CN116271048A - Application of SRC protein or down regulator of coding gene thereof in preparation of medicine for treating triple negative breast cancer - Google Patents

Abstract

The invention discloses an application of a down regulator of SRC protein or a coding gene thereof in preparing a medicament for treating triple negative breast cancer, and belongs to the technical field of biological medicines. The invention discloses that the erianin obviously inhibits the proliferation of two TNBC cells of MDA-MB-231 and 4T1, has time and concentration dependence, and also discovers that the erianin can obviously inhibit the growth of mouse transplantation tumor of two TNBC cells of MDA-MB-231 and 4T1 in mouse transplantation tumor experiments. It is known that erianin can exert a good activity against TNBC proliferation at the in vivo and in vitro levels. Further experiments prove that the erianin can reduce the cholesterol content by down-regulating SRC, thereby inhibiting proliferation of TNBC. The invention creatively discovers the effect of the erianin in TNBC progress, analyzes potential drug targets and downstream effect paths, and provides theoretical basis for TNBC targeted therapy.

Description

Application of SRC protein or down regulator of coding gene thereof in preparation of medicine for treating triple negative breast cancer

Technical Field

The invention relates to the technical field of biological medicine, in particular to application of SRC protein or a down regulator of a coding gene thereof in preparation of a medicine for treating triple negative breast cancer.

Background

Breast cancer is the most common female malignancy in the world, and more than 220 tens of thousands of lung cancers have become the most common cancer in the world. In China, the new cases and the death numbers of breast cancer respectively account for 12.2 percent and 9.6 percent of the total world, which forms a serious threat to the health of women. Triple Negative Breast Cancers (TNBC) do not express Estrogen Receptor (ER), progestogen Receptor (PR) and human epidermal growth factor receptor 2 (HER 2), accounting for 10% to 20% of all invasive breast cancers. Although the incidence of TNBC is not high, compared with other types of breast cancer, patients cannot receive endocrine and targeted therapy after surgery due to lack of definite drug treatment targets, the treatment effect is poor, the recurrence rate is high, and the survival rate is low. At present, chemotherapy means are commonly adopted at home and abroad, and paclitaxel and anthracycline drugs are common schemes for clinical treatment of TNBC. However, anthracyclines have irreversible toxic damage to the heart, which is not tolerated by many patients, and once patients develop chemotherapy resistance, tumors rapidly recur and metastasize. Therefore, there is an urgent need to explore more therapeutic agents in the field of TNBC.

Reprogramming lipid metabolism is a hallmark of cancer. Cholesterol is thought to be essential for proliferation and survival of cancer cells as an important component of lipids. Cholesterol is a precursor of bile acids, steroid hormones and vitamin D in addition to being a component of the cell membrane. It plays a key role in cell growth and differentiation. Mammalian cells maintain cholesterol homeostasis by regulating de novo synthesis, absorption, outflow and storage processes. Excessive activation of cholesterol biosynthesis is found in several cancers, which in turn promotes tumor growth, metastasis, stem cells and therapeutic resistance. In breast cancer, cholesterol and its metabolites have been found to promote tumor progression clinically. At the same time, TNBC shows a higher cholesterol biosynthesis than other subtypes, which may have profound biological functions.

Erianin is a small-molecule biphenyl compound extracted from plants of the genus dendrobium of the family orchidaceae, and has been found to have various functions including induction of apoptosis, inhibition of angiogenesis, antioxidation, etc. Previous studies have found that erianin can inhibit the growth of tumor cells by inducing apoptosis, autophagy, iron death, etc., and in the aspect of breast cancer, it has been reported that it can inhibit the growth, proliferation and migration of the breast cancer cell line T47D and induce apoptosis, but less studies on TNBC have been conducted.

Disclosure of Invention

The invention aims to provide an application of a down regulator of SRC protein or a coding gene thereof in preparing a medicine for treating triple negative breast cancer, so as to solve the problems in the prior art.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides an application of SRC protein or a down regulator of a coding gene thereof in preparing a medicament for treating triple negative breast cancer.

Preferably, the down-regulator includes:

a gene editing reagent for specifically knocking out the SRC encoding gene;

an interfering molecule that specifically interferes with the expression of the SRC-encoding gene;

small molecule compounds that specifically inhibit SRC proteins or genes encoding same;

or an antibody or ligand that specifically binds to the SRC protein.

Preferably, the small molecule compound that specifically inhibits SRC protein or gene encoding same comprises SRC specific inhibitor PP2.

Preferably, the antibody or ligand that specifically binds to SRC protein comprises erianin.

The invention also provides a pharmaceutical composition for treating triple negative breast cancer, which comprises the erianin and/or SRC specific inhibitor PP2.

Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, additives or auxiliaries.

Preferably, the pharmaceutical composition is administered by oral, systemic, enteral, parenteral, topical or buccal route.

The invention provides application of SRC in preparation of a triple negative breast cancer drug target.

The invention also provides an application of SRC protein in inhibiting cholesterol metabolism.

The invention discloses the following technical effects:

the invention discloses an application of SRC protein or a down regulator of a coding gene thereof in preparing a medicine for treating triple negative breast cancer, and experiments prove that the erianin remarkably inhibits proliferation of two TNBC cells of MDA-MB-231 and 4T1 and has time and concentration dependence. It is known that the erianin can exert better anti-TNBC proliferation activity on the in-vivo and in-vitro level, and has no obvious toxicity to the normal mouse organism. Further experiments prove that the erianin can reduce cholesterol content and further inhibit proliferation of TNBC by down-regulating expression of SRC. The invention creatively discovers the effect of the erianin in TNBC progress, analyzes potential drug targets and downstream effect paths, and provides theoretical basis for TNBC targeted therapy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1A shows the survival rate of erianin on MDA-MB-21 cell cells; b is the survival rate of the erianin acting on 4T1 cell cells;

fig. 2 a is an image of a nude mouse engrafted tumor after in-situ photographing of a erianin treatment; b is the MDA-MB-231 cells inoculated into Balb/c nude mice, and each group has tumor volume (N=5); c is a Balb/C normal mouse transplantation tumor image after in-situ shooting of the erianin treatment; d is 4T1 cells injected into Balb/c normal mice, each group tumor volume (n=5);

fig. 3 a is the change in body weight of nude mice after tumor-bearing on days 0 to 21; b is the weight change after tumor burden from day 0 to day 17 of Balb/c mice;

FIG. 4A is the effect of erianin on liver injury in Balb/c mice; b is the influence of the erianin on the content of glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase in whole blood of a Balb/c mouse;

FIG. 5A shows the expression of SRC after treatment of TNBC cells with WB; b is a statistical graph of the effect of erianin on SRC expression in TNBC cells; c is IHC to detect the expression condition of SRC in the mouse transplanted tumor after treatment of the erianin; d is a statistical graph of the effect of erianin on SRC expression in mouse transplantations;

FIGS. 6 and 7 are graphs showing the proliferation effect of SRC expression on TNBC; FIG. 6A shows the change in SRC protein levels following PP2 (200 nM) treatment of MDA-MB-231 and 4T1 cells; FIG. 6B shows the variation of SRC protein levels in two TNBC cells treated with PP 2; FIG. 6C shows the proliferation level of MDA-MB-231 and 4T1 cells detected by CCK-8 after 24 hours of PP2 (200 nM) action; FIG. 7A is an in situ image of a tumor transplanted in a Balb/c nude mouse treated with PP2 by injecting MDA-MB-231 cells in situ; b in fig. 7 is the tumor volume of each group (n=5);

c in fig. 7 is the tumor weight of each group (n=5); FIG. 7D is an image of a PP2 treated engrafted tumor obtained by injecting 4T1 cells into Balb/c mice; e in fig. 7 is the tumor volume of each group (n=5); f in fig. 7 is the tumor weight of each group (n=5); FIG. 7G is the expression level of SRC in two transplants; h in FIG. 7 is statistics of SRC expression levels;

FIGS. 8 and 9 are graphs showing that erianin inhibits proliferation of TNBC at in vitro and in vivo levels by lowering cholesterol levels; FIG. 8A is a KEGG pathway analysis diagram; FIG. 8B is a graph showing that treatment with erianin reduces cholesterol levels in MDA-MB-231 and 4T1 cells; FIG. 8C shows the expression levels of HMGCR, SERBP2, DHCR24 and LDLR detected by RT-PCR after treatment of MDA-MB-231 and 4T1 cells with erianin; fig. 8D shows that erianin reduces cholesterol levels in different mouse tumor models; FIG. 9A shows the change in cholesterol levels after cholesterol treatment of MDA-MB-231 and 4T1 cells; FIG. 9B shows the CCK-8 assay for cell proliferation in various groups 24 hours after cholesterol treatment of MDA-MB-231 and 4T1 cells; FIG. 9C is the effect of lovastatin on MDA-MB-231 and 4T1 intracellular cholesterol levels; FIG. 9D shows the detection of cell proliferation by CCK-8 after lovastatin treatment of MDA-MB-231 and 4T1 cells for 24 hours; FIG. 9E is an in situ image of an in situ engrafted tumor after cholesterol treatment by in situ seeding of MDA-MB-231 cells in Balb/c nude mice; f in fig. 9 is the tumor volume of each group (n=5); g in fig. 9 is the tumor weight of each group (n=5); FIG. 9H is a xenograft tumor after in situ imaging of cholesterol treatment by injection of 4T1 cells into Balb/c mice; i in fig. 9 is the tumor volume of each group (n=5); j in fig. 9 is the tumor weight of each group (n=5); k in FIG. 9 is the cholesterol content in the two model transplants;

FIG. 10 is the effect of SRC expression on cholesterol levels in TNBC cells and tumors; wherein A is PP2, which can reduce the cholesterol content of MDA-MB-231 and 4T1 cells; b is PP2, which can inhibit the expression of HMGCR, SERBP2, DHCR24 and LDLR in MDA-MB-231 and 4T1 cells; c is PP2, which can reduce the cholesterol content in a mouse transplanted tumor model;

FIG. 11 shows the relationship between SRC, cholesterol and erianin; a is the inhibition of MDA-MB-231 cell proliferation by the overexpression of SRC; b is the inhibition of the MDA-MB-231 cells by the over-expression SRC, which can relieve the cholesterol content of the erianin; c is high cholesterol content, which can relieve the inhibition of the erianin on MDA-MB-231 cell proliferation; d is high cholesterol and can reduce the inhibition of the content of cholesterol in MDA-MB-231 cells by the erianin.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, with reference to the examples using conventional methods, unless otherwise indicated, and with reference to reagents, either commercially available or formulated using conventional methods. The detailed description is not to be taken as limiting, but is to be understood as a more detailed description of certain aspects, features, and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The erianin of the invention is purchased from Shanghai Yuan Ye Biotechnology Co Ltd (B20844), the MDA-MB-231 and 4T1 cell lines are purchased from Beijing co-medicine college cell culture center (Beijing), the Balb/c immunodeficient mice are purchased from Yunnan university laboratory animal center, and the Balb/c normal female mice are obtained from Kunming university laboratory animal center.

EXAMPLE 1 Effect of erianin on TNBC cell growth

CCK-8 experiments were used to evaluate the effect of drug treatment on MDA-MB-231 and 4T1 cell proliferation and viability

MDA-MB-231 cells and 4T1 were assayed at 2X 10 per well ⁴ Inoculating the culture medium into a 48-well plate, when cells are fused by 70% -80%, treating the culture medium with different concentrations for a certain time, and sucking out the culture medium in the 48-well plate by using a liquid sucking pump, wherein each well200. Mu.L of CCK-8 dilution (180. Mu.L of complete medium and 20. Mu.L of CCK-8 working solution) was added and absorbance values at 450nm were measured in 48 well plates using a microplate reader. Cell viability (%) = (experimental group-blank group)/(negative control group-blank group) ×100%.

The results, assessed by CCK-8, showed that erianin significantly inhibited proliferation of both TNBC cells and had a time and concentration dependence (see fig. 1).

To explore the effect of erianin on TNBC tumor growth, we selected 6-8 week old female mice for the transplantation experiments using 3X 10 ⁵ 4T1 cells of (A) were inoculated into the left lower penultimate creamer pad, 3X 10, of Balb/c mice ⁵ MDA-MB-231 cells of Balb/c nude mice were inoculated into the left lower penultimate milk fat pad. When the tumor volume reaches 15mm ³ On the left and right, mice were randomly divided into control group (n=5) and experimental group (n=5), and weights of the mice were recorded. The mice of the experimental group were intraperitoneally injected with erianin 4mg/kg, the control group was injected with the same volume of solvent (1% dmso), the above treatment was performed once every 2 days, and the maximum and minimum diameters of the tumor were measured with a vernier caliper every two days. Tumor volume was calculated (tumor volume=0.5×long diameter×wide diameter ² ). After the end of the experiment (21 days of Balb/c nude mice treatment, 17 days of Balb/c mice treatment), mice were deeply anesthetized with 3% sodium pentobarbital, weighed, and sacrificed by means of mouse spinal cord dislocation, dissected, photographed, and weighed for tumor, and the results showed that erianin could significantly inhibit the growth of mouse tumors in both tumor-transplanted models (see fig. 2), and that erianin did not inhibit the increase of body weight in mice (see fig. 3).

To investigate the effect of erianin on the toxicity of mice, we selected 6-8 week old female Balb/c mice for toxicity experiments, the experimental group mice were intraperitoneally injected with erianin 4mg/kg, and the control group with the same volume of solvent (1% dmso). The above treatment was performed every 2 days. After the experiment is carried out for 17 days, 3% sodium pentobarbital is used for deeply anesthetizing the mice, eyeballs are used for taking blood from the mice, and the glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase detection kit is used for detecting the contents of the glutamic-oxaloacetic transaminase and the glutamic-pyruvic transaminase in the blood. And the mice are sacrificed by adopting a method of dislocation of the spinal cord of the mice, and the livers of the mice are dissected and taken out for HE staining. The results showed that the liver of the mice in the erianin group was not significantly damaged (see fig. 4 a) and the content of glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase in the blood of the mice was not significantly increased (see fig. 4B) compared with the normal group. It can be seen that the erianin can exert better activity of resisting TNBC proliferation on the in-vivo and in-vitro level, and has no obvious toxicity to the body of a normal mouse.

Example 2 target site of action of Maolansu against TNBC

In order to explore a specific mechanism of treating TNBC by using the erianin, screening and identifying target genes shared by the erianin and the TNBC through network pharmacology, obtaining 120 shared target genes, carrying out PPI enrichment analysis on proteins transcribed by the 120 shared target genes to find SRC and PI3KCA as proteins with the largest node number, and further carrying out molecular docking analysis to find that the erianin and the SRC have strong binding effect.

Next, the effect of the erianin on the expression of SRC in MDA-MB-231 and 4T1 cells was examined by WB, which showed that the erianin could significantly inhibit the expression of SRC in both TNBC cells (see A, B in fig. 5), while the expression of SRC in mouse engraftment tumors was examined by immunohistochemistry, resulting in the same results as WB (see C, D in fig. 5). It can be seen that erianin may affect TNBC progression by reducing SRC expression.

CCK-8 experiments were used to evaluate the effect of SRC on MDA-MB-231 and 4T1 cell proliferation and viability. MDA-MB-231 cells and 4T1 cells were plated at 2X 10 cells per well ⁴ The concentration of the SRC was inoculated into a 48-well plate, and MDA-MB-231 and 4T1 cells were treated with SRC-specific inhibitor PP2 when cells were 70% -80%, and the results showed that the expression level of SRC was significantly reduced after the MDA-MB-231 and 4T1 cells were treated with 200nM PP2 for 24 hours (see A, B in FIG. 6), while proliferation of MDA-MB-231 and 4T1 cells was significantly inhibited (see C in FIG. 6).

Further in vivo experiments show that the PP2 treatment group can significantly inhibit the growth of mouse tumors in two transplanted tumor models (see A-F in FIG. 7) and the expression level of SRC in tumor tissues (see G and H in FIG. 7).

Example 3 inhibition of tumor growth by erianin by lowering cholesterol levels

The mice were dissected for tumor transplants and total RNA was extracted with Trizol reagent. The integrity of ribonucleic acid was checked with an Agilent 2100 bioanalyzer according to the manufacturer's instructions, library

Ultra ^TM Directional RNALibrary Prep Kit for/>

And (5) construction. After library construction, the library was initially quantified using a qubit2.0 fluorescence analyzer, diluted to 1.5 ng/. Mu.L and the insert size and effective concentration of the library was measured using an Agilent 2100 bioanalyzer and qRT-PCR. Finally, the total RNA of the cells was mRNA sequenced on Illumina Novaseq 6000 platform. And (5) carrying out gene set enrichment analysis and signal drift diameter enrichment analysis after sequencing. The results showed a significant difference in total 873 genes following treatment with erianin, with 374 genes down-regulated and 499 genes up-regulated. KEGG pathway analysis showed that the steroid metabolic pathway was significantly enriched, while cholesterol was the largest class of steroids (see a in fig. 8).

To investigate the effect of erianin on cholesterol levels in TNBC.

We used TNBC cell and mouse engraftment models to examine the effect of treatment with erianin on cholesterol levels in TNBC, respectively. The results show that the erianin can significantly reduce the cholesterol content in MDA-MB-231 cells and 4T1 cells (see B in figure 8), and simultaneously inhibit the expression levels of HMGCR, SERBP2, DHCR24 and LDLR genes in cholesterol metabolic pathways (see C in figure 8), and further in vivo experiments show that the erianin can also significantly reduce the tumor cholesterol content in two transplanted tumor models (see D in figure 8).

The effect of cholesterol on TNBC proliferation was then investigated. We treated MDA-MB-231 cells and 4T1 cells with cholesterol (20 μm) and lovastatin (2 μm), a cholesterol synthesis inhibitor, respectively, and found that the cholesterol content in cells of the cholesterol-added group was significantly increased, and the proliferation capacity of cells was significantly enhanced, in contrast, the cholesterol content in cells of the lovastatin-treated group was significantly reduced, and the proliferation capacity of both TNBC cells was also significantly inhibited, as compared with the control group (see a-D in fig. 9). Further in vivo experiments show that cholesterol can significantly promote the growth of mouse tumors in two transplanted tumor models (see E-J in FIG. 9) and increase the cholesterol content in tumor tissues (see K in FIG. 9). The above results indicate that erianin can inhibit TNBC proliferation by reducing cholesterol levels.

Example 4 erianin affects proliferation of TNBC by down-regulating SRC to inhibit cholesterol metabolism

Treatment of MDA-MB-231 and 4T1 cells with 200nM PP2 for 24h revealed that the cell cholesterol content in the PP2 group was significantly reduced (see FIG. 10A) and the expression of genes involved in cholesterol synthesis and absorption was also significantly inhibited (see FIG. 10B) as compared with the control group. At the same time, we have also obtained the same results in the mouse engraftment experiments (see C in fig. 10).

Subsequently, a reversion experiment was performed in TNBC cells. MDA-MB-231 cells were first treated with 40nM of erianin while the SRC gene was overexpressed in the MDA-MB-231 cells. After 24 hours, the proliferation capacity and cholesterol content of the cells were examined. The results indicate that overexpression of the SRC gene reduces the inhibitory effect of erianin on MDA-MB-231 cell proliferation and cholesterol levels (see A, B in FIG. 11). Next, we treated MDA-MB-231 cells simultaneously with 40nM of erianin and 20. Mu.M of cholesterol. The results show that high cholesterol levels in cells can also partially slow down the inhibition of cell proliferation by erianin (see C, D in figure 11). It is suggested that erianin may affect proliferation of TNBC by down-regulating SRC to inhibit cholesterol metabolism.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (9)

1. An application of SRC protein or its coding gene in preparing the medicines for treating triple negative breast cancer.

2. The use according to claim 1, wherein the downregulator comprises:

a gene editing reagent for specifically knocking out the SRC encoding gene;

an interfering molecule that specifically interferes with the expression of the SRC-encoding gene;

small molecule compounds that specifically inhibit SRC proteins or genes encoding same;

or an antibody or ligand that specifically binds to the SRC protein.

3. The use according to claim 2, wherein the small molecule compound that specifically inhibits the SRC protein or gene encoding it comprises the SRC specific inhibitor PP2.

4. The use according to claim 2, wherein the antibody or ligand that specifically binds to SRC protein comprises erianin.

5. A pharmaceutical composition for the treatment of triple negative breast cancer, characterized in that the pharmaceutical composition comprises a pilin and/or SRC specific inhibitor PP2.

6. The pharmaceutical composition of claim 5, further comprising one or more pharmaceutically acceptable excipients, additives or auxiliaries.

7. The pharmaceutical composition according to any one of claims 5 to 6, wherein the pharmaceutical composition is administered by oral, systemic, enteral, parenteral, topical or buccal route.

Application of SRC in preparation of triple negative breast cancer drug targets.

Use of src protein in inhibiting cholesterol metabolism.

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