KR101911767B1 - Kidamycin derivatives L1-95-1 and composition for preventing or treating cancer comprising the same - Google Patents
Kidamycin derivatives L1-95-1 and composition for preventing or treating cancer comprising the same Download PDFInfo
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- KR101911767B1 KR101911767B1 KR1020170101802A KR20170101802A KR101911767B1 KR 101911767 B1 KR101911767 B1 KR 101911767B1 KR 1020170101802 A KR1020170101802 A KR 1020170101802A KR 20170101802 A KR20170101802 A KR 20170101802A KR 101911767 B1 KR101911767 B1 KR 101911767B1
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- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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Abstract
Description
본 발명은 키다마이신(kidamycin) 유도체 L1-95-1 및 이를 유효성분으로 함유하는 암 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a kidamycin derivative L1-95-1 and a composition for preventing or treating cancer comprising the same as an active ingredient.
암은 유전적 요인과 환경적 요인에 의해 세포의 정상적 세포분열이 조절되지 않아 비정상적인 세포분열을 하게 되고, 또 세포 분열 횟수의 한계를 넘어 죽지 않게 됨으로써 발생한다. 이렇게 형질이 전환된 세포는 계속 증식하여 암 덩어리가 되며, 암 덩어리의 내부 중심부는 산소의 공급과 암세포의 성장을 위한 영양분의 공급이 제한을 받게 된다. 산소의 공급이 제한되면 암세포는 HIF-1 단백질을 발현시켜 혈관 신생에 관여하는 단백질 및 에너지 대사와 관련된 단백질의 발현을 증가시킨다. 그 결과로 암 조직은 다시 성장한다.Cancer is caused by genetic and environmental factors leading to abnormal cell division because the normal cell division of the cells is not regulated, and it does not die beyond the limit of the number of cell divisions. These transformed cells continue to proliferate into cancerous masses, and the inner core of the cancerous mass is limited in the supply of oxygen and the supply of nutrients for the growth of cancer cells. When oxygen supply is limited, cancer cells express the HIF-1 protein and increase the expression of proteins involved in angiogenesis and proteins involved in energy metabolism. As a result, cancer tissue grows again.
일반적으로 유방암은 여성에서 가장 흔한 질병 중 하나이다. 미국의 경우 일년에 약 24만 명의 유방암 환자가 발생하며, 약 4만 명의 환자가 사망하는 것으로 보고되어 있다. 우리나라에서는 최근 식생활의 서구화 및 비만 인구의 증가로 인하여 유방암 환자가 급격히 증가하고 있으며, 일년에 약 1만 8천여명의 환자가 발생하는 것으로 추정된다. 유방암 세포의 전이는 다른 암 종에 비하여 높지 않은 특징을 갖고 있기 때문에 유방암을 조기에 발견하여 적절한 치료가 진행되면 약 93%의 5년 생존율 보일 만큼 완치율이 높은 암 종이다. 하지만 조기 발견과 적절한 치료가 이루어지지 않으면 혈관과 림프관을 통해 암의 전이가 일어나게 되며, 따라서 암에 의한 치사율은 높아진다. 실제로 제4기 유방암의 경우 5년간 생존율은 34%로 전체 유방암 생존율에 비하여 매우 낮다. In general, breast cancer is one of the most common diseases in women. In the United States, there are about 240,000 breast cancer cases a year and about 40,000 deaths are reported. In Korea, the number of breast cancer patients is rapidly increasing due to westernization of dietary habits and increase of obesity population, and it is estimated that about 18,000 patients are born in a year. Because the breast cancer cell metastasis is not as high as other cancer types, it is a cancer type with a high cure rate of about 93% 5 years survival rate when breast cancer is detected early and appropriate treatment is performed. However, if early detection and appropriate treatment are not done, cancer metastasis will occur through the blood vessels and lymphatic vessels, and the cancer mortality rate will increase. In fact, the survival rate for stage 4 stage breast cancer is 34%, which is much lower than the overall survival rate for breast cancer.
유방암을 탁솔(taxol)과 같은 항암제 또는 방사선으로 치료할 경우, 초기 암 조직의 성장이 멈춰지고, 암의 크기가 작아지는 등 치료가 되는 것으로 보이지만, 일정기간이 지나면 다시 암이 자라나오게 되는 경우가 있다. 다시 자라나온 암세포를 대상으로 다시 동일한 항암제를 처리할 경우 항암제가 더 이상 작용하지 않게 되어 내성을 갖게 되며, 암세포의 전이율이 높아지는 등 더욱 악성의 형질을 갖게 된다. 삼중음성 유방암세포는 에스트로겐(estrogen)과 프로게스테론(progesterone) 수용체가 결손 되어 있고(ER-/PR-), HER2가 발현 되지 않은(HER2-) 것으로 알려져 있어, 탁솔(taxol), 타목시펜(tamoxifen), Her 2 활성억제제(trastuzumab)에 내성을 갖는 특징을 갖고 있다. 더욱이 주로 50세 이하의 젊은 여성이나 African-American/Hispanic 여성 중 변이된 BRCA1를 가진 여성에게서 발생 빈도가 높다. 미국에서 전체 유방암 환자 중 삼중음성 유방암 환자가 약 12~17% 정도 차지하는 것으로 알려져 있으며, 한국인에서는 전체 유방암 환자 중 약 15.9% 정도가 삼중음성 유방암을 갖는 것으로 보고되어 있다. 삼중음성 유방암 환자의 5년 생존율은 약 77% 정도로 다른 종류의 유방암 환자에서 보이는 약 93%에 비해 낮은 것으로 보고되어 있다. 또한, 삼중음성 유방암은 다른 종류의 유방암에 비해 조직학적 등급(grade)이 높고 침윤성의 유관암(ductal carcinoma) 형태를 가지는 등 다른 유방암에 비해 공격적인 특징을 가지고 있으며, 다수의 삼중음성 유방암은 호르몬 치료(Hormone therapy) 또는 트라스트주맙(trastzumab) 등에 효과가 없고 염색체 이수성 정도가 높으며(Degree of aneuploidy), 유전자-발현 프로파일(gene-expression profile)이 유사한 점 등 기저-유사 유방암 서브타입(basal-like breast cancer subtype)과 유사한 특징을 가지고 있다.When breast cancer is treated with an anticancer agent such as taxol or radiation, the growth of the early cancer tissue is stopped and the size of the cancer is reduced. However, after a certain period of time, the cancer may grow again . When cancer cells that have regenerated again are treated with the same anticancer drug, the anticancer drug will no longer function, resulting in resistance, and a more malignant trait, such as a higher transfer rate of cancer cells. Triple-negative breast cancer cells are known to be deficient in estrogen and progesterone receptors (ER- / PR-) and not expressed in HER2 (HER2-), suggesting that taxol, tamoxifen, It has the property of being resistant to Her 2 active inhibitor (trastuzumab). Furthermore, it is more prevalent among younger women, mainly younger than 50 years, and women with BRCA1 mutated among African-American / Hispanic women. In the United States, triple negative breast cancer patients account for about 12-17% of all breast cancer patients, and about 15.9% of all breast cancer patients in Korea are reported to have triple negative breast cancer. The 5-year survival rate of triple-negative breast cancer patients is reported to be about 77%, which is lower than the 93% seen in other types of breast cancer patients. Triple-negative breast cancer is more aggressive than other types of breast cancer, such as having a histologic grade and invasive ductal carcinoma, compared with other types of breast cancer, and many triple- Like basal-like breast cancer, which has no effect on hormone therapy or trastzumab, has a high degree of chromosomal aberration (Degree of aneuploidy), similar gene-expression profile, cancer subtype).
삼중음성 유방암을 상대로 한 표적치료의 한계는 목표로 하고자 하는 표적을 찾기가 어렵고, 따라서 이와 관련한 유효한 표적치료가 없다는데 있다. 삼중음성 유방암 환자들에게 화학요법(chemotherapy)이 효과적이나 심장독성, 심부전, 고혈압, 피부병 등의 부작용을 감소시킬 수 있는 치료 개선 방안을 연구할 필요성이 있다.The limitation of target therapy for triple negative breast cancer is that it is difficult to find the target to target and therefore there is no effective target treatment related to it. Chemotherapy is effective for patients with triple-negative breast cancer, but there is a need to study treatment modalities that can reduce side effects such as cardiac toxicity, heart failure, hypertension, and skin disease.
키다마이신(kidamycin) 유도체인 L1-95-1은 Streptomyces sp . W2061로부터 추출한 천연물로서, 그 구조가 최근 본 연구진에 의하여 밝혀졌다. 따라서 L1-95-1에 대한 약리작용과 표적단백질, 그리고 암 예방 및 항암에 대한 효과는 알려진 바가 없다.Kida azithromycin (kidamycin) derivative L1-95-1 is Streptomyces sp . As a natural product extracted from W2061, its structure has been recently discovered by the present inventors. Therefore, the pharmacokinetics and target proteins for L1-95-1, and the effects on cancer prevention and anti-cancer are not known.
본 발명의 목적은 키다마이신(kidamycin) 유도체 L1-95-1 또는 이의 약학적으로 허용가능한 염 및 이를 유효성분으로 함유하는 암 예방, 개선 또는 치료용 약학조성물 또는 건강식품 조성물을 제공하는데 있다. It is an object of the present invention to provide a pharmaceutical composition or a health food composition for preventing, ameliorating or treating cancer, comprising a kidamycin derivative L1-95-1 or a pharmaceutically acceptable salt thereof and an effective ingredient thereof.
본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 제공한다.The present invention provides a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.
[화학식 1][Chemical Formula 1]
또한, 본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 암 예방 또는 치료용 약학조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
또한, 본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 암 예방 또는 개선용 건강식품 조성물을 제공한다.The present invention also provides a health food composition for preventing or ameliorating cancer, which comprises, as an active ingredient, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
본 발명은 키다마이신(kidamycin) 유도체 L1-95-1 및 이를 유효성분으로 함유하는 암 예방 또는 치료용 조성물에 관한 것으로서, L1-95-1은 JB6 Cl41 세포의 세포 증식 및 EGF에 의한 암화를 억제하였고, 유방암 세포 MCf-7 및 삼중음성 유방암 세포 MDA-MB-231 세포를 이용한 실험을 통하여 L1-95-1은 MDA-MB-231 세포에서 선택적으로 미토파지(mitophagy)를 유도하여 암세포의 증식 및 암 성장을 억제한다는 사실을 밝혀냈다. 이에, L1-95-1은 암의 치료를 위한 항암제 또는 암 개선용 건강식품으로써 활용가능성이 클 것으로 판단된다.The present invention relates to a kidamycin derivative L1-95-1 and a composition for preventing or treating cancer comprising the same as an active ingredient. L1-95-1 inhibits cell proliferation and EGF-induced cancer of JB6 Cl41 cells. L1-95-1 selectively induced mitophagy in MDA-MB-231 cells, and the proliferation and proliferation of cancer cells were suppressed by MDA-MB-231 cells. Cancer growth. Therefore, L1-95-1 may be used as an anticancer agent for the treatment of cancer or as a health food for cancer improvement.
도 1은 L1-95-1이 500 nM 이상에서 JB6Cl41의 세포증식과 EGF에 의한 형질전환을 억제한다는 결과를 나타낸다. (A) L1-95-1의 화학구조이다. (B) 마우스 피부 상피세포 JB6 Cl41 (1 × 103 cells)을 96-well 세포 배양 plate에 접종하고 세포증식을 24시간 간격으로 72시간 동안 MTS assay를 이용하여 측정하였다. 데이터 값은 4회의 측정치에 대한 평균값과 표준편차로 나타냈으며 통계적 유의도 측정을 위하여 Student's t-test를 사용하였다(* P < 0.05). (C) 마우스의 상피세포주 JB6 Cl41(8 × 103 cells)을 0.3% agar가 포함되어있고 10% FBS가 첨가된 BME 배양액에 섞어 현탁액을 만들고 6개 실험군 중 5개 실험군에 EGF (10 ng/ml)을 처리하였으며 EGF를 처리하지 않은 실험군에는 DMSO를 처리하고 EGF를 처리한 실험군에 각각에 DMSO, 0.05, 0.25, 0.5, 1.25 μM의 L1-95-1을 처리하였다. 세포는 37℃, 5% CO2 항온배양기에서 20일간 배양하였으며 형질전환 세포의 측정은 ECLIPSE Ti inverted microscope using the NIS-Elements AR (V. 4.0) computer software program을 이용하였다. 데이터 값은 3회의 측정치에 대한 평균값과 표준편차로 나타냈으며 통계적 유의도 측정을 위하여 Student's t-test를 사용하였다(* P < 0.05).
도 2는 L1-95-1이 사람 삼중음성 유방암 세포 MDA-MB-231에 특이적으로 세포증식억제 효과가 있으며 세포주기에 영향을 준다는 결과를 나타낸다. (A) 사람 유방암 세포 MCF-7 (2 × 103 cells)과 사람 삼중음성 유방암 세포 MDA-MB-231 (2 × 103 cells) 세포를 96-well 세포 배양 plate에 접종하고 L1-95-1을 농도 별로 처리한 후 세포증식을 24시간 간격으로 96시간 동안 MTS assay를 이용하여 측정하였다. 데이터 값은 4회의 측정치에 대한 평균값과 표준편차로 나타냈으며 통계적 유의도 측정을 위하여 Student's t-test를 사용하였다(* P < 0.05). (B) 사람 유방암 세포 MCF-7 (5 × 105 cells)과 사람 삼중음성 유방암 세포 MDA-MB-231(5 × 105 cells)을 60-mm dish에 접종하고 밤새 배양하였다. 세포 각각에 500 nM의 L1-95-1을 48시간 동안 처리한다. 세포들을 회수하고 70% 에탄올/1× PBS로 -20℃에서 2시간 동안 고정시킨다. 고정된 세포를 PBS로 wash하고 RNase A (200 μg/ml) 와 propidium iodide (20 μg/ml)를 15분간 처리하였다. 세포주기는 유세포 분석(flow cytometry)을 통하여 측정하였다. 데이터 값은 3회의 측정치에 대한 평균값과 표준편차로 나타냈으며 통계적 유의도 측정을 위하여 Student's t-test를 사용하였다(* P < 0.05). (C) 사람 유방암 세포 MCF-7 (5 × 105 cells)과 사람 삼중음성 유방암 세포 MDA-MB-231 (5 × 105 cells)을 60-mm dish에 접종하고 밤새 배양하였다. 세포 각각에 500 nM의 L1-95-1을 48시간 동안 처리하였다. 세포를 회수 70% 에탄올/1× PBS로 -20℃에서 2시간 동안 고정시켰다. 고정된 세포를 PBS로 wash하고 RNase A (200 μg/ml) 와 propidium iodide (20 μg/ml)를 15분간 처리하였다. Sub-G1은 유세포 분석(flow cytometry)을 통하여 측정하였다. 데이터 값은 3회의 측정치에 대한 평균값과 표준편차로 나타냈으며 통계적 유의도 측정을 위하여 Student's t-test를 사용하였다(* P < 0.05).
도 3은 L1-95-1이 MDA-MB-231 세포에서 오토파지(autophagy)를 유도한다는 결과를 나타낸다. (A) 사람 유방암 세포 MCF-7 (3 × 106 cells)과 사람 삼중음성 유방암 세포 MDA-MB-231 (3 × 106 cells)을 100-mm dish에 접종하고 밤샘 배양하였다. 세포 각각에 DMSO 및 500 nM의 L1-95-1을 처리하고 세포의 형태 변화를 24시간 간격으로 48시간 동안 광학 현미경을 이용하여 확인하였다. (B) 사람 유방암 세포 MCF-7(3 × 106)과 사람 삼중음성 유방암 세포 MDA-MB-231(3 × 106)을 100-mm dish에 접종하고 밤샘 배양하였다. 세포 각각에 DMSO, 100, 250, 500 nM의 L1-95-1을 처리하고 24시간 동안 37℃, 5% CO2 항온 배양기에서 배양하였다. 세포들을 회수하고 단백질을 추출한 후 LC3 항체를 이용하여 Western blotting을 수행하였다. β-actin은 각각 실험군 단백질의 양이 동량인지를 확인하기 위하여 내부 대조군(internal control)으로 사용하였다. (C) 사람 유방암 세포 MCF-7(2 × 104)및 사람 삼중음성 유방암 세포 MDA-MB-231(2 × 104)을 8-chamber slide에 접종하고 37℃, 5% CO2 항온배양기에서 밤샘 배양하였다. Chamber 각각에 DMSO 및 L1-95-1을 처리하고 24시간 동안 37℃, 5% CO2 항온배양기에서 배양하였다. Lysotracker를 1시간 동안 처리하고 Hoechst를 30분간 처리하였으며, ECLIPSE Ti inverted microscope와 the NIS-Elements AR (V.4.0) computer software program을 이용하여 형광을 관찰하였다. (D) 사람 유방암 세포 MCF-7(2 × 104) 및 사람 삼중음성 유방암 세포 MDA-MB-231(2 × 104)을 8-chamber slide에 접종하고 37℃, 5% CO2 항온배양기에서 밤샘 배양하였다. 각 세포에 각각에 mCherry plasmid를 도입하고 30시간 동안 37℃, 5% CO2 항온 배양기에서 배양하였다. Chamber 각각에 DMSO 및 L1-95-1 500 nM을 처리하였고 24시간 동안 37℃, 5% CO2 항온 배양기에서 배양하였으며, ECLIPSE Ti inverted microscope와 the NIS-Elements AR (V.4.0) computer software program을 이용하여 형광을 관찰하였다.
도 4는 L1-95-1이 MDA-MB-231 세포에서 선택적으로 미토파지(mitophagy)를 유도한다는 결과를 나타낸다. (A) 사람 유방암 세포 MCF-7 (3 × 106 cells)과 사람 삼중음성 유방암 세포 MDA-MB-231 (3 × 106 cells)을 100-mm dish에 접종하고 24시간 동안 37℃, 5% CO2 항온 배양기에서 밤샘 배양하였다. 세포 각각에 DMSO, 100, 250, 500 nM의 L1-95-1을 처리하고 3시간 동안 37℃, 5% CO2 항온 배양기에서 배양하였다. 세포들을 회수하고 단백질을 추출하였으며, 결과에 표기된 항체들을 이용하여 Western blotting을 수행하였다. β-actin은 각각 실험군 단백질의 양이 동량인지를 확인하기 위하여 사용하였다. (B) 사람 유방암 세포 MCF-7(3 × 106)과 사람 삼중음성 유방암 세포 MDA-MB-231(3 × 106)을 100-mm dish에 접종하고 24시간 동안 37℃, 5% CO2 항온 배양기에서 밤샘 배양하였다. 세포 각각에 DMSO, 100, 250, 500 nM의 L1-95-1을 처리하고 3시간 동안 37℃, 5% CO2 항온 배양기에서 배양하였다. 세포들을 회수하고 단백질을 결과에 표기된 항체들을 이용하여 Western blotting을 수행하였다. β-actin은 각각 실험군 단백질의 양이 동량인지를 확인하기 위하여 사용하였다. (C) 사람 유방암 세포 MCF-7(3 × 106)과 사람 삼중음성 유방암 세포 MDA-MB-231(3 × 106)을 100-mm dish에 접종하고 24시간 동안 37℃, 5% CO2 항온 배양기에서 밤새 배양하였다. 세포 각각에 DMSO, L1-95-1 500 nM을 처리 후, 24시간 동안 37℃, 5% CO2 항온 배양기에서 배양하였다. 세포들을 회수하고 단백질을 추출하여 phospho- 및 toal-AMPKα 항체를 이용하여 Western blotting을 수행하였다. β-actin은 각각 실험군 단백질의 양이 동량인지를 확인하기 위하여 사용하였다. (D) 사람 유방암 세포 MCF-7(2 × 104)및 사람 삼중음성 유방암 세포 MDA-MB-231(2 × 104)을 confocal dish에 접종하였고 5% CO2, 37℃ 항온 배양기에서 배양하였다. 각각의 세포에 DMSO 및 L1-95-1 500 nM, 1 mM 농도로 처리하고 24시간 동안 5% CO2, 37℃ 항온배양기에서 배양하였다. Lysotracker를 30 nM의 농도로 1시간 동안 처리하고 Mitotracker를 20 nM의 농도로 45분간 처리하였으며 Hoechst (1 ㎍/ml)를 30분간 처리하였다. 각각의 실험군은 LSM 710 laser scanning confocal microscope로 형광을 관찰하였다. 형광 Z-stack image를 LSM 710 laser scanning confocal microscope 의 3D image 프로그램을 이용하여 분석하였다.
도 5는 L1-95-1이 MDA-MB-231의 미토콘드리아에서 ROS 발생을 증가시킨다는 결과를 나타낸다. (A) 사람 유방암 세포 MCF-7 (2 × 104 cells)과 사람 삼중음성 유방암 세포 MDA-MB-231 (2 × 104 cells)을 confocal dish에 접종하고 5% CO2, 37℃ 항온 배양기에서 배양하였다. 각각의 세포에 DMSO 및 L1-95-1을 500 nM 또는 1 mM 농도로 처리하고 24시간 동안 5% CO2, 37℃ 항온 배양기에서 배양하였다. Mitosox™ Red를 5 μM 농도로 10분간 처리하고, 10분 후 각각의 실험군은 LSM 710 laser scanning confocal microscope를 이용하여 형광을 관찰하였다. (B) 사람 유방암 세포 MCF-7 (2 × 104 cells)과 사람 삼중음성 유방암 세포 MDA-MB-231 (2 × 104 cells)을 confocal dish 에 접종하고 5% CO2, 37℃ 항온 배양기에서 배양하였다. 각각의 세포에 DMSO 및 L1-95-1을 500 nM, 1 mM 농도로 처리하고 24시간 동안 5% CO2, 37℃ 항온 배양기에서 배양하였다. 24시간 후 MitoProbe™ JC-1을 2 μM의 농도로 15분간 처리하였으며, LSM 710 laser scanning confocal microscope로 형광을 관찰하였다. (C) 사람 유방암 세포 MCF-7 (8 × 103 cells) 및 삼중음성 유방암 세포 MDA-MB-231 (8 × 103 cells) 세포를 이용하여 표시된 농도의 L1-95-1이 포함된 soft agar와 혼합하여 동일한 L1-95-1이 포함된 bottom agar 위에서 굳히고, 5% CO2, 37℃ 항온 배양기에서 3주간 배양하였다. 성장한 콜로니(colony)는 ECLIPSE Ti inverted microscope와 the NIS-Elements AR (V.4.0) computer software program (NIKON Instruments Korea, Gangnam, Seoul, Korea)를 이용하여 분석하였다.Figure 1 shows that L1-95-1 inhibits cell proliferation of JB6Cl41 and transfection by EGF at 500 nM or more. (A) It is the chemical structure of L1-95-1. (B) Mouse skin epithelial cells JB6 Cl41 (1 × 10 3 cells) were inoculated on a 96-well cell culture plate and cell proliferation was measured by MTS assay for 72 hours at 24-hour intervals. Data were expressed as means and standard deviations for 4 measurements and Student's t- test was used for statistical significance (* P <0.05). (C) The epithelial cell line JB6 Cl41 (8 × 10 3 cells) of the mouse was mixed with BME culture medium containing 0.3% agar and 10% FBS to prepare a suspension. EGF (10 ng / ml), and in the experimental group without DMSO treatment, DMSO, 0.05, 0.25, 0.5, and 1.25 μM of L1-95-1 were administered to each experimental group treated with EGF. Cells were cultured in a 5% CO 2 incubator at 37 ° C for 20 days. Transfection cells were counted using an ECLIPSE Ti inverted microscope using the NIS-Elements AR (V. 4.0) computer software program. Data values were expressed as means and standard deviations for three measurements and Student's t- test was used for statistical significance (* P <0.05).
FIG. 2 shows that L1-95-1 specifically inhibits cell proliferation and affects the cell cycle of MDA-MB-231, a human triphasic breast cancer cell. (A) Human breast cancer cells MCF-7 (2 × 10 3 cells) and human triple-negative breast cancer cells MDA-MB-231 (2 × 10 3 cells) And the cell proliferation was measured by MTS assay for 96 hours at intervals of 24 hours. Data were expressed as means and standard deviations for 4 measurements and Student's t- test was used for statistical significance (* P <0.05). (B) Human breast cancer cells MCF-7 (5 × 10 5 cells) and human triple-negative breast cancer cells MDA-MB-231 (5 × 10 5 cells) were inoculated into a 60-mm dish and cultured overnight. Each of the cells is treated with 500 nM of L1-95-1 for 48 hours. The cells are harvested and fixed in 70% ethanol / 1 x PBS at -20 ° C for 2 hours. Fixed cells were washed with PBS and treated with RNase A (200 μg / ml) and propidium iodide (20 μg / ml) for 15 minutes. The cell cycle was measured by flow cytometry. Data values were expressed as means and standard deviations for three measurements and Student's t- test was used for statistical significance (* P <0.05). (C) Human breast cancer cells MCF-7 (5 × 10 5 cells) and human triple-negative breast cancer cells MDA-MB-231 (5 × 10 5 cells) were inoculated into a 60-mm dish and cultured overnight. Each of the cells was treated with 500 nM of L1-95-1 for 48 hours. Cells were fixed for 2 h at -20 < 0 > C with 70% ethanol / 1 x PBS. Fixed cells were washed with PBS and treated with RNase A (200 μg / ml) and propidium iodide (20 μg / ml) for 15 minutes. Sub-G1 was measured by flow cytometry. Data values were expressed as means and standard deviations for three measurements and Student's t- test was used for statistical significance (* P <0.05).
Figure 3 shows that L1-95-1 induces autophagy in MDA-MB-231 cells. (A) Human breast cancer cells MCF-7 (3 × 10 6 cells) and human triple-negative breast cancer cells MDA-MB-231 (3 × 10 6 cells) were inoculated into a 100-mm dish and cultured overnight. Each of the cells was treated with DMSO and 500 nM of L1-95-1, and morphological changes of the cells were confirmed by optical microscope for 48 hours at 24 hour intervals. (B) Human breast cancer cells MCF-7 (3 × 10 6 ) and human triple-negative breast cancer cells MDA-MB-231 (3 × 10 6 ) were inoculated into 100-mm dishes and cultured overnight. Cells were treated with DMSO, 100, 250, 500 nM of L1-95-1 and cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. Cells were recovered and protein was extracted and Western blotting was performed using LC3 antibody. β-actin was used as an internal control to confirm the amount of each protein in the experimental group. (C) Human breast cancer cells MCF-7 (2 × 10 4 ) and human triple-negative breast cancer cells MDA-MB-231 (2 × 10 4 ) were inoculated on an 8-chamber slide and incubated at 37 ° C in a 5% CO 2 incubator And cultured overnight. Each of the chambers was treated with DMSO and L1-95-1 and cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. Lysotracker was treated for 1 hour and Hoechst was treated for 30 minutes. Fluorescence was observed using an ECLIPSE Ti inverted microscope and the NIS-Elements AR (V.4.0) computer software program. (D) Human breast cancer cells MCF-7 (2 × 10 4 ) and human triple-negative breast cancer cells MDA-MB-231 (2 × 10 4 ) were inoculated into 8-chamber slides and incubated at 37 ° C in a 5% CO 2 incubator And cultured overnight. MCherry plasmids were introduced into each cell and cultured in a 5% CO 2 incubator at 37 ° C for 30 hours. The cells were treated with DMSO and L1-95-1 500 nM for 24 h at 37 ° C in a 5% CO 2 incubator. ECLIPSE Ti inverted microscope and the NIS-Elements AR (V.4.0) computer software program And fluorescence was observed.
Figure 4 shows that L1-95-1 selectively induces mitophagy in MDA-MB-231 cells. (A) Human breast cancer cells MCF-7 (3 × 10 6 cells) and human triple-negative breast cancer cells MDA-MB-231 (3 × 10 6 cells) were inoculated into 100- CO 2 incubator overnight. Cells were treated with DMSO, 100, 250, 500 nM of L1-95-1 and cultured in a 5% CO 2 incubator at 37 ° C for 3 hours. Cells were collected, proteins were extracted, and Western blotting was performed using the antibodies indicated in the results. β-actin was used to confirm the amount of protein in the experimental group. (B) human breast cancer cell MCF-7 (3 × 10 6 ) and human triple negative breast cancer cells MDA-MB-231 (3 × 10 6) inoculated and 37 ℃ for 24 hours in 100-mm dish, 5
Figure 5 shows that L1-95-1 increases ROS development in the mitochondria of MDA-MB-231. (A) Human breast cancer cells MCF-7 (2 × 10 4 cells) and human triple-negative breast cancer cells MDA-MB-231 (2 × 10 4 cells) were inoculated into confocal dishes and incubated in a 5% CO 2 , 37 ° C incubator Lt; / RTI > Each cell was treated with DMSO and L1-95-1 at 500 nM or 1 mM and cultured in a 5% CO 2 , 37 ° C incubator for 24 hours. Mitosox ™ Red was treated for 10 minutes at a concentration of 5 μM, and 10 minutes later, fluorescence was observed using LSM 710 laser scanning confocal microscope. (B) Human breast cancer cells MCF-7 (2 × 10 4 cells) and human triple-negative breast cancer cells MDA-MB-231 (2 × 10 4 cells) were inoculated into confocal dishes and incubated in a 5% CO 2 , 37 ° C incubator Lt; / RTI > Each cell was treated with 500 nM, 1 mM DMSO and L1-95-1 and cultured in a 5% CO 2 , 37 ° C incubator for 24 hours. Twenty-four hours later, MitoProbe ™ JC-1 was treated at a concentration of 2 μM for 15 minutes. Fluorescence was observed with a LSM 710 laser scanning confocal microscope. (C) human breast cancer cell MCF-7 (8 × 10 3 cells) and a triple negative breast cancer cells MDA-MB-231 (8 × 10 3 cells) The soft agar containing the indicated concentration of L1-95-1 using a cell Were mixed on a bottom agar containing the same L1-95-1, and cultured in a 5% CO 2 , 37 ° C incubator for 3 weeks. Growth colonies were analyzed using an ECLIPSE Ti inverted microscope and the NIS-Elements AR (V.4.0) computer software program (NIKON Instruments Korea, Gangnam, Seoul, Korea).
본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 제공한다.The present invention provides a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.
[화학식 1][Chemical Formula 1]
본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 암 예방 또는 치료용 약학조성물을 제공한다. The present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
상세하게는, 상기 약학조성물은 상피성장인자(epidermal growth factor; EGF)에 의해 유도되는 암세포로의 형질전환을 억제할 수 있고, 암세포에서 G1 세포주기는 감소시키고, S 세포주기는 증가시킬 수 있다. Specifically, the pharmaceutical composition can inhibit the transformation into cancer cells induced by epidermal growth factor (EGF), reduce the G1 cell cycle and increase the S cell cycle in cancer cells .
상세하게는, 상기 약학조성물은 암세포에서 오토파지(autophagy)를 유도할 수 있으며, 특히 미토파지(mitophagy)를 유도할 수도 있다.In particular, the pharmaceutical composition may induce autophagy in cancer cells, and in particular may induce mitophagy.
상세하게는, 상기 약학조성물은 암세포의 미토콘드리아에서 ROS(Reactive oxygen species) 발생을 유도할 수 있다.In detail, the pharmaceutical composition can induce ROS (Reactive oxygen species) generation in the mitochondria of cancer cells.
본 발명에서 언급되는 "L1-95-1"은 키다마이신의 유도체로서, 화학식 1로 표시되는 화합물이다. "L1-95-1" referred to in the present invention is a derivative of kidamycin, which is a compound represented by the formula (1).
< 화학식 1 >≪
한편, 상기 암은 폐암, 대장암, 간암, 위암, 식도암, 췌장암, 담낭암, 신장암, 방광암, 전립선암, 고환암, 자궁경부암, 자궁내막암, 융모암, 난소암, 유방암, 갑상선암, 뇌암, 두경부암, 악성흑색종, 림프종 및 혈액암로 이루어진 군에서 선택될 수 있으며, 바람직하게는 본 발명의 일실시예에서 기재한 바와 같이 유방암일 수 있으며, 가장 바람직하게는 삼중음성 유방암일 수 있다. The cancer may be selected from the group consisting of lung cancer, colon cancer, liver cancer, stomach cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, Cancer of the head, neck cancer, malignant melanoma, lymphoma, and blood cancer, and may be preferably breast cancer as described in one embodiment of the present invention, and most preferably triple negative breast cancer.
본 명세서에 있어서, "삼중음성 유방암"은 에스트로겐(estrogen) 및 프로게스테론(progesterone) 수용체가 결손되고(ER-/PR-), HER2 유전자가 발현되지 않는 것으로 알려져 있어, 탁솔(taxol), 타목시펜(tamoxifen), Her 2 활성억제제(trastuzumab)에 내성을 갖는 특징을 갖고 있다. 미국에서 전체 유방암 환자 중 삼중음성 유방암 환자가 약 12~17% 정도 차지하는 것으로 알려져 있으며, 한국인에서는 전체 유방암 환자 중 약 15.9% 정도가 삼중음성유방암을 갖는 것으로 보고되어 있다. 삼중음성 유방암 환자의 5년 생존율은 약 77% 정도로 다른 종류의 유방암 환자에서 보이는 약 93%에 비해 낮은 것으로 보고되어 있다.As used herein, " triple-negative breast cancer " is known to be deficient in the estrogen and progesterone receptors (ER- / PR-) and not in the expression of the HER2 gene, and taxol, tamoxifen ) And Her 2 activity inhibitor (trastuzumab). In the United States, triple negative breast cancer patients account for about 12-17% of all breast cancer patients, and about 15.9% of all breast cancer patients in Korea are reported to have triple negative breast cancer. The 5-year survival rate of triple-negative breast cancer patients is reported to be about 77%, which is lower than the 93% seen in other types of breast cancer patients.
본 명세서에 있어서, "세포사멸(apoptosis)"는 발생 및 노화, 그리고 조직 내에서 세포의 수를 일정하게 조절하여 항상성을 유지하는 기전으로 세포 속에 프로그램되어 있는 세포사멸의 과정을 말한다. 또한, 면역반응이나 질병 또는 유해한 물질에 의하여 세포가 손상을 받았을 때에도 세포의 방어기전으로 일어난다. Ionizing x-ray 또는 UV의 조사, 유전자에 손상을 주는 항암제를 이용한 약물 치료를 수행하였을 경우에도 유발되며, 이러한 세포사멸 세포(apoptotic cell)에서는 단백질분해(protein cleavage), 단백질 교차결합(protein cross-linking), DNA 분해(DNA breakdown), 식세포 인식(phagocytic recognition)과 같은 여러 가지 특징들이 나타난다. As used herein, the term " apoptosis " refers to a process of apoptosis programmed into a cell as a mechanism of maintaining homeostasis by regulating the number of cells in development, aging, and tissues. In addition, when the cells are damaged by an immune reaction, disease or harmful substance, they also develop before the defense of the cells. Ionizing is also induced by x-ray or UV irradiation, drug treatment using an anticancer drug that damages the gene, and apoptotic cells cause protein cleavage, protein cross- linking, DNA breakdown, and phagocytic recognition.
본 명세서에 있어서, "오토파지(autophagy)"는 세포 내에서 손상된 단백질이나 세포 내 소기관을 제거하여 세포가 건강한 상태를 유지하여 항상성을 유지하도록 하는 중요한 과정으로 일정 비율로 세포에서 항상 일어난다. 하지만, 세포에 영양공급이 불충분하거나, 세포 내 단백질 등이 손상을 받아 응집체(aggregates) 등을 형성하거나, 급격하게 세포 내에서 에너지가 필요한 상황에 처하거나, 변화하는 외부 환경에 세포가 적응하는 등 환경이 바뀌었을 경우 등에는 급격하게 증가한다.As used herein, " autophagy " is an important process that removes damaged proteins or intracellular organelles in a cell to maintain a healthy state and maintain the homeostasis. It always occurs in cells at a certain rate. However, when the cells are insufficiently fed with nutrients, proteins in the cells are damaged to form aggregates, or the like, the cells are required to be energized rapidly, or the cells are adapted to a changing external environment When the environment is changed, it suddenly increases.
본 명세서에 있어서, "미토파지(mitophagy)"는 선택적 오토파지(autophagy) 중 하나로서, 미토콘드리아(mitochondria)를 특이적으로 제거하기 위한 오토파지(autophagy)이며, 오래되거나 손상을 입은 미토콘드리아를 제거함으로써 미토콘드리아의 온전성을 유지할 수 있게 한다. 미토파지(mitophagy)는 비정상적인 기능을 하는 미토콘드리아를 정상적인 미토콘드리아로 회전시키는 것뿐만 아니라 저산소(hypoxia)와 기아와 같은 스트레스 환경에서 전체 미토콘드리아 양을 조절하는 역할을 하며 이로 인하여 ROS의 발생을 줄이고 산소로부터 중요한 영양분 등을 보호하며 이와 같은 기능으로 에너지와 관련된 스트레스 상황에서도 세포가 살아남을 수 있도록 하는 기능을 수행한다.As used herein, " mitophagy " is one of the autophagy, which is an autophagy for specifically removing mitochondria, and by removing old or damaged mitochondria To maintain the integrity of the mitochondria. Mitophagy not only rotates abnormal mitochondria into normal mitochondria but also plays a role in regulating total mitochondrial levels in hypoxia and starvation stress environments, thereby reducing the incidence of ROS, Nutrients, and so on. This function enables the cells to survive even under energy-related stress conditions.
본 발명의 조성물이 약학 조성물인 경우, 약학조성물의 제조에 통상적으로 사용하는 사용하는 적절한 담체, 부형제, 붕해제, 감미제, 피복제, 팽창제, 윤활제, 활택제, 향미제, 항산화제, 완충액, 정균제, 희석제, 분산제, 계면활성제, 결합제 및 윤활제로 이루어진 군에서 선택되는 하나 이상의 첨가제를 추가로 포함할 수 있다.When the composition of the present invention is a pharmaceutical composition, suitable carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, lubricants, flavors, antioxidants, buffers, , A diluent, a dispersant, a surfactant, a binder, and a lubricant.
구체적으로 담체, 부형제 및 희석제는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 사용할 수 있으며, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 있으며 흔히 사용되는 단순 희석제인 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, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기재로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.
본 발명의 다른 구체예에서, 상기 약학조성물은 통상적인 방법에 따라 과립제, 산제, 피복정, 정제, 환제, 캡슐제, 좌제, 겔, 시럽, 즙, 현탁제, 유제, 점적제 또는 액제로 제형화하여 사용할 수 있다.In another embodiment of the present invention, the pharmaceutical composition may be formulated into granules, powders, coated tablets, tablets, pills, capsules, suppositories, gels, syrups, juices, suspensions, emulsions, Can be used.
본 발명의 일실시예에 따르면, 상기 약학조성물은 정맥내, 동맥내, 복강내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 대상체로 투여할 수 있다.According to one embodiment of the present invention, the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, Can be administered to a subject in a conventional manner via the intradermal route.
상기 화합물의 바람직한 투여량은 대상체의 상태 및 체중, 질환의 종류 및 정도, 약물 형태, 투여경로 및 기간에 따라 달라질 수 있으며 당업자에 의해 적절하게 선택될 수 있다. 본 발명의 일실시예에 따르면 이에 제한되는 것은 아니지만 1일 투여량이 0.01 내지 200 mg/kg, 구체적으로는 0.1 내지 200 mg/kg, 보다 구체적으로는 0.1 내지 100 mg/kg 일 수 있다. 투여는 하루에 한 번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되는 것은 아니다.The preferred dosage of the compound may vary depending on the condition and body weight of the subject, the type and degree of disease, the drug form, the administration route and the period, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dose may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.
한편, 상기 약학적으로 허용 가능한 염은 특별히 제한되지 않고, 본 발명의 화합물을 유기 및 무기산을 이용해 형성할 수 있으며, 예컨대 염산, 브롬화수소산, 메탄술폰산, 히드록시에탄술폰산, 황산, 아세트산, 트리플루오로아세트산, 말레산, 벤젠술폰산, 톨루엔술폰산 및 기타 다양한 산으로 형성된 질산염, 인산염, 붕산염, 타르타르산염, 시트르산염, 숙신산염, 벤조산염, 아스코르브산염 또는 살리실산염 등이 될 수 있다.The pharmaceutically acceptable salt is not particularly limited, and the compound of the present invention can be formed using organic and inorganic acids. Examples of the pharmaceutically acceptable salt include hydrochloric acid, hydrobromic acid, methanesulfonic acid, hydroxyethanesulfonic acid, sulfuric acid, acetic acid, Nitrates, phosphates, borates, tartrates, citrates, succinates, benzoates, ascorbates or salicylates formed with hydrochloric acid, hydrochloric acid, hydrochloric acid, hydrobromic acid,
또한, 본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 암 예방 또는 개선용 건강식품 조성물을 제공한다.Also, the present invention provides a health food composition for preventing or ameliorating cancer, which comprises, as an active ingredient, a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
< 화학식 1 >≪
상기 건강식품 조성물은 분말, 과립, 정제, 캡슐, 시럽 또는 음료의 형태로 제공될 수 있으며, 상기 건강식품조성물은 유효성분인 본 발명에 따른 화합물 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적 예를 들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다.The health food composition may be provided in the form of powder, granules, tablets, capsules, syrups or beverages. The health food composition may be used in combination with food or food additives other than the compound according to the present invention, And the like. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.
상기 건강식품 조성물에 함유된 화합물의 유효용량은 상기 약학조성물의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the compound contained in the health food composition may be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range for health and hygiene purposes or long-term intake for health control purposes , It is clear that the active ingredient can be used in an amount exceeding the above range since there is no problem in terms of safety.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등을 들 수 있다.There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.
<< 실험예Experimental Example >>
하기의 실험예들은 본 발명에 따른 각각의 실시예에 공통적으로 적용되는 실험예를 제공하기 위한 것이다.The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.
1. 시약1. Reagents
버퍼를 만드는데 필요한 Tris, NaCl, sodium dodecyl sulfate (SDS) 등의 화학물질 등은 Sigma-Aldrich(St. Louis, MO, USA)에서 구입하여 사용하였다. L1-95-1은 한국생명공학연구원의 홍영수 박사 연구팀이 Streptomyces sp . W2061에서 추출한 것을 사용하였다. 세포배양을 위한 배지[Minimum Essential Medium Eagle(MEM), Dulbecco's Modification of Eagle's Medium(DMEM), Mammary Epithelium Basal Medium(MEBM) 등]는 Corning (Manassas, VA, USA), Lonza (Walkersville, MD, USA)에서 구입하여 사용하였다. 단백질 정량을 위한 DC protein assay kit는 Bio-Rad Laboratories (Hercules, CA, USA) 제품을 사용하였으며, 세포의 증식을 분석하기 위하여 Cell Titer 96 Aqueous One Solution Reagent [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)] kit를 Promega (Madison, WI, USA)에서 구입하여 사용하였다.Chemicals such as Tris, NaCl, and sodium dodecyl sulfate (SDS) required to make the buffer were purchased from Sigma-Aldrich (St. Louis, MO, USA). L1-95-1 is hongyoungsu's team of the Korea Research Institute of Bioscience and Biotechnology Streptomyces sp . W2061 was used. (Manassas, Va., USA) and Lonza (Walkersville, MD, USA) were used for cell culture. . The DC protein assay kit for protein determination was purchased from Bio-Rad Laboratories (Hercules, Calif., USA).
2. 항체2. Antibody
면역 Western blotting, 형광 현미경 및 공초점현미경 관찰을 위한 인산화 단백질 검출 항체(예: p-mTOR (S2448), p-Akt (T308), p-Akt (S473), p-P70S6K (T389), p-S6 (S235/236), p-MEK (S217/221), p-ERK (T202/Y204) 및 p-RSK (T359/S363)), 총량 단백질 검출용 항체(예: total-mTOR, total-Akt, total-P70S6K, total-S6, total-MEK, total ERK) 등의 항체는 Cell Signaling Technologies (Rockville, MD, USA)로부터 구입하여 사용하였으며, β-actin, total-RSK 및 anti-rabbit and anti-mouse 2차 항체는 Santa Cruz Biotechnology (Santa Cruz, CA, USA) 로부터 구입하여 사용하였다. P-Akt (T308), p-Akt (S473), p-P70S6K (T389), p-mTOR (S2448) S-6 (S235 / 236), p-MEK (S217 / 221), p-ERK (T202 / Y204) and p- RSK (T359 / S363) , total-RSK and anti-rabbit and anti-rabbit anti-rabbit antibodies were purchased from Cell Signaling Technologies (Rockville, Md., USA) mouse secondary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
3. 세포 배양3. Cell culture
마우스 상피 세포주(Mouse epidermal cell line; JB6 Cl41), 사람 삼중음성 유방암 세포주인 MDA-MB-231과 사람 유방암 세포주인 MCF-7은 American Type Culture Collection (ATCC, Manassas, VA, USA) 에서 구입하였다. JB6 Cl41은 5%의 영아소혈청(fetal bovine serum: FBS)과 항생제(100 Units/ml 페니실린, 100㎍/ml 스트렙토마이신)을 첨가한 minimum essential medium (MEM) (Corning; Manassas, VA, USA)을 사용하여 배양하였다. MCF-7은 minimum essential medium (MEM) (Corning; Manassas, VA, USA)에 10% 영아소혈청(FBS)과 항생제(100 Units/ml 페니실린, 100㎍/ml 스트렙토마이신)와 인슐린(10㎍/ml)을 첨가한 배지를 이용하여 배양하였고, MDA-MB-231은 Dulbecco's Modification of Eagle's Medium (DMEM) (Corning; Manassas, VA, USA)에 10% 영아소혈청(FBS)과 항생제 (100 Units/ml 페니실린, 100㎍/ml 스트렙토마이신)을 첨가한 배지를 이용하여 배양하였다. 이 세포들은 5%의 CO2가 공급되는 37℃ 항온배양기에서 배양하였으며, 2일 마다 배양액을 교체하였고, 세포가 배양용기의 표면적에 약 90% 정도 증식하면 계대하였다.MDA-MB-231, a human triple-negative breast cancer cell line, and MCF-7, a human breast cancer cell line, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). JB6 Cl41 was cultured in minimal essential medium (MEM) (Corning; Manassas, Va., USA) supplemented with 5% fetal bovine serum (FBS) and antibiotics (100 units / ml penicillin, 100 μg / ml streptomycin) Lt; / RTI > (100 units / ml penicillin, 100 μg / ml streptomycin) and insulin (10 μg / ml streptomycin) in a minimum essential medium (MEM) (Corning; Manassas, VA, USA) MDA-MB-231 was cultured in medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 Units / ml) in Dulbecco's Modification of Eagle's Medium (DMEM) ml penicillin, 100 占 퐂 / ml streptomycin). These cells were cultured in a 37 ° C incubator with 5% CO 2 , and the culture medium was changed every 2 days. When the cells were grown at about 90% of the surface area of the culture vessel, they were transplanted.
4. 세포 증식 분석 (MTS 분석)4. Cell proliferation assay (MTS assay)
세포들을 (JB6 Cl41; 1 × 103 cells/well, MCF-7; 2 × 103 cells/well, MDA-MB-231; 2 × 103 cells/well) 96-well plate에 접종하여 24시간 동안 배양하였다. 24시간 후 L1-95-1을 비처리군, 처리군(100 nM, 500 nM 및 1000 nM의 농도)으로 처리하였으며, 각각의 96-wll plate를 24, 48, 72, 96시간 별로 세포의 증식을 흡광도계를 이용하여 측정하였다. 흡광도를 측정하기 위하여 각 well에 20 ㎕의 MTS-based Cell Titer 96®Aqueous One Solution을 첨가 후 1시간 동안 37℃, 5% CO2 incubator에서 반응시켰으며, xMark™ Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA)를 이용하여 492 nm에서 흡광도를 측정하였다. L1-95-1에 의한 세포증식 억제 효과는 세포를 접종한 후 2시간째를 0시간으로 설정하고 24시간 간격으로 5일간 측정하였으며, 매 24시간째에 표시된 농도의 L1-95-1이 포함된 배지를 이용하여 배양액을 교체하였다.Cells (JB6 Cl41; 1 × 10 3 cells / well, MCF-7; 2 × 10 3 cells / well, MDA-MB-231; 2 × 10 3 cells / well) for 24 hours were inoculated in 96-well plate Lt; / RTI > After 24 hours, L1-95-1 was treated with untreated group, treated group (100 nM, 500 nM and 1000 nM concentration), and each 96-well plate was cultured for 24, 48, 72, Were measured using an absorbance meter. To measure the absorbance, 20 μl of MTS-based
5. 앵커리지 비의존적 세포 형질전환 분석(Anchorage-independent cell transformation assay)5. Anchorage-independent cell transformation assay (Anchorage-independent cell transformation assay)
1×BME, MEM, DMEM에 10% FBS와 0.5% agar 그리고 농도별 L1-95-1을 첨가한 3 ml의 bottom agar를 6 well plate에 분주하여 굳힌 후 마우스 피부 상피세포 JB6 Cl41 (8 × 103/well), 사람 유방암 세포 MCF-7 (8 × 103/well), 사람 삼중음성 유방암 세포 MDA-MB-231 (8 × 103/well)을 각각 1X BME, MEM, DMEM에 혼합하고 여기에 10% FBS 그리고 0.33% agar와 농도별 L1-95-1을 첨가하여 만든 1 ml top agar를 bottom agar 위에 분주하여 굳히고 약 20일간 37℃, 5% CO2 항온배양기에서 배양하였다. ECLIPSE Ti inverted microscope와 the NIS-Elements AR (V.4.0) computer software program (NIKON Instruments Korea, Gangnam, Seoul, Korea) 를 이용하여 콜로니(colony) 숫자를 측정하였다.3 ml of bottom agar containing 10% FBS, 0.5% agar and L1-95-1 at concentrations of 1 × BME, MEM, and DMEM were dispensed into 6 well plates and the cells were fixed with mouse skin epithelial cell JB6 Cl41 3 / well), human breast cancer cells MCF-7 (8 × 10 3 / well), human triple negative breast cancer cells MDA-MB-231 (8 × 10 3 / well) to each 1X BME, MEM, mixed with DMEM and here 1 ml top agar supplemented with 10% FBS, 0.33% agar and L1-95-1 by concentration was added to the bottom agar and incubated for about 20 days at 37 ° C in a 5% CO 2 incubator. Colony numbers were measured using an ECLIPSE Ti inverted microscope and the NIS-Elements AR (V.4.0) computer software program (NIKON Instruments Korea, Gangnam, Seoul, Korea).
6. 6. 웨스턴Western 블롯팅Blotting (Western blotting)(Western blotting)
100-mm dish에 사람 유방암 세포 MCF-7 (3 × 106)과 사람 삼중음성 유방암 세포 MDA-MB-231 (3 × 106)을 접종하였고 5% CO2가 공급되는 37℃ 항온 배양기에서 배양하였다. 24시간 후 L1-95-1을 [비처리군 (DMSO) 및 L1-95-1 처리군 (100 nM, 250 nM, 500 nM)] 배양액과 섞어 처리하였고 3시간 후 세포를 Scraper를 이용하여 harvest하였다. 단백질 현탁액은 세포 파쇄 버퍼 (cell lysis buffer) [50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40]에 protease inhibitor cocktail 정을 녹여 최종농도의 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate 및 1 mM phenyl methylsulfonyl fluoride (PMSF)]가 되도록 하여 이용하였다. 준비한 MCF-7 및 MDA-MB-231 세포에 각각 lysis buffer 100㎕를 넣고 freezing 과정과 thawing 과정으로 세포를 파쇄한 다음 4℃에서 12,000 rpm에 5분간 원심 분리하였다. 단백질 농도는 DC protein assay kit (Bio-Rad)의 프로토콜에 따라 수행하고 xMark™ Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA)를 이용하여 750 nm에서 흡광도를 측정하였고, 동일 양의 단백질을 이용하여 SDS-PAGE를 수행하고, 단백질을 transfer buffer [20 mM Tris-HCl (pH 8.0), containing 150 mM glycine and 20% (v/v) methanol]를 이용하여 폴리비닐리덴 플루오라이드(polyvinylidene fluoride; PVDF) 막에 옮겼다. 5%의 nonfat dry milk가 포함된 blocking buffer [1X TBS containing 0.05% Tween 20: (TBS-T)]로 blocking 한 다음, p-mTOR(S2448), p-Akt(T308), p-Akt(S473), p-p70S6K(T389), p-S6(S235/236), p-MEK(S217/221), p-ERK(T202/Y204), p-RSK(T359/S363), total-mTOR, total-Akt, total-P70S6K, total-S6, total-MEK, total ERK, total-RSK, β-actin 등의 1차 항체를 4℃에서 밤샘 hybridization 시켰다. 멤브레인(Membrane)을 3회 1X TBS-T buffer로 세척하였고 적절한 HRP-conjugated 2차 항체로 hybridization 하였다. 표적 단백질은 enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA)을 이용하여 Chemidoc XRS imager system (Bio-Rad Laboratories, Hercules, CA, USA)을 이용하여 검출하였다.Human breast cancer cells MCF-7 (3 × 10 6 ) on a 100-mm dish and human cultured in triple negative breast cancer cells MDA-MB-231 (3 × 10 6) of inoculum was 37 ℃ constant temperature incubator is 5% CO 2 is fed Respectively. After 24 hours, L1-95-1 was treated with the [untreated group (DMSO) and L1-95-1 treated group (100 nM, 250 nM, 500 nM)] and after 3 hours, cells were harvested using Scraper Respectively. Protein suspension was dissolved in cell lysis buffer [50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40] and the final concentration of 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, mM sodium vanadate and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. 100 μl of lysis buffer was added to the prepared MCF-7 and MDA-MB-231 cells, and the cells were disrupted by freezing and thawing, followed by centrifugation at 12,000 rpm for 5 minutes at 4 ° C. Protein concentration was measured according to the protocol of the DC protein assay kit (Bio-Rad) and absorbance was measured at 750 nm using a xMark ™ Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories, Hercules, Calif., USA) SDS-PAGE was carried out using polyvinylidene fluoride (SDS-PAGE) and the protein was eluted with a transfer buffer (20 mM Tris-HCl, pH 8.0, containing 150 mM glycine and 20% ; PVDF) membrane. (T248), p-Akt (T308), and p-Akt (S473) were blocked by blocking with 5% nonfat dry milk in blocking buffer (1X TBS containing 0.05% Tween 20 ), p-p70S6K (T389), p-S6 (S235 / 236), p-MEK (S217 / 221), p-ERK (T202 / Y204) Primary antibodies such as -Akt, total-P70S6K, total-S6, total-MEK, total ERK, total-RSK and β-actin were hybridized overnight at 4 ° C. Membranes were washed three times with 1X TBS-T buffer and hybridized with a suitable HRP-conjugated secondary antibody. Target proteins were detected using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA) using a Chemidoc XRS imager system (Bio-Rad Laboratories, Hercules, CA, USA).
7. 7. LysotrackerLysotracker 염색 분석 Dye analysis
8-chamber slide의 각 chamber에 사람 유방암 세포 MCF-7 (2 × 104 cells)과 사람 삼중음성 유방암 세포 MDA-MB-231(2 × 104 cells)을 접종하였고 5% CO2, 37℃ 항온배양기에서 배양하였다. 24시간 후 L1-95-1을 처리 후 24시간 동안 배양하였으며 LysoTracker® Red DND-99 (Life technologies, OR, USA) 를 30 nM의 농도로 각 세포의 배양액과 희석하여 1시간 동안 처리하였다. 칼슘-PBS로 3회 wash 후 4% 포르말린으로 고정한 다음 Hoechst (1 ㎍/ml)를 PBS에 희석하여 30분간 세포에 처리하였다. 그 후 8-chamber slide에 CLEAR-MOUNT with TRIS Buffer (Electron Microscopy Sciences, Hatfield, PA, USA)를 이용하여 커버슬라이드를 부착하였다. ECLIPSE Ti inverted microscope와 the NIS-Elements AR (V.4.0) computer software program (NIKON Instruments Korea, Gangnam, Seoul, Korea)을 이용하여 형광을 관찰하였다.8-chamber human breast cancer cells in each chamber of the slide MCF-7 (2 × 10 4 cells) and human triple negative breast cancer cells MDA-MB-231 (2 × 10 4 cells) the were inoculated with 5% CO 2, 37 ℃ constant temperature Lt; / RTI > Were cultured for 24 hours after treatment the L1-95-1 after 24 hours were treated by diluting the culture solution in each cell the LysoTracker ® Red DND-99 (Life technologies, OR, USA) at a concentration of 30 nM for 1 hour. The cells were washed three times with calcium-PBS, fixed with 4% formalin, and treated with Hoechst (1 ㎍ / ml) in PBS for 30 min. The cover slides were then attached to the 8-chamber slides using CLEAR-MOUNT with TRIS Buffer (Electron Microscopy Sciences, Hatfield, PA, USA). Fluorescence was observed using an ECLIPSE Ti inverted microscope and the NIS-Elements AR (V.4.0) computer software program (NIKON Instruments Korea, Gangnam, Seoul, Korea).
8. 8. MitotrackerMitotracker 염색 분석 Dye analysis
사람 유방암 세포 MCF-7 (2 × 104 cells)과 사람 삼중음성 유방암 세포 MDA-MB-231 (2 × 104 cells)을 confocal dish (SPL Life Sciences, Pocheon, South Korea)에 접종하고 5% CO2, 37℃ 항온배양기에서 배양하였다. 24시간 후 L1-95-1을 500 nM 농도로 각 세포의 배양액과 희석하여 처리 후 24시간 배양하였다. PBS로 3회 wash 후 Mitotracker® Green FM (Invitrogen, OR, USA)을 20 nM의 농도로 각 세포의 배양액에 희석하여 45분간 처리하였다. 칼슘-PBS로 3회 wash 후 Hoechst (1 ㎍/ml)를 PBS에 희석하여 30분간 세포에 처리하였다. 이후 칼슘-PBS로 배양액을 교체한 후 LSM 710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany)로 형광을 관찰하였다. Human breast cancer cells MCF-7 (2 × 10 4 cells) and human triple-negative breast cancer cells MDA-MB-231 (2 × 10 4 cells) were inoculated into a confocal dish (SPL Life Sciences, Pocheon, South Korea) 2 , and 37 ° C in a constant temperature incubator. After 24 hours, L1-95-1 was diluted with each cell culture at a concentration of 500 nM and cultured for 24 hours. After washing three times with PBS, Mitotracker ® Green FM (Invitrogen, OR, USA) was diluted in each cell culture medium at a concentration of 20 nM and treated for 45 minutes. After 3 washes with calcium-PBS, Hoechst (1 ㎍ / ml) was diluted with PBS and treated with cells for 30 minutes. After replacing the culture with calcium-PBS, fluorescence was observed with LSM 710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).
9. 9. mCherrymCherry 검출 분석 Detection analysis
8-chamber slide의 각 chamber에 사람 유방암 세포 MCF-7과 사람 삼중음성 유방암 세포 MDA-MB-231을 각각 2 × 104개의 세포를 접종하고 5% CO2, 37℃ 항온배양기에서 배양하였다. 24시간 후 jetPEI® DNA Transfection Reagent (Polypuls transfection, NY, USA)와 mCherry 벡터를 150 mM NaCl에 희석하여 20 ㎕씩 세포에 형질주입 하며 30시간 동안 배양하였다. 30시간 후 L1-95-1을 500 nM의 농도로 각 세포의 배양액에 희석하여 처리하였고 24시간 후 8-chamber slide에 CLEAR-MOUNT with TRIS Buffer (Electron Microscopy Sciences, Hatfield, PA, USA)를 이용하여 커버슬라이드를 부착하였다. ECLIPSE Ti inverted microscope와 the NIS-Elements AR (V.4.0) computer software program (NIKON Instruments Korea, Gangnam, Seoul, Korea)를 이용하여 형광을 관찰하였다. Human breast cancer cells MCF-7 and human triple-negative breast cancer cells MDA-MB-231 were inoculated at 2 × 10 4 cells in each chamber of an 8-chamber slide and cultured in a 5% CO 2, 37 ° C incubator. Twenty-four hours later, the cells were transfected with 20 μl of the gelpEI ® DNA Transfection Reagent (Polypuls transfection, NY, USA) and mCherry vector in 150 mM NaCl and cultured for 30 hours. After 30 hours, L1-95-1 was diluted to a concentration of 500 nM in each cell culture medium. After 24 hours, CLEAR-MOUNT with TRIS Buffer (Electron Microscopy Sciences, Hatfield, PA, USA) And a cover slide was attached. Fluorescence was observed using an ECLIPSE Ti inverted microscope and the NIS-Elements AR (V.4.0) computer software program (NIKON Instruments Korea, Gangnam, Seoul, Korea).
10. 미토콘드리아 막 전위 분석10. Mitochondrial membrane potential analysis
사람 유방암 세포 MCF-7 (2 × 104 cells)과 사람 삼중음성 유방암 세포 MDA-MB-231 (2 × 104 cells)을 confocal dish (SPL Life Sciences, Pocheon, South Korea)에 접종하고 5% CO2, 37℃ 항온배양기에서 배양하였다. 24시간 후 L1-95-1을 vehicle(DMSO), 100 nM, 250 nM, 500 nM의 농도로 각각 세포의 배양액에 희석하여 24시간 배양하였다. 그 후 양성 대조군(positive control)으로 사용하기 위해 DMSO를 처리한 세포에 CCCP (carbonyl cyanide 3-chlorophenylhydrazone)를 50 μM의 농도로 각각 세포의 배양액에 희석하여 처리하였고 MitoProbe™ JC-1 (Life technologies™, OR, USA)를 2 μM의 농도로 각각의 세포 배양액에 희석하여 15분간 처리하였다. 15분 후 칼슘-PBS로 배양액을 교체하였고, LSM 710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany)로 형광을 관찰하였다. Human breast cancer cells MCF-7 (2 × 10 4 cells) and human triple-negative breast cancer cells MDA-MB-231 (2 × 10 4 cells) were inoculated into a confocal dish (SPL Life Sciences, Pocheon, South Korea) 2 , and 37 ° C in a constant temperature incubator. After 24 hours, L1-95-1 cells were diluted in cell culture medium (DMSO), 100 nM, 250 nM and 500 nM, respectively, and cultured for 24 hours. Cells treated with DMSO were then treated with CCCP (carbonyl cyanide 3-chlorophenylhydrazone) at a concentration of 50 μM in a cell culture medium for the positive control, and treated with MitoProbe ™ JC-1 (Life technologies ™ , OR, USA) were diluted in each cell culture medium at a concentration of 2 μM and treated for 15 minutes. After 15 minutes, the culture medium was replaced with calcium-PBS, and fluorescence was observed with a LSM 710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).
11. 미토콘드리아 11. Mitochondria ROSROS 검출 분석 Detection analysis
사람 유방암 세포 MCF-7 (2 × 104 cells)과 사람 삼중음성 유방암 세포 MDA-MB-231 (2 × 104 cells)을 confocal dish (SPL Life Sciences, Pocheon, South Korea)에 접종하고 5% CO2, 37℃ 항온배양기에서 배양하였다. 24시간 후 DMSO, L1-95-1을 500 nM 농도로 각각 세포 배양액에 희석하여 처리 후 24시간 배양하였다. 여기에 MitoSOX™ Red (Invitrogen, OR, USA)를 5 μM 농도로 각각 세포 배양액에 희석하여 10분간 처리하였고 칼슘-PBS로 배양액을 교체한 후 LSM 710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany)로 형광을 측정하였다. Human breast cancer cells MCF-7 (2 × 10 4 cells) and human triple-negative breast cancer cells MDA-MB-231 (2 × 10 4 cells) were inoculated into a confocal dish (SPL Life Sciences, Pocheon, South Korea) 2 , and 37 ° C in a constant temperature incubator. After 24 hours, DMSO and L1-95-1 were diluted in the cell culture medium at a concentration of 500 nM, and cultured for 24 hours. After incubation for 10 min with MitoSOX ™ Red (Invitrogen, OR, USA) at a concentration of 5 μM, the culture medium was replaced with calcium-PBS and incubated with LSM 710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) To measure fluorescence.
12. 세포 주기 분석 12. Cell cycle analysis
사람 유방암 세포 MCF-7 (5 × 105 cells)과 사람 삼중음성 유방암 세포주 MDA-MB-231 (5 × 105 cells)을 60-mm dish에 접종 후 37℃, 5% CO2 항온배양기에서 24시간 배양하였다. 24시간 후 L1-95-1을 [비처리군 (DMSO) 및 처리군 (100 nM, 250 nM, 500 nM)]을 각 세포 배양액에 희석하여 24시간 동안 처리하였고, PBS로 wash 후 0.1% Trypsin-EDTA를 이용하여 세포를 회수한 다음 1× PBS로 wash 하였다. ice-cold 70% 에탄올(ethanol)로 -20℃에서 2시간 동안 고정하고, ×1000 g에서 각 세포들을 원심 분리하여 회수하였다. 250 ㎕의 0.6% Triton-X100-1X PBS로 세포를 부유하고 RNase A (200 μg/ml) 와 propidium iodide (20 μg/ml)를 첨가하여 4℃에서 15분간 방치하였다. 세포주기는 flow cytometry (BD FACS Calibur™ flow cytometry, Franklin Lakes, NJ, USA)를 이용하여 측정하였다. Human breast cancer cell MCF-7 (5 × 10 5 cells) and human triple-negative breast cancer cell line MDA-MB-231 (5 × 10 5 cells) were inoculated into a 60-mm dish and incubated at 37 ° C in a 5% CO 2 incubator Time. After 24 hours, L1-95-1 was diluted in each cell culture medium (DMSO) and treated group (100 nM, 250 nM, 500 nM) for 24 hours. After washing with PBS, 0.1% Trypsin Cells were recovered using EDTA and washed with 1 x PBS. The cells were fixed with ice-cold 70% ethanol at -20 ° C for 2 hours and recovered by centrifugation at × 1000 g. Cells were suspended in 250 μl of 0.6% Triton-X100-1X PBS and RNase A (200 μg / ml) and propidium iodide (20 μg / ml) were added and left at 4 ° C for 15 minutes. The cell cycle was measured by flow cytometry (BD FACS Calibur ™ flow cytometry, Franklin Lakes, NJ, USA).
13. 13. Streptomyces sp.Streptomyces sp. W2061 균주의 분리 Isolation of W2061 strain
본 균주는 2007년 8월 대전 연구소 인근 토양 시료로부터 분리하였다. 채집한 토양시료는 채집 즉시 멸균된 플라스틱 튜브에 넣어 보관하였으며 사용 전, 상온에서 48 시간 동안 자연 건조를 수행하였다. 토양 시료는 멸균된 증류수를 이용하여 100배 및 1000배 희석하였다. 희석액 0.5ml를 휴믹산 한천 (Humic acid agar) 배지 에 도말하여 28℃에서 50일간 배양하여 나타난 집락을 순수분리 하였다.This strain was isolated from soil samples near Daejeon Research Institute in August, 2007. Soil samples were collected and stored in sterilized plastic tubes. Natural drying was carried out at room temperature for 48 hours. Soil samples were diluted 100 times and 1000 times with sterilized distilled water. 0.5 ml of the diluted solution was plated on a humic acid agar medium and cultured at 28 ° C for 50 days to purify the colonies.
본 균주의 rRNA 서열 분석을 통하여 얻어진 염기서열의 GenBank 검색 결과, Streptomyces rubiginosohelvolus에 99%의 상동성을 보였다. 이에 본 균주를 Streptomyces 속으로 동정하였고 이를 Streptomyces sp. W2061로 명명하였다. As a result of GenBank search for the nucleotide sequence obtained through rRNA sequencing of this strain, 99% homology to Streptomyces rubiginosohelvolus was shown. This was identified the present strain as Streptomyces in this Streptomyces sp. W2061.
14. 14. StreptomycesStreptomyces spsp . . W2061 균주의 배양Culture of W2061 strain
본 방선균 균주를 배양하기 위하여, 통상적으로 미생물이 이용하는 영양원을 함유하는 배지를 준비하였다. 방선균의 조배지로서 YEME 배지(yeast extract-malt extract medium) 배지를 사용하였다. W2061 균주 포자를 이용하여 28℃에서 3일 동안 300 ㎖의 액체 그리세롤(glycerol)에 효모추출물-맥아추출물을 추가한 배지에서 1차 배양하였다. 상기 1차 배양액을 상기와 동일한 배지 300 ㎖를 가진 1ℓ 플라스크에 최종 5%가 되도록 접종한 후, 28 ℃에서 7일 동안 150 rpm에서 배양하였다. 이러한 플라스크 30개를 동시에 배양하여 약 10ℓ의 균주 배양액을 확보하였다.In order to cultivate the actinomycetes strain, a medium containing a nutrient source normally used by microorganisms was prepared. YEME medium (yeast extract-malt extract medium) was used as a culture medium for actinomycetes. W2061 strain spore for 3 days at 28 ° C in a medium supplemented with yeast extract-malt extract in 300 ml of liquid glycerol. The primary culture was inoculated in a 1 liter flask with 300 ml of the same medium as above to a final concentration of 5%, and then cultured at 28 ° C for 7 days at 150 rpm. Thirty flasks were cultured at the same time to obtain a culture solution of about 10 L of the strain.
15. 15. StreptomycesStreptomyces spsp . . W2061 균주로부터 L1-95-1 화합물의 분리 및 정제Isolation and purification of L1-95-1 compound from W2061 strain
본 발명에 따른 화학식 1(L1-95-1)의 화합물을 제조하기 위하여 다음과 같이 실시하였다. 상기에서 제조한 배양액 10ℓ를 동량의 EtOAc로 2회 추출하고, 이들 추출물을 여과하여 불용물을 제거하고, 농축한 후에 EtOAc와 물로 분획하여 유기상 추출물 7 g을 얻었다. 이 농축액을 실리카(Cosmosil 75C18-PREP)에 흡착시켜 역상컬럼(RediSep® Rf Reversed Phase C18 Column)을 통해 중압 액체 크로마토그라피(Medium pressure liquid chromatography)를 실시하였으며, 이때 메탄올/물을 혼합용매로 하여 단계적으로 메탄올 농도를 차등하게 비율을 증가시키면서 용출하였다. 이때 키다마이신계 화합물을 함유한 분획은 80% 메탄올에서 용출하였다. 이 분획을 감압 농축한 후, 고속액체크로마토그래피(칼럼: Cosmosil C18, 길이 250mm, 직경 10mm)를 이용하여 용매로 아세토나이트릴과 물을 30:70 80:20 비율 농도 구배로 용출 유속 3ml/min 조건으로 1차 용출하고 2차로 1차 용출물을 40:60 50:50 비율 농도 구배로 재용출하여 274nm의 UV 흡수 피크를 보이는 화합물 L1-95-1을 분리하였다.The preparation of the compound of formula (I) (L1-95-1) according to the present invention was carried out as follows. 10 L of the culture prepared above was extracted twice with EtOAc of the same amount and the extract was filtered to remove insolubles, and the filtrate was concentrated, and then fractionated with EtOAc and water to obtain 7 g of an organic phase extract. This concentrate was adsorbed onto silica (Cosmosil 75C18-PREP) and subjected to medium pressure liquid chromatography through a reversed phase column (RediSep® Rf Reversed Phase C18 Column) using methanol / To elute the methanol concentration while increasing the ratio. At this time, the fractions containing the kidamycin compound eluted from 80% methanol. This fraction was concentrated under reduced pressure, and acetonitrile and water were then eluted with a gradient of 30:70 to 80:20 by using high performance liquid chromatography (column: Cosmosil C18,
16. 16. StreptomycesStreptomyces spsp . . W2061 균주로부터 분리한 L1-95-1 화합물의 구조 분석Structural analysis of L1-95-1 compounds isolated from W2061 strain
상기 방선균 Streptomyces sp. W2061의 배양액으로부터 분리한 실시예의 화합물은 ESIMS 질량분석기(Electrospray Ionization mass spectrometer)를 사용하여 분자량 및 분자식을 측정하였다. 또한, 핵자기공명(NMR) 분석(Bruker Biospin Advance II 900 NMR spectrometer 및 Bruker AVANCE HD 800 NMR spectrometer)을 통하여 1H NMR, 13C NMR, COSY(Correlation Spectroscopy), HMBC(Heteronuclear Multiple-Bond Coherence), DEPT(Distortionless Enhancement by Polarization), NOESY(Nuclear Overhauser effect spectroscopy) 스펙트럼을 얻고, 화합물의 분자구조를 결정하였다. The Streptomyces sp. The compounds of the examples isolated from the culture medium of W2061 were measured for molecular weight and molecular formula using an ESIMS mass spectrometer (Electrospray Ionization mass spectrometer). Further, 1 H NMR, 13 C NMR, COZY (Correlation Spectroscopy), Heteronuclear Multiple-Bond Coherence (HMBC), and the like were performed through nuclear magnetic resonance (NMR) analysis (Bruker Biospin Advance II 900 NMR spectrometer and
측정 결과는 하기와 같으며, 상기 Streptomyces sp. W2061 균주의 배양액으로부터 분리한 물질은, 상기 화학식을 갖는 키다마이신과 신규한 L1-95-1 화합물로 동정하였으며, L1-95-1 화합물는 이미 보고 된 키다미이신 화합물 (Furukawa, M.; Hayakawa, I.; Ohta, G.; Iitaka, Y. Tetrahedron 1975, 31, 2989; Furukawa, M.; Iitaka, Y. Tetrahedron Lett. 1974, 15, 3287) 구조 중 angolosamine 당 구조에 이중결합 구조의 구조적인 차이를 보이는 것으로 확인하였다.The results of the measurement are as follows. Streptomyces sp. The substance isolated from the culture broth of the W2061 strain was identified as a new L1-95-1 compound with chidamycin having the above formula, and the L1-95-1 compound was identified by the previously reported chimeric compound (Furukawa, M .; Hayakawa, Iitaka, Y. Tetrahedron 1975, 31, 2989, Furukawa, M .; Iitaka, Y. Tetrahedron Lett 1974, 15, 3287) Structural differences of double bond structure in angolosamine sugar structure Respectively.
L1-95-1 화합물: 흰색 분말; UV(MeOH) λmax (log ε) 245nm, 275nm ;1H, 13C NMR 데이터는 하기 표 1에 나타내었음; HRESIMS m/z 685.3128 [M-H]- (calcd for C39H45N2O9, 685.3125).L1-95-1 Compound: white powder; UV (MeOH) [lambda] max (log [epsilon]) 245 nm, 275 nm; 1 H, < 13 > C NMR data are shown in Table 1 below; HRESIMS m / z 685.3128 [MH] - (calcd for C 39 H 45 N 2 O 9 , 685.3125).
40.2838.79
40.28
2.92 (s)2.87 (s)
2.92 (s)
2.38(dd,J=13.7,6.3Hz)2.15 (dd, J = 13.6, 7.7 Hz)
2.38 (dd, J = 13.7, 6.3 Hz)
37.1936.26
37.19
2.66(d,J=4.8Hz)2.73 (d, J = 4.7 Hz)
2.66 (d, J = 4.8 Hz)
<< 실시예Example 1> L1-95-1에 의한 JB6 1> JB6 by L1-95-1 Cl41Cl41 세포의 증식억제 및 Cell proliferation inhibition and EGF에EGF 의한 형질전환 억제 Inhibition of transfection by
L1-95-1은 본 발명자들에 의해 새롭게 구조가 밝혀진 새로운 물질로 연구가 전혀 이루어 지지 않았다.(도 1A). 본 발명에서는 마우스 피부 상피 세포인 JB61 Cl41 세포를 이용하여 L1-95-1이 세포의 증식과 암화에 미치는 영향을 검증하였다(도 1B). 그 결과 L1-95-1은 JB6 Cl41 세포의 증식을 농도 의존적으로 억제하였다(도 1B). 세포증식 억제의 효능을 비교하여 보면, L1-95-1은 1μM의 농도에서 JB6 Cl41 세포의 증식을 약 50% 억제하였다(도 1B). L1-95-1을 사용하여 앵커리지 비의존적(anchorage-independent) 조건에서 상피성장인자(epidermal growth factor; EGF)에 의해 유도되는 세포의 형질전환 억제능을 검증하였다. JB6 Cl41 세포는 EGF 자극에 의해 앵커리지 비의존적(anchorage-independent) 조건에서 콜로니(colony)를 형성하여 암세포로 형질이 전환되는 것을 확인하였다(도 1C). 그러나 L1-95-1을 EGF와 병용 처리하였을 경우, EGF에 의해 유도되는 JB6 Cl41 세포의 형질전환이 농도의존적으로 감소됨을 확인하였다(도 1C). 따라서 L1-95-1은 세포의 증식을 억제하며, 앵커리지 비의존적(anchorage-independent) 조건에서 EGF에 의해 유도되는 세포의 형질전환을 억제하는 효능이 있는 것을 확인하였다.L1-95-1 was not newly studied as a novel substance newly discovered by the present inventors (FIG. 1A). In the present invention, the effect of L1-95-1 on cell proliferation and carcinogenesis was examined using JB61 Cl41 cells as mouse skin epithelial cells (Fig. 1B). As a result, L1-95-1 inhibited the proliferation of JB6 Cl41 cells in a concentration-dependent manner (Fig. 1B). Comparing the efficacy of cell proliferation inhibition, L1-95-1 inhibited the proliferation of JB6 Cl41 cells by about 50% at a concentration of 1 μM (FIG. 1B). L1-95-1 was used to verify the ability of the cells to inhibit epidermal growth factor (EGF) -induced cell transformation under anchorage-independent conditions. JB6 Cl41 cells were colonized by anchorage-independent conditions by EGF stimulation and were transformed into cancer cells (Fig. 1C). However, when L1-95-1 was co-treated with EGF, it was confirmed that the transformation of JB6 Cl41 cells induced by EGF was reduced in a concentration-dependent manner (Fig. 1C). Thus, L1-95-1 inhibits cell proliferation and has the effect of inhibiting EGF-induced cell transformation under anchorage-independent conditions.
<< 실시예Example 2> L1-95-1에 의한 2> by L1-95-1 MCFMCF -7 및 -7 and MDAMDA -MB-231 세포서 in vitro 암 성장 억제-MB-231 inhibition of cancer growth in vitro
L1-95-1에 의한 항암효과를 검증하기 위하여 유방암 세포 MCF-7과 삼중음성 유방암 세포 MDA-MB-231을 이용하였다. L1-95-1을 MCF-7과 MDA-MB-231에 처리하여 세포의 증식억제 효과를 분석한 결과, MCF-7 유방암 세포는 1 μM 농도의 L1-95-1 처리에 의해서 약 50%의 세포증식을 억제하는 것을 확인하였다(도 2A, 왼쪽 그래프). L1-95-1을 삼중음성 유방암 세포 MDA-MB-231에 처리하였을 경우에는 500 nM 농도에서 약 60%의 세포증식을 억제하였으며, 1 μM 농도의 L1-95-1을 처리하였을 경우에는 세포증식을 완전하게 억제함을 확인하였다(도 2A, 오른쪽 그래프). 따라서 L1-95-1은 유방암 세포 중에서 삼중음성 유방암 세포에 특이성을 갖고 암세포의 증식을 효과적으로 억제하는 것을 확인하였다(도 2A). To investigate the anticancer effect of L1-95-1, breast cancer cell MCF-7 and triple negative breast cancer cell MDA-MB-231 were used. MCF-7 breast cancer cells were treated with 1 μM of L1-95-1 to inhibit proliferation of MCF-7 and MDA-MB-231. Inhibiting cell proliferation (Fig. 2A, Left graph). When L1-95-1 was treated with MDA-MB-231 for triple-negative breast cancer cells, the cell proliferation was inhibited by about 60% at a concentration of 500 nM. When L1-95-1 at a concentration of 1 μM was treated, (Fig. 2A, Right graph). Therefore, it was confirmed that L1-95-1 has specificity for triple negative breast cancer cells in breast cancer cells and effectively inhibits the proliferation of cancer cells 2A).
L1-95-1에 의한 세포증식 억제 효과와 세포주기와의 상관성을 확인하기 위하여 유세포 분석기를 이용하여 실험을 수행하였다. 그 결과 유방암 세포 MCF-7은 각각 500 nM의 L1-95-1 처리에 의해 세포주기의 변화가 관찰되지 않았다(도 2A). 그러나 삼중음성 유방암 세포 MDA-MB-231은 500 nM의 L1-95-1 처리에 의해 G1/G0 세포주기는 감소하고, S 세포주기는 증가하였으며, G2/M 세포주기는 변하지 않는 것을 관찰하였다(도 2B). L1-95-1 처리에 의해서 S 세포주기 집적화(accumulation)로 인하여 세포의 증식이 억제되는 것으로 판단하여 Sub-G1 세포주기를 분석하여 세포사멸(apoptosis) 유도가 일어나는지 예측하고자 하였다. 그러나 L1-95-1에 의해 MCF-7과 MDA-MB-231 세포에서 유의적인 sub-G1 세포주기의 집적화는 관찰되지 않았다(도 2C). 이상의 결과를 종합하면, L1-95-1은 S 세포주기의 집적을 통하여 세포의 증식을 억제하지만 세포사멸(apoptosis) 유도는 일어나지 않는 것으로 확인되었다.In order to confirm the cell proliferation inhibitory effect of L1-95-1 and its correlation with the cell cycle, experiments were carried out using a flow cytometer. As a result, no change in cell cycle was observed by treatment with 500 nM of L1-95-1 in breast cancer cells MCF-7 2A). However, in the triple-negative breast cancer cell line MDA-MB-231, the G1 / G0 cell cycle was decreased, the S cell cycle was increased, and the G2 / M cell cycle was not changed by treatment with 500 nM of L1-95-1 Degree 2B). The cells were incubated with L1-95-1 to inhibit the cell proliferation due to S-cell cycle accumulation, and the sub-G1 cell cycle was analyzed to predict whether induction of apoptosis would occur. However, no significant sub-G1 cell cycle integration was observed in MCF-7 and MDA-MB-231 cells by L1-95-1 2C). These results suggest that L1-95-1 inhibits cell proliferation through accumulation of S-cell cycle but does not induce apoptosis.
<< 실시예Example 3> L1-95-1에 의한 3> by L1-95-1 MDAMDA -MB-231 세포에서의 -MB-231 < / RTI > 오토파지Auto Phage (( autophagyautophagy ) 형성) formation
L1-95-1에 의해 세포의 증식과 암화, 암세포의 증식이 억제되고, S 세포주기의 집적현상이 관찰되지만 세포사멸은 관찰되지 않았다. 따라서 L1-95-1 처리에 의한 유방암 세포 MCF-7과 삼중음성 유방암 세포 MDA-MB-231 세포에서 형태 변화를 관찰하였다. MCF-7 유방암 세포는 L1-95-1 처리에 의해 세포의 형태 변화가 관찰되지 않았다(도 3A). 이에 반하여 MDA-MB-231 세포는 각 500 nM의 L1-95-1 처리에 의해 세포가 길쭉해지는 형태의 변화를 유도하였으며, 세포질에 오토파지(autophagy) 유도시에 의해 주로 관찰되는 작은 액포(vacuole)의 형성이 확인되었다(도 3A). 이를 바탕으로 MCF-7과 MDA-MB-231 세포에서 L1-95-1 처리에 의해 오토파지(autophagy)가 유도되는지 확인하기 위하여 오토파지(autophagy) 마커인 LC-3의 축적을 웨스턴 블랏(Western blotting)으로 확인하였다. 그 결과 유방암 세포 MCF-7에서는 L1-95-1 처리에 의하여 LC3-II의 축적이 일어나지 않은 반면, 삼중음성 유방암 세포 MDA-MB-231에서는 L1-95-1의 농도 증가에 따라 LC3-II의 양적 증가가 관찰되었다(도 3B). 그 변화를 정량화하여 분석한 결과 삼중음성 유방암 세포 MDA-MB-231에서는 L1-95-1의 농도 의존적으로 LC3-II가 점진적으로 증가하였으며, 500 nM의 농도에서 약 5.5배가 증가됨을 확인하였다(도 3B). 오토파지(autophagy)의 형성은 리소좀(lysosome)의 활성화와 연관되어 있음이 잘 알려져 있다. L1-95-1 처리에 의해 세포 내 리소좀(lysosome)의 활성을 검증하기 위하여 산성 상태에 놓인 리소좀(lysosome)을 염색하는 lysotracker를 이용하여 형광현미경으로 관찰하였다. 그 결과 유방암 세포 MCF-7은 500 nM의 L1-95-1 처리에 의해서 세포 내에 lysotracker의 염색이 보이지 않아 리소좀(lysosome)의 활성화가 유도되지 않음을 확인하였다(도 3C). 이와 반대로, 삼중음성 유방암 세포 MDA-MB- 231에서는 500 nM의 L1-95-1 처리에 의해 lysotracker의 염색이 강하게 유도되는 것이 관찰되었다(도 3C). 이러한 결과들을 종합하여 보면, L1-95-1은 삼중음성 유방암 세포에서 리소좀(lysosome)의 활성화와 LC3-II의 집적을 유도하여 오토파지(autophagy)를 유도함을 확인하였다. L1-95-1에 의한 액포(vacuole) 형성이 오토파지(autophagy)임을 확증하기 위하여 mCherry를 세포 내로 도입하고 형광현미경으로 분석하였다. L1-95-1은 유방암 세포 MCF-7에서 GFP와 RFP가 함께 검출되었다. 이와는 반대로 MDA-MB-231 세포의 경우 L1-95-1을 처리하지 않은 대조군 세포에서는 GFP와 RFP가 동시에 관찰되었지만, 500 nM의 L1-95-1을 처리한 MDA-MB-231 세포에서는 리소좀(lysosome)의 산성화에 의해 GFP는 관찰되지 않았고 RFP만 보여 리소좀(lysosome)의 활성화가 유도되어 오토파지(autophagy)가 발생함을 검증하였다(도 3D). 이상의 결과를 종합하면 L1-95-1은 삼중음성 유방암 세포 MDA-MB-231에서 선택적으로 오토파지(autophagy)를 형성하는 것을 증명하였다.L1-95-1 inhibited cell proliferation, carcinogenesis, and cancer cell proliferation, and accumulation of S-cell cycle was observed but no apoptosis was observed. Therefore, morphological changes were observed in L1-95-1-treated breast cancer cell MCF-7 and triple negative breast cancer cell MDA-MB-231 cells. Cell morphology was not observed in MCF-7 breast cancer cells by treatment with L1-95-1 (Fig. 3A). In contrast, MDA-MB-231 cells induced a change in the shape of cells elongated by treatment with 500 nM of L1-95-1, and they were found in the vacuole ) (Fig. 3A). Based on this, accumulation of LC-3, an autophagy marker, was examined by Western blotting to determine whether autophagy was induced by treatment with L1-95-1 in MCF-7 and MDA-MB-231 cells blotting). As a result, the accumulation of LC3-II was not observed in L1-95-1 treatment of breast cancer cell MCF-7, whereas in MDA-MB-231 of triple negative breast cancer cell, LC3-II A quantitative increase was observed (Figure 3B). Quantitative analysis of these changes revealed that LC3-II was gradually increased in MDA-MB-231, a triple-negative breast cancer cell, depending on the concentration of L1-95-1, and increased about 5.5-fold at a concentration of 500 nM 3B). It is well known that the formation of autophagy is associated with the activation of lysosomes. To examine the activity of lysosomes in cells by L1-95-1 treatment, lysotracker staining lysosomes in an acidic condition was observed with a fluorescence microscope. As a result, it was confirmed that lysotracker staining was not observed in the cells of the breast cancer cell MCF-7 by treatment with 500 nM of L1-95-1, and lysosome activation was not induced (Fig. 3C). In contrast, in the triple-negative breast cancer cell line MDA-MB-231, lysotracker staining was strongly induced by treatment with 500 nM of L1-95-1 (FIG. 3C). Taken together, these results indicate that L1-95-1 induces lysosomal activation and aggregation of LC3-II in triple-negative breast cancer cells, leading to autophagy. To confirm that the formation of vacuole by L1-95-1 is autophagy, mCherry was introduced into cells and analyzed by fluorescence microscopy. L1-95-1 detected both GFP and RFP in breast cancer cell MCF-7. In contrast, in MDA-MB-231 cells, GFP and RFP were observed in control cells without L1-95-1, whereas in MDA-MB-231 cells treated with 500 nM of L1-95-1, GFP was not observed by acidification of the lysosome, and only the RFP was visible, and activation of the lysosome was induced to induce autophagy (FIG. 3D). Taken together, these results demonstrate that L1-95-1 selectively forms autophagy in triple-negative breast cancer cell MDA-MB-231.
<< 실시예Example 4> L1-95-1에 의한 4> by L1-95-1 오토파지Auto Phage (( autophagyautophagy ) 유도 신호전달계) Inductive signal transmission system
삼중음성 유방암 세포 MDA-MB-231에서 L1-95-1에 의해 선택적으로 발생하는 오토파지(autophagy) 유도 신호전달계를 유방암 세포 MCF-7에서와 상호 비교 분석하였다. 정상적으로 배양하고 있는 유방암 세포 MCF-7과 삼중음성 유방암 세포 MDA-MB-231에 L1-95-1을 각각 농도 의존적으로 처리하고, 오토파지(autophagy) 유도와 관련된 PI3K-AKT-mTOR 신호전달계와 MAPK 신호전달계를 중심으로 웨스턴 블랏팅(Western blotting)으로 분석하였다. 그 결과, L1-95-1은 MDA-MB-231 세포에서 활성화된 mTORC2에 의해 인산화되는 Akt의 Ser473의 인산화를 농도 의존적으로 억제하였다(도 4A, 상단으로부터 4번째 패널). 이에 상응하여, 활성화된 Akt에 의해 인산화 표적 아미노산인 mTOR kinase의 Ser2448 이미노산의 인산화가 L1-95-1의 농도에 의존적으로 억제됨을 확인하였다(도 4A, 상단으로부터 1번째 레인). mTOR kinase C1의 하류 단백질인 p70S6K의 인산화는 MDA-MB-231에서 관찰되지 않았으나, p70S6K 및 p90RSK의 하류 단백질로 알려진 ribosomal S6 단백질의 Ser235/236 아미노산의 인산화가 L1-95-1의 농도에 의존적으로 억제되는 것을 관찰하였다(도 4A, 상단으로부터 6번째 및 8번째 레인). 또한, p90RSK에 의해 ribosomal S6 단백질의 Ser235/236 인산화가 변화될 수 있어 MAPK 신호전달계의 인산화를 확인하였다. 그 결과, p90RSK의 상위 인산화 효소인 MEK1/2 및 ERK1/2의 인산화 정도는 L1-95-1에 의해 변화되지 않았지만 p90RSK의 인산화는 L1-95-1의 농도에 의존적으로 감소하는 것을 확인하였다(도 4B). Akt/mTOR 신호전달계가 세포 내에서 에너지 감수성에 따라 인산화 및 활성도가 변화하는 것으로 알려져 있어, 세포 내 에너지 평형에 중요한 역할을 수행하는 AMPK의 인산화 정도를 검증하였다. 그 결과 MDA-MB-231세포에서 L1-95-1 처리에 의해 AMPKα의 Thr172 아미노산 인산화가 증가되는 것을 확인하였다(도 4C). 이 결과를 바탕으로, 세포 내의 에너지 균형을 유지하는 기관인 미토콘드리아(mitochondria)에 이상이 생긴 것으로 가설을 세웠다. 이를 검증하기 위하여 유방암 세포 MCF-7과 삼중음성 유방암 세포 MDA-MB-231에 500 nM의 L1-95-1을 각각 처리하고 lysotracker와 mitotracker를 이용하여 염색하여 활성화된 리소좀과 미토콘드리아가 함께 염색되는지를 공초점 현미경을 이용하여 분석하였다. 그 결과 유방암 세포 MCF-7에서는 L1-95-1에 의하여 미토콘드리아는 염색이 되었으나 lysotracker에 의해 활성화된 리소좀은 관찰되지 않았다(도 4D, 상단 왼쪽 패널). 이 결과와는 반대로 삼중음성 유방암 세포 MDA-MB-231에서는 500 nM의 L1-95-1에 의해 활성화된 lysotracker의 염색이 관찰되었으며, lysotracker에 의한 염색이 mitotracker와 동일한 장소에서 염색되는 것을 확인하였다(도 4D, 상단 오른쪽 패널). 활성화된 리소좀과 미토콘드리아가 함께 위치하는 것을 명확히 분석하기 위하여 공초점 현미경 결과를 3D 입체 형상으로 분석하였다. 그 결과 유방암 세포 MCF-7에서는 500 nM의 L1-95-1의 처리에 의해 lysotracker 염색이 관찰되지 않았으며, mitotracker와 핵이 분리되어 서로 다른 층(layer)에 위치하고 있음을 관찰하였다(도 4D, 하단 왼쪽 패널). 유방암 세포 MCF-7와는 달리 삼중음성 유방암 세포 MDA-MB-231에서는 500 nM의 L1-95-1의 처리에 의해 lysotracker에 의한 염색이 명확히 유도됨을 관찰하였으며, lysotracker와 mitotracker에 의한 동시에 염색되어 노란색 또는 오랜지색으로 보이는 functa가 관찰되었다(도 4D, 하단 오른쪽 패널). 더욱이 lysotracker와 mitotracker에 의해 염색된 functa가 삼중음성 유방암 세포 MDA-MB-231에서 선택적으로 관찰되었으며, 노란색 또는 오랜지색 functa가 파랗게 보이는 핵과 다른 layer에서 관찰되었다(도 4D, 하단 오른쪽 패널). 이상의 결과를 종합하면, L1-95-1은 삼중음성 유방암 세포 MDA-MB-231에서 선택적으로 미토파지(mitophagy)를 유도하는 것으로 판단된다. The autophagy-induced signal transduction pathway, which is selectively induced by L1-95-1 in triple-negative breast cancer cells MDA-MB-231, was compared with that of MCF-7 in breast cancer cells. L-95-1 was treated with MDA-MB-231 in triplicate negative breast cancer cell MCF-7 and the PI3K-AKT-mTOR signal transduction system associated with autophagy induction and MAPK And analyzed by Western blotting around a signal transduction system. As a result, L1-95-1 inhibited phosphorylation of Ser473 of Akt phosphorylated by mTORC2 activated in MDA-MB-231 cells in a concentration-dependent manner (Fig. 4A, fourth panel from top). Correspondingly, it was confirmed that the phosphorylation of Ser2448 imino acid of mTOR kinase, the phosphorylation target amino acid, was inhibited by activated Akt in a manner dependent on the concentration of L1-95-1 (Fig. 4A, the first lane from the top). The phosphorylation of p70S6K, a downstream protein of mTOR kinase C1, was not observed in MDA-MB-231 but phosphorylation of the Ser235 / 236 amino acid of the ribosomal S6 protein, known as the downstream protein of p70S6K and p90RSK, was dependent on the concentration of L1-95-1 (Fig. 4A, sixth and eighth lanes from the top). In addition, phosphorylation of the MAPK signaling system was confirmed by the change of Ser235 / 236 phosphorylation of ribosomal S6 protein by p90RSK. As a result, it was confirmed that the degree of phosphorylation of MEK1 / 2 and ERK1 / 2, which are the higher phosphorylating enzymes of p90RSK, was not changed by L1-95-1, but phosphorylation of p90RSK was decreased depending on the concentration of L1-95-1 4B). The Akt / mTOR signaling system has been known to change phosphorylation and activity depending on the energy sensitivity of the cells. Thus, the degree of phosphorylation of AMPK which plays an important role in intracellular energy equilibrium was verified. As a result, it was confirmed that the Thr172 amino acid phosphorylation of AMPKa was increased by L1-95-1 treatment in MDA-MB-231 cells (Fig. 4C). Based on these results, we hypothesized that mitochondria, an organ that maintains the balance of energy within the cell, has developed an abnormality. In order to verify this, 500 nM of L1-95-1 was treated with breast cancer cell MCF-7 and triple negative breast cancer cell MDA-MB-231, respectively, and stained with lysotracker and mitotracker to determine whether activated lysosomes and mitochondria were stained together And analyzed using a confocal microscope. As a result, mitochondria were stained by L1-95-1 in breast cancer cell MCF-7, but lysosomes activated by lysotracker were not observed (Fig. 4D, upper left panel). In contrast to this result, it was observed that lysotracker staining was activated by 500 nM of L1-95-1 in triple-negative breast cancer cell MDA-MB-231, and staining by lysotracker was stained in the same place as mitotracker 4D, upper right panel). Confocal microscopy results were analyzed in 3D solid form to clarify the co-location of activated lysosomes and mitochondria. As a result, lysotracker staining was not observed by treatment with 500 nM of L1-95-1 in breast cancer cell MCF-7, and it was observed that mitotracker and nucleus were separated and located in different layers (Fig. 4D, Bottom left panel). Unlike breast cancer cell MCF-7, lysotracker-induced staining was clearly induced by treatment with 500 nM of L1-95-1 in triple negative breast cancer cells MDA-MB-231, and was stained simultaneously with lysotracker and mitotracker, An orange-colored functa was observed (Fig. 4D, bottom right panel). Furthermore, functa stained with lysotracker and mitotracker was selectively observed in triple-negative breast cancer cell MDA-MB-231, and yellow or orange functa was observed in a different layer from the nucleus showing blue (Fig. 4D, lower right panel). These results suggest that L1-95-1 selectively induces mitophagy in triple-negative breast cancer cells MDA-MB-231.
<< 실시예Example 5> L1-95-1에 의한 5> By L1-95-1 ROSROS 형성과 미토콘드리아 막 전위차 형성에 미치는 영향 And formation of mitochondrial membrane potential
세포 내에는 미토콘드리아가 손상을 받아 미토콘드리아 내막에서 에너지를 효과적으로 생산하지 못하는 경우 손상된 미토콘드리아를 제거하기 위한 미토파지(mitophagy)를 유도한다는 사실이 알려져 있다. 따라서 삼중음성 유방암 세포 MDA-MB-231에서 L1-95-1에 의한 미토파지(mitophagy) 형성이 미토콘드리아의 손상과 관련이 있을 것으로 가설을 세우고, L1-95-1 처리에 의한 미토콘드리아에서의 ROS 생성을 mitosox로 염색한 후 공초점 현미경을 이용하여 관찰하였다. 예측했던 실험 결과와 동일하게 L1-95-1은 유방암 세포 MCF-7에서 염색되지 않았으며, 온전히 mitotracker에 의해서만 염색되었다(도 5A, 왼쪽 패널). 유방암 세포 MCF-7와는 반대로, 삼중음성 유방암 세포 MDA-MB-231에서는 500 nM의 L1-95-1에 의하여 mitosox에 의한 염색이 관찰하였으며, mitosox에 의한 염색이 mitotracker와 동일한 자리에서 함께 염색되는 것을 관찰하였다(도 5A, 오른쪽 패널). 삼중음성 유방암 세포 MDA-MB-231 세포에서 L1-95-1에 의해 생성된 ROS가 미토콘드리아의 손상을 유도하여 미토콘드리아 내막의 전위차에 영향을 주는지를 관찰하였다. 그 결과, 유방암 세포 MCF-7에서 JC-1/red의 염색이 MDA-MD-231 세포에 비하여 약하게 염색되었으며, JC-1/green 염색은 두 세포에서 동일하게 염색되었다(도 5B, 왼쪽 패널). 그러나 L1-95-1 처리에 의하여 JC-1/red 염색이 유방암 세포 MCF-7과 삼중음성 유방암 세포 MDA-MB-231 세포에서 감소하지 않았으며, L1-95-1을 처리하지 않은 대조군과 비교하여 양적 변화는 관찰되지 않았다(도 5B). 그러나 CCCP-1 처리에 의해서는 JC-1/red의 염색이 유방암 세포 MCF-7과 삼중음성 유방암 세포 MDA-MB-231 세포에서 사라짐이 관찰되어 실험이 정상적으로 수행되었음을 확인하였다(도 5B).It is known that if mitochondria are damaged in the cells and they do not produce energy efficiently in mitochondrial inner membrane, they induce mitophagy to remove damaged mitochondria. Therefore, it is hypothesized that mitophagy formation by L1-95-1 in triple-negative breast cancer cells MDA-MB-231 may be related to mitochondrial damage, and ROS production in mitochondria by L1-95-1 treatment Were stained with mitosox and observed using a confocal microscope. Like the predicted experimental results, L1-95-1 was not stained in breast cancer cell MCF-7 and stained only by mitotracker (Figure 5A, left panel). In contrast to breast cancer cell MCF-7, mitosoxin staining was observed with 500 nM of L1-95-1 in triple negative breast cancer cell MDA-MB-231, and mitosoxin staining was stained together with the mitotracker (Fig. 5A, right panel). We examined whether ROS produced by L1-95-1 in triple-negative breast cancer cells MDA-MB-231 cells induces mitochondrial damage and affects the potential difference of the mitochondrial inner membrane. As a result, JC-1 / red staining was weakly stained in MCF-7 breast cancer cell, MDA-MD-231 cells, and JC-1 / green staining was stained in both cells (FIG. 5B, left panel) . However, treatment with L1-95-1 did not reduce JC-1 / red staining in breast cancer cells MCF-7 and triple-negative breast cancer cells MDA-MB-231 cells, and compared with the control group without L1-95-1 No quantitative changes were observed (FIG. 5B). However, the CCCP-1 treatment showed that the JC-1 / red staining disappeared from the breast cancer cell MCF-7 and the triple-negative breast cancer cell MDA-MB-231 cells (FIG. 5B).
이상의 결과를 요약하면, L1-95-1은 삼중음성 유방암 세포 MDA-MB-231의 미토콘드리아에서 ROS 발생을 유도하며, ROS의 생성과 미토파지(mitophagy)의 유도가 서로 밀접한 관계가 있는 것으로 판단된다. 그러나 미토콘드리아에서 발생한 ROS가 미토콘드리아 내막에서 전위차는 발생시키지 않는 것으로 생각된다.These results suggest that L1-95-1 induces ROS generation in mitochondria of triple-negative breast cancer cell MDA-MB-231, and ROS production and induction of mitophagy are closely related to each other . However, it is thought that ROS originating from the mitochondria does not cause a potential difference in the mitochondrial inner membrane.
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