WO2017181943A1 - Composition pharmaceutique contenant du peitc et utilisation de cette dernière dans le traitement du cancer - Google Patents

Composition pharmaceutique contenant du peitc et utilisation de cette dernière dans le traitement du cancer 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
English (en)
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/zh
Publication of WO2017181943A1 publication Critical patent/WO2017181943A1/fr

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)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Public Health (AREA)
  • Emergency Medicine (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (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

La présente invention concerne une utilisation du PEITC pour préparer une préparation ou une composition. La préparation ou la composition est utilisée pour : (a) modifier le p53 mutant afin de restaurer l'activité de type sauvage; (b) inhiber la prolifération de cellules tumorales provoquée par le p53 mutant; (c) induire l'apoptose des cellules tumorales à p53 mutant; et/ou (d) prévenir ou traiter des maladies fondées sur des mutations de p53. La présente invention concerne également l'utilisation d'un réactif de détection du gène p53, un kit comprenant du PEITC et le réactif de détection du gène p53, et un procédé pour l'inhibition non thérapeutique de cellules tumorales in vitro.
PCT/CN2017/080960 2016-04-18 2017-04-18 Composition pharmaceutique contenant du peitc et utilisation de cette dernière dans le traitement du cancer WO2017181943A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201780037010.4A CN109562093A (zh) 2016-04-18 2017-04-18 含peitc的药物组合物及其在癌症治疗中的应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610240459.8 2016-04-18
CN201610240459.8A CN107303291A (zh) 2016-04-18 2016-04-18 含peitc的药物组合物及其在癌症治疗中的应用

Publications (1)

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

Family

ID=60115607

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/080960 WO2017181943A1 (fr) 2016-04-18 2017-04-18 Composition pharmaceutique contenant du peitc et utilisation de cette dernière dans le traitement du cancer

Country Status (2)

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

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220049274A1 (en) * 2018-09-12 2022-02-17 Institute For Basic Science Composition for inducing death of cells having mutated gene, and method for inducing death of cells having mutated gene by using composition
CN110241218A (zh) * 2019-07-11 2019-09-17 重庆大学 一种检测适用靶向p53的抗癌药的肿瘤患者的试剂盒
CN110538173A (zh) * 2019-08-20 2019-12-06 郑州大学第一附属医院 异硫氰酸酯类化合物在制备食管癌靶向药中的应用

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
CN109562093A (zh) 2019-04-02
CN107303291A (zh) 2017-10-31

Similar Documents

Publication Publication Date Title
Aggarwal et al. Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth
Subramanian et al. HDAC6 deacetylates Ku70 and regulates Ku70-Bax binding in neuroblastoma
Mondello et al. Dual inhibition of histone deacetylases and phosphoinositide 3-kinase enhances therapeutic activity against B cell lymphoma
Mohapatra et al. Quinacrine-mediated autophagy and apoptosis in colon cancer cells is through a p53-and p21-dependent mechanism
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
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
US20130288981A1 (en) Targeting senescent cells and cancer cells by interference with jnk and/or foxo4
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
Lin et al. Plumbagin induces autophagy and apoptosis of SMMC‐7721 cells in vitro and in vivo
WO2017181943A1 (fr) Composition pharmaceutique contenant du peitc et utilisation de cette dernière dans le traitement du cancer
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
Cherradi et al. Improving the response to oxaliplatin by targeting chemotherapy-induced CLDN1 in resistant metastatic colorectal cancer cells
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
Ou et al. Inhibition of autophagy and induction of glioblastoma cell death by NEO214, a perillyl alcohol-rolipram conjugate
US10206920B2 (en) Pharmaceutical composition for treating cancer and a method of using the same
Chikkegowda et al. Design, synthesis, characterization, and crystal structure studies of Nrf2 modulators for inhibiting cancer cell growth in vitro and in vivo
US20200101070A1 (en) Methods of treating cancer having an active wnt/beta-catenin pathway
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