KR20220138575A - Method for screening drugs for the treatment or prevention of translocation Renal Cell Carcinoma - Google Patents
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Abstract
Description
본 발명은 전좌 신장암(Translocation Renal Cell Carcinoma) 치료 또는 예방용 약물 스크리닝 방법에 관한 것으로, 보다 구체적으로 mTOR 억제제를 처리하여 TFE3 및 PD-L1을 발현시킨 전좌 신장암 세포주를 이용한 전좌 신장암 치료 또는 예방용 약물 스크리닝 방법에 관한 것이다.The present invention relates to a drug screening method for treating or preventing translocation Renal Cell Carcinoma, and more particularly, to a translocation Renal Cell Carcinoma treatment or It relates to a method of screening a drug for prophylaxis.
신장은 혈액을 여과하여 소변을 생성함으로써 생체 내의 노폐물을 체외로 배설하는 역할을 하는 중요한 비뇨기계 기관이다. 또한, 동시에 혈압을 조절하는 안지오텐신, 적혈구 조혈 인자인 에리스로포이에틴 등의 호르몬을 생산하는 중요한 내분비 기관이기도 하다. 이렇게 신장에 종양이 발생하는 것을 신장암이라 한다.The kidney is an important urinary system that filters blood and produces urine, thereby excreting wastes in the living body to the outside of the body. It is also an important endocrine organ that simultaneously produces hormones such as angiotensin that regulates blood pressure and erythropoietin, a erythropoietic factor. When a tumor develops in the kidney, it is called kidney cancer.
신장암(Renal Cell Carcinoma, RCC)은 일반적인 고형 악성 종양으로, 조직학적 유형(histologic subtypes)이 다른 이질성 질환(heterogeneous disease)으로 널리 알려져 있다. 신장암의 한 종류로 알려진 전좌 신장암(Translocation Renal Cell Carcinoma)은 신장암의 두문 유형(subtype)으로, 주로 소아 또는 청소년에서 발병하며, 최근 성인에서도 빈번히 발병되고 있다.Renal Cell Carcinoma (RCC) is a common solid malignant tumor and is widely known as a heterogeneous disease with different histologic subtypes. Translocation Renal Cell Carcinoma, known as a type of renal cancer, is a subtype of renal cancer, mainly occurring in children or adolescents, and recently occurring frequently in adults.
몇몇 연구들은 mTOR가 세포 성장, 증식 및 대사를 제어하는데 가장 중요한 역할을 하는 것을 입증하였다. mTOR는 번역, 전사, mRNA 대사회전(turnover), 단백질 안정성, 액틴 세포골격 구성(actin cytoskeletal organization) 및 자가소화작용(autophagy)을 포함하는, 광범위한 세포 기능을 조절한다. mTOR는 포스포이노시티드 키나제-관련 키나제(phosphoinositide kinase-related kinase, "PIKK") 군의 일원이다. 포유동물 세포에는 2종의 mTOR 복합체가 있다. mTOR 복합체 Ⅰ(mTORC1)은 라파마이신-민감성 방식으로 세포 성장을 주로 조절하는 랩터-mTOR 복합체(raptor-mTOR complex)인 반면, mTOR 복합체 Ⅱ(mTORC2)는 라파마이신-비민감성 방식으로 세포골격 구성을 조절하는 릭터-mTOR 복합체(rictor-mTOR complex)이다. Several studies have demonstrated that mTOR plays the most important role in controlling cell growth, proliferation and metabolism. mTOR regulates a wide range of cellular functions, including translation, transcription, mRNA turnover, protein stability, actin cytoskeletal organization and autophagy. mTOR is a member of the phosphoinositide kinase-related kinase (“PIKK”) family. There are two types of mTOR complexes in mammalian cells. mTOR complex I (mTORC1) is a raptor-mTOR complex that mainly regulates cell growth in a rapamycin-sensitive manner, whereas mTOR complex II (mTORC2) regulates cytoskeletal composition in a rapamycin-insensitive manner. It is the rictor-mTOR complex that regulates it.
mTOR 억제제는 신장암 치료제로 적용되고 있다. 다만, mTOR 억제제은 신장암 치료를 위해 FDA 승인을 받았음에도 불구하고, 특정 치료 상황에서 효능에 관한 많은 의문이 제기되고 있다.mTOR inhibitors are being applied as a treatment for kidney cancer. However, despite FDA approval for the treatment of kidney cancer, mTOR inhibitors raise many questions about their efficacy in certain therapeutic situations.
따라서, 전술한 문제점을 보완하기 위해 본 발명가들은 mTOR 억제제와 전위 신장암 사이의 관계를 입증하고, 이에 의해 발현되는 단백질을 이용하여 전좌 신장암 치료 또는 예방용 약물 스크리닝 방법의 개발이 시급하다 인식하여, 본 발명을 완성하였다.Therefore, in order to supplement the above problems, the present inventors demonstrate the relationship between an mTOR inhibitor and translocation renal cancer, and recognize that it is urgent to develop a drug screening method for treating or preventing translocation renal cancer using a protein expressed by this. , the present invention was completed.
본 발명의 목적은 mTOR 억제제를 처리하여 TFE3 및 PD-L1을 발현시킨 전좌 신장암 세포주를 이용한 전좌 신장암 치료 또는 예방용 약물 스크리닝 방법을 제공하는 것이다.It is an object of the present invention to provide a drug screening method for treating or preventing translocation renal cancer using a translocation renal cancer cell line expressing TFE3 and PD-L1 by treatment with an mTOR inhibitor.
발명이 이루고자 하는 기술적 과제들은 이상에서 언급한 기술적 과제들로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 본 발명의 기재로부터 당해 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있다.The technical problems to be achieved by the invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by those of ordinary skill in the art from the description of the present invention.
상기 목적을 달성하기 위하여, 본 발명은 전좌 신장암 치료 또는 예방용 약물 스크리닝 방법을 제공한다.In order to achieve the above object, the present invention provides a drug screening method for treating or preventing translocation renal cancer.
이하, 본 명세서에 대하여 더욱 상세하게 설명한다.Hereinafter, the present specification will be described in more detail.
본 발명은 하기의 단계를 포함하는 것을 특징으로 하는 전좌 신장암 치료 또는 예방용 약물 스크리닝 방법을 제공한다.The present invention provides a drug screening method for treating or preventing translocation renal cancer, comprising the following steps.
(S1) 전좌 신장암(Translocation Renal Cell Carcinoma) 세포주를 배양하는 단계;(S1) culturing a translocation Renal Cell Carcinoma cell line;
(S2) 상기 배양된 세포주에 mTOR 억제제를 처리하여 TFE3 및 PD-L1을 발현시키는 단계;(S2) treating the cultured cell line with an mTOR inhibitor to express TFE3 and PD-L1;
(S3) 상기 세포주에 후보물질을 처리하는 단계; 및(S3) treating the cell line with a candidate material; and
(S4) 상기 후보물질 처리 후, 상기 세포주의 TFE3 및 PD-L1 발현량을 측정하는 단계.(S4) After treatment with the candidate substance, measuring the expression levels of TFE3 and PD-L1 in the cell line.
본 발명에 있어서, 상기 전좌 신장암 세포주는 UOK146, UOK109 및 UOK120인 것을 특징으로 한다.In the present invention, the translocation renal cancer cell line is characterized in that UOK146, UOK109 and UOK120.
본 발명에 있어서, 상기 mTOR 억제제는 템시로리무스(temsirolimus), 에베로리무스(everolimus) 및 토린-1(torin-1)으로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 한다.In the present invention, the mTOR inhibitor is characterized in that at least one selected from the group consisting of temsirolimus, everolimus and torin-1.
본 발명에 있어서, 상기 (S4) 단계에서 상기 후보물질이 처리된 세포주의 TFE3 및 PD-L1 발현량이 상기 후보물질이 미처리된 세포주의 TFE3 및 PD-L1 발현량 보다 감소한 경우 상기 후보물질을 전좌 신장암 치료제로 선별하는 단계;를 추가적으로 포함하는 것을 특징으로 한다.In the present invention, when the expression levels of TFE3 and PD-L1 in the cell line treated with the candidate material in step (S4) are lower than the expression levels of TFE3 and PD-L1 in the cell line untreated with the candidate material, the candidate material is translocated and elongated. It is characterized in that it additionally comprises; selecting a cancer therapeutic agent.
본 발명의 전좌 신장암 치료 또는 예방용 약물 스크리닝 방법은 mTOR 억제제를 처리하여 TFE3 및 PD-L1을 발현시킨 전좌 신장암 세포주를 이용하여 보다 정확하기 신속하게 전좌 신장암의 치료제를 개발하는데 유용하게 적용할 수 있다.The drug screening method for the treatment or prevention of translocation kidney cancer of the present invention is useful for developing a therapeutic agent for translocation kidney cancer more accurately and quickly using a translocation kidney cancer cell line expressing TFE3 and PD-L1 by treatment with an mTOR inhibitor can do.
본 발명의 효과들은 이상에서 언급한 효과들로 제한되지 않으며, 언급되지 않은 또 다른 효과들은 청구범위의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.The effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the description of the claims.
도 1은 전좌 신장암 UOK146 세포주에서 mTOR 억제제 처리에 따른 TFE3 및 PD-L1이 발현을 확인한 이미지이다.1 is an image confirming the expression of TFE3 and PD-L1 according to mTOR inhibitor treatment in translocation renal cancer UOK146 cell line.
본 명세서에서 사용되는 용어는 본 발명에서의 기능을 고려하면서 가능한 현재 널리 사용되는 일반적인 용어들을 선택하였으나, 이는 당 분야에 종사하는 기술자의 의도 또는 판례, 새로운 기술의 출현 등에 따라 달라질 수 있다. 또한, 특정한 경우는 출원인이 임의로 선정한 용어도 있으며, 이 경우 해당되는 발명의 설명 부분에서 상세히 그 의미를 기재할 것이다. 따라서 본 발명에서 사용되는 용어는 단순한 용어의 명칭이 아닌, 그 용어가 가지는 의미와 본 발명의 전반에 걸친 내용을 토대로 정의되어야 한다.The terms used in this specification have been selected as currently widely used general terms as possible while considering the functions in the present invention, which may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like. In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not
수치 범위는 상기 범위에 정의된 수치를 포함한다. 본 명세서에 걸쳐 주어진 모든 최대의 수치 제한은 낮은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 낮은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 최소의 수치 제한은 더 높은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 높은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 수치 제한은 더 좁은 수치 제한이 명확히 쓰여져 있는 것처럼, 더 넓은 수치 범위 내의 더 좋은 모든 수치 범위를 포함할 것이다.Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. All numerical limitations given throughout this specification will include all numerical ranges that are better within the broader numerical limits, as if the narrower numerical limitations were expressly written.
이하, 본 발명의 실시예를 상세히 기술하나, 하기 실시예에 의해 본 발명이 한정되지 아니함은 자명하다.Hereinafter, examples of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.
전좌 신장암 치료 또는 예방용 약물 스크리닝 방법Drug screening method for treatment or prevention of translocated renal cancer
본 발명은 하기의 단계를 포함하는 전좌 신장암 치료 또는 예방용 약물 스크리닝 방법을 제공한다.The present invention provides a drug screening method for treating or preventing translocation renal cancer comprising the following steps.
(S1) 전좌 신장암(Translocation Renal Cell Carcinoma) 세포주를 배양하는 단계;(S1) culturing a translocation Renal Cell Carcinoma cell line;
(S2) 상기 배양된 세포주에 mTOR 억제제를 처리하여 TFE3 및 PD-L1을 발현시키는 단계;(S2) treating the cultured cell line with an mTOR inhibitor to express TFE3 and PD-L1;
(S3) 상기 세포주에 후보물질을 처리하는 단계; 및(S3) treating the cell line with a candidate material; and
(S4) 상기 후보물질 처리 후, 상기 세포주의 TFE3 및 PD-L1 발현량을 측정하는 단계.(S4) After treatment with the candidate substance, measuring the expression levels of TFE3 and PD-L1 in the cell line.
본 발명에 사용된 용어 “전좌(Translocation)”는 “전위”로도 불리며, 유전자 물질이 정상적인 장소를 이탈하는 것으로, 하나의 염색체에서 DNA가 떨어져 나와 다른 염색체에 붙는 것을 의미한다.As used herein, the term “translocation” is also referred to as “translocation”, which means that genetic material deviates from a normal place, meaning that DNA is detached from one chromosome and attached to another chromosome.
본 발명에 사용된 용어 “신장암(Renal Cell Carcinoma, RCC)”은 신장세포암으로도 불리며, 신장의 세뇨관(혈액을 여과하여 소변을 생성함) 내벽에서 시작된 암을 의미한다.As used herein, the term “renal cell carcinoma (RCC)” is also called renal cell carcinoma, and refers to cancer that begins in the inner wall of the renal tubule (which filters blood to produce urine).
상기 전좌 신장암 세포주는 UOK146, UOK109, UOK120일 수 있다.The translocation renal cancer cell line may be UOK146, UOK109, or UOK120.
상기 mTOR 억제제는 템시로리무스(temsirolimus), 에베로리무스(everolimus) 및 토린-1(torin-1)으로 이루어진 군으로부터 선택된 1종 이상일 수 있다.The mTOR inhibitor may be at least one selected from the group consisting of temsirolimus, everolimus and torin-1.
상기 (S2) 단계에서 상기 mTOR 억제제를 처리하여 TFE3 및 PD-L1을 발현된 세포주는 생리 활성을 안정하게 유지시키는 완충액 또는 반응액을 포함할 수 있다. The cell line expressing TFE3 and PD-L1 by treating the mTOR inhibitor in step (S2) may include a buffer or reaction solution for stably maintaining physiological activity.
상기 후보물질은 통상적인 선정방식에 따라 전좌 신장암의 치료체 또는 예방제로서의 가능성을 지닌 것으로 추정되거나 또는 무작위적으로 선정된 개별적인 핵산, 단백질, 기타 추출물 또는 천연물, 화합물 등이 될 수 있다.The candidate substances may be individual nucleic acids, proteins, other extracts, natural products, compounds, etc. estimated to have potential as therapeutic agents or prophylactic agents for translocation kidney cancer, or randomly selected according to a conventional selection method.
상기 발현량 측정은 RT(Reverse transcriptase)-PCR 또는 웨스턴 블롯팅(western blotting)을 수행하여 측정할 수 있으나, 이에 제한되는 것은 아니며 당업자에게 공지된 방법을 사용하여 수행할 수 있다.The expression level may be measured by performing reverse transcriptase (RT)-PCR or western blotting, but is not limited thereto and may be performed using methods known to those skilled in the art.
상기 (S4) 단계에서 상기 후보물질이 처리된 세포주의 TFE3 및 PD-L1 발현량이 상기 후보물질이 미처리된 세포주의 TFE3 및 PD-L1 발현량 보다 감소한 경우 상기 후보물질을 전좌 신장암 치료제로 선별하는 단계;를 추가적으로 포함할 수 있다.When the expression levels of TFE3 and PD-L1 of the cell line treated with the candidate material in step (S4) are lower than the expression levels of TFE3 and PD-L1 of the cell line untreated with the candidate material, the candidate material is selected as a translocation renal cancer therapeutic agent. step; may be additionally included.
보다 구체적으로, 상기 TFE3 및 PD-L1 발현을 증감소시키는 활성을 나타내는 후보물질은 전좌 신장암 치료제 후보물질이 될 수도 있고, 전좌 신장암의 치료제 후보물질이 될 수 있다.More specifically, the candidate substance exhibiting the activity of increasing or decreasing the expression of TFE3 and PD-L1 may be a translocation renal cancer therapeutic agent or a translocated renal cancer therapeutic agent candidate.
이와 같은 전좌 신장암의의 치료제 후보물질은 이후의 치료제 개발과정에서 선도물질(leading compound)로서 작용하게 되며, 선도물질의 구조를 변형시키고 최적화함으로써, 새로운 전좌 신장암을 치료제를 개발할 수 있다.Such a candidate drug for translocation kidney cancer acts as a leading compound in the process of developing a treatment thereafter, and by modifying and optimizing the structure of the lead material, a new therapeutic agent for translocation kidney cancer can be developed.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어 있는 실시예들을 참조하면 명확해 질 것이다. 그러나, 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 수 있으며, 단지 실시예들은 본 발명의 개시가 완전하도록 하고, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하세 알려 주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다.Advantages and features of the present invention, and methods for achieving them, will become apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, and only the embodiments allow the disclosure of the present invention to be complete, and common knowledge in the technical field to which the present invention pertains It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.
실시예 1. 전좌 신장암 세포주에서 mTOR 억제제 처리에 따른 TFE3 및 PD-L1이 발현 확인Example 1. Confirmation of expression of TFE3 and PD-L1 following mTOR inhibitor treatment in translocation renal cancer cell line
전좌 신장암(Translocation Renal Cell Carcinoma) UOK146 세포주를 배양하고 상기 배양된 UOK146 세포주에 mTOR 억제제로 토린-1(torin-1)을 처리하여 TFE3 및 PD-L1을 발현시켰으며, 이를 도 1에 나타내었다.Translocation Renal Cell Carcinoma UOK146 cell line was cultured, and TFE3 and PD-L1 were expressed by treating the cultured UOK146 cell line with torin-1 with an mTOR inhibitor, which is shown in FIG. 1 . .
도 1을 참조하면, 전좌 신장암 세포주(UOK146)와 비교하여 mTOR 억제제인 토린-1을 3시간 및 24시간 처리한 세포주에서 TFE3 및 PD-L1의 발현히 증가되었음을 확인할 수 있다.Referring to FIG. 1 , it can be seen that the expression of TFE3 and PD-L1 was increased in the cell lines treated with the mTOR inhibitor, Thorin-1, for 3 hours and 24 hours compared to the translocation renal cancer cell line (UOK146).
이상 설명으로부터, 본 발명에 속하는 기술 분야의 당업자는 본 발명의 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며, 한정적인 것이 아닌 것으로서 이해해야만 한다.From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.
Claims (4)
(S2) 상기 배양된 세포주에 mTOR 억제제를 처리하여 TFE3 및 PD-L1을 발현시키는 단계;
(S3) 상기 세포주에 후보물질을 처리하는 단계; 및
(S4) 상기 후보물질 처리 후, 상기 세포주의 TFE3 및 PD-L1 발현량을 측정하는 단계;를 포함하는 것을 특징으로 하는 전좌 신장암 치료 또는 예방용 약물 스크리닝 방법.(S1) culturing a translocation Renal Cell Carcinoma cell line;
(S2) treating the cultured cell line with an mTOR inhibitor to express TFE3 and PD-L1;
(S3) treating the cell line with a candidate material; and
(S4) after treatment with the candidate substance, measuring the expression levels of TFE3 and PD-L1 in the cell line; translocation renal cancer treatment or prevention drug screening method comprising the.
상기 전좌 신장암 세포주는 UOK146, UOK109 및 UOK120인 것을 특징으로 하는 전좌 신장암 치료 또는 예방용 약물 스크리닝 방법.The method of claim 1,
The translocation renal cancer cell line is UOK146, UOK109, and a drug screening method for the treatment or prevention of translocation renal cancer, characterized in that UOK120.
상기 mTOR 억제제는 템시로리무스(temsirolimus), 에베로리무스(everolimus) 및 토린-1(torin-1)으로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는 전좌 신장암 치료 또는 예방용 약물 스크리닝 방법.The method of claim 1,
The mTOR inhibitor is a translocation renal cancer treatment or prevention drug screening method, characterized in that at least one selected from the group consisting of temsirolimus, everolimus and torin-1.
상기 (S4) 단계에서 상기 후보물질이 처리된 세포주의 TFE3 및 PD-L1 발현량이 상기 후보물질이 미처리된 세포주의 TFE3 및 PD-L1 발현량 보다 감소한 경우 상기 후보물질을 전좌 신장암 치료제로 선별하는 단계;를 추가적으로 포함하는 것을 특징으로 하는 전좌 신장암 치료 또는 예방용 약물 스크리닝 방법.According to claim 1,
When the expression levels of TFE3 and PD-L1 of the cell line treated with the candidate material in step (S4) are lower than the expression levels of TFE3 and PD-L1 of the cell line untreated with the candidate material, the candidate material is selected as a translocation renal cancer therapeutic agent. A drug screening method for treatment or prevention of translocation renal cancer, characterized in that it additionally comprises a step.
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