Application of deoxyelephantopin in antitumor drugs

Abstract

The invention discloses an application of deoxyelephantopin in an antitumor drug. In particular to application of deoxyelephantopin or pharmaceutically acceptable salt thereof in preparation of drugs for resisting nervous system tumors, and the nervous system tumors comprise at least one of glioma, meningioma, medulloblastoma, tumor in a pineal body region, pituitary adenoma, neurofibroma, neurosheath tumor and parangganglioma. The glioma comprises at least one of astrocytoma, oligodendroglia and ventricular canal membrane tumor. The application of deoxyelephantopin can inhibit proliferation and invasion of glioma cells through a multi-target path and induce glioma cell apoptosis. In animal model experiments, it is found that deoxyelephantopin can inhibit growth of glioma in nude mice, has little toxic and side effects on the liver and kidney of nude mice, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to a new anti-tumor application of a traditional Chinese medicine component, and in particular to an application of deoxycholate in an anti-glioma drug. Background Art

[0002] Gliomas account for approximately 30% of all primary brain tumors and 80% of malignant brain tumors and are thought to develop from neural stem cells or progenitor cells that carry tumor-initiating genetic changes. According to the World Health Organization (WHO) grading system, they are divided into four histopathological grades, named I-IV. The least malignant of these is pilocytic astrocytoma, with a survival period of approximately 5-10 years (WHO grade I). The most malignant and most common (57.3%) is glioblastoma (GBM) (WHO grade IV). Due to its highly invasive nature, rapid progression, and high recurrence rate, the survival period after diagnosis of GBM is approximately 15 months, and the 5-year survival rate is only 6.8%.

[0003] At present, the treatment of glioma mainly includes surgical resection, temozolomide chemotherapy, radiotherapy, and targeted therapy. Although the current treatment can prolong the survival and prognosis of glioma patients to a certain extent. However, because glioma, especially GBM, is very easy to recur and develops drug resistance after secondary treatment, leading to faster progression, these treatments are generally unsatisfactory. Therefore, there is an urgent need to research and develop new treatment methods or find more effective anticancer drugs to improve patients' prognosis and increase survival.

[0004] Plant-derived natural compound monomers are currently one of the important means of cancer prevention and treatment. They are widely recognized for their multi-target anti-cancer characteristics. Deoxycholesterol is derived from the Asteraceae plant Euphorbia elegans. Euphorbia elegans has the effects of clearing away heat and detoxification, anti-inflammatory and anti-tumor.

[0005] Chinese patent CN101637468A discloses the use of isodeoxycholic acid in the preparation of anti-angiogenic drugs, and experimental results confirm that isodeoxycholic acid has the effect of inhibiting the proliferation and migration of human umbilical vein endothelial cells, thereby exerting an anti-angiogenic effect. However, it is not disclosed whether isodeoxycholic acid or its analogs have a direct inhibitory effect on tumor cell proliferation and migration. There is no report on the use of deoxycholic acid as an analog of isodeoxycholic acid to treat gliomas. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide an application of deoxycholate or a pharmaceutically acceptable salt thereof in the preparation of a drug against nervous system tumors, so as to solve the problem of how to treat nervous system tumors.

[0007] Technical solution: Use of deoxycholesterol or its pharmaceutically acceptable salt described in the present invention in the preparation of drugs for treating nervous system tumors.

[0008] Preferably, the nervous system tumor includes at least one of glioma, meningioma, medulloblastoma, pineal region tumor, pituitary adenoma, neurofibroma, neurilemmoma, and paraganglioma.

[0009] Preferably, the glioma includes at least one of astrocytoma, oligodendroglioma, and ependymoma.

[0010] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0011] Preferably, the dosage form of the drug includes at least one of tablets, capsules, granules, powders, solutions, suspensions, injections, powder injections, aerosols, and implants.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0013] 1. The present invention first discovered that deoxycholate can inhibit the proliferation and invasion of glioma cells through a multi-target pathway (EGFR / JUN) and induce glioma cell apoptosis.

[0014] 2. In animal model experiments, the present invention found that deoxycholate can inhibit the growth of gliomas in nude mice, and immunohistochemical experiments found that deoxycholate had little toxic side effects on the liver and kidneys of nude mice, indicating that deoxycholate is safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the structural formula of deoxycholate;

[0016] Figure 2 Results of tumor cell viability test;

[0017] Figure 3 The effect of deoxycholate on the proliferation of glioma cells;

[0018] Figure 4 The effect of deoxycholate on apoptosis of glioma cells;

[0019] Figure 5 The effect of deoxycholate on the invasion of glioma cells;

[0020] Figure 6 These are the results of animal experiments on the anti-glioma effect of deoxycholate;

[0021] Figure 7 The results of the liver and kidney toxicity test of deoxycholate on nude mice;

[0022] Figure 8 The results are a comparison of the inhibitory effects of deoxycholate on different tumor cells;

[0023] Fig. 9 The results are a comparison of the inhibitory effects of deoxycholate and its analogs on glioma cells;

[0024] Fig.10 These are the WB experimental results of the target sites of deoxycholate. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0026] Example 1: Deoxycholesterol was used to carry out anti-tumor cell experiments, the method is as follows:

[0027] like Figure 1 As shown, deoxycholesterol (CAS: 29307-03-7, purity ≥ 99%) was purchased from MCE Company (China), and deoxycholesterol was dissolved in DMSO for storage. The purchased product was verified by liquid chromatography, mass spectrometry and nuclear magnetic resonance detection. In the present invention, DET is used to represent deoxycholesterol.

[0028] Cell viability assessment: Glioma U87 cells were seeded on a 96-well plate at 5,000 cells per well. After the glioma cells adhered to the plate, they were treated with different final concentrations (5, 10, 20, and 40 μM) of deoxycholate. After 24 and 48 hours, the cell viability was determined using a CCK-8 kit, and the absorbance was measured at 450 nm using an ELISA reader. The results are shown in Table 1. Figure 2 As shown, Figure 2 It was shown that deoxycholate had a significant inhibitory effect on the growth of gliomas in a dose-dependent manner.

[0029] EDU experiment: U87 glioma cells were inoculated in a 96-well plate (8000 cells / well) and cultured to the normal stage, and incubated with deoxycholate (0, 10 and 20 μM) for 24 hours. Then, the thawed EDU dilution ratio was added to the deoxycholate-treated cell culture medium for labeling for 2 hours. After removing the waste liquid, the cells were fixed for 30 minutes, and then the click reaction solution was added, and incubated at room temperature in the dark for 20 minutes. Finally, after washing with washing solution 3 times (3 minutes / time), 5 μg / mL Hoechst33342 solution was added to each well and incubated at room temperature in the dark for 15 minutes. After completion, it was detected using a fluorescence microscope. The results are as follows Figure 3 As shown, Figure 3 It was shown that deoxycholate could inhibit the proliferation of glioma cells in a dose-dependent manner.

[0030] Apoptosis assessment: Glioma U87 cells were seeded on 6-well plates at 50,000 cells per well. After the cells adhered to the plate, 10 μM and 20 μM deoxycholate were added for 24 h. The number of apoptotic cells was measured by flow cytometry using the Annexin V-FITC / PI apoptosis detection kit. The control group was added with an equal amount of solvent DMSO as a blank control. The results are shown in Figure 2. Figure 4 As shown, Figure 4 It was shown that deoxycholate could promote the apoptosis of glioma cells in a dose-dependent manner.

[0031] Cell invasion assay: After treating U87 glioma cells with different concentrations of DET (10 μM and 20 μM), the glioma cell line U87 treated with different concentrations of DET was seeded in the upper compartment containing 300 μL of serum-free medium at 20,000 cells per well. 650 μL of culture medium containing 10% serum was placed in the lower chamber. 100 μL of matrix gel was added to each Transwell well in advance. After incubation for twenty-four hours under conventional cell culture conditions, the cells were fixed with 4% paraformaldehyde for 30 minutes. The number of invading glioma cells was quantified by taking pictures of the cells using a fluorescence microscope. The results are shown in Figure 2. Figure 5 As shown, Figure 5 It showed that deoxycholate could significantly inhibit the invasion and metastasis of glioma cells in a dose-dependent manner.

[0032] Example 2: Anti-tumor animal experiment using deoxycholate, the method is as follows:

[0033] Establishment and evaluation of glioma xenograft model: The experimental animals were obtained from the 83 Experimental Animal Center of Hangzhou Medical College. Four-week-old mice were kept in an 82 specific pathogen-free (SPF) environment. 7 U87-MG cells were subcutaneously injected into the left lower abdomen of each mouse at an inoculation dose of 100 cells / mouse to establish a glioblastoma xenograft model.

[0034] The mice were then divided into two groups, 5 in each group. The experimental group was given an intraperitoneal injection of deoxycholate (dosage 10 mg / kg, every other day) at week 3 after tumor cell inoculation, while the control group was given a saline injection. The longest and shortest tumor diameters were measured every three days for 18 days to track tumor growth. After being sacrificed on the 18th day, the tumors, livers, and kidneys were removed from the mice, weighed, and subjected to immunohistochemistry experiments. The results are shown in Figure 2. Figure 6 and Figure 7 As shown, Figure 6 and Figure 7 It was shown that deoxycholate could significantly inhibit the proliferation of gliomas in nude mice and had little toxicity to the liver and kidneys of nude mice.

[0035] Example 3: Inhibitory effect of deoxycholate on different tumor cells:

[0036] Glioma cells U87, ovarian cancer cells CAOV-3, and gastric cancer cells MKN-45 were seeded on 96-well plates at 5000 cells per well. After the cells adhered to the plate, they were treated with 10 μM deoxycholate. Cell viability was measured after 48 hours. The control group was glioma cells U87 without drug administration. Figure 8 As shown, Figure 8 It was shown that deoxycholic acid only has a good inhibitory effect on glioma cells, while other tumor cells such as gastric cancer and ovarian cancer are not sensitive to deoxycholic acid, and deoxycholic acid cannot effectively inhibit the growth of these cells. This shows that the anti-tumor effect of deoxycholic acid is specific.

[0037] Example 4: Comparison of antitumor effects of deoxycholate and its analogs:

[0038] Glioma cells U87 were seeded on 96-well plates at 5000 cells per well. After the cells adhered to the plate, they were treated with 10 μM deoxycholic acid and 10 μM isodeoxycholic acid (CAS: 38927-54-7). Cell viability was measured after 48 hours. The results are shown in Fig. 9 As shown, the Control group is glioma cells U87 without drug administration. Fig. 9 IDET represents the treatment with isodeoxyideoxytetracycline, and DET represents the treatment with deoxyideoxytetracycline. Fig. 9 It was shown that deoxycholate had a significant inhibitory effect on glioma cells, while isodeoxycholate had no direct inhibitory effect on tumor cell viability.

[0039] Example 5: Study on the anti-glioma mechanism of deoxycholate:

[0040] Glioma cells U87 were seeded on a 6-well plate at 50,000 cells per well. After the glioma cells adhered to the plate, they were treated with deoxycholesterol at a final concentration of 0 or 20 μM. After 24 hours, the cells were collected, lysed with cell lysis buffer, and centrifuged to obtain the supernatant for WB experiments. The results are shown in Fig.10 As shown, Fig.10 It was shown that deoxycholate could inhibit the proliferation and invasion of glioma cells and induce apoptosis of glioma cells through a multi-target pathway (EGFR / JUN).

Claims

1. Use of deoxycholate or its pharmaceutically acceptable salt in the preparation of drugs for treating nervous system tumors.

2. The use according to claim 1, characterized in that: The nervous system tumor includes at least one of glioma, meningioma, medulloblastoma, pineal region tumor, pituitary adenoma, neurofibroma, neurilemmoma, and paraganglioma.

3. The use according to claim 2, characterized in that: The glioma includes at least one of astrocytoma, oligodendroglioma, and ependymoma.

4. The use according to claim 1, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

5. The use according to claim 1, characterized in that: The dosage form of the drug includes at least one of tablets, capsules, granules, powders, solutions, suspensions, injections, powder injections, aerosols, and implants.

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