CN1314914A - Proteins for cancer cell specific induction of apoptosis and method for isolation thereof - Google Patents
Proteins for cancer cell specific induction of apoptosis and method for isolation thereof Download PDFInfo
- Publication number
- CN1314914A CN1314914A CN98813660A CN98813660A CN1314914A CN 1314914 A CN1314914 A CN 1314914A CN 98813660 A CN98813660 A CN 98813660A CN 98813660 A CN98813660 A CN 98813660A CN 1314914 A CN1314914 A CN 1314914A
- Authority
- CN
- China
- Prior art keywords
- cell
- protein
- apogen
- cells
- apoptosis
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 87
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 86
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 32
- 201000011510 cancer Diseases 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title abstract description 14
- 230000006882 induction of apoptosis Effects 0.000 title description 2
- 238000002955 isolation Methods 0.000 title 1
- 230000000694 effects Effects 0.000 claims abstract description 67
- 230000006907 apoptotic process Effects 0.000 claims abstract description 51
- 206010006187 Breast cancer Diseases 0.000 claims abstract description 24
- 208000026310 Breast neoplasm Diseases 0.000 claims abstract description 24
- 210000004072 lung Anatomy 0.000 claims abstract description 24
- 206010060862 Prostate cancer Diseases 0.000 claims abstract description 22
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims abstract description 22
- 208000032839 leukemia Diseases 0.000 claims abstract description 13
- 230000001640 apoptogenic effect Effects 0.000 claims description 21
- 239000000835 fiber Substances 0.000 claims description 21
- 239000012528 membrane Substances 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 7
- 210000004027 cell Anatomy 0.000 abstract description 227
- RDEIXVOBVLKYNT-VQBXQJRRSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(1-aminoethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2-yl]o Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)C(C)N)N)[C@@H](N)C[C@H]1N.O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-VQBXQJRRSA-N 0.000 abstract description 185
- 239000003636 conditioned culture medium Substances 0.000 abstract description 69
- 230000001939 inductive effect Effects 0.000 abstract description 66
- 206010009944 Colon cancer Diseases 0.000 abstract description 4
- 239000002246 antineoplastic agent Substances 0.000 abstract description 2
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 2
- 238000003782 apoptosis assay Methods 0.000 abstract description 2
- 230000005522 programmed cell death Effects 0.000 abstract description 2
- 210000002950 fibroblast Anatomy 0.000 abstract 4
- 208000029742 colonic neoplasm Diseases 0.000 abstract 3
- 230000030833 cell death Effects 0.000 description 62
- 235000018102 proteins Nutrition 0.000 description 43
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 36
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 32
- 238000013016 damping Methods 0.000 description 29
- 239000012530 fluid Substances 0.000 description 29
- 239000004480 active ingredient Substances 0.000 description 25
- 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 24
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 24
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 24
- 229920000669 heparin Polymers 0.000 description 24
- 210000004940 nucleus Anatomy 0.000 description 23
- 238000000926 separation method Methods 0.000 description 22
- 239000006228 supernatant Substances 0.000 description 21
- 229920000936 Agarose Polymers 0.000 description 20
- UYJXRRSPUVSSMN-UHFFFAOYSA-P ammonium sulfide Chemical compound [NH4+].[NH4+].[S-2] UYJXRRSPUVSSMN-UHFFFAOYSA-P 0.000 description 18
- 238000013467 fragmentation Methods 0.000 description 18
- 238000006062 fragmentation reaction Methods 0.000 description 18
- 239000002609 medium Substances 0.000 description 18
- 238000010828 elution Methods 0.000 description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 239000012141 concentrate Substances 0.000 description 16
- 238000004128 high performance liquid chromatography Methods 0.000 description 16
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 16
- 210000002966 serum Anatomy 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 12
- 230000001965 increasing effect Effects 0.000 description 12
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 12
- 208000002109 Argyria Diseases 0.000 description 11
- 238000005571 anion exchange chromatography Methods 0.000 description 11
- 238000001962 electrophoresis Methods 0.000 description 10
- 238000010867 Hoechst staining Methods 0.000 description 9
- 238000004043 dyeing Methods 0.000 description 9
- 238000001556 precipitation Methods 0.000 description 9
- 229920001184 polypeptide Polymers 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 8
- 238000012545 processing Methods 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 238000004062 sedimentation Methods 0.000 description 7
- 102000004877 Insulin Human genes 0.000 description 6
- 108090001061 Insulin Proteins 0.000 description 6
- 229930182555 Penicillin Natural products 0.000 description 6
- 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 6
- 102000009618 Transforming Growth Factors Human genes 0.000 description 6
- 108010009583 Transforming Growth Factors Proteins 0.000 description 6
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 6
- 229940125396 insulin Drugs 0.000 description 6
- 230000000877 morphologic effect Effects 0.000 description 6
- 229940049954 penicillin Drugs 0.000 description 6
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 6
- 230000000717 retained effect Effects 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 6
- 102000018594 Tumour necrosis factor Human genes 0.000 description 5
- 108050007852 Tumour necrosis factor Proteins 0.000 description 5
- 150000001413 amino acids Chemical group 0.000 description 5
- 229920002678 cellulose Polymers 0.000 description 5
- 239000001913 cellulose Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 239000012982 microporous membrane Substances 0.000 description 5
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 4
- 239000004793 Polystyrene Substances 0.000 description 4
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 4
- 102000013275 Somatomedins Human genes 0.000 description 4
- ZGSDJMADBJCNPN-UHFFFAOYSA-N [S-][NH3+] Chemical class [S-][NH3+] ZGSDJMADBJCNPN-UHFFFAOYSA-N 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 230000012292 cell migration Effects 0.000 description 4
- 230000003399 chemotactic effect Effects 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 239000007758 minimum essential medium Substances 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 108050002568 Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 3
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 3
- 210000000601 blood cell Anatomy 0.000 description 3
- 239000002975 chemoattractant Substances 0.000 description 3
- 238000005352 clarification Methods 0.000 description 3
- 230000001143 conditioned effect Effects 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 229920006267 polyester film Polymers 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 238000005096 rolling process Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- JVIPLYCGEZUBIO-UHFFFAOYSA-N 2-(4-fluorophenyl)-1,3-dioxoisoindole-5-carboxylic acid Chemical compound O=C1C2=CC(C(=O)O)=CC=C2C(=O)N1C1=CC=C(F)C=C1 JVIPLYCGEZUBIO-UHFFFAOYSA-N 0.000 description 2
- 108010081589 Becaplermin Proteins 0.000 description 2
- 229920001425 Diethylaminoethyl cellulose Polymers 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000007859 condensation product Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000011275 oncology therapy Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- 208000006332 Choriocarcinoma Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 201000010989 colorectal carcinoma Diseases 0.000 description 1
- 238000002316 cosmetic surgery Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000002308 embryonic cell Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 210000005095 gastrointestinal system Anatomy 0.000 description 1
- 230000002607 hemopoietic effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000012716 precipitator Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000004804 winding Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4747—Apoptosis related proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides the methods to isolate the proteins specifically induced apoptosis (programmed cell death) in prostate cancer cells (LNCAP), leukemia cells (HL-60), and breast cancer cells (MCF-70), but without effect in normal human lung fibroblast cells (CCD 39 Lu). P-1 has no effect on breast cancer cells. Five proteins have been isolated from the conditioned media of culture cells: (1) Apogen P-1: the proteins (Apogen P-1a, Apogen P-1b and Apogen P-1c) isolated from the conditioned medium of XC cells are able to induce apoptosis in prostate cancer cells (LNCAP) without effect in normal human lung fibroblast (CCD 39 Lu), colon cancer (T84), breast cancer (MCF-7) and leukemia (HL-60) cells. (2) Apogen P-2: the protein isolated from the conditioned medium of C3H1OT1/2 cells is able to induce apoptosis in prostate cancer cells (LNCAP) and breast cancer (MCF-7) without effect in normal human lung fibroblast (CCD 39 Lu) and colon cancer (T84) cells. (3) Apogen L: the protein isolated from the conditioned medium of XC cells is able to induce apoptosis in leukemia cells (HL-60), and breast cancer (MCF-7) without effect in normal human lung fibroblast (CCD 39 Lu), colon cancer (T84) and prostate cancer (LNCAP) cells. The invention may lead to the discovery of a novel class of anticancer drug that aims at prostate cancer, breast cancer, leukemia and other cancers by inducing apoptosis in cancer cells without affecting normal cells.
Description
Background technology of the present invention
For a long time, human and cancer is being done unremitting struggle always.Because this disease is so general, and merciless in every way map doing harm to human life, and therefore effectively the potential market of cancer treatment method is huge.Estimating at American more than 10,000,000 suffers from or suffered from cancer.American National institute of oncology (NCI) estimates that nearly 1,200,000 patients of the U.S. will suffer from cancer, and have 538,000 people will die from this disease in nineteen ninety-five.
Today, although combine surgical operation, chemotherapy, and emissivity therapy, the curative ratio of cancer is very low.Chemotherapeutic low success rate reason is that current chemotherapy is that tumour cell with rapid differentiation is a target.This method is inefficient to cancer cells that hide or poky.This therapy also influences the normal cell of rapid differentiation, causes deleterious side effect.
A kind of novel method for the treatment of cancer has just appearred recent years, this method is based on nearest firm found biological phenomena " apoptosis ", apoptosis be otherwise known as " programmed cell death of cell " or " cell suicide " (people such as Tomei, " apoptosis ": the molecular basis of necrocytosis.Press of cold spring harbor laboratory, 1991).Compare with the necrocytosis that cell injury causes, apoptosis is the cell active self-destruction process of gene mediated and is significant function service biologically (Kerr, J.F.R and J.Searle J.Pathol.107:41 1971).Apoptosis is that embryo's form forms (Michaelson at one of significant function example biologically, J.Biol.Rev.62:115,1987), increase or reduce argillous statue engraving process as needs, embryo's organ forms (form formation) and depends on the growth (increasing earth) of cell and the death (minimizing earth) of cell.All in human body, play a part key, (Wyllie, A.H.Int.Rev.Cytol.68:251,1980) from embryoplastic commitment to unavoidable old and feeble stage apoptosis.The normal function of immunity system, gastro-intestinal system and hemopoietic system depends on apoptotic normal effect.When apoptotic function is undesired, will cause such or such some diseases to produce, as cancer, virus infection, autoimmune disease/anaphylaxis, the neural inhibition and cardiovascular disorder.Because related apoptotic multifunctionality in human diseases, " apoptosis " becoming an outstanding industry term in the drug research field.People are used in a large amount of time and moneys in the research of its mechanism of action, study it and how to be suppressed or to be excited, and the research that this mechanism is applied to pharmacology is used.More such companies have been set up, it is devoted to develop in this new life field the product of the market competitiveness, the appearance of some new forces ingenious makes that in conjunction with the action of the trial of some strong major companies apoptosis--this nineteen nineties development is the swiftest and the most violent, has the research of the field of pharmacology of one of development potentiality to become possibility most like this.
Have a kind of like this viewpoint to think recently, cancer causes (Cope, F.O and Wille, J.j, " Apoptosis ": The molecular Basis of CellDeath, press of cold spring harbor laboratory, pp61,1991) by inadequate apoptosis.This viewpoint is that a kind of new thinking has been started in cancer therapy---cancer cells can be killed by the excitation apoptosis.Based on all processes in the normal development, apoptotic tunning effect is a kind of potential mechanism of controlling tumor cell proliferation.The Apoptosis Mechanism of recovering in the tumour cell is a kind of method of worth trial, because it will cause cell suicide death at least in theory.Yet, the treatment for cancer purpose is kill cancer cell and does not damage host cell, although Apoptosis Mechanism cell death inducing in cancer cells is that cancer therapy opens up a new way, successful treatment still depend on can be selectively in cancer cells inducing cell death and do not damage the operability of Normocellular medicament.In this part patent application, we introduce some proteinic separation methods, these protein cell death inducing but do not damage normal cell seriatim in cancer cells.These protein can provide a class new anti-cancer drug thing, they will be in cancer cells cell death inducing, thereby provide a breach for cancer therapy.
Detailed description of the present invention
Following five kinds of proteinic separation methods are introduced in this patent application: Apogen P-1a, Apogen 1b, Apogen 1c, Apogen P-2 and Apogen L.
(A) separation of Apogen P-1
(1) Apogen P-1 source
Apogen P-1 separates from the conditioned medium of the clone of a kind of XC by name, and this XC cell derives from the mouse tumour and can collect center (ATCC) from U.S.'s typical culture and buy.At first, allow the XC cell in the DMEM substratum that contains 10% N of tire serum (FBS), grow three days.Then, (3 * 100ml) clean the XC cell to remove serum deprivation, allow the XC cell cultivate 4 days in the DMEM that does not contain FBS again with PBS.Do not contain in the conditioned medium of serum at this, we detect a kind of in prostate cancer cell line (LNCAP) activity of cell death inducing.Yet normal people's lung fibre source clone (CCD39Lu) is not subjected to this activity influence with breast cancer cell (MCF-7).
(2) activity of Apogen P-1
(a) apoptosis induction activity
The activity of the natural conditioned medium of XC cell detects in following clone: JEG-3 (choriocarcinoma), G401 (Wilm ' s tumour), LNCAP (prostate cancer), T84 (colorectal carcinoma), HL-60 (leukemia), breast cancer cell (MCF-7) and CCD39Lu (normal lung fibre source cell).When cultivating 18 hours under the conditioned medium that is concentrated 10 times is having 5% serum to exist with above-mentioned clone the condition, this conditioned medium is cell death inducing in JEG-3 cell (35%), G401 cell (27%) and LNCAP (100%), and does not have activity in CCD39Lu (0%), T84 (0%), MCF-7 (0%) and HL-60 (0%).
Apoptosis is a kind of distinct necrocytosis type, and it is different from decline death or downright bad fully with regard to its nature and biological significance.Experiencing apoptotic cells and on morphology and biological chemistry, significant difference is being arranged with the cell that experiences necrosis.On morphology, in apoptosis, be the condensation product and the cytoplasmic condensation product of near the nuclear staining body homogeneous that nuclear membrane, is compressed to rapid winding with the detected early stage noticeable change of electron microscope.Shown that with phase microscope observation of cell apoptosis DNA is from being concentrated to division and cell from producing the whole process of apoptosis.
In order to contain the active ingredient of cell death inducing from morphology proof XC conditioned medium, the LNCAP cell is incubated in control medium and the conditioned medium each 15 hours, then with Hoechst stain dyeing 2 hours, shown in Figure 1A, with the LNCAP nucleus normal (A) of control medium cultivation.Yet replace the X20 substratum with the RPMI substratum and find that but features of apoptosis (Fig. 1 (B)) has appearred in the LNCAP nucleus.At first, relatively fluorescence intensity is higher with contrast nuclear phase among Fig. 1 (A), and this shows that conditioned medium can cause concentrating of caryoplasm.Secondly, the caryoplasm fragmentation shown in Fig. 1 (B) can prove that the fragmentation of DNA has also appearred in the spissated while of caryoplasm.Just as we state in front like that, caryoplasm concentrate with the DNA fragmentation be apoptotic morphological feature.These presentation of results contain from the conditioned medium of XC cell induces the apoptotic active ingredient of LNCAP.Yet in people's lung fibre source cell (CCD39Lu cell) and breast cancer cell (MCF-7), these conditioned mediums can not cell death inducing.As shown in Figure 2, all be the same (Fig. 2 (A) and Fig. 2 (B)) no matter whether the nucleus of CCD39Lu is cultivated with the XC cell conditioned medium.
(b) cell rejection activity
The Apogen P-1b of recent findings separated portions purifying as described below (Q2 anion-exchange chromatography) contains other active ingredients except that cell death inducing.We find that Apogen P-1b has the rejection cell activity.The activity of this activity and somatomedin is completely contradicted; Many somatomedins (as Thr6 PDGF BB (PDGF), Urogastron (EGF), fibre source cell growth factor (FOF) or transforming growth factor (TOF)) work as " chemical attractant ", this means that these somatomedins attract cell to draw close (Grotendorst towards them, O.R. wait people, Proc.NatI.Acad.Sci.78,3669,1981, Grant, people such as M.B., Invest.Ophthal.Visual Science.33,3292,1992).These discoveries mean by the isolating Apogen P-1b of the present invention to be played and the diametrically opposite effect of somatomedin.For example, growth of growth factor-induced cell and attraction cell, and Apogen P-1b inducing cell death and rejection cell.Apogen P-1b is that " chemotactic repellant " found in the modern biology field at first.
It is a kind of that (Cambridge, the tissue culture device that is called Transwell Insert MA) can be used for disclosing the activity of chemotactic repellant Apogen P-1b available from Costar.Thisly be widely used in studying the device that cell migration or cell invade and comprised a Shang Cang and a Lower Hold.Be that the aperture is many micropores polyester film that 3.0um allows cell to pass between these two bins.Tested cell is grown in last storehouse, and tested compound is placed on down in the storehouse.If tested compound is a chemical attractant, we should see that the comparison cell that product are many in the same old way passes film.In our experiment, the cell size is a membrane pore size 3-4 Hep02 cell (100 doubly, 000 cell) in last storehouse, cultivated 2 hours, will place down in the storehouse by the above-mentioned ammonium sulfide precipitation and the Apogen-1b (30 μ l) of Q2 HPLC chromatography separated portions purifying then.After 15 hours, handled film 30 minutes and collected the cell that passes through this film with the 0.2ML insulin solutions.Go out cell count in the 10 microlitre insulin solutions with blood cell register number.After repeating to test several times, we find that partially purified Apogen-1b contains a kind of active ingredient, and this composition reduces the cell quantity by film.For example, have in the experiment that partially purified Apogen-1b exists at one, the cell number in the 10 microlitre insulin solutions (by the cell quantity of film) is 24+-4, and the cell count by this film is 82+-27 in the contrast experiment.This result means that partially purified Apogen-1b stops Hep G2 cell to pass through film.In order clearly to prove the characteristic of Apogen P-1b rejection cell, we have then done an oppositely experiment, Apogen P-1b is transferred to the storehouse by following storehouse, implement same step, after 12 hours, find to have in per 10 microlitre insulin solutions 56+-19 cell by film, and control experiment there be 30+-1.7 cell to pass through film.By alternately Apogen P-1b being placed in the last storehouse or following storehouse of this tissue culture device, statistics is passed through the increase and decrease of the cell quantity of this film, thereby clearly proves the characteristic of Apogen P-1b rejection cell.
(3) from the XC conditioned medium, separate Apogen P-1
The Apogen P-1 that exists in conditioned medium can separate through the following steps:
Step 1: ammonium sulfide precipitation
Make Apogen P-1 sedimentation in the ammonium sulfide of 80% saturation ratio by in every liter of conditioned medium, adding 561 gram ammonium sulfides.By the protein bead of centrifugal collecting precipitation, and they are dissolved among the Tris-HCl (pH7.4) of 10mM.After removing ammonium sulfide, with the protein of Q2 HPLC chromatographic column separate dissolved by dialysis.
Step 2:Q2 HPLC chromatography
Be loaded on the Q2 post (BiO-Rad) through dissolving with after concentrating with the isolating protein of the ammonium sulfide precipitator method, utilize the HPLC of Bole biotech company chromatographic system by A damping fluid (10mM Tris-HCl then, pH7.4) and B damping fluid (10mM Tris-HCl, pH7.4,0.55M NaCl) constructed linear gradient is further launched.This linear gradient makes up (20 milliliters elution volumes) by making the B damping fluid progressively be increased to 100% from 0% in the A damping fluid in 10 minutes, use the 100%B damping fluid with this chromatographic column wash-out 5 minutes then.Color atlas as shown in Figure 3.
The activity of Apogen P-1 can be tested by cell death inducing in the LNCAP cell.Three active peaks are arranged on the chromatogram collection of illustrative plates.In 18 hours, a level part 5-7 can cause 70% necrocytosis, and a level part 8-10 can cause 65% necrocytosis, and a level part 11-14 can cause 90% necrocytosis.We collect level part 5-7 and with its called after ApogenP-1a, a level part 8-10 is named as Apogen P-1b, and a level part 11-14 is named as ApogenP-1c.These three kinds of Apogen P-1 are further purified by reversed-phase column.
Step 3: reverse-phase chromatography
Apogen P-1a, Apogen P-1b and Apogen P-1c are concentrated to 1.5 milliliters respectively.Add 1ml and contain the methanol solution of 0.05% trifluoroacetic acid in each sample, by this processing, amounts of protein is settled down, and the active ingredient of cell death inducing then is retained in the supernatant liquor.Then, supernatant liquor is applied to RP4 reverse-phase chromatographic column (MicroScientific Inc), and by solution A (H20,0.05%TFA) and solution B (Methanol, the linear gradient that 0.05%TFA) makes up expansion.This linear gradient by in 10 minutes, make B solution in the A damping fluid progressively from 0% being increased to and 100% making up (20 milliliters of elution volumes and with 100% solution B with this chromatographic column wash-out 5 minutes).
Step 4: preparation electrophoresis
(step 3) and preparation electrophoresis (the MiniPrep Gel electrophoresis apparatus of BIO--RED company) carry out purifying by reverse-phase chromatography to separate the Apogen 1c that obtains with anion-exchange chromatography.The reverse-phase chromatography figure of Apogen P-1a is shown in Fig. 4 (a).Level part 12-13 has the activity that can induce 80% LNCAP necrocytosis in 10 hours.The reverse-phase chromatography figure of Apogen P-1b is shown in Fig. 4 (b). Level parts 14 and 15 has the activity that can induce 45% LNCAP necrocytosis in 18 hours.The reverse-phase chromatography figure of Apogen P-1c is shown in Fig. 4 (c).Level parts 5 has the activity of inducing 52% LNCAP necrocytosis in 18 hours.
The purity of isolating Apogen P-1a, Apogen P-1b and Apogen P-1c can detect with the SDS polyacrylamide gel electrophoresis (SDSPAGE) of silver staining.
(1) Apogen P-1a: as shown in Figure 5, what obtained is that molecular weight is the polypeptide chain of 70KD.This result shows that on SDSPAGE molecular weight is that the ApogenP-1a of 70KD is almost by the purifying of success.
(2) Apogen P-1b: what obtained is that molecular weight is the polypeptide chain of 55KD, and this result shows that on SDSPAGE molecular weight is that the Apogen P-1b of 55KD is by the purifying of success (No data demonstration).
(3) Apogen P-1c: the protein chain separation that has the 70KD molecular weight by the Apogen 1c that is purified to reverse-phase chromatography uses the preparation electrophoretic method to obtain the protein chain of 57KD molecular weight.Shown in Fig. 6 (A), a main protein chain is the protein chain with 70KD molecular weight that is purified to through reverse-phase chromatography, and obtains the protein chain (Fig. 6 B) of 57KD molecular weight with the prerunning method.
Our further work is the whole protein chains of making great efforts to be devoted to obtain enough certain aminoacid sequences of tool with us.(B) separation of Apogen P-2
(1) source of Apogen P-2
Apogen P-2 separates from the conditioned medium of the clone that is called as C3H 1OT1/2.This origin of cell is bought in U.S. typical case culture collection center (ATCC) in the mouse embryonic cell.C3H 1OT1/2 cell was at first cultivated three days in the substratum (alpha-MEM) of the Eagle that contains 10% N of tire serum (FBS).(3 * 100ml) washed cells were cultivated 4 days in the alpha-MEM that does not contain FBS then to remove serum to use PBS then.We detect the activity of cell death inducing in prostate cancer cell line (LNCAP) from this conditioned medium that does not contain serum.Yet normal people's lung fibre source clone (CCD39Lu) but is not subjected to this active the influence.
(2) activity of Apogen P-2
(a) activity of cell death inducing
The activity of the natural condition substratum of C3H 1OT1/2 cell can detect based on following clone: LNCAP (prostate cancer cell), breast cancer cell (MCF-7), and CCD39Lu (normal people's lung fibre source cell).After cultivating 18 hours under 10 times of spissated conditioned mediums are having 5% serum to exist with above-mentioned cell the condition, this conditioned medium will be in LNCAP cell death inducing (100%), but in CCD39Lu, but do not have this activity (0%).In order to contain the activity of cell death inducing from morphology proof C3H IOT1/2 conditioned medium, with control medium or through the conditioned medium of above-mentioned processing with LNCAP cell cultures 15 hours, used the Hoechst dyeing then 2 hours.Shown in Fig. 7 A, the nucleus of the LNCAP cell of cultivating with control medium is normal (Fig. 7 A).But, the nucleus showed cell features of apoptotic (Fig. 7 B) of the LNCAP cell of cultivating with conditioned medium.At first, conditioned medium has caused nucleus to concentrate, and compares fluorescence intensity with the control cells nuclear shown in Fig. 7 A and suffices to show that this point more by force.Secondly, caryoplasm concentrates the fragmentation that is attended by DNA, and the nuclear fragmentation shown in Fig. 7 B will prove this point.As the above, the cell caryoplasm concentrates and the DNA fragmentation is the morphological feature of cell under the apoptotic state.Can use breast cancer cell (MCF-7) to prove this point equally, it exposes in P-2 will observe 85% apoptotic effect after 18 hours.These results show that the conditioned medium that comes from the C3H1OT1/2 cell contains the activity of cell death inducing in LNCAP and MCF-7 cell.On the other hand, this conditioned medium cell death inducing not in normal people's lung fibre source cell (CCD39Lu cell).As shown in Figure 8, whether the CCD39Lu cell no matter all keep same feature (Fig. 8 A and Fig. 8 B) with the conditioned medium cultivation of C3HIOT1/2 cell.
(b) cell rejection activity
The someone finds recently, and the Apogen P-2 that handles the separated portions purifying by ammonium sulfide sedimentation, hydroxylapatite and Vitrum AB comprises and the distinct activity of cell death inducing.Similar to Apogen P-1b, Apogen P-2 also has the rejection cell activity., (Cambridge, Transwell Insert MA) is used to detect the chemotactic repellant activity of Apogen P-2 available from Costar.Thisly be widely used in studying the equipment that cell migration or cell invade and comprised a Shang Cang and a Lower Hold.Be that an aperture is many micropores polyester film that 3.0 μ m allow cell to pass between these two bins.The cell of testing (HL-60) is grown in last storehouse, and miscellany to be tested (Apogen P-2) is placed on down in the storehouse.In our experiment, the cell size is that membrane pore size 2-3 HL-60 cell (100,000 cell) was doubly cultivated 2 hours in last storehouse.With the ammonium sulfide precipitation, after hydroxylapatite and Vitrum AB were handled, partial purification ApogenP-2 (301) was positioned over down in the storehouse then.After 6 hours, collect cell by microporous membrane.And then will descend centrifugal 10 minutes of culture (0.6ML) in the storehouse, collect HL-60 cell, and these cells are suspended among the PBS of 80 μ l again by film.With count cell number in per 10 microlitre PBS solution of blood cell register.After repeating to test several times, we find to contain among the partially purified ApogenP-2 a kind of active ingredient, and it can constantly reduce the cell quantity by film.For example, when having partially purified ApogenP-2 to exist, the cell number (by the cell number of film) in per 10 microlitre PBS solution is 47+-5.6, and the cell number by microporous membrane is 213+-40 in the contrast experiment.In this moment, features of apoptosis does not appear in the HL-60 cell in the last storehouse.These results prove that partially purified ApogenP-2 can hinder the HL-60 cell and pass through film.
(3) from the C3H1OT1/2 conditioned medium, separate Apogen P-2
Be present in Apogen P-2 in the conditioned medium and be carry out as follows isolating:
Step 1: ammonium sulfide precipitation
Make Apogen P-2 sedimentation in the ammonium sulfide of 80% saturation ratio by in every liter of conditioned medium, adding 561 gram ammonium sulfides,, and they are dissolved among the Tris-HCl (pH7.4) of 10mM by the protein bead of centrifugal collecting precipitation.
Step 2: handle with hydroxylapatite
Separate by 10mM Tris-HCl (pH7.5) dialysis and to remove after the ammonium sulfide, (Bio-Gel HTP gel cultivated 1 hour in Bio-Rad) at the hydroxylapatite gel with dissolved protein.By centrifugal remove the HTP gel after, it is found that the activity of cell death inducing in the LNCAP cell is present among the supernatant liquor, further handle this supernatant liquor with the Vitrum AB sepharose then.
Step 3: the Vitrum AB agarose is handled
To further cultivate 1 hour with Vitrum AB agarose (Sigma) from the supernatant liquor of step 2.By centrifugal remove the HTP gel after, the active ingredient of cell death inducing is present in the supernatant liquor in the LNCAP cell.
Step 4: reverse-phase chromatography
The Apogen P-2 that is present in step 3 in the supernatant liquor of Vitrum AB agarose further handles with reverse-phase chromatography.Apogen P-2 is concentrated into 1 milliliter.Add 1ml and contain the methanol solution of 0.05% trifluoroacetic acid.By this processing, most protein is by sedimentation, and the active ingredient of cell death inducing (P-2) is retained in the supernatant liquor.Then, this supernatant liquor is applied to RP4 reverse-phase chromatographic column (Micra Scientific Inc.) and by solution A (H
2O, 0.05%TFA) and solution B (methyl alcohol, 0.05%TFA) linear gradient that makes up is launched.This linear gradient is by making the solution B in the solution A progressively be increased to 100% (the 20 milliliters of elution volume and with 100% solution B with chromatographic column wash-out 5 minutes) that makes up from 0% in 10 minutes.
The reverse-phase chromatography of Apogen P-2 is illustrated in Fig. 9.Level part 12-14 contains in the LNCAP cell to induce the active ingredient of 80% necrocytosis in 12 hours.Separating the purity of back ApogenP-2 can measure with the SDS polyacrylamide gel electrophoresis of silver staining.What obtained is that molecular weight is the single protein chain (Figure 10) of 65Kd.(C) separation of Apogen L
(1) source of Apogen L
Apogen L separates from the conditioned medium available from the XC clone at U.S. typical case culture collection center (ATCC).The XC cell was cultivated four days in the Dulbecco modifier (DMEM) of the Eagle substratum that contains 10% N of tire serum (FBS).We detect the active ingredient of cell death inducing in the leukemia cell of HL-60 by name from this conditioned medium.On the other hand, normal people's lung fibre source cell (CCD39Lu) is not subjected to the influence of this active ingredient.
(2) from the XC conditioned medium, separate Apogen L
The Apogen L that is present in the XC conditioned medium can separate by following step.
Step 1:DE52 absorbs
(diethylaminoethylcellulose Whatman) is cultivated conditioned medium 1 hour with anionite DE52.Then, the mixture that centrifugal treating is cultivated is collected the DE52 that fetters Apogen L, and washs with the 10mM Tris-HCL (pH7.5) that contains 0.15M NaCL.Then, Apogen L is washed out from the DE52 Mierocrystalline cellulose with the 10mM Tris-HCl (pH7.5) that contains 0.5M NaCl.
Step 2: the Vitrum AB agarose absorbs
Further absorbed in 1 hour by cultivating through the isolating Apogen L of step 1 with the Vitrum AB agarose by Vitrum AB agarose (Sigma).By centrifugal collection Vitrum AB agarose, and wash with 10mM Tris-HCl (pH7.5).Then, the Apogen L that is absorbed by the Vitrum AB agarose with 2M NaCl elution.
Step 3:Q2 HPLC chromatography
Apogen L after separating as mentioned above is loaded on the Q2 chromatographic column (Bio-Rad) after further concentrating, this chromatographic column utilizes HPLC system of Bole biotech company to pass through by A damping fluid (10mM Tris-HCL, pH7.4) and the linear gradient that makes up of B damping fluid (10mM Tris-HCL, pH7.4.0.5M NaCL) further launch.This linear gradient makes up by making the B damping fluid progressively be increased to 100% from 0% in 10 minutes in the A damping fluid.Color atlas as shown in figure 12.The purity of Apogen L after the separation can be measured with the SDS polyacrylamide gel electrophoresis of silver staining.What obtained is that molecular weight is the single protein chain (Figure 11) of 55Kd.
(3) activity of Apogen L
The activity of isolating as mentioned above Apogen L is tested by following clone: HL-60 (leukemia) and CCD39Lu (normal lung fibre source cell).In order to contain the active ingredient of cell death inducing from morphology proof Apogen L.As above isolating Apogen L cultivates 15 hours then with Hoechst stain dyeing 2 hours with the HL-60 cell.As shown in FIG. 13A, the HL-60 nucleus of cultivating with control medium is very normal (Figure 13 A).The HL-60 nucleus of cultivating with Apogen L presents features of apoptosis (Figure 13 B).At first, Apogen L causes nucleus to concentrate, and this is confirmed by presenting stronger fluorescent with the control cells nuclear phase ratio shown in Figure 13 A.Secondly, the concentrated fragmentation that is attended by DNA of caryoplasm, this can prove by the nuclear fragmentation shown in Figure 13 B.As mentioned above, caryoplasm concentrates and the fragmentation of DNA is two morphological features that cell is in apoptotic state.These as a result none do not proved Apogen L contain can be in the HL-60 cell active ingredient of cell death inducing.Apogen L is cell death inducing in MCF-7 (breast cancer) cell also.Yet, this conditioned medium can not be in normal people's lung fibre source cell (CCD39Lu cell) cell death inducing.
Brief Description Of Drawings
Fig. 1: by conditioned medium cell death inducing in prostate cancer cell (LNCAP) of XC cell.The LNCAP cell was cultivated 15 hours with control medium or conditioned medium, then with Hoechst stain dyeing two hours.Shown in Figure 1A, the LNCAP nucleus of cultivating with control medium is normal, and the LNCAP nucleus of cultivating with conditioned medium (X20, exchanged to RPMI) presents features of apoptosis (Fig. 1 (B)).At first, conditioned medium can cause nucleus to concentrate, and this can be by being confirmed with the fluorescent stronger than presenting of the control cells nuclear phase shown in Fig. 1 (A).Secondly, caryoplasm concentrates the fragmentation that is attended by DNA, and this can be confirmed by the nucleus fragmentation shown in Fig. 1 (B).As mentioned above, caryoplasm concentrates and the fragmentation of DNA is two morphological features that cell is in apoptotic state.None has not proved that the conditioned medium that comes from the XC cell contains the active ingredient of cell death inducing in the LNCAP cell as a result for these.
Fig. 2: XC conditioned medium cell death inducing not in normal people's lung fibre source cell (CCD39Lu).The CCD39Lu cell was cultivated 15 hours with control medium or conditioned medium, then with 2 hours (as Fig. 1) of Hoechst stain dyeing.Cell looks it is very normal; Whether no matter cultivate with the XC cell conditioned medium, the nucleus of CCD39Lu all keeps equal state (Fig. 2 (A) and Fig. 2 (B)).These results proved the XC conditioned medium can not be in people's normal lung fibre source cell (CCD39Lu) cell death inducing.
Fig. 3: by the isolating Apogen P-1s of Q2 anion-exchange chromatography.Be concentrated by protein dissolved after the ammonium sulfide precipitate and separate, be loaded into then on the Q2 chromatographic column (Bio-Rad), this chromatographic column utilizes HPLC system of Bole biotech company by by A damping fluid (10mM Tris-HCL, pH7.4) and the linear gradient that makes up of B damping fluid (10mM Tris-HCL, pH7.4.0.55M NaCL) further launched.This linear gradient progressively makes up from being increased to 100% in the A damping fluid by made the B damping fluid in 10 minutes, and (20 milliliters of elution volumes) uses the 100%B damping fluid with this chromatographic column elution 5 minutes subsequently again.The activity of Apogen P-1 is tested by cell death inducing in the LNCAP cell.Three active peaks are arranged on the chromatogram collection of illustrative plates.In 18 hours, a level part 5-7 can cause 70% necrocytosis, and a level part 8-10 can cause 65% necrocytosis, and a level part 11-14 can cause 90% necrocytosis.We collect level part 5-7 and with its called after Apogen P-1a, a level part 8-10 is named as Apogen P-1b, and a level part 11-14 is named as Apogen P-1c.These three kinds of Apogen P-1s are further purified by reverse-phase chromatographic column.
Fig. 4: separate Apogen P-1 by reverse-phase chromatography.Apogen P-1a, ApogenP-1b and Apogen P-1c are concentrated into the 1.5ml volume respectively.In each sample, add 1 milliliter of methanol solution that contains 0.05% trifluoroacetic acid,, make most protein settling by this processing.And the active ingredient of cell death inducing is retained in the supernatant liquor.Then, supernatant liquor is applied to RP4 reverse-phase chromatographic column (Micra Scientific Inc), and by by solution A (H
2O, 0.05%TFA) and solution B (methyl alcohol, 0.05%TFA) linear gradient of Gou Jianing is unfolded.This linear gradient makes up (20 milliliters of elution volumes) by making solution B progressively be increased to 100% from 0% in solution A in 10 minutes, and uses 100% solution B with this chromatographic column elution 5 minutes subsequently.
The reverse-phase chromatography figure of Apogen P-1a is illustrated among Fig. 4 (a).A level part 12-13 has in the LNCAP cell with the activity of inducing 80% necrocytosis in 10 hours.
The reverse-phase chromatography figure of Apogen P-1b is illustrated among Fig. 4 (b).A level part 14-15 has in the LNCAP cell with the activity of inducing 45% necrocytosis in 18 hours.
The reverse-phase chromatography figure of Apogen P-1c is illustrated among Fig. 4 (c).A level part No.5 has in the LNCAP cell with the activity of inducing 52% necrocytosis in 18 hours.
The purity of separating back Apogen P-1a, Apogen P-1b and Apogen P-1c is to use the sds polyacrylamide gel electrophoresis of silver staining to detect.
Fig. 5: after the Apogen 1a that obtains by anion-exchange chromatography and reversed phase chromatography separation is concentrated again under the sex change condition experience by the electrophoresis of the Tris-Gly SDS-polyacrylamide gel of 4-20% gradient.This gel is by silver staining.What obtained is that molecular weight is the polypeptide chain of 70KD.This result shows that the Apogen P-1c that has the 70KD molecular weight on SDSPAGE is almost by purifying successfully.
Fig. 6: after the Apogen 1c that obtains by anion-exchange chromatography and reverse-phase chromatography and preparation electrophoretic separation is concentrated again under non-sex change condition experience by the SDS-polyacrylamide gel electrophoresis of 10% dissolving gel and 4% stacking gel.This gel is by silver staining.What obtained is that molecular weight is the polypeptide chain (Fig. 6 A) of 70KD.This result shows the purifying that the Apogen P-1c that has the 70KD molecular weight on SDSPAGE succeeds by reverse-phase chromatography.Causing molecular weight by the Apogen 1c for preparing the electrophoresis purifying is the proteinic separation (Fig. 6 B) of 57KD.These two kinds of protein are that the level part different same a kind of proteinic possibility of length during this time can't be affirmed.
Fig. 7: with conditioned medium cell death inducing in prostate cancer cell (LNCAP) of C3H IOTI/2 cell.After the LNCAP cell is cultivated 15 hours with control medium or conditioned medium, with Hoechst stain dyeing 2 hours.Shown in Fig. 7 A, the nucleus of the LNCAP cell of cultivating with control medium is normal.But, use the nucleus of the LNCAP cell of conditioned medium (X20 replaces to RPMI) cultivation to present features of apoptosis (Fig. 7 (B)).At first, conditioned medium causes nucleus to concentrate, and this can be proved by relatively having stronger fluorescent with the control cells nuclear shown in Fig. 7 (A).Secondly, caryoplasm concentrates the fragmentation that is attended by DNA simultaneously, and this can be proved by the nucleus fragmentation shown in Fig. 7 (B).As mentioned above, caryoplasm concentrates and the fragmentation of DNA is two morphological features of apoptosis state.None does not prove the active ingredient that contains cell death inducing in the LNCAP cell from the conditioned medium of XC cell as a result for these.
Fig. 8: C3H 1OT1/2 conditioned medium cell death inducing not in normal lung fibre source cell (CCD39Lu).As what in Fig. 1, introduce, after the CCD39Lu cell is cultivated 15 hours with control medium or conditioned medium, with Hoechst stain dyeing 2 hours.Cell looks it is very normal; Whether the nuclear of CCD39Lu cell no matter all keep equal state (Fig. 8 (A) and Fig. 8 (B)) with the conditioned medium cultivation of C3H10T112 cell.These results proof is C3H10T112 conditioned medium cell death inducing not in normal lung fibre source cell (CCD39Lu).
Fig. 9: the reverse-phase chromatography of Apogen P-2.Apogen P-2 by DE52 Mierocrystalline cellulose, hydroxylapatite and Vitrum AB agarose purifying will be further purified by reverse-phase chromatography.Apogen P-2 is concentrated into 1 milliliter.Add the methanol solution that 1ml contains 0.05% trifluoroacetic acid.Make most protein settling by this processing.But the active ingredient of cell death inducing (P-2) is retained in the supernatant liquor.Then, supernatant liquor is applied to that RP4 reverse-phase chromatographic column (Micra Scientific Inc) is gone up and by solution A (H20,0.05%TFA) and solution B (methyl alcohol, 0.05%TFA) linear gradient of Gou Jianing makes its expansion.This linear gradient makes up by making solution B progressively be increased to 100% from 0% in 10 minutes in solution A, (20 milliliters of elution volumes) and use 100% solution B with this chromatographic column elution 5 minutes subsequently.
Figure 10: after the Apogen P-2 that obtains by anion-exchange chromatography and reversed phase chromatography separation is concentrated again under the sex change condition experience by the electrophoresis of the SDS-polyacrylamide gel of 4-20% gradient.This gel is by silver staining.What obtained is that molecular weight is the polypeptide chain of 65KD.This result shows that on SDSPAGE molecular weight is that the Apogen P-2 of 65KD is by purifying successfully.
Figure 11: after the Apogen L that obtains by anion-exchange chromatography and reversed phase chromatography separation is concentrated again under the sex change condition experience by the electrophoresis of the SDS-polyacrylamide gel of 4-20% gradient.This gel is by silver staining.What obtained is that molecular weight is the polypeptide chain of 55KD.
Figure 12: the anion-exchange chromatography of Apogen L.Apogen L by DE52 Mierocrystalline cellulose and the resulting separation of Vitrum AB agarose is loaded into (BiO-Rad) on the Q2 chromatographic column after concentrating, and utilize HPLC system of Bole biotech company by A damping fluid (10mM Tris-HCL, pH7.4) and the linear gradient that makes up of B damping fluid (10mM Tris-HCL, pH7.4.0.55M NaCL) further launched.This linear gradient makes up by making the B damping fluid progressively be increased to 100% from 0% in 10 minutes in the A damping fluid.Level part 7 and 8 is included in the active ingredient of cell death inducing in the HL-60 cell.
Figure 13: with Apogen L cell death inducing in the HL-60 cell.Activity by the Apogen L of DE52 Mierocrystalline cellulose, Vitrum AB agarose and anion-exchange chromatography resulting separation is tested on following clone: HL-60 (leukemia) and CCD39Lu (normal lung fibre source cell).In order to contain the active ingredient of cell death inducing from morphology proof Apogen L, use isolating as mentioned above ApogenL to cultivate 15 hours in the HL-60 cell then with Hoechst stain dyeing 2 hours.As shown in FIG. 13A, the HL-60 nucleus of cultivating with control medium is very normal (A).But the HL-60 nucleus of cultivating with ApogenL presents features of apoptosis (Figure 13 B).At first, ApogenL can cause nucleus to concentrate, and this can be proved by relatively presenting stronger fluorescent with the control cells nuclear shown in Figure 13 A.Secondly, caryoplasm concentrates the fragmentation that is attended by DNA simultaneously, and this can be proved by the nuclear fragmentation shown in Figure 13 B.As mentioned above, caryoplasm concentrates and the fragmentation of DNA is two morphological features that cell is in apoptotic state.None has not proved that Apogen L contains the active ingredient of cell death inducing in the HL-60 cell as a result for these.
Figure 14: with Apogen L cell death inducing in MCF-7 (breast cancer cell).Activity by the Apogen L of DE52 Mierocrystalline cellulose, Vitrum AB agarose and anion-exchange chromatography resulting separation is checked on following clone: MCF-7 (breast cancer cell).For contain from morphology proof Apogen L can cell death inducing active ingredient, the MCF-7 cell was cultivated 15 hours with isolating Apogen L as mentioned above.Shown in Figure 14 A, the nuclear of the MCF-7 cell of cultivating with control medium is very normal.But the MCF-7 nucleus of cultivating with Apogen L presents features of apoptosis (Figure 14 B).None has not proved that Apogen L contains the active ingredient of cell death inducing in the MCF-7 cell as a result for these.
Figure 15: Apogen L does not induce normal laticiferous cell (Hs578Bst) apoptosis in MEM-Regular Insulin-10% blood serum medium.Hs578 Bst cell was cultivated 15 hours with control medium or conditioned medium.Cell looks it is normal; Whether no matter cultivate with Apogen L, the nucleus of Hs578Bst cell all keeps same feature.These results show Apogen L cell death inducing not in normal laticiferous cell (Hs578Bst).
The preparation of the preferred embodiments of the present invention A. method 1. conditioned mediums
A. separate the preparation of the XC conditioned medium of ApogenP-1
Apogen P-1 separates from the conditioned medium of the clone of a kind of XC of being called, and this clone derives from the mouse tumour and collects center (ATCC) available from U.S.'s typical culture.At first, XC cell inoculation (polystyrene, surface-area=850cm in rolling bottle
2, corning) and comprising CO
2, 10% N of tire serum (FBS), nonessential amino acid, penicillin and Streptomycin sulphate DMEM (=Dulbecco ' s ModificationofEagle ' s Medium (the Eagle substratum of Dulbecco modification)) in cultivated three days.Then, (3 * 100ml) washing XC cells do not comprise FBS (with CO at 100 milliliters then so that remove serum deprivation with PBS
2), cultivated 4 days among the DMEM of nonessential amino acid, penicillin and Streptomycin sulphate.By this conditioned medium of centrifugal collection and make it the clarification.
B. be used to separate the preparation of the C3H IOTI/2 conditioned medium of Apogen P-2
Apogen P-2 separates from the conditioned medium of the clone of a kind of C3H1OT1/2 of being called as, and this clone derives from the mouse embryo and can collect center (ATCC) available from U.S.'s typical culture.At first C3H 1OT1/2 cell inoculation (polystyrene, surface-area=850cm in rolling bottle
2, corning) and containing CO
2, 10% N of tire serum (FBS), penicillin and Streptomycin sulphate alpha-MEM (=alpha Modification ofEagle ' s Medium) in cultivated three days.(3 * 100ml) washing C3H1OT1/2 cells are to remove serum deprivation, and not comprising FBS at 100 milliliters then (has CO to use PBS then
2, penicillin and Streptomycin sulphate) alpha DMEM in cultivated 4 days.By this conditioned medium of centrifugal collection and make it the clarification.
C. be used to separate the preparation of the XC conditioned medium of Apogen L
Apogen L separates from the conditioned medium of the clone of a kind of XC of being called as, and this clone derives from the mouse tumour and can collect center (ATCC) available from U.S.'s typical culture.At first the XC cell inoculation to rolling (polystyrene, surface-area=850cm in the bottle
2, corning) and comprising CO
2, 10% N of tire serum (FBS), nonessential amino acid, penicillin and Streptomycin sulphate DMEM (=Dulbecco ' sModification of Eagle ' s Medium) in cultivated four days.By this conditioned medium of centrifugal collection and make it the clarification.2. experimentation
(a) necrocytosis (apoptosis) experiment
Prostate cancer cell line (LNCAP) is normally used for separating Apogen P-1 and Apogen P-2, and the leukemia cell is that HL-60 then is used to separate Apogen L.Experimental technique is as follows: 10 milliliters that are seeded in little plate (well of 25 microlitres, Robbins Scientific Corp.) at 37 degree a following LNCAP or HL-60 (1,000 cell) comprise 15% or 20% N of tire thrombotonin, penicillin, Streptomycin sulphate and 5%CO
2RPMI in.Spend 3-4 hour behind the inoculating cell and add 10 microlitre samples.After sample is cultivated 15 hours with cell, add 2 milliliters of Hoechst dye liquors (0.03ng/ml in PBS).After 2 hours, under luminescence microscope, observe with the painted cell of Hoechst stain.Make the identification easily of apoptotic cells nuclear with the dyeing of Hoechst stain, they show concentrating with broken of DNA.The per-cent of apoptotic cell calculates by following formula:
Percentage ratio=the DNA of apoptotic cell concentrates and broken cell count/total cell count
(b) cell rejection experiment
Select the HepG2 cell to study cell rejection activity two reasons are arranged: the first, the HepG2 cell is insensitive to Apogen P-1 aspect cell death inducing; The second, the size of Hep G2 cell is 3-4 a times of the membrane pore size size on the Transwell Insert, and this is very suitable cell size for research cell migration and cell intrusion.(Cambridge, MA), it is used to detect the activity of Apogen P-1b as chemotactic repellant available from Costar to be called as the tissue culture device of TranswellInsert.This device is extensively being used research cell migration or cell to invade, and it comprises a Shang Cang and a Lower Hold.Be that the aperture is many micropores polyester film that 3.0um, permission cell pass between these two bins.Cell to be tested is grown in last storehouse, and miscellany to be tested is placed in down in the storehouse.If this mixture to be tested is a chemical attractant, we should see has comparison to pass through microporous membrane according to the many cells of sample, in our experiment, cell size is equivalent to membrane pore size 3-4 HepG2 cell (100 doubly, 000 cell) (comprises 10%FBS at Shang Cang, PS and nonessential amino acid whose Minimum Essential Medium Eagle, 0.1 cultivated 2 hours milliliter), the ApogenP-1b (30 microlitre) by ammonium sulfide precipitation and aforesaid Q2 HPLC chromatography separated portions purifying be placed on comprise in 0.6 milliliter of same following storehouse that is fit to the substratum that the HepG2 cell grows then.After 15 hours, by handling the cell that microporous membrane was collected by microporous membrane in 30 minutes with the 0.2ml insulin solutions.With count cell number in the 10 microlitre insulin solutions of blood cell register.3. proteinic separation
A. separate Apogen P-1
Step 1: ammonium sulphate precipitation
Make Apogen P-1 by 80% saturated ammonium sulfide sedimentation by in every liter of XC conditioned medium, adding the 561g ammonium sulfide.By the tiny protein particulate of centrifugal collection, and they are dissolved among the 10mM Tris-HCl (pH7.4).Remove ammonium sulfide by dialysis, then by the protein of Q2 HPLC chromatographic column separate dissolved.
Step 2:Q2 HPLC high performance liquid chromatography
Be loaded on the Q2 chromatographic column (BiO-Rad) after concentrating by ammonium sulfide precipitate and separate and dissolved protein, the HPLC system that utilizes Bole biotech company is by A damping fluid (10mM Tris-HCl, pH7.4) and the constructed linear gradient of B damping fluid (10mM Tris-HCl, pH7.4.0.55M NaCl) further launched.This linear gradient is by making the B damping fluid in the A damping fluid progressively increase to 100% (the 20 milliliters of elution volume) that makes up from 0% in 10 minutes, and uses the 100%B damping fluid with this chromatographic column elution 5 minutes subsequently.Can verify the activity of Apogen P-1 by cell death inducing in the LNCAP cell.We find that three active peaks are arranged on the chromatogram collection of illustrative plates.In 18 hours, level part 5 to 7 can cause 70% necrocytosis, and a level part 8-10 can cause 65% necrocytosis, and a level part 11-14 can cause 90% necrocytosis.We collect level part 5-7 and with its called after Apogen P-1a, a level part 8-10 is named as Apogen P-1b, and a level part 11-14 is named as Apogen P-1c.By reverse-phase chromatography these three kinds of ApogenP-1 ' s are further purified.
Step 3: reverse-phase chromatography
Apogen P-1a, Apogen P-1b and Apogen P-1c are concentrated into 1.5 milliliters respectively.In each sample, add 1 milliliter of methanol solution that contains 0.05% trifluoroacetic acid, make most protein settling by this processing.But the active ingredient of cell death inducing is retained in the supernatant liquor.Then, supernatant liquor is applied to that RP4 reverse-phase chromatographic column (MicraScientific Inc) is gone up and by solution A (H2O, 0.05%TFA) and solution B (Methanol, 0.05%TFA) constructed linear gradient makes it expansion.This linear gradient is by making the solution B in the solution A progressively be increased to 100% (the 20 milliliters of elution volume) that makes up from 0% in 10 minutes, uses the 100%B damping fluid with this chromatographic column wash-out 5 minutes then.
Step 4: preparation electrophoresis
(step 3) and preparation electrophoresis (with the MiniPrep Gel electrophoresis apparatus of BIO--RED company) are purified by reverse-phase chromatography to separate the Apogen 1c that obtains with anion-exchange chromatography.The reverse-phase chromatography of Apogen P-1a is illustrated in Fig. 4 (a), and level part 12-13 has in the LNCAP cell activity with inductive 80% necrocytosis in 10 hours.
The reverse-phase chromatography of Apogen P-1b is illustrated in Fig. 4 (b). Level parts 14 and 15 has at the LNCAP cell with the activity of inducing 45% necrocytosis in 18 hours.
The reverse-phase chromatography of Apogen P-1c is illustrated in Fig. 4 (c).Level parts 5 has in the LNCAP cell to induce the activity of 52% necrocytosis in 18 hours.
Separated Apogen P-1a, Apogen P-1b, the purity of Apogen P-1c is to detect by the sds polyacrylamide gel electrophoresis of silver staining.
(1) Apogen P-1a: as shown in Figure 5, what obtained is that molecular weight is the polypeptide chain of 70KD.This result shows that molecular weight is that the ApogenP-1a of 70KD is by the purifying of success on SDSPAGE.
(2) Apogen P-1b: what obtained is that molecular weight is the polypeptide chain of 55KD.This result shows that on SDSPAGE molecular weight is that the Apogen P-1b of 55KD is by the purifying of success.
(3) Apogen P-1c: it is the protein chain of 70KD that the purifying of finishing Apogen 1c by reverse-phase chromatography causes molecular weight, otherwise it is the protein chain of 57KD that the purifying of finishing Apogen 1c by the preparation electrophoresis causes molecular weight.Shown in Fig. 6 (A), molecular weight is that the protein main chain of 70KD obtains by reverse-phase chromatography.On the other hand, molecular weight be 57KD protein chain by the preparation electrophoretic separation (Fig. 6 B).
The separation of B.Apogen P-2
The Apogen P-2 that is present in the C3H1OT1/2 conditioned medium can separate as follows:
Step 1: ammonium sulfide precipitation
Make Apogen P-2 by 80% saturated ammonium sulfide sedimentation by in every liter of conditioned medium, adding the 561g ammonium sulfide.By the tiny protein particulate of centrifugal collection, and they are dissolved among the 10mM Tris-HCl (pH7.4).
Step 2: handle with hydroxylapatite
After ammonium sulfide was removed in dialysis in 10mM Tris-HCL (pH7.5), (Bio-Gel HTP gel Bio-Rad) cultivated dissolved protein 1 hour with the phosphatic rock gel.Remove after the HTP gel by centrifugal, people will find that the activity of cell death inducing in the LNCAP cell is present in subsequently in the supernatant liquor that will further handle with the Vitrum AB sepharose.
Step 3: the Vitrum AB agarose is handled
Further use Vitrum AB agarose (Sigma) to cultivate 1 hour the supernatant liquor in second step.Remove after the HTP gel by centrifugal, people will find that the active ingredient of cell death inducing in the LNCAP cell is present in the supernatant liquor.
Step 4: reverse-phase chromatography
The Apogen P-2 that is present in the supernatant liquor of Vitrum AB agarose in the 3rd step is further purified by reverse-phase chromatography.Apogen P-2 is concentrated into 1 milliliter.Add 1ml and contain the methanol solution of 0.05% trifluoroacetic acid.Make the most protein sedimentation by this processing, and the active ingredient of cell death inducing (P-2) is retained in the supernatant liquor.Then, supernatant liquor is loaded into that RP4 reverse-phase chromatographic column (Micra Scientific Inc) is gone up and by solution A (water, 0.05%TFA) and solution B (methyl alcohol, 0.05%TFA) constructed linear gradient makes it to launch.This linear gradient is to make up by making solution B in the solution A progressively be increased to 100% from 0% in 10 minutes, and (20 milliliters of elution volumes) uses 100% solution B with this chromatographic column elution 5 minutes then.The reverse-phase chromatography of Apogen P-2 is illustrated in Fig. 9.A level part 12-14 has in the LNCAP cell with the activity of inducing 80% necrocytosis in 12 hours.The purity of Apogen P-2 after the separation is to measure by the SDS polyacrylamide gel electrophoresis of silver staining.What obtained is that molecular weight is the protein chain (Figure 10) of 65Kd.
The separation of C.Apogen L
The Apogen L that is present in the conditioned medium can separate through the following steps:
Step 1:DE52 absorbs
(diethylaminoethylcellulose Whatman) is cultivated conditioned medium 1 hour with anionite DE52.The mixture that centrifugal treating is cultivated is collected and is washed and Apogen L bonded DE52 with the 10mM Tris-HCl (pH7.5) that contains 0.15M NaCl.Then, with the 10mM Tris-HCl (pH7.5) that comprises 0.1 5M NaCl Apogen L elution from DE52 is come out.
Step 2: the Vitrum AB agarose absorbs
Further absorbed in 1 hour by it being cultivated through the described isolating Apogen L of step 1 with Vitrum AB agarose (Sigma) by the Vitrum AB agarose.Wash by centrifugal collection Vitrum AB agarose and with 10mM Tris-HCl (pH7.5).The Apogen L that is absorbed by the Vitrum AB agarose with the 2MNaCl elution then.
Step 3:Q2 HPLC high performance liquid chromatography
Isolating as mentioned above Apogen L is loaded on the Q2 chromatographic column (BiO-Rad) after concentrating, and utilize HPLC system of Bole biotech company by A damping fluid (10mM Tris-HCL, pH7.4) and the B damping fluid (10mM Tris-HCl, pH7.4.0.5MNaCL) constructed linear gradient is further launched.This linear gradient is by making the B damping fluid in the A damping fluid progressively be increased to 100% structure from 0% in 10 minutes.Color atlas as shown in figure 10.The purity of separating back Apogen L is to measure with the SDS polyacrylamide gel electrophoresis of silver staining.What obtained is to have the protein chain (Figure 11) that active molecular weight is approximately 55Kd.Discuss
The invention describes the separation method of five kinds of protein (Apogen P-1a, Apogen P-1b, ApogenP-1c, Apogen P-2 and Apogen L), these albumen mass-energy cell death inducing in following cell is included in (Apogen P-1 ' s) in the prostate cancer cell, in prostate cancer cell and breast cancer cell (Apogen P-2) and in leukemia and breast cancer cell (Apogen L).The protein that following evidence makes us be sure of these cell death inducings is novel and is never found by the people before this: tumour necrosis factor (TNF), transforming growth factor (TGF-Beta), Fas ligand and TRAIL are the protein (Lin that once was reported in cell death inducing in some clone, J.K. wait the people, Cancer Research, 52,385,1992.; Kawakawi, people such as M., J.of CellularPhysiology 138:1,1989; Wiley, people such as S.R., Immunity3,673,1995; People such as Tomei, " Apoptosis ": The molecular Basis of Cell Death, press of cold spring harbor laboratory, pp.87,1991).These evidences show that these five kinds of protein are different from the protein of these known cell death inducings fully:
(1) active different fully; In our experiment, even use very high dosage (10ng/ml), TNF and TGF be cell death inducing and do not have this effect in prostate cancer cell (LNCAP) in liver cancer cell only also.Otherwise, Apogen P-1 ' s and Apogen P-2 will be in prostate cancer (LNCAP) cell rather than in liver cancer cell cell death inducing.
(2) TRAIL and Fas are membrane-bound protein (wiley, people such as S.R., Immunity3,673,1995; People such as Tomei, " Apoptosis ": The molecularBasis of Cell Death (molecular basis of necrocytosis), press of cold spring harbor laboratory, PP.87,1991), and Apogen P-1a, Apogen P-1b, ApogenP-1c, ApogenP-2 and Apogen L are soluble protein rather than membrane bound protein.
(3) molecular weight of TNF, TGF and Fas ligand TRAIL is approximately 17 to 40Kd (TNF=17KD, TGF=24KD, TRAIL=32KD, Fas ligand=43KD) (McGrath, M.H.Clinics in Plastic surgery 17:421,1993; Wiley, people such as S.R., Immunity3,673,1995; People such as Tomei, " Apoptosis ": The molecular Basis of Cell Death (molecular basis of necrocytosis), press of cold spring harbor laboratory, PP.87,1991), otherwise the molecular weight of Apogen P-1a, Apogen P-1b, Apogen P-1c, Apogen P-2 and Apogen L is between 55 to 70Kd.
Claims (25)
1. the molecular weight protein between 57 to 70Kd greatly, described protein has apoptotic effect to other cancer cells except that lung fibre source cell (CCD39Lu) and breast cancer cell (MCF-7).
2. protein according to claim 1, the cancer cells of wherein observing the apoptosis effect is a prostate cancer cell.
3. protein according to claim 1, wherein said protein are non-membrane bound protein.
4. protein according to claim 1, wherein said protein have the feature of the cell of the cell mass that repels to come free HBPG-2 or HL-60 composition.
5. protein has following characteristics at least:
A. its molecular weight is greatly between 57 to 70Kd;
B. except lung fibre source cell (CCD39Lu) and breast cancer cell (MCF-7)
Other cancer cells all had the apoptosis effect outward; And
D. described protein is non-membrane-bound.
6. according to the described protein of claim 5, the cell of wherein observing the apoptosis effect is a prostate cancer cell.
7. according to the described protein of claim 5, wherein said protein has the feature of the cell of the cell mass that repels to come free HBPG-2 or HL-60 composition.
8. protein has following characteristics at least:
A. its molecular weight is greatly between 57 to 70Kd;
B. prostate cancer cell there is apoptotic effect;
C. has the cell that repels the cell mass that free HBPG-2 or HL-60 form
Feature; And
D. described protein is non-membrane-bound.
9. protein has following characteristics at least:
A. its molecular weight approximately is 65Kd; And
B. except lung fibre source cell (CCD39Lu), other cancer cells are all had carefully
Born of the same parents' apoptotic effect.
10. protein according to claim 9, the cell of wherein observing the apoptosis effect is a prostate cancer cell.
11. protein according to claim 9, the cell of wherein observing the apoptosis effect is a breast cancer cell.
12. protein according to claim 9, wherein said protein are non-membrane-bound.
13. protein according to claim 9, wherein said protein have the feature of the cell of the cell mass that repels to come free HBPG-2 or HL-60 composition.
14. a protein has following characteristics at least:
A. molecular weight approximately is 65Kd;
B. except lung fibre source cell (CCD39Lu), other cancer cells are all had carefully
Born of the same parents' apoptotic effect;
C. right and wrong are membrane-bound; And
D. has the cell that repels the cell mass that free HBPG-2 or HL-60 form
Feature.
15. protein according to claim 14, the cell of wherein observing the apoptosis effect is a prostate cancer cell.
16. protein according to claim 14, the cell of wherein observing the apoptosis effect is a breast cancer cell.
17. a protein has following characteristics at least:
A. molecular weight approximately is 65Kd;
B. prostate cancer cell and breast cancer cell there is apoptosis-induced effect;
C. right and wrong are membrane-bound; And
D. to come the cell of the cell mass that free HBPG-2 or HL-60 form be the spy to repel
Levy.
18. a protein shows at least as following feature:
A. its molecular weight approximately is 55Kd; And
B. except lung fibre source cell (CCD39Lu), other cancer cells are all lured
The guided cell effect of apoptosis.
19. protein according to claim 18, the cell of wherein observing the apoptosis effect is the leukemia cell.
20. protein according to claim 18, the cell of wherein observing the apoptosis effect is a breast cancer cell.
21. protein according to claim 18, wherein said protein right and wrong are membrane-bound.
22. a protein shows at least as following feature:
A. molecular weight approximately is 55Kd;
B. except lung fibre source cell (CCD39Lu), other cancer cells are all lured
The guided cell apoptotic effect, and
C. described protein right and wrong are membrane-bound.
23. protein according to claim 22, the cell of wherein observing the apoptosis effect is the leukemia cell.
24. protein according to claim 22, the cell of wherein observing the apoptosis effect is a breast cancer cell.
25. a protein presents following characteristics at least:
A. molecular weight approximately is 55Kd;
B. leukemia and breast cancer cell had the apoptosis effect; And
C. described protein right and wrong are membrane-bound.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US99343297A | 1997-12-18 | 1997-12-18 | |
| US08/993,432 | 1997-12-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1314914A true CN1314914A (en) | 2001-09-26 |
Family
ID=25539541
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98813660A Pending CN1314914A (en) | 1997-12-18 | 1998-12-18 | Proteins for cancer cell specific induction of apoptosis and method for isolation thereof |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP1037919A1 (en) |
| JP (1) | JP2002516821A (en) |
| KR (1) | KR20010033260A (en) |
| CN (1) | CN1314914A (en) |
| AU (1) | AU1933099A (en) |
| CA (1) | CA2325368A1 (en) |
| IL (1) | IL136850A0 (en) |
| MX (1) | MXPA00005954A (en) |
| NO (1) | NO20003099L (en) |
| PL (1) | PL343247A1 (en) |
| WO (1) | WO1999031135A1 (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997031107A2 (en) * | 1996-02-20 | 1997-08-28 | Coles John G | Human serum lectin - induced apoptosis and method for detecting apoptosis |
-
1998
- 1998-12-18 EP EP98964142A patent/EP1037919A1/en not_active Withdrawn
- 1998-12-18 JP JP2000539058A patent/JP2002516821A/en active Pending
- 1998-12-18 IL IL13685098A patent/IL136850A0/en unknown
- 1998-12-18 PL PL98343247A patent/PL343247A1/en unknown
- 1998-12-18 CA CA002325368A patent/CA2325368A1/en not_active Abandoned
- 1998-12-18 KR KR1020007006696A patent/KR20010033260A/en not_active Withdrawn
- 1998-12-18 AU AU19330/99A patent/AU1933099A/en not_active Abandoned
- 1998-12-18 MX MXPA00005954A patent/MXPA00005954A/en unknown
- 1998-12-18 CN CN98813660A patent/CN1314914A/en active Pending
- 1998-12-18 WO PCT/US1998/027108 patent/WO1999031135A1/en not_active Application Discontinuation
-
2000
- 2000-06-16 NO NO20003099A patent/NO20003099L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| IL136850A0 (en) | 2001-06-14 |
| CA2325368A1 (en) | 1999-06-24 |
| PL343247A1 (en) | 2001-07-30 |
| NO20003099L (en) | 2000-08-17 |
| WO1999031135A1 (en) | 1999-06-24 |
| AU1933099A (en) | 1999-07-05 |
| EP1037919A1 (en) | 2000-09-27 |
| MXPA00005954A (en) | 2002-09-18 |
| NO20003099D0 (en) | 2000-06-16 |
| JP2002516821A (en) | 2002-06-11 |
| KR20010033260A (en) | 2001-04-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112442116B (en) | Novel micro-peptide HMMW and application thereof | |
| CN104530199B (en) | A kind of tumor protein p53 and its preparation method and application | |
| Schmidt-Zachmann et al. | Identification and localization of a novel nucleolar protein of high molecular weight by a monoclonal antibody | |
| CN114349829B (en) | Identification and Application of ALV-J MHC-B2 Restricted Epitope Peptide | |
| CN110330551B (en) | Pancreatic cancer-specific binding peptide and its preparation method and use | |
| CN1314914A (en) | Proteins for cancer cell specific induction of apoptosis and method for isolation thereof | |
| CN105693838A (en) | Pinctada martensii antimicrobial peptide PmAMP and application thereof | |
| CN1679911A (en) | Method for producing antrodia camphorata culture and products obtained by said method | |
| US6258779B1 (en) | Method of using fetuin to induce apoptosis in cancer cells | |
| CN102172394B (en) | Application of a Ciona polypeptide | |
| CN107188930A (en) | A kind of polypeptide for suppressing tumour cell diffusion transfer and its preparation method and application | |
| US20030027767A1 (en) | Polypeptide for the treatment of cancer and a method for preparation thereof | |
| US6737402B2 (en) | Method of preparing fetuin to induce apoptosis | |
| CN1883703A (en) | Serine protease inhibitor of Rana grahami, its gene and application | |
| CN111778236B (en) | Shellfish genome DNA extraction method based on 3D printing special-shaped functional body, kit and application thereof | |
| CN104987381B (en) | Recombinant positively charged polypeptide interferon and its application in antitumor and antiviral therapy | |
| CN119912531B (en) | CB2 agonistic peptide for promoting bone formation and application thereof | |
| CN102174096B (en) | Ciona intestinalis polypeptide and preparation method thereof | |
| CN119015286B (en) | Use of Ispins combined with tamoxifen in the treatment of breast cancer | |
| CN117487750B (en) | Use of NK cells in the treatment of immune related disorders | |
| CN105238833B (en) | Application of bullacta oligopeptide in resisting prostatic cancer | |
| CN1840544A (en) | ESC42 protein, preparation method and use thereof | |
| CN118995682B (en) | Application of bacillus cereus 2, 3-phosphoglycerate mutase antibacterial peptide BM16 in preparation of antibacterial drugs | |
| CN114569709B (en) | Preparation method and application of melanoma autologous tumor vaccine with high ADAM-28 expression | |
| CN1313386A (en) | Kuhseng agglutinin and its zoophobous and disease-resistant coding gene and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| C10 | Entry into substantive examination | ||
| PB01 | Publication | ||
| 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 |