Application of compound 680C91 in preparation of drugs for inducing cell autophagy and / or inhibiting tumor cell migration and diffusion

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of a compound 680C91 in preparation of drugs for inducing cell autophagy and / or inhibiting tumor cell migration and diffusion. The compound 680C91 has the effects of inducing tumor cells to activate cell autophagy pathways and inhibiting migration and diffusion of the tumor cells, and can be used for research and development of related drugs.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and particularly to the use of compound 680C91 in the preparation of drugs for inducing autophagy and / or inhibiting the migration and spread of tumor cells. Background Art

[0002] Autophagy is an important metabolic process for maintaining intracellular homeostasis. In recent years, increasing evidence has shown that autophagy defects are associated with various clinical diseases, including cancer, infectious diseases, neurodegeneration, inflammation, and metabolic diseases, etc. This suggests that in the progression of diseases, developing inducers aimed at targeting and enhancing the autophagy function of tissues and organs is expected to become a new treatment strategy for autophagy-related diseases.

[0003] Compound 680C91 alias: 6-fluoro-3-[(1E)-2-(pyridin-3-yl)vinyl]-1H-indole, with its molecular formula C 15 H 11 FN 2 , and its molecular weight is 238.26. It is a fluoroindole derivative with a special chemical structure, characterized by pyridine and alkene properties. The special feature of compound 680C91 is that the hydrogen atom at the 3-position is replaced by 2-(pyridin-3-yl)vinyl, and its configuration is trans. Research indicates that 680C91 does not compete with the binding site of serum albumin to displace tryptophan. It mainly acts as a selective inhibitor, competitively inhibiting the function of tryptophan-2,3-dioxygenase (TDO), and in this way, it can effectively inhibit the conversion process of tryptophan to kynurenine (Kyn). As an inhibitor with relatively high oral bioavailability, the Ki value of 680C91 in experimental mice reaches 51 nM, and in in vitro experiments, its inhibitory effect on the activity of liver TDO2 is also significant, with a Ki value of approximately 30 nmol / l.

[0004] Currently, there has been no research report on the induction of autophagy by compound 680C91. Summary of the Invention

[0005] In view of the deficiencies of the above-mentioned prior art, the purpose of this application is to provide the use of compound 680C91 in the preparation of drugs for inducing autophagy and / or inhibiting the migration and spread of tumor cells, aiming to provide a new strategy for the research and development of drugs related to the induction of autophagy.

[0006] The technical solution of this application is as follows:

[0007] In the first aspect of this application, there is provided the use of compound 680C91 in the preparation of drugs for inducing autophagy and / or inhibiting the migration and spread of tumor cells.

[0008] Optionally, the tumor cells include malignant migratory tumors.

[0009] Optionally, the malignant migratory tumors include at least one of osteosarcoma, breast cancer, prostate cancer, neuroblastoma, pancreatic cancer, liver cancer, lung cancer, cervical squamous cell carcinoma, bladder cancer, colon cancer, gastric cancer, oral cancer, nasopharyngeal cancer, esophageal cancer, endometrial cancer, melanoma, adrenocortical carcinoma, and cholangiocarcinoma.

[0010] In a second aspect of the present application, a drug for inducing autophagy and / or inhibiting the migration and spread of tumor cells is provided, and the drug includes compound 680C91.

[0011] Optionally, the tumor cells include malignant migratory tumors.

[0012] Optionally, the malignant migratory tumors include at least one of osteosarcoma, breast cancer, prostate cancer, neuroblastoma, pancreatic cancer, liver cancer, lung cancer, cervical squamous cell carcinoma, bladder cancer, colon cancer, gastric cancer, oral cancer, nasopharyngeal cancer, esophageal cancer, endometrial cancer, melanoma, adrenocortical carcinoma, and cholangiocarcinoma.

[0013] Optionally, the drug includes pharmaceutically acceptable excipients.

[0014] Optionally, the excipients include at least one of carriers, osmotic pressure regulators, pH regulators, diluents, disintegrants, excipients, solubilizers, stabilizers, and preservatives.

[0015] Optionally, the dosage form of the drug includes at least one of tablets, capsules, solutions, granules, pills, powders, pills, suspensions, powders, injections, sustained-release agents, controlled-release agents, and targeted preparations.

[0016] Compared with the prior art, the present application has the following advantages:

[0017] Through transcriptome sequencing, the present application verified the effect of compound 680C91 in inducing tumor cells to activate the autophagy pathway. At the same time, through the experiment of compound 680C91 inducing the aggregation of mouse tumor cells, it was verified that compound 680C91 can inhibit the migration and spread of tumors, facilitate the removal of tumors, and thus improve the survival rate of mice. Therefore, compound 680C91 has the effect of promoting autophagy in various tumor cells and can be used for the research and development of related drugs. Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments.

[0019] Figure 1 It is a test chart of the activity of 680C91 on different tumor cells provided by the embodiments of the present application:

[0020] A shows the effect of 680C91 at different concentrations on the viability of 5637 cells detected by CCK-8; B shows the effect of 680C91 at different concentrations on the viability of MDA-MB-231 cells detected by CCK-8; C shows the effect of 680C91 at different concentrations on the viability of MCF-7 cells detected by CCK-8; D shows the effect of 680C91 at different concentrations on the viability of K7M2 cells detected by CCK-8;

[0021] Figure 2 This is the gene set enrichment analysis (GSEA) map of tumor cells treated with 680C91 provided in the examples of this application:

[0022] A is a scatter plot showing the GSEA analysis of all pathways from the MsigDB database. The x-axis represents the GSEA-normalized pathway score (NES), and the y-axis represents the p-value corresponding to the respective pathway; B is the GSEA order plot of AUTOPHAGIC_CELL_DEATH; C is a heat map showing the expression levels of six genes (Bmf, Trem2, Trp53inp1, Atp6v0c, Cdkn1b, and Lamp1) in three biological replicates in the Control control group and the 680C91 treatment group; D is the verification of gene expression levels by fluorescence quantitative PCR;

[0023] Figure 3 This is the analysis map of the formation of autophagolysosomes in tumor cells treated with 680C91 provided in the examples of this application:

[0024] A is the fluorescence map of lysosomes and mitochondria in K7M2 cells; B is the layer-by-layer imaging in the Z-axis direction by laser confocal microscopy, and the images of each layer are synthesized into a 3D stereoscopic image;

[0025] Figure 4 This is the electron microscopy morphological map of the formation of autophagolysosomes in tumor cells treated with 680C91 provided in the examples of this application;

[0026] Figure 5 This is the fluorescence map of the formation of autophagic flux in tumor cells treated with 680C91 provided in the examples of this application;

[0027] Figure 6 This is the detection map of the expression of autophagy key genes in tumor cells treated with 680C91 provided in the examples of this application:

[0028] A is for detecting the mRNA expression levels of autophagy key genes in tumor cells treated with different concentrations of 680C91 by qPCR; B is for detecting the protein expression levels of autophagy key genes in tumor cells treated with different concentrations of 680C91 by protein immunization; C is for detecting the protein expression levels of autophagy key genes in tumor cells treated with different concentrations of 680C91 by protein immunization; D is the immunofluorescence image of LCB;

[0029] Figure 7 This is the test chart for the effect of 680C91 provided in the embodiment of the present application in inhibiting tumor cell migration:

[0030] A is the fluorescence image of the cell scratch experiment; B is for statistically reflecting the migration rate of 680C91 in inhibiting tumor cells after 24 hours; C is the tumor cell migration image of the Transwell chamber (without Matrigel); D is the statistics of the number of tumor cell migrating cells;

[0031] Figure 8 This is the test chart for the effect of 680C91 provided in the embodiment of the present application in inhibiting tumor cell clone formation:

[0032] A is the crystal violet staining image of cell clones; B is the cell morphology image of cell clones under the microscope;

[0033] Figure 9 This is the test chart for the fact that the occurrence of autophagy induced by 680C91 in the embodiment of the present application does not depend on the activity of TDO2:

[0034] A is for detecting the gene expression level of LC3B in different cells by qPCR; B is for detecting the gene expression level of p62 in different cells by qPCR; C is for detecting the gene expression level of Beclin-1 in different cells by qPCR. Detailed implementation manners

[0035] To make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings and embodiments of the present application. Apparently, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application. Without conflict, the following embodiments and the features in the embodiments may be combined with each other.

[0036] It should be noted that if the implementation of this application involves descriptions such as "first" and "second", these descriptions are only for descriptive purposes and should not be construed as indicating or implying their relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one such feature. Additionally, the technical solutions between various embodiments can be combined with each other, but it must be based on the premise that those of ordinary skill in the art can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0037] An embodiment of this application provides an application of compound 680C91 in the preparation of a drug for inducing autophagy and / or inhibiting the migration and spread of tumor cells.

[0038] Through experiments, it is verified that compound 680C91 has the effect of inducing tumor cells to activate the autophagy pathway. At the same time, through the experiment of inducing the aggregation of mouse tumor cells by compound 680C91, it is verified that compound 680C91 can inhibit the migration and spread of tumors, which is beneficial to the removal of tumors.

[0039] In some embodiments, the tumor cells include malignant migratory tumors.

[0040] In some embodiments, the malignant migratory tumors include at least one of osteosarcoma, breast cancer, prostate cancer, neuroblastoma, pancreatic cancer, liver cancer, lung cancer, cervical squamous cell carcinoma, bladder cancer, colon cancer, gastric cancer, oral cancer, nasopharyngeal cancer, esophageal cancer, endometrial cancer, melanoma, adrenocortical carcinoma, and cholangiocarcinoma.

[0041] An embodiment of this application also provides a drug for inducing autophagy and / or inhibiting the migration and spread of tumor cells, and the drug includes compound 680C91.

[0042] In some embodiments, the tumor cells include malignant migratory tumors.

[0043] In some embodiments, the malignant migratory tumors include at least one of osteosarcoma, breast cancer, prostate cancer, neuroblastoma, pancreatic cancer, liver cancer, lung cancer, cervical squamous cell carcinoma, bladder cancer, colon cancer, gastric cancer, oral cancer, nasopharyngeal cancer, esophageal cancer, endometrial cancer, melanoma, adrenocortical carcinoma, and cholangiocarcinoma.

[0044] In some embodiments, the drug for inducing autophagy and / or inhibiting the migration and spread of tumor cells includes active ingredients with a compatibility and synergistic effect. Compatibility and synergy refer to ingredients with some commonalities in terms of performance and efficacy, which can enhance the effect of the main ingredient.

[0045] In some embodiments, the drugs that induce autophagy and / or inhibit the migration and spread of tumor cells include pharmaceutically acceptable excipients. Pharmaceutically acceptable means that it can be administered to humans and / or other animals as subjects without causing excessive adverse reactions or side effects (such as toxicity, irritation, allergic reactions, etc.). Excipients refer to auxiliary materials that coexist with the active ingredient in a pharmaceutical preparation and do not cause excessive adverse reactions or side effects. Excipients include carriers, osmotic pressure regulators, pH regulators, diluents, disintegrants, excipients, solubilizers, stabilizers, preservatives, etc. Pharmaceutically acceptable excipients refer to highly safe excipients that are suitable for specific pharmaceutical preparations and are conventionally used in pharmacy. Carriers include, but are not limited to, liposomes, ethosomes, polymeric micelles, nanostructured lipid carriers, solid lipid nanoparticles, mesoporous silica nanoparticles, etc.

[0046] In some embodiments, the dosage forms of the drugs that induce autophagy and / or inhibit the migration and spread of tumor cells include, but are not limited to, tablets, capsules, solutions, granules, pills, powders, pills, suspensions, powders, injections, sustained-release agents, controlled-release agents or targeted preparations.

[0047] In some embodiments, the drugs that induce autophagy and / or inhibit the migration and spread of tumor cells can be administered by injection, oral administration, rectal administration, etc.

[0048] The following is further illustrated by specific examples.

[0049] The sources of the materials used in the examples are as follows:

[0050] 680C91 (Catalog No.: HY-108681) is a product of MedChemExpress. Hoechst 33342 (Catalog No.: B2261) was purchased from Sigma-Aldrich. Cell culture medium DMEM, penicillin / streptomycin, and fetal bovine serum were purchased from ThermoFisher. The CCK-8 kit (Catalog No.: C0041), Lyso-Tracker Green (lysosome green fluorescent probe) (Catalog No.: C1047S), Mito-Tracker Red CMXRos (mitochondria red fluorescent probe) (Catalog No.: C1049B), and crystal violet staining solution (Catalog No.: C0121) are all products of Beyotime. Antibodies against LC3A / B (Catalog No.: 12741), p62 (Catalog No.: 5114), Beclin-1 (Catalog No.: 3738), and GAPDH (Catalog No.: 5174) are all products of Cell Signaling Technology. The cell RNA extraction kit (Catalog No.: 19221ES50), reverse transcription kit (Catalog No.: 11141ES10), and PCR amplification kit (Catalog No.: 11184ES08) are all products of Yeasen. Other reagents or instruments without specified manufacturers are all conventional products that can be obtained commercially.

[0051] The experimental methods in the examples are all conventional methods in the art unless otherwise specified.

[0052] Example 1

[0053] This example verifies the effect of 680C91 on the viability of tumor cells, including the following specific steps:

[0054] (1) Cell culture and treatment. Human bladder cancer cells 5637, human breast cancer cells MDA-MB-231, human breast cancer cells MCF-7, and mouse osteosarcoma cells K7M2 are all adherent cells and are cultured in DMEM medium containing 10% (v / v) fetal bovine serum, 100 μg / ml streptomycin, and 100 U / ml penicillin, and placed in a 37 °C, 5% CO 2 cell incubator for culture. Cells can be passaged after 2 - 3 days of culture. Inoculate the cells in a 96-well plate at a cell density of 5×10 4 cells / well, and then treat them with medium containing different concentrations of 680C91 (1.25, 2.5, 5, 10, 20, 40 μM) for 24 hours.

[0055] (2) Cell viability detection. Tumor cells were treated with 680C91 at different concentrations. After reacting for 24 hours, 10 μl of CCK8 reaction solution was added to each well and reacted at 37 °C for 30 minutes. The OD value was detected at a wavelength of 450 nm, and the cell viability was calculated by measuring the absorbance.

[0056] Treat different tumor cells with different concentrations of 680C91 for 24 hours, and use CCK8 to detect the cell viability. The results are shown in Figure 1 A, B, C and D in the figure. As can be seen from Figure 1 A, B, and C in the figure, 680C91 can dose-dependently inhibit the cell viability of human bladder cancer cell line 5637, human breast cancer cell line MDA-MB-231, and human breast cancer cell line MCF-7; in addition, as can be seen from Figure 1 D in the figure, 680C91 can dose-dependently inhibit the cell viability of mouse osteosarcoma cell line K7M2. Therefore, 680C91 can inhibit the viability of various tumor cells without species specificity.

[0057] Example 2

[0058] In this example, tumor cells were treated with 680C91, and the characteristics of autophagic cell death were revealed by gene set enrichment analysis (GSEA), including the following specific steps:

[0059] (1) Genes with changed expression related to transcriptome sequencing analysis. After treating tumor cells with 680C91 for 24 hours to induce a response, the mRNA of the cells was extracted for transcriptome sequencing. The data analysis steps were as follows: ① Obtaining the reference genome: Download the latest version of the mouse genome sequence and annotation information from the NCBI database, and use bowtie to build a genome index; ② Quality control of the original sequencing data: Use FASTAQC to detect the original sequencing quality, and use trim-galore to remove adapter sequences in the original reads. The criterion for determining adapter sequences in the reads was having 3 bp overlap with known adapter sequences; if the length of the reads was less than 20 bp after removing the adapters, this pair of reads was directly discarded; ③ Using Tophat with default parameters to align the filtered reads to the reference genome to obtain read-genome alignment information; ④ According to the genome annotation information, use FeatureCount to calculate the number of reads for each gene, and use RPKM to standardize the gene expression values; ⑤ Use DEseq2 to perform differential gene expression analysis on the number of reads for each gene to obtain differentially expressed genes; ⑥ DESeq performs normalization on RNA-Seq data to obtain the normalized expression level of each gene, and at the same time prepare the phenotypic information of the samples, including sample grouping (such as Control control group and 680C91 treatment group) and other relevant metadata for distinguishing gene expression patterns under different conditions in subsequent analysis. ⑦ Secondly, extract all gene sets from the MSigDB database (https: / / www.gsea-msigdb.org / gsea / msigdb / ), and use GSEA software to estimate the statistical significance of the enrichment score through random permutation tests.

[0060] (2) Detection of the expression of key genes by qPCR. After treating tumor cells with 680C91 for 24 hours, the mRNA of the cells was extracted for qPCR detection.

[0061] To study the pathways related to cell death under drug treatment, gene set enrichment analysis (GSEA) was performed on all pathways in the MSigDB database, and the results are shown in Figure 2 A as shown. Further analysis based on the results in Figure 2 A showed that the AUTOPHAGIC_CELL_DEATH (autophagic cell death) pathway was significantly enriched ( Figure 2 B as shown), suggesting that this pathway may be involved in the cell response induced by 680C91. Further analysis of AUTOPHAGEIC_CELL_DEATH found that the significant enrichment of this pathway was mainly driven by the following six key genes: Bmf, Trem2, Trp53inp1, Atp6v0c, Cdkn1b, and Lamp1. Subsequently, through a heatmap ( Figure 2Figure C) shows the differential expression of these genes between the control group and the 680C91 drug-treated group. The results show that the expression levels of these genes were significantly upregulated in the drug-treated group, indicating that they may play an important role in the process of autophagic cell death. To verify the expression differences of these genes, quantitative PCR (qPCR) experiments were performed. The qPCR results ( Figure 2 Figure D) further confirmed that the expression levels of Bmf, Trem2, Trp53inp1, Atp6v0c, Cdkn1b, and Lamp1 were all significantly upregulated under drug treatment, further supporting the key role of these genes in the autophagic cell death pathway. From the above results, it can be seen that 680C91 treatment significantly affected the AUTOPHAGIC_CELL_DEATH pathway and exerted its effect by regulating the above key genes.

[0062] Example 3

[0063] In this example, tumor cells were treated with 680C91 to induce the formation of autophagolysosomes, and corresponding detections were carried out. The specific steps are as follows:

[0064] (1) Cell culture and treatment.

[0065] Tumor cells were treated with different concentrations of 680C91. After reacting for 24 hours, Lyso-Tracker Green (lysosome green fluorescent probe, 5 μM) and Mito-Tracker Red CMXRos (mitochondria red fluorescent probe, 5 μM) were added, and incubated at 37 °C in the dark for 20 minutes. The morphology and degree of autophagolysosomes in the cells were photographed by an inverted fluorescence microscope and a laser confocal microscope.

[0066] The photographing results are as shown in Figure 3 Figures A and B. As can be seen from Figure 3 Figure A, when tumor cells were treated with different concentrations of 680C91, it was found that 680C91 could promote the formation of autophagolysosomes in a dose-dependent manner. Subsequently, layer-by-layer imaging in the Z-axis direction was performed by laser confocal microscopy ( Figure 3 Figure B), and three-dimensional imaging analysis at different angles was carried out, further indicating that 680C91 treatment could promote the formation of autophagolysosomes in tumor cells, and the formation of autophagolysosomes is a marker of the autophagy process, and further indicating that 680C91 has a unique activity of inducing autophagy in cells.

[0067] Example 4

[0068] In this example, tumor cells were treated with 680C91 to induce the formation of autophagolysosomes, and electron microscopy morphological detection was carried out. The specific steps are as follows:

[0069] (1) Cell culture and treatment.

[0070] Tumor cells were treated with 680C91 at different concentrations. After reacting for 24 hours, the culture medium was aspirated and discarded, and an electron microscopy fixative (2.5% glutaraldehyde) was added. Fix at room temperature in the dark for about 5 minutes, and gently scrape the cells in one direction with a cell scraper. Collect the cells into a centrifuge tube and centrifuge at 1500 rpm for 5 minutes. After fixation, dehydrate in gradients, embed, use an ultramicrotome to cut 70 nM sections, and stain; take pictures with a transmission electron microscope.

[0071] Detection of morphological ultrastructure by transmission electron microscopy is considered the gold standard for detecting autophagosomes. The results of transmission electron microscopy detection are as Figure 4 shown. As can be seen from Figure 4 , in tumor cells treated with 680C91, autophagosomes were visible to fuse with lysosomes under transmission electron microscopy, forming a single-membrane structure, which contained cytoplasmic components at different degradation stages. The electron density of the degradation products increased, forming black granular or amorphous aggregates, further indicating that 680C91 can indeed induce the occurrence of autophagy.

[0072] Example 5

[0073] In this example, tumor cells were treated with 680C91 to induce the formation of autophagic flux and fluorescence detection was performed. The specific steps are as follows:

[0074] (1) Establishment of the RFP-GFP-LC3 dual-fluorescence indicator system.

[0075] After the LC3B dual-fluorescence autophagy lentivirus was added to MDA-MB-231 cells for transfection for 48 hours, Puromycine (2 μg / ml) was added and the cells were cultured continuously to screen out tumor cells with positive expression and resistance. Subsequently, the tumor cells were treated with 680C91. After reacting for 24 hours, fluorescence images of GFP and RFP in the cells were taken with an inverted fluorescence microscope.

[0076] When autophagy forms in cells, the RFP-GFP-LC3 fusion protein transfers to the autophagosomal membrane of the cells, forming multiple bright yellow fluorescence spots under a fluorescence microscope. When autolysosomes are formed in cells, the acidic lysosomal environment quenches the GFP fluorescence, while the RFP fluorescence is not affected, and the autolysosomes show red fluorescence. Therefore, the autophagic flux in cells can be monitored through the LC3 fluorescence indicator system, and its principle is as shown in A of Figure 5 . The results of fluorescence imaging are as shown in B of Figure 5 . As can be seen from B of Figure 5 , after treating tumor cells with 680C91, the GFP fluorescence can be significantly quenched during the induction of autophagy, further proving that 680C91 can induce the occurrence of autophagy in cells.

[0077] Example 6

[0078] In this example, 680C91 was used to treat tumor cells, induce the expression of autophagy key genes and detect them. The specific steps are as follows:

[0079] (1) Detection of the mRNA expression level of autophagy key genes in cells. After K7M2 cells were treated with different concentrations of 680C91 (5, 10, 20 μM) for 24 hours, mRNA was extracted and separated. The cell culture medium was aspirated, 500 μl of lysis buffer was added, and the mixture was pipetted up and down repeatedly. It was added to a DNA removal / RNA adsorption universal column, centrifuged at 13000 rpm for 2 minutes, and the filtrate was collected. 500 μl of binding solution was added, and it was added to the RNA adsorption column, centrifuged at 13000 rpm for 1 minute, and the filtrate was discarded. 700 μl of protein removal solution was added, centrifuged at 13000 rpm for 1 minute, and the filtrate was discarded. After removing DNA, the adsorption universal column was placed back into a 2 ml absorption tube again. 500 μl of washing solution was added to the container, the rotation speed was adjusted to 1000 rpm, centrifuged for 1 minute, and it could be used after filtration, and washed 2 times. 60 μl of RNase-free H 2 O was added to the center of the filter membrane, allowed to stand for 2 minutes, centrifuged at 13000 rpm for 1 minute, and the filtrate was the RNA solution. cDNA was formed by reverse transcription, and finally qPCR amplification was performed to calculate the change in relative expression.

[0080] (2) Detection of the protein expression level of autophagy key genes in cells. After K7M2 cells were treated with different concentrations of 680C91 (5, 10, 20 μM) for 24 hours, proteins were extracted. The cell culture medium was aspirated, RIPA lysis buffer was added, and the mixture was pipetted up and down repeatedly. Lysed on ice for 15 minutes. Centrifuged at 13000 rpm for 10 minutes. The supernatant was taken, quantified, subjected to SDS electrophoresis, blocked for 1 hour, incubated with the primary antibody overnight, and incubated with the secondary antibody for 1 hour. Chemiluminescence was used to detect the expression level of the corresponding protein.

[0081] (3) Fluorescence detection of the LC3 protein of autophagy key genes in cells. After K7M2 cells were treated with 680C91 (20 μM) for 24 hours, paraformaldehyde was added for fixation for 15 minutes, washed, permeabilized for 10 minutes, and blocked with the blocking solution at room temperature for 1 hour. Anti-LC3A / B antibody was added, incubated overnight at 4 °C, secondary antibody was added, and incubated at room temperature in the dark for 1 hour. Hoechst 33342 (5 μg / ml) was added, and incubated at room temperature in the dark for 10 minutes. Confocal fluorescence imaging was performed.

[0082] As Figure 6As shown in A, qPCR detection revealed that after treating tumor cells with different concentrations of 680C91, it could promote the expression of LC3 and p62 in a dose-dependent manner, while the expression of beclin-1 was downregulated in a dose-dependent manner. The change in the LC3-B / A ratio was detected by Western blot to evaluate autophagy formation in cells. When autophagy occurs in cells, cytosolic LC3-A will enzymatically cleave a small polypeptide, and then bind to PE to transform into membrane-bound LC3-B. Therefore, the level of autophagy in cells can be estimated by the magnitude of the LC3-B / A ratio, and the results are as Figure 6 shown in B. As can be seen from Figure 6 B, treatment with different concentrations of 680C91 could promote the LC3B / A ratio, and the protein expression levels of p62 and beclin-1 were consistent with the mRNA levels. On the other hand, according to Figure 6 the immunofluorescence detection results shown in C, the increase in LC3-positive granules indicated an increase in the number of autophagosomes in cells. Based on the above results, it can be seen that 680C91 can induce autophagy at the gene and protein levels.

[0083] Example 7

[0084] In this example, the effect of 680C91 on inhibiting tumor cell migration was detected, including the following steps:

[0085] (1) Cell scratch assay. MDA-MB-231-GFP cells were seeded at a density of 5×10 5 in 6-well plates, incubated for 24 hours, scratched, and then treated with different concentrations of 680C91 (5, 10, 20 μM) for 24 hours. Images were taken under a fluorescence microscope and the width of the scratch was measured using Image J software to calculate the cell migration rate.

[0086] (2) Transwell migration assay. 500 μl of medium containing 10% fetal bovine serum was added to the lower chamber of the Transwell, gently added to adhere to the wall, avoiding the generation of bubbles. The plate was tilted, and the insert was picked up with sterile forceps and placed at an angle between the insert and the liquid level in the lower chamber to avoid the generation of bubbles between the bottom of the insert and the liquid surface. 200 μl of cell suspension (containing 20 μM 680C91) was added to the insert. After culturing for 24 hours, the cells on the upper layer of the insert were removed, fixed with 4% paraformaldehyde for 30 minutes, stained with crystal violet for 5 minutes, washed twice with PBS, and images were taken and cell counting was performed under a microscope.

[0087] Tumor cells grow rapidly and have the characteristic of migrating and spreading distantly. Therefore, inhibiting tumor cell migration is also an important anti-tumor strategy. The test results of cell migration are as Figure 7As shown in A - D, it can be seen that 680C91 can inhibit cell migration in a dose - dependent manner, thereby exerting an anti - tumor effect.

[0088] Example 8

[0089] In this example, the effect of 680C91 on inhibiting the colony formation of tumor cells was detected, including the following steps:

[0090] (1) Cell culture and treatment. Tumor cells MCF - 7 in the logarithmic growth phase were digested with trypsin and resuspended in a complete medium (basic medium + 10% fetal bovine serum) to form a cell suspension, and then counted. 300 cells / well were seeded in each well of a 6 - well culture plate, and different concentrations of 680C91 (5, 10, 20 μM) were added, and the cells were continuously cultured for 14 days or until the number of cells in the vast majority of single colonies was greater than 50. The medium was changed every 3 days during the process, and the cell status was observed. After colony formation, 1 ml of 4% paraformaldehyde was added to fix for 30 minutes, and then washed once with PBS; 1 ml of crystal violet staining solution was added to each well to stain the cells for 10 minutes. Digital camera photos were taken (photos of the entire six - well plate were taken respectively), and photos of cell colonies were taken under a microscope.

[0091] The cell colony formation experiment can detect the cell proliferation ability. The microscope photo results are as shown in Figure 8 A and B. As can be seen from Figure 8 A and B, different concentrations of 680C91 can inhibit the formation of cell colonies in a dose - dependent manner, indicating that 680C91 can inhibit the proliferation ability of tumors by inducing autophagy.

[0092] Example 9

[0093] (1) Establishment of stable cell lines with knockdown and overexpression of the TDO2 gene. Gene vectors were designed by shRNA and overexpression methods, viruses were packaged, cells were transfected, and stable resistant tumor cells (MDA - MB - 231) were screened. The knockdown and overexpression effects of the TDO2 gene were verified, and its stable inheritance was ensured. Subsequently, after the cells were treated with 680C91 (20 μM) for 24 hours, mRNA was collected and separated, and qPCR was performed to detect the expression of autophagy - related genes.

[0094] 680C91 is a selective inhibitor of tryptophan TDO. After treatment with 680C91, significant changes in autophagy genes can be observed in both shTDO2 cells with knockdown and cells with overexpression of TDO2. The qPCR detection results are as shown in Figure 9 A, B, and C. As can be seen from Figure 9As can be seen from A, B, and C, compared with the control group, there is no difference in the expression level of autophagy genes in cells with knockdown or overexpression of the TDO2 protein, indicating that the pharmacological activity of 680C91 in inducing autophagy does not depend on the activity of TDO2.

[0095] In summary, through cell viability detection, it was found that 680C91 can inhibit the viability of various tumor cells. Gene Set Enrichment Analysis (GSEA) was performed on all pathways in the MSigDB database. The analysis results showed that tumor cells treated with 680C91 were significantly enriched in the AUTOPHAGIC_CELL_DEATH pathway. Subsequently, through observation with the Lyso-Tracker Green fluorescent probe and transmission electron microscopy, it was found that 680C91 can significantly induce the formation of autophagolysosomes. Through the RFP-GFP-LC3 double-fluorescence indicator system, it was also found that 680C91 can significantly induce the occurrence of autophagic flux, and 680C91 can regulate the expression level of key autophagy in cells at the gene and protein levels. At the same time, through the cell scratch experiment and Transwell migration experiment, it was found that 680C91 has the effect of inhibiting the migration and diffusion of tumor cells and can be used for the research and development of drugs for inducing autophagy and / or inhibiting the migration and diffusion of tumor cells.

[0096] It should be understood that the application of this application is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above description, and all such improvements and transformations should fall within the protection scope of the appended claims of this application.

Claims

1. Use of compound 680C91 in the preparation of drugs for inducing cell autophagy and / or inhibiting the migration and spread of tumor cells.

2. The use according to claim 1, characterized in that: The tumor cells include malignant metastatic tumors.

3. The use according to claim 2, characterized in that: The malignant metastatic tumor includes at least one of osteosarcoma, breast cancer, prostate cancer, neuroblastoma, pancreatic cancer, liver cancer, lung cancer, cervical squamous cell carcinoma, bladder cancer, colon cancer, gastric cancer, oral cancer, nasopharyngeal carcinoma, esophageal cancer, endometrial cancer, melanoma, adrenocortical carcinoma and bile duct cancer.

4. A drug for inducing cell autophagy and / or inhibiting the migration and spread of tumor cells, characterized in that: The drugs include compound 680C91.

5. The drug according to claim 4, characterized in that The tumor cells include malignant metastatic tumors.

6. The drug according to claim 5, characterized in that The malignant metastatic tumor includes at least one of osteosarcoma, breast cancer, prostate cancer, neuroblastoma, pancreatic cancer, liver cancer, lung cancer, cervical squamous cell carcinoma, bladder cancer, colon cancer, gastric cancer, oral cancer, nasopharyngeal carcinoma, esophageal cancer, endometrial cancer, melanoma, adrenocortical carcinoma and bile duct cancer.

7. The drug according to claim 4, characterized in that The drug includes pharmaceutically acceptable excipients.

8. The drug according to claim 7, characterized in that The auxiliary materials include at least one of a carrier, an osmotic pressure regulator, a pH regulator, a diluent, a disintegrant, an excipient, a solubilizer, a stabilizer, and a preservative.

9. The drug according to claim 4, characterized in that The dosage form of the drug includes at least one of tablets, capsules, solutions, granules, pills, powders, pills, suspensions, powders, injections, sustained-release agents, controlled-release agents, and targeted preparations.

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