Application of isorhamnetin or its pharmaceutically acceptable salt in preparing medicine for treating esophageal cancer

Abstract

The present invention provides an application of isorhamnetin or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating esophageal cancer, belonging to the field of biomedicine technology. The present invention has found that isorhamnetin significantly inhibits the malignant growth of metastatic esophageal squamous cell carcinoma cells and ESCC metastases in vivo by inhibiting the de novo fatty acid synthesis pathway in tumor metabolism. Therefore, isorhamnetin can be used to develop drugs for anti-ESCC metastatic cell growth that target abnormally active de novo fatty acid synthesis enzymes.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an application of isorhamnetin or a pharmaceutically acceptable salt thereof in preparing a drug for treating esophageal cancer. Background Art

[0002] Metastasis is the main reason for treatment failure in cancer patients. The underlying mechanisms of cancer metastasis are largely unknown, involving many key signaling pathways and communication between multiple cell types. Currently, there is a lack of new effective and safe treatment strategies. Esophageal cancer is the eighth most common malignancy and the sixth most common cause of cancer death worldwide, with a survival rate of less than 20%. Esophageal squamous cell carcinoma (ESCC) is the main histological subtype of esophageal cancer. The high mortality rate of ESCC is mainly due to late diagnosis and metastasis. Despite recent advances in cancer treatment, treatment outcomes are still unsatisfactory. Therefore, there is an urgent need to elucidate the molecular mechanisms of ESCC occurrence and metastasis in order to develop effective anticancer drugs.

[0003] Metabolic reprogramming is one of the core malignant characteristics of tumors and plays an important role in promoting the malignant transformation of tumor cells and tumor progression. In recent years, studies have found that metabolic reprogramming also plays an important role in these steps of tumor metastasis. (1) Tumor cells with isocitrate dehydrogenase mutations can produce a large amount of the cancer-promoting metabolite 2-hydroxyglutarate (2-HG), which can induce epithelial-mesenchymal transition (EMT) in tumor cells, thereby acquiring invasive and metastatic abilities. (2) Asparagine promotes breast cancer metastasis by promoting the expression of EMT-related proteins; UDP-glucose can inhibit the EMT transformation of lung cancer cells by accelerating the degradation of SNAI1mRNA, thereby inhibiting lung cancer metastasis. (3) Tumor cells that highly express the fatty acid receptor protein CD36 can mediate and sense lipids in the diet and the extracellular lipid metabolite palmitic acid through CD36, thereby acquiring significant metastatic abilities. Therefore, elucidating the metabolic characteristics of metastatic cancer cells and searching for active ingredients of traditional Chinese medicine that can target and intervene in key metabolic pathways are expected to provide new means and strategies for the treatment of tumor metastasis. Summary of the invention

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

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

[0006] The present invention provides an application of isorhamnetin or a pharmaceutically acceptable salt thereof in preparing a medicine for treating esophageal cancer.

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

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

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

[0010] Preferably, the target protein of isorhamnetin in treating esophageal cancer is fatty acid synthase.

[0011] Preferably, the structural formula of isorhamnetin is

[0012] Preferably, the medicament comprises a pharmaceutically acceptable carrier.

[0013] Preferably, the mass percentage of isorhamnetin in the drug is greater than 50%.

[0014] Preferably, the pharmaceutically acceptable salt includes a pharmaceutically acceptable acid addition salt or a pharmaceutically acceptable base addition salt.

[0015] The present invention also provides a drug combination for treating esophageal cancer, wherein the active ingredients of the drug combination include isorhamnetin and at least one other active ingredient for treating esophageal cancer.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides an application of isorhamnetin or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating esophageal cancer. The present invention has found that isorhamnetin significantly inhibits the malignant growth of metastatic esophageal squamous cell carcinoma (ESCC) cells and ESCC metastases in vivo by inhibiting the de novo fatty acid synthesis pathway in tumor metabolism. Therefore, isorhamnetin can be used for the development of anti-ESCC metastatic cell growth drugs targeting abnormally active de novo fatty acid synthesis enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Isorhamnetin significantly inhibits the proliferation of metastatic esophageal cancer cells. A shows the changes in the expression of EMT-related proteins in KYSE150 induced by TGFβ2 at different time points. B shows the formation of metastatic esophageal cancer cells by KYSE150 induced by TGFβ2. C shows the effect of isorhamnetin on the proliferation 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 of the G2 phase of the cell cycle, in metastatic and non-metastatic esophageal cancer cells.

[0019] Figure 2 The effect of isorhamnetin on esophageal cancer lung metastasis in vivo. A is the lung image display and lung metastasis statistics of different groups. The red arrow indicates the esophageal cancer lung metastasis. B is the lung HE staining image of different groups. C is the effect of different groups on the survival of metastasis model mice.

[0020] Figure 3 Metabolomics analysis of potential targets of isorhamnetin. A shows the changes in the metabolic profile of tumor cells after 3 h of isorhamnetin treatment using the principal component analysis (PCA) algorithm. B shows the results of pathway enrichment analysis. C shows the pattern of de novo fatty acid synthesis. Red and blue words show upregulated and downregulated metabolites, respectively.

[0021] Figure 4 The results of the detection of isorhamnetin binding to FASN, the left figure in A shows the concentration of isorhamnetin entering tumor cells under different time conditions, the right figure 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 enzyme activity of FASN, D shows the molecular docking result that isorhamnetin can potentially bind to the four amino acid sites on the FASN thioesterase domain, E shows the results of the cell thermal migration experiment to detect the binding of isorhamnetin to FASN, and F shows the results of the surface plasmon resonance experiment to detect the binding of isorhamnetin to FASN;

[0022] Figure 5 is the activity of FASN-mediated fatty acid synthesis pathway in metastatic esophageal cancer cells and metastatic tumors, A is the mRNA expression level of FASN in non-metastatic and metastatic tumor cells, B is the protein expression level of FASN in non-metastatic and metastatic tumor cells, C is the change of FASN enzyme activity in non-metastatic and metastatic tumor cells, D is the change of free fatty acid content in non-metastatic and metastatic tumor cells, E is the protein expression of FASN in orthotopic subcutaneous transplanted tumors and lung metastases, F is the change of FASN enzyme activity in orthotopic subcutaneous transplanted tumors and lung metastases, G is the change of free fatty acid content in orthotopic subcutaneous transplanted tumors and lung metastases. DETAILED DESCRIPTION

[0023] The present invention provides an application of isorhamnetin or a pharmaceutically acceptable salt thereof in preparing a medicine for treating esophageal cancer.

[0024] In the present invention, according to the literature, the growth factor TGFβ2 is first used to construct in vitro metastatic ESCC cells, and combined with the literature and high-throughput screening technology, a Chinese medicine monomer with a more obvious growth inhibition on metastatic ESCC cells compared with non-metastatic cells is screened. Based on the above model, 30 Chinese medicine monomers were preliminarily screened, and the effects of Chinese medicine monomers on the proliferation, cycle and apoptosis of in vitro ESCC metastatic cell lines were detected. Finally, a Chinese medicine monomer I, isorhamnetin, which has a significantly enhanced growth inhibition effect on metastatic ESCC cells was obtained. The chemical structure of the isorhamnetin is

[0025] 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. The present invention has found that metabolomics detection of ESCC cells after isorhamnetin intervention has found that isorhamnetin can significantly inhibit the de novo fatty acid synthesis pathway of ESCC cells, while the free fatty acids in metastatic ESCC cells increase, and the expression of key de novo fatty acid synthesis enzymes is activated. Therefore, the isorhamnetin of the present invention can be used for the development of anti-ESCC metastatic cell growth drugs targeting abnormally active de novo fatty acid synthesis enzymes. The target protein for the isorhamnetin to play a role in treating esophageal cancer is fatty acid synthase.

[0026] In the present invention, the term "pharmaceutically acceptable salt" includes a pharmaceutically acceptable acid addition salt or a pharmaceutically acceptable base addition salt.

[0027] The "pharmaceutically acceptable acid addition salt" in the present invention refers to a salt formed with an inorganic acid or an organic acid that can retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, etc.; organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, caproate, caprylate, decanoate, 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, naphthalene disulfonate, etc. These salts can be prepared by methods known in the art.

[0028] The "pharmaceutically acceptable base addition salt" in the present invention refers to a salt formed with an inorganic base or an organic base that can maintain 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. The salt derived from organic base includes but is not limited to the following salt: primary amines, secondary amines and tertiary amines, substituted amines, including natural 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.

[0029] In the present invention, the drug refers to a preparation of the isorhamnetin of the present invention and a medium generally accepted in the art for delivering biologically active compounds to mammals (e.g., humans). The medium includes a pharmaceutically acceptable carrier. The purpose of the medium is to promote administration of the organism, facilitate the absorption of the active ingredient, and thus exert biological activity. The pharmaceutically acceptable carrier refers to a substance (such as 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 applied to an individual without causing adverse biological reactions or interacting with any component contained in the composition in an adverse manner. The pharmaceutically acceptable carrier includes, but is not limited to, pharmaceutically acceptable adjuvants, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers for use by humans or livestock.

[0030] In the present invention, the term "treatment" and other similar synonyms include the following meanings:

[0031] (i) preventing a disease or condition from occurring in a mammal, particularly where such mammal is susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition;

[0032] (ii) inhibiting a disease or condition, i.e. arresting its development;

[0033] (iii) the disease or condition is alleviated, even if the disease or condition is resolved; or

[0034] (iv) alleviating the symptoms caused by the disease or condition.

[0035] In the present invention, the mass percentage of isorhamnetin in the drug is preferably more than 50%. The above-mentioned therapeutic percentage is an effective amount that can achieve the treatment of esophageal cancer. The effective amount refers to the amount of at least one agent or compound that is sufficient to alleviate one or more symptoms of the disease or condition being treated to some extent after administration. The result can be the reduction and / or relief of signs, symptoms or causes, or any other desired changes in the biological system. For example, the "effective amount" used for treatment is the amount of the drug containing isorhamnetin disclosed in the present invention required to provide a significant symptom relief effect clinically. The effective amount suitable for any individual case can be determined using techniques such as a dose escalation test.

[0036] In the present invention, the "administration" refers to a method that can deliver a compound or composition to a desired site for biological action. These methods include, but are not limited to, oral route, intraduodenal 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 administration techniques that can be used for the isorhamnetin and methods described in the present invention. In a preferred embodiment, the isorhamnetin discussed in the present invention is administered orally.

[0037] The present invention also provides a drug combination for treating esophageal cancer, wherein the active ingredients of the drug combination include isorhamnetin and at least one other active ingredient for treating esophageal cancer.

[0038] In the present invention, the pharmaceutical combination refers to a pharmaceutical treatment obtained by mixing or combining more than one active ingredient, including fixed and non-fixed combinations of active ingredients. The term "fixed combination" refers to the simultaneous administration of at least one isorhamnetin according to the present invention and at least one synergistic agent to a patient in the form of a single entity or a single dosage form. The term "non-fixed combination" refers to the simultaneous administration of at least one isorhamnetin according to the present invention and at least one synergistic agent to a patient in the form of separate entities, in combination, or sequentially at variable intervals.

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] Example 1

[0041] In vitro study on the effect of isorhamnetin (ISO) on metastatic esophageal cancer

[0042] 1.1 Transwell cell transfer assay

[0043] (1) Human ESCC KYSE150 cells were digested, washed three times with PBS, centrifuged at 1200 rpm for 3 min, the supernatant was removed, and serum-free 1640 medium was added to resuspend the cells.

[0044] (2) Inoculating cells: Count the above cells and add TGFβ2 to the cell suspension to ensure that the final TGFβ2 concentration in the culture medium is 0 ng / mL and 30 ng / mL, respectively.

[0045] 200 μL of the cell suspension was added to each well of the Transwell chamber, and 600 μL of 1640 culture medium containing 20% ​​fetal bovine serum was added to the lower chamber, and the cells were cultured in a 37° C. incubator for 24 h.

[0046] (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 water in the upper chamber was gently wiped off with a cotton swab.

[0047] (4) Observe the cells in five random fields under a 400x microscope and count them.

[0048] 1.2 Western Blot Experiment

[0049] The human ESCC cells KYSE150 were digested as described in (1), washed three times with PBS, centrifuged at 1200 rpm for 3 min, the supernatant was removed, and serum-free 1640 medium was added to resuspend the cells.

[0050] (2) Inoculation of cells: Count the above cells according to 1×10 5 / 100 μL concentration was used to prepare cell suspension, and TGFβ2 was added to the cell suspension to ensure that the final TGFβ2 concentration in the culture medium was 0 ng / mL and 30 ng / mL, respectively. They were inoculated into 6-well plates, and cultured in a 37°C incubator for 0, 6, 12 and 24 h to obtain cells after culture for 0, 6, 12 and 24 h, respectively.

[0051] Proteins of cells were extracted after culturing for 0, 6, 12 and 24 hours, and the expression of proteins related to epithelial-mesenchymal transition (EMT) in cells was detected by Western Blot method. The Western Blot detection method is as follows:

[0052] (1) Load the prepared protein sample into the gel well. For the preparation of electrophoresis gel, see Recipe 1. Add 5 μL of protein marker to the leftmost well. Add the target protein sample from left to right in ascending time order. Load equal volumes.

[0053] (2) Pour an appropriate amount of electrophoresis buffer (see Recipe 2) into the bath, turn on the power supply, and perform electrophoresis at a constant voltage of 80 V.

[0054] (3) When the protein sample reaches the lower separation gel, switch the voltage to 120 V. After the protein sample reaches the desired position, turn off the power and remove the gel.

[0055] (4) Activate the PVDF membrane with methanol.

[0056] (5) Peel off the PAGE fast gel into a sandwich consisting of sponge and filter paper, apply an activated PVDF membrane, fix it, insert it into the transfer tank, add pre-cooled transfer buffer (see Recipe 3), add ice core, turn on the power, and transfer on ice for 1 h using a constant current of 300 mA.

[0057] (6) After the transfer, remove the PVDF membrane and place it in the pre-prepared TBST buffer (see Recipe 4) and 5% skim milk for blocking and incubation for 1 hour.

[0058] (7) Remove the PVDF membrane, wash the milk with TBST buffer, cut the target protein according to the molecular weight of the control protein marker, and place it in the antibody incubation box.

[0059] (8) Prepare the primary antibody of the target protein at a dilution ratio of 1:1000 in the primary antibody diluent, add it to the incubation box, and incubate it on a shaker at around 4°C overnight.

[0060] (9) The next day, the primary antibody was recovered and washed three times with TBST buffer solution.

[0061] (10) Add secondary antibody diluent with the same properties as the primary antibody and incubate at room temperature for 2 h.

[0062] (11) Wash three times with TBST buffer solution, prepare the color developing solution, and expose with a Bio-Rad photoexposed instrument.

[0063] Formula 110% -PAGE electrophoresis gel preparation:

[0064] The rapid gel kit includes: upper gel solution (2×), upper gel buffer (2×), lower gel solution (2×), lower gel buffer (2×), and improved coagulant.

[0065] (1) Use 1.0 mini thick glass plate to make glue.

[0066] (2) Take equal volumes of 2.5 mL of lower gel solution and lower gel buffer respectively, mix well by pipetting, then add 60 μL of improved coagulant according to the instructions, and mix well by pipetting again.

[0067] (3) Slowly inject the mixed solution into the glass plate until the distance between the liquid surface and the upper edge of the short glass plate is 0.7 cm longer than the comb teeth. Quickly add an appropriate amount of anhydrous ethanol to seal the lower layer of glue.

[0068] (4) Wait for 30 minutes until the lower layer of glue is completely solidified, then pour out the upper layer and use filter paper to absorb the remaining anhydrous ethanol.

[0069] (5) Take equal volumes of 1.5 mL of upper gel solution and upper gel buffer, mix well by pipetting, add 20 μL of improved coagulant according to the instructions, and mix well by pipetting again.

[0070] (6) The mixed solution was injected into a glass plate and a 15-hole comb was inserted.

[0071] (7) After 15 minutes, when the upper layer of gel solidifies, remove the comb teeth and use it for electrophoresis.

[0072] Formula 21×Running (electrophoresis buffer): During electrophoresis, take 100 mL of Tris / glycine / SDS electrophoresis buffer (10×), add 900 mL of ddH2O to make the volume to 1 L, prepare 1×Running Buffer, and store at room temperature.

[0073] Formula 31×Transfer Buffer: Take 100 mL of the rapid transfer buffer (10×), add 700 mL of ddH2O and 200 mL of methanol, and then make up to 1 L to prepare 1×Transfer Buffer, and store at 4°C.

[0074] Formula 4 1× TBST buffer: Take 100 mL of TBST buffer solution (10×), add 900 mL of ddH2O to make the volume to 1 L, prepare 1× TBST buffer, and store at room temperature.

[0075] 1.3 Evaluation of the proliferation activity of ISO on non-transferred KYSE150 and transferred KYSE150 using ATPlite chemiluminescence method

[0076] (1) Digest KYSE150 cells, wash once with PBS, centrifuge at 1200 rpm for 3 min, remove the supernatant, and add 3 mL of 1640 medium containing 10% fetal bovine serum to resuspend.

[0077] (2) Cell inoculation: Count the above cells and dispense 90 μL of the above cell suspension into each 96-well black plate. 3 / well and incubate in a 37°C incubator for 10 min.

[0078] (3) 10 μL of 0.9% saline and 30 ng / mL TGFβ2 were added to KYSE150 cells to construct non-transferred KYSE150 and transferred KYSE150 cells.

[0079] (4) 10 μL of ISO of different concentrations was added to the non-transferred and transferred KYSE150, respectively, to ensure that the final ISO concentration in the culture medium was 0 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively, and the cells were incubated in a 37°C incubator for 72 h.

[0080] (5) Aspirate the remaining medium in each well, add 50 μL / well of ATPlite, and oscillate at the maximum frequency on a micro-oscillator for 10 min.

[0081] (6) Fluorescence microplate reader detection: the fluorescence signal is an indicator of cell growth inhibition, proliferation and death. The stronger the signal, the more surviving cells there are, and vice versa, the number of cells decreases.

[0082] Proteins were extracted from the cells after incubation in step (4), and the expression of PCNA, a cell proliferation marker protein, was detected by Western Blot method. For the specific Western Blot method, see section 1.2.

[0083] 1.4 Flow cytometry cell cycle assay

[0084] (1) Digest KYSE150 cells, wash once with PBS, centrifuge at 1200 rpm for 3 min, remove the supernatant, and add 3 mL of 1640 medium containing 10% fetal bovine serum to resuspend.

[0085] (2) Inoculation of cells: Count the above cells and use 3×10 5 The cells were seeded at a concentration of 1,600 μL / well in a six-well plate, with each well containing 1,600 μL of cell suspension.

[0086] (3) 200 μL of 0.9% saline and 30 ng / mL TGFβ2 were added to KYSE150 cells to construct non-transferred KYSE150 and transferred KYSE150 cells.

[0087] (4) Add 200 μL of ISO of different concentrations to the non-transferred and transferred KYSE150, respectively, to ensure that the final ISO concentration in the culture medium is 0 μM and 70 μM, respectively, and incubate in a 37°C incubator for 48 h.

[0088] (5) After 48 hours, the cells were collected and transferred into flow cytometry tubes.

[0089] (6) Each flow tube was centrifuged at 1500 rpm for 5 min, resuspended and washed with PBS, and repeated twice; then, 3 mL of -20°C precooled 75% ethanol was added to each tube, pipetted evenly, sealed, and stored at -20°C overnight.

[0090] (7) Before detection, centrifuge at 1500 rpm for 5 min, resuspend and wash once with PBS; add 500 μL of cell cycle detection solution, incubate at room temperature and away from light for 30 min, and detect by flow cytometry.

[0091] (8) Collect the incubated cells, extract proteins, and use Western Blot to detect the expression of CyclinB1, a marker protein of the cell cycle G2 phase. For specific Western Blot methods, see Section 1.2.

[0092] Figure 1 The results showed that TGFβ2 inhibited the expression of E-cadherin, an epithelial cell marker of tumor cells, in a time-dependent manner, but upregulated the expression of N-cadherin and Fibronectin, markers of mesenchymal cells, indicating that TGFβ2 induced EMT transformation in tumor cells and transformed them into metastatic cells. Then, the study found that compared with non-metastatic KYSE150, metastatic KYSE150 was more sensitive to isorhamnetin treatment, as shown by more obvious proliferation inhibition, G2 phase arrest and cell apoptosis, indicating that isorhamnetin can significantly inhibit the growth of metastatic esophageal cancer. After incubation for 72 hours, the proliferation inhibition rates of 0μM, 25μM, 50μM, 100μM and 200μM isorhamnetin on non-metastatic KYSE150 were 0%, 2.79%, 31.98%, 49.27% ​​and 63.75%, respectively; the proliferation inhibition rates of 0μM, 25μM, 50μM, 100μM and 200μM isorhamnetin on metastatic KYSE150 were 0%, 46.92%, 66.99%, 82.68% and 93.69%, respectively.

[0093] Example 2

[0094] Animal experiments

[0095] (1) Six-week-old female BALB / c-nude mice were purchased from Shanghai Lingchang Co., Ltd. and housed under SPF conditions with free access to water and food. They were randomly assigned to three groups, namely, a placebo group (vehicle), a low-dose ISO treatment group, and a high-dose ISO treatment group, with 15 mice in each 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.

[0096] (2) Cultivate an appropriate amount of non-metastatic wild-type KYSE150 cells.

[0097] (3) When the cell volume is sufficient, digest the cells, collect them in a 50 mL centrifuge tube, centrifuge at 1200 rpm for 5 min, and discard the supernatant.

[0098] (4) Add PBS to resuspend and wash, centrifuge at 1200 rpm for 5 min, and discard the supernatant. Repeat twice to wash away the remaining culture medium.

[0099] (5) Count and take a sufficient amount of cells, resuspend them in PBS, and place them on ice for inoculation.

[0100] (6) Wild-type KYSE150 cells (2×10 6 / 100 μL per injection) to obtain tumor-bearing mice.

[0101] (7) One week later, tumor-bearing mice were intraperitoneally injected with drugs every two days. There were three groups: a placebo group (400 μL of 1 / 100 diluted DMSO solution), a low-dose ISO treatment group (400 μL of 15 mg / kg / day ISO suspension), and a high-dose ISO treatment group (400 μL of 30 mg / kg / day ISO suspension). The body weight and food intake of the mice were recorded every 4 days, and the mortality of the mice was also recorded.

[0102] (8) Two months later, the mice in the subgroup used to evaluate lung metastasis were killed and samples were collected:

[0103] (9) The lungs were fixed and stained with Bouins fixative for 30 min, and photographed. Next, the lungs were fixed with 4% paraformaldehyde, followed by H&E staining and paraffin blocks.

[0104] Figure 2 The results showed that intraperitoneal administration of isorhamnetin could significantly inhibit the malignant growth of lung metastases, and thus isorhamnetin could significantly prolong the survival of metastasis model mice. After isorhamnetin treatment, the median survival of mice was 78.5 days.

[0105] Example 3

[0106] Gas chromatography-high-throughput time-of-flight mass spectrometry (GC-TOFMS) was used to detect the changes in metabolites in human esophageal squamous cell carcinoma cell KYSE150 after ISO treatment.

[0107] (1) Digest non-translocated wild-type KYSE150 cells and centrifuge at 1200 rpm for 3 min.

[0108] (2) Discard the supernatant, add 1640 medium containing 10% FBS to resuspend the cells, and count them.

[0109] (3) Take an appropriate amount of cell suspension and add it to a 10 cm culture dish. The cell density is 1×10 per dish. 7 Cells were grown in 6 dishes in total and adhered to the plate for 24 hours before the next experiment.

[0110] (4) After 24 h, the six dishes of cells were divided into two groups, 3 dishes of the control group and 3 dishes of the drug-treated group. The control group (Vehicle) was added with 1 / 1000 DMSO, and ISO was added to the drug-treated group to a final concentration of 100 μM. Both groups were incubated in a 37°C incubator for 3 h.

[0111] (5) After 3 h, the cells were washed once with PBS, digested, and collected in a 1.5 mL centrifuge tube. The tubes were centrifuged at 1200 rpm for 3 min, and the supernatant was discarded. The tubes were washed once with PBS, and centrifuged at 1200 rpm for 3 min.

[0112] (6) Aspirate and discard the supernatant and store the cells at -80°C until detection.

[0113] The prepared KYSE150 was subjected to GC-TOFMS detection, and the relative content of various metabolites was obtained by comparison with metabolite standards. For this type of multidimensional data in metabolomics, the commonly used statistical analysis strategy is to first use multivariate statistical analysis methods for pattern recognition analysis after calibration and standardization, perform dimensionality reduction on the data, extract the multivariate characteristic standard spectrum information to a few new components, select two of the components, and plot each specimen on a two-dimensional graph according to the different values ​​of its two components, so that the differences between specimens or specimens of different categories can be intuitively analyzed.

[0114] (7) The experimental data were statistically analyzed and plotted using R software. For multivariate analysis of metabolomics data, the R software package mixOmics was used to fit the principal component analysis model (PCA) to display the metabolic characteristics of different groups. Combined with the previously reported algorithm, the differential abundance value (DA) of the metabolic pathway was calculated to evaluate the effect of ISO on the activity of the metabolic pathway. The data between the two groups were compared using the Student's t-test and two-tailed test, and the experimental data were expressed as mean ± standard deviation (mean ± SD).

[0115] In order to analyze the downstream targets of isorhamnetin in inhibiting the proliferation of metastatic tumors, we performed metabolomics analysis on isorhamnetin-treated tumor cells KYSE150 and control group cells.

[0116] Figure 3 The results showed that isorhamnetin treatment for only 3 hours could rapidly alter the metabolic profile of cells. Pathway enrichment analysis found that the de novo fatty acid synthesis pathway was the only metabolic pathway 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.

[0117] Example 4

[0118] In order to confirm whether FASN is the downstream target protein of isorhamnetin, the following experimental studies were carried out in this example.

[0119] 4.1 Detection of ISO concentration in KYSE150 cells by HPLC

[0120] (1) After cells were treated with different concentrations of ISO or 100 μM ISO, the supernatant was discarded, the cells were rinsed twice with PBS, lysis buffer was added to disrupt the cells, and then centrifuged at 12,000 rpm for 20 min to obtain the supernatant.

[0121] (2) Take 100 μL of the supernatant and add an equal volume of acetonitrile, mix well, centrifuge at 12000 rpm for 20 min, and take the supernatant for liquid chromatography analysis.

[0122] (3) Chromatographic analysis conditions: The analytical column was a Hedera C18 reverse phase column (150.0 mm×2.1 mm, 3 μm), the mobile phase was acetonitrile in phase A and 0.2% formic acid in water in phase B, and the flow rate was 0.4 mL / min.

[0123] (4) Gradient elution conditions: 0-2 min, 95% B-60% B; 2-6 min, 60%--5% B; 6-8 min, 5% B, 8.01-10 min, 95% B.

[0124] (5) The detector scanning wavelength range is 190 to 500 nm, and the detection wavelength is 290 nm.

[0125] 4.2 qPCR assay to detect FASN mRNA expression in KYSE150 cells after TGFβ2 treatment

[0126] KYSE150 cells were treated with 30 ng / mL TGFβ2 and an equal volume of 0.9% saline for 48 h.

[0127] (2) Add 1 mL of Trizol and pipette to mix well, then lyse for 5 minutes. Add 0.2 mL of chloroform and shake to mix well, incubate for 2 to 3 minutes, and centrifuge at 12,000 g for 15 minutes at 4°C. The solution will separate into three layers, and the upper colorless aqueous phase is the RNA layer. Transfer about 450 μL of the colorless aqueous phase containing RNA to a new 1.5 mL EP tube.

[0128] (3) Add 0.5 mL of isopropanol, mix thoroughly by inversion, and incubate at 4°C for 10 min. Centrifuge at 12000 g for 10 min at 4°C to obtain a white RNA precipitate. Discard the supernatant and add 1 mL of 75% ethanol to resuspend the precipitate (which can be stored at -20°C for 1 year). Centrifuge at 7500 g for 5 min at 4°C and discard the supernatant to obtain the RNA precipitate.

[0129] (4) Open the EP tube and allow the tube to dry naturally until the RNA precipitate becomes colorless and transparent. Add 20 to 100 μL of RNase-free water to resuspend the precipitate and mix well to measure the RNA concentration.

[0130] (5) Using PrimeScript TM RT Master Mix was used for reverse transcription to obtain cDNA, and then TB Premix Ex Taq TM Perform qPCR reactions.

[0131] (6) Primer sequences: FASN: forward primer 5'-AAGGACCTGTCTAGGTTTGATGC-3' (SEQ ID No. 1); reverse primer 5'-TGGCTTCATAGGTGACTTCCA-3' (SEQ ID No. 2).

[0132] 4.3 Western Blot analysis of FASN expression

[0133] (1) Digest KYSE150 cells and centrifuge at 1200 rpm for 3 min.

[0134] (2) Discard the supernatant, add 1640 medium containing 10% FBS to resuspend the cells, and count them.

[0135] (3) Take an appropriate amount of cell suspension and add it to a 10 cm culture dish. The cell density is 1×10 per dish. 7 Cells were grown in 6 dishes in total and adhered to the plate for 24 hours before the next experiment.

[0136] (4) After 24 h, the six dishes of cells were divided into two groups, 3 dishes of the control group and 3 dishes of the drug-treated group. The control group (Vehicle) was added with 1 / 1000 DMSO, and ISO was added to the drug-treated group to a final concentration of 100 μM. Both groups were incubated in a 37°C incubator for 3 h.

[0137] (5) After 3 h, the cells were washed once with PBS, digested, and collected in a 1.5 mL centrifuge tube. The tubes were centrifuged at 1200 rpm for 3 min, and the supernatant was discarded. The tubes were washed once with PBS, and centrifuged at 1200 rpm for 3 min.

[0138] (6) Aspirate and discard the supernatant and store the cells at -80°C until detection.

[0139] Then, 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).

[0140] 4.4 Molecular docking experiments

[0141] (1) The crystal structure of the protein was obtained from the Protein Data Bank (PDB) (FASN:1XKT).

[0142] (2) Docking process: Use Discovery Studio Client to perform protein dehydration and hydrogenation.

[0143] (3) Molecular docking was performed using Pyrx-0.8 and AutoDock Vina39, and mapping was performed using Pymol software.

[0144] 4.5 Cellular thermal shift assay (CETSA)

[0145] KYSE150 cell lysate was 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 for 20 min at 4°C, SDS-PAGE supernatant was added, boiled and stored at -20°C, and then Western blot was performed.

[0146] 4.6 Surface plasmon resonance analyses (SPR)

[0147] (1) The recombinant FASN protein was expressed in Escherichia coli BL21 (DE3) strain and purified using the His-tag protein purification method.

[0148] (2) The experiment was performed on a BIAcore 3000 instrument. According to 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, and the density was controlled to increase the response level by 3600-4000 response units (RU).

[0149] (3) Small compounds of different concentrations (3.12-100 μM) were diluted with 10 mM HEPES (pH 7.4), 4 mM EDTA, and 0.005% (v / v) P20 surfactant, and soaked for 5 min at a flow rate of 10 μL / min, with a dissociation time of 6 min. The sensor chip surface was regenerated by soaking in 100 μL / min PBS with 2.5 M NaCl for 5 min, and then washed twice with 100 μL / min PBS. The output sensor map was analyzed using BIAcore BIAnalysis software.

[0150] 4.7 FASN activity detection kit to detect FASN activity

[0151] (1) KYSE150 was treated with different concentrations of 0 μM, 50 μM, 100 μM, and 200 μM ISO for 3 h, and the cells were collected. The FASN activity detection kit (purchased from Solebol, catalog number: BC0550) was used for detection according to the following steps.

[0152] (2) Processing of cell samples: According to the number of cells (10 4 The extract was added in a ratio of 500 to 1000:1, and the cells were disrupted by ultrasonic wave in an ice bath (power 300 W, ultrasonic wave 3 seconds, interval 9 seconds, total time 5 min); centrifuged at 4°C, 12000g for 20 min, and the supernatant was taken and placed on ice for detection.

[0153] (3) Preheat the UV spectrophotometer for more than 30 minutes, adjust the wavelength to 340 nm, and zero it with distilled water.

[0154] (4) Reagent 3 should be preheated at 37°C (for mammals) or 25°C (for other species) for 15 min before use.

[0155] (5) Add samples according to Table 1 (add the following reagents to the cuvette).

[0156] Table 1 Information of added samples

[0157]

[0158] (6) FASN activity calculation: calculated by cell number, unit definition: at 37°C (mammals) or 25°C (other species), per 10 4 One unit of enzyme activity is when one cell oxidizes 1 nmol NADPH per minute. 4 cell)=1607.7×ΔA÷number of cells.

[0159] Figure 4The results showed that (1) Since FASN protein is mainly distributed in the cytoplasm, we first analyzed whether ISO could enter the cell. The results showed that when tumor cells were treated with a fixed concentration of isorhamnetin, the concentration of intracellular isorhamnetin increased with time and reached a peak at 1 hour; when tumor cells were treated with different concentrations of isorhamnetin, the concentration of intracellular isorhamnetin increased with the increase of extracellular isorhamnetin concentration. Therefore, ISO can enter the cell in a time-dependent and concentration-dependent manner. (2) The thioesterase domain on FASN plays a key role in FASN-catalyzed fatty acid synthesis. Molecular docking analysis found that ISO can potentially bind to the Glu2395 / Tyr2425 site of the A chain and the Glu2394 / Tyr2424 site of the B chain in the thioesterase domain of FASN, with a binding energy of -6.23 kcal / mol. (3) In vitro enzyme activity experiments found that ISO can quickly inhibit the activity of FASN. (4) Cellular Thermal Shift Assay (CETSA) results showed that ISO can bind to FASN. (5) Surface plasmon resonance assay (SPR) further confirmed that ISO can bind to FASN, and the binding constant (KD) value was 53.74 μM. Therefore, the target of isorhamnetin in the treatment of metastatic esophageal cancer is FASN.

[0160] Example 5

[0161] Study on the activity of FASN-mediated fatty acid synthesis pathway in metastatic esophageal cancer cells and esophageal cancer metastases

[0162] 5.1 Construction of KYSE150 orthotopic subcutaneous transplant tumor model

[0163] (1) Six-week-old female BALB / c-nude mice were purchased from Shanghai Lingchang Co., Ltd. and housed under SPF conditions with free access to water and food.

[0164] (2) Culture an appropriate amount of KYSE150 esophageal squamous cell carcinoma cells and inoculate subcutaneous transplanted tumors.

[0165] (3) When the cell volume is sufficient, digest the cells, collect them in a 50 mL centrifuge tube, centrifuge at 1200 rpm for 5 min, and discard the supernatant.

[0166] (4) Add PBS to resuspend and wash, centrifuge at 1200 rpm for 5 min, and discard the supernatant. Repeat twice to wash away the remaining culture medium.

[0167] (5) Count and take a sufficient amount of cells, resuspend them in PBS, and place them on ice for inoculation.

[0168] (6) KYSE150 cells (3×10 6 / 10μL per needle).

[0169] (7) One month after inoculation, the mice were killed and samples were collected. Part of the orthotopic subcutaneous transplanted tumor was placed at -80°C for WB, FASN activity, and free fatty acid content testing.

[0170] 5.2 Changes in FASN mRNA expression, protein expression, and enzyme activity in non-metastatic and metastatic esophageal cancer cells

[0171] (1) Digest KYSE150 cells, wash once with PBS, centrifuge at 1200 rpm for 3 min, remove the supernatant, and add 3 mL of 1640 medium containing 10% fetal bovine serum to resuspend.

[0172] (2) Cell inoculation: Count the above cells and dispense 90 μL of the above cell suspension into each 96-well black plate. 3 / well and incubate in a 37°C incubator for 10 min.

[0173] (3) 10 μL of 0.9% saline and 30 ng / mL TGFβ2 were added to KYSE150 cells to construct non-metastatic KYSE150 (Vehicle group) and metastatic KYSE150 (TGFβ2 group).

[0174] The detection of FASN mRNA expression is described in Section 4.2 of Example 4. The expression of FASN in non-metastatic and metastatic esophageal cancer cells was detected using the Western Blot method described in Section 1.2 of Example 1. The detection of enzyme activity is described in Section 4.7 of Example 4.

[0175] 5.3 Free fatty acid content detection kit to detect free fatty acids

[0176] (1) Treat KYSE150 with 30 ng / mL TGFβ2 and an equal volume of saline for 48 h, collect cells, and take about 0.1-0.2 g of KYSE150 orthotopic subcutaneous transplanted tumor tissue and lung metastasis tissue. Use a free fatty acid content determination kit (purchased from Qiyi Biological, catalog number: QYS-233086) to perform detection according to the following steps.

[0177] (2) Free fatty acid (FFA) sample extraction:

[0178] 1. Blood: After the blood is allowed to stand at room temperature for 1 hour, centrifuge it at 3500 rpm for 15 minutes at 4°C. Take 0.1 mL of the supernatant, add 1.2 mL of reagent 1, shake and extract for 3 hours, centrifuge at 8000g at 4°C for 10 minutes, and take the supernatant for testing.

[0179] 2. Tissue: After the tissue is rinsed with distilled water, absorb the surface moisture with absorbent paper, mash it and add reagent 1 according to the ratio of tissue mass (g): extraction solution volume (mL) of 1:5-12 (it is recommended to weigh about 0.1g tissue and add 1.2mL reagent 1), shake and extract for 3h, centrifuge at 8000g, 4℃ for 10min, and take the supernatant for testing.

[0180] 3. Bacteria and fungi: According to the number of cells (10 4 The volume of reagent 1 (mL) is 500-1000:1.2 (it is recommended to add 1.2 mL of reagent 1 for 5 million cells). Add reagent 1 and disrupt the cells by ultrasonic wave in an ice bath (power 300w, ultrasonic wave 2 seconds, interval 3 seconds, total time 3min); shake and extract for 3h, then centrifuge at 8000g, 4℃ for 10min, and take the supernatant for detection.

[0181] (3) Free fatty acid (FFA) content determination operation:

[0182] 1. Preheat the spectrophotometer for 30 minutes and adjust the wavelength to 715nm.

[0183] 2. Control tube: Take 1 mL of supernatant, add 0.5 mL of reagent 2, shake thoroughly for 5 min, let stand at room temperature for 5 min, take 0.8 mL of the upper layer into a 1 mL glass cuvette, and adjust to zero.

[0184] 3. Assay tube: Take 1 mL of supernatant, add 0.5 mL of reagent 3, shake thoroughly for 5 min, let stand at room temperature for 5 min, take 0.8 mL of the upper layer and put it in a 1 mL glass cuvette, measure the absorbance, and record it as A.

[0185] Note: The control tube needs to be measured once for each sample.

[0186] (4) Calculation formula for free fatty acid (FFA) content:

[0187] Standard curve: y = 0.0075x + 0.0055, R 2 =0.994.

[0188] 1. Calculation of FFA content in blood

[0189] FFA(nmol / mL)=(A-0.0055)÷0.0075×V1÷(V3×V1÷V2)=1600×(A-0.0055)

[0190] 2. Calculation of FFA content in tissues, bacteria or cells

[0191] (1) Calculated based on sample protein concentration

[0192] FFA(nmol / mg prot)=(A-0.0055)÷0.0075×V1÷(V1×Cpr)=133×(A-0.0055)÷Cpr

[0193] (2) Calculated by sample quality

[0194] FFA (nmol / g fresh weight)=(A-0.0055)÷0.0075×V1÷(V1÷V2×W)=160×(A-0.0055)÷W

[0195] (3) Calculated by the number of bacteria or cells

[0196] FFA (nmol / 10 4 cell)=(A-0.0055)÷0.0075×V1÷(V1÷V2×500)=0.32×(A-0.0055)

[0197] V1: added sample volume, 1 mL; V2: extract 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 lung metastasis (Me) and orthotopic subcutaneous transplanted tumor (Xe) in the lung metastasis model mice in Example 2 and the orthotopic subcutaneous transplanted tumor model mice constructed above were taken out respectively, and the protein expression, enzyme activity changes and free fatty acid content of FASN in the lung metastasis and orthotopic subcutaneous transplanted tumor were detected respectively.

[0198] Figure 5 The results showed that compared with non-metastatic esophageal cancer cells, the mRNA, protein and enzyme activity of FASN in metastatic esophageal cancer cells were significantly increased, and the level of free fatty acids in metastatic cells was also significantly increased. Secondly, compared with orthotopic subcutaneous transplanted tumors, the protein and enzyme activity of FASN in lung metastases were significantly upregulated, and the free fatty acid content in lung metastases was also significantly increased. These results confirm that the FASN-mediated fatty acid anabolic pathway is abnormally activated in metastatic tumor cells and metastases.

[0199] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of isorhamnetin or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating metastatic esophageal cancer, characterized in that: The esophageal cancer cell is esophageal squamous cell carcinoma KYSE150.

2. The use according to claim 1, characterized in that: The isorhamnetin exerts its anti-metastatic effect on esophageal cancer by inhibiting the de novo fatty acid synthesis pathway in tumor metabolism.

3. The use according to claim 1, characterized in that: The target protein of isorhamnetin for treating esophageal cancer is fatty acid synthase.

4. The use according to claim 1, characterized in that: The structural formula of isorhamnetin is 5. The use according to claim 1, characterized in that: The medicament includes a pharmaceutically acceptable carrier.

6. The use according to claim 1, characterized in that: The mass percentage of the isorhamnetin in the drug is more than 50%.

7. The use according to claim 1, characterized in that: The pharmaceutically acceptable salt includes a pharmaceutically acceptable acid addition salt or a pharmaceutically acceptable base addition salt.

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