KR102155713B1 - Composition for preventing or treating cancer comprising sea cucumber extracts or fraction thereof, and trail protein - Google Patents
Composition for preventing or treating cancer comprising sea cucumber extracts or fraction thereof, and trail protein Download PDFInfo
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- KR102155713B1 KR102155713B1 KR1020180060366A KR20180060366A KR102155713B1 KR 102155713 B1 KR102155713 B1 KR 102155713B1 KR 1020180060366 A KR1020180060366 A KR 1020180060366A KR 20180060366 A KR20180060366 A KR 20180060366A KR 102155713 B1 KR102155713 B1 KR 102155713B1
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- trail
- sea cucumber
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Abstract
본 발명은 해삼 추출물 또는 이의 분획물, 및 TRAIL 단백질을 포함하는 암 예방, 개선 또는 치료용 조성물에 관한 것이다.
본 발명에 따른 암 예방 또는 치료용 약학 조성물, TRAIL의 항암 효과 증진용 약학 조성물, 암세포 사멸 증진 방법, 암의 예방 또는 치료 방법 및 암 예방 또는 개선용 건강기능식품 조성물은 TRAIL 내성을 극복할 수 있는 해삼 추출물 또는 이의 분획물을 포함함으로써 TRAIL에 대한 내성을 보이는 암의 예방 또는 치료에 유용하게 사용될 수 있다.The present invention relates to a composition for preventing, improving or treating cancer comprising a sea cucumber extract or a fraction thereof, and TRAIL protein.
The pharmaceutical composition for preventing or treating cancer according to the present invention, a pharmaceutical composition for enhancing the anticancer effect of TRAIL, a method for promoting cancer cell death, a method for preventing or treating cancer, and a health functional food composition for preventing or improving cancer, can overcome TRAIL resistance. By including a sea cucumber extract or a fraction thereof, it can be usefully used in the prevention or treatment of cancer showing resistance to TRAIL.
Description
본 발명은 해삼 추출물 또는 이의 분획물, 및 TRAIL 단백질을 포함하는 암 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing, improving or treating cancer comprising a sea cucumber extract or a fraction thereof, and TRAIL protein.
암은 인류의 건강을 위협하는 최대의 질병 중의 하나로서, 세포주가 일련의 돌연변이 과정을 거쳐, 무제한적이고 비조절적인 방식으로 불사화 되어 발생하는 질병이다. 암 발생의 원인으로는 화학물질, 바이러스, 세균, 전리방사선 등의 환경적 또는 외적 요인과 선천성 유전자 변이 등의 내적 요인을 들 수 있다(Klaunig & Kamendulis, Annu Rev Pharmacol Toxicol., 44:239-267, 2004).Cancer is one of the greatest diseases that threaten human health, and it is a disease that occurs when cell lines undergo a series of mutations and become immortalized in an unlimited and unregulated manner. The causes of cancer include environmental or external factors such as chemicals, viruses, bacteria, and ionizing radiation, and internal factors such as congenital genetic mutations (Klaunig & Kamendulis, Annu Rev Pharmacol Toxicol. , 44:239-267 , 2004).
초기에 발견된 암일 경우 수술, 방사선 치료, 화학적 요법 등의 치료법이 있으나 그 부작용 또한 큰 문제로 대두되고 있으며, 말기 암이나 전이된 암의 경우 특별한 치료법 없이 시한부 인생으로 삶을 마감하는 상황이다. 또한, 암과 관련된 다양한 생화학적 기전이 규명되고 그에 따른 치료제가 개발되어 오고 있으나, 아직까지 암에 대한 근본적인 치료방법은 제시되지 않고 있다.In the case of early cancer, there are treatments such as surgery, radiation therapy, and chemotherapy, but the side effects are also emerging as a big problem, and in the case of terminal cancer or metastasized cancer, life ends with a limited time without special treatment. In addition, various biochemical mechanisms related to cancer have been identified and therapeutic agents have been developed accordingly, but a fundamental treatment method for cancer has not been suggested yet.
TRAIL(Tumor necrosis factor-related apoptosis inducing ligand)은 종양괴사인자(Tumor necrosis factor, TNF)의 일종으로 종양세포의 자가 세포사멸을 유도하는 리간드로 알려져 있다. TRAIL은 4개의 수용체를 갖고 있으며, 상기 수용체 중 사멸 수용체(death receptor, DR4, DR5)는 암 세포주에서, 디코이 수용체(Decoy receptor, DcR1, DcR2)는 정상 세포주에서 과발현된다. 상기 TRAIL은 종양세포의 표면에 있는 DR4 및 DR5와 같은 사멸 수용체에 결합하여 종양세포의 사멸을 유도할 수 있다. 상기 디코이 수용체의 경우는 c-터미널 말단에 사멸 도메인을 가지고 있지 않아 세포사멸 신호전달이 세포 내로 전해지지 않는다. 따라서, TRAIL을 기반으로 하는 치료는 정상세포에 대해서는 무해하면서 다양한 암 세포주에 대해서만 독성을 유도한다는 특징을 가지고 있어 매우 효과적인 차세대 항암 치료제이다.TRAIL (Tumor necrosis factor-related apoptosis inducing ligand) is a type of tumor necrosis factor (TNF) and is known as a ligand that induces autophagy of tumor cells. TRAIL has four receptors, of which death receptors (DR4, DR5) are overexpressed in cancer cell lines, and Decoy receptors (DcR1, DcR2) are overexpressed in normal cell lines. The TRAIL can induce death of tumor cells by binding to death receptors such as DR4 and DR5 on the surface of tumor cells. In the case of the decoy receptor, since it does not have a death domain at the end of the c-terminal, apoptosis signaling is not transmitted into the cell. Therefore, TRAIL-based treatment is a very effective next-generation anti-cancer treatment, as it is harmless to normal cells and induces toxicity only to various cancer cell lines.
그러나, TRAIL을 암세포주에 지속적으로 처리하면 TRAIL에 감수성을 보이던 암세포주도 점차 TRAIL에 대해 내성을 가지게 되는 것이 밝혀지게 되었다. 구체적으로, 많은 암세포는 사멸 수용체의 감소와 세포의 FLICE 유사 억제 단백질(cellular FLICE-like inhibitory protein, c-FLIP(L)), B-세포 림포마-2(B-cell lymphoma-2, Bcl-2), B-세포 림포마 엑스트라 라지(B-cell lymphoma-extra large, Bcl-xL) 또는 골수성 백혈병 세포-1(myeloid cell leukemia-1, Mcl-1)와 같은 항-세포사멸 단백질을 증가시키는 등 다양한 매커니즘을 통하여 TRAIL에 의해 유도된 세포사멸에 저항성을 나타내고 있다.However, it was found that if TRAIL was continuously treated with cancer cell lines, cancer cell lines that were sensitive to TRAIL also gradually became resistant to TRAIL. Specifically, many cancer cells have decreased death receptors, cellular FLICE-like inhibitory protein (c-FLIP(L)) and B-cell lymphoma-2 (Bcl-). 2), increasing anti-apoptotic proteins such as B-cell lymphoma-extra large (Bcl-xL) or myeloid cell leukemia-1 (Mcl-1) It shows resistance to apoptosis induced by TRAIL through various mechanisms, such as.
따라서, 암세포에 대해 선택적으로 세포사멸을 유도할 수 있는 TRAIL의 내성을 극복하여 암세포 증식을 막고 세포사멸을 촉진시킬 수 있는 방법에 대한 연구가 필요한 실정이다.Therefore, there is a need for research on a method capable of preventing cancer cell proliferation and promoting apoptosis by overcoming the resistance of TRAIL, which can selectively induce apoptosis against cancer cells.
이러한 배경 하에, 본 발명자들은 TRAIL에 저항성을 나타내는 암에 대하여도 효과적으로 작용하는 항암 조성물을 발명하고자 연구를 수행하였으며, 이에 해삼 추출물 또는 이의 분획물이 TRAIL 저항성 암세포주에서 TRAIL 세포독성에 대한 민감성을 증가시켜, 결과적으로 아폽토시스에 의한 세포사를 증가시키는 효능이 있음을 발견하고, 이를 활용한 암의 예방, 개선 또는 치료용 조성물을 완성하기에 이르렀다.Under this background, the present inventors conducted a study to invent an anticancer composition that works effectively against cancers that are resistant to TRAIL, so that sea cucumber extract or a fraction thereof increases the sensitivity to TRAIL cytotoxicity in TRAIL-resistant cancer cell lines. , As a result, it was found that there is an effect of increasing cell death by apoptosis, and a composition for the prevention, improvement or treatment of cancer using this was found.
본 발명의 목적은 정상세포에는 영향을 주지 않고 선택적으로 암세포의 세포사멸을 유도하는 TRAIL의 내성을 극복하여 효과적으로 암세포의 세포사멸을 유도함으로써 항암 효과를 증진시킬 수 있는 암 예방 또는 치료용 약학 조성물을 제공하는 데 있다.It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer capable of enhancing the anticancer effect by effectively inducing apoptosis of cancer cells by overcoming the resistance of TRAIL, which does not affect normal cells and selectively induces apoptosis of cancer cells. To provide.
상기한 목적을 달성하기 위하여 본 발명은 해삼 추출물 또는 이의 분획물, 및 TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand)을 유효성분으로 포함하는 암 예방 또는 치료용 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising a sea cucumber extract or a fraction thereof, and TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) as an active ingredient.
또한, 본 발명은 해삼 추출물 또는 이의 분획물을 유효성분으로 포함하며, TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand)의 내성을 감소시키는 것을 특징으로 하는 TRAIL의 항암 효과 증진용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for enhancing the anticancer effect of TRAIL, comprising a sea cucumber extract or a fraction thereof as an active ingredient, and reducing resistance of TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand).
또한, 본 발명은 해삼 추출물 또는 이의 분획물, 및 TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand; Trail)을 인간을 제외한 포유동물의 암세포에 병용 투여하는 것을 특징으로 하는 암세포 사멸 증진 방법을 제공한다.In addition, the present invention provides a method for promoting cancer cell death, characterized in that sea cucumber extract or a fraction thereof, and TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand; Trail) are administered in combination to cancer cells of mammals other than humans.
또한, 본 발명은 해삼 추출물 또는 이의 분획물, 및 TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand; Trail)을 인간을 제외한 개체에 투여하는 단계를 포함하는 암의 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating cancer comprising administering a sea cucumber extract or a fraction thereof, and TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand; Trail) to individuals other than humans.
아울러, 본 발명은 해삼 추출물 또는 이의 분획물, 및 TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand)을 유효성분으로 포함하는 암 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving cancer comprising a sea cucumber extract or a fraction thereof, and TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) as an active ingredient.
본 발명에 따른 암 예방 또는 치료용 약학 조성물, TRAIL의 항암 효과 증진용 약학 조성물, 암세포 사멸 증진 방법, 암의 예방 또는 치료 방법 및 암 예방 또는 개선용 건강기능식품 조성물은 TRAIL 내성을 극복할 수 있는 해삼 추출물 또는 이의 분획물을 포함함으로써 TRAIL에 대한 내성을 보이는 암의 예방 또는 치료에 유용하게 사용될 수 있다.The pharmaceutical composition for preventing or treating cancer according to the present invention, a pharmaceutical composition for enhancing the anticancer effect of TRAIL, a method for promoting cancer cell death, a method for preventing or treating cancer, and a health functional food composition for preventing or improving cancer, can overcome TRAIL resistance. By including a sea cucumber extract or a fraction thereof, it can be usefully used in the prevention or treatment of cancer showing resistance to TRAIL.
도 1은 TRAIL에 의한 세포사멸 경로를 나타내는 모식도이다.
도 2는 해삼 분획물이 대장암의 세포성장에 영향을 미치는 정도를 나타내는 세포성장(cell viability) 그래프이다.
도 3는 대장암 세포(DLD-1, HCT116, Colo205, HT29, SW620)에서 TRAIL이 세포성장(cell viability)에 영향을 미치는 정도를 나타내는 그래프이다.
도 4는 대장암 세포(DLD-1)에서 해삼 분획물(SC F2)과 TRAIL의 조합이 세포성장(cell viability)에 영향을 미치는 정도를 나타내는 그래프이다.
도 5는 대장암 세포주(DLD-1)에서 해삼 분획물(SC F2)을 투여한 후 TRAIL을 처리한 경우 세포사멸 관련 단백질의 발현이 증가되는 것을 나타내는 웨스턴 블랏 결과이다.
도 6은 대장암 세포주(DLD-1)에서 해삼분획물(SC F2)을 투여한 후 TRAIL을 처리한 경우 세포주의 몰폴로지가 세포 바디를 형성하지 못하고 세포 사멸이 유도되는 것을 현미경을 통해 나타낸 사진이다.
도 7은 해삼 분획물과 TRAIL을 투여한 세포의 콜로니 형성능 실험에서 콜로니가 형성된 모습을 나타내는 사진이다.
도 8은 해삼 분획물과 TRAIL을 투여한 세포의 콜로니 형성능 실험에서 콜로니의 수를 계수하여 나타낸 그래프이다.
도 9은 해삼 분획물이 투여된 대장암 세포주에서 TRAIL에 의한 apoptosis가 현저히 증가하는 것을 나타내는 그래프이다.
도 10은 해삼 분획물이 투여된 위암 세포주 AGS에서 TRAIL에 의한 apoptosis가 현저히 증가하는 것을 나타내는 그래프이고, 도 11은 해삼 분획물이 투여된 위암 세포주 SNU638에서 TRAIL에 의한 apoptosis가 현저히 증가하는 것을 나타내는 그래프이다.
도 12는 해삼 분획물이 투여된 유방암 세포주에서 TRAIL에 의한 apoptosis가 현저히 증가하는 것을 나타내는 그래프이다.
도 13은 해삼 분획물 투여시의 미토콘드리아의 세포사멸에 관련된 신호전달 단백질의 발현을 단백질 수준에서 확인한 결과이고, 도 14는 해삼 분획물 투여시의 TRAIL 신호전달에 관련된 인자들의 발현을 단백질 수준에서 확인한 결과이다.
도 15 및 도 16은 해삼 분획물에 의해 ER Stress 관련 인자가 증가하는지를 확인하는 웨스턴 블랏 실험 결과이다.
도 17은 해삼 분획물에 의한 ER stress의 증가가 ROS에 의한 것인지를 확인하기 위해, Flow cytometry를 이용하여 ROS를 측정한 결과를 나타내는 것이다.
도 18의 해삼 분획물에서 의해 ROS가 증가됨을 면역형광염색으로 나타낸 것이다.
도 19는 해삼 분획물 투여시에 mRNA 수준에서 XIAP의 발현량에 영향을 주지 않는다는 것을 확인한 PCR 결과이다.
도 20은 해삼 분획물 투여시에 mRNA 수준에서 XIAP의 발현량에 영향을 주지 않는다는 것을 확인한 RT-PCR 결과이다.
도 21은 해삼 분획물에 의해 XIAP의 ubiquitination이 증가하여, XIAP가 감소되는 것을 나타내는 Co-Immunoprecipitation 결과이다.1 is a schematic diagram showing a cell death pathway by TRAIL.
Figure 2 is a cell growth (cell viability) graph showing the extent to which the sea cucumber fraction affects the cell growth of colon cancer.
3 is a graph showing the extent to which TRAIL affects cell viability in colon cancer cells (DLD-1, HCT116, Colo205, HT29, SW620).
4 is a graph showing the extent to which the combination of sea cucumber fraction (SC F2) and TRAIL affects cell viability in colon cancer cells (DLD-1).
5 is a Western blot result showing that the expression of apoptosis-related protein is increased when TRAIL is treated after administration of a sea cucumber fraction (SC F2) in a colon cancer cell line (DLD-1).
6 is a photograph showing through a microscope that the morphology of the cell line does not form a cell body and cell death is induced when TRAIL is treated after administration of a sea cucumber fraction (SC F2) in a colon cancer cell line (DLD-1). .
7 is a photograph showing colonies formed in a colony-forming ability test of cells administered with a sea cucumber fraction and TRAIL.
8 is a graph showing the count of the number of colonies in a colony-forming ability experiment of a sea cucumber fraction and cells administered with TRAIL.
9 is a graph showing a remarkable increase in apoptosis by TRAIL in a colon cancer cell line administered with a sea cucumber fraction.
FIG. 10 is a graph showing a marked increase in apoptosis by TRAIL in the gastric cancer cell line AGS administered with a sea cucumber fraction, and FIG. 11 is a graph showing a marked increase in apoptosis by TRAIL in the gastric cancer cell line SNU638 to which the sea cucumber fraction was administered.
12 is a graph showing a marked increase in apoptosis by TRAIL in a breast cancer cell line administered with a sea cucumber fraction.
13 is a result of confirming the expression of signaling proteins related to mitochondrial apoptosis at the protein level when the sea cucumber fraction is administered, and FIG. 14 is a result of confirming the expression of factors related to TRAIL signaling at the protein level when the sea cucumber fraction is administered. .
15 and 16 are results of a Western blot experiment confirming whether the ER Stress related factor is increased by the sea cucumber fraction.
17 shows the results of measuring ROS using flow cytometry in order to confirm whether the increase in ER stress by the sea cucumber fraction is due to ROS.
It is shown by immunofluorescence staining that ROS is increased by the sea cucumber fraction of Figure 18.
19 is a PCR result confirming that the administration of the sea cucumber fraction does not affect the expression level of XIAP at the mRNA level.
Figure 20 is a result of RT-PCR confirming that the administration of the sea cucumber fraction does not affect the expression level of XIAP at the mRNA level.
21 is a result of Co-Immunoprecipitation showing that the ubiquitination of XIAP is increased and XIAP is decreased by the sea cucumber fraction.
이하, 도면을 참조하여 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail with reference to the drawings.
본 발명의 발명자들은 암세포에 선택적으로 세포사멸을 유도할 수 있는 TRAIL의 내성을 극복할 수 있는 방법에 대하여 연구하던 중, 해삼 추출물 또는 이의 분획물과 TRAIL을 암 세포에 병용 투여하는 경우, TRAIL에 의한 암 세포의 세포사멸이 증대되는 것을 확인하여 본 발명을 완성하였다.The inventors of the present invention are studying a method to overcome the resistance of TRAIL, which can selectively induce apoptosis in cancer cells, when sea cucumber extract or a fraction thereof and TRAIL are administered in combination to cancer cells, by TRAIL. The present invention was completed by confirming that the apoptosis of cancer cells is increased.
본 발명의 일 측면은 해삼 추출물 또는 이의 분획물, 및 TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand)을 유효성분으로 포함하는 암 예방 또는 치료용 약학 조성물에 관한 것이다.One aspect of the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising a sea cucumber extract or a fraction thereof, and TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) as an active ingredient.
본 발명에서 "해삼(Holothuroidea)"은 극피동물 해삼강에 속하는 해삼류의 총칭이다. 전세계적으로 분포하며 바다 밑바닥에 서식한다. 약효가 인삼과 같다고 하여 이름 지어졌다. 몸은 앞뒤로 긴 원통 모양이고, 등에 혹 모양의 돌기가 여러 개 나 있다. 해삼은 항암작용, 당뇨병, 천식(진액보호) 등에 예방, 개선 또는 치료 효과가 좋고, 칼슘과 조혈성분인 철의 함량이 높다. 알칼리 식품으로서 칼슘, 요오드, 알킨산이 특히 많아 체내에 신진대사 촉진 및 혈액을 정화하는 효능을 가지고 있다. 또한 해삼 성분 중 홀로테인은 피의 응고를 막고 균을 파괴시키며 항암작용을 하며, 해삼속의 지방산은 천식, 궤양성 대장암, 관절염 등에 효과가 있고 전립선 암세포주에서 성장을 억제하는 밝혀져 있다. 또한, 해삼에서 추출한 항산화된 사포닌이 혈관생성을 억제하고 항종양 작용을 한다고 알려져 있으나, TRAIL-저항성 암에 대한 치료 효과는 알려진 바 없다.In the present invention, "sea cucumber ( Holothuroidea )" is a generic term for sea cucumbers belonging to the sea cucumber river, an echinoderm. It is distributed worldwide and lives in the bottom of the sea. It is named because it has the same medicinal properties as ginseng. The body has a long cylindrical shape back and forth, and there are several hump-shaped projections on the back. Sea cucumber has good anti-cancer action, diabetes, asthma (treasure protection), and has good preventive, improved or therapeutic effects, and has a high content of calcium and iron, a hematopoietic component. As an alkaline food, it is particularly high in calcium, iodine and alkynic acid, and has the effect of promoting metabolism and purifying blood in the body. In addition, among sea cucumber components, holotein prevents blood clotting, destroys germs, and has anti-cancer activity, and fatty acids in sea cucumber are effective for asthma, ulcerative colon cancer, arthritis, etc., and have been found to inhibit growth in prostate cancer cell lines. In addition, it is known that the antioxidant saponin extracted from sea cucumber inhibits angiogenesis and has anti-tumor action, but the therapeutic effect on TRAIL-resistant cancer is unknown.
본 발명에서 "추출물"은 해삼의 추출 처리에 의하여 얻어지는 추출액, 상기 추출액의 희석액이나 농축액, 상기 추출액을 건조하여 얻어지는 건조물, 상기 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함한다.In the present invention, "extract" refers to an extract obtained by extracting sea cucumber, a diluted solution or a concentrate of the extract, a dried product obtained by drying the extract, a preparation or purified product of the extract, or a mixture thereof, the extract itself and the extract It includes extracts of all formulations that can be formed using.
상기 해삼의 추출물에 있어서, 상기 해삼을 추출하는 방법은 특별히 제한되지 않으며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 추출할 수 있다. 상기 추출 방법의 비제한적인 예로는, 초음파 추출법, 여과법, 환류 추출법 등을 들 수 있으며, 이들은 단독으로 수행되거나 2종 이상의 방법을 병용하여 수행될 수 있다.In the sea cucumber extract, a method of extracting the sea cucumber is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, which may be performed alone or in combination of two or more methods.
본 발명에서 상기 해삼을 추출하는 데에 사용되는 추출 용매의 종류는 특별히 제한되지 않으며, 당해 기술 분야에서 공지된 임의의 용매를 사용할 수 있다. 상기 추출 용매의 비제한적인 예로는 물, 알코올 또는 이들의 혼합 용매 등을 들 수 있으며, 알코올을 용매로 사용하는 경우에는 보다 바람직하게는 C1 내지 C4의 알코올(메탄올, 에탄올, 프로판올, 부탄올)을 사용할 수 있으며, 더욱 바람직하게는 에탄올을 사용할 수 있다.In the present invention, the type of the extraction solvent used to extract the sea cucumber is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent may include water, alcohol, or a mixed solvent thereof, and when alcohol is used as a solvent, more preferably C 1 to C 4 alcohol (methanol, ethanol, propanol, butanol ) May be used, and more preferably ethanol may be used.
본 발명에서 "분획물"은 여러 다양한 구성 성분들을 포함하는 혼합물로부터 특정 성분 또는 특정 성분 그룹을 분리하기 위하여 분획을 수행하여 얻어진 결과물을 의미한다.In the present invention, "fraction" refers to a product obtained by performing fractionation in order to separate a specific component or a specific component group from a mixture containing various various constituents.
본 발명에서 상기 분획물을 얻는 분획 방법은 특별히 제한되지 않으며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 수행될 수 있다. 상기 분획 방법의 비제한적인 예로는, 해삼을 추출하여 얻은 추출물에 소정의 용매를 처리하여 상기 추출물로부터 분획물을 수득할 수 있다.The fractionation method for obtaining the fraction in the present invention is not particularly limited, and may be performed according to a method commonly used in the art. As a non-limiting example of the fractionation method, a fraction can be obtained from the extract by treating the extract obtained by extracting sea cucumber with a predetermined solvent.
본 발명에서 상기 분획물을 얻는 데에 사용되는 분획 용매의 종류는 특별히 제한되지 않으며, 당해 기술 분야에서 공지된 임의의 용매를 사용할 수 있다. 상기 분획 용매의 비제한적인 예로는 물, 알코올 등의 극성 용매; 헥산(Hexane), 에틸 아세테이트(Ethyl acetate), 클로로포름(Chloroform), 디클로로메탄(Dichloromethane) 등의 비극성 용매 등을 들 수 있다. 이들은 단독으로 사용되거나 2종 이상 혼합하여 사용될 수 있다. 상기 분획 용매 중 알코올을 사용하는 경우에는 바람직하게는 C1 내지 C4의 알코올을 사용할 수 있다.In the present invention, the kind of fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include polar solvents such as water and alcohol; Non-polar solvents, such as hexane, ethyl acetate, chloroform, and dichloromethane, etc. are mentioned. These may be used alone or in combination of two or more. In the case of using an alcohol in the fractionation solvent, preferably a C 1 to C 4 alcohol may be used.
본 발명의 상기 해삼 추출물 또는 이의 분획물은 XIAP(X-chromosome linked inhibitor of apoptosis protein) 단백질의 발현 감소와 활성화된 카스파제 3(cleaved caspase 3) 및 활성화된 카스파제 9(cleaved caspase 9)의 발현 증가를 유도함으로써, 병용 투여된 TRAIL 단백질이 TRAIL-저항성 암에 대해서도 치료 효과를 나타낼 수 있도록 하는 것에 특징이 있다.The sea cucumber extract or a fraction thereof of the present invention decreases the expression of X-chromosome linked inhibitor of apoptosis protein (XIAP) protein and increases the expression of activated
또한, 본 발명의 상기 해삼 추출물 또는 이의 분획물은 상기 암 세포주의 세포사멸을 유도하는 항암 효능을 나타내며, 천연물로부터 유래된 것이어서 정상 세포에 심각한 자극을 가한다거나 유해한 작용을 유발함이 없이 안정하게 사용될 수 있는 이점이 있다.In addition, the sea cucumber extract or a fraction thereof of the present invention exhibits anti-cancer efficacy for inducing apoptosis of the cancer cell line, and is derived from a natural product, so it can be used stably without seriously stimulating normal cells or causing harmful effects. There is an advantage.
이에 따라, 해삼 추출물 또는 이의 분획물은 TRAIL과 함께 암 예방 또는 치료용 약학 조성물의 유효성분으로 유용하게 사용될 수 있다.Accordingly, sea cucumber extract or a fraction thereof may be usefully used as an active ingredient in a pharmaceutical composition for preventing or treating cancer together with TRAIL.
본 발명의 일 실시예에 의하면, 암 세포에 해삼 추출물 또는 이의 분획물, 및 TRAIL을 병용처리한 경우 세포사멸이 증가하는 것을 확인할 수 있다. According to an embodiment of the present invention, it can be seen that apoptosis is increased when a sea cucumber extract or a fraction thereof, and TRAIL are co-treated with cancer cells.
본 발명에서 사용되는 용어, "TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand) 단백질"은 종양괴사 인자(Tumor Necrosis Factor: TNF) 슈퍼 패밀리에 속하는 싸이토카인(cytokine) 단백질의 일원으로서, 그 서열 등은 공지의 데이터베이스인 NCBI의 GenBank 등에서 얻을 수 있다. 이와 같은 TRAIL 단백질은 차세대 항암제로 주목받고 있는, 정상세포에서는 거의 독성을 나타내지 않고 암세포만을 선택적으로 사멸시킨다. 정상세포에 무해한 특성은 일반 세포의 표면에만 존재하는 유인 수용체 1, 2(DcR1, DcR2)의 기능 때문으로, 상기 DcR1, DcR2는 TRAIL과 결합이 가능하지만, 세포내 신호 전달 부분이 결여되어 있어서 일반 세포들은 TRAIL에 의한 세포 사멸 유도가 일어나지 않는다. 이와 같이 TRAIL이 세포사멸을 유도하는 개략적인 과정을 도 1에 나타내었다. 본 발명에서, 상기 TRAIL은 일부 염기서열이 치환, 결실, 부가된 형태 등으로 변이되거나 조작(engineered)된 TRAIL을 포함한다.The term "TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) protein" used in the present invention is a member of a cytokine protein belonging to the Tumor Necrosis Factor (TNF) super family, and its sequence is It can be obtained from a well-known database such as GenBank of NCBI. Such TRAIL protein, which is attracting attention as a next-generation anticancer drug, exhibits little toxicity in normal cells and selectively kills only cancer cells. The harmless property of normal cells is due to the function of attracting
한편, 이러한 선택적 암세포 사멸 유도 특징을 가지고 있는 TRAIL 단백질은 현재 내성에 의해 치료 효과가 낮다는 문제점이 대두되고 있다. On the other hand, TRAIL protein, which has the characteristic of selective cancer cell death induction, has a problem that the therapeutic effect is low due to resistance.
이로 인해 본 발명자들이 새롭게 규명한 해삼 추출물 또는 이의 분획물의 병용 투여는 TRAIL-저항성 암에서도 효과적으로 TRAIL의 암세포 사멸 효과를 나타내는 특징이 있다.For this reason, the combined administration of sea cucumber extract or a fraction thereof newly identified by the present inventors is characterized by effectively showing the cancer cell killing effect of TRAIL even in TRAIL-resistant cancer.
본 발명에서 "암"은 세포의 사멸 조절과 관련된 질병으로서, 정상적인 아폽토시스(apoptosis)의 균형이 깨지는 경우 세포가 과다증식하게 되어 발병하는 질병을 총칭한다. 이러한 비정상적인 과다증식 세포들은 경우에 따라 주위 조직 및 장기에 침입하여 종괴를 형성할 수 있으며 체내의 정상적인 구조의 파괴나 변형을 유발할 수 있는데, 이러한 상태를 통칭하여 암이라고 한다.In the present invention, "cancer" refers to a disease related to the regulation of cell death, and refers to a disease caused by overproliferation of cells when the balance of normal apoptosis is broken. These abnormal hyperproliferative cells may invade surrounding tissues and organs in some cases to form masses, and may cause destruction or transformation of normal structures in the body, and such conditions are collectively referred to as cancer.
일반적으로 종양(tumor)이라 하면 신체 조직의 자율적인 과잉 성장에 의해 비정상적으로 자란 덩어리를 의미하며, 종양은 양성 종양(benign tumor)과 악성 종양(malignant tumor)으로 구분할 수 있다. 악성 종양은 양성 종양에 비해 성장 속도가 매우 빠르며 주변 조직에 침윤하면서 전이(metastasis)가 일어나 생명을 위협하게 된다. 이러한 악성 종양을 통상적으로 '암(cancer)'이라 한다.In general, a tumor means a mass that has grown abnormally due to the autonomous overgrowth of body tissues, and the tumor can be classified into a benign tumor and a malignant tumor. Malignant tumors grow faster than benign tumors, and metastases occur as they infiltrate the surrounding tissues, which is life-threatening. These malignant tumors are commonly referred to as'cancer'.
본 발명에서 상기 암의 종류는 특별히 제한되지 않지만, 본 발명의 목적상 TRAIL-저항성 암을 의미한다. 상기 암의 비제한적인 예로 대장암, 결장암, 췌장암, 간암, 자궁경부암, 신장암, 위암, 전립선암, 유방암, 뇌종양, 폐암, 자궁암, 방광암 및 혈액암 등을 들 수 있다.In the present invention, the type of cancer is not particularly limited, but for the purposes of the present invention, it means TRAIL-resistant cancer. Examples of the cancer include, but are not limited to, colon cancer, colon cancer, pancreatic cancer, liver cancer, cervical cancer, kidney cancer, stomach cancer, prostate cancer, breast cancer, brain tumor, lung cancer, uterine cancer, bladder cancer, and blood cancer.
본 발명에서 사용되는 용어, "TRAIL-저항성 암"은 TRAIL 단백질 처리에 의하여 암의 예방, 개선 또는 치료가 효과적으로 이루어지지 않는 암을 의미하는 것으로, 보다 구체적으로는 암세포 표면에 TRAIL 단백질 수용체가 적은 수준으로 발현되는 등, 처음부터 TRAIL 단백질에 대한 민감성이 낮아 TRAIL 단백질로 유도되는 세포 아폽토시스 기작이 효과적으로 이루어지지 않는 모든 종류의 암 뿐만 아니라, 초기에는 TRAIL 단백질 처리에 따른 항암 효과가 나타났으나, 반복적인 TRAIL 단백질 처리에 의해 점차로 TRAIL 단백질에 대한 항암 효과가 감소되거나 효과가 사라진 암을 모두 포함한다.As used herein, the term "TRAIL-resistant cancer" refers to cancer in which the prevention, improvement, or treatment of cancer is not effectively performed by treatment with TRAIL protein, and more specifically, the level of TRAIL protein receptor on the surface of cancer cells is low. As well as all types of cancers in which the cellular apoptosis mechanism induced by TRAIL protein is not effectively performed due to low sensitivity to the TRAIL protein from the beginning, anticancer effect was shown initially by TRAIL protein treatment, but repetitive It includes all cancers in which the anticancer effect on TRAIL protein is gradually decreased or the effect disappears by treatment with TRAIL protein.
본 발명에서 상기 TRAIL-저항성 암은 TRAIL에 대하여 저항성을 나타내는 암이라면 특별한 제한없이 모든 종류의 암을 포함한다. 상기 TRAIL-저항성 암의 비제한적인 예로는, 대장암, 위암, 유방암, 폐암, 간암, 전립선암, 신장암, 피부암, 신경교종암, 갑상선암, 혈액암 등을 들 수 있으며, 바람직하게는 대장암, 위암 및 유방암을 들 수 있다.In the present invention, the TRAIL-resistant cancer includes all types of cancer without particular limitation as long as it is a cancer that shows resistance to TRAIL. Non-limiting examples of the TRAIL-resistant cancer include colon cancer, stomach cancer, breast cancer, lung cancer, liver cancer, prostate cancer, kidney cancer, skin cancer, glioma cancer, thyroid cancer, blood cancer, and the like, preferably colon cancer, Gastric cancer and breast cancer.
본 발명의 일 실시예에서는 대표적인 TRAIL-저항성 암으로 대장암 세포주인 DLD-1에 대한 치료 효과, 위암 세포주인 AGS 및 SNU638에 대한 치료 효과 및 유방암 세포주인 MCF-7에 대한 치료 효과를 확인하였다. 본 발명의 해삼 추출물 또는 이의 분획물이 TRAIL과 병용 투여시 TRAIL-저항성 암에 치료효과가 있다는 상기의 구체적인 실험 결과로부터 다른 종류의 TRAIL-저항성 암세포에도 동일한 치료 효과가 있을 것임은 자명하다.In one embodiment of the present invention, a therapeutic effect for DLD-1, a colon cancer cell line, as a representative TRAIL-resistant cancer, a therapeutic effect for AGS and SNU638, which are gastric cancer cell lines, and a therapeutic effect for MCF-7, a breast cancer cell line, were confirmed. It is apparent from the above specific experimental results that the sea cucumber extract or a fraction thereof of the present invention has a therapeutic effect on TRAIL-resistant cancer when administered in combination with TRAIL. It is apparent that the same therapeutic effect will also be provided for other types of TRAIL-resistant cancer cells.
본 발명에서 "예방"은 본 발명의 상기 약학 조성물을 개체에 투여하여 암의 발명을 억제시키거나 지연시키는 모든 행위를 의미하고, "치료"는 본 발명의 상기 약학적 조성물을 개체에 투여하여 암의 증세가 호전되도록 하거나 이롭게 되도록 하는 모든 행위를 의미한다.In the present invention, "prevention" refers to any action that inhibits or delays the invention of cancer by administering the pharmaceutical composition of the present invention to an individual, and "treatment" refers to cancer by administering the pharmaceutical composition of the present invention to an individual. It refers to any action that makes the symptoms of a person better or beneficial.
본 발명의 상기 약학 조성물을 TRAIL-저항성 암세포에 처리하는 경우, 본 발명의 상기 해삼 추출물 또는 이의 분획물의 작용에 의하여 암세포에서 XIAP 단백질(X-chromosome linked inhibitor of apoptosis protein)의 발현이 감소하고, 활성화된 카스파제 3(cleaved caspase 3) 및 활성화된 카스파제 9(cleaved caspase 9)의 발현이 증가됨에 따라 TRAIL에 대한 민감성이 증가된 암세포에 TRAIL 단백질을 처리함으로써 종래 TRAIL에 저항성을 나타내었던 암을 효과적으로 예방, 개선 또는 치료할 수 있다.When the pharmaceutical composition of the present invention is treated on TRAIL-resistant cancer cells, the expression of XIAP protein (X-chromosome linked inhibitor of apoptosis protein) in cancer cells is reduced and activated by the action of the sea cucumber extract or a fraction thereof of the present invention. As the expression of
본 발명의 약학 조성물에 있어서, 상기 해삼 추출물 또는 이의 분획물은 상기 약학 조성물의 전체의 부피를 기준으로 바람직하게는 0.001 내지 300 ㎍/㎖로 함유될 수 있고, 보다 바람직하게는 1 내지 200 ㎍/㎖로 함유될 수 있다. 상기 범위 내에서 상기 해삼 추출물 또는 이의 분획물을 적절한 양으로 사용하면 경제적이면서도 상기 해삼 추출물 또는 이의 분획물에 따른 항암 효과가 충분히 발휘되어 본 발명의 목적을 달성하기에 보다 적합해지는 이점이 있다.In the pharmaceutical composition of the present invention, the sea cucumber extract or a fraction thereof may preferably be contained in an amount of 0.001 to 300 μg/ml based on the total volume of the pharmaceutical composition, more preferably 1 to 200 μg/ml It can be contained as. If the sea cucumber extract or a fraction thereof is used in an appropriate amount within the above range, it is economical and an anticancer effect according to the sea cucumber extract or a fraction thereof is sufficiently exhibited, thereby becoming more suitable for achieving the object of the present invention.
본 발명의 약학 조성물에 있어서, 상기 TRAIL 단백질은 상기 약학 조성물의 전체의 부피를 기준으로 바람직하게는 0.001 내지 200 ng/㎖로 함유될 수 있고, 보다 바람직하게는 1 내지 25 ng/㎖로 함유될 수 있다. 상기 범위 내에서, 상기 TRAIL 단백질을 적절한 양으로 사용하면 경제적이면서도 상기 TRAIL 단백질에 따른 아폽토시스 유도 효과를 비롯한 항암 효과가 충분히 발휘되어 본 발명의 목적을 달성하기에 보다 적합해지는 이점이 있다.In the pharmaceutical composition of the present invention, the TRAIL protein may preferably be contained in an amount of 0.001 to 200 ng/ml based on the total volume of the pharmaceutical composition, more preferably 1 to 25 ng/ml. I can. Within the above range, when the TRAIL protein is used in an appropriate amount, it is economical and the anti-cancer effect including the apoptosis inducing effect according to the TRAIL protein is sufficiently exhibited, thereby becoming more suitable for achieving the object of the present invention.
또한, 상기 약학 조성물은 해삼 추출물 또는 이의 분획물에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상을 함유할 수 있다.In addition, the pharmaceutical composition may contain one or more active ingredients exhibiting the same or similar function in addition to the sea cucumber extract or a fraction thereof.
본 발명에 따른 약학 조성물은 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 더 포함할 수 있다.The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent.
본 발명에서, 상기 "약학적으로 허용 가능"하다는 것은, 이를 투여시 생물체를 자극하지 않으면서, 투여되는 화합물의 생물학적 활성 및 특성을 저해하지 않는, 약학 분야에서 통상적으로 사용되는 것을 의미한다.In the present invention, the "pharmaceutically acceptable" means that it is commonly used in the field of pharmacy, which does not stimulate an organism when administered, and does not inhibit the biological activity and properties of the administered compound.
본 발명에서, 상기 담체의 종류는 특별히 제한되지 아니하며 당해 기술 분야에서 통상적으로 사용되는 담체라면 어느 것이든 사용할 수 있다. 상기 담체의 비제한적인 예로는, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사 용액, 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 말토 덱스트린, 글리세롤, 에탄올 등을 들 수 있다. 이들은 단독으로 사용되거나 2종 이상을 혼합하여 사용될 수 있다. In the present invention, the kind of the carrier is not particularly limited, and any carrier commonly used in the art may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. I can. These may be used alone or in combination of two or more.
또한, 본 발명의 상기 약학 조성물은 필요한 경우, 부형제, 희석제, 항산화제, 완충액 또는 정균제 등 기타 약학적으로 허용 가능한 첨가제들을 첨가하여 사용할 수 있으며, 충진제, 증량제, 습윤제, 붕해제, 분산제, 계면 활성제, 결합제 또는 윤활제 등을 부가적으로 첨가하여 사용할 수 있다. In addition, the pharmaceutical composition of the present invention may be used by adding other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, if necessary, and fillers, extenders, wetting agents, disintegrants, dispersants, surfactants , A binder or a lubricant may be additionally added and used.
본 발명의 상기 약학 조성물은 경구 투여 또는 비경구 투여를 위한 적합한 다양한 제형으로 제제화되어 사용될 수 있다.The pharmaceutical composition of the present invention may be formulated and used in various formulations suitable for oral administration or parenteral administration.
상기 경구 투여용 제제의 비제한적인 예로는, 트로키제(troches), 로젠지(lozenge), 정제, 수용성 현탁액, 유성 현탁액, 조제 분말, 과립, 에멀젼, 하드 캡슐, 소프트 캡슐, 시럽 또는 엘릭시르제 등을 들 수 있다. Non-limiting examples of formulations for oral administration include troches, lozenges, tablets, aqueous suspensions, oily suspensions, powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs, etc. Can be mentioned.
본 발명의 상기 약학 조성물을 경구 투여용으로 제제화하기 위하여, 락토오스, 사카로오스, 솔비톨, 만니톨, 전분, 아밀로펙틴(Amylopectin), 셀룰로오스(Cellulose) 또는 젤라틴(Gelatin) 등과 같은 결합제; 디칼슘 포스페이트(Dicalcium phosphate) 등과 같은 부형제; 옥수수 전분 또는 고구마 전분 등과 같은 붕괴제; 스테아르산마그네슘(Magnesium stearate), 스테아르산 칼슘(Calcium stearte), 스테아릴 푸마르산 나트륨(Sodium stearyl fumarate) 또는 폴리에틸렌 글리콜 왁스(Polyethylene glycol wax) 등과 같은 윤활유 등을 사용할 수 있으며, 감미제, 방향제, 시럽제 등도 사용할 수 있다. 나아가 캡슐제의 경우에는 상기 언급한 물질 외에도 지방유와 같은 액체 담체 등을 추가로 사용할 수 있다.In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricating oils such as magnesium stearate, calcium stearte, sodium stearyl fumarate or polyethylene glycol wax can be used, and sweeteners, fragrances, syrups, etc. can be used. I can. Furthermore, in the case of capsules, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be additionally used.
상기 비경구 제제의 비제한적인 예로는 주사액, 좌제, 호흡기 흡입용 분말, 스프레이용 에어로졸제, 연고, 도포용 파우더, 오일, 크림 등을 들 수 있다.Non-limiting examples of the parenteral preparation include injections, suppositories, respiratory inhalation powders, aerosols for sprays, ointments, powders for application, oils, creams, and the like.
본 발명의 상기 약학 조성물을 비경구 투여용으로 제제화하기 위하여, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 외용제 등을 사용할 수 있으며, 상기 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. In order to formulate the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, external preparations, etc. can be used. As the non-aqueous solvents and suspensions, propylene glycol, polyethylene Glycols, vegetable oils such as olive oil, injectable esters such as ethyloleate, and the like may be used.
또한, 보다 구체적으로 본 발명의 상기 약학 조성물을 주사액으로 제제화하는 경우, 본 발명의 상기 조성물을 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고 이를 앰플(ampoule) 또는 바이알(vial)의 단위 투여용으로 제제화할 수 있다. 또한, 본 발명의 상기 약학 조성물을 에어로졸제로 제제화하는 경우, 수분산된 농축물 또는 습윤 분말이 분산되도록 추진제 등이 첨가제와 함께 배합될 수 있다.In addition, more specifically, when the pharmaceutical composition of the present invention is formulated as an injection solution, the composition of the present invention is prepared as a solution or suspension by mixing in water with a stabilizer or a buffer, and this is used in an ampoule or vial. It can be formulated for unit administration. In addition, when the pharmaceutical composition of the present invention is formulated as an aerosol, a propellant or the like may be blended with an additive so that the aqueous concentrated or wet powder is dispersed.
또한, 본 발명의 상기 약학 조성물을 연고, 크림 등으로 제제화하는 경우에는, 동물성 오일, 식물성 오일, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크, 산화 아연 등을 담체로 사용하여 제제화할 수 있다.In addition, in the case of formulating the pharmaceutical composition of the present invention into an ointment, cream, etc., animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide It can be formulated using the like as a carrier.
본 발명의 상기 약학 조성물의 약학적 유효량, 유효 투여량은 상기 약학 조성물의 제제화 방법, 투여 방식, 투여 시간 및/또는 투여 경로 등에 의해 다양해질 수 있으며, 상기 약학 조성물의 투여로 달성하고자 하는 반응의 종류와 정도, 투여 대상이 되는 개체의 종류, 연령, 체중, 일반적인 건강 상태, 질병의 증세나 정도, 성별, 식이, 배설, 해당 개체에 동시 또는 이시에 함께 사용되는 약물 기타 조성물의 성분 등을 비롯한 여러 인자 및 의약 분야에서 잘 알려진 유사 인자에 따라 다양해질 수 있으며, 당해 기술 분야에서 통상의 지식을 가진 자는 목적하는 치료에 효과적인 투여량을 용이하게 결정하고 처방할 수 있다.The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, the mode of administration, the administration time and/or the route of administration of the pharmaceutical composition, and the response to be achieved by the administration of the pharmaceutical composition Including the type and degree, the type of the subject to be administered, the age, weight, general health condition, the symptom or degree of the disease, sex, diet, excretion, drugs used concurrently with the subject or at the same time, and other components of the composition. It may vary depending on various factors and similar factors well known in the medical field, and a person of ordinary skill in the art can easily determine and prescribe an effective dosage for a desired treatment.
본 발명의 상기 약학 조성물의 보다 바람직한 효과를 위한 투여량은, 바람직하게는 1일 10 mg/kg 내지 1,000 mg/kg, 보다 바람직하게는 20 mg/kg 내지 500 mg/kg일 수 있다. 본 발명의 상기 약학 조성물의 투여는 하루에 1회 투여될 수 있고, 수회에 나누어 투여될 수 있다. 따라서 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.A dosage for a more preferable effect of the pharmaceutical composition of the present invention may be preferably 10 mg/kg to 1,000 mg/kg per day, more preferably 20 mg/kg to 500 mg/kg. The administration of the pharmaceutical composition of the present invention may be administered once a day, or may be administered several times. Therefore, the dosage does not limit the scope of the present invention in any way.
본 발명의 상기 약학 조성물의 투여 경로 및 투여 방식은 각각 독립적일 수 있으며, 그 방식에 있어 특별히 제한되지 않으며, 목적하는 해당 부위에 상기 약학 조성물이 도달할 수 있는 한 임의의 투여 경로 및 투여 방식에 따를 수 있다. 상기 약학 조성물은 경구 투여 또는 비경구 투여 방식으로 투여할 수 있다.The route of administration and the method of administration of the pharmaceutical composition of the present invention may each be independent, and the method is not particularly limited, and any route and method of administration can be used as long as the pharmaceutical composition can reach the target site. You can follow. The pharmaceutical composition may be administered orally or parenterally.
상기 비경구 투여하는 방법으로는, 예를 들어 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내주사, 자궁내 경막주사, 뇌혈관내 주사 또는 흉부내 주사에 의해 투여될 수 있으며, 상기 조성물을 질환 부위에 도포하거나 분무, 흡입하는 방법 또한 이용할 수 있으나 이들에 제한되지 않는다.The parenteral administration method may be administered by, for example, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection, or intrathoracic injection, A method of applying, spraying, or inhaling the composition to a diseased area may also be used, but is not limited thereto.
본 발명의 상기 약학 조성물은 바람직하게는 경구 투여되거나 주사 투여될 수 있으며, 보다 바람직하게는 경구 투여될 수 있다.The pharmaceutical composition of the present invention may be preferably administered orally or administered by injection, and more preferably may be administered orally.
또한, 상기 약학 조성물은 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.In addition, the pharmaceutical composition may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers.
또한, 본 발명의 약학 조성물은 사람 또는 동물용으로 투여될 수 있다.In addition, the pharmaceutical composition of the present invention can be administered for humans or animals.
또한, 본 발명은 해삼 추출물 또는 이의 분획물을 유효성분으로 포함하며, TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand)의 내성을 감소시키는 것을 특징으로 하는 TRAIL의 항암 효과 증진용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for enhancing the anticancer effect of TRAIL, comprising a sea cucumber extract or a fraction thereof as an active ingredient, and reducing resistance of TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand).
상기 암은 대장암, 결장암, 췌장암, 간암, 자궁경부암, 신장암, 위암, 전립선암, 유방암, 뇌종양, 폐암, 자궁암, 방광암 및 혈액암으로 구성되는 군으로부터 선택되는 어느 하나인 것이 바람직하고, 위암, 유방암 또는 대장암인 것이 더욱 바람직하나 이에 한정되지 않으며, TRAIL에 대한 내성을 가진 암은 모두 포함되는 것이 바람직하다.The cancer is preferably any one selected from the group consisting of colon cancer, colon cancer, pancreatic cancer, liver cancer, cervical cancer, kidney cancer, stomach cancer, prostate cancer, breast cancer, brain tumor, lung cancer, uterine cancer, bladder cancer, and blood cancer, and gastric cancer , Breast cancer or colon cancer is more preferable, but is not limited thereto, and it is preferable that all cancers with resistance to TRAIL are included.
또한, 본 발명은 해삼 추출물 또는 이의 분획물, 및 TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand; Trail)을 인간을 제외한 포유동물의 암세포에 병용 투여하는 것을 특징으로 하는 암세포 사멸 증진 방법을 제공한다.In addition, the present invention provides a method for promoting cancer cell death, characterized in that sea cucumber extract or a fraction thereof, and TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand; Trail) are administered in combination to cancer cells of mammals other than humans.
또한, 본 발명은 해삼 추출물 또는 이의 분획물, 및 TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand; Trail)을 인간을 제외한 개체에 투여하는 단계를 포함하는 암의 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating cancer comprising administering a sea cucumber extract or a fraction thereof, and TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand; Trail) to individuals other than humans.
본 발명에서 "개체"는 쥐, 가축, 인간 등을 포함하는 포유동물을 비롯한 모든 동물을 의미한다.In the present invention, "individual" refers to all animals, including mammals, including mice, livestock, and humans.
본 발명의 상기 암의 예방 또는 치료 방법에서, 상기 해삼 추출물 또는 이의 분획물, 및 TRAIL의 투여량, 투여 경로, 투여 방식 등은 본 발명의 상기 약학 조성물과 관련하여 상기에서 설명한 바와 같다.In the method of preventing or treating cancer of the present invention, the sea cucumber extract or fraction thereof, and the dosage, route of administration, and mode of administration of TRAIL are as described above with respect to the pharmaceutical composition of the present invention.
또한, 본 발명은 해삼 추출물 또는 이의 분획물, 및 TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand)을 유효성분으로 포함하는 암 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving cancer comprising a sea cucumber extract or a fraction thereof, and TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) as an active ingredient.
본 발명에서 "개선"은 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.In the present invention, "improvement" means any action that at least reduces the severity of a parameter related to the condition being treated, for example, symptoms.
본 발명의 상기 건강기능식품 조성물을 식품 첨가물로 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다.When the health functional food composition of the present invention is used as a food additive, the composition may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method.
상기 식품의 종류는 특별히 제한되지 않으며, 통상적인 의미에서의 식품을 모두 포함한다. 상기 물질을 첨가할 수 있는 식품의 비제한적인 예로는, 육류, 소세지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 또는 비타민 복합제 등을 들 수 있다.The kind of the food is not particularly limited, and includes all foods in a conventional sense. Non-limiting examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, Tea, drinks, alcoholic beverages, or vitamin complexes.
본 발명의 상기 건강기능식품 조성물이 음료 조성물인 경우, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상기 천연 탄수화물의 비제한적인 예로 포도당, 과당과 같은 모노사카라이드; 말토스, 수크로오스와 같은 디사카라이드; 덱스트린, 사이클로덱스트린과 같은 천연 감미제; 사카린, 아스파르탐과 같은 합성 감미제 등을 들 수 있다. 상기 첨가되는 추가 성분의 비율은 당업자의 선택에 의해 적절하게 결정될 수 있다.When the health functional food composition of the present invention is a beverage composition, it may contain various flavoring agents or natural carbohydrates, etc. as an additional component like a normal beverage. Non-limiting examples of the natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin; And synthetic sweeteners such as saccharin and aspartame. The proportion of the additional ingredients to be added may be appropriately determined by the choice of a person skilled in the art.
본 발명의 건강기능식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴삼 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 박에 본 발명의 건강기능식품 조성물은 천연 과일 주스, 과일 음료 또는 야채 음료 등의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 사용되거나 2종 이상을 조합하여 사용할 수 있다. 이러한 첨가물의 비율 또한 당업자에 의해 적절히 선택될 수 있다. The health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acids and salts thereof, alginsam and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol, carbonates used in carbonated beverages, and the like. In the foil, the health functional food composition of the present invention may contain pulp for the production of natural fruit juices, fruit drinks or vegetable drinks. These components may be used independently or may be used in combination of two or more. The proportion of these additives can also be appropriately selected by those skilled in the art.
이하, 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail by examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.
실시예Example
제조예. 해삼 분획물의 제조Manufacturing example. Preparation of sea cucumber fraction
(1) 해삼 추출물의 제조(1) Preparation of sea cucumber extract
우선 해삼의 내장을 제거하고, 해삼 육부분만 취한 후, 잘 수세하였다. 수세된 해삼 육을 잘 교반한 후, 동결 건조하여 분말화하였다. 분말화된 해삼 시료 20g에 99% 에틸알코올 200mL를 첨가하여 70℃에서 2시간 동안 교반하면서 가열한 후, 원심분리하여 펠렛을 수득하였다. 이와 같은 과정을 2회 반복하고, 수득된 펠렛을 아세톤을 사용하여 펠렛에 남아있는 색소, 지질 및 저분자 단백질을 제거 하였다. 상기 색소, 지질 및 저분자 단백질이 제거된 시료를 원심분리하여 최종 펠렛을 수득한 후, 상온에서 18시간 건조하였다. First, the intestines of the sea cucumber were removed, and only six parts of the sea cucumber were taken, and then washed well. After the washed sea cucumber meat was stirred well, it was freeze-dried and powdered. 200 mL of 99% ethyl alcohol was added to 20 g of the powdered sea cucumber sample, heated while stirring at 70° C. for 2 hours, and centrifuged to obtain a pellet. This process was repeated twice, and the obtained pellet was removed using acetone to remove pigments, lipids, and low molecular protein remaining in the pellet. The sample from which the pigment, lipid and low molecular protein were removed was centrifuged to obtain a final pellet, and then dried at room temperature for 18 hours.
상기에서 수득된 시료 20g에 400mL의 증류수를 첨가하여 80℃에서 2시간동안 교반 가열 한 후, 원심분리하여 상등액을 수득하였다. 이와 같은 과정을 2회 반복하고, 수득된 상등액은 감압농축기를 이용하여 200mL로 농축하였다. 농축된 상등액이 총 알코올의 함량이 30%가 되게 에틸알코올을 첨가한 후, 4℃에서 4시간 동안 보관하고, 원심분리 후 상등액을 수득하였다. 수득된 상등액에 총 알코올의 함량이 80%가 되게 에틸알코올을 첨가한 후, 4℃에서 18시간 보관하였다. 알코올에 의해 침전된 침전물을 0.4μm 여과지로 여과하여 다당류를 수득하고, 이를 수세하여 최종적으로 해삼 유래 다당류를 수득하였다. 이와같이 수득한 다당류를 crude 다당류라 칭하였다. 400 mL of distilled water was added to 20 g of the sample obtained above, stirred and heated at 80° C. for 2 hours, and then centrifuged to obtain a supernatant. This process was repeated twice, and the obtained supernatant was concentrated to 200 mL using a vacuum concentrator. Ethyl alcohol was added to the concentrated supernatant so that the total alcohol content was 30%, and then stored at 4° C. for 4 hours, and a supernatant was obtained after centrifugation. Ethyl alcohol was added to the obtained supernatant so that the total alcohol content was 80%, and then stored at 4°C for 18 hours. The precipitate precipitated with alcohol was filtered through a 0.4 μm filter paper to obtain a polysaccharide, which was washed with water to finally obtain a sea cucumber-derived polysaccharide. The polysaccharide thus obtained was referred to as a crude polysaccharide.
(2) 해삼 분획물의 제조(2) Preparation of sea cucumber fraction
이온 교환 컬럼 크로마토그래피(DEAE Sepharose Fast Flow column)를 이용하여 상기 해삼 유래 crude 다당류를 정제하였다.The crude polysaccharide derived from sea cucumber was purified using ion exchange column chromatography (DEAE Sepharose Fast Flow column).
우선 상기 crude 다당류 200mg을 10mL 증류수에 용해한 후, 극초단파(microwave bomb)를 이용하여 더 작은 입자로 용해시켰다. 극초단파에 의해 가열되어 뜨거운 다당류를 이온 교환 컬럼 크로마토그래피에 주입한 후, 증류수를 2시간 동안 흘려주었다. 4시간 동안 용리한 용액에서 수득한 분획물을 F1 분획물이라 칭하고, 용매를 0.5 M NaCl 용액으로 바꾸어 준 후, 다시 4시간 후 동한 용리한 용액에서 수득한 분획물을 F2 분획물이라 칭하였으며, 1.0 M NaCl 용액으로 4시간 동안 용리한 용액에서 수득한 분획물을 F3 분획물이라 칭하였다. 또한, 1.5 M NaCl 용액으로 4시간 동안 용리한 용액에서 수득한 분획물을 F4 분획물이라 칭하였다. 각 용매에서 수득한 분획물을 Mw 8000의 투석막(dialysis membrane)에 주입하여, 일반수에서 2일간, 증류수에서 1일간 투석한 후 동결건조하여 최종 정제된 F1, F2, F3, F4 분획물을 수득하였다. 그리고, 상기 F1, F2, F3, F4 분획물을 혼합하여 본 발명의 해삼 분획물을 얻었다.First, 200 mg of the crude polysaccharide was dissolved in 10 mL of distilled water, and then dissolved into smaller particles using a microwave bomb. After heating by microwave and hot polysaccharide was injected into ion exchange column chromatography, distilled water was flowed for 2 hours. The fraction obtained from the solution eluted for 4 hours was called the F1 fraction, the solvent was changed to a 0.5 M NaCl solution, and the fraction obtained from the eluted solution after 4 hours was called the F2 fraction, and 1.0 M NaCl solution The fraction obtained from the solution eluted for 4 hours was referred to as an F3 fraction. In addition, a fraction obtained from a solution eluted with 1.5 M NaCl solution for 4 hours was referred to as an F4 fraction. Fractions obtained from each solvent were injected into a dialysis membrane of Mw 8000, dialyzed for 2 days in normal water and 1 day in distilled water, and then lyophilized to obtain final purified F1, F2, F3, F4 fractions. And, the F1, F2, F3, F4 fractions were mixed to obtain a sea cucumber fraction of the present invention.
실험예 1. 대장암 세포에서 TRAIL과 해삼 분획물이 세포 성장에 미치는 영향Experimental Example 1. Effect of TRAIL and sea cucumber fraction on cell growth in colon cancer cells
1-1. 대장암 세포주에서의 암세포 성장 억제효과 확인 - WST-1 assay1-1. Confirmation of cancer cell growth inhibitory effect in colon cancer cell line-WST-1 assay
대장암 세포주와 대장 정상세포에서 해삼 분획물 및/또는 TRAIL이 세포 성장에 미치는 영향을 조사하기 위하여 WST-1 assay를 수행하였다.WST-1 assay was performed to investigate the effect of sea cucumber fraction and/or TRAIL on cell growth in colon cancer cell lines and colon normal cells.
1-1-1. 해삼 분획물에 의한 Cell proliferation 확인1-1-1. Cell proliferation confirmation by sea cucumber fraction
먼저, 대장암 세포(DLD-1, HCT116)와 대장 정상세포(CCD18CO)를 96well에 각각 seeding한 후, 다음 날 해삼 분획물을 농도별(0, 100, 200, 300 ㎍/ml)로 처리하였다. First, colon cancer cells (DLD-1, HCT116) and colon normal cells (CCD18CO) were seeded in 96 wells, respectively, and then sea cucumber fractions were treated at different concentrations (0, 100, 200, 300 μg/ml) the next day.
그리고, 24시간 후에 WST-1 solution을 넣어 4시간 동안 반응시킨 후 450 nm에서 흡광도를 측정하여 살아있는 세포 수를 측정하여 도 2에 나타내었다.Then, after 24 hours, the WST-1 solution was added and reacted for 4 hours, and the absorbance was measured at 450 nm to measure the number of living cells, and it is shown in FIG. 2.
도 2는 해삼 분획물이 대장암의 세포성장에 영향을 미치는 정도를 나타내는 세포성장(cell viability) 그래프이다. 상기 도 2를 살펴보면, 해삼 분획물은 세포의 성장에 큰 영향이 없는 것을 확인할 수 있다. Figure 2 is a cell growth (cell viability) graph showing the extent to which the sea cucumber fraction affects the cell growth of colon cancer. Referring to FIG. 2, it can be seen that the sea cucumber fraction has no significant effect on the growth of cells.
1-1-2. TRAIL에 의한 Cell proliferation 확인1-1-2. Cell proliferation confirmation by TRAIL
또한, 대장암 세포(DLD-1, HCT116, Colo205, HT29, SW620)와 대장 정상세포(CCD18CO)를 96well에 각각 seeding한 후, 다음 날 TRAIL을 농도별(0, 10, 25, 50, 100 ng/ml)로 처리하였다. In addition, after seeding colon cancer cells (DLD-1, HCT116, Colo205, HT29, SW620) and normal colon cells (CCD18CO) in 96 wells, respectively, the next day, TRAIL was added by concentration (0, 10, 25, 50, 100 ng). /ml).
그리고, 2시간 후에 WST-1 solution을 넣어 4시간 동안 반응시킨 후 450 nm에서 흡광도를 측정하여 살아있는 세포 수를 측정하여 도 3에 나타내었다.Then, after 2 hours, the WST-1 solution was added and reacted for 4 hours, and the absorbance was measured at 450 nm, and the number of living cells was measured and shown in FIG. 3.
도 3은 대장암 세포(DLD-1, HCT116, Colo205, HT29, SW620)에서 TRAIL이 세포성장(cell viability)에 영향을 미치는 정도를 나타내는 그래프이다. 상기 도 3을 살펴보면, TRAIL은 대장암 세포의 성장에 미치는 영향이 대장암 세포의 종류에 따라 다른 것을 확인할 수 있다. 특히, TRAIL은 HCT116 세포의 성장에 큰 영향을 미치는 것을 알 수 있다. 3 is a graph showing the extent to which TRAIL affects cell viability in colon cancer cells (DLD-1, HCT116, Colo205, HT29, SW620). Referring to FIG. 3, it can be seen that the effect of TRAIL on the growth of colon cancer cells differs according to the type of colon cancer cells. In particular, it can be seen that TRAIL has a great influence on the growth of HCT116 cells.
1-1-3. 해삼 분획물과 TRAIL의 조합에 의한 Cell proliferation 확인1-1-3. Cell proliferation confirmation by combination of sea cucumber fraction and TRAIL
대장암 세포주(DLD-1)에서 해삼 분획물이 TRAIL의 민감성에 영향을 끼치는지 알아보고자 하였다.The purpose of this study was to find out whether the sea cucumber fraction affects the sensitivity of TRAIL in the colon cancer cell line (DLD-1).
대장암 세포(DLD-1)를 96well에 seeding한 후, 다음 날 해삼 분획물(200 ㎍/m)을 처리하였다. 그리고, 다음날, TRAIL(25 ng/ml)을 처리하고, 2시간 후에 WST-1 solution을 넣어 4시간 동안 반응시킨 후 450 nm에서 흡광도를 측정하여 살아있는 세포 수를 측정하여 도 4에 나타내었다.After seeding colon cancer cells (DLD-1) in 96 wells, the sea cucumber fraction (200 μg/m) was treated the next day. Then, the next day, TRAIL (25 ng/ml) was treated, and after 2 hours, WST-1 solution was added and reacted for 4 hours, and the absorbance was measured at 450 nm to measure the number of living cells, as shown in FIG. 4.
상기 도 4는 대장암 세포(DLD-1)에서 해삼 분획물(SC F2)과 TRAIL의 조합이 세포성장(cell viability)에 영향을 미치는 정도를 나타내는 그래프이다. 상기 도 4를 살펴보면, 대장암 세포(DLD-1)에 해삼 분획물이 투여된 경우 TRAIL에 의한 민감성이 더욱 높아진 것을 확인할 수 있다.4 is a graph showing the extent to which the combination of sea cucumber fraction (SC F2) and TRAIL affects cell viability in colon cancer cells (DLD-1). Referring to FIG. 4, when the sea cucumber fraction is administered to colon cancer cells (DLD-1), it can be seen that the sensitivity to TRAIL is further increased.
1-2. 해삼 분획물을 투여한 세포주에서 TRAIL에 대해 민감성이 증가함을 단백질 수준에서 확인 - Western blot1-2. Confirmation at the protein level that the sensitivity to TRAIL is increased in the cell line administered with the sea cucumber fraction-Western blot
대장암 세포주(DLD-1)에서 해삼 분획물을 투여한 후 TRAIL을 처리하는 경우에 세포 사멸 관련 단백질의 발현을 비교하기 위한 실험을 실시하였다. 이를 위해 먼저, 대장암 세포주(DLD-1)를 seeding한 후, 다음 날 해삼 분획물(200 ㎍/m)을 처리하고, 24시간 후에 TRAIL(25 ng/ml)을 처리하였다. 4 시간 후 harvest하여 lysis를 통하여 단백질을 모아 western blotting을 실시하였다. 그리고, 세포 사멸에 관련한 marker(PARP, Caspase)를 사용하여 발현의 변화를 확인하여 도 5에 나타내었다.An experiment was conducted to compare the expression of apoptosis-related proteins in the case of treatment with TRAIL after administering the sea cucumber fraction in the colon cancer cell line (DLD-1). To this end, first, after seeding the colon cancer cell line (DLD-1), the sea cucumber fraction (200 μg/m) was treated the next day, and TRAIL (25 ng/ml) was treated after 24 hours. After 4 hours, the protein was harvested, and the protein was collected through lysis and western blotting was performed. In addition, a change in expression was confirmed using markers related to cell death (PARP, Caspase) and shown in FIG. 5.
상기 도 5는 대장암 세포주(DLD-1)에서 해삼 분획물(SC F2)을 투여한 후 TRAIL을 처리한 경우 세포사멸 관련 단백질의 발현이 증가되는 것을 나타내는 웨스턴 블랏 결과이다. 상기 도 5를 살펴보면, 대장암 세포주(DLD-1)에 해삼 분획물이 투여한 후 TRAIL을 처리한 경우, Caspase 3, Caspase 9, PARP와 같은 세포 사멸에 관련된 단백질들의 발현이 증가하는 것을 확인하였다.5 is a Western blot result showing that the expression of apoptosis-related protein is increased when TRAIL is treated after administration of the sea cucumber fraction (SC F2) in the colon cancer cell line (DLD-1). Referring to FIG. 5, when the sea cucumber fraction was administered to the colon cancer cell line (DLD-1) and then treated with TRAIL, it was confirmed that the expression of proteins related to cell death such as
이러한 결과들을 통해, 해삼 분획물을 투여하는 경우 TRAIL에 대한 민감성이 증가하는 것을 세포 수준에서 확인할 수 있었다.Through these results, it was confirmed at the cellular level that the sensitivity to TRAIL is increased when the sea cucumber fraction is administered.
1-3. 해삼 분획물을 투여한 세포주에서 TRAIL에 대해 민감성이 증가함을 세포의 형태학적 변화를 통해 확인함1-3. It was confirmed through cell morphological changes that the sensitivity to TRAIL was increased in the cell line administered with the sea cucumber fraction.
해삼 분획물이 세포의 형태학적 변화에 끼치는 영향을 확인해 보고자 하였다.To confirm the effect of the sea cucumber fraction on the morphological change of cells.
이를 위하여, 대장암 세포주(DLD-1)를 6well에 seeding 한 후, 다음 날 해삼 분획물(200 ㎍/m)을 처리하였다. 그리고, 24시간 후에 TRAIL(25 ng/ml)을 처리하고, 4시간 후 세포의 모양을 광학 현미경으로 관찰하여 도 6에 나타내었다. To this end, after seeding the colon cancer cell line (DLD-1) in 6 wells, the sea cucumber fraction (200 μg/m) was treated the next day. Then, after 24 hours, TRAIL (25 ng/ml) was treated, and after 4 hours, the shape of the cells was observed with an optical microscope, and is shown in FIG. 6.
상기 도 6을 살펴보면, 해삼 분획물이 투여된 세포주에 TRAIL(25 ng/ml)을 처리한 실험군은 세포 형태가 파괴되어 떠 있는 모양으로 형성되는 것을 확인할 수 있다. Referring to FIG. 6, it can be seen that the cell line to which the sea cucumber fraction was administered was treated with TRAIL (25 ng/ml), and the cell morphology was destroyed to form a floating shape.
1-4. 해삼 분획물과 TRAIL을 투여한 세포의 콜로니 형성능력이 저해됨을 확인함 - colony formation assay1-4. Confirmed that the colony formation ability of the sea cucumber fraction and the cells administered with TRAIL is inhibited-colony formation assay
해삼 분획물이 세포 성장에 미치는 영향을 확인해 보고자 하였다. To confirm the effect of the sea cucumber fraction on cell growth.
이를 위한 콜로니 형성능을 확인하기 위하여, 대장암 세포주(DLD-1)를 seeding한 후, 다음 날 해삼 분획물(200 ㎍/m)을 처리하였다. 그리고, 24시간 후에 TRAIL(25 ng/ml)을 처리하고, 4시간 후 살아있는 세포를 다시 6well에 seeding하였다. 그리고, 14일 후 콜로니가 형성되면 염색하여 콜로니의 수를 확인하여 도 7 및 도 8에 나타내었다. In order to confirm the colony-forming ability for this, the colon cancer cell line (DLD-1) was seeded, and the sea cucumber fraction (200 μg/m) was treated the next day. Then, after 24 hours, TRAIL (25 ng/ml) was treated, and after 4 hours, the living cells were seeded into 6 wells again. And, when colonies are formed after 14 days, they were stained to check the number of colonies, and are shown in FIGS. 7 and 8.
도 7은 상기 실험에서 콜로니가 형성된 모습을 나타내는 사진이고, 도 8은 상기 실험에서 콜로니의 수를 계수하여 나타낸 그래프이다. 상기 도 7 및 도 8을 살펴보면, 해삼 분획물이 투여된 세포주에 TRAIL을 처리한 실험군은 콜로니가 덜 형성되는 것을 확인할 수 있다.7 is a photograph showing a state in which colonies were formed in the experiment, and FIG. 8 is a graph showing the count of the number of colonies in the experiment. 7 and 8, it can be seen that less colonies were formed in the experimental group treated with TRAIL in the cell line to which the sea cucumber fraction was administered.
1-5. 해삼 분획물을 투여한 세포주에서 TRAIL에 대해 민감성이 증가함을 확인 - Flow cytometry1-5. Confirmation that the sensitivity to TRAIL is increased in the cell line administered with the sea cucumber fraction-Flow cytometry
대장암 세포주(DLD-1)에서 해삼 분획물이 TRAIL의 민감성에 영향을 끼치는지 알아보고자 하였다.The purpose of this study was to find out whether the sea cucumber fraction affects the sensitivity of TRAIL in the colon cancer cell line (DLD-1).
이를 위하여 먼저, 대장암 세포주(DLD-1)를 seeding한 후, 다음 날 해삼 분획물(200 ㎍/m)을 처리하였다. 그리고, 24시간 후 TRAIL(20 ng/ml)을 처리하고, 4시간 후 cell을 harvest하였다. Annexin V-FITC, PI(propidium iodide)로 염색하고 Flow cytometry 측정 기계를 이용하여 세포 수를 측정하여 도 9에 나타내었다. To this end, first, after seeding the colon cancer cell line (DLD-1), the sea cucumber fraction (200 μg/m) was treated the next day. Then, after 24 hours, TRAIL (20 ng/ml) was treated, and the cells were harvested after 4 hours. Annexin V-FITC, PI (propidium iodide) was stained and the number of cells was measured using a flow cytometry measuring machine, and shown in FIG. 9.
도 9는 해삼 분획물이 투여된 대장암 세포주에서 TRAIL에 의한 apoptosis가 현저히 증가하는 것을 나타내는 그래프이다.9 is a graph showing a marked increase in apoptosis by TRAIL in a colon cancer cell line administered with a sea cucumber fraction.
상기 도 9를 살펴보면, 해삼 분획물이 투여된 대장암 세포주에서 TRAIL에 의한 민감성이 더욱 높은 것을 확인할 수 있다.9, it can be seen that the sensitivity to TRAIL is higher in the colon cancer cell line to which the sea cucumber fraction was administered.
실험예 2. 위암 및 유방암 세포에서 TRAIL과 해삼 분획물이 세포 성장에 미치는 영향Experimental Example 2. Effect of TRAIL and sea cucumber fraction on cell growth in gastric cancer and breast cancer cells
2-1. 해삼 분획물을 투여한 위암 세포주에서 TRAIL에 대해 민감성이 증가함을 확인 - Flow cytometry2-1. Confirmation of increased sensitivity to TRAIL in gastric cancer cell lines administered with sea cucumber fraction-Flow cytometry
위암 세포주에서 해삼 분획물이 TRAIL의 민감성에 영향을 끼치는지 알아보고자 하였다.The purpose of this study was to find out whether sea cucumber fractions affect the sensitivity of TRAIL in gastric cancer cell lines.
이를 위하여 먼저, 위암 세포주(AGS, SNU638)를 각각 seeding한 후, 다음 날 해삼 분획물(200 ㎍/m)을 처리하였다. 그리고, 24시간 후 TRAIL(20 ng/ml)을 처리하고, 4시간 후 cell을 harvest하였다. Annexin V-FITC, PI(propidium iodide)로 염색하고 Flow cytometry 측정 기계를 이용하여 세포 수를 측정하여 도 10 및 도 11에 나타내었다. For this, first, gastric cancer cell lines (AGS, SNU638) were each seeded, and then the sea cucumber fraction (200 μg/m) was treated the next day. Then, after 24 hours, TRAIL (20 ng/ml) was treated, and the cells were harvested after 4 hours. Annexin V-FITC and PI (propidium iodide) were stained and the number of cells was measured using a flow cytometry measuring machine, and are shown in FIGS. 10 and 11.
도 10은 해삼 분획물이 투여된 위암 세포주 AGS에서 TRAIL에 의한 apoptosis가 현저히 증가하는 것을 나타내는 그래프이고, 도 11은 해삼 분획물이 투여된 위암 세포주 SNU638에서 TRAIL에 의한 apoptosis가 현저히 증가하는 것을 나타내는 그래프이다.FIG. 10 is a graph showing a marked increase in apoptosis by TRAIL in the gastric cancer cell line AGS administered with a sea cucumber fraction, and FIG. 11 is a graph showing a marked increase in apoptosis by TRAIL in the gastric cancer cell line SNU638 to which the sea cucumber fraction was administered.
상기 도 10 및 도 11을 살펴보면, 해삼 분획물이 투여된 위암 세포주에서 TRAIL에 의한 민감성이 더욱 높은 것을 확인할 수 있다.10 and 11, it can be seen that the sensitivity to TRAIL is higher in the gastric cancer cell line to which the sea cucumber fraction was administered.
2-2. 해삼 분획물을 투여한 유방암 세포주에서 TRAIL에 대해 민감성이 증가함을 확인 - Flow cytometry2-2. Confirmation of increased sensitivity to TRAIL in breast cancer cell lines administered with sea cucumber fraction-Flow cytometry
유방암 세포주(MCF-7)에서 해삼 분획물이 TRAIL의 민감성에 영향을 끼치는지 알아보고자 하였다.We tried to find out whether the sea cucumber fraction affects the sensitivity of TRAIL in breast cancer cell line (MCF-7).
이를 위하여 먼저, 유방암 세포주(MCF-7)를 seeding한 후, 다음 날 해삼 분획물(200 ㎍/m)을 처리하였다. 그리고, 24시간 후 TRAIL(20 ng/ml)을 처리하고, 4시간 후 cell을 harvest하였다. Annexin V-FITC, PI(propidium iodide)로 염색하고 Flow cytometry 측정 기계를 이용하여 세포 수를 측정하여 도 12에 나타내었다. For this, first, the breast cancer cell line (MCF-7) was seeded, and then the sea cucumber fraction (200 μg/m) was treated the next day. Then, after 24 hours, TRAIL (20 ng/ml) was treated, and the cells were harvested after 4 hours. Annexin V-FITC and PI (propidium iodide) were stained and the number of cells was measured using a flow cytometry measuring machine, and shown in FIG. 12.
도 12는 해삼 분획물이 투여된 유방암 세포주에서 TRAIL에 의한 apoptosis가 현저히 증가하는 것을 나타내는 그래프이다.12 is a graph showing a marked increase in apoptosis by TRAIL in a breast cancer cell line administered with a sea cucumber fraction.
상기 도 12를 살펴보면, 해삼 분획물이 투여된 유방암 세포주에서 TRAIL에 의한 민감성이 더욱 높은 것을 확인할 수 있다.Referring to FIG. 12, it can be seen that the sensitivity to TRAIL is higher in the breast cancer cell line to which the sea cucumber fraction was administered.
실험예 3. 대장암 세포에서 해삼 분획물이 미치는 효과 확인Experimental Example 3. Check the effect of the sea cucumber fraction in colon cancer cells
3-1. 대장암 세포에서 해삼 분획물에 의한 세포사멸 관련 인자의 변화 확인3-1. Confirmation of changes in factors related to apoptosis by sea cucumber fraction in colon cancer cells
해삼 분획물이 대장암 세포주에 투여됐을 때의 세포사멸 신호전달에 관여한 인자들의 발현의 변화를 살펴보기 위해 pro-apoptotic, anti-apoptotic 단백질의 발현의 변화를 단백질 수준에서 살펴보기 위한 실험을 실시하였다. An experiment was conducted to examine the changes in the expression of pro-apoptotic and anti-apoptotic proteins at the protein level to examine the changes in the expression of factors involved in apoptosis signaling when the sea cucumber fraction was administered to a colon cancer cell line. .
이를 위하여, 대장암 세포주(DLD-1)를 seeding한 후, 다음 날 해삼 분획물(200 ㎍/m)을 처리하였다. 그리고, 4시간 후 cell을 harvest하고, lysis를 통하여 단백질을 모아 western blotting을 실시하였다. 세포사멸 관련 마커를 사용하여 확인하고, 이를 도 13 및 도 14에 나타내었다.To this end, after seeding the colon cancer cell line (DLD-1), the sea cucumber fraction (200 μg/m) was treated the next day. And, after 4 hours, the cells were harvested, and proteins were collected through lysis and western blotting was performed. It was confirmed using a cell death-related marker, which is shown in FIGS. 13 and 14.
도 13은 해삼 분획물 투여시의 미토콘드리아의 세포사멸에 관련된 신호전달 단백질의 발현을 단백질 수준에서 확인한 결과이고, 도 14는 해삼 분획물 투여시의 TRAIL 신호전달에 관련된 인자들의 발현을 단백질 수준에서 확인한 결과이다.13 is a result of confirming the expression of signaling proteins related to mitochondrial apoptosis at the protein level when the sea cucumber fraction is administered, and FIG. 14 is a result of confirming the expression of factors related to TRAIL signaling at the protein level when the sea cucumber fraction is administered. .
도 13을 살펴보면, 대장암 세포주에 해삼 분획물 투여시 anti-apoptosis 인자인 XIAP의 단백질 수준이 감소됨을 확인할 수 있었다. Referring to FIG. 13, it was confirmed that the protein level of the anti-apoptosis factor XIAP was decreased when the sea cucumber fraction was administered to the colon cancer cell line.
또한, 도 14를 통해 대장암 세포주에 해삼 분획물 투여시 TRAIL 신호전달에 관련된 인자인 caspase 3 및 caspase 9의 cleavage 폼이 증가한 것을 확인할 수 있었다.In addition, it was confirmed through FIG. 14 that the cleavage forms of
이러한 결과들은 해삼 분획물이 대장암 세포의 세포사멸을 유도한다는 것과 TRAIL에 의한 세포사멸 민감성을 증대시킨다는 것을 증명한다.These results prove that the sea cucumber fraction induces apoptosis of colon cancer cells and increases the sensitivity to apoptosis by TRAIL.
3-2. 대장암 세포에서 해삼 분획물에 의한 ER stress 증가 확인3-2. Confirmation of increase in ER stress by sea cucumber fraction in colon cancer cells
웨스턴 블랏 실험Western blot experiment
해삼 분획물이 대장암 세포주에 투여됐을 때의 ER stress의 변화를 확인하고자 하였다.It was attempted to confirm the change in ER stress when the sea cucumber fraction was administered to a colon cancer cell line.
이를 위하여 대장암 세포에서 해삼 분획물 투여에 의해 ER stress 관련 인자가 증가하는지를 확인하는 웨스턴 블랏 실험을 실시하였다. To this end, a Western blot experiment was conducted to confirm whether the factor related to ER stress increased by the administration of the sea cucumber fraction in colon cancer cells.
먼저, 대장암 세포주(DLD-1)를 seeding한 후, 다음 날 해삼 분획물(200 ㎍/m)을 처리하였다. 그리고, 4시간 후 cell을 harvest하였다. 그리고, lysis를 통하여 단백질을 모아 western blotting을 실시하고, 이를 도 15 및 도 16에 나타내었다.First, after seeding the colon cancer cell line (DLD-1), the sea cucumber fraction (200 μg/m) was treated the next day. And, after 4 hours, the cells were harvested. And, Western blotting was performed by collecting proteins through lysis, which are shown in FIGS. 15 and 16.
도 15 및 도 16은 해삼 분획물에 의해 ER Stress 관련 인자가 증가하는지를 확인하는 웨스턴 블랏 실험 결과이다.15 and 16 are results of a Western blot experiment confirming whether the ER Stress related factor is increased by the sea cucumber fraction.
상기 도 15을 통해, 해삼 분획물에 의해 ER Stress 관련 단백질 중 IRE-1α와 JNK의 인산화가 증가함을 확인할 수 있었다. 15, it was confirmed that phosphorylation of IRE-1α and JNK among ER Stress-related proteins was increased by the sea cucumber fraction.
그리고, 상기 도 16을 통해, 시간이 경과함에 따라 해삼 분획물에 의해 ER stress 신호전달의 마지막 전사인자인 CHOP가 증가함을 확인할 수 있었다. In addition, through FIG. 16, it was confirmed that CHOP, the last transcription factor of ER stress signaling, increased by the sea cucumber fraction as time passed.
이러한 결과들은 대장암 세포주에서 해삼 분획물이 ER stress를 증가시킴을 의미한다.These results imply that the sea cucumber fraction increases ER stress in colon cancer cell lines.
Flow cytometry, DCF-DA assayFlow cytometry, DCF-DA assay
상기 해삼 분획물에 의한 ER stress의 증가가 활성 산소(ROS, Reactive oxygen species)에 의한 것인지를 2,7-dicholorofluorescein diacetate(DCFH-DA) 염색을 통하여 Flow cytometry로 확인하고자 하였다. Whether the increase in ER stress caused by the sea cucumber fraction was due to reactive oxygen species (ROS) was confirmed by flow cytometry through 2,7-dicholorofluorescein diacetate (DCFH-DA) staining.
이를 위하여 먼저, 대장암 세포주(DLD-1)를 seeding한 후, 해삼 분획물(200 ㎍/m)을 3시간 동안 처리하였다. 그리고 형광 probe인 DCFH-DA(FITC conjugated) 시약으로 30분 동안 처리한 후, Flow cytometry를 이용하여 10,000 여개의 cell을 찍어 ROS 발생을 관찰하였다. To this end, first, after seeding the colon cancer cell line (DLD-1), the sea cucumber fraction (200 μg/m) was treated for 3 hours. Then, after treatment with a fluorescent probe DCFH-DA (FITC conjugated) reagent for 30 minutes, 10,000 cells were photographed using flow cytometry to observe the occurrence of ROS.
도 17은 해삼 분획물에 의한 ER stress의 증가가 ROS에 의한 것인지를 확인하기 위해, Flow cytometry를 이용하여 ROS를 측정한 결과를 나타내는 것이다. 상기 도 17을 통해, 해삼 분획물에 의해 ROS가 두 배 이상 증가되는 것을 확인할 수 있었다. 17 shows the results of measuring ROS using flow cytometry in order to confirm whether the increase in ER stress caused by the sea cucumber fraction is caused by ROS. Through the FIG. 17, it was confirmed that the ROS was increased more than twice by the sea cucumber fraction.
면역형광염색, DCF-DA assayImmunofluorescence staining, DCF-DA assay
대장암 세포주(DLD-1)를 seeding한 후, 해삼 분획물(200 ㎍/m)을 3시간 동안 처리하였다. 그리고 형광 probe인 DCFH-DA(FITC conjugated) 시약으로 30분 동안 처리한 후, 3.7% formaldehyde를 이용하여 fixiation 후 DAPI staining을 통해 핵을 염색하였다. mounting 과정을 통해 고정시켜 confocal microscopy를 통해 ROS의 발생을 확인하였다.After seeding the colon cancer cell line (DLD-1), the sea cucumber fraction (200 μg/m) was treated for 3 hours. Then, after treatment with a fluorescent probe DCFH-DA (FITC conjugated) reagent for 30 minutes, fixiation using 3.7% formaldehyde, and DAPI staining to stain the nuclei. It was fixed through the mounting process and the occurrence of ROS was confirmed through confocal microscopy.
도 18은 해삼 분획물에서 의해 ROS가 증가됨을 면역형광염색으로 나타낸 것이다. 18 shows the increase in ROS in the sea cucumber fraction by immunofluorescence staining.
이러한 결과들은 대장암 세포주에서 해삼 분획물이 활성산소(ROS)를 증가시키고, 이로 인해 ER stress가 증가됨을 의미한다.These results imply that the sea cucumber fraction increases active oxygen (ROS) in colorectal cancer cell lines, thereby increasing ER stress.
실험예 4. 대장암 세포에서 해삼 분획물에 의한 XIAP의 발현을 mRNA 수준에서 확인 Experimental Example 4. Expression of XIAP by sea cucumber fraction in colon cancer cells was confirmed at the mRNA level
4-1. 대장암 세포에서 해삼 분획물에 의한 XIAP의 RNA 수준 확인 - PCR4-1. Confirmation of RNA level of XIAP by sea cucumber fraction in colon cancer cells-PCR
해삼 분획물이 TRAIL에 의해 유도되는 세포 사멸을 증가시키는 과정에서 XIAP가 작용하는지를 알아보기 위하여, 세포 사멸에 관련한 인자인 XIAP의 변화를 RNA 수준에서 살펴보기 위한 실험을 실시하였다. In order to find out whether XIAP acts in the process of increasing apoptosis induced by TRAIL in the sea cucumber fraction, an experiment was conducted to examine changes in XIAP, a factor related to apoptosis, at the RNA level.
이를 위하여 먼저, 대장암 세포주(DLD-1)를 seeding한 후, 다음 날 해삼 분획물(200 ㎍/m)을 처리하였다. 그리고, 다음날 cell을 harvest한 후 RNA를 분리하고 TaqMan probe를 이용하여 PCR을 시행하였다. 그리고, 1% agarose gel을 사용하여 확인한 후 도 19에 나타내었다.To this end, first, after seeding the colon cancer cell line (DLD-1), the sea cucumber fraction (200 μg/m) was treated the next day. Then, after harvesting the cells the next day, RNA was isolated and PCR was performed using a TaqMan probe. And, it is shown in Fig. 19 after confirmation using 1% agarose gel.
도 19는 해삼 분획물 투여시에 RNA 수준에서 XIAP의 발현량에 영향을 주지 않는다는 것을 확인한 결과이다.Figure 19 shows the results confirming that the administration of the sea cucumber fraction does not affect the expression level of XIAP at the RNA level.
4-2. 대장암 세포에서 해삼 분획물에 의한 XIAP의 RNA 수준 확인 - Real time PCR4-2. Confirmation of RNA level of XIAP by sea cucumber fraction in colon cancer cells-Real time PCR
해삼 분획물이 TRAIL에 의해 유도되는 세포 사멸을 증가시키기 때문에 어떠한 인자가 작용하는 지를 알아보기 위하여, TRAIL 신호전달과 세포 사멸에 관련한 인자들을 RNA 수준에서 살펴보기 위한 실험을 실시하였다. In order to find out which factors act because the sea cucumber fraction increases apoptosis induced by TRAIL, an experiment was conducted to examine factors related to TRAIL signaling and apoptosis at the RNA level.
이를 위하여 먼저, 대장암 세포주(DLD-1)를 seeding한 후, 다음 날 해삼 분획물(200 ㎍/m)을 처리하였다. 그리고, 다음날 cell을 harvest한 후 RNA를 분리하고 TaqMan probe를 이용하여 qRT-PCR을 시행하였다. 그리고, CT값을 획득하여 발현양을 계산하여 분석하였다. To this end, first, after seeding the colon cancer cell line (DLD-1), the sea cucumber fraction (200 μg/m) was treated the next day. Then, after harvesting the cells the next day, RNA was isolated and qRT-PCR was performed using a TaqMan probe. Then, the CT value was obtained and the expression level was calculated and analyzed.
도 20은 해삼 분획물 투여시에 RNA 수준에서 XIAP의 발현량에 영향을 주지 않는다는 것을 확인한 결과이다.Figure 20 shows the results confirming that the administration of the sea cucumber fraction does not affect the expression level of XIAP at the RNA level.
이러한 결과들은 해삼 분획물이 XIAP의 RNA 수준을 조절하는 것이 아니라 단백질 수준을 조절하는 것을 의미한다. These results indicate that the sea cucumber fraction modulates the protein level, not the RNA level of XIAP.
실험예 5. 대장암 세포에서 해삼 분획물에 의한 XIAP ubiquitination 증가 확인 Experimental Example 5. Confirmation of increase in XIAP ubiquitination by sea cucumber fraction in colon cancer cells
Co-immunoprecipitationCo-immunoprecipitation
해삼 분획물이 XIAP의 ubiquitination에 관여하는지 알아보기 위해 Co-Immunoprecipitation 실험을 진행하였다.Co-Immunoprecipitation experiment was conducted to determine whether the sea cucumber fraction is involved in ubiquitination of XIAP.
먼저, 상기 실험을 실시하기 위해 대장암 세포주(DLD-1)를 seeding한 후, 다음 날 해삼 분획물(200 ㎍/m)을 처리하였다. 그리고, 4시간 후 cell을 harvest하여 lysis하였다. 1차로 XIAP antibody을 붙이고 난 후에, Western blotting과 같은 방법을 통하여 ubiquitination의 발현을 확인하였고, 그 결과를 도 21에 나타내었다.First, after seeding the colon cancer cell line (DLD-1) to perform the experiment, the sea cucumber fraction (200 μg/m) was treated the next day. And, after 4 hours, the cells were harvested and lysed. After attaching the XIAP antibody first, the expression of ubiquitination was confirmed through a method such as Western blotting, and the results are shown in FIG. 21.
도 21은 해삼 분획물에 의해 XIAP의 ubiquitination이 증가하여, XIAP가 감소되는 것을 나타내는 Co-Immunoprecipitation 결과이다.21 is a result of Co-Immunoprecipitation showing that the ubiquitination of XIAP is increased by the sea cucumber fraction, and XIAP is decreased.
상기 실험 결과를 통해, 해삼 분획물을 처리하였을 때 XIAP의 ubiquitination에 의한 감소를 통하여 TRAIL의 세포사멸 효과를 증가시켜 준다는 것을 알 수 있다.From the above experimental results, it can be seen that treatment of the sea cucumber fraction increases the apoptosis effect of TRAIL through the reduction of XIAP due to ubiquitination.
상기 실험 결과들을 통해, 암 세포주에서 해삼 분획물은 XIAP의 발현을 감소시키고, 이로 인해 TRAIL에 의해 유도되는 세포 사멸의 민감성을 증진시키는 것이 확인되었다.Through the above experimental results, it was confirmed that the sea cucumber fraction in cancer cell lines decreases the expression of XIAP, and thereby enhances the sensitivity of cell death induced by TRAIL.
이하, 바람직한 실시예를 들어 본 발명을 더욱 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이에 의하여 제한되지 않는다는 것은 당업계의 통상의 지식을 가진 자에게 자명할 것이다. Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples are for explaining the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.
Claims (7)
상기 해삼 추출물 유래 다당류는 해삼을 C1 내지 C4의 알코올로 추출한 후 남은 침전물을 수득하는 단계; 상기 침전물로부터 색소, 지질 및 저분자 단백질을 제거하는 단계; 및 상기 색소, 지질 및 단백질이 제거된 침전물로부터 다당류를 수득하는 단계;를 포함하는 방법으로 제조된 것이고,
상기 암은 TRAIL-내성 위암 및 TRAIL-내성 유방암 중에서 선택된 1종 이상인 것을 특징으로 하는 암 예방 또는 치료용 약학 조성물.As a pharmaceutical composition for preventing or treating cancer comprising a polysaccharide derived from sea cucumber extract and TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) as an active ingredient,
The polysaccharide derived from the sea cucumber extract is obtained by extracting the sea cucumber with alcohol of C1 to C4 and then obtaining a precipitate remaining; Removing pigments, lipids, and low molecular weight proteins from the precipitate; And obtaining a polysaccharide from the precipitate from which the pigments, lipids and proteins have been removed; and
The cancer is a pharmaceutical composition for preventing or treating cancer, characterized in that at least one selected from TRAIL-resistant gastric cancer and TRAIL-resistant breast cancer.
상기 해삼 추출물 유래 다당류는 XIAP(X-chromosome linked inhibitor of apoptosis protein) 단백질의 발현 감소와 활성화된 카스파제 3(cleaved caspase 3) 및 활성화된 카스파제 9(cleaved caspase 9)의 발현 증가를 유도하는 것을 특징으로 하는 조성물.The method of claim 1,
The polysaccharide derived from sea cucumber extract induces decreased expression of XIAP (X-chromosome linked inhibitor of apoptosis protein) protein and increased expression of activated caspase 3 and activated caspase 9. Composition characterized by.
상기 해삼 추출물 유래 다당류는 해삼을 C1 내지 C4의 알코올로 추출한 후 남은 침전물을 수득하는 단계; 상기 침전물로부터 색소, 지질 및 저분자 단백질을 제거하는 단계; 및 상기 색소, 지질 및 단백질이 제거된 침전물로부터 다당류를 수득하는 단계;를 포함하는 방법으로 제조된 것이고,
상기 암은 TRAIL-내성 위암 및 TRAIL-내성 유방암 중에서 선택된 1종 이상인 것을 특징으로 하는 TRAIL의 항암 효과 증진용 약학 조성물.As a pharmaceutical composition for enhancing the anticancer effect of TRAIL, comprising a polysaccharide derived from sea cucumber extract as an active ingredient, and reducing the resistance of TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand),
The polysaccharide derived from the sea cucumber extract is obtained by extracting the sea cucumber with alcohol of C1 to C4 and then obtaining a precipitate remaining; Removing pigments, lipids, and low molecular weight proteins from the precipitate; And obtaining a polysaccharide from the precipitate from which the pigments, lipids and proteins have been removed; and
The cancer is a pharmaceutical composition for enhancing the anticancer effect of TRAIL, characterized in that at least one selected from TRAIL-resistant gastric cancer and TRAIL-resistant breast cancer.
상기 해삼 추출물 유래 다당류는 해삼을 C1 내지 C4의 알코올로 추출한 후 남은 침전물을 수득하는 단계; 상기 침전물로부터 색소, 지질 및 저분자 단백질을 제거하는 단계; 및 상기 색소, 지질 및 단백질이 제거된 침전물로부터 다당류를 수득하는 단계;를 포함하는 방법으로 제조된 것이고,
상기 암은 TRAIL-내성 위암 및 TRAIL-내성 유방암 중에서 선택된 1종 이상인 것을 특징으로 하는 암세포 사멸 증진 방법.As a method for promoting cancer cell death, comprising administering a polysaccharide derived from sea cucumber extract and a Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in combination to cancer cells of mammals other than humans,
The polysaccharide derived from the sea cucumber extract is obtained by extracting the sea cucumber with alcohol of C1 to C4 and then obtaining a precipitate remaining; Removing pigments, lipids, and low molecular weight proteins from the precipitate; And obtaining a polysaccharide from the precipitate from which the pigments, lipids and proteins have been removed; and
The cancer is a method of promoting cancer cell death, characterized in that at least one selected from TRAIL-resistant gastric cancer and TRAIL-resistant breast cancer.
상기 해삼 추출물 유래 다당류는 해삼을 C1 내지 C4의 알코올로 추출한 후 남은 침전물을 수득하는 단계; 상기 침전물로부터 색소, 지질 및 저분자 단백질을 제거하는 단계; 및 상기 색소, 지질 및 단백질이 제거된 침전물로부터 다당류를 수득하는 단계;를 포함하는 방법으로 제조된 것이고,
상기 암은 TRAIL-내성 위암 및 TRAIL-내성 유방암 중에서 선택된 1종 이상인 것을 특징으로 하는 암의 예방 또는 치료 방법.A method for preventing or treating cancer comprising administering a polysaccharide derived from sea cucumber extract and a Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to an individual other than a human,
The polysaccharide derived from the sea cucumber extract is obtained by extracting the sea cucumber with alcohol of C1 to C4 and then obtaining a precipitate remaining; Removing pigments, lipids, and low molecular weight proteins from the precipitate; And obtaining a polysaccharide from the precipitate from which the pigments, lipids and proteins have been removed; and
The cancer is a method for preventing or treating cancer, characterized in that at least one selected from TRAIL-resistant gastric cancer and TRAIL-resistant breast cancer.
상기 해삼 추출물 유래 다당류는 해삼을 C1 내지 C4의 알코올로 추출한 후 남은 침전물을 수득하는 단계; 상기 침전물로부터 색소, 지질 및 저분자 단백질을 제거하는 단계; 및 상기 색소, 지질 및 단백질이 제거된 침전물로부터 다당류를 수득하는 단계;를 포함하는 방법으로 제조된 것이고,
상기 암은 TRAIL-내성 위암 및 TRAIL-내성 유방암 중에서 선택된 1종 이상인 것을 특징으로 하는 암 예방 또는 개선용 건강기능식품 조성물.As a health functional food composition for cancer prevention or improvement comprising a polysaccharide derived from sea cucumber extract and TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) as an active ingredient,
The polysaccharide derived from the sea cucumber extract is obtained by extracting the sea cucumber with alcohol of C1 to C4 and then obtaining a precipitate remaining; Removing pigments, lipids, and low molecular weight proteins from the precipitate; And obtaining a polysaccharide from the precipitate from which the pigments, lipids and proteins have been removed; and
The cancer is a health functional food composition for preventing or improving cancer, characterized in that at least one selected from TRAIL-resistant gastric cancer and TRAIL-resistant breast cancer.
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