Target degradation Bcr-Abl protein reagent and application of target degradation Bcr-Abl protein reagent in preparing Philadelphia chromosome positive tumor treatment medicine

A Philadelphia chromosome and bcr-abl technology, which is applied to a reagent for targeting degrading Bcr-Abl protein and its application in the preparation of a Philadelphia chromosome positive tumor treatment drug, and achieves the effects of inhibiting growth, good curative effect and difficult reversal.

Inactive Publication Date: 2013-01-30
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

There is no report about the application of oridonin in the degradation of Bcr-Abl protein and the preparation of drugs for the treatment of Philadelphia chromosome positive tumors

Method used

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  • Target degradation Bcr-Abl protein reagent and application of target degradation Bcr-Abl protein reagent in preparing Philadelphia chromosome positive tumor treatment medicine
  • Target degradation Bcr-Abl protein reagent and application of target degradation Bcr-Abl protein reagent in preparing Philadelphia chromosome positive tumor treatment medicine
  • Target degradation Bcr-Abl protein reagent and application of target degradation Bcr-Abl protein reagent in preparing Philadelphia chromosome positive tumor treatment medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Rubescensine A efficiently, rapidly and irreversibly degrades Bcr-Abl protein in Ph-positive tumor cells

[0034] We tested the effect of oridonin on Bcr-Abl fusion protein in the Ph positive tumor cell line K562. Each cell is divided into 3x10 5 Cells / mL were inoculated on cell culture plates, cultured for 24 hours to the logarithmic growth phase, and then treated with 20 μM oridonin and drug solvent DMSO. After 24 hours of induction, the cells were collected and the total protein was extracted by SDS method for Western blot detection. The results showed that compared with the control DMSO treatment, Rubescensin A treatment significantly reduced the Bcr-Abl and c-Abl protein levels in K562 cells, and almost completely degraded them, while the level of the internal control GAPDH protein had no significant change ( figure 1 ). It shows that Rubescensine A can specifically degrade the oncoprotein members in the Bcr-Abl protein family in K562 cells.

[0035...

Embodiment 2

[0039] Example 2 Rubescensine A targets ubiquitination to degrade Bcr-Abl protein

[0040] K562 cells were inoculated, and after 24 hours of culture, three proteasome inhibitors MG-132 (M, 10 μM), ALLN (A, 100 μM) or Lactacystin (L, 25 μM) were added for pretreatment for 2 hours, and then 20 μM winter Induction by iconidin. After 2 hours, the cells were collected and the total protein of the cells was extracted by SDS method for western blot detection. MG-132, ALLN and Lactacystin could completely inhibit the degradation of Bcr-Abl and c-Abl proteins by oridonin ( Figure 6 ), indicating that the degradation of Bcr-Abl and c-Abl by Rubescensin A depends on the ubiquitin-proteasome pathway.

[0041] K562 cells and KU812 cells were inoculated, cultured for 24 hours, and then pretreated with proteasome inhibitor MG-132 for 2 hours, and then induced with 20 μM oridonin. After 2 hours, the cells were collected and the total protein of the cells was extracted by SDS meth...

Embodiment 3

[0042] Example 3 Rubescensine A inhibits the growth of Ph-positive tumor cells and induces irreversible apoptosis

[0043] K562 cells were inoculated and treated with 0-20 μM oridonin after 24 hours of culture. After 24 hours of induction, the cell survival rate was detected by MTT, and the cells were collected to detect cell apoptosis by AnnexinV-FITC / PI staining. Under the treatment of different concentrations of oridonin, K562 cells and KU812 cells experienced different degrees of growth inhibition and apoptosis, and showed an obvious dose-dependent effect. 20 μM Oridonin A treatment for 24 hours, the growth of K562 cells and KU812 cells was significantly inhibited ( Figure 7 A, Figure 8 A), the survival rates were only 32% and 21%, respectively, and significant apoptosis occurred, the apoptosis rate was greater than 50% ( Figure 7 B, Figure 8 B).

[0044] K562 cells were inoculated and treated with 0-20 μM oridonin after 24 hours of culture. After 24 hours of i...

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Abstract

The invention discloses an oridonin which can be used for treating Philadelphia chromosome positive tumor through degradation of Bcr-Abl fusion protein and relates to an application of the oridonin in preparing a Philadelphia chromosome positive tumor treatment medicine. A large number of studies show that the oridonin is capable of activating Bcr-Abl specific ubiquitination degradation ways and rapidly and efficiently degrading Bcr-Abl oncoprotein, and thereby Philadelphia chromosome positive tumor cells are subjected to remarkable growth inhibition and irreversible cell apoptosis. In a mouse body, the oridonin also can degrade the Bcr-Abl protein and remarkably inhibit growth of the Philadelphia chromosome positive tumor. The oridonin is mainly derived from rabdosia rubescens which is a traditional Chinese medicine and the oridonin has slight toxic effect on human body and is easy to prepare and high in applicability. Besides, the action mechanism of the oridonin is completely different from STI-571 (gleevec) which is a first-line medicine used for leukemia clinical treatment presently, and thereby the oridonin can be used as a potential medicine for treating STI-571 tolerant Philadelphia chromosome positive tumor.

Description

technical field [0001] The invention discloses a method and reagent for targeted degradation of Bcr-Abl protein and its application in the preparation of Ph-positive tumor treatment drugs, belonging to the technical fields of medicine, biochemistry and cell biology, and specifically relates to the application of oridonin in pharmaceuticals and In clinical treatment, target ubiquitination to degrade pathogenic protein Bcr-Abl and inhibit and kill Ph-positive tumor cells, as well as in clinical and basic research as an inducer to analyze the biological functions of Bcr-Abl and its related signaling pathways. Background technique [0002] Tumor is the number one killer threatening human health. In 2008, there were 12.7 million new cancer patients and 7.6 million deaths worldwide, and these numbers are still growing. With the development of modern medicine, people have gradually realized that the formation of tumors is caused by gene mutations, and thus developed a variety of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/352A61P35/00A61P35/02
CPCA61K31/352A61P35/00A61P35/02
Inventor 黄慧琳翁桁游周惠屈良鹄
Owner SUN YAT SEN UNIV
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