KR101458272B1 - Inhibitor of cancer cell growth comprising retinoid and C-Jun N-terminal kinase inhibitor - Google Patents
Inhibitor of cancer cell growth comprising retinoid and C-Jun N-terminal kinase inhibitor Download PDFInfo
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- KR101458272B1 KR101458272B1 KR1020120084546A KR20120084546A KR101458272B1 KR 101458272 B1 KR101458272 B1 KR 101458272B1 KR 1020120084546 A KR1020120084546 A KR 1020120084546A KR 20120084546 A KR20120084546 A KR 20120084546A KR 101458272 B1 KR101458272 B1 KR 101458272B1
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- inhibitor
- metalloproteinase
- retinoic acid
- kinase
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/415—1,2-Diazoles
- A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/203—Retinoic acids ; Salts thereof
Abstract
본 발명은 레티노이드와 p38 키나제 억제제 또는 C-Jun N-말단 억제제를 포함하는 암세포의 성장 억제제에 관한 것으로, 상기 암세포 성장 억제제 투여시 메트릭스 메탈로프로테이나제의 발현과 활성이 증가하고 암세포의 성장을 억제하는 효과가 뛰어나 암세포의 성장을 억제하는 의약품으로 유용하게 사용될 수 있다.The present invention relates to a cancer cell growth inhibitor comprising a retinoid and a p38 kinase inhibitor or a C-Jun N-terminal inhibitor, wherein the expression and activity of the metrix metalloproteinase is increased and the growth of cancer cells is inhibited And thus can be usefully used as a medicine for inhibiting the growth of cancer cells.
Description
본 발명은 레티노이드와 p38 키나제 억제제, C-Jun N-말단 억제제를 포함하는 암세포의 성장 억제제에 관한 것이다. 또한 본 발명은 상기 암세포 성장 억제제 투여시 메트릭스 메탈로프로테이나제의 발현과 활성이 증가하는 암세포 성장 억제제에 관한 것이다.The present invention relates to a growth inhibitor of cancer cells comprising a retinoid and a p38 kinase inhibitor, a C-Jun N-terminal inhibitor. In addition, the present invention relates to a cancer cell growth inhibitor whose expression and activity of metrix metalloproteinase increases when the cancer cell growth inhibitor is administered.
레티노이드는 비타민 A의 구조적 유사체이고, 천연 화합물 및 합성 화합물 둘 다를 포함한다. 올-트랜스-레티노산(all-trans-retinoic acid, 'ATRA'), 9-시스-레티노산, 트랜스-3,4-디데하이드로레티노산, 4-옥소 레티노산, 13-시스-레티노산 및 레티놀과 같은 레티노이드 화합물은 수많은 염증, 면역 및 구조 세포에 영향을 주는 다형질발현성 조절 화합물이다. Retinoids are structural analogs of vitamin A and include both natural and synthetic compounds. Retinoic acid, 'ATRA', 9-cis-retinoic acid, trans-3,4-didehydrolate acid, 4-oxo retinoic acid, 13-cis- Retinoid compounds, such as retinol, are multimeric expression modulating compounds that affect a number of inflammatory, immune, and structural cells.
상기 레티노이드 중 레티노산은 연골과 골격 형성에 중요한 조절자로 알려져 있고 폐암, 유방암, 두경부암, 혈구암을 포함한 다양한 종류의 암치료에 있어 효과적인 화학 요법 물질의 파생물로 알려져 있다(Kakizuka A. et al., Cell, 1991, vol. 66, p663-671). 또한 레티노산은 폐암, 유방암, 백혈병, 횡문근육종을 포함한 여러 종류의 암에서 세포의 성장과 증식을 억제한다고 보고되었다(Sun SY et al., Cancer Res, 1997, vol. 57, p4931-4939). 하지만 항암제로서 레티노이드는 다른 항암제에 비해서 치료효율이 낮아 보다 높은 치료효율을 갖는 항암제가 필요하다.Among the retinoids, retinoic acid is known to be an important regulator of cartilage and skeletal formation and is known to be a derivative of effective chemotherapeutic agents in the treatment of various types of cancer, including lung, breast, head and neck cancer, and hematologic cancer (Kakizuka A. et al. Cell, 1991, vol. 66, p663-671). In addition, retinoic acid has been reported to inhibit cell growth and proliferation in a variety of cancers, including lung cancer, breast cancer, leukemia, and rhabdomyosarcoma (Sun SY et al., Cancer Res, 1997, vol. 57, p4931-4939). However, as an anticancer agent, retinoids have lower therapeutic efficiency than other anticancer drugs, and therefore, anticancer agents having higher therapeutic efficiency are required.
따라서 본 발명이 해결하고자 하는 과제는 기존의 암세포 성장 억제제보다 향상된 암세포 성장 억제제를 제공하는 것이다. 본 발명은 암세포에서 레티노이드와 유사분열물질-활성화 단백질 인산화효소 조절제의 동시 투여로 기존 레티노이드 단독 투여보다 높은 효과의 암세포 성장 억제제를 제공한다.Accordingly, an object of the present invention is to provide an agent for inhibiting growth of cancer cells, which is superior to an existing cancer cell growth inhibitor. The present invention provides a cancer cell growth inhibitor having a higher effect than a conventional retinoid alone by simultaneous administration of a retinoid and a mitogen-activator protein kinase modulator in cancer cells.
본 발명자는 레티노이드보다 높은 치료효율의 암세포 성장 억제제를 연구하던 중, 암세포에 레티노이드를 투여했을 때 메트릭스 메탈로프로테이나제의 조절제로서 작용하고, 또한 상기 레티노이드가 유사분열물질-활성화 단백질 인산화효소 신호전달 경로의 활성을 감소시키는 것을 발견했다. 유사분열물질-활성화 단백질 인산화효소(Mitogen-Activated Protein Kinase, MAPK)는 세포의 성장, 분화, 사멸 등 거의 모든 생리현상에 핵심적인 역할을 하는 중요한 신호전달 단백질로서 암세포의 성장에 중요한 역할을 한다. 상기 결과를 통해 항암효과가 입증된 레티노이드와 유사분열물질-활성화 단백질 인산화효소 조절제로 p38 키나제 억제제와 C-Jun N-말단 키나제 억제제의 동시투여가 레티노이드 또는 상기 억제제 중 하나의 투여보다 높은 효율로 암세포 성장을 억제할 수 있을 것으로 판단하고 이에 대한 연구를 계속했다. 상기 연구의 결과, 기존 레티노이드 단독 투여보다 레티노이드와 상기 억제제의 동시 투여가 높은 효율의 암세포 성장 억제 효과를 갖는 것을 놀랍게도 발견했다.The present inventors have been studying cancer cell growth inhibitors with higher therapeutic efficiency than retinoids, and have found that when retinoids are administered to cancer cells, they act as modulators of metrix metalloproteinases and that the retinoids also act as mitotic-activated protein kinase signaling Lt; RTI ID = 0.0 > activity. ≪ / RTI > Mitogen-Activated Protein Kinase (MAPK) plays an important role in the growth of cancer cells as an important signaling protein that plays a key role in almost all physiological phenomena such as cell growth, differentiation and death. As a result, the concurrent administration of p38 kinase inhibitor and C-Jun N-terminal kinase inhibitor as a retinoid and a mitogen-activated protein kinase regulator whose anticancer efficacy has been proven results in a cancer cell with a higher efficiency than the administration of one of retinoids or the inhibitor And that it would be possible to curb growth. As a result of the above research, it was surprisingly found that simultaneous administration of retinoid and the inhibitor is more effective in inhibiting cancer cell growth than existing retinoids alone.
본 발명의 암세포는 특별히 한정되지 않지만 섬유육종, 폐암, 유방암, 백혈병, 횡문근육종, 난소암, 간암, 췌장암, 전립선암 등의 암세포를 들 수 있다. 상기 섬유육종은 악성종양으로 신체의 어떤 부위에도 발생할 수 있으며, 상기 섬유육종의 예는 특별히 한정되지 않지만 HT1080을 포함한다.The cancer cells of the present invention are not particularly limited, but include cancer cells such as fibrosarcoma, lung cancer, breast cancer, leukemia, rhabdomyosarcoma, ovarian cancer, liver cancer, pancreatic cancer and prostate cancer. The fibrous sarcoma is a malignant tumor and may occur in any part of the body. Examples of fibrosarcoma include, but are not limited to, HT1080.
레티노이드는 4개의 이소프레노이드 단위가 헤드-투-테일(head-to-tail)식으로 연결된 골격을 가지는 화합물군 중 하나이다. 비타민A는 레티놀의 생물학적 활성을 정성적으로 가리키는 레티노이드의 일반적인 용어이다. 본 발명의 레티노이드는 암세포의 성장과정에서 메트릭스 메탈로프로테이나제의 발현과 활성을 증가시킬 수 있다. 상기 레티노이드는 특별히 한정되지 않고, 예를 들면 레티놀(retinol), 레티날(retinal), 레티노산(retinoic acid), 레티놀과 지방산의 에스터, 지방족알코올과 레티노산의 에스터 등의 레티노이드 유도체, 및 비타민A 유사체(analog)를 이용할 수 있다.Retinoids are one of a group of compounds in which four isoprenoid units are linked in a head-to-tail fashion. Vitamin A is a generic term for retinoids that qualitatively indicate the biological activity of retinol. The retinoid of the present invention can increase the expression and activity of the matrix metalloproteinase in the growth process of cancer cells. The retinoid is not particularly limited and includes, for example, retinol, retinal, retinoic acid, retinol and fatty acid esters, retinoid derivatives such as esters of aliphatic alcohol and retinoic acid, and vitamin A Analogs can be used.
본원에서 사용된 용어 "p38 키나제 억제제"는 p38 키나제를 표적으로 하거나, 저하시키거나 억제하는 화합물에 관련된다. p38 키나제는 사이토카인(cytokine)이나 자외선처리, 열충격, 삼투압충격 등 스트레스 자극에 반응하는 유사분열물질-활성화 단백질 인산화효소로 세포 분열과 사멸에 관여한다. p38 키나제 억제제의 예는 특별히 한정되지 않지만 하기 화학식 1로 표시되는 SB203580가 특히 바람직하다.The term "p38 kinase inhibitor " as used herein relates to compounds that target, decrease or inhibit p38 kinase. p38 kinase is a mitogen-activated protein kinase that responds to stress stimuli such as cytokines, ultraviolet light, thermal shock, and osmotic shock, and is involved in cell division and apoptosis. Examples of the p38 kinase inhibitor are not particularly limited, but SB203580 represented by the following formula (1) is particularly preferable.
본원에서 사용된 용어 "C-Jun N-말단 키나제(JNK) 억제제"는 C-Jun N-말단 키나제를 표적으로 하거나, 저하시키거나 억제하는 화합물에 관련된다. 세린-지정 단백질 키나제인 JNK는 c-Jun 및 ATF2의 인산화 및 활성화에 관여하고, 대사, 성장, 세포 분화 및 아팝토시스에서 중요한 역할을 한다. JNK 키나제 억제제의 예는 특별히 한정되지 않지만 하기 화학식 2로 표시되는 피라졸안트론이 특히 바람직하다.The term "C-Jun N-terminal kinase (JNK) inhibitor" as used herein relates to a compound that targets, decreases or inhibits C-Jun N-terminal kinase. JNK, a serine-specific protein kinase, is involved in the phosphorylation and activation of c-Jun and ATF2 and plays an important role in metabolism, growth, cell differentiation and apoptosis. Examples of the JNK kinase inhibitor are not particularly limited, but a pyrazolanthrone represented by the following
본원에서 사용된 용어 "메트릭스 메탈로프로테이나제(Matrix Metalloproteinases)"는 아연 의존성의 효소군으로 특이적인 구조를 가지며, 촉매의 성질이 있어 종양의 침윤과 전이 과정에 중요한 역할을 한다. 메트릭스 메탈로프로테이나제 체계는 태아 발달, 장기 형성, 신생혈관 형성, 뼈 성장, 연골 개조 등 신체의 결합조직 재구성을 조절하는 다양한 과정에 참여한다. 그러나 종양과 같은 병적인 상태에서는 메트릭스 메탈로프로테이나제 체계의 조절 기능이 파괴되어 광범위한 세포외 기질의 분해, 재구성 등이 발생하게 된다. 메트릭스 메탈로프로테이나제는 분해하는 각각의 기질 종류에 따라서 메트릭스 메탈로프로테이나제-1/메트릭스 메탈로프로테이나제-8/메트릭스 메탈로프로테이나제-13/메트릭스 메탈로프로테이나제-18(interstitial collagenases), 메트릭스 메탈로프로테이나제-2/메트릭스 메탈로프로테이나제-9(gelatinases), 메트릭스 메탈로프로테이나제-3/메트릭스 메탈로프로테이나제-10(stromelysins), 엘라스타아제(elastases), 막타입 메트릭스 메탈로프로테이나제(membrane-type MMP)로 분류할 수 있으며, 이들 중 젤라티나아제(gelatinases)인 메트릭스 메탈로프로테이나제-9와 메트릭스 메탈로프로테이나제-2가 종양의 침윤과 전이에 있어서 핵심적인 중요 조절자로 알려져 있다. 메트릭스 메탈로프로테이나제의 예는 특별히 한정되지 않지만 메트릭스 메탈로프로테이나제-2, 메트릭스 메탈로프로테이나제-9를 포함한다.As used herein, the term " Matrix Metalloproteinases "is a zinc-dependent family of enzymes that has a specific structure and catalytic properties and plays an important role in tumor invasion and metastasis. The Metrix metalloproteinase system participates in a variety of processes that regulate connective tissue remodeling in the body, including fetal development, organogenesis, neovascularization, bone growth, and cartilage remodeling. However, in pathological conditions such as tumors, the regulatory function of the matrix metalloproteinase system is destroyed, resulting in the degradation and reconstruction of a wide range of extracellular matrix. The matrix metalloproteinase is a matrix metalloproteinase-1 / Metrix metalloproteinase-8 / Metrix metalloproteinase-13 / Metrix metalloproteinase-18 interstitial collagenases, matrix metalloproteinase-2 / metrix metalloproteinase-9, matrix metalloproteinase-3 / metrix metalloproteinase-10, elastase elastases, and membrane-type matrix metalloproteinases (MMPs), among which gelatinases, Metrix metalloproteinase-9 and Metrix metalloproteinase-2 Is known to be a key regulator of tumor invasion and metastasis. Examples of the matrix metalloproteinase include, but are not limited to, Metrix metalloproteinase-2, Metrix metalloproteinase-9.
본원에서 사용된 용어 "암세포 성장 억제제"는 암의 성장에 관계되는 세포의 물리적, 화학적 및/또는 생리적인 작용 등을 직접 또는 간접적으로 억제하는 어떠한 약물일 수 있다. 상기 약물 중 항암제가 치료 효과 등의 측면에서 특히 바람직하다.The term "cancer cell growth inhibitor " as used herein may be any drug that directly or indirectly inhibits the physical, chemical and / or physiological actions of cells involved in the growth of cancer. Among these drugs, anticancer agents are particularly preferable in terms of therapeutic effects and the like.
본 발명에 따르면, 메트릭스 메탈로프로테이나제의 조절제로 레티노이드 및 유사분열물질-활성화 단백질 인산화효소 조절제로 p38 키나제 억제제 또는 C-Jun N-말단 키나제 억제제를 병용하여 기존 레티노이드 단독 투여시보다 높은 효율의 암세포 성장 억제 효과를 나타내었다.In accordance with the present invention, a combination of a p38 kinase inhibitor or a C-Jun N-terminal kinase inhibitor as a regulator of metrix metalloproteinase as a retinoid and a mitogen-activating protein kinase regulator, Cancer cell growth inhibitory effect.
도면 1은 섬유육종 HT1080에 24시간 동안 레티노산의 농도를 0, 10, 20, 30, 50 μM로 다양하게 처리하여 위상차 현미경으로 관찰한 사진이다.
도면2는 섬유육종 HT1080에 24시간 동안 레티노산의 농도를 0, 10, 20, 30, 50 μM로 다양하게 처리하여 세포증식 정도를 MTT 어세이를 통해 확인한 그래프이다.
도면 3은 섬유육종 HT1080에 레티노산 50μM를 0분, 10분, 30분, 1시간, 3시간, 6시간, 12시간, 24시간 동안 처리한 것과 섬유육종 HT1080에 24시간 동안 0, 10, 20, 30, 50μM를 처리한 한 후 젤라틴 자이모그래프를 통해 메트릭스 메탈로프로테이나제-2(MMP-2)와 메트릭스 메탈로프로테이나제-9(MMP-9)의 활성 정도를 확인한 결과이다.
도면 4는 도3의 처리 결과를 image J 프로그램을 통해 정량화한 그래프이다.
도면 5는 섬유육종 HT1080에 레티노산 50μM를 0분, 10분, 30분, 1시간, 3시간, 6시간, 12시간, 24시간 동안 처리한 것과 섬유육종 HT1080에 24시간 동안 0, 10, 20, 30, 50μM를 처리한 한 후 웨스턴블럿 분석를 통해 메트릭스 메탈로프로테이나제-2(MMP-2)와 메트릭스 메탈로프로테이나제-9(MMP-9), 액틴(Actin)의 발현량을 확인한 결과이다.
도면 6은 도5의 처리 결과를 image J 프로그램을 통해 정량화한 그래프이다.
도면 7은 섬유육종 HT1080에 레티노산 50μM을 0분, 10분, 30분, 1시간, 3시간, 6시간, 12시간, 24시간 동안 처리한 후 웨스턴블럿 분석을 통해 p38 키나제와 pp38 키나제, pJNK, C-Jun N-말단 키나제(JNK)의 발현량을 확인한 결과이다.
도면 8은 섬유육종 HT1080에 24시간 동안 0, 10, 20, 30, 50μM를 처리한 한 후 웨스턴블럿 분석을 통해 p38 키나제와 pp38 키나제, pJNK, C-Jun N-말단 키나제(JNK)의 발현량을 확인한 결과이다.
도면 9는 섬유육종 HT1080에 24시간 동안 어떤 시약도 처리하지 않은 대조군과 레티노산만 50μM 처리한 것, 레티노산 50μM과 p38 키나제 억제제인 SB203580(SB)을 처리한 것, 레티노산 50μM와 C-Jun N-말단 키나제 억제제인 SP600125(SP)를 처리한 것을 젤라틴 자이모그래프를 통해 메트릭스 메탈로프로테이나제-9(MMP-9), 메트릭스 메탈로프로테이나제-2(MMP-2)의 활성 정도를 확인한 결과이다.
도면 10은 도9의 처리 결과를 image J 프로그램을 통해 정량화한 그래프이다.
도면 11은 섬유육종 HT1080에 24시간 동안 어떤 시약도 처리하지 않은 대조군과 레티노산만 50μM 처리한 것, 레티노산 50μM과 p38 키나제 억제제인 SB203580을 처리한 것, 레티노산 50μM와 C-Jun N-말단 키나제 억제제인 SP600125를 처리한 것을 웨스턴블럿 분석을 통해 pp38 키나제, pJNK, 메트릭스 메탈로프로테이나제-9(MMP-9), 메트릭스 메탈로프로테이나제-2(MMP-2), 액틴(Actin)의 발현량을 확인한 결과이다.
도면 12는 도10의 처리 결과를 image J 프로그램을 통해 정량화한 그래프이다.FIG. 1 is a photograph of a fibrosarcoma HT1080 treated with various concentrations of retinoic acid at 0, 10, 20, 30, and 50 μM for 24 hours under a phase contrast microscope.
FIG. 2 is a graph showing the degree of cell proliferation by MTT assay in various concentrations of retinoic acid at 0, 10, 20, 30, and 50 μM for 24 h in fibrous sarcoma HT1080.
Figure 3 shows the results of treatment of fibrosarcoma HT1080 with 50 μM retinoic acid for 0, 10, 30, 1, 3, 6, 12, (MMP-2) and Metrix metalloproteinase-9 (MMP-9) through gelatin zymogens after 30, 50, and 50 μM, respectively.
4 is a graph obtained by quantifying the processing result of FIG. 3 through an image J program.
Figure 5 shows the results of treatment of fibrosarcoma HT1080 with 50 μM retinoic acid at 0, 10, 30, 1, 3, 6, 12 and 24 hours and fibrous sarcoma HT1080 at 0, 10 and 20 (MMP-2), Matrix metalloproteinase-9 (MMP-9), and Actin were determined by Western blot analysis after treatment with 30, 50, Results.
6 is a graph obtained by quantifying the processing result of FIG. 5 through the image J program.
FIG. 7 shows the results of Western blot analysis after treatment with 50 μM of retinoic acid in fibrosarcoma HT1080 at 0, 10, 30, 1, 3, 6, 12, and 24 hours, followed by p38 kinase and pp38 kinase, pJNK , And C-Jun N-terminal kinase (JNK).
Figure 8 shows the expression levels of p38 kinase, pp38 kinase, pJNK, and C-Jun N-terminal kinase (JNK) by Western blot analysis after treatment with fibrosarcoma HT1080 for 24 hours at 0, 10, 20, 30, .
Fig. 9 shows the results of treatment of fibrosarcoma HT1080 in a control group treated with 50 μM of retinoic acid only, a control group of 50 μM of retinoic acid and SB203580 (SB) of p38 kinase inhibitor, 50 μM of retinoic acid, - SP600125 (SP), an end-kinase inhibitor, was used to measure the activity of Metrix metalloproteinase-9 (MMP-9) and Metrix metalloproteinase-2 (MMP-2) The result is confirmed.
FIG. 10 is a graph obtained by quantifying the processing result of FIG. 9 through an image J program.
Figure 11 shows that fibrosarcoma HT1080 was treated with 50 μM of retinoic acid, 50 μM of retinoic acid and SB203580, a p38 kinase inhibitor, 50 μM of retinoic acid and C-Jun N-terminal kinase (MMP-9), Matrix metalloproteinase-2 (MMP-2), and Actin (MMP-9) by Western blot analysis using the inhibitor SP600125 And the amount of expression was confirmed.
12 is a graph obtained by quantifying the processing result of FIG. 10 through an image J program.
이하 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 그러나, 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화될 수도 있다.
Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms.
실시예 1-1Example 1-1
본 실시예에서 사용된 인간 섬유육종 HT1080은 아메리칸 타입 컬처 콜렉션( American Type Culture Collection, 미국 버지니아주 마나사스 소재)에서 구매하였다. 상기 인간 섬유육종 HT1080은 10% 소태아혈청(Fetal bovine serum, 인비트로젠(invitrogen, 캐나다 벌링턴 소재)에서 구매)과 50㎍/㎖ 스트렙토마이신(streptomycin), 50units/㎖의 페니실린(penicillin)이 포함된 RPMI-1640(인비트로젠(incitrogen, 캐나다 벌링턴 소재)에서 구매) 배지에 37℃가 유지되는 5% 이산화탄소(CO2) 인큐베이터에서 배양했으며 밀도는 1.2×105 cell/dish의 밀도로 배양했다.Human fibrosarcoma HT1080 used in this example was purchased from the American Type Culture Collection (Manassas, Va.). The human fibrosarcoma HT1080 contains 10% fetal bovine serum (purchased from Invitrogen, Burlington, CA), 50 μg / ml streptomycin and 50 units / ml penicillin (Purchased from incitrogen, Burlington, CA) medium in a 5% carbon dioxide (CO 2 ) incubator maintained at 37 ° C and the density was cultured at a density of 1.2 × 10 5 cells / dish .
실시예 1-2Examples 1-2
실시예 1-1에서 배양된 인간 섬유육종 HT1080에 24시간 동안 레티노산의 농도를 다르게 처리하여 세포의 모양 변화를 위상차 현미경으로 관찰하였으며 MTT(3-(4,5-디메틸티아졸-2-닐)-2,5-디페닐테트라졸륨 브로마이드) 어세이(assay)를 통해 세포증식 정도를 확인하였다(도 1, 도 2). 상기 MTT 어세이는 하기의 방식으로 진행했다.The morphology of the cells was observed by phase contrast microscope by treating the human fibrosarcoma HT1080 cultured in Example 1-1 with different concentrations of retinoic acid for 24 hours, and MTT (3- (4,5-dimethylthiazol-2-yl ) -2,5-diphenyltetrazolium bromide) assay (FIG. 1, FIG. 2). The MTT assay proceeded in the following manner.
실시예 1-1에서 배양된 섬유육종 HT1080을 96웰-플레이트(well-plate)에 웰당 1.2×105셀의 밀도로 분주하여 37℃가 유지되는 5% 이산화탄소 인큐베이터에서 배양하였다. 세포가 70~80% 자랐을 때 레티노산을 처리하여 24시간 동안 배양하였으며, 배양 후 MTT 시약 Ⅰ(10㎎/㎖)를 웰당 10㎕씩 처리하여 4시간 배양하였다. 그 후 다시 MTT 시약 Ⅱ를 웰당 100㎕씩 처리하여 12시간 배양한 후 ELISA reader를 이용하여 550~600nm의 흡광도값을 측정하였다. 상기 MMT 시약 Ⅱ는 10% SDS(도데실 황산 나트륨, Sodium dodecyl sulfate)와 0.01 N 염화수소(HCl), 디메틸설폭사이드(Dimethylsulfoxide, DMSO)를 혼합한 용액이다.Examples 1-1 The fibrosarcoma HT1080 cultured in 96-well-seeded at a density of 1.2 × 10 5 cells per well in the plate (well-plate) and cultured in 5% carbon dioxide incubator that is kept 37 ℃. When cells were grown at 70 ~ 80%, retinoic acid was treated and cultured for 24 hours. After culturing, MTT reagent I (10 mg / ml) was treated with 10 쨉 l per well and cultured for 4 hours. After that, MTT reagent II was treated with 100 μl per well and cultured for 12 hours, and the absorbance value at 550 to 600 nm was measured using an ELISA reader. The MMT reagent II is a solution prepared by mixing 10% SDS (sodium dodecyl sulfate), 0.01 N HCl, and dimethylsulfoxide (DMSO).
상기 섬유육종 HT1080에 24시간 동안 레티노산의 농도를 0, 10, 20, 30, 50 μM로 다양하게 처리하여 확인한 결과 레티노산 농도 의존적으로 세포의 증식이 억제되는 것이 위상차 현미경으로 관찰되었다(도 1). 이에 세포증식 정도를 상기 MTT 어세이를 통해 확인한 결과 레티노산 처리 농도 의존적으로 세포의 증식이 억제되었으며, 특히 레티노산 50μM에서의 세포의 증식이 50%정도 억제됨을 확인하였다(도 2).The fibrous sarcoma HT1080 was treated with various concentrations of retinoic acid at concentrations of 0, 10, 20, 30, and 50 μM for 24 hours, and it was observed as a phase contrast microscope that cell proliferation was inhibited depending on the concentration of retinoic acid ). As a result, the cell proliferation was inhibited dependently on retinoic acid treatment concentration, and cell proliferation was specifically inhibited by 50% at 50 μM of retinoic acid (FIG. 2).
실시예 1-3Example 1-3
실시예 1-1에서 배양된 섬유육종 HT1080에 레티노산을 시간과 농도를 다양하게 처리한 후 메트릭스 메탈로프로테이나제의 활성 정도를 젤라틴 자이모그래프(Gelatin zymography)를 통해 확인하였다. 상기 젤라틴 자이모그래프는 하기의 방식으로 진행된다.The activity of the matrix metalloproteinase was confirmed by gelatin zymography after treating the fibrous sarcoma HT1080 cultured in Example 1-1 with various concentrations of time and concentration of retinoic acid. The gelatin zymograph proceeds in the following manner.
상기 젤라틴 자이모그래프는 10% 소태아혈청(Fetal bovine serum, 인비트로젠(invitrogen, 캐나다 벌링턴 소재)에서 구매)이 포함된 RPMI-1640(인비트로젠(incitrogen, 캐나다 벌링턴 소재)에서 구매)를 이용하여 섬유육종 HT1080을 부착시킨 후 2% 소태아혈청이 포함된 RPIM-1640으로 교환하여 12시간 배양하였다. 세포가 70~80% 자랐을 때 2% 소태아혈청이 포함된 RPMI-1640을 이용하여 레티노산을 처리하였다. 24시간 후 상등액만을 따로 모아 0.1%의 젤라틴(gelatin)이 함유된 SDS-폴리아크릴아미드 겔(SDS-polyacrylamide gel)에 전기영동하여 단백질들을 크기별로 분리하였다. 그 후 2.5% 트리톤(triton) X-100에 30분씩 3번 씻은 후 증류수로 씻어주어 SDS를 제거하였으며 5mM 염화칼슘(CaCl2), 0.2M 염화나트륨(NaCl), 50mM 트리스(히드록시메틸)아미노메탄(tris-(hydroxymethyl)-aminomethane)가 함유된 젤라틴 인큐베이션 버퍼(pH7.5)에 담가 20~24시간 동안 37℃ 인큐베이터에서 70rpm으로 쉐이킹하여 반응시켰다. 반응 후 겔을 쿠마시블루 용액으로 염색하였다.The gelatin zymogram was purchased from RPMI-1640 (purchased from incitrogen, Burlington, Canada) containing 10% fetal bovine serum (purchased from Invitrogen, Burlington, Canada) Fibrosarcoma HT1080 was stained with RPMI-1640 supplemented with 2% fetal bovine serum and cultured for 12 hours. When cells grew 70-80%, retinoic acid was treated with RPMI-1640 containing 2% fetal bovine serum. After 24 hours, only the supernatant was collected separately and electrophoresed on SDS-polyacrylamide gel containing 0.1% of gelatin to separate the proteins by size. After washing with 2.5% Triton X-100 three times for 30 minutes, the cells were washed with distilled water to remove the SDS. Then, 5 mM calcium chloride (CaCl 2 ), 0.2 M sodium chloride (NaCl), and 50 mM tris (hydroxymethyl) tris- (hydroxymethyl) -aminomethane) in a gelatin incubation buffer (pH 7.5) and shaking at 70 rpm in a 37 ° C incubator for 20 to 24 hours. After the reaction, the gel was stained with Coomassie blue solution.
또한 상기 섬유육종 HT1080에 메트릭스 메탈로프로테이나제의 발현량을 웨스턴블럿 분석(Western blot analysis)를 수행하였다. 먼저 단백질의 정량분석을 위해 50mM 트리스-염화수소(Tris-HCl) pH 7.4, 150mM 염화나트륨, 1% SDS가 함유된 세포 라이시스 버퍼(세포용해제)(cell lysis buffer)에 단백질 분해효소 억제제(10㎍/㎖ 아프로티닌, 10㎍/㎖ 류펩틴, 10㎍/㎖ 펩스타틴, 1mM 4-(2-아미노에틸) 벤젠셀포닐 플루오라이드)와 인산 분해효소 억제제(1mM 소듐 오르토 바나데이트(sodium orthovanadate), 1mM 플루오르나트륨)를 첨가하여 세포 단백질을 추출하였다. 추출한 단백질은 SDS-폴리아크릴 아미드 겔에 전기영동하여 크기별로 분리하였으며, 이를 니트로 셀룰로오즈 막으로 이동시켰다. 그 후 각 단백질 발현의 분석을 위하여 각 단백질에 특이적인 항체인 메트릭스 메탈로프로테이나제-9(미국 캘리포니아주 산타 크루즈 소재 산타 크루즈 바이오 테크놀로지에서 구매), 메트릭스 메탈로프로테이나제-2(미국 캘리포니아주 산타 크루즈 소재 산타 크루즈 바이오 테크놀로지에서 구매), pp38 키나제(미국 매사추세츠주 댄버스 소재 셀 시그널링 테크놀로지에서 구매), p38 키나제(미국 캘리포니아주 산타 크루즈 소재 산타 크루즈 바이오 테크놀로지에서 구매), pJNK 키나제(미국 매사추세츠주 댄버스 소재 셀 시그널링 테크놀로지에서 구매), C-Jun N-말단 키나제(미국 캘리포니아주 산타 크루즈 소재 산타 크루즈 바이오 테크놀로지에서 구매), 액틴(미국 캘리포니아주 산타 크루즈 소재 산타 크루즈 바이오 테크놀로지에서 구매)을 사용하였다. Western blot analysis was also performed on the amount of expression of matrix metalloproteinase in the fibrous sarcoma HT1080. First, a proteinase inhibitor (10 μg / ml) was added to a cell lysis buffer (cell lysis buffer) containing 50 mM Tris-HCl pH 7.4, 150 mM sodium chloride, and 1% SDS for quantitative analysis of protein. (1mM sodium orthovanadate, 1mM sodium dithiothreitol, 10μg / ml leupeptin, 10μg / ml pepstatin, 1mM 4- (2-aminoethyl) benzenesulfonyl fluoride) Fluorosodium) was added to extract the cell protein. The extracted proteins were separated by size by electrophoresis on SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Then, for the analysis of each protein expression, each protein-specific antibody, Metrix Metaloproteinase-9 (purchased from Santa Cruz Biotechnology, Santa Cruz, CA), Metrix Metaloproteinase-2 (Purchased from Santa Cruz Biotechnology, Santa Cruz, CA), pp38 kinase (purchased from Cell Signaling Technologies, Danvers, Mass.), P38 kinase (purchased from Santa Cruz Biotechnology, Santa Cruz, CA), pJNK kinase Purchased from C-Jun N-terminal kinase (purchased from Santa Cruz Biotechnology, Santa Cruz, CA), and Actin (purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA) Respectively.
실시예 1-1에서 배양된 섬유육종 HT1080에 레티노산 50μM을 0분, 10분, 30분, 1시간, 3시간, 6시간, 12시간, 24시간별로 처리하여 메트릭스 메탈로프로테이나제 활성을 확인한 결과 시간 의존적으로 메트릭스 메탈로프로테이나제-9와 메트릭스 메탈로프로테이나제-2의 활성이 증가하였으며, 레티노산을 0, 10, 20, 30, 50μM농도로 24시간 처리하여 확인한 결과 농도 의존적으로 메트릭스 메탈로프로테이나제-9와 메트릭스 메탈로프로테이나제-2의 발현량이 증가함을 확인하였다(도3). 도면 3을 image J 프로그램을 이용하여 그래프로 정량화하였다(도4). 메트릭스 메탈로프로테이나제-2, 메트릭스 메탈로프로테이나제-9의 발현 정도를 상기 웨스턴블럿 분석을 통해 확인한 결과 레티노산 50μM에서 시간 의존적으로 메트릭스 메탈로프로테이나제-2, 메트릭스 메탈로프로테이나제-9의 발현량이 증가하였으며, 농도 의존적으로 메트릭스 메탈로프로테이나제-2, 메트릭스 메탈로프로테이나제-9의 발현량이 증가함을 확인할 수 있었다(도5). 상기 웨스턴블럿 분석을 수행하여 얻은 결과값은 image J 프로그램을 통해 수치화하였다(도6). 결과적으로, 레티노산은 섬유육종 HT1080에서 시간과 농도 의존적으로 메트릭스 메탈로프로테이나제-2, 메트릭스 메탈로프로테이나제-9의 활성과 발현을 증가시킴을 알 수 있었다.
The fibrous sarcoma HT1080 cultured in Example 1-1 was treated with 50 μM retinoic acid at 0, 10, 30, 1, 3, 6, 12, and 24 hours to determine the metrix metalloproteinase activity As a result, the activity of Metrix metalloproteinase-9 and Metrix metalloproteinase-2 was increased in a time-dependent manner. The concentration of retinoic acid was found to be 0, 10, 20, 30 and 50 μM for 24 hours, And the expression level of Metrix metalloproteinase-9 and Metrix metalloproteinase-2 was increased (FIG. 3). Figure 3 was quantified as a graph using the image J program (Figure 4). The degree of expression of Metrix metalloproteinase-2 and Metrix metalloproteinase-9 was examined by Western blot analysis. As a result, it was found that the concentration of Metrix metalloproteinase-2 and Metrix metalloproteinase-9 in a time- And the expression level of Metrix metalloproteinase-2 and Metrix metalloproteinase-9 was increased in a concentration-dependent manner (FIG. 5). The results obtained by performing the Western blot analysis were quantified through the image J program (Fig. 6). As a result, it was found that retinoic acid increases the activity and expression of Metrix metalloproteinase-2, Metrix metalloproteinase-9 in time and concentration-dependent manner in fibrosarcoma HT1080.
실시예 2-1Example 2-1
실시예 1-1에서 배양된 섬유육종 HT1080에서 레티노산 50μM을 다양한 시간별로 처리하여 실시예 1-3에서 사용한 웨스턴블럿 분석을 수행한 결과 시간 의존적으로 p38 키나제와 C-Jun N-말단 키나제의 활성이 감소함을 확인할 수 있었다(도7). 레티노산의 농도를 0, 10, 20, 30, 50μM로 점차 증가시켜 24시간 동안 처리한 결과, 농도 의존적으로 p38 키나제와 C-Jun N-말단 키나제의 활성이 감소함을 확인할 수 있었다(도8). 반면, ERK의 활성과 농도에 비의존적인 활성을 보였다.Western blot analysis using the fibrous sarcoma HT1080 cultured in Example 1-1 with 50 μM of retinoic acid for various time periods was carried out. As a result, the activity of p38 kinase and C-Jun N-terminal kinase (Fig. 7). When the concentration of retinoic acid was gradually increased to 0, 10, 20, 30, and 50 μM for 24 hours, it was confirmed that the activity of p38 kinase and C-Jun N-terminal kinase decreased in a concentration-dependent manner ). On the other hand, the activity and concentration of ERK were independent.
상기 실험의 결과, 레티노산과 p38 키나제, C-Jun N-말단 키나제 억제제를 함께 사용시 메트릭스 메탈로프로테이나제의 활성이 더 증가할 것이라는 가설을 가지고 다음을 진행하였다. As a result of the above experiment, the following was conducted with the hypothesis that the activity of matrix metalloproteinase would be further increased when retinoic acid and p38 kinase and C-Jun N-terminal kinase inhibitor were used together.
레티노산과 p38 키나제의 활성 억제제인 SB203580(SB)과 C-Jun N-말단 키나제의 활성 억제제인 SP600125(SP)를 처리하여 메트릭스 메탈로프로테이나제-2와 메트릭스 메탈로프로테이나제-9의 활성과 발현 정도를 실시예 1-3에서 사용한 젤라틴 자이모그래프와 웨스턴블럿 분석을 통해 확인하였다. 젤라틴 자이모그래프를 수행한 결과 섬유육종 HT1080에 어떤 시약도 처리하지 않은 대조군과 비교하여 레티노산 50μM 단독 처리시 메트릭스 메탈로프로테이나제-9, 메트릭스 메탈로프로테이나제-2의 활성이 2배 이상 증가하였고, SP600125와 SB203580을 함께 처리하여 p38 키나제와 C-Jun N-말단 키나제를 억제시켰을 때 레티노산 단독처리보다 약 1.6배 많이 메트릭스 메탈로프로테이나제-9와 메트릭스 메탈로프로테이나제-2의 활성이 증가함을 확인할 수 있었다(도9). 웨스턴블럿 분석을 수행한 결과 또한 섬유육종 HT1080에 어떤 시약도 처리하지 않은 대조군과 비교하여 레티노산 50μM 단독 처리시 메트릭스 메탈로프로테이나제-9와 메트릭스 메탈로프로테이나제-2의 발현량이 2배 이상 증가하였고, SB203580과 SP600125를 함께 처리하여 p38 키나제와 C-Jun N-말단 키나제의 활성을 저해시켰을 때 레티노산 단독 처리보다 약 1.6배 많이 메트릭스 메탈로프로테이나제-9와 메트릭스 메탈로프로테이나제-2의 발현량이 증가함을 알 수 있었다(도 11). 상기 젤라틴 자이모그래프와 상기 웨스턴블럿 분석의 결과를 image J 프로그램을 이용하여 그래프로 나타냈다(도10, 도12).
SB203580 (SB), an activity inhibitor of retinoic acid and p38 kinase, and SP600125 (SP), an activity inhibitor of C-Jun N-terminal kinase, were treated to determine the activity of Metrix metalloproteinase-2 and Metrix metalloproteinase- And degree of expression were confirmed by gelatin zymography and western blot analysis as used in Examples 1-3. As a result of gelatin zymography, the activity of matrix metalloproteinase-9 and matrix metalloproteinase-2 in fibrosarcoma HT1080 treated with 50 μM of retinoic acid alone was found to be twice as high . When SP600125 and SB203580 were treated together to inhibit p38 kinase and C-Jun N-terminal kinase, about 1.6 times more amount of metrix metalloproteinase-9 and metrix metalloproteinase- 2 was increased (Fig. 9). Western blot analysis also showed that the amount of expression of Metrix metalloproteinase-9 and Metrix metalloproteinase-2 in the treatment of fibrosarcoma HT1080 alone with 50 μM of retinoic acid was 2 times , And when SB203580 and SP600125 were treated together to inhibit the activity of p38 kinase and C-Jun N-terminal kinase, about 1.6 times more than the treatment with retinoic acid alone, Metrix metalloproteinase-9 and Metrix metalloproteinase -2 expression level was increased (FIG. 11). The results of the gelatin zymograph and Western blot analysis were plotted using the image J program (Figs. 10 and 12).
이와 같이 암세포에 레티노산, 및 SB 또는 SP를 투여했을 때 레티노산 단독 처리보다 높은 효율의 항암 작용을 나타냄을 알 수 있다.As described above, when retinoic acid and SB or SP are administered to cancer cells, it shows higher anticancer activity than retinoic acid alone treatment.
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