CN107823205A

CN107823205A - Application of the jamaicin in anti-acute lymphoblastic leukemia with positive Philadelphia chromosome medicine is prepared

Info

Publication number: CN107823205A
Application number: CN201711318456.2A
Authority: CN
Inventors: 费嘉; 费雲昊; 阴钊
Original assignee: Guangzhou Disheng Biological Medicine Technology Co Ltd
Current assignee: Guangzhou Disheng Biological Medicine Technology Co Ltd
Priority date: 2017-12-12
Filing date: 2017-12-12
Publication date: 2018-03-23

Abstract

The present invention discloses a kind of application of jamaicin in anti-acute lymphoblastic leukemia with positive Philadelphia chromosome medicine is prepared, and belongs to biomedicine field.Present invention demonstrates that jamaicin can be directly targeted BCR ABL albumen, BCR ABL albumen in degrade Ph+ drug-resistant cell strains and non-resistant cell strain.BCR ABL protein degradation mechanism is further studied, it is found that jamaicin can be targetted with reference to BCR ABL albumen, and passes through autophagy approach degraded BCR ABL albumen.And horizontal in toy, jamaicin can also suppress T315I tumour growths, and only not traditional Chinese medicine jamaicin have found new target spot, also a kind of possible therapeutic scheme has been looked for for Ph+ALL treatments.

Description

Jamaicin is in anti-acute lymphoblastic leukemia with positive Philadelphia chromosome medicine is prepared Application

Technical field

The invention belongs to biomedicine field, and in particular to a kind of jamaicin is preparing the acute leaching of anti-Philadelphia Chromosome Positive Application in bar chronic myeloid leukemia medicine.

Background technology

Acute lymphoblastic leukemia with positive Philadelphia chromosome (Ph+ALL) accounts for the 20%~40% of adult ALL, has The Ph chromosomes and/or BCR/ABL1 of characteristic, the clinical characters such as conventional chemotherapy remission rate is low, recurrence rate is high.Philadelphia chromosome Positive Acute Lymphoblastic Leukemia (Ph+ALL), it is one of grade malignancy highest leukaemia, its complete remission rate is low and holds The continuous time is short, although 2 years disease-free survival rates are up to 50% after allo-HSCT transplanting, high recurrence rate is up to 30%.Although junket ammonia Acid kinase inhibitor (Tyrosine kinase inhibitor, TKI) Small dose injection chemotherapy significantly improves Ph+ALL's Remission rate, but alleviate that the duration is short, high recurrence rate, and also also serious influence be present in the highly resistance to TKI such as T315I mutation TKI treats Ph+ALL long-term efficacy [5], therefore there is an urgent need to find new therapy target and strategy.

Jamaicin is also known as berberine, is the main active of Chinese Drug Rhizomes of Coptis, is that one kind is stabilized alkaloid, extensively It is present in such as among Berberidaceae, Ranunculaceae various plants, and is easily obtained [1].Jamaicin is (such as acute in hematological system tumor Myelogenous leukemia, AML) in show strong anti-tumour cell proliferative activity [2].There is allusion quotation in jamaicin energy inducing leukemia cell The apoptosis morphology change of type, for example there are the regular fractures of DNA, the exemplary ribbon change [3] of apoptosis is presented in electrophoresis.

There is lot of documents to report that it has multiple biological activities and drug effect, and jamaicin is early widely used to face Bed.Clinical practice at present is widely the functions such as its clearing heat and detoxicating and antibacterial, and its blood sugar reducing function is that have diabetes in treatment Diarrhea patient is chanced on.Lot of experiments shows that jamaicin can improve the expression [6] of insulin receptor, promote glycolysis [7], strengthen insulin sensitivity [8] etc., there is good hypoglycemic effect, natural, toxic side effect is small standby because jamaicin has It is concerned.There is document report to 32 high cholesterol patient's oral hydrochloride jamaicin pieces, 0.5g/ times, 2 times/d, treat 3 months After can make serum total cholesterol (TC), LDL-C (LDL-C) and triacylglycerol (TG) are horizontal to reduce respectively 29%th, 25%, 35%, result of study shows that jamaicin lipid-lowering effect is ideal [9].

Jamaicin has had certain basis to the research in leukaemia.Studies have found that jamaicin can influence it is white The distribution of blood disease cell cycle, jamaicin can not influence cell periodic protein B 1 (CyclinB) mRNA expression in the case of, Reduce CyclinB protein levels and increase the expression of Wee1 albumen, so that cyclin Cdc25c activity reduces, make Leukaemia is arrested in the G2/M phases [10].Jamaicin can cause active oxygen with the apoptosis of inducing leukemia cell, jamaicin Release, cause injury of mitochondria, discharge CytoC, activate caspsae3, so as to trigger the Apoptosis way of caspase dependences Footpath [11]；Also detect that suppression apoptotic proteins Survivin expression quantity is lowered in the K562 cells of barberry alkali process [12], illustrate that jamaicin may promote the apoptosis of leukaemia by number of ways.Jamaicin can also cooperate with neoplasm necrosis The apoptosis induction ligand related inducing acute Lymphoblastic Leukemia Cells apoptosis of the factor, and tumor necrosis factor α is in tumour phase It is considered as a key regulatory molecule to close in the cell factor of proinflammatory cytokine secretion, and the apoptosis-induced mechanism of jamaicin may relate to And suppress Nuclear factor kappa B/p65 expression and caspase-3, caspase-8 shearing activation.Research finds that jamaicin can lure The differentiation of leukaemia is led, jamaicin activity is that 1~8mg/L has more significant induction differentiation to human leukemia cell Effect, with the differentiation of cell, its multiplication capacity also declines [13] therewith.

Autophagy cell death, it is a kind of apoptosis independent of Caspase approach different from apoptosis The complicated catalytic process that approach is internal unnecessary protein and subcellular component is degraded in lysosome, has physiology and disease The double action of reason.Autophagy mainly has 3 types：Huge autophagy, miniature autophagy, the autophagy of molecular chaperones mediation, it is significantly special Sign is the formation of " autophagic vacuole ".

Cell autophagy is the complicated catalytic process that protein unnecessary in vivo is degraded in lysosome.Autophagy process is by a system Row autophagy GAP-associated protein GAP (Atg) is mediated to complete, and these protein play a role in the different phase that autophagosome is formed.It is such as micro- Pipe associated protein 1 light chain 3 (microtubule-associated protein 1light chain 3, LC3/Atg8) is certainly Bite the labelled protein on body film.The intracellular LC3 albumen that two kinds of forms be present, i.e. LC3- I and LC3- II.LC3 albumen is synthesizing Its C-terminal becomes LC3- I by the cutting of Atg4 protease afterwards, and LC3- I, which is dispersed in, to be distributed in cytoplasm.After autophagosome is formed, LC3- I and phosphatidyl-ethanolamine (phosphatidylethanolamine, PE) coupling form LC3- II and are positioned at autophagosome Inner membrance and outer membrane, and the all-the-time stables of LC3- II be retained on autophagosome film until with lysosome fusion, therefore be used to make For the mark of autophagosome, LC3- II level reflects the quantity of autophagosome, the mark [14] formed as autophagy. Beclin1 is a required molecule in autophagosome (autophagosome) forming process, and it can participate in the formation of autophagosome. It is advantageous to the aggregation and assembling of phagocytic vacuole, this formation [15] of effect dependent on Beclin1-PI3KC3 complexs.Beclin1 Other autophagy GAP-associated protein GAPs can also be mediated to be positioned at phagocytic vacuole (phagophore), so as to regulate and control mammal autophagosome Formed and maturation.The expression of Beclin1 albumen often rises in autophagy process.P62/SQSTM1 is a kind of multi-functional Ubiquitin-binding proteins, participate in Ubiquitin-proteasome systerm (ubiquitin-proteasome system, UPS) and autophagy-lyase System two kinds of albumen degradation processes [16] of system.Autophagy substrate protein white matter generally in the more poly-ubiquitin chains of K63 or K27 position marks, and Interacted with P62/SQSTM1 UBA domains, substrate is loaded into P62/SQSTM1 oligomer, it is few to form P62/SQSTM1 Aggressiveness and autophagy substrate protein white matter or the aggregation of organelle, autophagosome is entered by P62/SQSTM1 LIR regions targeting, Degraded is in the selective autophagy process of some protein in lysosome, 403 serines of P62/SQSTM1 UBA domains Residue phosphorylation can substantially speed up autophagy process [17].Autophagy generation after, be positioned at autophagosome inner membrance LC3-II and from Bite and tolerant degraded [18] by lysosomal enzyme together in vivo.Therefore, P62/SQSTM1 albumen can also be degraded in autophagy process, table Reduction is showed up to level.Chloroquine is autophagy inhibitor, the chloroquine induction pH enhancings of protonation, causes lysosome activity to be pressed down System, so as to block the degraded of autophagy lysosome.

With inducing leukemia cell autophagy cell death can occur for many medicines of studies have shown that.Such as resveratrol (RSV) p62/SQSTM1 overexpression is mediated with the activation of AMPK approach by JNK approach to promote CML that autophagy cell occurs Dead [19], and curcumin can by the time and it is dose-dependent in a manner of suppress the activity of K562 cells, further research confirms Curcumin can be added after handling cell with autophagy inhibitor with inducing cell apoptosis and autophagy, and its cell death induced is reduced [20]。

Autophagy is played an important role in tumor development, turns into the new focus of current cancer research, may be pernicious The treatment of tumour plays an important role, and may provide a kind of new approach for the treatment of malignant tumour.Autophagy can promote The death of tumour cell, existing evidence show that autophagy death is probably the main side of death of neoplastic cells in some tumours Formula, such as arsenic trioxide (As₂O₃) can be by raising Beclin1 expression, the autophagy of inducing cell is dead, so as to slow Solve the state of an illness [4] of lymphocytic leukemia and Huppert's disease.

The content of the invention

The shortcomings that in order to overcome prior art and deficiency, it is an object of the invention to provide a kind of jamaicin to prepare anti-expense Application in the Chromosome Positive Acute lymphocytic leukemia medicine of city.

The purpose of the present invention is achieved through the following technical solutions：

The present invention provides a kind of jamaicin answering as the direct target of the BCR-ABL protein of ALL With；Particularly as acute lymphoblastic leukemia with positive Philadelphia chromosome BCR-ABL protein direct target application.

Further, jamaicin answering in anti-acute lymphoblastic leukemia with positive Philadelphia chromosome medicine is prepared With.

Described anti-acute lymphoblastic leukemia with positive Philadelphia chromosome medicine, including jamaicin or its can pharmaceutically connect The salt received, and the solvated compoundses of jamaicin or its pharmaceutically acceptable salt, enantiomter, diastereoisomer, mutually The mixture of tautomeric or its arbitrary proportion, including racemic mixture；

Described anti-acute lymphoblastic leukemia with positive Philadelphia chromosome medicine can also contain one kind either at least Two kinds of pharmaceutically acceptable carriers；

Described carrier be preferably sustained release agent, excipient, filler, adhesive, wetting agent, disintegrant, sorbefacient, Absorption carrier, surfactant or lubricant etc.；

Injection, piece can be further made in described anti-acute lymphoblastic leukemia with positive Philadelphia chromosome medicine The diversified forms such as agent, granula or capsule, the medicine of various formulations can be prepared according to the conventional method of pharmaceutical field；

Application of the above-mentioned jamaicin in BCR-ABL protein degradation products are prepared.

Inquire into whether traditional Chinese medicine monomer jamaicin is directly targeted degraded BCR-ABL, and its Mechanism Study of degraded.

Method：

1st, the present invention probes into degradation of the jamaicin to BCR-ABL protein first, with the barberry alkali process of 5 μM of concentration BaF3-P210, BaF3-P210-T315I cell, Western blot detect the change of BCR-ABL and ABL1 protein levels.

2nd, then we employ molecular simulation docking (softwares of Discovery Studio 4.5) and surface plasma is total to Whether imaging (SPRi) technology for detection of shaking jamaicin directly acts on ABL1 albumen.

3rd, in order to further probe into the mechanism of barberry alkaline degradation BCR-ABL protein, we use the jamaicin of various concentrations Processing 24h has been carried out to BaF3-P210 and BaF3-P210-T315I cells, then detected using Western blot method The expression of autophagy GAP-associated protein GAP in barberry alkali process cell.

4th, we have been carried out pre- using autophagy inhibitor chloroquine (CQ) to BaF3-P210 and BaF3-P210-T315I cells Processing, then using the above-mentioned cell 48h of barberry alkali process, Western blot detection BCR-ABL protein expressions.

5th, we use nude mice into tumour caused by T315I in the model construction animal body of knurl, and carry out the dry of jamaicin Pretreatment, the influence of BCR-ABL protein in influence and tumour of the observation jamaicin to tumour caused by T315I.

As a result：

1st, the BCR-ABL protein that jamaicin is significantly reduced in BaF3-P210 and BaF3-P210-T315I cells is horizontal.

2nd, molecular docking simulation is carried out to jamaicin and BCR-ABL protein using the softwares of Discocery Studio 4.5, As a result show that BCR-ABL shares 2 position (SH₂Region and F- actins join domain) can be with jamaicin molecular docking.

3rd, surface plasma resonance imaging (SPRi) confirms that jamaicin can be with three domain (SH of ABL1 albumen₃- SH₂, PTK and F-actin) directly in conjunction with.

4th, after (0,1,3, the 5 μM) effect of various concentrations jamaicin, to CML cells BaF3-P210 and BaF3-P210-T315I Autophagy level is detected in cell, it is found that it is horizontal jamaicin significantly promotes the autophagy in Ph (+) cell.

5th, BaF3-P210 and BaF3-P210-T315I cells are pre-processed using autophagy inhibitor chloroquine (CQ) Afterwards, the processing of jamaicin is carried out, it is found that the BCR-ABL of jamaicin mediation degraded is inhibited, illustrates jamaicin to BCR- In ABL degradation process, autophagy played an important role.

6th, jamaicin can significantly inhibit the growth of nude mouse tumor caused by T315I, and SABC detection jamaicin The expression of tumour BCR-ABL protein after effect, it is found that it is horizontal jamaicin can significantly reduce BCR-ABL in tumour.

Conclusion：Jamaicin has targetted ABL1 albumen, and by autophagy approach degraded BCR-ABL protein, in Mice Body Jamaicin can significantly inhibit the growth of tumour caused by T315I

The present invention is had the following advantages relative to prior art and effect：

Present invention demonstrates that jamaicin can be directly targeted BCR-ABL protein, degrade Ph+ drug-resistant cell strains and non-resistant cell BCR-ABL protein in strain.BCR-ABL protein degradation mechanism is further studied, it is found that jamaicin can be targetted with reference to BCR-ABL eggs In vain, and pass through autophagy approach degrade BCR-ABL protein.And horizontal in toy, jamaicin can also suppress the life of T315I tumours Long, only not traditional Chinese medicine jamaicin have found new target spot, also look for a kind of possible therapeutic scheme for Ph+ALL treatments.

Brief description of the drawings

Fig. 1 be molecular simulation docking prediction jamaicin directly and ABL1 Protein Ss H₃-SH₂, the combination of F-actin domains；Its In, A：The F-actin join domains (1ZZP) of BCR-ABL protein carboxyl terminal (1519aa-1644aa) and jamaicin simulation pair Connect；B：The SH of ABL1 albumen₃Domain (4J9I) docks with jamaicin simulation.

Fig. 2 is the combination between surface plasma resonance imaging (SPRi) checking jamaicin and ABL1 domains；Wherein, A： ABL1 protein structure domain protein purification result figures；B：The positive control figure of jamaicin chip；C：Jamaicin and ABL1 domain proteins It is white to combine figure；D：The SH of jamaicin and various concentrations₃-SH₂With reference to SH₃-SH₂Set gradually from below to up for 250nM, 500nM, 1000nM、2000nM；E：Jamaicin and various concentrations PTK protein bindings, PTK set gradually from below to up for 100nM, 200nM, 400nM、800nM；F：Jamaicin and various concentrations F-actin protein bindings, F-actin set gradually from below to up for 100nM, 200nM、400nM、800nM。

Fig. 3 is the influence horizontal to BCR-ABL protein of Western blot detection jamaicins；Wherein, A：Barberry alkali process BaF3-P210 cells 12,24,48h；B：Barberry alkali process BaF3-P210-T315I cells 12,24,48h.

Fig. 4 is the BCR-ABL protein in jamaicin autophagy approach degraded CML cells；Wherein, A：Various concentrations jamaicin is made With rear, the change of autophagy GAP-associated protein GAP (LC3, Beclin1, P62) in BaF3-P210 cells；B：Various concentrations jamaicin acts on Afterwards, in BaF3-P210-T315I cells autophagy GAP-associated protein GAP (LC3, Beclin1, P62) change.

Fig. 5 is the influence of degraded of the jamaicin to BCR-ABL protein；Wherein, A：BaF3-P210 cells first use CQ (10 μ M), 4h is pre-processed, then using barberry alkali process 48h, detects the change that BCR-ABL protein is expressed in cell；B：BaF3- P210-T315I cells first use CQ (10 μM), pre-process 4h, then using barberry alkali process 48h, detect BCR-ABL in cell The change of protein expression.

Fig. 6 is jamaicin to the suppression under T315I cell skins into knurl；Wherein, A：Each group processing is to Balb/c nude mouse The influence of subcutaneous T315I tumor sizes；B：Influence of each group processing to the subcutaneous T315I tumour growths of Balb/c nude mouse；C： Tumour is directed to ABL1 ImmunohistochemistryResults Results between each group；D：Influence of each group processing to ABL1 protein expressions in mouse tumor.

Embodiment

With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited In this.

Hyclone and RPMI-1640 culture mediums are purchased from Gibco companies；RIPA cell pyrolysis liquids are purchased from green skies company； Bradford determination of protein concentration kit is century biology Co., Ltd purchased from Beijing section；C-ABL monoclonal antibodies are purchased from U.S. SANTA CRUZ companies of state；P62 monoclonal antibodies are purchased from Sigma companies；Beclin1 monoclonal antibodies are purchased from CST companies； GAPDH and β-actin antibody is century purchased from health；Mouse source secondary antibody IgG-HRP is purchased from SANTA CRUZ companies of the U.S.；Anti-Bcr Antibody is purchased from American AB CAM companies.

BaF3-P210, BaF3-P210-T315I cell line are teaching and research by medical test system of Medical University Of Chongqing clinical hematology Room provides (Effect study [D] Chongqing medical courses in general of the Chen Xi interference Apg-2 genes in BaF3-p210 and BaF3-p210-T3151 University, 2012.)

Whether the molecular simulation docking of embodiment 1 and surface plasma resonance imaging (SPRi) checking jamaicin are directly targeted BCR-ABL1 albumen

1st, molecular simulation is docked

The PDB files of ABL1 protein three-dimensional space structures are found from Protein Data Bank (Protein data bank)； The three-D space structure PDB files of jamaicin are found in drug data base.Free use is downloaded from Discovery official website The softwares of Dicovery Studio 4.5.After having downloaded software Discovery Studio 4.5, by jamaicin and ABL1 albumen The three-dimensional structure of domain, which imports, to be entered in software, using receptor-ligand binding modules, observation jamaicin and ABL1 albumen SH₃-SH₂, PTK, F-actin domain combination situations, use 2d display modules, 3d display modules, display jamaicin and ABL1 eggs The amino acid that white 3 domains are combined.

In order to probe into whether jamaicin directly acts on ABL1 albumen, we employ molecular simulation docking and surface etc. from The technology of sub-resonance imaging is studied it, as shown in figure 1, the software predictions of Discovery studio 4.5, jamaicin Can be with the SH in BCR-ABL₃-SH₂Region and F-actin calmodulin binding domain CaMs are to connecting.

2nd, the combination situation of surface plasma resonance imaging (SPRi) checking tri- domains of jamaicin and ABL1

(1) ABL1 SH₃-SH₂, PTK, and the expression of F-actin domain proteins

ABL1 protein structure domain plasmids are synthesized in reactivation gene

1) the μ L of plasmid 1 are added in 100 μ L Arctic Expression (DE3) competence bacteriums, be placed on ice 20min；

2) 42 DEG C of heat shocks 90 seconds, are placed on rapidly 3min in ice；Add 600 μ L LB nutrient solutions；

3) 37 DEG C, 220rpm shaking culture 1h, 50 μ L bacterium solutions are taken to be coated on the LB flat boards containing 50 μ g/mL Kan, 37 DEG C are fallen Put overnight incubation；

4) monoclonal on picking in the next morning flat board is inoculated in the test tube of the 4mL LB nutrient solutions containing 50 μ g/mL Kan In, 37 DEG C, 220rpm shakings culture to 1 point or so of afternoon, OD about 0.6；

5) 1 is pressed：250 ratios, it is inoculated in 50 μ g/mL Kan 1L LB nutrient solutions, 37 DEG C, 220rpm shaken to thalline OD600 is 0.5~0.6 (about 3h)；

6) derivant IPTG to final concentration of 0.1mM, 16 DEG C of cultures of 220rpm are proportionally added in 1L fermentation mediums Overnight；

7) 5000rpm, 5min centrifugation go supernatant to collect fermentation thalli, and -20 DEG C preserve purifying to be broken.

(2) broken and albumen the purifying of thalline

1) ultrasonication bacterial cell disruption condition：350W power, 4s is crushed, be spaced 6s, totally 180 circulation, break bacterium buffer solution： 20mM Tris, 500mM NaCl, pH8.0.

2) the thalline 12000rpm after Ni-IMAC (5mL) purifying is broken, 20min is centrifuged, 4 DEG C, supernatant is collected and carries out nickel post Purifying, wire feeding Ni-IMAC.Balancing formula of liquid is：20mM Tris, 500mM NaCl, pH8.0, eluent：20mM Tris, 500mM NaCl, 500mM imidazole, pH8.0.Respectively using containing 20mM/50mM/200mM/500mM Imidazole 4 kinds of eluents are eluted, and are collected eluent according to absorption peak, after protein purification comes out, are carried out SPRi examinations Test.

(3) surface plasma resonance imaging experiment (SPRi)

By Berberine hydrochloride point sample on 3D photo-crosslinking chips, as antigen, the positive of chip is used as using rapamycin Control, using the albumen being purified into as mobile phase, is circulated, flux buffer is 1 × PBS (pH=7.4), by SH₃-SH₂If 250nM, 500nM, 1000nM, 2000nM, 4 concentration gradients are put to be circulated, PTK sets 100nM, 200nM, 400nM, 800nM, 4 concentration gradients are circulated, and F-actin sets 100nM, 200nM, 400nM, and 800nM is circulated, and merchantable thing enters Sample speed is 2 μ Ls^-1, time 300s, react and carried out under conditions of 25 DEG C, analysis SPRi analysis traditional Chinese medicine monomer jamaicins With SH₃-SH₂Deng the kinetic parameter of albumen, analyze it and combine situation.

The sample and information used in the SPRi of table 1 analyses

The experimental method of table 2 and parameter designing

Using the method for protein purification by ABL1 3 domain (SH₃-SH₂/ PTK/F-actin) protein purification is carried out, Using surface plasma resonance imaging technology, transactional analysis experiment is carried out to ABL1 and jamaicin, as a result such as Fig. 2 is shown, Jamaicin can directly and ABL1 3 domains combine.

Table 3 analyzes jamaicin and SH by SPRi₃-SH₂Etc. protein bound dynamics, affine profile

In summary, in the jamaicin target intracellular to Ph+ALL is probed into, this research uses Discovry Studio 4.5 softwares carry out molecular docking simulation to characteristic protein BCR-ABL in jamaicin and Ph+ cells, detect barberry base molecule knot Structure and BCR-ABL protein binding ability and avtive spot.As a result as Fig. 1 shows that ABL1 shares 3 domain (SH₂Region, albumen Kinase region and F- actins join domain), jamaicin can be with SH therein₂Domain, F-actin domains are mutually tied Close.Then we employ the checking that the technology of surface plasma resonance imaging is tested to it, and we are by ABL1 albumen 3 domains be purified, as shown in Figure 2 A, tri- domain purifying protein purity of ABL1 have all reached more than 80%, symbol Surface plasma resonance imaging requirement of experiment is closed, we are tested by surface plasma resonance imaging, and jamaicin can be with ABL1 3 domains are combined, with reference to quick and stably, these results indicate that jamaicin can directly and ABL1 albumen phases With reference to.

Embodiment 2

1st, Western Blot detect BCR-ABL protein expression in BaF3-P210 and BaF3-P210-T315I

Experiment divides jamaicin group (jamaicin treatment group is divided into 12h, 24h, 48h treatment groups again) and blank group, jamaicin dense 5 μM of degree selection.Cell kind is in six orifice plates.

1) after acting on 48h, cell is collected in 15mL centrifuge tubes, cell is collected by centrifugation in 1500rpm, 5min, abandons supernatant.

2) PBS washes cell, and piping and druming is uniform, and cell is collected by centrifugation in 1500rpm, 5min.

3) according to cell precipitation amount, add appropriate cell pyrolysis liquid and crack cell completely.30min is cracked on ice.

4) 4 DEG C of high speed centrifugations, 12000rpm centrifugations 30min.

5) supernatant is drawn into another clean EP pipe, according to the specification of Coomassie brilliant blue determination of protein concentration kit Determine protein concentration.Sample-loading buffer is added in proportion, calculates the applied sample amount needed for 50 μ g albumen.

6) after adding sample-loading buffer, 5min is boiled in boiling water bath.

7) SDS-PAGE glue, loading, 80V electrophoresis 30min, 120V electrophoresis 1h are prepared.

8) electrophoresis terminates, transferring film.According to being followed successively by from top to bottom in wet rotary device：Filter paper, film, gel, filter paper, use glass Glass rod catches up with bubble of calming the anger, and carries out wet turn.The albumen of different molecular weight carries out transferring film using different transferring film conditions.

9) after transferring film terminates, closing.Pvdf membrane is put into the confining liquid of 5% skimmed milk power, under room temperature condition, in shaking table On shake 1h.

10) after closing terminates, primary antibody is incubated.Antibody is proportionally diluted, in the antibody that pvdf membrane is put into.On 4 DEG C of shaking tables It is incubated overnight.

11) it is incubated after terminating, TBST washings pvdf membrane is three times.5min is washed on each shaking table.

12) it is incubated at room temperature secondary antibody.The secondary antibody of proper ratio concentration is chosen, is diluted with antibody diluent.Pvdf membrane is put into In the secondary antibody diluted.

13) it is incubated after terminating, TBST washings pvdf membrane is three times.5min is washed on shaking table.

14) chemiluminescence：Luminescent solution (A, B liquid 1 is prepared to specifications:1 mixes), luminescent solution is uniformly added drop-wise to PVDF On film, it is put into the gel imaging systems of ALLIANCE 4.7 and is taken pictures.

15) the albumen relative expression levels such as BCR-ABL are compared with internal reference albumen (β-actin or GAPDH).

After barberry alkali concn chooses 5 μM of processing BaF3-P210 and BaF3-P210-T315I cells 12,24,48h, extraction is total Albumen, Western blot detect the jamaicin influence horizontal to BCR-ABL protein.As a result as shown in figures 3 a-b, jamaicin shows Writing reduces the level of intracellular BCR-ABL protein.

(2) it is horizontal to detect the intracellular autophagy of BaF3-P210 and BaF3-P210-T315I by Western blot

After various concentrations jamaicin (0,1,3,5 μM) processing BaF3-P210 and BaF3-P210-T315I cells 24h, 24h Total protein is extracted, Western blot detect LC3- II, the P62 in cell, and Beclin1 protein expression.

In order to explore the mechanism that jamaicin causes BCR-ABL protein to be degraded, Western blot check the autophagy water of cell It is flat.Jamaicin handles BaF3-P210/BaF3-P210-T315I cell 12h, 24h and 48h respectively, as a result as shown in figures 4 a-b. The albumen of LC3- II has increased trend as jamaicin processing time increases in figure, and Beclin1 albumen has increased trend, P62 The trend that albumen is reduced.As a result it is horizontal to show that jamaicin promotes the autophagy in CML cells.

The effect that detection autophagy is degraded to BCR-ABL protein：

Using autophagy inhibitor chloroquine (5 μM) pretreatment cell 4h, then with 5 μM of barberry alkali process BaF3-P210 and BaF3-P210-T315I cell 48h, then extract total protein, and Western blot detect the change of BCR-ABL protein in cell Change.

After using autophagy inhibitor chloroquine pretreatment 4h, barberry alkali process BaF3-P210/BaF3-P210-T315I is thin Born of the same parents 48h, as a result as shown in figures 5 a-b, there occurs inhibitory action for degraded of the jamaicin to BCR-ABL protein.

(3) nude mice by subcutaneous is tested into knurl confirms that jamaicin can suppress the growth of tumour caused by T315I

It is 8 × 10 by density to probe into influence of the jamaicin to Balb/c nude mouse T315I tumor sizes⁶Individual/ ML BaF3-P210-T315I cells are inoculated with 200 μ L to Balb/c nude mouse left lower extremities oxters, observe it into knurl situation, Mouse to carrying out even group-division into knurl mouse, and carries out jamaicin (15mg/kg/d) and Imatinib (50mg/kg/ into after knurl D) intraperitoneal injection experiment, the daily change for measuring mouse tumor size, and take mouse tumor to carry out immune group after 15d is treated Change experiment.Such as Fig. 6 A, B, jamaicin significantly inhibits the growth of the mouse tumor caused by nude mice by subcutaneous T315I, Fig. 6 C, D degradeds BCR-ABL and ABL1 expression in nude mouse tumor caused by T315I.

In summary, further study show that, jamaicin can reduce Ph+ALL cells BaF3-P210 and BaF3-P210- BCR-ABL protein expression quantity such as Fig. 3 A, B in T315I cells.As illustrated, Ph+ cell lines are after barberry alkali process, BCR-ABL protein has obvious degradation phenomenon.In order to probe into the degradation mechanism in Ph+ cells Berberine to BCR-ABL protein, We are detected to the autophagy level in Ph+ cells, such as Fig. 4 A, shown in B, it has been found that with jamaicin activity Increase, autophagy marker protein LC3- II increases in cell, Beclin1 increases, and autophagy substrate connection albumen P62 is reduced, and is said It is horizontal that bright jamaicin promotes autophagy overall in cell.

In order to probe into whether barberry alkaline degradation BCR-ABL process has the participation of autophagy approach, the suppression of autophagy is employed Agent chloroquine (CQ) is suppressed to the autophagy approach of cell, such as Fig. 5 A, shown in B, it is found that small after autophagy inhibitor is added Degraded of the bark of a cork tree alkali to BCR-ABL is inhibited by, and illustrates jamaicin in BCR-ABL degraded, autophagy approach plays important work With.

The influence for the Ph+ cells that jamaicin is mutated to T315I is probed into animal level, we use nude mice by subcutaneous into knurl The Ph+ mouse models jamaicin and jamaicin and Imatinib Combined Treatment of the T315I mutation of structure, can be significantly inhibited naked The growth of the subcutaneous T315I tumours of mouse, and the BCR-ABL protein in the T315I tumours that can significantly degrade, such as Fig. 6 C, shown in D, Illustrate in model of the nude mice by subcutaneous into knurl, jamaicin plays inhibitory action and the degraded to BCR-ABL protein to T315I cells Important role.

In a word, jamaicin can be directly targeted BCR-ABL protein, in degrade Ph+ drug-resistant cell strains and non-resistant cell strain BCR-ABL protein.BCR-ABL protein degradation mechanism is further studied, it is found that jamaicin can be targetted with reference to BCR-ABL protein, And pass through autophagy approach degraded BCR-ABL protein.And horizontal in toy, jamaicin can also suppress T315I tumour growths, It is not only that traditional Chinese medicine jamaicin have found new target spot, has looked for a kind of possible therapeutic scheme for Ph+ALL treatments yet.

Finally, draw the following conclusions：

1st, jamaicin, which is directly targeted, acts on BCR-ABL；

2nd, in the BCR-ABL of jamaicin mediation degraded, autophagy plays an important role；

3rd, for mouse subcutaneously into knurl model, jamaicin can significantly inhibit the life of the nude mice by subcutaneous tumour caused by T315I It is long, and the expression of BCR-ABL protein in tumour can be suppressed.

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Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

Claims

1. application of the jamaicin as the direct target of the BCR-ABL protein of ALL.

2. application according to claim 1, it is characterised in that：

Application of the jamaicin as the direct target of the BCR-ABL protein of acute lymphoblastic leukemia with positive Philadelphia chromosome.

3. application of the jamaicin in anti-acute lymphoblastic leukemia with positive Philadelphia chromosome medicine is prepared.

4. application according to claim 3, it is characterised in that：

Described anti-acute lymphoblastic leukemia with positive Philadelphia chromosome medicine, including jamaicin or its is pharmaceutically acceptable Salt, and the solvated compoundses of jamaicin or its pharmaceutically acceptable salt, enantiomter, mutually diastereoisomer, variation The mixture of structure body or its arbitrary proportion, including racemic mixture.

5. the application according to claim 3 or 4, it is characterised in that：

Described anti-acute lymphoblastic leukemia with positive Philadelphia chromosome medicine contains a kind of either at least two pharmaceutically Acceptable carrier.

6. application according to claim 5, it is characterised in that：

Described carrier is sustained release agent, excipient, filler, adhesive, wetting agent, disintegrant, sorbefacient, absorption load Body, surfactant or lubricant.

7. the application according to claim 3 or 4, it is characterised in that：

Described anti-acute lymphoblastic leukemia with positive Philadelphia chromosome medicine be further made injection, tablet, granula or Capsule.

8. application of the jamaicin in BCR-ABL protein degradation products are prepared.