CN115444937A - Application of combination of CD317 inhibitor and proteasome inhibitor in preparation of antitumor drugs - Google Patents

Abstract

The invention discloses an application of a combination of a CD317 inhibitor and a proteasome inhibitor in preparing an anti-tumor medicament. The research of the application finds that the CD317 has the effect of maintaining the protein homeostasis of the tumor cells, and the CD317 inhibitor is utilized to knock down the expression of the CD317 of the tumor cells, so that the protein homeostasis of the cancer cells is broken, the sensitivity of the cancer cells to a proteasome inhibitor is enhanced, and the anti-tumor effect of the proteasome inhibitor is enhanced. Therefore, the combined application of the CD317 inhibitor and the proteasome inhibitor is expected to solve the problems of clinical drug resistance and insensitivity to solid tumors.

Description

Application of combination of CD317 inhibitor and proteasome inhibitor in preparation of antitumor drugs

Technical Field

The invention belongs to the technical field of tumor treatment, and relates to an application of a CD317 inhibitor and a proteasome inhibitor in preparation of an anti-tumor medicament.

Background

Tumors (tumors) are a non-genetic disease of the gene. Under the action of tumorigenic factors, the gene of normal cells is changed, and the normal regulation and control of the growth of the cells are lost, so that abnormal hyperplasia is caused. Tumor cells have three significant basic features: immobility, migration and loss of contact inhibition. The core idea of tumor therapy is to kill and eliminate tumor cells. The current tumor treatment means commonly used in clinic are chemotherapy, radiotherapy, hormone therapy and the like. However, almost all drugs, over the course of time of use by the patient, develop resistance to the drug, causing the drug to cease acting on the cancer cells. This acquired resistance of tumor cells to drug therapy not only severely limits the efficacy of clinical therapy, but also is the molecular basis for tumor recurrence. Therefore, we need to develop new treatment methods or optimize existing methods, on one hand, to break drug resistance and enhance antitumor effect, on the other hand, to enrich the "weapon base" of our antitumor, and to broaden the selection range of tumor treatment strategies.

Bortezomib (BTZ) or (PS-341) is a proteasome (proteasome) inhibitor with reversibility and selectivity, which effectively inhibits the 20S proteasome by targeting threonine residues, thus disrupting protein homeostasis, affecting cell cycle, or even directly inducing apoptosis. Bortezomib was approved for the treatment of refractory multiple myeloma in 2003 and for the treatment of multiple myeloma patients who received at least one treatment in 2005. On 23.6.2008, bortezomib was approved by the FDA for the treatment of multiple myeloma patients as the initial treatment for the patients. Bortezomib was approved in 2006 for the treatment of relapsed or refractory mantle cell lymphoma, and in 2014 for untreated mantle cell lymphoma patients. Bortezomib, when used as a single agent or in combination with other anticancer agents, has significant antimyeloma activity in previously untreated myeloma patients and in relapsed/refractory myeloma patients. However, the generation of drug resistance and the poor effect on solid tumors limit the clinical application effect. Therefore, there is an urgent need for a method capable of breaking the drug resistance or enhancing the anti-solid tumor effect thereof.

Disclosure of Invention

In order to solve the problems in the background art, the present invention aims to provide an application of a CD317 inhibitor and a proteasome inhibitor in combination in preparing an anti-tumor drug.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: in one aspect, the invention provides an application of a CD317 inhibitor as a proteasome inhibitor sensitizer.

On the other hand, the invention provides an application of the CD317 inhibitor in preparing a medicament for improving the sensitivity of tumor cells to proteasome inhibitors.

On the other hand, the invention provides an application of the combination of the CD317 inhibitor and the proteasome inhibitor in preparing an antitumor medicament.

In yet another aspect, the present invention provides a method of increasing the sensitivity of a tumor cell to a proteasome inhibitor, comprising the steps of: the expression of CD317 in tumor cells is knocked down using a CD317 inhibitor.

In yet another aspect, the present invention provides a pharmaceutical composition comprising a proteasome inhibitor, and at least one CD317 inhibitor.

In a further aspect, the invention provides a proteasome inhibitor sensitizer comprising a CD317 inhibitor.

Further, the proteasome inhibitor comprises bortezomib, MG132.

Further, the CD317 inhibitor comprises CD317 siRNA, CD317 shRNA, coding DNA of shRNA, CD317 sgRNA, PROTAC, antibody or blocking peptide;

preferably, the CD317 inhibitor is CD317 siRNA designed according to human CD317 gene transcript NM _ 004335.3;

more preferably, the CD317 siRNA comprises a sense strand of a nucleotide sequence shown in SEQ ID NO. 1 and an antisense strand of a nucleotide sequence shown in SEQ ID NO. 2, and/or a sense strand of a nucleotide sequence shown in SEQ ID NO. 3 and an antisense strand of a nucleotide sequence shown in SEQ ID NO. 4.

Further, the tumor or tumor cells include solid tumors and non-solid tumors.

Further, the solid tumors include human cervical cancer, human non-small cell lung adenocarcinoma, liver cancer, human breast cancer;

the non-solid tumor comprises chronic myelogenous leukemia, acute T lymphocyte leukemia and myeloma.

The invention has the beneficial effects that: the research of the application finds that the CD317 has the effect of maintaining the protein homeostasis of the tumor cells, and the CD317 inhibitor is utilized to knock down the expression of the CD317 of the tumor cells, so that the protein homeostasis of the cancer cells is broken, the sensitivity of the cancer cells to a proteasome inhibitor is enhanced, and the anti-tumor effect of the proteasome inhibitor is enhanced. Therefore, the combined application of the CD317 inhibitor and the proteasome inhibitor is expected to solve the problems of clinical drug resistance and insensitivity to solid tumors.

Drawings

FIG. 1 is a graph showing the silencing effect of siRNA specific to CD 317;

FIG. 2 is a graph of the effect of CD317 on BTZ-induced blood tumor cell death;

FIG. 3 is a graph of the effect of CD317 knockdown on tumor cell misfolded protein levels;

figure 4 is a graph of the effect of CD317 on BTZ-induced solid tumor cell death.

Detailed Description

The present invention is described in detail below with reference to specific examples, which are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. The test methods in the following examples are, unless otherwise specified, all conventional methods, and the test materials used are commercially available products.

Example 1siRNA design

According to the basic principle of siRNA target sequence, 2siRNA sequences of 21 nucleotides, namely SiR-1 and SiR-2, including a sense strand and an antisense strand, are designed aiming at a human CD317 gene transcript (NM-004335.3), and the base sequences are as follows:

siR-1

a sense strand: 5'-CCAGGUCUUAAGCGUGAGAdTdT-3' SEQ ID NO 1

Antisense strand: 5 '-UCUCACGCUUAAGACCUGdTdT-3'; SEQ ID NO 2

siR-2

A sense strand: 5 '-UCGCGGACAAGAAGUAGUACUTdT-3' and SEQ ID NO 3

Antisense strand: 5 '-UAGUACUUUGUCCGAdTd-3'; SEQ ID NO 4

The base sequence of the Negative Control (NC) siRNA selected in this example was as follows (siCT):

sense strand: 5 '-UUCCCGAACGUCACGUdTdT-3', SEQ ID NO 5

Antisense strand: 5'-ACGUGACACGUUCGGAGAAdTdT-3'; SEQ ID NO 6

Two deoxyribonucleotides in a single-chain suspension structure are added at the 3' end of the interference fragment, so that the stability of siRNA in vivo and in vitro is enhanced, and degradation is prevented. The siRNA was synthesized by Shanghai Ji Ma.

According to the preferred embodiment of the present invention, the currently more common cationic liposome Lipofectamine3000 was selected as the transfection reagent.

Example 2siRNA transfection and interference Effect verification

The cell lines selected by the invention are a chronic myelogenous leukemia cell line k562 and an acute leukemia T cell line Jurkat cell line which highly express human CD 317. The transfection method and results were verified as follows:

(1) Cell transfection

1) According to the siRNA synthesis report, adding a proper amount of DEPC water (DEPC, diethyl pyrocarbonate) to prepare a 20 mu M stock solution;

2) Inoculating the cells into a 12-hole plate, wherein the density is preferably that the cell confluence degree reaches 60-80% after overnight culture;

3) Diluting 4. Mu.L Lipofectamine3000 transfection reagent with 50. Mu.L Opti-MEM medium, mixing well, and standing at room temperature for 5min;

4) Diluting 2. Mu.L siRNA with 50. Mu.L Opti-MEM medium, mixing well, and standing at room temperature for 5min;

5) Mixing the diluent obtained in the step 3) and the diluent obtained in the step 4), fully and uniformly mixing, and standing at room temperature for 10 minutes. At this time, the ratio of siRNA to Lipofectamine3000 in the mixed solution is 1:2;

6) Dropwise adding the transfection mixed solution obtained in the step 5) into a cell culture hole, and uniformly mixing by a cross method;

7) The cells were cultured for an additional 36 hours for subsequent experiments.

(2) Interference effect detection

1) RNA extraction (using the TransZol Up Plus RNA Kit (Cat. No.: ER 501-01) from Beijing Quanyujin biol Ltd.;

a) After transfection of siRNA for 36h, the medium was removed, washed once with PBS, and 1mL of Trizol was added to lyse the cells sufficiently;

b) The lysate was collected in a 1.5mL EP tube and 200. Mu.L of chloroform was added. Mixing for 30s, standing at room temperature for 3min, centrifuging at 10000Xg and 4 deg.C for 15min;

c) Sucking 400 mu L of supernatant into a new centrifuge tube, adding 400 mu L of anhydrous ethanol with the same volume, uniformly mixing, transferring into a centrifugal column, centrifuging for 30s at room temperature of 12000x g, and discarding an effluent;

d) Adding 500 μ L CB9, room temperature 12000x g, centrifuging for 30s;

e) Repeating step d) once;

f) Adding 500. Mu.L of WB9, at room temperature 12000x g, and centrifuging for 30s;

g) Repeating step f) once;

h) At room temperature 12000x g, centrifuging for 2min, and completely removing residual ethanol;

i) Drying at room temperature for 2min, adding 30 μ L RNA-free Water in the center of the centrifugal column, and standing at room temperature for 1min;

j) Room temperature 12000x g, centrifugating for 1min, eluting RNA;

k) RNA concentration and purity were determined using Nanodrop.

2) The RNA was reverse-transcribed into cDNA (using a Kyoto Kagaku Kogyo Co., ltd.)

One-Step gDNA Removal and cDNA Synthesis SuperMix(Cat.No.:AE311-02)

a) Preparing reverse transcription reaction solution A according to the following table, uniformly mixing, and placing on ice for later use;

b) Preparing a reverse transcription reaction B solution, wherein the system is as follows:

c) Mixing solution A and solution B, incubating at 42 deg.C for 30min, heating at 85 deg.C for 5s for inactivation

RT/RI and Remover, stop the reaction.

3) Real-time quantitative PCR measurement of AMPK expression (Using from Beijing Quanjin Bio Inc.)

Tip Green qPCR SuperMix(Cat.No.:AQ141-03)

a) A PCR reaction solution in a nuclease-free PCR reaction tube is prepared as follows:

b) PCR amplification was performed on a Bio-Rad CFX96 real-time fluorescent quantitation PCR instrument under the following conditions: denaturation at 94 ℃ for 30s; circulating for 39 times, detecting [94 ℃ 10s,60 ℃ 20s and 72 ℃ 20 s/fluorescence signal ]. Times.39 cycles, and after the amplification is finished, analyzing and collecting a melting curve at 50-95 ℃;

c) By using 2 ^-△△CT The PCR result is semi-quantitatively analyzed, the experimental result is shown in FIG. 1, and it can be seen from FIG. 1 that the two siRNAs involved in this example can be efficiently silencedThe expression of CD317 in k562 cells (left side) and jurkat cells (right side) can achieve the silencing effect of more than 70 percent at most.

Example 3 flow cytometry analysis of the Effect of CD317 silencing plus BTZ on apoptosis of hematological tumor cells (k 562 cells, jurkat cells)

(1) Cell pretreatment

1) After 36h of cell transfection with siRNA, the cells were resuspended to 3X 10 ⁵ cells/mL, seeded in 12-well plates;

2) Adding PS-341, and setting up a drug-free group as a control;

(2) Flow cytometry analysis of apoptosis

1) Collecting the cell suspension;

2) Centrifuging the cell suspension at 500g and 4 deg.C for 5min;

3) Washing the cells with precooled PBS for 3 times, then adding PI staining solution or Annexin V-FITC/PI double staining solution, mixing lightly, and reacting at room temperature in a dark place for 15min;

5) After incubation, detecting the apoptosis condition by a flow cytometer: the cells with single positive of Annexin V-FITC are early apoptosis cells, and the cells with double positive of Annexin V-FITC/PI are late apoptosis cells. The experimental results are shown in fig. 2, and it can be seen from fig. 2 that the PI positive cell number of the siR-1 and siR-2 groups is significantly higher than that of the siCT group, indicating that CD317 knockdown promotes BTZ-induced k562 cell death (a panel); consistent with this, CD317 knockdown also promoted BTZ-induced cell death in Jurkat cells, as indicated by an increase in the total number of early and late apoptotic cells in the knockdown cells after receiving BTZ treatment (panel B).

Example 4 Effect of CD317 knockdown on tumor cell misfolded protein levels

(1) Cell pretreatment and sample preparation

1) Cells (Hela and MCF-7) were plated at 5X 10 per well ⁵ The cell number of (2) was plated in a 6-well plate;

2) Plating for 12h, and then transfecting siRNA;

3) Adding PS-341 or MG132 after transfection for 36-48h, and setting up a drug-free group as a control;

4) After 6h of drug treatment, digesting adherent cells by 0.25% (mass volume percentage) of pancreatin, and centrifugally collecting the cells at 3200r/5 min;

5) After the cell pellet was washed once with PBS, the appropriate amount of NS buffer (50 mM Tris, 100mM NaCl, 5mM MgCl. RTM.) was added ₂ 0.5% (volume percent) NP-40, 2mM DTT, 250IU/ml Benzonase, 1mM PMSF, 1 XRoche protease inhibitor, 20mM NEM), on ice for 30 minutes. The whole cell lysate was centrifuged at 13,000rpm for 15 minutes at 4 ℃ and the supernatant (NP-40 soluble protein) was aspirated; taking part of the supernatant, detecting protein concentration by using BCA, adding other protein solutions into protein denaturation buffer solution, and heating at 100 ℃ for 6-8min to prepare NS component;

6) The pellet was further added with the appropriate amount of SS buffer (20 mM Tris, 15mM MgCl2, 2mM DTT, 250IU/ml Benzonase, 1mM PMSF, 1 XRoche protease inhibitor, 20mM NEM) and incubated on ice for 30min. Then, in a buffer containing 2% SDS (mass volume percent, 2g SDS dissolved in 100mL double distilled water) and 50mM DTT, boiling at 100 ℃ for 6-8min to prepare an SS fraction (SDS-soluble protein)

(2) Western blot detection of tumor cell protein level changes

1) Equal amounts of NS and SS fractions were separated by SDS-PAGE, respectively;

5) Film transfer: transferring the protein from SDS-PAGE to a PVDF membrane for 80min under the condition of constant current of 400 mA;

6) Sealing the film: PVDF membrane soaked in 5% (v/v) BSA (mass volume percentage, i.e. 5g BSA in 100mL PBS) and blocked for 1h at room temperature;

7) Primary antibody incubation overnight at 4 ℃: the antibody comprises Anti-K48 Ubiquitin (Abcam), anti-beta-actin (Sigma), anti-CD317 (Abcam);

8) PBST washing 3 times, each time 6min;

9) Incubating the secondary antibody at room temperature for 45min, wherein the secondary antibody is goat anti-rabbit or anti-IgG labeled by horseradish peroxidase (respectively purchased from EARTHOX and KPL);

10 PBST wash 3 times, 6min each time;

11 Enhanced chemiluminescence detection kit (Milipore) was used to detect the corresponding protein signal on PVDF membrane. The results are shown in fig. 3, that CD317 knockdown promotes accumulation of misfolded proteins in tumor cells, as evidenced by increased K48 polyubiquitination modification in the SS fraction.

Example 5 flow cytometry analysis of the Effect of CD317 silencing plus BTZ on apoptosis of solid tumor cells (Hela, MCF7, hepG2, H1975)

(1) Cell pretreatment

1) Cells were plated at 2X 10 per well ⁵ The cell number of (2) was plated in a 12-well plate;

2) Plating for 12h, and then transfecting siRNA;

3) BTZ is added after transfection for 36h, and a drug-free group is set as a control;

(2) Flow cytometry analysis of apoptosis

1) After BTZ treatment for 24h, collecting cell supernatant, centrifuging at 3200rpm for 5min, and collecting floating dead cells in the supernatant;

2) Adding pancreatin to the cells adhering to the wall for digesting for a proper time and then collecting;

3) Mixing the cells of steps 1) and 2), washing 3 times with pre-cooled PBS, and then resuspending with 500 μ L PBS;

4) Adding 5 mu L of PI dye solution into each sample, mixing the mixture evenly, and reacting the mixture for 15min at room temperature in a dark place;

5) After the incubation was completed, the apoptosis was detected by flow cytometry. The results are shown in FIG. 4, and the percentage of PI-positive cells is significantly increased after the CD317 knockdown cells are treated by BTZ, which indicates that the CD317 knockdown promotes BTZ-induced solid tumor cell death (A-D are the results of Hela, MCF-7, hepG2 and H1975 in sequence).

The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.

SEQUENCE LISTING

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Application of combination of <120> CD317 inhibitor and proteasome inhibitor in preparation of antitumor drugs

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Claims (10)

1. Application of CD317 inhibitor as proteasome inhibitor sensitizer.
2. Use of a CD317 inhibitor in the preparation of a medicament for increasing the sensitivity of tumor cells to proteasome inhibitors.
3. Application of combination of CD317 inhibitor and proteasome inhibitor in preparation of antitumor drugs.
4. 4. A method of increasing the sensitivity of a tumor cell to a proteasome inhibitor, comprising the steps of: the expression of CD317 in tumor cells is knocked down using a CD317 inhibitor.
5. 5. A pharmaceutical composition comprising a proteasome inhibitor and at least one CD317 inhibitor.
6. 6. A proteasome inhibitor sensitizer which is characterized by comprising a CD317 inhibitor.
7. 7. The use according to any one of claims 1 to 3, the method according to claim 4, the pharmaceutical composition according to claim 5 or the proteasome inhibitor sensitizer according to claim 6, wherein said proteasome inhibitor comprises bortezomib, MG132.
8. 8. The use according to any one of claims 1 to 3, the method according to claim 4, the pharmaceutical composition according to claim 5 or the proteasome inhibitor sensitizer of claim 6, wherein the CD317 inhibitor comprises CD317 siRNA, CD317 shRNA, DNA encoding shRNA, CD317 sgRNA, PROTAC, an antibody or a blocking peptide;

   preferably, the CD317 inhibitor is CD317 siRNA designed according to human CD317 gene transcript NM _ 004335.3;

   more preferably, the CD317 siRNA comprises a sense strand of a nucleotide sequence shown in SEQ ID NO. 1 and an antisense strand of a nucleotide sequence shown in SEQ ID NO. 2, and/or a sense strand of a nucleotide sequence shown in SEQ ID NO. 3 and an antisense strand of a nucleotide sequence shown in SEQ ID NO. 4.
9. 9. The use of any one of claims 1-3 or the method of claim 4, wherein the tumor or tumor cells comprise solid and non-solid tumors.
10. 10. The use or method of claim 9, wherein the solid tumor comprises human cervical cancer, human non-small cell lung adenocarcinoma, liver cancer, human breast cancer;

    the non-solid tumor comprises chronic myelogenous leukemia, acute T lymphocyte leukemia and myeloma.

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