Use of isorhamnetin or pharmaceutically acceptable salt thereof in preparation of medicine for treating esophageal cancer

Abstract

To provide use of isorhamnetin or pharmaceutically acceptable salts thereof in preparation of drugs for treating esophageal cancer in the technical field of biological medicines.SOLUTION: It has been found that isorhamnetin significantly inhibits malignant growth of metastatic esophageal squamous carcinoma cells and in-vivo ESCC metastatic tumors by inhibiting a fatty acid de novo synthesis pathway in tumor metabolism. Therefore, isorhamnetin can be used for developing an anti-ESCC metastatic cell growth medicine targeting an abnormally active fatty acid de novo anabolic enzyme.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the use of isorhamnetin or its pharmaceutically acceptable salt in the manufacture of a medicament for treating esophageal cancer. [Background technology]

[0002] Metastasis is a major cause of treatment failure in cancer patients. The underlying mechanisms of cancer metastasis are largely unknown, involving many important signaling pathways and communication between multiple cell types. Currently, effective and safe new therapeutic strategies are lacking. Esophageal cancer is the eighth most common malignant tumor and the sixth leading cause of cancer death worldwide, with a survival rate of less than 20%. Esophageal squamous cell carcinoma (ESCC) is the major histological subtype of esophageal cancer. The high mortality rate of ESCC is primarily due to delayed diagnosis and metastasis. Despite recent advances in cancer treatment, treatment outcomes remain unsatisfactory. Therefore, elucidating the molecular mechanisms of ESCC development and metastasis is urgently needed to develop effective anticancer drugs.

[0003] Metabolic reprogramming is one of the core malignant properties of tumors and plays a crucial role in promoting tumor cell transformation and tumor progression. Recent studies have revealed that metabolic reprogramming also plays an important role in these stages of tumor metastasis. (1) Tumor cells with isocitrate dehydrogenase mutations can produce large amounts of the cancer-promoting metabolite 2-hydroxyglutarate (2-HG), which can induce epithelial-mesenchymal transition (EMT) in tumor cells, thereby acquiring invasive and metastatic potential. (2) Asparagine promotes breast cancer metastasis by promoting the expression of EMT-related proteins, while UDP-glucose inhibits EMT in lung cancer cells by accelerating the degradation of SNAI1 mRNA, further inhibiting lung cancer metastasis. (3) Tumor cells with high expression of the fatty acid receptor protein CD36 can acquire significant metastatic potential by mediating and sensing dietary fat and the extracellular lipid metabolite palmitate via CD36. Therefore, elucidating the metabolic characteristics of metastatic tumor cells and exploring active ingredients in traditional Chinese medicines that can target and intervene in key metabolic pathways within them is expected to provide new tools and strategies for the treatment of tumor metastasis. Summary of the Invention [Problem to be solved by the invention]

[0004] In view of this, the object of the present invention is to provide the use of isorhamnetin or a pharmaceutically acceptable salt thereof in the manufacture of a drug for treating esophageal cancer, wherein said isorhamnetin can significantly inhibit the growth of esophageal cancer, particularly the growth of metastatic esophageal cancer. [Means for solving the problem]

[0005] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of isorhamnetin or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating esophageal cancer.

[0007] Preferably, the esophageal cancer is metastatic esophageal cancer or non-metastatic esophageal cancer.

[0008] Preferably, the esophageal cancer cells include esophageal squamous cell carcinoma cells.

[0009] Preferably, said isorhamnetin exerts anti-metastatic esophageal cancer activity by inhibiting the de novo fatty acid synthesis pathway in tumor metabolism.

[0010] Preferably, the target protein for isorhamnetin to play a role in the treatment of esophageal cancer is fatty acid synthase.

[0011] Preferably, the structural formula of the isorhamnetin is:

[0012] [ka]

[0013] Preferably, the medicament comprises a pharmaceutically acceptable carrier.

[0014] Preferably, the mass percentage of the isorhamnetin in the drug is 50% or more.

[0015] Preferably, the pharmaceutically acceptable salt comprises a pharmaceutically acceptable acid addition salt or a pharmaceutically acceptable base addition salt.

[0016] The present invention further provides a drug combination for treating esophageal cancer, wherein the active ingredients of said drug combination comprise isorhamnetin and at least one other active ingredient for treating esophageal cancer. [Effects of the Invention]

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the use of isorhamnetin or a pharmaceutically acceptable salt thereof in the manufacture of a drug for treating esophageal cancer. Research in the present invention has revealed that isorhamnetin significantly inhibits the malignant growth of metastatic esophageal squamous cell carcinoma (ESCC) cells and in vivo ESCC metastatic tumors by inhibiting the de novo fatty acid synthesis pathway in tumor metabolism. Therefore, isorhamnetin can be used to develop anti-ESCC metastatic cell growth drugs that target abnormally active fatty acid de novo synthesis metabolic enzymes. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 shows that isorhamnetin significantly inhibits the growth of metastatic esophageal cancer cells. A shows the changes in the expression of EMT-related proteins at different time points in KYSE150 induced by TGFβ2 treatment. B shows the formation of metastatic esophageal cancer cells by KYSE150 induced by TGFβ2 treatment. C shows the effect of isorhamnetin on the growth of metastatic and non-metastatic esophageal cancer cells. D shows the effect of isorhamnetin on the cell cycle of metastatic and non-metastatic esophageal cancer cells. E shows the effect of isorhamnetin on the expression of PCNA, a proliferation marker protein, and CyclinB1, a marker protein for the G2 phase of the cell cycle, in metastatic and non-metastatic esophageal cancer cells. [Figure 2] Figure 1 shows the effect of isorhamnetin on metastatic lung tumors of esophageal cancer in vivo. A shows photographs of the lungs of different groups and statistical graphs of metastatic lung tumors. The red arrows indicate metastatic lung tumors of esophageal cancer. B shows HE staining graphs of the lungs of different groups. C shows the effect of different groups on the survival time of metastatic model mice. [Figure 3]Potential targets of action of isorhamnetin are shown by metabolomics analysis. A shows the analysis of changes in the metabolic profile of tumor cells after 3 h of treatment with isorhamnetin using the principal component analysis (PCA) algorithm. B shows the results of pathway enrichment analysis. C shows a schematic diagram of de novo fatty acid synthesis. Red and blue letters indicate up-regulated and down-regulated metabolites, respectively. [Figure 4] Figure 1 shows the results of detecting the binding of isorhamnetin to FASN. The left panel of A shows the concentration of isorhamnetin that entered tumor cells under different time conditions. The right panel shows the concentration of isorhamnetin in tumor cells after treatment with different concentrations of isorhamnetin. B shows the protein level of FASN after treatment with isorhamnetin. C shows the effect of isorhamnetin on the enzymatic activity of FASN. D shows the molecular docking results showing that isorhamnetin can potentially bind to four amino acid sites on the FASN thioesterase domain. E shows the results of detecting the binding of isorhamnetin to FASN using a cellular thermal shift assay. F shows the results of detecting the binding of isorhamnetin to FASN using a surface plasmon resonance assay. [Figure 5] Figure 1 shows the activity of the FASN-mediated fatty acid synthesis pathway in metastatic esophageal cancer cells and metastatic tumors. A shows the FASN mRNA expression level in non-metastatic and metastatic tumor cells. B shows the FASN protein expression level in non-metastatic and metastatic tumor cells. C shows the changes in FASN enzyme activity in non-metastatic and metastatic tumor cells. D shows the changes in free fatty acid content in non-metastatic and metastatic tumor cells. E shows the FASN protein expression in orthotopic subcutaneously implanted tumors and metastatic lung tumors. F shows the changes in FASN enzyme activity in orthotopic subcutaneously implanted tumors and metastatic lung tumors. G shows the changes in free fatty acid content in orthotopic subcutaneously implanted tumors and metastatic lung tumors. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides the use of isorhamnetin or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating esophageal cancer.

[0020] In this study, based on literature, we first constructed in vitro metastatic ESCC cells using the growth factor TGFβ2, and then combined literature with high-throughput screening technology to screen for Chinese herbal medicine monomers that significantly inhibited the proliferation of metastatic ESCC cells compared with non-metastatic cells. Based on the above method, we preliminarily screened 30 Chinese herbal medicine monomers to detect their effects on the proliferation, cycle, and apoptosis of in vitro ESCC metastatic cell lines. Finally, we identified the Chinese herbal medicine monomer I-isorhamnetin, which has significantly enhanced growth inhibitory effects on metastatic ESCC cells. The chemical structure of the isorhamnetin is as follows:

[0021] [ka]

[0022] In the present invention, the esophageal cancer is preferably metastatic esophageal cancer and non-metastatic esophageal cancer, such as metastatic esophageal squamous cell carcinoma. The esophageal cancer cells preferably include esophageal squamous cell carcinoma cells. In the present study, metabolomics detection of ESCC cells after isorhamnetin treatment revealed that isorhamnetin significantly inhibits the de novo fatty acid synthesis pathway in ESCC cells, increases free fatty acids in metastatic ESCC cells, and activates the expression of key de novo fatty acid synthesis metabolic enzymes. Therefore, the isorhamnetin of the present invention can be used to develop anti-ESCC metastatic cell proliferation drugs that target abnormally active de novo fatty acid synthesis metabolic enzymes. The target protein for isorhamnetin's role in the treatment of esophageal cancer is fatty acid synthase.

[0023] In the present invention, the term "pharmaceutically acceptable salts" includes pharmaceutically acceptable acid addition salts or pharmaceutically acceptable base addition salts.

[0024] In the present invention, the term "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that can retain the biological effectiveness of the free base without other adverse effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc. Organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, caproate, caprylate, caprate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalenedisulfonate, etc. These salts can be prepared by methods known in the art.

[0025] In the present invention, the term "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that can retain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary amines, secondary amines, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0026] In the present invention, the drug refers to a formulation of the isorhamnetin of the present invention with a vehicle generally accepted in the art for delivering a biologically active compound to a mammal (e.g., a human). The vehicle includes a pharmaceutically acceptable carrier. The purpose of the vehicle is to facilitate administration to an organism, favor absorption of the active ingredient, and exert biological activity. The pharmaceutically acceptable carrier refers to a substance (e.g., a carrier or diluent) that does not affect the biological activity or properties of the isorhamnetin of the present invention and is relatively non-toxic; that is, the substance can be administered to an individual without causing an adverse biological response or interacting with any components contained in the composition in an inappropriate manner. The pharmaceutically acceptable carrier includes, but is not limited to, adjuvants, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that are pharmaceutically acceptable for use in humans or veterinary animals.

[0027] In the present invention, the term "treatment" and other similar synonyms include: (i) preventing the occurrence of a disease or condition in a mammal, particularly where such mammal is susceptible to the disease or condition but has not yet been diagnosed as suffering from the disease or condition; (ii) inhibiting the disease or condition, i.e., arresting its progression; (iii) alleviating the disease or condition, i.e., causing the disease or condition to disappear; or (iv) Relieving symptoms caused by the disease or condition.

[0028] In the present invention, the mass percentage of the isorhamnetin in the drug is preferably 50% or more. The therapeutic percentage is an effective amount capable of achieving treatment of esophageal cancer. The effective amount refers to the amount of at least one drug or compound sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated after administration. The result may be reduction and / or alleviation of signs, symptoms, or etiology, or any other desired change in a biological system. For example, a "therapeutic effective amount" is the amount of the drug, including isorhamnetin, disclosed in the present invention required to provide a clinically significant symptom-alleviating effect. The effective amount appropriate for any individual case can be determined using techniques such as a dose escalation study.

[0029] In the present invention, the term "administration" refers to the method that can deliver a compound or composition to the desired site of biological action.These methods include, but are not limited to, oral route, transduodenal route, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration.Those skilled in the art are familiar with the application techniques that can be used for the isorhamnetin and methods described in the present invention, and in a preferred embodiment, the isorhamnetin discussed herein is administered by oral administration.

[0030] The present invention further provides a drug combination for treating esophageal cancer, wherein the active ingredients of said drug combination comprise isorhamnetin and at least one other active ingredient for treating esophageal cancer.

[0031] In the present invention, the drug combination refers to the drug treatment obtained by mixing or combining multiple active ingredients, and includes fixed and non-fixed combinations of active ingredients.The term "fixed combination" refers to at least one isorhamnetin and at least one synergist according to the present invention being administered to patients simultaneously in the form of a single entity or a single dosage form.The term "non-fixed combination" refers to at least one isorhamnetin and at least one synergist according to the present invention being administered to patients simultaneously, in combination, or sequentially at variable intervals in the form of separate entities.

[0032] The technical solutions provided by the present invention are described in detail below with reference to examples, which should not be construed as limiting the protection scope of the present invention.

[0033] Example 1 In vitro study of the effects of isorhamnetin (ISO) on metastatic esophageal cancer

[0034] 1.1 Transwell cell metastasis assay

[0035] (1) Human-derived ESCC cells KYSE150 were digested, washed three times with PBS, centrifuged at 1200 rpm for 3 minutes, the supernatant was removed, and the cells were resuspended in serum-free 1640 medium.

[0036] (2) Cell inoculation: The cells were counted, and TGFβ2 was added to the cell suspension so that the final TGFβ2 concentrations in the medium were 0 ng / mL and 30 ng / mL, respectively. 200 μL of the cell suspension was added to each well of the Transwell chamber, and 600 μL of 1640 medium containing 20% ​​fetal bovine serum was added to the lower chamber, followed by static culture in a 37° C. incubator for 24 hours.

[0037] (3) After 24 hours, the chamber was removed and fixed with 4% formaldehyde for 30 minutes, then stained with 0.05% crystal violet for 1 hour, and washed three times with PBS. The moisture in the upper chamber was gently wiped off with a cotton swab.

[0038] (4) Cells were observed and counted in five randomly selected fields under a microscope at 400x magnification.

[0039] 1.2 Western Blot Experiment

[0040] (1) Human-derived ESCC cells KYSE150 were digested, washed three times with PBS, centrifuged at 1200 rpm for 3 minutes, the supernatant was removed, and the cells were resuspended in serum-free 1640 medium.

[0041] (2) Cell inoculation: Count the cells and inoculate them to 1 x 10 5 Cell suspensions were prepared at a concentration of 0 ng / mL / 100 μL, and TGFβ2 was added to the cell suspensions to make the final TGFβ2 concentrations in the medium 0 ng / mL and 30 ng / mL, respectively. Each was seeded into a 6-well plate and cultured statically in a 37°C incubator for 0, 6, 12, and 24 hours to obtain cells cultured for 0, 6, 12, and 24 hours, respectively.

[0042] Proteins were extracted from cells cultured for 0, 6, 12, and 24 hours, and the expression of epithelial-mesenchymal transition (EMT)-related proteins in the cells was detected by Western blotting. The Western blotting method was as follows.

[0043] (1) Load the prepared protein samples into the gel wells. Refer to recipe 1 for preparing the electrophoresis gel. Add 5 μL of protein marker to the leftmost loading well, and then add the target protein samples sequentially from left to right in a time-incremental sorting manner, loading them in equal volumes.

[0044] (2) An appropriate amount of electrophoresis buffer (see recipe 2) was poured into the electrophoresis tank, the power was turned on, and electrophoresis was carried out at a constant voltage of 80V.

[0045] (3) When the protein sample reached the lower separation gel, the voltage was switched to 120 V. After the protein sample had migrated to the desired position, the power was turned off and the gel was removed.

[0046] (4) The PVDF membrane was activated with methanol.

[0047] (5) The PAGE Fast gel was peeled into a sandwich structure consisting of a sponge and filter paper, coated with an activated PVDF membrane, fixed, and inserted into a transfer chamber. Pre-cooled transfer buffer was added (see recipe 3), an ice core was added, the power was turned on, and the transfer was performed on ice for 1 h using a constant current of 300 mA.

[0048] (6) After the transfer was completed, the PVDF membrane was removed and placed in the previously prepared TBST buffer (see recipe 4), and then placed in the prepared 5% skim milk and incubated in a sealed state for 1 hour.

[0049] (7) The PVDF membrane was removed, and the milk was thoroughly washed with TBST buffer. The target protein was then excised to match the molecular weight of the protein marker and placed in an antibody incubator.

[0050] (8) The primary antibody for the target protein was prepared at a dilution ratio of 1:1000 in primary antibody diluent, added to an incubator, and incubated overnight on a shaker at approximately 4°C.

[0051] (9) The next day, the primary antibody was collected and washed three times with TBST buffer.

[0052] (10) A diluted secondary antibody solution with the same properties as the primary antibody was added and incubated at room temperature for 2 hours.

[0053] (11) After washing three times with TBST buffer, a color-developing solution was prepared and exposed using a BioRad exposure device.

[0054] Recipe 1: Preparation of 10%-PAGE electrophoresis gel:

[0055] The FastGel kit includes upper gel solution (2x), upper gel buffer (2x), lower gel solution (2x), lower gel buffer (2x), and improved coagulant.

[0056] (1) The gel was prepared using a 1.0-mini thick glass plate for preparing gels.

[0057] (2) Equal volumes of 2.5 mL of the lower gel solution and the lower gel buffer were taken and mixed uniformly by pipetting. Then, 60 μL of the improved coagulant was added according to the instructions, and the mixture was again pipetted and mixed uniformly.

[0058] (3) The above mixed solution was gradually poured onto the glass plate for gel preparation, and the distance between the liquid surface and the upper edge of the short glass plate was set to 0.7 cm longer than the comb teeth. An appropriate amount of absolute ethanol was quickly added to seal the lower gel.

[0059] (4) After waiting for 30 minutes, when the lower gel layer had completely solidified, the upper layer was poured off and the remaining absolute ethanol was absorbed with filter paper.

[0060] (5) Equal volumes of 1.5 mL of the upper gel solution and the upper gel buffer were taken and mixed uniformly by pipetting. 20 μL of the improved coagulant was added according to the instructions, and the mixture was again pipetted and mixed uniformly.

[0061] (6) The above mixed solution was poured into a glass plate for gel preparation, and a 15-hole comb was inserted.

[0062] (7) After 15 minutes, the upper gel layer solidified, and the comb teeth could be removed and used for electrophoresis.

[0063] Formulation 2 1x Running (Electrophoresis Buffer): For electrophoresis, 100 mL of Tris / glycine / SDS electrophoresis buffer (10x) was taken, and 900 mL of ddH2O was added to make a final volume of 1 L to prepare 1x Running Buffer. This was then stored at room temperature.

[0064] Formulation 3 1x Transfer Buffer (transfer solution): At the time of transfer, 100 mL of high-speed transfer buffer (10x) was taken, and 700 mL of ddH2O and 200 mL of methanol were added to make a final volume of 1 L to prepare 1x Transfer Buffer. This was then cooled and stored at 4°C.

[0065] Formulation 4 1x TBST buffer: 100 mL of TBST buffer (10x) was taken, and 900 mL of ddH2O was added to make up the total volume to 1 L to prepare 1x TBST buffer, which was then stored at room temperature.

[0066] 1.3 Evaluation of the proliferation activity of ISO against non-metastatic KYSE150 and metastatic KYSE150 cells using ATPlite chemiluminescence assay

[0067] (1) KYSE150 cells were digested, washed once with PBS, centrifuged at 1200 rpm for 3 minutes, the supernatant was removed, and the cells were resuspended in 3 mL of 1640 medium containing 10% fetal bovine serum.

[0068] (2) Cell inoculation: Count the cells and inoculate 3 × 10 cells per well of a 96-well black plate with 90 μL of the cell suspension. 3 The mixture was dispensed at 1 / well and incubated in an incubator at 37°C for 10 minutes.

[0069] (3) KYSE150 cells were treated with 10 μL of 0.9% saline and 30 ng / mL of TGFβ2 to construct non-metastatic KYSE150 and metastatic KYSE150 cells, respectively.

[0070] (4) 10 μL of ISO at different concentrations was added to non-metastatic and metastatic KYSE150 cells, respectively, so that the final ISO concentrations in the medium were 0 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively, and the cells were co-incubated in a 37°C incubator for 72 hours.

[0071] (5) The remaining medium in each well was aspirated, and 50 μL / well of ATPlite was added. The plate was then shaken for 10 minutes at the maximum shaking frequency in a microshaker.

[0072] (6) The fluorescent signal was detected using a fluorescent microplate reader. It is an indicator of cell growth inhibition, proliferation, and death. The stronger the signal, the higher the number of surviving cells, and conversely, the lower the number of cells.

[0073] Proteins were extracted from the cells incubated in step (4), and the expression of PCNA, a cell proliferation marker protein, was detected by Western blotting. For details of the Western blotting method, see Section 1.2.

[0074] 1.4 Cell cycle detection experiment using a flow cytometer

[0075] (1) KYSE150 cells were digested, washed once with PBS, centrifuged at 1200 rpm for 3 minutes, the supernatant was removed, and the cells were resuspended in 3 mL of 1640 medium containing 10% fetal bovine serum.

[0076] (2) Cell inoculation: Count the cells and inoculate 3 x 10 5 Cells were seeded into 6-well plates at a cell concentration of 1600 μL / well, with each well containing 1600 μL of cell suspension.

[0077] (3) KYSE150 cells were treated with 200 μL of 0.9% saline and 30 ng / mL of TGFβ2 to construct non-metastatic KYSE150 and metastatic KYSE150 cells, respectively.

[0078] (4) 200 μL of ISO at different concentrations was added to non-metastatic and metastatic KYSE150 cells, respectively, so that the final ISO concentrations in the medium were 0 μM and 70 μM, respectively, and the cells were co-incubated in a 37°C incubator for 48 hours.

[0079] (5) After 48 h, the cells were collected and transferred to a flow tube.

[0080] (6) Each flow tube was centrifuged at 1500 rpm for 5 minutes, resuspended in PBS, and washed twice. After this, 3 mL of 75% ethanol pre-cooled at -20°C was added to each tube, pipetted evenly, sealed, and stored at -20°C overnight.

[0081] (7) Before detection, the cells were centrifuged at 1500 rpm for 5 minutes, resuspended in PBS and washed once, and then 500 μL of cell cycle detection solution was added. The cells were incubated at room temperature in the dark for 30 minutes and detected using a flow cytometer.

[0082] (8) The incubated cells were collected, proteins were extracted, and the expression of CyclinB1, a marker protein for the G2 phase of the cell cycle, was detected by Western blotting. For specific Western blotting procedures, see Section 1.2.

[0083] The results in Figure 1 show that TGF-β2 inhibited the expression of the epithelial cell marker E-cadherin in tumor cells in a time-dependent manner, while upregulating the expression of mesenchymal cell markers N-cadherin and fibronectin, indicating that TGF-β2 induces tumor cells to undergo EMT and become metastatic. Next, the study found that metastatic KYSE150 cells were more sensitive to isorhamnetin treatment than non-metastatic KYSE150 cells, manifesting as more pronounced growth inhibition, G2 arrest, and apoptosis, thus demonstrating that isorhamnetin significantly inhibits the growth of metastatic esophageal cancer. After 72 hours of incubation, the growth inhibition rates of 0 μM, 25 μM, 50 μM, 100 μM, and 200 μM isorhamnetin against non-metastatic KYSE150 were 0%, 2.79%, 31.98%, 49.27%, and 63.75%, respectively. The growth inhibition rates of 0 μM, 25 μM, 50 μM, 100 μM, and 200 μM isorhamnetin against metastatic KYSE150 were 0%, 46.92%, 66.99%, 82.68%, and 93.69%, respectively.

[0084] Example 2 Animal experiments

[0085] (1) Six-week-old female BALB / c nude mice were purchased from Shanghai Lingchun Liability Co., Ltd., housed under specific-pathogen-free conditions with free access to water and food. They were randomly assigned to three groups (15 mice per group): a placebo group (vehicle), a low-dose ISO treatment group, and a high-dose ISO treatment group. Each treatment group was randomly divided into two subgroups. One subgroup (n = 10) was used to record overall survival (OS), defined as the time from cell injection to mouse death, and the other subgroup (n = 5) was used to evaluate lung metastasis.

[0086] (2) Non-metastatic wild-type KYSE150 cells were cultured in appropriate amounts.

[0087] (3) When the cell amount was sufficient, the cells were digested, collected in a 50 mL centrifuge tube, and centrifuged at 1200 rpm for 5 minutes, after which the supernatant was aspirated and discarded.

[0088] (4) The cells were washed by adding PBS and resuspending them. After centrifugation at 1200 rpm for 5 minutes, the supernatant was aspirated and discarded. This was repeated twice to wash away the remaining medium.

[0089] (5) After counting, a sufficient amount of cells was resuspended in PBS and placed on ice in preparation for inoculation.

[0090] (6) Wild-type KYSE155 cells (2 × 10) were injected into the mouse tail vein. 6 / 100 μL / needle) to obtain tumor-bearing mice.

[0091] (7) One week later, tumor-bearing mice were intraperitoneally injected every two days. The placebo group (400 μL of 1 / 100 diluted DMSO solution), low-dose ISO treatment group (400 μL of ISO suspension at 15 mg / kg / day), and high-dose ISO treatment group (400 μL of ISO suspension at 30 mg / kg / day) had their weights and food intake recorded every four days, and the mortality of the mice was also recorded.

[0092] (8) Two months later, a subgroup of mice for evaluation of lung metastasis was sacrificed and samples were collected.

[0093] (9) The lungs were fixed and stained with Bouins fixative for 30 minutes, photographed, and then fixed in 4% paraformaldehyde for further H&E staining and preparation of paraffin blocks.

[0094] The results in Figure 2 show that intraperitoneal administration of isorhamnetin can significantly inhibit the malignant growth of metastatic lung tumors, and therefore isorhamnetin can significantly prolong the survival time of metastatic model mice, with the median survival time of mice being 78.5 days after treatment with isorhamnetin.

[0095] Example 3 Detection of metabolite changes in human esophageal squamous cell carcinoma cells KYSE150 after ISO treatment by gas chromatography-high-throughput time-of-flight mass spectrometry (GC-TOFMS)

[0096] (1) Non-metastatic wild-type KYSE150 cells were digested and centrifuged at 1200 rpm for 3 min.

[0097] (2) The supernatant was aspirated and discarded, and the cells were resuspended in 1640 medium containing 10% FBS and counted.

[0098] (3) Add an appropriate amount of cell suspension to a 10 cm culture dish to achieve a cell density of 1 × 10 per dish. 7 The cells were separated into 1000 cells, and a total of 6 plates were prepared. The cells were allowed to adhere to the plate wall for 24 hours before being seeded onto the plate and the next experiment was started.

[0099] (4) After 24 hours, the six dishes of cells were divided into two groups: three dishes as a control group and three dishes as a treatment group. 1 / 1000 DMSO was added to the control group (vehicle), and ISO was added to the treatment group to a final concentration of 100 μM. The two groups were incubated in a 37°C incubator for 3 hours.

[0100] (5) After 3 h, the cells were washed once with PBS, digested, collected in a 1.5 mL centrifuge tube, centrifuged at 1200 rpm for 3 min, the supernatant was aspirated and discarded, and then washed once with PBS and centrifuged at 1200 rpm for 3 min.

[0101] (6) The supernatant was aspirated and discarded, and the cells were stored at −80°C to await detection.

[0102] The KYSE150 prepared above was subjected to GC-TOFMS detection, and the relative abundance of various metabolites was determined by comparison with metabolite standards. A commonly used statistical analysis strategy for such multidimensional data in metabolomics is as follows: After calibration and normalization, multivariate statistical analysis is first used to perform pattern recognition analysis, which then reduces the data to extract the multivariate feature standard spectral information into a small number of new components. Two of these components are then selected, and each sample is plotted on a two-dimensional diagram based on the differential values ​​of these two components. This allows for intuitive analysis of differences between individual samples or between samples in different categories.

[0103] (7) Experimental data were statistically analyzed and graphed using R software. For multivariate analysis of metabolomics data, the R software package mixOmics was used to fit a principal component analysis (PCA) model to show the metabolic characteristics of different groups. Combined with a previously reported algorithm, differential abundance values ​​(DA) of metabolic pathways were calculated to evaluate the effect of ISO on metabolic pathway activity. Data between two groups were compared using Student's t-test and two-tailed test. Experimental data were expressed as mean ± standard deviation (mean ± SD).

[0104] To analyze the downstream action targets of isorhamnetin, which inhibits metastatic tumor growth, metabolomics detection was performed on isorhamnetin-treated KYSE150 tumor cells and control cells.

[0105] The results in Figure 3 demonstrate that treatment with isorhamnetin for only 3 hours can rapidly alter the metabolic profile of cells. Pathway enrichment analysis revealed that the de novo fatty acid synthesis pathway was specifically and significantly inhibited by isorhamnetin. Further detailed analysis of the fatty acid synthesis pathway suggested that fatty acid synthase (FASN) may be a potential target of isorhamnetin.

[0106] Example 4 In order to confirm whether FASN is a downstream target protein acted upon by isorhamnetin, the following experimental studies were carried out in this example.

[0107] 4.1 Detection of ISO concentration in KYSE150 cells by high-performance liquid chromatography

[0108] (1) After treating cells with different concentrations of ISO or 100 μM ISO, the supernatant was discarded, the cells were washed twice with PBS, and the lysis solution was added to disrupt the cells. The cells were then centrifuged at 12,000 rpm for 20 minutes, and the supernatant was collected.

[0109] (2) 100 μL of the supernatant was taken, an equal volume of acetonitrile was added, and the mixture was mixed uniformly. The mixture was centrifuged at 12,000 rpm for 20 minutes, and the supernatant was collected and analyzed by liquid chromatography.

[0110] (3) Chromatographic analysis conditions: The analytical column was a Hedera C18 reverse-phase chromatography column (150.0 mm × 2.1 mm, 3 μm), the mobile phase was acetonitrile for phase A and 0.2% formic acid water for phase B, and the flow rate was 0.4 mL / min.

[0111] (4) Gradient elution conditions were: 0–2 min, 95% B–60% B; 2–6 min, 60% B–5% B; 6–8 min, 5% B; and 8.0–10 min, 95% B.

[0112] (5) The scanning wavelength range of the detector was 190 to 500 nm, and the detection wavelength was 290 nm.

[0113] 4.2 Detection of FASN mRNA expression in KYSE150 after TGFβ2 treatment by qPCR experiments

[0114] KYSE150 cells were treated with 30 ng / mL TGFβ2 and an equal volume of 0.9% saline for 48 h.

[0115] (2) 1 mL of Trizol was added, pipetted evenly, and dissolved for 5 minutes. 0.2 mL of chloroform was added, and the mixture was shaken to mix evenly. The mixture was then incubated for 2-3 minutes, and then centrifuged at 12,000 g for 15 minutes at 4°C to separate the solution into three layers. The upper colorless aqueous layer was the RNA layer. Approximately 450 μL of the colorless aqueous layer containing the RNA was transferred to a new 1.5 mL EP tube.

[0116] (3) 0.5 mL of isopropanol was added, the mixture was inverted to mix evenly, and then incubated at 4°C for 10 minutes. The mixture was centrifuged at 12,000 g for 10 minutes at 4°C to obtain a white RNA precipitate. The supernatant was discarded, and 1 mL of 75% ethanol was added to resuspend the precipitate (it can be stored at -20°C for one year). The mixture was centrifuged at 7,500 g for 5 minutes at 4°C, and the supernatant was discarded to obtain the RNA precipitate.

[0117] (4) The EP tube was opened and the RNA precipitate was allowed to air dry until it became colorless and transparent. 20 to 100 μL of RNase-free water was added to resuspend the precipitate, which was then mixed uniformly and the RNA concentration was measured.

[0118] (5) PrimeScript TM cDNA was obtained by reverse transcription using RT Master Mix, and then purified with TB Green R Premix Ex Taq TM qPCR reactions were performed using

[0119] (6) Primer sequences: FASN: forward primer 5'-AAGGACCTGTCTAGGTTTGATGC-3' (SEQ ID No. 1); reverse primer 5'-TGGCTTCATAGGTGACTTCCA-3' (SEQ ID No. 2).

[0120] 4.3 Detection of FASN expression by Western blotting

[0121] (1) KYSE150 cells were digested and centrifuged at 1200 rpm for 3 minutes.

[0122] (2) The supernatant was aspirated and discarded, and the cells were resuspended in 1640 medium containing 10% FBS and counted.

[0123] (3) Add an appropriate amount of cell suspension to a 10 cm culture dish to achieve a cell density of 1 × 10 per dish. 7 The cells were separated into 1000 cells, and a total of 6 plates were prepared. The cells were allowed to adhere to the plate wall for 24 hours before being seeded onto the plate and the next experiment was started.

[0124] (4) After 24 hours, the six dishes of cells were divided into two groups: three dishes as a control group and three dishes as a treatment group. 1 / 1000 DMSO was added to the control group (vehicle), and ISO was added to the treatment group to a final concentration of 100 μM. The two groups were incubated in a 37°C incubator for 3 hours.

[0125] (5) After 3 h, the cells were washed once with PBS, digested, collected in a 1.5 mL centrifuge tube, centrifuged at 1200 rpm for 3 min, the supernatant was aspirated and discarded, and the cells were washed once again with PBS and centrifuged at 1200 rpm for 3 min.

[0126] (6) The supernatant was aspirated and discarded, and the cells were stored at −80°C to await detection.

[0127] Next, the Western Blot method described in Section 1.2 of Example 1 was used to detect the expression of FASN in the cells of step (6).

[0128] 4.4 Molecular docking experiments

[0129] (1) The crystal structure of the protein was obtained from the Protein Data Bank (PDB) (FASN: 1XKT).

[0130] (2) Docking process: Protein dehydration and hydrogenation were performed using Discovery Studio Client.

[0131] (3) Molecular docking was performed using Pyrx-0.8 and AutoDock Vina39, and graphs were generated using Pymol software.

[0132] 4.5 Cellular thermal shift assay (CETSA)

[0133] KYSE150 cell lysates were incubated with 70 μM ISO or an equal volume of DMSO for 2 h, then heated at different temperatures (25–60°C) for 3 min and stored at room temperature for 3 min. After centrifugation at 12,000 rpm at 4°C for 20 min, the supernatant was added to SDS-PAGE, boiled, and stored at -20°C for 20 min before Western blot analysis.

[0134] 4.6 Surface plasmon resonance analyzes (SPR)

[0135] (1) Recombinant FASN protein was expressed in Escherichia coli BL21(DE3) strain and purified using the His-tag protein purification method.

[0136] (2) Experiments were performed on a BIAcore 3000 instrument. Following the standard amine coupling protocol, FASN recombinant protein was immobilized on the surface of a CM5 sensor chip with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at a rate of 10 μL / min. The density was controlled so that the response level increased by 3600–4000 response units (RU).

[0137] (3) Small compounds were diluted at different concentrations (3.12–100 μM) in 10 mM HEPES (pH 7.4), 4 mM EDTA, and 0.005% (v / v) P20 surfactant and immersed for 5 min at a flow rate of 10 μL / min, followed by a dissociation time of 6 min. The sensor chip surface was regenerated by immersing in 2.5 M NaCl PBS at 100 μL / min for 5 min and then washing twice with PBS at 100 μL / min. The output sensor maps were analyzed using BIAcore BIAnalysis software.

[0138] 4.7 Detection of FASN activity using a FASN activity detection kit

[0139] (1) KYSE150 cells were treated with ISO at different concentrations (0 μM, 50 μM, 100 μM, and 200 μM) for 3 h, and then the cells were harvested. FASN activity was detected using a FASN activity detection kit (purchased from Solarbio, catalog number: BC0550) according to the following procedure.

[0140] (2) Cell sample processing: Cell count (10 4 The extract was added at a ratio of 500-1000:1 to the volume of the extract (mL), and the cells were disrupted by ultrasonication in an ice bath (power 300W, ultrasonic wave 3 seconds, interval 9 seconds, total time 5 minutes), centrifuged at 4°C and 12000g for 20 minutes, and the supernatant was removed and placed on ice for measurement.

[0141] (3) Preheat the UV spectrophotometer for 30 minutes or more, adjust the wavelength to 340 nm, and zero the sample with distilled water.

[0142] (4) Reagent 3 was preheated to 37°C (mammals) or 25°C (other species) for 15 min before use.

[0143] (5) The sample was added according to the method in Table 1 (the following reagents were added to the cuvette):

[0144] [Table 1]

[0145] (6) FASN activity calculation: Calculated by cell number. Unit definition: 10 per minute under conditions of 37°C (mammals) or 25°C (other species). 4 The oxidation of 1 nmol of NADPH per 10 cells was defined as one enzyme activity unit. 4 cell)=1607.7×ΔA÷number of cells.

[0146] The results in Figure 4 demonstrate the following: (1) Because FASN protein is primarily distributed in the cytoplasm, we first analyzed whether ISO could enter cells. The results showed that when tumor cells were treated with a constant concentration of isorhamnetin, the intracellular isorhamnetin concentration increased over time, reaching a peak at 1 hour. When tumor cells were treated with different concentrations of isorhamnetin, the intracellular isorhamnetin concentration increased with increasing extracellular isorhamnetin concentration, indicating that ISO could enter cells in a time- and concentration-dependent manner. (2) The thioesterase domain of FASN plays an important role in the catalysis of fatty acid synthesis by FASN. Molecular docking analysis revealed that ISO could potentially bind to the Glu2395 / Tyr2425 site of the A chain and the Glu2394 / Tyr2424 site of the B chain in the FASN thioesterase domain, with a binding energy of -6.23 kcal / mol. (3) In vitro enzyme activity experiments demonstrated that ISO could rapidly inhibit the activity of FASN. (4) Cellular thermal shift assay (CETSA) results indicated that ISO could bind to FASN. (5) Surface plasmon resonance assay (SPR) further confirmed that ISO could bind to FASN, with a binding constant (KD) of 53.74 μM. Therefore, the target of action of isorhamnetin in the treatment of metastatic esophageal cancer was FASN.

[0147] Example 5 Study on the activity of the FASN-mediated fatty acid synthesis pathway in metastatic esophageal cancer cells and metastatic tumors

[0148] 5.1 Construction of the KYSE150 orthotopic subcutaneous tumor model

[0149] (1) Six-week-old female BALB / c nude mice were purchased from Shanghai Lingchun Liability Co., Ltd., and were housed under SPF conditions with free access to water and food.

[0150] (2) An appropriate amount of KYSE150 esophageal squamous cell carcinoma cells was cultured and inoculated into subcutaneously transplanted tumors.

[0151] (3) When the cell amount was sufficient, the cells were digested, collected in a 50 mL centrifuge tube, and centrifuged at 1200 rpm for 5 minutes, after which the supernatant was aspirated and discarded.

[0152] (4) The cells were washed by adding PBS and resuspending them. After centrifugation at 1200 rpm for 5 minutes, the supernatant was aspirated and discarded. This was repeated twice to wash away the remaining medium.

[0153] (5) After counting, a sufficient amount of cells was resuspended in PBS and placed on ice in preparation for inoculation.

[0154] (6) KYSE150 cells (3 × 10 6 / 10 μL / needle) was inoculated.

[0155] (7) One month after inoculation, the mice were sacrificed and samples were collected. A portion of the orthotopic subcutaneously implanted tumor was placed at −80°C and used for WB, FASN activity, and free fatty acid content detection.

[0156] 5.2 Changes in FASN mRNA expression, protein expression, and enzyme activity in non-metastatic and metastatic esophageal cancer cells

[0157] (1) KYSE150 cells were digested, washed once with PBS, centrifuged at 1200 rpm for 3 minutes, the supernatant was removed, and the cells were resuspended in 3 mL of 1640 medium containing 10% fetal bovine serum.

[0158] (2) Cell inoculation: Count the cells and inoculate 3 × 10 cells per well of a 96-well black plate with 90 μL of the cell suspension. 3 The mixture was dispensed at 1 / well and incubated in an incubator at 37°C for 10 minutes.

[0159] (3) KYSE150 cells were treated with 10 μL of 0.9% saline and 30 ng / mL TGFβ2 to construct non-metastatic KYSE150 (Vehicle group) and metastatic KYSE150 (TGFβ2 group).

[0160] Here, for detection of FASN mRNA expression, see Section 4.2 of Example 4. FASN expression in non-metastatic and metastatic esophageal cancer cells was detected using the Western blot method described in Section 1.2 of Example 1. For detection of enzyme activity, see Section 4.7 of Example 4.

[0161] 5.3 Detection of free fatty acids using a free fatty acid content detection kit

[0162] (1) KYSE150 cells were treated with 30 ng / mL TGFβ2 and an equal volume of saline for 48 h, and then harvested. Approximately 0.1–0.2 g of KYSE150 orthotopic subcutaneously implanted tumor tissue and metastatic lung tumor tissue were collected. Detection was performed using a free fatty acid content measurement kit (purchased from Saiichi Biosciences, catalog number: QYS-233086) according to the following procedure.

[0163] (2) Extraction of free fatty acid (FFA) samples:

[0164] 1. Blood: The collected blood was left to stand at room temperature for 1 hour, then centrifuged at 3500 rpm for 15 minutes at 4°C. 0.1 mL of the supernatant was taken, to which 1.2 mL of reagent 1 was added, and the mixture was shaken for 3 hours to extract. The blood was then centrifuged at 8000 g for 10 minutes at 4°C, and the supernatant was taken for measurement.

[0165] 2. Tissue: After rinsing the tissue with distilled water, absorb the surface moisture with absorbent paper and grind it. After that, add Reagent 1 at a tissue mass (g): extraction liquid volume (mL) ratio of 1:5-12 (it is recommended to weigh approximately 0.1 g of tissue and add 1.2 mL of Reagent 1). Shake for 3 hours to extract, then centrifuge at 8000 g and 4°C for 10 minutes, and collect the supernatant for measurement.

[0166] 3. Bacteria, fungi: cell number (10 4 Depending on the cells, Reagent 1 was added at a volume (mL) ratio of 500-1000:1.2 (it is recommended to add 1.2 mL of Reagent 1 to 5 million cells), and the cells were disrupted by ultrasonication in an ice bath (power 300W, ultrasonic wave 2 seconds, interval 3 seconds, total time 3 minutes), shaken for 3 hours, and then centrifuged at 8000 g for 10 minutes at 4°C. The supernatant was collected and prepared for measurement.

[0167] (3) Measurement of free fatty acid (FFA) content:

[0168] 1. The spectrophotometer was preheated for 30 minutes and the wavelength was adjusted to 715 nm.

[0169] 2. Control tube: 1 mL of the supernatant was taken, 0.5 mL of Reagent 2 was added, the mixture was thoroughly shaken for 5 minutes, and left to stand at room temperature for 5 minutes. 0.8 mL of the upper layer was placed in a 1 mL glass cuvette and adjusted to zero.

[0170] 3. Measurement tube: 1 mL of the supernatant was taken, 0.5 mL of Reagent 3 was added, and the mixture was thoroughly shaken for 5 minutes and allowed to stand at room temperature for 5 minutes. 0.8 mL of the upper layer was placed in a 1 mL glass cuvette, and the absorbance value was measured and recorded as A.

[0171] NOTE: The control tube had to be measured once per sample.

[0172] (4) Formula for calculating free fatty acid (FFA) content: Calibration curve: y = 0.0075x + 0.0055, R 2 =0.994.

[0173] 1. Calculation of blood FFA content FFA(nmol / mL)=(A-0.0055)÷0.0075×V1÷(V3×V1÷V2)=1600×(A-0.0055)

[0174] 2. Calculation of FFA content in tissues, bacteria, or cells

[0175] (1) Calculated based on the protein concentration of the sample FFA(nmol / mg prot)=(A-0.0055)÷0.0075×V1÷(V1×Cpr)=133×(A-0.0055)÷Cpr

[0176] (2) Calculate based on sample mass FFA (nmol / g fresh weight)=(A-0.0055)÷0.0075×V1÷(V1÷V2×W)=160×(A-0.0055)÷W

[0177] (3) Calculation by bacterial or cell count FFA (nmol / 10 4 cell)=(A-0.0055)÷0.0075×V1÷(V1÷V2×500)=0.32×(A-0.0055)

[0178] V1: added sample volume, 1 mL; V2: extraction liquid volume, 1.2 mL; V3: added serum (plasma) volume, 0.1 mL; Cpr: sample protein concentration, mg / mL; W: sample mass, g; 500: total number of bacteria or cells, 5 million. The metastatic lung tumors (Me) and orthotopic subcutaneously implanted tumors (Xe) in the metastatic lung tumor model mice in Example 2 and the orthotopic subcutaneously implanted tumor model mice constructed above were removed, respectively, and FASN protein expression, enzyme activity changes, and free fatty acid content in the metastatic lung tumors and orthotopic subcutaneously implanted tumors were detected, respectively.

[0179] The results in Figure 5 show that FASN mRNA, protein, and enzyme activity were all significantly increased in metastatic esophageal cancer cells compared with non-metastatic esophageal cancer cells, and free fatty acid levels were also significantly increased in metastatic cells. Next, compared with orthotopic subcutaneously implanted tumors, FASN protein and enzyme activity were significantly upregulated in metastatic lung tumors, and the free fatty acid content in metastatic lung tumors was also significantly increased. These results demonstrate that the FASN-mediated fatty acid synthesis pathway is abnormally activated in metastatic tumor cells and tumors.

[0180] The above are only preferred embodiments of the present invention, and those skilled in the art can make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications are within the protection scope.

Claims

1. 1. Use of isorhamnetin or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating metastatic esophageal cancer, comprising: The metastatic esophageal cancer cells are esophageal squamous cell carcinoma KYSE150.

10. The use characterized by:

2. Isorhamnetin exerts anti-metastatic effects on esophageal cancer by inhibiting the de novo fatty acid synthesis pathway in tumor metabolism.

2. The use according to claim 1.

3. The target protein for isorhamnetin's role in treating esophageal cancer is fatty acid synthase.

2. The use according to claim 1.

4. The structural formula of isorhamnetin is: 【Chemistry 1】 is 2. The use according to claim 1.

5. The drug comprises a pharmaceutically acceptable carrier.

2. The use according to claim 1.

6. The mass percentage of the isorhamnetin in the drug is 50% or more.

2. The use according to claim 1.

7. The pharmaceutically acceptable salts include pharmaceutically acceptable acid addition salts or pharmaceutically acceptable base addition salts.

2. The use according to claim 1.

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