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Synergistic apoptosis induced by a targeted combination treatment with midostaurin and ABT199 in the FMS-like tyrosine kinase (FLT)3-internal tandem duplication (ITD)-positive and BCL2-overexpressing cell line MV4-11
MV4-11 (FMS-like tyrosine kinase (FLT)3-internal tandem duplication (ITD) 변이 및 BCL2 과발현) 세포주에서 midostaruin과 ABT199에 의한 표적 항암치료

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dc.contributor.advisorDiederich, Marc-
dc.contributor.author이소미-
dc.date.accessioned2019-05-07T04:10:14Z-
dc.date.available2019-05-07T04:10:14Z-
dc.date.issued2019-02-
dc.identifier.other000000155408-
dc.identifier.urihttps://hdl.handle.net/10371/151376-
dc.description학위논문 (석사)-- 서울대학교 대학원 : 약학대학 약학과, 2019. 2. Diederich, Marc.-
dc.description.abstract급성 골수성 백혈병 (Acute myeloid leukemia, AML)은 골수성 혈액 암으로서, 유전학적, 생리학적 이상으로 발병되는 것으로 보고되어 있다. 특히, FMS-like tyrosine kinase 3 (FLT3)는 세포 생존, 증식 및 분화에 핵심적인 역할을 하는 수용체로, 특히 재발환자에서 나타나는 FLT3-ITD 돌연변이는 그 예후를 악화한다.
본 연구에서는 단백질의 기초 발현 수준을 다양한 AML 세포주에서 확인하고 FLT3 인산화 및 Bcl-2 관련 단백질의 과발현 특성을 가진 MV4-11 세포주를 선택하였다.
보다 효율적인 표적 화학 요법을 제안하기 위해 FLT3-ITD와 BCL-2를 선택적으로 억제하는 midostaurin (Rydapt)과 ABT199 (Venetoclax)의 sub-toxic한 농도를 산출하였다.
두 화합물의 Sub-toxic한 농도는 각각 MV4-11 세포주에서 생존능력에 큰 영향을 미치지 않은 것과 대조적으로, 동반처리 후 caspase의존성 세포사멸기전을 유도함으로써 유의미한 상승효과를 확인하였다. 뿐만 아니라, 이 조합은 정상 세포주에서 유의미한 세포독성을 나타내지 않았다. 이 결과를 바탕으로 통계적으로 Chou-Talalay 의 CompuSyn 소프트웨어를 이용하여 combination index를 산출하였다.
또한, 두 조합에 의한 세포독성을 annexin V/PI 염색을 통해 flow cytometry로 정량 하였고, 전체 caspase 를 억제하는 z-VAD-FMK 처리를 통해 Caspase 의존성 세포사멸 기전을 검증하였다.
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dc.description.abstractAcute myeloid leukemia (AML) is a heterogenous disease with low survival rate, and relapsed AML with FMS-like tyrosine kinase (FLT)3 (FLT3)-internal tandem duplication (ITD) mutations worsens the prognosis of the patients. After an initial profiling of AML cell lines, we selected the cell line MV4-11 presenting both the FLT3-ITD mutation as well as overexpression of the antiapoptotic BCL2 resistance protein. In an attempt to suggest a more efficient targeted chemotherapeutic approach, we determined sub-toxic doses of midostaurin targeting the FLT3-ITD and ABT199 (Venetoclax), a specific inhibitor of BCL2. Whereas subtoxic doses of both compounds did not strongly affect viability of MV4-11 cells, a combination treatment synergistically triggered caspase-dependent apoptotic cell death compared to AML cells lines without FLT3 mutation or cells with a healthy phenotype. Our results could be validated by calculation of the combination index according to Chou-Talalay. Moreover, the cytotoxic effect of the combination treatment was evaluated by Annexin-PI staining and flow cytometry and detection of caspase activation with or without pre-treatment of the pan-caspase inhibitor z-VAD-FMK. Importantly, the combination of midostaurin and ABT199 reduced the tumor formation in in vitro colony formation assays and zebrafish xenografts. Altogether, we suggest a novel, more efficient chemotherapeutic approach for AML patients expressing both elevated levels of antiapoptotic BCL2 and mutated FLT3.-
dc.description.tableofcontentsTable of Contents

1. INTRODUCTION 1
2. MATERIAL AND METHODS 4
2.1 Compounds 4
2.2 Cell culture and treatments 4
2.3 Cell Proliferation and viability 5
2.4 Protein extraction and western blots 5
2.5 Quantification of apoptosis 5
2.6 Colony formation assay 6
2.7 Investigation of cellular morphology 6
2.8 Zebrafish xenografts 7
2.9 Statistical analysis 8
3. RESULTS 9
3.1 Effect of midostaurin on the viability of AML cell lines with differential phosphorylation levels of FLT3 and BCL2 protein patterns 9
3.2 Midostaurin and ABT199 induce caspase-dependent apoptosis 18
3.3 Synergistic apoptotic effect of a combination of midostaurin and ABT199 21
3.4 Validation of synergistic combination treatments by colony formation assays and in vivo zebrafish xenografts 28
4. DISCUSSION 31
5. REFERENCES 37
6. 국문초록 40
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dc.language.isoeng-
dc.publisher서울대학교 대학원-
dc.subject.ddc615-
dc.titleSynergistic apoptosis induced by a targeted combination treatment with midostaurin and ABT199 in the FMS-like tyrosine kinase (FLT)3-internal tandem duplication (ITD)-positive and BCL2-overexpressing cell line MV4-11-
dc.title.alternativeMV4-11 (FMS-like tyrosine kinase (FLT)3-internal tandem duplication (ITD) 변이 및 BCL2 과발현) 세포주에서 midostaruin과 ABT199에 의한 표적 항암치료-
dc.typeThesis-
dc.typeDissertation-
dc.description.degreeMaster-
dc.contributor.affiliation약학대학 약학과-
dc.date.awarded2019-02-
dc.contributor.major의약생명과학-
dc.identifier.uciI804:11032-000000155408-
dc.identifier.holdings000000000026▲000000000039▲000000155408▲-
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College of Pharmacy (약학대학)Dept. of Pharmacy (약학과)Theses (Master's Degree_약학과)
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