CN104395754A - Trop-2作为对基于cd9、akt和四跨膜蛋白信号传导网络分子的抑制剂的抗肿瘤疗法的应答的预测性标记的用途 - Google Patents
Trop-2作为对基于cd9、akt和四跨膜蛋白信号传导网络分子的抑制剂的抗肿瘤疗法的应答的预测性标记的用途 Download PDFInfo
- Publication number
- CN104395754A CN104395754A CN201380025540.9A CN201380025540A CN104395754A CN 104395754 A CN104395754 A CN 104395754A CN 201380025540 A CN201380025540 A CN 201380025540A CN 104395754 A CN104395754 A CN 104395754A
- Authority
- CN
- China
- Prior art keywords
- trop
- cell
- protein
- akt
- intracellular signaling
- 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.)
- Pending
Links
- 239000003112 inhibitor Substances 0.000 title claims description 11
- 230000000259 anti-tumor effect Effects 0.000 title claims description 4
- 230000004044 response Effects 0.000 title abstract description 7
- 230000011664 signaling Effects 0.000 title abstract 5
- 102000043977 Tetraspanins Human genes 0.000 title abstract 3
- 108700031126 Tetraspanins Proteins 0.000 title abstract 3
- 238000002560 therapeutic procedure Methods 0.000 title description 9
- 239000003550 marker Substances 0.000 title description 3
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 64
- 150000001875 compounds Chemical class 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 38
- 239000000090 biomarker Substances 0.000 claims abstract description 24
- 239000003814 drug Substances 0.000 claims abstract description 21
- 201000011510 cancer Diseases 0.000 claims abstract description 11
- 238000011319 anticancer therapy Methods 0.000 claims abstract description 10
- 229940079593 drug Drugs 0.000 claims abstract description 9
- 238000012216 screening Methods 0.000 claims abstract description 7
- 239000002246 antineoplastic agent Substances 0.000 claims abstract 2
- 108090000623 proteins and genes Proteins 0.000 claims description 58
- 230000004068 intracellular signaling Effects 0.000 claims description 54
- 102000004169 proteins and genes Human genes 0.000 claims description 48
- 108091008611 Protein Kinase B Proteins 0.000 claims description 47
- 102000035160 transmembrane proteins Human genes 0.000 claims description 36
- 108091005703 transmembrane proteins Proteins 0.000 claims description 36
- 238000011282 treatment Methods 0.000 claims description 28
- 230000014509 gene expression Effects 0.000 claims description 27
- 230000000694 effects Effects 0.000 claims description 24
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 17
- 108020004999 messenger RNA Proteins 0.000 claims description 13
- 230000004614 tumor growth Effects 0.000 claims description 13
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 12
- -1 AT7867 Chemical compound 0.000 claims description 9
- 108091034117 Oligonucleotide Proteins 0.000 claims description 9
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 9
- 239000004473 Threonine Substances 0.000 claims description 9
- 230000010261 cell growth Effects 0.000 claims description 9
- 230000004663 cell proliferation Effects 0.000 claims description 8
- 238000000338 in vitro Methods 0.000 claims description 8
- 102000001253 Protein Kinase Human genes 0.000 claims description 7
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims description 7
- 239000012660 pharmacological inhibitor Substances 0.000 claims description 7
- 108060006633 protein kinase Proteins 0.000 claims description 7
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 6
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 claims description 6
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 claims description 6
- 230000001093 anti-cancer Effects 0.000 claims description 6
- 229940124650 anti-cancer therapies Drugs 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 6
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 6
- 238000001262 western blot Methods 0.000 claims description 6
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 claims description 5
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 claims description 5
- 230000005764 inhibitory process Effects 0.000 claims description 5
- 101100464197 Caenorhabditis elegans pak-1 gene Proteins 0.000 claims description 4
- 102000011068 Cdc42 Human genes 0.000 claims description 4
- 238000002965 ELISA Methods 0.000 claims description 4
- 102100022623 Hepatocyte growth factor receptor Human genes 0.000 claims description 4
- 101710184069 Hepatocyte growth factor receptor Proteins 0.000 claims description 4
- 101001077604 Homo sapiens Insulin receptor substrate 1 Proteins 0.000 claims description 4
- 102100025087 Insulin receptor substrate 1 Human genes 0.000 claims description 4
- 108010055717 JNK Mitogen-Activated Protein Kinases Proteins 0.000 claims description 4
- 102000019145 JUN kinase activity proteins Human genes 0.000 claims description 4
- 108700027648 Mitogen-Activated Protein Kinase 8 Proteins 0.000 claims description 4
- 102100037808 Mitogen-activated protein kinase 8 Human genes 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 claims description 4
- 229940100514 Syk tyrosine kinase inhibitor Drugs 0.000 claims description 4
- 102100033444 Tyrosine-protein kinase JAK2 Human genes 0.000 claims description 4
- 101710112791 Tyrosine-protein kinase JAK2 Proteins 0.000 claims description 4
- 102100033019 Tyrosine-protein phosphatase non-receptor type 11 Human genes 0.000 claims description 4
- 101710116241 Tyrosine-protein phosphatase non-receptor type 11 Proteins 0.000 claims description 4
- 102100032791 cAMP-dependent protein kinase catalytic subunit alpha Human genes 0.000 claims description 4
- 101710110809 cAMP-dependent protein kinase catalytic subunit alpha Proteins 0.000 claims description 4
- 230000003197 catalytic effect Effects 0.000 claims description 4
- 108010051348 cdc42 GTP-Binding Protein Proteins 0.000 claims description 4
- 229940000406 drug candidate Drugs 0.000 claims description 4
- 238000003757 reverse transcription PCR Methods 0.000 claims description 4
- 229940126638 Akt inhibitor Drugs 0.000 claims description 3
- 230000004069 differentiation Effects 0.000 claims description 3
- 239000003197 protein kinase B inhibitor Substances 0.000 claims description 3
- GRZXWCHAXNAUHY-NSISKUIASA-N (2S)-2-(4-chlorophenyl)-1-[4-[(5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl]-1-piperazinyl]-3-(propan-2-ylamino)-1-propanone Chemical compound C1([C@H](C(=O)N2CCN(CC2)C=2C=3[C@H](C)C[C@@H](O)C=3N=CN=2)CNC(C)C)=CC=C(Cl)C=C1 GRZXWCHAXNAUHY-NSISKUIASA-N 0.000 claims description 2
- BPNUQXPIQBZCMR-IBGZPJMESA-N (2s)-1-{[5-(3-methyl-1h-indazol-5-yl)pyridin-3-yl]oxy}-3-phenylpropan-2-amine Chemical compound C([C@H](N)COC=1C=NC=C(C=1)C1=CC=C2NN=C(C2=C1)C)C1=CC=CC=C1 BPNUQXPIQBZCMR-IBGZPJMESA-N 0.000 claims description 2
- AVLYNJZZQYEFEA-XDJHFCHBSA-N (2z)-2-(3h-1,3-benzothiazol-2-ylidene)-2-[2-(2-pyridin-3-ylethylamino)pyrimidin-4-yl]acetonitrile Chemical compound N/1C2=CC=CC=C2SC\1=C(/C#N)C(N=1)=CC=NC=1NCCC1=CC=CN=C1 AVLYNJZZQYEFEA-XDJHFCHBSA-N 0.000 claims description 2
- JVVRJMXHNUAPHW-UHFFFAOYSA-N 1h-pyrazol-5-amine Chemical class NC=1C=CNN=1 JVVRJMXHNUAPHW-UHFFFAOYSA-N 0.000 claims description 2
- HNFMVVHMKGFCMB-UHFFFAOYSA-N 3-[3-[4-(1-aminocyclobutyl)phenyl]-5-phenylimidazo[4,5-b]pyridin-2-yl]pyridin-2-amine Chemical compound NC1=NC=CC=C1C1=NC2=CC=C(C=3C=CC=CC=3)N=C2N1C1=CC=C(C2(N)CCC2)C=C1 HNFMVVHMKGFCMB-UHFFFAOYSA-N 0.000 claims description 2
- JDUBGYFRJFOXQC-KRWDZBQOSA-N 4-amino-n-[(1s)-1-(4-chlorophenyl)-3-hydroxypropyl]-1-(7h-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide Chemical compound C1([C@H](CCO)NC(=O)C2(CCN(CC2)C=2C=3C=CNC=3N=CN=2)N)=CC=C(Cl)C=C1 JDUBGYFRJFOXQC-KRWDZBQOSA-N 0.000 claims description 2
- BYWWNRBKPCPJMG-UHFFFAOYSA-N 4-dodecyl-n-(1,3,4-thiadiazol-2-yl)benzenesulfonamide Chemical compound C1=CC(CCCCCCCCCCCC)=CC=C1S(=O)(=O)NC1=NN=CS1 BYWWNRBKPCPJMG-UHFFFAOYSA-N 0.000 claims description 2
- ZBNZXTGUTAYRHI-UHFFFAOYSA-N Dasatinib Chemical compound C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1Cl ZBNZXTGUTAYRHI-UHFFFAOYSA-N 0.000 claims description 2
- KGPGFQWBCSZGEL-ZDUSSCGKSA-N GSK690693 Chemical compound C=12N(CC)C(C=3C(=NON=3)N)=NC2=C(C#CC(C)(C)O)N=CC=1OC[C@H]1CCCNC1 KGPGFQWBCSZGEL-ZDUSSCGKSA-N 0.000 claims description 2
- BYTORXDZJWWIKR-UHFFFAOYSA-N Hinokiol Natural products CC(C)c1cc2CCC3C(C)(CO)C(O)CCC3(C)c2cc1O BYTORXDZJWWIKR-UHFFFAOYSA-N 0.000 claims description 2
- 239000012825 JNK inhibitor Substances 0.000 claims description 2
- 229940118135 JNK inhibitor Drugs 0.000 claims description 2
- 239000002067 L01XE06 - Dasatinib Substances 0.000 claims description 2
- HOGVTUZUJGHKPL-UHFFFAOYSA-N LSM-1546 Chemical compound C=12C3=NC=NC=1N(C)N=C(N)C2=CN3C1OC(CO)C(O)C1O HOGVTUZUJGHKPL-UHFFFAOYSA-N 0.000 claims description 2
- 229940121849 Mitotic inhibitor Drugs 0.000 claims description 2
- AFJRDFWMXUECEW-LBPRGKRZSA-N N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-2-methyl-3-pyrazolyl)-2-thiophenecarboxamide Chemical compound CN1N=CC(Cl)=C1C1=C(Cl)SC(C(=O)N[C@H](CN)CC=2C=C(F)C=CC=2)=C1 AFJRDFWMXUECEW-LBPRGKRZSA-N 0.000 claims description 2
- 229940079156 Proteasome inhibitor Drugs 0.000 claims description 2
- 229940122924 Src inhibitor Drugs 0.000 claims description 2
- 101710183280 Topoisomerase Proteins 0.000 claims description 2
- XSYODYDCSYFRRB-QXLMEFJZSA-N [(2r)-2-methoxy-3-octadecoxypropyl] [(1r,2r,3s,4r)-2,3,4-trihydroxycyclohexyl] hydrogen phosphate Chemical compound CCCCCCCCCCCCCCCCCCOC[C@@H](OC)COP(O)(=O)O[C@@H]1CC[C@@H](O)[C@H](O)[C@H]1O XSYODYDCSYFRRB-QXLMEFJZSA-N 0.000 claims description 2
- 229940100198 alkylating agent Drugs 0.000 claims description 2
- 239000002168 alkylating agent Substances 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 239000003242 anti bacterial agent Substances 0.000 claims description 2
- 230000000340 anti-metabolite Effects 0.000 claims description 2
- 229940088710 antibiotic agent Drugs 0.000 claims description 2
- 229940100197 antimetabolite Drugs 0.000 claims description 2
- 239000002256 antimetabolite Substances 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims description 2
- 239000012472 biological sample Substances 0.000 claims description 2
- 229960001467 bortezomib Drugs 0.000 claims description 2
- GXJABQQUPOEUTA-RDJZCZTQSA-N bortezomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)B(O)O)NC(=O)C=1N=CC=NC=1)C1=CC=CC=C1 GXJABQQUPOEUTA-RDJZCZTQSA-N 0.000 claims description 2
- XDLYKKIQACFMJG-WKILWMFISA-N chembl1234354 Chemical compound C1=NC(OC)=CC=C1C(C1=O)=CC2=C(C)N=C(N)N=C2N1[C@@H]1CC[C@@H](OCCO)CC1 XDLYKKIQACFMJG-WKILWMFISA-N 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000003246 corticosteroid Substances 0.000 claims description 2
- 229960002448 dasatinib Drugs 0.000 claims description 2
- 229940121647 egfr inhibitor Drugs 0.000 claims description 2
- 239000002532 enzyme inhibitor Substances 0.000 claims description 2
- 229940125532 enzyme inhibitor Drugs 0.000 claims description 2
- FVYXIJYOAGAUQK-UHFFFAOYSA-N honokiol Chemical compound C1=C(CC=C)C(O)=CC=C1C1=CC(CC=C)=CC=C1O FVYXIJYOAGAUQK-UHFFFAOYSA-N 0.000 claims description 2
- VVOAZFWZEDHOOU-UHFFFAOYSA-N honokiol Natural products OC1=CC=C(CC=C)C=C1C1=CC(CC=C)=CC=C1O VVOAZFWZEDHOOU-UHFFFAOYSA-N 0.000 claims description 2
- 238000003364 immunohistochemistry Methods 0.000 claims description 2
- PQLXHQMOHUQAKB-UHFFFAOYSA-N miltefosine Chemical compound CCCCCCCCCCCCCCCCOP([O-])(=O)OCC[N+](C)(C)C PQLXHQMOHUQAKB-UHFFFAOYSA-N 0.000 claims description 2
- 229960003775 miltefosine Drugs 0.000 claims description 2
- 238000004393 prognosis Methods 0.000 claims description 2
- 239000003207 proteasome inhibitor Substances 0.000 claims description 2
- 230000002829 reductive effect Effects 0.000 claims description 2
- HOGVTUZUJGHKPL-HTVVRFAVSA-N triciribine Chemical compound C=12C3=NC=NC=1N(C)N=C(N)C2=CN3[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O HOGVTUZUJGHKPL-HTVVRFAVSA-N 0.000 claims description 2
- 229950003873 triciribine Drugs 0.000 claims description 2
- AAALVYBICLMAMA-UHFFFAOYSA-N 4,5-dianilinophthalimide Chemical compound C=1C=CC=CC=1NC=1C=C2C(=O)NC(=O)C2=CC=1NC1=CC=CC=C1 AAALVYBICLMAMA-UHFFFAOYSA-N 0.000 claims 1
- 229940127089 cytotoxic agent Drugs 0.000 claims 1
- 238000002651 drug therapy Methods 0.000 claims 1
- 229940041181 antineoplastic drug Drugs 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 103
- 102100037904 CD9 antigen Human genes 0.000 description 44
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 44
- 235000018102 proteins Nutrition 0.000 description 41
- 238000006366 phosphorylation reaction Methods 0.000 description 24
- 230000026731 phosphorylation Effects 0.000 description 21
- 238000001890 transfection Methods 0.000 description 20
- 230000002018 overexpression Effects 0.000 description 19
- 230000008859 change Effects 0.000 description 17
- 230000012010 growth Effects 0.000 description 17
- 102100024924 Protein kinase C alpha type Human genes 0.000 description 15
- 101710109947 Protein kinase C alpha type Proteins 0.000 description 15
- 230000000875 corresponding effect Effects 0.000 description 15
- 230000002441 reversible effect Effects 0.000 description 15
- 108091027967 Small hairpin RNA Proteins 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 11
- 230000000692 anti-sense effect Effects 0.000 description 11
- 210000000170 cell membrane Anatomy 0.000 description 11
- 230000003993 interaction Effects 0.000 description 11
- 230000008569 process Effects 0.000 description 11
- 230000019491 signal transduction Effects 0.000 description 11
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 230000000638 stimulation Effects 0.000 description 8
- 230000001629 suppression Effects 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 210000004881 tumor cell Anatomy 0.000 description 8
- 108090000315 Protein Kinase C Proteins 0.000 description 7
- 102000003923 Protein Kinase C Human genes 0.000 description 7
- 230000003321 amplification Effects 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 206010067472 Organising pneumonia Diseases 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 208000023367 bronchiolitis obliterans with obstructive pulmonary disease Diseases 0.000 description 6
- 238000000749 co-immunoprecipitation Methods 0.000 description 6
- 201000009805 cryptogenic organizing pneumonia Diseases 0.000 description 6
- 230000001419 dependent effect Effects 0.000 description 6
- 238000001493 electron microscopy Methods 0.000 description 6
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 6
- 238000003119 immunoblot Methods 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 102100035893 CD151 antigen Human genes 0.000 description 5
- 102100027221 CD81 antigen Human genes 0.000 description 5
- 206010009944 Colon cancer Diseases 0.000 description 5
- 101000946874 Homo sapiens CD151 antigen Proteins 0.000 description 5
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 241001529936 Murinae Species 0.000 description 5
- 108020004459 Small interfering RNA Proteins 0.000 description 5
- 230000005907 cancer growth Effects 0.000 description 5
- 235000012000 cholesterol Nutrition 0.000 description 5
- 210000005220 cytoplasmic tail Anatomy 0.000 description 5
- 239000012636 effector Substances 0.000 description 5
- 239000010931 gold Substances 0.000 description 5
- 229910052737 gold Inorganic materials 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- YZOUYRAONFXZSI-SBHWVFSVSA-N (1S,3R,5R,6R,8R,10R,11R,13R,15R,16R,18R,20R,21R,23R,25R,26R,28R,30R,31S,33R,35R,36R,37S,38R,39S,40R,41S,42R,43S,44R,45S,46R,47S,48R,49S)-5,10,15,20,25,30,35-heptakis(hydroxymethyl)-37,39,40,41,42,43,44,45,46,47,48,49-dodecamethoxy-2,4,7,9,12,14,17,19,22,24,27,29,32,34-tetradecaoxaoctacyclo[31.2.2.23,6.28,11.213,16.218,21.223,26.228,31]nonatetracontane-36,38-diol Chemical compound O([C@@H]([C@H]([C@@H]1OC)OC)O[C@H]2[C@@H](O)[C@@H]([C@@H](O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3O)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O3)O[C@@H]2CO)OC)[C@H](CO)[C@H]1O[C@@H]1[C@@H](OC)[C@H](OC)[C@H]3[C@@H](CO)O1 YZOUYRAONFXZSI-SBHWVFSVSA-N 0.000 description 4
- 239000012103 Alexa Fluor 488 Substances 0.000 description 4
- 206010006187 Breast cancer Diseases 0.000 description 4
- 208000026310 Breast neoplasm Diseases 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 101710138460 Leaf protein Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 4
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 4
- 101150117918 Tacstd2 gene Proteins 0.000 description 4
- 102100027212 Tumor-associated calcium signal transducer 2 Human genes 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
- 239000006185 dispersion Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 231100000588 tumorigenic Toxicity 0.000 description 4
- 230000000381 tumorigenic effect Effects 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- ZAINTDRBUHCDPZ-UHFFFAOYSA-M Alexa Fluor 546 Substances [H+].[Na+].CC1CC(C)(C)NC(C(=C2OC3=C(C4=NC(C)(C)CC(C)C4=CC3=3)S([O-])(=O)=O)S([O-])(=O)=O)=C1C=C2C=3C(C(=C(Cl)C=1Cl)C(O)=O)=C(Cl)C=1SCC(=O)NCCCCCC(=O)ON1C(=O)CCC1=O ZAINTDRBUHCDPZ-UHFFFAOYSA-M 0.000 description 3
- 102100027217 CD82 antigen Human genes 0.000 description 3
- 206010057248 Cell death Diseases 0.000 description 3
- QRLVDLBMBULFAL-UHFFFAOYSA-N Digitonin Natural products CC1CCC2(OC1)OC3C(O)C4C5CCC6CC(OC7OC(CO)C(OC8OC(CO)C(O)C(OC9OCC(O)C(O)C9OC%10OC(CO)C(O)C(OC%11OC(CO)C(O)C(O)C%11O)C%10O)C8O)C(O)C7O)C(O)CC6(C)C5CCC4(C)C3C2C QRLVDLBMBULFAL-UHFFFAOYSA-N 0.000 description 3
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 3
- 102000001267 GSK3 Human genes 0.000 description 3
- 108060006662 GSK3 Proteins 0.000 description 3
- 101000914469 Homo sapiens CD82 antigen Proteins 0.000 description 3
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 3
- 102100020944 Integrin-linked protein kinase Human genes 0.000 description 3
- 108700020796 Oncogene Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 108091000080 Phosphotransferase Proteins 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 230000000711 cancerogenic effect Effects 0.000 description 3
- 231100000315 carcinogenic Toxicity 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- UVYVLBIGDKGWPX-KUAJCENISA-N digitonin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)C[C@@H](O)[C@H](O[C@H]5[C@@H]([C@@H](O)[C@@H](O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O)CO7)O)[C@H](O)[C@@H](CO)O6)O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O7)O)[C@@H](O)[C@@H](CO)O6)O)[C@@H](CO)O5)O)C[C@@H]4CC[C@H]3[C@@H]2[C@@H]1O)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 UVYVLBIGDKGWPX-KUAJCENISA-N 0.000 description 3
- UVYVLBIGDKGWPX-UHFFFAOYSA-N digitonine Natural products CC1C(C2(CCC3C4(C)CC(O)C(OC5C(C(O)C(OC6C(C(OC7C(C(O)C(O)CO7)O)C(O)C(CO)O6)OC6C(C(OC7C(C(O)C(O)C(CO)O7)O)C(O)C(CO)O6)O)C(CO)O5)O)CC4CCC3C2C2O)C)C2OC11CCC(C)CO1 UVYVLBIGDKGWPX-UHFFFAOYSA-N 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 235000015220 hamburgers Nutrition 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- NFVJNJQRWPQVOA-UHFFFAOYSA-N n-[2-chloro-5-(trifluoromethyl)phenyl]-2-[3-(4-ethyl-5-ethylsulfanyl-1,2,4-triazol-3-yl)piperidin-1-yl]acetamide Chemical compound CCN1C(SCC)=NN=C1C1CN(CC(=O)NC=2C(=CC=C(C=2)C(F)(F)F)Cl)CCC1 NFVJNJQRWPQVOA-UHFFFAOYSA-N 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 102000020233 phosphotransferase Human genes 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000002626 targeted therapy Methods 0.000 description 3
- 238000007492 two-way ANOVA Methods 0.000 description 3
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- WEVYNIUIFUYDGI-UHFFFAOYSA-N 3-[6-[4-(trifluoromethoxy)anilino]-4-pyrimidinyl]benzamide Chemical group NC(=O)C1=CC=CC(C=2N=CN=C(NC=3C=CC(OC(F)(F)F)=CC=3)C=2)=C1 WEVYNIUIFUYDGI-UHFFFAOYSA-N 0.000 description 2
- 102100037263 3-phosphoinositide-dependent protein kinase 1 Human genes 0.000 description 2
- 235000010894 Artemisia argyi Nutrition 0.000 description 2
- 101000741929 Caenorhabditis elegans Serine/threonine-protein phosphatase 2A catalytic subunit Proteins 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 102000009193 Caveolin Human genes 0.000 description 2
- 108050000084 Caveolin Proteins 0.000 description 2
- 108010077544 Chromatin Proteins 0.000 description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 101000600756 Homo sapiens 3-phosphoinositide-dependent protein kinase 1 Proteins 0.000 description 2
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 2
- 101001117146 Homo sapiens [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 1, mitochondrial Proteins 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical group OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 102000038030 PI3Ks Human genes 0.000 description 2
- 108091007960 PI3Ks Proteins 0.000 description 2
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 2
- 102100023085 Serine/threonine-protein kinase mTOR Human genes 0.000 description 2
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 description 2
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical compound C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 description 2
- 101710120037 Toxin CcdB Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 244000030166 artemisia Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 229960005395 cetuximab Drugs 0.000 description 2
- 210000003483 chromatin Anatomy 0.000 description 2
- 239000003818 cinder Substances 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000008034 disappearance Effects 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 229940011871 estrogen Drugs 0.000 description 2
- 239000000262 estrogen Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 229960002949 fluorouracil Drugs 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 230000012447 hatching Effects 0.000 description 2
- 210000003128 head Anatomy 0.000 description 2
- 229940022353 herceptin Drugs 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 238000010185 immunofluorescence analysis Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 2
- 229960000485 methotrexate Drugs 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000009826 neoplastic cell growth Effects 0.000 description 2
- 231100001223 noncarcinogenic Toxicity 0.000 description 2
- 230000000474 nursing effect Effects 0.000 description 2
- 238000011275 oncology therapy Methods 0.000 description 2
- 230000004850 protein–protein interaction Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000007634 remodeling Methods 0.000 description 2
- 239000004055 small Interfering RNA Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003153 stable transfection Methods 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229940121358 tyrosine kinase inhibitor Drugs 0.000 description 2
- 239000005483 tyrosine kinase inhibitor Substances 0.000 description 2
- 150000004917 tyrosine kinase inhibitor derivatives Chemical class 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- TZJUVVIWVWFLCD-UHFFFAOYSA-N 1,1-dioxo-2-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-1,2-benzothiazol-3-one Chemical compound O=S1(=O)C2=CC=CC=C2C(=O)N1CCCCN(CC1)CCN1C1=NC=CC=N1 TZJUVVIWVWFLCD-UHFFFAOYSA-N 0.000 description 1
- HJTAZXHBEBIQQX-UHFFFAOYSA-N 1,5-bis(chloromethyl)naphthalene Chemical compound C1=CC=C2C(CCl)=CC=CC2=C1CCl HJTAZXHBEBIQQX-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- IJJWOSAXNHWBPR-HUBLWGQQSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-n-(6-hydrazinyl-6-oxohexyl)pentanamide Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)NCCCCCC(=O)NN)SC[C@@H]21 IJJWOSAXNHWBPR-HUBLWGQQSA-N 0.000 description 1
- WYWHKKSPHMUBEB-UHFFFAOYSA-N 6-Mercaptoguanine Natural products N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 1
- VHRSUDSXCMQTMA-PJHHCJLFSA-N 6alpha-methylprednisolone Chemical compound C([C@@]12C)=CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2[C@@H](O)C[C@]2(C)[C@@](O)(C(=O)CO)CC[C@H]21 VHRSUDSXCMQTMA-PJHHCJLFSA-N 0.000 description 1
- 102100027211 Albumin Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- MLDQJTXFUGDVEO-UHFFFAOYSA-N BAY-43-9006 Chemical compound C1=NC(C(=O)NC)=CC(OC=2C=CC(NC(=O)NC=3C=C(C(Cl)=CC=3)C(F)(F)F)=CC=2)=C1 MLDQJTXFUGDVEO-UHFFFAOYSA-N 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 1
- 102000000905 Cadherin Human genes 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- GAGWJHPBXLXJQN-UORFTKCHSA-N Capecitabine Chemical compound C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 GAGWJHPBXLXJQN-UORFTKCHSA-N 0.000 description 1
- GAGWJHPBXLXJQN-UHFFFAOYSA-N Capecitabine Natural products C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1C1C(O)C(O)C(C)O1 GAGWJHPBXLXJQN-UHFFFAOYSA-N 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 241001111317 Chondrodendron tomentosum Species 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- PTOAARAWEBMLNO-KVQBGUIXSA-N Cladribine Chemical compound C1=NC=2C(N)=NC(Cl)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 PTOAARAWEBMLNO-KVQBGUIXSA-N 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 102100026359 Cyclic AMP-responsive element-binding protein 1 Human genes 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 1
- 108010092160 Dactinomycin Proteins 0.000 description 1
- 241000582790 Dactylorhiza viridis Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 244000148064 Enicostema verticillatum Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 238000000729 Fisher's exact test Methods 0.000 description 1
- 108091006027 G proteins Proteins 0.000 description 1
- 102000030782 GTP binding Human genes 0.000 description 1
- 108091000058 GTP-Binding Proteins 0.000 description 1
- 206010051066 Gastrointestinal stromal tumour Diseases 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010009202 Growth Factor Receptors Proteins 0.000 description 1
- 102000009465 Growth Factor Receptors Human genes 0.000 description 1
- 101150072436 H1 gene Proteins 0.000 description 1
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 1
- 101000855516 Homo sapiens Cyclic AMP-responsive element-binding protein 1 Proteins 0.000 description 1
- 101000692455 Homo sapiens Platelet-derived growth factor receptor beta Proteins 0.000 description 1
- 101100368708 Homo sapiens TACSTD2 gene Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- VSNHCAURESNICA-UHFFFAOYSA-N Hydroxyurea Chemical compound NC(=O)NO VSNHCAURESNICA-UHFFFAOYSA-N 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 239000005517 L01XE01 - Imatinib Substances 0.000 description 1
- 239000005411 L01XE02 - Gefitinib Substances 0.000 description 1
- 239000005551 L01XE03 - Erlotinib Substances 0.000 description 1
- 239000002147 L01XE04 - Sunitinib Substances 0.000 description 1
- 239000005511 L01XE05 - Sorafenib Substances 0.000 description 1
- 208000030984 MIRAGE syndrome Diseases 0.000 description 1
- 235000006679 Mentha X verticillata Nutrition 0.000 description 1
- 235000002899 Mentha suaveolens Nutrition 0.000 description 1
- 235000001636 Mentha x rotundifolia Nutrition 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 241000209094 Oryza Species 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241001319148 Pharmacis Species 0.000 description 1
- 102100026547 Platelet-derived growth factor receptor beta Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 102000016971 Proto-Oncogene Proteins c-kit Human genes 0.000 description 1
- 108010014608 Proto-Oncogene Proteins c-kit Proteins 0.000 description 1
- 102000014450 RNA Polymerase III Human genes 0.000 description 1
- 108010078067 RNA Polymerase III Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- NCYCYZXNIZJOKI-OVSJKPMPSA-N Retinaldehyde Chemical compound O=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-OVSJKPMPSA-N 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- BPEGJWRSRHCHSN-UHFFFAOYSA-N Temozolomide Chemical compound O=C1N(C)N=NC2=C(C(N)=O)N=CN21 BPEGJWRSRHCHSN-UHFFFAOYSA-N 0.000 description 1
- 102100032802 Tetraspanin-8 Human genes 0.000 description 1
- 101710151636 Tetraspanin-8 Proteins 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 108091008605 VEGF receptors Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 1
- LUTSRLYCMSCGCS-BWOMAWGNSA-N [(3s,8r,9s,10r,13s)-10,13-dimethyl-17-oxo-1,2,3,4,7,8,9,11,12,16-decahydrocyclopenta[a]phenanthren-3-yl] acetate Chemical compound C([C@@H]12)C[C@]3(C)C(=O)CC=C3[C@@H]1CC=C1[C@]2(C)CC[C@H](OC(=O)C)C1 LUTSRLYCMSCGCS-BWOMAWGNSA-N 0.000 description 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229940045799 anthracyclines and related substance Drugs 0.000 description 1
- 229940045719 antineoplastic alkylating agent nitrosoureas Drugs 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- GOLCXWYRSKYTSP-UHFFFAOYSA-N arsenic trioxide Inorganic materials O1[As]2O[As]1O2 GOLCXWYRSKYTSP-UHFFFAOYSA-N 0.000 description 1
- 238000011717 athymic nude mouse Methods 0.000 description 1
- 229960000397 bevacizumab Drugs 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 229960002092 busulfan Drugs 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 229960004117 capecitabine Drugs 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 235000021256 carbohydrate metabolism Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229960004630 chlorambucil Drugs 0.000 description 1
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 1
- 229960002436 cladribine Drugs 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 229960000928 clofarabine Drugs 0.000 description 1
- WDDPHFBMKLOVOX-AYQXTPAHSA-N clofarabine Chemical compound C1=NC=2C(N)=NC(Cl)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@@H]1F WDDPHFBMKLOVOX-AYQXTPAHSA-N 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 229960003901 dacarbazine Drugs 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 229960003668 docetaxel Drugs 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000010218 electron microscopic analysis Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229960001433 erlotinib Drugs 0.000 description 1
- AAKJLRGGTJKAMG-UHFFFAOYSA-N erlotinib Chemical compound C=12C=C(OCCOC)C(OCCOC)=CC2=NC=NC=1NC1=CC=CC(C#C)=C1 AAKJLRGGTJKAMG-UHFFFAOYSA-N 0.000 description 1
- FRPJXPJMRWBBIH-RBRWEJTLSA-N estramustine Chemical compound ClCCN(CCCl)C(=O)OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 FRPJXPJMRWBBIH-RBRWEJTLSA-N 0.000 description 1
- 229960001842 estramustine Drugs 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 229960005420 etoposide Drugs 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229960000961 floxuridine Drugs 0.000 description 1
- ODKNJVUHOIMIIZ-RRKCRQDMSA-N floxuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ODKNJVUHOIMIIZ-RRKCRQDMSA-N 0.000 description 1
- 229960000390 fludarabine Drugs 0.000 description 1
- GIUYCYHIANZCFB-FJFJXFQQSA-N fludarabine phosphate Chemical compound C1=NC=2C(N)=NC(F)=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O GIUYCYHIANZCFB-FJFJXFQQSA-N 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 description 1
- 229960002584 gefitinib Drugs 0.000 description 1
- XGALLCVXEZPNRQ-UHFFFAOYSA-N gefitinib Chemical compound C=12C=C(OCCCN3CCOCC3)C(OC)=CC2=NC=NC=1NC1=CC=C(F)C(Cl)=C1 XGALLCVXEZPNRQ-UHFFFAOYSA-N 0.000 description 1
- 229960005277 gemcitabine Drugs 0.000 description 1
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 101150090422 gsk-3 gene Proteins 0.000 description 1
- 102000046001 human TACSTD2 Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 229960001330 hydroxycarbamide Drugs 0.000 description 1
- 229960001101 ifosfamide Drugs 0.000 description 1
- HOMGKSMUEGBAAB-UHFFFAOYSA-N ifosfamide Chemical compound ClCCNP1(=O)OCCCN1CCCl HOMGKSMUEGBAAB-UHFFFAOYSA-N 0.000 description 1
- KTUFNOKKBVMGRW-UHFFFAOYSA-N imatinib Chemical compound C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 KTUFNOKKBVMGRW-UHFFFAOYSA-N 0.000 description 1
- 229960002411 imatinib Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 102000035035 immunoglobulin-like cell adhesion molecules Human genes 0.000 description 1
- 108091005483 immunoglobulin-like cell adhesion molecules Proteins 0.000 description 1
- 239000012133 immunoprecipitate Substances 0.000 description 1
- 238000012750 in vivo screening Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 230000005732 intercellular adhesion Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229950003599 ipsapirone Drugs 0.000 description 1
- 229960004768 irinotecan Drugs 0.000 description 1
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229960004961 mechlorethamine Drugs 0.000 description 1
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical compound ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 229960004584 methylprednisolone Drugs 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 1
- 229960001156 mitoxantrone Drugs 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 108091006026 monomeric small GTPases Proteins 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- LBWFXVZLPYTWQI-IPOVEDGCSA-N n-[2-(diethylamino)ethyl]-5-[(z)-(5-fluoro-2-oxo-1h-indol-3-ylidene)methyl]-2,4-dimethyl-1h-pyrrole-3-carboxamide;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.CCN(CC)CCNC(=O)C1=C(C)NC(\C=C/2C3=CC(F)=CC=C3NC\2=O)=C1C LBWFXVZLPYTWQI-IPOVEDGCSA-N 0.000 description 1
- 239000002077 nanosphere Substances 0.000 description 1
- 238000003012 network analysis Methods 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 201000005443 oral cavity cancer Diseases 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000003068 pathway analysis Methods 0.000 description 1
- 229960005079 pemetrexed Drugs 0.000 description 1
- QOFFJEBXNKRSPX-ZDUSSCGKSA-N pemetrexed Chemical compound C1=N[C]2NC(N)=NC(=O)C2=C1CCC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 QOFFJEBXNKRSPX-ZDUSSCGKSA-N 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 150000003906 phosphoinositides Chemical class 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- TVLSRXXIMLFWEO-UHFFFAOYSA-N prochloraz Chemical compound C1=CN=CN1C(=O)N(CCC)CCOC1=C(Cl)C=C(Cl)C=C1Cl TVLSRXXIMLFWEO-UHFFFAOYSA-N 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 235000020945 retinal Nutrition 0.000 description 1
- 239000011604 retinal Substances 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 102000030938 small GTPase Human genes 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229960003787 sorafenib Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940034785 sutent Drugs 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 229960004964 temozolomide Drugs 0.000 description 1
- NRUKOCRGYNPUPR-QBPJDGROSA-N teniposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@@H](OC[C@H]4O3)C=3SC=CC=3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 NRUKOCRGYNPUPR-QBPJDGROSA-N 0.000 description 1
- 229960001278 teniposide Drugs 0.000 description 1
- 229930192474 thiophene Natural products 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 229960003087 tioguanine Drugs 0.000 description 1
- MNRILEROXIRVNJ-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=NC=N[C]21 MNRILEROXIRVNJ-UHFFFAOYSA-N 0.000 description 1
- 229960000303 topotecan Drugs 0.000 description 1
- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 102000027257 transmembrane receptors Human genes 0.000 description 1
- 108091008578 transmembrane receptors Proteins 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 238000001419 two-dimensional polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- GBABOYUKABKIAF-GHYRFKGUSA-N vinorelbine Chemical compound C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC([C@]23[C@H]([C@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC GBABOYUKABKIAF-GHYRFKGUSA-N 0.000 description 1
- 229960002066 vinorelbine Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
-
- 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
-
- 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
- G01N2333/82—Translation products from oncogenes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
-
- 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
Abstract
本发明包含诊断和治疗癌症的方法,其特征在于基于以下事实,肿瘤中Trop-2的水平与相应正常组织中Trop-2的水平相比的增加构成了生物标记,其可以预测所述肿瘤对于用针对Trop-2信号传导网络的组分的药物的抗癌疗法的应答,所述药物包括但不限于抑制CD9、Akt和四跨膜蛋白信号传导网络分子的药物。更具体来说,发明涉及生物标记Trop-2在筛选新化合物中的用途,并且在临床环境下用作靶向Trop-2信号传导网络分子的抗癌药物的用途的指示,所述抗癌药物包括但不限于抑制CD9、Akt和四跨膜蛋白信号传导网络分子的药物。
Description
技术领域
本发明涉及在带有过度表达Trop-2的肿瘤的患者中进行诊断和抗癌治疗,其借助于针对Trop-2信号传导网络的组分的药物来进行,所述药物包括但不限于抑制CD9、Akt和四跨膜蛋白信号传导网络分子的药物。Trop-2过度表达还可以开发用于体外和体内筛选新化合物的抗癌活性。
背景技术
最新一代的靶向抗癌疗法利用酪氨酸激酶抑制剂(TKI)和/或单克隆抗体(mAb),其是具有显性致癌激酶和/或生长因子受体作为其靶标。然而,与这些药剂相关的临床获益一般来说限制于经治疗的患者的子集,在许多情况下通过肿瘤中的特定分子变化(突变、扩增/缺失、表达的增加/减少)进行界定。这个发现已经突出了肿瘤基因型/表型作为的决定性因素的可能重要性,所述决定性因素是对特异性治疗的灵敏度的决定性因素和患者在治疗之前基于用作生物标记的分子变化的所得分层的决定性因素。这些生物标记经研究以便用作“预测性标记”,即,能够预测对靶向疗法的应答或耐受,其目的在于通过有效个性化治疗来优化临床结果。
预测性生物标记当前在临床中用作选择极少的靶向治疗的指南。实例包括雌激素(ER)和孕酮的受体,其用以选择患有乳腺癌的患者,以便用激素疗法治疗;HER-2的扩增,以便用曲妥珠单抗治疗;慢性骨髓性白血病中的BCR-ABL的易位和胃肠道间质瘤中的c-kit的突变,以便用伊马替尼治疗;肺癌中的EGFR突变,以便用吉非替尼/埃罗替尼治疗;结肠直肠癌中的KRAS突变,以便用西妥昔单抗(Cetuximab)治疗。
“富集生物标记”,即,可以预测哪些患者子组可能对治疗进行应答的标记,其是至关重要的需要。实际上,其可以用以使实验聚焦于可以适用的子组,增加新药物的成功的可能性和试验的有效性。HER2/Herceptest/曲妥珠单抗的组合提供了靶向疗法的诊断工具的成功使用的一个实例。在其它情况下,尽管不是许多情况下,预测性并且富集的标记支持了这个策略的应用。
然而,在大多数情况下,当前可用的生物标记是指治疗靶标本身或主要信号传导通路的个别组分,认为靶标通过其起作用。这些策略的共同限制在于将单一信号传导通路的改变视为造成肿瘤生长的唯一改变。在肿瘤发生的基础上,实际上,存在多阶段进展过程。多重证据,包括从对整个外显子组测序获得的数据和肿瘤的基因表达谱的微阵列分析,都揭示了极其大量的不同突变和肿瘤中的不同信号传导通路之间的相互作用的巨大复杂性。这些研究将肿瘤分为分化的临床上相关的子组,并且突出了在命中多种信号传导通路或最终共同通路以便获得有效治疗的重要性。
经典地参与肿瘤发生的基因变化涉及致癌基因,例如PI3K、Ras、BCR-ABL、EGFR、HER-2(“致癌基因成瘾性”),并且对于触发和维持致癌状况是必需的,并且因此是疗法的逻辑靶标。然而,致瘤状态还取决于多种参与正常细胞功能的基因和通路,其呈现改变的调节,但其本质上并不致癌(非致癌基因成瘾性)。这种成瘾性可以提供在正常细胞与肿瘤细胞之间的差异性毒性的许多药物靶标:其中,一些靶标正在临床前模型上进行研究。这些靶标中仅有一些靶标正在临床试验中进行测试或已经在治疗中使用(VEGF,例如用于用贝伐单抗或索拉非尼/舒尼替尼的治疗)。
因此,为了改进现有疗法的有效性并且鉴别新治疗靶标,经典致癌基因的分析必须伴随有正常信号传导通路的研究。然而,这些“正常”标记虽然呈现为支持肿瘤发生的重要因子,但仍极少用于预测人类对于疗法的应答。
Trop-2(AC:P09758)是一种能够转导细胞质钙增加信号的跨膜糖蛋白[日帕尼(Ripani),1998#648],参与上皮组织中的细胞间粘附,并且驱动了诱导肿瘤生长的信号传导网络(格拉,特雷罗托拉等人2013)(Guerra,Trerotola et al.2013)。Trop-2的结构特征不同于粘附分子的四个经典家族(即整合素、钙粘素、选择素和Ig-CAM)的结构特征。Trop-2的胞外域含有富含半胱氨酸的球状部分,其具有EGF样域(GA733I型域(庄(Chong)和施派歇尔(Speicher)2001)(Chong and Speicher 2001))和甲状腺球蛋白域(林嫩巴赫(Linnenbach),森(Seng)等人1993;福尔纳罗(Fornaro),戴尔阿尔奇普雷特(Dell'Arciprete)等人1995;艾尔斯维迪(El Sewedy),福尔纳罗等人1998)(Linnenbach Seng et al 1993;Fornaro,Dell'Arciprete et al1995;El Sewedy Fornaro et al 1998),其对于同源多聚体的形成是必需的(巴尔扎尔(Balzar),布里亚尔-德布鲁因(Briaire-de Bruijn)等人2001)(Balzar,Briaire-de Bruijn et al 2001)。不具有半胱氨酸的区(“茎干”)将Trop-2的球状部分连接到疏水性跨膜螺旋。Trop-2的胞内部分由缺乏酶促活性、含有磷酸肌醇结合HIKE的26个aa的尾区组成(艾尔斯维迪,福尔纳罗等人1998;奇卡雷利(Ciccarelli),阿恰里托(Acciarito)等人2000)(ElSewedy,Fornaro et al.1998;Ciccarelli,Acciarito et al.2000)。HIKE发现于信号-转导分子中并且可以充当效应子/调节分子/例如G蛋白、PKC、钙对接位点(阿尔贝蒂(Alberti)1999)(Alberti 1999)。HIKE还包括PKC磷酸化位点(S303)(巴苏(Basu),戈登伯格(Goldenberg)等人1995)(Basu,Goldenberget al.1995)。
Trop-2表达已经与实验模型中的肿瘤生长相关联(克莱因(Klein),哈特曼(Hartmann)等人1990;巴苏,戈登伯格等人1995;王(Wang),戴(Day)等人2008;古巴斯(Cubas),张(Zhang)等人2010;戈德斯坦(Goldstein),斯托亚诺娃(Stoyanova)等人2010)(Klein,Hartmann et al.1990;Basu,Goldenberg et al.1995;Wang,Day et al.2008;Cubas,Zhang et al.2010;Goldstein,Stoyanova et al.2010)。在人类肿瘤中,Trop-2的过度表达是胰腺、胃、口腔、卵巢、肺和结肠直肠癌的肿瘤的消极预兆因子(特雷罗托拉,坎它内利(Cantanelli)等人2013)(Trerotola,Cantanelli et al.2013)。此外,Trop-2还是前列腺癌中的祖细胞的标记(戈德斯坦,斯托亚诺娃等人2010;特雷罗托拉,拉索(Rathore)等人2010)(Goldstein,Stoyanova et al.2010;Trerotola,Rathore et al.2010)。
本发明的作者先前已经展现,Trop-2在人类的大多数肿瘤和肿瘤细胞系以高水平表达(特雷罗托拉,坎它内利等人2013)(Trerotola,Cantanelli et al.2013)。具体来说,作者展现,Trop-2即使当与已经表达其的起源组织相比时在癌细胞中也过度表达(S.阿尔贝蒂“抗Trop-2单克隆抗体和其在治疗和诊断肿瘤中的用途”,—PCT/IT2009/000035),表明增加的表达向肿瘤细胞赋予选择性优势(特雷罗托拉,坎它内利等人2013)(Trerotola,Cantanelli etal.2013)。
本发明的作者先前已经展现了Trop-2的刺激对于体外和体内肿瘤发展的功能。Trop-2的过度表达刺激了永生化细胞和肿瘤细胞的生长,使细胞周期的S期中细胞的比例增加。另一方面,针对Trop-2的小干扰(si)RNA的使用消除了表达Trop-2的乳腺癌和结肠癌细胞系的增殖。类似结果也已经在实验肿瘤中获得:Trop-2的表达显著增加肿瘤生长,并且这个生长与表达水平直接成正比。
Trop-2的细胞质尾区的完整性是必需的以便具有生长刺激:整个尾区或HIKE域的缺失、丝氨酸303(由da PKCα磷酸化)和322的点突变、四个谷氨酸残基的置换,所述置换是其沿着α-螺旋一侧的整个长度与赖氨酸正残基交换负电荷,都致使Trop-2刺激活性损失(图1)。
体外和体内细胞生长的刺激已经通过正常Trop-2的过度表达来获得。与这个一致,本发明的作者尚未鉴别出肿瘤中的TROP2基因的突变或结构变化,所述突变或结构变化是在编码区中和5'和3'非翻译区(UTR)中或者接近于启动子的区中(特雷罗托拉,坎它内利等人2013)(Trerotola,Cantanelliet al.2013)。
这些结果表明,在不存在突变下,Trop-2表达增加对于刺激癌细胞生长是必需并且充足的(特雷罗托拉,坎它内利等人2013)(Trerotola,Cantanelliet al.2013)。因此,Trop-2是一种“非致癌基因”,肿瘤细胞的生长对于其过度表达上瘾(特雷罗托拉,坎它内利等人2013)。这种生长刺激与组织型(癌瘤、肉瘤)无关并且与物种(人类、鼠类)无关,这表明Trop-2作用的信号传导网络(格拉,特雷罗托拉等人2013)(Guerra,Trerotola et al.2013)是高度保守的(特雷罗托拉,坎它内利等人2013)(S.阿尔贝蒂“抗Trop-2单克隆抗体和其在治疗和诊断肿瘤中的用途”,PCT/IT2009/000035)。
这个信号传导网络现在将由作者进一步定义,以便鉴别表达Trop-2的肿瘤中特异地并且差异性地活化的效应分子,其用作Trop-2依赖性抗癌疗法的靶标。
作者通过电子显微镜进行的分析显示,Trop-2定位于细胞的顶端表面处的大绒毛的聚集体中、延伸于细胞之间的绒毛中和细胞膜的分散区中(图2)。进一步研究已经将这些聚集体鉴别为四跨膜蛋白网络,即细胞膜中的可以引导信号传导蛋白质之间的相互作用的分子网络的结构平台。通过共焦显微镜对Trop-2与这些大分子平台的特异性标记之间的相互作用的系统分析(巴雷罗(Barreiro),扎迈(Zamai)等人2008)(Barreiro,Zamai et al.2008)展现Trop-2与四跨膜蛋白和与其相关的分子(CD9)在完整细胞(图3a)中和在“细胞足迹”中的共定位(哈科莫里(Hakomori)2002)(Hakomori 2002)(图3c)。通过共帽化和共免疫沉淀的技术还展现,在人类和鼠类细胞中在Trop-2与CD9、CD81、CD82、CD151之间存在直接物理相互作用(图3b,e)。另一方面,Trop-2不与小窝蛋白共定位,所述小窝蛋白是脂质筏的特征标记(图3a-c)。
已知,四跨膜蛋白与其搭配物的结合是胆固醇依赖性的(哈科莫里2002)(Hakomori 2002):Trop-2是四跨膜蛋白平台的整体组分的事实的进一步证实由甲基-β-环糊精和毛地黄皂苷的能力提供,其分别能够从细胞膜去除胆固醇并且使其沉淀(哈科莫里2002,查林(Charrin),2003#17269)(Hakomori 2002,Charrin,2003#17269)、从而诱导培养基中Trop-2的释放和Trop-2从BRIJ中的细胞膜的溶解物的沉淀(勒纳乌尔(Le Naour),安德烈(Andre)等人2006)(Le Naour,Andre et al.2006)(图3f)。
四跨膜蛋白网络向活化脂质、蛋白激酶和胞内效应子的不同细胞表面受体提供了支持(赫姆勒(Hemler)2003;勒纳乌尔,安德烈等人2006)(Hemler2003;Le Naour,Andre et al.2006)。因此,本发明的作者已经展示,Trop-2能够调节跨膜酪氨酸激酶受体(PDGFR、Met、Ret、VEGFR)、酪氨酸和丝氨酸/苏氨酸激酶、磷酸酶、细胞周期和细胞凋亡的调节分子的表达(格拉,特雷罗托拉等人2013)(Guerra,Trerotola et al.2013)(S.阿尔贝蒂“抗Trop-2单克隆抗体和其在治疗和诊断肿瘤中的用途”,PCT/IT2009/000035)。这些结果此处通过新蛋白质组分析而扩展(图4)。生物信息学工具用于蛋白质间接触的统计分析(明格斯(Minguez),戈茨(Gotz)等人2009)(Minguez,Gotzet al.2009)和通路分析(Ingenuity Pathways Analysis软件)的应用使得我们可定义Trop-2的主要信号传导网络(格拉,特雷罗托拉等人2013)(Guerra,Trerotola et al.2013)(图5)。由Trop-2驱动的分子包括信号转导子(跨膜受体、细胞质酪氨酸和丝氨酸/苏氨酸激酶、磷酸酶)、蛋白质合成、折叠和降解的组分、参与核酸合成和碳水化合物代谢的酶。由Trop-2调节的所有蛋白质中超过三分之二(114/165)(图4)通过蛋白质间的接触而连接(图5a)。蛋白质间接触的这个网络已经被鉴别为癌症生长的重要信号传导通路(图5b),其包括PKC、Akt/Gsk3β/S6K和ERK通路以及效应子NF-kB、Jun、CREB1、STAT、Rb和p53(格拉,特雷罗托拉等人2013)(Guerra,Trerotolaet al.2013)。
因此,这些数据已经使本发明的作者能够展现PKC是Trop-2信号传导网络中的关键介体。Trop-2一共调节7种PKC底物和PKC的55种底物/调节分子(图4和5)。PKCα展示在表达Trop-2的细胞中的其活化位点S657的磷酸化的最高增加(图4a),这表明其活化是Trop-2依赖性的。这与ERK信号传导通路的活化以及与由Trop-2诱导的PP2A(参与使PKCα失活的磷酸酶)和PDK1(在其活化位点中使PKCα磷酸化)的调节是一致。PKCα经常过度表达于肿瘤中,并且引导参与癌症的信号传导过程。PKCα还是CD9的主要效应子之一(张,邦特拉杰(Bontrager)等人2001)(Zhang,Bontrageret al.2001),并且募集到四跨膜蛋白复合物(张,邦特拉杰等人2001;罗斯(Rosse),林奇(Linch)等人2010)(Zhang,Bontrager et al.2001;Rosse,Linchet al.2010),在此通过二酰基甘油(DAG)活化(帕雷克(Parekh),齐格勒(Ziegler)等人2000)(Parekh,Ziegler et al.2000)。
基于这些指示,本发明的作者已经展示,CD9微结构域充当PKCα与Trop-2之间的连接体的功能:PKCα与CD81和CD151四跨膜蛋白共免疫沉淀并且与CD9更大程度地共免疫沉淀(图3e)。以类似方式,Trop-2与CD9、CD81、CD82和CD151共免疫沉淀(图3e)。此外,CD9、Trop-2和PKCα共定位于不同的膜子域中并且这种共定位是磷酸化依赖性的(图3c,d),因此证实Trop-2和PKCα都通过与CD9的相互作用而募集到四跨膜蛋白微结构域。
这个信号传导网络的生物信息学分析随后已经使得作者认定Akt信号传导通路为细胞生长由Trop-2的刺激中的关键通路(图6),其是通过GSK3和S6K臂,而不是mTOR组的。Akt是PI3K的主要下游效应子。Akt T308和S473活化位点是磷酸化的靶标。rictor-mTOR复合物与ILK(库尔(Koul),沈(Shen)等人2005)(Koul,Shen et al.2005)一起使S473上的Akt磷酸化(萨尔巴索夫(Sarbassov),古尔汀(Guertin)等人2005)(Sarbassov,Guertin etal.2005),但在T308磷酸化中和在GSK3和S6K效应子的活化中不具有作用(雅辛托(Jacinto),法奇内提(Facchinetti)等人2006)(Jacinto,Facchinetti etal.2006).。与这个一致,Trop-2诱导Akt-T308的磷酸化,但不诱导Akt-S473的磷酸化(图6b),并且调节GSK3和S6K的磷酸化(图4a),但不调节mTOR的磷酸化。先前由本发明的作者在蛋白质组芯片上所展现的ILK的下调以及Akt-S473磷酸化通过Trop-2的减少(因PMA进一步增加)(图6b),与由PKC诱导的ILK和Akt-S473磷酸化的下调一致(温(Wen),黄(Huang)等人2003)(Wen,Huang et al.2003).。另一方面,Trop-2诱导Akt-T308磷酸化(图6b),这也与先前在蛋白质组芯片上所展现的PP2A催化和调节亚单位的化学计量减少一致(PPA是对T308作用的Ca2+依赖性磷酸酶)(弗雷利(Freeley),帕克(Park)等人2007)(Freeley,Park et al.2007),而非与PDK1的诱导一致(图4a)。与PKCα非依赖性机制一致,PMA对Akt-T308磷酸化水平不具有作用(图6b)。
Trop-2与四跨膜蛋白膜复合物并且具体来说与CD9的相互作用活化了具有Akt信号传导通路作为关键组分的复合物下游信号传导网络,并且刺激细胞增殖。
作者随后研究在表达Trop-2的增殖细胞中CD9和Akt的选择性抑制的作用。CD9通过siRNA所介导的下调(图7a,b)导致表达Trop-2的MTE 4-14细胞的增殖显著减少,但对于Trop-2阴性细胞基本上不具有影响(图7d)。这个减少与由Trop-2沉默所造成的减少相当(图7d),并且与Trop-2在癌细胞生长中的基本作用一致。此外,考虑到CD9的表达不受Trop-2影响(图7c),这些结果还表明CD9对于肿瘤生长由Trop-2的刺激是必需的,但另外对于基线细胞生长是非活跃的。通过沉默或通过特异性药物对Akt抑制以剂量依赖性方式诱导了表达Trop-2的细胞的细胞增殖的显著减少,而其对于对照细胞不具有作用(图8)。与这个一致,动物模型中皮下注射的表达Trop-2的肿瘤在用抑制Akt活性的特异性药物治疗之后展示出生长的显著减少(图9)。因此,Akt在介导Trop-2依赖性生长刺激方面具有重要功能作用,并且这种分子的药理学抑制在临床前模型中具有抗癌治疗作用。
发明内容
本发明的作者已经鉴别了新颖的信号传导网络,其刺激肿瘤生长并且通过与CD9的相互作用和Akt信号传导通路的活化由Trop-2特异性地活化。Trop-2过度表达指示对于Akt和CD9抑制剂的灵敏度,所述抑制剂能够选择性地终止表达Trop-2的细胞的生长。这些结果为个人化抗癌疗法的初始模式铺设了道路。
因此,本发明的一个具体实施方式是一种体外预测抗癌疗法的结果的方法,所述的抗癌疗法是用抑制Trop-2信号传导网络的组分的活性的药物,其中所述组分选自由以下物质组成的群组:CD9、Akt和四跨膜蛋白信号传导网络的分子,所述方法包含以下步骤或由以下步骤组成:测定生物样品中Trop-2蛋白质或相应mRNA的表达水平,当检测到Trop-2蛋白质或相应mRNA的表达水平与相应正常组织中的水平相比增加时,有效结果出现。
如通过本发明的作者鉴别的是,四跨膜蛋白信号传导网络的分子属于Trop-2网络,并且还是四跨膜蛋白信号传导网络的一部分的分子,并且具体来说是表皮生长因子受体(EGFR)、酪氨酸蛋白激酶Met、丝氨酸/苏氨酸蛋白激酶c-RAF、原癌基因酪氨酸蛋白激酶Src、小GTP结合蛋白CDC42、酪氨酸蛋白激酶JAK2、cAMP依赖性蛋白激酶催化亚单位α(PKA C-α)、酪氨酸蛋白磷酸酶非受体11型(SHP2)、胰岛素受体底物1(IRS1)、丝氨酸/苏氨酸蛋白激酶PAK 1、丝裂原活化蛋白激酶8(JNK)。
在一个具体实施方式中,生物标记Trop-2的阳性意指肿瘤中的Trop-2mRNA或蛋白质的增加与相同受试者的相应正常组织相比等于或大于10%。在一个具体实施方式中,所述阳性用以选择出于治疗性目的(包括临床试验)使用抗癌药物的患者,所述药物针对Trop-2信号传导网络的组分,包括但不限于CD9、Akt和四跨膜蛋白信号传导网络的分子。因此,本发明教示了一种新颖的生物标记,所述标记特征在于Trop-2的过度表达。这是对特异性疗法的应答的预测性标记,并且是可以鉴别潜在响应性的患者子组的富集标记,并且可以在针对由Trop-2驱动的分子(尤其但不限于CD9和Akt)的药物的临床试验中进行募集。所述生物标记可以单独或与其它生物标记组合使用。
在另一个具体实施方式中,本发明提供一种体外筛选用于治疗或预防癌症或抑制癌细胞生长的候选药物的方法,其中所述药物针对Trop-2信号传导网络的组分,其中所述组分选自由以下物质组成的群组:CD9、Akt和四跨膜蛋白信号传导网络的分子,即,如由本发明的作者鉴别为属于Trop-2网络并且还是四跨膜蛋白信号传导网络的一部分的分子,并且具体来说是表皮生长因子受体(EGFR)、酪氨酸蛋白激酶Met、丝氨酸/苏氨酸蛋白激酶c-RAF、原癌基因酪氨酸蛋白激酶Src、小GTP结合蛋白CDC42、酪氨酸蛋白激酶JAK2、cAMP依赖性蛋白激酶催化亚单位α(PKA C-α)、酪氨酸蛋白磷酸酶非受体11型(SHP2)、胰岛素受体底物1(IRS1)、丝氨酸/苏氨酸蛋白激酶PAK 1、丝裂原活化蛋白激酶8(JNK)。这种方法包含以下步骤:将无菌生理盐水溶液中的待测试化合物给予到表达Trop-2和不表达Trop-2的细胞;将单独的无菌生理盐水溶液平行给予到表达Trop-2和不表达Trop-2的细胞。与化合物接触的不表达细胞用于控制化合物本身的作用的特异性,而与单独的无菌生理盐水溶液接触的表达和不表达Trop-2的细胞用于控制化合物本身的活性。随后,测量化合物的生物活性,即,表达Trop-2的细胞的增殖,将其与经历治疗的不表达Trop-2的细胞以及与不经历治疗(即与单独的生理盐水溶液接触)的表达和不表达Trop-2的细胞相比。最终,选择在表达Trop-2并且经历治疗的细胞中能够减少细胞增殖至少10%的化合物,与不表达Trop-2并且经历治疗的细胞和不经历治疗的细胞的增殖相比。
本发明还提供一种体内筛选用于治疗或预防癌症或抑制癌细胞生长的候选药物的方法,其中所述药物是针对Trop-2信号传导网络的组分,包括但不限于CD9、Akt和四跨膜蛋白信号传导网络的分子。这种方法包含以下步骤:将无菌生理盐水溶液中的待测试化合物给予到临床前模型和临床模型中的对于生物标记Trop-2呈阳性(即过度表达)或阴性(即不过度表达)的肿瘤;将单独的无菌生理盐水溶液平行给予到过度表达或不过度表达Trop-2的肿瘤。与化合物接触的非过度表达肿瘤用于控制化合物本身的作用的特异性,而与单独的无菌生理盐水溶液接触的过度表达或不过度表达Trop-2的肿瘤用于控制化合物本身的活性。随后,测量化合物的生物活性,即过度表达Trop-2的肿瘤的生长,将其与经历治疗的不过度表达Trop-2的肿瘤以及与不经历治疗(即与单独的生理盐水溶液接触)的过度表达或不过度表达Trop-2的肿瘤相比。最终,选择在过度表达Trop-2并且经历治疗的肿瘤中能够减少肿瘤生长至少10%的化合物,其是与经治疗并且未经治疗的对照的肿瘤生长相比。
另外,本发明涉及一种抑制Trop-2信号传导网络的组分的活性的化合物,其中所述组分选自由以下物质组成的群组:CD9、Akt和四跨膜蛋白信号传导网络的分子,其用于治疗Trop-2蛋白质或相应mRNA的表达水平高于正常组织的肿瘤。如上所述,四跨膜蛋白信号传导网络的分子是如由本发明的作者鉴别为属于Trop-2网络并且还是四跨膜蛋白信号传导网络的一部分的分子,并且具体来说是表皮生长因子受体(EGFR)、酪氨酸蛋白激酶Met、丝氨酸/苏氨酸蛋白激酶c-RAF、原癌基因酪氨酸蛋白激酶Src、小GTP结合蛋白CDC42、酪氨酸蛋白激酶JAK2、cAMP依赖性蛋白激酶催化亚单位α(PKA C-α)、酪氨酸蛋白磷酸酶非受体11型(SHP2)、胰岛素受体底物1(IRS1)、丝氨酸/苏氨酸蛋白激酶PAK 1、丝裂原活化蛋白激酶8(JNK)。
一些已经已知的抑制性分子是:MK-2206、A6730、哌立福新、GSK690693、GSK2110183、GDC-0068、AT7867、ARQ092、AZD5363、A-674563、PHT-427、PF-04691502、SureCN1078972、842148-40-7、AC1NX3D3、MLS002702033、SureCN10005574、SureCN1559590、SureCN570829、SH-5、SH-6、和厚朴酚、米替福新、磷酸曲西立滨(Akt抑制剂)、K00598a、DAPH 2、SureCN238877(EGFR抑制剂)、SureCN4269573(EGFR、c-RAF、Src的抑制剂)、达沙替尼(Src抑制剂)、SureCN1518805、1,9-吡唑蒽酮、AS-601245、氨基吡啶衍生物2(JNK抑制剂)。
任选地,以上化合物可以与其它疗法组合使用,所述疗法例如化学疗法、烷化剂(例如二氯甲二乙胺苯丁酸氮芥、环磷酰胺、异环磷酰胺、美法仑、亚硝基脲、白消安、达卡巴嗪(DTIC)、替莫唑胺、噻替派和六甲蜜胺)、抗代谢剂(例如5-氟尿嘧啶(5-FU)、6-巯基嘌呤(6-MP)、卡培他滨、克拉屈滨、氯法拉滨、阿糖胞苷、氟尿苷、氟达拉滨、吉西他滨、羟基脲、甲氨蝶呤、培美曲塞、喷司他汀、硫鸟嘌呤)、抗肿瘤抗生素(例如蒽环霉素、放射菌素-D、博莱霉素、丝裂霉素-C)、拓扑异构酶抑制剂(例如拓扑替康、伊立替康、依托泊苷、替尼泊苷、米托蒽醌)、有丝分裂抑制剂(例如太平洋紫杉醇、多西他赛、伊沙匹隆、长春碱、长春新碱、长春瑞滨、雌莫司汀)、皮质类固醇(例如强的松、甲基泼尼松龙、地塞米松)、分化剂(例如类视黄素、维甲酸/ATRA、贝萨罗汀和三氧化二砷)、激素疗法、靶向激酶抑制剂、蛋白酶体抑制剂硼替佐米。
根据本发明使用的根据本发明的化合物可以选自由以下物质组成的群组:寡核苷酸;充当“显性阴性”分子的对应于CD9、Akt和四跨膜蛋白信号传导网络的分子的工程化分子;单克隆抗体;药理学抑制剂;小分子化学化合物;或其组合。
如上所述,可以获得以上分子的活性的抑制,作为一非限制性实例,借助于充当“显性阴性物”的相应工程化分子,即类似于正常分子但缺乏生物活性的分子;这些显性阴性分子置换了正常分子并且消除了其在细胞过程中的功能。生物活性的抑制还可以通过用与正常分子的催化和/或调节位点结合并且抑制其活性的特异性单克隆抗体获得。生物活性的抑制还可以用药理学抑制剂获得。主要实例之一是MK-2206和A6730,Akt的药理学抑制剂,对于其来说,Trop-2表达构成了根据本发明的抗癌用途的指示。
本发明还涉及能够抑制患者中的肿瘤生长的寡核苷酸(RNA或DNA,双或单链),这种抑制是通过抑制Trop-2的信号传导通路的组分的表达来实现的,这些肿瘤对于生物标记Trop-2是阳性的,所述组分包括但不限于CD9、Akt和四跨膜蛋白信号传导网络的分子。作为一个实例,一种能够抑制CD9表达的寡核苷酸包含在靶向CD9的沉默的RNA(siRNA)内,其中正义链中的靶标序列含于CD9的mRNA(基因库(GenBank)登记号:NM_007657.3)中,包括但不限于以下序列:
正义5'GGTAGCAAGT GCATCAAAT 3'(SEQ ID NO:1),
反义5'ATTTGATGCA CTTGCTACC 3'(SEQ ID NO:2)。
这些沉默的靶标序列还可以与其它序列融合以便获得当插入到适当载体中时转录到发夹RNA的寡核苷酸,其具有以下序列作为一个实例,其中正义和反义定向中的靶标序列加下划线:
正向5'GATCCCCGGT AGCAAGTGCA TCAAATTTCA AGAGAATTTG ATGCACTTGC TACCTTTTTG GAAA 3'(SEQ ID NO:3),
反向5'AGCTTTTCCA AAAAGGTAGC AAGTGCATCA AATTCTCTTGAAATTTGATG CACTTGCTAC CGGG 3'(SEQ ID NO:4)。
作为一个实例,一种抑制AKT表达的方法在于靶向AKT的siRNA,其中正义链中的靶标序列含于AKT的mRNA(NM_009652.3)中,包括但不限于以下序列:
正义5'GCACCTTTAT TGGCTACAA 3'(SEQ ID NO:5),
反义5'TTGTAGCCAA TAAAGGTGC 3'(SEQ ID NO:6),
和转录到发夹RNA的相应寡核苷酸:
正向5'GATCCCCGCA CCTTTATTGG CTACAATTCA AGAGATTGTA GCCAATAAAG GTGCTTTTTC 3'(SEQ ID NO:7),
反向5'TCGAGAAAAA GCACCTTTAT TGGCTACAAT CTCTTGAATT GTAGCCAATA AAGGTGCGGG 3'(SEQ ID NO:8);
或
正义5'GGAAGGTGAT TCTGGTGAA 3'(SEQ ID NO:9),
反义5'TTCACCAGAA TCACCTTCC 3'(SEQ ID NO:10),
和转录到发夹RNA的相应寡核苷酸:
正向5'GATCCCCGGA AGGTGATTCT GGTGAATTCA AGAGATTCAC CAGAATCACC TTCCTTTTTC 3'(SEQ ID NO:11),
反向5'TCGAGAAAAA GGAAGGTGAT TCTGGTGAAT CTCTTGAATT CACCAGAATC ACCTTCCGGG 3'(SEQ ID NO:12)。
根据本发明,序列SEQ ID NO:1到SEQ ID NO:12可以作为单链寡核苷酸或双链寡核苷酸的形式使用。所述序列可以插入克隆载体中。
工业应用和实施方式
本发明可以在临床上用于诊断过度表达Trop-2的肿瘤并且用于治愈带有所述肿瘤的患者,其中Trop-2过度表达构成了生物标记,其引导对于靶向Trop-2信号传导网络的组分的化合物和/或治疗的治疗选择,所述组分包括但不限于CD9、Akt和四跨膜蛋白信号传导网络的分子。本发明的另一个应用在于筛选用于癌症疗法的新药物和疗法,其是使用过度表达Trop-2并且体外和体内对针对属于Trop-2信号传导网络的分子中所鉴别的靶标的物质和/或治疗灵敏的细胞和肿瘤,所述分子具体来说是CD9、Akt和四跨膜蛋白信号传导网络的分子。
肿瘤细胞中与相应对照细胞相比的TROP2 mRNA增加可以在培养物中的细胞和由新鲜或冷冻细胞和组织组成的活体组织切片中进行定量。mRNA可以由本领域技术人员通过应用可用技术来提取和纯化。用于提取和纯化的试剂盒和仪器可以从商业来源获取。如本发明中所述,TROP2 mRNA通过cDNA合成和PCR或实时定量PCR的检测和定量可以根据本领域中已知的技术来进行。用于实时定量RT-PCR的试剂和仪器是市场上可获得的。
肿瘤细胞中与相应对照细胞相比的Trop-2蛋白质增加可以借助于免疫组织化学、ELISA分析、蛋白质印迹法(Western blotting)或本领域中已知的其它等效技术在培养物中的细胞和由新鲜、冷冻或福尔马林固定的细胞和组织组成的活体组织切片中进行定量。用于执行这些测试和测量的Trop-2反应性抗体、试剂盒和设备是市场上可获得的。
永生化鼠类细胞系MTE 4-14(纳凯,勒珀桑等人1989)(Naquet,Lepesantet al.1989)可以根据本领域中已知的技术进行转染。用Trop-2的表达载体和用空载载体转染的MTE 4-14细胞可以体外生长并且用于筛选靶向于Trop-2信号传导网络的组分的化合物,所述化合物包括CD9、Akt和四跨膜蛋白信号传导网络的分子的抑制剂。细胞增殖可以根据本领域中已知的技术通过直接计数或染色用于细胞成分或代谢分析来测量。
待用于体内筛选直接针对Trop-2信号传导网络的组分的化合物的临床前模型由根据本领域中已知的方法进行皮下注射有过度表达或不过度表达Trop-2的细胞(异种移植物)的免疫功能不全小鼠和类似模型组成,所述化合物包括CD9、Akt和四跨膜蛋白信号传导网络的分子的抑制剂。
本发明现将具体参考附后的附图的详细实施例和附图,根据但不限于其一些优选具体实施方式以以说明和实例方式描述。
附图说明
图1:负责生长刺激的Trop-2细胞质尾区决定子。(a)Trop-2的C端部分(aa 275-323)的二级结构预测;带负电的氨基酸加下划线。所用的预测软件在左侧指示,h(以深灰色突出):α-螺旋,e(以黑色突出):β-链;c(以淡灰色突出):连接区(环路)。根据预测的共同二级结构在底部指示,逐残基分数是1到10。预测准确性分数≥80%(cubic.bioc.columbia.edu)。(b)Trop-2细胞质尾区突变体的序列;正常序列(wt)在顶部展示。HIKE域以灰色方框突出显示;假定的丝氨酸磷酸化位点的位置以白色突出。S303A,S322A:丝氨酸303和322分别变为丙氨酸的点突变;E→K:谷氨酸310、313、316和320变为赖氨酸的点突变;ΔΗΙΚΕ:HIKE区缺失;Δcyto:整个细胞质尾区缺失。(c)Trop-2细胞质尾区突变对于Trop-2依赖性生长的作用。结果用空载载体或用wt Trop-2或突变Trop-2转染的MTE 4-14细胞的生长曲线表示。wt与突变Trop-2之间的差异:P<0.0001(双向ANOVA)。星形指示个别时间点的统计显著差异(用于多个比较的邦弗朗尼检验(Bonferroni test)):****≤0.0001。条状物:平均标准差。
图2:Trop-2信号传导簇。Trop-2表达的电子显微镜分析。(a)用结合到金纳米球的抗Trop-2抗体染色的MCF7乳腺癌细胞(黑点)。Trop-2经鉴别在细长绒毛突起和大绒毛中(箭头)并且在分散细胞膜区域中。白色圆圈(直径30nm)集中在个别金颗粒上并且涵盖发现结合到Trop-2的抗体分子的机率最高的区域。条状物:50nm。(b)用结合到免疫过氧化酶的抗Trop-2抗体染色的Trop-2表达细胞(深色颗粒状沉积物)。Trop-2转染的293细胞(i),OVCA-432卵巢癌细胞(ii),MCF-7乳腺癌细胞(iii)。Trop-2定位于分散细胞膜区域(箭头)中,在细胞间接触点处(i插图:放大的接触区域),在绒毛突起中(ii),和在分散细胞膜区域中(iii)。还在胞质内囊泡的层面(箭头)处检测到Trop-2存在。
图3:Trop-2和PKCα与四跨膜蛋白分子和CD9的相互作用。(a-d)对下列结构进行共焦显微镜分析:完整细胞膜(a);显示共帽化的细胞膜区域(b);用Trop-2(a-c,d顶部)或用空载载体(d底部)转染的MTE 4-14细胞的细胞膜的足迹(c,d),同时用与荧光分子结合的抗Trop-2和抗CD9(四跨膜蛋白网的成员)或抗小窝蛋白(阴性对照)(a-c)或抗CD9和抗PKCα(d)染色。共定位的区域由箭头指示。(e)Trop-2、PKCα、CD9和四跨膜蛋白的免疫共沉淀(IP)。使用针对鼠类CD9、CD81和CD151(MTE 4-14细胞)或人类CD9、CD82和CD151(MCF7细胞)的初级抗体进行免疫沉淀反应;免疫沉淀物中Trop-2和PKCα的存在通过蛋白质印迹法检测。用抗Trop-2的IP:阳性对照。(f)Trop-2/胆固醇关联。在用甲基-β-环糊精(MβCD)处理之后培养基中Trop-2的释放(左)。用毛地黄皂苷从BRIJ溶解物的沉淀Trop-2(右)。对用Trop-2转染的MTE4-14细胞进行处理,并且通过蛋白质印迹法评估可溶和不溶部分中Trop-2的存在。
图4:Trop-2蛋白质组。(a)通过蛋白质印迹法测量的MTE 4-14转染子中信号传导分子的量和磷酸化。V:单独的载体;T2:Trop-2。具有四个条带的图展示了两对独立测量。(b)来自用单独的载体(左)或Trop-2(右)转染的MTE 4-14细胞的蛋白质提取物的2D凝胶;圆圈表示通过Trop-2表达抑制(R,左)或诱导(I,右)的蛋白质。
图5:Trop-2信号传导网络。(a)由Trop-2驱动的蛋白质间相互作用的网络(SNOW分析)。圆圈:可以彼此间进行物理上相互作用的由Trop-2调节的所有蛋白质(所鉴别的165种蛋白质中的114种)。矩形:两种蛋白质之间的“连接体”。线(“边缘”)表示蛋白质间接触。(b)由Trop-2驱动的癌症信号传导网络。使用程序Ingenuity Pathway Analysis对由Trop-2调节的蛋白质(呈黑色)进行映射定位。六边形:激酶;梯形:小G蛋白和其相互作用体;矩形:细胞凋亡因子;椭圆形:转录因子/染色质重塑蛋白质;双圆圈:蛋白质复合物。以灰色突出的是信号传导分子,其不是蛋白质。粗灰色线:细胞膜;灰色方框:细胞核和线粒体腔室。P=6.19×10-45。
图6:由Trop-2的控制的Akt信号传导网络。(a)Trop-2/Akt信号传导网络的映射定位。使用Ingenuity Pathway Analysis软件对由Trop-2调节的蛋白质(呈黑色)进行映射定位。六边形:激酶;矩形:细胞凋亡因子;菱形:磷酸酶;椭圆形:转录因子/染色质重塑蛋白质;双圆圈:蛋白质复合物。以灰色突出的是信号传导分子,其不是蛋白质。粗灰色线:细胞膜。(b)通过Trop-2的调节Akt苏氨酸308和丝氨酸473处的磷酸化水平。在存在或不存在PMA下用单独的载体或用Trop-2转染的MTE 4-14细胞的蛋白质印迹分析;对于每个条带,表示相对于对照标准化的强度。
图7:Trop-2影响对CD9抑制剂的灵敏度。(a,b)CD9沉默在MTE4-14/载体和MTE4-14/Trop-2中的效率的控制。(a)在用无效siRNA(对照)或特异性抗CD9siRNA转染之后24小时通过定量实时RT-PCR对CD9的mRNA水平的测量。条状物:测量的标准差。(b)在用无效siRNA(对照)或特异性抗CD9siRNA转染之后48小时对CD9水平的流式细胞计数分析。白色轮廓:未染色的对照抗体;灰色轮廓:结合到Alexa-488的抗CD9抗体。(c)用Trop-2(MTE 4-14/Trop-2、IGROV/Trop-2、KM12-SM/Trop-2)或内源性表达Trop-2(HCT116U5.5、MCF7、HT29)稳定转染的鼠类细胞或人类细胞中的CD9水平的流式细胞计数分析。白色轮廓:未染色的对照抗体;灰色轮廓:结合到Alexa-488的抗CD9抗体。(d)用无效siRNA(对照)或特异性抗CD9或抗Trop-2siRNA转染的MTE4-14/载体(左)和MTE4-14/Trop-2(右)的生长曲线。条状物:平均标准误差。
图8:Trop-2影响对Akt抑制剂的灵敏度。用单独的载体或用Trop-2转染并且使用400nM的剂量或如所指示的不同剂量的无效siRNA(对照)或特异性抗AKT siRNA(左)或用单独的溶剂或Akt的药理学抑制剂(中,右)处理的MTE 4-14细胞的生长曲线。条状物:平均标准误差。
图9:通过Akt的抑制对表达Trop-2的肿瘤的生长进行抑制。用单独的溶剂或用Akt的MK-2206药理学抑制剂处理的用Trop-2转染的Colo205结肠癌细胞(上)或MTE 4-14细胞(下)的体内生长曲线。用空载载体转染的MTE 4-14对照细胞在这个模型中不具有致瘤能力。对照与经治疗的肿瘤之间的差异:P<0.0001(双向ANOVA)。星形表示个别时间点的统计显著差异(用于多个比较的邦弗朗尼校验(Bonferroni adjustment)):*≤0.05,**≤0.01,***≤0.001≤0.001****。条状物:平均标准误差。插图,肿瘤细胞和转染子中的Trop-2表达的流式细胞计数分析:白色轮廓,未染色的对照抗体;灰色轮廓,结合到Alexa-488的抗Trop-2。
实施例
细胞培养
将人类MCF-7和OVCA-432细胞系在RPMI 1640培养基(GibcoBRL,佩斯利,苏格兰)(GibcoBRL,Paisley,Scotland)中生长。将人类293和KM12SC以及鼠类永生化MTE 4-14(纳凯,勒珀桑等人1989)(Naquet,Lepesant et al.1989)和L细胞系维持在DMEM中。所有培养基都补充有10%胎牛血清(GibcoBRL)、100IU/ml青霉素和100μg/ml链霉素(优洛克隆,米兰,意大利)(Euroclone,Milano,Italy)。
DNA转染
将细胞用DNA在脂质体(Lipofectamine)2000或LTX(英杰,卡尔斯巴德,加利福尼亚州)(Invitrogen,Carlsbad,CA)中根据制造商的说明书转染。选择在含G-418的培养基中的稳定转染子。
免疫荧光分析
用与Alexa Fluor-488/546/633结合的指定抗体或用由与AlexaFluor-488/546/633结合的二级抗体检测的相同未结合的抗体对涂布在玻璃盖玻片上的细胞染色。将细胞在固定之前在37℃下用10%FBS在培养基中活染色5分钟,或在固定之后通过在室温下在PBS中与相关抗体一起孵育而染色30分钟。将细胞用4%多聚甲醛在PBS中固定20分钟。用10%FCS、0.1%皂苷进行非特异性相互作用的透化和封闭。随后将载玻片在PBS中洗涤,并且固定以便通过LSM-510META共焦显微镜(蔡司,上科亨,德国)(Zeiss,Oberkochen,Germany).分析。
细胞足迹
“细胞足迹”是在用EDTA分离细胞之后组织培养皿上的细胞膜残余物(哈科莫里2002)(Hakomori 2002)。基本上如所描述进行细胞处理和免疫荧光分析(哈科莫里2002)。
免疫共沉淀分析
将细胞溶解于BRIJ缓冲液(150mM NaCl,50mM Tris-HCl,pH 7.5,1mM MgCl2,1mM CaCl2,1%BRIJ,蛋白酶抑制剂)中。通过在4℃下与蛋白G琼脂糖凝胶(通用电气医疗集团,皮斯卡塔威,新泽西州)(GE Healthcare,Piscataway,NJ)一起孵育1小时使细胞溶解物澄清。将澄清的溶解物在4℃下与针对假定Trop-2相互作用体的初级抗体一起孵育2小时,随后与蛋白G琼脂糖凝胶一起孵育(在4℃下,1小时)。将蛋白质复合物用0.1M甘氨酸在pH 2.5下从树脂上洗脱,并且通过蛋白质印迹法用所指定的特异性抗体探测。
胆固醇从细胞膜的消耗和沉淀
为了消耗细胞膜上的胆固醇,将完整细胞在PBS中洗涤三次以去除血清,并且在37℃下在DMEM中用10mM甲基-β-环糊精孵育45分钟。将对照细胞在不存在甲基-β-环糊精的条件下平行处理。将样品在2000g下离心以去除细胞和细胞碎片。将上清液中的蛋白质沉淀于三氯乙酸中,并且通过蛋白质印迹法分析。
还将细胞通过刮擦而收集并且溶解于具有1%BRIJ的缓冲液中。将上清液与1/10(体积/体积)甲醇(对照)或1/10(体积/体积)甲醇中的10%毛地黄皂苷混合,并且在冰上孵育10分钟。将细胞膜通过在12,000g下离心15分钟而恢复。将团粒在冷丙酮中洗涤,再悬浮于勒姆利缓冲液(Laemmlibuffer)中,并且通过蛋白质印迹法分析。
免疫电子显微术
为进行免疫金电子显微术(EM),将细胞在37℃下固定于4%甲醛、0.05%戊二醛、0.15M HEPES,pH 7.3中10分钟,并且在室温下进行后固定于4%多聚甲醛、0.15M HEPES,pH 7.3中50分钟,如所描述(波利修克,波利修克等人2000)(Polishchuk,Polishchuk et al.2000)。阳离子化金、蛋白A-金和金结合的抗兔抗体(10nm胶态金颗粒)来自英国BioCell(加的夫,英国)(Cardiff,UK)。免疫过氧化酶EM如所描述进行(布朗(Brown)和法科1989)(Brown and Farquhar 1989)。假定抗体分子的平均长度是11nm,进行最小标记面积的估计。
体外细胞生长分析
将用空载载体或用Trop-2转染的MTE 4-14细胞以1.5-3×103个细胞/孔接种于96孔板中(每个数据点五个复孔)。通过MTT比色分析(罗氏分子生物化学品(Roche Molecular Biochemicals),曼海姆(Mannheim),德国)或通过用结晶紫染色来定量细胞数目。将细胞数目针对连续稀释的细胞样品的参考标准曲线进行标准化。
抗体
通过用已经在细菌中产生的重组Trop-2皮下免疫来产生兔多克隆抗Trop-2抗血清(福尔纳罗,戴尔阿尔奇普雷特等人1995)(Fornaro,Dell'Arciprete et al.1995)。将Trop-2反应性抗体通过与固定到NHS-琼脂糖凝胶柱(法玛西,乌普萨拉,瑞典)(Pharmaci,Uppsala,Sweden)上的重组Trop-2结合来纯化,并且用0.2M甘氨酸,在pH 2.5洗脱。山羊抗Trop-2多克隆抗体AF650获自研发系统公司(R&D Systems,Inc.)(明尼阿波利斯,明尼苏达州)(Minneapolis,MN)。针对鼠类分子的其它抗体是:大鼠单克隆抗CD9(sc-18869);兔单克隆抗Akt(ser473)(D9E)(细胞信号技术,马萨诸塞州)(Cell Signal Technology,MA);山羊多克隆抗CD151(sc-18753)、抗磷酸化PKCα(S657)(sc-12356)抗Akt(sc-1618);兔多克隆抗小窝蛋白-1(sc-894)、抗PKCα(sc-208)、抗磷酸化Akt(Thr 308)(sc-16646-R)(圣克鲁兹生物技术,圣克鲁兹,加利福尼亚州)(Santa Cruz Biotechnology,SantaCruz,CA);仓鼠多克隆抗CD81(GTX75430)(基因文本,欧文,加利福尼亚州)(GeneText,Irvine,CA)。针对相应人类分子的小鼠单克隆抗体是:抗CD9(14-0098)、抗CD98(14-0982)(电子生物科学,圣地亚哥,加利福尼亚州)(eBioscience,San Duego,CA)、抗CD151(H00000977-M02)(亚诺法,台湾,中国)(Abnova,Taiwan,China)和抗CD81(TS81)(由F.兰扎(F.Lanza)博士慷慨供应)(generously supplied by Dr.F.Lanza)。二级Alexa Fluor-488/546/633结合的抗体来自英杰。
药理学抑制剂
将细胞用50-100-200-400nM A6730_SIGMA(西格马技术公司,圣路易斯,密苏里州63178-9916)(Sigma Technical Company,St Louis,MO 63178-9916)或用400nM MK-2206(赛莱克化学品,休斯顿,德克萨斯州)(SelleckChemicals,Houston,TX)处理以抑制Akt。将储备溶液于DMSO中制备并且用乙醇或水稀释,DMSO和乙醇的最终浓度分别决不超过0.1%和1%。对照细胞接受单独的媒剂。在接种之后24小时进行第二处理。
体内模型:无胸腺裸小鼠中的异种移植体
将用Trop-2转染的MTE 4-14永生化细胞系(阿尔贝蒂,纽蒂尼等人1994)(Alberti,Nutini et al.1994)或内源性表达Trop-2的Colo205结肠癌细胞皮下注射(4×106个细胞/注射)到裸小鼠组(8周大的雌性CD1-Foxn1 nu/nu小鼠(查尔斯河实验室,卡尔科,莱科,意大利)(Charles River Laboratories,Calco,Lecco,Italy)中。将用Trop-2转染的MTE 4-14细胞与基质胶(低生长因子基质,碧迪生物科学,富兰克林湖,美国新泽西州)(growth factor reducedmatrix,BD Biosciences,Franklin Lakes,NJ USA)以1:3体积/体积的比例共注射。
在使用时将M-2206于DMSO中的储备溶液用不致热生理盐水溶液中稀释到0.7mg/ml的最终浓度,并且从肿瘤变得摸得出的时间开始,将药物以每天每小鼠0.35mg的剂量腹膜内给予。将对照组小鼠用单独的媒剂处理。每5-7天目测检验小鼠,并且测量实验肿瘤的大直径和小直径。使用用于椭球体积的式子(D×d2/2)计算肿瘤体积。用空载载体转染的MTE 4-14细胞在这个模型中不致瘤。涉及动物和其护理的程序按照制度指南、国家法律和国际方案(D.L.第116期,G.U.,增刊40,1992年2月18日;第8期,G.U.,1994年7月;关于实验瘤形成中的动物福利的UKCCCR指南(UKCCCR Guidelinesfor the Welfare of Animals in Experimental Neoplasia);EEC委员会指令(EECCouncil Directive)86/609,OJ L 358.1,1987年12月12日;实验动物的护理和使用指南(Guide for the Care and Use of Laboratory Animals),美国国家研究委员会(United States National Research Council),1996)进行。
抗体介导的共帽化
将细胞使用胰蛋白酶从培养板分离,并且与初级抗Trop-2抗体(T16)一起在冰上孵育20分钟(阿尔贝蒂,苗蒂等人1992)(Alberti,Miotti et al.1992)。在洗涤之后,在37℃下将细胞与二级Alexa Fluor 488结合的抗体一起孵育10分钟,以使靶标分子交联并且诱导抗原-抗体复合物的帽化(利维和肖哈姆2005)(Levy and Shoham 2005)。将‘帽化的’细胞在室温下用1%多聚甲醛固定10分钟。将多聚甲醛用FBS淬灭,并且将细胞用针对所关注的膜蛋白质的抗体染色。
蛋白质印迹分析
如先前所述进行蛋白质印迹法(艾尔斯维迪,福尔纳罗等人1998)(ElSewedy,Fornaro et al.1998)。简单来说,将细胞溶解物通过在变性聚丙烯酰胺凝胶上电泳(SDS-PAGE)来分析,并且转移到硝化纤维素滤膜上。将滤膜与相关初级抗体(艾宾,剑桥,英国;圣克鲁兹)(Abeam,Cambridge,UK;Santa Cruz)一起在具有5%脱脂奶粉的TBS中孵育。将杂交的滤膜在TBS,0.1%Tween-20中进行洗涤。通过化学发光(ECL,安玛西亚,艾尔斯伯里,英国)(ECL,Amersham,Aylesbury,UK)使用辣根过氧化酶结合的抗小鼠或抗兔二级抗体(卡尔生物化学,拉荷亚,加利福尼亚州)(Calbiochem,La Jolla,CA)揭示抗体结合。用NIH-图像1.62、使用柯达(Kodak)灰阶标准功率曲线(www.kodak.com)作为参考来定量信号强度。
质粒
pEYFP表达载体获自克隆科技(Clontech)(帕洛阿尔托,加利福尼亚州)(Palo Alto,CA)。缺乏EYFP的编码序列的相应载体用以表达野生型或诱变处理的Trop-2cDNA。将野生型人类TROP2的编码序列通过PCR用正向引物5'GCGATTCTCG AGTCCGGTCC GCGTTCC 3'(SEQ ID NO:13)和反向引物5'GCGCCGGTAC CAAGCTCGGT TCCTTTC 3'(SEQ ID NO:14)扩增,并且亚克隆到pEYFP-N1载体中。表达载体pCMV-SPORT6和CD316pCMV-SPORT6获自伊马基因(Imagenes)(柏林,德国)(Berlin,Germany)。按照标准程序构筑CD9(EcoRI/BamHI)、CD316(EcoRI/AgeI)与mCherry之间的嵌合蛋白。
siRNA
按照根据以下的程序使用四种设计策略:突施尔准则(Tuschl criteria)(艾尔巴希尔,哈博特等人2001)(Elbashir,Harborth et al.2001):英杰(rnaidesigner.invitrogen.com/rnaiexpress/),怀特黑德研究所(WhiteheadInstitute)网站(jura.wi.mit.edu/bioc/siRNAext/)(塞米扎罗夫,弗罗斯特等人2003)(Semizarov,Frost et al.2003);松哈默(Sonnhammer)(sonnhammer.cgb.ki.se/siSearch)(查克,瓦勒斯泰特等人2004)(Chalk,Wahlestedt et al.2004)。所选择的siRNA是通过以上方法中的超过一者鉴别或由其中的至少一者视为最优的siRNA。对所关注的基因具有特异性的来自相应siRNA的发夹RNA(shRNA)的编码序列(艾尔巴希尔,哈博特等人2001)(Elbashir,Harborth et al.2001)已经在针对RNA聚合酶-III H1基因的启动子的控制下亚克隆于pSUPER载体(布鲁梅尔坎普,伯纳兹等人2002)(Brummelkamp,Bernards et al.2002)中。借助于抗CD9siRNA,通过靶标正义序列5'GGTAGCAAGT GCATCAAAT 3'(SEQ ID NO:1)和反义序列5'ATTTGATGCA CTTGCTACC 3'(SEQ ID NO:2)获得CD9的抑制,作为一个实例包括于以下序列中用于转录发夹RNA,其中正义和反义定向中的靶标序列加下划线:
正向5'GATCCCCGGT AGCAAGTGCA TCAAATTTCA AGAGAATTTG ATGCACTTGC TACCTTTTTG GAAA 3'(SEQ ID NO:3),
反向5'AGCTTTTCCA AAAAGGTAGC AAGTGCATCA AATTCTCTTGAAATTTGATG CACTTGCTAC CGGG 3'(SEQ ID NO:4)。
通过借助于抗AKT siRNA已经获得Akt抑制,靶标正义序列5'GCACCTTTAT TGGCTACAA 3'(SEQ ID NO:5)和反义序列5'TTGTAGCCAA TAAAGGTGC 3'(SEQ ID NO:6)作为一个实例包括于以下序列中用于转录发夹RNA:
正向5'GATCCCCGCA CCTTTATTGG CTACAATTCA AGAGATTGTA GCCAATAAAG GTGCTTTTTC 3'(SEQ ID NO:7),
反向5'TCGAGAAAAA GCACCTTTAT TGGCTACAAT CTCTTGAATT GTAGCCAATA AAGGTGCGGG 3'(SEQ ID NO:8);
或靶标正义序列5'GGAAGGTGAT TCTGGTGAA 3'(SEQ ID NO:9)、反义序列5'TTCACCAGAA TCACCTTCC 3'(SEQ ID NO:10)作为一个实例包括于以下序列中用于转录发夹RNA:
正向5'GATCCCCGGA AGGTGATTCT GGTGAATTCA AGAGATTCAC CAGAATCACC TTCCTTTTTC 3'(SEQ ID NO:11),
反向5'TCGAGAAAAA GGAAGGTGAT TCTGGTGAAT CTCTTGAATT CACCAGAATC ACCTTCCGGG 3'(SEQ ID NO:12)。
将相应构筑体瞬时转染到MTE 4-14细胞中,并且评估其对于细胞增殖的作用。通过实时RT-PCR定量沉默之后的转录水平。针对无关靶标的siRNA用作阴性对照,在充分校验对于细胞生长不存在假性作用之后进行选择。以下给出相应序列;对于每种siRNA,用根据参考序列(RefSeq)数据库(基因库)的标识码指定相应基因。
针对鼠类Co-029/Tspan8的siRna(NM_146010.2)用作人类细胞的阴性对照,其具有靶标序列正义5'CTTTCAAACC TGAGTATAA 3'(SEQ ID NO:15)、反义5'TTATACTCAG GTTTGAAAG 3'(SEQ ID NO:16)作为一个实例包括于以下序列中用于转录发夹RNA:
正向5'GATCCCCCTT TCAAACCTGA GTATAATTCA AGAGATTATA CTCAGGTTTG AAAGTTTTTG GAAA 3'(SEQ ID NO:17),
反向5'AGCTTTTCCA AAAACTTTCA AACCTGAGTA TAATCTCTTGAATTATACTC AGGTTTGAAA GGGG 3'(SEQ ID NO:18)。
针对人类CD133的siRNA(NM_006017.2)用作鼠类细胞的阴性对照,其具有靶标序列正义5'CTTGACAACG TTAATAACG 3'(SEQ ID NO:19)、反义5'CGTTATTAAC GTTGTCAAG 3'(SEQ ID NO:20)作为一个实例包括于以下序列中用于转录发夹RNA:
正向5'GATCCCCCTT GACAACGTTA ATAACGTTCA AGAGACGTTA TTAACGTTGT CAAGTTTTTG GAAA 3'(SEQ ID NO:21),
反向5'AGCTTTTCCA AAAACTTGAC AACGTTAATA ACGTCTCTTGAACGTTATTA ACGTTGTCAA GGGG 3'(SEQ ID NO:22)。
针对人类CD316的siRNA(NM_052868.2)用作鼠类细胞的阴性对照,其具有靶标序列正义5'TGCAGGAAGTG GTGGGAAT 3'(SEQ ID NO:23)、反义5'ATTCCCACCA CTTCCTGCA 3'(SEQ ID NO:24)作为一个实例包括于以下序列中用于转录发夹RNA:
正向5'GATCCCCTGC AGGAAGTGGT GGGAATTTCA AGAGAATTCC CACCACTTCC TGCATTTTTG GAAA 3'(SEQ ID NO:25),
反向5'AGCTTTTCCA AAAATGCAGG AAGTGGTGGG AATTCTCTTGAAATTCCCAC CACTTCCTGC AGGG 3'(SEQ ID NO:26)。
如先前所述获得TROP2沉默(特雷罗托拉,坎它内利等人2013)(Trerotola,Cantanelli et al.2013)。
Trop-2的细胞质域的诱变
通过PCR用以下引物获得Trop-2的细胞质域的突变体:
Trop-2 Δcyto
正向5'CTCGGATCCA CCATGGCTCG GGGCCCC 3'(SEQ ID NO:27),
反向5'CTCGAATTCC CGGTTGGTGA TCACCAG 3'(SEQ ID NO:28)。
Trop-2E→K
正向5'GCGGAATTCC GTCCGGTCCG CGTTCCT 3'(SEQ ID NO:29),
反向5'GCGTCTAGAC TACAAGCTCG GTTTCTTTCT CAACTTCCCCAGTTTCTTGA TCTTCACCTT CTTG 3'(SEQ ID NO:30)。
Trop-2 ΔΗΙΚΕ
正向5'GCGGAATTCC GTCCGGTCCG CGTTCCT 3'(SEQ ID NO:31),
反向5'GCGTCTAGAC TACAAGCTCG GTTCCTTTCT CAACTCCCCCAGTTCCTTGA TCTCCACTCT CCGGTTGGTG ATCAC 3'(SEQ ID NO:32)。
Trop-2 S303A
正向5'CTCGGATCCA CCATGGCTCG GGGCCCC 3'(SEQ ID NO:33),
反向5'GCGTCTAGAC TACAAGCTCG GTTCCTTTCT CAACTCCCCCAGTTCCTTGA TCTCCACCTT CTTGTACTTC CCCGCCTTTC TCCGG 3'(SEQ ID NO:34)。
Trop-2 S322A
正向5'CTCGGATCCA CCATGGCTCG GGGCCCC 3'(SEQ ID NO:35),
反向5'GCGTCTAGAC TACAAGGCCG GTTCCTTTCT CAACTCCCC 3'(SEQ ID NO:36)。
对所有扩增区域完全测序以验证通过Taq聚合酶诱导的突变的不存在。
实时定量RT-PCR
在Trizol(英杰)中按照制造商的说明书从指定细胞系中提取总RNA。将一μg总RNA根据标准方案使用ImProm-II逆转录酶(普洛麦格(Promega))用于每个逆转录(RT)反应。使用ABI-PRISM 7900HT序列检测系统(PE应用生物系统,福斯特市,加利福尼亚州)(PE Applied Biosystems,Foster City,CA)用特异性引物(Hs99999905;CD9:Mm00514275_g1;B2M:Mm00437762_m1)(应用生物系统)和相应(罗氏(Roche))荧光探针根据制造商说明书进行实时定量PCR反应(朱列蒂,奥弗伯格等人2001)(Giulietti,Overbergh et al.2001)。一式三份地分析每种样品,并且将2-ΔΔCT方法用以计算基因表达的相对变化(利瓦克和施米特根2001)(Livak andSchmittgen 2001)。使用1.834的更准确基准(格拉,特雷罗托拉等人2008)(Guerra,Trerotola et al.2008),其需要1.1个周期来使扩增物质加倍。GAPDH和B2M管家基因用作内部控制。对于每种cDNA,计算ΔCT(CT,靶标基因-CT,GAPDH/B2M):使用最小二乘线性回归分析。由于扩增效率在所用RNA量的范围内是线性的,所以将扩增曲线用以计算经siRNA处理的样品的交叉点值。通过使用非逆转录的RNA作为PCR反应的模板,常规地评估每种样品的基因组DNA污染。
2D凝胶和质谱法
通过如所描述进行的2D-PAGE获得整个基因组的幻像(瑞士生物信息学研究所(Swiss Institute of Bioinformatics,SIB),http://us.expasy.org/ch2d/protocols/)。在约克大学(University of York)进行质谱分析(www.york.ac.uk/depts/biol/tf/proteomics/)。
Trop-2信号传导网络的通路分析和鉴别
使用SNOW(snow.bioinfo.cipf.es/cgi-bin/snow.cgi)和Ingenuity PathwaysAnalysis(IPA,Ingenuity系统,www.ingenuity.com)生物信息学工具分析Trop-2的蛋白质组。SNOW(snow.bioinfo.cipf.es/cgi-bin/snow.cgi)构建了蛋白质间相互作用的网络,使蛋白质映射定位于参考相互作用组上。参考相互作用组是一组逐对直接蛋白质相互作用,其中节点是蛋白质本身并且连接边缘是相互作用事件。使用HPRD(人类蛋白质参考数据库)、IntAct、BIND(生物分子相互作用网络数据库)、DIP(相互作用蛋白质的数据库)和MINT(分子相互作用数据库)数据库构建Trop-2依赖性相互作用组。将这些数据库用以构建最小连接网络(MCN)。基于以下评估拓扑网络:节点连接程度(即每个节点边缘的数目);中间性(即节点向心性及其分布的量度);簇聚系数分布(即每个节点的邻近的连接性);网络的组分的数目(即在网络分析中产生的节点的不同群组);双组分(即通过单一边缘连接到另一个节点群组的节点群组的数目);和关节点(即在网络中加入双组分的边缘)。使用迪杰斯特拉(Dijkstra)算法、应用如下严格选项来鉴别MCN:允许在所分析的任何输入蛋白质对之间引入仅一个外部连接蛋白质。以这种方式,可能获得将经历分析的由Trop-2调节的165种蛋白质中的114种与22种连接体组合的单一网络。相较于整个参考相互作用组上产生的网络并且相较于随机组成的网络验证来说具有统计上的显著性。为了使用Ingenuity PathwaysAnalysis软件分析,将表示为Trop-2与对照转染子之间的标准化平均信号强度之间的比率的蛋白质表达值转化为倍数变化值,其中对于0与1之间的值,获取负倒数(-1/x)。在过滤数据集时,不设定临限取舍点。将所有分子(Swiss-Prot标识符)映射定位到Ingenuity Knowledge Base上作为聚焦点。Ingenuity Knowledge Base将蛋白质相互作用组织为实体格式,如通过人工管理从公开的实验结果提取。通过使特定连接性最大化来产生聚焦分子的网络,所述特定连接性是即分子之间的实际互连性相较于Knowledge Base中的所有分子的互连性。通过分数(通过右尾费舍尔精确检验(Fisher's exact test)计算的p值的负对数)对网络分级,其考虑网络中的合格分子的数目和其最终大小以及输入数据集大小和包括于网络中的Ingenuity Knowledge Base中的分子的总数目。分数越高,随机相互作用网络的机率越低。随后将聚焦分子映射定位到Ingenuity典型路径上。
统计分析
使用双向ANOVA检验来比较肿瘤生长曲线(罗西,迪丽娜等人2008)(Rossi,Di Lena et al.2008)。
参考文献
Alberti,S.(1999)."HIKE,a candidate protein binding site for PH domains,is amajor regulatory region of Gbeta proteins."Proteins 35:360-363.(阿尔贝蒂S.(1999).“HIKE,PH域的候选蛋白结合位点是Gβ蛋白的主要调节区。”蛋白 质35:360-363。)
Alberti,S.,S.Miotti,et al.(1992)."Biochemical characterization of Trop-2,acell surface molecule expressed by human carcinomas:formal proof that themonoclonal antibodies T16 and MOv-16 recognize Trop-2."Hvbridoma 11:539-5.(阿尔贝蒂S.,S.苗蒂等人(1992).“Trop-2的生物化学表征,一种由人类癌表达的细胞表面分子:单克隆抗体T16和MOv-16识别Trop-2的正式证据。”融合瘤11:539-5。)
Albert.,S.,M.Nutini,et al.(1994)."DNA methylation prevents theamplification of TROP1,a tumor associated cell surface antigen gene."Proc.Natl. Acad.Sci.USA 91:5833-7.(阿尔贝蒂S.,M.纽蒂尼等人(1994).“DNA甲基化防止TROP1的扩增,一种肿瘤相关的细胞表面抗原基因。”美国国家科学院 院刊91:5833-7。
Balzar,M.,I.H.Briaire-de Bruijn,et al.(2001)."Epidermal growth factor-likerepeats mediate lateral and reciprocal interactions of Ep-CAM molecules inhomophilic adhesions."Mol.Cell.Biol.21(7):2570-80.(巴尔扎尔M.,I.H.布里亚尔-德布鲁因等人(2001).“表皮生长因子样重复介导同嗜性粘着中的Ep-CAM分子的横向和交互相互作用。”分子与细胞生物学21(7):2570-80。
Barreiro,O.,M.Zamai,et al.(2008)."Endothelial adhesion receptors arerecruited to adherent leukocytes by inclusion in preformed tetraspaninnanoplatforms."J Cell Biol 183(3):527-42.(巴雷罗O.,M.扎迈等人(2008).“内皮粘附受体通过包括于预先形成的四跨膜蛋白纳米平台而募集到粘附白细胞。”细胞生物学杂志183(3):527-42。
Basu,A.,D.M.Goldenberg,et al.(1995)."The epithelial/carcinoma antigenEGP-1,recognized by monoclonal antibody RS7-3G11,is phosphorylated on serine303."Int.J.Cancer 62:472-479.(巴苏,A.,D.M.戈登伯格等人(1995).“由单克隆抗体RS7-3G11识别的上皮/癌抗原EGP-1在丝氨酸303上磷酸化。”国 际癌症杂志62:472-479。
Brown,W.J.and M.G.Farquhar(1989)."Immunoperoxidase methods for thelocalization of antigens in cultured cells and tissue sections by electron microscopy."Methods Cell Biol 31:553-69.(布朗,W.J.和M.G.法科(1989).“通过电子显微术定位培养的细胞和组织切片中的抗原的免疫过氧化酶方法。”细胞生物学 方法31:553-69。)
Brummelkamp,T.R.,R.Bernards,et al.(2002)."A System for StableExpression of Short Interfering RNAs in Mammalian Cells."Science 296(5567):550-553.(布鲁梅尔坎普T.R.,R.伯纳兹等人(2002).“一种用于在哺乳动物细胞中稳定表达短干扰RNA的系统。”科学296(5567):550-553。)
Chalk,A.M.,C.Wahlestedt,et al.(2004)."Improved and automated predictionof effective siRNA."Biochem Biophvs Res Commun 319(1):264-74.(查克A.M.,C.瓦勒斯泰特等人(2004).“有效siRNA的改进和自动化预测。”生物化学与 生物物理研究通讯(Biochem Biophys Res Commun)319(1):264-74。)
Chong,J.M.and D.W.Speicher(2001)."Determination of Disulfide BondAssignments and N-Glycosylation Sites of the Human Gastrointestinal CarcinomaAntigen GA733-2(CO 17-1A,EGP,KS1-4,KSA,and Ep-CAM)."J.Biol.Chem.276(8):5804-5813.(庄J.M.和D.W.施派歇尔(2001).“测定人类胃肠道癌抗原GA733-2(CO17-1A、EGP、KS1-4、KSA和Ep-CAM)的二硫键分配和N-糖基化位点。”生物化学杂志276(8):5804-5813。)
Ciccarelli,F.,A.Acciarito,et al.(2000)."Large and diverse numbers of humandiseases with HIKE mutations."Hum.Mol.Genet.9:1001-7.(奇卡雷利F.,A.阿恰里托等人(2000).“庞大而多样化的具有HIKE突变的人类疾病。”人类分 子遗传学9:1001-7。)
Cubas,R.,S.Zhang,et al.(2010)."Trop2expression contributes to tumorpathogenesis by activating the ERK MAPK pathway."Mol Cancer 9(1):253.(古巴斯R.,S.张等人(2010).“Trop2表达对通过活化ERK MAPK通路而促进肿瘤的发病机制。”分子癌症9(1):253。)
El Sewedy,T.,M.Fornaro,et al.(1998)."Cloning of the murine Trop2 gene:conservation of a PIP2-binding sequence in the cytoplasmic domain of Trop-2."Int J Cancer 75(2):324-30.(艾尔斯维迪,T.,M.福尔纳罗等人(1998).“鼠类Trop2基因的克隆:Trop-2的细胞质域中的PIP2结合序列的保守。”国际癌症杂志75(2):324-30。)
Elbashir,S.M.,J.Harborth,et al.(2001)."Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells."Nature 411(6836):494-8.(艾尔巴希尔S.M.,J.哈博特等人(2001).“21-核苷酸RNA的双螺旋介导人造哺乳动物细胞中的RNA干扰。”自然411(6836):494-8。)
Fornaro,M.,R.Dell'Arciprete,et al.(1995)."Cloning of the gene encodingTROP-2,a cell-surface glycoprotein expressed by human carcinomas."Int.J.Cancer62:610-8.(福尔纳罗M.,R.戴尔阿尔奇普雷特等人(1995).“编码Trop-2的基因的克隆,一种由人类癌瘤表达的细胞表面糖蛋白。”国际癌症杂志62:610-8。)
Freeley,M.,J.Park,et al.(2007)."Loss of PTEN expression does not contributeto PDK-1activity and PKC activation-loop phosphorylation in Jurkat leukaemic Tcells."Cell Signal 19(12):2444-57.(弗雷利M.,J.帕克等人(2007).“PTEN表达的损失不会有助于Jurkat白血病性T细胞中的PDK-1活性和PKC活化环路磷酸化。”细胞信号19(12):2444-57。)
Giulietti,A.,L.Overbergh,et al.(2001)."An overview of real-time quantitativePCR:applications to quantify cytokine gene expression."Methods 25(4):386-401.(朱列蒂A.,L.奥弗伯格等人(2001).“实时定量PCR的概述:用以定量细胞因子基因表达的应用。”方法25(4):386-401。)
Goldstein,A.S.,T.Stoyanova,et al.(2010)."Primitive origins of prostatecancer:In vivo evidence for prostate-regenerating cells and prostate cancer-initiatingcells."Mol Oncol.(戈德斯坦A.S.,T.斯托亚诺娃等人(2010).“前列腺癌的原始来源:前列腺再生细胞和前列腺癌引发细胞的体内证据。”分子肿瘤学(Mol Oncol)。)
Guerra,E.,M.Trerotola,et al.(2013)."The Trop-2signalling network in cancergrowth."Oncogene doi:10.1038/onc.2012.151.[Epub ahead of print].(格拉E.,M.特雷罗托拉等人(2013).“癌症生长中的Trop-2信号传导网络。”致癌基因doi:10.1038/onc.2012.151.[印刷前电子出版]。)
Guerra,E.,M.Trerotola,et al.(2008)."A bi-cistronic CYCLIN D1-TROP2mRNA chimera demonstrates a novel oncogenic mechanism in human cancer."Cancer Res 68(19):8113-8121.(格拉E.,M.特雷罗托拉等人(2008).“双顺反子CYCLIN D1-TROP2 mRNA嵌合体展示了人类癌症的一种新颖的致癌机制。”癌症研究68(19):8113-8121。
Hakomori,S.I.(2002)."Inaugural Article:The glycosynapse."Proc Natl Acad Sci USA 99(1):225-32.(哈科莫里S.I.(2002).“就职论文:糖突触。”美国国 家科学院院刊99(1):225-32。)
Hemler,M.E.(2003)."Tetraspanin proteins mediate cellular penetration,invasion,and fusion events and define a novel type of membrane microdomain."Annu Rev Cell Dev Biol 19:397-422.(赫姆勒M.E.(2003).“四跨膜蛋白蛋白质介导细胞穿透、侵袭和融合事件并且定义一类新颖的细胞膜微结构域。”细 胞与发育生物学年评19:397-422。)
Jacinto,E.,V.Facchinetti,et al.(2006)."SIN1/MIP1maintains rictor-mTORcomplex integrity and regulates Akt phosphorylation and substrate specificity."Cell127(1):125-37.(雅辛托E.,V.法奇内提等人(2006).“SIN1/MIP1维持rictor-mTOR复合物完整性并且调节Akt磷酸化和底物特异性。”细胞127(1):125-37。)
Klein,C.E.,B.Hartmann,et al.(1990)."Expression of 38-kD cell-surfaceglycoprotein in transformed human keratinocyte cell lines,basal cell carcinomas,andepithelial germs."J.Invest.Dermatol.95:74-82.(克莱因C.E.,B.哈特曼等人(1990).“38-kD细胞表面糖蛋白在转型的人类角质细胞细胞系、基细胞癌和上皮生殖细胞中的表达。”研究性皮肤病学杂志95:74-82。)
Koul,D.,R.Shen,et al.(2005)."Targeting integrin-linked kinase inhibits Aktsignaling pathways and decreases tumor progression of human glioblastoma."Mol Cancer Ther 4(11):1681-8.(库尔D.,R.沈等人(2005).“靶向整合素连接的激酶抑制Akt信号传导通路并且降低人类成胶质细胞瘤的肿瘤进展。”分子癌 症治疗学4(11):1681-8。)
Le Naour,F.,M.Andre,et al.(2006)."Membrane microdomains andproteomics:lessons from tetraspanin microdomains and comparison with lipid rafts."Proteomics 6(24):6447-54.(勒纳乌尔,F.,M.安德烈等人(2006).“细胞膜微结构域和蛋白质组学:来自四跨膜蛋白微结构域和与脂质筏比较的启示。”蛋 白质组学6(24):6447-54。)
Levy,S.and T.Shoham(2005)."The tetraspanin web modulates immune-signalling complexes."Nat Rev Immunol 5(2):136-48.(利维S.和T.肖哈姆(2005).“四跨膜蛋白网调节免疫信号传导复合物。”自然免疫学综述5(2):136-48。)
Linnenbach,A.J.,B.A.Seng,et al.(1993)."Retroposition in a family ofcarcinoma-associated antigen genes."Mol.Cell.Biol.13:1507-1515.(林嫩巴赫A.J.,B.A.森等人(1993).“癌瘤相关的抗原基因的家族中的逆转录转座。”分子 与细胞生物学13:1507-1515。)
Livak,K.J.and T.D.Schmittgen(2001)."Analysis of relative gene expressiondata using real-time quantitative PCR and the 2(-Delta Delta C(T))Method."Methods 25(4):402-8.(利瓦克K.J.和T.D.施米特根(2001).“使用实时定量PCR和2(-ΔΔC(T))方法对相对基因表达数据进行分析。”方法25(4):402-8。)
Minguez,P.,S.Gotz,et al.(2009)."SNOW,a web-based tool for the statisticalanalysis of protein-protein interaction networks."Nucleic Acids Res 37(Web Serverissue):W 109-14.(明格斯P.,S.戈茨等人(2009).“SNOW,一种用于对蛋白质间相互作用网络进行统计分析的Web化的工具。”核酸研究37(网络服务器期号):W 109-14。)
Naquet,P.,H.Lepesant,et al.(1989)."Establishment and characterization ofmouse thymic epithelial cell lines."Thymus 13(3-4):217-26.(纳凯P.,H.勒珀桑等人(1989).“小鼠胸腺上皮细胞系的建立和表征。”胸腺13(3-4):217-26。)
Parekh,D.B.,W.Ziegler,et al.(2000)."Multiple pathways control proteinkinase C phosphorylation."EMBO J 19(4):496-503.(帕雷克D.B.,W.齐格勒等人(2000).“多个通路控制蛋白激酶C的磷酸化。”欧洲分子生物学杂志(EMBO J)19(4):496-503。)
Polishchuk,R.S.,E.V.Polishchuk,et al.(2000)."Correlative light-electronmicroscopy reveals the saccular-tabular ultrastructure of carriers operating betweenthe Golgi apparatus and the plasma membrane."J.Cell Biol.148:45-58.(波利修克R.S.,E.V.波利修克等人(2000).“相关光电子显微术揭示了在Golgi设备与质膜之间操作的载剂的囊片状超微结构。”细胞生物学杂志(J.Cell Biol.)148:45-58。)
Rosse,C,M.Linch,et al.(2010)."PKC and the control of localized signaldynamics."Nat Rev Mol Cell Biol 11(2):103-12.(罗斯C,M.林奇等人(2010).“定位信号动力学的PKC和控制。”自然分子与细胞生物学综述(Nat Rev Mol Cell Biol)11(2):103-12。)
Rossi,C,A.Di Lena,et al.(2008)."Intestinal tumour chemoprevention with theantioxidant lipoic acid stimulates the growth of breast cancer."Eur J Cancer 44:2696-2704.(罗西C,A.迪丽娜等人(2008).“用抗氧化剂硫辛酸刺激了乳腺癌的生长的肠道肿瘤化学预防。”欧洲癌症杂志(Eur J Cancer)44:2696-2704。)
Sarbassov,D.D.,D.A.Guertin,et al.(2005)."Phosphorylation and regulationof Akt/PKB by the rictor-mTOR complex."Science 307(5712):1098-101.(萨尔巴索夫D.D.,D.A.古尔汀等人(2005).“Akt/PKB通过rictor-mTOR复合物的磷酸化和调节。”科学307(5712):1098-101。)
Semizarov,D.,L.Frost,et al.(2003)."Specificity of short interfering RNAdetermined through gene expression signatures."Proc Natl Acad Sci USA100(11):6347-52.(塞米扎罗夫D.,L.弗罗斯特等人(2003).“通过基因表达特征确定的短干扰RNA的特异性。”美国国家科学院院刊100(11):6347-52。)
Trerotola,M.,P.Cantanelli,et al.(2013)."Up-regulation of Trop-2quantitatively stimulates human cancer growth."Oncogene 32 222-233.(特雷罗托拉M.,P.坎它内利等人(2013).“Trop-2的上调定量刺激人类癌症生长。”致癌 基因32222-233。)
Trerotola,M.,S.Rathore,et al.(2010)."CD133,Trop-2and alpha2betal integrinsurface receptors as markers of putative human prostate cancer stem cells."Am J Transl Res 2(2):135-44.(特雷罗托拉M.,S.拉索等人(2010).“作为假定的人类前列腺癌干细胞的标记的CD133、Trop-2和α2β1整合素表面受体。”美国转 译研究杂志(Am J Transl Res)2(2):135-44。)
Wang,J.,R.Day,et al.(2008)."Identification of Trop-2as an oncogene and anattractive therapeutic target in colon cancers."Mol Cancer Ther 7(2):280-5.(王J.,R.戴等人(2008).“Trop-2鉴别为致癌基因和结肠癌中的诱人治疗靶标。”分子 癌症治疗学7(2):280-5。)
Wen,H.C,W.C.Huang,et al.(2003)."Negative regulation ofphosphatidylinositol 3-kinase and Akt signalling pathway by PKC."Cell Signal15(1):37-45.(温H.C,W.C.黄等人(2003).“磷脂酰肌醇3-激酶和Akt信号传导通路通过PKC的负调节。”细胞信号15(1):37-45。)
Zhang,X.A.,A.L.Bontrager,et al.(2001)."Transmembrane-4 superfamilyproteins associate with activated protein kinase C(PKC)and link PKC to specificbeta(l)integrins."J Biol Chem 276(27):25005-13.(张X.A.,A.L.邦特拉杰等人(2001).“跨膜-4超家族蛋白质与活化蛋白激酶C(PKC)相关并且使PKC连接到特定性β(1)整合素。”生物化学杂志276(27):25005-13。)
Claims (10)
1.一种体外预测抗癌疗法的结果的方法,所述抗癌疗法是用抑制Trop-2信号传导网络组分的活性的药物治疗癌症,其中所述组分选自由以下物质组成的群组:CD9、Akt和四跨膜蛋白信号传导网络分子,所述方法包含以下步骤或由以下步骤组成:测定生物样品中Trop-2蛋白质或相应mRNA的表达水平,当检测到Trop-2蛋白质或所述相应mRNA的所述表达水平与相应正常组织中的水平相比增加时,有效结果出现。
2.根据权利要求1所述的方法,其中如借助于RT-PCR或实时定量RT-PCR所定量,肿瘤组织中Trop-2的mRNA与相应正常组织相比的增加等于或大于10%。
3.根据权利要求1所述的方法,其中如借助于免疫组织化学、ELISA分析、蛋白质印迹法定量,肿瘤组织中Trop-2蛋白质与相应正常组织相比的增加等于或大于10%。
4.一种体外筛选用于治疗或预防癌症或抑制癌细胞生长的候选药物的方法,其中所述药物针对Trop-2信号传导网络组分,其中所述组分选自由以下物质组成的群组:CD9、Akt和四跨膜蛋白信号传导网络分子,所述方法包含以下步骤或由以下步骤组成:
a.将无菌生理盐水溶液中的待测试化合物给予到表达Trop-2和不表达Trop-2的细胞,其中所述不表达细胞充当特异性的对照;将单独的无菌生理盐水溶液平行给予到表达Trop-2和不表达Trop-2的细胞(未经治疗的对照),其中所述单独的无菌生理盐水溶液充当活性的对照;
b.检测步骤(a)的化合物在表达Trop-2的细胞的生物活性,将其与不表达Trop-2的经治疗的对照以及表达和不表达Trop-2的未经治疗的对照相比,其中所述生物活性是表达Trop-2的细胞的细胞增殖减少,其是与不表达Trop-2的经治疗的对照细胞相比以及与表达和不表达Trop-2的未经治疗的对照细胞相比;
c.选择步骤(a)的在表达Trop-2的经治疗的细胞中细胞增殖减少至少10%的化合物,其是与不表达Trop-2的经治疗的细胞以及表达和不表达Trop-2的未经治疗的细胞中的细胞增殖相比。
5.一种抑制Trop-2信号传导网络的组分的活性的化合物,其中所述组分选自由以下物质组成的群组:CD9、Akt和四跨膜蛋白信号传导网络的分子,其用于治疗Trop-2蛋白质或相应mRNA表达水平高于正常组织的肿瘤。
6.根据权利要求5所述的化合物,其在根据权利要求5应用,其中所述化合物选自由以下物质组成的群组:寡核苷酸;充当“显性阴性”分子的对应于CD9、Akt和所述四跨膜蛋白信号传导网络的分子的工程化分子;单克隆抗体;药理学抑制剂;小分子化学化合物;或其组合。
7.根据权利要求5到6中任一项所述的化合物,其在根据权利要求5到6中任一项所述的应用,其中所述化合物选自由以下物质组成的群组:MK-2206、A6730、哌立福新、GSK690693、GSK2110183、GDC-0068、AT7867、ARQ092、AZD5363、A-674563、PHT-427、PF-04691502、SureCN1078972、842148-40-7、AC1NX3D3、MLS002702033、SureCN10005574、SureCN1559590、SureCN570829、SH-5、SH-6、和厚朴酚、米替福新、磷酸曲西立滨(Akt抑制剂)、K00598a、DAPH 2、SureCN238877(EGFR抑制剂)、SureCN4269573(EGFR、c-RAF、Src的抑制剂)、达沙替尼(Src抑制剂)、SureCN1518805、1,9-吡唑蒽酮、AS-601245、氨基吡啶衍生物2(JNK抑制剂)。
8.根据权利要求5到7中任一项所述的化合物,其在根据权利要求5到7中任一项所述的应用,其与选自由以下物质组成的群组的药物组合:化学治疗药物、烷基化剂、抗代谢物、抗肿瘤抗生素、拓扑异构酶抑制剂、有丝分裂抑制剂、皮质类固醇、分化剂、激素疗法、靶向激酶抑制剂、蛋白酶体抑制剂硼替佐米。
9.根据权利要求5到8中任一项所述的化合物,其在根据权利要求5到8中任一项所述的应用,其中所述四跨膜蛋白信号传导网络分子选自由以下分子组成的群组:表皮生长因子受体(EGFR)、酪氨酸蛋白激酶Met、丝氨酸/苏氨酸蛋白激酶c-RAF、原癌基因酪氨酸蛋白激酶Src、小GTP结合蛋白CDC42、酪氨酸蛋白激酶JAK2、cAMP依赖性蛋白激酶催化亚单位α(PKA C-α)、酪氨酸蛋白磷酸酶非受体11型(SHP2)、胰岛素受体底物1(IRS1)、丝氨酸/苏氨酸蛋白激酶PAK 1、丝裂原活化蛋白激酶8(JNK)。
10.一种体内筛选用于治疗或预防癌症或抑制癌细胞生长的候选药物的方法,其中所述药物针对Trop-2信号传导网络组分,包括CD9、Akt和四跨膜蛋白信号传导网络的分子,所述方法包含以下步骤:
a.将无菌生理盐水溶液中的针对所述Trop-2信号传导网络的组分的待测试化合物给予到临床前模型和临床模型中的对权利要求1所述的Trop-2生物标记呈阳性或阴性的肿瘤,其中所述阴性肿瘤充当特异性的对照,所述Trop-2信号传导网络的组分包括CD9、Akt和所述四跨膜蛋白信号传导网络分子;将单独的无菌生理盐水溶液平行地给予到对所述Trop-2生物标记呈阳性或阴性的肿瘤(未经治疗的对照),其中所述单独的无菌生理盐水溶液充当活性的对照;
b.检测步骤(a)的化合物对针对所述Trop-2生物标记呈阳性的肿瘤的生物活性,将其与对所述Trop-2生物标记呈阴性的经治疗的对照以及对所述Trop-2生物标记呈阳性和阴性的未经治疗的对照相比,其中生物活性是对所述Trop-2生物标记呈阳性的肿瘤中的肿瘤生长的减少,其是与对所述Trop-2生物标记呈阴性的对照肿瘤相比以及与对于所述Trop-2生物标记呈阳性和阴性的未经治疗的对照肿瘤相比;
c.选择步骤(a)的在对所述Trop-2生物标记阳性的肿瘤中减少肿瘤生长至少10%的化合物,其是与对所述Trop-2生物标记呈阴性的经治疗的对照肿瘤以及对所述Trop-2生物标记呈阳性和阴性的未经治疗的对照肿瘤相比。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ITCH2012A000008 | 2012-05-16 | ||
IT000008A ITCH20120008A1 (it) | 2012-05-16 | 2012-05-16 | Uso di trop-2 come marcatore predittivo di risposta a terapia anti-tumorale basata su inibitori di cd9, akt e vie di segnale collegate |
PCT/IT2013/000139 WO2013171777A2 (en) | 2012-05-16 | 2013-05-16 | Use of trop-2 as predictive marker of response to anti-tumor therapy based on inhibitors of cd9, akt and molecules of the tetraspanin signalling network |
Publications (1)
Publication Number | Publication Date |
---|---|
CN104395754A true CN104395754A (zh) | 2015-03-04 |
Family
ID=46832698
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201380025540.9A Pending CN104395754A (zh) | 2012-05-16 | 2013-05-16 | Trop-2作为对基于cd9、akt和四跨膜蛋白信号传导网络分子的抑制剂的抗肿瘤疗法的应答的预测性标记的用途 |
Country Status (9)
Country | Link |
---|---|
US (1) | US20210311061A1 (zh) |
EP (1) | EP2850433B1 (zh) |
JP (1) | JP2015524051A (zh) |
CN (1) | CN104395754A (zh) |
AU (1) | AU2013261018A1 (zh) |
CA (1) | CA2873017A1 (zh) |
IN (1) | IN2014DN10081A (zh) |
IT (1) | ITCH20120008A1 (zh) |
WO (1) | WO2013171777A2 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106467914A (zh) * | 2015-08-18 | 2017-03-01 | 华东理工大学 | 靶向人TSPAN8基因的siRNA及其应用 |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ITUB20155701A1 (it) | 2015-11-19 | 2017-05-19 | Saverio Alberti | Uso di Trop-2 circolante sierico come nuovo biomarcatore tumorale |
KR101806294B1 (ko) * | 2016-03-22 | 2017-12-07 | 주식회사 대웅제약 | 고형암 환자의 항암제 치료 반응성 예측용 마커 |
WO2023175483A1 (en) * | 2022-03-16 | 2023-09-21 | Astrazeneca Uk Limited | A scoring method for an anti-trop2 antibody‑drug conjugate therapy |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011057222A1 (en) * | 2009-11-06 | 2011-05-12 | Infinity Pharmaceuticals, Inc. | Oral formulations of a hedgehog pathway inhibitor |
WO2011085276A2 (en) * | 2010-01-09 | 2011-07-14 | The Translational Genomics Research Institute | Methods and kits to predict prognostic and therapeutic outcome in small cell lung cancer |
CA2798778A1 (en) * | 2010-05-17 | 2011-11-24 | Livtech, Inc. | Anti-human trop-2 antibody having anti-tumor activity in vivo |
CN102448492A (zh) * | 2009-02-05 | 2012-05-09 | 温科克斯有限公司 | 抗trop-2单克隆抗体及其在治疗和诊断肿瘤中的用途 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8383357B2 (en) * | 2005-03-16 | 2013-02-26 | OSI Pharmaceuticals, LLC | Biological markers predictive of anti-cancer response to epidermal growth factor receptor kinase inhibitors |
DE102005052384B4 (de) * | 2005-10-31 | 2009-09-24 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Verfahren zur Erkennung, Markierung und Behandlung von epithelialen Lungentumorzellen sowie Mittel zur Durchführung des Verfahrens |
US20080131428A1 (en) * | 2006-02-24 | 2008-06-05 | Arius Research, Inc. | Cytotoxicity mediation of cells evidencing surface expression of TROP-2 |
-
2012
- 2012-05-16 IT IT000008A patent/ITCH20120008A1/it unknown
-
2013
- 2013-05-16 CA CA2873017A patent/CA2873017A1/en not_active Abandoned
- 2013-05-16 IN IN10081DEN2014 patent/IN2014DN10081A/en unknown
- 2013-05-16 WO PCT/IT2013/000139 patent/WO2013171777A2/en active Application Filing
- 2013-05-16 AU AU2013261018A patent/AU2013261018A1/en not_active Abandoned
- 2013-05-16 US US14/401,456 patent/US20210311061A1/en not_active Abandoned
- 2013-05-16 CN CN201380025540.9A patent/CN104395754A/zh active Pending
- 2013-05-16 JP JP2015512199A patent/JP2015524051A/ja active Pending
- 2013-05-16 EP EP13739819.4A patent/EP2850433B1/en not_active Not-in-force
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102448492A (zh) * | 2009-02-05 | 2012-05-09 | 温科克斯有限公司 | 抗trop-2单克隆抗体及其在治疗和诊断肿瘤中的用途 |
WO2011057222A1 (en) * | 2009-11-06 | 2011-05-12 | Infinity Pharmaceuticals, Inc. | Oral formulations of a hedgehog pathway inhibitor |
WO2011085276A2 (en) * | 2010-01-09 | 2011-07-14 | The Translational Genomics Research Institute | Methods and kits to predict prognostic and therapeutic outcome in small cell lung cancer |
CA2798778A1 (en) * | 2010-05-17 | 2011-11-24 | Livtech, Inc. | Anti-human trop-2 antibody having anti-tumor activity in vivo |
Non-Patent Citations (7)
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106467914A (zh) * | 2015-08-18 | 2017-03-01 | 华东理工大学 | 靶向人TSPAN8基因的siRNA及其应用 |
Also Published As
Publication number | Publication date |
---|---|
IN2014DN10081A (zh) | 2015-08-21 |
WO2013171777A3 (en) | 2014-03-20 |
US20210311061A1 (en) | 2021-10-07 |
AU2013261018A1 (en) | 2014-12-04 |
EP2850433A2 (en) | 2015-03-25 |
EP2850433B1 (en) | 2018-08-15 |
JP2015524051A (ja) | 2015-08-20 |
WO2013171777A2 (en) | 2013-11-21 |
CA2873017A1 (en) | 2013-11-21 |
ITCH20120008A1 (it) | 2013-11-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Ibrahim et al. | Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways | |
Tao et al. | Cancer-associated fibroblasts treated with cisplatin facilitates chemoresistance of lung adenocarcinoma through IL-11/IL-11R/STAT3 signaling pathway | |
Lu et al. | ARK5 promotes glioma cell invasion, and its elevated expression is correlated with poor clinical outcome | |
Chen et al. | L1cam promotes tumor progression and metastasis and is an independent unfavorable prognostic factor in gastric cancer | |
Grass et al. | CD147, CD44, and the epidermal growth factor receptor (EGFR) signaling pathway cooperate to regulate breast epithelial cell invasiveness | |
Herling et al. | High TCL1 expression and intact T-cell receptor signaling define a hyperproliferative subset of T-cell prolymphocytic leukemia | |
Magkou et al. | Expression of the epidermal growth factor receptor (EGFR) and the phosphorylated EGFR in invasive breast carcinomas | |
Ayala et al. | Stromal antiapoptotic paracrine loop in perineural invasion of prostatic carcinoma | |
Kato et al. | Semaphorin 4D, a lymphocyte semaphorin, enhances tumor cell motility through binding its receptor, plexinB1, in pancreatic cancer | |
KR20170094792A (ko) | 환자를 진단 및 치료하기 위한 신호전달 경로 활성의 측정 방법 | |
KR20130095183A (ko) | 약물 감응성, 내성, 및 질환 진행성을 예측하기 위한 조성물 및 방법 | |
Pan et al. | ATP synthase ecto-α-subunit: a novel therapeutic target for breast cancer | |
Leech et al. | Cleavage of the extracellular domain of junctional adhesion molecule-A is associated with resistance to anti-HER2 therapies in breast cancer settings | |
Martini et al. | Cortactin, a Lyn substrate, is a checkpoint molecule at the intersection of BCR and CXCR4 signalling pathway in chronic lymphocytic leukaemia cells | |
CN103502470A (zh) | 嗅介蛋白-4蛋白(olfm4)在结肠直肠癌诊断中的用途 | |
Hu et al. | The O-glycosylating enzyme GALNT2 suppresses the malignancy of gastric adenocarcinoma by reducing EGFR activities | |
You et al. | Galectin-1 promotes metastasis in gastric cancer through a sphingosine-1-phosphate receptor 1-dependent mechanism | |
Hamel et al. | Both t-Darpp and DARPP-32 can cause resistance to trastuzumab in breast cancer cells and are frequently expressed in primary breast cancers | |
Matušan-Ilijaš et al. | EGFR expression is linked to osteopontin and Nf-κB signaling in clear cell renal cell carcinoma | |
Li et al. | Phospholipase Cγ1 (PLCG1) overexpression is associated with tumor growth and poor survival in IDH wild-type lower-grade gliomas in adult patients | |
Gao et al. | Expression of Lewis y antigen and integrin αv, β3 in ovarian cancer and their relationship with chemotherapeutic drug resistance | |
CN104395754A (zh) | Trop-2作为对基于cd9、akt和四跨膜蛋白信号传导网络分子的抑制剂的抗肿瘤疗法的应答的预测性标记的用途 | |
Ma et al. | Clinical implications of Ezrin and CD44 co‑expression in breast cancer | |
Pan et al. | Cathepsin L promotes angiogenesis by regulating the CDP/Cux/VEGF-D pathway in human gastric cancer | |
Glorieux et al. | Regulation of CD137 expression through K-Ras signaling in pancreatic cancer cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150304 |