CN116870162A - Application of STAT3 inhibitor combined with oxaliplatin in preparation of medicament for treating colorectal cancer - Google Patents
Application of STAT3 inhibitor combined with oxaliplatin in preparation of medicament for treating colorectal cancer Download PDFInfo
- Publication number
- CN116870162A CN116870162A CN202311004597.2A CN202311004597A CN116870162A CN 116870162 A CN116870162 A CN 116870162A CN 202311004597 A CN202311004597 A CN 202311004597A CN 116870162 A CN116870162 A CN 116870162A
- Authority
- CN
- China
- Prior art keywords
- oxaliplatin
- stat3
- colorectal cancer
- hct
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 title claims abstract description 132
- 229960001756 oxaliplatin Drugs 0.000 title claims abstract description 130
- 206010009944 Colon cancer Diseases 0.000 title claims abstract description 88
- 208000001333 Colorectal Neoplasms Diseases 0.000 title claims abstract description 85
- 102000004495 STAT3 Transcription Factor Human genes 0.000 title claims abstract description 67
- 108010017324 STAT3 Transcription Factor Proteins 0.000 title claims abstract description 67
- 239000003112 inhibitor Substances 0.000 title claims abstract description 32
- 239000003814 drug Substances 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 21
- 230000004913 activation Effects 0.000 claims abstract description 5
- 150000001875 compounds Chemical class 0.000 claims description 47
- 239000000203 mixture Substances 0.000 claims description 6
- 230000000903 blocking effect Effects 0.000 claims description 5
- 230000019491 signal transduction Effects 0.000 claims description 5
- 102000039446 nucleic acids Human genes 0.000 claims description 2
- 108020004707 nucleic acids Proteins 0.000 claims description 2
- 150000007523 nucleic acids Chemical class 0.000 claims description 2
- 101000997832 Homo sapiens Tyrosine-protein kinase JAK2 Proteins 0.000 claims 1
- 102100033444 Tyrosine-protein kinase JAK2 Human genes 0.000 claims 1
- 101100311214 Xenopus laevis stat3.1 gene Proteins 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 15
- 230000035755 proliferation Effects 0.000 abstract description 10
- 230000002401 inhibitory effect Effects 0.000 abstract description 8
- 230000009545 invasion Effects 0.000 abstract description 6
- 238000001727 in vivo Methods 0.000 abstract description 5
- 230000005012 migration Effects 0.000 abstract description 5
- 238000013508 migration Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 230000002195 synergetic effect Effects 0.000 abstract description 5
- 230000012010 growth Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 124
- 239000002609 medium Substances 0.000 description 48
- 108020004459 Small interfering RNA Proteins 0.000 description 36
- 238000002474 experimental method Methods 0.000 description 17
- 230000006907 apoptotic process Effects 0.000 description 16
- 229940079593 drug Drugs 0.000 description 15
- 238000011534 incubation Methods 0.000 description 10
- 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 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 108010082117 matrigel Proteins 0.000 description 9
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 8
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 230000002829 reductive effect Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 206010052358 Colorectal cancer metastatic Diseases 0.000 description 7
- 239000003560 cancer drug Substances 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 238000003556 assay Methods 0.000 description 6
- 230000003833 cell viability Effects 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 239000002033 PVDF binder Substances 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 230000004791 biological behavior Effects 0.000 description 5
- 239000013078 crystal Substances 0.000 description 5
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 5
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 102000006503 Janus Kinase 2 Human genes 0.000 description 4
- 108010019437 Janus Kinase 2 Proteins 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 230000030279 gene silencing Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000002147 killing effect Effects 0.000 description 4
- 238000011580 nude mouse model Methods 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 230000035899 viability Effects 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- YXTKHLHCVFUPPT-YYFJYKOTSA-N (2s)-2-[[4-[(2-amino-5-formyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid;(1r,2r)-1,2-dimethanidylcyclohexane;5-fluoro-1h-pyrimidine-2,4-dione;oxalic acid;platinum(2+) Chemical compound [Pt+2].OC(=O)C(O)=O.[CH2-][C@@H]1CCCC[C@H]1[CH2-].FC1=CNC(=O)NC1=O.C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 YXTKHLHCVFUPPT-YYFJYKOTSA-N 0.000 description 3
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 230000012292 cell migration Effects 0.000 description 3
- 238000002512 chemotherapy Methods 0.000 description 3
- 238000009643 clonogenic assay Methods 0.000 description 3
- 231100000096 clonogenic assay Toxicity 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 241000283707 Capra Species 0.000 description 2
- 102000016362 Catenins Human genes 0.000 description 2
- 108010067316 Catenins Proteins 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 230000002424 anti-apoptotic effect Effects 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- KVUAALJSMIVURS-ZEDZUCNESA-L calcium folinate Chemical compound [Ca+2].C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C([O-])=O)C=C1 KVUAALJSMIVURS-ZEDZUCNESA-L 0.000 description 2
- 235000008207 calcium folinate Nutrition 0.000 description 2
- 239000011687 calcium folinate Substances 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000003570 cell viability assay Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229960002949 fluorouracil Drugs 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000000123 paper Substances 0.000 description 2
- 239000011087 paperboard Substances 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000000902 placebo Substances 0.000 description 2
- 229940068196 placebo Drugs 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000003026 viability measurement method Methods 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 1
- 102000000412 Annexin Human genes 0.000 description 1
- 108050008874 Annexin Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 206010055114 Colon cancer metastatic Diseases 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 208000007433 Lymphatic Metastasis Diseases 0.000 description 1
- 238000011794 NU/NU nude mouse Methods 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 206010064390 Tumour invasion Diseases 0.000 description 1
- 206010049040 Weight fluctuation Diseases 0.000 description 1
- ZSTCHQOKNUXHLZ-PIRIXANTSA-L [(1r,2r)-2-azanidylcyclohexyl]azanide;oxalate;pentyl n-[1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate;platinum(4+) Chemical compound [Pt+4].[O-]C(=O)C([O-])=O.[NH-][C@@H]1CCCC[C@H]1[NH-].C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 ZSTCHQOKNUXHLZ-PIRIXANTSA-L 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000006909 anti-apoptosis Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- FCPVYOBCFFNJFS-LQDWTQKMSA-M benzylpenicillin sodium Chemical compound [Na+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 FCPVYOBCFFNJFS-LQDWTQKMSA-M 0.000 description 1
- 229960000397 bevacizumab Drugs 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 230000005773 cancer-related death Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 229960005395 cetuximab Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000009104 chemotherapy regimen Methods 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 201000002758 colorectal adenoma Diseases 0.000 description 1
- 238000009096 combination chemotherapy Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000012137 double-staining Methods 0.000 description 1
- 230000000431 effect on proliferation Effects 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000000771 oncological effect Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000011125 single therapy Methods 0.000 description 1
- 229960002385 streptomycin sulfate Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000012447 xenograft mouse model Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
-
- 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
- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention discloses application of STAT3 inhibitor combined with oxaliplatin in preparation of a medicine for treating p-STAT3 activation or p-STAT3 low-expression solid tumors. The p-STAT3 activated solid tumor is sensitive colorectal cancer. The solid tumor with low expression of the p-STAT3 is oxaliplatin-resistant colorectal cancer. The combination of the STAT3 inhibitor and the oxaliplatin has a good synergistic effect on treating p-STAT3 activated colorectal cancer, and meanwhile, the combination of the STAT3 inhibitor and the oxaliplatin can increase the sensitivity of colorectal cancer resistant cells with low expression of p-STAT3 to the oxaliplatin, enhance the effect of the oxaliplatin on inhibiting CRC proliferation, invasion and migration, obviously inhibit the growth rate of CRC tumors in vivo and have a good synergistic effect on the oxaliplatin resistant colorectal cancer with low expression of p-STAT 3.
Description
Technical Field
The invention belongs to the field of biological medicines, relates to application of STAT3 inhibitors combined with oxaliplatin in preparation of medicines for treating colorectal cancer, and in particular relates to application of STAT3 inhibitors combined with oxaliplatin in preparation of medicines for treating colorectal cancer with oxaliplatin resistance.
Background
Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths worldwide, with 140000 new diagnosed cases in 2023 and 53200 deaths. Many CRC patients are diagnosed at an advanced stage where lymph node metastasis or distant metastasis occurs. Despite significant improvements in surgery, chemotherapy, molecular targeted drugs, and immunotherapy of patients with metastatic colorectal cancer (mCRC), the survival rate of patients for 3 years or no disease is still low.
Oxaliplatin (Oxaliplatin, OXA) is a third generation platinum (Pt) -based chemotherapeutic drug, and has a structure shown in formula (II):
the combined use of oxaliplatin with 5-fluorouracil and calcium folinate (FOLFOX) is a classical first-line approach to the clinical treatment of metastatic colon cancer, with nearly 50% remission rates, increasing the 5-year survival and quality of life of the patient, but still about 40% of the patients are insensitive to the chemotherapy regimen. Studies have suggested that the combined use of cetuximab, calcium folinate, 5-fluorouracil and Oxaliplatin (FOLFOX-4) may lead to good clinical outcome for RAS wild-type mCRC patients, increasing disease-free survival (DFS) and Overall Survival (OS) rates. However, in mCRC patients receiving Oxaliplatin-based chemotherapy (CapeOx or FOLFOX in combination with bevacizumab), median DFS was only 9.4 months and median OS was only 21.3 months. Furthermore, researchers found that mCRC patients had an overall response rate to FOLFOX <50%. Thus, resistance to OXA-based chemotherapy remains a major obstacle to the treatment of mCRC patients, and research into oxaliplatin resistance mechanisms has become a clinically urgent problem, and finding strategies to delay or overcome resistance are also necessary to improve prognosis in colorectal cancer patients.
The family of signal transduction and transcription activator (STAT) proteins is one of the most important transcription factors in the biological immune system to activate transcription factors. In all colorectal tumors, the abnormal expression rate of STAT3 of colon cancer reaches 72%, while in colorectal adenomas it accounts for 18%. Therefore, blocking the STAT3 related signal transduction pathway or effectively inhibiting malignant biological behaviors such as proliferation, angiogenesis, invasion, metastasis and the like of colorectal cancer cells, and STAT3 can assist cancer cells to escape drug killing, and the action mechanism of STAT3 in the tumor drug resistance process can be related to the STAT3 regulation cell cycle and apoptosis genes.
A total of 14 clinical trials of treatment of tumors with STAT3 inhibitors, including single or combination chemotherapy, for early, late or metastatic colorectal cancer, are reported worldwide. However, no report of STAT3 inhibitors in preparing oxaliplatin-resistant colorectal cancer drugs is found, and no report of STAT3 inhibitors combined with oxaliplatin-resistant colorectal cancer drugs is found.
Disclosure of Invention
The inventors have found a way to exert anticancer effects by modulating STAT 3. The effect of platinum drugs on STAT3 pathway is associated with colorectal cancer resistance, and oxaliplatin-resistant colorectal cancer cells have low expressed levels of phosphorylated STAT3 (p-STAT 3), which is associated with inhibition of STAT3 by oxaliplatin. Based on this finding, the inventors propose the treatment of colorectal cancer cells highly expressed by p-STAT3 and oxaliplatin resistant colorectal cancer under-expressed by p-STAT3 with STAT3 inhibitors. The results of a cell CCK-8 proliferation experiment, a clone formation experiment, an apoptosis experiment and a mouse in-vivo tumorigenesis experiment show that the STAT3 inhibitor LY1 can obviously inhibit colorectal cancer cells activated by p-STAT3, thereby achieving the effect of targeted inhibition of colorectal cancer. Further, LY1 combined with oxaliplatin can obviously inhibit the growth rate of oxaliplatin-resistant colorectal cancer cells in a nude mouse, and the combined use of the STAT3 inhibitor and the oxaliplatin has a good synergistic effect on both p-STAT3 high-expression colorectal cancer cells and p-STAT3 low-expression oxaliplatin-resistant colorectal cancer.
Thus, the present invention proposes a strategy for targeted treatment of colorectal cancer cells with high p-STAT3 expression (i.e. p-STAT3 activation) and oxaliplatin-resistant colorectal cancer with low p-STAT3 expression by using a combination of STAT3 inhibitor and oxaliplatin.
The invention aims to provide application of STAT3 inhibitor combined with oxaliplatin in preparation of a medicine for treating p-STAT3 activation or p-STAT3 low-expression solid tumors.
The JAK2/STAT3 signal pathway is activated JAK2 (Janus Kinase 2) phosphorylation modified STAT3 (Signal transducer and activator of Transcription 3), and the phosphorylated STAT3 enters the nucleus in a dimer form to be combined with a target gene so as to regulate the transcription of the gene.
The STAT3 inhibitor is a small molecular compound, an antibody or a nucleic acid medicament for blocking a JAK2/STAT3 signal pathway.
Preferably, the STAT3 inhibitor is selected from a compound LY1 with a structure shown as a formula (I):
the p-STAT3 activated solid tumor is sensitive colorectal cancer.
The solid tumor with low expression of the p-STAT3 is oxaliplatin-resistant colorectal cancer.
The ratio of the STAT3 inhibitor to oxaliplatin is 1:2-1:10, preferably 1:3-1:5.
Another object of the invention is to provide the use of STAT3 inhibitors in combination with oxaliplatin for the preparation of a medicament for the treatment of oxaliplatin-resistant colorectal cancer.
It is another object of the present invention to provide a composition for treating colorectal cancer that is activated by p-STAT3 or has low expression of p-STAT3, which comprises STAT3 inhibitor and oxaliplatin as main active ingredients.
Compared with the prior art, the invention has the following beneficial effects:
the combination of the STAT3 inhibitor and the oxaliplatin has a good synergistic effect on treating p-STAT3 activated colorectal cancer, and meanwhile, the combination of the STAT3 inhibitor and the oxaliplatin can increase the sensitivity of colorectal cancer resistant cells with low expression of p-STAT3 to the oxaliplatin, enhance the effect of the oxaliplatin on inhibiting CRC proliferation, invasion and migration, obviously inhibit the growth rate of CRC tumors in vivo and have a good synergistic effect on the oxaliplatin resistant colorectal cancer with low expression of p-STAT 3.
Drawings
FIG. 1 shows the cell morphology of colorectal cancer sensitive cell lines HCT 116 and HCT8 and colorectal cancer oxaliplatin resistant cell lines HCT 116L and HCT 8L.
FIG. 2 shows the results of a clonogenic assay of colorectal cancer-sensitive cell lines HCT 116 and HCT8 and colorectal cancer oxaliplatin-resistant cell lines HCT 116L and HCT 8L.
FIG. 3 shows the results of CCK8 experiments on oxaliplatin by colorectal cancer-sensitive cell lines and colorectal cancer oxaliplatin-resistant cell lines; wherein, A is the experimental result of HCT 116 and HCT 116L on CCK8 of oxaliplatin; b is the CCK8 experimental result of HCT8 and HCT8L on oxaliplatin.
FIG. 4 shows the results of cell invasion experiments on colorectal cancer drug-resistant cell line HCT 116L and colorectal cancer sensitive cell line HCT 116.
FIG. 5 shows the results of cell migration experiments for colorectal cancer drug-resistant cell lines HCT8L and colorectal cancer susceptible cell lines HCT 8.
FIG. 6 shows the comparison of anti-apoptotic capacity of colorectal cancer resistant and susceptible cell lines; wherein P <0.05 compared to the control group; * P <0.01; * P <0.001.
FIG. 7 shows STAT3-siRNA silencing efficacy screening results.
FIG. 8 is the effect of siRNA knockdown on invasion and resistance of oxaliplatin resistant cell lines; wherein A is the sensitivity investigation result of HCT8L to oxaliplatin after siRNA knockdown; b is the result of examination of sensitivity of HCT 116L cells to oxaliplatin after STAT3 knockdown.
Fig. 9 is the effect on proliferation inhibition of colon cancer resistant strains after STAT3 knockdown.
FIG. 10 is the effect of siRNA knockdown on invasion of oxaliplatin resistant cell lines.
Fig. 11 shows results of flow cytometry analysis of apoptosis of HCT 116L and HCT8L after STAT3 knockdown.
FIG. 12 shows that compound LY1 gradient inhibits expression of phosphorylated Stat 3.
Fig. 13 shows the effect of different concentrations of compound LY1, oxaliplatin on the cell viability of HCT 116L, HCT8L cells, respectively.
FIG. 14 is a graph showing the IC of a combination of LY1 and Oxaliptin for HCT 116L and HCT8L cell lines 50 。
FIG. 15 shows the effect of LY1, oxaliptin, and combinations of both on cell status and cell viability after HCT 116L and HCT8L, respectively.
FIG. 16 shows the results of the combination of compound LY1 and Oxaliptin on inhibition of colorectal cancer resistant strain HCT8L clones.
FIG. 17 shows the results of combination of compound LY1 and Oxaliptin for inhibiting migration of colorectal cancer resistant strain HCT 8L.
FIG. 18 is a graph showing the results of an investigation of apoptosis in HCT 116/L and HCT 8/L cells using a combination of compounds LY1 and Oxaliptin.
FIG. 19 is the effect of combination of compound LY1 and Oxaliptin on the volume of subcutaneous HCT8L xenograft tumors in nude mice; a is tumor volume, and B is analysis result.
Fig. 20 is the change in nude mice body weight (n=6) following the combination of compound LY1 and Oxaliplatin.
Detailed Description
The technical solution of the present invention is further described below with reference to specific examples and drawings in the specification, but the examples are not limited in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1
Oxaliplatin colorectal cancer drug resistant cell lines are more resistant to oxaliplatin than more sensitive cell lines
Differences in the biological properties of oxaliplatin resistant cell lines for colorectal cancer and oxaliplatin sensitive cell lines for colorectal cancer were studied in vitro by comparing the biological behavior of oxaliplatin resistant cell lines for colorectal cancer HCT 116L, HCT L and oxaliplatin sensitive cell lines for colorectal cancer HCT 116, HCT 8.
(1) Morphology observation: the above cells were inoculated into a 6-well plate at 100000 cells/well, and the whole medium of 1640 containing 10% FBS and 1% PS (penicillin-streptomycin solution (double antibody), penicillin G sodium salt 10kU/mL, streptomycin sulfate 10mg/mL, the same applies hereinafter) was cultured for 3 days, and cell forms of HCT 116, HCT 116L, HCT, and HCT8L were photographed with a living cell workstation.
(2) Cell plate cloning experiments: the cells were inoculated at 1000 cells/well in 6-well plates, respectively, and cultured in 1640 complete medium containing 10% FBS and 1% PS for 7 days, after PBS washing, fixed with methanol, and stained with 0.1% crystal violet, and the cell clone formation numbers and sizes of the respective groups were counted and compared.
(3) Four strains of cells (HCT 116, HCT 116L, HCT, HCT 8L) were seeded in 96-well plates, 5000 cells per well, 1640 complete medium containing 10% FBS and 1% PS incubated for 24 hours; oxaliptin was diluted to final concentrations of 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7, 700. Mu.M with 1640 complete medium, incubated with the above-described Oxaliptin-containing 1640 complete medium for 48h, and viability was identified using the CCK8 assay. Absorbance was read at 490 nm.
The morphology of the colorectal cancer oxaliplatin resistant strains HCT 116L and HCT8L is greatly changed compared with that of the sensitive strain, and the oxaliplatin resistant cell strain is changed from the round shape of the sensitive cell strain to a long spindle shape (figure 1). However, drug-resistant cell lines were less proliferative than sensitive cell lines (FIG. 2). As can be seen from fig. 3 and table 1, the drug resistant cell line HCT 116L, HCT L had significantly improved resistance to oxaliplatin. The results show that the morphology of the oxaliplatin colorectal cancer drug-Resistant strain is greatly changed compared with that of a sensitive strain, the proliferation capacity is weakened, but the resistance of the drug-Resistant cell strain HCT 116L, HCT L to oxaliplatin (resistance index) is respectively improved by 17.69 times and 23.40 times compared with that of the sensitive cell strain. The oxaliplatin colorectal cancer drug-resistant cell strain is more resistant to oxaliplatin than the sensitive cell strain.
TABLE 1 drug resistance coefficients of HCT 116L and HCT8L
| Cell line | IC 50 (μM) | Resistant index |
| HCT 116 | 2.95 | - |
| HCT 116L | 52.19 | 17.69 |
| HCT 8 | 5.44 | - |
| HCT 8L | 127.32 | 23.40 |
Example 2
Oxaliplatin colorectal cancer drug-resistant cell strain is more sensitive to attack and has stronger anti-apoptosis capability
Differences in biological properties were studied in vitro by comparing the biological behavior of HCT 116L, HCT L and HCT 116, HCT 8.
(1) Invasion experiments: matrigel (BD bioscience, usa) thawed overnight at 4 ℃. Before plating, the matrigel was diluted with cold serum-free growth medium at a volume ratio of 1640 serum-free growth medium to matrigel of 1:8. The cooled diluted matrigel (70 μl) was placed in the center of the upper chamber; the plates were placed in an incubator for 60 minutes to gel the diluted matrix. Cells (1X 10) 5 Each/each chamber) was inoculated into the serum-free medium in the upper chamber 1640, and the medium 1640 containing 15% fbs was added to the lower chamber, and the migrated cells passed through the membrane and adhered to the lower chamber side of the membrane. After 48 hours of incubation, the migrated cells were stained with crystal violet and counted.
As can be seen from fig. 4, oxaliplatin colorectal cancer resistant cell line HCT 116L has a greater invasive capacity than the sensitive line.
(2) Migration experiment: HCT8 and HCT8L cells were seeded in six well plates, cells were cultured with 1640 complete medium containing 10% FBS and 1% PS, when the cells reached 100% confluence, cells were scraped using a yellow tip and washed twice to remove non-adherent cells, cells were allowed to migrate to the scratch area for 24 hours, and photographs were observed under a living cell workstation, and the percentage of wound healing was calculated.
Average of wound healing percentage = [ (gap area: 0 hour) - (gap area: 24 hours) ]/(gap area: 0 hour)
As can be seen from fig. 5, the mobility of oxaliplatin colorectal cancer resistant cell lines and sensitive lines did not differ much.
(3) HCT 116, HCT 116L, HCT, HCT8L were inoculated overnight in 6-well plates, respectively. Control group was given 1640 complete medium for culture, HCT 116 and HCT 116L administration groups were given 1640 complete medium containing 15. Mu.M oxaliplatin (formulated in 5% dextrose solution), HCT8 and HCT8L administration groups were given 1640 complete medium containing 25. Mu.M oxaliplatin (formulated in 5% dextrose solution), after 24h incubation of each group of cells, V-FITC and PI were stained separately and together for 15min, flow cytometry detection was performed on samples, cell membrane catenin V-FITC at early apoptosis was positive, PI was negative, and cells at late apoptosis were both stained positive. As can be seen from fig. 6, the apoptosis resistance of oxaliplatin colorectal cancer resistant cell lines was significantly improved compared with those of sensitive lines.
The above results indicate that: oxaliplatin colorectal cancer resistant cell lines have higher invasive and anti-apoptotic capacity than sensitive lines.
Example 3
Effects of siRNA STAT3 knockdown on biological behavior of colorectal cancer oxaliplatin-resistant cell lines
The effect of STAT3 knockdown on colorectal cancer resistant cell growth was studied in vitro by siRNA knockdown of HCT 116L and HCT8L colorectal cancer cell lines.
(1) SiRNA STAT3 silencing efficiency study
Inoculating HCT 116L and HCT8L cells in logarithmic growth phase into 6-well plate after pancreatin digestion treatment, and inoculating at a density of 5×10 5 3 duplicate wells were placed in each group, 1640 complete medium containing 10% FBS and 1% PS was used for culturing, and when 80-90% cells were fused, control group, positive Control group and 3 ST AT3 siRNA groups were placed, respectively, and cells in Positive Control group, positive Control group and STAT3 siRNA group were transfected. Firstly, 18 mu L of Lipofectamine 3000 and 750 mu L of 1640 basal medium are gently mixed, and the mixture is left at room temperature for 5min, and then the 750 mu L of 1640 basal medium is mixed with 4.5 mu L of Control siRNA (GAPDH siRNA) or STAT3 siRNA (siR NA 1729,siRNA 1272,siRNA 1878, all ddH) 2 O dissolved) solution, and standing at room temperature for 20min to form a Lipofectamine 3000 and control siRNA or STAT3 siRNA complex solution. The cell culture medium was changed to 1.5mL of fresh medium, 500. Mu.L of the complex solution was added, the cell culture plate was gently shaken, and the sample was collected after 48 hours. Control group cells were incubated in 1640 complete medium containing 10% FBS and 1% PS; the Negative control group: the medium was replaced with 1.5mL of fresh medium, and 500. Mu.L of 1640 complete medium was added for cultivation (18. Mu.L of Lipofectamine 3000 and750 μl of 1640 medium containing 10% FBS and 1% PS). The final concentration of all siRNAs was 20nM (same applies below).
siRNA 1729 sequence: sense (5 '-3') GGGACCUGGUGUGAAUUAUTT; anti (5 '-3') AUA AUUCACACCAGGUCCCTT;
siRNA 1272 sequence: sense (5 '-3') CCCGGAAAUUUAACAUUCUTT; anti (5 '-3') AGA AUGUUAAAUUUCCGGGTT;
siRNA 1878 sequence: sense (5 '-3') GGUACAUCAUGGGCUUUAUTT; anti (5 '-3') AUA AAGCCCAUGAUGUACCTT.
As shown in fig. 7, three unknown siRNA fragments were screened by WB, STAT3 in both HCT 116L and HCT8L was significantly reduced after siRNA knockdown, and the highest selective silencing efficiency of siRNA 1878 was used, and therefore, the siRNA 1878 fragment with the highest silencing efficiency was selected for subsequent experiments.
(2) Cell viability assay: HCT 116L, HCT L was seeded into 96-well plates, 5000 cells per well, incubated with 1640 complete medium (containing 10% FBS and 1% PS) for 24 hours, oxaliplatin was diluted with 1640 complete medium to 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7, 700 μm respectively, and the cells were co-treated with siRNA STAT3, incubated for 48 hours, and viability was identified using the CCK8 assay. Absorbance was read at 490 nm.
The results are shown in FIG. 8, which shows the IC of oxaliplatin to HCT8L after siRNA knockdown 50 From 127.32. Mu.M down to 12.38. Mu.M, for HCT 116L IC 50 The reduction from 52.19 mu M to 22.84 mu M suggests that STAT3 knockdown can enhance the killing power of oxaliplatin on colorectal cancer oxaliplatin resistant cell lines.
(3) Cell plate cloning experiments: HCT8L cells were seeded at 1000/well overnight in six well plates. A Control group, an Oxaliptin group (Oxaliptin concentration: 25. Mu.M), an siRNA group, and an siRNA+Oxaliptin group (Oxaliptin concentration: 25. Mu.M) were set, respectively. After the complete medium 1640 (containing 10% FBS and 1% PS) was used to dilute the drugs (siRNA STAT3 and Oxaliptin), the culture was continued with the complete medium 1640 containing the drugs, and the complete medium 1640 was used as a Control group, after 10 days of culture, PBS was washed, and then fixed with methanol, stained with 0.1% crystal violet, and counted, and the number and size of cell clones in each group were compared.
The cloning experiment results are shown in fig. 9, which indicate that after siRNA knockdown, the proliferation capacity of the drug-resistant cell strain of colorectal cancer oxaliplatin is obviously reduced, and the inhibition of the proliferation capacity of the drug-resistant cell strain of colorectal cancer oxaliplatin can be obviously enhanced by knocking down STAT3 and adding low-concentration oxaliplatin.
The above results indicate that: oxaliplatin resistant cell lines of colorectal cancer have increased sensitivity to oxaliplatin (fig. 7, 8), and drug-resistant lines have reduced proliferation capacity after knockout of STAT3 (fig. 9).
Example 4
Effects of siRNA STAT3 knockdown on biological behavior of colorectal cancer oxaliplatin-resistant cell lines
The effect of STAT3 knockdown on colorectal cancer resistant cell growth was studied in vitro by siRNA knockdown of HCT 116L and HCT8L colorectal cancer cell lines.
(1) Matrigel (BD bioscience, usa) thawed overnight at 4 ℃. The matrigel was diluted with cold 1640 serum-free growth medium at a volume ratio of 1640 serum-free growth medium to matrigel of 1:8. The cooled diluted matrigel (70 μl) was placed in the center of the upper chamber, and the plate was placed in an incubator for 60 minutes to gel the diluted matrigel. Cells (1X 10) 5 Each chamber) was inoculated into the upper chamber serum-free medium, and to the lower chamber of the Control group, the Oxaliplatin group, the sirna+oxaliplatin group, 1640 complete medium containing 15% fbs, 1640 complete medium containing 15 μm Oxaliplatin, siRNA STAT3 were added, respectively, and incubated for 48 hours, the migrated cells penetrated the membrane and adhered to the lower chamber side of the membrane, stained with crystal violet, and the migrated cells were counted (fig. 10).
(2) HCT 116L was inoculated in 6-well plates overnight. Control group cells were given 1640 complete medium for culture, oxaliptin group cells were given 1640 complete medium containing 15. Mu.M Oxaliplatin (5% dextrose solution), and siRNA+Oxaliptin group cells were given 1640 complete medium containing siRNA STAT3 and 15. Mu.M Oxaliplatin (5% dextrose solution). After each group of cells is incubated for 24 hours, the V-FITC and PI are respectively and jointly stained for 15 minutes, the samples are subjected to flow cytometry detection, the cell membrane catenin V-FITC in early apoptosis is positive, the PI is negative, and the cells in late apoptosis are positive.
The results are shown in fig. 10 and 11, which demonstrate that oxaliplatin-resistant cell lines of colorectal cancer have an increased ability to inhibit tumor invasion by oxaliplatin after siRNA knockdown (fig. 10), and that drug-resistant lines have a decreased ability to resist apoptosis after knockdown (fig. 11).
Example 5
Evaluation was performed by a cell CCK-8 proliferation assay, a clonogenic assay, an apoptosis assay, and a mouse in vivo oncological assay using a STAT3 inhibitor (Compound LY 1).
(one), the inhibition of p-STAT3 by compound LY1 (0. Mu.M, 1. Mu.M, 2. Mu.M, 4. Mu.M) at gradient concentration was investigated by Western Blot (WB) Western Blot experiments, by reference to the "molecular cloning Experimental guidelines (third edition) (scientific Press), as follows:
(1) Preparation of 7.5% SDS-PAGE gel electrophoresis
Rapid preparation of the kit Using the Yase PAGE gel A7.5% SDS-PAGE gel was prepared and according to the kit instructions a 7.5% SDS-PAGE gel was prepared. And injecting 10 mu L of each prepared protein sample and 5 mu L of protein pre-dyeing Marker into the gel hole by using a 10 mu L pipetting gun, carrying out electrophoresis for 45min at 75V voltage, adjusting the voltage to 110V, and continuing electrophoresis until bromophenol blue completely goes out of the gel.
(2) Film transfer
Before electrophoresis is finished, a PVDF film and two pieces of thick filter paper boards which are almost the same as gel are sheared by scissors, the PVDF film is soaked in methanol for 1min for activation, and meanwhile, the filter paper boards and the PVDF film are soaked in 1X film transferring buffer solution for balancing for 5min. After electrophoresis, SDS-PAGE gel was carefully removed and the gel was equilibrated by immersing in 1 Xtransfer buffer for 5 minutes. The membrane was then placed on a semi-dry membrane transfer apparatus in the order anode-filter paper board-PVDF membrane-gel-filter paper board-cathode, carefully removed air bubbles, and transferred at 25V for 30min.
(3) Sealing
After the transfer, taking out the PVDF membrane, cutting out bands of molecular weight ranges of STAT3 (88 kDa), p-STAT3 (88 kDa) and GAPDH (36 kDa) according to a molecular weight range indicated by a protein pre-dyeing Marker, immersing the bands in a sealing liquid of 5% nonfat milk powder, sealing the bands on a horizontal shaking table at room temperature for 1h.
(4) Incubation of primary antibody
The blocking solution was removed by pipetting, and anti-E-cadherin and anti-vimentin monoclonal antibodies diluted 1000-fold with 5% goat serum blocking solution and 20000-fold diluted anti-GAPDH monoclonal antibodies were added to the strips, respectively, and incubated overnight on a horizontal shaker at 4 ℃.
(5) Incubation of secondary antibody
The used primary antibody was recovered by pipetting and stored at 4 ℃. The strip was washed 3 times for 10 minutes with 1 TBST. Goat anti-rabbit secondary antibodies (STAT 3, p-STAT3 and GAPDH) were labeled with 1000-fold horseradish peroxidase diluted with 1×tbst, respectively, and incubated in a horizontal shaker for 1h at room temperature.
(6) Color development
The secondary antibody was discarded and the strip was washed 3 times with 1 XTBE solution, 8min each time for the first three times and 15min for the last time. The strips were removed, and A, B drops of ECL chromogenic substrate premixed at a volume ratio of 1:1 were added dropwise to the strips and imaged with a gel imaging system (ChemiDoc MPTM Imaging System, BIO-RAD).
The results are shown in fig. 12, which shows that: compound LY1 has no inhibitory effect on total protein of STAT3, and can inhibit phosphorylation of STAT3 in a gradient.
Screening of the dose of Compound LY1, oxaliplatin
Cell viability assay: HCT 116L, HCT L cells were individually seeded in 96-well plates, 5000 cells per well, 1640 complete medium (containing 10% FBS and 1% PS) incubated for 24 hours; oxaliptin was diluted to 23, 24, 25, 26. Mu.M using 1640 complete medium to administer HCT 8L; oxaliptin was diluted to 13, 14, 15, 16. Mu.M using 1640 complete medium to administer HCT 116L; compound LY1 to 1, 1.5, 2, 2.5 μm were diluted with 1640 complete medium to administer HCT 116L and HCT8L, respectively. After 48h incubation with the drug-containing medium, cell viability was identified using the CCK8 assay. Absorbance was read at 490 nm.
As a result, see fig. 13, the cell viability of compound LY1 and oxaliplatin on HCT 116L, HCT L cells was selected to be around 80% as the subsequent experimental dose: compound LY1 was 2 μm, oxaliplatin was 15 μm (HCT 116L) and 25 μm (HCT 8L).
(III), cell viability measurement: HCT 116L, HCT L cells were individually seeded in 96-well plates, 5000 cells per well, 1640 complete medium (containing 10% FBS and 1% PS) incubated for 24 hours; oxaliptin (HCT 116L: 15. Mu.M, HCT 8L: 25. Mu.M) was diluted with 1640 complete medium, compound LY1 was diluted to 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40. Mu.M, and after incubation with medium containing both Oxaliptin and compound LY1 for 48h, viability was identified using CCK8 assay. Absorbance was read at 490 nm.
(IV), cell viability measurement: HCT 116L, HCT L cells were individually seeded in 96-well plates, 5000 cells per well, 1640 complete medium (containing 10% FBS and 1% PS) incubated for 24 hours; after compound LY1 was diluted to 2 μm using 1640 complete medium, oxaliplatin was diluted to 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7, 700 μm and viability was identified using CCK8 assay after 48h incubation in medium containing both compound LY1 and Oxaliplatin. Absorbance was read at 490 nm.
The results show that: IC of Compound LY1 against HCT 116L, HCT L cell line 50 4.44. Mu.M and 13.53. Mu.M respectively; after the compound LY1 was combined with Oxaliptin (HCT 116L: 15. Mu.M, HCT 8L: 25. Mu.M), IC was performed on HCT L, HCT L cells 50 Reduced to 1.34. Mu.M and 4.01. Mu.M, respectively; compound LY1 (2. Mu.M) was used in combination with gradient Oxaliptin, oxaliplatin versus HCT 116L IC 50 IC for HCT8L with a decrease in value from 52.19. Mu.M to 16.34. Mu.M 50 The value was reduced from 84.78. Mu.M to 20.91. Mu.M (FIG. 14). It was demonstrated that compound LY1 in combination with oxaliplatin significantly enhances the inhibition of colorectal cancer resistant cell lines by oxaliplatin compared to compound LY1 or oxaliplatin alone. And from the curve it is deduced that: the compound LY1 is combined with the Oxaliptin with different concentrations, and the ratio of the mass concentration of the compound LY1 to the mass concentration of the Oxaliptin is 1:2.
When compound LY1 (2 μm) and Oxaliplatin ((HCT 116L:15 μm, HCT 8L:25 μm)) were used in combination for HCT 116L and HCT8L, respectively, a decrease in the number of cells and an increase in apoptotic body production were observed in the combination as compared with Oxaliplatin alone, suggesting that compound LY1 could significantly enhance the killing power of Oxaliplatin against colorectal cancer Oxaliplatin-resistant strains (fig. 15).
Example 6
Evaluation was performed by a clonogenic assay, cell migration assay, using STAT3 inhibitor compound LY 1.
(1) Cloning experiments: cells were seeded in 6-well plates, 1000 cells per well, after 24 hours of culture, the cell culture medium was replaced with fresh 1640 complete medium (10% FBS, 1% PS) containing compound LY1 (2 μm), fresh 1640 complete medium containing Oxaliplatin (HCT 8l:25 μm), fresh 1640 complete medium containing the combination of compound LY1 and Oxaliplatin (HCT 8l:2 μm ly1+25 μm Oxaliplatin), and after 10 days of incubation with PBS, the medium was removed and washed twice with PBS, fixed with pre-chilled paraformaldehyde for 20 minutes, crystal violet stained for 10 minutes, the cells were washed, and counted.
(2) Cell migration experiments: cells (5X 10 per well) 5 ) Inoculating the plate in 6 holes, scraping with yellow tip when the cells reach 100% confluence, and cleaning twice to remove non-adhesive cells; 1640 complete medium containing compound LY1 (2. Mu.M), 1640 complete medium containing Oxaliptin (HCT 8L: 25. Mu.M) (10% FBS, 1% PS), 1640 complete medium containing a combination of compound LY1 and Oxaliplatin (HCT 8L: 2. Mu.M LY1+25. Mu.M Oxaliptin) were each administered, and the Control group (Control) was taken as 1640 complete medium, observed after 24 hours, and photographed by a fluorescent inverted microscope.
As shown in fig. 16, a significant decrease in the number of HCT8L cells after the combination of low concentration of compound LY1 with oxaliplatin compared to oxaliplatin alone suggests that compound LY1 can enhance the proliferation inhibitory effect of oxaliplatin on colorectal cancer oxaliplatin-resistant cell lines. As shown in fig. 17, in the scratch test, the mobility of HCT8L cells was significantly reduced after the combination of compound LY1 and oxaliplatin, as compared with oxaliplatin alone. It is suggested that low concentrations of compound LY1 can enhance the inhibitory effect of oxaliplatin on the migration and proliferation of colorectal cancer oxaliplatin-resistant strains.
Example 7
The effect of LY1 and Oxaliptin on colorectal cancer Oxaliplatin resistant cell lines was investigated using xenograft mouse models.
(1) Flow detection of apoptosis using annexin v and PI double staining: cells were seeded in 12-well plates, after 24h incubation in 1640 complete medium (10% FBS, 1% PS) were replaced with 1640 complete medium containing compound LY1 (2. Mu.M), 1640 complete medium containing Oxaliptin (HCT 116L: 15. Mu.M, HCT 8L: 25. Mu.M), and 1640 complete medium with a combination of both (HCT 116L: 2. Mu.M LY1+15. Mu.M Oxaliptin, HCT 8L: 2. Mu.M LY1+25. Mu.M Oxaliptin), respectively and after 24h incubation, V-FITC and PI were stained together for 15min, respectively, and samples were subjected to flow cytometry detection, with early apoptosis, with V-FITC positive, and PI negative, and late apoptosis cells were stained positive.
As shown in fig. 18, the apoptosis rates of HCT 116L and HCT8L after Oxaliplatin administration alone were 4.76% and 7%, respectively, and the apoptosis rates of HCT 116L and HCT8L after LY1 administration alone were 4.38% and 7.75%, respectively, and the apoptosis rates of HCT 116L and HCT8L after the same dose of Oxaliplatin was combined with low concentration LY1 (2 μm) were 18.93% and 28.5%, suggesting that compound LY1 can significantly improve the killing effect of Oxaliplatin on Oxaliplatin-resistant cell lines of colorectal cancer.
(2) Xenograft tumor mice, 4-6 week old BALB/c nu/nu nude mice, were inoculated subcutaneously in the left armpit with 1X 10 6 individual/HCT 8L cells only. The nude mice after inoculation were randomly divided into placebo group (0.9% physiological saline), STAT3 inhibitor group (compound LY1 administration dose: 5mg/kg,0.9% physiological saline formulation), oxaliptin group (Oxa administration dose: 10mg/kg,5% glucose solution formulation) and combination group (compound LY1 administration dose: 5mg/kg, oxa administration dose: 10 mg/kg), 5 animals per group, and the tumor growth was reached to 2X 2mm 3 After that, oxaliplatin tail was intravenously injected twice a week, compound LY1 was orally administered every other day, and the tumor size was measured. At the end of the experiment (day 21)Mice were sacrificed to separate tumor tissues, counted and weighed.
As shown in fig. 19, the tumor weight (fig. 19A) and tumor volume (fig. 19B) were significantly reduced in the combination group compared to the two separate groups, suggesting that compound LY1 was able to enhance the effect of oxaliplatin in vivo in inhibiting oxaliplatin-resistant colorectal cancer. As shown in fig. 20, animals were gradually reduced in weight over time following oxaliplatin alone, but the mice in the combination group were not different in weight fluctuation from the placebo group, suggesting that compound LY1 might be able to reduce oxaliplatin toxicity.
Claims (9)
- Use of STAT3 inhibitor in combination with oxaliplatin for the preparation of a medicament for the treatment of p-STAT3 activation or p-STAT3 low expression solid tumors.
- 2. The use according to claim 1, characterized in that: the p-STAT3 activated solid tumor is sensitive colorectal cancer.
- 3. The use according to claim 1, characterized in that: the solid tumor with low expression of the p-STAT3 is oxaliplatin-resistant colorectal cancer.
- Use of stat3 inhibitor in combination with oxaliplatin for the preparation of a medicament for the treatment of oxaliplatin-resistant colorectal cancer.
- 5. Use according to claim 1 or 4, characterized in that: the STAT3 inhibitor is a small molecular compound, an antibody or a nucleic acid medicament for blocking a JAK2/STAT3 signal pathway.
- 6. The use according to claim 5, characterized in that: the STAT3 inhibitor is selected from a compound LY1 with a structure shown in a formula I:
- 7. use according to claim 1 or 4, characterized in that: the ratio of the STAT3 inhibitor to the oxaliplatin is 1:2-1:10.
- 8. Use according to claim 1 or 4, characterized in that: the ratio of the STAT3 inhibitor to the oxaliplatin is 1:3-1:5.
- 9. A composition for treating colorectal cancer that is p-STAT3 activated or p-STAT3 underexpressed, characterized by: the composition comprises STAT3 inhibitor and oxaliplatin as main effective components.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311004597.2A CN116870162A (en) | 2023-08-10 | 2023-08-10 | Application of STAT3 inhibitor combined with oxaliplatin in preparation of medicament for treating colorectal cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311004597.2A CN116870162A (en) | 2023-08-10 | 2023-08-10 | Application of STAT3 inhibitor combined with oxaliplatin in preparation of medicament for treating colorectal cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116870162A true CN116870162A (en) | 2023-10-13 |
Family
ID=88266420
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311004597.2A Pending CN116870162A (en) | 2023-08-10 | 2023-08-10 | Application of STAT3 inhibitor combined with oxaliplatin in preparation of medicament for treating colorectal cancer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116870162A (en) |
-
2023
- 2023-08-10 CN CN202311004597.2A patent/CN116870162A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Down-regulating IL-6/GP130 targets improved the anti-tumor effects of 5-fluorouracil in colon cancer | |
| Gao et al. | Long non‑coding RNA FER1L4 inhibits cell proliferation and metastasis through regulation of the PI3K/AKT signaling pathway in lung cancer cells | |
| Zeng et al. | Dasatinib ameliorates chronic pancreatitis induced by caerulein via anti-fibrotic and anti-inflammatory mechanism | |
| Min et al. | Astragaloside IV inhibits the progression of liver cancer by modulating macrophage polarization through the TLR4/NF-κB/STAT3 signaling pathway | |
| Zhang et al. | TIPE2 suppresses the tumorigenesis, growth and metastasis of breast cancer via inhibition of the AKT and p38 signaling pathways | |
| Wei et al. | Low expression of TUG1 promotes cisplatin sensitivity in cervical cancer by activating the MAPK pathway | |
| Kim et al. | The coffee diterpene kahweol inhibits metastasis by modulating expressions of MMPs and VEGF via STAT3 inactivation | |
| Liang et al. | RASSF6-mediated inhibition of Mcl-1 through JNK activation improves the anti-tumor effects of sorafenib in renal cell carcinoma | |
| Huang et al. | Metformin and MiR-365 synergistically promote the apoptosis of gastric cancer cells via MiR-365-PTEN-AMPK axis | |
| Zhang et al. | Epigallocatechin-3-gallate inhibits the formation of neutrophil extracellular traps and suppresses the migration and invasion of colon cancer cells by regulating STAT3/CXCL8 pathway | |
| WO2021223713A1 (en) | Application of sms2 inhibitor in preparation of drug for treatment of highly invasive breast cancer | |
| US20210263039A1 (en) | Application of niemann-pick c1 protein in diagnosis and treatment of cancer | |
| Paradziej-Łukowicz et al. | Anticancer imidazoacridinone C-1311 inhibits hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and angiogenesis | |
| CN112891354B (en) | Application of MDM2 inhibitor Nutlin-3a in preparation of medicine for activating cancer cell apoptosis induced by endoplasmic reticulum stress | |
| CN116870162A (en) | Application of STAT3 inhibitor combined with oxaliplatin in preparation of medicament for treating colorectal cancer | |
| CN109260197B (en) | Application of indirubin compound and bortezomib in preparation of medicine for treating multiple myeloma | |
| CN113440519A (en) | Application of mycophenolic acid and derivatives thereof in preparation of drugs for targeted therapy of cancers | |
| CN107529558B (en) | New application of polynucleotide-5 'kinase-3' phosphatase | |
| WO2019184306A1 (en) | Target for drug treatment of tumor metastasis and use thereof | |
| KR20160093637A (en) | Methods of determining interferon having direct inhibitory effects on tumors and uses thereof | |
| CN111419832A (en) | Pharmaceutical composition and application thereof in preparing medicines for treating tumors | |
| Han et al. | STAT3 inhibitor enhances chemotherapy drug efficacy by modulating mucin 1 expression in non-small cell lung carcinoma | |
| KR102249814B1 (en) | Composition for inhibiting cancer metastasis comprising kalkitoxin thioamide alcohol or salt thereof as an active ingredient | |
| Lan et al. | Yulangsan polysaccharide inhibits epithelial-mesenchymal transition and invasion in NSCLC by attenuating the TGF-β1/ERK signaling pathway | |
| KR101950386B1 (en) | Pharmaceutical composition comprising 15-keto prostaglandin e2 for treatment of cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |