급성골수성백혈병세포에서 에리불린에 의한 세포사 기전 연구
- Abstract
- 배경 : 급성골수성백혈병은 골수성 백혈구의 분화 단계 중 초기 단계에 있는 전구세포인 골수 모세포가 미분화 상태로서 축적하고 비정상적으로 증식하는 혈액 악성종양이다. 시타라빈과 안트라사이클린 계열 항암제가 주된 치료제로 사용되고 있으나, 50~70%의 환자들은 재발을 겪는다. 에리불린은 전이성 유방암과 지방육종의 치료에 쓰이고 있는 약물로 해면 스펀지에서 추출 및 분리한 halichondrin B를 기본으로 한 합성의약품으로 급성골수성백혈병에서의 효과는 아직 알려지지 않았다. 본 연구에서는 급성골수성백혈병 세포에서의 에리불린의 효능을 평가하고 신호기전에 대한 연구를 진행하였다.
방법 : 급성골수성백혈병 세포주인 HL60에 에리불린을 처리한 후 MTS Assay와 Annexin V 염색을 사용하여 세포 생존율과 세포사를 확인하였고 세포사멸이 일어나는 경로를 Caspase 발현도 및 활성도와 DiOC6(3) 염색을 통한 미토콘드리아 막 전위를 통해 확인하였다. 세포주기의 변화를 보기 위해 Propidium idodide(PI) 염색을 통해 확인하였다. 세포사와 관련된 단백질들의 발현을 Western blot Assay를 통해 확인하였고 혈액 세포의 분화와 성숙에 관련된 표지자인 여러 표면 항원들의 발현도를 확인하였다.
결과 : 에리불린을 HL60 세포에 처리하였을 때 에리불린의 농도가 높아질수록 세포 생존율이 감소하는 것을 확인하였고 세포사멸이 일어나는 세포의 비율은 증가하는 것을 확인하였다. 세포사멸의 경로를 확인하기 위한 실험에서 에리불린의 농도가 높아질수록 Caspase 활성도가 증가하고 미토콘드리아 막 전위는 감소하는 것을 확인하였다. 세포주기를 분석한 결과 에리불린에 의해서 G2/M기의 정지가 유도됨을 확인하였고 그와 관련된 단백질 발현 또한 에리불린에 의해 조절되는 것을 확인하였다. 또한 혈액 세포의 분화와 성숙에 관련된 표지자인 CD11b, CD14, CCR1, CCR2, ICAM-1, HLA-DR의 발현 정도를 확인하였을 때 에리불린의 농도가 높아질수록 발현이 증가하는 것을 확인하였다.
결론 : 에리불린은 급성골수성백혈병 세포에서 세포독성과 세포사멸 유도 효과가 있으며 미성숙 백혈병 세포의 분화를 유도하여, 새로운 백혈병 치료제로서의 가능성이 있다.|Background : Acute myeloid leukemia is a hematological malignant tumor in which bone marrow cells, which are progenitor cells in the early stage of myelogenous leukocyte differentiation, accumulate in an undifferentiated state and proliferate abnormally. Cytarabine and anthracycline-based anticancer drugs are used as the main treatment, but 50~70% of patients experience recurrence. Eribulin is a drug used to treat metastatic breast cancer and liposarcoma. It is a natural compound obtained from halichondrin B extracted and isolated with a marine sponge. Its effect on acute myeloid leukemia is not yet known. In this study, the efficacy of eribulin in acute myeloid leukemia cells was evaluated and the signaling mechanism was studied.
Method : After eribulin was treated with HL60, a cell line for acute myeloid leukemia, cell viability and cell death were confirmed using MTS Assay and Annexin V staining. The pathway in which apoptosis occurs was confirmed by Caspase expression and activity and mitochondrial membrane potential through DiOC6(3) staining. Propidium iodide (PI) staining was used to confirm the change in the cell cycle. The expression of proteins related to cell death was confirmed through Western blot assay, and the expression levels of various surface antigens, which are markers related to the differentiation and maturation of blood cells, were confirmed.
Result : When eribulin was treated at different concentrations in HL60, it was confirmed that the cell viability decreased as the concentration of eribulin was arranged, and the proportion of cells in which apoptosis occurred was confirmed that the presence of eribulin concentration increased. Later, in an experiment to confirm the pathway of apoptosis, it was confirmed that the Caspase activity increased as the concentration of eribulin increased and the mitochondrial membrane potential decreased. As a result of checking the cell cycle, it was confirmed that the G2/M phase was stopped in eribulin, and the expression of the related protein was also regulated by eribulin. In addition, when the level of expression of several surface antigens, which are markers related to the differentiation and maturation of blood cells, was confirmed, the expression increased as the concentration of eribulin increased.
Conclusion : It has been confirmed that eribulin induces the differentiation and maturation of immature cells in acute myeloid leukemia cells, inhibits cell proliferation, and finally leads to apoptosis, so it may be used as a new therapeutic agent.
- Author(s)
- 김도경
- Issued Date
- 2021
- Awarded Date
- 2021-08
- Type
- Dissertation
- Keyword
- Acute myeloid leukemia; Eribulin; Cell death; Differentiation
- URI
- https://oak.ulsan.ac.kr/handle/2021.oak/5775
http://ulsan.dcollection.net/common/orgView/200000506807
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