Application of cefathiamidine in preparation of antitumor drugs

Abstract

The invention relates to a new application of cefathiamidine in preparation of antitumor drugs, in particular to an application in preparation of drugs for resisting lung cancer, esophageal cancer, breast cancer and gastric cancer. Experimental results show that the compound has a remarkable inhibition effect on cell proliferation of lung cancer cell lines A549 and H460, an esophageal cancer cell line KYSE450, a breast cancer cell line BT549 and a gastric cancer cell line BGC803, and flat plate cloning formation and soft agar cloning formation of the lung cancer cell lines A549 and H460; the cell cycle G2 phase retardation of lung cancer cell lines A549 and H460 is promoted. Therefore, the cefathiamidine can be used as a lead compound for developing a new anti-tumor drug, and can also be used for preparing anti-tumor drugs, especially anti-lung cancer, anti-esophageal cancer, anti-breast cancer and anti-gastric cancer drugs, and a new source is provided for seeking the anti-lung cancer, anti-esophageal cancer, anti-breast cancer and anti-gastric cancer drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical application technology, specifically relating to the application of cefotaxime (cephalosporin 18) in the preparation of antitumor drugs. Background Technology

[0002] Cefoperazone, a first-generation cephalosporin antibiotic, can be used to treat respiratory, hepatobiliary, ENT, and urinary tract infections, as well as endocarditis and sepsis caused by susceptible bacteria. For a long time, it has primarily been used to treat bacterial infections such as respiratory and genitourinary tract infections caused by susceptible bacteria. Its mechanism of action mainly involves inhibiting bacterial cell wall synthesis to achieve a bactericidal effect. The molecular formula of cefoperazone is C6H2O. 19 H 28 The structural formula of cefotaxime (N4O6S2, molecular weight 472.58) is shown below: .

[0003] The blood concentration of cefotiam is positively correlated with the dose. The product information states that a 1g intravenous infusion at the standard dose can achieve a peak blood concentration of (146.04±14.53) μmol / L, effectively covering the minimum inhibitory concentration (MIC) of most susceptible bacteria. However, there are no existing literature reports on its antitumor activity, and related studies have not established a link between it and anticancer treatment; its clinical application has remained limited to the field of antibacterial therapy.

[0004] Based on this, this application was developed. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects of the prior art and provide a new application of cefotiam in the preparation of antitumor drugs. That is, this invention discovers a new use of cefotiam in antitumor, aiming to break through the traditional antibacterial application of cefotiam, explore its potential antitumor activity, and expand its medicinal value.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides the application of cefotaxime in the preparation of antitumor drugs.

[0007] Specifically, the above-mentioned application refers to the use of cefotiam in the preparation of anticancer drugs.

[0008] Furthermore, the aforementioned applications refer to the use of cefotiam in the preparation of drugs for treating lung cancer, esophageal cancer, breast cancer, or gastric cancer.

[0009] Furthermore, in the above applications, cefoperazone can inhibit the proliferation of lung cancer, esophageal cancer, breast cancer, or gastric cancer cell lines.

[0010] Furthermore, in the above applications, cefoperazone can inhibit the formation of lung cancer plate colonies and soft agar colonies, promote G2 phase arrest of lung cancer cell lines, and inhibit AKT1 / 2 phosphatase activity.

[0011] When cefotiam is used in the preparation of antitumor drugs, the dosage of cefotiam is 40-160 μM, preferably 80 μM.

[0012] This invention discloses the application of cefotiam in the preparation of antitumor drugs, particularly in the preparation of drugs for treating lung cancer, esophageal cancer, breast cancer, and gastric cancer. Cytotoxicity experiments and cell cycle analysis verified the significant inhibitory effect of cefotiam on lung cancer cells, esophageal cancer cells, breast cancer cells, and gastric cancer cells.

[0013] The present invention was verified using the following experimental methods: 1) MTT assay: By detecting the activity of intracellular mitochondrial enzymes (succinate dehydrogenase), the cell proliferation capacity is indirectly reflected. The cytotoxicity of lung cancer cell lines A549 and H460, esophageal cancer cell line KYSE450, breast cancer cell line BT549, and gastric cancer cell line BGC803 is evaluated. 2) Plate colony formation assay: to evaluate the inhibitory effect of cefoperazone on colony formation of lung cancer cells under adherent conditions; 3) Soft agar cloning assay: to evaluate the ability of cefoperazone to inhibit the malignant proliferation of lung cancer cells under non-adhesion-dependent conditions; 4) Cell flow cytometry: to analyze the effect of cefoperazone on the cell cycle of lung cancer cells.

[0014] The experimental results of this invention show that: In the MTT assay, cefoperazone showed concentration-dependent inhibitory effects on the proliferation of lung cancer cell lines A549 and H460, esophageal cancer cell line KYSE450, breast cancer cell line BT549, and gastric cancer cell line BGC803. In plate colony experiments, the number of cell colonies formed after treatment with cefotaxime was significantly reduced; In the soft agar assay, lung cancer cell colony formation was significantly inhibited, suggesting that it has an inhibitory effect on tumor cell growth in a non-dependent manner. In flow cytometry cell cycle analysis, cefoperazone significantly increased the proportion of lung cancer cells in the G2 phase, suggesting that it inhibits cell division by inducing G2 phase arrest. In in vitro kinase assays, cefotiamidine inhibited the kinase activity of AKT1 / 2.

[0015] In summary, cefotiam has a significant inhibitory effect on the proliferation and colony formation of lung cancer cells, and its anti-cancer mechanism may be related to the inhibition of AKT1 / 2 kinase activity and cell cycle regulation.

[0016] The objective of this invention is to provide a novel use of cefotiam in the preparation of antitumor drugs, particularly in the application of anti-lung cancer, anti-esophageal cancer, anti-breast cancer, or anti-gastric drugs. This aims to provide new ideas, new pathways, and new lead compounds for the research and development of antitumor drugs, solve the technical problems of high toxicity, easy drug resistance, and poor applicability of existing anticancer drugs, and meet the urgent clinical demand for safe and effective antitumor drugs.

[0017] Compared with the prior art, the present invention has the following significant advantages and beneficial effects: 1. Highly innovative: This is the first discovery that cefotaxime has antitumor activity, breaking through the boundaries of its antibacterial drug application and giving existing drugs new uses; 2. Low R&D risk: Cefoperazone is an antibiotic that has been on the market for many years. Its toxicology, safety and pharmacokinetic characteristics are well-defined, which is conducive to its rapid conversion into an anti-tumor drug. Attached Figure Description

[0018] Figure 1 The purpose of this study was to detect the effect of cefoperazone on the proliferation of lung cancer (cell lines H460, A549), esophageal cancer (cell line KYSE450), breast cancer (cell line BT549), and gastric cancer (cell line BGC803) using the MTT assay. Figure 2 The effects of cefotaxime on colony formation in lung cancer cell lines were investigated. A. Inhibition of colony formation in lung cancer cell line H460 after treatment with cefotaxime at 40 μM, 80 μM, and 160 μM, respectively. B. Inhibition of colony formation in lung cancer cell line A549 after treatment with cefotaxime at 40 μM, 80 μM, and 160 μM, respectively. Figure 3 The effects of cefotaxime on soft agar colony formation in lung cancer cell lines were investigated. A. Inhibition of soft agar colony formation in lung cancer cell line H460 after treatment with cefotaxime at 40 μM, 80 μM, and 160 μM, respectively. B. Inhibition of soft agar colony formation in lung cancer cell line A549 after treatment with cefotaxime at 40 μM, 80 μM, and 160 μM, respectively. Figure 4The effects of cefotaxime on the cell cycle of lung cancer cell lines: A. Effects of cefotaxime treatment at 40 μM, 80 μM, and 160 μM on the cell cycle in lung cancer cell line H460; B. Effects of cefotaxime treatment at 40 μM, 80 μM, and 160 μM on the cell cycle in lung cancer cell line A549. Figure 5 To detect the effect of cefotaxime on the kinase activity of AKT1 / 2 in an in vitro kinase assay: A. Effect of treatment with 40 μM and 80 μM cefotaxime on the kinase activity of AKT1; B. Effect of treatment with 40 μM and 80 μM cefotaxime on the kinase activity of AKT2; The gray values ​​of the immunoblot bands analyzed using ImageJ software are marked below the corresponding positions in the images. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0020] For any experimental procedures not detailed or described in the following experiments, conventional techniques in this field can be used. 1. Materials

[0021] 1.1 Reagents Cefoperazone was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number: S26179; purity ≥97%; Human lung cancer cell lines H460 and A549, esophageal cancer cell line KYSE450, breast cancer cell line BT549, and gastric cancer cell line BGC803 were purchased from ATCC. RPMI 1640 culture medium was purchased from Biological Industries (BI) in Israel. Penicillin / streptomycin, trypsin digestion solution, 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT), crystal violet, Triton-100, and propidium iodide (PI) solution were purchased from Beijing Solarbio Co., Ltd. AKT1 / 2 kinase and GSK3b protein were purchased from Sinocare. Adenosine triphosphate (ATP) was purchased from MCE; The soft agar gel was purchased from BD Biosciences.

[0022] 1.2 Instruments and Equipment: ELISA reader (BD company); Inverted microscope (OLYPUMS); Haier Medical Low Temperature Storage Box (Qingdao Haier Special Electric Appliances Co., Ltd.); Analytical balance (Mettler-Toledo Instruments Shanghai Co., Ltd.); Thermo Clean Bench; IVC System (Suzhou Fengshi Experimental Animal Equipment Co., Ltd.) Pipettes, pipettes (5 ml, 10 ml, 25 ml), centrifuge tubes (15 ml / 50 ml); 6-well plates, 96-well plates (Corning). Electronic balance (Mettler-Toledo Instruments Shanghai Co., Ltd.); Haier Medical Low Temperature Storage Box (Qingdao Haier Special Electric Appliances Co., Ltd.); Flow cytometer (Agilent).

[0023] 2. Experimental Procedure: 2.1 Detection of the effects of cefoperazone on the proliferation of lung cancer, esophageal cancer, breast cancer, and gastric cancer cell lines: First, cefotaxime powder was prepared into a 200 mM cefotaxime stock solution using dimethyl sulfoxide. Lung cancer cell lines H460 and A549, esophageal cancer cell line KYSE450, breast cancer cell line BT549, and gastric cancer cell line BGC803 were seeded into 96-well plates at 2000 cells per well and cultured at 37°C for 24 hours in a 5% CO2 cell culture incubator (complete culture medium consisted of RPMI 1640 + 10% fetal bovine serum + 1% penicillin / streptomycin). Then, 0, 40, 80, and 160 μM cefotaxime stock solutions were added, and the cells were cultured for 48 hours. Afterward, 20 ml of 5 mg / ml MTT solution was added to each well, and the cells were incubated at 37°C for 2 hours. The OD value at 570 nm was measured using a microplate reader. The results are shown below. Figure 1 .

[0024] The results showed that, compared with the control group without cefoperazone, three repeated assays showed that 40, 80, and 160 μM cefoperazone significantly inhibited the proliferation of lung cancer cells H460 and A549, as well as esophageal cancer cells KYSE450, breast cancer cells BT549, and gastric cancer cells BGC803. (See details below.) Figure 1 .

[0025] 2.2 Detection of the effect of cefotaxime on colony formation of lung cancer cell lines on plate: First, lung cancer cell lines H460 and A549 were seeded in 6-well plates at 800 cells per well and cultured at 37°C for 24 hours in a 5% CO2 incubator (complete culture medium consisted of RPMI 1640 + 10% fetal bovine serum + 1% penicillin / streptomycin). Then, 0, 40, 80, and 160 μM cefoperazone stock solution were added, and the cells were cultured for 7 days. After each well, the cells were washed once with PBS, fixed with pre-cooled methanol at 4°C for 10 minutes, stained with 1 ml of 0.4% crystal violet for 20 minutes, washed with PBS, air-dried, and photographed. The data were then statistically analyzed. Results are shown below. Figure 2 .

[0026] The results showed that, compared with the control group without cefotaxime, 40, 80, and 160 μM cefotaxime significantly inhibited the formation of plate colonies of lung cancer cells H460 and A549. (See attached results for details.) Figure 2 .

[0027] 2.3 Detection of the effect of cefoperazone on soft agar colony formation of lung cancer cell lines: First, prepare 0.6% and 1.2% soft agar gels (BD Biosciences, 214010) and autoclave them. After melting 1.2% and 0.6% soft agar gels by heating, the mixture was placed in a 43°C water bath. 40 ml of 2×RPMI 1640 medium was mixed with 20 mg / ml gentamicin and 10 ml fetal bovine serum (FBS). 0, 40, 80, and 160 μM cefoperazone stock solution were added, and the mixture was aliquoted into 5 ml tubes. 5 ml of 1.2% soft agar gel was added to each tube, and the mixture was thoroughly mixed. 3 ml of the mixture was then spread into each well of a 6-well plate and incubated at room temperature for 1 hour. Subsequently, lung cancer cell lines H460 and A549 were digested with 1 ml of 0.25% trypsin, followed by termination of digestion with 3 ml of complete medium (RPMI 1640 + 10% FBS + 1% penicillin / streptomycin). The cells were centrifuged at 1000 rpm for 3 min at room temperature, resuspended in complete medium, and counted. Cells were counted at 8000 cells per well, with 3 replicates per sample, for a total count of 4×10⁻⁶ cells. 4 Prepare 5 replicates per cell.

[0028] Next, 40,000 cells were added to culture medium containing 0, 40, 80, and 160 μM cefotaxime (40 ml 2×RPMI 1640 medium with 20 mg / ml gentamicin and 10 ml FBS) to a final volume of 2.5 ml. 2.5 ml of 0.6% soft agar gel was added and mixed thoroughly. 1 ml of the mixture was then seeded into each well of a 6-well plate and incubated at room temperature for 1 hour before being transferred to a 37°C incubator for 10 days. Finally, four random fields of view from each well were photographed using a fluorescence inverted microscope, and the colony count was determined using IPP 6.0. Differences between groups were analyzed using statistical methods. Results are shown below. Figure 3 .

[0029] The results showed that, compared with the control group without cefotaxime, 40, 80, and 160 μM cefotaxime significantly inhibited soft agar colony formation in H460 and A549 cells. (See details below.) Figure 3 Images of soft clone formation and statistical analysis results.

[0030] 2.4 Detection of the effect of cefoperazone on the cell cycle of lung cancer cell lines: First, lung cancer cells H460 and A549 were seeded in 6-well plates at 1200 cells per well and cultured at 37°C for 24 hours in a 5% CO2 incubator (complete culture medium consisted of RPMI 1640 + 10% fetal bovine serum + 1% penicillin / streptomycin). Then, 0, 40, 80, and 160 μM cefoperazone stock solution were added, and the cells were cultured for 48 hours. After washing each well with PBS, the cells were collected and resuspended in 70% ethanol with 30% PBS. The cells were incubated at -20°C for 24 hours, and the supernatant was discarded. 250 μl of 0.6% Triton X-100 and 5 μl of 200 mg / ml RNase A were added, respectively, and the cells were incubated at room temperature for 1 hour. Then, 5 μl of 20 μg / ml PI solution was added, and the cells were incubated at 4°C in the dark for 15 minutes. Cell cycle analysis was performed using flow cytometry. Results are shown below. Figure 4 .

[0031] Cell cycle analysis included the number of cells in G1, G2, and S phases. Results showed that cefotaxime treatment significantly promoted G2 phase arrest in H460 and A549 cells. (See details...) Figure 4 .

[0032] 2.5 In vitro kinase assay to detect the effect of cefotiam on AKT1 / 2 kinase activity: First, a blank control (without AKT1 / 2 kinase), 0 μM, 40 μM, and 80 μM cefoperazone stock solutions, and 50 ng of AKT1 / 2 kinase were reacted with the cefoperazone solution at room temperature for 15 minutes. After the reaction, adenosine triphosphate (ATP) to a final concentration of 25 μM and 100 ng of GSK3b substrate protein were added sequentially, and the mixture was reacted at 30°C for 30 minutes. The reaction mixture was then subjected to high-temperature denaturation at 95°C for 5 minutes using SDS-PAGE loading buffer. Finally, Western blotting was used to verify whether cefoperazone could inhibit AKT1 / 2 phosphatase activity using a phosphorylated GSK3b antibody. The results are shown below. Figure 5 .

[0033] The results showed that, compared with the control group without cefotaxime, both 40 and 80 μM cefotaxime could inhibit AKT1 / 2 phosphatase activity. See below for details. Figure 5 .

[0034] In summary, the cefotiam described in this invention can inhibit the proliferation and colony formation of non-small cell lung cancer cells; at the same time, cefotiam inhibits AKT1 / 2 phosphatase activity, and this invention is particularly suitable for non-small cell lung cancer.

[0035] In summary, this invention employed the MTT assay to conduct cytotoxic activity experiments on cefoperazone using lung cancer cell lines A549 and H460, esophageal cancer cell line KYSE450, breast cancer cell line BT549, and gastric cancer cell line BGC803; it also used the plate colony method and soft agar method to conduct cytotoxic activity experiments on cefoperazone using lung cancer cell lines A549 and H460; and it used flow cytometry to detect cell cycle activity of cefoperazone. The results showed that cefoperazone significantly inhibited cell proliferation in lung cancer cell lines A549 and H460, esophageal cancer cell line KYSE450, breast cancer cell line BT549, and gastric cancer cell line BGC803, as well as plate colony formation and soft agar colony formation in lung cancer cell lines A549 and H460; and promoted G2 phase arrest in lung cancer cell lines A549 and H460. Therefore, cefotiam can serve as a lead compound for the development of new antitumor drugs, and can also be used to prepare antitumor drugs, especially for lung cancer, esophageal cancer, breast cancer, and gastric cancer. This invention provides a new source for seeking drugs for lung cancer, esophageal cancer, breast cancer, and gastric cancer.

Claims

1. Use of cefathiamidine in the preparation of an anti-tumor drug.

2. The use of cefathiamidine according to claim 1 for the preparation of an antitumor medicament, characterized in that, Use of cefathiamidine in the preparation of an anti-cancer drug.

3. The use of cefathiamidine according to claim 2 for the preparation of an antitumor medicament, characterized in that, Use of cefathiamidine in the preparation of an anti-lung cancer, anti-esophageal cancer, anti-breast cancer, or anti-gastric cancer drug.

4. The use of cefathiamidine according to claim 2 for the preparation of an antitumor medicament, characterized in that, Cefathiamidine can inhibit the proliferation of lung cancer, esophageal cancer, breast cancer, or gastric cancer cell lines.

5. The use of cefathiamidine according to claim 4 for the preparation of antitumor medicaments, characterized in that, Cefathiamidine can inhibit lung cancer plate colony formation and soft agar colony.

6. The use of cefathiamidine according to claim 4 for the preparation of an antitumor medicament, characterized in that, Cefathiamidine can promote lung cancer cell line cell cycle G2 phase arrest.

7. The use of cefathiamidine according to claim 4 for the preparation of antitumor medicaments, characterized in that, Cefathiamidine can inhibit AKT1 / 2 phosphatase activity.

8. The use of cefathiamidine according to claim 2 for the preparation of an antitumor medicament, characterized in that, The dosage of cefathiamidine is 40-160 μM.

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