KR101315897B1 - Gintonin with anti-metastasis activity and composition containing the gintonin for anti-cancer and anti-metastasis - Google Patents
Gintonin with anti-metastasis activity and composition containing the gintonin for anti-cancer and anti-metastasis Download PDFInfo
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- KR101315897B1 KR101315897B1 KR1020110128734A KR20110128734A KR101315897B1 KR 101315897 B1 KR101315897 B1 KR 101315897B1 KR 1020110128734 A KR1020110128734 A KR 1020110128734A KR 20110128734 A KR20110128734 A KR 20110128734A KR 101315897 B1 KR101315897 B1 KR 101315897B1
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- Prior art keywords
- gintonin
- cancer
- cells
- metastasis
- autotaxin
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Abstract
본 발명은 인삼에서 분리 동정한 당지질단백질(glycolipoprotein)인 진토닌(gintonin)의 신규 용도에 관한 것으로, 더욱 상세하게는 오토탁신(autotaxin) 혹은 리소포스포리파제 D(lisophospholipase D)의 활성 억제를 통해 암전이(cancer metastasis)를 억제하는 작용을 하는 진토닌과 상기 진토닌을 유효성분으로 함유하는 항암 및 암전이 억제용 조성물에 관한 것이다.
본 발명에 따른 진토닌은, 인삼에 존재하는 당지질단백질로서, 세포 독성은 거의 없는 반면, 암 세포에 대한 독성이 강하고, 암 세포에의 성장 및 증식에는 많은 영향을 미치지 않는 반면, 오토탁신(autotaxin) 혹은 리소포스포리파제 D(lisophospholipase D)의 활성을 강력하게 억제함으로써 암전이(cancer metastasis)를 억제하므로 항암 및 암전이 예방 및 치료에 유용하게 사용될 수 있다.The present invention relates to a novel use of gintonin, a glycolipid protein (glycolipoprotein) isolated from ginseng, and more particularly through inhibition of the activity of autotaxin or lysophospholipase D. It relates to a composition for inhibiting cancer and cancer metastasis containing gintonin and the gintonin as an active ingredient to inhibit cancer metastasis (cancer metastasis).
Gintonin according to the present invention is a glycolipid protein present in ginseng, while having little cytotoxicity, strong toxicity to cancer cells, and little effect on growth and proliferation to cancer cells, while autotaxin ) Or inhibits cancer metastasis by strongly inhibiting the activity of lisophospholipase D may be useful in the prevention and treatment of cancer and cancer metastasis.
Description
본 발명은 인삼에서 분리 동정한 당지질단백질(glycolipoprotein)인 진토닌(gintonin)의 신규 용도에 관한 것으로, 더욱 상세하게는 오토탁신(autotaxin) 활성 억제를 통해 암전이(cancer metastasis)를 억제하는 작용을 하는 진토닌과 상기 진토닌을 유효성분으로 함유하는 항암 및 암전이 억제용 조성물에 관한 것이다.The present invention relates to a novel use of gintonin, a glycolipid protein (glycolipoprotein) isolated from ginseng, and more particularly, inhibits cancer metastasis through autotaxin activity inhibition. It relates to a composition for inhibiting anticancer and cancer metastasis comprising gintonin and the gintonin as an active ingredient.
예부터 인삼은 일반적으로 장수/생명연장을 위한 강장제(tonic)로 이용되거나, 또는 아답토겐 시약(adaptogenic agent)으로서 각종 스트레스, 피로, 암, 당뇨병에 대항하여 생체기능을 향상시킨다고 알려져 있다. 이러한 인삼은 한국뿐만 아니라 중국과 일본에서도 천년 이상 동안 사용해 왔는데, 최근 인삼은 세계에서 소비되는 약초로 가장 유명한 것 중 하나이다(Tyler, J. Pharm. Technol. 11, 214-220, 1995).Since ancient times, ginseng is generally used as a tonic for longevity / life extension, or as an adaptogenic agent, and is known to improve biological function against various stresses, fatigue, cancer, and diabetes. These ginsengs have been used for more than a thousand years in Korea and also in China and Japan. Recently, ginseng is one of the most famous herbs consumed in the world (Tyler, J. Pharm. Technol. 11 , 214-220, 1995).
인삼 성분 중 인삼 사포닌으로 불리는 진세노사이드(Ginsenosides)는 1960년대 초반에 분리되어 생리학적/약리학적 연구에서 대표적인 성분으로 알려진 뒤로 연구에 많이 이용되었으며(Shibata, et al. Tetraheadron Letters 1962, 1239-1245, 1963; Wagner-Jauregg and Roth, Pharm Acta Helv 37, 352-357, 1962), 이외에도 많은 연구를 통해 인삼에 다당류(polysaccharides), 폴리아세틸렌류(polyacetylenes), 수용성 단백질(proteins) 등 알려지지 않은 다른 성분들이 함유되어 있는 것이 밝혀졌다(Nah, Kor. J. Ginseng Sci. 21, 1-12, 1997).
Ginsenosides, called ginseng saponins, were isolated in the early 1960s and were widely used in research since it was known as a representative ingredient in physiological and pharmacological studies (Shibata, et al. Tetraheadron Letters 1962 , 1239-1245). , 1963; Wagner-Jauregg and Roth, Pharm Acta Helv 37 , 352-357, 1962), as well as other unknowns, including polysaccharides, polyacetylenes, and water-soluble proteins in ginseng. Were found (Nah, Kor. J. Ginseng Sci. 21, 1-12, 1997).
또한, 앞서 본 발명자들은 인삼에서 당, 지방 및 단백질 성분으로 구성되어 있는 신규 당지질단백질(glycolipoproteins)의 분리 동정에 성공하였으며, 이를 진토닌(gintonin)이라 명명하였다. In addition, the present inventors have successfully identified and identified new glycolipoproteins composed of sugar, fat, and protein components in ginseng, and named them gintonin.
진토닌은 인삼 기존에 알려진 인삼 사포닌(진세노사이드)와는 달리 아직 확인/규명 안 된 세포막 단백질과 상호 작용을 통한 신호 경로(membrane signal transduction pathway)를 통해 그 활성이 나타나는 것으로 밝혀졌는데, 예를 들면, 마우스 EAT(Ehrlich Ascites Tumor) 세포에 진토닌의 처리는 G 단백질 공역 수용체(G protein coupled receptors, GPCRs 혹은 7TM 수용체)들이 활성될 때 이루어지는 신호 전달 경로와 같이 인지질분해효소(phospholipase C, PLC)에 연결되어 있는 신호전달 경로를 통해 세포질내 유리 Ca2+(Free Ca2+)의 증가를 일시적으로 유발하고, 또한 진토닌을 개구리알(Xenopus oocytes)에 처리할 경우 PTX에 민감하지 않은 G 단백질→PLC→IP3→칼슘 저장고인 세포질세망(endoplasmic reticulum, ER)에서의 칼슘 방출을 통하여 칼슘 의존성 염소이온 채널(Ca2+ activated Chloride Channel, CaCC)이 활성화 된다(국내특허등록 제 10-0973202호; Pyo et al., J Ginseng Res, 35, 92-103, 2011).
Ginseng, unlike ginseng saponins (ginsenosides), is known to be active through a signal transduction pathway that interacts with cell membrane proteins that have not yet been identified or identified. The treatment of gintonin in mouse EAT (Ehrlich Ascites Tumor) cells is directed to phospholipase C (PLC), a signaling pathway that occurs when G protein coupled receptors (GPCRs or 7TM receptors) are activated. G signaling, which is insensitive to PTX when transiently triggering an increase in free cytosolic Ca 2+ (Free Ca 2+ ) in the cytoplasm through the signaling pathways associated with it, and also treating gintonin in Xenopus oocytes → Calcium-dependent chlorine ion channel (Ca 2+ activated Chloride Channel (CaCC)) is activated through calcium release from PLC → IP 3 → calcium reservoir endoplasmic reticulum (ER) (Domestic Patent Registration No. 10-0973202; Pyo et al., J Ginseng Res, 35, 92-103, 2011).
글리세롤(Glycerol-) 및 스핑고신-기반 인지질(sphingosine-based phospholipids)들은 세포막에 아주 풍부하며, 주로 막을 구성하는 구조적인 성분들이다. 또한, 이들 성분들은 혈액에도 존재하며, 대사과정을 거칠 경우 일부는 리소인지질(lysophospholipids)이 형성된다(Okudaira et al., Biochimie 92, 698-706. 2010). Glycerol- and sphingosine-based phospholipids are very abundant in cell membranes and are primarily structural components that make up the membrane. In addition, these components are also present in the blood, and some of them form lysophospholipids after metabolism (Okudaira et al., Biochimie 92, 698-706. 2010).
이들 리소인지질 중에 리소포스파티딜콜린(lysophosphatidylcholine, LPC)이 있는데 LPC가 오토탁신(autotaxin)으로도 알려져 있는 리소포스포리파제 D(lysophospholipase D)라는 효소의 작용을 받아 리소포스파티딘산(lysophosphatidic acid, LPA; 1- or 2-acyl-sn-glycerol-3-phosphate)이 생성된다(Aoki, Seminars Cell & Dev. Biol. 15, 477-489, 2004; Okudaira et al., Biochimie 92, 698-706. 2010). 초기에는 LPA가 혈소판이 활성될 때 생성되는 것으로 알려져 지혈, 상처 치유 및 조직 재생과 관련이 있는 것으로 보고되었으나, 최근의 연구에 따르면 LPA는 혈소판 외에, 혈장, 혈청(serum), 타액, 정액(seminal fluid), 난포액(follicular fluid) 등에 존재하며, 혈장에서는 80-100 nM, 혈청에서는 1-5 μM 농도로 존재하는 것으로 알려졌다(Aoki, Seminars Cell & Dev. Biol. 15, 477-489, 2004). Lysophosphatidylcholine (LPC) is one of these lysophosphatidylcholines (LPC), LPC is also known as autotaxin (lysophospholipase D) under the action of an enzyme called lysophospholipase D (lysophosphatidic acid, LPA; 1 or 2-acyl-sn-glycerol-3-phosphate) (Aoki, Seminars Cell & Dev. Biol. 15, 477-489, 2004; Okudaira et al., Biochimie 92, 698-706. 2010). Initially, LPA is known to be produced when platelets are activated and has been reported to be associated with hemostasis, wound healing and tissue regeneration, but recent studies have shown that LPA is in addition to platelets, plasma, serum, saliva, and semen. fluids, follicular fluids, etc., 80-100 nM in plasma and 1-5 μM in serum (Aoki, Seminars Cell & Dev. Biol. 15, 477-489, 2004).
또한, 지방 세포, 섬유아 세포(fibroblast), 뇌 및 여러 기관들과 같은 다양한 세포들에 널리 분포하는 것으로 밝혀졌다(Pages et al., Prostaglandins 64, 1-10, 2001).
It has also been found to be widely distributed in various cells such as fat cells, fibroblasts, brain and various organs (Pages et al., Prostaglandins 64, 1-10, 2001).
혈액에 존재하는 LPA는 주로 혈장 단백질(예, 알부민)과 결합할 경우 더 안정하며, LPA는 주로 혈장 단백질(주로 알부민)과 결합하여 혈액 순환시 혈장 단백질과 같이 순환하면서 타겟 장기에 존재하는 LPA 수용체와 결합하여 그 효과를 발휘한다(Croset et al., Biochem J 345, 61-67, 2000). LPA의 인산기(phosphate group)와 글리세롤 골격(glycerol backbone) 부위가 LPA 작용에 중요한 역할을 하며(Jalink et al., Biochem J. 307, 609-615, 1995), 인산기가 혈액 중에 존재하는 LPA는 1~5 μM 정도로 존재할지라도, 혈액 혹은 세포막에 있는 리소인지질 인산분해효소(lysophospholipid phosphatase)라는 효소에 의해 LPA의 인산기가 아주 짧은 시간에 제거되어 LPA가 불활성화 되기 때문에, 효소의 영향을 받아 쉽게 대사되지 않고 오랫동안 작용하는 LPA 유사체(LPA analogs)의 합성 혹은 개발에 많은 연구를 진행하고 있으나 아직 임상적 응용에 적합한 화합물은 발견되지 않았다(Pilquil et al., Prostaglandins. 64, 83-92, 2001; Brindley and Pilquil, J Lipid Res. 50 SupplS225-230, 2009; Croset et al., Biochem J 345. 61-67, 2000; Deng et al., Gastroenterology 132. 1834-1851, 2007).
LPA present in the blood is more stable when it is mainly associated with plasma proteins (eg, albumin), and LPA is mainly associated with plasma protein (mainly albumin), which is present in the target organs as it circulates with plasma proteins in the blood circulation. And exert its effect (Croset et al., Biochem J 345, 61-67, 2000). The phosphate group and glycerol backbone of LPA play an important role in LPA action (Jalink et al., Biochem J. 307, 609-615, 1995). Although present in the order of ~ 5 μM, LPA is inactivated by the enzyme lysophospholipid phosphatase in blood or cell membranes in a very short time. While many studies have been conducted on the synthesis or development of long-acting LPA analogs (LPA analogs), no suitable compounds have yet been found for clinical applications (Pilquil et al., Prostaglandins. 64, 83-92, 2001; Brindley and Pilquil, J Lipid Res. 50 Suppl S225-230, 2009; Croset et al., Biochem J 345. 61-67, 2000; Deng et al., Gastroenterology 132. 1834-1851, 2007).
상기에서 언급한 바와 같이, LPA는 리소포스파티딜콜린(lysophosphatidylcholine, LPC)에 오토탁신(autotaxin)으로도 알려져 있는 리소포스포리파제 D(lysophospholipase D)라는 효소의 작용을 받아 리소포스파티딘산(lysophosphatidic acid, LPA; 1- or 2-acyl-sn-glycerol-3-phosphate)이 생성되는데, 이렇게 생성된 LPA는 각종 생리활성에 관여한다(Aoki, Seminars Cell & Dev. Biol. 15, 477-489, 2004; Okudaira et al., Biochimie 92, 698-706. 2010).As mentioned above, LPA is lysophosphatidyl acid (LPA) under the action of an enzyme called lysophospholipase D (lysophospholipase D), also known as autotaxin, to lysophosphatidylcholine (LPC). 1- or 2-acyl-sn-glycerol-3-phosphate), which is involved in various physiological activities (Aoki, Seminars Cell & Dev. Biol. 15, 477-489, 2004; Okudaira et al., Biochimie 92, 698-706. 2010).
흥미로운 것은 신장암(renal cell carcinoma), 전이가 심한 난소암 및 유방암, 갑상선암(thyroid carcinoma), 호지킨 림프종(Hodgkin lymphomas), 신경모세포종(neuroblastoma), 침습성 다형성 교모세포종(invasive glioblastoma multiforme), 전립선암, 및 피부암(melanomas)등과 같은 암 환자의 암세포에서 오토탁신(autotaxin)이 과다하게 생성 분비되고, 분비된 오토탁신은 LPC를 LPA로 전환시키는데(Xie et al., BBA, 1582, 270-281, 2002; Sengupta et al., Seminars in Cells & Dev. Biol. 15, 503-512, 2004; David, et al., PLoS One 5, e9741, 2010; Zeng et al, Prostate 69, 283??292, 2009; Kishi, et al., J. Biol. Chem. 281, 17492??17500, 2006; Hoelzinger et al, Neoplasia 7, 7??16, 2005; Kehlen, et al., Int. J. Cancer 109, 833??838, 2004; Black et al, Oncogene 23, 2357??2366, 2004; Yang et al, Clin. Exp. Metastasis 19, 603??608, 2002; Nam et al, Oncogene 19, 241??247, 2000; Kawagoe et al., Cancer Res. 57, 2516??2521, 1997), 이렇게 생성된 LPA들 중, 특히 LPA C16:0가 가장 많이 만들어지는 것으로 알려져 있다(Meleh et al.,J Chromatogr B Analyt Technol Biomed Life Sci. 858, 287-291, 2007; Yoon et al., J. Chromatogr. B, 788, 85-92, 2003). Interesting are renal cell carcinoma, highly metastatic ovarian and breast cancer, thyroid carcinoma, Hodgkin lymphomas, neuroblastoma, invasive glioblastoma multiforme, prostate cancer , And excessive production of autotaxin in cancer cells of cancer patients such as skin cancer (melanomas), and secreted autotaxin converts LPC into LPA (Xie et al., BBA, 1582, 270-281, 2002; Sengupta et al., Seminars in Cells & Dev. Biol. 15, 503-512, 2004; David, et al., PLoS One 5, e9741, 2010; Zeng et al, Prostate 69, 283 ?? 292, 2009 Kishi, et al., J. Biol. Chem. 281, 17492 ?? 17500, 2006; Hoelzinger et al, Neoplasia 7, 7 ?? 16, 2005; Kehlen, et al., Int. J. Cancer 109, 833 838, 2004; Black et al, Oncogene 23, 2357 ?? 2366, 2004; Yang et al, Clin.Exp. Metastasis 19, 603 ?? 608, 2002; Nam et al, Oncogene 19, 241 ?? 247, 2000; Kawagoe et al., Cancer Res. 57, 2516 ?? 2521, 1997) Of these LPAs, LPA C 16: 0 is known to produce the most (Meleh et al., J Chromatogr B Analyt Technol Biomed Life Sci. 858, 287-291, 2007; Yoon et al., J. Chromatogr.B , 788, 85-92, 2003).
그리고, 이 LPA들은 다양한 암세포의 성장(growth), 이동(migration) 및 전이(metastasis)를 촉진시키는 것으로 보고되고 있다(Xie et al., 277, 32516-32526, 2002; Meleh et al., J Chromagraph B, 858, 287-291, 2007). In addition, these LPAs are reported to promote growth, migration and metastasis of various cancer cells (Xie et al., 277, 32516-32526, 2002; Meleh et al., J Chromagraph). B, 858, 287-291, 2007).
그러나, 정상적인 경우에는, 오토탁신(autotaxin)에 LPA 혹은 S1P 결합자리가 존재하고 있어서, 이들 결합자리에 LPA나 S1P가 결합할 경우 오토탁신의 활성을 억제하여 LPA가 과도하게 생성되는 것을 차단하는 것으로 보고되고 있다(van Meeteren et al., J Biol Chem 280,21155-21161, 2005). 따라서, LPA는 양날의 칼의 작용을 지닌 물질이라고 비유하는데, 정상적인 경우에 LPA는 각종 생리 활성작용에 관여하지만, 암환자에게는 오토탁신의 작용에 의하여 과도하게 생성된 LPA가 암세포 증식, 전이(migration), 침입(invasion), 콜로니 형성(colony formation), 암 전이 촉진 및 이로 인한 사망의 주요 원인이 되는 것으로 알려져 있다(Li et al., Mol Cancer Ther 1692-1701, 2009; Tokumura, BBA 1582, 18-25, 2002; Lin et al., Prostaglandins & other lipid mediators 91, 130-138, 2010; Tania et al., BBRC, 401, 493-497, 2010).
In normal cases, however, LPA or S1P binding sites are present in autotaxin, and when LPA or S1P is bound to these binding sites, it inhibits the activity of autotaxin to block excessive production of LPA. Reported (van Meeteren et al., J Biol Chem 280,21155-21161, 2005). Therefore, LPA is analogous to a double-edged knife. In normal cases, LPA is involved in various physiological activities, but in patients with cancer, LPA produced excessively by the action of autotaxin causes cancer cell proliferation and migration. ), Invasion, colony formation, promoting cancer metastasis and resulting death (Li et al., Mol Cancer Ther 1692-1701, 2009; Tokumura, BBA 1582, 18 -25, 2002; Lin et al., Prostaglandins & other lipid mediators 91, 130-138, 2010; Tania et al., BBRC, 401, 493-497, 2010).
오토탁신의 활성을 억제하는 LPA 유사체(LPA analogue)의 합성을 통하여 암전이를 억제하거나, 차단하는 약물 연구가 진행 중에 있다(Anna et al., Cancer Metastasis Rev. Published online, Oct 16, 2011; Mukai et al., Int J Cancer 81, 918-922, 1999; Baker et al., J Biol. Chem 281, 22786-22793, 2006; Zhang et al., Cancer Res. 69, 5441-5449, 2009). Drug research is underway to inhibit or block cancer metastasis through the synthesis of LPA analogues that inhibit the activity of autotaxin (Anna et al., Cancer Metastasis Rev. Published online, Oct 16, 2011; Mukai et al., Int J Cancer 81, 918-922, 1999; Baker et al., J Biol. Chem 281, 22786-22793, 2006; Zhang et al., Cancer Res. 69, 5441-5449, 2009).
또한, LPA에 의한 오토탁신(리소포스포리파제 D)의 활성 억제 정도는 LPA의 아실기(acyl groups)에 부착되어 있는 지방산의 종류에 따라 현저하게 차이가 있으며, 여러 LPAs 중 LPA C18:2가 오토탁신의 활성을 가장 강력하게 억제하는 것으로 보고되었다(Liu et al., J Agri Food Chem 55, 8717-8722, 2007). In addition, the degree of inhibition of autotaxin (lysophospholipase D) activity by LPA is significantly different depending on the type of fatty acid attached to the acyl groups of LPA, among which LPA C 18: 2 Has been reported to most potently inhibit the activity of autotaxin (Liu et al., J Agri Food Chem 55, 8717-8722, 2007).
선행연구에서, 본 발명자들은 최초로 인삼에서 생리학적/약리학적 활성이 있는 GPCR 리간드인 리소포스파티딘산들(LPAs), 특히 LPA C18:2이 다량 존재하며, 일시적으로 세포질내 유리 Ca2+(Free Ca2+)의 증가 활성을 유발하고, 유리 LPA(free LPA)에 비하여 세포질내 유리 Ca2+(Free Ca2+)의 증가 활성을 오랫동안 유지하여 주는 안정된 형태의 당 및 단백질과 복합체(complex)로 되어 있는 것을 확인하였다(특허 출원 제10-2011-0744070호 "인삼으로부터 분리 동정한 리소포스파티딘산 및 그 제조 방법").
In a previous study, we first found that lysophosphatidine acids (LPAs), especially LPA C 18: 2 , GPGP ligands with physiological / pharmacological activity in ginseng, are present in large amounts and are temporarily suspended in cytoplasmic free Ca 2+ ( induce increased activity of free Ca 2+), and the glass LPA (free LPA) within the cellular free Ca 2+ (free Ca 2+) and protein form stable complexes with sugar to maintain for a long time by the increased activity compared to the (complex ) (Patent Application No. 10-2011-0744070 "Lisophosphatidic acid identified and separated from ginseng and its manufacturing method").
LPA C18:2는 다른 여러 LPAs에 비하여 오토탁신 활성을 아주 낮은 농도(3-62배 낮은 농도)에서 억제하는 것으로 보고되었는데(Liu et al., J Agri Food Chem 55, 8717-8722, 2007), 진토닌은 LPA 수용체들을 활성시키는 작용도 있지만, 진토닌 특징들 중의 하나는 LPA C18:2가 다른 LPAs에 비하여 현저하게 많이 함유되어 있다는 점이다(특허 출원 제 10-2011-0744070호 참조).
LPA C 18: 2 has been reported to inhibit autotaxin activity at very low concentrations (3-62 fold lower concentrations) than many other LPAs (Liu et al., J Agri Food Chem 55, 8717-8722, 2007). Although gintonin also acts to activate LPA receptors, one of the features of gintonin is that LPA C 18: 2 contains significantly more than other LPAs (see patent application 10-2011-0744070). .
본 발명에서는 in vitro 연구로서 상기 진토닌이 오토탁신 활성을 억제하고, 마우스 암세포의 일종인 흑색종 세포(B16/F10)의 성장(growth)에는 거의 영향을 미치지 않지만 암세포의 전이(migration) 및 상처 치유(scratch wound healing)를 강력하게 억제하는 것을 발견하였다. 또한, 실험동물을 이용한 in vivo 연구에서 진토닌이 마우스 꼬리 정맥에 주입된 흑색종 세포(B16/F10)의 폐(lung)로의 전이를 차단함을 발견함으로써, 진토닌이 세포 독성(cell toxicity) 없이 암전이(metastasis)를 선택적으로 억제함을 확인하고 본 발명을 완성하였다.In the present invention, as an in vitro study, the gintonin inhibits autotaxin activity and hardly affects the growth of melanoma cells (B16 / F10), a kind of mouse cancer cells, but the migration and wounds of cancer cells. It has been found to strongly inhibit scratch wound healing. In vivo studies using experimental animals also found that gintonin blocks the metastasis of melanoma cells (B16 / F10) injected into the mouse tail vein into the lungs, resulting in cell toxicity. The present invention was confirmed to selectively inhibit metastasis without the present invention.
결국, 본 발명의 주된 목적은 인삼에서 분리 동정한 당지질단백질(glycolipoprotein)인 진토닌(gintonin)의 신규 용도를 제공하는데 있다.After all, the main object of the present invention is to provide a novel use of gintonin, a glycoliplipoprotein isolated from ginseng.
상기 목적을 달성하기 위하여, 본 발명은 암전이를 억제하는 진토닌을 제공한다.In order to achieve the above object, the present invention provides gintonin to inhibit cancer metastasis.
본 발명에서, 상기 진토닌은 암전이 효소로 알려진 오토탁신(autotaxin) 혹은 리소포스포리파제 D(lisophospholipase D)의 활성을 억제함으로써 암전이를 억제하는 것을 특징으로 한다.
In the present invention, the gintonin is characterized by inhibiting cancer metastasis by inhibiting the activity of autotaxin or lisophospholipase D known as cancer metastasis enzyme.
또한, 본 발명은 상기 진토닌을 유효성분으로 함유하는 항암 및 암전이 억제용 조성물을 제공한다.The present invention also provides an anticancer and cancer metastasis inhibiting composition containing the gintonin as an active ingredient.
본 발명에 있어서, 상기 조성물은 오토탁신 활성에 기인하여 암전이가 촉진되는 것으로 알려진 각종 암 전이의 예방 및 억제를 위한 약학적 조성물 및 건강기능식품 조성물을 포함한다.In the present invention, the composition comprises a pharmaceutical composition and a nutraceutical composition for the prevention and inhibition of various cancer metastases known to promote cancer metastasis due to autotaxin activity.
또한, 상기 조성물은 신장암(renal cell carcinoma), 전이가 심한 난소암 및 유방암, 갑상선암(thyroid carcinoma), 호지킨 림프종(Hodgkin lymphomas), 신경모세포종(neuroblastoma), 침습성 다형성 교모세포종(invasive glioblastoma multiforme), 전립선암, 및 피부암(melanomas)등 각종 암질환을 예방 및 치료하거나 암전이를 예방 및 억제하는데 있어 유용하다.In addition, the composition is renal cancer (renal cell carcinoma), severe metastatic ovarian cancer and breast cancer, thyroid carcinoma (thyroid carcinoma), Hodgkin lymphomas, neuroblastoma, invasive glioblastoma multiforme (invasive glioblastoma multiforme) It is useful in preventing and treating various cancer diseases, such as prostate cancer, and skin cancer (melanomas) or preventing and inhibiting cancer metastasis.
또한, 본 발명의 조성물은 단독 또는 병행하여 사용이 가능하며, 기존의 항암제와 병행 사용할 경우 항암제에 의한 암성장 억제와 본 발명의 진토닌에 의한 암전이 억제 효과를 얻을 수 있어서 암 치료 효과가 극대화될 수 있다.
In addition, the composition of the present invention can be used alone or in combination, and when used in combination with the existing anticancer drugs can be inhibited cancer growth by the anticancer drugs and cancer metastasis inhibition by the gintonin of the present invention to maximize the cancer treatment effect Can be.
인삼은 오랫동안 생약으로 사용되어 오던 것으로 인삼에서 분리해 낸 본 발명의 진토닌 역시 독성 및 부작용 등의 문제가 전혀 없다.Ginseng has been used as a herbal medicine for a long time, the gintonin of the present invention isolated from ginseng also has no problems such as toxicity and side effects.
본 발명의 진토닌을 유효성분으로 함유하는 항암 및 암전이 억제용 조성물은, 조성물 총 중량에 대하여 상기 진토닌을 0.0001 내지 10중량%, 바람직하게는 0.001 내지 1중량%로 포함한다. The anticancer and cancer metastasis inhibiting composition containing the gintonin of the present invention as an active ingredient comprises 0.0001 to 10% by weight, preferably 0.001 to 1% by weight, based on the total weight of the composition.
또한, 본 발명의 진토닌을 함유하는 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있고, 본 발명의 진토닌의 약학적 투여 형태는 단독으로 또는 타 약리적 활성 화합물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다.In addition, the composition containing gintonin of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, and the pharmaceutical dosage form of gintonin of the present invention may be used alone or in other forms. It can be used in an appropriate combination as well as in combination with pharmacologically active compounds.
본 발명에 따른 진토닌을 함유하는 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상기 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알제네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸셀룰로오스, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 진토닌 또는 분획물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘 카보네이트, 수크로오스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면, 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있고, 좌제의 기제로는 위텝솔(witepsol), 마크로골(macrogol), 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The composition containing gintonin according to the present invention is in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alzenate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used, and suppository bases such as witepsol, macrogol, Tween 61, cacao butter, laurin, glycerogelatin and the like can be used.
본 발명의 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나, 바람직한 효과를 위해서, 본 발명의 조성물은 1일 0.0001 내지 100 ㎎/㎏으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.
본 발명의 약학 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내(intracerebroventricular) 주사에 의해 투여될 수 있다.The pharmaceutical composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.
본 발명의 진토닌을 함유하는 조성물은 상기와 같은 제형으로 항암 및 암전이 예방 및 치료를 위한 약제, 식품 및 음료 등에 다양하게 이용될 수 있고, 상기 진토닌은 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강기능성 식품류 등이 있다.
The composition containing the gintonin of the present invention can be used in various formulations, such as drugs, foods and beverages for the prevention and treatment of cancer and cancer metastasis, the gintonin is a food that can be added, for example For example, there are various foods, beverages, gum, tea, vitamin complexes, health functional foods and the like.
본 발명의 진토닌은 독성 및 부작용이 거의 없으므로 예방 목적으로 장기간 복용시에도 안심하고 사용할 수 있다.Gintonin of the present invention has little toxicity and side effects, and therefore can be used safely even for prolonged administration for prophylactic purposes.
본 발명의 진토닌은 각종 암 및 암전이 예방을 목적으로 식품 또는 음료에 첨가될 수 있는데, 이때 식품 또는 음료 중의 상기 진토닌의 양은 전체 식품 중량의 0.01 내지 15중량%로 가할 수 있으며, 건강음료 조성물은 100 ㎖를 기준으로 0.02 내지 5 g, 바람직하게는 0.3 내지 1 g의 비율로 가할 수 있다.Gintonin of the present invention can be added to food or beverage for the purpose of preventing various cancers and cancer metastasis, wherein the amount of gintonin in the food or beverage can be added to 0.01 to 15% by weight of the total food weight, health drinks The composition can be added at a ratio of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml.
본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 진토닌을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 더 포함할 수 있다. 이때, 상기 천연 탄수화물은 예를 들어, 포도당, 과당 등의 모노사카라이드; 말토오스, 수크로오스 등의 디사카라이드; 및 덱스트린, 시클로덱스트린 등의 폴리사카라이드 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알코올 등이 있다. 또한, 향미제로는 천연 향미제(타우마틴, 스테비아 추출물, 예를 들어, 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율을 본 발명의 조성물 100 ㎖당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다.The functional beverage composition of the present invention is not particularly limited to other ingredients other than containing the gintonin as an essential ingredient in the indicated ratio, and may further include various flavors or natural carbohydrates, such as ordinary drinks. At this time, the natural carbohydrate is, for example, monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; And conventional sugars such as polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents (tauumatin, stevia extract, for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 추출물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 증진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 추출물들은 천연 과일 주스 및 과일 주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있으며, 이러한 성분을 독립적으로 또는 조합하여 사용할 수 있다. 또한, 상기 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 추출물 100중량부당 0 내지 약 20중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the extract of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks, and these ingredients may be used independently or in combination. In addition, the ratio of the additive is not so important, but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.
상기와 같은 본 발명에 따른 진토닌은, 인삼에 존재하는 당지질단백질로서, 세포 독성이 거의 없는 반면, 암 세포에 대한 독성이 강하고, 암 세포에의 성장 및 증식에는 많은 영향을 미치지 않는 반면, 오토탁신(autotaxin) 혹은 리소포스포리파제 D(lisophospholipase D)의 활성을 강력하게 억제함으로써 암전이(cancer metastasis)를 억제하므로 항암 및 암전이 예방 및 치료에 유용하게 사용될 수 있다.Gintonin according to the present invention as described above, as a glycolipid protein present in ginseng, while having little cytotoxicity, strong toxicity to cancer cells, does not significantly affect the growth and proliferation to cancer cells, Otto It inhibits cancer metastasis by strongly inhibiting the activity of taxin (autotaxin) or lysophospholipase D, and thus may be useful for preventing and treating cancer and metastasis.
도 1은 본 발명에 따른 진토닌의 오토탁신(autotaxin) 활성 억제 효과를 오토탁신 분석 키트(autotaxin assay kit)를 이용하여 확인한 결과이다.
도 2는 본 발명에 따른 진토닌의 인간 흑색종 세포 MDA-MB-435(A)와 마우스 흑색종 세포 B16/F10(B) 배지 유래 오토탁신(lysophospholipase D)의 활성 억제 효과를 확인한 결과이다(*p<0.05, **p<0.01, ***p<0.001, 진토닌 미처리 시료 대비).
도 3은 본 발명에 따른 진토닌 및 Brp-LPA의 마우스 흑색종 세포 B16/F10에서의 상처 치유 효과를 확인한 결과이다(*p<0.05, **p<0.01, ***p<0.001, 진토닌을 투여하지 않은 세포 대비; Brp: Brp-LPA, 10μM).
도 4는 본 발명에 따른 진토닌의 마우스 흑색종 세포 B16/F10의 세포 이동 혹은 세포 운동성(motility) 억제 효과를 보이든 챔버(Boyden chamber)를 이용하여 확인한 결과이다(*p<0.05, **p<0.001, 진토닌 미처리군과 비교; GT: 진토닌; Brp: Brp-LPA, 10μM).
도 5는 본 발명에 따른 진토닌의 배양된 마우스 흑색종 세포 B16/F10 사멸 및 증식 억제 효과를 확인한 결과이다(*p<0.05, **p<0.01, ***p<0.001, 진토닌을 투여하지 않은 세포 대비; Brp: Brp-LPA, 10μM).
도 6 내지 도 8은 본 발명에 따른 진토닌의 마우스 흑색종 폐전이에서 암전이 예방 및 억제 효과를 확인한 사진이다.1 is a result of confirming the effect of inhibiting autotaxin (autotaxin) activity of gintonin according to the present invention using an autotaxin assay kit (autotaxin assay kit).
Figure 2 shows the results of confirming the inhibitory effect of the activity of human melanoma cells MDA-MB-435 (A) and mouse melanoma cells B16 / F10 (B) medium-derived autotaxin (lysophospholipase D) according to the present invention ( * p <0.05, ** p <0.01, *** p <0.001 compared to untreated gintonin sample).
Figure 3 is a result confirming the wound healing effect in the mouse melanoma cells B16 / F10 of gintonin and Brp-LPA according to the present invention ( * p <0.05, ** p <0.01, *** p <0.001, Jinto Compared to cells not administered nin; Brp: Brp-LPA, 10 μΜ).
4 is a result of confirming the cell migration or cell motility inhibitory effect of the mouse melanoma cells B16 / F10 of gintonin according to the present invention using the Boyden chamber ( * p <0.05, ** p <0.001 compared with untreated gintonin; GT: gintonin; Brp: Brp-LPA, 10 μM).
5 is a result confirming the effect of inhibiting the growth and proliferation of cultured mouse melanoma cells B16 / F10 in accordance with the present invention ( * p <0.05, ** p <0.01, *** p <0.001, gintonin Relative to unadministered cells: Brp: Brp-LPA, 10 μΜ).
6 to 8 are photographs confirming the cancer metastasis prevention and suppression effect in the mouse melanoma lung metastasis of gintonin according to the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
실시예 1. 진토닌의 오토탁신 활성 억제 확인Example 1. Confirmation of inhibition of autotaxin activity of gintonin
1-(1). 오토탁신 활성 키트(kit)를 이용한 확인1- (1). Identification with Autotaxin Activation Kit
본 발명에서는 진토닌에 의한 오토탁신(autotaxin) 활성 억제 효과를 확인하기 위해, 오토탁신 억제제 스크리닝 키트(Autotaxin Inhibitor Screening Kit; Echelon, Salt Lake City, UT, USA)를 사용하였다.In the present invention, autotaxin inhibitor screening kit (Autotaxin Inhibitor Screening Kit; Echelon, Salt Lake City, UT, USA) was used to confirm the effect of inhibiting autotaxin activity by gintonin.
상기 키트는 옥토톡신이 기질 Fluorescent autotaxin substrate(FS-3) (Echelon, Salt Lake City, UT, USA)를 분해하여 형광을 발하게 하는 현상을 이용하여(Ferguson CG et al. Org Lett 8, 2023-2026, 2006), 분해산물의 형광강도를 측정(excitation 파장: 450 ㎚, emission 파장: 528 ㎚)함으로써 오토톡신의 활성을 측정하는 원리를 이용한 것이다.
The kit utilizes the phenomenon that octotoxins decompose and fluoresce the substrate Fluorescent autotaxin substrate (FS-3) (Echelon, Salt Lake City, UT, USA) (Ferguson CG et al .
진토닌 또는 키트에 포함된 양성 오토탁신 억제제인 Brp-LPA와 오토탁신을 함유하는 반응액을 혼합하여 10분간 실온에서 반응시킨 다음, 제조사의 사용 설명에 따라 오토탁신 활성을 측정하고, 진토닌의 오토탁신 활성 억제 효과를 산출하였다.Gintonin or Brp-LPA, a positive autotaxin inhibitor included in the kit, and the reaction solution containing autotaxin were mixed and reacted at room temperature for 10 minutes, followed by measuring the autotaxin activity according to the manufacturer's instructions. The effect of inhibiting autotaxin activity was calculated.
도 1B는 진토닌 처리 농도별 오토탁신 활성 억제를 나타낸 것으로, 진토닌에 의한 오토톡신 활성 억제 IC50은 346±88 ng/㎖로 나타났으며, 대조 약물인 Brp-LPA역시 오토탁신 활성을 차단하였으며, 이때 IC50은 462±119 nM로 나타났다(도 1A 참조).
1B shows the inhibition of autotaxin activity according to the concentration of gintonin. The autotoxin activity inhibition IC 50 by gintonin was found to be 346 ± 88 ng / ml, and the control drug Brp-LPA also blocked autotaxin activity. In this case, the IC 50 was found to be 462 ± 119 nM (see FIG. 1A).
1-(2). 오토탁신을 분비하는 세포에서 분리된 오토탁신 활성에 대한 진토닌 의 영향 확인1- (2). Identification of the Effect of Gintonine on Autotaxin Activity Isolated from Autotaxin-secreting Cells
진토닌과 인간 흑색종 세포인 MDA-MB-435와 마우스 흑색종 세포 B16/F10에서 얻은 오토탁신을 반응용액 TBS(Tris-buffered saline solution; 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 50 mM Tris-HCl, pH 8.0)에서 희석 혼합하여 96 웰 플레이트에서 10분간 반응시켰다.Autotaxin obtained from gintonin and human melanoma cells MDA-MB-435 and mouse melanoma cells B16 / F10 was treated with Tris-buffered saline solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1). mM MgCl 2 , 50 mM Tris-HCl, pH 8.0) was mixed and reacted for 10 minutes in a 96 well plate.
여기에, 1 ㎎/㎖ 소혈청 알부민(bovine serum albumin, BSA)를 함유한 TBS(pH 8.0)에 희석한 FS-3(12.5 μM)를 넣어(60℃에서 10분간 가열) 각 웰의 FS-3 최종 농도를 2.5 μM이 되게 하고, 37℃에서 배양한 후, FS-3 분해 산물의 형광 강도를 측정하여(excitation 파장: 450 ㎚, emission 파장: 528 ㎚), 진토닌의 오토탁신 활성 억제 효과를 산출하였다.
To this, FS-3 (12.5 μM) diluted in TBS (pH 8.0) containing 1 mg / ml bovine serum albumin (BSA) was added (heated at 60 ° C. for 10 minutes). 3 The final concentration was 2.5 μM, and after culturing at 37 ° C., the fluorescence intensity of the FS-3 degradation product was measured (excitation wavelength: 450 nm, emission wavelength: 528 nm) to inhibit the autotaxin activity of gintonin. Was calculated.
그 결과, 도 2A에서 보는 바와 같이, 오토탁신을 생성 분비하는 MDA-MB-435 세포에서 진토닌은 처리 농도별로 오토탁신 활성을 억제하는 것으로 나타났으며, 진토닌에 의한 오토탁신 활성 억제 IC50은 2.39±2.28 ㎍/㎖로 나타났다. 또한, B16/F10세포에서 얻어진 오토탁신에 대해서도, 진토닌은 처리 농도별로 활성 억제 효과를 나타내었다.
As a result, as shown in Fig. 2A, in the MDA-MB-435 cells that produce and secrete autotaxin, gintonin was shown to inhibit the autotaxin activity by treatment concentrations, the autotaxin activity inhibitory IC 50 Was 2.39 ± 2.28 μg / ml. In addition, for autotaxin obtained in B16 / F10 cells, gintonin showed an activity inhibitory effect by treatment concentration.
측정예Measurement example
(1) MDA-MB-435 및 B16/F10 세포 배양(1) MDA-MB-435 and B16 / F10 Cell Culture
MDA-MB-435 세포 및 B16/F10 세포를 열로 비활성화된(heat-inactivated) 10% 소태아 혈청(fetal bovine serum, FBS) 및 스트렙토마이신(streptomycin)과 페니실린(penicillin)을 첨가한 DMEM 배지를 사용하여 5% CO2/95% 공기 배양기에서 배양하였다.
Using DMEM medium containing 10% fetal bovine serum (FBS) heat-inactivated MDA-MB-435 cells and B16 / F10 cells and streptomycin and penicillin Incubated in a 5% CO 2 /95% air incubator.
(2) MDA-MB-435 및 B16/F10 세포 배지로부터 오토탁신의 분리(2) Isolation of Autotaxin from MDA-MB-435 and B16 / F10 Cell Media
MDA-MB-435 세포를 100 ㎜ 접시(dish)에 키워, 세포가 접시에 60~70% 찼을 때, 배지를 제거한 다음, DMEM으로 세포를 1회 씻어낸 후, 0.1% BSA 함유 DMEM을 접시 당 10 ㎖씩 넣어 48시간 배양하였다.MDA-MB-435 cells were grown in a 100 mm dish, when the cells were 60-70% full in the dish, the medium was removed, the cells washed once with DMEM, and then DMEM containing 0.1% BSA per dish. 10 ml each was incubated for 48 hours.
배양 후 얻어진 배지를 모아 1500g, 4℃에서 10분간 원심분리하여, 상등액을 취해, 아미콘(Amicon)사의 초원심분리 여과 장치(Ultra Centrifugal Filter Device; Millipore)를 사용해서 100배 농축하였다.The culture medium obtained after the cultivation was collected, centrifuged at 1500 g and 4 ° C for 10 minutes, the supernatant was collected, and concentrated 100 times using an Ultracon centrifugal filter device (Millipore) manufactured by Amicon.
농축배지는 보존액(storage buffer; 50 mM Tris, 20% ethyleneglycol, pH 7.5)으로 3회 세척한 후, 다시 바인딩액(binding buffer; 50 mM Tris, 0.1 M NaCl, 0.01 M CaCl2, 20% ethyleneglycol, pH 7.2)로 세척한 다음, 다시 바인딩 완충액 6 ㎖를 첨가하고, 2 ㎖ 콘카나발린 A 아가로스 비즈 컬럼(concanavalin A agarose beads column)에 넣어 4℃에서 2시간 배양하여 단백질을 컬럼에 결합시켰다.The concentrated medium was washed three times with storage buffer (50 mM Tris, 20% ethyleneglycol, pH 7.5), and then again bound with binding buffer (50 mM Tris, 0.1 M NaCl, 0.01 M CaCl 2 , 20% ethyleneglycol, pH 7.2), and then 6 ml of binding buffer was added again, and placed in a 2 ml concanavalin A agarose beads column and incubated at 4 ° C. for 2 hours to bind the protein to the column.
비즈에 결합한 단백질은 용출액(elution buffer; 50 mM Tris, 0.1 M NaCl, 0.01 M CaCl2, 20% ethyleneglycol, 100 mM α-D-mannopyranoside, pH 7.2)으로 용출시켜 초원심분리 여과 장치를 사용하여 보존액으로 세척, 농축하였다.
Proteins bound to beads were eluted with elution buffer (50 mM Tris, 0.1 M NaCl, 0.01 M CaCl 2 , 20% ethyleneglycol, 100 mM α-D-mannopyranoside, pH 7.2) and preserved using ultracentrifugal filtration. Washed with and concentrated.
실시예 2. 진토닌의 상처 치유 효과 확인Example 2 Confirming the Wound Healing Effect of Gintonin
In vitro 암세포 전이 연구에 널리 쓰이는 상처 치유(scratch wound healing) 방법을 이용하여 진토닌의 효과를 확인하였다(Dua and Gude, Eur J Cancer 44, 1587-1595, 2008; Zhang et al., Cancer Res. 69, 5441-5449, 2009 ;Goldsmith et al., Genes and Cancer 2, 563-575, 2011).The effect of gintonin was confirmed using the wound wound healing method widely used in in vitro cancer cell metastasis studies (Dua and Gude, Eur J Cancer 44, 1587-1595, 2008; Zhang et al., Cancer Res. 69, 5441-5449, 2009; Goldsmith et al., Genes and Cancer 2, 563-575, 2011).
B16/F10 세포를 2.5×105 웰의 농도로, 24-웰 플레이트에 분주하여 24시간 배양한 다음, 다시 0.2% FBS 함유 DMEM 배지에서 6시간 배양하였다.B16 / F10 cells were aliquoted into 24-well plates at a concentration of 2.5 × 10 5 wells for 24 hours and then incubated for 6 hours in DMEM medium containing 0.2% FBS.
200 ㎕ 파이펫 팁으로 세포가 자란 각 웰의 중앙에 선을 그어 상처를 낸 다음, 부유 세포를 제거하기 위해 0.2% FBS 함유 DMEM 배지로 2회 세척하였다.Lines were wound in the center of each well in which cells were grown with 200 μl pipette tips, and then washed twice with DMEM medium containing 0.2% FBS to remove suspended cells.
상기 세포들에 진토닌(1~30 ㎍/㎖)을 처리한 후 배양하여, 각 웰의 진토닌 처리 이전과 이후의 세포 모양을 인버티드 형광 현미경(Inverted Fluorescence microscope; AxioVert200; Carl Zeiss)으로 관찰, 사진을 촬영하여(100×), 진토닌을 처리하지 않은 세포와 비교하였다.The cells were treated with gintonin (1-30 μg / ml) and then cultured, and the cell shapes before and after gintonin treatment of each well were observed with an Inverted Fluorescence microscope (AxioVert200; Carl Zeiss). Photographs were taken (100 ×) and compared with cells treated with gintonin.
파이펫 팁으로 상처 낸 부분에서 세포가 자라는 정도의 분석은, AxioVision Rel 4.7(Carl Zeiss, Germany)을 이용하여 세포가 회복되지 않은 상처 부위 면적을 측정하였다.
Analysis of the extent of cell growth in the wound portion with the pipette tip was performed using AxioVision Rel 4.7 (Carl Zeiss, Germany) to determine the area of the wound where the cell was not recovered.
도 3에서 보는 바와 같이, 상처를 낼 경우, 약물을 처리하지 않은 그룹은 20시간이 지나면서 세포들이 이동하여 상처를 낸 부분(스크래치 부분)을 메꾸어 주지만, 진토닌을 처리한 경우에는 이러한 작용이 투여 농도별로 억제되는 것으로 확인되었다.As shown in Fig. 3, when wounding, the group not treated with the drug fills the wounded part (scratch part) after 20 hours, but the action is performed when gintonin is treated. It was confirmed to be inhibited by the dose concentration.
또한, 양성 대조군으로 사용한 Brp-LPA 역시 B16/F10 세포 이동을 억제하는 것으로 나타났다.
In addition, Brp-LPA used as a positive control also appeared to inhibit B16 / F10 cell migration.
실시예 3. 진토닌의 B16/F10 세포 이동 효과 확인 Example 3. Confirmation of G16 Toner B16 / F10 Cell Migration Effect
본 발명에서는 세포 이동 효과를 측정하기 위해, 보이든(Boyden) 챔버(48웰; Neuro Probe Inc., Gaithersburg, MD, USA)를 사용하는 방법을 이용하였다(Kim et al. Biol Pharm Bull 30: 1674-1679, 2007; Lee et al. Am J Physiol Cell Physiol 278:C612-C618, 2000).In the present invention, a method using a Boyden chamber (48 well; Neuro Probe Inc., Gaithersburg, MD, USA) was used to measure the effect of cell migration (Kim et al . Biol Pharm Bull 30: 1674). -1679, 2007; Lee et al. Am J Physiol Cell Physiol 278: C612-C618, 2000).
보이든 챔버의 하단의 웰에 DMEM 배지(0.2% FBS, 0.1% BSA 함유)에 녹인 진토닌을 넣고, 그 위에 피브로넥틴(fibronectin)으로 코팅한 폴리카보네이트 멤브레인(polycarbonate membrane, 8-㎛ porse)을 놓은 후, 챔버를 조립하였다.Gintonin dissolved in DMEM medium (containing 0.2% FBS, 0.1% BSA) was placed in a well at the bottom of the Boyden chamber, and a polycarbonate membrane (8-μm porse) coated with fibronectin was placed thereon. The chamber was then assembled.
또한, 챔버 상단의 웰에는 B16/F10 현탁액(5×104 세포/웰)을 넣은 후, 37℃에서 3시간 배양하여, 이동 세포가 멤브레인 위쪽에서부터 아래쪽으로 이동하도록 하였다.In addition, a B16 / F10 suspension (5 × 10 4 cells / well) was added to the well at the top of the chamber, and then incubated at 37 ° C. for 3 hours to allow the moving cells to move from the top to the bottom of the membrane.
챔버를 해체하여, 멤브레인에 있는 세포를 Diff Quik(Sysmax, Kobe, Japan)으로 고정 및 염색하고, 멤브레인에 붙어 있는 채로 슬라이드 위에 붙인 다음, 이동하지 않은 세포를 닦아 제거하고, 현미경(Dark Field microscope, Eclipse 80i, Nikon)을 이용하여 사진 촬영하여 이동된 세포의 수를 세어 비교하였다(배율: ×200).
The chamber was disassembled to fix and stain the cells on the membrane with Diff Quik (Sysmax, Kobe, Japan), adhere to the membrane while attached to the membrane, wipe off the unmoved cells, and remove the dark field microscope (Dark Field microscope, Eclipse 80i, Nikon) was photographed and counted by comparing the number of cells moved (magnification: x 200).
그 결과, 도 4에서 보는 바와 같이, 진토닌을 처리하지 않은 대조군에서는 B16/F10 세포 이동이 활발하게 일어난 반면, 진토닌을 처리할 경우에는 세포 이동이 진토닌 투여 농도별로 억제되는 것으로 나타났다.As a result, as shown in FIG. 4, B16 / F10 cell migration occurred actively in the control group not treated with gintonin, while cell migration was inhibited by the concentration of gintonin when treated with gintonin.
또한, 양성 대조군으로 사용한 Brp-LPA와 LPA에서도 B16/F10 세포 이동을 억제하는 것으로 확인되었다.
In addition, Brp-LPA and LPA used as positive controls were also found to inhibit B16 / F10 cell migration.
실시예Example 4. 4. 진토닌의Gintonin B16/F10 세포에서의 세포 증식 및 세포 사멸 효과 Cell proliferation and apoptosis effects in B16 / F10 cells 확인Confirm
상기 실시예 2 및 3에서 확인한 진토닌의 효과가, 세포 사멸 혹은 세포 증식에 영향을 미쳐서 나타나는 현상인지를 확인하기 위하여, 다음과 같은 실험을 실시하였다.In order to confirm whether the effect of the gintonin confirmed in Examples 2 and 3 is a phenomenon occurring by affecting cell death or cell proliferation, the following experiment was conducted.
BrDU는 세포 증식 과정에서 새로운 DNA가 합성될 때, DNA에 삽입되는 것으로 알려져 있으며(Porstmann T. et al. Immunol. Methods 82, 169-179, 1985), XTT는 세포내 미토콘드리아 효소에 의해 오렌지색 염을 형성하는 것으로 알려져 있어서(Cancer Res 48, 4827-4833, 1988), 이들 모두 세포 증식이나 세포 활성을 측정하는데 사용된다.BrDU is known to be inserted into DNA when new DNA is synthesized during cell proliferation (Porstmann T. et al. Immunol. Methods 82, 169-179, 1985), and XTT is responsible for the addition of orange salts by intracellular mitochondrial enzymes. Known to form (Cancer Res 48, 4827-4833, 1988), these are all used to measure cell proliferation and cell activity.
본 발명에서는 진토닌의 B16/F10 세포 증식에 대한 효과를 알아보고자 BrDU 방법과 XTT 방법을 사용하였다.
In the present invention, the BrDU method and the XTT method were used to determine the effect of gintonin on B16 / F10 cell proliferation.
먼저, 원 등이 제안한 BrDU 방법(Won et al. J Pharmacol Sci 108, 372-379, 2008; Chen et al. Cell Physiol Biochem 22, 307-314, 2008)에 따라 BrDU 세포 증식 ELISA 키트(BrDU cell proliferation ELISA kit; Roche, 독일)를 사용하여 세포에 삽입된 BrDU의 상대량을 측정하였다.First, BrDU cell proliferation ELISA kit (BrDU cell proliferation) according to the BrDU method proposed by Won et al. (Won et al. J Pharmacol Sci 108, 372-379, 2008; Chen et al. Cell Physiol Biochem 22, 307-314, 2008) ELISA kit (Roche, Germany) was used to measure the relative amount of BrDU inserted into the cells.
이때, 진토닌 처리 전날, 세포를 96 웰 플레이트에 각 웰당 3×103씩 분주하고, 24시간 후에 DMEM 배지(0.2% FBS)로 교환하여 6시간 동안 배양한 다음, 다시 배지를 새로운 DMEM 배지(0.2% FBS)로 교체하여 진토닌을 처리하고 24시간 배양하였다. 세포에 BrDU 라벨링(labelling) 시약을 넣고, 다시 24시간 배양한 다음, 세포의 배지를 제거하고 10% FBS 함유 DMEM로 세척하여 고정액으로 고정한 후, 항-Br여-과산화효소(anti-BrDU-peroxidase) 항체와 광도계(luminometer; Veritas, Turner Biosystems사, 미국)를 사용하여 세포 내에 들어간 BrDU의 상대적인 양을 정량하였다.
At this time, the day before the gintonin treatment, the cells were dispensed 3 × 10 3 per well into 96 well plate, and after 24 hours, exchanged with DMEM medium (0.2% FBS) and incubated for 6 hours, and then the medium was replaced with fresh DMEM medium ( 0.2% FBS) was treated with gintonin and incubated for 24 hours. BrDU labeling reagent was added to the cells, incubated for another 24 hours, and then the medium of the cells was removed, washed with DMEM containing 10% FBS, fixed in fixed solution, followed by anti-BrDU-peroxidase. ) The relative amount of BrDU in the cells was quantified using an antibody and a luminometer (Veritas, Turner Biosystems, USA).
다음으로는, XTT 방법(Hwang et al., Mol Cancer Ther 7, 559-568, 2008; Scudiero et al., Cancer Res 48, 4827-4833, 1988)에 따른 세포활성 측정을 위해, 상기와 같이 진토닌을 처리한 세포의 배지를 페놀 레드와 FBS가 들어있지 않은 배지로 교환한 후(200 ㎕/웰), XTT 반응액(1 ㎎/㎖ XTT와 0.0306 ㎎/㎖ PMS를 함유하는 DMEM 배지)을 넣어(50 ㎕/웰), 2시간 동안 배양하여 세포 중의 효소와 반응하게 하여 만들어진 오렌지색 수용성 염의 상대적인 농도를 파장 450 ㎚에서 흡광도를 청구하여 비교하였다(Spectramax 190 plate reader, Molecular Devices, Sunnyvale, CA, USA).
Next, for the measurement of cell activity according to the XTT method (Hwang et al.,
그 결과, 도 5A에서 보는 바와 같이, 진토닌에 의한 세포 증식에 미치는 영향에 있어서, 10 ㎍/㎖ 미만의 진토닌 처리에서는 거의 효과가 없는 것으로 나타난 반면, 10 ㎍/㎖ 이상에서는 BrDU 편입(incorporation)을 억제하는 것으로 나타났다.As a result, as shown in Fig. 5A, the effect on the cell proliferation by gintonin was found to be almost ineffective in the gintonin treatment of less than 10 ㎍ / ㎖, while BrDU incorporation (incorporation above 10 ㎍ / ㎖) ).
또한, XTT assay를 이용한 세포 사멸에 대한 진토닌의 영향에서도, 10 ㎍/㎖에서 진토닌은 미약하게 세포 사멸 효과가 있는 것으로 나타났고, 그 이상에서는 그 효과가 점차 크게 나타났다(도 5B 참조).In addition, the effect of gintonin on apoptosis using the XTT assay, jintonin was found to have a slight cell death effect at 10 ㎍ / ㎖, the effect was gradually greater than that (see Figure 5B).
특히, 30 ㎍/㎖에서는 BrDU 편입을 각각 약 40%(BrCU incorporation assay) 및 약 30%(XTT assay) 억제하여 세포 증식 억제 효과가 있음을 확인할 수 있었다.
In particular, at 30 μg / ml, BrDU incorporation was inhibited by about 40% (BrCU incorporation assay) and about 30% (XTT assay), respectively.
이와 같이, 10 ㎍/㎖에서 진토닌은 세포 증식이나 세포 사멸에는 미약한 효과를 발휘하나, 상처 치유 및 세포 이동에서는 현저하게 억제하는 것으로 보아(도 3 및 도 4 참조), 진토닌은 세포 사멸 및 증식에는 거의 영향을 미치지 않으나 암세포의 상처 치유나 세포 이동에는 영향을 미치는 것으로 나타나 암전이 억제 작용이 있는 것으로 확인되었다.
As such, gintonin exerts a slight effect on cell proliferation and cell death at 10 ㎍ / ml, but is significantly inhibited in wound healing and cell migration (see FIGS. 3 and 4). And it has little effect on the proliferation, but appears to affect the wound healing or cell migration of cancer cells, it was confirmed that the cancer metastasis has an inhibitory action.
실시예 5. 마우스 흑색종 폐전이에서 진토닌의 암전이 예방 및 억제 효과 확인Example 5 Confirmation of the Cancer Metastasis Prevention and Inhibition Effect of Gintonin in Mouse Melanoma Lung Metastasis
B16/F10 세포에 트립신(trypsin; 0.05% trypsin with EDTA, Invitrogen)을 처리하여 배양 플라스크(culture flask)로부터 세포를 떼어 세포 현탁액을 만든 후, 원심분리기를 이용하여 세포를 PBS(pH 7.4)로 세척하였다.Treatment of B16 / F10 cells with trypsin (0.05% trypsin with EDTA, Invitrogen) removes the cells from the culture flask to form a cell suspension, and the cells are washed with PBS (pH 7.4) using a centrifuge. It was.
세포를 다시 1×106/㎖가 되도록 PBS에 현탁하여, 마우스 꼬리 정맥(tail lateral vein)을 통해 주입하였다(세포 1×105/0.1 ㎖/마우스).
The cells were suspended in PBS again to 1 × 10 6 / ml and injected via mouse tail lateral vein (
진토닌의 암전이 예방 효과 확인을 위해, B16/F10 세포를 주입하기 3일 전부터 1일 1회 진토닌(0, 25, 50, 및 100 ㎎/㎏, p.o)을 경구 투여하기 시작하여 B16/F10 세포 주입 후부터 다시 21일간 진토닌을 더 투여하였다(Baker et al., J Biol Chem. 281, 22786-22793, 2006; Gupte et al., Bioorg Med Chem Lett 20, 7525-7528, 2010).To confirm the cancer metastasis prevention effect of gintonin, B16 / F10 cells were orally administered with gintonin (0, 25, 50, and 100 mg / kg, po) once a day from 3 days before B16 / F10 cells were injected. Gintonin was administered for another 21 days after F10 cell injection (Baker et al., J Biol Chem. 281, 22786-22793, 2006; Gupte et al., Bioorg
B16/F10 세포 주입 후 21일째 되는 날, 졸레틸(40 ㎎/㎏ of Zoletil™, Virbac Laboratories)과 럼푼(5 ㎎/㎏ of Rompun™, Bayer Korea)으로 마우스를 마취시킨 후 경추탈골 치사시켜, 폐 등 각 장기의 종양을 관찰하고, 종양의 수를 계산(count)하여 대조군과 투여군 간의 종양 수를 비교하였다.
On day 21 after B16 / F10 cell injection, mice were anesthetized with zoletil (40 mg / kg of Zoletil ™, Virbac Laboratories) and lumpoons (5 mg / kg of Rompun ™, Bayer Korea) and killed by cervical distal bone. Tumors of each organ such as the lung were observed, and the number of tumors was counted to compare the number of tumors between the control group and the administration group.
또한, 진토닌의 폐전이 흑색종의 치료 효과를 확인하기 위해, B16/F10 세포를 꼬리 정맥에 주입한 후, 1, 4, 및 7일 뒤에 진토닌 100 ㎎/㎏, p.o을 경구 투여하고 B16/F10 세포를 주입하여 14일 경과 후 폐에 전이되어 생긴 종양의 수를 비교하였다(Park et al., Biol Pharm Bull 31, 1802-1805, 2008). In addition, to confirm the therapeutic effect of pulmonary metastatic melanoma of gintonin, after injection of B16 / F10 cells into the tail vein, 1, 4, and 7 days later, oral administration of gintonin 100 mg / kg, po and B16 / F10 cells were injected and compared with the number of tumors that metastasized to the lung after 14 days (Park et al., Biol Pharm Bull 31, 1802-1805, 2008).
도 6A는 진토닌을 경구로 3일 전부터 투여한 다음, 배양된 B16/F10 세포(1×105)를 꼬리 정맥에 주입하고 나서 21일 후 폐에 생긴 종양 노듈수(nodules)를 확인한 것으로, 폐에 생긴 종양 노듈의 수는 진토닌 투여량(0, 25, 50, 및 100 ㎎/㎏)에 따라 각각 26.2±3.2, 24.8±5.8, 10.2±3.4, 및 1.8±0.7으로 나타났다(*p<0.01, 진토닌을 투여하지 않은 동물; n=5; 각 처리 그룹).6A is an oral administration of gintonin from 3 days before, followed by injecting cultured B16 / F10 cells (1 × 10 5 ) into the tail vein to confirm tumor nodules in the lung 21 days later. The number of tumor nodules in the lung was 26.2 ± 3.2, 24.8 ± 5.8, 10.2 ± 3.4, and 1.8 ± 0.7, respectively, according to the gintonin dose (0, 25, 50, and 100 mg / kg) ( * p < 0.01, animals not receiving gintonin; n = 5; each treatment group).
또한, 도 6B는 B16/F10 세포(1×105)를 마우스 꼬리 정맥에 주입한 후, 1, 4, 및 7일 뒤에 진토닌 100 ㎎/㎏을 경구로 투여한 다음, B16/F10 세포를 주입하여 14일 뒤에 폐에 생긴 종양 노듈수를 계산한 것으로, 생리식염수를 투여한 그룹(vehicle)과 1, 4, 및 7일 후 진토닌을 경구투여한 그룹에서 폐에 생긴 종양 노듈수가 각각 26.4±4.7과 10.5±3.6, 13.3±2.3, 및 16.3±3.4로 나타났다(*p<0.01, 진토닌을 투여하지 않은 동물; n=5; 각 처리 그룹).6B shows that B16 / F10 cells (1 × 10 5 ) were injected into the mouse tail vein, followed by oral administration of 100 mg / kg of gintonin after 1, 4, and 7 days, and then B16 / F10 cells. The number of tumor nodules in the lungs was calculated 14 days after injection, and the number of tumor nodules in the lungs was 26.4 in the group administered with saline and the oral administration of gintonin after 1, 4, and 7 days, respectively. ± 4.7 and 10.5 ± 3.6, 13.3 ± 2.3, and 16.3 ± 3.4 ( * p <0.01, animals without gintonin; n = 5; each treatment group).
한편, 도 6C와 D에는 각각 도 6A와 B에서 얻은 폐의 무게를 그룹별로 측정하여 나타내었다(*p<0.05, 진토닌을 투여하지 않은 동물).
On the other hand, Figure 6C and D shows the weight of the lungs obtained in Figures 6A and B, respectively, measured by group ( * p <0.05, animals not administered gintonin).
도 7은 진토닌을 투여하지 않은 마우스에서 암세포를 주입하기 3일전 진토닌(0, 25, 50, 100 ㎎/㎏, p.o)을 마우스에 경구 투여한 후 배양된 B16/F10 세포(1×105)를 꼬리 정맥에 주입하고 나서 21일 후 폐에 생긴 종양 노듈을 촬영한 사진으로, 각각 A는 생리 식염수(진토닌 0 ㎎/㎏, p.o), B는 진토닌 25 ㎎/㎏, C는 진토닌 50 ㎎/㎏ 및 D는 진토닌 100 ㎎/㎏을 마우스에 경구투여한 것이며, 화살표는 폐에 존재하는 종양 노듈을 가리키고 있다. FIG. 7 shows B16 / F10 cells (1 × 10) cultured after oral administration of gintonin (0, 25, 50, 100 mg / kg, po) to
이는, 도 6A의 결과와 같이, 진토닌 투여량이 증가함에 따라 폐에 생긴 종양 노듈의 수가 감소했음을 시각적으로 보여 준다.
This visually shows that as the result of FIG. 6A, the number of tumor nodules in the lungs decreased as the gintonin dose increased.
한편, 도 8A는 진토닌을 투여하지 않을 마우스에서 배양된 B16/F10 세포(1×105)를 꼬리 정맥에 주입하고 나서 14일째 되는 날, 폐에 생긴 종양 노듈을 촬영한 사진이며, B 내지 D는 배양된 B16/F10 세포(1×105)를 꼬리 정맥에 주입한 다음, 각각 1, 4 및 7일 후부터 진토닌 100 ㎎/㎏을 마우스에 경구투여하고 B16/F10 세포 주입 후 14일째 폐에 생긴 종양 노듈을 촬영한 것이다. 또한, 화살표는 폐에 존재하는 종양 노듈을 가리킨다.On the other hand, Fig. 8A is a photograph of tumor nodules formed in the lung on day 14 after injection of B16 / F10 cells (1 × 10 5 ) cultured in mice not to be administered gintonin into the tail vein. D was injected with cultured B16 / F10 cells (1 × 10 5 ) into the tail vein, followed by oral administration of 100 mg / kg of gintonin to mice from
이는, 상기 도 6B의 결과와 같이, 진토닌 투여량이 증가함에 따라 폐에 생긴 종양 노듈의 수가 감소했음을 보여 주고 있다.
This shows that as the result of FIG. 6B, the number of tumor nodules in the lung decreased as the gintonin dose increased.
상기의 B16/F10 세포 주입 마우스를 이용한 동물 실험 결과, 진토닌에는 마우스 흑색종 세포의 폐전이를 농도 의존적으로 억제하는 작용이 있으며, 진토닌 투여 시기에 따라 그 효과에 차이가 있음을 알 수 있다.
As a result of animal experiments using the B16 / F10 cell-injected mice, it can be seen that gintonin has a function of inhibiting lung metastasis of mouse melanoma cells in a concentration-dependent manner. .
이상, 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific descriptions are only for the preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (10)
Anticancer and cancer metastasis inhibiting composition containing gintonin as an active ingredient.
상기 진토닌은 인삼으로부터 분리 동정한 당지질단백질(glycolipoprotein)인 것을 특징으로 하는 항암 및 암전이 억제용 조성물.
The method of claim 2,
The gintonin is anti-cancer and cancer metastasis inhibiting composition, characterized in that the glycolipoprotein (glycolipoprotein) isolated from ginseng.
상기 진토닌은 오토탁신(autotaxin) 혹은 리소포스포리파제 D(lisophospholipase D)의 활성을 억제함으로써 암전이를 억제하는 것을 특징으로 하는 항암 및 암전이 억제용 조성물.
The method of claim 2,
The gintonin is anti-cancer and cancer metastasis inhibiting composition, characterized in that to suppress the cancer metastasis by inhibiting the activity of autotaxin (lytaxin) or lysophospholipase D (lisophospholipase D).
상기 암은 신장암(renal cell carcinoma), 난소암, 유방암, 갑상선암(thyroid carcinoma), 호지킨 림프종(Hodgkin lymphomas), 신경모세포종(neuroblastoma), 침습성 다형성 교모세포종(invasive glioblastoma multiforme), 전립선암, 및 피부암(melanomas)에서 선택되는 것을 특징으로 하는 항암 및 암전이 억제용 조성물.
The method of claim 2,
The cancer may include renal cell carcinoma, ovarian cancer, breast cancer, thyroid carcinoma, Hodgkin lymphomas, neuroblastoma, invasive glioblastoma multiforme, prostate cancer, and Anticancer and cancer metastasis inhibiting composition, characterized in that selected from skin cancer (melanomas).
상기 조성물은 단독 또는 병행하여 사용하는 것을 특징으로 하는 항암 및 암전이 억제용 조성물.
The method of claim 2,
The composition for anticancer and cancer metastasis suppression, characterized in that used alone or in parallel.
Anticancer agent containing gintonin as an active ingredient.
Cancer metastasis inhibitor containing gintonin as an active ingredient.
Anti-cancer health functional food containing gintonin as an active ingredient.
Health functional food for cancer metastasis containing gintonin as an active ingredient.
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| KR100973202B1 (en) * | 2009-11-17 | 2010-07-30 | 건국대학교 산학협력단 | Novel glycolipoprotein from ginseng and manufacturing method thereof |
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