Application of riboflavin in preparation of medicine for treating colorectal cancer

Abstract

The invention discloses application of riboflavin in preparation of medicines for treating colorectal cancer, belongs to the field of development of new application of medicines, and particularly relates to application of riboflavin to in-vitro stimulation of primary CD8 + T cells and improvement of the activity of the CD8 + T cells to improve the capacity of killing colorectal cancer cell lines. Meanwhile, the invention also proves that the activity of immune cells in the body of a colorectal cancer patient can be obviously activated through a conventional drinking water preparation and riboflavin in an animal model for the first time, particularly through the activity of CD8 + T cells, so that the capability of killing tumors is improved, and the volume and weight of the tumors are obviously reduced; the riboflavin can be used for preparing the medicine for treating the colorectal cancer.

Description

Technical Field

[0001] The invention relates to the field of development of new drug uses, and in particular to the use of riboflavin in the preparation of drugs for treating colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is the most common malignant tumor of the digestive system. According to the latest data released by the World Health Organization, its global incidence ranks third among all malignant tumors, and its cancer-related mortality ranks second. In addition to the strong plasticity and heterogeneity of tumors, colorectal tumors are also affected by the complex intestinal environment. Although many treatment strategies have been clinically transformed and a large number of potential intervention targets have been continuously revealed, there is still no effective way to truly conquer intestinal tumors.

[0003] Theoretically, tumor cells, as abnormal cells in the human body, will be recognized by the immune system and further eliminated through the tumor immune cycle in the body. + T cells can secrete a large number of effector factors and are considered to be the executors and core effector members of anti-tumor immunity. However, tumor cells have a variety of immune escape mechanisms that can inhibit anti-tumor immune responses. Among them, nutritional competition is a key means for tumor cells to escape immune responses. With their strong nutrient uptake ability, tumor cells preferentially consume key nutrients in the microenvironment, thereby inhibiting CD8 + T cell function and proliferation.

[0004] Riboflavin, also known as vitamin B2, is an important component of cellular energy metabolism, antioxidant defense, and cell proliferation. It participates in multiple enzyme reactions in the body, promotes the metabolism of fats, proteins, and carbohydrates, and is an essential nutrient for the body. Previous studies compared the culture medium of normal intestinal epithelial cells with that of intestinal tumor cells and found that riboflavin was most significantly downregulated in the tumor culture environment and was also the nutrient with the most significant changes. Therefore, in view of the riboflavin-deficient microenvironment formed by the significant upregulation of riboflavin uptake by tumor cells, intervention measures were designed to restore CD8 + The powerful functions of T cells themselves have potential application value and prospects in the treatment of colorectal cancer. Summary of the invention

[0005] In view of this, the main purpose of the present invention is to provide a use of riboflavin in preparing a preparation for enhancing the immune system to fight against tumors.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows: The first aspect of the present invention is to provide the use of riboflavin in preparing a preparation for improving the immune activity of a subject, wherein the subject is a tumor patient; Furthermore, the subject is a colorectal cancer patient or a potential colorectal cancer cell carrier; Furthermore, the preparation is a daily drinking preparation containing riboflavin.

[0007] Furthermore, the immune activity is to enhance the activity of CD8+T cells.

[0008] Furthermore, the administration concentration of the riboflavin is 0.02-0.04 mg / mL.

[0009] The second aspect of the present invention is to provide the use of riboflavin in the preparation of a preparation for eliminating tumor cells inhibiting the activity of immune cells, wherein the immune cells are CD8+ T cells; Furthermore, the riboflavin is used at a concentration of 0.5-10 μM.

[0010] The third aspect of the present invention is to provide the use of riboflavin in the preparation of a preparation for enhancing the activity of immune cells in killing tumor cells, wherein the application is an in vitro application; Furthermore, the tumor cells are colorectal cancer cells; and the immune cells are CD8+ T cells.

[0011] Furthermore, the concentration of riboflavin used is 0.5~10μM.

[0012] The beneficial effects of the present invention include: 1) Based on the previous discovery that colorectal cancer cell lines and normal intestinal epithelial cells have huge differences in their metabolic requirements for riboflavin, the present invention designed experiments to confirm that riboflavin can stimulate primary CD8+T cells in vitro and enhance the activity of CD8+T cells to enhance their ability to kill colorectal cancer cell lines; 2) The present invention also confirmed for the first time in an animal model that riboflavin can significantly activate the activity of immune cells in colorectal cancer patients through conventional drinking water preparations, specifically through the activity of CD8+T cells, thereby enhancing their ability to kill tumors and significantly reducing the size and weight of tumors; suggesting that riboflavin can be used to prepare drugs for the treatment of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The effect of riboflavin treatment on the viability of primary CD8+T cells; Figure 2 Effects of riboflavin treatment on proliferation (A) and effector function (B) of primary CD8+T cells; Figure 3 Differences in riboflavin uptake between direct co-culture of MC38 cells and CD8+ T cells; Figure 4The effect of riboflavin supplementation on CD8+T cells in the transwell co-culture system of MC38 and primary CD8+T cells, A is a schematic diagram of the experimental process, B is a diagram of cell proliferation results, and C is a diagram of effector function results; Figure 5 This is a schematic diagram of the intervention of riboflavin drinking preparation on MC38 injected mice; Figure 6 The effect of riboflavin drinking preparation intervention on tumor size; Figure 7 The effect of riboflavin dietary intervention on intratumoral CD8+T cells. DETAILED DESCRIPTION

[0014] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0015] Example 1 In vitro experiment of riboflavin on CD8+ T cells killing colorectal cancer lines

[0016] 1.1 Primary mouse CD8 + T cell extraction and culture (1) WT mice were euthanized, the spleen was removed by dissection, rinsed in pre-cooled PBS, ground in 5 mL PBS using a pestle, and the cell suspension was passed through a 40 uM cell sieve; (2) Centrifuge at 300 × g for 5 min, pour off the liquid, add 1 mL of red blood cell lysis buffer (Biyuntian, C3702), let stand for 5 min, and add 5 mL of FACS (PBS containing 2.5% FBS) to terminate lysis; (3) Centrifuge at 300 × g for 5 min, pour off the liquid, and extract CD8 using EasySep Mouse CD8+ TCell Isolation Kit (Stemcell, 19853). + T cells; (4) Centrifuge at 300 × g for 5 min, pour off the liquid, and the precipitate is CD8+ T cells, which are cultured in RPMI-1640 medium containing 10% FBS, 2 mM HEPES (Gibico, 15630080), 200 μM sodium pyruvate (Gibico, 11360070), 50 μM 2-mercaptoethanol (Gibico, 31350010), and 20 ng / mL Recombinant Mouse IL-2 Protien (PeproTech, 51061-MNAE), and add 5 μg / mL anti-CD3 (Biolegend,) and anti-CD28 (Biolegend,) to activate CD8 + T cells; (4) After 48 hours, the cells were expanded and treated with different concentrations of riboflavin; (5) Determination of various indicators after 72 hours;

[0017] 1.2 Culture of mouse colorectal cancer cell line MC38 (1) Cells were cultured in RPMI-1640 (Roswell Park Memorial Institute medium 1640) containing 10% FBS. (2) If there are more than 80% adherent cell clusters, they can be subcultured. Remove the culture medium, add 1 mL of preheated sterile PBS for washing, digest with 1 mL of 0.25% trypsin at room temperature for 2 min, add 2 mL of complete culture medium to terminate, transfer to a sterile enzyme-free 15 mL centrifuge tube, and centrifuge at 500.g for 5 min. Remove the supernatant, add 2 mL of preheated complete culture medium to resuspend and blow evenly, divide evenly into multiple culture bottles according to a certain subculture ratio, add preheated complete culture medium to 5 mL / bottle, and shake crosswise to mix well.

[0018] (3) Culture in a 37°C, 5% CO2 cell culture incubator. Subculture at a 1:2 or 1:3 ratio every 2-3 days; (4) MC38 cells were seeded in 96-well plates; (5) After 24 hours, the cells were allowed to adhere to the wall and different concentrations of riboflavin were added for treatment; (6) Measure each index after 24 hours;

[0019] 1.3 Direct co-culture of MC38 and primary CD8+ T cells (1) MC38 cells were passaged and plated into 48-well plates; (2) 24 hours later, add 100 ng / mL Mouse IFN-gamma Recombinant Protein (3) After 24 hours, the MC38 medium was replaced and primary CD8 cells that had been pre-activated for 48 hours with anti-CD3 and anti-CD28 were added at a ratio of 1:1. + T cells were treated with different concentrations of riboflavin; (4) Measure each index after 72 hours;

[0020] 1.4 Transwell co-culture of MC38 and primary CD8+ T cells (1) MC38 cells were passaged and plated into the lower chamber of a 24-well Transwell; (2) 24 hours later, add 100 ng / mL Mouse IFN-gamma Recombinant Protein; (3) After 24 hours, the MC38 medium was replaced and primary CD8 cells pre-activated with anti-CD3 and anti-CD28 for 48 hours were added to the upper chamber at a ratio of 1:2. + T cells, and different concentrations of riboflavin were added to the upper and lower chambers; (4) After 72 hours, MC38 cells in the lower chamber and primary CD8 + T cells, to measure various indicators;

[0021] 1.5 Cell flow cytometry analysis (1) Collect cells; (2) Centrifuge at 300 × g for 5 min, remove the supernatant, resuspend in PBS and add fluorescently labeled antibodies, incubate at 4°C in the dark for 20 min to stain cell membrane proteins; (3) Centrifuge at 300 × g for 5 min, remove the supernatant, resuspend in PBS and add Zombie Aqua™ Fixable Viability Kit, and incubate at room temperature in the dark for 20 min; (4) Centrifuge at 300 × g for 5 min, remove the supernatant, and use Cyto-Fast™ Fix / Perm Buffer Set for fixation and permeabilization; Centrifuge at 300×g for 5 min, remove the supernatant, resuspend with PBS and add fluorescent-labeled antibodies, incubate at room temperature in the dark for 30 min to stain intracellular proteins.

[0022] 1.6 Detection of riboflavin uptake by cells (1) Collect cells treated with riboflavin; (2) Centrifuge at 300 × g for 5 min, remove the supernatant and resuspend in PBS; (3) Pass through a 40 μm cell sieve and transfer to a flow cytometry tube; (4) Since riboflavin has its own FITC fluorescence, flow cytometry was used to detect the FITC fluorescence of cells; (5) Calculate the average FITC fluorescence intensity using Flowjo.

[0023] 1.7 Cell proliferation and activity assay (1) Cells treated with the indicated treatments were seeded into 96-well plates; (2) Replace the culture medium with 10% CCK-8 solution (Beyotime, C0038); (3) After the cells were cultured for another 2 h at 37 °C, the absorbance was measured at 450 nm using a microplate reader;

[0024] 1.8 Results (1) Effect of riboflavin treatment on primary mouse CD8 + Effects on T cell activity: Figure 1~2 As shown in the figure, as the concentration of riboflavin increased (range 0.5~10μM), the relative cell viability of the isolated primary CD8+ T cells also increased significantly, and increased by nearly 1 times at 10μM. Figure 2 It showed that with the increase of riboflavin concentration, the activity of CD+8T cells was significantly improved. Figure 2 The relative number of Ki67+ cells in A increased with the increase of riboflavin concentration, and in the range of 2-10 μM, it increased by more than 50% compared with the control group, indicating that CD8+T proliferation was obvious. Figure 2 Middle B shows that the relative number of IFNγ+ cells increases with the increase of riboflavin concentration, indicating that CD8+T cells are activated and the cellular effect is significantly enhanced.

[0025] (2) MC38 and primary CD8 + Riboflavin uptake by T cells during direct co-culture: Results Figure 3 As shown, MC38 cells have a stronger uptake capacity for riboflavin than primary CD8+T cells; (3) Analysis of the activity and lethality of MC38 and primary CD8+T cells in Transwell co-culture. Figure 4 As shown, specifically, Figure 4 A represents the experimental process, Figure 4 B and Figure 4 C indicates that MC38 has a remote inhibitory effect on the proliferation and effector function of primary CD8+T cells, and this inhibitory effect will be eliminated by adding riboflavin to the co-culture system.

[0026] Example 2 Effect of riboflavin drinking water on inhibiting colorectal cancer in mice

[0027] 2.1 Experimental animals C57BL / 6J strain WT (wild type) mice and Apc min / + (miR-29ab1 whole body knockout) mice were housed in an SPF animal room at a room temperature of 20-22°C, a humidity of 50-60%, a 12 h light / dark cycle, free access to water, and fed with normal chow;

[0028] 2.2 MC38 subcutaneous injection mouse model and drinking water intervention program (1) Shave the back of 8-week-old C57BL / 6J WT mice; (2) Inject 1 × 10 5 MC38 cells (schematic diagram as shown Figure 5 shown); (3) On the day of injection, the drinking water of the experimental group mice was replaced with sterile pure water containing riboflavin. The low-dose group consumed 3 mg / kg per day, and the high-dose group consumed 6 mg / kg per day. The drinking water of the control group mice was sterile pure water without riboflavin. (4) Use a vernier caliper to measure the length, width, and height of the tumor daily; (5) After 14 days, the mice were euthanized and tissues were collected;

[0029] 2.3 Flow cytometry analysis of tumor tissue (1) Take a portion of the tumor, cut it into pieces with scissors, transfer it into a 15 mL sterile enzyme-free centrifuge tube, and rinse it several times with PBS pre-cooled at 4°C; (2) Add 5 mL of digestion solution (DMEM / F12 medium containing 300 U / mL type II collagenase (Gibico, 17101015) and 0.1 U / mL dispase® II (Roche, 04942078001)) preheated at 37°C to the centrifuge tube containing the tissue and digest for 30 min in a shaking incubator at 37°C; (3) Pour off the digestion solution and gently rinse with PBS to remove the digestion solution, then add 10 mL of PBS and shake vigorously to separate the cells, then pass the cell suspension through a 40 uM cell sieve to obtain a single cell suspension; (4) Centrifuge at 300 × g for 5 min. Subsequent staining steps are the same as in 2.8.

[0030] 2.4 Results (1) Results of tumor volume and weight in mice Figure 6 As shown in the figure, it can be seen that after feeding drinking water containing riboflavin, the tumor volume and weight of mice were significantly lower than those of the control group, and had a dose-dependent characteristic. Figure 6 A in the middle is a visual measurement diagram of tumor size. Figure 6B in the figure is the result of counting the tumor volume. Figure 6 C in the figure is the result of counting tumor weight; (2) Figure 7 A in the middle shows that the proportion of effector CD8+T cells in mouse tumors increased after riboflavin intervention. Figure 7 Figure B shows the improvement of the effector function of CD8+T cells after riboflavin intervention.

[0031] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. Use of riboflavin in preparing a preparation for improving the immune activity of a subject, characterized in that: The subject is a tumor patient.

2. The use according to claim 1, characterized in that The subject is a colorectal cancer patient or a potential colorectal cancer cell carrier.

3. The use according to claim 1 or 2, characterized in that The preparation is a daily drinking preparation containing riboflavin.

4. The use according to claim 3, characterized in that The immune activity is to enhance the activity of CD8+T cells.

5. The use according to claim 3, characterized in that: The administration concentration of riboflavin is 0.02-0.04 mg / mL.

6. Use of riboflavin in the preparation of a preparation for eliminating tumor cells inhibiting the activity of immune cells, characterized in that: The immune cells are CD8+ T cells.

7. The use according to claim 6, characterized in that: The riboflavin is used at a concentration of 0.5-10 μM.

8. The use of riboflavin in the preparation of a preparation for enhancing the activity of immune cells in killing tumor cells, characterized in that: The application described is an in vitro application.

9. The use according to claim 8, characterized in that: The tumor cells are colorectal cancer cells; the immune cells are CD8+ T cells.

10. The use according to claim 8 or 9, characterized in that: The concentration of riboflavin used is 0.5~10μM.

Images