[보고서]세포괴사를 이용한 암 내성 세포 극복 전략

김태형

세포괴사를 이용한 암 내성 세포 극복 전략 원문보기

보고서 정보
주관연구기관	조선대학교 Chosun University
연구책임자	김태형
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2017-12
과제시작연도	2016
주관부처	보건복지부 [Ministry of Health & Welfare(MW)(MW)
등록번호	TRKO201900001752
과제고유번호	1465021860
사업명	질환극복기술개발
DB 구축일자	2019-06-01
키워드	세포괴사.세포사멸.암 내성 세포.엠티디.세포사.Necrosis.Apoptosis.Resistant cancer cells.MTD.Cell death.

초록

- 세포사멸 내성 세포 확립
- 세포괴사 유도 펩타이드 R8:MTD에 의한 세포사멸 내성 세포의 세포괴사 유도
- ErbB2 표적 세포괴사 유도 펩타이드 개발 (특허 출원)
- MTD 유사 도메인 B1MLM 발견 (특허 출원)

(출처 : 요약서 3p)

Abstract ▼

□ Purpose&Contents
Final goal: The ultimate goal is to develop a strategy to overcome anticancer drug resistance using cytotoxic peptide-based anticancer drugs.
Currently, the cancer treatment methods used in clinical practice are 1) surgery,2) chemotherapy, 3) radiation therapy, 4) hematopoietic stem cell transplantation therapy, and 5) immunotherapy. In particular, after treatment with chemotherapy, many cases of recurrence of cancer and multidrug - resistant cancer show resistance to other chemotherapeutic agents. These recurrent cancers are very difficult to treat, and the treatment itself is often insignificant. Therefore, it is urgent to develop strategies to overcome cancer-resistant cancer.
This project aims to establish a strategy to overcome the anticancer drug resistance by using a Necrosis-inducing substance which is a method different from the currently used anticancer drug.

□ Results
The differences between the conventional drug-resistance overcoming strategy and the method pursued by the present invention are 1) the difference of cell death induction modes, namely, necrosis rather than apoptosis, 2) a substance that uses a peptide rather than a chemical substance, and induces cell necrosis strongly and rapidly.
Most of the existing anticancer drugs cause apoptosis, and recurring drug-resistant cells have the multidrug-resistant characteristics due to the resistance to cell death signaling pathway; another words, cell death cannot be occured even when several other anticancer drugs are used. The existing approach to overcome cancer tolerance using other inducers of cell death is not appropriate as a strategy to overcome anticancer drug resistance.
This research was carried out in three stages. First, cancer cells resistant to anticancer drugs like doxorubicin and afatinib were established. Secondly, it was confirmed that these drug-resistant cells underwent necrosis by R8:MTD, a necrosis-inducing substance. Third, it was confirmed that cancer tissues bearing the drug-resistant cell were killed by cell necrosis in animal models. In addition, we have developed a novel necrosis-inducing peptide targeting ErbB2, and have demonstrated that it can kill many cancer cells expressing ErbB2. Also, we found a domain B1MLM similar to MTD and confirmed that this domain caused necrosis.
The ErbB2 target peptide and the B1MLM domain are applying for patents. The results of this project are in the process for publication and patents.

□ Expected Contribution
1. Technical aspects
1.1 Proof of anticancer drugs using cell necrosis rather than apoptosis
1.2 Development of ErbB2 Target Necrosis-Inducing Peptide – Paper Publication and Patent Application
1.3 Provide a new alternative to cancer therapy in anticancer drugs.

2. Economic-Social aspects
2.1 Obtaining intellectual property rights for ErbB2 target peptide
2.2 MTD-like domain B1MLM securing intellectual property rights
2.3 Accumulation of cell necrotic anticancer technology

(출처 : SUMMARY 5p)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
국문 요약문 ... 4
SUMMARY ... 5
목차 ... 6
1. 연구개발과제의 개요 ... 7
1-1. 연구개발 목적 ... 7
1-2. 연구개발의 필요성 ... 7
1-3. 연구개발 범위 ... 9
2. 국내외 기술개발 현황 ... 14
3. 연구수행 내용 및 결과 ... 17
1. 화학항암제 저항 세포주 확립 ... 17
2. EGFR 표적 항암제 저항 세포주 확립 ... 20
3. 화학 항암제에 대한 암 내성 극복전략 ... 25
4. EGFR 표적 항암제에 대한 암 내성 극복전략 ... 31
5. 동물 모델에서 화학 항암제에 대한 암 내성 극복전략 연구결과 ... 35
6. EGFR 표적 세포 괴사 펩타이드를 이용한 항암치료 연구결과 ... 37
7. 동물모델에서 ErbB2 표적 세포 괴사 펩타이드를 이용한 항암치료 연구결과 ... 43
8. MTD 유사 도메인 BNIP1 MTD-Like Motif (B1MLM) 발견 ... 46
4. 목표달성도 및 관련분야 기여도 ... 52
4-1. 목표달성도 ... 52
4-2. 관련분야 기여도 ... 52
5. 연구결과의 활용계획 ... 53
6. 연구과정에서 수집한 해외과학기술정보 ... 54
7. 연구개발성과의 보안등급 ... 54
8. 국가과학기술종합정보시스템에 등록한 연구시설·장비 현황 ... 54
9. 연구개발과제 수행에 따른 연구실 등의 안전조치 이행실적 ... 54
10. 연구개발과제의 대표적 연구실적 ... 55
11. 기타사항 ... 56
12. 참고문헌 ... 58
별첨: 실적 증빙자료 ... 59
별첨: 기타 첨부서류 ... 65
끝페이지 ... 75

표/그림 (34)

표 B16F10 parental 세포와 B16F10 5’FU-Resistant 세포에 5’FU (왼쪽 패널), MTD 펩타이드 (오른쪽 패널)를 처리하여 세포의 생존율을 조사하였다
표 CT26 parental 세포와 CT26 5‘FU–Resistant 세포에 5’FU (왼쪽 패널), MTD 펩타이드 (오른쪽 패널)를 처리하여 세포의 생존율을 조사하였다
표 MCF7 parental 세포와 MCF7 5‘FU–Resistant 세포에 5’FU (왼쪽 패널), MTD 펩타이드 (오른쪽 패널)를 처리하여 세포의 생존율을 조사하였다
표 HeLa parental 세포와 HeLa Apatinib–Resistant 세포에 Apatinib를 처리하여 Caspase-3, -9, Mcl-1 등의 단백질의 변화를 조사하였다
표 HeLa parental 세포와 HeLa Apatinib–Resistant 세포에 Apatinib (왼쪽 패널), MTD 펩타이드 (오른쪽 패널)를 처리하여 세포의 생존율을 조사하였다
표 HeLa parental 세포와 HeLa Apatinib–Resistant 세포에 Apatinib (왼쪽 패널), MTD 펩타이드 (오른쪽 패널)를 처리하여 세포의 생존율을 조사하였다
표 (A) Parental and Dox-Res CT26 cells were treated in triplicate with indicated concentrations of doxorubicin for 48 hours, and viability was measured using an XTT assay; *** p <0.001 and ** p <0.01 compared to no treatment. (B) Bright field images (630 × magnification) of cells were taken using white-light microscopy
표 Cell lysates were prepared from parental and Dox-ResCT26 cells treated with 15 uM doxorubicin for 48 hours. Immunoblot analysis was performed using anti-caspase-3, anti-caspase-8, anti-caspase-9, anti-MCL-1, anti-BAD, anti-BAX, and anti-actin antibodies
표 Activities of caspase-3 and –8 were measured using colorimetric assay kits (n = 3). Samples were prepared from parental and Dox-Res CT26 cells treated with 15 mM doxorubicin for 48 hours; ** p <0.01 and *** p <0.001. All data are mean ± SD
표 Parental and the Dox-Res CT26 cells were treated with (A) carboplatin and (B) 5ʹFU (B) for 24 hours. Cell viability was determined using an XTT assay. All data are presented as the mean ± SD (n=3). **p<0.01, *** P<0.001 compared to no treatment
표 Parental and Dox-Res CT26 cells were treated with R8:MTD peptide for 1 hour and cell viability was measured using an XTT assay; **NS, non-significant
표 Parental and Dox-Res CT26 cells were treated with R8:MTD peptide (10 uM) for 30 minutes in serum-free medium and the conditioned medium was collected for protein precipitation using TCA. TCA-precipitants were analyzed using immunoblot using anti-HMGB1antibody
표 Parental and Dox-Res CT26 cells were treated with R8:MTD peptide (10 uM) for 1 hour. Bright images (630× magnification) were taken, and the fluorescenceimages (630× magnification) were taken after dead cell staining using Green Dead Cell Stain kit
표 Parental and Dox-Res CT26 cells were treated with R8:MTD peptide (10 uM) for 1 hour in the presence orabsence of zVAD-fmk (15 mM). Cell lysates were analyzed using immunoblot using anti-caspase-3, -8, and –9, as well as anti-actin antibodies
표 Parental and Dox-Res CT26 cells were cultured on a glass slide. After cell confluency had reached appropriate cell numbers, cells were loaded with Fluo-4-AM at 5 μM for 10 minutes, followed by washing with calcium-free HBSS buffer and treatment with 5 mM R8:MTD peptide. Time-lapse images were obtained at 488 nm excitation wavelength using Argon laser scanning confocal microscope at 5-second intervals for 10 min to visualize Fluo-4-AM. (A) Relative fluorescence intensity (F1/F0) of ROI were charted and (B) representative fluorescence images are presented
표 Dose depentent cell viability curve for HeLa parental and Afa-Res HeLa cell lines. Cells were cultured in 96-well plate, on the second day, cells were treated with afatinib or DMSO for 24 h. XTT assay were perfomed to measure the cell viability
표 Cell lysates were prepared from parental and Afa-Res HeLa cells treated with 16 uM Afatinib for 48 hours. Immunoblot analysis was performed using anti-caspase-3, anti-caspase-9, anti-MCL-1, and anti-GAPDH antibodies
표 Parental and the Afa-Res HeLa cells were treated with (A) Lapatinib and (B) TRAIL for 24 hours. Cell viability was determined using an XTT assay. All data are presented as the mean ±SD (n=3). **p<0.01, *** P<0.001 compared to no treatment
표 Parental and Afa-Res HeLa cells were treated with R8:MTD peptide for 1 hour and cell viability was measured using an XTT assay; **NS, non-significant
표 Parental and Afa-Res HeLa cells were treated with R8:MTD peptide (10 uM) for 2 hours. Cell lysates were analyzed using immuno-blot using anti-caspase-3, anti-actin antibodies
표 Intratumorla injection of Doxorubicin and R8:MTD Tumors were established in 6 week-old BALB/c mice by injecting 2.5 × 105 parental or Dox-Res CT26 cells into subcutaneous layer of mice backs. Then, 100μl each of R8:MTD(100 μM), Doxorubicin (20 μM), and saline were separately injected directly into tumor tissues once daily for 3 consecutive days. Then, 24 hours after final R8:MTD, Doxorubicin, and saline injections, tumor tissues were processed for hematoxylin and eosin staining after 4% formaldehyde fixation and paraffin embedding Images of whole tumor tissues (magnification 40x) were taken using a light microscope and phto-merged using Adobe Photoshop CSA5
표 A431 cells(2x10^4 cells/well) were treated with KCCYSL alone, MTD alone, and KCCYSL: MTD (0~80uM) for 2hours, and cell viability was measured by MTS assay. All data are presented as the mean ± SD (n=3). **p<0.01, *** P<0.001 compared to no treatment
표 Table was described that KCCYSL sequence modified by replacement or indel mutation. additionally EC50(concentration to induce 50% inhibition) was calculated by based on cell viability(A).The patterns of cell killing effects in FaDu and MCF7. All peptides was given to cells(2x10^4 cells/well) each concentration of 0,10,20,40,80 uM, and Cell viability was described by color density(B)
표 Killing activities of KCCYSL:MTD, KWCYS:MTD, and KWSY;MTD in ErbB2-high-expressing or ErbB2-low-expressing cells. (A) Indicated cell lines were treated with KCCYSL:MTD, KWCYS:MTD, or KWSY:MTD, and color density presents relative cell viability. (B)The IC50 density for peptides in indicated cells is presented as color density; black indicates no killing activity (Top panel).Lysates from cell lines indicated on toppanel were subjected to westernblot analysis using anti-ErbB2 and anti-GAPDHantibodies(bottom panel)
표 Blocking of KWSY:MTD –induced cell death by anti-ErbB2 antibody 20uM of KWSY:MTD was added to FaDu(2x10^4 cells/well) after pre-treatment of ErbB2 antibody(1ug/ml) or control antibody(1ug/ml) for 1hour. After 1hour, cell death was detected by MTS assay (A), and all images were taken using inverted optical microscopy(B)
표 KWSY:MTD induces necrosis. 4T1 cells were treated with the indicated peptides (20 μM) for 2 hours. PI positive cells were counted (A), and PI fluorescence images were obtained with Olympus fluorescence microcopy (B). HMGB1 in conditioned media from cells treated with peptides was detected by western blotting using an anti-HMGB1 antibody (1:1000) after precipitation with TCA and acetone (C)
표 Isolated mitochondria from mouse livers were treated with the indicated peptides (20μM), calcium ions (200 μM), or CsA (20 μM), and optical densities at 540 nm were monitored every 10 minutes
표 CT26 and 4T1 cells were inoculated into backs of BalB/C mice. Tumors were formed after 12days. KWSY:MTD(0.2 mM, 200μl) or PBS was I.v. injected into the tail vein of (A)CT26 tumorigenic mice(n=10) or (B) 4T1 tumorigenic mice(n=10) every 2days interval total 4times(as indicated arrows)
표 FaDu cells were inoculated into backs of SCID mice. Tumors wer formed after 10days. (A) KWSY:MTD(0.4 mM, 200μl) or PBS were I.v. injected into the tail vain of FADU tumorigenic mice(n=6) every 2days interval total 4times(as indicadted arrows). (B) Isolated tumors of FaDu tumorigenic mice were stained with H&E, and images taken(magnification 400x) and merged using Adobe Photoshop Cs5
표 Isolated livers and kidney from FaDu tumorigenic mice were stained with H&E, and images taken(magnification 400x)
표 Induction of cell death by R8:B1MLM.A, The amino acid sequence of B1MLM has six identical residues foundin the MTD of NOXA. CPP (cell penetrating peptide 8-arginine) was fused to the N-terminalof B1MLM. B, The cell-killingactivity of R8:B1MLM in HeLa cells was measured by MTS assay. C, Morphological changes of HeLa cellstreated with 10 μM B1MLM (DIC, for10 minutes)
표 Intracellular Ca2+alteration by R8:B1MLM. A–B,Intracellular Ca2+alteration in HeLa cells was measured with Fluo4 after treatment with R8:B1MLMin HBSS buffer (A) or in HBSS bufferwithout Ca2+ (B). C–D, Morphological changes and Fluo4fluorescence of HeLa cells treated with B1MLM in HBSS buffer (C) or in HBSS buffer without Ca2+(D)
표 Opening of the mitochondrial permeability transition pore by R8:B1MLM.A, HeLa cells were pre-loaded withCalcein AM and Co2+ for 20 minutes and MitoTracker for 2 minutes. Cellswere treated with 10 μM R8:B1MLM and time-lapse images were taken for 10 minutes. Green fluorescentsignal (Calcein AM) indicates the mitochondrial calcium ion. Red fluorescentsignal (MitoTracker Red) shows mitochondrial morphological changes. B, Time-lapse images of non-treated HeLa cells were taken for 10 minutes
표 ROSgeneration by R8:B1MLM.A, Cytosolic ROS generation in HeLa cell was measured with DCFafter treatment with R8:B1MLM. B,Mitochondrial ROS generation in HeLa cells was measured with MitoSOX aftertreatment with R8:B1MLM. C, Morphologicalchanges and DCF fluorescence of HeLa cells treated with R8:B1MLM. D, Morphological changes and MitoSOXfluorescence of HeLa cells treated with R8:B1MLM

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총연구비 (DetailSeriesProject) :	-
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과제수행기간(LeadAgency) :	-
연구목표(Goal) :	-
연구내용(Abstract) :	-
기대효과(Effect) :	-

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