KR20210020849A - Pharmaceutical composition for preventing or treating ovarian cancer or breast cancer comprising chrysophanol - Google Patents
Pharmaceutical composition for preventing or treating ovarian cancer or breast cancer comprising chrysophanol Download PDFInfo
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- KR20210020849A KR20210020849A KR1020200103533A KR20200103533A KR20210020849A KR 20210020849 A KR20210020849 A KR 20210020849A KR 1020200103533 A KR1020200103533 A KR 1020200103533A KR 20200103533 A KR20200103533 A KR 20200103533A KR 20210020849 A KR20210020849 A KR 20210020849A
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- chrysophanol
- ovarian cancer
- breast cancer
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- NZPQWZZXRKZCDU-UHFFFAOYSA-N chrysophanol Natural products Cc1cc(O)c2C(=O)c3c(O)cccc3Oc2c1 NZPQWZZXRKZCDU-UHFFFAOYSA-N 0.000 title claims abstract description 117
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Abstract
Description
본 발명은 크리소파놀을 유효성분으로 포함하는 조성물에 관한 것으로, 보다 상세하게는 크리소파놀을 유효성분으로 포함하는 난소암 또는 유방암의 예방 또는 치료용 약학적 조성물 및 크리소파놀을 유효성분으로 포함하는 난소암 또는 유방암의 예방 또는 개선용 건강기능식품 조성물에 관한 것이다.The present invention relates to a composition comprising chrysophanol as an active ingredient, and more specifically, a pharmaceutical composition for the prevention or treatment of ovarian cancer or breast cancer comprising chrysophanol as an active ingredient, and chrysophanol as an active ingredient. It relates to a health functional food composition for preventing or improving ovarian cancer or breast cancer comprising.
난소암은 조기 진단 기술의 부재와 화학요법에 대한 내성으로 인해 발병률 대비 사망률이 매우 높은 편이다 (Kobayashi et al., Journal of Translational Medicine, 2014). 현재 종양감축술 치료 이후 백금계, 탁산계 }항암화학요법 또는 방사선치료가 난소암 치료의 표준으로 여겨지고 있지만 말기 난소암 환자의 경우 재발률이 대략 70%에 달한다 (Capriglione et al., Medical Oncology, 2017).Ovarian cancer has a very high mortality rate versus incidence due to the lack of early diagnosis technology and resistance to chemotherapy (Kobayashi et al. , Journal of Translational Medicine, 2014). Currently, after tumor reduction treatment, platinum-based, taxane-based} chemotherapy or radiation therapy is considered the standard of ovarian cancer treatment, but the recurrence rate is approximately 70% in terminal ovarian cancer patients (Capriglione et al. , Medical Oncology, 2017). ).
유방암은 전 세계 여성에게서 발병하는 악성 종양 중 2위의 발병률을 보이며, 일생 동안 8명중 1명꼴로 발병하는 사망률 2위의 치명적인 질환이다 (Siegel et al., CA Cancer J., 2018, 68: p.7-30). BRCA 유전자의 돌연변이가 유방암 발병에 주요한 원인이라고 알려져 있지만, 그 수치는 유방암 환자중 5-10%에 그친다. 또한, 수유를 하지 않거나 수유 기간이 짧은 여성이 유방암 발병률이 높다고 알려져 있다. (Collaborative Group on Hormonal Factors in Breast, C. Lancet, 2002, 360: p.187-195.) 일반적으로 유방암에 대해서는 전체 종양을 외과적으로 제거하는 치료법을 사용한다. 하지만, 최근까지 개선된 수술법이나 화학적 항암요법들은 병기가 진행된 환자들에게 효과를 보이지 못하며 부작용만 심화시킨다는 문제점이 있다 (Aghapour et al., Biochem. Biophy. Res. Commun, 2018, 500: p.860-865). Breast cancer has the 2nd incidence rate among malignant tumors in women worldwide, and is the 2nd most fatal disease with a mortality rate of 1 in 8 people throughout life (Siegel et al., CA Cancer J., 2018, 68: p. .7-30). Although mutations in the BRCA gene are known to be the leading cause of breast cancer, the number is only 5-10% of breast cancer patients. In addition, it is known that women who do not lactate or who have a short lactation period have a high incidence of breast cancer. (Collaborative Group on Hormonal Factors in Breast, C. Lancet, 2002, 360 : p.187-195.) In general, for breast cancer, a treatment that surgically removes the entire tumor is used. However, until recently, improved surgical methods or chemotherapy have a problem that they do not show any effect in patients with advanced stage and only worsen side effects (Aghapour et al. , Biochem. Biophy. Res. Commun, 2018, 500: p.860: p.860) -865).
한편, 크리소파놀은 한약의 재료로 쓰이는 대황에 주로 함유된 안트라퀴논 계열의 천연물이다. 대표적인 안트라퀴논 계열 화합물인 에모딘, 레인 등은 다양한 암종의 세포사멸을 유도하고 침습성을 억제하는 것으로 알려져있는 반면, 크리소파놀의 항암 효과 특히, 대표적인 부인과 질환인 난소암 및 유방암 치료 효과에 대해서는 아직까지 보고된바 없다. On the other hand, chrysophanol is an anthraquinone-based natural product mainly contained in rhubarb used as an ingredient in herbal medicine. While representative anthraquinone-based compounds such as emidine and lane are known to induce apoptosis and inhibit invasiveness of various carcinomas, the anticancer effect of chrysophanol, in particular, the treatment effect of ovarian cancer and breast cancer, which are representative gynecological diseases, is still Has not been reported.
전술한 기술적 배경하에서 본 발명자들은 천연물 유래 물질을 이용한 새로운 난소암 및 유방암 개발하기 위하여 예의 노력한 결과, 크리소파놀이 난소암 또는 유방암의 세포 증식을 억제하고, 세포 사멸 유도 효과가 있음을 확인하고, 본 발명을 완성하였다.Under the above-described technical background, the present inventors made diligent efforts to develop new ovarian cancer and breast cancer using natural substances, and as a result, chrysophanol inhibited cell proliferation of ovarian cancer or breast cancer and confirmed that it has an effect of inducing cell death. The invention was completed.
본 발명은 크리소파놀을 유효성분으로 포함하는 난소암 또는 유방암의 예방 또는 치료용 약학적 조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of ovarian cancer or breast cancer comprising chrysophanol as an active ingredient.
본 발명은 또한, 크리소파놀을 유효성분으로 포함하는 난소암 또는 유방암의 예방 또는 개선용 건강기능식품 조성물을 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a health functional food composition for preventing or improving ovarian cancer or breast cancer comprising chrysophanol as an active ingredient.
본 발명은 또한, 상기 약학적 조성물 또는 건강기능식품 조성물을 이용하여 난소암 또는 유방암 세포주의 증식 능력을 억제시키는 방법을 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a method of inhibiting the proliferation ability of ovarian cancer or breast cancer cell lines by using the pharmaceutical composition or health functional food composition.
본 발명은 또한, 상기 약학적 조성물 또는 건강기능식품 조성물을 이용하여 난소암 또는 유방암 세포주의 사멸 효과를 향상시키는 방법을 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a method of improving the killing effect of ovarian cancer or breast cancer cell lines by using the pharmaceutical composition or health functional food composition.
본 발명은 크리소파놀을 유효성분으로 포함하는 난소암 또는 유방암의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating ovarian cancer or breast cancer comprising chrysophanol as an active ingredient.
본 발명은 또한, 크리소파놀을 유효성분으로 포함하는 난소암 또는 유방암의 예방 또는 개선용 건강기능식품 조성물을 제공한다.The present invention also provides a health functional food composition for preventing or improving ovarian cancer or breast cancer comprising chrysophanol as an active ingredient.
본 발명은 또한, 상기 약학적 조성물 또는 건강기능식품 조성물을 이용하여 난소암 또는 유방암 세포주의 증식 능력을 억제시키는 방법을 제공한다.The present invention also provides a method of inhibiting the proliferation ability of ovarian cancer or breast cancer cell lines by using the pharmaceutical composition or health functional food composition.
본 발명은 또한, 상기 약학적 조성물 또는 건강기능식품 조성물을 이용하여 난소암 또는 유방암 세포주의 사멸 효과를 향상시키는 방법을 제공한다.The present invention also provides a method of improving the killing effect of ovarian cancer or breast cancer cell lines by using the pharmaceutical composition or health functional food composition.
본 발명에 따른 크리소파놀을 유효성분으로 포함하는 조성물은 식물에서 추출된 것으로서 화학적 합성물에 비해 체내 독성 내지 부작용을 최소화할 수 있으며, 난소암 및 유방암 세포의 증식력을 억제하고 용량 의존적으로 난소암 및 유방암 세포주 내 사멸 세포의 수를 증가시킬 뿐만 아니라, PI3K/AKT, MAPK 신호전달경로를 억제하는 타겟 억제제와 병용 처리하는 경우 세포 사멸의 시너지 효과를 얻을 수 있는바, 생명과학, 의약학 등 다양한 산업 분야에 널리 활용될 수 있다.The composition comprising chrysophanol according to the present invention as an active ingredient is extracted from plants and can minimize toxicity or side effects in the body compared to chemical compounds, inhibit proliferation of ovarian cancer and breast cancer cells, and dose-dependently, ovarian cancer and In addition to increasing the number of dead cells in breast cancer cell lines, synergistic effects of cell death can be obtained when combined treatment with target inhibitors that inhibit PI3K/AKT and MAPK signaling pathways, and various industries such as life science and medicine It can be widely used in
도 1은 크리소파놀에 의한 난소암 및 유방암 세포 증식력 감소 효과를 측정한 결과를 나타낸 것이다.
도 2는 크리소파놀에 의한 난소암 및 유방암 세포 사멸 유도 효과를 측정한 결과를 나타낸 것이다.
도 3은 크리소파놀에 의한 난소암 및 유방암 세포 내 산화 스트레스 변화를 측정한 결과를 나타낸 것이다.
도 4는 크리소파놀에 의한 난소암 및 유방암 세포 내 미토콘드리아 기능장애 유도 효과를 측정한 결과를 나타낸 것이다.
도 5는 크리소파놀에 의한 난소암 및 유방암 내 신호전달기전 조절 양상을 분석한 결과를 나타낸 것이다.
도 6은 크리소파놀과 신호전달경로 억제제 처리에 따른 난소암 및 유방암 세포 증식력 변화를 측정한 결과를 나타낸 것이다.
도 7은 크리소파놀에 의한 난소암 세포 이주 능력 감소 효과를 측정한 결과를 나타낸 것이다.
도 8은 크리소파놀의 난소암에 대한 항암기전을 나타낸 것이다.
도 9는 크라이소파놀의 유방암 세포에 대한 항암 작용 기전을 나타낸 것이다.Figure 1 shows the results of measuring the effect of reducing the proliferation of ovarian cancer and breast cancer cells by chrysophanol.
Figure 2 shows the results of measuring the effect of inducing ovarian cancer and breast cancer cell death by chrysophanol.
3 shows the results of measuring changes in oxidative stress in ovarian cancer and breast cancer cells by chrysophanol.
Figure 4 shows the results of measuring the mitochondrial dysfunction induction effect in ovarian cancer and breast cancer cells by chrysophanol.
5 shows the results of analyzing the regulation of signaling mechanisms in ovarian cancer and breast cancer by chrysophanol.
6 shows the results of measuring changes in ovarian cancer and breast cancer cell proliferation capacity according to treatment with chrysophanol and a signaling pathway inhibitor.
7 shows the results of measuring the effect of reducing the ovarian cancer cell migration ability by chrysophanol.
Figure 8 shows the anticancer mechanism of chrysophanol against ovarian cancer.
9 shows the mechanism of anticancer action of chrysophanol on breast cancer cells.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 가진다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.
본 발명에서는 크리소파놀의 난소암 또는 유방암 세포주의 증식 억제 및 세포 사멸 기전을 밝혔다(도 8, 도9).In the present invention, chrysophanol inhibited the proliferation of ovarian cancer or breast cancer cell lines and revealed a mechanism of cell death (FIGS. 8 and 9 ).
본 발명은 일 관점에서 크리소파놀을 유효성분으로 포함하는 난소암 및 유방암 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention or treatment of ovarian cancer and breast cancer comprising chrysophanol as an active ingredient in one aspect.
본 발명은 다른 관점에서 크리소파놀을 유효성분으로 포함하는 난소암 및 유방암 예방 또는 개선용 건강기능식품 조성물에 관한 것이다.The present invention relates to a health functional food composition for preventing or improving ovarian cancer and breast cancer comprising chrysophanol as an active ingredient from another viewpoint.
본 발명에서 사용된 용어 "조성물"은 특정 성분을 포함하는 산물뿐만 아니라, 특정 성분의 배합에 의해 직접 또는 간접적으로 만들어지는 임의의 산물을 포함하는 것으로 간주된다.The term "composition" as used in the present invention is considered to include not only products containing specific ingredients, but also any products made directly or indirectly by the combination of specific ingredients.
본 발명에 있어서, 상기 난소암 또는 유방암 세포는 포유동물 유래의 세포일 수 있으며, 상기 포유동물은 설치목(예를 들어, 생쥐, 쥐, 햄스터, 게르빌루스 및 기니피그), 우제목(예를 들어, 소, 양, 돼지, 염소, 사슴, 기린 및 영양), 기제목(예를 들어, 말, 당나귀, 코뿔소 및 맥), 식육목(예를 들어, 개, 고양이, 호랑이, 늑대, 여우, 사자, 치타, 표범, 너구리, 오소리, 퓨마, 재규어 및 삵쾡이), 토끼목(토끼 및 우는 토끼), 식충목(예를 들어, 고슴도치, 두더지 및 솔레노돈) 및 영장목(예를 들어, 침팬지, 오랑우탄, 고릴라, 보노보노, 일본원숭이, 붉은털원숭이)일 수 있다.In the present invention, the ovarian cancer or breast cancer cells may be cells derived from mammals, and the mammals are rodents (eg, mice, rats, hamsters, gerbils, and guinea pigs), right heads (eg For example, cattle, sheep, pigs, goats, deer, giraffes and antelopes), base titles (e.g. horses, donkeys, rhinos and tapirs), carnivores (e.g. dogs, cats, tigers, wolves, foxes, lions) , Cheetahs, leopards, raccoons, badgers, cougars, jaguars and wildcats), hare (rabbits and crying rabbits), carnivores (e.g. hedgehogs, moles and solenoidons) and primates (e.g. chimpanzees, orangutans , Gorilla, bono bono, Japanese monkey, rhesus monkey).
본 발명에 있어서, 약학적 조성물은 약제학적으로 허용가능한 담체를 함유하는 것을 특징으로 할 수 있고, 상기 담체는 이온 교환 수지, 알루미나, 알루미늄 스테아레이트, 레시틴, 혈청 단백질, 완충 물질, 물, 염, 전해질, 교질성 실리카, 마그네슘 트리실리케이트, 폴리비닐피롤리돈, 셀룰로즈계 기질, 폴리에틸렌 글리콜, 나트륨 카르복시메틸셀룰로즈, 폴리아릴레이트, 왁스, 폴리에틸렌 글리콜 및 양모지로 구성된 군에서 선택되는 하나 이상인 것을 특징으로 할 수 있다.In the present invention, the pharmaceutical composition may be characterized in that it contains a pharmaceutically acceptable carrier, wherein the carrier is an ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer material, water, salt, Electrolyte, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol and wool paper, characterized in that at least one selected from the group consisting of I can.
본 발명에 있어서, 약학적 조성물은 정맥내, 복강내, 근육내, 동맥내, 구강, 심장내, 골수내, 경막내, 경피, 장관, 피하, 설하 또는 국부 투여용으로 제형화하는 것을 특징으로 할 수 있고, 완충제, 항균성 보존제, 계면활성제, 산화방지제, 긴장성 조정제, 방부제, 증점제 및 점도 개질제로 구성된 군에서 선택된 어느 하나 이상의 보조제를 추가로 함유하는 것을 특징으로 할 수 있으며, 용액, 현탁액, 에멀젼, 겔 및 분말로 구성된 군에서 선택되는 제형을 가지는 것을 특징으로 할 수 있다.In the present invention, the pharmaceutical composition is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intrathecal, transdermal, intestinal, subcutaneous, sublingual or topical administration. It can be characterized in that it further contains any one or more auxiliary agents selected from the group consisting of buffers, antimicrobial preservatives, surfactants, antioxidants, tonicity modifiers, preservatives, thickeners and viscosity modifiers, solutions, suspensions, emulsions , It may be characterized in that it has a formulation selected from the group consisting of gels and powders.
본 발명의 약학적 조성물의 적합한 투여량은 증상의 경중도, 환자의 체중, 연령, 성, 투여 방식 및 투여시간 등과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정할 수 있다.A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the severity of symptoms, weight, age, sex, mode of administration and time of administration of the patient, and generally skilled physicians are effective in the desired treatment or prevention. The dosage can be easily determined.
본 발명에 있어서, 건강기능식품은 건강기능식품에 관한 법률 제6722호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 식품을 의미한다.In the present invention, the health functional food refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No. 6722, and contains nutrients for the structure and function of the human body. It refers to foods consumed for the purpose of controlling or obtaining beneficial effects for health purposes such as physiological effects.
본 발명의 건강기능식품 조성물은 당해 기술분야에 공지되어 있는 통상적인 건강기능식품의 제형으로 제제화될 수 있고, 과립제, 정제, 환제, 현탁액, 에멀젼, 시럽제, 껌, 차, 젤리, 각종 음료수, 드링크제, 알코올 음료 등으로 제조될 수 있으며, 상기 건강기능식품의 종류에는 특별한 제한이 없다.The health functional food composition of the present invention may be formulated into a formulation of a general health functional food known in the art, and granules, tablets, pills, suspensions, emulsions, syrups, gums, teas, jellies, various beverages, and drinks. , Alcoholic beverages, etc., and there is no particular limitation on the kind of the health functional food.
본 발명의 건강기능식품 조성물은 인체를 비롯한 동물 신체에 투여하기 적합한 임의의 생약 형태, 더욱 구체적으로는 경구 투여에 통상적인 임의의 형태, 예를 들어 식품 또는 사료, 식품 또는 사료의 첨가제 및 보조제, 강화된 식품 또는 사료, 정제, 환제, 과립, 캡슐 및 발포 배합물 등과 같은 고체 형태 또는 용액, 현탁액, 유화액, 음료, 페이스트 등과 같은 액체형태 일 수 있고, 영양제, 비타민, 전해질, 감미제, 착색제, 유기산, 방부제 등을 함유할 수 있으며, 이러한 성분들을 독립적으로 또는 조합하여 사용할 수 있다.The health functional food composition of the present invention is in any herbal form suitable for administration to an animal body, including the human body, more specifically any form conventional for oral administration, for example, food or feed, additives and adjuvants for food or feed, It may be in a solid form such as fortified food or feed, tablets, pills, granules, capsules and foam formulations, or in liquid form such as solutions, suspensions, emulsions, beverages, pastes, etc., and nutrients, vitamins, electrolytes, sweeteners, colorants, organic acids, It may contain a preservative or the like, and these ingredients may be used independently or in combination.
본 발명에 따르면, 상기 크리소파놀은 PI3K/AKT 및 MAPK 신호전달기전을 조절하는 것을 특징으로 할 수 있다.According to the present invention, the chrysophanol may be characterized in that it regulates PI3K/AKT and MAPK signaling mechanisms.
또한, 본 발명에 따르면, PI3K/AKT 및 MAPK 신호전달경로를 억제하는 타겟 억제제를 더 포함하는 것을 특징으로 할 수 있다.In addition, according to the present invention, it may be characterized in that it further comprises a target inhibitor that inhibits the PI3K/AKT and MAPK signaling pathways.
본 발명에 따르면, 상기 크리소파놀은 산화스트레스 유도를 통해 난소암, 유방암을 예방 또는 치료할 수 있다.According to the present invention, the chrysophanol may prevent or treat ovarian cancer and breast cancer through induction of oxidative stress.
본 발명에 따르면, 상기 크리소파놀은 미토콘드리아 기능장애 유도를 통해 난소암, 유방암을 예방 또는 치료할 수 있다.According to the present invention, the chrysophanol may prevent or treat ovarian cancer and breast cancer through induction of mitochondrial dysfunction.
본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와 순차적 또는 동시에 투여될 수 있다.The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent.
본 발명에 있어서, 시스플라틴 또는 파클리탁셀을 더 포함하는 것을 특징으로 할 수 있으나, 이에 한정되는 것은 아니고 난소암 또는 유방암 치료제로 사용되는 화학치료제를 더 포함할 수 있다.In the present invention, cisplatin or paclitaxel may be further included, but the present invention is not limited thereto, and a chemotherapeutic agent used as a therapeutic agent for ovarian cancer or breast cancer may be further included.
본 발명은 또 다른 관점에서, 크리소파놀을 유효성분으로 포함하는 조성물을 이용하여 인간을 제외한 포유동물 유래 난소암 세포주 또는 인간을 제외한 포유동물 유래 유방암 세포주의 증식 능력을 억제시키는 방법에 관한 것이다.In another aspect, the present invention relates to a method of inhibiting the proliferation ability of a mammalian-derived ovarian cancer cell line excluding humans or a mammalian-derived breast cancer cell line excluding humans by using a composition comprising chrysophanol as an active ingredient.
본 발명은 또 다른 관점에서, 크리소파놀을 유효성분으로 포함하는 조성물을 이용하여 인간을 제외한 포유동물 유래 난소암 세포주 또는 인간을 제외한 포유동물 유래 유방암 세포주의 사멸 효과를 향상시키는 방법에 관한 것이다.In another aspect, the present invention relates to a method for improving the killing effect of a mammalian-derived ovarian cancer cell line excluding humans or a mammalian-derived breast cancer cell line excluding humans by using a composition comprising chrysophanol as an active ingredient.
[실시예][Example]
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.
실험방법Experiment method
실험동물 및 세포배양Experimental animals and cell culture
ES2, OVCAR3 세포는 American Type Culture Collection에서 구매하여 사용하였으며, 세포의 단층배양을 위해서 ES2는 McCoy’s 5A 배지에, OVCAR3는 25mM HEPES가 포함된 RPMI-1640 배지에 10%의 소태아혈청(fetal bovine serum, FBS)을 함께 혼합하여 사용하였다. 유방암 세포주인 BT-474, MCF-7는 한국세포주은행 (KCLB)에서 구입하였으며, 정상 유선 세포인 MCF-12A는 American Type Culture Collection에서 구매하여 사용하였다. 유방암 세포의 단층배양을 위해서 RPMI1640 (+25mM HEPES) 배지에 10%의 소태아혈청(fetal bovine serum, FBS)을 추가한 배양용 배지에 배양하였으며, 정상 유선세포는 DMEM/F12 배지에 20ng/ml hEGF, 0.01mg/ml bovine insulin, 500ng/ml hydrocortisone 및 5% horse serum을 섞은 배양용 배지에서 배양하였다.ES2 and OVCAR3 cells were purchased from the American Type Culture Collection and used.For monolayer culture of cells, ES2 was used in McCoy's 5A medium, and OVCAR3 was used in RPMI-1640 medium containing 25 mM HEPES, and 10% fetal bovine serum. , FBS) were mixed together and used. Breast cancer cell lines BT-474 and MCF-7 were purchased from the Korea Cell Line Bank (KCLB), and normal mammary cells, MCF-12A, were purchased from the American Type Culture Collection and used. For monolayer culture of breast cancer cells, 10% fetal bovine serum (FBS) was added to RPMI1640 (+25mM HEPES) medium, and normal mammary cells were 20 ng/ml in DMEM/F12 medium. HEGF, 0.01mg/ml bovine insulin, 500ng/ml hydrocortisone, and 5% horse serum were mixed in culture medium.
실험 재료Experiment material
후보 조성물질인 크리소파놀은 Sigma-Aldrich, Inc로부터 구매하여 사용하였으며, 크라이신에 의한 신호전달메커니즘을 확인하기 위하여 phospho-AKT (Ser473), ERK1/2 (Thr202/Tyr204), JNK (Thr183/Tyr185), P70S6K (Thr421/Ser424), P90RSK (Thr573), S6 (Ser235 /236), P38 (Thr180/Tyr182) 단백질 및 total-AKT, ERK1/2, JNK, P70S6K, P90RSK, S6, P38에 대한 항체를 Cell Signaling Techonology사로부터 구매하였다. 또한 타겟 신호전달과정을 억제함에 따른 효과를 규명하기 위하여 PI3K 억제제인 LY294002를 Cell Signaling Technology사로부터, ERK1/2 억제제인 U0126, P38 억제제인 SB203580 및 JNK 억제제인 SP600125를 Enzo Life Science사로부터 구매하여 사용하였다.Chrysophanol, a candidate composition, was purchased and used from Sigma-Aldrich, Inc, and phospho-AKT (Ser 473 ), ERK1/2 (Thr 202 /Tyr 204 ), JNK (Thr 183 /Tyr 185 ), P70S6K (Thr 421 /Ser 424 ), P90RSK (Thr 573 ), S6 (Ser 235 /236 ), P38 (Thr 180 /Tyr 182 ) protein and total-AKT, ERK1/2, JNK , P70S6K, P90RSK, S6, P38 antibodies were purchased from Cell Signaling Techonology. In addition, in order to investigate the effect of inhibiting the target signaling process, PI3K inhibitor LY294002 was purchased from Cell Signaling Technology, ERK1/2 inhibitor U0126, P38 inhibitor SB203580 and JNK inhibitor SP600125 were purchased from Enzo Life Science. I did.
BrdU를BrdU 이용한 세포 증식 능력 분석 Cell proliferation ability analysis
난소암 세포 및 유방암 세포의 증식 능력에 크리소파놀이 미치는 영향을 확인하기 위하여 FBS 기아 조건으로 배양한 5×103개의 세포와 배지 100 ㎕를 96 well에 분주하고 크리소파놀을 용량의존적으로 처리하여 48시간 동안 배양한 다음, BrdU 키트 (Cat No: 1167229001, Roche)를 사용하여 제조사의 매뉴얼에 따라 실험을 수행하였다. 48시간 인큐베이션 후, 10 μM BrdU를 각 well에 추가적으로 넣어 37℃/5% CO2 인큐베이터 내에서 2시간 동안 배양하였다. 세포에 BrdU를 labeling 하고 세포를 고정하여 anti-BrdU-POD 용액을 상온에서 90분 인큐베이션 시킨 후 3차례 씻어주고, 마지막으로 100 μl의 3,3’,5,5’-tetramethylbenzidine substrate으로 세포를 반응시키고, ELISA 리더기를 사용하여 370 nm, 492 nm 내 흡광도를 측정하여 세포 증식 능력을 분석하였다.To confirm the effect of chrysophanol on the proliferation ability of ovarian cancer cells and breast cancer cells, 5×10 3 cells cultured under FBS starvation and 100 μl of medium were dispensed into 96 wells and treated with chrysophanol dose-dependently. After incubation for 48 hours, the experiment was performed according to the manufacturer's manual using the BrdU kit (Cat No: 1167229001, Roche). After incubation for 48 hours, 10 μM BrdU was additionally added to each well and incubated for 2 hours in a 37° C./5% CO 2 incubator. The cells are labeled with BrdU and the cells are fixed, incubated with anti-BrdU-POD solution for 90 minutes at room temperature, washed three times, and finally reacted with 100 μl of 3,3',5,5'-tetramethylbenzidine substrate. And, using an ELISA reader, the absorbance within 370 nm and 492 nm was measured to analyze the cell proliferation ability.
면역형광법을Immunofluorescence 이용한 세포 증식 Cell proliferation used 마커Marker 확인 Confirm
3×104 개의 BT-474 및 MCF-7 세포를 confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea)에 분주하여 배양한 뒤, 24시간 FBS 기아상태로 추가로 배양하여 크라이소파놀 100 μM을 24시간 동안 처리한 뒤 메탄올로 10분간 세포를 고정하고, 2 μg/ml의 PCNA 항체를 처리하여 4℃에서 16시간 인큐베이션 하였다. 이후, 0.1% BSA (bovine serum albumin)이 포함된 PBS로 2번의 워싱과정을 거쳐 2차 항체로는 goat anti-mouse IgG Alexa 488 (catalog number: A-11001, Invitrogen, Carlsbad, CA, USA)을 antibody dilution buffer에 1:200으로 희석하여 상온에서 1시간 동안 배양하였다. 세포를 0.1% BSA-PBS로 워싱한 다음 DAPI 염색을 추가적으로 시행하여 핵을 동시에 관찰할 수 있도록 하였다. 실험 종료 후 LSM710 (Carl Zeiss, Thornwood, NY, USA) 공초점 현미경을 이용하여 세포를 관찰 및 촬영하였다.3×10 4 BT-474 and MCF-7 cells were dispensed and cultured in a confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea), and then further cultured in FBS starvation state for 24 hours. After treatment with 100 μM for 24 hours, the cells were fixed with methanol for 10 minutes, and 2 μg/ml of PCNA antibody was treated and incubated at 4° C. for 16 hours. After that, after washing twice with PBS containing 0.1% BSA (bovine serum albumin), goat anti-mouse IgG Alexa 488 (catalog number: A-11001, Invitrogen, Carlsbad, CA, USA) was used as the secondary antibody. It was diluted 1:200 in antibody dilution buffer and incubated for 1 hour at room temperature. After washing the cells with 0.1% BSA-PBS, DAPI staining was additionally performed so that the nuclei could be observed simultaneously. After the end of the experiment, the cells were observed and photographed using an LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope.
AnnexinAnnexin V와 V and propidiumpropidium iodide 염색을 통한 세포사멸 분석 Apoptosis analysis through iodide staining
크리소파놀에 의한 난소암 및 유방암 세포의 사멸 효과를 확인하기 위하여 FITC Annexin V 세포 사멸 진단 키트 I (BD Biosciences)를 사용하여 실험을 진행하였다. 먼저 5×105 개의 세포를 6 well에 배양하고 70 ~ 80% 배양 접시에 세포가 찼을 때 24시간 FBS 기아상태로 추가 배양하였다. 이후, 크리소파놀을 용량의존적으로 처리하여 48시간 동안 37℃/5% CO2 인큐베이터 내에서 배양하였다. 이후, 트립신을 사용하여 세포를 배양 접시에서 떼어 PBS로 워싱을 진행하고 1 mL의 1× binding buffer를 사용하여 세포를 천천히 혼합하고 원심분리하여 세포 pellet을 얻은 다음, 200 μL의 1× binding buffer으로 세포현탁배양하여 브라운 1.5 mL 튜브에 100 μL 넣고 Annexin V 5 μL, PI 5 μL를 함께 혼합하여 세포를 15분 동안 실온에 두어 염색하였다. 이후, 1× binding buffer를 400 μL 추가하고, 5 mL FACS 튜브에 염색된 용액을 옮겨 유세포 분석기를 사용하여 형광 강도를 분석하여 사멸된 세포의 수를 측정하였다.In order to confirm the killing effect of ovarian cancer and breast cancer cells by chrysophanol, an experiment was conducted using FITC Annexin V Apoptosis Diagnostic Kit I (BD Biosciences). First, 5×10 5 cells were cultured in 6 wells and further cultured in FBS starvation for 24 hours when the cells were filled in a 70-80% culture dish. Thereafter, chrysophanol was dose-dependently treated and cultured in a 37° C./5
TUNELTUNEL assay를 통한 DNA DNA through assay 분절화Segmentation 측정 Measure
3×104개의 BT-474 및 MCF-7 세포를 confocal dish (catalog number: 100350, SPL) 에 배양한 후, 24시간 serum 기아상태로 추가로 배양하여 크라이소파놀 100 μM을 48시간 동안 처리하였다. 이후, 세포를 에어드라이 시키고 4% paraformaldehyde로 상온에서 1시간 인큐베이션하여 세포를 고정시켰다. 고정된 세포는 PBS로 한번 헹궈내고 0.1% sodium citrate에 0.1% Triton X-100가 함유된 용액을 사용하여 아이스 위에서 2분간 인큐베이션 시켰다. 다음으로 In Situ Cell Death Detection Kit, TMR red (Roche) 에 포함된 TUNEL staining mixture를 사용하여 37℃/5% CO2 인큐베이터 내에서 1시간 동안 배양하였다. 마지막으로 PBS로 헹구고 DAPI를 염색한 후, LSM710 (Carl Zeiss, Thornwood, NY, USA) 공초점 현미경을 이용하여 세포를 관찰 및 촬영하였다.After culturing 3×10 4 BT-474 and MCF-7 cells in a confocal dish (catalog number: 100350, SPL), they were further cultured in serum starvation for 24 hours and treated with 100 μM of chrysophanol for 48 hours. . Thereafter, the cells were air-dried and incubated for 1 hour at room temperature with 4% paraformaldehyde to fix the cells. The fixed cells were rinsed once with PBS and incubated on ice for 2 minutes using a solution containing 0.1% sodium citrate and 0.1% Triton X-100. Next, using the TUNEL staining mixture included in In Situ Cell Death Detection Kit, TMR red (Roche), it was incubated for 1 hour in a 37°C/5% CO2 incubator. Finally, after rinsing with PBS and staining with DAPI, cells were observed and photographed using an LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope.
DCFHDCFH -DA을 이용한 활성산소 형성 분석-Analysis of active oxygen formation using DA
활성산소 형성 분석은 2’,7’-dichlorofluorescin diacetate(DCFH-DA, Sigma-Aldrich)을 통해 진행하였다. 100π 배양 접시에 70 ~ 80% 세포가 찼을 때, 트립신을 사용하여 세포를 배양 접시에서 떼어 PBS로 워싱을 진행하고 원심분리 하여 세포 pellet을 얻었다. 세포에 10 μM DCFH-DA을 처리해 37℃/5% CO2 인큐베이터 내에서 30분 동안 배양하였다. 이후, PBS로 워싱을 진행한 뒤 크리소파놀을 용량의존적으로 처리하여 1시간 동안 37℃/5% CO2 인큐베이터 내에서 배양하였다. 세포를 다시 원심분리 하여 PBS로 워싱한 후 유세포 분석기를 사용하여 형광 강도를 분석하였다. Reactive oxygen formation analysis was performed through 2',7'-dichlorofluorescin diacetate (DCFH-DA, Sigma-Aldrich). When the 100π culture dish was filled with 70 to 80% cells, the cells were removed from the culture dish using trypsin, washed with PBS, and centrifuged to obtain a cell pellet. Cells were treated with 10 μM DCFH-DA and incubated for 30 minutes in a 37° C./5
지질과산화 분석Lipid peroxidation analysis
난소암 및 유방암 세포내 지질과산화 정도를 분석하기 위해 Click-iT lipid peroxidation imaging kit (Invitrogen)을 사용하였다. 3×104개의 세포를 10% FBS가 포함 된 배지 300 μl와 함께 confocal dish 에 분주하여 배양한 뒤, 크리소파놀 100 μM과 함께 37℃ 인큐베이터 내에서 2시간 동안 인큐베이션 하였다. 3.7% formaldehyde로 세포를 고정하고 0.5% Triton X-100을 이용하여 premeablization한 후 Alexa Fluor 488 Azide로 상온에서 30분 동안 형광염색하였다. 마지막으로 PBS로 헹구고 DAPI를 염색한 후, LSM710 (Carl Zeiss, Thornwood, NY, USA) 공초점 현미경을 이용하여 세포를 관찰 및 촬영하였다.Click-iT lipid peroxidation imaging kit (Invitrogen) was used to analyze the degree of lipid peroxidation in ovarian cancer and breast cancer cells. 3 × 10 4 cells were cultured in a confocal dish with 300 μl of a medium containing 10% FBS, and then incubated with 100 μM of chrysophanol in an incubator at 37° C. for 2 hours. Cells were fixed with 3.7% formaldehyde, premeablized with 0.5% Triton X-100, and fluorescently stained with
FluoFluo -4와 -4 and RhodRhod -2를 이용한 With -2 세포내Intracellular CaCa 22 ++ 농도 측정 Concentration measurement
세포 내 Ca2 + 농도에 크리소파놀이 미치는 영향을 확인하기 위하여 먼저 5×105 세포를 6 well에 배양하고 70 ~ 80% 배양 접시에 세포가 찼을 때 24시간 FBS 기아상태로 추가 배양하였다. 이후, 크리소파놀을 처리하여 48시간 동안 37℃/5% CO2 인큐베이터 내에서 배양하였다. 이후, 트립신을 사용하여 세포를 배양 접시에서 떼어 Fluo-4 AM (Invitrogen)을 37℃ 인큐베이터 내에서 20분 동안 인큐베이션 하였다. 염색된 세포를 PBS로 한 번 워싱하고 유세포 분석기를 사용하여 형광 강도를 분석하였다. 미토콘드리아 특이적 Ca2 + 농도 측정을 위해 Rhod-2 AM (Invitrogen)을 HBSS에 희석한 후 4℃에서 30분 동안 인큐베이션 하였다. 염색된 세포를 HBSS로 워싱하고 유세포 분석기를 사용하여 형광 강도를 분석하였다.In order to confirm the effect of chrysophanol on the intracellular Ca 2 + concentration, 5 × 10 5 cells were first cultured in 6 wells and further cultured in FBS starvation for 24 hours when the cells were filled in a 70-80% culture dish. Thereafter, chrysophanol was treated and cultured in a 37° C./5
JCJC -1 염색을 통한 미토콘드리아 Mitochondria through -1 staining 막전위Membrane potential 측정 Measure
JC-1 미토콘드리아 막 전위 (MMP) 변화는 mitochondria staining kit (Cat No: CS0390, Sigma-Aldrich)를 사용하여 측정하였다. 5×105개의 난소암 및 유방암 세포를 6 well에 배양하고 70 ~ 80% 배양 접시에 세포가 찼을 때 24시간 FBS 기아상태로 추가 배양하였다. 이후, 크리소파놀을 용량의존적으로 (0, 20, 50, 100 μM) 처리하여 48시간 동안 37℃/5% CO2 인큐베이터 내에서 배양하였다. 이후, 트립신을 사용하여 세포를 배양 접시에서 떼어 원심분리 하여 세포 pellet을 얻었다. 세포를 JC-1 staining solution 에 풀어준 후 37℃/5% CO2 인큐베이터 내에서 20분 간 인큐베이션 하였다. 염색된 세포를 다시 원심분리하여 1x JC-1 staining buffer로 워싱한 후 유세포 분석기를 사용하여 형광 강도를 분석하였다.JC-1 mitochondrial membrane potential (MMP) change was measured using a mitochondria staining kit (Cat No: CS0390, Sigma-Aldrich). 5×10 5 ovarian cancer and breast cancer cells were cultured in 6 wells and further cultured in FBS starvation for 24 hours when the cells were filled in 70-80% culture dishes. Thereafter, chrysophanol was dose-dependently treated (0, 20, 50, 100 μM) and cultured in a 37° C./5% CO 2 incubator for 48 hours. Thereafter, the cells were removed from the culture dish using trypsin and centrifuged to obtain a cell pellet. The cells were released in JC-1 staining solution and incubated for 20 minutes in a 37°C/5% CO2 incubator. The stained cells were centrifuged again, washed with 1x JC-1 staining buffer, and the fluorescence intensity was analyzed using a flow cytometer.
단백질 발현 분석 (Protein expression analysis ( 웨스턴블롯Western Blot ))
난소암 및 유방암 세포에 크리소파놀을 처리한 다음 전체 단백질을 추출하여 Bradford protein assay (Bio-Rad, Hercules, CA, USA)로 단백질을 정량하였다. 이 후, 추출한 단백질을 95℃에서 5분간 변성하였으며 10% SDS/PAGE 젤을 이용하여 전기영동 수행한 뒤, nitrocellulose membrane으로 옮겨주고, 1차 항체와 2차 항체를 차례로 인큐베이션 시킨 다음 chemiluminescence detection (SuperSignal West Pico, Pierce, Rockford, IL, USA) 시약 및 ChemiDoc EQ system과 Quantity One software (Bio-Rad) 기기를 사용하여 타겟 단백질의 발현을 분석하였다. Ovarian cancer and breast cancer cells were treated with chrysophanol, and then total protein was extracted, and the protein was quantified by Bradford protein assay (Bio-Rad, Hercules, CA, USA). After that, the extracted protein was denatured at 95°C for 5 minutes, electrophoresis was performed using 10% SDS/PAGE gel, transferred to a nitrocellulose membrane, and the primary antibody and the secondary antibody were incubated sequentially, followed by chemiluminescence detection (SuperSignal West Pico, Pierce, Rockford, IL, USA) reagent, and ChemiDoc EQ system and Quantity One software (Bio-Rad) instrument were used to analyze the expression of the target protein.
세포 cell 침습성Invasive 분석 analysis
난소암 세포의 세포 침습성에 있어 크리소파놀이 미치는 영향을 확인하기 위하여 Migration culture dish에 Matrigel을 코팅한 후 FBS 기아 조건으로 배양한 1×105개의 난소암 세포를 8-mm transwell inserts (Corning, Inc., Corning, NY, USA)에 분주하고 크리소파놀을 처리하여 12시간 (ES2) 또는 16시간 (OVCAR3) 동안 배양한 다음, 메탄올로 10분간 trans-membrane을 이동한 세포를 고정하여, 건조 및 헤마톡실린 (Sigma Aldrich, Inc.) 염색을 30분간 수행하고, 씻어주었다. 슬라이드 글라스에 막을 올려주고 퍼마운트 용액(Permount solution)으로 고정하여 세포 이주 능력을 광학현미경 DM3000 (Leica)로 관찰, 촬영 및 계산하였다. 또한 Ibidi migration inserts를 통해 세포의 이주성 변화를 분석하기 위하여 2×105개/ml의 난소암 세포가 포함된 배지 70 μl를 inserts에 배양한 후 24시간 동안 기아 상태를 유지하였다. 이후 inserts를 제거하고 크리소파놀을 처리한 후 세포군 간의 평균 길이를 측정하였다.To confirm the effect of chrysophanol on the cellular invasiveness of ovarian cancer cells, 1×10 5 ovarian cancer cells cultured under FBS starvation conditions after coating Matrigel on migration culture dishes were inserted into 8-mm transwell inserts (Corning, Inc. ., Corning, NY, USA), treated with chrysophanol, incubated for 12 hours (ES2) or 16 hours (OVCAR3), and then fixed the cells transferred trans-membrane with methanol for 10 minutes, dried and Hematoxylin (Sigma Aldrich, Inc.) staining was performed for 30 minutes and washed. The membrane was placed on a slide glass and fixed with a Permount solution, and the cell migration ability was observed, photographed and calculated with an optical microscope DM3000 (Leica). In addition, in order to analyze the migration change of cells through Ibidi migration inserts, 70 μl of a medium containing 2×10 5 cells/ml of ovarian cancer cells was cultured in inserts, and starvation was maintained for 24 hours. After removing the inserts and treating with chrysophanol, the average length between cell groups was measured.
통계분석Statistical analysis
본 실험결과는 SAS (statistical analysis system) 통계프로그램을 이용하여 평균과 표준오차를 계산하였고, 일원배치분산분석 (one-way ANOVA)을 실시하였다. P < 0.05 수준에서 유의성 검정을 실시하였다. The mean and standard error were calculated using SAS (statistical analysis system) statistical program, and one-way ANOVA was performed. A significance test was conducted at the P <0.05 level.
결과 및 고찰Results and Discussion
크리소파놀에To chrysophanol 의한 난소암 및 유방암 세포의 증식 억제 및 사멸 유도 효과 Inhibition of proliferation and death of ovarian cancer and breast cancer cells
난소암 세포주인 ES2 세포와 OVCAR3 세포의 증식력에 크리소파놀이 영향을 미치는지 알아보기 위해 크리소파놀을 용량의존적(0, 5, 10, 20, 50, 100 μM)으로 처리한 후 BrdU ELISA assay를 수행하였다. 그 결과, 크리소파놀을 처리한 실험군은 DMSO를 처리한 대조군에 비해 용량의존적으로 세포 증식이 감소하는 경향을 보였다 (도 1A). 100 μM의 크리소파놀은 ES2 세포와 OVCAR3 세포의 증식력을 각각 52% (p < 0.001), 55% (p < 0.001) 감소시켰다. 또한 크리소파놀이 유방암 세포주 BT-474 및 MCF-7 세포 성장에 미치는 영향을 확인하기 위하여 크리소파놀을 용량의존적으로 (0, 5, 10, 20, 50, 100 μM) 처리한 배양액에서 48시간 배양하였으며, 이후 BrdU를 이용하여 세포 증식 정도를 측정하였다. 그 결과, 두 세포주에서 모두 20 μM부터 세포 증식이 유의적으로 감소하는 것을 확인할 수 있었다 (도 1B). 반면에 정상 유선 세포주인 MCF-12A에는 동일한 농도의 크리소파놀을 처리하여도 세포 증식 정도가 유의적으로 감소하지 않음을 확인하였다 (도 1C). 이러한 결과에 따라 100 μM 농도의 크리소파놀을 처리하여 핵 내 증식 마커인 PCNA의 발현률을 유방암 세포주에서 확인하였으며, 용매만 처리한 세포주에 비해 크리소파놀을 처리한 세포주에서 발현이 감소하는 것을 확인하였다 (도 1D). To determine whether chrysophanol affects the proliferation of ovarian cancer cell lines, ES2 cells and OVCAR3 cells, dose-dependent treatment with chrysophanol (0, 5, 10, 20, 50, 100 μM) and then BrdU ELISA assay I did. As a result, the experimental group treated with chrysophanol showed a tendency to decrease cell proliferation in a dose-dependent manner compared to the control group treated with DMSO (FIG. 1A). 100 μM of chrysophanol decreased the proliferation capacity of ES2 cells and OVCAR3 cells by 52% ( p <0.001) and 55% ( p <0.001), respectively. In addition, in order to determine the effect of chrysophanol on the growth of breast cancer cell lines BT-474 and MCF-7 cells, it was cultured for 48 hours in a culture medium treated with chrysophanol dose-dependently (0, 5, 10, 20, 50, 100 μM). Then, the degree of cell proliferation was measured using BrdU. As a result, it was confirmed that the cell proliferation significantly decreased from 20 μM in both cell lines (Fig. 1B). On the other hand, it was confirmed that the normal mammary cell line MCF-12A did not significantly decrease the degree of cell proliferation even when treated with the same concentration of chrysophanol (FIG. 1C). According to these results, the expression rate of PCNA, an intranuclear proliferation marker, was confirmed in breast cancer cell lines by treatment with 100 μM concentration of chrysophanol, and it was confirmed that the expression decreased in the cell line treated with chrysophanol compared to the cell line treated with only solvent. (Fig. 1D).
다음으로, 크리소파놀이 난소암 세포의 사멸을 유도할 수 있는지 알아보기 위해 ES2 세포 및 OVCAR3 세포에 FITC가 결합된 Annexin V 및 PI 염색을 수행하였다. 그 결과, 크리소파놀은 용량의존적으로 ES2와 OVCAR3 세포의 사멸을 유도하였다 (도 2A). ES2 세포에 100 μM의 크리소파놀 처리시 Annexin V와 PI가 모두 염색된 세포의 비율은 대조군에 비해 약 8배 (p < 0.001) 증가하였다. 마찬가지로 100 μM의 크리소파놀은 OVACR3 세포의 사멸을 약 11배 (p < 0.001) 증가하였다. 이러한 결과는 크리소파놀이 난소암 세포의 증식력을 억제하며 항암효과를 지닐 수 있음을 암시한다. 또한 크리소파놀이 유방암 세포주 BT-474 및 MCF-7 세포 사멸에 미치는 영향을 확인하기 위하여, 크리소파놀을 용량 의존적으로 (0, 10, 20, 50, 100 μM) 처리한 배양액을 48시간 처리한 후 Annexin V & PI 염색을 통해 사멸한 세포의 수를 확인하였다. 그 결과 두 세포주에서 모두 크리소파놀에 의해 세포 사멸이 점진적으로 증가하였다 (도 2B). 반면, 정상 유선 세포주인 MCF-12A세포 에서는 최고 농도의 크리소파놀 (100 μM) 처리시에만 유의적인 세포 사멸을 보였으며, 최저 농도의 크리소파놀 (10 μM)을 처리하였을때는 오히려 세포 사멸이 감소하였다 (도 2C). 이는 크리소파놀이 유방암 세포주의 DNA에 분절화를 유도하여 일어나는 것임을 TUNEL assay를 통하여 확인하였다 (도 2D).Next, to determine whether chrysophanol can induce the death of ovarian cancer cells, FITC-conjugated Annexin V and PI staining were performed on ES2 cells and OVCAR3 cells. As a result, chrysophanol induced the death of ES2 and OVCAR3 cells in a dose-dependent manner (FIG. 2A). When ES2 cells were treated with 100 μM of chrysophanol, the proportion of cells stained with both Annexin V and PI increased about 8 times ( p <0.001) compared to the control group. Similarly, 100 μM of chrysophanol increased the death of OVACR3 cells by about 11 times ( p <0.001). These results suggest that chrysophanol may inhibit the proliferation of ovarian cancer cells and have anticancer effects. In addition, in order to confirm the effect of chrysophanol on the apoptosis of breast cancer cell lines BT-474 and MCF-7, a culture medium treated with chrysophanol dose-dependently (0, 10, 20, 50, 100 μM) was treated for 48 hours. Afterwards, the number of dead cells was confirmed through Annexin V & PI staining. As a result, cell death was gradually increased by chrysophanol in both cell lines (FIG. 2B ). On the other hand, in MCF-12A cells, which is a normal mammary cell line, significant apoptosis was shown only when the highest concentration of chrysophanol (100 μM) was treated. Decreased (Fig. 2C). It was confirmed through the TUNEL assay that chrysophanol occurs by inducing segmentation in the DNA of the breast cancer cell line (Fig. 2D).
크리소파놀에To chrysophanol 의한 난소암 세포 내 산화 스트레스 유도 효과 Inducing oxidative stress in ovarian cancer cells
크리소파놀이 난소암 세포 내에서 산화스트레스를 유도할 수 있는지 알아보기 위해 DCF 녹색 형광의 강도를 측정하였다 (도 3A). ES2와 OVCAR3 세포는 크리소파놀 (0, 20, 50, 100 μM)이 포함된 배지에서 2시간 동안 배양되었다. 또한 양성 대조군으로는 0.03% 과산화수소가 사용되었다. ES2 세포 내 ROS의 생성은 크리소파놀에 의해 용량의존적으로 증가하였으며 최고 농도에서 약 24배 (p < 0.001)까지 증가하였다. 하지만 OVCAR3 세포에서는 크리소파놀에 의해 ROS의 생성이 유의적으로 증가하지는 않았다. 세포 내 ROS 생성에 의한 하위 생리학적 현상인 지질과산화 역시 비슷한 경향을 보였다 (도 3B). 크리소파놀은 OVCAR3 세포에서는 지질과산화 정도를 변화시키지 못했지만 ES2 세포에서는 대조군에 비해 약 12배 (p < 0.001) 가량 지질과산화를 증가시켰다. 뿐만 아니라, 크리소파놀에 의해 BT-474 및 MCF-7에서 모두 활성산소가 증가하였으며 (도 3C) 특히 세포질에서의 지질 인산화가 관찰되었다 (도 3D).To determine whether chrysophanol can induce oxidative stress in ovarian cancer cells, the intensity of DCF green fluorescence was measured (FIG. 3A). ES2 and OVCAR3 cells were cultured for 2 hours in a medium containing chrysophanol (0, 20, 50, 100 μM). In addition, 0.03% hydrogen peroxide was used as a positive control. The production of ROS in ES2 cells was dose-dependently increased by chrysophanol and increased by about 24 times ( p <0.001) at the highest concentration. However, in OVCAR3 cells, the production of ROS by chrysophanol was not significantly increased. Lipid peroxidation, a sub-physiological phenomenon caused by intracellular ROS production, also showed a similar tendency (FIG. 3B). Chrysophanol did not change the degree of lipid peroxidation in OVCAR3 cells, but increased lipid peroxidation in ES2 cells by about 12 times (p <0.001) compared to the control. In addition, free radicals were increased in both BT-474 and MCF-7 by chrysophanol (FIG. 3C), and lipid phosphorylation in the cytoplasm was observed (FIG. 3D).
크리소파놀에To chrysophanol 의한 난소암 세포 내 미토콘드리아 기능 장애 유도 효과 Induction of mitochondrial dysfunction in ovarian cancer cells
미토콘드리아로의 과도한 칼슘 이온 유입에 따른 기능 장애는 세포 사멸로 이어지는 대표적인 생리학적 기전이다. 따라서 크리소파놀에 의해 난소암 세포 안과 미토콘드리아 특이적 칼슘 이온 농도 변화를 측정하기 위해 각각 fluo-4와 rhod-2 염색을 수행하였다. 그 결과, 크리소파놀 (0, 20, 50, 100 μM)은 ES2 세포와 OVCAR3 세포 내 칼슘 이온 농도에 큰 영향을 미치지 않는 것으로 확인되었다 (도 4A). 하지만 rhod-2로 염색된 미토콘드리아 특이적 칼슘 이온의 농도는 크리소파놀 처리에 따라 용량의존적으로 증가하였다 (도 4B). 100 μM의 크리소파놀에 의해 ES2 세포와 OVCAR3 세포 내에서 미토콘드리아 특이적 칼슘 이온은 각각 8.5배 (p < 0.001), 2.5배 (p < 0.001) 증가하였다. 다음으로 크리소파놀에 의한 미토콘드리아 막전위 변화 양상을 확인하기 위해 크리소파놀을 용량의존적 (0, 20, 50, 100 μM)으로 처리한 후 JC-1 염색을 수행하였다. 그 결과, 크리소파놀에 의해 난소암 세포주 내 미토콘드리아 막 전위가 감소하는 것으로 나타났다 (도 4C). 100 μM의 크리소파놀은 ES2와 OVCAR3 세포 내 미토콘드리아 막전위를 대조군에 비해 각각 4.2배 (p < 0.001), 2.7배 (p < 0.001) 감소시켰다. 이러한 결과는 크리소파놀은 난소암 세포 내에서 미토콘드리아 내 칼슘 이온 유입 증가에 따른 기능 장애를 유발할 수 있음을 암시한다. 뿐만 아니라, JC-1 염색을 통해 유방암 세포주 내 크리소파놀 처리에 의해, 마이토콘드리아 막 전위가 감소함을 확인하였다 (도 4D). 또한, BT-474, MCF-7 세포에서 모두 크리소파놀의 용량의존적 처리에 따라 세포질 내 칼슘 이온이 증가하는 것을 확인하였다 (도 4E). 이러한 크라이소파놀의 미토콘드리아에 대한 작용은 미토콘드리아 의존성 세포 사멸 단백질인 Bax, Bak, cytochrome c의 발현을 증가시키는 것 또한 확인할 수 있었다 (도 4F).Dysfunction caused by excessive calcium ion influx into mitochondria is a representative physiological mechanism leading to cell death. Therefore, fluo-4 and rhod-2 staining were performed, respectively, in order to measure changes in the concentration of intraocular and mitochondrial-specific calcium ions by chrysophanol. As a result, it was confirmed that chrysophanol (0, 20, 50, 100 μM) did not significantly affect the calcium ion concentration in ES2 cells and OVCAR3 cells (Fig. 4A). However, the concentration of mitochondrial-specific calcium ions stained with rhod-2 increased dose-dependently with chrysophanol treatment (FIG. 4B). By 100 μM of chrysophanol, mitochondrial-specific calcium ions increased 8.5-fold ( p <0.001) and 2.5-fold ( p <0.001), respectively, in ES2 cells and OVCAR3 cells. Next, JC-1 staining was performed after treatment with chrysophanol dose-dependently (0, 20, 50, 100 μM) in order to confirm the change of mitochondrial membrane potential by chrysophanol. As a result, it was found that the mitochondrial membrane potential in the ovarian cancer cell line was decreased by chrysophanol (Fig. 4C). 100 μM of chrysophanol reduced mitochondrial membrane potential in ES2 and OVCAR3 cells by 4.2 times ( p <0.001) and 2.7 times ( p <0.001), respectively, compared to the control group. These results suggest that chrysophanol may induce dysfunction due to increased influx of calcium ions into mitochondria in ovarian cancer cells. In addition, it was confirmed that mitochondrial membrane potential was reduced by treatment with chrysophanol in breast cancer cell lines through JC-1 staining (FIG. 4D). In addition, it was confirmed that calcium ions in the cytoplasm increased according to the dose-dependent treatment of chrysophanol in both BT-474 and MCF-7 cells (Fig. 4E). It was also confirmed that the action of this chrysophanol on mitochondria increases the expression of mitochondrial-dependent cell death proteins Bax, Bak, and cytochrome c (FIG. 4F).
크리소파놀과Chrysophanol and 신호전달기전 억제제를 통한 난소암 세포의 증식 변화 양상 분석 Analysis of changes in proliferation of ovarian cancer cells through signal transduction mechanism inhibitors
크리소파놀이 난소암 세포의 생존 및 증식에 관여하는 대표적인 신호전달 경로인 MAPK 및 PI3K/AKT 신호전달경로의 활성화에 미치는 영향을 분석하기 위해 크리소파놀은 시간의존적(0, 5, 15, 30, 60분)으로 처리한 후 신호전달경로 단백질의 인산화를 웨스턴블롯을 통해 확인하였다 (도 5A-5F). 그 결과, MAPK 신호전달 단백질인 ERK1/2의 경우 크리소파놀을 5분 처리 시 인산화가 증가하며 이후 시간이 지나며 감소하는 모습을 보였다. 또한 ERK1/2의 하위 신호전달 단백질인 P90RSK는 이보다 느린 15분(ES2) 또는 60분(OVCAR3)에서 가장 인산화 정도가 높았다. 또다른 MAPK 단백질인 JNK와 그 하위 신호전달 단백질인 c-Jun의 난소암 세포 내 인산화 역시 5분 또는 60분의 크리소파놀 처리 시 가장 높게 나타났다. 또한 AKT의 인산화도 크리소파놀 5분 처리시 가장 높게 나타나며 이후 감소하는 양상을 보였으며 AKT의 하위 신호전달 단백질인 P70S6K 역시 ES2에서는 크리소파놀 5분 처리시 가장 인산화 정도가 높았으며 OVCAR3에서는 60분까지 점진적으로 증가하였다. 뿐만 아니라, 유방암 세포주 BT-474 및 MCF-7에서도 크리소파놀 처리에 의한 세포 내 신호전달기전 단백질의 활성 변화를 확인하기 위하여 웨스턴블롯을 통해 MAPK 및 PI3K/AKT 경로 단백질을 검출하였다 (도 5G-5L). 그 결과, 크리소파놀에 의해 두 세포주에서 ERK1/2의 인산화가 감소하였으며, P38 및 JNK의 인산화는 BT-474에서 감소한 반면 MCF-7에서는 증가하였고, 크리소파놀의 용량 의존적 처리는 AKT, P70S6K, S6의 인산화를 모두 감소시키는 것으로 나타났다. 이러한 결과를 통해, 크리소파놀이 유방암 세포주에서도 세포 신호전달 단백질의 인산화를 조절할 수 있음을 확인하였다.To analyze the effect of chrysophanol on the activation of MAPK and PI3K/AKT signaling pathways, which are representative signaling pathways involved in the survival and proliferation of ovarian cancer cells, chrysophanol was time-dependent (0, 5, 15, 30, 60 minutes), the phosphorylation of the signaling pathway protein was confirmed through Western blot (Figs. 5A-5F). As a result, in the case of ERK1/2, the MAPK signaling protein, phosphorylation increased after 5 minutes treatment with chrysophanol, and then decreased with time. In addition, P90RSK, a sub-signaling protein of ERK1/2, showed the highest degree of phosphorylation at a slower 15 minutes (ES2) or 60 minutes (OVCAR3). The phosphorylation of another MAPK protein, JNK and its sub-signaling protein, c-Jun, in ovarian cancer cells was also highest after 5 or 60 minutes of chrysophanol treatment. In addition, phosphorylation of AKT was highest when treated with chrysophanol for 5 minutes and decreased thereafter.P70S6K, a lower signaling protein of AKT, also showed the highest degree of phosphorylation when treated with chrysophanol for 5 minutes in ES2 and 60 minutes in OVCAR3. Gradually increased to. In addition, in breast cancer cell lines BT-474 and MCF-7, MAPK and PI3K/AKT pathway proteins were detected through Western blot in order to confirm the change in the activity of intracellular signaling mechanisms by chrysophanol treatment (Fig. 5G- 5L). As a result, phosphorylation of ERK1/2 was decreased by chrysophanol in both cell lines, phosphorylation of P38 and JNK decreased in BT-474, whereas increased in MCF-7, and dose-dependent treatment of chrysophanol was AKT, P70S6K. , It has been shown to reduce all of the phosphorylation of S6. Through these results, it was confirmed that chrysophanol can regulate phosphorylation of cell signaling proteins in breast cancer cell lines.
다음으로, PI3K/AKT 및 MAPK 경로 단백질에 대한 활성 억제제와 크리소파놀을 병용 처리하였을 시 난소암 세포 증식력 변화를 야기하는지 알아보기 위해 크리소파놀 (100 μM)과 LY294002 (AKT 억제제, 20 μM), U0126 (ERK1/2 억제제, 20 μM), SP600125 (JNK 억제제, 20 μM), SB203580 (P38 억제제, 20 μM)를 병용처리하였다 (도 6A). 그 결과, ES2 세포에서는 크리소파놀에 의한 항증식 효과가 U0126 처리에 의해서는 더욱 강화되었으며 (p < 0.05) SB203580 처리에 의해서는 완화되었다 (p < 0.05). OVCAR3 세포에서는 크리소파놀에 의해 감소한 증식력이 모든 억제제와의 병용처리에서 더욱 감소하는 양상을 보였다. 뿐만 아니라, 유방암 세포주 내 크리소파놀 처리에 의해 조절되는 신호 전달 매커니즘을 분석하기 위하여 크리소파놀과 LY294002, U0126, SB203580, SP600125를 병용 처리하여 세포주들의 증식력 변화를 확인한 결과 (도 6B), BT-474와 MCF-7 유방암 세포주 모두 LY294002를 크리소파놀과 병용처리 하였을 때, 세포 증식이 추가적으로 억제되었던 반면 SP600125와 SB203580을 병용처리 하였을 때, 세포 증식이 다시 증가하는 것을 확인할 수 있었다. 이러한 결과를 통해, 난소암 및 유방암 세포 내에서 크리소파놀에 의해 조절받는 신호전달경로가 실제로 세포 증식력을 조절할 수 있음을 확인할 수 있었다.Next, to investigate whether the combination treatment with an inhibitor of the activity of PI3K/AKT and MAPK pathway proteins and chrysophanol causes changes in ovarian cancer cell proliferation, chrysophanol (100 μM) and LY294002 (AKT inhibitor, 20 μM) , U0126 (ERK1/2 inhibitor, 20 μM), SP600125 (JNK inhibitor, 20 μM), SB203580 (P38 inhibitor, 20 μM) were co-treated (Fig. 6A). As a result, in ES2 cells, the antiproliferative effect of chrysophanol was further enhanced by U0126 treatment ( p <0.05) and alleviated by SB203580 treatment ( p <0.05). In OVCAR3 cells, the proliferative capacity decreased by chrysophanol was further decreased in combination treatment with all inhibitors. In addition, in order to analyze the signal transduction mechanism regulated by the treatment of chrysophanol in breast cancer cell lines, the results of confirming the change in the proliferative capacity of the cell lines were confirmed by treatment with chrysophanol and LY294002, U0126, SB203580, SP600125 in combination (Fig. 6B), BT- In both 474 and MCF-7 breast cancer cell lines, when LY294002 was treated in combination with chrysophanol, cell proliferation was additionally inhibited, whereas when SP600125 and SB203580 were co-treated, cell proliferation was increased again. Through these results, it was confirmed that the signaling pathway regulated by chrysophanol in ovarian cancer and breast cancer cells can actually regulate cell proliferation.
크리소파놀에To chrysophanol 의한 난소암 세포의 이주 능력 억제 효과 Inhibiting the migration ability of ovarian cancer cells
난소암 세포의 이주 및 침습 능력은 난소암의 전이 정도를 결정하는데 중요한 특성이다. 따라서, 크리소파놀에 의한 난소암 세포의 이주 능력 변화를 측정하기 위해 transwell 과 Ibidi 배양 접시에 ES2 세포와 OVCAR3 세포를 배양하였다. 그 결과, 크리소파놀 100 μM 처리 시 transwell을 통과하는 ES2 및 OVCAR3 세포의 수가 각각 69% (p < 0.001), 52% (p < 0.001) 감소하였다 (도 7A). 또한 크리소파놀에 의해 ibidi plate 내 세포 군집 간의 넓이는 ES2 세포에서 약 2배 (p < 0.01), OVCAR3 세포에서 약 1.5배 (p < 0.05) 만큼 차이를 보였다 (도 7B). 이러한 결과는 크리소파놀이 난소암 세포의 이주 능력을 현저하게 감소시킬 수 있음을 암시한다.The ability of ovarian cancer cells to migrate and invade is an important characteristic in determining the degree of metastasis of ovarian cancer. Therefore, ES2 cells and OVCAR3 cells were cultured in transwells and Ibidi culture dishes to measure changes in the migration ability of ovarian cancer cells by chrysophanol. As a result, when
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시형태일 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.As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereby. something to do. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.
Claims (6)
상기 크리소파놀은 PI3K/AKT 및 MAPK 신호전달기전을 조절하는 것을 특징으로 하는, 난소암 및 유방암 예방 또는 치료용 약학적 조성물.The method of claim 1,
The chrysophanol is characterized in that it regulates PI3K/AKT and MAPK signaling mechanisms, a pharmaceutical composition for preventing or treating ovarian cancer and breast cancer.
PI3K/AKT 및 MAPK 신호전달경로를 억제하는 타겟 억제제를 더 포함하는 것을 특징으로 하는, 난소암 및 유방암 예방 또는 치료용 약학적 조성물.The method of claim 1,
A pharmaceutical composition for the prevention or treatment of ovarian cancer and breast cancer, characterized in that it further comprises a target inhibitor that inhibits the PI3K/AKT and MAPK signaling pathways.
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