Application of chrysin-sensitized cis-platinum in treatment of esophageal squamous cell carcinoma

Abstract

The invention relates to application of chrysin-sensitized cis-platinum in treatment of esophageal squamous cell carcinoma. It is found that chrysin can sensitize the growth inhibition effect, invasion inhibition effect and apoptosis induction effect of cis-platinum-mediated esophageal squamous carcinoma cells in a dose-dependent manner, and can effectively enhance the growth inhibition and apoptosis promotion effect of cis-platinum-mediated tumors to inhibit the increase of the tumor volume. The inhibition of cis-platinum on lymphatic metastasis of esophageal squamous carcinoma cells is remarkably enhanced, and a synergistic anti-cancer effect is generated through combination of cis-platinum and cis-platinum.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical compositions, and in particular to application of chrysin in sensitizing cisplatin in treating esophageal squamous cell carcinoma. Background Art

[0002] Esophageal cancer is mainly divided into two histological types: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. In my country, more than 95% of esophageal cancers are ESCC, and the 5-year survival rate is less than 15%. ESCC is very prone to malignant progression and metastasis. More than 50% of ESCC patients cannot undergo surgical resection or have metastases at the time of initial diagnosis, and the 5-year survival rate of patients with advanced ESCC with metastasis is less than 5%. With the advancement of diagnosis and treatment, the 5-year survival rate of patients with malignant tumors has been significantly improved, but the mortality rate of patients with metastasis is still extremely high, with no significant improvement compared with the past. The main reason is the lack of comprehensive understanding of the mechanism of tumor occurrence and development. The first-line drug for the clinical treatment of esophageal squamous cell carcinoma is platinum compounds, but it is very easy to develop drug resistance, and tumor recurrence is prone to occur after treatment. In addition, cisplatin can also produce toxic side effects such as hepatotoxicity and nephrotoxicity while fighting tumors, which limits its clinical application.

[0003] In recent years, Chinese medicine has been widely used by researchers to combine with other drugs to enhance synergy and reduce toxicity due to its comprehensive effects of multiple components, multiple pathways and multiple targets. Some Chinese medicines also have anti-tumor effects, so the research on the anti-tumor effect of Chinese medicine-assisted chemotherapy drugs has become a current hot topic.

[0004] Chrysin (CHR) is a type of flavonoid small molecule compound, mainly distributed in Oroxylum indica and propolis of Bignoniaceae, and its structural formula is as follows:

[0005]

[0006] Chrysin has a wide range of biological activities, such as anti-inflammatory, anti-oxidation, prevention of cardiovascular and cerebrovascular diseases, etc. In recent years, it has also been reported that chrysin has an anti-cancer effect by inhibiting the growth of tumor cells, but whether chrysin enhances the anti-cancer effect of cisplatin still needs further study. Summary of the invention

[0007] The present invention finds that when used in the treatment of esophageal squamous cell carcinoma, chrysin can significantly enhance the anti-cancer effect of cisplatin, and the two achieve an obvious synergistic effect.

[0008] Therefore, the present invention provides a use of chrysin or a pharmaceutically acceptable salt thereof in enhancing the anti-esophageal squamous cell carcinoma effect of cisplatin.

[0009] The present invention also provides a use of chrysin or a pharmaceutically acceptable salt thereof in preparing a drug for enhancing the anti-esophageal squamous cell carcinoma effect of cisplatin.

[0010] In another aspect, the present invention provides a pharmaceutical composition comprising chrysin or a pharmaceutically acceptable salt thereof and cisplatin.

[0011] The present invention also provides a kit comprising chrysin or a pharmaceutically acceptable salt thereof and cisplatin.

[0012] In one embodiment, the ratio of chrysin or a pharmaceutically acceptable salt thereof to cisplatin is 0.3-50:1, based on the mass of chrysin and cisplatin.

[0013] Preferably, the ratio of chrysin or a pharmaceutically acceptable salt thereof to cisplatin, based on the mass of chrysin and cisplatin, is 0.3:1, 1:1, 1.7:1, 2.1:1, 3.4:1, 5:1, 10:1, 15:1, 17.5:1, 20:1, 25:1, 30:1, 40:1, 50:1; preferably, it is 1-20:1, or 1-17.5:1; most preferably, it is 1-3.4:1, or 17.5:1.

[0014] At the same time, the present invention also provides the use of the pharmaceutical composition of the present invention in preparing a drug for treating esophageal squamous cell carcinoma.

[0015] In the present invention, the treatment of esophageal squamous cell carcinoma includes at least one of inhibiting the growth of esophageal squamous cell carcinoma cells, inhibiting the invasion of esophageal squamous cell carcinoma cells, inducing apoptosis of esophageal squamous cell carcinoma cells, and inhibiting the metastasis of esophageal squamous cell carcinoma cells.

[0016] Preferably, the metastasis comprises lymphatic metastasis.

[0017] 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.

[0018] In one embodiment, the pharmaceutically acceptable salt of chrysin includes an acid addition salt or a base addition salt of chrysin. In one embodiment, the pharmaceutically acceptable salt of chrysin is hydrochloride, hydrobromide, sulfate, phosphate, metaphosphate, acetate, propionate, caproate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, L-malate, maleate, oxalate, fumarate, trifluoroacetate, tartrate, L-tartrate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, cinnamate , mandelate, methanesulfonate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, toluenesulfonate, 2-naphthalenesulfonate, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylate, glucoheptonic acid, 3-phenylpropionic acid, pivalate, tert-butylacetate, lauryl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate, stearate, muconate, butyrate, phenylacetate, phenylbutyrate or valproate.

[0019] Preferably, the medicine or pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0020] Preferably, the chrysin or its pharmaceutically acceptable salt and cisplatin in the pharmaceutical composition of the present invention can be administered in the same or different pharmaceutical preparations. The pharmaceutical dosage forms of chrysin or its pharmaceutically acceptable salt and cisplatin can be the same or different. Chrysin or its pharmaceutically acceptable salt and cisplatin can be administered simultaneously or sequentially.

[0021] In the medical uses described above, the administration time, number of administrations and frequency of administration of chrysin or its pharmaceutically acceptable salt and cisplatin, etc., need to be determined according to the specific diagnosis results of the disease, which is within the technical scope mastered by those skilled in the art.

[0022] For example, when a treatment regimen for mice or rats is applied to humans, the effective doses of all drugs for humans can be converted by the effective doses of the drugs for mice or rats, which is also easy to achieve for ordinary technicians in this field.

[0023] As used herein, "synergy", "synergistic action", "synergistic effect" and the like are used to mean that the result of the combination of two compounds, components or targeting agents is greater than the sum of each compound, component or targeting agent alone. This improvement in the disease, condition or disorder being treated is a "synergistic" effect.

[0024] As used herein, "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

[0025] The pharmaceutically acceptable carrier may comprise any conventional pharmaceutical carrier or excipient. The choice of carrier and / or excipient depends to a large extent on factors such as the particular mode of administration, the effect of the carrier or excipient on solubility and stability, and the nature of the dosage form.

[0026] Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents (such as hydrates and solvates). If necessary, the pharmaceutical composition may contain additional ingredients, such as flavorings, adhesives, excipients, etc. Therefore, for oral administration, tablets containing various excipients (such as citric acid) can be used with various disintegrants (such as starch, alginic acid and some composite silicates) and with adhesives (such as sucrose, gelatin and gum arabic). The example of excipients includes calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols without limitation. In addition, lubricants such as magnesium stearate, sodium lauryl sulfate and talc are often used for tableting purposes. Solid compositions of similar types can also be used in soft and hard filled gelatin capsules. Therefore, the non-limiting example of material includes lactose and high molecular weight polyethylene glycol. When an aqueous suspension or elixir is desired for oral administration, the active compound therein may be combined with various sweetening or flavoring agents, coloring or dyes and, if desired, emulsifying or suspending agents and diluents such as water, ethanol, propylene glycol, glycerin or combinations thereof.

[0027] In the present invention, the pharmaceutical composition can be, for example, in a form suitable for oral administration (as tablets, capsules, pills, powders, solutions, suspensions), a form suitable for parenteral injection (as sterile solutions, suspensions or emulsions), a form suitable for topical administration (as ointments or creams) or a form suitable for rectal administration (as suppositories).

[0028] In the present invention, the pharmaceutical composition may be in unit dosage form suitable for single administration of precise dosage.

[0029] The pharmaceutical compositions of the present invention may be administered orally. Oral administration may include swallowing, whereby the compound enters the gastrointestinal tract; or buccal or sublingual administration may be employed, whereby the compound enters the bloodstream directly from the mouth.

[0030] Formulations suitable for oral administration include solid preparations such as tablets; capsules containing microparticles, liquids or powders; lozenges; chewables; multi-microparticles and nanoparticles; gels; solid solutions; liposomes; films; ovoids; sprays and liquid preparations. Liquid preparations include suspensions, solutions, syrups and elixirs. Such preparations can be used as fillers in soft or hard capsules and typically include a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or a suitable oil, and one or more emulsifiers and / or suspending agents. Liquid preparations can also be prepared by reconstituting a solid.

[0031] For tablet dosage forms, depending on the dosage, the drug can account for 1% to 80% by weight of the dosage form, more typically 5% to 60% by weight of the dosage form. In addition to the drug, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, cross-linked sodium carboxymethylcellulose, crospovidone, polyvinyl pyrrolidone, methylcellulose, microcrystalline cellulose, low alkyl substituted hydroxypropyl cellulose, starch, pregelatinized starch and sodium alginate. Typically, the disintegrant accounts for 1% to 25% by weight of the dosage form, preferably 5% to 20% by weight.

[0032] Binders are generally used to impart cohesiveness to tablet formulations. Suitable binders include microcrystalline cellulose, gelatin, sugar, polyethylene glycol, natural and synthetic gums, polyvinyl pyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous lactose, etc.), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.

[0033] Tablets may also optionally include surfactants, such as sodium lauryl sulfate and polysorbate 80, and glidants, such as silicon dioxide and talc. When present, the amount of surfactant is generally 0.2% to 5% by weight of the tablet, and the amount of glidant is generally 0.2% to 1% by weight of the tablet.

[0034] Tablets generally also contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and a mixture of magnesium stearate and sodium lauryl sulfate. Lubricants are generally present in an amount of 0.25% to 10% by weight, preferably 0.5% to 3% by weight of the tablet.

[0035] Other conventional ingredients include antioxidants, colorants, flavorings, preservatives and taste-masking agents.

[0036] Exemplary tablets contain up to about 80% by weight of the drug, about 10% to about 90% by weight of the binder, about 0% to about 85% by weight of the diluent, about 2% to about 10% by weight of the disintegrant, and about 0.25% to about 10% by weight of the lubricant.

[0037] Tablet blends can be compressed directly or by rollers to form tablets. Alternatively, tablet blends or portions of blends can be wet, dry or melt granulated, melt solidified or extruded and then tableted. The final formulation can include one or more layers and can be coated or uncoated; or encapsulated.

[0038] Solid dosage forms for oral administration can be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained pulsed, controlled, targeted, and programmed release.

[0039] The pharmaceutical compositions of the present invention can also be administered directly into the bloodstream, muscle or internal organs. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) syringes, needle-free syringes and infusion techniques.

[0040] The pharmaceutical composition of the present invention can also be applied topically to the skin or mucous membrane, that is, applied transdermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages, and microemulsions. Liposomes can also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerol, polyethylene glycol, and propylene glycol.

[0041] The pharmaceutical compositions of this invention may also be administered intranasally or by inhalation, typically in dry powder form from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, sprayer, nebulizer or nebulizer.

[0042] Beneficial effects:

[0043] The present invention provides the use of chrysin to enhance cisplatin in the treatment of esophageal squamous cell carcinoma. As evaluated by the MTS method, chrysin can dose-dependently enhance the growth inhibition of esophageal squamous cell carcinoma cells KYSE410 and KYSE450 mediated by cisplatin. As evaluated by the Transwell method, chrysin can dose-dependently enhance the invasion inhibition of esophageal squamous cell carcinoma cells mediated by cisplatin. Chrysin significantly enhances the apoptosis-inducing effect of cisplatin on esophageal squamous cell carcinoma cell lines KYSE410 and KYSE450, and can significantly induce cisplatin to activate the apoptosis-related protein PARP. In the KYSE450 tumor-bearing mouse model, chrysin effectively enhances the tumor growth inhibition and apoptosis promotion mediated by cisplatin to inhibit the growth of tumor volume. Chrysin can also significantly enhance the lymphatic metastasis of esophageal squamous cell carcinoma cell line KYSE450 in the cisplatin-inhibited mouse model. Experiments have shown that chrysin can produce a synergistic anti-esophageal squamous cell carcinoma effect with cisplatin. The combined use of the two is beneficial to improving the anti-tumor effect, thereby reducing the dosage of drugs, reducing the occurrence of toxic side effects, and reducing patients' resistance to cisplatin. BRIEF DESCRIPTION OF THE DRAWINGS Description of the drawings:

[0045] Figure 1 The figure is a schematic diagram showing the results that chrysin in Example 1 can dose-dependently enhance the inhibitory effect of cisplatin on the growth of esophageal squamous cell carcinoma cell lines.

[0046] Figure 2 The figure is a schematic diagram showing the result that chrysin of Example 2 can dose-dependently enhance the inhibition of cisplatin on the invasion of esophageal squamous cell carcinoma cell lines.

[0047] Figure 3 This is a schematic diagram of the results of Example 3 showing that chrysin dose-dependently enhances cisplatin-induced apoptosis of multiple esophageal squamous cell carcinoma cell lines and up-regulation of cleaved PARP expression.

[0048] Figure 4 This is a schematic diagram of the results of Example 4 showing that chrysin enhances cisplatin's ability to inhibit the growth of KYSE450 tumors and the expression of Ki67, CD31 and LYVE1 in a xenogeneic tumor-bearing mouse model; and significantly upregulates cisplatin's inducing effect on the expression of cleaved PARP in KYSE450 tissues.

[0049] Figure 5 This is a schematic diagram of the results of Example 5 in which chrysin enhances cisplatin to inhibit the lymphatic metastasis of KYSE450 tumors in a xenogeneic tumor-bearing mouse model.

[0050] Figure 6 This is a schematic diagram showing the results of Example 6 showing that chrysin can achieve a more effective anti-cancer effect of sensitizing cisplatin in esophageal squamous cell carcinoma cells than in other types of tumor cells.

[0051] Figure 7This is a schematic diagram of the results of Example 7 showing that chrysin is more effective than apigenin in enhancing the effect of cisplatin against esophageal squamous cell carcinoma.

[0052] In the above figures, *** represents P < 0.001. DETAILED DESCRIPTION

[0053] 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.

[0054] 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. Specific implementation method:

[0056] Example 1: Chrysin enhances the growth inhibitory effect of cisplatin on esophageal squamous cell carcinoma cell lines

[0057] 1. Cell Culture

[0058] Human esophageal squamous cell carcinoma cell lines KYSE410 and KYSE450 were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The incubator temperature was 37°C and contained 5% CO2.

[0059] 2. Cell Growth Ability Determination

[0060] KYSE410 and KYSE450 cell lines were cultured at 3×10 3 Cells were inoculated at a density of 100 μM / well in a 96-well plate. After the cells adhered to the wall, different concentrations of chrysin (12.5, 20 μM) and different concentrations of cisplatin were added, used alone or in combination. 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. The absorbance value was measured at 490 nm by an enzyme reader. The growth inhibition rate of different concentrations of cisplatin compared with the control group was input into the GraphPad Prism software, and the IC was analyzed using the "Dose-response inhibition" model. 50 The value of fitting.

[0061] Compare with Figure 1 The results showed that chrysin could dose-dependently enhance the inhibitory effect of cisplatin on the growth of esophageal squamous cell carcinoma cell lines.

[0062] Example 2: Chrysin enhances the inhibitory effect of cisplatin on the invasion of esophageal squamous cell carcinoma cell lines

[0063] Cell invasion ability assay

[0064] KYSE410 and KYSE450 cell lines were cultured in serum-free medium for 24 hours. Using a transwell chamber with an 8 μm pore size, 100 μL of matrigel was added to the upper chamber of the chamber and incubated at 37°C for 1 hour to solidify the matrigel. The prepared KYSE410 and KYSE450 cell lines were inoculated in the upper chamber of the transwell chamber, and 800 μL of RPMI 1640 medium containing 20% ​​fetal bovine serum was added to the lower chamber, and different concentrations of chrysin (12.5, 20 μM) and cisplatin (10 μM) were added, used alone or in combination, and the transwell chamber was placed in an incubator and incubated for 24 hours. After removal, the inner wall of the upper chamber was wiped clean with a cotton swab, and the invasive cells in the lower chamber were separated by dissociation solution and then cell dye was added, and the invasion cell rate was calculated by an ELISA instrument.

[0065] Compare with Figure 2 The results showed that chrysin could dose-dependently enhance the inhibitory effect of cisplatin on the invasion of esophageal squamous cell carcinoma cell lines.

[0066] Example 3: Chrysin enhances the apoptosis-promoting effect of cisplatin on esophageal squamous cell carcinoma cell lines

[0067] 1. Determination of cell apoptosis index

[0068] KYSE410 and KYSE450 cell lines were inoculated in RPMI 1640 medium containing 10% fetal bovine serum. After the cells adhered, different concentrations of chrysin (concentrations: 12.5, 20μM) and cisplatin (5μM) 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 (100μL / well) was added, and after shaking for 15 minutes, the 96-well plate was tapped and the absorbance value was measured at 405nm.

[0069] 2. Detection of key apoptosis proteins in esophageal squamous cell carcinoma cell lines

[0070] KYSE410 and KYSE450 cell lines were inoculated in RPMI 1640 medium containing 10% fetal bovine serum. After the cells adhered to the wall, different concentrations of chrysin (concentrations: 12.5, 20μM) and cisplatin (5μM) 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, 12000rpm 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, 12000rpm 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.

[0071] Compare with Figure 3 The results showed that chrysin dose-dependently enhanced cisplatin-induced apoptosis and upregulation of cleaved PARP expression in multiple esophageal squamous cell carcinoma cell lines.

[0072] Example 4: Chrysin enhances the anti-tumor growth and apoptosis-promoting effects of cisplatin on esophageal squamous cell carcinoma cell line KYSE450 tumor-bearing mice

[0073] KYSE450 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 KYSE450 tumor-bearing mice were randomly divided into four groups: control group, chrysin group, cisplatin group, and chrysin plus cisplatin group. The chrysin group (12.5 mg / kg) was intragastrically administered once a day, the cisplatin group (5 mg / kg) was intraperitoneally injected once a week, and the chrysin plus cisplatin group was intraperitoneally injected with cisplatin (5 mg / kg, once a week) on the basis of chrysin (12.5 mg / kg, intragastrically administered, once a day). After a total of 3 weeks, the tumor volume was measured. The tumor volume calculation formula is: Volume = Tumor Long Diameter × Tumor Transverse Diameter 2× 0.5. Tumor tissue lysates were collected, and the expression of proliferation marker Ki67, angiogenesis marker CD31, lymphangiogenesis marker LYVE1, and apoptosis key protein (cleaved PARP) in KYSE450 tissues was observed by quantitative ELISA.

[0074] Compare with Figure 4 The results showed that chrysin enhanced the inhibitory effect of cisplatin on the growth of KYSE450 tumors and the expression of Ki67, CD31 and LYVE1 in a xenograft-bearing mouse model; and significantly upregulated the induction effect of cisplatin on the expression of cleaved PARP in KYSE450 tissues.

[0075] The Jin Zhengjun Q value judgment method was used to evaluate the synergistic effect of the combination of chrysin and cisplatin in inhibiting tumor growth. The Q value was calculated using the following formula: Q = E a+b / (E a +E b -E a ×E b ). a+b is the inhibition rate of the drug combination, E a and E b It is the inhibition rate of drug treatment when administered alone. Q<0.85 indicates antagonism, 0.85≤Q<1.15 indicates additive effect, and Q≥1.15 indicates synergistic effect.

[0076] After calculation, the Q value for tumor volume was 1.34; for Ki67, the Q value was 1.49; for CD31, the Q value was 1.44; for LYVE1, the Q value was 1.50; and for cleaved PARP, the Q value was 1.34.

[0077] Example 5: Chrysin enhances the anti-tumor invasion promoting effect of cisplatin on esophageal squamous cell carcinoma cell line KYSE450 tumor-bearing mice

[0078] KYSE450 cells were inoculated into the footpads of nude mice (4-week-old female BALB / c-nu nude mice (average weight 14-15 g), purchased from Vital River Company). Chrysin (12.5 mg / kg / day) and / or cisplatin (5 mg / kg / week) were administered orally about 1 week later. The experiment lasted for 5 weeks. The popliteal lymph nodes were collected and the lymph node volume was calculated. The volume calculation formula is: Volume = Long diameter × Transverse diameter 2 ×0.5.

[0079] Compare with Figure 5 The results showed that chrysin enhanced the inhibition of cisplatin on lymphatic metastasis of KYSE450 tumors in a xenograft-bearing mouse model.

[0080] Similarly, Jin Zhengjun's Q value method was used to evaluate the synergistic effect of the combination of chrysin and cisplatin in inhibiting tumor lymphatic metastasis, and the Q value was 1.16.

[0081] Example 6: Chrysin can achieve a more effective anti-cancer effect of sensitizing cisplatin in esophageal squamous cell carcinoma cells than other types of tumor cells

[0082] Cell growth ability assay:

[0083] KYSE450 cell line, non-small cell lung cancer A549 and H520 were inoculated in 96-well plates, and after the cells adhered to the wall, chrysin (12.5μM) and different concentrations of cisplatin were added. 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.

[0084] Compare with Figure 6 The results showed that chrysin can achieve a more effective anti-cancer effect of sensitizing cisplatin in esophageal squamous cell carcinoma cells than other types of tumor cells.

[0085] Example 7: Chrysin is more effective than apigenin in enhancing the effect of cisplatin against esophageal squamous cell carcinoma

[0086] Cell growth ability assay:

[0087] KYSE410 cells were inoculated in 96-well plates, and after the cells adhered to the wall, chrysin or apigenin (12.5 μM) and different concentrations of cisplatin were added. 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. The absorbance value was measured at 490 nm by an ELISA reader.

[0088] Compare with Figure 7 , the results showed that chrysin was more effective than apigenin in enhancing the effect of cisplatin against esophageal squamous cell carcinoma.

[0089] 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. A use of chrysin or a pharmaceutically acceptable salt thereof in enhancing the anti-esophageal squamous cell carcinoma effect of cisplatin.

2. Use of chrysin or a pharmaceutically acceptable salt thereof in the preparation of a drug for enhancing the anti-esophageal squamous cell carcinoma effect of cisplatin.

3. A pharmaceutical composition comprising chrysin or a pharmaceutically acceptable salt thereof and cisplatin.

4. The pharmaceutical composition according to claim 3, characterized in that The ratio of chrysin or its pharmaceutically acceptable salt to cisplatin is 0.3-50:1, based on the mass of chrysin and cisplatin.

5. The pharmaceutical composition according to claim 3, characterized in that The ratio of chrysin or a pharmaceutically acceptable salt thereof to cisplatin is 1-20:1, based on the mass of chrysin and cisplatin.

6. The pharmaceutical composition according to claim 3, characterized in that The ratio of chrysin or a pharmaceutically acceptable salt thereof to cisplatin is 1-3.4:1, or 17.5:1, based on the mass of chrysin and cisplatin.

7. A kit comprising chrysin or a pharmaceutically acceptable salt thereof and cisplatin.

8. Use of the pharmaceutical composition according to any one of claims 1 to 7 in the preparation of a drug for treating esophageal squamous cell carcinoma.

9. The use according to claim 8, characterized in that: The treatment of esophageal squamous cell carcinoma includes at least one of inhibiting the growth of esophageal squamous cell carcinoma cells, inhibiting the invasion of esophageal squamous cell carcinoma cells, inducing apoptosis of esophageal squamous cell carcinoma cells, and inhibiting the metastasis of esophageal squamous cell carcinoma cells.

10. The use according to claim 9, characterized in that: The metastasis includes lymphatic metastasis.

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