Application of G-749 in preparation of medicine for treating and / or preventing esophageal cancer

Abstract

The invention discloses application of G-749 in preparation of a medicine for treating and / or preventing esophageal cancer, and belongs to the technical field of biological medicine. It is found for the first time that the small molecule compound G-749 can inhibit invasion and metastasis of esophagus cancer, and particularly, invasion and metastasis of esophagus cancer are inhibited by targeting NAT10. As a medicine for treating and / or preventing esophageal cancer, the small molecule compound G-749 has good safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of G-749 in preparing drugs for treating and / or preventing esophageal cancer. Background Art

[0002] Esophageal cancer is a disease that seriously threatens human physical and mental health. Its morbidity and mortality rate rank 7th and 6th among all malignant tumors, of which 90% of the mortality rate is caused by tumor metastasis. Tumor metastasis is a multi-factor, multi-link process under gene regulation, and its molecular mechanism is complex. Insufficient understanding of the metastasis process and its molecular mechanism of tumors, especially esophageal squamous cell carcinoma, and the lack of targeted therapeutic drugs are common difficulties and urgent problems faced by current basic research and clinical treatment. Therefore, a comprehensive investigation of the key driving factors and signaling pathways that regulate cancer metastasis will provide new clues for cancer treatment.

[0003] Although some progress has been made in the diagnosis and treatment of esophageal cancer in recent years, the most effective treatment methods are still surgery, radiotherapy, chemotherapy, and molecular targeted drugs. Due to the side effects and drug resistance of chemotherapy drugs, the treatment effect and prognosis of esophageal cancer are still poor, and the 5-year survival rate is low. Small molecule drugs can specifically block the signal transduction pathways necessary for tumor proliferation and metastasis, thereby achieving the purpose of treatment. Therefore, the development of small molecule compounds as cancer treatment drugs has great advantages in efficacy and price.

[0004] G-749 (Denfivontinib, CAS No.: 1457983-28-6) is a small molecule compound. Currently, there is no application of G-749 in inhibiting cancer cells, especially inhibiting the invasion and metastasis of cancer cells. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide the use of G-749 in the preparation of drugs for treating and / or preventing and inhibiting esophageal cancer metastasis.

[0006] The above-mentioned purpose of the present invention is achieved by the following technical solutions: The use of G-749 in the preparation of a drug for treating and / or preventing esophageal cancer, wherein the structural formula of the G-749 is as shown in formula (I): Formula (I).

[0007] Furthermore, the effective concentration of G-749 is 0.1 to 5 μM, preferably 1.25 to 5 μM.

[0008] Furthermore, the drug for treating and / or preventing esophageal cancer is a drug that can inhibit the invasion and / or metastasis of esophageal cancer.

[0009] Furthermore, the metastasis includes but is not limited to lung metastasis.

[0010] Furthermore, the esophageal cancer includes but is not limited to esophageal squamous cell carcinoma.

[0011] Furthermore, the drug for treating and / or preventing esophageal cancer also contains pharmaceutically acceptable excipients.

[0012] Furthermore, the pharmaceutically acceptable excipient is preferably at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant and a lubricant.

[0013] Furthermore, the administration methods of the drug for treating and / or preventing esophageal cancer include but are not limited to oral administration and injection administration.

[0014] Compared with the prior art, the present invention has the following advantages and effects: The present invention first discovered that the small molecule compound G-749 can inhibit the invasion and metastasis of esophageal cancer, specifically by targeting NAT10. The small molecule compound G-749, as a drug for treating and / or preventing esophageal cancer, has good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the structural formula of G-749.

[0016] Figure 2 This figure shows the effect of NAT10 gene knockout on the invasion ability of highly metastatic esophageal squamous cell carcinoma cells KYSE150-Luc-LM5 and KYSE410-Luc-I3.

[0017] Figure 3 This is the result of NAT10 gene expression on lung metastasis of highly metastatic esophageal squamous cell carcinoma cell KYSE150-Luc-LM5 marked with luciferase.

[0018] Figure 4 This is a graph showing the results of the cell thermal shift assay analysis of G-749 and NAT10 genes.

[0019] Figure 5 This is a graph showing the effects of different concentrations of G-749 on the invasive ability of esophageal squamous cell carcinoma cells.

[0020] Figure 6 This figure shows the effect of G-749 on the invasion ability of NAT10 gene-knockout highly metastatic esophageal squamous cell carcinoma cells KYSE150-Luc-LM5.

[0021] Figure 7 This is the result of the effect of G-749 on the lung metastasis ability of NAT10 gene-knockout, luciferase-labeled, highly metastatic esophageal squamous cell carcinoma cells KYSE150-Luc-LM5.

[0022] Figure 8 This is the H&E result of G-749 inoculating the lungs of mice with NAT10 gene knockout, luciferase-labeled, highly metastatic esophageal squamous cell carcinoma cells KYSE150-Luc-LM5.

[0023] Fig. 9 This is the H&E result of G-749 on the liver, lung and kidney functions of mice. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0025] G-749 was synthesized by Shanghai Taosu Biochemical Technology Co., Ltd., and its structural formula is as follows Figure 1 As shown; KYSE150 and KYSE410 esophageal squamous cell carcinoma cells were purchased from German Microbiology and Cell Culture Co., Ltd.; LB medium was purchased from Sangon Biotechnology (Shanghai) Co., Ltd., and 0.25% trypsin was purchased from Gibco, USA. Experimental male mice (NOD-Prkdcem26Cd52Il2rgem26Cd22, NCG) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0026] Human esophageal squamous cell carcinoma luciferase-labeled cells KYSE150-LUC have been published in the literature “Wen Wen Xu et al. Direct Targeting of CREB1 with Imperatorin Inhibits TGFβ2-ERK SignalingtoSuppress Esophageal Cancer Metastasis, Advanced Science, 2020. DOI: 10.1002 / advs.202000925”.

[0027] Human esophageal squamous cell carcinoma luciferase-labeled cells KYSE410-Luc have been disclosed in the literature "Yu XM, Li SJ, Yao ZT,Xu JJ, Zheng CC, Liu ZC, Ding PB, Jiang ZL, Wei X, Zhao LP,Shi XY, Li ZG, XuWW, Li B. N4-acetylcytidine modification of lncRNA CTC-490G23.2 promotes cancer metastasis through interacting with PTBP1 to increase CD44 alternative splicing. Oncogene. 2023 Mar;42(14):1101-1116. doi: 10.1038 / s41388-023-02628-3."

[0028] KYSE150-Luc-LM5 and KYSE410-Luc-I3 (highly metastatic esophageal squamous cell carcinoma) cell construction method: Luciferase-labeled esophageal squamous cell carcinoma cells KYSE150-Luc were digested into single cells with 0.25% trypsin, and KYSE150-Luc cells were diluted to 10 6 The cells were resuspended in PBS at 100 µL and injected into the tail vein of mice. After 4-5 weeks of feeding, D-Luciferin (purchased from GOLDBIO) was injected intraperitoneally and in vivo fluorescence photography was performed. The mice with lung metastasis were dissected and the lungs with KYSE150-Luc cell metastasis were removed. The lungs were minced into minced meat and resuspended in culture medium. After standing for one week, PBS was washed twice, and Blasticidin (5 µg / mL, purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) was added. After screening for one week until there was no cell death, these cells were KYSE150-Luc-LM1. Then, 0.25% trypsin was used to digest the KYSE150-Luc-LM1 cells into single cells and the cells were cultured at 10 6 The cells were resuspended in PBS at 100 µL per 100 μL and injected into the mice through the tail vein. After 4-5 weeks of feeding, D-Luciferin was injected intraperitoneally and in vivo fluorescence photography was performed. The mice with lung metastasis were dissected and the lungs with KYSE150-Luc-LM1 cells were removed. The lungs were minced again and resuspended in culture medium. After standing for a week, the cells were washed twice with PBS and Blasticidin was added. After screening for a week until there was no cell death, these cells were KYSE150-Luc-LM2. This cycle was repeated five times to obtain KYSE150-Luc-LM5 cells. The construction method of KYSE410-Luc-I3 cells was the same as above.

[0029] Based on the highly metastatic esophageal squamous cell carcinoma cells KYSE150-Luc-LM5 / KYSE410-Luc-I3, the specific method for constructing NAT10 gene knockout esophageal squamous cell carcinoma luciferase-labeled cells (i.e., KYSE150-Luc-LM5-sgNAT10 and KYSE410-Luc-I3-sgNAT10) is as follows: The sgNAT10 plasmid (sgRNA: 5'-AACTGAGGCCCATCAGGATG-3') was constructed using CRISPR-Cas9 gene knockout technology, and then the constructed plasmid was transformed into 293T cells. The 293T cells with good growth status were digested and plated on a 6-well plate. When the cells grew to 50%-60%, the sgNAT10 plasmid was transfected. After 48 hours, the cell medium was replaced and the supernatant was collected. Centrifugation was performed and filtered with a 0.45 µM filter membrane. The supernatants were added to KYSE150-Luc-LM5 / KYSE410-Luc-I3 cells, and the cells were transferred from the six-well plate to the culture dish 48 hours after infection. After the cells adhered to the wall, puromycin (purchased from Sigma-Aldrich) with a working concentration of 1µg / mL was added for screening. After 1-2 weeks, when the cells no longer died, the drug addition was stopped, and the NAT10 gene knockout esophageal squamous cell carcinoma luciferase-labeled cells KYSE150-Luc-LM5-sgNAT10 / KYSE410-Luc-I3-sgNAT10 were obtained. Western blot identified that the cells could reduce the NAT10 content and almost did not express it.

[0030] Example 1: NAT10 gene can enhance the metastatic ability of esophageal squamous cell carcinoma cells NAT10 was knocked out in esophageal squamous cell carcinoma cells, and then the cell invasion and metastasis abilities were detected through invasion and in vivo mouse experiments.

[0031] 1.1 Invasion Experiment (1) Chamber treatment: Add 100 μL of 20% matrix gel diluted with basic culture medium to the upper layer of the chamber and place it in a 37°C incubator for 1 h.

[0032] (2) Cell plating: NAT10 knockout and non-knockout highly invasive esophageal squamous cell carcinoma cells KYSE150-Luc-LM5 and KYSE410-Luc-I3 were digested and resuspended into single cells, diluted to 400,000 / well, respectively, and added to the upper layer of the chamber. Different concentrations of G-749 (0, 1.25, 2.5, and 5 μM) were added dropwise. 600 μL of complete culture medium was added to the lower layer and cultured at 37°C, 5% CO2 for 24 h.

[0033] (3) Crystal violet staining: Take out the chamber, wash twice with PBS, put it into a 12-well plate containing 600 μL methanol, fix it for 10 min, remove the methanol, and then stain it with 0.2% crystal violet for 10 min. After removing the crystal violet, wash away the residual crystal violet with clean water, dry it and take a picture.

[0034] The results are as follows Figure 2 As shown, NAT10 knockout can reduce the invasive ability of esophageal squamous cell carcinoma cells.

[0035] 1.2 In vivo experiments Twelve 6-week-old male NCG mice were selected, 6 in each control group and experimental group, to construct a tumor metastasis model.

[0036] Esophageal squamous cell carcinoma luciferase-labeled cells KYSE150-Luc-LM5 and KYSE150-Luc-LM5-sgNAT10 were digested into single cells and then 6 The cells were diluted and resuspended in PBS at a ratio of 100 µL / 100 µL. The vascular part of the mouse tail can be clearly observed using a mouse tail vein injection imaging device. The NCG mice were fixed in the device and the tail was disinfected with an alcohol cotton ball. The cells were resuspended and injected into the tail vein of the mice with an insulin syringe, for a total of 12 mice. After 4 weeks, D-Luciferin was injected intraperitoneally, and in vivo fluorescence photography was performed to observe the metastasis of cancer cells.

[0037] The results are as follows Figure 3 As shown, NAT10 knockout can reduce the lung metastasis of human esophageal squamous cell carcinoma luciferase-labeled cells KYSE150-Luc-LM5. The bioluminescence is caused by the enzymatic reaction of D-Luciferin in esophageal squamous cell carcinoma KYSE150-Luc-LM5 cells with D-Luciferin in body fluids, and the intensity of fluorescence reflects the density of KYSE150-Luc-LM5 cells. Therefore, the number of lung metastases in esophageal cancer cells in the NAT10 knockout group was significantly less than that in the control group.

[0038] Example 2: Cellular thermal shift assay to analyze the binding of NAT10 and compound G-749 After 12 h of treatment with DMSO and G-749 (5 μM), equal numbers of cells were collected, resuspended in PBS containing protease inhibitors, and transferred to PCR tubes. Highly metastatic esophageal squamous cell carcinoma cells KYSE150-Luc-LM5 were treated with DMSO or G-749 at the specified temperature in a water bath. After 12 h, equal numbers of cells were collected and resuspended in PBS containing protease inhibitors. The two groups of cell suspensions were respectively distributed into 6 PCR tubes, with a concentration of 100 μL in each tube. Proteins were heat-shocked in a metal bath at the specified temperature (45, 50, 55, 60, 65, and 70°C) for 3 min to denature the proteins and cooled at room temperature for 3 min. Then, the cells were repeatedly frozen and thawed by liquid nitrogen three times to lyse the cells, and the supernatant was collected by high-speed centrifugation at 4°C. Protein samples were prepared with 5× loading buffer and boiled. Changes in the amount of NAT10 directly bound to G-749 were analyzed by Western blot.

[0039] Figure 4 The results of the cell thermal shift assay showed that G-749 could bind to NAT10 protein. G-749 could enhance the thermal stability of NAT10.

[0040] Example 3: G-749 Functional Verification 1. Specific use of experimental drugs G-749 drug: Weigh an appropriate amount of the drug, dissolve it in dimethyl sulfoxide (DMSO) solution, prepare a 10 mM stock solution, divide it into portions, and store it in a -20°C refrigerator. When adding the drug, dilute it with complete culture medium to 1.25, 2.5, and 5 μM working solutions.

[0041] 2. Experimental methods: 2.1 Invasion Experiment Esophageal squamous cell carcinoma cells were treated with different concentrations of G-749, and then the invasion ability of esophageal squamous cell carcinoma cells was detected to study the effect of different concentrations of G-749 on the invasion ability of esophageal squamous cell carcinoma cells.

[0042] (1) Chamber treatment: Add 100 μL of 20% matrix gel diluted with basic culture medium to the upper layer of the chamber and place it in a 37°C incubator for 1 h.

[0043] (2) Cell plating: Highly invasive esophageal squamous cell carcinoma cells KYSE150-Luc-LM5 and KYSE410-Luc-I3 were digested and resuspended into single cells, diluted to 400,000 per well, respectively, and added to the upper layer of the chamber. Different concentrations of G-749 (0, 1.25, 2.5, and 5 μM) were added dropwise. 600 μL of complete culture medium was added to the lower layer and cultured at 37°C and 5% CO2 for 24 h.

[0044] (3) Crystal violet staining: Take out the chamber, wash twice with PBS, put it into a 12-well plate containing 600 μL methanol, fix it for 10 min, remove the methanol, and then stain it with 0.2% crystal violet for 10 min. After removing the crystal violet, wash away the residual crystal violet with clean water, dry it and take a picture.

[0045] The results are as follows Figure 5 As shown, the invasion ability of esophageal squamous cell carcinoma cells was weakened with the increase of G-749 concentration.

[0046] Example 4: G-749 can enhance the metastasis and invasion ability of esophageal squamous cell carcinoma cells through NAT10 gene NAT10 was knocked out in esophageal squamous cell carcinoma cells, and G-749 was administered on this basis. The cell invasion ability was then detected through invasion experiments and in vivo mouse experiments.

[0047] 1. Invasion Experiment 1) Chamber treatment: Add 100 μL of 20% Matrigel to the clean invasion chamber and place it in a 37°C incubator for 30 min.

[0048] 2) Cell plating: NAT10 knockout highly invasive esophageal squamous cell carcinoma cells KYSE150-Luc-LM5-sgNAT10 and wild-type KYSE150-Luc-LM5 were digested and resuspended into single cells, counted and diluted to 400,000 / well, and added to the upper layer of the chamber, and different concentrations of G-749 (0, 5 μM) were added dropwise; 600 μL of complete culture medium was added to the lower layer and cultured at 37°C, 5% CO2 for 24 h.

[0049] 3) Crystal violet staining: Take out the chamber, wash twice with PBS, add 500 μL methanol to the lower layer, fix for 10 min, remove the methanol, add 0.2% crystal violet to stain for 10 min, remove the crystal violet and wash away the residual crystal violet with clean water, dry and take pictures.

[0050] Depend on Figure 6 The results of in vitro experiments showed that the invasive ability of NAT10 knockout esophageal squamous cell carcinoma cells was not affected by the drug G-749.

[0051] 2. In vivo experiments Twenty-four 6-week-old male NCG mice were selected and divided into four groups, with 6 mice in each control group and experimental group, to construct a tumor metastasis model.

[0052] NAT10 knockout highly invasive esophageal squamous cell carcinoma cells KYSE150-Luc-LM5-sgNAT10 and wild-type KYSE150-Luc-LM5 were digested into single cells and cultured at 10 6The cells were resuspended in PBS at a ratio of 100 μL / 100 μL. The resuspended cells were injected into the tail vein of mice with an insulin syringe. One week later, the mice were divided into four groups, two groups of each type of cell, and each group had 6 mice, for a total of 24 mice. G-749 was diluted and dissolved in physiological saline, and the concentrations were set to 0 mg / kg (control group) and 5 mg / kg (experimental group). It was administered by gavage 3 times a week for 3 consecutive weeks. After 3 weeks, D-Luciferin was injected intraperitoneally, and in vivo fluorescence photography was performed to observe the metastasis of cancer cells.

[0053] from Figure 7 and Figure 8 The experimental results show that the bioluminescence in the lungs of mice decreased after G-749 treatment, while the bioluminescence in the lungs of mice in the NAT10 knockout group did not change after G-749 treatment. Therefore, in the G-749 treatment group, the number of lung metastases of esophageal cancer cells was significantly less than that of the control group, while the metastasis of esophageal squamous cell carcinoma cells with NAT10 knockout was not affected by the drug G-749.

[0054] The liver, lungs, and kidneys of mice treated with G-749 (5 mg / kg) were removed for immunohistochemistry. The results showed that G-749 had no effect on the liver, lung, and kidney functions of mice ( Fig. 9 ).

[0055] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of G-749 in the preparation of a drug for treating and / or preventing esophageal cancer, characterized in that: The structural formula of G-749 is shown in formula (I): Formula (I).

2. The use of G-749 according to claim 1 in preparing a drug for treating and / or preventing esophageal cancer, characterized in that: The effective concentration of G-749 is 0.1 to 5 μM.

3. The use of G-749 according to claim 1 in preparing a drug for treating and / or preventing esophageal cancer, characterized in that: The effective concentration of G-749 is 1.25-5 μM.

4. The use of G-749 according to claim 1 in preparing a drug for treating and / or preventing esophageal cancer, characterized in that: The drug for treating and / or preventing esophageal cancer is a drug that can inhibit the invasion and / or metastasis of esophageal cancer.

5. The use of G-749 according to claim 4 in preparing a drug for treating and / or preventing esophageal cancer, characterized in that: The metastasis includes lung metastasis.

6. Use of G-749 according to any one of claims 1 to 5 in the preparation of a drug for treating and / or preventing esophageal cancer, characterized in that: The esophageal cancer includes esophageal squamous cell carcinoma.

7. Use of G-749 according to any one of claims 1 to 5 in the preparation of a drug for treating and / or preventing esophageal cancer, characterized in that: The medicine for treating and / or preventing esophageal cancer further comprises pharmaceutically acceptable excipients.

8. The use of G-749 according to claim 7 in preparing a drug for treating and / or preventing esophageal cancer, characterized in that: The pharmaceutically acceptable excipient is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant and a lubricant.

9. The use of G-749 according to claim 7 in preparing a drug for treating and / or preventing esophageal cancer, characterized in that: The administration method of the medicine for treating and / or preventing esophageal cancer is oral administration.

10. The use of G-749 according to claim 7 in preparing a drug for treating and / or preventing esophageal cancer, characterized in that: The administration method of the medicine for treating and / or preventing esophageal cancer is injection.

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