Use of isorhamnetin or pharmaceutically acceptable salts in the manufacture of drugs for the treatment of 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 field of biopharmaceutical technology and, in particular, relates to the use of isorhamnetin or a pharmaceutically acceptable salt thereof in the manufacture of drugs for the treatment of esophageal cancer. [Background technology]

[0002] Metastasis is a major cause of treatment failure in cancer patients, and the potential mechanisms of cancer metastasis are largely unknown, involving many important signaling pathways and communication between multiple cell types, resulting in a lack of effective and safe new treatment strategies. Esophageal cancer is the eighth most common malignant tumor worldwide and the sixth leading cause of cancer death, 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 mainly 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 characteristics of tumor malignancy, promoting the transformation of tumor cells and playing a crucial role in tumor progression. Recent studies have also found that metabolic reprogramming plays a vital role in these stages of tumor metastasis. (1) Tumor cells with mutations in isocitrate dehydrogenase can produce large amounts of 2-hydroxyglutarate (2-HG), a cancer-promoting metabolite, which can induce epithelial-mesenchymal transition (EMT) in tumor cells, enabling them to acquire invasive and metastatic capabilities. (2) Asparagine promotes breast cancer metastasis by promoting the expression of EMT-related proteins, while UDP-glucose can inhibit EMT conversion in lung cancer cells by accelerating the degradation of SNAI1 mRNA, thereby further inhibiting lung cancer metastasis. (3) Tumor cells that highly express the fatty acid receptor protein CD36 can acquire significant metastatic capabilities by mediating and sensing dietary fats and the extracellular lipid metabolite palmitic acid via CD36. Therefore, elucidating the metabolic characteristics of metastatic tumor cells and searching for active ingredients in traditional Chinese medicine that can target and intervene in important metabolic pathways within them is expected to provide new means and strategies for treating tumor metastasis. [Overview of the Initiative] [Problems that the invention aims to solve]

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

[0005] To achieve the objectives of the above invention, the present invention provides the following technical solutions.

[0006] This invention provides the use of isorhamnetin or a pharmaceutically acceptable salt in the manufacture of a drug 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, isorhamnetin exerts its anti-metastatic esophageal cancer effect by inhibiting the de novo synthesis pathway of fatty acids 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 isorhamnetin is as follows.

[0012] [ka]

[0013] Preferably, the drug includes a pharmaceutically acceptable carrier.

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

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

[0016] The present invention further provides a combination of drugs 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. [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. According to the research of the present invention, isorhamnetin has been found to significantly inhibit the malignant growth of metastatic esophageal squamous cell carcinoma (ESCC) cells and ESCC metastatic tumors in vivo by inhibiting the de novo synthesis pathway of fatty acids in tumor metabolism. Therefore, isorhamnetin can be used in the development of anti-ESCC metastatic cell growth drugs targeting abnormally active fatty acid de novo synthesis metabolic enzymes.

Brief Description of the Drawings

[0018] [Figure 1] It 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 of 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. E shows the effect of isorhamnetin on the expression of PCNA, a growth marker protein of metastatic and non-metastatic esophageal cancer cells, and CyclinB1, a marker protein of the G2 phase of the cell cycle. [Figure 2] It shows the effect of isorhamnetin on metastatic lung tumors of esophageal cancer in vivo. A shows the photos of the lungs of different groups and the statistical graph of metastatic lung tumors. The red arrow indicates the metastatic lung tumors of esophageal cancer. B shows the HE staining graph of the lungs of different groups. C shows the effect of different groups on the survival period of metastatic model mice. [Figure 3]Showing potential action targets of isolamnetin by metabonomics analysis. A shows analyzing the changes in the metabolic profiles of tumor cells using the principal component analysis (PCA) algorithm after treatment with isolamnetin for 3 h. B shows the results of pathway enrichment analysis. C shows a schematic diagram of de novo synthesis of fatty acids. Red and blue letters indicate upregulated and downregulated metabolites, respectively. [Figure 4] Showing the detection results of the binding between isolamnetin and FASN. The left figure in A shows the concentration of isolamnetin invading tumor cells under different time conditions, and the right figure shows the results of the concentration of isolamnetin in tumor cells after treatment with different concentrations of isolamnetin. B shows the protein level of FASN after treatment with isolamnetin. C shows the effect of isolamnetin on the enzyme activity of FASN. D shows the molecular docking results that isolamnetin can potentially bind to four amino acid sites on the FASN thioesterase domain. E shows the results of detecting the binding between isolamnetin and FASN by cell thermal shift assay. F shows the results of detecting the binding between isolamnetin and FASN by surface plasmon resonance assay. [Figure 5] Showing the activity status of the fatty acid synthesis pathway mediated by FASN in metastatic esophageal cancer cells and metastatic tumors. A shows the mRNA expression levels of FASN in non-metastatic and metastatic tumor cells. B shows the protein expression levels of FASN 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 the free fatty acid content in non-metastatic and metastatic tumor cells. E shows the protein expression of FASN in orthotopic subcutaneous transplanted tumors and metastatic lung tumors. F shows the changes in FASN enzyme activity in orthotopic subcutaneous transplanted tumors and metastatic lung tumors. G shows the changes in the free fatty acid content in orthotopic subcutaneous transplanted tumors and metastatic lung tumors.

Modes for Carrying Out the Invention

[0019] This invention provides the use of isorhamnetin or a pharmaceutically acceptable salt in the manufacture of a drug for treating esophageal cancer.

[0020] In this invention, based on the literature, in vitro metastatic ESCC cells were first constructed using the growth factor TGFβ2, and herbal medicine monomers that more significantly inhibit the proliferation of metastatic ESCC cells compared to non-metastatic cells were screened by combining the literature with high-throughput screening technology. Based on the above mode, 30 herbal medicine monomers were preliminaryly screened, and the effects of the herbal medicine monomers on the proliferation, cycle, and apoptosis of the in vitro ESCC metastatic cell line were detected, and finally, the herbal medicine monomer I-isorhamnetin, which has a significantly enhanced growth inhibitory effect on metastatic ESCC cells, was obtained. The chemical structure of isorhamnetin is as follows.

[0021] [ka]

[0022] In the present invention, the esophageal cancer is preferably metastatic esophageal cancer and non-metastatic esophageal cancer, for example, metastatic squamous cell carcinoma of the esophagus. The esophageal cancer cells preferably include squamous cell carcinoma cells of the esophagus. Research of the present invention has shown that metabolomics detection of ESCC cells after isorhamnetin intervention 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 important de novo fatty acid synthesis metabolic enzymes. Therefore, the isorhamnetin of the present invention can be used in the development of anti-ESCC metastatic cell proliferation agents that target abnormally active de novo fatty acid synthesis metabolic enzymes. The target protein for which isorhamnetin plays a role in the treatment of esophageal cancer is fatty acid synthase.

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

[0024] In this invention, "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that can retain the biological efficacy of a free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochloride salts, hydrobromide salts, sulfate salts, nitrate salts, and phosphate salts. Organic salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, caproate, caprylate, caprine, undecylenate, glycolate, gluconate, lactate, sebacinate, adipine, 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, and naphthalenedisulfonate. These salts can be produced by methods known in the art.

[0025] In this invention, "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that can retain the biological efficacy 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, and aluminum salts. 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 and tertiary amines, substituted amines including naturally substituted amines, cyclic amines, and basic ion exchange resins, such as salts of 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, purines, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be produced by methods known in the art.

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

[0027] In this invention, the term "treatment" and other similar synonyms are defined as follows: (i) To prevent the occurrence of diseases or conditions in mammals, in particular, in mammals that are susceptible to such diseases or conditions but have not yet been diagnosed with them. (ii) To inhibit a disease or symptom, that is, to prevent its progression, (iii) To alleviate a disease or symptom, that is, to resolve the state of the disease or symptom, (iv) This includes alleviating the symptoms caused by the disease or condition.

[0028] In the present invention, the mass percentage of isorhamnetin in the drug is preferably 50% or more. The 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 drug or compound that, after administration, is sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated. The result may be a reduction and / or mitigation of signs, symptoms or etiology, or any other desired change in the biological system. For example, the “effective amount” for treatment is the amount of drug containing isorhamnetin disclosed in the present invention that is required to provide a clinically significant symptom-relieving effect. An effective amount suitable for any individual case can be determined using techniques such as dose escalation studies.

[0029] In the present invention, “administration” refers to a method by which a compound or composition can be delivered to a desired site for biological action. These methods include, but are not limited to, oral routes, transduodenal routes, parenteral injections (including intravenous, subcutaneous, intraperitoneal, intramuscular, intra-arterial injections or infusions), topical administration, and transrectal administration. Those skilled in the art will be familiar with the administration techniques that can be used with isorhamnetin and the methods described in the present invention, and in preferred embodiments, the isorhamnetin discussed herein is administered by oral administration.

[0030] The present invention further provides a combination of drugs 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.

[0031] In the present invention, the combination of drugs refers to the therapeutic use of drugs obtained by mixing or combining multiple active ingredients, and includes fixed and unfixed combinations of active ingredients. The term "fixed combination" refers to the simultaneous administration of at least one isorhamnetin and at least one synergistic agent described in the present invention to a patient in the form of a single entity or a single dosage form. The term "unfixed combination" refers to the simultaneous administration, combined use, or sequential administration at variable intervals of time of at least one isorhamnetin and at least one synergistic agent described in the present invention to a patient in the form of separate entities.

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

[0033] Example 1 In vitro study on 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 above cells were counted, and TGFβ2 was added to the cell suspension so that the final TGFβ2 concentrations in the culture 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. The cells were then incubated statically in a 37°C incubator for 24 hours.

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

[0038] (4) Cells in 5 random fields were observed and counted using a 400x microscope.

[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) Inoculation of cells: Count the above cells and 1 × 10 5 Cell suspensions were prepared at a concentration of 0 ng / mL / 100 μL, and TGFβ2 was added to the cell suspensions to achieve final TGFβ2 concentrations of 0 ng / mL and 30 ng / mL, respectively. These were then inoculated into 6-well plates and incubated 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 within the cells was detected using the Western Blot method. The Western Blot detection method is as follows:

[0043] (1) The prepared protein samples were loaded into the gel wells. Following the instructions in Formulation 1 for preparing the electrophoresis gel, 5 μL of protein marker was added to the leftmost loading well. Target protein samples were then added sequentially from left to right using a time-increasing sorting method, and the gel was loaded in equivolume.

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

[0045] (3) When the protein sample reached the lower layer of separation gel, the voltage was switched to 120V. After the protein sample had moved 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 sponge and filter paper, coated with an activated PVDF film, fixed in place, and inserted into the transfer tank. Pre-cooled transfer buffer was added (see Formulation 3), an ice core was added, the power was turned on, and the transfer was performed on ice for 1 hour using a constant current of 300 mA.

[0048] (6) After the transfer was complete, the PVDF membrane was removed, placed in the pre-prepared TBST buffer (see Formulation 4), and then placed in the prepared 5% skim milk, sealed, and incubated for 1 hour.

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

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

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

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

[0053] (11) Washed three times with TBST buffer to prepare the color developing solution, and exposed with a BioRad exposure system.

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

[0055] The Fast Gel Kit includes the upper layer gel solution (2x), upper layer gel buffer (2x), lower layer gel solution (2x), lower layer gel buffer (2x), and an improved coagulant.

[0056] (1) Gel preparation was performed using a 1.0 mini thick glass plate for gel preparation.

[0057] (2) Take 2.5 mL equal volumes each of the lower gel solution and the lower gel buffer, pipette to mix them uniformly, then add 60 μL of the improved coagulant as instructed in the manual, and pipette again to mix them uniformly.

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

[0059] (4) Wait 30 minutes, and once the lower layer of gel has completely solidified, pour out the upper layer and absorb the remaining anhydrous ethanol with filter paper.

[0060] (5) Take 1.5 mL each of the upper gel solution and the upper gel buffer, pipette to mix them uniformly, add 20 μL of the improved coagulant as instructed in the manual, and pipette again to mix them uniformly.

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

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

[0063] Formulation 2 1× Running (Electrophoresis Buffer): Take 100 mL of Tris / Glycine / SDS electrophoresis buffer (10×) during electrophoresis, add 900 mL of ddH2O to bring the volume to 1 L, prepare 1× Running Buffer, and store at room temperature.

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

[0065] Formulation 4 1×TBST buffer: Take 100 mL of TBST buffer (10×), add 900 mL of ddH2O to bring the volume to 1 L, prepare 1×TBST buffer, and store at room temperature.

[0066] 1.3 Evaluation of ISO growth activity against non-transitional and transitional KYSE150 by ATPlite chemiluminescence

[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 above cells and inoculate each 96-well black plate with 3 × 10⁶ cells, 90 μL per well. 3 The mixture was dispensed into one well and incubated in a 37°C incubator for 10 minutes.

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

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

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

[0072] (6) Detected with a fluorescence microplate reader, the fluorescence signal is an indicator of cell growth inhibition, proliferation, and death. A stronger signal indicates a higher number of viable cells, while a stronger signal indicates a lower number of viable cells.

[0073] Proteins were extracted from the cells incubated in step (4), and the expression of PCNA, a cell proliferation marker protein, was detected using the Western Blot method. For details on the Western Blot method, please refer to Section 1.2.

[0074] 1.4 Cell cycle detection experiment using flow cytometry

[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) Inoculation of cells: Count the above cells and 3 × 10 5 Cells were inoculated into a 6-well plate at the cell concentration per well, with 1600 μL of cell suspension in each well.

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

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

[0079] (5) After 48 hours, 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, and this process was repeated twice. Then, 3 mL of 75% ethanol pre-cooled to -20°C was added to each tube, the contents were pipetted uniformly, sealed, and stored overnight at -20°C.

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

[0082] (8) Incubated cells were collected, proteins were extracted, and the expression of Cyclin B1, a marker protein for the G2 phase of the cell cycle, was detected using the Western Blot method. See Section 1.2 for details on the Western Blot method.

[0083] The results in Figure 1 show that TGFβ2 inhibits the expression of E-cadherin, an epithelial cell marker in tumor cells, in a time-dependent manner, while upregulating the expression of N-cadherin and fibronectin, markers for mesenchymal cells. This indicates that TGFβ2 induces tumor cells to undergo EMT conversion, leading to metastatic cell formation. Next, the study found that metastatic KYSE150 cells were more sensitive to isorhamnetin treatment than non-metastatic KYSE150 cells, exhibiting more pronounced growth inhibition, G2 phase arrest, and apoptosis. Thus, isorhamnetin significantly inhibits the growth of metastatic esophageal cancer. After 72 hours of incubation, the inhibition rates of isorhamnetin growth against non-metastatic KYSE150 were 0%, 2.79%, 31.98%, 49.27%, and 63.75% for 0 μM, 25 μM, 50 μM, 100 μM, and 200 μM for 0 μM, 25 μM, 50 μM, 100 μM, and 200 μM for metastatic KYSE150.

[0084] Example 2 Animal experiments

[0085] (1) Six-week-old female BALB / c nude mice were purchased from Shanghai Lingchang Co., Ltd., reared under SPF conditions, and given free access to water and food. They were randomly assigned to one of three groups: 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) recorded overall survival (OS), defined as the time from cell injection to mouse death, and the other subset (n=5) was used to evaluate lung metastasis.

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

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

[0088] (4) Add PBS, resuspend and wash, then centrifuge at 1200 rpm for 5 minutes, aspirate and discard the supernatant, repeat twice, and wash away the remaining culture medium.

[0089] (5) Count the cells, add a sufficient amount to PBS and resuspend, then place on ice in preparation for inoculation.

[0090] (6) Wild-type KYSE150 cells (2 × 10) were injected into the tail vein of mice. 6 Tumor-carrying mice were obtained by inoculating them with (100 μL / needle).

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

[0092] (8) Two months later, mice from a subgroup were sacrificed and samples were collected to evaluate lung metastases.

[0093] (9) The lungs were fixed and stained with Bouins fixative for 30 minutes, photographs were taken, then fixed with 4% paraformaldehyde, and further H&E staining experiments and paraffin block preparation were carried out.

[0094] The results in Figure 2 show that intraperitoneal administration of isorhamnetin significantly inhibits the malignant growth of metastatic lung tumors, thus significantly extending the survival time of metastatic model mice. After treatment with isorhamnetin, the median survival time of mice was 78.5 days.

[0095] Example 3 Detection of metabolite changes in human esophageal squamous cell carcinoma cells KYSE150 after ISO treatment using 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 minutes.

[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, and set the cell density to 1 × 10 per dish. 7 The cells were separated into individual cells, totaling six plates. They were left attached to the wall for 24 hours before being seeded on the plates and awaited the next experiment.

[0099] (4) After 24 hours, the cells from the 6 dishes were divided into two groups: 3 dishes as the control group and 3 dishes as the treatment group. 1 / 1000 DMSO was added to the control group (Vehicle), and ISO was added to the treatment group, bringing the final concentration to 100 μM. Both groups were incubated in a 37°C incubator for 3 hours.

[0100] (5) After 3 hours, wash once with PBS to digest the cells, collect the cells in a 1.5 mL centrifuge tube, centrifuge at 1200 rpm for 3 minutes, aspirate and discard the supernatant, wash once with PBS again, and centrifuge at 1200 rpm for 3 minutes.

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

[0102] GC-TOFMS detection was performed on the KYSE150 prepared as described above, and the relative content of various metabolites was obtained by comparison with metabolite standards. The statistical analysis strategies commonly used for such multidimensional data in metabolomics are as follows: After calibration and standardization, pattern recognition analysis is first performed using multivariate statistical analysis methods, dimensionality reduction is performed on the data, standard spectral information of multivariate features is extracted into a few new components, two of these components are selected, and each sample is graphed in a 2D figure based on the different values ​​of these two components, thus allowing for intuitive analysis of differences between samples or between samples of different categories.

[0103] (7) Experimental data were statistically analyzed and graphed using R software. For multivariate analysis of metabolomics data, a principal component analysis model (PCA) was fitted using the R software package mixOmics to show the metabolic characteristics of different groups. In combination with previously reported algorithms, 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 tests, and experimental data were expressed as mean ± standard deviation (mean ± SD).

[0104] To analyze the downstream targets of isorhamnetin, which inhibits the proliferation of metastatic tumors, metabolomics detection was performed on tumor cells (KYSE150) treated with isorhamnetin and control cells.

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

[0106] Example 4 To confirm whether FASN is a downstream target protein acted upon by isorhamnetin, this example conducted the following experimental studies.

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

[0108] (1) After treating the cells with ISO at different concentrations or 100 μM ISO, the supernatant was discarded, the cells were washed twice with PBS, the lysant was added to disrupt the cells, and then the cells were centrifuged at 12000 rpm for 20 minutes and the supernatant was taken.

[0109] (2) Take 100 μL of the supernatant, add an equal volume of acetonitrile and mix uniformly, then centrifuge at 12000 rpm for 20 minutes, take the supernatant and perform liquid chromatography analysis.

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

[0111] (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.

[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 experiment

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

[0115] (2) Add 1 mL of Trizol and pipette uniformly, then dissolve for 5 minutes. Add 0.2 mL of chloroform and shake to mix uniformly, then incubate for 2-3 minutes. Centrifuge at 12000 g at 4°C for 15 minutes to separate the solution into three layers, with the upper colorless aqueous phase being the RNA layer. Transfer approximately 450 μL of the colorless aqueous phase containing RNA to a new 1.5 mL EP tube.

[0116] (3) Add 0.5 mL of isopropanol, invert the mixture to mix uniformly, and incubate at 4°C for 10 minutes. Centrifuge at 12000 g at 4°C for 10 minutes to obtain a white RNA precipitate. Discard the supernatant and resuspend the precipitate with 1 mL of 75% ethanol (can be stored at -20°C for 1 year). Centrifuge at 7500 g at 4°C for 5 minutes, discard the supernatant to obtain the RNA precipitate.

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

[0118] (5) PrimeScript TM After reverse transcription using RT Master Mix to obtain cDNA, then TB Green R Premix Ex Chapter TM The qPCR reaction was performed using [the specified method].

[0119] (6) Primer sequence: 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 using the Western Blot method

[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 and counted by adding Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS).

[0123] (3) An appropriate amount of the cell suspension was added to a 10-cm culture dish to adjust the cell density to 1×10 7 cells per dish, for a total of 6 dishes. The cells were allowed to adhere to the wall for 24 h and then seeded onto the plate, waiting for the next experiment.

[0124] (4) After 24 h, the 6 dishes of cells were divided into 2 groups, with 3 dishes as the control group and 3 dishes as the administration group. 1 / 1000 DMSO was added to the control group (Vehicle), and isoproterenol (ISO) was added to the administration group to a final concentration of 100 μM. The two groups were incubated in an incubator at 37 °C for 3 h.

[0125] (5) After 3 h, the cells were washed once with PBS, digested, and collected into 1.5-mL centrifuge tubes. The cells were 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, waiting for detection.

[0127] Next, the expression of FASN in the cells from step (6) was detected using the Western Blot method in section 1.2 of Example 1.

[0128] 4.4 Molecular docking experiment

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

[0130] (2) Docking process: The protein was dehydrated and hydrogenated 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 hours, then heated at different temperatures (25-60°C) for 3 minutes, and stored at room temperature for 3 minutes. After centrifugation at 4°C and 12000 rpm for 20 minutes, SDS-PAGE supernatant was added, boiled, and stored at -20°C before Western blot detection.

[0134] 4.6 Surface plasmon resonance analyzes (SPR)

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

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

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

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

[0139] (1) Cells were collected after being treated with KYSE150 at different concentrations of ISO (0 μM, 50 μM, 100 μM, and 200 μM) for 3 hours. FASN activity was detected using a FASN activity detection kit (supplier: Solarbio, catalog number: BC0550) following the procedure below.

[0140] (2) Processing of cell samples: Number of cells (10 4 (per sample): The extract was added in a ratio of 500-1000:1 of the extract volume (mL), cells were disrupted by ultrasound in an ice bath (power 300W, ultrasound 3 seconds, interval 9 seconds, total time 5 min), centrifuged at 12000g at 4°C for 20 min, the supernatant was taken and placed on ice for measurement.

[0141] (3) Preheat the ultraviolet spectrophotometer for more than 30 minutes, adjust the wavelength to 340 nm, and then zero out with distilled water.

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

[0143] (5) Samples were 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 in terms of 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 cell was defined as one enzyme activity unit. FASN(U / 10 4 cell)=1607.7×ΔA÷number of cells.

[0146] The results in Figure 4 show the following: (1) Since FASN proteins are 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 constant concentration of isorhamnetin, the intracellular isorhamnetin concentration increased with time and reached a peak value at 1 hour. On the other hand, when tumor cells were treated with different concentrations of isorhamnetin, the intracellular isorhamnetin concentration increased with increasing extracellular isorhamnetin concentration, indicating that ISO can enter the cell in a time-dependent and concentration-dependent manner. (2) The thioesterase domain on FASN plays an important role in the catalytic action of fatty acid synthesis by FASN. Molecular docking analysis revealed that ISO can potentially bind to the Glu2395 / Tyr2425 site on the A chain and the Glu2394 / Tyr2424 site on the B chain of the FASN thioesterase domain, with a binding energy of -6.23 kcal / mol. (3) In vitro enzyme activity experiments revealed that ISO can rapidly inhibit FASN activity. (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, with a binding constant (KD) value of 53.74 μM. Therefore, the target of action of isorhamnetin in the treatment of metastatic esophageal cancer was FASN.

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

[0148] 5.1 Construction of a KYSE150 orthotopic subcutaneous transplant tumor model

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

[0150] (2) An appropriate amount of KYSE150 esophageal squamous cell carcinoma cells were cultured and inoculated with subcutaneous transplant tumors.

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

[0152] (4) Add PBS, resuspend and wash, then centrifuge at 1200 rpm for 5 minutes, aspirate and discard the supernatant, repeat twice, and wash away the remaining culture medium.

[0153] (5) Count the cells, add a sufficient amount to PBS and resuspend, then place on ice in preparation for inoculation.

[0154] (6) KYSE150 cells (3 × 10) are placed on the subcutaneous fat side of the abdomen of each mouse. 6 (10 μL / needle) was administered.

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

[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 above cells and inoculate each 96-well black plate with 3 × 10⁶ cells, 90 μL per well. 3 The mixture was dispensed into one well and incubated in a 37°C incubator for 10 minutes.

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

[0160] Here, for the detection of FASN mRNA expression, refer to Section 4.2 of Example 4. The Western Blot method described in Section 1.2 of Example 1 was used to detect FASN expression in non-metastatic and metastatic esophageal cancer cells, and for the detection of enzyme activity, refer to 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 was treated with 30 ng / mL TGFβ2 and an equal volume of physiological saline for 48 hours, and cells were collected. Approximately 0.1-0.2 g of KYSE150 orthotopic subcutaneous transplant tumor tissue and metastatic lung tumor tissue were taken. Free fatty acid content was detected using a kit (purchased from Saiichi Seibutsu, 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 in a centrifuge at 4°C. 0.1 mL of the supernatant was taken, 1.2 mL of reagent 1 was added, and the mixture was shaken for 3 hours to extract. The mixture was then centrifuged at 8000 g at 4°C for 10 minutes, and the supernatant was taken for measurement.

[0165] 2. Tissue: After washing the tissue with distilled water, the surface moisture was absorbed with absorbent paper, and the tissue was ground up. Reagent 1 was added in a ratio of tissue mass (g):extraction liquid volume (mL) of 1:5 to 12 (it was recommended to weigh approximately 0.1g of tissue and add 1.2mL of reagent 1), and the mixture was shaken for 3 hours to extract. The mixture was then centrifuged at 8000g, 4°C for 10min, and the supernatant was taken for measurement.

[0166] 3. Bacteria, fungi: cell number (10 4 Depending on the number of cells: Add reagent 1 in a ratio of 500-1000 mL to 1.2 (it was recommended to add 1.2 mL of reagent 1 to 5 million cells), disrupt the cells with ultrasound in an ice bath (power 300 watts, ultrasound for 2 seconds, interval 3 seconds, total time 3 min), extract by shaking for 3 hours, then centrifuge at 8000 g and 4°C for 10 mins, and take the supernatant for measurement.

[0167] (3) Procedure for measuring 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: Take 1 mL of the supernatant, add 0.5 mL of reagent 2, shake well for 5 minutes, let stand at room temperature for 5 minutes, and then transfer 0.8 mL of the upper layer to a 1 mL glass cuvette and adjust to zero.

[0170] 3. Measurement tube: Take 1 mL of the supernatant, add 0.5 mL of reagent 3, shake well for 5 minutes, let stand at room temperature for 5 minutes, place 0.8 mL of the upper layer into a 1 mL glass cuvette, measure the absorbance and label it A.

[0171] Note: The control tube needed to be measured once for each 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) Calculate using 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) Calculated using sample mass FFA (nmol / g fresh weight)=(A-0.0055)÷0.0075×V1÷(V1÷V2×W)=160×(A-0.0055)÷W

[0177] (3) Calculated by number of bacteria or cells FFA(nmol / 10 4 cell)=(A-0.0055)÷0.0075×V1÷(V1÷V2×500)=0.32×(A-0.0055)

[0178] V1: Volume of added sample, 1 mL; V2: Volume of extracted liquid, 1.2 mL; V3: Volume of added serum (plasma), 0.1 mL; Cpr: Sample protein concentration, mg / mL; W: Sample mass, g; 500: Total number of bacteria or cells, 5 million. Metastatic lung tumors (Me) and orthotopic subcutaneous transplant tumors (Xe) were isolated from the metastatic lung tumor model mouse in Example 2 and the orthotopic subcutaneous transplant tumor model mouse constructed above, respectively, and the protein expression of FASN, changes in enzyme activity, and free fatty acid content were detected in the metastatic lung tumors and orthotopic subcutaneous transplant tumors, respectively.

[0179] The results in Figure 5 show that, compared to non-metastatic esophageal cancer cells, FASN mRNA, protein, and enzyme activity were significantly increased in metastatic esophageal cancer cells, and free fatty acid levels in metastatic cells were also significantly increased. Next, compared to orthotopic subcutaneous transplant tumors, FASN protein and enzyme activity were significantly upregulated in metastatic lung tumors, and at the same time, 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 metastatic tumors.

[0180] The foregoing are merely preferred embodiments of the present invention, and those skilled in the art can make some improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are within the scope of protection.

Claims

1. A drug for treating metastatic esophageal squamous cell carcinoma KYSE150, It contains isorhamnetin or a pharmaceutically acceptable salt as an active ingredient. The isorhamnetin is present in the drug at a mass percentage of 50% or more. A drug characterized by the following features.

2. The aforementioned isorhamnetin exerts anti-metastatic esophageal cancer effects by inhibiting the de novo synthesis pathway of fatty acids in tumor metabolism. The drug according to claim 1.

3. The target protein for isorhamnetin's role in treating esophageal cancer is fatty acid synthase. The drug according to claim 1.

4. The drug comprises a pharmaceutically acceptable carrier. The drug according to claim 1.

5. The pharmaceutically acceptable salts include pharmaceutically acceptable acid addition salts or pharmaceutically acceptable base addition salts. The drug according to claim 1.

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