Use of chrysolaminarin in preparation of drug for promoting aggregation of tumor cells and inhibiting migration and spread of tumor cells

Abstract

The present invention relates to use of chrysolaminarin in the preparation of a drug for promoting the aggregation of tumor cells and inhibiting the migration and spread of tumor cells. Chrysolaminarin has the effects of promoting the aggregation of tumor cells and inhibiting the migration and spread of tumor cells, has a potential therapeutic effect in the clinical treatment of tumor metastasis diseases, and can be used for the research and development of related drugs.

Description

Use of chrysolaminarin in preparation of medicine for promoting tumor cell aggregation and inhibiting tumor cell migration and diffusion TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to use of chrysolaminarin in preparation of medicine for promoting tumor cell aggregation and inhibiting tumor cell migration and diffusion. BACKGROUND

[0002] Tumor metastasis is a complex biological process in the late stage of tumor development, which involves migration of cancer cells from the primary tumor site to other parts of the body to form new tumor foci. The incidence and mortality of malignant tumors are increasing, and metastasis is the most deadly feature of malignant tumors, accounting for more than 90% of tumor-related deaths. After metastasis, malignant tumors spread throughout the body, cannot be treated by surgery and are prone to recurrence. At present, tumor metastasis cannot be predicted and intervened in the early stage, and there is a lack of effective drugs for treatment, which is a difficult problem to be solved in clinical practice. SUMMARY

[0003] In view of the deficiencies of the prior art, the present application aims to provide use of chrysolaminarin in preparation of medicine for promoting tumor cell aggregation and inhibiting tumor cell migration and diffusion, and aims to solve the problem that tumor metastasis cannot be intervened at present.

[0004] The technical scheme of the present application is as follows:

[0005] In a first aspect, the present application provides use of chrysolaminarin in preparation of medicine for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion.

[0006] Optionally, the tumor cells include malignant metastatic tumors.

[0007] Optionally, the malignant metastatic tumors include at least one of 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, adrenal cortex cancer, and bile duct cancer.

[0008] In a second aspect, the present application provides a medicine composition for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion, wherein the medicine composition comprises chrysolaminarin.

[0009] Optionally, the tumor cells include malignant metastatic tumors.

[0010] Optionally, the malignant metastatic tumor includes at least one of breast cancer, prostate cancer, neuroblastoma, pancreatic cancer, liver cancer, lung cancer, cervical squamous carcinoma, bladder cancer, colon cancer, gastric cancer, oral cancer, nasopharyngeal cancer, esophageal cancer, endometrial cancer, melanoma, adrenal cortex cancer, and bile duct cancer.

[0011] Optionally, the pharmaceutical composition includes effective components with compatibility synergy.

[0012] Optionally, the pharmaceutical composition includes a pharmaceutically acceptable excipient.

[0013] Optionally, the excipient includes 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.

[0014] Optionally, the dosage form of the pharmaceutical composition includes at least one of a tablet, a capsule, a solution, a granule, a pill, a powder, a pill, a suspension, a powder, a needle, a sustained-release preparation, a controlled-release preparation, and a targeted preparation.

[0015] Advantages of the present application: The laminarin has the function of specifically inducing the aggregation of various tumor cells, has no effect on the activity and proliferation ability of tumor cells, is non-toxic to normal immune cells, and has the effect of inhibiting the migration or invasion of tumor cells. Through transcriptome sequencing, it is verified that the laminarin induces the aggregation effect of tumor cells, and it is found that the laminarin induces the aggregation of tumor cells by activating the NRF2-Keap1-NQO1 pathway and up-regulating the expression of E-cadherin protein, and reverses the epithelial-mesenchymal transition; at the same time, through the experiment of inducing the aggregation of tumor cells in mice by the laminarin, it is verified that the laminarin can inhibit the migration and diffusion of tumors in vivo, is beneficial to the removal of tumors, and further improves the survival rate of mice. Therefore, the laminarin has the effects of promoting the aggregation of tumor cells and inhibiting the migration and diffusion of tumor cells, and can be used for the research and development of related drugs. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows.

[0017] FIG. 1 is a test diagram of the effect of the laminarin provided by the present application on the activity and proliferation ability of tumor cells:

[0018] A is the CCK-8 detection of the effect of the laminarin on the activity of tumor cells; B is the detection of lactate dehydrogenase released in the culture supernatant of tumor cells treated by the laminarin; C is the fluorescence diagram of EdU-Azide555 cell proliferation detection of the effect of the laminarin on tumor proliferation; and D is a histogram of the percentage of EdU positive cells in total cells.

[0019] Figure 2 is a morphological detection chart of tumor cell aggregation induced by laminarin according to an embodiment of the present application;

[0020] A is a fluorescence chart of SHSY-5Y-GFP cell aggregation; B is a total area statistics of cell clusters of SHSY-5Y-GFP aggregation; C is a 3D stereoscopic image obtained by imaging each layer in the Z-axis direction layer by layer through laser confocal; D is a cell aggregation condition photographed by crystal violet staining and microscope;

[0021] Figure 3 is a morphological detection chart of human and mouse tumor cell aggregation induced by laminarin according to an embodiment of the present application;

[0022] A is a fluorescence chart of human tumor cell lines SHSY-5Y-GFP (neuroblastoma cells), MDA-MB-231-GFP (breast cancer cells) and PC-3-GFP (prostate cancer cells) cell aggregation; B is a fluorescence chart of mouse tumor cell lines B16-F10 (melanoma) and Neuro-2A (neuroblastoma cells) cell aggregation;

[0023] Figure 4 is a cell fluorescence micrograph of human THP-1 cells treated with laminarin according to an embodiment of the present application;

[0024] Figure 5 is a cell fluorescence micrograph of mouse dendritic cells treated with laminarin according to an embodiment of the present application;

[0025] Figure 6 is an effect test chart of tumor cell aggregation, inhibition of tumor cell migration and invasion induced by laminarin according to an embodiment of the present application:

[0026] A is a fluorescence chart of cell scratch experiment; B is a statistics of 24 hours and 48 hours of laminarin promoting tumor cell migration rate; C is a tumor cell migration chart of Transwell chamber (without matrix glue); D is a statistics of tumor cell migration cell number; E is a tumor cell invasion chart of Transwell chamber (with matrix glue); F is a statistics of tumor cell invasion cell number;

[0027] Figure 7 is a verification chart of NRF2-Keap-1-NQO1 pathway activated by laminarin treatment according to an embodiment of the present application;

[0028] A is a heatmap of NRF2-Keap-1-NQO1 pathway key genes; B is a box plot of NRF2-Keap-1-NQO1 pathway key genes; C is the expression change of different genes in the NRF-ARE signaling pathway; D is the mRNA expression level of NRF2-Keap-1-NQO1 key genes verified by qPCR; E is the protein expression level of NRF2-Keap-1-NQO1 key genes verified by Western blotting; F is the gray value of the protein in E is counted and analyzed;

[0029] Figure 8 is a verification diagram of the induction of laminarin on cell adhesion gene expression provided by the embodiments of the present application;

[0030] A is Mfuzz clustering analysis; B is a heatmap of cell adhesion genes with concentration-dependent increase; C is the mRNA expression level of cell adhesion genes verified by qPCR;

[0031] Figure 9 is a verification diagram of the effect of knocking down NQO1 gene on the tumor aggregation effect induced by laminarin provided by the embodiments of the present application:

[0032] A is the knocking down efficiency of NQO1 detected by Western blotting; B is the gray scale analysis of NQO1 protein; C is a schematic diagram of mixed culture of NC siRNA-GFP and NQO1-siRNA-RFP cells in a ratio of 1:1; D is a fluorescence diagram of cell aggregation taken by an inverted fluorescence microscope after being treated with laminarin (4 mg / mL) for 24 hours;

[0033] Figure 10 is a fluorescence diagram of tumor cell aggregation promoted by laminarin provided by the embodiments of the present application:

[0034] A is a schematic diagram of tumor cell migration; B is the expression and distribution of E-cadherin protein detected by immunofluorescence after being treated with laminarin for 24 hours;

[0035] Figure 11 is a test diagram of laminarin inhibiting tumor migration and diffusion in mice provided by the embodiments of the present application:

[0036] A is a schematic diagram of laminarin administration; B is a diagram of body weight change of mice intraperitoneally injected with laminarin for 7 consecutive days; C is the distribution of tumor cells in the abdominal cavity of mice observed by an imaging system; D is the total number of tumor cells in the abdominal cavity of mice; E is the number of cells in the largest tumor cell aggregation in the abdominal cavity of mice;

[0037] Figure 12 is a test diagram of the survival rate of mice after tumor cell removal by laminarin to promote tumor cell aggregation, which is beneficial to tumor removal surgery provided by the embodiments of the present application:

[0038] A is the administration diagram of chrysolaminarin; B is the CT imaging system for observing the distribution of tumor cells in the mouse in vivo; C is the mouse tumor tissue removed by surgery; D is the survival curve of the mouse after surgery. DETAILED DESCRIPTION

[0039] In order 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 described clearly and completely below with reference to the drawings and embodiments of the present application. Obviously, 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 work fall within the protection scope of the present application. The embodiments below and the features in the embodiments can be combined with each other without conflict.

[0040] It should be noted that if the present application is described as "first", "second" and the like in the implementation, the "first", "second" and the like are only for the purpose of description, and cannot be understood as indicating or implying the relative importance and implicitly indicating the number of the indicated technical features. Therefore, the features with "first", "second" can be explicitly or implicitly included at least one of the features. In addition, the technical solutions of each embodiment can be combined with each other, but the combination of the technical solutions must enable the person skilled in the art to realize the basis, and when the combination of the technical solutions is contradictory or cannot be realized, it should be considered that the combination of the technical solutions does not exist, and is not within the protection scope claimed by the present application.

[0041] Tumor metastasis refers to the process that the cells of malignant tumor, from the location where the lesion just begins, through lymphatic vessels, blood vessels or body cavities, and then transfer to other parts to erode other parts to continue to grow, which is a complex biological process. Epithelial-mesenchymal transition (EMT) is a prerequisite for tumor invasion and metastasis. When tumor cells invade and metastasize, they change from epithelial phenotype to mesenchymal phenotype, fall off from the primary lesion into the metastasis pathway, and the transformed cancer cells eventually metastasize to other organs through the lymphatic system, blood system or other metastasis pathways. This process is accompanied by the degradation of epithelial cadherin E-cadherin (a hallmark event of EMT), which mediates the adhesion between epithelial cells and is an effective way to inhibit tumor cell invasion and metastasis. Therefore, E-cadherin is considered an important biological marker of cell differentiation degree and tumor metastasis potential, that is, the degree of its reduction reflects the severity of cell dedifferentiation and the possibility of tumor cell invasion and metastasis. E-cadherin has been very clear as a target for inhibiting tumor invasion and metastasis, and currently there is a need to develop a drug preparation that can specifically up-regulate the expression of E-cadherin with less toxicity to improve the survival rate and quality of life of cancer patients.

[0042] Based on this, the first aspect of the embodiments of the present application provides an application of chrysolaminarin in the preparation of a drug for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion. The drug for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion can be used for clinical treatment of tumor metastasis-related symptoms.

[0043] The extreme environment of the ocean breeds a large number of structure-specific marine natural products, especially the development, research and utilization of seaweed, which makes seaweed become the most mature marine plant for human research and development. The chrysolaminarin in the present application is obtained by separation and purification from algae, and the algae used include but are not limited to cuppulina, phaeodactylum tricornutum, chlorella variabilis, etc. The chrysolaminarin has the effects of inducing different tumor cells to aggregate, up-regulating E-cadherin protein expression, reversing epithelial-mesenchymal transition, and then promoting tumor cell aggregation and inhibiting tumor cell migration and diffusion.

[0044] In some embodiments, the derivative of chrysolaminarin can be applied to the preparation of a drug for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion, and the derivative of chrysolaminarin includes a polymorph of chrysolaminarin, or a sulfated derivative, a phosphorylated derivative, a carboxymethylated derivative, etc. of chrysolaminarin.

[0045] In some embodiments, the tumor cells include malignant migratory tumors.

[0046] Further, the malignant migratory tumors include 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, adrenal cortex cancer, cholangiocarcinoma, etc.

[0047] The second aspect of the embodiments of the present application provides a drug composition for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion, which includes chrysolaminarin.

[0048] In some embodiments, the drug composition for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion includes effective ingredients with compatibility synergy. Compatibility synergy refers to ingredients with certain common properties in performance and efficacy, which can improve the effect of main ingredients.

[0049] In some embodiments, the pharmaceutical composition for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion comprises a pharmaceutically acceptable excipient. Pharmaceutically acceptable refers to the ability to be administered to a human and / or other animals as a subject, and does not produce excessive adverse reactions or side effects (such as toxicity, irritation, allergic reaction, etc.). Excipient refers to the auxiliary material that exists in the pharmaceutical preparation together with the active ingredient, and does not produce excessive adverse reactions or side effects, and the excipient includes carrier, osmotic pressure regulator, pH regulator, diluent, disintegrant, excipient, solubilizer, stabilizer, preservative, etc. The pharmaceutically acceptable excipient refers to the high-safety excipient suitable for a specific pharmaceutical preparation and commonly used in pharmacy. The carrier includes but is not limited to liposome, alcoholosome, polymeric micelle, nanostructured lipid carrier, solid lipid nanoparticle, mesoporous silica nanoparticle, etc.

[0050] In some embodiments, the dosage form of the pharmaceutical composition for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion includes but is not limited to tablet, capsule, solution, granule, pill, powder, pill, suspension, powder, needle, sustained-release preparation, controlled-release preparation or targeted preparation.

[0051] In some embodiments, the pharmaceutical composition for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion can be administered by injection, oral administration, rectal administration, etc.

[0052] The following is further illustrated by specific examples.

[0053] The materials used in the examples are as follows:

[0054] Propidium iodide (PI) (catalog number: P4170), Hoechst 33342 (catalog number: B2261) were purchased from Sigma-Aldrich. Cell culture medium RPMI1640, penicillin / streptomycin, fetal bovine serum were purchased from ThermoFisher. CCK-8 kit (catalog number: C0041), lactate dehydrogenase cytotoxicity detection kit (catalog number: C0017), BeyoClick TMEdU-555 Cell Proliferation Detection Kit (Cat. No. C0075L) and Crystal Violet Staining Solution (Cat. No. C0121) were products of Biyun Tian Company. Anti-NRF2 (Cat. No. 12721), KEAP1 (Cat. No. 8047), HO-1 (Cat. No. 43966), NQO1 (Cat. No. 62262), E-Cadherin (Cat. No. 3135) and GAPDH (Cat. No. 5174) antibodies were products of Cell Signaling Technology Company. Cell RNA extraction kit (Cat. No. 19221ES50), reverse transcription kit (Cat. No. 11141ES10) and PCR amplification kit (Cat. No. 11184ES08) were products of Yeasen Company. Matrigel (Cat. No. 356234) was a product of solarbio Company.

[0055] The experimental steps in the examples were the conventional methods in the art, unless otherwise specified.

[0056] Example 1

[0057] The effect of chrysiogenin on the viability and proliferation of tumor cells included the following specific steps:

[0058] (1) Cell culture and treatment. Human neuroblastoma cells SHSY-5Y were adherent cells, which were cultured in RPMI1640 medium containing 10% (v / v) fetal bovine serum, 100 μg / mL streptomycin and 100 U / mL penicillin, and placed in a 37℃, 5% CO2 cell incubator. The cells were subcultured after 2-3 days of culture. Cells were seeded in 96-well plates at a cell density of 5×10 4 mg / mL) for 24 hours.

[0059] (2) Cell viability detection. After treatment of tumor cells with different concentrations of chrysiogenin, CCK8 (10 μL) reaction solution was added to each well after 24 hours and 48 hours of reaction, respectively, and the OD value was detected at 450 nm after 37℃ reaction for 30 minutes. The viability of the cells was calculated by measuring the absorbance.

[0060] (3) Cell death detection. After treatment of tumor cells with different concentrations of chrysiogenin, the culture supernatant of the cells was collected after 24 hours and 48 hours of reaction, respectively, and the release of lactate dehydrogenase (LDH) was detected to reflect the cell death rate.

[0061] (4) Cell proliferation detection. After tumor cells were treated with different concentrations of Laminarin for 24 hours, EdU-Azide 555 (10 mM) was added for Click reaction. Fluorescence microscopy was used to take pictures and calculate the percentage of EdU-positive cells in total cells, and then the proliferation rate of cells was calculated.

[0062] (5) Results: Drugs that inhibit tumor cell proliferation or induce tumor cell death can exert anti-tumor effects. After tumor cells were treated with different concentrations of Laminarin for 24 hours or 48 hours, CCK8 was used to detect cell viability and lactate dehydrogenase release method was used to detect cell death. As shown in Figures 1A and 1B, Laminarin did not affect cell viability and had no killing function on tumor cells. At the same time, through cell proliferation experiment (EdU-555 probe), it was found from the results shown in Figures 1C and 1D that Laminarin treatment also did not affect the proliferation of tumor cells. Therefore, Laminarin is different from general anti-tumor drugs and has no effect on the activity and proliferation ability of tumor cells.

[0063] Example 2

[0064] Morphological detection of tumor cell aggregation induced by Laminarin includes the following steps:

[0065] (1) Cell culture and treatment. In order to better observe the aggregation of cells, green fluorescent protein (GFP) plasmid was transfected into human neuroblastoma cells SHSY-5Y, which were cultured for 48 hours and screened by puromycin to construct SHSY-5Y-GFP cells. The constructed SHSY-5Y-GFP cells were cultured in RPMI1640 medium containing 10% (v / v) fetal bovine serum, 100 pg / mL streptomycin and 100 U / mL penicillin in a 37°C, 5% CO2 cell incubator. Cells can be passaged after 2-3 days of culture. After plating, the cells were treated with different concentrations of Laminarin (1, 2, 4, 6 mg / mL) for 24 hours.

[0066] (2) Detection of cell aggregation morphology. The aggregation morphology and degree of cells were photographed by inverted fluorescence microscope and laser confocal, and further showed the aggregation morphology of cells by crystal violet staining.

[0067] (3)Results: Figure 2A is a fluorescence image of tumor cells (SHSY-5Y-GFP) treated with different concentrations of laminarin, and the SHSY-5Y-GFP cells aggregated after 24 hours of reaction. As shown in Figure 2A and B, although laminarin does not have the efficacy of inhibiting tumor cell viability and proliferation or promoting cell death as general anti-tumor drugs, tumor cells treated with laminarin can induce tumor cell aggregation in a dose-dependent manner. The use of green fluorescent protein GFP expressed in tumor cells can more clearly see the aggregation of tumor cells. Through laser confocal imaging in the Z-axis direction (Figure 2C), different angle stereoscopic imaging analysis and crystal violet staining (Figure 2D), it is further verified that laminarin treatment can promote tumor cells to aggregate in space, indicating that laminarin has unique pharmacological activity.

[0068] Example 3

[0069] Morphological detection of laminarin-induced aggregation of human and mouse tumor cells includes the following steps:

[0070] (1) Cell culture and treatment. In order to better observe the aggregation of cells, green fluorescent protein (GFP) plasmid was transfected into human neuroblastoma cells SHSY-5Y, human breast cancer cells MDA-MB-231, human prostate cancer PC-3, mouse melanoma cells B16-F10 and mouse neuroblastoma cells Neuro-2A, respectively, and cultured for 48 hours. Through puromycin screening, SHSY-5Y-GFP, MDA-MB-231-GFP, PC-3-GFP, B16-F10-GFP and Neuro-2A-GFP cells were constructed. The constructed cells were cultured in RPMI1640 medium containing 10% (v / v) fetal bovine serum, 100 μg / mL streptomycin and 100 U / mL penicillin, and placed in a 37°C, 5% CO2 cell incubator. Cells can be passaged after 2-3 days of culture. After plating, the cells were treated with laminarin (4 mg / mL) for 24 hours.

[0071] (2) Detection of cell aggregation morphology. The aggregation morphology and degree of different tumor cells were photographed by inverted fluorescence microscope.

[0072] (3) Results: The photographs of the aggregation morphology and degree of different tumor cells are shown in Figure 3. In Figure 3A, the fluorescence photographs of the aggregation of human tumor cell lines SHSY-5Y-GFP (neuroblastoma cells), MDA-MB-231-GFP (breast cancer cells), and PC-3-GFP (prostate cancer cells) are shown. In Figure 3B, the fluorescence photographs of the aggregation of mouse tumor cell lines B16-F10 (melanoma) and Neuro-2A (neuroblastoma cells) are shown. As can be seen from the results in Figures 3A and 3B, the treatment with laminarin promotes the aggregation of different tumor cells into clusters in both humans and mice, further indicating that the effect of laminarin in promoting the aggregation of tumor cells into clusters is effective for both human and mouse cells, and is a broad-spectrum pharmacological property without species specificity.

[0073] Example 4

[0074] Morphological detection of the aggregation of human THP-1 cells induced by laminarin, including the following steps:

[0075] (1) Cell culture and treatment. Human acute monocytic leukemia cells THP-1 are suspension cells, which are cultured in RPMI1640 medium containing 10% (v / v) fetal bovine serum, 100 μg / mL streptomycin, 100 U / mL penicillin, and 50 μM β-mercaptoethanol, and are placed in a 37°C, 5% CO2 cell incubator for culture. The cells can be passaged after being cultured for 2-3 days. After plating, the cells are treated with medium containing different concentrations of laminarin (1, 2, and 4 mg / mL) for 24 hours.

[0076] (2) Detection of cell aggregation morphology and death. The aggregation morphology and degree of THP-1 cells are photographed by inverted fluorescence microscopy. Finally, PI (2 μg / mL) and Hoechst 33342 (5 μg / mL) are added, and the cells are stained at room temperature for 10 minutes in the dark, and the cell death is detected.

[0077] (3) Results: The fluorescence microscopic observation of human THP-1 cells treated with laminarin is shown in Figure 4. After treatment with laminarin, no obvious aggregation of THP-1 cells occurs, and after the addition of PI and Hoechst 33342, no obvious cell death is observed, indicating that the treatment with laminarin does not induce the aggregation of human THP-1 cells, and laminarin also has no cytotoxicity to normal human immune cells.

[0078] Example 5

[0079] Morphological detection of the aggregation of mouse dendritic cells induced by laminarin, including the following steps:

[0080] (1) Cell culture and treatment. Mouse dendritic cells are adherent cells, which are cultured in DMEM medium containing 10% (v / v) fetal bovine serum, 100 μg / mL streptomycin, 100 U / mL penicillin, and placed in a 37°C, 5% CO2 cell incubator. The cells can be passaged after 2-3 days of culture. After plating, the cells are treated with medium containing different concentrations of laminarin (1, 2, 4 mg / mL) for 24 hours.

[0081] (2) Detection of cell aggregation morphology and death. The aggregation morphology and degree of mouse dendritic cells are photographed by inverted fluorescence microscope. PI (2 μg / mL) and Hoechst 33342 (5 μg / mL) are finally added, and the cells are stained at room temperature for 10 minutes in the dark. The cell death is detected.

[0082] (3) Results:

[0083] The fluorescence microscopic observation of mouse dendritic cells treated with laminarin is shown in Figure 5. After treatment with laminarin, mouse dendritic cells do not show obvious aggregation, and after adding PI and Hoechst 33342, no obvious cell death is found, indicating that laminarin treatment does not induce mouse dendritic cell aggregation, and laminarin has no cytotoxicity to normal mouse immune cells.

[0084] Combining Example 4 and Example 5 can further illustrate that laminarin has the function of specifically inducing tumor cell aggregation, has no effect on normal immune cells and no toxicity.

[0085] Example 6

[0086] The tumor aggregation induced by laminarin is beneficial for the detection of cell migration and invasion, including the following steps:

[0087] (1) Wound-healing assay. SHSY-5Y-GFP cells are seeded in a 6-well plate at a cell density of 5 x 10 5 cells / well, and plated for 24 hours. After scratch treatment, different concentrations of laminarin (1, 2, 4 mg / mL) are added for 24 hours and 48 hours. The width of the scratch is measured by fluorescence microscopy and Image J software, and the cell migration rate is calculated.

[0088] (2) Transwell migration experiment. 500 μL of culture medium containing 10% fetal bovine serum was added to the lower chamber of the Transwell, and the cells were gently added to avoid the generation of bubbles. The chamber was placed in the lower chamber at an angle to avoid the generation of bubbles between the bottom of the chamber and the liquid surface. 200 μL of cell suspension (containing laminarin, 4 mg / mL) was added to the chamber, and the cells were cultured for 24 hours. After removing the cells on the upper chamber, the cells were fixed with 4% paraformaldehyde for 30 minutes, stained with crystal violet for 5 minutes, washed with PBS twice, and observed under a microscope to collect images and count the cells.

[0089] (3) Transwell invasion experiment. Dilution of Matrigel: Matrigel and serum-free medium were diluted at a ratio of 1:8. 60 μL of diluted Matrigel was added vertically to the upper chamber of the Transwell and evenly spread on the bottom. The chamber was incubated in an incubator for 3 hours to allow the Matrigel to polymerize into a film. After incubation, the excess liquid in the upper chamber was removed, and 100 μL of serum-free medium was added to each well. The chamber was placed in the incubator for 30 minutes to hydrate the basement membrane. The liquid in the upper chamber was removed, and the presence of liquid passing through the chamber into the lower chamber was checked. If there was no liquid, cells could be inoculated. The remaining steps were the same as the Transwell migration experiment described above.

[0090] (4) Results: Tumor cells grow rapidly and have the characteristics of migration, invasion, and metastasis. Therefore, inhibiting the migration and invasion of tumor cells is also an important anti-tumor strategy. After treating tumor cells with different concentrations of laminarin for 24 and 48 hours, cell scratch and Transwell experiments (results shown in Figures 6A-D) showed that laminarin can dose-dependently inhibit the migration and invasion of tumor cells during the process of inducing tumor cell aggregation. This indicates that laminarin can induce tumor cell aggregation, which is beneficial for inhibiting cell migration and invasion, thereby exerting an anti-tumor effect. This is also a new mechanism for inhibiting the migration and invasion of tumor cells.

[0091] Example 7

[0092] Detection of NRF2-Keap-1-NQO1 pathway activation by laminarin, including the following steps:

[0093] (1) Transcriptome sequencing analysis of related changed genes. After tumor cells were induced to aggregate by different concentrations of laminarin (1, 2, 4 mg / mL), the mRNA of the cells was extracted and subjected to transcriptome sequencing. The analysis steps of the data are as follows: ① Acquisition of reference genome: the latest version of human genome sequence and annotation information were downloaded from the NCBI database, and the genome index was established by using bowtie; ② Quality control of raw sequencing data: the raw quality of sequencing was detected by using FASTAQC, and the adapter sequences in the raw reads were removed by using trim-galore; the standard for judging the adapter sequences in the reads was that there was an overlap of 3 bp with the known adapter sequences; if the length of the reads after removing the adapter was less than 20 bp, the pair of reads was directly discarded; ④ Using Tophat, the filtered reads were aligned to the reference genome using the default parameters to obtain the read alignment information with the genome; ⑤ According to the genome annotation information, the read number of each gene was calculated by using FeatureCount, and the gene expression value was standardized by using RPKM; ⑥ The differentially expressed genes were obtained by using DEseq2 for gene differential expression analysis of the read number of each gene; ⑦ The differentially expressed genes were subjected to functional enrichment and pathway analysis by using ClusterProfiler and KEGG database; ⑧ The gene expression changes after laminarin treatment were analyzed and compared, and the expression of related genes in NRF2-ARE regulation was analyzed by KEGG.

[0094] (2) qPCR and Western blotting were used to detect the expression of NRF2-Keap-1-NQO1 key genes. After tumor cells were induced to aggregate by different concentrations of laminarin, the mRNA and protein of the cells were extracted and subjected to qPCR and Western blotting detection, respectively.

[0095] (3) Results: Transcriptome sequencing was used to analyze the gene expression differences and pathways of tumor cells treated with different concentrations of laminarin. The results are shown in Figures 7A, 7B and 7C. As shown in Figures 7A, 7B and 7C, the NRF2-Keap-1-NQO1 signaling pathway was significantly activated under different concentrations of laminarin treatment. Further, qPCR and Western blotting were used to verify the expression levels of key genes in the pathway, and the results are shown in Figures 7D, 7E and 7F. As shown in Figures 7D, 7E and 7F, laminarin can dose-dependently up-regulate the expression levels of key genes in the NRF2-ARE pathway. This indicates that laminarin may promote the aggregation behavior of tumor cells by regulating the NRF2-Keap1-NQO1 pathway.

[0096] Example 8

[0097] Analysis and detection of laminarin-induced expression of cell adhesion genes, including the following steps:

[0098] (1) Transcriptome sequencing combined with cell adhesion gene database analysis. Systematic information retrieval was performed from the "GeneCards" database (website: https: / / www.genecards.org / ). The keyword "Cell adhesion" was selected, and a set of 13876 genes directly related to cell adhesion function was collected. Combined with the transcriptome sequencing data after laminarin treatment, in-depth cluster analysis was performed. A cluster of genes was found whose expression levels showed an upward trend with increasing laminarin concentration. Further analysis of the expression values of this gene cluster found that this gene cluster was composed of 22 genes, and qPCR verified NQO1 and CDH1 (encoding E-cadherin protein) as key genes involved in cell aggregation and adhesion.

[0099] (2) Results: Combined with the clustering analysis results (Fig. 8 A and B) and qPCR verification results (Fig. 8 C), it was found that CDH1 and NQO1 expression values were significantly increased after laminarin treatment. Further, it was found that NQO1 played an important role in the process of laminarin promoting cell adhesion and aggregation.

[0100] Example 9

[0101] Detection of NQO1 gene knockdown significantly reversing the tumor aggregation phenomenon induced by laminarin, including the following steps:

[0102] (1) Knockdown of NQO1 gene and treatment. NQO1 gene knockdown tumor cells were constructed by siRNA technology, negative control siRNA (Negative control siRNA) was transfected into SHSY-5Y-GFP cells, and NQO1 siRNA was transfected into SHSY-5Y-RFP cells. After 48 hours of transfection, NC-siRNA-GFP and NQO1-siRNA-RFP cells were mixed and cultured in a 1:1 ratio (as shown in Fig. 9 C), and then laminarin was added for 24 hours. The aggregation was detected by fluorescence microscopy.

[0103] (2) Results: The knockdown efficiency of NQO1 protein was detected by Western blotting, and the results are shown in Fig. 9 A and B. As can be seen from Fig. 9 A and B, the NQO1 gene in tumor cells was successfully knocked down by siRNA technology. After knocking down NQO1 protein, laminarin was added for 24 hours, and the results of fluorescence microscopy detection are shown in Fig. 9 D. As can be seen from Fig. 9 D, knocking down NQO1 protein can significantly reverse the tumor cell aggregation effect induced by laminarin. Further, it was found that NQO1 protein was involved in the tumor cell aggregation induced by laminarin.

[0104] Example 10

[0105] The detection of the promotion of tumor cell aggregation by laminarin through up-regulation of E-cadherin protein expression includes the following steps:

[0106] (1) Detection of E-cadherin protein expression. Tumor cells were inoculated in a glass-bottomed culture dish (at a density of 5 x 10 5 cells / well), and after overnight plating, complete medium containing laminarin was added for 24 hours. Finally, the culture medium was aspirated, 1 mL of 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 15 minutes. Then, 2 mL of cold methanol was added to each well, and the cells were permeabilized at -20°C for 10 minutes. After blocking with blocking solution at room temperature for 1 hour, the primary antibody E-cadherin (100 μL / well) was added, and the cells were incubated at 4°C overnight. After washing, the non-cross-reactive CF568-goat rabbit anti-IgG (Biotium, USA) was added, and the cells were incubated at room temperature for 1 hour. Hoechst 33342 staining was performed in the dark for 10 minutes, and the cells were observed and photographed under a Zeiss inverted fluorescence microscope.

[0107] (2) Results: The results of fluorescence microscopic observation are shown in Figure 10B. As can be seen from Figure 10B, with increasing amounts of laminarin, the tumor aggregation-promoting effect was observed, and E-cadherin protein was also expressed in the aggregated tumor cells. This indicates that laminarin can significantly induce the expression of E-cadherin protein in tumor cells, especially in the aggregated cell clusters, and that E-cadherin expression is increased between cells, and the connection is tight. It can be seen that E-cadherin protein is likely to be a key execution molecule for the induction of tumor cell aggregation by laminarin, and the relationship between E-cadherin and tumor cell aggregation is shown in Figure 10A.

[0108] Further studies have shown that, in addition to NQO1, the key member of the gene expression regulation network related to cell adhesion, the expression of CDH1 gene (encoding E-cadherin protein) is also significantly up-regulated. E-cadherin protein plays an important role in inhibiting tumor cell invasion and metastasis. If E-cadherin expression decreases, epithelial cells will lose normal polarity and adhesion, leading to a transformation from an epithelial phenotype to a mesenchymal phenotype, i.e., epithelial-mesenchymal transition (EMT), and EMT is the basis for the invasion and metastasis of tumor cells.

[0109] Example 11

[0110] The detection of the promotion of tumor cell aggregation by laminarin and the inhibition of tumor migration and diffusion in mice includes the following steps:

[0111] (1) The construction and processing of tumor migration and diffusion model. The specific process is shown in Figure 11A. C57BL / 6 mice were injected intraperitoneally with melanoma B16-F10-Luc cells to construct a tumor migration and diffusion model. Three days after injection, laminarin (10 mg per mouse per day) was injected intraperitoneally for 7 consecutive days. The same volume of PBS was injected as Vehicle (solvent control group). The body weight changes of mice were detected after 7 days of continuous administration. The distribution of tumor cells in the peritoneal cavity was observed by a mouse in vivo imaging system. The observation was performed on the 3rd, 7th and 10th days after modeling, respectively. Each mouse was injected intraperitoneally with 150 μL of luciferin substrate (30 mg / mL) before observation, and in vivo imaging was performed 25 minutes after injection. The total number of tumor cells in the peritoneal cavity and the number of cells in the largest tumor cell aggregation were counted.

[0112] (2) Results: C57BL / 6 mice were injected intraperitoneally with B16-F10-Luc cells to construct a tumor migration and diffusion animal model. The body weight of mice was measured after intraperitoneal injection of laminarin for 7 consecutive days; the observation was performed on the 3rd, 7th and 10th days after modeling, respectively. Each mouse was injected intraperitoneally with 150 μL of luciferin substrate (30 mg / mL) before observation, and in vivo imaging was performed 25 minutes after injection. The statistical results are shown in Figures 11B, C, D and E. As shown in Figures 11B, C and D, there was no significant difference in the body weight of mice after treatment with laminarin, and there was also no significant difference in the total number of tumor cells in the peritoneal cavity compared with the solvent control group, indicating that laminarin did not affect the proliferation of tumor cells in vivo, which was consistent with the results of the in vitro cell experiment in Example 1. In addition, as shown in Figures 11C and D, the fluorescence density of tumor cells in the peritoneal cavity of mice in the laminarin treatment group was higher, and the aggregation degree was larger (showing obvious aggregation distribution). The number of cells in the largest tumor cell aggregation was statistically analyzed (Figure 11E). On the 7th day, there was a significant difference, and on the 10th day, the difference was more obvious. Combined with the above results, it was shown that in the tumor migration and diffusion animal model, laminarin promoted the aggregation of tumor cells into clusters and inhibited the migration and diffusion of tumors in mice. Further, it was shown that the pharmacological activity of laminarin in promoting tumor cell aggregation could play a role in vivo and in vitro.

[0113] Example 12

[0114] The detection of laminarin promoting the aggregation of tumor cells, facilitating the performance of tumor removal surgery, and improving the survival rate of mice included the following steps:

[0115] (1) The construction and processing of tumor migration and diffusion model, the specific process is shown in Figure 12A. The method of construction and processing of tumor migration and diffusion model is the same as "Example 11". On the 10th day, the mouse abdominal tumor removal operation was performed. After the operation, the mice were continued to be adaptively fed for 28 days, and the survival rate of the mice was observed.

[0116] (2) Results: The distribution of tumor cells in the mouse body was observed by the mouse in vivo CT imaging system (the results are shown in Figure 12B), and the observation results showed that the tumor cells were diffusely distributed in the solvent group, and migrated and diffused into the whole abdominal cavity. In the laminarin treatment group, the tumor cells aggregated into groups, and no diffuse diffusion occurred. Therefore, on the 10th day, the mouse abdominal tumor removal operation was performed, and the tumor cell groups visible to the naked eye were removed (the results are shown in Figure 12C), and sutured. Continue to feed for 28 days and observe the survival of the mice. The results are shown in Figure 12D. As can be seen from Figure 12D, the survival rate of the laminarin group (80%) is 60% higher than that of the solvent group (20%).

[0117] Therefore, laminarin promotes the aggregation of tumor cells in the mouse body, facilitates the performance of tumor removal operation, and improves the survival rate of the mouse. Further, it is also indicated that although laminarin does not inhibit the proliferation of tumor cells, promoting the aggregation of tumor cells into groups is beneficial to the performance of tumor removal operation.

[0118] In summary, through cell viability and proliferation ability detection, it is found that laminarin is different from general anti-tumor drugs, and has no effect on the activity and proliferation ability of tumor cells. Through transcriptome sequencing, it is verified that laminarin induces tumor cell aggregation effect, and it is also verified that the specificity of laminarin inducing tumor cell aggregation has no effect on normal immune cells and no toxicity; it is further found that it depends on the activation of NRF2-Keap1-NQO1 pathway and up-regulation of E-cadherin protein expression, and reverses epithelial-mesenchymal transition; at the same time, through the experiment of laminarin inducing tumor cell aggregation in vivo, it is verified that laminarin can inhibit the migration and diffusion of tumor in vivo, which is beneficial to the performance of tumor removal, and further improves the survival rate of the mouse. Laminarin has the effects of promoting tumor cell aggregation and inhibiting tumor cell migration and diffusion, and can be used for the research and development of related drugs.

[0119] It should be understood that the application of the present application is not limited to the above examples, and those of ordinary skill in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the claims attached to the present application.

Claims

1. Use of laminarin in the preparation of a drug for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion.

2. Use according to claim 1, characterized in that, The tumor cells include malignant migratory tumors.

3. Use according to claim 2, characterized in that, The malignant migratory tumors include at least one of 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, adrenal cortex cancer, and bile duct cancer.

4. A pharmaceutical composition for promoting aggregation of tumor cells and / or inhibiting migration and diffusion of tumor cells, characterized by, The pharmaceutical composition includes laminarin.

5. The pharmaceutical composition of claim 4, wherein, The tumor cells include malignant migratory tumors.

6. The pharmaceutical composition of claim 4, wherein, The malignant migratory tumors include at least one of 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, adrenal cortex cancer, and bile duct cancer.

7. The pharmaceutical composition of claim 4, wherein, The pharmaceutical composition includes effective ingredients with compatibility synergy.

8. The pharmaceutical composition of claim 4, wherein, The pharmaceutical composition includes pharmaceutically acceptable excipients.

9. The pharmaceutical composition of claim 8, wherein, The excipients include at least one of carriers, osmotic pressure regulators, pH regulators, diluents, disintegrants, excipients, solubilizers, stabilizers, and preservatives.

10. The pharmaceutical composition of claim 4, wherein, The dosage form of the pharmaceutical composition includes at least one of tablets, capsules, solutions, granules, pills, powders, beads, suspensions, powders, injections, sustained-release preparations, controlled-release preparations, and targeted preparations.

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