Application of GABA-A receptor negative regulator in preparation of medicine for treating panthenic cancer

Abstract

The invention provides an application of a GABA-A receptor negative regulator in preparation of a medicine for treating pan cancer, and belongs to the technical field of medicines. The research finds that in the tumor microenvironment of panthenic cancer, the GABA-A receptor negative regulator acts on the key cancer-promoting gene target of cancer cells for drug therapy, abnormal overexpression of the key cancer-promoting gene causing cancer occurrence and development can be controlled, the inhibition effect of the cancer cells on immune cells is reduced, and the cancer-promoting effect of the cancer cells is improved. The medicine can be used for treating cancer cells, so that an immunosuppressive tumor microenvironment beneficial to cancer cell growth is destroyed, immune infiltration of the cancer tumor microenvironment is readjusted, a normal immune system of an organism is activated to treat the cancer cells, and the medicine can also be used for reducing NF-kb signal channels of the cancer cells to reduce inflammatory factor expression of the cancer cells and promote apoptosis of the cancer cells. The GABA-A receptor negative regulator acts on key cancer promoting gene targets of cancer cells, so that accurate and effective targeted therapy can be carried out on cancers on the molecular level.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the application of a GABA-A receptor negative regulator in the preparation of a drug for treating pan-cancer. Background Art

[0002] The incidence of cancer continues to rise worldwide, becoming one of the major public health problems. Although cancer treatment methods have continued to advance in recent years, including surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, some remarkable successes have been achieved, especially in some types of cancer, where survival rates have improved. However, current treatments still face multiple limitations. First, individual responses to treatment vary greatly, and some patients are resistant to existing therapies or have poor treatment effects. Second, the heterogeneity of cancer makes tumor treatment more challenging, and cancer cells in different patients may respond very differently to the same drug. In addition, side effects during treatment, such as nausea, immunosuppression, and fatigue, often affect patients' quality of life, and in some cases, severe side effects may lead to treatment interruption. Therefore, despite important progress in cancer treatment, there is still a need to further explore more effective and more accessible treatment options to improve patients' prognosis and quality of life. Pan-cancer is a new systematic research method across multiple cancer types in the modernization of medicine, which aims to discover common biomarkers, molecular mechanisms, and therapeutic targets through comprehensive analysis of multiple types of cancer genomic data. Pan-cancer research focuses on common features of different types of cancer, such as gene mutations, epigenetic changes, signaling pathways, immune responses, etc., and is committed to providing broader insights into cancer treatment and promoting the development of personalized and precision treatments.

[0003] Cancer immunotherapy is an innovative treatment method that aims to use the patient's own immune system to identify and attack cancer cells. This treatment method mainly includes immune checkpoint inhibitors, CAR-T cell therapy, monoclonal antibodies and vaccines. Immune checkpoint inhibitors enhance the anti-tumor activity of T cells by blocking molecules that inhibit immune responses (such as PD-1 and CTLA-4), and have shown significant efficacy in a variety of cancers such as melanoma, lung cancer and kidney cancer. CAR-T cell therapy genetically modifies the patient's T cells to enable them to specifically recognize and attack tumor cells, especially in certain hematological tumors. In addition, monoclonal antibodies can mark cancer cells to promote recognition and clearance by the immune system, and vaccines are used to prevent cancers caused by certain viruses (such as cervical cancer). Although immunotherapy has shown broad prospects, it still faces some challenges and limitations, such as limited response to treatment in some patients, possible serious immune-related side effects (such as autoimmune reactions), and high treatment costs. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide the use of GABA-A receptor negative regulators in the preparation of drugs for the treatment of pan-cancer. By using GABA-A receptor negative regulators to act on key oncogene targets of cancer cells for drug treatment, a new theoretical basis can be provided for cancer immunotherapy. In controlling the abnormal overexpression of oncogenes of cancer cells, drugs are used to effectively inhibit the expression products of key oncogenes at the oncogene targets of cancer cells in pan-cancer at the molecular level, so as to carry out precise and effective treatment, thereby improving the treatment effect of cancer patients.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] Application of GABA-A receptor negative regulators in the preparation of pan-cancer treatment drugs.

[0007] Preferably, the GABA-A receptor negative regulator includes aconitum biflavone and β-carboline.

[0008] Preferably, the GABA-A receptor negative modulator inhibits cancer cell growth in pan-cancer.

[0009] Preferably, the cancer cells in pan-cancer include human U-87 glioblastoma cells, human Bcap-37 breast cancer cells, human Detroit 562 throat cancer cells, human 143B osteosarcoma cells, human HGC-27 gastric cancer cells, human CW-2 colon cancer cells, human HeLa 229 cervical cancer cells and human Caov-3 ovarian cancer cells.

[0010] Preferably, the GABA-A receptor negative modulator induces apoptosis of cancer cells.

[0011] Preferably, the GABA-A receptor negative modulator reduces NF-κb-mediated anti-apoptotic signaling in glioblastoma.

[0012] Preferably, the active ingredient of the drug includes a GABA-A receptor negative regulator.

[0013] Preferably, the drug is added with pharmaceutically acceptable excipients to prepare powders, decoctions, pills, capsules, tablets and granules.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides that in a pan-cancer tumor microenvironment, by using a GABA-A receptor negative regulator, the abnormal overexpression of key oncogenes that cause cancer occurrence and development can be controlled, thereby destroying the tumor microenvironment that is conducive to the growth of cancer cells, re-regulating the immune infiltration of the cancer tumor microenvironment, activating the body's normal immune system, and regulating the NF-κb signaling pathway of cancer cells to promote apoptosis of cancer cells, and can play a comprehensive therapeutic role on cancer cells in pan-cancer. The GABA-A receptor negative regulator can effectively and accurately immunotherapy the cancer-promoting gene drug treatment targets at the molecular level. . BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The MTT cell proliferation assay was used to observe the effects of different concentrations of flavonoids from Acanthocephalum on the viability of U-87 cells; **p<0.01 indicates the statistical value compared with the blank control group;

[0017] Figure 2 The inhibitory effect of spicate biflavonoids on human U87 glioblastoma in cell experiments, where A is the proportion of cell cycle arrest in the sub-G1 phase; B is the expression level of caspase-3, which is an active protease; **p<0.01 indicates the statistical value compared with the blank control group;

[0018] Figure 3 The figure shows the inhibitory effect of sedum biflavonoids on the growth of human Bcap-37 breast cancer cells, wherein A is the experimental flow chart; B is the effect of sedum biflavonoids on the TNBC tumor volume of mice (n=3); C is the effect of sedum biflavonoids on the TNBC tumor volume of mice (n=6); *p<0.05 represents the statistical value compared with the blank control group, **p<0.01 represents the statistical value compared with the blank control group, ***p<0.001 represents the statistical value compared with the blank control group, and ns represents that there is no significant difference in the statistical value compared with the blank control group. DETAILED DESCRIPTION

[0019] The present invention provides the use of a GABA-A receptor negative regulator in preparing a drug for treating pan-cancer.

[0020] The present invention has found that the GABA-A receptor gene is a key gene for cancer cells to escape immune attack in pan-cancer. The present invention uses a GABA-A receptor negative regulator to control the abnormal expression of key genes that cause cancer, thereby destroying the tumor microenvironment that is conducive to the growth of cancer cells, re-regulating the immune infiltration of the cancer tumor microenvironment, activating the body's normal immune system, and regulating the NF-κb signaling pathway of cancer cells to promote apoptosis of cancer cells. It can play a comprehensive therapeutic role on cancer cells in pan-cancer. The GABA-A receptor negative regulator can effectively and accurately immunotherapy the cancer-promoting target gene drug treatment targets of cancer at the molecular level. .

[0021] In the present invention, the GABA-A receptor negative regulator includes aconitum biflavonoids and β-carboline, both of which act on the oncogene targets of cancer cells to effectively exert anti-tumor therapeutic pharmacological effects, and also have oral active drug monomers. They can be extracted from natural products and are small molecule compounds, which have multiple advantages in cancer treatment, such as multi-target effects, lower side effects, stronger immunomodulatory effects, etc., and their rich sources and strong structural diversity provide a wide range of research and application space for cancer targeted therapy. These advantages make it one of the important directions in modern cancer treatment research.

[0022] The chemical structure of Amentoflavone is shown in Structural Formula 1, molecular formula: C 30 H 18 O 10 , molecular weight: 538.46, can be extracted from the Chinese medicine Selaginella.

[0023] Molecular structure formula 1.

[0024] The chemical structure of the β-carboline (ZDLD20) is shown in Structural Formula 2, molecular formula: C 22 H 22 N6O, molecular weight: 386.45, can be extracted from the traditional Chinese medicine Camel Pecan.

[0025] Molecular structure formula 2.

[0026] In the present invention, the GABA-A receptor negative regulator inhibits the growth of cancer cells, and the GABA-A receptor can target and control the abnormal expression of oncogenes, thereby achieving the effect of inhibiting the growth of cancer cells. Preferably, the cancer cells include pan-cancer cancer cells including human U-87 glioblastoma cells, human Bcap-37 breast cancer cells, human Detroit 562 pharyngeal cancer cells, human 143B osteosarcoma cells, human HGC-27 gastric cancer cells, human CW-2 colon cancer cells, human HeLa 229 cervical cancer cells and human Caov-3 ovarian cancer cells.

[0027] In the present invention, the GABA-A receptor negative regulator induces apoptosis of cancer cells.

[0028] In the present invention, the GABA-A receptor negative regulator can reduce the anti-apoptotic signal regulated by NF-κb of glioblastoma.

[0029] In the present invention, the active ingredient of the drug includes a GABAA receptor negative regulator. The drug of the present invention also includes pharmaceutically acceptable excipients, and different dosage forms are prepared by adding the excipients, including powders, decoctions, pills, capsules, tablets and granules.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example 1

[0032] Effects of GABA-A receptor negative modulators on glioblastoma.

[0033] 1. Test materials:

[0034] Human U-87 glioblastoma cells were purchased from the Cell Bank of the Chinese Academy of Sciences. MTT (thiazolyl blue) was purchased from MCE (MedChemExpress). Propidium iodide (PI) and RNase were purchased from Biovision (Mountainview, CA, USA) and Thermo Fisher Scientific, respectively.

[0035] 2. Test steps:

[0036] (1) Cell culture: Human U-87 glioblastoma cells were cultured in Minimum Essential Medium Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin (10,000 U / mL) and 1% sodium pyruvate (100 mM) at 37° C. with 5% CO 2 .

[0037] (2) Cytotoxicity MTT assay: U-87 cells were cultured in 96-well plates at 2 × 10 4 Cells / well were cultured overnight. Aquifolium flavonoids were dissolved in DMSO (dimethyl sulfoxide) to prepare different concentrations (0-200 μM) of aquifolium flavonoids solutions. After treating cells with aquifolium flavonoids solution for 48 h, the cell viability was detected by MTT method. The absorbance of each well (including blank) was measured at 570 nm using Multiskan GO microplate spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA).

[0038] The results are as follows Figure 1 As shown in the results, sedum biflavonoids induced cytotoxicity in U-87 glioblastoma cells, and the MTT method was used to observe the effect of sedum biflavonoids on the viability of U-87 cells. The results showed that compared with the control group (treated with 0μM sedum biflavonoids), 50-200μM sedum biflavonoids treated for 48h significantly inhibited the viability of U-87MG cells, with an inhibition rate of 23-71%. Under 48h treatment, the half inhibitory concentration (IC 50 ) is 100 μM.

[0039] (3) Flow cytometry experiment: used to detect late stage of cell apoptosis. U-87 cells (5×10 5 ) were inoculated in 6-well plates and treated with 50 or 100 μmol / L of flavonoids for 48 h. The cells were then fixed with 70% ethanol overnight and stained in P / RNase solution for 30 minutes. The P / RNase solution was a phosphate buffered saline (PBS) containing a working concentration of 40 μg / mL PI+100 μg / mL RNase and 1% TritonX-100. Cell samples in the PI / RNase solution were analyzed by flow cytometry, and at least 10,000 single cells were saved in each group. The percentage of cells was measured by FlowJo 7.6.1 software (FlowJo LLC, Ashland, OR, USA).

[0040] The results are as follows Figure 2 As shown in the figure, in U-87 glioblastoma cells, apoptosis induced by aconitum biflavonoids depends on both intrinsic and extrinsic apoptosis signaling pathways. The cell cycle and caspase-3 flow cytometry experimental methods were used to detect and analyze the apoptosis signaling pathway induced by aconitum biflavonoids. Figure 2 Figures A and B show that compared with the control group, spike-flower biflavonoids can significantly induce cell cycle arrest of U87 cells in the sub-G1 phase after 48 hours of treatment of U87 cells, and increase the level of caspase-3, an important execution protease activity in the apoptosis process of U87 cells, thereby promoting apoptosis of U87 cells.

[0041] Cell experiments showed that aconite biflavone could not only induce intrinsic and extrinsic apoptosis of glioblastoma cells, but also reduce the anti-apoptotic signal regulated by NF-κb of glioblastoma in vitro.

[0042] Example 2

[0043] Therapeutic effects of GABAA receptor negative modulators on breast cancer.

[0044] 1. Experimental materials:

[0045] Human Bcap-37 breast cancer cells (TNBC cells) were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM, a basic medium supplemented with 10% FBS and 1% penicillin / streptomycin.

[0046] SPF-grade BALB / cNude female nude mice (16-18 g), 6-8 weeks old, were purchased from Beijing Weitonglihua Laboratory Animal Company.

[0047] 2. Experimental methods:

[0048] The number is (1×10 5 / mL) of human Bcap-37 breast cancer cells and Matrix-Gel produced by Bio-Tech TM The cells were mixed with matrix gel (high concentration, without phenol red, Bio-Sky) and implanted into the mammary fat pad of BALB / c Nude mice to establish a breast cancer model of TNBC cells. The mice were randomly divided into a spicate-flavonoids drug treatment group and a blank drug control group (Control).

[0049] Experiment 1 (n=3): When the breast cancer tumor to be established was 3mm×3mm, the sedum biflavonoids drug treatment group (Treatment Group) was intraperitoneally injected with sedum biflavonoids at a dose of 25 mg / kg. The tumor was measured every other day. After 19 days of the experiment, the mice were humanely euthanized and the tumor size was measured.

[0050] Experiment 2 (n=6): When the breast cancer tumor to be established was 3mm×3mm, the sedum biflavonoids drug treatment group was divided into two groups, and sedum biflavonoids were injected intraperitoneally, treatment group 1: injection dose was 25mg / kg, treatment group 2: injection dose was 50mg / kg. After that, the tumor was measured every other day, and the mice were humanely euthanized after 19 days of the experiment and the tumor size was measured.

[0051] The experimental process is as follows Figure 3 As shown in A, TNBC tumor cells were inoculated into mice on day 0, drug treatment was started on day 7, and tumor volume was measured and analyzed on day 19 and thereafter.

[0052] The results are as follows Figure 3 As shown in B, the X-axis is the number of days relative to treatment (from -2 days to 12 days), and the Y-axis is the tumor volume (mm 3), the red curve is the tumor growth curve of the control group. Control group (Control): black curve, no treatment, rapid tumor growth. Treatment group (Treatment Group): blue curve, drug treatment effectively inhibited tumor growth. After the start of treatment (from day 0), the tumor volume of the treatment group was significantly lower than that of the control group. The results showed that the treatment group had a significant anti-tumor effect compared with the control group.

[0053] The results are as follows Figure 3 As shown in C, the X-axis is the number of days relative to treatment (from -2 days to 12 days), and the Y-axis is the tumor volume (mm 3 ), the red curve is the tumor growth curve of the control group. Control group (Control): black curve, rapid growth of tumor volume. Treatment Group 1 (Treatment Group 1): green curve, tumor growth was inhibited, but the effect was weak, and the statistical marker showed ns (no significant difference) at certain time points, indicating that the treatment effect was not significant. Treatment Group 2 (Treatment Group 2): blue curve, tumor growth was significantly inhibited, and the effect was better than that of Treatment Group 1. Treatment Group 2 showed a stronger anti-tumor effect than Treatment Group 1.

[0054] Animal experiments have shown that the biflavonoids of Achyranthes bidentata can effectively inhibit the growth of the Bcap-37 breast cancer (TNBC) cell tumor model in BALB / c Nude female nude mice.

[0055] The present invention studies the anti-cancer effect of GABA-A receptor negative regulators in pan-cancer and finds that in the pan-cancer tumor microenvironment, the abnormal expression of key oncogenes of cancer cells can be inhibited by using GABA-A receptor negative regulators. In this newly discovered therapeutic target, the abnormal overexpression of key oncogenes that lead to cancer occurrence, development and immune escape can be controlled, and the inhibitory effect of cancer cells on immune cells can be reduced, thereby destroying the immunosuppressive tumor microenvironment that is conducive to the growth of cancer cells, re-regulating the immune infiltration of the cancer tumor microenvironment, activating the body's normal immune system to treat cancer cells, and inhibiting the NF-κb signaling pathway of cancer cells to reduce the release of inflammatory factors of cancer cells and promote apoptosis of cancer cells, and can exert a comprehensive therapeutic effect on cancer cells in pan-cancer. Therefore, the GABA-A receptor negative regulator acts on the target of the key oncogene of cancer cells in pan-cancer, effectively inhibits the expression product of the key oncogene, and can effectively and accurately treat the cancer oncogene targeting at the molecular level.

[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of GABA-A receptor negative regulators in the preparation of pan-cancer drugs.

2. The use according to claim 1, characterized in that: The GABA-A receptor negative regulators include aconitum biflavone and β-carboline.

3. The use according to claim 1, characterized in that: The GABA-A receptor negative modulators inhibit cancer cell growth.

4. The use according to claim 3, characterized in that: The cancer cells in pan-cancer include human U-87 glioblastoma cells, human Bcap-37 breast cancer cells, human Detroit 562 pharyngeal cancer cells, human 143B osteosarcoma cells, human HGC-27 gastric cancer cells, human CW-2 colon cancer cells, human HeLa 229 cervical cancer cells, and human Caov-3 ovarian cancer cells.

5. The use according to claim 1, characterized in that: The GABA-A receptor negative modulator induces apoptosis in cancer cells.

6. The use according to claim 1, characterized in that: The GABA-A receptor negative modulator reduces NF-κB-regulated anti-apoptotic signaling in glioblastoma.

7. The use according to claim 1, characterized in that: The active ingredient of the drug includes a GABA-A receptor negative regulator.

8. The use according to claim 7, characterized in that: The drug is added with pharmaceutically acceptable adjuvants to prepare powders, decoctions, pills, capsules, tablets and granules.

Images