KR101818187B1 - Pharmaceutical composition containing a fibulin-3 protein for inhibiting the metastasis of lung cancer - Google Patents
Pharmaceutical composition containing a fibulin-3 protein for inhibiting the metastasis of lung cancer Download PDFInfo
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- KR101818187B1 KR101818187B1 KR1020120129418A KR20120129418A KR101818187B1 KR 101818187 B1 KR101818187 B1 KR 101818187B1 KR 1020120129418 A KR1020120129418 A KR 1020120129418A KR 20120129418 A KR20120129418 A KR 20120129418A KR 101818187 B1 KR101818187 B1 KR 101818187B1
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- cells
- protein
- lung cancer
- metastasis
- cancer
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Abstract
본 발명은 피불린-3(fibulin-3) 단백질을 유효성분으로 함유하는 폐암 전이 억제용 약학적 조성물에 관한 것으로, 더욱 구체적으로는 피불린-3 단백질에 의해서 암세포 전이의 중요한 경로 중의 하나인 EMT(epithelial-mesenchymal transition) 기능을 현저히 감소시켜 이동성 같은 세포의 운동성을 저해시킴으로써, 상기 피불린-3를 폐암 전이 억제용 약학적 조성물의 유효성분으로 유용하게 사용할 수 있다.TECHNICAL FIELD The present invention relates to a pharmaceutical composition for inhibiting lung cancer metastasis comprising fibulin-3 protein as an active ingredient, and more particularly, to a pharmaceutical composition for inhibiting metastasis of lung cancer, 3 can be usefully used as an effective ingredient of a pharmaceutical composition for inhibiting lung cancer metastasis by significantly reducing the epithelial-mesenchymal transition function and inhibiting cell mobility such as mobility.
Description
본 발명은 피불린-3(fibulin-3) 단백질을 유효성분으로 함유하는 폐암 전이 억제용 약학적 조성물 및 건강식품용 조성물에 관한 것이다.
The present invention relates to a pharmaceutical composition for inhibiting lung cancer metastasis and a composition for health food, which contains fibulin-3 protein as an active ingredient.
폐암(lung cancer)은 남녀 모두의 성별에서 두 번째로 흔히 발생하는 암으로서, 모든 암에서 15%를 차지한다. 2011년 미국 암학회(American cancer society)의 보고에 따르면, 한 해 22만건 이상이 폐암으로 진단을 받으며, 이 중 약 70%가 사망에 이르는데 이는 암 사망 중에서 27%를 차지한다. 폐암 중에서 비소세포성 폐암(non small lung cancer)은 상피성 암(carcinoma)의 일종으로 폐소성암(small lung cancer)이 아닌 모든 상피성 폐암(epithelial lung cancer)을 일컫으며, 폐암 전체의 약 85% 내지 90%를 차지한다. 비소세포성 폐암은 폐소성암에 비해 상대적으로 화학요법(chemotherapy)에 덜 민감하며, TNM 분류법에 기초하여 암의 단계를 나눈다: 종양의 크기(the size of tumor), 국소 림프절(reginal lymph node)로의 암 확산 정도 및 암 전이(metastasis)의 유무. 비소세포성 폐암의 치료에 있어서, 초기의 비전이성(non-metastatic) 비소세포성 폐암은 화학요법 및 방사선에 대한 민감도가 매우 낮기 때문에, 일반적으로 백금을 함유하는 시스플라틴(cisplatin)과 관련된 보조적인 화학요법(ancillary chemotherapy)와 함께 수술을 한다. 반면에 초기 단계를 지나, 전이성 비소세포성 폐암인 경우에는, 다양한 화학요법 및 방사선 치료가 사용된다. 비소세포성 폐암의 증상는 지속적인 기침, 흉부 통증, 체중감소, 손톱 손상, 관절 통증, 호흡의 단기화(shortness of breath) 등이 있으나, 비소세포성 폐암은 일반적으로 천천히 진행되기 때문에 초기에는 그 증상을 거의 보이지 않는다. 따라서, 비소세포성 폐암의 조기 발견 및 치료가 어려우며, 뼈, 간, 소장, 및 뇌 등 전신에 전이된 후에 발견할 가능성이 높다. 따라서, 높은 발병률과 사망률에도 불구하고 아직 비소세포성 폐암을 극복할 수 있는 어떠한 약물 또는 치료방법이 개발되지 않았으며, 그 필요성이 대두되고 있다. 비소세포성폐암은 암세포의 크기, 모양 및 화학적 구성에 따라 몇가지 하위 종류로 나뉘며, 대표적으로는 선암(adenocarcinoma), 편평상피암(squamous cell carcinoma), 대세포암(large cell carcinoma)등이 있다. 선암은 전체 폐암의 40%이상을 차지할 정도로 가장 높은 빈도로 발생하는 폐암으로서, 폐의 바깥부위(outer region)에서 발견되며 다른 폐암보다 천천히 진행되는 경향이 있으나, 초기에 높은 전이 경향을 보이고 또한 높은 방사선 저항성을 보인다. 편평상피암은 전체 폐암의 25-30%를 차지하는 비소세포성 폐암의 한 종류로서, 기도(airway)를 이루고 있는 세포의 초기 단계(early version)에서 시작되며, 상기 암은 주로 흡연자에게서 높은 발병률을 보인다. 또한 전체 폐암의 10-15% 정도를 차지하는 대세포암은 폐의 어느 부위에서나 발병할 수 있으며, 그 진행속도가 소세포성 폐암(small cell lung cancer)과 유사할만큼 빠르기 때문에 그 치료는 현재까지도 난제로 떠오르고 있다.
Lung cancer is the second most common cancer in men and women, accounting for 15% of all cancers. According to the American Cancer Society in 2011, more than 220,000 cases of lung cancer are diagnosed each year, of which about 70% are deaths, accounting for 27% of cancer deaths. Among non-small cell lung cancer, non small cell lung cancer is a type of carcinoma. It refers to all epithelial lung cancer that is not small lung cancer. Approximately 85% To 90%. Non-small cell lung cancer is less sensitive to chemotherapy relative to pulmonary metastases and divides the stages of cancer based on the TNM classification: the size of tumor, the size of the tumor, The extent of cancer spread and the presence or absence of metastasis. In the treatment of non-small cell lung cancer, early non-metastatic non-small cell lung cancer is very susceptible to chemotherapy and radiation, and thus is generally associated with platinum-containing cisplatin- Surgery is done with ancillary chemotherapy. On the other hand, in the case of metastatic non-small cell lung cancer, various chemotherapy and radiotherapy are used beyond the initial stage. Symptoms of non-small cell lung cancer include persistent cough, chest pain, weight loss, nail damage, joint pain, and shortness of breath. Non-small cell lung cancer, however, I can not see. Therefore, early detection and treatment of non-small cell lung cancer is difficult, and it is highly likely to be detected after metastasis to bone, liver, small intestine, and brain. Thus, despite the high incidence and mortality rates, no drugs or treatment methods have yet been developed that can overcome non-small cell lung cancer, and the need for this has arisen. Non-small cell lung cancer is classified into several subtypes according to the size, shape and chemical composition of cancer cells. Typical examples are adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Adenocarcinoma is the most frequently occurring lung cancer, accounting for more than 40% of all lung cancers. It is found in the outer region of the lungs and tends to progress more slowly than other lung cancers, It shows radiation resistance. Squamous cell carcinoma is a type of non-small cell lung cancer that accounts for 25-30% of all lung cancers, beginning with an early version of airway cells, which are predominantly in smokers . In addition, large-cell lung cancer, which accounts for about 10-15% of all lung cancers, can develop in any part of the lungs, and its progression rate is so fast that it is similar to small cell lung cancer, .
암의 가장 중요한 생물학적 특성의 하나는 전이를 일으킬 수 있다는 것이며, 이는 암을 치료하는데 가장 큰 장애물로 여겨지고 있다. 실제로 모든 고형 종양 환자의 약 60%는 이미 원발종양 진단시 미세하게나마 임상적으로 전이된 암을 가지고 있으며, 대부분의 암환자들의 중요한 사망 원인이 암 전이임은 이미 잘 알려져 있다. One of the most important biological characteristics of cancer is that it can cause metastasis, which is considered the biggest obstacle to cancer treatment. In fact, about 60% of all solid tumor patients already have clinically metastasized tumors at the time of primary tumor diagnosis and it is well known that the major cause of death in most cancer patients is cancer metastasis.
전이가 일어나는 과정 가운데 암의 국소조직의 침윤과 동반되어 나타나는 현상이 신생혈관형성인데, 여기에는 종양맥관형성인자 등이 관여하며, 종양에 의해 새로 생겨난 혈관들은 결함이 많기 때문에 암세포들에 의해 쉽게 침범된다. 또한, 암의 침습 및 전이 과정에는 조직의 기질 및 기저막에 유착하는데 필요한 라미닌 수용체와 같은 수많은 암세포 표면의 수용체들, 제 IV형 콜라게나제(collagenase), 플라스미노겐(plasminogen) 활성 인자 및 카텝신 D 등과 같은 정상조직의 간질을 용해시키는데 필요한 수많은 효소들, 성장인자, 자분비 운동인자(autocrine motility factor) 및 암 유전자 등의 발현이 필요하다.In the process of metastasis, the phenomenon that is accompanied by the infiltration of the local tissue of the cancer is neovascularization, in which tumor vasculature is involved, and newly formed blood vessels are defective. do. In addition, the invasion and metastasis of cancer include a number of cancer cell surface receptors, such as laminin receptors required to adhere to the matrix and basement membrane of the tissue, collagenase type IV collagenase, plasminogen activator, and cathepsin D, etc., growth factors, autocrine motility factors, and cancer genes, which are necessary for dissolving epilepsy of normal tissues.
현재까지 이러한 암 전이 억제 작용을 가지는 물질의 개발에 대한 기대는 매우 크지만, 실제로 암 전이 억제를 목적으로 한 물질의 개발예는 극히 적다. 황산화 다당류, N-디아조아세틸글리신 유도체, 노이라미니타제 및 피브로넥틴 분해효소(FNS) 등이 이러한 억제 작용을 갖는다고 보고되어 있지만 아직 그 실용화의 보고는 없으며 그들 자체가 암 전이 억제 효과를 갖는다는 보고도 없다. 따라서, 암의 전이를 효과적으로 억제할 수 있는 방법을 개발하면 암 전이에 의한 사망을 효과적으로 제어할 수 있는 유용한 치료법의 개발이 가능하게 된다.
Although there is a great expectation for the development of a substance having such a cancer-metastasis-inhibiting action to date, the development of a substance for suppressing cancer metastasis is extremely small. It has been reported that sulfated polysaccharides, N-diazoacetylglycine derivatives, noirminitase, and fibronectinase (FNS) have such an inhibitory effect, but there is no report on practical use thereof, and they have an effect of inhibiting cancer metastasis There is no report. Therefore, if a method capable of effectively inhibiting metastasis of cancer is developed, a useful therapeutic method capable of effectively controlling cancer metastasis can be developed.
피불린(fibulin) 단백질군은 엘라스틱 세포외기질(extracellular matrix, ECM)과 결합된 단백질로서 세포간 상호작용 또는 세포와 기질간 상호작용에 기여하는 단백질로서 타입 1부터 타입 6까지의 6 종류가 알려져 있다. 이러한 피불린 단백질은 종류에 따라 종양억제를 하는 새로운 유전자 표적으로 제안되고 있다. 최근에, 피불린-5와 상동의 피불린-3는 세포 성장 조절 및 몇몇의 종양에 작용한다고 알려져 있다. 최근에 피불린-5는 종양억제는 물론이고 반대로 세포증식, 침투, 혈관 내피 성장인자와 종양 혈관 신생에 관련이 있다고 보고되었다. 한편, 피불린-3도 세포 성장 조절과 종양 혈관 신생 및 특정 종양을 감소시키는 것이 알려져 있으나, 신경 교종 세포 및 췌장암 세포에서는 종양세포의 전이를 촉진시킨다는 보고도 있다. 따라서, 피불린-3는 암 종류에 따라 종양억제 및 전이억제 기능이 상반될 수 있어, 각각의 종양에 따라 피불린-3 기능의 규명이 필요하다.
The fibulin protein group is a protein associated with the extracellular matrix (ECM), which is a protein that contributes to intercellular interactions or interactions between cells and substrates. Six types of proteins, type 1 to type 6, are known have. These proteins are proposed as new tumor targets that inhibit tumor growth. Recently, it has been reported that pFB-5 and pFB-3 of homology act on cell growth control and some tumors. Recently, P-5 has been reported to be involved in cell proliferation, invasion, vascular endothelial growth factor and tumor angiogenesis as well as tumor suppression. On the other hand, it has been reported that PfB-3 also regulates cell growth and decreases tumor angiogenesis and specific tumors, but it has also been reported to promote metastasis of tumor cells in glioma cells and pancreatic cancer cells. Therefore, pibulin-3 may be conflicting with tumor suppression and metastasis-suppressing function depending on the type of cancer, and identification of pibulin-3 function is required depending on each tumor.
EMT(epithelial-mesenchymal transition)는 상피세포가 이동성이 증가된 중간엽성 세포(mesenchymal cell)로 변화하는 현상으로 배아 발달 및 기관 형성, 상처 치유, 조직 재생, 기관 섬유화 및 종양 악성화 및 전이 등 다양한 생명 현상 및 질병 발생과 관련이 있다. EMT 과정에는 몇몇의 RTK(receptor tyrosine kinase)와 TGF-β(transforming growth factor-β)가 관련되어 있고, 이 신호 전달의 하위 부분에 있는 세포의 상호 작용에 관련된 상피성 세포 마커인 E-카데린(cadherin) 유전자의 전사 억제자(Snail(스네일), Twist(트위스트), Zeb(젭) 등)들이 활성화되어 결국 상피성 표현형이 없어지게 되고 중간엽성 세포의 마커인 vimentin(비멘틴), fibronectin(피브로넥틴) 등은 증가하게 된다. 이는 발암 과정 동안 E-카데린의 발현과 기능에 관련이 있고 이 과정은 EMT에서 E-카데린의 발현이 줄어드는 양상을 보이기 때문에 발생과 암 형성 연구에 E-카데린의 조절이 중요하게 여겨진다. 그리고 E-카데린과 N-카데린의 발현이 쥐 배아 형성 과정에서 형태 형성의 조절 인자로 중요한 역할을 한다는 보고와 함께 상피성(epithelial)과 중간엽성(mesenchymal) 조직에서 대표적인 카데린으로 여겨지고 있다. 또한 E-카데린은 상실배를 비롯한 상피세포의 기관 형성에 중요하고 EMT 과정 동안 사라진다. N-카데린은 심장 발생과 체절 발생 그리고 신경체계의 올바른 발달에 필요하다. EMT가 일어날 때, E-에서 N-으로 바뀌고 이와 같은 과정에 있어서 어떤 특정 카데린을 올리고 내림에 따라 세포-세포 접착력이 변함을 알았으며 카데린의 전사 단계에서 발현이 조절되고 그 안정성에 영향을 주는 신호 전달에 의해 기능이 조절됨을 생각할 수 있다. EMT 유도는 조직의 기계적, 생리적 보전성을 훼손함으로써 질병을 야기할 수 있으며 대표적 질환으로 주요 장기의 섬유화 및 경변화를 포함하는 조직 섬유화증과 이행성 암 형성 및 발달을 들 수가 있다.
EMT (epithelial-mesenchymal transition) is a phenomenon in which epithelial cells are transformed into mesenchymal cells with increased mobility, and various life phenomena such as embryo development and organ formation, wound healing, tissue regeneration, organ fibrosis and tumor malignancy and metastasis And disease outbreaks. In the EMT process, several receptor tyrosine kinases (RTKs) and TGF-β (transforming growth factor-β) have been implicated and E-cadherin, an epithelial cell marker involved in cell interactions in the lower part of this signal transduction the transcriptional repressors of the cadherin gene (Snail, Twist, Zeb, etc.) are activated and eventually the epithelial phenotype disappears, and the markers of mesenchymal cells, vimentin (fibronectin) (Fibronectin) and the like are increased. This is related to the expression and function of E-cadherin during the carcinogenesis process, and the regulation of E-cadherin is considered to be important in the development and oncogenesis studies since this process appears to decrease the expression of E-cadherin in EMT. And the expression of E-cadherin and N-cadherin is considered to be a typical cadherin in epithelial and mesenchymal tissues with reports that it plays an important role as a regulator of morphogenesis in mouse embryogenesis . E-cadherin is also important for the organs of epithelial cells, including the recipient embryo, and disappears during the EMT process. N-cadherin is necessary for cardiac development, development of chracterism, and proper development of the nervous system. When EMT occurs, it changes from E- to N-, and in this process it is known that cell-cell adhesion changes with increasing or decreasing specific cadherin, and its expression is regulated at the transcriptional stage of cadherin and its stability is affected It is conceivable that the function is regulated by signaling. EMT induction can cause diseases by damaging the mechanical and physiological integrity of tissues. Representative diseases include fibrosis of the main organs, and fibrosis including transitional changes and transitional cancer formation and development.
암 줄기세포(cancer stem cell, CSCs)의 기원에 대해서는 잘 알려져 있지 않지만 상피세포에 TGF-β로 자극하여 EMT를 유도하면 암 줄기세포와 유사한 표현형을 보이므로 EMT가 암 줄기세포의 생성과 작용에도 관여할 것으로 추측하고 있다. 또한 EMT 특성을 보이는 일부 종양 세포는 항암제에 대한 내성을 보임으로써 원래 종양 조직에서 멀리 떨어진 다른 조직으로의 이동 및 전이 증거가 난소암, 대장암, 유방암 등의 종양에서 밝혀지면서 EMT는 항암제 대한 내성을 유도한다는 사실이 밝혀졌다.
Although the origin of cancer stem cells (CSCs) is not well known, induction of EMT by stimulation with TGF-β in epithelial cells produces similar phenotypes to cancer stem cells. Therefore, EMT is involved in the production and action of cancer stem cells I think it will be involved. In addition, some tumor cells showing EMT characteristics are resistant to anticancer agents, and the evidence for migration and metastasis to other tissues far from the original tumor tissue is revealed in tumors such as ovarian cancer, colorectal cancer, and breast cancer. It was revealed that it induces.
이에 본 발명자들은 피불린-3 단백질이 암세포 전이의 중요한 경로 중의 하나인 EMT 기능을 현저히 감소시켜 이동성과 같은 운동성을 저해시킴으로써 폐암 전이 억제용 조성물의 유효성분으로 사용될 수 있음을 확인함으로써 본 발명을 완성하였다.
Accordingly, the inventors of the present invention confirmed that the pibulin-3 protein can be used as an effective ingredient of a composition for inhibiting lung cancer metastasis by inhibiting mobility such as mobility by significantly reducing EMT function, which is one of important pathways of cancer cell metastasis Respectively.
본 발명의 목적은 피불린-3 단백질을 유효성분으로 하는 폐암 전이 억제용 조성물을 제공하기 위한 것이다.
It is an object of the present invention to provide a composition for inhibiting lung cancer metastasis comprising a pibulin-3 protein as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 피불린-3 단백질을 유효성분으로 함유하는 폐암 전이 억제용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for inhibiting lung cancer metastasis comprising a pibuline-3 protein as an active ingredient.
아울러, 본 발명은 피불린-3 단백질을 유효성분으로 함유하는 폐암 전이 억제용 건강식품 조성물을 제공한다.In addition, the present invention provides a health food composition for inhibiting lung cancer metastasis comprising a pibuline-3 protein as an active ingredient.
본 발명의 피불린-3(fibulin-3) 단백질은 암세포 전이의 중요한 경로 중의 하나인 EMT 기능을 현저히 감소시켜 이동성 같은 세포의 운동성을 저해시킴으로써, 폐암 전이 억제용 약학적 조성물로 유용하게 사용될 수 있다.
The fibulin-3 protein of the present invention significantly inhibits EMT function, which is one of important pathways of cancer cell metastasis, and inhibits mobility of cells such as mobility, thereby being useful as a pharmaceutical composition for inhibiting lung cancer metastasis .
도 1은 폐암 세포주(A549, H460 세포)에서 피불린-3(fibulin-3) 유전자가 과발현과 저해되었을 때 상피성 세포에서 중간엽성 세포로의 전이(EMT)와의 상관성을 보기 위해 EMT 마커를 사용하여 웨스턴 블롯을 통해 확인한 도이다:
A549: A549 세포;
pcDNA: 공벡터를 과발현시킨 대조군;
FBLN3(+): 피불린-3 벡터를 과발현시킨 세포;
H460: H460 세포;
si-ctl: si컨트롤 벡터를 발현시킨 대조군; 및
si-FBLN3: 피불린-3 발현 억제 벡터를 발현.
도 2는 A549 세포에 피불린-3 유전자를 과발현시켜서 EMT 마커인 E-카데린, N-카데린, 비멘틴, 스네일1의 발현을 형광 현미경을 사용하여 확인한 도이다:
DAPI: 4',6-디아미디노-2-페닐인돌 (4',6-diamidino-2-phenylindole); 및
FITC: 플루오레세인 이소티오시아네이트(fluorescein isothio-cyanate).
도 3은 피불린-3 유전자 과발현시 세포의 형태 변화를 확인한 도이다:
A549: A549 세포;
pcDNA: 공벡터를 과발현시킨 대조군;
si-ctl: si컨트롤 벡터를 발현시킨 대조군;
si-FBLN3: 피불린-3 발현 억제 벡터를 발현
Phase contrast: 위상차; 및
Migration: 이동.
도 4는 피불린-3 유지전의 GSK3β/β-catenin 경로와 IGF1R과 AKT/GSK3β경로에서의 역할을 확인한 도이다:
A549: A549 세포;
V.C: 컨트롤 벡터를 발현시킨 대조군;
FBLN3(+): 피불린-3 과발현 벡터를 발현;
H460: H460 세포;
si-ctl: si컨트롤 벡터를 발현시킨 대조군;
si-FBLN3: 피불린-3 발현 억제 벡터를 발현;
si-akt1: akt-1 발현 억제 벡터를 발현;
AG-1024: 3-Bromo-5-t-butyl-4-hydroxy-benzylidenemalonitrile, IGFR1 저해제; 및
A549/ALDH(+): ALDH1(aldehyde dehydrogenase 1) 활성을 갖는 A549 세포.
도 5는 폐암 세포주에 CHO 세포에서 합성된 피불린-3 단백질을 처리하였을 때 세포의 형태 변화를 확인한 도이다:
A549: A549 세포;
A549/FBLN3: A549 세포에 피불린-3 단백질 처리;
Phase contrast: 위상차;
Migration: 이동; 및
Invasion: 침윤.
도 6은 ALDH (+)세포와 ALDH (-)세포에서의 EMT 마커인 스네일, 트위스트, 젭1, 슬러그과 IGF1R/AKT/GSK3β 및 β-카테닌 단백질 발현의 변화를 확인한 도이다:
AL(+): ALDH1 활성세포; 및
AL(-): ALDH1 비활성세포.
도 7은 ALDH(+) 활성 폐암 줄기세포 전이 활성화를 확인한 도이다:
A549: A549 세포;
AL(+): ALDH1 활성세포;
AL(+)/FBLN3: ALDH1 활성 세포에 피불린-3 단백질을 처리;
Invasion: 침윤; 및
Migration: 이동.
도 8은 ALDH1(+) 세포에 CHO 세포에서 합성된 피불린-3에 의해 EMT 마커인 스네일, 트위스트, 젭1, 슬러그와 IGF1R/AKT/GSK3β 및 β-카테닌 단백질 발현의 변화를 확인한 도이다:
AL(+): ALDH1 활성세포; 및
FBLN3: 피불린-3 단백질. Figure 1 illustrates the use of EMT markers to look at the correlation of epithelial to mesenchymal cell metastasis (EMT) when the overexpression and inhibition of the fibulin-3 gene in lung cancer cell lines (A549, H460 cells) It is also confirmed by Western blot:
A549: A549 cells;
pcDNA: a control group overexpressing the empty vector;
FBLN3 (+): cells overexpressing the pibulin-3 vector;
H460: H460 cells;
si-ctl: si control vector; And
si-FBLN3: Expression of the pibulin-3 expression inhibition vector.
FIG. 2 shows the expression of EMT markers E-cadherin, N-cadherin, visentin and snail 1 by overexpressing the pibulin-3 gene in A549 cells using a fluorescence microscope:
DAPI: 4 ', 6'-diamidino-2-phenylindole; And
FITC: fluorescein isothio-cyanate.
FIG. 3 shows changes in cell morphology upon overexpression of the pibulin-3 gene:
A549: A549 cells;
pcDNA: a control group overexpressing the empty vector;
si-ctl: si control vector;
si-FBLN3: expression of the pibulin-3 expression inhibiting vector
Phase contrast: phase difference; And
Migration: Move.
Figure 4 shows the role of GSK3? /? - catenin pathway and IGF1R and AKT / GSK3? Pathways before pibuline-3 maintenance:
A549: A549 cells;
VC: control group expressing control vector;
FBLN3 (+) expresses the over-3 overexpressed vector;
H460: H460 cells;
si-ctl: si control vector;
si-FBLN3: expressing the pibulin-3 expression inhibiting vector;
Expression of si-akt1: akt-1 expression inhibition vector;
AG-1024: 3-Bromo-5-t-butyl-4-hydroxy-benzylidenemalonitrile, IGFR1 inhibitor; And
A549 / ALDH (+): A549 cells with ALDH1 (aldehyde dehydrogenase 1) activity.
FIG. 5 shows changes in cell morphology upon treatment of pibulin-3 protein synthesized from CHO cells in a lung cancer cell line:
A549: A549 cells;
A549 / FBLN3: A549 cells were treated with pibilin-3 protein;
Phase contrast: phase difference;
Migration: Moving; And
Invasion.
FIG. 6 shows changes in the expression of sNail, twist, 젭 1, slug and IGF1R / AKT / GSK3β and ß-catenin proteins, which are EMT markers in ALDH (+) and ALDH
AL (+): ALDH1 active cells; And
AL (-): ALDH1 inactive cells.
FIG. 7 shows activation of ALDH (+) activated lung cancer stem cell metastasis;
A549: A549 cells;
AL (+): ALDH1 active cells;
AL (+) / FBLN3: treatment of PBL-3 protein with ALDH1 active cells;
Invasion: invasion; And
Migration: Move.
8 is a graph showing changes in the expression of EMT markers snail, twist, 젭 1, slug and IGF1R / AKT / GSK3β and β-catenin protein by pibulin-3 synthesized in CHO cells in ALDH1 (+) cells :
AL (+): ALDH1 active cells; And
FBLN3: Pibulin-3 protein.
이하, 본 발명을 상세히 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명은 피불린-3(fibulin-3) 단백질을 유효성분으로 함유하는 폐암 전이 억제용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for inhibiting lung cancer metastasis comprising fibulin-3 protein as an active ingredient.
상기 피불린-3 단백질은 하기 1) 내지 3)의 아미노산 서열로부터 선택되는 어느 하나의 아미노산 서열을 갖는 것이 바람직하나 이에 한정되지 않는다:The pibulin-3 protein preferably has an amino acid sequence selected from the amino acid sequences of the following 1) to 3), but is not limited thereto:
1) 서열번호 1로 기재되는 아미노산 서열;1) an amino acid sequence represented by SEQ ID NO: 1;
2) 서열번호 1의 일부분으로 나타내는 아미노산 서열; 및2) an amino acid sequence represented by a part of SEQ ID NO: 1; And
3) 서열번호 1과 80 % 이상의 상동성을 나타내는 아미노산 서열.3) an amino acid sequence having 80% or more homology with SEQ ID NO: 1.
상기 암은 폐암인 것이 바람직하고, 비소세포성 폐암인 것이 더욱 바람직하나 이에 한정되지 않는다. The cancer is preferably lung cancer, more preferably non-small cell lung cancer, but is not limited thereto.
상기 피불린-3 단백질은 EMT(epithelial-mesenchymal transition) 억제 활성을 갖는 것이 바람직하나, 이에 한정되지 않는다.
The pibulin-3 protein preferably has an epithelial-mesenchymal transition (EMT) inhibitory activity, but is not limited thereto.
본 발명의 구체적인 실시예에 있어서, 본 발명자들은 비소세포성 폐암 세포주에서 EMT 마커 발현량을 확인하기 위하여, 피불린-3 발현량이 상대적으로 낮은 A549 세포에는 피불린-3 과발현용 벡터를 형질도입하여 상기 유전자의 과발현을 유도하였고(A549/FBLN3(+)), 피불린-3 발현량이 높은 H640 세포에는 피불린-3 유전자의 siRNA를 형질도입하여 상기 유전자의 발현을 억제하였다(H640/si-FBLN3). 웨스턴 블롯(western blotting) 및 형광 현미경 관찰을 실시하여 EMT marker들의 단백질 발현량을 비교하였다. 그 결과, A549 세포에서는 피불린-3 유전자가 과발현 되었을 경우 EMT 마커의 종류로 중간엽성 세포의 단백질 마커인 N-카데린, 비멘틴(vimentin), 트위스트(twist), 젭1(Zeb1), 스네일1(snail1), 슬러그(slug) 단백질의 발현량이 감소하였고, 피불린-3 유전자가 억제되었을 경우 중간엽성 세포의 단백질 마커인 E-카데린의 발현량이 증가됨을 확인하였다(도 1 및 2 참조). 이러한 결과를 통하여, 피불린-3 유전자가 EMT 마커의 종류 중 중간엽성 세포의 단백질 마커인 N-카데린, 비멘틴, 트위스트, 젭1, 스네일1, 슬러그 단백질의 발현량은 감소시키고, 상피성 세포의 단백질 마커인 E-카데린 단백질의 발현량은 증가시키는 것을 알 수 있다.In a specific example of the present invention, in order to confirm the expression level of EMT marker in the non-small cell lung cancer cell line, the inventors transfected a vector for overexpression of pibuline-3 in A549 cells having a relatively low expression level of pibulin-3 (H640 / si-FBLN3 (+)) was induced by overexpression of the gene (A549 / FBLN3 (+)), and siRNA of the pibulin- ). Western blotting and fluorescence microscopy were performed to compare protein expression levels of EMT markers. As a result, in A549 cells, when the pibulin-3 gene was overexpressed, the types of EMT markers were N-cadherin, vimentin, twist, Zeb1, It was confirmed that the expression level of nail 1 (snail1) and slug protein decreased and that the expression of E-cadherin, a protein marker of mesenchymal cells, was increased when the pibulin-3 gene was inhibited (see FIGS. 1 and 2 ). These results indicate that the expression of the PfuBuRin-3 gene decreases the expression level of N-cadherin, visentin, twist, 젭 1, snail 1, and slug protein, which are protein markers of mesenchymal cells among the types of EMT markers, The expression level of E-cadherin protein, which is a protein marker of sperm cells, is increased.
또한, 본 발명자들은 세포 형태(morphology) 변화를 확인하기 위하여, A549세포에는 피불린-3 유전자를 과발현시키고, H640 세포에는 피불린-3 유전자를 억제시킨 다음, 48시간 후에 A549/FBLN3(+) 및 H460/si-FBLN3 세포를 현미경으로 관찰한 결과, A549/FBLN3(+) 세포는 상피세포와 같은 형태(epithelium-like morphology)로 변화하였고, H460/si-FBLN3 세포는 스핀들 모양과 같은 형태(spindle-shaped-like morphology)로 변화됨을 확인하였다. 또한, 세포의 이동성을 통하여 A549/FBLN3(+) 세포의 경우 운동성이 현저하게 감소되었고, H460/si-FBLN3 세포의 경우에는 운동성이 증가함을 확인함으로써, 피불린-3가 EMT가 유도되었을 때 세포의 형태가 변화해 세포-세포 부착성에도 영향을 미쳐, 그것이 세포의 이동성을 저해되는 것을 알 수 있다(도 3 참조). 또한, 비소세포성 폐암 세포에서 피불린-3에 의한 종양발생 및 증식에 중요한 GSK3β/β-카테닌, IGF1R 및 AKT/GSK3β 단백질에 대한 항체를 사용하여 웨스턴 블롯을 실시한 결과, 피불린-3가 과발현되면 IGF1R/AKT/GSK3β의 인산화 단백질의 발현이 감소하고, β-카테닌의 발현도 감소하였다. 반면에, 피불린-3가 저해되면 상기 단백질들의 발현이 증가됨을 알 수 있다. 또한, AKT의 발현을 저해시켰을 때, IGF1R/AKT/GSK3β, β-카테닌, 스네일1 모두 감소하며 IGF1R 억제제인 AG1024를 처리하였을 경우, A549 세포와 ALDH1(+) 활성세포에서 IGF1R/AKT/GSK3β, 스네일1이 모두 현저하게 감소됨을 확인하였다. 이러한 결과를 통하여, 피불린-3은 GSK3β/베타-카테닌 경로의 중개자이며 IGF1R과 AKT/GSK3β의 상위 조절자로, 피불린-3에 의해 저해됨을 알 수 있다(도 4 참조).In order to confirm morphology changes, the inventors of the present invention overexpressed the pibulin-3 gene in A549 cells, suppressed the pibulin-3 gene in H640 cells, and then transfected with A549 / FBLN3 (+ And H460 / si-FBLN3 cells underwent microscopic observation. As a result, A549 / FBLN3 (+) cells changed into epithelium-like morphology, and H460 / si-FBLN3 cells showed a spindle- spindle-shaped-like morphology). In addition, the mobility of A549 / FBLN3 (+) cells was markedly decreased by the mobility of cells, and the motility was increased in H460 / si-FBLN3 cells. When PbuLin-3 was induced by EMT The shape of the cell changes to affect the cell-cell adhesion, and it can be seen that it inhibits cell mobility (see FIG. 3). In addition, Western blotting using anti-GSK3? /? -Catenin, IGF1R and AKT / GSK3? Proteins, which are important for tumorigenesis and proliferation by pibuline-3 in non-small cell lung cancer cells, , The expression of the phosphorylated protein of IGF1R / AKT / GSK3β was decreased and the expression of β-catenin was also decreased. On the other hand, inhibition of pibuline-3 indicates that the expression of the proteins is increased. In addition, when IGF1R / AKT / GSK3β, β-catenin, and Snale 1 decreased and AG1024, an IGF1R inhibitor, was treated with inhibition of AKT expression, IGF1R / AKT / GSK3β , And snail 1 were all significantly reduced. These results indicate that pibulin-3 is mediated by the GSK3? / Beta-catenin pathway and is an upstream regulator of IGF1R and AKT / GSK3?, And is inhibited by pibulin-3 (see FIG. 4).
본 발명자들은 세포 형태, 이동 및 침윤 분석을 통하여 CHO 세포에서 합성된 피불린-3 단백질을 폐암 세포주 처리하였을 때 변화를 확인한 결과, 스핀들 모양과 같은 형태가 상피성 세포 형태로 변화가 진행되며 이동성과 침윤된 세포수가 현저히 감소함을 확인하였다(도 5 참조). 또한, 폐암 줄기세포의 활성을 가지고 있는 ALDH(+) 세포와 활성을 가지고 있지 않은 ALDH(-) 세포에서 EMT 마커의 발현량을 확인한 결과, 폐암 줄기세포의 활성을 가지고 있는 ALDH(+) 세포에서 스네일1, 트위스트, 젭1, 슬러그의 단백질 발현이 증가하였고, IGF1R/AKT/GSK3β과 베타-카테닌 발현도 증가됨을 확인함으로써, 이동성과 침윤이 증가되는 것을 알 수 있었다(도 6 참조). 또한, ALDH(+) 활성 폐암 줄기세포 전이 활성화을 매트리젤 키트(matrigel kit)를 사용하여 확인하였다. 그 결과, A549 세포보다 폐암 줄기세포의 활성을 가지고 있는 ALDH(+) 활성 세포에서 침윤과 이동성이 현저히 증가하였고, CHO 세포에서 합성된 피불린-3 단백질을 ALDH(+) 활성 세포에 처리하였을 때, 세포의 침윤 및 이동성이 현저히 감소함을 확인한 결과, 폐암 줄기 세포에서도 피불린-3가 EMT 감소에 깊은 연관성이 있음을 알 수 있다. 또한, 피불린-3에 의해 EMT 마커와 관련된 단백질 발현량을 확인한 결과, 폐암줄기세포의 활성을 가지고 있는 ALDH(+) 활성세포에서도 CHO 세포에서 합성된 피불린-3에 의해 스네일1, 스위스트, 젭1, 슬러그의 단백질 발현이 저해되는 것을 알 수 있었고, IGF1R/AKT/GSK3β와 β-카테닌 단백질 발현도 저해되는 것을 알 수 있었다. 이러한 결과를 통하여, ALDH(+)의 활성을 기반으로 분리된 폐암 줄기세포에서도 CHO 세포에서 합성된 피불린-3는 EMT의 기능을 감소시키는데 관련이 있음을 알 수 있다.The inventors of the present invention found that the pibulin-3 protein synthesized in CHO cells through cell type, migration and infiltration analysis was changed when treated with a lung cancer cell line. As a result, the morphology of the spindle shape changed into the epithelial cell shape, And the number of infiltrated cells was markedly decreased (see FIG. 5). In addition, the expression level of EMT markers in ALDH (+) cells that have lung cancer stem cell activity and ALDH (-) cells that do not have activity showed that ALDH (+) cells having lung cancer stem cell activity It was found that the protein expression of snail 1, twist, 젭 1, and slug was increased, and IGF1R / AKT / GSK3β and β-catenin expression were also increased, thereby increasing mobility and invasion (see FIG. 6). Activation of ALDH (+) active lung cancer stem cell transfection was also confirmed using a matrigel kit. As a result, infiltration and mobility were significantly increased in ALDH (+) -activated cells having lung cancer stem cell activity than A549 cells. When the PBO-3 protein synthesized in CHO cells was treated with ALDH (+) activated cells , And cell infiltration and mobility were significantly reduced. As a result, it was also found that P-Bull-3 was strongly related to EMT reduction in lung cancer stem cells. In addition, as a result of confirming the amount of EMT marker-related protein expressed by pibuline-3, ALDH (+) -activated cells having the activity of lung cancer stem cells were also found to be able to express sNail 1, And IGF1R / AKT / GSK3β and β-catenin protein were also inhibited. Based on these results, it can be seen that pibulin-3 synthesized in CHO cells is also involved in reducing the function of EMT in isolated lung cancer stem cells based on the activity of ALDH (+).
따라서, 본 발명의 피불린-3 단백질은 암세포 전이의 중요한 경로 중의 하나인 EMT 기능을 현저히 감소시켜 이동성 같은 세포의 운동성을 저해시킴으로써, 이를 암 전이 억제용 약학적 조성물로서 유용하게 사용될 수 있다.
Therefore, the fibrin-3 protein of the present invention significantly reduces the EMT function, which is one of important pathways of cancer cell metastasis, and inhibits the mobility of cells such as mobility, thus being useful as a pharmaceutical composition for inhibiting cancer metastasis.
본 발명의 조성물의 치료적으로 유효한 양은 여러 요소, 예를 들면 투여방법, 목적부위, 환자의 상태 등에 따라 달라질 수 있다. 따라서, 인체에 사용 시 투여량은 안전성 및 효율성을 함께 고려하여 적정량으로 결정되어야 한다. 동물실험을 통해 결정한 유효량으로부터 인간에 사용되는 양을 추정하는 것도 가능하다. 유효한 양의 결정시 고려할 이러한 사항은, 예를 들면 Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; 및 E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.에 기술되어있다.The therapeutically effective amount of the composition of the present invention may vary depending on a variety of factors, such as the method of administration, the site of administration, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined in consideration of safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. Such considerations in determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman ' s Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E.W. Martin ed., Remington ' s Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
본 발명의 조성물은 또한 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 약제학적으로 허용 가능한 담체는 조성물을 생체 내 전달에 적합한 것이면 특별히 제한되지 않으며, 예를 들면, Merck Index, 13th ed., Merck & Co. Inc.에 기재된 화합물, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.Compositions of the present invention may also include carriers, diluents, excipients, or a combination of two or more thereof commonly used in biological formulations. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, for example, Merck Index, 13th ed., Merck & Sodium chloride, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. If necessary, antioxidants, buffers, And other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules or tablets. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) in a suitable manner in the art.
본 발명의 조성물에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. 본 발명의 조성물은, 조성물 총 중량에 대하여 상기 단백질을 0.0001 내지 10 중량 %로, 바람직하게는 0.001 내지 1 중량 %를 포함한다. The composition of the present invention may further contain one or more active ingredients showing the same or similar functions. The composition of the present invention contains 0.0001 to 10% by weight, preferably 0.001 to 1% by weight of the protein, based on the total weight of the composition.
본 발명의 조성물은 목적하는 방법에 따라 비 경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 적용)하거나 경구 투여할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 다양하다. 본 발명에 따른 조성물의 일일 투여량은 0.0001 ~ 10 ㎎/㎖이며, 바람직하게는 0.0001 ~ 5 ㎎/㎖이며, 하루 일 회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다.
The composition of the present invention may be administered orally or non-orally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally administered in accordance with a desired method, and the dose may be appropriately determined depending on the patient's body weight, The range varies depending on diet, time of administration, method of administration, excretion rate, and severity of the disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, more preferably administered once to several times a day.
또한, 본 발명은 피불린-3 단백질을 유효성분으로 함유하는 폐암 전이 억제용 건강식품 조성물을 제공한다.In addition, the present invention provides a health food composition for inhibiting lung cancer metastasis comprising a pibuline-3 protein as an active ingredient.
상기 피불린-3 단백질은 하기 1) 내지 3)의 아미노산 서열로부터 선택되는 어느 하나의 아미노산 서열을 갖는 것이 바람직하나 이에 한정되지 않는다:The pibulin-3 protein preferably has an amino acid sequence selected from the amino acid sequences of the following 1) to 3), but is not limited thereto:
1) 서열번호 1로 기재되는 아미노산 서열;1) an amino acid sequence represented by SEQ ID NO: 1;
2) 서열번호 1의 일부분으로 나타내는 아미노산 서열; 및2) an amino acid sequence represented by a part of SEQ ID NO: 1; And
3) 서열번호 1과 80 % 이상의 상동성을 나타내는 아미노산 서열.3) an amino acid sequence having 80% or more homology with SEQ ID NO: 1.
상기 암은 폐암인 것이 바람직하고, 비소세포성 폐암인 것이 더욱 바람직하나 이에 한정되지 않는다. The cancer is preferably lung cancer, more preferably non-small cell lung cancer, but is not limited thereto.
상기 피불린-3 단백질은 EMT(epithelial-mesenchymal transition) 억제 활성을 갖는 것이 바람직하나, 이에 한정되지 않는다.
The pibulin-3 protein preferably has an epithelial-mesenchymal transition (EMT) inhibitory activity, but is not limited thereto.
본 발명의 피불린-3 단백질은 암세포 전이의 중요한 경로 중의 하나인 EMT 기능을 현저히 감소시켜 이동성 같은 세포의 운동성을 저해시킴으로써, 이를 폐암 전이 억제용 건강식품 조성물로서 유용하게 사용될 수 있다.
The pibulin-3 protein of the present invention significantly reduces EMT function, which is one of important pathways of cancer cell metastasis, and inhibits mobility of cells such as mobility, and thus can be usefully used as a health food composition for inhibiting lung cancer metastasis.
이하, 본 발명을 실시예, 실험예 및 제조예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.
단, 하기 실시예, 실험예 및 제조예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예, 실험예 및 제조예에 의해 한정되는 것은 아니다.
However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples, Experimental Examples and Preparation Examples.
<< 실시예Example 1> 1> 피불린Blood -3 유전자의 과발현 또는 발현 억제 유도 -3 gene overexpression or suppression of expression
<1-1> A549 세포에서의 ≪ 1-1 > 피불린Blood -3 유전자의 과발현 유도-3 Overexpression of the gene
A549 세포에서 피불린-3 유전자를 인위적으로 과발현시키기 위하여, 피불린-3 유전자의 과발현 벡터를 제작하여 이를 A549 세포에 형질도입하였다. A549 cells were transfected with A549 cells by overexpressing the pibulin-3 gene to artificially overexpress the pibulin-3 gene in A549 cells.
구체적으로, 폐암세포 H460(ATCC no. HTB-177)로부터 High Pure RNA Isolation Kit(Roche, Germany)를 이용하여 전체 RNA를 추출한 뒤, Transcriptor First Strand cDNA Synthesis Kit(Roche, Germany)를 이용하여 전체 RNA 1 ㎍에 대해 역전사 중합효소연쇄반응(reverse transcription polymerase chain reaction, RT-PCR)을 수행하였다. PCR 조건은 55℃에서 30분, 및 85℃에서 5분간으로 하여, cDNA를 합성함으로써 수득하였다. 상기 cDNA를 주형으로 하여, 5'-말단에 HindⅢ 제한효소인식서열과 3'-말단에 XhoI 제한효소인식서열을 포함하도록 제작된 정방향 프라이머 5'-ATATAAGCTTATGTTGAAAGCCC-3'(서열번호 2) 및 역방향 프라이머(5'-ATATCTCGAGCTAAAATGAAAATGGCCCC-3' (서열번호 3)의 프라이머쌍을 이용하여 94℃에서 5분간 전변성시킨 후, 94℃에서 1분 변성, 56℃에서 1분간 풀림, 72℃에서 1분 30초간 신장을 수행하는 조건으로 30회 반복한 다음, 72℃에서 5분 동안 후신장을 수행하였다. 이와 같은 조건으로 PCR을 수행함으로써, 1,482bp의 피불린-3 유전자를 수득하였다. 상기 수득한 PCR 산물 및 pcDNA3.1(invitrogen, USA) 벡터를 각각 제한효소로 처리한 후, 혼합하여 리가아제로 연결시킴으로써 피불린-3 과발현용 pcDNA3.1/fibulin-3 벡터를 제작하였다. 그리고 나서, 1.5×105/㎖의 A549 세포에 상기 피불린-3 과발현용 벡터 2㎍을 Lipofecamine 2000을 이용하여 페니실린-스트렙토마이신(penicillin- streptomycin solution)(Hyclone)이 첨가되지 않은 배지에서 형질도입하였고, 4 ~ 6시간 반응시킨 후, 100 units/㎖ 페니실린-스트렙토마이신을 첨가한 배지로 교체하여 48시간 배양하였다.
Specifically, total RNA was extracted from lung cancer cell line H460 (ATCC no. HTB-177) using High Pure RNA Isolation Kit (Roche, Germany), and then total RNA was extracted using Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany) (RT-PCR) was performed on 1 μg of the reaction mixture. The PCR conditions were obtained by synthesizing cDNA at 55 캜 for 30 minutes and at 85 캜 for 5 minutes. (SEQ ID NO: 2) and a forward primer 5'-ATATAAGCTTATGTTGAAAGCCC-3 '(SEQ ID NO: 2) prepared to include a Hind III restriction enzyme recognition sequence at the 5'-end and an Xho I restriction enzyme recognition sequence at the 3'- Denatured at 94 ° C for 5 minutes using a primer pair of reverse primer (5'-ATATCTCGAGCTAAAATGAAAATGGCCCC-3 '(SEQ ID NO: 3)), denatured at 94 ° C for 1 minute, annealed at 56 ° C for 1 minute, The PCR was carried out under the same conditions as above for 30 minutes, followed by extension at 72 ° C for 5 minutes. Thus, a 1,482 bp pibulin-3 gene was obtained. PCR products and pcDNA3.1 (Invitrogen, USA) vectors were treated with restriction enzymes, respectively, and ligated with ligase to construct pcDNA3.1 / fibulin-3 vector for overexpression of pbuLin-3. × 10 5 / ㎖ of vectors for over-expressing -3 referred to the blood in A549 cells 2 을 was transfected using Lipofecamine 2000 in a medium not supplemented with penicillin-streptomycin solution (Hyclone), reacted for 4 to 6 hours, added with 100 units / ml penicillin-streptomycin And the cells were cultured for 48 hours.
<1-2> <1-2> siRNAsiRNA 를 이용한 Using H460H460 세포에서의 In the cell 피불린Blood -3 유전자의 발현 억제-3 gene expression inhibition
H460 세포에 대하여 피불린-3 siRNA를 사용하여 피불린-3의 발현을 억제하였다. The expression of piburin-3 was inhibited by using piburin-3 siRNA against H460 cells.
구체적으로, 피불린-3 유전자에 대한 siRNA는 Invitrogen사에 유전자 서열정보를 제공한 후, 맞춤 서비스로 제작 주문한 정방향 프라이머 5'-UAGAAUGUAGGGAUCUUGACAAGG-3' (서열번호 4)와 역방향 프라이머 5'-CCUUGUCAAGAUCCCUACAUUCUAA-3' (서열번호 5)인 각각 25머(mer)인 fibulin-3 stealth-RNAi를 사용하였다. 상기 siRNA의 세포 내 도입은 H460 세포 2×105 개당 각각의 RNAi를 80 nM씩 취해 Lipofectamine RNAi MAX(invitrogen)를 이용하여 실시하였고, Scrambled StealthTM RNA 분자(molecule)을 도입한 세포(음성 대조군; siControl)는 Scrambled StealthTM RNA 분자를 80 nM 취해 Lipofectamine RNAi MAX(invitrogen)를 이용하여 형질도입하였다. 피불린-3 siRNA 이중 올리고리보뉴클레오티드는 페니실린-스트렙토마이신이 첨가되지 않은 배지에서 세포에 형질도입하여 4~6시간 반응시킨 후, 100 units/㎖ 페니실린-스트렙토마이신을 첨가한 배지로 교체하여 72시간 배양하였다.
Specifically, the siRNA for the pibulin-3 gene was amplified by PCR using the forward primer 5'-UAGAAUGUAGGGAUCUUGACAAGG-3 '(SEQ ID NO: 4) and the reverse primer 5'-CCUUGUCAAGAUCCCUACAUUCUAA- Fibulin-3 stealth-RNAi, which is 25 mer each of 3 '(SEQ ID NO: 5), was used. Intracellular introduction of the siRNA was carried out by taking 80 nM of each RNAi per 2 × 10 5 H460 cells using Lipofectamine RNAi MAX (Invitrogen), and cells transfected with a Scrambled Stealth ™ RNA molecule (negative control; siControl) was transfected with 80 nM of Scrambled Stealth ™ RNA molecules using Lipofectamine RNAi MAX (Invitrogen). The pibulin-3 siRNA double oligoribonucleotide was transfected into cells in a medium supplemented with penicillin-streptomycin, allowed to react for 4 to 6 hours, replaced with medium supplemented with 100 units / ml penicillin-streptomycin, Lt; / RTI >
<< 실시예Example 2> 2> 피불린Blood -3 단백질 발현과 -3 protein expression and EMTEMT (( epithelialepithelial -- mesenchymalmesenchymal transitiontransition ) ) 마커Marker 확인 Confirm
상기 <실시예 1>에서 제조한 피불린-3 유전자를 과발현시킨 A549 세포 및 발현 억제시킨 H460 세포에 대하여, EMT marker와의 상관성을 보기 위하여 웨스턴 블롯(western blot)을 실시하였다. A549 cells overexpressing the pibulin-3 gene prepared in Example 1 and H460 cells inhibited from expression were subjected to western blotting to examine their correlation with EMT markers.
구체적으로, 1 × 107개의 A549 또는 H460 세포를 PBS로 2회 세척한 후, 단백질 분해효소 억제복합체[2 mM AEBSF, 1 mM EDTA, 130 μM Bestatin, 1 μM leupeptin, 14 μM E-64, 0.3 μM Aprotinin] 및 인산화분해효소 억제제(Roche)를 포함하는 RIPA 버퍼를 이용하여 부유하는 상기 세포를 얼음에 20분간 방치하였다가 빠르게 복텍싱(vortexing)하는 과정을 연속하여 3회 반복하였다. 그런 다음, 12,000 rpm으로 4℃에서 20분 동안 원심분리하고, 상층액을 수거한 뒤, 다시 12,000 rpm으로 4℃에서 20분 동안 원심분리하여 깨끗한 상층액만을 수득하였다. 단백질을 로우리 방법(Lowry method)으로 정량한 뒤, 10~15% 아크릴아마이드 겔로 분리하여 니트로셀룰로스(nitrocellulose) 멤브레인(Hybond; Amersham Pharmacia,USA)에 옮겼다. 단백질이 옮겨진 멤브레인에 소혈청 알부민(Bovin serum albumin, BSA, Amesco)을 사용하여 단백질 표면에 비특이적으로 항체가 결합하지 않도록 막을 입히고 1차 항체 Fibulin-3, Twist, Zeb1(santa cruz), Vimetin(thermo), Snail, E-cadherin, Slug의 1차 항체(primary antibody)를 각각 1:1000의 비율로 희석하여 4℃에서 철야(overnight) 반응 뒤 2차 항체(secondary antibody)인 토끼(rabbit)와 마우스(mouse)를 각 1:10000의 비율로 희석하여 1시간 반응 후, 필름에 감광하였다. Specifically, 1 × 10 7 A549 or H460 cells were washed twice with PBS and then treated with protease inhibitor complex [2 mM AEBSF, 1 mM EDTA, 130 μM Bestatin, 1 μM leupeptin, 14 μM E-64, 0.3 The cells were suspended in ice for 20 minutes using a RIPA buffer containing [mu] M aprotinin and a phosphorylated protease inhibitor (Roche), and vortexing was repeated three times in succession. Then, centrifugation was performed at 12,000 rpm at 4 DEG C for 20 minutes, and the supernatant was collected and centrifuged again at 12,000 rpm for 20 minutes at 4 DEG C to obtain a clean supernatant. Proteins were quantified by the Lowry method and then separated into 10-15% acrylamide gels and transferred to a nitrocellulose membrane (Hybond; Amersham Pharmacia, USA). Bovine serum albumin (BSA, Amesco) was added to the membranes from which the proteins were transferred, and the membranes were coated so that the antibodies did not bind nonspecifically on the protein surface. The primary antibodies Fibulin-3, Twist, Zeb1 (santa cruz), Vimetin ), Snail, E-cadherin, and Slug were diluted at a ratio of 1: 1000, respectively, and incubated overnight at 4 ° C. Then, secondary antibodies (rabbit and mouse) (mouse) was diluted at a ratio of 1: 10000 for 1 hour, and then sensitized to a film.
그 결과, 도 1에 나타낸 바와 같이, 피불린-3 유전자를 과발현시킨 A549 세포에서 중간엽성(mesenchymal) 마커인 N-카데린(cadherin), 비멘틴(Vimentin), 트위스트(Twist), 젭1(Zeb1), 스네일1(Snail1), 슬러그(Slug)의 발현이 감소하였고, 반대로 피불린-3 유전자를 억제시킨 H460 세포에서 상기 중간엽성(mesenchymal) 단백질 마커들이 증가함을 확인하였다(도 1).
As a result, as shown in Fig. 1, mesenchymal markers such as cadherin, vimentin, twist, and 젭 1 (Fig. 1) were detected in A549 cells over- The expression of Zeb1, Snail1, and Slug was decreased and the mesenchymal protein markers were increased in H460 cells inhibiting the pibulin-3 gene (FIG. 1) .
<< 실시예Example 3> 3> 피불린Blood -3의 과발현시 Overexpression of -3 EMTEMT markermarker 발현 감소의 재확인 Reaffirmation of decreased expression
상기 <실시예 1>에서 제조한 피불린-3 유전자를 과발현시킨 5 × 105 개의 5A549 세포에 대하여, EMT 마커인 E-카데린, N-카데린, 비멘틴, 스네일1의 발현을 형광 현미경을 통하여 확인하였다. The expression of E-cadherin, N-cadherin, visentin, and snail 1 as EMT markers was detected in 5 × 10 5 5A549 cells overexpressing the pibulin-3 gene prepared in Example 1, It was confirmed through a microscope.
구체적으로, 피불린-3 유전자 4㎍을 과발현 후, 포르말린 용액을 사용하여 세포를 고정시킨 뒤 Triton X-100을 이용해 세포막을 얇게 만든 후 BSA를 이용하여 블로킹(blocking)하였다. 각각의 항체를 1:200로 희석시킨 후 4℃에서 철야(overnight)하였다. FITC가 붙어있는 2차 토끼(rabbit) 항체를 사용하였고 DAPI를 1:1000로 희석하여 10분 반응시켰다. PBS로 여러번 세척 후 형광 현미경을 사용하여 관찰하였다.Specifically, after overexpression of 4 피 of PfuLin-3 gene, the cells were fixed with formalin solution, and the cell membrane was thinned using Triton X-100, followed by blocking with BSA. Each antibody was diluted 1: 200 and then overnight at 4 ° C. A secondary rabbit antibody with FITC was used and DAPI was diluted to 1: 1000 and reacted for 10 minutes. After washing several times with PBS, they were observed using fluorescence microscope.
그 결과, 도 2에 나타낸 바와 같이, 피불린-3 유전자가 과발현하면 대조군보다 중간엽성(mesenchymal) 세포의 단백질 마커인 N-카데린, 비멘틴, 스네일1의 발현이 감소되는 것을 확인하였고, 상피성(epithelial) 세포의 단백질 마커인 E-카데린의 발현이 증가되는 것을 확인하였다(도 2).
As a result, as shown in FIG. 2, it was confirmed that the expression of N-cadherin, visentin and snail 1, which are protein markers of mesenchymal cells, was decreased when the pibulin-3 gene was overexpressed, It was confirmed that expression of E-cadherin, which is a protein marker of epithelial cells, is increased (Fig. 2).
<< 실시예Example 4> 4> 피불린Blood -3 유전자에 의한 세포 형태 변화와 이동 확인-3 Changes in cell shape and migration by gene
피불린-3 유전자와 세포 운동성과의 상관성을 확인하기 위하여, 상기 <실시예 1>과 동일한 방법으로 피불린-3 유전자를 A549 세포에서는 과발현시키고, H640 세포에는 발현을 억제시킨 후 48시간 후에 현미경을 사용해 세포 형태의 변화를 관찰하였다. 또한, 24 well migration kit(BD)를 사용하여 각각 5ⅹ105개를 well에 분주하고 24시간 후에 크리스탈 바이올렛 용액을 10분 반응시킨 후 3차 증류수로 3회 세척하여 현미경으로 세포의 이동성을 관찰하였다.3 gene was overexpressed in A549 cells and suppressed in H640 cells in the same manner as in Example 1 to confirm the correlation between the pibulin-3 gene and cell motility, and after 48 hours, Were used to observe changes in cell morphology. In addition, 5 × 10 5 cells were each seeded into 5 wells using a 24 well migration kit (BD). After 24 hours, the cells were reacted with crystal violet solution for 10 minutes, washed three times with distilled water and observed with a microscope.
그 결과, 도 3에 나타낸 바와 같이, 피불린-3 유전자를 과발현시킨 A549 세포는 상피세포와 같은 형태(epithelium-like morphology)로 변화하였고, 피불린-3 유전자를 억제시킨 H640 세포는 스핀들 모양 형태(spindle-shaped morphology)로 변화하는 것을 확인하였다. 또한, 피불린-3 유전자를 과발현시킨 A549 세포는 운동성이 현저히 감소되었고, 피불린 유전자를 억제시킨 H640 세포에서는 운동성이 증가함을 확인하였다(도 3).As a result, as shown in FIG. 3, A549 cells overexpressing the pibulin-3 gene changed into epithelium-like morphology, and H640 cells suppressed the pibulin-3 gene showed spindle-shaped (spindle-shaped morphology). In addition, A549 cells overexpressing the pibulin-3 gene showed markedly decreased mobility and increased motility in H640 cells inhibiting the phylogenetic gene (FIG. 3).
따라서, 피불린-3가 EMT와 상관성이 높아 EMT가 유도되었을 때 세포의 형태가 변화해 세포-세포 부착성에도 변화가 있으며, 이는 세포의 운동성과 깊은 관련이 있음을 확인하였다.
Therefore, it was confirmed that pibulin - 3 correlated well with EMT, and when EMT was induced, cell morphology changed and cell - cell adhesion was also changed.
<< 실시예Example 5> 5> IGF1RIGF1R // AKTAKT 의 억제에 의한 By inhibition of 피불린Blood -3의 -3 of GSK3GSK3 β 인산화 감소 확인β phosphorylation decrease confirmation
A549 세포에 피불린-3 유전자를 상기 <실시예 1>의 방법으로 과발현시키고 반대로 H460 세포에는 발현을 억제시켜 GSK3β/β-catenin 경로와 IGF1R 및 AKT/GSK3 경로에서의 피불린-3의 역할을 알아보기 위해 웨스턴 블롯을 실시하였다. p-IGF1R, IGF1R, p-AKT, AKT, p-GSK3β, GSK3β(cell signaling), Snail1(Abcam), β-catenine, cyclin D1(santa cruz)의 1차 항체를 1:1000의 비율로 희석하고 토끼와 마우스의 2차 항체로 확인하였다. 또한 바이오니아에 의뢰하여 AKT의 siRNA 뉴클레오티드를 제작하였고 A549 세포에 100nM 사용하여 발현을 저해시켰다. 그리고 5 × 105개의 A549 세포와 ALDH 활성 세포를 접종한 뒤 세포가 부착되면 24시간 동안 영양분이 없는 배지로 키워준 후 IGF1R 억제제인 AG1024를 5시간 동안 처리하여 단백질의 발현을 확인하였다.A549 cells were overexpressed by the method of Example 1 and inhibited expression in H460 cells to inhibit the GSK3β / β-catenin pathway and the role of pibulin-3 in the IGF1R and AKT / GSK3 pathway Western blotting was performed to find out. The primary antibody of p-IGF1R, IGF1R, p-AKT, AKT, p-GSK3β, GSK3β (cell signaling), Snail1 (Abcam), β-catenine and cyclin D1 (santa cruz) And secondary antibody of rabbit and mouse. In addition, AKT siRNA nucleotides were prepared by Bioneer and 100 nM was used for A549 cells to inhibit the expression. Then, 5 × 10 5 A549 cells and ALDH active cells were inoculated, and when the cells were attached, the cells were grown in a nutrient-free medium for 24 hours, and the protein expression was confirmed by treating the IGF1R inhibitor AG1024 for 5 hours.
그 결과, 도 4에 나타낸 바와 같이, 피불린-3 유전자를 과발현시킨 A549 세포에서는 IGF1R/AKT/GSK3β 단백질의 인산화가 감소하고 피불린-3 유전자를 억제시킨 H460 세포에서는 IGF1R/AKT/GSK3β 단백질의 인산화가 증가함 확인하였다. 또한, AKT 발현을 억제시킨 A549 세포에서 IGF1R/AKT/GSK3β, β-카테닌, 및 스네일1 모두 감소하고, IGF1R 억제제인 AG1024를 처리하였을 때, A549 세포 및 ALDH1(+) 활성세포에서도 IGF1R/AKT/GSK3β, β-카테닌, 및 스네일1 모두에서 현저히 감소함을 확인하였다.As a result, as shown in FIG. 4, in the A549 cells overexpressing the pibulin-3 gene, the phosphorylation of IGF1R / AKT / GSK3β protein decreased and the IGF1R / AKT / GSK3β protein Phosphorylation was increased. In addition, IGF1R / AKT / GSK3β, β-catenin, and Snale 1 decreased in A549 cells inhibiting AKT expression and IGF1R / AKT was also observed in A549 and ALDH1 (+) activated cells when IGF1R inhibitor AG1024 was treated / GSK3 [beta], [beta] -catenin, and snail 1, respectively.
따라서, 피불린-3는 GSK3β/β-카테닌 경로의 중개자이며 IGF1R/AKT/GSK3β의 상위 조절자로 피불린-3에 의해 저해됨을 확인하였다(도 4).
Thus, it was confirmed that pibulin-3 is mediated by the GSK3? /? -Catenin pathway and is inhibited by p-benzene-3 as an upstream regulator of IGF1R / AKT / GSK3? (FIG.
<< 실시예Example 6> 6> CHOCHO 세포에서 합성된 Synthesized in cells 피불린Blood -3 단백질에 의한 A549 세포 형태 변화와 이동 및 침윤 확인-3 Protein-mediated A549 cell morphology, migration and invasion
피불린 단백질을 추출해내기 위하여 his(히스)단백질을 피불린-3 유전자에 염기서열을 첨가한 히스-피불린-3를 CHO세포에 과발현시켜 100mm 플레이트를 5개 배양시킨 뒤 트립신(trypsin)을 이용해 부착된 세포를 거두어 내고 PBS로 씻어낸 뒤 1㎖의 단백질 용해버퍼를 넣어 여러번 피펫팅 후 얼음에 15분간 반응시킨 뒤 4℃에 10분 동안 원심분리하여 단백질을 추출하였다. 그런 다음, 4℃에 보관되어있던 Ni-NTA 비드(bead)를 PBS에 3번 씻어낸뒤 적당량을 덜어 피블린 단백질에 섞어주고 4℃에서 2시간동안 붙여준뒤 차가운 PBS를 이용하여 3~4번 씻어준다. 상층액을 모두 제거하고 비드만을 얻어낸 뒤 용출버퍼(Elution buffer) (250mM Imidazole buffer) 100㎕넣고 튜브를 여러번 툭툭 쳐준 뒤 상온에서 10분 반응시킨다. 4℃에서 원심분리한 후 상층액만 새로운 튜브로 옮겨 주고 4℃에서 보관한다. 추출된 피불린 단백질은 정량하여 사용하였다. CHO 세포에서 합성된 피불린-3 단백질을 A549 세포에 처리하고 48시간 후에 현미경을 사용해 세포 형태의 변화를 관찰하였다. 또한, 24 well migration kit(BD)를 사용하여 각각 5 × 105개를 well에 분주하고 24시간 후에 크리스탈 바이올렛 용액을 10분 반응시킨 후 3차 증류수로 3회 세척하여 현미경으로 세포의 이동성을 관찰하였다.In order to extract the protein to be phylogenetic, the His (Heath) protein was overexpressed in CHO cells by adding HeS-Piburin-3 with the nucleotide sequence to the P-3 gene. Five 100 mm plates were cultured and trypsin The attached cells were harvested and washed with PBS, pipetted several times with 1 ml of protein lysis buffer, allowed to react on ice for 15 minutes, and then centrifuged at 4 ° C for 10 minutes to extract the proteins. Then, wash the Ni-NTA beads stored at 4 ° C three times in PBS, add the appropriate amount to the piblene protein, paste at 4 ° C for 2 hours, wash 3 ~ 4 times with cold PBS give. Remove the supernatant and collect only the beads. Add 100 μl of elution buffer (250 mM Imidazole buffer). Tube the tube several times and incubate at room temperature for 10 minutes. After centrifugation at 4 ° C, transfer only the supernatant to a new tube and store at 4 ° C. The extracted pibuline protein was used in a quantitative manner. A549 cells were treated with pibolin-3 protein synthesized in CHO cells, and after 48 hours, changes in cell morphology were observed using a microscope. Using a 24 well migration kit (BD), 5 × 10 5 cells were added to each well. After 24 hours, the cells were reacted with a crystal violet solution for 10 minutes, washed three times with distilled water and observed with a microscope. Respectively.
그 결과, 도 5에 나타낸 바와 같이, CHO 세포에서 합성된 피불린-3 단백질을 A549 세포에 처리하면 대조군에 비해 세포 형태의 변화가 스핀들 모양 형태(spindle-shaped morphology)가 상피성 세포 형태로 변화하고 이동 및 침윤 능력이 현저히 감소함을 확인하였다(도 5).
As a result, as shown in FIG. 5, when the pibulin-3 protein synthesized in CHO cells was treated with A549 cells, a change in cell morphology was observed in spindle-shaped morphology, And the migration and infiltration ability was significantly reduced (FIG. 5).
<< 실시예Example 7> A549 세포에서 7> in A549 cells ALDH1ALDH1 활성 세포와 비활성 세포의 분리 Isolation of active and inactive cells
본 발명자들은 Aldehyde dehydrogenase(ALDH) 활성에 기초한 stem cell회사의 ALDEFLUOR 제품을 사용해 비소세포성 폐암 세포인 A549 세포에서 암 줄기세포를 구별 및 선별하였다. We used the ALDEFLUOR product from the stem cell company based on Aldehyde dehydrogenase (ALDH) activity to differentiate and select cancer stem cells from non-small cell lung cancer A549 cells.
구체적으로, 건조된 ALDEFLUOR 시약을 25 ㎕의 DMSO(Dimethylsulphoxide)를 넣어 1분 동안 상온에서 반응시키고 시약을 적주황색에서 밝은 연노랑색이 되도록 활성화하였다. 활성화된 시약에 2 M의 염산(Hydrocholoric acid)을 25 ㎕ 넣고 15분 동안 상온에서 반응시킨 뒤 360 ㎕ ALDEFLUOR assay 버퍼를 넣고 냉장보관하여 ALDEFLUOR 기질을 만들었다. 이 후, 1×106 개의 A549 세포를 원심분리기로 상층액을 분리하였다. 상층액은 버리고 ALDEFLUOR assay 버퍼 1 ml을 섞어준다. 빈 튜브를 두 개 준비해 상기의 세포와 ALDEFLUOR assay 버퍼가 섞인 시료를 각각 500 ㎕ 넣고, 상기의 한 튜브에는 활성화된 ALDEFLUOR 기질을 5 ㎕넣고 다른 한 튜브의 대조군에는 DEAB(Diethylaminobenzaldehyde) 용액을 5 ㎕넣고 섞어주었다. 두 개의 튜브를 37 ℃에서 반응한 뒤 원심분리기를 이용해 상층액을 제거하고 세포에 500 ㎕의 ALDEFLUOR assay 버퍼를 넣고 4 ℃에서 24시간 반응시켰다. 그 후 음전하 ALDH-기질인 BAAA는 수동적인 확산을 통해 살아있는 세포 내로 침투되면 세포 내의 ALDH에 의해 BAA-로 전환되면서 형광 빛을 발산하는데, 이를 FACS sorter기계(BD bioscience, United states)로 형광을 발산하는 세포를 활성 세포로 형광이 없는 세포를 비활성 세포로 분리하였다.
Specifically, the dried ALDEFLUOR reagent was reacted with 25 μl of DMSO (Dimethylsulphoxide) at room temperature for 1 minute, and the reagent was activated so as to have a bright pale yellow color in an orange-red color. 25 μl of 2 M hydrochloric acid was added to the activated reagent, and reacted at room temperature for 15 minutes. Then, 360 μl of ALDEFLUOR assay buffer was added and refrigerated to prepare ALDEFLUOR substrate. Thereafter, 1x10 6 A549 cells were separated by centrifugation. Discard the supernatant and mix 1 ml of ALDEFLUOR assay buffer. Prepare two empty tubes, add 500 μL each of the above cells and ALDEFLUOR assay buffer, add 5 μL of activated ALDEFLUOR substrate to one of the tubes, add 5 μL of DEAB (Diethylaminobenzaldehyde) solution to the other tube, I mixed it. Two tubes were incubated at 37 ° C, and the supernatant was removed using a centrifuge. 500 μl of ALDEFLUOR assay buffer was added to the cells and incubated at 4 ° C for 24 hours. Subsequently, BAAA, which is a negatively charged ALDH-substrate, is permeated into living cells through passive diffusion and is converted to BAA- by the intracellular ALDH, which emits fluorescence light, which is then emitted to a FACS sorter machine (BD bioscience, United states) Cells were separated into active cells and non - fluorescent cells into inactive cells.
<7-1> <7-1> ALDH1ALDH1 활성세포와 비활성세포에서 In active and inactive cells EMTEMT 확인 Confirm
A549 세포에서 분리한 ALDH1(+) 활성세포에서 EMT marker의 발현 변화를 상기 <실시예 1>의 방법에 따라 웨스턴 블롯을 수행하였다.The expression of the EMT marker in ALDH1 (+) -activated cells isolated from A549 cells was subjected to western blotting according to the method of Example 1 above.
그 결과, 도 6에 나타낸 바와 같이, 암 줄기세포의 활성을 가지고 있는 ALDH1(+) 세포에서 ALDH(-) 비활성세포에서 보다 EMT 마커인 스네일1, 트위스트, 젭1, 슬러그의 발현이 증가함 확인하였고, IGF1R/AKT/GSK3β 및 β-카테닌 발현도 증가함을 확인하였다(도 6).
As a result, as shown in FIG. 6, the expression of SNT 1, twist, 젭 1, and slug, which are EMT markers, increased in ALDH1 (+) cells having cancer stem cell activity than in ALDH , And IGF1R / AKT / GSK3? And? -Catenin expression were also increased (FIG. 6).
<7-2> <7-2> ALDH1ALDH1 (+) 활성세포에서 침윤((+) Active cells invasioninvasion )과 이동() And movement migrationmigration ) 확인) Confirm
A549 세포와 ALDH1(+) 활성세포를 각각 24 well migration/invasion kit(BD)를 사용하여 각각 1 × 105개를 well에 분주하고 24시간 후에 크리스탈 바이올렛 용액을 10분 반응시킨 후 3차 증류수로 3회 세척하여 현미경으로 결과를 확인하였고 동시에 ALDH1 활성세포에 피불린-3를 처리하고 24시간 후에 24 well migration kit에 1 × 105개를 well에 분주하고 24시간 후에 같은 방법을 통해 현미경으로 관찰하였다.A549 cells and ALDH1 (+) active cells were each seeded at 1 × 10 5 cells / well in a 24 well migration / invasion kit (BD), and after 24 hours, the cells were reacted with crystal violet solution for 10 minutes. The cells were washed three times and examined by microscopy. At the same time, PBDE-3 was treated with ALDH1 active cells. After 24 hours, 1 × 10 5 cells were placed in a 24-well migration kit and 24 hours later, Respectively.
그 결과, 도 7에 나타낸 바와 같이, A549 세포보다 암 줄기세포의 활성을 가지고 있는 ALDH1(+) 세포에서 이동과 침윤이 현저히 증가함을 확인하였고, ALDH1(+) 활성세포에 CHO 세포에서 합성된 피불린-3 단백질을 처리하면 이동 및 침윤이 현저히 감소함을 확인하였다.As a result, as shown in FIG. 7, it was confirmed that migration and invasion were significantly increased in ALDH1 (+) cells having cancer stem cell activity than A549 cells, and that ALDH1 (+) activated cells It was confirmed that migration and infiltration were significantly reduced when the PfuBuRin-3 protein was treated.
따라서, 암 줄기세포에서도 피불린-3는 EMT 감소에 영향을 미치는 것을 확인하였다(도 7).
Thus, it was also confirmed that P-benzyl-3 affects EMT reduction in cancer stem cells (Fig. 7).
<7-3> <7-3> ALDH1ALDH1 활성세포에서의 In active cells 피불린Blood -3과 -3 and EMTEMT 확인 Confirm
A549 세포에서 분리한 ALDH1(+) 활성세포에 CHO 세포에서 합성된 피불린-3 단백질을 처리한 후, EMT 마커의 변화를 상기 <실시예 1>의 방법에 따라 웨스턴 블롯을 수행하였다.The ALDH1 (+) -activated cells isolated from A549 cells were treated with pibuline-3 protein synthesized in CHO cells, and then the EMT marker was changed by Western blotting according to the method of Example 1 above.
그 결과, 도 8에 나타낸 바와 같이, 암 줄기세포의 활성을 가지고 있는 ALDH1(+) 세포에 피불린-3를 처리하면 스네일1, 트위스트, 젭1, 슬러그의 발현이 감소함을 확인하였고, IGF1R/AKT/GSK3β 및 β-카테닌 발현도 감소함을 확인하였다.As a result, as shown in FIG. 8, it was confirmed that the expression of Snail 1, twist, 젭 1, and slug was reduced by treatment of PBMC -3 with ALDH1 (+) cells having cancer stem cell activity, IGF1R / AKT / GSK3β and β-catenin expression were also decreased.
따라서, ALDH1(+)의 활성을 기반으로 분리된 암 줄기세포에서도 피불린-3는 EMT 기능을 감소시키는 것을 확인하였다.
Therefore, it was confirmed that PbuLin-3 also reduced EMT function in isolated cancer stem cells based on the activity of ALDH1 (+).
<< 제조예Manufacturing example 1> 약학적 제제의 제조 1> Preparation of pharmaceutical preparations
<1-1> <1-1> 산제의Sanje 제조 Produce
본 발명의 피불린-3 단백질 2 g2 g < RTI ID = 0.0 > of < / RTI &
유당 1 gLactose 1 g
상기의 성분을 혼합하고 기밀포에 충진하여 산제를 제조하였다.
The above components were mixed and packed in airtight bags to prepare powders.
<1-2> 정제의 제조<1-2> Preparation of tablets
본 발명의 피불린-3 단백질 100 ㎎100 mg < RTI ID = 0.0 > of <
옥수수전분 100 ㎎
유당 100 ㎎
스테아린산 마그네슘 2 ㎎2 mg of magnesium stearate
상기의 성분을 혼합한 후, 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다.
After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.
<1-3> 캡슐제의 제조≪ 1-3 > Preparation of capsules
본 발명의 피불린-3 단백질 100 ㎎100 mg < RTI ID = 0.0 > of <
옥수수전분 100 ㎎
유당 100 ㎎
스테아린산 마그네슘 2 ㎎2 mg of magnesium stearate
상기의 성분을 혼합한 후, 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.
After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.
<1-4> 환의 제조≪ 1-4 >
본 발명의 피불린-3 단백질 1 g1 g < RTI ID = 0.0 > of < / RTI &
유당 1.5 gLactose 1.5 g
글리세린 1 gGlycerin 1 g
자일리톨 0.5 g0.5 g of xylitol
상기의 성분을 혼합한 후, 통상의 방법에 따라 1환 당 4 g이 되도록 제조하였다.
After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.
<1-5> 과립의 제조<1-5> Preparation of granules
본 발명의 피불린-3 단백질 150 ㎎150 mg < RTI ID = 0.0 > of <
대두추출물 50 ㎎Soybean extract 50 mg
포도당 200 ㎎200 mg of glucose
전분 600 ㎎600 mg of starch
상기의 성분을 혼합한 후, 30 % 에탄올 100 ㎎을 첨가하여 섭씨 60 ℃에서 건조하여 과립을 형성한 후 포에 충진하였다.After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.
<110> Korea Atomic Energy Research Institute <120> Pharmacetical composition containing a fibulin-3 protein for inhibiting the metastasis of lung cancer <130> 12p-09-23 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 202 <212> PRT <213> Homo sapiens <400> 1 Met Cys Thr Gly Gly Cys Ala Arg Cys Leu Gly Gly Thr Leu Ile Pro 1 5 10 15 Leu Ala Phe Phe Gly Phe Leu Ala Asn Ile Leu Leu Phe Phe Pro Gly 20 25 30 Gly Lys Val Ile Asp Asp Asn Asp His Leu Ser Gln Glu Ile Trp Phe 35 40 45 Phe Gly Gly Ile Leu Gly Ser Gly Val Leu Met Ile Phe Pro Ala Leu 50 55 60 Val Phe Leu Gly Leu Lys Asn Asn Asp Cys Cys Gly Cys Cys Gly Asn 65 70 75 80 Glu Gly Cys Gly Lys Arg Phe Ala Met Phe Thr Ser Thr Ile Phe Ala 85 90 95 Val Val Gly Phe Leu Gly Ala Gly Tyr Ser Phe Ile Ile Ser Ala Ile 100 105 110 Ser Ile Asn Lys Gly Pro Lys Cys Leu Met Ala Asn Ser Thr Trp Gly 115 120 125 Tyr Pro Phe His Asp Gly Asp Tyr Leu Asn Asp Glu Ala Leu Trp Asn 130 135 140 Lys Cys Arg Glu Pro Leu Asn Val Val Pro Trp Asn Leu Thr Leu Phe 145 150 155 160 Ser Ile Leu Leu Val Val Gly Gly Ile Gln Met Val Leu Cys Ala Ile 165 170 175 Gln Val Val Asn Gly Leu Leu Gly Thr Leu Cys Gly Asp Cys Gln Cys 180 185 190 Cys Gly Cys Cys Gly Gly Asp Gly Pro Val 195 200 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> fibulin-3 forward primer <400> 2 atataagctt atgttgaaag ccc 23 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> fibulin-3 reverse primer <400> 3 atatctcgag ctaaaatgaa aatggcccc 29 <210> 4 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> fibulin-3 Stealth-RNAi forward primer <400> 4 uagaauguag ggaucuugac aagg 24 <210> 5 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> fibulin-3 Stealth-RNAi reverse primer <400> 5 ccuugucaag aucccuacau ucuaa 25 <110> Korea Atomic Energy Research Institute <120> Pharmacetical composition containing a fibulin-3 protein for inhibiting the metastasis of lung cancer <130> 12p-09-23 <160> 5 <170> Kopatentin 1.71 <210> 1 <211> 202 <212> PRT <213> Homo sapiens <400> 1 Met Cys Thr Gly Gly Cys Ala Arg Cys Leu Gly Gly Thr Leu Ile Pro 1 5 10 15 Leu Ala Phe Phe Gly Phe Leu Ala Asn Ile Leu Leu Phe Phe Pro Gly 20 25 30 Gly Lys Val Ile Asp Asp Asn Asp His Leu Ser Gln Glu Ile Trp Phe 35 40 45 Phe Gly Gly Ile Leu Gly Ser Gly Val Leu Met Ile Phe Pro Ala Leu 50 55 60 Val Phe Leu Gly Leu Lys Asn Asn Asp Cys Cys Gly Cys Cys Gly Asn 65 70 75 80 Glu Gly Cys Gly Lys Arg Phe Ala Met Phe Thr Ser Thr Ile Phe Ala 85 90 95 Val Val Gly Phe Leu Gly Ala Gly Tyr Ser Phe Ile Ile Ser Ala Ile 100 105 110 Ser Ile Asn Lys Gly Pro Lys Cys Leu Met Ala Asn Ser Thr Trp Gly 115 120 125 Tyr Pro Phe His Asp Gly Asp Tyr Leu Asn Asp Glu Ala Leu Trp Asn 130 135 140 Lys Cys Arg Glu Pro Leu Asn Val Val Pro Trp Asn Leu Thr Leu Phe 145 150 155 160 Ser Ile Leu Leu Val Val Gly Gly Ile Gln Met Val Leu Cys Ala Ile 165 170 175 Gln Val Val Asn Gly Leu Leu Gly Thr Leu Cys Gly Asp Cys Gln Cys 180 185 190 Cys Gly Cys Cys Gly Gly Asp Gly Pro Val 195 200 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> fibulin-3 forward primer <400> 2 atataagctt atgttgaaag ccc 23 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> fibulin-3 reverse primer <400> 3 atatctcgag ctaaaatgaa aatggcccc 29 <210> 4 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> fibulin-3 Stealth-RNAi forward primer <400> 4 uagaauguag ggaucuugac aagg 24 <210> 5 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> fibulin-3 Stealth-RNAi reverse primer <400> 5 ccuugucaag aucccuacau ucuaa 25
Claims (7)
A composition for inhibiting epithelial-mesenchymal transition (EMT) in vitro comprising fibulin-3 protein as an active ingredient.
2. The composition for inhibiting epithelial-mesenchymal transition (EMT) in vitro according to claim 1, wherein the fibrin-3 protein has the amino acid sequence of SEQ ID NO: 1.
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Non-Patent Citations (2)
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| Cancer Res. 66(5): 2621-2629 (2006. 3. 1.)* |
| Lung Cancer. 69(2): 239-244(2010. 8.) |
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