WO2017181943A1 - Pharmaceutical composition containing peitc and use thereof in cancer treatment - Google Patents

Pharmaceutical composition containing peitc and use thereof in cancer treatment Download PDF

Info

Publication number
WO2017181943A1
WO2017181943A1 PCT/CN2017/080960 CN2017080960W WO2017181943A1 WO 2017181943 A1 WO2017181943 A1 WO 2017181943A1 CN 2017080960 W CN2017080960 W CN 2017080960W WO 2017181943 A1 WO2017181943 A1 WO 2017181943A1
Authority
WO
WIPO (PCT)
Prior art keywords
peitc
cells
mutant
cancer
tumor
Prior art date
Application number
PCT/CN2017/080960
Other languages
French (fr)
Chinese (zh)
Inventor
宗方隆
程景才
阿加沃尔M
Original Assignee
无锡杰西医药股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 无锡杰西医药股份有限公司 filed Critical 无锡杰西医药股份有限公司
Priority to CN201780037010.4A priority Critical patent/CN109562093A/en
Publication of WO2017181943A1 publication Critical patent/WO2017181943A1/en

Links

Images

Classifications

    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/26—Cyanate or isocyanate esters; Thiocyanate or isothiocyanate esters
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of medicines, and more particularly to pharmaceutical compositions containing PEITC and their use in the prevention and treatment of cancer and other diseases caused by p53 mutations.
  • the present patent application is a subsequent patent of CN200510040865.1, CN200610126892.5, CN200910052231.6, CN201310205609.8, CN201310352414.6, CN201310364101.2 and CN201410346419.2, and US8039511B2, US8410170B2, EP06817815.1, CA2630262 and JP5308160.
  • Cancer is a major disease that threatens human health. According to the Global Cancer Report 2014, there were 14 million new cancer cases worldwide and 8.2 million deaths in 2012. Among them, China added 3.07 million cancer patients and caused about 2.2 million deaths, accounting for 21.9% and 26.8% of the global total, respectively.
  • FLS Fomany Syndrome
  • PEITC for the preparation of a formulation or composition for (a) altering (or activating) a mutant p53, (b) inhibiting a mutation Proliferation of p53 tumor cells, (c) induction of apoptosis in mutant p53 tumor cells, and/or (d) prevention or treatment of diseases caused by p53 mutations.
  • the mutant p53 has a mutation at a site selected from the group consisting of R175, C176, Y220, P223, C242, G245, R248, R249, R273, V274, P278, R282 or a combination thereof .
  • the mutant p53 is selected from the group consisting of mutant p53 R175 , mutant p53 C176 , and mutant p53 C242 .
  • the mutant p53 is a mutant p53 R175 .
  • the mutant p53 is mutant p53 R175H .
  • said altering (or activating) comprises inducing a mutant p53 to re-establish a conformation or function of a wild-type p53.
  • said wild-type means that the conformation or function of the mutant p53 after alteration (or activation) is similar to the conformation or function of wild-type p53 by ⁇ 90%, preferably ⁇ 95%, More preferably ⁇ 99%, optimally ⁇ 99.9%.
  • the function comprises activating phosphorylated ATM/CHK2, delaying S and G2/M phases, and/or inducing apoptosis.
  • the PEITC comprises a naturally occurring PEITC or a synthetic PEITC.
  • the naturally occurring PEITC comprises PEITC of natural food source.
  • the natural food comprises a cruciferous plant.
  • the cruciferous plant is selected from the group consisting of:
  • the PEITC is extracted from a cruciferous plant.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition is for preventing and/or treating cancer.
  • the cancer is selected from the group consisting of:
  • breast cancer pancreatic cancer, liver cancer, prostate cancer, cervical cancer, ovarian cancer, oral cancer, esophageal cancer, gastric cancer, colorectal cancer, nasopharyngeal cancer, lung cancer, bladder cancer, soft tissue sarcoma, brain tumor, lymphocyte tumor, osteosarcoma Tumor or a combination thereof.
  • the pharmaceutical composition is in the form of an injection, a suppository, an implant, an ointment, a solution, and an oral dosage form.
  • the oral dosage form comprises a tablet, a capsule, a film, an oral solution, and a granule.
  • the pharmaceutical composition comprises a sustained release dosage form, and a non-slow release dosage form.
  • the pharmaceutical composition may further comprise other anti-tumor active ingredients.
  • the pharmaceutical composition may further comprise active ingredients Nutlin, MG132, and/or Zn 2+ .
  • the pharmaceutical composition is a unit dosage form, and the content of PEITC in each unit dosage form is about 0.1 to 1 (or 0.25-1, or 0.5-1) of the daily dose, wherein the daily dose It is 5 to 500 mg, preferably 20 to 200 mg, more preferably 60 to 180 mg.
  • a p53 gene detecting reagent for preparing a diagnostic reagent or a diagnostic kit for (a) determining a PEITC therapeutic effect, and/or (b) Determine whether a tumor patient is suitable for treatment with PEITC.
  • the reagent comprises a protein chip, a nucleic acid chip, or a combination thereof.
  • the determination includes an auxiliary determination and/or a pre-treatment determination.
  • the diagnostic reagent or diagnostic kit detects a p53 gene mutation selected from the group consisting of:
  • the diagnostic reagent or diagnostic kit detects the 175th R ⁇ H mutation of the p53 gene.
  • the diagnostic reagent or diagnostic kit is for detecting a sample selected from the group consisting of a surgically removed tissue sample, a paraffin section tissue sample, a biopsy tissue sample, a blood sample, or a combination thereof.
  • the p53 gene mutation detecting reagent is selected from the group consisting of a p53 gene, a p53 protein, a p53 protein-specific antibody, or a combination thereof.
  • the p53 protein or a specific antibody thereof is conjugated with or with a detectable label.
  • the detectable label is selected from the group consisting of a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.
  • kits comprising:
  • the instructions indicate the following:
  • the instructions also specify the following:
  • a method of non-therapeutic inhibition of tumor cells in vitro comprising the steps of:
  • step (i) providing a tumor cell, detecting the mutation of the tumor cell p53 gene, if the tumor cell p53 is a mutant type, performing step (ii);
  • the concentration of the PEITC is from 1 to 100 ⁇ M, preferably from 4 to 50 ⁇ M, more preferably from 5 to 20 ⁇ M.
  • a pharmaceutical composition comprising the active ingredient (a) PEITC, active ingredient (b) Nutlin, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may further contain Zn 2+ , or MG132.
  • the pharmaceutical composition is for (a) altering (or activating) a mutant p53, (b) inhibiting proliferation of a mutant p53 tumor cell, and (c) inducing a mutant p53 tumor cell. Death, and / or (d) prevention or treatment of diseases caused by p53 mutations.
  • Figure 1 shows that PEITC inhibits cell proliferation and induces apoptosis of p53 mutant cell DU145.
  • Figure 1A shows the results of measuring the cell proliferation rate by the WST-1 method. These included DU145 cells treated with DMSO (control) or PEITC for 3 days.
  • Figure 1B shows the effect of PEITC on apoptosis. This included treatment of DU145 cells for 3 days via DMSO (control) or PEITC. Detection of histone-associated DNA fragments indicates apoptosis.
  • Figure 2 shows that PEITC inhibits mutant p53 R175- dependent cell proliferation and induces apoptosis.
  • Figure 2a respectively, with DMSO (control) or PEITC treatment with p53 hotspot gene mutations and wild-type p53 Human tumor cell line for 3 days.
  • Figure 2b shows the results of measuring the cell proliferation rate by the WST-1 method. These included SK-BR-3 cells and A549 cells transfected with siRNA for 3 days with DMSO or PEITC.
  • Figure 2c shows the effect of PEITC on apoptosis.
  • Figure 2d shows the results of measuring the cell proliferation rate by the WST-1 method. These included H1299 cells transfected with DMSO or PEITC for 3 days transfected with pcDNA3, pcDNA3-p53R175, pcDNA3-p53R273 or pcDNA3-wtp53.
  • Figure 2e shows the effect of PEITC on apoptosis.
  • Figure 3 shows the conformational change of PEITC-induced mutant p53 R175 protein into a "wild-like type”.
  • Figure 3a shows the effect of PEITC on the conformation of recombinant purified GST-p53 R175H by ELISA using PAb240 antibody (mutant) and PAb1620 antibody (wild type).
  • Figures 3b and 3c show the results of the immunofluorescence assay. This included SK-BR-3 cells treated with DMSO or 4 ⁇ M PEITC for 6 hours. Immunofluorescence of cells was performed using PAb240 and PAb1620 antibodies. The A549 cell line served as a control, indicating that the conformation of the wild type of p53 gene is not altered by PEITC. The H1299 cell line served as an anti-p53 antibody control. The threshold for all assays was limited to 20 ⁇ M. *** indicates that PAB240 is compared with PAB1620, p ⁇ 0.0001.
  • Figure 3d shows the results of immunoprecipitation of mutant p53 protein in SK-BR-3 cell lysate using PAb240 antibody and detection with p53 antibody (fl393).
  • Figure 4 shows that PEITC enables the mutant p53 R175 protein to reactivate its wild-type transcriptional activation.
  • Figure 4a shows that PEITC induces binding of mutant p53 protein to chromatin.
  • SK-BR-3 cells were treated with PEITC for 4 hours, and chromatin binding and nuclear soluble components were analyzed by immunoblotting.
  • Histone H3 and topoisomerase IIB serve as markers for chromatin and nuclear soluble components, respectively.
  • Figure 4b shows the results of RNA extraction and detection of gene expression levels using the TaqMan gene expression detection kit.
  • SK-BR-3, H1299 and A549 cells were treated with DMSO or 4 ⁇ M PEITC for 4 hours, and the p53 regulatory gene was amplified by qRT-PCR.
  • Figure 4c shows the results of RNA extraction and detection of gene expression levels using the TaqMan gene expression detection kit.
  • SK-BR-3 cells transfected with p53 siRNA or NS siRNA were treated with DMSO or 4 ⁇ M PEITC for 4 hours, and the p53 regulatory gene was amplified by qRT-PCR.
  • Figure 4d shows the results of luciferase assay.
  • plasmids 16451 were transfected with SK-BR-3, HOP92, AU565, H1299 and MEF [(10)3/175 and (10)3/273] cells, and PEITC (4 or 6 ⁇ M) was treated for 24 hours. Perform luciferase assay.
  • Figure 4e shows the results of Western blotting to determine protein levels.
  • p21 expression levels were analyzed by Western blot.
  • A549 cells were treated with 4 ⁇ M PEITC for 4 hours as a control. Protein levels were determined by Western blotting using p53DO-1 and GAPDH antibodies.
  • Figure 5 shows the results of detection of gene expression levels.
  • PEITC induced the expression of a typical p53 target gene p21 in DU145 cells.
  • DU145 cells were treated with DMSO (control) or PEITC for 4 hours.
  • RNA was extracted and the expression level of the gene was detected using a TaqMan gene expression kit.
  • FIG. 6 shows that ITCs restore the transcriptional activation function of p53.
  • Figure 6A shows the results of immunoblotting analysis of p53, p21 expression levels.
  • SCC114 cells were treated with different concentrations of DMSO (control) or ITCs for 24 hours.
  • 40 mg of total cell protein was extracted and assayed on polyacrylamide gel electrophoresis and detected with P21 antibody.
  • the blot was stripped and detected again with p53 (DO-1) antibody and anti-GAPDH antibody.
  • Figure 6B shows the results of chromatin immunoprecipitation (CHIP).
  • CHIP chromatin immunoprecipitation
  • Figure 7 shows that the proteasome degrades the p53 protein after PEITC treatment of SK-BR-3 and A549 cells.
  • Figure 7a shows the degradation of p53 protein after treatment of SK-BR-3 cells with different concentrations of phenethyl isothiocyanate and inhibitor (10 ⁇ M Nutlin-3 or 20 ⁇ M MG132) for 4 hours.
  • Figure 7b shows the degradation of p53 protein after SK-BR-3 cells were treated with PEITC (4 ⁇ M or 8 ⁇ M), 20 ⁇ M MG132, or both for 4 hours.
  • Figure 7c shows the degradation of p53 protein after SK-BR-3 cells were treated with PEITC (4 ⁇ M or 8 ⁇ M), 10 ⁇ M MG132, or both for 4 hours.
  • Figure 7d shows the degradation of p53 protein after treatment of A549 cells with different concentrations of phenethyl isothiocyanate and inhibitor (10 ⁇ M Nutlin-3 or 20 ⁇ M MG132) for 24 hours.
  • the cells were collected and cell lysates were prepared.
  • the lysate was electrophoresed by SDS-PAGE and detected with p53DO-1 antibody.
  • Figure 7e shows the degradation of p53 protein after 4 hours of treatment of SK-BR-3 cells with different concentrations of PEITC or DMSO.
  • the cells are collected to prepare soluble components and insoluble components. 30 ⁇ g of soluble or insoluble lysate was detected by SDS-PAGE and detected with p53DO-1 antibody.
  • Figure 8 shows that pPI R175 protein is autophagy by cells after PEITC treatment of SK-BR-3 cells.
  • Figure 8a shows the autophagy of SK-BR-3 cells treated with PEITC (4 ⁇ M or 8 ⁇ M), CHQ (50 ⁇ M), or both for 4 hours.
  • the lysate was electrophoresed by SDS-PAGE and detected with p53DO-1 antibody.
  • Figure 8b shows the autophagy of cells after transfection of SK-BR-3 cells with ATG5 siRNA or NS siRNA. 30 ⁇ g of the lysate was subjected to SDS-PAGE electrophoresis and detected with an anti-ATG5 antibody. After stripping the blot, re-binding with anti-GAPDH antibody.
  • Figure 8c shows the autophagy of SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA for 4 hours after treatment with DMSO or PEITC. Western blotting of eggs using p53DO-1 and GAPDH antibodies White level.
  • Figure 8d shows the results of measuring the cell proliferation rate by the WST-1 method.
  • SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA were treated with DMSO or PEITC for 3 days.
  • Figure 8e shows the effect of PEITC on apoptosis.
  • SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA were treated with DMSO or PEITC for 3 days. Detection of histone-associated DNA fragments is indicative of apoptosis.
  • Figure 9 shows the effect of zinc and redox agents on PEITC-induced reactivation of p53 R175 protein.
  • Figure 9a shows the effect of zinc on PEITC activity.
  • SK-BR-3 cells were co-treated with PEITC, zinc or both.
  • the cell proliferation rate was measured by the WST-1 method.
  • PEITC activity is expressed as the IC50 value of the growth inhibition rate.
  • Figure 9b shows the effect of co-treatment of zinc or zinc and PEITC on the conformation of recombinant purified GST-p53 R175H by ELISA using PAb240 antibody (mutant) and PAb1620 antibody (wild type).
  • Figure 9c shows the effect of PEITC on the reduction of glutathione by SK-BR-3 cells.
  • SK-BR-3 cells were treated with PEITC (4 ⁇ M or 8 ⁇ M) or DMSO for 4 hours.
  • the ratio of reducing agent GSH to oxidant GSSG was measured using a GSH/GSSG GLO glutathione kit.
  • Figure 9d shows the effect of NAC on PEITC activity.
  • SK-BR-3 was treated with different concentrations of PEITC or PEITC in combination with 3 ⁇ M NAC for 3 days.
  • the cell proliferation rate was measured by the WST-1 method.
  • Figure 9e shows the results of measuring the cell proliferation rate by the WST-1 method.
  • PEITC co-treated SK-BR-3 cells alone or in combination with 2 mM ATZ or 500 units of PEG catalase for 3 days.
  • Figure 9f shows the effect of each component on apoptosis.
  • DMSO, ATZ, NAC or PEITC were treated alone, or PEITC co-treated with untransfected or siRNA transfected SK-BR-3 cells for 3 days with ATZ or NAC.
  • Apoptosis was detected by Annexin-V staining using a BD lsrfortessa instrument.
  • FIG. 10 shows that PEITC induces H2AX foci, activates ATM and Chk2, blocks G2/M phase and S phase, and induces apoptosis.
  • PEITC or DMSO treated SK-BR-3 cells and A549 cells for 3 days, and stained with anti- ⁇ -H2AX antibody.
  • Figure 10a shows a combined image of cell anti-gamma-H2AX antibody staining (green) and DAPI (blue). Among them, the threshold of all detections was limited to 20 ⁇ M.
  • Figure 10b shows the percentage of gamma-H2AX foci cells ( ⁇ 10 or > 10, as shown).
  • Figure 10c shows the results of immunoblot analysis of SK-BR-3 cells and A549 cells treated with PEITC or DMSO for 4 hours. Among them, immunoblotting was carried out using anti-pATM S1981 and anti-pCHK2Thr68 antibodies. After blotting, the anti-ATM and anti-CHK2 antibodies recombine.
  • Figure 10d and Figure 10e show the results of flow cytometry analysis of SK-BR-3(d) or A549(e) cells after PEITC, 10 ⁇ M Nutlin-3 or co-treatment for 24 h, respectively.
  • Figure 10f shows the results of apoptosis detected by Annexin-V staining using the BD lsrfortessa instrument, including SK-BR-3 cells and A549 cells treated with 4 ⁇ M PEITC, 10 ⁇ M Nutlin-3 or co-treated for 24 hours.
  • FIG 11 shows that ITCs restore the cell cycle arrest function of the p53 mutein.
  • Figure 11A and Figure 11B show the analysis of SCC003 cells treated with BITC (5 ⁇ m or 10 ⁇ M) or DMSO (control) by flow cytometry.
  • FIG 11C and Figure 11D show the analysis of SCC003 cells treated with PEITC (5 ⁇ or 10 ⁇ M) or DMSO (control) by flow cytometry.
  • Figure 11E and Figure 11F show the analysis of SCC114 cells treated with BITC (5 ⁇ or 10 ⁇ M) or DMSO (control) by flow cytometry.
  • FIG 11G and Figure 11H show the analysis of SCC114 cells treated with PEITC (5 ⁇ or 10 ⁇ M) or DMSO (control) by flow cytometry.
  • Figure 12 shows that ITCs induce mutant p53-dependent cell cycle arrest.
  • Figure 12A shows the results of immunoblot analysis of DMSO control group (untransfected), non-specific siRNA (N) group and mtp53 siRNA (P) group SCC114 cells cultured for 24 hours, 48 hours and 72 hours. Among them, the expression of p53 protein in the mtp53 siRNA transfected group was decreased after transfection, but not in the control group.
  • Figure 12B shows the results of the WST-1 assay to determine the proliferation rate of siRNA transfected cells. Among them, mutant p53 cells were able to proliferate after treatment with PEITC/BITC.
  • Figures 12C, 12D, and 12E show the results of flow cytometry analysis.
  • DMSO control group untransfected
  • N non-specific siRNA
  • P mtp53 siRNA
  • Figures 12F, 12G, and 12H show the results of flow cytometry analysis.
  • DMSO control group untransfected
  • non-specific (scrambled) siRNA (N) group and mtp53 siRNA (P) group transfected SCC114 cells were treated with PEITC (5 ⁇ M or 10 ⁇ M) or DMSO (control), and then flowed. Cell technology analysis.
  • Figure 13 shows that PEITC induces the reactivation of mutant p53 R175 protein in vivo and inhibits the growth of xenografts in nude mice.
  • Figure 13a shows a typical image of a mouse mammary fat pad (top panel) and H&E staining (bottom panel). The threshold for all assays was limited to 20 ⁇ M.
  • Figure 13c shows the change in body weight per week.
  • Figure 13f shows the results of immunoblot analysis of p53 expression levels in the PEITC group and the control group in nude mice xenografts.
  • the blot is a representative picture of 12 tumor tissue lysates per group.
  • Figure 13h shows an immunoblot of p21 and Bax in SK-BR-3 swollen xenografts.
  • Figure 14 shows that PEITC inhibits the growth of transplanted tumors of DU145 cells in nude mice.
  • Figure 14A shows a representative image of the side of the mouse.
  • Figure 14C shows the change in body weight (grams) per week.
  • WT indicates wild type
  • WB indicates immunoblotting
  • WCL indicates whole cell lysate
  • AIN-93M indicates AIN-93M standard feed.
  • PEITC can restore mutant p53 to its wild-type activity (activate wild-type p53 activity), inhibit tumor cell proliferation induced by p53 mutation, and induce apoptosis.
  • mutant p53 transformation especially mutant p53 R175 , p53 P223L , p53 V274F , and p53 R248 re-establish the conformation or function of wild-type p53, thereby restoring activation of wild-type p53 targets, such as phosphorylated ATM /CHK2, blocks S and G2/M phases, induces apoptosis.
  • the present invention has been completed on this basis.
  • PEITC can restore the wild-type conformation and transcriptional activation function of mutant p53 with missense mutations in hot spot mutations R175, C176, Y220, P223, C242, G245, R248, R249, R273, V274, P278, R282, and induce expression Apoptosis of hot mutant mutant p53 cells.
  • PEITC can inhibit mutant p53 R175- expressing breast cancer cells SK-BR-3, AU565, mutant p53 R248- expressing oral cancer cell SCC114 and mutant p53 P223L or p53 V274F- expressing prostate cancer cell DU145 proliferation.
  • mutant p53 R175 breast cancer cells SK-BR-3, AU565, expressing mutant p53 P223L or p53 V274F DU145 prostate cancer cells expressing mutant oral cancer cells in SCC114 p53 R248, can be of PEITC
  • the mutant conformation transforms into a wild-type conformation, revives transcriptional activation, activates wild-type p53 targets, such as phosphorylation of ATM/CHK2, blocks S and G2/M phases, and induces apoptosis.
  • PEITC can increase the sensitivity of breast cancer cell SK-BR-3, AU565 mutant p53 R175 protein to the degradation of proteasome and autophagy.
  • Zinc ion can enhance the reactivation of PEITC-induced breast cancer cell SK-BR-3 and AU565 mutant p53 R175 .
  • Redox changes are important for reactivation of p53 R175 and inhibition of growth, but not for restoration of p53 R175 conformation.
  • the term "conformational and/or functional of wild-type p53 (wt-like p53)” refers to a conformation and/or function substantially possessing wild-type p53, a conformation of mutant p53 after alteration (activation) and/or Or the degree of similarity to the conformation and/or function of wild-type p53 is > 90%, preferably > 95%, more preferably > 99%, optimally > 99.9%.
  • the conformation and/or function of wild-type p53 is the conformation and/or function of human wild-type p53.
  • altering mutant p53 means altering (reactivation) a mutant p53 to restore its wild-type activity and/or conformation (activating wild-type p53 activity), inhibiting proliferation of tumor cells caused by p53 mutation and Inducing its apoptosis.
  • p53 and "p53 gene” are used interchangeably and refer to the tumor suppressor gene p53, a mutation in the p53 gene which is a very common phenomenon in human cancer. Most p53 gene mutations are missense mutations and can be further subdivided into contact mutations (which directly disrupt p53 binding to DNA) and conformational mutations (destruction of p53 conformation). Both of these mutations result in the inactivation of normal wild-type p53. Studies have shown that certain specific small molecules can achieve tumor suppressive function by altering (activating) mutant p53 for the treatment of cancer.
  • the p53 gene is localized to human chromosome 17p13.1, which encodes a 53 kD nuclear-phosphorylated protein consisting of 393 amino acids, which is called p53 protein.
  • the wild-type p53 protein is extremely unstable, has a half-life of only a few minutes, and has a transactivation function and a broad-spectrum tumor suppressive effect. Mutant p53 protein has increased stability and extended half-life and can be detected by immunohistochemistry.
  • wtp53 wild-type
  • HDM2/MDM2 degrades p53 through transcriptional repression and E3 function.
  • hdm2/mdm2 is the target gene of p53.
  • This negative feedback mechanism formed by p53-HDM2/MDM2 maintains wtp53 activity at a lower level in the cell.
  • p53 has been known to regulate more than 150 genes, forming a fine and complex p53 regulatory network, which plays an important role in maintaining genomic stability.
  • p53 is closely related to the development of cancer, and there are p53 mutations in about 50% of human cancers.
  • the dominant mutations caused by point mutations account for about 80% of the total.
  • the point mutation rate occurring in the DBD region was as high as 97%.
  • every amino acid in the DBD region of p53 can be mutated to form a corresponding mutant, but mutations in the following 12 sites occur at high frequency in cancer.
  • hot spot mutations they are: R175, C176, Y220, P223, C242, G245, R248, R249, R273, V274, P278, R282.
  • PEITC Phenethyl isothiocyanate
  • phenethyl isothiocyanate phenethyl isothiocyanate
  • watercress cruciferous vegetables
  • PEITC Animal experiments have shown that PEITC has chemopreventive effects on cancer, and epidemiological studies support the efficacy of such compounds against human cancer. In fact, PEITC has completed Phase I clinical trials for lung cancer prevention in healthy populations, and Phase II clinical trials are underway. PEITC-induced oxidative stress contributes to apoptosis, however, the exact mechanism and molecular targets of this effect are not well understood.
  • PEITC can alter (reactivate) mutant p53 in vitro and in vivo, revealing a novel mechanism of action of natural food-derived compounds.
  • PEITC has a stronger inhibitory activity on p53 mutants, such as p53 R175 , p53 R248 , p53 P223, and p53 V274 , with the most common "hot spot" mutations.
  • p53 R175 has the strongest inhibitory activity.
  • Mechanistic studies have shown that PEITC induces apoptosis of mutant p53 R175 by restoring p53 wild-type conformation and transcriptional activation.
  • the altered mutant p53 R175 induces apoptosis by activating the wild-type p53 target, ie, phosphorylating ATM/CHK2, delaying the S and G2/M phases. Further studies of the mechanism indicate that this growth inhibition of PEITC is affected to some extent by the concentration of zinc ions and the redox state of cells.
  • PEITC sensitizes the p53 R175 mutant to degradation by proteasome and autophagy, and its sensitivity is related to concentration. PEITC induces changes in p53 R175 and increases its sensitivity to degradation pathways, which is likely to contribute to its anticancer activity.
  • PEITC is the only compound found to treat tumors with a pure natural food source targeting mutant P53;
  • PEITC is a food-derived compound, its safety is very high. Moreover, PEITC can be used not only as a drug for treating tumors, but also as a health care product for preventing tumors;
  • PEITC is not susceptible to drug resistance even after long-term use, and for other anti-tumor drugs Tumor patients who have developed drug resistance can still achieve good results with PEITC treatment;
  • PEITC can be used alone or in combination with other anti-tumor drugs
  • PEITC can also have a good therapeutic effect on other diseases caused by P53 mutation
  • HOP92, OVCAR3 and SW620 were purchased from NCI DTP, DCDT Tumor Repository, Fredrick, Maryland.
  • H1299, HT29, A549, MDA-MB-231, AU565, SK-BR-3, DU145, SCC003, SCC016, SCC114, SCC122, and MCF7 are from Tissue Culture Source Resource, Georgetown University, Washington, DC. All cell lines were mycoplasma negative and were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS).
  • Normal colon cell CCD841 purchased from ATCC was cultured in Eagle's basal medium containing 10% FBS.
  • 3T3Balb/c fibroblasts (p53 +/+ ) were cultured in Dulbecco's modified Eagle's medium containing 10% FBS. (10) 3 (p53 -/- ) mouse embryonic fibroblasts (MEFs) and (10) 3 derived p53 mutant MEFs [(10) 3/175 and (10) 3/273] in 10% FBS Incubate in Dulbecco's modified Eagle's medium with 400 ⁇ g/mL G418. MEF (10) 3 and its derived human p53 residues R175 and R273 mutant cell lines were presented by Dr. Spotify R. Carpizo.
  • PEITC effect of PEITC on tumor cell proliferation was determined by the WST-1 test (Roche). Briefly, an appropriate amount of PEITC was diluted with DMSO to allow 10 ⁇ l of the drug stock to contain the desired drug concentration in a final volume of 1 ml of SK-BR-3 cells (40000 cells/ml) and a DMSO concentration of 1%. .
  • the PEITC-containing SK-BR-3 cell culture was added to a 96-well microplate at 4000 cells per well. 1% DMSO was used as a control, and a cell-free medium was used as a blank control. The plate was incubated at 37 ° C for 3 days, followed by incubation with WST-1 reagent for 2 hours.
  • PEITC ratio in treated cells and control cells OD 450 value of OD 450 value of the percentage of cell proliferation is calculated DMSO.
  • a similar test method was used to determine the effect of PEITC on cell proliferation: p53 siRNA or NS siRNA transfected H1299, HOP92, AU565, OVCAR3, SW620, HT29, A549, MCF7, CCD841, SK-BR-3, DU145, SCC003 , SCC016, SCC114, SCC122 cells.
  • siRNA was purchased from SMARTpool (Thermo Scientific/Dharmacon, Lafayette, CO, USA). siRNA was transfected with Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Briefly, before transfection, cells were cultured for 24 hours in a 10 cm dish with a cell confluence of 50-60%. siRNA (0.430 nmol) and 43 ⁇ L Lipofectamine 2000 were taken and mixed with 1 mL of Opti-MEM (Invitrogen). After the mixture was added to the cell culture medium, it was cultured for 6 hours. After 24 hours, a second similar transfection was performed.
  • the pcDNA3, pcDNA3-wtp53, pcDNA3-p53 R175 and pcDNA3-p53 R273 plasmids were transfected with Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Briefly, before transfection, cells were cultured for 24 hours in a 10 cm dish with a cell confluence of 50-60%. Plasmid (14 ⁇ g) and 43 ⁇ L of Lipofectamine 2000 were mixed with 1 mL of Opti-MEM (Invitrogen). The mixture was added to the cell culture medium and cultured for 6 hours. After 24 hours, the transfected cells were treated with PEITC and subjected to WST-1 assay or Annexin V staining as described above. Transfected cells were maintained in RPMI 1640 medium containing 10% FBS and 400 ⁇ g/mL G418.
  • the pGeX4T1-mutp53 R175H plasmid-transformed E. coli cells ( BL21DE3) were cultured in LB medium containing 100 ⁇ g/mL ampicillin at 37 ° C until the OD value at 600 nm was 0.4. At the same temperature, 0.5 mM isopropyl-1-thio- ⁇ -galactosidin (IPTG) was added, and the flask was continuously shaken for 3 hours to induce expression of the recombinant protein.
  • IPTG isopropyl-1-thio- ⁇ -galactosidin
  • lysis buffer 250 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 0.1% Tween 20, 0.2% SDS.
  • the cells were lysed by 1 M DTT and protease inhibitors and repeatedly thawed three times and then sonicated (three cycles, one minute each). After sonication, the mixture was centrifuged at 18,500 x g for 30 minutes at 4 ° C, and the solution became clear. Transfer the supernatant to a new tube and store.
  • the pellet was resuspended in sodium lauryl sarcosinate buffer (lysis buffer + 2% sodium lauryl sarcosinate) and sonicated with a probe (three cycles, one minute each).
  • the supernatant fraction obtained in the above two steps was diluted in a ratio of 1:1 with 1 ⁇ PBS, and then incubated with glutathione-agarose beads ( ⁇ , G4510) at a constant rate at 4 ° C for 2 hours.
  • the protein was eluted with an elution buffer (100 mM Tris-HCl, pH 8.0, 10 mM GSH ( ⁇ , G4251), 300 mM NaCl, 1 mM dithiothreitol (DTT) and protease inhibitor).
  • an elution buffer 100 mM Tris-HCl, pH 8.0, 10 mM GSH ( ⁇ , G4251), 300 mM NaCl, 1 mM dithiothreitol (DTT) and protease inhibitor.
  • Another 25 ng of recombinant GST-mutant p53 R175H was diluted with 100 mM Tris-Cl, 300 mM NaCl buffer, pH 7.5. Treat with DMSO or 4 ⁇ M PEITC and incubate for 1 hour on ice. The resulting protein samples were added to an ELISA plate and incubated for 2 hours at 4 °C.
  • 1x PBST wash (containing 0.05% Tween-20), blocked with 5% skim milk for 4 hours at 4 °C.
  • the mouse primary antibody PAB240 or PAB1620
  • a horseradish peroxidase-labeled anti-mouse secondary antibody was added at 4 ° C for 1 hour.
  • 1x PBST was washed, substrate (SuperSignal ELISA Pico Chemiluminescent Substrate, Thermo Scientific) was added, and chemiluminescence intensity was measured at 450 nM.
  • Annexin V staining was performed according to the manufacturer's instructions (Biolegend). Briefly, cells to be tested were treated with PEITC or DMSO as a control, and cells were collected 3 days later, washed once with 1 x PBS, and resuspended in 0.5 mL of Annexin V binding buffer. The cells were collected by centrifugation, 5 ⁇ L of Annexin V-conjugated fluorescent dye was added to the residual buffer, and incubated for 15 minutes at room temperature in the dark, followed by 0.5 mL of Annexin V binding buffer and 5 ⁇ L of 0.1 ⁇ g/mL of PI staining solution. The cells were analyzed by BD LSRFORTESSA flow meter (BD Biosciences).
  • cells to be tested were treated with the indicated concentrations of PEITC, Nutlin-3 alone, or both, or DMSO for 24 or 72 hours.
  • the treated cells were stained with Annexin V as previously described.
  • the cells to be tested are treated with PEITC, a reducing agent or an oxidizing agent alone, or with PEITC in combination with a reducing agent or an oxidizing agent.
  • the treated cells were stained with Annexin V as previously described.
  • ATG5 siRNA or NS siRNA transfected cells were treated with DMSO or the indicated concentrations of PEITC for 72 hours.
  • a cytoplasmic histone-associated DNA fragment in the apoptotic phase was quantified using a cell death assay ELISA in combination with photometric enzyme assay (Roche).
  • the cells to be tested were treated with PEITC (4 or 6 ⁇ M) or 1% DMSO as a control in a four-well slide (Lab-Tek) for 6 hours.
  • the cells were washed twice with 1 x PBS and then fixed with formaldehyde (3.7%) for 15 minutes at room temperature (RT).
  • the fixed cells were treated with 0.5% Triton X-100 ( ⁇ ) for 5 minutes at room temperature.
  • the cells were washed four times with 1 x PBS containing 0.5% Tween-20 and blocked overnight with 10% goat serum ( ⁇ ) at 4 °C.
  • Immunofluorescence analysis was performed using a Zeiss LSM 510 NLO with a Plan-Apochromat 63 x 1.4-aperture oil mirror and an Axiovert 200M inverted laser scanning microscope. Images were acquired with a Photomultiplier Tubes (PMT) detector and analyzed by Image J software. Fluorescence staining intensity was quantified using Metamorph software.
  • PMT Photomultiplier Tubes
  • p53 siRNA or NS siRNA The transfected cells to be tested were treated with 4 ⁇ M PEITC or 1% DMSO as a control at 37 ° C for 3 days.
  • a mouse anti- ⁇ -H2AX monoclonal antibody (1:300, Upstate) was used as a primary antibody, and the cells were fixed with formaldehyde, and ⁇ -H2AX was detected by immunostaining.
  • Cells to be tested were treated with PERTC at the indicated concentrations or 1% DMSO as control for 6 hours.
  • To prepare cell lysates the harvested cells were washed once with 1 ⁇ PBS, using a lysis buffer containing protease inhibitor (Roche Molecular Biochemicals) (20 mM Tris-Cl (pH 8.0), 137 mM sodium chloride, 10% glycerol, 1
  • the cell pellet was resuspended in %NP-40, 2 mM EDTA and incubated on ice for 30 minutes.
  • the cell suspension was centrifuged at 18,500 x g, 4 ° C for 10 minutes in a centrifuge, and the supernatant was collected.
  • the supernatant was diluted with lysis buffer, and an appropriate amount of proteinG Agarose (Roche) was added to 200 ⁇ g of the lysate and gently shaken at 4 ° C for 1 hour.
  • the pre-purified lysate was gently shaken for 2 hours at 4 ° C by adding mouse-derived PAB240 antibody (2 ⁇ g, Calbiochem).
  • ProteinG Agarose- was then added to the suspension and incubated for 2 hours at 4 °C.
  • the pellet was washed four times with a lysis buffer supplemented with a protease inhibitor, and the immunoprecipitate was eluted by boiling in Laemml i buffer, followed by 4-12% SDS-PAGE electrophoresis.
  • the immunoprecipitated p53 protein was detected by immunoblotting using FL393 (Santa Cruz Biotechnology) as a primary antibody.
  • the secondary antibody was a peroxidase-labeled anti-mouse IgG (1:2000, GE healthcare).
  • Western blots were detected using the ECL Prime Western Blot assay kit according to the manufacturer's instructions (Amersham). As a control, the blot was removed and re-detected with anti-p53 (DO-1) antibody (1:1000, Santa Cruz Biotechnology) or anti-GAPDH antibody (1:2000, Novus Biologicals).
  • the density of the p53 bands in the PEITC-treated samples relative to the DMSO control samples was determined using the Gene Tools software.
  • lysates were used to prepare soluble, insoluble, and whole cell lysing components.
  • the cells were washed twice with 1 x PBS and the cells were collected for preparation of lysate (soluble).
  • RIPA buffer (10 mM sodium phosphate (pH 7.2), 300 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate, 2 mM EDTA) was added to the cells and allowed to stand in an ice bath for 30 minutes. The cell suspension was then centrifuged at 18,500 x g for 10 minutes at 4 ° C, and the supernatant was collected unless otherwise mentioned.
  • the precipitate is an insoluble component.
  • the insoluble fraction was dissolved in a lysis buffer containing 2% SDS (65 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2% SDS, 50 mM DTT).
  • the harvested cell pellet was dissolved in 2% SDS lysis buffer as described above to prepare a whole cell lysing fraction.
  • the fraction was centrifuged at 18,500 x g for 4 minutes to collect the components therein. Then 30-50 ⁇ g of lysate was taken for 4–12% SDS/PAGE electrophoresis. Proteins were transferred to PVDF membranes and Western blot assays were performed according to the manufacturer's (Amersham) instructions ECL Prime Western blot.
  • Antibodies for p21, Bax, ATM, pATM S1981, CHK2, pCHK2Thr68 and p53 (DO-1) were purchased from Santa Cruz Biotechnology, respectively, and GAPDH antibodies were purchased from Novus Biologicals.
  • ATG5 (1:1000, cell signaling) antibodies were donated by Dr. Shivendra Singh.
  • the cells to be tested were treated with the indicated concentration of PEITC or DMSO as a control for 4 hours, respectively.
  • the cells were trypsinized and collected by centrifugation at 500 xg for 5 minutes.
  • the cell pellet was washed once with ice-cold PBS and transferred to a 1.5 mL microcentrifuge tube and centrifuged at 500 xg for 2 minutes.
  • the cell pellet was stored at -80 ° C and the chromatin-soluble fraction and chromatin-binding protein fraction were separated in the chromatin according to the manufacturer's instructions (Subcel lular protein fractionation kit, Thermo Scientific).
  • the cells to be detected were extracted with the Qiagen RNA extraction kit, and cDNA was synthesized using a high-capacity RNA-cDNA conversion kit (Applied Biosystems, Invitrogen), and quantified by TaqMan RT-PCR (qRT-PCR) (Applied Biosystems, Invitrogen). The gene expression level was determined. Normalized by GAPDH, the results were expressed as the mean and the standard deviation of the three replicates. RNA of allograft tumor tissues was also extracted with QIAGEN kit, followed by qRT-PCR, and gene expression levels were determined by GAPDH normalization. The fold change in the expression level of each tumor gene in the PEITC treatment group and the control group was calculated and expressed as mean and standard deviation.
  • GSH reduced glutathione
  • GSSG oxidized glutathione
  • GSH/GSSG-GLO glutathione detection kit Promega. Briefly, cells to be tested were treated with PEITC or DMSO as a control for 4 hours, and cells were treated according to the manufacturer's (Promega) instructions to determine glutathione.
  • the cell lysate transduction gene luciferase activity was assayed according to the manufacturer's (Luciferase assay, Promega) instructions.
  • the cells to be tested were treated with PEITC, Nutlin-3 or both for 24 or 72 hours, respectively, with DMSO as a control.
  • the collected cells were subjected to cell cycle detection analysis by flow cytometry. Briefly, the cells were washed with PBS containing no calcium or magnesium ions, trypsinized for 5 minutes, and the cells were collected and centrifuged at 190 x g for 3 minutes at 4 °C. The collected cells were washed once with PBS, resuspended in 1 ml of 70% ethanol, and stored at 20 ° C overnight. Then, at 420 ⁇ g, after centrifugation for 10 minutes, the precipitated cell pellets were collected and pre-cooled with 1 ml of ice.
  • the PBS was washed once and resuspended in 1 ml of freshly prepared PI staining solution (PBS containing 0.1% Triton X-100, 0.05 ⁇ g/mL propidium iodide, 0.1 mg/mL RNase (Sigma)).
  • PBS PI staining solution
  • the cell suspension was first placed in the dark at room temperature for 30 minutes and then placed at 4 ° C for 30 minutes. Samples were tested with a Becton Dickinson FACS and analyzed by the Mod Fit program (Verity Software House).
  • the cells to be tested were treated with DMSO, ATZ, NAC, PEG-catalase or a single PEITC or with ATZ or NAC or PEG-catalase for 4 hours in combination with PEITC.
  • the cells were then collected at 1600 x g, 4 ° C, and centrifuged for 10 minutes, washed once with PBS, and resuspended in RIPA buffer containing a mixture of protease and phosphatase inhibitor (10 mM sodium phosphate (pH 7.2) 300 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate, and 2 mM EDTA) were placed on ice for 30 min, centrifuged at 18500 x g for 10 minutes at 4 ° C, and the supernatant was collected.
  • protease and phosphatase inhibitor 10 mM sodium phosphate (pH 7.2) 300 mM NaCl, 0.1% SDS, 1% Nonidet P
  • mice Twenty female athymic nu/nu BALB/C mice (CAnN.Cg-Foxn1nu/Crl, 4-6 weeks old) were purchased from Charles River Laboratories (Wilmington, MA). All in vivo studies and tumor collections are in accordance with the procedures and guidelines of the Laboratory Animal Protection and Use Committee (IACUC). The mice were weighed and placed in a polycarbonate cage (five/cage, with the same average body weight and variance for each cage) for one week. The mice were given water ad libitum and the feed was AIN-93M.
  • IACUC Laboratory Animal Protection and Use Committee
  • mice feed is added every other day.
  • 2 ⁇ 10 6 tumor cells (suspended in 50 ⁇ L Matrigel) in the exponential growth phase were injected into the mice and left and right after one week of feeding and PEITC mice.
  • Breast fat pad position ("cancer chemoprevention" setting). No mice died during the experiment.
  • Tumor size was measured externally using a vernier caliper weekly to assess tumor formation and growth, and 10 bioassay cycles were continued.
  • the tumor volume was calculated according to the formula L x W 2 x 0.523.
  • H&E hematoxylin and eosin
  • H&E stained tissue section pathology was used to determine if it was a tumor. Immunohistochemistry was performed according to the Georgetown University Histopathology and Tissue Shared Resources standard. Briefly, the tissue was cut into 5 ⁇ m slices, dewaxed with xylene and dehydrated with a gradient alcohol, and the tissue sections were immersed in 10 nM citrate buffer (pH 6) containing 0.05% Tween at 98 ° C for 20 minutes. Heat-induced antigen retrieval (HIER). Immunohistochemical staining was performed using Dako's horseradish peroxidase-labeled polymer (k4001, k4003) according to the manufacturer's instructions.
  • HIER Heat-induced antigen retrieval
  • the sections were microscopically examined at a magnification of 200 times under an Olympus BX61 microscope. Representative images of whole tumor tissue sections were photographed using a DP70 camera and processed with DP70 software. Images were analyzed using Image J software. In addition, due to the small tumor volume, four sections were used for each tumor analysis to determine the number of cells. The sections stained with each antibody were taken in different regions to take twenty pictures, and the total number of cells was counted. The data for each tumor is expressed as the average of the total number of cells stained with different antibodies.
  • tumors were randomly grouped for western blotting and qRT-PCR analysis.
  • PEITC can reduce the proliferation of various mutant p53 cells.
  • PEITC had the strongest inhibitory effect on human breast cancer cells SK-BR-3, AU565 and human non-small cell lung cancer HOP92 expressing p53 R175 mutant. These tumor cells as compared to other hot PEITC the IC 50 mutant cells decreased approximately 2.5-5 fold ( Figure 2a). However, no significant inhibition of proliferation was observed in cells treated with PEITC expressing wild-type p53.
  • PEITC targets the mutant p53 as a target to inhibit the proliferation of tumor cell lines.
  • PEITC inhibits tumor cell proliferation and induces apoptosis in a mutant p53-dependent manner
  • 1.PEITC inhibits the proliferation of breast cancer cell SK-BR-3 and induces apoptosis in a p53 R175 or p53 R248 -dependent manner.
  • PEITC inhibits proliferation and induces apoptosis of prostate cancer cell line DU145 in a p53 P223 or p53 V274 -dependent manner.
  • PEITC restores "wild-type" conformation and transcriptional activation of mutant p53
  • FIG. 4a is a nuclear chromatin binding portion of SK-BR-3 cells treated with PEITC and the results show a dose-dependent increase in p53 R175 .
  • 4 ⁇ M of PEITC increased the expression of p53 target genes in SK-BR-3 cells, particularly p21, MDM2, PUMA, NOXA, BCL2, BAX (Fig. 4b).
  • No significant changes were observed in SK-BR-3 cells treated with PEITC for reduced expression of A549, H1299 or p53 R175 . This indicates that the PEITC-induced p53 target is p53 R175 -dependent (Fig. 4b and Fig. 4c).
  • PEITC acts on prostate cancer cell line DU145 mutant p53 P223L or p53 V274F to restore its "wild-type" conformation and transcriptional activation
  • PEITC is capable of inducing DU145 cells expressing mutant p53 protein Apoptosis, we conclude that it is also a pathway that restores the wild-type function of p53. Therefore, we investigated its effect on the expression of the p53 target gene p21. The results showed that DU145 cells treated with PEITC (8 ⁇ M) did enhance the expression of p21 (Fig. 5). This result indicates that PEITC is able to restore the "wild-type" conformation and transcriptional activation of p53 mutants.
  • the present invention conducted a luciferase reporter assay.
  • the results showed that luciferase activity was increased by about 2-2.5 fold in cells treated with 4 ⁇ M PEITC (Fig. 4d).
  • PEITC (4 ⁇ M) is capable of inducing expression of the p21 gene in SK-BR-3 cells, whereas the DNA damaging agent, etoposide, cannot be achieved (Fig. 4e). This suggests that this induction is mutant p53 dependent.
  • PEITC degrades p53 R175 protein expressed by breast cancer SK-BR-3 cells by proteasome and autophagy
  • Examples 1-3 show that PEITC ( ⁇ 10 ⁇ M) selectively degrades the mutant p53 protein, but not the wild-type p53 protein. Since PEITC is able to restore p53 R175 to a "wild-type" state, whereas wild-type p53 is regulated by MDM2, the decreased stability of p53 R175 after recovery may be due to degradation of the MDM2-dependent proteasome resulting in ubiquitinated protein. The accumulation of dissolved parts. To verify this, SK-BR-3 cells were treated with PEITC, the proteasome inhibitor MG132, or a specific MDM2 inhibitor, Nutlin-3, alone or in combination.
  • the present invention further investigates the effect of PEITC on autophagy of SK-BR-3 cells.
  • the results showed that treatment of SK-BR-3 cells with 8 ⁇ M PEITC and 50 ⁇ M autophagy inhibitor chloroquine (CHQ) significantly increased the content of p53 in WCL compared with cells treated with PEITC alone.
  • CHQ chloroquine
  • Zinc-enhanced PEITC induces reactivation of p53 R175 in breast cancer SK-BR-3 cells
  • Redox changes are important for altering p53 R175 and inhibiting tumor cell proliferation, but not for restoring p53 R175 conformation
  • PEITC induces the production of reactive oxygen species by inactivating the glutathione antioxidant system in cancer cells.
  • the redox changes affect the conformation of the wild-type p53 protein.
  • the results of glutathione expression levels showed that glutathione levels were decreased in SK-BR-3 cells treated with PEITC (4 or 8 ⁇ M) compared to the DMSO control group (Fig. 9c).
  • FIG. 10 is a comparison of pATM-S1981 and pCHK2/Thr68 in SK-BR-3 cells treated with 4 ⁇ M PEITC and DMSO control cells. The results indicate that ATM/CHK2 inhibition in the absence of p53 R175H results in reactivation of the DNA damage response.
  • Treatment of A549 cells without pATM-S1981 and pCHK2-Thr68 with PEITC was consistent with gamma-H2AX focus data ( Figures 10a and 10b). This suggests that PEITC restores DNA damage repair depending on the redox state of the cells.
  • a modified trial of the effect of mutant p53 R175 on cell cycle progression revealed that treatment of SK-BR-3 cells with 4 ⁇ M PEITC for 24 hours significantly delayed G2/M and S phases (Fig. 10d); this indicates that PEITC inhibits cell proliferation, not only delaying G2 /M period, also delayed S period.
  • A549 cells were treated with 4 ⁇ M PEITC for 24 hours, and the G1 phase was postponed. This indicates that the delay in cell cycle progression is associated with p53 R175 .
  • SK-BR-3 cells co-treated with 10 ⁇ M Nutlin-3 and 4 ⁇ M PEITC for 24 and 72 hours compared with PEITC or Nutlin-3 alone showed a significant increase in the number of S phases (Fig.
  • PEITC induces G1 phase cell cycle arrest by mutant p53 R248 acting on oral cancer cell line SCC114
  • SCC114 cells expressing mutant P53 were able to restore the cells from the G1 phase to the S phase after treatment with different concentrations of PEITC (P ⁇ 0.05) (Fig. 11E, 11F, 11G, 11H).
  • the difference between the experimental group and the control group was not statistically significant (P>0.05) (Fig. 11A, 11B, 11C, 11D).
  • the results showed that PEITC induced cell cycle arrest in G1 phase was dependent on p53 R248 mutein.
  • the inventors further analyzed the cell cycle by transfecting SCC114 cells with siRNA to determine whether mutant p53 plays a role in restoring G1 phase arrest. Flow cytometry and immunoblot analysis showed that when the mutant P53 gene was silenced by siRNA, cell cycle arrest was no longer observed (Fig. 12).
  • PEITC inhibits the growth of allograft tumors by changing mutant p53 proteins of different hot spots
  • Altering mutant p53 by transactivation provides a promising direction for tumor-targeted therapy. Alterations in mutant p53 have been demonstrated in mouse models of different tumors, respectively. According to the literature, artificially designed small molecules have been reported to restore the reverse transcription of mutant p53. However, no studies have been published on the alteration of mutant p53 by natural food-derived compounds. Studies of the present invention have shown that PEITC selectively degrades mutant p53 protein without affecting the wild type. Specifically, PEITC inhibits cell proliferation and induces apoptosis by altering mutant p53 R175 , resulting in selective clearance of such cells. This is a new mechanism by which natural food-derived compounds induce apoptosis.
  • PEITC may aggravate PEITC-induced oxidative stress by increasing reactive oxygen species in mutant p53 cells. Although induction of reactive oxygen species had no effect on restoring the p53 R175H conformation, increasing oxidative stress helped restore p53 R175 activity and induce apoptosis. ATZ enhanced the anti-proliferative ability of PEITC, while PEG-catalase or NAC played an inhibitory role, which provided evidence for the above conclusions from both positive and negative aspects.
  • PEITC a natural food source
  • the results of the analysis showed that the concentration of ITC in the blood samples fed PEITC mice was 1.13 ⁇ 0.15 ⁇ M, which was related to the results of human pharmacokinetics--approximately 50 g of raw watercress (about 40 mg of PEITC) volunteers.
  • the peak concentration of PEITC in plasma was approximately 1 ⁇ M.
  • the inhibitory effect of PEITC on tumors was confirmed by the "chemoprevention setting" method. Specifically, the test animals were fed a feed containing PEITC prior to injection of mutant p53 cells and tumor formation. At the same time, the injected mutant p53 cells were classified into two types: "starting phase” or "cancerousized”. The results showed that the mutant p53 was degraded, inhibited in proliferation and significantly reduced in tumor volume in mice fed PEITC. In addition, elevated p53 target gene mRNA provides evidence of reactivation of p53 R175H in mice fed PEITC feed.
  • the present invention clarifies the novel mechanism by which PEITC prevents and treats cancer.
  • Mutations in the p53 gene may occur at different stages of cancer, such as in the early stages of breast cancer (DCIS, Ductal carcinoma in situ) and liver cancer, late stages of pancreatic cancer, hepatocellular carcinoma, prostate cancer, and the like.
  • the DCIS stage is the early stage of development of invasive breast cancer.
  • the role of PEITC in this phase of the p53 mutant gene is fully applicable to the prevention and early intervention of breast cancer.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Oncology (AREA)
  • Organic Chemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • Veterinary Medicine (AREA)
  • Emergency Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Public Health (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Provided in the present invention is a use of PEITC for preparing a preparation or composition. The preparation or composition is used for: (a) altering the mutant p53 to restore the wild-type activity; (b) inhibiting the tumour cell proliferation caused by the mutant p53; (c) inducing apoptosis of mutant p53 tumour cells; and/or (d) preventing or treating diseases based on p53 mutations. Also provided in the present invention are a use of a p53 gene detection reagent, a kit comprising PEITC and the p53 gene detection reagent, and a method for non-therapeutically inhibiting tumour cells in vitro.

Description

含PEITC的药物组合物及其在癌症治疗中的应用Pharmaceutical composition containing PEITC and its application in cancer treatment 技术领域Technical field
本发明涉及药物领域，更具体地涉及含PEITC的药物组合物及其在癌症和其他基于p53突变引起的疾病的预防与治疗中的应用。The present invention relates to the field of medicines, and more particularly to pharmaceutical compositions containing PEITC and their use in the prevention and treatment of cancer and other diseases caused by p53 mutations.
背景技术Background technique
本专利申请是CN200510040865.1、CN200610126892.5、CN200910052231.6、CN201310205609.8、CN201310352414.6、CN201310364101.2和CN201410346419.2以及US8039511B2、US8410170B2、EP06817815.1、CA2630262和JP5308160的后续专利。The present patent application is a subsequent patent of CN200510040865.1, CN200610126892.5, CN200910052231.6, CN201310205609.8, CN201310352414.6, CN201310364101.2 and CN201410346419.2, and US8039511B2, US8410170B2, EP06817815.1, CA2630262 and JP5308160.
癌症(Cancer)，是威胁人类健康的重大疾病。根据《全球癌症报告2014》，2012年全世界共新增1400万癌症病例并有820万人死亡。其中，中国新增307万癌症患者并造成约220万人死亡，分别占全球总量的21.9％和26.8％。Cancer is a major disease that threatens human health. According to the Global Cancer Report 2014, there were 14 million new cancer cases worldwide and 8.2 million deaths in 2012. Among them, China added 3.07 million cancer patients and caused about 2.2 million deaths, accounting for 21.9% and 26.8% of the global total, respectively.
虽然由于流行病学的进步，早期诊断和治疗方法的进一步改良，肿瘤的治疗和预防近些年来已取得显著的成就，然而肿瘤仍然是威胁人民生命的主要疾病之一。发掘新型高效低毒的抗癌、防癌新药，仍然是药物研究的重要课题。Although advances in early diagnosis and treatment have been further improved due to advances in epidemiology, and the treatment and prevention of cancer has made remarkable achievements in recent years, tumors are still one of the major diseases that threaten people's lives. Discovering new high-efficiency and low-toxic anticancer and anticancer drugs is still an important topic in drug research.
佛美尼综合症(LFS)是一种由p53基因的胚系突变所引起的常染色体显性遗传疾病，该类病人患癌症的风险明显增加。因此，以突变型p53为治疗靶点，为肿瘤预防开辟了一条前途径。Fomany Syndrome (LFS) is an autosomal dominant genetic disease caused by germline mutations in the p53 gene, which has a significantly increased risk of cancer. Therefore, the use of mutant p53 as a therapeutic target opens up a promising approach for tumor prevention.
然而，以突变型p53为靶点的癌症治疗和预防研究才刚刚起步。随着很多小分子药物筛选方法不断被发现和运用，目前已有以p53为靶点的新型化合物的研究报道，尤其是以突变型p53为靶点的化合物APR-246已进入临床II期试验，这给癌症治疗带来了新的希望。However, cancer treatment and prevention research targeting mutant p53 has just begun. With the discovery and application of many small molecule drug screening methods, there have been reports on novel compounds targeting p53, especially the compound APR-246 targeting mutant p53 has entered the clinical phase II trial. This brings new hope to cancer treatment.
但是，人工设计的小分子药物普遍采用单靶点筛选方法。由于单靶点药物仅对一个突变的基因有效，一旦被单靶点药物阻拦生长通道，肿瘤细胞会主动选择其他通路配合自身的生长。只要成功找到其他通路，单靶点药物就会失去作用，这就是这类药物在癌症治疗最令人头痛的耐药性的问题。一旦出现耐药性，即意味着癌症治疗的预后不良。突破靶向药物治疗的耐药性是全球医学界的研究焦点之一，解决耐药性，必须寻找到有效的多靶点抑制剂。However, artificially designed small molecule drugs generally use a single target screening method. Since a single-target drug is only effective against one mutated gene, once the single-target drug blocks the growth channel, the tumor cell will actively select other pathways to match its own growth. As long as other pathways are successfully found, single-target drugs will lose their effect, which is the most headache-causing drug problem in cancer treatment. Once drug resistance occurs, it means that the prognosis of cancer treatment is poor. Breaking through the drug resistance of targeted drug therapy is one of the research focuses of the global medical community. To solve drug resistance, effective multi-target inhibitors must be found.
综上所述，本领域迫切需要开发不易产生耐药性的、以突变型p53为靶点的癌症治疗和预防药物。In summary, there is an urgent need in the art to develop cancer therapeutic and prophylactic drugs that are resistant to drug resistance and target mutant p53.
发明内容Summary of the invention
本发明的目的在于提供涉及含PEITC的药物组合物及其在癌症和其他由于 p53突变引起的疾病的预防与治疗，尤其是以突变型p53为靶点的癌症预防和治疗中的应用。It is an object of the present invention to provide a pharmaceutical composition comprising PEITC and its use in cancer and other Prevention and treatment of diseases caused by p53 mutations, especially in cancer prevention and treatment targeting mutant p53.
在本发明的第一方面，提供了一种PEITC的用途，用于制备一制剂或组合物，所述制剂或组合物用于(a)改变(或激活)突变型p53，(b)抑制突变型p53肿瘤细胞的增殖，(c)诱导突变型p53肿瘤细胞的凋亡，和/或(d)预防或治疗基于p53突变引起的疾病。In a first aspect of the invention, there is provided a use of PEITC for the preparation of a formulation or composition for (a) altering (or activating) a mutant p53, (b) inhibiting a mutation Proliferation of p53 tumor cells, (c) induction of apoptosis in mutant p53 tumor cells, and/or (d) prevention or treatment of diseases caused by p53 mutations.
在另一优选例中，所述的突变型p53在选自下组的位点具有突变：R175、C176、Y220、P223、C242、G245、R248、R249、R273、V274、P278、R282或其组合。In another preferred embodiment, the mutant p53 has a mutation at a site selected from the group consisting of R175, C176, Y220, P223, C242, G245, R248, R249, R273, V274, P278, R282 or a combination thereof .
在另一优选例中，所述的突变型p53选自下组：突变型p53R175、突变型p53C176、突变型p53C242。In another preferred embodiment, the mutant p53 is selected from the group consisting of mutant p53 R175 , mutant p53 C176 , and mutant p53 C242 .
在另一优选例中，所述的突变型p53为突变型p53R175。In another preferred embodiment, the mutant p53 is a mutant p53 R175 .
在另一优选例中，所述的突变型p53为突变型p53R175H。In another preferred embodiment, the mutant p53 is mutant p53 R175H .
在另一优选例中，所述的改变(或激活)包括诱导突变型p53重新具备类野生型p53的构象或功能。In another preferred embodiment, said altering (or activating) comprises inducing a mutant p53 to re-establish a conformation or function of a wild-type p53.
在另一优选例中，所述的类野生型指改变(或激活)后的突变型p53的构象或功能与野生型p53的构象或功能的相似程度≥90％，较佳地≥95％，更佳地≥99％，最佳地≥99.9％。In another preferred embodiment, said wild-type means that the conformation or function of the mutant p53 after alteration (or activation) is similar to the conformation or function of wild-type p53 by ≥90%, preferably ≥95%, More preferably ≥99%, optimally ≥99.9%.
在另一优选例中，所述的功能包括激活磷酸化ATM/CHK2、推迟S和G2/M期、和/或诱导细胞凋亡。In another preferred embodiment, the function comprises activating phosphorylated ATM/CHK2, delaying S and G2/M phases, and/or inducing apoptosis.
在另一优选例中，所述的PEITC包括天然存在的PEITC或人工合成的PEITC。In another preferred embodiment, the PEITC comprises a naturally occurring PEITC or a synthetic PEITC.
在另一优选例中，所述天然存在的PEITC包括天然食物来源的PEITC。In another preferred embodiment, the naturally occurring PEITC comprises PEITC of natural food source.
在另一优选例中，所述的天然食物包括十字花科植物。In another preferred embodiment, the natural food comprises a cruciferous plant.
在另一优选例中，所述的十字花科植物选自下组：In another preferred embodiment, the cruciferous plant is selected from the group consisting of:
青菜、萝卜、大白菜、西洋菜、西蓝花、胡萝卜、甘蓝、辣根、芥末、花椰菜、覆盆子或其组合。Greens, radishes, Chinese cabbage, watercress, broccoli, carrots, kale, horseradish, mustard, broccoli, raspberries or combinations thereof.
在另一优选例中，所述的PEITC提取自十字花科植物。In another preferred embodiment, the PEITC is extracted from a cruciferous plant.
在另一优选例中，所述的组合物为药物组合物。In another preferred embodiment, the composition is a pharmaceutical composition.
在另一优选例中，所述的药物组合物用于预防和/或治疗癌症。In another preferred embodiment, the pharmaceutical composition is for preventing and/or treating cancer.
在另一优选例中，所述的癌症选自下组：In another preferred embodiment, the cancer is selected from the group consisting of:
乳腺癌、胰腺癌、肝癌、***癌、***、卵巢癌、口腔癌、食道癌、胃癌、结直肠癌、鼻咽癌、肺癌、膀胱癌、软组织肉瘤、脑瘤、淋巴细胞肿瘤、成骨肉瘤或其组合。Breast cancer, pancreatic cancer, liver cancer, prostate cancer, cervical cancer, ovarian cancer, oral cancer, esophageal cancer, gastric cancer, colorectal cancer, nasopharyngeal cancer, lung cancer, bladder cancer, soft tissue sarcoma, brain tumor, lymphocyte tumor, osteosarcoma Tumor or a combination thereof.
在另一优选例中，所述的药物组合物的剂型包括注射剂、栓剂、植入剂、膏剂、溶液剂和口服剂型。 In another preferred embodiment, the pharmaceutical composition is in the form of an injection, a suppository, an implant, an ointment, a solution, and an oral dosage form.
在另一优选例中，所述口服剂型包括片剂、胶囊剂、膜剂、口服液剂和颗粒剂。In another preferred embodiment, the oral dosage form comprises a tablet, a capsule, a film, an oral solution, and a granule.
在另一优选例中，所述的药物组合物的剂型包括缓释型剂型、和非缓释型剂型。In another preferred embodiment, the pharmaceutical composition comprises a sustained release dosage form, and a non-slow release dosage form.
在另一优选例中，所述的药物组合物中还可含有其他抗肿瘤活性成分。In another preferred embodiment, the pharmaceutical composition may further comprise other anti-tumor active ingredients.
在另一优选例中，所述的药物组合物中还可含有活性成分Nutlin、MG132、和/或Zn2+ In another preferred embodiment, the pharmaceutical composition may further comprise active ingredients Nutlin, MG132, and/or Zn 2+ .
在另一优选例中，所述的药物组合物为单元剂型，每个单元剂型中PEITC的含量约为日剂量的0.1至1(或0.25-1，或0.5-1)，其中所述日剂量为5-500mg，较佳地为20-200mg，更佳地为60-180mg。In another preferred embodiment, the pharmaceutical composition is a unit dosage form, and the content of PEITC in each unit dosage form is about 0.1 to 1 (or 0.25-1, or 0.5-1) of the daily dose, wherein the daily dose It is 5 to 500 mg, preferably 20 to 200 mg, more preferably 60 to 180 mg.
在本发明的第二方面，提供了一种p53基因检测试剂的用途，用于制备诊断试剂或诊断试剂盒，所述诊断试剂或诊断试剂盒用于(a)判断PEITC治疗效果，和/或(b)判断肿瘤患者是否适合用PEITC进行治疗。In a second aspect of the present invention, there is provided a use of a p53 gene detecting reagent for preparing a diagnostic reagent or a diagnostic kit for (a) determining a PEITC therapeutic effect, and/or (b) Determine whether a tumor patient is suitable for treatment with PEITC.
在另一优选例中，所述的试剂包括蛋白芯片、核酸芯片、或其组合。In another preferred embodiment, the reagent comprises a protein chip, a nucleic acid chip, or a combination thereof.
在另一优选例中，所述的判断包括辅助判断和/或治疗前判断。In another preferred embodiment, the determination includes an auxiliary determination and/or a pre-treatment determination.
在另一优选例中，所述的诊断试剂或诊断试剂盒检测选自下组的p53基因突变：In another preferred embodiment, the diagnostic reagent or diagnostic kit detects a p53 gene mutation selected from the group consisting of:
p53R175、p53C176、p53C242、p53Y220、p53P223、p53C242、p53G245、p53R248、p53R249、p53R273、p53V274、p53P278、p53R282或其组合。P53 R175 , p53 C176 , p53 C242 , p53 Y220 , p53 P223 , p53 C242 , p53 G245 , p53 R248 , p53 R249 , p53 R273 , p53 V274 , p53 P278 , p53 R282 or combinations thereof.
在另一优选例中，所述的诊断试剂或诊断试剂盒检测p53基因第175位R→H突变。In another preferred embodiment, the diagnostic reagent or diagnostic kit detects the 175th R→H mutation of the p53 gene.
在另一优选例中，所述的诊断试剂或诊断试剂盒用于检测选自下组的样本：手术切除组织样本、石蜡切片组织样本、活检穿刺组织样本、血液样本、或其组合。In another preferred embodiment, the diagnostic reagent or diagnostic kit is for detecting a sample selected from the group consisting of a surgically removed tissue sample, a paraffin section tissue sample, a biopsy tissue sample, a blood sample, or a combination thereof.
在另一优选例中，所述的p53基因突变检测试剂选自下组：p53基因、p53蛋白、p53蛋白特异性抗体、或其组合。In another preferred embodiment, the p53 gene mutation detecting reagent is selected from the group consisting of a p53 gene, a p53 protein, a p53 protein-specific antibody, or a combination thereof.
在另一优选例中，所述的p53蛋白或其特异性抗体偶联有或带有可检测的标记物。In another preferred embodiment, the p53 protein or a specific antibody thereof is conjugated with or with a detectable label.
在另一优选例中，所述可检测的标记物选自下组：生色团、化学发光基团、荧光团、同位素或酶。In another preferred embodiment, the detectable label is selected from the group consisting of a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.
在本发明的第三方面，提供了一种试剂盒，所述试剂盒包括：In a third aspect of the invention, a kit is provided, the kit comprising:
(a)容器A，以及位于容器A中的PEITC或含PEITC的药物；(a) container A, and PEITC or PEITC-containing drug located in container A;
(b)容器B，以及位于容器B中的p53基因检测试剂； (b) container B, and a p53 gene detection reagent located in container B;
(c)说明书。(c) Instructions.
在另一优选例中，所述的说明书注明以下内容：In another preferred embodiment, the instructions indicate the following:
(i)如果检测对象中肿瘤细胞的p53为突变型，则提示PEITC治疗效果较好，和/或所述肿瘤患者适合用PEITC进行治疗；(i) If the p53 of the tumor cells in the test subject is a mutant type, it indicates that the PEITC treatment is better, and/or the tumor patient is suitable for treatment with PEITC;
在另一优选例中，所述的说明书还注明以下内容：In another preferred embodiment, the instructions also specify the following:
(ii)如果检测对象中肿瘤细胞的p53为野生型，则提示PEITC治疗效果较差，和/或所述肿瘤患者不适合用PEITC进行治疗。(ii) If the p53 of the tumor cells in the test subject is wild type, the PEITC treatment effect is poor, and/or the tumor patient is not suitable for treatment with PEITC.
在本发明的第四方面，提供了一种体外非治疗性地抑制肿瘤细胞的方法，所述方法包括步骤：In a fourth aspect of the invention, a method of non-therapeutic inhibition of tumor cells in vitro is provided, the method comprising the steps of:
(i)提供一肿瘤细胞，检测所述肿瘤细胞p53基因的突变情况，如果所述肿瘤细胞的p53为突变型时，进行步骤(ii)；(i) providing a tumor cell, detecting the mutation of the tumor cell p53 gene, if the tumor cell p53 is a mutant type, performing step (ii);
(ii)在PEITC存在的条件下，培养所述肿瘤细胞。(ii) culturing the tumor cells in the presence of PEITC.
在另一优选例中，所述的PEITC的浓度为1-100μM，较佳地为4-50μM，更佳地为5-20μM。In another preferred embodiment, the concentration of the PEITC is from 1 to 100 μM, preferably from 4 to 50 μM, more preferably from 5 to 20 μM.
在本发明的第五方面，提供了一种药物组合物，所述的药物组合物含有活性成分(a)PEITC，活性成分(b)Nutlin，和药学上可接受的载体。In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising the active ingredient (a) PEITC, active ingredient (b) Nutlin, and a pharmaceutically acceptable carrier.
在另一优选例中，所述的药物组合物还可含有Zn2+、或MG132。In another preferred embodiment, the pharmaceutical composition may further contain Zn 2+ , or MG132.
在另一优选例中，所述的药物组合物用于(a)改变(或激活)突变型p53，(b)抑制突变型p53肿瘤细胞的增殖，(c)诱导突变型p53肿瘤细胞的凋亡，和/或(d)预防或治疗基于p53突变引起的疾病。In another preferred embodiment, the pharmaceutical composition is for (a) altering (or activating) a mutant p53, (b) inhibiting proliferation of a mutant p53 tumor cell, and (c) inducing a mutant p53 tumor cell. Death, and / or (d) prevention or treatment of diseases caused by p53 mutations.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.
附图说明DRAWINGS
图1显示PEITC抑制p53突变型细胞DU145的细胞增殖和诱导细胞凋亡。Figure 1 shows that PEITC inhibits cell proliferation and induces apoptosis of p53 mutant cell DU145.
图1A显示了WST-1法测定细胞增殖率的结果。其中包括经DMSO(对照)或PEITC处理3天的DU145细胞。Figure 1A shows the results of measuring the cell proliferation rate by the WST-1 method. These included DU145 cells treated with DMSO (control) or PEITC for 3 days.
图1B显示了PEITC对细胞凋亡的影响。其中包括经DMSO(对照)或PEITC处理3天DU145细胞。检测组蛋白相关DNA片段指示细胞凋亡。Figure 1B shows the effect of PEITC on apoptosis. This included treatment of DU145 cells for 3 days via DMSO (control) or PEITC. Detection of histone-associated DNA fragments indicates apoptosis.
图2显示PEITC抑制突变型p53R175依赖的细胞增殖和诱导细胞凋亡。Figure 2 shows that PEITC inhibits mutant p53 R175- dependent cell proliferation and induces apoptosis.
图2a分别用DMSO(对照)或PEITC处理拥有p53热点基因突变和野生型p53 的人肿瘤细胞株3天。Figure 2a, respectively, with DMSO (control) or PEITC treatment with p53 hotspot gene mutations and wild-type p53 Human tumor cell line for 3 days.
图2b显示了WST-1法测定细胞增殖率的结果。其中包括DMSO或PEITC处理3天的转染siRNA的SK-BR-3细胞和A549细胞。Figure 2b shows the results of measuring the cell proliferation rate by the WST-1 method. These included SK-BR-3 cells and A549 cells transfected with siRNA for 3 days with DMSO or PEITC.
图2c显示了PEITC对细胞凋亡的影响。其中包括用DMSO或4μM PEITC处理3天的未转染的或转染siRNA的SK-BR-3细胞和A549细胞。使用BD lsrfortessa仪器经Annexin-V染色检测凋亡。Figure 2c shows the effect of PEITC on apoptosis. These included SK-BR-3 cells and A549 cells that were untransfected or transfected with siRNA for 3 days with DMSO or 4 μM PEITC. Apoptosis was detected by Annexin-V staining using a BD lsrfortessa instrument.
图2d显示了WST-1法测定细胞增殖率的结果。其中包括用DMSO或PEITC处理3天的转染pcDNA3、pcDNA3-p53R175、pcDNA3-p53R273或pcDNA3-wtp53的H1299细胞。Figure 2d shows the results of measuring the cell proliferation rate by the WST-1 method. These included H1299 cells transfected with DMSO or PEITC for 3 days transfected with pcDNA3, pcDNA3-p53R175, pcDNA3-p53R273 or pcDNA3-wtp53.
图2e显示了PEITC对细胞凋亡的影响。其中包括用DMSO或8μM PEITC处理3天的转染pcDNA3、pcDNA3-p53R175、pcDNA3-p53R273或pcDNA3-wtp53的H1299细胞。使用BD lsrfortessa仪器经Annexin-V染色检测凋亡。Figure 2e shows the effect of PEITC on apoptosis. These included H1299 cells transfected with pcDNA3, pcDNA3-p53R175, pcDNA3-p53R273 or pcDNA3-wtp53 treated with DMSO or 8 μM PEITC for 3 days. Apoptosis was detected by Annexin-V staining using a BD lsrfortessa instrument.
图3显示了PEITC诱导突变型p53R175蛋白转变成“类野生型”的构象变化。Figure 3 shows the conformational change of PEITC-induced mutant p53 R175 protein into a "wild-like type".
图3a显示了应用PAb240抗体(突变型)和PAb1620抗体(野生型)经ELISA方法确定PEITC对重组纯化的GST-p53R175H的构象的影响。Figure 3a shows the effect of PEITC on the conformation of recombinant purified GST-p53 R175H by ELISA using PAb240 antibody (mutant) and PAb1620 antibody (wild type).
图3b和图3c显示了免疫荧光试验的检测结果。其中包括用DMSO或4μM PEITC处理6小时的SK-BR-3细胞。细胞的免疫荧光使用PAb240和PAb1620抗体进行。A549细胞株作为对照，表明p53基因野生型的构象不会被PEITC改变。H1299细胞系作为抗p53抗体对照。所有检测的阈值限定为20μM。***表示PAB240和PAB1620相比，p≤0.0001。Figures 3b and 3c show the results of the immunofluorescence assay. This included SK-BR-3 cells treated with DMSO or 4 μM PEITC for 6 hours. Immunofluorescence of cells was performed using PAb240 and PAb1620 antibodies. The A549 cell line served as a control, indicating that the conformation of the wild type of p53 gene is not altered by PEITC. The H1299 cell line served as an anti-p53 antibody control. The threshold for all assays was limited to 20 μM. *** indicates that PAB240 is compared with PAB1620, p ≤ 0.0001.
图3d显示了使用PAb240抗体对SK-BR-3细胞裂解物中的突变型p53蛋白进行免疫沉淀，并用p53抗体(fl393)进行检测的结果。Figure 3d shows the results of immunoprecipitation of mutant p53 protein in SK-BR-3 cell lysate using PAb240 antibody and detection with p53 antibody (fl393).
图4显示PEITC能使突变型p53R175蛋白复活其野生型的转录激活作用。Figure 4 shows that PEITC enables the mutant p53 R175 protein to reactivate its wild-type transcriptional activation.
图4a显示PEITC诱导突变型p53蛋白与染色质结合。其中，以PEITC处理SK-BR-3细胞4小时，染色质结合和核可溶性组分用免疫印迹分析。组蛋白H3和拓扑异构酶IIB分别作为染色质和核可溶性组分的标记物。Figure 4a shows that PEITC induces binding of mutant p53 protein to chromatin. Among them, SK-BR-3 cells were treated with PEITC for 4 hours, and chromatin binding and nuclear soluble components were analyzed by immunoblotting. Histone H3 and topoisomerase IIB serve as markers for chromatin and nuclear soluble components, respectively.
图4b显示了提取RNA并利用TaqMan基因表达检测试剂盒检测基因表达水平的结果。其中，以DMSO或4μM PEITC处理SK-BR-3、H1299和A549细胞4小时，qRT-PCR扩增p53调节基因。Figure 4b shows the results of RNA extraction and detection of gene expression levels using the TaqMan gene expression detection kit. Among them, SK-BR-3, H1299 and A549 cells were treated with DMSO or 4 μM PEITC for 4 hours, and the p53 regulatory gene was amplified by qRT-PCR.
图4c显示了提取RNA并利用TaqMan基因表达检测试剂盒检测基因表达水平的结果。其中，以DMSO或4μM PEITC处理p53 siRNA转染或NS siRNA转染的SK-BR-3细胞4小时，qRT-PCR扩增p53调节基因。Figure 4c shows the results of RNA extraction and detection of gene expression levels using the TaqMan gene expression detection kit. Among them, SK-BR-3 cells transfected with p53 siRNA or NS siRNA were treated with DMSO or 4 μM PEITC for 4 hours, and the p53 regulatory gene was amplified by qRT-PCR.
图4d显示了荧光素酶检测的结果。其中，用质粒16451转染SK-BR-3、HOP92、AU565、H1299和MEF[(10)3/175和(10)3/273]细胞后，PEITC(4或6μM)处理24小时，再分别进行荧光素酶检测。 Figure 4d shows the results of luciferase assay. Among them, plasmids 16451 were transfected with SK-BR-3, HOP92, AU565, H1299 and MEF [(10)3/175 and (10)3/273] cells, and PEITC (4 or 6 μM) was treated for 24 hours. Perform luciferase assay.
图4e显示了Western blotting测定蛋白水平的结果。其中，在4μM PEITC或20μM依托泊苷处理SK-BR-3细胞4小时后，用Western blot分析p21表达水平。4μM PEITC处理A549细胞4小时作为对照。用p53DO-1和GAPDH抗体采用Western blotting测定蛋白水平。Figure 4e shows the results of Western blotting to determine protein levels. Among them, after treatment of SK-BR-3 cells with 4 μM PEITC or 20 μM etoposide for 4 hours, p21 expression levels were analyzed by Western blot. A549 cells were treated with 4 μM PEITC for 4 hours as a control. Protein levels were determined by Western blotting using p53DO-1 and GAPDH antibodies.
图5显示了基因表达水平的检测结果。其中，PEITC诱导DU145细胞中典型p53靶基因p21的表达。DU145细胞经DMSO(对照)或PEITC处理4小时。提取RNA，应用TaqMan基因表达试剂盒检测基因的表达水平。Figure 5 shows the results of detection of gene expression levels. Among them, PEITC induced the expression of a typical p53 target gene p21 in DU145 cells. DU145 cells were treated with DMSO (control) or PEITC for 4 hours. RNA was extracted and the expression level of the gene was detected using a TaqMan gene expression kit.
图6显示ITCs恢复p53的转录激活功能。Figure 6 shows that ITCs restore the transcriptional activation function of p53.
图6A显示了免疫印迹分析p53、p21表达水平的结果。其中，SCC114细胞用不同浓度的DMSO(对照)或ITCs处理24小时。提取40mg细胞总蛋白，在聚丙烯酰胺凝胶电泳上测定，用P21抗体检测。剥离印迹，再次用p53(DO-1)抗体和抗GAPDH抗体检测。Figure 6A shows the results of immunoblotting analysis of p53, p21 expression levels. Among them, SCC114 cells were treated with different concentrations of DMSO (control) or ITCs for 24 hours. 40 mg of total cell protein was extracted and assayed on polyacrylamide gel electrophoresis and detected with P21 antibody. The blot was stripped and detected again with p53 (DO-1) antibody and anti-GAPDH antibody.
图6B显示了染色质免疫沉淀反应(CHIP)的结果。其中，经过PEITC处理24小时的SCC-114细胞的裂解液与抗p53抗体(DO-1)进行免疫沉淀反应，然后对p53识别元素p21、PUMA和MDM2进行PCR。Figure 6B shows the results of chromatin immunoprecipitation (CHIP). Among them, the lysate of SCC-114 cells treated with PEITC for 24 hours was subjected to immunoprecipitation with anti-p53 antibody (DO-1), and then PCR was carried out on p53 recognition elements p21, PUMA and MDM2.
图7显示PEITC处理SK-BR-3和A549细胞后，蛋白酶体降解p53蛋白。Figure 7 shows that the proteasome degrades the p53 protein after PEITC treatment of SK-BR-3 and A549 cells.
图7a显示了用不同浓度的异硫氰酸苯乙酯和抑制剂(10μM Nutlin-3或20μM MG132)处理SK-BR-3细胞4小时后的p53蛋白降解情况。Figure 7a shows the degradation of p53 protein after treatment of SK-BR-3 cells with different concentrations of phenethyl isothiocyanate and inhibitor (10 μM Nutlin-3 or 20 μM MG132) for 4 hours.
图7b显示了SK-BR-3细胞经PEITC(4μM或8μM)，20μM MG132，或两者共处理4小时后的p53蛋白降解情况。Figure 7b shows the degradation of p53 protein after SK-BR-3 cells were treated with PEITC (4 μM or 8 μM), 20 μM MG132, or both for 4 hours.
图7c显示了SK-BR-3细胞经PEITC(4μM或8μM)，10μM MG132，或两者共处理4小时后的p53蛋白降解情况。Figure 7c shows the degradation of p53 protein after SK-BR-3 cells were treated with PEITC (4 μM or 8 μM), 10 μM MG132, or both for 4 hours.
图7d显示了用不同浓度的异硫氰酸苯乙酯和抑制剂(10μM Nutlin-3或20μM MG132)处理A549细胞24小时后的p53蛋白降解情况。收集细胞，制备细胞裂解物。裂解物经SDS-PAGE电泳后，用p53DO-1抗体检测。Figure 7d shows the degradation of p53 protein after treatment of A549 cells with different concentrations of phenethyl isothiocyanate and inhibitor (10 μM Nutlin-3 or 20 μM MG132) for 24 hours. The cells were collected and cell lysates were prepared. The lysate was electrophoresed by SDS-PAGE and detected with p53DO-1 antibody.
图7e显示了不同浓度的PEITC或DMSO处理SK-BR-3细胞4小时后的p53蛋白降解情况。收集细胞制备可溶性组分和不溶性组分。30μg可溶性或不溶性裂解物经SDS-PAGE电泳后，用p53DO-1抗体检测。Figure 7e shows the degradation of p53 protein after 4 hours of treatment of SK-BR-3 cells with different concentrations of PEITC or DMSO. The cells are collected to prepare soluble components and insoluble components. 30 μg of soluble or insoluble lysate was detected by SDS-PAGE and detected with p53DO-1 antibody.
图8显示PEITC处理SK-BR-3细胞后，p53R175蛋白被细胞自噬。Figure 8 shows that pPI R175 protein is autophagy by cells after PEITC treatment of SK-BR-3 cells.
图8a显示了以PEITC(4μM或8μM)、CHQ(50μM)、或两者共处理SK-BR-3细胞4小时后的细胞自噬情况。裂解物经SDS-PAGE电泳后，用p53DO-1抗体检测。Figure 8a shows the autophagy of SK-BR-3 cells treated with PEITC (4 μM or 8 μM), CHQ (50 μM), or both for 4 hours. The lysate was electrophoresed by SDS-PAGE and detected with p53DO-1 antibody.
图8b显示了用ATG5siRNA或NS siRNA转染SK-BR-3细胞后的细胞自噬情况。30μg裂解物经SDS-PAGE电泳后，用抗ATG5抗体检测。剥离印迹后，用抗GAPDH抗体重新结合。Figure 8b shows the autophagy of cells after transfection of SK-BR-3 cells with ATG5 siRNA or NS siRNA. 30 μg of the lysate was subjected to SDS-PAGE electrophoresis and detected with an anti-ATG5 antibody. After stripping the blot, re-binding with anti-GAPDH antibody.
图8c显示了用DMSO或PEITC处理转染ATG5siRNA或NS siRNA的SK-BR-3细胞4小时后的细胞自噬情况。用p53DO-1和GAPDH抗体采用Western blotting测定蛋 白水平。Figure 8c shows the autophagy of SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA for 4 hours after treatment with DMSO or PEITC. Western blotting of eggs using p53DO-1 and GAPDH antibodies White level.
图8d显示了WST-1法测定细胞增殖率的结果。其中，用DMSO或PEITC处理转染ATG5siRNA或NS siRNA的SK-BR-3细胞3天Figure 8d shows the results of measuring the cell proliferation rate by the WST-1 method. Among them, SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA were treated with DMSO or PEITC for 3 days.
图8e显示了PEITC对细胞凋亡的影响。其中，以DMSO或PEITC处理转染ATG5siRNA或NS siRNA的SK-BR-3细胞3天。检测组蛋白相关的DNA片段指示细胞凋亡。Figure 8e shows the effect of PEITC on apoptosis. Among them, SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA were treated with DMSO or PEITC for 3 days. Detection of histone-associated DNA fragments is indicative of apoptosis.
图9显示了锌和氧化还原剂对PEITC诱导p53R175蛋白复活作用的影响。Figure 9 shows the effect of zinc and redox agents on PEITC-induced reactivation of p53 R175 protein.
图9a显示了锌对PEITC活性的影响。其中，以PEITC、锌或两者共处理SK-BR-3细胞。WST-1法测定细胞增殖率。PEITC活性以生长抑制率的IC50值来表示。Figure 9a shows the effect of zinc on PEITC activity. Among them, SK-BR-3 cells were co-treated with PEITC, zinc or both. The cell proliferation rate was measured by the WST-1 method. PEITC activity is expressed as the IC50 value of the growth inhibition rate.
图9b显示了应用PAb240抗体(突变型)和PAb1620抗体(野生型)经ELISA方法确定锌或锌和PEITC共处理对重组纯化的GST-p53R175H的构象的影响。Figure 9b shows the effect of co-treatment of zinc or zinc and PEITC on the conformation of recombinant purified GST-p53 R175H by ELISA using PAb240 antibody (mutant) and PAb1620 antibody (wild type).
图9c显示了PEITC对SK-BR-3细胞降低谷胱甘肽的影响。其中，SK-BR-3细胞经PEITC(4μM或8μM)或DMSO处理4小时。利用GSH/GSSG GLO谷胱甘肽试剂盒检测还原剂GSH和氧化剂GSSG的比值。Figure 9c shows the effect of PEITC on the reduction of glutathione by SK-BR-3 cells. Among them, SK-BR-3 cells were treated with PEITC (4 μM or 8 μM) or DMSO for 4 hours. The ratio of reducing agent GSH to oxidant GSSG was measured using a GSH/GSSG GLO glutathione kit.
图9d显示了NAC对PEITC活性的影响。其中，以不同浓度的PEITC或PEITC结合3μM NAC处理SK-BR-3细3天。WST-1法测定细胞增殖率。Figure 9d shows the effect of NAC on PEITC activity. Among them, SK-BR-3 was treated with different concentrations of PEITC or PEITC in combination with 3 μM NAC for 3 days. The cell proliferation rate was measured by the WST-1 method.
图9e显示了WST-1法测定细胞增殖率的结果。其中，PEITC单独或联合2mM ATZ或500单位的PEG过氧化氢酶共处理SK-BR-3细胞3天。Figure 9e shows the results of measuring the cell proliferation rate by the WST-1 method. Among them, PEITC co-treated SK-BR-3 cells alone or in combination with 2 mM ATZ or 500 units of PEG catalase for 3 days.
图9f显示了各成分对细胞凋亡的影响。其中，DMSO、ATZ、NAC或PEITC单独处理，或PEITC与ATZ或NAC共处理未转染的或siRNA转染的SK-BR-3细胞3天。使用BD lsrfortessa仪器经Annexin-V染色检测凋亡。Figure 9f shows the effect of each component on apoptosis. Among them, DMSO, ATZ, NAC or PEITC were treated alone, or PEITC co-treated with untransfected or siRNA transfected SK-BR-3 cells for 3 days with ATZ or NAC. Apoptosis was detected by Annexin-V staining using a BD lsrfortessa instrument.
图10显示PEITC诱导H2AX灶，激活ATM和Chk2，阻滞G2/M期和S期，诱导细胞凋亡。其中，PEITC或DMSO处理SK-BR-3细胞和A549细胞3天，抗γ–H2AX抗体染色。Figure 10 shows that PEITC induces H2AX foci, activates ATM and Chk2, blocks G2/M phase and S phase, and induces apoptosis. Among them, PEITC or DMSO treated SK-BR-3 cells and A549 cells for 3 days, and stained with anti-γ-H2AX antibody.
图10a显示了细胞抗γ–H2AX抗体染色(绿色)和DAPI(蓝色)的合并图像。其中，所有检测的阈值限定为20μM。Figure 10a shows a combined image of cell anti-gamma-H2AX antibody staining (green) and DAPI (blue). Among them, the threshold of all detections was limited to 20 μM.
图10b显示了γ-H2AX灶细胞百分比(≤10或＞10，如图所示)。Figure 10b shows the percentage of gamma-H2AX foci cells (≤ 10 or > 10, as shown).
图10c显示了PEITC或DMSO处理4小时的SK-BR-3细胞和A549细胞的免疫印迹分析结果。其中，免疫印迹使用抗pATM S1981和抗pCHK2Thr68抗体进行。印迹剥离后，抗ATM和抗CHK2抗体重新结合。Figure 10c shows the results of immunoblot analysis of SK-BR-3 cells and A549 cells treated with PEITC or DMSO for 4 hours. Among them, immunoblotting was carried out using anti-pATM S1981 and anti-pCHK2Thr68 antibodies. After blotting, the anti-ATM and anti-CHK2 antibodies recombine.
图10d和图10e分别显示了SK-BR-3(d)或A549(e)细胞经PEITC，10μM Nutlin-3或共处理24h后，采用流式细胞仪分析的结果。Figure 10d and Figure 10e show the results of flow cytometry analysis of SK-BR-3(d) or A549(e) cells after PEITC, 10 μM Nutlin-3 or co-treatment for 24 h, respectively.
图10f显示了使用BD lsrfortessa仪器经Annexin-V染色检测凋亡的结果，其中包括以4μM PEITC，10μM Nutlin-3或共处理24小时的SK-BR-3细胞和A549细胞。 Figure 10f shows the results of apoptosis detected by Annexin-V staining using the BD lsrfortessa instrument, including SK-BR-3 cells and A549 cells treated with 4 μM PEITC, 10 μM Nutlin-3 or co-treated for 24 hours.
图11显示ITCs恢复p53突变蛋白的细胞周期阻滞功能。Figure 11 shows that ITCs restore the cell cycle arrest function of the p53 mutein.
图11A和图11B显示了用流式细胞术分析经BITC(5μm或10μM)或DMSO(对照)处理的SCC003细胞。Figure 11A and Figure 11B show the analysis of SCC003 cells treated with BITC (5 μm or 10 μM) or DMSO (control) by flow cytometry.
图11C和图11D显示了用流式细胞术分析经PEITC(5μm或10μM)或DMSO(对照)处理的SCC003细胞。Figure 11C and Figure 11D show the analysis of SCC003 cells treated with PEITC (5 μιη or 10 μM) or DMSO (control) by flow cytometry.
图11E和图11F显示了用流式细胞术分析经BITC(5μm或10μM)或DMSO(对照)处理的SCC114细胞。Figure 11E and Figure 11F show the analysis of SCC114 cells treated with BITC (5 μιη or 10 μM) or DMSO (control) by flow cytometry.
图11G和图11H显示了用流式细胞术分析经PEITC(5μm或10μM)或DMSO(对照)处理的SCC114细胞。Figure 11G and Figure 11H show the analysis of SCC114 cells treated with PEITC (5 μιη or 10 μM) or DMSO (control) by flow cytometry.
图12显示ITCs诱导突变型p53依赖型细胞周期阻滞。Figure 12 shows that ITCs induce mutant p53-dependent cell cycle arrest.
图12A显示了DMSO对照组(未转染)，非特异性siRNA(N)组和mtp53siRNA(P)组SCC114细胞培养24小时、48小时和72小时后的免疫印迹分析结果。其中，转染后mtp53siRNA转染组的细胞p53蛋白表达减少，而对照组没有。Figure 12A shows the results of immunoblot analysis of DMSO control group (untransfected), non-specific siRNA (N) group and mtp53 siRNA (P) group SCC114 cells cultured for 24 hours, 48 hours and 72 hours. Among them, the expression of p53 protein in the mtp53 siRNA transfected group was decreased after transfection, but not in the control group.
图12B显示了WST-1试验测定siRNA转染细胞增殖率的结果。其中，用PEITC/BITC处理后，突变型p53细胞能够增殖。Figure 12B shows the results of the WST-1 assay to determine the proliferation rate of siRNA transfected cells. Among them, mutant p53 cells were able to proliferate after treatment with PEITC/BITC.
图12C、图12D、图12E显示了流式细胞术分析结果。其中，用BITC(5μM或10μM)或DMSO(对照)处理DMSO对照组(未转染)、非特异性siRNA(N)组和mtp53siRNA(P)组转染的SCC114细胞，然后用流式细胞术分析。Figures 12C, 12D, and 12E show the results of flow cytometry analysis. Among them, DMSO control group (untransfected), non-specific siRNA (N) group and mtp53 siRNA (P) group transfected SCC114 cells were treated with BITC (5 μM or 10 μM) or DMSO (control), and then analyzed by flow cytometry. .
图12F、图12G、图12H显示了显示了流式细胞术分析结果。其中，用PEITC(5μM或10μM)或DMSO(对照)处理DMSO对照组(未转染)、非特异性(scrambled)siRNA(N)组和mtp53siRNA(P)组转染的SCC114细胞，然后用流式细胞技术分析。Figures 12F, 12G, and 12H show the results of flow cytometry analysis. Among them, DMSO control group (untransfected), non-specific (scrambled) siRNA (N) group and mtp53 siRNA (P) group transfected SCC114 cells were treated with PEITC (5 μM or 10 μM) or DMSO (control), and then flowed. Cell technology analysis.
图13显示PEITC在体内诱导突变型p53R175蛋白复活和抑制裸鼠移植瘤的生长。Figure 13 shows that PEITC induces the reactivation of mutant p53 R175 protein in vivo and inhibits the growth of xenografts in nude mice.
图13a显示了小鼠乳腺脂肪垫(上图)和H&E染色(下图)的典型图像。所有检测的阈值限定为20μM。Figure 13a shows a typical image of a mouse mammary fat pad (top panel) and H&E staining (bottom panel). The threshold for all assays was limited to 20 μM.
图13b显示了肿瘤体积测量结果。用游标卡尺测量肿瘤，计算肿瘤体积。肿瘤体积计算公式：L x W2x 0.523(**，p≤0.009，*，p≤0.03)(n＝7)。Figure 13b shows the tumor volume measurement results. Tumors were measured with a vernier caliper and the tumor volume was calculated. Tumor volume calculation formula: L x W 2 x 0.523 (**, p ≤ 0.009, *, p ≤ 0.03) (n = 7).
图13c显示了每周动物体重的变化情况。Figure 13c shows the change in body weight per week.
图13d显示了根据对照组和PEITC组每个组织切片的肿瘤细胞的平均数量对动物进行分配的情况(***，p≤0.00026)(n＝7)。Figure 13d shows the distribution of animals according to the average number of tumor cells per tissue section of the control and PEITC groups (***, p ≤ 0.00026) (n = 7).
图13e显示了移植瘤组织中代表性图像Ki67(**，p≤0.007)和p53(*，p≤0.033)染色(上图)和阳性细胞计数(下图)(n＝7)。**表示Ki67p≤0.007，*表示p53p≤0.033。结果以均值显示。所有检测的阈值限定为200μM。Figure 13e shows representative images Ki67 (**, p ≤ 0.007) and p53 (*, p ≤ 0.033) staining (top panel) and positive cell count (bottom panel) (n = 7) in transplanted tumor tissues. ** indicates that Ki67p ≤ 0.007, and * indicates p53p ≤ 0.033. The results are shown as the mean. The threshold for all assays was limited to 200 μM.
图13f显示了免疫印迹分析PEITC组和对照组裸鼠移植瘤中p53的表达水平的结果。印迹为每组12个肿瘤组织裂解物的代表性图片。 Figure 13f shows the results of immunoblot analysis of p53 expression levels in the PEITC group and the control group in nude mice xenografts. The blot is a representative picture of 12 tumor tissue lysates per group.
图13g显示了PEITC组和对照组动物的p53调节基因qRT-PCR结果(n＝4)。Figure 13g shows the results of p53 regulatory gene qRT-PCR in the PEITC group and control animals (n=4).
图13h显示了SK-BR-3肿移植瘤中p21和Bax的免疫印迹。Figure 13h shows an immunoblot of p21 and Bax in SK-BR-3 swollen xenografts.
图14显示PEITC抑制DU145细胞裸鼠移植瘤的生长。Figure 14 shows that PEITC inhibits the growth of transplanted tumors of DU145 cells in nude mice.
图14A显示了小鼠侧面的代表性图像。Figure 14A shows a representative image of the side of the mouse.
图14B显示了肿瘤体积测量结果。用游标卡尺测量肿瘤，计算肿瘤体积。公式L×W2×0.523(**，P≤0.003；***，P≤0001)(n＝7)。Figure 14B shows the tumor volume measurement results. Tumors were measured with a vernier caliper and the tumor volume was calculated. The formula L × W 2 × 0.523 (**, P ≤ 0.003; ***, P ≤ 0001) (n = 7).
图14C显示了每周动物体重(克)的变化情况。Figure 14C shows the change in body weight (grams) per week.
各附图中，WT表示野生型，WB表示免疫印迹，WCL表示全细胞裂解物，AIN-93M表示AIN-93M标准饲料。In the drawings, WT indicates wild type, WB indicates immunoblotting, WCL indicates whole cell lysate, and AIN-93M indicates AIN-93M standard feed.
具体实施方式detailed description
本发明人经过广泛而深入地研究，首次意外地发现，PEITC能够使突变型p53恢复其野生型活性(激活拟野生型p53活性)，抑制p53突变引起的肿瘤细胞的增殖并诱导其凋亡。实验表明，PEITC能够诱导突变型p53转型，尤其是突变型p53R175、p53P223L、p53V274F、p53R248重新具备类野生型p53的构象或功能，进而恢复激活野生型p53靶点，如磷酸化ATM/CHK2，阻滞S和G2/M期，诱导细胞凋亡。在此基础上完成本发明。The present inventors have extensively and intensively studied, and for the first time, unexpectedly found that PEITC can restore mutant p53 to its wild-type activity (activate wild-type p53 activity), inhibit tumor cell proliferation induced by p53 mutation, and induce apoptosis. Experiments have shown that PEITC can induce mutant p53 transformation, especially mutant p53 R175 , p53 P223L , p53 V274F , and p53 R248 re-establish the conformation or function of wild-type p53, thereby restoring activation of wild-type p53 targets, such as phosphorylated ATM /CHK2, blocks S and G2/M phases, induces apoptosis. The present invention has been completed on this basis.
本发明人研究发现：The inventors have found that:
1、PEITC可以恢复热点突变点R175、C176、Y220、P223、C242、G245、R248、R249、R273、V274、P278、R282错义突变的突变型p53的野生型构象和转录激活功能，诱导表达上述热点突变型p53细胞的凋亡。1. PEITC can restore the wild-type conformation and transcriptional activation function of mutant p53 with missense mutations in hot spot mutations R175, C176, Y220, P223, C242, G245, R248, R249, R273, V274, P278, R282, and induce expression Apoptosis of hot mutant mutant p53 cells.
2、PEITC能够抑制突变型p53R175表达型的乳腺癌细胞SK-BR-3、AU565、突变型p53R248表达型的口腔癌细胞SCC114和突变型p53P223L或p53V274F表达型的***癌细胞DU145的增殖。2. PEITC can inhibit mutant p53 R175- expressing breast cancer cells SK-BR-3, AU565, mutant p53 R248- expressing oral cancer cell SCC114 and mutant p53 P223L or p53 V274F- expressing prostate cancer cell DU145 proliferation.
3、体内动物实验证明PEITC能够抑制乳腺癌细胞SK-BR-3和***癌细胞DU145异体移植肿瘤的生长。3. In vivo animal experiments demonstrate that PEITC can inhibit the growth of breast cancer cell SK-BR-3 and prostate cancer cell line DU145.
4、在表达突变型p53R175的乳腺癌细胞SK-BR-3、AU565，表达突变型p53P223L或p53V274F的***癌细胞DU145和表达突变型p53R248的口腔癌细胞SCC114中，PEITC可以通过将突变型构象转变成类野生型构象，复活转录激活功能，激活野生型p53靶点，如磷酸化ATM/CHK2，阻滞S和G2/M期，诱导细胞凋亡。4, the expression of mutant p53 R175 breast cancer cells SK-BR-3, AU565, expressing mutant p53 P223L or p53 V274F DU145 prostate cancer cells expressing mutant oral cancer cells in SCC114 p53 R248, can be of PEITC The mutant conformation transforms into a wild-type conformation, revives transcriptional activation, activates wild-type p53 targets, such as phosphorylation of ATM/CHK2, blocks S and G2/M phases, and induces apoptosis.
4、PEITC可以增加乳腺癌细胞SK-BR-3、AU565突变型p53R175蛋白对蛋白酶体和自噬降解的敏感性。4, PEITC can increase the sensitivity of breast cancer cell SK-BR-3, AU565 mutant p53 R175 protein to the degradation of proteasome and autophagy.
5、锌离子能增强PEITC诱导乳腺癌细胞SK-BR-3、AU565突变型p53R175的复活作用。 5. Zinc ion can enhance the reactivation of PEITC-induced breast cancer cell SK-BR-3 and AU565 mutant p53 R175 .
6、氧化还原变化对重新激活p53R175和抑制增长很重要，而对恢复p53R175构象不重要。6. Redox changes are important for reactivation of p53 R175 and inhibition of growth, but not for restoration of p53 R175 conformation.
术语the term
如本文所用，术语“类野生型p53(wt-like p53)的构象和/或功能”是指基本具备野生型p53的构象和/或功能，改变(激活)后的突变型p53的构象和/或功能与野生型p53的构象和/或功能的相似程度≥90％，较佳地≥95％，更佳地≥99％，最佳地≥99.9％。较佳地，类野生型p53的构象和/或功能为人野生型p53的构象和/或功能。As used herein, the term "conformational and/or functional of wild-type p53 (wt-like p53)" refers to a conformation and/or function substantially possessing wild-type p53, a conformation of mutant p53 after alteration (activation) and/or Or the degree of similarity to the conformation and/or function of wild-type p53 is > 90%, preferably > 95%, more preferably > 99%, optimally > 99.9%. Preferably, the conformation and/or function of wild-type p53 is the conformation and/or function of human wild-type p53.
如本文所用，术语“改变突变型p53”是指改变(重新激活)突变型p53恢复其野生型活性和/或构型(激活拟野生型p53活性)，抑制p53突变引起的肿瘤细胞的增殖并诱导其凋亡。As used herein, the term "altering mutant p53" means altering (reactivation) a mutant p53 to restore its wild-type activity and/or conformation (activating wild-type p53 activity), inhibiting proliferation of tumor cells caused by p53 mutation and Inducing its apoptosis.
p53基因P53 gene
如本文所用，术语“p53”、“p53基因”可互换使用，是指肿瘤抑制基因p53，p53基因的突变是人类癌症中十分普遍的现象。大多数的p53基因突变是错义突变，进一步可细分为接触突变(直接破坏p53与DNA的结合)和构象突变(破坏p53的构象)。这两种突变均造成正常的野生型p53的失活。研究表明，某些特定的小分子可以通过改变(激活)突变型p53来达到肿瘤抑制功能，用于癌症的治疗。As used herein, the terms "p53" and "p53 gene" are used interchangeably and refer to the tumor suppressor gene p53, a mutation in the p53 gene which is a very common phenomenon in human cancer. Most p53 gene mutations are missense mutations and can be further subdivided into contact mutations (which directly disrupt p53 binding to DNA) and conformational mutations (destruction of p53 conformation). Both of these mutations result in the inactivation of normal wild-type p53. Studies have shown that certain specific small molecules can achieve tumor suppressive function by altering (activating) mutant p53 for the treatment of cancer.
p53基因定位于人类染色体17p13.1，由其编码的包括393个氨基酸组成的53kD的核内磷酸化蛋白，被称为p53蛋白。野生型p53蛋白极不稳定，半衰期仅数分钟，并具有反式激活功能和广谱的肿瘤抑制作用。突变型p53蛋白稳定性增加，半衰期延长，可被免疫组化方法检测出来。The p53 gene is localized to human chromosome 17p13.1, which encodes a 53 kD nuclear-phosphorylated protein consisting of 393 amino acids, which is called p53 protein. The wild-type p53 protein is extremely unstable, has a half-life of only a few minutes, and has a transactivation function and a broad-spectrum tumor suppressive effect. Mutant p53 protein has increased stability and extended half-life and can be detected by immunohistochemistry.
通常状况下,野生型(wtp53)在细胞内的活性水平是被严格控制的,蛋白半衰期很短。HDM2/MDM2作为最主要的p53负调控分子,通过转录抑制和行使E3功能降解p53。同时,hdm2/mdm2又是p53的靶基因。p53-HDM2/MDM2所形成的这一负反馈机制使wtp53活性在细胞内维持在较低水平。p53作为体内信号通路的重要网络节点分子,现已知道超过150多种基因受其调控,形成一个精细复杂的p53调控网络,在维护基因组稳定性中发挥了重要作用。Under normal conditions, the activity level of wild-type (wtp53) in cells is strictly controlled, and the protein half-life is very short. As the most important p53 negative regulatory molecule, HDM2/MDM2 degrades p53 through transcriptional repression and E3 function. At the same time, hdm2/mdm2 is the target gene of p53. This negative feedback mechanism formed by p53-HDM2/MDM2 maintains wtp53 activity at a lower level in the cell. As an important network node molecule in the in vivo signaling pathway, p53 has been known to regulate more than 150 genes, forming a fine and complex p53 regulatory network, which plays an important role in maintaining genomic stability.
p53作为最重要的抑癌基因之一,与癌症的发生发展联系紧密,大约50％的人类癌症中存在着p53突变。在所有p53突变形式中,占主导地位的还是因点突变引起的错义突变,其比例约占总体的80％。而在这些p53错义突变中,发生在DBD区的点突变比例高达97％。实际上,p53的DBD区每一个氨基酸都可发生点突变而形成相应的突变体,但是以下12个位点的突变在癌症中高频率出现, 与癌症进程紧密关联,被称为热点突变,它们分别是:R175、C176、Y220、P223、C242、G245、R248、R249、R273、V274、P278、R282。As one of the most important tumor suppressor genes, p53 is closely related to the development of cancer, and there are p53 mutations in about 50% of human cancers. Among all p53 mutant forms, the dominant mutations caused by point mutations account for about 80% of the total. In these p53 missense mutations, the point mutation rate occurring in the DBD region was as high as 97%. In fact, every amino acid in the DBD region of p53 can be mutated to form a corresponding mutant, but mutations in the following 12 sites occur at high frequency in cancer. Closely related to the cancer process, known as hot spot mutations, they are: R175, C176, Y220, P223, C242, G245, R248, R249, R273, V274, P278, R282.
PEITCPEITC
如本文所用，PEITC(Phenethyl isothiocyanate)，即苯乙基异硫氰酸酯，西洋菜和其他十字花科蔬菜中富含PEITC。动物实验表明，PEITC对癌症具有化学预防作用，流行病学研究也支持此类化合物预防人类癌症的效果。事实上，PEITC用于健康人群肺癌预防的I期临床研究已经完成，II期临床正在进行中。PEITC诱导的氧化应激有助于细胞凋亡，然而，这一作用的确切机制和分子靶点还不是很清楚。As used herein, PEITC (Phenethyl isothiocyanate), phenethyl isothiocyanate, watercress and other cruciferous vegetables are rich in PEITC. Animal experiments have shown that PEITC has chemopreventive effects on cancer, and epidemiological studies support the efficacy of such compounds against human cancer. In fact, PEITC has completed Phase I clinical trials for lung cancer prevention in healthy populations, and Phase II clinical trials are underway. PEITC-induced oxidative stress contributes to apoptosis, however, the exact mechanism and molecular targets of this effect are not well understood.
已有的研究表明，PEITC和顺铂联用时，通过p53途径诱导肿瘤细胞凋亡。但是上述研究并没有明确诱导凋亡的分子机理，尚不清楚通过p53DBD区哪几种热点突变起作用。Previous studies have shown that when PEITC is combined with cisplatin, tumor cell apoptosis is induced by the p53 pathway. However, the above studies did not clarify the molecular mechanism of induction of apoptosis, and it is unclear which kinds of hotspot mutations play a role in the p53DBD region.
本发明人非常意外地发现，PEITC可以在体外和体内改变(重新激活)突变型p53，揭示了天然食物来源化合物的一种新的作用机理。在含有突变型p53的细胞中，PEITC对p53突变序列中较为常见的“热点”突变，如：p53R175、p53R248、p53P223和p53V274抑制活性较强，其中对最常见的“热点”突变之一p53R175的抑制活性最强。机理研究显示，PEITC通过恢复p53类野生型构象和转录激活功能诱导突变型p53R175的细胞凋亡。具体而言，在用PEITC处理的细胞内，改变的突变型p53R175通过激活野生型p53靶点，即磷酸化ATM/CHK2，推迟S和G2/M期，诱导细胞凋亡。机理的进一步研究表明，PEITC的这种生长抑制作用，在一定程度上受锌离子浓度和细胞氧化还原状态的影响。此外，PEITC通过蛋白酶体和自噬使p53R175突变体对降解敏感，其敏感程度与浓度相关。PEITC诱导p53R175的改变，提升其对降解途径的敏感性，很可能有助于其抗癌活性。以异体移植的小鼠模型抑制肿瘤生长的研究进一步证实，天然食物来源的化合物PEITC也能够在体内复活突变型p53R175的野生型功能，诱导细胞凋亡。这些前所未有的发现证明了突变型p53可以被天然食物来源化合物改变，对癌症预防和治疗有重大的意义。The inventors have surprisingly discovered that PEITC can alter (reactivate) mutant p53 in vitro and in vivo, revealing a novel mechanism of action of natural food-derived compounds. In cells containing mutant p53, PEITC has a stronger inhibitory activity on p53 mutants, such as p53 R175 , p53 R248 , p53 P223, and p53 V274 , with the most common "hot spot" mutations. One of p53 R175 has the strongest inhibitory activity. Mechanistic studies have shown that PEITC induces apoptosis of mutant p53 R175 by restoring p53 wild-type conformation and transcriptional activation. Specifically, in cells treated with PEITC, the altered mutant p53 R175 induces apoptosis by activating the wild-type p53 target, ie, phosphorylating ATM/CHK2, delaying the S and G2/M phases. Further studies of the mechanism indicate that this growth inhibition of PEITC is affected to some extent by the concentration of zinc ions and the redox state of cells. In addition, PEITC sensitizes the p53 R175 mutant to degradation by proteasome and autophagy, and its sensitivity is related to concentration. PEITC induces changes in p53 R175 and increases its sensitivity to degradation pathways, which is likely to contribute to its anticancer activity. The study of tumor growth inhibition by a mouse model of allogeneic transplantation further confirmed that the natural food-derived compound PEITC can also reactivate the wild-type function of mutant p53 R175 in vivo and induce apoptosis. These unprecedented findings demonstrate that mutant p53 can be altered by natural food-derived compounds and is of great significance for cancer prevention and treatment.
本发明的主要优点包括：The main advantages of the invention include:
(a)PEITC是目前发现的唯一针对突变型P53作为靶点的纯天然食物来源***的化合物；(a) PEITC is the only compound found to treat tumors with a pure natural food source targeting mutant P53;
(b)由于PEITC是食物来源化合物，因此其安全性非常高。并且，PEITC不仅可以作为***的药物，而且可以作为预防肿瘤的保健品使用；(b) Since PEITC is a food-derived compound, its safety is very high. Moreover, PEITC can be used not only as a drug for treating tumors, but also as a health care product for preventing tumors;
(c)即使长期服用，PEITC也不易产生耐药性，并且对于其他抗肿瘤药物 已经产生耐药性的肿瘤患者，使用PEITC治疗，仍然能够获得良好的效果；(c) PEITC is not susceptible to drug resistance even after long-term use, and for other anti-tumor drugs Tumor patients who have developed drug resistance can still achieve good results with PEITC treatment;
(d)PEITC既可以单独使用，也可以与其它抗肿瘤药物配合使用；(d) PEITC can be used alone or in combination with other anti-tumor drugs;
(e)PEITC对于其它由于P53突变引起的疾病也能起到很好的治疗效果；(e) PEITC can also have a good therapeutic effect on other diseases caused by P53 mutation;
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.
通用材料和方法General materials and methods
细胞系Cell line
HOP92，OVCAR3和SW620购自NCI DTP,DCDT Tumor Repository,Fredrick,Maryland。H1299、HT29、A549、MDA-MB-231、AU565、SK-BR-3、DU145、SCC003、SCC016、SCC114、SCC122、和MCF7来自Tissue Culture Source Resource,Georgetown University,Washington,DC。所有细胞系都是支原体阴性的，以含10％胎牛血清(FBS)的RPMI1640培养基培养。从ATCC购买的正常的结肠细胞CCD841，用含10％FBS的Eagle’s基础培养基培养。3T3Balb/c纤维母细胞(p53+/+)，则在含10％FBS的Dulbecco改良的Eagle’s培养基中培养。(10)3(p53-/-)小鼠胚胎成纤维细胞(MEFs)和(10)3衍生的p53突变的MEFs[(10)3/175和(10)3/273]在含10％FBS和400μg/mL G418的Dulbecco改良的Eagle’s培养基中培养。MEF(10)3及其衍生的人p53残基R175和R273突变细胞系，由Darren R.Carpizo博士赠送。HOP92, OVCAR3 and SW620 were purchased from NCI DTP, DCDT Tumor Repository, Fredrick, Maryland. H1299, HT29, A549, MDA-MB-231, AU565, SK-BR-3, DU145, SCC003, SCC016, SCC114, SCC122, and MCF7 are from Tissue Culture Source Resource, Georgetown University, Washington, DC. All cell lines were mycoplasma negative and were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS). Normal colon cell CCD841 purchased from ATCC was cultured in Eagle's basal medium containing 10% FBS. 3T3Balb/c fibroblasts (p53 +/+ ) were cultured in Dulbecco's modified Eagle's medium containing 10% FBS. (10) 3 (p53 -/- ) mouse embryonic fibroblasts (MEFs) and (10) 3 derived p53 mutant MEFs [(10) 3/175 and (10) 3/273] in 10% FBS Incubate in Dulbecco's modified Eagle's medium with 400 μg/mL G418. MEF (10) 3 and its derived human p53 residues R175 and R273 mutant cell lines were presented by Dr. Darren R. Carpizo.
细胞增殖试验Cell proliferation assay
如前所述，PEITC对肿瘤细胞增殖的作用通过WST-1试验(Roche)测定。简单的说，取适量PEITC，用DMSO稀释，以使10μl的药物储备液在最终体积为1ml的SK-BR-3细胞(40000细胞/ml)中含有所需的药物浓度并且DMSO浓度为1％。将含PEITC的SK-BR-3细胞培养物加入96孔微量培养板中，每孔为4000个细胞。以1％DMSO作为对照，同时以不含细胞的培养液作为空白对照。培养板在37℃下培养3天，随后添加WST-1试剂孵育2小时。随后以微孔板酶标仪(Bio-Rad)测定OD450。以PEITC处理过的细胞OD450数值及DMSO对照细胞的OD450数值的比值来计算细胞增殖百分比。以类似的试验方法，测定PEITC对以下细胞增殖的影响：p53 siRNA或NS siRNA转染的H1299，HOP92，AU565，OVCAR3，SW620，HT29，A549，MCF7，CCD841，SK-BR-3、DU145、SCC003、SCC016、SCC114、SCC122细胞。As previously stated, the effect of PEITC on tumor cell proliferation was determined by the WST-1 test (Roche). Briefly, an appropriate amount of PEITC was diluted with DMSO to allow 10 μl of the drug stock to contain the desired drug concentration in a final volume of 1 ml of SK-BR-3 cells (40000 cells/ml) and a DMSO concentration of 1%. . The PEITC-containing SK-BR-3 cell culture was added to a 96-well microplate at 4000 cells per well. 1% DMSO was used as a control, and a cell-free medium was used as a blank control. The plate was incubated at 37 ° C for 3 days, followed by incubation with WST-1 reagent for 2 hours. Followed by a microtiter plate spectrophotometer (Bio-Rad) was measured OD 450. PEITC ratio in treated cells and control cells OD 450 value of OD 450 value of the percentage of cell proliferation is calculated DMSO. A similar test method was used to determine the effect of PEITC on cell proliferation: p53 siRNA or NS siRNA transfected H1299, HOP92, AU565, OVCAR3, SW620, HT29, A549, MCF7, CCD841, SK-BR-3, DU145, SCC003 , SCC016, SCC114, SCC122 cells.
细胞转染Cell transfection
p53 siRNA购自SMARTpool(Thermo Scientific/Dharmacon,Lafayette,CO, USA)。siRNA按制造商(Invitrogen)的说明书采用Lipofectamine 2000转染。简单地说，转染前，细胞于10cm培养皿中培养24小时，细胞汇合度为50-60％。取siRNA(0.430nmol)和43μL Lipofectamine 2000，与1mL的Opti-MEM(Invitrogen)混合。将混合物加入到细胞培养基后，培养6小时。24小时后，进行第二次类似的转染。于初次转染的72小时后，收获细胞，制备裂解物或用指定浓度的PEITC或DMSO处理，再用如前所述的WST-1试剂(Roche)测定肿瘤细胞的增殖。对于SK-BR-3细胞中ATG5的敲低实验，ATG5siRNA(Santa Cruz Biotechnology)转染采用如前所述的方法，但单次转染为6小时。24小时后，收获转染细胞以制备裂解物或用PEITC处理转染细胞4小时后制备裂解物。P53 siRNA was purchased from SMARTpool (Thermo Scientific/Dharmacon, Lafayette, CO, USA). siRNA was transfected with Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Briefly, before transfection, cells were cultured for 24 hours in a 10 cm dish with a cell confluence of 50-60%. siRNA (0.430 nmol) and 43 μL Lipofectamine 2000 were taken and mixed with 1 mL of Opti-MEM (Invitrogen). After the mixture was added to the cell culture medium, it was cultured for 6 hours. After 24 hours, a second similar transfection was performed. After 72 hours of initial transfection, cells were harvested, lysates were prepared or treated with the indicated concentrations of PEITC or DMSO, and tumor cell proliferation was determined using WST-1 reagent (Roche) as previously described. For the knockdown assay of ATG5 in SK-BR-3 cells, ATG5 siRNA (Santa Cruz Biotechnology) transfection was performed as previously described, but with a single transfection for 6 hours. After 24 hours, transfected cells were harvested to prepare lysates or transfected cells were treated with PEITC for 4 hours to prepare lysates.
按制造商(Invitrogen)的说明书，用Lipofectamine 2000转染pcDNA3、pcDNA3-wtp53、pcDNA3-p53R175和pcDNA3-p53R273质粒。简单地说，转染前，细胞于10cm培养皿中培养24小时，细胞汇合度为50-60％。将质粒(14μg)和43μLLipofectamine 2000，与1mL的Opti-MEM(Invitrogen)混合。添加混合物到细胞培养基中，培养6小时。24小时后，转染细胞用PEITC处理，进行如前所述的WST-1试验或Annexin V染色。转染细胞保存在含10％FBS和400μg/mL G418的RPMI 1640培养基中。The pcDNA3, pcDNA3-wtp53, pcDNA3-p53 R175 and pcDNA3-p53 R273 plasmids were transfected with Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Briefly, before transfection, cells were cultured for 24 hours in a 10 cm dish with a cell confluence of 50-60%. Plasmid (14 μg) and 43 μL of Lipofectamine 2000 were mixed with 1 mL of Opti-MEM (Invitrogen). The mixture was added to the cell culture medium and cultured for 6 hours. After 24 hours, the transfected cells were treated with PEITC and subjected to WST-1 assay or Annexin V staining as described above. Transfected cells were maintained in RPMI 1640 medium containing 10% FBS and 400 μg/mL G418.
ELISAELISA
以含100μg/mL氨苄青霉素的LB培养基，于37℃下培养pGeX4T1-mutp53R175H质粒转化后的大肠杆菌细胞(BL21DE3)，直至600nm处OD值为0.4。在同一温度下，添加0.5mM异丙基-1-硫代-β-半乳糖皮蒽(IPTG)，持续摇瓶3小时，以诱导重组蛋白表达。细菌沉淀后，加入裂解缓冲液(250mM Tris-HCl，pH7.5，1mM EDTA，150mM NaCl，1％的Triton X-100，0.5％的Nonidet P-40，0.1％的Tween 20，0.2％SDS，1M DTT和蛋白酶抑制剂)使细胞裂解，并反复冻融三次，然后超声破碎(三个循环，每次一分钟)。超声破碎后，于4℃下18500×g离心30分钟，溶液变澄清。将上清液转移一支新管内，保存。以十二烷基肌氨酸钠缓冲液(裂解缓冲液+2％十二烷基肌氨酸钠)重悬沉淀，再用探针超声破碎(三个循环，每次一分钟)。将上述两步得到的上清液部分，以1x PBS按1:1的比例稀释后，用谷胱甘肽-琼脂糖小球(σ，G4510)，4℃下匀速旋转，孵育2小时。用PBS清洗几次后，以洗脱缓冲液(100mM Tris-HCl，pH 8.0，10mM GSH(σ，G4251)，300mM NaCl，1mM二硫苏糖醇(DTT)和蛋白酶抑制剂)洗脱蛋白质。另取25ng重组GST-突变体p53R175H，以pH 7.5的100mM Tris-Cl、300mM NaCl缓冲液稀释。用DMSO或4μM PEITC处理，在冰上孵育1小时。得到的蛋白样本加入ELISA板，4℃下孵育2小时。1x PBST清洗(含有0.05％Tween-20)，用5％的脱脂牛奶4℃封闭2小时。清洗后，添加以1x PBST按1:1000的比例稀释的小鼠一抗(PAB240或PAB1620)，继续于4℃下孵育过夜。1x PBST清洗，于4℃下添加辣根过氧化酶 标记的抗鼠的二抗反应1小时。1x PBST清洗，加入底物(SuperSignal ELISA Pico Chemiluminescent Substrate,Thermo Scientific)，再于450nM测定化学发光强度。纯化的GST和纯化的野生型p53(Thermo Scientific)作为对照。为了确定ZnCl2的影响，另取25ng重组GST-突变体p53R175H，以pH 7.5的100mM Tris-Cl、300mM NaCl缓冲液稀释，用2.5μM ZnCl2或4μM PEITC单独处理或共处理后，冰上孵育1小时。然后按上述进行ELISA试验。The pGeX4T1-mutp53 R175H plasmid-transformed E. coli cells ( BL21DE3) were cultured in LB medium containing 100 μg/mL ampicillin at 37 ° C until the OD value at 600 nm was 0.4. At the same temperature, 0.5 mM isopropyl-1-thio-β-galactosidin (IPTG) was added, and the flask was continuously shaken for 3 hours to induce expression of the recombinant protein. After bacterial precipitation, lysis buffer (250 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 0.1% Tween 20, 0.2% SDS, The cells were lysed by 1 M DTT and protease inhibitors and repeatedly thawed three times and then sonicated (three cycles, one minute each). After sonication, the mixture was centrifuged at 18,500 x g for 30 minutes at 4 ° C, and the solution became clear. Transfer the supernatant to a new tube and store. The pellet was resuspended in sodium lauryl sarcosinate buffer (lysis buffer + 2% sodium lauryl sarcosinate) and sonicated with a probe (three cycles, one minute each). The supernatant fraction obtained in the above two steps was diluted in a ratio of 1:1 with 1×PBS, and then incubated with glutathione-agarose beads (σ, G4510) at a constant rate at 4 ° C for 2 hours. After washing several times with PBS, the protein was eluted with an elution buffer (100 mM Tris-HCl, pH 8.0, 10 mM GSH (σ, G4251), 300 mM NaCl, 1 mM dithiothreitol (DTT) and protease inhibitor). Another 25 ng of recombinant GST-mutant p53 R175H was diluted with 100 mM Tris-Cl, 300 mM NaCl buffer, pH 7.5. Treat with DMSO or 4 μM PEITC and incubate for 1 hour on ice. The resulting protein samples were added to an ELISA plate and incubated for 2 hours at 4 °C. 1x PBST wash (containing 0.05% Tween-20), blocked with 5% skim milk for 4 hours at 4 °C. After washing, the mouse primary antibody (PAB240 or PAB1620) diluted 1:1000 in 1x PBST was added and incubation was continued at 4 °C overnight. After washing with 1 x PBST, a horseradish peroxidase-labeled anti-mouse secondary antibody was added at 4 ° C for 1 hour. 1x PBST was washed, substrate (SuperSignal ELISA Pico Chemiluminescent Substrate, Thermo Scientific) was added, and chemiluminescence intensity was measured at 450 nM. Purified GST and purified wild-type p53 (Thermo Scientific) served as controls. To determine the effect of ZnCl 2 , another 25 ng of recombinant GST-mutant p53 R175H was diluted with 100 mM Tris-Cl, pH 7.5, 300 mM NaCl buffer, treated with 2.5 μM ZnCl 2 or 4 μM PEITC alone or co-treated, on ice. Incubate for 1 hour. The ELISA assay was then performed as described above.
Annexin V染色Annexin V staining
按制造商(Biolegend)的说明书进行Annexin V染色。简言之，以PEITC或作为对照的DMSO处理待检测细胞，3天后收集细胞，以1x PBS洗一次，用0.5mL Annexin V结合缓冲液重悬。离心收集细胞，将5μL结合Annexin V的荧光染料添加到残留的缓冲液中，在避光条件下室温孵化15分钟，随后加入0.5mL Annexin V结合缓冲液和5μL 0.1μg/mL的PI染色液。再以BD LSRFORTESSA流式仪(BD Biosciences)分析细胞。为了明确Nutlin-3处理对细胞诱导凋亡的影响，用指定浓度的PEITC、Nutlin-3单独处理、或两者共处理、或DMSO处理待检测细胞24或72小时。如前所述，处理好的细胞用Annexin V染色。为了确定还原剂NAC或PEG-过氧化氢酶对PEITC诱导细胞凋亡的效果，单独用PEITC、还原剂或氧化剂、或用PEITC结合还原剂或氧化剂，处理待检测细胞。如前所述，处理好的细胞用Annexin V染色。用DMSO或指定浓度的PEITC处理ATG5siRNA或NS siRNA转染的细胞72小时。为了检测细胞凋亡，用细胞死亡检测ELISA结合光度酶分析法(Roche)，定量测定处于细胞凋亡期的胞浆组蛋白相关DNA片段。Annexin V staining was performed according to the manufacturer's instructions (Biolegend). Briefly, cells to be tested were treated with PEITC or DMSO as a control, and cells were collected 3 days later, washed once with 1 x PBS, and resuspended in 0.5 mL of Annexin V binding buffer. The cells were collected by centrifugation, 5 μL of Annexin V-conjugated fluorescent dye was added to the residual buffer, and incubated for 15 minutes at room temperature in the dark, followed by 0.5 mL of Annexin V binding buffer and 5 μL of 0.1 μg/mL of PI staining solution. The cells were analyzed by BD LSRFORTESSA flow meter (BD Biosciences). To clarify the effect of Nutlin-3 treatment on cell-induced apoptosis, cells to be tested were treated with the indicated concentrations of PEITC, Nutlin-3 alone, or both, or DMSO for 24 or 72 hours. The treated cells were stained with Annexin V as previously described. To determine the effect of reducing agent NAC or PEG-catalase on PEITC-induced apoptosis, the cells to be tested are treated with PEITC, a reducing agent or an oxidizing agent alone, or with PEITC in combination with a reducing agent or an oxidizing agent. The treated cells were stained with Annexin V as previously described. ATG5 siRNA or NS siRNA transfected cells were treated with DMSO or the indicated concentrations of PEITC for 72 hours. To detect apoptosis, a cytoplasmic histone-associated DNA fragment in the apoptotic phase was quantified using a cell death assay ELISA in combination with photometric enzyme assay (Roche).
免疫荧光染色Immunofluorescence staining
在四孔的载玻片(Lab-Tek)中，用PEITC(4或6μM)或作为对照的1％DMSO，处理待检测细胞6小时。细胞用1x PBS清洗两次，然后用甲醛(3.7％)在室温(RT)下固定15分钟。固定的细胞用0.5％的Triton X-100(σ)室温下处理5分钟。细胞以含0.5％Tween-20的1x PBS清洗四次，用10％的山羊血清(σ)4℃下封闭过夜。用0.1％的Tween-20清洗细胞四次后，用能区分突变型或野生型p53的小鼠源一抗PAB240(1:300,Calbiochem)或PAB1620(1:300,Calbiochem)，4℃下孵育过夜。以0.1％的Tween-20清洗四次后,细胞用Alexa Fluor 488-标记的山羊抗小鼠IgG(1:400,Invitrogen)，于室温下孵育2小时。以0.1％的Tween-20清洗细胞四次，用含DAPI的Prolong Gold Anti-Fade试剂(Invitrogen)染核。盖上盖玻片,细胞在避光室温下处理24小时。用Zeiss LSM 510 NLO带Plan-Apochromat 63×1.4-孔径油镜，Axiovert 200M倒置激光扫描显微镜进行免疫荧光分析。图像用Photomultiplier Tubes(PMT)检测器采集，Image J软件分析。荧光染色强度用Metamorph软件定量。The cells to be tested were treated with PEITC (4 or 6 μM) or 1% DMSO as a control in a four-well slide (Lab-Tek) for 6 hours. The cells were washed twice with 1 x PBS and then fixed with formaldehyde (3.7%) for 15 minutes at room temperature (RT). The fixed cells were treated with 0.5% Triton X-100 (σ) for 5 minutes at room temperature. The cells were washed four times with 1 x PBS containing 0.5% Tween-20 and blocked overnight with 10% goat serum (σ) at 4 °C. After washing the cells four times with 0.1% Tween-20, they were incubated with mouse-derived PAB240 (1:300, Calbiochem) or PAB1620 (1:300, Calbiochem), which can distinguish between mutant or wild-type p53, at 4 °C. overnight. After washing four times with 0.1% Tween-20, cells were incubated with Alexa Fluor 488-labeled goat anti-mouse IgG (1:400, Invitrogen) for 2 hours at room temperature. The cells were washed four times with 0.1% Tween-20 and stained with DAPI-containing Prolong Gold Anti-Fade reagent (Invitrogen). The coverslips were covered and the cells were treated for 24 hours at room temperature in the dark. Immunofluorescence analysis was performed using a Zeiss LSM 510 NLO with a Plan-Apochromat 63 x 1.4-aperture oil mirror and an Axiovert 200M inverted laser scanning microscope. Images were acquired with a Photomultiplier Tubes (PMT) detector and analyzed by Image J software. Fluorescence staining intensity was quantified using Metamorph software.
为了测定肿瘤细胞中PEITC对γ-H2AX灶形成的影响，p53 siRNA或NS siRNA 转染的待检测细胞，以4μM PEITC或作为对照的1％DMSO在37℃下处理3天。如前所述，以小鼠抗-γ-H2AX单克隆抗体(1:300,Upstate)作一抗，细胞用甲醛固定处理，以免疫染色检测γ-H2AX灶。To determine the effect of PEITC on the formation of γ-H2AX foci in tumor cells, p53 siRNA or NS siRNA The transfected cells to be tested were treated with 4 μM PEITC or 1% DMSO as a control at 37 ° C for 3 days. As described above, a mouse anti-γ-H2AX monoclonal antibody (1:300, Upstate) was used as a primary antibody, and the cells were fixed with formaldehyde, and γ-H2AX was detected by immunostaining.
免疫共沉淀Immunoprecipitation
待检测细胞以指定浓度的PEITC或作为对照的1％DMSO处理6小时。为了制备细胞裂解物，将收获的细胞以1x PBS清洗一次，用含有蛋白酶抑制剂(Roche Molecular Biochemicals)的裂解缓冲液(20mM Tris-Cl(pH 8.0)，137mM氯化钠，10％甘油，1％NP-40，2mM EDTA)将细胞沉淀重新混悬，冰上孵育30分钟。以离心机于18500×g、4℃下将细胞悬液离心10分钟，收集上清液。上清液用裂解缓冲液稀释，向200μg裂解物中加入适量proteinG Agarose(Roche)于4℃轻轻地摇晃1小时。预纯化后的裂解物，于4℃下加入小鼠源PAB240抗体(2μg，Calbiochem)轻轻摇晃2小时。然后将proteinG Agarose-加入悬浮液，4℃下孵育2小时。沉淀用添加有蛋白酶抑制剂的裂解缓冲液清洗四次，免疫沉淀物通过在Laemml i缓冲液中沸腾进行洗脱，然后进行4-12％的SDS-PAGE电泳。免疫沉淀的p53蛋白以FL393(Santa Cruz Biotechnology)作为一抗，进行免疫印迹检测。二抗采用过氧化物酶标记的抗小鼠IgG(1:2000，GE healthcare)。根据制造商的说明书(Amersham)，使用ECL Prime Western Blot检测试剂盒检测蛋白印迹。作为对照，清除印迹后，再以抗p53(DO-1)抗体(1:1000，Santa Cruz Biotechnology)或抗GAPDH抗体(1:2000，Novus Biologicals)重新检测。用Gene Tools软件测定经PEITC处理后的样本中，相对于DMSO对照样品，其p53条带的密度。Cells to be tested were treated with PERTC at the indicated concentrations or 1% DMSO as control for 6 hours. To prepare cell lysates, the harvested cells were washed once with 1×PBS, using a lysis buffer containing protease inhibitor (Roche Molecular Biochemicals) (20 mM Tris-Cl (pH 8.0), 137 mM sodium chloride, 10% glycerol, 1 The cell pellet was resuspended in %NP-40, 2 mM EDTA and incubated on ice for 30 minutes. The cell suspension was centrifuged at 18,500 x g, 4 ° C for 10 minutes in a centrifuge, and the supernatant was collected. The supernatant was diluted with lysis buffer, and an appropriate amount of proteinG Agarose (Roche) was added to 200 μg of the lysate and gently shaken at 4 ° C for 1 hour. The pre-purified lysate was gently shaken for 2 hours at 4 ° C by adding mouse-derived PAB240 antibody (2 μg, Calbiochem). ProteinG Agarose- was then added to the suspension and incubated for 2 hours at 4 °C. The pellet was washed four times with a lysis buffer supplemented with a protease inhibitor, and the immunoprecipitate was eluted by boiling in Laemml i buffer, followed by 4-12% SDS-PAGE electrophoresis. The immunoprecipitated p53 protein was detected by immunoblotting using FL393 (Santa Cruz Biotechnology) as a primary antibody. The secondary antibody was a peroxidase-labeled anti-mouse IgG (1:2000, GE healthcare). Western blots were detected using the ECL Prime Western Blot assay kit according to the manufacturer's instructions (Amersham). As a control, the blot was removed and re-detected with anti-p53 (DO-1) antibody (1:1000, Santa Cruz Biotechnology) or anti-GAPDH antibody (1:2000, Novus Biologicals). The density of the p53 bands in the PEITC-treated samples relative to the DMSO control samples was determined using the Gene Tools software.
裂解液的制备及Western blot分析Preparation of lysate and Western blot analysis
采用不同的裂解液以制备可溶性、不溶性和全细胞裂解组分。以1x PBS清洗两次，收集细胞，用来制备裂解物(可溶的)。将RIPA缓冲液(10mM磷酸钠(pH 7.2)，300mMNaCl，0.1％SDS，1％Nonidet P-40，1％脱氧胆酸盐，2mM EDTA)加入细胞中，于冰浴中放置30分钟。然后将细胞悬液于18500×g在4℃下离心10分钟，收集上清液，除非另有提及。沉淀物即为不溶性组分。将不溶性组分溶解在含有2％SDS的裂解缓冲液中(65mM Tris-HCl(pH 8.0),150mM NaCl,2％SDS,50mM DTT)。如上所述，将所收获细胞沉淀以2％的SDS裂解缓冲液溶解以制备全细胞裂解组分。将该组分于18500×g4℃下离心10分钟收集其中组分。然后取30-50μg的裂解物进行4–12％SDS/PAGE电泳。将蛋白质转移到PVDF膜上，根据制造商(Amersham)的说明书ECL Prime Western blot进行蛋白印迹检测。其中p21、Bax、ATM、pATM S1981、CHK2、pCHK2Thr68和p53(DO-1)的抗体分别购自于Santa Cruz Biotechnology，GAPDH抗体购自Novus Biologicals。ATG5(1:1000，细胞信号传导)的抗体由Dr.Shivendra Singh赠送。 Different lysates were used to prepare soluble, insoluble, and whole cell lysing components. The cells were washed twice with 1 x PBS and the cells were collected for preparation of lysate (soluble). RIPA buffer (10 mM sodium phosphate (pH 7.2), 300 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate, 2 mM EDTA) was added to the cells and allowed to stand in an ice bath for 30 minutes. The cell suspension was then centrifuged at 18,500 x g for 10 minutes at 4 ° C, and the supernatant was collected unless otherwise mentioned. The precipitate is an insoluble component. The insoluble fraction was dissolved in a lysis buffer containing 2% SDS (65 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2% SDS, 50 mM DTT). The harvested cell pellet was dissolved in 2% SDS lysis buffer as described above to prepare a whole cell lysing fraction. The fraction was centrifuged at 18,500 x g for 4 minutes to collect the components therein. Then 30-50 μg of lysate was taken for 4–12% SDS/PAGE electrophoresis. Proteins were transferred to PVDF membranes and Western blot assays were performed according to the manufacturer's (Amersham) instructions ECL Prime Western blot. Antibodies for p21, Bax, ATM, pATM S1981, CHK2, pCHK2Thr68 and p53 (DO-1) were purchased from Santa Cruz Biotechnology, respectively, and GAPDH antibodies were purchased from Novus Biologicals. ATG5 (1:1000, cell signaling) antibodies were donated by Dr. Shivendra Singh.
染色质分离Chromatin separation
将待检测细胞分别用指定浓度PEITC或作为对照的DMSO处理4小时。细胞用胰蛋白酶消化后于500×g离心5分钟收集。细胞沉淀以冰预冷的PBS洗涤一次，并转移至1.5mL微量离心管中于500×g离心2分钟。细胞沉淀于-80℃储存，然后按照生产商的说明书(Subcel lular protein fractionation kit,Thermo Scientific)分离染色质中核可溶性部分和染色质结合蛋白部分。取10微克来自PEITC或DMSO处理后的细胞样品可溶性核提取物与染色质结合核提取物蛋白进行4-12％SDS-PAGE电泳，并将蛋白转移到PVDF膜。用p53(DO-1)抗体(1:1000，Santa Cruz Biotechnology)进行蛋白免疫印迹测定p53含量。以组蛋白H3和TopoII B作为染色质和可溶性核组分的内标。组蛋白H3多克隆抗体购于(Thermo Scientific)和，鼠抗topoii B单克隆抗体购于(Santa Cruz Biotechnology)。The cells to be tested were treated with the indicated concentration of PEITC or DMSO as a control for 4 hours, respectively. The cells were trypsinized and collected by centrifugation at 500 xg for 5 minutes. The cell pellet was washed once with ice-cold PBS and transferred to a 1.5 mL microcentrifuge tube and centrifuged at 500 xg for 2 minutes. The cell pellet was stored at -80 ° C and the chromatin-soluble fraction and chromatin-binding protein fraction were separated in the chromatin according to the manufacturer's instructions (Subcel lular protein fractionation kit, Thermo Scientific). Ten micrograms of cell extracts from PEITC or DMSO treated with soluble nuclear extract and chromatin-bound nuclear extract protein were subjected to 4-12% SDS-PAGE electrophoresis and the protein was transferred to a PVDF membrane. The p53 content was determined by Western blotting with p53 (DO-1) antibody (1:1000, Santa Cruz Biotechnology). Histone H3 and TopoII B were used as internal standards for chromatin and soluble nuclear components. Histone H3 polyclonal antibodies were purchased from (Thermo Scientific) and mouse anti-topoii B monoclonal antibodies were purchased from (Santa Cruz Biotechnology).
RNA提取和定量RT-PCRRNA extraction and quantitative RT-PCR
待检测细胞用QiagenRNA提取试剂盒提取细胞RNA，采用高容量的RNA-cDNA转换试剂盒合成cDNA(Applied Biosystems，Invitrogen)，采用TaqMan RT-PCR(qRT-PCR)(Applied Biosystems，Invitrogen公司)定量方法测定基因表达水平。并以GAPDH归一化，结果以平均值及三个重复测定的标准偏差来表示。异体移植瘤组织的RNA亦以QIAGEN试剂盒提取，然后进行qRT-PCR，经GAPDH归一化处理测定基因表达水平。计算PEITC处理组与对照组的每个肿瘤基因表达水平的变化倍数，并以平均值及标准偏差表示。The cells to be detected were extracted with the Qiagen RNA extraction kit, and cDNA was synthesized using a high-capacity RNA-cDNA conversion kit (Applied Biosystems, Invitrogen), and quantified by TaqMan RT-PCR (qRT-PCR) (Applied Biosystems, Invitrogen). The gene expression level was determined. Normalized by GAPDH, the results were expressed as the mean and the standard deviation of the three replicates. RNA of allograft tumor tissues was also extracted with QIAGEN kit, followed by qRT-PCR, and gene expression levels were determined by GAPDH normalization. The fold change in the expression level of each tumor gene in the PEITC treatment group and the control group was calculated and expressed as mean and standard deviation.
谷胱甘肽水平测定Glutathione level determination
使用GSH/GSSG-GLO谷胱甘肽检测试剂盒(Promega)，测定还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)的水平。简而言之，用PEITC或作为对照的DMSO处理待检测细胞4小时，根据制造商(Promega)的说明书处理细胞，测定谷胱甘肽。The levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined using the GSH/GSSG-GLO glutathione detection kit (Promega). Briefly, cells to be tested were treated with PEITC or DMSO as a control for 4 hours, and cells were treated according to the manufacturer's (Promega) instructions to determine glutathione.
转导基因荧光素酶活性的检测Detection of transduction gene luciferase activity
将Bert Vogelstein(Addgene plasmid#16451)赠送的编码有能与p53 WT蛋白特定位点结合的p21启动子序列的WWP-Luc(p21/WAF1启动子)质粒，转染到待检测细胞中，以4或6μM PEITC分别处理24小时。按照制造商(Luciferase assay,Promega)说明书，检测细胞裂解物转导基因荧光素酶活性。A WWP-Luc (p21/WAF1 promoter) plasmid encoding a p21 promoter sequence capable of binding to a specific site of the p53 WT protein, which was donated by Bert Vogelstein (Addgene plasmid #16451), was transfected into the cells to be detected to 4 Or 6μM PEITC for 24 hours. The cell lysate transduction gene luciferase activity was assayed according to the manufacturer's (Luciferase assay, Promega) instructions.
细胞周期分析Cell cycle analysis
待检测细胞分别以PEITC、Nutlin-3或两者合用处理24或72小时，以DMSO作为对照。收集细胞以流式细胞仪进行细胞周期检测分析。简之，细胞以不含钙、镁离子的PBS清洗，胰蛋白酶消化5分钟，收集细胞并于190xg、4℃离心3分钟。所收集细胞以PBS洗涤一次，重新悬浮于1毫升70％乙醇中，–20℃储存过夜。然后，在420×g，离心10分钟后收集沉淀的细胞颗粒，以1毫升冰预冷 的PBS清洗一次，重悬于1毫升新鲜制备的PI染色液(PBS含0.1％Triton X-100、0.05μg/mL碘化丙啶，0.1mg/mL的RNase(Sigma))中。细胞悬液先在暗处、室温放置30分钟，然后于4℃放置30分钟。以Becton Dickinson FACS检测样品，并以Mod Fit程序(Verity Software House)进行数据分析。The cells to be tested were treated with PEITC, Nutlin-3 or both for 24 or 72 hours, respectively, with DMSO as a control. The collected cells were subjected to cell cycle detection analysis by flow cytometry. Briefly, the cells were washed with PBS containing no calcium or magnesium ions, trypsinized for 5 minutes, and the cells were collected and centrifuged at 190 x g for 3 minutes at 4 °C. The collected cells were washed once with PBS, resuspended in 1 ml of 70% ethanol, and stored at 20 ° C overnight. Then, at 420 × g, after centrifugation for 10 minutes, the precipitated cell pellets were collected and pre-cooled with 1 ml of ice. The PBS was washed once and resuspended in 1 ml of freshly prepared PI staining solution (PBS containing 0.1% Triton X-100, 0.05 μg/mL propidium iodide, 0.1 mg/mL RNase (Sigma)). The cell suspension was first placed in the dark at room temperature for 30 minutes and then placed at 4 ° C for 30 minutes. Samples were tested with a Becton Dickinson FACS and analyzed by the Mod Fit program (Verity Software House).
PEITC处理后的ATM和CHK2检测ATM and CHK2 detection after PEITC processing
待检测细胞以DMSO、ATZ、NAC、PEG-过氧化氢酶或单一的PEITC或以ATZ或NAC或PEG-过氧化氢酶与PEITC联合处理4小时。然后于1600×g、4℃、离心10分钟后收集细胞，PBS洗涤一次，用含有蛋白酶和磷酸酶抑制剂混合物的RIPA缓冲液重悬(10mM磷酸钠(pH 7.2)300mM NaCl,0.1％SDS,1％Nonidet P-40,1％脱氧胆酸盐，和2mM EDTA)，冰上放置30min，于18500×g、4℃离心10分钟，收集上清液。取200μg的裂解物进行4-12％SDS/PAGE电泳。电泳后，蛋白转移到PVDF膜上，以anti-pATM Ser1981抗体(1:500)(Santa Cruz Biotechnology)或anti-pCHK2Thr68抗体(1:500)(Santa Cruz Biotechnology)进行蛋白印迹检测。二抗为过氧化物酶标记的抗小鼠IgG(1:1000，GE Healthcare)。用ECL Prime Western Blot Detection Kit，按照制造商(Amersham)的说明书，显示印迹。作为对照，剥离印迹后，用anti-ATM抗体(1:500，Santa Cruz Biotechnology)或Chk2抗体(Santa Cruz Biotechnology)重新检测。The cells to be tested were treated with DMSO, ATZ, NAC, PEG-catalase or a single PEITC or with ATZ or NAC or PEG-catalase for 4 hours in combination with PEITC. The cells were then collected at 1600 x g, 4 ° C, and centrifuged for 10 minutes, washed once with PBS, and resuspended in RIPA buffer containing a mixture of protease and phosphatase inhibitor (10 mM sodium phosphate (pH 7.2) 300 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate, and 2 mM EDTA) were placed on ice for 30 min, centrifuged at 18500 x g for 10 minutes at 4 ° C, and the supernatant was collected. 200 μg of the lysate was taken for 4-12% SDS/PAGE electrophoresis. After electrophoresis, the protein was transferred to a PVDF membrane and subjected to Western blot detection with anti-pATM Ser1981 antibody (1:500) (Santa Cruz Biotechnology) or anti-pCHK2Thr68 antibody (1:500) (Santa Cruz Biotechnology). The secondary antibody was a peroxidase-labeled anti-mouse IgG (1:1000, GE Healthcare). The blot was visualized using the ECL Prime Western Blot Detection Kit according to the manufacturer's instructions (Amersham). As a control, after stripping the blot, it was re-detected with an anti-ATM antibody (1:500, Santa Cruz Biotechnology) or Chk2 antibody (Santa Cruz Biotechnology).
小鼠移植瘤模型Mouse xenograft model
二十只雌性无胸腺nu/nu BALB/C小鼠(CAnN.Cg-Foxn1nu/Crl,4-6周龄)购自Charles River实验室(Wilmington，MA)。所有体内研究和肿瘤收集符合实验动物保护及使用委员会(IACUC)的程序和指导原则。将小鼠称重后置于聚碳酸酯笼中(五只/笼，每笼动物平均体重和方差相同)一周。小鼠随意取食水，饲料为AIN-93M。隔离一周后，将小鼠随机分配到AIN-93M饲料对照组或含有PEITC的AIN-93M饲料组(5μmol PEITC/g饲料，10只/组)。这2种饲料均由Research Diets(New Brunswick,NJ)提供。所采用的动物数是根据以前试验结果决定的，该试验已证明所采用的SK-BR-3细胞移植瘤小鼠模型的动物数所获结果具有统计学意义。饲料中所含的PEITC浓度也基于之前的小鼠生物试验结果。如前所述，采用乙酸乙酯萃取饲料中的PEITC后，加入1,2-苯二硫酚与ITC环合反应测定，实测的浓度为4.97±0.16μmol/g。采用单因素方差分析，计算样品之间PEITC的差异，表明无统计学意义(α＝0.05)。Twenty female athymic nu/nu BALB/C mice (CAnN.Cg-Foxn1nu/Crl, 4-6 weeks old) were purchased from Charles River Laboratories (Wilmington, MA). All in vivo studies and tumor collections are in accordance with the procedures and guidelines of the Laboratory Animal Protection and Use Committee (IACUC). The mice were weighed and placed in a polycarbonate cage (five/cage, with the same average body weight and variance for each cage) for one week. The mice were given water ad libitum and the feed was AIN-93M. One week after isolation, mice were randomly assigned to the AIN-93M feed control group or the AIN-93M feed group containing PEITC (5 μmol PEITC/g feed, 10/group). Both feeds were supplied by Research Diets (New Brunswick, NJ). The number of animals used was determined based on the results of previous tests which have demonstrated statistically significant results for the number of animals in the SK-BR-3 cell xenograft mouse model. The concentration of PEITC contained in the feed is also based on previous mouse bioassay results. As described above, after extracting PEITC from the feed with ethyl acetate, 1,2-benzenedithiol was added to the ITC cyclization reaction, and the measured concentration was 4.97±0.16 μmol/g. One-way ANOVA was used to calculate the difference in PEITC between samples, indicating no statistical significance (α=0.05).
小鼠饲料隔天添加一次。在人乳腺癌异体移植瘤小鼠模型中，对照组与PEITC组小鼠饲喂一周后，将处于指数生长期的2x106的肿瘤细胞(悬浮于50μL Matrigel中)，注射到小鼠左、右乳腺脂肪垫位置(“癌症化学预防”设置)。实验过程中无小鼠死亡。每周用游标卡尺从外部测定肿瘤大小，评估肿瘤的形成和生长，持续进行10个生物测定周期。按照公式L x W2x 0.523计算肿瘤体 积。Mouse feed is added every other day. In a mouse model of human breast xenograft tumor, 2×10 6 tumor cells (suspended in 50 μL Matrigel) in the exponential growth phase were injected into the mice and left and right after one week of feeding and PEITC mice. Breast fat pad position ("cancer chemoprevention" setting). No mice died during the experiment. Tumor size was measured externally using a vernier caliper weekly to assess tumor formation and growth, and 10 bioassay cycles were continued. The tumor volume was calculated according to the formula L x W 2 x 0.523.
在***癌体外移植瘤小鼠模型中，由于肿瘤位置的特殊性，并且小鼠的肿瘤体积小，不容易从外部测量。还是由于体积小，一些小鼠的肿瘤在数周体积的测量值仍然参差不齐。为此，最终的肿瘤体积计算，只采纳符合生长模式、无异常波动的动物肿瘤数据(n＝7)。采用GraphPad软件没有分析到异常数据。其余操作与人乳腺癌异体移植瘤模型一致。In the mouse model of prostate cancer in vitro xenografts, due to the particularity of the tumor location, and the tumor volume of the mouse is small, it is not easy to measure from the outside. Or because of the small size, the measurement of tumors in some mice in several weeks is still uneven. To this end, the final tumor volume was calculated using only animal tumor data consistent with growth patterns and no abnormal fluctuations (n=7). Abnormal data was not analyzed using the GraphPad software. The rest of the procedure was consistent with the human breast xenograft model.
所有的动物处死后，采集肿瘤，按下文描述的方法用苏木精和伊红(H&E)染色。试验过程中，每周称量动物体重一次。按前述以1,2-本二硫酚与ITCS环合反应，测定尸检时收集的动物血液中的ITC浓度。治疗组及对照组动物的血液中PEITC浓度分别为1.13±0.15μm(n＝3)和0.37±0.03μm(n＝2)。由于各组动物饲喂不同的饲料，试验无法按盲法进行；但是试验中，采用盲法完成肿瘤样本采集和组织病理学分析。病理学家按盲法检测H&E染色组织切片中的肿瘤发生率。After all animals were sacrificed, tumors were harvested and stained with hematoxylin and eosin (H&E) as described below. During the test, the animals were weighed once a week. The concentration of ITC in the blood of the animals collected at the time of autopsy was determined by cyclization of 1,2-dithiol with ITC S as described above. The PEITC concentrations in the blood of the treatment group and the control group were 1.13 ± 0.15 μm (n = 3) and 0.37 ± 0.03 μm (n = 2), respectively. Because the animals in each group were fed different feeds, the trial could not be performed blindly; however, in the trial, tumor sample collection and histopathological analysis were performed blindly. Pathologists blinded the incidence of tumors in H&E-stained tissue sections.
移植瘤的组织病理学分析、免疫组织化学、Western blot和qRT-PCR分析Histopathological analysis, immunohistochemistry, Western blot and qRT-PCR analysis of transplanted tumors
以H&E染色组织切片病理学检查来确定其是否为肿瘤。按照乔治城大学Histopathology and Tissue Shared Resources标准进行免疫组织化学检查。简之，将组织切成5μm的薄片，以二甲苯脱蜡和梯度醇脱水，再将组织切片放入98℃含0.05％Tween的10nM柠檬酸盐缓冲液(pH 6)中浸泡20分钟，进行热诱导抗原修复(HIER)。免疫组化染色采用Dako的辣根过氧化物酶标记的聚合物(k4001，k4003)，根据制造商指南进行。简之，将切片以3％过氧化氢和10％正常山羊血清处理各10分钟，然后加入一抗p53(DO-7)(1:800，Dako)，在室温(RT)放置1小时，或加入Ki-67(1:15，NovusBiologicals)抗体,于4℃放置过夜。然后将切片暴露于适当的HRP标记的聚合物30分钟和DAB显色液中(Dako)5分钟。切片再以苏木精复染(Fisher,Harris Modified Hematoxylin)、在1％氢氧化铵中变蓝，脱水，以Acrymount固定。以未经一抗孵育的切片作为阴性对照。切片在奥林巴斯BX61显微镜下以放大200倍进行镜检。整个肿瘤组织切片中有代表性的图像使用DP70相机照相并以DP70软件处理。图像采用Image J软件分析。此外，由于肿瘤体积小，每个肿瘤分析采用四张切片来确定细胞数。将每种抗体染色的切片，取不同区域拍摄二十张图片，计算细胞总数。每个肿瘤的数据以不同抗体染色的细胞总数的平均值表示。H&E stained tissue section pathology was used to determine if it was a tumor. Immunohistochemistry was performed according to the Georgetown University Histopathology and Tissue Shared Resources standard. Briefly, the tissue was cut into 5 μm slices, dewaxed with xylene and dehydrated with a gradient alcohol, and the tissue sections were immersed in 10 nM citrate buffer (pH 6) containing 0.05% Tween at 98 ° C for 20 minutes. Heat-induced antigen retrieval (HIER). Immunohistochemical staining was performed using Dako's horseradish peroxidase-labeled polymer (k4001, k4003) according to the manufacturer's instructions. Briefly, sections were treated with 3% hydrogen peroxide and 10% normal goat serum for 10 minutes, then a primary anti-p53 (DO-7) (1:800, Dako) was added and allowed to stand at room temperature (RT) for 1 hour, or Ki-67 (1:15, Novus Biologicals) antibody was added and allowed to stand overnight at 4 °C. The sections were then exposed to the appropriate HRP-labeled polymer for 30 minutes and DAB chromogenic solution (Dako) for 5 minutes. The sections were further counterstained with hematoxylin (Fisher, Harris Modified Hematoxylin), turned blue in 1% ammonium hydroxide, dehydrated, and fixed with Acrymount. Sections that were not incubated with primary antibodies were used as negative controls. The sections were microscopically examined at a magnification of 200 times under an Olympus BX61 microscope. Representative images of whole tumor tissue sections were photographed using a DP70 camera and processed with DP70 software. Images were analyzed using Image J software. In addition, due to the small tumor volume, four sections were used for each tumor analysis to determine the number of cells. The sections stained with each antibody were taken in different regions to take twenty pictures, and the total number of cells was counted. The data for each tumor is expressed as the average of the total number of cells stained with different antibodies.
因为得到的肿瘤组织尤其是来自补充有PEITC小鼠的肿瘤组织数量有限，把肿瘤随机地分组，进行western blotting和qRT-PCR分析。对于Western blot，各组的SK-BR-3移植瘤(n＝12)裂解液按瘤组织和裂解缓冲液(Pierce)20w/v的比例制成匀浆。如前述方法，取25μg的裂解物进行4–12％SDS/PAGE电泳，将蛋白转移到PVDF膜上，以p53(DO-1)抗体进行蛋白免疫印迹试验。 Because of the limited number of tumor tissues obtained, especially from tumors supplemented with PEITC mice, tumors were randomly grouped for western blotting and qRT-PCR analysis. For Western blot, the SK-BR-3 xenograft (n=12) lysate of each group was homogenized according to the ratio of tumor tissue and lysis buffer (Pierce) at 20 w/v. 25 μg of the lysate was subjected to 4–12% SDS/PAGE electrophoresis as described above, and the protein was transferred to a PVDF membrane and subjected to Western blotting with p53 (DO-1) antibody.
统计分析Statistical Analysis
采用双尾t检验法检验肿瘤体积和生物端点的统计差异。P值≤0.05被认为具有统计学意义。所有统计分析均为双侧检验。Statistical differences in tumor volume and biological endpoints were examined using a two-tailed t-test. A P value of ≤ 0.05 was considered statistically significant. All statistical analyses were two-sided.
实施例1Example 1
PEITC对突变型p53细胞增殖的抑制作用Inhibition of PEITC on proliferation of mutant p53 cells
1.PEITC通过作用于***癌细胞DU145突变型p53抑制其增殖1.PEITC inhibits proliferation by acting on prostate cancer cell line DU145 mutant p53
通过WST-1试验研究了PEITC对***癌细胞DU145增殖的影响。为此，用不同浓度的PEITC处理DU145细胞72小时。之后通过检测各组细胞的OD450数值，计算对应的IC50。结果显示PEITC减少了DU145细胞系的增殖(IC50s＝～8μM)(图1A)。The effect of PEITC on the proliferation of prostate cancer cell line DU145 was investigated by WST-1 assay. To this end, DU145 cells were treated with different concentrations of PEITC for 72 hours. The corresponding IC50 was calculated by measuring the OD450 value of each group of cells. The results showed that PEITC reduced the proliferation of the DU145 cell line (IC 50 s = ~ 8 μM) (Fig. 1A).
2.PEITC通过作用于乳腺癌细胞SK-BR-3、AU565突变型p53R175抑制其增殖2.PEITC inhibits proliferation by acting on breast cancer cells SK-BR-3 and AU565 mutant p53 R175
WST-1试验研究表明，PEITC能降低各种表达突变型p53细胞的增殖。其中，PEITC对表达p53R175突变型的人乳腺癌细胞SK-BR-3、AU565和人非小细胞肺癌HOP92的抑制作用最强。这些肿瘤细胞对PEITC的IC50相比其它热点突变细胞下降了约2.5-5倍(图2a)。而用PEITC处理表达野生型p53的细胞，没有观察到明显的增殖抑制。WST-1 experimental studies have shown that PEITC can reduce the proliferation of various mutant p53 cells. Among them, PEITC had the strongest inhibitory effect on human breast cancer cells SK-BR-3, AU565 and human non-small cell lung cancer HOP92 expressing p53 R175 mutant. These tumor cells as compared to other hot PEITC the IC 50 mutant cells decreased approximately 2.5-5 fold (Figure 2a). However, no significant inhibition of proliferation was observed in cells treated with PEITC expressing wild-type p53.
3.PEITC通过作用于口腔癌细胞SCC114突变型p53R248抑制其增殖3.PEITC inhibits proliferation by acting on oral cancer cell line SCC114 mutant p53 R248
同样，在口腔癌细胞系SCC003，SCC016，SCC114和SCC122中进行的WST-1试验结果显示，在表达R248突变型p53的SCC114细胞中，经过PEITC处理后，观察到了最大的抑制率，而在表达野生型p53的SCC003细胞中却没有发现这种现象。Similarly, the WST-1 test performed in the oral cancer cell lines SCC003, SCC016, SCC114 and SCC122 showed that the maximum inhibition rate was observed in the SCC114 cells expressing the R248 mutant p53 after PEITC treatment. This phenomenon was not observed in wild-type p53 SCC003 cells.
上述结果表明，PEITC以突变型p53为靶点抑制肿瘤细胞系的增殖。The above results indicate that PEITC targets the mutant p53 as a target to inhibit the proliferation of tumor cell lines.
实施例2Example 2
PEITC以突变型p53依赖性方式抑制肿瘤细胞增殖并诱导细胞凋亡PEITC inhibits tumor cell proliferation and induces apoptosis in a mutant p53-dependent manner
1.PEITC以p53R175或p53R248依赖性方式抑制乳腺癌细胞SK-BR-3的增殖并诱导细胞凋亡1.PEITC inhibits the proliferation of breast cancer cell SK-BR-3 and induces apoptosis in a p53 R175 or p53 R248 -dependent manner.
为了确定PEITC抑制肿瘤细胞增殖的作用是否是通过使p53R175转型改变野生型p53活性所介导的，转染p53 siRNA后的WST-1试验显示：p53R175表达减少的SK-BR-3细胞(图2b)显著降低了PEITC抑制增殖的敏感性，而用NS siRNA转染的细胞则保持高敏感性。然而同样的试验在A549细胞中却没有观察到显著的增殖差异(图2b)。上述结果表明PEITC诱导的增殖抑制，依赖于突变型p53蛋白。To determine whether the effect of PEITC inhibition of tumor cell proliferation is mediated by the transformation of p53 R175 to alter wild-type p53 activity, the WST-1 assay after transfection of p53 siRNA revealed that SK-BR-3 cells with reduced p53 R175 expression ( Figure 2b) significantly reduced the sensitivity of PEITC to inhibit proliferation, while cells transfected with NS siRNA remained highly sensitive. However, the same experiment did not observe significant proliferation differences in A549 cells (Fig. 2b). The above results indicate that PEITC-induced inhibition of proliferation is dependent on mutant p53 protein.
为了确定PEITC是否通过改变突变型p53蛋白而诱导肿瘤细胞凋亡，通过流式细胞技术进一步检测了表达不同突变型p53蛋白的肿瘤细胞系，试验结果显 示与MDA-MB-231(p53R280)或A549细胞(图2c)相比，以4μM PEITC处理的SK-BR-3细胞，发生凋亡的细胞百分比与对照相比增加了约3倍(图2c)。值得注意的是，用PEITC或DMSO处理p53R175表达减少的SK-BR-3的细胞，其凋亡率没有显著的差异(图2c)，这证明PEITC依赖于突变型p53诱导细胞凋亡。To determine whether PEITC induces tumor cell apoptosis by altering the mutant p53 protein, tumor cell lines expressing different mutant p53 proteins were further detected by flow cytometry, and the results were shown with MDA-MB-231 (p53 R280 ) or Compared to A549 cells (Fig. 2c), the percentage of cells that developed apoptosis in SK-BR-3 cells treated with 4 μM PEITC was approximately 3-fold higher than that of the control (Fig. 2c). Notably, there was no significant difference in apoptotic rates between cells treated with PEITC or DMSO for SK-BR-3 with reduced expression of p53 R175 (Fig. 2c), demonstrating that PEITC is dependent on mutant p53 to induce apoptosis.
2.PEITC以p53P223或p53V274依赖性方式抑制***癌细胞系DU145的增殖并诱导细胞凋亡2. PEITC inhibits proliferation and induces apoptosis of prostate cancer cell line DU145 in a p53 P223 or p53 V274 -dependent manner.
在***癌细胞系DU145中，细胞死亡酶联免疫吸附试验结合光度酶测定试验结果显示：与经DMSO处理的对照细胞组相比，用8μM PEITC处理的DU145细胞其凋亡率增加了约2倍(图1B)In the prostate cancer cell line DU145, the cell death enzyme-linked immunosorbent assay combined with photometric enzyme assay showed that the apoptosis rate of DU145 cells treated with 8 μM PEITC increased about 2 times compared with the DMSO-treated control cell group. (Figure 1B)
本实施例中的结果说明，PEITC通过介导突变型p53的改变抑制了肿瘤细胞的增殖，并诱发了肿瘤细胞的凋亡。The results in this example demonstrate that PEITC inhibits tumor cell proliferation by mediating changes in mutant p53 and induces apoptosis of tumor cells.
实施例3Example 3
PEITC恢复突变型p53的“类野生型”构象和转录激活功能PEITC restores "wild-type" conformation and transcriptional activation of mutant p53
1.PEITC作用于乳腺癌细胞SK-BR-3突变型p53R175H，恢复其“类野生型”构象和转录激活功能1.PEITC acts on breast cancer cell SK-BR-3 mutant p53 R175H , restores its "wild-type" conformation and transcriptional activation
由于PEITC依赖于突变型p53诱导细胞凋亡，有理由推断可能是通过恢复p53的野生型功能来实现的。为此，用具有特定构象的抗p53抗体的酶联免疫吸附试验(ELISA)测定PEITC对p53R175构象的影响。结果表明，用PEITC孵育谷胱甘肽-S-转移酶(GST)-p53R175H重组体，PAB1620(野生型特有的)部分增加了约2.8倍，而PAB240(突变型特有的)部分减少了约2.6倍(图3a)。用PEITC处理的SK-BR-3细胞的免疫荧光试验显示，PAB1620抗体的荧光强度增加了约2倍，而PAB240抗体的荧光强度出现了下降(图3b和图3c)。重要的是,用PEITC处理的SK-BR-3细胞(图3d)的p53R175的免疫沉淀反应显示PAB240的免疫反应性降低了95％以上。这些结果证明，PEITC诱导了p53R175的“类野生型”构象。Since PEITC relies on mutant p53 to induce apoptosis, it is reasonable to conclude that it may be achieved by restoring the wild-type function of p53. To this end, the effect of PEITC on the conformation of p53 R175 was determined by an enzyme-linked immunosorbent assay (ELISA) with an anti-p53 antibody of a specific conformation. The results showed that the glutathione-S-transferase (GST)-p53 R175H recombinant was incubated with PEITC, and the PAB1620 (wild-type-specific) portion was increased by about 2.8-fold, while the PAB240 (mutant-specific) was partially reduced. 2.6 times (Figure 3a). Immunofluorescence assays of SK-BR-3 cells treated with PEITC showed that the fluorescence intensity of the PAB1620 antibody increased approximately 2-fold, while the fluorescence intensity of the PAB240 antibody decreased (Fig. 3b and Fig. 3c). Importantly, immunoprecipitation of p53 R175 in SK-BR-3 cells treated with PEITC (Fig. 3d) showed a 95% reduction in immunoreactivity of PAB240. These results demonstrate that PEITC induces a "wild-like" conformation of p53 R175 .
能否恢复p53R175与DNA结合的能力，对p53突变型肿瘤的抑制至关重要。为此，进一步研究PEITC是否能使核染色质富集p53R175。图4a是用PEITC处理的SK-BR-3细胞的核染色质结合部分，结果显示了p53R175剂量依赖性增加。与此一致的是，4μM的PEITC增加了SK-BR-3细胞中p53靶基因的表达，特别是p21、MDM2、PUMA、NOXA、BCL2、BAX(图4b)。而用PEITC处理A549、H1299或p53R175表达减少的SK-BR-3细胞，没有观察到明显变化。这表明PEITC诱导p53靶点是p53R175依赖性的(图4b和图4c)。The ability to restore p53 R175 binding to DNA is critical for inhibition of p53 mutant tumors. To this end, further study whether PEITC can enrich nuclear chromatin for p53 R175 . Figure 4a is a nuclear chromatin binding portion of SK-BR-3 cells treated with PEITC and the results show a dose-dependent increase in p53 R175 . Consistent with this, 4 μM of PEITC increased the expression of p53 target genes in SK-BR-3 cells, particularly p21, MDM2, PUMA, NOXA, BCL2, BAX (Fig. 4b). No significant changes were observed in SK-BR-3 cells treated with PEITC for reduced expression of A549, H1299 or p53 R175 . This indicates that the PEITC-induced p53 target is p53 R175 -dependent (Fig. 4b and Fig. 4c).
2.PEITC作用于***癌细胞DU145突变型p53P223L或p53V274F，恢复其“类野生型”构象和转录激活功能2.PEITC acts on prostate cancer cell line DU145 mutant p53 P223L or p53 V274F to restore its "wild-type" conformation and transcriptional activation
在***癌研究中，由于PEITC能够诱导表达突变型p53蛋白的DU145细胞 凋亡，我们推断它也是通过恢复p53的类野生型功能的途径。因此，我们研究了它对典型的p53靶基因p21表达的影响。结果显示，用PEITC(8μM)处理的DU145细胞确实增强了p21的表达(图5)。这一结果表明，PEITC能够恢复p53突变体“类野生型”构象和转录激活功能。In prostate cancer research, PEITC is capable of inducing DU145 cells expressing mutant p53 protein Apoptosis, we conclude that it is also a pathway that restores the wild-type function of p53. Therefore, we investigated its effect on the expression of the p53 target gene p21. The results showed that DU145 cells treated with PEITC (8 μM) did enhance the expression of p21 (Fig. 5). This result indicates that PEITC is able to restore the "wild-type" conformation and transcriptional activation of p53 mutants.
3.PEITC作用于口腔癌细胞SCC114突变型p53R248，恢复其“类野生型”构象和转录激活功能3.PEITC acts on oral cancer cell line SCC114 mutant p53 R248 , restoring its "wild-type" conformation and transcriptional activation
同样，在口腔癌SCC114细胞中，用PEITC处理后，随着药物浓度的增加，突变型P53蛋白表达水平逐渐下降，而P21蛋白表达水平逐渐上升(图6A)。染色质免疫共沉淀实验进一步证实了经PEITC处理过的SCC114细胞能够恢复突变型P53蛋白结合到基因p21、PUMA和MDM2启动子特定位点的能力。(图6B)。Similarly, in oral cancer SCC114 cells, after treatment with PEITC, the expression level of mutant P53 protein gradually decreased with the increase of drug concentration, while the expression level of P21 protein gradually increased (Fig. 6A). Chromatin immunoprecipitation experiments further confirmed that PEITC-treated SCC114 cells were able to restore the ability of mutant P53 proteins to bind to specific sites of the gene p21, PUMA and MDM2 promoters. (Figure 6B).
为了提供更多PEITC能恢复p53转录激活功能的证据，本发明进行了荧光素酶报告基因检测试验。结果表明，用4μM PEITC处理过的细胞，荧光素酶活性增加了约2-2.5倍(图4d)。这证明PEITC(4μM)能够诱导SK-BR-3细胞中p21基因的表达，而DNA损伤剂——依托泊苷无法做到(图4e)。这表明，这种诱导是突变型p53依赖性的。In order to provide more evidence that PEITC can restore p53 transcriptional activation, the present invention conducted a luciferase reporter assay. The results showed that luciferase activity was increased by about 2-2.5 fold in cells treated with 4 μM PEITC (Fig. 4d). This demonstrates that PEITC (4 μM) is capable of inducing expression of the p21 gene in SK-BR-3 cells, whereas the DNA damaging agent, etoposide, cannot be achieved (Fig. 4e). This suggests that this induction is mutant p53 dependent.
本实施例中的所有结果均显示，PEITC能够恢复突变型p53蛋白的“类野生型”构象并获得转录激活功能。All the results in this example show that PEITC is able to restore the "wild-like" conformation of the mutant p53 protein and obtain transcriptional activation.
实施例4Example 4
PEITC使乳腺癌SK-BR-3细胞表达的p53R175蛋白通过蛋白酶体和自噬降解PEITC degrades p53 R175 protein expressed by breast cancer SK-BR-3 cells by proteasome and autophagy
实施例1-3表明，PEITC(≥10μM)选择性降解突变型p53蛋白，而不是野生型p53蛋白。由于PEITC能够恢复p53R175到“类野生型”状态，而野生型p53受MDM2调控，因此恢复后的p53R175的稳定性下降可能是由于MDM2依赖的蛋白酶体的降解导致了泛素化蛋白在不溶解部分的积累。为了验证这一点，分别用PEITC、蛋白酶体抑制剂MG132或特定的MDM2抑制剂Nutlin-3单独或联合处理SK-BR-3细胞。结果显示MG132或Nutlin-3均无法防止p53R175的降低(图7a)。与单独用PEITC或MG132处理的细胞相比较，用4或8μM PEITC和20μM MG132联合处理的SK-BR-3细胞，全细胞裂解物(WCL)溶解部分和不溶解部分的p53显著积累(图7b)。同样，用4或8μM PEITC和20μM Nutlin联合处理的SK-BR-3全细胞裂解物(WCL)中的p53显著增加(图7c)，而A549中没有观察到p53积累的差别(图7d)。单独用8μM PEITC处理的SK-BR-3细胞中的p53存在于全细胞裂解物中(图7b)。为了观察到p53R175的聚集，用不同浓度(1-16μM)的PEITC处理SK-BR-3细胞。较高浓度(≥8μM)时，观察到p53R175的积累(图7e)。上述结果表明，改变的p53R175稳定性的降低是由于蛋白酶体的降解。Examples 1-3 show that PEITC (≥10 μM) selectively degrades the mutant p53 protein, but not the wild-type p53 protein. Since PEITC is able to restore p53 R175 to a "wild-type" state, whereas wild-type p53 is regulated by MDM2, the decreased stability of p53 R175 after recovery may be due to degradation of the MDM2-dependent proteasome resulting in ubiquitinated protein. The accumulation of dissolved parts. To verify this, SK-BR-3 cells were treated with PEITC, the proteasome inhibitor MG132, or a specific MDM2 inhibitor, Nutlin-3, alone or in combination. The results showed that neither MG132 nor Nutlin-3 prevented the reduction of p53 R175 (Fig. 7a). Compared with cells treated with PEITC or MG132 alone, SK-BR-3 cells treated with 4 or 8 μM PEITC and 20 μM MG132 showed significant accumulation of p53 in the lysed and insoluble fractions of whole cell lysate (WCL) (Fig. 7b) ). Similarly, p53 was significantly increased in SK-BR-3 whole cell lysate (WCL) treated with 4 or 8 μM PEITC and 20 μM Nutlin (Fig. 7c), while no difference in p53 accumulation was observed in A549 (Fig. 7d). P53 in SK-BR-3 cells treated with 8 μM PEITC alone was present in whole cell lysates (Fig. 7b). To observe the aggregation of p53 R175 , SK-BR-3 cells were treated with different concentrations (1-16 μM) of PEITC. At higher concentrations (≥ 8 μM), accumulation of p53 R175 was observed (Fig. 7e). The above results indicate that the altered stability of p53 R175 is due to degradation of the proteasome.
由于蛋白质也可能通过自噬过程清除，因此，本发明进一步研究了PEITC 对SK-BR-3细胞自噬的影响。结果表明，和单独用PEITC处理的细胞相比，用8μM PEITC和50μM自噬抑制剂氯喹(CHQ)联合处理SK-BR-3细胞，明显增加了WCL中p53的含量。但4μM PEITC和50μM CHQ联合处理与单独用PEITC处理的细胞没有观察到显著差异，表明较高浓度的PEITC能够诱导自噬的发生(图8a)。由于自噬的形成需要自噬蛋白5(ATG5)的参与，因此，我们通过向SK-BR-3细胞中转染ATG5 siRNA或NS siRNA，以检测细胞对PEITC的敏感性。结果发现，和用NS siRNA转染的细胞相比，ATG5表达减少的SK-BR-3细胞，用8μM或16μM浓度PEITC处理时表现出较高水平的p53R175(图8b)，而2μM到4μM浓度PEITC处理则没有明显变化(图8c)。这说明ATG5表达减少的SK-BR-3细胞具有抵抗PEITC的抗恶性肿瘤细胞增殖的作用，而NS SiRNA转染细胞仍然保持高度敏感(图8d和图8e)，这表明在这些细胞中自噬消极地控制细胞生长。Since proteins may also be cleared by autophagy, the present invention further investigates the effect of PEITC on autophagy of SK-BR-3 cells. The results showed that treatment of SK-BR-3 cells with 8 μM PEITC and 50 μM autophagy inhibitor chloroquine (CHQ) significantly increased the content of p53 in WCL compared with cells treated with PEITC alone. However, no significant difference was observed between the combination of 4 μM PEITC and 50 μM CHQ and cells treated with PEITC alone, indicating that higher concentrations of PEITC were able to induce autophagy (Fig. 8a). Since the formation of autophagy requires the involvement of autophagy protein 5 (ATG5), we tested the sensitivity of cells to PEITC by transfecting ATG5 siRNA or NS siRNA into SK-BR-3 cells. It was found that SK-BR-3 cells with reduced ATG5 expression showed higher levels of p53 R175 when treated with 8 μM or 16 μM PEITC compared to cells transfected with NS siRNA (Fig. 8b), and 2 μM to 4 μM. There was no significant change in the concentration of PEITC treatment (Fig. 8c). This indicates that SK-BR-3 cells with reduced ATG5 expression have anti-tumor cell proliferation resistance against PEITC, while NS SiRNA transfected cells remain highly sensitive (Fig. 8d and Fig. 8e), indicating autophagy in these cells. Negatively control cell growth.
本实施例中的试验结果证明PEITC通过两种不同的途径降解细胞中的突变型p53R175蛋白，分别为：MDM2依赖型蛋白酶体降解途径和通过自噬过程降解p53R175蛋白。The results of the experiments in this example demonstrate that PEITC degrades the mutant p53 R175 protein in cells by two different pathways: MDM2-dependent proteasome degradation pathway and degradation of p53 R175 protein by autophagy.
实施例5Example 5
锌离子增强PEITC诱导乳腺癌SK-BR-3细胞p53R175的复活能力Zinc-enhanced PEITC induces reactivation of p53 R175 in breast cancer SK-BR-3 cells
p53野生型蛋白质的适当折叠构象需要锌离子。p53R175是无法与锌直接结合的。通过用氯化锌(ZnCl2)和最佳浓度范围(10-20μM)的PEITC联合处理表达突变型p53R175的SK-BR-3细胞，发现氯化锌将PEITC抑制肿瘤细胞增殖的作用增强了约3.3倍(图9a)，但单独用ZnCl2没有发现这样的效果。同样，用PEITC(4μM)和ZnCl2(2.5μM)孵化GST-p53R175H导致PAB1620部分的增加非常显著，而PAB240没有明显变化(图9b)。这些结果证明锌离子有助于帮助PEITC重建p53R175的“类野生型”构象。The proper folded conformation of the p53 wild-type protein requires zinc ions. P53 R175 is not directly bindable to zinc. By treating the SK-BR-3 cells expressing mutant p53 R175 with zinc chloride (ZnCl 2 ) and PEITC at the optimal concentration range (10-20 μM), it was found that zinc chloride enhanced the inhibitory effect of PEITC on tumor cell proliferation. About 3.3 times (Fig. 9a), but no such effect was observed with ZnCl 2 alone. Similarly, incubation of GST-p53 R175H with PEITC (4 μM) and ZnCl 2 (2.5 μM) resulted in a significant increase in the PAB1620 fraction, while PAB240 did not change significantly (Fig. 9b). These results demonstrate that zinc ions help to help PEITC reconstruct the "wild-like" conformation of p53 R175 .
实施例6Example 6
氧化还原变化对改变p53R175和抑制肿瘤细胞增殖很重要，而对恢复p53R175构象不重要Redox changes are important for altering p53 R175 and inhibiting tumor cell proliferation, but not for restoring p53 R175 conformation
研究表明，PEITC通过使癌细胞中的谷胱甘肽抗氧化***失活，诱导产生活性氧。而氧化还原变化影响野生型p53蛋白的构象。谷胱甘肽表达水平检测结果表明：和DMSO对照组相比，用PEITC(4或8μM)处理过的SK-BR-3细胞中谷胱甘肽水平出现了下降(图9c)。用PEITC和还原剂(3mM N-乙酰基半胱氨酸(NAC)或500单位PEG-过氧化氢酶)联合处理SK-BR-3细胞后，减弱了PEITC抑制肿瘤细胞增殖和诱导凋亡的作用，而过氧化氢酶特异性抑制剂3-氨基-1,2,4-***(ATZ)则增强了PEITC抑制肿瘤细胞增殖和凋亡的作用(图9d,图9e和图9f)。单 独用PEITC处理或PEITC结合ATZ或NAC处理p53R175表达减少的SK-BR-3细胞，在细胞凋亡上没有显著的差异(图9f)。这些结果表明氧化还原变化对改变p53R175和抑制肿瘤细胞增殖很重要，而对恢复p53R175构象不重要。Studies have shown that PEITC induces the production of reactive oxygen species by inactivating the glutathione antioxidant system in cancer cells. The redox changes affect the conformation of the wild-type p53 protein. The results of glutathione expression levels showed that glutathione levels were decreased in SK-BR-3 cells treated with PEITC (4 or 8 μM) compared to the DMSO control group (Fig. 9c). Treatment of SK-BR-3 cells with PEITC and a reducing agent (3 mM N-acetylcysteine (NAC) or 500 units of PEG-catalase) attenuated PEITC inhibition of tumor cell proliferation and induction of apoptosis The action, while the catalase-specific inhibitor 3-amino-1,2,4-triazole (ATZ) enhanced the effect of PEITC on tumor cell proliferation and apoptosis (Fig. 9d, Fig. 9e and Fig. 9f). There was no significant difference in apoptosis between SK-BR-3 cells treated with PEITC alone or PEITC in combination with ATZ or NAC to reduce p53 R175 expression (Fig. 9f). These results indicate that redox changes are important for altering p53 R175 and inhibiting tumor cell proliferation, but not for restoring p53 R175 conformation.
本实施例中的结果证明锌离子和氧化还原变化对PEITC诱导的p53R175复活具有重要的影响。The results in this example demonstrate that zinc ion and redox changes have an important effect on PEITC-induced reactivation of p53 R175 .
实施例7Example 7
PEITC对细胞周期和ATM/CHK2激活的影响Effect of PEITC on cell cycle and ATM/CHK2 activation
1.PEITC复活乳腺癌细胞SK-BR-3突变型p53R175的反式激活功能,从而激活细胞凋亡的DNA损伤应答1.PEITC reactivates the transactivation function of SK-BR-3 mutant p53 R175 , which activates DNA damage response of apoptosis
通过使SK-BR-3细胞产生氧化应激压力，以评估PEITC对DNA损伤修复的影响。结果发现，和DMSO对照组相比，用4μM PEITC处理的SK-BR-3细胞中的γ–H2AX焦点增加了约1.8倍，这代表了DNA双链断裂(DSB’s)的积累，而同样的试验在A549细胞中没有检测到差异(图10a和图10b)。这表明DSB’s的积累是与p53R175相关的。The effect of PEITC on DNA damage repair was assessed by generating oxidative stress in SK-BR-3 cells. As a result, it was found that the γ-H2AX focus in SK-BR-3 cells treated with 4 μM PEITC was increased by about 1.8-fold compared with the DMSO control group, which represents the accumulation of DNA double-strand breaks (DSB's), and the same test. No differences were detected in A549 cells (Fig. 10a and Fig. 10b). This indicates that the accumulation of DSB's is related to p53 R175 .
为了进一步研究将突变型p53R175改变为“类野生型”p53后，是否丧失其抑制ATM/检验点激酶2(CHK2)途径的能力。图10是经4μM PEITC处理后的SK-BR-3细胞与DMSO对照组细胞中pATM-S1981和pCHK2/Thr68的比较,结果表明缺乏p53R175H的ATM/CHK2抑制导致DNA损伤应答的重新激活。用PEITC处理没有pATM-S1981和pCHK2-Thr68的A549细胞结果与γ–H2AX焦点数据一致(图10a和图10b)。这表明PEITC恢复DNA损伤修复取决于细胞的氧化还原状态。To further investigate whether the mutant p53 R175 was changed to "wild-like" p53, its ability to inhibit the ATM/Checkpoint Kinase 2 (CHK2) pathway was lost. Figure 10 is a comparison of pATM-S1981 and pCHK2/Thr68 in SK-BR-3 cells treated with 4 μM PEITC and DMSO control cells. The results indicate that ATM/CHK2 inhibition in the absence of p53 R175H results in reactivation of the DNA damage response. Treatment of A549 cells without pATM-S1981 and pCHK2-Thr68 with PEITC was consistent with gamma-H2AX focus data (Figures 10a and 10b). This suggests that PEITC restores DNA damage repair depending on the redox state of the cells.
改变的突变型p53R175对细胞周期进程影响的试验发现用4μM PEITC处理SK-BR-3细胞24小时，G2/M和S期显著延迟(图10d)；这表明PEITC抑制细胞增殖，不仅延迟G2/M期，也延迟S期。用4μM PEITC处理A549细胞24小时，G1期延期。这表明细胞周期进程的延迟是与p53R175相关的。另外，与单独应用PEITC或Nutlin-3相比较，10μM Nutlin-3和4μM PEITC共同处理的SK-BR-3细胞24和72小时，结果表明，S期的数量显著增加(图10d)、细胞凋亡数量显著增加(图10f),说明了Nutlin-3的协同效应。而Nutlin-3单独或联合PEITC处理A549细胞24小时，显著推迟G1期；72小时，推迟G1和G2/M期(图10e)。证明观察到的Nutlin-3效果是野生型p53特有的。A modified trial of the effect of mutant p53 R175 on cell cycle progression revealed that treatment of SK-BR-3 cells with 4 μM PEITC for 24 hours significantly delayed G2/M and S phases (Fig. 10d); this indicates that PEITC inhibits cell proliferation, not only delaying G2 /M period, also delayed S period. A549 cells were treated with 4 μM PEITC for 24 hours, and the G1 phase was postponed. This indicates that the delay in cell cycle progression is associated with p53 R175 . In addition, SK-BR-3 cells co-treated with 10 μM Nutlin-3 and 4 μM PEITC for 24 and 72 hours compared with PEITC or Nutlin-3 alone showed a significant increase in the number of S phases (Fig. 10d). The number of deaths increased significantly (Fig. 10f), illustrating the synergistic effect of Nutlin-3. Treatment of A549 cells with Nutlin-3 alone or in combination with PEITC for 24 hours significantly delayed the G1 phase; 72 hours, delayed G1 and G2/M phases (Fig. 10e). It was demonstrated that the observed effect of Nutlin-3 is unique to wild-type p53.
总之,上述研究结果表明,PEITC在恢复突变型p53R175的反式激活功能的同时，复活细胞凋亡的DNA损伤应答。In summary, the above results indicate that PEITC revives the DNA damage response of apoptosis while restoring the transactivation function of mutant p53 R175 .
2.PEITC通过作用于口腔癌细胞SCC114的突变型p53R248诱导G1期细胞周期阻滞2. PEITC induces G1 phase cell cycle arrest by mutant p53 R248 acting on oral cancer cell line SCC114
在口腔癌细胞中，表达突变型P53的SCC114细胞，经过不同浓度的PEITC处 理后，能够恢复细胞从G1期进入S期的过程(P<0.05)(图11E,11F,11G,11H)。但是,对于表达野生型P53的SCC003细胞，实验组和对照组的差异没有统计学意义(P>0.05)(图11A,11B,11C,11D)。实验结果表明：PEITC诱导G1期细胞周期阻滞依赖于p53R248突变蛋白。另外，发明人通过向SCC114细胞转染siRNA进一步分析细胞周期，以确定是否是突变型p53在恢复G1期周期阻滞中发挥作用。流式细胞术和免疫印迹分析表明，当通过siRNA使突变型P53基因沉默时，就不再出现细胞周期阻滞现象(图12)。In oral cancer cells, SCC114 cells expressing mutant P53 were able to restore the cells from the G1 phase to the S phase after treatment with different concentrations of PEITC (P<0.05) (Fig. 11E, 11F, 11G, 11H). However, for SCC003 cells expressing wild-type P53, the difference between the experimental group and the control group was not statistically significant (P>0.05) (Fig. 11A, 11B, 11C, 11D). The results showed that PEITC induced cell cycle arrest in G1 phase was dependent on p53 R248 mutein. In addition, the inventors further analyzed the cell cycle by transfecting SCC114 cells with siRNA to determine whether mutant p53 plays a role in restoring G1 phase arrest. Flow cytometry and immunoblot analysis showed that when the mutant P53 gene was silenced by siRNA, cell cycle arrest was no longer observed (Fig. 12).
本实施例试验结果证明PEITC通过作用于突变型p53蛋白诱导肿瘤细胞产生G1期阻滞；同时，PEITC通过作用于ATM/CHK2激酶恢复细胞的DNA损伤修复。The results of the experiments in this example demonstrate that PEITC induces G1 arrest by tumor cells by acting on mutant p53 protein; meanwhile, PEITC restores DNA damage repair by acting on ATM/CHK2 kinase.
实施例8Example 8
PEITC通过改变不同热点的突变型p53蛋白，抑制异体移植肿瘤的生长PEITC inhibits the growth of allograft tumors by changing mutant p53 proteins of different hot spots
1.PEITC抑制人乳腺癌SK-BR-3异体移植肿瘤的生长1.PEITC inhibits the growth of human breast cancer SK-BR-3 xenograft tumor
人乳腺癌动物模型试验结果显示，与对照饮食组相比，PEITC饮食组(5μmol/g AIN-93M)小鼠肿瘤生长受到明显的抑制(p<0.05)(图13a和图13b)。六周后对照组肿瘤体积的减小，可以解释为非侵犯表型的SK-BR-3细胞。两组间体重没有差异(图13c)。这些结果说明PEITC在SK-BR-3异体移植肿瘤模型上显示了抗肿瘤活性。Human breast cancer animal model test results showed that tumor growth was significantly inhibited in the PEITC diet group (5 μmol/g AIN-93M) compared with the control diet group (p<0.05) (Fig. 13a and Fig. 13b). The reduction in tumor volume in the control group after six weeks can be interpreted as a non-invasive phenotype of SK-BR-3 cells. There was no difference in body weight between the two groups (Fig. 13c). These results indicate that PEITC shows anti-tumor activity on the SK-BR-3 xenograft tumor model.
进一步的组织学检查表明，PEITC组肿瘤细胞显著减少(图13a和图13d)。同时，肿瘤Ki67和p53突变的染色细胞也显著减少(图13e)。为了评估PEITC是否复活体内突变型p53R175H,对肿瘤组织中的p53蛋白表达水平进行了检测。结果表明，尽管存在可能源自于试验用小鼠模型固有的特性，PEITC组p53R175H的水平存在差异，但和对照组相比较，PEITC组突变型p53蛋白水平显著降低(图13f)。另外，PEITC组p53调控基因p21和Bax的mRNA的蛋白质表达都有所增加(图13g和图13h)。这些结果提供了PEITC抑制SK-BR-3异体移植肿瘤生长、复活体内突变型p53的证据。Further histological examination revealed a significant reduction in tumor cells in the PEITC group (Fig. 13a and Fig. 13d). At the same time, stained cells of tumor Ki67 and p53 mutations were also significantly reduced (Fig. 13e). To assess whether PEITC reactivates mutant p53 R175H in vivo, p53 protein expression levels in tumor tissues were examined. The results showed that although there were differences in the levels of p53 R175H in the PEITC group, the level of mutant p53 protein in the PEITC group was significantly lower than that in the control group (Fig. 13f). In addition, protein expression of the p53 regulatory genes p21 and Bax mRNA in the PEITC group was increased (Fig. 13g and Fig. 13h). These results provide evidence that PEITC inhibits SK-BR-3 xenograft tumor growth and reactivates mutant p53 in vivo.
2.PEITC抑制人***癌DU145异体移植肿瘤的生长2.PEITC inhibits the growth of human prostate cancer DU145 xenograft tumor
***癌动物模型实验结果显示：与对照组动物相比，喂饲PEITC的动物组中观察到了有统计学意义的肿瘤生长抑制(p<0.05)(图14A和14B)。在生物测定期，两组动物的体重未观察到显著差异(图14C)。这些结果证明，PEITC在DU145移植瘤模型内有抗肿瘤活性。The results of the animal model of prostate cancer showed that statistically significant tumor growth inhibition (p < 0.05) was observed in the animals fed PEITC compared with the control animals (Figs. 14A and 14B). No significant difference in body weight was observed between the two groups of animals during the bioassay period (Fig. 14C). These results demonstrate that PEITC has antitumor activity in the DU145 xenograft model.
本实施例结果说明PEITC能够改变p53R175H，抑制体内异体移植瘤的生长。The results of this example demonstrate that PEITC can alter p53 R175H and inhibit the growth of allograft tumors in vivo.
讨论discuss
通过转活途径改变突变型p53，为肿瘤靶向治疗提供了一种前景光明的方 向。突变型p53的改变已经在不同肿瘤的小鼠模型上分别得到验证。根据文献，已有人工设计的小分子恢复突变型p53反向转录的报道。然而,尚未见天然食物来源化合物改变突变型p53的研究报告。本发明的研究表明，PEITC选择性降解突变型p53蛋白而不影响野生型。具体而言，PEITC通过改变突变型p53R175，抑制细胞增殖和诱导细胞凋亡，导致选择性清除这类细胞。这是天然食物来源化合物诱导细胞凋亡的新机理。研究PEITC对细胞中最终导致细胞死亡的系列不同基因之间的相互影响是非常有趣的。尽管p53通过转活多个相关、不相关的因子调节抗凋亡调节因子BCL2家族，PEITC除了影响BCL2外,还通过诱导多个启动凋亡的靶点表达,包括BH3-only类成员，恢复p53R175转录激活功能。重要的是,BCL2不能抑制PEITC诱导的细胞凋亡。Altering mutant p53 by transactivation provides a promising direction for tumor-targeted therapy. Alterations in mutant p53 have been demonstrated in mouse models of different tumors, respectively. According to the literature, artificially designed small molecules have been reported to restore the reverse transcription of mutant p53. However, no studies have been published on the alteration of mutant p53 by natural food-derived compounds. Studies of the present invention have shown that PEITC selectively degrades mutant p53 protein without affecting the wild type. Specifically, PEITC inhibits cell proliferation and induces apoptosis by altering mutant p53 R175 , resulting in selective clearance of such cells. This is a new mechanism by which natural food-derived compounds induce apoptosis. It is interesting to study the interaction between PEITC and the different genes in the cell that ultimately lead to cell death. Although p53 regulates the anti-apoptotic regulatory factor BCL2 family by transduction of multiple related, unrelated factors, in addition to affecting BCL2, PEITC also restores p53 by inducing expression of multiple apoptotic targets, including BH3-only class members. R175 transcriptional activation function. Importantly, BCL2 did not inhibit PEITC-induced apoptosis.
本发明的研究结果显示,改变的p53R175可以通过适当的折叠与锌结合，而ZnCl2显著增加了野生型p53特有的PAB1620，提示ZnCl2增强了PEITC的抗细胞增殖活性。The results of the present study showed that the altered p53 R175 can bind to zinc by appropriate folding, while ZnCl 2 significantly increased PAB1620 specific to wild-type p53, suggesting that ZnCl 2 enhances the anti-cell proliferation activity of PEITC.
PEITC可能通过增加突变型p53细胞活性氧，加剧PEITC诱导的氧化应激。尽管诱导活性氧对恢复p53R175H构象没有影响,但提高氧化应激有助于恢复p53R175活性、诱导细胞凋亡。ATZ增强了PEITC抗增殖能力,而PEG-catalase或NAC起抑制作用，分别从正反两个方面为上述结论提供佐证。PEITC may aggravate PEITC-induced oxidative stress by increasing reactive oxygen species in mutant p53 cells. Although induction of reactive oxygen species had no effect on restoring the p53 R175H conformation, increasing oxidative stress helped restore p53 R175 activity and induce apoptosis. ATZ enhanced the anti-proliferative ability of PEITC, while PEG-catalase or NAC played an inhibitory role, which provided evidence for the above conclusions from both positive and negative aspects.
本发明的体内研究，证实了天然食物来源的PEITC可以通过改变试验小鼠突变型p53蛋白，诱导肿瘤细胞凋亡。分析结果表明，喂食PEITC小鼠的血液样本中ITC浓度为1.13±0.15μM，这与人体药代动力学的研究结果-——食用约50克生西洋菜(大约相当于40毫克PEITC)志愿者的血浆中PEITC峰值浓度约1μM一致。In vivo studies of the present invention demonstrate that PEITC, a natural food source, can induce tumor cell apoptosis by altering the mutant p53 protein of the test mouse. The results of the analysis showed that the concentration of ITC in the blood samples fed PEITC mice was 1.13±0.15 μM, which was related to the results of human pharmacokinetics--approximately 50 g of raw watercress (about 40 mg of PEITC) volunteers. The peak concentration of PEITC in plasma was approximately 1 μM.
本发明的研究,按“化学预防设置”方式，证实PEITC对肿瘤的抑制作用。具体而言，在注射突变型p53细胞和肿瘤形成前，就给试验动物喂饲含PEITC的饲料。与此同时，注射的突变型p53细胞分为“启动阶段”或“已癌化”两种。研究结果表明，喂饲含PEITC饲料的小鼠，体内突变型p53得到降解、增殖抑制、肿瘤体积显著缩小。此外，p53靶基因mRNA的升高提供了喂饲含PEITC饲料小鼠p53R175H复活的证据。In the study of the present invention, the inhibitory effect of PEITC on tumors was confirmed by the "chemoprevention setting" method. Specifically, the test animals were fed a feed containing PEITC prior to injection of mutant p53 cells and tumor formation. At the same time, the injected mutant p53 cells were classified into two types: "starting phase" or "cancerousized". The results showed that the mutant p53 was degraded, inhibited in proliferation and significantly reduced in tumor volume in mice fed PEITC. In addition, elevated p53 target gene mRNA provides evidence of reactivation of p53 R175H in mice fed PEITC feed.
医药界已经进行了很多研究，力图以改变突变型p53为靶点，从化学品库寻找可能作为肿瘤治疗药物的小分子化合物。然而,以天然食物来源化合物，预防为靶向的研究甚少。A lot of research has been done in the medical community to try to change the mutant p53 as a target, and to find small molecule compounds that may be used as tumor therapeutic drugs from the chemical library. However, research on prevention as a target for natural food-derived compounds is scarce.
通过靶向p53R175，本发明阐明了PEITC预防和治疗癌症的新机理。p53基因的突变可能发生在癌症的不同阶段,如在乳腺癌(DCIS,Ductal carcinoma in situ)和肝癌的早期，胰腺癌、肝细胞性肝癌、***癌等的晚期,。就乳腺癌而言，DCIS阶段是发展为浸润性乳腺癌的前期。PEITC对这一阶段p53突变基因 的作用,完全可用于乳腺癌的预防和早期干预。恢复p53R175“类野生型”构象和功能的发现，结合天然食物来源化合物特有的安全性，开辟了靶向性癌症预防和治疗的新途径。By targeting p53 R175 , the present invention clarifies the novel mechanism by which PEITC prevents and treats cancer. Mutations in the p53 gene may occur at different stages of cancer, such as in the early stages of breast cancer (DCIS, Ductal carcinoma in situ) and liver cancer, late stages of pancreatic cancer, hepatocellular carcinoma, prostate cancer, and the like. In the case of breast cancer, the DCIS stage is the early stage of development of invasive breast cancer. The role of PEITC in this phase of the p53 mutant gene is fully applicable to the prevention and early intervention of breast cancer. The discovery of p53 R175 "wild-type" conformation and function, combined with the unique safety of natural food-derived compounds, opens up new avenues for targeted cancer prevention and treatment.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。 All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (10)

  1. 一种PEITC的用途，其特征在于，用于制备一制剂或组合物，所述制剂或组合物用于(a)改变(或激活)突变型p53，(b)抑制突变型p53肿瘤细胞的增殖，(c)诱导突变型p53肿瘤细胞的凋亡，和/或(d)预防或治疗基于p53突变引起的疾病。Use of a PEITC for the preparation of a formulation or composition for (a) altering (or activating) a mutant p53, and (b) inhibiting proliferation of a mutant p53 tumor cell (c) inducing apoptosis of mutant p53 tumor cells, and/or (d) preventing or treating diseases caused by p53 mutations.
  2. 如权利要求1所述的用途，其特征在于，所述的突变型p53在选自下组的位点具有突变：R175、C176、Y220、P223、C242、G245、R248、R249、R273、V274、P278、R282或其组合。The use according to claim 1, wherein said mutant p53 has a mutation at a site selected from the group consisting of R175, C176, Y220, P223, C242, G245, R248, R249, R273, V274, P278, R282 or a combination thereof.
  3. 如权利要求1所述的用途，其特征在于，所述的突变型p53为突变型p53R175。The use according to claim 1, wherein said mutant p53 is mutant p53 R175 .
  4. 如权利要求1所述的用途，其特征在于，所述的PEITC提取自十字花科植物。The use according to claim 1 wherein said PEITC is extracted from a cruciferous plant.
  5. 如权利要求4所述的用途，其特征在于，所述的十字花科植物选自下组：The use according to claim 4, wherein the cruciferous plant is selected from the group consisting of:
    青菜、萝卜、大白菜、西洋菜、西蓝花、胡萝卜、甘蓝、辣根、芥末、花椰菜、覆盆子或其组合。Greens, radishes, Chinese cabbage, watercress, broccoli, carrots, kale, horseradish, mustard, broccoli, raspberries or combinations thereof.
  6. 如权利要求1所述的用途，其特征在于，所述的组合物为药物组合物，较佳地，所述的药物组合物用于预防和/或治疗癌症。The use according to claim 1, wherein the composition is a pharmaceutical composition, preferably the pharmaceutical composition is for preventing and/or treating cancer.
  7. 如权利要求6所述的用途，其特征在于，所述的癌症选自下组：The use according to claim 6 wherein said cancer is selected from the group consisting of:
    乳腺癌、胰腺癌、肝癌、***癌、***、卵巢癌、口腔癌、食道癌、胃癌、结直肠癌、鼻咽癌、肺癌、膀胱癌、软组织肉瘤、脑瘤、淋巴细胞肿瘤、成骨肉瘤或其组合。Breast cancer, pancreatic cancer, liver cancer, prostate cancer, cervical cancer, ovarian cancer, oral cancer, esophageal cancer, gastric cancer, colorectal cancer, nasopharyngeal cancer, lung cancer, bladder cancer, soft tissue sarcoma, brain tumor, lymphocyte tumor, osteosarcoma Tumor or a combination thereof.
  8. 一种p53基因检测试剂的用途，其特征在于，用于制备诊断试剂或诊断试剂盒，所述诊断试剂或诊断试剂盒用于(a)判断PEITC治疗效果，和/或(b)判断肿瘤患者是否适合用PEITC进行治疗。Use of a p53 gene detecting reagent for preparing a diagnostic reagent or a diagnostic kit for (a) determining a PEITC treatment effect, and/or (b) determining a tumor patient Is it suitable for treatment with PEITC?
  9. 一种试剂盒，其特征在于，所述试剂盒包括：A kit, characterized in that the kit comprises:
    (a)容器A，以及位于容器A中的PEITC或含PEITC的药物；(a) container A, and PEITC or PEITC-containing drug located in container A;
    (b)容器B，以及位于容器B中的p53基因检测试剂；(b) container B, and a p53 gene detection reagent located in container B;
    (c)说明书。(c) Instructions.
  10. 一种体外非治疗性地抑制肿瘤细胞的方法，其特征在于，所述方法包括步骤： A method for non-therapeutic inhibition of tumor cells in vitro, characterized in that the method comprises the steps of:
    (i)提供一肿瘤细胞，检测所述肿瘤细胞p53基因的突变情况，如果所述肿瘤细胞的p53为突变型时，进行步骤(ii)；(i) providing a tumor cell, detecting the mutation of the tumor cell p53 gene, if the tumor cell p53 is a mutant type, performing step (ii);
    (ii)在PEITC存在的条件下，培养所述肿瘤细胞。 (ii) culturing the tumor cells in the presence of PEITC.
PCT/CN2017/080960 2016-04-18 2017-04-18 Pharmaceutical composition containing peitc and use thereof in cancer treatment WO2017181943A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201780037010.4A CN109562093A (en) 2016-04-18 2017-04-18 Pharmaceutical composition containing PEITC and its application in cancer treatment

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610240459.8A CN107303291A (en) 2016-04-18 2016-04-18 Pharmaceutical composition containing PEITC and its application in treatment of cancer
CN201610240459.8 2016-04-18

Publications (1)

Publication Number Publication Date
WO2017181943A1 true WO2017181943A1 (en) 2017-10-26

Family

ID=60115607

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/080960 WO2017181943A1 (en) 2016-04-18 2017-04-18 Pharmaceutical composition containing peitc and use thereof in cancer treatment

Country Status (2)

Country Link
CN (2) CN107303291A (en)
WO (1) WO2017181943A1 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112955550A (en) * 2018-09-12 2021-06-11 基础科学研究院 Composition for inducing death of cell having mutant gene and method for inducing death of cell having mutant gene by using the same
CN110241218A (en) * 2019-07-11 2019-09-17 重庆大学 It is a kind of to detect the kit for being applicable in the tumor patient of anticarcinogen of targeting p53
CN110538173A (en) * 2019-08-20 2019-12-06 郑州大学第一附属医院 Application of isothiocyanate compounds in preparation of esophageal cancer targeted drugs

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130158111A1 (en) * 2011-12-20 2013-06-20 Georgetown University Compositions and Methods for Depleting Mutant p53

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130158111A1 (en) * 2011-12-20 2013-06-20 Georgetown University Compositions and Methods for Depleting Mutant p53

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WANG, XIANTAO ET AL.: "Selective depletion of mutant p53 by cancer chemopreventive isothiocyanates and their structure - activity relationships", J MED CHEM., vol. 54, no. 3, 10 February 2011 (2011-02-10), pages 809 - 816, XP055602292, ISSN: 0022-2623, DOI: 10.1021/jm101199t *
YAO-TSUNG: "Phenethyl isothiocyanate induces DNA damage-associated G2/ M arrest and subsequent apoptosis in oral cancer cells with varying p53 mutations", FREE RADICAL BIOLOGY AND MEDICINE, vol. 74, 19 June 2014 (2014-06-19), pages 1 - 13, XP055602296, ISSN: 0891-5849, DOI: 10.1016/j.freeradbiomed.2014.06.008 *

Also Published As

Publication number Publication date
CN107303291A (en) 2017-10-31
CN109562093A (en) 2019-04-02

Similar Documents

Publication Publication Date Title
Aggarwal et al. Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth
Wei et al. Melatonin induces apoptosis of colorectal cancer cells through HDAC 4 nuclear import mediated by C a MKII inactivation
Subramanian et al. HDAC6 deacetylates Ku70 and regulates Ku70-Bax binding in neuroblastoma
Li et al. Anti-cancer efficacy of SREBP inhibitor, alone or in combination with docetaxel, in prostate cancer harboring p53 mutations
Mohapatra et al. Quinacrine-mediated autophagy and apoptosis in colon cancer cells is through a p53-and p21-dependent mechanism
Mondello et al. Dual inhibition of histone deacetylases and phosphoinositide 3-kinase enhances therapeutic activity against B cell lymphoma
US20130288980A1 (en) Targeting senescent and cancer cells for selective killing by interference with foxo4
Göttgens et al. Inhibition of CDK4/CDK6 enhances radiosensitivity of HPV negative head and neck squamous cell carcinomas
US8759097B2 (en) Inhibition of dynamin related protein 1 to promote cell death
US20130288981A1 (en) Targeting senescent cells and cancer cells by interference with jnk and/or foxo4
Morelli et al. Axitinib induces senescence-associated cell death and necrosis in glioma cell lines: The proteasome inhibitor, bortezomib, potentiates axitinib-induced cytotoxicity in a p21 (Waf/Cip1) dependent manner
Ramezani et al. Rolipram potentiates bevacizumab-induced cell death in human glioblastoma stem-like cells
Grassi et al. SP600125 has a remarkable anticancer potential against undifferentiated thyroid cancer through selective action on ROCK and p53 pathways
Aggarwal et al. p53 mutant-type in human prostate cancer cells determines the sensitivity to phenethyl isothiocyanate induced growth inhibition
WO2017181943A1 (en) Pharmaceutical composition containing peitc and use thereof in cancer treatment
Zhang et al. Antifungal agent Terbinafine restrains tumor growth in preclinical models of hepatocellular carcinoma via AMPK-mTOR axis
US9492450B2 (en) Inhibition of dynamin related protein 1 to promote cell death
Lone et al. Non-POU Domain-Containing Octomer-Binding (NONO) protein expression and stability promotes the tumorigenicity and activation of Akt/MAPK/β-catenin pathways in human breast cancer cells
Wang et al. Antitumor effects of a covalent cyclin-dependent kinase 7 inhibitor in colorectal cancer
US10206920B2 (en) Pharmaceutical composition for treating cancer and a method of using the same
Cherradi et al. Improving the response to oxaliplatin by targeting chemotherapy-induced CLDN1 in resistant metastatic colorectal cancer cells
US20200101070A1 (en) Methods of treating cancer having an active wnt/beta-catenin pathway
Chikkegowda et al. Design, synthesis, characterization, and crystal structure studies of Nrf2 modulators for inhibiting cancer cell growth in vitro and in vivo
US20220040134A1 (en) Electrophiles and electrophile pro-drugs as rad51 inhibitors
Biswas et al. Phytochemical inhibition of multidrug resistance protein-1 as a therapeutic strategy for hemangioendothelioma

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17785427

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17785427

Country of ref document: EP

Kind code of ref document: A1