CN111956656B - Application of OGA inhibitors in the preparation of antitumor drugs - Google Patents
Application of OGA inhibitors in the preparation of antitumor drugs Download PDFInfo
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- CN111956656B CN111956656B CN202010885117.8A CN202010885117A CN111956656B CN 111956656 B CN111956656 B CN 111956656B CN 202010885117 A CN202010885117 A CN 202010885117A CN 111956656 B CN111956656 B CN 111956656B
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Abstract
The invention provides application of an OGA inhibitor in preparation of an anti-tumor medicament, wherein the anti-tumor medicament is a medicament for treating tumor cells carrying DNA damage repair factor defects. The OGA inhibitor is Thiamet G (TMG) and/or (Z) -PUGNAC. The tumor cells carrying the defects of the DNA damage repair factors comprise BRCA defect tumor cells and ATM defect tumor cells. The OGA inhibitor has synthetic lethal effect in tumors with DNA damage repair defects, tumor cells with DNA damage repair function defects are particularly sensitive to TMG treatment, and experimental results show that the OGA inhibitor can be used as a new medicine for treating the tumors with DNA damage repair defects, and lay a foundation for treating the tumors with DNA damage repair defects by using the OGA inhibitor.
Description
Technical Field
The invention relates to the technical field of antitumor drugs, in particular to application of an OGA inhibitor in preparation of antitumor drugs.
Background
The discovery of new drug action targets is a key for guiding the design and research and development of novel antitumor drugs.
When cells are subjected to various exogenous and exogenous hazards, various types of DNA damage can be caused, and the DNA repair response system can repair the DNA damage in time and maintain the stability of a genome. This process, if deregulated, causes genomic instability leading to the development of a variety of tumors. At present, the development of anti-tumor drugs aiming at DNA damage response has become a hot spot.
O-GlcNAc glycosylation widely modifies many proteins in the nucleus and cytoplasm, and participates in many vital activities such as regulation of gene transcription, signal transduction, and DNA damage response. O-GlcNAc glycosylation is a dynamic reversible post-translational modification, synergistically regulated by two enzymes of opposite function, N-acetylglucosaminyltransferase (O-GlcNAc transferase, OGT) and N-acetylglucosaminidase (O-GlcNAcase, OGA). OGT catalyzes the transfer of GlcNAc groups from the glycosyl donor UDP-GlcNAc to the Ser or Thr residue of the acceptor protein, while OGA catalyzes the dissociation of O-GlcNAc from the modified protein. OGT and OGA are the only one pair of enzymes for regulating and controlling reversible O-GlcNAc glycosylation modification, and the OGT and the OGA jointly regulate the balance of protein O-GlcNAc in a body and play an important role in maintaining normal life activities.
At present, there are some reports on the relation between O-GlcNAc glycosylation and antitumor effect, for example, Shandong university teacher in Kangzhou has studied the effect of protein O-GIcNAc glycosylation on paclitaxel antitumor activity, and in this study, it was found that OGA inhibitors TMG and PUGNAc have no significant proliferation inhibitory effect on breast cancer cells MDA-MB-231, and that TMG or PUGNAc combined with paclitaxel has no significant difference on the proliferation inhibitory effect on cells from the group treated with paclitaxel alone. At present, no report that the OGA inhibitor can be applied to antitumor drugs is available.
Disclosure of Invention
The invention aims to provide application of an OGA inhibitor in preparation of an anti-tumor medicament, and provides a new idea and scheme for research, development and production of the anti-tumor medicament.
The technical scheme adopted by the invention is as follows: an application of an OGA inhibitor in preparing an anti-tumor medicament, wherein the anti-tumor medicament is a medicament for treating tumor cells carrying DNA damage repair factor defects.
The OGA inhibitor is Thiamet G (TMG) and/or (Z) -PUGNAC. Wherein the structures of Thiamet G and (Z) -PUGNAC are respectively as follows:
the tumor cells carrying the defects of the DNA damage repair factors comprise BRCA defect tumor cells and ATM defect tumor cells.
The BRCA-deficient tumor cells include BRCA 1-or BRCA 2-deficient tumor cells.
The BRCA1 or BRCA2 deficient tumor cells comprise BRCA1 or BRCA2 deficient breast cancer cells and BRCA1 or BRCA2 deficient ovarian cancer cells.
The medicament is a pharmaceutical composition which takes an OGA inhibitor or pharmaceutically acceptable salts thereof as a main component.
The dosage form of the medicine is tablets, powder, injection, granules, syrup, capsules, solution, suppository or ointment.
The invention clarifies the molecular mechanism of OGA-mediated O-GlcNAc glycosylation for regulating DNA damage response and clinical transformation research based on DNA damage repair defect tumor treatment.
(1) Elucidating the mechanism of action of OGA-mediated deglycosylation modification in DNA damage repair: the invention discovers for the first time that OGA-mediated O-GlcNAc glycosylation can promote DNA damage repair. By studying the functional interaction between de-O-GlcNAc glycosylation and ubiquitination in DNA damage response, the results are expected to potentially elucidate a completely new molecular mechanism that regulates DNA damage response.
(2) The OGA inhibitor can be used in the drugs for clinical tumor treatment: the xenograft tumor animal model is used for detecting whether the OGA inhibitor can inhibit the tumor growth in vivo, and the expected research result can possibly reveal a brand-new treatment strategy for clinical tumor treatment in the future.
The OGA inhibitor has synthetic lethal effect in tumors with DNA damage repair defects, tumor cells with DNA damage repair function defects are particularly sensitive to TMG treatment, and experimental results show that the OGA inhibitor can be used as a new medicine for treating the tumors with DNA damage repair defects, and lay a foundation for treating the tumors with DNA damage repair defects by using the OGA inhibitor.
Drawings
FIG. 1: results of changes in the level of modification of O-GlcNAc glycosylation in cells induced by Ionizing Radiation (IR) induced DNA damage. Among them, Poly ADP ribosylation (ADP-ribosylation) modification induced by DNA damage was used as an effective treatment control for DNA damage, and GAPDH was used as an internal reference protein for unifying protein loading.
FIG. 2: results of changes in the level of modification of intracellular O-GlcNAc glycosylation induced by Etoposide (Etoposide) induced DNA damage. Among them, Poly ADP-ribosylation (Poly (ADP-Ribosyl) -ation, PARylation) modification induced by DNA damage was used as an effective treatment control for DNA damage, and GAPDH was used as an internal reference protein to unify the amount of protein load.
FIG. 3: loss of OGA function results in aberrant expression of intracellular O-GlcNAc glycosylation modifications following DNA damage.
FIG. 4: graph of results of action of OGA with green fluorescent label recruited to DNA damage site when DNA damage is formed by laser micro-irradiation (laser).
FIG. 5: and (3) detecting an influence result diagram of OGA expression deletion on the cell DNA damage repair function by a neutral comet assay.
FIG. 6: and (3) detecting an influence result diagram of OGA expression deletion on the cell DNA damage repair function by a clone formation experiment.
FIG. 7: graph of the effect of OGA inhibitor treatment on DNA damage repair non-terminal homologous recombination repair (NHEJ) pathway and Homologous Recombination (HR) repair pathway.
FIG. 8: neutral comet assay results plot analysis of the effect of OGA inhibitor (TMG/(Z) -PUGNAC) treatment on DNA damage repair function.
FIG. 9: clone formation experiments analysis results plot the effect of OGA inhibitor (TMG/(Z) -PUGNAC) treatment on DNA damage repair function.
FIG. 10: graph of the effect of OGA inhibitor (TMG) on DNA damage repair deficient tumor cells (BRCA 1-/-).
FIG. 11: graph of the effect of OGA inhibitor (TMG) on DNA damage repair deficient tumor cells (ATM-/-).
FIG. 12: results of selective killing of BRCA1/BRCA2 mutant tumor cells by OGA inhibitors (TMG) are shown. Among them, HCC1937, HCC1395 and SUM149PT are triple negative breast cancer cells deficient in BRCA1, and PEO1 is ovarian cancer cells deficient in BRCA 2.
Detailed Description
The present invention is further illustrated by the following examples, in which the operations not described are performed according to the conventional operations in the art, and the reagents not described are all conventional and commercially available reagents.
Some reagents and terms in the examples of the present invention are described below:
1 × PBS buffer: 137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4，1.4mM KH2PO4。
NETN 300 buffer: 300mM NaCl, 20mM Tris, 1mM Na2EDTA，0.5％NP-40，pH 8.0。
BCA protein quantification method: BCA protein quantification is a commonly used protein quantification method in the laboratory. Under alkaline conditions, the protein converts Cu into2+Reduction to Cu+，Cu+The complex interacts with the BCA reagent to form a purple complex, the water-soluble complex shows strong light absorption at 562nm, and the light absorption and the protein concentration have good linear relation in a wide range, so the protein concentration can be calculated according to the light absorption value.
Transfer buffer (Transfer buffer): 48mM Tris, 39mM Glycine, 0.037% SDS, 20% Methanol.
1 × TBST buffer: 20mM Tris, 150mM NaCl, 5mM KCl, 0.2% Tween-20, pH 7.4.
Confining liquid (5% skim milk): 5g of skimmed milk powder was dissolved in 100ml of 1 XTSST buffer to obtain a blocking solution.
Developing solution: SuperSignal product of Thermo was usedTMWest Pico PLUS cheminescent Substrate (34577) was developed.
Poly ADP ribosylation (Poly (ADP-Ribosyl) -ation, PARylation) modification: ADP ribosylation refers to the covalent linkage reaction of ADP ribose moiety in nicotinamide adenine dinucleotide with amino acid residues of certain proteins, thereby affecting the function of the protein. ADP ribosylation is achieved by ADP ribose transferase (ADPRTS), and in this gene family, there are poly ADP ribose group transfer activities (poly ADP ribosylation) which modify substrate by poly ADP ribosylation; some have single ADP ribose group transfer activity (momo-ADP ribosylation) and perform single ADP ribosylation modification on the substrate.
GAPDH: glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase) is abbreviated in English. Is a key enzyme participating in glycolysis, consists of 4 subunits of 30-40kDa, has the molecular weight of 146kDa, and has a detection band of about 36 kDa. The GAPDH gene is expressed at high levels in almost all tissues and is widely used as an internal reference for protein normalization in western blot experiments. Since the protein expression level of GAPDH as a housekeeping gene in the same kind of cells or tissues is generally constant, when a GAPDH internal control antibody is used, the content of the target protein measured in each sample is divided by the content of GAPDH in the present sample to obtain the relative content of the target protein in each sample, and then the comparison between the samples is performed.
Dot blotting: the method comprises the steps of adding a denatured sample to be tested on a nitrocellulose membrane (or a nylon membrane or an NC membrane), hybridizing with a labeled probe, washing the membrane (removing the unconjugated probe), and performing autoradiography to judge whether hybridization exists and the hybridization strength.
Western blotting: the protein sample separated by PAGE is transferred to a solid phase carrier (such as nitrocellulose membrane), and the solid phase carrier adsorbs the protein in a non-covalent bond form and can keep the type of the polypeptide separated by electrophoresis and the biological activity of the polypeptide unchanged. Taking protein or polypeptide on a solid phase carrier as an antigen, carrying out immunoreaction with a corresponding antibody, then carrying out reaction with a second antibody labeled by enzyme or isotope, and carrying out substrate chromogenic or autoradiography to detect the protein component expressed by the specific target gene separated by electrophoresis.
OGA-WT: wild-type full-length OGA, comprising three domains: an N-terminal catalytic domain responsible for O-GlcNAc sugar chain removal; an intermediate Stalk domain; the C-terminal pseudohistone acetyltransferase (HAT) domain, whose acetyltransferase activity is lost due to the lack of a key catalytic site for binding to acetyl-coa.
N-OGA: is composed of amino acids located at positions 1 to 700 of OGA.
C-OGA: is composed of amino acids from 701 th to 916 th of OGA.
Neutral comet assay: comet experiments are also called single cell gel electrophoresis experiments, when various endogenous and exogenous DNA damage factors induce cell DNA chain breakage, the supercoiled structures of the cells are damaged, membrane structures such as cell membranes, nuclear membranes and the like are damaged under the action of cell lysate, proteins, RNA and other components in the cells are diffused into electrolyte, and nuclear DNA cannot enter gel due to large molecular weight and is left in situ. Under neutral conditions, DNA fragments can migrate into the gel, and negatively charged DNA can migrate away from the nuclear DNA toward the positive electrode during electrophoresis to form a "comet" image, while the undamaged DNA portion remains spherical. The more damaged the DNA, the more broken strands and fragments are produced, and the longer the DNA is, the more DNA is migrated under the same electrophoresis conditions, and the longer the migration distance is. The degree of DNA damage of a single cell can be determined by measuring the optical density or migration length of a DNA migration part, so that the relation between the acting dose of the test object and the DNA damage effect can be determined.
1% low melting agar: 1g of low melting point agar was dissolved in 100ml of 1 XPBS to obtain 1% low melting point agar.
Lysis buffer (neutral comet assay): 90Mm Tris, 90mM boric acid, 2mM Na2EDTA，pH 8.5。
Running buffer (neutral comet assay): 90Mm Tris, 90mM boric acid, 2mM Na2EDTA，pH 8.5。
Propidium Iodide (PI): cannot penetrate into intact living cell membrane, and after the permeability of the cell membrane is increased, propidium iodide can enter into cells to be combined with DNA.
Clone formation experiments: the cell clone formation rate, i.e., the cell seeding survival rate, indicates the number of adherent cells surviving and forming clones after seeding the cells. Cells after attachment do not necessarily have to be each capable of proliferating and forming clones, while cells forming clones must be adherent and viable. The clonogenic rate reflects two important traits, cell population dependence and proliferative capacity.
Homologous Recombination (HR): the homologous sequence of sister chromatids is used as a template for repairing, and the repair method is an error-free DNA double bond fracture repair approach.
Non-homologous end recombination (NHEJ): the DNA broken ends are directly connected without homology between the broken ends of the recombinant DNA, and the method is an error-prone DNA double-strand break repair way. Homologous Recombination (HR) and non-homologous end recombination (NHEJ) competitively repair DNA double strand breaks.
JS-20 plasmid: carrying an I-SceI expression gene, expressing an I-SceI endonuclease in a cell, and recognizing an I-SceI site in the cell for cutting to form DNA double-strand break damage in the cell.
BRCA 1: breast cancer susceptibility gene (BRCA) 1.
BRCA 2: breast cancer susceptibility gene (BRCA) 2.
ATM: ataxia telangiectasia mutated gene (ataxia telangiectasia-mutated gene).
BRCA 1-/-: indicating a BRCA1 gene knockout.
ATM-/-: indicating ATM gene knock-out.
Example 1 detection of the level of modification of intracellular O-GlcNAc glycosylation after DNA damage has occurred.
To detect changes in the level of intracellular O-GlcNAc glycosylation following the onset of DNA damage, the inventors treated cells for different DNA damage and sampled cells at different time points after induction of DNA damage for O-GlcNAc glycosylation level detection and analyzed the changes in the level of intracellular O-GlcNAc glycosylation at different time points after induction of DNA damage.
I. Experimental methods, results and analysis of the study of the level of O-GlcNAc glycosylation in cells following treatment for laser radiation (IR) induced DNA damage (FIG. 1).
Grouping experiments:
normal U2OS cells were obtained from American Type Culture Collection (ATCC). Cells were equally randomly assigned to 8 groups: 1 control group (labeled "No IR" in the figure) and 7 experimental groups (labeled "cell recovery time (min) after IR treatment: 0, 5, 10, 30, 60, 120, 240" in the figure). Each test sample was set up for 3 replicates for testing.
Treating by using a medicine:
cells were subjected to DNA damage treatment using 10Gy of laser radiation (IR), after which the cells were allowed to recover and cell sample collection was performed at the corresponding recovery time points.
Collecting and processing a sample:
washing cells for 2 times by using precooled 1 XPBS buffer solution, collecting the cells, fully cracking the cells by using NETN 300 buffer solution, centrifuging for 10min at 13000rpm, taking supernatant as cell total protein extract, carrying out quantitative treatment on the protein by using BCA method, and storing for later use at-80 ℃ after quick freezing by using liquid nitrogen.
Dot blot the samples were subjected to O-GlcNAc glycosylation modification detection analysis:
after equilibration of the nylon membrane (NC) with the Transfer buffer, the membrane was dried in an oven at 37 ℃. Spotting is performed according to consistency of total protein amount → oven treatment at 55 ℃ for 15min for crosslinking → 5% skim milk blocking solution: 30min → 1 XTSST buffer wash → O-GlcNAc glycosylation modification detection antibody (RL2) (Santa Cruz Biotechnology) incubation at room temperature for 1h → 1 XTSST buffer wash 3 times, 10min → horseradish peroxidase (HRP) labeled secondary antibody incubation at room temperature for 45min → 1 XTSST buffer wash 3 times, 10min → developer for development.
Dot blot samples were subjected to Poly ADP ribosylation (Poly (ADP-Ribosyl) -ation, PARylation) modification detection:
after equilibration of the nylon membrane (NC) with the Transfer buffer, the membrane was dried in an oven at 37 ℃. Spotting is performed according to consistency of total protein amount → oven treatment at 55 ℃ for 15min for crosslinking → 5% skim milk blocking solution: 30min → 1 × TBST buffer washing → Poly ADP ribosylation (Poly (ADP-Ribosyl) -ation, PARylation) modified antibody (Trevigen) was incubated at room temperature for 1h → 1 × TBST buffer washing for 3 times, 10min → horseradish peroxidase (HRP) labeled secondary antibody was incubated at room temperature for 45min → 1 × TBST buffer washing for 3 times, 10min → developer for development.
Protein immunoblotting (Western blot) samples were subjected to the detection of the internal reference protein GAPDH:
equal amounts of samples were run on SDS-PAGE gel → 0.3A, constant flow membrane transfer 60min → 5% skim milk blocking solution: 30min → washing with 1 XTSST buffer → incubation of GAPDH detection antibody (Proteintech) at room temperature for 1h → washing with 1 XTSST buffer for 3 times, 10min → incubation with horseradish peroxidase (HRP) labeled secondary antibody at room temperature for 45min → washing with 1 XTSST buffer for 3 times, 10min → developing with developer.
Experimental results and analysis:
as shown in FIG. 1, Dot blot (Dot blot) and Western blot (Western blot) showed that the level of modification of intracellular O-GlcNAc glycosylation gradually decreased toward normal levels after effective treatment with laser radiation (IR) for DNA damage. The method shows that the cells generate more O-GlcNAc glycosylation modifications after the cells generate DNA damage, and the intracellular O-GlcNAc glycosylation modifications gradually tend to the normal level after the cells perform DNA damage repair. And (4) carrying out metering analysis on the marking result by using software Image J to obtain a time variation trend graph.
Experimental methods, results and analysis of the study of the level of O-GlcNAc glycosylation in cells after treatment for Etoposide-induced DNA damage (figure 2).
Grouping experiments:
normal U2OS cells were obtained from American Type Culture Collection (ATCC). Cells were equally randomly assigned to 8 groups: 1 control group (marked as "No Etoposide" in the figure) and 7 experimental groups (marked as "cell recovery time (min):0, 5, 10, 30, 60, 120, 240" after Etoposide treatment in the figure). Each set of experiments was set up with 3 parallel experiments for detection.
Treating by using a medicine:
the experimental cells were treated for DNA damage with 1mM Etoposide (Etoposide) for 30min, after which the cells were allowed to recover by withdrawal of the drug and cell sample collection was performed at the corresponding recovery time points.
Collecting and processing a sample:
the sample collection and processing method is the same as above (FIG. 1).
Experimental results and analysis:
as shown in FIG. 2, Dot blot (Dot blot) and Western blot (Western blot) showed that the intracellular O-GlcNAc glycosylation modification level gradually decreased toward the normal level after the effective treatment of Etoposide (Etoposide) for DNA damage. The method shows that the cells generate more O-GlcNAc glycosylation modifications after the cells generate DNA damage, and the intracellular O-GlcNAc glycosylation modifications gradually tend to the normal level after the cells perform DNA damage repair. And (4) carrying out metering analysis on the marking result by using software Image J to obtain a time variation trend graph.
Example 2 detection of the level of modification of intracellular O-GlcNAc glycosylation following treatment with OGA-deficient DNA damage.
Grouping experiments:
normal U2OS cells were obtained from American Type Culture Collection (ATCC). Cells were equally randomly assigned to 3 groups (7 subgroups were equally randomly assigned to each group of cells): 1 control group (labeled "WT" in the figure), OGA inhibitor (TMG) -treated experimental group (labeled "TMG" in the figure) and siRNA-knockdown endogenous OGA expression experimental group (labeled "siOGA" in the figure), each group contained 1 control group (labeled "No IR" in the figure) that was not treated with DNA damage and 6 experimental groups that were treated with DNA damage (labeled "cell recovery time (min):0, 10, 30, 60, 120, 240" after IR treatment). Each test sample was set up for 3 replicates for testing.
Treating by using a medicine:
after treating the cells with the OGA inhibitor TMG (20mM) for 12h, the cells were subjected to DNA damage treatment.
Transfection of cells with siRNA induced endogenous OGA expression reduction, and cells were treated for DNA damage 48h after transfection.
Cells were subjected to DNA damage treatment using 10Gy of laser radiation (IR), after which the cells were allowed to recover and cell sample collection was performed at the corresponding recovery time points.
Collecting and processing a sample:
the sample collection and processing method is the same as above (FIG. 1).
Experimental results and analysis:
as shown in FIG. 3, Dot blot (Dot blot) and Western blot (Western blot) showed that the level of modification of cellular O-GlcNAc glycosylation was increased after DNA damage by cells treated with the OGA inhibitor TMG. After the siRNA treatment reduces the expression level of endogenous OGA in cells and the DNA damage treatment is carried out, the O-GlcNAc glycosylation modification level of the cells is increased, which indicates that OGA plays a role in removing O-GlcNAc glycosylation modification in the repair process after the DNA damage occurs. And (4) carrying out metering analysis on the marking result by using software Image J to obtain a time variation trend graph.
Example 3 localization of OGA after DNA damage occurred.
Collecting and processing a sample:
an OGA expression vector with a green fluorescent protein (EGFP) label is constructed, 24h after cell transfection (marked as OGA FL in the figure), cells which successfully express EGFP-OGA are subjected to DNA damage treatment by using a laser micro-radiation (laser) system, the cells before the DNA damage treatment (marked as before in the figure), and the cells after the DNA damage treatment are subjected to image acquisition at different time points (marked as time (min) in the figure: 0, 5, 10, 20, 30, 60). The cells to be detected are set in 3 parallel experiments for detection.
Experimental results and analysis:
as shown in fig. 4, green fluorescence signals indicate that after DNA damage occurs, OGAs are gradually recruited to the site of DNA damage to play a role. And (3) carrying out metrological analysis on a fluorescence signal formed by OGA at the DNA damage site by using software Image J to obtain a time variation trend graph.
Example 4 detection of the Effect of deletion of OGA expression on the repair function of cellular DNA damage.
I. The effect of the deletion of OGA expression on the repair function of cellular DNA damage was examined by a neutral comet assay (fig. 5).
Grouping experiments:
normal U2OS cells were obtained from American Type Culture Collection (ATCC). Cells were equally randomly assigned to 5 groups (4 subgroups with equal random assignment of cells in each group): 1 control group (labeled "siCon" in the figure) and 4 experimental groups (labeled "siOGA", "siOGA + OGA-WT", "siOGA + N-OGA" and "siOGA + C-OGA", respectively), and 4 groups of equal amounts of intracellular random distribution were: 1 control group (labeled "untreated") and 3 experimental groups (labeled "0 h, 2h and 4h after IR (10Gy) treatment, respectively).
Treating by using a medicine:
constructing cells with specific background requirements by transfecting siRNA knocking down OGA endogenous expression and corresponding exogenous expression plasmids, wherein the siRNA comprises the following components:
and (3) siCon: siRNA-Control was transfected as a Control group.
siOGA: transfection of siRNA-OGA knockdown endogenous OGA expression in cells.
siOGA + OGA-WT: transfection of siOGA-OGA knockdown of endogenous OGA expression in cells, followed by transfection of the full-length OGA expression plasmid.
siOGA + N-OGA: after transfection of siOGA-OGA knockdown of endogenous OGA expression in cells, N-terminal OGA (1-700aa) expression plasmids were transfected.
siOGA + C-OGA: transfection of siOGA-OGA knockdown of endogenous OGA expression in cells was followed by transfection of the C-terminal OGA (701-916aa) expression plasmid.
The cells of the DNA damage treatment experiment group are treated by laser radiation (IR) (10Gy) and subjected to DNA damage repair for 0h, 2h and 4h respectively, and cell samples are stored in precooled 1 XPBS.
Collecting and processing a sample:
preparation of 1% Low melting agar (preservation in a 37 ℃ Water bath) → lysis of a cell sample preserved in precooled 1 XPBS in 1% Low melting agar → extraction of 1X 10 cells lysed in 1% Low melting agar4One performs glue spreading → the glue is left to dry sufficiently → lysis: the gel plate was placed in a freshly prepared precooled lysis buffer and lysed at 4 ℃ in the dark for 12h → wash: electrophoresis buffer washing at room temperature for 30min, washing 3 times → electrophoresis: electrophoresis in precooled electrophoresis buffer (7mA/20V) for 1h → washing: pure water wash 3 times → staining: propidium iodide staining solution (2.5 μ g/ml) was stained for 20min → washing: pure water washing 3 times → observation: random selection using fluorescence microscopyCells were recorded by photography.
Experimental results and analysis:
as shown in fig. 5, the cells were observed with a microscope, photographed with a camera, sampled, and subjected to scatter statistics mapping for cell tailing conditions, thereby obtaining the tailing cell rate. The expression of endogenous OGA of the cells is knocked down, the cell tailing is obviously enhanced, and the damage repair function of cell DNA is inhibited. The expression of endogenous OGA of the cells is knocked down and then transferred into exogenous full-length OGA expression plasmid, and the cell tailing is obviously shortened, which indicates that the repair function of the inhibited DNA damage is recovered. After the expression of endogenous OGA of cells is knocked down, the exogenous expression N-terminal OGA (1-700aa) or C-terminal OGA (701-916aa) plasmid is transferred, and the inhibited DNA damage repair function can not be completely recovered according to the cell tailing condition.
Clone formation experiments examine the effect of deletion of OGA expression on cellular DNA damage repair function (fig. 6).
Grouping experiments:
normal U2OS cells were obtained from American Type Culture Collection (ATCC). Cells were equally randomly assigned to 5 groups (4 subgroups with equal random assignment of cells in each group): 1 control group (labeled "siCon" in the figure) and 4 experimental groups (labeled "siOGA", "siOGA + OGA-WT", "siOGA + N-OGA" and "siOGA + C-OGA", respectively), and 4 groups of equal amounts of intracellular random distribution were: 1 control group (marked "IR: 0 Gy" in the figure, i.e., cells not treated for DNA damage) and 3 experimental groups (marked "IR: 1Gy, IR: 2Gy, IR: 4 Gy" in the figure, respectively).
Treating by using a medicine:
constructing cells with specific background requirements by transfecting siRNA knocking down OGA endogenous expression and corresponding exogenous expression plasmids, wherein the siRNA comprises the following components:
and (3) siCon: siRNA-Control was transfected as a Control group.
siOGA: transfection of siRNA-OGA knockdown endogenous OGA expression in cells.
siOGA + OGA-WT: transfection of siOGA-OGA knockdown of endogenous OGA expression in cells, followed by transfection of the full-length OGA expression plasmid.
siOGA + N-OGA: after transfection of siOGA-OGA knockdown of endogenous OGA expression in cells, N-terminal OGA (1-700aa) expression plasmids were transfected.
siOGA + C-OGA: transfection of siOGA-OGA knockdown of endogenous OGA expression in cells was followed by transfection of the C-terminal OGA (701-916aa) expression plasmid.
The cells were subjected to DNA damage treatment with laser radiation (IR), respectively: laser radiation (IR) treatment 0Gy (i.e.cells not treated for DNA damage), laser radiation (IR) treatment 1Gy, laser radiation (IR) treatment 2Gy, laser radiation (IR) treatment 4 Gy.
Collecting and processing a sample:
plate clone formation experiments different pre-treated cell samples were analyzed. Cell counting: cells were counted and three counts per group were averaged → plated: 1000 cells were sampled per group and evenly plated in a 3.5cm dish → cell culture: same conditions (37 ℃, 5% CO)2And saturation humidity) static culture of cells for 10-14 days → termination of culture: terminating the culture → washing after the cells find a cell mass visible to the naked eye: the medium was removed and the cells were carefully rinsed with 1 × PBS → cell fixation: cell fixation with pure methanol for 15min before removal → cell staining: cell staining with crystal violet 15min → wash dry: the staining solution was slowly washed off with running water, air dried → clonal statistics were performed.
Experimental results and analysis:
the plate was inverted and overlaid with a piece of transparent film with a grid, and the clones were counted directly with the naked eye, with a clone formation rate (number of clones/number of seeded cells) × 100%. As shown in fig. 6, by knocking down the endogenous OGA expression in cells, the number of clones formed by the cells after DNA damage is obviously reduced, indicating that the DNA damage repair function of the cells is inhibited. The expression of endogenous OGA of the cells is knocked down and then transferred into the plasmid of exogenous expression full-length OGA, the clone quantity formed by the cells after DNA damage is obviously increased and is equal to that of a control group, and the repair function of the inhibited DNA damage is restored. After the expression of endogenous OGA of cells is knocked down, the exogenous expression plasmids of N-terminal OGA (1-700aa) or C-terminal OGA (701-916aa) are transferred, and the inhibited DNA damage repair function can not be completely recovered according to the quantity formed by cell cloning.
Example 5 Effect of OGA inhibitor (TMG/(Z) -PUGNAC) treatment on cellular DNA damage repair function.
I. The effect of OGA inhibitor (TMG/(Z) -PUGNAC) treatment on cellular DNA damage repair pathway was analyzed using a DNA damage repair pathway Homologous Recombination (HR)/non-homologous end recombination (NHEJ) detection reporter method (fig. 7).
Grouping experiments:
taking a normal U2OS (EJ-5-GFP) cell line (the cell line is specially used for detecting a non-homologous recombination end repair (NHEJ) pathway after DNA damage), dividing the cell into 3 groups at random according to the equivalent weight: 1 control group (labeled "DMSO" in the figure) and two experimental groups (labeled "TMG" and "(Z) -PUGNAC) to which OGA inhibitors were added.
Taking a normal U2OS (DR-GFP) cell line (the cell line is specially used for detecting a Homologous Recombination (HR) repair pathway after DNA damage), equally dividing the cells into 3 groups randomly: 1 control group (labeled "DMSO" in the figure) and two experimental groups (labeled "TMG" and "(Z) -PUGNAC) to which OGA inhibitors were added.
Treating by using a medicine:
cells with specific background requirements were constructed by corresponding drug treatments, respectively:
TMG: cells were treated at 20mM for 12 h.
(Z) -PUGNAC: cells were treated at 20mM for 12 h.
DMSO, DMSO: the cells were added in the same volume as TMG/(Z) -PUGNAC and treated for the same time.
Collecting and processing a sample:
equal amount of cells are randomly and evenly distributed for plating, corresponding medicines are added for treatment for 12 hours after the cells are attached to the wall, and then the cells are transferred into JS-20 plasmid to induce the cells to generate DNA double-chain damage. After the plasmid was transferred for 48h, the medium was removed, the cells were collected with pre-cooled 1 × PBS after washing, and placed on ice for use. If the cells can repair the damage, the cells will generate green fluorescence signals, and then the flow cytometry is used to perform the 2 × 10 analysis4Individual cell fluorescent signals were analyzed. 3 replicates were performed.
Experimental results and analysis:
as shown in fig. 7, the fluorescence signal of the cells in the non-homologous recombination end repair (NHEJ) detection pathway is significantly reduced after the addition of the OGA inhibitor TMG or (Z) -PUGNAC, and the fluorescence signal of the cells in the Homologous Recombination (HR) detection pathway is not significantly changed, indicating that the non-homologous recombination end repair (NHEJ) pathway after DNA damage of the cells is significantly inhibited and the Homologous Recombination (HR) repair pathway after DNA damage of the cells is not significantly affected after the addition of the OGA inhibitor TMG or (Z) -PUGNAC.
Neutral comet assay analysis of the effect of OGA inhibitor (TMG/(Z) -PUGNAC) treatment on DNA damage repair function (fig. 8).
Grouping experiments:
normal U2OS cells were obtained from American Type Culture Collection (ATCC). Cells were equally randomized into 3 groups: 1 control group (marked "DMSO" in the figure) and two experimental groups (marked "TMG" and "(Z) -PUGNAC") to which OGA inhibitors were added (4 groups were randomly allocated in equal amounts within each group). The 4 groups, which were equally randomly distributed within cells of each group, were: 1 control group (marked as "untreated") and 3 experimental groups (marked as "0 h, 2h and 4h after IR (10Gy) treatment", respectively)
Treating by using a medicine:
cells with specific background requirements were constructed by corresponding drug treatments, respectively:
TMG: cells were treated at 20mM for 12 h.
(Z) -PUGNAC: cells were treated at 20mM for 12 h.
DMSO, DMSO: the cells were added in the same volume as TMG/(Z) -PUGNAC and treated for the same time.
The cells of the DNA damage treatment experiment group are treated by laser radiation (IR) (10Gy) and subjected to DNA damage repair for 0h, 2h and 4h respectively, and cell samples are stored in precooled 1 XPBS.
Collecting and processing a sample:
preparation of 1% Low melting agar (preservation in a 37 ℃ Water bath) → lysis of a cell sample preserved in precooled 1 XPBS in 1% Low melting agar → extraction of 1X 10 cells lysed in 1% Low melting agar4Spreading glue → drying glue fully→ lysis: the gel plate was placed in a freshly prepared precooled lysis buffer and lysed at 4 ℃ in the dark for 12h → wash: electrophoresis buffer washing at room temperature for 30min, washing 3 times → electrophoresis: electrophoresis in precooled electrophoresis buffer (7mA/20V) for 1h → washing: pure water wash 3 times → staining: propidium iodide staining solution (2.5 μ g/ml) was stained for 20min → washing: pure water washing 3 times → observation: cells were randomly selected using a fluorescence microscope for photographic recording.
Experimental results and analysis:
as shown in fig. 8, the cells were observed with a microscope, photographed with a camera, sampled, and subjected to scatter statistics mapping for cell tailing conditions, thereby obtaining the tailing cell rate. After the cells treated by the OGA inhibitor TMG or (Z) -PUGNAC are damaged by DNA, the cell tailing is obviously enhanced, which indicates that the DNA damage repair function of the cells is inhibited, and proves that OGA plays an important role in the DNA damage repair process.
Clone formation experiments analyze the effect of OGA inhibitor (TMG/(Z) -PUGNAC) treatment on DNA damage repair function (fig. 9).
Grouping experiments:
normal U2OS cells were obtained from American Type Culture Collection (ATCC). Cells were equally randomized into 3 groups: 1 control group (marked "DMSO" in the figure) and two experimental groups (marked "TMG" and "(Z) -PUGNAC") to which OGA inhibitors were added (4 groups were randomly allocated in equal amounts within each group). The 4 groups, which were equally randomly distributed within cells of each group, were: 1 control group (marked "IR: 0 Gy" in the figure, i.e.cells not treated for DNA damage) and 3 experimental groups (marked "IR: 1Gy, IR: 2Gy, IR: 4 Gy" in the figure, respectively).
Treating by using a medicine:
cells with specific background requirements were constructed by corresponding drug treatments, respectively:
TMG: cells were treated at 20mM for 12 h.
(Z) -PUGNAC: cells were treated at 20mM for 12 h.
DMSO, DMSO: the cells were added in the same volume as TMG/(Z) -PUGNAC and treated for the same time.
The cells were subjected to DNA damage treatment with laser radiation (IR), respectively: laser radiation (IR) treatment 0Gy (i.e.cells not treated for DNA damage), laser radiation (IR) treatment 1Gy, laser radiation (IR) treatment 2Gy, laser radiation (IR) treatment 4 Gy.
Collecting and processing a sample:
plate clone formation experiments different pre-treated cell samples were analyzed. Cell counting: cells were counted and three counts per group were averaged → plated: 1000 cells were sampled per group and evenly plated in a 3.5cm dish → cell culture: the cells were cultured under the same conditions (37 ℃, 5% CO2 and saturation humidity) for 10-14 days → termination of culture: terminating the culture → washing after the cells find a cell mass visible to the naked eye: the medium was removed and the cells were carefully rinsed with 1 × PBS → cell fixation: cell fixation with pure methanol for 15min before removal → cell staining: cell staining with crystal violet 15min → wash dry: the staining solution was slowly washed off with running water, air dried → clonal statistics were performed.
Experimental results and analysis:
the plate was inverted and overlaid with a piece of transparent film with a grid, and the clones were counted directly with the naked eye, with a clone formation rate (number of clones/number of seeded cells) × 100%. As shown in FIG. 9, the significant decrease in the number of clones formed by cells after DNA damage occurred in cells treated with the OGA inhibitor TMG or (Z) -PUGNAC indicates that the function of cellular DNA damage repair is inhibited, demonstrating that OGA plays an important role in the process of DNA damage repair.
Example 6 Effect of OGA inhibitor treatment on tumor cells deficient in DNA damage repair.
I. Clonogenic experiments analyzed the effect of OGA inhibitor (TMG) treatment on DNA damage repair-deficient tumor cells (BRCA1-/-) (fig. 10).
Grouping experiments:
normal U2OS cells (labeled "Mock") were obtained from American Type Culture Collection (ATCC). Cells were equally randomized into 4 groups: 1 control group (marked "OGA inhibitor: 0 mM", i.e.without inhibitor treatment) and 3 groups treated with OGA inhibitor (marked "OGA inhibitor: 1 mM", "OGA inhibitor: 2 mM" and "OGA inhibitor: 3 mM" in the figures).
A BRCA1 knock-out U2OS cell line (labeled "BRCA 1-/-" in the figure) was established, and cells were divided into 4 groups at random in equal amounts: 1 control group (marked "OGA inhibitor: 0 mM", i.e.without inhibitor treatment) and 3 groups treated with OGA inhibitor (marked "OGA inhibitor: 1 mM", "OGA inhibitor: 2 mM" and "OGA inhibitor: 3 mM" in the figures).
Treating by using a medicine:
cells with specific background requirements were constructed by treatment with the corresponding drug (OGA inhibitor TMG), respectively:
TMG: cells were treated at 1 mM.
TMG: cells were treated at 2 mM.
TMG: cells were treated at 3 mM.
Collecting and processing a sample:
plate clone formation experiments different pre-treated cell samples were analyzed. Cell counting: cells were counted and three counts per group were averaged → plated: 1000 cells were sampled per group and evenly plated in a 3.5cm dish → cell culture: the cells were cultured under the same conditions (37 ℃, 5% CO2 and saturation humidity) for 10-14 days → termination of culture: terminating the culture → washing after the cells find a cell mass visible to the naked eye: the medium was removed and the cells were carefully rinsed with 1 × PBS → cell fixation: cell fixation with pure methanol for 15min before removal → cell staining: cell staining with crystal violet 15min → wash dry: the staining solution was slowly washed off with running water, air dried → clonal statistics were performed.
Experimental results and analysis:
the plate was inverted and overlaid with a piece of transparent film with a grid, and the clones were counted directly with the naked eye, with a clone formation rate (number of clones/number of seeded cells) × 100%. As shown in fig. 10, the normal U2OS cells had a reduced colony formation rate after DNA damage by OGA inhibitor TMG treatment, indicating that the cellular DNA damage repair function was inhibited. Compared with cells in a normal state, the clone formation rate of the cells (BRCA1-/-) with DNA damage repair defects is obviously reduced after the cells are treated by OGA inhibitor TMG for DNA damage, which shows that the DNA damage repair function of the cells is inhibited to a higher degree, and proves that the tumor cells with DNA damage repair defects are more sensitive to the treatment of the OGA inhibitor.
Clone formation experiments analyze the effect of OGA inhibitor (TMG) treatment on DNA damage repair-deficient tumor cells (ATM-/-) (see figure 11).
Grouping experiments:
normal U2OS cells (labeled "Mock") were obtained from American Type Culture Collection (ATCC). Cells were equally randomized into 4 groups: 1 control group (marked "OGA inhibitor: 0 mM", i.e.without inhibitor treatment) and 3 groups treated with OGA inhibitor (marked "OGA inhibitor: 1 mM", "OGA Ainhibitor: 2 mM" and "OGA inhibitor: 3 mM" in the figure).
An ATM knockout U2OS cell line (labeled "ATM-/-" in the figure) was established and the cells were divided into 4 groups at random in equal amounts: 1 control group (marked "OGA inhibitor: 0 mM", i.e.without inhibitor treatment) and 3 groups treated with OGA inhibitor (marked "OGA inhibitor: 1 mM", "OGA inhibitor: 2 mM" and "OGA inhibitor: 3 mM" in the figures).
Treating by using a medicine:
cells with specific background requirements were constructed by treatment with the corresponding drug (OGA inhibitor TMG), respectively:
TMG: cells were treated at 1 mM.
TMG: cells were treated at 2 mM.
TMG: cells were treated at 3 mM.
Collecting and processing a sample:
plate clone formation experiments different pre-treated cell samples were analyzed. Cell counting: cells were counted and three counts per group were averaged → plated: 1000 cells were sampled per group and evenly plated in a 3.5cm dish → cell culture: same conditions (37 ℃, 5% CO)2And saturation humidity) static culture of cells for 10-14 days → termination of culture: terminating the culture → washing after the cells find a cell mass visible to the naked eye: the medium was removed and the cells carefully rinsed with 1 XPBS → the cells were fixedDetermining: cell fixation with pure methanol for 15min before removal → cell staining: cell staining with crystal violet 15min → wash dry: the staining solution was slowly washed off with running water, air dried → clonal statistics were performed.
Experimental results and analysis:
the plate was inverted and overlaid with a piece of transparent film with a grid, and the clones were counted directly with the naked eye, with a clone formation rate (number of clones/number of seeded cells) × 100%. As shown in fig. 11, the normal U2OS cells had a reduced colony formation rate after DNA damage by OGA inhibitor TMG treatment, indicating that the cellular DNA damage repair function was inhibited. Compared with cells in a normal state, the clone formation rate of cells in a DNA damage repair defect type (ATM-/-) after DNA damage is carried out by OGA inhibitor TMG treatment is obviously reduced, which shows that the DNA damage repair function of the cells is inhibited to a higher degree, and the tumor cells with DNA damage repair defects are proved to be more sensitive to the OGA inhibitor treatment.
Effect of oga inhibitor TMG treatment on BRCA1 or BRCA2 deficient tumor cells (fig. 12).
Grouping experiments:
1) two groups of cells: BRCA 1-deficient triple-negative breast cancer cells (labeled "HCC 1937" in the figure) and BRCA 1-deficient triple-negative breast cancer cells transformed with a normal BRCA 1-expressing gene (labeled "HCC 1937+ BRCA 1" in the figure), each group of cells was equally divided into 3 groups on average for treatment with OGA inhibitor (TMG) (labeled "TMG: 0 mM", "TMG: 1 mM" and "TMG: 2 mM" in the figure).
2) Two groups of cells: BRCA 1-deficient triple-negative breast cancer cells (labeled "HCC 1395" in the figure) and normal mammary epithelial cells (labeled "MCF-10A" in the figure), and each group of cells was equally divided into 3 groups on average and treated with OGA inhibitor (TMG) (labeled "TMG: 0 mM", "TMG: 1 mM" and "TMG: 2 mM" in the figure).
3) Two groups of cells: BRCA 2-deficient ovarian cancer cells (labeled "PEO-1" in the figure) and non-BRCA 2-deficient ovarian cancer cells (labeled "PEO-4" in the figure), each group of cells was equally divided into 3 groups on average (labeled "TMG: 0 mM", "TMG: 1 mM" and "TMG: 2 mM") and treated with OGA inhibitor (TMG).
4) Two groups of cells: BRCA 1-deficient triple-negative breast cancer cells (labeled "SUM 149 PT" in the figure) and normal mammary epithelial cells (labeled "MCF-10A" in the figure), each group of cells was equally divided into 3 groups on average (labeled "TMG: 0 mM", "TMG: 1 mM", and "TMG: 2 mM") and treated with OGA inhibitor (TMG).
Treating by using a medicine:
DMSO, DMSO: same time as dose addition treatment, and TMG: 0mM cell treatment group.
TMG: cells were treated at 1mM for the same time.
TMG: cells were treated at 2mM for the same time.
Collecting and processing a sample:
equal amounts of different background cells were taken in the same incubator (37 ℃, 5% CO)2Humidity saturation) environment, and counting the cell survival rate.
Experimental results and analysis:
as shown in fig. 12, the survival of BRCA 1-deficient triple-negative breast cancer cells (HCC1937) after treatment with different doses of OGA inhibitor (TMG) was significantly reduced in BRCA 1-deficient triple-negative breast cancer cells (HCC1937) and BRCA 1-deficient triple-negative breast cancer cells (HCC1937+ BRCA1) that have transferred the normal BRCA1 expression gene; BRCA 1-deficient triple-negative breast cancer cells (HCC1395) and normal mammary epithelial cells (MCF-10A), with significantly reduced survival of BRCA 1-deficient triple-negative breast cancer cells (HCC1395) after treatment with different doses of OGA inhibitor (TMG); BRCA 2-deficient ovarian cancer cells (PEO-1) and non-BRCA 2-deficient ovarian cancer cells (labeled PEO-4 in the figure), the survival of BRCA 2-deficient ovarian cancer cells (PEO-1) is significantly reduced after treatment with different doses of OGA inhibitor (TMG); the survival of BRCA 1-deficient triple-negative breast cancer cells (SUM149PT) and normal breast epithelial cells (MCF-10A), and BRCA 1-deficient triple-negative breast cancer cells (SUM149PT) was significantly reduced. The OGA inhibitor TMG treatment was shown to be particularly sensitive to BRCA1 or BRCA2 deficient tumor cells, demonstrating that OGA inhibition may be a novel approach for treating tumors with DNA damage repair defects.
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| CN107253970A (en) * | 2006-08-31 | 2017-10-17 | 西蒙·弗雷瑟大学 | Selective glycosidase inhibitors and application thereof |
| CN103339138A (en) * | 2010-12-23 | 2013-10-02 | 阿勒克图治疗公司 | Selective glycosidase inhibitors and uses thereof |
Non-Patent Citations (4)
| Title |
|---|
| O-GlcNAcylation-Inducing Treatments Inhibit Estrogen Receptor α Expression and Confer Resistance to 4-OH-Tamoxifen in Human Breast Cancer-Derived MCF-7 Cells;Shahzina Kanwal等;《PLOS ONE》;20130711;第8卷(第7期);第1-17页 * |
| O-GLcNAc糖基化修饰对卵巢癌细胞增殖的影响;马春;《万方学位论文》;20181129;第1-55页 * |
| ROCK2 mediates osteosarcoma progression and TRAIL resistance by modulating O-GlcNAc transferase degradation;Xueqiang Deng等;《Am J Cancer Res.》;20200301;第10卷(第3期);第781-798页 * |
| 蛋白质O-GlcNAc糖基化在紫杉醇抗肿瘤活性中的作用研究;张肖冰;《万方学位论文》;20150925;第1-91页 * |
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