Application of luteolin glycoside combined with dacomitinib or ponatinib in blocking the progression of esophageal squamous cell carcinoma

Abstract

The present invention provides the application of marigrin in combination with defactinib or ponatinib in blocking the progression of esophageal squamous cell carcinoma. The combination of marigrin with the FAK inhibitor defactinib or the Src family kinase inhibitor ponatinib can inhibit the metastasis of esophageal squamous cell carcinoma, especially lymphatic metastasis, and can inhibit the growth of esophageal squamous cell carcinoma cells in the primary focus and metastatic foci, and has a synergistic anti-cancer effect.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceuticals. Specifically, the present invention relates to the use of quercetagitrin in combination with the FAK inhibitor defactinib or the Src family kinase inhibitor ponatinib to block the progression of esophageal squamous cell carcinoma. Background Art

[0002] Esophageal squamous cell carcinoma (ESCC) is one of the high-incidence and specific solid tumors in China. Due to its complex pathological mechanism, high malignancy, and difficulty in molecular typing, targeted drugs have not been able to achieve effective anti-cancer effects in the treatment of esophageal squamous cell carcinoma.

[0003] Clinical specimen studies have shown that phosphorylated FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, and Lyn tyrosine Y397 are highly expressed in esophageal squamous cell carcinoma tissues, are closely correlated with tumor malignant progression, and are negatively correlated with patient survival. In the past few years, clinical trials of FAK and Src family kinase inhibitors have been carried out. However, in the treatment of solid tumors, these FAK and Src family kinase inhibitors still cannot be used as single drugs for anti-cancer. This suggests whether the combination of certain small molecule compounds can effectively enhance the anti-cancer effect of targeted drugs.

[0004] There have been many research reports on small molecule monomer compounds sensitizing anti-cancer drugs, including targeted drugs, chemoradiotherapy drugs, and immunotherapy. Quercetagitrin is isolated from Tagetes erecta L., and its structural formula is:

[0005]

[0006] It has the biological activities of anti-inflammatory and antioxidant, and is widely used in cardiovascular and cerebrovascular diseases and other diseases related to inflammation and oxidation. In recent years, there have also been reports that quercetagitrin has an anti-cancer effect by inhibiting the growth of tumor cells. However, whether quercetagitrin enhances the effects of the FAK inhibitor defactinib and the Src family kinase inhibitor ponatinib against esophageal squamous cell carcinoma remains to be further studied. Summary of the Invention

[0007] The present invention discovers that quercetagitrin can significantly enhance the anti-esophageal squamous cell carcinoma effects of the FAK inhibitor defactinib and the Src family kinase inhibitor ponatinib, and has a synergistic anti-cancer effect.

[0008] The FAK inhibitor defactinib and the Src family kinase inhibitor ponatinib can treat the progression of tumors (including esophageal squamous cell carcinoma). However, the efficacy of these targeted drugs used alone as single agents in clinical applications is poor. The reason is closely related to the complexity of the downstream signaling network. Through immunohistochemical experiments, the present invention found that in in-situ esophageal squamous cell carcinoma tissues, FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, and Lyn tyrosine Y397 were positively correlated with downstream proliferation-related molecules, including MCM3, MCM4, MCM5, MCM6, MCM7, CDC45, CDK7, and CDK9. In lymphatic metastasis esophageal squamous cell carcinoma tissues, FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, and Lyn tyrosine Y397 were positively correlated with downstream invasion-promoting and stemness-forming related markers, such as SDC2, ITGA5, GALK1, AXL, Myc, Sox2, and OCT4. The present invention constructed a nude mouse popliteal lymph node tumor cell metastasis model to observe the effect of tagetesin in enhancing the anti-esophageal squamous cell carcinoma progression effect of the FAK inhibitor defactinib and the Src family kinase inhibitor ponatinib. The results showed that tagetesin could significantly enhance the inhibition of lymphatic metastasis of esophageal squamous cell carcinoma cell lines KYSE410 and KYSE510 in the mouse model by defactinib and ponatinib, and inhibit the expression of MCM3, MCM4, MCM5, MCM6, MCM7, CDC45, CDK7, CDK9, and the proliferation marker Ki67, and the lymphangiogenesis marker LYVE1 in in-situ carcinoma tissues. Tagetesin could significantly enhance the inhibition of the expression of SDC2, ITGA5, GALK1, AXL and the transcriptional activities of Myc, Sox2, and OCT4 in metastatic tumors in the mouse model by defactinib and ponatinib.

[0009] Therefore, in a first aspect, the present invention provides a pharmaceutical composition comprising tagetesin or a pharmaceutically acceptable salt, prodrug, metabolite thereof and a FAK inhibitor or a Src family kinase inhibitor.

[0010] In addition, the present invention also provides a kit comprising tagetesin or a pharmaceutically acceptable salt, prodrug, metabolite thereof and a FAK inhibitor or a Src family kinase inhibitor.

[0011] In one embodiment, the weight ratio of tagetesin or a pharmaceutically acceptable salt, prodrug, metabolite thereof to a FAK inhibitor or a Src family kinase inhibitor is 0.1-10:1, preferably 1-5:1, more preferably 2-3:1.

[0012] Preferably, the weight ratio of tagetesin or its pharmaceutically acceptable salt, prodrug, metabolite to the FAK inhibitor is 3 - 9:3, preferably 4 - 7:3, more preferably 5:3.

[0013] Preferably, the weight ratio of tagetesin or its pharmaceutically acceptable salt, prodrug, metabolite to the Src family kinase inhibitor is 1 - 4:1, preferably 2 - 3:1, more preferably 2.5:1.

[0014] In one embodiment, the FAK inhibitor includes defactinib or its pharmaceutically acceptable salt; the Src family kinase inhibitor includes ponatinib or its pharmaceutically acceptable salt.

[0015] In one embodiment, the ratio of tagetesin or its pharmaceutically acceptable salt, prodrug, metabolite to defactinib or its pharmaceutically acceptable salt or ponatinib or its pharmaceutically acceptable salt, based on the mass of tagetesin and defactinib or ponatinib, is 0.1 - 10:1.

[0016] Preferably, the ratio of tagetesin or its pharmaceutically acceptable salt, prodrug, metabolite to defactinib or its pharmaceutically acceptable salt or ponatinib or its pharmaceutically acceptable salt, based on the mass of tagetesin and defactinib or ponatinib, is 1 - 5:1.

[0017] In a preferred embodiment, the ratio of tagetesin or its pharmaceutically acceptable salt, prodrug, metabolite to defactinib or its pharmaceutically acceptable salt, based on the mass of tagetesin and defactinib, is 3 - 9:3, preferably 4 - 7:3, more preferably 5:3.

[0018] In a preferred embodiment, the ratio of tagetesin or its pharmaceutically acceptable salt, prodrug, metabolite to ponatinib or its pharmaceutically acceptable salt, based on the mass of tagetesin and ponatinib, is 1 - 4:1, preferably 2 - 3:1, more preferably 2.5:1.

[0019] In a second aspect, the present invention also provides the use of the said pharmaceutical composition in the preparation of a drug for blocking the progression of esophageal squamous cell carcinoma.

[0020] In a third aspect, the present invention also provides the use of tagetesin or its pharmaceutically acceptable salt, prodrug, metabolite in enhancing the blocking of the progression of esophageal squamous cell carcinoma by a FAK inhibitor or an Src family kinase inhibitor.

[0021] In a fourth aspect, the present invention also provides the use of tagetesin or its pharmaceutically acceptable salt, prodrug, metabolite in the preparation of a drug for enhancing the blocking of the progression of esophageal squamous cell carcinoma by a FAK inhibitor or an Src family kinase inhibitor.

[0022] In one embodiment, the blocking of the progression of esophageal squamous cell carcinoma includes inhibiting the metastasis of esophageal squamous cell carcinoma cells, inhibiting the growth of primary esophageal squamous cell carcinoma cells, and inhibiting the growth of metastatic esophageal squamous cell carcinoma cells.

[0023] In a preferred embodiment, the metastasis refers to lymphatic metastasis.

[0024] In a preferred embodiment, the esophageal squamous cell carcinoma cells include esophageal squamous cell carcinoma KYSE410, esophageal squamous cell line KYSE450, esophageal squamous cell carcinoma KYSE510, and more preferably esophageal squamous cell carcinoma KYSE410 and esophageal squamous cell carcinoma KYSE510.

[0025] In the present invention, the pharmaceutically acceptable salts of tagetesin include salts formed by tagetesin and pharmaceutically acceptable acids or bases.

[0026] In the present invention, the prodrug refers to a derivative that can directly or indirectly provide tagetesin after being administered to a patient, such as an ester of tagetesin.

[0027] In the present invention, the metabolite refers to a pharmaceutically acceptable metabolic derivative form of tagetesin or its pharmaceutically acceptable salt.

[0028] Preferably, the drug or pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable adjuvant.

[0029] Preferably, the tagetesin or its pharmaceutically acceptable salt, prodrug, metabolite, and FAK inhibitor or Src family kinase inhibitor in the pharmaceutical composition of the present invention can be administered in the same or different pharmaceutical formulations. The pharmaceutical dosage forms of tagetesin or its pharmaceutically acceptable salt, prodrug, metabolite, and FAK inhibitor or Src family kinase inhibitor can be the same or different. The tagetesin or its pharmaceutically acceptable salt, prodrug, metabolite, and FAK inhibitor or Src family kinase inhibitor can be administered simultaneously or sequentially.

[0030] In the above-mentioned pharmaceutical use, for the administration time, administration frequency, and dosing frequency of tagetesin or its pharmaceutically acceptable salt, prodrug, metabolite, and FAK inhibitor or Src family kinase inhibitor, etc., it needs to be determined according to the specific diagnosis results of the disease condition, which is within the technical scope mastered by those skilled in the art.

[0031] For example, when applying the treatment plan for mice or rats to humans, the effective dose of all drugs for humans can be converted from the effective dose of the drug for mice or rats, which is also easily achievable for those of ordinary skill in the art.

[0032] As pharmaceutically acceptable adjuvants, any adjuvants known to be suitable for the preparation of specific drugs or pharmaceutical compositions can be used. Examples include, but are not limited to, solvents, excipients, dispersants, emulsifiers, solubilizers, gel formers, ointment bases, antioxidants, preservatives, stabilizers, carriers, fillers, binders, thickeners, complexing agents, disintegrants, buffers, penetration enhancers, polymerizing agents, lubricants, coating agents, propellants, tonicity regulators, surfactants, colorants, flavoring agents, sweeteners, and dyes. Adjuvants of the type suitable for the desired formulation and the desired mode of administration are particularly used.

[0033] The drug or pharmaceutical composition of the present invention can be one of tablets, powders, granules, pills, capsules, solutions, eye drops, emulsions, suspensions, ointments, oils, pastes, foams, sprays, injections, skin patches, suppositories, liposome preparations, microparticle preparations, and microcapsule preparations.

[0034] The drug or pharmaceutical composition of the present invention is suitable for oral, rectal, nasal, topical (including transdermal, buccal, and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), or pulmonary administration.

[0035] The drug or pharmaceutical composition of the present invention can be produced in a manner known to those skilled in the art, such as by dissolution, mixing, granulation, sugar coating preparation, grinding, emulsification, capsule formation, encapsulation, or freeze-drying processes.

[0036] In the drug or pharmaceutical composition of the present invention, the amount of tageteside or its pharmaceutically acceptable salt, prodrug, metabolite, and drafatinib and ponatinib can account for 0.01% to 50% of the total weight, preferably 0.1% to 10%, more preferably 0.5% to 5%, and most preferably 1% to 2%.

[0037] The therapeutic dose can be determined empirically and will vary with the pathology being treated, the weight of the subject being treated, and the efficacy and toxicity of the agent. Similarly, those skilled in the art can readily determine the appropriate dosage formulation and method of administering the reagent. For example, for adult patients, the pharmaceutical composition of the present invention can be administered orally or non-orally at a dose of 0.001 mg to 500 mg per day, once a day or divided into several times.

[0038] Beneficial effects:

[0039] The present invention provides the application of marigrin in combination with the FAK inhibitor defactinib or the Src family kinase inhibitor ponatinib in blocking the progression of esophageal squamous cell carcinoma. The experimental results show that marigrin can inhibit the proliferation and invasion of esophageal squamous cell carcinoma cells. When combined with the FAK inhibitor defactinib or the Src family kinase inhibitor ponatinib, it can inhibit the metastasis of esophageal squamous cell carcinoma, especially lymphatic metastasis, and can inhibit the growth of esophageal squamous cell carcinoma cells in the primary focus and metastatic foci, showing a synergistic anti-cancer effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the results of the dose-dependent inhibition of the proliferation and invasion of the esophageal squamous cell carcinoma cell line KYSE510 by marigrin in Example 1.

[0041] Figure 2 Schematic diagram of the results that marigrin (Quer) more effectively inhibits the invasion of the esophageal squamous cell carcinoma cell line KYSE30 than quercetin in Example 2.

[0042] Figure 3 Schematic diagram of the results of the positive correlation expression of FAK tyrosine 576 / 577, Src kinase family members Src tyrosine Y419, Fyn tyrosine Y420, and Lyn tyrosine Y397 with downstream proliferation-related molecules such as MCM3, MCM4, MCM5, MCM6, MCM7, CDC45, CDK7, and CDK9 in Example 3.

[0043] Figure 4 Schematic diagram of the results of the positive correlation expression of FAK tyrosine 576 / 577, Src kinase family members Src tyrosine Y419, Fyn tyrosine Y420, and Lyn tyrosine Y397 with downstream invasion-promoting and stemness-related markers such as SDC2, ITGA5, GALK1, AXL, Myc, Sox2, and OCT4 in lymphatic metastatic foci of esophageal squamous cell carcinoma in Example 4.

[0044] Figure 5 Schematic diagram of the results that marigrin significantly enhances the anti-esophageal squamous cell carcinoma progression effect of defactinib and ponatinib in Example 5.

[0045] Figure 6 Schematic diagram of the results that marigrin significantly enhances the inhibitory effect of defactinib and ponatinib on the expression of MCMs / CDC45 molecules in primary esophageal squamous cell carcinoma tissues in Example 6.

[0046] Figure 7 Schematic diagram of the results that marigrin significantly enhances the inhibitory effect of defactinib and ponatinib on the expression of proliferation markers CDK7, CDK9, and Ki67 and lymphangiogenesis marker LYVE1 in primary esophageal squamous cell carcinoma tissues in Example 7.

[0047] Figure 8 Schematic diagram of the results that luteoloside in Example 8 significantly enhanced the inhibitory effects of dacomitinib and ponatinib on the molecular expressions of SDC2, ITGA5, GALK1, and AXL in metastatic esophageal squamous cell carcinoma tissues.

[0048] Figure 9 Schematic diagram of the results that luteoloside in Example 9 significantly enhanced the inhibitory effects of dacomitinib and ponatinib on the transcriptional activities of Myc, Sox2, and OCT4 in metastatic esophageal squamous cell carcinoma tissues.

[0049] In each of the above figures, * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; n.s represents no statistical difference. Detailed implementation mode

[0050] The preferred examples of the invention will be described in detail below. The examples are given to better state the content of the invention, and the content of the invention is not limited to the examples. Non-essential improvements and adjustments to the implementation schemes according to the content of the invention still belong to the scope of the invention.

[0051] In the following examples, the experimental methods are all conventional methods unless otherwise specified. For those not indicating specific techniques or conditions in the examples, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications.

[0052] Example 1: Luteoloside inhibits the growth and invasion of esophageal squamous cell carcinoma cell line KYSE510

[0053] 1. Cell proliferation assay

[0054] The KYSE510 cell line was inoculated into a 96-well plate at a density of 3×10 3 cells / well. After the cells adhered, luteoloside (concentrations: 1, 2.5, 5, 10, 25, 50, 100 μM) was added. After 72 hours, a 10% MTS solution was prepared with RPMI 1640 medium. The cultured cells were taken out, and after discarding the upper layer of the culture medium, the prepared MTS was added and incubated for 2 hours. The absorbance value was measured at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0055] 2. Detection of cell invasion ability

[0056] The KYSE510 cell line was cultured in serum-free medium for 24 hours. A transwell chamber with an 8-μm pore size was used. 100 μL of Matrigel was added to the upper chamber of the chamber, incubated at 37 °C for 1 hour to solidify the Matrigel. The prepared KYSE510 cell line was inoculated into 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 marigrin (concentrations: 10, 25, 50 μM) was added. 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. 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-linked immunosorbent assay (ELISA) reader.

[0057] Control attached Figure 1 , marigrin dose-dependently inhibited the proliferation and invasion of esophageal squamous cell carcinoma cell line KYSE510.

[0058] Example 2: Marigrin more effectively inhibits the invasion of esophageal squamous cell carcinoma than quercetin

[0059] Detection of cell invasion ability

[0060] The KYSE30 cell line was cultured in serum-free medium for 24 hours. A transwell chamber with an 8-μm pore size was used. 100 μL of Matrigel was added to the upper chamber of the chamber, incubated at 37 °C for 1 hour to solidify the Matrigel. The prepared KYSE30 cell line was inoculated into 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 marigrin or quercetin (concentration 25 μM) was added. 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. 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-linked immunosorbent assay (ELISA) reader.

[0061] Control attached Figure 2 , the results showed that marigrin more effectively inhibited the invasion of esophageal squamous cell carcinoma cell line KYSE30 than quercetin.

[0062] Example 3: Detection of the expression of FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, and Lyn tyrosine Y397 and downstream proliferation-related molecules in primary esophageal squamous cell carcinoma

[0063] 1. Immunohistochemistry

[0064] The pathological sections of 18 esophageal squamous cell carcinoma tissues with primary foci were routinely dewaxed and antigen repaired, and then incubated with antibodies against FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, Lyn tyrosine Y397, MCM3, MCM4, MCM5, MCM6, MCM7, CDC45, CDK7, and CDK9 respectively, followed by color development. The expression intensity of the corresponding proteins in the local tissues was observed by immunohistochemistry.

[0065] 2. According to the immunohistochemical results, it was judged that there was a positive correlation between FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, Lyn tyrosine Y397 and downstream proliferation-related molecules.

[0066] The immunohistochemical results were calculated by multiplying the staining intensity (distinguished by four degrees: 0, 1, 2, 3) by the proportion of positive cells in the local tissues (distinguished by five degrees: 0, 1, 2, 3, 4). A product greater than 3 was considered high expression (positive), and less than or equal to 3 was considered low expression (negative). According to Pearson correlation analysis, the expression correlation between FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, Lyn tyrosine Y397 and downstream proliferation-related molecules was observed.

[0067] Control appendix Figure 3 , there was a positive correlation between FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, Lyn tyrosine Y397 and downstream proliferation-related molecules, such as MCM3, MCM4, MCM5, MCM6, MCM7, CDC45, CDK7, and CDK9.

[0068] Example 4: Detection of the expression of FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, Lyn tyrosine Y397 and downstream invasion-promoting and stemness-related markers in esophageal squamous cell carcinoma with lymphatic metastasis foci

[0069] 1. Immunohistochemistry

[0070] The pathological sections of 20 esophageal squamous cell carcinoma tissues with lymphatic metastasis foci were routinely dewaxed and antigen repaired, and then incubated with antibodies against FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, Lyn tyrosine Y397, SDC2, ITGA5, GALK1, AXL, Myc, Sox2, and OCT4 respectively, followed by color development. The expression intensity of the corresponding proteins in the local tissues was observed by immunohistochemistry.

[0071] 2. Judging from the immunohistochemical results, the expressions of FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, and Lyn tyrosine Y397 are positively correlated with the downstream molecules related to promoting invasion and stemness formation.

[0072] The immunohistochemical results are obtained by multiplying the staining intensity (distinguished by four degrees: 0, 1, 2, 3) by the proportion of positive cells in the local tissue (distinguished by five degrees: 0, 1, 2, 3, 4). A product greater than 3 indicates high expression (positive), and a product less than or equal to 3 indicates low expression (negative). According to Pearson correlation analysis, the expression correlations of FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, and Lyn tyrosine Y397 with the downstream molecules related to promoting invasion and stemness formation were observed.

[0073] Control attached Figure 4 , in esophageal squamous cell carcinoma with lymphatic metastasis foci, the expressions of FAK tyrosine 576 / 577, Src kinase family member Src tyrosine Y419, Fyn tyrosine Y420, and Lyn tyrosine Y397 are positively correlated with the downstream markers related to promoting invasion and stemness formation, such as SDC2, ITGA5, GALK1, AXL, Myc, Sox2, and OCT4.

[0074] Example 5: Inhibiting lymphatic metastasis of esophageal squamous cell carcinoma by marigrin combined with FAK inhibitor defactinib and Src family kinase inhibitor ponatinib

[0075] Verifying the inhibition of lymphatic metastasis of esophageal squamous cell carcinoma by marigrin combined with FAK inhibitor defactinib and Src family kinase inhibitor ponatinib in an animal model

[0076] After inoculating KYSE410 and KYSE510 cells into the footpads of nude mice (4-week-old female BALB / c-nu nude mice (average body weight 14 - 15 g), purchased from Vital River Laboratories), after about 1 week, gavages were given marigrin (25 mg / kg / day), defactinib (15 mg / kg / day), or ponatinib (10 mg / kg / day) alone or in combination, and the experiment lasted for 5 weeks. The popliteal lymphatics were collected, and the volume calculation formula is: volume = long diameter × cross diameter 2 × 0.5.

[0077] Control attached Figure 5 , the results showed that marigrin significantly enhanced the effects of defactinib and ponatinib on suppressing the progression of esophageal squamous cell carcinoma.

[0078] Example 6: Inhibiting the expression of MCMs / CDC45 molecules in primary esophageal squamous carcinoma cells by combining calenduloside with FAK inhibitor defactinib and Src family kinase inhibitor ponatinib

[0079] Animal model verification of the inhibition of lymphatic metastasis of esophageal squamous carcinoma cells by combining calenduloside with FAK inhibitor defactinib and Src family kinase inhibitor ponatinib

[0080] After inoculating KYSE410 and KYSE510 cells into the footpads of nude mice (4-week-old female BALB / c-nu nude mice (average weight 14 - 15 g), purchased from Vital River), after about 1 week, gavaged with calenduloside (25 mg / kg / day), defactinib (15 mg / kg / day) or ponatinib (10 mg / kg / day) alone or in combination, and the experiment lasted for 5 weeks. After collecting the primary esophageal squamous carcinoma tissues, they were prepared into paraffin sections, and MCM3, MCM4, MCM5, MCM6, MCM7, and CDC45 were observed by immunohistochemistry.

[0081] Control attached Figure 6 , and the results showed that calenduloside significantly enhanced the inhibitory effect of defactinib and ponatinib on the expression of MCMs / CDC45 molecules in primary esophageal squamous carcinoma tissues.

[0082] Example 7: Inhibiting the expression of CDK7, CDK9, Ki67 and YVE1 in primary esophageal squamous carcinoma cells by combining calenduloside with FAK inhibitor defactinib and Src family kinase inhibitor ponatinib

[0083] After inoculating KYSE410 and KYSE510 cells into the footpads of nude mice (4-week-old female BALB / c-nu nude mice (average weight 14 - 15 g), purchased from Vital River), after about 1 week, gavaged with calenduloside (25 mg / kg / day), defactinib (15 mg / kg / day) or ponatinib (10 mg / kg / day) alone or in combination, and the experiment lasted for 5 weeks. After collecting the primary esophageal squamous carcinoma tissues, they were prepared into protein lysates. The expression levels of proliferation markers CDK7, CDK9 and Ki67 and lymphangiogenesis marker LYVE1 in local tumor tissues were observed by high-throughput enzyme-linked immunosorbent assay (ELISA).

[0084] Control attached Figure 7 , and the results showed that calenduloside significantly enhanced the inhibitory effect of defactinib and ponatinib on the expression of proliferation markers CDK7, CDK9 and Ki67 and lymphangiogenesis marker LYVE1 in primary esophageal squamous carcinoma tissues.

[0085] Example 8: Inhibitory effect of calenduloside A combined with FAK inhibitor defactinib and Src family kinase inhibitor ponatinib on the expression of SDC2, ITGA5, GALK1, and AXL molecules in esophageal squamous cell carcinoma cells with lymphatic metastasis

[0086] Animal model verification of the inhibitory effect of calenduloside A combined with FAK inhibitor defactinib and Src family kinase inhibitor ponatinib on the expression of SDC2, ITGA5, GALK1, and AXL molecules in esophageal squamous cell carcinoma cells with lymphatic metastasis

[0087] After inoculating KYSE410 and KYSE510 cells into the footpads of nude mice, after about 1 week, gavaged with calenduloside A (25 mg / kg / day), defactinib (15 mg / kg / day), or ponatinib (10 mg / kg / day) alone or in combination, and the experiment lasted for 5 weeks. After collecting the esophageal squamous cell carcinoma tissues with lymphatic metastasis, paraffin sections were prepared, and the expression of SDC2, ITGA5, GALK1, and AXL was observed by immunohistochemistry.

[0088] Control attached Figure 8 The results showed that calenduloside A significantly enhanced the inhibitory effect of defactinib and ponatinib on the expression of SDC2, ITGA5, GALK1, and AXL molecules in esophageal squamous cell carcinoma tissues with lymphatic metastasis.

[0089] Example 9: Inhibitory effect of calenduloside A combined with FAK inhibitor defactinib and Src family kinase inhibitor ponatinib on the transcriptional activities of Myc, Sox2, and OCT4 molecules in esophageal squamous cell carcinoma cells with lymphatic metastasis

[0090] Animal model verification of the inhibitory effect of calenduloside A combined with FAK inhibitor defactinib and Src family kinase inhibitor ponatinib on the transcriptional activities of Myc, Sox2, and OCT4 molecules in esophageal squamous cell carcinoma cells with lymphatic metastasis

[0091] After inoculating KYSE410 and KYSE510 cells into the footpads of nude mice (4-week-old female BALB / c-nu nude mice (average body weight of 14 - 15 g), purchased from Vital River Laboratories), after about 1 week, gavaged with calenduloside A (25 mg / kg / day), defactinib (15 mg / kg / day), or ponatinib (10 mg / kg / day) alone or in combination, and the experiment lasted for 5 weeks. After collecting the esophageal squamous cell carcinoma tissues with lymphatic metastasis, nuclear proteins were extracted, and the transcriptional activities of Myc, Sox2, and OCT4 molecules were observed by transcriptional activity assay.

[0092] Control attached Figure 9, The results showed that tagetesin significantly enhanced the inhibitory effects of defactinib and ponatinib on the transcriptional activities of Myc, Sox2, and OCT4 in metastatic esophageal squamous cell carcinoma tissues.

[0093] Example 10:

[0094] Tagetesin was respectively encapsulated in liposomes with defactinib and ponatinib (mass ratios = 5:3 and 5:2 respectively) to prepare liposomal drugs as drugs for treating esophageal squamous cell carcinoma.

[0095] Example 11:

[0096] Tagetesin was respectively mixed with defactinib and ponatinib in proportion (mass ratios = 5:3 and 5:2 respectively), and excipients were added in the proportion required for the preparation to make tablets.

[0097] Example 12:

[0098] Tagetesin was respectively mixed with defactinib and ponatinib in proportion (mass ratios = 5:3 and 5:2 respectively), dissolved in water, finely filtered, sealed and sterilized to make injections.

[0099] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. 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 tagetesin or a pharmaceutically acceptable salt thereof and a FAK inhibitor or a Src family kinase inhibitor, wherein the FAK inhibitor is selected from defactinib or a pharmaceutically acceptable salt thereof; the Src family kinase inhibitor is selected from ponatinib or a pharmaceutically acceptable salt thereof, and the ratio of tagetesin or a pharmaceutically acceptable salt thereof to defactinib or a pharmaceutically acceptable salt thereof or ponatinib or a pharmaceutically acceptable salt thereof, based on the mass of tagetesin and defactinib or ponatinib, is 0.1 - 10:

1.

2. A kit comprising tagetesin or a pharmaceutically acceptable salt thereof and a FAK inhibitor or a Src family kinase inhibitor, wherein the FAK inhibitor is selected from defactinib or a pharmaceutically acceptable salt thereof; the Src family kinase inhibitor is selected from ponatinib or a pharmaceutically acceptable salt thereof, and the ratio of tagetesin or a pharmaceutically acceptable salt thereof to defactinib or a pharmaceutically acceptable salt thereof or ponatinib or a pharmaceutically acceptable salt thereof, based on the mass of tagetesin and defactinib or ponatinib, is 0.1 - 10:

1.

3. Use of the pharmaceutical composition according to claim 1 in the preparation of a drug for blocking the progression of esophageal squamous cell carcinoma.

4. The application according to claim 3, wherein The blocking of the progression of esophageal squamous cell carcinoma includes inhibiting the metastasis of esophageal squamous cell carcinoma cells, inhibiting the growth of primary esophageal squamous cell carcinoma cells, and inhibiting the growth of metastatic esophageal squamous cell carcinoma cells.

5. The application according to claim 4, wherein The metastasis refers to lymphatic metastasis.

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