[보고서]곤충 단백질로부터 비소세포폐암에 대한 표적 항암 펩타이드 개발

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곤충 단백질로부터 비소세포폐암에 대한 표적 항암 펩타이드 개발
Development of targeted anticancer peptides for non-small cell lung cancer from insect proteins 원문보기

보고서 정보
주관연구기관	계명대학교 KeiMyung University
연구책임자	이승욱
참여연구자	유미희 , 정연섭 , 이효선 , 류희정 , 박지현
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2019-02
과제시작연도	2018
주관부처	농촌진흥청 Rural Development Administration(RDA)
등록번호	TRKO201900015996
과제고유번호	1395053915
사업명	농업첨단핵심기술개발사업(R&D)
DB 구축일자	2019-10-19
키워드	식용곤충.단백가수분해물.펩타이드.항암.비소세포폐암.Edible insect.Protein hydrolysate.Peptide.Anti-cancer.Non-small cell lung cancer.
DOI	https://doi.org/10.23000/TRKO201900015996

초록 ▼

3종의 식약용 곤충(갈색거저리 유충, 흰점박이꽃무지 유충, 장수풍뎅이 유충)으로부터 단백가수분해물 제조를 위한 최적의 효소제로 alcalase, flavourzyme 및 neutrase 선정하였으며, 각 곤충별과 효소제별로 단백가수분해물 제조 완료
단백가수분해물을 대상으로 NR4A1에 대한 저해 활성 탐색 결과, 갈색거저리 유충 flavourzyme 단백가수분해물을 선발함
선발된 단백가수분해물로부터 분리한 MPF8(EEVDDPK)를 고활성 저해제로 최종 선발함
MPF8의 NR4A1 저해기전(AB 도메인을 통한 NR4

Abstract ▼

□ Purpose&Contents
The aim of this study was to identify the peptides that inhibit the nuclear receptor NR4A1, a novel target for the treatment of non-small cell lung cancer, from the edible insects currently being farmed in Korea, to provide basic data on the development of anti-cancer peptides targeting NR4A1, and to contribute to the activation of related research. In addition, by developing new high-value biomaterials from insects, we aimed to activate the insect industry in Korea. In this study, therefore, we carried out multiple experiments as shown below.
• Selection of optimal enzymes and preparation of protein hydrolysates from various edible insects
• Screening of orphan nuclear receptor NR4A1 inhibitors from protein hydrolysates
• Purification and amino acid sequence of NR4A1 inhibitory peptides from a selected hydrolysate
• Selection of the most active NR4A1 inhibitory peptide and investigation of its mechanisms of NR4A1 inhibition
• Examination of anti-cancer activity of the peptide in multiple cancer cells overexpressing NR4A1
• Examination of NR4A1-mediated anti-cancer mechanisms of the peptide in non-small cell lung cancer (NSCLC) cells

□ Results
• Selection of three optimal enzymes (alcalase, flavourzyme, and neutrase) and production of protein hydrolyastes using the enzymes from three different edible insects (Protaetia brevitarsis larvae, Allomyrina dichotoma larvae, and Tenebrio molitor larvae)
• Selection of the flavourzyme hydrolysate from Tenebrio molitor larvae as the most active NR4A1 inhibitor
• Final selection of a potent NR4A1 inhibtory peptide, MPF8(EEVDDPK), from the flavourzyme hydrolysate
• Understanding of the molecular mechanisms of NR4A1 inhibition by MPF8 (regulation of transcriptional activity of nuclear NR4A1 via its AB domain) and confirmation of anti-proliferative effects of MPF8 on 9 human cancer cell lines from 6 different origins
• Confirmation of NR4A1-mediated mechanisms of apoptosis induced by MPF8 in human NSCLC cells
• 2 Patents (registration), 6 Patents (pending), 4 Papers published (2 SCI and 2 KCI)

□ Expected Contribution
• Providing a new treatment option for human NSCLC and critical preliminary data that would help to developNR4A1-targeting anti-cancer peptides
• Providing valuable information for potential medical application of MPF8 itself or in combination with anticancer drugs to treat NSCLC and other types of cancers overexpressing NR4A1
• NR4A1-inhibiting peptides may be used to develop new drugs for metabolic diseases since NR4A1 is known to play a critical role in hepatic gluconeogenesis
• Transfer of the patents and technology to nutraceutical companies is now ongoing
• It is expected to maximize the use of insect resources through the exploration of these new biological activities of edible insects and to activate domestic insect industry and increase income of insect farmers

(출처 : SUMMARY 5p)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
국 문 요 약 문 ... 4
Summary ... 5
목차 ... 7
제 1 장 연구 개발 과제의 개요 ... 8
제1절 연구 개발 목적 ... 8
제2절 연구 개발의 필요성 ... 8
제3절 연구 개발 범위 ... 10
제 2 장 연구 수행 내용 및 결과 ... 12
제1절 : 1차년도 연구 수행 내용 및 결과 ... 12
제2절 : 2차년도 연구 수행 내용 및 결과 ... 19
제3절 : 3차년도 연구 수행 내용 및 결과 ... 24
제4절 : 기타 연구 수행 내용 및 결과 ... 30
제 3 장 목표달성도 및 관련분야 기여도 ... 34
제1절 : 목표대비 달성도 ... 34
제2절 : 정량적 성과(논문게재, 특허출원, 기타)를 기술 ... 35
제 4 장 연구 결과의 활용 계획 ... 36
제1절 : 연구개발 결과의 활용방안 ... 36
제2절 : 추가적인 논문게재 및 산업재산권 활용 방안 ... 36
제 5 장 연구개발과제의 대표적 연구실적 ... 37
제 6 장 참고문헌 ... 39
끝페이지 ... 41

표/그림 (27)

표 Effects of five different proteases on SDS-PAGE profiles of larvae protein hydrolysates. NE, no enzyme; A, alcalase; B, bromelain; F, flavourzyme; N, neutrase; P,papain
표 Degree of hydrolysis of Tenebrio molitor larvae by five different proteases at 55℃. Each value is mean±SE (n≥3)
표 Degree of hydrolysis of P rotaetia brevitarsis larvae by five different proteases at 55℃. Each value is mean±SE (n≥3)
표 Degree of hydrolysis of Allomyrina dichotoma larvae by five different proteases at 55℃. Each value is mean±SE (n≥3)
표 Yields of protein hydrolysates (< 3 kDa) prepared from dried Tenebrio molitor larvae, P rotaetia brevitarsis larvae, and Allomyrina dichotoma larvae
표 Protective effects of three different protein hydrolysates from Tenebrio molitor larvae against H2O2-induced cytotoxicity in AML-12 cells. After 24 h treatment with different concentrations of each hydrolysate, the media were replaced with new media containing H2O2 (7 mM) and the cells were incubated for another 5 hours. Cell viability was then measured by an MTT assay. Each value is mean±SE (n≥3) and different superscripts are significantly different at P <0.05
표 Scavenging effects of trolox and the alcalase protein hydrolysates from Tenebrio molitor larvae against DPPH radicals, ABTS radical, and H2O2
표 Inhibitory effects of trolox and the alcalase protein hydrolysates from Tenebrio molitor larvae on linoleic acid peroxidation. Each value is mean±SE (n≥3) and different superscripts at the same day are significantly different at P <0.05
표 Effects of different protein hydrolysates from Tenebrio molitor larvae or P rotaetia brevitarsis larvae on transcriptional activity of NR4A1. MiaPaCa-2 cells were cotransfected with NBRE-Luc (25 ng) and Flag-NR4A1 (12.5 ng) for 5 h, and treated with indicated concentrations of each protein hydrolysate for 18 h. Luciferase activity(relative β-galactosidase) was determined and the results are presented as means±SE. *P <0.05, **P <0.01, ***P <0.005, and #P <0.001 vs. buffer control. C, buffer control; A,alcalase; F, flavourzyme; N, neutrase
표 Effects of the flavourzyme hydrolysates from Tenebrio molitor larvae or the alcalase hydrolysates from P rotaetia brevitarsis larvae on cell proliferation in A549 human non-small cell lung cancer cells. Cells were treated with variouconcentrations of protein hydrolysates for 24 h, and cell viability was determined by an MTT assay. The results are presented as means±SE. *P <0.05, ***P <0.005, and #P <0.001vs. buffer control
표 Effects of the flavourzyme hydrolysates from Tenebrio molitor larvae or the alcalase hydrolysates from P rotaetia brevitarsis larvae on cell proliferation in H460 human non-small cell lung cancer cells. Cells were treated with various concentrations of protein hydrolysates for 24 h, and cell viability was determined by an MTT assay. The results are presented as means±SE. *P <0.05 and #P <0.001 vs. buffer control
표 Effects of the flavourzyme hydrolysates from Tenebrio molitor larvae (A) or the alcalase hydrolysates from P rotaetia brevitarsis larvae (B) on H460 cell morphology. Cells were treated with various concentrations of protein hydrolysates for 24h, and cells were photographed at visible light (x200)
표 Effect of mealworm protein hydrolysates with different molecular weight on transcriptional activity of NR4A1. MiaPaCa-2 cells were cotransfected with NBRE-Luc (25 ng) and Flag-NR4A1 (12.5 ng) for 5 h, and treated with indicated concentrations of each protein hydrolysate for 18 h. Luciferase activity (relative β-galactosidase) was determined and the results are presented as means±SE (n≥3).**P <0.01, ***P <0.005, and #P <0.001 vs. control
$Effect of mealworm protein hydrolysate fractions with different polarity on transcriptional activity of NR4A1 and cell viability of H460 human non-small cell lung cancer cells. A, MiaPaCa-2 cells were cotransfected with NBRE-Luc (25 ng) and Flag-NR4A1 (12.5 ng) for 5 h, and treated with indicated concentrations of each fraction for 18 h. Luciferase activity (relative β-galactosidase) was determined and the results are presented as means±SE (n≥3). B, H460 cells were treated with SFM0 for 24 h and cell viability was determined by an MTT assay. ***P <0.005, and #P <0.001 vs. control$ 표 Effect of mealworm protein hydrolysate fractions with different polarity on transcriptional activity of NR4A1 and cell viability of H460 human non-small cell lung cancer cells. A, MiaPaCa-2 cells were cotransfected with NBRE-Luc (25 ng) and Flag-NR4A1 (12.5 ng) for 5 h, and treated with indicated concentrations of each fraction for 18 h. Luciferase activity (relative β-galactosidase) was determined and the results are presented as means±SE (n≥3). B, H460 cells were treated with SFM0 for 24 h and cell viability was determined by an MTT assay. ***P <0.005, and #P <0.001 vs. control
$Reversed-phase HPLC chromatogram of the mealworm protein hydrolysate fractions with different polarity$ 표 Reversed-phase HPLC chromatogram of the mealworm protein hydrolysate fractions with different polarity
$Reversed-phase HPLC chromatogram showing peptide separation of SFM0 into four fractions and effect of the fractions on transcriptional activity of NR4A1. B and C, MiaPaCa-2 cells were cotransfected with NBRE-Luc (25 ng) and Flag-NR4A1 (12.5 ng) for 5 h, and treated with indicated concentrations of each fraction for 18 h. Luciferase activity (relative β-galactosidase) was determined. *P <0.05 and #P <0.001 vs. control$ 표 Reversed-phase HPLC chromatogram showing peptide separation of SFM0 into four fractions and effect of the fractions on transcriptional activity of NR4A1. B and C, MiaPaCa-2 cells were cotransfected with NBRE-Luc (25 ng) and Flag-NR4A1 (12.5 ng) for 5 h, and treated with indicated concentrations of each fraction for 18 h. Luciferase activity (relative β-galactosidase) was determined. *P <0.05 and #P <0.001 vs. control
표 Effect of synthesized mealworm peptides on transcriptional activity of NR4A1. A and B, MiaPaCa-2 cells were cotransfected with NBRE-Luc (25 ng) and Flag-NR4A1 (12.5 ng) for 5 h, and treated with indicated concentrations of each peptide for 18 h. Luciferase activity (relative β-galactosidase) was determined and the results are presented as means±SE (n≥3). Different superscripts (a-e or a-f) are significantly different at P <0.05 by Duncan’s multiple range test
표 Inhibitory mechanisms of MPF8 on NR4A1 transactivity. A and B, NBRE-Luc (25 ng) was cotransfected with 12.5 ng of each Flag-NR4A1-WT (wild type), Flag-NR4A1-AD (AD domain deletion mutant), Flag-NR4A1-CF (CF domain deletion mutant), or Flag-NR4A2 into MiaPaCa-2 cells for 5 h, and treated with indicated concentrations of each peptide for 18 h. Luciferase activity (relative β-galactosidase) was determined and the results are presented as means±SE (n≥3). ***P <0.005 vs. control. C, Subcellular localizaton of NR4A1. MiaPaCa-2 cells were treated with MPF8 or PMA for 6h, and endogenous NR4A1 was stained with anti-rabbit IgG conjugated to Alexa Fluor 488. Nuclei were counterstained with DAPI, and PMA was used as a positive control for inducing nuclear export of NR4A1
표 IC50 values for MPF8 against various cancer and normal cell lines
표 Effect of MPF8 on cell proliferation in various cancer and normal cell lines. Cells were treated with different concentrations of MPF8 for 24 h, and the cell viability was measured by the MTT assay. A and B, solid tumor cell lines. C, leukemia cell lines. D, normal cell lines. The results are presented as means±SE (n≥3). *P <0.05,**P <0.01, ***P <0.005, and #P <0.001 vs. control
표 MPF8 induces apoptosis in lung cancer cells. A, Cells were treated with different concentrations of MPF8 for 24 h, and then the cells were fixed and stained with DAPI. Fluorescence microscopy images (left, 400x) and percentage of apoptotic cells on total cell number (right). *P<0.05 and #P<0.001 vs. control. B, Cells were treated with different concentrations of MPF8 for 24 h, and whole-cell lysates were analysed by western blot analysis
표 Schematic diagram illustrating two known mechanisms of nuclear NR4A1-mediated apoptosis
표 Effects of MPF8 on ROS production, Sp1-dependent transactivation, and survivin expression in lung cancer cells. A, Cells were treated with different concentrations of MPF8 for 12 h and intracellular ROS level was determined using DCF-DA assay in fluorescence microscopy (100x). B and C, H460 cells were transfected with 25 ng of pGL3-(GC)3-Luc or pGL3-SVV(-269)-Luc for 5 h and then treated with MPF8 for 18 h. Luciferase activity (relative β-galactosidase) was determined. *P <0.05 and #P <0.001 vs. control. D, H460 cells were treated with different concentrations of MPF8 for 24 h, and whole-cell lysates were analysed by western blot analysis. GAPDH was used as a loading control
표 Protective effect of mealworm-alcalase hydrolysates with different molecular weight against H2O2-induced cytotoxicity in AML-12 cells. After 24 h treatment with different concentrations of each hydrolysate, the media were replaced with new media containing H2O2 (7 mM) and the cells were incubated for another 5 hours. Cell viability was then measured by an MTT assay. The results are presented as mean±SE (n≥3) and different superscripts are significantly different at P <0.05. *P <0.05, **P <0.01,***P <0.005, and #P <0.001 vs. H2O2 control
표 Effects of mealworm-alcalase hydrolysate (MW<1 kDa) on H2O2–induced ROS production and mRNA expression of antioxidant-related genes in AML-12 cells. A, Intracellular ROS production. After 24 h treatment with different concentrations of the hydrolysate, the media were replaced with new media containing H2O2 (7 mM) and the cells were incubated for another 1 h. B, Cells were treated with the hydrolysate for 18 h, and mRNA expression levels were determined by qPCR. Tbp was used as an internal control and the results are presented as mean±SE (n≥3). *P <0.05, **P <0.01, ***P <0.005, and #P <0.001 vs. control
표 Effects of mealworm-alcalase hydrolysate (MW<1 kDa) on total GSH content, catalase activity, and HO-1 protein expression in AML-12 cells. A and B, Cells were treated with the hydrolysate for 24 h, and total GSH content and catalase activity were measured. C, Whole cell lysates were analyzed by western blot analysis, and β-actin was used as a loading control. The results are presented as mean±SE (n≥3). *P <0.05 vs. control
표 Effects of mealworm-alcalase hydrolysate (MW<1 kDa) on protein expression and activation of Nrf2 in AML-12 cells. A, Cells were treated with the hydrolysate for 4 h, and whole cell lysates were analyzed by western blot analysis, and β -actin was used as a loading control. B, Subcellular localization of Nrf2. Cells were treated with the hydrolysate (5 mg/mL) for 4 h, and endogenous Nrf2 protein was detected by indirect immunofluorescence staining with anti-Nrf2 antibody. Sulforaphane (10 μM), a known Nrf2 activator, was used as a positive control

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곤충 단백질로부터 비소세포폐암에 대한 표적 항암 펩타이드 개발
Development of targeted anticancer peptides for non-small cell lung cancer from insect proteins 원문보기