Application of gallium chelate in preparation of antitumor drugs

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of a gallium chelate in preparation of antitumor drugs. The structural formula of the gallium chelate Ga-cooulibactin E (1) is as shown in the specification, and the structural formula of the gallium chelate Ga-cooulibactin E (1) is as shown in the specification. The Ga-coulibactin E (1) can obviously inhibit proliferation of tumor cells, retard cell cycles, interfere mitochondrial functions and remarkably promote cell apoptosis so as to cause death of the tumor cells, and the Ga-coulibactin E (1) is low in use dosage, can be used as a novel anti-tumor drug, is applied to treatment of tumors, and is simple in medication route and easy to operate.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of a gallium chelate in the preparation of anti-tumor drugs. Background Art

[0002] In 2020, there were 19.29 million new cancer cases and 9.96 million cancer deaths worldwide, of which 4.57 million new cases and 3 million deaths were reported in my country. Cancer has become one of the leading causes of death in the world and a major obstacle to improving human life expectancy. Existing cancer treatments include surgical resection, radiotherapy, chemotherapy, and immunotherapy, but due to the high incidence of cancer, low cure rate, and poor prognosis, the effects of existing treatments are still unsatisfactory. Therefore, it is urgent to develop new treatment strategies to improve the survival rate of cancer patients. In recent years, metal chemotherapy drugs such as cisplatin and oxaliplatin have been widely used in tumor treatment, further promoting the research and development of metal anticancer drugs. Gallium (Ga) ions have been approved by the US Food and Drug Administration (FDA) for use in the medical field. Gallium ions not only have antibacterial, anti-inflammatory, immunomodulatory, anti-hypercalcemia and analgesic activities, but also show anti-tumor potential. As an anticancer drug, gallium compounds have been used in clinical research for a long time. With the development of new gallium preparations, their anticancer effects have become more and more significant. For example, gallium nitrate is an effective treatment for multiple myeloma. Compared with traditional organic small molecule anticancer drugs, gallium chelates have unique reaction and coordination chemical properties at their metal centers, providing a rich mechanism of action for cancer treatment. Therefore, it is of great scientific significance and clinical value to find gallium chelates with significant effects, unique mechanisms, and few toxic side effects as anticancer drugs. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art clinical anti-tumor drugs, such as high dosage, unsatisfactory efficacy, lack of selectivity, drug resistance, and toxic side effects that inhibit the hematopoietic system, digestive system, immune system, nervous system and endocrine system, and to provide an application of a gallium chelate in the preparation of an anti-tumor drug.

[0004] The purpose of the present invention is achieved through the following solutions: A gallium chelate is used in the preparation of an anti-tumor drug, wherein the gallium chelate is Ga-coublibactin E (1), and its structural formula is as follows: .

[0005] The anti-tumor drug is preferably an anti-leukemia drug or an anti-lymphoma drug; The gallium chelate Ga-coublibactin E (1) can be used as a new type of anti-tumor drug for the treatment of tumors; An antitumor drug containing the gallium chelate.

[0006] Principle of the present invention: The essential characteristic of tumor cells is malignant proliferation. Inhibiting tumor cell proliferation, blocking cell cycle and inducing cell apoptosis are key links in curbing tumor growth. The present invention discovers and confirms that Ga-coublibactin E (1) can inhibit tumor cell proliferation and significantly promote tumor cell apoptosis, and the dosage of Ga-coublibactin E (1) is low.

[0007] Compared with the prior art, the present invention has the following advantages and effects: (1) The present invention first discovered that the gallium chelate Ga-coublibactin E (1) can significantly inhibit the proliferation of tumor cells in vitro.

[0008] (2) Ga-coublibactin E (1) causes tumor cell death mainly by blocking the cell cycle, interfering with mitochondrial function, inducing tumor cell apoptosis, and changing the expression of related proteins.

[0009] (3) The dosage of Ga-coublibactin E (1) is relatively low. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is the structural formula of the gallium chelate.

[0011] Figure 2 The results of the anti-tumor activity test of Ga-coublibactin E (1) are analyzed as follows: (a) The effect of different concentrations of compound 1 on the viability of three tumor cells (NB4, Farage and SU-DHL-2) detected by CCK-8 method; (b, c) The effect of compound 1 on the proliferation of NB4 cells (EdU fluorescence staining); (d, e) The effect of compound 1 on the cell cycle distribution of NB4 cells; (f) The effect of compound 1 on the expression level of NB4 cell cycle-related proteins; (g) The effect of compound 1 on the mitochondrial membrane potential of NB4 cells; (h) The effect of compound 1 on the ROS level of NB4 cells; (i, j) The effect of compound 1 on the apoptosis of NB4 cells; (k) The effect of compound 1 on the expression level of apoptosis-related proteins in NB4 cells; (e, f, j) * P <0.05,** P <0.01,*** P <0.001; (f, k) GAPDH was the internal reference protein. DETAILED DESCRIPTION

[0012] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0013] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0014] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0015] In the embodiment, human acute promyelocytic leukemia cell line NB4 and human diffuse large B-cell lymphoma cell lines Farage and SU-DHL-2 were purchased from Wuhan Pronocell Life Science Co., Ltd.; Compound 1 (structural formula: Figure 1 (shown) was prepared from Nocardia coubleae DSM 44960 and derivatized with gallium sulfate.

[0016] Example 1 Antitumor activity test of compound 1 1. Experimental Methods (1) Cell culture and cytotoxic activity assay: Human acute promyelocytic leukemia cells NB4 and human diffuse large B-cell lymphoma cells Farage and SU-DHL-2 were cultured in RPMI-1640 medium containing 10% fetal bovine serum; culture conditions: 37 °C, CO2 volume fraction and 5% saturated humidity in an incubator. The cytotoxic activity of compound 1 was determined by the CCK-8 method. Cells were seeded at a density of 10,000 cells / well on a 96-well plate, and different concentrations of compound 1 (0.625, 1.25, 2.5, 5, 10 and 20 μM) were added and cultured for 48 h, then 10 μl of CCK-8 solution was added to each well and incubated at 37 °C for another 4 h. The absorbance at a wavelength of 490 nm was measured by an enzyme reader. The IC of the compound was calculated using the analysis software GraphPad v.10 50 value.

[0017] (2) EdU experiment: NB4 cells were cultured at 5×10 4 Cells were seeded at a density of 100 cells / well in a 12-well plate, and 2 μM and 4 μM compound 1 and 200 nM doxorubicin (Dox) were added and cultured for 24 h. EdU working solution was added and cultured for another 2 h. After fixation and permeabilization, EdU reaction complex was added and incubated at room temperature in the dark for 30 min. The cell nucleus was counterstained with DAPI, and the fluorescence intensity was detected by confocal microscopy and flow cytometry.

[0018] (3) Cell cycle and apoptosis detection: NB4 cells were cultured at 5×10 4 Cells were seeded at a density of 100 cells / well in a 12-well plate, and 2 μM and 4 μM compound 1 and 200 nM doxorubicin were added and cultured for 48 h. For cell cycle detection, cells were fixed with 70% pre-cooled ethanol, washed with PBS, and incubated with a staining solution containing 50 μg / mL PI, 100 μg / mL RNase, and 0.2% Triton-X100 at 37 °C in the dark for 30 minutes, and detected by flow cytometry. For apoptosis detection, cells were washed with PBS and resuspended in a staining solution containing Annexin V-FITC and PI, incubated at room temperature for 10 minutes, and detected by flow cytometry.

[0019] (4) Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) detection: NB4 cells were cultured at 5×10 4 Cells were seeded at a density of 100 cells / well in a 12-well plate, and 2 μM and 4 μM compound 1 and 200 nM doxorubicin were added and cultured for 24 h. After washing with PBS, fluorescent dye TMRE or DCFH-DA was added, and after incubation at 37 °C for 20 minutes, flow cytometry was used for detection.

[0020] (5) Western blot: NB4 cells were treated with 2 μM, 4 μM compound 1 and 200 nM doxorubicin for 48 h, and proteins were extracted using RIPA lysis buffer containing phosphatase inhibitor cocktail (50x) and PMSF (100x). The protein concentration was determined using the BCA protein concentration assay kit (enhanced), and the proteins were separated by 10% or 15% SDS-PAGE gel electrophoresis, transferred to a 0.2 μm NC membrane, blocked with skim milk at room temperature for 1 h, then incubated with the primary antibody at 4 °C overnight, and then incubated with the secondary antibody at room temperature for 1 h, and finally detected using a near-infrared fluorescence imaging system.

[0021] 2. Experimental results Different concentrations of compound 1 were used to treat NB4, Farage and SU-DHL-2 cells for 48 h, and the CCK-8 method was used to analyze the effect of compound 1 on cell viability. The results showed that as the concentration of compound 1 increased, cell viability gradually decreased. The IC values ​​of compound 1 on the three tumor cell lines NB4, Farage and SU-DHL-2 were 50 The values ​​were 1.35±0.23, 4.32±0.63 and 5.87±0.76 μM ( Figure 2a). NB4 cells are most sensitive to compound 1, so NB4 cells were selected for further study. NB4 cells were treated with 2 μM (low dose, L) and 4 μM (high dose, H) of compound 1 for 24 h, and the cell proliferation rate was analyzed by EdU assay. The results showed that compared with the control group, compound 1 significantly reduced the proliferation rate of NB4 cells ( Figure 2 b and 2c). To investigate whether compound 1 inhibits the growth of NB4 cells by arresting the cell cycle, NB4 cells were treated with different concentrations of compound 1, and then the cell cycle distribution was detected by flow cytometry. The results are shown in Figure 2 As shown in d and 2e, compared with the control group, compound 1 significantly increased the ratio of G0 / G1 phase and decreased the ratio of S phase and G2 / M phase, indicating that compound 1 can block NB4 cells in G0 / G1 phase. Western blot detection results showed ( Figure 2 f) Compared with the control group, the expression levels of cell cycle-related proteins Cyclin D2, CDK2 and CDK6 in NB4 cells treated with compound 1 were reduced, and the expression levels of p-Chk2 and p21 were increased. After 24 h of treatment of NB4 cells with compound 1, the mitochondrial membrane potential of the cells was significantly increased ( Figure 2 g), and reduce ROS levels ( Figure 2 h), indicating that compound 1 caused damage to mitochondrial function in NB4 cells. The results of cell apoptosis experiments showed that ( Figure 2 i and 2j), compared with the control group, compound 1 significantly increased the apoptosis rate of NB4 cells. After 48 h of treatment with 4 μM compound 1, the apoptosis rate of NB4 cells reached about 70%. At the same time, compound 1 dose-dependently increased the expression levels of apoptosis-related proteins cleaved-PARP, Bak, γ-H2A.X and cleaved-caspase 3 / 8 / 9 ( Figure 2 k).

[0022] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A gallium chelate for use in the preparation of an anti-tumor drug, characterized in that: The gallium chelate is Ga-coublibactin E (1), and its structural formula is shown below: 。 2. The use of the gallium chelate according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is an anti-leukemia drug or an anti-lymphoma drug.

3. An anti-tumor drug, characterized in that: Contains the gallium chelate according to claim 1.

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