WO2017186103A1 - Pde3a在判断阿那格雷治疗肿瘤效果中的应用 - Google Patents

Pde3a在判断阿那格雷治疗肿瘤效果中的应用 Download PDF

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WO2017186103A1
WO2017186103A1 PCT/CN2017/081908 CN2017081908W WO2017186103A1 WO 2017186103 A1 WO2017186103 A1 WO 2017186103A1 CN 2017081908 W CN2017081908 W CN 2017081908W WO 2017186103 A1 WO2017186103 A1 WO 2017186103A1
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pde3a
anagrelide
tumor
protein
cells
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French (fr)
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俞强
刘珏妤
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中国科学院上海药物研究所
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Priority to CN201780025830.1A priority Critical patent/CN109072313A/zh
Priority to US16/097,076 priority patent/US20190145978A1/en
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    • 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
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    • 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
    • G01N33/53—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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to the field of tumor molecular detection. More specifically, it relates to the use of PDE3A in determining the effect of anagrelide in treating tumors.
  • Targeted therapy and the use of tumor-specific gene mutations and gene function "target therapy” is currently a hot spot in cancer drug research.
  • Molecular target treatment is a key molecule that specifically promotes tumor growth and survival in different tumor cells, and achieves an anti-tumor effect of inhibiting tumor cell growth or promoting apoptosis.
  • Representative target drugs such as Gefitinib, a receptor tyrosine kinase inhibitor, Lapatinib, Crizotinib, etc. and the antibody drug Avastin ), cetuximab, etc.
  • inhibitors targeting other kinases ubiquitin-proteasome inhibitors, and histone deacetylase (HDAC) inhibitors and the like.
  • HDAC histone deacetylase
  • Anagrelide is a phosphodiesterase inhibitor for the treatment of anti-thrombocytopenia. Studies have shown that anagrelide or its pharmaceutically acceptable preparations have new applications in inhibiting tumors, but anagrelide does not have a good effect on all types of tumors, and there is still no clear diagnostic method to guide Ana Gray's medication. There is an urgent need in the art to develop biomarkers capable of determining the efficacy of anagrelide in the treatment of tumors.
  • the diagnostic reagent comprises a protein chip, a nucleic acid chip, or a combination thereof.
  • the diagnostic reagents are used in PCR, immunoblotting, and immunohistochemistry.
  • the determination includes an auxiliary determination and/or a prior (pre-treatment) determination.
  • the sensitivity refers to the sensitivity of the tumor cells in the presence of anagregrein at a concentration of from 0.001 to 0.25 ⁇ M, preferably from 0.01 to 0.1 ⁇ M, more preferably 0.02. -0.08 ⁇ M.
  • the sensitivity refers to the sensitivity of the tumor cells in the presence of anagregrein at a concentration of from 0.25 to 10 ⁇ M, preferably from 0.5 to 5 ⁇ M, more preferably from 0.8 to 3 ⁇ M. .
  • the sensitivity includes sensitivity of the tumor cells under in vitro culture conditions, and/or sensitivity of the tumor cells in vivo.
  • the anagrelide is administered in an amount of from 1 mg to 500 mg, preferably from 10 mg to 250 mg, more preferably from 30 to 100 mg per day.
  • the PDE3A gene sequence comprises a coding sequence and/or a non-coding sequence of PDE3A.
  • the PDE3A gene sequence comprises genomic DNA, cDNA and/or mRNA sequences.
  • the PDE3A nucleic acid detection reagent is coupled or carried with a detectable label.
  • the PDE3A protein detecting reagent comprises a specific antibody against a PDE3A protein, a protein chip.
  • the PDE3A protein detection reagent is coupled or carried 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.
  • the PDE3A protein comprises a full length PDE3A protein, or a secreted protein thereof.
  • the PDE3A gene sequence and/or PDE3A protein is used as a standard or control in the kit.
  • the PDE3A nucleic acid detection reagent comprises a primer, a probe, or a nucleic acid chip.
  • the tumor patient comprises a non-hematologic tumor patient or a solid tumor patient.
  • the tumor is selected from the group consisting of lung cancer, liver cancer, gastric cancer, esophageal cancer, intestinal cancer, nasopharyngeal cancer, breast cancer, lymphoma, renal cancer, pancreatic cancer, bladder cancer, ovarian cancer, and uterus.
  • the tumor is selected from the group consisting of lung cancer, melanoma, renal cancer, colon cancer, glioma, and cervical cancer.
  • the diagnostic reagent is a monoclonal and polyclonal antibody or a combination thereof.
  • the diagnostic reagent or diagnostic kit is for detecting a sample selected from the group consisting of a surgically removed tissue sample, a biopsy tissue sample, a tumor tissue lysate, a blood sample, a cell sample, a body fluid sample, Urine sample, or a combination thereof.
  • the diagnostic kit is for detecting a slice made from a tissue sample.
  • the section comprises a paraffin section, or a frozen section.
  • the blood sample is detected by detecting the expression level of PDE3A in circulating tumor cells in the peripheral blood.
  • the diagnostic kit includes an immunoblot analysis kit and/or a PCR assay kit.
  • a diagnostic kit for detecting the effect of anagrelide in treating a tumor comprising:
  • the label or instructions indicate that the kit is used to (a) determine the effect of anagrelide in treating a tumor, and/or (b) determine whether a tumor patient is suitable for treatment with anagrelide.
  • the detection reagent comprises: a primer, a probe, an antibody, a nucleic acid chip, Protein chip or a combination thereof.
  • the first container comprises one or more first containers.
  • the PDE3A protein or a specific antibody thereof is conjugated with or with a detectable label.
  • the standard or control comprises a PDE3A nucleic acid sequence, a PDE3A protein, or a combination thereof.
  • the diagnostic kit further comprises a second container containing reagents for treating tumor tissue.
  • the diagnostic kit further contains reagents for performing immunoblot detection.
  • the label or the description states the following:
  • said positive expression of PDE3A means that the tumor cells express PDE3A and/or have PDE3A protein activity.
  • the negative expression of PDE3A means that the tumor cells have low or no expression of PDE3A and/or no PDE3A protein activity.
  • a detection reagent for detecting a PDE3A gene sequence or protein for preparing a kit for (a) determining the effect of anagrelide in treating a tumor, And/or (b) a marker for determining whether a tumor patient is suitable for treatment with anagrelide.
  • a method of determining the sensitivity of a tumor cell to anagrelide in vitro comprising the steps of:
  • the low or no expression of PDE3A means that the ratio of the mRNA level M1 of PDE3A to the mRNA level M2 of ⁇ -actin in the tumor cells is R1 ⁇ 0.2, preferably ⁇ 0.1.
  • said low or no expression of PDE3A means that the ratio of PDE3A protein level P1 in said tumor cells to PDE3A protein level P2 in Bel7404 is R2 ⁇ 0.5, preferably ⁇ 0.2.
  • "having no PDE3A protein activity” means that the ratio of the activity A1 of PDE3A in the tumor cell to the PDE3A protein activity A2 in Bel7404 is R3 ⁇ 0.5, preferably ⁇ 0.2.
  • the expression of PDE3A means that the ratio of the mRNA level M1 of PDE3A to the mRNA level M2 of ⁇ -actin in the tumor cells is R1 ⁇ 0.3, preferably ⁇ 0.5.
  • the expression of PDE3A means that the ratio of PDE3A protein level P1 in the tumor cell to PDE3A protein level P2 in Bel7404 is R2 ⁇ 0.6, preferably ⁇ 0.8.
  • "having PDE3A protein activity” means that the ratio of the activity A1 of PDE3A in the tumor cells to the PDE3A protein activity A2 in Bel7404 is R3 ⁇ 0.6, preferably ⁇ 0.8.
  • the method further comprises: detecting whether the PDE3A of the tumor cell to be tested has a mutation.
  • the method further includes:
  • the tumor cells to be tested which are sensitive to anagrelide in the previous step of in vitro culture, and observe the growth of the tumor cells, thereby verifying that the tumor cells are anagrelide Sensitivity.
  • the concentration of the anagrelide is from 0.001 to 0.25 ⁇ M, preferably from 0.01 to 0.1 ⁇ M, more preferably from 0.02 to 0.08 ⁇ M.
  • the concentration of the anagrelide is 0.25 to 10 ⁇ M, preferably 0.5 to 5 ⁇ M, more preferably 0.8 to 3 ⁇ M.
  • step (iii) the growth of the tumor cells is observed, including observing the apoptosis and/or migration of the tumor cells.
  • the method is non-diagnostic and non-therapeutic.
  • a method of treating a tumor comprising the steps of: administering anagrelide to a subject in need thereof, wherein the subject is a tumor patient, and the tumor cell expresses PDE3A and/or has PDE3A protein activity .
  • the method further comprises detecting the PDE3A expression or activity of the subject prior to administering the anagrelide.
  • the anagrelide comprises a compound of the structure of formula II, or a pharmaceutically acceptable salt or prodrug or derivative thereof, or a formulation thereof.
  • Figure 1 shows the expression of PDE3A protein in different cell lines.
  • ⁇ -Tubulin and GAPDH are internal reference proteins.
  • unidentified are anagrelide insensitive cells R.
  • Figure 2 shows that anagrelide selectively inhibits tumor cell growth from different tissue sources. Among them, treatment with anagrelide (1 ⁇ M) for 72 hours, MTT assay for cell survival rate, the cells were classified by the test results: sensitive (survival rate ⁇ 40%), moderately sensitive (survival rate 40-75%) ), resistance (survival rate >75%), the cell name in the figure is marked with a circle and a triangle respectively to identify the sensitive group and the moderately sensitive group.
  • Figure 3 shows the expression of PDE3A mRNA in different cells. Among them, RT-PCR was used to detect PDE3A mRNA levels in nine cells; ordinate: quantitative results relative to ⁇ -actin.
  • Figure 4 shows the role of PDE3A protein in tumor cell growth.
  • Figure 4A shows the expression of PDE3A protein in HeLa cells by immunoblotting 48 hours after pde3a siRNA 1/2 transfection.
  • Figure 4B shows the RTCA assay for monitoring the growth of HeLa cells before and after transfection of pde3a siRNA.
  • NC negative control
  • KD1 and KD2 PDE3A knockdown group.
  • Figure 5 shows the relationship between the expression of PDE3A protein and cytotoxicity of anagrelide.
  • Figure 5A shows that siRNA was transfected with HeLa for 48 hours, and the RTCA method recorded cell growth curves before and after treatment with different anagrelide.
  • NC control group
  • KD PDE3A knockdown group.
  • Figure 5B shows HeLa cells treated with anagrelide or DMSO for 36 hours before and after transfection, stained with PI for 20 minutes, and photographed by fluorescence microscopy.
  • Figure 5C shows the results of counting PI positive cells using the Cell Profiler software.
  • Figure 5D shows the results of ELISA treatment of cells for 48 hours with anagrelide.
  • Figure 6 shows the mRNA expression of other PDEs in the anagrelide-insensitive cell line, and the relative expression level was obtained based on the PDE3A expression of the moderately sensitive cell Bel7404.
  • PDE3A biomarker
  • the present invention establishes a safe and effective diagnostic and typing method based on the expression of PDE3A in tumor cells, and can guide anagrelide clinically on individualized treatment of tumor patients in a simple, rapid and economical manner.
  • PDE3A ELISA kits on the market, but there is no method for the diagnosis and treatment of tumors using PDE3A as a tumor marker.
  • the method of immunoblotting and PCR of the present invention for detecting the expression of PDE3A in tumor samples and applying it to guide the application of anagrelide in treating tumors is the first in technology and application.
  • the present invention found for the first time that about 30% of tumor cell lines are positive for PDE3A protein or mRNA expression, and PDE3A-positive tumor cells exhibit different degrees of sensitivity to Anagrelide, while anagrelide is negative for PDE3A.
  • the tumor cells do not have any inhibitory effect.
  • Animal experiments indicate that Anagrelide is safe at doses that inhibit tumor cell growth (10 mg-200 mg/kg, preferably 10-20 mg/kg), so PDE3A serves as a biomarker for the drug to guide anagrelide. There are theoretical basis and application value for tumor treatment.
  • PDE3A phosphodiesterase 3A
  • phosphodiesterase 3A is a member of the family of serine kinases that terminates these by hydrolyzing cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in vivo.
  • cAMP cyclic adenosine monophosphate
  • cGMP cyclic guanosine monophosphate
  • the biosignal transmitted by the second messenger regulates life activities.
  • PDE3A is mainly distributed in heart, platelets, vascular smooth muscle and egg cells; the hydrolysis efficiency of cAMP is 10 times that of cGMP.
  • the terms "protein of the present invention”, “PDE3A protein”, and “PDE3A polypeptide” are used interchangeably and refer to a protein or polypeptide having the amino acid sequence of human PDE3A protein (UniProtKB/Swiss-Prot: Q14432). Includes PDE3A protein with or without the initial methionine. In addition, the term also includes full length PDE3A and fragments thereof, particularly secretory fragments (or secreted proteins).
  • the PDE3A protein referred to in the present invention includes its complete amino acid sequence, its secreted protein, its mutant and its function. Active fragment.
  • PDE3A proteins of the invention include glycosylated and non-glycosylated proteins.
  • PDE3A gene ID: 5139
  • PDE3A polynucleotide refers to a nucleic acid sequence having a human PDE3A nucleotide sequence (NC_000012.12). It will be appreciated that the substitution of a nucleotide in a codon is acceptable when encoding the same amino acid. It will also be appreciated that nucleotide substitutions are also acceptable when substituted by nucleotides to produce conservative amino acid substitutions.
  • nucleic acid sequence encoding the same can be constructed therefrom, and a specific probe can be designed based on the nucleotide sequence.
  • the full-length nucleotide sequence or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed according to the PDE3A nucleotide sequences disclosed herein, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • DNA sequences encoding the proteins (or fragments, derivatives thereof) of the present invention can be obtained completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • polynucleotide sequences of the present invention can be used to express or produce recombinant PDE3A polypeptides by conventional recombinant DNA techniques.
  • antibody of the present invention and “specific antibody against PDE3A” are used interchangeably.
  • the invention also encompasses polyclonal and monoclonal antibodies, particularly monoclonal antibodies, that are specific for human PDE3A polypeptides.
  • specificity means that the antibody binds to a human PDE3A gene product or fragment.
  • the antibodies of the present invention include those capable of binding to and inhibiting the human PDE3A protein, as well as those which do not affect the function of the human PDE3A protein.
  • the invention also includes those that can be repaired An antibody that binds to the unmodified form of the human PDE3A gene product.
  • the invention encompasses not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) 2 fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules ( Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have murine antibody binding specificity but still retain antibody portions from humans.
  • immunologically active antibody fragments such as Fab' or (Fab) 2 fragments
  • antibody heavy chains such as antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules ( Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have murine antibody binding specificity but still retain antibody portions from humans.
  • Antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, a purified human PDE3A gene product or a fragment thereof that is antigenic can be administered to an animal to induce polyclonal antibody production. Similarly, cells expressing human PDE3A protein or antigenic fragments thereof can be used to immunize animals to produce antibodies.
  • the antibody of the invention may also be a monoclonal antibody. Such monoclonal antibodies can be prepared using hybridoma technology.
  • the antibodies of the present invention include antibodies that block the function of the human PDE3A protein as well as antibodies that do not affect the function of the human PDE3A protein.
  • the various antibodies of the invention can be obtained by conventional immunological techniques using fragments or functional regions of the human PDE3A gene product.
  • fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer.
  • An antibody that binds to an unmodified form of the human PDE3A gene product can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (eg, E. coli); an antibody that binds to a post-translationally modified form (eg, glycosylated or phosphorylated)
  • a protein or polypeptide can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell, such as a yeast or insect cell.
  • Antibodies against human PDE3A protein can be used in immunohistochemistry to detect human PDE3A protein in specimens, particularly tumor tissues.
  • anagrelide As used herein, the terms “anagrelide”, “Anagrelide”, “Anguining”, “ANA” are used interchangeably and refer to a compound having the structure shown in the following formula, or a pharmaceutically acceptable salt or prodrug thereof. Or a derivative, or a form of its preparation:
  • Anagrelide is a phosphodiesterase inhibitor for the treatment of anti-thrombocytopenia. Studies have shown that anagrelide or its pharmaceutically acceptable formulations have new applications in inhibiting tumors. Experiments have shown that the IC50 of anagrelide in inhibiting platelets in vitro is 0.27-1 ⁇ M, while the IC50 of anagrelide in sensitive tumor cells is less than 0.03 ⁇ M.
  • the anagrelide is 6,7-dichloro-1,5-dihydroimidazole [2,1-b]quinazoline-2(3H)one.
  • anagrelide is clinically used as a phosphodiesterase inhibitor for the treatment of anti-thrombocytopenia. Studies have shown that anagrelide or its pharmaceutically acceptable formulations have new uses in inhibiting tumors, specifically including
  • the anagrelide or a pharmaceutical composition thereof can be administered at a lower concentration.
  • the anagrelide can act on a subject cell at a concentration of ⁇ 1 ⁇ M and produce a desired effect.
  • the derivative of anagrelide has the structure shown in formula I below:
  • R 1 to R 8 are each independently selected from the group consisting of a hydrogen atom, a halogen atom, an amino group, a hydroxyl group, a cyano group, an aldehyde group, a nitro group, a carboxyl group (-COOH), a substituted or unsubstituted C1-C10 alkyl group, and a substitution.
  • C3-C10 cycloalkyl substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C1 a C10 heteroaryl group (such as a substituted or unsubstituted 5- or 6-membered heterocyclic ring, an 8- to 10-membered heteroaryl bicyclic ring system), a substituted or unsubstituted C1-C10 alkoxy group, a substituted or unsubstituted C6-C10 aryl-oxy, substituted or unsubstituted C1-C10 heteroaryl-oxy, substituted or unsubstituted acyl (preferably -CO-C1-C10 alkyl), substituted or unsubstituted ester group (preferably C1 ⁇ C10 alkyl group -COO
  • R 9 is selected from the group consisting of a hydrogen atom, an oxygen atom, a substituted or unsubstituted C1-C10 alkyl group, a substituted or unsubstituted C3-C10 cycloalkyl group, a substituted or unsubstituted C6-C10 aryl group, a substituted or not Substituted C1-C10 heteroaryl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C6-C10 aryl-oxy, substituted or unsubstituted acyl (preferably -CO-C1-C10) An alkyl group, a substituted or unsubstituted C1 to C10 sulfonyl group;
  • one or more hydrogen atoms on the substituent group are substituted with a substituent selected from the group consisting of C1-C10 alkyl, C3-C10 cycloalkyl, C1-C10 alkoxy, halogen, hydroxy, carboxy ( -COOH), C1-C10 aldehyde group, C2-C10 acyl group, C2-C10 ester group, amino group, phenyl group;
  • the phenyl group includes an unsubstituted phenyl group or a substituted phenyl group having 1 to 3 substituents selected from the group consisting of halogen, C1-C10 alkyl, cyano, OH, nitro, C3 to C10. Cycloalkyl, C1-C10 alkoxy, amino.
  • each of R 1 to R 8 is independently selected from the group consisting of a hydrogen atom, a halogen atom, an amino group, a hydroxyl group, a cyano group, a nitro group, an amino group, an aldehyde group, a carboxyl group, a substituted or unsubstituted group.
  • C1-C5 alkyl substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C5 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C6-C10 aryl , substituted or unsubstituted C1-C6 heteroaryl, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C6-C10 aryl-oxy, substituted or unsubstituted C1-C6 heteroaryl - group, a substituted or unsubstituted -CO-C1 ⁇ C5 alkyl group, a substituted or unsubstituted C1 ⁇ C5 alkyl group -COO-, substituted or unsubstituted C1 ⁇ C5 sulfonyl group; or R 1 and R 2, R 3 and R 4 together constitute a
  • R 9 is selected from the group consisting of a hydrogen atom, an oxygen atom, a substituted or unsubstituted C1-C5 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted C6-C10 aryl group, a substituted or not Substituted C1-C10 heteroaryl, substituted or unsubstituted C1-C5 alkoxy, substituted or unsubstituted C6-C10 aryl-oxy, substituted or unsubstituted-CO-C1-C5 alkyl, substituted Or unsubstituted C1-C5 sulfonyl;
  • R 1 to R 8 are each independently selected from the group consisting of a hydrogen atom, a halogen atom, a cyano group, a substituted or unsubstituted C1-C5 alkyl group, a substituted or unsubstituted C3-C6 naphthenic ring. base;
  • R 1 and R 2 , R 3 and R 4 together constitute a group selected from the group consisting of a substituted or unsubstituted C1-C5 cycloalkyl group, a substituted or unsubstituted C1-C5 heterocycloalkyl group, a carbonyl group;
  • R 9 is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted C1-C5 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group;
  • 1 to 8 of the above R 1 to R 9 are a hydrogen atom, and preferably 2 to 7 are a hydrogen atom.
  • 1 to 8 of the above R 1 to R 8 are a halogen atom.
  • R 1 to R 8 are a halogen atom, and the other R 1 to R 8 are each a hydrogen atom.
  • the compound of formula I has the structure shown in formula II:
  • the pharmaceutically acceptable salt is a salt selected from the group consisting of hydrochloride, acetate, phosphate, or a combination thereof.
  • Western blot analysis can be performed by taking a small amount of tumor tissue from a tumor patient by biopsy or surgery. If the patient's PDE3A protein expression is positive (PDE3A+), then Anagrelide can be considered for treatment, the effective rate is close to 70%; if PDE3A negative (PDE3A-), the drug is not used, the accuracy is excluded. 100%.
  • PDE3A mRNA The expression of PDE3A mRNA can be detected by taking a small amount of tumor tissue from a tumor patient by biopsy or surgery, extracting mRNA, and performing RT-PCR reaction.
  • PDE3A mRNA expression is positive (PDE3A+), you can consider the use of Anagrelide for treatment, the efficiency is close to 70%; if the PDE3A mRNA is negative (PDE3A-), the drug is not used, the exclusion rate is 100 %.
  • this method can be used to detect this PDE3A protein.
  • the expression level of PDE3A in circulating tumor cells in peripheral blood can also be detected.
  • the invention also provides a test kit for treating tumor effect of anagrelide, wherein the kit comprises a container a containing a PDE3A gene sequence, a protein or a specific antibody thereof; and a label or a manual,
  • the label or instructions indicate that the kit is used to (a) determine the effect of anagrelide in treating a tumor, and/or (b) determine whether a tumor patient is suitable for treatment with anagrelide.
  • it comprises an immunoglobulin or immunoconjugate against PDE3A, or an active fragment thereof.
  • the above cell lines were cultured in a cell culture incubator containing 5% CO 2 at 37 ° C, and the experiment was performed while the cells were in the logarithmic growth phase.
  • the sample was dissolved in a suitable loading buffer, and after mixing, it was allowed to stand for 3 hours to fully dissolve the protein in the sample, and it was collected in 5 minutes, and collected by centrifugation at 4 degrees.
  • Laemmli sample buffer was added (the concentration of the protein sample was 1 : 1 or 1: 2 ratio mixing) Strongly mix, the sample is placed in a water bath of 100 degrees for 3-5 minutes. Centrifuge at 10000g for 10 minutes, take the supernatant, and transfer it to another clean tube. At this point, the electrophoresis sample is ready (the sample can be used immediately or frozen, and the sample stored at -20 °C can be used.) Stable for months.)
  • the cells were continuously diluted geometrically with a cell maintenance solution to 1:10, 1:100, 1:1,000, 1:10,000, and a normal cell control group was added. Each drug of different concentrations was added to a 96-well monolayer. In the plate, inoculate 3 replicate wells per dilution, add 100 ⁇ l per well, incubate in a 37 ° C carbon dioxide incubator for 72 h, and add 5 ⁇ g/ml MTT 25 ⁇ L.
  • cell viability % (mean value of experimental well OD measured value / mean value of control well OD measured value) X 100%, using probability unit regression method, calculate the drug concentration at 50% cell poisoning (IC50 ).
  • SDA-PAGE SDS-polyacrylamide
  • Monolayer cells plus TRIzol 1ml/10cm diameter small dish Monolayer cells plus TRIzol 1ml/10cm diameter small dish; TRIzol evenly covered in small dish, placed on ice for 5 minutes, collected cells and transferred to 1.5ml RNAase free EP tube; add 200 ⁇ L chloroform to EP tube, shake vigorously for 15s Place on ice for 3 minutes; centrifuge at 12000g in a low-temperature high-speed centrifuge, centrifuge at 15°C for 15 minutes, transfer the upper layer to a new EP tube; add 500 ⁇ L of isopropanol to the EP tube, mix and place on ice for 10 minutes; 12,000 g of a high-speed centrifuge, centrifuged at 4 ° C for 10 minutes, see a white solid sinking at the bottom of the EP tube, and discard the supernatant.
  • the reaction system is as follows:
  • the Real-time PCR primer sequences are as follows:
  • StepOnePlus TM Real-Time PCR System (Applied biosystem) system analysis software to ⁇ -Actin as a reference, determined according to the relative expression of PDE3A 2 ⁇ Ct method.
  • Lipofectamine TM 2000 The amount and experimental operation of Lipofectamine TM 2000 are referred to the specification.
  • the logarithmic growth of HeLa cells was inoculated into 96-well plates or RTCA plates at about 6000/well, and the cells were over 70-80% overnight, and the cells were in good condition; a certain amount of serum-free and antibiotic-free medium was added to each of the two cells.
  • the EP tube add appropriate amount of siRNA to one tube and mix well, add another amount of Lipofectamine TM 2000 and mix at room temperature for 5-10 min. Add the medium supplemented with Lipofectamine TM reagent to the siRNA-containing EP tube and mix at room temperature.
  • the cells were allowed to stand for 15 min; during the time of washing, the cells were washed once with a serum-free and non-antibody-resistant medium, and then 75 ⁇ L of the medium was added for use. After 15 minutes, the mixed medium was added to the cell culture dish, 25 ⁇ L/well, and gently shaken and mixed; after culturing at 37 ° C, 5% CO 2 for 6 hours, it was replaced with fresh medium containing 10% FBS; 24- After 72h, the expression of RNAi interference protein was detected by western blot.
  • RTCA Cell real-time monitor
  • the cells in the logarithmic growth phase were inoculated into a 16 or 96-well plate equipped with a real-time monitor of the cells at 3-7 ⁇ 10 4 /mL per well, and incubated at 37 ° C overnight, followed by dosing or transfection.
  • the real-time monitor automatically monitors cell growth for more than 4 days, depending on the amount of cells that form a resistance after spreading. The larger the Cel lIndex value, the faster the cells grow and the more cells.
  • PI stain 25 ug/mL
  • PDE3A protein was positively expressed in SMMC7721, FHCC98, H4, HeLa, Bel7404, A498, SW1116 and other cell lines, and negatively expressed in the other 20 cell lines.
  • PDE3A mRNA levels of some cell lines were detected by RT-PCR; RQ: quantitative results relative to ⁇ -actin.
  • Example 1 The cell strains of Example 1 were subjected to activity experiments using Anagrelide at a concentration of 1 ⁇ M, and the cell lines were classified into three categories according to cell viability: sensitive (S), moderately sensitive (M), and insensitive ( R), and further determine the IC50 value of anagrelide for inhibition of each cell line.
  • S sensitive
  • M moderately sensitive
  • R insensitive
  • Table 1 shows the IC50 values of anagrelide-suppressed sensitive cell lines.
  • the cells expressing SM-positive PDM3A, SMMC7721, FHCC98, H4, and HeLa are sensitive cells of anagrelide, and anagrelide has excellent properties. Inhibition effect, IC50 value is less than 30nM; Pel3A positive cell line Bel7404, A498 and SW1116 are moderately sensitive cells of anagrelide, and anagrelide has a long-lasting inhibitory effect, its IC50 value is 0.4 ⁇ -16 ⁇ M .
  • Twenty strains of cells expressing PDE3A negatively were not sensitive to anagrelide and showed tolerance to anagrelide. The IC50 values were all >50 ⁇ M, ie anagrelide could not inhibit its growth.
  • Example 1 and Example 2 illustrate the relationship between PDE3A protein and growth inhibition from the viewpoint of the compound inhibiting protein activity, but the compound may cause a difference in cell selectivity due to its different structure and different mode of action with the target.
  • siRNA interference technology Two pde3a siRNAs (different efficiencies) were tested separately. After 48 hours of siRNA transfection, cells were plated on RTCA plates for long-term growth monitoring.
  • PDE3A protein is involved in the regulation of apoptosis induced by anagrelide
  • the KD group was HeLa cells transfected with pde3a siRNA, and 1 and 2 represent two siRNAs, respectively.
  • the NC group was HeLa cells transfected with siRNA without knockout effect, blank control.
  • kits for detecting the effect of anagrelide in treating tumors comprising:
  • kits (b) and a label or instructions indicating that the kit is for detecting the effect of anagrelide in treating a tumor.
  • Western blotting was used to detect the protein expression levels of other members of the PED family, including PDE1A, PDE3A, PDE3B, PDE4A, PDE7A, and PDE11A, in cell lines SW480 and MCF-7.
  • Anagrelide has been used as a first-line treatment for thrombocytopenia for more than 20 years, and has always been a blank in the history of solid tumor treatment; previous laboratory studies have shown for the first time that anagrelide can be an excellent choice.
  • Antitumor drugs are used clinically. After deepening its mechanism research, it was found that PDE3A protein can be used as the first biomarker to assist the diagnosis and treatment of anagrelide in clinical tumor patients. This is the first time that the compound has been researched and targeted for individualized tumor treatment.
  • anagrelide showed strong inhibition on the growth of a variety of tumor cells (liver cancer, melanoma, kidney cancer, colon cancer, glioma, cervical cancer), and has not revealed its mechanism of action.
  • tumor cells liver cancer, melanoma, kidney cancer, colon cancer, glioma, cervical cancer
  • anagrelide caused cell cycle arrest and apoptosis, and the biomarker PDE3A protein was found to guide clinical diagnosis and typing. Individualized targeted therapy for cancer patients.

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Abstract

一种磷酸二酯酶PDE3A及其基因在判断阿那格雷(Anagrelide)治疗肿瘤效果中的应用。具体提供了PDE3A基因序列、蛋白、或抗PDE3A蛋白的特异性抗体在制备诊断试剂或诊断试剂盒中的用途。所述诊断试剂或试剂盒用于(a)判断阿那格雷治疗肿瘤的效果，和/或(b)判断肿瘤患者是否适合用阿那格雷进行治疗。还提供了相应检测试剂盒。

Description

PDE3A在判断阿那格雷治疗肿瘤效果中的应用 技术领域
本发明涉及肿瘤分子检测领域。更具体地涉及PDE3A在判断阿那格雷治疗肿瘤的效果中的应用。
背景技术
肿瘤的发生、发展是多基因，多环境因素共同作用的结果，肿瘤患者遗传背景的多样性决定了肿瘤治疗的复杂性，也同时表明该疾病需进行个体化治疗。应用靶向技术向肿瘤病灶区精确递送药物的“靶向治疗”和利用肿瘤特异的基因突变和基因功能的“靶点治疗”是目前肿瘤药物研究的热点。分子靶点治疗是针对不同肿瘤细胞中特异的促进肿瘤生长和存活的关键分子，实现抑制肿瘤细胞生长或促进凋亡的抗肿瘤作用。代表性的靶点药物如靶向受体酪氨酸激酶的抑制剂吉非替尼(Gefitinib)，拉帕替尼(Lapatinib)，克唑替尼(Crizotinib)等和抗体药物阿瓦斯汀(Avastin)，西妥昔单抗(Cetuximab)等。此外，还有靶向其它激酶的抑制剂、泛素－蛋白酶体抑制剂、以及组蛋白去乙酰化酶(HDAC)抑制剂等。
与传统细胞毒化疗药物不同，肿瘤分子靶点药物具有特异性或选择性抗肿瘤作用和减低药物毒性作用，大大提高了肿瘤治疗的精准性，延长了患者生存期和生活质量。但目前的抗肿瘤药物靶点和靶点药物还远远不够。阿那格雷作为磷酸二酯酶抑制剂，用于治疗抗血小板增多症。有研究表明阿那格雷或其药学上可接受的制剂在抑制肿瘤方面具有新用途，但是阿那格雷并非对所有类型的肿瘤都有比较好的效果，目前仍缺乏明确的诊断方法以指导阿那格雷的用药。本领域迫切需要开发能够判断阿那格雷治疗肿瘤效果的生物标记物。
发明内容
本发明的目的在于提供判断阿那格雷治疗肿瘤的效果的标记物PDE3A，以及PDE3A在判断阿那格雷治疗肿瘤的效果中的应用。
在本发明的第一方面，提供了一种PDE3A基因序列、PDE3A核酸检测试剂、 PDE3A蛋白、和/或PDE3A蛋白检测试剂的用途，用于制备诊断试剂或诊断试剂盒，所述诊断试剂或试剂盒用于：
(a)判断阿那格雷(Anagrelide)治疗肿瘤的效果，和/或
(b)判断肿瘤患者是否适合用阿那格雷进行治疗，和/或
(c)判断肿瘤细胞对阿那格雷的敏感性。
在另一优选例中，所述的诊断试剂包括蛋白芯片、核酸芯片、或其组合。
在另一优选例中，所述的诊断试剂用于PCR、免疫印迹和免疫组化。
在另一优选例中，所述的判断包括辅助判断和/或预先(治疗前)判断。
在另一优选例中，所述的敏感性是指所述肿瘤细胞在以下浓度的阿那格雷存在下的敏感性：0.001-0.25μM，较佳地为0.01-0.1μM，更佳地为0.02-0.08μM。
在另一优选例中，所述的敏感性是指所述肿瘤细胞在以下浓度的阿那格雷存在下的敏感性：0.25-10μM，较佳地为0.5-5μM，更佳地为0.8-3μM。
在另一优选例中，所述的敏感性包括肿瘤细胞在体外培养条件下的敏感性，和/或肿瘤细胞在体内的敏感性。
在另一优选例中，所述的阿那格雷治疗肿瘤的用量为每天1mg-500mg，较佳地为10mg-250mg，更佳地为30-100mg。
在另一优选例中，所述的PDE3A基因序列包括PDE3A的编码序列和/或非编码序列。
在另一优选例中，所述的PDE3A基因序列包括基因组DNA、cDNA和/或mRNA序列。
在另一优选例中，所述的PDE3A核酸检测试剂偶联有或带有可检测的标记物。
在另一优选例中，所述的PDE3A蛋白检测试剂包括抗PDE3A蛋白的特异性抗体、蛋白芯片。
在另一优选例中，所述的PDE3A蛋白检测试剂偶联有或带有可检测的标记物。
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。
在另一优选例中，所述的PDE3A蛋白包括全长PDE3A蛋白、或其分泌性蛋白。
在另一优选例中，所述的PDE3A基因序列和/或PDE3A蛋白被作为所述试剂盒中的标准品或对照。
在另一优选例中，所述的PDE3A核酸检测试剂包括引物、探针、或核酸芯片。
在另一优选例中，所述肿瘤患者包括非血液肿瘤患者或实体瘤患者。
在另一优选例中，所述肿瘤选自下组：肺癌、肝癌、胃癌、食道癌、肠癌、鼻咽癌、乳腺癌、淋巴癌、肾癌、胰腺癌、膀胱癌、卵巢癌、子宫癌、骨癌、胆囊癌、唇癌、黑色素瘤、舌癌、喉癌、白血病、前列腺癌、脑癌、鳞癌、皮肤癌、血管瘤、脂肪瘤、甲状腺癌、神经胶质瘤、宫颈癌、或其组合。
在另一优选例中，所述肿瘤选自下组：肺癌、黑色素瘤、肾癌、结肠癌、神经胶质瘤、宫颈癌。
在另一优选例中，所述诊断试剂是单克隆和多克隆抗体或其组合。
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、活检穿刺组织样本、肿瘤组织裂解液、血液样本、细胞样本、体液样本、尿液样本、或其组合。
在另一优选例中，所述诊断试剂盒用于检测组织样本制成的切片。
在另一优选例中，所述切片包括石蜡切片、或冷冻切片。
在另一优选例中，所述的血液样本的检测是检测外周血中循环肿瘤细胞中PDE3A的表达量。
在另一优选例中，所述诊断试剂盒包括免疫印迹分析试剂盒和/或PCR检测试剂盒。
在本发明的第二方面，提供了一种用于检测阿那格雷治疗肿瘤的效果的诊断试剂盒，所述的试剂盒含有：
(a)第一容器，所述第一容器中含有用于检测PDE3A表达和/或PDE3A蛋白的检测试剂；
(b)标签或说明书；和
(c)任选的标准品或对照品，
其中，所述标签或说明书注明所述试剂盒用于(a)判断阿那格雷治疗肿瘤的效果，和/或(b)判断肿瘤患者是否适合用阿那格雷进行治疗。
在另一优选例中，所述的检测试剂包括：引物、探针、抗体、核酸芯片、 蛋白芯片或其组合。
在另一优选例中，所述的第一容器包括一个或多个第一容器。
在另一优选例中，所述的PDE3A蛋白或其特异性抗体偶联有或带有可检测的标记物。
在另一优选例中，所述标准品或对照品包括PDE3A核酸序列、PDE3A蛋白、或其组合。
在另一优选例中，所述诊断试剂盒还含有第二容器，所述第二容器中含有对肿瘤组织进行处理的试剂。
在另一优选例中，所述诊断试剂盒还含有用于进行免疫印迹检测的试剂。
在另一优选例中，所述的标签或说明书中注明以下内容：
(i)肿瘤组织中PDE3A表达为阳性，预测提示阿那格雷治疗肿瘤的效果好，和/或所述肿瘤患者适合用阿那格雷进行治疗；和
(ii)肿瘤组织中PDE3A表达为阴性，预测提示阿那格雷治疗肿瘤的效果差，和/或所述肿瘤患者不适合用阿那格雷进行治疗。
在另一优选例中，所述的PDE3A表达为阳性是指肿瘤细胞表达PDE3A和/或具有PDE3A蛋白活性。
在另一优选例中，所述的PDE3A表达为阴性是指肿瘤细胞低表达或不表达PDE3A和/或不具有PDE3A蛋白活性
在另一优选例中，提供了一种用于检测PDE3A基因序列或蛋白的检测试剂的用途，用于制备一试剂盒，所述试剂盒用于(a)判断阿那格雷治疗肿瘤的效果，和/或(b)判断肿瘤患者是否适合用阿那格雷进行治疗的标志物。
在本发明的第三方面，提供了一种体外判断肿瘤细胞对阿那格雷敏感性的方法，包括步骤：
(i)提供一待测肿瘤细胞；
(ii)在体外检测待测肿瘤细胞中PDE3A的表达和/或活性，其中，如果所述待测肿瘤细胞表达PDE3A和/或具有PDE3A蛋白活性，则表示所述待测肿瘤细胞对阿那格雷敏感；如果所述待测肿瘤细胞低表达或不表达PDE3A和/或不具有PDE3A蛋白活性，则表示所述待测肿瘤细胞对阿那格雷不敏感。
在另一优选例中，所述的低表达或不表达PDE3A指所述肿瘤细胞中PDE3A的mRNA水平M1与β－肌动蛋白的mRNA水平M2之比R1≤0.2，较佳地≤0.1。
在另一优选例中，所述的低表达或不表达PDE3A指所述肿瘤细胞中PDE3A蛋白水平P1与Bel7404中PDE3A蛋白水平P2之比R2≤0.5，较佳地≤0.2。
在另一优选例中，“不具有PDE3A蛋白活性”指所述肿瘤细胞中PDE3A的活性A1与Bel7404中PDE3A蛋白活性A2之比R3≤0.5，较佳地≤0.2。
在另一优选例中，所述的表达PDE3A指所述肿瘤细胞中PDE3A的mRNA水平M1与β－肌动蛋白的mRNA水平M2之比R1≥0.3，较佳地≥0.5。
在另一优选例中，所述的表达PDE3A指所述肿瘤细胞中PDE3A蛋白水平P1与Bel7404中PDE3A蛋白水平P2之比R2≥0.6，较佳地≥0.8。
在另一优选例中，“具有PDE3A蛋白活性”指所述肿瘤细胞中PDE3A的活性A1与Bel7404中PDE3A蛋白活性A2之比R3≥0.6，较佳地≥0.8。
在另一优选例中，所述的方法还包括：检测待测肿瘤细胞的PDE3A是否有突变。
在另一优选例中，所述的方法还包括：
(iii)在阿那格雷存在下，在体外培养上一步骤中确定为对阿那格雷敏感的待测肿瘤细胞，并观察所述肿瘤细胞的生长情况，从而验证所述肿瘤细胞对阿那格雷的敏感性。
在另一优选例中，所述步骤(iii)中，所述的阿那格雷的浓度为0.001-0.25μM，较佳地为0.01-0.1μM，更佳地为0.02-0.08μM。
在另一优选例中，所述步骤(iii)中，所述的阿那格雷的浓度为0.25-10μM，较佳地为0.5-5μM，更佳地为0.8-3μM。
在另一优选例中，在步骤(iii)中，观察所述肿瘤细胞的生长情况，包括观察肿瘤细胞的凋亡情况和/或迁移情况。
在另一优选例中，所述方法是非诊断性和非治疗性的。
在本发明的第四方面，提供了一种治疗肿瘤的方法，包括步骤：给需要的对象施用阿那格雷，其中所述的对象为肿瘤患者，且肿瘤细胞表达PDE3A和/或具有PDE3A蛋白活性。
在另一优选例中，所述的方法还包括：在施用阿那格雷之前，对所述对象进行PDE3A表达或活性的检测。
在另一优选例中，所述的阿那格雷包括式II所示结构的化合物，或其药学上可接受的盐或前药或衍生物，或其制剂形式。
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。
附图说明
图1显示了PDE3A蛋白在不同来源细胞株中的表达情况。其中，以α-Tubulin、GAPDH为内参蛋白。S:阿那格雷敏感细胞；M：阿那格雷中度敏感细胞；未标明的均为阿那格雷不敏感细胞R。
图2显示阿那格雷选择性抑制不同组织来源肿瘤细胞生长。其中，用阿那格雷(1μM)处理72小时，MTT法检测细胞存活率的结果，以检测结果对细胞进行了分类：敏感(存活率<40％)，中度敏感(存活率40-75％)，抵抗(存活率>75％)，图中细胞名称前用圆形、三角分别标明了敏感组和中度敏感组。
图3显示了PDE3A mRNA在不同细胞中的表达。其中，利用RT-PCR对九株细胞的PDE3A mRNA水平进行检测；纵坐标:相对β-actin的定量结果。
图4显示了PDE3A蛋白在肿瘤细胞生长中起到的作用。
图4A显示了pde3a siRNA1/2转染48小时后，免疫印迹法检测PDE3A蛋白在HeLa细胞中的表达情况。
图4B显示了RTCA法监测HeLa细胞在转染pde3a siRNA前后的生长状况。其中，NC:阴性对照；KD1和KD2:PDE3A敲减组。
图5显示了PDE3A蛋白的表达与阿那格雷对细胞毒性的关系。
图5A显示了siRNA转染HeLa 48小时，RTCA法记录不同阿那格雷处理前后的细胞生长曲线。其中，NC：对照组，KD：PDE3A敲减组。
图5B显示了阿那格雷或者DMSO处理转染前后的HeLa细胞36小时，PI染色20分钟，荧光显微镜拍照结果。
图5C显示了利用Cell Profiler软件对PI阳性细胞进行计数的结果。
图5D显示了阿那格雷处理细胞48小时，MTT法检测细胞生存率的结果。
图6显示了对阿那格雷不敏感细胞株的其他PDE的mRNA表达情况，以中度敏感细胞Bel7404的PDE3A表达为基准，得到相对表达量。
具体实施方式
本发明人经过广泛而深入地研究，首次发现一种能够指导阿那格雷(Anagrelide)治疗肿瘤效果的生物标记物(PDE3A)。实验表明，如果肿瘤组织及外周血循环肿瘤细胞阳性表达PDE3A，则提示阿那格雷治疗肿瘤的效果好，和/或肿瘤患者适合用阿那格雷进行治疗。在此基础上完成了本发明。
具体地，本发明根据PDE3A在肿瘤细胞中的表达建立了一种安全有效的诊断和分型方法、能简单、快速、经济的指导阿那格雷在临床上对肿瘤患者进行个体化治疗。目前市场上有PDE3A的ELISA检测试剂盒，但还没有运用PDE3A作为肿瘤标记物对肿瘤进行诊断和治疗的方法。本发明的免疫印迹法和PCR法等方法检测PDE3A在肿瘤样本中的表达并运用于指导阿那格雷在治疗肿瘤中的应用在技术上和应用上皆属于首创。
本发明首次发现，肿瘤细胞株中约30％为PDE3A蛋白或mRNA的表达阳性，PDE3A阳性的肿瘤细胞中约对阿那格雷(Anagrelide)表现出不同程度的敏感性，而阿那格雷对PDE3A阴性的肿瘤细胞无任何抑制作用。动物实验表明阿那格雷(Anagrelide)在抑制肿瘤细胞生长的剂量(10mg-200mg/kg,较佳地10-20mg/kg)时具有安全性，因此PDE3A作为该药的生物标记物指导阿那格雷进行肿瘤治疗具有理论依据和应用价值。
PDE3A蛋白和基因序列
如本文所用，术语“PDE3A”即磷酸二酯酶3A，是丝苏氨酸激酶家族的一员，通过水解生物体内的环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)，终止这些第二信使所传递的生物信号，调控生命活动。PDE3A主要分布在心脏、血小板、血管平滑肌和卵细胞；对cAMP水解效率是cGMP的10倍。
在本发明中，术语“本发明蛋白”、“PDE3A蛋白”、“PDE3A多肽”可互换使用，都指具有人PDE3A蛋白的氨基酸序列(UniProtKB/Swiss-Prot:Q14432)的蛋白或多肽。包括含有或不含起始甲硫氨酸的PDE3A蛋白。此外，该术语还包括全长的PDE3A及其片段，尤其是分泌性片段(或分泌性蛋白)。本发明所指的PDE3A蛋白包括其完整的氨基酸序列、其分泌蛋白、其突变体以及其功能上 活性的片段。
此外，本发明的PDE3A蛋白包括糖基化和非糖基化的蛋白。
在本发明中，术语“PDE3A基因(ID：5139)”、“PDE3A多核苷酸”可互换使用，都指具有人PDE3A核苷酸序列(NC_000012.12)的核酸序列。需理解的是，当编码相同的氨基酸时，密码子中核苷酸的取代是可接受的。另外需理解的是，由核苷酸取代而产生保守的氨基酸取代时，核苷酸的变换也是可被接受的。
在得到了PDE3A的氨基酸片段的情况下，可根据其构建出编码它的核酸序列，并且根据核苷酸序列来设计特异性探针。核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的PDE3A核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(如载体)和细胞中。
通过常规的重组DNA技术，可利用本发明的多核苷酸序列可用来表达或生产重组的PDE3A多肽。
特异性抗体
在本发明中，术语“本发明抗体”和“抗PDE3A的特异性抗体”可互换使用。
本发明还包括对人PDE3A多肽具有特异性的多克隆抗体和单克隆抗体，尤其是单克隆抗体。这里，“特异性”是指抗体能结合于人PDE3A基因产物或片段。较佳地，指那些能与人PDE3A基因产物或片段结合但不识别和结合于其它非相关抗原分子的抗体。本发明中抗体包括那些能够结合并抑制人PDE3A蛋白的分子，也包括那些并不影响人PDE3A蛋白功能的抗体。本发明还包括那些能与修 饰或未经修饰形式的人PDE3A基因产物结合的抗体。
本发明不仅包括完整的单克隆或多克隆抗体，而且还包括具有免疫活性的抗体片段，如Fab’或(Fab)2片段；抗体重链；抗体轻链；遗传工程改造的单链Fv分子(Ladner等人，美国专利No.4,946,778)；或嵌合抗体，如具有鼠抗体结合特异性但仍保留来自人的抗体部分的抗体。
本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。例如，纯化的人PDE3A基因产物或者其具有抗原性的片段，可被施用于动物以诱导多克隆抗体的产生。与之相似的，表达人PDE3A蛋白或其具有抗原性的片段的细胞可用来免疫动物来生产抗体。本发明的抗体也可以是单克隆抗体。此类单克隆抗体可以利用杂交瘤技术来制备。本发明的抗体包括能阻断人PDE3A蛋白功能的抗体以及不影响人PDE3A蛋白功能的抗体。本发明的各类抗体可以利用人PDE3A基因产物的片段或功能区，通过常规免疫技术获得。这些片段或功能区可以利用重组方法制备或利用多肽合成仪合成。与人PDE3A基因产物的未修饰形式结合的抗体可以用原核细胞(例如E.Coli)中生产的基因产物来免疫动物而产生；与翻译后修饰形式结合的抗体(如糖基化或磷酸化的蛋白或多肽)，可以用真核细胞(例如酵母或昆虫细胞)中产生的基因产物来免疫动物而获得。
抗人PDE3A蛋白的抗体可用于免疫组织化学技术中，检测标本(尤其是肿瘤组织)中的人PDE3A蛋白。
阿那格雷及其用途
如本文所用，术语“阿那格雷”、“Anagrelide”、“安归宁”、“ANA”可互换使用，均指具有如下式所示结构的化合物，或其药学上可接受的盐或前药或衍生物，或其制剂形式：
Figure PCTCN2017081908-appb-000001
阿那格雷是一种磷酸二酯酶抑制剂，用于治疗抗血小板增多症。有研究表明阿那格雷或其药学上可接受的制剂在抑制肿瘤方面具有新用途。实验表明，阿那格雷体外抑制血小板的IC50为0.27-1μM，而阿那格雷在体外对敏感性肿瘤细胞的IC50均小于0.03μM。
在本发明的优选例中，所述的阿那格雷为6,7-二氯-1,5-二氢咪唑并 [2,1-b]喹唑啉-2(3H)酮。
目前，在临床上阿那格雷作为磷酸二酯酶抑制剂，用于治疗抗血小板增多症。有研究表明阿那格雷或其药学上可接受的制剂在抑制肿瘤方面具有新用途，具体地包括
(a)作为抗肿瘤药物，用于肿瘤或癌症的治疗或抑制；
(b)在体外或体内选择性地用于抑制肿瘤细胞增殖，或者诱导细胞凋亡；
(c)在体外或体内选择性地调控肿瘤细胞的周期，诱导细胞产生G1周期阻滞和G2周期阻滞。
(d)在体外或体内选择性地用于抑制肿瘤细胞迁移。
所述的阿那格雷或其药物组合物可以在较低浓度下作用，较佳地，所述阿那格雷可以在≤1μM的浓度下作用于对象细胞，并产生所需的作用。
优选地，阿那格雷的衍生物具有如下式I所示结构：
Figure PCTCN2017081908-appb-000002
其中，
R1～R8各自独立地选自下组：氢原子、卤素原子、氨基、羟基、氰基、醛基、硝基、羧基(-COOH)、取代或未取代的C1～C10烷基、取代或未取代的C3～C10环烷基、取代或未取代的C2～C10烯基、取代或未取代的C2～C10炔基、取代或未取代的C6～C10芳基、取代或未取代的C1～C10杂芳基(如取代或未取代的5元或6元杂环、8元至10元杂芳二环环系)、取代或未取代的C1～C10烷氧基、取代或未取代的C6～C10芳基-氧基、取代或未取代的C1～C10杂芳基-氧基、取代或未取代的酰基(优选为-CO-C1～C10烷基)、取代或未取代的酯基(优选为C1～C10烷基-COO-)、取代或未取代的C1～C10磺酰基(-SO2-C1～C10烷基)；
或R1和R2、R3和R4共同构成选自下组的基团：取代或未取代的C3～C20环烷基(优选为C3～C10环烷基)、取代或未取代的C1～C20杂环烷基(优选为取代或未取代的5元或6元杂环、8元至12元杂芳二环环系)、羰基(＝O)；
R9选自下组：氢原子、氧原子、取代或未取代的C1～C10烷基、取代或未取代的C3～C10环烷基、取代或未取代的C6～C10芳基、取代或未取代的C1～C10杂芳基、取代或未取代的C1～C10烷氧基、取代或未取代的C6～C10芳基-氧基、取代或未取代的酰基(优选为-CO-C1～C10烷基)、取代或未取代的C1～C10磺酰基；
其中，取代指基团上的一个或多个氢原子被选自下组的取代基取代：C1～C10烷基、C3～C10环烷基、C1～C10烷氧基、卤素、羟基、羧基(-COOH)、C1～C10醛基、C2～C10酰基、C2～C10酯基、氨基、苯基；
所述的苯基包括未取代的苯基或具有1-3个取代基的取代苯基，所述取代基选自：卤素、C1-C10烷基、氰基、OH、硝基、C3～C10环烷基、C1～C10烷氧基、氨基。
在另一优选例中，所述R1～R8各自独立地选自下组：氢原子、卤素原子、氨基、羟基、氰基、硝基、氨基、醛基、羧基、取代或未取代的C1～C5烷基、取代或未取代的C3～C6环烷基、取代或未取代的C2～C5烯基、取代或未取代的C2～C5炔基、取代或未取代的C6～C10芳基、取代或未取代的C1～C6杂芳基、取代或未取代的C1～C5烷氧基、取代或未取代的C6～C10芳基-氧基、取代或未取代的C1～C6杂芳基-氧基、取代或未取代的-CO-C1～C5烷基、取代或未取代的C1～C5烷基-COO-、取代或未取代的C1～C5磺酰基；或R1和R2、R3和R4共同构成选自下组的基团：取代或未取代的C3～C10环烷基、取代或未取代的C1～C10杂环烷基、羰基；
R9选自下组：氢原子、氧原子、取代或未取代的C1～C5烷基、取代或未取代的C3～C6环烷基、取代或未取代的C6～C10芳基、取代或未取代的C1～C10杂芳基、取代或未取代的C1～C5烷氧基、取代或未取代的C6～C10芳基-氧基、取代或未取代的-CO-C1～C5烷基、取代或未取代的C1～C5磺酰基；
其中，取代的定义如上所述。
在另一优选例中，R1～R8各自独立地选自下组：氢原子、卤素原子、氰基、取代或未取代的C1～C5烷基、取代或未取代的C3～C6环烷基；
或R1和R2、R3和R4共同构成选自下组的基团：取代或未取代的C1～C5环烷基、取代或未取代的C1～C5杂环烷基、羰基；
R9选自下组：氢原子、取代或未取代的C1～C5烷基、取代或未取代的C3～C6环烷基；
其中，取代的定义如上所述。
在另一优选例中，所述的R1～R9中的1～8个为氢原子，较佳地2～7个为氢原子。
在另一优选例中，所述的R1～R8中的1～8个为卤素原子。
在另一优选例中，所述的R1～R8中的1～8个为卤素原子，且其他的R1～R8均为氢原子。
在另一优选例中，所述的式I化合物具有如式II所示的结构：
Figure PCTCN2017081908-appb-000003
在另一优选例中，所述的药学上可接受的盐为选自下组的盐：盐酸盐、醋酸盐、磷酸盐，或其组合。
检测方案
方案(1)
通过活检或手术取少量肿瘤患者的肿瘤组织，即可进行免疫印迹法(western blot)检测。如果该患者的PDE3A的蛋白表达为阳性(PDE3A+)，即可考虑选用阿那格雷(Anagrelide)进行治疗，有效率接近70％；若是PDE3A阴性(PDE3A-)，就不选用该药，排除准确率100％。
方案(2)
通过活检或手术取少量肿瘤患者的肿瘤组织，提取mRNA，进行RT-PCR反应，即可检测PDE3A的mRNA的表达。PDE3A的mRNA表达为阳性(PDE3A+)，即可考虑选用阿那格雷(Anagrelide)进行治疗，有效率接近70％；若是PDE3A的mRNA为阴性(PDE3A-)，就不选用该药，排除准确率100％。对肝癌、神经胶质瘤、宫颈癌患者，可以优先考虑使用该方法对这PDE3A蛋白进行检测。
方案(3)
在本发明中，还可检测外周血中循环肿瘤细胞中PDE3A的表达量。
检测试剂盒
本发明还提供了一种阿那格雷治疗肿瘤效果的检测试剂盒，所述的试剂盒含有一容器a，所述容器a中含有PDE3A基因序列、蛋白或其特异性抗体；以及标签或说明书，所述标签或说明书注明所述试剂盒用于(a)判断阿那格雷治疗肿瘤的效果，和/或(b)判断肿瘤患者是否适合用阿那格雷进行治疗。较佳地，含有抗PDE3A的免疫球蛋白或免疫偶联物，或其活性片段。
本发明的主要优点包括：
(a)提供了预测阿那格雷治疗肿瘤效果的标志物，有助于患者治疗方案的选择。
(b)提前分析患者对阿那格雷的敏感性，避免无效治疗。
(c)检测方便、快捷、经济，可以降低患者医治成本。
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。
通用材料
1.药品和试剂
实施例中涉及的药品和试剂如下表所示：
Figure PCTCN2017081908-appb-000004
Figure PCTCN2017081908-appb-000005
2.细胞与细胞培养基
实施例中涉及的细胞与细胞培养基如下表所示：
Figure PCTCN2017081908-appb-000006
Figure PCTCN2017081908-appb-000007
以上细胞株皆置于37℃含5％CO2的细胞培养箱培养，待细胞处于对数生长期时进行实验。
通用方法
1.组织蛋白样品的准备
手术切除的组织块迅速置于预冷的0.95生理盐水中，漂洗数次，以清洁表面的血迹，将组织称量后切成几个较小的组织块放入机戒组织匀浆器中，按组织净重，裂解液＝1：10的比例，加入相应体积的裂解液进行匀浆，离心收集上清(如有粘稠物可超声处理，也可以冷冻干燥降解核酸后，将冻干的蛋白质样品溶解在适当的上样buffer中，混匀后静置3小时使样品中的蛋白质充分的溶解，有5分钟即可，4度离心收集)加入Laemmli样品缓冲液(视蛋白样品浓度，以1：1或1：2的比例混合)强力混匀，样品置100度的水浴箱水浴加热3-5分钟， 10000g离心10分钟，取上清液，将其转入另一洁净的试管中，至此，电泳样品已准备就绪(样品即可立即使用也可也可以分装冻存，-20℃存放的样品可稳定保持数月。)
2.组织RNA的提取
组织(<100mg)加TRIzol1ml至1.5ml无RNAase的EP管中，置于冰上5分钟，加200μL氯仿，剧烈振摇15s后置于冰上3分钟；低温高速离心机12000g，4℃离心15分钟，将上层移至新的EP管中；向EP管中加500μL异丙醇，混匀后置于冰上10分钟；低温高速离心机12000g，4℃，离心10分钟，见白色固体沉于EP管底部，弃上清。向EP管中加入1ml 75％乙醇(DEPC水配置)，振荡混匀；低温高速离心机7500g，4℃离心5分钟，弃上清，尽量吸干乙醇；将EP管中的RNA置于空气中，干燥至透明，用DEPC水30-50μL溶解RNA，-80℃保存。
3.药物对细胞的半数中毒剂量的测定(MTT法)
用细胞维持液将药液连续做几何级稀释为1∶10，1∶100，1∶1,000，1∶10,000，设正常细胞对照组，将不同浓度的各药分别加入已长成单层96孔板中，每个稀释度接种3个复孔，每孔加入100μl，置于37℃二氧化碳培养箱中培养72h，加入5mg/ml MTT 25μL，
继续培养3.5h，取出弃上清，每孔加溶解液DMSO 150μl，振荡5～10min，待结晶完全溶解，酶标仪测570nm的OD值。计算各药物稀释度的细胞存活率：细胞存活率％＝(实验孔OD测定值均值/对照孔OD测定值均值)X100％，采用概率单位回归法，计算50％细胞中毒时的药物浓度(IC50)。
4.免疫印迹法(western blot)分析
8％-10％SDS-聚丙烯酰胺(SDA-PAGE)分离胶，5％浓缩胶。浓缩胶电压为80V，分离胶电压为120V，溴酚蓝指示剂跑至底部停止；采用半干法转膜，转膜电压为10-15V，转膜时间根据蛋白分子的大小进行调整；转膜结束后用丽春红预染硝酸纤维膜，剪膜；用含有5％脱脂牛奶的TBST置于脱色摇床封闭1小时后，TBST冲洗一遍，加入一抗结合，一抗1:2000-1:5000稀释于含有5％BSA的TBST中，室温置于脱色摇床结合1小时后，4℃过夜。次日在室温下平衡1小时后，TBST洗三次，每次10分钟。加入二抗结合，二抗1:3000-1:5000稀释于含有5％BSA的TBST 中，室温置于脱色摇床结合1小时，TBST洗三次，每次10分钟。与ECL试剂盒底物作用2-3分钟后，曝光于化学发光仪。
5.RT-PCR反应
1)Trizol提取细胞总RNA
单层细胞加TRIzol 1ml/10cm直径小皿；TRIzol均匀覆盖于小皿，置于冰上5分钟，收集细胞并转移至1.5ml RNAase free EP管中；向EP管中加200μL氯仿，剧烈振摇15s后置于冰上3分钟；低温高速离心机12000g，4℃离心15分钟，将上层移至新的EP管中；向EP管中加500μL异丙醇，混匀后置于冰上10分钟；低温高速离心机12000g，4℃，离心10分钟，见白色固体沉于EP管底部，弃上清。向EP管中加入1ml 75％乙醇(DEPC水配置)，振荡混匀；低温高速离心机7500g，4℃离心5分钟，弃上清，尽量吸干乙醇；将EP管中的RNA置于空气中，干燥至透明，用DEPC水30-50μL溶解RNA，-80℃保存。
2)反转录反应(20μL反应体系，冰上操作)，反应体系如下：
Figure PCTCN2017081908-appb-000008
3)实时定量PCR(Real-time PCR)StepOnePlusTM Real-Time PCR System
反应体系如下：
试剂 使用量
SYBRPremix Ex TaqTM(2×) 10.0μL
PCR Forward Primer(10μM) 0.4μL
PCR Reverse Primer(10μM) 0.4μL
ROX Reference Dye(50×) 0.4μL
DNA模版 2.0μL
dH2O(灭菌蒸馏水) 6.8μL
Total 20.0μL
                          ↓
Stage 1:95℃，30s(Replicate:1)
Stage 2:95℃，5s；60℃，30s(Replicate:40)
Stage 3:95℃，15s；60℃，1min；95℃，15s(Replicate:1)
Real-time PCR引物序列如下：
Figure PCTCN2017081908-appb-000009
根据StepOnePlusTM Real-Time PCR System(Applied biosystem)系统自带分析软件，以β-Actin为参比，根据2△△Ct法求得PDE3A相对表达量。
6.RNAi干扰
LipofectamineTM2000的用量及实验操作参照其说明书。
对数期生长的HeLa细胞约6000/孔接种于96孔板或者RTCA板，过夜贴壁长满约70-80％，细胞状态良好；取一定量的无血清无抗生素的培养基分别加入两个EP管中，向一管加入适量siRNA混匀，另一管加入适量LipofectamineTM2000混匀，室温放置5-10min；将加有LipofectamineTM试剂的培养基加入含siRNA的EP管中混匀，室温放置15min；放置期间，用无血清无双抗的培养基洗细胞一次，再加入75μL该培养基备用。15分钟到，将混匀后的培养基加入细胞培养皿，25μL/孔，轻轻振荡混匀；37℃，5％CO2培养6小时后，换成含10％FBS的新鲜培养基；24-72h后免疫印迹法(western blot)检测RNAi干扰蛋白表达情况。
7.细胞实时监控仪(RTCA)监测细胞生长
将处于对数生长期的细胞以每孔3-7×104/mL接种于细胞实时监控仪配套的16或者96孔板中，37℃孵育过夜后，加药或者转染处理。实时监控仪自动监测细胞生长4天以上，根据细胞铺展后形成电阻的大小反应细胞数量。Cel lIndex值越大，细胞生长的越快，细胞数越多。
8.PI检测凋亡细胞
在96孔板中，不同化合物对细胞进行处理24-36小时，2μL的PI染液(25ug/mL)加到96孔板中，孵育细胞20min,荧光显微镜下拍照，观察红色荧光的细胞数目。
实施例1
肿瘤细胞株中PDE3A蛋白表达情况的检测
为了研究PDE3A蛋白在肿瘤细胞中的表达情况，用免疫印迹法对通用材料中列举的细胞株进行PDE3A蛋白表达水平的检测。
结果如图1所示，PDE3A蛋白在SMMC7721、FHCC98、H4、HeLa、Bel7404、A498、SW1116等细胞株中为阳性表达，在其他20种细胞株中为阴性表达。
进一步地，利用RT-PCR对部分细胞株的PDE3A mRNA水平进行检测；RQ:相对β－actin的定量结果。
结果如图3所示，SMMC7721、FHCC98、H4、HeLa、Bel7404、A498和SW1116中均有PDE3A mRNA表达，H522和SKOV3中几乎无PDE3A mRNA表达，与免疫印迹的检测结果一致。
实施例2
药物的抗肿瘤活性实验
利用浓度为1μM的阿那格雷(Anagrelide)对实施例1中的细胞株进行活性实验，根据细胞存活率，将细胞株分成三类：敏感(S)、中度敏感(M)、不敏感(R)，并进一步测定阿那格雷抑制各细胞株的IC50值。
结果如图2所示，测试细胞株中共有7株敏感细胞和20株不敏感细胞(图2)。表1显示了阿那格雷抑制敏感细胞株的IC50值，其中，阳性表达PDE3A的细胞株SMMC7721，FHCC98，H4，HeLa为阿那格雷的敏感细胞，阿那格雷对其有极好的 抑制效果，其IC50值均小于30nM；阳性表达PDE3A的细胞株Bel7404，A498和SW1116为阿那格雷的中度敏感细胞，阿那格雷对其有持久的抑制效果，其IC50值为0.4μΜ-16μM。20株阴性表达PDE3A的细胞株对阿那格雷均不敏感，表现出对阿那格雷的耐受，IC50值均＞50μM，即阿那格雷不能抑制其生长。
表1 7株敏感细胞的IC50值
细胞株 Hela H4 FHCC98 SMMC7721 Bel7404 A498 SW1116
IC50(μM) 0.022 0.023 0.008 0.015 0.428 15.900 14.510
综上，PDE3A阳性细胞中，在阿那格雷处理后出现不同程度的生长抑制。此外，所有PDE3A阴性细胞均对阿那格雷不敏感。
实施例3
PDE3A蛋白敲除对敏感细胞生长的影响
实施例1和实施例2从化合物抑制蛋白活性的角度来说明PDE3A蛋白与生长抑制的关系，但是化合物由于结构不同、与靶点作用方式不一样，会造成细胞选择性的差异。于是，在获得PDE3A蛋白细胞表达谱数据之后，利用siRNA干扰技术，考察敲除PDE3A蛋白对细胞生长影响。用两个pde3a siRNA(效率不同)分别进行试验，在siRNA转染细胞48小时后，将细胞铺在RTCA板上，进行长时间的生长监控。
结果如图4所示：PDE3A蛋白的表达量(图4A)与细胞生长的快慢(图4B)成正相关，表明PDE3A蛋白在阿那格雷敏感的肿瘤细胞生长中起到重要的作用。
实施例4
PDE3A蛋白参与调控阿那格雷引起的凋亡
为研究PDE3A蛋白本身是否参与调控阿那格雷引起的凋亡，先用siRNA干扰技术，将pde3a siRNA转染到敏感细胞HeLa进行敲除，再用阿那格雷处理细胞，考察PDE3A蛋白与细胞对阿那格雷敏感性之间的关系。
其中，KD组为转染了pde3a siRNA的HeLa细胞，1和2分别代表两个siRNA
NC组为转染了无敲除效应的siRNA的HeLa细胞，空白对照。
结果表明，未加入阿那格雷情况下，PDE3A的蛋白水平与HeLa细胞的生长成正相关，而阿那格雷加入之后，PDE3A蛋白水平低的KD2存活率最高，而NC组的最低(图5A)。PDE3A敲除48小时后，阿那格雷处理HeLa细胞36小时，KD组 的细胞也比对照组有较少的PI阳性细胞数(图5B和图5C)；同步进行MTT的检测，也说明HeLa细胞的PDE3A蛋白水平越低，越有利于对阿那格雷产生抵抗(图5D)。上述结果表明，PDE3A蛋白确实参与了阿那格雷对细胞凋亡的调控，PDE3A蛋白表达水平可以作为阿那格雷治疗治疗效果的标志物。
实施例5
判断阿那格雷治疗肿瘤效果的诊断试剂盒的制备
制备检测阿那格雷治疗肿瘤效果的试剂盒，所述试剂盒包括:
(a)容器，以及位于容器内的特异性针对PDE3A的抗体：兔抗PDE3A抗体；和
(b)以及标签或说明书，所述标签或说明书注明所述试剂盒用于检测阿那格雷治疗肿瘤的效果。
实施例6
免疫印迹法检测细胞株SW480和MCF-7中，PED家族的其他成员，包括PDE1A、PDE3A、PDE3B、PDE4A、PDE7A、PDE11A的蛋白表达水平。
结果如图6所示，SW480和MCF-7中的PDE3B表达水平相对比较高，而PDE3A以及检测的PED家族的其他成员的表达水平均比较低。
讨论
阿那格雷作为一线治疗血小板增多症的药物已有二十多年的历史，而在实体肿瘤的治疗史上始终是空白；先前实验室的研究第一次证明阿那格雷可以作为一个选择性极佳的抗肿瘤药物用于临床。深入其机制研究后，发现PDE3A蛋白可以作为第一生物标记物辅助阿那格雷在临床肿瘤患者的诊断和治疗，这是该化合物研究至今，第一次有明确的靶标辅助个体化肿瘤治疗。这一发现在一系列分子、细胞水平的试验中得以证实——利用siRNA干扰技术敲除敏感细胞中的PDE3A蛋白，同时用同类抑制剂做竞争性结合实验，最终证明阿那格雷与靶蛋白PDE3A结合是该药物诱导肿瘤细胞凋亡必需的。
本发明发现大约三分之一的肿瘤患者是PDE3A高表达的，PDE3A阳性表达的肿瘤细胞均对阿那格雷表现出不同程度的敏感性，而PDE3A阴性的肿瘤对阿那格雷不敏感。总结如下：
研究发现阿那格雷对多种肿瘤细胞(肝癌、黑色素瘤、肾癌、结肠癌、神经胶质瘤、宫颈癌)的生长显示出较强的抑制作用，而至今仍未揭示其作用机制。经过本发明的研究发现阿那格雷造成细胞周期阻滞、细胞凋亡的原因，并找到了生物标记物PDE3A蛋白指导临床诊断、分型。进行肿瘤患者的个体化靶向治疗。
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。

Claims (10)

  1. 一种PDE3A基因序列、PDE3A核酸检测试剂、PDE3A蛋白、和/或PDE3A蛋白检测试剂的用途，其特征在于，用于制备诊断试剂或诊断试剂盒，所述诊断试剂或试剂盒用于：
    (a)判断阿那格雷(Anagrelide)治疗肿瘤的效果，
    (b)判断肿瘤患者是否适合用阿那格雷进行治疗，和/或
    (c)判断肿瘤细胞对阿那格雷的敏感性。
  2. 如权利要求1所述的用途，其特征在于，所述的判断包括辅助判断和/或预先判断。
  3. 如权利要求1所述的用途，其特征在于，所述的敏感性是指所述肿瘤细胞在以下浓度的阿那格雷存在下的敏感性：0.001-0.25μM，较佳地为0.01-0.1μM，更佳地为0.02-0.08μM。
  4. 如权利要求1所述的用途，其特征在于，所述的PDE3A核酸检测试剂、或PDE3A蛋白检测试剂偶联有或带有可检测的标记物。
  5. 如权利要求4所述的用途，其特征在于，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。
  6. 如权利要求1所述的用途，其特征在于，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、活检穿刺组织样本、肿瘤组织裂解液、血液样本、细胞样本、体液样本、尿液样本、或其组合。
  7. 一种用于检测阿那格雷治疗肿瘤的效果的诊断试剂盒，其特征在于，所述的试剂盒含有：
    (a)第一容器，所述第一容器中含有用于检测PDE3A表达和/或PDE3A蛋白的检测试剂；
    (b)标签或说明书；和
    (c)任选的标准品或对照品，
    其中，所述标签或说明书注明所述试剂盒用于(a)判断阿那格雷治疗肿瘤的效果，和/或(b)判断肿瘤患者是否适合用阿那格雷进行治疗。
  8. 如权利要求7所述的试剂盒，其特征在于，所述的标签或说明书中注明以下内容：
    (i)肿瘤组织中PDE3A表达为阳性，预测提示阿那格雷治疗肿瘤的效果好， 和/或所述肿瘤患者适合用阿那格雷进行治疗；和
    (ii)肿瘤组织中PDE3A表达为阴性，预测提示阿那格雷治疗肿瘤的效果差，和/或所述肿瘤患者不适合用阿那格雷进行治疗。
  9. 一种体外判断肿瘤细胞对阿那格雷敏感性的方法，其特征在于，包括步骤：
    (i)提供一待测肿瘤细胞；
    (ii)在体外检测待测肿瘤细胞中PDE3A的表达和/或活性，其中，如果所述待测肿瘤细胞表达PDE3A和/或具有PDE3A蛋白活性，则表示所述待测肿瘤细胞对阿那格雷敏感；如果所述待测肿瘤细胞低表达或不表达PDE3A和/或不具有PDE3A蛋白活性，则表示所述待测肿瘤细胞对阿那格雷不敏感。
  10. 如权利要求9所述的方法，其特征在于，所述的方法还包括：
    (iii)在阿那格雷存在下，在体外培养上一步骤中确定为对阿那格雷敏感的待测肿瘤细胞，并观察所述肿瘤细胞的生长情况，从而验证所述肿瘤细胞对阿那格雷的敏感性。
PCT/CN2017/081908 2016-04-26 2017-04-25 Pde3a在判断阿那格雷治疗肿瘤效果中的应用 WO2017186103A1 (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015055898A2 (en) * 2013-10-17 2015-04-23 Sihto Harri Compositions comprising phosphodiesterase inhibitors for use in the treatment of a solid tumor in a human patient
WO2017027854A1 (en) * 2015-08-13 2017-02-16 The Broad Institute, Inc. Compositions and methods for cancer expressing pde3a or slfn12

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104161759B (zh) * 2013-05-16 2019-10-08 中国科学院上海药物研究所 阿那格雷及其衍生物的抗肿瘤用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015055898A2 (en) * 2013-10-17 2015-04-23 Sihto Harri Compositions comprising phosphodiesterase inhibitors for use in the treatment of a solid tumor in a human patient
WO2017027854A1 (en) * 2015-08-13 2017-02-16 The Broad Institute, Inc. Compositions and methods for cancer expressing pde3a or slfn12

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WALL, L. D. ET AL.: "Identification of cancer-cytotoxic modulators of PDE3A by predictive chemogenomics", NATURE CHEMICAL BIOLOGY, vol. 12, 14 December 2015 (2015-12-14), pages 102 - 107, XP055364721 *

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