Application of indobufen in enhancing antitumor activity of natural killer cells

Abstract

The invention provides application of indobufen in enhancing the antitumor activity of natural killer cells, which is characterized in that the indobufen is added into a solution, suspension or culture medium containing the natural killer cells, so that the concentration of the indobufen is not higher than 10 mu M. Indobufen can enhance the anti-tumor activity of NK cells under the condition of lower concentration, firstly, the indobufen is not limited to specific tumor cells aiming at the tumor cells and has broad spectrum, and secondly, the enhancement effect of the indobufen on the killing activity of the NK cells does not depend on the effect-target ratio.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to a use of indobufen in enhancing the anti-tumor activity of natural killer cells. Background Art

[0002] Natural killer cells (NK cells) are core cells of the innate immune system. They have direct cytotoxicity and participate in adaptive immune responses, and they occupy an important position in the entire immune network. Studies have shown that the killing activity of NK cells and the number of NK cells infiltrating into tumors are significantly correlated with the risk of cancer and the prognosis of cancer treatment. Therefore, improving the anti-tumor activity of NK cells has always been one of the research focuses of NK cell immunotherapy strategies.

[0003] Indobufen, chemical formula is C 18 H 17 NO3, Indobufen is an antiplatelet aggregation drug, mainly used to prevent and treat ischemic cardiovascular disease, ischemic cerebrovascular disease and venous thrombosis caused by arteriosclerosis, and can also be used to prevent thrombosis during hemodialysis. It works by inhibiting platelet cyclooxygenase, reducing platelet factor levels and reducing platelet aggregation. There are currently no reports on the anti-tumor activity of indobufen affecting natural killer cells. Summary of the invention

[0004] In view of this, the purpose of the embodiments of the present application is to provide a use of indobufen in enhancing the anti-tumor activity of natural killer cells, so as to improve the anti-tumor activity of NK cells.

[0005] According to a first aspect of an embodiment of the present invention, there is provided a use of indobufen in enhancing the anti-tumor activity of natural killer cells, wherein indobufen is added to a solution, suspension or culture medium containing natural killer cells so that the concentration of indobufen is no higher than 10 μM.

[0006] Furthermore, the number ratio of the natural killer cells to tumor cells, i.e., the effector-target ratio, is (1:1) to (50:1).

[0007] Furthermore, the effect-target ratio is (5:1) to (50:1).

[0008] Furthermore, the natural killer cells are NK-92mi cells.

[0009] Furthermore, the indobufen concentration is 10 nM-10 μM.

[0010] According to a second aspect of an embodiment of the present invention, there is provided a drug for enhancing the anti-tumor activity of natural killer cells, the drug comprising indobufen, wherein the indobufen is in a free form or in the form of a pharmaceutically acceptable compound; and a pharmaceutically acceptable carrier.

[0011] Furthermore, the carrier is any one or more of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant.

[0012] Furthermore, the drug is in the form of oral tablets, granules, injections or capsules.

[0013] According to a third aspect of an embodiment of the present invention, there is provided a use of indobufen in preparing an anti-tumor preparation, the anti-tumor preparation comprising: indobufen, human natural killer cells and a pharmaceutically acceptable carrier; Alternatively, the anti-tumor preparation comprises: natural killer cells treated with indobufen and a pharmaceutically acceptable carrier; the natural killer cells are obtained by in vitro culture.

[0014] According to a fourth aspect of an embodiment of the present invention, a culture medium for enhancing the anti-tumor activity of NK cells is provided, wherein the culture medium comprises a basal nutrient medium and indobufen, wherein the basal nutrient medium is a culture medium for maintaining basal nutrition of NK cells.

[0015] It should be noted that the antitumor activity of NK cells in the present application refers to the ability of NK cells to kill tumor cells, that is, the ability to cause tumor cell growth stagnation or death by inhibiting tumor cell proliferation or tissue growth. The tumor is a tumor that can be recognized by natural killer cells, including but not limited to PANC-1 cells and Hep3B cells.

[0016] The technical solution provided by the embodiments of the present application may have the following beneficial effects: It can be seen from the above technical scheme that indobufen can enhance the anti-tumor activity of NK cells from various sources at a relatively low concentration (starting from 10nM), and enhance the anti-tumor activity of NK cells on the basis of ensuring the high survival of NK cells to achieve the anti-tumor effect; firstly, the targeted tumor cells are not limited to specific tumor cells and have a broad spectrum; secondly, under different effector-target ratios, the enhancing effect of indobufen on the killing activity of NK cells shows an increasing trend. Under the condition of 1:1 effector-target ratio, the killing rate of cultured NK cells on PANC-1 cells is increased by an average of 8.84%, and under the condition of 5:1 effector-target ratio, the killing rate of cultured NK cells on PANC-1 cells is increased by 1.5%. The killing rate of cultured NK cells on PANC-1 cells was increased by an average of 18.70% under the condition of 10:1 effector-target ratio, and by an average of 21.70% under the condition of 50:1 effector-target ratio, and by an average of 27.49% under the condition of 1:1 effector-target ratio. The killing rate of cultured NK cells on Hep3B cells was increased by an average of 8.82% under the condition of 1:1 effector-target ratio, and by an average of 12.39% under the condition of 5:1 effector-target ratio, and by an average of 20.82% under the condition of 10:1 effector-target ratio, and by an average of 33.92% under the condition of 50:1 effector-target ratio.

[0017] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0019] Figure 1 1 is the effect of different concentrations of indobufen on the growth activity of PANC-1 cells shown in an example of the present invention.

[0020] Figure 2 1 is the effect of different concentrations of indobufen on the growth activity of Hep3B cells shown in an example of the present invention.

[0021] Figure 3 FIG. 1 is the effect of different concentrations of indobufen on the growth activity of NK-92mi cells shown in an example of the present invention.

[0022] Figure 4 1 is a graph showing the corresponding relationship between the killing rate of NK-92mi cells killing PANC-1 cells and the effector-target ratio after being treated with 10 nM indobufen in an embodiment of the present invention.

[0023] Figure 5 1 is a graph showing the corresponding relationship between the killing rate of Hep3B cells killed by NK-92mi cells and the effector-target ratio after being treated with 10 nM indobufen in an embodiment of the present invention.

[0024] Figure 6 The real-time quantitative PCR analysis of the mRNA expression levels of perforin and granzyme B is shown in one embodiment of the present invention. DETAILED DESCRIPTION

[0025] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0026] Table 1 Experimental reagents name Specification factory Indobufen 1G，>98.0% Shanghai THIAI Natural Killer Cells (NK-92mi) - Punosa CCK-8 5mL APExBIO LDH Cytotoxicity Detection Kit 500 assays Shanghai Grandview Hotel Human pancreatic cancer PANC-1 cells - Punosa Human Hep 3B Cells - Punosa RNA Extraction Kit 50 rxns Nanjing Novozyme Reverse transcription master mix 100 rxns Nanjing Novozyme qPCR dye method fluorescence quantitative special premix 500 RXNS (20 μL / RXN) Nanjing Novozyme All experimental data below are expressed as mean ± standard error (mean ± SE), and Origin (9.8) is used for data plotting. SPSS Statistics 27 was used for data analysis, and Student's T test was used for significance analysis between two groups, and one-way analysis of variance was used for significance analysis between multiple groups. P>0.05 is not statistically different (nosignificant, ns), P<0.05 is statistically different (*), P<0.01 is significantly different (**), and P<0.001 is extremely significantly different (***).

[0027] Cell culture media used in the following experiments: Among them, the components of the NK-92mi cell complete culture medium include: MEMa; Inositol, FolicAcid; 0.1mMβ-mercaptocthano; 12.5% ​​HS, 12.5% ​​FBS; 1% P / S.

[0028] The complete culture medium for PANC-1 cells includes: DMEM; 10% FBS.

[0029] The complete culture medium for Hep 3B cells includes: MEM; 10% FBS.

[0030] Experiment 1: Cytotoxicity of indobufen on PANC-1 cells, Hep 3B cells or NK-92mi cells The effects of indobufen on the cell activities of PANC-1 cells, Hep 3B cells and NK-92mi cells were measured respectively to determine the cytotoxicity of indobufen on these three cells. CCK-8, i.e. Cell Counting Kit-8 reagent, was used to measure cell activity.

[0031] Specifically, the cytotoxicity test of indobufen on PANC-1 cells, Hep 3B cells and NK-92mi cells was carried out according to the following operating steps. Different groups were set up for the toxicity test of each cell: a blank control group containing only culture medium, a cell group without drug addition and a drug addition group, with 6 parallel samples in each group. The specific operation was as follows: resuspend the cells in the logarithmic growth phase and adjust the cell concentration to 5×10 4 / mL, 100μL per well was plated in a 96-well flat-bottom culture plate overnight. The next day, for the adherent PANC-1 cells and Hep 3B cells, the culture medium was discarded and washed once with PBS; this step was omitted for the suspended NK-92mi cells; and the drug concentration was given according to the above; then the cells were cultured for 24h in a 37℃, 5% volume concentration CO2 incubator. After 24h, 20μL of CCK-8 reagent was added to each well, mixed on a shaker, and then placed in a 37℃, 5% volume concentration CO2 incubator for another 2h. The absorbance S103 photometry, i.e., OD value, was measured at 450nm using an enzyme reader.

[0032] The cytotoxicity of indobufen on PANC-1 cells, Hep 3B cells or NK-92mi cells was calculated as follows: cell viability (%) = (OD of drug-treated group-OD of blank control group) / (OD of non-drug-treated group-OD of blank control group) × 100%.

[0033] The results are as follows Figure 1 The figure shows the effect of different concentrations of indobufen on the growth activity of PANC-1 cells. It can be seen that within the concentration of 1 μM, indobufen has almost no cytotoxicity to PANC-1 cells; Figure 2 The figure shows the effect of different concentrations of indobufen on the growth activity of Hep 3B cells. It can be seen that within the concentration of 1 μM, indobufen has almost no cytotoxicity to Hep 3B cells; it can be determined that the cell viability of the two cancer cells is above 85% at a final concentration of 1 μM indobufen. Figure 3 Shown is the effect of different concentrations of indobufen on the growth activity of NK-92mi cells; it can be seen that within the concentration of 1 μM, indobufen has almost no cytotoxicity to NK-92mi cells.

[0034] Experiment 2: Effect of indobufen on the cytotoxic activity of NK-92mi cells This experiment used a lactate dehydrogenase cytotoxicity assay kit, i.e., LDH reagent, to determine the effect of indobufen on the cell killing activity of NK-92mi cells, and the steps were as follows: Seven experimental groups were set up: in a 96-well flat-bottom culture plate, the groups were set as: effector cell natural release group, target cell natural release group without drug treatment, target cell maximum enzyme activity group without drug treatment, effector cell killing cancer cell group without drug treatment, target cell release group treated with drug, effector cell killing cancer cell group treated with drug, and target cell maximum enzyme activity group treated with drug; each group had 3 parallel samples.

[0035] (1) Effect of cytotoxic activity of NK cells without drug treatment The groups were divided into the effector cell natural release group, the target cell natural release group without drug treatment, the target cell maximum enzyme activity group without drug treatment, and the effector cell killing cancer cell group without drug treatment. The specific treatment steps were as follows: the cells in the logarithmic growth phase were resuspended and the cell concentration was adjusted to 5×10 4 / mL, in a 96-well flat-bottom culture plate, 100μL per well was plated overnight; NK-92mi cells were not treated for the time being. NK-92mi effector cells in the logarithmic growth phase were resuspended and the cell concentration was adjusted and plated in a 96-well plate, 100μL per well; effector cells in the logarithmic growth phase were resuspended and the cell concentration was adjusted and plated in the effector cell killing cancer cell group; the other two groups were not treated for the time being. Then continue to culture in a 37°C, 5% volume concentration CO2 incubator for 24 h. 1 hour before the scheduled time point, take out the 96-well plate, add 20 μL of LDH release reagent to the target cell maximum enzyme activity group, and mix it by blowing several times repeatedly; 1 hour later, take 120 μL of the supernatant from each well and add it to a new 96-well plate, and then perform sample determination; according to the number of samples to be determined, prepare the LDH detection working solution in a volume ratio of lactic acid solution: INT solution: enzyme solution = 1:1:1; add 60 μL of LDH detection working solution to each well, mix it on a shaker, and incubate it at room temperature in the dark for 30 min. Use an enzyme reader to measure the absorbance at 490 nm.

[0036] (2) Effects of drugs on NK cell activity The cells were divided into the effector cell natural release group, the drug-treated target cell (PANC-1 and Hep3B cell) natural release group, the drug-treated target cell maximum enzyme activity group, and the drug-treated effector cell killing cancer cell group. The specific treatment steps were to resuspend the cells in the logarithmic growth phase and adjust the cell concentration to 5×10 4 / mL, in a 96-well flat-bottom culture plate, 100μL per well was plated overnight; NK-92mi cells were not treated for the time being. The next day, the NK-92mi effector cells in the logarithmic growth phase were resuspended and the cell concentration was adjusted (the ratio of NK-92mi cells: target cells was 1:1-50:1) and plated in a 96-well plate, 100μL per well; the effector cells in the logarithmic growth phase were resuspended and the cell concentration was adjusted to plate in the effector cell killing cancer cell group; the other two groups were not treated for the time being; and indobufen was added at a final concentration of 10 nM; and then cultured for 24 h in a 37°C, 5% volume concentration CO2 incubator. 1 hour before the scheduled time point, take out the 96-well plate, add 20 μL of LDH release reagent to the target cell maximum enzyme activity group, and mix it by blowing several times repeatedly; 1 hour later, take 120 μL of the supernatant from each well and add it to a new 96-well plate, and then perform sample determination; according to the number of samples to be determined, prepare the LDH detection working solution in a volume ratio of lactic acid solution: INT solution: enzyme solution = 1:1:1; add 60 μL of LDH detection working solution to each well, mix it on a shaker, and incubate it at room temperature in the dark for 30 minutes. Use an enzyme reader to measure the absorbance at 490 nm.

[0037] The killing rate of NK against cancer cells was calculated as follows: The killing rate of NK-92mi against cancer cells (%) = (OD of the effector cell killing cancer cell group without drug treatment - OD of the target cell release group without drug treatment - OD of the effector cell natural release group) / (OD of the target cell maximum enzyme activity group without drug treatment - OD of the target cell natural release group without drug treatment) × 100%.

[0038] The effect of the drug on NK cell activity was calculated as follows: NK-92mi killing rate of cancer cells (%) = (OD of drug-treated effector cell killing cancer cell group - OD of drug-treated target cell release group - OD of effector cell natural release group) / (OD of drug-treated target cell maximum enzyme activity group - OD of drug-treated target cell natural release group) × 100%.

[0039] Figure 4 and Figure 5The figures are the effects of indobufen on the killing activity of NK-92mi cells against PANC-1 cells and Hep 3B cells. It can be seen that the enhancement effect of indobufen on the killing activity of NK-92mi cells is not limited to one type of cancer cell, but has a broad spectrum. Secondly, under different effector-target ratios, the enhancing effect of indobufen on the killing activity of NK cells showed an increasing trend. Under the condition of 1:1 effector-target ratio, the killing rate of cultured NK cells on PANC-1 cells was increased by 8.84% on average, under the condition of 5:1 effector-target ratio, the killing rate of cultured NK cells on PANC-1 cells was increased by 18.70% on average, under the condition of 10:1 effector-target ratio, the killing rate of cultured NK cells on PANC-1 cells was increased by 21.70% on average, and under the condition of 50:1 effector-target ratio, the killing rate of cultured NK cells on PANC-1 cells was increased by 27.49% on average; under the condition of 1:1 effector-target ratio, the killing rate of cultured NK cells on Hep3B cells was increased by 8.82% on average, under the condition of 5:1 effector-target ratio, the killing rate of cultured NK cells on Hep3B cells was increased by 12.39% on average, and under the condition of 10:1 effector-target ratio, the killing rate of cultured NK cells on Hep3B cells was increased by 20.82% on average. Under the condition of 50:1 effector-target ratio, the killing rate of cultured NK cells on Hep3B cells was increased by an average of 33.92%.

[0040] Experiment 3: Analysis of mRNA expression levels of perforin and granzyme B using real-time PCR NK-92 mi cells were treated with indobufen at 0, 10 nM, 100 nM and 1000 nM for 24 h, and total RNA was extracted using an RNA kit and reverse transcribed into cDNA. Fluorescence quantitative PCR was used to detect the expression levels of perforin mRNA and granzyme B mRNA. Figure 6 The real-time quantitative PCR analysis of the mRNA expression levels of perforin and Granzyme B shown in one embodiment of the present invention. Figure 6 It can be seen that indobufen can significantly increase the mRNA expression levels of perforin and Granzyme B in NK-92mi cells within the dose range of 10nM-1000nM.

[0041] Those skilled in the art will readily appreciate other embodiments of the present application after considering the description and practicing the contents disclosed herein. The present application is intended to cover any modification, use or adaptation of the present application, which follows the general principles of the present application and includes common knowledge or customary techniques in the art that are not disclosed in the present application. The description and examples are intended to be exemplary only, and the true scope and spirit of the present application are indicated by the claims.

[0042] It should be understood that the present application is not limited to the precise structures that have been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present application is limited only by the appended claims.

Claims

1. Use of indobufen in enhancing the anti-tumor activity of natural killer cells, characterized in that: Indobufen is added to a solution, suspension or culture medium containing natural killer cells so that the concentration of indobufen is no higher than 10 μM.

2. The use according to claim 1, characterized in that The number ratio of the natural killer cells to the tumor cells, ie, the effect-target ratio, is (1:1) to (50:1).

3. The use according to claim 2, characterized in that The effector-target ratio is (5:1) to (50:1).

4. The use according to claim 1, characterized in that The natural killer cells are NK-92mi cells.

5. The use according to claim 1, characterized in that The indobufen concentration is 10 nM-10 μM.

6. A drug for enhancing the anti-tumor activity of natural killer cells, the drug comprising indobufen, wherein the indobufen is in a free form or in the form of a pharmaceutically acceptable compound; and comprising a pharmaceutically acceptable carrier.

7. The drug according to claim 6, characterized in that The carrier is any one or more of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant.

8. The drug according to claim 6, characterized in that The drug is in the form of oral tablets, granules, injections or capsules.

9. Use of indobufen in the preparation of an anti-tumor preparation, characterized in that: The anti-tumor preparation comprises: indobufen, human natural killer cells and a pharmaceutically acceptable carrier; Alternatively, the anti-tumor preparation comprises: natural killer cells treated with indobufen and a pharmaceutically acceptable carrier; the natural killer cells are obtained by in vitro culture.

10. A culture medium for enhancing the anti-tumor activity of NK cells, characterized in that: The culture medium comprises a basic nutrient medium and indobufen, and the basic nutrient medium is a culture medium for maintaining basic nutrition of NK cells.

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