WO2019091013A1 - Use of no donor compound for preparing drug for inhibiting invasion and metastasis ability of tumor cell rich in mercapto molecules - Google Patents

Use of no donor compound for preparing drug for inhibiting invasion and metastasis ability of tumor cell rich in mercapto molecules Download PDF

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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
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cells
rich
thiol
molecules
quinone
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贾力
余素红
贺苏丹
李书慧
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福州大学
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    • 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.

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Abstract

Provided is the use of a NO donor compound for preparing a drug for inhibiting the invasion and metastasis ability of tumor cells rich in mercapto molecules. The NO donor compound comprises one or more of mercaptonitroso glutathione, nitric oxide, sodium nitrite, nitroglycerin, mercaptonitroso cysteine, mercaptonitroso captopril, mercaptonitroso-N-acetylpenicillamine, monosaccharide-mercaptonitroso-N-acetylpenicillamine and the conjugate of monosaccharide-mercaptonitroso-N-acetylpenicillamine.

Description

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Application of a NO donor compound in preparing drugs for inhibiting invasion and metastasis of tumor cells rich in thiol molecules 技术领域Technical field
本发明涉及一种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。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.
背景技术Background technique
肿瘤转移是恶性肿瘤的重要生物学特征，大多数癌症患者并非死于原发性癌而是死于转移性癌。肿瘤转移的发生涉及到肿瘤细胞及其所处的微环境中复杂的、微妙的变化，这些变化导致了肿瘤的转移、侵袭和在血液/淋巴循环***中存活，以及在转移靶部位的生长过程。原发灶肿瘤中只有很少一部分细胞具有转移能力。在动物模型中，只有0.01%甚至更少的细胞可以进入循环***进行转移肿瘤细胞。内源性的基因组不稳定性增加了其获得转移能力的可能性。基因组不稳定及异质性的肿瘤细胞具有染色体缺失、易位、重排等与癌症相关的特征。每个组织/器官都有自身的结构，肿瘤细胞侵入这种器官就必须应对环境的压力，包括：氧气和营养的缺乏，低pH，活性氧自由基和炎症反应调节因子等。经过微环境的影响，肿瘤细胞获得恶性的表型。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.
1992年Stamler等研究发现，蛋白质巯基被一氧化氮（NO）或其衍生物修饰可以发挥一氧化氮的生物活性，并且比一氧化氮更稳定。1994年，Stamler首次提出了蛋白质巯亚硝基化修饰的概念，即NO 作用于蛋白质半胱氨酸上巯基（ -SH），生成巯亚硝基（ -SNO）的反应。并强调 NO 与蛋白质发生巯亚硝基化修饰可以调控氧化还原信号传导。已有的研究表明，蛋白质的巯亚硝基化修饰可以影响蛋白质的功能、活性以及蛋白-蛋白相互作用等，如蛋白质巯亚硝基化修饰可以活化钙离子通道、调控酶的活性、诱导细胞凋亡，改变蛋白质在细胞内转运，影响蛋白质的相关信号通路，改变亚细胞定位等。In 1992, 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. In 1994, 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. Previous studies have shown that the quinone nitrosylation of proteins can affect protein function, activity and protein-protein interactions, such as protein nitrosation modification can activate calcium channels, regulate enzyme activity, induce cells Apoptosis, altering the transport of proteins in cells, affecting protein-related signaling pathways, and altering subcellular localization.
蛋白质的巯亚硝基化主要分为两种：第一种是 NO 作用于蛋白质的直接亚硝基化作用，其可能的反应为：2NO+1/2O 2→N 2O 3+RSH→RSNO+2NO 2 -+2H +。第二种是蛋白质与低分子量的巯基亚硝基化合物（R-SNO）发生亚硝基化作用，其可能的反应可以简单表示为: R 1-SNO+R 2-SH ←→ R 1-SH + R 2-SNO。细胞内的绝大多数的蛋白质巯亚硝基化修饰是通过第二种方式进行的。首先，NO与小分子物质上的半胱氨酸发生反应，生成小分子亚硝基硫醇。 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). 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. First, NO reacts with cysteine on a small molecule to form a small molecule of nitrosothiol.
当含有半胱氨酸（Cys）的体内三肽物质谷胱甘肽（Glutathione，GSH）与NO直接结合发生巯亚硝基化反应时，生成的物质为巯亚硝基谷胱甘肽（S-nitrosoglutathione，GSNO），该物质是体内主要的一氧化氮供体。GSNO作为一氧化氮供体，可以临时储存NO，然后将 NO转移到其它蛋白质中半胱氨酸的巯基（-SH）上，完成巯亚硝基化修饰。有研究表示，体内 GSNO 的浓度可以间接反映出体内蛋白质巯亚硝基化程度。蛋白质经巯亚硝基化修饰后的产物比一氧化氮本身更稳定，RS-NO键对光及强还原性的物质（如抗坏血酸盐等）非常敏感，尤其对金属离子（如铜离子、铁离子等）极其敏感。When the in vivo tripeptide substance glutathione (GSH) containing cysteine (Cys) is directly bound to NO to form a quinone nitrosylation reaction, the resulting substance is nitrosonitroglutathione (S). -nitrosoglutathione, GSNO), which is the major nitric oxide donor in the body. As a nitric oxide donor, 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. Studies have shown that the body GSNO The concentration can indirectly reflect the degree of nitrosylation of the protein in the body. 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.
蛋白质的巯亚硝基化修饰是一种可逆的特异性蛋白质翻译后的再修饰。蛋白质巯亚硝基化的位点主要发生在蛋白质的半胱氨酸上，由于蛋白质的空间结构复杂，只有特殊位置的半胱氨酸能够被巯亚硝基化修饰，这些能够被巯亚硝基化作用的关键巯基，一般都具有特殊的生物功能。蛋白质巯亚硝基化修饰大多存在共同的一级序列（motif)，其序列包括一个碱性的疏水氨基酸残基和一个酸性的氨基酸残基，同时中间的Cys处于疏水环境中。此外，蛋白质巯亚硝基化修饰能否成功还取决于蛋白质所处的空间环境。The quinone nitrosylation modification of the protein is a reversible modification of the reversible specific protein after translation. The site of nitrosylation of protein 主要 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. In addition, the success of protein nitrosation modification depends on the spatial environment in which the protein is located.
蛋白质中的巯基在维持蛋白质的构象、功能方面具有重要作用。细胞中的某些蛋白质巯基还与细胞的粘附、侵袭功能相关。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.
目前，没有针对改变富含巯基分子的蛋白质的巯基状态，以抑制肿瘤细胞侵袭和转移的技术。本发明者首次创造性地提出了一种利用巯亚硝基化反应，达到抑制富含巯基分子的肿瘤细胞的侵袭和转移能力的方法及其应用。Currently, there is no technique for inhibiting the thiol state of a protein rich in sulfhydryl molecules to inhibit tumor cell invasion and metastasis. The present inventors have for the first time creatively proposed a method for inhibiting the invasion and metastasis of tumor cells rich in sulfhydryl molecules by using a quinone nitrosylation reaction and an application thereof.
技术问题technical problem
本发明的目的在于针对现有技术的不足，提供一种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。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.
技术解决方案Technical solution
为实现上述发明目的，本发明的具体内容如下：In order to achieve the above object, the specific contents of the present invention are as follows:
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：所述的NO供体化合物包括巯亚硝基谷胱甘肽、一氧化氮、亚硝酸钠、***、巯亚硝基谷胱甘肽、巯亚硝基半胱氨酸、巯亚硝基卡托普利、巯亚硝基-N-乙酰青霉胺、单糖-巯亚硝基-N-乙酰青霉胺和单糖-巯亚硝基-N-乙酰青霉胺的偶合物中的一种或多种。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: 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.
所述的巯基分子包括富含巯基的蛋白质分子（如人富半胱氨酸蛋白61（cysteine-rich 61, CYR61）、富含半胱氨酸的酸性蛋白（Secreted protein acidic and rich in cysteine，Sparc）、牛血清白蛋白（Bovine serumalbumin，BSA）、肌酸激酶（Creatine kinase，CK）、铁硫蛋白（Fe/S protein）、金属硫蛋白（metallothionesins，MTs）、肝细胞生成素 (Hepatopietin)（HPO和HPO 20 5）、富含巯基的核酸“5-巯基胞嘧啶多核苷酸”(5-Mercaptopolycytidylic acid,MPC）”、富含巯基的氨基酸“同型半胱氨酸”（homocysteine）、富含巯基的多肽“奥曲肽”中的一种或多种。 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".
所述的富含巯基分子的肿瘤细胞包括MDA-MB-231、HLE、HepG2、HelaPc-3 DU-145中的一种或多种。这些癌细胞富含还原型巯基（-SH）的蛋白质。将癌细胞在注射前用一氧化氮供体预处理，相对未处理的癌细胞，巯亚硝基化后的癌细胞造成的裸鼠转移率大幅降低、死亡率也明显降低。本研究说明了，富含巯基的蛋白质分子巯亚硝基化修饰作用抑制了高转移型癌细胞的侵袭转移。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.
所述的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，其具体作用机制为：NO供体化合物与肿瘤细胞中的巯基分子发生巯亚硝基反应，使巯基转化为巯亚硝基，从而抑制富含巯基分子的肿瘤细胞的侵袭和转移能力。The use of the NO donor compound 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.
NO供体化合物的剂量范围在1-30 μM。The dose of the NO donor compound ranges from 1 to 30 μM.
有益效果Beneficial effect
本申请通过癌细胞与细胞外基质的粘附实验发现，过表达富含巯基的蛋白质分子的癌细胞其基质粘附率比正常癌细胞的基质粘附率高，而富含巯基的蛋白质分子表达受干扰后，其基质粘附率明显小于正常癌细胞。说明富含巯基的蛋白质分子表达量升高能促进癌细胞的基质粘附，而表达量减少，能降低癌细胞的基质粘附能力。In the present application, 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.
本申请通过癌细胞与内皮细胞的粘附实验，检测富含巯基的蛋白质分子过表达/沉默对癌细胞内皮粘附性能的影响。人脐静脉内皮细胞取自健康产妇分娩的新生儿脐带，并按照我们建立的方法(Lu, et al. Sci Rep, 4:4344, 2014)进行分离、传代培养与鉴定。研究结果显示，与正常癌细胞相比，过表达富含巯基的蛋白质分子的癌细胞与内皮细胞的粘附率显著增加，而富含巯基的蛋白质分子表达量降低，则癌细胞与内皮细胞的粘附性能显著下降。说明富含巯基的蛋白质分子的表达高低与癌细胞与内皮细胞的粘附力成正相关。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.
本申请采用细胞划痕实验来研究细胞迁移能力。细胞划痕实验是用于研究细胞运动和修复能力的方法，在细胞生长区域划线，通过对比细胞对划线区域的修复能力，判断细胞的运动迁移能力。研究结果表明，富含巯基的蛋白质分子过表达的癌细胞的迁移距离显著增加，富含巯基的蛋白质分子表达量降低后，细胞的迁移距离显著下降，说明癌细胞的迁移率与富含巯基的蛋白质分子表达量成正相关。This application uses cell scratch assays to study cell migration ability. 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.
本申请采用Transwell侵袭实验来研究癌细胞的侵袭能力。在Transwell小室内的底部铺一层Matrigel，安装在孔板内，用于体外模拟细胞外基质。细胞在低营养培养基中，会主动往高营养的培养基迁移。由于中间有细胞外基质阻隔，细胞必须消化基质才能穿过。检测穿过Matrigel的细胞量，即可得出细胞的侵袭能力。与正常癌细胞相比，过表达富含巯基的蛋白质分子后，细胞的侵袭率显著增加，降低富含巯基的蛋白质分子的表达，则细胞侵袭率显著下降。说明癌细胞的侵袭力与富含巯基的蛋白质分子表达量成正相关。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.
本申请采用从人新鲜血液中分离血小板，在孔板上铺一层癌细胞，血小板染色后与癌细胞共同孵育，用荧光显微镜检测粘附在癌细胞上的血小板的数量，从而分析癌细胞与血小板粘附的能力。研究结果得出，过表达富含巯基的蛋白质分子的癌细胞，其血小板粘附率比正常癌细胞血小板粘附率高。而降低富含巯基的蛋白质分子表达后的癌细胞，其血小板粘附率比正常癌细胞血小板粘附率低。说明了癌细胞与血小板的粘附率与富含巯基的蛋白质分子表达量成正相关。In the present application, 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.
本申请提出了一氧化氮供体物质对富含巯基蛋白分子巯亚硝基化作用后，对肿瘤细胞的侵袭和转移能力的抑制作用。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.
本申请检测富含巯基的蛋白质分子过表达/沉默癌细胞及正常癌细胞，分别与一氧化氮供体孵育后，巯亚硝基化对癌细胞与细胞基质粘附性能的影响。研究结果表明，正常癌细胞与一氧化氮供体孵育后，癌细胞与细胞基质的粘附力明显降低；富含巯基的蛋白质分子表达量高的癌细胞与一氧化氮供体孵育后，癌细胞的基质黏附能力降低显著；富含巯基的蛋白质分子表达量低的癌细胞与一氧化氮供体孵育后，癌细胞的基质黏附力变化不大。说明一氧化氮对富含巯基蛋白分子的癌细胞基质黏附力有明显抑制作用。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.
本申请通过癌细胞与内皮细胞的粘附实验，检测富含巯基的蛋白质分子过表达/沉默癌细胞及正常癌细胞，分别与一氧化氮供体孵育后，巯亚硝基化对癌细胞与内皮细胞粘附性能的影响。研究结果表明：正常癌细胞与一氧化氮供体孵育后，癌细胞与内皮细胞的粘附力明显降低；富含巯基的蛋白质分子表达量高的癌细胞与一氧化氮供体孵育后，癌细胞与内皮细胞的粘附力降低显著；富含巯基的蛋白质分子表达量低的癌细胞与一氧化氮供体孵育后，癌细胞与内皮细胞的粘附力变化不大。说明一氧化氮供体对富含巯基蛋白分子的癌细胞与内皮细胞的粘附力有明显抑制作用。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.
本申请通过划痕实验，分析富含巯基的蛋白质分子过表达/沉默癌细胞及正常癌细胞，分别与一氧化氮供体孵育后，巯亚硝基化修饰对癌细胞迁移能力的影响。研究结果表明：正常癌细胞与一氧化氮供体孵育后，癌细胞的迁移力明显降低；富含巯基的蛋白质分子表达量高的癌细胞与一氧化氮供体孵育后，癌细胞迁移力降低显著；富含巯基的蛋白质分子表达量低的癌细胞与一氧化氮供体孵育后，癌细胞迁移力变化不大。说明一氧化氮供体对富含巯基蛋白分子的癌细胞迁移力有明显抑制作用。In the present application, 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. Significantly; 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.
本申请通过Transwell侵袭实验，分析富含巯基的蛋白质分子过表达/沉默癌细胞及正常癌细胞，分别与一氧化氮供体孵育后，巯亚硝基化对癌细胞侵袭力的影响。研究结果表明：正常癌细胞与一氧化氮供体孵育后，癌细胞的侵袭力明显降低；富含巯基的蛋白质分子表达量高的癌细胞与一氧化氮供体孵育后，癌细胞侵袭力降低显著；富含巯基的蛋白质分子表达量低的癌细胞与一氧化氮供体孵育后，癌细胞侵袭力变化不大。说明一氧化氮供体对富含巯基蛋白分子的癌细胞侵袭力有明显抑制作用。In the present application, 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. Significantly; 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.
本申请检测富含巯基的蛋白质分子过表达/沉默癌细胞及正常癌细胞，分别与一氧化氮供体孵育后，巯亚硝基化对癌细胞与血小板粘附性能的影响。研究结果表明，正常癌细胞与一氧化氮供体孵育后，癌细胞与血小板的粘附力明显降低；富含巯基的蛋白质分子表达量高的癌细胞与一氧化氮供体孵育后，癌细胞与血小板的粘附能力降低显著；富含巯基的蛋白质分子表达量低的癌细胞与一氧化氮供体孵育后，癌细胞与血小板的粘附力变化不大。说明一氧化氮对富含巯基蛋白分子的癌细胞与血小板的粘附力有明显抑制作用。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.
细胞的形态与其生物活性密切相关，细胞形态如果发生改变，细胞的生物功能就会受到影响。取对数生长的癌细胞，铺于共聚焦小皿内，轻轻晃动，使细胞均匀分布，于细胞培养箱内培养48 h。清洗后，加入一定量的一氧化氮供体。使用激光共聚焦显微镜定时拍照，观察同一位置细胞形态的改变。研究结果发现，加入一氧化氮供体后，细胞形态发生了改变。如正常MDA-MB-231细胞是一种间叶型细胞，细胞突出伸展，体型细长，呈梭形，具有纤维细胞的形态特点。当一氧化氮供体处理后，细胞内蛋白发生巯亚硝基化反应，细胞外观突触不明显，细胞呈椭圆状。说明细胞内富含巯基的蛋白分子的巯亚硝基化修饰，影响了细胞的形态。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. For example, normal MDA-MB-231 cells are a kind of mesenchymal cells. The cells are prominent and stretched, and the body is slender and fusiform. It has the morphological characteristics of fibroblasts. When the nitric oxide donor is treated, the intracellular protein undergoes quinone nitrosylation, and the cell appearance is not obvious, and the cells are oval. It indicates that the quinone nitrosylation modification of thiol-rich protein molecules in cells affects the morphology of cells.
本申请同时检测了一氧化氮供体 “巯亚硝基谷胱甘肽（S-nitrosoglutathione, GSNO）对正常人脐静脉内皮细胞HUVEC存活力的影响。结果表明，GSNO对正常人脐静脉内皮细胞HUVEC的存活力无显著影响。This application also detects the effect of nitric oxide donor “S-nitrosoglutathione (GSNO) on the viability of normal human umbilical vein endothelial cells HUVEC. The results show that GSNO is normal human umbilical vein endothelial cells. The viability of HUVEC has no significant effect.
附图说明DRAWINGS
图1为MDA-MB-231细胞（人乳腺癌细胞）中CYR61蛋白的巯亚硝基化反应流程：细胞与一氧化氮供体物质（GSNO）共孵、超声破碎细胞提取蛋白、生物素标记巯亚硝基化蛋白、收集标记蛋白、Western blot检测蛋白；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;
图2为MDA-MB-231细胞中富含半胱氨酸蛋白的巯亚硝基化；A.巯亚硝基化程度随时间的变化， B.蛋白质巯亚硝基化验证（Western Blot实验结果），C，D.细胞转染后CYR61巯亚硝基化的验证；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, D. Verification of CYR61巯 nitrosylation after cell transfection;
图3为CYR61蛋白表达量变化对MDA-MB-231细胞与细胞基质、内皮细胞的粘附作用的影响，以及一氧化氮供体物质（GSNO）的巯亚硝基化修饰对MDA-MB-231细胞与细胞基质、内皮细胞粘附的抑制作用；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;
图4为CYR61蛋白表达量变化对MDA-MB-231细胞侵袭（A、B）、迁移（C、D）作用的影响，以及一氧化氮供体物质（GSNO）的巯亚硝基化修饰对MDA-MB-231细胞侵袭（A、B）、迁移（C、D）的抑制作用；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) 巯 nitrosylation modification Inhibition of MDA-MB-231 cell invasion (A, B), migration (C, D);
图5为CYR61蛋白表达量变化对MDA-MB-231细胞与血小板粘附力的影响（A、B）、一氧化氮供体物质（GSNO）的巯亚硝基化修饰对MDA-MB-231细胞与血小板粘附力的抑制作用（A、B）、以及一氧化氮供体物质（GSNO）的巯亚硝基化修饰对MDA-MB-231细胞形态的影响；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) 巯 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;
图6为MDA-MB-231细胞中CYR61蛋白的巯亚硝基化修饰抑制了小鼠的肺转移：A，B、不同处理组乳腺癌细胞在小鼠肺部聚集变化。C，D、不同处理组乳腺癌细胞在小鼠肺部生成结节数的差异；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;
图7为GSNO对HUVEC存活力的影响。Figure 7 shows the effect of GSNO on HUVEC viability.
本发明的最佳实施方式BEST MODE FOR CARRYING OUT THE INVENTION
以下通过具体实例对本发明进行说明。以下实例用来说明本发明，但不限于本发明的实施范围。（以下具体实施方案中，肿瘤细胞均以高转移型乳腺癌细胞“MDA-MB-231”为例，富含巯基的蛋白质分子均以“CYR61”为例，一氧化氮供体均以“巯亚硝基谷胱甘肽（S-nitrosoglutathione, GSNO）”为例）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. (In the following specific embodiments, 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 "巯". Sodium glutathione (S-nitrosoglutathione, GSNO)" as an example)
实施例Example 1 1
以富含巯基的蛋白质分子“CYR61”为例：CYR61蛋白质的巯亚硝基化反应：MDA-MB-231细胞清洗后加入巯亚硝基谷胱甘肽（S-nitrosoglutathione, GSNO），使其终浓度为30μM，于37℃避光培养30min。细胞悬浮液中加入200μL HENS缓冲溶液，将离心管放在有碎冰的小盒内，用细胞破碎仪中超声破碎30 min。于4℃下10000 g离心10 min，吸取上清液收集于离心管中，用BCA法测蛋白浓度。在蛋白样品中加入2 μL 1 M MMTS，涡旋震荡1 min，使其充分与蛋白质混匀，室温下避光孵育30 min。加入600 μL预冷的丙酮，于-20℃冰箱避光静置1 h。于4℃，10000 g离心10 min，轻轻倒去丙酮，晾干。用100 μL HENS缓冲液重悬沉淀，分到两个离心管中。每个离心管各加1 μL标记试剂，短暂涡旋混合。再各加入2 μL 1 M 抗坏血酸试剂，短暂涡旋混合，室温下避光孵育2 h。加入300 μL预冷的丙酮，于-20℃冰箱避光静置1 h。4℃，10,000 g离心10 min，轻轻倒去丙酮，晾干。用100 μL HENS缓冲液重悬沉淀，相同样品合并一管，Western Blot实验，一抗工作液为TMT抗体工作液，验证细胞内蛋白质发生巯亚硝基化反应。在新的纯化柱中加入50 μL抗TMT树脂，1,000 g离心1 min，去除保存液。用50 μL TBS洗树脂3次，每次1 min，1,000 g离心1 min。加入标记TMT的样品，置于4℃恒温摇床上，摇晃过夜，1,000 g离心1 min。用50 μL TBS洗五次，每次五分钟，1,000 g离心1 min。用50 μL TBS二次水洗树脂，1,000 g离心1 min。加入200 μL TMT Elution Buffer，1,000 g离心1 min，收集洗脱液。Western Blot实验，一抗工作液为CYR61抗体工作液，本实施例描述了富含巯基分子（如CYR61）的乳腺癌细胞MDA-MB-231的巯亚硝基化反应过程（见图1、2）。Take the thiol-rich protein molecule "CYR61" as an example: 巯 nitrosylation of CYR61 protein: MDA-MB-231 cells are washed and added with S-nitrosoglutathione (GSNO) to make it The final concentration was 30 μM and incubated at 37 ° C for 30 min in the dark. Add 200 μL 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. Add 2 μL of 1 M MMTS to the protein sample, vortex for 1 min, mix well with the protein, incubate at room temperature in the dark 30 Min. 600 μL of pre-cooled acetone was added and allowed to stand in the refrigerator at -20 ° C for 1 h. After centrifugation at 10,000 g for 10 min at 4 ° C, the acetone was gently decanted and allowed to dry. The pellet was resuspended in 100 μL of HENS buffer and divided into two centrifuge tubes. Add 1 μL of labeling reagent to each tube and mix briefly by vortexing. Add 2 μL of 1 M ascorbic acid reagent, briefly vortex and incubate for 2 h at room temperature in the dark. Add 300 μL of pre-cooled acetone and let stand for 1 h in the refrigerator at -20 °C. Centrifuge at 10,000 g for 10 min at 4 ° C, gently decanter the acetone and allow to dry. Resuspend the pellet in 100 μL HENS buffer and combine the same sample into one tube, Western In the Blot experiment, the primary anti-working solution was a TMT antibody working solution to verify the nitrosation reaction of the intracellular protein. Add 50 μL of anti-TMT resin to the new purification column and centrifuge at 1,000 g for 1 min to remove the preservation solution. The resin was washed 3 times with 50 μL of TBS, 1 min each, and centrifuged at 1,000 g for 1 min. A sample labeled with TMT was added, placed on a 4 ° C constant shaker, shaken overnight, and centrifuged at 1,000 g for 1 min. Wash five times with 50 μL of TBS for five minutes and centrifuge at 1,000 g for 1 min. The resin was washed twice with 50 μL of TBS and centrifuged at 1,000 g for 1 min. Add 200 μL TMT Elution Buffer, centrifuged at 1,000 g for 1 min, and the eluate was collected. In the Western Blot experiment, 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). ).
本发明的实施方式Embodiments of the invention
实施例Example 2 2
CYR61对乳腺癌细胞MDA-MB-231粘附细胞基质的影响，以及巯亚硝基谷胱甘肽（S-nitrosoglutathione）对富含巯基蛋白分子（如CYR61）的乳腺癌细胞MDA-MB-231的巯亚硝基化作用。这种作用降低了癌细胞与细胞基质的粘附力。Effect of CYR61 on the adhesion of breast cancer cell MDA-MB-231 to cell matrix, and S-nitrosoglutathione to breast cancer cell MDA-MB-231 rich in thiol protein molecules (such as CYR61) Nitrosation of hydrazine. This action reduces the adhesion of cancer cells to the cell matrix.
铺明胶：取24孔板，每孔加入200μL明胶，十字摇匀，均匀分布在孔内。放入培养箱37℃孵育过夜。吸干多余的明胶，每孔加入200μL含2%BSA的PBS，置于培养箱内37℃封闭 1 h。吸干液体，用PBS 清洗三次。细胞计数：MDA-MB-231、OE-CYR61、MDA-MB-231、Si-CYR61 MDA-MB-231细胞清洗消化后，用PBS重悬2次。吸取2份12μL细胞分别于新EP管中，一份加入12μL试剂T，一份加入12 μL试剂NC，混匀，加至细胞计数板的上样孔中。将细胞计数板水平***细胞计数仪，运行程序。活细胞数/mL=细胞总数-死亡细胞数。细胞染色：每组取 6 万细胞分于新的1.5EP离心管中，稀释至1mL。加入5μL罗丹明123，避光静置30min。用PBS清洗细胞，重悬2次。Spread gelatin: Take 24 well plates, add 200 μL gelatin to each well, shake well and evenly distribute in the wells. Incubate overnight at 37 ° C in an incubator. The excess gelatin was blotted, 200 μL of 2% BSA in PBS was added to each well, and the cells were blocked in an incubator at 37 ° C for 1 h. The liquid was blotted and washed three times with PBS. Cell count: MDA-MB-231, OE-CYR61, MDA-MB-231, Si-CYR61 MDA-MB-231 cells were washed and digested, and resuspended twice with PBS. Pipette 2 portions of 12 μL cells into a new EP tube, add 12 μL of reagent T to one part, add 12 μL of reagent NC to one part, mix and add to the well of the cell counting plate. Insert the cell counting plate horizontally into the cell counter and run the program. Viable cell count / mL = total number of cells - number of dead cells. Cell staining: 60,000 cells in each group were dispensed into a new 1.5 EP centrifuge tube and diluted to 1 mL. Add 5 μL of Rhodamine 123 and let stand for 30 min in the dark. The cells were washed with PBS and resuspended twice.
铺板：在以上各组乳腺癌细胞中分别加入巯亚硝基谷胱甘肽使其终浓度分别为0、10、30μM，混匀。将巯亚硝基谷胱甘肽处理后的细胞铺于 24 孔板，摇匀，使细胞均匀分布在孔内，孵育2h。观察：用 PBS清洗细胞2次，除去未黏附的细胞和杂质。将板放在荧光倒置显微镜下观察拍照，每个孔随机选取十个视野拍照，记录每个视野的细胞数，计算不同实验组乳腺癌的基质黏附率。结果发现，CYR61表达量升高，提高了MDA-MB-231细胞与基质的粘附力；CYR61表达量降低，降低了MDA-MB-231细胞与基质的粘附力。结果还发现，MDA-MB-231细胞与巯亚硝基谷胱甘肽共孵育后，随巯亚硝基谷胱甘肽浓度的升高，癌细胞与基质的粘附率逐渐降低。并且，巯亚硝基谷胱甘肽与CYR61表达量高的细胞孵育后，对癌细胞与基质的粘附力的影响大，与CYR61表达量低的细胞孵育后，对癌细胞与基质的粘附力影响小。本实施例表明，癌细胞中富含巯基的关键蛋白（如CYR61）的巯亚硝基化反应可抑制癌细胞与基质的粘附作用（见图3）。Plating: In the above groups of breast cancer cells, nitrosyl glutathione was added to a final concentration of 0, 10, 30 μM, 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. The results showed that the expression of CYR61 increased, which increased the adhesion of MDA-MB-231 cells to the matrix; the expression of CYR61 decreased, which decreased the adhesion of MDA-MB-231 cells to the matrix. The results also showed that the adhesion rate of cancer cells to matrix decreased with the increase of nitrosoglutathione concentration in MDA-MB-231 cells after co-incubation with nitroso-nitroglutathione. Moreover, after nitroso-nitroglutathione is incubated with cells with high expression of CYR61, it has a great influence on the adhesion of cancer cells to the matrix, and is viscous with cancer cells and cells after incubation with cells with low expression of CYR61. The influence of the attached force is small. This example demonstrates that the guanidine nitrosylation of a thiol-rich key protein (such as CYR61) in cancer cells inhibits the adhesion of cancer cells to the matrix (see Figure 3).
实施例Example 3 3
CYR61影响乳腺癌细胞MDA-MB-231粘附于内皮细胞，而巯亚硝基谷胱甘肽（S-nitrosoglutathione）对富含巯基分子（如CYR61）的乳腺癌细胞MDA-MB-231的巯亚硝基化修饰，抑制了肿瘤细胞MDA-MB-231与内皮细胞的粘附作用。CYR61 affects the adhesion of breast cancer cells MDA-MB-231 to endothelial cells, whereas 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.
内皮细胞铺板：取对数生长的脐静脉内皮细胞，用PBS清洗消化，将细胞铺于24孔板内，待其长满一层内皮细胞。PBS洗细胞三次，加入含有 10 μg/mL TNF-α 的ECM培养基，37℃培养箱孵育4 h，激活内皮细胞。MDA-MB-231、OE-CYR61 MDA-MB-231、Si-CYR61 MDA-MB-231 细胞用罗丹明b染色、铺板、观察。取对数生长的脐静脉内皮细胞，接种于铺有明胶的24孔板中，待内皮细胞铺满瓶底后，加刺激因子IL-1β (1ng/ml)作用4小时后，加入0，10，30μmol/L的巯亚硝基谷胱甘肽处理过的MBA-MD-231，OE-CYR61MBA-MD-231,si-CYR61MBA-MD-231细胞。罗丹明-123染色肿瘤细胞。二种细胞共孵育，分别培养1 h和12 h后，将24孔板中的培养基吸走，用PBS清洗3遍，每孔加入500 μL的单细胞悬液。在37℃、5 %CO 2的条件下孵育1 h，用PBS清洗3遍后，加入无血清无酚红的培养基500 μL。然后在荧光显微镜下进行拍照，并计算不同作用时间下，巯亚硝基谷胱甘肽对内皮细胞的粘附抑制率。研究结果显示，CYR61的表达量增加能够促进MDA-MB-231细胞与内皮细胞的粘附作用。而CYR61的表达量降低抑制了MDA-MB-231细胞与内皮细胞的粘附作用。对富含巯基分子的MDA-MB-231细胞的巯亚硝基化修饰降低了MDA-MB-231细胞与内皮细胞之间的粘能力。并且，巯亚硝基谷胱甘肽与CYR61表达量高的细胞孵育后，对其内皮黏附能力的影响较大，与CYR61表达量低的细胞孵育后，对其内皮黏附能力的影响较小。本实施例表明，细胞中富含巯基的关键蛋白（如CYR61）的巯亚硝基化反应可抑制癌细胞与内皮细胞的粘附作用（见图3）。 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 μg/mL TNF-α, 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-1β (1 ng/ml) was added for 4 hours, and then 0, 10 was added. 30 μmol/L of 巯-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 μL of single cell suspension was added to each well. Incubate for 1 h at 37 ° C, 5% CO 2 , wash 3 times with PBS, and add 500 μL of serum-free phenol red free medium. Then, photographs were taken under a fluorescence microscope, and the adhesion inhibition rate of nitroso-nitroglutathione to endothelial cells was calculated under different action times. The results showed that increased expression of CYR61 promoted the adhesion of MDA-MB-231 cells to endothelial cells. The decrease in the expression level of CYR61 inhibited the adhesion of MDA-MB-231 cells to endothelial cells. The guanidine nitrosylation modification of MDA-MB-231 cells rich in sulfhydryl molecules reduced the adhesion between MDA-MB-231 cells and endothelial cells. Moreover, after incubation with cells with high expression of CYR61, nitroso-nitroglutathione has a greater effect on endothelial adhesion, and incubation with cells with low expression of CYR61 has little effect on endothelial adhesion. This example demonstrates that the quinone nitrosylation of a thiol-rich key protein (such as CYR61) in cells inhibits the adhesion of cancer cells to endothelial cells (see Figure 3).
实施例Example 4 4
CYR61对乳腺癌细胞MDA-MB-231迁移力的影响，以及巯亚硝基谷胱甘肽（S-nitrosoglutathione）对富含巯基分子（如CYR61）的乳腺癌细胞MDA-MB-231的巯亚硝基化修饰，降低了乳腺癌细胞MDA-MB-231的迁移力。The effect of CYR61 on the migration of breast cancer cells MDA-MB-231, and the sputum of S-nitrosoglutathione on breast cancer cells MDA-MB-231 rich in sulfhydryl molecules (such as CYR61) Nitrification modification reduced the migration of breast cancer cells MDA-MB-231.
取对数生长的MDA-MB-231、OE-CYR61 MDA-MB-231、Si-CYR61 MDAMB-231细胞，消化后加入L-15完全培养基吹打成细胞悬液，铺于 24 孔板内，置于37℃培养箱培养，待其长至 90%密度后，进行下一步实验。用10μL枪头倾斜45°的角度在每个孔内划四道平行的直线，用黑色标记笔，于孔板外底部，划一道与四条线垂直且平分的直线。每个孔内加500μL PBS轻柔洗细胞3遍，洗去掉落的细胞和杂物。每个孔内加入500mL L-15 培养基（含 1%胎牛血清）。将24孔板放置于荧光倒置显微镜下，用5倍镜观察，找到四条平行线与黑色标记笔相交的4个点，分别选取四个点的上下方共八个视野，记录坐标，拍照。孔板内分别加入巯亚硝基谷胱甘肽使其终浓度分别为 0、10、30μM，混匀。置于37℃培养箱内培养。培养 24、48  h 后分别对记录下的八个视野拍照，测量划痕距离。研究结果发现，CYR61的表达量增加促进 MDA-MB-231细胞的迁移，而CYR61的表达量下降则抑制了MDA-MB-231细胞的迁移。此外，GSNO的巯亚硝基化作用降低了乳腺癌细胞的迁移能力。并且，GSNO与CYR61表达量高的细胞孵育后，对其运动迁移能力的影响较大，与CYR61表达量低的细胞孵育后，对其运动迁移能力的影响较小。本实施例表明，细胞中富含巯基的关键蛋白（如CYR61）的巯亚硝基化反应可抑制癌细胞的迁移（见图4）。Logarithmic growth of MDA-MB-231, OE-CYR61 MDA-MB-231, Si-CYR61 MDAMB-231 cells, after digestion, add L-15 complete medium and blow into a cell suspension, place in a 24-well plate, and incubate in a 37 °C incubator until it grows to 90 After the % density, the next experiment was carried out. Draw four parallel straight lines in each hole with a 10 μL tip tilted at an angle of 45°. Use a black marker pen to draw a straight line that is perpendicular to the four lines at the bottom of the hole plate. The cells were gently washed with 500 μL of PBS in each well for 3 times to wash away the dropped cells and debris. Add 500 mL of L-15 medium (containing 1% fetal bovine serum) to each well. Place the 24-well plate under a fluorescent inverted microscope and observe with 5 times the mirror to find 4 points where the four parallel lines intersect the black marker pen. Select eight fields from the top and bottom of the four points, record the coordinates, and take a picture. The quinone nitrosoglutathione was added to the well plates to a final concentration of 0, 10, and 30 μM, respectively, and mixed. Incubate in a 37 ° C incubator. After 24 and 48 hours of incubation, the eight fields of view were taken and the scratch distance was measured. The results showed that the increased expression of CYR61 promoted the migration of MDA-MB-231 cells, while the decreased expression of CYR61 inhibited the migration of MDA-MB-231 cells. In addition, the nitrosation of GSNO reduces the migration capacity of breast cancer cells. Moreover, GSNO and cells with high expression of CYR61 had a greater effect on their migration and migration ability, and they had less effect on the migration ability of cells after incubation with cells with low expression of CYR61. This example demonstrates that the guanidine nitrosylation of a thiol-rich key protein (such as CYR61) in cells inhibits migration of cancer cells (see Figure 4).
实施例Example 5 5
CYR61对乳腺癌细胞MDA-MB-231侵袭力的影响，以及巯亚硝基谷胱甘肽（S-nitrosoglutathione）对富含巯基分子（如CYR61）的乳腺癌细胞MDA-MB-231的巯亚硝基化，抑制了乳腺癌细胞MDA-MB-231的侵袭力。The effect of CYR61 on the invasiveness of breast cancer cells MDA-MB-231, and the sputum of S-nitrosoglutathione on breast cancer cells MDA-MB-231 rich in sulfhydryl molecules (such as CYR61) Nitrification inhibits the invasiveness of breast cancer cell MDA-MB-231.
采用Transwell侵袭实验来研究乳腺癌细胞MDA-MB-231的侵袭能力。Transwell小室安装于24孔板内，在冰盒上预冷10min。按照Matrigel比无血清无酚红培养基为1:8的比例稀释Matrigel。在Transwell小室内加入每孔100μL稀释的Matrigel，于37℃培养箱孵育30min。取出24孔板，置于操作台内，照紫外过夜。用移液枪吸干小室内液体，加入50μL不完全L-15培养基，置于 37℃培养箱内平衡30min。MDA-MB-231、OE-CYR61  MDA-MB-231、Si-CYR61MDA-MB-231细胞用PBS清洗，加入不完全L-15培养基，37℃培养箱孵育24h。用PBS清洗消化后，用不完全L-15培养基吹打成细胞悬液，细胞计数，于小室内加入 200μL的细胞悬液（100 个细胞/μL），每孔小室内细胞数为2万，分别加入巯亚硝基谷胱甘肽使其终浓度分别为0、10、30μM，混匀。于24孔板中加入含20%胎牛血清的L-15培养基，每孔500μL，37℃培养箱孵育24h。吸干孔板内的培养基，加入500μLPBS，洗小室下部3遍。吸干孔板内的PBS，加入500 μL 4%多聚甲醛，放入小室，固定20min。吸干小室内培养基，用棉签擦去小室内部的细胞和Matrigel，每个小室至少擦拭两遍。于小室内加入200μLPBS，清洗小室内部2次。吸干孔板内的4%多聚甲醛，加入500μLPBS，洗小室下部2遍。吸干孔板内的PBS，晾干小室10min。在孔板内加入0.1%结晶紫，每孔500μL，孵育30min。吸干孔板内的结晶紫，加入500μLPBS，洗小室下部3遍。小心取下小室底部的膜，置于载玻片上，滴一滴甘油，放上盖玻片，晾干10min。于正置显微镜下观察拍照。研究结果显示，随着CYR61的表达量增加，MDA-MB-231细胞的侵袭力显著增加，CYR61的表达量降低，MDA-MB-231细胞的侵袭力也随之降低。并且，MDA-MB-231、OE-CYR61 MDA-MB-231、si-CYR61 MDA-MB-231细胞分别与巯亚硝基谷胱甘肽孵育后，随着GSNO浓度增加，细胞内蛋白巯亚硝基化程度增加，细胞的侵袭率显著下降。并且，巯亚硝基谷胱甘肽与CYR61表达量高的细胞孵育后，对其侵袭能力的影响较大，与CYR61表达量低的细胞孵育后，对其侵袭能力的影响较小。（本实施例表明，细胞中富含巯基的关键蛋白（如CYR61）的巯亚硝基化可抑制癌细胞的侵袭力见图4）。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 μL 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 μL 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 μL of cell suspension (100 cells/μL) 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 μM, and mix. L-15 medium containing 20% fetal calf serum was added to a 24-well plate, 500 μL per well, and incubated at 37 ° C for 24 h. The medium in the well plate was blotted, 500 μL of PBS was added, and the lower part of the chamber was washed 3 times. The PBS in the well plate was blotted, 500 μL 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 μL 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 μL 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. 0.1% crystal violet was added to the well plate, 500 μL per well, and incubated for 30 min. The crystal violet in the well plate was blotted, 500 μL of PBS was added, and the lower part of the chamber was washed 3 times. Carefully remove the membrane at the bottom of the chamber, place it on a glass slide, drop a drop of glycerin, place a cover slip, and let it dry for 10 min. Take a photo under a stereo microscope. The results showed that with the increase of CYR61 expression, the invasiveness of MDA-MB-231 cells increased significantly, the expression of CYR61 decreased, and the invasiveness of MDA-MB-231 cells decreased. And, 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, 巯-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).
实施例Example 6 6
CYR61干预乳腺癌细胞MDA-MB-231与血小板的粘附作用，以及巯亚硝基谷胱甘肽（S-nitrosoglutathione）对富含巯基分子（如CYR61）的巯亚硝基化修饰，抑制了肿瘤细胞MDA-MB-231与血小板的粘附作用。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.
取人新鲜血液（含抗凝剂）5mL，移置10mLEP离心管，200g离心10min。吸取上清液移至新的10 mLEP离心管中，1500g，离心15min，弃上清。加入1mL PBS 吹打重悬沉淀，加入 2μL CFSE，37℃避光孵育30min。1500g 离心15min，弃上清，用PBS洗沉淀一遍。用800μL PBS重悬沉淀，加入200μLADP，37℃孵育30min，活化血小板。1500g离心15min，弃上清，用1mLPBS吹打重悬沉淀。取对数生长的MDA-MB-231、OE-CYR61MDA-MB-231、Si-CYR61MDAMB-231细胞，消化后加入L-15完全培养基吹打成细胞悬液，铺于24孔板内，置于37℃培养箱培养，待其长置90%密度。清洗细胞，分别加入巯亚硝基谷胱甘肽使其终浓度分别为0、10、30 μM，混匀，37℃培养箱孵育30 min。每孔加入等量血小板，37℃避光孵育15min。用PBS清洗两次，除去未与乳腺癌细胞聚集的血小板和杂质，加入少量无血清无酚红培养基保持细胞活性，置于荧光倒置显微镜下观察拍照。研究结果显示，CYR61的表达量增加，促进了MDA-MB-231细胞与血小板粘附作用。而CYR61的表达量降低，则明显抑制了MDA-MB-231细胞与血小板粘附作用。此外，结果还发现，MDA-MB-231细胞与巯亚硝基谷胱甘肽共孵育后，随着巯亚硝基谷胱甘肽浓度的升高，细胞的血小板粘附率逐渐降低，并且GSNO与CYR61表达量高的细胞孵育后，对其与血小板粘度能力的影响较大，与CYR61表达量低的细胞孵育后，对其与血小板粘度能力的影响较小。本实施例表明，细胞中富含巯基的关键蛋白（如CYR61）的巯亚硝基化反应可抑制癌细胞与血小板的粘附作用（见图5）。Take 5 mL of fresh blood (including anticoagulant), displace 10 mL EP centrifuge tube, and centrifuge at 200 g for 10 min. The supernatant was pipetted into a new 10 mL EP centrifuge tube, 1500 g, centrifuged for 15 min, and the supernatant was discarded. The pellet was resuspended by pipetting with 1 mL of PBS, 2 μL of CFSE was added, and incubation was carried out for 30 min at 37 ° C in the dark. Centrifuge at 1500 g for 15 min, discard the supernatant, and wash the pellet with PBS. The pellet was resuspended in 800 μL of PBS, added to 200 μL 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 μM, 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. In addition, it was found that after MDA-MB-231 cells were co-incubated with quinone nitrosoglutathione, the platelet adhesion rate of cells gradually decreased with the increase of nitroso-nitroglutathione concentration. When GSNO was incubated with cells with high expression of CYR61, it had a greater effect on the viscosity of platelets. After incubation with cells with low expression of CYR61, the effect of GSNO on platelet viscosity was small. This example demonstrates that the quinone nitrosylation of a thiol-rich key protein (such as CYR61) in cells inhibits the adhesion of cancer cells to platelets (see Figure 5).
实施例Example 77
巯亚硝基谷胱甘肽（S-nitrosoglutathione）对富含巯基分子（如CYR61）的巯亚硝基化修饰，改变了肿瘤细胞MDA-MB-231形态。S-nitrosoglutathione nitrofluorination of sulfhydryl-rich molecules (such as CYR61) alters the morphology of tumor cells MDA-MB-231.
取对数生长的MDA-MB-231细胞，清洗消化后，铺于共聚焦小皿内，轻轻晃动，使细胞均匀分布，于细胞培养箱内培养48h。清洗后，加入30μM的巯亚硝基谷胱甘肽。使用激光共聚焦显微镜定时拍照，观察同一位置细胞形态的改变。加入巯亚硝基谷胱甘肽后，立即拍摄一张为0min，之后每30min拍摄一张。结果发现，细胞与巯亚硝基谷胱甘肽共孵后，其形态发生了改变，由正常的间叶型细胞（细胞突出伸展，体型细长，呈梭形，具有纤维细胞的形态特点），变为椭圆状细胞（细胞外观突触不明显）。本实施例表明，细胞中富含巯基的关键蛋白（如CYR61）的巯亚硝基化可改变细胞的形态（见图5）。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 μM 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. It was found that the morphology of the cells was changed after co-incubation with nitroso-nitroglutathione, which was composed of normal mesenchymal cells (cells protruding, slender, fusiform, with morphological characteristics of fibroblasts). , becomes elliptical cells (cell appearance is not obvious). This example demonstrates that quinone nitrosylation of a thiol-rich key protein (such as CYR61) in cells can alter cell morphology (see Figure 5).
实施例Example 88
体内富含巯基的蛋白质分子（如CYR61）的巯亚硝基化修饰对小鼠体内乳腺癌转移的抑制作用。Inhibition of breast cancer metastasis in mice by quinone nitrosylation of thiol-rich protein molecules (such as CYR61) in vivo.
取对数生长的MDA-MB-231细胞，用PBS清洗后消化，用完全L-15培养基吹打成细胞悬液，移入1.5ml EP离心管。分别加入巯亚硝基谷胱甘肽使其终浓度分别为0、10、30 nM，混匀，37℃培养箱孵育30 min。PBS清洗一次后，重悬，细胞计数，调整细胞的浓度为 5×10 6/200μL，通过尾静脉注射的方法，将细胞注射到裸鼠体内，每只注射200μL。取MDA-MB-231、OE-CYR61 MDA-MB-231、Si-CYR61 MDA-MB-231细胞，清洗后消化，分别加入巯亚硝基谷胱甘肽使其终浓度分别为0、10、30μM，混匀，37℃培养箱孵育30 min。PBS清洗一次后，加入1mLPBS吹打重悬沉淀，加入2μLCFSE，37℃避光孵育 30 min。1500g离心15 min，弃上清，用PBS 洗沉淀一遍。用800μLPBS重悬沉淀，通过尾静脉注射到裸鼠体内，每只注射200μL。4h后，按照动物保护守则等有关规定予以处死动物，取肺部组织，用生理盐水清洗2次，于4%多聚甲醛内避光浸泡24h。用冷冻切片机切取肺部组织薄片，置于载玻片上，滴一滴抗荧光猝灭剂于组织薄片上，盖上盖玻片。用荧光共聚焦显微镜观察、拍照。将细胞通过尾静脉注射的方法，注射到裸鼠体内。10周后，按照动物保护守则等有关规定予以处死动物，取裸鼠肺部组织，用生理盐水清洗2次，于布氏固定液浸泡24 h。将肺部组织转至脱色液中浸泡，每 24h换一次脱色液，连续浸泡一周。统计每组裸鼠肺部组织表面的肺部结节数。 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. After washing once with PBS, the cells were resuspended, the cells were counted, and the concentration of the cells was adjusted to 5 × 10 6 /200 μL. The cells were injected into the nude mice by tail vein injection, and each injection was 200 μL. Take MDA-MB-231, OE-CYR61 MDA-MB-231, Si-CYR61 MDA-MB-231 cells, wash and digest, add nitrosonitroglutathione to a final concentration of 0, 10, respectively. Mix at 30 μM and incubate in a 37 ° C incubator for 30 min. After washing once with PBS, the pellet was resuspended by adding 1 mL of PBS, added with 2 μL 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 μL of PBS and injected into the nude mice through the tail vein, each injection of 200 μL. 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. After 10 weeks, the animals were sacrificed according to the animal protection regulations and other related regulations. 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.
将固定后的肺部组织切成厚度约为0.3cm的肺部组织块，依次浸泡于不同比例的乙醇中梯度脱水，每个浓度浸泡1 h。脱水完成后，浸泡于二甲苯中40min，组织成透明状。将组织放到60℃的石蜡中，包埋3h。用镊子取出包埋的肺部组织块，用切片机将组织切成厚度约4 µm的组织薄片。40℃的水浴锅中加热，使肺部组织薄片展开，铺于有赖氨酸的载玻片上，放到50℃的恒温烤片盒中，待组织薄片烤干后，将组织薄片固定于载玻片上。将含有组织薄片的载玻片依次于二甲苯、100 %乙醇、95 %乙醇、80%乙醇和70%乙醇中，分别浸泡10min，再浸泡于超纯水中10min。将水化后的切片浸泡于苏木精中染色5min，用超纯水洗2次，放入伊红溶液中复染3min，用超纯水清洗掉多余的入伊红溶液。将染色后的切片依次浸泡在不同比例的乙醇中2min，梯度脱水，于二甲苯中浸泡1min，用中性树脂封片，放置盖玻片，防止气泡产生。研究结果发现，CYR61表达量高的细胞 OE-CYR61  MDA-MB-231在裸鼠体内肺部聚集的数量和程度比MDA-MB-231细胞多，因此CYR61的过表达，可以显著提高细胞在体内肺部聚集的数量和程度。沉默CYR61表达的细胞 si-CYR61MDA-MB-231 体内肺部聚集的数量和程度显著低于MDA-MB-231细胞，因此，沉默CYR61的表达，能够显著降低细胞肺部聚集的数量和程度。由此得出，CYR61 的表达量与乳腺癌细胞在肺部的聚集密切相关，CYR61表达量越高，细胞在肺部聚集的数量和程度就越大。经过巯亚硝基化修饰后，MDA-MB-231 细胞在肺部的聚集显著降低。说明蛋白质的巯亚硝基化可以降低乳腺癌细胞在肺部的聚集。与对照组相比，经过巯亚硝基化修饰的乳腺癌细胞在裸鼠肺部形成转移灶的数量和大小显著降低，从HE染色切片可以看出，巯亚硝基化修饰程度高的细胞在裸鼠肺部形成数目较少大小较小的微小转移灶，巯亚硝基化修饰程度低的细胞相较于对照组转移现象也有明显改善。本实施例表明，细胞中富含巯基的关键蛋白（如CYR61）的巯亚硝基化可抑制癌细胞在动物体内的转移（见图6）。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 μm 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. a. 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. Therefore, silencing CYR61 expression significantly reduced the number and extent of cellular lung accumulation. From this, 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. From HE staining, it can be seen that the cells with high degree of nitrosation modification In the lungs of nude mice, a small number of small metastases with smaller size were formed, and the cells with low degree of nitrosylation were significantly improved compared with the control group. This example demonstrates that guanidine nitrosylation of a thiol-rich key protein (such as CYR61) in cells inhibits the metastasis of cancer cells in animals (see Figure 6).
实施例Example 99
巯亚硝基谷胱甘肽（S-nitrosoglutathione, GSNO）对正常人脐静脉内皮细胞HUVEC存活力的影响。S-nitrosoglutathione (S-nitrosoglutathione, Effect of GSNO) on HUVEC viability in normal human umbilical vein endothelial cells.
取处于对数生长期、状态良好的人脐静脉内皮细胞HUVEC转入96孔板培养，使细胞数达到5000-8000 cells/ 100 μL。细胞贴壁过夜后弃去培养基，PBS清洗3遍后加入含不同浓度梯度GSNO（0、1、10、100、500、1000 μM）的培养基，并设置空白对照组和阴性对照组，每孔一律要求设置3-5个复孔。培养板分别孵育24 hh后，弃去培养基，每孔加入无血清无酚红M199培养基稀释后（M199：MTT=9:1）的MTT溶液（5 mg/mL），100 μL/well，37℃培养继续培养至少4 h。弃去MTT溶液，加入DMSO后室温摇床震摇10-30 min，在酶标仪570 nm处检测吸光度值，根据吸光度值计算细胞增值抑制率，并绘制增殖抑制率随浓度和时间的变化关系图。本实施例表明，GSNO对正常人脐静脉内皮细胞存活力无显著影响。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 μL. 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 μM) was added, and a blank control group and a negative control group were set. The hole is required to set 3-5 double holes. After incubating the plates for 24 h, the medium was discarded, and MTT solution (5 mg/mL) diluted in serum-free phenol red-free M199 medium (M199:MTT=9:1) was added to each well, 100 μL/well. 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.
虽然以上描述了本发明的具体实施方式，但是熟悉本技术领域的技术人员应当理解，我们所描述的具体的实施例只是说明性的，而不是用于对本发明的范围的限定，熟悉本领域的技术人员在依照本发明的精神所作的等效的修饰以及变化，都应当涵盖在本发明的权利要求所保护的范围内。While the invention has been described with respect to the preferred embodiments of the embodiments of the invention Equivalent modifications and variations of the skilled person in accordance with the spirit of the invention are intended to be included within the scope of the appended claims.

Claims (5)

  1. 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，其特征在于：所述的NO供体化合物包括巯亚硝基谷胱甘肽、一氧化氮、亚硝酸钠、***、巯亚硝基谷胱甘肽、巯亚硝基半胱氨酸、巯亚硝基卡托普利、巯亚硝基-N-乙酰青霉胺、单糖-巯亚硝基-N-乙酰青霉胺和单糖-巯亚硝基-N-乙酰青霉胺的偶合物中的一种或多种。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, characterized in that the NO donor compound comprises quinone nitroglutathione and nitric oxide , sodium nitrite, nitroglycerin, quinone nitrosoglutathione, quinone nitrosocysteine, quinone nitro-captopril, quinone nitroso-N-acetylpenicillamine, monosaccharide- One or more of a conjugate of quinone nitro-N-acetylpenicillamine and monosaccharide-nonylnitroso-N-acetylpenicillamine.
  2. 根据权利要求1所述的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，其特征在于：所述的巯基分子包括含还原型巯基的蛋白质、富含半胱氨酸的酸性蛋白、牛血清白蛋白、肌酸激酶、金属硫蛋白、肝细胞生成素、5-巯基胞嘧啶多核苷酸、富含巯基的氨基酸、富含巯基的多肽中的一种或多种。The use of the NO donor compound according to claim 1 for the preparation of a medicament for inhibiting invasion and metastasis of tumor cells rich in sulfhydryl molecules, characterized in that the thiol molecule comprises a protein containing a reduced thiol group, which is rich in One of cysteine acidic protein, bovine serum albumin, creatine kinase, metallothionein, hepatocyte stimulating hormone, 5-mercapto-cytosine polynucleotide, thiol-rich amino acid, thiol-rich polypeptide Or a variety.
  3. 根据权利要求1所述的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，其特征在于：所述的富含巯基分子的肿瘤细胞包括MDA-MB-231、HLE、HepG2、HelaPc-3 DU-145中的一种或多种。The use of an NO donor compound according to claim 1 for the preparation of a medicament for inhibiting invasion and metastasis of tumor cells rich in sulfhydryl molecules, characterized in that said tumor cells rich in sulfhydryl molecules include MDA-MB- 231, one or more of HLE, HepG2, HelaPc-3 DU-145.
  4. 根据权利要求1所述的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，其特征在于：NO供体化合物与肿瘤细胞中的巯基分子发生巯亚硝基反应，使巯基转化为巯亚硝基，从而抑制富含巯基分子的肿瘤细胞的侵袭和转移能力。The use of the NO donor compound according to claim 1 for the preparation of a medicament for inhibiting invasion and metastasis of tumor cells rich in sulfhydryl molecules, characterized in that the NO donor compound and the sulfhydryl molecule in the tumor cell are sputum nitrous oxide The base reaction converts the thiol group into a quinone nitroso group, thereby inhibiting the invasion and metastasis ability of tumor cells rich in thiol molecules.
  5. 根据权利要求1所述的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，其特征在于：NO供体化合物的剂量范围在1-30 μM。The use of an NO donor compound according to claim 1 for the preparation of a medicament for inhibiting invasion and metastasis of tumor cells rich in sulfhydryl molecules, characterized in that the dose of the NO donor compound is in the range of 1-30 μM.
PCT/CN2018/076220 2017-11-07 2018-02-11 Use of no donor compound for preparing drug for inhibiting invasion and metastasis ability of tumor cell rich in mercapto molecules WO2019091013A1 (en)

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CN112107595A (en) * 2019-06-19 2020-12-22 华南理工大学 Aryl sulfatase and glucuronidase inhibitor as well as preparation method and application thereof
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1326785A (en) * 2000-06-01 2001-12-19 孔庆忠 Anti-cancer medicine composition
CN101001623A (en) * 2004-06-10 2007-07-18 日本化药株式会社 Anticancer effect enhancer
WO2008013633A2 (en) * 2006-07-26 2008-01-31 Duke University Treating sex steroid responsive disorders
CN104262614A (en) * 2014-08-22 2015-01-07 华中科技大学 Compounds with anticancer activity and anticancer composition

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2001247579B2 (en) * 2000-03-20 2005-01-06 Novovascular, Inc. Matrices containing nitric oxide donors and reducing agents and their use
US20100087370A1 (en) * 2007-02-14 2010-04-08 The General Hospital Corporation Modulation of nitric oxide signaling to normalize tumor vasculature
CN101613393A (en) * 2009-05-25 2009-12-30 中国药科大学 Synthetic and the application in medication preparation of ursolic acid derivative
WO2013103924A2 (en) * 2012-01-05 2013-07-11 Guilford Frederick Timothy 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 (en) * 2000-06-01 2001-12-19 孔庆忠 Anti-cancer medicine composition
CN101001623A (en) * 2004-06-10 2007-07-18 日本化药株式会社 Anticancer effect enhancer
WO2008013633A2 (en) * 2006-07-26 2008-01-31 Duke University Treating sex steroid responsive disorders
CN104262614A (en) * 2014-08-22 2015-01-07 华中科技大学 Compounds with anticancer activity and anticancer composition

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) *

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