Application of nobiletin in preparation of esophageal squamous carcinoma inhibitor

Abstract

The invention relates to application of nobiletin in preparation of an esophageal squamous carcinoma inhibitor. In a cell experiment, nobiletin can inhibit the progress of esophageal squamous carcinoma cells in a plurality of links including growth, invasion inhibition, apoptosis induction and the like. In a tumor-bearing mouse model, nobiletin can effectively inhibit the increase of tumor volume, and has a good anti-cancer effect. Therefore, nobiletin can be used for preparing the medicine for preventing and treating esophageal squamous carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of nobiletin in the preparation of esophageal squamous cell carcinoma inhibitors. Background Art

[0002] Esophageal squamous cell carcinoma (ESCC) is a common digestive tract malignancy in clinical practice, which is extremely invasive and lethal. There are no typical symptoms in the early stage of ESCC, which often leads to the phenomenon that ESCC is diagnosed in the late stage. At present, in most clinical trials, a combination of chemotherapy drugs such as platinum, paclitaxel, and fluorouracil is often used to treat ESCC. Although these combination regimens can achieve certain effects in a short period of time, drug resistance is very likely to occur, resulting in rapid progression of ESCC and inability to treat. In recent years, research related to immunotherapy has begun to receive widespread attention, and its role in the treatment of ESCC has also been studied, but due to the lack of effective molecular typing of ESCC, it is difficult to evaluate the therapeutic response of ESCC to immunotherapy. The application of these esophageal squamous cell carcinoma therapies still needs further research and clarification.

[0003] A large number of flavonoids have strong medicinal value and great prospects for drug development. They have antioxidant and anti-inflammatory biological effects and low toxicity. These flavonoids can be used to prevent and treat cardiovascular and cerebrovascular diseases, prevent and treat hypertension in the elderly, cerebral hemorrhage, angina pectoris, dilate coronary arteries, increase blood flow, etc. They can also resist fungi, free radicals and oxidation, and thus treat autoimmune diseases and other diseases. Niboletin is a polymethoxyflavonoid compound with the structural formula:

[0004]

[0005] Nobiletin has multiple biological activities such as anti-cancer, anti-inflammatory and antioxidant, and is an important flavonoid small molecule compound. There are few reports on the anti-tumor effect of Nobiletin, and its role in preventing the malignant progression of esophageal squamous cell carcinoma has not been reported. Summary of the invention

[0006] The present invention finds that nobiletin has an excellent effect on inhibiting esophageal squamous cell carcinoma and can be used to prepare an esophageal squamous cell carcinoma inhibitor.

[0007] Therefore, the present invention provides a use of nobiletin or a pharmaceutically acceptable salt thereof in the preparation of an esophageal squamous cell carcinoma inhibitor.

[0008] In one embodiment, the esophageal squamous cell carcinoma inhibitor can inhibit the growth of esophageal squamous cell carcinoma cells, inhibit the invasion of esophageal squamous cell carcinoma cells and / or induce apoptosis of esophageal squamous cell carcinoma cells.

[0009] In one embodiment, the esophageal squamous cell carcinoma cells include esophageal squamous cell carcinoma cells KYSE410, esophageal squamous cell carcinoma cells KYSE450, and esophageal squamous cell carcinoma cells KYSE510.

[0010] The present invention also provides a pharmaceutical composition for inhibiting esophageal squamous cell carcinoma, comprising nobiletin or a pharmaceutically acceptable salt thereof.

[0011] In one embodiment, the pharmaceutical composition further comprises other drugs that are helpful for the treatment of esophageal squamous cell carcinoma.

[0012] Preferably, the other drugs are selected from platinums, paclitaxel, and fluorouracils; more preferably, the other drugs include at least one of cisplatin, paclitaxel, and fluorouracil.

[0013] Preferably, the mass ratio of nobiletin or its pharmaceutically acceptable salt to other drugs is 1-10:10-1.

[0014] In one embodiment, the "pharmaceutically acceptable salt" refers to an addition salt formed by Nobiletin and a pharmaceutically acceptable acid or base, which is relatively non-toxic to the patient and has harmless activity, and its side effects do not reduce the beneficial effects of Nobiletin. Representative acids and bases that can be used to prepare pharmaceutically acceptable salts include the following: acids, including acetic acid, 2,2-dichloroacetic acid, acylated amino acids, fatty acids, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid Acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, α-oxo-glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, maleic acid, (-)-L-malic acid , malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, and undecylenic acid; and bases, including ammonia, L-arginine, calcium hydroxide, choline, decanol, diethanolamine, diethylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylenediamine, N-methyl-glucamine, hepamine, 1H-imidazole, L-lysine, magnesium hydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassium hydroxide, 1-(2-hydroxyethyl)-pyrrolidine, secondary amines, sodium hydroxide, triethanolamine, tromethamine, and zinc hydroxide.

[0015] In one embodiment, the inhibitor or pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier or excipient.

[0016] The inhibitor or pharmaceutical composition can be prepared according to methods known in the art. Any dosage form suitable for human or animal use can be prepared by combining the active compound with a pharmaceutically acceptable carrier or excipient. The content of nobiletin or its pharmaceutically acceptable salt in the pharmaceutical composition is usually 0.1-95% by weight.

[0017] Nobiletin or its pharmaceutically acceptable salt in the form of dosage units can be administered once or multiple times at appropriate intervals per day. Conveniently, the dosage unit preparation contains 0.1 mg to 1000 mg, preferably 1 mg to 100 mg, such as 5-50 mg of Nobiletin or its pharmaceutically acceptable salt. The optimal dose for administration can be easily determined by professionals in the field and will vary depending on the specific compound used, the mode of administration, the strength of the preparation, the mode of administration, and the progression of the disease condition. In addition, factors related to the specific patient being treated, including patient age, weight, diet, and number of administrations, will produce a need to adjust the dose.

[0018] Such formulations include, for example, those suitable for oral (including sustained release or timed release), rectal, parenteral (including subcutaneous, intraperitoneal, intramuscular, intraarticular and intravenous), transdermal, ocular, topical, dermal, nasal, buccal or intradermal administration.

[0019] The dosage form can be a liquid dosage form, a solid dosage form or a semisolid dosage form. The liquid dosage form can be a solution (including a true solution and a colloidal solution), an emulsion (including an o / w type, a w / o type and a multiple emulsion), a suspension, an injection (including a water injection, a powder injection and an infusion), an eye drop, a nasal drop, a lotion and an liniment, etc.; the solid dosage form can be a tablet (including a regular tablet, an enteric-coated tablet, a lozenge, a dispersible tablet, a chewable tablet, an effervescent tablet, an orally disintegrating tablet), a capsule (including a hard capsule, a soft capsule, an enteric-coated capsule), a powder, a granule, a powder, a micropill, a dropping pill, a suppository, a film, a patch, an aerosol, a spray, etc.; the semisolid dosage form can be an ointment, a gel, a paste, etc. The dosage form can also be a liposome.

[0020] Examples of preparations for oral administration are tablets, pills, hard / soft capsules, solutions, suspensions, emulsions, syrups, powders, granules, etc. These preparations may contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine) and lubricants (e.g., silicon dioxide, talc, stearates and their magnesium or calcium salts, and / or polyethylene glycol) in addition to the active ingredient. Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinyl pyrrolidone, and, if necessary, may also contain disintegrants such as starch, agarose, alginic acid or its sodium salt or azeotropic mixture, and / or adsorbents, colorants, flavoring agents and sweeteners may also be included therein.

[0021] The inhibitor or pharmaceutical composition of the present invention can be administered orally or parenterally and used in the general form of pharmaceutical preparations, but is not limited thereto.

[0022] Beneficial effects:

[0023] The present invention provides the use of Nobiletin in the preparation of esophageal squamous cell carcinoma inhibitors. In cell experiments, Nobiletin can inhibit the progression of esophageal squamous cell carcinoma cells in multiple aspects, including growth, invasion inhibition and apoptosis induction. The experimental results show that Nobiletin can inhibit the growth and non-fixation-dependent growth of esophageal squamous cell carcinoma cell lines KYSE410, KYSE450 and KYSE510 in a dose-dependent manner as evaluated by MTS and soft agar cloning methods. As evaluated by the Transwell method, Nobiletin can inhibit the invasion of esophageal squamous cell carcinoma cell lines KYSE410, KYSE450 and KYSE510 in a dose-dependent manner. Nobiletin significantly induces an upregulation of apoptosis index in KYSE410, KYSE450 and KYSE510 cells, and significantly activates apoptosis-related protein PARP. In the KYSE510 tumor-bearing mouse model, Nobiletin can effectively inhibit the growth of tumor volume and play a good anti-cancer effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram showing the results that the nobiletin of Example 1 can dose-dependently inhibit the growth of various esophageal squamous cell carcinoma cell lines.

[0026] Figure 2 This is a schematic diagram showing the results that nobiletin of Example 2 can dose-dependently inhibit the invasion of various esophageal squamous cell carcinoma cell lines.

[0027] Figure 3 Schematic diagram of the results of Example 3 showing that nobiletin dose-dependently induced apoptosis of multiple esophageal squamous cell carcinoma cell lines and up-regulated expression of cleaved PARP.

[0028] Figure 4 This is a schematic diagram showing the results of Nobiletin significantly inhibiting the growth of KYSE510 tumors in a xenogeneic tumor-bearing mouse model in Example 4. Nobiletin significantly inhibited the expression of Ki67, CD31 and LYVE1 in KYSE510 tumors; and significantly upregulated the expression of cleaved PARP in KYSE510 tumor tissues.

[0029] Figure 5 The nobiletin of Example 5 can achieve a more effective tumor growth inhibition effect in esophageal squamous cell carcinoma cells than in other types of tumor cells.

[0030] In the above figures, * represents P < 0.05; ** represents P < 0.01; ** represents P < 0.001; ns represents no statistical difference. DETAILED DESCRIPTION

[0031] The preferred embodiments of the invention are described in detail below. The examples are provided to better illustrate the content of the invention, but the content of the invention is not limited to the examples. Non-essential improvements and adjustments to the implementation scheme according to the content of the invention still fall within the scope of the invention.

[0032] The experimental methods in the following examples are all conventional methods unless otherwise specified. If no specific techniques or conditions are specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0033] Example 1: Growth inhibition effect of nobiletin on esophageal squamous cell carcinoma cell line

[0034] 1. Cell Culture

[0035] Human esophageal squamous cell carcinoma cell lines KYSE410, KYSE450 and KYSE510 were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The incubator temperature was 37°C and 5% CO 2 .

[0036] 2. MTS Assay

[0037] KYSE410, KYSE450 and KYSE510 cell lines were cultured at 3×10 3 The cells were inoculated at a density of 100 μM / well in a 96-well plate, and different concentrations of nobiletin (1, 5, 25, 50 μM) were added after the cells adhered to the wall. After 72 hours, 10% MTS solution was prepared with RPMI 1640 medium, the cultured cells were taken out, the upper medium was discarded, and the prepared MTS was added and incubated for 2 hours, and the absorbance at 490 nm was measured with a spectrophotometer.

[0038] 3. Soft agar colony formation assay

[0039] The lower agarose layer was prepared in a 96-well plate. After paving, the plate was placed at 4°C for 15 minutes, and then the upper agarose layer containing KYSE410, KYSE450 and KYSE510 cell lines was prepared. After paving, different doses of nobiletin (1, 5, 25, 50 μM) were added. After incubation for 8 days, the color working solution was added, incubated at 37°C for 4 hours, and the absorbance at 450 nm was measured using a spectrophotometer.

[0040] Compare with Figure 1 The results showed that nobiletin could dose-dependently inhibit the growth of multiple esophageal squamous cell carcinoma cell lines.

[0041] Example 2: Inhibitory effect of nobiletin on esophageal squamous cell carcinoma cell line invasion

[0042] 1. Cell invasion ability detection

[0043] KYSE410, KYSE450 and KYSE510 cell lines were cultured in serum-free medium for 24 hours. 100 μL of Matrigel was added to the upper chamber of the chamber using a transwell with an 8 μm pore size and incubated at 37°C for 1 hour to allow the Matrigel to solidify. The prepared KYSE410, KYSE150, KYSE450 and KYSE510 cell lines were cultured at 1×10 5 The density of cells / well was inoculated in the upper chamber of the transwell chamber, and 1 mL of RPMI 1640 medium containing 20% ​​fetal bovine serum was added to the lower chamber, and different concentrations of nobiletin (5, 25, 50 μM) were added. The transwell chamber was placed in an incubator and incubated for 24 hours. After taking it out, the inner wall of the upper chamber was wiped clean with a cotton swab, and the invading cells in the lower chamber were separated by dissociation solution and then added with cell dye, and the invasion cell rate was calculated by an ELISA instrument.

[0044] Compare with Figure 2 The results showed that nobiletin could dose-dependently inhibit the invasion of multiple esophageal squamous cell carcinoma cell lines.

[0045] Example 3: Nobiletin induces apoptosis in esophageal squamous cell carcinoma cell lines

[0046] 1. Determination of cell apoptosis index

[0047] KYSE410, KYSE150, KYSE450 and KYSE510 cell lines were inoculated in RPMI1640 medium containing 10% fetal bovine serum. After the cells adhered, different concentrations of nobiletin (5, 25, 50 μM) were added. After incubation for 24 hours, the supernatant was collected by centrifugation. The supernatant was added to the wells of the 96-well plate apoptosis detection kit, sealed with a blocking membrane, incubated at room temperature for 1 hour, and the sample was removed. After repeatedly washing the 96-well plate, the substrate solution (100 μL / well) was added, and after shaking for 15 minutes, the 96-well plate was tapped and the absorbance value was measured at 405 nm.

[0048] 2. Detection of key apoptosis proteins in esophageal squamous cell carcinoma cell lines

[0049] KYSE410, KYSE450 and KYSE510 cell lines were inoculated in RPMI 1640 medium containing 10% fetal bovine serum. After the cells adhered to the wall, different concentrations of nobiletin (5, 25, 50 μM) were added. After incubation for 24 hours, the cells were collected together with the supernatant, and the precipitate was collected after centrifugation at 4°C, 12000 rpm for 10 minutes. RIPA lysis buffer was added and lysed on ice for 1 hour, with shaking every 10 minutes. After the lysis was completed, the supernatant was collected after centrifugation at 4°C, 12000 rpm for 20 minutes to obtain the protein lysate. The cleaved poly ADP-ribose polymerase (PARP) was further observed by high-throughput enzyme-linked immunosorbent assay (ELISA). PARP is a key protein for apoptosis, and the expression of cleaved PARP can clearly measure the degree of cell apoptosis. Add samples (100 μL / well) to the 96-well plate of sheared PARP ELISA in sequence and incubate at 37°C. After incubation, wash the 96-well plate repeatedly. Add the sheared PARP antibody to the 96-well plate, incubate at room temperature for 1 hour, wash repeatedly and discard. Add the reaction substrate to each well, incubate at room temperature in the dark, add the reaction stop solution, and measure the absorbance value at 450nm using an ELISA reader.

[0050] Compare with Figure 3 The results showed that nobiletin dose-dependently induced apoptosis of multiple esophageal squamous cell carcinoma cell lines and upregulated the expression of cleaved PARP.

[0051] Example 4: Antitumor effect of nobiletin on KYSE510 esophageal squamous cell carcinoma cell line in mice

[0052] 2.5×10 6 KYSE510 cells were injected into the subcutaneous tissue of the upper limbs of nude mice (4-week-old female BALB / c-nu nude mice (average body weight 14-15 g), purchased from Vital River Company) until the tumor grew to about 100 mm. 3 After about 3 weeks, the KYSE510 tumor-bearing mice were randomly divided into two groups, a control group and a Nobiletin group. The Nobiletin (50 mg / kg) group was given oral administration once a day for 3 weeks, and then the tumor volume was measured. The tumor volume calculation formula is: Volume = Tumor Long Diameter × Tumor Transverse Diameter 2 ×0.5. Tumor tissues were collected on day 28, protein lysates were prepared, and ELISA experiments were performed to observe the expression of key proliferation proteins (Ki67), angiogenesis markers (CD31), lymphangiogenesis markers (LYVE1), and apoptosis key proteins (cleaved PARP) in KYSE510 tissues.

[0053] Compare with Figure 4 The results showed that nobiletin significantly inhibited the growth of KYSE510 tumors in xenograft-bearing mouse models. Nobiletin significantly inhibited the expression of Ki67, CD31, and LYVE1 in KYSE510 tumors; and significantly upregulated the expression of cleaved PARP in KYSE510 tumor tissues.

[0054] Example 5: Nobiletin can achieve more effective tumor growth inhibition in esophageal squamous cell carcinoma cells than in other types of tumor cells

[0055] KYSE450, A549, MDA231, FaDu and MGC803 cell lines were inoculated in 96-well plates, and nobiletin was added after the cells adhered to the wall. After 72 hours, 10% MTS solution was prepared with RPMI 1640 medium, the cultured cells were taken out, the upper medium was discarded, and the prepared MTS was added and incubated for 2 hours, and the absorbance value was measured at 490nm by a microplate reader.

[0056] Compare with Figure 5 The results showed that nobiletin can achieve more effective tumor growth inhibition in esophageal squamous cell carcinoma cells than in other types of tumor cells.

[0057] Embodiment 6:

[0058] The liposomal nobiletin is prepared by encapsulating the nobiletin in liposomes and used as a drug for treating esophageal squamous cell carcinoma.

[0059] Embodiment 7:

[0060] The nobiletin is added to the excipients according to the required proportion of the preparation to prepare tablets.

[0061] Embodiment 8:

[0062] The nobiletin is added to the excipients according to the required proportion of the preparation to prepare a powder.

[0063] Embodiment 9:

[0064] The granules are obtained by extracting the nobiletin, spray granulating, drying and granulating, and are used as medicine and health care product for preventing and treating esophageal squamous cell carcinoma.

[0065] Embodiment 10:

[0066] The injection is prepared by dissolving the nobiletin in water, filtering it finely, sealing and sterilizing it.

[0067] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Use of nobiletin or a pharmaceutically acceptable salt thereof in the preparation of an esophageal squamous cell carcinoma inhibitor.

2. The use according to claim 1, characterized in that: The esophageal squamous cell carcinoma inhibitor can inhibit the growth of esophageal squamous cell carcinoma cells, inhibit the invasion of esophageal squamous cell carcinoma cells and / or induce apoptosis of esophageal squamous cell carcinoma cells.

3. The use according to claim 2, characterized in that: The esophageal squamous cell carcinoma cells include esophageal squamous cell carcinoma cell KYSE410, esophageal squamous cell carcinoma cell KYSE450, and esophageal squamous cell carcinoma cell KYSE510.

4. The use according to any one of claims 1 to 3, characterized in that: The inhibitors are suitable for oral, rectal, parenteral, transdermal, ocular, topical, cutaneous, nasal, buccal or intradermal administration.

5. The use according to any one of claims 1 to 3, characterized in that: The inhibitor is in the form of a liquid dosage form, a solid dosage form, a semisolid dosage form, or a liposome.

6. The use according to any one of claims 1 to 3, characterized in that: The inhibitor is in the form of solution, emulsion, suspension, injection, eye drops, nasal drops, lotion, liniment, tablet, capsule, powder, granule, granule, pellet, drop pill, suppository, film, patch, aerosol, spray, ointment, gel, or paste.

7. A pharmaceutical composition for inhibiting esophageal squamous cell carcinoma, comprising nobiletin or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition also contains other drugs that are helpful for the treatment of esophageal squamous cell carcinoma.

9. The pharmaceutical composition according to claim 8, characterized in that The other drugs are selected from platinum, paclitaxel, and fluorouracil.

10. The pharmaceutical composition according to claim 8, characterized in that The mass ratio of nobiletin or its pharmaceutically acceptable salt to other drugs is 1-10:10-1.

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