Application of crotonic acid and / or crotonyl-coenzyme A in preparation of medicine for treating tumors

Abstract

The invention belongs to the technical field of biological medicines, particularly relates to the technical direction of cooperation of medical oncology treatment and immunotherapy, and provides application of crotonic acid and / or crotonyl-coenzyme A in preparation of medicines for treating tumors. It is confirmed for the first time that crotonic acid can inhibit proliferation and migration of various solid tumor cells including breast cancer, liver cancer and colorectal cancer, the inhibition effect of crotonyl coenzyme A on breast cancer metastasis is defined, the research blank of crotonyl coenzyme A and crotonyl coenzyme A in the field of tumor treatment is filled, and short-chain fatty acid and short-chain fatty-acyl coenzyme A anti-tumor substance libraries are enriched. Meanwhile, it is proved that crotonic acid can up-regulate expression of tumor tissue PD-L1, when the crotonic acid is combined with the PD-L1 monoclonal antibody, the cancer suppression effect is remarkably better than that of independent medication, the immunotherapy effect is improved, the applicable crowd is expanded, and a novel treatment substance with a broad-spectrum cancer suppression effect and a synergistic treatment strategy are provided.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically involving the technical direction of synergistic treatment of tumors with immunotherapy, and provides the application of crotonic acid and / or crotonyl-CoA in the preparation of tumor treatment drugs. Background Technology

[0002] Malignant tumors (such as breast cancer, cervical cancer, esophageal cancer, and colorectal cancer) are major diseases threatening human health worldwide. Their high proliferative and metastatic potential is the core reason for poor clinical treatment outcomes and poor patient prognosis. Currently, although cancer treatment has formed a system combining surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy, it still faces many unresolved technical bottlenecks. The limitations of existing treatment regimens are becoming increasingly apparent, necessitating the development of new mechanisms of action, new therapeutic substances, or new combination therapy strategies to overcome clinical challenges.

[0003] In the field of tumor metabolism-related therapeutic research, short-chain fatty acids (aliphatic carboxylic acids composed of 1-6 carbon atoms, such as acetic acid, propionic acid, butyric acid, and valeric acid) have become a research hotspot in recent years due to their potential to inhibit cancer cell proliferation and migration. Existing studies have confirmed that supplementing with short-chain fatty acids from dietary sources or those produced by gut bacteria metabolism can inhibit various cancer cells by regulating tumor cell energy metabolism and cell cycle, with butyrate showing the most significant inhibitory effect. However, the short-chain fatty acid family includes many members, each with significantly different biological functions. Current research on short-chain fatty acids mainly focuses on widely studied types such as acetic acid, propionic acid, and butyric acid, while research on crotonic acid, another short-chain fatty acid, has long been lacking. Existing technologies have neither clarified whether crotonic acid possesses activity in inhibiting in situ tumor growth and metastasis, nor explored its potential application in tumor treatment. This has resulted in the untapped potential of short-chain fatty acids in tumor treatment, hindering the provision of more novel treatment options based on short-chain fatty acids for clinical practice.

[0004] Meanwhile, the role of acyl-CoA (a thioester derivative formed by the combination of fatty acyl groups and coenzyme A in fatty acid metabolism) in tumor metabolic regulation has gradually gained attention. Acyl-CoA can be classified into short-chain, medium-chain, and long-chain types according to fatty acid chain length, and is widely distributed in the cytoplasm and mitochondria, participating in key physiological processes such as cellular energy metabolism and lipid synthesis. Studies have found that some acyl-CoA (such as acetyl-CoA) can play a pro-cancer role in tumor development and progression by accelerating energy supply to tumor cells and promoting tumor cell proliferation. Furthermore, targeted drugs based on the mechanism of "inhibiting acetyl-CoA-mediated fatty acid oxidation" have been successfully marketed. However, apart from acetyl-CoA, the roles and mechanisms of other short-chain acyl-CoA (such as crotonyl-CoA) in tumors have not been studied: existing technologies are unclear whether crotonyl-CoA participates in the process of tumor development, have not revealed its regulatory effects on tumor cell proliferation and migration, and have not explored its application value as a tumor therapeutic substance. This results in an incomplete regulatory network of acyl-CoA in tumor treatment, making it impossible to form a treatment strategy that covers more acyl-CoA members, thus limiting the development of tumor treatment technologies based on the acyl-CoA metabolic pathway.

[0005] In the field of tumor immunotherapy, PD-L1 monoclonal antibodies, as typical immune checkpoint inhibitors, can exert good therapeutic effects on some cancer patients by blocking the PD-L1 / PD-1 signaling pathway and relieving tumor cell immune escape. However, in clinical applications, PD-L1 monoclonal antibody monotherapy has problems such as limited response rate and some patients not benefiting due to low PD-L1 expression in tumor tissue. There is an urgent need to find substances that can synergize with PD-L1 monoclonal antibodies to improve the therapeutic effect of PD-L1 monoclonal antibodies and expand their applicable population through "combination therapy" strategies. However, current technology has not yet discovered short-chain fatty acids or their related metabolites that can regulate PD-L1 expression in tumor tissue and thus produce a synergistic anti-cancer effect with PD-L1 monoclonal antibodies. This results in a limited number of PD-L1 monoclonal antibody combination therapy options, making it difficult to meet the clinical demand for highly effective and broad-spectrum tumor immunotherapy combination therapy.

[0006] In summary, existing tumor treatment technologies have significant gaps and deficiencies in the application of short-chain fatty acids, the exploration of the function of short-chain acyl-CoA, and the development of combined treatment strategies with immune checkpoint inhibitors. There is an urgent need to develop new substances with anti-cancer activity (such as specific short-chain fatty acids or their metabolites) and new combination treatment regimens to address the limited efficacy and narrow applicability of existing treatments and to provide new technological pathways for tumor treatment. Summary of the Invention

[0007] The purpose of this invention is to address the technical problems in existing tumor treatment technologies, such as the lack of application of short-chain fatty acids and short-chain acyl-CoA substances and the inadequacy of tumor immunotherapy combined treatment programs, and to provide novel therapeutic substances and synergistic treatment strategies with broad-spectrum anti-cancer effects.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] The first aspect of the present invention provides the use of crotonic acid and / or crotonyl-CoA in the preparation of a tumor-treating drug.

[0010] Furthermore, the tumor is a solid tumor, including breast cancer, liver cancer, and colorectal cancer.

[0011] Furthermore, the drug is used to inhibit the proliferation or migration of tumor cells.

[0012] Furthermore, the tumor cells include breast cancer cells MDA-MB-231, breast cancer cells SUM159, breast cancer cells py8119, breast cancer cells MCF7, liver cancer cells HepG2, or colorectal cancer cells SW620.

[0013] A second aspect of the present invention provides a pharmaceutical composition for treating tumors, comprising crotonic acid and / or crotonyl-CoA.

[0014] Furthermore, the pharmaceutical composition also contains a PD-L1 monoclonal antibody.

[0015] Preferably, in the pharmaceutical composition, the mass ratio of crotonic acid to PD-L1 monoclonal antibody is 8:1, and the tumor inhibition rate is significantly improved when the two are used in combination compared with the use of PD-L1 monoclonal antibody alone.

[0016] Furthermore, the tumor is a solid tumor, including breast cancer.

[0017] Furthermore, the pharmaceutical composition is used to inhibit the in situ growth or metastasis of tumor cells. The advantages of this invention compared to the prior art are as follows:

[0018] 1. Filling a technological gap: This application is the first to demonstrate that crotonic acid can inhibit the proliferation and migration of various solid tumor cells, such as breast cancer and cervical cancer, and clarifies the inhibitory effect of crotonyl-CoA on breast cancer metastasis, filling the research gap between the two in the field of tumor treatment and enriching the library of anti-tumor substances such as short-chain fatty acids and short-chain acyl-CoA.

[0019] 2. Excellent anti-cancer effect: It can simultaneously inhibit the in situ growth and metastasis of tumors. Crotonic acid can reduce the volume of in situ tumors in mice by gavage. Both crotonic acid and crotonyl-CoA can reduce lung and liver metastases of breast cancer. Moreover, crotonic acid is effective against a variety of solid tumors and has a wide range of applications.

[0020] 3. Breaking through the bottleneck of immunotherapy: Crotonic acid can upregulate the expression of PD-L1 in tumor tissues. When used in combination with PD-L1 monoclonal antibodies, the anti-cancer effect is significantly better than that of monotherapy, which can improve the effect of immunotherapy and expand the applicable population.

[0021] 4. Easy to translate into clinical practice: In animal experiments, the effective dose did not cause abnormal weight in mice, and the safety was good; crotonic acid can be administered by gavage, and crotonate has good solubility and stability, making it easy to prepare oral formulations, which is simple to operate and has high patient compliance. Attached Figure Description

[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0023] Figure 1 This study demonstrates the inhibition of breast cancer cell proliferation and migration by crotonic acid. Specifically, (A) the WST1 assay detected the effect of different concentrations of sodium crotonate (2.5, 5, 10, 15, 20 mmol / L) on the proliferation of MDA-MB-231 cells. Differential analysis was performed using two-way ANOVA. (B) A plate colony assay detected the effect of different concentrations of sodium crotonate (2.5, 5, 10, 15, 20, 25 mmol / L) on the colony-forming ability of MDA-MB-231 cells. Cells in three replicates of each plate colony were quantified and statistically analyzed using ImageJ, followed by statistical analysis using Graphpad Prism 9. (C) A Transwell assay detected the changes in the motility of MDA-MB-231 cells after 72 h of treatment with different concentrations of sodium crotonate in (B). Five different fields of view were taken from each group under a microscope, and the number of migrating cells was quantified and statistically analyzed using ImageJ, followed by statistical analysis using Graphpad Prism 9. (D) Transwell assay was used to detect the effect of 20 mmol / L sodium crotonate on the migration ability of various breast cancer cell lines, liver cancer cell lines, and colorectal cancer cell lines. Statistical methods were the same as in (C). (E) After treating MDA-MB-231 cells with 20 mmol / L sodium crotonate for 72 h, the migration trajectories of individual cells were plotted using TAXIScan-FL software. Approximately 12 cells were selected in each group, and the cell migration trajectories were plotted starting from the same point. Using the same horizontal line as the starting point of cell migration, the direction of the serum concentration gradient from low to high perpendicular to the starting horizontal line was used as the direction of cell migration. The straight-line distance of cell migration in the vertical direction was measured as the effective migration distance. The average migration distance of all selected cells in each group was calculated. Based on the migration distance and movement time of each cell, the migration speed of each cell was calculated.

[0024] Figure 2This study demonstrates the ability of crotonyl-CoA to inhibit breast cancer cell migration. Specifically, (A) after treating SUM159 cells with 60 μmol / L crotonyl-CoA for 72 h, the movement of individual cells was observed in real-time using TAXIScan-FL software, and trajectory curves were plotted. The length of the trajectory of each individual cell was measured and statistically analyzed to calculate the average migration speed of each cell. (B) Using the same horizontal line as the starting point of cell migration, the direction of the serum concentration gradient from low to high, perpendicular to the starting horizontal line, was used as the direction of cell migration. The straight-line distance of cell movement in the vertical direction was measured as the effective migration distance. The effective migration distance and migration speed were calculated.

[0025] Figure 3 This study demonstrates the ability of crotonic acid to inhibit in situ tumorigenesis of breast cancer cells. (A) Flowchart of the mouse in situ tumorigenesis experiment. MDA-MB-231 cells were injected into the right fourth pair of mammary fat pads of BALB / c-nu nude mice. After approximately 28 days, most mice showed visible tumors. The tumor-bearing mice were divided into two groups based on tumor size, and each group was treated with saline and sodium crotonate (60 mg / kg) by gavage every two days. The mice were sacrificed on day 54 for subsequent experiments. py8119 cells were injected into the right fourth pair of mammary fat pads of C57BL / 6N mice. After approximately 2 days, most mice showed visible tumors. Subsequent experiments were conducted using the same method, with sodium crotonate administered at a concentration of 200 mg / kg by gavage. The mice were sacrificed on day 14 for tissue collection. (B) Tumor size in BALB / c-nu nude mice was recorded every three days from day 36 to day 54, and growth curves were plotted. All tumor conditions are shown in the figure, n=5. (C) On day 54, the mice in both groups were weighed to analyze whether there was a statistically significant difference in body weight after administration of sodium crotonate. (D) Tumor size in C57BL / 6N mice was recorded every 2-3 days, and growth curves were plotted. All tumor data are shown in the figure, n=5. (E) On day 14, the mice in both groups were weighed to analyze whether there was a statistically significant difference in body weight after administration of sodium crotonate.

[0026] Figure 4The diagram shows the inhibition of lung and liver metastases of breast cancer cells by crotonic acid. (A) Flowchart of the BALB / c-nu nude mouse breast cancer lung metastasis experiment. 4-6 week old BALB / c-nu nude mice were injected intravenously with (iv) MDA-MB-231-luci cell line. The mice were then divided into groups and administered physiological saline or different concentrations of sodium crotonate by gavage every two days. Lung metastasis of breast cancer was observed on days 35, 42, and 49. The mice were then sacrificed for tissue collection for subsequent experiments. (B) IVIS imaging results of the tail vein mouse model. Mice underwent IVIS in vivo imaging on days 35, 42, and 49 after cell injection via the tail vein to observe and photograph the lung metastasis of breast cancer cells in the mice. (C) Fluorescence intensity was calculated for each IVIS imaging photograph of the lung-metastatic mice, and statistical differences between different groups were compared. Lung tissue was collected after sacrifice, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). One mouse from each group was selected for display. (D) Flowchart of the BALB / c-nu nude mouse breast cancer systemic metastasis model experiment. 4-6 week old BALB / c-nu nude mice were cardiacly injected with the (ici) MDA-MB-231-luci cell line. The mice were then divided into groups and administered physiological saline or different concentrations of sodium crotonate by gavage every two days. Lung metastasis of breast cancer was observed on days 21 and 28. The mice were then sacrificed for tissue collection for subsequent experiments. (E) IVIS imaging results of the cardiac injection mouse model. Mice underwent IVIS in vivo imaging on days 21 and 28 after cardiac cell injection to observe liver metastasis of breast cancer cells. (F) Fluorescence intensity was calculated from the IVIS images taken on day 28 of (E), and statistical differences between groups were compared. Liver tissue was collected after sacrifice, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). One mouse from each group was selected for display. (G) IVIS imaging results of the tail vein mouse model. C57BL / 6N mice were injected with py8119-luci cells via the tail vein and then divided into groups. Every two days, the mice were administered physiological saline or sodium crotonate (200 mg / kg) by gavage. On day 14, in vivo IVIS imaging was performed to observe and capture images of lung metastasis of breast cancer cells in the mice. (H) Fluorescence intensity was calculated for the IVIS images of mice in (G). After the mice were sacrificed, lung tissue was collected, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). One mouse from each group was selected for display. (I) IVIS imaging results of the cardiac injection mouse model. C57BL / 6N mice were injected with py8119-luci cells via the heart and then divided into groups. Every two days, the mice were administered physiological saline or sodium crotonate (200 mg / kg) by gavage. On day 14, in vivo IVIS imaging was performed to observe and capture images of systemic metastasis of breast cancer cells in the mice. (J) Fluorescence intensity was calculated for the IVIS images of mice in (I).After the mice were sacrificed, lung tissue was taken, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). One mouse from each group was selected for display.

[0027] Figure 5 This study demonstrates how crotonyl-CoA inhibits lung and liver metastasis of breast cancer cells. (A) IVIS imaging results in a tail vein mouse model. 4-6 week old BALB / c-nu nude mice were injected intravenously with (iv) MDA-MB-231-luci cell line. The mice were then divided into groups and administered either saline or crotonyl-CoA (60 μg / kg) by gavage every two days. On day 42, in vivo IVIS imaging was performed to observe and capture images of lung metastasis of breast cancer cells in the mice. (B) Fluorescence intensity calculation of the IVIS images of the mice in (A). (C) After euthanasia, lung tissue was collected, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). One mouse from each group was selected for presentation. (D) IVIS imaging results in a mouse model with cardiac injection. After injecting MDA-MB-231-luci cells into the hearts of 4-6 week old BALB / c-nu nude mice, the mice were divided into groups and administered physiological saline or crotonyl-CoA (60 μg / kg) by gavage every two days. On day 28, in vivo small animal imaging (IVIS) was performed to observe and capture images of systemic metastasis of breast cancer cells in the mice. (E) Fluorescence intensity was calculated from the IVIS images of the mice in (D). (F) After the mice were sacrificed, liver tissue was collected, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). One mouse from each group was selected for display.

[0028] Figure 6This study demonstrates that crotonic acid combined with PD-L1 monoclonal antibody can better inhibit breast cancer proliferation and metastasis. Specifically, (A) tumor tissues from mice in the crotonic acid gavage group and control group in the in situ tumorigenesis experiment of C57BL / 6N mouse mammary fat pads were extracted and a portion was analyzed by flow cytometry using the antibody shown in the figure to analyze the difference in the number of immune cells in the tumor tissues of the two groups. (B) Tumor tissues from (A) were analyzed by flow cytometry using PD-L1 antibody. (C) 0 and 20 mmol / L crotonic acid were added to py8119, MCF7, and SUM159 cells, respectively, for 72 h, and flow cytometry was performed using PD-L1 antibody. (D) Sodium crotonate was added to the MDA-MB-231 cell line at concentration gradients of 0, 5, 15, and 25 mmol / L. After 72 h, whole-cell protein lysates were extracted and Western blotting was performed using the antibody shown in the figure. (E) Py8119 cells were injected into the right fourth pair of mammary fat pads of C57BL / 6N mice. Tumors were visible to the naked eye in most mice approximately two days later. The tumor-forming mice were divided into four groups based on tumor size, and administered saline, sodium crotonate (60 mg / kg), PD-L1 monoclonal antibody, and a combination of both drugs, respectively, for 18 consecutive days. Mice were sacrificed on day 21 for tumor analysis. All tumor characteristics are shown in the figure (n=10). (F) Tumor size in C57BL / 6N mice was recorded every four days from day 5 to day 21, and growth curves were plotted. (G) After cardiac injection of Py8119-luci cells, C57BL / 6N mice were divided into groups and administered saline, sodium crotonate (60 mg / kg), PD-L1 monoclonal antibody, and a combination of both drugs, respectively, for 14 consecutive days. IVIS small animal in vivo imaging was performed on day 16 to observe and photograph the systemic metastasis of breast cancer cells in the mice.

[0029] Detailed Implementation

[0030] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0031] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0032] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention. All experimental operations comply with bioethical guidelines (ethics approval number: LA2020183).

[0033] Example 1: In vitro experiment on the inhibition of breast cancer cell proliferation and migration by crotonic acid

[0034] 1. Experimental materials

[0035] Cell lines: human breast cancer cells MDA-MB-231 and SUM159 (purchased from ATCC);

[0036] Reagents: Sodium crotonate (purity ≥98%), DMEM medium containing 0.5% FBS, DMEM medium containing 20% ​​FBS, WST-1 assay kit, crystal violet dye, 4% paraformaldehyde;

[0037] Instruments: CO2 incubator, microplate reader, inverted microscope, TAXIScan-FL cell dynamics analysis system.

[0038] 2. Experimental Methods and Results

[0039] (1) WST-1 assay for cell proliferation

[0040] Digest logarithmic growth phase MDA-MB-231 cells, resuspend in DMEM medium containing 0.5% FBS, and adjust the concentration to [missing value]. Inoculate 100 μl into each well of a 96-well plate, and add PBS to the blank wells around the perimeter to prevent evaporation;

[0041] After the cells adhered, sodium crotonate was added at final concentrations of 0, 2.5, 5, 10, 15, and 20 mmol / L, respectively. Each group was divided into four replicates and incubated at 37°C in a 5% CO2 incubator for 72 h.

[0042] Prepare the working solution according to the WST-1 kit instructions. After discarding the culture medium in each well, add 100 μl of working solution and incubate for 2 hours. Measure the absorbance at 480 nm using a microplate reader.

[0043] Results: As shown in Figure 1A, sodium crotonate ( 15 mmol / L significantly inhibited the proliferation of MDA-MB-231 cells (p<0.0001).

[0044] (2) Plate cloning assay to detect cell colony formation ability

[0045] After digestion and counting, MDA-MB-231 cells were resuspended in DMEM medium containing 10% FBS, and the concentration was adjusted to 800 cells / well, and seeded in 6-well plates.

[0046] After cell adhesion, sodium crotonate was added at final concentrations of 0, 2.5, 5, 10, 15, 20, and 25 mmol / L, with 3 replicates per group, and cultured for 14 days, with culture medium added as needed during the period;

[0047] Discard the culture medium, wash twice with PBS, fix with 4% paraformaldehyde for 15 min, stain with crystal violet for 15 min, wash away the residue with water, and take pictures after air drying.

[0048] Results: As shown in Figure 1B, sodium crotonate ( 15 mmol / L significantly inhibited the clonogenic ability of MDA-MB-231 cells (p<0.01).

[0049] (3) Transwell assay for cell migration

[0050] Adjust the cell concentration according to Table 1 below, and resuspend in medium containing 0.5% FBS;

[0051] Table 1

[0052]

[0053] The Transwell chambers were pre-activated with 100 μl of "three-free" medium for 30 min. After removing the medium, 100 μl of cell suspension (containing 0, 2.5-25 mmol / L sodium crotonate) was added to the upper chamber, and 800 μl of medium containing 20% ​​FBS was added to the lower chamber. The cells were then cultured for the appropriate time.

[0054] Remove the chamber, wipe away the unmigrated cells in the upper chamber with a cotton swab, fix with 4% paraformaldehyde for 15 min, stain with crystal violet for 15 min, and count the cells in 5 fields of view under an inverted microscope.

[0055] Results: As shown in Figure 1C, low concentrations of sodium crotonate ( At a concentration of 2.5 mmol / L, sodium crotonate significantly inhibited the migration of breast cancer cells (p<0.0001). As shown in Figure 1D, sodium crotonate significantly inhibited the migration ability of various tumor cells, including breast cancer cells, liver cancer cells, and colorectal cancer cells.

[0056] (4) TAXIScan-FL detects cell movement trajectory

[0057] MDA-MB-231 cells were treated with 20 mmol / L sodium crotonate for 72 h, then digested and counted, and the concentration was adjusted with PBS. ;

[0058] 1-2 μl of cell suspension was loaded onto the TAXIScan-FL system, and a concentration gradient was formed using 40% FBS as a chemotactic agent. Cell movement trajectories were recorded for 5 h.

[0059] The analysis software calculates cell movement speed and effective migration distance (vertical straight-line distance).

[0060] Results: As shown in E in Figure 1, crotonic acid significantly inhibited the migration distance and migration speed of MDA-MB-231 cells (p<0.0001).

[0061] Example 2: In vitro experiment on the inhibition of breast cancer cell migration by crotonyl-CoA

[0062] 1. Experimental materials

[0063] Cell line: Human breast cancer cell line SUM159;

[0064] Reagents: Crotonyl-CoA (purity ≥95%), PBS, TAXIScan-FL special culture medium;

[0065] Instruments: TAXIScan-FL cell dynamics analysis system, inverted microscope.

[0066] 2. Experimental Methods and Results

[0067] SUM159 cells were treated with 60 μmol / L crotonyl-CoA for 72 h, digested and counted, and then the concentration was adjusted with PBS. ;

[0068] Samples were loaded onto the TAXIScan-FL system, with 40% FBS as a chemotactic agent, and cell motility was recorded for 5 hours.

[0069] Analyze the length, speed, and effective migration distance of cell movement trajectories;

[0070] Results: As shown in Figure 2, crotonic acid significantly inhibited the migration distance (p<0.0001) and migration speed (p<0.0001) of MDA-MB-231 cells.

[0071] Example 3: In vivo experiment on the inhibition of in situ tumorigenesis of breast cancer by crotonic acid

[0072] 1. Experimental materials

[0073] Animals: 20 4-6 week old BALB / c-nu nude mice (athymic, immunodeficient) and 20 C57BL / 6N mice (immune normal);

[0074] Cell lines: human breast cancer cell line MDA-MB-231, mouse breast cancer cell line py8119;

[0075] Reagents: Sodium crotonate, physiological saline, 4% paraformaldehyde;

[0076] Instruments: electronic balance, vernier calipers, pathology slide cutter.

[0077] 2. Experimental Methods and Results

[0078] (1) BALB / c-nu nude mouse in situ tumor formation experiment

[0079] MDA-MB-231 cells were digested and counted, and the concentration was adjusted to [specific value]. 100 μl of cell suspension was injected into the fat pad of the fourth mammary gland on the right side of nude mice;

[0080] After 28 days, the tumor-bearing nude mice (tumor volume approximately 100 mm³) were randomly divided into two groups (n=5): control group (administered by gavage with physiological saline) and treatment group (administered by gavage with 60 mg / kg sodium crotonate), once every 2 days for 26 days;

[0081] Tumor volume was measured every 3 days (volume = length × width). 2 / 2), mouse body weight;

[0082] Mice were sacrificed on day 54, and tumors were photographed and weighed.

[0083] Results: As shown in Figures 3B and 3C, sodium crotonate (60 mg / kg) significantly inhibited the growth of breast cancer cells in BALB / c-nu nude mice (p<0.0001) and had no obvious toxicity.

[0084] (2) In situ tumor formation experiment in C57BL / 6N mice

[0085] The concentration of py8119 cells was adjusted to... 100 μl was injected into the fat pad of the right mammary gland of C57BL / 6N mice;

[0086] Two days later, the tumor-forming mice were randomly divided into two groups (n=6): control group (saline by gavage) and treatment group (200 mg / kg sodium crotonate by gavage), once every 2 days for 12 days;

[0087] Tumor volume and mouse weight were measured every 2-3 days.

[0088] Mice were sacrificed on day 14, and tumors were collected for analysis.

[0089] Results: As shown in Figures 3D and 3E, sodium crotonate (200 mg / kg) significantly inhibited the growth of breast cancer cells in C57BL / 6N mice (p<0.0001) and had no obvious toxicity.

[0090] Example 4: In vivo experiment on the inhibition of breast cancer metastasis by crotonic acid

[0091] 1. Experimental materials

[0092] Animals: 4-6 week old BALB / c-nu nude mice, C57BL / 6N mice, and BALB / c mice;

[0093] Cell lines: MDA-MB-231-luci (luciferase-labeled), py8119-luci, 4T1-luci;

[0094] Reagents: Sodium crotonate, potassium D-fluorescein, 4% paraformaldehyde, HE staining kit;

[0095] Instruments: IVIS small animal in vivo imaging system, pathology slide machine, microscope.

[0096] 2. Experimental Methods and Results

[0097] (1) Nude mouse tail vein injection lung transfer model

[0098] MDA-MB-231-luci cell concentration adjustment 100 μl was injected into the tail vein of nude mice;

[0099] The patients were randomly divided into 3 groups (n=5): control group (physiological saline), low-dose group (20mg / kg sodium crotonate), and high-dose group (60mg / kg sodium crotonate), and were given gavage once every 2 days.

[0100] On days 35, 42, and 49, mice were intraperitoneally injected with D-fluorescein potassium salt (150 mg / kg), and lung fluorescence signals were detected by IVIS imaging.

[0101] Mice were sacrificed on day 49, and their lungs were weighed and stained with hematoxylin and eosin (HE).

[0102] Results: As shown in Figures 4B and 4C, both low and high doses of sodium crotonate significantly inhibited lung metastasis of breast cancer cells in nude mice (p<0.01). HE staining showed that the number and size of lung metastases were smaller in the treated group.

[0103] (2) Nude mouse left ventricular injection liver metastasis model

[0104] MDA-MB-231-luci cell concentration adjustment 100 μl was injected into the left ventricle under ultrasound guidance;

[0105] Groups were administered gavage (same as the nude mouse tail vein lung metastasis model in Example (1)), and IVIS imaging was performed on days 21 and 28.

[0106] Mice were sacrificed on day 28, and livers were collected for HE staining.

[0107] Results: As shown in Figures 4E and F, sodium crotonate significantly inhibited liver metastasis of breast cancer cells in nude mice (p<0.001), and HE staining showed that the treated group had fewer liver metastases and smaller areas.

[0108] (3) C57BL / 6N mouse transfer model

[0109] py8119-luci cells were injected via tail vein ( / each) or left ventricular injection ( / (each) to C57BL / 6N mice;

[0110] The patients were divided into two groups (n=6): control group (physiological saline) and treatment group (200mg / kg sodium crotonate by gavage).

[0111] IVIS imaging, weighing, and lung harvesting and weighing were performed on day 14 in the tail vein injection group; IVIS imaging was performed on day 14 in the left ventricle injection group.

[0112] Results: As shown in G and H in Figure 4, sodium crotonate significantly inhibited lung metastasis of breast cancer cells in C57BL / 6N mice (p<0.001); as shown in I and J in Figure 4, sodium crotonate significantly inhibited liver metastasis of breast cancer cells in C57BL / 6N mice (p<0.001).

[0113] Example 5: In vivo experiment on the inhibition of breast cancer metastasis by crotonyl-CoA

[0114] 1. Experimental materials

[0115] Animals: 4-6 week old BALB / c-nu nude mice;

[0116] Cell line: MDA-MB-231-luci;

[0117] Reagents: Crotonyl-CoA, D-fluorescein potassium salt, HE staining kit;

[0118] Instruments: IVIS imaging system, pathology slide machine.

[0119] 2. Experimental Methods and Results

[0120] (1) Lung metastasis experiment

[0121] Nude mice were injected via tail vein with MDA-MB-231-luci cells ( The patients were randomly divided into two groups (n=5): a control group (physiological saline) and a treatment group (60 μg / kg crotonyl-CoA by gavage), once every 2 days.

[0122] On day 42, IVIS imaging was performed, and mice were sacrificed to obtain lung samples for HE staining.

[0123] Results: As shown in Figure 5 (AC), crotonyl-CoA significantly inhibited lung metastasis of breast cancer cells in nude mice (p<0.01), and HE staining showed that the treated group had fewer lung metastases and smaller areas.

[0124] (2) Liver metastasis experiment

[0125] Nude mice were injected with MDA-MB-231-luci cells into the left ventricle. ), grouped by gavage (same as lung metastasis experiment in Example (1));

[0126] On day 28, IVIS imaging was performed, and the mice were sacrificed and their livers were stained with hematoxylin and eosin (HE).

[0127] Results: As shown in Figure 5 (DF), crotonyl-CoA significantly inhibited liver metastasis of breast cancer cells in nude mice (p<0.05), and HE staining showed that the treated group had fewer liver metastases and smaller areas.

[0128] Example 6: Experiment on the inhibition of breast cancer by crotonic acid combined with PD-L1 monoclonal antibody

[0129] 1. Experimental materials

[0130] Animals: 4-6 week old C57BL / 6N mice;

[0131] Cell lines: py8119, MCF7, SUM159, MDA-MB-231;

[0132] Reagents: Sodium crotonate, PD-L1 monoclonal antibody (anti-mouse PD-L1 antibody), flow cytometry antibody (anti-CD16 / 32, anti-PD-L1), Western Blot antibody (PD-L1, β-actin);

[0133] Instruments: Flow cytometer, Western blotting electrophoresis apparatus, IVIS imaging system.

[0134] 2. Experimental Methods and Results

[0135] (1) Crotonic acid upregulates PD-L1 expression

[0136] Flow cytometry analysis: In situ tumor formation was performed in C57BL / 6N mice (py8119 cells). Tumor tissues were collected from the sodium crotonate gavage group and the control group, digested into single-cell suspensions, and Fc receptors were blocked with anti-CD16 / 32. Anti-PD-L1 antibody staining was performed, and flow cytometry was used for detection. At the same time, py8119, MCF7, and SUM159 cells were treated with 20 mmol / L sodium crotonate for 72 h, and PD-L1 expression was detected by flow cytometry.

[0137] Western Blot: MDA-MB-231 cells were treated with sodium crotonate at concentrations of 0, 5, 15, and 25 mmol / L for 72 h, proteins were extracted, incubated with PD-L1 antibody, and then visualized.

[0138] Results: As shown in Figure 6A, CD8 in the tumor tissue of the crotonic acid-treated group... + The number of T cells increased significantly, while the number of other immune cells did not change significantly; as shown in Figure 6B, PD-L1 in the tumor tissue of the crotonic acid treatment group increased significantly. + The number of cells increased significantly; as shown in Figure 6C, PD-L1 in crotonic acid-treated breast cancer cells... + The number of cells increased significantly; as shown in Figure 6D, the level of PD-L1 protein increased in breast cancer cells treated with crotonic acid.

[0139] (2) Combined therapy tumor suppression experiment

[0140] C57BL / 6N mice mammary fat pads injected with py8119 cells ( Two days later, the tumor-forming mice were randomly divided into four groups (n=10): control group (physiological saline), sodium crotonate group (60 mg / kg by gavage), PD-L1 monoclonal antibody group (300 μg / mouse by intraperitoneal injection, once every 4 days), and combination group (sodium crotonate + PD-L1 monoclonal antibody), and the administration was continued for 18 days;

[0141] Tumor volume was measured every 4 days, and mice were sacrificed on day 21 to photograph the tumor.

[0142] Results: As shown in E and F of Figure 6, the tumor volume in the combined treatment group was significantly smaller than that in the control group, sodium crotonate group, and PD-L1 monoclonal antibody group (p<0.0001), confirming that the combined treatment had the best effect in inhibiting tumor growth.

[0143] (3) Combined therapy anti-metastasis experiment

[0144] C57BL / 6N mice were injected with py8119-luci cells into the left ventricle. Two days later, the mice were randomly divided into 6 groups (n=5): control group (physiological saline), sodium crotonate group (60 mg / kg by gavage), crotonyl-CoA group (60 μg / kg by gavage), PD-L1 monoclonal antibody group (300 μg / mouse by intraperitoneal injection), combination group 1 (sodium crotonate + PD-L1 monoclonal antibody), and combination group 2 (crotonyl-CoA + PD-L1 monoclonal antibody), for 14 days.

[0145] IVIS imaging on day 16;

[0146] Results: As shown in Figure 6 (G), the tumor cell metastasis signal in the two combined groups was significantly weaker than that in the control group, sodium crotonate group, crotonyl-CoA group, and PD-L1 monoclonal antibody group, confirming that the combined treatment had the best effect in inhibiting metastasis.

[0147] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. The use of crotonic acid and / or crotonyl-CoA in the preparation of drugs for treating tumors.

2. The application according to claim 1, characterized in that, The tumors referred to are solid tumors, including breast cancer, liver cancer, and colorectal cancer.

3. The application according to claim 1 or 2, characterized in that, The drug is used to inhibit the proliferation or migration of tumor cells.

4. The application according to claim 3, characterized in that, The tumor cells include breast cancer cells MDA-MB-231, breast cancer cells SUM159, breast cancer cells py8119, breast cancer cells MCF7, liver cancer cells HepG2, or colorectal cancer cells SW620.

5. A pharmaceutical composition for treating tumors, characterized in that, The pharmaceutical composition contains crotonic acid and / or crotonyl-CoA.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition also contains a PD-L1 monoclonal antibody.

7. The pharmaceutical composition according to claim 6, characterized in that, In the pharmaceutical composition, the mass ratio of crotonic acid to PD-L1 monoclonal antibody is 8:

1.

8. The pharmaceutical composition according to any one of claims 5 to 7, characterized in that, The tumor is a solid tumor, including breast cancer.

9. The pharmaceutical composition according to any one of claims 5 to 7, characterized in that, The pharmaceutical composition is used to inhibit the in situ growth or metastasis of tumor cells.

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