CN111773215A

CN111773215A - Medicine for treating AML and application thereof

Info

Publication number: CN111773215A
Application number: CN202010752873.3A
Authority: CN
Inventors: 曾辉; 王惠雯; 展会恩; 姜欣雅
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-07-30
Filing date: 2020-07-30
Publication date: 2020-10-16

Abstract

The invention discloses a medicine for treating AML, which is an AP-1 inhibitor T5224, and the application of the AP-1 inhibitor T5224 in preparing an anti-AML medicine; the AP-1 inhibitor T5224 can inhibit the proliferation of cells and promote the apoptosis of the cells in an AML cell strain, and does not cause obvious influence on the proliferation of a normal cell strain; the T5224 combined with cytarabine acts on AML cell strains, and the inhibition on AML cell proliferation is obviously enhanced compared with the inhibition on AML cell proliferation by singly using cytarabine; t5224 can enhance the sensitivity of AML cells to cytarabine, a chemotherapeutic drug, and promote the killing effect of cytarabine on AML cells.

Description

Medicine for treating AML and application thereof

Technical Field

The invention relates to the technical field of antitumor drugs, and particularly relates to a drug for treating AML and application thereof.

Background

Acute Myeloid Leukemia (AML) is a group of highly heterogeneous hematological malignancies caused by clonal proliferation of immature myeloid progenitor cells, and the standard treatment is induction of remission followed by consolidation chemotherapy or hematopoietic stem cell transplantation. Although as early as the 70's of the 20 th century, the combined use of the induction chemotherapy drugs daunorubicin and cytarabine could achieve Complete Remission (CR) in a large number of AML patients; however, in the last 40 years, treatment of AML has progressed slowly, with the exception of Acute Promyelocytic Leukemia (APL), and patient prognosis has remained poor. Therefore, new, effective therapeutic approaches are crucial for improving the prognosis of AML.

Human activin 1 (AP-1) is a homodimer or heterodimer formed by protein families of Jun (C-Jun, Jun-B, Jun-D), Fos (C-Fos, Fos-B, Fra-1, Fra-2), ATF (ATF2, ATF3, LRF1, B-ATF, JDP1, JDP2) and MAF (C-Maf, MafB, MafA, Maf) polygenes. AP-1 is an important transcription factor, participates in gene transcription of various growth factors and cytokines in cells, plays an important role in biological processes such as cell proliferation, apoptosis, differentiation, survival, migration, transformation and the like, and is increased in expression of various solid tumors and blood system tumors. Many studies in the early days have been devoted to the detection of AP-1 activity in tumor cells, but the results showed that its activity did not affect tumor cell survival. Therefore, recent studies have focused on the expression and activity of members of the JUN and Fos families in tumors. Among them, Fra-1 and c-Fos in Fos family are the most widely studied functions in tumors. Previous studies have shown that in hematological tumors, the expression levels of c-Fos, Jun and AP-1 are elevated in AML. Compared with the extensive research data of Fra-1 and c-Fos, FosB, Fra-2, FosB2 and delta FosB2 have been studied in tumors, and the expression and biological effect of FosB in AML has not been clarified. Importantly, FosB, as a proto-oncogene, can increase the invasive potential of breast cancer cells and promote the progression of pancreatic cancer. In pancreatic cancer, miR-144-3p targets proto-oncogene FosB, inhibiting proliferation, metastasis and invasion of pancreatic malignant tumor cells. T5224 is a specific non-peptide small molecule AP-1 inhibitor, which can inhibit the binding of the heterodimer AP-1 formed by Fos and Jun protein and the binding site of the promoter region AP-1, thereby inhibiting the activation of the AP-1 signaling pathway, but the effect in acute myelogenous leukemia is not reported yet.

Disclosure of Invention

The invention discovers that FosB in an AP-1 signaling pathway is remarkably improved in the refractory and recurrent groups of AML by detecting bone marrow samples of patients with acute myelogenous leukemia. The invention thus envisages: whether the AP-1 signaling pathway regulates the proliferation of AML can affect the sensitivity of AML cells to chemotherapeutic drugs. Aiming at the assumption, the invention takes acute myeloid leukemia cell strains and clinical samples as research objects to prove the assumption through experiments, and then, aiming at the proving fact, the invention takes the acute myeloid leukemia cell strains and clinical samples as research objects to discuss the effect of FosB in acute myeloid leukemia cells and the influence of FosB on the sensitivity of chemotherapeutic drug cytosine arabinoside (Ara-C), and verifies that AP-1 inhibitor T5224 can inhibit the proliferation of cells and promote the apoptosis of the cells in the acute myeloid leukemia cell strains under the condition of not obviously influencing the proliferation of normal cell strains, and can enhance the sensitivity of AML cell strains to Ara-C.

The invention aims to solve the problem of providing a new application of T5224, namely a new application in pharmacy. The invention aims to provide application of T5224 in preparing medicines for resisting acute myeloid leukemia and chemotherapy sensitization.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a medicament for treating AML which is the AP-1 inhibitor T5224, said T5224 having the formula:

preferably, the medicament further comprises at least cytarabine.

Preferably, the application of the medicine AP-1 inhibitor T5224 in preparing anti-AML medicines.

Preferably, the medicine takes T5224 as an active ingredient, and is added with one or more pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials comprise diluents, excipients, fillers, adhesives, wetting agents, absorption promoters, surfactants, lubricants, stabilizers, flavoring agents, sweetening agents and pigments which are conventional in the pharmaceutical field.

Preferably, the drug is a sensitizer that increases the sensitivity of AML cells to chemotherapeutic drugs, such as cytarabine.

Preferably, the medicament is a pharmaceutical composition for treating AML, and the composition contains a pharmaceutically effective amount of T5224, a pharmaceutically effective amount of cytarabine and a pharmaceutically acceptable carrier.

Preferably, the medicine is prepared into any pharmaceutically acceptable dosage form, and the dosage form comprises tablets, capsules, granules, oral liquid, sustained release preparations, nano preparations and injections.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention detects the cell killing effect of T5224 on cell strains, and finds that T5224 does not cause obvious influence on the proliferation of human umbilical vein endothelial cells;

2. t5224 can inhibit cell proliferation and promote apoptosis in acute myelogenous leukemia cell strain;

3. the T5224 combined with cytarabine acts on AML cell strains, and the inhibition on AML cell proliferation is obviously enhanced compared with the inhibition on AML cell proliferation by singly using cytarabine; t5224 can promote the killing effect of cytarabine on AML cells, and enhance the sensitivity of AML cells to chemotherapeutic drug cytarabine;

4. the invention provides a new drug for treating acute myeloid leukemia, T5224, which has no toxic or side effect on human body, and can be used together with chemotherapeutic drug cytarabine to promote the killing effect of cytarabine on AML cells, reduce the dose of cytarabine and reduce the toxic or side effect of chemotherapeutic drug on human body.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph of FosB expression in AML patients;

FIG. 2 is a graph showing the expression of FosB in normal cell lines and AML cell lines;

FIG. 3 is a graph showing the results of the proliferation of the stem cells of human umbilical vein endothelial cell line and AML cell line;

FIG. 4 is a graph showing the results of apoptosis of AML cells treated with different concentrations of T5224;

FIG. 5 is a graph showing the results of T5224 in combination with cytarabine on the proliferation of AML cells.

Detailed Description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein the description is for illustrative purposes only and is not intended to limit the scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

A medicament for treating AML which is the AP-1 inhibitor T5224, said T5224 having the formula:

preferably, the medicament further comprises at least cytarabine.

Example 1

First, expression of FosB in AML cell lines and clinical patients

1. Experimental materials:

cell line

Human AML cell lines HL-60 and THP-1 and human umbilical vein endothelial cell line (HUV-EC-E) were purchased from the cell center of university of Central and south China.

Clinical samples

Fresh bone marrow specimens of 72 AML patients were collected using heparin sodium anticoagulant-containing blood collection tubes in an amount of about 2-3mL per specimen.

2. The main experimental method comprises the following steps:

clinical specimen collection

From 2017 to 2020, a total of 72 myeloid cell samples from AML patients were collected, and the diagnosis standard of acute myeloid leukemia was determined according to NCCN Guidelines from 2019. Sample men were 40, women 32, with an age range of 17-67 years, with an average age of 42.1 years. Bone marrow samples of AML patients were obtained from the department of housing and outpatient service of xiangya hospital, central and south university and passed ethical audits. The collected bone marrow cell samples were divided into complete Remission (AML-CR) and relapse/Refractory (delayed/Refractry AML, AML-RR) groups, as shown in Table 1; a total of 34 AML-CR patients and 38 AML-RR patients were collected from the 72 AML patients collected from the specimens.

TABLE 1 basic information of AML patients

Cell culture

Culturing suspension cells THP-1 and HL-60 in a complete culture medium (containing 10% FBS) prepared by RPMI-1640, culturing adherent cells HUV-EC-E in a complete culture medium (containing 10% FBS) prepared by DMEM, and placing the cells in a cell culture box at 37 ℃ and containing 5% saturated humidity carbon dioxide for culturing and subculturing; the growth state of the cells and whether the cells are polluted or not are observed, and the solution is changed according to the cell metabolism condition, and the solution is changed every two days generally.

Extraction of mononuclear cells from bone marrow specimens

(1) Collecting 2-3mL of fresh bone marrow samples of AML patients into a heparan violaceum anticoagulation tube;

(2) mixing fresh bone marrow sample with PBS (precooled at 4 deg.C) in equal volume;

(3) taking a clean 15mL centrifuge tube, adding 2mL human lymphocyte separation liquid into the tube, sucking the diluted bone marrow sample by using a 1000uL pipette, fixing the centrifuge tube at an inclination angle of at least 45 ℃ to avoid shaking, and slowly adding the bone marrow sample onto the lymphocyte separation liquid along the tube wall of the centrifuge tube to ensure that obvious layering occurs between the separation liquid and the liquid level of the bone marrow liquid.

(4) The tube was placed in a centrifuge and centrifuged at 0.4rcf/min for 20 minutes.

(5) Take out the centrifuging tube gently, avoid shaking, liquid divides 4 layers in the visible centrifuging tube, and from the bottom up is in proper order: red blood cell layer, lymphocyte separation liquid layer, milky white mononuclear cell layer and plasma layer. Adding 2mL PBS (precooling at 4 ℃) into another 15mL sterile centrifuge tube, carefully sucking the second mononuclear cell layer by using a 1000uL pipettor, adding the second mononuclear cell layer into the tube, and uniformly mixing the second mononuclear cell layer and the second mononuclear cell layer by blowing;

(6) placing the centrifugal tube in a centrifuge, centrifuging for 5 minutes at a set parameter of 0.2rcf/min, discarding the supernatant to show that white cells are deposited at the tube bottom, and if the cells are reddish, standing and lysing the erythrocyte lysate for 5-8 minutes, repeatedly washing with 4 ℃ precooled PBS solution, and centrifuging for 2 times for later use.

Extraction of RNA

(1) Firstly, collecting AML cell strains which are in logarithmic growth phase and have good growth state, placing the AML cell strains into a 1.5mL sterile EP tube, centrifuging the AML cell strains in a centrifuge at the rotating speed of 1000rpm/min for 5 minutes, then discarding supernatant, and placing the AML cell strains on ice for storage;

(2) adding Trizol with the volume of 1mL into the EP tube after centrifugation, and repeatedly blowing and beating the mixture evenly for a plurality of times by using a 1mL pipette;

(3) adding 200 mu L of chloroform into the mixed solution of the EP tube, manually turning upside down and uniformly mixing for 30 seconds, placing the mixture in room temperature for standing reaction for 5 minutes, and then centrifuging the mixture for 15 minutes at 12000rpm/min at 4 ℃;

(4) the centrifuged EP tube was taken out and placed on ice, and the liquid in the EP tube was divided into 3 layers: the upper layer is clear RNA dissolving solution, the middle layer is flocculent DNA and protein, and the bottom layer is organic solvent such as light red Trizol; carefully pipette the supernatant liquid in an amount of about 400. mu.L into another 1.5mL sterile EP tube;

(5) adding 400 mu L of isopropanol into the EP tube after the supernatant liquid is absorbed, gently shaking for a plurality of times, standing at room temperature for about 10 minutes, and centrifuging at 4 ℃ and 12000rpm/min for 10 minutes; the EP tube is taken out gently to avoid oscillation, and the milky white floccule deposited at the bottom of the EP tube wall is RNA;

(6) discarding the supernatant in the EP tube after centrifugation, adding 1mL of the 75% ethanol which is prepared in situ and precooled at 4 ℃ into the EP tube, placing the EP tube into a centrifuge after slight shaking, centrifuging the EP tube at 4 ℃ of 7500rpm/min for 5 minutes, and taking out the EP tube; removing liquid in the tube, and sucking residual small amount of liquid by using a 10 mu L liquid shifter to avoid sucking precipitated RNA;

(7) placing the liquid-removed EP tube at room temperature, air-drying for about 15-20 min, adding dd water (about 20 μ L), and mixing;

(8) and (3) uniformly oscillating the RNA sample in the EP tube on an oscillator, and detecting the A260/A280 ratio and the RNA concentration on a machine.

Reverse transcription of RNA

(1) Preparing an ice box, placing all components of a reverse transcription reagent (purchased from Guangzhou multifunctional Gene Co., Ltd.: Cat. No. QP007) on ice to be fully dissolved, then gently shaking and uniformly mixing, and placing on ice for standby after short-time centrifugation in a centrifuge;

(2) an RNA-Primer premix was prepared and the reagent components were added to RNase free clean EP tubes according to Table 2.

TABLE 2 RNA-Primer premix reagent Components

(3) Gently shaking and uniformly mixing the RNA-Primer premixed solution, placing the premixed solution in a centrifuge for short-time centrifugation, incubating the premixed solution at the constant temperature of 65 ℃ for 10min, and immediately placing the incubated solution on ice for later use;

(4) preparing a reverse transcription reaction solution, and adding each reagent component into the mixed solution on ice according to the content in the table 3;

TABLE 3 reagent Components of reverse transcription reaction solution

(5) Gently shaking the reverse transcription reaction solution, mixing well, placing in a centrifuge for short-time centrifugation, incubating at 37 ℃ for 60min, and incubating at 85 ℃ for 5min to terminate the reverse transcription reaction;

(6) the cDNA obtained after the reaction was stored on ice for further use.

RT-qPCR quantitative reaction

(1) QPCR SYBR Green Master Mix (purchased from Shanghai assist in san Biotech Co., Ltd., cat # 11202ES08) in the kit was dissolved well on ice and gently shaken well, centrifuged briefly in a centrifuge and placed on ice for use.

(2) Preparing a qPCR reaction system with the volume of 20ul on ice, adding each reagent component into a 96-well nuclease-free plate according to the table 4, centrifuging and mixing uniformly to deposit the reagents at the bottom of the tube, wherein the FosB primer and the GAPDH primer are constructed by Guangzhou multifunctional gene GmbH;

TABLE 4 qPCR reaction System reagent Components

(3) Setting qPCR reaction conditions

The first step, 5 minutes at 95 ℃ and 1 cycle.

The second step is that the temperature is 95 ℃ for 10 seconds, 60 ℃ for 20 seconds and 72 ℃ for 20 seconds, and the circulation is carried out for 40 times.

The third step: dissolution curve generation-cycle 1.

Clinical sample protein sample preparation

(1) Collecting the cell strain in logarithmic growth phase and good growth state, placing in 15mL centrifuge tube, centrifuging in a centrifuge at 1000rpm/min for 5min, sucking off supernatant with a pipette,

(2) washing the cells with 4 deg.C precooled PBS for 2 times, transferring to 1.5mL EP, centrifuging at 1000rpm/min for 5 minutes in a centrifuge, and then removing the supernatant with a pipette;

(3) dissolving a protease inhibitor PMSF preserved at the temperature of minus 20 ℃ and a protein lysate NP40 on ice, and then adding a solvent according to the weight ratio of 1: 100, 1mL of mixed solution is evenly mixed and then placed on ice.

(4) Adding a proper amount of the mixed solution into a 1.5mL centrifuge tube according to the cell amount, standing and cracking on ice, shaking once every 10min, shaking for 10s every time, and shaking for 3 times in total;

(5) after standing and shaking for 30 minutes, carrying out ultrasonic treatment on the lysate for 3 times by using an ultrasonic crusher, wherein each time is 10s, so that the liquid in the EP tube becomes viscous after DNA is cracked;

(6) centrifuging the cracked EP tube at 12000rpm/min at 4 deg.C for 15 min to deposit white precipitate (DNA fragment) on the bottom of the tube;

(7) the EP tube is taken out, the supernatant fluid, namely the protein, is sucked by a pipette gun and is transferred to a new sterile 1.5mL EP tube, and then the protein concentration is measured, or the tube is placed in a refrigerator at the temperature of minus 80 ℃ for storage.

BCA method for determining protein content

(1) Taking out a standard protein sample stored in a refrigerator at the temperature of-80 ℃, and fully dissolving the standard protein sample on ice;

(2) calculating the total amount of the BCA working solution according to the total amount of samples and 200ul per well, and paying attention to the fact that the loss is increased by 1-2 wells, wherein the total amount of the BCA reagent is fully mixed according to the volume ratio of 1:50 and then placed on ice for standby (the BCA reagent comprises the reagent B and the reagent A, wherein the reagent A is 1% BCA disodium salt, 2% anhydrous sodium carbonate, 0.16% sodium tartrate, 0.4% sodium hydroxide and 0.95% sodium bicarbonate, the pH value is adjusted to 11.25 by mixing, and the reagent B is 4% copper sulfate);

(3) adding standard protein samples into a 96-well plate in the volume of 0 μ L, 1 μ L, 2 μ L, 4 μ L, 8 μ L, 12 μ L, 16 μ L, 18 μ L, 19 μ L and 20 μ L; the volumes of the corresponding enzyme-free water were 20. mu.L, 19. mu.L, 18. mu.L, 16. mu.L, 12. mu.L, 8. mu.L, 4. mu.L, 2. mu.L, 1. mu.L, and 0. mu.L, respectively, so that the total volume of each well was 20. mu.L.

(4) On the 96-well plate, 1 μ L of protein sample to be detected is added into each protein sample well to be detected, and then 19 μ L of enzyme-free water is added respectively, so that the total volume of each well is 20 μ L.

(5) Three multiple holes are arranged in each protein sample adding hole to be measured so as to ensure that the measurement result is accurate;

(6) adding 200 mul of the BCA working solution prepared in the step (2) into each hole of the 96-hole plate, mixing uniformly, placing in a 37 ℃ thermostat, and incubating for 30 minutes;

(7) taking out the 96-well plate, placing the 96-well plate in a normal temperature, cooling to room temperature, placing the 96-well plate in an enzyme-labeling instrument, setting parameters, and detecting the absorbance (OD value) of a protein sample at a wavelength of 562 nm;

(8) and drawing a standard curve according to the reading measured by the standard protein pore, and substituting the reading of the protein pore to be measured into a regression equation to calculate the protein concentration of each protein sample to be measured and the sample loading volume of each pore in the WB experiment.

Gel electrophoresis

(1) Cleaning two glass plates with different thicknesses by using clear water, drying the glass plates, aligning and clamping the two glass plates by using a glue making clamp, fixedly clamping the glue making clamp on a WB glue making frame with a leakage-proof glue strip at the bottom, and horizontally placing the glue making frame;

(2) preparing separation gel according to SDS-PAGE separation gel (10%), mixing the components uniformly, injecting glue horizontally along the long and short glass gaps with a 1000-microliter pipette at a constant speed, then sealing the glue quickly with ddH2O, standing at room temperature for about 30min-1h, and allowing the separation gel to solidify;

(3) preparing concentrated glue, slowly and gently pouring the upper layer of ddH2O after a clear boundary line between ddH2O and the separation glue appears, and carefully sucking up the residual ddH2O in the glue tank by using clean filter paper; preparing upper layer concentrated glue according to SDS-PAGE concentrated glue (5%), mixing all components uniformly, horizontally, rapidly and uniformly pouring glue, immediately horizontally inserting into a comb, keeping the lower edge of comb teeth horizontal to avoid bubbles, placing a glue making frame on a horizontal desktop, and standing for about 15-30min at room temperature;

(4) after the concentrated glue colloid is solidified, taking out, clamping a glass plate (a short plate is close to the inside) in an electrophoresis clamp, putting the glass plate into an electrophoresis tank (the tank is filled with electrophoresis buffer solution), and slightly pulling out the comb by pinching the two ends of the comb with hands to avoid generating bubbles and broken glue to damage the adhesive tape;

(5) taking out a protein sample with the determined protein concentration from a refrigerator at the temperature of-80 ℃, dissolving the protein sample on ice, adding a sample buffer SDS (sodium dodecyl sulfate) with the volume of 1/4 protein samples into the protein sample, fully shaking and uniformly mixing the protein sample, placing the mixture into a constant temperature heater at the temperature of 100 ℃, continuously heating the mixture for 10 minutes to denature the protein, taking out the sample, and placing the sample on ice for later use;

(6) shaking the mixed protein Marker, adding 5 mu L of the protein Marker along the 1 st sample loading hole on the left side of the glue groove, fully shaking the mixed protein sample, and slowly adding the protein sample into the sample loading hole by using a 10 mu L pipette according to the calculated equal-mass protein sample loading volume; during sample loading, the generation of bubbles is avoided, and the protein sample is prevented from being added out of the hole;

(7) and switching on a power supply after the core of the electrophoresis tank is aligned with the positive electrode and the negative electrode, setting the parameters to be constant voltage of 90V for electrophoresis, changing to constant voltage of 120V and continuing electrophoresis when the bromophenol blue strip descends to be close to the boundary of the separation gel and the concentration gel, and switching off the power supply to stop electrophoresis when the bromophenol blue strip runs to the bottom of the separation gel.

Rotary film

(1) Taking out the glass plates in the electrophoresis tank, fully soaking the glass plates in the pre-cooled in-situ prepared membrane transferring liquid in a refrigerator at 4 ℃, carefully separating the two glass plates to avoid the breakage of the adhesive tape, and soaking the adhesive tape in the membrane transferring liquid; cutting an adhesive tape with proper size according to the corresponding range of the protein Marker display strip and the protein molecular weight, cutting a PVDF film according to the size of the adhesive tape, soaking the PVDF film in a methanol solution for 5 minutes, taking out the PVDF film, activating the white PVDF film to become transparent, covering the PVDF film on the adhesive tape without a film transfer liquid, and softly removing residual bubbles between the PVDF film and the adhesive by using a plastic tool;

(2) the sandwich is made by placing related articles in the following order: and (3) clamping a red positive electrode, sponge, PVDF (polyvinylidene fluoride) membrane, adhesive tape, filter paper, sponge, a black negative electrode and a clamping rotating membrane after stacking, and placing the clamping rotating membrane in a membrane transferring solution for later use. (ii) a

(3) Preparing a foam box, paving a layer of ice block at the bottom, placing a film transfer groove in the foam box, putting a sandwich into the film transfer groove after distinguishing the sequence of the positive and negative electrodes, paying attention to avoid mistaking the positive and negative electrodes, pouring 4 ℃ precooled film transfer liquid into the film transfer groove to enable the film transfer liquid to be submerged at the upper part of the sandwich, closing a cover of the film transfer groove, covering the whole film transfer groove with ice, sealing a gap between the ice block and the film transfer groove with ice water, setting a film transfer instrument to be constant-voltage film transfer, and setting the voltage to be 110V and the duration to be about 90 minutes;

(4) and after the film is rotated, closing the film rotating instrument, taking out the PVDF film, discarding the adhesive tape, distinguishing the front side and the back side of the PVDF film, and cutting a small angle at one corner of the PVDF film by using scissors to mark.

Immune response

(1) And (3) sealing: pouring the rapid sealing liquid into a sealing box, putting the PVDF membrane into the sealing box, taking attention to the fact that the sealing liquid covers the whole PVDF membrane, and placing the sealing box on a shaking table to slowly shake and seal for about 15 minutes at room temperature;

(2) washing the membrane: recovering the blocking solution, pouring 1XTBST buffer solution into the blocking box to cover the whole PVDF membrane, and quickly shaking and cleaning for 3 times in a shaking table for 10 minutes each time;

(3) incubating primary antibody: placing the cleaned PVDF membrane in an anti-incubation box, adding primary antibody with proper concentration (diluted by primary antibody diluent according to the antibody specification) to cover the whole PVDF membrane, placing in a refrigerator at 4 ℃, and slowly shaking on a shaking table overnight;

(4) washing a primary antibody: recovering a first antibody, taking out the PVDF membrane, putting the PVDF membrane into a membrane washing box, pouring 1XTBST buffer solution into the membrane washing box to cover the whole PVDF membrane, and quickly shaking and cleaning the PVDF membrane for 3 times in a shaking table for 10 minutes each time;

(5) and (4) hatching a secondary antibody. The washed PVDF membrane is put into a secondary antibody incubation box, a secondary antibody with proper concentration (diluted by a secondary antibody diluent according to the antibody specification) is added, the mixture is slowly shaken on a shaking table, and the mixture is incubated for 2 hours at room temperature.

(6) Washing a secondary antibody: recovering the secondary antibody, taking out the PVDF membrane, putting the PVDF membrane into a membrane washing box, pouring 1XTBST buffer solution into the membrane washing box to cover the whole PVDF membrane, quickly shaking on a shaking table, and fully washing for 3 times, each time for 10 minutes.

Chemiluminescence imaging

(1) Fully sucking the liquid on the PVDF membrane by using filter paper, distinguishing the front side and the back side, placing the membrane in a clean culture dish, adding a proper amount of luminous liquid to cover the whole PVDF membrane, and slightly discharging bubbles below the membrane by using a plastic tool;

(2) placing the culture dish in a gel imager, and acquiring an imaging strip by using imaging software (Image Lab);

statistical analysis

Images were analyzed using Image Lab imaging software, statistical charts and data analysis were performed using GraphPad Prism 9. The two sample means were compared using the t-test method. P <0.05 results were statistically significant.

3. The invention proves the effect of the FosB gene in acute myeloid leukemia cells and the influence on drug sensitivity through the experimental steps as follows:

A. FosB increased expression in AML patients in the refractory/relapsed group compared to the remitted group;

the qPCR method is used for detecting the expression difference of FosB of 34 cases of patients with acute myeloid leukemia with complete remission (AML-CR) and 38 cases of patients with refractory/relapsed acute myeloid leukemia (AML-RR); the results are shown in FIG. 1, the expression of FosB in the bone marrow sample of AML-RR patients is obviously higher than that of AML-CR patients; from the above experimental results, compared with patients who are refractory/relapsed in acute myeloid leukemia, the gene expression of FosB of remitting patients is obviously increased, which indicates that FosB is related to the disease progression of acute myeloid leukemia;

B. FosB expression in AML cell line is higher than that of human umbilical vein endothelial cell line

The experiment detects the expression difference of FosB in a human normal cell strain (HUV-EC-E) and a human acute myeloid leukemia cell strain (THP-1 and HL-60) by applying an qPCR and WB experimental method; the results are shown in fig. 2A, the expression of FosB in human AML cell lines was significantly increased compared to human normal cell lines at the gene expression level; meanwhile, at the protein expression level as shown in fig. 2B, FosB was also expressed in human AML cell line significantly higher than that of the normal human cell line.

4. Conclusion

Acute Myeloid Leukemia (AML) is a group of hematological malignancies caused by uncontrolled clonal proliferation of hematopoietic stem cells, the occurrence and development of which are closely related to the dysregulation of various signaling pathways. AP-1 is a key transcription factor for regulating cell proliferation, differentiation and apoptosis, and the activity disorder of AP-1 related signal channels is related to the occurrence and development of various autoimmune diseases and tumors. Abnormal expression of members of the AP-1-associated signaling pathway is found in a variety of hematological tumors, such as AML, Chronic Myelogenous Leukemia (CML), Hodgkin's lymphoma (HD), and Anaplastic Large Cell Lymphoma (ALCL); through gene expression analysis of AML patients bone marrow samples, the STAber PB et al found that the RAF/MEK/ERK pathway was activated when the disease recurred, as well as the MKP-1, c-jun, c-fos and egr-1mRNA expression levels increased, suggesting that the c-jun, c-fos in the AP-1 family are associated with poor prognosis. Whereas FosB, a member of the AP-1 signaling pathway, has been studied only rarely in AML.

According to the experiment, the gene expression level of FosB is detected by collecting bone marrow samples of 72 clinical patients by using a qPCR method, and the result indicates that the gene expression of FosB in an AML-RR group is obviously increased compared with that of AML-CR group patients. At the same time, FosB expression was also increased in AML cell lines relative to human normal cell lines. The conclusion is that FosB is possibly closely related to the state of acute myeloid leukemia, high expression of FosB in AML patients possibly prompts poor prognosis of the patients, laboratory indexes prompting the prognosis are expected to be provided for the AML patients, and experimental basis is also provided for new target point research in targeted therapy of acute myeloid leukemia.

Compared with AML-CR group patients, the expression of FosB in the AML-RR group is obviously increased, and simultaneously, compared with a normal human cell line, the expression of FosB in the AML cell line is also increased, which indicates that the FosB can be connected with relapse and drug resistance of AML to some extent.

Example 2

Effect of the AP-1 inhibitor T5224 on proliferation and drug resistance in AML cells

1. Main experimental method

Cell culture

Culturing suspension cells THP-1 and HL-60 in a complete culture medium (containing 10% FBS) prepared by RPMI-1640, culturing adherent cells HUV-EC-E in a complete culture medium (containing 10% FBS) prepared by DMEM, and placing the cells in a cell culture box at 37 ℃ and containing carbon dioxide with five percent of saturation humidity for culturing and passaging; the growth state of the cells and the presence or absence of pollution are observed, and the solution is changed according to the cell metabolism, generally every two days.

Detection of cell proliferation

FosB inhibitor T5224 treated cells

(1) After cell counting, HUV-EC-E, THP-1 and HL60 cell strains are respectively inoculated in a 96-well plate according to the cell inoculation density of 5 multiplied by 104/mL, the cells which are treated differently are provided with 3 auxiliary wells, and inoculation is carried out according to the volume of 90 mu L per well;

(2) mu.l of FosB inhibitor T5224 (purchased from Selleck Biotechnology Co., Ltd., USA, product number: S8966) was added to each well according to the final concentration gradient of 0. mu.M, 10. mu.M, 20. mu.M, 40. mu.M, 60. mu.M and 80. mu.M in Table 5, and after gentle shaking and mixing, the 96-well plate was placed in a cell incubator for 72 hours;

TABLE 5 Final concentration gradient of FosB inhibitor T5224

(3) Adding 10 mu L of CCK-8 reagent into each hole to be detected respectively when culturing for 0, 24, 48 and 72 hours;

(4) after uniformly mixing, continuously putting the 96-hole plate into a constant-temperature incubator at 37 ℃ for incubation for 3-4 h;

(5) and (3) putting the 96-well plate into an enzyme-labeling instrument, setting the wavelength of the enzyme-labeling instrument to be 450nm, measuring the OD value, and calculating the average value of the OD values of the three multiple wells.

Cytarabine-treated cells

(1) After cell counting, THP-1 cells are inoculated in a 96-well plate according to the cell inoculation density of 5 multiplied by 104/mL, 3 auxiliary wells are arranged for differently treated cells, and the total volume of the cell suspension is 90 ul;

(2) adding 10 μ l cytarabine (purchased from national medicine Yixin pharmacy Co., Ltd., national medicine Standard H20055127) into each well according to final concentration gradient of 0.625 μ M,1.25 μ M,2.5 μ M,5 μ M,10 μ M,20 μ M and 0 μ M, slightly shaking and uniformly mixing, and placing a 96-well plate into a cell culture box for further culture for 72H;

TABLE 6 Cytarabine Final concentration gradient

(3) Adding 10 mu L of CCK-8 reagent into each to-be-detected hole respectively when culturing for 0, 24, 48 and 72 hours;

(5) and (3) putting the 96-well plate into an enzyme labeling instrument, setting the wavelength to be 450nm, measuring the OD value, and calculating the average value of the OD values of the three multiple wells.

T5224 treatment of cells in combination with cytarabine

(1) After cell counting, THP-1 cells are inoculated in a 96-well plate according to the cell inoculation density of 5 multiplied by 104/mL, 3 auxiliary wells are arranged for differently treated cells, and the total volume of cell suspension is 80 ul;

(2) taking 10ul cytarabine, adding the 10ul cytarabine into each hole according to the final concentration gradient of 0.625 mu M,1.25 mu M,2.5 mu M,5 mu M,10 mu M,20 mu M and 0 mu M in the table 7, and slightly shaking and uniformly mixing;

TABLE 7 Cytarabine Final concentration gradient

(3) Adding FosB inhibitor T522410 mu L into each hole to enable the final concentration to be 40 mu mol/L, slightly shaking and uniformly mixing, and putting the 96-hole plate into an incubator to continue culturing for 24 hours;

(4) adding 10 mu L of CCK-8 reagent (purchased from Shanghai Qihaifutai Biotechnology limited company, the product number is C008-3) into each hole to be detected, slightly mixing uniformly, putting the 96-hole plate into a cell culture box, and continuously culturing for 3-4 h;

Detection of apoptosis

(1) After cell counting, HL-60 cells were seeded at a cell seeding density of 5X 106/mL into 6-well plates, and FosB inhibitor T5224 was added at final concentrations of 0. mu.M, 40. mu.M, and 80. mu.M, respectively, and then placed in a cell incubator for further incubation for 24 hours. Cells treated differently were provided with 3 secondary wells.

(2) After 24 hours of incubation, the cells were pipetted into a 1.5mL sterile EP tube using a 1mL pipette, the tube was placed in a centrifuge, centrifuged at 1.0rcf/min for 5 minutes, the supernatant was discarded, washed repeatedly with 4 ℃ pre-cooled PBS and centrifuged 2 times, and the supernatant was discarded.

(3) Pre-cooled double distilled water at 4 ℃ was mixed with 5 × Binding Buffer as 4: 1 to obtain 1 XBinding Buffer, taking a proper amount of 1 XBinding Buffer to re-suspend cells, counting the cells, and adjusting the cell density to be 1 XBinding 106 cells/mL

(4) Taking 4 1.5ml sterile EP tubes, respectively taking 100 mu L of resuspended cell sap, and respectively adding each reagent component into the 4 EP tubes according to the content in the following table (AnnexinV-APC/7AAD apoptosis kit is purchased from Hangzhou Unico-technology corporation, Cat: AP 104-30);

TABLE 8 annexin V-APC/7AAD apoptosis experimental groups

(5) Gently shaking the components in each tube, mixing well, placing in room temperature, and incubating for about 15 minutes in dark;

(6) adding 300 mu L of 1 × Binding Buffer precooled at 4 ℃ into each tube;

(7) the sample is pipetted into a flow tube using a pipette, the fluorescence channel pressure, SSC, FSC are adjusted using a blank control tube in a flow cytometer, and the compensation of the fluorescence channel is adjusted using a single standard tube at this pressure.

Statistical method

Data calculation and software mapping were performed using FlowJo 7.6, and data analysis and statistical charting were performed using GraphPad Prism 7. The two sample means were compared using the t-test method. P <0.05 results were statistically significant.

2. The invention proves the effect of T5224 in acute myeloid leukemia cells and the influence on drug sensitivity by the above experimental steps as follows:

A. effect of T5224 on cell line proliferation

Detecting the cell proliferation condition after adding inhibitor T5224 into human normal cell strain (HUV-EC-E) and AML cell strain (HL-60, THP-1) respectively by using a CCK-8 method for intervening 24h, wherein T5224 can inhibit the cell proliferation in AML without affecting the proliferation of the normal cell strain as shown in figure 3;

B. effect of T5224 on apoptosis of AML cell lines

Detecting the apoptosis rate of HL-60 cell strains after being treated for 24 hours by adopting an Annexin V-APC/7-AAD kit with different concentrations of T5224; the results are shown in fig. 4, T5224 has a promoting effect on apoptosis of acute myeloid leukemia cell lines, and the higher the concentration is, the more significant the promoting effect is;

C. effect of T5224 in combination with Cytarabine on proliferation of AML cell lines

The experimental group is provided with two groups in total, THP-1 cells are taken, cytarabine with different concentrations is added into the first group, and FosB inhibitor T5224(40 mu M) with the same concentration is added into the treatment group added with cytarabine with different concentrations; the cells are respectively cultured for 24h, and relative cell viability is detected by adopting a CCK-8 kit, and the result is shown in figure 5, and the inhibition effect of T5224 and cytarabine on AML cell proliferation is stronger.

3. Conclusion

Acute Myeloid Leukemia (AML) is a hematological malignancy, highly heterogeneous, with its high morbidity and mortality seriously threatening human health. With the continuous efforts of clinicians and scientists, the prognosis of AML patients is continuously improved, with the use of retinoic acid and arsenous acid healing more than 90% of APL patients. In the case of non-APL patients, hematopoietic stem cell transplantation also has the potential to radically cure leukemia. In recent years, new molecular targeted drugs and results of clinical trials of immunotherapy have been encouraging, opening a new chapter on AML therapy. Nevertheless, relapsed and refractory acute myeloid leukemia is still a difficult problem in clinical treatment, and with the emergence of multiple drug resistance, the search for new treatment modes is still a research hotspot in the field of current blood diseases.

Considering the relationship between the disease states of FosB and AML patients in earlier experiments, the experiments provide experimental bases for exploring the effect of inhibiting FosB in AML and providing experimental researches for the treatment research of targeting FosB in AML, the experiments detect the proliferation and apoptosis conditions by applying FosB inhibitor T5224 to an AML normal human umbilical vein endothelial cell strain and an AML cell strain, find that the inhibition of FosB can reduce the cell activity and proliferation speed of AML cells, promote the apoptosis of AML cells, and has small inhibition on the growth of the normal cell strain, preliminarily suggest the effectiveness and safety of the treatment of FosB in AML, but the experimental results are to be further confirmed in animal experiments. Meanwhile, the effect of treating AML cells by the combination of T5224 and cytarabine is more obvious than the effect of treating growth inhibition by cytarabine alone, which suggests that the combined application of T5224 and cytarabine has the effect of synergistically inhibiting the proliferation of AML cells and can improve the drug sensitivity of cytarabine. In a related research of gastric cancer, in a vincristine-resistant cell strain SGC7901/VCR, an AP-1 inhibitor BATF2 can improve the drug resistance of the resistant cell strain by reducing the expression of c-Fos and c-Jun, and whether T5224 can overcome the chemotherapy resistance of AML in primary AML resistant cells or AML resistant cell strains is continuously explored in the next experiment; for the function regulation mechanism of FosB, Shidan Liu et al find that miR-144-3p targets and down-regulates FosB in pancreatic cancer, and can up-regulate the expression of COX2, VEGF, MMP2 and MMP9 after over-expressing FosB, wherein COX2 mediated signal pathway can regulate cell proliferation, VEGF is one of important angiogenesis factors and can promote the proliferation and invasion of tumor blood vessels, and MMP2 and MMP9 are members of matrix metalloproteinase gene family (MMPs) and participate in the invasion and migration of tumors. The function regulation mechanism of Fos B in AML is to be further researched by establishing FosB over-expression cell strain in AML at later stage. The results provide experimental basis for researching research and development of FosB inhibitor as a medicine for treating acute myeloid leukemia, and are expected to provide a new treatment option for cytarabine-resistant AML-RR patients.

Therefore, the invention proves that T5224 can inhibit the proliferation of cells and promote the apoptosis of the cells in acute myeloid leukemia cell strains; and does not have a significant effect on the proliferation of normal cell lines. T5224 can enhance the sensitivity of AML cell line to the chemotherapeutic drug cytarabine.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. A medicament for treating AML, which is the AP-1 inhibitor T5224, wherein T5224 has the formula:

2. the medicament of claim 1, further comprising at least cytarabine.

3. Use of the drug AP-1 inhibitor T5224 according to claim 1 for the preparation of an anti-AML drug.

4. The use as claimed in claim 3, wherein the medicament comprises T5224 as the active ingredient, together with one or more pharmaceutically acceptable excipients selected from diluents, excipients, fillers, binders, wetting agents, absorption enhancers, surfactants, lubricants, stabilizers, flavoring agents, sweeteners and pigments, which are conventional in the pharmaceutical arts.

5. The use according to claim 4, wherein the medicament is a sensitizer for increasing the sensitivity of AML cells to chemotherapeutic drugs, such as cytarabine.

6. The use of claim 4, wherein the medicament is a pharmaceutical composition against AML comprising a pharmaceutically effective amount of T5224 and a pharmaceutically effective amount of cytarabine and a pharmaceutically acceptable carrier.

7. The use of any one of claims 4 to 6, wherein the medicament is formulated into any one of pharmaceutically acceptable dosage forms, including tablets, capsules, granules, oral liquids, sustained release preparations, nano preparations, injections.