[보고서]항암제 doxorubicin에 의한 혈소판 감소증(thrombocytopenia)의 분자독성   기전 및 개선 약물개발 연구

정진호

[국가R&D연구보고서] 항암제 doxorubicin에 의한 혈소판 감소증(thrombocytopenia)의 분자독성 기전 및 개선 약물개발 연구
Molecular Mechanism for Doxorubicin-Induced Thrombocytopenia 원문보기

보고서 정보
주관연구기관	서울대학교 산학협력단
연구책임자	정진호
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2011-02
과제시작연도	2010
주관부처	교육과학기술부
과제관리전문기관	한국연구재단 National Research Foundation of Korea
등록번호	TRKO201200002042
과제고유번호	1345120364
사업명	중견연구자지원
DB 구축일자	2013-05-20
키워드	혈소판 감소증.혈소판.세포독성.혈전.항암제.Thrombocytopenia.Platelets.Doxorubicin.Thrombosis.Cytotoxicity.Anti-cancer agent.Procoagulant activity.Apoptosis.

초록 ▼

◦ Drug-induced thrombocytopenia 연구에 활용
직접적으로 혈소판 독성을 유발하거나 thrombocytopenia를 보이는 약물은 상당히 많은데, 항생제인 ristocetin 항암제인 bleomycin 등이 알려져 있다. 본 연구를 통하여 기전이 확립되면 thrombocytopenia를 경감시킬수 있는 약제 개발에 활용이 가능하다.
◦ 암환자의 부작용 경감을 통한 삶의 질 개선
많은 임상적 보고에서 doxorubicin을 투여한 환자에서 부작용으로 thrombocytopenia, thrombosis 가 나타나기 때문에 부작용의 원인을 제시함으로써 효율적인 항암제 투여를 통한 암환자들에게 삶의 질 개선이 가능하다. ◦ 항암제 약물 개발을 통한 경제적 부가가치 창출 따라서 항암제 약물 screening에 본 연구의 작용기전을 활용하여 효능이 뛰어나며 부작용을 최소화 시킨, 부가가치가 높은 약제 개발에 활용할 수 있다.

Abstract ▼

◦ New Insight on Drug-Induced Thrombocytopenia and thrombosis : Since there are many drugs to induce thrombocytopenia in vivo, our study will provide novel methods to reduce thrombocytopenia in patients by developing new drug candidate.
◦ Application to Cancer Patients : Considering the repetitive use of DOX for the cancer treatment, cumulative DOX concentration might become much higher and this could increase the chance of thrombosis and embolism further. Incidentally, the risk of cardiotoxicity of DOX is being regularly checked by monitoring cardiac function during DOX treatment. we suggest that platelet procoagulant activity should be also checked as an important diagnostic marker for monitoring the risk of DOX-associated thrombosis.

목차 Contents

중견연구자지원사업(핵심연구) 최종보고서 ... 1
목차 ... 3
Ⅰ. 연구계획 요약문 ... 4
1. 국문요약문 ... 4
Ⅱ. 연구결과 요약문 ... 5
1. 국문요약문 ... 5
2. 영문요약문 ... 6
Ⅲ. 연구내용 및 결과 ... 7
1. 연구개발과제의 개요 ... 7
2. 국내외 기술개발 현황 ... 9
3. 연구수행 내용 및 결과 ... 12
4. 목표달성도 및 관련분야에의 기여도 ... 38
5. 연구결과의 활용계획 ... 39
6. 연구과정에서 수집한 해외과학기술정보 ... 40
7. 주관연구책임자 대표적 연구실적 ... 42
8. 참고문헌 ... 43
9. 연구성과 ... 49
10. 기타사항 ... 54

표/그림 (35)

표 Effects of doxorubicin(DOX) on LDH leakage in platelets. Platelet-rich plasma (PRP) were incubated with doxorubin and then LDH lactate dehydrogenase) leakage was determined.
표 Time- and concentration-dependent LDH leakage by DOX in washed platelets.Washed platelets were incubated with doxorubin and then release of LDH (lactate dehydrogenase) was determined.
표 Effects of doxorubicin(DOX) on platelet counts.Washed platelets were incubated with doxorubin and then decrease of platelet counts was determined.
표 Effects of doxorubicin (DOX) on free radical generation.Washed platelets were incubated with DOX and then free radical generation was determined
표 Effects of doxorubicin(DOX) on GSH/GSSG ratio.Wahsed platelets were incubated with doxorubicin and then glutathione levels were determined. Values are means ± SEM of three to six independent experiments.
표 Effects of doxorubicin (DOX) on protein thiol levels.Washed platelets were incubated with doxorubicin and then protein thiol levels were determined. Values are means ± SEM of three to four independent experiments.
표 Effects of doxorubicin (DOX) on caspase-3 activity.Flow cytometric studies of caspase-3 activation using PhiPhiLuxG1D2.
표 Effects of doxorubicin (DOX) on glutathiolated procaspase-3. Glutathiolation of procaspase-3 significantly decreased by DOX in a time dependent manner.
표 Effects of doxorubicin (DOX) on mitochondrial transmembrane potential.Flow cytometric studies of mitochondrial transmembrane potential using JC-1.
표 Effects of doxorubicin (DOX) on phosphatidylserine(PS) exposure.Flow cytometric studies phosphatidylserine exposure induced by DOX using of annexin V binding.
표 Effects of caspase inhibitors and thiol supplements on doxorubicin (DOX)-induced platelet lysis.
표 Effects of caspase inhibitors and thiol supplements on doxorubicin (DOX)-induced thiol depletion and caspase-3 activation.. Platelets were pretreated with caspase inhibitor 100 μM z-DEVD-FMK, z-VAD-FMK or thiol supplement, 10 mM L-cysteine, 2 mM DTT or 5 mM NAC 30 min prior to incubation.
표 Effect of shear rate on DOX-induced LDH leakage. Shear stress was applied to platelets with DOX or vehicle using cone-plate
표 Synergistic effect of shear stress on doxorubicin-induced platelet cytotoxicity.
표 In vitro and in vivo effects of doxorubicin (DOX) treatment on platelets in rats.
표 Schematic Diagram for Doxorubicin-Induced CytotoxicityProcaspase-3-SSG; S-glutathiolated procaspase-3, PS; Phosphatidylserine, Procaspase-3-SSG; S-glutathiolated procaspase-3, PS; Phosphatidylserine, ROS; reactive oxygen species. Blue colored arrow; effect by shear stress.
표 Enhancement of procoagulant activity by doxorubicin(DOX) in human WP.
표 Effects of doxorubicin(DOX) on LDH leakage and platelet aggregation in human WP. After incubation with 0, 30, 60, and 100 mM of doxorubicin for 1 hr,
표 Potentiation of phosphatidylserine exposure by doxorubicin(DOX) in humanWP.　In flow cytometry analysis, doxorubicin treatment enhanced phosphatidylserine(PS) exposure of human WP.
표 PS-dependency of doxorubicin(DOX)-enhanced procoagulant activity．　Effect of doxorubicin was attenuated by purified annexin V which blocks the interaction of PS with coagulation enzymes. Values are mean ± SEM of 3 independent experiments.
표 Enhanced generation of microparticles by doxorubicin(DOX) from human WP. After treatment of doxorubicin, microparticles (MP) generation was analyzed by flow cytometry.
표 Identification of generated MPs using confocal microscopyExposure of doxorubicin to adherent platelets on fibrinogen-coated coverslips to generate MPs.
표 Procoagulant activity and phosphatidylserine exposure of isolated MPs. After treatment of doxorubicin in human WPs, generated MPs were isolated as described in Materials and Methods. Procoagulant activities and phosphatidylserine exposure of isolated MPs were examined using prothombinase complex assay and inflow cytometry analysis. Values are mean ± SEM of 3-4 independent experiments.
표 Kinetic studies of intracellular calcium levels in doxorubicin (DOX) treated platelets. To examine the effect of doxorubicin on intracellular calcium levels, fluo-3 loaded platelets were exposed to doxorubicin. The fluorescence intensities were measured continuously. Tracings of representative data of 3-4 independent experiments are shown.
표 After platelets were treated with DOX, the extents of phospholipid translocation were measured. (A) C6-NBDPC translocation by scramblase was measured. (B) C6-NBDPS translocation by flippase was determined.
표 The levels of intracellular ATP were measured by luciferin-luciferase assay.
표 DOX-enhanced PS exposure and MP generation were examined after pretreatment with EGTA (5 mM) for 5 min.
표 Reactive oxygen generation was measured in flow cytometry using DCF-DA (5 μM). Cells were considered to be positive when the fluorescence intensity was > 99% of the signal from the DCF-unloaded control group.
표 Change of mitochondrial membrane potential (Δψm) was determined by flow cytometry using JC-1, which shows green fluorescence (FL-1) in collapsed Δψm. Representative histograms from three independent experiments are shown.
표 Activation of caspase-3 was measured by flow cytometry using PhiPhiLux-G1D2, a fluorescent substrate for caspase-3. Data are expressed as percentage of positive cells, of which fluorescence intensity was > 99 % of the signal from negative control group without PhiPhiLux-G1D2.
$Cytochrome c (cyt c) release and mitochondrial translocation of Bax were evaluated by measuring their levels in platelet mitochondrial fraction and cytosolic fraction using western blot.$ 표 Cytochrome c (cyt c) release and mitochondrial translocation of Bax were evaluated by measuring their levels in platelet mitochondrial fraction and cytosolic fraction using western blot.
표 Washed platelets were pretreated with caspase inhibitor (0.5 mM QVD) or anti-oxidants [5 mM Vitamin C (Vit C) or 0.1 mM trolox] for 30 min, and then were incubated with 60 μM DOX for 1 hr. Procoagulant activity and phosphatidylserine exposure was determined.
표 One hour after intravenous administration of 2.5 or 7.5 mg kg-1 DOX or vehicle(saline),
표 After intravenous infusion of DOX, FeCl3 was infused for thrombus formation. Stasis-induced thrombosis was induced in the vena cava, and then isolated thrombus was weighed.
표 Suggested mechanism of DOX-induced platelet procoagulant activity.

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연합인증

[국가R&D연구보고서] 항암제 doxorubicin에 의한 혈소판 감소증(thrombocytopenia)의 분자독성 기전 및 개선 약물개발 연구
Molecular Mechanism for Doxorubicin-Induced Thrombocytopenia 원문보기