CN113456625A - Application of pterocarpin in inhibiting Pim-1 expression - Google Patents
Application of pterocarpin in inhibiting Pim-1 expression Download PDFInfo
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- CN113456625A CN113456625A CN202110863809.7A CN202110863809A CN113456625A CN 113456625 A CN113456625 A CN 113456625A CN 202110863809 A CN202110863809 A CN 202110863809A CN 113456625 A CN113456625 A CN 113456625A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention belongs to the technical field of application of medicines, and particularly relates to application of pterocarpumine in preparation of a medicine for inhibiting Pim-1 expression. Pharmacodynamic experiments show that: the pterocarpumine can obviously reduce the expression level of Pim-1 in HepG2 and SMMC-7721 cells, and the effect of over-expression of Pim-1 can be effectively reversed after the pterocarpumine is dried; the pterocarpin can effectively inhibit the growth of liver cancer cells in vivo, down-regulate protein expression of Pim-1, N-cadherin and Vimentin, and up-regulate protein expression of E-cadherin; the pterocarpin can effectively inhibit the growth and migration invasion of liver cancer cells, and reverse epithelial-mesenchymal transition of the liver cancer cells by down-regulating Pim-1. The pterocarpumine disclosed by the invention can be used for preparing a medicament for inhibiting Pim-1 expression, can be used for developing an anti-tumor medicament, has a wide application prospect, and provides a new idea for treating liver cancer by the pterocarpumine.
Description
Technical Field
The invention relates to the technical field of application of medicines, in particular to application of pterocarpumine in preparation of a medicine for inhibiting Pim-1 expression.
Background
Drupe, latin scientific name:Ventilago leiocarpa Benthit is a seed of Rhamnaceae and Pterocarpus, and grows in sparse forest or bush at elevation below 1500 m. The medicine has the effects of invigorating qi and blood, relaxing muscles and tendons, and activating collaterals, and has certain curative effects on deficiency of qi and blood, menoxenia, rheumatalgia, numbness of limbs, and traumatic injury. Guangxi folks are used for radically treating anemia, rheumatic arthritis, lumbar muscle strain and other diseases. The plant of the genus Pterocarpus contains abundant quinone compounds, and seven crystal components are obtained from ethanol extract of Pterocarpus heterophyllus root produced in Guangxi, and are respectively identified as emodin-methyl ether, emodin, 1, 2, 4, 8-tetrahydroxy-3-methylanthraquinone, Pterocarpin-I and a new naphthoquinone compound named as Pterocarpin (Ventilaglolin) according to physicochemical properties and spectral data. The pterocarpin belongs to 1, 4-naphthoquinone compounds, and the previous research of the applicant shows that the pterocarpin has good anti-tumor activity, can obviously inhibit the proliferation activity of liver cancer cells and promote the apoptosis. The pterocarpin has a certain effect of resisting liver cancer, but the deep action mechanism of the pterocarpin is not further clarified.
Epithelial-mesenchymal transition (EMT) is one of the important mechanisms for tumor cell invasion and metastasis. The EMT refers to the phenomenon that epithelial cells are transformed into mesenchymal cells under specific physiological and pathological conditions, in the process, the polarity of the epithelial cells is destroyed and finally transformed into mesenchymal cells with migration capability and infiltration capability, and the EMT is mainly characterized in that the expression of cellular cadherin is abnormal, the expression of adhesion molecules E-cadherin (E-cadherin) representing the characteristics of the epithelial cells is reduced, and the expression of markers vimentin (vimentin) and N-cadherin (N-cadherin) representing the characteristics of the mesenchymal cells is increased, and researches show that the EMT plays an important role in the processes of invasion and metastasis of liver cancer cells.
The serine/threonine kinase coded by the Pim protooncogene acts on a wide range of substrates in the cell and participates in the occurrence and development processes of various malignant solid tumors, including the proliferation, cycle arrest, apoptosis, migration, invasion, drug resistance and the like of tumor cells. Pim kinases are overexpressed in many human tumors, for example Pim-1 and Pim-2 proteins are common in hematological malignancies and prostate tumors. Pim-1 is the most popular molecular target in the Pim kinase family, and research shows that Pim-1 can activate downstream transcription factors to induce the generation of EMT, thereby enhancing the malignant activity of renal clear cell carcinoma.
At present, no document discloses the application of the pterocarpin in inhibiting Pim-1 expression.
Disclosure of Invention
The invention aims to provide application of pterocarpumin in inhibiting Pim-1 expression and application of the pterocarpumin in preparing a medicament for inhibiting Pim-1 expression.
The technical scheme adopted by the invention is as follows:
application of pterocarpin in preparing medicine for inhibiting Pim-1 expression is provided.
The drug for inhibiting Pim-1 expression is a drug capable of reducing Pim-1 expression in liver cancer cells.
The drug for inhibiting Pim-1 expression is a drug capable of inhibiting epithelial-mesenchymal transition of liver cancer cells.
The drug for inhibiting Pim-1 expression can reverse epithelial-mesenchymal transition of liver cancer cells and inhibit migration and invasion of the liver cancer cells by reducing Pim-1 expression.
Furthermore, the medicament for inhibiting Pim-1 expression is prepared by taking the pterocarpin as an active ingredient and a medicament carrier.
The chemical structure of the pterocarpin of the invention is as follows:
pharmacodynamic experiments show that:
(1) the pterocarpin inhibits the migration and invasion of liver cancer cells. The pterocarcinol shows proliferation inhibition effect on human liver cancer cells HepG2 and SMMC-7721 in a dose-dependent mode, and the results of a scratch experiment and a Transwell chamber experiment show that the pterocarcinol can obviously inhibit the migration and invasion capacity of HepG2 and SMMC-7721. The result shows that the pterocarpin can effectively inhibit the growth and migration invasion of human liver cancer cells and has a potential liver cancer resistance effect.
(2) The pterocarpin inhibits epithelial-mesenchymal transition of liver cancer cells. Western blot analysis shows that the pterocarpin can reduce the expression of N-cadherin and Vimentin in HepG2 and SMMC-7721 cells and improve the expression of E-cadherin. The result shows that the pterocarpin can inhibit epithelial-mesenchymal transition of the liver cancer cells.
(3) The pterocarpin can regulate the expression of liver cancer cell Pim-1. The expression of Pim-1 was down-regulated by pterocarcinolin at the mRNA and protein levels in a dose-dependent manner, indicating that Pim-1 is regulated by pterocarcinolin.
(4) The pterocarpin inhibits the migration invasion and EMT of liver cancer cells by regulating Pim-1. The pterocarpin can reversely over-express the promotion effect of Pim-1 on cell migration and invasion. Western blot analysis shows that the pterocarpin can reverse the promotion effect of over-expressed Pim-1 on N-cadherin and Vimentin and the inhibition effect on E-cadherin. The results suggest that pterocarpin inhibits malignant progression of liver cancer, such as migration, invasion and epithelial-mesenchymal transition of cells, by negatively regulating Pim-1.
(5) The pterocarpin can inhibit the growth of transplanted tumor of nude mouse with liver cancer. Compared with the blank control group, the pterocarpin group has obviously reduced tumor size and weight. After the treatment of the pterocarpin, cell degeneration and necrosis with different degrees can be seen in tumor tissues, and lymphocyte infiltration is obvious. In addition, compared with a blank control group, the expression levels of Pim-1, N-cadherin and Vimentin in the tumor tissue of the pterocarcinolin treatment group are obviously reduced, and the expression level of E-cadherin is obviously increased. These results indicate that the pterocarpin inhibits the growth of liver cancer in vivo.
Compared with the prior art, the invention has the following positive effects:
pharmacodynamic experiments show that: the pterocarpumine can obviously reduce the expression level of Pim-1 in HepG2 and SMMC-7721 cells, and the effect of over-expressing Pim-1 can be effectively reversed after the pterocarpumine is dried. In vivo experiments show that the pterocarpin can effectively inhibit the growth of liver cancer cells in vivo, down-regulate protein expression of Pim-1, N-cadherin and Vimentin, and up-regulate protein expression of E-cadherin. Therefore, the pterocarpin can effectively inhibit the growth and migration invasion of the liver cancer cells, and reverse epithelial-mesenchymal transition of the liver cancer cells by down-regulating Pim-1. The pterocarpumine disclosed by the invention can be used for preparing a medicament for inhibiting Pim-1 expression, can be used for developing an anti-tumor medicament, has a wide application prospect, and provides a new idea for treating liver cancer by the pterocarpumine.
Drawings
FIG. 1 shows the cell inhibition rate of different doses of prussian in HepG2 cells for 24h, measured by CCK-8 method;
FIG. 2 shows the cell inhibition rate of SMMC-7721 cells treated with different doses of pterocarpin for 24h by the CCK-8 method;
FIG. 3 is a graph of scratch experiments performed after treatment of HepG2 and SMMC-7721 cells with 30. mu.M and 60. mu.M pterocarcinol;
FIG. 4 is a graph of experimental data obtained from scratch experiments performed on HepG2 and SMMC-7721 cells treated with 30. mu.M and 60. mu.M pterocarcinol;
FIG. 5 Experimental graphs (20X) of invasion assays performed after treatment of HepG2 and SMMC-7721 cells with 30. mu.M and 60. mu.M pterocarosin;
FIG. 6 is a graph of experimental data obtained from invasion assay experiments performed after treatment of HepG2 and SMMC-7721 cells with 30. mu.M and 60. mu.M pterocarosin;
FIG. 7 is a graph of the protein levels of E-cadherin (E-Ca), N-cadherin (N-Ca), Vimentin and GAPDH determined by treating HepG2 cells with 30. mu.M and 60. mu.M pterocarcinol for 24 h;
FIG. 8 is a graph of experimental data obtained by treating HepG2 cells with 30. mu.M and 60. mu.M pterosin for 24h and determining the protein levels of E-cadherin (E-Ca), N-cadherin (N-Ca), Vimentin and GAPDH;
FIG. 9 protein levels of E-cadherin (E-Ca), N-cadherin (N-Ca), Vimentin and GAPDH determined by treating SMMC-7721 cells with 30 μ M and 60 μ M pterosin for 24 h;
FIG. 10 is a graph of experimental data obtained by treating SMMC-7721 cells with 30. mu.M and 60. mu.M pterosin for 24h and determining the protein levels of E-cadherin (E-Ca), N-cadherin (N-Ca), Vimentin and GAPDH;
FIG. 11 expression levels of Pim-1mRNA and protein detected by qRT-PCR and Western blot for 24h of HepG2 and SMMC-7721 cells treated with 30. mu.M and 60. mu.M of delphinidin;
FIG. 12 is a graph of experimental data obtained by measuring the expression level of Pim-1mRNA from HepG2 and SMMC-7721 cells treated with 30. mu.M and 60. mu.M pterocarosin for 24 h;
FIG. 13 is a graph of experimental data obtained by measuring protein expression levels by treating HepG2 and SMMC-7721 cells with 30. mu.M and 60. mu.M pterocarosin for 24 h;
FIG. 14 Pim-1mRNA and protein expression levels of control, NC (negative control) and Pim-1 OE (overexpression) groups;
FIG. 15 is a graph showing experimental data on the Pim-1mRNA expression level in the control group, NC (negative control) group and Pim-1 OE (overexpression) group;
FIG. 16 is a graph showing experimental data on protein expression levels in a control group, an NC (negative control) group and a Pim-1 OE (overexpression) group;
FIG. 17 is a graph of a scratch experiment performed after grouping HepG2 cells;
FIG. 18 is a graph of a scratch test performed after treatment of SMMC-7721 cells in groups;
FIG. 19 is a graph of experimental data obtained from a scratch experiment conducted on grouped HepG2 and SMMC-7721 cells;
FIG. 20 is a graph of an invasion assay performed after treatment of HepG2 and SMMC-7721 cells in groups;
FIG. 21 is a graph of experimental data obtained from invasion assay experiments performed after treatment of HepG2 and SMMC-7721 cells in groups;
FIG. 22 Western blot analysis of protein levels of E-cadherin (E-Ca), N-cadherin (N-Ca), Vimentin and GAPDH from different treatment groups of HepG2 cells;
FIG. 23 is a graph of experimental data for detecting E-cadherin (E-Ca), N-cadherin (N-Ca) and Vimentin protein levels in HepG2 cells of different treatment groups by Western blotting;
FIG. 24 Western blot analysis of the protein levels of E-cadherin (E-Ca), N-cadherin (N-Ca), Vimentin and GAPDH in SMMC-7721 cells from different treatment groups;
FIG. 25 is a graph of experimental data for Western blotting of E-cadherin (E-Ca), N-cadherin (N-Ca) and Vimentin protein levels in SMMC-7721 cells from different treatment groups;
FIG. 26 is a graph comparing the tumor size of nude mice treated with different treatments of 3 groups of high and low doses of pterocarpin (12, 6 mg/Kg) and physiological saline;
FIG. 27 is a graph comparing the tumor mass weight in nude mice treated with different treatments of 3 groups of high and low doses of pterocarpin (12, 6 mg/Kg) and physiological saline;
FIG. 28 is a graph of tumor tissue in nude mice treated with different groups of high and low doses of delphinidin (12, 6 mg/Kg) and physiological saline 3;
FIG. 29 expression of Pim-1, E-cadherin, N-cadherin, Vimentin in tumor tissue of 3 different treatment-treated nude mice tested by IHC (bar =50 μm).
Detailed Description
The action effect of the pterocarpin in inhibiting Pim-1 expression provided by the invention is further illustrated by combining specific pharmacodynamic experiments.
Material
1.1 cell lines human hepatoma HepG2 and SMMC-7721 cells were purchased from Guangzhou Suyao Biotech limited (STR identified by).
An animal SPF-grade female BALB/c-nu nude mouse (15.5-17.5 g) is 5 weeks old and purchased from Jiangsu Jiejiekang Biotechnology Co., Ltd [ SCXK (su) 2018-.
The medicine and reagent pterocarpin are provided by Guangxi institute of traditional Chinese medicine chemistry, purple red needle crystal, and dimethyl sulfoxide (DMSO) are dissolved into 100 μmol/mL-1Filtering and sterilizing the stock solution for later use; DMEM high-glucose medium (batch 8119431, Gibco, usa); 0.25% trypsin (batch No. 2120734, Gibco, usa); cell proliferation assay Kit (Cell Counting Kit-8, CCK-8, batch number KN658, Japan Dojinye); transwell chamber (08620032, Corning, usa); TRIzol (262312, Life, usa); real-time fluorescent quantitative polymerase chain reaction (qPCR) primers were synthesized by bio-engineering (shanghai) gmbh; hiscript
RT Supermix for qPCR (7E 402G0 Nanjing Nozao Zan)(ii) a ChamQ Universal SYBR qPCR Master Mix (7E 362L9, nuo zha); pim-1 overexpression lentivirus (a lentivirus vector carrying a protein coding sequence of Pim-1 gene, H17971) and control empty vector virus (GL 107) were purchased from Heyu Biotechnology (Shanghai) GmbH; puromycin (107R 0432, Solarbio, beijing); Omni-Easy PAGE12.5%, 7.5% gel Rapid preparation kit (02511100, 024B1030, Shanghai Yazyme Biotech Co., Ltd.); rabbit anti-human Pim-1 monoclonal antibody (HM 0826, Novus, usa); a murine anti-human E-cadherin (E-cadherin) monoclonal antibody (GR 3315795-1, abcame, USA); a rabbit anti-human N-cadherin (N-cadherin) monoclonal antibody (GR 73907-13, Abcame, USA); a mouse anti-human Vimentin (Vimentin) monoclonal antibody (GR 3361308-1, Abcame, USA); a murine anti-human GAPDH monoclonal antibody (GR 3235553, abcame, usa); horse radish peroxidase-labeled goat anti-rabbit IgG and goat anti-mouse IgG (GR 3307268-2, GR3257574-1, U.S. abcame); sheep serum (20110601, sequoia jezoides); mouse/rabbit enhanced polymer assay detection system (2021D 1111, sequoia jessamine); DAB color kit (2010A 1118, China fir gold bridge).
An instrument biosafety cabinet (ESCO AC2-4S1, Singapore); CO 22Cell culture incubator (MCO-18 AIC, Japan Song Fang); multifunctional detectors (Synergy H1, Biotek, usa); a micro nucleic acid protein analyzer (Nanodrop One, Thermo Fisher, usa); real-time fluorescent quantitative PCR instrument (LightCycler 480 II, switzerland); inverted fluorescence microscope (DMi 8, come card, germany); vertical electrophoresis and transfer system (Mini-PROTEAN Tetra/Mini Trans-Blot Module, Bio-Rad, USA) multifunctional imaging analysis system (Protein simple Fluorchem R, U.S.A.);
2 method
2.1 cell culture of human hepatoma HepG2 and SMMC-7721 cells in DMEM high-glucose medium containing 10% FBS at 37 deg.C and 5% CO2Culturing under the condition, and digesting and passaging by using 0.25 percent trypsin when the cell fusion rate reaches 80-90 percent.
Detecting cell proliferation capacity by 5 × 104Inoculating one cell/well in 96-well plate, growing adherentThe length is 24 h. Adding different concentrations of drupellin (200. mu. mol/L, 100. mu. mol/L, 50. mu. mol/L, 25. mu. mol/L, 12.5. mu. mol/L, 6.25. mu. mol/L), setting 4 multiple wells for each concentration, taking a culture medium without liquid medicine as a blank control, adding 10. mu.L of CCK-8 into each well after acting for 24h, continuing incubating for 2 h, detecting the absorbance (OD value) of each well by a microplate reader at a wavelength of 450 nm, calculating the cell activity inhibition rate, and drawing a growth curve. Inhibition (%) =1- (experimental OD value/control OD value) × 100%.
And (3) detecting the cell migration capacity by a scratch experiment, digesting and counting the cells to be detected, inoculating the cells into a 6-well plate, and carrying out adherent growth for 24 hours to ensure that the cell confluency reaches 95%. Marking a straight line wound on the cell surface, washing by PBS to remove floating cells, adding culture solution, taking a picture under a microscope and recording the position, adding different concentrations of the pterocarpin (60 mu mol/L and 30 mu mol/L) into a drug action group, adding an equal volume of culture solution into a blank control group, and continuously culturing for 24h to take a picture at the same position. The distance between scratches was measured by Image J software, and the cell mobility (%) = (distance before cell migration — distance after cell migration)/distance before cell migration was calculated.
Cell invasion capacity detection in Chamber experiment to count the number of cells to be detected, and adjusting the concentration to 5 × 10 with serum-free DMEM medium5And each well is inoculated into an upper chamber of a Transwell chamber covered by Matrigel glue according to 0.2 mL/hole, pterocarpin (60 mu mol/L and 30 mu mol/L) with different concentrations is added into a drug action group, a DMEM high-sugar culture medium containing 20% FBS is added into a lower chamber, the chamber is taken out after being cultured for 24 hours, PBS is used for washing for 2-3 times, residual cells on the upper layer of a microporous filter membrane are wiped off by a cotton swab, 4% paraformaldehyde is used for fixing cells for 15 min, 0.1% crystal violet is used for staining for 30min, the cell is washed by running water and dried, a photo is taken under a light lens, 3% acetic acid is used for decolorizing, and the absorbance (OD value) of a decolorizing solution is detected by a microplate reader at the wavelength of 570 nm.
Real-time fluorescent quantitative PCR (polymerase chain reaction) is carried out on cells to be detected according to the proportion of 1 multiplied by 106One cell/well is inoculated in a 6-well plate, adherent growth is carried out for 24h, and different concentrations of the pterocarpin (60 mu mol/L and 30 mu mol/L) are added into the drug action group for 24h of action. Extracting total RNA from cells by Trizol method, detecting RNA purity and concentration by micro nucleic acid analyzer, conventionally reverse transcribing cDNA, adding Pim-1 gene primerGAPDH as internal reference, amplified by SYBR qPCR using 2—△△CTThe relative expression level of the gene is calculated. The primer sequences are shown in Table 1.
2.6 Western blot for detecting Pim-1 and EMT marker protein expression level6One cell/well is inoculated in a 6-well plate, adherent growth is carried out for 24h, and different concentrations of the pterocarpin (60 mu mol/L and 30 mu mol/L) are added into the drug action group for 24h of action. Extracting total cell protein by RIPA lysate, and determining protein concentration by BCA protein kit. 20 g of total protein is subjected to SDS-PAGE electrophoresis by 12 percent separation gel and 5 percent concentration gel, transferred to a PVDF membrane, and is sealed by PBST containing 5 percent skim milk powder for 1h, and then is respectively incubated with rabbit anti-human Pim-1 (1: 2000 dilution), mouse anti-human E-cadherin (1: 1000 dilution), rabbit anti-human N-cadherin (1: 1000 dilution), mouse anti-human Vimentin (1: 2000 dilution) and mouse anti-human GAPDH (1: 5000 dilution) at 4 ℃ overnight, PBST is washed for 3 times, HRP-labeled goat anti-rabbit IgG and goat anti-mouse IgG (1: 2000 dilution) are incubated for 2 h at normal temperature, PBST is washed for 3 times, ECL luminescent substrate is soaked for 1 min, and then a protein simple multifunctional imaging analysis system is used for collecting pictures.
Construction of liver cancer cell line with Pim-1 gene over-expression by lentivirus infection method HepG2 and SMMC-7721 cells at 5 × 104One dish was inoculated into a 60mm petri dish and cultured for 24h before lentivirus infection (lentivirus vector pSLenti-EF1-EGFP-P2A-Puro-CMV-Pim-1-3 xFLAG-WPRE). The experiment was divided into a blank control group (control), a negative control group (NC) and a Pim-1 overexpression group (Pim-1 OE), each dish was filled with 3mL of complete medium, the negative control group was filled with an empty vector lentivirus (vector pSLenti-EF 1-EGFP-P2A-Puro-CMV-MCS-3 FLAG) and 15. mu.l Polybrene (1 mg/mL), the Pim-1 overexpression group was filled with a recombinant lentivirus and 15. mu.l Polybrene (1 mg/mL), and the lentivirus addition volume was calculated from the multiplicity of infection (MOI) value of the pre-experimental viruses. After 2-3 days of virus infection, the medium containing 2 mug/mL puromycin is replaced for screening positive cells of virus infectionAnd maintaining the condition for continuous culture for 3-4 days, monitoring the cell state and GFP expression level, extracting total RNA and protein of the cell, detecting the expression level of Pim-1mRNA and protein in the cell by adopting a fluorescence quantitative PCR and Western blot method, and verifying the infection efficiency. And (5) carrying out passage on the cells in a good state, freezing and storing the cells for subsequent experiments.
Establishment of nude mouse transplanted tumor model SMMC-7721 cells in logarithmic growth phase were taken and the concentration was adjusted to 1X 10 with serum-free medium7Each mouse is inoculated into the subcutaneous part of the right back of a nude mouse according to 0.2mL, and when the maximum diameter of a tumor body reaches (6 +/-0.5) mm, the tumor bodies are randomly divided into 3 groups, and each group comprises 6 mice: the blank control group, the high dose group (12 mg/Kg) of the pterocarpin and the low dose group (6 mg/Kg) of the pterocarpin are intraperitoneally injected for 1 time every day, the blank control is given with normal saline with corresponding volume for continuous administration for 2 weeks, and the general state and the tumor formation condition of the nude mice are observed every day. All animals were approved by the ethical committee of the institute of traditional Chinese medicine, Guangxi (ethical number 2020111601).
Nude mice were sacrificed by cervical dislocation 2 weeks after histological and Immunohistochemical (IHC) administration, tumor masses were dissected, and tumor mass weight and volume were measured. Fixing tumor blocks in 10% paraformaldehyde, embedding with conventional paraffin, slicing, dewaxing to water, performing conventional HE staining, and observing histopathological changes under a light microscope. Dewaxing paraffin section to water, repairing antigen by citrate solution microwave method, and 3% H2O2Blocking the activity of endogenous peroxidase, sealing goat serum for 1h, incubating at 37 ℃ for 1h, labeling with secondary antibody, developing DAB, counterstaining cell nucleus with hematoxylin, sealing with neutral resin, and observing the expression conditions of Pim-1, E-cadherin, N-cadherin and Vimentin under a light microscope.
Results
3.1 the inhibition of migration and invasion of liver cancer cells by pterocarpumin we assessed the anti-liver cancer activity of pterocarpumin by detecting its effect on the proliferation ability of liver cancer cells. The pterocarcinol shows the proliferation inhibiting effect on human liver cancer cells HepG2 and SMMC-7721 in a dose-dependent mode, and the IC of the pterocarcinol in the cells HepG2 and SMMC-772150112.38 (FIG. 1) and 59.41. mu.M (FIG. 2), respectively. In order to further verify the anti-liver cancer effect of the pterocarpin, the migration of the pterocarpin on HepG2 and SMMC-7721 cells is detectedAnd invasion ability, the scratch test and Transwell cell test results show that the pterocarcinol can significantly inhibit the migration and invasion ability of HepG2 and SMMC-7721 (fig. 3-6). These results show that the pterocarpin can effectively inhibit the growth and migration invasion of human liver cancer cells, and has a potential liver cancer resistance effect.
FIG. 1-FIG. 6 pterocarosin inhibits growth, migration and invasion of hepatoma cells. (FIG. 1, FIG. 2) HepG2 and SMMC-7721 cells were treated with different doses of drupellin for 24h, and the cell inhibition rate was measured by CCK-8 method. (FIGS. 3-6) scratch experiments (10 fields) and invasion assays (20X) were performed after treatment of HepG2 and SMMC-7721 cells with 30. mu.M and 60. mu.M pterocarosin; the data are expressed as the mean of three independent experiments. + -. standard deviation, and respectivelyP< 0.05 andPwhen the concentration is less than 0.01, the difference is obvious compared with a control group.
The fact that the pterocarpin inhibits epithelial-mesenchymal transition (EMT) of the liver cancer cells is of great importance in the process of generating and developing tumors, and in order to explore whether the pterocarpin influences the epithelial-mesenchymal transition of the liver cancer cells, the expression condition of the pterocarpin on EMT marker protein in the liver cancer cells is detected. Western blot analysis shows that the pterocarpin can reduce the expression of N-cadherin and Vimentin in HepG2 and SMMC-7721 cells and increase the expression of E-cadherin (figure 7-figure 10). The result indicates that the pterocarpin can inhibit epithelial-mesenchymal transition of the liver cancer cells.
FIG. 7-FIG. 10 pterocarpin inhibits epithelial-mesenchymal transition of hepatoma cells. (FIGS. 7-10) HepG2 and SMMC-7721 cells were treated with 30. mu.M and 60. mu.M of pterosin for 24h and the protein levels of E-cadherin (E-Ca), N-cadherin (N-Ca), Vimentin and GAPDH were determined by Western blotting. Data are expressed as mean. + -. standard deviation of three independent experiments, relative expression is analyzed by ProteinSimple analysis software, andP< 0.05 andPwhen the concentration is less than 0.01, the difference is obvious compared with a control group.
The expression Pim kinase family of the pterocarpin for down regulating liver cancer cell Pim-1 is closely related to the development process of malignant tumor, Pim-1 is a key member of the family, is highly expressed in the malignant tumor, and can induce the generation of EMT by activating related transcription factors. To verify whether Pim-1 is regulated by pterocarcinolin, we treated HepG2 and SMMC-7721 cells with different concentrations of pterocarcinolin for 24h, which showed that pterocarcinolin down-regulates Pim-1 expression at the mRNA and protein levels in a dose-dependent manner (fig. 11-13). Indicating that Pim-1 is regulated by the pterocarpin.
FIG. 11-FIG. 13 pterocarpin reduces the expression of Pim-1 from hepatoma cells. (FIGS. 11-13) cells of HepG2 and SMMC-7721 were treated with 30. mu.M and 60. mu.M of pterocarcinol for 24h, expression levels of Pim-1mRNA and protein were measured by qRT-PCR and Western blot, GAPDH was used as an internal reference, data were expressed as the mean. + -. standard deviation of three independent experiments, relative expression was analyzed by ProteinSimple analysis software, andP< 0.05 andPwhen the concentration is less than 0.01, the difference is obvious compared with a control group.
To investigate whether pterocarcinol inhibits the migratory invasion and EMT of liver cancer cells by regulating Pim-1, we constructed lentiviral vectors overexpressing Pim-1 expression in HepG2 and SMMC-7721 cells (FIGS. 14-16). We found that the pterocarpin can reverse over-express Pim-1 to promote cell migration and invasion (FIGS. 17-21). Western blot analysis showed that the promotion effect of over-expressed Pim-1 on N-cadherin and Vimentin and the inhibition effect on E-cadherin can be reversed by the pterocarpin (FIGS. 22-23). The results suggest that pterocarpin inhibits malignant progression of liver cancer, such as migration, invasion and epithelial-mesenchymal transition of cells, by negatively regulating Pim-1.
FIG. 14-FIG. 25 show that effective reverse overexpression of Pim-1 in pterocarpumine has effects of promoting migration and invasion of liver cancer cells and promoting EMT. (FIGS. 14-16) the control, NC (negative control) and Pim-1 OE (over-expressed) groups were tested for Pim-1mRNA and protein expression levels by qRT-PCR and Western blot. (FIGS. 17-21) scratch experiments (10 fields) and invasion assays (20X) were performed on HepG2 and SMMC-7721 cells in groups and data are presented as the mean of three independent experiments. + -. standard deviation. (FIGS. 22-25) different treatment groups of HepG2 and SMMC-7721 cells were tested by Western blotting for E-cadherin (E-Ca), N-cadherin (N-Ca), Vimentin and GAPDH protein levels, the data are expressed as the mean of three independent experiments. + -. standard deviation, and the relative expression was analyzed by ProteinSimple analysis software.
In order to further study the effect of the pterocarpin in liver cancer growth and epithelial-mesenchymal transition, a liver cancer cell nude mouse subcutaneous transplantation tumor model is constructed, high-dose and low-dose (12 mg/Kg) treatment of the pterocarpin is given, and the tumor growth is monitored. The sarcomere size and weight of the pterocarpin group were significantly reduced compared to the blank control group (FIGS. 26-27). After treatment with pterocarpin, various degrees of cell degeneration and necrosis were observed in tumor tissues, with significant lymphocyte infiltration (fig. 28). Moreover, the expression levels of Pim-1, N-cadherin and Vimentin in the tumor tissues of the pterocarpin-treated group were significantly decreased and the expression level of E-cadherin was significantly increased, as compared with the blank control group (FIG. 29). These results indicate that the pterocarpin inhibits the growth of liver cancer in vivo.
FIGS. 26-29 Forbinol inhibited tumor growth in vivo. (FIG. 26) SMMC-7721 cells were injected subcutaneously into the outer back of nude mice, and when the maximum diameter of tumor reached (6 + -0.5) mm, the tumor was randomly divided into 3 groups of 6 mice each, which were administered with sarcodictyin (12 mg/Kg, 6 mg/Kg) by intraperitoneal injection and a corresponding volume of physiological saline 1 time a day, and after 2 weeks, nude mice were sacrificed, and the tumor mass was removed. (FIG. 27) tumor mass is weighed, and represented byP< 0.05 andP< 0.01, compared to control group. (FIG. 28) histopathological changes were observed by HE staining. (FIG. 29) tumor tissue expression of Pim-1, E-cadherin, N-cadherin, Vimentin was detected by IHC (bar =50 μm). Analysis of the percentage of positive expression regions by Image J software, which are expressed separatelyP< 0.05 andP< 0.01, compared to control group.
Through the pharmacodynamic test, the technical scheme of the invention is realized:
application of pterocarpin in preparing medicine for inhibiting Pim-1 expression is provided.
The drug for inhibiting Pim-1 expression is a drug capable of reducing Pim-1 expression in liver cancer cells.
The drug for inhibiting Pim-1 expression is a drug capable of inhibiting epithelial-mesenchymal transition of liver cancer cells.
The drug for inhibiting Pim-1 expression can reverse epithelial-mesenchymal transition of liver cancer cells and inhibit migration and invasion of the liver cancer cells by reducing Pim-1 expression.
Furthermore, the medicament for inhibiting Pim-1 expression is prepared by taking the pterocarpin as an active ingredient and a medicament carrier.
Claims (5)
1. Application of pterocarpin in preparing medicine for inhibiting Pim-1 expression is provided.
2. Use according to claim 1, characterized in that: the drug for inhibiting Pim-1 expression is a drug capable of reducing Pim-1 expression in liver cancer cells.
3. Use according to claim 1, characterized in that: the drug for inhibiting Pim-1 expression is a drug capable of inhibiting epithelial-mesenchymal transition of liver cancer cells.
4. Use according to claim 1, characterized in that: the drug for inhibiting Pim-1 expression can reverse epithelial-mesenchymal transition of liver cancer cells and inhibit migration and invasion of the liver cancer cells by reducing Pim-1 expression.
5. Use according to claim 1, characterized in that: the medicament for inhibiting Pim-1 expression is prepared by taking the pterocarpin as an active ingredient and a medicament carrier.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103212064A (en) * | 2012-01-19 | 2013-07-24 | 中国科学院上海巴斯德研究所 | Application of phosphorylation-pathway-related factor in regulating regulatory T-cell function |
| CN103271901A (en) * | 2013-04-03 | 2013-09-04 | 广东医学院附属医院 | Application of dihydromyricetin (DHM) in preparation of anti-hepatoma medicines |
| CN105687182A (en) * | 2016-01-26 | 2016-06-22 | 广西壮族自治区中医药研究院 | Anti-tumor application of ventilago leiocarpa extract |
| CN106377522A (en) * | 2016-08-18 | 2017-02-08 | 天承南运(天津)科技有限公司 | Applications of apigenin for preparation of medicines inhibiting liver cancer epithelial-mesenchymal transition |
| CN107137392A (en) * | 2017-06-02 | 2017-09-08 | 南方医科大学 | Neo-garcinolic acid is preparing the application in suppressing hepatoma cell proliferation, migration and liver cancer apoptosis reducing medicine |
-
2021
- 2021-07-29 CN CN202110863809.7A patent/CN113456625A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103212064A (en) * | 2012-01-19 | 2013-07-24 | 中国科学院上海巴斯德研究所 | Application of phosphorylation-pathway-related factor in regulating regulatory T-cell function |
| CN103271901A (en) * | 2013-04-03 | 2013-09-04 | 广东医学院附属医院 | Application of dihydromyricetin (DHM) in preparation of anti-hepatoma medicines |
| CN105687182A (en) * | 2016-01-26 | 2016-06-22 | 广西壮族自治区中医药研究院 | Anti-tumor application of ventilago leiocarpa extract |
| CN106377522A (en) * | 2016-08-18 | 2017-02-08 | 天承南运(天津)科技有限公司 | Applications of apigenin for preparation of medicines inhibiting liver cancer epithelial-mesenchymal transition |
| CN107137392A (en) * | 2017-06-02 | 2017-09-08 | 南方医科大学 | Neo-garcinolic acid is preparing the application in suppressing hepatoma cell proliferation, migration and liver cancer apoptosis reducing medicine |
Non-Patent Citations (6)
| Title |
|---|
| 刘瑛，等: "翼核果素对肝癌HepG2细胞凋亡及细胞骨架蛋白F-actin的影响", 《中国中医基础医学杂志》 * |
| 徐宏，等: "翼核果素对人肝癌细胞HepG2裸鼠移植瘤的抑制作用及其机制探讨", 《广西医科大学学报》 * |
| 成军，编著: "《肿瘤相关基因》", 31 January 2000, 北京医科大学出版社 * |
| 王晓炜，等: "翼核果及其同属植物的化学成分的研究进展", 《沈阳药科大学学报》 * |
| 王雪芬，等: "翼核果化学成分的研究", 《药学学报》 * |
| 赵锦燕，等: "熊胆粉对肝癌移植瘤裸鼠Pim1、Pim2、bcl-xl的影响", 《福建中医药大学学报》 * |
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