[보고서]암세포 및 면역세포의 방사선 반응 조절

김정애

암세포 및 면역세포의 방사선 반응 조절
Regulation of radiation response in cancer cells and immune cells 원문보기

보고서 정보
주관연구기관	영남대학교 YeungNam University
연구책임자	김정애
참여연구자	박진우 , 이영섭
보고서유형	1단계보고서
발행국가	대한민국
언어	한국어
발행년월	2011-03
과제시작연도	2010
주관부처	교육과학기술부
사업 관리 기관	한국연구재단
등록번호	TRKO201200003172
과제고유번호	1345120814
사업명	원자력연구기반확충사업
DB 구축일자	2013-05-20
키워드	방사선 암치료.방사선민감화제.산화환원.cancer radiotherapy.radiosensitizer.STAT3.EGFR.DR.redox status.

초록 ▼

방사선에 의한 암 치료 (cancer radiotherapy) 시 나타나는 가장 큰 부작용은 지속적인 적용에 의한 정상 세포에 대한 손상임. 그러므로 방사선조사의 dose를 최대한 줄이고 정상세포의 손상을 최소화하는 효과적인 치료를 위해 정상세포의 보호를 위한 radioprotector의 발굴과 암세포의 방사선에 대한 민감화 (radiosensitization)가 유용한 방법임.
방사선조사는 활성산소종 (ROS)을 생성하므로 ROS에 의한 암 세포의 cell death가 항암 효과의 기작으로 추정되며, 방사선조사에 의한 면역세포와 암세포의 반응을 신호전달계의 변화와 proteome의 변화 측면에서 규명하고 방사선방어제와 민감화제의 처리에 의한 암 세포와 정상세포 간의 생존율, redox status의 변화를 관찰하고 이러한 산화환원상태의 변화가 세포사멸을 유발하는 과정을 다양한 세포사멸 markers로 검토하고, 아울러 세포사멸에 수반되는 미토콘드리아의 손상을 mitochondria permeability transition (MPT)와 ROS level의 변화로 검토하고 Bcl-2의 과 발현과 Bax의 발현저해 세포조건에서 apoptosis의 기작을 규명함으로써 세포사멸의 경로를 파악하고자 하였음. 또한 NFκB pathway, MAPK 신호전달계, Akt, p53의 연계성을 검토하여 방사선 방어와 민감화의 기작을 규명하고자 하였음.

Abstract ▼

The use of ionizing radiation (IR) is essential for the treatment of many human cancers. However, radioresistance markedly impairs the efficacy of tumor radiotherapy. In a variety of cells IR has been shown to enhance the production of reactive oxygen species (ROS), which are determinant components in the induction of apoptosis. In order to improve its therapeutic ratio, there has been much interest in augmenting the effect of radiation in tumors by combining it with radiosensitizers. In the current study, the radiosensitizing effects of xanthohumol, ursolic acid, resveratrol and piperine on cancer cells were evaluated. Cancer cell lines treated with these natural products exhibited significantly augmented IR-induced apoptosis and loss of mitochondrial membrane potential presumably through enhancing the generation of ROS. The application of natural products as a sensitizer for IR-induced apoptotic cell death offers a promising therapeutic approach for the treatment of cancer. The present study showed that cancer cells which have drug resistance (adriamycin-resistant MCF-7 cells) are also radioresistant, and p53-mutant HT29 cells are more resistant to the radiation treatment than p53-wild type HCT116 cells. Treatment of these cancer cells with xanthohumol recovered radiosensitivity. One of the important molecules mediating this response was signal transducer and activator of transcription-3 (STAT-3). These results provide the foundation for effective screening of radiosensitizer by identifying molecules to control STAT-3.

목차 Contents

표지 ... 1
제출문 ... 2
보고서 요약서 ... 3
요약문 ... 4
SUMMARY ... 6
목차 ... 8
제 1 장 연구개발과제의 개요 ... 9
제 2 장 국내외 기술개발 현황 ... 11
제 3 장 연구개발수행 내용 및 결과 ... 14
1. 연구내용 ... 14
2. 연구방법 ... 14
3. 연구결과 ... 16
제 4 장 목표달성도 및 관련분야에의 기여도 ... 42
1. 기술적 측면 ... 42
2. 경제·산업적 측면 ... 43
3. 사회·문화적 측면 ... 43
제 5 장 연구개발결과의 활용계획 ... 44
제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 45
제 7 장 참고문헌 ... 46
연구성과(연구마루 입력성과) ... 49
자체평가의견서 ... 54

표/그림 (17)

표 Radio-resistant property of doxorubicin-resistant MCF-7 breast cancer cells (MCF-7/ADR).
표 Screening of radiation-sensitizing agents in MCF-7 and MCF-7/ADR human breast cancer cells.
표 Xanthohumol induces radiation-induced apoptosis of MCF-7 and MCF-7/ADR cells. The degree of apoptosis is represented as the DNA content measured by flow cytometry. Cells were pre-treated with or without xanthohumol (10 μM) for 24 h. Then cells were irradiated by γ-ray and incubated 48 h in fresh medium. Then apoptosis was measured by flow cytometric analysis. The region to the left of the sub-G1 peak, designated M1, was defined as cell undergoing apoptosis-associated DNA degradation. In bar graph, the data represent the mean values of three independent experiments with bars indicating SEM. *P<0.05 compared to vehicle-treated control group. #P<0.05 compared to 10Gy-irradiated group.
표 ROS generated by xanthohumol and radiation is involved in cell viability decrease of MCF-7 and MCF-7/ADR cells. (A) Cells were stimulated with XN alone, γ-radiation alone or combination of both XN and γ-radiation. After 48 h, ROS generation (fluorescence) was measured. (B) Cells were preincubated with 10 μM of N-Acetylcystein (NAC) for 90 min then treated with xanthohumol alone (10 μM), γ-radiation alone (10 Gy) or combination of xanthohumol and γ-radiation. After 48 h, cell viability was measured by MTT assay. Data are expressed as the mean S.E.M. of three independent experiments. *P < 0.05, compared to vehicle-treated control group, #P<0.05, compared to radiation alone group and &P<0.05 compared to XN+10Gy group.
표 Xanthohumol and γ-radiation synergistically downregulate STAT3 expression and its phosphorylation only in MCF-7/ADR cells.
표 XN enhances γ-radiation-induced downregulation of Bcl-2 and IAP family members. The cells were treated with XN alone (10 μM), γ-radiation alone (10 Gy) or combination of XN and γ-radiation.
표 Xanthohumol enhances radiation-induced DR4 and DR5 expression in MCF-7 and MCF-7/ADR cell
표 Xanthohumol and γ-radiation synergistically downregulate EGFR expression in MCF-7/ADR cells. Cells were treated with γ-radiation alone (10 Gy) or combination of both XN and γ-radiation. After 1 h, whole cell lysates were prepared and western blot analysis was performed using EGFR antibodies. The data represent a typical experiment conducted three times with similar results.
표 Cell viability and morphological change of DU145 cells exposed to IR. (A) After DU145 cells were exposed to 5 Gy of γ-irradiation and 30 μM ursolic acid (UA) for 24 h, viability of the cells were determined by trypan blue dye exclusion assay. Each value represents the mean + S.D. from three independent experiments. *p<0.01 versus unirrated cells exposed to ursolic acid. (B) DU145 cells were treated with 5 Gy γ-irradiation and 30 μM ursolic acid for 24 h. Morphologic changes were examined by Hoechst 33258 staining and observed by fluorescence microscope.
표 IR-induced apoptosis in DU145 cells. (A) Cell cycle profiles with cellular DNA contents were examined by flow cytometry. The number of each panel refers to the percentage of apoptotic cells. (B) Agarose gel electrophoresis of nuclear DNA fragments of DU145 cells exposed to IR (5 Gy) and 30 μM ursolic acid (UA) for 24 h (left). DNA fragmentation was determined using diphenylamine assay. Each value represents the mean + S.D. from three independent experiments. *p<0.01 versus control cells exposed to ursolic acid (right). (C) Activation of caspase-3 in DU145 cells exposed to 5 Gy of γ-irradiation and 30 μM ursolic acid for 24 h. DU145 cells were lysed and centrifuged. The supernatant was then added to Ac-DEVD-pNA and subjected to caspase colorimetric activity. Protease activity of caspase-3 was calculated by monitoring the absorbance at 405 nm. Each value represents the mean + S.D. from three independent experiments. *p<0.01 versus control cells exposed to ursolic acid. (D) Cell extracts were subjected to western blot analysis with antibodies against cleaved PARP, caspase 8, Bid and lamin B. β-Actin was run as an internal control.
표 Western blot analysis of apoptotic marker proteins in CT26 cells exposed to 15 Gy of γ-irradiation and 15 μM ursolic acid (UA), 20 μM resveratrol (RE) or 40 μM piperine (PI) for 24 h. β-Actin was run as an internal control.
표 Western blot analysis of apoptotic marker proteins in B16F10 cells exposed to 5 Gy of γ-irradiation and 10 μM ursolic acid (UA), 10 μM resveratrol (RE), or 30 μM piperine (PI) for 24 h. β-Actin was run as an internal control.
표 MMP of cancer cells was measured by the incorporation of rhodamine123 dye into mitochondria. The average of fluorescence intensity was calculated as described (Sundaresan et al., 1995). Open and shaded bars represent cancer cells unexposed and exposed to IR, respectively. Each value represents the mean + S.D. from three independent experiments. *p<0.01 versus IR irradiated cells untreated with natural products.
표 The levels of intracellular ROS in cancer cells. (A) DCF fluorescence was measured in cancer cells exposed to IR and natural products by a fluorescence microscope. (B) The average of fluorescence intensity was calculated as described (Sundaresan et al., 1995). Open and shaded bars represent cancer cells unexposed and exposed to IR, respectively. Each value represents the mean + S.D. from three independent experiments. *p<0.01 versus IR irradiated cells untreated with natural products.
표 Cell viability of HCT15 cells exposed to 131I radiation. After HCT15 cells were exposed to various doses or times of 131I, viability of the cells were determined by MTT assay. Each value represents the mean from three independent experiments.
표 Effects of radiosensitizer on the radiation-induced cell death. HCT15 cells were cultured for 24 hr in the absence (control) or presence of 20 μM curcumin (A), 15 μM ursolic acid (B), 15 μM resveratol (C), or 20 μM piperine (D). After cells were exposed to 300 μCi of 131I, viability of the cells were determined by MTT assay.
표 Effects of piperine on the radiation-induced cell death. HCT15 cells were cultured for in the presence of 40 μM (A) or 60 μM piperine (B). After cells were exposed to 500 μCi of 131I, viability of the cells were determined by MTT assay.

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연합인증

암세포 및 면역세포의 방사선 반응 조절
Regulation of radiation response in cancer cells and immune cells 원문보기