CN114081954B

CN114081954B - Pharmaceutical composition for glioblastoma treatment and diagnostic reagent for prognosis

Info

Publication number: CN114081954B
Application number: CN202111278584.5A
Authority: CN
Inventors: 王岩; 卞修武; 盖曲倞; 吕胜青
Original assignee: First Affiliated Hospital of Army Medical University
Current assignee: First Affiliated Hospital of Army Medical University
Priority date: 2021-10-30
Filing date: 2021-10-30
Publication date: 2023-02-28
Anticipated expiration: 2041-10-30
Also published as: CN114081954A

Abstract

The invention provides a pharmaceutical composition for treating glioblastoma, which comprises an inhibitor of ephrin A type receptor 2 (EPHA 2) expression and an inhibitor of platelet-derived growth factor receptor (PDGFRA) expression. The present invention also provides a diagnostic reagent for monitoring prognosis of glioblastoma, comprising: a first composition for detecting the expression level of the EPHA2 gene, and a second composition for detecting the expression level of the PDGFRA encoding gene. The invention firstly provides a scheme for treating GBM by jointly inhibiting PDGFRA and EPHA2, and solves the problem that the tumor resistance cannot be achieved in clinical application by only using a single PDGFRA inhibitor.

Description

Pharmaceutical composition for glioblastoma treatment and diagnostic reagent for prognosis

Technical Field

The invention relates to the technical field of biomedicine, in particular to a pharmaceutical composition for treating glioblastoma and a prognostic diagnostic reagent.

Background

Gliomas are the most common brain tumors, with central nervous system tumors classified into four grades (grade I-IV) according to WHO 2016 tumor pathological grading criteria. Although a small amount of low-grade glioma progresses slowly and has good prognosis, most of the tumors are malignant tumors and have the characteristics of high invasive growth and obviously shortened survival period. Taking glioblastoma multiforme (grade IV) as an example, it is the most malignant form of glioma, and despite the ongoing progress of surgical and pharmaceutical treatments, it is still difficult to cure.

Platelet-derived growth factor receptors α (PDGFRA) and β (PDGFRB) belong to the family of Receptor Tyrosine Kinases (RTKs) and are receptors for platelet-derived growth factor (PDGF). 4 PDGF genes (PDGFA, PDGFB, PDGFC, and PDGFD) have been identified and encoded in mammals. The specificity of PDGF ligand interaction with PDGFR receptor has been elucidated many times, with PDGFRA being the only receptor mediated by PDGFA.

PDGFA and PDGFRA have been found to play important roles in both glioma formation and tumor progression: in one aspect, PDGFA is ubiquitous in glioblastoma and is also one of the marker genes for classical glioblastoma multiforme (GBM); on the other hand, copy number amplification and mRNA overexpression of PDGFRA are typical features of the probucol type GBM. In experiments, successful overexpression of PDGFA and PDGFRA induced GBM development in mouse models, and these results all indicate a significant role for PDGFRA in GBM and identify the PDGFA/PDGFRA axis as a potential therapeutic target for GBM. However, single PDGFRA inhibitors did not show anti-tumor effects in clinical trials.

Disclosure of Invention

In view of the above-mentioned problem that a single PDGFRA inhibitor cannot achieve the anti-tumor purpose, the present inventors found that the simultaneous inhibition of EPHA2 and PDGFRA has a synergistic in vitro and in vivo inhibition effect on GBM cells, and proposed a scheme for treating GBM by jointly inhibiting PDGFRA and EPHA2, so as to solve the problem that a single PDGFRA inhibitor cannot effectively prevent tumor.

The invention firstly provides a pharmaceutical composition for treating glioblastoma, which comprises an ephrin A type receptor 2 (EPHA 2) expression inhibitor and a platelet-derived growth factor receptor (PDGFRA) expression inhibitor.

In one embodiment according to the present invention, the inhibitor of ephrin-a type receptor 2 (EPHA 2) expression is selected from one or more of DNA, RNA, mircoRNA, siRNA, peptide fragment or protein that interferes with or blocks the transcription or translation process of EPHA2 gene.

In one embodiment according to the invention, the inhibitor of platelet-derived growth factor receptor (PDGFRA) expression is selected from one or more of DNA, RNA, mircoRNA, siRNA, peptide fragment or protein that interferes with or blocks the process of transcription or translation of the PDGFRA-encoding gene.

The present invention also provides a diagnostic reagent for monitoring the prognosis of glioblastoma comprising:

a first composition for detecting the expression level of the EPHA2 gene, and a second composition for detecting the expression level of the PDGFRA-encoding gene.

In one embodiment according to the present invention, the first composition is selected from one or more of a primer set or probe for detecting the copy number of the EPHA2 gene, a primer set or probe for detecting the mRNA content transcribed from the EPHA2 gene, and a probe or antibody for detecting the EPHA2 content.

In one embodiment according to the invention, the second composition is selected from one or more of a primer set or probe for detecting PDGFRA coding gene copy number, a primer set or probe for detecting mRNA content of PDGFRA coding gene transcription, and a probe or antibody for detecting PDGFRA content.

The technical scheme of the invention has the following beneficial effects:

aiming at the problems in the technology, the invention firstly provides a scheme of jointly inhibiting PDGFRA and EPHA2 as GBM treatment, and solves the problem that the tumor resistance cannot be achieved in clinical application by only using a single PDGFRA inhibitor.

Drawings

FIG. 1 is a graph showing the results of detection in example 1; wherein the content of the first and second substances,

a is a transient expression profile of PDGFA-induced associated proteins in LN18 cells detected by Western blot;

b, detecting a transient expression pattern of related proteins in PDGFA-induced LN18 stem cells by using Western blot;

c, detecting a transient expression profile of related proteins in LN18 cells induced by PDGFA after pretreatment of dimethyl sulfoxide (DMSO) and MK2206 (AKT inhibitor) by using Western blot;

d is knocking off the expression of PDGFRA in LN18 cells, and detecting the transient expression pattern of related proteins in the LN18 cells induced by PDGFA by using Western blot;

e is a Western blot used for detecting a transient expression profile of related proteins in the PDGFA-induced LN18 cells after pretreatment of dimethyl sulfoxide (DMSO) and IMA (PDGFRA inhibitor);

f is an immunofluorescence imaging profile in GBM cells using EPHA2, EEA1 and DAPI antibodies;

g is an expression profile of EPHA2 in LN18 cells treated by using Co-IP and Western blot;

h is a simulation diagram of interaction between PDGFA and EPHA2 in a three-dimensional structure of an extracellular domain;

i is a thermodynamic map of the interaction between the extracellular domain of the recombinant human EPHA2 and the recombinant human PDGF-AA;

j is a result map obtained by performing three modes of no treatment, PDGFA treatment for 15 minutes and PDGFA treatment for 15 minutes after the pretreatment of the extracellular domain of the recombinant PDGFRA on LN18 respectively, and gray-white signals represent the interaction between EPHA2 and PDGFA;

k is to knock down the expression of EPHA2 in LN18 cells, and Western blot is used to detect the transient expression pattern of PDGFA-induced associated proteins in LN18 cells.

FIG. 2 is a graph showing the results of detection in example 2; wherein, the first and the second end of the pipe are connected with each other,

a is representative immunohistochemical images of EPHA2 and PDGFRA proteins on serial tissue sections;

b is PDGFRA high expression/EPHA 2 high expression (PDGFRA) in glioma cases ^High /EPHA2 ^High ) And PDGFRA high expression/EPHA 2 low expression (PDGFRA) ^High /EPHA2 ^Low ) A comparative survival analysis profile;

c is comparison of PDGFRA in glioma cases ^High /EPHA2 ^High Survival profiles of groups versus all other cases;

d is in case of glioma, PDGFRA ^High /EPHA2 ^High The proportion map of the group in each grade of glioma;

e is according to the TCGA-GBM database, PDGFRA ^High /EPHA2 ^High Survival analysis graphs of different treatment modes are assembled;

f is according to TCGA-GBM database, non-PDGFRA ^High /EPHA2 ^High Survival analysis graphs of different treatment modes are assembled;

FIG. 3 is a graph showing the results of detection in example 3; wherein the content of the first and second substances,

a is a half inhibition concentration graph of high expression (left graph) of EPHA2 and low expression (right graph) of EPHA2 LN18 cells to IMA detected by MTT method;

b is an antibody chip analysis of LN18 cells treated with DMSO (control), ALW (EPHA 2 inhibitor) and IMA (PDGFRA inhibitor), respectively, and proteins with significant changes are labeled with a frame and listed separately;

c is a graph of the synergistic effect of two medicines of ALW and IMA on four GBM cells detected by an MTT method;

d is a representative in vivo imaging plot of U251 cells grown in situ tumors treated with DMSO, IMA, ALW, and IMA + ALW, respectively;

e is a statistical chart for calculating the size of the tumor by using the intensity of the bioluminescent signal;

fig. 4 is a functional diagram of EPHA2 and PDGFRA-regulated PDGFA in GBM cells plotted against the conclusions drawn from the above examples. Wherein both PDGFRA and EPHA2 mediate PDGFA function, promoting invasive growth and drug resistance of GBM cells; due to the existence of a compensatory pathway, inhibition of EPHA2 or PDGFRA by a single agent is not effective in inhibiting PDGFA activity, but inhibition of both EPHA2 and PDGFRA is effective in blocking PDGFA signaling (below).

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.

Unless otherwise indicated, the instruments, materials and reagents referred to in the following examples are commercially available.

Example 1

The results can be concluded from western blotting and immunofluorescence imaging experiments as shown in fig. 1: in GBM cells, PDGFA can activate EPHA2 independent of PDGFRA.

1) Western blotting (Western blot) FIGS. 1A-E, G, K

(1) And (3) extracting total cell protein: the medium was aspirated, 5ml of PBS was added, the medium was gently shaken to wash out, and the PBS was aspirated and discarded. After addition of 1ml of PBS, the cells were scraped off using a cell scraper and transferred to a 1.5ml centrifuge tube using a pipette gun. Centrifugation was carried out at 800g for 3min at 4 ℃ and the supernatant was aspirated off, leaving a cell pellet. Cell pellets of about 10 volumes of RIPA lysate containing PMSF were added and lysed on ice for 30min. After lysis, the cell lysate was placed in a 4 ℃ centrifuge and centrifuged at 25000g for 15min. And transferring the centrifuged supernatant into a new 1.5ml centrifuge tube in a subpackage manner to obtain the total cell protein.

(2) Protein sample preparation: after determining the concentration of each protein using the BCA protein quantification kit, 4 loading buffers of 1/3 protein volume were added to the protein, and then 1 loading buffer was used to make up until the concentration of each protein was the same. And (3) putting the mixed protein sample into a water bath kettle at the temperature of 95 ℃ for 5min to complete denaturation.

(3) SDS-PAGE electrophoresis: the gel is placed in an electrophoresis tank and sufficient electrophoresis solution is added. A30. Mu.g protein sample was added to each well. After the voltage is 120V for 20min, the voltage is changed to 160V for about 40min until bromophenol blue just runs out of the gel, the electrophoresis is stopped, and the membrane is changed.

(4) Film transfer: the filter paper, the PVDF membrane and the electrophoresis gel are sequentially placed into a clamp by using the Sanming method, the clamp is placed into an electric rotating tank, and the membrane is rotated for 2 hours at a constant current of 0.36A.

(5) And (3) immune reaction: the membrane was placed in 7.5% skimmed milk and the shaker closed for 1h at room temperature. The membrane was washed with PBST and placed in primary antibody and shaken overnight at 4 ℃. After the primary antibody incubation, the membrane was removed from the antibody and placed in PBST, and washed 3 times in a room temperature water flat shaker for 10min each. Then the membrane is put into a second antibody and incubated for 1h in a shaking table at room temperature. The secondary antibody was then washed 3 times for 10min in the same manner.

(6) Chemiluminescence: and (3) preparing a developing solution, draining the redundant liquid on the film, dripping a proper amount of the developing solution, putting the film into a developing instrument for developing and photographing.

2) Immunofluorescence imaging FIGS. 2F, J

(1) Spreading appropriate amount of cells in immunofluorescence chamber, culturing overnight in incubator, taking out, rinsing with PBS 3 times, fixing 4% paraformaldehyde at room temperature for 15min, rinsing with PBS 3 times, each time for 5min.

(2) 0.3% Triton X-100 for 15min, disrupting cell membranes, and rinsing with PBS for 5min 3 times.

(3) Blocking with 10% goat serum at 37 deg.C for 30min.

(4) Diluted antibody was added and incubated overnight at 4 ℃.

(5) Rinsing with PBS for 3 times, 5min each time, adding diluted fluorescent secondary antibody mixture, and incubating at 37 deg.C for 30min.

(6) The cells were rinsed with PBS 3 times for 5min each, stained with DAPI, and incubated at 37 deg.C for 15min.

(7) PBS rinse 3 times, each for 5min, seal with anti-fluorescence quenching sealing agent, store in dark wet box at 4 ℃.

(8) And observing and acquiring images by using a laser confocal microscope.

3) Protein interaction simulation scheme 1H

Searching a three-dimensional protein structure in a website UniProt (https:// www. UniProt. Org /), and introducing the protein structure into an analysis website ClusPro protein-protein gating (https:// ClusPro. Bu. Edu/login. Phpredir =/results. Phpoffset = 9) to obtain a simulation graph of protein interaction.

4) Thermodynamic measurement FIG. 1I

Thermodynamic measurements were performed using Microcal iTC 200. The protein was purchased from Yi Qiao Shen.

Example 2

The results from immunohistochemical analysis experiments, as shown in fig. 2, conclude the clinical therapeutic significance of EPHA2 and PDGFRA in GBM.

1) Immunohistochemical analysis FIG. 2A

(1) Paraffin section: and (3) placing the paraffin specimen into a refrigerator at the temperature of-20 ℃ for precooling, and then carrying out slicing, spreading, air drying and other steps.

(2) Dewaxing: and (3) placing the paraffin sections in a 60 ℃ oven for baking for 30min, and then carrying out dewaxing: xylene I15 min, xylene II 15min,100% alcohol 10min,95% alcohol 5min,85% alcohol 5min,75% alcohol 5min, tap water washing 5min, PBS 5min.

(3) Antigen retrieval: the sections were rinsed thoroughly with PBS for 15min. Preparing a repairing solution corresponding to the antibody, adding a proper amount of water into an autoclave, heating to boil, and placing the slices into the autoclave for high pressure for 2min 30s. And immediately washing the cooling pressure cooker with tap water, taking out the slices, putting the slices into a repair box filled with repair liquid, and naturally cooling to room temperature.

(4) Inactivation of endogenous catalase: taking out the slices, rinsing with PBS for 3 times, 5min each time, adding 3% of H2O2, and acting at 37 deg.C for 30min.

(5) And (3) sealing: the sections were removed and rinsed 3 times with PBS for 5min each time. After the section is gently spun off, the section is placed in a wet box, and goat serum sealer is dripped onto the tissue. Incubate in oven at 37 ℃ for 30min.

(6) Primary antibody incubation: spin-dry the sections gently, drip the diluted antibody onto the sections, cover the wet box lid, incubate overnight in a refrigerator at 4 ℃.

(7) And (3) secondary antibody incubation: taking out the slices, rewarming to room temperature, placing the slices in a repair box, and rinsing with PBS for 3 times, 5min each time. After the PBS was gently spun off, the cells were placed in a wet box, and the immunohistochemical secondary antibody was added dropwise to the sections, which were incubated in an oven at 37 ℃ for 30min.

(8) Color development: the wet box was removed and the sections were placed in a repair box and rinsed 3 times with PBS for 5min each time. Taking out the slices, placing the slices under a microscope, and dropwise adding DAB color development liquid. When the color developed to an appropriate degree, the reaction was immediately stopped by putting it in water.

(9) Counterdyeing: washing the slices in tap water for 5min, staining in hematoxylin staining solution for 20s, washing with tap water, differentiating in hydrochloric acid alcohol for 1s, and washing with tap water until the slices turn blue.

R dehydrated, transparent: the slices are sequentially soaked in 75% alcohol for 5min,85% alcohol for 5min,95% alcohol for 5min,100% alcohol for 10min, xylene for 15min and fresh xylene for 15min.

Sealing: placing the slices in a fume hood for natural drying, dripping 50-100 μ L of neutral gum into each slice, and sealing with a cover glass. The slices can be placed in a 37 ℃ oven to remove air bubbles.

Scanning and storing: the slice scanner scans the slices and saves the images.

2) Survival analysis chart 2B, C, E, F

Inputting data to be analyzed into the SPSS, selecting survival analysis-related variables-defined event values-comparison factors, analyzing and then exporting the picture.

3) Data statistics FIG. 2D

And inputting the data into analysis software, and selecting an output image mode to obtain the image.

Example 3

The results of MTT experiments and mouse model experiments, as shown in fig. 3, conclude that jointly IMA and ALW inhibiting the expression of PDGFRA and EPHA2 can inhibit GBM cells.

1) MTT assay FIGS. 3A and C

(1) An appropriate amount of cells was seeded in a 96-well plate, 3000-5000 cells per well, 100. Mu.L of medium. After the cells are adhered to the wall, the medicine is added for treatment, and the cells are cultured for 72 hours in an incubator at 37 ℃.

(2) The 96-well plate was removed, the old medium was aspirated off, 100. Mu.L of medium containing MTT solution was added, incubation was continued for 3h at 37 ℃ and the culture was terminated, the medium was carefully aspirated off in wells, 100. Mu.L of LDMSO was added to each well, and shaking was carried out until the crystals were sufficiently thawed.

(3) OD value is measured by a microplate reader at 570nm, and the result is recorded.

2) Antibody Array FIG. 3B

Using the antibody array kit from R & D Systems, the cells were scraped into 1.5ml centrifuge tubes, centrifuged at 800g at 4 ℃ for 3min, and the supernatant was aspirated off to leave a cell pellet. The development was performed in a developer after the procedure according to the kit instructions.

3) Mouse model construction of orthotopic transplantation tumor FIGS. 3D and E

(1) Experimental animals: female NOD/SCID mice of 4-6 weeks old were bred using standard pathogen-free conditions.

(2) Animal anesthesia: the experimental mice were anesthetized by intraperitoneal injection at 50mg/kg body weight using a 0.8% chloral hydrate solution.

(3) Inoculating intracranial in-situ transplantation tumor: the needle insertion site 0.5mm before the coronal suture and 2mm to the right of bregma was selected from the mice, sterilized with 75% alcohol and fully exposed. Injecting by using a sterilized 20 mul sample injector, wherein the needle insertion depth is about 1 mm-2 mm and has breakthrough feeling, and slowly injecting for 1min; after injection, the sample injector is stopped for 1min and then slowly withdrawn, and the needle inlet is sterilized by using 75% alcohol solution again. Each mouse was inoculated with 5X 10 ⁵ And (4) cells.

(4) And 7 days after the transplantation of the tumor, performing living body imaging on the small animals, injecting 200 mu L of luciferase substrate into the abdominal cavity of each mouse, performing isoflurane anesthesia, and then placing the mice in an IVIS living body imaging instrument for observation and photographing. After photographing, the tumor size was calculated according to the intensity of the bioluminescent signal of the living body image, and the mice were equally divided into four groups.

(5) The four groups of mice are PBS control group, IMA single medicine group, ALW single medicine group and IMA + ALW double medicine group respectively. The IMA injection concentration is 25mg/kg, and the ALW injection concentration is 10mg/kg. IMA was injected weekly and friday with ALW, together with an equal volume of PBS.

(6) Live imaging of small animals and observation of the photographs were performed once a week, and changes in tumor size were calculated by analyzing the bioluminescent signal intensity. Four groups of mice were euthanized 4 weeks after drug injection and brain tissue harvested for immunohistochemical analysis.

And (4) conclusion: both PDGFRA and EPHA2 mediate PDGFA function, promoting invasive growth and drug resistance of GBM cells; due to the presence of the compensatory pathway, a single drug inhibiting EPHA2 or PDGFRA did not effectively inhibit PDGFA activity, but inhibiting both EPHA2 and PDGFRA simultaneously effectively blocked PDGFA signaling (below), see fig. 4.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A pharmaceutical composition for the treatment of glioblastoma comprising the inhibitor of ephrin-a receptor type 2 (EPHA 2) expression ALW and the inhibitor of Platelet Derived Growth Factor Receptor (PDGFRA) expression IMA.

2. The pharmaceutical composition of claim 1, wherein the inhibitor of ephrin-a receptor 2 (EPHA 2) expression is selected from one or more of DNA, RNA, mircoRNA, siRNA, peptide fragments, or proteins that interfere with or block the transcription or translation process of EPHA2 gene.

3. The pharmaceutical composition of claim 1, wherein the inhibitor of platelet-derived growth factor receptor (PDGFRA) expression is selected from one or more of DNA, RNA, mircoRNA, siRNA, peptide or protein that interferes with or blocks the process of transcription or translation of PDGFRA-encoding genes.

4. A diagnostic reagent for monitoring the prognosis of glioblastoma, characterized by consisting of a first composition for detecting the expression level of EPHA2 gene and a second composition for detecting the expression level of PDGFRA encoding gene.

5. The diagnostic reagent for monitoring prognosis of glioblastoma according to claim 4, wherein the first composition is selected from one or more of a primer set or a probe for detecting copy number of EPHA2 gene, a primer set or a probe for detecting mRNA content transcribed from EPHA2 gene, and a probe or an antibody for detecting EPHA2 content.

6. The diagnostic reagent for monitoring prognosis of glioblastoma according to claim 4, wherein said second composition is selected from one or more of a primer set or a probe for detecting PDGFRA-encoding gene copy number, a primer set or a probe for detecting PDGFRA-encoding gene transcribed mRNA content, and a probe or an antibody for detecting PDGFRA content.