WO2020073593A1 - Utilisation d'un inhibiteur de sérine protéase kazal de type 1 dans la préparation d'un agent pour diagnostiquer ou réguler la sénescence cellulaire et les tumeurs - Google Patents

Utilisation d'un inhibiteur de sérine protéase kazal de type 1 dans la préparation d'un agent pour diagnostiquer ou réguler la sénescence cellulaire et les tumeurs Download PDF

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WO2020073593A1
WO2020073593A1 PCT/CN2019/076946 CN2019076946W WO2020073593A1 WO 2020073593 A1 WO2020073593 A1 WO 2020073593A1 CN 2019076946 W CN2019076946 W CN 2019076946W WO 2020073593 A1 WO2020073593 A1 WO 2020073593A1
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spink1
factor
serine protease
protease inhibitor
tumor
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孙宇
陈斐
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中国科学院上海生命科学研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7042—Aging, e.g. cellular aging

Definitions

  • the present invention belongs to the field of disease diagnosis and regulation. More specifically, the present invention relates to the use of serine protease inhibitor Kazal type 1 factor (SPINK1) in the preparation of diagnostic or regulatory agents for cell aging and tumors.
  • SPINK1 serine protease inhibitor Kazal type 1 factor
  • Cell senescence manifests as inward nuclear membrane folding, chromatin condensation, lipofuscin accumulation, increased cell volume, enlarged nuclear nuclei, increased ⁇ -galactosidase activity, and secretion of various factors.
  • Cell senescence is triggered by one or more factors, activating downstream signaling pathways including p53, p16 INK4A / Rb, PI3K / Akt, FoxO transcription factor, and mitochondrial SIRT1.
  • senescent cells are often associated with many pathological features, including local inflammation. Cell aging occurs in damaged cells and prevents them from multiplying in the organism. Under the influence of various external stimuli and internal factors, cell damage can cause obvious signs of cell aging; when the damage accumulates and reaches a certain limit, the tissue presents various visually identifiable tissue degeneration changes and physiological aging phenotypes.
  • SASP senescence-associated secretion phenotype
  • exocrine proteins produced by senescent cells often depends on the genetic background and species source of aging tumor cells.
  • SASP is of great significance to tumor biology, it is still not clear how it controls tumors.
  • studies have focused on anti-aging targeting the upstream signaling pathway of SASP, drugs or genetic specific inhibition of IKK / NF- ⁇ B, mTOR, p38MAPK, JAK / STAT, etc. in senescent cells, which can inactivate the side effects caused by SASP Secretory effect, thereby improving the aging state of cells and body.
  • the purpose of the present invention is to provide the application of serine protease inhibitor Kazal type 1 factor in the preparation of diagnostic or regulatory agents for cell aging and tumors.
  • a pharmaceutical composition for inhibiting tumors or reducing tumor resistance comprising: an antibody that specifically inhibits the serine protease inhibitor Kazal type 1 factor, and chemotherapy drug.
  • the chemotherapy drugs are genotoxic drugs; preferably, the chemotherapy drugs include: mitoxantrone, doxorubicin, bleomycin, sabplatin, cisplatin, carboplatin, Daunomycin, nogamycin, arubicin, epirubicin, doxorubicin, cytarabine, capecitabine, gemcitabine, 5-fluorouracil.
  • the pharmaceutical composition includes: an antibody that specifically inhibits the serine protease inhibitor Kazal1 type factor and mitoxantrone, and the mass ratio of the two is 1: 0.005 to 1: 2.
  • the ground is 1: 0.01 ⁇ 1: 1.0; more preferably 1: 0.02 ⁇ 1: 0.6, such as 1: 0.2.
  • the pharmaceutical composition includes: an antibody that specifically inhibits the serine protease inhibitor Kazal1 type factor and doxorubicin, and the mass ratio of the two is 1: 0.02 to 1: 1.5; preferably 1: 0.05 ⁇ 1: 0.8; more preferably 1: 0.06 ⁇ 1: 0.3, such as 1: 0.1.
  • the pharmaceutical composition includes: an antibody that specifically inhibits the serine protease inhibitor Kazal1 factor and bleomycin, and the mass ratio of the two is 1: 0.02 to 1.5; preferably 1: 0.05 to 1: 0.8; more preferably 1: 0.06 to 1: 0.3, such as 1: 0.1.
  • the pharmaceutical composition includes: an antibody that specifically inhibits the serine protease inhibitor Kazal1 factor and one or more selected from saplatin, cisplatin, and carboplatin, and the antibody and the latter
  • the mass ratio is 1: 0.02 to 1.5; preferably 1: 0.05 to 1: 0.8; more preferably 1: 0.06 to 1: 0.3, such as 1: 0.1.
  • the antibody that specifically inhibits the serine protease inhibitor Kazal type 1 factor is secreted by the hybridoma cell line CCTCC NO: C2018213.
  • an antibody that specifically inhibits the serine protease inhibitor Kazal type 1 factor reduces the tumor resistance by inhibiting the serine protease inhibitor Kazal type 1 factor expressed by stromal cells in the tumor microenvironment Medicinal properties.
  • the tumors include: prostate cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, and lung cancer.
  • the tumor resistance is the resistance of the tumor to chemotherapy drugs.
  • an antibody that specifically inhibits the serine protease inhibitor Kazal type 1 factor, which is secreted by the hybridoma cell line CCTCC NO: C2018213.
  • an antibody that specifically inhibits the serine protease inhibitor Kazal type 1 factor in the preparation of an antibody drug is used in combination with a chemotherapeutic drug to inhibit or eliminate tumor resistance; or It is used to eliminate the resistance of tumor cells to chemotherapy drugs.
  • a hybridoma cell line SP2 / 0-01-SPINK1-SUN is provided, and its deposit number in the Chinese Type Culture Collection Center is CCTCC NO: C2018213.
  • kits for inhibiting tumors or reducing tumor resistance comprising: an antibody that specifically inhibits the serine protease inhibitor Kazal type 1 factor, or a cell line that produces the antibody.
  • the pharmaceutical composition includes: the kit further includes: chemotherapy drugs; preferably the chemotherapy drugs are genotoxic drugs; preferably, the chemotherapy drugs include: rice Toxantrone, doxorubicin, bleomycin, sabplatin, cisplatin, carboplatin, daunorubicin, nogamycin, arubicin, epirubicin, doxorubicin, arabinocytoma Glycosides, capecitabine, gemcitabine, 5-fluorouracil.
  • chemotherapy drugs include: rice Toxantrone, doxorubicin, bleomycin, sabplatin, cisplatin, carboplatin, daunorubicin, nogamycin, arubicin, epirubicin, doxorubicin, arabinocytoma Glycosides, capecitabine, gemcitabine, 5-fluorouracil.
  • the use of a serine protease inhibitor Kazal type 1 factor in the preparation of a diagnostic reagent for the evaluation of tumor chemotherapy prognosis is provided, wherein the serine protease inhibitor Kazal type 1 factor is in the tumor microenvironment The serine protease inhibitor Kazal type 1 factor produced by stromal cells.
  • the serine protease inhibitor Kazal1 type factor produced by stromal cells in the tumor microenvironment can be isolated from the sample tissue by conventional isolation means.
  • the use of a reagent that specifically recognizes the serine protease inhibitor Kazal type 1 factor in the preparation of a diagnostic reagent for prognostic evaluation of tumor chemotherapy or pathological grading wherein the serine protease inhibitor Kazal Type 1 factor is the Kazal type 1 factor, a serine protease inhibitor produced by stromal cells in the tumor microenvironment.
  • the reagents that specifically recognize the serine protease inhibitor Kazal type 1 factor include antibody reagents, primers, and probes.
  • a method for screening potential substances that inhibit tumors or reduce tumor resistance includes: (1) treating an expression system with candidate substances that expresses NF- ⁇ B and serine Protease inhibitor Kazal type 1 factor, and there is a NF- ⁇ B binding site upstream of the gene encoding the serine protease inhibitor Kazal type 1 factor; and (2) Detection of NF- ⁇ B in the system for the serine protease inhibitor Kazal type 1 factor If the candidate substance statistically inhibits the transcriptional regulation of the serine protease inhibitor Kazal1 type factor by NF- ⁇ B, it indicates that the candidate substance is a potential substance that inhibits tumors or reduces tumor resistance.
  • step (1) includes: adding the candidate substance to the expression system in the test group; and / or step (2) includes: detecting the inhibition of serine protease by NF- ⁇ B in the system of the test group
  • the transcriptional regulation of factor Kazal type 1 factor is compared with the control group, where the control group is an expression system that does not add the candidate substance; if the NF- ⁇ B in the test group is for the transcription of serine protease inhibitor Kazal type 1 factor Regulation is significantly inhibited (eg, inhibition by more than 20%, better inhibition by more than 50%; better inhibition by more than 80%), indicating that the candidate substance is a potential substance that inhibits tumors or reduces tumor resistance.
  • the NF- ⁇ B binding site includes: Serine protease inhibitor Kazal type 1 factor encoding gene upstream -3902, -1851, -1763, -362, +51 position.
  • a method for screening potential substances that inhibit tumors or reduce tumor drug resistance includes: (1) treating an expression system with a candidate substance that expresses EGFR-mediated signals Pathway and serine protease inhibitor Kazal type 1 factor; and (2) detection of the activation effect of serine protease inhibitor Kazal type 1 factor on EGFR-mediated signaling pathway in the system; if the candidate substance statistically inhibits this Activation indicates that the candidate substance is a potential substance that inhibits tumors or reduces tumor drug resistance.
  • step (1) includes: adding the candidate substance to the expression system in the test group; and / or step (2) includes: detecting the serine protease inhibitor Kazal type 1 in the system of the test group
  • the effect of factor on the activation of EGFR-mediated signaling pathway is compared with the control group, where the control group is an expression system that does not add the candidate substance; if the serine protease inhibitor Kazal type 1 factor in the test group is The activation of the signal pathway induced by the pathway is significantly inhibited (eg, inhibition by more than 20%, preferably by more than 50%; more preferably by more than 80%), indicating that the candidate substance is a potential for inhibiting tumors or reducing tumor resistance substance.
  • Figure 1 Human prostate prostate stromal cell line PSC27 gene expression profile heat map after chemotherapy and radiation treatment. CTRL, control. BLEO, bleomycin. HP, hydrogen peroxide. RAD, radiation. Red arrow, SPINK1.
  • FIG. 1 DNA damage response (DDR) of PSC27 cells after various conditions.
  • A Representative pictures after immunofluorescence detection, red fluorescence is ⁇ H2AX, blue is DAPI.
  • B DDR statistics comparison analysis.
  • MIT mitoxantrone.
  • SAT satraplatin.
  • RAD radiation.
  • DOX doxorubicin.
  • BLEO bleomycin.
  • Figure 3 PSC27 aging detection after various conditions in Figure 2.
  • A Representative image of bright field microscope after SA-B-Gal staining.
  • B Statistical analysis of SA-B-Gal staining positive cells.
  • Figure 4 Analysis of the rate of DNA intercalation in cells after PSC27 was treated with various conditions in Figure 2.
  • A Representative diagram after BrdU staining, green fluorescence is BrdU.
  • B BrdU statistical analysis after various drug treatments.
  • FIG. 1 SPINK1 expression in stromal cells.
  • A SPINK1 transcript expression level in PSC27 cells after various conditions.
  • B Western blot analysis of SPINK1 protein expression.
  • FIG. 1 PSC27 stromal cells expressing several typical factors of SASP after bleomycin treatment. Collected on days 1 ("2"), 3 ("3"), 5 ("4"), 7 ("5"), 10 ("6") and 15 (“7") after the drug injury The stromal cells and their total RNA were obtained for RT-PCR detection. The data at each time point is the same as that of the control (non-medicated group, "1") and is used for graphing.
  • Figure 7 Comparative analysis of SPINK1 transcript expression levels in prostate stromal cells and cancer cells after treatment with several drugs.
  • the protein samples of each cell line used in FIG. 8 and FIG. 7 after bleomycin treatment were analyzed by Western blot to determine the change of SPINK1 expression.
  • IC intracellular protein.
  • CM conditioned media.
  • GAPDH loading control.
  • Figure 9 DNA damage of human breast stromal cell line HBF1203 after treatment with chemotherapy drugs.
  • A Representative graph of immunofluorescence staining results, red fluorescence is ⁇ H2AX, blue is DAPI.
  • B Statistical analysis of DDR signals.
  • CIS Cisplatin
  • CARB Carboplatin.
  • Figure 10 HBF1203 senescence cell analysis and detection after various conditions in Figure 9.
  • A Representative image of bright field microscope after SA-B-Gal staining.
  • B Statistical analysis of SA-B-Gal staining positive cells.
  • FIG. 11 HBF1203 DNA insertion analysis in cells after various drug treatments.
  • A Representative diagram after BrdU staining, green fluorescence is BrdU.
  • B BrdU statistical analysis after various drug treatments.
  • FIG. 12 The expression of SPINK1 in HBF1203. The transcript expression level of SPINK1 in cells after various conditions.
  • Figure 13 Comparative analysis of SPINK1 transcript expression levels in breast stromal cells and cancer cells after treatment with several drugs.
  • the protein samples of each cell line used in FIG. 14 and FIG. 13 after doxorubicin treatment were analyzed by Western blot to determine the change of SPINK1 expression.
  • IC intracellular protein.
  • CM conditioned media.
  • GAPDH loading control.
  • Figure 15 The stable transgenic lines established based on PSC27 and HBF1203 were used to determine the expression level of SPINK1 protein by Western blot.
  • Native original stroma cell.
  • Vector subline translated with control vector.
  • SPINK1 subline overexpressing exogenous SPINK1.
  • GAPDH loading control.
  • EGF epidermal growth factor.
  • FIG. 16 Various phenotype analyses of PSC27 and HBF1203 subline after treatment with DNA-damaging chemotherapy drugs.
  • A DDR statistics.
  • B DNA synthesis analysis based on BrdU staining.
  • C Cell senescence analysis based on SA-B-gal staining.
  • DMSO control.
  • Figure 17 Proliferation potential analysis of stromal cells after drug treatment.
  • A The time growth curve of each subline of PSC27 cells.
  • B HBF1203 growth curve.
  • Drug treatment Drug treatment
  • drug treatment respectively for bleomycin and adriamycin dosing time point.
  • Figure 18 Comparative analysis of histopathology of primary lesions in patients with prostate cancer before and after chemotherapy. On the left, representative pictures of histochemical staining. On the right, representative pictures of H & E staining.
  • Figure 19 Statistical comparison analysis after pathological grading based on the results of SPINK1 staining in tumor tissue of prostate cancer patients.
  • A Statistics, the number of patients without chemotherapy and those who have experienced chemotherapy are 42 and 48, respectively.
  • B representative pictures of pathological grading.
  • EL expression level.
  • Figure 20 Comparative analysis of SPINK1 transcript expression of stromal cells and epithelial cells after laser capture microdissection (LCM) separation.
  • Figure 21 Analysis of SPINK1 transcript expression in stromal cells and cancer cells based on a single patient. The number of patients in each group is 10. Left, stromal cells. Right, epithelial cells.
  • Figure 22 Comparative analysis of SPINK1, IL-8 and WNT16B protein expression in tumor stromal cells of patients with prostate cancer after chemotherapy.
  • the pathological score of each factor comes from the histochemical staining of each factor, and each reading is the average of 3 pathological blind readings.
  • Figure 23 Representative graph of histochemical staining based on SPINK1, IL-8 and WNT16B.
  • the histopathological staining series of the three factors comes from 3 consecutive slices of a patient after treatment.
  • Figure 24 Analysis of protein expression relationships between SPINK1 and IL-8, and between SPINK1 and WNT16B in patients after chemotherapy. The value of each factor comes from three pathological blind readings. Among them, r, R 2 , slope and P value are all from Pearson correlation analysis. Left, SPINK1-IL-8. Right, SPINK1-WNT16B.
  • Figure 25 Survival curve (Kaplan Meier) analysis based on the expression level of SPINK1 in the lesions of patients after chemotherapy. Number of patients in SPINK1 low expression group, 20, green curve. SPINK1 high expression group patients, 28, red curve.
  • Figure 26 Comparative analysis of histopathology of primary lesions in breast cancer patients before and after chemotherapy. On the left, representative pictures of histochemical staining. On the right, representative pictures of H & E staining.
  • Figure 27 Statistical comparison analysis after pathological grading based on the results of SPINK1 staining in the tumor tissue of breast cancer patients. The number of patients without chemotherapy and those undergoing chemotherapy were 68 and 62, respectively.
  • Figure 28 Comparative analysis of SPINK1 expression between different types of cells.
  • A Comparative analysis of SPINK1 transcript expression of stromal cells and epithelial cells separated by laser capture microdissection (LCM).
  • B Based on the expression analysis of SPINK1 transcripts of stromal cells of a single patient, the number of patients in each group is 10.
  • C Based on a similar analysis of cancer cells, the number of patients in each group is 10.
  • Figure 29 Kaplan Meier analysis based on the expression level of SPINK1 in breast cancer patients after chemotherapy. Number of patients in SPINK1 low expression group, 26, green curve. SPINK1 high expression group patients, 36, red curve.
  • Figure 30 Comparative analysis of histopathology of primary lesions in patients with colorectal cancer before and after chemotherapy. On the left, representative pictures of histochemical staining. On the right, representative pictures of H & E staining.
  • Figure 31 Statistical comparison analysis after pathological grading based on the results of SPINK1 staining in the tumor tissue of colorectal cancer patients. The number of patients who have not received chemotherapy and those who have undergone chemotherapy are both 40.
  • Figure 32 Comparative analysis of SPINK1 expression between different types of cells.
  • SPINK1 transcript expression analysis of colorectal cancer stromal cells and epithelial cells after laser capture microdissection (LCM) separation In the analysis of SPINK1 transcript expression of stromal cells of a single patient, the number of patients in each group was 10. Right, based on a similar analysis of cancer cells, the number of patients in each group is 10.
  • Figure 33 Survival curve (Kaplan Meier) analysis based on the expression level of SPINK1 in patients with colorectal cancer after chemotherapy. Number of patients in SPINK1 low expression group, 14, blue curve. SPINK1 high expression group patients, 26, purple curve.
  • Figure 34 Shengxin analysis of the NF-kB binding site within 4000bp upstream of the SPINK1 promoter.
  • A Schematic diagram of the typical binding site of mammalian NF-kB subunit p65.
  • B A schematic diagram of a set of expression vectors constructed based on the speculative NF-kB binding site in the SPINK1 promoter region.
  • Figure 35 After the four reporter expression vectors in Figure 34B were transformed into 293 cells and stimulated with TNF ⁇ , the luciferase activity was detected. NAT11-Luc2CP, positive control vector.
  • Figure 36 After the four vectors used in Figure 35 were transferred into PSC27 stromal cells and treated with 50 ⁇ g / ml bleomycin, the luciferase signal intensity was compared and analyzed.
  • FIG. 37 ChIP-PCR analysis of the PCR signal intensity of the 4 speculative NF-kB binding sites on the SPINK1 promoter in the fraction precipitated by the NF-kB specific antibody.
  • Both IL-6-p1 and IL-8-p1 are NF-kB sites with known sequences and are used here as a positive control.
  • FIG. 38 NF-kB to the nucleus after three kinds of mutations after chemotherapy treatment, SPINK1 and expression levels of PSC27 IkB ⁇ IL-8 cell subline.
  • Figure 39 After transfecting GL-SPINK1-P04 into PSC27 cells, and then treated with bleomycin and NF-kB, c / EBP, and AP-1 inhibitors, respectively, the luciferase signals obtained were compared. BAY, NF-kB inhibitor. BA, c / EBP inhibitor. T5224 and SR, AP-1 inhibitors.
  • FIG. 40 After PSC27 cells were treated with bleomycin and NF-kB, c / EBP and AP-1 inhibitors respectively, the expression of several SASP components transcripts A, SPINK1. B, IL-6. C, IL-8.
  • Figure 41 Analysis of protein expression of SPINK1 in the SPINK1 overexpression subline and knockout subline of PSC27 and the effect on the cell itself.
  • A Western blot detected SPINK1 expression.
  • GAPDH loading control.
  • B Statistical analysis of SA- ⁇ -Gal staining.
  • C representative pictures.
  • Figure 42 Proliferation analysis of prostate cancer cells after CM treatment by SPINK1 overexpression group and knockout group of PSC27, respectively.
  • Hela cells are a positive control.
  • the mitoxantrone drug concentration is set near the IC50 value of each cancer cell line.
  • Figure 46 Prostate cancer cell line DU145 in the presence of SPINK1 and / or the use of chemotherapeutic drugs, the analysis of the expression of the complete form of caspase3 and its cleaved form.
  • Figure 47 Comparative analysis of apoptosis of prostate cancer cell line PC3 under the action of mitoxantrone and apoptosis inhibitor (QVD-OPH, ZVAD / FMK) or activator (PAC1, GA).
  • QVD-OPH, ZVAD / FMK mitoxantrone and apoptosis inhibitor
  • PAC1, GA activator
  • A Paclitaxel (DOC).
  • B Mitoxantrone (MIT).
  • FIG 48 Analysis of the activation of EGFR and its downstream molecules under the action of stromal cell-derived SPINK1 in prostate cancer cell lines PC3 and DU145.
  • EGF as an endogenous growth factor for detectable cancer cells.
  • the bottom value is the relative calculated concentration of SPINK1 in stromal cell CM (ELISA reading).
  • Figure 49 The prostate cancer cell lines PC3 and DU145 were subjected to stromal cell-derived SPINK1, and the expression of each molecule in Figure 48 was again analyzed by Western blot.
  • Figure 50 IP and Western blot analysis based on SPINK1-specific antibodies.
  • IgG control antibody.
  • S SPINK1 monoclonal antibody.
  • Figure 51 The effect of SPINK1 knockout in stromal cells on the senescence of the cells themselves.
  • A Statistical analysis after SA-B-Gal staining.
  • B Representative cell staining pictures.
  • FIG 52 The CM produced by PSC27 (PSC27-BLEO) after bleomycin treatment was used to treat prostate cancer cells, and the proliferation rate of cancer cells was analyzed with or without SPINK1 knockout.
  • Fig. 55 and Fig. 54 The experimental conditions in Fig. 55 and Fig. 54 are similar, but the EGFR inhibitors AG-1478 (2 ⁇ M), Cetuximab (50 ⁇ g / ml) and SPINK1 mAb (1 ⁇ g / ml) are used to detect cell resistance.
  • the MIT drug concentration is designed to approximate the actual MIT concentration in the plasma of prostate cancer patients under clinical administration conditions.
  • Figure 57 Cell resistance curves of human breast cancer cells MDA-MB-231 and stromal cells HBF1203 after various treatments similar to those in Figure 56. DOX, doxorubicin.
  • Figure 58 Measurement of terminal tumor volume of mice at the end of the 8th week after immunodeficient mice were inoculated with PC3 / PSC27 subcutaneously. On the left, a statistical comparison analysis of the five groups of samples. On the right, a representative tumor picture.
  • Figure 59 Schematic diagram of tumor growth, administration and detection in mice. In the third week after PC3 / PSC27 subcutaneous injection, single or multi-drug treatment was started.
  • Figure 60 Schematic diagram of mouse treatment mode under pre-clinical conditions. The upper part is each processing method, and the lower part is distributed at each time point.
  • Figure 61 Statistical analysis of tumor terminal volume of mice after PC3 / PSC27 inoculation after 8 weeks of MIT preclinical administration. On the left, statistical comparison. On the right, a representative tumor picture.
  • Figure 62 Expression analysis of SASP representative factors and cell aging markers after mouse tumors were microdissected by laser capture after stromal cells and cancer cells were specifically separated.
  • A-H are IL-8, WNT16B, SPINK1, MMP2, AREG, ANGPTL4, p16 and p21 respectively
  • FIG. 63 Expression of SPINK1 in mouse tumor tissues treated with placebo and mitoxantrone, respectively.
  • Figure 64 Statistical analysis of tumor terminal volume in mice after mitoxantrone and therapeutic antibody Cetuximab or SPINK1 mAb monotherapy or multi-drug treatment.
  • Figure 65 Detection and analysis of luciferase expression in mice after subcutaneous inoculation based on PC3-luc / PSC27.
  • Figure 66 Comparative analysis of DNA damage and apoptosis of cancer cells in mouse tumors 7 days after preclinical administration. On the left, statistical comparison. On the right, a representative histochemical staining picture (cleaved caspase 3).
  • Figure 67 Analysis of SPINK1 protein levels in mouse plasma under several treatment conditions. The test results are from ELISA.
  • Figure 68 Statistical analysis of the terminal tumor volume of mice at the end of mitoxantrone treatment at the 8th week after inoculation of LNCaP / PSC27 in immunodeficient mice.
  • Figure 69 Statistical analysis of tumor terminal volume at the end of paclitaxel treatment at week 8 after immunodeficiency mice were inoculated with breast cancer cell lines MDA-MB-231 and / or HBF1203.
  • Figure 70 Comparative analysis of PC3 mice's body weight (A) and peripheral blood creatinine (B), urea (C), ALP (D) and ALT (E) levels at the end of preclinical studies.
  • FIG. 71 MDA-MB-231 mice at the end of MIT pre-clinical treatment at the end of body weight (A) and peripheral blood creatinine (B), urea (C), ALP (D) and ALT (E) levels of comparative analysis.
  • FIG 72 Body weight (A) and peripheral blood creatinine (B), urea (C), ALP (D) and ALT (E) levels of immune intact mice (C57BL / 6) at the end of DOX preclinical treatment Comparative analysis. At the same time, the unit volume of hemoglobin (F), white blood cells (G), lymphocytes (H) and platelets (I) in the plasma was also detected.
  • A peripheral blood creatinine
  • C urea
  • D ALP
  • E ALT
  • Figure 73 Analysis of SPINK1 and IL-8 protein levels in plasma of clinical prostate patients and their relationship.
  • A SPINK1 level readings obtained by ELISA (20 patients before and after chemotherapy).
  • B SPINK1 level readings obtained by ELISA test (20 patients before and after chemotherapy).
  • C Pearson analyzed the statistical relationship between SPINK1 and IL-8.
  • Figure 74 A-C, similar to the clinical data analysis of Figure 73, a sample of breast cancer patients.
  • Figure 75 A-C, similar to the analysis of clinical data in Figure 72 and Figure 73, a sample of colorectal cancer patients.
  • Figure 76 Western blot detection of SPINK1 and IL-8 levels in plasma of prostate cancer patients before and after chemotherapy. Six patients before and after chemotherapy. Albumin, plasma loading control.
  • Figure 77 Correlation analysis of the expression levels of SPINK1 and IL-8 in the primary tumor tissue and peripheral blood of patients after chemotherapy. A total of 20 patients.
  • FIG 78 Expression analysis of multiple SASP factors in stromal cells in the lesion tissue of 20 prostate cancer patients in Figure 77.
  • IL-2 / 3/5/12 are SASP non-related interleukins (or pro-inflammatory factors), which are experimental controls.
  • Figure 79 Correlation between plasma levels of SPINK1 and disease-free survival in 20 prostate cancer patients after chemotherapy. Ten patients with low level SPINK1, cyan curve. SPINK1 patients with high levels, purple curve.
  • Figure 80 Statistical graph of SPINK1 overexpression in ovarian cancer patients. Each red dot represents a patient with ovarian cancer, and each black dot represents a healthy volunteer.
  • Figure 81 Statistical comparison analysis of mutation, amplification and deletion of SPINK1 in breast cancer patients in TCGA database.
  • Figure 82 Statistical comparison analysis of mutation, amplification and deletion of SPINK1 in patients with prostate cancer in the TCGA database.
  • Figure 83 SPINK1 expression and secretion in stromal cells in the tumor microenvironment during clinical treatment, the pathological effects on peripheral cancer cells, and the working mode diagram of entering peripheral blood to participate in circulation.
  • SPINK1 serine protease inhibitor Kazal type 1 factor
  • SPINK1 serine protease inhibitor Kazal type 1
  • PSTI pancreatic secretory pancreatin inhibitor
  • TATI tumor-associated pancreatin inhibitor
  • the SPINK family is closely related to diseases such as chronic pancreatitis, Netherton syndrome, and esophageal cancer.
  • the amino acid sequence of human SPINK1 is as follows:
  • SPINK1 overexpression is positively correlated with the poor clinical outcome of prostate cancer patients.
  • the gene expression of SPINK1 in liver cancer tissues was significantly up-regulated, which may be due to tissue destruction caused by tumor invasion inducing an acute phase reaction.
  • Interleukin 6 (IL-6) and interleukin 1 (IL-1) induced high expression of SPINK1.
  • the high expression of SPINK1 in hepatocellular carcinoma tissues shows its potential as a tumor marker for hepatocellular carcinoma.
  • plasma SPINK1 has the potential as a serum tumor marker of hepatocellular carcinoma, and the level of plasma SPINK1 expression is significantly related to the tumor stage of patients with hepatocellular carcinoma.
  • the synergistic effect of the antibody that specifically inhibits SPINK1 and the chemotherapeutic drug is through the following mode of action: the antibody that specifically inhibits SPINK1 inhibits its activity by binding to SPINK1 derived from the tumor microenvironment (especially the stromal cells), reversing the tumor for chemotherapy Drug resistance, which makes the effect of chemotherapy drugs more ideal.
  • the present invention provides a drug combination or composition for inhibiting tumors or reducing tumor drug resistance
  • the drug combination or composition includes: specific inhibition of serine protease inhibitor Kazal type 1 factor Antibodies, as well as chemotherapy drugs.
  • the tumor may be a tumor in situ or a metastatic tumor, which includes refractory tumors that have drug resistance, especially tumors that are resistant to genotoxic chemotherapy drugs.
  • the tumor is a solid tumor.
  • the tumors include: prostate cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, lung cancer, etc.
  • an anti-SPINK1 monoclonal antibody that is particularly effective for inhibiting tumors or reducing tumor resistance.
  • the anti-SPINK1 monoclonal antibody has high specificity for SPINK1 and does not bind to proteins other than SPINK1 . Also, when used in combination with chemotherapeutic drugs to suppress tumors, its effect is extremely excellent.
  • the anti-SPINK1 monoclonal antibody of the present invention is prepared using hybridoma technology.
  • the deposit number of the hybridoma cell strain in the Chinese Type Culture Collection Center is CCTCC NO: C2018213.
  • the hybridoma cells can be cultured and expanded in vitro according to a conventional animal cell culture method, so that the anti-SPINK1 monoclonal antibody is secreted.
  • the anti-SPINK1 monoclonal antibody can be prepared by the following preparation methods: (1) mice pre-treated with adjuvant; (2) inoculation of the hybridoma cells in the abdominal cavity of the mouse and secretion of monoclonal antibodies Clone the antibody; (3) draw ascites and isolate the monoclonal antibody.
  • the monoclonal antibody isolated from ascites fluid is further purified to obtain high-purity antibodies.
  • the monoclonal antibodies of the present invention can also be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Those skilled in the art understand that after the monoclonal antibody hybridoma cell line is obtained or the monoclonal antibody is known by sequencing and other means, those skilled in the art can easily obtain the antibody.
  • Antibodies and chemotherapy drugs that specifically inhibit SPINK1 can be administered as a pharmaceutical composition, or the two can be present separately in a kit.
  • the antibodies and chemotherapeutics that specifically inhibit SPINK1 are effective amounts.
  • the antibody that specifically inhibits SPINK1 is also mixed with a pharmaceutically acceptable carrier.
  • the term "effective amount” or “effective dose” refers to a human and / or animal that can produce a function or activity and can be accepted by a human and / or animal as used herein.
  • A is the body surface area, calculated in m 2 ; W is the body weight, calculated in g; K is a constant, which varies with the type of animal, in general, mouse and rat 9.1, guinea pig 9.8, rabbit 10.1, cat 9.9, Dog 11.2, monkey 11.8, human 10.6. It should be understood that the conversion of the administered dose can be changed according to the difference of the drug and clinical situation and the evaluation of an experienced pharmacist.
  • pharmaceutically acceptable ingredients are suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergies), that is, substances with a reasonable benefit / risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
  • the present invention provides a kit for inhibiting tumors or reducing tumor drug resistance.
  • the kit includes antibodies and chemotherapy drugs (such as mitoxantrone, doxorubicin, bleomycin) that specifically inhibit SPINK1 Vegetarian, satraplatin, paclitaxel). More preferably, the kit further includes instructions for use to guide the clinician to use the medicine in a correct and reasonable manner.
  • chemotherapy drugs such as mitoxantrone, doxorubicin, bleomycin
  • the kit further includes instructions for use to guide the clinician to use the medicine in a correct and reasonable manner.
  • the combination of the antibody that specifically inhibits SPINK1 and chemotherapy drugs can be made into unit dosage form and placed in the kit.
  • chemotherapy drugs such as mitoxantrone, doxorubicin, bleomycin, sabplatin, paclitaxel
  • antibodies or chemotherapy drugs that exist independently of each other can be made into unit dosage form and placed in the kit.
  • Unit dosage form refers to the preparation of the medicine into a dosage form required for single administration for the convenience of taking, including but not limited to various solid agents (such as tablets), liquid agents, capsules, and sustained-release agents.
  • SPINK1 can be used as a marker for prognostic evaluation in the post-chemotherapy stage of tumor: (i) disease classification, differential diagnosis, and / or disease-free survival analysis in post-chemotherapy stage of tumor; ii) Evaluate the tumor treatment drugs, drug efficacy, prognosis of the relevant population, and select appropriate treatment methods.
  • diseases classification, differential diagnosis, and / or disease-free survival analysis in post-chemotherapy stage of tumor ii) Evaluate the tumor treatment drugs, drug efficacy, prognosis of the relevant population, and select appropriate treatment methods.
  • people with abnormal expression of the SPINK1 gene in the tumor microenvironment, especially in stromal cells can be isolated for more targeted treatment.
  • the prognosis of the tumor of the subject who provided the sample to be evaluated can be predicted by judging the expression or activity of SPINK1 in the sample to be evaluated (stromal cells), and selecting a suitable drug for treatment.
  • a threshold of SPINK1 can be specified. When the expression of SPINK1 is higher than the specified threshold, consider using SPINK1 suppression treatment.
  • the threshold is easily determined by those skilled in the art. For example, the expression of SPINK1 in the microenvironment of normal human tissues can be compared with the expression of SPINK1 in the microenvironment of tumor patients to obtain abnormal expression of SPINK1 Threshold.
  • the present invention provides the use of SPINK1 gene or protein for preparing reagents or kits for tumor prognosis evaluation.
  • Various techniques known in the art can be used to detect the presence or expression of the SPINK1 gene and these techniques are all included in the present invention. For example, existing techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR, etc., can be used in combination.
  • the invention also provides reagents for detecting the presence or absence and expression of SPINK1 gene in analytes.
  • primers that specifically amplify SPINK1 may be used; or probes that specifically recognize SPINK1 to determine the presence or absence of the SPINK1 gene; when performing protein-level detection, specificity may be used.
  • the antibody or ligand of the protein encoded by SPINK1 is combined to determine the expression of SPINK1 protein.
  • the kit can also include various reagents required for DNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution, display solution Color, lotion, etc.
  • the kit can also include instructions for use and / or nucleic acid sequence analysis software.
  • the present invention provides a method for screening potential substances that inhibit tumors or reduce tumor resistance.
  • the method includes: treating a system for expressing NF- ⁇ B and SPINK1 with a candidate substance, and NF- is present upstream of the gene encoding SPINK1 ⁇ B binding site; and detection of the regulatory effect of NF- ⁇ B on SPINK1 in the system; if the candidate substance statistically inhibits the transcriptional regulation of SPINK1 by NF- ⁇ B, it indicates that the candidate substance inhibits or reduces tumors Potential substances for drug resistance.
  • a control group in the screening, in order to more easily observe the transcriptional regulation of SPINK1 by NF- ⁇ B and the change of the expression or activity of SPINK1, a control group may also be set, and the control group may not be added The expression system of the candidate substance.
  • the present invention provides a method for screening potential substances that inhibit tumors or reduce tumor resistance.
  • the method includes: treating an expression system with candidate substances that expresses EGFR-mediated signaling pathways and SPINK1; and detection
  • SPINK1 activates the EGFR-mediated signaling pathway; if the candidate substance statistically inhibits the activation, it indicates that the candidate substance is a potential substance that inhibits tumors or reduces tumor resistance.
  • a control group when performing screening, in order to more easily observe the activation effect of SPINK1 on the EGFR-mediated signaling pathway and changes in the expression or activity of SPINK1, a control group may also be provided, and the control group may It is an expression system that does not add the candidate substance.
  • the method further includes: performing further cell experiments and / or animal experiments on the obtained potential substances to further select and determine substances that are truly useful for inhibiting tumors or reducing tumor drug resistance.
  • Normal human-derived primary prostate stromal cell line PSC27 and human-derived primary breast stromal cell line HBF1203 were propagated and passaged in PSCC complete culture medium.
  • Benign prostate epithelial cell lines BPH1, prostate cancer epithelial cell lines M12, DU145, PC3, LNCaP and VCaP, breast cancer epithelial cell lines MCF-7, MDA-MB-231, MDA-MB-468, T47D and BT474 purchased from ATCC Both were cultured in 5% FBS RPMI-1640 complete culture medium in an incubator at 37 ° C and 5% CO 2 .
  • PSC27-Pre 100nM docetaxel (DTX), 100nM paclitaxel (PTX), and 200nM vincristine (VCR) are added to the culture medium.
  • the full-length human SPINK1 was cloned between the lentiviral expression vector pLenti-CMV / To-Puro-DEST2 (Invitrogen) restriction sites BamHI and XbaI.
  • the packaging line 293FT is used for cell transfection and lentivirus manufacturing.
  • the small hairpin RNAs (shRNAs) sense strands used to knock out SPINK1 are GAAGAGAGGCCAAATGTTATTCAAGAGATAACATTTGGCCTCTCTTCTTT (SEQ ID NO: 2) and CCAAGATATATGACCCTGTTTCAAGAGAACAGGGTCATATATCTTGGTTTTT (SEQ ID NO: 3).
  • Mouse monoclonal antibody anti-phospho-Histone H2A.X (Ser139) (clone JBW301, Millipore) and mouse monoclonal antibody anti-SPINK1 (Cat # H00006690-M01 (clone 4D4), Abnova), and secondary antibody Alexa 488 (or 594) -conjugated F (ab ') 2 was added to the slides covered with fixed cells in order.
  • the nuclei were counterstained with 2 ⁇ g / ml of 4 ', 6-diamidino-2-phenylindole (DAPI). Select the most representative one of the three observation fields for data analysis and result display.
  • the FV1000 laser scanning confocal microscope (Olympus) was used to acquire confocal fluorescence images of cells.
  • the SPINK1 antibody used in the IHC staining of clinical prostate cancer, breast cancer and colorectal cancer patients was the same as that purchased from Abnova.
  • the specific steps are as follows: conventional dewaxing, incubation with 0.6% H2O2 methanol at 37 ° C for 30min, then repair with 0.01M pH6.0 citrate buffer for 20min, and cooling at room temperature for 30min. Block with normal goat serum for 20 min, incubate with SPINK1 primary antibody (1: 200) at 37 °C for 1h, and move to 4 °C refrigerator overnight. The next day, it was washed three times with TBS, incubated with a secondary antibody (HRP-conjugated goat anti-rabbit) at 37 ° C for 45 min, washed with TBS three times, and finally developed with DAB.
  • conventional dewaxing incubation with 0.6% H2O2 methanol at 37 ° C for 30min, then repair with 0.01M pH6.0 citrate buffer for 20min, and cooling at room
  • PSC27 cells were cultured with DMEM + 0.5% FBS medium for 3 days, and then washed with 1 times PBS to wash the full abundance of the cell population. After simple centrifugation, the supernatant was collected and stored as conditioned medium at –80 ° C or used directly.
  • Prostate epithelial cells were continuously cultured in this conditioned medium for 3 days to carry out in vitro experiments.
  • epithelial cell lines are cultured in low serum DMEM (0.5% FBS) (referred to as "DMEM"), or conditioned medium, while mitoxantrone (MIT) is used to treat cells for 1 to 3 days, The concentration was close to the IC 50 value of each cell line, and then observed under a bright field microscope.
  • the total RNA of growing cells was extracted with Trizol reagent to perform reverse transcription reaction.
  • the reverse transcription reaction product cDNA was diluted 50 times as a template, and RT-PCR was performed.
  • the analysis of the amplification of each gene is analyzed through the software, the corresponding threshold cycle number is derived, and the relative expression level of each gene is calculated using the 2- ⁇ Ct method.
  • the peaks and waveforms of the melting curve are analyzed to determine whether the amplified product is a specific single-purpose fragment.
  • the antiviral vector pBabe-Puro-I ⁇ B ⁇ -Mut (superrepressor) containing the two IKK phosphorylation mutation sites S32A and S34A on the I ⁇ B ⁇ protein sequence was used to transfect the lentiviral packaging cell line PHOENIX.
  • the lentivirus was then used to infect the PSC27 stromal cell line, and 1 ⁇ g / ml puromycin was used to screen positive clones.
  • a 5 ⁇ M small molecule inhibitor Bay 11-7082 (available from Selleck) was used for NF- ⁇ B activity control.
  • the stromal cells were subsequently exposed to several different forms of cytotoxicity, and the resulting phenotypes were recorded in time to analyze the expression of related genes.
  • the cells treated in this way, the conditioned medium produced is collected and used for various detections of epithelial cells.
  • the human SPINK1 gene (Gene ID 6690, Genbank accession NM_001354966.1) was analyzed using software CONSITE to discover potential core NF- ⁇ B binding sites.
  • primer # 1 (-482 ⁇ -259): forward 5'-CTACTGAAATCACAGTGAAGTATAG-3 '(SEQ ID NO: 4), reverse 5'-CTGTTCATTGCATCCTGCTAT-3 '(SEQ ID NO: 5); primer set # 2 (-1870 ⁇ -1625): forward 5'-GACCAGTCTGGCCAACATGG-3' (SEQ ID NO: 6) , Reverse 5'-CCTCATGCTGTATGTTAGATATTCAGAC-3 '(SEQ ID NO: 7); primer set # 3 (-1917 ⁇ -1773): forward 5'-TACTTTGGGAGGCCGAGGCAG
  • IL-6 forward 5'-AAATGCCCAACAGAGGTCA-3 '(SEQ ID NO: 12), reverse 5'-CACGGCTCTAGGCTCTGAAT-3' (SEQ ID NO: 13) and IL8: forward 5'-ACAGTTGAAAACTATAGGAGCTACATT-3 '(SEQ ID NO: 14), reverse 5'-TCGCTTCTGGGCAAGTACA-3' (SEQ ID NO: 15) promoter sequence (all known positive controls).
  • ChIP analysis was performed on early passage PSC27 cells (such as p8) and PSC27 cells treated with bleomycin (50ug / ml).
  • the chromosomes fixed in vitro were settled using mouse monoclonal antibody anti-p65antibody (F-6, Santa Cruz), and DNA was extracted for amplification.
  • Reporter expression vectors carrying multiple NF- ⁇ B binding site mutations are designed and generated by the site-directed mutagenesis (Stratagene) method. In addition, it covers multiple NF- ⁇ B binding sites and the optimized IL-2 minimum promoter as the reporting vector for NF- ⁇ B activated transgenic system (NAT system) NAT11-Luc2CP-IRES-nEGFP (Dr. Hatakeyama friendship of Hokkaido University, Japan Provided), used as a positive control in the experiment.
  • Each reporter vector was co-transfected with pRL-TK vector (Addgene) for signal standardization.
  • the chemotherapy regimen is based on patients with castration-resistant prostate cancer (clinical trial registration number NCT03258320), patients with osmotic catheter breast cancer (clinical trial registration number NCT02897700) and patients with non-metastatic colorectal cancer (clinical trial registration number NCT00643877) Pathological characteristics are specified. Patients with a clinical stage of primary cancer above I subtype A (IA) (T1a, N0, M0) but no obvious distant metastatic lesions were recruited into the clinical cohort. At the same time, patients aged 40-75 years who were clinically diagnosed with PCa, or those older than 18 years who had been confirmed to have osmotic BCa by tissues, and patients younger than 75 years who were clinically diagnosed with CRC were recruited.
  • IA I subtype A
  • ICR / SCID mice (about 25g in weight) of immunodeficient mice aged about 6 weeks are used in the animal experiments related to the present invention.
  • the stromal cells PSC27 and epithelial cells are mixed in a ratio of 1: 4, and each graft contains 1.25 ⁇ 10 6 cells for tissue reconstruction.
  • the transplanted tumors were implanted into mice by subcutaneous transplantation, and the animals were euthanized 8 weekends after the transplantation.
  • breast cancer xenografts are formed by MDA-MB-231 (triple negative, highly malignant breast cancer cell line) and HBF1203 (breast fibroblast cell line) through tissue remodeling.
  • mice subcutaneously transplanted were given standard experimental diets, and the chemotherapy drugs mitoxantrone (0.2 mg / kg dose) or doxorubicin (1.0 mg / kg dose) were administered intraperitoneally after 2 weeks .
  • the FDA approved therapeutic antibody Cetuximab (10.0 mg / kg dose, 200 ⁇ l / dose) or SPINK1 mAb (10.0 mg / kg dose, 200 ⁇ l / dose) after strict purification was administered intravenously as a single or double agent.
  • the time point was the first day of the 3rd, 5th, and 7th weeks. There were 3 cycles of drug administration throughout the course of treatment, and each cycle was 2 weeks.
  • mice were collected for tumor measurement and histological analysis. Each mouse received cumulatively mitoxantrone 0.6 mg / kg body weight, or doxorubicin 3.0 mg / kg body weight.
  • the chemotherapy experiment was carried out until the end of the 8th week. The mice were dissected immediately after sacrifice, and the transplanted tumors were collected and used for pathological analysis. Part of the mice 7 days after the administration was used for histochemical evaluation of the caspase 3 activity at the tissue level.
  • mice were weighed once a week; after the completion of chemotherapy as a whole, the mice were weighed again and their blood was collected by cardiac puncture and placed in an ice bath for 45 minutes. Peripheral blood was immediately centrifuged at 8000 rpm for 10 minutes at 4 ° C, and about 50 ⁇ l was sucked by the VetTest pipette tip for detection by the IDEXX VetTest 8008 chemical analyzer. Liver function measurement items include creatinine, urea, alkaline phosphatase and alanine aminotransferase.
  • Genotoxic drugs can induce high expression of SPINK1 in human stromal cells
  • the inventors have noticed that the human prostate stromal cell line PSC27 (mainly composed of fibroblasts), after being treated with cytotoxic, especially genotoxic chemotherapy drugs or ionizing radiation, will generate a large amount of SASP factors, and SPINK1 appears to upregulate the expression amplitude Among the highest group of proteins ( Figure 1).
  • the inventors subsequently used a set of DNA-damaging drugs, including mitoxantrone (MIT), sabplatin (SAT), gamma rays (RAD), doxorubicin (DOX) Treat stromal cells with Bleomycin (BLEO).
  • MIT mitoxantrone
  • SAT sabplatin
  • RAD gamma rays
  • DOX doxorubicin
  • the expression pattern of SPINK1 in stromal cells is very similar to several other SASP markers such as MMP1, WNT16B, SFRP2 and MMP12, that is, the expression level gradually increases after the cells are damaged by drugs until The cells reached a plateau after 7-10 days and maintained a secreted state for a long time (Figure 6).
  • Example 2 The expression of SPINK1 in the tumor microenvironment is significantly negatively correlated with the survival of patients after chemotherapy
  • SPINK1 is highly expressed in tumors after chemotherapy. It is a pre-established pathological detection system that can quantitatively evaluate its expression level in tissues according to the histochemical staining intensity of specific proteins. Analysis and further determination (Figure 19). After the microscopic technique of laser capture microdissection, the inventors found that SPINK1 in tissues is more inclined to induce inducible expression in stromal cell populations rather than epithelial cell populations (FIG. 20). In order to confirm the drug-inducibility of SPINK1, the inventors selected a group of patients whose tissue samples were obtained and saved before and after chemotherapy, and found that in any of them, SPINK1 stromal cells instead of epithelia after chemotherapy Highly expressed in cells (Figure 21).
  • Example 3 The expression of SPINK1 in stromal cells is regulated by transcription factors such as NF-kB
  • the NF- ⁇ B complex plays an important role in the process of cell senescence caused by oncogene induction or therapeutic injury.
  • NF- ⁇ B mediates the expression of SPINK1 in stromal cells after DNA damage The analysis revealed that there were several NF- ⁇ B binding sites in the 4000 bp upstream of SPINK1 ( Figure 34), and subsequent fluorescence detection based on the reporter vector confirmed the importance of these several sites.
  • BA betulinic acid
  • T-5224 which is an AP-1 selective inhibitor
  • SPINK1 in stromal cells under genotoxic background is mainly regulated by NF- ⁇ B.
  • drugs that inhibit tumors by affecting the interaction between the two can be screened. Some drugs that can inhibit or prevent the interaction of the two may potentially be beneficial to tumor treatment.
  • Example 4 The functional impact of SPINK1 on cancer cells is mainly controlled by EGFR and its downstream signaling pathways
  • CM conditioned medium
  • PSC27 SPINK1 conditioned medium
  • PC3, DU145, LNCaP and M12 including upregulated proliferation Rate, mobility and invasiveness
  • SPINK1 significantly reversed after SPINK1 was knocked out of stromal cells
  • SPINK1 significantly increased the resistance of prostate cancer cells to the clinical chemotherapy drug mitoxantrone (MIT) ( Figure 45).
  • MIT induces apoptosis by causing caspase 3 cleavage in cells, but this process can be significantly weakened by SPINK1, and SPINK1 knocked out of stromal cells can restore this effect of MIT ( Figure 46).
  • the inventors subsequently used QVD-OPH and ZVAD-FMK, two broad-spectrum caspase 3 inhibitors, and PAC1 and gambogic acid (GA), two caspase 3 activators, to treat cells at MIT, respectively Previously used for cell culture.
  • the inventors found that the degree of apoptosis was significantly reduced in the presence of QVD-OPH or VAD-FMK, even if SPINK1 was used to culture cancer cells (FIG. 47).
  • SPINK1 shares about 50% sequence homology with EGF
  • the inventors first determined the effect of SPINK1 as an EGF analog growth factor on cancer cell signaling pathways.
  • SPINK1 highly expressed stromal cells PSC27 SPINK1
  • the inventors found that the latter showed multiple protein molecule changes, mainly including EGFR (Y845), Akt (S473) and mTOR (S2448), etc.
  • Phosphorylation at the site implies activation of PI3K / Akt / mTOR signaling pathway mediated by SPINK1 ( Figure 48).
  • phosphorylation of Erk (T202 / Y204) and Stat3 (S727) indicates the activation of MAPK meridian in these cells.
  • the phenotype change of cancer cells caused by SPINK1 is mainly achieved through the activation of EGFR-mediated signaling pathways, although the participation of other receptor molecules in this process cannot be ruled out.
  • the activation of this series of signaling pathways of cancer cells basically disappeared ( Figure 49), further confirming that SPINK1 caused the activation of multiple downstream signaling pathways through EGFR receptors.
  • the inventors conducted IP experiments using SPINK1 specific antibodies. The results show that there is a physical direct interaction between the two molecules of SPINK1 and EGFR, and the IP signal can be easily found in cancer cell samples treated with PSC27 SPINK1 instead of PSC27 Vector CM ( Figure 50).
  • the inventors asked another key question, whether SPINK1 plays a central role in the process of SASP driving cancer cell malignant progression.
  • the inventors constructed the PSC27-shRNA SPINK1 stability subline and collected its CM after DNA damage treatment.
  • the inventors noticed that after SPINK1 was knocked out, the senescence of cells that PSC27 originally appeared under DNA damage conditions was neither delayed nor accelerated, and the SA- ⁇ -Gal positive rate was unchanged ( Figure 51).
  • the CM produced by PSC27-BLEO cultured cancer cells the latter's proliferation rate, migration rate and invasiveness were significantly increased, and the knockout of SPINK1 from stromal cells can greatly reduce the increase in this series of malignant phenotypes ( Figures 52, 53).
  • the present inventors found that after SPINK1 was knocked out, the resistance of prostate cancer cells conferred by PSC27-BLEO to mitoxantrone significantly decreased (FIG. 54). Similarly, under the culturing conditions of PSC27-BLEO CM, the drug resistance of cancer cells treated by the EGFR inhibitor AG-1478 was also significantly reduced (Figure 55).
  • Cetuximab a monoclonal antibody that specifically approved by the FDA to specifically inhibit EGFR in the clinic. The inventors found that Cetuximab can significantly down-regulate the drug resistance of stromal cells to cancer cells, and the effect is close to AG-1478 (Figure 54).
  • Example 5 Targeting SPINK1 in vivo can delay tumor progression and increase tumor sensitivity to chemotherapy drugs
  • microenvironment SASP in the clinical anticancer process can accelerate many malignant events, including tumorigenesis, local inflammation and therapeutic resistance.
  • this trend toward malignancy can be controlled by specifically controlling the core factors of SASP in the microenvironment, and how to effectively inhibit SASP in the in vivo microenvironment activated by anti-cancer therapies have always been a problem in the scientific community.
  • the inventors In order to simulate clinical conditions as much as possible, the inventors inoculated a subpopulation of immunodeficient mice with a mixed cell population of PSC27 and PC3, and after 8 weeks, the inventors stopped the experiment and analyzed.
  • mice inoculated with PSC27 SPINK1 increased significantly, but the tumor volume formed under the screening pressure caused by the intraperitoneal administration of the chemotherapy drug mitoxantrone was significantly reduced, proving that chemotherapy itself can effectively block tumor development. (Figure 61).
  • the tumors of the control group PSC27 Vector mice
  • the tumors of PSC27 SPINK1 residual mice still increased significantly, suggesting the pathological role of the microenvironment throughout the chemotherapy.
  • the present inventors confirmed the obvious expression of SPINK1 in tumor tissue under pre-clinical treatment conditions by histochemical staining (FIG. 63).
  • the inventors then used Cetuximab or SPINK1 mAb in combination with mitoxantrone.
  • Cetuximab or SPINK1 mAb in combination with mitoxantrone.
  • the concurrent administration of Cetuximab therapeutic antibody did not further reduce the mass (Figure 64), suggesting PC3 Tumors basically progress in a microenvironment independent of the EGF / EGFR signal axis.
  • the present inventors used the PC3 cell line (PC3-luc) expressing luciferase and found that the relative intensity of the bioluminescence signal detected under the conditions in mice in vivo among the animals in each group was similar to the tumor terminal detected above.
  • the volume basically corresponds, and the possibility of ectopic organ metastasis in cancer cells is excluded (Figure 65).
  • LNCaP another classic prostate epithelial cancer cell line, which was inoculated with PSC27 to form tumors under the skin of mice.
  • LNCaP itself expresses the androgen receptor AR and exhibits androgen-dependent growth.
  • the present inventors did not take castration treatment, but followed a series of steps in PC3 mouse pre-clinical experiments.
  • the terminal tumor volume of LNCaP / PSC27 mice decreased significantly after the combination of chemotherapeutic drugs and therapeutic antibodies (36.7%, cetuximb; 50.7%, SPINK1 mAb) (Figure 68).
  • the inventors then expanded the research to human breast cancer. After inoculating MDA-MB-231 cancer cells and HBF1203 breast-derived stromal cells subcutaneously in mice, the inventors found that MDA-MB-231 / HBF1203 tumors showed a trend very similar to the data of prostate cancer mice. On the basis of element, combined use of cetuximb further inhibited tumor volume by 26.6%, and combined use of SPINK1 mAb further inhibited tumor volume by 39.5%) ( Figure 69). Therefore, the drug resistance antagonistic data caused by targeting SPINK1 indicates that controlling the effect of SPINK1 in the microenvironment on tumor treatment is organ-independent and a means applicable to a variety of solid tumors.
  • SPINK1 is a novel biomarker for judging the occurrence and development of SASP in patients under clinical conditions
  • the inventors then determined whether SPINK1 could be detected in cancer peripheral blood after clinical chemotherapy using conventional techniques. To this end, the inventors collected plasma samples of two groups of prostate cancer patients, including a group of patients undergoing chemotherapy and another group of patients without any treatment. After ELISA-based protein detection, the present inventors found that the level of SPINK1 in the blood of patients after chemotherapy was significantly higher than that of patients without chemotherapy ( Figure 73). Interestingly, this trend is very similar to IL-8, a typical factor of SASP.
  • the inventors conducted a longitudinal analysis based on the patient's clinical specimens.
  • the inventors were surprised to note that the two SASP related factors SPINK1 and IL-8 can be clearly displayed on the Western blot, and only after chemotherapy A signal appeared in the patient's plasma sample ( Figure 76).
  • Figure 77 there is a clear correlation between these two factors.
  • the present inventors used laser capture microdissection technology to isolate stromal cells in the lesion tissue of prostate cancer patients and analyzed their transcript levels.
  • the hybridoma cell line SP2 / 0-01-SPINK1-SUN of the present invention is deposited at the Chinese Type Culture Collection (CCTCC, Wuhan, China), with the deposit number CCTCC NO: C2018213, and the deposit date is October 10, 2018.

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Abstract

L'invention concerne l'utilisation d'inhibiteur de sérine protéase Kazal de type 1 (SPINK1) dans la préparation d'un agent pour diagnostiquer ou réguler la sénescence cellulaire et les tumeurs. L'inhibiteur SPINK1 joue un rôle biologique important dans le phénotype SASP et le microenvironnement tumoral, et est étroitement corrélé avec le pronostic établi après chimiothérapie. Ainsi, l'inhibiteur SPINK1 peut représenter une cible pour la recherche sur la régulation du phénotype SASP et la recherche anti-tumorale basée sur le microenvironnement tumoral, en tant que marqueur pour l'évaluation pronostique et le grade histologique de tumeur après chimiothérapie, et en tant que cible pour le développement d'un médicament inhibiteur de tumeur.
PCT/CN2019/076946 2018-10-11 2019-03-05 Utilisation d'un inhibiteur de sérine protéase kazal de type 1 dans la préparation d'un agent pour diagnostiquer ou réguler la sénescence cellulaire et les tumeurs WO2020073593A1 (fr)

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CN201811184715.1A CN111110849B (zh) 2018-10-11 2018-10-11 丝氨酸蛋白酶抑制因子Kazal 1型在制备细胞衰老及肿瘤诊断或调控制剂中的应用

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113774025A (zh) * 2021-08-26 2021-12-10 湖南丰晖生物科技有限公司 一种结直肠癌顺铂耐药株的构建方法

Citations (2)

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Publication number Priority date Publication date Assignee Title
CN101652484A (zh) * 2006-11-08 2010-02-17 密歇根大学董事会 前列腺癌标志物spinki及其应用
WO2011102999A2 (fr) * 2010-02-19 2011-08-25 The Regents Of The University Of Michigan Thérapie ciblant spink1

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CN101988110A (zh) * 2009-08-07 2011-03-23 芮屈生物技术（上海）有限公司 一种Spink1基因的原位杂交检测试剂盒及其检测方法和应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101652484A (zh) * 2006-11-08 2010-02-17 密歇根大学董事会 前列腺癌标志物spinki及其应用
WO2011102999A2 (fr) * 2010-02-19 2011-08-25 The Regents Of The University Of Michigan Thérapie ciblant spink1

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113774025A (zh) * 2021-08-26 2021-12-10 湖南丰晖生物科技有限公司 一种结直肠癌顺铂耐药株的构建方法
CN113774025B (zh) * 2021-08-26 2023-07-14 湖南丰晖生物科技有限公司 一种结直肠癌顺铂耐药株的构建方法

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