CN1329083C - Method for degrading AML 1-ETO fusion protein and used reagent - Google Patents
Method for degrading AML 1-ETO fusion protein and used reagent Download PDFInfo
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- CN1329083C CN1329083C CNB2004100168916A CN200410016891A CN1329083C CN 1329083 C CN1329083 C CN 1329083C CN B2004100168916 A CNB2004100168916 A CN B2004100168916A CN 200410016891 A CN200410016891 A CN 200410016891A CN 1329083 C CN1329083 C CN 1329083C
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- 208000026784 acute myeloblastic leukemia with maturation Diseases 0.000 description 3
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention a method for degrading AML1-ETO fusion protein and a used reagent. Oridonin with the concentration of 0.5 to 10 muM is used for processing Kasumi-1 cells, and after 2 to 72 hours, the degradation of the AML1-ETO fusion protein can be evoked. After the transfection of the U937 cells of AML1-ETO fusion genes, the oridonin can also degrade the AML1-ETO fusion protein. The present invention lays a foundation for the development of leukemia targeted therapy drugs with t (8; 21) chromosomal translocation, simultaneously provides a new path and a reagent for the research of the biological functions of the AML1-ETO fusion protein, and provides a basis for the widening of the clinical / experimental research functions of the oridonin.
Description
Technical field
The present invention discloses a kind of method and agents useful for same of the AML1-ETO of degraded fusion rotein.
Background technology
Leukemic generation is how relevant with genomic abnormal change, a certain (or some) unusual molecule can be by the malignant proliferation of the final leukemogenesis cell of number of mechanisms, the generation that differentiation is obstructed, apoptosis is suppressed to cause disease.The unusual molecule of targeting degraded this (or these) becomes a kind of strategy of leukemia treating naturally, and this targeted therapies often has the curative effect height and the low characteristics of toxic and side effects, is that the mankind are rely and conquered leukemic strong instrument.Typical example is differentiation and the apoptosis that all-trans-retinoic acid (ATRA), arsenic trioxide (ATO) are induced acute promyelocytic leukemia (APL) leukaemia by degraded PML-RAR alpha fusion protein, improved APL patient's prognosis greatly, made APL become a kind of leukemia of being cured of being hopeful.M2 type acute myeloid leukaemia (AML M2) accounts for 25% of all acute myeloid leukaemias (AML), to have t (8; 21) chromosome translocation is a feature, and the AML1-ETO fusion rotein of its formation is the pathogenic factor of AML M2.This disease clinical manifestation is symptom and signs such as heating, anemia, hemorrhage and granulocyte sarcoma, and often has leukocyte to increase in the peripheral blood, erythrocyte, thrombocytopenia.Based on chemotherapy such as cytosine arabinoside, daunorubicins, and lack special effective Therapeutic Method in the treatment at the AML1-ETO fusion rotein.Exploitation targeting degraded AML1-ETO fusion rotein is also induced t (8; 21) medicine of apoptosis of leukemia/differentiation has great importance to the further AML of improvement M2 patient's clinical efficacy.
Summary of the invention
The objective of the invention is to propose a kind of method of the AML1-ETO of degraded fusion rotein.
Another object of the present invention is to propose the used reagent of a kind of AML1-ETO of degraded fusion rotein.
Technical characterictic of the present invention is to adopt the rubescensine A processing of 0.5～10 μ M concentration to contain t (8; 21) the Kasumi-1 cell of chromosome translocation can cause the degraded of AML1-ETO fusion rotein after 2～72 hours; Handle the U937 cell 2～72 hours that the AML1-ETO fusion gene is crossed in transfection with the rubescensine A of 0.5～10 μ M concentration, also can cause the degraded of AML1-ETO fusion rotein.The approach of rubescensine A degraded AML1-ETO fusion rotein has two kinds, and the one, caspases is passed through in activation, and the 2nd, by the ubiquitin approach.
Discover that rubescensine A all has inhibited proliferation to leukaemias such as Kasumi-1, NB4, HL-60, U937, K562, but the most obvious to the Kasumi-1 cytosis with AML1-ETO fusion rotein, IC
50Minimum; And rubescensine A does not have Degradation to the BCR-ABL fusion rotein, illustrates that its degraded to the AML1-ETO fusion rotein has certain specificity.
More than be found to be exploitation and have t (8; 21) the leukemic targeted therapies of chromosome translocation is laid a good foundation, and also the biological function for research AML1-ETO fusion rotein provides new approach and reagent.
Compare prior art, the present invention has following technical characterstic:
1, a kind of method of the AML1-ETO fusion rotein of degrading and the used reagent of AML1-ETO fusion rotein of degrading are proposed, disclosed the new biological function of tetracyclic diterpene compounds.For exploitation has t (8; 21) the leukemic target therapeutic agent of chromosome translocation is laid a good foundation; For the biological function of studying the AML1-ETO fusion rotein provides new approach and reagent.
2, found the biological effect that rubescensine A is new.Rubescensine A degradable AML1-ETO fusion rotein is also induced AML M2 apoptosis of leukemia, adds that its toxic and side effects is low and cheap, and therefore comparatively broad clinical application prospect is arranged.
Description of drawings
Fig. 1 rubescensine A is to the Degradation of AML1-ETO fusion rotein
Fig. 2 rubescensine A is to the effect of BCR-ABL fusion rotein.Show the rubescensine A BCR-ABL fusion rotein of can not degrading, and ATO can degrade to it.
The specific embodiment
The rubescensine A (0.5～5 μ M concentration) of I, our usefulness variable concentrations is handled the Kasumi-1 cell and is extracted its protein after 24,48 hours, carry out the Western blot hybridization, find that rubescensine A can be with the AML1-ETO protein degradation of 94kDa, the degradation fragment size is respectively 70kDa, 25kDa left and right sides (see figure 1).(1 is contrast, and 2～4 are respectively 0.5,2,5 μ M rubescensine A effects proteic variation of Kasumi-1 cell AML-ETO after 24 hours, and 5～7 is that 0.5,2,5 μ M rubescensine A are handled the proteic variation of Kasumi-1 cell AML-ETO after 48 hours.Upper arrow is depicted as the AML1-ETO fusion rotein, and middle below arrow is depicted as catabolite.)
II, we detect AML1-ETO and the proteic distribution situation of ETO in rubescensine A is handled back Kasumi-1 cell with the method for immunofluorescence, find that the interior proteic expression of AML1-ETO, ETO of Kasumi-1 cell obviously reduces.
III, handle the U937 cell that the AML1-ETO fusion gene is crossed in transfection, extract its albumen again and carry out the test of Western blot hybridization, also find rubescensine A degradable AML1-ETO fusion rotein with rubescensine A.
IV, we have compared rubescensine A to Kasumi-1, NB4, HL-60, U937, the isocellular inhibited proliferation of K562, find that rubescensine A is the most obvious to the Kasumi-1 cytosis of tool AML1-ETO fusion rotein, IC50 minimum (table 1).And rubescensine A does not have Degradation (Fig. 2) to the BCR-ABL fusion rotein, illustrates that its degraded to the AML1-ETO fusion rotein has certain specificity.
Table 1 rubescensine A is to different leukaemias' inhibited proliferation
| Cell type | Kasumi-1 | NB4 | HL-60 | U937 | K672 |
| IC 50(μM) | 0.34 | 2.04 | 2.72 | 1.22 | 2.21 |
V, we handle the Kasumi-1 cell with rubescensine A (0.5～5 μ M concentration), find that but apoptotic body and DNA " trapezoidal " band appear in its inducing leukemia cell, the Phosphatidylserine tableization detects positive with the original position apoptosis of terminal deoxyribotide transferase mediation, inferior G1 peak appears in cell cycle, and this effect is dosage-time dependence.Rubescensine A also can cause the disintegrate of Kasumi-1 cell mitochondrial transmembrane potential, activation and the following degradation of bcl-2 cancer protein expression of caspases.To other leukemia cell line such as NB4, HL-60, cells such as K562, U937, rubescensine A also can be induced its apoptosis, but desired concn is higher, between 5～15 μ M.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2004100168916A CN1329083C (en) | 2004-03-11 | 2004-03-11 | Method for degrading AML 1-ETO fusion protein and used reagent |
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| CNB2004100168916A CN1329083C (en) | 2004-03-11 | 2004-03-11 | Method for degrading AML 1-ETO fusion protein and used reagent |
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| CN1666780A CN1666780A (en) | 2005-09-14 |
| CN1329083C true CN1329083C (en) | 2007-08-01 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100370981C (en) * | 2005-11-07 | 2008-02-27 | 上海第二医科大学附属瑞金医院 | The application of calyxin in pharmacy |
| CN1994293A (en) * | 2006-08-18 | 2007-07-11 | 上海交通大学医学院附属瑞金医院 | Application of oridonin in pharmacy |
| CN102895221A (en) * | 2012-09-29 | 2013-01-30 | 中山大学 | Target degradation Bcr-Abl protein reagent and application of target degradation Bcr-Abl protein reagent in preparing Philadelphia chromosome positive tumor treatment medicine |
| WO2025087245A1 (en) * | 2023-10-23 | 2025-05-01 | 深圳领济生物科技有限公司 | New recruitment element for ubiquitin ligase and use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1255502A (en) * | 1999-01-18 | 2000-06-07 | 郑州大学 | Rebescensine A derivatives and preparing process thereof |
| WO2003075943A2 (en) * | 2002-03-06 | 2003-09-18 | The Medical Research And Education Trust | Botanical extract compositions with anti-cancer or phytoestrogenic activity comprising wogonin, isoliquiritigenin and/or coumestrol |
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- 2004-03-11 CN CNB2004100168916A patent/CN1329083C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1255502A (en) * | 1999-01-18 | 2000-06-07 | 郑州大学 | Rebescensine A derivatives and preparing process thereof |
| WO2003075943A2 (en) * | 2002-03-06 | 2003-09-18 | The Medical Research And Education Trust | Botanical extract compositions with anti-cancer or phytoestrogenic activity comprising wogonin, isoliquiritigenin and/or coumestrol |
Non-Patent Citations (3)
| Title |
|---|
| AML1-ETO基础表达质粒的构建 郭萌等，厦门大学学报（自然科学版），第42卷第3期 2003 * |
| AML1-ETO基础表达质粒的构建 郭萌等，厦门大学学报（自然科学版），第42卷第3期 2003;冬凌草甲素的药学感觉进展 张典瑞等，中国药学杂志，第38卷第11期 2003 * |
| 冬凌草甲素的药学感觉进展 张典瑞等，中国药学杂志，第38卷第11期 2003 * |
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