Application of chrysophyceae laminarin in preparation of medicine for promoting tumor cell aggregation and inhibiting tumor cell migration and diffusion

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of chrysophyceae laminarin in preparation of medicines for promoting tumor cell aggregation and inhibiting tumor cell migration and diffusion. The chrysophyceae laminarin provided by the invention has the effects of promoting tumor cell aggregation and inhibiting tumor cell migration and diffusion, has a potential treatment effect on clinical treatment of tumor metastasis diseases, and can be used for research and development of related drugs.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to the application of kelp polysaccharide in the preparation of drugs that promote tumor cell aggregation and inhibit tumor cell migration and spread. Background Art

[0002] Tumor metastasis is a complex biological process in the late stages of tumor development, which involves the 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. Metastasis is the most deadly characteristic of malignant tumors, accounting for more than 90% of tumor-related deaths. After metastasis, malignant tumors spread throughout the body, cannot be treated surgically and are prone to recurrence. At present, it is impossible to predict and intervene in tumor metastasis early in clinical practice, and there is a lack of effective drugs for treatment, which is a difficult problem that needs to be solved urgently in clinical practice. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide an application of kelp polysaccharide in the preparation of drugs that promote tumor cell aggregation and inhibit tumor cell migration and spread, aiming to solve the current problem of being unable to intervene in tumor metastasis in clinical practice.

[0004] The technical solution of this application is as follows:

[0005] In a first aspect of the present application, there is provided a use of kelp polysaccharide in the preparation of a drug for promoting tumor cell aggregation and / or inhibiting tumor cell migration and spread.

[0006] Optionally, the tumor cells comprise malignant migratory tumors.

[0007] Optionally, the malignant migratory tumor includes 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 carcinoma, esophageal cancer, endometrial cancer, melanoma, adrenal cortical carcinoma, and bile duct cancer.

[0008] In a second aspect of the present application, a pharmaceutical composition for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion is provided, wherein the pharmaceutical composition comprises kelp polysaccharide.

[0009] Optionally, the tumor cells comprise malignant migratory tumors.

[0010] Optionally, the malignant migratory tumor includes 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 carcinoma, esophageal cancer, endometrial cancer, melanoma, adrenal cortical carcinoma, and bile duct cancer.

[0011] Optionally, the pharmaceutical composition comprises active ingredients having synergistic effects.

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

[0013] Optionally, the auxiliary material 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 tablets, capsules, solutions, granules, pills, powders, pills, suspensions, powders, injections, sustained-release agents, controlled-release agents, and targeted preparations.

[0015] Beneficial effects of the present application: Golden algae 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. Transcriptome sequencing was used to verify the effect of golden algae laminarin in inducing tumor cell aggregation, and it was found that its induction of tumor cell aggregation depends on activating the NRF2-Keap1-NQO1 pathway and upregulating the expression of E-cadherin protein, reversing epithelial-mesenchymal transition; at the same time, through the experiment of golden algae laminarin inducing tumor cell aggregation in mice, it was verified that golden algae laminarin can inhibit the migration and spread of tumors in vivo, facilitate tumor removal, and thus improve the survival rate of mice. Therefore, golden algae laminarin has the effect of promoting tumor cell aggregation and inhibiting the migration and spread of tumor cells, and can be used for the research and development of related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments.

[0017] Figure 1 This is a test chart showing the effect of kelp polysaccharide on the activity and proliferation of tumor cells provided in the examples of this application:

[0018] A is the effect of golden algae laminarin on tumor cell viability detected by CCK-8; B is the detection of lactate dehydrogenase released from the culture supernatant of tumor cells treated with golden algae laminarin; C is the fluorescence image of EdU-Azide555 cell proliferation detection of golden algae laminarin on tumor proliferation; D is the statistical graph of the percentage of EdU positive cells in total cells;

[0019] Figure 2 Morphological detection diagram of tumor cell aggregation induced by kelp polysaccharide provided in the examples of this application:

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

[0021] Figure 3 Morphological detection diagram of human and mouse tumor cell aggregation induced by golden algae laminarin polysaccharide provided in the examples of this application:

[0022] A is a fluorescence image of cell 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); B is a fluorescence image of cell aggregation of mouse tumor cell lines B16-F10 (melanoma) and Neuro-2A (neuroblastoma cells);

[0023] Figure 4 This is a cell fluorescence micrograph of human THP-1 cells treated with kelp polysaccharide provided in the examples of the present application;

[0024] Figure 5 This is a cell fluorescence micrograph of mouse dendritic cells treated with kelp polysaccharide provided in the examples of the present application;

[0025] Figure 6 The test diagram of the effect of kelp polysaccharide in inducing tumor cell aggregation and inhibiting tumor cell migration and invasion provided in the examples of this application:

[0026] A is a fluorescence image of the cell scratch test; B is a statistical analysis of the migration rate of tumor cells promoted by kelp polysaccharide at 24 and 48 hours; C is a tumor cell migration image in the Transwell chamber (without matrix gel); D is a statistical analysis of the number of tumor cell migration cells; E is a tumor cell invasion image in the Transwell chamber (with matrix gel); F is a statistical analysis of the number of tumor cell invasion cells;

[0027] Figure 7 Verification diagram of the activation of NRF2-Keap-1-NQO1 pathway by kelp polysaccharide treatment provided in the examples of this application:

[0028] A is a heat map of key genes in the NRF2-Keap-1-NQO1 pathway; B is a box plot of key genes in the NRF2-Keap-1-NQO1 pathway; C is the expression changes of different genes in the NRF-ARE signaling pathway; D is the mRNA expression level of the key gene NRF2-Keap-1-NQO1 verified by qPCR; E is the protein expression level of the key gene NRF2-Keap-1-NQO1 verified by protein immunoblotting; F is the statistics and analysis of the gray value of the protein in E;

[0029] Figure 8 Verification diagram of the expression of cell adhesion genes induced by kelp polysaccharide provided in the examples of this application:

[0030] A is the Mfuzz cluster analysis; B is the heat map of the concentration-dependent increase in cell adhesion genes; C is the mRNA expression level of cell adhesion genes verified by qPCR;

[0031] Fig. 9 This is a verification diagram of the effect of knocking down the NQO1 gene on the tumor aggregation induced by kelp polysaccharide provided in the examples of this application:

[0032] A is the knockdown efficiency of NQO1 detected by protein immunoblotting; B is the grayscale analysis of NQO1 protein; C is a schematic diagram of the mixed culture of NCsiRNA-GFP and NQO1-siRNA-RFP cells in a ratio of 1:1; D is a fluorescence image of cell aggregation taken by inverted fluorescence microscope after treatment with kelp polysaccharide (4 mg / mL) for 24 hours;

[0033] Fig.10 Fluorescence image of the golden algae laminarin promoting tumor cell aggregation provided in the examples of this 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 24 hours of treatment with kelp polysaccharide;

[0035] Fig.11 This is a test chart of the effect of kelp polysaccharide provided in the examples of this application on the inhibition of tumor migration and spread in mice:

[0036] A is a schematic diagram of administration of kelp polysaccharide; B is a statistical diagram of weight changes of mice after intraperitoneal injection of kelp polysaccharide for 7 consecutive days; C is the distribution of tumor cells in the peritoneal cavity of living mice observed by an imaging system; D is the total number of tumor cells in the peritoneal cavity of mice; E is the number of cells with the largest tumor cell aggregation in the peritoneal cavity of mice;

[0037] Fig.12The present invention provides an example of promoting the aggregation of tumor cells by using kelp polysaccharide, which is beneficial for tumor removal surgery to remove tumor cells from mice. The following is a test chart of the survival rate of mice:

[0038] A is a schematic diagram of the administration of kelp polysaccharide; B is the distribution of tumor cells in living mice observed by the CT imaging system; C is the tumor tissue of mice removed by surgery; D is the survival curve of mice after surgery. DETAILED DESCRIPTION

[0039] In order to make the purpose, 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 combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0040] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0041] Tumor metastasis refers to the process in which malignant tumor cells move from the initial lesion site to other sites through lymphatic channels, blood vessels or body cavities, and then invade other sites to continue growing. It is a complex biological process. Among them, 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 and enter the metastatic pathway. The transformed cancer cells pass through the lymphatic system, blood system or other metastatic pathways and eventually metastasize to other organs. This process is accompanied by the degradation of epithelial cadherin E-cadherin (a landmark event in EMT), and the adhesion between epithelial cells mediated by it is an effective way to inhibit tumor cell invasion and metastasis. Therefore, E-cadherin is considered to be an important biological marker of cell differentiation 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. It is very clear that E-cadherin is a target for inhibiting tumor invasion and metastasis. Currently, there is a need to develop a drug preparation that can specifically upregulate the expression of E-cadherin and has less toxicity to improve the survival rate and quality of life of cancer patients.

[0042] Based on this, the first aspect of the embodiment of the present application provides a use of chrysolaminarin in the preparation of a drug that promotes tumor cell aggregation and / or inhibits tumor cell migration and spread. The drug that promotes tumor cell aggregation and / or inhibits tumor cell migration and spread can be used in the clinical treatment of symptoms related to tumor metastasis.

[0043] The extreme environment of the ocean has bred a large number of marine natural products with specific structures, especially the development, research and utilization of seaweed, making seaweed the most mature marine plant for human research and development. The golden algae laminarin polysaccharide in the present invention is separated and purified from algae, and the algae used include but are not limited to cup brown flagella, triangular brown finger algae, and isochrysis. Golden algae laminarin polysaccharide has the effect of inducing the aggregation of different tumor cells, upregulating the expression of E-cadherin protein, and reversing epithelial-mesenchymal transition, thereby promoting the aggregation of tumor cells and inhibiting the migration and spread of tumor cells.

[0044] In some embodiments, derivatives of golden algae laminarin can be used to prepare drugs that promote tumor cell aggregation and / or inhibit tumor cell migration and spread. The derivatives of golden algae laminarin include polymorphs of golden algae laminarin, or sulfated derivatives, phosphorylated derivatives, carboxymethylated derivatives, etc. of golden algae laminarin.

[0045] In some embodiments, the tumor cells comprise malignant metastatic tumors.

[0046] Furthermore, malignant metastatic 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 carcinoma, esophageal cancer, endometrial cancer, melanoma, adrenocortical carcinoma, bile duct cancer, etc.

[0047] According to a second aspect of the embodiments of the present application, a pharmaceutical composition is provided for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion, including kelp polysaccharide.

[0048] In some embodiments, the pharmaceutical composition for promoting tumor cell aggregation and / or inhibiting tumor cell migration and spread includes active ingredients with synergistic effects. Synergistic effects refer to ingredients that have some commonality in performance and efficacy, which can enhance the effect of the main ingredient.

[0049] In some embodiments, the pharmaceutical composition that promotes tumor cell aggregation and / or inhibits tumor cell migration and diffusion includes pharmaceutically acceptable excipients. Pharmaceutically acceptable refers to being able to be applied to humans and / or other animals as subjects, and will not produce excessive adverse reactions or side effects (such as toxicity, irritation, allergic reactions, etc.). Excipients refer to auxiliary materials that exist in the pharmaceutical preparation at the same time as the active ingredient and will not produce 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 high-safety excipients that are suitable for specific pharmaceutical preparations and are routinely used in pharmacy. Carriers include but are not limited to liposomes, alcohol bodies, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, mesoporous silica nanoparticles, etc.

[0050] In some embodiments, the dosage form of the pharmaceutical composition for promoting tumor cell aggregation and / or inhibiting tumor cell migration and spread includes but is not limited to tablets, capsules, solutions, granules, pills, powders, pills, suspensions, powders, injections, sustained-release agents, controlled-release agents or targeted preparations.

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

[0052] The following is further described by means of specific examples.

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

[0054] Propidium iodide (PI) (catalog number: P4170) and Hoechst 33342 (catalog number: B2261) were purchased from Sigma-Aldrich. Cell culture medium RPMI1640, penicillin / streptomycin, and fetal bovine serum were purchased from ThermoFisher. CCK-8 kit (catalog number: C0041), lactate dehydrogenase cytotoxicity detection kit (catalog number: C0017), BeyoClick TM EdU-555 cell proliferation detection kit (Catalog No.: C0075L) and crystal violet staining solution (Catalog No.: C0121) are all products of Beyotime. Antibodies against NRF2 (Catalog No.: 12721), KEAP1 (Catalog No.: 8047), HO-1 (Catalog No.: 43966), NQO1 (Catalog No.: 62262), E-Cadherin (Catalog No.: 3135) and GAPDH (Catalog No.: 5174) are all products of Cel1 Signaling Technology. 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. Matrigel (basal glue) (Catalog No.: 356234) is a product of solarbio.

[0055] Unless otherwise specified, the experimental steps in the examples are conventional methods in the art.

[0056] Example 1

[0057] The effect of kelp polysaccharide on the viability and proliferation of tumor cells includes the following specific steps:

[0058] (1) Cell culture and treatment. Human neuroblastoma SHSY-5Y cells are adherent cells and were cultured in RPMI1640 medium containing 10% (v / v) fetal bovine serum, 100 μg / mL streptomycin and 100 U / mL penicillin in a 37°C, 5% CO2 cell culture incubator. Cells can be passaged after 2-3 days of culture. 5×10 4 The cells were inoculated at a density of 100 cells / well in a 96-well plate and then treated with culture medium containing different concentrations of kelp polysaccharide (1, 2, 4, 6 mg / mL) for 24 hours.

[0059] (2) Cell viability detection. Tumor cells were treated with different concentrations of kelp polysaccharide. After 24 hours and 48 hours of reaction, CCK8 (10 μL) 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.

[0060] (3) Cell death detection. Tumor cells were treated with different concentrations of kelp polysaccharide. After 24 hours and 48 hours of reaction, the culture supernatant of the cells was collected and the release of lactate dehydrogenase (LDH) was detected to reflect the cell death rate.

[0061] (4) Cell proliferation detection. Tumor cells were treated with different concentrations of kelp polysaccharide. After 24 hours of reaction, EdU-Azide 555 (10 μM) was added to perform a click reaction. Fluorescence microscopy was used to photograph the percentage of EdU-positive cells in the total cells and calculate the cell proliferation rate.

[0062] (5) Results: Drugs that inhibit tumor cell proliferation or induce tumor cell death can play an anti-tumor role. Tumor cells were treated with different concentrations of kelp polysaccharide for 24 hours or 48 hours; CCK8 was used to detect cell viability and lactate dehydrogenase release method was used to detect cell death; the results are as follows Figure 1 Figures A and B show that golden algae kelp polysaccharide does not affect cell viability and has no killing function on tumor cells. At the same time, through cell proliferation experiments (EdU-555 probe), Figure 1 The results shown in C and D show that the treatment with kelp polysaccharide did not affect the proliferation of tumor cells. Therefore, kelp polysaccharide is different from general anti-tumor drugs and has no effect on the activity and proliferation of tumor cells.

[0063] Example 2

[0064] The morphological detection of tumor cell aggregation induced by golden algae laminarin polysaccharide includes the following steps:

[0065] (1) Cell culture and treatment. In order to better observe the aggregation of cells, the green fluorescent protein (GFP) plasmid was transfected into human neuroblastoma cells SHSY-5Y, cultured for 48 hours, and selected 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 μg / mL streptomycin and 100 U / mL penicillin, and cultured in a cell culture incubator at 37°C and 5% CO2. The cells can be passaged after 2-3 days of culture. After plating, they were treated with culture medium containing different concentrations of kelp polysaccharide (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 using an inverted fluorescence microscope and laser confocal microscopy, and the images were taken after crystal violet staining to further demonstrate the aggregation morphology of cells.

[0067] (3) Results: Figure 2 A is a fluorescence image of SHSY-5Y-GFP cell aggregation after 24 hours of treatment with different concentrations of golden algae laminarin polysaccharide. Figure 2 As shown in A and B, although golden algae laminarin does not have the effect of inhibiting tumor cell vitality and proliferation or promoting cell death like general anti-tumor drugs, tumor cells treated with golden algae laminarin can induce tumor cell aggregation in a dose-dependent manner. By expressing green fluorescent protein GFP in tumor cells, the aggregation of tumor cells can be more clearly seen. Through laser confocal imaging in the Z-axis direction ( Figure 2 C), stereoscopic imaging analysis at different angles and crystal violet staining ( Figure 2 Middle D) further verified that the treatment with golden algae laminarin can promote the spatial aggregation of tumor cells, indicating that golden algae laminarin has unique pharmacological activity.

[0068] Example 3

[0069] The morphological detection of human and mouse tumor cell aggregation induced by kelp polysaccharide includes the following steps:

[0070] (1) Cell culture and treatment. In order to better observe the aggregation of cells, green fluorescent protein (GFP) plasmids were 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 selection, 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 cultured in a cell culture incubator at 37°C and 5% CO2. Cells can be passaged after 2-3 days of culture. After plating, they were treated with culture medium containing kelp polysaccharide (4 mg / mL) for 24 hours.

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

[0072] (3) Results: The images of different tumor cell aggregation morphologies and degrees are shown in the figure below. Figure 3 As shown. Figure 3A in the middle is a fluorescence image of cell 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). Figure 3 B is a fluorescent image of the cell aggregation of mouse tumor cell lines B16-F10 (melanoma) and Neuro-2A (neuroblastoma cells). Figure 3 It can be seen from the results of A and B that the treatment with golden algae laminarin can promote the aggregation of different tumor cells in humans and mice, which further illustrates that the effect of golden algae laminarin in promoting the aggregation of tumor cells is effective for both human and mouse cells, has a broad-spectrum pharmacological property and is not species-specific.

[0073] Example 4

[0074] The morphological detection of human THP-1 cell aggregation induced by kelp polysaccharide includes the following steps:

[0075] (1) Cell culture and treatment. Human acute monocytic leukemia THP-1 cells were suspension cells 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 cultured in a cell culture incubator at 37°C and 5% CO2. Cells can be passaged after 2-3 days of culture. After plating, they were treated with culture medium containing different concentrations of kelp polysaccharide (1, 2, 4 mg / mL) for 24 hours.

[0076] (2) Detection of cell aggregation morphology and cell death. The aggregation morphology and degree of THP-1 cells were photographed using an inverted fluorescence microscope. Finally, PI (2 μg / mL) and Hoechst33342 (5 μg / mL) were added, and the cells were stained in the dark at room temperature for 10 minutes to detect cell death.

[0077] (3) Results: Fluorescence microscopic observation of human THP-1 cells treated with kelp polysaccharide Figure 4 After treatment with golden algae laminarin, THP-1 cells did not show obvious aggregation, and after adding PI and Hoechst33342, no obvious cell death was found, indicating that golden algae laminarin treatment does not induce human THP-1 cell aggregation, and golden algae laminarin has no cytotoxicity to normal human immune cells.

[0078] Example 5

[0079] The morphological detection of dendritic cell aggregation induced by golden algae laminarin in mice includes the following steps:

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

[0081] (2) Detection of cell aggregation morphology and cell death. The aggregation morphology and degree of dendritic cells of mice were photographed by inverted fluorescence microscopy. Finally, PI (2 μg / mL) and Hoechst33342 (5 μg / mL) were added, and the cells were stained in the dark at room temperature for 10 minutes to detect cell death.

[0082] (3) Results:

[0083] Fluorescence microscopic observation of mouse dendritic cells treated with golden algae laminarin Figure 5 After treatment with golden algae laminarin, mouse dendritic cells did not show obvious aggregation, and after adding PI and Hoechst33342, no obvious cell death was found, indicating that golden algae laminarin treatment does not induce mouse dendritic cell aggregation, and golden algae laminarin has no cytotoxicity to normal mouse immune cells.

[0084] Combining Example 4 and Example 5, it can be further illustrated that the kelp polysaccharide has the function of specifically inducing tumor cell aggregation, has no effect on normal immune cells and is non-toxic.

[0085] Example 6

[0086] Tumor aggregation induced by kelp polysaccharides is beneficial for the detection of inhibition of cell migration and invasion, including the following steps:

[0087] (1) Wound-healing assay. 5 SHSY-5Y-GFP cells were inoculated into 6-well plates at a cell density of 100 cells / well, plated for 24 hours, and then scratched. Different concentrations of kelp polysaccharide (1, 2, 4 mg / mL) were added for 24 hours and 48 hours. Fluorescence microscopy was used to observe and the width of the scratch was measured using Image J software to calculate the migration rate of the cells.

[0088] (2) Transwell migration assay. Pipette 500 μL of culture medium containing 10% fetal bovine serum and add it to the lower chamber of the Transwell. Add it gently to the wall to avoid bubbles. Tilt the well plate and use sterile tweezers to pick up the chamber. Place the chamber at an angle to the liquid surface of the lower chamber to avoid bubbles between the bottom of the chamber and the liquid surface. Add 200 μL of cell suspension (containing kelp polysaccharide, 4 mg / mL) to the chamber, culture for 24 hours and then test. After removing the upper layer of cells in the chamber, fix with 4% paraformaldehyde for 30 minutes; stain with crystal violet for 5 minutes; wash twice with PBS, take pictures under a microscope to observe and collect images and count the cells.

[0089] (3) Transwell invasion assay. Dilute Matrigel: First dilute Matrigel and serum-free culture medium at a ratio of 1:8, aspirate 60 μL of diluted Matrigel, add it vertically to the upper chamber of Transwell, and evenly spread it on the bottom. Incubate in an incubator for 3 hours to allow Matrigel to polymerize into a thin film. After incubation, aspirate the excess liquid in the upper chamber, add 100 μL of serum-free culture medium to each well, and place it in the incubator for 30 minutes to hydrate the basement membrane. Aspirate the liquid in the upper chamber and check whether there is liquid passing through the small chamber into the lower chamber. If not, cells can be inoculated. The remaining steps are the same as the above-mentioned Transwell migration assay.

[0090] (4) Results: Tumor cells grow rapidly and have the characteristics of migration, diffusion and invasion to distant sites. Therefore, inhibiting the migration and invasion of tumor cells is also an important anti-tumor strategy. Tumor cells were treated with different concentrations of kelp polysaccharide for 24 hours and 48 hours. Cell scratch and Transwell experiments (results as shown in Figure 6 (shown in AD), it was found that during the process of kelp-induced tumor cell aggregation, the migration and invasion ability of cells can be inhibited in a dose-dependent manner. This indicates that kelp-induced tumor cell aggregation is beneficial to inhibit cell migration and invasion, thereby exerting an anti-tumor effect; this is also a new mechanism for inhibiting the migration and spread of tumor cells.

[0091] Example 7

[0092] The detection of the activation of NRF2-Keap-1-NQO1 pathway by kelp polysaccharide includes the following steps:

[0093] (1) Transcriptome sequencing analysis of related changed genes. After different concentrations of kelp polysaccharide (1, 2, 4 mg / mL) were treated to induce tumor cell aggregation, the cell mRNA was extracted and transcriptome sequencing was performed. The steps of data analysis are as follows: ① Acquisition of reference genome: Download the latest version of human genome sequence and annotation information from NCBI database, and use bowtie to establish genome index; ② Quality control of raw sequencing data: Use FASTAQC to detect the original sequencing quality, use trim-galore to remove the adapter sequence in the original read, and the standard for determining the adapter sequence in the read is 3bp overlap with the known adapter sequence; if the read length is less than 20bp after removing the adapter, the pair of reads will be directly discarded; ④ Use Tophat to match the filtered reads to the reference genome with default parameters to obtain the read and genome alignment information; ⑤ According to the genome annotation information, FeatureCount is used to calculate the number of reads of each gene, and RPKM is used to standardize the gene expression value; ⑥ DEseq2 is used to analyze the number of reads of each gene for gene differential expression to obtain differentially expressed genes; ⑦ ClusterProfiler and KEGG database are used to perform functional enrichment and pathway analysis on differentially expressed genes; ⑧ Analysis and comparison of gene expression changes after treatment with golden algae kelp polysaccharide, and KEGG analysis of the expression of related genes in NRF2-AREregulation.

[0094] (2) qPCR and protein immunoblotting were used to detect the expression of the key genes NRF2-Keap-1-NQO1. After different concentrations of kelp polysaccharides were used to induce tumor cell aggregation, the mRNA and protein of the cells were extracted and detected by qPCR and protein immunoblotting, respectively.

[0095] (3) Results: Transcriptome sequencing was used to analyze gene expression differences and pathways in tumor cells treated with different concentrations of kelp polysaccharides. Figure 7 As shown in A, B and C, from Figure 7 As shown in Figures A, B, and C, the NRF2-Keap-1-NQO1 signaling pathway was significantly activated under treatment with different concentrations of kelp polysaccharide. qPCR and protein immunoblotting were further used to verify the expression levels of key genes in the pathway. The results are shown in Figure 2. Figure 7 As shown in D, E, and F, Figure 7 As shown in D, E, and F, golden algae laminarin can dose-dependently upregulate the expression levels of key genes in the NRF2-ARE pathway, indicating that golden algae laminarin may promote the aggregation behavior of tumor cells by regulating the NRF2-Keap1-NQO1 pathway.

[0096] Example 8

[0097] The analysis and detection of the expression of cell adhesion genes induced by golden algae laminarin polysaccharide includes the following steps:

[0098] (1) Joint analysis of transcriptome sequencing and cell adhesion gene database. A systematic information search was performed from the "GeneCards" database (URL: https: / / www.genecards.org / ). "Cell adhesion" was selected as the keyword, and a set of 13,876 genes directly related to cell adhesion function was identified. In-depth clustering analysis was performed based on the transcriptome sequencing data after treatment with kelp polysaccharide. A class of gene clusters was identified whose expression levels increased with increasing concentrations of kelp polysaccharide. Further analysis of the expression values ​​of this class of genes revealed that this gene cluster consisted of 22 genes, and qPCR was used to verify that NQO1 and CDH1 (encoding E-cadherin protein) were key genes involved in cell aggregation and adhesion.

[0099] (2) Results: Combined with the cluster analysis results ( Figure 8 A and B) and qPCR validation results ( Figure 8 Middle C) shows that the expression values ​​of CDH1 and NQO1 increased significantly after treatment with kelp polysaccharide, which further illustrates the important role of NQO1 in the process of kelp polysaccharide promoting cell adhesion and aggregation.

[0100] Example 9

[0101] The detection of knocking down the NQO1 gene to significantly reverse the tumor aggregation phenomenon induced by kelp polysaccharide in the golden algae includes the following steps:

[0102] (1) Knockdown and treatment of NQO1 gene. Tumor cells with NQO1 gene knockdown were constructed by siRNA technology. Negative control siRNA was transfected into SHSY-5Y-GFP cells, and NQO1 siRNA was transfected into SHSY-5Y-RFP cells. 48 hours after transfection, NC-siRNA-GFP and NQO1-siRNA-RFP cells were mixed and cultured in a ratio of 1:1 (e.g. Fig. 9 Then, kelp polysaccharide was added to treat the cells for 24 hours, and the aggregation was detected by fluorescence microscopy.

[0103] (2) Results: Western blotting was used to detect the knockout efficiency of NQO1 protein. The results are as follows: Fig. 9 As shown in A and B, from Fig. 9 As shown in Figures A and B, the siRNA technology was used to successfully knock down the NQO1 gene in tumor cells. After knocking down the NQO1 protein, the cells were treated with kelp polysaccharide for 24 hours. The results of fluorescence microscopy were as follows: Fig. 9As shown in D, from Fig. 9 As shown in D, knocking down NQO1 protein can significantly reverse the tumor cell aggregation effect induced by kelp polysaccharide. This further proves that NQO1 protein is involved in the tumor cell aggregation induced by kelp polysaccharide.

[0104] Example 10

[0105] The detection of promoting the aggregation of tumor cells by inducing the upregulation of E-cadherin protein expression by golden algae kelp polysaccharide includes the following steps:

[0106] (1) Detection of E-cadherin protein expression. Tumor cells were inoculated in a glass-bottomed culture dish (density of 5×10 5 / well), after plating overnight, add complete medium containing kelp polysaccharide for 24 hours, finally discard the medium, add 1mL 4% paraformaldehyde to each well, fix at room temperature for 15 minutes, then add 2mL cold methanol to each well for permeabilization at -20℃ for 10 minutes, and then block with blocking solution at room temperature for 1 hour, add primary antibody E-cadherin (100μL / well), incubate at 4℃ overnight, wash and add non-cross-reactive CF568-goat rabbit anti-IgG (Biotium, USA), incubate at room temperature for 1 hour, stain with Hoechst33342 in the dark for 10 minutes, observe and take pictures with Zeiss inverted fluorescence microscope.

[0107] (2) Results: The results of fluorescence microscopy observation are as follows Fig.10 As shown in B, from Fig.10 As shown in Figure B, with the increase of the amount of golden algae laminarin added, it has the effect of promoting tumor aggregation. At the same time, E-cadherin protein is also expressed in the aggregated tumor cells, indicating that golden algae laminarin can significantly induce the E-cadherin protein of tumor cells. ca The expression of dherin protein increased, especially in aggregated cell clusters, and E- ca Dherin expression is increased between cells, and the connection is tight and visible. ca Dherin protein may be the key executive molecule of kelp polysaccharide-induced tumor cell aggregation. Its relationship with tumor cell aggregation is as follows: Fig.10 As shown in A.

[0108] It was further shown that, in addition to NQO1, the CDH1 gene (encoding E-cadherin protein), a key member of the gene expression regulatory network related to cell adhesion, was significantly upregulated. ca dheri nThe protein plays an important role in inhibiting the invasion and metastasis of tumor cells. If the expression of E-cadherin decreases, the normal polarity between epithelial cells will be lost, and adhesion will be lost, leading to the transition from epithelial phenotype to mesenchymal phenotype, that is, epithelial-mesenchymal transition (EMT). The occurrence of EMT in tumor cells is the basis for their ability to invade and metastasize.

[0109] Embodiment 11

[0110] The detection of the golden algae laminarin promoting the aggregation of tumor cells and inhibiting the migration and spread of tumors in mice includes the following steps:

[0111] (1) Construction and processing of tumor migration and diffusion model. The specific process is as follows Fig.11 As shown in A. C57BL / 6 mice were intraperitoneally injected with melanoma B16-F10-Luc cells to construct a model of tumor migration and diffusion. Three days after the injection, golden algae laminarin (10 mg / mouse / day) was intraperitoneally injected for 7 consecutive days. An equal volume of PBS was injected as the Vehicle (solvent control group). The weight changes of mice after 7 consecutive days of administration were detected. The distribution of tumor cells in the peritoneal cavity was observed by the mouse in vivo imaging system. Observations were carried out on the 3rd, 7th and 10th days of modeling. Before the observation, each mouse was intraperitoneally injected with 150 μL of fluorescein substrate (30 mg / mL), and in vivo imaging was performed 25 minutes after the injection. The total number of tumor cells in the peritoneal cavity and the number of cells with the largest tumor cell aggregation were counted.

[0112] (2) Results: An animal model of tumor migration and spread was established by intraperitoneal injection of B16-F10-Luc cells in C57BL / 6 mice. The body weight of mice was measured for 7 consecutive days after intraperitoneal injection of golden algae laminarin polysaccharide; observations were performed on the 3rd, 7th and 10th days of modeling. Before observation, each mouse was intraperitoneally injected with 150 μL of luciferin substrate (30 mg / mL), and in vivo imaging observations were performed 25 minutes after injection. The statistical results are as follows: Fig.11 As shown in B, C, D and E, from Fig.11 As shown in Figures B, C and D, after the treatment with kelp polysaccharide, there was no significant difference in the weight of the mice, and the total number of tumor cells in the abdominal cavity was also no significant difference compared with the solvent control group, indicating that kelp polysaccharide did not affect the proliferation of mouse tumor cells in vivo, which is consistent with the results of the in vitro cell experiment in Example 1. Fig.11 In C and D, it can be clearly observed that the fluorescence density of tumor cells in the peritoneal cavity of mice in the group treated with kelp polysaccharide was higher and the degree of aggregation was greater (showing obvious aggregation distribution). The number of cells in the largest tumor cell aggregation was statistically analyzed ( Fig.11In the middle E), there was a significant difference on the 7th day, and the difference was more obvious on the 10th day. Combined with the above results, it shows that in the animal model of tumor migration and spread, golden algae laminarin promotes tumor cell aggregation and inhibits the migration and spread of tumors in mice. This further shows that the pharmacological activity of golden algae laminarin, which promotes tumor cell aggregation, can play a role both in vivo and in vitro.

[0113] Example 12

[0114] The detection of whether golden algae kelp polysaccharide promotes the aggregation of tumor cells, facilitates tumor removal surgery, and improves the survival rate of mice includes the following steps:

[0115] (1) Construction and processing of tumor migration and diffusion model. The specific process is as follows Fig.12 As shown in A. The construction and treatment methods of the tumor migration and diffusion model were the same as those in Example 11. On the 10th day, the abdominal tumors of mice were removed. After surgery, the mice were adaptively fed for 28 days and the survival rate was observed.

[0116] (2) Results: The distribution of tumor cells in mice was observed using a mouse in vivo CT imaging system (results as shown in Fig.12 The results showed that the tumor cells in the solvent group were diffusely distributed and migrated to the entire abdominal cavity. In the group treated with kelp polysaccharide, the tumor cells aggregated into clusters and did not diffuse. Therefore, on the 10th day, the mouse abdominal tumor was removed and the tumor cell clusters visible to the naked eye were removed (the results are shown in Fig.12 The mice were fed for 28 days and their survival was observed. Fig.12 As shown in D, from Fig.12 As shown in Figure D, the survival rate of the kelp group (survival rate 80%) was increased by 60% compared with the solvent group (survival rate 20%).

[0117] Therefore, golden algae laminarin promotes the aggregation of tumor cells in mice, facilitates tumor removal surgery, and improves the survival rate of mice. This also shows that although golden algae laminarin does not inhibit the proliferation of tumor cells, it promotes the aggregation of tumor cells, which is beneficial to the tumor removal surgery.

[0118] In summary, this application has found through cell viability and proliferation ability detection that golden algae laminarin polysaccharide is different from general anti-tumor drugs and has no effect on the activity and proliferation ability of tumor cells. Transcriptome sequencing has verified the effect of golden algae laminarin polysaccharide in inducing tumor cell aggregation, and also verified the specificity of golden algae laminarin polysaccharide in inducing tumor cell aggregation. It has no effect on normal immune cells and is non-toxic; it was further found that it depends on activating the NRF2-Keap1-NQO1 pathway and upregulating the expression of E-cadherin protein to reverse epithelial-mesenchymal transition; at the same time, through the experiment of golden algae laminarin polysaccharide inducing tumor cell aggregation in a few bodies, it was verified that golden algae laminarin polysaccharide can inhibit the migration and spread of tumors in the body, which is beneficial to tumor removal, and thus improves the survival rate of mice. Golden algae laminarin polysaccharide has the effect of promoting the aggregation of tumor cells and inhibiting the migration and spread of tumor cells, 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. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. Application of kelp polysaccharide in the preparation of drugs that promote tumor cell aggregation and / or inhibit tumor cell migration and spread.

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 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 carcinoma, esophageal cancer, endometrial cancer, melanoma, adrenal cortical carcinoma, and bile duct cancer.

4. A pharmaceutical composition for promoting tumor cell aggregation and / or inhibiting tumor cell migration and diffusion, characterized in that: The pharmaceutical composition comprises chrysophyte laminarin.

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

6. The pharmaceutical composition according to claim 4, characterized in that 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 carcinoma, esophageal cancer, endometrial cancer, melanoma, adrenal cortical carcinoma, and bile duct cancer.

7. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition comprises effective ingredients having synergistic effects.

8. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition includes pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, 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.

10. The pharmaceutical composition according to claim 4, characterized in that The dosage form of the pharmaceutical composition 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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