CN115414353A - Application of brucesin D in inhibiting CAFs (adenosine triphosphate) triple negative breast cancer metastasis under TNF (tumor necrosis factor) -alpha action - Google Patents
Application of brucesin D in inhibiting CAFs (adenosine triphosphate) triple negative breast cancer metastasis under TNF (tumor necrosis factor) -alpha action Download PDFInfo
- Publication number
- CN115414353A CN115414353A CN202210620484.4A CN202210620484A CN115414353A CN 115414353 A CN115414353 A CN 115414353A CN 202210620484 A CN202210620484 A CN 202210620484A CN 115414353 A CN115414353 A CN 115414353A
- Authority
- CN
- China
- Prior art keywords
- breast cancer
- metastasis
- cafs
- brucesin
- alpha
- 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
- 206010027476 Metastases Diseases 0.000 title claims abstract description 61
- 230000009401 metastasis Effects 0.000 title claims abstract description 57
- 108060008682 Tumor Necrosis Factor Proteins 0.000 title claims abstract description 42
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 title claims abstract description 24
- 208000022679 triple-negative breast carcinoma Diseases 0.000 title claims abstract description 24
- 230000009471 action Effects 0.000 title claims abstract description 14
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 14
- 102000003390 tumor necrosis factor Human genes 0.000 title description 4
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 title 1
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 title 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims abstract description 38
- 206010006187 Breast cancer Diseases 0.000 claims abstract description 35
- 208000026310 Breast neoplasm Diseases 0.000 claims abstract description 35
- 239000003814 drug Substances 0.000 claims abstract description 26
- 230000009545 invasion Effects 0.000 claims abstract description 16
- 210000004072 lung Anatomy 0.000 claims abstract description 12
- 210000004185 liver Anatomy 0.000 claims abstract description 10
- 210000000481 breast Anatomy 0.000 claims abstract description 8
- 230000008595 infiltration Effects 0.000 claims abstract description 8
- 238000001764 infiltration Methods 0.000 claims abstract description 8
- 208000030776 invasive breast carcinoma Diseases 0.000 claims abstract description 7
- 206010055113 Breast cancer metastatic Diseases 0.000 claims abstract description 6
- 230000001737 promoting effect Effects 0.000 claims abstract description 5
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims abstract 5
- 230000014509 gene expression Effects 0.000 claims description 20
- 238000013508 migration Methods 0.000 claims description 12
- 229940079593 drug Drugs 0.000 claims description 11
- 230000005012 migration Effects 0.000 claims description 11
- 210000002966 serum Anatomy 0.000 claims description 11
- 108010035532 Collagen Proteins 0.000 claims description 10
- 102000008186 Collagen Human genes 0.000 claims description 10
- 229920001436 collagen Polymers 0.000 claims description 10
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 claims description 9
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 claims description 9
- 102100026802 72 kDa type IV collagenase Human genes 0.000 claims description 8
- 101000627872 Homo sapiens 72 kDa type IV collagenase Proteins 0.000 claims description 8
- 239000003550 marker Substances 0.000 claims description 8
- 208000020816 lung neoplasm Diseases 0.000 claims description 7
- 208000037841 lung tumor Diseases 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 206010019695 Hepatic neoplasm Diseases 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 208000014018 liver neoplasm Diseases 0.000 claims description 6
- 230000006907 apoptotic process Effects 0.000 claims description 5
- 230000012010 growth Effects 0.000 claims description 5
- 230000002685 pulmonary effect Effects 0.000 claims description 5
- 230000002440 hepatic effect Effects 0.000 claims description 2
- 230000001394 metastastic effect Effects 0.000 claims 1
- 206010061289 metastatic neoplasm Diseases 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 64
- 230000000694 effects Effects 0.000 abstract description 32
- 238000003501 co-culture Methods 0.000 abstract description 8
- 238000011161 development Methods 0.000 abstract description 4
- 230000002269 spontaneous effect Effects 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract description 3
- 238000001727 in vivo Methods 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- 241000699670 Mus sp. Species 0.000 description 52
- 210000004027 cell Anatomy 0.000 description 50
- 210000001519 tissue Anatomy 0.000 description 34
- 241000699666 Mus <mouse, genus> Species 0.000 description 31
- 238000002474 experimental method Methods 0.000 description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 238000005406 washing Methods 0.000 description 15
- 238000011065 in-situ storage Methods 0.000 description 14
- 230000002829 reductive effect Effects 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 241001533159 Brucea javanica Species 0.000 description 12
- 235000010889 Rhus javanica Nutrition 0.000 description 12
- 239000003153 chemical reaction reagent Substances 0.000 description 12
- 239000001963 growth medium Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 9
- RRKTZKIUPZVBMF-IBTVXLQLSA-N brucine Chemical compound O([C@@H]1[C@H]([C@H]2C3)[C@@H]4N(C(C1)=O)C=1C=C(C(=CC=11)OC)OC)CC=C2CN2[C@@H]3[C@]41CC2 RRKTZKIUPZVBMF-IBTVXLQLSA-N 0.000 description 9
- RRKTZKIUPZVBMF-UHFFFAOYSA-N brucine Natural products C1=2C=C(OC)C(OC)=CC=2N(C(C2)=O)C3C(C4C5)C2OCC=C4CN2C5C31CC2 RRKTZKIUPZVBMF-UHFFFAOYSA-N 0.000 description 9
- 229960003668 docetaxel Drugs 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 206010006500 Brucellosis Diseases 0.000 description 8
- 235000019441 ethanol Nutrition 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 238000011160 research Methods 0.000 description 8
- 210000004881 tumor cell Anatomy 0.000 description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 5
- 239000006285 cell suspension Substances 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- 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 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 238000005138 cryopreservation Methods 0.000 description 4
- 235000005911 diet Nutrition 0.000 description 4
- 230000037213 diet Effects 0.000 description 4
- 235000020188 drinking water Nutrition 0.000 description 4
- 239000003651 drinking water Substances 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 238000004393 prognosis Methods 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 239000008096 xylene Substances 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 230000012292 cell migration Effects 0.000 description 3
- 210000003855 cell nucleus Anatomy 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 108010082117 matrigel Proteins 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000000465 moulding Methods 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 241000158621 Brucea Species 0.000 description 2
- OQWSVCXXAYKEFF-UHFFFAOYSA-N Brucin Natural products COc1cc2N3C4C5C(CC3=O)OC=CC6CN7CCC4(C7CC56)c2cc1OC OQWSVCXXAYKEFF-UHFFFAOYSA-N 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 206010027457 Metastases to liver Diseases 0.000 description 2
- 206010027458 Metastases to lung Diseases 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000004709 cell invasion Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 208000037976 chronic inflammation Diseases 0.000 description 2
- 230000006020 chronic inflammation Effects 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 210000005075 mammary gland Anatomy 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000010827 pathological analysis Methods 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 210000002536 stromal cell Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 241000283707 Capra Species 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 101000851181 Homo sapiens Epidermal growth factor receptor Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 1
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 241001093962 Simaroubaceae Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 229940045799 anthracyclines and related substance Drugs 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000000729 antidote Substances 0.000 description 1
- 229940075522 antidotes Drugs 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000004791 biological behavior Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 238000010523 cascade reaction Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 102000015694 estrogen receptors Human genes 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000000556 factor analysis Methods 0.000 description 1
- 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 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 238000007489 histopathology method Methods 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 150000003057 platinum Chemical class 0.000 description 1
- 239000000583 progesterone congener Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- IOSXSVZRTUWBHC-UHFFFAOYSA-N quassin Natural products C1C(C23C)OC(=O)CC3C(C)=C(OC)C(=O)C2C2(C)C1C(C)C=C(OC)C2=O IOSXSVZRTUWBHC-UHFFFAOYSA-N 0.000 description 1
- -1 quassin lactone compounds Chemical class 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 102000037983 regulatory factors Human genes 0.000 description 1
- 108091008025 regulatory factors Proteins 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 238000013424 sirius red staining Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 210000004981 tumor-associated macrophage Anatomy 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 230000029663 wound healing Effects 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
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Oncology (AREA)
- Epidemiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses application of brucesin D in promoting triple negative breast cancer metastasis by CAFs under the action of inhibiting TNF-alpha. The invention provides an application of brucesin D in preparing a medicament for preventing and/or treating invasive/metastatic breast cancer, wherein the invasive/metastatic breast cancer comprises local infiltration and remote metastasis of the breast cancer; local infiltration and remote metastasis of breast cancer are invasion and metastasis of breast cancer cells promoted by CAFs under the action of TNF-alpha; the distant metastasis is liver metastasis or lung metastasis. The invention adopts a co-culture system of TNF-alpha stimulated CAFs and triple negative breast cancer cells to construct an in vitro co-culture triple negative breast cancer cell metastasis promotion model and a mouse spontaneous breast 'in situ-lung/liver' tumor metastasis model under in vivo CAFs/4T1 co-culture, and explores the effect of brucesin D in inhibiting CAFs to promote invasion and metastasis of triple negative breast cancer. The invention provides experimental data support for the effective prevention and treatment of local infiltration and remote metastasis of the triple negative breast cancer by the brucesin D and the development of the medicine for preventing and treating the triple negative breast cancer.
Description
Technical Field
The invention relates to an application of brucesin D in promoting triple negative breast cancer metastasis by CAFs under the action of inhibiting TNF-alpha, belonging to the technical field of tumor treatment drug development.
Background
The breast cancer is one of high-risk malignant tumors of women all over the world, and the mortality rate of the breast cancer is the first malignant tumor of women all over the world. Triple-negative Breast Cancer (TNBC) lacking estrogen receptors, progestogen receptors and human epidermal growth factor receptors 2 is one of the most aggressive subtypes of Breast Cancer, and because of the clinical characteristics of easy early transfer, easy relapse after operation and the like, the prognosis of Breast Cancer subtype patients is poor, and the mortality is high. Although some TNBC patients are sensitive to chemotherapy schemes mainly comprising anthracyclines, taxoids and platinum complexes, some patients still have poor clinical treatment responsiveness due to the complexity of the pathological characteristics of tumors, and are prone to various toxic and side effects and drug resistance phenomena, so that the survival and prognosis of the patients are seriously affected. Research shows that distant organ metastasis is an important reason for the treatment and prognosis of TNBC patients. Therefore, how to control the metastasis of the tumor cells with the strong invasive breast cancer subtype so as to improve the prognosis of the TNBC patient is a difficult problem to be solved clinically.
Recent studies have demonstrated that tumor progression is significantly regulated by Tumor Microenvironment (TME) and does not depend entirely on autonomous deficiency of tumor cells. Tumor-associated fibroblasts (CAFs) are used as antidotes for "tumor wound healing" and are mainly activated or transdifferentiated from normal fibroblasts or mesenchymal stem cells in the microenvironment of chronic tissue injury. The CAFs are used as a main matrix cell component in a tumor microenvironment, regulate the biological behaviors of tumor cells and other matrix cells through intercellular contact, release a large number of regulatory factors, synthesize and remodel extracellular matrix, and therefore influence the occurrence and development of tumors.
Studies have shown that chronic inflammation is considered an important biological feature of malignancies. Tumor necrosis factor-alpha (TNF-alpha) is an important proinflammatory cytokine found in the microenvironment of breast cancer, participates in various stages of tumor development and plays an important role in tumor metastasis, and can be secreted by various cells in TME, such as tumor-associated macrophages, stromal cells, cancer cells and the like, so as to induce cascade reactions of other inflammatory cytokines and chemokines and expand the generation of chronic inflammation in the tumor microenvironment. Clinical experiments show that the content of TNF-alpha in the serum of a breast cancer patient is positively correlated with clinical stages. And researches show that TNF-alpha in the tumor microenvironment can enhance the interactive conversation between stromal cells and tumor cells and accelerate the proliferation and the remote metastasis of the tumor cells.
The brucea javanica is a mature fruit of brucea javanica of Simaroubaceae, mainly produced in two regions with different natures, is bitter and cold in nature, and has the effects of clearing heat and removing toxicity, preventing malaria, stopping dysentery, corroding proud warts and the like. Brucine D belongs to quassin lactone compounds, which are marked secondary metabolites of brucea plants and are also main compounds in brucea. Researches show that the brucesin D can play an anticancer role in ways of promoting cancer cell apoptosis, reducing intracellular glutathione level, hindering inflammation cascade amplification, inhibiting cancer cell invasion and migration and the like. However, whether brucellolicin D can weaken the tumor cell metastasis promotion effect of CAFs under the action of inflammatory mediators TNF-alpha and inhibit the metastasis effect of mouse orthotopic tumors to distant organs (lung/liver) has not been reported, so that the research on the application of brucellolicin D is necessary.
Disclosure of Invention
The invention aims to provide brucesin D for inhibiting CAFs promotes triple negative breast cancer cell metastasis under the action of TNF-alpha and inhibiting spontaneous breast orthotopic-lung/liver tumor metastasis of mice.
The invention firstly provides the application of brucesin D in preparing the medicine for preventing and/or treating breast cancer.
The invention also provides application of the brucesin D in preparing a medicament for preventing and/or treating invasive/metastatic breast cancer.
The breast cancer is triple negative breast cancer;
the invasive/metastatic breast cancer comprises local infiltration and distant metastasis of breast cancer;
the local infiltration and the remote metastasis of the breast cancer are the invasion and metastasis of breast cancer cells promoted by CAFs under the action of TNF-alpha;
the breast cancer cell is MDA-MB-231 or 4T1;
the distant metastasis is hepatic metastasis;
the distant metastasis is pulmonary metastasis.
The invention further provides the application of the brucesin D in preparing the medicine for preventing and/or treating the breast cancer metastasis;
the breast cancer metastasis is a liver metastasis and/or a lung metastasis.
The application of the brucine D in any one of the following substances also belongs to the protection scope of the invention:
1) Preparing a medicament for inhibiting the growth of breast cancer cells and promoting the apoptosis of the breast cancer cells;
2) Preparing a medicament for inhibiting invasion and migration of breast cancer cells;
3) Drugs that inhibit the formation of mammary orthotopic-lung tumor metastases;
4) Drugs that inhibit the formation of breast orthotopic-hepatic tumor metastases;
5) A drug for reducing the content of TNF-alpha in serum;
6) A drug for reducing the content of TNF-alpha protein in breast tumor tissues and the expression of a CAFs marker alpha-SMA;
7) A medicament for reducing the expression of MMP2, MMP9 and collagen in breast tumor tissues.
The invention adopts a co-culture system of TNF-alpha stimulated CAFs and triple negative breast cancer cells (MDA-MB-231 and 4T 1), constructs an in vitro co-culture triple negative breast cancer cell metastasis promotion model and a mouse spontaneous breast 'in situ-lung/liver' tumor metastasis model under in vivo CAFs/4T1 co-culture, and explores the effect of brucellosis D on inhibiting invasion and metastasis of the CAFs triple negative breast cancer. The invention provides experimental data support for the effective prevention and treatment of local infiltration and remote metastasis of the triple negative breast cancer by the brucesin D and the development of the medicine for preventing and treating the triple negative breast cancer.
Drawings
FIG. 1 is a graph of the effect of brucine D on the growth of MDA-MB-231 and 4T1 cells.
FIG. 2 shows the effect of Brucesin D (BD) on the migration and invasion capacity of "CAFs + MDA-MB-231/CAFs +4T1" co-cultured cells under the action of TNF- α.
FIG. 3 shows the effect of brucesin D on mouse orthotopic tumors, wherein FIG. 3A shows the effect of brucesin D on mouse orthotopic tumors ((C))
n = 6); FIG. 3B is a graph of the effect of brucesin D on tumor size in situ in mice (
n = 6); FIG. 3C is a pathological analysis of brucine D on mouse orthotopic tumors (
n = 3); FIG. 3D: effect of brucin D on Ki67 protein of orthotopic tumor in mice: (
n＝3)。
FIG. 4 shows the effect of brucesin D on lung/liver metastasis in mice, wherein FIGS. 4A and 4B show the effect of brucesin D on pulmonary metastasis nodules in mice (B)
n = 6); FIG. 4C is a graph of the effect of brucesin D on lung/liver metastases in mice.
FIG. 5 shows the effect of brucesin D on TNF- α content in mouse serum (
n＝6)。
FIG. 6 shows the effect of brucesin D on TNF- α and CAFs marker expression in mouse orthotopic tumor tissues, wherein FIG. 6A shows the effect of brucesin D on TNF- α in mouse orthotopic tumor tissues (C:)
n = 3); FIG. 6B is a drawing of brucine D binding to mouse orthotopic tumor tissueEffect of the CAFs marker α -SMA (
n = 3); FIG. 6C shows the effect of brucesin D on co-localization expression of TNF- α and CAFs marker α -SMA in mouse orthotopic tumor tissue (
n＝3)。
FIG. 7 shows the effect of brucellosis D on MMP2, MMP9 and collagen expression in mouse orthotopic tumor tissues, wherein FIG. 7A shows the effect of brucellosis D on MMP2 and MMP9 secreted by CAFs in mouse orthotopic tumor tissues: (
n = 3); FIG. 7B shows the effect of brucesin D on the secretion of collagen by CAFs in mouse orthotopic tumor tissues (
n＝3)。
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 study of proliferation, invasion and migration capabilities of Coultured cells of "CAFs + MDA-MB-231/4T1" under the action of brucesin D in inhibiting TNF-alpha
1. Experimental methods
1.1 preparation of experiment reagent and reagent
1.1.1 cell culture Medium containing 15% FBS
42.5mL of DMEM/F12 medium is added into a 50mL sterile centrifuge tube, then 7.5mL of LFBS and 500. Mu.L of a mixture of penicillin and streptomycin are added into the sterile centrifuge tube, and the mixture is uniformly mixed and stored at 4 ℃ for later use.
1.1.2 cell culture Medium containing 10% FBS
45mL of DMEM culture medium is added into a 50mL sterile centrifuge tube, then 5mL of FBS and 500 mu L of mixed solution of penicillin and streptomycin are added into the sterile centrifuge tube, and the mixture is uniformly mixed and stored at 4 ℃ for later use.
1.1.3 brucea javanica picrin D stock solution
Precisely weighing 5.47mg brucea javanica picrin D, dissolving in 20 μ L DMSO to prepare 667mM/L stock solution, and storing at-20 deg.C for use.
1.1.4 TNF-alpha solution
Centrifuging 10 mu g of TNF-alpha (10000 rpm/min,1 min), adding 100 mu L of dissolving solution into an ultra-clean workbench for natural dissolution, adding 900 mu L of diluent (PBS containing 5% trehalose), uniformly mixing to obtain 10 mu g/mL of TNF-alpha solution, and storing at-20 ℃ for later use without repeated freeze thawing.
1.2 Experimental cell culture
1.2.1 recovery of cell lines
MDA-MB-231 cells and 4T1 cells were purchased from the cell bank of the Committee for type culture Collection of the national academy of sciences. The frozen MDA-MB-231 or 4T1 cells were removed from the liquid nitrogen tank and rapidly shaken in a water bath at 37 ℃ to rapidly dissolve them. Adding 2mL of serum-containing culture medium into 4mL centrifuge tube, transferring cells into the centrifuge tube, blowing, mixing, centrifuging (1000 rpm/min, 5 min), removing supernatant, adding fresh culture medium, blowing to obtain cell suspension, and inoculating to 100mm 3 In the culture dish, 37 ℃ and 5% of CO 2 The incubator of (2).
1.2.1 culture and passage of cell lines
After the recovered cell strain grows for 1-2 days, the original culture medium is discarded, and 1mL of PBS is added into the culture medium after being washed twice. When the cell growth density reached 85%, 1mL of PBS was washed twice and 1mL of trypsin was added to digest the cells. When observing that the cell antenna shrinks, the shape becomes round, the gap is enlarged and the single cells are scattered under a microscope, immediately adding 2mL of fresh culture medium to stop digestion, blowing the bottom of a culture dish by using a pipette to ensure that the cells are completely fallen off, collecting cell suspension in a 4mL sterile centrifuge tube, centrifuging (1000 rpm/min and 5 min), removing supernatant, adding the fresh culture medium to blow and beat into cell suspension, inoculating according to the density required by the experiment, and replacing the fresh culture medium for 2-3 times every week.
1.2.3 cryopreservation of cell lines
Digesting and centrifuging the mixture, collecting cells, adding 1mL of prepared cryopreservation liquid, uniformly mixing the mixture, transferring the mixture into a sterile cryopreservation tube, marking the cell name and the cryopreservation date, sealing the tube by a sealing film, standing in a gradient cooling box at-80 ℃ overnight, and transferring the tube into a liquid nitrogen tank for long-term storage.
1.3MTT assay to determine the Effect of brucine D on MDA-MB-231 and 4T1 cell inhibition
Cell suspensions of MDA-MB-231 and 4T1 in logarithmic growth phase were taken and adjusted to a concentration of 1X10 5 Perml, 100. Mu.L of cell suspension per well and 200. Mu.L of PBS per peripheral well were added to the assay wells set in the 96-well plate. When the cells grow to a proper density, removing the original culture medium, adding brucesin D with different concentrations to treat the cells for 24 hours, 48 hours and 72 hours respectively, removing the drug-containing culture medium, adding 100 mu L of serum-free culture medium and 10 mu L of MTT solution into each hole, placing the holes in an incubator for 4 hours, absorbing the culture solution, adding 150 mu L of DMSO solution into each hole, shaking the holes in an enzyme-linked immunosorbent assay at 37 ℃ for 10 minutes, measuring the OD value at 490nm, and repeating the experiment for 3 times (cell inhibition rate%) =1- (experiment group OD value-blank group OD value)/(control group OD value-blank group OD value).
1.4 migration and invasion experiments to test the influence of brucine D on the migration and invasion abilities of "CAFs + MDA-MB-231/CAFs +4T1" co-cultured cells under the action of TNF-alpha
To a 24-well plate was added 600. Mu.L of a medium containing 10% FBS (DMEM: DMEM/F12= 3. In the Transwell migration experiment, the Transwell chamber was placed on top and 200. Mu.L of TNF-. Alpha.affected tumor cells were co-cultured with CAFs at a density of 1X10 (ratio 10 5 one/mL of the cells were added to a Transwell chamber and placed in an incubator for 24 hours. After the culture is finished, pouring out the culture solution in the upper chamber, gently wiping off the cells in the upper chamber by using a cotton swab, placing the small chamber in 4% paraformaldehyde for fixation for 15min, washing twice by using PBS, and carrying out crystal violet staining for 20min. After staining was completed, excess staining solution was washed out with PBS, migrated cells were photographed at the bottom of the Transwell chamber under a microscope, and the number of migrated cells was counted with Image J software. In the Transwell invasion test, 100. Mu.L of matrigel gel (matrigel: medium =1 = 30) was coated on the upper part of a Transwell chamber in advance, placed in an incubator 12h, and then aspiratedThe unset matrigel, the subsequent operation with Transwell migration experiment, each experiment repeated 3 times.
2. Results of the experiment
All data were analyzed using SPSS 24.0 software. The data measurement results are all as follows
It is shown that the multiple sample data comparison is performed with the normality and homogeneity of variance tests, both of which are in accordance with the t test or the one-factor analysis of variance. If the normality or homogeneity of variance is not met, the statistical difference is considered to be present by using Kruskal-Wallis rank sum test with a =0.05 as the test level and P < 0.05.
2.1 Effect of brucin D on MDA-MB-231 and 4T1 cell growth
The results show that the inhibition rate of BD on MDA-MB-231 and 4T1 cells is gradually increased along with the increase of the BD concentration, and is time-dose dependent. IC50 values of each group were calculated from Graphpad prism 8.0, and the results showed that the IC50 values of BD on MDA-MB-231 cells were 37.883. Mu.M (24 h), 8.146. Mu.M (48 h), and 2.622. Mu.M (72 h), respectively; the IC50 values of BD on 4T1 cells were 59.305. Mu.M (24 h), 36.264. Mu.M (48 h), 22.637. Mu.M (72 h), respectively. The results are shown in FIG. 1.
2.2 Effect of brucesin D on Co-culture cell migration and Capacity invasion of "CAFs + MDA-MB-231/CAFs +4T1" under TNF-alpha
The results show that the number of migrating and invading cells decreased (P < 0.05) after administration of different concentrations of brucesin D in a co-cultured cell model of "CAFs + MDA-MB-231" under the action of TNF-alpha. The results are shown in FIG. 2.
3. Conclusion
In a cytotoxicity experiment, the inhibition rate of brucesin D on MDA-MB-231 and 4T1 cells is time-dose dependent. According to the guiding principle of the pharmacodynamics of the antitumor drug, when the drug acts with human cancer cell strains for 48 to 72 hours, the IC50 is less than 20 mug/mL, which shows that the antitumor drug has antitumor research value. According to the standard, brucesin D has certain research value.
In cell migration and invasion experiments, brucesin D inhibits migration and capacity invasion of 'CAFs + MDA-MB-231/CAFs +4T 1' co-culture cells under the action of TNF-alpha, and the number of the migration and invasion cells is reduced along with the increase of concentration.
Although the in vitro cytotoxicity and migration invasion experiments can quickly and economically obtain the anti-tumor research value of brucesin D, the influence on tumor cells under the whole environment is lacked, so that a BALB/C mouse 'breast in situ-lung/liver' tumor metastasis model is established, and the anti-triple negative breast cancer tumor metastasis characteristic and the research value of brucesin D are determined.
Example 2 brucesin D inhibits mouse "Breast in situ-Lung/liver" tumor metastasis
1. Experimental materials and methods
1.1 Experimental animals
50 BALB/C female mice, 4-6 weeks old, were purchased, weighing 16-18 g, and all animals were housed in the Ningxia university of medicine animal center SPF-grade barrier (ethical # IACUC-NYLAC-2021-158).
1.2 preparation of the test reagents
1.2.1 preparation of brucea javanica picrin D stock solution
Accurately weighing 8.00mg of brucea javanica picrin D, dissolving in 4mL of physiological saline, shaking for dissolving, and preparing for use.
1.2.2 preparation of brucea javanica picrin D working solution
As shown in table 1.
TABLE 1 brucea javanica bitter D working solution
1.2.3 preparation of docetaxel solution
Taking the docetaxel injection out of a refrigerator, standing for 5min to room temperature. 2mL of solvent was poured into the solution and mixed by hand inversion for 1min without shaking. 0.24mL of the extract is added into 20mL of physiological saline to be mixed evenly and is ready to use.
1.3 Experimental reagents and materials
1.3.1 Primary reagents and materials
TABLE 2 Main reagents and materials
1.3.2 antibodies
TABLE 3 antibodies
1.4 establishment of spontaneous mammary gland in situ-lung metastasis model of BALB/C mice
Resuscitating and culturing 4T1 cells and 4T1 and CAFs co-cultured cells (4T 1/CAFs is 10 6 0.1mL of the cells were inoculated into the penultimate pair of mammary gland fat pads of BALB/C mice for molding. All mice developed tumors after 10 days and were randomized and administered intraperitoneally according to the dosing schedule. (1) normal group: no treatment is carried out; (2) 4T1 group: after molding, giving physiological saline/2 d; (3) 4T1+ CAFs group: after molding, giving normal saline/2 days; (4) Docetaxel group (Docetaxel): 3mg/kg/2d; (5) brucea javanica picrin D1:1.5mg/kg/2d; (6) brucea javanica picrin D2:3mg/kg/2d. During the administration period, the mice were observed daily for drinking water, hair and activity, and the weight change of the mice was recorded. The experiment was terminated 24 days after administration, and each group of mice was sacrificed by taking blood from the eyeballs and removing the necks, and tumor tissues of the mice were peeled off, weighed after photographing, and fixed in 4% paraformaldehyde for the subsequent experiment.
1.5H & E staining
After the tissue was soaked in 4% paraformaldehyde for 24h, the gradient alcohol was sequentially dehydrated and cleared in xylene, followed by preparation of paraffin sections. Drying the paraffin sections in a 70 ℃ oven for 2h, respectively drying the paraffin sections with dimethylbenzene I and II for 20min, respectively drying with absolute ethyl alcohol, 95% and 90% ethyl alcohol for 5min, respectively drying with 80% and 70% ethyl alcohol for 3min, respectively washing with flowing water, then washing with flowing water, differentiating with hydrochloric acid and alcohol for several seconds, then washing with eosin for 5min, and moving up with alcohol to dehydrate and seal the sections.
1.6 immunohistochemistry
After conventional dewaxing and rehydration, the slices are placed in a sodium citrate buffer solution to be boiled for 10min, and tissues are circled by using an immunohistochemical pen. Reagent one was added dropwise, incubated at 37 ℃ for 10min, washed with PBS 2 min. Times.3 times. Goat serum was added dropwise, incubated at 37 ℃ for 20min and washed slightly. Primary antibody incubation, 4 ℃ overnight, PBS washing 2min x 3 times. Adding reagent II dropwise, incubating at 37 deg.C for 20min, washing with PBS for 2min × 3 times. Adding reagent III dropwise, incubating at 37 deg.C for 20min, washing with PBS for 2min × 3 times. DAB color development, flushing with tap water, dripping hematoxylin staining solution on the tissue, adding ethanol to xylene, and sealing.
1.7Masson staining
Slices were soaked overnight in Masson A solution after conventional deparaffinization and rehydration. Sections were then incubated in Masson A for 30min at 65 ℃ and washed with water for 30s. The sections were dip-stained for 1min in a mixture (Masson B: massonC volume ratio 1, ready for use) and washed slightly with running water. The slices were differentiated by 1% hydrochloric acid alcohol for 1min, washed with tap water, and stained with Masson D for 6min. After washing with water, the mixture is soaked in Masson E for 1-2min and directly dyed in Masson F liquid for 30s without washing with water. Rinsing the slices with 3 continuous cylinders of 1% glacial acetic acid, dehydrating with 3 continuous cylinders of anhydrous ethanol for 8s for 5s, 10s and 30s, dehydrating with two cylinders of n-butanol for 30s and 2min, removing xylene, and sealing.
1.8Sirius Red Staining
After conventional dewaxing and rehydration, the Tianlang chimpanzee is stained by liquid drop staining for 1h. Washing with flowing water for a while, if necessary, washing with 0.5% glacial acetic acid for 1-2 times. And (3) dyeing with hematoxylin liquid drops for 10min, then washing with running water for 10min, dehydrating and sealing with neutral resin.
1.9 immunofluorescence assay
And (3) after conventional dewaxing and rehydration, putting the tissue slices into heated EDTA antigen repairing liquid for treatment for 20min, naturally cooling, putting the tissue slices on a shaking table, washing the tissue slices for 5min by 3 times by using PBS, and drawing circles by using a composition pen after the tissue slices are slightly dried. BSA was added to the tissue and incubated for 30min. The first antibody was added overnight in a wet box at 4 ℃. Corresponding secondary antibody was added and incubated at room temperature for 50min. After PBS is washed, CY3 reagent is dripped, the tissue slices are incubated for 15min at room temperature in a dark place, and then the tissue slices are placed in EDTA antigen repairing liquid, heated for 20min in a microwave oven, naturally cooled and then placed on a shaking bed to be washed by PBS. And (3) dropwise adding a second primary antibody and a second secondary antibody: the same as the above-mentioned primary antibody method. Adding FITC reagent dropwise, incubating for 15min at room temperature in dark place, and washing with TBST for 5min on a shaking bed. And (3) dropwise adding an autofluorescence quenching agent for 5min, washing with running water for 10min, and soaking the wafer in deionized water for 10min. And (3) dehydrating: after the cell nuclei were counterstained with DAPI by ascending ethanol to xylene (same as above), the cell nuclei were washed with PBS and after slight drying, the cell nuclei were mounted on a mounting plate with an anti-fluorescence quenching mounting plate agent for photographing.
1.10ELISA assay
Blood is taken from the orbit of the mouse, and serum is taken and placed in a refrigerator at the temperature of 20 ℃ below zero for standby after centrifugation at 12000r/min for 5min. TNF-alpha detection was performed according to the ELISA kit instructions.
2. Results of the experiment
The statistical method is as above.
2.1 Effect of brucesin D on general State and weight changes in mice
All mice were successfully modeled and dosed. During the experiment, the mice in the normal group drink water normally, the state is good, and the weight is steadily increased. Mice in groups 4T1 and 4T1+ CAF do not die during the experiment, the early mice have normal active diet and hair, the mice have reduced diet drinking water with tumor enlargement, slow reaction, disordered hair and no desire to lie asleep in the later period, and the mice in the group 4T1 and 4T1+ CAF appear earlier. No death and no obvious change of body weight were observed in the mice in the Docetaxel group during the experiment, and the water and hair of the other mice were normal except for the abnormality of individual mice. 3mg/kg and 1.5mg/kg bruceanin D mice do not die during the experiment, the body weight does not change obviously, the diet drinking water and hair of the early-stage mice are normal, the diet drinking water of the later-stage mice is slightly reduced, the reaction is slightly blunted, the hair is disordered, but the state is obviously superior to that of mice in the group of 4T1+ CAF.
2.2 Effect of brucesin D on mouse orthotopic tumors
The mice in the group of 4T1+ CAFs have larger in-situ tumors than the mice in the group of 4T1 (P is less than 0.001), and the weights of the in-situ tumors of the mice are reduced after the administration of Docetaxel or 1.5mg/kg and 3mg/kg of brucea javanica picrin D (P is less than 0.001, P is less than 0.01 and P is less than 0.01). See fig. 3A, B. Histopathological analysis of mouse in-situ tumor discovers that 4T1 and 4T1+ CAFs group tumor nuclei are large and round, and the nuclear division phenomenon is obvious. After administration of Docetaxel or 1.5mg/kg and 3mg/kg brucesin D, tumors showed different degrees of necrosis and vacuole apoptosis which were significantly higher than that of group 4T1+ CAFs (P < 0.05), and positive expression of Ki67 in mouse orthotopic tumor tissues was reduced (P < 0.001, P < 0.01), as shown in FIG. 3D. The results show that 3mg/kg and 1.5mg/kg of brucesin D can inhibit the growth of in-situ tumor of mouse and promote the necrotic apoptosis.
2.3 Effect of brucesin D on mouse Lung/liver metastases
By stripping and counting lung organs of each group of tumor-bearing mice, the number of pulmonary nodules of the mice in the group of 4T1+ CAFs is more than that of the mice in the group of 4T1 (P < 0.05), and the number of pulmonary nodules of the mice is reduced (P < 0.05, P < 0.05 and P < 0.01) after the mice are administered with Docetaxel or 1.5mg/kg and 3mg/kg of brucesin D. See fig. 4A, 4B. Pathological analysis is carried out on the lung and the liver of each group of tumor-bearing mice to find that the lung/liver tumor metastasis of the mice in the group of 4T1+ CAFs is more than that of the mice in the group of 4T1, and the lung/liver tumor metastasis of the mice is reduced after the administration of Docetaxel or 1.5mg/kg and 3mg/kg of brucellosis D, which indicates that the brucellosis D can obviously inhibit the formation of the breast orthotopic-lung/liver tumor metastasis of the tumor-bearing mice.
2.4 Effect of brucesin D on TNF-alpha content in mouse serum
Through the detection of TNF-alpha in the serum of each group of mice, the 4T1 group and the 4T1+ CAFs group are increased compared with the normal group (P is less than 0.05, P is less than 0.001); compared with the group of 4T1+, CAFs, the content of TNF-alpha in the serum of mice is reduced to different degrees after 1.5mg/kg and 3mg/kg of brucesin D are given (P is less than 0.05). The brucesin D is shown to reduce the content of TNF-alpha in the serum of mice, and the result is shown in figure 5.
2.5 Effect of brucesin D on TNF-alpha protein content and CAFs marker alpha-SMA expression in mouse orthotopic tumor tissue
After detecting mouse in-situ tumor tissue TNF-alpha protein, the positive expression of mice in the group of 4T1+ CAFs is higher than that in the group of 4T1 (P is less than 0.01), and after 1.5mg/kg and 3mg/kg of brucellosis D are given, the positive expression of mouse in-situ tumor tissue is reduced (P is less than 0.01, P is less than 0.001), and the result is shown in figure 6A. Meanwhile, after we detect the marker alpha-SMA of CAFs, the positive expression of mice in the group of 4T1+ CAFs is higher than that in the group of 4T1 (P is less than 0.001), and after 1.5mg/kg and 3mg/kg of brucellosis D are given, the positive expression of in-situ tumor tissues of the mice is reduced (P is less than 0.01), and the result is shown in the attached figure 6B. In addition, the double immunofluorescence of alpha-SMA (green) and TNF-alpha (red) shows that TNF-alpha and CAFs coexist simultaneously, TNF-alpha is dispersed in the whole tumor tissue, and more TNF-alpha is distributed around the CAFs. And after different groups are analyzed, positive expression of mice in the group of 4T1+ CAFs is higher than that in the group of 4T1 (P is less than 0.01, P is less than 0.05), and positive expression of in-situ tumor tissues of the mice is reduced (P is less than 0.01 ) after 1.5mg/kg and 3mg/kg of bruceojavanil D are administered, and the result is shown in figure 6C. The results show that the brucesin D can simultaneously reduce the content of TNF-alpha protein in the mouse orthotopic tumor tissue and the expression of CAFs marker alpha-SMA.
2.6 Effect of brucesin D on MMP2, MMP9, and collagen expression in mouse orthotopic tumor tissues
The result shows that the positive expressions of MMP2 and MMP9 of mice in the group of 4T1+ CAFs are higher than those of the group of 4T1 (P is less than 0.05, and P is less than 0.05), and after 1.5mg/kg and 3mg/kg of brucellosis D are administered, the positive expressions of MMP2 and MMP9 of in-situ tumor tissues of the mice are reduced (P is less than 0.05, and P is less than 0.01), and the result is shown in figure 7A. Then, after the collagen of the mouse in-situ tumor tissue is detected by Masson staining and Sirius Red staining respectively, the collagen volume of the mouse in-situ tumor tissue is found to be higher than that of the 4T1 group (P < 0.001 and P < 0.001) in the group of 4T1+ CAFs, and the collagen volume of the mouse in-situ tumor tissue is reduced (P < 0.01 and P < 0.001) after 1.5mg/kg and 3mg/kg of brucea javanica D are given, and the results are shown in FIG. 7B and FIG. 7C. The results show that brucellosidine D can reduce the contents of MMP2, MMP9 and collagen which are mainly secreted by CAFs in the mouse orthotopic tumor tissues.
3. Conclusion
Brucellosid D inhibits the growth of in-situ tumors of mice and the formation of lung/liver tumor metastasis, weakens the expression of proinflammatory factors TNF-alpha and the abundance of CAFs in tumor tissues simultaneously with brucellosid, and inhibits the inseparable tumor promotion function (MMP 2, MMP9 and collagen) of the CAFs.
Claims (11)
1. Application of brucesin D in preparing medicine for preventing and/or treating breast cancer is provided.
2. Application of brucesin D in preparation of medicines for preventing and/or treating invasive/metastatic breast cancer.
3. Use according to claim 1 or 2, characterized in that: the breast cancer is triple negative breast cancer.
4. Use according to claim 2, characterized in that: the invasive/metastatic breast cancer includes localized infiltration and distant metastasis of breast cancer.
5. Use according to claim 2, characterized in that: the local infiltration and the remote metastasis of the breast cancer are the invasion and metastasis of breast cancer cells promoted by CAFs under the action of TNF-alpha.
6. Use according to claim 4 or 5, characterized in that: the distant metastasis is hepatic metastasis.
7. Use according to claim 4 or 5, characterized in that: the distant metastasis is pulmonary metastasis.
8. Application of brucesin D in preparing medicine for preventing and/or treating metastatic focus of breast cancer is disclosed.
9. Use according to claim 8, characterized in that: the breast cancer metastasis is a liver metastasis and/or a lung metastasis.
10. Use according to claim 8 or 9, characterized in that: the breast cancer is triple negative breast cancer.
11. The application of brucesin D in any one of the following:
1) Preparing a medicament for inhibiting the growth of breast cancer cells and promoting the apoptosis of the breast cancer cells;
2) Preparing a medicament for inhibiting invasion and migration of breast cancer cells;
3) Drugs that inhibit the formation of mammary orthotopic-lung tumor metastases;
4) Drugs that inhibit the formation of breast in situ-liver tumor metastases;
5) A drug for reducing the content of TNF-alpha in serum;
6) A drug for reducing the content of TNF-alpha protein in breast tumor tissues and the expression of a CAFs marker alpha-SMA;
7) A medicament for reducing the expression of MMP2, MMP9 and collagen in breast tumor tissues.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210620484.4A CN115414353A (en) | 2022-06-02 | 2022-06-02 | Application of brucesin D in inhibiting CAFs (adenosine triphosphate) triple negative breast cancer metastasis under TNF (tumor necrosis factor) -alpha action |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210620484.4A CN115414353A (en) | 2022-06-02 | 2022-06-02 | Application of brucesin D in inhibiting CAFs (adenosine triphosphate) triple negative breast cancer metastasis under TNF (tumor necrosis factor) -alpha action |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115414353A true CN115414353A (en) | 2022-12-02 |
Family
ID=84196690
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210620484.4A Pending CN115414353A (en) | 2022-06-02 | 2022-06-02 | Application of brucesin D in inhibiting CAFs (adenosine triphosphate) triple negative breast cancer metastasis under TNF (tumor necrosis factor) -alpha action |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115414353A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115919845A (en) * | 2023-02-08 | 2023-04-07 | 中南大学湘雅二医院 | Application of brucea javanica element A as medicine for treating liver cancer |
| CN117357515A (en) * | 2023-11-27 | 2024-01-09 | 宁夏医科大学 | Application of brucea javanica kurrow kurarine D in preparing medicament for inhibiting tumor angiogenesis |
| CN117398378A (en) * | 2023-11-27 | 2024-01-16 | 宁夏医科大学 | Application of brucea javanica kurrow kurarine D in preparation of medicines for improving tumor microenvironment |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107261061A (en) * | 2017-06-23 | 2017-10-20 | 邓圣平 | A kind of Chinese medicine composition for treating malignant tumour, preparation method and its preparation |
| CN113616652A (en) * | 2020-05-06 | 2021-11-09 | 石家庄以岭药业股份有限公司 | Application of SMS2 inhibitor in preparation of medicine for treating highly invasive breast cancer |
-
2022
- 2022-06-02 CN CN202210620484.4A patent/CN115414353A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107261061A (en) * | 2017-06-23 | 2017-10-20 | 邓圣平 | A kind of Chinese medicine composition for treating malignant tumour, preparation method and its preparation |
| CN113616652A (en) * | 2020-05-06 | 2021-11-09 | 石家庄以岭药业股份有限公司 | Application of SMS2 inhibitor in preparation of medicine for treating highly invasive breast cancer |
Non-Patent Citations (1)
| Title |
|---|
| 罗璨: "鸦胆子苦素D对人三阴性乳腺癌MDA-MB-231细胞迁移和侵袭的影响及其机制", 中国优秀硕士学位论文全文数据库，医药卫生科技辑 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115919845A (en) * | 2023-02-08 | 2023-04-07 | 中南大学湘雅二医院 | Application of brucea javanica element A as medicine for treating liver cancer |
| CN117357515A (en) * | 2023-11-27 | 2024-01-09 | 宁夏医科大学 | Application of brucea javanica kurrow kurarine D in preparing medicament for inhibiting tumor angiogenesis |
| CN117398378A (en) * | 2023-11-27 | 2024-01-16 | 宁夏医科大学 | Application of brucea javanica kurrow kurarine D in preparation of medicines for improving tumor microenvironment |
| CN117357515B (en) * | 2023-11-27 | 2024-06-14 | 宁夏医科大学 | Application of brucea javanica kurrow kurarine D in preparing medicament for inhibiting tumor angiogenesis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115414353A (en) | Application of brucesin D in inhibiting CAFs (adenosine triphosphate) triple negative breast cancer metastasis under TNF (tumor necrosis factor) -alpha action | |
| CN104856996B (en) | PQQ, its derivative and/or the new pharmaceutical usage and Pharmaceutical composition of salt | |
| TW200932245A (en) | Methods and compositions for inhibiting angiogenesis | |
| Jia et al. | KGF is delivered to inflammatory and induces the epithelial hyperplasia in trinitrobenzene sulfonic acid-induced ulcerative colitis rats | |
| WO2019233469A1 (en) | Use of pdgfr signaling pathway inhibitor for preparation of drug for treating intestinal inflammatory diseases | |
| JP7555142B2 (en) | Stem Cell Medicines to Treat Diabetes | |
| CN114010630A (en) | Application of oxygen methyl modifier of quercetin in preparation of medicine for inhibiting tumor cell proliferation | |
| Li et al. | Microfluidic printed 3D bioactive scaffolds for postoperative treatment of gastric cancer | |
| Hu et al. | Salvianolic acid B-loaded polydopamine-modified hollow mesoporous organic silica nanoparticles for treatment of breast cancer metastasis via suppressing cancer-associated fibroblasts | |
| CN104997781B (en) | The application of legalon or its salt in enhancing ALK-TKI curative effects are prepared, delay its drug resistant medicine | |
| WO2024031928A1 (en) | Use of a-mangostin in preparing medicament for treating melanoma and medicament | |
| CN111870639A (en) | Application of Tibetan medicine scindapsus aureus in preparing medicine for treating atherosclerosis | |
| CN108714149A (en) | Purposes of the garden burnet active constituent in preparing anti-tumor drug | |
| CN109260228A (en) | A kind of compound for treating tumour | |
| CN109276572B (en) | Application of loganin and 5-fluorouracil in combination in treatment of gastric cancer | |
| US10426785B2 (en) | C-19 steroids for inhibiting neovascularization | |
| CN105030777B (en) | Enhancing ALK TKI curative effects, the compound and its preparation for delaying its resistance | |
| Phung et al. | In-vitro and in-vivo assays for angiogenesis-modulating drug discovery and development | |
| CN109106710B (en) | Use of compounds | |
| CN113181166A (en) | Application of curzerene alcohol in preparation of anti-lung cancer drugs and anti-lung cancer drugs | |
| CN105396136A (en) | Application of CCN1(Cyr61) to treatment of diseases related to skin injuries and atrophoderma | |
| CN117357515B (en) | Application of brucea javanica kurrow kurarine D in preparing medicament for inhibiting tumor angiogenesis | |
| CN109745314A (en) | Application of the iron chelating agent Deferasirox (DFX) in the drug for the treatment of cervical carcinoma | |
| CN110393716A (en) | Application of the fangchinoline in the drug that preparation inhibits lung cancer metastasis | |
| CN109700791B (en) | Application of neosinomenine in preparing medicine for treating benign prostatic hyperplasia |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20221202 |