KR101708032B1 - Pharmaceutical composition for preventing or treating cancer comprising CysA5W peptide as effective component - Google Patents
Pharmaceutical composition for preventing or treating cancer comprising CysA5W peptide as effective component Download PDFInfo
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- KR101708032B1 KR101708032B1 KR1020150040812A KR20150040812A KR101708032B1 KR 101708032 B1 KR101708032 B1 KR 101708032B1 KR 1020150040812 A KR1020150040812 A KR 1020150040812A KR 20150040812 A KR20150040812 A KR 20150040812A KR 101708032 B1 KR101708032 B1 KR 101708032B1
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- South Korea
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- cancer
- cysa5w
- peptide
- microrna
- protein
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- C—CHEMISTRY; METALLURGY
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
본 발명은 CysA5W 펩타이드를 유효성분으로 함유하는 암 예방 또는 치료용 약학 조성물, 상기 암 예방 또는 치료용 약학 조성물을 인간을 제외한 포유동물 내에 투여하는 단계를 포함하는, 암세포에 대한 세포사멸 증진 방법 및 CysA5W 펩타이드를 유효성분으로 함유하는 항암보조제용 건강기능식품을 제공한다.The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising CysA5W peptide as an active ingredient, a method for promoting apoptosis of cancer cells and a method for promoting apoptosis of CysA5W The present invention provides a health functional food for anti-cancer adjuvants containing a peptide as an active ingredient.
Description
본 발명은 CysA5W 펩타이드를 유효성분으로 함유하는 암 예방 또는 치료용 약학 조성물에 관한 것으로, 더욱 상세하게는 CysA5W 펩타이드를 유효성분으로 함유하는 암 예방 또는 치료용 약학 조성물, 상기 암 예방 또는 치료용 약학 조성물을 인간을 제외한 포유동물 내에 투여하는 단계를 포함하는, 암세포에 대한 세포사멸 증진 방법 및 CysA5W 펩타이드를 유효성분으로 함유하는 항암보조제용 건강기능식품에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising CysA5W peptide as an active ingredient, and more particularly, to a pharmaceutical composition for preventing or treating cancer comprising CysA5W peptide as an active ingredient, a pharmaceutical composition for preventing or treating cancer A method for promoting apoptosis of cancer cells, and a health functional food for anticancer adjuvants containing CysA5W peptide as an active ingredient.
최근 펩타이드, 단백질, 핵산 등의 고분자물질을 포함하는 바이오 의약품의 중요성이 점점 부각되고 있다. 그 중 펩타이드는 아미노산의 펩타이드 결합(Peptide bond)으로 연결된 생합성 물질로, 일반 화학약품에 비해 생체 내에서 독성이 낮고, 높은 안정성을 보인다. 일반적으로 펩타이드는 100개의 아미노산 이하로 이루어진 것으로 비교적 작은 크기와 생체막을 잘 통과한다는 장점이 있어, 약품전달물질로 많이 이용되고 있다. 또한 간이나 신장 같은 조직에 축적되지 않기 때문에 면역 반응이 일어나지 않아 부작용이 적은 의약품으로써 펩타이드 신약개발이 활발히 진행되고 있다. 저분자 물질에 비해 큰 분자량을 가지고 있어, 다른 생체 분자들을 특이적으로 강력하게 조절할 수 있다. 진핵 생물체에서 자신의 신체 기능 조절을 위해 여러 종류의 생리활성을 가진 펩타이드를 분비한다. 이를 이용하여 인유두종 바이러스(Human papillomavirus; HPV)의 치료를 위한 펩타이드 백신의 연구 및 개발이 진행 중에 있다. 그 밖에 여러 질병을 치료할 의약품으로써의 역할로 펩타이드가 각광받고 있다. 이러한 펩타이드 중에 알파 나선(α-helix) 구조를 가지는 짧은 길이의 아미노산으로 이루어진 펩타이드가 스템-루프(Stem-loop) 이차 구조로 이루어진 RNA에 특이적으로 작용할 수 있다는 연구 결과들이 있다. 특히, 스템-루프(Stem-loop)로 이루어진 microRNA에 선택적으로 작용할 수 있다고 알려졌다.Recently, the importance of biopharmaceuticals including high molecular substances such as peptides, proteins and nucleic acids has been increasingly emphasized. Among them, peptides are biosynthetic substances that are linked to peptide bonds of amino acids. They are less toxic in vivo than normal chemicals and show high stability. In general, peptides are composed of less than 100 amino acids, and they are used as drug delivery materials because they have a relatively small size and good ability to pass through biological membranes. In addition, the development of peptide drugs is actively underway as a drug that does not accumulate in tissues such as the liver or kidney, and thus has no side effects due to no immune response. Has a larger molecular weight than a low molecular substance, and can specifically and strongly regulate other biomolecules. In eukaryotes, various kinds of physiologically active peptides are secreted to regulate their body functions. Research and development of peptide vaccines for the treatment of human papillomavirus (HPV) are under way. Peptides are in the spotlight as a drug to treat a variety of other diseases. Among such peptides, there are researches that peptides composed of short amino acids having an alpha-helix structure can specifically act on RNA composed of a stem-loop secondary structure. In particular, it is known that it can selectively act on a microRNA composed of a stem-loop.
MicroRNA는 일반적으로 18~25개 정도의 뉴클레오타이드(nucleotide)로 이루어지며 생체 내에 존재하여 유전자 발현을 전사 후 수준에서 조절하는 기능을 가진 단일 염기가닥의 작은 RNA 조각을 일컫는다. MicroRNA가 생성되는 과정은 먼저 microRNA 유전자에서 RNA 중합효소 II에 의해 전사되어 primary-miRNA을 생성한 후, RNase III 타입인 Drosha 라는 효소와 그 파트너 단백질들에 의해 약 70 nt 길이의 전구체-miRNA(precursor-miRNA)가 생성된다. Exportin-5라는 단백질에 의해 핵 밖으로 나온 전구체-miRNA는 RNase III 타입인 Dicer에 의해 microRNA duplex가 생성된다. 이 두 가닥의 microRNA duplex는 ARGONAUT 단백질을 포함하는 RNA-induced silencing complex(RISC)에 결합하고 이 중 한 가닥만이 선택되어 표적 유전자의 상보적인 부위에 결합하여 mRNA를 자르거나(mRNA cleavage), 번역 저해(translational inhibition)를 일으켜 표적 유전자의 발현을 억제한다. 수많은 microRNA들이 진핵생물체에 존재하고 있으며, 발생에서의 유전자 발현을 조절할 뿐만 아니라 암, 백혈병 등을 비롯한 여러 질병에서도 microRNA의 중요한 역할이 밝혀지고 있다. 특히, microRNA-29가 종양 억제 유전자(tumor suppressor gene)인 p53을 조절할 수 있다고 알려져 있다. MicroRNAs generally consist of about 18 to 25 nucleotides and are small RNA fragments of single stranded strand that are present in vivo and function to regulate gene expression at post-transcriptional level. The microRNA is firstly transcribed from the microRNA gene by RNA polymerase II to generate primary-miRNA, which is then transformed into an RNase III-type Drosha enzyme and its partner proteins to form a precursor-miRNA -mRNA) is generated. The precursor-miRNA exiting the nucleus by a protein called Exportin-5 is produced by Dicer of RNase III type. These two strands of microRNA duplex bind to the RNA-induced silencing complex (RISC) containing the ARGONAUT protein and only one of them is selected to mRNA cleavage by binding to the complementary site of the target gene (mRNA cleavage) Inhibit the expression of target genes by causing translational inhibition. Numerous microRNAs are present in eukaryotes, not only regulating gene expression in development, but also of microRNAs in many diseases including cancer, leukemia, and the like. In particular, microRNA-29 is known to regulate p53, a tumor suppressor gene.
암 발생에 중요한 열쇠가 될 수 있는 종양 억제 단백질(tumor suppressor protein) 중 하나인 p53은 세포의 생존 여부를 결정하는 감시인(guardian)의 역할을 한다. DNA 손상, 산화적 스트레스 같은 여러 타입의 스트레스에 빠르게 반응하여 세포주기 정지(cell-cycle arrest) 또는 세포사멸(apoptosis)을 일으킨다. 이러한 기능들이 망가지게 되면 비정상적인 세포 증식을 막지 못하여 암 발생이 일어나게 된다. 실제 연구보고들에 의하면, 암 발생의 절반 이상이 p53 단백질의 이상으로 생긴다. 그렇기 때문에, 암 연구에 있어서 p53은 매우 중요한 단백질로 항암 신약 개발에 가장 중요한 표적 단백질이라 할 수 있다. P53, one of the tumor suppressor proteins that can be a key to cancer development, acts as a guardian to determine cell viability. DNA damage, and oxidative stress, leading to cell-cycle arrest or apoptosis. When these functions are broken, abnormal cell proliferation can not be prevented and cancer development occurs. According to actual research reports, more than half of the cancer cases arise from p53 protein abnormalities. Therefore, p53 is a very important protein in cancer research, and it is the most important target protein for the development of anti-cancer drugs.
지금까지 p53 단백질을 이용한 의약품들이 많이 개발되어 왔고, 대부분 작은 분자(small molecule)로 이루어진 화학약품이다. 앞서 언급했듯이, 기존에 사용되고 있는 의약품들은 독성뿐만 아니라 표적에 대한 특이성이 떨어지므로 부작용들이 많다. Up to now, many p53 protein medicines have been developed and are mostly small molecules. As mentioned earlier, existing drugs have many side effects because they are not only toxic but also specific for the target.
한편, 한국공개특허 제1998-0702283호에서는 'ｐ53 기능을 활성화시키는 사람 ｐ53과 유사한 구조를 갖는 펩티드 및 펩티도미메틱'이 개시되어 있고, 한국공개특허 제2010-0064516호에서는 '마이크로ＲＮＡ를 유효성분으로 포함하는 항암제 및 그 제조방법'이 개시되어 있으나, 본 발명에서와 같이 CysA5W 펩타이드를 유효성분으로 함유하는 암 예방 또는 치료용 약학 조성물에 관해서는 밝혀진 바가 전혀 없다.Korean Patent Laid-Open Publication No. 1998-0702283 discloses a peptide having a structure similar to that of human p53 that activates p53 function and peptidomimetic, and in Korean Patent Publication No. 2010-0064516, Discloses an anticancer agent and a method for producing the same. However, as described in the present invention, a pharmaceutical composition for preventing or treating cancer containing CysA5W peptide as an active ingredient has never been disclosed.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명에서는 81개의 무작위 펩타이드들 중 한 개의 펩타이드가 microRNA-29 패밀리 중 microRNA-29b의 Dicer 작용을 좀 더 효율적으로 만들어 microRNA-29b 전구체에 특이적으로 작용하여 성숙된 microRNA-29b 생성을 증가시키고, 종양 억제 단백질 p53의 안정성을 더 증진시켜 세포사멸을 더 유발한다는 것을 확인하였다. 이를 통해, 본 발명은 생체 독성이 낮고 세포침투가 용이하며 표적에 대한 특이성이 높은 펩타이드를 기반으로 하여 항암 신약 개발에 새로운 접근방식을 제공할 수 있을 것으로 기대함으로써, 본 발명을 완성하였다.The present invention has been made in view of the above-mentioned needs. In the present invention, one of the 81 random peptides makes the microRNA-29b dicer action of the microRNA-29 family more efficient, And increased mature microRNA-29b production and further enhanced the stability of tumor suppressor protein p53, leading to further apoptosis. Accordingly, the present invention has been accomplished based on the expectation that the present invention can provide a novel approach to the development of anti-cancer drugs based on peptides having low bio-toxicity, easy penetration into cells, and high specificity to targets.
상기 과제를 해결하기 위해, 본 발명은 서열번호 1의 아미노산 서열로 이루어진 CysA5W 펩타이드를 제공한다.In order to solve the above problems, the present invention provides a CysA5W peptide consisting of the amino acid sequence of SEQ ID NO: 1.
또한, 본 발명은 CysA5W 펩타이드를 유효성분으로 함유하는 암 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising CysA5W peptide as an active ingredient.
또한, 본 발명은 상기 암 예방 또는 치료용 약학 조성물을 인간을 제외한 포유동물 내에 투여하는 단계를 포함하는, 암세포에 대한 세포사멸 증진 방법을 제공한다.In addition, the present invention provides a method for promoting apoptosis in cancer cells, comprising the step of administering the pharmaceutical composition for cancer prevention or treatment to a mammal other than a human.
또한, 본 발명은 CysA5W 펩타이드를 유효성분으로 함유하는 항암보조제용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for anticancer drugs containing CysA5W peptide as an active ingredient.
본 발명은 기존에 항암 작용이 있는 의약품과는 달리 특정 펩타이드가microRNA-29b 전구체에 특이적으로 작용하여 그 생성을 증가시키고, 그렇게 생성된 성숙된 microRNA-29b가 종양 억제 단백질인 p53 단백질의 안정성을 증진시켜 세포사멸을 일으킨다는 것을 새롭게 확인함으로써, 생체 독성이 낮고 세포침투가 용이하며 표적에 대한 특이성이 높은 펩타이드를 기반으로 한 새로운 항암 치료제를 제공할 수 있어, 획기적인 발명으로 기대된다.The present invention is based on the finding that a specific peptide acts specifically on the precursor of microRNA-29b to increase its production, unlike conventional drugs having anticancer activity, and the mature microRNA-29b thus produced produces the stability of the tumor suppressor protein p53 protein The present invention is expected to be a breakthrough invention because it is possible to provide a novel anticancer drug based on a peptide having low bio-toxicity, easy cell penetration and high specificity to a target.
도 1은 81개의 무작위 펩타이드에서 p53 활성으로 인한 세포사멸을 증가시키는 펩타이드를 선별하기 위해 자궁경부암 세포인 HeLa 세포의 생존율을 실험한 결과를 나타낸다. 물을 처리한(mock) 대조구 샘플을 100%로 기준하여 각각의 생존율을 계산하였다. 그 값들을 내림차순으로 정렬한 뒤, 별(*)표로 표시한 9개의 펩타이드를 우선 선별하였다.
도 2는 선별된 9개 펩타이드 중 microRNA-29b와 p53 활성을 증가시키는 펩타이드를 선별하였고, 그것의 아미노산 서열(C)을 나타낸다. (A) 그래프는 Taqman microRNA 분석을 통해 microRNA-29b 양을 측정한 것이고, (B) 그래프는 루시퍼라제 분석(luciferase assay)을 통해 p53 활성을 측정한 것이다.
도 3은 microRNA 생성인자인 Drosha와 Dicer 활성 테스트를 시험관 분석(in vitro processing assay)을 통해, 선별된 CysA5W 펩타이드의 작용을 확인한 결과이다.(A-C) 젤 사진은 Dicer 활성을 나타낸 것이고, (D)는 Drosha 활성을 나타낸 것이다. CysA5W 펩타이드 뿐만 아니라 대조구로써 다른 펩타이드(13Aad-14W, HelixA)도 함께 비교하였고, 처리한 농도와 반응 시간은 도면에 나타내었다. 젤 사진 아래에 나타낸 값은 전체 전구체 microRNA에서 생성된 microRNA의 밴드 강도(band intensity) 비율을 나타낸 것이다. (D)의 아래에 나타낸 값은 전체 primary microRNA에서 생성된 전구체 microRNA 비율을 나타낸 것이다. 각각의 값들은 각 실험에서 실시한 mock control을 1로 기준으로 하여 표준화한(normalized) 것이다.
도 4는 CysA5W 펩타이드가 전구체 microRNA-29b와 Dicer 복합체 형성에 어떠한 작용을 하는지 확인한 실험 결과이다. (A)는 microRNA-29a 전구체와 microRNA-29b 전구체의 이차구조를 나타낸 것이다. 파란색으로 표시된 서열이 passenger strand이며, 빨간색으로 표시된 부분이 guide strand이다. (B)는 젤 쉬프트 분석(gel shift assay) 결과이다. 실험에 이용된 재조합 Dicer의 양은 30 nM이며, 펩타이드 농도는 도 3에서 처리했던 농도와 동일하고, 반응 시간은 20분이다. 젤 사진 아래에 나타낸 값은 전체 전구체 RNA에서 Dicer와 복합체를 이룬 RNA 강도(intensity)의 비율을 나타낸 것이다. 각각의 값들은 CysA5W 펩타이드를 처리하지 않고 Dicer만 존재하였을 때의 값을 1로 각각 기준으로 하여 표준화한 것이다.
도 5는 선별된 CysA5W 펩타이드가 다른 펩타이드들(13Aad-14W, HelixA)과는 달리 p53 단백질을 안정성을 증진시켜 암세포의 세포사멸이 증가한 것을 나타낸 결과이다. (A)는 단백질 생합성을 억제시키는 사이클로헥시마이드(Cycloheximide; CHX)을 처리하여 웨스턴 블럿 분석(western blot analysis)으로 p53 단백질 수준을 확인하였다. GAPDH 단백질은 로딩 대조구(loading control)로써 사용되었다. (B)는 프로피디움 요오드화물(propidium iodide)을 이용한 세포사멸 집단(apoptotic cell population)을 측정할 수 있는 유세포 분류기(Fluorescence-activated cell sorting; FACS)의 분석 결과이다. 50 μM 에토포시드(Etoposide)는 p53에 의한 세포사멸이 잘 일어나는 양성 대조구로써 이용되었다.
도 6은 CysA5W 펩타이드가 다른 암세포인 MCF7 유방암세포에서도 microRNA-29b와 p53 단백질 안정성을 증진시켜 세포사멸이 유발되는 것을 나타낸 결과이다. (A) 그래프는 Taqman microRNA 분석으로 microRNA-29b의 상대 정량을 나타낸 것이고, (B)는 웨스턴 블럿 분석을 통해 p53 단백질 안정성을 확인한 것이다. GAPDH 단백질은 로딩 대조구(loading control)로써 사용되었다. (C)는 프로피디움 요오드화물을 이용한 세포사멸 집단(apoptotic cell population)을 측정할 수 있는 유세포 분리기(FACS)의 분석 결과이다. 50 μM 에토포시드(Etoposide)는 p53에 의한 세포사멸이 잘 일어나는 양성 대조구로써 이용되었다.
도 7은 CysA5W 펩타이드가 p53 활성을 증가시키기 위해 반드시 microRNA-29b 생성을 통해서만 증진시킬 수 있음을 증명한 실험 결과이다. microRNA-29b의 안티센스 RNA에 2'O-메틸화시킨 RNA(항-miR29b)를 제작하여, HeLa와 MCF7 세포에 각각 처리하여 내생의 miR29b(endogenous miR29b)의 수준을 억제시킨 후, CysA5W 펩타이드로 인해 p53 단백질이 안정화되는지 웨스턴 블럿으로 확인하였다. GAPDH 단백질은 로딩 대조구(loading control)로써 사용되었다.FIG. 1 shows the results of experiments on the survival rate of HeLa cells as cervical cancer cells in order to screen peptides that increase apoptosis due to p53 activity in 81 random peptides. The survival rate of each mock control sample was calculated on the basis of 100%. The values were sorted in descending order, and then the nine peptides indicated by asterisk (*) were first selected.
Fig. 2 shows microRNA-29b and peptides that increase p53 activity among nine selected peptides, and shows the amino acid sequence (C) thereof. (A) graph shows the amount of microRNA-29b measured by Taqman microRNA analysis, and (B) graph shows p53 activity measured by luciferase assay.
Figure 3 shows the result of confirming the action of the selected CysA5W peptide through the in vitro processing assay of Drosha and Dicer activity tests of the microRNA producers. (AC) The gel photograph shows the Dicer activity, (D) Indicates Drosha activity. CysA5W peptides as well as other peptides (13Aad-14W, HelixA) as a control were also compared, and the concentrations treated and the reaction times are shown in the figure. Gel images The values shown below represent the band intensity ratios of the microRNAs generated from the entire precursor microRNA. The values shown below in (D) represent the percentage of precursor microRNAs produced in the entire primary microRNA. Each value is normalized based on the mock control performed in each experiment.
Figure 4 shows the results of experiments to determine how the CysA5W peptide interacts with the precursor microRNA-29b and Dicer complexes. (A) shows the secondary structure of the microRNA-29a precursor and the microRNA-29b precursor. The sequence shown in blue is the passenger strand, and the red portion is the guide strand. (B) is a result of gel shift assay. The amount of recombinant Dicer used in the experiment is 30 nM, the peptide concentration is the same as the concentration treated in FIG. 3, and the reaction time is 20 minutes. Gel image The values shown below represent the ratio of the intensity of RNA complexes with Dicer in total precursor RNA. Each value was normalized based on the value obtained when only the Dicer was present without treating the CysA5W peptide.
FIG. 5 shows that the selected CysA5W peptide increased the stability of the p53 protein, unlike the other peptides (13Aad-14W, HelixA), and increased cell death of cancer cells. (A) was treated with cycloheximide (CHX), which inhibits protein biosynthesis, and Western blot analysis confirmed p53 protein levels. The GAPDH protein was used as a loading control. (B) is the result of analysis of fluorescence-activated cell sorting (FACS) which can measure the apoptotic cell population using propidium iodide. 50 μM Etoposide was used as a positive control for apoptosis by p53.
FIG. 6 shows that CysA5W peptide promotes apoptosis by enhancing microRNA-29b and p53 protein stability in MCF7 breast cancer cells, which are different cancer cells. (A) graph shows the relative quantitation of microRNA-29b by Taqman microRNA analysis, and (B) the p53 protein stability by Western blot analysis. The GAPDH protein was used as a loading control. (C) is the result of the flow cytometer separator (FACS) which can measure the apoptotic cell population using propidium iodide. 50 μM Etoposide was used as a positive control for apoptosis by p53.
Figure 7 shows the results of experiments demonstrating that the CysA5W peptide can be promoted only through microRNA-29b production to increase p53 activity. (anti-miR29b) was prepared by 2'O-methylation on antisense RNA of microRNA-29b, treated with HeLa and MCF7 cells, respectively, to inhibit the level of endogenous miR29b (endogenous miR29b) It was confirmed by Western blot that the protein was stabilized. The GAPDH protein was used as a loading control.
본 발명의 목적을 달성하기 위하여, 본 발명은 서열번호 1의 아미노산 서열로 이루어진 CysA5W 펩타이드를 제공한다.In order to achieve the object of the present invention, the present invention provides a CysA5W peptide consisting of the amino acid sequence of SEQ ID NO: 1.
본 발명에서 용어, "펩타이드(peptide)"란 아미드 결합(또는 펩타이드 결합)으로 연결된 아미노산으로 이루어진 폴리머를 의미한다. 본 발명의 목적상, microRNA-29b 생성을 증가시키고, 종양 억제 단백질 p53의 안정성을 증진시키는 펩타이드를 의미한다.In the present invention, the term "peptide " means a polymer composed of amino acids linked by an amide bond (or peptide bond). For purposes of the present invention, it is meant peptides that increase microRNA-29b production and enhance the stability of tumor suppressor protein p53.
본 발명에 따른 CysA5W 펩타이드의 범위는 서열번호 1로 표시되는 아미노산 서열을 갖는 펩타이드의 기능적 동등물을 포함한다. "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 1로 표시되는 아미노산 서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더 바람직하게는 90% 이상, 더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 1로 표시되는 펩타이드와 실질적으로 동질의 생리활성을 나타내는 펩타이드를 말한다. "실질적으로 동질의 생리활성"이란 암 예방 또는 치료에 대한 활성을 의미한다.The range of the CysA5W peptide according to the present invention includes functional equivalents of the peptide having the amino acid sequence shown in SEQ ID NO: 1. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 90% or more, more preferably 90% or more, more preferably 90% or more, Refers to a peptide having substantially the same physiological activity as the peptide represented by SEQ ID NO: 1 and having a sequence homology of 95% or more. "Substantially homogenous physiological activity" means an activity for preventing or treating cancer.
또한, 본 발명은 CysA5W 펩타이드를 유효성분으로 함유하는 암 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising CysA5W peptide as an active ingredient.
본 발명의 일 구현 예에 따른 암 예방 또는 치료용 약학 조성물에서, 상기 CysA5W 펩타이드는 서열번호 1의 아미노산 서열로 이루어진 것이나, 이에 제한되지 않는다.In the pharmaceutical composition for preventing or treating cancer according to an embodiment of the present invention, the CysA5W peptide is composed of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
본 발명의 일 구현 예에 따른 암 예방 또는 치료용 약학 조성물에서, 상기 암은 후두암, 폐암, 식도암, 췌장암, 대장암, 간암, 위암, 설암, 피부암, 뇌종양, 자궁암, 유방암, 자궁경부암, 난소암, 신장암, 담낭암, 구강암, 결장암, 간암, 방광암 및 대장암으로 이루어진 군으로부터 선택되는 것일 수 있고, 바람직하게는 자궁경부암 또는 유방암일 수 있으나, 이에 제한되지 않는다.In the pharmaceutical composition for preventing or treating cancer according to an embodiment of the present invention, the cancer may be cancer of the larynx, lung, esophagus, pancreas, colon, liver, stomach, , Kidney cancer, gallbladder cancer, oral cancer, colon cancer, liver cancer, bladder cancer and colorectal cancer, preferably, but not limited to, cervical cancer or breast cancer.
본 발명의 일 구현 예에 따른 암 예방 또는 치료용 약학 조성물에서, 상기 CysA5W 펩타이드는 MicroRNA-29b 생성을 증가시켜 p53 단백질을 안정화시킴으로써 항암 효과를 유발하는 것일 수 있다.In the pharmaceutical composition for preventing or treating cancer according to an embodiment of the present invention, the CysA5W peptide may induce an anti-cancer effect by stabilizing the p53 protein by increasing the production of MicroRNA-29b.
본 발명에 따른 CysA5W 펩타이드를 포함하는 암 예방 또는 치료용 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The pharmaceutical composition for preventing or treating cancer comprising the CysA5W peptide according to the present invention may be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups or aerosols, May be formulated in the form of injection solutions.
상세하게는, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 CysA5W 펩타이드에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose), 락토오스(lactose), 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween)61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.In detail, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.
본 발명에 따른 CysA5W 펩타이드 투여량은 환자의 나이, 성별, 체중에 따라 달라질 수 있다. 또한 CysA5W 펩타이드의 투여량은 투여경로, 질병의 정도, 성별, 체중, 나이 등에 따라서 증감될 수 있다. 따라서 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The dosage of CysA5W peptide according to the present invention may vary depending on the patient's age, sex, and body weight. The dose of CysA5W peptide can also be increased or decreased depending on route of administration, degree of disease, sex, weight, age, and the like. Accordingly, the dosage is not limited in any way to the scope of the present invention.
또한, 본 발명은 상기 암 예방 또는 치료용 약학 조성물을 인간을 제외한 포유동물 내에 투여하는 단계를 포함하는, 암세포에 대한 세포사멸 증진 방법을 제공한다.In addition, the present invention provides a method for promoting apoptosis in cancer cells, comprising the step of administering the pharmaceutical composition for cancer prevention or treatment to a mammal other than a human.
또한, 본 발명은 CysA5W 펩타이드를 유효성분으로 함유하는 항암보조제용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for anticancer drugs containing CysA5W peptide as an active ingredient.
본 발명의 일 구현 예에 따른 항암보조제용 건강기능식품에서, 상기 건강 기능성 식품의 종류에는 특별한 제한은 없다. 상기 CysA5W 펩타이드를 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올음료 및 비타민 복합제 등이 있으며, 환제, 분말, 과립, 침제, 정제, 캡슐 또는 음료인 형태로 사용할 수 있고 통상적인 의미에서의 건강 기능성 식품을 모두 포함한다.In the health functional food for anticancer adjuvant according to an embodiment of the present invention, there is no particular limitation on the kind of health functional food. Examples of foods to which the CysA5W peptide can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, soups, , An alcoholic beverage and a vitamin complex, and can be used in the form of pills, powders, granules, infusions, tablets, capsules or beverages, and includes health functional foods in a conventional sense.
본 발명의 일 구현 예에 따른 항암보조제용 건강기능식품에서, 상기 CysA5W 펩타이드는 서열번호 1의 아미노산 서열로 이루어진 것일 수 있으나, 이에 제한되지 않는다. In the health functional food for anti-cancer adjuvant according to an embodiment of the present invention, the CysA5W peptide may be composed of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
본 발명의 일 구현 예에 따른 항암보조제용 건강기능식품에서, 상기 암은 후두암, 폐암, 식도암, 췌장암, 대장암, 간암, 위암, 설암, 피부암, 뇌종양, 자궁암, 유방암, 자궁경부암, 난소암, 신장암, 담낭암, 구강암, 결장암, 간암, 방광암 및 대장암으로 이루어진 군으로부터 선택되는 것일 수 있고, 바람직하게는 자궁경부암 또는 유방암일 수 있으나, 이에 제한되지 않는다.In the health functional food for anticancer adjuvant according to an embodiment of the present invention, the cancer is selected from the group consisting of laryngeal cancer, lung cancer, esophageal cancer, pancreatic cancer, colon cancer, liver cancer, gastric cancer, stomach cancer, skin cancer, brain tumor, uterine cancer, breast cancer, cervical cancer, Kidney cancer, gallbladder cancer, oral cancer, colon cancer, liver cancer, bladder cancer and colorectal cancer, preferably, but not limited to, cervical cancer or breast cancer.
본 발명의 건강 음료 조성물은 지시된 비율로 필수 성분으로서 상기 CysA5W 펩타이드를 함유하는 외에는 액체성분에는 특별한 제한은 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예로는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린; 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등)) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.The health beverage composition of the present invention is not particularly limited to liquid ingredients other than those containing the CysA5W peptide as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrins; And sugar alcohols such as xylitol, sorbitol, and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have.
상기 외에 본 발명의 건강 기능성 식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다.In addition to the above, the health functional food of the present invention may contain various kinds of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.
본 발명의 일 구현 예에 따른 항암보조제용 건강기능식품에서, 상기 CysA5W 펩타이드는 MicroRNA-29b 생성을 증가시켜 p53 단백질을 안정화시킴으로써 항암 효과를 유발하는 것일 수 있다.
In the health functional food for anticancer adjuvants according to an embodiment of the present invention, the CysA5W peptide may induce an anti-cancer effect by stabilizing the p53 protein by increasing the production of MicroRNA-29b.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
재료 및 방법Materials and methods
세포 배양Cell culture
본 실험에서 이용된 세포는 자궁경부암세포인 HeLa와 유방암세포인 MCF7이다. HeLa 세포는 microRNA-29의 발현이 매우 낮아 독시사이클린(doxycycline; dox)에 의해 유도되는 시스템이 구축되어있는 세포주(inducible miR29a/b HeLa)를 이용하였다. 두 세포 모두 DMEM(Welgene) 배지에 10%(v/v) Fetal Bovine Serum(FBS, Welgene)을 첨가한 배지에 넣고, 37℃, 5% CO2 인큐베이터에서 배양하였다.
The cells used in this experiment are cervical cancer cell line HeLa and breast cancer cell MCF7. HeLa cells were cultured with inducible miR29a / b HeLa, a system in which the expression of microRNA-29 was very low and induced by doxycycline (dox). Both cells were placed in DMEM (Welgene) medium supplemented with 10% (v / v) Fetal Bovine Serum (FBS, Welgene) and incubated at 37 ° C in a 5% CO 2 incubator.
펩타이드Peptides 합성 synthesis
본 실험에서 이용된 펩타이드는 일반적인 고체상 펩타이드 합성(solid-phase peptide synthesis)을 통해 합성되었다. 합성된 펩타이드의 N-말단에 아세틸화(acetylation) 시킨 후, HPLC(Agilent 1100)로 분리하여 MALDI-TOF/TOF로 확인한 후, 정제하였다.
The peptides used in this experiment were synthesized by the general solid-phase peptide synthesis. The synthesized peptides were acetylated at the N-terminus and then separated by HPLC (Agilent 1100) and identified by MALDI-TOF / TOF and purified.
세포 생존율 측정Cell viability measurement
본 실험에서 우선 p53 활성으로 인한 세포사멸을 증가시키는 항암 펩타이드를 선별하기 위해 세포 생존율 측정(MTS assay) 실험을 실시하였다. 알파 나선구조를 가지며 16~18개의 무작위 아미노산 서열을 가진 펩타이드 81개를 유도성 miR29a/b HeLa 세포에 독시사이클린(dox)과 함께 처리한 후, 72시간 뒤에 MTS 용액(Promega)을 넣는다. 한 시간 후에 마이크로플레이트 리더(microplate reader) 기계로 490 nm 파장에서 시그널을 검출한다.
In this experiment, the cell survival rate (MTS) assay was performed to select anti-cancer peptides that increase apoptosis due to p53 activity. Eighty-eight (81) peptides with alpha helical structure and 16 to 18 random amino acid sequences are treated with doxycycline (dox) in inducible miR29a / b HeLa cells, followed by MTS solution (Promega) 72 hours later. One hour later, the signal is detected at a wavelength of 490 nm with a microplate reader machine.
정량적 실시간 Quantitative real-time PCR(TaqmanPCR (Taqman microRNAmicroRNA assayassay ) )
본 실험에서 유도성 miR29a/b HeLa 세포를 24-웰 플레이트에 심고 펩타이드(1 μM)와 독시사이클린(dox)을 처리한 24시간 후에 TriReagent(Ambion) 시약을 이용하여 총 RNA를 추출하였다. Applied Biosystem 사의 Taqman microRNA assay Kit을 이용하여 microRNA-29b 특이적으로 cDNA를 합성하였다. 합성된 cDNA를 Taqman microRNA assay kit에 있는 마스터 믹스(master mix)로 PCR 반응을 Light Cycler II 480(Roche) 기계로 수행하여 Ct값을 얻었다. U6 snRNA는 내부 대조구(internal control)로써 이용되었고, microRNA-29b의 발현 값을 U6 발현 값으로 표준화하였다.
In this experiment, in vitro miR29a / b HeLa cells were seeded in 24-well plates and total RNA was extracted using TriReagent (Ambion) reagent 24 hours after treatment with peptide (1 μM) and doxycycline (dox). CDNA was specifically synthesized with microRNA-29b using Applied Biosystem's Taqman microRNA assay kit. The synthesized cDNA was subjected to PCR using a Light Cycler II 480 (Roche) machine with a master mix in the Taqman microRNA assay kit to obtain the Ct value. U6 snRNA was used as an internal control and the expression level of microRNA-29b was normalized to U6 expression value.
p53p53 활성을 측정할 수 있는 The ability to measure activity 루시퍼라제Luciferase 분석( analysis( luciferaseluciferase assayassay ))
본 실험에서는 p53 활성을 측정하기 위해 루시퍼라제 유전자 업스트림(upstream)에 13개의 p53-결합 자리가 존재하는 플라스미드(pG13-luciferase plasmid)를 이용하여 실험을 수행하였다. 먼저 유도성 miR29a/b HeLa 세포를 24-웰 플레이트에 심고, 펩타이드(1μM)와 독시사이클린(dox)을 처리한 3시간 후에 pG13-루시퍼라제 플라스미드(Firefly luciferase)와 Renilla 루시퍼라제 벡터를 함께 세포 안으로 주입시켰다. 24시간 배양 후에 Promega사의 듀얼-루시퍼라제 분석 키트(Dual-luciferase assay kit)로 루시퍼라제 활성을 측정하였다. 화이어플라이(Firefly) 루시퍼라제(Firefly luciferase) 활성 값을 Renilla 루시퍼라제 활성 값으로 표준화하였다.
In this experiment, the p53-luciferase plasmid was used to measure the p53 activity. The p53-luciferase plasmid has 13 p53-binding sites upstream of the luciferase gene. First, inducible miR29a / b HeLa cells were seeded in a 24-well plate and pG13-firefly luciferase and Renilla luciferase vectors were injected into the cells together three hours after treatment with the peptide (1 μM) and doxycycline (dox) . After 24 hours of incubation, luciferase activity was measured with a Dual-luciferase assay kit from Promega. Firefly luciferase activity values were normalized to Renilla luciferase activity values.
시험관 내 In vitro DicerDicer 또는 or DroshaDrosha 프로세싱 분석( Processing analysis ( DicerDicer oror DroshaDrosha processingprocessing assayassay ))
본 실험에서 선별된 CysA5W 펩타이드가 microRNA 생성과정에서 어떤 단계에서 영향을 미치는지 알아보기 위해 시험관 분석(in vitro processing assay)를 수행하였다. 이 실험을 수행하기 위해 primary microRNA와 microRNA 전구체를 합성하였다. Primary microRNA는 일반적인 시험관 내 전사(in vitro transcription) 방법으로 합성하였는데, primary microRNA-29b 서열을 포함한 게놈 DNA 부위를 PCR로 증폭시킨 후, T7 프로모터가 포함되어 있는 벡터에 클로닝하였다. 이때 이용된 프라이머들은 다음과 같다: 정방향 프라이머, 5'-CTTTTCCTTTCTAGGTTGTCTTGGG-3'(서열번호 2), 역방향 프라이머, 5'-AAGGGGTCAGCTACATGTGA-3'(서열번호 3). 클로닝된 플라스미드를 선형(linear)으로 만들어 준 후, T7 RNA 폴리머라제와 NTP mix(각각 10 mM ATP, GTP, CTP 및 1 mM UTP), [α-32P]UTP을 사용하여 37℃에서 3시간 반응시켰다. 그 후, 12% 우레아-폴리아크릴아미드 겔(urea-PAGE) 상에서 전기영동 하여 primary microRNA-29b 전사체를 분리 정제하였다. MicroRNAs 전구체들은 두 가닥의 단일 가닥 RNA을 바이오니어사에서 합성하여, 한가닥의 RNA로 연결(ligation)시켰다. 그 후, T4 Polynucleotide kinase(Takara)와 [γ-32P]ATP을 이용하여 5' 말단 라벨링을 하였다. 12% 우레아-PAGE 상에서 전기 영동하여 microRNA 전구체들을 분리 정제하였다. In vitro processing assay was performed to investigate the effect of selected CysA5W peptides on microRNA production. Primary microRNA and microRNA precursors were synthesized to perform this experiment. The primary microRNA is a normal in vitro transcript ( in vitro transcription. The genomic DNA region including the primary microRNA-29b sequence was amplified by PCR and cloned into a vector containing the T7 promoter. The primers used were: forward primer, 5'-CTTTTCCTTTCTAGGTTGTCTTGGG-3 '(SEQ ID NO: 2), reverse primer, 5'-AAGGGGTCAGCTACATGTGA-3' (SEQ ID NO: 3). The cloned plasmids were linearly transformed with T7 RNA polymerase and NTP mix (10 mM ATP, GTP, CTP and 1 mM UTP, respectively) and [α- 32 P] UTP at 37 ° C for 3 hours Lt; / RTI > Thereafter, primary microRNA-29b transcripts were separated and purified by electrophoresis on 12% urea-polyacrylamide gel (urea-PAGE). MicroRNAs precursors synthesized two stranded single stranded RNAs from Biona, and ligated them into one strand of RNA. Subsequently, 5 'end labeling was performed using T4 Polynucleotide kinase (Takara) and [γ- 32 P] ATP. The microRNA precursors were separated and purified by electrophoresis on 12% urea-PAGE.
다음, 본 발명에서 이용된 Dicer와 Drosha는 IP(immunoprecipitation) 방법을 통해 얻어내었다. Flag tag으로 연결되어 있는 Dicer 또는 Drosha 단백질을 세포 내에서 과발현시킨 후, 세포 용해 버퍼(20 mM Tris-Cl at pH 8.0, 500 mM NaCl, 1 mM EDTA, 1%(v/v) Triton X-100)를 넣고 음파처리(sonication)로 세포질을 깨서 세포 용해물을 얻어내었다. 그 후, anti-Flag bead(anti-Flag M2 affinity gel)을 이용하여 4℃에서 한 시간 동안 IP(immunoprecipitation)하였다. 세포 용해 버퍼로 3번, 버퍼 D(20 mM Tris-Cl at pH 8.0, 200 mM KCl, 0.2 mM EDTA, 20%(v/v) Glycerol, 1 mM DTT)로 4번 세척한 후, 시험관 내 Dicer 분석 실험(in vitro Dicer processing assay)을 수행하였다. 그리고 Drosha는 세포 용해 버퍼(20 mM Tris-Cl at pH8.0, 100 mM KCl, 0.2 mM EDTA)로 7번 세척 후, 시험관 내 Drosha 분석 실험(in vitro Drosha processing assay)을 실시하였다. 시험관 내 Dicer 반응은 총 10 ㎕에서 20 mM MgCl2 1 ㎕, 20 mM DTT 0.5 ㎕, 32P-labeled 전구체 RNAs 0.5 ㎕, Dicer-IP 5㎕에 펩타이드(CysA5W, 13Aad-14W, HelixA)와 함께 37℃에서 반응시켰다. 그렇게 반응시킨 RNA들을 12% 우레아-PAGE 상에서 전기 영동하여 분리시킨 후, 이미지 필름에 노출시킨 뒤 BAS-2500(Fuji) 기계로 시그널을 검출하였다. 시험관 내 Drosha 반응은 총 10 ㎕에서 64 mM MgCl2 1 ㎕, 32P-labeled primary microRNA-29b 0.5 ㎕, Drosha-IP 5㎕에 펩타이드(CysA5W, 13Aad-14W, HelixA)와 함께 37℃에서 반응시켰다. 그렇게 반응시킨 RNA들을 10% 우레아-PAGE 상에서 전기영동하여 분리시킨 후, 이미지 필름에 노출시킨 뒤 BAS-2500(Fuji) 기계로 시그널을 검출하였다.
Next, Dicer and Drosha used in the present invention were obtained by an IP (immunoprecipitation) method. Dicer or Drosha protein linked by a Flag tag was overexpressed in a cell and then lysed in a cell lysis buffer (20 mM Tris-Cl at pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% (v / v) Triton X-100 ) Was added and the cell lysate was obtained by sonication by sonication. Then, IP (immunoprecipitation) was performed at 4 ° C for one hour using anti-Flag bead (anti-Flag M2 affinity gel). After washing 4 times with
전구체 Precursor microRNAmicroRNA -29b와 -29b and DicerDicer 복합체 형성 실험인 겔 The complex formation experiment gel 쉬프트Shift 분석( analysis( gelcome shiftshift assayassay ))
본 실험에서 CysA5W 펩타이드가 전구체 microRNA-29b와 Dicer 단백질의 복합체에 어떤 영향을 미치는지 확인하기 위해 겔 쉬프트 분석을 실시하였다. 이 실험에 이용된 전구체 microRNA는 앞서 언급했듯이 5'-말단 라벨링을 통해 준비되었고, Dicer 단백질은 재조합 Dicer로 실험에 사용되었다. 분석 반응은 30 nM 재조합 Dicer, ~0.2 nM의 32P-labeled 전구체 microRNA-29a와 microRNA-29b, 1 ㎍ BSA(Takara), 1 mM DTT, 결합 버퍼(20 mm HEPES, pH7.4, 50 mM KCl, 0.5 mM EDTA, 10%(v/v) Glycerol)와 CysA5W 펩타이드를 넣고 30℃에서 반응시켰다. 그 후 반응물들을 6% non-denaturing PAGE에 분리하여 이미지 필름에 노출시킨 뒤 BAS-2500(Fuji) 기계로 시그널을 검출하였다.
In this experiment, gel shift analysis was performed to determine the effect of CysA5W peptide on the complex of precursor microRNA-29b and Dicer protein. The precursor microRNA used in this experiment was prepared by 5'-end labeling as previously mentioned, and the Dicer protein was used in the experiment as a recombinant Dicer. The assay was performed with 30 nM recombinant Dicer, ~ 0.2 nM 32 P-labeled precursor microRNA-29a and microRNA-29b, 1 ug BSA (Takara), 1 mM DTT, binding buffer (20 mM HEPES, pH 7.4, 50 mM KCl , 0.5 mM EDTA, 10% (v / v) Glycerol) and CysA5W peptide were added and reacted at 30 ° C. The reactions were then separated on a 6% non-denaturing PAGE, exposed to imaging film and detected by BAS-2500 (Fuji) machine.
p53p53 단백질 안정성을 확인하는 To confirm protein stability 웨스턴Western 블럿Blot 실험 Experiment
본 실험에서 microRNA-29b가 표적으로 하는 암 발생에 중요한 열쇠가 되는 p53 단백질의 활성을 확인하기 위해 단백질 양을 확인할 수 있는 웨스턴 블럿 실험을 실시하였다. 유도성 miR29a/b HeLa 또는 MCF7 세포를 60 mm 디쉬에 심고, 펩타이드(CysA5W, 13Aad-14W, HelixA)를 1 μM을 처리하였다. 24 시간 후에 PBS로 세포를 모았다. RIPA 버퍼로 총 단백질을 뽑아 10% SDS-PAGE 젤에 전기영동으로 분리하여, PVDF 막(Millipore)에 옮겼다. 0.1%(v/v) Tween-20이 포함된 PBS(PBS-T)에 5%(w/v) 스킴 밀크(BD)로 만든 것으로 블럭킹하고, 산타크루즈사의 항-p53 마우스 항체(anti-p53 mouse antibody)를 1%(w/v) BSA에 담아 결합시켰다. Jackson 사의 항-마우스 IgG HRP-콘쥬케이트 항체(Donkey anti-mouse antibody)를 이차로 붙였다. ECL 용액(Thermo Scientific)으로 반응시켜 X-레이 필름에 노출시킨 후 밴드를 확인하였다. 로딩 대조구로써 항-GAPDH 마우스 항체(anti-GAPDH mouse antibody)가 이용되었고, 동일한 이차항체로 실험을 진행하여 단백질을 검출하였다.
In order to confirm the activity of p53 protein, which is an important key for cancer development targeted by microRNA-29b, Western blot test was performed to confirm the amount of protein. Inducible miR29a / b HeLa or MCF7 cells were seeded in 60 mm dishes and treated with 1 μM of peptide (CysA5W, 13Aad-14W, HelixA). After 24 hours, cells were collected with PBS. The total protein was extracted with RIPA buffer, electrophoresed on a 10% SDS-PAGE gel, and transferred to a PVDF membrane (Millipore). The cells were blocked with 5% (w / v) skim milk (BD) in PBS (PBS-T) containing 0.1% (v / v) Tween-20 and incubated with Santa Cruz anti-p53 mouse anti- mouse antibody was bound to 1% (w / v) BSA. Jackson-type anti-mouse IgG HRP-conjugate antibody (Donkey anti-mouse antibody). ECL solution (Thermo Scientific), exposed to X-ray film, and the band was confirmed. Anti-GAPDH mouse antibody was used as a loading control, and the same secondary antibody was used to detect the protein.
세포사멸 집단(Cell death group Apoptotic cellApoptotic cell populationpopulation )을 측정할 수 있는 FACS(Fluorescence-activated FACS (Fluorescence-activated cellcell sortingsorting ))
본 실험에서 CysA5W 펩타이드로 인해 p53 활성이 증가되어 세포사멸이 높게 유도되는지 확인하기 위해 세포사멸 집단(apoptotic cell population)을 측정할 수 있는 FACS를 수행하였다. 유도성 miR29a/b HeLa 또는 MCF7 세포를 100 mm 디쉬에 심고, 1 μM의 펩타이드(CysA5W, 13Aad-14W, HelixA)와 양성 대조구인 50 μM 에토포시드(Sigma)를 각각 처리하고 48시간 후에 PBS로 세포를 모았다. 70%(v/v) 에탄올로 4℃에서 16시간 세포 고정을 시킨 후, 프로피디움 요오드화물(50 ㎍/ml)과 RNase A(5 ㎍/ml)를 처리하여 세포 염색을 시켰다. 염색된 세포들을 FACS AriaII(Becton Dickinson) 기계로 DNA 함량을 분석하였고, FlowJo 소프트웨어를 이용하여 그래프로 나타내었다. In this experiment, FACS was performed to measure the apoptotic cell population in order to confirm the induction of apoptosis by increasing the p53 activity due to the CysA5W peptide. Inducible miR29a / b HeLa or MCF7 cells were seeded in 100 mm dishes and treated with 1 μM peptide (CysA5W, 13Aad-14W, HelixA) and 50 μM ethoposide (Sigma) Cells were collected. Cells were stained with 70% (v / v) ethanol at 4 ° C for 16 hours and treated with propidium iodide (50 μg / ml) and RNase A (5 μg / ml) for cell staining. The stained cells were analyzed for DNA content using a FACS AriaII (Becton Dickinson) machine and plotted using FlowJo software.
내생의 Endogenous microRNAmicroRNA -29b 발현을 -29b expression 억제시키기Suppress 위해 for 안티센스Antisense microRNAmicroRNA -29b을 이용한 세포주입(Cell infusion with -29b ( transfectiontransfection ) 실험 ) Experiment
본 실험에서 사용된 안티센스 microRNA-29b는 Bioneer 사에서 합성하였고, 2'-O-메틸화한 RNA(anti-miR29b)로 microRNA-29b 수준을 감소시키고자 하였다. 먼저 유도성 miR29a/b HeLa 또는 MCF7 세포를 60 mm 디쉬에 심고, 1 μM CysA5W 처리와 동시에 항-miR29b(anti-miR29b) 또는 대조구로써 이용된 항-siGFP(anti-siGFP)를 각각 세포 안에 리포펙타민 2000(Lipofectamine, Invitrogen)으로 주입시켰다. 24시간 동안 배양한 후, 총 RNA을 분리 정제하여 Taqman microRNA 분석을 실시하거나 단백질을 뽑아 웨스턴 블럿 분석을 수행하였다. 이 실험에 이용된 항-RNA의 서열은 다음과 같다: 항-miR29b, 5'-AACACUGAUUUCAAAUGGUGCUA-3'(서열번호 4), 항-siGFP, 5'-AACAUCGCCAUCUAAUUCA-3'(서열번호 5).
The antisense microRNA-29b used in this experiment was synthesized by Bioneer and was intended to reduce the level of microRNA-29b with 2'-O-methylated RNA (anti-miR29b). First, inducible miR29a / b HeLa or MCF7 cells were seeded in a 60 mm dish and anti-miR29b (anti-miR29b) or anti-siGFP (anti-siGFP) used as a control simultaneously with 1 μM CysA5W treatment Gt; 2000 < / RTI > (Lipofectamine, Invitrogen). After culturing for 24 hours, the total RNA was isolated and purified, subjected to Taqman microRNA analysis, or proteins were extracted and Western blot analysis was performed. The sequence of the anti-RNA used in this experiment is as follows: anti-miR29b, 5'-AACACUGAUUUCAAAUGGUGCUA-3 '(SEQ ID NO: 4), anti-siGFP, 5'-AACAUCGCCAUCUAAUUCA-3' (SEQ ID NO: 5).
실시예Example 1. One. p53 p53 활성으로 인한 세포사멸을 증진시키는 Promoting apoptosis due to activation 펩타이드Peptides 1차 선별 Primary selection
본 실험에서 알파 나선 구조를 가지는 81개의 다른 펩타이드에서 p53 활성으로 인한 세포사멸을 증진시킬 수 있는 펩타이드를 선별하기 위해 세포 생존율 실험을 실시하였다. 독시사이클린에 의해 microRNA-29 발현이 유도되는 시스템이 구축된 HeLa 세포(inducible miR29a/b HeLa)에 펩타이드를 각각 처리한 후, 72시간이 지난 뒤 MTS 분석을 통해 세포생존율을 측정하였다. 생존율이 가장 낮은 9개의 후보 펩타이드를 우선적으로 선별하였다(도 1).
In this experiment, cell viability experiments were performed to select peptides capable of promoting apoptosis due to p53 activity in 81 different peptides having an alpha helical structure. The cell viability was measured by MTS analysis after 72 hours of treatment of the peptide with HeLa cells (inducible miR29a / b HeLa) in which the system for inducing microRNA-29 expression was induced by doxycycline. Nine candidate peptides with the lowest survival rate were preferentially screened (Fig. 1).
실시예Example 2. 1차 선별된 9개 2. The first nine selected 펩타이드Peptides 중 medium microRNAmicroRNA -29b와 -29b and p53p53 활성을 증가시키는 2차 Secondary to increase activity 펩타이드Peptides 선별 Selection
우선적으로 선별된 9개의 후보 펩타이드 중 microRNA-29b와 p53 활성을 모두 증가시키는 펩타이드를 선별하기 위해 Taqman microRNA 분석(도 2A)과 p53 루시퍼라제 분석(도 2B)을 수행하였다. 각 실험에서 공통적으로 선별된 펩타이드는 CysA5W로, 그 서열은 도 2C에 나타내었다.
Taqman microRNA analysis (FIG. 2A) and p53 luciferase analysis (FIG. 2B) were performed to select peptides that increased both microRNA-29b and p53 activity among nine preferentially selected candidate peptides. The peptide selected commonly in each experiment is CysA5W, and its sequence is shown in Fig. 2C.
실시예Example 3. 선별된 3. Selected CysA5WCysA5W 펩타이드가The peptide 전구체 Precursor microRNAmicroRNA -29b에서 At -29b DicerDicer 활성을 증진시켜 Promoting activity 성숙된Mature microRNAmicroRNA -29b의 생성을 촉진시켰다.-29b. ≪ / RTI >
본 실험에서는 선별된 CysA5W 펩타이드가 microRNA 생성과정에서 어떤 기작으로 작용하는지 알아보기 위해, 시험관내 분석(in vitro processing assay)을 수행하였다. MicroRNA 생성과정에 중요한 역할을 하는 두 단백질(Drosha와 Dicer)의 작용을 확인하고자 하였다. 먼저 Drosha 작용에서 CysA5W 펩타이드가 다른 펩타이드(13Aad-14W, HelixA)와 mock control 결과와 유사함을 확인하였다(도 3D). 이 결과로 볼 때, CysA5W 펩타이드는 Drosha 활성과 무관함을 알 수 있다. 다음 Dicer 작용에서 CysA5W 펩타이드의 영향을 알아보았다(도 3A-C). MicroRNA-29a 또는 let-7a과는 달리 microRNA-29b에서만 CysA5W 펩타이드가 존재할 때, Dicer 활성이 증가함을 보였다(도 3A-C). 이 결과들로 보아 CysA5W 펩타이드는 특이적으로 전구체 microRNA-29b의 Dicer 작용만 증진시킴을 알 수 있다.
To see if this experiment, selected CysA5W peptides which act as Mechanism in microRNA generation process, in vitro assays (in vitro processing assay. (Drosha and Dicer), which play an important role in the microRNA production process. First, it was confirmed that the CysA5W peptide in the Drosha action was similar to other peptide (13Aad-14W, HelixA) and mock control results (FIG. 3D). As a result, the CysA5W peptide is independent of Drosha activity. We next examined the effect of CysA5W peptide on Dicer action (Figure 3A-C). Dicer activity was increased when CysA5W peptide was present only in microRNA-29b, unlike MicroRNA-29a or let-7a (Fig. 3A-C). These results suggest that the CysA5W peptide specifically promotes the Dicer action of the precursor microRNA-29b.
실시예Example 4. 선별된 4. Selected CysA5WCysA5W 펩타이드가The peptide 전구체 Precursor microRNAmicroRNA -29b와 -29b and DicerDicer 복합체를 좀 더 효율적으로 형성하도록 To form the complex more efficiently 도움주었다Helped ..
CysA5W 펩타이드가 특이적으로 전구체 microRNA-29b에 대한 Dicer processing이 어떤 메커니즘으로 작용하는지 알아보기 위해 젤 쉬프트 분석(gel shift assay)을 수행하였다. 서열이 거의 유사한 전구체 microRNA-29a와 전구체 microRNA-29b를 비교하였을 때, 이차 구조에서 루프(loop) 부분은 유사하지만 스템(stem) 부분에서 차이를 보였다(도 4A). 이러한 차이로 CysA5W 펩타이드가 선택적으로 전구체 microRNA-29b에 작용하여 Dicer와 복합체를 좀 더 효율적으로 형성되도록 도움을 준다는 것을 알 수 있었다(도 4B).
A gel shift assay was performed to investigate the mechanism by which the CysA5W peptide specifically acts on the precursor microRNA-29b for dicer processing. When comparing the precursor microRNA-29a and the precursor microRNA-29b, which are similar in sequence, the loop part is similar but the stem part is different in the secondary structure (FIG. 4A). These differences suggest that the CysA5W peptide selectively acts on the precursor microRNA-29b to help complexes with Dicer form more efficiently (FIG. 4B).
실시예Example 5. 선별된 5. Selective CysA5WCysA5W 펩타이드가The peptide 다른 Other 펩타이드와는With peptides 달리 Otherwise p53p53 단백질의 안정성을 증진시켜 세포사멸을 높게 유발시켰다. The stability of the protein was enhanced to induce high cell death.
일반적으로 p53 단백질은 매우 불안정하기 때문에 CysA5W 펩타이드가 존재하였을 때 어떻게 영향을 받는지 확인해 보기 위해, 단백질 생합성을 억제시키는 사이클로헥사마이드(Cycloheximide, CHX)을 이용하여 p53 단백질 안정성을 테스트 하였다. 단백질은 웨스턴 블럿 분석으로 확인하였다. 도 5A에서 보면, 다른 펩타이드(13Aad-14W, HelixA)와는 달리 CysA5W 펩타이드를 처리하였을 때에 p53 단백질은 안정화되었다. 이 결과를 기반으로 CysA5W 펩타이드가 세포사멸을 더 많이 유발시키는지 확인해 보기 위해 FACS 실험을 실시하였다. 그 결과, CysA5W 펩타이드를 처리한 샘플에서 양성 대조구로써 이용된 에토포시드와 비슷하게 높은 세포사멸 집단(apoptotic cell population)이 측정되었다(도 5B).
In general, the p53 protein is highly unstable, so to determine how it is affected when the CysA5W peptide is present, p53 protein stability was tested using Cycloheximide (CHX), which inhibits protein biosynthesis. Proteins were identified by Western blot analysis. 5A, unlike the other peptides (13Aad-14W, HelixA), the p53 protein was stabilized when the CysA5W peptide was treated. Based on these results, FACS experiments were conducted to confirm whether CysA5W peptides induce apoptosis more. As a result, a high apoptotic cell population similar to the etoposide used as a positive control in samples treated with CysA5W peptide was measured (FIG. 5B).
실시예Example 6. 6. CysA5WCysA5W 펩타이드가The peptide 다른 암세포인 Other cancer cells MCF7MCF7 에서도 On microRNAmicroRNA -29b와 -29b and p53p53 단백질 활성을 모두 증진시켜 세포사멸을 유도하였다. All of the protein activities were enhanced to induce apoptosis.
CysA5W 펩타이드가 HeLa 세포뿐만 아니라 다른 암세포주인 유방암세포 MCF7에서도 microRNA-29b와 p53 단백질 안정성을 모두 증진시켜 세포사멸을 유도하는지 확인하였다(도 6). 그 결과, CysA5W 펩타이드가 존재할 때 microRNA-29b 생성이 증가되고(도 6A), p53 단백질도 안정화되었다(도 6B). 더불어 세포사멸 집단도 증가함을 확인할 수 있었다(도 6C).
CysA5W peptide promoted cell death by enhancing both microRNA-29b and p53 protein stability in not only HeLa cells but also other cancer cell lines, breast cancer cells MCF7 (FIG. 6). As a result, microRNA-29b production was increased when the CysA5W peptide was present (Fig. 6A), and p53 protein was also stabilized (Fig. 6B). In addition, it was confirmed that the cell death group also increased (FIG. 6C).
실시예Example 7. 7. CysA5WCysA5W 펩타이드는Peptides microRNAmicroRNA -29b 생성을 통해서 Through the generation of -29b p53p53 단백질을 안정화시킬 수 있음을 확인하였다. It was confirmed that the protein could be stabilized.
본 실험에서 CysA5W 펩타이드가 microRNA-29b 생성을 증진시킴으로써 p53 활성에 영향을 주는 것인지 아닌지 확인하기 위해 내생의 microRNA-29b 수준을 억제시켜 p53 단백질 양상을 웨스턴 블럿으로 확인하였다. 유도성 miR29a/b HeLa 또는 MCF7 세포에 CysA5W 펩타이드를 처리하고, 항-miR29b를 주입시켰다. 24시간 후, 총 RNA을 뽑아 Taqman microRNA 분석을 수행하여 microRNA-29b 수준을 확인하였다(도 7A). 그 결과 대조구로써 이용된 항-siGFP를 처리한 샘플과 비교하였을 때, 성공적으로 microRNA-29b가 억제된 것을 확인할 수 있었다(도 7A). 다음, p53 단백질의 안정성 양상을 테스트해 본 결과, CysA5W 펩타이드가 존재하여도 microRNA-29b 생성이 제대로 이루어지지 않았을 때 다른 대조구 샘플들과 유사하게 p53 단백질이 분해된다는 것을 알 수 있었다(도 7B). 이를 통해, CysA5W 펩타이드는 microRNA-29b의 생성을 통해서 p53 단백질을 안정화시킬 수 있음을 확인하였다. In this experiment, to confirm whether CysA5W peptide promotes microRNA-29b production and thus affect p53 activity, endogenous microRNA-29b level was inhibited and p53 protein pattern was confirmed by Western blot. Inducible miR29a / b HeLa or MCF7 cells were treated with CysA5W peptide and injected with anti-miR29b. After 24 hours, total RNA was extracted and Taqman microRNA analysis was performed to confirm microRNA-29b levels (Fig. 7A). As a result, it was confirmed that microRNA-29b was successfully inhibited when compared with the sample treated with anti-siGFP used as a control (Fig. 7A). Next, the stability of the p53 protein was tested. As a result, it was found that when the CysA5W peptide was present but the microRNA-29b was not produced properly, the p53 protein was degraded similarly to other control samples (FIG. 7B). Thus, it was confirmed that CysA5W peptide can stabilize p53 protein through the production of microRNA-29b.
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