WO2019091013A1 - Utilisation d'un composé donneur de no pour préparer un médicament afin d'inhiber la capacité d'invasion et de métastase de cellules tumorales riches en molécules mercapto - Google Patents

Utilisation d'un composé donneur de no pour préparer un médicament afin d'inhiber la capacité d'invasion et de métastase de cellules tumorales riches en molécules mercapto Download PDF

Info

Publication number
WO2019091013A1
WO2019091013A1 PCT/CN2018/076220 CN2018076220W WO2019091013A1 WO 2019091013 A1 WO2019091013 A1 WO 2019091013A1 CN 2018076220 W CN2018076220 W CN 2018076220W WO 2019091013 A1 WO2019091013 A1 WO 2019091013A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
rich
thiol
molecules
quinone
Prior art date
Application number
PCT/CN2018/076220
Other languages
English (en)
Chinese (zh)
Inventor
贾力
余素红
贺苏丹
李书慧
Original Assignee
福州大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 福州大学 filed Critical 福州大学
Publication of WO2019091013A1 publication Critical patent/WO2019091013A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides

Definitions

  • the present invention relates to the use of an NO donor compound for the preparation of a medicament for inhibiting the invasion and metastasis of tumor cells rich in sulfhydryl molecules.
  • Tumor metastasis is an important biological feature of malignant tumors. Most cancer patients do not die from primary cancer but die from metastatic cancer. The occurrence of tumor metastasis involves complex, subtle changes in the tumor cells and the microenvironment in which they occur, which leads to tumor metastasis, invasion, and survival in the blood/lymphatic circulatory system, as well as the growth process at the target site. . Only a small percentage of cells in primary tumors have metastatic potential. In animal models, only 0.01% or less of cells can enter the circulatory system to metastasize tumor cells. Endogenous genomic instability increases the likelihood that it will gain metastasis. Genomically unstable and heterogeneous tumor cells have cancer-related features such as chromosomal deletions, translocations, and rearrangements. Each tissue/organ has its own structure, and tumor cells must cope with environmental stresses, including oxygen and nutrient deficiencies, low pH, reactive oxygen species and inflammatory response modifiers. After the microenvironment, tumor cells acquire a malignant phenotype.
  • Stamler et al. found that protein sulfhydryl groups can be modified by nitric oxide (NO) or its derivatives to exert the biological activity of nitric oxide and be more stable than nitric oxide.
  • NO nitric oxide
  • Stamler first proposed the concept of protein nitrosation modification, that is, NO acts on the protein cysteine sulfhydryl group (-SH) to form quinone nitroso (-SNO). It is also emphasized that nitric acid nitrosylation of NO and protein can regulate redox signaling.
  • the quinone nitrosylation of proteins is mainly divided into two types: the first one is the direct nitrosylation of NO on proteins, and the possible reactions are: 2NO+1/2O 2 ⁇ N 2 O 3 +RSH ⁇ RSNO +2NO 2 - +2H + .
  • the second is the nitrosylation of protein with a low molecular weight mercapto nitroso compound (R-SNO).
  • R-SNO mercapto nitroso compound
  • the possible reactions can be simply expressed as: R 1 -SNO+R 2 -SH ⁇ R 1 -SH + R 2 -SNO.
  • the vast majority of proteins in the cell are nitrosylated and modified in the second way.
  • NO reacts with cysteine on a small molecule to form a small molecule of nitrosothiol.
  • GSNO nitrosonitroglutathione
  • S nitrosonitroglutathione
  • GSNO nitrosonitroglutathione
  • GSNO can temporarily store NO, and then transfer NO to the sulfhydryl group (-SH) of cysteine in other proteins to complete the quinone nitrosylation modification.
  • -SH sulfhydryl group
  • the product modified by nitrosation of the protein is more stable than the nitric oxide itself.
  • the RS-NO bond is very sensitive to light and strongly reducing substances (such as ascorbate, etc.), especially to metal ions (such as copper ions, iron). Ions, etc.) are extremely sensitive.
  • the quinone nitrosylation modification of the protein is a reversible modification of the reversible specific protein after translation.
  • the site of nitrosylation of protein ⁇ mainly occurs on the cysteine of protein. Due to the complex spatial structure of the protein, only the cysteine at a specific position can be modified by nitrosation, which can be nitrous oxide.
  • the key sulfhydryl groups of basicization generally have special biological functions.
  • Most of the protein quinone nitrosylation modifications share a common primary sequence, which consists of a basic hydrophobic amino acid residue and an acidic amino acid residue, while the intermediate Cys is in a hydrophobic environment.
  • the success of protein nitrosation modification depends on the spatial environment in which the protein is located.
  • the sulfhydryl groups in proteins play an important role in maintaining the conformation and function of proteins. Certain protein sulfhydryl groups in cells are also associated with cell adhesion and invasion functions.
  • the object of the present invention is to provide an application of an NO donor compound for the preparation of a medicament for inhibiting the invasion and metastasis of tumor cells rich in sulfhydryl molecules, in view of the deficiencies of the prior art.
  • the NO donor compound includes quinone nitrosity glutathione, nitric oxide, sodium nitrite , nitroglycerin, quinone nitrosoglutathione, quinone nitrosocysteine, quinone nitro-captopril, quinone nitro-N-acetylpenicillamine, monosaccharide-quinone nitroso One or more of a conjugate of -N-acetylpenicillamine and a monosaccharide-nonylnitroso-N-acetylpenicillamine.
  • the sulfhydryl molecule comprises a thiol-rich protein molecule (such as human cysteine protein 61 (cysteine-rich 61, CYR61), cysteine-rich acidic protein (Secreted protein acidic and rich in cysteine, Sparc) ), Bovine serum albumin (BSA), Creatine kinase (CK), Fe/S protein, metallothiones (MTs), Hepatopietin (Hepatopietin) HPO and HPO 2 0 5 ), thiol-rich nucleic acid "5-Mercaptopolycytidylic acid (MPC)", thiol-rich amino acid "homocysteine", rich One or more of the thiol-containing polypeptide "octreotide”.
  • a thiol-rich protein molecule such as human cysteine protein 61 (cysteine-rich 61, CYR61), cysteine-rich acidic protein (Secre
  • the thiol-rich molecule-containing tumor cells include one or more of MDA-MB-231, HLE, HepG2, HelaPc-3 DU-145. These cancer cells are rich in reduced thiol (-SH) proteins.
  • the cancer cells were pretreated with a nitric oxide donor before injection, and the metastasis rate of the nude mice caused by the nitrosylated cancer cells was significantly reduced and the mortality was significantly reduced compared with the untreated cancer cells. This study demonstrates that the nitrosation modification of thiol-rich protein molecules inhibits the invasion and metastasis of highly metastatic cancer cells.
  • NO donor compound used in the preparation of a drug for inhibiting the invasion and metastasis of tumor cells rich in sulfhydryl molecules, the specific mechanism of action is: NO donor compound and sulfhydryl reaction in sulfhydryl molecules in tumor cells The thiol group is converted to a quinone nitroso group, thereby inhibiting the invasion and metastasis ability of tumor cells rich in sulfhydryl molecules.
  • the dose of the NO donor compound ranges from 1 to 30 ⁇ M.
  • adhesion experiments of cancer cells with extracellular matrix revealed that cancer cells overexpressing thiol-rich protein molecules have higher matrix adhesion rate than normal cancer cells, and thiol-rich protein molecules are expressed. After being disturbed, the matrix adhesion rate was significantly lower than that of normal cancer cells. It indicated that the increased expression of thiol-rich protein molecules can promote the matrix adhesion of cancer cells, while the expression level is reduced, which can reduce the matrix adhesion ability of cancer cells.
  • the present application detects the effect of overexpression/silencing of thiol-rich protein molecules on endothelial adhesion properties of cancer cells by adhesion experiments of cancer cells and endothelial cells.
  • Human umbilical vein endothelial cells were obtained from neonatal umbilical cords delivered by healthy mothers and isolated, subcultured and identified according to our established method (Lu, et al. Sci Rep, 4:4344, 2014).
  • the results showed that compared with normal cancer cells, the adhesion rate of cancer cells overexpressing thiol-rich protein molecules to endothelial cells was significantly increased, while the expression of thiol-rich protein molecules was decreased, and cancer cells and endothelial cells were Adhesion performance is significantly reduced. It indicates that the expression level of thiol-rich protein molecules is positively correlated with the adhesion of cancer cells to endothelial cells.
  • the cell scratch test is a method for studying the cell movement and repair ability, and is streaked in the cell growth region, and the cell migration ability is judged by comparing the repair ability of the cell to the streak region.
  • the results showed that the migration distance of cancer cells overexpressing thiol-rich protein molecules was significantly increased, and the migration distance of thiol-rich protein molecules decreased significantly, indicating that the migration rate of cancer cells was rich in sulfhydryl groups.
  • the expression level of protein molecules is positively correlated.
  • This application uses Transwell invasion assay to study the invasive ability of cancer cells.
  • a layer of Matrigel was placed on the bottom of the Transwell chamber and mounted in the well plate for in vitro simulation of the extracellular matrix. Cells in the low nutrient medium will actively migrate to the highly nutrient medium. Because of the extracellular matrix barrier in the middle, the cells must digest the matrix to pass through. By detecting the amount of cells passing through Matrigel, the invasive ability of the cells can be obtained. Compared with normal cancer cells, when the protein molecule rich in thiol is overexpressed, the cell invasion rate is significantly increased, and the expression of the thiol-rich protein molecule is decreased, and the cell invasion rate is significantly decreased. This indicates that the invasiveness of cancer cells is positively correlated with the expression of thiol-rich protein molecules.
  • platelets are separated from human fresh blood, and a layer of cancer cells are plated on the well plate, and the platelets are stained and then incubated with the cancer cells, and the number of platelets adhering to the cancer cells is detected by a fluorescence microscope, thereby analyzing the cancer cells and The ability of platelets to adhere.
  • the results of the study showed that cancer cells overexpressing thiol-rich protein molecules had higher platelet adhesion rates than normal cancer cells.
  • the cancer cells with reduced expression of thiol-rich protein molecules have lower platelet adhesion rate than normal cancer cells. It is indicated that the adhesion rate of cancer cells to platelets is positively correlated with the expression of thiol-rich protein molecules.
  • the present application proposes an inhibitory effect of a nitric oxide donor substance on the invasion and metastasis ability of tumor cells after nitrosation of a thiol-rich protein molecule.
  • the present application detects the effect of guanidine nitrosylation on the adhesion of cancer cells to cell matrix after over-expressing/silencing cancer cells and normal cancer cells, which are rich in thiol-rich protein molecules.
  • the results showed that the adhesion of cancer cells to the cell matrix was significantly reduced after incubation of normal cancer cells with nitric oxide donors. Cancer cells with high expression of thiol-rich protein molecules were incubated with nitric oxide donors. The matrix adhesion ability of the cells was significantly reduced; when the cancer cells with low expression of thiol-rich protein molecules were incubated with the nitric oxide donor, the matrix adhesion of the cancer cells did not change much. This indicates that nitric oxide has a significant inhibitory effect on the adhesion of cancer cell matrix rich in thiol protein molecules.
  • the present application detects adhesion of thiol-rich protein molecules to overexpressing/silencing cancer cells and normal cancer cells by adherence experiments of cancer cells and endothelial cells, respectively, after incubation with nitric oxide donors, nitrosation of ruthenium on cancer cells and The effect of endothelial cell adhesion properties.
  • the results showed that the adhesion of cancer cells to endothelial cells was significantly reduced after incubation of normal cancer cells with nitric oxide donors. Cancer cells with high expression of thiol-rich protein molecules were incubated with nitric oxide donors.
  • the adhesion of cells to endothelial cells was significantly reduced; the adhesion of cancer cells to endothelial cells was not changed after the cancer cells with low expression of thiol-rich protein molecules were incubated with nitric oxide donors. This indicates that the nitric oxide donor has a significant inhibitory effect on the adhesion of cancer cells rich in thiol-protein molecules to endothelial cells.
  • the application of the thiol-rich protein molecules overexpressing/silencing cancer cells and normal cancer cells by sputum-based protein molecules, and the effects of nitrosation modification on cancer cell migration ability after incubation with nitric oxide donors were analyzed.
  • the results showed that the migration of cancer cells was significantly decreased after the normal cancer cells were incubated with the nitric oxide donors.
  • the cancer cells with high expression of thiol-rich protein molecules were incubated with nitric oxide donors, and the migration of cancer cells decreased.
  • cancer cells with low expression of thiol-rich protein molecules had little change in cancer cell migration after incubation with nitric oxide donors. This indicates that the nitric oxide donor has a significant inhibitory effect on the migration of cancer cells rich in thiol protein molecules.
  • the Transwell invasion assay was used to analyze the effect of nitrosation on the invasiveness of cancer cells after incubated with nitric oxide donors by over-expressing/silencing cancer cells and normal cancer cells.
  • the results showed that the invasiveness of cancer cells was significantly decreased after normal cancer cells were incubated with nitric oxide donors.
  • the cancer cells with high expression of thiol-rich protein molecules were incubated with nitric oxide donors, and the invasiveness of cancer cells decreased.
  • cancer cells with low expression of thiol-rich protein molecules had little change in cancer cell invasiveness after incubation with nitric oxide donors. This indicates that the nitric oxide donor has a significant inhibitory effect on the invasiveness of cancer cells rich in thiol protein molecules.
  • the present application detects the effect of guanidine nitrosylation on the adhesion properties of cancer cells and platelets after the thiol-rich protein molecules overexpress/silence cancer cells and normal cancer cells, respectively, after incubation with a nitric oxide donor.
  • the results showed that the adhesion of cancer cells to platelets was significantly reduced after normal cancer cells were incubated with nitric oxide donors. Cancer cells with high expression of thiol-rich protein molecules were incubated with nitric oxide donors. The adhesion to platelets was significantly reduced; the adhesion of cancer cells to platelets did not change much after cancer cells with low expression of thiol-rich protein molecules were incubated with nitric oxide donors. This indicates that nitric oxide has a significant inhibitory effect on the adhesion of cancer cells rich in thiol-protein molecules to platelets.
  • the morphology of the cells is closely related to its biological activity. If the cell morphology changes, the biological function of the cells will be affected.
  • the logarithmic growth of cancer cells was placed in a confocal dish and gently shaken to evenly distribute the cells for 48 h in a cell culture incubator. After washing, a certain amount of nitric oxide donor is added. Time-lapse photographing was performed using a laser confocal microscope to observe changes in cell morphology at the same location. The study found that the morphology of the cells changed after the addition of nitric oxide donor.
  • normal MDA-MB-231 cells are a kind of mesenchymal cells. The cells are prominent and stretched, and the body is slender and fusiform.
  • This application also detects the effect of nitric oxide donor “S-nitrosoglutathione (GSNO) on the viability of normal human umbilical vein endothelial cells HUVEC.
  • GSNO nitric oxide donor “S-nitrosoglutathione
  • Figure 1 shows the quinone nitrosylation reaction of CYR61 protein in MDA-MB-231 cells (human breast cancer cells): cells co-incubated with nitric oxide donor material (GSNO), sonicated cell extract protein, biotin label Nitrosylated protein, collected marker protein, Western Detection of protein by blot;
  • GSNO nitric oxide donor material
  • Figure 2 shows the nitrosation of cysteine-rich protein in MDA-MB-231 cells;
  • A The degree of nitrosylation over time,
  • B Verification of nitrosylation of protein West (Western) Blot test results),
  • C Verification of CYR61 ⁇ nitrosylation after cell transfection;
  • Figure 3 shows the effect of CYR61 protein expression on the adhesion of MDA-MB-231 cells to cell matrix and endothelial cells, and the nitroso-nitrogenation of nitric oxide donor material (GSNO) to MDA-MB- Inhibition of adhesion of 231 cells to cell matrix and endothelial cells;
  • GSNO nitric oxide donor material
  • Figure 4 shows the effect of CYR61 protein expression on MDA-MB-231 cell invasion (A, B), migration (C, D), and nitric oxide donor material (GSNO) ⁇ nitrosylation modification Inhibition of MDA-MB-231 cell invasion (A, B), migration (C, D);
  • Figure 5 shows the effect of CYR61 protein expression on the adhesion of MDA-MB-231 cells to platelets (A, B), nitric oxide donor substance (GSNO) ⁇ nitrosylation modification to MDA-MB-231 The inhibition of cell-platelet adhesion (A, B), and the effect of nitroso-nitrogenation of nitric oxide donor material (GSNO) on the morphology of MDA-MB-231 cells;
  • Figure 6 shows that guanidine nitrosylation of CYR61 protein in MDA-MB-231 cells inhibits lung metastasis in mice: A, B, breast cancer cells in different treatment groups aggregated in the lungs of mice. C, D, the difference in the number of nodules produced by breast cancer cells in the lungs of different treatment groups;
  • Figure 7 shows the effect of GSNO on HUVEC viability.
  • the invention is illustrated by the following specific examples.
  • the following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.
  • the tumor cells are all based on the high metastatic breast cancer cell line "MDA-MB-231", and the thiol-rich protein molecules are all exemplified by "CYR61”, and the nitric oxide donors are all " ⁇ ”.
  • CYR61 ⁇ nitrosylation of CYR61 protein: MDA-MB-231 cells are washed and added with S-nitrosoglutathione (GSNO) to make it The final concentration was 30 ⁇ M and incubated at 37 ° C for 30 min in the dark. Add 200 ⁇ L of HENS buffer solution to the cell suspension, place the tube in a small box with crushed ice, and ultrasonically disrupt it with a cell disrupter. Min. 10000 at 4 ° C After centrifugation for 10 min, the supernatant was aspirated and collected in a centrifuge tube, and the protein concentration was measured by BCA method.
  • GSNO S-nitrosoglutathione
  • the primary anti-working solution was a TMT antibody working solution to verify the nitrosation reaction of the intracellular protein.
  • the primary antibody working solution is the CYR61 antibody working solution.
  • This example describes the quinone nitrosylation reaction of breast cancer cell MDA-MB-231 rich in sulfhydryl molecules (such as CYR61) (see Figures 1, 2). ).
  • Plating In the above groups of breast cancer cells, nitrosyl glutathione was added to a final concentration of 0, 10, 30 ⁇ M, respectively, and mixed. The cells treated with nitroso-nitroglutathione were plated in a 24-well plate, shaken, and the cells were evenly distributed in the wells and incubated for 2 hours. Observation: The cells were washed twice with PBS to remove unadhered cells and impurities. The plates were placed under a fluorescent inverted microscope to observe photographs. Ten wells were randomly selected from each well to record the number of cells in each field of view, and the matrix adhesion rate of breast cancer in different experimental groups was calculated.
  • CYR61 affects the adhesion of breast cancer cells MDA-MB-231 to endothelial cells
  • S-nitrosoglutathione is a target for breast cancer cells MDA-MB-231 rich in sulfhydryl molecules (such as CYR61).
  • the nitrosylation modification inhibits the adhesion of tumor cells MDA-MB-231 to endothelial cells.
  • Endothelial cell plating Logarithmically grown umbilical vein endothelial cells were harvested and washed with PBS, and the cells were plated in 24-well plates until they were covered with endothelial cells. The cells were washed three times with PBS, added to ECM medium containing 10 ⁇ g/mL TNF- ⁇ , and incubated for 4 h in a 37 ° C incubator to activate endothelial cells. MDA-MB-231, OE-CYR61 MDA-MB-231, and Si-CYR61 MDA-MB-231 cells were stained with rhodamine b, plated, and observed.
  • Logarithmically grown umbilical vein endothelial cells were seeded in a 24-well plate covered with gelatin. After the endothelial cells were covered with the bottom of the bottle, the stimulation factor IL-1 ⁇ (1 ng/ml) was added for 4 hours, and then 0, 10 was added. 30 ⁇ mol/L of ⁇ -nitrosoglutathione-treated MBA-MD-231, OE-CYR61MBA-MD-231, si-CYR61MBA-MD-231 cells. Rhodamine-123 stained tumor cells. The two cells were incubated for 1 h and 12 h, respectively, and the medium in the 24-well plate was aspirated, washed 3 times with PBS, and 500 ⁇ L of single cell suspension was added to each well.
  • Transwell invasion assay was used to study the invasive ability of breast cancer cell MDA-MB-231.
  • the Transwell chamber was mounted in a 24-well plate and pre-cooled for 10 min on an ice box.
  • Matrigel was diluted 1:8 in a ratio of Matrigel to serum-free phenol red free medium. 100 ⁇ L of diluted Matrigel per well was added to the Transwell chamber and incubated for 30 min at 37 ° C in an incubator.
  • the 24-well plate was taken out, placed in the console, and exposed to ultraviolet light overnight.
  • the liquid in the chamber was blotted with a pipette, and 50 ⁇ L of incomplete L-15 medium was added and equilibrated in a 37 ° C incubator for 30 min.
  • MDA-MB-231, OE-CYR61 MDA-MB-231 and Si-CYR61MDA-MB-231 cells were washed with PBS, added to incomplete L-15 medium, and incubated at 37 ° C for 24 h. After washing with PBS, the cells were suspended by incomplete L-15 medium, and the cells were counted. 200 ⁇ L of cell suspension (100 cells/ ⁇ L) was added to the chamber, and the number of cells per well was 20,000. Add quinone nitrosoglutathione to a final concentration of 0, 10, 30 ⁇ M, and mix. L-15 medium containing 20% fetal calf serum was added to a 24-well plate, 500 ⁇ L per well, and incubated at 37 ° C for 24 h.
  • the medium in the well plate was blotted, 500 ⁇ L of PBS was added, and the lower part of the chamber was washed 3 times.
  • the PBS in the well plate was blotted, 500 ⁇ L of 4% paraformaldehyde was added, and the chamber was placed and fixed for 20 min. Drain the chamber medium, wipe the cells inside the chamber and Matrigel with a cotton swab, and wipe each chamber at least twice. 200 ⁇ L of PBS was added to the chamber, and the inside of the chamber was washed twice. Drain the 4% paraformaldehyde in the well plate, add 500 ⁇ L of PBS, and wash the lower part of the chamber 2 times. The PBS in the well plate was blotted and the chamber was dried for 10 min.
  • MDA-MB-231, OE-CYR61 After MDA-MB-231 and si-CYR61 MDA-MB-231 cells were incubated with nitroso-nitroglutathione, the degree of nitrosylation of intracellular peptone increased with the increase of GSNO concentration, and the cell invasion rate was significant. decline. Moreover, ⁇ -nitrosoglutathione and cells with high expression of CYR61 had a greater influence on the invasive ability, and the effect on the invasive ability was less after incubation with cells with low expression of CYR61. (This example demonstrates that guanidine nitrosylation of a thiol-rich key protein (such as CYR61) in cells inhibits the invasiveness of cancer cells (see Figure 4).
  • a thiol-rich key protein such as CYR61
  • CYR61 interferes with the adhesion of breast cancer cells MDA-MB-231 to platelets, and nitrosyl glutathione (S-nitrosoglutathione) inhibits quinone nitrosylation of sulfhydryl-rich molecules (such as CYR61). Adhesion of tumor cell MDA-MB-231 to platelets.
  • the pellet was resuspended in 800 ⁇ L of PBS, added to 200 ⁇ L of LADP, and incubated at 37 ° C for 30 min to activate platelets. After centrifugation at 1500 g for 15 min, the supernatant was discarded and the pellet was resuspended by blowing with 1 mL of PBS.
  • Logarithmically grown MDA-MB-231, OE-CYR61MDA-MB-231, Si-CYR61MDAMB-231 cells were digested and added to L-15 complete medium to make a cell suspension, which was placed in a 24-well plate. Incubate in a 37 ° C incubator until it is 90% dense.
  • the cells were washed, and nitroso-nitroglutathione was added to a final concentration of 0, 10, and 30 ⁇ M, respectively, and mixed, and incubated at 37 ° C for 30 min. Equal amounts of platelets were added to each well and incubated for 15 min at 37 ° C in the dark. Wash twice with PBS to remove platelets and impurities that did not accumulate with breast cancer cells, add a small amount of serum-free phenol red medium to maintain cell viability, and observe under a fluorescent inverted microscope. The results showed that the expression of CYR61 increased, which promoted the adhesion of MDA-MB-231 cells to platelets. The decrease in the expression level of CYR61 significantly inhibited the adhesion of MDA-MB-231 cells to platelets.
  • S-nitrosoglutathione nitrofluorination of sulfhydryl-rich molecules alters the morphology of tumor cells MDA-MB-231.
  • the logarithmically grown MDA-MB-231 cells were washed and digested, placed in a confocal dish, and gently shaken to evenly distribute the cells, and cultured in a cell culture incubator for 48 hours. After washing, 30 ⁇ M of quinone nitrosoglutathione was added. Time-lapse photographing was performed using a laser confocal microscope to observe changes in cell morphology at the same location. Immediately after the addition of nitroso-nitroglutathione, one shot was taken for 0 min, and then one shot was taken every 30 min.
  • Logarithmically grown MDA-MB-231 cells were washed with PBS, digested, pipetted into complete cell suspension with L-15 medium, and transferred to a 1.5 ml EP centrifuge tube.
  • the nitroso-nitroglutathione was added to a final concentration of 0, 10, 30 nM, respectively, and mixed, and incubated at 37 ° C for 30 min.
  • the cells were resuspended, the cells were counted, and the concentration of the cells was adjusted to 5 ⁇ 10 6 /200 ⁇ L.
  • the cells were injected into the nude mice by tail vein injection, and each injection was 200 ⁇ L.
  • the pellet was resuspended by adding 1 mL of PBS, added with 2 ⁇ L of LCFSE, and incubated at 37 ° C for 30 min in the dark. Centrifuge at 1500g for 15 min, discard the supernatant, and wash the pellet with PBS.
  • the pellet was resuspended in 800 ⁇ L of PBS and injected into the nude mice through the tail vein, each injection of 200 ⁇ L. After 4 hours, the animals were sacrificed according to the animal protection regulations and other relevant regulations.
  • the lung tissues were taken, washed twice with normal saline, and soaked in 4% paraformaldehyde for 24 hours. The lung tissue was cut out with a cryostat, placed on a glass slide, and a drop of anti-fluorescent quencher was applied to the tissue sheet, and a cover slip was placed. Observed and photographed with a fluorescence confocal microscope. The cells were injected into the nude mice by tail vein injection.
  • the lung tissues of nude mice were taken, washed twice with normal saline, and soaked in Brinell's fixative for 24 h. The lung tissue was transferred to a decolorizing solution and soaked, and the decolorizing solution was changed every 24 hours for one week. The number of lung nodules on the surface of the lung tissue of each group of nude mice was counted.
  • the fixed lung tissue was cut into pieces of lung tissue with a thickness of about 0.3 cm, and then immersed in different proportions of ethanol for gradient dehydration, and each concentration was immersed for 1 h. After the dehydration was completed, it was immersed in xylene for 40 min, and the structure was transparent. The tissue was placed in paraffin at 60 ° C and embedded for 3 h. The embedded lung tissue pieces were removed with forceps and the tissue was cut into tissue sheets having a thickness of about 4 ⁇ m using a microtome.
  • the tissue was heated in a 40 ° C water bath to spread the tissue of the lung tissue, placed on a lysine-loaded glass slide, placed in a constant temperature baking box at 50 ° C, and the tissue sheet was fixed on the glass after the tissue was baked.
  • the slides containing the tissue sheets were sequentially soaked in xylene, 100% ethanol, 95% ethanol, 80% ethanol and 70% ethanol for 10 min, and then immersed in ultrapure water for 10 min.
  • the hydrated sections were immersed in hematoxylin for 5 min, washed twice with ultrapure water, and then counterstained for 3 min in an eosin solution, and the excess iin solution was washed away with ultrapure water.
  • the stained sections were sequentially immersed in different proportions of ethanol for 2 min, dehydrated by gradient, soaked in xylene for 1 min, and sealed with a neutral resin to place a cover slip to prevent the generation of bubbles.
  • the results showed that the number of OE-CYR61 MDA-MB-231 cells with high expression of CYR61 was more abundant and more than that of MDA-MB-231 cells in nude mice. Therefore, overexpression of CYR61 can significantly increase cells in vivo.
  • the amount and extent of lung accumulation Silencing CYR61-expressing cells si-CYR61MDA-MB-231 The amount and extent of lung accumulation in vivo was significantly lower than that of MDA-MB-231 cells.
  • CYR61 The expression level is closely related to the accumulation of breast cancer cells in the lungs. The higher the expression of CYR61, the greater the number and extent of cell aggregation in the lungs. MDA-MB-231 after nitrosation modification The accumulation of cells in the lungs is significantly reduced. This indicates that the nitrosidization of proteins can reduce the accumulation of breast cancer cells in the lungs. Compared with the control group, the number and size of metastases formed in the lungs of nude mice were significantly decreased in the nitroso-modified breast cancer cells.
  • S-nitrosoglutathione S-nitrosoglutathione, Effect of GSNO on HUVEC viability in normal human umbilical vein endothelial cells.
  • Human umbilical vein endothelial cells HUVEC in logarithmic growth phase and in good condition were transferred to 96-well plate culture, and the number of cells reached 5000-8000 cells/ 100 ⁇ L. After the cells were attached to the wall overnight, the medium was discarded, and the medium was washed three times with PBS, and then a medium containing different concentrations of GSNO (0, 1, 10, 100, 500, 1000 ⁇ M) was added, and a blank control group and a negative control group were set. The hole is required to set 3-5 double holes.
  • the culture was continued at 37 ° C for at least 4 h. Discard the MTT solution, add DMSO, shake at room temperature for 10-30 min, measure the absorbance at 570 nm, calculate the cell proliferation inhibition rate according to the absorbance value, and plot the inhibition rate of proliferation with concentration and time.
  • Figure. This example shows that GSNO has no significant effect on the viability of normal human umbilical vein endothelial cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

L'invention concerne l'utilisation d'un composé donneur de NO pour préparer un médicament afin d'inhiber la capacité d'invasion et de métastase de cellules tumorales riches en molécules mercapto. Le composé donneur de NO comprend un ou plusieurs des éléments suivants: mercaptonitroso glutathione, oxyde nitrique, nitrite de sodium, nitroglycérine, mercaptonitroso cystéine, mercaptonitroso captopril, mercaptonitroso-N-acétylpénicillamine, monosaccharide-mercaptonitroso-N-acétylpénicillamine et le conjugué de monosaccharide-mercaptonitroso-N-acétylpénicillamine.
PCT/CN2018/076220 2017-11-07 2018-02-11 Utilisation d'un composé donneur de no pour préparer un médicament afin d'inhiber la capacité d'invasion et de métastase de cellules tumorales riches en molécules mercapto WO2019091013A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201711086282.1 2017-11-07
CN201711086282.1A CN107823644B (zh) 2017-11-07 2017-11-07 一种no供体化合物在制备抑制富含巯基分子的肿瘤细胞的侵袭和转移能力药物中的应用

Publications (1)

Publication Number Publication Date
WO2019091013A1 true WO2019091013A1 (fr) 2019-05-16

Family

ID=61654786

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/076220 WO2019091013A1 (fr) 2017-11-07 2018-02-11 Utilisation d'un composé donneur de no pour préparer un médicament afin d'inhiber la capacité d'invasion et de métastase de cellules tumorales riches en molécules mercapto

Country Status (2)

Country Link
CN (1) CN107823644B (fr)
WO (1) WO2019091013A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112107595A (zh) * 2019-06-19 2020-12-22 华南理工大学 一种芳基硫酸酯酶和葡萄糖苷醛酸酶抑制剂及其制备方法和应用
CN112220791A (zh) * 2020-11-25 2021-01-15 福州大学 Cyr61蛋白巯基亚硝基化修饰位点的应用
CN113588954B (zh) * 2021-07-29 2023-04-21 福州大学 血清cyr61-sno蛋白作为标志物在制备新型乳腺癌快速检测试剂盒中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1326785A (zh) * 2000-06-01 2001-12-19 孔庆忠 一种抗癌药物组合物
CN101001623A (zh) * 2004-06-10 2007-07-18 日本化药株式会社 抗癌效果增强剂
WO2008013633A2 (fr) * 2006-07-26 2008-01-31 Duke University Traitement de troubles sensibles aux stéroïdes sexuels
CN104262614A (zh) * 2014-08-22 2015-01-07 华中科技大学 一种具有抗癌活性的化合物及抗癌组合物

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003527209A (ja) * 2000-03-20 2003-09-16 ノボバスキュラー インコーポレーテッド 一酸化窒素供与体及び還元剤を含有するマトリックス並びにその使用
WO2008100591A2 (fr) * 2007-02-14 2008-08-21 The General Hospital Corporation Modulation de la signalisation de l'oxyde nitrique pour normaliser le système vasculaire tumoral
CN101613393A (zh) * 2009-05-25 2009-12-30 中国药科大学 熊果酸衍生物的合成及其在药物制备中的应用
US20150030668A1 (en) * 2012-01-05 2015-01-29 Frederick Timothy Guilford Liposomally encapsulated reduced glutathione for management of cancer, including with other pharmaceutical compositions

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1326785A (zh) * 2000-06-01 2001-12-19 孔庆忠 一种抗癌药物组合物
CN101001623A (zh) * 2004-06-10 2007-07-18 日本化药株式会社 抗癌效果增强剂
WO2008013633A2 (fr) * 2006-07-26 2008-01-31 Duke University Traitement de troubles sensibles aux stéroïdes sexuels
CN104262614A (zh) * 2014-08-22 2015-01-07 华中科技大学 一种具有抗癌活性的化合物及抗癌组合物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG, YANGDONG ET AL.: "Effects of S-nitrosoglutathione on the proliferation of tumor cells", CHINESE JOURNAL OF CLINICAL REHABILITATION, vol. 9, no. 2, 14 January 2005 (2005-01-14) *

Also Published As

Publication number Publication date
CN107823644B (zh) 2021-03-02
CN107823644A (zh) 2018-03-23

Similar Documents

Publication Publication Date Title
Liu et al. Collagen-based three-dimensional culture microenvironment promotes epithelial to mesenchymal transition and drug resistance of human ovarian cancer in vitro
WO2019091013A1 (fr) Utilisation d'un composé donneur de no pour préparer un médicament afin d'inhiber la capacité d'invasion et de métastase de cellules tumorales riches en molécules mercapto
Uyama et al. Fascin, a novel marker of human hepatic stellate cells, may regulate their proliferation, migration, and collagen gene expression through the FAK-PI3K-Akt pathway
Shatz et al. Caveolin-1 mutants P132L and Y14F are dominant negative regulators of invasion, migration and aggregation in H1299 lung cancer cells
CN111057680A (zh) 用于肺部肿瘤细胞的培养基和三维培养方法
EP3361251A1 (fr) Procédé de détection de cellules cancéreuses, réactif pour introduire une substance dans des cellules cancéreuses et composition pour traiter le cancer
Yu et al. The feasibility of ROS-and GSH-responsive micelles for treating tumor-initiating and metastatic cancer stem cells
Han et al. Netrin-1 promotes the collective cell migration of liver cancer cells in a 3D cell culture model
Zhao et al. Metformin reduces pancreatic cancer cell proliferation and increases apoptosis through MTOR signaling pathway and its dose-effect relationship.
Shah et al. Role of stiffness and physico-chemical properties of tumour microenvironment on breast cancer cell stemness
CN103463619B (zh) 骨形成蛋白4在抑制癌症中的应用
CN108866064A (zh) 一种靶向转移性人乳腺癌细胞的核酸适配体及其应用
CN110384680B (zh) 一种温度/pH响应性双载药复合纳米粒子及其制备方法和用途
CN111228266A (zh) Gw8510在制备哺乳动物自然衰老时延长寿命、提高认知能力等药物中的应用
Wang et al. Vimentin‐Rab7a Pathway Mediates the Migration of MSCs and Lead to Therapeutic Effects on ARDS
CN107144695A (zh) Arl13b蛋白在癌症诊断中的应用
US9138422B2 (en) Method of using antimycin A to downregulate Wnt/b-catenin pathway to treat gefitinib resistant cancer
Orozco-García et al. Angiogenesis is promoted by hypoxic cervical carcinoma-derived extracellular vesicles depending on the endothelial cell environment
CN110305962A (zh) DKC1与HIF-1α在协同治疗结直肠癌中的应用
JP2021516695A (ja) 腫瘍転移の薬物治療における標的、及びその使用
US20220401504A1 (en) Use of magnolia figo extract in the manufacture of compound for inhibiting growth of lung cancer cells
CN114107507B (zh) Zswim1蛋白在调控肺腺癌细胞增殖和转移中的应用
CN117805376B (zh) CD44和Lgr5作为标记物在筛选胃癌肿瘤干细胞中的应用
Zhivkova et al. Meloxicam and its metal complexes: cytotoxic activity and ability to induce autophagy in human triple negative breast cancer cells
Yu et al. Activation of Src tyrosine kinase in esophageal carcinoma cells in different regulatory environments and corresponding occurrence mechanism

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18876281

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18876281

Country of ref document: EP

Kind code of ref document: A1