performed in triplicate. All primer sets amplified a 200 base pair long fragment. The primer sequences used are as follows. beta-actin, forward direction, 50-TTG CCG ACA GGA TGC AGA AGG A-30, reverse direction, 50-AGG TGG ACA GCG AGG CCA GGA T-30 and Gli1, forward direction, 50-GGT TCA AGA GCC TG T-30, reverse direction, 50-GGC AGC ATT CTC AGT GAT GCT G-30. The amount of gene in a sample was normalized to the amount of β-actin in the sample (Kameda C, Nakamura M, Tanaka H, Yamazaki A, Kubo M, Tanaka M, et al. Oestrogen receptor-alpha. (see contributes to the regulation of the hedgehog signaling pathway in ER alphapositive gastric cancer. Br J Cancer. 2010; 102: 738-47.).
The results are shown in FIGS. 4A and 4B. The relative mRNA level of Gli1 is more significantly suppressed in AsPC1 cells cultured with peptides B, C and G compared to AsPC1 cells cultured with peptide A or without interacting peptide (see NC) (See FIG. 4A). It is also shown that the amount of mRNA in SUIT2 cells cultured with peptides B, C, F and G is significantly suppressed compared to the amount of mRNA in SUIT2 cells cultured with peptide A or without interacting peptide. (See FIG. 4B). This result strongly suggests that the inhibitory effect on the growth of cancer cells of the peptide interacting with Ptch1 is induced by attenuating the (transcription) activity of the hedgehog signaling pathway.
Example 4
(Immunostaining of sonic hedgehog (Shh) of cultured cells)
Since the interacting peptide for Ptch1 was too small to be stained by the antibody, the specificity of the interacting peptide for the Ptch1 protein was confirmed by the effect of the peptide competitively inhibiting the interaction between Ptch1 and Shh. AsPC1 cells (2 × 10 4 / well) were cultured overnight according to the cell culture conditions of Example 2 on a 24-well plate cover glass (manufactured by AGC Techno Glass Co., Ltd.). The medium was then replaced with fresh medium containing 10 μg / ml interacting peptides (Peptide A, Peptide B, Peptide C, Peptide G) to Ptch1. After 24 hours incubation, the slides were air dried and soaked in 8% formaldehyde for 30 minutes. The primary antibody was soaked overnight at 4 ° C. The primary antibody used was Shh at a concentration of 1: 250 (N-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibody (rabbit anti-goat immune globulin; manufactured by Nichirei Co., Ltd.) reacted for 1 hour at room temperature. After mounting on a vector shield mounting medium (Vectashield mounting medium, Vector Laboratories, Burlingame, California, USA), the sample was a laser scanning confocal fluorescence microscope (LSM-GB200 System, Inc .; Olympus Optical Co., Ltd .; Visualization under).
The results are shown in FIG. The upper panel of FIG. 5 shows the level of staining for Shh around AsPC1 cells with interacting peptides (Peptides B, C and G) against Patched-1 (Ptch1). Staining of cells with Peptide A (control peptide) It is lower than the level. This supports that peptides B, C and G interact specifically with Ptch1 at a given binding site with Shh. The lower panel is an image (phase) based on the phase difference of peptides A, B and G (from left to right), and shows the outline of the cells. Compared with peptide A, in the cells to which peptides B, C, and D were added, a stained image according to the cell shape observed in the phase contrast image was not observed.
Example 5
(Animal model and AsPC1 scalp subcutaneous procedure Follow the guidelines for animal experiments at Kyushu University for the management and use of laboratory animals and the basic guidelines for conducting animal experiments at the Ministry of Education, Culture, Sports, Science and Technology) 4-6 weeks old female NOD-SCID (NOD / severe combined immunofidelity) mice were purchased from Japan SLC Co., Ltd. under the condition of specific pathogen removal in a facility approved by Kyushu University. The AsPC1 or SUIT2 cells were housed in a horizontal laminar flow cabinet in a serum-free RPMI / Matrigel (Nippon Becton Dickinson) mixture (1: 1 volume, 1 × 10 5 cells / 0.4 ml total). Suspend, then use a # 27 needle on the mouse buttock After tumor formation, 200 μg of interacting peptide (peptide A, peptide C for AsPC1 cells, peptide A, peptide G for SUIT2 cells) was placed on the tumor site using a 27th diameter needle. Tumor formation, tumor size and mouse body weight were measured every other day for 46 days (Akiyoshi T, Nakamura M, Yanai K, Nagai S, Wadaj, Koga K, et al. Gamma-secretase inhibitors enhance taxane-induced mitotic array and apoptosis in colon cancer cells.Gastroenterology.2008; 134: 131-44. Carried out was).
The results are shown in FIGS. 6A and 6B. 6A and 6B show that peptide C tends to suppress the growth of pancreatic tumors in vivo by AsPC1 cell xenograft as compared to peptide A. FIG.
(Quantification of expression level of Gli1 mRNA using RT-PCR)
The expression level of Gli1 mRNA was quantified by real-time PCR described in Example 3 using the tumor tissue excised and collected on the last day (46th day) after the subcutaneous transplantation of the buttocks.
The result is shown in FIG. 6C. As is clear from FIG. 6C, the expression level of Gli1 in AsPC1 tumor cells injected with peptide C was significantly reduced compared to AsPC1 tumor cells injected with peptide A. In addition, as shown in FIG. 6D, the expression level of Gli1 in SUIT2 tumor cells injected with peptide G is significantly reduced as compared with tumor cells injected with peptide A. This result strongly suggests that the inhibitory effect of Ptch1 interacting peptides (peptide C and peptide G) on the growth of cancer cells is induced by attenuating the (transcription) activity of the hedgehog signaling pathway. .
(Statistical analysis)
Student t test was used for statistical analysis. All calculations were performed using Statview 5.0J software (StatView 5.0J software, Abacus Concepts, Berkeley, CA, USA). P values less than 0.05 were considered significant (Kameda C, Nakamura M, Tanaka H, Yamazaki A, Kubo M, Tanaka M, et al. Oestrogen receptor-alpha conjugation to the tributaries. ER alphapositive gastric cancer.Br J Cancer.2010; 102: 738-47.).
本発明に係る新規ペプチドは、ヒト膵臓癌細胞系（ＡｓＰＣ１およびＳＵＩＴ２）に対して試験管内（ｉｎ ｖｉｔｒｏ）および生体内（ｉｎ ｖｉｖｏ）においてＰａｔｃｈｅｄ−１蛋白質と相互作用することにより、そのヘッジホッグシグナル伝達系の活性を抑制し、膵臓癌細胞の増殖を抑制する傾向を示す。また、該ペプチドは膵臓癌に対し抗癌性を有するペプチド開発のリード化合物としても利用することができる。従って、本発明は、膵臓癌の治療に利用することができ、該産業分野の発展に大きく貢献することができる。
［配列表］
The novel peptide according to the present invention interacts with the Patched-1 protein in vitro and in vivo against human pancreatic cancer cell lines (AsPC1 and SUIT2), thereby producing its hedgehog signal. It shows a tendency to suppress the activity of the transmission system and suppress the proliferation of pancreatic cancer cells. The peptide can also be used as a lead compound for peptide development having anticancer activity against pancreatic cancer. Therefore, the present invention can be used for the treatment of pancreatic cancer and can greatly contribute to the development of the industrial field.
[Sequence Listing]
Claims (4)
An anticancer agent for pancreatic cancer comprising the peptide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt of the peptide.
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| JPN6012046074; 中村雅史， ｅｔ ａｌ．: 'Ｈｅｄｇｅｈｏｇシグナルを標的とした癌治療法の可能性' 福岡医学雑誌 Ｖｏｌ．９９， Ｎｏ．５, 2008, ｐ．１０２-１０６ * |
| JPN6012046076; 中村雅史， ｅｔ ａｌ．: 'Ｈｅｄｇｅｈｏｇシグナルを利用した癌分子標的治療' 癌と化学療法 Ｖｏｌ．３４， Ｎｏ．１２, 2007, ｐ．１９１４-１９１６ * |
| JPN6012046078; ＨＡＨＮ， Ｈ．， ｅｔ ａｌ．: 'Ａ ｍａｍｍａｌｉａｎ ｐａｔｃｈｅｄ ｈｏｍｏｌｏｇ ｉｓ ｅｘｐｒｅｓｓｅｄ ｉｎ ｔａｒｇｅｔ ｔｉｓｓｕｅｓ ｏｆ ｓｏｎｉｃ ｈｅｄｇｅｈｏｇ ａｎｄ ｍａｐｓ ｔｏ ａ ｒｅｇｉｏｎ ａｓ' Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ Ｖｏｌ．２７１， Ｎｏ．２１, 1996, ｐ．１２１２５-１２１２８ * |
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| JPWO2013186934A1 (en) | 2016-02-01 |
| WO2013186934A1 (en) | 2013-12-19 |
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