서울대에서 연구는 종양 Microenvironment 글로벌 핵심 연구 센터 (GCRC) 그랜트, [허가 번호 2011-0030001];에 대 한 한국 개 국립 연구 재단 (NRF)에 의해 지원 됩니다. 서울 국가 대학 연구 보조금 및 두뇌 한국 (BK21) 프로그램 플러스.

1. 식민지 대형 분석 실험
<ol><li>20000 셀/cm2 RPMI1640의 15 ml에서의 농도에서 문화 A549 세포 세포 배양 10 보충 %FBS (태아 둔감 한 혈 청)과 37 ° C, 5% CO2CO2 배양 기에서 75 c m2 플라스 크에 1% 항생제.</li>
	<li>실험의 날에는 hemocytometer를 사용 하 여 셀 농도 결정 합니다. 1.5 mL microcentrifuge 튜브에서 7 분 400 x g 에서 원심 분리 하 여 50, 000 셀을 수집 하 고 다시 1 x 살 균 PBS (인산 염 버퍼) 1.5 mL 튜브에의 100 µ L에서 세포를 일시 중단.</li>
	<li>multipipette와 함께 15 mL 튜브에서 반 고체 스 기반 매체 (MCBM)의 1.1 mL을 분배. 각 조건에 대 한 튜브를 준비 합니다.</li>
	<li>P200 micropipette를 사용 하 여 MCBM의 1.1 ml FBS의 110 µ L를 추가 합니다.</li>
	<li>1, 000 셀/mL에서 씨앗 셀입니다. 재고 솔루션 (50000 셀 1 x PBS의 100 µ L)에서 2.4 µ L 세포 현 탁 액의 수집 P2 micropipette 사용 하 고 10% 보충 하는 MCBM의 1.1 mL에 추가 FBS.</li>
	<li>셀, 추가한 후 소용돌이 MCBM와 셀을 잘 혼합 하 최고 속도로 그들을 수직으로 잡고 1 분에 대 한 관.</li>
	<li>테스트 화합물 (OT48 혼자 또는 조합에서 A1210477으로)을 추가 합니다. 제어로 별도 튜브 준비 용 매 사용에 대 한 (여기 디 메 틸 sulfoxide (DMSO)). 각 조건에 대 한 추가 볼륨과 화합물의 농도 따라 용 컨트롤 용 매 화합물, 테스트의 희석에 대 한 사용 하는 용 매로 인 한 부작용에 대 한 평가의 가장 높은 농도 포함 됩니다.</li>
	<li>1 분 동안 소용돌이 튜브.</li>
	<li>각 분석 결과 대 한 12 음 셀 문화 접시의 센터 P1000 micropipette와 혼합물의 1 mL를 추가 합니다.</li>
	<li>살 균 물의 1 mL와 함께 빈 우물 또는 습도 안정 시키기 위해 1 x PBS를 채우십시오.</li>
	<li>37 ° C, 5% CO2배양 기에서 10 일 동안 배양 배지를 품 어.</li>
	<li>10 일 후 부 화, 각 잘 1 mg/mL의 최종 농도 도달 하는 MTT (methylthiazolyldiphenyl tetrazolium 브 로마 이드) 재고 솔루션 (5 mg/mL)의 200 µ L를 추가 합니다.</li>
	<li>37 ° C, 5% CO2배양 기에서 10 분을 품 어. 실행 가능한 식민지 바뀝니다 바이올렛.</li>
	<li>흰색 배경 플레이트 (라스 화이트 디 아 트레이)를 사용 하 여 디지털 카메라를 사용 하 여 이미지를 캡처하십시오. 다음 설정을 사용 합니다: 방법, 디지털화; 노출 유형, 정밀; 노출 시간, 수동, 1 초; 감도/해상도, 높은 해상도입니다. Tiff 형식에 이미지를 저장 합니다.</li>
	<li>ImageJ 소프트웨어를 사용 하 여 가능한 식민지를 계량. 선택 메뉴 "이미지 | 유형 | 8 비트 "입니다. 선택 메뉴 "조정 | 임계값 "입니다. 제어 임계값을 설정 하 고 모든 조건;에 대 한 일정 하 게 유지 선택 메뉴 "분석 | 입자 분석 ". 데이터를 저장 하 고 그래픽 표현에 대 한 통계 소프트웨어와 분석.</li>
</ol>2. 회전 타원 체 형성 분석 결과
<ol><li>10 %FBS 1% 항생제로 보충 RPMI 1640 매체의 15 mL와 75 c m2 플라스 크에 20000 셀/c m2 의 농도에 문화 A549 세포.</li>
	<li>실험 당일 준비는 96 U-바닥판 잘.</li>
	<li>이미 관심 (여기 50 µ M OT48) 및 A1210477의 20 µ M의 화합물을 포함 하는 우물으로 접시 10, 000 셀입니다. 세포 배양의 100 µ L 최종 볼륨을 조정 합니다. 그대로 외부 경계와 균일 한 spheroids를 형성 하는 세포의 수는 양식 spheroids에 적절 한 간주 됩니다.</li>
	<li>37 ° C, 5% CO2에 인큐베이터에서 3 일 동안 품 어.</li>
	<li>3 일 후 부 화, 4 x 확대와 함께 가벼운 현미경을 사용 하 여 spheroids의 이미지를 캡처하십시오.</li>
</ol>3. 제 브라가 종이 식 분석 결과
참고:이 기술 접근 회로도 (그림 1)으로 시각 이다.
<ol><li>
재고 시 약의 준비
	<ol>
<li>Danieau의 솔루션 x 30의 1 리터를 준비: 1740 mM NaCl, 21 m KCl m, 12 mM MgSO4∙7H2O, 18 m m Ca (3)2및 150 mM HEPES. 7.6 pH 미터를 사용 하 여 버퍼의 pH를 조정 합니다.</li>
		<li>1% (50 x) tricaine 솔루션 준비: 20mg 에틸 3-aminobenzoate methanesulfonate (tricaine) 2 mL의 증류수에 녹.</li>
		<li>1% 페 놀 레드-PBS 솔루션 준비: 198 µ L의 1.5 µ L 튜브의 소독된 PBS 솔루션 들을 페 놀 레드 나트륨 소금 솔루션의 추가 2 µ L.</li>
		<li>3% 스 준비: Danieau의 솔루션 x 1의 100 mL에 스의 3 세대를 추가.</li>
	</ol>
</li>
	<li>
Zebrafish 계란 생산 및 부 화
	<ol>
<li>별도 한 여성 및 파티션 탱크에 한 남성 zebrafish 어둠 속에서 28.5 ° C에서 UV 살 균 및 필터링 된 수돗물으로 가득합니다. 분리의 24 h 후 물고기 짝짓기를 시작 하는 두 남녀의 혼합. 짝짓기 탱크의 수정 된 난 자 신선한 Danieau 솔루션의 5 mL를 포함 하는 배양 접시에 전송.</li>
		<li>세 번 Danieau의 솔루션의 5 mL와 함께 계란을 세척. 세척, 피 펫 및 신선한 솔루션의 5 mL에 계란을 발음 신중 하 게. 8 h 28.5 ° c.에 대 한 품 어<ol>
<li>Danieau의 솔루션에 외피의 8 h 후 0.03 %1-페 닐-2-thiourea 착 색을 억제 (PTU)를 포함 하는 Danieau의 솔루션을 변경 합니다. 오염을 방지 하기 위해 불투명 계란 (unfertilized 또는 포함 미 개발된 태아) 밖으로 정렬.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
태아 dechorionation
	<ol>
<li>24 시간 수정 (hpf), dechorionate zebrafish 태아 날카로운 집게를 사용 하 여 게시할 수 있습니다. 0.03 %Danieau 솔루션에서 해칭 태아를 품 어 48까지 28.5 ° C에서 통해 hpf.</li>
	</ol>
</li>
	<li>
복합 치료 세포 준비
	<ol>
<li>20000 셀/cm2 25 cm2 문화 플라스 크 씨 A549 세포. 뿌리기의 24 h 후 추가 화합물 (50 µ M에서 여기 OT48 및 또는 20 µ M에서 A1210477) 37 ° C, 5% CO2배양 기에서 24 h에 대 한. 하지만 세포 죽음으로 그들을 저지 하는 세포의 생존 능력을 유지 하기 위해 각 화합물에 대 한 치료 시간을 설정 합니다. 이 시간은 개별적으로 설정 해야 포인트 기준 세포 생존 능력, 형태학 및 분자 마커.</li>
		<li>치료의 끝 전에 2 h, 얼룩 세포 형광 셀 추적기의 4 µ M로 37 ° C, 5% CO2CM Dil 염료. 외피의 2 시간 후 0.05% 트립 신-EDTA, 셀 trypsinize 그리고 106 세포 주입 전에 페 놀 레드-PBS 솔루션의 50 µ L 희석.</li>
	</ol>
</li>
	<li>
이 종이 식 형성에 대 한 암 세포의 마이크로 주입
	<ol>
<li>그들은 한 접시에 정착 될 때까지 5 분 0.02 %tricaine 솔루션에서 zebrafish를 anesthetize.</li>
		<li>(외부 직경 (OD) 유리 모 세관) 1.0 m m는 micropipette 끌어당기는 당겨 (설정 프로그램: p: 300, 열: 260, 당겨: 60, 벨: 50, 및 시간: 200). 주사기는 날카로운 가장자리를 생산 하는 microcapillary를 잘라. 로드 셀/페 놀 레드의 20 µ L 함께 microcapillaries-PBS 솔루션. 사출 압력을 설정 하 고 하는 걸 2 시간 주입 당 nL.</li>
		<li>주사 6 ~ 10 여 른 골목에 100-200 셀 주사 3 ~ 5 주사 통해 nL. 주입 후 물고기를 통해 솔루션을 포함 하는 신선한 Danieau 솔루션의 5 mL에 10 ~ 30 분에 대 한 복구할 수 있습니다. 디스패치 통해 솔루션을 포함 하는 Danieau의 솔루션의 1 mL 24-잘 접시에는 생선과 28.5 ° c.에서 72 h에 대 한 품 어</li>
		<li>외피의 72 h 후 물고기 0.02 %tricaine 솔루션에서 anesthetize 고 스 3%의 하락을 가진 유리 슬라이드에 고정. 620 750의 파장에서 형광 현미경 검사 법에 의해 5 x 사진을 찍어 nm.</li>
		<li>ImageJ 소프트웨어에 의해 형광 종양의 크기를 계량. 선택 메뉴 "분석 | 측정 "입니다. 형광 신호에 해당 하는 값을 저장 하 고 그래픽 표현에 대 한 통계 소프트웨어와 분석.</li>
	</ol>
</li>
</ol>

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저자는 공개 없다.

MCBM으로 얻은 식민지 형성의 속도 셀 형식에 따라 달라 집니다. 일반적으로, 비 부착 한 세포에 대 한 식민지의 수는 부착 세포에 비해 훨씬 높은. 우리가 관찰 A549 세포 10 일 후 30 ~ 40 식민지를 형성 했다. 이전 우리 보고 다른 백혈병 세포에 대 한 식민지의 수 200 ~ 2509사이입니다. 우리의 결과 OT48 혼자 혼자 식민지의 수의 상당한 감소를 생성 하지 않았다 보여주었다. 그러나, BH3 ?...

암은 세포 돌연변이 의해 발생 하 고 결과적으로 생 화 확 적인 신호 경로 통제 세포 분열 및 세포 죽음에 저항을 트리거링 혼란 된다. 종양 소화, 신경, 순환 시스템 및 이후에 이웃 조직1의 생리 기능을 방해합니다. 광범위 한 연구 노력에도 불구 하 고 암2세계 가장 널리 퍼진 생명이 질병에 남아 있다. 정밀 의학 미래 암 치료의 기초로 인식 하고있다. 임상 결과 개선 하기 위해 기존 약물과 함께에서 새로운 분자 엔티티를 정기적으로 테스트 합니다.
그러나, 새로운 효율적인 표적으로 한 치료의 개발과 관련 된 중요 한 제한 중 약물 노출3의 정확한 생물 학적 결과 시뮬레이션 하기 위해 일반적으로 사용 되는 분석의 실패 이다. 암 약물 발견 여전히 대부분 암 세포 선에서4임상 시험 확인 하기 어려운 차원 단층 문화에서 교양 치료제의 효능을 테스트에 의존 합니다. 따라서, 더 나은 종양 vivo에서5의 기본 기능을 모방 하는 더 나은 암 모델을 개발 하는 관심이 있다. 최근 몇 년 동안, 3D 문화 모델에 증가 관심 3D 종양 모델5생산 향상된 방법론의 개발에 결과.
여기, 우리는 깊이 비보에 분석 실험에 함께 더 전에 BH3 mimetics hydroxycoumarin OT486 의 힘의 이해를 향상 시킬 수 있도록 세 개의 다른 3D 셀 문화 기술로 접근을 제시. 우리의 방법은 식민지 및 SFAs는 vivo에서 종양 형성에 따라 검사 추가 폐 암 세포에서 OT48 및 BH3 mimetics의 조합의 효능을 확인 하 고 생활에서 암 진행 모니터링 zebrafish 모델에 결합의 구성 유기 체입니다.
식민지 대형 분석 실험 정기적으로 모두 고체 뿐만 아니라 조 혈 암에 대 한 항 암 제의 효능을 평가 하는 데 사용 됩니다. 분석 결과 무한정 증식 하 고 식민지7을 형성 하는 세포의 능력을 결정 합니다. 셀의 능력을 형성 하는 식민지에는 항 암 제의 효과 수의 감소 및 식민지의 크기에 의해 결정 됩니다.
Spheroids 종양 모델 생체 외에서 나타내고 제 에이전트는 저가 심사 플랫폼 역할. Spheroids는에 성장 하는 셀의 집계 또는 3D 매트릭스에 포함 된. 이 접근은 마약 검사 및 종양 성장, 확산 및 면역 상호 작용8의 연구에 널리 사용 됩니다.
완전히 새로운 약물의 속성 이해, vivo에서 실험 설치류에 필수적 이다. 그러나,이 전통적인 메서드는 비용과 시간이 소요 됩니다. 최근 몇 년 동안, zebrafish (Danio rerio) 널리 공부 실험실 유기 체를 저렴 하 고 빠르게 인상 되었다. 종양은 zebrafish 대표 3D 셀 문화 접근에서 하지만 척추9의 vivo에서 설정 내에서 개발.
전부, 사용 여기 CFAs, SFAs zebrafish vivo에서 종양 형성 등 3 가지 3D 문화 접근 BH3 mimetics 함께에서 폐 암 A549 세포 모델에서 hydroxycoumarin OT48의 항 암 제 용량을 설명 합니다.

<table><tbody><tr><td>&lt;strong&gt;Materials required for colony formation assays&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td>37 C&amp;deg;</td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>30096</td><td>4 C&amp;deg;</td></tr><tr><td>FBS</td><td>Biowest</td><td>S1520-500</td><td>-20 C&amp;deg;</td></tr><tr><td>Penicillin-Streptomycin</td><td>Lonza</td><td>17-602E</td><td>-20 C&amp;deg;</td></tr><tr><td>Cell culture flask T75</td><td>SPL</td><td>70075</td><td>RT</td></tr><tr><td>PBS solution</td><td>Hyclone</td><td>SH30256.02</td><td>RT</td></tr><tr><td>1.5ml tube</td><td>Extragene</td><td>Tube-170-C</td><td>RT</td></tr><tr><td>15 ml tube</td><td>Hyundai Micro</td><td>H20015</td><td>RT</td></tr><tr><td>12 well plate</td><td>SPL</td><td>30012</td><td>RT</td></tr><tr><td>MethoCult</td><td>StemCell technologies</td><td>4230</td><td>-20 C&amp;deg;</td></tr><tr><td>Thiazolyl Blue Tetrazolium Bromide (MTT powder)</td><td>Sigma</td><td>M5622</td><td>4 C&amp;deg;</td></tr><tr><td>LAS4000</td><td>GE Healthcare Technologies</td><td></td><td>RT</td></tr><tr><td>&lt;strong&gt;Materials required for spheroid formation assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td>37 C&amp;deg;</td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>30096</td><td>4 C&amp;deg;</td></tr><tr><td>FBS</td><td>Biowest</td><td>S1520-500</td><td>-20 C&amp;deg;</td></tr><tr><td>Penicillin-streptomycin</td><td>Lonza</td><td>17-602E</td><td>-20 C&amp;deg;</td></tr><tr><td>Cell culture flask T75</td><td>SPL</td><td>70075</td><td>RT</td></tr><tr><td>PBS solution</td><td>Hyclone</td><td>SH30256.02</td><td>RT</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25-300-054</td><td>4 C&amp;deg;</td></tr><tr><td>Corning costar ultra low attachemnt 96 well plate</td><td>Corning</td><td>3474</td><td>RT</td></tr><tr><td>Microscopy</td><td>Nikon</td><td>Eclipse TS100</td><td>RT</td></tr><tr><td>&lt;strong&gt;Materials required for zebrafish xenografts&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td>37 C&amp;deg;</td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>30096</td><td>4 C&amp;deg;</td></tr><tr><td>FBS</td><td>Biowest</td><td>S1520-500</td><td>-20 C&amp;deg;</td></tr><tr><td>Penicillin-streptomycin</td><td>Lonza</td><td>17-602E</td><td>-20 C&amp;deg;</td></tr><tr><td>Cell culture flask T25</td><td>SPL</td><td>70025</td><td>RT</td></tr><tr><td>Cell culture flask T75</td><td>SPL</td><td>70075</td><td>RT</td></tr><tr><td>Cell culture flask T175</td><td>SPL</td><td>71175</td><td>RT</td></tr><tr><td>1.5ml tube</td><td>Extragene</td><td>Tube-170-C</td><td>RT</td></tr><tr><td>24 well plate</td><td>SPL</td><td>30024</td><td>RT</td></tr><tr><td>Petridish</td><td>SPL</td><td>10100</td><td>RT</td></tr><tr><td>PBS solution</td><td>Hyclone</td><td>SH30256.02</td><td>RT</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25-300-054</td><td>4 C&amp;deg;</td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>71382</td><td>RT</td></tr><tr><td>Potassium chloride (KCL)</td><td>Sigma-Aldrich</td><td>P9541</td><td>RT</td></tr><tr><td>Magnesium sulfate heptahydrate (MgSO4.7H2O)</td><td>Sigma-Aldrich</td><td>M2773</td><td>RT</td></tr><tr><td>Calcium nitrate tetrahydrate (Ca(NO3)2)</td><td>Sigma-Aldrich</td><td>C1396</td><td>RT</td></tr><tr><td>HEPES solution</td><td>Sigma-Aldrich</td><td>H0887</td><td>RT</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td><td>Sigma-Aldrich</td><td>E10521</td><td>RT</td></tr><tr><td>Phenol Red solution</td><td>Sigma-Aldrich</td><td>P0290</td><td>RT</td></tr><tr><td>Methylcellulose</td><td>Sigma-Aldrich</td><td>M0512</td><td>RT</td></tr><tr><td>1-phenyl-2-thiourea (PTU)</td><td>Sigma-Aldrich</td><td>P7629</td><td>RT</td></tr><tr><td>CM-Dil dye</td><td>Invitrogen</td><td>C7001</td><td>-20 C&amp;deg;</td></tr><tr><td>Glass capillary</td><td>World Precision Instruments</td><td>TW 100F-4</td><td>RT</td></tr><tr><td>Micropipette puller</td><td>Shutter instrument, USA</td><td>P-97</td><td>RT</td></tr><tr><td>Micro injector</td><td>World Precision Instruments</td><td>PV820</td><td>RT</td></tr><tr><td>Syringe</td><td>KOVAX</td><td>1ml</td><td>RT</td></tr><tr><td>Micro loader</td><td>Eppendorf</td><td>5242956003</td><td>RT</td></tr><tr><td>Glass slide</td><td>Marienfeld</td><td>HSU-1000612</td><td>RT</td></tr><tr><td>Fluorescence microscopy</td><td>Leica</td><td>DE/DM 5000B</td><td>RT</td></tr></tbody></table>

preclinical assessment of the bioactivity of the anticancer coumarin ot48 by spheroids, colony formation assays, and zebrafish xenografts

생체 외에서 그리고 vivo에서 새로운 치료제의 전 임상 심사 암 약물 발견에 필수적인 도구입니다. 비록 인간의 암 세포 선 2 차원 (차원) 단층 세포 배양에서 치료 화합물에 대응, 더 순수 관련 모델에서 약물의 효능을 이해 하 3 차원 배양 시스템 개발 되었다. 최근 몇 년 동안, 패러다임의 변화는 3D 문화 시스템, 더 정확 하 게 종양 microenvironment를 흉내 낸 새로운 분자의 힘을 확인 하기 위해 전 임상 연구에서 관찰 되었다. 이러한 시스템 보다 생리 적으로 관련 된 방식으로 질병 상태를 특성화 하 고 더 나은 기계적 통찰력 및 주어진된 분자의 약리 효능을 이해 하는 데 도움이. 또한, 암 모델 vivo에서 개선 추세와 함께 zebrafish vivo에서 종양의 형성을 평가 하 고 치료제의 효과 연구 하는 중요 한 척추 모델로 떠오르고 있다. 여기, 우리 조사 hydroxycoumarin OT48의 치료 효능 하거나 폐 암 세포 라인 A549 BH3 mimetics와 함께 식민지 대형 분석 실험 (CFA), 회전 타원 체 형성 분석 결과 (SFA) 등 세 가지 다른 3D 문화 시스템을 사용 하 여 및 zebrafish xenografts vivo에서 .

그림 2, 폐암 A549 했다 시드 MCBM에서 식민지를 형성 하 OT48와 치료 후 또는 표시 농도에서 BH3 모방 A1210477와 선 세포. 결과 조합 크게 감소 번호, 크기 및 식민지의 총 면적 외피의 10 일 후에 나타났다.
그림 3에서는 A549 세포 BH3 모방 A1210477와 혼자 또는 조합에서 OT48로 치료 했다 고 양?...

Watch this Scientific Journal Video about Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts at JoVE.com

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

Spheroids, 식민지 대형 분석 실험, 그리고 Zebrafish Xenografts 제 Coumarin OT48의 Bioactivity의 전 임상 평가

여기, 선물이 3D 문화와 제 브라를 사용 하 여 항 암 제 coumarins의 전 임상 심사.

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

preclinical-assessment-bioactivity-anticancer-coumarin-ot48-spheroids

Research

JoVE Journal

Cancer Research

8.4K 조회수.  Seoul National University. 여기, 선물이 3D 문화와 제 브라를 사용 하 여 항 암 제 coumarins의 전 임상 심사.

비디오: Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

MTT의 분석에 의해 합성 및 세포 독성 평가 : 항암 금속 합성물

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

연구

화학

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

암 연구학

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

질량 분석 및 Luminogenic 기반의 접근 방법은 단계 I 대사 역량의의 특성을<em&gt; 체외</em&gt; 세포 배양

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

생화학

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

형성 화학식 DNA Adducts 효소 활성화 된 발암 물질 및 생체 외에서 마약 및 32P postlabeling에 의해 그들의 결정에 의해

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

환자 유래 종양 오르가노이드를 사용하여 시험관 분석에서 높은 처리량

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

암 약물 과민 반응의 효과를 평가<em&gt; 체외</em&gt; 및<em&gt; 생체</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

의학

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic chemotherapy due to their precise spatiotemporal activation by directing a benign, non-ionizable light to a specific location within the patient&#39;s body. This paper presents a set of methods to evaluate the in vitro potency and ex vivo efficiency of the photoactivation of photocontrolled, biologically active compounds as well as the in vivo efficacy at early stages of drug development. The methodology is applied to anticancer cytotoxic peptides, namely, the diarylethene-containing analogs of a known antibiotic, gramicidin S. The experiments are performed using 2D (adherent cells) and 3D (spheroids) cell cultures of a cancer cell line (Lewis lung carcinoma, LLC), live tissue surrogates (pork meat mince), and an allograft cancer model (subcutaneous LLC) in immunocompetent mice. The selection of the most effective compounds and estimation of realistic phototherapeutic windows are performed via automated fluorescence microscopy. The photoactivation efficiency at varying illumination regimens is determined at different depths in a model tissue, and the optimal light dosage is applied in the final therapeutic in vivo experiment.

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

This protocol presents a set of experiments adopted for the evaluation of photoswitchable anticancer peptides, that can be used in the preclinical screening of such compounds. This includes cytotoxicity assessment in 2D and 3D cell cultures, the evaluation of ex vivo (model tissue) photoisomerization efficiency, and in vivo efficacy.

광제어 생물학적 활성 화합물의 시험관 내 및 생체 내 평가 - 암 광약리학을 위한 잠재적 약물 후보

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

소프트 한천 콜로니 형성 분석의 활용은 유방암 세포에서 종양 유발의 억제를 확인하는

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

테스트를위한 병아리 심장 침략 분석 암 세포의 침윤과 잠재적 안티 침략 화합물의 활동

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

유해 및 잠재적으로 유해한 구성 성분의 독성 효과를 평가하기 위해 높은 콘텐츠 심사 분석 (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

Spheroids, 식민지 대형 분석 실험, 그리고 Zebrafish Xenografts 제 Coumarin OT48의 Bioactivity의 전 임상 평가

요약

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