KR20230051965A - Composition for Enhancing Efficacy of Radiotherapy of Cancer Comprising Butyrate as Active Ingredient - Google Patents
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Abstract
Description
본 발명은 부티르산를 유효성분으로 포함하는 암의 방사선 치료 감수성 향상용 조성물에 관한 것이다. The present invention relates to a composition for improving sensitivity to radiotherapy of cancer, comprising butyric acid as an active ingredient.
암은 세계적으로 사망률 1위의 질환으로 암의 치료와 생존율 향상을 위해 다양한 치료법이 시도되고 있다. 암의 치료에 널리 사용되고 있는 방사선치료법은 주변 정상조직의 손상들을 일으키는 심각한 부작용을 일으킬 수 있어 이러한 부작용을 줄이고 암치료 효율을 높일 수 있는 새로운 전략이 요구되고 있는 상황이다. 치료 효율성을 높이기 위한 다양한 접근방법중 하나로 주목받고 있는 것이 방사선 민감성을 높이는 새롭고 안전한 방사선 치료 민감제(radiosensitizer)의 사용을 통해 동일한 치료효과를 좀 더 낮고 안전한 양의 방사선치료로 얻어내는 방법이다. 기존 다양한 연구결과를 통해 Fluorouracil (5-FU)를 비롯하여 다양한 방사선 치료 민감제가 연구되어 사용해 왔으나 여전히 정상세포의 손상으로 인한 부작용은 계속되고 있는 상태이다. 따라서 부작용을 최소화하고 암치료효과를 높일 수 있는 안전한 약제의 개발의 필요성은 계속되고 있는 상태이다. Cancer is a disease with the highest mortality worldwide, and various therapies are being attempted to treat cancer and improve survival rates. Radiation therapy, which is widely used in the treatment of cancer, can cause serious side effects that cause damage to surrounding normal tissues, and thus, a new strategy capable of reducing these side effects and increasing the efficiency of cancer treatment is required. One of the various approaches to increase treatment efficiency is a method of obtaining the same treatment effect with a lower and safer amount of radiation treatment through the use of a new and safe radiosensitizer that increases radiation sensitivity. Various radiotherapy sensitizers, including Fluorouracil (5-FU), have been studied and used through various research results, but side effects due to damage to normal cells still continue. Therefore, there is an ongoing need to develop safe drugs capable of minimizing side effects and increasing cancer treatment effects.
탄소수가 6개 이하로 작은 지방산인 짧은사슬지방산(Short chain fatty acids, SCFAs)은 장내 미생물의 주요 대사 산물로 소장까지 소화 및 흡수되지 않은 탄수화물을 대사하는 과정 중에 생성된다. 장에서 검출되는 짧은사슬지방산은 아세트산(acetate, acetic acid), 프로피온산(propionate, propionic acid), 부티르산(butyrate, butyric acid)이 있으며 주로 장관내 pH를 낮춰 산성 환경을 유지하여 유해균의 정착을 막고, 영양분 흡수를 돕는 것으로 알려져 있다. Short chain fatty acids (SCFAs), which are small fatty acids with less than 6 carbon atoms, are major metabolites of intestinal microorganisms and are produced during the metabolism of undigested and unabsorbed carbohydrates to the small intestine. Short-chain fatty acids detected in the intestine include acetic acid (acetate, acetic acid), propionate (propionic acid), and butyrate (butyric acid). They mainly lower the pH in the intestine to maintain an acidic environment to prevent the establishment of harmful bacteria, It is known to aid in nutrient absorption.
본 발명자들은 암의 방사선 치료 감수성을 높이는 물질을 개발하기 위해 연구 노력한 결과, 장내 미생물의 주요대사산물인 짧은사슬지방산(Short Chain Fatty Acids, SCFAs) 중 하나인 부티르산(butyrate)이 방사선 치료의 감수성을 향상시킨다는 것을 암환자유래 조직 오가노이드를 사용하여 규명하였다. As a result of research efforts to develop a substance that increases the sensitivity of cancer radiation therapy, the present inventors found that butyrate, one of the short chain fatty acids (SCFAs), which is a major metabolite of intestinal microbes, increases the sensitivity of radiation therapy. It was identified using tissue organoids derived from cancer patients.
따라서, 본 발명의 목적은 부티르산을 유효성분으로 포함하는 암의 방사선 치료 감수성 향상용 조성물을 제공하는 것에 있다. Accordingly, an object of the present invention is to provide a composition for improving sensitivity to radiotherapy of cancer containing butyric acid as an active ingredient.
본 발명의 다른 목적은 부티르산을 유효성분으로 포함하는 암의 방사선 치료 감수성 향상용 약학적 조성물을 제공하는 것에 있다. Another object of the present invention is to provide a pharmaceutical composition for improving sensitivity to radiotherapy of cancer containing butyric acid as an active ingredient.
본 발명의 일 양태에 따르면, 부티르산(butyrate)을 유효성분으로 포함하는 암의 방사선 치료 감수성 향상용 조성물을 제공한다. According to one aspect of the present invention, there is provided a composition for improving cancer radiation therapy sensitivity comprising butyrate as an active ingredient.
본 명세서에서 용어 "방사선 치료"는 방사선을 조사하여 암 세포의 DNA 손상을 통해 세포분열을 억제함으로써 세포사멸을 유도하는 기전을 통해 암을 치료하는 치료 방법을 의미한다. 상기 방사선 치료는 고형암의 근치적(curative), 보조적(adjuvant), 고식적(palliative) 목적으로 단독으로 또는 수술적 치료와 함께, 또는 항암 화학요법이나 방사선 민감제(radiation sensitizer)와의 병용 요법으로 시행될 수 있다. As used herein, the term "radiation therapy" refers to a treatment method of treating cancer through a mechanism of inducing apoptosis by irradiating radiation to inhibit cell division through DNA damage of cancer cells. The radiation therapy may be performed alone or together with surgical treatment for curative, adjuvant, and palliative purposes of solid cancer, or as combination therapy with chemotherapy or radiation sensitizer. can
본 명세서에서 사용되는 용어, "방사선 조사(irradiation)"란, 방사선의 치료적 작용에 대한 노출을 의미한다. As used herein, the term "irradiation" refers to exposure to the therapeutic action of radiation.
본 명세서에서 사용되는 용어 "방사선"은 원자핵에서 방출되는 특정한 전자기파 또는 고에너지를 갖는 미세한 입자를 의미하며, 예를 들면, X-선, 알파선, 감마선, 베타선, 중성자선, 양성자선을 포함할 수 있다. As used herein, the term "radiation" refers to specific electromagnetic waves emitted from atomic nuclei or fine particles having high energy, and may include, for example, X-rays, alpha rays, gamma rays, beta rays, neutron rays, and proton rays. .
본 명세서에서 사용되는 용어 "방사선 치료 감수성"은 세포가 방사선 조사에 의해 쉽게 영향을 받으며, 방사선에 노출시 세포 복구 또는 증식에 변화를 보이는 상태를 의미한다. As used herein, the term "radiation treatment sensitivity" refers to a state in which cells are easily affected by radiation and show changes in cell repair or proliferation when exposed to radiation.
본 명세서에서 사용되는 용어 "방사선 치료 감수성 향상"은 암 환자를 대상으로 방사선치료를 수행할 때, 방사선 치료와 동시, 방사선 치료 전, 또는 방사선 치료 후에 복용 또는 투여함으로써 치료 효과를 향상시키는 행위를 의미한다. As used herein, the term "improvement in radiation therapy sensitivity" refers to an act of improving a therapeutic effect by taking or administering radiation therapy to cancer patients simultaneously with radiation therapy, before radiation therapy, or after radiation therapy. do.
본 발명에서 치료 대상 질환인 "암"은 종양, 신생물과 상호 호환적으로 사용되며, 생장, 증식 또는 생존이 대조군으로 활용되는 정상 세포의 생장, 증식 또는 생존 보다 우수한 세포 또는 세포 집단, 예를 들어, 세포 증식성 또는 분화성 장애를 의미한다. 전형적으로, 상기 생장은 통제를 벗어난다. 구체적으로 상기 암은 유방암, 폐암, 대장암, 전립선암, 비소세포성 폐암, 결장암, 골암, 췌장암, 피부암, 두부 또는 경부 암, 피부 또는 안구내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문부근암, 결장암, 나팔관암, 자궁내막암, 자궁경부암, 질암, 음문암, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS; central nervous system) 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종 등일 수 있으며, 구체적으로 대장암일 수 있다. In the present invention, "cancer", which is a disease to be treated, is used interchangeably with tumor and neoplasia, and is a cell or cell population whose growth, proliferation, or survival is superior to that of normal cells used as a control group, such as For example, it means a cell proliferative or differentiation disorder. Typically, the growth gets out of control. Specifically, the cancer is breast cancer, lung cancer, colon cancer, prostate cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anus Adrenal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, or It may be a pituitary adenoma or the like, and specifically, it may be colon cancer.
일 구현예에서, 상기 암의 암세포는 FOXO3A 유전자가 발현되는 암세포일 수 있다. In one embodiment, the cancer cell of the cancer may be a cancer cell in which the FOXO3A gene is expressed.
본 발명의 다른 양태에 따르면, (i) 부티르산(butyrate)의 약학적 유효량 및 (ii) 약학적으로 허용되는 담체를 포함하는 암에 대한 방사선 치료 감수성 향상용 약학적 조성물을 제공한다. According to another aspect of the present invention, there is provided a pharmaceutical composition for improving sensitivity to radiation therapy for cancer, comprising (i) a pharmaceutically effective amount of butyrate and (ii) a pharmaceutically acceptable carrier.
본 발명의 암에 대한 방사선 치료 감수성 향상용 조성물은 약학적 조성물의 형태로 제공될 수 있다. The composition for improving sensitivity to radiation therapy for cancer of the present invention may be provided in the form of a pharmaceutical composition.
본 발명의 부티르산은 방사선의 암 치료의 감수성을 향상시키므로 방사선을 이용하는 항암 치료에 치료 효율을 향상시키는 보조적 약물로 유용하게 사용될 수 있다. Since the butyric acid of the present invention improves the sensitivity of radiation cancer treatment, it can be usefully used as an adjuvant drug to improve the treatment efficiency of cancer treatment using radiation.
본 발명에서 "부티르산의 약학적 유효량"은 부티르산을 투여하여 치료적 효능을 발휘하는 적절하고 최소한의 양을 의미한다. In the present invention, "pharmaceutically effective amount of butyric acid" means an appropriate and minimal amount that exhibits therapeutic efficacy by administering butyric acid.
본 발명의 약학적 조성물은 상기 활성성분인 부티르산 이외에 약제학적으로 허용되는 담체를 추가로 포함할 수 있으며, 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 탄수화물류 화합물(예: 락토스, 아밀로스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 셀룰로스, 등), 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 염 용액, 알코올, 아라비아 고무, 식물성 기름(예: 옥수수 기름, 목화 종자유, 두유, 올리브유, 코코넛유), 폴리에틸렌 글리콜, 메틸 셀룰로스, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier in addition to the active ingredient, butyric acid, and the pharmaceutically acceptable carrier is one commonly used in formulation and includes a carbohydrate compound (e.g., lactose). , amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc.), acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup , salt solution, alcohol, gum arabic, vegetable oil (e.g. corn oil, cottonseed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, stearic acid magnesium and mineral oil; and the like, but are not limited thereto.
본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 문헌 (Remington's Pharmaceutical Science, 최근, Mack Publishing Company, Easton PA)에 상세히 기재되어 있다. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Science (currently, Mack Publishing Company, Easton PA).
본 발명의 약학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity. It can be.
한편, 본 발명의 약학적 조성물의 경구 투여량은 바람직하게는 1일 당 0.00001 - 1000 mg/kg(체중)이다. On the other hand, the oral dosage of the pharmaceutical composition of the present invention is preferably 0.00001 - 1000 mg/kg (body weight) per day.
본 발명의 약학적 조성물은 조사되는 방사선의 세기, 방사선에 대한 암의 치료 효과, 또는 정상 세포에 대한 손상 등을 감안하여 적절한 투여량을 결정할 수 있다. An appropriate dose of the pharmaceutical composition of the present invention may be determined in consideration of the intensity of radiation, the cancer treatment effect of radiation, or damage to normal cells.
본 발명의 약학적 조성물은 경구 또는 비경구로 투여할 수 있다. 비경구로 투여되는 경우, 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 투여 등으로 투여할 수 있다. The pharmaceutical composition of the present invention may be administered orally or parenterally. In the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.
본 발명의 약학적 조성물이 암에 대한 방사선 감수성을 향상시키는 것을 목적으로 투여되는 것을 고려하면, 암세포에 대한 방사선 조사의 전, 후 또는 동시에 투여될 수 있다. Considering that the pharmaceutical composition of the present invention is administered for the purpose of improving radiation sensitivity to cancer, it may be administered before, after, or simultaneously with radiation to cancer cells.
본 발명의 약학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.
일 구현에서, 본 발명의 약학적 조성물의 치료 대상 암은 유방암, 폐암, 대장암, 전립선암, 비소세포성 폐암, 결장암, 골암, 췌장암, 피부암, 두부 또는 경부 암, 피부 또는 안구내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문부근암, 결장암, 나팔관암, 자궁내막암, 자궁경부암, 질암, 음문암, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS; central nervous system) 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종일 수 있다. In one embodiment, the cancer to be treated by the pharmaceutical composition of the present invention is breast cancer, lung cancer, colorectal cancer, prostate cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, Uterine cancer, ovarian cancer, rectal cancer, gastric cancer, perianal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer , adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary center It can be a lymphoma of the nervous system, a tumor of the spinal cord, a brainstem glioma or a pituitary adenoma.
일 구현예에서, 본 발명의 약학적 조성물이 적용되는 상기 방사선은 X-선, 알파선, 감마선, 베타선, 중성자선 또는 양성자선일 수 있다. In one embodiment, the radiation to which the pharmaceutical composition of the present invention is applied may be X-rays, alpha rays, gamma rays, beta rays, neutron rays or proton rays.
일 구현예에서, 본 발명의 약학적 조성물이 적용되는 상기 암의 암세포는 FOXO3A 유전자가 발현되는 암세포일 수 있다. In one embodiment, the cancer cell of the cancer to which the pharmaceutical composition of the present invention is applied may be a cancer cell in which the FOXO3A gene is expressed.
본 발명의 약학적 조성물과 상술한 본 발명의 일 양태인 방사선 치료 감수성 향상용 조성물 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위해 그 기재를 생략한다. Descriptions of common contents between the pharmaceutical composition of the present invention and the composition for improving sensitivity to radiation therapy, which is one aspect of the present invention described above, are omitted to avoid excessive complexity in the present specification.
본 발명은 부티르산을 유효성분으로 포함하는 암의 방사선 치료 감수성 향상용 조성물에 관한 것이다. 본 발명의 부티르산은 정상세포에는 영향을 주지 않고 암세포에만 선택적으로 방사선 치료 감수성 향상 효과를 보여주므로, 방사선 치료 효과를 높일 수 있는 방사선 치료 민감제로 효과적으로 활용될 수 있다. The present invention relates to a composition for improving cancer radiation therapy sensitivity, comprising butyric acid as an active ingredient. Since butyric acid of the present invention shows an effect of selectively improving radiation therapy sensitivity only to cancer cells without affecting normal cells, it can be effectively used as a radiation therapy sensitizer that can increase the radiation therapy effect.
다만, 본 출원의 효과는 상기에서 언급한 효과로 제한되지 아니하며, 언급되지 않은 또 다른 효과들은 하기의 기재로부터 당업자에게 명확히 이해될 수 있을 것이다. However, the effects of the present application are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.
도 1은 부티레이트(butyrate)가 CRCPDO에 선택적 항암 효과를 나타내는 것을 보여주는 도면이다. (A) 알려진 주요 암 유발인자 유전자의 대부분을 포함하여 5번과 10번 계대에 속하는 CRC-PDO #60 및 CRC-조직 #60의 체세포암 유발인자 변경의 종양 플롯(Oncoplot). (B) 상응하는 H&E 염색과 함께 CK19, CK20 및 CDX2에 대한 오르가노이드 및 상응하는 조직의 FFPE 섹션의 면역조직화학적 프로파일. 배율, x40. 스케일 바(bar), 50μm. (C) CHIR99021 및 R-spondin(CR+)이 있거나 없는(CR) normalPDO #8 및 CRCPDO #3 형태의 이미지. 스케일 바, 400μm. (D) normal PDO #8 및 CRCPDO #3의 형광 현미경 이미지. 파란색, DAPI; 빨간색, Ki67; 녹색, Ecadherin. 스케일 바, 100μm. (E) 3일 후 SCFA로 처리된 CRCPDO #3의 형태. 스케일 바, 200μm. (F) SCFA 치료 3일 후 상대적인 CRCPDO #3 크기(n=3). 데이터는 평균 ± SEM으로 표시함. *P<0.05, **P<0.01. (G) 3일 후 SCFA로 처리된 normal-PDO #8의 형태. 스케일 바, 200μm. (H) SCFA 치료 3일 후의 상대적 normal-PDO #8 크기(n=3). 데이터는 평균±평균의 표준오차로 표시함. *P<0.05, **P<0.01. ns (유의성 없음); H&E, 헤마톡실린 및 에오신; SCFA, 단쇄 지방산; CRC, 결장직장암; PDO, 환자 유래 오르가노이드; CK, 사이토케라틴.
도 2는 부티레이트가 CRCPDO에서 방사선 감수성을 향상시킨다는 것을 보여준다. (A) 5 Gy로 3회 조사된 CRCPDO #7의 부티레이트 유무에 따른 형태. (B) (A)의 오르가노이드 크기(n=3). 스케일 바, 200μm. (C) (A)의 오르가노이드의 MTT 세포 생존능 분석 (n=3). (D) 2차 계대 후 오르가노이드의 이미지. 스케일 바, 400μm. (E) S 단계에서 CRCPDO의 백분율(n=3). (F) 왼쪽: 5Gy로 3회 조사된 오르가노이드의 부티레이트 유무에 따른 형광 현미경 이미지. 파란색, DAPI; 빨간색, Ki67. 스케일 바, 200μm. 오른쪽: DAPI 염색 세포당 Ki67 양성 세포를 나타내는 통계 분석(n=3). (G) γH2AX 초점을 보여주는 형광 현미경 이미지. 파란색, DAPI; 녹색, γH2AX. 오른쪽: γH2AX 초점을 나타내는 통계 분석(n=3). (H) CRC-PDO에서 PARP, cPARP, caspase 3, ccaspase 3, pJNK, JNK, pp38 및 p38의 발현 수준. β-액틴은 대조군. 데이터는 평균±평균의 표준오차로 표시. *P<0.05, **P<0.01. CRC, 결장직장암; PDO, 환자 유래 오르가노이드; PARP, poly-ADP-ribose polymerase; c-, cleaved; p-, 인산화; IR, 조사
도 3은 부티레이트가 normal-PDO의 방사선 감수성에는 영향을 미치지 않는 것을 보여주는 결과들이다. (A) 오르가노이드 형태 및 (B) 5 Gy로 3회 조사된 normalPDO #8의 부티레이트 유무에 따른 오르가노이드 크기(n=3). 스케일 바, 400μm. (C) (A)의 오르가노이드의 MTT 세포 생존능 분석 결과 (n=3). (D) 2차 계대 후 오르가노이드의 이미지. 스케일 바, 400μm. 데이터는 평균±평균의 표준오차로 표시. ns, 유의성 없음; PDO, 환자 유래 오르가노이드; IR, 조사.
도 4는 부티레이트의 방사선 감수성 향상 효과는 포도당 농도에 따라 차이가있음을 보여주는 결과이다. (A) 저혈당 또는 고혈당 조건에서 성장한 CRCPDO #7의 젖산 수치(n=3). (B) 부티레이트가 있거나 없는 5Gy로 3회 조사된 저- 또는 고- 포도당 조건에서 성장한 CRCPDO #7. 스케일 바, 400μm. (C) (B)에 나타낸 오르가노이드의 MTT 세포 생존능 분석(n=3). (D) 2차 계대 후 오르가노이드의 이미지. 스케일 바, 1,000μm. (E) 저- 또는 고-포도당 조건에서 배양된 CRCPDO #7에 EdU 혼입(incorporation)의 형광 현미경 이미지 *P<0.05, **P<0.01. 스케일 바, 200μm. 파란색, DAPI; 레드, EdU. CRC, 결장직장암; PDO, 환자 유래 오르가노이드; IR, 조사.
도 5는 부티레이트가 FOXO3A를 통해 CRCPDO의 방사선 감수성 향상 효과를 발휘하는 것을 보여주는 결과이다. (A) FOXO 억제제에 따른 IR 및 부티레이트로 처리된 CRCPDO #7의 형태 및 (B) 오르가노이드 크기(n=3). 스케일 바, 200μm. (C) (A)에 기재된 오르가노이드의 MTT 세포 생존능 분석결과 (n=3). (D) 2차 계대 후 오르가노이드의 이미지. 스케일 바, 400μm. (E) 왼쪽: FOXO 억제제의 유무에 따른 IR 및 부티레이트로 처리된 오르가노이드의 형광 현미경 이미지. 파란색, DAPI; 빨간색, Ki67. 오른쪽: Ki67 양성 오르가노이드를 나타내는 통계 분석(n=3). (F)역전사-정량 PCR에 의해 분석된 FOXO 억제제의 유무에 따른 IR 및 부티레이트로 처리된 CRC-PDO #7의 세포주기 관련 유전자 발현 수준. 표적 mRNA의 수준은 GAPDH mRNA의 수준으로 정규화됨(n=3). 데이터는 평균±평균의 표준 오차로 표시. *P<0.05, **P<0.01. CRC, 결장직장암; PDO, 환자 유래 오르가노이드; IR, 조사.
도 6은 부티레이트의 방사선 감수성 향상 효과는 CRCPDO의 FOXO3A 발현 수준에 따라 다르게 나타남을 보여주는 결과이다. (A) 5Gy로 3회 조사된 CRCPDO #7의 부티레이트 유무에 따른 이미지. 스케일 바, 200μm. (B) 부티레이트 반응성-CRC-PDO #7 및 비반응성-CRC-PDO #11에 EdU 혼입의 형광현미경 이미지. 스케일 바, 200μm. 파란색, DAPI; 빨간색, EdU. (C) 반응성-CRC-PDO #7 및 비반응성-CRC-PDO #11 조직내 FOXO3a의 IHC 및 형광 현미경 이미지, 면역조직화학 스케일 바, 50μm; 형광 현미경 스케일 바, 100 μm. 파란색, DAPI; 빨간색, FOXO3A. (D) CRCPDO의 핵 분획에서 FOXO3A의 단백질 발현양. LaminB는 핵 분획에 대한 마커임. (E) 반응성-CRC-PDO의 5가지 경우와 비반응성-CRC-PDO의 3가지 경우에서 FOXO3A 발현의 Box-and-whisker 플롯. (F) CRCPDO #7을 24시간에 수득하고 세포 주기 관련 유전자에 대해 역전사 정량-PCR로 분석함. 표적 mRNA의 발현 수준은 GAPDH mRNA의 발현 수준으로 정규화함(n=3). 데이터는 평균±평균의 표준오차로 표시. *P<0.05, **P<0.01. CRC, 결장직장암; PDO, 환자 유래 오르가노이드; IR, 조사; IHC, 면역조직화학. 1 is a diagram showing that butyrate exhibits a selective anticancer effect on CRCPDO. (A) Tumor plots (Oncoplot) of somatic carcinoma inducer alterations in CRC-PDO #60 and CRC-
Figure 2 shows that butyrate enhances radiosensitivity in CRCPDO. (A) Morphology with and without butyrate of
3 shows results showing that butyrate does not affect the radiation sensitivity of normal-PDO. (A) Organoid morphology and (B) organoid size with and without butyrate in
Figure 4 is a result showing that the radiosensitivity enhancing effect of butyrate is different depending on the glucose concentration. (A) Lactate levels of
5 is a result showing that butyrate exerts the effect of improving the radiation sensitivity of CRCPDO through FOXO3A. (A) Morphology and (B) organoid size (n=3) of
6 is a result showing that the effect of improving radiosensitivity of butyrate appears differently depending on the FOXO3A expression level of CRCPDO. (A) Images with and without butyrate of
이하, 본 출원을 실시예에 의하여 상세히 설명한다. 단, 하기 실시예는 본 출원을 구체적으로 예시하는 것이며, 본 출원의 내용이 하기 실시예에 의해 한정되지 아니한다. Hereinafter, the present application will be described in detail by examples. However, the following examples specifically illustrate the present application, and the content of the present application is not limited by the following examples.
실시예 Example
실험재료 및 실험 방법 Test materials and test methods
1. 암 세포 샘플 1. Cancer cell samples
2017년 12월부터 2018년 5월 사이에 원자력병원(서울, 한국)에서 CRC로 진단된 8명의 환자로부터 총 7개의 직장암 샘플과 1개의 대장암 종양 샘플 및 이와 짝을 이루는 건강한 조직 샘플을 얻었다. 모든 종양은 American Joint Committee on Cancer의 병리학적 종양/결절/전이 분류(8판)에 따라 병기(stage)를 분류하였다. 외과적으로 절제된 내시경 생검 샘플은 공지된 방법에 따라 헤마톡실린(hematoxylin) 및 에오신(eosin) 염색을 통해 병리학적으로 확인하였다. 7개의 직장 샘플은 내시경 생검에서 얻었고, 1개의 대장암 샘플은 하부 전방 절제술로 얻었다. 환자의 평균 연령은 61.5세(범위, 53-85세)이었고, 남자가 4명, 여자가 4명이었다. Between December 2017 and May 2018, a total of 7 rectal cancer samples, 1 colorectal cancer tumor sample, and a paired healthy tissue sample were obtained from 8 patients diagnosed with CRC at Atomic Energy Hospital (Seoul, Korea). All tumors were staged according to the pathological tumor/nodule/metastasis classification of the American Joint Committee on Cancer (8th edition). Surgically excised endoscopic biopsy samples were pathologically confirmed by hematoxylin and eosin staining according to a known method. Seven rectal samples were obtained by endoscopic biopsy and one colorectal sample was obtained by lower anterior resection. The mean age of the patients was 61.5 years (range, 53-85 years), and there were 4 males and 4 females.
2. 오가노이드 배양(organoid culture) 2. Organoid culture
종양 및 이에 인접한 정상 조직의 두 가지 모두를 수집하고, 이것으로부터 건강한 선와(crypt) 및 종양 세포를 분리하였다. 간략하게 설명하면, 정상 장 조직 단편을 얼음 위에서 20분 동안 8mM EDTA-인산염완충식염수(PBS)에서 배양하고, 37℃에서 8분 동안 추가로 배양하였다. 이러한 분해 조건하에서 결장 선와(colonic crypt)는 약하게 분해되어 바닥 부분과 상단 부분으로 물리적으로 분리되었다. 암 조직을 II형 콜라게나제(SigmaAldrich; Merck KGaA), II형 디스파제(dispase)(Roche Applied Science) 및 Y27632(BioVision, Inc.)와 함께 37℃에서 30분 동안 배양하였다. Both the tumor and its adjacent normal tissue were collected, from which healthy crypts and tumor cells were isolated. Briefly, normal intestinal tissue fragments were incubated in 8 mM EDTA-phosphate buffered saline (PBS) for 20 min on ice and further incubated for 8 min at 37°C. Under these disintegration conditions, the colonic crypt was weakly disintegrated and physically separated into a bottom and top portion. Cancer tissues were incubated at 37° C. for 30 minutes with type II collagenase (SigmaAldrich; Merck KGaA), type II dispase (Roche Applied Science) and Y27632 (BioVision, Inc.).
분리된 선와(crypt) 및 세포를 PBS로 세척하고 실온에서 3분 동안 300xg에서 원심분리하였다. 이어서, 세포를 얼음 위의 Matrigel(성장 인자 감소, 페놀 레드 없음, Corning, Inc.)에 포매하고, 24웰 플레이트에 분주한 다음, 배양 배지를 추가하였다. CRC-PDO 배양 배지의 조성은 1xB27(Gibco; Thermo Fisher Scientific, Inc.), 1.25mM N-아세틸시스테인(United States Pharmacopeia), 50ng/ml 인간 EGF(BioVision, Inc.), 50ng/ml 인간 Noggin(PeproTech, Inc.), 10nM 가스트린(gastrin)(SigmaAldrich; Merck KGaA), 500nM A8301(BioVision, Inc.) 및 100mg/ml 프리모신(InvivoGen)으로 사용하였다. The isolated crypts and cells were washed with PBS and centrifuged at 300xg for 3 minutes at room temperature. Cells were then embedded in Matrigel (growth factor reduced, no phenol red, Corning, Inc.) on ice, seeded into 24-well plates, and culture medium was added. The composition of the CRC-PDO culture medium was 1xB27 (Gibco; Thermo Fisher Scientific, Inc.), 1.25mM N-acetylcysteine (United States Pharmacopeia), 50ng/ml human EGF (BioVision, Inc.), 50ng/ml human Noggin ( PeproTech, Inc.), 10 nM gastrin (SigmaAldrich; Merck KGaA), 500 nM A8301 (BioVision, Inc.) and 100 mg/ml primosin (InvivoGen).
와버그(Warburg) 효과를 배제한 실험에서 오르가노이드는 동일한 조성에서, 고포도당(3.151g/l)에서 저포도당(1g/l) 배지로 성장시켰다. Wnt 신호 관련 인자는 건강한 결장 오르가노이드(정상-PDO)의 성장에 필수적이었다. 그러나, 대부분의 경우 CRC는 Wnt 신호 전달 경로를 비정상적으로 활성화하는 돌연변이와 관련이 있다. 따라서, 종양세포 선택을 위해 CRC-PDO를 Wnt 관련 인자없이 배양 배지에서 배양하여 정상 조직 유래 오르가노이드가 종양조직을 오염시키지 않도록 하였다. 정상 PDO를 IntestiCultTM Organoid Growth Medium(Stemcell Technologies, Inc.) 및 CHIR99021(Stemgent; ReproCELL) 및 Rspondin1(R&D Systems, Inc.)이 포함된 CRCPDO 배양 배지에서 배양하였다. 아노이키스(anoikis)를 방지하기 위해 10μM Y-27632를 최초 2-3일 동안 배양 배지에 첨가하였다. 오르가노이드의 유지 및 동결은 공지된 방법을 약간 수정하여 수행하였다. 오르가노이드가 >200μm일 때에, 제조사의 지침에 따라 Gentle Cell Dissociation Reagent(Stemcell Technologies, Inc.)를 사용하여 피펫팅하여 계대하였다. In experiments excluding the Warburg effect, organoids were grown in high glucose (3.151 g/l) to low glucose (1 g/l) medium at the same composition. Wnt signaling-related factors were essential for the growth of healthy colon organoids (normal-PDO). However, in most cases CRC is associated with mutations that aberrantly activate the Wnt signaling pathway. Therefore, for tumor cell selection, CRC-PDO was cultured in a culture medium without Wnt-related factors to prevent normal tissue-derived organoids from contaminating the tumor tissue. Normal PDO was cultured in CRCPDO culture medium containing IntestiCult TM Organoid Growth Medium (Stemcell Technologies, Inc.) and CHIR99021 (Stemgent; ReproCELL) and Rspondin1 (R&D Systems, Inc.). To prevent anoikis, 10 μM Y-27632 was added to the culture medium for the first 2-3 days. Maintenance and freezing of organoids was performed using known methods with minor modifications. When organoids were >200 μm, they were passaged by pipetting using Gentle Cell Dissociation Reagent (Stemcell Technologies, Inc.) according to the manufacturer's instructions.
3. 오가노이드 생존능 측정 실험 3. Experiments to measure organoid viability
단일 세포로의 추가 분리를 위해 오르가노이드를 p200 피펫으로 피펫팅하여 TrypLE Express(Thermo Fisher Scientific, Inc.)에 재현탁하고 37℃에서 10분 동안 배양하였다. 이어서, 세포를 수회 피펫팅하여 균질한 재현탁액으로 형성하고, 4℃에서 600xg에서 5분 동안 원심분리한 다음 상층액을 버리고 펠렛화된 세포를 얻었다. 펠릿을 Matrigel로 재현탁하고 96웰-플레이트에 분주하였습니다(웰당 5,000개 세포/10μl Matrigel). Matrigel이 중합된 후, 100㎕ 배양 배지를 첨가하였다. 오르가노이드 생존능은 약간 수정한 공지된 방법을 통해 평가하였다. 간략히 설명하면, 3-[4,5-디메틸티아졸-2-일]-2,5 디페닐 테트라졸륨 브로마이드(MTT) 용액을 500㎍/ml의 최종 농도로 오르가노이드 배양물에 첨가하였다. 3시간 동안 인큐베이션한 후, 배지를 버리고 20㎕의 2% 소디엄도데실설페이트 용액을 첨가하여 2시간 동안 Matrigel을 용해시켰다. 이어서, 100㎕ DMSO를 1시간 동안 첨가하여 환원된 MTT를 용해시키고, 광학밀도(optical density)를 BioTek Eon 마이크로플레이트 흡광도 판독기(BioTek Instruments, Inc.)을 사용하여 562 nm에서 측정하였다. 오르가노이드가 없는 Matrigel(10μl)을 대조군으로 사용하였다. For further dissociation into single cells, organoids were resuspended in TrypLE Express (Thermo Fisher Scientific, Inc.) by pipetting with a p200 pipette and incubated at 37° C. for 10 minutes. The cells were then pipetted several times to form a homogenous resuspension, centrifuged at 4°C at 600xg for 5 minutes, then the supernatant was discarded to obtain pelleted cells. Pellets were resuspended in Matrigel and dispensed into 96-well-plates (5,000 cells per well/10 μl Matrigel). After Matrigel was polymerized, 100 μl culture medium was added. Organoid viability was assessed via a known method with minor modifications. Briefly, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) solution was added to organoid cultures at a final concentration of 500 μg/ml. After incubation for 3 hours, the medium was discarded and 20 μl of a 2% sodium dodecyl sulfate solution was added to dissolve Matrigel for 2 hours. Then, 100 μl DMSO was added for 1 hour to dissolve the reduced MTT, and the optical density was measured at 562 nm using a BioTek Eon microplate absorbance reader (BioTek Instruments, Inc.). Matrigel (10 μl) without organoids was used as a control.
4. 화합물 및 방사선으로 오가노이드 처리 4. Treatment of organoids with compounds and radiation
방사선 감수성을 테스트하기 위해, CRC-PDO(Colorectal cancer Patient derived organoid)에, 1mM 아세테이트(Sigma-Aldrich; Merck KGaA), 부티레이트(Sigma-Aldrich; Merck KGaA), 프로피오네이트(Sigma-Aldrich, Merck) 및/또는 100nM FOXO 억제제, AS1842856(EMD Millipore)으로 37℃에서 6시간 동안 처리한 후에, 플레이트를 Matrigel과 함께 137Cs γ-선 소스(Atomic Energy of Canada Ltd.)를 사용하여 3.81Gy/min의 선량률로 조사하였다. 1mM SCFA의 농도는 공지된 기술에 따라 선택하였다. 오르가노이드에 5Gy의 3분획(1일 1분획)을 조사하고, 0일과 3일에 ImageJ 소프트웨어 1.48V(National Institutes of Health)로 이들의 크기를 분석하였다. 2차 계대(passage)에서 분석하기 위해, 오가노이드를 부티레이트 및/또는 이온화 방사선(IR)을 조사하였다. 72시간 후, 1:2-1:4 분할 비율로 Gentle Cell Dissociation Reagent를 사용하여 피펫팅하여 오르가노이드를 계대하였다. 72시간 후, 오가노이드를 EVOS FL 세포 이미징 시스템(Thermo Fisher Scientific, Inc.)을 사용하여 평가하였다. To test for radiosensitivity, colorectal cancer patient derived organoid (CRC-PDO), 1 mM acetate (Sigma-Aldrich; Merck KGaA), butyrate (Sigma-Aldrich; Merck KGaA), propionate (Sigma-Aldrich, Merck) and/or 100 nM FOXO inhibitor, AS1842856 (EMD Millipore) at 37°C for 6 hours, then the plates were treated with Matrigel using a 137 Cs γ-ray source (Atomic Energy of Canada Ltd.) at a dose rate of 3.81 Gy/min. was investigated. The concentration of 1 mM SCFA was selected according to known techniques. Organoids were irradiated with 3 fractions (1 fraction per day) of 5 Gy, and their size was analyzed with ImageJ software 1.48V (National Institutes of Health) on
5. 면역세포화학 및 면역조직화학 분석 실험 5. Immunocytochemistry and immunohistochemical assays
장 줄기세포는 술잔세포(goblet cell)(mucin 2+, Muc2+), 장내분비세포(entero-endocrine cell)(chromogranin A+, ChgA+) 및 장세포(enterocyte)(VL1+)를 포함한 장 상피의 모든 세포 계통으로 분화할 수 있다. 한편, 모든 증식 세포는 Ki67을 발현한다. PDO를 실온에서 24시간 동안 4% 파라포름알데히드에 고정하고 파라핀에 포매한 다음, 절편(section)(5μm)으로 절개하였다. 실온에서 30분 동안 Smartblock(CANDOR Bioscience)으로 처리한 후, 슬라이드를 항-Ki-67(1:200; cat. no. ab16667; Abcam)항체, 항-Muc2(1:100; 카탈로그 번호 ab90007; Abcam)항체 및 항-ChgA(1:100; 카탈로그 번호 20086; ImmunoStar)항체의 1차 항체와 4℃에서 인큐베이션하였다. 이어서, 슬라이드를 토끼 IgG(H+L)에 대한 Alexa Fluor 594 염소 항체(1:200; 카탈로그 번호 A11012; Thermo Fisher Scientific, Inc.)와 함께 실온에서 1시간 동안 인큐베이션하였다. EVOS FL 세포 이미징 시스템(Thermo Fisher Scientific, Inc.)을 사용하여 이미지를 획득하였다. Intestinal stem cells are all cell lineages of the intestinal epithelium, including goblet cells (
이중가닥 DNA 파손(DSB) 및 DNA 손상 반응을 인식하는 세포의 효능은 γ-H2AX의 초점을 관찰하여 평가할 수 있다. γH2AX의 검출을 위해, 오르가노이드를 8-웰 유리 챔버 슬라이드(LabTek Services, Ltd.)에 분주하였다. 세포를 실온에서 30분 동안 4% 파라포름알데히드로 고정하고, PBS 중 0.1% Triton X-100으로 투과화하였다. 오르가노이드상의 비특이적 에피토프는 Tris-완충 식염수(PBS)에서 실온에서 1시간 동안 5% 소혈청알부민(Gene Depot)으로 차단하고, 항-γ-H2AX(1:100, 카탈로그 번호 05636, EMD Millipore)항체와 함께 밤새 인큐베이션하고, 실온에서 1시간 동안 마우스 IgG(H+L)에 대한 Alexa Fluor 488 염소 항체(1:200, 카탈로그 번호 A11001, Thermo Fisher Scientific, Inc.)와 함께 인큐베이션하였다. The efficacy of cells to recognize double-stranded DNA breaks (DSBs) and DNA damage responses can be assessed by observing foci of γ-H2AX. For detection of γH2AX, organoids were seeded onto 8-well glass chamber slides (LabTek Services, Ltd.). Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS. Non-specific epitopes on organoids were blocked with 5% bovine serum albumin (Gene Depot) for 1 hour at room temperature in Tris-buffered saline (PBS) and incubated with anti-γ-H2AX (1:100, catalog number 05636, EMD Millipore) antibody. and incubated with Alexa Fluor 488 goat antibody (1:200, catalog number A11001, Thermo Fisher Scientific, Inc.) to mouse IgG (H+L) for 1 hour at room temperature.
오르가노이드와 그 기원 조직을 특성 분석하기 위해 결장직장 마커 사이토케라틴(CK)19, CK20 및 CDX2를 사용하여 면역조직화학을 수행하였다. 오르가노이드와 조직을 실온에서 4% 파라포름알데히드에 밤새 고정하고 파라핀 블록에 포매하였다. 그 후, 3μm 절편(section)을 크실렌으로 탈랍하고 등급 지정된 일련의 알코올을 통해 수화하였다. 내인성 퍼옥시다제 활성은 절편을 0.3% H2O2에서 30분 동안 인큐베이션하여 켄칭하고, Smartblock(CANDOR Bioscience)에서 실온에서 30분 동안 인큐베이션하여 비특이적 결합을 감소시켰다. 절편을 항-CDX2 항체(1:200, 카탈로그 번호 235R-16, Cell Marque, SigmaAldrich, Merck KGaA), 항-CK20 항체(1:500, 카탈로그 번호 320M16, Cell Marque, SigmaAldrich, Merck KGaA) 및 항-CK19항체(1:400, 카탈로그 번호 ab15463, Abcam)와 함께 37*에서 1시간 동안 인큐베이션하였다. Envision/Horseradish Peroxidase system(Dako; Agilent Technologies, Inc.)에서 검출을 수행하고 실온에서 10분 동안 헤마톡실린으로 대조염색하였다. 마지막으로, 절편을 등급된 일련의 알코올을 통해 탈수하고, 크실렌에서 제거하여 장착하였다. 이미지는 IX73 도립 현미경(Olympus Corporation; 배율, x40)을 사용하여 획득하였다. Immunohistochemistry was performed using the colorectal markers cytokeratin (CK) 19, CK20 and CDX2 to characterize organoids and their tissues of origin. Organoids and tissues were fixed overnight in 4% paraformaldehyde at room temperature and embedded in paraffin blocks. The 3 μm sections were then dewaxed in xylene and hydrated through a graded series of alcohols. Endogenous peroxidase activity was quenched by incubating the sections in 0.3% H2O2 for 30 minutes, and non-specific binding was reduced by incubating for 30 minutes at room temperature in a Smartblock (CANDOR Bioscience). Sections were incubated with anti-CDX2 antibody (1:200, catalog number 235R-16, Cell Marque, SigmaAldrich, Merck KGaA), anti-CK20 antibody (1:500, catalog number 320M16, Cell Marque, SigmaAldrich, Merck KGaA) and anti- Incubation with CK19 antibody (1:400, catalog number ab15463, Abcam) at 37* for 1 hour. Detection was performed on an Envision/Horseradish Peroxidase system (Dako; Agilent Technologies, Inc.) and counterstained with hematoxylin for 10 minutes at room temperature. Finally, sections were dehydrated through a graded series of alcohols, cleared in xylene, and mounted. Images were acquired using an IX73 inverted microscope (Olympus Corporation; magnification, x40).
6. EdU 염색 6. EdU staining
EdU 염색은 ClickiT Plus EdU FACS 키트(카탈로그 번호 C10424; Thermo Fisher Scientific, Inc.) 및 ClickiT® Plus EdU 이미징 키트(카탈로그 번호 C10639, Thermo Fisher Scientific, Inc.)를 사용하여 수행하였다. 제조사의 지침에 따라, EVOS FL Cell Imaging 시스템 형광 현미경(Thermo Fisher Scientific, Inc.; 배율, x200)을 사용하여 이미지를 획득하였다. EdU staining was performed using the ClickiT Plus EdU FACS Kit (catalog number C10424; Thermo Fisher Scientific, Inc.) and the ClickiT® Plus EdU Imaging Kit (catalog number C10639, Thermo Fisher Scientific, Inc.). Images were acquired using an EVOS FL Cell Imaging System fluorescence microscope (Thermo Fisher Scientific, Inc.; magnification, x200) according to the manufacturer's instructions.
7. 젖산(lactate) 분석 7. Lactate analysis
젖산 농도는 제조사의 지침에 따라, 젖산 분석 키트(카탈로그 번호 K607-100, BioVision)를 사용하여 결정하였다. 기질 첨가 후 30분에 570 nm에서 BioTek Eon 마이크로플레이트 흡광도 판독기(BioTek Instruments, Inc.)에서 광학 밀도를 측정하였다. 젖산 농도의 평균값은 세 번의 독립적인 실험에서 얻은 데이터를 기반으로 각 조건에 대해 계산하였다. Lactic acid concentration was determined using a lactic acid assay kit (catalog number K607-100, BioVision), according to the manufacturer's instructions. Optical density was measured on a BioTek Eon microplate absorbance reader (BioTek Instruments, Inc.) at 570
8. 역전사-정량 PCR (RTqPCR) 분석 8. Reverse transcription-quantitative PCR (RTqPCR) analysis
오가노이드를 차가운 PBS로 세척하고, 4℃에서 1시간 동안 세포복구 용액(Corning, Inc.)에서 배양하여 Matrigel에서 수득하였다. 총 RNA는 RNeasy 미니 키트(Qiagen GmbH)를 사용하여 분리하였다. 이어서, 2μg RNA를 사용하여, 제조사의 프로토콜에 따라 RevertAid First Strand cDNA 키트(Thermo Fisher Scientific, Inc.)를 사용하여 cDNA를 합성하였다. 유전자 발현 수준은 SYBR Green PCR 키트(Qiagen)를 사용하여 정량화하였고, qPCR은 ABI7500 Real-Time PCR 시스템(Applied Biosystems, Thermo Fisher Scientific, Inc.)를 사용하여 실행하였다. qPCR 써멀 싸이클 조건은 94℃에서 5분, 이어서 94℃에서 30초, 60℃에서 30초를 30회 반복하고, 최종 연장 단계는 72℃에서 10분 수행하였다. 표적 mRNA의 양은 GAPDH mRNA의 양으로 정규화하였고, 공지된 방법을 사용하여 분석하였다. Organoids were washed with cold PBS and incubated in a cell recovery solution (Corning, Inc.) for 1 hour at 4° C., and obtained from Matrigel. Total RNA was isolated using the RNeasy mini kit (Qiagen GmbH). Then, using 2 μg RNA, cDNA was synthesized using the RevertAid First Strand cDNA kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Gene expression levels were quantified using the SYBR Green PCR kit (Qiagen), and qPCR was performed using the ABI7500 Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific, Inc.). The qPCR thermal cycle conditions were 5 minutes at 94°C, followed by 30 repetitions of 94°C for 30 seconds and 60°C for 30 seconds, and a final extension step was performed at 72°C for 10 minutes. The amount of target mRNA was normalized to the amount of GAPDH mRNA and analyzed using a known method.
본 실험에 사용한 프라이머는 다음 표 1에 표시하였다. Primers used in this experiment are shown in Table 1 below.
사이클 임계값(Cq)은 해당 PCR 기기 소프트웨어(Applied Biosystems; Thermo Fisher Scientific, Inc.)에 의해 설정된 동일한 프라이머의 모든 앰플리콘에 적용된 자동 기준선을 사용하여 얻었다. >0.5의 Cq값 차이를 갖는 3회 반복은 고려되지 않았고 분석에서 제외하였다. 유전자 발현의 상대적 변화를 분석하기 위한 cDNA의 상대적 농도는 2-△△Cq 방법을 사용하여 계산하였다. Cycle thresholds (Cq) were obtained using an automated baseline applied to all amplicons of the same primer set by the corresponding PCR instrument software (Applied Biosystems; Thermo Fisher Scientific, Inc.). Three replicates with a Cq value difference of >0.5 were not considered and excluded from the analysis. The relative concentration of cDNA to analyze the relative change in gene expression was calculated using the 2 -ΔΔCq method.
9. 웨스턴 블로팅 9. Western blotting
웨스턴 블로팅 분석을 위해 오르가노이드를 차가운 PBS로 세척하고 RIPA 완충액(Thermo Fisher Scientific, Inc.)에 용해시켰다. 단백질은 Bradford 방법을 사용하여 정량화하고, 20μg 단백질/레인을 8-13.5% SDS-PAGE로 분석하였다. 멤브레인은 다음의 단백질들에 대한 항체와 함께 4℃에서 밤새 배양하였다: 절단된 poly-ADP-ribose polymerase(c-PARP;1:1,000;카탈로그 번호 5625; Cell Signaling Technology, Inc.), PARP(1:1,000, 카탈로그 번호 9542; Cell Signaling Technology, Inc.), cleaved caspase 3(c-caspase 3, 1:1,000, 카탈로그 번호 9664, Cell Signaling Technology, Inc.), Caspase 3(1:1,000, 카탈로그 번호 9662, Cell Signaling) Technology, Inc.), 인산화(p)-JNK(1:1,000, 카탈로그 번호 9251, Cell Signaling Technology, Inc.), JNK(1:1,000, 카탈로그 번호 9252, Cell Signaling Technology, Inc.), pp38(1:1,000, 카탈로그 번호 9211, Cell Signaling Technology, Inc.), p38(1:1,000, cat. 9212, Cell Signaling Technology, Inc.), FOXO3A(1:1,000, 카탈로그 번호 2499, Cell Signaling Technology, Inc.), Lamin B(1:1,000, 카탈로그 번호 SC-6216, Santa Cruz Biotechnology, Inc.) 및 β-액틴(1:5,000, 카탈로그 번호 A5441, SigmaAldrich, Merck KGaA). 이어서, 해당 호스래디쉬퍼옥시다제-컨쥬게이트된 2차 항체(1:2,000, 카탈로그 번호 SC-2357 및 SC-516102, Santa Cruz Biotechnology, Inc.)와 실온에서 1시간 동안 인큐베이션하였다. 단백질은 enhanced chemi-liminescence(Thermo Fisher Scientific, Inc.)을 사용하여 시각화하였다. 웨스턴 블롯 이미지는 BioRad ChemiDoc(BioRad Laboratories, Inc.)를 사용하여 분석하였다. For Western blot analysis, organoids were washed with cold PBS and dissolved in RIPA buffer (Thermo Fisher Scientific, Inc.). Protein was quantified using the Bradford method and 20 μg protein/lane was analyzed by 8-13.5% SDS-PAGE. Membranes were incubated overnight at 4°C with antibodies against the following proteins: cleaved poly-ADP-ribose polymerase (c-PARP; 1:1,000; catalog number 5625; Cell Signaling Technology, Inc.), PARP (1 :1,000, catalog number 9542; Cell Signaling Technology, Inc.), cleaved caspase 3 (c-
10. 표적화 Next Generation Sequencing 분석 10. Targeted Next Generation Sequencing Analysis
조직 및 오르가노이드의 돌연변이 상태를 분석하기 위해 세포 회수 용액을 사용하여 수득하였다. DNA 추출 및 라이브러리 구축은 170개의 암 관련 유전자로 구성된 Macrogen with Axen Cancer Panel 2(Macrogen)에서 수행하였다. 라이브러리는 NextSeq 500 시스템(Illumina, Inc.)에서 합성 기술을 사용하여, ~2,000x의 깊이 범위까지 대용량(high-throughput) 시퀀싱을 통해 페어드엔드 시퀀싱하였다(2x150bp). Tissues and organoids were obtained using a cell recovery solution to analyze the mutational status. DNA extraction and library construction were performed in Macrogen with Axen Cancer Panel 2 (Macrogen) consisting of 170 cancer-related genes. Libraries were paired-end sequenced (2x150bp) by high-throughput sequencing to a depth range of ~2,000x using synthetic technology on a NextSeq 500 system (Illumina, Inc.).
11. 통계 분석 11. Statistical Analysis
최소 3회의 독립적인 실험에서 얻은 데이터는 평균±표준편차로 표시하였다. Unpaired two-tailed Student's t-검정을 적용하여 두 그룹 간의 유의한 차이를 확인하였다. 다중 비교를 수행할 때, 그룹 간의 평균을 비교하기 위해 One-way ANOVA에 이어 Tukey의 검정을 수행하였다. P<0.05는 통계적으로 유의한 차이를 나타내는 것으로 간주하였다. 통계 분석은 GraphPad Prism 7(GraphPad Software, Inc.)을 사용하여 수행하였고, 돌연변이 주석이 달린 파일의 분석은 figure oncoplot의 생성을 포함하는 maftools R 패키지 V 3.5.2를 사용하여 수행하였다. Data from at least 3 independent experiments are expressed as mean±standard deviation. Unpaired two-tailed Student's t-test was applied to confirm significant differences between the two groups. When performing multiple comparisons, one-way ANOVA was followed by Tukey's test to compare means between groups. P<0.05 was considered to represent a statistically significant difference. Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, Inc.), and analysis of mutation annotated files was performed using maftools R package V 3.5.2 including generation of figure oncoplot.
실험결과 Experiment result
1. 부티르산은 CRC-PDO에 대해 특이적 항암 활성을 나타내었다. 1. Butyric acid showed specific anticancer activity against CRC-PDO.
면역세포화학분석을 통해 확립된 CRC-PDO가 장 상피 세포 계통의 의 모든 마커(Muc2+, ChgA+, VL1+, Ki-67+)에 대해 양성임을 확인하였다. 또한, CRCPDO는 종양 플롯 분석(oncoplot analysis)에서 입증된 바와 같이, 매칭되는 종양 조직과 동일한 유전적 지형을 가지고 있었다(도 1A). 면역조직화학 분석에서 CK19, CK20 및 CDX2는 CRC-PDO #11 원래 조직 및 오르가노이드에서 발현되는 것으로 확인하였다(도 1B). 한편, 다른 조직 및 오르가노이드는 CK19에 대해 약한 양성인 반면, CK20 및 CDX2의 강한 발현은 CRC-PDO #3에서 검출되었다. 이러한 특징은 환자 조직 샘플에 존재하는 특징을 반영한다. 종양 오르가노이드는 배양 배지에서 Wnt 관련 인자를 제외하여 선택적으로 확장되었으며(도 1C), 면역세포화학 분석 결과, CRC-PDO가 정상 PDO에 비해 더 많은 증식 세포를 포함하는 것으로 나타났다(P<0.001; 도 1D). 오르가노이드 배양은 1차 조직의 특성을 반영한다. 스크리닝된 3개의 SCFA 중 부티레이트만이 오르가노이드 크기를 유의하게 감소시킨 반면(P=0.038), 프로피오네이트와 아세테이트는 효과가 없었다(도 1E 및 도 1F). 대조적으로, 부티레이트(butyrate)는 정상-PDO에 어떠한 부작용도 나타내지 않았다(도 1G 및 도 1H). 이와 같은 결과는 부티레이트가 CRC-PDO에 대해 선택적 항종양 활성을 나타낼 수 있음을 시사한다. It was confirmed that the CRC-PDO established through immunocytochemical analysis was positive for all markers (Muc2+, ChgA+, VL1+, Ki-67+) of the intestinal epithelial cell lineage. In addition, CRCPDO had the same genetic topography as the matched tumor tissue, as demonstrated by oncoplot analysis (FIG. 1A). Immunohistochemical analysis confirmed that CK19, CK20 and CDX2 were expressed in CRC-
2. 부티르산은 CRC-PDO에서 방사선 감수성을 증대시켰다. 2. Butyric acid increased radiosensitivity in CRC-PDO.
부티레이트와 IR을 함께 처리한 오르가노이드는 IR만 처리한 것과 비교하여 오르가노이드 크기가 1.1배에서 0.6배로 유의하게 감소한 것으로 나타났다(P=0.04, 도 2A 및 도 2B). 이러한 결과는 MTT 분석 결과에 의해 뒷받침되었으며, 부티레이트 및 IR의 조합 처리 후에 오르가노이드 생존능의 상당한 감소가 관찰되었다(도 2C). 암은 종양유발성 암세포와 비종양유발성 암세포로 구성되어 있으므로 부티레이트의 항종양 효과를 더 평가하기 위해 PDO 분열 후 2차 계대(2nd passage)에서 종양 오르가노이드의 수를 계수하였다. 분열 후, 총 3일 후에 형성된 종양 오르가노이드의 수는 IR 단독 처리군과 비교하여 부티레이트(butyrate) 및 IR 처리 후에 유의하게 감소하였다(도 2D). 이러한 결과는 부티레이트가 CRC 오르가노이드에 대한 방사선의 효과를 향상시킨다는 것을 시사한다. 부티레이트 및 IR 노출 후 세포 생존능의 손실이 세포 주기 정지의 변화를 동반하는지 더 확인하기 위해 유세포 분석으로 EdU 혼입 정도를 측정했으며, 12%의 세포가 IR 후 S기에 있는 것으로 확인하였다. 부티레이트와 IR 병합치료 후에 5%만이 S기에 있었다(P=0.003; 도 2E). 이어서, CRCPDO의 성장에 대한 부티레이트의 영향을 평가하였다. 부티레이트와 IR로 처리한 후 오르가노이드는 IR만 처리한 것보다 Ki-67+ 세포 수가 상당히 낮게 나타났다(P=0.005, 도 2F). Organoids treated with butyrate and IR showed a significant reduction in organoid size from 1.1-fold to 0.6-fold compared to those treated with IR only (P=0.04, Figure 2A and Figure 2B). These results were supported by the results of the MTT assay, in which a significant decrease in organoid viability was observed after combined treatment with butyrate and IR (Figure 2C). Since cancer is composed of tumorigenic cancer cells and non-tumorigenic cancer cells, the number of tumor organoids was counted at the second passage after PDO division to further evaluate the antitumor effect of butyrate. After division, the number of tumor organoids formed after a total of 3 days was significantly reduced after butyrate and IR treatment compared to the IR alone treatment group (Fig. 2D). These results suggest that butyrate enhances the effect of radiation on CRC organoids. To further confirm that the loss of cell viability following exposure to butyrate and IR was accompanied by changes in cell cycle arrest, we measured the extent of EdU incorporation by flow cytometry and found that 12% of the cells were in S phase after IR. Only 5% were in S phase after butyrate plus IR combination (P=0.003; Fig. 2E). The effect of butyrate on the growth of CRCPDO was then evaluated. Organoids after treatment with butyrate and IR showed significantly lower Ki-67+ cell numbers than those treated with IR only (P=0.005, Figure 2F).
방사선 유발 DNA 손상에 대한 부티레이트의 영향을 조사하기 위해 γ-H2AX를 분석한 결과, post-RT 6시간 및 24시간 후 부티레이트와 방사선을 병용한 치료가 방사선 단독 치료에 비해 더 높은 수준의 γ-H2AX를 나타내는 것으로 확인되었다. 방사선 조사 후, 암 오르가노이드의 18.3%가 γ-H2AX 병소에 대해 양성인 반면, 병용 치료는 오르가노이드의 58.6%가 γ-H2AX 병소를 형성하도록 유도하여, DSB의 수가 훨씬 더 많아졌음을 확인하였다(P<0.001, 도 2G). As a result of analyzing γ-H2AX to investigate the effect of butyrate on radiation-induced DNA damage, the combination of butyrate and radiation treatment at 6 and 24 hours post-RT showed higher levels of γ-H2AX than radiation alone. It was confirmed that it represents After irradiation, 18.3% of cancer organoids were positive for γ-H2AX foci, whereas the combination treatment induced 58.6% of organoids to form γ-H2AX foci, confirming a much higher number of DSBs ( P<0.001, Fig. 2G).
다음으로, 방사선에 대한 세포 반응을 평가하기 위해 세포사멸(apoptosis) 관련 단백질 수준을 분석하였다. 세포사멸(apoptosis)의 특징으로 간주되는 cPARP와 cCaspase 3의 수치는 IR 단독 치료 후의 각각의 수치에 비해 IR 및 부티레이트 조합 치료 후에 증가하였다. 또한, p-JNK 수준은 병용 치료 후에 증가하였다. p-p38의 발현수준에는 변화가 없었다(도 2H). 이러한 결과들은 부티레이트가 CRCPDO에서 방사선 감수성 향상제로 작용함을 시사한다. Next, the level of apoptosis-related proteins was analyzed to evaluate the cellular response to radiation. The levels of cPARP and
3. 부티르산은 정상-PDO에서 방사선 감수성 향상제로 작용하지 않는다. 3. Butyric acid does not act as a radiosensitivity enhancer in normal-PDO.
부티레이트의 안전성을 평가하기 위해 정상(normal) PDO에서 부티레이트의 반응을 추가로 확인하였다. 방사선 조사 후에 오르가노이드의 크기가 감소하였지만, 부티레이트는 정상 PDO를 IR에 의한 손상으로부터 보호하였다(도 3A 및 도 3B). MTT 분석과 2차 계대는 이러한 결과를 뒷받침하였다(도 3C 및 도 3D). 이러한 결과는 부티레이트가 정상(normal)-PDO에서 방사선 감수성에 영향을 미치지 않음을 시사한다. To evaluate the safety of butyrate, the reaction of butyrate in normal PDO was further confirmed. Although the size of organoids decreased after irradiation, butyrate protected normal PDO from IR-induced damage (Figures 3A and 3B). MTT assay and second passage supported these results (Fig. 3C and Fig. 3D). These results suggest that butyrate does not affect radiosensitivity in normal-PDO.
4. 부티르산은 와버그 효과(Warburg effect)를 통해 방사선 감수성을 향상시킨다. 4. Butyric acid enhances radiation sensitivity through the Warburg effect.
부티레이트는 정상결장 세포와 결장암 세포 사이의 와버그 효과(Warburg effect)의 차이로 인해 암세포주의 증식을 선택적으로 억제한다. 따라서, 와버그 효과가 부티레이트의 차등적인 방사선 감수성 향상 효과를 설명할 수 있는지 여부를 테스트하기 위해, CRC-PDO의 와버그 효과는 배양 배지를 고포도당(3.151g/l)에서 저포도당(1g/l)으로 변경하여 억제되었다. 이러한 조건에서 포도당 수준은 호기성 해당 작용을 지원하기에는 너무 낮았는데, 이는 무시할 수 있는 수준의 젖산, 즉 해당 작용의 최종 생성물의 검출에 의해 입증되었다(P=0.01, 도 4A). 저포도당 상태에서 와버그 효과가 없으면, 부티레이트는 방사선 조사 후 오르가노이드 크기와 생존능에 더 이상 영향을 미치지 않는다(도 4B 및 도 4C). 또한, 형성된 두 번째 계대 오르가노이드의 수는 저포도당 조건에서 IR 및 부티레이트 처리 후에도 변하지 않았다(도 4D). EdU 염색에서 알 수 있듯이 고포도당 배지에서 IR 및 부티레이트 처리 후 S기 세포의 비율은 감소하였지만(P<0.001) 저포도당 배지에서는 감소하지 않았다(도 4E). 이러한 결과는 부티레이트가 와버그 효과 의존 방식으로 CRC-PDO에서 방사선 민감도를 증가시킨다는 것을 보여준다. Butyrate selectively inhibits the proliferation of cancer cell lines due to the difference in the Warburg effect between normal colon cells and colon cancer cells. Therefore, to test whether the Warburg effect could explain the differential radiosensitivity enhancing effect of butyrate, the Warburg effect of CRC-PDO was varied by varying the culture medium from high glucose (3.151 g/l) to low glucose (1 g/l). l) was suppressed. Glucose levels in these conditions were too low to support aerobic glycolysis, as evidenced by the detection of negligible levels of lactic acid, i.e., the end product of glycolysis (P=0.01, Figure 4A). Without the Warburg effect in the low glucose condition, butyrate no longer affects organoid size and viability after irradiation (Figure 4B and Figure 4C). In addition, the number of second passage organoids formed did not change after IR and butyrate treatment under low glucose conditions (Fig. 4D). As can be seen from EdU staining, the percentage of S-phase cells decreased after IR and butyrate treatment in high-glucose medium (P<0.001), but not in low-glucose medium (Fig. 4E). These results show that butyrate increases the radiation sensitivity in CRC-PDO in a Warburg effect dependent manner.
5. 부티르산은 CRC-PDO에서 FOXO3A를 통해 방사선 반응을 조절한다. 5. Butyric acid modulates radiation response through FOXO3A in CRC-PDO.
부티레이트가 방사선 감수성에 미치는 잠재적 효과의 메커니즘을 조사하기 위해 세포주기 유전자를 조절하는 전사인자, FOXO3A의 영향을 조사하였다. FOXO 억제제와 함께 CRCPDO의 치료는 CRCPDO에서 부티레이트의 방사선 민감 효과를 감소시켰다(P<0.001; 도 5의 A 및 B). 또한, MTT 분석 결과, 병용 치료 후 오르가노이드 생존력의 현저한 감소가, 계대에 의해 분석된 오르가노이드 재생 능력과 함께, FOXO 억제제에 의해 구제되는 것이 확인되었다((P=0.03; 도 5C; 도 5D). 또한, FOXO 억제제는 방사선 조사 후 부티레이트(butyrate)의 증식억제 효과를 반전시켰다(P<0.001; 도 5E). FOXO3A에 의해 조절되는 세포주기 관련 유전자, 즉 GADD45, p21 및 p57의 발현 수준을 조사하기 위해 역전사 정량 PCR을 수행하였다. 실험결과, 부티레이트와 IR이 CRC-PDO에서 GADD45(P<0.001), p21(P<0.001) 및 p57(P<0.001)의 mRNA 발현 수준을 증가시켰으나, 이는 모두 FOXO 억제제를 처리한 후에 감소하였다는 것을 보여준다(도 5F). 이러한 결과는 부티레이트가 FOXO3A에 의해 조절되는 p21, p57 및 GADD45의 유도를 통해 방사선 감수성을 증가시킨다는 것을 시사한다. To investigate the mechanism of the potential effect of butyrate on radiosensitivity, the effect of FOXO3A, a transcription factor that regulates cell cycle genes, was investigated. Treatment of CRCPDO with a FOXO inhibitor reduced the radiosensitizing effect of butyrate on CRCPDO (P<0.001; Fig. 5A and B). MTT assay also confirmed that the significant decrease in organoid viability after combination treatment was rescued by FOXO inhibitors, along with organoid regeneration assayed by passage ((P=0.03; Fig. 5C; Fig. 5D) In addition, FOXO inhibitors reversed the antiproliferative effect of butyrate after irradiation (P<0.001; Fig. 5E) Examining the expression levels of cell cycle-related genes regulated by FOXO3A, namely GADD45, p21 and p57 As a result, butyrate and IR increased the mRNA expression levels of GADD45 (P<0.001), p21 (P<0.001), and p57 (P<0.001) in CRC-PDO, but these were all 5F), suggesting that butyrate increases radiosensitivity through the induction of p21, p57 and GADD45 regulated by FOXO3A.
6. 부티르산의 방사선 감수성 향상효과는 FOXO3A 발현에 의존적이다. 6. The radiosensitivity enhancement effect of butyric acid is dependent on FOXO3A expression.
부티레이트와 IR 치료를 조합한 후 오르가노이드 크기가 전반적으로 감소했지만 일부 경우에서는 차이가 관찰되지 않았다(이하, 비반응성 CRC-PDO라고 함, 도 6A). 따라서, 반응성 및 비반응성 CRC-PDO의 증식 능력을 비교하기 위해 EdU 염색을 수행하였다. 부티레이트와 IR 치료 후에, 비반응성 CRC-PDO 그룹은 반응성 CRC-PDO 그룹보다 더 많은 EdU-혼입된 세포를 가졌다(P<0.001; 도 6B). 또한, 반응성 CRCPDO와, 이의 유래 원래 조직은 FOXO3A에 대해, 비반응성 CRCPDO 보다 더 강한 핵 염색을 보였다(도 6C). 또한, 웨스턴 블로팅 분석결과, 5개의 반응성 CRC-PDO 사례 모두에서 비반응성 CRC-PDO에 비해 FOXO3A의 발현 증가가 나타났고(도 6D), FOXO3A 발현의 강도는 3개의 비반응성 CRC-PDO에 비해 유의하게 증가하였다(P=0.0084; 도 6E). 또한, 역전사 정량 PCR 분석결과, 부티레이트와 IR 조합 치료는, 반응성 CRCPDO에서 p21(P<0.001), p57(P<0.001) 또는 GADD45(P=0.048)의 발현 수준에 영향을 미치는 반면, 비반응성 CRC-PDO에서는 발현수준에 영향을 미치지 않는 것으로 나타났다(도 6F). 이러한 결과들은 부티레이트의 방사선 감수성 향상 효과가 FOXO3A 발현 수준에 의존할 수 있음을 시사한다. Although organoid size decreased overall after combining butyrate and IR treatment, no difference was observed in some cases (hereafter referred to as non-reactive CRC-PDO, Figure 6A). Therefore, EdU staining was performed to compare the proliferative abilities of reactive and non-reactive CRC-PDO. After butyrate and IR treatment, the non-reactive CRC-PDO group had more EdU-incorporated cells than the responsive CRC-PDO group (P<0.001; Fig. 6B). In addition, reactive CRCPDO and its native tissue showed stronger nuclear staining for FOXO3A than non-reactive CRCPDO (Fig. 6C). In addition, as a result of Western blotting analysis, FOXO3A expression increased compared to non-reactive CRC-PDO in all 5 reactive CRC-PDO cases (Fig. 6D), and the intensity of FOXO3A expression was higher than that of 3 non-reactive CRC-PDO. significantly increased (P=0.0084; Fig. 6E). In addition, as a result of reverse transcription quantitative PCR analysis, butyrate and IR combination treatment affected the expression level of p21 (P<0.001), p57 (P<0.001) or GADD45 (P=0.048) in reactive CRCPDO, whereas non-reactive CRC -PDO did not appear to affect the expression level (FIG. 6F). These results suggest that the radiosensitivity enhancement effect of butyrate may depend on the FOXO3A expression level.
상기에서는 본 출원의 대표적인 실시예를 예시적으로 설명하였으나, 본 출원의 범위는 상기와 같은 특정 실시예만 한정되지 아니하며, 해당 분야에서 통상의 지식을 가진 자라면 본 출원의 청구범위에 기재된 범주 내에서 적절하게 변경이 가능할 것이다. In the above, representative embodiments of the present application have been described as examples, but the scope of the present application is not limited to the specific embodiments as described above, and those skilled in the art are within the scope described in the claims of the present application. will be able to change accordingly.
<110> Korea Institute of Radiological and Medical Sciences <120> Composition for Enhancing Efficacy of Radiotherapy of Cancer Comprising Butyrate as Active Ingredient <130> 2021-DPA-4256 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for p21 <400> 1 tgtccgtcag aacccatgc 19 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for p21 <400> 2 aaagtcgaag ttccatcgct c 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for p57 <400> 3 gcggcgatca agaagctgt 19 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for p57 <400> 4 gcttggcgaa gaaatcggag a 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Gadd45 <400> 5 tgctgtgaca acgacatcaa c 21 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Gadd45 <400> 6 gtgagggttc gtgaccagg 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GAPDH <400> 7 tgcaccacca actgcttagc 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GAPDH <400> 8 ggcatggact gtggtcatga g 21 <110> Korea Institute of Radiological and Medical Sciences <120> Composition for Enhancing Efficacy of Radiotherapy of Cancer Comprising Butyrate as Active Ingredient <130> 2021-DPA-4256 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> artificial sequence <220> <223> Forward primer for p21 <400> 1 tgtccgtcag aacccatgc 19 <210> 2 <211> 21 <212> DNA <213> artificial sequence <220> <223> Reverse primer for p21 <400> 2 aaagtcgaag ttccatcgct c 21 <210> 3 <211> 19 <212> DNA <213> artificial sequence <220> <223> Forward primer for p57 <400> 3 gcggcgatca agaagctgt 19 <210> 4 <211> 21 <212> DNA <213> artificial sequence <220> <223> Reverse primer for p57 <400> 4 gcttggcgaa gaaatcggag a 21 <210> 5 <211> 21 <212> DNA <213> artificial sequence <220> <223> Forward primer for Gadd45 <400> 5 tgctgtgaca acgacatcaa c 21 <210> 6 <211> 19 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Gadd45 <400> 6 gtgagggttc gtgaccagg 19 <210> 7 <211> 20 <212> DNA <213> artificial sequence <220> <223> Forward primer for GAPDH <400> 7 tgcaccacca actgcttagc 20 <210> 8 <211> 21 <212> DNA <213> artificial sequence <220> <223> Reverse primer for GAPDH <400> 8 ggcatggact gtggtcatga g 21
Claims (8)
A composition for improving sensitivity to radiation therapy for cancer, comprising butyrate as an active ingredient.
상기 암은 유방암, 폐암, 대장암, 전립선암, 비소세포성 폐암, 결장암, 골암, 췌장암, 피부암, 두부 또는 경부 암, 피부 또는 안구내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문부근암, 결장암, 나팔관암, 자궁내막암, 자궁경부암, 질암, 음문암, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS; central nervous system) 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종인 것인, 암에 대한 방사선 치료 감수성 향상용 조성물.
The method of claim 1,
The cancer is breast cancer, lung cancer, colorectal cancer, prostate cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, cervical cancer, ovarian cancer, rectal cancer, stomach cancer, cancer near the anus , colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma Which would, a composition for improving the sensitivity of radiation therapy for cancer.
상기 방사선은 X-선, 알파선, 감마선, 베타선, 중성자선 또는 양성자선인 것인, 암에 대한 방사선 치료 감수성 향상용 조성물.
The method of claim 1,
Wherein the radiation is X-ray, alpha ray, gamma ray, beta ray, neutron ray or proton ray, a composition for improving sensitivity to radiation therapy for cancer.
상기 암의 암세포는 FOXO3A 유전자가 발현되는 것인, 암에 대한 방사선 치료 감수성 향상용 조성물.
The method of claim 1,
The cancer cells of the cancer is the FOXO3A gene is expressed, a composition for improving the sensitivity to radiation therapy for cancer.
(i) butyric acid (butyrate) of a pharmaceutically effective amount and (ii) a pharmaceutical composition for improving sensitivity to radiation therapy for cancer comprising a pharmaceutically acceptable carrier.
상기 암은 유방암, 폐암, 대장암, 전립선암, 비소세포성 폐암, 결장암, 골암, 췌장암, 피부암, 두부 또는 경부 암, 피부 또는 안구내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문부근암, 결장암, 나팔관암, 자궁내막암, 자궁경부암, 질암, 음문암, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS; central nervous system) 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종인 것인, 암에 대한 방사선 치료 감수성 향상용 약학적 조성물.
The method of claim 5,
The cancer is breast cancer, lung cancer, colorectal cancer, prostate cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, cervical cancer, ovarian cancer, rectal cancer, stomach cancer, cancer near the anus , colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma Which is, a pharmaceutical composition for improving sensitivity to radiation therapy for cancer.
상기 방사선은 X-선, 알파선, 감마선, 베타선, 중성자선 또는 양성자선인 것인, 암에 대한 방사선 치료 감수성 향상용 약학적 조성물.
The method of claim 5,
Wherein the radiation is X-ray, alpha ray, gamma ray, beta ray, neutron ray or proton ray, a pharmaceutical composition for improving sensitivity to radiation therapy for cancer.
상기 암의 암세포는 FOXO3A 유전자가 발현되는 것인, 암에 대한 방사선치료 감수성 향상용 약학적 조성물. The method of claim 5,
The cancer cells of the cancer is a pharmaceutical composition for improving the sensitivity of radiation therapy for cancer, which FOXO3A gene is expressed.
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| KR20110012812A (en) | 2009-07-31 | 2011-02-09 | 재단법인 한국원자력의학원 | A pharmaceutical composition for enhancing the radiotherapy of cancer and a method of screening an active material for enhancing the radiotherapy of cancer |
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| KR20110012812A (en) | 2009-07-31 | 2011-02-09 | 재단법인 한국원자력의학원 | A pharmaceutical composition for enhancing the radiotherapy of cancer and a method of screening an active material for enhancing the radiotherapy of cancer |
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