CN114306617A - Small molecule inhibitor for promoting glioma stem cell apoptosis and application thereof - Google Patents
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Abstract
The invention provides a small molecule inhibitor for promoting glioma stem cell apoptosis and application thereof. The small molecule inhibitor is OSMI-1, and the invention proves the new application of the small molecule inhibitor OSMI-1 in promoting the apoptosis of glioma stem cells, so that the small molecule inhibitor OSMI-1 can be used for preparing a pharmaceutical composition for treating glioblastoma.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a small molecule inhibitor for promoting Glioma Stem Cell (GSC) apoptosis and application thereof.
Background
Glioblastoma (GBM) is the most common primary malignant tumor in the cranium, accounting for 57% of all gliomas and 48% of primary malignant tumors in the Central Nervous System (CNS). Although multi-modal complex treatments for GBM, including surgery, radiation therapy, systemic therapy (chemotherapy, targeted therapy), have advanced greatly in recent years, GBM has a short overall survival time and long-term survival patients are rare. Furthermore, the method is simple. The nerve dysfunction and the life quality reduction caused by the tumor bring destructive impact to patients and family members.
The glioma stem cells are a key cell group of the glioblastoma and are closely related to the occurrence, development and malignancy of the glioblastoma. An increasing number of studies indicate that Glioma Stem Cells (GSCs) are a critical cell population against which glioblastoma treatments are resistant. It is different from common tumor cells which have the characteristics of rapid division, sensitivity to anticancer drugs, no self-renewal capability and the like. The glioma stem cells are a small number of cell subsets with stem cell characteristics, namely self-renewal capacity, expression of dry markers CD133, nestin and the like and high tumor forming capacity, are considered as 'seed' cells in tumors, are the root of tumorigenesis, metastasis and relapse, and chemoradiotherapy tolerance, and are the most main reasons of tumor treatment failure and death. It has the following characteristics: glioma stem cells are usually in a quiescent state and insensitive to anticancer drugs. In radiotherapy, a large number of tumor cells are killed and glioma stem cells survive to differentiate into common tumor cells, which in turn cause the tumor to form again, i.e., to recur. Therefore, to cure cancer, not only general tumor cells but also glioma stem cells causing tumor proliferation are killed and killed by specific therapy. However, specific therapeutic targets for tumor stem cells are lacking at present. Therefore, the effective drug for killing the glioma stem cells can be selected to enhance the effect of radiotherapy, and a new possibility is provided for treating the glioblastoma.
OSMI-1 is an inhibitor of cell permeable O-GlcNAc transferase (OGT) and has been reported primarily for inhibiting abnormally high levels of O-GlcNAcylation and thus tumor development in liver, breast and colon cancers. However, there is no report on the use of OSMI-1 in glioma stem cell therapy.
Disclosure of Invention
The invention aims to provide a new application of OSMI-1 in killing glioma stem cells.
In order to achieve the above purpose, the invention provides the following scheme:
in one aspect, the invention provides the use of an inhibitor of a targeted O-linked N-acetylglucosamine transferase for the preparation of a pharmaceutical composition for the treatment of a brain tumor.
In some embodiments, the brain tumor is a glioblastoma.
In some embodiments, the inhibitor targeting an O-linked N-acetylglucosamine transferase is OSMI-1.
In another aspect, the invention provides the use of the small molecule inhibitor OSMI-1 in the preparation of a pharmaceutical composition for killing glioma stem cells.
Preferably, the small molecule inhibitor OSMI-1 treats cancer by promoting apoptosis of glioma stem cells, thereby killing glioma stem cells.
Preferably, the small molecule inhibitor OSMI-1 kills glioma stem cells concentration-dependently.
Preferably, the molecular formula of the small molecule inhibitor OSMI-1 is C28H25N3O6S2。
In another aspect, the invention provides the use of the small molecule inhibitor OSMI-1 for the preparation of a pharmaceutical composition for increasing the effectiveness of radiation therapy.
In some embodiments, the radiation therapy is radiation therapy directed to glioma stem cells.
In some embodiments, the radiation therapy is radiation therapy directed to glioblastoma.
In another aspect, the present invention provides a pharmaceutical composition for treating glioblastoma comprising an effective amount of OSMI-1 and a pharmaceutically acceptable excipient.
In another aspect, the present invention provides a pharmaceutical composition for killing glioma stem cells comprising an effective amount of OSMI-1 and a pharmaceutically acceptable excipient.
Preferably, the small molecule inhibitor OSMI-1 induces glioma stem cell apoptosis by disrupting glioma stem cell homeostasis by inhibiting high levels of O-GlcNAcylation in glioma stem cells.
In another aspect, the invention provides a method of treating glioblastoma comprising administering to a subject in need thereof an effective amount of an inhibitor targeting an O-linked N-acetylglucosamine transferase.
In another aspect, the invention provides a method of killing glioma stem cells, said method comprising administering to a subject in need thereof an effective amount of an inhibitor targeting an O-linked N-acetylglucosaminyltransferase.
In some embodiments, the method further comprises irradiating the subject.
Definition of
OSMI-1: a small molecule inhibitor of cell permeability targeting O-linked N-acetylglucosamine transferase (OGT) has molecular formula C28H25N3O6S2。
PUGNAC: a targeted O-linked N-acetylglucosaminidase (OGA) inhibitor with molecular formula of C15H19N3O7。
CCK-8: reagents for simple and accurate cell proliferation and toxicity analysis.
IC50: drug concentration at which the small molecule inhibitor kills glioma stem cells by 50%.
Small molecule inhibitors: the organic compound molecules with the molecular weight less than 1000 daltons can be targeted on proteins, reduce protein activity or block biochemical reaction, and are widely applied to the research of signal paths.
Advantageous effects
The invention proves the new application of a small molecular inhibitor OSMI-1 in promoting glioma stem cell apoptosis. The discovery of the drug can provide a new idea for understanding the treatment resistance of the glioma stem cells and a new treatment method for solving the treatment resistance of the glioma stem cells.
Drawings
FIG. 1 shows the CCK8 test of cell proliferation and activity of small molecule inhibitor OSMI-1 treated glioma stem cells (GBM-GSC 1);
FIG. 2 shows the CCK8 test of cell proliferation and activity of glioma stem cells GBM-GSC1 and GBM-GSC2 treated with DMSO and the small molecule inhibitor OSMI-1, respectively. FIG. 2A is a diagram showing CCK8 testing the cell proliferation and activity of glioma stem cell GBM-GSC1 treated with DMSO and the small molecule inhibitor OSMI-1; FIG. 2B is a CCK8 assay for cell proliferation and activity of glioma stem cell GBM-GSC2 treated with DMSO and the small molecule inhibitor OSMI-1.
FIG. 3 ("-" represents no inhibitor added "+" represents both inhibitors added) is a flow analysis of small molecule inhibitor OSMI-1 labeled with Annexin V-FITC/PI and combined irradiation treatment of glioma stem cells for apoptosis; (the first two groups are the addition of inhibitors, the last two groups are the combined irradiation of inhibitors, i.e. IR (4 Gy)); FIG. 3A is a schematic diagram of Annexin V-FITC/PI label flow analysis; FIG. 3B shows the proportion of apoptotic cells of GBM-GSC 1; FIG. 3C shows the proportion of apoptotic cells of GBM-GSC 2.
FIG. 4 ("-" for no inhibitor addition "+" for inhibitor addition) is a schematic representation of the small molecule inhibitor OSMI-1 disrupting glioma stem cell homeostasis by inhibiting high O-GlcNAcylation levels of glioma stem cells, thereby inducing glioma stem cell apoptosis. FIG. 4A is a Western blot to detect changes in O-GlcNAcylation levels following treatment of glioma stem cells GBM-GSC1 with inhibitor OSMI-1; FIG. 4B is a comparison of Western blotting to detect O-GlcNAcylation levels of glioma stem cells GBM-GSC1, GBM-GSC2 and GBM-GSC3 compared to NPC; FIG. 4C is a graph of the change in the O-GlcNAcylation level of the small molecule inhibitor PUGNAC treated glioma stem cell GBM-GSC 2; FIG. 4D is a schematic diagram of the apoptosis of Annexin V-FITC/PI labeled flow analysis small molecule inhibitors PUGNAC treated glioma stem cells GBM-GSC1 and GBM-GSC2 and combined irradiation treated glioma stem cells (the first two groups are the addition of the inhibitor and the latter two groups are the combined irradiation of the inhibitor, i.e., IR (4 Gy); FIG. 4E shows the proportion of apoptotic cells of the glioma stem cells GBM-GSC1 treated with the inhibitor PUGNAC, and FIG. 4F shows the proportion of apoptotic cells of the glioma stem cells GBM-GSC2 treated with the inhibitor PUGNAC.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
The main materials used in the invention are as follows:
OSMI-1 and PUGNAC were purchased from Sigma-Aldrich; the BCA protein quantification kit is purchased from Solario company; WB chemiluminescent chromogenic substrates (Super Signal West cheminescent substrates) were purchased from Biosharp corporation; the CCK8 kit was purchased from Vazyme; FITC-annexin V/PI Apoptosis Detection kit (FITC annexin V Apoptosis Detection kit) from BD Biosciences; primary anti-O-GlcNAc was purchased from Cell Signaling Technology; anti-beta-actin is available from TRANS; anti- α -tubulin available from Cell Signaling Technology; the secondary antibody Anti-mouse and Anti-rabbitt were obtained from Cell Signaling Technology;
the statistical analysis involved in the invention was performed using Graph Pad Prism8 statistical software, the experimental results were expressed as mean ± standard deviation (x ± s), two sets of mean comparisons were performed using t-test, multiple sets of mean comparisons were performed using one-way anova, and p <0.05 was considered to have statistical difference and was expressed as "+". In fig. 1-4, statistical differences (p <0.05) are indicated; indicates significant differences (p < 0.01); marked differences (p < 0.001); indicates very significant differences (p < 0.0001).
Example 1: the small molecule inhibitor OSMI-1 can effectively kill glioma stem cells and is in a concentration dependence.
Glioma stem cells GBM-GSC1 (cell line preparation method see Li Y, He ZC, Zhang XN, et al Stanniiocalcin-1 augments stem-like tracks of gliobastoma cells through binding and activating NOTCH1.cancer Lett.2018Mar 1; 416:66-74) (5000 cells/well, 100ul) were seeded in 96-well plates, six wells per group, cells were treated with different concentrations of OSMI-1(10uM, 15uM, 20uM, 30uM) for 24hr, respectively, and DMSO was used as a control. CCK-8 was added to each of the cells to measure absorbance (A450) at a wavelength of 450nm, and a cell growth curve was prepared. Detection of the killing effect of OSMI-1 on glioma stem cells GBM-GSC1 (CCK8 is commonly used for detecting the killing of cells by drugs)Action) and calculate IC50The results are shown in FIG. 1 (FIG. 1, cell viability; concentration).
The results show that OSMI-1 can effectively kill glioma stem cell GBM-GSC1, and presents concentration dependence, IC50It was 15 uM.
Example 2: the small molecule inhibitor OSMI-1 can obviously inhibit the proliferation capacity of glioma stem cells GBM-GSC1 and GBM-GSC2
Two different glioma stem cells (GBM-GSC1 and GBM-GSC2) (cell line preparation methods are described in Li Y, He ZC, Zhang XN, et al.Stanniiocalcin-1 antigens stem-like peptides of gliobastoma cells through binding and activating NOTCH1.cancer Lett.2018Mar 1; 416:66-74) (5000 cells/well, 100ul) were seeded in 96-well plates, six wells per group, and GBM-GSC1 and GBM-GSC2 cells were treated with 20uM OSMI-1 for 96hr, DMSO as a control. CCK-8 was added to measure absorbance (A450) at a wavelength of 450nm, and each group was normalized with absorbance of 0hr to plot a histogram of proliferation. The results are shown in FIG. 2 (FIG. 2, cell viability).
The results show that OSMI-1 can remarkably inhibit the proliferation capacity of glioma stem cells GBM-GSC1 and GBM-GSC2 (p < 0.0001).
Example 3: killing glioma stem cells by inducing glioma stem cell apoptosis (apoptosis) through small-molecule inhibitor OSMI-1
Two different glioma stem cells (GBM-GSC1 and GBM-GSC2) (5X 10)5Cells/well, 6mL) were seeded in 10cm dishes and GBM-GSC1 and GBM-GSC2 cells were pre-treated with 20uM OSMI-1 for 12hr, DMSO as a control. Then, each group was irradiated (4Gy) and cells were collected 24hr after irradiation for AnnexinV-FITC/PI labeling flow analysis of apoptotic cell ratio. The staining procedure is (is a standard procedure of the kit, and only reasonable changes are made on the dosage):
(1) utilizing an enzymolysis method (commonly used enzyme accutase for dissociating glioma stem cells, removing supernatant after cell centrifugation, adding 1ml of enzyme, digesting for 5 minutes), dissociating the glioma stem cells into single cells (the glioma stem cells can form balls when growing, and need to be dissociated into single cells when dyeing the glioma stem cells), centrifuging at 1000rpm for 3min, and then discarding the supernatant;
(2) PBS washing twice, cell heavy suspension in 1 x buffer, adjusting cell concentration to 1 x 107Cells/well;
(3) taking 10ul of cell resuspension during staining, staining 5ul of PI (propidium iodide) for 30min, staining 2ul of FITC for 15min (FITC-Annexin V/PI apoptosis detection kit is a relatively conventional means for detecting cell apoptosis, and the basic principle is that phosphatidylserine at the inner side of a cell membrane can be transferred from the inside of the cell to the outside of the cell membrane and can be stained by Annexin-V combination when the cell undergoes apoptosis at the early stage, the cell membrane can be damaged when the cell undergoes apoptosis at the late stage, PI can enter the cell to stain the cell, and the two dyes can be used jointly to determine the apoptosis state through flow analysis);
(4) blank tubes (blank tubes are not dyed tubes) and single positive tubes (single dyed tubes are added for adjusting compensation, namely preventing signal crosstalk of two dyes) are arranged as required, and finally 400ul of 1 × buffer is added into each dyed tube and is tested on a machine.
The results are shown in FIG. 3. The results show that the OSMI-1 can promote the apoptosis of glioma stem cells GBM-GSC1 and GBM-GSC2, the proportion of apoptotic cells is equivalent to that of a single irradiation group, and the proportion of apoptotic cells is further increased after the inhibitor and the irradiation are simultaneously treated. FIGS. 3B and 3C are statistical graphs with the ordinate representing the proportion of apoptotic cells, and according to the graphs, "-" represents no addition of inhibitor OSMI-1, "+" represents addition of inhibitor OSMI-1, and the left two groups are comparisons of addition of inhibitor alone, and the latter two groups are addition of 4Gy irradiation based on inhibitor addition.
Example 4: inhibitor OSMI-1 promotes the development of apoptosis by inhibiting O-GlcNAcylation of glioma stem cells
Considering that the small molecule inhibitor OSMI-1 mainly targets O-linked N-acetylglucosamine transferase (OGT), which is the only modifying enzyme mediating O-GlcNAcylation, we speculate that the killing and pro-apoptotic effects of the small molecule inhibitor OSMI-1 on glioma stem cells may exert anti-tumor activity by inhibiting its O-GlcNAcylation, thereby breaking the homeostasis of glioma stem cells.
We therefore first examined changes in O-GlcNAcylation levels following treatment of glioma stem cells with inhibitor OSMI-1. After treating glioma stem cell GBM-GSC 124 hr with small molecule inhibitor OSMI-1, collecting protein, and incubating antibody according to Western Blot procedure for detection.
(1) Adding RIPA protein lysate to respectively extract proteins of the glioma stem cells GBM-GSC 1;
(2) quantifying the protein concentration of the sample according to the BCA method, and preparing a protein sample from 4 xSDS;
(3) preparing 8% separation gel (prepared according to the instruction), loading according to 20ug of protein, and performing 100V constant voltage electrophoresis;
(4) cutting a PVDF membrane, soaking in methanol, placing in an electric transfer liquid, installing electric transfer clamps according to the sequence of black clamp-sponge-filter paper-glue-PVDF membrane-filter paper-sponge-white clamp, and carrying out constant-current 250mA ice-bath wet transfer for 90 min;
(5) after the electrotransformation is finished, putting the PVDF membrane into 5% skimmed milk powder, and sealing at room temperature for 1 hr;
(6) incubating anti-O-GlcNAc primary antibody and anti-beta-actin primary antibody (a common internal reference protein) in a shaking table at 4 ℃ for overnight incubation;
(7) after the primary antibody incubation is finished, washing the membrane for 3 times by using TBST (conventional membrane washing solution), and washing for 5min each time;
(8) incubating a secondary antibody (the secondary antibody is an antibody combined with the primary antibody and provided with an enzyme capable of reacting with the substrate in the luminous solution) at room temperature for 90min, and washing the membrane for 3 times by using TBST after finishing the incubation, wherein each time is 5 min;
(9) adding luminous liquid, and developing and exposing.
The results are shown in fig. 4A, indicating that small molecule inhibitor OSMI-1 can significantly reduce the O-GlcNAcylation level of glioma stem cells; the black shading is the result after WB exposure, indicating the number of proteins modified by O-GlcNAc glycosylation, and the black shading with the addition of the inhibitor group is significantly reduced, indicating a significant reduction in the number of proteins modified by O-GlcNAc glycosylation.
Since the small molecule inhibitor OSMI-1 can significantly reduce the O-GlcNAcylation level of glioma stem cells and finally achieve the effect of killing glioma stem cells, the fact indicates that O-GlcNAcylation in glioma stem cells can be a protective measure for cells. We therefore followed by Western Blot to detect the O-GlcNAcylation levels of glioma stem cells (GBM-GSC1, GBM-GSC2 and GBM-GSC3) and neuroprogenitor NPC. (NPC is a normal stem cell type, comparison of glioma stem cells with NPC led to our mechanistic approach) (cell line preparation methods see Li Y, He ZC, Zhang XN, et al.Stanniiocalcin-1 augments stem-like traits of globustomas cells through binding and activating NOTCH1.Cancer Lett.2018Mar 1; 416:66-74 and Shi Y, Guryanova OA, ZHou W, et al.Ibrutinib inactivates BMX-STAT3 in glioma cell impair polypeptide Med.Sci Trans.2018 May 30; 10 (681443): eaah 6.) procedure is as above, and GlcNO-resistant and anti-buanti- α line are incubated.
As a result, as shown in FIG. 4B, the O-GlcNAcylation level was higher in glioma stem cells (GBM-GSC1, GBM-GSC2 and GBM-GSC3) than in Neuro Progenitor Cells (NPC). Therefore we have reason to speculate that abnormally high levels of O-GlcNAcylation are an important mechanism for glioma stem cells to maintain their own properties. (α -tubulin in FIG. 4B is a commonly used internal reference protein) to further confirm this hypothesis, the present invention uses another small molecule inhibitor, PUGNAC, which targets mainly O-linked N-acetylglucosaminidase (OGA), which is the only modifying enzyme that mediates O-GlcNAcylation, simultaneously, so that this small molecule inhibitor can increase O-GlcNAcylation of glioma stem cells, theoretically, can produce a biological function opposite to that of the inhibitor OSMI-1, i.e., can play a role in supporting survival of tumor stem cells. First we measured the O-GlcNAcylation level of glioma stem cells (GBM-GSC2) 24hr after treatment of the small molecule inhibitor PUGNAC with Western Blot. The steps are the same as above, and the primary antibodies for incubation are anti-O-GlcNAc primary antibodies and anti-alpha-tubulin primary antibodies.
FIG. 4C shows that the small molecule inhibitor PUGNAC can significantly increase the O-GlcNAcylation level of glioma stem cells. The black shading is the result after WB exposure, indicating the number of proteins modified by O-GlcNAc glycosylation.
Glioma stem cells GBM-GSC1 and GBM-GSC 212 hr were then treated with the small molecule inhibitor 2. mu.M PUGNAC, DMSO being used as a control. Then, each group was irradiated (4Gy) and cells were collected 24hr after irradiation for AnnexinV-FITC/PI labeling flow analysis of apoptotic cell ratio. The results shown in fig. 4D, fig. 4E and fig. 4F indicate that the small molecule inhibitor PUGNAC can save the proportion of cells undergoing apoptosis after irradiation to some extent, although the treatment group of the small molecule inhibitor PUGNAC alone does not show a reduction in the proportion of apoptotic cells, which may be related to the strong self-protective function of glioma stem cells themselves. Fig. 4D is a schematic diagram of flow-based detection of apoptosis, fig. 4E and 4F are statistical diagrams, the ordinate of the statistical diagram is the proportion of apoptotic cells, "-" indicates that no inhibitor punmac is added, "+" indicates that inhibitor punmac is added, and IR (4Gy) indicates that irradiation is performed, so that the first two groups on the statistical diagram are single inhibitor groups, and the last two groups are inhibitor and irradiation combined groups, and it can be seen that the proportion of apoptotic cells can be reduced by the addition of inhibitor punmac in combination with the irradiation group. It can be seen from the correspondence of FIGS. 4A and 4C that the inhibitors OSMI-1 and PUGNAC are biologically opposite in that the former reduces the O-GlcNAc glycosylation modification and the latter increases the O-GlcNAc glycosylation modification. FIG. 3 demonstrates that the addition of the small molecule inhibitor OSMI-1, modified to reduce O-GlcNAc glycosylation, increases the proportion of apoptosis in the cells upon cumulative irradiation. Therefore, in combination, the modification of O-GlcNAc glycosylation is a protective mechanism for glioma stem cells. The suppression thereof can enhance the effect of irradiation.
The combined results show that glioma stem cells have significantly higher levels of O-GlcNAcylation than normal stem cells, i.e. neural progenitor cells, which may be a key mechanism for glioma stem cells to resist radiotherapy. The small molecule inhibitor OSMI-1 can achieve the effect of killing glioma stem cells by promoting the apoptosis of the glioma stem cells, the small molecule inhibitor OSMI-1 can obviously reduce the O-GlcNAcylation level of the glioma stem cells, and the small molecule inhibitor PUGNAC with opposite biological functions can save the apoptosis ratio of the glioma stem cells after radiotherapy to a certain extent, so that the killing effect of the small molecule inhibitor on the glioma stem cells can be presumed to play an anti-tumor activity effect by inhibiting the O-GlcNAcylation so as to break the homeostasis of the glioma stem cells.
In conclusion, the invention provides the micromolecule inhibitor OSMI-1 for the first time, which can be used for preparing a novel anti-cancer medicament for killing glioma stem cells, and the anti-tumor effect of the micromolecule inhibitor OSMI-1 can enhance the apoptosis of tumor cells after irradiation. Provides a new direction for curing glioblastoma and improving the survival rate of glioblastoma.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. Use of an inhibitor of a targeted O-linked N-acetylglucosamine transferase for the preparation of a pharmaceutical composition for the treatment of brain tumors.
2. The use according to claim 1, wherein the brain tumor is a glioblastoma.
3. Use according to claim 1 or 2, wherein the inhibitor targeting the O-linked N-acetylglucosaminyltransferase is OSMI-1.
4. Use of small molecule inhibitor OSMI-1 in preparing pharmaceutical composition for killing glioma stem cells.
5. Use of small molecule inhibitor OSMI-1 in preparing pharmaceutical composition for increasing radiotherapy effect.
6. Use according to claim 5, wherein the radiotherapy is radiotherapy directed against glioma stem cells.
7. A pharmaceutical composition for the treatment of glioblastoma comprising an effective amount of OSMI-1 and a pharmaceutically acceptable excipient.
8. A pharmaceutical composition for killing glioma stem cells comprising an effective amount of OSMI-1 and a pharmaceutically acceptable excipient.
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|---|---|---|---|---|
| US20210113540A1 (en) * | 2018-06-06 | 2021-04-22 | National University Corporation Hokkaido University | Treatment agent and pharmaceutical composition for glioma |
| CN113368249A (en) * | 2021-06-07 | 2021-09-10 | 中国人民解放军军事科学院军事医学研究院 | OGT inhibitor and application thereof |
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| CN113368249A (en) * | 2021-06-07 | 2021-09-10 | 中国人民解放军军事科学院军事医学研究院 | OGT inhibitor and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 黄晓敏: "N-乙酰氨基葡萄糖转移酶-V通过改变上皮生长因子受体的糖基化水平增加爱必妥在鼻咽癌细胞中的放疗敏感性", 《中国优秀硕博士学位论文全文数据库（硕士）医药卫生科技辑》 * |
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|---|---|---|---|---|
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