CN111228269A - Application of cepharanthine and its salt as iron death inducer in preparing antitumor drugs - Google Patents
Application of cepharanthine and its salt as iron death inducer in preparing antitumor drugs Download PDFInfo
- Publication number
- CN111228269A CN111228269A CN202010256288.4A CN202010256288A CN111228269A CN 111228269 A CN111228269 A CN 111228269A CN 202010256288 A CN202010256288 A CN 202010256288A CN 111228269 A CN111228269 A CN 111228269A
- Authority
- CN
- China
- Prior art keywords
- hydrochloride
- cepharanthine
- cells
- prostate cancer
- stephanine
- 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
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 title claims abstract description 69
- 229910052742 iron Inorganic materials 0.000 title claims abstract description 35
- 230000034994 death Effects 0.000 title claims abstract description 32
- 239000000411 inducer Substances 0.000 title claims abstract description 12
- 150000003839 salts Chemical class 0.000 title claims abstract description 11
- 239000002246 antineoplastic agent Substances 0.000 title claims abstract description 6
- 229940041181 antineoplastic drug Drugs 0.000 title claims abstract description 6
- VQAWRQZAAIQXHM-UHFFFAOYSA-N Cepharanthine Natural products O1C(C=C2)=CC=C2CC(C=23)N(C)CCC3=CC=3OCOC=3C=2OC(=CC=23)C(OC)=CC=2CCN(C)C3CC2=CC=C(O)C1=C2 VQAWRQZAAIQXHM-UHFFFAOYSA-N 0.000 title claims description 40
- YVPXVXANRNDGTA-WDYNHAJCSA-N cepharanthine Chemical compound C1C(C=C2)=CC=C2OC(=C2)C(OC)=CC=C2C[C@H](C2=C3)N(C)CCC2=CC(OC)=C3OC2=C(OCO3)C3=CC3=C2[C@H]1N(C)CC3 YVPXVXANRNDGTA-WDYNHAJCSA-N 0.000 title claims description 40
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 23
- 239000003814 drug Substances 0.000 claims abstract description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 58
- 210000004881 tumor cell Anatomy 0.000 claims description 8
- 208000023958 prostate neoplasm Diseases 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- UEAPAHNNFSZHMW-UHFFFAOYSA-N stepahnine Natural products COC1=CC=CC(C2=C34)=C1CC3N(C)CCC4=CC1=C2OCO1 UEAPAHNNFSZHMW-UHFFFAOYSA-N 0.000 abstract description 34
- UEAPAHNNFSZHMW-CQSZACIVSA-N stephanine Chemical compound CN([C@@H]1CC2=C(C3=C11)C=CC=C2OC)CCC1=CC1=C3OCO1 UEAPAHNNFSZHMW-CQSZACIVSA-N 0.000 abstract description 33
- 229940079593 drug Drugs 0.000 abstract description 8
- 238000011282 treatment Methods 0.000 abstract description 7
- 230000001093 anti-cancer Effects 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 85
- 206010060862 Prostate cancer Diseases 0.000 description 44
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 44
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 21
- 239000001963 growth medium Substances 0.000 description 16
- 230000000694 effects Effects 0.000 description 15
- 230000006907 apoptotic process Effects 0.000 description 12
- 230000030833 cell death Effects 0.000 description 11
- -1 iron ions Chemical class 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 239000003112 inhibitor Substances 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 101100173542 Caenorhabditis elegans fer-1 gene Proteins 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- UJHBVMHOBZBWMX-UHFFFAOYSA-N ferrostatin-1 Chemical compound NC1=CC(C(=O)OCC)=CC=C1NC1CCCCC1 UJHBVMHOBZBWMX-UHFFFAOYSA-N 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 108010033024 Phospholipid Hydroperoxide Glutathione Peroxidase Proteins 0.000 description 3
- 102100023410 Phospholipid hydroperoxide glutathione peroxidase Human genes 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- OEUUFNIKLCFNLN-LLVKDONJSA-N chembl432481 Chemical compound OC(=O)[C@@]1(C)CSC(C=2C(=CC(O)=CC=2)O)=N1 OEUUFNIKLCFNLN-LLVKDONJSA-N 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- BKQFRNYHFIQEKN-UHFFFAOYSA-N erastin Chemical compound CCOC1=CC=CC=C1N1C(=O)C2=CC=CC=C2N=C1C(C)N1CCN(C(=O)COC=2C=CC(Cl)=CC=2)CC1 BKQFRNYHFIQEKN-UHFFFAOYSA-N 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 239000012894 fetal calf serum Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 230000035508 accumulation Effects 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 230000004770 neurodegeneration Effects 0.000 description 2
- 208000015122 neurodegenerative disease Diseases 0.000 description 2
- 230000006610 nonapoptotic cell death Effects 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000004017 serum-free culture medium Substances 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 239000004156 Azodicarbonamide Substances 0.000 description 1
- 238000011729 BALB/c nude mouse Methods 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 230000005971 DNA damage repair Effects 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 101150117869 Hras gene Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 241000304195 Salvia miltiorrhiza Species 0.000 description 1
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 208000004631 alopecia areata Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 229940124650 anti-cancer therapies Drugs 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000011319 anticancer therapy Methods 0.000 description 1
- 229910052785 arsenic Inorganic materials 0.000 description 1
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 1
- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 description 1
- 229930101531 artemisinin Natural products 0.000 description 1
- 229960004191 artemisinin Drugs 0.000 description 1
- 230000009789 autophagic cell death Effects 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- 235000019399 azodicarbonamide Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000006721 cell death pathway Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000001808 exosome Anatomy 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 201000002364 leukopenia Diseases 0.000 description 1
- 231100001022 leukopenia Toxicity 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004066 metabolic change Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000004984 non-apoptotic programmed cell death Effects 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000000861 pro-apoptotic effect Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 125000003698 tetramethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- 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)
- 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)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Dermatology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to the technical field of medicines, and provides application of stephanine and salts thereof as an iron death inducer in preparation of antitumor drugs. Therefore, the stephanine and the salt thereof have good anticancer effect and provide a new effective treatment medicine for patients with tumor diseases.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of stephanine and salt thereof serving as an iron death inducer in preparation of antitumor drugs.
Background
Apoptosis is the most deeply studied type of cell death, however, in recent years, in addition to apoptosis, many other forms of cell death have been discovered, including autophagic cell death, iron death, pyro-death, glutamate apoptosis and exosome-associated cell death. Traditional cancer treatments are primarily directed at apoptosis, however, many cancer cells are chemoresistant and have defects in apoptosis induction, most of the mechanisms controlling apoptotic and non-apoptotic cell death are also independently regulated, and the cells may also experience various stresses leading to apoptotic and non-apoptotic cell death. Most tumors are treated by surgical resection and radiotherapy at an early stage, and are treated in multiple modes in combination with surgery and chemotherapy at a later stage. However, some chemotherapy-resistant tumor cells can escape apoptotic and other death signals through drug efflux, metabolic changes, alterations in DNA damage repair mechanisms, or other unknown mechanisms, and in addition cancer cells also exhibit higher ROS levels than normal cells and develop resistance to pro-inflammatory and pro-apoptotic molecules to maintain self-survival. Iron death (ferrotosis), a novel cell death mode which is independent from apoptosis and other modes in a mechanism, and a novel iron-dependent cell death mode which is found by high-throughput screening of lethal effects of more than 2 ten thousand compounds by DolmaS and the like in 2003 and is used for targeted killing of HRas overexpressed tumor cells induced by Erastin, wherein the novel iron-dependent cell death mode is greatly different from classical cell death modes such as apoptosis, necrosis, autophagy and the like in aspects of morphology, biochemistry and the like. In 2012, Dixon SJ named this cell death as iron death, when cells died iron, mitochondria condensed in cytoplasm, inner membrane ridges decreased or even disappeared, outer membrane density increased, ruptured, intracellular active divalent iron ions increased, and finally lethal lipid peroxide accumulated to cause cell death. Researches show that the iron death inducer Erastin has better cancer inhibition effect in cancers such as liver cancer, breast cancer, ovarian cancer and the like, and more recent researches show that iron death is not only related to tumors, but also plays an important role in diseases such as neurodegenerative diseases, cardiovascular diseases, diabetes, inflammation and the like.
However, few drugs have been found to induce iron death, such as Erastin and RSL-3, an inhibitor of glutathione peroxidase 4. Although there have been more and more recent studies reporting that some natural products, molecules can induce iron death in different cells, such as: artemisinin, salvia miltiorrhiza, arsenic and the like, but the clinical application of the substances through an iron death mechanism is not proved, and more iron death inducers are still to be developed. There is increasing evidence that compounds from natural sources can induce non-apoptotic programmed cell death in cancer cells, and natural compounds have gained wide promise for future anti-cancer therapies.
Cepharanthine (CEP) is a monomeric compound extracted from Cepharanthine belonging to genus Cepharanthaceae, and belongs to the class of alkaloids, and can be used for clinically treating leukopenia, alopecia areata, etc. Cepharanthine Hydrochloride (CH), a semisynthetic compound obtained by salinization of cepharanthine extracted from cepharanthus spicatus of cepharanthus, has a structural formula:
at present, the effect of stephanine as an iron death inducer on tumor-related aspects is not reported at home and abroad.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the application of stephanine and salt thereof as an iron death inducer in preparing antitumor drugs, and experiments show that: the cepharanthine and salts thereof can induce the cell to generate iron death in the tumor cell to play a role in treating the tumor, and provide a new technical means for treating tumor patients.
Further, the drug is administered by injection.
Further, the tumor is a prostate tumor.
Further, the concentration of stephanine and the salt thereof for inducing the death of the prostate tumor by iron is 1-100 mu M.
Experiments show that the stephanine hydrochloride induces the cells to die by iron in the tumor cells so as to play a role in resisting cancer, and the influence of the single use of the stephanine hydrochloride on the growth of different prostate cancer cells is detected for the first time. The inventor finds that the cells die but not die after the cepharanthine hydrochloride is added, detects the change of the morphology of the prostate cancer cells through an electron microscope, and finds that the mitochondrial structure is changed after the cepharanthine treatment.
The invention selects 1-100 mu M cepharanthine hydrochloride, and determines the drug toxicity of cepharanthine hydrochloride to prostate cancer DU145, LNcap, PC-3 and PC-3M cell strains in the range. After the cepharanthine hydrochloride is added to treat the cells, the active oxygen level in four cell strains is improved, the lipid peroxide accumulation and the expression level of GPX4 (glutathione peroxidase 4) are reduced, and the anti-cancer effect is obvious.
The prostate cancer LNcap cells have the highest sensitivity to the cepharanthine hydrochloride, and the concentration of the cepharanthine hydrochloride is 20 mu M, so that the LNcap cells can be induced to generate iron death.
The invention also provides a tumor cell iron death inducer, which comprises cepharanthine hydrochloride. The cepharanthine hydrochloride has therapeutic potential for neurodegenerative diseases, cardiovascular diseases, etc.
In summary, the invention includes at least one of the following beneficial technical effects:
the stephanine and the salt thereof can induce the cell to generate iron death in the tumor cell and induce the death of apoptosis-tolerant tumor cells, have obvious anti-cancer and anti-drug resistance effects and provide more effective treatment schemes for a large number of tumor patients. Meanwhile, the cepharanthine is used as a clinical medicine, has good safety, wide sources of the cepharanthine hydrochloride, simple and economic acquisition mode and good application prospect in the technical field of anti-tumor.
Drawings
FIG. 1 is a graph of the survival rate of various strains of prostate cancer cells inhibited by stephanine hydrochloride at various concentrations in example 1;
FIG. 2 is a graph of apoptosis of prostate cancer cells following cepharanthine hydrochloride treatment in example 2;
FIG. 3 is a graph showing the change in reactive oxygen species in prostate cancer cells after cepharanthine hydrochloride treatment in example 3;
FIG. 4 is a graph showing the change in intracellular lipid peroxide levels of prostate cancer cells in example 3 after treatment with stephanine hydrochloride and the iron death inhibitor, Fer-1;
FIG. 5 is a graph of the effect of LNcap cells on prostate cancer cell viability following the combination of stephaglabrin hydrochloride with the iron chelator DFO or with the iron death inhibitor Fer-1 of example 3.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Example 1 Effect of varying concentrations of stephanine hydrochloride on prostate cancer cell survival
S1, weighing cepharanthine hydrochloride powder by using an electronic balance, placing the cepharanthine hydrochloride powder into a centrifugal tube, adding dimethyl sulfoxide (DMSO) to dissolve the cepharanthine hydrochloride powder to prepare a mother solution with the concentration of 100mM, then diluting the mother solution with DMSO to different concentrations, wherein the concentrations of the cepharanthine hydrochloride are 1 mu M, 20 mu M, 40 mu M, 80 mu M and 100 mu M respectively, adding the cepharanthine hydrochloride powder into a DMEM/F12 culture medium containing 10% fetal calf serum respectively, and performing an experiment for later use;
s2, selecting four strains of prostate cancer cells DU145 (purchased from an ATCC cell bank), LNcap (purchased from an ATCC cell bank), PC-3 (purchased from an ATCC cell bank) and PC-3M (purchased from a national experiment cell resource sharing platform), digesting the prostate cancer cells by using a digestive juice containing 0.075 wt.% of I-type collagenase, 0.5 wt.% of II-type collagenase and 0.1 wt.% of trypsin, digesting at 37 ℃ for 1min, repeatedly blowing the digested suspension by using a straw after digestion, plating the suspension on a corresponding culture dish for subculture or drug experiments, and observing the cell morphology and growth condition under an inverted microscope every day;
s3, inoculating the suspension prepared in the step S2 into a 96-well plate, wherein each well is inoculated with 0.8 multiplied by 10 cells4Culturing prostate cancer cells by using 5 groups of culture media containing drugs with different concentrations prepared in the step S1, setting 20 groups of experimental groups, adding 4 groups of DMSO groups with the same dosage into DMEM/F12 culture media containing 10% fetal calf serum to culture prostate cancer cells as a control group, after overnight culture and wall attachment, respectively adding 10 mu l of 5mg/ml tetramethyl azozolium (MTT) during 24h and 48h of culture, sucking the DMEM/F12 culture media and MTT after 4h of incubation at 37 ℃, adding DMSO, reacting for 30min at room temperature on a shaking table, then measuring an absorbance value at a wavelength of 570nm by using a chemiluminescence instrument, and finally drawing a fitting curve of drug concentration corresponding to cell survival rate by using GraphPad Prism 8.
The invention adopts an MTT method to detect the toxicity of the stephanine hydrochloride to prostate cancer cells DU145, LNcap, PC-3 and PC-3M four strains of cells for 24h and 48h, and calculates to obtain the IC50 value of the stephanine hydrochloride to different prostate cancer cells. The results show that LNcap cells are most sensitive to cepharanthine hydrochloride, see figure 1.
In this example, concentrations of cepharanthine hydrochloride of 1. mu.M, 40. mu.M, 80. mu.M and 100. mu.M, the effect of cepharanthine hydrochloride on prostate cancer cell survival was similar and the fitted curves were similar.
Example 2 testing the Effect of stephanine hydrochloride on the cell death pathway of prostate cancer
S1, adopting the mother liquor with the concentration of 100mM prepared in the embodiment 1, then diluting the mother liquor with a culture medium into different concentrations, wherein the final concentrations of stephanine hydrochloride are respectively 10 mu M and 20 mu M, respectively culturing cells into two groups, setting a group of DMSO as a blank control group (represented by control), wherein the dose of the added DMSO is the same as that of the stephanine hydrochloride, and performing an experiment for later use;
s2, inoculating the suspension of prostate cancer cells LNcap prepared in step S2 of example 1 into a 6-well plate, wherein each well is inoculated with 30X 10 cells4And (2) culturing the prostate cancer cells by using 3 groups of culture media prepared in step S1 of the embodiment 2, collecting the prostate cancer cells in a centrifuge tube when the cells are cultured for 24 hours and 48 hours, centrifuging the cells for 20 minutes at a speed of 1000rpm/min, removing supernatant after centrifugation, re-suspending the cells by using DMEM/F12 culture medium containing 10% fetal calf serum, discarding the culture medium by centrifugation, repeatedly washing the cells for 2 times by using phosphate buffer, then adding binding buffer containing 5 mul of annexin V dye for re-suspending, then adding 10 mul of PI dye, incubating the cells for 5 minutes at room temperature, detecting the apoptosis by using a flow cytometer, and respectively recording the apoptosis of the prostate cancer cells at 24 hours and 48 hours, and referring to FIG. 2.
As can be seen from fig. 2, when the concentration of cepharanthine hydrochloride was 20 μ M, significant cell death was induced in LNcap prostate cancer cells, but the results showed that the proportion of apoptotic cells was not high, suggesting that cepharanthine hydrochloride induces other forms of cell death.
Example 3 determination of the Effect of stephanine hydrochloride on reactive oxygen species in LNcap of prostate cancer cells
S1, miningThe method comprises diluting the suspension of the prostate cancer cells prepared in example 2 to obtain the concentrations of stephanine hydrochloride of 10 μ M and 20 μ M, adding the stephanine hydrochloride into DMEM/F12 culture medium containing 10% fetal bovine serum, dividing into two groups, setting a group of DMSO as a blank control group (indicated by control), adding the DMSO in the same amount as the stephanine hydrochloride, inoculating the suspension of the prostate cancer cells LNcap prepared in example 2 into 6-well plates, inoculating 30 × 10 cells per well4Culturing prostate cancer cells by using 3 groups of culture media prepared in the embodiment, and performing experiments for standby;
s2, diluting the active oxygen detection probe DCFH-DA by using a serum-free culture medium according to the ratio of 1:1000 so that the final concentration is 10 mu M. Removing the culture medium of prostate cancer cells LNcap cultured in a 6-well plate, and adding 2ml of diluted DCFH-DA solution into each well;
s3, putting the 6-hole plate into a cell culture box at 37 ℃ for incubation for 20min, and then washing the cells three times by using a serum-free culture medium to sufficiently remove the DCFH-DA probe which does not enter the cells. Collecting prostate cancer cells LNcap by pancreatin, centrifuging for 25min at the speed of 800rpm/min, re-suspending and cleaning by PBS after centrifuging, centrifuging again for 15min at the speed of 500rpm/min, discarding the supernatant, re-suspending the cells by 200 μ l PBS, and then detecting the fluorescence intensity of DCFH-DA in the cells by a flow analyzer under the conditions of 488nm excitation wavelength and 525nm emission wavelength, referring to FIG. 3.
As can be seen from fig. 3, the concentration of stephanine hydrochloride of 20 μ M has a greater effect on the active oxygen in the prostate cancer cells LNcap, the active oxygen in the prostate cancer cells LNcap is increased more, and the concentration of stephanine hydrochloride of 10 μ M has a slightly lower effect on the active oxygen in the prostate cancer cells LNcap.
Example 4 determination of the Effect of stephanine hydrochloride on the level of lipid peroxides in LNcap of prostate cancer cells
S1, taking the cepharanthine hydrochloride with the concentration of 20 mu M obtained by dilution in the example 3 as one group and taking the solution of the cepharanthine hydrochloride combined inhibitor Fer-1 with the concentration of 20 mu M as one group, in the example, the two groups are respectively added into DMEM/F12 culture medium containing 10% fetal bovine serum, and a group of DMSO (expressed by control) is further set as a blank control group to be addedThe dose of the physiological saline is the same as that of the stephanine hydrochloride, and the suspension of prostate cancer cells LNcap prepared in example 3 is inoculated into a 6-well plate, and each well is inoculated with 30X 10 cells4Culturing the prostate cancer cells in LNcap by using 3 groups of culture media prepared in the step of the embodiment, wherein the experiments are ready for use;
s2, detecting the level of lipid peroxide by using BODIPY C11 as a probe, adding 5 mu M BODIPYC11 into each well of a 6-well plate, incubating in a cell culture box at 37 ℃ for 30min, collecting prostate cancer cells LNcap into a centrifuge tube by trypsinization, centrifuging for 3min at the speed of 1200rpm/min, re-suspending and cleaning with PBS after centrifugation, centrifuging again for 5min at the speed of 700rpm/min, discarding the supernatant, re-suspending the cells with 200 mu l PBS, and further analyzing the fluorescence intensity of the cells under the excitation wavelength of 488nm by using a flow cytometer, wherein the reference is shown in FIG. 4.
As can be seen from fig. 4, stephanine hydrochloride with a concentration of 20 μ M has a greater effect on the LNcap lipid peroxide level of prostate cancer cells than the stephanine hydrochloride combined inhibitor Fer-1, and the lipid peroxide accumulation is higher than that of the LNcap lipid peroxide level of prostate cancer cells in the blank control group compared with that in the other two groups. This result suggests that cepharanthine hydrochloride induces iron death.
Example 5 determination of the Effect of stephanine hydrochloride on inhibiting the viability of prostate cancer LNcap cells by iron death
S1, taking the cepharanthine hydrochloride with the concentration of 20 mu M obtained by dilution in the example 3 as a group, taking the solution of the cepharanthine hydrochloride with the inhibitor Fer-1 with the concentration of 20 mu M as a group, taking the solution of the cepharanthine hydrochloride with the inhibitor iron chelator DFO with the concentration of 20 mu M as a group, respectively adding the three groups into DMEM/F12 culture medium containing 10% fetal bovine serum, inoculating the prostate cancer cell LNcap suspension prepared in the example 3 into a 6-well plate, and inoculating 30 multiplied by 10 cells into each well4Culturing the prostate cancer cells in LNcap by using 3 groups of culture media prepared in the step of the embodiment, wherein the experiments are ready for use;
s2, after overnight culture and adherence, respectively adding 10 mu l of 5mg/ml tetramethyl azodicarbonamide (MTT) during 24h and 48h of culture, after incubation for 4h at 37 ℃, absorbing DMEM/F12 culture medium and MTT, adding DMSO, and reacting for 30min at room temperature on a shaking table;
s3, detecting the level of lipid peroxide by using BODIPY C11 as a probe, adding 5 mu M BODIPYC11 into each well of a 6-well plate, incubating in a cell culture box at 37 ℃ for 30min, collecting prostate cancer cells LNcap into a centrifuge tube by trypsinization, centrifuging for 3min at the speed of 1200rpm/min, re-suspending and cleaning with PBS after centrifugation, centrifuging again for 5min at the speed of 700rpm/min, discarding the supernatant, re-suspending the cells with 200 mu l PBS, and further analyzing the fluorescence intensity of the cells under the excitation wavelength of 488nm by using a flow cytometer, wherein the reference is made to FIG. 5.
As can be seen from fig. 5, the iron death inhibitor Fer-1 or the iron chelator DFO can reverse the inhibition of stephaglabrin hydrochloride on the viability of prostate cancer LNcap cells, and this result demonstrates that stephaglabrin hydrochloride is an iron death inducer.
Example 6 tumor formation test
Selecting male atmoic BALB/c nude mouse (4-6 weeks old), purchasing from Chinese laboratory animal science institute, placing in sterile environment, subcutaneously injecting prostate cancer cell LNcap suspended in 200 μ l matrix gel/PBS (5mg/ml) onto mouse body to construct nude mouse tumor model, and when the tumor volume in mouse body grows to 100mm3Randomly dividing the mice into 4 groups, namely a model group, a stephanine hydrochloride low-dose group of 15mg/kg, a stephanine hydrochloride medium-dose group of 30mg/kg and a stephanine hydrochloride high-dose group of 40mg/kg, measuring the weights of the mice and the sizes of tumors every other day by adopting a mode of intraperitoneal injection every day, killing the mice on day 13 to collect the tumors, wherein the weight of the tumors of each group of mice after the test is shown in a table 1, and the tumor growth rate of each group of mice after the test is shown in a table 2.
TABLE 1
TABLE 2
According to the data in tables 1 and 2, the tumor weight of the stephanine hydrochloride mice is far lower than that of the model mice, which indicates that the stephanine hydrochloride can inhibit tumor growth, and especially the low-dose stephanine hydrochloride group has the best tumor inhibition effect.
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Claims (5)
1. Application of cepharanthine and its salt as iron death inducer in preparing antitumor drugs is provided.
2. Use according to claim 1, characterized in that: the medicament is administered by injection.
3. Use according to claim 1, characterized in that: the tumor is prostate tumor.
4. Use according to claim 3, characterized in that: the concentration of the cepharanthine and salts thereof for inducing the death of the prostate tumor by iron is 1-100 mu M.
5. An agent for inducing iron death in tumor cells, comprising: the iron death inducer comprises cepharanthine hydrochloride.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010256288.4A CN111228269A (en) | 2020-04-02 | 2020-04-02 | Application of cepharanthine and its salt as iron death inducer in preparing antitumor drugs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010256288.4A CN111228269A (en) | 2020-04-02 | 2020-04-02 | Application of cepharanthine and its salt as iron death inducer in preparing antitumor drugs |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111228269A true CN111228269A (en) | 2020-06-05 |
Family
ID=70869956
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010256288.4A Pending CN111228269A (en) | 2020-04-02 | 2020-04-02 | Application of cepharanthine and its salt as iron death inducer in preparing antitumor drugs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111228269A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111838074A (en) * | 2020-07-15 | 2020-10-30 | 浙江大学 | Method for constructing iron death mouse model by using iron death inducer Erastin and application of method |
| CN113244252A (en) * | 2021-06-09 | 2021-08-13 | 广西农业职业技术学院 | Application of saponin compound in preparation of iron death inducer |
| CN113337569A (en) * | 2021-05-26 | 2021-09-03 | 深圳市人民医院 | Method for screening antitumor drugs based on induction of cell inflammatory death |
| CN113552353A (en) * | 2021-07-12 | 2021-10-26 | 江南大学 | Magnetic particle chemiluminescence kit for diagnosis of PCa and CRPC diseases |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101507725A (en) * | 2008-12-31 | 2009-08-19 | 河南省医药科学研究院 | Use of stephanine and hydrochloride thereof in preparing anti-hepatitis virus medicine |
| US20150190392A1 (en) * | 2014-01-03 | 2015-07-09 | Macau University Of Science And Technology | Group of Alkaloids, the Novel Autophagic Enhancers for Treatment of Cancers and Neurodegenerative Conditions Thereof |
| CN104922677A (en) * | 2015-05-27 | 2015-09-23 | 广州暨南生物医药研究开发基地有限公司 | Application of drug combination containing cepharanthine hydrochloride in preparing drug for treating leukopenia |
| CN110496225A (en) * | 2019-09-28 | 2019-11-26 | 广州暨南生物医药研究开发基地有限公司 | Stephanine and autophagy inhibitor are combined the application in preparation treatment liver-cancer medicine |
-
2020
- 2020-04-02 CN CN202010256288.4A patent/CN111228269A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101507725A (en) * | 2008-12-31 | 2009-08-19 | 河南省医药科学研究院 | Use of stephanine and hydrochloride thereof in preparing anti-hepatitis virus medicine |
| US20150190392A1 (en) * | 2014-01-03 | 2015-07-09 | Macau University Of Science And Technology | Group of Alkaloids, the Novel Autophagic Enhancers for Treatment of Cancers and Neurodegenerative Conditions Thereof |
| CN104922677A (en) * | 2015-05-27 | 2015-09-23 | 广州暨南生物医药研究开发基地有限公司 | Application of drug combination containing cepharanthine hydrochloride in preparing drug for treating leukopenia |
| CN110496225A (en) * | 2019-09-28 | 2019-11-26 | 广州暨南生物医药研究开发基地有限公司 | Stephanine and autophagy inhibitor are combined the application in preparation treatment liver-cancer medicine |
Non-Patent Citations (2)
| Title |
|---|
| ITA等: "Remarkable enhancement of cytotoxicity of onconase and cepharanthine when used in combination on various tumor cell lines", 《CANCER BIOLOGY & THERAPY》 * |
| 潘柏申等: "《2018检验医学进展》", 31 August 2018, 中华医学电子音像出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111838074A (en) * | 2020-07-15 | 2020-10-30 | 浙江大学 | Method for constructing iron death mouse model by using iron death inducer Erastin and application of method |
| CN113337569A (en) * | 2021-05-26 | 2021-09-03 | 深圳市人民医院 | Method for screening antitumor drugs based on induction of cell inflammatory death |
| CN113244252A (en) * | 2021-06-09 | 2021-08-13 | 广西农业职业技术学院 | Application of saponin compound in preparation of iron death inducer |
| CN113552353A (en) * | 2021-07-12 | 2021-10-26 | 江南大学 | Magnetic particle chemiluminescence kit for diagnosis of PCa and CRPC diseases |
| CN113552353B (en) * | 2021-07-12 | 2023-08-25 | 江南大学 | Magnetic particle chemiluminescence kit for PCa and CRPC disease diagnosis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111228269A (en) | Application of cepharanthine and its salt as iron death inducer in preparing antitumor drugs | |
| Sun et al. | Discovery and development of tumor glycolysis rate-limiting enzyme inhibitors | |
| CN109320570A (en) | A kind of icariside I class compound, derivative, officinal salt and application | |
| CN111658655A (en) | Application of cucurbitacin B in preparation of iron death inducer and anti-nasopharyngeal carcinoma drug | |
| CN113181166B (en) | Application of curcumenol in preparing anti-lung cancer medicine | |
| CN111166754A (en) | Application of cryptotanshinone in preparation of medicine for preventing and treating cachexia skeletal muscle atrophy | |
| CN104817608A (en) | Cordycepin salt containing selenium compound, preparation method of cordycepin salt and application | |
| CN112870189B (en) | Application of 2-acyl-1-dimethylaminomethyl ferrocene derivative in preparation of drug for targeted therapy of hepatocellular carcinoma | |
| CN112402413B (en) | Application of lindley eupatorium herb sesquiterpene lactone B in preparation of anti-liver cancer medicine and anti-liver cancer medicine | |
| CN111393278A (en) | Usnea longissima derivative and application thereof in preparation of medicine for treating gallbladder cancer | |
| CN111789830A (en) | Application of 2- (2,2, 2-trifluoroethylene) -1, 3-dione compound in preparation of anti-lung cancer drugs | |
| CN112336716A (en) | Application of vitamin C and disulfiram in preparation of anti-tumor combined medicine | |
| CN112791081A (en) | Application of white ketone in preparation of lung cancer treatment medicine | |
| CN111000850A (en) | Application of cryptotanshinone in preparation of drug for overcoming drug resistance of EGFR-TKI used for treating NSCLC | |
| CN114366740B (en) | Application of compound A-6 in preparation of broad-spectrum anticancer drugs | |
| Cao et al. | Study On Anticancer Effect of Synthetic Biogenic Source of Germacrene A | |
| CN105969725B (en) | The purposes of fructus lycii red pigment | |
| CN115645532B (en) | Application of isoxazole derivative in preparing brain glioma radiotherapy sensitization drugs | |
| CN104127394A (en) | Application of above one of alpha-pinene, beta-pinene, 1,8-eudesmol and camphene in preparation of human blood vessel endothelial cell damage treatment medicines | |
| CN113908148B (en) | Application of nobiletin in preparation of anti-cholangiocarcinoma drugs | |
| CN113876774B (en) | Application of neoliensinine in preparing medicine for treating leukemia | |
| CN104337807B (en) | Nanometer liposome of load paclitaxel and sensitizer and preparation method thereof and application altogether | |
| CN112274514B (en) | Application of sinomenine compounds in preparation of medicines for preventing or treating glioma | |
| CN108379282A (en) | A kind of drug and application thereof | |
| CN116173008B (en) | Application of nardostachyne as iron death inducer |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200605 |