KR102250925B1 - Pharmaceutical composition for preventing or treating radiation-resistant cancer comprising lotus root or lotus extract - Google Patents
Pharmaceutical composition for preventing or treating radiation-resistant cancer comprising lotus root or lotus extract Download PDFInfo
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- KR102250925B1 KR102250925B1 KR1020190130961A KR20190130961A KR102250925B1 KR 102250925 B1 KR102250925 B1 KR 102250925B1 KR 1020190130961 A KR1020190130961 A KR 1020190130961A KR 20190130961 A KR20190130961 A KR 20190130961A KR 102250925 B1 KR102250925 B1 KR 102250925B1
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Abstract
MAP4K4, ACSL4 및 모에신 유전자의 발현 또는 활성을 억제하는 제제를 유효성분으로 포함하는 방사선 내성 암 치료용 약학적 조성물이 개시된다.Disclosed is a pharmaceutical composition for the treatment of radiation-resistant cancer comprising, as an active ingredient, an agent that inhibits the expression or activity of MAP4K4, ACSL4 and moesin genes.
Description
본 발명은 연자육 또는 연자방 추출물을 포함하는 방사선 내성 암 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention or treatment of radiation-resistant cancer comprising the extract of yeonjayuk or yeonjabang.
암 치료에 주로 사용되고 있는 방법으로는 외과적 수술, 화학 요법, 방사선 요법 등이 있다. 이 중 방사선 치료는 조사부위에 효과가 있는 국소요법으로서 두경부암, 후두암, 자궁경부암, 유방암, 대장암 등에 시행되고 있다. 방사선 치료의 효과는 암의 성질, 환자, 및 기타 치료와 조합에 따라 달라질 수 있으며 방사선 치료시 반응이 좋지 않거나, 초기 반응이 좋더라도 재발하는 경우가 빈번하다. Methods commonly used for cancer treatment include surgical surgery, chemotherapy, and radiation therapy. Among them, radiation therapy is a topical therapy that is effective in the irradiated area and is being used for head and neck cancer, laryngeal cancer, cervical cancer, breast cancer, and colon cancer. The effect of radiation therapy may vary depending on the nature of the cancer, the patient, and other treatments and combinations, and there are frequent cases of poor response or recurrence even if the initial response is good.
따라서 환자별로 방사선 치료의 감수성 차이가 존재하므로 이를 확인하고 적절한 치료제를 선택해야 하지만, 방사선 치료를 실시하기 전에는 방사선에 대한 내성을 나타내는지 예측하기가 어려운 실정이다.Therefore, since there is a difference in sensitivity to radiation therapy for each patient, it is necessary to confirm this and select an appropriate therapeutic agent, but it is difficult to predict whether or not the patient exhibits resistance to radiation before radiation therapy is performed.
연자육 및 연자방은 설사, 심신 불안 증세 등을 치료하기 위한 약제로 사용되는 것으로 알려져 있으나(출처: 식품의약품안전처 생약종합정보), 방사선 내성 암에 대한 치료 효과는 알려진 바가 없다.Yeonjayuk and Yeonjabang are known to be used as drugs to treat diarrhea, mental and physical anxiety symptoms, etc. (Source: General information on herbal medicines from the Ministry of Food and Drug Safety), but the therapeutic effect on radiation-resistant cancer is unknown.
일 구체예에 따르면, MAP4K4, ACSL4 및 모에신 유전자의 발현 또는 활성을 억제하는 제제를 포함하는 방사선 내성 암 치료용 약학적 조성물을 제공한다. According to one embodiment, it provides a pharmaceutical composition for the treatment of radiation-resistant cancer comprising an agent that inhibits the expression or activity of MAP4K4, ACSL4 and moesin genes.
일 구체예에 따르면, 연자육 또는 연자방 추출물을 포함하는 방사선 내성 암 치료용 약학적 조성물을 제공한다.According to one embodiment, it provides a pharmaceutical composition for the treatment of radiation-resistant cancer comprising the extract of yeonjayuk or yeonjabang.
일 양상은 MAP4K4, ACSL4, 및 모에신 유전자의 발현 또는 활성을 억제하는 제제를 유효성분으로 포함하는 방사선 내성 암 치료용 약학적 조성물을 제공한다. One aspect provides a pharmaceutical composition for the treatment of radiation-resistant cancer comprising, as an active ingredient, an agent that suppresses the expression or activity of MAP4K4, ACSL4, and moesin genes.
본 발명에서 용어"방사선 내성"은 방사선 조사에 의해 받는 영향이 일반적인 암에 대한 것보다 적으며, 방사선에 노출시 세포 복구 또는 증식에 변화가 적은 상태를 의미한다. 방사선은 X-선, 자외선, 알파 입자, 또는 감마선을 포함하는 전자기 방사선일 수 있다. In the present invention, the term "radiation resistance" refers to a state in which the effect of radiation irradiation is less than that for general cancer, and there is little change in cell repair or proliferation upon exposure to radiation. The radiation may be electromagnetic radiation including X-rays, ultraviolet rays, alpha particles, or gamma rays.
본 발명에서 용어 "그레이(Gy)"는 생물학 조직 1kg에서 1J을 방출하는 방사선의 양을 의미한다. In the present invention, the term "Gy" refers to the amount of radiation that emits 1J from 1 kg of biological tissue.
본 발명에서 용어 "바이오마커"는 암세포 또는 암 질환을 가진 개체를 방사선 저항성 세포 또는 개체로 구분하여 진단할 수 있는 물질로서, 정상 암세포에 비하여 방사선 저항성을 가진 암세포 또는 개체에서 증가하는 폴리펩티드, 단백질, 또는 핵산(mRNA 등) 등과 같은 유기 생체 분자들을 포함한다. In the present invention, the term "biomarker" is a substance capable of diagnosing cancer cells or individuals with cancer diseases as radiation-resistant cells or individuals, and increases in cancer cells or individuals with radiation resistance compared to normal cancer cells, Or organic biomolecules such as nucleic acids (mRNA, etc.).
본 발명자는 인간 유방암 세포에 방사선을 지속적으로 조사하여 방사선 내성을 갖는 암세포를 분리하고 유전자 발현을 측정한 결과, 일반 암세포보다 MAP4K4, ACSL4 및 모에신 유전자의 발현이 현저히 증가하는 점을 확인하였으며, 이들 유전자의 발현을 억제하는 경우 방사선 내성 특이적인 세포 사멸효과가 있음을 확인하였으므로 MAP4K4, ACSL4 및 모에신 유전자의 발현 또는 활성을 억제하는 제제를 포함하는 조성물은 방사선 내성 암 치료제로서 유용하게 사용될 수 있다. The present inventors have continuously irradiated human breast cancer cells with radiation to isolate radiation-resistant cancer cells, and as a result of measuring gene expression, it has been confirmed that the expression of MAP4K4, ACSL4 and Moesin genes is significantly increased compared to general cancer cells. When suppressing the expression of the gene, it has been confirmed that there is a radiation-resistant specific apoptosis effect, so a composition comprising an agent that suppresses the expression or activity of MAP4K4, ACSL4, and moesin genes can be usefully used as a radiation-resistant cancer therapeutic agent.
상기 MAP4K4 유전자는 Mitogen-activated protein kinase kinase kinase kinase 4로서 세포의 이동, 증식, 및 접착을 포함하는 광범위한 생리학적 과정에 관여하는 것으로 알려져 있고, 일반적인 세포보다 암에서 상향 조절되는 것으로 알려져 있다. 그러나 일반적인 암세포와 방사선 내성 암에서의 발현량 차이 및 방사선 치료에 대한 내성과의 관련성에 대해서는 알려진 바가 없다. The MAP4K4 gene, as Mitogen-activated protein kinase
상기 ACSL4 유전자는 long chain fatty acid CoA ligase 4를 암호화하는 유전자로서, 방사선 치료에 대한 내성과의 관련성은 보고된 바가 없다. The ACSL4 gene is a gene encoding long chain fatty
상기 모에신(Moesin)은 MSN 유전자에 의해 암호화되는 단백질로서, ERM(Ezrin-radixin-moesin) 패밀리 단백질로 분류되며 액틴 세포 골격을 원형질막에 연결하는 것으로 알려져 있다. 상기 모에신 유전자는 MSN 유전자일 수 있다. 상기 모에신의 발현과 방사선 치료에 대한 내성과의 관련성은 보고된 바가 없다. Moesin is a protein encoded by the MSN gene, is classified as an ERM (Ezrin-radixin-moesin) family protein, and is known to link the actin cytoskeleton to the plasma membrane. The moesin gene may be an MSN gene. The relationship between the expression of the moesin and resistance to radiation therapy has not been reported.
상기 MAP4K4 유전자는 서열번호 1의 염기서열로 이루어진 폴리뉴클레오티드일 수 있으며, 상기 MAP4K4 단백질은 서열번호 4의 펩타이드 서열로 이루어진 폴리펩타이드일 수 있으며, 상기 ACSL4 유전자는 서열번호 2의 염기서열로 이루어진 폴리뉴클레오티드일 수 있으며, 상기 ACSL4 단백질은 서열번호 5의 펩타이드 서열로 이루어진 폴리펩타이드일 수 있으며, 상기 Moesin 유전자는 서열번호 3의 염기서열로 이루어진 폴리뉴클레오티드일 수 있으며, 상기 Moesin 단백질은 서열번호 6의 펩타이드 서열로 이루어진 폴리펩타이드일 수 있다. 상기 서열번호 1 내지 4는 하기 표 1에 개시되어 있다.The MAP4K4 gene may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, the MAP4K4 protein may be a polypeptide consisting of the peptide sequence of SEQ ID NO: 4, and the ACSL4 gene is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 The ACSL4 protein may be a polypeptide consisting of a peptide sequence of SEQ ID NO: 5, the Moesin gene may be a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 3, and the Moesin protein is a peptide sequence of SEQ ID NO: 6 It may be a polypeptide consisting of. The SEQ ID NOs: 1 to 4 are disclosed in Table 1 below.
번호order
number
상기 발현은 mRNA 발현 또는 단백질 발현을 의미하는 것일 수 있다. The expression may mean mRNA expression or protein expression.
일 구체예에서, 상기 제제는 연자육 또는 연자방 추출물일 수 있다. In one embodiment, the formulation may be an extract of yeonjayuk or yeonjabang.
상기 연자육(Nelumbinis Semen)은 연꽃(Nelumbo nucifera Gaertner)의 잘익은 씨앗으로, 타원형 또는 구형에 가깝고 길이는 12 내지 18mm, 지름은 8 내지 14mm이다. 바깥면은 단단하고 짙은 회갈색으로 가느다란 세로무늬와 비교적 넓은 맥문이 있으며 냄새가 거의 없고 맛은 달다. 상기 연자방은 연방으로도 부르며 연꽃의 씨방을 말린 것으로서 연자육을 제외한 것을 의미한다. 본 발명자는 연자육 또는 연자방의 에탄올 추출물이 방사선 내성 암에서 증가하는 MAP4K4, ACSL4, 및 Moesin의 발현을 억제함으로서 방사선 내성 암 특이적인 세포 사멸 효과가 있음을 확인하였다. The lotus (Nelumbinis Semen) is a well-ripe seed of a lotus (Nelumbo nucifera Gaertner ), elliptical or close to a spherical shape, the length is 12 to 18 mm, the diameter is 8 to 14 mm. The outer surface is hard, dark grayish brown, with thin vertical patterns and relatively wide veins, almost no smell, and sweet taste. The lotus leaf is also referred to as federation, and refers to the dried ovary of a lotus, excluding lotus meat. The present inventors have confirmed that the ethanol extract of yeonjayuk or yeonjabang suppresses the expression of MAP4K4, ACSL4, and Moesin, which are increased in radiation-resistant cancer, and thus has a radiation-resistant cancer-specific cell killing effect.
연자육 추출물 또는 연자방 추출물은 당업자에게 알려진 방법을 이용하여 준비할 수 있으며, 시중에서 구입한 제품을 사용할 수도 있다.The lotus meat extract or the lotus leaf extract may be prepared using a method known to those skilled in the art, and a commercially available product may be used.
상기 연자육 또는 연자방 추출물의 용매는 물, 에탄올, 메탄올, 프로판올과 같은 극성 용매 또는 헥산, 에테르 등의 비극성 용매를 가하여 실온에서 침적 추출하거나 가온 추출한 것일 수 있으며, 바람직하게는 에탄올을 용매로 하여 상온 추출한 것일 수 있다. 상온 추출은 연자육 또는 연자방을 1 내지 4일, 또는 3일간 20 내지 30℃에서 용매에 침지시켜 추출하는 것일 수 있다. The solvent of the lotus meat or lotus leaf extract may be immersion extraction or warm extraction at room temperature by adding a polar solvent such as water, ethanol, methanol, or propanol, or a non-polar solvent such as hexane or ether. It may be extracted. Room temperature extraction may be extracted by immersing the lotus meat or lotus leaf in a solvent at 20 to 30°C for 1 to 4 days, or 3 days.
상기 연자육 또는 연자방 추출물의 용매는 연자육의 4 내지 6배, 또는 5배 부피의 에탄올을 사용하여 추출한 것일 수 있으며, 에탄올은 30% 에탄올, 60% 에탄올, 또는 100% 에탄올일 수 있으며, 바람직하게는 70% 에탄올일 수 있다. The solvent of the lotus root or lotus leaf extract may be extracted using 4 to 6 times or 5 times the volume of ethanol, and the ethanol may be 30% ethanol, 60% ethanol, or 100% ethanol, preferably May be 70% ethanol.
일 구체예에서, 상기 암은 MAP4K4, ACSL4 및 모에신 발현 차이에 의해 구별할 수 있는 암이라면 특별히 제한되지 않으며, 예를 들면, 두경부암, 후두암, 인두암, 폐암, 뇌암, 유방암, 또는 대장암일 수 있으며, 바람직하게는 유방암일 수 있다. 상기 유방암 세포는 Her2 과발현 유방암, 또는 호르몬 수용체 양성 유방암 세포일 수 있으며, 상기 호르몬 수용체 양성은 에스트로겐 또는 프로게스테론 수용체의 발현인 것일 수 있다. In one embodiment, the cancer is not particularly limited as long as it is a cancer that can be distinguished by a difference in expression of MAP4K4, ACSL4 and moesin, for example, head and neck cancer, laryngeal cancer, pharyngeal cancer, lung cancer, brain cancer, breast cancer, or colon cancer. It may be, preferably breast cancer. The breast cancer cells may be Her2 overexpressing breast cancer or hormone receptor-positive breast cancer cells, and the hormone receptor-positive may be expression of an estrogen or progesterone receptor.
상기 방사선 내성 암은 MAP4K4, ACSL4, 및 모에신 유전자의 발현이 SK-BR-3 또는 MCF-7 암세포의 발현량보다 증가한 것일 수 있다. In the radiation-resistant cancer, the expression of MAP4K4, ACSL4, and moesin genes may be increased than that of SK-BR-3 or MCF-7 cancer cells.
상기 약학적 조성물은 통상적으로 사용되는 부형제, 붕해제, 감미제, 활택제, 향미제 등을 추가로 포함할 수 있다. 상기 붕해제로는 전분글리콜산나트륨, 크로스포비돈, 크로스카멜로스나트륨, 알긴산, 카르복시메틸셀룰로오스 칼슘, 카르복시 메틸셀룰로오스 나트륨, 키토산, 구아검, 마그네슘 알루미늄 실리케이트, 폴라크릴린 칼륨 등이 있다.The pharmaceutical composition may further include commonly used excipients, disintegrants, sweeteners, lubricants, flavoring agents, and the like. The disintegrants include sodium starch glycolate, crospovidone, croscarmellose sodium, alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium, chitosan, guar gum, magnesium aluminum silicate, and polyacrylic potassium.
상기 약학적 조성물은 약제학적으로 허용가능한 첨가제를 더 포함할 수 있으며, 이때 약제학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨, 탈크 등이 사용될 수 있다. 본 발명에 따른 약제학적으로 허용가능한 첨가제는 상기 약학적 조성물에 대해 0.1~90 중량부 포함되는 것이 바람직하다.The pharmaceutical composition may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose , Mannitol, syrup, arabic rubber, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose , Sorbitol, talc, and the like may be used. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the pharmaceutical composition.
경구투여를 위한 고형제제에는 산제, 과립제, 정제, 캡슐제, 연질캅셀제, 환 등이 포함된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제, 에어로졸 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Solid preparations for oral administration include powders, granules, tablets, capsules, soft capsules, and pills. Liquid preparations for oral use include suspensions, solvents, emulsions, syrups, aerosols, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives are included. I can.
비경구 투여를 위한 제제로는 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 멸균된 수용액, 액제, 비수성용제, 현탁제, 에멀젼, 시럽, 좌제, 에어로졸 등의 외용제 및 멸균 주사제제의 형태로 제형화하여 사용될 수 있으며, 바람직하게는 크림, 젤, 패취, 분무제, 연고제, 경고제, 로션제, 리니멘트제, 파스타제 또는 카타플라스마제의 피부 외용 약학적 조성물을 제조하여 사용할 수 있으나, 이에 한정하는 것은 아니다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Formulations for parenteral administration include powders, granules, tablets, capsules, sterilized aqueous solutions, solutions, non-aqueous solutions, suspensions, emulsions, syrups, suppositories, aerosols, etc. Formulated in a form and used, preferably, a pharmaceutical composition for external use of the skin of cream, gel, patch, spray, ointment, warning agent, lotion, liniment, pasta, or cataplasma may be prepared and used. , But is not limited thereto. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
상기 약학적 조성물의 바람직한 투여량은 체내에서 활성성분의 흡수도, 불활성화율 및 배설속도, 환자의 연령, 성별 및 상태, 치료할 질병의 중증 정도에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나 바람직한 효과를 위해서, 경구 투여제의 경우 일반적으로 성인에게 1일에 체중 1 kg당 본 발명의 조성물을 1일 0.0001 내지 100 ㎎/㎏으로, 바람직하게는 0.001 내지 100 ㎎/㎏으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다.The preferred dosage of the pharmaceutical composition varies depending on the degree of absorption, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, and the severity of the disease to be treated, but may be appropriately selected by those skilled in the art. However, for a desirable effect, in the case of oral administration, it is generally recommended to administer the composition of the present invention per 1 kg of body weight per day to adults at 0.0001 to 100 mg/kg, preferably 0.001 to 100 mg/kg. good. Administration may be administered once a day, or may be divided several times.
다른 양상은, MAP4K4, ACSL4 및 모에신 유전자의 발현 또는 활성을 억제하는 제제를 포함하는 방사선 내성 암 개선용 건강기능식품을 제공한다. Another aspect provides a health functional food for improving radiation-resistant cancer comprising an agent that inhibits the expression or activity of MAP4K4, ACSL4 and moesin genes.
상기 MAP4K4, ACSL4, 및 모에신에 대한 설명은 상술한 내용과 동일하다. Descriptions of the MAP4K4, ACSL4, and Moesin are the same as those described above.
상기 제제는 연자육 또는 연자방 추출물일 수 있다. 연자육 또는 연자방 및 이들의 추출물에 대한 설명은 상술한 내용과 동일하다. The formulation may be an extract of yeonjayuk or yeonjabang. The description of the yeonjayuk or yeonjabang and their extracts is the same as described above.
상기 암 및 방사선 내성 암에 대한 내용은 상술한 내용과 동일하다. The contents of the cancer and radiation-resistant cancer are the same as those described above.
상기 건강 기능 식품은 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 사용 목적(예방, 건강 또는 위생)에 따라 적합하게 결정될 수 있다.The health functional food may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or hygiene).
상기 건강 기능 식품의 형태 및 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 건강 기능 식품의 형태는 정제, 캅셀, 분말, 과립, 액상 및 환 등일 수 있고, 건강 기능 식품의 종류는 버터, 요구르트, 치즈를 포함한 유제품, 아이스크림류를 포함한 낙농제품, 유산균 제제, 발효유, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있다.There is no particular limitation on the form and type of the health functional food. The form of the health functional food to which the above substances can be added may be tablets, capsules, powders, granules, liquids and pills, and the types of health functional foods are dairy products including butter, yogurt, cheese, dairy products including ice cream, There are lactic acid bacteria preparations, fermented milk, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes.
본 발명의 건강 기능 식품은 통상의 건강 기능 식품과 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성감미제 등을 사용할 수 있다.The health functional food of the present invention may contain various flavoring agents or natural carbohydrates as an additional component, like a normal health functional food. The natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used.
상기한 것 외에 본 발명의 건강 기능 식품은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다.In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, It may contain glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like.
일 구체예에 따른 방사선 내성 암 치료용 약학적 조성물은 방사선 내성 암에서 과발현되는 MAP4K4, ACSL4, 및 모에신 유전자의 mRNA 또는 이의 단백질의 발현을 억제하는 제제를 포함함으로써 방사선 내성 암을 선택적으로 사멸시킬 수 있으므로 방사선 내성 암 치료 용도로 유용하게 사용될 수 있다. The pharmaceutical composition for the treatment of radiation-resistant cancer according to an embodiment comprises an agent that suppresses the expression of MAP4K4, ACSL4, and mRNA of the moesin gene or protein thereof, which are overexpressed in radiation-resistant cancer, thereby selectively killing radiation-resistant cancer. Since it can be used, it can be usefully used for radiation-resistant cancer treatment.
일 구체예에 따른 방사선 내성 암 개선용 건강기능식품은 방사선 내성 암에서 과발현되는 MAP4K4, ACSL4, 및 모에신 유전자의 mRNA 또는 이의 단백질의 발현을 억제하는 제제를 포함함으로써 방사선 내성 암을 선택적으로 사멸시킬 수 있으므로 방사선 내성 암 개선 용도로 유용하게 사용될 수 있다. The health functional food for improving radiation-resistant cancer according to an embodiment may selectively kill radiation-resistant cancer by including an agent that suppresses the expression of MAP4K4, ACSL4, and mRNA of the moesin gene or protein thereof that are overexpressed in radiation-resistant cancer. Since it can be used, it can be usefully used for improving radiation-resistant cancer.
도 1은 유방암 세포 SK-BR-3 및 MCF-7로부터 방사선 내성 유방암 세포인 SR 및 MR을 구축하는 과정을 도시한 것이다.
도 2는 방사선 내성 암세포주인 SR 및 MR이 일반 암세포주인 SK-BR-3 및 MCF-7 보다 방사선 조사 후 조직 생존율이 높음을 확인한 결과이다.
도 3은 방사선 내성 암세포주인 SR이 표적치료제(트라스트주맙과 라파티닙) 및 일반항암제(독소루비신)에 대해 내성이 있음을 확인한 결과를 나타낸 것이다.
도 4는 방사선 내성 암세포주인 SR에서 MAP4K4, ACSL4, 및 Moesin 유전자의 발현이 증가함을 cDNA 마이크로어레이 분석을 통해 확인한 결과이다.
도 5는 방사선 내성 암세포주인 SR 및 MR에서 MAP4K4, ACSL4, 및 Moesin mRNA의 발현이 증가함을 확인한 결과이다.
도 6은 방사선 내성 암세포주인 SR 및 MR에서 MAP4K4, ACSL4 및 Moesin 단백질의 발현이 증가함을 확인한 결과이다.
도 7의 A는 방사선 내성 암세포 SR에서 ACSL1, ACSL3, ACSL4, ACSL5, 및 ACSL6 중에서 ACSL4 유전자의 발현이 특이적으로 증가함을 확인한 결과이며, 도 7의 B는 방사선 내성 암세포 SR 및 MR에서 ACSL 이성체 중 ACSL4 단백질의 발현만 특이적으로 증가함을 확인한 결과이다.
도 8은 수용성 연자육 추출물이 방사선 내성 암세포인 SR에 대해 세포 독성이 없음을 확인한 결과이다.
도 9는 본 발명의 실시예에 따른 연자육의 70% 에탄올 추출물을 제조하는 과정을 도시한 것이다.
도 10은 연자육의 70% 에탄올 추출물이 방사선 내성 암세포 SR 및 MR에 대해 선택적으로 높은 세포사멸효과를 나타냄을 확인한 결과이다.
도 11은 방사선 내성 암세포 SR 및 MR에서 증가한 MAP4K4, ACSL4, 및 Moesin 발현이 연자육 70% 에탄올 추출물에 의해 효과적으로 억제됨을 확인한 결과이다.
도 12는 본 발명의 실시예에 따른 연자방의 70% 에탄올 추출물을 제조하는 과정을 도시한 것이다.
도 13의 A는 연자방 70% 에탄올 추출물이 방사선 내성 암세포(SR)에 대해 선택적으로 높은 세포사멸효과를 나타냄을 확인한 결과이며, 도 13의 B는 연자방 70% 에탄올 추출물이 방사선 내성 암세포(SR)에서 증가한 MAP4K4, ACSL4, 및 Moesin 단백질의 발현을 억제함을 확인한 결과이다.1 shows a process of constructing radiation-resistant breast cancer cells SR and MR from breast cancer cells SK-BR-3 and MCF-7.
2 is a result of confirming that the radiation-resistant cancer cell lines SR and MR have higher tissue survival rates after irradiation than normal cancer cell lines SK-BR-3 and MCF-7.
Figure 3 shows the results of confirming that the radiation-resistant cancer cell line SR is resistant to target therapeutic agents (trastzumab and lapatinib) and general anticancer drugs (doxorubicin).
4 is a result of confirming through cDNA microarray analysis that the expression of MAP4K4, ACSL4, and Moesin genes is increased in SR, which is a radiation-resistant cancer cell line.
5 is a result of confirming that the expression of MAP4K4, ACSL4, and Moesin mRNA is increased in SR and MR, which are radiation-resistant cancer cell lines.
6 is a result of confirming that the expression of MAP4K4, ACSL4 and Moesin proteins is increased in SR and MR, which are radiation-resistant cancer cell lines.
7A is a result of confirming that the expression of the ACSL4 gene is specifically increased among ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6 in radiation-resistant cancer cells SR, and FIG. 7B is an ACSL isomer in radiation-resistant cancer cells SR and MR. This is the result of confirming that only the expression of the ACSL4 protein increases specifically.
Figure 8 is a result of confirming that the water-soluble lotus root extract has no cytotoxicity to SR, which is a radiation-resistant cancer cell.
9 shows a process for preparing a 70% ethanol extract of lotus meat according to an embodiment of the present invention.
10 is a result of confirming that the 70% ethanol extract of yeonjayuk selectively exhibits a high apoptosis effect on radiation-resistant cancer cells SR and MR.
11 is a result of confirming that the increased expression of MAP4K4, ACSL4, and Moesin in radiation-resistant cancer cells SR and MR is effectively suppressed by the 70% ethanol extract of Yeonjayuk.
12 shows a process for preparing a 70% ethanol extract of Yeonjabang according to an embodiment of the present invention.
13A is a result of confirming that the 70% ethanol extract of yeonjabang exhibits a selectively high apoptosis effect on radiation-resistant cancer cells (SR), and FIG. ) Is the result of confirming that the expression of MAP4K4, ACSL4, and Moesin proteins increased in is suppressed.
이하 하나 이상의 구체예를 실시예를 통해 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1: 방사선 내성 종양모델 구축Example 1: Construction of radiation-resistant tumor model
SK-BR-3 및 MCF-7 암세포주를 준비하였다. SK-BR-3은 Her2 과발현 subtype의 인간 유방암 세포이며, MCF-7은 호르몬 수용체 양성 subtype의 인간 유방암 세포이다. SK-BR-3 and MCF-7 cancer cell lines were prepared. SK-BR-3 is a human breast cancer cell of the Her2 overexpression subtype, and MCF-7 is a human breast cancer cell of a hormone receptor positive subtype.
SK-BR-3 및 MCF-7 암세포주에 (a) 2 내지 5 Gy의 방사선을 주 2회 반복 조사하였으며 (b) 총 4 내지 10 Gy의 방사선이 조사된 암세포를 총 6주간 계대배양 하였다. 상기 (a) 단계 및 (b) 단계를 6회 내지 10 회 반복함으로써 방사선 내성 암세포를 확립하였다. 방사선 저항성 유무를 판단하기 위해 집락 형성능 분석(colony forming assay)를 시행하여 저항성 획득 여부를 판단하였다. 상기 각 일반 유방암 세포 SK-BR-3 및 MCF-7로부터 확립된 각각의 방사선 내성 인간 유방암 세포를 SR 및 MR로 명명하였다(도 1 참조). SK-BR-3 and MCF-7 cancer cell lines were (a) irradiated with 2 to 5 Gy of radiation twice a week, and (b) cancer cells irradiated with a total of 4 to 10 Gy of radiation were subcultured for a total of 6 weeks. Radiation-resistant cancer cells were established by repeating steps (a) and (b) 6 to 10 times. To determine the presence or absence of radiation resistance, a colony forming assay was performed to determine whether or not resistance was obtained. Each radiation-resistant human breast cancer cell established from each of the general breast cancer cells SK-BR-3 and MCF-7 was designated as SR and MR (see Fig. 1).
실시예 2: SR 및 MR 세포의 방사선 내성 획득 확인 Example 2: Confirmation of obtaining radiation resistance of SR and MR cells
SR 및 MR 세포의 방사선 내성 획득 여부를 조사하기 위해 집락 형성능 분석을 실시하여 일반 유방암 세포(이하 일반 암세포) SK-BR-3 및 MCF-7과 조직 생존율(survival fraction)을 비교 평가하였다. 먼저 300 내지 900개의 세포를 10% fetal bovine serum이 포함된 DMEM(이하 10% FBS DMEM)에 현탁한 후 6 well plate에 plating 하였다. 24시간 동안 배양한 다음 각각 0, 2, 4 Gy로 방사선을 조사한 후 37도, 5% CO2가 유지되는 배양기에서 10 내지 30일 동안 배양하였다. 형성된 집락을 10% formalin으로 15분 동안 고정한 다음 0.01% crystal violet으로 염색한 후 counting 하여 조직 생존율을 산출하였다. To investigate whether SR and MR cells acquired radiation resistance, colony-forming ability analysis was performed to compare and evaluate the tissue survival rate with normal breast cancer cells (hereinafter, normal cancer cells) SK-BR-3 and MCF-7. First, 300 to 900 cells were suspended in DMEM (hereinafter referred to as 10% FBS DMEM) containing 10% fetal bovine serum, and then plated on a 6 well plate. After incubation for 24 hours, radiation was irradiated with 0, 2, and 4 Gy, respectively, and cultured for 10 to 30 days in an incubator maintained at 37 degrees and 5% CO 2. The formed colonies were fixed with 10% formalin for 15 minutes, stained with 0.01% crystal violet, and counted to calculate the tissue survival rate.
도 2의 A 및 B에 나타낸 바와 같이, 방사선 내성 암세포인 SR 및 MR의 조직 생존율이 SR-BR-3 및 MCF-7보다 높은 것으로 확인되었으며, SR 및 MR 모두 방사선에 대한 저항성을 획득하였음을 확인하였다. 2A and 2B, it was confirmed that the tissue survival rates of SR and MR, which are radiation-resistant cancer cells, are higher than that of SR-BR-3 and MCF-7, and that both SR and MR have acquired resistance to radiation. I did.
실시예 3: 방사선 내성 암세포의 항암제에 대한 내성 획득 확인 Example 3: Confirmation of acquisition of resistance to anticancer agents of radiation-resistant cancer cells
임상에서 유방암 치료제로 사용 중인 HER2 표적 치료제 트라스트주맙(trastuzumab), EGFR/HER2 이중표적 치료제 라파티닙(lapatinib), 및 일반 항암제 독소루비신(doxorubicin)의 방사선 내성 암세포에 대한 항암 효과를 확인하였다. 각 항암제를 일반 암세포 (SK-BR-3) 및 방사선 내성 암세포(SR)에 72시간 동안 처리한 후 MTT assay를 실시하였다. The anticancer effect of the HER2 target treatment trastuzumab, the EGFR/HER2 dual target treatment lapatinib, and the general anticancer drug doxorubicin on radiation-resistant cancer cells was confirmed in clinical practice. Each anticancer agent was treated on general cancer cells (SK-BR-3) and radiation-resistant cancer cells (SR) for 72 hours, followed by MTT assay.
구체적으로, 일반 암세포 SK-BR-3 (5,000개/well)와 방사선 내성 암세포 SR (2,000개/well)를 10% FBS DMEM배지에 현탁한 후 96-well plate에 seeding하고 24시간 배양하였다. 그 후 다양한 농도의 trastuzumab (3, 6, 12.5, 25, 50 μg/mL), lapatinib (0.01, 0.05, 0.1, 0.5, 1, 5 μM) 또는 doxorubicin (0.05, 0.1, 0.2, 0.4, 0.6 μM)을 처리한 다음 3일(약 72 시간) 동안 배양하였다. 배양이 끝난 세포에 MTT 시약을 첨가하고 4시간 동안 배양 한 다음 570nm에서 흡광값을 측정하여 일반 암세포와 방사선 내성 암세포의 생존율을 비교 분석하였다Specifically, normal cancer cells SK-BR-3 (5,000 cells/well) and radiation-resistant cancer cells SR (2,000 cells/well) were suspended in 10% FBS DMEM medium, seeded in a 96-well plate, and cultured for 24 hours. Afterwards, various concentrations of trastuzumab (3, 6, 12.5, 25, 50 μg/mL), lapatinib (0.01, 0.05, 0.1, 0.5, 1, 5 μM) or doxorubicin (0.05, 0.1, 0.2, 0.4, 0.6 μM) And then incubated for 3 days (about 72 hours). MTT reagent was added to the cultivated cells and incubated for 4 hours, and then the absorbance value was measured at 570 nm to compare and analyze the survival rates of normal cancer cells and radiation-resistant cancer cells.
도 3의 A 및 B에 나타낸 바와 같이, 표적치료제인 트라스트주맙과 라파티닙에 의한 세포 생존율은 일반 암세포인 SK-BR-3 보다 방사선 내성 암세포인 SR에서 월등히 높게 나타나는 것으로 나타났다. 뿐만 아니라 일반 항암제인 독소루비신에 의한 세포 생존율 또한 일반 암세포 보다 방사선 내성 암세포에서 높은 것으로 나타났다(도 3의 C). 이러한 결과는 방사선 내성 암세포가 방사선 내성뿐만 아니라 표적 치료제 (trastuzumab 및 lapatinib)와 일반 항암제(doxorubicin)에 대한 내성 또한 획득하였음을 보여주고 있다. As shown in Fig. 3A and 3B, it was found that the cell survival rate by the target therapeutic agents trastzumab and lapatinib was significantly higher in SR, which is radiation-resistant cancer cells, than in SK-BR-3, which is a general cancer cell. In addition, it was found that the cell survival rate by doxorubicin, a general anticancer drug, was also higher in radiation-resistant cancer cells than in general cancer cells (FIG. 3C). These results show that radiation-resistant cancer cells acquired not only radiation resistance, but also resistance to target therapeutics (trastuzumab and lapatinib) and general anticancer drugs (doxorubicin).
특히, 상기 결과는 방사선 내성 유방암의 효율적 치료를 위하여 방사선 내성을 유도하는 핵심 인자 즉, 방사선 내성 인자를 발굴하여 이를 표적으로 하는 새로운 치료 전략을 개발할 필요가 있음을 보여주고 있다. In particular, the above results show that for the efficient treatment of radiation-resistant breast cancer, it is necessary to discover a key factor inducing radiation resistance, that is, a radiation resistance factor, and develop a new treatment strategy targeting it.
실시예 4: 방사선 내성 암세포에서 MAP4K4, ACSL4 및 Moesin 유전자 발현 증가 확인Example 4: Confirmation of increased expression of MAP4K4, ACSL4 and Moesin genes in radiation-resistant cancer cells
방사선 내성 암세포(SR)에서 방사선 내성 획득에 따른 유전자의 발현 변화를 분석하기 위해서 cDNA microarray를 실시하였다. 먼저, easy-BLUE total RNA extraction kit (iNtRON Biotechnology, Sungnam, Korea)를 이용하여 각 세포로부터 total RNA를 추출한 다음 (주)디앤피바이오택 (Daegu, Korea)에 cDNA microarray 분석을 의뢰하여 유전자의 발현 변화를 비교 분석하였다.A cDNA microarray was performed to analyze the gene expression change according to the acquisition of radiation resistance in radiation-resistant cancer cells (SR). First, total RNA was extracted from each cell using the easy-BLUE total RNA extraction kit (iNtRON Biotechnology, Sungnam, Korea), and then the gene expression was changed by requesting cDNA microarray analysis to D&P Biotech (Daegu, Korea). Were compared and analyzed.
분석 결과, 일반 암세포 (SK-BR-3)와 방사선 내성 암세포(SR) 사이에서는 약 2,000개의 유전자의 발현에 변화가 있음을 확인하였다, 특히 도 4에 나타낸 바와 같이, SR 세포주는 SK-BR-3 세포주보다 MAP4K4 유전자의 발현이 6배 이상, ACSL4 유전자의 발현이 5배 이상, Moesin 유전자는 4.6배 이상 증가하였음을 확인하였다.As a result of the analysis, it was confirmed that there was a change in the expression of about 2,000 genes between normal cancer cells (SK-BR-3) and radiation-resistant cancer cells (SR). In particular, as shown in FIG. 4, the SR cell line SK-BR- It was confirmed that the expression of the MAP4K4 gene increased by 6 times or more, the expression of the ACSL4 gene by more than 5 times, and the Moesin gene by 4.6 times or more than that of the 3 cell line.
실시예 5: 방사선 내성 암세포에서 MAP4K4, ACSL4 및 Moesin mRNA 발현 증가 확인Example 5: Confirmation of increased expression of MAP4K4, ACSL4 and Moesin mRNA in radiation-resistant cancer cells
일반 암세포(SK-BR-3와 MCF-7)와 방사선 내성 암세포 (SR과 MR)에서 MAP4K4, ACSL4, 및 Moesin의 mRNA의 발현을 비교 분석하기 위해서 realtime PCR을 수행하였다. 먼저, 상기 실시예 4와 동일한 방법으로 total RNA 추출한 다음 reverse-transcriptase를 이용하여 cDNA를 제조하였다. 제조한 cDNA를 주형으로 하여 MAP4K4 프라이머 (Forward 5'-GAGAGGCTCCAGAGGCAGTTGCA-3', Reverse 5'-CCACCTCTCGCGCTCGGTCAG-3'), ACSL4 프라이머 (Forward 5'-GGGAAGAAGGACAGCCTTGGG-3', Reverse 5'-TCGGCCCTGGTCTCACAGAA-3'), Moesin 프라이머 (Forward 5'-GCTGTGGAGTGGCAGCAGAA-3', Reverse 5'-CGTTCCTCCTCACTGCGGTC-3')와 18s rRNA 프라이머(Forward 5'-GTAACCCGTTGAACCCCATT-3', Reverse 5'-CCATCCAATCGGTAGTAGCG-3')를 이용하여 변성단계(95℃, 10초), 어닐링단계(60℃, 20초) 및 연장단계(72℃, 20초)를 1 cycle로 하여 45 cycles를 실시하여 realtime PCR을 시행한 후 방사선 내성 암세포와 일반 암세포에서 나타난 ACSL4와 Moesin mRNA의 발현을 비교 분석하였다. 프라이머 및 서열목록 정보는 하기 표 2에 기재되어 있다.Real-time PCR was performed to compare and analyze the expression of MAP4K4, ACSL4, and Moesin mRNAs in normal cancer cells (SK-BR-3 and MCF-7) and radiation-resistant cancer cells (SR and MR). First, total RNA was extracted in the same manner as in Example 4, and then cDNA was prepared using reverse-transcriptase. Using the prepared cDNA as a template, MAP4K4 primer (Forward 5'-GAGAGGCTCCAGAGGCAGTTGCA-3', Reverse 5'-CCACCTCTCGCGCTCGGTCAG-3'), ACSL4 primer (Forward 5'-GGGAAGAAGGACAGCCTTGGG-3', Reverse 5'-TCGGCCCTGGTCTCACAGAA-3') , Moesin primer (Forward 5'-GCTGTGGAGTGGCAGCAGAA-3', Reverse 5'-CGTTCCTCCTCACTGCGGTC-3') and 18s rRNA primer (Forward 5'-GTAACCCGTTGAACCCCATT-3', Reverse 5'-CCATCCAATCGGTAGTAGCG-3') (95℃, 10 seconds), annealing stage (60℃, 20 seconds) and extension stage (72℃, 20 seconds) were performed as 1 cycle, 45 cycles, and real-time PCR was performed. The expressions of ACSL4 and Moesin mRNA were compared and analyzed. Primer and sequence listing information is shown in Table 2 below.
도 5의 A, B, 및 C에 나타낸 바와 같이, 방사선 내성 암세포(SR과 MR)가 일반 암세포(SK-BR-3, MCF-7)보다 MAP4K4, ACSL4, 및 Moesin mRNA의 발현이 월등이 높음을 확인할 수 있었다.5A, B, and C, radiation-resistant cancer cells (SR and MR) have significantly higher expression of MAP4K4, ACSL4, and Moesin mRNA than normal cancer cells (SK-BR-3, MCF-7). Was able to confirm.
실시예 6: 방사선 내성 암세포에서 MAP4K4, ACSL4, 및 Moesin 단백질의 발현 증가 확인 Example 6: Confirmation of increased expression of MAP4K4, ACSL4, and Moesin proteins in radiation-resistant cancer cells
일반 암세포(SK-BR-3와 MCF-7)와 방사선 내성 암세포 (SR과 MR)에서 MAP4K4, ACSL4, 및 Moesin 단백질의 발현을 분석하기 위해서 western blot assay를 수행하였다. Protease inhibitor와 phosphatase inhibitor를 1:100의 비율로 혼합한 RIPA lysis buffer를 세포에 넣고 상온에서 5분 동안 반응시킨 후 13,000 rpm으로 10분 동안 원심분리하여 획득한 상층액을 단백질 샘플로 사용하였다. Western blot assay was performed to analyze the expression of MAP4K4, ACSL4, and Moesin proteins in normal cancer cells (SK-BR-3 and MCF-7) and radiation resistant cancer cells (SR and MR). A RIPA lysis buffer in which a protease inhibitor and a phosphatase inhibitor were mixed at a ratio of 1:100 was added to the cells, reacted at room temperature for 5 minutes, and then centrifuged at 13,000 rpm for 10 minutes, and the obtained supernatant was used as a protein sample.
각 단백질 샘플은 BSA (Bovine serum와albumin)로 단백질 정량을 하고, 동일한 양의 단백질을 8%의 SDS-PAGE (Sodium dodecylsulfate-polyacrylamide gel electrophoresis)로 분리한 후 PVDF (polyvinylidene fluoride) 막으로 옮겨서 ACSL4 또는 Moesin antibody를 사용하여 발현변화를 분석하였다. 단백질의 loading control로는 β-actin을 사용하였다. 2차 항체(Secondary antibody)는 horseradish peroxidase (HRP)-conjugated anti-rabbit IgG 또는 HRP-conjugated anti-mouse IgG를 사용하였고, 단백질 밴드는 ECL (Enhanced chemiluminescence) solution으로 발색한 후 LAS-4000 (Fujifilm, Tokyo, Japan)으로 확인하였다. Each protein sample was quantified with BSA (Bovine serum and albumin), and the same amount of protein was separated by 8% SDS-PAGE (Sodium dodecylsulfate-polyacrylamide gel electrophoresis), transferred to PVDF (polyvinylidene fluoride) membrane, and transferred to ACSL4 or Expression changes were analyzed using Moesin antibody. As a protein loading control, β-actin was used. Secondary antibody was horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or HRP-conjugated anti-mouse IgG, and protein bands were colored with ECL (Enhanced chemiluminescence) solution and then LAS-4000 (Fujifilm, Tokyo, Japan).
도 6의 A, B, 및 C에 나타낸 바와 같이, 일반 암세포(SK-BR-3와 MCF-7)에서는 MAP4K4, ACSL4, 및 Moesin 단백질의 발현이 나타나지 않는 반면, 방사선 내성 암세포인 SR과 MR에서는 뚜렷하게 증가하는 것을 확인할 수 있었다.As shown in Fig. 6A, B, and C, the expression of MAP4K4, ACSL4, and Moesin proteins was not observed in normal cancer cells (SK-BR-3 and MCF-7), whereas in SR and MR, radiation-resistant cancer cells It was confirmed that there was a distinct increase.
실시예 7: 방사선 내성 암세포에서 5가지 ACSL 이성체 중 ACSL 4의 단독 발현 증가 확인Example 7: Confirmation of increased expression of
7-1. 방사선 내성 암세포에서 ACSL 4 유전자의 단독 발현 증가 확인7-1. Confirmation of increased expression of
상기 실시예 4와 동일한 방법으로 cDNA microarray 분석을 실시 한 후 ACSL1, ACSL3, ACSL4, ACSL5, 및 ACSL6의 발현을 분석하였다.After performing cDNA microarray analysis in the same manner as in Example 4, the expression of ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6 was analyzed.
도 7의 A에서 나타낸 바와 같이, ACSL1, ACSL3, ACSL5, 및 ACSL6의 유전자발현은 방사선 내성 세포 SR에서 일반 암세포(SK-BR-3) 보다 감소하거나 2 배 이하의 낮은 수준으로 증가하는 것으로 나타났다. 반면 ACSL4의 경우, 방사선 내성 암세포인 SR에서 일반 암세포 보다 발현이 5.5배 증가하는 것으로 나타났다.As shown in Fig. 7A, the gene expression of ACSL1, ACSL3, ACSL5, and ACSL6 was decreased or increased to a lower level of 2 times or less than that of normal cancer cells (SK-BR-3) in radiation-resistant cells SR. On the other hand, in the case of ACSL4, it was found that the expression of SR, which is a radiation-resistant cancer cell, was 5.5 times higher than that of normal cancer cells.
7-2. 방사선 내성 암세포에서 ACSL4 단백질의 단독 발현 증가 확인7-2. Confirmation of increased expression of ACSL4 protein alone in radiation-resistant cancer cells
상기 실시예 6과 동일한 방법으로 준비한 단백질 샘플을 이용하여 ACSL1, ACSL3, ACSL4, 및 ACSL5의 발현을 분석하였다.Expression of ACSL1, ACSL3, ACSL4, and ACSL5 was analyzed using protein samples prepared in the same manner as in Example 6.
도 7의 B에 나타낸 바와 같이, 방사선 내성 암세포인 SR과 MR은 4가지 이성체 ACSL1, ACSL3, ACSL4, 및 ACSL5 중에서 ACSL4의 단백질만 특이적으로 발현이 증가함을 알 수 있다. 따라서 실시예 7-1 및 실시예 7-2의 결과를 바탕으로 방사선 내성 암세포에서 ACSL 4만 특이적으로 발현이 증가함을 알 수 있다.As shown in B of FIG. 7, it can be seen that among the four isomers ACSL1, ACSL3, ACSL4, and ACSL5, only the protein of ACSL4 is specifically increased in expression of the radiation-resistant cancer cells SR and MR. Therefore, based on the results of Example 7-1 and Example 7-2, it can be seen that
실시예 8: 수용성 연자육의 방사선 내성 암세포에 대한 무독성 확인Example 8: Confirmation of non-toxicity of water-soluble soft meat against radiation-resistant cancer cells
본 실험에 사용된 수용성 연자육 추출물은 시중에서 고형차 식품유형으로 판매되고 있는 연자육 제품을 구매하여 실험에 사용하였다. 방사선 내성 암세포에 대한 수용성 연자육 추출물의 선택적 세포사멸효과를 분석하기 위해서 일반 암세포(SK-BR-3 및 MCF-7)와 방사선 내성 암세포 (SR 및 MR)에 다양한 농도의 수용성 연자육 추출물을 처리한 후 SRB (Sulfohodamin B) assay를 실시하였다.The water-soluble lotus meat extract used in this experiment was used in the experiment by purchasing a lotus meat product sold as a solid tea food type on the market. To analyze the selective apoptosis effect of the water-soluble lotus root extract on radiation-resistant cancer cells, normal cancer cells (SK-BR-3 and MCF-7) and radiation-resistant cancer cells (SR and MR) were treated with various concentrations of water-soluble lotus meat extract. SRB (Sulfohodamin B) assay was performed.
실시예 3과 동일한 방법으로 세포를 seeding한 후 24시간 동안 배양하였다. 그 후 1, 5, 10, 50, 100, 및 200 μg/mL의 수용성 연자육 추출물을 처리하고 48시간 동안 배양하였다. 상층액을 버리고 10% (w/v) TCA (Trichloroacetic acid) 용액을 100 μL 넣고 4℃에서 한 시간 동안 배양한 후 천천히 흐르는 수돗물을 이용하여 4회 세척하였다. Plate를 상온에 방치하여 물기를 모두 건조시킨 후 0.4% (w/v) SRB 용액 100 μL를 넣고 상온에서 30분 동안 반응 시킨 후 1% (v/v) acetic acid 용액을 이용하여 세포단백질에 결합하지 않은 여분의 SRB를 제거하였다. 상온에 방치하여 plate를 완전히 건조 시킨 후 200 μL의 10 mM Tris base 용액 (pH 10.5)를 넣어 세포단백질에 결합한 SRB를 용해시킨 후 510 nm에서 흡광값을 측정하여 세포의 생존율을 산출 하여 비교 분석하였다.After seeding the cells in the same manner as in Example 3, the cells were cultured for 24 hours. Then, 1, 5, 10, 50, 100, and 200 μg/mL of water-soluble lotus extract were treated and cultured for 48 hours. The supernatant was discarded, and 100 μL of a 10% (w/v) TCA (Trichloroacetic acid) solution was added and incubated at 4° C. for an hour, followed by washing 4 times with slow flowing tap water. After leaving the plate at room temperature to dry all the water, add 100 μL of 0.4% (w/v) SRB solution, react at room temperature for 30 minutes, and bind to the cell protein using 1% (v/v) acetic acid solution. The excess SRB that did not do was removed. After standing at room temperature to completely dry the plate, 200 μL of 10 mM Tris base solution (pH 10.5) was added to dissolve the SRB bound to the cell protein, and the absorbance value was measured at 510 nm to calculate the viability of cells and analyzed for comparison. .
도 8에 나타낸 바와 같이, 수용성 연자육 추출물은 일반 암세포 뿐만 아니라 방사선 내성 암세포에 대한 세포 독성이 없는 것으로 나타났다.As shown in Figure 8, it was found that the water-soluble lotus extract was not cytotoxic to radiation-resistant cancer cells as well as general cancer cells.
실시예 9: 70% 에탄올 연자육 추출물 제조Example 9: Preparation of 70% ethanol lotus meat extract
도 9를 참조하여 제조방법을 설명한다. 연자육은 경상북도 경산에서 재배된 것을 구입하여 사용하였다. 70% 에탄올을 이용한 연자육 추출물을 제조하기 위해 연자육 20g에 5배 부피의 70% 에탄올(100 mL)을 넣고 3일간 상온에서 추출하였다. 동일한 방법으로 2회 반복 추출하여 추출액을 분리한 다음 여과하고 회전 감압 농축기를 이용하여 고형이 될 때까지 농축하였다. 추출물은 DMSO (Dimethyl sulfoxide)에 고농도로 녹인 다음 -80℃에 보관하며 실험에 사용하였다.The manufacturing method will be described with reference to FIG. 9. Yeonjayuk was purchased and used in Gyeongsan, Gyeongsangbuk-do. To prepare a lotus extract using 70% ethanol, 5 times the volume of 70% ethanol (100 mL) was added to 20 g of lotus leaf and extracted at room temperature for 3 days. Extraction was repeated twice in the same manner to separate the extract, filtered, and concentrated until solid using a rotary vacuum concentrator. The extract was dissolved in DMSO (dimethyl sulfoxide) at a high concentration, then stored at -80°C and used in the experiment.
실시예 10: 연자육 70% 에탄올 추출물의 방사선 내성 암세포에 대한 선택적 세포 사멸 및 MAP4K4, ACSL 4, 및 Moesin의 발현 억제효과 확인Example 10: Confirmation of selective cell death and inhibition of expression of MAP4K4,
10-1. 연자육 70% 에탄올 추출물의 방사선 내성 암세포에 대한 선택적 세포사멸효과 분석10-1. Analysis of Selective Apoptosis Effect of 70% Ethanol Extract of Yeonjayuk on Radiation-resistant Cancer Cells
연자육 70% 에탄올 추출물의 방사선 내성 암세포에 대한 선택적 세포사멸효과를 분석하기 위해서 일반 암세포(SK-BR-3 및 MCF-7)와 방사선 내성 암세포(SR 및 MR)에 다양한 농도의 연자육 70% 에탄올 추출물을 처리한 후 SRB (Sulfohodamin B) assay를 실시하였다. In order to analyze the selective apoptosis effect of 70% ethanol extract on radiation-resistant cancer cells, 70% ethanol extracts of 70% soft tea in various concentrations on normal cancer cells (SK-BR-3 and MCF-7) and radiation-resistant cancer cells (SR and MR) After treatment, SRB (Sulfohodamin B) assay was performed.
상기 실시예 8과 동일한 방법으로 세포를 seeding한 후 24시간 동안 배양하였다. 그 후 20∼120 μg/mL 또는 10∼200 μg/mL의 연자육 70% 에탄올 추출물을 처리하고 24 또는 48시간 동안 배양한 후 세포 고정, SRB 염색 후 510nm에서 흡광값 측정을 하여 세포의 생존율을 산출하여 비교 분석하였다. After seeding the cells in the same manner as in Example 8, the cells were cultured for 24 hours. Thereafter, 20 to 120 μg/mL or 10 to 200 μg/mL of 70% ethanol extract was treated and cultured for 24 or 48 hours, followed by cell fixation, SRB staining, and measuring the absorbance at 510 nm to calculate the survival rate of cells. And compared and analyzed.
도 10의 A 및 B에 나타낸 바와 같이, 연자육 70% 에탄올 추출물은 방사선 내성 암세포인 SR 및 MR에 대해 농도 의존적으로 높은 선택적 세포 사멸 효과를 나타냄을 알 수 있다.As shown in A and B of Fig. 10, it can be seen that the 70% ethanol extract of yeonjayuk exhibits a high selective apoptosis effect in a concentration-dependent manner for the radiation-resistant cancer cells SR and MR.
10-2. 연자육 70% 에탄올 추출물의 방사선 내성 암세포에서 증가된 MAP4K4, ACSL4, 및 Moesin 단백질에 대한 발현 억제 효과 분석10-2. Analysis of the Inhibitory Effect of 70% Ethanol Extract of Lotus Silk on the Increased Expression of MAP4K4, ACSL4, and Moesin Proteins in Radiation-resistant Cancer Cells
연자육 70% 에탄올 추출물을 처리한 방사선 내성 암세포에서 MAP4K4, ACSL4, 및 Moesin 단백질의 발현 변화를 분석하기 위해 western blot assay를 실시하였다. 방사선 내성 암세포(SR 및 MR)에 10, 25, 50 및 100 μg/mL 또는 20, 40, 80, 120 μg/mL의 연자육 70% 에탄올 추출물을 24시간 동안 처리한 후 상기 실시예 6과 동일한 방법으로 단백질 샘플을 준비하여 실험에 사용하였다. Western blot assay was performed to analyze the expression changes of MAP4K4, ACSL4, and Moesin proteins in radiation-resistant cancer cells treated with 70% ethanol extract of lotus root. The same method as in Example 6 after treating radiation-resistant cancer cells (SR and MR) with 10, 25, 50, and 100 μg/mL or 20, 40, 80, 120 μg/mL of soft-grown 70% ethanol extract for 24 hours. A protein sample was prepared and used in the experiment.
도 11의 A 및 B에 나타낸 바와 같이, 연자육 70% 에탄올 추출물의 처리 농도에 의존적으로 MAP4K4, ACSL4, 및 Moesin 단백질의 발현이 효과적으로 저해됨을 알 수 있다. 이는 연자육 70% 에탄올 추출물의 방사선 내성 암세포인 SR과 MR에 대한 선택적 세포사멸 효과는 MAP4K4, ACSL4, 및 Moesin의 발현 억제에 의한 것임을 보여는 결과이다.11A and 11B, it can be seen that the expression of MAP4K4, ACSL4, and Moesin proteins is effectively inhibited depending on the treatment concentration of the 70% ethanol extract of lotus root. This is a result of demonstrating that the selective apoptosis effect of the 70% ethanol extract of Yeonjayuk on SR and MR, which are radiation-resistant cancer cells, is due to the inhibition of the expression of MAP4K4, ACSL4, and Moesin.
실시예 11: 연자방 70% 에탄올 추출물의 조제Example 11: Preparation of 70% ethanol extract of Yeonjabang
도 12를 참조하여 연자방 추출물 제조방법을 설명한다. 연자방은 경상북도 경산에서 재배된 것을 구입하여 사용하였다. 70% 에탄올을 이용한 연자방 추출물을 제조하기 위해 연자방 214.3 g에 5배 부피의 70% 에탄올(1,100 mL)을 넣고 3일간 상온에서 추출하였다. 동일한 방법으로 2회 반복 추출하여 추출액을 분리한 다음 여과하고 회전 감압 농축기를 이용하여 고형이 될 때까지 농축하였고 잔류 수분은 동결건조기를 이용하여 제거하였다. 추출물은 DMSO에 고농도로 녹인 다음 -80℃에 보관하며 실험에 사용하였다.With reference to Figure 12 will be described a method for producing a lotus leaf extract. Yeonjabang was purchased and used in Gyeongsan, Gyeongsangbuk-do. To prepare a lotus leaf extract using 70% ethanol, 5 times the volume of 70% ethanol (1,100 mL) was added to 214.3 g of lotus leaf and extracted at room temperature for 3 days. Extraction was repeated twice in the same way to separate the extract, filtered, and concentrated until solid using a rotary vacuum concentrator, and residual moisture was removed using a freeze dryer. The extract was dissolved in DMSO at a high concentration and then stored at -80°C and used in the experiment.
실시예 12: 연자방 70% 에탄올 추출물에 의한 방사선 내성 암세포의 선택적 세포 사멸 및 MAP4K4, ACSL4, 및 Moesin의 발현 억제효과 확인Example 12: Selective apoptosis of radiation-resistant cancer cells by 70% ethanol extract of Yeonjabang and confirming the effect of inhibiting the expression of MAP4K4, ACSL4, and Moesin
12-1. 방사선 내성 암세포에 대한 연자방 70% 에탄올 추출물의 선택적 세포사멸효과 분석 12-1. Analysis of Selective Apoptosis Effect of 70% Ethanol Extract of Yeonjabang on Radiation-resistant Cancer Cells
연자방 70% 에탄올 추출물의 방사선 내성 암세포에 대한 선택적 세포사멸효과를 분석하기 위해서 일반 암세포(SK-BR-3) 및 방사선 내성 암세포 (SR)에 다양한 농도의 연자방 70% 에탄올 추출물을 처리한 후 SRB assay를 실시하였다. In order to analyze the selective apoptosis effect of the 70% ethanol extract of Yeonjabang on radiation-resistant cancer cells, general cancer cells (SK-BR-3) and radiation-resistant cancer cells (SR) were treated with 70% ethanol extract of Yeonjabang at various concentrations. SRB assay was performed.
상기 실시예 8과 동일한 방법으로 세포를 seeding한 후 24시간 동안 배양하였다. 그 후 5, 7.5, 10, 20, 30, 40, 및 50 μg/mL 의 연자방 70% 에탄올 추출물을 48시간 동안 처리한 후 세포 고정, SRB 염색 후 510nm에서 흡광값 측정을 하여 세포의 생존율을 산출하여 비교 분석하였다.After seeding the cells in the same manner as in Example 8, the cells were cultured for 24 hours. After that, 5, 7.5, 10, 20, 30, 40, and 50 μg/mL of 70% ethanol extract of Yeonjabang were treated for 48 hours, and then the cells were fixed and the absorbance value was measured at 510 nm after SRB staining to determine the survival rate of the cells. It was calculated and analyzed for comparison.
도 13의 A에 나타낸 바와 같이, 연자방 70% 에탄올 추출물의 농도에 의존적으로 방사선 내성 암세포인 SR에 선택적으로 높은 세포 사멸 효과를 나타냄을 알 수 있다.As shown in Fig. 13A, it can be seen that selectively exhibiting a high apoptosis effect on SR, which is radiation-resistant cancer cells, depending on the concentration of the 70% ethanol extract of yeonjabang.
12-2. 방사선 내성 암세포에서 증가된 MAP4K4, ACSL4, 및 Moesin 단백질 발현에 대한 연자방 70% 에탄올 추출물의 발현 억제 효과 분석12-2. Analysis of the Inhibitory Effect of 70% Ethanol Extract of Yeonjabang on the Increased Expression of MAP4K4, ACSL4, and Moesin Proteins in Radiation-resistant Cancer Cells
연자방 70% 에탄올 추출물을 처리한 방사선 내성 암세포에서 MAP4K4, ACSL4, 및, Moesin 단백질의 발현 변화를 분석하기 위해 western blot assay를 실시하였다. 방사선 내성 암세포(SR)에 5, 10, 20, 및 30 μg/mL의 연자방 70% 에탄올 추출물을 24시간 동안 처리한 후 상기 실시예 6과 동일한 방법으로 단백질 샘플을 준비하여 실험에 사용하였다. Western blot assay was performed to analyze the expression changes of MAP4K4, ACSL4, and Moesin proteins in radiation-resistant cancer cells treated with 70% ethanol extract of Yeonjabang. After treating radiation-resistant cancer cells (SR) with 5, 10, 20, and 30 μg/mL of 70% ethanol extract of Yeonjabang for 24 hours, a protein sample was prepared in the same manner as in Example 6 and used in the experiment.
도 13의 B에 나타낸 바와 같이, MAP4K4, ACSL4, 및 Moesin 단백질의 발현은 연자방 70% 에탄올 추출물의 처리 농도에 의존적으로 발현이 저해됨을 알 수 있다. 이는 방사선 내성 암세포인 SR에 대한 연자방 70% 에탄올 추출물의 선택적 세포사멸 효과는 MAP4K4, ACSL4, 및 Moesin의 발현 억제에 의한 것임을 보여는 결과이다.13B, it can be seen that the expression of MAP4K4, ACSL4, and Moesin proteins was inhibited depending on the concentration of the 70% ethanol extract of Yeonjabang. This is a result showing that the selective apoptosis effect of the 70% ethanol extract of Yeonjabang on SR, which is a radiation-resistant cancer cell, is due to the inhibition of the expression of MAP4K4, ACSL4, and Moesin.
<110> Dongguk University Gyeongju Campus Industry-Academy Cooperation Foundation <120> Pharmaceutical composition for preventing or treating radiation-resistant cancer comprising lotus root or lotus extract <130> PN190309 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 3498 <212> DNA <213> Artificial Sequence <220> <223> MAP4K4 gene <400> 1 atggcgaacg actcccctgc aaaaagtctg gtggacatcg acctctcctc cctgcgggat 60 cctgctggga tttttgagct ggtggaagtg gttggaaatg gcacctatgg acaagtctat 120 aagggtcgac atgttaaaac gggtcagttg gcagccatca aagttatgga tgtcactgag 180 gatgaagagg aagaaatcaa actggagata aatatgctaa agaaatactc tcatcacaga 240 aacattgcaa catattatgg tgctttcatc aaaaagagcc ctccaggaca tgatgaccaa 300 ctctggcttg ttatggagtt ctgtggggct gggtccatta cagaccttgt gaagaacacc 360 aaagggaaca cactcaaaga agactggatc gcttacatct ccagagaaat cctgagggga 420 ctggcacatc ttcacattca tcatgtgatt caccgggata tcaagggcca gaatgtgttg 480 ctgactgaga atgcagaggt gaaacttgtt gactttggtg tgagtgctca gctggacagg 540 actgtggggc ggagaaatac gttcataggc actccctact ggatggctcc tgaggtcatc 600 gcctgtgatg agaacccaga tgccacctat gattacagaa gtgatctttg gtcttgtggc 660 attacagcca ttgagatggc agaaggtgct ccccctctct gtgacatgca tccaatgaga 720 gcactgtttc tcattcccag aaaccctcct ccccggctga agtcaaaaaa atggtcgaag 780 aagtttttta gttttataga agggtgcctg gtgaagaatt acatgcagcg gccctctaca 840 gagcagcttt tgaaacatcc ttttataagg gatcagccaa atgaaaggca agttagaatc 900 cagcttaagg atcatataga tcgtaccagg aagaagagag gcgagaaaga tgaaactgag 960 tatgagtaca gtgggagtga ggaagaagag gaggaagtgc ctgaacagga aggagagcca 1020 agttccattg tgaacgtgcc tggtgagtct actcttcgcc gagatttcct gagactgcag 1080 caggagaaca aggaacgttc cgaggctctt cggagacaac agttactaca ggagcaacag 1140 ctccgggagc aggaagaata taaaaggcaa ctgctggcag agagacagaa gcggattgag 1200 cagcagaaag aacagaggcg acggctagaa gagcaacaaa ggagagagcg ggaagctaga 1260 aggcagcagg aacgtgaaca gcgaaggaga gaacaagaag aaaagaggcg tctagaggag 1320 ttggagagaa ggcgcaaaga agaagaggag aggagacggg cagaagaaga aaagaggaga 1380 gttgaaagag aacaggagta tatcaggcga cagctagaag aggagcagcg gcacttggaa 1440 gtccttcagc agcagctgct ccaggagcag gccatgttac tgcatgacca taggaggccg 1500 cacccgcagc actcgcagca gccgccacca ccgcagcagg aaaggagcaa gccaagcttc 1560 catgctcccg agcccaaagc ccactacgag cctgctgacc gagcgcgaga ggttcctgtg 1620 agaacaacat ctcgctcccc tgttctgtcc cgtcgagatt ccccactgca gggcagtggg 1680 cagcagaata gccaggcagg acagagaaac tccaccagta ttgagcccag gcttctgtgg 1740 gagagagtgg agaagctggt gcccagacct ggcagtggca gctcctcagg gtccagcaac 1800 tcaggatccc agcccgggtc tcaccctggg tctcagagtg gctccgggga acgcttcaga 1860 gtgagatcat catccaagtc tgaaggctct ccatctcagc gcctggaaaa tgcagtgaaa 1920 aaacctgaag ataaaaagga agttttcaga cccctcaagc ctgctggcga agtggatctg 1980 accgcactgg ccaaagagct tcgagcagtg gaagatgtac ggccacctca caaagtaacg 2040 gactactcct catccagtga ggagtcgggg acgacggatg aggaggacga cgatgtggag 2100 caggaagggg ctgacgagtc cacctcagga ccagaggaca ccagagcagc gtcatctctg 2160 aatttgagca atggtgaaac ggaatctgtg aaaaccatga ttgtccatga tgatgtagaa 2220 agtgagccgg ccatgacccc atccaaggag ggcactctaa tcgtccgcca gactcagtcc 2280 gctagtagca cactccagaa acacaaatct tcctcctcct ttacaccttt tatagacccc 2340 agattactac agatttctcc atctagcgga acaacagtga catctgtggt gggattttcc 2400 tgtgatggga tgagaccaga agccataagg caagatccta cccggaaagg ctcagtggtc 2460 aatgtgaatc ctaccaacac taggccacag agtgacaccc cggagattcg taaatacaag 2520 aagaggttta actctgagat tctgtgtgct gccttatggg gagtgaattt gctagtgggt 2580 acagagagtg gcctgatgct gctggacaga agtggccaag ggaaggtcta tcctcttatc 2640 aaccgaagac gatttcaaca aatggacgta cttgagggct tgaatgtctt ggtgacaata 2700 tctggcaaaa aggataagtt acgtgtctac tatttgtcct ggttaagaaa taaaatactt 2760 cacaatgatc cagaagttga gaagaagcag ggatggacaa ccgtagggga tttggaagga 2820 tgtgtacatt ataaagttgt aaaatatgaa agaatcaaat ttctggtgat tgctttgaag 2880 agttctgtgg aagtctatgc gtgggcacca aagccatatc acaaatttat ggcctttaag 2940 tcatttggag aattggtaca taagccatta ctggtggatc tcactgttga ggaaggccag 3000 aggttgaaag tgatctatgg atcctgtgct ggattccatg ctgttgatgt ggattcagga 3060 tcagtctatg acatttatct accaacacat gtaagaaaga acccacactc tatgatccag 3120 tgtagcatca aaccccatgc aatcatcatc ctccccaata cagatggaat ggagcttctg 3180 gtgtgctatg aagatgaggg ggtttatgta aacacatatg gaaggatcac caaggatgta 3240 gttctacagt ggggagagat gcctacatca gtagcatata ttcgatccaa tcagacaatg 3300 ggctggggag agaaggccat agagatccga tctgtggaaa ctggtcactt ggatggtgtg 3360 ttcatgcaca aaagggctca aagactaaaa ttcttgtgtg aacgcaatga caaggtgttc 3420 tttgcctctg ttcggtctgg tggcagcagt caggtttatt tcatgacctt aggcaggact 3480 tctcttctga gctggtag 3498 <210> 2 <211> 2136 <212> DNA <213> Artificial Sequence <220> <223> ACSL4 gene <400> 2 atgaaactta agctaaatgt gctcaccatt attttgctgc ctgtccactt gttaataaca 60 atatacagtg cccttatatt tattccatgg tattttctta ccaatgccaa gaagaaaaac 120 gctatggcaa agagaataaa agctaagccc acttcagaca aacctggaag tccatatcgc 180 tctgtcacac acttcgactc actagctgta atagacatcc ctggagcaga tactctggat 240 aaattatttg accatgctgt atccaagttt gggaagaagg acagccttgg gaccagggaa 300 atcctaagtg aagaaaatga aatgcagcca aatggaaaag tttttaagaa gttaattctt 360 gggaattata aatggatgaa ctatcttgaa gtgaatcgca gagtgaataa ctttggtagt 420 ggactcactg cactgggact aaaaccaaag aacaccattg ccatcttctg tgagaccagg 480 gccgaatgga tgattgcagc acagacctgc tttaagtaca actttcctct tgtgacttta 540 tatgccacac ttggcaaaga agcagtagtt catgggctaa atgaatctga ggcttcctat 600 ctgattacca gtgttgaact tctggaaagt aaacttaaga ctgcattgtt agatatcagt 660 tgtgttaaac atatcattta tgtggacaat aaggctatca ataaagcaga gtaccctgaa 720 ggatttgaga ttcacagcat gcaatcagta gaagagttgg gatctaaccc agaaaacttg 780 ggcattcctc caagtagacc aacgccttca gacatggcca ttgttatgta tactagtggt 840 tctactggcc gacctaaggg agtgatgatg catcatagca atttgatagc tggaatgaca 900 ggccagtgtg aaagaatacc tggactggga ccgaaggaca catatattgg ctacttgcct 960 ttggctcatg tgctagaact gacagcagag atatcttgct ttacctatgg ctgcaggatt 1020 ggatattctt ctccgcttac actctctgac cagtccagca aaattaaaaa aggaagcaaa 1080 ggagactgta ctgtactgaa gcccacactt atggctgctg ttccggaaat catggataga 1140 atttataaga atgttatgag caaagtccaa gagatgaatt atattcagaa aactctgttc 1200 aagatagggt atgattacaa attggaacag atcaaaaagg gatatgatgc acctctttgc 1260 aatctgttac tgtttaaaaa ggtcaaggcc ctgctgggag ggaatgtccg catgatgctg 1320 tctggagggg ccccgctatc tcctcagaca caccgattca tgaatgtctg cttctgctgc 1380 ccaattggcc agggttatgg actgacagaa tcatgtggtg ctgggacagt tactgaagta 1440 actgactata ctactggcag agttggagca cctcttattt gctgtgaaat taagctaaaa 1500 gactggcaag aaggcggtta tacaattaat gacaagccaa accccagagg tgaaatcgta 1560 attggtggac agaacatctc catgggatat tttaaaaatg aagagaaaac agcagaagat 1620 tattctgtgg atgaaaatgg acaaaggtgg ttttgcactg gtgatattgg agaattccat 1680 cccgatggat gtttacagat tatagatcgt aagaaagatc tagtgaagtt acaagcagga 1740 gagtatgtat ctcttgggaa agtagaagct gcactgaaga attgtccact tattgacaac 1800 atctgtgctt ttgccaaaag tgatcagtcc tatgtgatca gttttgtggt tcctaaccag 1860 aaaaggttga cacttttggc acaacagaaa ggggtagaag gaacttgggt tgatatctgc 1920 aataatcctg ctatggaagc tgaaatactg aaagaaattc gagaagctgc aaatgccatg 1980 aaattggagc gatttgaaat tccaatcaag gttcgattaa gcccagagcc atggacccct 2040 gaaactggtt tggtaactga tgctttcaaa ctgaaaagga aggagctgag gaaccattac 2100 ctcaaagaca ttgaacgaat gtatgggggc aaataa 2136 <210> 3 <211> 1734 <212> DNA <213> Artificial Sequence <220> <223> Moesin gene <400> 3 atgcccaaaa cgatcagtgt gcgtgtgacc accatggatg cagagctgga gtttgccatc 60 cagcccaaca ccaccgggaa gcagctattt gaccaggtgg tgaaaactat tggcttgagg 120 gaagtttggt tctttggtct gcagtaccag gacactaaag gtttctccac ctggctgaaa 180 ctcaataaga aggtgactgc ccaggatgtg cggaaggaaa gccccctgct ctttaagttc 240 cgtgccaagt tctaccctga ggatgtgtcc gaggaattga ttcaggacat cactcagcgc 300 ctgttctttc tgcaagtgaa agagggcatt ctcaatgatg atatttactg cccgcctgag 360 accgctgtgc tgctggcctc gtatgctgtc cagtctaagt atggcgactt caataaggaa 420 gtgcataagt ctggctacct ggccggagac aagttgctcc cgcagagagt cctggaacag 480 cacaaactca acaaggacca gtgggaggag cggatccagg tgtggcatga ggaacaccgt 540 ggcatgctca gggaggatgc tgtcctggaa tatctgaaga ttgctcaaga tctggagatg 600 tatggtgtga actacttcag catcaagaac aagaaaggct cagagctgtg gctgggggtg 660 gatgccctgg gtctcaacat ctatgagcag aatgacagac taactcccaa gataggcttc 720 ccctggagtg aaatcaggaa catctctttc aatgataaga aatttgtcat caagcccatt 780 gacaaaaaag ccccggactt cgtcttctat gctccccggc tgcggattaa caagcggatc 840 ttggccttgt gcatggggaa ccatgaacta tacatgcgcc gtcgcaagcc tgataccatt 900 gaggtgcagc agatgaaggc acaggcccgg gaggagaagc accagaagca gatggagcgt 960 gctatgctgg aaaatgagaa gaagaagcgt gaaatggcag agaaggagaa agagaagatt 1020 gaacgggaga aggaggagct gatggagagg ctgaagcaga tcgaggaaca gactaagaag 1080 gctcagcaag aactggaaga acagacccgt agggctctgg aacttgagca ggaacggaag 1140 cgtgcccaga gcgaggctga aaagctggcc aaggagcgtc aagaagctga agaggccaag 1200 gaggccttgc tgcaggcctc ccgggaccag aaaaagactc aggaacagct ggccttggaa 1260 atggcagagc tgacagctcg aatctcccag ctggagatgg cccgacagaa gaaggagagt 1320 gaggctgtgg agtggcagca gaaggcccag atggtacagg aagacttgga gaagacccgt 1380 gctgagctga agactgccat gagtacacct catgtggcag agcctgctga gaatgagcag 1440 gatgagcagg atgagaatgg ggcagaggct agtgctgacc tacgggctga tgctatggcc 1500 aaggaccgca gtgaggagga acgtaccact gaggcagaga agaatgagcg tgtgcagaag 1560 cacctgaagg ccctcacttc ggagctggcc aatgccagag atgagtccaa gaagactgcc 1620 aatgacatga tccatgctga gaacatgcga ctgggccgag acaaatacaa gaccctgcgc 1680 cagatccggc agggcaacac caagcagcgc attgacgaat ttgagtctat gtaa 1734 <210> 4 <211> 1165 <212> PRT <213> Artificial Sequence <220> <223> MAP4K4 protein <400> 4 Met Ala Asn Asp Ser Pro Ala Lys Ser Leu Val Asp Ile Asp Leu Ser 1 5 10 15 Ser Leu Arg Asp Pro Ala Gly Ile Phe Glu Leu Val Glu Val Val Gly 20 25 30 Asn Gly Thr Tyr Gly Gln Val Tyr Lys Gly Arg His Val Lys Thr Gly 35 40 45 Gln Leu Ala Ala Ile Lys Val Met Asp Val Thr Glu Asp Glu Glu Glu 50 55 60 Glu Ile Lys Leu Glu Ile Asn Met Leu Lys Lys Tyr Ser His His Arg 65 70 75 80 Asn Ile Ala Thr Tyr Tyr Gly Ala Phe Ile Lys Lys Ser Pro Pro Gly 85 90 95 His Asp Asp Gln Leu Trp Leu Val Met Glu Phe Cys Gly Ala Gly Ser 100 105 110 Ile Thr Asp Leu Val Lys Asn Thr Lys Gly Asn Thr Leu Lys Glu Asp 115 120 125 Trp Ile Ala Tyr Ile Ser Arg Glu Ile Leu Arg Gly Leu Ala His Leu 130 135 140 His Ile His His Val Ile His Arg Asp Ile Lys Gly Gln Asn Val Leu 145 150 155 160 Leu Thr Glu Asn Ala Glu Val Lys Leu Val Asp Phe Gly Val Ser Ala 165 170 175 Gln Leu Asp Arg Thr Val Gly Arg Arg Asn Thr Phe Ile Gly Thr Pro 180 185 190 Tyr Trp Met Ala Pro Glu Val Ile Ala Cys Asp Glu Asn Pro Asp Ala 195 200 205 Thr Tyr Asp Tyr Arg Ser Asp Leu Trp Ser Cys Gly Ile Thr Ala Ile 210 215 220 Glu Met Ala Glu Gly Ala Pro Pro Leu Cys Asp Met His Pro Met Arg 225 230 235 240 Ala Leu Phe Leu Ile Pro Arg Asn Pro Pro Pro Arg Leu Lys Ser Lys 245 250 255 Lys Trp Ser Lys Lys Phe Phe Ser Phe Ile Glu Gly Cys Leu Val Lys 260 265 270 Asn Tyr Met Gln Arg Pro Ser Thr Glu Gln Leu Leu Lys His Pro Phe 275 280 285 Ile Arg Asp Gln Pro Asn Glu Arg Gln Val Arg Ile Gln Leu Lys Asp 290 295 300 His Ile Asp Arg Thr Arg Lys Lys Arg Gly Glu Lys Asp Glu Thr Glu 305 310 315 320 Tyr Glu Tyr Ser Gly Ser Glu Glu Glu Glu Glu Glu Val Pro Glu Gln 325 330 335 Glu Gly Glu Pro Ser Ser Ile Val Asn Val Pro Gly Glu Ser Thr Leu 340 345 350 Arg Arg Asp Phe Leu Arg Leu Gln Gln Glu Asn Lys Glu Arg Ser Glu 355 360 365 Ala Leu Arg Arg Gln Gln Leu Leu Gln Glu Gln Gln Leu Arg Glu Gln 370 375 380 Glu Glu Tyr Lys Arg Gln Leu Leu Ala Glu Arg Gln Lys Arg Ile Glu 385 390 395 400 Gln Gln Lys Glu Gln Arg Arg Arg Leu Glu Glu Gln Gln Arg Arg Glu 405 410 415 Arg Glu Ala Arg Arg Gln Gln Glu Arg Glu Gln Arg Arg Arg Glu Gln 420 425 430 Glu Glu Lys Arg Arg Leu Glu Glu Leu Glu Arg Arg Arg Lys Glu Glu 435 440 445 Glu Glu Arg Arg Arg Ala Glu Glu Glu Lys Arg Arg Val Glu Arg Glu 450 455 460 Gln Glu Tyr Ile Arg Arg Gln Leu Glu Glu Glu Gln Arg His Leu Glu 465 470 475 480 Val Leu Gln Gln Gln Leu Leu Gln Glu Gln Ala Met Leu Leu His Asp 485 490 495 His Arg Arg Pro His Pro Gln His Ser Gln Gln Pro Pro Pro Pro Gln 500 505 510 Gln Glu Arg Ser Lys Pro Ser Phe His Ala Pro Glu Pro Lys Ala His 515 520 525 Tyr Glu Pro Ala Asp Arg Ala Arg Glu Val Pro Val Arg Thr Thr Ser 530 535 540 Arg Ser Pro Val Leu Ser Arg Arg Asp Ser Pro Leu Gln Gly Ser Gly 545 550 555 560 Gln Gln Asn Ser Gln Ala Gly Gln Arg Asn Ser Thr Ser Ile Glu Pro 565 570 575 Arg Leu Leu Trp Glu Arg Val Glu Lys Leu Val Pro Arg Pro Gly Ser 580 585 590 Gly Ser Ser Ser Gly Ser Ser Asn Ser Gly Ser Gln Pro Gly Ser His 595 600 605 Pro Gly Ser Gln Ser Gly Ser Gly Glu Arg Phe Arg Val Arg Ser Ser 610 615 620 Ser Lys Ser Glu Gly Ser Pro Ser Gln Arg Leu Glu Asn Ala Val Lys 625 630 635 640 Lys Pro Glu Asp Lys Lys Glu Val Phe Arg Pro Leu Lys Pro Ala Gly 645 650 655 Glu Val Asp Leu Thr Ala Leu Ala Lys Glu Leu Arg Ala Val Glu Asp 660 665 670 Val Arg Pro Pro His Lys Val Thr Asp Tyr Ser Ser Ser Ser Glu Glu 675 680 685 Ser Gly Thr Thr Asp Glu Glu Asp Asp Asp Val Glu Gln Glu Gly Ala 690 695 700 Asp Glu Ser Thr Ser Gly Pro Glu Asp Thr Arg Ala Ala Ser Ser Leu 705 710 715 720 Asn Leu Ser Asn Gly Glu Thr Glu Ser Val Lys Thr Met Ile Val His 725 730 735 Asp Asp Val Glu Ser Glu Pro Ala Met Thr Pro Ser Lys Glu Gly Thr 740 745 750 Leu Ile Val Arg Gln Thr Gln Ser Ala Ser Ser Thr Leu Gln Lys His 755 760 765 Lys Ser Ser Ser Ser Phe Thr Pro Phe Ile Asp Pro Arg Leu Leu Gln 770 775 780 Ile Ser Pro Ser Ser Gly Thr Thr Val Thr Ser Val Val Gly Phe Ser 785 790 795 800 Cys Asp Gly Met Arg Pro Glu Ala Ile Arg Gln Asp Pro Thr Arg Lys 805 810 815 Gly Ser Val Val Asn Val Asn Pro Thr Asn Thr Arg Pro Gln Ser Asp 820 825 830 Thr Pro Glu Ile Arg Lys Tyr Lys Lys Arg Phe Asn Ser Glu Ile Leu 835 840 845 Cys Ala Ala Leu Trp Gly Val Asn Leu Leu Val Gly Thr Glu Ser Gly 850 855 860 Leu Met Leu Leu Asp Arg Ser Gly Gln Gly Lys Val Tyr Pro Leu Ile 865 870 875 880 Asn Arg Arg Arg Phe Gln Gln Met Asp Val Leu Glu Gly Leu Asn Val 885 890 895 Leu Val Thr Ile Ser Gly Lys Lys Asp Lys Leu Arg Val Tyr Tyr Leu 900 905 910 Ser Trp Leu Arg Asn Lys Ile Leu His Asn Asp Pro Glu Val Glu Lys 915 920 925 Lys Gln Gly Trp Thr Thr Val Gly Asp Leu Glu Gly Cys Val His Tyr 930 935 940 Lys Val Val Lys Tyr Glu Arg Ile Lys Phe Leu Val Ile Ala Leu Lys 945 950 955 960 Ser Ser Val Glu Val Tyr Ala Trp Ala Pro Lys Pro Tyr His Lys Phe 965 970 975 Met Ala Phe Lys Ser Phe Gly Glu Leu Val His Lys Pro Leu Leu Val 980 985 990 Asp Leu Thr Val Glu Glu Gly Gln Arg Leu Lys Val Ile Tyr Gly Ser 995 1000 1005 Cys Ala Gly Phe His Ala Val Asp Val Asp Ser Gly Ser Val Tyr Asp 1010 1015 1020 Ile Tyr Leu Pro Thr His Val Arg Lys Asn Pro His Ser Met Ile Gln 1025 1030 1035 1040 Cys Ser Ile Lys Pro His Ala Ile Ile Ile Leu Pro Asn Thr Asp Gly 1045 1050 1055 Met Glu Leu Leu Val Cys Tyr Glu Asp Glu Gly Val Tyr Val Asn Thr 1060 1065 1070 Tyr Gly Arg Ile Thr Lys Asp Val Val Leu Gln Trp Gly Glu Met Pro 1075 1080 1085 Thr Ser Val Ala Tyr Ile Arg Ser Asn Gln Thr Met Gly Trp Gly Glu 1090 1095 1100 Lys Ala Ile Glu Ile Arg Ser Val Glu Thr Gly His Leu Asp Gly Val 1105 1110 1115 1120 Phe Met His Lys Arg Ala Gln Arg Leu Lys Phe Leu Cys Glu Arg Asn 1125 1130 1135 Asp Lys Val Phe Phe Ala Ser Val Arg Ser Gly Gly Ser Ser Gln Val 1140 1145 1150 Tyr Phe Met Thr Leu Gly Arg Thr Ser Leu Leu Ser Trp 1155 1160 1165 <210> 5 <211> 711 <212> PRT <213> Artificial Sequence <220> <223> ACSL4 protein <400> 5 Met Lys Leu Lys Leu Asn Val Leu Thr Ile Ile Leu Leu Pro Val His 1 5 10 15 Leu Leu Ile Thr Ile Tyr Ser Ala Leu Ile Phe Ile Pro Trp Tyr Phe 20 25 30 Leu Thr Asn Ala Lys Lys Lys Asn Ala Met Ala Lys Arg Ile Lys Ala 35 40 45 Lys Pro Thr Ser Asp Lys Pro Gly Ser Pro Tyr Arg Ser Val Thr His 50 55 60 Phe Asp Ser Leu Ala Val Ile Asp Ile Pro Gly Ala Asp Thr Leu Asp 65 70 75 80 Lys Leu Phe Asp His Ala Val Ser Lys Phe Gly Lys Lys Asp Ser Leu 85 90 95 Gly Thr Arg Glu Ile Leu Ser Glu Glu Asn Glu Met Gln Pro Asn Gly 100 105 110 Lys Val Phe Lys Lys Leu Ile Leu Gly Asn Tyr Lys Trp Met Asn Tyr 115 120 125 Leu Glu Val Asn Arg Arg Val Asn Asn Phe Gly Ser Gly Leu Thr Ala 130 135 140 Leu Gly Leu Lys Pro Lys Asn Thr Ile Ala Ile Phe Cys Glu Thr Arg 145 150 155 160 Ala Glu Trp Met Ile Ala Ala Gln Thr Cys Phe Lys Tyr Asn Phe Pro 165 170 175 Leu Val Thr Leu Tyr Ala Thr Leu Gly Lys Glu Ala Val Val His Gly 180 185 190 Leu Asn Glu Ser Glu Ala Ser Tyr Leu Ile Thr Ser Val Glu Leu Leu 195 200 205 Glu Ser Lys Leu Lys Thr Ala Leu Leu Asp Ile Ser Cys Val Lys His 210 215 220 Ile Ile Tyr Val Asp Asn Lys Ala Ile Asn Lys Ala Glu Tyr Pro Glu 225 230 235 240 Gly Phe Glu Ile His Ser Met Gln Ser Val Glu Glu Leu Gly Ser Asn 245 250 255 Pro Glu Asn Leu Gly Ile Pro Pro Ser Arg Pro Thr Pro Ser Asp Met 260 265 270 Ala Ile Val Met Tyr Thr Ser Gly Ser Thr Gly Arg Pro Lys Gly Val 275 280 285 Met Met His His Ser Asn Leu Ile Ala Gly Met Thr Gly Gln Cys Glu 290 295 300 Arg Ile Pro Gly Leu Gly Pro Lys Asp Thr Tyr Ile Gly Tyr Leu Pro 305 310 315 320 Leu Ala His Val Leu Glu Leu Thr Ala Glu Ile Ser Cys Phe Thr Tyr 325 330 335 Gly Cys Arg Ile Gly Tyr Ser Ser Pro Leu Thr Leu Ser Asp Gln Ser 340 345 350 Ser Lys Ile Lys Lys Gly Ser Lys Gly Asp Cys Thr Val Leu Lys Pro 355 360 365 Thr Leu Met Ala Ala Val Pro Glu Ile Met Asp Arg Ile Tyr Lys Asn 370 375 380 Val Met Ser Lys Val Gln Glu Met Asn Tyr Ile Gln Lys Thr Leu Phe 385 390 395 400 Lys Ile Gly Tyr Asp Tyr Lys Leu Glu Gln Ile Lys Lys Gly Tyr Asp 405 410 415 Ala Pro Leu Cys Asn Leu Leu Leu Phe Lys Lys Val Lys Ala Leu Leu 420 425 430 Gly Gly Asn Val Arg Met Met Leu Ser Gly Gly Ala Pro Leu Ser Pro 435 440 445 Gln Thr His Arg Phe Met Asn Val Cys Phe Cys Cys Pro Ile Gly Gln 450 455 460 Gly Tyr Gly Leu Thr Glu Ser Cys Gly Ala Gly Thr Val Thr Glu Val 465 470 475 480 Thr Asp Tyr Thr Thr Gly Arg Val Gly Ala Pro Leu Ile Cys Cys Glu 485 490 495 Ile Lys Leu Lys Asp Trp Gln Glu Gly Gly Tyr Thr Ile Asn Asp Lys 500 505 510 Pro Asn Pro Arg Gly Glu Ile Val Ile Gly Gly Gln Asn Ile Ser Met 515 520 525 Gly Tyr Phe Lys Asn Glu Glu Lys Thr Ala Glu Asp Tyr Ser Val Asp 530 535 540 Glu Asn Gly Gln Arg Trp Phe Cys Thr Gly Asp Ile Gly Glu Phe His 545 550 555 560 Pro Asp Gly Cys Leu Gln Ile Ile Asp Arg Lys Lys Asp Leu Val Lys 565 570 575 Leu Gln Ala Gly Glu Tyr Val Ser Leu Gly Lys Val Glu Ala Ala Leu 580 585 590 Lys Asn Cys Pro Leu Ile Asp Asn Ile Cys Ala Phe Ala Lys Ser Asp 595 600 605 Gln Ser Tyr Val Ile Ser Phe Val Val Pro Asn Gln Lys Arg Leu Thr 610 615 620 Leu Leu Ala Gln Gln Lys Gly Val Glu Gly Thr Trp Val Asp Ile Cys 625 630 635 640 Asn Asn Pro Ala Met Glu Ala Glu Ile Leu Lys Glu Ile Arg Glu Ala 645 650 655 Ala Asn Ala Met Lys Leu Glu Arg Phe Glu Ile Pro Ile Lys Val Arg 660 665 670 Leu Ser Pro Glu Pro Trp Thr Pro Glu Thr Gly Leu Val Thr Asp Ala 675 680 685 Phe Lys Leu Lys Arg Lys Glu Leu Arg Asn His Tyr Leu Lys Asp Ile 690 695 700 Glu Arg Met Tyr Gly Gly Lys 705 710 <210> 6 <211> 577 <212> PRT <213> Artificial Sequence <220> <223> Moesin protein <400> 6 Met Pro Lys Thr Ile Ser Val Arg Val Thr Thr Met Asp Ala Glu Leu 1 5 10 15 Glu Phe Ala Ile Gln Pro Asn Thr Thr Gly Lys Gln Leu Phe Asp Gln 20 25 30 Val Val Lys Thr Ile Gly Leu Arg Glu Val Trp Phe Phe Gly Leu Gln 35 40 45 Tyr Gln Asp Thr Lys Gly Phe Ser Thr Trp Leu Lys Leu Asn Lys Lys 50 55 60 Val Thr Ala Gln Asp Val Arg Lys Glu Ser Pro Leu Leu Phe Lys Phe 65 70 75 80 Arg Ala Lys Phe Tyr Pro Glu Asp Val Ser Glu Glu Leu Ile Gln Asp 85 90 95 Ile Thr Gln Arg Leu Phe Phe Leu Gln Val Lys Glu Gly Ile Leu Asn 100 105 110 Asp Asp Ile Tyr Cys Pro Pro Glu Thr Ala Val Leu Leu Ala Ser Tyr 115 120 125 Ala Val Gln Ser Lys Tyr Gly Asp Phe Asn Lys Glu Val His Lys Ser 130 135 140 Gly Tyr Leu Ala Gly Asp Lys Leu Leu Pro Gln Arg Val Leu Glu Gln 145 150 155 160 His Lys Leu Asn Lys Asp Gln Trp Glu Glu Arg Ile Gln Val Trp His 165 170 175 Glu Glu His Arg Gly Met Leu Arg Glu Asp Ala Val Leu Glu Tyr Leu 180 185 190 Lys Ile Ala Gln Asp Leu Glu Met Tyr Gly Val Asn Tyr Phe Ser Ile 195 200 205 Lys Asn Lys Lys Gly Ser Glu Leu Trp Leu Gly Val Asp Ala Leu Gly 210 215 220 Leu Asn Ile Tyr Glu Gln Asn Asp Arg Leu Thr Pro Lys Ile Gly Phe 225 230 235 240 Pro Trp Ser Glu Ile Arg Asn Ile Ser Phe Asn Asp Lys Lys Phe Val 245 250 255 Ile Lys Pro Ile Asp Lys Lys Ala Pro Asp Phe Val Phe Tyr Ala Pro 260 265 270 Arg Leu Arg Ile Asn Lys Arg Ile Leu Ala Leu Cys Met Gly Asn His 275 280 285 Glu Leu Tyr Met Arg Arg Arg Lys Pro Asp Thr Ile Glu Val Gln Gln 290 295 300 Met Lys Ala Gln Ala Arg Glu Glu Lys His Gln Lys Gln Met Glu Arg 305 310 315 320 Ala Met Leu Glu Asn Glu Lys Lys Lys Arg Glu Met Ala Glu Lys Glu 325 330 335 Lys Glu Lys Ile Glu Arg Glu Lys Glu Glu Leu Met Glu Arg Leu Lys 340 345 350 Gln Ile Glu Glu Gln Thr Lys Lys Ala Gln Gln Glu Leu Glu Glu Gln 355 360 365 Thr Arg Arg Ala Leu Glu Leu Glu Gln Glu Arg Lys Arg Ala Gln Ser 370 375 380 Glu Ala Glu Lys Leu Ala Lys Glu Arg Gln Glu Ala Glu Glu Ala Lys 385 390 395 400 Glu Ala Leu Leu Gln Ala Ser Arg Asp Gln Lys Lys Thr Gln Glu Gln 405 410 415 Leu Ala Leu Glu Met Ala Glu Leu Thr Ala Arg Ile Ser Gln Leu Glu 420 425 430 Met Ala Arg Gln Lys Lys Glu Ser Glu Ala Val Glu Trp Gln Gln Lys 435 440 445 Ala Gln Met Val Gln Glu Asp Leu Glu Lys Thr Arg Ala Glu Leu Lys 450 455 460 Thr Ala Met Ser Thr Pro His Val Ala Glu Pro Ala Glu Asn Glu Gln 465 470 475 480 Asp Glu Gln Asp Glu Asn Gly Ala Glu Ala Ser Ala Asp Leu Arg Ala 485 490 495 Asp Ala Met Ala Lys Asp Arg Ser Glu Glu Glu Arg Thr Thr Glu Ala 500 505 510 Glu Lys Asn Glu Arg Val Gln Lys His Leu Lys Ala Leu Thr Ser Glu 515 520 525 Leu Ala Asn Ala Arg Asp Glu Ser Lys Lys Thr Ala Asn Asp Met Ile 530 535 540 His Ala Glu Asn Met Arg Leu Gly Arg Asp Lys Tyr Lys Thr Leu Arg 545 550 555 560 Gln Ile Arg Gln Gly Asn Thr Lys Gln Arg Ile Asp Glu Phe Glu Ser 565 570 575 Met <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MAP4K4 FW Primer <400> 7 gagaggctcc agaggcagtt gca 23 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MAP4K4 BW Primer <400> 8 ccacctctcg cgctcggtca g 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ACSL4 FW Primer <400> 9 gggaagaagg acagccttgg g 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ACSL4 BW Primer <400> 10 tcggccctgg tctcacagaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Moesin FW Primer <400> 11 gctgtggagt ggcagcagaa 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Moesin BW Primer <400> 12 cgttcctcct cactgcggtc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s rRNA FW Primer <400> 13 gtaacccgtt gaaccccatt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s rRNA BW Primer <400> 14 ccatccaatc ggtagtagcg 20 <110> Dongguk University Gyeongju Campus Industry-Academy Cooperation Foundation <120> Pharmaceutical composition for preventing or treating radiation-resistant cancer comprising lotus root or lotus extract <130> PN190309 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 3498 <212> DNA <213> Artificial Sequence <220> <223> MAP4K4 gene <400> 1 atggcgaacg actcccctgc aaaaagtctg gtggacatcg acctctcctc cctgcgggat 60 cctgctggga tttttgagct ggtggaagtg gttggaaatg gcacctatgg acaagtctat 120 aagggtcgac atgttaaaac gggtcagttg gcagccatca aagttatgga tgtcactgag 180 gatgaagagg aagaaatcaa actggagata aatatgctaa agaaatactc tcatcacaga 240 aacattgcaa catattatgg tgctttcatc aaaaagagcc ctccaggaca tgatgaccaa 300 ctctggcttg ttatggagtt ctgtggggct gggtccatta cagaccttgt gaagaacacc 360 aaagggaaca cactcaaaga agactggatc gcttacatct ccagagaaat cctgagggga 420 ctggcacatc ttcacattca tcatgtgatt caccgggata tcaagggcca gaatgtgttg 480 ctgactgaga atgcagaggt gaaacttgtt gactttggtg tgagtgctca gctggacagg 540 actgtggggc ggagaaatac gttcataggc actccctact ggatggctcc tgaggtcatc 600 gcctgtgatg agaacccaga tgccacctat gattacagaa gtgatctttg gtcttgtggc 660 attacagcca ttgagatggc agaaggtgct ccccctctct gtgacatgca tccaatgaga 720 gcactgtttc tcattcccag aaaccctcct ccccggctga agtcaaaaaa atggtcgaag 780 aagtttttta gttttataga agggtgcctg gtgaagaatt acatgcagcg gccctctaca 840 gagcagcttt tgaaacatcc ttttataagg gatcagccaa atgaaaggca agttagaatc 900 cagcttaagg atcatataga tcgtaccagg aagaagagag gcgagaaaga tgaaactgag 960 tatgagtaca gtgggagtga ggaagaagag gaggaagtgc ctgaacagga aggagagcca 1020 agttccattg tgaacgtgcc tggtgagtct actcttcgcc gagatttcct gagactgcag 1080 caggagaaca aggaacgttc cgaggctctt cggagacaac agttactaca ggagcaacag 1140 ctccgggagc aggaagaata taaaaggcaa ctgctggcag agagacagaa gcggattgag 1200 cagcagaaag aacagaggcg acggctagaa gagcaacaaa ggagagagcg ggaagctaga 1260 aggcagcagg aacgtgaaca gcgaaggaga gaacaagaag aaaagaggcg tctagaggag 1320 ttggagagaa ggcgcaaaga agaagaggag aggagacggg cagaagaaga aaagaggaga 1380 gttgaaagag aacaggagta tatcaggcga cagctagaag aggagcagcg gcacttggaa 1440 gtccttcagc agcagctgct ccaggagcag gccatgttac tgcatgacca taggaggccg 1500 cacccgcagc actcgcagca gccgccacca ccgcagcagg aaaggagcaa gccaagcttc 1560 catgctcccg agcccaaagc ccactacgag cctgctgacc gagcgcgaga ggttcctgtg 1620 agaacaacat ctcgctcccc tgttctgtcc cgtcgagatt ccccactgca gggcagtggg 1680 cagcagaata gccaggcagg acagagaaac tccaccagta ttgagcccag gcttctgtgg 1740 gagagagtgg agaagctggt gcccagacct ggcagtggca gctcctcagg gtccagcaac 1800 tcaggatccc agcccgggtc tcaccctggg tctcagagtg gctccgggga acgcttcaga 1860 gtgagatcat catccaagtc tgaaggctct ccatctcagc gcctggaaaa tgcagtgaaa 1920 aaacctgaag ataaaaagga agttttcaga cccctcaagc ctgctggcga agtggatctg 1980 accgcactgg ccaaagagct tcgagcagtg gaagatgtac ggccacctca caaagtaacg 2040 gactactcct catccagtga ggagtcgggg acgacggatg aggaggacga cgatgtggag 2100 caggaagggg ctgacgagtc cacctcagga ccagaggaca ccagagcagc gtcatctctg 2160 aatttgagca atggtgaaac ggaatctgtg aaaaccatga ttgtccatga tgatgtagaa 2220 agtgagccgg ccatgacccc atccaaggag ggcactctaa tcgtccgcca gactcagtcc 2280 gctagtagca cactccagaa acacaaatct tcctcctcct ttacaccttt tatagacccc 2340 agattactac agatttctcc atctagcgga acaacagtga catctgtggt gggattttcc 2400 tgtgatggga tgagaccaga agccataagg caagatccta cccggaaagg ctcagtggtc 2460 aatgtgaatc ctaccaacac taggccacag agtgacaccc cggagattcg taaatacaag 2520 aagaggttta actctgagat tctgtgtgct gccttatggg gagtgaattt gctagtgggt 2580 acagagagtg gcctgatgct gctggacaga agtggccaag ggaaggtcta tcctcttatc 2640 aaccgaagac gatttcaaca aatggacgta cttgagggct tgaatgtctt ggtgacaata 2700 tctggcaaaa aggataagtt acgtgtctac tatttgtcct ggttaagaaa taaaatactt 2760 cacaatgatc cagaagttga gaagaagcag ggatggacaa ccgtagggga tttggaagga 2820 tgtgtacatt ataaagttgt aaaatatgaa agaatcaaat ttctggtgat tgctttgaag 2880 agttctgtgg aagtctatgc gtgggcacca aagccatatc acaaatttat ggcctttaag 2940 tcatttggag aattggtaca taagccatta ctggtggatc tcactgttga ggaaggccag 3000 aggttgaaag tgatctatgg atcctgtgct ggattccatg ctgttgatgt ggattcagga 3060 tcagtctatg acatttatct accaacacat gtaagaaaga acccacactc tatgatccag 3120 tgtagcatca aaccccatgc aatcatcatc ctccccaata cagatggaat ggagcttctg 3180 gtgtgctatg aagatgaggg ggtttatgta aacacatatg gaaggatcac caaggatgta 3240 gttctacagt ggggagagat gcctacatca gtagcatata ttcgatccaa tcagacaatg 3300 ggctggggag agaaggccat agagatccga tctgtggaaa ctggtcactt ggatggtgtg 3360 ttcatgcaca aaagggctca aagactaaaa ttcttgtgtg aacgcaatga caaggtgttc 3420 tttgcctctg ttcggtctgg tggcagcagt caggtttatt tcatgacctt aggcaggact 3480 tctcttctga gctggtag 3498 <210> 2 <211> 2136 <212> DNA <213> Artificial Sequence <220> <223> ACSL4 gene <400> 2 atgaaactta agctaaatgt gctcaccatt attttgctgc ctgtccactt gttaataaca 60 atatacagtg cccttatatt tattccatgg tattttctta ccaatgccaa gaagaaaaac 120 gctatggcaa agagaataaa agctaagccc acttcagaca aacctggaag tccatatcgc 180 tctgtcacac acttcgactc actagctgta atagacatcc ctggagcaga tactctggat 240 aaattatttg accatgctgt atccaagttt gggaagaagg acagccttgg gaccagggaa 300 atcctaagtg aagaaaatga aatgcagcca aatggaaaag tttttaagaa gttaattctt 360 gggaattata aatggatgaa ctatcttgaa gtgaatcgca gagtgaataa ctttggtagt 420 ggactcactg cactgggact aaaaccaaag aacaccattg ccatcttctg tgagaccagg 480 gccgaatgga tgattgcagc acagacctgc tttaagtaca actttcctct tgtgacttta 540 tatgccacac ttggcaaaga agcagtagtt catgggctaa atgaatctga ggcttcctat 600 ctgattacca gtgttgaact tctggaaagt aaacttaaga ctgcattgtt agatatcagt 660 tgtgttaaac atatcattta tgtggacaat aaggctatca ataaagcaga gtaccctgaa 720 ggatttgaga ttcacagcat gcaatcagta gaagagttgg gatctaaccc agaaaacttg 780 ggcattcctc caagtagacc aacgccttca gacatggcca ttgttatgta tactagtggt 840 tctactggcc gacctaaggg agtgatgatg catcatagca atttgatagc tggaatgaca 900 ggccagtgtg aaagaatacc tggactggga ccgaaggaca catatattgg ctacttgcct 960 ttggctcatg tgctagaact gacagcagag atatcttgct ttacctatgg ctgcaggatt 1020 ggatattctt ctccgcttac actctctgac cagtccagca aaattaaaaa aggaagcaaa 1080 ggagactgta ctgtactgaa gcccacactt atggctgctg ttccggaaat catggataga 1140 atttataaga atgttatgag caaagtccaa gagatgaatt atattcagaa aactctgttc 1200 aagatagggt atgattacaa attggaacag atcaaaaagg gatatgatgc acctctttgc 1260 aatctgttac tgtttaaaaa ggtcaaggcc ctgctgggag ggaatgtccg catgatgctg 1320 tctggagggg ccccgctatc tcctcagaca caccgattca tgaatgtctg cttctgctgc 1380 ccaattggcc agggttatgg actgacagaa tcatgtggtg ctgggacagt tactgaagta 1440 actgactata ctactggcag agttggagca cctcttattt gctgtgaaat taagctaaaa 1500 gactggcaag aaggcggtta tacaattaat gacaagccaa accccagagg tgaaatcgta 1560 attggtggac agaacatctc catgggatat tttaaaaatg aagagaaaac agcagaagat 1620 tattctgtgg atgaaaatgg acaaaggtgg ttttgcactg gtgatattgg agaattccat 1680 cccgatggat gtttacagat tatagatcgt aagaaagatc tagtgaagtt acaagcagga 1740 gagtatgtat ctcttgggaa agtagaagct gcactgaaga attgtccact tattgacaac 1800 atctgtgctt ttgccaaaag tgatcagtcc tatgtgatca gttttgtggt tcctaaccag 1860 aaaaggttga cacttttggc acaacagaaa ggggtagaag gaacttgggt tgatatctgc 1920 aataatcctg ctatggaagc tgaaatactg aaagaaattc gagaagctgc aaatgccatg 1980 aaattggagc gatttgaaat tccaatcaag gttcgattaa gcccagagcc atggacccct 2040 gaaactggtt tggtaactga tgctttcaaa ctgaaaagga aggagctgag gaaccattac 2100 ctcaaagaca ttgaacgaat gtatgggggc aaataa 2136 <210> 3 <211> 1734 <212> DNA <213> Artificial Sequence <220> <223> Moesin gene <400> 3 atgcccaaaa cgatcagtgt gcgtgtgacc accatggatg cagagctgga gtttgccatc 60 cagcccaaca ccaccgggaa gcagctattt gaccaggtgg tgaaaactat tggcttgagg 120 gaagtttggt tctttggtct gcagtaccag gacactaaag gtttctccac ctggctgaaa 180 ctcaataaga aggtgactgc ccaggatgtg cggaaggaaa gccccctgct ctttaagttc 240 cgtgccaagt tctaccctga ggatgtgtcc gaggaattga ttcaggacat cactcagcgc 300 ctgttctttc tgcaagtgaa agagggcatt ctcaatgatg atatttactg cccgcctgag 360 accgctgtgc tgctggcctc gtatgctgtc cagtctaagt atggcgactt caataaggaa 420 gtgcataagt ctggctacct ggccggagac aagttgctcc cgcagagagt cctggaacag 480 cacaaactca acaaggacca gtgggaggag cggatccagg tgtggcatga ggaacaccgt 540 ggcatgctca gggaggatgc tgtcctggaa tatctgaaga ttgctcaaga tctggagatg 600 tatggtgtga actacttcag catcaagaac aagaaaggct cagagctgtg gctgggggtg 660 gatgccctgg gtctcaacat ctatgagcag aatgacagac taactcccaa gataggcttc 720 ccctggagtg aaatcaggaa catctctttc aatgataaga aatttgtcat caagcccatt 780 gacaaaaaag ccccggactt cgtcttctat gctccccggc tgcggattaa caagcggatc 840 ttggccttgt gcatggggaa ccatgaacta tacatgcgcc gtcgcaagcc tgataccatt 900 gaggtgcagc agatgaaggc acaggcccgg gaggagaagc accagaagca gatggagcgt 960 gctatgctgg aaaatgagaa gaagaagcgt gaaatggcag agaaggagaa agagaagatt 1020 gaacgggaga aggaggagct gatggagagg ctgaagcaga tcgaggaaca gactaagaag 1080 gctcagcaag aactggaaga acagacccgt agggctctgg aacttgagca ggaacggaag 1140 cgtgcccaga gcgaggctga aaagctggcc aaggagcgtc aagaagctga agaggccaag 1200 gaggccttgc tgcaggcctc ccgggaccag aaaaagactc aggaacagct ggccttggaa 1260 atggcagagc tgacagctcg aatctcccag ctggagatgg cccgacagaa gaaggagagt 1320 gaggctgtgg agtggcagca gaaggcccag atggtacagg aagacttgga gaagacccgt 1380 gctgagctga agactgccat gagtacacct catgtggcag agcctgctga gaatgagcag 1440 gatgagcagg atgagaatgg ggcagaggct agtgctgacc tacgggctga tgctatggcc 1500 aaggaccgca gtgaggagga acgtaccact gaggcagaga agaatgagcg tgtgcagaag 1560 cacctgaagg ccctcacttc ggagctggcc aatgccagag atgagtccaa gaagactgcc 1620 aatgacatga tccatgctga gaacatgcga ctgggccgag acaaatacaa gaccctgcgc 1680 cagatccggc agggcaacac caagcagcgc attgacgaat ttgagtctat gtaa 1734 <210> 4 <211> 1165 <212> PRT <213> Artificial Sequence <220> <223> MAP4K4 protein <400> 4 Met Ala Asn Asp Ser Pro Ala Lys Ser Leu Val Asp Ile Asp Leu Ser 1 5 10 15 Ser Leu Arg Asp Pro Ala Gly Ile Phe Glu Leu Val Glu Val Val Gly 20 25 30 Asn Gly Thr Tyr Gly Gln Val Tyr Lys Gly Arg His Val Lys Thr Gly 35 40 45 Gln Leu Ala Ala Ile Lys Val Met Asp Val Thr Glu Asp Glu Glu Glu 50 55 60 Glu Ile Lys Leu Glu Ile Asn Met Leu Lys Lys Tyr Ser His His Arg 65 70 75 80 Asn Ile Ala Thr Tyr Tyr Gly Ala Phe Ile Lys Lys Ser Pro Pro Gly 85 90 95 His Asp Asp Gln Leu Trp Leu Val Met Glu Phe Cys Gly Ala Gly Ser 100 105 110 Ile Thr Asp Leu Val Lys Asn Thr Lys Gly Asn Thr Leu Lys Glu Asp 115 120 125 Trp Ile Ala Tyr Ile Ser Arg Glu Ile Leu Arg Gly Leu Ala His Leu 130 135 140 His Ile His His Val Ile His Arg Asp Ile Lys Gly Gln Asn Val Leu 145 150 155 160 Leu Thr Glu Asn Ala Glu Val Lys Leu Val Asp Phe Gly Val Ser Ala 165 170 175 Gln Leu Asp Arg Thr Val Gly Arg Arg Asn Thr Phe Ile Gly Thr Pro 180 185 190 Tyr Trp Met Ala Pro Glu Val Ile Ala Cys Asp Glu Asn Pro Asp Ala 195 200 205 Thr Tyr Asp Tyr Arg Ser Asp Leu Trp Ser Cys Gly Ile Thr Ala Ile 210 215 220 Glu Met Ala Glu Gly Ala Pro Pro Leu Cys Asp Met His Pro Met Arg 225 230 235 240 Ala Leu Phe Leu Ile Pro Arg Asn Pro Pro Pro Arg Leu Lys Ser Lys 245 250 255 Lys Trp Ser Lys Lys Phe Phe Ser Phe Ile Glu Gly Cys Leu Val Lys 260 265 270 Asn Tyr Met Gln Arg Pro Ser Thr Glu Gln Leu Leu Lys His Pro Phe 275 280 285 Ile Arg Asp Gln Pro Asn Glu Arg Gln Val Arg Ile Gln Leu Lys Asp 290 295 300 His Ile Asp Arg Thr Arg Lys Lys Arg Gly Glu Lys Asp Glu Thr Glu 305 310 315 320 Tyr Glu Tyr Ser Gly Ser Glu Glu Glu Glu Glu Glu Val Pro Glu Gln 325 330 335 Glu Gly Glu Pro Ser Ser Ile Val Asn Val Pro Gly Glu Ser Thr Leu 340 345 350 Arg Arg Asp Phe Leu Arg Leu Gln Gln Glu Asn Lys Glu Arg Ser Glu 355 360 365 Ala Leu Arg Arg Gln Gln Leu Leu Gln Glu Gln Gln Leu Arg Glu Gln 370 375 380 Glu Glu Tyr Lys Arg Gln Leu Leu Ala Glu Arg Gln Lys Arg Ile Glu 385 390 395 400 Gln Gln Lys Glu Gln Arg Arg Arg Leu Glu Glu Gln Gln Arg Arg Glu 405 410 415 Arg Glu Ala Arg Arg Gln Gln Glu Arg Glu Gln Arg Arg Arg Glu Gln 420 425 430 Glu Glu Lys Arg Arg Leu Glu Glu Leu Glu Arg Arg Arg Lys Glu Glu 435 440 445 Glu Glu Arg Arg Arg Ala Glu Glu Glu Lys Arg Arg Val Glu Arg Glu 450 455 460 Gln Glu Tyr Ile Arg Arg Gln Leu Glu Glu Glu Gln Arg His Leu Glu 465 470 475 480 Val Leu Gln Gln Gln Leu Leu Gln Glu Gln Ala Met Leu Leu His Asp 485 490 495 His Arg Arg Pro His Pro Gln His Ser Gln Gln Pro Pro Pro Pro Gln 500 505 510 Gln Glu Arg Ser Lys Pro Ser Phe His Ala Pro Glu Pro Lys Ala His 515 520 525 Tyr Glu Pro Ala Asp Arg Ala Arg Glu Val Pro Val Arg Thr Thr Ser 530 535 540 Arg Ser Pro Val Leu Ser Arg Arg Asp Ser Pro Leu Gln Gly Ser Gly 545 550 555 560 Gln Gln Asn Ser Gln Ala Gly Gln Arg Asn Ser Thr Ser Ile Glu Pro 565 570 575 Arg Leu Leu Trp Glu Arg Val Glu Lys Leu Val Pro Arg Pro Gly Ser 580 585 590 Gly Ser Ser Ser Gly Ser Ser Asn Ser Gly Ser Gln Pro Gly Ser His 595 600 605 Pro Gly Ser Gln Ser Gly Ser Gly Glu Arg Phe Arg Val Arg Ser Ser 610 615 620 Ser Lys Ser Glu Gly Ser Pro Ser Gln Arg Leu Glu Asn Ala Val Lys 625 630 635 640 Lys Pro Glu Asp Lys Lys Glu Val Phe Arg Pro Leu Lys Pro Ala Gly 645 650 655 Glu Val Asp Leu Thr Ala Leu Ala Lys Glu Leu Arg Ala Val Glu Asp 660 665 670 Val Arg Pro Pro His Lys Val Thr Asp Tyr Ser Ser Ser Ser Glu Glu 675 680 685 Ser Gly Thr Thr Asp Glu Glu Asp Asp Asp Val Glu Gln Glu Gly Ala 690 695 700 Asp Glu Ser Thr Ser Gly Pro Glu Asp Thr Arg Ala Ala Ser Ser Leu 705 710 715 720 Asn Leu Ser Asn Gly Glu Thr Glu Ser Val Lys Thr Met Ile Val His 725 730 735 Asp Asp Val Glu Ser Glu Pro Ala Met Thr Pro Ser Lys Glu Gly Thr 740 745 750 Leu Ile Val Arg Gln Thr Gln Ser Ala Ser Ser Thr Leu Gln Lys His 755 760 765 Lys Ser Ser Ser Ser Phe Thr Pro Phe Ile Asp Pro Arg Leu Leu Gln 770 775 780 Ile Ser Pro Ser Ser Gly Thr Thr Val Thr Ser Val Val Gly Phe Ser 785 790 795 800 Cys Asp Gly Met Arg Pro Glu Ala Ile Arg Gln Asp Pro Thr Arg Lys 805 810 815 Gly Ser Val Val Asn Val Asn Pro Thr Asn Thr Arg Pro Gln Ser Asp 820 825 830 Thr Pro Glu Ile Arg Lys Tyr Lys Lys Arg Phe Asn Ser Glu Ile Leu 835 840 845 Cys Ala Ala Leu Trp Gly Val Asn Leu Leu Val Gly Thr Glu Ser Gly 850 855 860 Leu Met Leu Leu Asp Arg Ser Gly Gln Gly Lys Val Tyr Pro Leu Ile 865 870 875 880 Asn Arg Arg Arg Phe Gln Gln Met Asp Val Leu Glu Gly Leu Asn Val 885 890 895 Leu Val Thr Ile Ser Gly Lys Lys Asp Lys Leu Arg Val Tyr Tyr Leu 900 905 910 Ser Trp Leu Arg Asn Lys Ile Leu His Asn Asp Pro Glu Val Glu Lys 915 920 925 Lys Gln Gly Trp Thr Thr Val Gly Asp Leu Glu Gly Cys Val His Tyr 930 935 940 Lys Val Val Lys Tyr Glu Arg Ile Lys Phe Leu Val Ile Ala Leu Lys 945 950 955 960 Ser Ser Val Glu Val Tyr Ala Trp Ala Pro Lys Pro Tyr His Lys Phe 965 970 975 Met Ala Phe Lys Ser Phe Gly Glu Leu Val His Lys Pro Leu Leu Val 980 985 990 Asp Leu Thr Val Glu Glu Gly Gln Arg Leu Lys Val Ile Tyr Gly Ser 995 1000 1005 Cys Ala Gly Phe His Ala Val Asp Val Asp Ser Gly Ser Val Tyr Asp 1010 1015 1020 Ile Tyr Leu Pro Thr His Val Arg Lys Asn Pro His Ser Met Ile Gln 1025 1030 1035 1040 Cys Ser Ile Lys Pro His Ala Ile Ile Ile Leu Pro Asn Thr Asp Gly 1045 1050 1055 Met Glu Leu Leu Val Cys Tyr Glu Asp Glu Gly Val Tyr Val Asn Thr 1060 1065 1070 Tyr Gly Arg Ile Thr Lys Asp Val Val Leu Gln Trp Gly Glu Met Pro 1075 1080 1085 Thr Ser Val Ala Tyr Ile Arg Ser Asn Gln Thr Met Gly Trp Gly Glu 1090 1095 1100 Lys Ala Ile Glu Ile Arg Ser Val Glu Thr Gly His Leu Asp Gly Val 1105 1110 1115 1120 Phe Met His Lys Arg Ala Gln Arg Leu Lys Phe Leu Cys Glu Arg Asn 1125 1130 1135 Asp Lys Val Phe Phe Ala Ser Val Arg Ser Gly Gly Ser Ser Gln Val 1140 1145 1150 Tyr Phe Met Thr Leu Gly Arg Thr Ser Leu Leu Ser Trp 1155 1160 1165 <210> 5 <211> 711 <212> PRT <213> Artificial Sequence <220> <223> ACSL4 protein <400> 5 Met Lys Leu Lys Leu Asn Val Leu Thr Ile Ile Leu Leu Pro Val His 1 5 10 15 Leu Leu Ile Thr Ile Tyr Ser Ala Leu Ile Phe Ile Pro Trp Tyr Phe 20 25 30 Leu Thr Asn Ala Lys Lys Lys Asn Ala Met Ala Lys Arg Ile Lys Ala 35 40 45 Lys Pro Thr Ser Asp Lys Pro Gly Ser Pro Tyr Arg Ser Val Thr His 50 55 60 Phe Asp Ser Leu Ala Val Ile Asp Ile Pro Gly Ala Asp Thr Leu Asp 65 70 75 80 Lys Leu Phe Asp His Ala Val Ser Lys Phe Gly Lys Lys Asp Ser Leu 85 90 95 Gly Thr Arg Glu Ile Leu Ser Glu Glu Asn Glu Met Gln Pro Asn Gly 100 105 110 Lys Val Phe Lys Lys Leu Ile Leu Gly Asn Tyr Lys Trp Met Asn Tyr 115 120 125 Leu Glu Val Asn Arg Arg Val Asn Asn Phe Gly Ser Gly Leu Thr Ala 130 135 140 Leu Gly Leu Lys Pro Lys Asn Thr Ile Ala Ile Phe Cys Glu Thr Arg 145 150 155 160 Ala Glu Trp Met Ile Ala Ala Gln Thr Cys Phe Lys Tyr Asn Phe Pro 165 170 175 Leu Val Thr Leu Tyr Ala Thr Leu Gly Lys Glu Ala Val Val His Gly 180 185 190 Leu Asn Glu Ser Glu Ala Ser Tyr Leu Ile Thr Ser Val Glu Leu Leu 195 200 205 Glu Ser Lys Leu Lys Thr Ala Leu Leu Asp Ile Ser Cys Val Lys His 210 215 220 Ile Ile Tyr Val Asp Asn Lys Ala Ile Asn Lys Ala Glu Tyr Pro Glu 225 230 235 240 Gly Phe Glu Ile His Ser Met Gln Ser Val Glu Glu Leu Gly Ser Asn 245 250 255 Pro Glu Asn Leu Gly Ile Pro Pro Ser Arg Pro Thr Pro Ser Asp Met 260 265 270 Ala Ile Val Met Tyr Thr Ser Gly Ser Thr Gly Arg Pro Lys Gly Val 275 280 285 Met Met His His Ser Asn Leu Ile Ala Gly Met Thr Gly Gln Cys Glu 290 295 300 Arg Ile Pro Gly Leu Gly Pro Lys Asp Thr Tyr Ile Gly Tyr Leu Pro 305 310 315 320 Leu Ala His Val Leu Glu Leu Thr Ala Glu Ile Ser Cys Phe Thr Tyr 325 330 335 Gly Cys Arg Ile Gly Tyr Ser Ser Pro Leu Thr Leu Ser Asp Gln Ser 340 345 350 Ser Lys Ile Lys Lys Gly Ser Lys Gly Asp Cys Thr Val Leu Lys Pro 355 360 365 Thr Leu Met Ala Ala Val Pro Glu Ile Met Asp Arg Ile Tyr Lys Asn 370 375 380 Val Met Ser Lys Val Gln Glu Met Asn Tyr Ile Gln Lys Thr Leu Phe 385 390 395 400 Lys Ile Gly Tyr Asp Tyr Lys Leu Glu Gln Ile Lys Lys Gly Tyr Asp 405 410 415 Ala Pro Leu Cys Asn Leu Leu Leu Phe Lys Lys Val Lys Ala Leu Leu 420 425 430 Gly Gly Asn Val Arg Met Met Leu Ser Gly Gly Ala Pro Leu Ser Pro 435 440 445 Gln Thr His Arg Phe Met Asn Val Cys Phe Cys Cys Pro Ile Gly Gln 450 455 460 Gly Tyr Gly Leu Thr Glu Ser Cys Gly Ala Gly Thr Val Thr Glu Val 465 470 475 480 Thr Asp Tyr Thr Thr Gly Arg Val Gly Ala Pro Leu Ile Cys Cys Glu 485 490 495 Ile Lys Leu Lys Asp Trp Gln Glu Gly Gly Tyr Thr Ile Asn Asp Lys 500 505 510 Pro Asn Pro Arg Gly Glu Ile Val Ile Gly Gly Gln Asn Ile Ser Met 515 520 525 Gly Tyr Phe Lys Asn Glu Glu Lys Thr Ala Glu Asp Tyr Ser Val Asp 530 535 540 Glu Asn Gly Gln Arg Trp Phe Cys Thr Gly Asp Ile Gly Glu Phe His 545 550 555 560 Pro Asp Gly Cys Leu Gln Ile Ile Asp Arg Lys Lys Asp Leu Val Lys 565 570 575 Leu Gln Ala Gly Glu Tyr Val Ser Leu Gly Lys Val Glu Ala Ala Leu 580 585 590 Lys Asn Cys Pro Leu Ile Asp Asn Ile Cys Ala Phe Ala Lys Ser Asp 595 600 605 Gln Ser Tyr Val Ile Ser Phe Val Val Pro Asn Gln Lys Arg Leu Thr 610 615 620 Leu Leu Ala Gln Gln Lys Gly Val Glu Gly Thr Trp Val Asp Ile Cys 625 630 635 640 Asn Asn Pro Ala Met Glu Ala Glu Ile Leu Lys Glu Ile Arg Glu Ala 645 650 655 Ala Asn Ala Met Lys Leu Glu Arg Phe Glu Ile Pro Ile Lys Val Arg 660 665 670 Leu Ser Pro Glu Pro Trp Thr Pro Glu Thr Gly Leu Val Thr Asp Ala 675 680 685 Phe Lys Leu Lys Arg Lys Glu Leu Arg Asn His Tyr Leu Lys Asp Ile 690 695 700 Glu Arg Met Tyr Gly Gly Lys 705 710 <210> 6 <211> 577 <212> PRT <213> Artificial Sequence <220> <223> Moesin protein <400> 6 Met Pro Lys Thr Ile Ser Val Arg Val Thr Thr Met Asp Ala Glu Leu 1 5 10 15 Glu Phe Ala Ile Gln Pro Asn Thr Thr Gly Lys Gln Leu Phe Asp Gln 20 25 30 Val Val Lys Thr Ile Gly Leu Arg Glu Val Trp Phe Phe Gly Leu Gln 35 40 45 Tyr Gln Asp Thr Lys Gly Phe Ser Thr Trp Leu Lys Leu Asn Lys Lys 50 55 60 Val Thr Ala Gln Asp Val Arg Lys Glu Ser Pro Leu Leu Phe Lys Phe 65 70 75 80 Arg Ala Lys Phe Tyr Pro Glu Asp Val Ser Glu Glu Leu Ile Gln Asp 85 90 95 Ile Thr Gln Arg Leu Phe Phe Leu Gln Val Lys Glu Gly Ile Leu Asn 100 105 110 Asp Asp Ile Tyr Cys Pro Pro Glu Thr Ala Val Leu Leu Ala Ser Tyr 115 120 125 Ala Val Gln Ser Lys Tyr Gly Asp Phe Asn Lys Glu Val His Lys Ser 130 135 140 Gly Tyr Leu Ala Gly Asp Lys Leu Leu Pro Gln Arg Val Leu Glu Gln 145 150 155 160 His Lys Leu Asn Lys Asp Gln Trp Glu Glu Arg Ile Gln Val Trp His 165 170 175 Glu Glu His Arg Gly Met Leu Arg Glu Asp Ala Val Leu Glu Tyr Leu 180 185 190 Lys Ile Ala Gln Asp Leu Glu Met Tyr Gly Val Asn Tyr Phe Ser Ile 195 200 205 Lys Asn Lys Lys Gly Ser Glu Leu Trp Leu Gly Val Asp Ala Leu Gly 210 215 220 Leu Asn Ile Tyr Glu Gln Asn Asp Arg Leu Thr Pro Lys Ile Gly Phe 225 230 235 240 Pro Trp Ser Glu Ile Arg Asn Ile Ser Phe Asn Asp Lys Lys Phe Val 245 250 255 Ile Lys Pro Ile Asp Lys Lys Ala Pro Asp Phe Val Phe Tyr Ala Pro 260 265 270 Arg Leu Arg Ile Asn Lys Arg Ile Leu Ala Leu Cys Met Gly Asn His 275 280 285 Glu Leu Tyr Met Arg Arg Arg Lys Pro Asp Thr Ile Glu Val Gln Gln 290 295 300 Met Lys Ala Gln Ala Arg Glu Glu Lys His Gln Lys Gln Met Glu Arg 305 310 315 320 Ala Met Leu Glu Asn Glu Lys Lys Lys Arg Glu Met Ala Glu Lys Glu 325 330 335 Lys Glu Lys Ile Glu Arg Glu Lys Glu Glu Leu Met Glu Arg Leu Lys 340 345 350 Gln Ile Glu Glu Gln Thr Lys Lys Ala Gln Gln Glu Leu Glu Glu Gln 355 360 365 Thr Arg Arg Ala Leu Glu Leu Glu Gln Glu Arg Lys Arg Ala Gln Ser 370 375 380 Glu Ala Glu Lys Leu Ala Lys Glu Arg Gln Glu Ala Glu Glu Ala Lys 385 390 395 400 Glu Ala Leu Leu Gln Ala Ser Arg Asp Gln Lys Lys Thr Gln Glu Gln 405 410 415 Leu Ala Leu Glu Met Ala Glu Leu Thr Ala Arg Ile Ser Gln Leu Glu 420 425 430 Met Ala Arg Gln Lys Lys Glu Ser Glu Ala Val Glu Trp Gln Gln Lys 435 440 445 Ala Gln Met Val Gln Glu Asp Leu Glu Lys Thr Arg Ala Glu Leu Lys 450 455 460 Thr Ala Met Ser Thr Pro His Val Ala Glu Pro Ala Glu Asn Glu Gln 465 470 475 480 Asp Glu Gln Asp Glu Asn Gly Ala Glu Ala Ser Ala Asp Leu Arg Ala 485 490 495 Asp Ala Met Ala Lys Asp Arg Ser Glu Glu Glu Arg Thr Thr Glu Ala 500 505 510 Glu Lys Asn Glu Arg Val Gln Lys His Leu Lys Ala Leu Thr Ser Glu 515 520 525 Leu Ala Asn Ala Arg Asp Glu Ser Lys Lys Thr Ala Asn Asp Met Ile 530 535 540 His Ala Glu Asn Met Arg Leu Gly Arg Asp Lys Tyr Lys Thr Leu Arg 545 550 555 560 Gln Ile Arg Gln Gly Asn Thr Lys Gln Arg Ile Asp Glu Phe Glu Ser 565 570 575 Met <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MAP4K4 FW Primer <400> 7 gagaggctcc agaggcagtt gca 23 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MAP4K4 BW Primer <400> 8 ccacctctcg cgctcggtca g 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ACSL4 FW Primer <400> 9 gggaagaagg acagccttgg g 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ACSL4 BW Primer <400> 10 tcggccctgg tctcacagaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Moesin FW Primer <400> 11 gctgtggagt ggcagcagaa 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Moesin BW Primer <400> 12 cgttcctcct cactgcggtc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s rRNA FW Primer <400> 13 gtaacccgtt gaaccccatt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s rRNA BW Primer <400> 14 ccatccaatc ggtagtagcg 20
Claims (7)
상기 방사선 내성 유방암은 MAP4K4, ACSL4, 및 모에신 유전자의 발현이 일반 유방암 세포보다 증가한 것인,
방사선 내성 유방암 치료용 약학적 조성물.
As a pharmaceutical composition for the treatment of radiation-resistant breast cancer comprising an ethanol extract of yeonjayuk or yeonjabang as an active ingredient,
In the radiation-resistant breast cancer, the expression of MAP4K4, ACSL4, and moesin genes is increased than that of normal breast cancer cells,
Pharmaceutical composition for the treatment of radiation-resistant breast cancer.
상기 방사선 내성 유방암은 MAP4K4, ACSL4, 및 모에신 유전자의 발현이 일반 유방암 세포보다 증가한 것인,
방사선 내성 유방암 개선용 건강기능식품.
As a health functional food for improving radiation-resistant breast cancer comprising an ethanol extract of Yeonjayuk or Yeonjabang,
In the radiation-resistant breast cancer, the expression of MAP4K4, ACSL4, and moesin genes is increased than that of normal breast cancer cells,
Health functional food for improving radiation-resistant breast cancer.
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KR20240010982A (en) | 2022-07-18 | 2024-01-25 | 한국원자력의학원 | Pharmaceutical composition for preventing and treating radioresistant cancer comprising a compounds for inhibiting rip1 |
KR20240010983A (en) | 2022-07-18 | 2024-01-25 | 한국원자력의학원 | Pharmaceutical composition for preventing and treating radioresistant cancer |
KR20240012151A (en) | 2022-07-20 | 2024-01-29 | 한국원자력의학원 | Pharmaceutical composition for preventing and treating radioresistant cancer comprising a rip1 inhibitor |
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US20090214683A1 (en) | 2008-02-27 | 2009-08-27 | Chau-Jong Wang | Extracts of Sacred Water Lotus for the Treatment of Cancer |
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KR101244402B1 (en) * | 2009-12-29 | 2013-03-18 | 한국원자력의학원 | A composition for enhancing the radiotherapy of cancer |
KR101952649B1 (en) | 2016-05-17 | 2019-02-27 | 울산대학교 산학협력단 | Biomarker composition for diagnosing radiation resistant cancer or predicting prognosis of radiation therapy comprising LRP-1 |
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US20090214683A1 (en) | 2008-02-27 | 2009-08-27 | Chau-Jong Wang | Extracts of Sacred Water Lotus for the Treatment of Cancer |
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2017년도 한국생명과학괴 제58회 정기총회 및 국제학술대회 초록집 pp. 225* |
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KR20240010982A (en) | 2022-07-18 | 2024-01-25 | 한국원자력의학원 | Pharmaceutical composition for preventing and treating radioresistant cancer comprising a compounds for inhibiting rip1 |
KR20240010983A (en) | 2022-07-18 | 2024-01-25 | 한국원자력의학원 | Pharmaceutical composition for preventing and treating radioresistant cancer |
KR20240012151A (en) | 2022-07-20 | 2024-01-29 | 한국원자력의학원 | Pharmaceutical composition for preventing and treating radioresistant cancer comprising a rip1 inhibitor |
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