CN113391069B - Use of chkα -based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction - Google Patents
Use of chkα -based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction Download PDFInfo
- Publication number
- CN113391069B CN113391069B CN202110602766.7A CN202110602766A CN113391069B CN 113391069 B CN113391069 B CN 113391069B CN 202110602766 A CN202110602766 A CN 202110602766A CN 113391069 B CN113391069 B CN 113391069B
- Authority
- CN
- China
- Prior art keywords
- site
- chkα
- chk
- alpha
- phosphorylation
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
- 238000004393 prognosis Methods 0.000 title claims abstract description 12
- 206010028980 Neoplasm Diseases 0.000 title abstract description 51
- 238000011282 treatment Methods 0.000 title abstract description 32
- 201000011510 cancer Diseases 0.000 title abstract description 27
- 230000007102 metabolic function Effects 0.000 title abstract description 9
- 238000003745 diagnosis Methods 0.000 title abstract description 8
- 230000026731 phosphorylation Effects 0.000 claims description 78
- 238000006366 phosphorylation reaction Methods 0.000 claims description 78
- 238000006640 acetylation reaction Methods 0.000 claims description 16
- 230000021736 acetylation Effects 0.000 claims description 15
- 201000007983 brain glioma Diseases 0.000 claims description 12
- 230000002596 correlated effect Effects 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 238000010837 poor prognosis Methods 0.000 claims description 3
- 102100031065 Choline kinase alpha Human genes 0.000 abstract description 67
- 101000777314 Homo sapiens Choline kinase alpha Proteins 0.000 abstract description 61
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 abstract description 22
- 230000004614 tumor growth Effects 0.000 abstract description 15
- 230000015572 biosynthetic process Effects 0.000 abstract description 12
- 229960003180 glutathione Drugs 0.000 abstract description 11
- 230000004130 lipolysis Effects 0.000 abstract description 11
- 238000003786 synthesis reaction Methods 0.000 abstract description 11
- 108010024636 Glutathione Proteins 0.000 abstract description 9
- 238000007254 oxidation reaction Methods 0.000 abstract description 5
- 101710106334 Choline kinase alpha Proteins 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 4
- 230000034659 glycolysis Effects 0.000 abstract description 4
- 230000007246 mechanism Effects 0.000 abstract description 3
- 230000023852 carbohydrate metabolic process Effects 0.000 abstract description 2
- 230000033095 regulation of phosphatidylcholine biosynthetic process Effects 0.000 abstract description 2
- 101001059535 Homo sapiens Megakaryocyte-associated tyrosine-protein kinase Proteins 0.000 description 58
- 210000004027 cell Anatomy 0.000 description 49
- 102100034976 Cystathionine beta-synthase Human genes 0.000 description 37
- 108010073644 Cystathionine beta-synthase Proteins 0.000 description 37
- 230000035772 mutation Effects 0.000 description 30
- 150000002632 lipids Chemical class 0.000 description 29
- 238000004458 analytical method Methods 0.000 description 28
- 230000027455 binding Effects 0.000 description 26
- 102100038910 Alpha-enolase Human genes 0.000 description 23
- 238000003119 immunoblot Methods 0.000 description 21
- 102000017794 Perilipin-2 Human genes 0.000 description 18
- 108010067163 Perilipin-2 Proteins 0.000 description 18
- 101000882335 Homo sapiens Alpha-enolase Proteins 0.000 description 17
- VLMZMRDOMOGGFA-WDBKCZKBSA-N festuclavine Chemical compound C1=CC([C@H]2C[C@H](CN(C)[C@@H]2C2)C)=C3C2=CNC3=C1 VLMZMRDOMOGGFA-WDBKCZKBSA-N 0.000 description 17
- 230000004083 survival effect Effects 0.000 description 15
- 210000001519 tissue Anatomy 0.000 description 15
- 238000001114 immunoprecipitation Methods 0.000 description 14
- 230000001404 mediated effect Effects 0.000 description 13
- 210000004881 tumor cell Anatomy 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 239000003112 inhibitor Substances 0.000 description 12
- 102100034582 E3 ubiquitin/ISG15 ligase TRIM25 Human genes 0.000 description 10
- 101000848655 Homo sapiens E3 ubiquitin/ISG15 ligase TRIM25 Proteins 0.000 description 10
- 101001129187 Homo sapiens Patatin-like phospholipase domain-containing protein 2 Proteins 0.000 description 10
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 description 10
- 102100031248 Patatin-like phospholipase domain-containing protein 2 Human genes 0.000 description 10
- 102000001253 Protein Kinase Human genes 0.000 description 10
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 description 10
- 108091027967 Small hairpin RNA Proteins 0.000 description 10
- 102000004243 Tubulin Human genes 0.000 description 10
- 108090000704 Tubulin Proteins 0.000 description 10
- 238000003556 assay Methods 0.000 description 10
- 238000001514 detection method Methods 0.000 description 10
- 108060006633 protein kinase Proteins 0.000 description 10
- 239000004055 small Interfering RNA Substances 0.000 description 10
- 102000004072 Beclin-1 Human genes 0.000 description 9
- 108090000524 Beclin-1 Proteins 0.000 description 9
- 239000011324 bead Substances 0.000 description 9
- 238000006664 bond formation reaction Methods 0.000 description 9
- 230000007812 deficiency Effects 0.000 description 9
- 239000003550 marker Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 208000032612 Glial tumor Diseases 0.000 description 8
- 206010018338 Glioma Diseases 0.000 description 8
- 230000036542 oxidative stress Effects 0.000 description 8
- 101150070696 CHKA gene Proteins 0.000 description 7
- 108010068633 Perilipin-3 Proteins 0.000 description 7
- 102000001486 Perilipin-3 Human genes 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 101710165425 Alpha-enolase Proteins 0.000 description 6
- 101150015836 ENO1 gene Proteins 0.000 description 6
- 101710184673 Enolase 1 Proteins 0.000 description 6
- 230000002950 deficient Effects 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 102220275498 rs1555871913 Human genes 0.000 description 6
- 230000034512 ubiquitination Effects 0.000 description 6
- 238000010798 ubiquitination Methods 0.000 description 6
- 229920000936 Agarose Polymers 0.000 description 5
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 5
- 238000011717 athymic nude mouse Methods 0.000 description 5
- 230000007423 decrease Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000010166 immunofluorescence Methods 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 238000000021 kinase assay Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000012130 whole-cell lysate Substances 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 4
- 229960004308 acetylcysteine Drugs 0.000 description 4
- 230000008045 co-localization Effects 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 206010061309 Neoplasm progression Diseases 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 239000000090 biomarker Substances 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000005754 cellular signaling Effects 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 229960004679 doxorubicin Drugs 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000006303 immediate early viral mRNA transcription Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- -1 sterol esters Chemical class 0.000 description 3
- 230000005751 tumor progression Effects 0.000 description 3
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 2
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 102100036009 5'-AMP-activated protein kinase catalytic subunit alpha-2 Human genes 0.000 description 2
- CIVGYTYIDWRBQU-UFLZEWODSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid;pyrrole-2,5-dione Chemical compound O=C1NC(=O)C=C1.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 CIVGYTYIDWRBQU-UFLZEWODSA-N 0.000 description 2
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 2
- 108010018888 Choline kinase Proteins 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 108010053070 Glutathione Disulfide Proteins 0.000 description 2
- 102000012215 HSC70 Heat-Shock Proteins Human genes 0.000 description 2
- 108010036652 HSC70 Heat-Shock Proteins Proteins 0.000 description 2
- 101000783681 Homo sapiens 5'-AMP-activated protein kinase catalytic subunit alpha-2 Proteins 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 2
- 239000005642 Oleic acid Substances 0.000 description 2
- 102000011755 Phosphoglycerate Kinase Human genes 0.000 description 2
- 101001099217 Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) Triosephosphate isomerase Proteins 0.000 description 2
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical group C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 2
- 230000004900 autophagic degradation Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- 230000001713 cholinergic effect Effects 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 230000007946 glucose deprivation Effects 0.000 description 2
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000002991 immunohistochemical analysis Methods 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 230000004576 lipid-binding Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000002493 microarray Methods 0.000 description 2
- 238000003032 molecular docking Methods 0.000 description 2
- 238000000329 molecular dynamics simulation Methods 0.000 description 2
- 210000000607 neurosecretory system Anatomy 0.000 description 2
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- YHHSONZFOIEMCP-UHFFFAOYSA-O phosphocholine Chemical compound C[N+](C)(C)CCOP(O)(O)=O YHHSONZFOIEMCP-UHFFFAOYSA-O 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 230000000865 phosphorylative effect Effects 0.000 description 2
- 229950004354 phosphorylcholine Drugs 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 description 2
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 230000007115 recruitment Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 108010051423 streptavidin-agarose Proteins 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-N 0.000 description 1
- MPLLLQUZNJSVTK-UHFFFAOYSA-N 5-[3-[4-[2-(4-fluorophenyl)ethoxy]phenyl]propyl]furan-2-carboxylic acid Chemical compound O1C(C(=O)O)=CC=C1CCCC(C=C1)=CC=C1OCCC1=CC=C(F)C=C1 MPLLLQUZNJSVTK-UHFFFAOYSA-N 0.000 description 1
- 206010002660 Anoxia Diseases 0.000 description 1
- 241000976983 Anoxia Species 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- RZZPDXZPRHQOCG-OJAKKHQRSA-M CDP-choline(1-) Chemical compound O[C@@H]1[C@H](O)[C@@H](COP([O-])(=O)OP([O-])(=O)OCC[N+](C)(C)C)O[C@H]1N1C(=O)N=C(N)C=C1 RZZPDXZPRHQOCG-OJAKKHQRSA-M 0.000 description 1
- 206010058019 Cancer Pain Diseases 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 208000003445 Mouth Neoplasms Diseases 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 206010031096 Oropharyngeal cancer Diseases 0.000 description 1
- 206010057444 Oropharyngeal neoplasm Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 208000000821 Parathyroid Neoplasms Diseases 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 208000007913 Pituitary Neoplasms Diseases 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 102000013009 Pyruvate Kinase Human genes 0.000 description 1
- 108020005115 Pyruvate Kinase Proteins 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 102000008078 Sterol Regulatory Element Binding Protein 1 Human genes 0.000 description 1
- 108010074436 Sterol Regulatory Element Binding Protein 1 Proteins 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 208000024313 Testicular Neoplasms Diseases 0.000 description 1
- 206010057644 Testis cancer Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000008385 Urogenital Neoplasms Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 238000001772 Wald test Methods 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000035508 accumulation Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 208000024447 adrenal gland neoplasm Diseases 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000007953 anoxia Effects 0.000 description 1
- 238000011319 anticancer therapy Methods 0.000 description 1
- 238000011394 anticancer treatment Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000002886 autophagic effect Effects 0.000 description 1
- 210000004957 autophagosome Anatomy 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 201000009036 biliary tract cancer Diseases 0.000 description 1
- 208000020790 biliary tract neoplasm Diseases 0.000 description 1
- 230000008436 biogenesis Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008668 cellular reprogramming Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 229960001284 citicoline Drugs 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 206010016629 fibroma Diseases 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000012296 in situ hybridization assay Methods 0.000 description 1
- 238000010832 independent-sample T-test Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000010150 least significant difference test Methods 0.000 description 1
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 description 1
- 230000006372 lipid accumulation Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000001325 log-rank test Methods 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 208000026045 malignant tumor of parathyroid gland Diseases 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000010436 membrane biogenesis Effects 0.000 description 1
- 206010027191 meningioma Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000007814 microscopic assay Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000011201 multiple comparisons test Methods 0.000 description 1
- 238000000491 multivariate analysis Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 201000006958 oropharynx cancer Diseases 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 201000002530 pancreatic endocrine carcinoma Diseases 0.000 description 1
- FIKAKWIAUPDISJ-UHFFFAOYSA-L paraquat dichloride Chemical compound [Cl-].[Cl-].C1=C[N+](C)=CC=C1C1=CC=[N+](C)C=C1 FIKAKWIAUPDISJ-UHFFFAOYSA-L 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 208000028591 pheochromocytoma Diseases 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 1
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 1
- 108091005981 phosphorylated proteins Proteins 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 208000010916 pituitary tumor Diseases 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 238000010379 pull-down assay Methods 0.000 description 1
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 1
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 1
- 229960001327 pyridoxal phosphate Drugs 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 125000000946 retinyl group Chemical group [H]C([*])([H])/C([H])=C(C([H])([H])[H])/C([H])=C([H])/C([H])=C(C([H])([H])[H])/C([H])=C([H])/C1=C(C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])C1(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 238000000528 statistical test Methods 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 201000003120 testicular cancer Diseases 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 206010044285 tracheal cancer Diseases 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 238000007473 univariate analysis Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- 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
- G01N33/57488—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 involving compounds identifable in body fluids
-
- 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
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N2333/91205—Phosphotransferases in general
- G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
- G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Hospice & Palliative Care (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Oncology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of oncology, and relates to application of non-metabolic function based on CHK alpha as a target for cancer treatment, diagnosis and prognosis prediction. The invention provides a new action target for cancer treatment, diagnosis and prognosis prediction by analyzing the mechanism of occurrence and development of choline kinase alpha (CHK alpha) in human cancers and utilizing the actions of non-metabolic functions of the CHK alpha in the regulation of phosphatidylcholine synthesis, glycometabolism (glycolysis), glutathione synthesis, lipid-droplet lipolysis, beta-oxidation and tumor growth.
Description
Technical Field
The invention belongs to the technical field of oncology, and relates to application of non-metabolic function based on CHK alpha as a target for cancer treatment, diagnosis and prognosis prediction.
Background
The choline kinase catalyzes the conversion of free choline to phosphorylcholine, which is further converted to cytidine diphosphate choline by phosphorylcholine cytidylyltransferase, and to phosphatidylcholine by cholinergic transferase. Choline kinase alpha promotes proliferation and survival of tumor cells, is highly expressed in 40% -60% of human tumors, is positively correlated with the difference prognosis of a plurality of tumors, and plays a role in the occurrence and development of tumors. However, the specific mechanism by which choline kinase alpha (chkα) enhances tumor progression is not yet defined.
Reprogramming of cell metabolism is a feature of tumor cells. The non-metabolic function of many metabolic enzymes in tumor cells is becoming more and more interesting and plays an important role in the development and progression of tumors. Such as the protein kinase function of Pyruvate Kinase (PKM), the protein kinase function of phosphoglycerate kinase (PGK), the protein kinase function of Phosphoenolpyruvate Carboxykinase (PCK), etc.
Active oxygen and oxidative stress are increased in tumor cells, glutathione synthesis can counteract oxidative stress and promote tumor progression, and phosphorylation of Cystathionine Beta Synthase (CBS) can promote glutathione synthesis.
The lipid droplets are surrounded by a polar amphiphilic monolayer of phospholipids, the structural protein of which is lipid droplet coating Protein (PLIN). Lipid droplets regulate the hydrolysis of neutral lipids, such as triglycerides, sterol esters and retinyl esters, which are used for energy production of fatty acid oxidation, membrane biogenesis, protein modification, etc., and are thus critical for the growth of tumor cells.
Disclosure of Invention
The invention aims at analyzing the mechanism of the occurrence and development of choline kinase alpha (CHK alpha) in human cancers, utilizing the effect of the non-metabolic function of the CHK alpha in the regulation of phosphatidylcholine synthesis, glycometabolism (glycolysis), glutathione synthesis, lipid-droplet lipolysis, beta-oxidation and tumor growth, and proposing the application of the non-metabolic function based on the CHK alpha regulation as a target for cancer treatment, diagnosis and prognosis prediction.
The invention is realized by adopting the following technical scheme:
the invention provides an application of CHK alpha-based non-metabolic function as a target for cancer treatment and diagnosis, which comprises the following steps:
(1) Determining the detection of cancer cells in the patient or blood in the patient comprises: elevated CBS Y484 site phosphorylation levels, chkαs279 site phosphorylation levels, chkαk247 site acetylation levels, PLIN2Y232 site phosphorylation levels, PLIN3Y251 site phosphorylation levels, enolase 1 (ENO 1) Y44 site phosphorylation levels, intramolecular disulfide bond formation between C303 and C307 of chkα; and
(2) Blocking one or more than two states of CHK alpha protein kinase activity activation, CBS Y484 site phosphorylation, CHK alpha S279 site phosphorylation, CHK alpha K247 site acetylation, PLIN2Y232 site phosphorylation, PLIN3Y251 site phosphorylation, enolase 1 (ENO 1) Y44 site phosphorylation level and intramolecular disulfide bond formation between C303 and C307 of CHK alpha by using an inhibition method; and/or
(3) Predicting a favorable response of the patient to the method of treatment;
the reference level is a level from a non-cancerous or early stage cancerous cell or the patient's blood.
Preferably, the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer or hematopoietic cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, renal cancer, biliary tract cancer, pheochromocytoma, islet cell cancer, li-furo Mei Niliu, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.
Preferably, the method of determining comprises performing ELISA, immunoassay, radioimmunoassay, immunohistochemistry, immunoradiometric assay, fluoroimmunoassay, gel electrophoresis, immunoblot analysis, in situ hybridization, flow cytometry or microscopic assay using a phospho-specific antibody.
Preferably, the inhibition method comprises using a chkα inhibitor, CBS Y484 inhibitor, chkαs279 inhibitor, chkαk247 inhibitor, PLIN2Y232 inhibitor, PLIN3Y251 inhibitor, or any other method of inhibiting chkα protein kinase activity, or any other method of inhibiting CBS Y484 site phosphorylation, chkαs279 site phosphorylation, chkαk247 site acetylation, PLIN2Y232 site phosphorylation, PLIN3Y251 site phosphorylation, enolase 1 (ENO 1) Y44 site phosphorylation level, intramolecular disulfide bond formation between C303 and C307 of chkα.
Preferably, the chka inhibitor comprises a small molecule inhibitor of chka protein kinase activity, CBS Y484, chka S279, chka K247, PLIN2Y232, PLIN3Y251 inhibitor comprises a polypeptide, small molecule inhibitor or complementary inhibitory polynucleotide that selectively targets CBS Y484 site phosphorylation, chka S279 site phosphorylation, chka K247 site acetylation, PLIN2Y232 site phosphorylation, PLIN3Y251 site phosphorylation, enolase 1 (ENO 1) Y44 site phosphorylation level, intramolecular disulfide bond formation between C303 and C307 of chka.
Preferably, the beneficial response comprises a decrease in tumor size or burden, a retardation of tumor growth, a decrease in tumor-associated pain, a decrease in cancer-associated pathological conditions, a decrease in cancer-associated symptoms, no progression of cancer, no prolongation of disease intervals, prolonged progression time, induced remission, reduced metastasis, prolonged patient survival or increased sensitivity of the tumor to anti-cancer treatment.
The invention also provides the use of chkα -based protein kinase activity as a target for prognosis prediction of cancer, the use comprising:
(1) Determining whether detecting cancer cells in the patient or blood in the patient comprises: elevated CBS Y484 site phosphorylation levels, chkαs279 site phosphorylation levels, chkαk247 site acetylation levels, PLIN2Y232 site phosphorylation levels, PLIN3Y251 site phosphorylation levels, and elevated levels of various combinations of these five;
(2) Predicting that the patient has an aggressive cancer if the cancer cells or the patient's blood contain an elevated level of any of (1);
(3) Predicting that an aggressive cancer that the patient has is in a stage of progression if the cancer cells or the patient's blood contain an elevated level of any of (1);
(4) Predicting that the patient has a poor prognosis if the cancer cells or the patient's blood comprise an elevated level of any of (1);
the reference level is a level from a non-cancerous or early stage cancerous cell or the patient's blood.
Further, if the subject has invasive cancer, inhibitor anti-cancer therapy is performed by blocking one or more of CHK alpha protein kinase activity activation, CBS Y484 site phosphorylation, CHK alpha S279 site phosphorylation, CHK alpha K247 site acetylation, PLIN2Y232 site phosphorylation, PLIN3Y251 site phosphorylation, enolase 1 (ENO 1) Y44 site phosphorylation level, formation of intramolecular disulfide bond between C303 and C307 of CHK alpha.
The beneficial effects of the invention are as follows: the present invention utilizes any upstream signal that activates oxidative stress signals, which results in intramolecular disulfide bond formation between C303 and C307 of CHKα, thereby phosphorylating CBS Y484 site, enhancing binding of CBS to pyridoxal phosphate, and promoting de novo synthesis of glutathione synthesis in tumor cells, cell proliferation and tumor growth. The present invention utilizes any signal upstream of chkα binding lipid droplets (including phosphorylation at chkαs279 and acetylation at chkαk247 due to glucose deprivation) that, upon activation, results in conformational changes in the catalytic domain of chkα with protein kinase activity to phosphorylate the PLIN2Y232 and PLIN3Y251 sites. Phosphorylated PLIN2/3 dissociates from lipid droplets and is degraded by Hsc 70-mediated autophagy, thereby promoting lipid droplet lipolysis, beta-oxidation and tumor growth. The importance of the newly identified protein kinase activity of choline kinase chkα in de novo synthesis of glutathione synthesis, lipid droplet lipolysis, β -oxidation and tumor growth is emphasized. Importantly, the expression levels of CBS Y484 site phosphorylation, chkαs279 site phosphorylation, chkαk247 site acetylation, PLIN2Y232 site phosphorylation, PLIN3Y251 site phosphorylation were significantly up-regulated in tumor tissue, expression levels were significantly positively correlated with each other, correlated with tumor progression, correlated with poor prognosis of tumors. The present invention utilizes any signal that can bind chkα to ENO1 and phosphorylate enolase 1 (ENO 1) Y44 site (including its binding site mutation chkαf199N/P200N and ENO 1Y 44F mutation) to block TRIM25 binding and polyubiquitination chkα (K195 site) and proteasome degradation chkα, thereby promoting cholinergic phospholipid metabolism and tumor growth. Importantly, the expression level of ENO 1Y 44 site phosphorylation was significantly up-regulated in tumor tissue, inversely correlated with longer survival of tumor patients.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
In the drawings:
FIG. 1 is H of example 1 2 O 2 LN229 cells were treated and subjected to biotin pulldown experiments, followed by immunizationBlot analysis, using tubulin as marker; wherein, WLC, whole cell lysate; cyto, cytoplasmic extract; nuc, nuclear extract.
FIG. 2 is a three-dimensional pattern of human CHK alpha protein structure of example 1.
FIG. 3 is a biotin pulldown test of example 1; wherein WLC, whole cell lysate.
FIG. 4 is a Ni-NTA pulldown test, H, of example 2 2 O 2 After treatment, flag, his immunoblot analysis patterns, tubulin was used as a marker.
FIG. 5 is an in vitro kinase assay of example 2, H 2 O 2 After treatment, CHKα, CBS, 32 Immunoblot analysis of P-CBS and CBS pY 484.
FIG. 6 shows the pattern for filling the in vitro kinase assay (left side) and CBS activity assay (right side) of example 3.
Fig. 7 is a bar-change pattern for filling of ROS level detection experiment (left side) and GSH/GSSG ratio detection experiment (right side) of example 4.
Fig. 8 shows the tumor size detection assay of example 5, P <0.001 (two-tailed student t-test). C1, clone 1; c2, clone 2. Pattern for filling column-changing pattern
FIG. 9 is a survival curve of the phosphorylation level of CBS Y484 site in the brain glioma patients of example 6.
FIG. 10 shows the detection of CHK alpha and CHK beta expression by immunoblot analysis following treatment of tumor cells with glucose deprivation in example 7, using tubulin as a marker.
FIG. 11 shows the co-localization of CHKα, ATGL, beclin1 and lipid droplets after treatment of tumor cells with sugar deficiency in example 8.
FIG. 12 is an immunoblot analysis of CHKα binding to lipid droplets using tubulin as a marker in example 9.
FIG. 13 is an immunoblot analysis of CHKα binding to lipid droplets using tubulin as a marker in example 10.
FIG. 14 shows immunoprecipitation and immunoblotting of example 11 for CHKα binding to PLIN2 and PLIN3 using tubulin as a marker.
FIG. 15 shows immunoprecipitation and immunoblot analysis of CHKα binding to PLIN2/3, example 12, using tubulin as a marker.
FIG. 16 shows the CHK alpha phosphorylated PLIN2Y232 and PLIN3Y251 sites of Ni-NTA agarose beads of example 13 for pulldown analysis and immunoblotting analysis. Tubulin was used as a marker.
FIG. 17 is a molecular dynamics simulation analysis CHKα of example 14.
FIG. 18 shows the immunoprecipitation and immunoblot analysis of PLIN2/3 interaction with Hsc70, example 15, using tubulin as a marker.
FIG. 19 shows the co-localization of PLIN2/3 with lipid droplets by immunofluorescence assay of example 16.
FIG. 20 shows the co-localization of ATGL, beclin1, LC3B and lipid droplets for the immunofluorescence assay of example 16.
FIG. 21 is a statistical graph of tumor cell proliferation detected under 2-DG treatment conditions of example 17.
FIG. 22 shows the measurement results of the glioma in mice of example 18.
FIG. 23 shows the results of lipid droplet accumulation assay in a mouse brain glioma tissue sample of example 18.
FIG. 24 is an immunohistochemical assay of the ACC S79 phosphorylated antibody, CHK.alpha.S 279 phosphorylated antibody, CHK.alpha.K 247 acetylated antibody, PLIN2Y232 phosphorylated antibody and PLIN3Y251 phosphorylated antibody of example 19 in 100 human glioma samples.
FIG. 25 is a correlation of expression levels of the tumor markers shown in example 19 in 60 human glioma samples.
FIG. 26 is a survival curve of ACC S79 phosphorylation level, CHK αS279 phosphorylation level, CHK αK247 acetylation level, PLIN2Y232 phosphorylation level and PLIN3Y251 phosphorylation level of the brain glioma patient of example 19.
FIG. 27 shows the binding of ENO1 to CHK.alpha.in the GST pulldown assay of example 20 and the immunoblot analysis.
FIG. 28 shows the ubiquitination of CHK.alpha.by immunoprecipitation and immunoblotting analysis in example 21.
FIG. 29 shows the binding of TRIM25 to CHK.alpha.by immunoprecipitation and immunoblotting of example 22.
FIG. 30 shows the ubiquitination site of CHK.alpha.by immunoprecipitation and immunoblotting analysis in example 22.
FIG. 31 shows the binding of TRIM25 to CHK.alpha.by immunoprecipitation and immunoblotting of example 23.
FIG. 32 shows the measurement of intracellular phosphatidylcholine production in example 24.
FIG. 33 is an in vitro kinase assay of example 25.
FIG. 34 is a graph showing the measurement of lactic acid production in example 26.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings, in order to make the objects and technical solutions of the present invention more apparent. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to product specifications; the reagents and materials, unless otherwise specified, are commercially available.
1. The detection method comprises the following steps:
1. detection of the level of phosphorylated proteins:
by contacting the sample with an antibody that specifically binds to the phosphorylated polypeptide and determining the amount of antibody bound, for example, by detecting or measuring the travel of the complex between the antibody and the polypeptide. Antibodies can be labeled (radioactive, fluorescent, etc.) to facilitate detection of the complex.
The detection system for polypeptide level of the present invention comprises: autoradiography immunoassay RIA, immunofluorescence, gel electrophoresis, western immunoblotting assay.
Antibody used (added): tubulin (sc-8035) was purchased from Santa Cruz Biotechnology (Santa Cruz, calif.); anti-CHKα, ENO1, LC3B, ATGL, beclin1, ACC pS79, CHK Anti-bodies are purchased from Cell Signaling Technology (Danvers, mass.); rabbit antibodies that recognize CHK, CBS (pY 484), CBS, CHKαpS279, CHKαAcK247, PLIN2 pY232, PLIN3 pY251 are purchased from Signalway Biotechnology (Pearland, TX); mouse monoclonal anti-Flag (F1804), rabit anti-Flag (F7425), anti-His (SAB 1305538) anti-bodies were purchased from Sigma-Aldrich (St. Louis, mo.).
2. Mouse tumor sample acquisition and analysis:
huh7 cells (1×106) were injected intracranially into athymic nude mice (n=7/group). Mice were euthanized 28 days after injection and examined for HCC tumor growth.
3. Immunohistochemical IHC staining detection of proteins in cells (tissue microarray construction):
formalin-fixed paraffin-embedded tissue was obtained by surgical excision and stained with Mayer's hematoxylin and eosin (H & E; biogenex laboratories, san Raymond, calif.).
Tissue Microarray (TMA) treatment was performed on cancer case tumor samples and normal tissue samples. Using automatic tissue array instrumentAlphelys, plaisir, france), cancer tissue (2 mm diameter, selected by pathologists) was extracted from each specimen and fixed in paraffin blocks. After quality control, TMA blocks were cut into sections for immunohistochemical analysis.
Secondary antibodies anti-Rabbit IgG heavy chain (HRP) (ab 99702) anti-bodies were purchased from Abcam (Cambridge, mass.).
4. Pearson correlation test
The present invention uses pearson correlation assays to correlate protein expression in human brain glioma specimens.
Immunohistochemical analysis was performed according to previous publications (see nucleic-Translocated ACSS2Promotes Gene Transcription for Lysosomal Biogenesis and Autophagy. Molecular cell.2017;66 (5): 684-97e 9). After deparaffinization, rehydration and antigen retrieval, TMA slides were incubated overnight at 4 ℃ with primary anti-rabbit anti-human AKT pS473 (dilution 1:200), primary anti-rabbit anti-human phosphorylated PCK 1pS 90 (dilution 1:200), primary anti-rabbit anti-human incubations ins ig1pS207 and ins ig2 pS151 (dilution 1:500), primary anti-rabbit anti-human SREBP1 (dilution 1:100) or non-specific IgG (as a negative control). The slides were then incubated with anti-rabbit secondary antibodies (ready-to-use solution; cell Signaling Technology; # 8114), followed by secondary Diaminobenzidine (DAB) staining (Cell Signaling Technology) and hematoxylin staining, and fixed on xylene. The tissue slides were scored quantitatively under a microscope based on the percentage of positive cells and staining intensity. The invention assigns the following ratio scores: 0,0% of cells were positive; 1,0% to 1%;2,2% to 10%; 3. 11% to 30%; 4. 31% to 70%; and 5, from 71% to 100%. Also 0 to 3: a scale of 0 (negative) rates the staining intensity. 1, weak; 2, moderate; 3, strengthening. The ratio and intensity scores are then added to obtain a total score (range 0-8), as described in the literature previously. Two pathologists unaware of the clinical information independently verified the repeatability of the scoring system.
5. Total survival Kaplan-Meier plot for patients
Data analysis was performed using SPSS version 20.0 software (SPSS inc., chicago, il.a.). The expression levels of the biomarkers in tumor and normal tissues were compared using independent sample t-test. Multiple comparisons and least significant difference tests were performed on the correlation between the expression levels of the biomarkers and the clinical pathology features of the patients using one-way analysis of variance (ANOVA, post hoc Bonferroni test). The Pearson correlation coefficient was used to analyze the correlation between biomarker expression levels. Total survival (OS) is defined as the duration from the day of diagnosis to the day of death or last follow-up. The expression levels of the relevant markers were classified using a K-means cluster analysis, a Kaplan-Meier method was used to draw a survival curve, a log rank test to compare survival rates, a Cox regression model with a bi-directional Wald test to calculate Hazard Ratios (HR) and 95% Confidence Intervals (CIs) for survival analysis. The censored data is for patients who were alive in the last follow-up or lost due to the follow-up. Univariate analysis variables with P values less than 0.05 were included in the multivariate analysis. P <0.05 is considered statistically significant. All statistical tests are double-sided.
2. The materials used in the following examples are as follows:
1. cell type:
huh7 cells (human hepatoma cell line Huh7 cells), LN229 cells (human glioma cells), from ATCC;
2. the athymic nude mice are BALB/c athymic nude mice;
3. patient samples:
samples of surgically resected, formalin fixed, paraffin embedded NSCLC tissue were retrospectively collected from the biological library of the university of Zhejiang transformation institute (Hangzhou, china). Tissue samples from 100 patients pathologically diagnosed with brain glioma who had not received surgical treatment were selected as independent cohorts. Clinical data was obtained by reviewing the patient's medical history. The pathological stage was assessed by the united states joint committee for cancer/the international association for cancer control TNM classification system, 8 th edition.
4. The shRNA sequence used for gene knockout is as follows:
CHKα：TGATACTAAAGACGGTATTAA
PLIN2：CAGAAGCTAGAGCCGCAAATT
PLIN3：CTGGACCACATGGTGGAATAT
ATGL：CCTGCCACTCTATGAGCTTAA
Beclin 1：GCTTGGGTGTCCTCACAATTT
ENO1：CGCATTGGAGCAGAGGTTTAC
TRIM25：CCGGAACAGTTAGTGGATTTA
example 1 CHK alpha is a direct oxidative stress sensor
1. Intramolecular disulfide bond formation of chkα is a direct event in cells that respond to oxidative stress
With 50 mu M H 2 O 2 LN229 cells were treated. Oxidized proteins were labeled with biotin-maleimide and purified with streptavidin-agarose beads. As shown in FIG. 1, H 2 O 2 The chkα intramolecular disulfide bond formation was observed at 2 minutes of treatment.
2. Intramolecular disulfide bond formation between C303 and C307
(1) As shown in FIG. 2, the human CHKα structure (PDB: 2 CKO) shows the spatial positions of C303 and C307. The surrounding areas of C303 and C307 are framed and enlarged. Intramolecular disulfide bonds are formed between the sulfur atoms of C303 and C307.
(2) HA-tagged wild-type chkα, chkαc303A, or chkαc307A were expressed in LN229 cells. Cells were treated with the reducing agent N-acetylcysteine (NAC) for 30 min and then 50. Mu. M H 2 O 2 Treatment was carried out for 10 minutes. Oxidized proteins were labeled with biotin-maleimide and purified with streptavidin-agarose beads. WLC, whole cell lysate. As shown in FIG. 3, C303 or C307 of CHKα in LN229 cells (CHKα structure is only separated) The mutations of (2) eliminate disulfide bond formation and faster chkα migration.
Example 2 oxidative stress results in chkα binding and phosphorylating CBS
1.H 2 O 2 Chkα binding to CBS after treatment
LN229 cells expressing Flag-tagged CHKα and His-tagged CBS were treated with NAC for 10 min and then 50 μ M H 2 O 2 Treatment was carried out for 10 minutes. The experimental results of Ni-NTA show that H 2 O 2 Treatment resulted in chkα binding to CBS, which was blocked by NAC treatment. (FIG. 4)
2. Oxidative stress phosphorylates chkα to the Y484 site of CBS
Purified wild-type Flag-CHKα was immobilized on magnetic beads, and H was added 2 O 2 Treatment for 10 minutes, PBS rinse, followed by incubation with purified wild-type His-CBS or His-CBS Y484F in the presence of 32P-ATP, was used for in vitro kinase assay. The results are shown in FIG. 5,H 2 O 2 Treatment phosphorylates CBS, while the Y484F mutation of CBS blocks this phosphorylation.
Example 3 chkα mediated phosphorylation of CBS activated CBS
Purified wild-type Flag-CHK alpha or its mutein was immobilized on magnetic beads, and 50. Mu. M H was added 2 O 2 Treatment for 10 min, 10mM DTT for 30 min, PBS rinsing, and subsequent incubation with purified wild-type His-CBS or His-CBS Y484F in the presence of ATP for in vitro kinase assayAssay (left) and CBS activity assay (right). As a result, FIG. 6, Y484 phosphorylation of CBS enhanced CBS activity.
Example 4 chkα mediated CBS phosphorylation enhanced de novo synthesis of glutathione
LN229 wild-type cells and CHKαC307A, CHK αS298A/P299A or CBS Y484F mutant LN229 cells were treated with 0.25mM paraquat for 6 hours, and intracellular ROS levels (left side), GSH/GSSG ratios (right side) were examined. As shown in fig. 7, chkα -mediated phosphorylation of CBS Y484 site activates CBS, increasing the de novo synthesis of glutathione (a direct response of tumor cells to counteract oxidative stress).
Example 5 Synthesis of CHK alpha-mediated glutathione promoted tumor growth
LN229 wild type cells and CHKαC307A, CHK αS298A/P299A or CBS Y484F mutant LN229 cells were injected intracranially into athymic nude mice (14 per group) once every three days (four total) after one week with liposomal doxorubicin (20 mg/kg) (7 per group). Tumor size was measured after one month. As shown in FIG. 8, doxorubicin inhibited tumor growth, whereas CHKαC307A, CHK αS298A/P299A, or CBS Y484F mutant expression reduced tumor growth and enhanced the effect of doxorubicin on tumor growth.
Example 6, negative correlation of the phosphorylation level at the CBS Y484 site with the survival of glioma patients
The expression of the phosphorylation level of the CBS Y484 site in the glioma sample is divided into high expression and low expression, and a Kaplan-Meier diagram of the overall survival of the patient is drawn. As shown in fig. 9, the level of CBS Y484-site phosphorylation was inversely related to the survival of glioma patients.
EXAMPLE 7 CHK alpha binds to lipid droplets and is required for lipolysis of the lipid droplets
Huh7, U87, GP06, GP08 cells were treated for 1 hour for sugar deficiency and chkα expression in whole cell lysates, cytoplasm, lipid droplets was detected. As shown in fig. 10, the sugar-deficient treatment allows a small fraction of cytoplasmic chkα, but not chkβ, to bind to lipid droplets.
EXAMPLE 8 CHK alpha is required for lipolysis of lipid droplets
Huh7 cells with or without CHKα or CHKβ knockdown were treated for 1 hour with sugar deficiency and immunofluorescence was performed with BODIPY or DAPI or antibodies recognizing Beclin1 or ATGL. As shown in FIG. 11, only CHK alpha knockdown inhibited the co-localization of ATGL and autophagic protein Beclin1 with lipid droplets induced by the sugar deficiency treatment. This result demonstrates that chkα is required for lipolysis of lipid droplets in the absence of sugar.
EXAMPLE 9 AMPK mediated phosphorylation of CHK alpha S279 at CHK alpha binding lipid droplets under sugar deficiency conditions
Huh7 cells were treated with 5. Mu.M Compound C (AMPK inhibitor) for 30 min, with 1 h of sugar-deficient treatment, or with 0.5mM A769662 (AMPK activator) for 30 min. The lipid droplets were purified and subjected to immunoblot analysis. As shown in fig. 12, compound C treatment blocked sugar-deficient induced chkα binding to lipid droplets, whereas a769662 treatment promoted such binding even in the absence of sugar.
Example 10 phosphorylation of CHK alpha S279 site results in KAT 5-mediated acetylation of CHK alpha K247 site and recruitment of CHK alpha to lipid droplets
Huh7 cells expressing wild-type Flag-CHKα or Flag-CHKαK247R mutations were treated for 1 hour for sugar deficiency, lipid droplets were purified, and immunoprecipitation analysis was performed using Flag antibodies. As shown in fig. 13, the k247R mutant chkα (chkαs279 site phosphorylation was not affected) did not translocate to lipid droplets.
EXAMPLE 11 CHK alpha binding to PLIN2/3
By immunoprecipitation and immunoblot analysis, chkα binds to PLIN2 and PLIN3 from 1 hour of sugarless treatment of Huh7 cells as shown in FIG. 14.
EXAMPLE 12 monomer CHKα binding to PLIN2/3
Huh7 cells expressing Flag-PLIN2, flag-PLIN3, wild His-CHKα or CHKα mutation were treated with sugar deficiency for 1 hour to obtain whole cell lysates. Pull down analysis was performed with Flag antibody agarose beads and Ni-NTA agarose beads. As shown in fig. 15, chkα converts from dimer to monomer so that it can bind to PLIN2 and PLIN3.
EXAMPLE 13 CHK alpha phosphorylation of PLIN2Y232 and PLIN3Y251 sites
Huh7 cells expressing wild type His-PLIN2, his-PLIN 2Y 232F mutation, wild type His-PLIN3, his-PLIN 3Y251F mutation were treated anoxia for 1 hour and were subjected to pulldown analysis using Ni-NTA agarose beads. As shown in FIG. 16, the sugar-deficient treatment resulted in phosphorylation of PLIN2Y232 and PLIN3Y251 sites, which was blocked by PLIN2Y 232F mutation and PLIN3Y251F mutation.
EXAMPLE 14 CHKαK247 site-acetylation-mediated CHKαMonognizing alters the Structure of its catalytic Domain and phosphorylates PLIN2Y232 and PLIN3Y251 sites
Molecular dynamics simulation analysis of chkα dimer and monomer, results are shown in fig. 17, with the overall profile of the monomer chkα docking PLIN2Y232 (LHSRAYQQALS) peptide or PLIN3Y251 (LRQHAYEHSLG) peptide shown above. Below is the magnified image. The distance between the gamma-phosphate of ATP and the OH roots of PLIN2Y232 or PLIN3Y251 was measured. Computer docking analysis results showed that only the monomeric, expanded choline binding pocket of chkα, such that the PLIN2Y232 or PLIN3Y251 peptide interacted with the catalytic domain of chkα; and PLIN2Y232Or PLIN3Y 251->The distance between the OH groups of (c) and the gamma-phosphate groups of ATP is close enough to allow for phosphate group transfer.
EXAMPLE 15 PLIN2/3 phosphorylated CHK alpha under sugar deficient conditions promotes PLIN2/3 binding to Hsc70
Huh7 cells expressing HA-Hsc70, wild-type Flag-PLIN2, wild-type Flag-PLIN3, PLIN2Y 232F mutation or PLIN3Y251F mutation, were treated with BSA-conjugated oleic acid for 12 hours and with sugar deficiency for 1 hour, followed by immunoprecipitation analysis with Flag agarose beads. As shown in FIG. 18, PLIN2Y 232F mutation or PLIN3Y251F mutation blocked PLIN2/3 interaction with Hsc 70.
EXAMPLE 16 CHKα -mediated PLIN2/3 phosphorylation under sugarless conditions resulted in the recruitment of ATGL and autophagosomes to lipid droplets for lipid droplet lipolysis
U87 wild-type and U87 cells with CHKαS279A mutation, CHKαK247R mutation, PLIN2Y 232F mutation or PLIN3Y251F mutation were treated with BSA-conjugated oleic acid for 12 hours. Followed by sugar-deficient treatment for 2 hours. Cells were stained with BODIPY, DAPI, PLIN antibody, PLIN3 antibody and immunofluorescence analyzed. Results as shown in fig. 19 and 20, chkαs279A mutation, chkαk247R mutation, PLIN2Y 232F mutation or PLIN3Y251F mutation blocked sugar deficiency induced dissociation of PLIN2/3 from lipid droplets (fig. 19), and ATGL, beclin1, LC3B from lipid droplets (fig. 20).
EXAMPLE 17 CHK alpha-mediated lipolysis to promote tumor cell survival
Cells expressing CHKαshRNA, PLIN2 shRNA, PLIN3 shRNA, reconstituted wild-type Flag-rCHKα, reconstituted wild-type Flag-rPLIN2, reconstituted wild-type HA-PLIN3, or Huh7 cells mutated as shown were treated with 25mM 2-DG for 48 hours before cell count statistics were performed. As shown in FIG. 21, under 2-DG treatment conditions, mutations expressing CHK.alpha.or PLIN2/3 further reduced cell proliferation.
EXAMPLE 18 CHK alpha-mediated lipolysis to promote brain tumor growth
U87 cells and U87 cells expressing CHKαS279A mutation, CHKαK247R mutation, PLIN2Y 232F/PLIN 3Y251F mutation (PLIN 2/3 Mut), ATGL shRNA or Beclin1 shRNA were intraperitoneally injected into athymic nude mice, 0.2ml 2-DG (500 mg/kg) was daily injected into the abdominal cavity after two weeks for 14 days, after which the tumor size was examined (FIG. 22), and the left HE staining results showed representative tumor xenografts, and the right results were tumor volume examination. Tumor tissues were frozen and subsequently stained with red O and the percentage of red O stained areas was counted (see fig. 23). The results show that expression of these mutations, either ATGL shRNA or Beclin1 shRNA, inhibited tumor growth and increased lipid accumulation.
Example 19 chkα mediated lipolysis plays a key role in the malignant clinical manifestations of brain gliomas
Immunohistochemistry was performed in 100 brain glioma samples with the antibodies shown in fig. 24, and the results showed that ACC S79 phosphorylation levels, chkαs279 phosphorylation levels, chkαk247 acetylation levels, PLIN2Y232 phosphorylation levels, and PLIN3Y251 phosphorylation levels were positively correlated with each other (see fig. 25). In the samples of 60 patients, the ACC S79 phosphorylation level, chkαs279 phosphorylation level, chkαk247 acetylation level, PLIN2Y232 phosphorylation level and PLIN3Y251 phosphorylation level were divided into high expression and low expression, respectively, and the total survival time Kaplan-Meier chart was drawn, and the results were shown in fig. 26, in which chkαs279 phosphorylation level, chkαk247 acetylation level, PLIN2Y232 phosphorylation level and PLIN3Y251 phosphorylation level were positively correlated with the difference prognosis of brain glioma patients.
EXAMPLE 20 binding of ENO1 to CHK alpha
After incubation of purified GST-CHKα with purified ENO1, the binding between NO1 and CHKα was found to exist as shown in FIG. 27 by GST pull down test.
EXAMPLE 21 inhibition of ubiquitination of CHK alpha by ENO1
Flag-CHKα, his-Ub and ENO1 shRNA were expressed in U87 cells, and by immunoprecipitation and immunoblot analysis, knocking down ENO1 enhanced ubiquitination of CHKα as shown in FIG. 28.
Example 22 TRIM25 binding and ubiquitination of the K195 site of CHK alpha
Expressing Flag-chkα, HA-TRIM25, chkαk195R, CHK αk273R, CHK αk276R, CHK αk325R mutations in U87 cells, by immunoprecipitation and immunoblot analysis, as in fig. 29, trim25 binds chkα; as shown in fig. 30, chkαk195R mutation reduced its ubiquitination level.
Example 23 binding of ENO1 to CHK alpha reduced binding of TRIM25 to CHK alpha
Expression of HA-TRIM25, flag-CHKα, flag-CHKαF199N, flag-CHKαP200N in U87 cells, binding site mutation of ENO1 to CHKα increased the binding of TRIM25 to CHKα by immunoprecipitation and immunoblotting analysis, as shown in FIG. 31.
EXAMPLE 24 upregulation of ENO1 CHK alpha facilitates phosphatidylcholine production
Expressing ENO1 shRNA and TRIM25 shRNA in U87 cells, and detecting the content of phosphatidylcholine in the cells. As shown in fig. 32, knocking down ENO1 reduced the yield of phosphatidylcholine, while knocking down TRIM25 enhanced the yield of phosphatidylcholine; on the basis of knocking down TRIM25, knocking down ENO1 weakens the yield of phosphatidylcholine.
EXAMPLE 25 CHK alpha phosphorylation of the ENO 1Y 44 site
The chkα phosphorylates the ENO 1Y 44 site as shown in fig. 33 by in vitro kinase assay, immunoprecipitation and immunoblotting experiments.
Example 26 CHK alpha-mediated phosphorylation of ENO1 enhanced tumor cell glycolysis
As shown in fig. 34, either the chkα knockout or the ENO1 knockout resulted in a decrease in lactate production by tumor cell LN229, while decreasing glycolysis.
It should be understood that the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited to the above-described embodiment, but may be modified or substituted for some of the features described in the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. An application of a reagent for detecting the phosphorylation level of CBS Y484 site in preparing a brain glioma prognosis product.
2. The use according to claim 1, wherein the level of phosphorylation at CBS Y484 site is positively correlated with the poor prognosis of a brain glioma patient.
3. A reagent for detecting the phosphorylation level of CBS Y484 site and an application of the reagent for detecting the phosphorylation level of CHK alpha S279 site, the acetylation level of CHK alpha K247 site, the phosphorylation level of PLIN2Y232 site and the phosphorylation level of PLIN3Y251 site in preparing brain glioma prognosis products.
4. The use according to claim 3, wherein the CBS Y484 site phosphorylation level, chkαs279 site phosphorylation level, chkαk247 site acetylation level, PLIN2Y232 site phosphorylation level, PLIN3Y251 site phosphorylation level is positively correlated with the differential prognosis of a brain glioma patient.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311546211.0A CN117849343A (en) | 2021-05-31 | 2021-05-31 | Use of chkα -based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction |
| CN202110602766.7A CN113391069B (en) | 2021-05-31 | 2021-05-31 | Use of chkα -based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110602766.7A CN113391069B (en) | 2021-05-31 | 2021-05-31 | Use of chkα -based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311546211.0A Division CN117849343A (en) | 2021-05-31 | 2021-05-31 | Use of chkα -based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113391069A CN113391069A (en) | 2021-09-14 |
| CN113391069B true CN113391069B (en) | 2023-10-27 |
Family
ID=77619658
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110602766.7A Active CN113391069B (en) | 2021-05-31 | 2021-05-31 | Use of chkα -based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction |
| CN202311546211.0A Pending CN117849343A (en) | 2021-05-31 | 2021-05-31 | Use of chkα -based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311546211.0A Pending CN117849343A (en) | 2021-05-31 | 2021-05-31 | Use of chkα -based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN113391069B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101405403A (en) * | 2005-04-13 | 2009-04-08 | 科学研究高等机关 | In vitro cancer therapy compound identification method |
| CN105886625A (en) * | 2016-04-28 | 2016-08-24 | 北京泱深生物信息技术有限公司 | Application of CHKA gene to preparation of esophageal cancer diagnosis and treatment product |
| CN106290895A (en) * | 2016-07-26 | 2017-01-04 | 王红阳 | CHKA albumen application, test kit and method in preparing prognosis in hcc assessment test kit |
| CN107249692A (en) * | 2015-01-05 | 2017-10-13 | 得克萨斯州大学系统董事会 | As for treatment of cancer and the protein kinase activity of the phosphoglyceric kinase 1 of the target of diagnosis |
| CN111505292A (en) * | 2020-04-03 | 2020-08-07 | 青岛大学附属医院 | Use of PCK 1-based modulation of lipid metabolism as a target for cancer therapy, diagnosis and prognosis prediction |
-
2021
- 2021-05-31 CN CN202110602766.7A patent/CN113391069B/en active Active
- 2021-05-31 CN CN202311546211.0A patent/CN117849343A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101405403A (en) * | 2005-04-13 | 2009-04-08 | 科学研究高等机关 | In vitro cancer therapy compound identification method |
| CN103215351A (en) * | 2005-04-13 | 2013-07-24 | 科学研究高等机关 | In vitro cancer therapy compound identification method |
| CN107249692A (en) * | 2015-01-05 | 2017-10-13 | 得克萨斯州大学系统董事会 | As for treatment of cancer and the protein kinase activity of the phosphoglyceric kinase 1 of the target of diagnosis |
| CN105886625A (en) * | 2016-04-28 | 2016-08-24 | 北京泱深生物信息技术有限公司 | Application of CHKA gene to preparation of esophageal cancer diagnosis and treatment product |
| CN106290895A (en) * | 2016-07-26 | 2017-01-04 | 王红阳 | CHKA albumen application, test kit and method in preparing prognosis in hcc assessment test kit |
| CN111505292A (en) * | 2020-04-03 | 2020-08-07 | 青岛大学附属医院 | Use of PCK 1-based modulation of lipid metabolism as a target for cancer therapy, diagnosis and prognosis prediction |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113391069A (en) | 2021-09-14 |
| CN117849343A (en) | 2024-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lee et al. | Altered proteome of extracellular vesicles derived from bladder cancer patients urine | |
| Lam et al. | Comprehensive identification and modified-site mapping of S-nitrosylated targets in prostate epithelial cells | |
| CN101971026B (en) | Detect composition and the method for TIABS | |
| Ma et al. | Overexpression of Arginase-1 is an indicator of poor prognosis in patients with colorectal cancer | |
| Kim et al. | Prognostic impact and clinicopathological correlation of CD133 and ALDH1 expression in invasive breast cancer | |
| RU2434946C2 (en) | Method of in vitro determination of prognosis of disease development in patient with cancer and method of in vitro monitoring effect of therapy administered to patient with cancer | |
| Zhang et al. | CXCL5 overexpression predicts a poor prognosis in pancreatic ductal adenocarcinoma and is correlated with immune cell infiltration | |
| Tickoo et al. | Immunohistochemical expression of hypoxia inducible factor-1α and its downstream molecules in sarcomatoid renal cell carcinoma | |
| Zuo et al. | Positive expression of SMYD2 is associated with poor prognosis in patients with primary hepatocellular carcinoma | |
| Zuckerman et al. | Expression and function of lysophosphatidic acid receptors (LPARs) 1 and 3 in human hepatic cancer progenitor cells | |
| Streckfus et al. | Salivary protein profiles among HER2/neu‐receptor‐positive and‐negative breast cancer patients: support for using salivary protein profiles for modeling breast cancer progression | |
| Qu et al. | Down-regulation of salt-inducible kinase 1 (SIK1) is mediated by RNF2 in hepatocarcinogenesis | |
| US10480034B2 (en) | Cancer biomarker and diagnostic | |
| Mei et al. | Phospho-histone H2AX is a diagnostic and prognostic marker for epithelial ovarian cancer | |
| Aras et al. | Mitochondrial autoimmunity and MNRR1 in breast carcinogenesis | |
| TWI567391B (en) | Biomarker for identifying subgroup of early-stage lung adenocarcinoma patients | |
| Fourneaux et al. | Identifying and targeting cancer stem cells in leiomyosarcoma: prognostic impact and role to overcome secondary resistance to PI3K/mTOR inhibition | |
| Lin et al. | The prognostic role of expression of nectin-4 in esophageal cancer | |
| Byström et al. | Affinity proteomics exploration of melanoma identifies proteins in serum with associations to T-stage and recurrence | |
| Yin et al. | Association of PKCζ expression with clinicopathological characteristics of breast cancer | |
| Luo et al. | PAD4-dependent citrullination of nuclear translocation of GSK3β promotes colorectal cancer progression via the degradation of nuclear CDKN1A | |
| Leung et al. | Pin1 overexpression is associated with poor differentiation and survival in oral squamous cell carcinoma | |
| CN108508212A (en) | The marker of high-level serous ovarian cancer targeted therapy and Index for diagnosis | |
| CN113391069B (en) | Use of chkα -based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction | |
| OĞUZTÜZÜN et al. | GST isoenzymes in matched normal and neoplastic breast tissue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |