Anti-tumor combined medicine composition for preventing and / or treating esophageal squamous carcinoma and application of anti-tumor combined medicine composition

Abstract

The invention relates to an anti-tumor combined medicine composition for preventing and / or treating esophageal squamous carcinoma and application of the anti-tumor combined medicine composition, and belongs to the technical field of anti-tumor medicines. The invention provides an application of a resting sulfydryl oxidase QSOX2 inhibitor in preparation of a medicine for preventing / and / or treating esophageal squamous carcinoma. The invention finds that the resting sulfydryl oxidase (QSOX2) gene is highly expressed in esophageal squamous cell carcinoma, and the QSOX2 up-regulates the expression of mammal rapamycin target protein (mTOR) and stem cell related transcription factor (c-Myc), thereby enhancing the tumor dryness. Therefore, the QSOX2 is a novel target spot for tumor stemness regulation and control. The invention proves that the combined administration of the QSOX2 inhibitor (Ebseen), the mTOR inhibitor (Rapamycin) and the chemotherapeutic drug cisplatin (Cisplatin) can effectively lower the c-Myc expression of the esophageal squamous carcinoma and induce the dormancy of the xenotransplantation tumor.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and in particular to an anti-tumor combined drug composition for preventing and / or treating esophageal squamous cell carcinoma and an application thereof. Background Art

[0002] Esophageal squamous cell carcinoma (ESCC) is one of the most common and highly lethal malignant tumors worldwide. Because its early symptoms are not obvious, most patients are in the middle and late stages when diagnosed, making treatment difficult. Currently, surgery, radiotherapy, chemotherapy and targeted therapy are the main treatments for ESCC. However, the treatment effect is often unsatisfactory, and the 5-year survival rate of patients is still low. There is a small number of tumor cells with stemness characteristics in ESCC, namely tumor stem cells. These cells have the potential for self-renewal and differentiation, can resist conventional treatment, and cause tumor recurrence and metastasis. Therefore, effective inhibition strategies for the stemness of ESCC are of great significance for improving treatment effects and prolonging patient survival.

[0003] Chemotherapeutic drug resistance is an important factor leading to treatment failure and tumor recurrence in esophageal squamous cell carcinoma. This indicates that existing drug administration regimens cannot eliminate drug-resistant tumor stem cells, and it is urgent to screen new anti-tumor drug targets that inhibit tumor stemness. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an anti-tumor combined pharmaceutical composition for preventing and / or treating esophageal squamous cell carcinoma and its application.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is: In a first aspect, the present invention provides the use of a quiescent sulfhydryl oxidase QSOX2 inhibitor in the preparation of a medicament for preventing and / or treating esophageal squamous cell carcinoma.

[0006] The present invention found that the quiescent sulfhydryl oxidase (QSOX2) gene is highly expressed in esophageal squamous cell carcinoma, and QSOX2 upregulates the expression of mammalian target of rapamycin (mTOR) and stem cell-related transcription factor (c-Myc), thereby enhancing tumor stemness, indicating that QSOX2 is a novel target for regulating stemness of esophageal squamous cell carcinoma.

[0007] In a specific embodiment of the present invention, the resting sulfhydryl oxidase QSOX2 inhibitor includes ebselen.

[0008] In a specific embodiment of the present invention, the drug is a drug that inhibits the proliferation of esophageal squamous cell carcinoma cells and / or promotes the apoptosis of esophageal squamous cell carcinoma cells.

[0009] Furthermore, the drug is a drug that inhibits the stemness of esophageal squamous cell carcinoma cells and / or induces dormancy of esophageal squamous cell carcinoma cells.

[0010] In a second aspect, the present invention provides a pharmaceutical composition for preventing and / or treating esophageal squamous cell carcinoma, the pharmaceutical composition comprising a quiescent sulfhydryl oxidase QSOX2 inhibitor, a mammalian target of rapamycin mTOR inhibitor, and a chemotherapeutic drug.

[0011] The present invention confirms that a drug combination of a QSOX2 inhibitor combined with an mTOR inhibitor and a chemotherapeutic drug effectively downregulates c-Myc expression in esophageal squamous cell carcinoma and induces xenograft tumor dormancy, providing a new drug administration scheme for solving the drug resistance of esophageal squamous cell carcinoma.

[0012] Furthermore, the resting sulfhydryl oxidase QSOX2 inhibitor includes ebselen.

[0013] In a specific embodiment of the present invention, ebselen is used as an example for verification as a QSOX2 inhibitor.

[0014] Furthermore, the mammalian rapamycin target protein mTOR inhibitor includes rapamycin, everolimus and temsirolimus.

[0015] In a specific embodiment of the present invention, rapamycin is used as an mTOR inhibitor for verification.

[0016] Furthermore, the chemotherapy drugs include platinum-based, taxane-based and fluorouracil-based chemotherapy drugs.

[0017] As a preferred embodiment, the platinum chemotherapy drug includes at least one of cisplatin, carboplatin, nedaplatin, oxaliplatin and lopoplatin.

[0018] In the specific implementation manner of the present invention, cisplatin is used as a chemotherapy drug for example for verification.

[0019] Furthermore, the mass ratio of the resting sulfhydryl oxidase QSOX2 inhibitor, the mammalian target of rapamycin protein mTOR inhibitor and the chemotherapy drug is resting sulfhydryl oxidase QSOX2 inhibitor: mammalian target of rapamycin protein mTOR inhibitor: chemotherapy drug = 0.5-2:1:1.

[0020] In a specific embodiment of the present invention, the mass ratio of the resting sulfhydryl oxidase QSOX2 inhibitor, the mammalian target of rapamycin mTOR inhibitor and the chemotherapeutic drug is 1:1:1 for verification.

[0021] Furthermore, the drug is a pharmaceutical preparation prepared by using the resting sulfhydryl oxidase QSOX2 inhibitor, mammalian rapamycin target protein mTOR inhibitor and chemotherapy drug as active ingredients and adding pharmaceutically acceptable excipients.

[0022] Furthermore, the medicine or pharmaceutical preparation includes tablets, granules or liquid preparations.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention found that the quiescent sulfhydryl oxidase (QSOX2) gene is highly expressed in esophageal squamous cell carcinoma, and QSOX2 upregulates the expression of mammalian rapamycin target protein (mTOR) and stem cell-related transcription factor (c-Myc), thereby enhancing tumor stemness. This shows that QSOX2 is a new target for tumor stemness regulation. At the same time, the present invention confirmed in a mouse model that the combination of QSOX2 inhibitor (Ebselen), mTOR inhibitor (Rapamycin) and chemotherapy drug cisplatin (Cisplatin) effectively downregulates esophageal squamous cell carcinoma c-Myc expression and induces xenograft tumor dormancy. The present invention provides a new dosing regimen for solving tumor resistance, which has important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The expression levels of quiescent sulfhydryl oxidase (QSOX2) in normal esophageal tissue and esophageal squamous cell carcinoma tissue. A is the electrophoresis of protein immunoblotting (Western blot); B is the score of Western blot experiment; C is the photo of immunohistochemical staining experiment; D is the score of immunohistochemical staining experiment.

[0025] Figure 2 The combined use of QSOX2 inhibitor Ebselen and mammalian target of rapamycin (mTOR) inhibitor Rapamycin inhibits tumor stemness. A shows the changes in the mTOR / stem cell-related transcription factor (c-Myc) pathway after QSOX2 overexpression in esophageal squamous cell carcinoma cells; B shows the changes in the mTOR / c-Myc pathway after QSOX2 knockdown in esophageal squamous cell carcinoma cells; C shows the changes in the mTOR / c-Myc pathway when QSOX2 inhibitors and mTOR inhibitors are used alone or in combination.

[0026] Figure 3The figure shows the inhibition of tumors by QSOX2 inhibitors, mTOR inhibitors and chemotherapy drugs. A is the experimental process of mice; B is the photos of tumor weight in different groups; C is the change of tumor weight in different groups over time; D is multiple immunofluorescence staining of tumor cells to show tumor markers; green represents tumor cell proliferation marker (Ki67); red represents activated caspase 3 (Cleaved caspase-3); blue represents cell nuclei marked by 4',6-diamidino-2-phenylindole (DAPI); E is the Ki67 positive tumor cell rate in different groups; F is the Cleaved caspase-3 positive tumor cell rate in different groups.

[0027] Figure 4 The figure shows the inhibition of tumor stemness by QSOX2 inhibitors, mTOR inhibitors and chemotherapy drugs. A is a multiple immunofluorescence staining of tumor cells showing tumor stemness markers; green represents tumor cells labeled by pan-keratin (pan-CK); red represents tumor stem cells labeled by c-Myc; blue represents cell nuclei labeled by DAPI; B is the rate of c-Myc-positive tumor cells; C is a multiple immunofluorescence staining of tumor cells showing tumor dormancy markers; gray represents tumor cells labeled by pan-CK; green represents dormant tumor cells labeled by phosphorylated p38 mitogen-activated protein kinase (p-P38); red represents tumor cells in a proliferative state labeled by phosphorylated extracellular signal-regulated kinase (p-ERK); blue represents cell nuclei labeled by DAPI; D is the fluorescence value of p-P38 / p-ERK in different groups. An increase in the ratio of p-P38 / p-ERK indicates that tumor cells have entered a dormant state. DETAILED DESCRIPTION

[0028] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0029] Example 1: QSOX2 protein is highly expressed in esophageal squamous cell carcinoma 1. Experimental Methods Western blot and immunohistochemical staining were performed on esophageal squamous cell carcinoma tissue and normal esophageal tissue samples. The tissue samples were from the Cancer Center of Sun Yat-sen University. Western blot or immunohistochemical staining was performed on 5 esophageal squamous cell carcinoma tissues and 5 normal esophageal tissues. The Gene ID of QSOX2 in NCBI is 169714.

[0030] 1. Western blot experimental steps: (1) Use RIPA Lysis Buffer (Radio Immunoprecipitation Assay; Brand: NCM Biotech; Product No.: WB3100) lysis buffer to extract total protein from the tissue sample to be tested.

[0031] (2) The total protein of step (1) is separated by electrophoresis using 10% (w / v) sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel, and then electro-transferred to a polyvinylidene fluoride membrane to obtain a polyvinylidene fluoride membrane for electro-transferred proteins.

[0032] (3) The polyvinylidene fluoride membrane to which the protein was electrotransferred in step (2) was blocked with 5% (w / v) bovine serum albumin (BSA) at room temperature for 1 hour to obtain a blocked polyvinylidene fluoride membrane to which the protein was electrotransferred.

[0033] (4) The polyvinylidene fluoride membrane on which the electrotransferred protein was blocked in step (3) was incubated with QSOX2 primary antibody (brand: abcam; catalog number: ab121376; 1:2000 dilution, i.e., the original QSOX2 primary antibody was diluted 2000 times with 5% w / v bovine serum albumin BSA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primary antibody (brand: Servicebio; catalog number: GB15002; 1:10000 dilution, i.e., the original GAPDH primary antibody was diluted 10000 times with 5% w / v BSA) at 4° C. overnight to obtain a polyvinylidene fluoride membrane on which the protein was electrotransferred after primary antibody incubation.

[0034] (5) Wash the polyvinylidene fluoride membrane of the electrotransferred protein after incubation with the primary antibody in step (4) three times with Tris-buffered saline (TBST) containing Tween 20, and incubate with the corresponding species secondary antibody at room temperature for 1 hour to obtain the polyvinylidene fluoride membrane of the electrotransferred protein after incubation with the secondary antibody. The secondary antibodies are anti-rabbit IgG, horseradish peroxidase (HRP)-linked antibody (Anti-rabbit IgG HRP-linked Antibody; brand: CST; item number: 7074S) and anti-mouse IgG, HRP-linked antibody (Anti-mouse IgG HRP-linked Antibody; brand: CST; item number: 7076S), 1:4000 dilution, that is, the secondary antibody is diluted 4000 times with 5% w / v BSA.

[0035] (6) Wash the polyvinylidene fluoride membrane with the electrotransferred protein after incubation with the secondary antibody in step (5) three times with TBST, and develop the immunoreactive bands with ultrasensitive electrochemiluminescence immunoassay (ECL) detection reagent (brand: Meilunbio; catalog number: #MA0186-2).

[0036] 2. Experimental steps of immunohistochemistry: (1) Fix the tissue sample to be tested with a 4% (w / v) paraformaldehyde solution for 24 hours to obtain a fixed tissue sample to be tested, dehydrate the fixed tissue sample to be tested, embed it in paraffin, and cut it into 5 μm slices to obtain paraffin slices.

[0037] (2) Dewax the paraffin sections with xylene I and II for 10 min, hydrate with gradient alcohol concentrations (100%, 95%, 85%, 75% and 50% v / v) for 5 min, and perform microwave heating for antigen retrieval. After cooling at room temperature, soak the sections in 3% H2O2 for 10 min to block endogenous peroxidase activity.

[0038] (3) The slices treated in step (2) were incubated with QSOX2 primary antibody (brand: abcam; catalog number: ab121376; 1:100 dilution, i.e., the original QSOX2 primary antibody was diluted 100 times) at 4°C overnight to obtain slices incubated with the primary antibody.

[0039] (4) After washing the slices incubated with the primary antibody in step (3) three times with PBS (phosphate buffered saline), they were incubated with the secondary antibody (brand: Zhongshan Jinqiao; item number: PV-6000-55) at 37° C. for 30 min to obtain the slices incubated with the secondary antibody.

[0040] (5) The target protein QSOX2 was detected using a diaminobenzidine (DAB) substrate kit (brand: Zhongshan Jinqiao; catalog number: PV-6000-55) and observed under an optical microscope.

[0041] (6) After the color development in step (5) is completed, the slices are washed with water twice, each time for 3 minutes, and the cell nuclei of the slices are counterstained with hematoxylin for 1 minute to obtain hematoxylin-stained slices; the hematoxylin-stained slices are washed with water for 10 minutes, and gradient dehydration is performed: 75% (v / v) alcohol dehydration for 3 minutes, 85% (v / v) alcohol dehydration for 3 minutes, 95% (v / v) alcohol dehydration for 3 minutes, anhydrous ethanol dehydration for 3 minutes, and xylene clearing for 5 minutes; air-dried to obtain dehydrated clear slices, and the dehydrated clear slices are sealed with neutral gum and observed and photographed.

[0042] 2. Experimental Results like Figure 1 A and Figure 1 As shown in Figure B, Western blot showed that QSOX2 was highly expressed in esophageal squamous cell carcinoma tissues. Figure 1 C and Figure 1 As shown in D, immunohistochemical staining experiments showed that QSOX2 was highly expressed in esophageal squamous cell carcinoma tissues.

[0043] Example 2 Overexpression of QSOX2 activates the expression of mammalian target of rapamycin (mTOR) and stem cell-related transcription factor (c-Myc) in esophageal squamous cell carcinoma cells 1. Experimental Methods The expression of QSOX2 was overexpressed or knocked down in esophageal squamous cell carcinoma cells (ESCC cells). Western blot experiments were performed on ESCC cells according to the method of Example 1.

[0044] 1. Plasmid construction: The full-length coding gene of QSOX2 was connected to the empty lentiviral vector pEZ-Lv105 to obtain a QSOX2 overexpression plasmid (manufacturer: GeneCopoeia; catalog number: EX-Y4543-Lv105, and the empty lentiviral vector pEZ-Lv105 (manufacturer: GeneCopoeia; catalog number: EX-SV40T-Lv105) was used as a control.

[0045] The sequence of shRNA1 for QSOX2 knockdown is: 5'-GGCTATTGTCTTTGAAAGCAA-3' (SEQ ID NO: 1); the sequence of shRNA2 for QSOX2 knockdown is: 5'-GCGGATTTCTGGAATATTCCT-3' (SEQ ID NO: 2). shRNA1 and 2 were connected to the lentiviral empty vector psi-LVRU6P, respectively, to obtain QSOX2 knockdown plasmids, namely shQSOX2#1 (manufacturer: GeneCopoeia; catalog number: HSH004239-LVRU6P-a) and shQSOX2#2 (manufacturer: GeneCopoeia; catalog number: HSH004239-LVRU6P-b), and the lentiviral empty vector psi-LVRU6P (manufacturer: GeneCopoeia; catalog number: CSHCTR001-LVRU6P) was used as a control.

[0046] 2. Transfection: (1) Grow 293FT cells in a 10 cm dish until the cell density reaches 90%. Replace the medium with 293FT complete medium 2 hours before transfection.

[0047] The 293FT complete culture medium is DMEM medium (Dulbecco's Modified Eagle Medium; brand: ThermoFisher; catalog number: C11995500BT) supplemented with 10% (v / v) fetal bovine serum (FBS), 1% (w / v) double-antibody (100U / mL penicillin, 100μg / mL streptomycin), 1% (v / v) non-essential amino acids (brand: GIBCO; catalog number: 11140050) and 1% (w / v) glutamine.

[0048] (2) Add 900 μL of DMEM medium to a 1.5 mL EP tube (a small centrifuge tube), and then add 5 μg each of the lentiviral packaging plasmids pLP1, pLP2, pLP-VSVG, and the target plasmid (each QSOX2 knockdown plasmid, QSOX2 overexpression plasmid, or control plasmid), and mix well to obtain a plasmid mixture.

[0049] (3) Add 40 μL of 1 mg / mL PEI MAX-Transfection Grade Linear Polyethylenimine (brand: Polysciences; catalog number: 24765-100) transfection reagent to the plasmid mixture in step (2), mix well, let stand for 15 min, add to 293FT cells, culture in a 37°C, 5% CO2 incubator for 8 h, replace with 293FT complete medium, culture for 48 to 72 h, collect the supernatant, and filter through a 0.45 μm filter (brand: Pall; catalog number: PN4614-1EA) to obtain the virus solution.

[0050] 3. Infection: (1) ESCC cells were seeded into a 6 cm dish in advance until the cell density reached 90%.

[0051] (2) After removing the supernatant of step (1), add 2 mL of virus solution and 1 mL of complete tumor cell culture medium, and add 3 μL of polybrene (brand: Beyotime; item number: C0351), culture in a 37°C, 5% CO2 incubator for 10 h, change to DMEM culture medium, culture for 48 h, add 1 μg / mL of puromycin for selection, and culture for 7 days to obtain the target cells.

[0052] KYSE140 and KYSE510 are esophageal squamous cell carcinoma cells overexpressing QSOX2; KYSE30 and KYSE180 are esophageal squamous cell carcinoma cells with QSOX2 knockdown.

[0053] The complete culture medium for tumor cells was DMEM supplemented with 10% (v / v) FBS and 1% (w / v) dual antibody.

[0054] 4. Adding drugs: The corresponding esophageal squamous cell carcinoma cells were plated in a 6-well plate and cultured in a 37°C, 5% CO2 incubator for 12 hours. The QSOX2 inhibitor Ebselen (Brand: Selleck; Catalog No.: S6676) or the mTOR inhibitor Rapamycin (Brand: Selleck; Catalog No.: S1039) were added to the culture medium, and Ebselen and Rapamycin were added to the culture medium. The final concentration of Ebselen was 100 μM, and the final concentration of Rapamycin was 10 μM. After 24 hours, the proteins were collected for Western blot experiments.

[0055] 2. Experimental Results Western blot experiments demonstrated that in ESCC cells overexpressing QSOX2, the phosphorylation of mTOR and the expression of stem cell-related transcription factor (c-Myc) were increased ( Figure 2 In contrast, in QSOX2 knockdown ESCC cells, both mTOR phosphorylation and c-Myc expression were decreased ( Figure 2 B). The combination of QSOX2 inhibitor ebselen and mTOR inhibitor rapamycin can significantly downregulate c-Myc expression in KYSE140 and KYSE510 cells ( Figure 2 C).

[0056] Example 3 Combination of QSOX2 inhibitor, mTOR inhibitor and chemotherapy drugs to inhibit tumors 1. Experimental Methods 1. If Figure 3 As shown in A, mice (nude mice) were subcutaneously injected with KYSE510 cells of Example 2, with each mouse injected with 3×10 6 cells, 12 days after cell injection, mice with tumors less than 0.5 mm in diameter were intraperitoneally injected with 5 mg / kg of QSOX2 inhibitor ebselen, 5 mg / kg of mTOR inhibitor rapamycin, 5 mg / kg of chemotherapy drug cisplatin (Cisplatin; brand: Selleck; item number: S1166) or 5 mg / kg of ebselen, 5 mg / kg of rapamycin and 5 mg / kg of cisplatin every three days, for a total of three times. At the same time, the control group was given the same volume of control solvent, which was a mixed solution composed of 5% (v / v) dimethyl sulfoxide (DMSO), 40% (v / v) polyethylene glycol (PEG300), 5% (v / v) Tween-80 (Tween-80) and 50% (v / v) saline. The tumor volume was measured at the same time of drug administration.

[0057] 2. Perform multiple immunofluorescence staining on the tumors of the different treatment groups in step 1 using a multiplex immunohistochemistry (IHC) kit (brand: Panovue; catalog number: 10001100020).

[0058] 2. Experimental Results like Figure 3 B and Figure 3 As shown in Figure C, compared with the control group, the tumor volume of the chemotherapy drug group was smaller, while the tumor volume of the combination group of QSOX2 inhibitor, mTOR inhibitor and chemotherapy drug was even smaller. This indicates that inhibiting QSOX2 and mTOR can improve the efficacy of chemotherapy, and the tumor volume of the combination group is the smallest.

[0059] As shown in Table 1, the rates of tumor cells positive for tumor cell proliferation marker (Ki67) and activated caspase 3 (Cleaved caspase-3) were shown.

[0060] Table 1 like Figure 3 D in Figure 3 E and Figure 3 As shown in F, compared with other groups, the combined drug group significantly inhibited tumor cell proliferation and promoted tumor cell apoptosis.

[0061] Example 4: Combination administration of QSOX2 inhibitors, mTOR inhibitors and chemotherapeutic drugs inhibits tumor stemness and induces tumor dormancy 1. Experimental Methods Multiple immunofluorescence staining of stemness markers was performed on the tumors of different treatment groups in step 1 of Example 3.

[0062] Multiple immunofluorescence staining of dormancy markers was performed on the tumors of different treatment groups in step 1 of Example 3.

[0063] In summary, the present invention provides a novel therapeutic target QSOX2 for tumors and a very promising anti-tumor combined drug composition (QSOX2 inhibitor, mTOR inhibitor and chemotherapeutic drug) for inhibiting tumor stemness, which can be used to inhibit tumor resistance, prevent tumor recurrence, and is expected to improve patient prognosis.

[0064] 2. Experimental Results like Figure 4 A and Figure 4 As shown in B, compared with other groups, the combined drug group significantly reduced the rate of tumor stem cells and the rate of c-Myc positive cells was greatly reduced.

[0065] As shown in Table 2, it is the rate of c-Myc positive tumor cells.

[0066] Table 2 like Figure 4 C and Figure 4 As shown in D, compared with other groups, the combined drug group induced tumor cells to enter a dormant state, and the fluorescence value of p-P38 / p-ERK (ratio of phosphorylated p38 mitogen-activated protein kinase / phosphorylated extracellular signal-regulated kinase, p-P38 / p-ERK) increased.

[0067] As shown in Table 3, these are the average fluorescence values ​​of p-P38, p-ERK, and p-p38 / p-ERK.

[0068] Table 3 Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a quiescent sulfhydryl oxidase QSOX2 inhibitor in the preparation of a drug for preventing and / or treating esophageal squamous cell carcinoma.

2. The use according to claim 1, characterized in that The resting sulfhydryl oxidase QSOX2 inhibitors include ebselen.

3. The use according to claim 1, characterized in that The drug is a drug that inhibits the proliferation of esophageal squamous cell carcinoma cells and / or promotes the apoptosis of esophageal squamous cell carcinoma cells.

4. The use according to claim 1, characterized in that The drug is a drug for inhibiting the stemness of esophageal squamous cell carcinoma cells and / or inducing dormancy of esophageal squamous cell carcinoma cells.

5. A pharmaceutical composition for preventing and / or treating esophageal squamous cell carcinoma, characterized in that: The pharmaceutical composition comprises a resting sulfhydryl oxidase QSOX2 inhibitor, a mammalian rapamycin target protein mTOR inhibitor and a chemotherapeutic drug.

6. The pharmaceutical composition for preventing and / or treating esophageal squamous cell carcinoma according to claim 5, characterized in that: The resting sulfhydryl oxidase QSOX2 inhibitors include ebselen.

7. The pharmaceutical composition for preventing and / or treating esophageal squamous cell carcinoma according to claim 5, characterized in that: The mammalian target of rapamycin (mTOR) inhibitor comprises at least one of rapamycin, everolimus and temsirolimus.

8. The pharmaceutical composition for preventing and / or treating esophageal squamous cell carcinoma according to claim 5, characterized in that: The chemotherapy drugs include platinum, taxane and fluorouracil chemotherapy drugs.

9. The pharmaceutical composition for preventing and / or treating esophageal squamous cell carcinoma according to claim 5, characterized in that: The platinum chemotherapy drug includes at least one of cisplatin, carboplatin, nedaplatin, oxaliplatin and lopoplatin.

10. The pharmaceutical composition according to any one of claims 5 to 9, characterized in that The mass ratio of the resting sulfhydryl oxidase QSOX2 inhibitor, the mammalian target of rapamycin protein mTOR inhibitor and the chemotherapy drug is resting sulfhydryl oxidase QSOX2 inhibitor: mammalian target of rapamycin protein mTOR inhibitor: chemotherapy drug = 0.5-2:1:1.

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