KR20220054174A - Composition for preventing or treating liver cancer comprising OSMI-1 and antitumor agent - Google Patents
Composition for preventing or treating liver cancer comprising OSMI-1 and antitumor agent Download PDFInfo
- Publication number
- KR20220054174A KR20220054174A KR1020210101696A KR20210101696A KR20220054174A KR 20220054174 A KR20220054174 A KR 20220054174A KR 1020210101696 A KR1020210101696 A KR 1020210101696A KR 20210101696 A KR20210101696 A KR 20210101696A KR 20220054174 A KR20220054174 A KR 20220054174A
- Authority
- KR
- South Korea
- Prior art keywords
- osmi
- liver cancer
- dox
- confirmed
- pharmaceutical composition
- Prior art date
Links
- IYIGLWQQAMROOF-HHHXNRCGSA-N OSMI-1 Chemical compound COC1=C(C=CC=C1)[C@@H](NS(=O)(=O)C1=CC2=C(NC(=O)C=C2)C=C1)C(=O)N(CC1=CC=CO1)CC1=CC=CS1 IYIGLWQQAMROOF-HHHXNRCGSA-N 0.000 title claims abstract description 132
- 201000007270 liver cancer Diseases 0.000 title claims abstract description 58
- 208000014018 liver neoplasm Diseases 0.000 title claims abstract description 58
- 239000002246 antineoplastic agent Substances 0.000 title claims abstract description 34
- 239000000203 mixture Substances 0.000 title abstract description 9
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 claims abstract description 290
- 229960004679 doxorubicin Drugs 0.000 claims abstract description 144
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 claims abstract description 68
- 230000006907 apoptotic process Effects 0.000 claims abstract description 47
- 230000000694 effects Effects 0.000 claims abstract description 45
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 23
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 14
- 239000004480 active ingredient Substances 0.000 claims abstract description 12
- 230000001093 anti-cancer Effects 0.000 claims abstract description 12
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 claims description 64
- 230000014509 gene expression Effects 0.000 claims description 33
- 108010057466 NF-kappa B Proteins 0.000 claims description 15
- 102000003945 NF-kappa B Human genes 0.000 claims description 15
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 claims description 8
- 229960004562 carboplatin Drugs 0.000 claims description 8
- 190000008236 carboplatin Chemical compound 0.000 claims description 8
- 229960004316 cisplatin Drugs 0.000 claims description 8
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 8
- 229960002949 fluorouracil Drugs 0.000 claims description 8
- 229960005277 gemcitabine Drugs 0.000 claims description 8
- 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 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 229960001756 oxaliplatin Drugs 0.000 claims description 8
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical group O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 claims description 8
- 229960005079 pemetrexed Drugs 0.000 claims description 8
- 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 claims description 8
- 239000002671 adjuvant Substances 0.000 claims description 7
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 claims description 6
- 206010059866 Drug resistance Diseases 0.000 claims description 6
- 229960004397 cyclophosphamide Drugs 0.000 claims description 6
- 239000003642 reactive oxygen metabolite Substances 0.000 claims description 5
- 101100452784 Caenorhabditis elegans ire-1 gene Proteins 0.000 claims description 4
- 229930012538 Paclitaxel Natural products 0.000 claims description 4
- 230000004611 cancer cell death Effects 0.000 claims description 4
- 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 claims description 4
- 229960005420 etoposide Drugs 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 229960001592 paclitaxel Drugs 0.000 claims description 4
- 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 claims description 4
- 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 claims description 4
- 229960004528 vincristine Drugs 0.000 claims description 4
- 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 claims description 4
- 230000001737 promoting effect Effects 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 2
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 abstract description 15
- 101150017888 Bcl2 gene Proteins 0.000 abstract description 15
- 230000002195 synergetic effect Effects 0.000 abstract description 5
- 230000002438 mitochondrial effect Effects 0.000 abstract description 4
- 230000037361 pathway Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 117
- 238000011282 treatment Methods 0.000 description 59
- 230000001965 increasing effect Effects 0.000 description 35
- 206010028980 Neoplasm Diseases 0.000 description 32
- 102100031929 UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110 kDa subunit Human genes 0.000 description 28
- 101710117112 UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110 kDa subunit Proteins 0.000 description 28
- 102100035100 Transcription factor p65 Human genes 0.000 description 25
- 230000006271 O-GlcNAcylation Effects 0.000 description 22
- 238000001262 western blot Methods 0.000 description 22
- 238000011284 combination treatment Methods 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 16
- 102100034174 Eukaryotic translation initiation factor 2-alpha kinase 3 Human genes 0.000 description 14
- 108091008010 PERKs Proteins 0.000 description 14
- 102000003952 Caspase 3 Human genes 0.000 description 13
- 108090000397 Caspase 3 Proteins 0.000 description 13
- 230000011664 signaling Effects 0.000 description 13
- 230000003833 cell viability Effects 0.000 description 12
- 230000003247 decreasing effect Effects 0.000 description 12
- 101710179684 Poly [ADP-ribose] polymerase Proteins 0.000 description 11
- 229920000776 Poly(Adenosine diphosphate-ribose) polymerase Polymers 0.000 description 11
- 108020004459 Small interfering RNA Proteins 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 11
- 201000011510 cancer Diseases 0.000 description 11
- 230000001419 dependent effect Effects 0.000 description 11
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 10
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 description 10
- 230000004913 activation Effects 0.000 description 10
- 230000008859 change Effects 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 230000002829 reductive effect Effects 0.000 description 9
- 230000035899 viability Effects 0.000 description 9
- -1 and O-GlcNAc Proteins 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 238000012986 modification Methods 0.000 description 8
- 102100022900 Actin, cytoplasmic 1 Human genes 0.000 description 7
- 108010085238 Actins Proteins 0.000 description 7
- 210000003494 hepatocyte Anatomy 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 101000891649 Homo sapiens Transcription elongation factor A protein-like 1 Proteins 0.000 description 6
- 102000055102 bcl-2-Associated X Human genes 0.000 description 6
- 108700000707 bcl-2-Associated X Proteins 0.000 description 6
- 238000001114 immunoprecipitation Methods 0.000 description 6
- 238000011580 nude mouse model Methods 0.000 description 6
- 108091006081 Inositol-requiring enzyme-1 Proteins 0.000 description 5
- 101000596402 Mus musculus Neuronal vesicle trafficking-associated protein 1 Proteins 0.000 description 5
- 101000800539 Mus musculus Translationally-controlled tumor protein Proteins 0.000 description 5
- 101000781972 Schizosaccharomyces pombe (strain 972 / ATCC 24843) Protein wos2 Proteins 0.000 description 5
- 101001009610 Toxoplasma gondii Dense granule protein 5 Proteins 0.000 description 5
- 102100040250 Transcription elongation factor A protein-like 1 Human genes 0.000 description 5
- 238000012790 confirmation Methods 0.000 description 5
- 231100000673 dose–response relationship Toxicity 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 239000000546 pharmaceutical excipient Substances 0.000 description 5
- 230000026731 phosphorylation Effects 0.000 description 5
- 238000006366 phosphorylation reaction Methods 0.000 description 5
- 230000035755 proliferation Effects 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 230000035882 stress Effects 0.000 description 5
- 102000002804 Ataxia Telangiectasia Mutated Proteins Human genes 0.000 description 4
- 108010004586 Ataxia Telangiectasia Mutated Proteins Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 238000011278 co-treatment Methods 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 4
- 238000003119 immunoblot Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 230000016914 response to endoplasmic reticulum stress Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 101100508533 Drosophila melanogaster IKKbeta gene Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 3
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 3
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 230000003828 downregulation Effects 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 3
- 230000002055 immunohistochemical effect Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000000314 lubricant Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000005937 nuclear translocation Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 239000000829 suppository Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000012827 ATM inhibitor Substances 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 201000009030 Carcinoma Diseases 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102100030013 Endoribonuclease Human genes 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 241000711549 Hepacivirus C Species 0.000 description 2
- 101001010783 Homo sapiens Endoribonuclease Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 102000005520 O-GlcNAc transferase Human genes 0.000 description 2
- 108010077991 O-GlcNAc transferase Proteins 0.000 description 2
- 108091006041 O-GlcNAcylated proteins Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- 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 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 101710120037 Toxin CcdB Proteins 0.000 description 2
- 102000004243 Tubulin Human genes 0.000 description 2
- 108090000704 Tubulin Proteins 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000005754 cellular signaling Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 230000005757 colony formation Effects 0.000 description 2
- 238000010293 colony formation assay Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000003599 food sweetener Nutrition 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 239000012660 pharmacological inhibitor Substances 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000000861 pro-apoptotic effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000007781 signaling event Effects 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 239000003765 sweetening agent Substances 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 239000000454 talc Substances 0.000 description 2
- 229910052623 talc Inorganic materials 0.000 description 2
- 235000012222 talc Nutrition 0.000 description 2
- 230000005740 tumor formation Effects 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- QAPSNMNOIOSXSQ-YNEHKIRRSA-N 1-[(2r,4s,5r)-4-[tert-butyl(dimethyl)silyl]oxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O[Si](C)(C)C(C)(C)C)C1 QAPSNMNOIOSXSQ-YNEHKIRRSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- HSTOKWSFWGCZMH-UHFFFAOYSA-N 3,3'-diaminobenzidine Chemical compound C1=C(N)C(N)=CC=C1C1=CC=C(N)C(N)=C1 HSTOKWSFWGCZMH-UHFFFAOYSA-N 0.000 description 1
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- 235000006491 Acacia senegal Nutrition 0.000 description 1
- 229930195730 Aflatoxin Natural products 0.000 description 1
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 1
- 208000022309 Alcoholic Liver disease Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- PTHCMJGKKRQCBF-UHFFFAOYSA-N Cellulose, microcrystalline Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC)C(CO)O1 PTHCMJGKKRQCBF-UHFFFAOYSA-N 0.000 description 1
- 102100033270 Cyclin-dependent kinase inhibitor 1 Human genes 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 208000018565 Hemochromatosis Diseases 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108010047294 Lamins Proteins 0.000 description 1
- 102000006835 Lamins Human genes 0.000 description 1
- 239000012098 Lipofectamine RNAiMAX Substances 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 1
- 101710089543 Nitric oxide synthase, inducible Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 102100030122 Protein O-GlcNAcase Human genes 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 238000010818 SYBR green PCR Master Mix Methods 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 244000299461 Theobroma cacao Species 0.000 description 1
- 235000005764 Theobroma cacao ssp. cacao Nutrition 0.000 description 1
- 235000005767 Theobroma cacao ssp. sphaerocarpum Nutrition 0.000 description 1
- 102000005747 Transcription Factor RelA Human genes 0.000 description 1
- 108010031154 Transcription Factor RelA Proteins 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000005409 aflatoxin Substances 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000005775 apoptotic pathway Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 235000001046 cacaotero Nutrition 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 1
- 239000012830 cancer therapeutic Substances 0.000 description 1
- 210000001715 carotid artery Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 230000010001 cellular homeostasis Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000010109 chemoembolization Effects 0.000 description 1
- 208000006990 cholangiocarcinoma Diseases 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 230000001447 compensatory effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000003111 delayed effect Effects 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
- 229960003957 dexamethasone Drugs 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 238000010185 immunofluorescence analysis Methods 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 201000007450 intrahepatic cholangiocarcinoma Diseases 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 210000005053 lamin Anatomy 0.000 description 1
- VMPHSYLJUKZBJJ-UHFFFAOYSA-N lauric acid triglyceride Natural products CCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCC)COC(=O)CCCCCCCCCCC VMPHSYLJUKZBJJ-UHFFFAOYSA-N 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229960003511 macrogol Drugs 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 239000000845 maltitol Substances 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 231100000324 minimal toxicity Toxicity 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000030648 nucleus localization Effects 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 108700025694 p53 Genes Proteins 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000001686 pro-survival effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 108091006091 regulatory enzymes Proteins 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 230000011506 response to oxidative stress Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 238000011272 standard treatment Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012956 testing procedure Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000012130 whole-cell lysate Substances 0.000 description 1
- 238000012447 xenograft mouse model Methods 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—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
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
-
- 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
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
본 발명은 OSMI-1 및 항암제를 유효성분으로 함유하는 간암 예방 또는 치료용 약학조성물 및 항암보조제에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating liver cancer containing OSMI-1 and an anticancer agent as active ingredients, and an anticancer adjuvant.
간암(Hepatoma)은 전 세계적으로 가장 흔한 암 중의 하나로, OECD 국가 중 우리나라는 간암 발생률 1위로서 2011년 전체 사망자 중 27.8%가 암으로 사망하였고 이중 폐암은 22.2%로 1위, 간암은 15.3%로 2위를 기록하였다. 최근 간암에 대한 치료법(간절제술, 간이식 및 항암요법 등)이 괄목할만하게 발전하고 있음에도 불구하고 간암의 5년 생존율은 췌장암, 폐암에 이어 세 번째로 낮은 것으로 보고되고 있고, 상대 생존율 변화를 보면 26.7%의 증가 추세를 보이고 있지만 수술 후 5년 내 재발률이 70% 이상으로 보고되고 있어 난치성 암으로 분류되고 있다.Hepatoma is one of the most common cancers worldwide. Among OECD countries, Korea has the highest incidence of liver cancer, accounting for 27.8% of all deaths in 2011. ranked second. In spite of the remarkable development of treatments for liver cancer (liver resection, liver transplantation, and chemotherapy, etc.) in recent years, the 5-year survival rate for liver cancer is reported to be the third lowest after pancreatic cancer and lung cancer. Although it shows an increasing trend of 26.7%, the recurrence rate within 5 years after surgery is reported to be over 70%, so it is classified as intractable cancer.
간의 대부분을 차지하는 간세포에서 기원하는 악성 종양을 말한다. 넓은 의미로는 간에 생기는 모든 종류의 악성 종양(예를 들면 간내 담관암)이나 다른 기관의 암이 간에 전이되어 발생하는 전이성 간암까지도 포함하지만, 간세포암종이 간암 중 가장 흔하기 때문에 일반적으로는 간세포에서 발생하는 간세포암종만을 의미한다.It refers to a malignant tumor originating from hepatocytes, which occupy most of the liver. In a broad sense, it includes all types of malignant tumors of the liver (e.g., intrahepatic cholangiocarcinoma) and even metastatic liver cancer that occurs when cancers of other organs have metastasized to the liver. means hepatocellular carcinoma only.
간암의 주요 원인은 B형 간염바이러스(HBV), C형 간염바이러스(HCV)에 의한 급성 및 만성 감염이며, 이들 바이러스에 의한 만성 감염의 결과 간경변증이 발생하고 장기간 염증이 진행하면서 간암으로 진행되는 경우가 대부분이다. 이외에도 알코올성 간질환, 혈색소증, 흡연, 아플라톡신(aflatoxin)과 같은 독소에 의한 간 손상 등의 만성 간 질환이 모두 간암의 원인이 될 수 있다.The main cause of liver cancer is acute and chronic infection by hepatitis B virus (HBV) and hepatitis C virus (HCV). is mostly In addition, chronic liver diseases such as alcoholic liver disease, hemochromatosis, smoking, and liver damage caused by toxins such as aflatoxin can all cause liver cancer.
간암은 진행 속도가 매우 빠르므로 조기 검진이 무엇보다 중요하다. 간암의 치료방법으로는 수술로 암 조직을 제거하는 방법, 경동맥 화학 색전술, 경피적 에탄올 주입술, 고주파 응고 치료를 이용하여 암세포를 사멸시키는 방법 등이 있으나, 이들 방법은 간암의 크기가 너무 크거나, 큰 혈관이 침범되어 있거나 다른 장기에 암이 전이되어 있는 경우 효과적이지 못하거나 값이 비싼 단점이 있다.Liver cancer progresses very quickly, so early detection is of utmost importance. Treatment methods for liver cancer include surgical removal of cancerous tissue, carotid artery chemoembolization, percutaneous ethanol injection, and a method of killing cancer cells using radiofrequency coagulation therapy. When blood vessels are invaded or cancer has metastasized to other organs, it is ineffective or expensive.
간암을 치료하기 위해서는 항암제를 단독 또는 복합으로 주사하여 치료하는 것이 가장 효과적인 방법이다. 그러나 기존에 쓰이고 있는 알킬화제나 항대사제, 항암 항생제, 알칼로이드 등 항암제들은 대부분 간암에 대해서는 작용효과를 나타내지 못하며 심지어 이들 중 일부는 다른 암세포에는 효과적이나 간 기능에는 오히려 장애를 가져오는 경우도 있다.In order to treat liver cancer, the most effective method is to inject an anticancer agent alone or in combination. However, most of the existing anticancer drugs such as alkylating agents, antimetabolites, anticancer antibiotics, and alkaloids do not show an effect on liver cancer, and some of these drugs are effective on other cancer cells, but there are cases where they interfere with liver function.
또한, 대부분의 간세포 암종은 통상적인 화학요법제에 대한 내성을 가지고 있으므로, 표준적인 치료법이 없는 상황에서, 부작용이 적고 치료 효능이 높은 간 표적 약물의 발굴은 간암 치료를 위한 약물 개발 시 충족되지 않은 요구사항이며, 그 필요성이 절실하게 대두되고 있다.In addition, since most hepatocellular carcinomas have resistance to conventional chemotherapeutic agents, in the absence of a standard treatment, the discovery of liver-targeting drugs with few side effects and high therapeutic efficacy has not been met when developing drugs for the treatment of liver cancer. It is a requirement, and the need is urgently emerging.
본 발명은 간암 세포의 항암제 내성 문제를 해결하고 항암제의 감수성을 증가시켜 간암 세포의 세포사멸 효과를 향상시키기 위해 OSMI-1 및 항암제를 유효성분으로 함유하는 항암치료용 약학조성물 및 항암 보조제를 제공하고자 한다.The present invention solves the problem of anticancer drug resistance of liver cancer cells and increases the sensitivity of anticancer drugs to improve the apoptosis effect of liver cancer cells. do.
본 발명은 OSMI-1 및 항암제를 유효성분으로 함유하는 간암 예방 또는 치료용 약학조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating liver cancer containing OSMI-1 and an anticancer agent as active ingredients.
또한, 본 발명은 OSMI-1을 유효성분으로 함유하며, p53 야생형 간암 세포 사멸 증진용 항암 보조제를 제공한다.In addition, the present invention provides an anticancer adjuvant containing OSMI-1 as an active ingredient and promoting apoptosis of p53 wild-type liver cancer.
본 발명에 따르면, OSMI-1 또는 독소루비신이 단독 처리된 간암 세포와 비교하여 OSMI-1 및 독소루비신을 병용처리된 간암 세포에서 p53 및 미토콘드리아 Bcl2 경로가 활성화되고 이를 통하여 상승적 세포사멸 효과가 확인되었으며, 특히 OSMI-1은 p53 야생형 간암 세포 특이적으로 항암제 유도성 세포사멸 효과를 증가시키는 것이 확인됨에 따라, OSMI-1 및 독소루비신을 유효성분으로 함유하는 조성물은 p53 야생형 간암 예방 또는 치료용 약학조성물로 제공될 수 있으며, OSMI-1은 p53 야생형 간암 세포에 대한 항암제의 항암효과를 상승시키기 위한 항암 보조제로 제공될 수 있다.According to the present invention, compared to liver cancer cells treated with OSMI-1 or doxorubicin alone, p53 and mitochondrial Bcl2 pathways are activated in liver cancer cells treated with OSMI-1 and doxorubicin alone, and a synergistic apoptosis effect was confirmed, particularly As it was confirmed that OSMI-1 increases the anticancer drug-induced apoptosis effect specifically for p53 wild-type liver cancer cells, a composition containing OSMI-1 and doxorubicin as an active ingredient may be provided as a pharmaceutical composition for preventing or treating p53 wild-type liver cancer. In addition, OSMI-1 may be provided as an anticancer adjuvant to enhance the anticancer effect of the anticancer agent on p53 wild-type liver cancer cells.
도 1은 간세포 암종 (HCC) 세포의 세포 생존력에 대한 OSMI-1 및 DOX의 효과를 확인한 결과로, 도 1A는 불활성화된 인간 간 세포(AML12) 및 인간 HCC 세포 (Hep3B, Huh7 및 HepG2)에서 p53, OGT, OGA, 및 O-GlcNAc 발현 수준을 확인한 웨스턴블롯 분석결과로, β-액틴을 로딩 대조군으로 사용하였으며, 도 1B는 AML12, Hep3B, Huh7 및 HepG2 세포에 0.4 μM DOX 및 20 μM OSMI-1을 15시간 동안 처리한 후 세포 생존도를 확인한 MTT 분석결과이며 (***p < 0.005 DOX 처리군과 비교), 도 1C의 왼쪽 그래프는 OSMI-1 (20 μM) 존재하거나 존재하는 않는 조건에서 HepG2 세포에 다양한 농도의 DOX (0.1 내지 1 μM)을 15시간 동안 처리하고 세포 생존도를 확인한 MTT 분석결과이며, 오른쪽 그래프는 DOX (0.4 μM) 존재하거나 존재하는 않는 조건에서 HepG2 세포에 다양한 농도의 OSMI-1 (10 내지 40 μM)을 15시간 동안 처리하고 세포 생존도를 확인한 MTT 분석결과로, 데이터는 3번의 독립적인 실험 결과를 나타내며 (***p < 0.005는 DOX 단독 또는 OSMI-1 단독 처리군과 비교), 도 1D는 세포콜로니 형성분석을 수행한 결과로, DOX (0.4 또는 4 μM)이 처리되거나 처리되지 않았거나 또는 OSMI-1 (20 μM)의 병용처리하에서 HepG2 세포의 생존력을 확인하기 위해 8-10일 간 배양 후 플레이트를 크리스탈 바이올렛으로 염색한 결과이다.
도 2는 HepG2 세포에서 p53 신호전달을 통한 OSMI-1의 DOX 유도 세포사멸 향상 효과를 확인한 결과로, 도 2A는 15시간 동안 0, 0.1, 0.4 및 1μM DOX이 처리된 HepG2 세포에서 p53 및 p21 단백질 수준을 확인한 웨스턴 블롯 분석 결과로, 오른쪽 그래프는 배 증가에 따른 상대적인 단백질량을 나타내는 결과이며 (***p < 0.005 대조군과 비교), 동일한 방법으로 다양한 농도의 OSMI-1 (1-40 μM)이 처리된 HepG2 세포에서 p53 수준을 확인한 확인하였으며, β-액틴을 내부대조군으로 사용하였다. 도 2B는 DOX (0.4 μM) 처리된 HepG2 세포에 다양한 농도의 OSMI-1 (2-40 μM)를 15시간 처리하고 p53 및 절단된 caspase-3의 수준을 확인한 웨스턴 블롯 분석 결과이며, 도 2C는 HepG2 및 AML12 세포에 15시간 동안 DOX (0.4 μM) 단독 또는 OSMI-1 (20 μM)와 병용처리 후 p53 및 PARP 수준을 확인한 웨스턴 블롯 분석결과이며, 도 2D의 상단 도면은 0.4 μM DOX 및 20 μM OSMI-1를 단독 또는 병용처리한 HepG2 세포에 10 μM ku55933를 처리하고 phospho-ATM 및 절단된 PARP에 대한 항체를 이용하여 웨스턴 블롯 분석을 수행한 결과이고, 하단 도면은 대조군 또는 p53 siRNA가 형질 주입된 HepG2 세포에서 15시간 동안 DOX (0.4 μM) 단독 또는 OSMI-1 (20 μM) 병용처리 후 phospho-ATM, OGT 및 OGA 항체를 이용하여 웨스턴 블롯 분석을 수행한 결과로, β-액틴을 내부대조군으로 사용하였으며, 결과 값은 3번의 독립적인 실험 결과의 평균 ± SD로 나타내었다.
도 3은 OSMI-1의 ER 스트레스 반응 자극을 확인한 결과로, 도 3A는 15시간 동안 20 μM OSMI-1이 처리되거나 처리되지 않은 HepG2 및 AML12 세포에서 PERK 및 IRE1α의 수준을 확인한 웨스턴 블롯 분석 결과이며, 오른쪽 그래프는 배 증가에 따른 상대적인 단백질량을 나타내는 결과이며 (***p < 0.005 대조군과 비교), 도 3B는 DOX (0.4 μM) 또는 OSMI-1 (20 및 40 μM)을 15시간 처리 후 HepG2 세포에서 O-GlcNAcylated proteins, PERK, IRE1α, XBP 및 Bcl2의 수준을 확인한 웨스턴 블롯 분석 결과이며, 도 3C는 HepG2 세포에 15 시간 동안 DOX (0.4 μM) 단독 또는 OSMI-1 (20 μM)와 병용처리 후 Bcl2, Bax, 절단된 PARP, caspase-3 및 O-GlcNAcylated proteins에 대한 항체를 이용하여 웨스턴 블롯 분석을 수행한 결과로, β-액틴을 내부 대조군으로 사용하였다. 도 3D는 HepG2 세포에 0.4 μM DOX 단독 또는 다양한 농도의 OSMI-1 (2, 20, or 40 μM)와 병용처리 후 qRT-PCR을 수행하여 Bcl2 및 CHOP mRNA의 수준을 확인하고, 대조군 시료에 대하여 표준화하여 평균 ± SD (n = 6)으로 값을 나타내었다 (***p < 0.005 대조군과 비교).
도 4는 DOX 및 OSMI-1에 의한 염증성 NF-κB 신호전달을 확인한 결과로, 도 4A의 왼쪽 도면은 DOX (0.4 μM)이 처리된 HepG2 세포에서 phospho-p65의 수준을 최대 12시간 동안 확인한 웨스턴 블롯 분석결과이며, 오른쪽 도면은 OSMI-1 (20 μM)이 존재하거나 존재하지 않는 조건에서 최대 60분 동안 DOX (0.4 μM)를 처리한 HepG2 세포에서 phospho-p65 수준을 확인한 결과이며, 하단의 두 개의 그래프는 상기 단백질의 배 증가에 따른 상대적 발현량을 나타낸 결과로, 3번의 독립적인 실험 결과 값으로 나타내었으며 (***p < 0.005 대조군과 비교), 도 4B는 DOX (0 내지 4 μM)이 1시간 동안 처리된 HepG2 세포에서 phospho-p65, phospho-IKKα/β, phospho-IκB, 및 p53에 대한 항체를 이용하여 웨스턴 블롯 분석을 수행한 결과이며, 도 4C는 대조군 또는 OGT siRNA를 형질 주입한 HepG2 세포에 DOX (0.1 and 0.4 μM) 처리을 1시간 동안 처리한 후 세포 내 phospho-p65 및 phospho-IKKα/β의 수준을 확인한 웨스턴 블롯 분석 결과이며, 하단의 도면은 IKKβ 항체를 이용하여 면역침강을 수행하고 GlcNAc 항체를 이용하여 면역블롯팅을 수행한 결과로, 면역침강 분석은 전체 세포 용해물을 이용하여 수행되었으며, GlcNAc 수준은 동량의 침전물을 이용하여 수행되었다. 도 4D는 OSMI-1 (20 μM)이 전처리되거나 처리되지 않은 HepG2 세포에 DOX (0.4 μM)을 1시간 동안 처리한 후 핵 내 및 세포질 분획에서 p65 및 p53 수준을 확인한 웨스턴 블롯 분석 결과로, 핵 및 세포질 단백질에 대한 로딩 대조군으로 각각 LaminB 및 tubulin을 사용하였으며, 하단의 도면은 세포 용해물로 부터 분리된 핵 분획에 항-p65 항체를 이용하여 면역침강을 수행하고 항-O-GlcNAc 항체를 이용하여 웨스턴 블롯 분석을 수행한 결과이며, 도 4E는 scramble-siRNA 또는 OGT-siRNA 형질 주입 후 15 시간 동안 DOX (0.4 μM)을 처리하거나 처리하지 않은 HepG2 세포의 전체 용해물에 각각의 항체를 이용하여 웨스턴 블롯 분석을 수행한 결과이다.
도 5는 in vivo 에서 DOX 및 OSMI-1 병용처리가 인간 HCC 이종이식 모델의 종양 성장을 상승적으로 억제하는 효과를 확인한 결과로, 도 5A는 HepG2 세포를 누드 마우스 피하에 접종하고 32일 동안 종양 성장을 확인하였다. vehicle (DMSO, 100 mm3), DOX (0.1mg/kg) 및 OSMI-1 (1 mg/kg)을 3주 동안 3일에 한번 정맥주사로 단독 또는 병용처리한 후 각 종양의 성장을 기록하고 표시된 시간마다 종양 부피(mm3)의 변화를 확인하였으며, 3회 독립적인 실험의 평균 표준편차로 값을 표시한 결과이며 (**p < 0.01 대조군과 비교), 오른쪽 도면은 누드 마우스로부터 분리된 종양의 대표 이미지이며, 도 5B는 누드 마우스 유래 절개된 종양 조직으로부터 동량의 전체 조직 단백질을 이용하여 p53, p-p65, p65, 절단된 Caspase-3 및 PARP의 발현 수준을 확인한 웨스턴 블롯 분석 결과로, 발현 수준을 β-액틴에 대해 표준화하였으며, 오른쪽 도면은 측정된 단백질의 상대적 발현량을 배수 증가로 나타낸 그래프이며, 도 5C는 도 5A와 같이 HepG2 이종이식 마우스에 피하 주사를 통하여 DOX (1 mg/kg) 또는 OSMI-1 (5 mg/kg) 단독 또는 병용처리하고 종양 성장을 32일간 확인한 결과로, 이종이식 종양의 부피를 측정하고 처리 후 표시된 시간마다 플로팅하였으며, 결과 값은 평균 ± SD (n = 4)로 나타내었으며 (*p < 0.05, **p < 0.01, ***p < 0.005 대조군과 비교), 오른쪽 도면은 누드 마우스로부터 유래된 절개된 종양의 대표이미지이다. 도 5D는 누드 마우스로부터 유래된 절개된 종양 조직에서 p53, p21, Bax, cleaved Caspase-3, IRE1 및 PERK의 발현 수준을 확인한 웨스턴 블롯 분석결과로, 동량의 전체 조직 단백질을 사용하였으며, 발현 수준을 β-액틴에 대해 표준화하였으며, 오른쪽 도면은 측정된 단백질의 상대적 발현량을 배수 증가로 나타낸 그래프이다.
도 6A는 DMSO, DOX, OSMI-1 및 DOX+OSMI-1이 처리된 절개된 종양 절편에서 절단된 caspase-3 Ki-67, p53, p65 및 PERK에 대한 항체를 이용하여 면역조직화학 분석을 수행한 결과이며, 도 6B는 치료 약물의 신호전달 과정을 나타낸 모식도로, 화살표와 막대는 각각 활성 및 억제를 나타내고, ER 스트레스는 아폽토시스 및 염증과 같은 과정을 활성화시키고, 에스트로겐 유도성 아폽토시스 매개를 위한 ER 스트레스 센서 IRE1 및 PERK 활성 동안 전사 인자인 NF-κB의 전환은 활성화된다. 그러나 OSMI-1은 NF-κB 신호전달을 억제하여 DOX에 의한 염증을 현저하게 감소시키고 IKK β는 세포내 p53 안정성을 차단할 수 있다.Figure 1 is the result of confirming the effect of OSMI-1 and DOX on the cell viability of hepatocellular carcinoma (HCC) cells, Figure 1A is in inactivated human liver cells (AML12) and human HCC cells (Hep3B, Huh7 and HepG2) As a result of Western blot analysis confirming the expression levels of p53, OGT, OGA, and O-GlcNAc, β-actin was used as a loading control, and FIG. 1B shows 0.4 μM DOX and 20 μM OSMI- in AML12, Hep3B, Huh7 and HepG2 cells. 1 is the result of MTT analysis confirming cell viability after treatment for 15 hours (***p < 0.005 compared to the DOX treatment group), and the graph on the left of FIG. 1C shows the presence or absence of OSMI-1 (20 μM) In HepG2 cells, various concentrations of DOX (0.1 to 1 μM) were treated for 15 hours and cell viability was checked for MTT analysis results, and the graph on the right shows various concentrations of HepG2 cells in the presence or absence of DOX (0.4 μM). of OSMI-1 (10 to 40 μM) was treated for 15 hours and cell viability was confirmed by MTT analysis, and the data represent the results of three independent experiments (***p < 0.005 is DOX alone or OSMI-1 Compared with the single treatment group), FIG. 1D shows the results of cell colony formation assay. Viability of HepG2 cells with or without DOX (0.4 or 4 μM) or OSMI-1 (20 μM) co-treatment This is the result of staining the plate with crystal violet after 8-10 days of incubation to confirm.
Figure 2 is the result of confirming the effect of improving DOX-induced apoptosis of OSMI-1 through p53 signaling in HepG2 cells. Figure 2A shows p53 and p21 proteins in HepG2 cells treated with 0, 0.1, 0.4 and 1 μM DOX for 15 hours. As a result of Western blot analysis that confirmed the level, the graph on the right shows the relative protein amount according to the fold increase (***p < 0.005 compared to the control), and OSMI-1 at various concentrations (1-40 μM) in the same way The p53 level was confirmed in the treated HepG2 cells, and β-actin was used as an internal control. Figure 2B is a Western blot analysis result confirming the levels of p53 and cleaved caspase-3 after treating various concentrations of OSMI-1 (2-40 μM) in DOX (0.4 μM)-treated HepG2 cells for 15 hours, Figure 2C is Western blot analysis confirming the p53 and PARP levels after treating HepG2 and AML12 cells alone or in combination with DOX (0.4 μM) or OSMI-1 (20 μM) for 15 hours, the upper figure of FIG. 2D shows 0.4 μM DOX and 20 μM HepG2 cells treated with OSMI-1 alone or in combination were treated with 10 μM ku55933 and Western blot analysis was performed using an antibody against phospho-ATM and cleaved PARP. The lower figure is a control or p53 siRNA transfection Western blot analysis was performed using phospho-ATM, OGT and OGA antibodies after DOX (0.4 μM) alone or OSMI-1 (20 μM) combination treatment was performed on the HepG2 cells for 15 hours. As a result, β-actin was used as an internal control group. was used, and the result value was expressed as the mean ± SD of the results of three independent experiments.
Figure 3 is the result of confirming the ER stress response stimulation of OSMI-1, Figure 3A is the result of Western blot analysis confirming the levels of PERK and IRE1α in HepG2 and AML12 cells treated with or without 20 μM OSMI-1 for 15 hours. , The graph on the right is the result showing the relative protein amount according to the fold increase (***p < 0.005 compared to the control), and Figure 3B is DOX (0.4 μM) or OSMI-1 (20 and 40 μM) after 15 hours of treatment Western blot analysis confirming the levels of O-GlcNAcylated proteins, PERK, IRE1α, XBP and Bcl2 in HepG2 cells, Figure 3C is DOX (0.4 μM) alone or in combination with OSMI-1 (20 μM) in HepG2 cells for 15 hours After treatment, Western blot analysis was performed using antibodies against Bcl2, Bax, cleaved PARP, caspase-3 and O-GlcNAcylated proteins. As a result, β-actin was used as an internal control. 3D shows HepG2 cells with 0.4 μM DOX alone or in combination with OSMI-1 (2, 20, or 40 μM) at various concentrations, followed by qRT-PCR to confirm the levels of Bcl2 and CHOP mRNA, and for control samples Values are expressed as mean ± SD (n = 6) by normalization (***p < 0.005 compared to control).
Figure 4 is a result of confirming the inflammatory NF-κB signaling by DOX and OSMI-1, the left diagram of Figure 4A is a Western confirming the level of phospho-p65 in HepG2 cells treated with DOX (0.4 μM) for up to 12 hours Blot analysis results, and the figure on the right is the result of confirming phospho-p65 levels in HepG2 cells treated with DOX (0.4 μM) for up to 60 minutes in the presence or absence of OSMI-1 (20 μM). The graph of the dog shows the relative expression level according to the fold increase of the protein, and is shown as the result of three independent experiments (***p < 0.005 compared to the control), Figure 4B is DOX (0 to 4 μM) Western blot analysis was performed using antibodies against phospho-p65, phospho-IKKα/β, phospho-IκB, and p53 in HepG2 cells treated for 1 hour. FIG. 4C is control or OGT siRNA transfection. The results of Western blot analysis confirming the levels of phospho-p65 and phospho-IKKα/β in one HepG2 cell after treatment with DOX (0.1 and 0.4 μM) for 1 hour. The figure at the bottom is immunoprecipitation using IKKβ antibody As a result of performing immunoblotting using GlcNAc antibody, immunoprecipitation analysis was performed using whole cell lysates, and GlcNAc levels were performed using the same amount of precipitate. Figure 4D shows the results of Western blot analysis confirming the levels of p65 and p53 in the nuclear and cytoplasmic fractions after treating HepG2 cells pre-treated with or without OSMI-1 (20 μM) with DOX (0.4 μM) for 1 hour. And LaminB and tubulin were used as loading controls for cytoplasmic proteins, respectively. The figure at the bottom shows immunoprecipitation using anti-p65 antibody on nuclear fraction isolated from cell lysate and anti-O-GlcNAc antibody The results of western blot analysis were performed, and FIG. 4E shows the total lysate of HepG2 cells treated with or without DOX (0.4 μM) for 15 hours after scramble-siRNA or OGT-siRNA transfection using each antibody. This is the result of performing Western blot analysis.
5 is a result confirming the effect of in vivo DOX and OSMI-1 combination treatment to synergistically inhibit tumor growth in a human HCC xenograft model. was confirmed. The growth of each tumor was recorded after vehicle (DMSO, 100 mm 3 ), DOX (0.1 mg/kg) and OSMI-1 (1 mg/kg) were administered intravenously or in combination once every 3 days for 3 weeks. The change in tumor volume (mm 3 ) was confirmed at each indicated time, and the value was expressed as the average standard deviation of three independent experiments (**p < 0.01 compared to the control group), and the figure on the right is It is a representative image of a tumor, and FIG. 5B is a Western blot analysis result confirming the expression levels of p53, p-p65, p65, cleaved Caspase-3 and PARP using the same amount of whole tissue protein from the dissected tumor tissue derived from a nude mouse. , the expression level was normalized to β-actin, the right figure is a graph showing the relative expression level of the measured protein as a fold increase, and Figure 5C is DOX (1 mg /kg) or OSMI-1 (5 mg/kg) alone or in combination, and as a result of confirming tumor growth for 32 days, the volume of xenograft tumors was measured and plotted at every indicated time after treatment, and the result value was mean ± SD ( n = 4) (*p < 0.05, **p < 0.01, ***p < 0.005 compared to the control group), and the figure on the right is a representative image of a dissected tumor derived from a nude mouse. 5D is a Western blot analysis result confirming the expression levels of p53, p21, Bax, cleaved Caspase-3, IRE1 and PERK in dissected tumor tissue derived from nude mice. The same amount of whole tissue protein was used, and the expression level was It was normalized to β-actin, and the figure on the right is a graph showing the relative expression level of the measured protein as a fold increase.
6A is an immunohistochemical analysis performed using antibodies against caspase-3 Ki-67, p53, p65 and PERK cut in the dissected tumor section treated with DMSO, DOX, OSMI-1 and DOX+OSMI-1. As a result, FIG. 6B is a schematic diagram showing the signaling process of a therapeutic drug, arrows and bars indicate activation and inhibition, respectively, ER stress activates processes such as apoptosis and inflammation, and ER for mediating estrogen-induced apoptosis During stress sensor IRE1 and PERK activation, the conversion of transcription factor NF-κB is activated. However, OSMI-1 inhibits NF-κB signaling to significantly reduce DOX-induced inflammation, and IKKβ can block intracellular p53 stability.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 발명자들은 효과적인 간암 치료제 개발을 위해 연구를 진행하던 중 OSMI-1 또는 독소루비신이 단독 처리된 간암 세포와 비교하여 병용처리된 간암 세포에서 p53 및 미토콘드리아 Bcl2 경로가 활성화되고 이를 통하여 상승적 세포사멸 효과가 나타나는 것을 확인하였으며, 특히 OSMI-1은 p53 야생형 간암 세포 특이적으로 항암제 유도성 세포사멸 효과를 증가시키는 것을 확인함에 따라 본 발명을 완성하였다.While the inventors of the present invention were conducting research for the development of an effective liver cancer therapeutic agent, the p53 and mitochondrial Bcl2 pathways were activated in the liver cancer cells treated in combination with OSMI-1 or doxorubicin alone compared to the liver cancer cells treated alone, and thereby a synergistic apoptosis effect was confirmed to appear, and in particular, the present invention was completed by confirming that OSMI-1 increased the anticancer drug-induced apoptosis effect specifically for p53 wild-type liver cancer cells.
본 발명은 OSMI-1 및 항암제를 유효성분으로 함유하는 간암 예방 또는 치료용 약학조성물을 제공할 수 있다.The present invention can provide a pharmaceutical composition for preventing or treating liver cancer containing OSMI-1 and an anticancer agent as active ingredients.
상기 간암은 p53 야생형 (p53 wild type) 간암 세포에 의해 유도되는 것일 수 있다.The liver cancer may be induced by p53 wild-type liver cancer cells.
상기 약학조성물은 p53 및 PERK/IRE-1 발현을 증가시켜 항암제의 간암 세포 사멸을 증가시키는 것일 수 있으며, 활성산소(ROS) 생성 및 ER 스트레스를 유도하여 항암제의 간암 세포 사멸을 증가시키는 것일 수 있다.The pharmaceutical composition may increase the expression of p53 and PERK / IRE-1 to increase the liver cancer cell death of the anticancer agent, it may be to increase the liver cancer cell death of the anticancer agent by inducing the production of reactive oxygen species (ROS) and ER stress .
상기 OSMI-1은 NF-κB 활성을 억제시켜 간암 세포의 항암제 내성을 저해하는 것일 수 있다.The OSMI-1 may inhibit NF-κB activity to inhibit anticancer drug resistance of liver cancer cells.
본 발명의 실시예에 따르면, 암세포주 Hep3B, Huh7 및 HepG2 (p53 야생형)에서 변형 및 촉매 효소 OGT의 수준을 확인하였으며, 비 암종 간세포주 AML12와 비교하였다. According to an example of the present invention, the levels of the modified and catalytic enzyme OGT were confirmed in the cancer cell lines Hep3B, Huh7 and HepG2 (p53 wild-type), and compared with the non-carcinoma hepatocyte line AML12.
그 결과, 도 1A와 같이 Huh7 및 HepG2 세포에서 p53 발현뿐만 아니라 O-GlcNAc 변형 및 OGT 수준이 Hep3B 및 AML12 세포보다 높게 나타나는 것을 확인할 수 있었다.As a result, as shown in FIG. 1A , it was confirmed that p53 expression as well as O-GlcNAc modification and OGT levels in Huh7 and HepG2 cells were higher than in Hep3B and AML12 cells.
또한, OGT의 약리학적 억제제인 OSMI-1이 세포 사멸을 자극할 수 있지를 확인하기 위해, 저용량 DOX (0.4 μM)을 단독 또는 OSMI-1과 병용처리한 간세포의 생존도를 MTT 분석으로 확인한 결과, 도 1B와 같이 다른 세포주 보다 HepG2 세포의 DOX 처리군과 DOX 및 OSMI-1 병용처리군 간의 생존도 차이가 매우 현저하게 나타나는 것이 확인되었다. In addition, to confirm that OSMI-1, a pharmacological inhibitor of OGT, can stimulate apoptosis, the viability of hepatocytes treated with low-dose DOX (0.4 μM) alone or in combination with OSMI-1 was confirmed by MTT analysis. , It was confirmed that the difference in viability between the DOX-treated group and the DOX and OSMI-1 combination treatment group of HepG2 cells was significantly more significant than that of other cell lines, as shown in FIG. 1B.
다음으로 DOX 및 OSMI-1 농도 범위에 대한 반응으로 HepG2 세포의 생존도를 확인한 결과, 도 1C와 같이 DOX 반응에 대한 세포 생존도는 농도의존적으로 감소하였으나, 0.1 내지 1 μM 범위의 저농도에서는 80% 미만의 생존도로 유의한 효과가 나타나지 않았다. 그러나 OSMI-1 (20 μM)를 함께 사용한 경우, DOX의 농도의존적으로 현저하게 세포사멸 효과가 증가되었으며, HepG2의 증식 활성이 30% 더 억제된 것을 확인할 수 있었다. Next, as a result of confirming the viability of HepG2 cells in response to the DOX and OSMI-1 concentration range, as shown in FIG. 1C, the cell viability to the DOX response decreased in a concentration-dependent manner, but at a low concentration ranging from 0.1 to 1 μM, 80% There was no significant effect with less than the viability. However, when OSMI-1 (20 μM) was used together, the apoptosis effect was significantly increased in a concentration-dependent manner of DOX, and it was confirmed that the proliferative activity of HepG2 was further inhibited by 30%.
상기 결과로부터 OSMI-1 및 DOX의 병용처리는 상승적으로 p53 야생형 HepG2 세포의 증식을 억제하고, 세포 사멸을 촉진시키는 것이 확인되었다.From the above results, it was confirmed that the combined treatment of OSMI-1 and DOX synergistically inhibited the proliferation of p53 wild-type HepG2 cells and promoted cell death.
상기 항암제는 옥살리플라틴 (Oxaliplatin), 페메트렉시드 (Pemetrexed), 시스플라틴 (Cisplatin), 젬시타빈 (Gemcitabine), 카보플라틴 (Carboplatin), 플루오로우라실 (5-FU), 시클로포스파미드 (Cyclophosphamide), 파클리탁셀 (Paclitaxel), 빈크리스틴 (Vincristine), 에토포사이드 (Etoposide) 및 독소루비신 (Doxorubicin)으로 이루어진 군에서 선택되는 것일 수 있다.The anticancer agent is oxaliplatin (Oxaliplatin), pemetrexed (Pemetrexed), cisplatin (Cisplatin), gemcitabine (Gemcitabine), carboplatin (Carboplatin), fluorouracil (5-FU), cyclophosphamide (Cyclophosphamide), It may be one selected from the group consisting of paclitaxel, vincristine, etoposide, and doxorubicin.
상기 약학조성물은 약학조성물 총 100 중량부에 대하여, OSMI-1 0.05 내지 5 중량부 및 항암제 0.1 내지 10 중량부를 포함하는 것일 수 있다.The pharmaceutical composition may include 0.05 to 5 parts by weight of OSMI-1 and 0.1 to 10 parts by weight of the anticancer agent based on 100 parts by weight of the total pharmaceutical composition.
본 발명의 한 구체예에서, 상기 OSMI-1 및 항암제를 유효성분으로 함유하는 간암 예방 또는 치료용 약학조성물은 통상적인 방법에 따라 주사제, 과립제, 산제, 정제, 환제, 캡슐제, 좌제, 겔, 현탁제, 유제, 점적제 또는 액제로 이루어진 군에서 선택된 어느 하나의 제형을 사용할 수 있다.In one embodiment of the present invention, the pharmaceutical composition for the prevention or treatment of liver cancer containing OSMI-1 and an anticancer agent as an active ingredient is prepared by injection, granule, powder, tablet, pill, capsule, suppository, gel, Any one formulation selected from the group consisting of suspensions, emulsions, drops or solutions may be used.
본 발명의 다른 구체예에서, OSMI-1 및 항암제를 유효성분으로 함유하는 간암 예방 또는 치료용 약학조성물은 약학조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제, 붕해제, 감미제, 피복제, 팽창제, 활택제, 향미제, 항산화제, 완충액, 정균제, 희석제, 분산제, 계면활성제, 결합제 및 윤활제로 이루어진 군에서 선택되는 하나 이상의 첨가제를 추가로 포함할 수 있다.In another embodiment of the present invention, the pharmaceutical composition for preventing or treating liver cancer containing OSMI-1 and an anticancer agent as an active ingredient is an appropriate carrier, excipient, disintegrant, sweetener, coating agent, and swelling agent commonly used in the manufacture of pharmaceutical compositions. , lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders and lubricants may further include one or more additives selected from the group consisting of.
구체적으로 담체, 부형제 및 희석제는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 사용할 수 있으며, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 있으며 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기재로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Specifically, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agent and the like, and the solid preparation may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, solutions, emulsions, syrups, etc., and various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.
본 발명의 일실시예에 따르면 상기 약학 조성물은 정맥내, 동맥내, 복강내, 근육내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 대상체로 투여할 수 있다.According to an embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject.
상기 OSMI-1 및 항암제의 바람직한 투여량은 대상체의 상태 및 체중, 질환의 종류 및 정도, 약물 형태, 투여경로 및 기간에 따라 달라질 수 있으며 당업자에 의해 적절하게 선택될 수 있다. 본 발명의 일실시예에 따르면 이에 제한되는 것은 아니지만 1일 투여량이 0.01 내지 200 mg/kg, 구체적으로는 0.1 내지 200 mg/kg, 보다 구체적으로는 0.1 내지 100 mg/kg 일 수 있다. 투여는 하루에 한 번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되는 것은 아니다.Preferred dosages of OSMI-1 and the anticancer agent may vary depending on the subject's condition and weight, the type and extent of disease, drug form, administration route and period, and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although not limited thereto, the daily dose may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be administered in several divided doses, thereby not limiting the scope of the present invention.
본 발명에 있어서, 상기 '대상체'는 인간을 포함하는 포유동물일 수 있으나, 이들 예에 한정되는 것은 아니다.In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.
또한, 본 발명은 OSMI-1을 유효성분으로 함유하며, p53 야생형 간암 세포 사멸 증진용 항암 보조제를 제공할 수 있다.In addition, the present invention can provide an anticancer adjuvant containing OSMI-1 as an active ingredient and promoting apoptosis of p53 wild-type liver cancer.
상기 OSMI-1은 p53 야생형 간암세포의 항암제 내성을 억제하고, 항암제 감수성을 증진시키는 것일 수 있다.The OSMI-1 may inhibit the anticancer drug resistance of p53 wild-type liver cancer cells and enhance the anticancer drug sensitivity.
상기 항암제는 옥살리플라틴 (Oxaliplatin), 페메트렉시드 (Pemetrexed), 시스플라틴 (Cisplatin), 젬시타빈 (Gemcitabine), 카보플라틴 (Carboplatin), 플루오로우라실 (5-FU), 시클로포스파미드 (Cyclophosphamide), 파클리탁셀 (Paclitaxel), 빈크리스틴 (Vincristine), 에토포사이드 (Etoposide) 및 독소루비신 (Doxorubicin)으로 이루어진 군에서 선택되는 것일 수 있다.The anticancer agent is oxaliplatin (Oxaliplatin), pemetrexed (Pemetrexed), cisplatin (Cisplatin), gemcitabine (Gemcitabine), carboplatin (Carboplatin), fluorouracil (5-FU), cyclophosphamide (Cyclophosphamide), It may be one selected from the group consisting of paclitaxel, vincristine, etoposide, and doxorubicin.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.
<실험예><Experimental example>
하기의 실험예들은 본 발명에 따른 각각의 실시예에 공통적으로 적용되는 실험예를 제공하기 위한 것이다.The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.
1. 세포 용해물 및 웨스턴 블롯 분석1. Cell Lysate and Western Blot Analysis
50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40 및 단백질분해효소 억제제 칵테일 타블렛 (Roche, Mannheim, Germany)이 포함된 NP-40 용해 버퍼를 이용하여 AML12, Hep3B, Huh7 및 HepG2 세포 용해물을 얻었다.AML12, Hep3B; Huh7 and HepG2 cell lysates were obtained.
단백질을 8-15% SDS-PAGE 겔에서 전기영동으로 분리시킨 후 니트로셀룰로스 막(GE Healthcare Life science)에 옮겼다. 5% 탈지유로 실온에서 1시간 동안 블로킹 후 막을 1차 항체와 4℃에서 하룻밤동안 인큐베이션하였으며, 1차 항체로 O-GlcNAc (Sigma-Aldrich, St Louis, MO, USA), OGA 및 IL-1β (Abcam, Cambridge, UK), PARP (Invitrogen, Waltham, MA, USA), cleaved caspase-3, IRE1α, P-p65, p65, P-IKKα/β, IKKα/β 및 P-IκB (Cell Signaling Technology, Danvers, MA, USA), OGT, p53, p21, PERK, P-ATM, XBP, Bcl2, Bax, lamin, α-tubulin 및 β-actin (Santa Cruz, Dallas, TX, USA)을 이용하였다. Proteins were separated by electrophoresis on an 8-15% SDS-PAGE gel and transferred to a nitrocellulose membrane (GE Healthcare Life science). After blocking with 5% skim milk at room temperature for 1 hour, the membrane was incubated with a primary antibody overnight at 4°C, and the primary antibody was O-GlcNAc (Sigma-Aldrich, St Louis, MO, USA), OGA and IL-1β ( Abcam, Cambridge, UK), PARP (Invitrogen, Waltham, MA, USA), cleaved caspase-3, IRE1α, P-p65, p65, P-IKKα/β, IKKα/β and P-IκB (Cell Signaling Technology, Danvers) , MA, USA), OGT, p53, p21, PERK, P-ATM, XBP, Bcl2, Bax, lamin, α-tubulin and β-actin (Santa Cruz, Dallas, TX, USA) were used.
TBS-T로 세척 후 적합한 호래디쉬 퍼옥시다아제 (HRP)가 결합된 이차 항체로 실온에서 30분간 인큐베이션하였다. 마지막으로 ECL Plus Western Blotting Detection System (GE Healthcare Life Sciences)를 이용하여 단백질 밴드를 시각화하였으며, ImageJ software로 신호를 정량하였다.After washing with TBS-T, it was incubated for 30 minutes at room temperature with an appropriate horadish peroxidase (HRP)-conjugated secondary antibody. Finally, protein bands were visualized using ECL Plus Western Blotting Detection System (GE Healthcare Life Sciences), and signals were quantified using ImageJ software.
2. 생체 내 종양형성 분석 (In Vivo Xenograft Assay)2. In Vivo Xenograft Assay
5주령 암컷 BALB/c-Foxn1nu 누드마우스를 JANVIER Laboratory (France)에서 구입하였다. 약 5×106 HepG2 세포를 수집하고 50% PBS 및 50% Matrigel (BD Bio-science) 혼합물 100μl에 재현탁시키고 각 마우스의 측면 피하에 주사하였다. 이종이식 일주일 후 종양이 형성된 누드 마우스를 저농도 처리군 (0.1mg/kg DOX and/or 1mg/kg OSMI-1(Sigma-Aldrich, St Louis, MO, USA)) 및 고농도 처리군 (1mg/kg DOX and/or 5mg/kg OSMI-1)으로 무작위 분리하고 각 그룹을 다음과 같은 4개의 하위 그룹으로 구분하였다: 그룹 1) 대조군으로 DMSO를 투여한 마우스, 그룹 2) DOX을 투여한 마우스, 그룹 3) OSMI-1을 투여한 마우스, 그룹 4) DOX 및 OSMI-1을 투여한 마우스.Five-week-old female BALB/c-Foxn1nu nude mice were purchased from JANVIER Laboratory (France). About 5×10 6 HepG2 cells were collected and resuspended in 100 μl of a mixture of 50% PBS and 50% Matrigel (BD Bio-science) and injected subcutaneously on the lateral side of each mouse. Nude mice with tumors one week after xenograft were treated with a low dose (0.1mg/kg DOX and/or 1mg/kg OSMI-1 (Sigma-Aldrich, St Louis, MO, USA)) and a high dose group (1mg/kg DOX). and/or 5 mg/kg OSMI-1) and each group was divided into four subgroups as follows: Group 1) DMSO-administered mice as controls, Group 2) DOX-administered mice, and Group 3 ) mice administered OSMI-1, group 4) mice administered DOX and OSMI-1.
각 그룹은 4주간 격일로 정맥 투여하였으며, 4일에 한 번씩 디지털 캘리퍼 (Mitutoyo, Tokyo, Japan)를 이용하여 피하 종양의 크기를 확인하였다. 또한, 같은 날 각 마우스의 체중을 내약성 지표로 기록하였다. Each group was administered intravenously every other day for 4 weeks, and the size of the subcutaneous tumor was checked using a digital caliper (Mitutoyo, Tokyo, Japan) once every 4 days. In addition, the body weight of each mouse on the same day was recorded as a tolerability index.
종양 부피(V)는 하기 계산식을 이용하여 확인하였다.The tumor volume (V) was confirmed using the following formula.
V = (L × W2)/2 (mm3)V = (L × W 2 )/2 (mm 3 )
상기 종양 길이(L)은 가장 큰 직경이며, 종양 폭(W)은 수직 직경이다.The tumor length (L) is the largest diameter, and the tumor width (W) is the vertical diameter.
실험 종료 시점에 마우스를 안락사시키고 종양 덩어리를 절제하고 무게를 측정하였다. 종양 덩어리를 4% 파라포름알데하이드 완충액으로 고정하고 면역조직화학(IHC) 염색을 수행하였다. 상기 동물 실험 과정은 경북대학교 동물 관리위원회에서 승인된 동물 프로토콜 및 지침에 따라 수행되었다 (No. KNU 2019-0002). At the end of the experiment, the mice were euthanized and the tumor mass was excised and weighed. Tumor masses were fixed with 4% paraformaldehyde buffer and immunohistochemical (IHC) staining was performed. The animal testing procedure was performed according to animal protocols and guidelines approved by the Animal Care Committee of Kyungpook National University (No. KNU 2019-0002).
3. 면역조직화학 (Immunohistochemistry)3. Immunohistochemistry
이종이식 종양 조직을 마우스로부터 추출하고, 종양 조직을 4% 파라포름알데하이드로 고정시킨 후 파라핀에 포매시켰다.The xenograft tumor tissue was extracted from the mouse, and the tumor tissue was fixed with 4% paraformaldehyde and then embedded in paraffin.
절편의 파라핀을 제거하고 항원 검색을 위해 시트르산 완충액 (pH 6.0)에서 끓였다. 실온에서 냉각시킨 후 절편을 3% BSA가 포함된 고트 혈청으로 1시간 동안 불활성화시켰다. Sections were deparaffinized and boiled in citrate buffer (pH 6.0) for antigen retrieval. After cooling to room temperature, the sections were inactivated with goat serum containing 3% BSA for 1 hour.
조직화학 분석을 위한 일차 항체로 절단된 capase-3, Ki-67, p53 및 p65를 사용하였다. 이후 절편을 적합한 이차 항체와 인큐베이션하고, 3, 3-diaminobenzidine (DAB)를 이용하여 면역조직화학 반응을 시각화하였다. The cleaved capase-3, Ki-67, p53 and p65 as primary antibodies for histochemical analysis were used. The sections were then incubated with a suitable secondary antibody, and the immunohistochemical reaction was visualized using 3,3-diaminobenzidine (DAB).
4. 세포 배양 및 형질 주입4. Cell Culture and Transfection
살아있는 암세포주 HepG2 (p53 wild type), Hep3B 및 Huh7 (p53 mutant type)을 Korean Cell Line Bank (Seoul, Korea)에서 얻었다. 마우스 간세포 유래 AML12 세포를 Dr. Jae Man Lee (KNUM, Korea)로부터 제공받았다. HepG2 및 Hep3B 세포주는 DMEM에서 배양하였으며, Huh7 세포는 10% 태아소혈청(fetal bovine serum, FBS) 및 1% 페니실린이 포함된 RPMI-1640 배지에서 배양하였다. AML12 세포주는 10% FBS, 1% 페니실린, 1% ITS, 및 40 ng/ml 덱사메타손이 포함된 RPMI-1640 배지에서 배양하였다. Live cancer cell lines HepG2 (p53 wild type), Hep3B and Huh7 (p53 mutant type) were obtained from the Korean Cell Line Bank (Seoul, Korea). Mouse hepatocyte-derived AML12 cells were treated with Dr. Provided by Jae Man Lee (KNUM, Korea). HepG2 and Hep3B cell lines were cultured in DMEM, and Huh7 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin. The AML12 cell line was cultured in RPMI-1640 medium containing 10% FBS, 1% penicillin, 1% ITS, and 40 ng/ml dexamethasone.
세포를 가습조건의 37℃ 및 5% CO2 배양기에서 배양하였다. 리포펙타민 RNAi MAX (Invitrogen)를 이용하여 대조군, p53 및 OGT siRNA (santa Cruz)를 HepG2 세포에 형질 주입하였다. Cells were cultured in a humidified condition at 37° C. and 5% CO 2 incubator. Control, p53 and OGT siRNAs (santa Cruz) were transfected into HepG2 cells using Lipofectamine RNAi MAX (Invitrogen).
NP-40 용해 완충액으로 세포를 균질화시키고, 면역블롯 과정은 상기 실험예와 같다. OGT 및 p53 일차 항체 (Cell Signaling Technology)를 단백질 검출을 위해 사용하였다. The cells were homogenized with NP-40 lysis buffer, and the immunoblotting procedure was the same as in the above experimental example. OGT and p53 primary antibodies (Cell Signaling Technology) were used for protein detection.
5. 면역침강분석 (Immunoprecipitation)5. Immunoprecipitation
HepG2 세포로부터 추출된 전체 단백질을 p65 또는 IKKβ 항체 및 단백질 G IP kit (Thermo Fisher Scientific)의 DynabeadsTM와 인큐베이션하였다. Total protein extracted from HepG2 cells was incubated with p65 or IKKβ antibody and Dynabeads ™ of Protein G IP kit (Thermo Fisher Scientific).
2×샘플 버퍼로 끓여 단백질 G와 침강된 면역복합체로부터 단백질을 추출하였다. 단백질 샘플을 SDS-PAGE로 분리시키고 막 위에 옮긴 후 웨스턴 블롯을 수행하여 침강물을 분석하였다. Protein G and protein were extracted from the precipitated immunocomplex by boiling with 2x sample buffer. Protein samples were separated by SDS-PAGE, transferred onto a membrane, and Western blot was performed to analyze the sediment.
6. 실시간 PCR (Real-time PCR)6. Real-time PCR
TRIzol 시약 (Invitrogen)을 이용하여 전체 RNA를 추출하였다. 간략하게, 용해물을 클로로포름과 혼합하고 4℃에서 12,000×g로 10분간 원심분리하였다. Total RNA was extracted using TRIzol reagent (Invitrogen). Briefly, the lysate was mixed with chloroform and centrifuged at 12,000×g at 4° C. for 10 min.
상층액을 동량의 이소프로판올과 혼합하고, 4℃에서 12,000×g로 10분간 원심분리하여 RNA를 펠렛화하였다. The supernatant was mixed with an equal amount of isopropanol, and centrifuged at 12,000×g at 4° C. for 10 minutes to pellet RNA.
샘플을 70% 에탄올로 두 번 세척하고, 최종 농도가 1 μg/μl이 되도록 DEPC water로 RNA를 용해시켜 역전사 PCR을 수행하였다.Samples were washed twice with 70% ethanol, and RNA was dissolved in DEPC water to a final concentration of 1 μg/μl, followed by reverse transcription PCR.
cDNA synthesis kit (Invitrogen)를 이용하여 cDNA를 얻었다. cDNA was obtained using a cDNA synthesis kit (Invitrogen).
각 qPCR 반응을 스탁 cDNA 2 μl, 1 μM 정방향 및 역방향 프라이머 믹스 및 power SYBR Green PCR Master Mix (Thermo Fischer Scientific) 5 μl를 포함하였다. PCR 프라이머는 다음과 같다: CHOP, 정방향 (Forward): 5’-AAGGAAAGTGGCACAGCTAGCT -3’, 역방향 (reverse): 5’-CTGGTCAGGCGCTCGATTT -3’; Bcl2, 정방향 (Forward): 5’-TTGTGGCCTTCTTTGAGTTCGGTG -3’, 역방향 (reverse): 5’-GGTGCCGGTTCAGGTACTCAGTCA -3’.Each qPCR reaction contained 2 μl of stock cDNA, 1 μM forward and reverse primer mix and 5 μl of power SYBR Green PCR Master Mix (Thermo Fischer Scientific). PCR primers are as follows: CHOP, Forward: 5'-AAGGAAAGTGGCACAGCTAGCT -3', Reverse: 5'-CTGGTCAGGCGCTCGATTT -3'; Bcl2, Forward: 5'-TTGTGGCCTTCTTTGAGTTCGGTG -3', Reverse: 5'-GGTGCCGGTTCAGGTACTCAGTCA -3'.
7. 세포 생존력 확인7. Check cell viability
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)를 이용하여 HepG2 및 AML12 세포 주의 세포 생존력을 확인하였다. 96웰 플레이트에 5 × 103 cells/well 세포와 다양한 농도의 DOX를 분주하였다. MTT 분석은 DOX 처리 후 15시간 동안 수행되었다. 처리 기간 후 세포를 PBS로 잠시 세척하고 Mixing solution (2 mg of MTT (Sigma-Aldrich) in 1 ml PBS)를 각 웰에 첨가한 후 37℃에서 4시간 동안 인큐베이션하였다. MTT 용액을 제거하고, 각 웰에 DMSO 100 μl를 첨가하였다. 이후 플레이트를 37℃에서 30분간 유지시키고 spectrophotometric microplate reader (Molecular Devices, San Jose, CA, USA)를 이용하여 580 nm에서 웰의 OD 값을 확인하였으며, 세포 생존의 백분율로 결과를 나타내었다.Cell viability of HepG2 and AML12 cell lines was confirmed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT). In a 96-well plate, 5 × 10 3 cells/well cells and various concentrations of DOX were dispensed. MTT analysis was performed for 15 hours after DOX treatment. After the treatment period, the cells were briefly washed with PBS, and a mixing solution (2 mg of MTT (Sigma-Aldrich) in 1 ml PBS) was added to each well and incubated at 37° C. for 4 hours. The MTT solution was removed and 100 μl of DMSO was added to each well. Thereafter, the plate was maintained at 37° C. for 30 minutes, and the OD value of the well was checked at 580 nm using a spectrophotometric microplate reader (Molecular Devices, San Jose, CA, USA), and the result was expressed as a percentage of cell viability.
8. 콜로니 형성 분석8. Colony Formation Assay
6웰 플레이트에 총 1000개의 HepG2 세포를 접종하고, 4일 마다 새배지로 교체하였다. A total of 1000 HepG2 cells were inoculated in a 6-well plate, and a fresh medium was replaced every 4 days.
37℃에서 8-10일간 배양한 후 세포를 수집하고, PBS로 두번 세척하였다. 그 후 100% 메탄올로 콜로니를 고정하고, 세포를 PBS로 세척한 후 0.5% 크리스탈 바이올렛으로 염색하여 광학 현미경하에서 콜로니를 계수하였다. 모든 실험을 3번 반복 수행되었다. After culturing for 8-10 days at 37°C, cells were collected and washed twice with PBS. Thereafter, colonies were fixed with 100% methanol, cells were washed with PBS, and then stained with 0.5% crystal violet, and colonies were counted under an optical microscope. All experiments were repeated three times.
9. 전체 세포 및 핵 추출9. Whole Cell and Nuclear Extraction
HepG2 세포를 1 × 105 cells/well로 분주하고 DOX (Sigma-Aldrich, St Louis, MO, USA) 및/또는 OSMI-1을 처리하였다. 처리 후 원심분리하여 세포를 수집하고, 얼음 같이 차가운 PBS로 두 번 세척하였다. NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Inc.)를 이용하여 제조사의 설명서에 따라 세포질 및 핵단백질을 추출하였다.HepG2 cells were seeded at 1 × 10 5 cells/well and treated with DOX (Sigma-Aldrich, St Louis, MO, USA) and/or OSMI-1. After treatment, the cells were collected by centrifugation and washed twice with ice-cold PBS. Cytoplasmic and nuclear proteins were extracted using NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Inc.) according to the manufacturer's instructions.
<실시예 1> 독소루비신 (DOX) 및 O-GlcNAc 전이효소 (O-GlcNAc transferase, OGT) 저해제 OSMI-1 병용처리에 따른 세포사멸 효과 확인<Example 1> Confirmation of apoptosis effect of doxorubicin (DOX) and O-GlcNAc transferase (O-GlcNAc transferase, OGT) inhibitor OSMI-1 combination treatment
먼저, HepG2 암세포 (HCC)에서 O-글루넥당화 (O-GlcNAcylation, O-GlcNAc) 수준이 독소루비신에 의해 유도되는 세포 사멸과 상관관계를 나타내는지 확인하였다.First, it was confirmed whether the level of O-GlcNAcylation (O-GlcNAc) in HepG2 cancer cells (HCC) correlates with apoptosis induced by doxorubicin.
여러 보고서에 따르면, 많은 암에서 O-GlcNAc 수준이 증가하는 것으로 보고되었으나, O-GlcNAc 수준과 간암 세포 사멸간의 연관성은 명확하지 않다.According to several reports, it has been reported that O-GlcNAc levels are increased in many cancers, but the association between O-GlcNAc levels and hepatocellular death is not clear.
이를 확인하기 위해, 암세포 주 Hep3B, Huh7 및 HepG2에서 변형 및 촉매 효소 OGT의 수준을 확인하였으며, 비 암종 간세포주 AML12와 비교하였다. To confirm this, the levels of the modifying and catalytic enzyme OGT in the cancer cell lines Hep3B, Huh7 and HepG2 were checked and compared with the non-carcinoma hepatocyte line AML12.
그 결과, 도 1A와 같이 Huh7 및 HepG2 세포에서 p53 발현뿐만 아니라 O-GlcNAc 변형 및 OGT 수준이 Hep3B 및 AML12 세포보다 높게 나타나는 것을 확인할 수 있었다.As a result, as shown in FIG. 1A , it was confirmed that p53 expression as well as O-GlcNAc modification and OGT levels in Huh7 and HepG2 cells were higher than in Hep3B and AML12 cells.
또한, OGT의 약리학적 억제제인 OSMI-1이 세포 사멸을 자극할 수 있지를 확인하기 위해, 저용량 DOX (0.4 μM)을 단독 또는 OSMI-1과 병용처리한 간세포의 생존도를 MTT 분석으로 확인하였다.In addition, to confirm that OSMI-1, a pharmacological inhibitor of OGT, can stimulate apoptosis, the viability of hepatocytes treated with low-dose DOX (0.4 μM) alone or in combination with OSMI-1 was confirmed by MTT analysis. .
그 결과, 도 1B와 같이 다른 세포주 보다 HepG2 세포의 DOX 처리군과 DOX 및 OSMI-1 병용처리군 간의 생존도 차이가 현저하게 나타나는 것을 확인할 수 있었다. As a result, it was confirmed that the difference in viability between the DOX-treated group and the DOX and OSMI-1 combination treatment group of HepG2 cells was significantly different than that of other cell lines, as shown in FIG. 1B.
다음으로 DOX 및 OSMI-1 농도 범위에 대한 반응으로 HepG2 세포의 생존도를 확인하였다. Next, the viability of HepG2 cells was confirmed in response to DOX and OSMI-1 concentration ranges.
그 결과, 도 1C와 같이 DOX 반응에 대한 세포 생존도는 농도 의존적으로 감소하였으나, 0.1 내지 1 μM 범위의 저농도에서는 80% 미만의 생존도로 유의한 효과가 나타나지 않았다. 그러나 OSMI-1 (20 μM)를 병용 처리한 경우, DOX의 농도 의존적으로 현저하게 세포사멸 효과가 증가되었으며, HepG2의 증식 활성이 30% 더 억제된 것을 확인할 수 있었다. As a result, as shown in FIG. 1C , the cell viability for the DOX response decreased in a concentration-dependent manner, but at a low concentration ranging from 0.1 to 1 μM, there was no significant effect with a viability of less than 80%. However, when OSMI-1 (20 μM) was co-treated, the apoptosis effect was significantly increased in a concentration-dependent manner of DOX, and it was confirmed that the proliferation activity of HepG2 was further inhibited by 30%.
한편, OGT siRNA을 사용하여 DOX의 농도 의존적 세포 생존력이 OGT 발현 수준과 관련성이 있는 지를 확인하였으며, DOX/OSMI-1 병용처리 결과와 같이 OGT 넉다운과 DOX 병용 처리는 세포 생존도를 현저하게 감소시켰다. On the other hand, using OGT siRNA, it was confirmed whether the concentration-dependent cell viability of DOX was related to the OGT expression level. As shown in the results of the DOX/OSMI-1 combination treatment, the OGT knockdown and DOX combination treatment significantly reduced cell viability. .
또한, 도 1C의 두 번째 도면과 같이 OGT 농도 의존적으로도 유사한 결과가 확인되었다. 농도 의존적으로 OSMI-1을 단독 처리한 경우, DOX과 비교하여 매우 낮은 세포사멸 효과가 확인되었으며, DOX과 병용처리한 결과와 비교하여 생존에는 거의 영향을 미치지 않는 것을 확인할 수 있었다. 0.4 μM DOX이 단독 처리된 대조군에서는 세포 생존도 수준이 약간 감소하였다. In addition, similar results were confirmed in dependence on the OGT concentration as shown in the second figure of FIG. 1C. When OSMI-1 was treated alone in a concentration-dependent manner, a very low apoptosis effect was confirmed compared to DOX, and it was confirmed that there was little effect on survival compared to the result of co-treatment with DOX. The cell viability level was slightly decreased in the control group treated with 0.4 μM DOX alone.
상기 결과로부터 OSMI-1 및 DOX의 병용처리는 상승적으로 HepG2 세포의 증식을 억제하고, 세포 사멸을 촉진시키는 것으로 제안될 수 있다.From the above results, it can be suggested that the combined treatment of OSMI-1 and DOX synergistically inhibits the proliferation of HepG2 cells and promotes apoptosis.
이를 확인하기 위해, DOX 단독 또는 OSMI-1와의 병용처리 후 세포 콜로니 형성 분석을 수행하여 HepG2 세포의 증식을 확인하였다. 8-10일 배양 후 크리스탈 바이올렛 염색으로 세포 콜로니를 시각화한 결과, 도 1D와 같이 MTT 분석 결과와 유사하게 병용 처리군에서 콜로니 형성이 현저하게 감소된 것을 확인할 수 있었다.To confirm this, cell colony formation analysis was performed after treatment with DOX alone or in combination with OSMI-1 to confirm the proliferation of HepG2 cells. As a result of visualizing cell colonies with crystal violet staining after 8-10 days of culture, it was confirmed that colony formation was significantly reduced in the combination treatment group, similar to the MTT analysis result as shown in FIG. 1D.
상기 결과로부터 DOX와 OSMI-1의 병용처리는 각각의 단독처리 보다 매우 효과적인 것을 확인할 수 있었다. From the above results, it was confirmed that the combination treatment of DOX and OSMI-1 was more effective than each treatment alone.
<실시예 2> OSMI-1의 p53 활성화를 통한 DOX 유도 세포사멸 증가 확인<Example 2> Confirmation of increase in DOX-induced apoptosis through p53 activation of OSMI-1
DOX는 생체 내 간암세포에 p53 과발현을 유도하는 것으로 보고됨에 따라, HepG2 세포에서 DOX의 세포사멸 효과를 평가하기 위해, 15시간 동안 다양한 농도의 DOX을 단독 또는 OSMI-1과 병용처리하였다.As it was reported that DOX induces p53 overexpression in liver cancer cells in vivo, to evaluate the apoptosis effect of DOX in HepG2 cells, various concentrations of DOX were treated alone or in combination with OSMI-1 for 15 hours.
그 결과, 도 2A와 같이 p21과 함께, p53의 수준은 DOX 용량 의존적으로 상향 조절되는 것을 확인할 수 있었다. 그러나 OSMI-1 단독 처리군에서는 p53 발현에 어떠한 영향도 나타나지 않는 것이 확인되었다. As a result, it was confirmed that the level of p53 was up-regulated in a dose-dependent manner, along with p21, as shown in FIG. 2A. However, it was confirmed that there was no effect on p53 expression in the group treated with OSMI-1 alone.
또한, HepG2 세포의 DOX 유도 세포사멸에 대하여 OSMI-1에 의해 유도된 감작효과의 영향이 용량 의존적인지 추가로 확인하였다. In addition, it was further confirmed whether the effect of the sensitization effect induced by OSMI-1 on DOX-induced apoptosis of HepG2 cells was dose-dependent.
그 결과, 도 2B와 같이 0.4 μM DOX 단독 처리군에서는 명확하게 세포 사멸이 유도되지 않은 반면, OSMI-1과의 병용 처리군에서는 절단된 caspase-3 신호가 용량 의존적으로 현저하게 증가하였다. As a result, as shown in FIG. 2B, apoptosis was not clearly induced in the group treated with 0.4 μM DOX alone, whereas the cleaved caspase-3 signal was significantly increased in a dose-dependent manner in the group treated with OSMI-1 in combination.
또한, OSMI-1은 p53 발현을 DOX 단독으로 유도한 것보다 더욱 향상시킨 것이 확인되었다. 도 2C를 참고하면, p53 발현 및 pro-apoptotic PARP 신호가 OSMI-1 및 DOX 병용처리된 HepG2 암세포에서 증가한 반면, AML12 정상세포에서는 어떠한 변화도 확인되지 않았다.In addition, it was confirmed that OSMI-1 further improved p53 expression than that induced by DOX alone. Referring to FIG. 2C , while p53 expression and pro-apoptotic PARP signal were increased in OSMI-1 and DOX co-treated HepG2 cancer cells, no change was observed in AML12 normal cells.
상기 결과들로부터 OSMI-1은 HepG2 세포에서 DOX에 의해 유도되는 세포 사멸을 향상시킨 반면, AML12 정상세포에서는 세포 사멸을 유도시키지 않는 것이 확인되었다. From the above results, it was confirmed that OSMI-1 enhanced apoptosis induced by DOX in HepG2 cells, but did not induce apoptosis in normal AML12 cells.
이후 최소 독성 및 치료적 효과를 고려하여 저용량의 DOX (0.4 μM)과 OSMI-1 (20 μM)을 모든 세포 배양 실험에 적용하였다.Thereafter, low doses of DOX (0.4 μM) and OSMI-1 (20 μM) were applied to all cell culture experiments in consideration of minimal toxicity and therapeutic effect.
한편, p53이 DOX/OSMI-1 병용 처리에 의한 세포사멸을 조절하는 지를 확인하기 위해, ATM 억제제 ku55933 및 p53 siRNA를 이용하였다. Meanwhile, to determine whether p53 regulates apoptosis by DOX/OSMI-1 combination treatment, ATM inhibitors ku55933 and p53 siRNA were used.
DOX 처리에 의해 세포 DNA가 손상되면 ATM이 인산화되고 활성화되어 결국 p53 발현이 증가한다는 것은 잘 알려져 있다. 도 2D를 참고하면, DOX이 단독 처리된 경우 p-ATM 및 PARP 수준이 증가하였으나, OSMI-1와 병용처리된 실험군에서 더욱 증가된 것을 확인할 수 있었다. 그러나 왼쪽 패널과 같이 하향 작동인자인 절단된 PARP에 대한 효과는 ATM 억제제 ku55933 처리에 의해 감소되었다. 이와 유사하게 p53-특이적 siRNA 처리는 향상된 아폽토시스 세포사멸 마커의 신호를 현저하게 감소시키는 것이 확인되었다.It is well known that damage to cellular DNA by DOX treatment leads to phosphorylation and activation of ATM, eventually leading to an increase in p53 expression. Referring to FIG. 2D, when DOX was treated alone, p-ATM and PARP levels were increased, but it was confirmed that it was further increased in the experimental group treated in combination with OSMI-1. However, as shown in the left panel, the effect on the down-effector cleaved PARP was reduced by treatment with the ATM inhibitor ku55933. Similarly, it was confirmed that p53-specific siRNA treatment significantly reduced the signal of enhanced apoptosis markers of apoptosis.
상기 결과로부터 DOX/OSMI-1 병용처리의 세포사멸 효과는 p53 신호와 직접적으로 관련있는 것이 확인되었다.From the above results, it was confirmed that the apoptosis effect of the DOX/OSMI-1 combination treatment was directly related to the p53 signal.
HepG2 세포는 야생형 p53 세포이며, Huh7 세포는 P53 Y220C이 돌연변이된 형태로 P21의 발현을 제한하는 세포주이다. 또한, Hep3B는 p53 유전자가 결핍되어 발현되지 않는 세포주로, 앞선 실험에서 확인된 도 1B와 같이 DOX/OSMI-1 병용처리는 야생형 p53이 발현되어 있는 HepG2 세포에서 가장 우수한 세포사멸 효과가 확인됨에 따라, DOX/OSMI-1 병용처리는 p53 신호 특이적으로 세포사멸 효과를 향상시키는 것이 확인되었다.HepG2 cells are wild-type p53 cells, and Huh7 cells are a cell line in which P53 Y220C is mutated and the expression of P21 is restricted. In addition, Hep3B is a cell line that is not expressed due to a deficiency in the p53 gene. As shown in FIG. 1B confirmed in the previous experiment, the DOX/OSMI-1 combination treatment confirmed the best apoptosis effect in HepG2 cells expressing wild-type p53. , It was confirmed that the DOX/OSMI-1 combination treatment enhanced the apoptosis effect specifically for the p53 signal.
한편, ATM은 같은 인산화 잔기에 대한 경쟁을 통해 활성화를 음성적으로 조절하는 O-GlcNAc에 의해 변형되는 것으로 알려져 있다.On the other hand, ATM is known to be modified by O-GlcNAc, which negatively regulates activation through competition for the same phosphorylation residue.
도 2E를 참고하면, DOX 단독 처리는 ATM의 인산화를 분명하게 유도하였으나, OSMI-1와의 병용처리는 비 처리된 HepG2 세포와 비교하여 ATM의 인산화 수준을 더욱 현저하게 증가시켰다. 또한, OGT 및 OGA와 같은 O-GlcNAcylation 조절 효소의 발현 수준을 확인한 결과, OGA 수준은 DOX 또는 OSMI-1 처리와 상관없이 거의 일정하게 나타났으나, OGT 발현은 DOX에 의해 상향 조절되었으며 DOX/OSMI-1 병용처리에 의 더욱 현저하게 증가된 것을 확인할 수 있었다.Referring to FIG. 2E , treatment with DOX alone clearly induced phosphorylation of ATM, but co-treatment with OSMI-1 significantly increased the phosphorylation level of ATM compared to untreated HepG2 cells. In addition, as a result of confirming the expression level of O-GlcNAcylation regulatory enzymes such as OGT and OGA, OGA levels were almost constant regardless of DOX or OSMI-1 treatment, but OGT expression was upregulated by DOX and DOX/OSMI -1 It was confirmed that the increase was significantly increased by the combined treatment.
상기 결과로부터 OGT 발현 변화는 세포 항상성 유지에 기여하는 것으로 제안될 수 있다.From the above results, it can be suggested that changes in OGT expression contribute to the maintenance of cell homeostasis.
<실시예 3> OSMI-1에 의해 유도되는 ER 스트레스 반응 확인<Example 3> Confirmation of ER stress response induced by OSMI-1
먼저, OSMI-1 존재 또는 비존재 조건에서 간세포 주의 PERK 및 IRE1α 단백질의 기초 발현 수준을 확인하였다. First, the basal expression levels of PERK and IRE1α proteins in hepatocytes were confirmed in the presence or absence of OSMI-1.
그 결과, 도 3A와 같이 OSMI-1이 처리된 HepG2 세포에서 PERK 및 IRE1α 발현이 상향조절된 것이 확인된 반면, AML12 세포에서는 유의한 변화가 나타나지 않았다. As a result, it was confirmed that PERK and IRE1α expression was upregulated in OSMI-1 treated HepG2 cells as shown in FIG. 3A, whereas no significant change was observed in AML12 cells.
상기 결과로부터 OSMI-1은 특히 간암 세포주에서 ERK 및 IRE1α-XBP1 신호의 활성화를 유도하는 것을 확인할 수 있었다.From the above results, it was confirmed that OSMI-1 induced the activation of ERK and IRE1α-XBP1 signals, especially in liver cancer cell lines.
또한, HepG2 세포에서 O-GlcNAcylation의 억제가 ER 스트레스 반응을 통하여 세포 사멸 신호를 조절하는지 여부를 확인하였다.In addition, it was confirmed whether the inhibition of O-GlcNAcylation in HepG2 cells regulates the apoptosis signal through the ER stress response.
그 결과, 도 3B와 같이 저용량 DOX 처리 후 O-GlcNAcylation 수준 변화는 확인되지 않았으나 OSMI-1 처리군에서는 용량 의존적으로 감소 효과가 확인되었다. O-GlcNAcylation 수준 변화와 다르게 PERK 및 IRE1α-XBP의 발현은 DOX 처리에 의해 약간 증가한 반면, OSMI-1 처리 시 용량 의존적으로 유의하게 증가되는 것을 확인할 수 있었다. 하향 조절 타겟 Bcl2는 DOX 및 OSMI-1 처리 모두에서 감소되었다. As a result, as shown in FIG. 3B , no change in O-GlcNAcylation level was observed after low-dose DOX treatment, but a dose-dependent reduction effect was confirmed in the OSMI-1 treatment group. Unlike the change in O-GlcNAcylation level, the expression of PERK and IRE1α-XBP was slightly increased by DOX treatment, but it was confirmed that it was significantly increased in a dose-dependent manner during OSMI-1 treatment. The down-regulation target Bcl2 was reduced in both DOX and OSMI-1 treatment.
OSMI-1이 세포를 DOX 유도 세포사멸에 민감하게 만드는 메커니즘을 추가로 확인하였다. The mechanism by which OSMI-1 makes cells susceptible to DOX-induced apoptosis was further identified.
그 결과, 도 3C와 같이 0.4 μM DOX 단독 처리군에서는 Bcl2 수준이 약하게 감소하였으나 Bax를 증가시키지 못하여 아폽토시스 세포사멸이 충분하지 못한 것을 확인할 수 있었다. 그러나 DOX 및 OSMI-1의 병용 처리는 HepG2 세포에서 절단된 caspase-3 및 PARP를 급격히 증가시켰다. 또한, DOX 처리된 세포에서 유의한 O-GlcNAcylation 수준 변화가 나타나지 않았으나, DOX 및 OSMI-1 병용처리된 세포에서는 유의한 감소가 확인되었다. As a result, as shown in FIG. 3C, in the group treated with 0.4 μM DOX alone, the Bcl2 level was weakly decreased, but Bax could not be increased, so it was confirmed that apoptotic cell death was not sufficient. However, co-treatment with DOX and OSMI-1 dramatically increased cleaved caspase-3 and PARP in HepG2 cells. In addition, there was no significant change in O-GlcNAcylation level in DOX-treated cells, but a significant decrease was confirmed in DOX and OSMI-1 co-treated cells.
상기 결과로부터 OSMI-1은 DOX으로 유도되는 세포 사멸을 자극할 수 있음이 확인되었다.From the above results, it was confirmed that OSMI-1 can stimulate DOX-induced apoptosis.
CHOP는 pro-survival 단백질 Bcl2의 하향조절을 통하여 미토콘드리아 매개성 아폽토시스를 촉진시키는 것으로 잘 알려져있으며, 도 2D와 같이 OSMI-1의 농도 의존적으로 CHOP mRNA 수준이 점진적으로 증가되는 것을 확인하였다. 그러나 이와 대조적으로 Bcl2의 수준은 OSMI-1에 의해 유의하게 감소되는 것을 확인할 수 있었다.CHOP is well known to promote mitochondrial-mediated apoptosis through downregulation of the pro-survival protein Bcl2, and it was confirmed that the CHOP mRNA level was gradually increased in a concentration-dependent manner of OSMI-1 as shown in FIG. 2D. However, in contrast to this, it was confirmed that the level of Bcl2 was significantly reduced by OSMI-1.
상기 결과로부터 DOX 존재하에서 OSMI-1은 ER 스트레스 반응의 활성을 상승적으로 향상시켜 Bcl2/Bax 신호경로를 통하여 세포사멸을 유도하는 것이 확인되었다.From the above results, it was confirmed that OSMI-1 synergistically enhanced the activity of the ER stress response in the presence of DOX to induce apoptosis through the Bcl2/Bax signaling pathway.
<실시예 4> DOX 처리된 HepG2 세포에서 OSMI-1에 의해 조절되는 NF-κB 신호 전달 확인<Example 4> Confirmation of NF-κB signaling regulated by OSMI-1 in DOX-treated HepG2 cells
산화스트레스에 대한 반응에 있어서 NF-κB는 매우 중요한 조절 유전자로, 염증 반응과 관련성이 있다. In response to oxidative stress, NF-κB is a very important regulatory gene and is associated with an inflammatory response.
HepG2 세포에서 DOX 처리 기간에 따른 NF-κB p65 서브유닛의 활성화를 확인하였다. 도 4A를 참고하면, 왼쪽 도면에서 확인되는 바와 같이 60분 후 인산화-p65 수준이 증가하여 정점에 도달하였다가 감소하였다. Activation of the NF-κB p65 subunit according to the period of DOX treatment was confirmed in HepG2 cells. Referring to FIG. 4A , as shown in the left figure, the phosphorylation-p65 level increased after 60 minutes, reached a peak, and then decreased.
또한, p65 수준을 60분 동안 DOX 단독 처리군과 OSMI-1 (20 μM)와 병용처리군에서 비교한 결과, 도 4A의 오른쪽 도면과 같이 DOX 처리된 세포에서 시간 경과와 함께 인산화된 p65 수준이 점진적으로 증가하였으나, OSMI-1와 함께 처리된 세포군에서는 인산화가 완전히 억제되는 것을 확인할 수 있었다. In addition, as a result of comparing the p65 level in the group treated with DOX alone and OSMI-1 (20 μM) for 60 minutes, the phosphorylated p65 level over time in the DOX-treated cells as shown in the right figure of FIG. 4A was increased. Although it increased gradually, it was confirmed that phosphorylation was completely inhibited in the cell group treated with OSMI-1.
상기 결과로부터 DOX로 인한 손상이 초기에 염증 또는 세포 생존을 유도하지만 OSMI-1 존재하에서는 억제되는 것이 확인되었다. 이에 따라, DOX에 의해 유도된 p65 활성은 OSMI-1에 의한 음성적 조절을 보상하며, p65 활성은 O-GlcNAc 변형과 직간접적으로 관련되어 있음이 제안될 수 있다.From the above results, it was confirmed that damage caused by DOX initially induces inflammation or cell survival, but is inhibited in the presence of OSMI-1. Accordingly, it could be suggested that p65 activity induced by DOX compensates for negative regulation by OSMI-1, and that p65 activity is directly or indirectly related to O-GlcNAc modification.
또한, 도 4B와 같이 NF-κB 및 p53 신호의 활성은 DOX 농도 의존적인 것이 확인되었다. p65, IKK 및 IκB와 같은 NF-κB 신호 증가 작용기들은 낮은 농도의 DOX (최대 0.4 μM)에 따라 점진적으로 활성화된 반면, 높은 농도에서는 활성이 점진적으로 감소되었다. 그러나 이와 다르게, p53 발현 수준은 DOX을 최대 4 μM까지 처리할 경우 지속적으로 상향 조절되는 것을 확인할 수 있었다.In addition, it was confirmed that the activity of NF-κB and p53 signaling was DOX concentration-dependent as shown in FIG. 4B. NF-κB signaling-increasing groups such as p65, IKK and IκB were gradually activated in response to a low concentration of DOX (up to 0.4 μM), whereas their activity was progressively decreased at a high concentration. However, unlike this, it was confirmed that the p53 expression level was continuously upregulated when DOX was treated up to 4 μM.
상기 결과들로부터 NF-κB 신호 전달은 낮은 농도의 DOX 처리 후 p53 신호 전달과 함께 활성화되는 것이 확인되었다. 그러나 DOX에 의한 손상이 농도 의존적으로 증가함에 따라, NF-κB 신호 전달의 영향이 점차 감소하였으며, p53 신호전달은 급격히 증가하였다.From the above results, it was confirmed that NF-κB signaling was activated together with p53 signaling after treatment with a low concentration of DOX. However, as DOX-induced damage increased in a concentration-dependent manner, the effect of NF-κB signaling gradually decreased, and p53 signaling rapidly increased.
DOX에 의해 유도된 NF-κB 신호전달의 관여를 추가로 확인하기 위해, HepG2 세포를 각각 대조군 또는 OGT siRNA로 형질전환 후 DOX (0.1 및 0.4 μM)를 처리하였다. To further confirm the involvement of DOX-induced NF-κB signaling, HepG2 cells were treated with DOX (0.1 and 0.4 μM) after transformation with control or OGT siRNA, respectively.
그 결과, 도 4C와 같이 OGT 넉다운 세포에서 p65 및 IKK의 활성이 완전히 억제된 반면, 대조군 siRNA 형질전환된 세포에서는 농도 의존적으로 발현이 증가하는 것이 확인되었다. As a result, it was confirmed that the activity of p65 and IKK was completely inhibited in the OGT knockdown cells as shown in FIG. 4C, whereas the expression increased in a concentration-dependent manner in the control siRNA-transformed cells.
또한, IKK가 OGT에 의해 O-GlcNAc화될 수 있음이 보고됨에 따라, DOX 처리에 의한 IKK의 활성화가 O-GlcNAcylation과 관련이 있는지 확인하기 위해, 면역 침전 후 면역블로팅을 수행하여 O-GlcNAcylated IKK의 수준을 확인하였다.In addition, as it has been reported that IKK can be O-GlcNAcized by OGT, to confirm whether activation of IKK by DOX treatment is related to O-GlcNAcylation, immunoblotting was performed after immunoprecipitation to O-GlcNAcylated IKK level was confirmed.
그 결과, 도 4C의 하단 도면과 같이 IKK의 GlcNAc 변형 수준이 DOX 농도와 함께 비례하여 증가하였으며, OGT siRNA로 형질전환된 세포에서는 어떠한 변화도 확인되지 않았다.As a result, as shown in the lower figure of FIG. 4C , the GlcNAc modification level of IKK increased in proportion to the DOX concentration, and no change was observed in cells transformed with OGT siRNA.
상기 결과로부터 IKK 단백질의 O-GlcNAc 변형은 DOX 농도 의존적으로 증가되는 것이 확인되었다. From the above results, it was confirmed that the O-GlcNAc modification of the IKK protein was increased in a DOX concentration-dependent manner.
한편, 몇몇의 보고에 따르면, DOX로 세포를 자극할 경우 p65가 핵에 축적되는 것으로 보고되었다. 이에 따라, O-GlcNAc 변형이 p65의 핵 위치에 미치는 영향을 확인하였다.On the other hand, according to several reports, it was reported that p65 accumulates in the nucleus when cells are stimulated with DOX. Accordingly, the effect of O-GlcNAc modification on the nuclear localization of p65 was confirmed.
그 결과, 도 4D와 같이 DOX 처리는 p65의 핵 전좌를 빠르게 자극하였지만 OSMI-1 처리는 p65 핵 전좌를 유의하게 저해하였다. 이와 반대로, p53 발현 수준은 예상한 바와 같이 DOX 단독 처리로 상향 조절되었으나, OSMI-1 병용 처리에 의해 더욱 증가되는 것을 확인할 수 있었다.As a result, as shown in FIG. 4D , DOX treatment rapidly stimulated p65 nuclear translocation, but OSMI-1 treatment significantly inhibited p65 nuclear translocation. On the contrary, p53 expression level was up-regulated by DOX alone treatment as expected, but it was confirmed that it was further increased by OSMI-1 combined treatment.
또한, 면역형광분석 결과 역시, 상기 결과와 유사하게 핵 p53의 수준이 DOX 단독 처리군보다 OSMI-1과 병용처리된 실험군에서 유의하게 증가된 것을 확인할 수 있었다.In addition, the immunofluorescence analysis result also confirmed that, similar to the above results, the level of nuclear p53 was significantly increased in the experimental group treated with OSMI-1 in combination with DOX alone than in the DOX alone group.
다음으로, p65의 O-GlcNAc을 확인하기 위해, p65 항체를 이용하여 면역침강을 수행한 후 O-GlcNAc 항체로 면역블로팅을 수행한 결과, p65의 O-GlcNAc 변형 수준은 DOX 처리에 의해 증가하였으나 OSMI-1 처리에 의해 감소하였다.Next, to confirm the O-GlcNAc of p65, immunoprecipitation was performed using the p65 antibody and then immunoblotting was performed with the O-GlcNAc antibody. As a result, the level of O-GlcNAc modification of p65 was increased by DOX treatment. However, it was decreased by OSMI-1 treatment.
상기 결과로부터 p65의 O-GlcNAc 변형은 핵 전좌와 관련있음이 확인되었다.From the above results, it was confirmed that the O-GlcNAc modification of p65 is related to nuclear translocation.
DOX 처리 또는 OGT siRNA가 형질 주입된 HepG2 세포에서 염증 및 세포 사멸을 추가로 확인하였다.Inflammation and apoptosis were further confirmed in HepG2 cells transfected with DOX-treated or OGT siRNA.
그 결과, 도 4E와 같이 대조군 siRNA 형질 주입된 세포에서 DOX 처리에 의해 IL-1 및 iNOS의 상향조절이 확인되었으나 OGT 넉다운 세포에서는 DOX 처리에도 증가가 나타나지 않았다. 반면, Bcl2, 절단된 PARP 및 caspase-3과 같은 전세포사멸 신호는 대조군 세포에서 DOX 처리 후 약하게 감소되거나 거의 검출되지 않았다. 그러나 OGT siRNA가 형질 주입된 세포에서는 Bcl2가 현저하게 감소되었으며, 절단된 PARP 및 caspase-3이 명확하게 확인되었다.As a result, upregulation of IL-1 and iNOS was confirmed by DOX treatment in control siRNA-transfected cells as shown in FIG. 4E, but no increase was observed in DOX treatment in OGT knockdown cells. On the other hand, proapoptotic signals such as Bcl2, cleaved PARP and caspase-3 were weakly decreased or hardly detected after DOX treatment in control cells. However, in cells transfected with OGT siRNA, Bcl2 was significantly reduced, and cleaved PARP and caspase-3 were clearly identified.
상기 결과들로부터 DOX로 유도된 세포 손상은 약물 내성 활성과 관련있는 NF-κB 활성에 의해 부분적으로 완화되는 것이 확인됨에 따라, OSMI-1와 DOX의 병용처리는 약물 내성을 현저하게 감소시켜 세포 손상을 증가시킴으로서 향상된 세포 사멸 효과를 유도할 수 있음이 확인되었다.From the above results, it was confirmed that DOX-induced cell damage was partially alleviated by NF-κB activity related to drug resistance activity. It was confirmed that by increasing the it can induce an improved apoptosis effect.
<실시예 5> HepG2 이종이식 마우스에서 OSMI-1과 DOX 병용처리에 따른 항암활성 시너지 효과 확인<Example 5> Confirmation of anticancer activity synergistic effect according to OSMI-1 and DOX combination treatment in HepG2 xenograft mice
앞서 확인된 생체 외 (in vitro) 실험 결과를 바탕으로, NF-κB 및 OGT의 이중 억제가 생체 내에서 DOX의 효과를 증가시킬 수 있는 지 여부를 확인하였다.Based on the previously confirmed in vitro test results, it was confirmed whether the dual inhibition of NF-κB and OGT could increase the effect of DOX in vivo.
낮은 용량의 DOX (0.1 mg/kg) 및 OSMI-1 (1 mg/kg) 병용처리가 잠재적인 치료 효과를 나타낼 수 있는 지 확인하기 위해 피하에 HepG2가 이종 이식된 마우스 모델을 사용하였다. HepG2 세포를 누드 마우스의 왼쪽 축면 피하에 주사하였으며, 7일 후 종양이 주사 부위에서 만져졌다. 상기 마우스를 4개의 처리군으로 구분하고 DOX 또는 OSMI-1 단독 처리군 또는 병용군으로 32일간 관리한 후 다른 시간 마다 종양 부피를 확인하였다.To determine whether the low-dose DOX (0.1 mg/kg) and OSMI-1 (1 mg/kg) combination treatment could have a potential therapeutic effect, a subcutaneously HepG2 xenograft mouse model was used. HepG2 cells were injected subcutaneously on the left axon of nude mice, and the tumor was palpable at the injection site 7 days later. The mice were divided into 4 treatment groups, and after 32 days of administration as a DOX or OSMI-1 treatment group or a combined group, the tumor volume was checked at different times.
그 결과, 도 5A와 같이 OSMI-1과 병용처리된 DOX 군에서는 종양 형성이 4.5 배 감소하는 것이 확인된 반면, OSMI-1 또는 DOX 단독 처리군에서는 대조군과 다른 차이가 나타나지 않았다. As a result, as shown in FIG. 5A , in the DOX group treated with OSMI-1 in combination, it was confirmed that the tumor formation was reduced by 4.5 times, whereas in the group treated with OSMI-1 or DOX alone, there was no difference from the control group.
또한, 웨스턴 블롯 분석을 수행하여 아폽토시스 세포 사멸과 관련된 단백질의 수준을 확인하였다. In addition, Western blot analysis was performed to confirm the level of a protein associated with apoptotic cell death.
그 결과, 도 5B와 같이 비이클(vehicle)이 처리된 이종 이식 마우스와 비교하여 DOX 단독 처리군에서는 p53 및 phospho-p65의 발현이 증가하였으나, OSMI-1 단독 처리군에서는 약간 감소하거나 전혀 변화가 나타나지 않았다.As a result, the expression of p53 and phospho-p65 was increased in the DOX-only group compared to the vehicle-treated xenograft mice as shown in FIG. 5B, but a slight decrease or no change was observed in the OSMI-1 single-treated group. didn't
그러나 병용 처리군에서는 DOX 단독 처리군보다 p53 수준이 매우 증가하였다. 반면, 활성 p65 수준은 대조군과 유사한 수준으로 회복되는 것이 확인됨에 따라, 세포 사멸 경로의 시너지 활성화가 나타나는 것이 확인되었다.However, the p53 level was significantly increased in the combined treatment group than in the DOX alone treatment group. On the other hand, as it was confirmed that the active p65 level was restored to a level similar to that of the control group, it was confirmed that synergistic activation of the apoptosis pathway appeared.
상기 결과들은 DOX과 OSMI-1의 보상작용으로 인해 p65 신호 활성이 감소하고, 그 결과 p53 신호 작용을 통해 아폽토시스 세포사멸이 시너지적으로 증가하는 것을 확인한 시험관 내 실험 결과와 일치하는 것을 확인할 수 있었다.The above results were confirmed to be consistent with the results of in vitro experiments confirming that the p65 signaling activity was decreased due to the compensatory action of DOX and OSMI-1, and as a result, apoptotic cell death was synergistically increased through the p53 signaling action.
상기 결과를 추가로 확인하기 위해, 고용량의 화학치료법 효과를 확인하였다. 고용량의 DOX (1mg/kg) 및 OSMI-1 (5 mg/kg)의 병용 처리 방법이 잠재적인 치료 효과를 나타낼 수 있는지 확인하기 위해, 다른 시간 마다 종양을 추출하여 부피를 확인하였다.To further confirm the above results, the effect of high-dose chemotherapy was confirmed. In order to determine whether the combined treatment method of high-dose DOX (1 mg/kg) and OSMI-1 (5 mg/kg) could have a potential therapeutic effect, tumors were extracted at different times and the volume was checked.
그 결과, 도 5C와 같이 HepG2 세포 이종이식 모델에서 대조군과 비교하여 OSMI-1과 병용처리된 DOX 군에서는 종양 형성이 20배 감소한 반면, DOX 단독 처리군에서는 1.8배 감소한 것을 확인할 수 있었다. 또한, 처리 기간 동안 마우스에서 급성 또는 지연된 독성의 다른 신호는 전혀 나타나지 않았다.As a result, compared to the control group in the HepG2 cell xenograft model, as shown in FIG. 5C , tumor formation was reduced by 20 times in the DOX group treated with OSMI-1, whereas it was confirmed that the DOX alone treatment group decreased by 1.8 times. In addition, there were no other signs of acute or delayed toxicity in mice during the treatment period.
한편, 웨스턴 블론 분석을 수행하여 아폽토시스 세포 사멸과 연관된 단백질 수준을 확인하였다.Meanwhile, Western blot analysis was performed to determine the protein level associated with apoptotic cell death.
그 결과, 도 5D와 같이 비이클 처리된 마우스와 비교하여 DOX 또는 OSMI-1 단독 처리군에서 p53 및 Bax의 발현 증가가 확인되었으나, 병용 처리 후 더욱 증가된 발현 수준이 확인되었다. p21 및 절단된 caspase-3은 DOX 단독 및 병용 처리군 에서 증가하였으나, OSMI-1 단독 처리군에서는 대조군과 비교하여 변화가 나타나지 않았다. 특히, 절단된 caspase-3의 급격한 증가가 확인됨에 따라, 병용 처리군에서만 아폽토시스 연속단계가 나타난 것이 확인되었다.As a result, as shown in FIG. 5D , it was confirmed that the expression of p53 and Bax was increased in the group treated with DOX or OSMI-1 alone compared to the vehicle-treated mice, but the expression level was further increased after the combined treatment. p21 and cleaved caspase-3 were increased in the DOX alone and combined treatment groups, but there was no change in the OSMI-1 alone treatment group compared to the control group. In particular, as the rapid increase in cleaved caspase-3 was confirmed, it was confirmed that the apoptosis continuum appeared only in the combination treatment group.
또한, 예상한 바와 같이 IRE-1 및 PERK의 발현 수준은 OSMI-1 처리에 의해 유의하게 증가하였다. 각각의 처리 후 이종이식의 표현형의 특징을 확인하기 위해, 면역조직화학을 수행하였다. Also, as expected, the expression levels of IRE-1 and PERK were significantly increased by OSMI-1 treatment. To characterize the phenotype of the xenografts after each treatment, immunohistochemistry was performed.
그 결과, 도 6A와 같이 세포 증식 마커인 Ki-67은 DOX 또는 OSMI-1이 단독 처리된 종양세포의 증식을 약간 감소시켰으나, 병용 처리군에서는 매우 우수한 감소 효과가 확인되었다. As a result, as shown in FIG. 6A , Ki-67, a cell proliferation marker, slightly decreased the proliferation of tumor cells treated with DOX or OSMI-1 alone, but a very good reduction effect was confirmed in the combined treatment group.
웨스턴 블롯 분석 결과에서 확인된 바와 같이 DOX 및 OSMI-1 병용 처리의 경우, 절단된 caspase-3, p53 및 PERK의 발현 수준이 다른 처리군과 비교하여 가장 높은 증가를 나타내었으나, 그와 반대로 핵 p65의 수준은 가장 낮게 나타났다.As confirmed by Western blot analysis, in the case of DOX and OSMI-1 combined treatment, the expression levels of cleaved caspase-3, p53 and PERK showed the highest increase compared to other treatment groups, but on the contrary, nuclear p65 level was the lowest.
DOX 단독 처리의 경우, 핵 p65의 발현 수준은 매우 높은 반면, PERK의 수준은 변화가 나타나지 않았다. 또한, OSMI-1 단독 처리군에서는 p65 및 PERK의 변화가 쉽게 확인되었으나, 세포사멸 관련 신호는 전혀 확인되지 않았다.In the case of DOX alone treatment, the expression level of nuclear p65 was very high, whereas the level of PERK did not change. In addition, changes in p65 and PERK were easily confirmed in the OSMI-1 alone treatment group, but apoptosis-related signals were not confirmed at all.
상기 결과들로부터 도 6B와 같이 DOX 및 OSMI-1의 병용 처리는 ROS 생산 및 ER 스트레스 유도를 통하여 상승적으로 아폽토시스 세포 사멸을 유도하는 것으로 제안될 수 있으며, 각 약물의 주요 하위 작용기는 p53 및 PERK/IRE-1의 활성화였으나, NF-κB 신호전달은 ER 스트레스에 의해 조절되거나 동시에 p53 신호전달을 억제하였다.From the above results, it can be suggested that the combined treatment of DOX and OSMI-1 synergistically induces apoptotic cell death through ROS production and ER stress induction, as shown in FIG. Although activation of IRE-1, NF-κB signaling was regulated by ER stress or simultaneously inhibited p53 signaling.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (10)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20200137991 | 2020-10-23 | ||
| KR1020200137991 | 2020-10-23 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20220054174A true KR20220054174A (en) | 2022-05-02 |
| KR102685770B1 KR102685770B1 (en) | 2024-07-18 |
Family
ID=81593524
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020210101696A KR102685770B1 (en) | 2020-10-23 | 2021-08-03 | Composition for preventing or treating liver cancer comprising OSMI-1 and antitumor agent |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR102685770B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115068479A (en) * | 2022-06-20 | 2022-09-20 | 无锡市妇幼保健院 | Carrier-free double-medicine nano assembly and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150136818A (en) | 2014-05-28 | 2015-12-08 | 연세대학교 산학협력단 | Composition for treating hepatic cancer comprising mesenchymal stem cell transfected with IL-12 expressing vector and the treatment methods using the same |
-
2021
- 2021-08-03 KR KR1020210101696A patent/KR102685770B1/en active IP Right Grant
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150136818A (en) | 2014-05-28 | 2015-12-08 | 연세대학교 산학협력단 | Composition for treating hepatic cancer comprising mesenchymal stem cell transfected with IL-12 expressing vector and the treatment methods using the same |
Non-Patent Citations (2)
| Title |
|---|
| International Journal of Toxicology, 39(6), pp. 586-593(2020.08.27.) 1부.* * |
| Oncology Reports, 40, pp.2193-2205(2018) 1부.* * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115068479A (en) * | 2022-06-20 | 2022-09-20 | 无锡市妇幼保健院 | Carrier-free double-medicine nano assembly and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR102685770B1 (en) | 2024-07-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Curcumin derivative WZ35 inhibits tumor cell growth via ROS-YAP-JNK signaling pathway in breast cancer | |
| Zou et al. | ROS generation mediates the anti-cancer effects of WZ35 via activating JNK and ER stress apoptotic pathways in gastric cancer | |
| Kim et al. | Esculetin suppresses tumor growth and metastasis by targeting Axin2/E-cadherin axis in colorectal cancer | |
| Rajendran et al. | Suppression of signal transducer and activator of transcription 3 activation by butein inhibits growth of human hepatocellular carcinoma in vivo | |
| Zeng et al. | Thymol Isolated from Thymus vulgaris L. inhibits colorectal cancer cell growth and metastasis by suppressing the Wnt/β-catenin pathway | |
| US9597325B2 (en) | Inhibitors of late SV40 factor (LSF) as cancer chemotherapeutics | |
| Kim et al. | Artesunate suppresses tumor growth and induces apoptosis through the modulation of multiple oncogenic cascades in a chronic myeloid leukemia xenograft mouse model | |
| Zhang et al. | Dehydroeffusol inhibits gastric cancer cell growth and tumorigenicity by selectively inducing tumor-suppressive endoplasmic reticulum stress and a moderate apoptosis | |
| Yang et al. | Sinomenine, a COX-2 inhibitor, induces cell cycle arrest and inhibits growth of human colon carcinoma cells in vitro and in vivo | |
| Chen et al. | A novel MyD88 inhibitor LM9 prevents atherosclerosis by regulating inflammatory responses and oxidative stress in macrophages | |
| Jiang et al. | Safrana l prevents prostate cancer recurrence by blocking the Re-activation of quiescent cancer cells via downregulation of S-phase kinase-associated protein 2 | |
| Zhang et al. | JMJD3 promotes survival of diffuse large B-cell lymphoma subtypes via distinct mechanisms | |
| Abdullah et al. | A plant alkaloid, veratridine, potentiates cancer chemosensitivity by UBXN2A-dependent inhibition of an oncoprotein, mortalin-2 | |
| Wang et al. | Ursolic acid inhibits breast cancer metastasis by suppressing glycolytic metabolism via activating sp1/caveolin-1 signaling | |
| Li et al. | ZNF32 protects against oxidative stress-induced apoptosis by modulating C1QBP transcription | |
| Wang et al. | Synergistic anti-breast cancer effect of pulsatilla saponin D and camptothecin through interrupting autophagic–lysosomal function and promoting p62-mediated ubiquitinated protein aggregation | |
| US11260062B2 (en) | Pharmaceutical composition for treatment of lung cancer comprising glucocorticoid-based compound | |
| KR101978629B1 (en) | Pharmaceutical Composition for Treating Non-small Cell lung Cancer Comprising Glucocorticoids | |
| Wang et al. | Homoharringtonine could induce quick protein synthesis of PSMD11 through activating MEK1/ERK1/2 signaling pathway in pancreatic cancer cells | |
| Chen et al. | Jervine exhibits anticancer effects on nasopharyngeal carcinoma through promoting autophagic apoptosis via the blockage of Hedgehog signaling | |
| Zhao et al. | As a downstream target of the AKT pathway, NPTX1 inhibits proliferation and promotes apoptosis in hepatocellular carcinoma | |
| Russell et al. | Bromodomain and extraterminal (BET) proteins regulate hepatocyte proliferation in hepatocyte-driven liver regeneration | |
| Wen et al. | Sesamin exerts anti‐tumor activity in esophageal squamous cell carcinoma via inhibition of TRIM44 and NF‐κB signaling | |
| Bai et al. | Modulating MGMT expression through interfering with cell signaling pathways | |
| KR102685770B1 (en) | Composition for preventing or treating liver cancer comprising OSMI-1 and antitumor agent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right |