Application of luteoloside-sensitized cis-platinum in preparation of esophageal squamous cell carcinoma treatment medicine

Abstract

The invention relates to an application of galuteolin-sensitized cis-platinum in preparation of a medicine for treating esophageal squamous cell carcinoma. It is found that the galuteolin can block the progress of esophageal squamous carcinoma cells in multiple links including apoptosis induction, growth and invasion inhibition and the like, the galuteolin can also significantly enhance inhibition of cis-platinum on lymphatic metastasis of esophageal squamous carcinoma cells in a mouse model, and the galuteolin and the cis-platinum have a significant synergistic effect.

Description

Technical Field

[0001] The present invention relates to a combination drug, and in particular to the use of luteolin to enhance the sensitivity of cisplatin in the preparation of a drug for treating esophageal squamous cell carcinoma. Background Art

[0002] Esophageal cancer is a malignant tumor of the digestive tract with high morbidity and mortality. Esophageal squamous cell carcinoma (ESCC) is usually diagnosed with advanced disease or metastasis, and cannot be treated with radical surgery. The currently recommended standard first-line treatment for this advanced or metastatic disease is chemotherapy, including fluoropyrimidine- and platinum-based treatment regimens. Adjuvant or neoadjuvant therapy based on cisplatin has become the standard treatment for advanced esophageal cancer. Cisplatin alone is prone to drug resistance and is the main cause of chemotherapy failure. The reason is related to the activation of multiple key cancer-promoting molecules and the signaling pathways they regulate. Therefore, based on drug resistance considerations, the combination of drugs is an important strategy to combat drug resistance.

[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] Cynoside (Cy), also known as luteolin-7-O-glucoside, is a flavonoid glycoside compound widely found in plants, mainly in honeysuckle, snow chrysanthemum, brocade lantern and celery. It has antioxidant, antitussive, anti-inflammatory, liver protection, anti-tumor and other biological activities. The structural formula of cynoside is as follows:

[0005]

[0006] However, whether luteolin combined with cisplatin can enhance the effect of cisplatin against esophageal squamous cell carcinoma still needs further study. Summary of the invention

[0007] The present invention finds that the combination of luteolin and cisplatin can well enhance the anti-esophageal squamous cell carcinoma effect of cisplatin, and the combination shows an unexpected synergistic effect.

[0008] Therefore, in a first aspect, the present invention provides a pharmaceutical composition comprising luteolin or a pharmaceutically acceptable salt, prodrug, metabolite thereof and cisplatin.

[0009] In addition, the present invention also provides a kit comprising luteolin or a pharmaceutically acceptable salt thereof, a prodrug, a metabolite and cisplatin.

[0010] In one embodiment, the ratio of luteolin or a pharmaceutically acceptable salt, prodrug, metabolite thereof to cisplatin is 0.5-60:1, based on the mass of luteolin and cisplatin.

[0011] Preferably, the ratio of luteolin or its pharmaceutically acceptable salt, prodrug, metabolite to cisplatin, based on the mass of luteolin and cisplatin, is 0.5:1, 1:1, 1.5:1, 3:1, 10:1, 20:1, 30:1, 35:1, 40:1, 50:1, 60:1, preferably, it is 1-40:1, or 1.5-35:1, most preferably, it is 1.5-3:1, or 35:1.

[0012] In a second aspect, the present invention also provides use of the pharmaceutical composition of the present invention in preparing a drug for treating esophageal squamous cell carcinoma.

[0013] 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.

[0014] Preferably, the metastasis comprises lymphatic metastasis.

[0015] 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.

[0016] In a third aspect, the present invention also provides a use of luteolin or a pharmaceutically acceptable salt, prodrug, or metabolite thereof in enhancing the anti-esophageal squamous cell carcinoma effect of cisplatin.

[0017] The present invention also provides a use of luteolin or a pharmaceutically acceptable salt prodrug or metabolite thereof in the preparation of a drug for enhancing the anti-esophageal squamous cell carcinoma effect of cisplatin.

[0018] In the present invention, the pharmaceutically acceptable salt of luteolin includes acid addition salt or base addition salt of luteolin. In one embodiment, the pharmaceutically acceptable salt of luteolin 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] In the present invention, the prodrug refers to a derivative that can directly or indirectly provide luteolin after being administered to a patient, such as an ester of tagetoidin.

[0020] In the present invention, the metabolite refers to a pharmaceutically acceptable metabolic derivative form of luteolin or a pharmaceutically acceptable salt thereof.

[0021] Preferably, the medicine or pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0022] Preferably, the luteolin or its pharmaceutically acceptable salt, prodrug, metabolite 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 luteolin or its pharmaceutically acceptable salt, prodrug, metabolite and cisplatin can be the same or different. Luteolin or its pharmaceutically acceptable salt, prodrug, metabolite and cisplatin can be administered simultaneously or sequentially.

[0023] In the medical uses described above, the administration time, number of administrations and frequency of administration of luteolin or its pharmaceutically acceptable salts, prodrugs, metabolites 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] In the present invention, the drug or 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).

[0030] In the present invention, the drug or pharmaceutical composition may be in a unit dosage form suitable for single administration of a precise dosage.

[0031] The medicament or pharmaceutical composition of the present invention can 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] Other conventional ingredients include antioxidants, colorants, flavorings, preservatives and taste-masking agents.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The medicament or pharmaceutical composition of the present invention can also be directly administered 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.

[0042] The medicine or pharmaceutical composition of the present invention can also be applied topically to the skin or mucous membrane, that is, applied dermally 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.

[0043] The medicament or pharmaceutical composition of the present 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.

[0044] Beneficial effects:

[0045] The present invention provides the use of luteolin sensitizing cisplatin in the preparation of a drug for treating esophageal squamous cell carcinoma. Luteolin can block the progression of esophageal squamous cell carcinoma cells in multiple links, including apoptosis induction, growth and invasion inhibition. MTS results show that luteolin enhances the growth of cisplatin-resistant KYSE450 cells. Transwell experimental results show that luteolin enhances cisplatin's anti-KYSE450 cell invasion. Luteolin enhances the apoptosis-inducing effect of cisplatin in KYSE450 cells and promotes the activation of PARP. In the KYSE450 tumor-bearing mouse model, luteolin combined with cisplatin effectively inhibits the increase of tumor cell volume, inhibits the expression of multiple tumor markers, promotes the apoptosis of tumor cells; and enhances the anti-cancer effect of cisplatin. At the same time, luteolin can significantly enhance the lymphatic metastasis of esophageal squamous cell carcinoma cell line KYSE450 in the mouse model inhibited by cisplatin. There is a significant synergistic effect between luteolin and cisplatin in anti-esophageal squamous cell carcinoma. The combination of the two can effectively improve the anti-tumor effect, reduce the dosage of the drug, reduce the toxic side effects and reduce the occurrence of drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS Description of the drawings:

[0047] Figure 1 This is a schematic diagram of the results showing that luteolin in Example 1 can enhance the effect of cisplatin on the growth inhibition of esophageal squamous cell carcinoma cell lines.

[0048] Figure 2 This is a schematic diagram of the results showing that luteolin in Example 2 can enhance the effect of cisplatin on the invasion of esophageal squamous cell carcinoma cell lines.

[0049] Figure 3 This is a schematic diagram of the results of Example 3 in which luteolin enhances cisplatin-induced apoptosis of esophageal squamous cell carcinoma cell lines and upregulation of cleavage-type PARP expression.

[0050] Figure 4 This is a schematic diagram of the results of Example 4 showing that luteolin enhances the effect of cisplatin in inhibiting the growth of KYSE450 tumors and the expression of multiple cancer-promoting markers in a xenogeneic tumor-bearing mouse model; and significantly upregulating the induction effect of cisplatin on the expression of cleaved PARP in KYSE450 tissues.

[0051] Figure 5 This is a schematic diagram of the results of Example 5 in which luteolin enhances cisplatin to inhibit the lymphatic metastasis of KYSE450 tumors in a xenogeneic tumor-bearing mouse model.

[0052] Figure 6 The luteolin in Example 6 can achieve a more effective anti-cancer effect of sensitizing cisplatin in esophageal squamous cell carcinoma cells than in other types of tumor cells.

[0053] In the above figures, ** represents P < 0.01; *** represents P < 0.001. DETAILED DESCRIPTION

[0054] 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.

[0055] 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:

[0057] Example 1: Luteolin enhances the growth inhibitory effect of cisplatin on esophageal squamous cell carcinoma cell lines

[0058] Cell growth capacity assay

[0059] KYSE450 cells 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, luteolin (10 μM) and different concentrations of cisplatin were added, either 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 GraphPadPrism software, and IC was analyzed using the "Dose-response inhibition" model. 50 The value of the fit.

[0060] Compare with Figure 1 , the results showed that luteolin could enhance the inhibitory effect of cisplatin on the growth of esophageal squamous cell carcinoma cell lines.

[0061] Example 2: Luteolin enhances the inhibitory effect of cisplatin on esophageal squamous cell carcinoma cell line invasion

[0062] Cell invasion ability assay

[0063] The KYSE450 cell line was cultured in serum-free medium for 24 hours. Using a transwell chamber with an 8 μm pore size, 100 μL of matrix gel was added to the upper chamber of the chamber and incubated at 37°C for 1 hour to solidify the matrix gel. The prepared KYSE450 cell line was 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 luteolin (10 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 taking it out, 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 enzyme plate reader.

[0064] Compare with Figure 2 , the results showed that luteolin could enhance the cisplatin-inhibitory effect on the invasion of esophageal squamous cell carcinoma cell lines.

[0065] Example 3: Luteolin enhances the apoptosis-promoting effect of cisplatin on esophageal squamous cell carcinoma cell lines

[0066] 1. Determination of cell apoptosis index

[0067] The KYSE450 cell line was inoculated in RPMI 1640 medium containing 10% fetal bovine serum. After the cells adhered, luteolin (10μM) and cisplatin (5μM) were added, used alone or in combination. 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.

[0068] 2. Detection of key apoptosis proteins in esophageal squamous cell carcinoma cell lines

[0069] The KYSE450 cell line was inoculated in RPMI 1640 medium containing 10% fetal bovine serum. After the cells adhered, luteolin (10μM) and cisplatin (5μM) were added, either alone or in combination. 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 lysis, 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.

[0070] Compare with Figure 3 The results showed that luteolin enhanced cisplatin-induced apoptosis of esophageal squamous cell carcinoma cell lines and upregulation of cleaved PARP expression.

[0071] Example 4: Luteolin enhances the anti-tumor growth and apoptosis-promoting effects of cisplatin on esophageal squamous cell carcinoma cell line KYSE450 tumor-bearing mice

[0072] 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, luteolin group, cisplatin group, and luteolin plus cisplatin group. The luteolin group (25 mg / kg) was given by oral gavage once a day, the cisplatin group (5 mg / kg) was injected intraperitoneally once a week, and the luteolin plus cisplatin group was injected intraperitoneally with cisplatin (5 mg / kg, intraperitoneal injection, once a week) on the basis of luteolin (25 mg / kg, oral gavage, once a day) for a total of 3 weeks. The control group was given the same dose of normal saline by oral gavage every day for 3 consecutive weeks. The formula for calculating tumor volume 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), and key apoptosis proteins (cleaved PARP) in KYSE450 tumors.

[0073] Compare with Figure 4 The results showed that luteolin enhanced the effect of cisplatin in inhibiting the growth of KYSE450 tumors and the expression of multiple cancer-promoting markers in a xenograft-bearing mouse model; and significantly upregulated the induction effect of cisplatin on the expression of cleaved PARP in KYSE450 tissues.

[0074] The Jin Zhengjun Q value judgment method was used to evaluate the synergistic effect of the combination of luteolin 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.

[0075] After calculation, the Q value for tumor volume was 1.34; for Ki67, the Q value was 1.51; for CD31, the Q value was 1.39; and for cleaved PARP, the Q value was 1.44.

[0076] Example 5: Luteolin enhances the anti-tumor invasion promoting effect of cisplatin on esophageal squamous cell carcinoma cell line KYSE450 tumor-bearing mice

[0077] 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). After about 1 week, luteolin (25 mg / kg / day) and / or cisplatin (5 mg / kg / week) were administered by gavage. 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.

[0078] Compare with Figure 5 .The results showed that luteolin enhanced the effect of cisplatin in inhibiting the lymphatic metastasis of KYSE450 tumors in a xenograft-bearing mouse model.

[0079] Similarly, Jin Zhengjun's Q value judgment method was used to evaluate the synergistic effect of the combination of luteolin and cisplatin in inhibiting tumor lymph node metastasis, and the Q value was 1.16.

[0080] Example 6: Luteolin can achieve a more effective anti-cancer effect of sensitizing cisplatin in esophageal squamous cell carcinoma cells than other types of tumor cells

[0081] Cell growth ability assay:

[0082] KYSE450 cell line and nasopharyngeal carcinoma cell line C666-1 were inoculated in 96-well plates. After the cells adhered to the wall, luteolin (10 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 a microplate reader.

[0083] Compare with Figure 6 The results showed that luteolin could achieve a more effective anti-cancer effect of sensitizing cisplatin in esophageal squamous cell carcinoma cells than other types of tumor cells.

[0084] 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 pharmaceutical composition comprising luteolin or a pharmaceutically acceptable salt, prodrug, metabolite thereof and cisplatin.

2. The pharmaceutical composition according to claim 1, It is characterized in that The ratio of luteolin or its pharmaceutically acceptable salt, prodrug, metabolite to cisplatin is 0.5-60:1 based on the mass of luteolin and cisplatin.

3. The pharmaceutical composition according to claim 1, It is characterized in that The ratio of luteolin or its pharmaceutically acceptable salt, prodrug, metabolite to cisplatin is 1-40:1 based on the mass of luteolin and cisplatin.

4. The pharmaceutical composition according to claim 1, It is characterized in that The ratio of luteolin or its pharmaceutically acceptable salt, prodrug, metabolite to cisplatin is 1.5-3:1, or 35:1, based on the mass of luteolin and cisplatin.

5. A kit comprising luteolin or a pharmaceutically acceptable salt, prodrug, metabolite thereof and cisplatin.

6. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a drug for treating esophageal squamous cell carcinoma.

7. The use according to claim 6, It is 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.

8. The use according to claim 7, It is characterized in that The metastasis includes lymphatic metastasis.

9. Use of luteolin or its pharmaceutically acceptable salt, prodrug, metabolite in enhancing the anti-esophageal squamous cell carcinoma effect of cisplatin.

10. Use of luteolin or its pharmaceutically acceptable salt prodrug or metabolite in the preparation of a drug for enhancing the anti-esophageal squamous cell carcinoma effect of cisplatin.

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