Application of small molecule metabolite quinine nucleobase in preparation of anti-colorectal cancer drugs

Abstract

The invention relates to an application of a small molecule metabolite quinine nucleobase or pharmaceutically acceptable salts, esters and solvates thereof in preparation of anti-colorectal cancer drugs. The structure of the small molecule metabolite quinine nucleobase is shown as a formula I in the specification. The invention develops new application of the small molecule metabolite quinine nucleobase or pharmaceutically acceptable salts, esters and solvates thereof, and proves that the quinine nucleobase can inhibit the proliferation, invasion and migration ability of colorectal cancer cell strains and influence the change of cell cycles at the molecular level, and finally inhibits the occurrence and development process of colorectal cancer. Meanwhile, animal experiments also prove that the small molecule metabolite quinine nucleobase can effectively inhibit tumor growth of colorectal cancer mice.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of the small molecule metabolite queuine or its pharmaceutically acceptable salt, ester, or solvate in the preparation of anti-colorectal cancer drugs. Background Art

[0002] Colorectal cancer (CRC) is a common malignant tumor in the gastrointestinal tract, a cancer that affects the colon or rectum and can cause serious harm and death. The occurrence and development of CRC are the result of the long-term interaction of multiple factors, including genetic susceptibility, dietary imbalance, and unhealthy lifestyle, etc., which together lead to the imbalance of the intestinal internal environment homeostasis. The main current clinical treatment methods for CRC are surgical resection, adjuvant chemotherapy, molecular targeted therapy, and neoadjuvant therapy. Although these treatment methods have improved the survival rate of patients to a certain extent, the treatment of CRC still faces many challenges, including tumor recurrence, metastasis, and drug resistance to treatment, etc. Therefore, the treatment of colorectal cancer remains an important research direction, and it is necessary to continue to optimize the existing treatment methods and combination strategies, and continuously explore new treatment targets and innovative therapies.

[0003] In recent years, the role of the gut microbiota in cancer occurrence and treatment has received increasing attention. Combining the gut microbiota with cancer treatment methods can improve the treatment effect. The gut microbiota is a complex microecosystem that has established a stable symbiotic relationship with the host during long-term evolution. This symbiotic relationship is crucial for the health of the host, and the activities of the microbiota directly affect the physiological functions and disease states of the host. More and more studies have shown that the gut microbiota affects the host organism mainly through its metabolites, and these metabolites play a key role in regulating intestinal health and cancer occurrence.

[0004] The gut microbiota mainly exerts its biological effects through the metabolites it produces. Among the numerous gut microbiota metabolites, the small molecule metabolite queuine is a small molecule metabolite synthesized by dominant bacteria, with a molecular weight of 277.28. Animal, plant, and fungal species can obtain this substance through recycling. Since animals lack a pathway for synthesizing queuine, they must obtain it from the diet or the gut microbiota. The queuine produced by the gut microbiota can be absorbed by humans through the intestinal epithelium and is widely distributed and enriched in tissues, playing an important role in human health. Existing studies have shown that queuine participates in a variety of cellular functions in the body, such as regulating cell proliferation, cell signal transduction, and altering the expression of growth-related proto-oncogenes. Queuine shows potential therapeutic effects in human inflammatory bowel disease (IBD) by regulating intestinal cell proliferation and connection and reducing inflammation of epithelial cells.

[0005] However, so far, no one has reported the application of the small molecule metabolite queuine in the preparation of anti-colorectal cancer drugs. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an application of a small molecule metabolite queuine or a pharmaceutically acceptable salt, ester, or solvate thereof in the preparation of anti-colorectal cancer drugs. Queuine can inhibit the proliferation, invasion, and migration abilities of human colorectal cancer cell lines HCT116 and / or HT29 and affect the changes in the cell cycle, thereby playing an anti-colorectal cancer role and effectively inhibiting tumor growth.

[0007] To achieve the purpose of this invention, the following technical solutions are adopted:

[0008] In the first aspect, the present invention provides an application of a small molecule metabolite queuine or a pharmaceutically acceptable salt, ester, or solvate thereof in the preparation of anti-colorectal cancer drugs.

[0009] The structure of the small molecule metabolite queuine is shown in Formula I.

[0010]

[0011] The present invention has developed a new use of the small molecule metabolite queuine, proving that it can inhibit the proliferation, invasion, and migration abilities of colorectal cancer cell lines and affect the changes in the cell cycle at the molecular level, ultimately inhibiting the occurrence and development process of colorectal cancer. At the same time, in animal experiments, it has also been proven that the small molecule metabolite queuine can effectively inhibit tumor growth in colorectal cancer mice.

[0012] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0013] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of fillers, binders, wetting agents, disintegrants, solubilizers, osmotic pressure regulators, coating materials, colorants, pH regulators, antioxidants, or bacteriostatic agents.

[0014] In the second aspect, the present invention provides an application of a small molecule metabolite queuine or a pharmaceutically acceptable salt, ester, or solvate thereof in the preparation of a colorectal cancer cell line proliferation inhibitor.

[0015] The structure of the small molecule metabolite queuine is shown in Formula I.

[0016]

[0017] Preferably, the colorectal cancer cell line includes HCT116 cells and / or HT29 cells.

[0018] In a third aspect, the present invention provides an application of a small molecule metabolite queuine or a pharmaceutically acceptable salt, ester, or solvate thereof in the preparation of an inhibitor of colorectal cancer cell line invasion.

[0019] The structure of the small molecule metabolite queuine is shown in Formula I.

[0020]

[0021] Preferably, the colorectal cancer cell line includes HCT116 cells.

[0022] In a fourth aspect, the present invention provides an application of a small molecule metabolite queuine or a pharmaceutically acceptable salt, ester, or solvate thereof in the preparation of an inhibitor of colorectal cancer cell line migration.

[0023] The structure of the small molecule metabolite queuine is shown in Formula I.

[0024]

[0025] Preferably, the colorectal cancer cell line includes HCT116 cells and / or HT29 cells.

[0026] In a fifth aspect, the present invention provides an application of a small molecule metabolite queuine or a pharmaceutically acceptable salt, ester, or solvate thereof in the preparation of a cell cycle blocker for colorectal cancer cell lines.

[0027] The structure of the small molecule metabolite queuine is shown in Formula I.

[0028]

[0029] Preferably, the colorectal cancer cell line includes HCT116 cells and / or HT29 cells.

[0030] In a sixth aspect, the present invention further provides an application of a small molecule metabolite queuine or a pharmaceutically acceptable salt, ester, or solvate thereof in the preparation of a reagent having the efficacy of inhibiting the proliferation of colorectal cancer cell lines, inhibiting the invasion of colorectal cancer cell lines, inhibiting the migration of colorectal cancer cell lines, or blocking the cell cycle progression of colorectal cancer cell lines.

[0031] The structure of the small molecule metabolite queuine is shown in Formula I.

[0032]

[0033] Preferably, the colorectal cancer cell line includes HCT116 cells and / or HT29 cells.

[0034] According to the research results of the present invention, it is found that the small molecule metabolite queuine or its pharmaceutically acceptable salt, ester, or solvate has the effects of inhibiting the proliferation, invasion, and migration of colorectal cancer cell lines and blocking the cell cycle progression of colorectal cancer cell lines. Because it can also be used as a single scientific research reagent for basic scientific research, for example, to explore the related mechanisms of colorectal cancer cells.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention has developed a new use of the small molecule metabolite queuine, and has proved that it can inhibit the proliferation, invasion, and migration abilities of colorectal cancer cell lines at the molecular level and affect the changes in the cell cycle, ultimately inhibiting the occurrence and development process of colorectal cancer. At the same time, in animal experiments, it is further proved that the small molecule metabolite queuine can effectively inhibit the tumor growth of colorectal cancer mice, thereby enhancing the anti-tumor effect.

[0037] Compared with the existing colorectal cancer treatment drugs, the small molecule metabolite queuine is derived from diet or the gut microbiota, can be absorbed by humans through the intestinal epithelium, and is widely distributed and enriched in tissues, with higher safety. Description of the Drawings

[0038] Figure 1 It is a diagram showing the effect of queuine on the proliferation of human colorectal cancer cells HCT116 and HT29.

[0039] Figure 2 It is a diagram showing the effect of queuine on the invasion of human colorectal cancer cells HCT116.

[0040] Figure 3 It is a diagram showing the effect of queuine on the migration of human colorectal cancer cells HCT116.

[0041] Figure 4 It is a diagram showing the effect of queuine on the migration of human colorectal cancer cells HT29.

[0042] Figure 5 It is a diagram showing the effect of queuine on the cell cycle of human colorectal cancer cells HCT116.

[0043] Figure 6 It is a diagram showing the effect of queuine on the cell cycle of human colorectal cancer cells HT29.

[0044] Figure 7It is a diagram showing the effect of queuine nucleobase on nude mice with colorectal cancer. Among them, A is the intuitive view of the tumor in nude mice; B is the statistical chart of the tumor weight / mouse body weight; C is the tumor growth curve chart. Specific implementation manners

[0045] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only helpful for understanding the present invention and should not be regarded as specific limitations on the present invention.

[0046] The drug queuine nucleobase involved in the following embodiments was purchased from Toronto Research Chemicals, with the product number Q525000-0.5mg, and was diluted with sterile water to a concentration of 1 mM.

[0047] Example 1

[0048] Effect of queuine nucleobase on the proliferation of human colorectal cancer HCT116 cells and HT29 cells:

[0049] (1) Effect of queuine nucleobase on the proliferation of HCT116 cells

[0050] Experimental method:

[0051] (1.1) Take HCT116 cells in good growth state, digest them with 1 mL of trypsin, and inoculate the cells into a 96-well plate at a ratio of 2000 cells per 100 μL by counting.

[0052] (1.2) Divide the above cells into a control group and an experimental group:

[0053] Queuine nucleobase was added to the experimental group, and the added volume was 0.3 μL of queuine nucleobase / 100 μL of DMEM medium; 0.3 μL of deionized water / 100 μL of DMEM medium was added to the control group correspondingly.

[0054] (1.3) Add 100 μL of PBS around the wells to be measured to reduce the evaporation of the medium.

[0055] (1.4) After 12 h, 24 h, 48 h, and 72 h respectively, add 10 μL of CCK-8 solution to the 96-well plate and incubate for 1.5 h.

[0056] (1.5) After the incubation is completed, measure the optical density value of each well in an enzyme-linked immunosorbent assay (ELISA) reader, and set the measurement wavelength to 450 nm to evaluate the proliferation effect of HCT116 cells.

[0057] (2) Effect of queuine nucleobase on the proliferation of HT29 cells

[0058] Experimental method: Refer to step (1), the difference from step (1) is only that HCT116 cells are replaced with HT29 cells, and DMEM medium is replaced with an equal volume of RPMI 1640 medium.

[0059] (3) Experimental results

[0060] The results of the effect of quinoline nucleobase on the proliferation of human colorectal cancer HCT116 cells and HT29 cells are as Figure 1 shown, demonstrating that quinoline nucleobase can inhibit the growth of HCT116 cells and HT29 cells.

[0061] Example 2

[0062] Transwell experiment to analyze the effect of quinoline nucleobase on the invasion of human colorectal cancer HCT116 cells:

[0063] (1) Experimental method:

[0064] (1.1) Remove the medium from well-grown HCT116 cells, and add serum-free DMEM medium to starve for 24 h;

[0065] (1.2) Place the Matrigel at 4 °C overnight to melt. On ice, dilute the Matrigel with serum-free medium to 1 mg / mL and mix well;

[0066] (1.3) Take 120 μL of the above mixed solution and vertically add it into the Transwell chamber, spread the gel evenly, and incubate in a 37 °C incubator for 2 h; aspirate the unbound Matrigel, add 200 μL of serum-free DMEM medium, and incubate at 37 °C for 30 min for hydration;

[0067] (1.4) Remove the liquid in the chamber, take 800 μL of DMEM medium containing 10% FBS and add it to the lower chamber of the 12-well plate, and place the chamber in the 12-well plate;

[0068] (1.5) Digest the starved HCT116 cells, resuspend them with serum-free DMEM medium, and add 200 μL of 5×10 4 cells per well to the upper chamber of the 12-well plate. Divide into experimental group and control group. Add quinoline nucleobase to the experimental group, and the added volume is 3 μL of quinoline nucleobase / 1 mL of serum-free DMEM medium; correspondingly, add 3 μL of deionized water / 1 mL of serum-free DMEM medium to the control group;

[0069] (1.6) Place the 12-well plate in a cell culture incubator and culture for 48 h;

[0070] (1.7) Take out the Transwell chamber, remove the culture medium, and gently wipe the Matrigel and cells in the chamber with a cotton swab or cotton moistened with PBS; add 600 μL of 4% paraformaldehyde fixative to a clean well of a 12-well plate, and place the chamber in it for fixation for 30 min. Discard the fixative, and wash the inside and outside of the chamber once with PBS. Add 600 μL of crystal violet staining solution to a clean well of a 12-well plate, and place the chamber in it for staining for 10 min. Take out the chamber, and wash the inside and outside of the chamber 3 times with PBS;

[0071] (1.8) After appropriate air drying, observe under a microscope, take pictures, and count with ImageJ.

[0072] (2) Experimental results:

[0073] The results of the invasion of HCT116 cells are as Figure 2 shown. It can be seen that the number of HCT116 cells in the experimental group is significantly lower than that in the control group, indicating that quinone nucleobase has an obvious inhibitory effect on the invasion ability of HCT116 cells.

[0074] Example 3

[0075] Analysis of the effect of quinone nucleobase on the migration of human colorectal cancer HCT116 cells and HT29 cells by cell scratch assay:

[0076] (1) Study on the migration of quinone nucleobase on HCT116 cells

[0077] Experimental method:

[0078] (1.1) Draw a line on the back of a six-well plate with a marker pen. Take HCT116 cells in good growth state, digest them with 1 mL of trypsin. After digestion, inoculate 5×10 5 cells per well into the six-well plate by counting, and draw an "8" to evenly distribute the cells in the six-well plate;

[0079] (1.2) After overnight culture, use a 200 μL pipette tip to scratch the cells vertically along the ruler on the back of the six-well plate in the culture dish, and then add PBS adherently to the six-well plate to wash off the detached cells;

[0080] (1.3) Divide into an experimental group and a control group: add quinone nucleobase to the experimental group, with an added volume of 3 μL of quinone nucleobase / 1 mL of serum-free DMEM medium; correspondingly, add 3 μL of deionized water / 1 mL of serum-free DMEM medium to the control group;

[0081] (1.4) Take pictures of the cell scratch area using an inverted microscope at 0, 24, 48, and 72 h respectively, and calculate the cell migration rate;

[0082] Cell migration rate = (S 0 -St ) / S 0 × 100%, where S 0 is the scratch area at 0 h, and S t is the scratch area at 24 h, 48 h, or 72 h.

[0083] (2) Study on the effect of quinone nucleobase on the migration of HT29 cells

[0084] Experimental method: Referring to step (1), the difference from step (1) is only that HCT116 cells are replaced with HT29 cells, and DMEM medium is replaced with an equal volume of RPMI 1640 medium.

[0085] (3) Experimental results:

[0086] The results of the migration of human colorectal cancer HCT116 cells and HT29 cells are respectively as Figure 3 and Figure 4 shown. It can be seen that the cell migration in the experimental group is significantly lower than that in the control group, indicating that quinone nucleobase has an obvious inhibitory effect on the migration of HCT116 cells and HT29 cells.

[0087] Example 4

[0088] Flow cytometry analysis of the effect of quinone nucleobase on the cell cycle of human colorectal cancer HCT116 cells and HT29 cells:

[0089] (1) Study on the effect of quinone nucleobase on the cell cycle of HCT116 cells

[0090] Experimental method:

[0091] (1.1) Take HCT116 cells in good growth state, add 3×10 5 cells per well to a six-well plate, gently shake well and place in a cell incubator for overnight culture;

[0092] (1.2) After overnight culture, discard the old medium, and divide it into an experimental group and a control group: The experimental group is added with quinone nucleobase, and the added volume is 3 μL of quinone nucleobase / 1 mL of DMEM medium, and placed in a cell incubator for 24 h; Correspondingly, 3 μL of deionized water / 1 mL of DMEM medium is added to the control group;

[0093] (1.3) Collect the cell supernatant into a 15 mL centrifuge tube. Digest the cells with trypsin. After digestion, pipette the cells at the bottom of the dish and collect them into the 15 mL centrifuge tube. Gently pipette to mix evenly to make a single cell suspension; centrifuge at 1000 g for 5 min, discard the supernatant, add 1 mL of pre-cooled PBS, resuspend the cells, and transfer them into a 1.5 mL centrifuge tube. Centrifuge at 1000 g for 5 min, discard the supernatant, then add 1 mL of pre-cooled 70% ethanol, gently pipette to mix evenly, and fix overnight at 4°C; centrifuge at 1000 g for 5 min, discard the supernatant, add 1 mL of pre-cooled PBS, resuspend the cells, centrifuge at 1000 g for 5 min, and discard the supernatant;

[0094] (1.4) Add 500 μL of PI / RNase staining solution to each tube, slowly and thoroughly resuspend the cell pellet, incubate in the dark at 37°C for 30 minutes, and then perform on-machine detection.

[0095] (2) Study on the effect of quinone nucleobase on the cell cycle of HT29 cells

[0096] Experimental method: Refer to step (1), the difference from step (1) is only that HCT116 cells are replaced with HT29 cells, and DMEM medium is replaced with an equal volume of RPMI 1640 medium.

[0097] (3) Experimental results:

[0098] The flow cytometry results and cell cycle conditions of human colorectal cancer HCT116 cells are as Figure 5 shown, and the flow cytometry results and cell cycle conditions of HT29 cells are as Figure 6 shown. It can be seen that for both HCT116 cells and HT29 cells, the proportion of cells in the G2 phase in the experimental group decreased significantly, and the proportion of G1 phase cells increased, indicating that quinone nucleobase caused more cells to arrest in the G1 phase and significantly inhibited the proliferation of human colorectal cancer cells.

[0099] Example 5

[0100] Analyze the effect of quinone nucleobase on the tumor growth of nude mice with colorectal cancer through animal experiments:

[0101] (1) Experimental method:

[0102] (1.1) Take HCT116 cells in good growth state, collect and resuspend the cultured HCT116 cells to prepare a cell suspension with a concentration of 1×10 7 cells / 200 μL; Take 20 6-week-old BALB / c nude mice (purchased from Guangdong Medicilon Biotech Co., Ltd.), and subcutaneously inject 200 μL of cell suspension with a concentration of 1×10 7A suspension of HCT116 cells at a density of 1 cell / 200 μL was used to establish a subcutaneous tumor model, and the mice were waited for three days until the tumor tissues were visible to the naked eye;

[0103] (1.2) The above 20 nude mice were randomly divided into an experimental group and a control group (10 mice in each group): The experimental group was intragastrically administered 100 μL of an aqueous solution of quinolinic acid at a concentration of 100 μmol / L every day. Correspondingly, the control group was intragastrically administered 100 μL of sterile water every day for 16 consecutive days; During the experiment, the body weight and tumor volume of the nude mice were recorded every 2 days; After 16 days of drug administration, the surviving mice were dissected, the tumor parts were excised, and the tumors were weighed;

[0104] Among them, the maximum longitudinal diameter (length) and maximum transverse diameter (width) of the tumor were measured with an electronic vernier caliper, and then the tumor volume was calculated using the modified ellipsoid formula: tumor volume = 1 / 2 × (length × width 2 ).

[0105] (2) Experimental results:

[0106] The effects of quinolinic acid on nude mice with colorectal cancer are as Figure 7 shown. Among them, A is a visual diagram of the tumors of the nude mice in the control group and the experimental group; B is a test of the ratio of tumor weight to nude mouse weight after 16 days of the experiment; C is the monitoring result of the ratio of tumor volume to nude mouse body weight during 16 days of drug administration, which is used to evaluate the disease progression of the nude mice. It can be seen that due to the intragastric administration of quinolinic acid, the growth rate of the tumors in the experimental group of nude mice decreased significantly, indicating that quinolinic acid can effectively inhibit the growth of tumors in mice with colorectal cancer.

[0107] The applicant declares that the present invention illustrates the technical solutions of the present invention through the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0108] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0109] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. Use of small molecule metabolites of quinine nucleobase or pharmaceutically acceptable salts, esters, and solvates thereof in the preparation of anti-colorectal cancer drugs; The structure of the small molecule metabolite quinone nucleobase is shown in Formula I; 2. The use according to claim 1, characterized in that: The drug also contains pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include any one of fillers, binders, wetting agents, disintegrants, solubilizers, osmotic pressure regulators, coating materials, colorants, pH regulators, antioxidants or antibacterial agents, or a combination of at least two thereof.

3. Use of small molecule metabolites of quinine nucleobase or pharmaceutically acceptable salts, esters, and solvates thereof in the preparation of colorectal cancer cell line proliferation inhibitors; The structure of the small molecule metabolite quinone nucleobase is shown in Formula I; 4. The use according to claim 3, characterized in that: The colorectal cancer cell lines include HCT116 cells and / or HT29 cells.

5. Use of small molecule metabolites quinone nucleobases or pharmaceutically acceptable salts, esters, and solvates thereof in the preparation of colorectal cancer cell line invasion inhibitors; The structure of the small molecule metabolite quinone nucleobase is shown in Formula I; 6. The use according to claim 5, characterized in that: The colorectal cancer cell line includes HCT116 cells.

7. Use of small molecule metabolites quinone nucleobases or pharmaceutically acceptable salts, esters, and solvates thereof in the preparation of inhibitors of migration of colorectal cancer cell lines; The structure of the small molecule metabolite quinone nucleobase is shown in Formula I; 8. The use according to claim 7, characterized in that: The colorectal cancer cell lines include HCT116 cells and / or HT29 cells.

9. Use of small molecule metabolites of quinine nucleobase or pharmaceutically acceptable salts, esters, and solvates thereof in the preparation of colorectal cancer cell line cycle arrest agents; The structure of the small molecule metabolite quinone nucleobase is shown in Formula I; 10. The use according to claim 9, characterized in that: The colorectal cancer cell lines include HCT116 cells and / or HT29 cells.

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