CN110693862A - Application of protocatechualdehyde in inhibiting CtBP1 - Google Patents

Abstract

The invention relates to application of protocatechualdehyde in preparation of a CtBP1 inhibitor, and functional research shows that PA can inhibit proliferation and migration of breast cancer cells. In addition, we found that in breast cancer cells, PA can increase the expression level of target genes P21 and E-cadherin of CtBP1, and can reduce the CtBP1 binding strength of P21 and E-cadherin promoter regions. However, in the breast cancer cells with CtBP1 gene knockout constructed by using CRISPR/Cas9, PA can not influence the expression level of P21 and E-cadherin. In addition, resistance to inhibition of PA-induced proliferation and migration was observed in CtBP1 knock-out breast cancer cells. Our research results show that PA can be directly combined with CtBP1, and one of molecular mechanisms of the anticancer activity of PA is targeting CtBP1, which shows that PA is a potential CtBP1 inhibitor.

Description

Application of protocatechualdehyde in inhibiting CtBP1

Technical Field

The invention relates to the technical field of new indications of medicines, in particular to application of protocatechualdehyde in inhibiting CtBP 1.

Background

Carboxy-terminal binding proteins (CtBP) are evolutionarily conserved transcription co-repressors. The study shows that CtBP is related to tumorigenesis and tumor progression, and the CtBP can promote epithelial-mesenchymal transition (EMT) and is used as an apoptosis antagonist to participate in the inhibition of a plurality of tumor suppressor genes. Deletion of epithelial cadherin (E-cadherin) expression is a characteristic molecular event of various epithelial tumor EMTs, CtBP-mediated E-cadherin inhibition shows its important role in promoting tumor EMT, which is a step leading to malignant characteristics of tumor cells: loss of cell adhesion molecules to the tumor, acquisition of migration and erosion phenotypes, resistance to apoptosis, and the like.

CtBP of vertebrate is coded by CtBP1 gene and CtBP2 gene, and expressed to produce two proteins CtBP1 and CtBP 2. C-terminal binding protein 1 (CtBP 1), a transcriptional co-repressor, is overexpressed in a variety of cancers. CtBP1 can promote cancer cell proliferation, migration, invasion and apoptosis by inhibiting a plurality of tumor inhibiting factors.

Protocatechualdehyde (PA), with chemical name of 3, 4-dihydroxybenzaldehyde, is derived from water-soluble extract of blood circulation promoting and blood stasis removing medicine (such as Saviae Miltiorrhizae radix, folium Ilicis Purpureae, and herba Salvia officinalis). Because the antioxidant activity of protocatechualdehyde is derived from the mother nucleus of its catechol, it has various physiological activities, such as the actions of dilating artery, reducing myocardial oxygen consumption, inhibiting platelet aggregation, resisting lipid peroxidation and scavenging free radicals.

In recent years, as more and more protocatechualdehyde is researched and reported, new pharmacological effects of protocatechualdehyde are discovered successively. XuY et al found that bolus tail vein injection of PA reduced the mortality of caecal ligation and perforation induced sepsis rats by blocking the inflammatory site through HMGBl and NF-jB signaling pathways. ZouZ and the like firstly prove that PA has the effect of resisting HBV, can effectively inhibit the replication of the HBV of the cell strain in vitro and can also effectively inhibit the replication of DHBV in vivo. Gao J et al found that PA protected human neuroblastoma cells by modulating the DJ-1 gene. Chinese patent document CN201910615961.6 discloses the application of protocatechualdehyde in preparing inhibitory drugs for bacterial quorum sensing systems. Chinese patent document CN 201610711489.2 discloses the application of protocatechualdehyde in the preparation of medicine for treating acute kidney injury. However, the action mechanism of PA on CtBP1 has not been reported yet.

Disclosure of Invention

The invention aims to overcome the defects and technical shortcomings of the prior art and provide an application of protocatechualdehyde in preparation of a CtBP1 inhibitor.

The invention also aims to provide a new application of protocatechuic aldehyde in inhibiting cell proliferation and migration related to CtBP1 overexpression and/or treating diseases of CtBP1 overexpression.

It is still another object of the present invention to provide a CtBP1 inhibitor.

In order to achieve the first purpose, the invention adopts the technical scheme that:

application of protocatechualdehyde in preparation of CtBP1 inhibitor is provided.

After learning the inhibitory effect of the protocatechualdehyde of the present invention on CtBP1, one skilled in the art can use various conventional methods to process other protocatechualdehyde-containing materials into pharmaceutically acceptable salts and esters, optionally substituted analogs of synthetic or semi-synthetic protocatechualdehyde, or combinations of one or more compounds comprising protocatechualdehyde, including derivatives of protocatechualdehyde. Such processing includes, but is not limited to: pulverizing, extracting with water, extracting with organic solvent, etc. More specifically, the process comprises, for example, the steps of: weighing, pulverizing, decocting, etc.

Preferably, the protocatechualdehyde is used as a single active ingredient of a medicament or is used as an active ingredient together with other CtBP1 inhibitors.

In order to achieve the second object, the invention adopts the technical scheme that:

application of protocatechualdehyde in preparation of medicines for inhibiting cell proliferation and migration related to CtBP1 overexpression and/or treating diseases of CtBP1 overexpression.

Preferably, the protocatechualdehyde is used as a single active ingredient of a medicament or as an active ingredient in combination with other medicaments.

Preferably, the disease in which CtBP1 is overexpressed is a tumor.

The invention also discloses the application of protocatechuic aldehyde in designing, developing and preparing related inhibitors aiming at CtBP1 overexpression or using the same as biochemical reagents, analytical reagents or detection reagents, or the application of protocatechuic aldehyde in a novel method and a technical aspect for researching cell signaling pathway regulated by CtBP1 overexpression and related target protein biological functions.

In order to achieve the third object, the invention adopts the technical scheme that:

an inhibitor of CtBP1, the active ingredient of which comprises a therapeutically effective amount of protocatechualdehyde.

Preferably, the medicament also comprises a component which has positive effects on inhibiting cell proliferation and migration related to the over-expression of CtBP1 and/or treating a medicament for a disease of the over-expression of CtBP1 after being simultaneously administered with protocatechualdehyde and/or a pharmaceutically acceptable carrier for improving the stability of protocatechualdehyde.

Preferably, the pharmaceutically acceptable carrier includes an emulsifier, excipient, filler, binder, humectant, disintegrant, absorption enhancer, flavoring agent, coloring agent or solubilizing agent

By "pharmaceutically acceptable" is meant a substance that is not substantially biologically or otherwise undesirable, i.e., the substance can be administered to an individual without causing any substantially undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The "carrier", also referred to as "excipient", includes any commonly used excipient in pharmacy and should be selected based on compatibility and the desired release profile properties of the dosage form. Exemplary carrier materials include, for example, emulsifiers, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, flavoring agents, coloring agents, or solubilizing agents, and the like. "pharmaceutically acceptable carriers" may include, for example, gum arabic, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, dextrin-maltose complexing agent, glycerin, magnesium silicate, sodium caseinate, soybean lecithin, sodium chloride, tricalcium phosphate, dipotassium hydrogen phosphate, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, and the like. The pharmaceutical composition can be prepared into oral preparations, such as tablets, hard capsules, soft capsules, dripping pills, granules, micro-capsule tablets, suspensions, dripping pills or oral liquids, by adopting methods known in the art.

Functional studies have shown that PA inhibits proliferation and migration of breast cancer cells. In addition, we found that in breast cancer cells, PA can increase the expression level of target genes P21 and E-cadherin of CtBP1, and can reduce the CtBP1 binding strength of P21 and E-cadherin promoter regions. However, in the breast cancer cells with CtBP1 gene knockout constructed by using CRISPR/Cas9, PA can not influence the expression level of P21 and E-cadherin. In addition, resistance to inhibition of PA-induced proliferation and migration was observed in CtBP1 knock-out breast cancer cells. Our research results show that PA can be directly combined with CtBP1, and one of molecular mechanisms of the anticancer activity of PA is targeting CtBP1, which shows that PA is a potential CtBP1 inhibitor.

Drawings

FIG. 1 is a graphical representation of PA in combination with CtBP 1. Wherein A is a butt joint mode of PA and CtBP1, CtBP1 is in a mode diagram, a is PA, b is phenylpyruvate, and a dashed line is a distance; b is a binding affinity map for detecting PA to CtBP1 using the MST assay.

Figure 2A is a growth curve of MDA-MB-231 and MCF-7 cells treated with 0.1% DMSO or 100 μ MPA, respectively, indicating P < 0.05; b is an image of the ability of breast cancer cells to migrate as measured by the scratch test, showing scratches with or without PA treatment. The percentage of scratch closure area shown in the bar graph is the mean ± standard deviation from triplicate experimental data, representing P < 0.05; c is the ability of breast cancer cells to migrate as measured by transwell experiments, the number of migrated cells was calculated from 3 randomly selected microscopic fields, the results shown in the bar graph are the mean ± standard deviation of data from triplicates, indicating P < 0.05.

FIG. 3A is the expression of P21 and E-cadherin mRNA from each group of cells after 48 hours of drug treatment; b is the expression level of P21 and E-cadherin protein after 48 hours of PA treatment; c is CtBP1 chromatin co-immunoprecipitation plot, P < 0.05.

FIG. 4A shows Crispr-Cas9 construction of MDA-MB-231 and MCF-7CtBP1 knock-out cell Sanger sequencing, confirming 2 knock-out cell lines with nucleotide 'A' insertion between the 57 th and 58 th nucleotides of the 2 nd CDS exon of CtBP 1; b is the mRNA and protein expression level of P21 and E-cadherin after the CtBP1 is knocked out by breast cancer cells, and represents that P is less than 0.05; c is a complete knock-out of CtBP1 in MDA-MB-231 and MCF-7 cells increased the IC50 value of PA-treated breast cancer cells; d is an image for detecting the migration capacity of the MDA-MB-231 and MCF-7 cells knocked out by CtBP1 through a scratch test, and E is an image for detecting the migration capacity of the MDA-MB-231 and MCF-7 cells knocked out by CtBP1 through a transwell test; f is the coverage area and the percentage of migrated cells for the DMSO group/100 μ MPA group, calculated from the mean of the groups; g is the expression level of P21 and E-cadherin proteins in CtBP1 knockout cells.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention; furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1

1 methods and materials

1.1 cell culture reagents

Will knock out or not knock out CMDA-MB-231 and MCF-7 cells of the tBP1 gene were cultured in appropriate medium containing 10% FBS, and cell culture incubator conditions were set to: 37 ℃ and 5% CO₂The following steps. Protocatechualdehyde was purchased from Sigma (Cat n.od108405, st.louis, MO, USA) and dissolved in DMSO.

1.2 Structure-based network pharmacology computational screening

From the Protein Data Bank, 4CtBP1 structures (PDB codes: 1MX3,4LCE, 4U6Q and 4U6S) were retrieved with a resolution better than 2.50. Using the OPLS _2005 force field, using Epik 3.4(

LLC, New York, USA) pairs Ligprep 3.6(

LLC, New York, NY, USA) to produce appropriate protonation states at ph7.0 ± 2.0. The substrate phenyl pyruvate in 4U6S was selected to define a docking grid. Computer docking of 8000 internal compounds with 4CtBP1 constructs based on default parameters was performed by Glide 6.9(

LLC, New York, NY, USA) with Standard Precision (SP). Based on manual examination of the docking conformation, PA was selected for further in vitro analysis.

1.3 micro-calorimetric surge detection

Microcalorimetric surge assays (MST) were applied to determine the binding affinity of CtBP1 to PA. Recombinant human CtBP1 protein (Abnova Cat N.O H00001487-P01, Taiwan, China) was labeled with a fluorescent dye by using a whole-body protein labeling kit (nanotemperers Technologies Cat N.O L001, Germany). The assay was performed in pH7.4 buffer containing 20mM Tris, 0.3M Nac1, 5% glycerol, 3% DMSO and 0.05% Tween-20. After protein labeling was complete, the samples were loaded into Monolith NT.115(NanoTemper technologies, Germany) standard glass capillary tubes. During the MST experiment, the 3.15 μ M labeled CtBP1 remained constant, while the concentration of PA continued to be 1: 2 dilutions, a total of 16 dilutions of PA were prepared in a gradient from a maximum final concentration of 0.66mM to a minimum concentration of 0.05nM and mixed with labeled CTBP 1. After the completion of the sample loading, the detection was performed on the machine in a Monolith NT.115 apparatus.

1.4 Western blot analysis

Cells were lysed with RIPA150 lysis buffer (Cat N.O 04693116001; Sigma, st. louis, MO, USA) containing a 1X protease inhibitor. Then, total cell lysate samples were separated by SDS-PAGE and transferred to PVDF membrane (Cat N.O162-0177; Bio-Rad, Calif., USA), which was incubated with primary and secondary antibodies. Signals were detected using enhanced chemiluminescence reagents (Cat N.O34577; Thermo Scientific, Rockford, IL, USA). Primary antibodies used for Western blot analysis were rabbit anti-P21 (Cat N.O2947; Cell Signaling Technology, USA), rabbit anti-CtBP 1(Cat NOab 129181; Abcam, Cambridge, UK), murine anti-GAPDH (Cat N.O60004-1-Ig protein Tech, Wuhan, China) and rabbit anti-E-Cadherin (Cat N.O20874-1-AP; protein Tech, Wuhan, China).

1.5 quantitative RT-PCR

Trizol (Cat N.O 15596026; Invitrogen, Carlsbad, Calif., USA) was used to extract total RNA. 100ng of total RNA was used for reverse transcription into cDNA by cDNA synthesis kit (Cat N.O K1622; Thermo Scientific, Rockford, Ill., USA). E-cadherin and P21 expression were detected by the following primers:

e-cadherin forward primer: 5'-GCAGCCAAAGACAGAGCGGAAC-3' the flow of the air in the air conditioner,

e-cadherin reverse primer: 5 'ACCCACCTCAATCATCCTCAGCA-3';

p21 forward primer: 5'-GCTATTTTGTCCTTGGGCTG-3' the flow of the air in the air conditioner,

p21 reverse primer: 5 'ATTAGCGCATCACAGTCG-3';

18S expression was used as an internal control with the sequence:

18S forward primer: 5'-TGACGGAAGGGCACCACCAG-3' the flow of the air in the air conditioner,

18S reverse primer: 5 'GCACCACCACCCACGGAATC-3'.

Relative expression levels were calculated by normalization with an internal control.

1.6 chromatin Co-immunoprecipitation (ChIP)

MDA-MB-231 and MCF-7 cells, with or without 100uM PA treatment, were used in the ChIP assay. Cells that were 80% confluent were cross-linked with 1% formaldehyde for 15 minutes and then terminated with 0.125M glycine. The cell pellet was sonicated in lysis buffer. Fragmented DNA was precipitated with CtBP1 antibody (Cat N.O ab 129181; Abcam, Cambridge, UK) and protein A beads (CatN.O 26159; Thermo Scientific, Rockford, IL, USA). The precipitated protein/DNA complexes were reverse cross-linked with additional 350mM NaCl at 65 ℃ for 6 hours. The DNA fragment was then purified and used for PCR analysis. The primers used to detect the E-cadherin and P21 promoters are as follows.

E-cadherin forward primer: 5'-GGCGTCGGAACTGCAAAGCA-3' the flow of the air in the air conditioner,

e-cadherin reverse primer: 5'-GGAGCGGGCTGGAGTCTGAA-3' are provided.

P21 forward primer: 5'-TCTTTTCAGCTGCATTGGG-3' the flow of the air in the air conditioner,

p21 reverse primer: 5'-TTGGAGAATGAGTTGGCAC-3' are provided.

Line1 was used as an internal control, and the primer sequences were:

line1 forward primer: 5'-GCGCAAGGGGTCAGGGAGTT-3' the flow of the air in the air conditioner,

line1 reverse primer: 5'-CTCGTGGTGCGCCGTTTCTT-3' are provided.

1.7MTT assay

MTT assay for detecting cell growth and IC₅₀. MTT assay kit was purchased from Sigma (CatN.O11465007001; Sigma, St.Louis, MO, USA) and performed according to the manufacturer's recommended protocol. Each assay was repeated three times.

1.8CRISPR/Cas 9-mediated CtBP1 knockout

We constructed CtBP1 knock-out cells by lentivirus-mediated knock-out. The sgRNA sequences used in the experiments were:

a forward direction 5'-CACCGGTCCGACCTCCGATCATGAA-3';

and reverse direction 5'-AAACTTCATGATCGGAGGTCGGACC-3'.

The treated sgRNA oligomers were ligated into the lentiCRISPR vector obtained from Addgene (plasmid # 52961). The lentiCRISPR vector constructed to contain CtBP1 sgRAN was transduced into 293T cells with the packaging plasmids psPAX2 and pMD2 to produce lentiviruses. MDA-MB-231 and MCF-7 cells were infected with lentivirus for 24 hours and then screened with 1. mu.g/ml puromycin for 14 days. The selected cells were seeded into 96-well plates at 1 cell/well. After single cells were amplified until clones were formed, a portion of the cells were taken to verify the expression level of CtBP1 by western blotting. Clones without CtBP1 protein expression were sent to Sanger sequencing for further confirmation.

1.9 cell scratch test

Cells in 6-well plates were cultured in medium with or without PA. Uniform scratches were made in a monolayer of cells by a 200 μ l pipette. The percentage of healed area was measured by Image-J software after 24 hours of culture of MAD-MB-231or 48 hours of culture of MCF-7 cells. The assay was repeated three times.

1.10Transwell test

Transfected 1X105 cells were seeded on the upper side of a polycarbonate transwell chamber (Cat N.O 353097; Corning Costar, USA) containing serum-free DMEM medium, and the lower side of the transwell chamber was filled with DMEM medium containing 10% FBS. For MDA-MB-231 cells, 50. mu.M or 100. mu.M PA was added to the upper side of the chamber and cultured for 48 hours. For MCF-7 cells, 50. mu.M or 100. mu.M PA was added to the upper side of the chamber and cultured for 72 hours. After incubation, the cells left on the upper side of the chamber were removed with a cotton swab. The chamber was washed 2 times with 1X PBS and the passed cells were fixed to the underside of the chamber with 95% ethanol for 20 minutes. Fixed cells were stained with 0.1% crystal violet. Images were taken with an Olympus LX73 microscope. (Olympus corporation, Tokyo, Japan)

2 results of the experiment

2.1 direct binding of PA to CtBP1

As shown in FIG. 1, PA bound directly to CtBP1 with a binding affinity of 612 nM. + -. 13.7 nM.

2.2PA inhibits growth and migration of Breast cancer cells

As shown in FIG. 2, MDA-MB-231 and MCF-7 cells treated with 0.1% DMSO showed increasing relative growth rates, and cells treated with 100. mu.M PA showed inhibited growth (FIG. 2A); the migration ability of breast cancer cells was examined by scratch test, and PA showed inhibition at 50. mu.M concentration for both MDA-MB-231 and MCF-7 cells (FIG. 2B); the ability of breast cancer cells to migrate was tested by transwell assay, and the number of migrated cells was counted from 3 randomly selected microscopic fields, consistent with the scratch assay (fig. 2C).

2.3PA downregulated expression of P21 and E-cadherin in MDA-MB-231 and MCF-7 cells

The results of the experiments are shown in fig. 3, and show that PA up-regulated the expression of P21 and E-cadherin mRNA and the expression levels of P21 and E-cadherin protein in a dose-dependent manner after 48 hours of dosing (fig. 3A, B); CtBP1 chromatin co-immunoprecipitation assay showed that PA treatment reduced CtBP1 binding strength in the promoter regions of P21 and E-cadherin.

(FIG. 3C)

2.4CtBP1 knock-out increases resistance to PA

The experimental results are shown in fig. 4, when criprpr-Cas 9 constructs MDA-MB-231 and MCF-7CtBP1 knock-out cells, Sanger sequencing confirmed that there was a nucleotide 'a' insertion between the 57 th and 58 th nucleotides of the 2 nd CDS exon of CtBP1 in 2 knock-out cell lines (fig. 4A); knock-out of CtBP1 in breast cancer cells increased P21 and E-cadherin mRNA and protein expression levels (FIG. 4B); the complete knock-out of CtBP1 in MDA-MB-231 and MCF-7 cells increased the IC50 value in PA-treated breast cancer cells (FIG. 4C); the CtBP1 knockout was shown to be resistant to PA-induced migration inhibition in MDA-MB-231 and MCF-7 cells. 50 μ M PA did not have any inhibitory effect on knockdown cells, but 100 μ MPA still showed slight inhibition (FIG. 4D, E); compared with CtBP1 expressing cells, 100 mu M PA has reduced effect of inhibiting migration of CtBP1 knockout cells. The relative inhibitory effect is represented by the coverage area or the percentage of migrated cells of the DMSO/100 μ MPA group, calculated by the mean of the groups (fig. 4F). The expression levels of P21 and E-cadherin proteins in CtBP1 knock-out cells were not responsive to PA treatment (FIG. 4G).

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims (9)

1. Application of protocatechualdehyde in preparation of CtBP1 inhibitor is provided.

2. The use according to claim 1, wherein the protocatechualdehyde further comprises pharmaceutically acceptable salts and esters thereof, optionally substituted analogs thereof, or combinations of one or more compounds comprising protocatechualdehyde, further comprising derivatives of protocatechualdehyde.

3. Use according to claim 1 or 2, characterized in that the protocatechualdehyde is used as single active ingredient of a medicament or as active ingredient together with other CtBP1 inhibitors.

4. Application of protocatechualdehyde in preparation of medicines for inhibiting cell proliferation and migration related to CtBP1 overexpression and/or treating diseases of CtBP1 overexpression.

5. Use according to claim 4, wherein the protocatechuic aldehyde is used as the sole active ingredient of the medicament or together with other medicaments as the active ingredient.

6. The use of claim 4, wherein the disease in which CtBP1 is overexpressed is a tumor.

7. An inhibitor of CtBP1, wherein the active ingredient of the inhibitor comprises a therapeutically effective amount of protocatechualdehyde.

8. The inhibitor according to claim 7, wherein the medicament further comprises a component which has a positive effect on the inhibition of cell proliferation and migration associated with the overexpression of CtBP1 and/or a medicament for treating diseases in which CtBP1 is overexpressed, and/or a pharmaceutically acceptable carrier for improving the stability of protocatechualdehyde, when the medicament is administered simultaneously with protocatechualdehyde.

9. The inhibitor according to claim 7, wherein the pharmaceutically acceptable carrier comprises an emulsifier, excipient, filler, binder, humectant, disintegrant, absorption enhancer, flavoring agent, coloring agent, or solubilizing agent.

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