WO2022111670A1 - Tipranavir在制备杀伤肿瘤干细胞和肿瘤细胞的癌症治疗药物中的用途 - Google Patents
Tipranavir在制备杀伤肿瘤干细胞和肿瘤细胞的癌症治疗药物中的用途 Download PDFInfo
- Publication number
- WO2022111670A1 WO2022111670A1 PCT/CN2021/133781 CN2021133781W WO2022111670A1 WO 2022111670 A1 WO2022111670 A1 WO 2022111670A1 CN 2021133781 W CN2021133781 W CN 2021133781W WO 2022111670 A1 WO2022111670 A1 WO 2022111670A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- tipranavir
- cells
- tumor
- stem cells
- cancer
- Prior art date
Links
- SUJUHGSWHZTSEU-FYBSXPHGSA-N tipranavir Chemical compound C([C@@]1(CCC)OC(=O)C([C@H](CC)C=2C=C(NS(=O)(=O)C=3N=CC(=CC=3)C(F)(F)F)C=CC=2)=C(O)C1)CC1=CC=CC=C1 SUJUHGSWHZTSEU-FYBSXPHGSA-N 0.000 title claims abstract description 198
- SUJUHGSWHZTSEU-UHFFFAOYSA-N Tipranavir Natural products C1C(O)=C(C(CC)C=2C=C(NS(=O)(=O)C=3N=CC(=CC=3)C(F)(F)F)C=CC=2)C(=O)OC1(CCC)CCC1=CC=CC=C1 SUJUHGSWHZTSEU-UHFFFAOYSA-N 0.000 title claims abstract description 193
- 229960000838 tipranavir Drugs 0.000 title claims abstract description 193
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 186
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 171
- 210000004881 tumor cell Anatomy 0.000 title claims abstract description 75
- 239000003814 drug Substances 0.000 title claims abstract description 66
- 229940079593 drug Drugs 0.000 title claims abstract description 43
- 230000002147 killing effect Effects 0.000 title claims abstract description 43
- 238000011275 oncology therapy Methods 0.000 title abstract 2
- 201000011510 cancer Diseases 0.000 claims abstract description 46
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 12
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 154
- 206010017758 gastric cancer Diseases 0.000 claims description 154
- 201000011549 stomach cancer Diseases 0.000 claims description 154
- 210000004027 cell Anatomy 0.000 claims description 138
- 230000014509 gene expression Effects 0.000 claims description 67
- 229960004316 cisplatin Drugs 0.000 claims description 65
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 65
- 150000003839 salts Chemical class 0.000 claims description 53
- 150000002148 esters Chemical class 0.000 claims description 52
- 238000011282 treatment Methods 0.000 claims description 43
- 201000010099 disease Diseases 0.000 claims description 34
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 34
- 206010060862 Prostate cancer Diseases 0.000 claims description 33
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 33
- 101001069710 Homo sapiens Serine protease 23 Proteins 0.000 claims description 29
- 102100033835 Serine protease 23 Human genes 0.000 claims description 29
- 230000002401 inhibitory effect Effects 0.000 claims description 24
- 230000002062 proliferating effect Effects 0.000 claims description 23
- 229930012538 Paclitaxel Natural products 0.000 claims description 19
- 229960001592 paclitaxel Drugs 0.000 claims description 19
- 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 19
- 108090000623 proteins and genes Proteins 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 17
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 claims description 13
- 238000002512 chemotherapy Methods 0.000 claims description 12
- 229940044683 chemotherapy drug Drugs 0.000 claims description 12
- 229960002949 fluorouracil Drugs 0.000 claims description 12
- 102000003898 interleukin-24 Human genes 0.000 claims description 12
- 108090000237 interleukin-24 Proteins 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 11
- 201000005202 lung cancer Diseases 0.000 claims description 11
- 208000020816 lung neoplasm Diseases 0.000 claims description 11
- 206010006187 Breast cancer Diseases 0.000 claims description 10
- 208000026310 Breast neoplasm Diseases 0.000 claims description 10
- 206010009944 Colon cancer Diseases 0.000 claims description 10
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 10
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 10
- 201000004101 esophageal cancer Diseases 0.000 claims description 10
- 201000007270 liver cancer Diseases 0.000 claims description 9
- 208000014018 liver neoplasm Diseases 0.000 claims description 9
- 238000001959 radiotherapy Methods 0.000 claims description 7
- 208000037819 metastatic cancer Diseases 0.000 claims description 6
- 208000011575 metastatic malignant neoplasm Diseases 0.000 claims description 6
- 206010061289 metastatic neoplasm Diseases 0.000 claims description 6
- 238000011127 radiochemotherapy Methods 0.000 claims description 6
- 230000000306 recurrent effect Effects 0.000 claims description 6
- 230000004663 cell proliferation Effects 0.000 claims description 5
- 238000002560 therapeutic procedure Methods 0.000 claims description 5
- 230000000694 effects Effects 0.000 abstract description 49
- 231100000331 toxic Toxicity 0.000 abstract description 20
- 230000002588 toxic effect Effects 0.000 abstract description 20
- 239000004480 active ingredient Substances 0.000 abstract description 7
- 229940126585 therapeutic drug Drugs 0.000 abstract description 7
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 6
- 239000003937 drug carrier Substances 0.000 abstract description 4
- 101000853009 Homo sapiens Interleukin-24 Proteins 0.000 description 46
- 102100036671 Interleukin-24 Human genes 0.000 description 46
- 230000006907 apoptotic process Effects 0.000 description 41
- 230000005775 apoptotic pathway Effects 0.000 description 22
- 230000002438 mitochondrial effect Effects 0.000 description 22
- 102000002574 p38 Mitogen-Activated Protein Kinases Human genes 0.000 description 16
- 108010068338 p38 Mitogen-Activated Protein Kinases Proteins 0.000 description 16
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- 102000055574 bcl-2 Homologous Antagonist-Killer Human genes 0.000 description 15
- 108700039689 bcl-2 Homologous Antagonist-Killer Proteins 0.000 description 15
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 13
- 102000055102 bcl-2-Associated X Human genes 0.000 description 12
- 108700000707 bcl-2-Associated X Proteins 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- 230000001419 dependent effect Effects 0.000 description 10
- 230000002496 gastric effect Effects 0.000 description 10
- 230000012010 growth Effects 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 230000001105 regulatory effect Effects 0.000 description 10
- 101100325747 Mus musculus Bak1 gene Proteins 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 9
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 9
- 229920000776 Poly(Adenosine diphosphate-ribose) polymerase Polymers 0.000 description 9
- 108010087230 Sincalide Proteins 0.000 description 9
- 238000010609 cell counting kit-8 assay Methods 0.000 description 9
- 210000002919 epithelial cell Anatomy 0.000 description 9
- 102100023275 Dual specificity mitogen-activated protein kinase kinase 3 Human genes 0.000 description 8
- 101001115394 Homo sapiens Dual specificity mitogen-activated protein kinase kinase 3 Proteins 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 210000003734 kidney Anatomy 0.000 description 8
- 230000002018 overexpression Effects 0.000 description 8
- 102100030497 Cytochrome c Human genes 0.000 description 7
- 108010075031 Cytochromes c Proteins 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 230000001093 anti-cancer Effects 0.000 description 7
- 210000004185 liver Anatomy 0.000 description 7
- 210000003470 mitochondria Anatomy 0.000 description 7
- 210000000952 spleen Anatomy 0.000 description 7
- 102000043136 MAP kinase family Human genes 0.000 description 6
- 108091054455 MAP kinase family Proteins 0.000 description 6
- 210000004072 lung Anatomy 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 230000001640 apoptogenic effect Effects 0.000 description 5
- 210000002216 heart Anatomy 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 4
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 4
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 description 4
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 description 4
- 108020004459 Small interfering RNA Proteins 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000012830 cancer therapeutic Substances 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000002648 combination therapy Methods 0.000 description 4
- 210000000805 cytoplasm Anatomy 0.000 description 4
- 238000011354 first-line chemotherapy Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000009456 molecular mechanism Effects 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000004614 tumor growth Effects 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 206010027476 Metastases Diseases 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 3
- 206010029155 Nephropathy toxic Diseases 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 238000003559 RNA-seq method Methods 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 239000002253 acid Chemical class 0.000 description 3
- 230000005754 cellular signaling Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000009950 gastric cancer growth Effects 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 230000009401 metastasis Effects 0.000 description 3
- 230000007694 nephrotoxicity Effects 0.000 description 3
- 231100000417 nephrotoxicity Toxicity 0.000 description 3
- 238000011580 nude mouse model Methods 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 231100000588 tumorigenic Toxicity 0.000 description 3
- 230000000381 tumorigenic effect Effects 0.000 description 3
- YKJYKKNCCRKFSL-RDBSUJKOSA-N (-)-anisomycin Chemical compound C1=CC(OC)=CC=C1C[C@@H]1[C@H](OC(C)=O)[C@@H](O)CN1 YKJYKKNCCRKFSL-RDBSUJKOSA-N 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- YKJYKKNCCRKFSL-UHFFFAOYSA-N Anisomycin Natural products C1=CC(OC)=CC=C1CC1C(OC(C)=O)C(O)CN1 YKJYKKNCCRKFSL-UHFFFAOYSA-N 0.000 description 2
- 108090000397 Caspase 3 Proteins 0.000 description 2
- 102100029855 Caspase-3 Human genes 0.000 description 2
- 108010092160 Dactinomycin Proteins 0.000 description 2
- CTKXFMQHOOWWEB-UHFFFAOYSA-N Ethylene oxide/propylene oxide copolymer Chemical compound CCCOC(C)COCCO CTKXFMQHOOWWEB-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-AHCXROLUSA-N Iodine-123 Chemical compound [123I] ZCYVEMRRCGMTRW-AHCXROLUSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 206010066901 Treatment failure Diseases 0.000 description 2
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 2
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 229930183665 actinomycin Natural products 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 150000001447 alkali salts Chemical class 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003560 cancer drug Substances 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000007541 cellular toxicity Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 210000002249 digestive system Anatomy 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 229960003722 doxycycline Drugs 0.000 description 2
- XQTWDDCIUJNLTR-CVHRZJFOSA-N doxycycline monohydrate Chemical compound O.O=C1C2=C(O)C=CC=C2[C@H](C)[C@@H]2C1=C(O)[C@]1(O)C(=O)C(C(N)=O)=C(O)[C@@H](N(C)C)[C@@H]1[C@H]2O XQTWDDCIUJNLTR-CVHRZJFOSA-N 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000005003 heart tissue Anatomy 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- -1 inorganic bases Acids Chemical class 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 210000005084 renal tissue Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000005740 tumor formation Effects 0.000 description 2
- PNDPGZBMCMUPRI-HVTJNCQCSA-N 10043-66-0 Chemical compound [131I][131I] PNDPGZBMCMUPRI-HVTJNCQCSA-N 0.000 description 1
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 1
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- ZFVRYNYOPQZKDG-UHFFFAOYSA-N 4-[6,6-dimethyl-4-oxo-3-(trifluoromethyl)-5,7-dihydroindazol-1-yl]-2-[(4-hydroxycyclohexyl)amino]benzamide Chemical compound O=C1CC(C)(C)CC2=C1C(C(F)(F)F)=NN2C(C=1)=CC=C(C(N)=O)C=1NC1CCC(O)CC1 ZFVRYNYOPQZKDG-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-OUBTZVSYSA-N Ammonia-15N Chemical compound [15NH3] QGZKDVFQNNGYKY-OUBTZVSYSA-N 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- AXRYRYVKAWYZBR-UHFFFAOYSA-N Atazanavir Natural products C=1C=C(C=2N=CC=CC=2)C=CC=1CN(NC(=O)C(NC(=O)OC)C(C)(C)C)CC(O)C(NC(=O)C(NC(=O)OC)C(C)(C)C)CC1=CC=CC=C1 AXRYRYVKAWYZBR-UHFFFAOYSA-N 0.000 description 1
- 108010019625 Atazanavir Sulfate Proteins 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 238000011729 BALB/c nude mouse Methods 0.000 description 1
- OKTJSMMVPCPJKN-IGMARMGPSA-N Carbon-12 Chemical compound [12C] OKTJSMMVPCPJKN-IGMARMGPSA-N 0.000 description 1
- OKTJSMMVPCPJKN-OUBTZVSYSA-N Carbon-13 Chemical compound [13C] OKTJSMMVPCPJKN-OUBTZVSYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-NJFSPNSNSA-N Carbon-14 Chemical compound [14C] OKTJSMMVPCPJKN-NJFSPNSNSA-N 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 108090000567 Caspase 7 Proteins 0.000 description 1
- 102100038902 Caspase-7 Human genes 0.000 description 1
- 102100026550 Caspase-9 Human genes 0.000 description 1
- 108090000566 Caspase-9 Proteins 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical compound [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 108010040476 FITC-annexin A5 Proteins 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- QJGQUHMNIGDVPM-BJUDXGSMSA-N Nitrogen-13 Chemical compound [13N] QJGQUHMNIGDVPM-BJUDXGSMSA-N 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-OUBTZVSYSA-N Phosphorus-32 Chemical compound [32P] OAICVXFJPJFONN-OUBTZVSYSA-N 0.000 description 1
- OAICVXFJPJFONN-NJFSPNSNSA-N Phosphorus-33 Chemical compound [33P] OAICVXFJPJFONN-NJFSPNSNSA-N 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- NINIDFKCEFEMDL-NJFSPNSNSA-N Sulfur-34 Chemical compound [34S] NINIDFKCEFEMDL-NJFSPNSNSA-N 0.000 description 1
- NINIDFKCEFEMDL-AKLPVKDBSA-N Sulfur-35 Chemical compound [35S] NINIDFKCEFEMDL-AKLPVKDBSA-N 0.000 description 1
- NINIDFKCEFEMDL-RNFDNDRNSA-N Sulfur-36 Chemical compound [36S] NINIDFKCEFEMDL-RNFDNDRNSA-N 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- QPMSXSBEVQLBIL-CZRHPSIPSA-N ac1mix0p Chemical compound C1=CC=C2N(C[C@H](C)CN(C)C)C3=CC(OC)=CC=C3SC2=C1.O([C@H]1[C@]2(OC)C=CC34C[C@@H]2[C@](C)(O)CCC)C2=C5[C@]41CCN(C)[C@@H]3CC5=CC=C2O QPMSXSBEVQLBIL-CZRHPSIPSA-N 0.000 description 1
- 150000008044 alkali metal hydroxides Chemical class 0.000 description 1
- 229910001860 alkaline earth metal hydroxide Inorganic materials 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000036436 anti-hiv Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229960003277 atazanavir Drugs 0.000 description 1
- AXRYRYVKAWYZBR-GASGPIRDSA-N atazanavir Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)OC)C(C)(C)C)[C@@H](O)CN(CC=1C=CC(=CC=1)C=1N=CC=CC=1)NC(=O)[C@@H](NC(=O)OC)C(C)(C)C)C1=CC=CC=C1 AXRYRYVKAWYZBR-GASGPIRDSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 description 1
- 229940092714 benzenesulfonic acid Drugs 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- OKTJSMMVPCPJKN-BJUDXGSMSA-N carbon-11 Chemical compound [11C] OKTJSMMVPCPJKN-BJUDXGSMSA-N 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 229960005107 darunavir Drugs 0.000 description 1
- CJBJHOAVZSMMDJ-HEXNFIEUSA-N darunavir Chemical compound C([C@@H]([C@H](O)CN(CC(C)C)S(=O)(=O)C=1C=CC(N)=CC=1)NC(=O)O[C@@H]1[C@@H]2CCO[C@@H]2OC1)C1=CC=CC=C1 CJBJHOAVZSMMDJ-HEXNFIEUSA-N 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 229910052805 deuterium Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 108010030074 endodeoxyribonuclease MluI Proteins 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 229960003142 fosamprenavir Drugs 0.000 description 1
- MLBVMOWEQCZNCC-OEMFJLHTSA-N fosamprenavir Chemical compound C([C@@H]([C@H](OP(O)(O)=O)CN(CC(C)C)S(=O)(=O)C=1C=CC(N)=CC=1)NC(=O)O[C@@H]1COCC1)C1=CC=CC=C1 MLBVMOWEQCZNCC-OEMFJLHTSA-N 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000003481 heat shock protein 90 inhibitor Substances 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 229940071870 hydroiodic acid Drugs 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- XMBWDFGMSWQBCA-NJFSPNSNSA-N iodane Chemical compound [129IH] XMBWDFGMSWQBCA-NJFSPNSNSA-N 0.000 description 1
- XMBWDFGMSWQBCA-YPZZEJLDSA-N iodane Chemical compound [125IH] XMBWDFGMSWQBCA-YPZZEJLDSA-N 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 229940044173 iodine-125 Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000011242 molecular targeted therapy Methods 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- QVGXLLKOCUKJST-BJUDXGSMSA-N oxygen-15 atom Chemical compound [15O] QVGXLLKOCUKJST-BJUDXGSMSA-N 0.000 description 1
- QVGXLLKOCUKJST-OUBTZVSYSA-N oxygen-17 atom Chemical compound [17O] QVGXLLKOCUKJST-OUBTZVSYSA-N 0.000 description 1
- QVGXLLKOCUKJST-NJFSPNSNSA-N oxygen-18 atom Chemical compound [18O] QVGXLLKOCUKJST-NJFSPNSNSA-N 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229940097886 phosphorus 32 Drugs 0.000 description 1
- 229920001993 poloxamer 188 Polymers 0.000 description 1
- 229940044519 poloxamer 188 Drugs 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- PUZPDOWCWNUUKD-ULWFUOSBSA-M sodium;fluorine-18(1-) Chemical compound [18F-].[Na+] PUZPDOWCWNUUKD-ULWFUOSBSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- NINIDFKCEFEMDL-IGMARMGPSA-N sulfur-32 atom Chemical compound [32S] NINIDFKCEFEMDL-IGMARMGPSA-N 0.000 description 1
- NINIDFKCEFEMDL-OUBTZVSYSA-N sulfur-33 atom Chemical compound [33S] NINIDFKCEFEMDL-OUBTZVSYSA-N 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- AYUNIORJHRXIBJ-TXHRRWQRSA-N tanespimycin Chemical compound N1C(=O)\C(C)=C\C=C/[C@H](OC)[C@@H](OC(N)=O)\C(C)=C\[C@H](C)[C@@H](O)[C@@H](OC)C[C@H](C)CC2=C(NCC=C)C(=O)C=C1C2=O AYUNIORJHRXIBJ-TXHRRWQRSA-N 0.000 description 1
- 229950007866 tanespimycin Drugs 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 210000001835 viscera Anatomy 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
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/4433—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
-
- 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
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the invention relates to the technical field of medicine, in particular to the use of Tipranavir in the preparation of a cancer therapeutic drug for killing tumor stem cells and tumor cells.
- GC gastric cancer
- my country is a high-incidence area of gastric cancer, with high morbidity and mortality, ranking second in malignant tumors.
- the principle of gastric cancer treatment is comprehensive treatment based on surgery. In my country, the rate of early diagnosis of gastric cancer is still relatively low. Most patients are diagnosed with advanced tumors and lose the chance of surgical cure. Most patients die due to tumor recurrence and metastasis.
- Tumor cells in tumors can be divided into two categories: one is ordinary tumor cells, and the other is tumor stem cells.
- Ordinary tumor cells have the characteristics of rapid division, sensitivity to anticancer drugs, and no self-renewal ability.
- Cancer stem cells are a small number of cell subsets in tumors that have the characteristics of stem cells, that is, self-renewal ability and can produce tumor cells of different degrees of differentiation. Metastasis and recurrence, as well as the root cause of chemoradiotherapy resistance, are also the main reasons for tumor treatment failure and death. It has the following characteristics: it is usually in a resting state, insensitive to anticancer drugs, and has the ability to self-renew, that is, to have the ability to proliferate indefinitely.
- tumor chemotherapy In tumor chemotherapy, a large number of tumor cells are killed (sensitive to chemotherapeutic drugs), while tumor stem cells survive (insensitive to chemotherapeutic drugs).
- many treatments can control and shrink tumors in a short period of time, but most patients experience recurrence and metastasis, which leads to treatment failure. Therefore, tumor recurrence is due to the ineffectiveness of tumor chemotherapeutics on tumor stem cells.
- current tumor chemotherapeutics mainly target tumor cells rather than tumor stem cells.
- most tumor chemotherapy drugs are only effective on tumor cells, but not on tumor stem cells. Therefore, to achieve the purpose of curing cancer, it is not only necessary to eliminate ordinary tumor cells, but more importantly, to kill tumor stem cells that cause tumor proliferation through specific treatment.
- Tipranavir (tipranavir, PNU-140690, CAS No.: 174484-41-4) is a clinical anti-HIV drug and the first approved non-peptide protease inhibitor. Its mechanism of action is mainly by inhibiting protease Play a role. Studies have shown that Tipranavir can inhibit HIV virus that is resistant to commercially available drugs, and is suitable for HIV-infected patients with signs of viral replication who are resistant to multiple protease inhibitors. However, at present, the application of Tipranavir in the preparation of cancer stem cells and their tumor cell drugs has not been reported in literature, and has not been used in anti-tumor.
- the main purpose of the present invention is to provide a new use of Tipranavir (tipranavir, PNU-140690) for preparing a cancer therapeutic drug for killing tumor stem cells and tumor cells.
- the human gastric cancer stem cells were treated with Tipranavir, and the human gastric cancer stem cells were treated with different concentrations of Tipranavir and at different times.
- the CCK-8 kit was used to detect the survival of the cells. The results showed that Tipranavir could effectively kill human gastric cancer stem cells in a time- and concentration-dependent manner.
- Tipranavir and the current first-line chemotherapy drugs for gastric cancer fluorouracil (fluorouracil, 5-FU) and cisplatin (cisplatin)
- fluorouracil fluorouracil, 5-FU
- cisplatin cisplatin
- Tipranavir treats different cancer cell lines, including lung cancer cells (PC9), prostate cancer cells (PC3), paclitaxel-resistant prostate cancer cells (PC3/Tax), esophageal cancer cells (KYSE180 and KYSE520), colorectal cancer cells ( HCT116), breast cancer cells (MDA-MB-231), liver cancer cells (Huh7), and the cell survival was detected using CCK-8 kit.
- lung cancer cells PC9
- prostate cancer cells PC3
- paclitaxel-resistant prostate cancer cells PC3/Tax
- esophageal cancer cells KYSE180 and KYSE520
- colorectal cancer cells HCT116
- breast cancer cells MDA-MB-231
- liver cancer cells Huh7
- Tipranavir treatment can significantly inhibit the growth of transplanted tumors, and its effect is significantly better than the combination of 5-FU and cisplatin (cisplatin), and has no obvious toxic and side effects on lung, liver, spleen, kidney, and heart tissue.
- 5-FU and cisplatin cisplatin
- the combined use of 5-FU and cisplatin (cisplatin) has nephrotoxicity and has toxic side effects on the kidneys.
- Apoptosis detection experiment To analyze the mechanism of Tipranavir killing gastric cancer stem cells, the results show that: Tipranavir can kill gastric cancer stem cells by inducing apoptosis of gastric cancer stem cells (apoptosis).
- RNA-seq analysis compared the gene expression changes of gastric cancer stem cells treated with Tipranavir and not treated, and searched for the differential genes of gastric cancer stem cells treated with Tipranavir, and verified the expression of related differential genes by qPCR and Western blotting. The results showed that the gastric cancer stem cells treated with Tipranavir were significantly up-regulated IL24 expression, IL24 may be the target of Tipranavir. IL24 has been confirmed as a tumor cell-specific tumor suppressor gene, its high expression can significantly inhibit tumor growth, and its high expression in normal cells has no effect on normal cells.
- Tipranavir killing gastric cancer stem cells is as follows: Tipranavir up-regulates IL24, then up-regulates Bax and Bak proteins, activates the mitochondrial apoptosis pathway (mitochondrial apoptosis pathway), and induces gastric cancer stem cell apoptosis (apoptosis), thereby Kill gastric cancer stem cells.
- Tipranavir inhibited the expression of IL24 induced by PRSS23 and promoted the apoptosis of gastric cancer stem cells.
- the increase strongly affects the expression of pro-apoptotic proteins Bax and Bak, thereby activating the mitochondrial apoptosis pathway and promoting gastric cancer stem cell apoptosis.
- the present invention confirms that Tipranavir has the effect of killing gastric cancer stem cells and gastric cancer cells, as well as other new functions of cancer stem cells and tumor cells, and can be used to prepare anti-gastric cancer and other anti-tumor therapeutic drugs.
- the present invention provides a method of applying Tipranavir or its salts, esters, isotopes to kill or inhibit tumor stem cells or tumor cells by inhibiting or reducing PRSS23 protein or gene expression in tumor stem cells or tumor cells or by upregulating tumor stem cells or IL24 protein or gene expression levels in tumor cells.
- the present invention provides methods of applying Tipranavir or its salts, esters, isotopes to kill or inhibit tumor stem cells or quiescent tumor cells by inhibiting or reducing PRSS23 protein or gene expression in tumor stem cells or tumor cells or via up-regulation thereof IL24 protein or gene expression levels in cancer stem cells or tumor cells.
- IL24 is up-regulated, which in turn up-regulates Bax and Bak proteins, activates the mitochondrial apoptosis pathway of cancer stem cells or tumor cells, and promotes the apoptosis of cancer stem cells or tumor cells.
- the present invention also provides various uses of Tipranavir or its salts, esters, isotopes:
- Tipranavir or its salts, esters and isotopes in killing or inhibiting tumor stem cells or quiescent tumor cells.
- Tipranavir or a salt, ester or isotope thereof in the preparation of a medicament for killing or inhibiting tumor stem cells or quiescent tumor cells.
- Tipranavir or its salts, esters and isotopes in killing or inhibiting tumor stem cells or quiescent tumor cells when used alone or in combination with chemotherapeutic drugs.
- Tipranavir or its salts, esters and isotopes in the preparation of drugs for killing or inhibiting tumor stem cells or quiescent tumor cells when used alone or in combination with chemotherapeutic drugs.
- Tipranavir or a salt, ester or isotope thereof in inhibiting or reducing the expression of PRSS23 protein or gene in cells, preferably tumor stem cells or quiescent tumor cells.
- Tipranavir or a salt, ester or isotope thereof in the preparation of a medicament for inhibiting or reducing the expression of PRSS23 protein or gene in cells, preferably tumor stem cells or quiescent tumor cells.
- Tipranavir or its salts, esters and isotopes for up-regulating IL24 protein or gene expression levels in cells, preferably tumor stem cells or quiescent tumor cells.
- Tipranavir or a salt, ester or isotope thereof in the preparation of a medicament for up-regulating IL24 protein or gene expression level in cells, preferably tumor stem cells or quiescent tumor cells.
- the salt of Tipranavir described in the present invention may be a basic salt or an acid salt.
- Basic salts include Tipranavir with inorganic bases (such as alkali metal hydroxides, alkaline earth metal hydroxides, etc.) or with organic bases (such as monoethanolamine, diethanolamine or triethanolamine, etc.) salts;
- acid salts include Tipranavir with inorganic bases Acids (such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, perchloric acid, sulfuric acid or phosphoric acid, etc.) or with organic acids (such as methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluene sulfonic acid, fumaric acid, oxalic acid, maleic acid, citric acid, etc.).
- the ester of Tipranavir described in the present invention may be an ester formed with an organic acid or an inorganic acid. Obviously, the ester can be normally metabolized into Tipranavir in animals or humans.
- Isotopes of Tipranavir refer to isotopes that contain unnatural proportions at one or more of the atoms that make up Tipranavir. Including, but not limited to, hydrogen ( 1H ), deuterium (2H), tritium ( 3H ), carbon-11 ( 11C ), carbon-12 ( 12C ), carbon-13 ( 13C ), carbon- 14( 14C ), nitrogen- 13 (13N), nitrogen-14( 14N ), nitrogen-15( 15N ), oxygen- 14 (14O), oxygen- 15 (15O), oxygen-16( 16 O), oxygen-17 ( 17 O), oxygen-18 ( 18 O), fluorine-17 ( 17 F), fluorine-18 ( 18 F), phosphorus-31 ( 31 P), phosphorus-32 ( 32 P ), phosphorus-33 ( 33 P), sulfur-32 ( 32 S), sulfur-33 ( 33 S), sulfur-34 ( 34 S), sulfur-35 ( 35 S), sulfur-36 ( 36 S), Chloro- 35 ( 35Cl), Chloro- 36 ( 36Cl),
- the single use according to the present invention is the single drug treatment.
- Combination therapy refers to the use of only one anticancer drug in a course of treatment. Of course, it also includes the administration of another drug alone after resistance to one drug develops.
- Combination therapy refers to the simultaneous or sequential use of two or more anticancer drugs in a course of treatment.
- combination therapy needs to explore different dosages and periods of administration according to the characteristics of the disease and the types of combined drugs. Only based on the above conditions can the explored combination therapy regimens achieve better therapeutic effects than single drug therapy.
- the present invention provides Tipranavir or its salts, esters and isotopes for treating diseases with low levels of IL24 protein or gene expression.
- the present invention provides a medicament for treating diseases with low IL24 protein or gene expression levels, which contains Tipranavir or its salts, esters and isotopes.
- the present invention provides Tipranavir or its salts, esters and isotopes for treating diseases with excessive PRSS23 protein or gene expression levels.
- the present invention provides a medicament for treating diseases with excessive PRSS23 protein or gene expression level, which contains Tipranavir or its salts, esters and isotopes.
- the present invention provides Tipranavir or its salts, esters and isotopes for the treatment of early stage tumors or cancer or cell proliferative diseases.
- the present invention provides Tipranavir or its salts, esters, isotopes for the treatment of metastatic tumors or cancers or cell proliferative diseases.
- the present invention provides Tipranavir or its salts, esters, isotopes for the treatment of recurrent tumors or cancer or cell proliferative diseases.
- the present invention provides Tipranavir or its salts, esters and isotopes for treating tumors or cancers or cell proliferative diseases resistant to radiotherapy or chemotherapy or radiochemotherapy.
- the drugs used for chemotherapy include 5-FU, cisplatin and paclitaxel.
- the present invention provides the use of Tipranavir or its salts, esters and isotopes for preparing a medicament for treating early-stage tumors or cancer or cell proliferative diseases.
- the present invention provides the use of Tipranavir or its salts, esters and isotopes for preparing a medicament for treating metastatic tumors or cancers or cell proliferative diseases.
- the present invention provides the use of Tipranavir or its salts, esters and isotopes for preparing a medicament for treating recurrent tumors or cancer or cell proliferative diseases.
- the present invention provides the use of Tipranavir or its salts, esters and isotopes for preparing a medicament for treating tumors, cancers or cell proliferative diseases resistant to radiotherapy or chemotherapy or radiotherapy and chemotherapy.
- the present invention provides a medicament containing Tipranavir or a salt, ester or isotope thereof for use in therapy
- Metastatic tumor or cancer or cell proliferative disease or
- Radiotherapy or chemotherapy or radiochemotherapy-resistant tumor or cancer or cell proliferative disease are examples of Radiotherapy or chemotherapy or radiochemotherapy-resistant tumor or cancer or cell proliferative disease.
- the drugs used in chemotherapy include 5-FU, cisplatin, and paclitaxel, and the tumor or cancer is prostate cancer.
- tumor stem cells and tumor cells are tumor stem cells or tumor cells of gastric cancer, lung cancer, prostate cancer, esophageal cancer, colorectal cancer, breast cancer or liver cancer, preferably gastric cancer stem cells and gastric cancer cells;
- the tumor stem cells or tumor cells are prostate cancer stem cells or prostate cancer cells that are resistant to paclitaxel;
- Tipranavir kills or inhibits gastric cancer stem cells or gastric cancer cells by inducing apoptosis of gastric cancer stem cells or gastric cancer cells;
- Tipranavir induces apoptosis of gastric cancer stem cells and gastric cancer cells through the mitochondrial apoptosis pathway, and then kills gastric cancer stem cells and gastric cancer cells. That is, Tipranavir promotes the release of Cytochrome c in mitochondria and promotes Cleaved caspas9, Cleaved caspas7, Cleaved caspas3 by up-regulating IL24. , the expression of Cleaved PARP, and at the same time, up-regulated Bax and Bak proteins.
- Tipranavir is used in killing or inhibiting tumor stem cells or tumor cells.
- Cancer stem cells and tumor cells are time- and concentration-dependent, preferably, Tipranavir kills or inhibits gastric cancer stem cells and gastric cancer cells in a time- and concentration-dependent manner.
- the present invention also provides a variety of medicines:
- the drug can inhibit or reduce the expression of PRSS23, and contains the active ingredient Tipranavir.
- the drug can up-regulate the expression of IL24 and contains the active ingredient Tipranavir.
- the drug is capable of killing or inhibiting tumor stem cells or tumor cells, and contains the active ingredient Tipranavir, preferably, killing or inhibiting gastric cancer stem cells or gastric cancer cells.
- the various medicines provided by the present invention also contain a pharmaceutically acceptable carrier.
- the carrier described in the present invention is a pharmaceutically acceptable carrier, which refers to: one or more compatible solid or liquid fillers or gel substances. They are suitable for human use and must be sufficiently pure and sufficiently low in toxicity. "Compatibility” as used herein means that the components of the composition can be blended with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients.
- the carrier includes but is not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, binders, sprays, transdermal absorption agents, moisturizing agents agents, disintegrants, absorption enhancers, surfactants, colorants, flavors, or adsorption carriers.
- the invention provides the use of Tipranavir in preparing a cancer therapeutic drug for killing tumor stem cells and tumor cells.
- the present invention provides the use of Tipranavir in preparing a drug for inhibiting tumor stem cells and tumor cell proliferation.
- the present invention also provides the use of Tipranavir in the preparation of a drug for reducing the tumorigenic ability of tumor stem cells and tumor cells in vivo, which includes reducing the tumor volume formed by the tumor stem cells, reducing the tumor weight, and slowing down the growth rate of the tumor formed by the tumor stem cells, At the same time, it has no obvious toxic and side effects on lung, liver, spleen, kidney and heart tissue.
- Tipranavir treats cancer by inducing apoptosis of gastric cancer stem cells, thereby killing gastric cancer stem cells.
- Tipranavir induces apoptosis of gastric cancer stem cells through the mitochondrial apoptosis pathway, and then kills gastric cancer stem cells, that is, Tipranavir promotes the release of Cytochrome c in mitochondria and the expression of Cleaved caspas9, Cleaved caspas7, Cleaved caspas3, and Cleaved PARP by up-regulating IL24, At the same time, Bax and Bak proteins were up-regulated.
- Tipranavir kills human gastric cancer stem cells in a concentration-dependent manner, and has no toxic and side effects on normal gastric epithelial cells.
- Tipranavir is used to prepare a drug for the treatment of prostate cancer that is resistant to paclitaxel.
- the present invention also provides a drug for the treatment of cancer, which can kill tumor stem cells and tumor cells; the drug uses Tipranavir as the main active ingredient and contains a pharmaceutically acceptable carrier.
- the drug can be prepared including but not limited to microinjection, dosage form suitable for transfection, injection, tablet, powder, granule, and capsule.
- dosage form suitable for transfection injection
- tablet powder
- granule granule
- capsule granule, and capsule.
- the medicines in the above-mentioned various dosage forms can be prepared according to the conventional methods in the pharmaceutical field.
- Cancer stem cell cancers mentioned in the present invention include gastric cancer and other cancers.
- gastric cancer is the most common malignant tumor of the digestive system, it has a high recurrence rate and a low cure rate, and has a high mortality rate. Therefore, research on its survival regulation mechanism and molecular targeted therapy can be used as experimental basis and theoretical reference for other tumors.
- cancer stem cells of different solid tumors have common characteristics: they all have the ability of self-renewal, proliferation and self-differentiation, and they can all lead to tumorigenesis in vivo. Different solid tumor stem cells have the same or similar molecular regulatory pathways.
- Tipranavir also has anti-cancer effects on other cancer stem cells and tumor cells except gastric cancer stem cells, and is used to prepare cancer therapeutic drugs against cancer stem cells and tumor cells.
- Cancers of the tumor stem cells and tumor cells include gastric cancer, lung cancer, prostate cancer, esophageal cancer, colorectal cancer, breast cancer, and liver cancer.
- the cancer of the tumor stem cells and tumor cells is gastric cancer.
- the beneficial effects of the present invention are as follows: the present invention provides for the first time that Tipranavir can be used to prepare an anticancer drug that kills tumor stem cells and tumor cells, and its anticancer effect is significantly better than the existing first-line treatment drugs for gastric cancer (combination of 5-FU and cisplatin) , and no obvious toxic side effects, the toxic side effects are significantly less than the first-line treatment drugs for gastric cancer (the combination of 5-FU and cisplatin). It is of great significance to improve the quality of life of patients.
- Figure 1 shows the cell proliferation and activity of gastric cancer stem cells (GCSC1 and GCSC2) treated with Tipranavir by CCK8;
- FIG. 2 shows the cell proliferation and activity of gastric cancer stem cells (GCSC1 and GCSC2) treated with Tipranavir, fluorouracil (5-FU) and cisplatin (cisplatin) in CCK8 detection, and Control is the control group;
- FIG. 3 shows CCK8 detection of gastric cancer cell lines (AGS, HGC-27, MGC-803, BGC-823) and normal gastric epithelial cells treated with Tipranavir, fluorouracil (5-FU) and cisplatin (cisplatin), respectively. and activity;
- Figure 4 shows the detection of CCK8 in lung cancer cells (PC9), prostate cancer cells (PC3), paclitaxel-resistant prostate cancer cells (PC3/Tax), esophageal cancer cells (KYSE180 and KYSE520), and colorectal cancer cells (HCT116) treated with Tipranavir , the cell proliferation and activity of breast cancer cells (MDA-MB-231) and liver cancer cells (Huh7), Control is the control group;
- Figure 5 Tipranavir inhibits the growth of gastric cancer stem cell transplanted tumors in nude mice:
- Figure 5a is an in vivo tumorigenic experiment in mice;
- Figure 5b shows the Tipranavir group, the 5-FU and cisplatin combination group (5-FU+Cis) and the control group (Control)
- Figure 5c shows the weight change of the transplanted tumor formed by the gastric cancer stem cells treated by the Tipranavir group, the 5-FU and cisplatin combination group (5-FU+Cis) and the control group (Control);
- Figure 5d is the growth curve of the transplanted tumor formed by the gastric cancer stem cells in the Tipranavir group, the 5-FU and cisplatin combination group (5-FU+Cis) and the control group (Control);
- Figure 5e is the staining detection of lung cancer in each administration group , the pathological characteristics of heart, liver, spleen and kidney
- Figure 6 Tipranavir kills gastric cancer stem cells by inducing apoptosis of gastric cancer stem cells:
- Figure 6a shows the apoptosis morphology of gastric cancer stem cells (GCSC1 and GCSC2) treated with Tipranavir, and Control is the control group;
- Figure 6b Figure 6c are Annexin V-FITC/PI labeling flow cytometry analysis of apoptosis rate of gastric cancer stem cells (GCSC1 and GCSC2);
- Figure 6d Figure 6e TUNEL detection and analysis of gastric cancer stem cells (GCSC1 and GCSC2) apoptosis;
- Figure 6f Western blotting method Detect the distribution of Cytochrome c and the expression of apoptosis markers (Bax, Bak, Cleaved caspas9, Cleaved caspas7, Cleaved caspas3, Cleaved PARP);
- Figure 7 Tipranavir up-regulates IL24, then up-regulates Bax and Bak proteins, activates the mitochondrial apoptosis pathway, and induces gastric cancer stem cell apoptosis, thereby killing gastric cancer stem cells:
- Figure 7a, Figure 7b RNA-seq analysis compares Tipranavir-treated and untreated gastric cancer Stem cell gene expression changes, looking for the differential genes of gastric cancer stem cells treated with Tipranavir, it was found that Tipranavir gastric cancer stem cells significantly up-regulated the expression of IL24;
- Figures 7c and 7d were verified by qPCR and Western blotting respectively.
- Figure 7f The expression of IL24 in gastric cancer stem cells increased with the increase of Tipranavir concentration, showing a concentration-dependent expression;
- Figure 8 After knockdown of IL24 by siRNA in gastric cancer stem cells, the gastric cancer stem cells were then treated with Tipranavir, which confirmed that Tipranavir induced gastric cancer stem cell apoptosis through IL24-Bax/Bak mitochondrial apoptosis pathway, thereby killing gastric cancer stem cells:
- Figure 8a siRNA interference knockdown in gastric cancer stem cells After low IL24, the killing effect of Tipranavir on gastric cancer stem cells was significantly alleviated;
- Figure 8B Western blotting was used to detect the expression of related proteins in gastric cancer stem cells (siIL24-1, siIL24-2) after siRNA interference knockdown of IL24 in Tipranavir-treated gastric cancer stem cells.
- Gastric cancer stem cells with knockdown of IL24 are the control group;
- Figure 8c The effect of IL24 overexpression in gastric cancer stem cells on gastric cancer stem cells, GCSC-IL24 is the IL24 overexpression group;
- GCSC-vec is the control group with normal expression of IL24;
- Figure 8d Gastric cancer stem cells Overexpression of IL24 in gastric cancer stem cells affects the expression of mitochondrial apoptosis pathway proteins and apoptosis markers in gastric cancer stem cells.
- GCSC-IL24 is the IL24 overexpression group;
- GCSC-vec is the control group with normal expression of IL24;
- Figure 9 PRSS23/MKK3/p38 MAPK pathway activates the IL24-dependent mitochondrial apoptosis pathway:
- Figure 9a Phosphorylated p38 MAPK (p-p38 MAPK) and total p38 MAPK (p38 MAPK) expression, GAPDH as control;
- Figure 9b relative mRNA levels of IL24 after GCSC1 cells were treated with SB203580 (10 ⁇ M), anisomycin (5 ⁇ M) and tipranavir (20 ⁇ M) for 3 hours, respectively;
- Figure 9c actinomycin (10 ⁇ g/ml) IL24 expression after pretreatment of GCSC1 cells for 1 hour and treatment with culture medium or SB203580 (10 ⁇ M) and tipranavir (20 ⁇ M) for 2 hours;
- Figure 9d from PRSS23-silenced GCSC1 cells, or GCSC2 cells (GCSC-Vec) and PRSS23 Expression of PRSS23, phosphorylated p38 MAPK (p-p38 MA
- the experimental method of unreceipted specific conditions in the embodiment is usually a routine method in the field, such as according to routine conditions such as Sambrook et al., Molecular Cloning, Experiment Manual (Third Edition) (Science Press, 2002) The condition described in , or as recommended by the reagent manufacturer.
- the main materials used in the present invention are as follows:
- Atazanavir, Darunavir, Fosamprenavir, Tipranavir, 5-FU and cisplatin were purchased from Med Chemexpress Company; Poloxamer 188 (F68) was purchased from Shenyang Jiqi Pharmaceutical Company; RNA extraction kit (RNeasy Mini Kit) was purchased from QIAGEN Company; Kit and Tag DNA polymerase were purchased from Life Technology; reverse transcriptase M-MLV and PCR kit were purchased from TIANGEN; BCA protein quantification kit was purchased from Thermo Fischer Scientific; WB chemiluminescence substrate (Immobilon Western Chemiluminescent HRP substrate) was purchased from Millipore; WB chemiluminescence substrate (Super Signal West Chemiluminescent substrate) was purchased from Thermo Fischer Scientific; CCK-8 kit was purchased from Tongren Institute of Chemistry; FITC-annexin V/PI apoptosis detection The kit (FITC Annexin V Apoptosis Detection kit) was purchased from BD Biosciences
- Secondary antibody Anti-rabbit was purchased from Cell Signaling Technology; BCA protein quantification kit was purchased from Thermo Fischer Scientific; restriction enzymes EcoRI and MluI were purchased from NEB; HSP90 inhibitors 17-AAG and SNX2112 were purchased from Selleck Chemicals Company; DMSO, puromycin (puromycin), doxycycline (Doxycycline, Dox), primary antibody Anti- ⁇ -actin were purchased from Sigma Company; primary antibody Anti-Clusterin- ⁇ , secondary antibody Anti-goat were purchased from Santa Cruz Biotechnologies; primary antibody Anti-HSP90 was purchased from Abcam; primary antibody Anti-Sox2, anti-Cleaved PARP, anti-pSer807/Ser811-Rb, anti-AKT, anti-CDK4, anti-HER2, anti-c-Raf, Anti-EGFR, anti-IGF-1R, secondary antibody Anti-mouse, and secondary antibody Anti-rabbit were purchased from Cell Signaling Technology.
- the statistical analysis method involved in the present invention adopts SPSS12.0 statistical software, the experimental results are expressed as mean ⁇ standard deviation (x ⁇ s), t test is used for the comparison of the means of two groups, and one-way analysis of variance is used for the comparison of the means of multiple groups , p ⁇ 0.05 was considered statistically significant, and was indicated by "*".
- * indicates a statistically significant difference (p ⁇ 0.05); ** indicates a significant difference (p ⁇ 0.01); *** indicates a significant difference (p ⁇ 0.001); **** indicates a significant difference (p ⁇ 0.001); Very significant difference (p ⁇ 0.0001).
- the tumor stem cells and tumor cells used in the examples of the present invention were purchased from the cell bank of the Institute of Biochemical Cells, Chinese Academy of Sciences, and the cell culture method was a conventional method in the art.
- Cellular proteins were extracted from RIPA cell lysate, and the protein concentration was determined by BCA method. The same amount of protein samples were subjected to 10% SDS-PAGE electrophoresis, transferred to membrane, blotted, blocked, incubated with primary antibody overnight at 4°C, washed with secondary antibody, incubated at room temperature for 1 h, washed The membrane was developed with ECL (Super Signal West chemiluminescent substrates, Thermo Fisher Scientific).
- ECL Super Signal West chemiluminescent substrates, Thermo Fisher Scientific.
- the in situ end labeling kit (Insitu Cell Death Detection Kit, Cat. No. 11684817910) produced by Roche Molecular Biochemicals was used, according to the kit instructions: (1) 4% paraformaldehyde fixed for 1 h, rinsed with PBS buffer; (2) 3 %H 2 O 2 blocking solution at room temperature for 10 min, rinsed with PBS; (3) 0.1% Triton X-100 permeate on ice for 2 min, rinsed with PBS; (4) Add 50 ⁇ l TUNEL reaction mixture, in a wet box at 37°C for 1 h, rinsed with PBS ; (5) Mount the slide with DAPI anti-quencher, observe and record under microscope, all cell nuclei are blue, and apoptotic cells are green fluorescence, select a certain number of fields under light microscope to count apoptotic cells, and count apoptotic cells (FITC Green fluorescent cells) as a percentage of all cells (DAPI stained blue cells).
- FITC Green fluorescent cells a percentage
- Example 1 Tipranavir can effectively kill human gastric cancer stem cells at low concentrations in a time- and concentration-dependent manner.
- GCSC1 and GCSC2 Two gastric cancer stem cells (denoted as GCSC1 and GCSC2) derived from different gastric cancer patients were taken, and the cell concentrations were both (1 ⁇ 10 5 cells/mL, 100 ⁇ l), and were inoculated into 96-well plates. Concentrations of Tipranavir (0uM, 0.5uM, 1.0uM, 2.5uM, 5.0uM, 10uM, 20uM, 40uM, 100uM) were used to treat GCSC1 and GCSC2 cells for 24hr, 48hr, 72hr, and DMSO was used as a control. CCK-8 was added to measure the absorbance value (A450) at a wavelength of 450 nm, and the cell growth curves of each group were drawn. The killing effect of Tipranavir on gastric cancer stem cells (GCSC1 and GCSC2) was detected, and its IC 50 was calculated. The results are shown in Figure 1 ( Figure 1, cell viability cell activity; concentration concentration).
- Tipranavir can effectively kill human gastric cancer stem cells (GCSC1 and GCSC2) at low concentrations in a time- and concentration-dependent manner, with IC 50 of 4.7uM and 6.4uM, respectively.
- Example 2 The effect of Tipranavir in killing gastric cancer stem cells was significantly better than the combination of the existing first-line drug 5-FU and cisplatin.
- Two gastric cancer stem cells (denoted as GCSC1 and GCSC2) (1 ⁇ 10 5 cells/mL, 100 ⁇ l) derived from different gastric cancer patients were inoculated into 96-well plates, three duplicate wells in each group, respectively treated with Tipranavir (10 uM) and 5- GCSC1 and GCSC2 cells were treated with FU+cisplatin (5-FU: 2.5 ⁇ M; cisplatin: 4 ⁇ M) for 72 hr, and DMSO was used as a control. CCK-8 was added to measure the absorbance value (A450) at a wavelength of 450 nm, and the cell growth curves of each group were drawn. The killing effects of Tipranavir and 5-FU+cisplatin on gastric cancer stem cells (GCSC1 and GCSC2) were compared. The results are shown in Figure 2 ( Figure 2, cell viability).
- Example 3 Tipranavir can effectively kill gastric cancer cells, and its effect is also significantly better than the combination of 5-FU and cisplatin, and has no toxic and side effects on normal gastric epithelial cells, while the combination of 5-FU and cisplatin has no effect on normal gastric epithelium. Cell toxicity.
- Tipranavir can effectively kill gastric cancer stem cells and gastric cancer cells, which is significantly better than the combination of 5-FU and cisplatin, and has no toxic and side effects on normal gastric epithelial cells.
- Example 4 Tipranavir against other different cancer cells, including lung cancer cells (PC9), prostate cancer cells (PC3), Paclitaxel-resistant prostate cancer cells (PC3/Tax), esophageal cancer cells (KYSE180 and KYSE520), colorectal cancer Cells (HCT116), breast cancer cells (MDA-MB-231), liver cancer cells (Huh7) and various tumor cells have anti-cancer effects and can be used to prepare broad-spectrum tumor therapy drugs; at the same time, Tipranavir is resistant to paclitaxel-resistant prostate cancer cells The killing effect of (PC3/Tax) is significantly stronger than that of ordinary prostate cancer cells (PC3), and can be used as a drug for the preparation of paclitaxel-resistant prostate cancer drugs.
- PC3/Tax is significantly stronger than that of ordinary prostate cancer cells (PC3), and can be used as a drug for the preparation of paclitaxel-resistant prostate cancer drugs.
- Lung cancer cells PC9, prostate cancer cells (PC3), paclitaxel-resistant prostate cancer cells (PC3/Tax), esophageal cancer cells (KYSE180 and KYSE520), colorectal cancer cells (HCT116), breast cancer cells (MDA- MB-231), hepatoma cells (Huh7) (1 ⁇ 10 5 cells/mL, 100 ⁇ l) were seeded in 96-well plates, 3 replicate wells in each group, respectively treated with Tipranavir (10uM) and 5-FU+cisplatin (5-FU) : 2.5 ⁇ M; cisplatin: 4 ⁇ M) to treat each strain of cells for 72 hr, and DMSO as a control.
- Tipranavir 10uM
- 5-FU+cisplatin 5-FU+cisplatin
- cisplatin 4 ⁇ M
- CCK-8 was added to measure the absorbance value (A450) at a wavelength of 450 nm, and the cell growth curves of each group were drawn. The killing effects of Tipranavir and 5-FU+cisplatin on various tumor cells were compared. The results are shown in Figure 4 ( Figure 4, cell viability).
- Tipranavir can effectively kill various tumor cells such as gastric cancer, lung cancer, prostate cancer, esophageal cancer, colorectal cancer, breast cancer, liver cancer, etc., and its effect is also significantly better than the combination of 5-FU and cisplatin, and Tipranavir is resistant to paclitaxel.
- the killing effect of the drug-treated prostate cancer cells (PC3/Tax) is significantly stronger than that of ordinary prostate cancer cells (PC3), and it has a significant effect on the prostate cancer patients who are resistant to paclitaxel.
- Tipranavir has anticancer effects on various tumor cells such as gastric cancer, lung cancer, prostate cancer, esophageal cancer, colorectal cancer, breast cancer, and liver cancer, and can be used as a broad-spectrum tumor therapy drug.
- Tipranavir is resistant to paclitaxel
- the killing effect of the prostate cancer cells (PC3/Tax) is significantly stronger than that of ordinary prostate cancer cells (PC3), and can be used as a drug for the preparation of taxol-resistant prostate cancer drugs.
- Example 5 Tipranavir can significantly inhibit the growth of gastric cancer stem cell transplanted tumors in nude mice, and its effect is significantly better than the combination of 5-FU and cisplatin, and there is no obvious toxic and side effects.
- the combination of 5-FU and cisplatin (cisplatin) has nephrotoxicity, and there are toxic side effects on the kidneys.
- mice Female Balb/C nude mice (5 weeks old, body weight 16.0 ⁇ 2.0 g) were cultured in a sterile environment. Mice were divided into 3 groups, Tipranavir treatment group (Tipranavir), 5-FU and cisplatin combination group (5-FU+Cis) and control group (Control), 4 mice in each group, subcutaneous injection of 4 ⁇ 10 4 GCSCs in the lateral flank The cell suspension was used to establish a subcutaneous transplanted tumor model. After 10 days of tumor formation, related treatments were started.
- Tipranavir treatment group Tipranavir was intraperitoneally injected with Tipranavir (F68solution: 1 mg/ml in normal saline, 25 mg/kg/mouse); 5-FU and cisplatin combined treatment group (5 -FU+Cis) intraperitoneal injection of 5-FU+Cis (5FU: 20mg/kg/mouse in F68 solution; cisplatin: 2mg/kg/mouse in F68 solution); control group (Control) by intraperitoneal injection of F68 solution (1mg/ml in normal saline), injected every 2 days. The growth of subcutaneous tumors was closely observed, and the tumor size was measured with a vernier caliper every 2 days.
- the index also confirmed that Tipranavir had no obvious toxic and side effects on important organs of the heart, liver, spleen, lung and kidney (Figure 5g); while the combined treatment of 5-FU and cisplatin significantly affected mice and affected the growth of mice (Figure 5f).
- the organ index also confirmed that the combined use of 5-FU and cisplatin had obvious toxic and side effects on the kidneys and had nephrotoxicity (Fig. 5g).
- WB analysis of tumors showed that Tipranavir up-regulated IL24, Bak expression, up-regulated Cleaved caspase3, Cleaved PARP, activated mitochondrial apoptosis pathway, and induced apoptosis, thereby inhibiting tumor growth (Figure 5h).
- Example 6 Tipranavir kills gastric cancer stem cells by inducing apoptosis of gastric cancer stem cells.
- Tipranavir treatment of gastric cancer stem cells resulted in cytoplasm concentration, reduced volume, fragmented and scattered nuclei, and showed apoptosis in the form of apoptotic bodies ( Figure 6a).
- Tipranavir significantly induced the apoptosis of gastric cancer stem cells ( Figure 6b, 6c); TUNEL test also confirmed that Tipranavir significantly induced the apoptosis of gastric cancer stem cells ( Figure 6d, 6e);
- Western blot also found that Tipranavir treated the mitochondrial apoptosis pathway proteins in gastric cancer stem cells and its apoptotic markers (Bax, Bak, Cleaved caspas9, Cleaved caspas7, Cleaved caspas3, Cleaved PARP) were significantly increased, Cytochrome c, a marker of activation of the circuit body apoptotic pathway, flowed from mitochondria into the cytoplasm, and Cytochrome c decreased in mitochondria (
- Tipranavir can up-regulate IL24, then up-regulate Bax and Bak proteins, activate the mitochondrial apoptosis pathway (mitochondrial apoptosis pathway), and induce gastric cancer stem cell apoptosis (apoptosis), thereby killing gastric cancer stem cells.
- RNA-seq analysis compared the gene expression changes of Tipranavir-treated and untreated gastric cancer stem cells, looking for differential genes in Tipranavir-treated gastric cancer stem cells, and found that Tipranavir gastric cancer stem cells significantly up-regulated the expression of IL24 (Figure 7a, 7b), and by qPCR (Figure 7c), Western The blotting was validated (Fig. 7d). And the expression of IL24 in gastric cancer stem cells increased with the increase of Tipranavir concentration, showing a concentration-dependent expression ( Figure 7e, 7f). IL24 has been confirmed as a tumor cell-specific tumor suppressor gene, its high expression can significantly inhibit tumor growth, and its high expression in normal cells has no effect on normal cells.
- IL24 was further knocked down by siRNA interference in gastric cancer stem cells.
- the Tipranavir cell activity assay showed that knockdown of IL24 significantly alleviated the killing effect of Tipranavir on gastric cancer stem cells (8a).
- the mitochondrial apoptosis pathway proteins and their apoptosis markers (Bax, Bak, Cleaved caspas9, Cleaved caspas7, Cleaved caspas3, Cleaved PARP) were up-regulated, inhibited the flow of Cytochrome c in mitochondria to the cytoplasm, and significantly inhibited the activation of mitochondrial apoptosis pathway (8b ).
- IL24 in gastric cancer stem cells without the addition of Tipranavir can also significantly inhibit the growth of gastric cancer stem cells and induce apoptosis of gastric cancer stem cells (8c, GCSC-IL24 is the IL24 overexpression group; GCSC-vec is the control group with normal expression of IL24)
- GCSC-IL24 is the IL24 overexpression group
- GCSC-vec is the control group with normal expression of IL24
- mitochondrial apoptosis pathway proteins and their apoptosis markers (Bax, Bak, Cleaved caspas9, Cleaved caspas7, Cleaved caspas3, Cleaved PARP) were significantly increased in gastric cancer stem cells, which promoted the flow of Cytochrome c in mitochondria to the cytoplasm and activated mitochondrial apoptosis.
- GCSC-IL24 is the IL24 overexpression group; GCSC-vec is the control group with normal IL24 expression
- Tipranavir induces gastric cancer stem cell apoptosis (apoptosis) through the IL24-Bax/Bak mitochondrial apoptosis pathway, thereby killing gastric cancer stem cells.
- Example 8 Tipranavir inhibits PRSS23-induced IL24 expression and promotes gastric cancer stem cell apoptosis.
- the PRSS23/MKK3/p38 MAPK pathway activates the IL24-dependent mitochondrial apoptosis pathway:
- Figure 9a Phosphorylated p38 MAPK (p-p38 MAPK) and total p38 MAPK (p38 MAPK) after 48 h treatment with the indicated concentrations of tiponavir , GAPDH was used as control;
- Figure 9b relative mRNA levels of IL24 after GCSC1 cells were treated with SB203580 (10 ⁇ M), anisomycin (5 ⁇ M) and tipranavir (20 ⁇ M) for 3 hours, respectively;
- Figure 9c actinomycin (10 ⁇ g/ml) pretreatment was observed IL24 expression in GCSC1 cells 1 hour after treatment with culture medium or SB203580 (10 ⁇ M), tipranavir (20 ⁇ M) for 2 hours;
- Figure 9d from PRSS23-silenced GCSC1 cells, or GCSC2 cells (GCSC-Vec) and PRSS23 overexpression Expression of PRSS
- Example 7 The above results combined with Example 7 can show that tiponavir induces the expression of IL24 by reducing the expression of PRSS23, and the increase of the expression of IL24 strongly affects the expression of the pro-apoptotic proteins Bax and Bak, thereby activating the mitochondrial apoptosis pathway and promoting the Apoptosis of gastric cancer stem cells.
- the present invention provides for the first time that Tipranavir can be used to prepare a new anti-cancer effect of killing tumor stem cells and tumor cells, and its anti-cancer effect is significantly better than the existing first-line treatment drugs for gastric cancer (combination of 5-FU and cisplatin), It has no obvious toxic and side effects, and the toxic and side effects are obviously less than that of first-line treatment drugs for gastric cancer (combination of 5-FU and cisplatin). It provides a new direction for curing gastric cancer and improving the survival rate of gastric cancer patients.
Abstract
Tipranavir在制备杀伤肿瘤干细胞和肿瘤细胞的癌症治疗药物中的用途。一种癌症的治疗药物，所述药物能够杀伤肿瘤干细胞和肿瘤细胞；所述药物以Tipranavir为主要的活性成分，并含有药学上可接受的载体。Tipranavir可以用于制备杀伤肿瘤干细胞和肿瘤细胞的抗癌药物，无明显毒副作用。
Description
本发明涉及医药技术领域，尤其涉及Tipranavir在制备杀伤肿瘤干细胞和肿瘤细胞的癌症治疗药物中的用途。
恶性肿瘤是严重危害人类健康的高发病率和高致死率疾病。例如，胃癌(Gastric Cancer,GC)是最常见的消化系统恶性肿瘤，发病率在全球范围内位列所有恶性肿瘤的第五位，死亡率位居恶性肿瘤死因第三位。我国是胃癌高发区，发病率、死亡率居高不下，均居恶性肿瘤第2位。胃癌治疗原则是以手术治疗为主的综合治疗。在我国，胃癌早诊率仍然相对低下，患者诊断时多为晚期肿瘤，失去手术治愈的机会，大多数患者由于肿瘤复发转移而死亡。
肿瘤中的肿瘤细胞可分为两类：一类为普通的肿瘤细胞，一类为肿瘤干细胞。普通肿瘤细胞具有快速分裂，对抗癌药敏感，无自我更新能力等特点。而肿瘤干细胞(Cancer stem cells,CSCs)是肿瘤中少数具有干细胞特性即自我更新能力并能产生不同分化程度的肿瘤细胞的细胞亚群，被认为是肿瘤中的“种子”细胞，是肿瘤发生、转移和复发，及放化疗耐受的根源，也是肿瘤治疗失败和死亡的最主要原因。其具有以下特点：通常情况下处于静息状态，对抗癌药不敏感，具有自我更新能力，即具有无限增殖的能力。在肿瘤化疗中，大量的肿瘤细胞被杀死(对化疗药敏感)，然而肿瘤干细胞会生存下来(对化疗药不敏感)。目前，很多治疗手段都能使肿瘤在短时间内得到控制和缩小，但大多数患者会出现复发和转移，从而导致治疗失败。因此，肿瘤的复发是由于肿瘤化疗药对肿瘤干细胞无效，换言之，目前的肿瘤化疗药主要是针对肿瘤细胞，而不是肿瘤干细胞。事实上，大多数肿瘤化疗药只对肿瘤细胞有效，而对肿瘤干细胞无效。因此，要达 到治愈癌症的目的，不仅要消灭普通的肿瘤细胞，更重要的是通过特异性治疗杀死引起肿瘤增殖的肿瘤干细胞。但目前针对肿瘤干细胞的特异性治疗靶点缺乏，缺乏针对肿瘤干细胞的药物，尤其是同时杀伤肿瘤干细胞和肿瘤细胞的药物。因此，寻找并建立能有效杀伤肿瘤干细胞和肿瘤细胞的治疗药物对于控制进而治愈肿瘤，改善癌症患者预后有重要意义。
Tipranavir(替拉那韦，PNU-140690，CAS No.:174484-41-4)为临床抗HIV药物，也是第一个批准上市的非肽类蛋白酶抑制剂，其作用机制主要为通过抑制蛋白酶而发挥作用。研究表明，Tipranavir可以抑制对市售药物产生耐药性的HIV病毒，适用于对多种蛋白酶抑制剂耐药的有病毒复制迹象的HIV感染者。但目前，Tipranavir在制备治疗肿瘤干细胞及其肿瘤细胞药物中的应用未见文献报道，未用于抗肿瘤中
发明内容
本发明的主要目的在于提供Tipranavir(替拉那韦，PNU-140690)在用于制备杀伤肿瘤干细胞和肿瘤细胞的癌症治疗药物的新用途。
因此，本发明进行了以下研究：
1、Tipranavir处理人胃癌干细胞并检测不同浓度Tipranavir及不同时间处理人胃癌干细胞，使用CCK-8试剂盒检测细胞生存情况。结果显示：Tipranavir能有效杀伤人胃癌干细胞，且呈现时间和浓度依赖性。
2、Tipranavir与目前胃癌一线化疗药物fluorouracil(氟尿嘧啶，5-FU)和cisplatin(顺铂)联用分别处理人胃癌干细胞，使用CCK-8试剂盒检测细胞生存情况，比较Tipranavir和目前胃癌一线化疗药物fluorouracil(氟尿嘧啶，5-FU) 和cisplatin(顺铂)联用对胃癌干细胞的作用。结果显示：胃癌干细胞对5-FU和顺铂联用抵抗，5-FU和顺铂不能有效杀伤胃癌干细胞，而Tipranavir能有效杀伤胃癌干细胞，效果显著优于5-FU和顺铂联用。
3、Tipranavir和目前胃癌一线化疗药物fluorouracil(氟尿嘧啶，5-FU)和cisplatin(顺铂)联用分别处理四种胃癌细胞系(AGS，HGC-27，MGC-803，BGC-823)，及其正常胃上皮细胞(GES-1)，使用CCK-8试剂盒检测细胞生存情况，比较Tipranavir和目前胃癌一线化疗药物fluorouracil(氟尿嘧啶，5-FU)和cisplatin(顺铂)联用对胃癌细胞的杀伤作用及其对正常细胞的毒副作用。结果显示：Tipranavir杀伤胃癌细胞的作用也显著优于5-FU和顺铂联用，且对正常胃上皮细胞无毒副作用，而5-FU和顺铂联用存在对正常胃上皮细胞的毒副作用。
4、Tipranavir处理不同癌细胞系，包括肺癌细胞(PC9)，前列腺癌细胞(PC3)，紫杉醇耐药的前列腺癌细胞(PC3/Tax)，食管癌细胞(KYSE180和KYSE520)，结直肠癌细胞(HCT116)，乳腺癌细胞(MDA-MB-231)，肝癌细胞(Huh7)，使用CCK-8试剂盒检测细胞生存情况。结果表明：Tipranavir能有效杀伤各种肿瘤细胞，包括肺癌、前列腺癌、食管癌、结直肠癌、乳腺癌、肝癌，表明Tipranavir对其他各种肿瘤具有抗癌作用，可用做制备广谱肿瘤治疗药物；同时，Tipranavir对紫杉醇耐药的前列腺癌细胞(PC3/Tax)的杀伤作用显著强于普通前列腺癌细胞(PC3)，表明Tipranavir对紫杉醇耐药的前列腺癌病人具有显著作用，可用做制备紫杉醇耐药的前列腺癌治疗药物。
5、裸鼠成瘤实验检测动物水平上Tipranavir抗胃癌干细胞作用，并比较其与一线胃癌化疗药物5-FU和cisplatin(顺铂)联用治疗胃癌干细胞移植瘤作用。人胃癌干细胞注射于裸鼠皮下，待其成瘤后Tipranavir或5-FU和cisplatin(顺铂)联用腹腔注射，通过检测移植瘤体积，瘤重及瘤体大小分析其对人胃癌干细胞始动的胃癌发生和生长的作用。结果显示：Tipranavir处理能明显抑制移植瘤的生长，其作用显著优于5-FU和cisplatin(顺铂)联用，且对肺、肝脏、脾脏、肾、心脏组织无明显毒副作用。而5-FU和cisplatin(顺铂)联用具有肾毒性， 存在对肾脏的毒副作用。
6、凋亡检测实验分析Tipranavir杀伤胃癌干细胞的作用机制，结果表明：Tipranavir通过诱导胃癌干细胞凋亡(apoptosis)实现杀伤胃癌干细胞。
7、RNA-seq分析比较Tipranavir处理和不处理的胃癌干细胞基因表达变化，寻找Tipranavir处理胃癌干细胞的差异基因，并通过qPCR，Western blotting验证相关差异基因的表达，结果表明：Tipranavir处理胃癌干细胞显著上调IL24表达，IL24可能为Tipranavir作用靶点。IL24已被证实为肿瘤细胞特异性抑癌基因，其高表达能显著抑制肿瘤生长，且其在正常细胞高表达对正常细胞无影响。
8、分子机制上，Tipranavir杀伤胃癌干细胞的分子机制具体为：Tipranavir通过上调IL24，进而上调Bax和Bak蛋白，激活细胞线粒体凋亡通路(mitochondrial apoptotic pathway)，诱导胃癌干细胞凋亡(apoptosis)，从而杀伤胃癌干细胞。
9、通过进一步研究Tipranavir杀伤胃癌干细胞的分子机制，发现Tipranavir抑制PRSS23诱导IL24表达，促进胃癌干细胞凋亡，具体为：替普那韦通过降低PRSS23的表达，从而诱导IL24的表达，而IL24表达的增加强烈影响促凋亡蛋白Bax和Bak的表达，从而激活线粒体凋亡通路，促进胃癌干细胞凋亡。
根据上述研究结果，本发明确认了Tipranavir具有杀伤胃癌干细胞和胃癌细胞的作用，及其它肿瘤干细胞和肿瘤细胞的新作用，可以用于制备抗胃癌及其它抗肿瘤治疗药物。
本发明提供了施加Tipranavir或其盐、酯、同位素杀死或抑制肿瘤干细胞或肿瘤细胞的方法，其经由抑制或减少肿瘤干细胞或肿瘤细胞中PRSS23蛋白或基因表达来实现或其经由上调肿瘤干细胞或肿瘤细胞中IL24蛋白或基因表达水平来实现。
本发明提供施加Tipranavir或其盐、酯、同位素杀死或抑制肿瘤干细胞或静息状态的肿瘤细胞的方法，其经由抑制或减少肿瘤干细胞或肿瘤细胞中PRSS23蛋白或基因表达来实现或其经由上调肿瘤干细胞或肿瘤细胞中IL24蛋白或基因 表达水平来实现。
一般地，在PRSS23被抑制或减少后，IL24表达上调，进而上调Bax和Bak蛋白，激活肿瘤干细胞或肿瘤细胞线粒体凋亡通路，促进肿瘤干细胞或肿瘤细胞的凋亡。
本发明还提供Tipranavir或其盐、酯、同位素的多种用途：
(1)Tipranavir或其盐、酯、同位素在杀死或抑制肿瘤干细胞或静息状态的肿瘤细胞中的用途。
(2)Tipranavir或其盐、酯、同位素在制备杀死或抑制肿瘤干细胞或静息状态的肿瘤细胞的药物中的用途。
(3)Tipranavir或其盐、酯、同位素在与化疗药物单用或联用杀死或抑制肿瘤干细胞或静息状态的肿瘤细胞中的用途。
(4)Tipranavir或其盐、酯、同位素在制备与化疗药物单用或联用杀死或抑制肿瘤干细胞或静息状态的肿瘤细胞的药物中的用途。
(5)Tipranavir或其盐、酯、同位素在抑制或减少细胞中PRSS23蛋白或基因表达中的用途，所述细胞优选为肿瘤干细胞或静息状态的肿瘤细胞。
(6)Tipranavir或其盐、酯、同位素在制备抑制或减少细胞中PRSS23蛋白或基因表达的药物中的用途，所述细胞优选为肿瘤干细胞或静息状态的肿瘤细胞。
(7)Tipranavir或其盐、酯、同位素在上调细胞中IL24蛋白或基因表达水平的用途，所述细胞优选为肿瘤干细胞或静息状态的肿瘤细胞。
(8)Tipranavir或其盐、酯、同位素在制备上调细胞中IL24蛋白或基因表达水平的药物中的用途，所述细胞优选为肿瘤干细胞或静息状态的肿瘤细胞。
本发明所述Tipranavir的盐可以是碱式盐或酸式盐。碱式盐包括Tipranavir与无机碱(例如碱金属氢氧化物、碱土金属氢氧化物等)或与有机碱(例如单乙醇胺、二乙醇胺或三乙醇胺等)形成的盐；酸式盐包括Tipranavir与无机酸(例如盐酸、氢溴酸、氢碘酸、硝酸、高氯酸、硫酸或磷酸等)或与有机酸(例如甲 磺酸、三氟甲磺酸、乙磺酸、苯磺酸、对甲苯磺酸、富马酸、草酸、马来酸、柠檬酸等)形成的盐。
本发明所述Tipranavir的酯，可以是与有机酸或无机酸形成的酯，显然，该酯在动物体内或人体内能够正常的代谢为Tipranavir。
本发明所述Tipranavir的同位素是指在组成Tipranavir的原子中的一或多者处含有非天然比例的同位素。包括(但不限于)氢(
1H)、氘(
2H)、氚(
3H)、碳-11(
11C)、碳-12(
12C)、碳-13(
13C)、碳-14(
14C)、氮-13(
13N)、氮-14(
14N)、氮-15(
15N)、氧-14(
14O)、氧-15(
15O)、氧-16(
16O)、氧-17(
17O)、氧-18(
18O)、氟-17(
17F)、氟-18(
18F)、磷-31(
31P)、磷-32(
32P)、磷-33(
33P)、硫-32(
32S)、硫-33(
33S)、硫-34(
34S)、硫-35(
35S)、硫-36(
36S)、氯-35(
35Cl)、氯-36(
36Cl)、氯-37(
37Cl)、溴-79(
79Br)、溴-81(
81Br)、碘-123(
123I)、碘-125(
125I)、碘-127(
127I)、碘-129(
129I)及碘-131(
131I)。
本发明所述单用，即单药治疗。联用，即联合用药治疗。单药治疗是指在一个疗程中仅使用一种抗癌药物，当然，也包括在对某一药物产生耐药性后，再单独给予另一种药物治疗。联合治疗是指在一个疗程中同时或先后使用两种或两种以上的抗癌药物。
一般而言，联合治疗需要根据病情特点、联用药物种类探索不同的给药剂量、给药周期，只有根据上述情况，探索得到的联合用药治疗方案才可能取得较单一用药治疗好的治疗效果。
本发明提供Tipranavir或其盐、酯、同位素用于治疗IL24蛋白或基因表达水平低下疾病。
本发明提供用于治疗IL24蛋白或基因表达水平底下疾病的药物，其含有Tipranavir或其盐、酯、同位素。
本发明提供Tipranavir或其盐、酯、同位素用于治疗PRSS23蛋白或基因表达水平过高疾病。
本发明提供用于治疗PRSS23蛋白或基因表达水平过高疾病的药物，其含有Tipranavir或其盐、酯、同位素。
本发明提供Tipranavir或其盐、酯、同位素用于治疗早期肿瘤或癌症或细胞增生性疾病。
本发明提供Tipranavir或其盐、酯、同位素用于治疗发生转移的肿瘤或癌症或细胞增生性疾病。
本发明提供Tipranavir或其盐、酯、同位素用于治疗复发肿瘤或癌症或细胞增生性疾病。
本发明提供Tipranavir或其盐、酯、同位素用于治疗放疗或化疗或放化疗耐受的肿瘤或癌症或细胞增生性疾病，优选的，化疗使用的药物包括5-FU、顺铂、紫杉醇。
本发明提供Tipranavir或其盐、酯、同位素用于制备治疗早期肿瘤或癌症或细胞增生性疾病药物中的用途。
本发明提供Tipranavir或其盐、酯、同位素用于制备治疗发生转移的肿瘤或癌症或细胞增生性疾病药物中的用途。
本发明提供Tipranavir或其盐、酯、同位素用于制备治疗复发肿瘤或癌症或细胞增生性疾病药物中的用途。
本发明提供Tipranavir或其盐、酯、同位素用于制备治疗放疗或化疗或放化疗耐受的肿瘤或癌症或细胞增生性疾病药物中的用途。
本发明提供含有Tipranavir或其盐、酯、同位素的药物，其用于治疗
早期肿瘤或癌症或细胞增生性疾病；或
发生转移的肿瘤或癌症或细胞增生性疾病；或
复发肿瘤或癌症或细胞增生性疾病；或
放疗或化疗或放化疗耐受的肿瘤或癌症或细胞增生性疾病。
进一步，化疗使用的药物包括5-FU、顺铂、紫杉醇，肿瘤或癌症为前列腺癌。
进一步地，上述肿瘤干细胞和肿瘤细胞为胃癌、肺癌、前列腺癌、食管癌、结直肠癌、乳腺癌或肝癌的肿瘤干细胞或肿瘤细胞，优选为胃癌肿瘤干细胞、胃 癌细胞；
和/或，肿瘤干细胞或肿瘤细胞为对紫杉醇耐药的前列腺癌干细胞或前列腺癌细胞；
和/或，Tipranavir通过诱导胃癌干细胞或胃癌细胞凋亡从而杀死或抑制胃癌干细胞或胃癌细胞；
进一步地，Tipranavir通过线粒体凋亡途径诱导胃癌干细胞和胃癌细胞凋亡，进而杀伤胃癌干细胞和胃癌细胞，即，Tipranavir通过上调IL24，促进线粒体中Cytochrome c的释放并促进Cleaved caspas9、Cleaved caspas7、Cleaved caspas3、Cleaved PARP的表达，同时，上调Bax和Bak蛋白。
进一步地，本发明提供的用Tipranavir或其盐、酯、同位素杀死或抑制肿瘤干细胞或肿瘤细胞的方法和提供的Tipranavir或其盐、酯、同位素的多种用途中，Tipranavir在杀死或抑制肿瘤干细胞和肿瘤细胞时具有时间和浓度依赖性，优选地，Tipranavir在杀死或抑制胃癌干细胞和胃癌细胞时具有时间和浓度依赖性。
本发明还提供多种药物：
A.该药物能够抑制或减少PRSS23表达，并且含有活性成分Tipranavir。
B.该药物能够上调IL24表达，并且含有活性成分Tipranavir。
C.该药物能够杀死或抑制肿瘤干细胞或肿瘤细胞，并且含有活性成分Tipranavir，优选地，杀死或抑制胃癌干细胞或胃癌细胞。
进一步地，本发明提供的多种药物中，还含有药学上可接受的载体。
本发明所述的载体为药学上可以接受的载体，其指的是：一种或多种相容性固体或液体填料或凝胶物质。它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组分能和本发明的活性成分以及它们之间相互掺和，而不明显降低活性成分的药效。
优选的，所述载体包括但不限于：稀释剂、缓冲剂、混悬剂、乳剂、颗粒剂、包囊剂、赋形剂、填充剂、粘合剂、喷雾剂、透皮吸收剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、着色剂、矫味剂或吸附载体。
本发明提供了Tipranavir在制备杀伤肿瘤干细胞和肿瘤细胞的癌症治疗药物中的用途。
进一步地，本发明提供了Tipranavir在制备抑制肿瘤干细胞和肿瘤细胞增殖药物中的用途。
进一步地，本发明还提供了Tipranavir在制备降低肿瘤干细胞和肿瘤细胞体内成瘤能力药物中的用途，其包括减少肿瘤干细胞形成的肿瘤体积、减轻瘤重、以及减缓肿瘤干细胞形成肿瘤的生长速度，同时，其对肺、肝脏、脾脏、肾、心脏组织无明显毒副作用。
优选地，Tipranavir通过诱导胃癌干细胞凋亡从而杀伤胃癌干细胞来治疗癌症。
优选地，Tipranavir通过线粒体凋亡途径诱导胃癌干细胞凋亡，进而杀伤胃癌干细胞，即，Tipranavir通过上调IL24，促进线粒体中Cytochrome c的释放并促进Cleaved caspas9、Cleaved caspas7、Cleaved caspas3、Cleaved PARP的表达，同时，上调Bax和Bak蛋白。
优选地，Tipranavir浓度依赖性地杀伤人胃癌干细胞，且对正常胃上皮细胞无毒副作用。
优选地，Tipranavir用于制备紫杉醇耐药的前列腺癌治疗药物。
更进一步地，本发明还提供一种癌症的治疗药物，所述药物能够杀伤肿瘤干细胞和肿瘤细胞；所述药物以Tipranavir为主要的活性成分，并含有药学上可接受的载体。
优选的，所述药物可以制成包括但不限于显微注射剂、适于转染的剂型、注射液、片剂、粉剂、粒剂、胶囊剂。上述各种剂型的药物均可以按照药学领域的常规方法制备。
本发明提到的肿瘤干细胞癌症包括胃癌以及其他癌症。
进一步地，由于胃癌是最常见的消化系统恶性肿瘤，具有高复发率和低治愈率，且死亡率很高。因此，对其生存调控机制及分子靶向治疗进行的研究可作为 其他肿瘤的实验依据和理论参考。同时，不同实体瘤的肿瘤干细胞具有共同的特点：均具有自我更新、增殖和自我分化的能力，都能导致体内成瘤性等，不同实体瘤干细胞具有相同或相似的分子调控通路。因此，根据本发明的记载和本领域的公知常识，可以推测Tipranavir同样对除胃癌干细胞以外的其他肿瘤干细胞和肿瘤细胞具有抗癌作用，用于制备抗肿瘤干细胞和肿瘤细胞的癌症治疗药物。
所述肿瘤干细胞和肿瘤细胞的癌症包括胃癌、肺癌、前列腺癌、食管癌、结直肠癌、乳腺癌、肝癌。
优选地，所述肿瘤干细胞和肿瘤细胞的癌症为胃癌。
本发明的有益效果在于：本发明首次提供了Tipranavir可以用于制备杀伤肿瘤干细胞和肿瘤细胞的抗癌药物，其抗胃癌效果明显优于现有胃癌一线治疗药物(5-FU和cisplatin联用)，且无明显毒副作用，毒副作用明显小于胃癌一线治疗药物(5-FU和cisplatin联用)，将为靶向肿瘤干细胞的肿瘤治疗及其治愈肿瘤提供了新的药物和方案，对提高肿瘤治疗效果，改善患者生存质量具有重要意义。
图1为CCK8检测用Tipranavir处理胃癌干细胞(GCSC1和GCSC2)的细胞增殖和活性情况；
图2为CCK8检测分别用Tipranavir、fluorouracil(氟尿嘧啶，5-FU)和cisplatin(顺铂)处理胃癌干细胞(GCSC1和GCSC2)的细胞增殖和活性情况，Control为对照组；
图3为CCK8检测分别用Tipranavir、fluorouracil(氟尿嘧啶，5-FU)和cisplatin(顺铂)处理胃癌细胞株(AGS，HGC-27，MGC-803，BGC-823)和正常胃上皮细胞的细胞增殖和活性情况；
图4为CCK8检测用Tipranavir处理肺癌细胞(PC9)、前列腺癌细胞(PC3)、紫杉醇耐药的前列腺癌细胞(PC3/Tax)、食管癌细胞(KYSE180和KYSE520)、 结直肠癌细胞(HCT116)、乳腺癌细胞(MDA-MB-231)及肝癌细胞(Huh7)的细胞增殖和活性情况，Control为对照组；
图5 Tipranavir抑制裸鼠中胃癌干细胞移植瘤生长：图5a小鼠体内成瘤实验；图5b为Tipranavir组、5-FU和顺铂联用组(5-FU+Cis)和对照组(Control)处理的胃癌干细胞形成的移植瘤形状变化；图5c为Tipranavir组、5-FU和顺铂联用组(5-FU+Cis)和对照组(Control)处理的胃癌干细胞形成的移植瘤重量变化；图5d为Tipranavir组、5-FU和顺铂联用组(5-FU+Cis)和对照组(Control)处理的胃癌干细胞形成的移植瘤生长曲线；图5e为染色检测各给药组中肺、心脏、肝脏、脾脏、肾脏器组织的病理学特征；图5f为给药后的小鼠体重变化；图5g为各给药组中肺、心脏、肝脏、脾脏、肾脏器组织的脏器指数；图5h为Tipranavir组、5-FU和顺铂联用组(5-FU+Cis)和对照组(Control)处理的胃癌干细胞形成的移植瘤瘤体的WB分析；
图6 Tipranavir通过诱导胃癌干细胞凋亡实现杀伤胃癌干细胞：图6a为Tipranavir处理胃癌干细胞(GCSC1和GCSC2)的胃癌干细胞(GCSC1和GCSC2)凋亡形态，Control为对照组；图6b、图6c为Annexin V-FITC/PI标记流式分析胃癌干细胞(GCSC1和GCSC2)的细胞凋亡率；图6d、图6e为TUNEL检测分析胃癌干细胞(GCSC1和GCSC2)的细胞凋亡；图6f为蛋白质免疫印迹法检测Cytochrome c的分布和凋亡标志物(Bax、Bak、Cleaved caspas9、Cleaved caspas7、Cleaved caspas3、Cleaved PARP)的表达；
图7 Tipranavir通过上调IL24，进而上调Bax和Bak蛋白，激活细胞线粒体凋亡通路，诱导胃癌干细胞凋亡，从而杀伤胃癌干细胞：图7a、图7b用RNA-seq分析比较Tipranavir处理和不处理的胃癌干细胞基因表达变化，寻找Tipranavir处理胃癌干细胞的差异基因，发现Tipranavir胃癌干细胞显著上调IL24表达；图7c、图7d为分别用qPCR、Western blotting验证了Tipranavir处理的胃癌干细胞显著上调IL24表达；图7e，图7f胃癌干细胞中IL24表达随Tipranavir浓度的升高而增加，呈现浓度依赖性表达；
图8胃癌干细胞中siRNA干扰敲低IL24后，再用Tipranavir处理胃癌干细胞，证实Tipranavir通过IL24-Bax/Bak线粒体凋亡通路诱导胃癌干细胞凋亡，从而杀伤胃癌干细胞：图8a胃癌干细胞中siRNA干扰敲低IL24后，显著缓解Tipranavir对胃癌干细胞的杀伤作用；图8B用Western blotting检测Tipranavir处理胃癌干细胞中siRNA干扰敲低IL24后的胃癌干细胞(siIL24-1、siIL24-2)的相关蛋白表达情况，未敲IL24的胃癌干细胞(siNC)为对照组；图8c胃癌干细胞中过表达IL24对胃癌干细胞的影响，GCSC-IL24为IL24过表达组；GCSC-vec为IL24正常表达的对照组；图8d胃癌干细胞中过表达IL24，对胃癌干细胞中线粒体凋亡通路蛋白及其凋亡标志物表达的影响，GCSC-IL24为IL24过表达组；GCSC-vec为IL24正常表达的对照组；
图9 PRSS23/MKK3/p38 MAPK通路激活IL24依赖性线粒体凋亡通路：图9a，使用指定浓度的替普那韦处理48小时后，磷酸化p38 MAPK(p-p38 MAPK)和总p38 MAPK(p38 MAPK)的表达，GAPDH作为对照；图9b，分别用SB203580(10μM)、anisomycin(5μM)和tipranavir(20μM)处理GCSC1细胞3小时后IL24的相对mRNA水平；图9c，观察actinomycin(10μg/ml)预处理GCSC1细胞1小时后，用培养基或SB203580(10μM)、tipranavir(20μM)处理2小时后IL24的表达情况；图9d，从PRSS23沉默的GCSC1细胞，或GCSC2细胞(GCSC-Vec)和PRSS23过表达的GCSC2细胞(GCSC-PRSS23)和对照细胞中，在WCE中PRSS23、磷酸化的p38 MAPK(p-p38 MAPK)、总p38 MAPK(p38 MAPK)的表达；图9e，MKK3沉默的GCSC1细胞和对照组细胞在接受或不接受tipranavir治疗48小时后的WCE中MKK3、PRSS23、p-p38 MAPK、p38 MAPK和IL24的表达；图9f，在使用或不使用tipranavir处理48小时后，从PRSS23沉默的GCSC1(si-PRSS23-1)细胞和对照细胞中，MKK3与PRSS23或MKK3与p-p38 MAPK结合。
下面将结合本发明实施例中的附图，对实施例中的技术方案进行清楚、完整地描述。显然，以下将描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
可以理解地，以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用的试剂可以商业购买得到。
实施例中未注明具体条件的实验方法，通常为本领域常规方法，如按照常规条件如Sambrook等人，分子克隆，实验手册(第三版)(科学出版社，2002)中的所述条件，或按照试剂制造厂家所建议的条件。
本发明所用的主要材料如下：
Atazanavir,Darunavir,Fosamprenavir,Tipranavir,5-FU and cisplatin购自Med Chemexpress公司；Poloxamer 188(F68)购自Shenyang Jiqi Pharmaceutical Company公司；RNA抽提试剂盒(RNeasy Mini Kit)购自QIAGEN公司；逆转录试剂盒、Tag DNA聚合酶购自Life Technology公司；逆转录酶M-MLV、PCR试剂盒购自TIANGEN公司；BCA蛋白定量试剂盒购自Thermo Fischer Scientific公司；WB化学发光显色底物(Immobilon Western Chemiluminescent HRP substrate)购自Millipore公司；WB化学发光显色底物(Super Signal West Chemiluminescent substrate)购自Thermo Fischer Scientific公司；CCK-8试剂盒购自同仁化学研究所；FITC-annexin V/PI凋亡检测试剂盒(FITC Annexin V Apoptosis Detection kit)购自BD Biosciences公司；TUNEL凋亡检测试剂盒(Insitu Cell Death Detection Kit)购自Roche Molecular Biochemicals公司；线粒体抽提试剂盒(Mitochondria Isolation Kit)购自Thermo Fischer Scientific公司；细胞转染试剂盒(Lipofectamine 3000)购自Thermo Fischer Scientific公司；一抗anti-Bcl-2,anti-Bcl-xL,anti-Bax,anti-Bak,anti-Cleaved caspase-9,anti-Cleaved caspase-7,anti-Cleaved caspase-3,anti-Cleaved PARP,anti-GAPDH,anti-VDAC购自Cell Signaling Technology公司； anti-IL-24购自Proteintech公司；anti-Cytochrome C购自BD Biosciences公司；二抗Anti-mouse、二抗Anti-rabbit购自Cell Signaling Technology公司；BCA蛋白定量试剂盒购自Thermo Fischer Scientific公司；限制性内切酶EcoRI、MluI购自NEB公司；HSP90抑制剂17-AAG、SNX2112购自Selleck Chemicals公司；DMSO、嘌呤霉素(puromycin)、强力霉素(Doxycycline，Dox)、一抗Anti-β-actin购自Sigma公司；一抗Anti-Clusterin-α、二抗Anti-goat购自Santa Cruz Biotechnologies公司；一抗Anti-HSP90购自Abcam公司；一抗Anti-Sox2、anti-Cleaved PARP、anti-pSer807/Ser811-Rb、anti-AKT、anti-CDK4、anti-HER2、anti-c-Raf、anti-EGFR、anti-IGF-1R、二抗Anti-mouse、二抗Anti-rabbit购自Cell Signaling Technology公司。
本发明涉及到的统计分析方法是采用SPSS12.0统计学软件，实验结果以均值±标准差(x±s)表示，两组均数比较采用t检验，多组均数比较采用单因素方差分析，p＜0.05认为具有统计学差异，并用“*”表示。图1-8中，*表示有统计学差异(p＜0.05)；**表示有显著差异(p＜0.01)；***表示有明显显著差异(p＜0.001)；****表示有极为显著差异(p＜0.0001)。
本发明实施例所用肿瘤干细胞和肿瘤细胞均购自中科院生化细胞所细胞库，细胞培养方法为本领域常规方法。
本发明实施例中涉及的Western blot检测方法，如无特殊说明，其具体操作如下：
RIPA细胞裂解液提取细胞蛋白，BCA法测定蛋白浓度，等量蛋白样品经10％SDS-PAGE电泳后转膜印迹，封闭，一抗4℃过夜孵育，洗膜加二抗，室温孵育1h，洗膜后用ECL(Super Signal West chemiluminescent substrates，Thermo Fisher Scientific)显色。
本发明实施例中涉及的TUNEL检测GCSC细胞凋亡方法，如无特殊说明，其具体操作如下：
采用罗氏公司(Roche Molecular Biochemicals)生产的原位末端标记试剂盒 (Insitu Cell Death Detection Kit，Cat.No.11684817910)，参照试剂盒说明书进行：⑴4％多聚甲醛固定1h，PBS缓冲液漂洗；⑵3％H
2O
2阻断液室温10min，PBS漂洗；⑶0.1％Triton X-100渗透液冰上作用2min，PBS漂洗；⑷加50μl TUNEL反应混合液，湿盒中37℃作用1h，PBS漂洗；⑸含DAPI抗猝灭剂封片，显微镜观察记录，所有细胞核呈蓝色，凋亡细胞呈绿色荧光，在光镜下任选一定数目的视野进行凋亡细胞计数，统计凋亡细胞(FITC绿色荧光细胞)占所有细胞(DAPI染色的蓝色细胞)的百分率。
实施例1：Tipranavir低浓度时就能有效杀伤人胃癌干细胞，且呈现时间和浓度依赖性。
取两株不同胃癌病人来源的胃癌干细胞(记为GCSC1和GCSC2)，细胞浓度均为(1×10
5cells/mL，100μl)，接种于96孔板，每组3个复孔，分别用不同浓度Tipranavir(0uM,0.5uM,1.0uM,2.5uM,5.0uM,10uM,20uM,40uM,100uM)处理GCSC1和GCSC2细胞24hr,48hr,72hr，DMSO作为对照。分别加入CCK-8测定波长450nm的吸光值(A450)，绘制各组细胞生长曲线。检测Tipranavir对胃癌干细胞(GCSC1和GCSC2)的杀伤作用，并计算其IC
50，结果见图1(图1，cell viability细胞活性；concentration浓度)。
结果表明Tipranavir低浓度时能有效杀伤人胃癌干细胞(GCSC1和GCSC2)，且呈现时间和浓度依赖性，IC
50分别为4.7uM和6.4uM。
实施例2：Tipranavir杀伤胃癌干细胞的作用显著优于现有一线用药5-FU和顺铂联用。
取两株不同胃癌病人来源的胃癌干细胞(记为GCSC1和GCSC2)(1×10
5cells/mL，100μl)接种于96孔板，每组3个复孔，分别用Tipranavir(10uM)和5-FU+cisplatin(5-FU:2.5μM；cisplatin:4μM)处理GCSC1和GCSC2细胞72hr，DMSO作为对照。分别加入CCK-8测定波长450nm的吸光值(A450)，绘制各 组细胞生长曲线。比较Tipranavir和5-FU+cisplatin对胃癌干细胞(GCSC1和GCSC2)的杀伤作用。结果见图2(图2，cell viability细胞活性)。
结果表明Tipranavir杀伤胃癌干细胞的作用显著优于现有胃癌一线用药5-FU和顺铂联用。
实施例3：Tipranavir能有效杀伤胃癌细胞，其作用也显著优于5-FU和顺铂联用，且对正常胃上皮细胞无毒副作用，而5-FU和顺铂联用存在对正常胃上皮细胞的毒副作用。
取四种胃癌细胞株(AGS，HGC-27，MGC-803，BGC-823)和正常胃上皮细胞系(GES-1)(1×10
5cells/mL，100μl)接种于96孔板，每组3个复孔，分别用Tipranavir(10uM)和5-FU+cisplatin(5-FU:2.5μM；cisplatin:4μM)处理各株细胞72hr，DMSO作为对照。分别加入CCK-8测定波长450nm的吸光值(A450)，绘制各组细胞生长曲线。比较Tipranavir和5-FU+cisplatin对胃癌细胞(AGS,HGC-27,MGC-803,BGC-823)和正常胃上皮细胞(GES-1)的杀伤作用及毒副作用。结果见图3(图3，cell viability细胞活性，+表示有，-表示无)。
结果表明Tipranavir能有效杀伤各种胃癌细胞，其作用也显著优于5-FU和顺铂联用，且对正常胃上皮细胞无毒副作用，而5-FU和顺铂联用存在对正常胃上皮细胞的毒副作用。
以上结果表明，Tipranavir能有效杀伤胃癌干细胞和胃癌细胞，显著优于5-FU和顺铂联用，且对正常胃上皮细胞无毒副作用。
实施例4：Tipranavir对其他不同癌细胞，包括肺癌细胞(PC9)，前列腺癌细胞(PC3)，紫杉醇耐药的前列腺癌细胞(PC3/Tax)，食管癌细胞(KYSE180和KYSE520)，结直肠癌细胞(HCT116)，乳腺癌细胞(MDA-MB-231)，肝癌细胞(Huh7)各种肿瘤细胞具有抗癌作用，可用做制备广谱肿瘤治疗药物；同时，Tipranavir对紫杉醇耐药的前列腺癌细胞(PC3/Tax)的杀伤作用显著强 于普通前列腺癌细胞(PC3)，可用做制备紫杉醇耐药的前列腺癌治疗药物。
取肺癌细胞(PC9)，前列腺癌细胞(PC3)，紫杉醇耐药的前列腺癌细胞(PC3/Tax)，食管癌细胞(KYSE180和KYSE520)，结直肠癌细胞(HCT116)，乳腺癌细胞(MDA-MB-231)，肝癌细胞(Huh7)(1×10
5cells/mL，100μl)接种于96孔板，每组3个复孔，分别用Tipranavir(10uM)和5-FU+cisplatin(5-FU:2.5μM；cisplatin:4μM)处理各株细胞72hr，DMSO作为对照。分别加入CCK-8测定波长450nm的吸光值(A450)，绘制各组细胞生长曲线。比较Tipranavir和5-FU+cisplatin对各种肿瘤细胞的杀伤作用。结果见图4(图4，cell viability细胞活性)。
结果表明Tipranavir能有效杀伤胃癌、肺癌、前列腺癌、食管癌、结直肠癌、乳腺癌、肝癌等各种肿瘤细胞，其作用也显著优于5-FU和顺铂联用，且Tipranavir对紫杉醇耐药的前列腺癌细胞(PC3/Tax)的杀伤作用显著强于普通前列腺癌细胞(PC3)，其对紫杉醇耐药的前列腺癌病人具有显著作用。
本实施例表明Tipranavir对胃癌、肺癌、前列腺癌、食管癌、结直肠癌、乳腺癌、肝癌等各种肿瘤细胞具有抗癌作用，可用作广谱肿瘤治疗药物，同时，Tipranavir对紫杉醇耐药的前列腺癌细胞(PC3/Tax)的杀伤作用显著强于普通前列腺癌细胞(PC3)，可用做制备紫杉醇耐药的前列腺癌治疗药物。
实施例5：Tipranavir能显著抑制裸鼠中胃癌干细胞移植瘤生长，其作用显著优于5-FU和顺铂联用，且无明显毒副作用。而5-FU和cisplatin(顺铂)联用具有肾毒性，存在对肾脏的毒副作用。
雌性Balb/C裸鼠(5周龄，体重16.0±2.0g)，培养于无菌环境。小鼠分为3组，Tipranavir处理组(Tipranavir)、5-FU和顺铂联用组(5-FU+Cis)和对照组(Control)，每组4只，侧肋皮下注射含4x10
4GCSC细胞悬液，建立皮下移植瘤模型。10天待其成瘤后开始相关处理，其中Tipranavir处理组(Tipranavir)腹腔注射Tipranavir(F68solution：1mg/ml in normal saline，25mg/kg/mouse)； 5-FU和顺铂联用处理组(5-FU+Cis)腹腔注射5-FU+Cis(5FU:20mg/kg/mouse in F68 solution；cisplatin:2mg/kg/mouse in F68 solution)；对照组(Control)腹腔注射F68 solution(1mg/ml in normal saline)，每2天注射一次。密切观察皮下肿瘤生长情况，每2天用游标卡尺测量肿瘤大小，按公式：V＝1/2长径×短径
2，计算肿瘤体积，并绘制皮下移植瘤生长曲线。25天处死老鼠取瘤体拍照并称重，取心、肝、脾、肺、肾内脏称重，并固定做免疫组化分析。结果见图5。
图5中结果表明体内成瘤实验发现，较对照组(Control)及5-FU和顺铂联用处理组(5-FU+Cis)相比，Tipranavir处理(Tipranavir)的胃癌干细胞形成的移植瘤明显缩小(图5b)，瘤重显著减轻(图5c)，肿瘤生长曲线也表明Tipranavir处理(Tipranavir)的胃癌干细胞形成的移植瘤生长明显抑制(图5d)。而比较5-FU和顺铂联用处理组(5-FU+Cis)和对照组(Control)发现，两者不论在移植瘤大小、瘤重及生长无明显差异，表明5-FU和顺铂联用对胃癌干细胞无杀伤或抑制作用。进一步，毒副作用分析表明，Tipranavir对心、肝、脾、肺、肾重要脏器无明显毒副作用(图5e)，亦对小鼠体重无影响，不影响小鼠生长(图5f)，脏器指数也证实Tipranavir对心、肝、脾、肺、肾重要脏器无明显毒副作用(图5g)；而5-FU和顺铂联用处理显著影响小鼠，影响小鼠生长(图5f)，脏器指数也证实5-FU和顺铂联用对肾有明显毒副作用，具有肾毒性(图5g)。进一步，对瘤体进行WB分析表明，Tipranavir上调IL24，Bak表达，上调Cleaved caspase3,Cleaved PARP，激活线粒体凋亡通路，诱导凋亡，从而达到抑制肿瘤生长(图5h)。
实施例6：Tipranavir通过诱导胃癌干细胞凋亡(apoptosis)实现杀伤胃癌干细胞。
Tipranavir处理胃癌干细胞导致细胞质浓缩，体积变小，细胞核断裂分散，呈现凋亡小体形态，胃癌干细胞凋亡(图6a)；Annexin V-FITC/PI标记流式分析细胞凋亡表明，凋亡细胞显著增加，Tipranavir显著诱导胃癌干细胞凋亡(图 6b,6c)；TUNEL检测也证实Tipranavir极为显著诱导胃癌干细胞凋亡(图6d、6e)；Western blot也发现Tipranavir处理胃癌干细胞中线粒体凋亡通路蛋白及其凋亡标志物(Bax、Bak、Cleaved caspas9、Cleaved caspas7、Cleaved caspas3、Cleaved PARP)明显增加，线路体凋亡通路激活标志Cytochrome c从线粒体流进细胞质，线粒体中Cytochrome c减少(图6f)。因此，Tipranavir通过线粒体凋亡通路诱导胃癌干细胞凋亡(apoptosis)实现杀伤胃癌干细胞。
实施例7：分子机制上，Tipranavir通过上调IL24，进而上调Bax和Bak蛋白，激活细胞线粒体凋亡通路(mitochondrial apoptotic pathway),诱导胃癌干细胞凋亡(apoptosis),从而杀伤胃癌干细胞。
RNA-seq分析比较Tipranavir处理和不处理的胃癌干细胞基因表达变化，寻找Tipranavir处理胃癌干细胞的差异基因，发现Tipranavir胃癌干细胞显著上调IL24表达(图7a,7b)，并通过qPCR(图7c)，Western blotting得到验证(图7d)。且胃癌干细胞中IL24表达随Tipranavir浓度的升高而增加，呈现浓度依赖性表达(图7e,7f)。IL24已被证实为肿瘤细胞特异性抑癌基因，其高表达能显著抑制肿瘤生长，且其在正常细胞高表达对正常细胞无影响。
进一步在胃癌干细胞中用siRNA干扰敲低IL24，通过Tipranavir细胞活性实验显示，敲低IL24之后，显著缓解Tipranavir对胃癌干细胞的杀伤作用(8a)；WB检测显示，敲低IL24之后，显著降低Tipranavir引起的线粒体凋亡通路蛋白及其凋亡标志物(Bax、Bak、Cleaved caspas9、Cleaved caspas7、Cleaved caspas3、Cleaved PARP)的上调，抑制线粒体中Cytochrome c流向细胞质，显著抑制线粒体凋亡通路的活化(8b)。而在胃癌干细胞中过表达IL24，不加入Tipranavir，同样可显著抑制胃癌干细胞的生长，诱导胃癌干细胞凋亡(8c，GCSC-IL24为IL24过表达组；GCSC-vec为IL24正常表达的对照组)；同样地，胃癌干细胞中线粒体凋亡通路蛋白及其凋亡标志物(Bax、Bak、Cleaved caspas9、Cleaved caspas7、Cleaved caspas3、Cleaved PARP)明显增加，促进线粒体中Cytochrome c流向细胞质，激活线粒体凋亡通路，结果见图8d(GCSC-IL24为IL24过表达组；GCSC-vec为IL24正常表达的对照组)，其与Tipranavir处理胃癌干细胞结果相似(8b)。因此，Tipranavir通过IL24-Bax/Bak线粒体凋亡通路诱导胃癌干细胞凋亡(apoptosis)，从而杀伤胃癌干细胞。
实施例8：Tipranavir抑制PRSS23诱导IL24表达，促进胃癌干细胞凋亡。
以与上述相同的实验方法处理Tipranavir抑制PRSS23，从而诱导IL24表达的实验，得到图9所示结果。
PRSS23/MKK3/p38 MAPK通路激活IL24依赖性线粒体凋亡通路：图9a，使用指定浓度的替普那韦处理48小时后，磷酸化p38 MAPK(p-p38 MAPK)和总p38 MAPK(p38 MAPK)的表达，GAPDH作为对照；图9b，分别用SB203580(10μM)、anisomycin(5μM)和tipranavir(20μM)处理GCSC1细胞3小时后IL24的相对mRNA水平；图9c，观察actinomycin(10μg/ml)预处理GCSC1细胞1小时后，用培养基或SB203580(10μM)、tipranavir(20μM)处理2小时后IL24的表达情况；图9d，从PRSS23沉默的GCSC1细胞，或GCSC2细胞(GCSC-Vec)和PRSS23过表达的GCSC2细胞(GCSC-PRSS23)和对照细胞中，在WCE中PRSS23、磷酸化的p38 MAPK(p-p38 MAPK)、总p38 MAPK(p38 MAPK)的表达；图9e，MKK3沉默的GCSC1细胞和对照组细胞在接受或不接受tipranavir治疗48小时后的WCE中MKK3、PRSS23、p-p38 MAPK、p38 MAPK和IL24的表达；图9f，在使用或不使用tipranavir处理48小时后，从PRSS23沉默的GCSC1(si-PRSS23-1)细胞和对照细胞中，MKK3与PRSS23或MKK3与p-p38 MAPK结合。上述实验以GAPDH作为对照，实验数据具有显著性。
以上结果结合实施例7可以表明，替普那韦通过降低PRSS23的表达，从而诱导IL24的表达，而IL24表达的增加强烈影响促凋亡蛋白Bax和Bak的表达，从而激活线粒体凋亡通路，促进胃癌干细胞凋亡。
综上所述，本发明首次提供了Tipranavir可以用于制备杀伤肿瘤干细胞和肿瘤细胞的抗癌新作用，其抗胃癌效果明显优于现有胃癌一线治疗药物(5-FU和cisplatin联用)，且无明显毒副作用，毒副作用明显小于胃癌一线治疗药物(5-FU和cisplatin联用)，可用于制备抗肿瘤治疗药物，解决了目前胃癌一线治疗药物无法根除胃癌及复发耐药的短板，为治愈胃癌及提高胃癌患者的生存率提供新的方向。
在上述实施例中，对各个实施例的描述都各有侧重，某个实施例中没有详细描述的部分，可以参见其他实施例的相关描述。
以上所述，为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到各种等效的修改或替换，这些修改或替换都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以权利要求的保护范围为准。
Claims (25)
- 一种施加Tipranavir或其盐、酯、同位素杀死或抑制肿瘤干细胞或肿瘤细胞的方法，其经由抑制或减少肿瘤干细胞或肿瘤细胞中PRSS23蛋白或基因表达来实现或其经由上调肿瘤干细胞或肿瘤细胞中IL24蛋白或基因表达水平来实现。
- 一种施加Tipranavir或其盐、酯、同位素杀死或抑制肿瘤干细胞或静息状态的肿瘤细胞的方法，其经由抑制或减少肿瘤干细胞或肿瘤细胞中PRSS23蛋白或基因表达来实现或其经由上调肿瘤干细胞或肿瘤细胞中IL24蛋白或基因表达水平来实现。
- Tipranavir或其盐、酯、同位素在杀死或抑制肿瘤干细胞或静息状态的肿瘤细胞中的用途。
- Tipranavir或其盐、酯、同位素在制备杀死或抑制肿瘤干细胞或静息状态的肿瘤细胞的药物中的用途。
- Tipranavir或其盐、酯、同位素在与化疗药物单用或联用杀死或抑制肿瘤干细胞或静息状态的肿瘤细胞中的用途。
- Tipranavir或其盐、酯、同位素在制备与化疗药物单用或联用杀死或抑制肿瘤干细胞或静息状态的肿瘤细胞的药物中的用途。
- Tipranavir或其盐、酯、同位素在抑制或减少细胞中PRSS23蛋白或基因表达中的用途，所述细胞优选为肿瘤干细胞或静息状态的肿瘤细胞。
- Tipranavir或其盐、酯、同位素在制备抑制或减少细胞中PRSS23蛋白或基因表达的药物中的用途，所述细胞优选为肿瘤干细胞或静息状态的肿瘤细胞。
- Tipranavir或其盐、酯、同位素在上调细胞中IL24蛋白或基因表达水平的用途，所述细胞优选为肿瘤干细胞或静息状态的肿瘤细胞。
- Tipranavir或其盐、酯、同位素在制备上调细胞中IL24蛋白或基因表达水平的药物中的用途，所述细胞优选为肿瘤干细胞或静息状态的肿瘤细胞。
- Tipranavir或其盐、酯、同位素用于治疗IL24蛋白或基因表达水平低下疾病。
- 用于治疗IL24蛋白或基因表达水平低下疾病的药物，其含有Tipranavir或其盐、酯、同位素。
- Tipranavir或其盐、酯、同位素用于治疗PRSS23蛋白或基因表达水平过高疾病。
- 用于治疗PRSS23蛋白或基因表达水平过高疾病的药物，其含有Tipranavir或其盐、酯、同位素。
- Tipranavir或其盐、酯、同位素用于治疗早期肿瘤或癌症或细胞增生性疾病。
- Tipranavir或其盐、酯、同位素用于治疗发生转移的肿瘤或癌症或细胞增生性疾病。
- Tipranavir或其盐、酯、同位素用于治疗复发肿瘤或癌症或细胞增生性疾病。
- Tipranavir或其盐、酯、同位素用于治疗放疗或化疗或放化疗耐受的肿瘤或癌症或细胞增生性疾病，优选的，化疗使用的药物包括5-FU、顺铂、紫杉醇。
- Tipranavir或其盐、酯、同位素用于制备治疗早期肿瘤或癌症或细胞增生性疾病药物中的用途。
- Tipranavir或其盐、酯、同位素用于制备治疗发生转移的肿瘤或癌症或细胞增生性疾病药物中的用途。
- Tipranavir或其盐、酯、同位素用于制备治疗复发肿瘤或癌症或细胞增生性疾病药物中的用途。
- Tipranavir或其盐、酯、同位素用于制备治疗放疗或化疗或放化疗耐受的肿瘤或癌症或细胞增生性疾病药物中的用途。
- 含有Tipranavir或其盐、酯、同位素的药物，其用于治疗早期肿瘤或癌症或细胞增生性疾病；或发生转移的肿瘤或癌症或细胞增生性疾病；或复发肿瘤或癌症或细胞增生性疾病；或放疗或化疗或放化疗耐受的肿瘤或癌症或细胞增生性疾病。
- 如权利要求23所述的药物，其中，化疗使用的药物包括5-FU、顺铂、紫杉醇，肿瘤或癌症为前列腺癌。
- 如权利要求1-2任一项所述的方法或权利要求3-10、19-22中任一项所述的用途，其中，所述肿瘤干细胞和肿瘤细胞为胃癌、肺癌、前列腺癌、食管癌、结直肠癌、乳腺癌或肝癌的肿瘤干细胞或肿瘤细胞，优选为胃癌肿瘤干细胞、胃癌细胞。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011362686.0 | 2020-11-27 | ||
| CN202011362686.0A CN112263578B (zh) | 2020-11-27 | 2020-11-27 | Tipranavir在制备杀伤肿瘤干细胞和肿瘤细胞的癌症治疗药物中的用途 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2022111670A1 true WO2022111670A1 (zh) | 2022-06-02 |
Family
ID=74350023
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2021/133781 WO2022111670A1 (zh) | 2020-11-27 | 2021-11-28 | Tipranavir在制备杀伤肿瘤干细胞和肿瘤细胞的癌症治疗药物中的用途 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN112263578B (zh) |
| WO (1) | WO2022111670A1 (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112263578B (zh) * | 2020-11-27 | 2022-02-11 | 深圳大学 | Tipranavir在制备杀伤肿瘤干细胞和肿瘤细胞的癌症治疗药物中的用途 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105769863A (zh) * | 2015-04-01 | 2016-07-20 | 重庆理工大学 | 替拉那韦在抗癌症药物中的应用及抗癌症药物 |
| CN112263578A (zh) * | 2020-11-27 | 2021-01-26 | 深圳大学 | Tipranavir在制备杀伤肿瘤干细胞和肿瘤细胞的癌症治疗药物中的用途 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008011045A2 (en) * | 2006-07-18 | 2008-01-24 | Cytovia, Inc. | 3-ARYL-6-ARYL-7H-[1,2,4]TRIAZOLO[3,4-b][1,3,4]THIADIAZINES AND ANALOGS AS ACTIVATORS OF CASPASES AND INDUCERS OF APOPTOSIS AND THE USE THEREOF |
-
2020
- 2020-11-27 CN CN202011362686.0A patent/CN112263578B/zh active Active
-
2021
- 2021-11-28 WO PCT/CN2021/133781 patent/WO2022111670A1/zh active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105769863A (zh) * | 2015-04-01 | 2016-07-20 | 重庆理工大学 | 替拉那韦在抗癌症药物中的应用及抗癌症药物 |
| CN112263578A (zh) * | 2020-11-27 | 2021-01-26 | 深圳大学 | Tipranavir在制备杀伤肿瘤干细胞和肿瘤细胞的癌症治疗药物中的用途 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112263578A (zh) | 2021-01-26 |
| CN112263578B (zh) | 2022-02-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | AT-533, a novel Hsp90 inhibitor, inhibits breast cancer growth and HIF-1α/VEGF/VEGFR-2-mediated angiogenesis in vitro and in vivo | |
| Zhang et al. | Harmine induces apoptosis and inhibits tumor cell proliferation, migration and invasion through down-regulation of cyclooxygenase-2 expression in gastric cancer | |
| KR102490334B1 (ko) | 암 치료용 아젤니디핀 약학 조성물의 새로운 적응증 | |
| Liu et al. | RBCK1 contributes to chemoresistance and stemness in colorectal cancer (CRC) | |
| Li et al. | A novel imidazopyridine derivative, HS-106, induces apoptosis of breast cancer cells and represses angiogenesis by targeting the PI3K/mTOR pathway | |
| RU2602937C2 (ru) | Комбинация сиросингопина и митохондриальных ингибиторов для лечения рака и для иммуносупрессии | |
| CN103347520A (zh) | 用作癌症化学治疗的晚期sv40因子（lsf）的抑制剂 | |
| BR112013024211B1 (pt) | Tratamento de tumores sólidos | |
| US20100015042A1 (en) | Combine radiation therapy and chemotherapy for treating cancer | |
| Song et al. | Magnolin targeting of ERK1/2 inhibits cell proliferation and colony growth by induction of cellular senescence in ovarian cancer cells | |
| EP2968379A1 (en) | Etoposide and prodrugs thereof for use in targeting cancer stem cells | |
| TW201607531A (zh) | 癌幹細胞之增殖抑制劑 | |
| Tian et al. | Fangchinoline targets PI3K and suppresses PI3K/AKT signaling pathway in SGC7901 cells Retraction in/10.3892/ijo. 2023.5547 | |
| Wang et al. | E platinum, a newly synthesized platinum compound, induces apoptosis through ROS‐triggered ER stress in gastric carcinoma cells | |
| Boran et al. | Extended regorafenib treatment can be linked with mitochondrial damage leading to cardiotoxicity | |
| WO2022111670A1 (zh) | Tipranavir在制备杀伤肿瘤干细胞和肿瘤细胞的癌症治疗药物中的用途 | |
| Russell et al. | Bromodomain and extraterminal (BET) proteins regulate hepatocyte proliferation in hepatocyte-driven liver regeneration | |
| EP3429582B1 (en) | Combination therapy for proliferative diseases | |
| Jung et al. | Suppression of tumor proliferation and angiogenesis of hepatocellular carcinoma by HS-104, a novel phosphoinositide 3-kinase inhibitor | |
| Hong et al. | Genipin suppression of growth and metastasis in hepatocellular carcinoma through blocking activation of STAT-3 | |
| Luo et al. | Ursolic acid augments the chemosensitivity of drug-resistant breast cancer cells to doxorubicin by AMPK-mediated mitochondrial dysfunction | |
| Natu et al. | Mitochondrial dysfunction and chromatin changes with autophagy-mediated survival in doxorubicin resistant cancer cell lines | |
| JP2024023269A (ja) | 抗腫瘍剤及び配合剤 | |
| Zhang et al. | Kaempferol 3‐O‐gentiobioside, an ALK5 inhibitor, affects the proliferation, migration, and invasion of tumor cells via blockade of the TGF‐β/ALK5/Smad signaling pathway | |
| Li et al. | Mitochondrial fission factor promotes cisplatin resistancein hepatocellular carcinoma: Mff promotes cisplatin resistance in HCC |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21897170 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 21897170 Country of ref document: EP Kind code of ref document: A1 |