Application of duloxetine in preparation of medicine for preventing or treating glioblastoma

Abstract

The invention provides an application of duloxetine in preparation of a drug for preventing or treating glioblastoma, the active ingredient of the drug is duloxetine, and the duloxetine realizes an anti-tumor effect by inhibiting the proliferation activity of tumor cells through experiments; duloxetine realizes an anti-tumor effect by reducing the migration and invasion ability of tumor cells; and duloxetine intervenes in tumor cell autophagy and cell cycle signal pathways to play an anti-tumor role.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of duloxetine in preparing a drug for preventing or treating glioblastoma. Background Art

[0002] Gliomas are the most common primary tumors of the central nervous system, accounting for about 80% of primary malignant brain tumors. According to the World Health Organization classification, gliomas are divided into grades I, II, III, and IV. Among them, grade IV gliomas, namely glioblastomas (GBM), are the most malignant and have the worst prognosis. Because glioblastomas grow very fast and easily invade surrounding brain tissue, they are usually difficult to completely remove by surgery, and the possibility of recurrence after treatment is very high. Among all primary malignant intracranial tumors and other central nervous system tumors, glioblastomas account for about 48.6%, and the median survival of patients is only 8 months. On the one hand, due to the incomplete differentiation of the tumor, it often grows in an invasive manner, and tumor cells penetrate deep into the nerve tissue, making it difficult to completely remove the tumor tissue; on the other hand, due to the obstruction of the blood-brain barrier and the heterogeneity and intrinsic drug resistance of brain tumors, traditional chemotherapy drugs are prone to side effects, and it is difficult to achieve good results in treatment. The first-line treatment for GBM is radiotherapy and chemotherapy after surgical resection. Because surgery cannot guarantee the complete resection of tumor tissue, patients are prone to recurrence after surgery. Temozolomide (TMZ) is the first-line chemotherapy drug for the clinical treatment of GBM. However, since GBM cells are very susceptible to TMZ resistance, it is crucial to find new drugs for the treatment of GBM and to explore their targets and mechanisms.

[0003] Duloxetine, chemically known as (R)-(-)-N-methyl-3-(1-naphthyloxy)-3-(2-thiophene)-propylamine, is a selective 5-hydroxytryptamine (5-HT) and norepinephrine (NE) reuptake inhibitor used to treat major depression and neuropathic pain and other related diseases. As an approved drug, the safety of duloxetine has been certified by the U.S. Food and Drug Administration (FDA).

[0004] So far, its anti-glioblastoma (GBM) effect has not been reported. Summary of the invention

[0005] The present invention aims to provide a method for preparing a drug for preventing or treating glioblastoma, which achieves anti-tumor effect by inhibiting the proliferation activity of glioblastoma cells, reducing the migration and invasion ability of glioblastoma cells, and intervening in autophagy and cell cycle pathways in tumor cells.

[0006] The present invention provides the use of duloxetine as shown in formula I in preparing a drug for preventing or treating glioblastoma. Formula I.

[0007] Optionally, in the above-mentioned use of duloxetine in the preparation of a drug for preventing or treating glioblastoma, the effective concentration of duloxetine in the drug for preventing or treating glioblastoma is 1 μM to 100 μM.

[0008] Optionally, in the above-mentioned use of duloxetine in the preparation of a drug for preventing or treating glioblastoma, the effective concentration of duloxetine in the drug for preventing or treating glioblastoma is 5 μM to 60 μM.

[0009] Optionally, in the use of the above-mentioned duloxetine in the preparation of a drug for preventing or treating glioblastoma, the drug for preventing or treating glioblastoma inhibits the proliferation activity of glioblastoma cells, reduces the migration and invasion ability of glioblastoma cells, and intervenes in the autophagy and cell cycle pathways in tumor cells.

[0010] Optionally, in the above-mentioned use of duloxetine in the preparation of a drug for preventing or treating glioblastoma, the drug for preventing or treating glioblastoma comprises an effective dose of duloxetine and a pharmaceutically acceptable excipient and / or carrier.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention proposes that duloxetine exerts anti-tumor effects by reducing GBM cell activity and inhibiting its migration and invasion ability, which has not been reported. Duloxetine has been verified through experiments that duloxetine achieves anti-tumor effects by inhibiting tumor cell proliferation activity (i.e., reducing GBM cell activity); duloxetine achieves anti-tumor effects by reducing tumor cell migration and invasion ability; and duloxetine intervenes in tumor cell autophagy and cell cycle signaling pathways to exert anti-tumor effects. Therefore, duloxetine has obvious anti-glioblastoma activity and can be used as an active ingredient of a candidate drug for the treatment of glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0013] Figure 1 It shows the changes in cell survival rate of U87 and U251 cells after 24 hours of treatment with different concentrations of duloxetine; Figure 2 The half inhibitory concentration (IC) of duloxetine on U87 and U251 cells after 24, 48 and 72 h 50 ); Figure 3It shows the changes in the migration and invasion abilities of U87 and U251 cells after 24 hours of exposure to different concentrations of duloxetine; Figure 4 It is a volcano map showing differentially expressed genes in U87 cells after 48 hours of exposure to different concentrations of duloxetine; Figure 5 It is a cluster diagram showing differentially expressed genes in U87 cells after 48 hours of exposure to different concentrations of duloxetine; Figure 6 It is a KEGG scatter plot showing differentially expressed genes in U87 cells after 48 hours of exposure to different concentrations of duloxetine; Figure 7 It shows the number of genes significantly enriched in the top 20 pathways after U87 cells were treated with different concentrations of duloxetine for 48 hours. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0015] The biological materials, drugs and experimental methods used in the following examples are as follows: Cells: Human glioblastoma cell lines U87 and U251 were provided by the Institute of Materia Medica, Chinese Academy of Medical Sciences.

[0016] Drugs: Duloxetine was purchased from MedChemExpress and prepared into a 10 mmol / L duloxetine solution with dimethyl sulfoxide (as a diluent solvent for duloxetine) and stored at -80°C.

[0017] CCK-8 assay: cells were cultured in DMEM medium containing 10% (v / v) fetal bovine serum and antibiotics (100 μg / ml penicillin-streptomycin) and stored in a 37°C, 5% CO2 incubator. After trypsinization, cells were counted using a cell counting plate. 100 μL of cell suspension (3×10 3 U87 and U251 cells were inoculated into 96-well plates and placed in an incubator (37°C, 5% CO2) for overnight culture. The original culture medium was discarded, and DMEM culture medium with different concentrations of duloxetine (0-100μM) was added to each well and incubated in the incubator for 24h, 48h and 72h. The culture medium was discarded, and 10μl CCK-8 solution was added to each well. After continuing to incubate in the incubator for 0.5~1h, the absorbance was detected at 450nm using an enzyme reader to calculate the IC of duloxetine. 50 value.

[0018] Transwell experiment: The effect of duloxetine on the migration and invasion ability of U87 and U251 cells was evaluated by Transwell experiment. In the invasion experiment, pre-cooled Matrigel and serum-free DMEM medium were diluted in a ratio of 1:7, and 60 μL of diluted Matrigel was added vertically to each chamber, and the chamber was placed in an incubator (37°C, 5% CO2) for 4 hours to allow the Matrigel to polymerize into a thin film. After incubation, 100 µL of serum-free medium was added to each well and placed in the incubator for 30 minutes. This step is not required for the migration experiment. U87 and U251 cells were digested with trypsin, centrifuged and the culture medium was discarded, washed once with PBS, and resuspended in serum-free DMEM medium. Add 200 μL of cell suspension (density of 4×10 4 / ml). Incubate in the incubator for 4 hours to allow the cells to adhere to the bottom of the chamber. Replace the culture medium in the chamber with 200μL of serum-free DMEM containing different concentrations of duloxetine (0, 10, 20, 30μM), add 1mL of DMEM medium containing 10% FBS to the chamber and incubate in the incubator. After incubation for 19 hours in the migration experiment and 24 hours in the invasion experiment, take out the chamber and wipe with a cotton swab to remove the culture medium. Fix the chamber in 4% paraformaldehyde for 15 minutes, wash with phosphate buffered solution (PBS), stain with 0.1% crystal violet for 30 minutes, wash with PBS, wipe with a cotton swab, and dry, and take pictures under a microscope.

[0019] RNA extraction and transcriptome sequencing: RNA-seq detection of gene expression changes: 5 ml cell suspension (5 × 10 6 U87 cells were seeded in 6 cm culture dishes (100 μM, 20 μM, 30 μM duloxetine) and treated with 0, 20, and 30 μM duloxetine for 48 h. The cells were broken with Trizol reagent, and total RNA was collected and stored at -80 °C. The samples were sent to Beijing Yikebaid Technology Co., Ltd. for RNA sequencing. The RNA quality was verified by Nanodrop and Agilent 2100, and the library was established using NEBNext MultiplexOligos. The library was standardized, mixed, and sequenced using the Illumina Novaseq 6000 sequencing platform.

[0020] Example 1 (1) The survival rates of tumor cells after different concentrations of duloxetine were applied to U87 and U251 cells for 24, 48 and 72 hours, respectively. Figure 1 A, B and Table 1, Table 2. Specifically, Figure 1A and B in the figure respectively show the changes in cell viability of U87 and U251 cells after 24 hours of action of duloxetine at different concentrations, and Tables 1 and 2 list the cell inhibition rates of U87 and U251 cells at different dosages and at different times.

[0021] Table 1 The cell inhibition rate of U87 cells at different dosages and time of duloxetine Table 2 The cell inhibition rate of U251 cells at different dosages of duloxetine at different times It can be seen that duloxetine inhibits the proliferation of glioblastoma U87 and U251 cells in a concentration- and time-dependent manner, and the half inhibitory concentration (IC 50 ) are 20.64 μM, 14.08 μM, 12.01 μM and 34.20 μM, 24.45 μM, 23.64 μM respectively.

[0022] (2) After different concentrations of duloxetine were applied to U87 and U251 cells for 24 hours, the changes in cell migration and invasion abilities were shown in Figure 3 .

[0023] These results suggest that duloxetine inhibits the migration and invasion abilities of U87 and U251 cells in a concentration-dependent manner.

[0024] (3) RNA was extracted and sequenced from U87 cells treated with 0 μM (control group), 20 μM, and 30 μM duloxetine for 48 h. The differentially expressed genes were Figure 4 , 5 , 6, and 7. Compared with control cells, there were 2069 upregulated genes and 1676 downregulated genes in cells treated with 30 μM duloxetine (|log2(FoldChange)|>1 and qvalue(padj)<0.005). KEGG pathway enrichment analysis showed that autophagy, mitophagy, and protein processing in the endoplasmic reticulum were enhanced, while cell cycle, DNA replication, and focal adhesion were weakened, indicating that duloxetine exerts an anti-glioblastoma effect by intervening in the autophagy, mitophagy, and cell cycle signaling pathways of U87 cells.

[0025] The above embodiments are only specific implementation methods of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the field should understand that any technician familiar with the technical field can still modify or improve the technical solutions recorded in the above embodiments within the technical scope disclosed by the present invention, or replace some of the technical features therein by equivalents; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. Use of duloxetine as shown in formula I in the preparation of a drug for preventing or treating glioblastoma, Formula I.

2. The use of duloxetine according to claim 1 in the preparation of a drug for preventing or treating glioblastoma, characterized in that: In the drug for preventing or treating glioblastoma, the effective concentration of duloxetine is 1 μM to 100 μM.

3. The use of duloxetine according to claim 1 in the preparation of a drug for preventing or treating glioblastoma, characterized in that: In the drug for preventing or treating glioblastoma, the effective concentration of duloxetine is 5 μM to 60 μM.

4. The use of duloxetine according to claim 1 in the preparation of a drug for preventing or treating glioblastoma, characterized in that: The drug for preventing or treating glioblastoma inhibits the proliferation activity of glioblastoma cells, reduces the migration and invasion ability of glioblastoma cells, and intervenes in the autophagy and cell cycle pathways in tumor cells.

5. The use of duloxetine according to claim 1 in the preparation of a drug for preventing or treating glioblastoma, characterized in that: The drug for preventing or treating glioblastoma comprises an effective dose of duloxetine and pharmaceutically acceptable excipients and / or carriers.

Images