Use of K279-0738 in the preparation of a medicament for treating and / or preventing tumors

Abstract

The present invention provides the use of K279-0738 in the preparation of a medicament for treating and / or preventing tumors, which belongs to the field of biomedicine. The structural formula of K279-0738 is shown in Formula (I), and the effective concentration of K279-0738 is 2.5-5 μM. The small molecule compound K279-0738 can inhibit the invasion and metastasis of esophageal cancer, and specifically, it has been discovered for the first time to inhibit esophageal cancer invasion and metastasis by targeting KCTD4. As a promising antitumor drug, the small molecule compound K279-0738 has a certain degree of safety and, further, significant advantages in efficacy and price.

Description

[Technical Field]

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the use of K279-0738 in the preparation of a medicament for treating and / or preventing tumors. [Background technology]

[0002] Esophageal cancer is one of the most lethal cancers in the world, ranking among the top 10 in terms of both incidence and mortality in China. 90% of deaths are due to tumor metastasis. The metastasis process is complex and difficult to study, and there is little information and functional validation on metastasis-related genes, especially in esophageal squamous cell carcinoma (ESCC). Therefore, comprehensive investigation of the key drivers and signaling pathways controlling cancer metastasis will provide new insights for cancer treatment.

[0003] Surgery, radiation therapy, chemotherapy, and molecular-targeted drugs are the main treatments for esophageal cancer. Surgery and radiation therapy are localized treatments, while chemotherapy and molecular-targeted drugs are systemic treatments. However, commonly used anticancer drugs often have specific toxicities and side effects, and resistance during chemotherapy can lead to postoperative recurrence and poor patient outcomes, making treatment more challenging. Small molecule drugs can specifically inhibit signaling pathways necessary for tumor growth and proliferation, thereby achieving therapeutic goals. Small molecule inhibitors have several advantages: (1) high oral bioavailability, (2) good permeability through physiological barriers, allowing them to cross cell membranes and act on intracellular targets, and (3) low cost. Therefore, the development of small molecule compounds as cancer therapeutics offers significant advantages in terms of efficacy and price.

[0004] K279-0738 (N-(4-Methoxybenzyl)-3-{5-[(3-Nitrobenzyl)thio]-3-oxo-2,3-dihydroimidazo[1,2-C]quinazolin-2-yl}propanamide) is a small molecule compound with the following structural formula: [ka]

[0005] At present, there are no examples of K279-0738 being applied to inhibit cancer cells, particularly to inhibit cancer cell invasion and metastasis. Summary of the Invention [Problem to be solved by the invention]

[0006] The main object of the present invention is to overcome the drawbacks and deficiencies of the prior art and to provide a use of K279-0738 in the preparation of a medicament for treating and / or preventing esophageal cancer. [Means for solving the problem]

[0007] The above object of the present invention is achieved by the following technical solutions.

[0008] Use of K279-0738 in the preparation of a medicament for treating and / or preventing esophageal cancer, wherein the structural formula of said K279-0738 is represented by formula (I). [ka]

[0009] Furthermore, the effective concentration of K279-0738 is 2.5 to 5 μM.

[0010] Furthermore, the drug for treating and / or preventing esophageal cancer is a drug that can inhibit the invasion and / or metastasis of esophageal cancer cells.

[0011] Furthermore, the esophageal cancer includes, but is not limited to, esophageal squamous cell carcinoma.

[0012] Furthermore, K279-0738 inhibits the invasion and / or metastasis of esophageal cancer by targeting KCTD4. The nucleotide sequence encoding KCTD4 is shown in the gene sequence of NCBI Gene bank under accession number 386618.

[0013] A drug for treating and / or preventing esophageal cancer, comprising K279-0738, the structural formula of which is represented by formula (I). [ka]

[0014] Furthermore, the medicament for treating and / or preventing esophageal cancer further comprises a pharmaceutically acceptable auxiliary material.

[0015] Furthermore, the pharmaceutically acceptable auxiliary material is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant, and a lubricant.

[0016] Furthermore, the method of administering the drug for treating and / or preventing esophageal cancer is at least one of oral administration, intragastric administration, and injection administration. [Effects of the Invention]

[0017] Compared with the prior art, the present invention has the following advantages and effects.

[0018] The present invention is the first to discover that the small molecule compound K279-0738 can inhibit the invasion and metastasis of esophageal cancer, specifically by targeting KCTD4. As a promising antitumor drug, the small molecule compound K279-0738 has a certain degree of safety and, furthermore, has significant advantages in efficacy and price. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the structural formula of K279-0738. [Figure 2] FIG. 1 shows the results of a study on the effect of the KCTD4 gene on the invasive ability of luciferase-labeled human esophageal squamous cell carcinoma cells KYSE150-LUC and human esophageal squamous cell carcinoma KYSE410. [Figure 3] FIG. 1 shows the results of a study on the effect of the KCTD4 gene on the lung metastatic potential of luciferase-labeled human esophageal squamous cell carcinoma cells, KYSE150-LUC. [Figure 4] FIG. 1 shows the results of a study on the effects of different concentrations of K279-0738 on the invasive potential of esophageal squamous cell carcinoma. [Figure 5] FIG. 1 shows the results of a fluorescence detection study of the effect of K279-0738 on the lung metastatic potential of esophageal squamous cell carcinoma. [Figure 6] FIG. 1 shows the results of immunohistochemical studies of the effects of K279-0738 on liver, lung, kidney, and spleen function in mice. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will be described in detail below with reference to examples and drawings, but the embodiments of the present invention are not limited to these.

[0021] K279-0738 was synthesized by Shanghai Topscience Chemical Technology Co., Ltd. (Topscience) and its structural formula is as follows: [ka]

[0022] Human esophageal squamous cell carcinoma cells KYSE150, KYSE410, and KYSE270 were purchased from German Microorganisms and Cell Cultures Co., Ltd. LB medium was purchased from Sangon Biotech (Shanghai), and 0.25% trypsin was purchased from Gibco (USA). Female experimental mice (NOD-SCID) were purchased from GemPharmatech (Jiangsu).

[0023] Luciferase-labeled human esophageal squamous cell carcinoma cells, KYSE150-LUC, are described in the paper "Wen WenXu et al. Direct Targeting of CREB1 with Imperatorin Inhibits TGFβ2-ERK Signaling to Suppress Esophageal Cancer Metastasis," Advanced Science, 2020. DOI: 10.1002 / advs.202000925."

[0024] KYSE150-LUC-LM3 cell construction method: Human esophageal squamous cell carcinoma luciferase-labeled cells, KYSE150-LUC, were digested into single cells using 0.25% trypsin. 6100 μL of luciferase-labeled human esophageal squamous cell carcinoma (KYSE150-LUC) cells were resuspended in 100 μL of PBS buffer and injected into the tail vein of mice. After 5–6 weeks of incubation, D-luciferin (purchased from GOLDBIO) was injected intraperitoneally, and in vivo fluorescence photography was performed. Mice with lung metastases were dissected, and the lungs containing KYSE150-LUC cells were removed. The lungs were minced and resuspended in medium. After allowing to stand for one week, the cells were washed twice with PSB buffer, and blasticidin (5 μg / mL, purchased from Beyotime Biotechnology (Shanghai) Co., Ltd.) was added. Screening was continued for one week until cell death ceased. These cells were designated KYSE150-LUC-LM1. Subsequently, 0.25% trypsin was used to digest the luciferase-labeled human esophageal squamous cell carcinoma (KYSE150-LUC-LM1) cells into single cells, and 10 6 Luciferase-labeled human esophageal squamous cell carcinoma (KYSE150-LUC-LM1) cells were resuspended in 100 μL of PBS buffer and injected into mice via the tail vein. After 5–6 weeks of incubation, D-luciferin (purchased from GOLDBIO) was injected intraperitoneally, and in vivo fluorescence images were taken. Mice with lung metastases were dissected, and the lungs containing KYSE150-LUC-LM1 cells were removed. The lungs were minced and resuspended in medium. After allowing to stand for one week, the cells were washed twice with PSB buffer and blasticidin (5 μg / mL, purchased from Beyotime Biotechnology, Shanghai) was added. Screening was continued for one week until no cell death was observed. These cells were designated KYSE150-LUC-LM2. Three cycles of this procedure were repeated to obtain KYSE150-LUC-LM3 cells.

[0025] Construction of luciferase-labeled human esophageal squamous cell carcinoma cells KYSE150-LUC (i.e., KYSE150-LUC-KCTD4 cells) stably overexpressing the KCTD4 gene and human esophageal squamous cell carcinoma cells KYSE410 (i.e., KYSE410-KCTD4 cells) : The KCTD4 gene (gene ID: 386618) was constructed on the pTSB-CMV-MCS-SBP-3Flag-copGFP-F2A-PuroR expression vector (TranSheepBio), and the KCTD4 eukaryotic expression plasmid was obtained (construction was requested from Shanghai Quanyang Biotechnology Co., Ltd.). The constructed plasmid was transformed into 293T cells, and well-growing 293T cells were digested and seeded into 6-well plates. When the cells reached 50%-60% proliferation, they were transfected with the KCTD4 eukaryotic expression plasmid. After 24 and 48 hours, the cell culture medium was replaced and the supernatant was collected. The supernatant was centrifuged and filtered through a 0.45 μM filter. The supernatant was then added to KYSE150-LUC cells and human esophageal carcinoma KYSE410 cells, respectively. After 48 hours of infection, the cells were transferred from the 6-well plate to vials. After adhesion, puromycin (purchased from Sigma-Aldrich) was added at a working concentration of 1 μg / mL for screening. After 1–2 weeks, when cells no longer died, the drug addition was discontinued to obtain luciferase-labeled human esophageal squamous cell carcinoma cells KYSE150-LUC (i.e., KYSE150-LUC-KCTD4 cells) stably overexpressing the KCTD4 gene, and human esophageal squamous cell carcinoma cells KYSE410 (i.e., KYSE410-LUC-KCTD4 cells) stably overexpressing the KCTD4 gene. These cells were able to overexpress KCTD4 as identified by Western blotting.

[0026] Example 1: KCTD4 gene enhances metastatic and invasive potential of human esophageal squamous cell carcinoma cells

[0027] 1. We stably overexpressed KCTD4 in esophageal squamous cell carcinoma cells, and then detected the cell invasive ability through invasion experiments and in vivo experiments in mice.

[0028] (1) Cell invasion experiment

[0029] 1) Chamber treatment: 100 μL of 5% matrix gel was added to a clean invasion chamber and left in an incubator at 37° C. for 30 minutes.

[0030] 2) Cell seeding: Human esophageal squamous cell carcinoma luciferase-labeled cells KYSE150-LUC (i.e., KYSE150-LUC-KCTD4 cells) stably overexpressing the KCTD4 gene and human esophageal squamous cell carcinoma cells KYSE410 (i.e., KYSE410-KCTD4 cells) stably overexpressing the KCTD4 gene were digested with 0.25% trypsin to form single suspended cells. After cell counting, 500,000 KYSE150-LUC-KCTD4 cells and 200,000 KYSE410-KCTD4 cells were added to the upper chamber of each chamber, and 500 μL of complete medium was added to the lower chamber. The cells were cultured at 37°C and 5% CO2 for 24 hours. Simultaneously, the above experiments were performed using human esophageal squamous cell carcinoma luciferase-labeled cells KYSE150-LUC and human esophageal squamous cell carcinoma cells KYSE410 as controls.

[0031] 3) Crystal violet staining: The chamber was removed and washed twice with PBS buffer (pH 7.4, purchased from Sigma-Aldrich; the same applies below). 500 μL of methanol was added to the bottom of the chamber and fixed for 10 minutes. The methanol was then aspirated off, and 0.2% crystal violet was added and stained for 10 minutes. After aspirating off the crystal violet, the remaining crystal violet was removed with clean water, the chamber was dried, and photographed.

[0032] (2) In vivo experiments

[0033] Twelve 6-week-old female mice (NOD-SCID) (6 in the control group and 6 in the experimental group) were selected to establish a tumor metastasis model.

[0034] (1) Human esophageal squamous cell carcinoma luciferase-labeled cells KYSE150-LUC and KYSE150-LUC-KCTD4 were digested into single cells using 0.25% trypsin. 10 6 Luciferase-labeled human esophageal squamous cell carcinoma cells KYSE150-LUC and KYSE150-LUC-KCTD4 were resuspended in 100 μL of PBS buffer.

[0035] (2) Before the tail vein experiment, mice were anesthetized, and the degree of anesthesia was assessed using painless and painful stimuli to confirm that the mice were in an anesthetized state.

[0036] (3) The cells were resuspended in a microsyringe with a 25G needle and injected into the tail vein of a total of 12 mice.

[0037] After 5 to 6 weeks of breeding, D-luciferin (purchased from GOLDBIO) was injected intraperitoneally, and in vivo fluorescent photographs were taken to observe lung metastasis of cancer cells.

[0038] The results are shown in Figures 2 and 3. The in vitro experimental results in Figure 2 demonstrate that the KCTD4 gene can enhance the invasive ability of luciferase-labeled human esophageal squamous cell carcinoma cells KYSE150-LUC and KYSE410-LUC. The in vivo experimental results in Figure 3 demonstrate that the KCTD4 gene can promote lung metastasis of luciferase-labeled human esophageal squamous cell carcinoma cells KYSE150-LUC in mice. Example 2: Functional verification of K279-0738

[0039] (1) Infiltration experiment

[0040] We investigated the effects of different concentrations of K279-0738 on the invasive ability of esophageal squamous cell carcinoma cells by treating the cells with different concentrations of K279-0738 and then detecting the invasive ability of the cells.

[0041] 1) Chamber treatment: 100 μL of 5% Matrigel was added to a clean invasion chamber and left in an incubator at 37° C. for 30 minutes.

[0042] 2) Cell seeding: Highly invasive human esophageal squamous cell carcinoma cells, KYSE150-LUC-LM3 and KYSE270, were digested with 0.25% trypsin to form single-cell suspensions. After cell counting, 500,000 KYSE150-LUC-LM3 cells and 200,000 KYSE270 cells were added to the upper chamber of each chamber, followed by the addition of different concentrations of K279-0738 (final concentrations of 0, 2.5, and 5 μM, respectively). 500 μL of complete medium was added to the lower chamber, and the cells were cultured at 37°C and 5% CO2 for 24 hours.

[0043] 3) Crystal violet staining: The chamber was removed and washed twice with PBS buffer. 500 μL of methanol was added to the bottom of the chamber and fixed for 10 minutes. The methanol was then aspirated off, and 0.2% crystal violet was added and stained for 10 minutes. After aspirating off the crystal violet, the remaining crystal violet was removed with clean water, dried, and photographed.

[0044] The in vitro test results in Figure 4 show that the invasive ability of esophageal squamous cell carcinoma cells weakened with increasing K279-0738 concentration.

[0045] (2) In vivo experiments

[0046] Eighteen 6-week-old female mice (NOD-SCID) (6 in the control group and 12 in the experimental group) were selected to establish a tumor metastasis model.

[0047] 1) Luciferase-labeled human esophageal squamous cell carcinoma cells, KYSE150-LUC-LM3 cells, were digested into single cells with 0.25% trypsin and incubated for 10 min. 6 Luciferase-labeled human esophageal squamous cell carcinoma cells KYSE150-LUC-LM3 were resuspended in 100 μL of PBS buffer.

[0048] 2) Before the experiment, mice were anesthetized, and the degree of anesthesia was assessed using painless and painful stimuli to confirm that the mice were in an anesthetized state.

[0049] 3) The cells were resuspended in a microsyringe with a 25G needle and injected into the tail vein of a total of 18 mice.

[0050] 4) K279-0738 treatment: One week after tail vein injection of KYSE150-LUC-LM3 esophageal squamous cell carcinoma cells, mice were administered the drug via intragastric administration. K279-0738 was dissolved in PBS buffer and administered to six mice per group at concentrations of 0 mg / kg (i.e., control group), 2.5 mg / kg (i.e., experimental group 1), and 5 mg / kg (i.e., experimental group 2) once weekly. Five to six weeks later, D-luciferin (purchased from GOLDBIO) was injected intraperitoneally, and in vivo fluorescence photography was performed to observe cancer cell metastasis.

[0051] The in vivo experimental results shown in Figure 5 revealed reduced bioluminescence in the lungs of mice treated with K279-0738. This bioluminescence is caused by an enzymatic reaction between luciferase in KYSE150-LUC-LM3 esophageal squamous cell carcinoma cells and D-luciferin in the body fluids, and the intensity of the fluorescence reflects the density of KYSE150-LUC-LM3 cells. Therefore, the number of lung metastases of esophageal cancer cells in the K279-0738-treated group (i.e., the experimental group) was significantly lower than that in the control group.

[0052] The liver, lung, kidney, and spleen of mice treated with K279-0738 (2.5 mg / kg and 5 mg / kg) were isolated and subjected to immunohistochemical staining. The results (see Figure 6) showed that K279-0738 did not affect the function of the liver, lung, kidney, or spleen of mice.

[0053] The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention are equivalent replacement methods and fall within the protection scope of the present invention.

Claims

1. 1. Use of K279-0738 in the preparation of a medicament for treating and / or preventing esophageal cancer, wherein the structural formula of said K279-0738 is represented by formula (I). 【Chemistry 1】

2. The use of K279-0738 in preparing a medicament for treating and / or preventing esophageal cancer according to claim 1, wherein the effective concentration of K279-0738 is 2.5-5 μM.

3. The use of K279-0738 in the preparation of a drug for treating and / or preventing esophageal cancer according to claim 1 or 2, 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 cells.

4. The use of K279-0738 in the preparation of a medicament for treating and / or preventing esophageal cancer according to claim 1 or 2, wherein the esophageal cancer is esophageal squamous cell carcinoma.

5. The use of K279-0738 in the preparation of a medicament for treating and / or preventing esophageal cancer according to claim 1 or 2, characterized in that K279-0738 inhibits the invasion and / or metastasis of esophageal cancer by targeting the KCTD4 gene.

6. A drug for treating and / or preventing esophageal cancer, comprising K279-0738, the structural formula of which is represented by formula (I). 【Chemistry 2】

7. The drug for treating and / or preventing esophageal cancer according to claim 6, characterized in that the drug for treating and / or preventing esophageal cancer further comprises a pharmaceutically acceptable auxiliary material.

8. The drug for treating and / or preventing esophageal cancer according to claim 7, wherein the pharmaceutically acceptable auxiliary material is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant, and a lubricant.

9. The drug for treating and / or preventing esophageal cancer according to any one of claims 6 to 8, characterized in that the method of administration of the drug for treating and / or preventing esophageal cancer is oral administration, intragastric administration, or injection administration.

Images