CN107007601A - Applications of the AF38469 in anti-glioma drugs or health products are prepared - Google Patents
Applications of the AF38469 in anti-glioma drugs or health products are prepared Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses applications of sorting protein (sortilin) the antagonist AF38469 in anti-glioma drugs or health products are prepared, expand AF38469 application, improve its application value, treatment or health care to glioma bring new hope, and, using AF38469 as lead compound, activity or reduction side effect are improved by structural modification or transformation, additionally aids and further develops new anti-glioma drugs or health products.
Description
Technical field
The invention belongs to the pharmaceutical product technical field containing organic effective component, it is related to AF38469 in medicine or health care
The new application of product preparation field.
Background technology
Glioma is a kind of most common primary malignant brain tumor of current China.Account for intracranial tumors 35.26%~
60.96%, average 44.69%, the incidence of disease was in increased trend year by year in the last few years.Due to the invasive growth side of glioma
Formula, is obscured with normal cerebral tissue's boundary of surrounding, it is difficult to be removed by surgery.Glioma currently for Postoperative Residual is thin
Born of the same parents, are mainly killed using measures such as radiotherapy, chemotherapy, but these Postoperative Residual lesions are to the measure table such as radiotherapy, chemotherapy
Reveal significant repellence, often lead to tumor recurrence, patient's prognosis is not good.Therefore, new effective therapeutic strategy and curative
The research of thing will produce actively impact to the treatment of glioblastoma.
Sorting protein (sortilin) is the newfound I receptoroids of a class, in cardiovascular, intestines and stomach and central nervous system
Disease in play a role.AF38469 is newfound oral sortilin selective depressants, currently as a kind of science
Investigational agent is mainly used in the research of sortilin blocking aftereffect, and whether it has antitumor action there is not yet document report
Road.
The content of the invention
In view of this, it is an object of the invention to investigate effects of the AF38469 to glioma, to develop new anticol
The medicine or health products of matter knurl, so as to provide more effective drug candidates or guarantor for the clinical treatment or daily health caring of glioma
Strong product.
Through experiment, the present invention provides following technical scheme：
Applications of the AF38469 in anti-glioma drugs or health products are prepared.
Further, the anti-glioma is to suppress propagation and the migration of glioma cell.
Further, the anti-glioma is by blocking GSK-3 β/β-catenin signal paths to suppress glioma cell
Propagation and migration.
Sortilin about 80%-90% in glioma cell are distributed in transhipment golgiosome system and nucleus, participate in
The sorting and exchange of a large amount of intracellular proteins, and about 10%-20% is distributed on cell membrane and plays the part of receptor acting, receptor acting mould
Formula is mainly the endocytosis of mediating ligand.Present invention firstly discovers that sortilin antagonists AF38469 has resistance glioblastoma
Function, mainly by blocking GSK-3 β/β-catenin signal paths, and then suppress propagation and the migration of glioma cell.
Above-mentioned conclusion is also confirmed in further glioma animal model experiment.Therefore, AF38469 can be used for preparing and resist
The medicine or health products of glioma.
The beneficial effects of the present invention are be used to prepare anticol matter the invention discloses sortilin antagonists AF38469
The medicine of knurl or the new application of health products, not only expand AF38469 application, its application value are improved, to colloid
The treatment or health care of knurl bring new hope, also, using AF38469 as lead compound, pass through structural modification or transformation
Activity or reduction side effect are improved, additionally aids and further develops new anti-glioma drugs or health products.
Brief description of the drawings
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below and carried out
Explanation：
Fig. 1 shows that AF38469 can effectively suppress the invasion and attack of glioma cell, and wherein A is scratch experiment result (p ＜
0.01vs control groups), B is Transwell cells Matrigel result (p ＜ 0.01vs control groups).
Fig. 2 shows that AF38469 can effectively suppress the propagation of glioma cell, and wherein A is fluidic cell cycle detection
As a result (p ＜ 0.01vs control groups), B is CCK-8 experimental results (p ＜ 0.01vs control groups), and C is EdU cell proliferation experiment knots
Really (p ＜ 0.01vs control groups).
Fig. 3 shows that AF38469 is m- when glioma cell can suppress GSK-3 β/β-catenin signal paths and have
Dose relationship, wherein A are the immune protein marking result of 1 μM of AF38469 processing cell different times, and B is various concentrations
Immune protein marking result after AF38469 processing cells.
Fig. 4 show AF38469 on the nude mice model of subcutaneous vaccination and in-situ inoculating have suppress glioma growth and
The function of migration, wherein A are the subcutaneous gross tumor volume image into knurl of mouse, and B is that mouse is subcutaneous into knurl volume curve, and C is encephalic
The HE coloration results of dead mouse brain in situ into knurl, D is the encephalic mouse survival curve in situ into knurl.
Embodiment
The present invention is described in detail below in conjunction with accompanying drawing.The experiment side of unreceipted actual conditions in embodiment
Method, generally according to normal condition, or according to the condition progress proposed by manufacturer.
First, AF38469 can effectively suppress the invasion and attack of glioma cell
The invasive ability of glioma cell is detected by scratch experiment and Transwell cells culture cell penetration test,
Analyze influences of the AF38469 to invasion of glioma cells ability.
Scratch experiment：People's glioblastoma U87 cells of 2 hours are handled with 0.25% pancreatin digestion mitomycin,
Count after appropriate cell number is resuspended with complete medium, add in 6 orifice plates and cultivate, to next day cell it is completely adherent after, PBS is washed
Floating cells are removed, are divided into two groups (experimental group and control groups), every group of 3 holes make 3 cuts per hole and (cover rifle with 10 μ l pipettors
Head is leaned on ruler makees cut), experimental group adds 1 μM of AF38469 (solvent is DMSO) per hole, the body such as control group is added per hole
Long-pending solvent DMSO, two groups added serum-free F12/DMEM culture mediums per hole and continue to cultivate, respectively at 0 hour and 24 after cut
Hour is taken pictures under microscope, and 5 visuals field are randomly selected per hole, utilizes Image J to analyze the healing area of cell cut.As a result
After seeing that Figure 1A, U87 cells are handled 24 hours through 1 μM of AF38469, scratch area reduces 15%, and control group scratch area contracts
It is small by 38%.
Transwell cells culture cell penetration test：Handled 2 hours with 0.25% pancreatin digestion mitomycin
U87 cells, count appropriate cell number and are resuspended with serum free medium, cell density is reached 1 × 106Individual/ml, is divided into two groups
(experimental group and control group), every group of 3 cells (8.0um), the upper indoor addition of the cell of each pre-coated matrigel is above-mentioned thin
The μ l and 1 μM of AF38469 (solvent is DMSO) of 10% blood serum medium of indoor addition 600, right under the μ l of born of the same parents' suspension 200, experimental group
According to the μ l and isometric solvent DMSO of 10% blood serum medium of the lower indoor addition of group 600, cell is taken out after 36 hours, will with cotton swab
Small indoor matrigel and the cell not passed through all are wiped, and cell is put into 4% paraformaldehyde and fixes 15 minutes, PBS is washed
Wash 2 times, penetrating 10 minutes of methanol, PBS is washed 2 times, violet staining 5 minutes, PBS is washed 2 times, after cell back-off is dried,
The careful film for removing cell lower end, uses resinene mounting, in micro- Microscopic observation, the image in 5 visuals field is taken at random,
Image-pro-plus 6.0 counts cell.As a result after seeing that Figure 1B, U87 cells are handled 36 hours through 1 μM of AF38469, pass through
The cell quantity of Transwell micropores reduces 39% compared with control group.
2nd, AF38469 can effectively suppress the propagation of glioma cell
The propagation feelings of glioma cell are detected by fluidic cell cycle detection, CCK-8 experiments and EdU cell proliferation experiments
Condition, influences of the analysis AF38469 to glioma.
Fluidic cell cycle detection：U87 cells are pressed 1 × 106Individual cells/well culture is in 6 orifice plates, and next day pastes in cell
After wall, six holes are divided into two groups, experimental group adds 1 μM of AF38469 (solvent is DMSO), control group body such as addition per hole per hole
Long-pending solvent DMSO, discards culture medium after 24 hours, PBS is washed 2 times, and collected by trypsinisation cell, PBS is washed 2 times, with 4 DEG C
70% ethanol of precooling is resuspended and fixes cell, 4 DEG C of refrigerated overnights, and next day takes out cell suspension, and fixer is removed in centrifugation, and cell sinks
Shallow lake is washed 2 times with PBS, and with the propidium iodide stain liquid in cell cycle determination kit, lucifuge is dyed 30 minutes at room temperature
Afterwards, cell cycle, the change of analysis cell cycle are detected on flow cytometer.As a result Fig. 2A is seen, U87 cells are through 1 μM
After AF38469 is handled 24 hours, relative to control group, the cell cycle is arrested in the S/G2 phases.
CCK-8 is tested：U87 cells point four groups (experimental group 1,2,3 and control groups) are inoculated in 96 porocyte culture plates, often
5 multiple holes of group, cell density is 2 × 103Individual cells/well, the DMEM/F12 culture mediums 100 containing 10% hyclone are added per hole
μ l, in 37 DEG C of CO2gas incubator overnight incubations, culture medium is replaced by serum-free DMEM/F12 culture mediums by next day, continues to train
After supporting 12 hours, add 10 to experimental group 1,2,3 respectively, 100,1000nM AF38469 (solvent is DMSO), control group adds
Isometric solvent DMSO, then respectively 0, the same time point of 1,3,5 and 7 days add CCK-8 into 5 multiple holes of each group
(Cell Counting Kit-8), determines light absorption value of the cell at wavelength 450nm, utilizes after being incubated 2 hours
GRAPHPAD5.0 draws cell growth curve.As a result Fig. 2 B are seen, in AF38469 respectively to U87 cells processing 3, the feelings of 5,7 days
Under condition, compared to control group, 100nM and 1000nM AF38469 can significantly suppress the propagation of U87 cells, and with
The increase of AF38469 concentration, progressively strengthens the inhibition that U87 cells are bred, shows increasings of the AF38469 to glioma cell
Grow with obvious inhibitory action, and dose-dependent effect is presented.
EdU cell proliferation experiments：U87 cells are pressed 2 × 103Individual cells/well is inoculated in 96 porocyte culture plates, to next day
After cell is completely adherent, it is divided into two groups, experimental group adds 1 μM of AF38469 (solvent is DMSO), control group adds isometric
Solvent DMSO, after handling 48 hours, discards culture medium, adds the working solution being diluted to the A liquid in EdU staining kits, after
Continuous culture 2 hours, discards working solution, and PBS is washed 2 times, 30 minutes are fixed with 4% paraformaldehyde, fixer is discarded, with PBS and
2mg/ml glycine washes away remnants paraformaldehyde, then penetrating 10 minutes with 0.5%triton, discards penetrating liquid, PBS washings 2
It is secondary, add the dyeing working solution being configured to B, C, D, E liquid in EdU staining kits and dye 30 minutes, with containing 0.5%
Triton PBS and methanol is respectively washed 1 time, and PBS is washed 2 times, then carries out with Hoechst33342 core DNA fluorescent staining, and PBS is washed
Wash after 2 times, image is gathered under fluorescence microscope and is analyzed.As a result Fig. 2 C are seen, compared with control group, U87 cells are through 1 μM
After AF38469 is handled 48 hours, EdU positive rates are substantially reduced, and are shown 1 μM of AF38469 and can significantly be suppressed glioma
The propagation of cell.
3rd, AF38469 suppresses propagation and the migration of glioma cell by GSK-3 β/β-catenin signal paths
By western-blot immune proteins blotting experiments detect AF38469 handle U87 cells after intracellular GSK-3 β/
The change of β-catenin signal paths.
U87 cells are handled with 100nM AF38469, the total egg of cell is extracted respectively at processing 0,4,8,16,24,48 hours
In vain, concentration rear electrophoresis, transferring film, closing are determined, then uses GSK-3 β, P-GSK3 β (ser9) and β-catenin antibody to carry out respectively
Immuning hybridization, is incubated and band is shown after secondary antibody, and band gray value is calculated with Image-J softwares, draws curve.As a result Fig. 3 A, U87 are seen
After cell is handled 16 hours through 100nM AF38469, intracellular β-catenin expression is significantly reduced, GSK-3 β 9 silk ammonia
The phosphorylation of acid is significantly reduced, and shows that GSK-3 β/β-catenin signal paths are substantially suppressed.
Respectively with 0,1,10,100,1000,10000nM AF38469 handle U87 cells after 24 hours, collect cell total
Albumen, determines concentration rear electrophoresis, transferring film, closing, then use GSK-3 β, P-GSK3 β (ser9) and β-catenin antibody to enter respectively
Row immuning hybridization, is incubated and band is shown after secondary antibody, and band gray value is calculated with Image-J softwares, draws curve.As a result Fig. 3 B are seen,
After AF38469 of the U87 cells through various concentrations is handled 24 hours, intracellular GSK-3 β/β-catenin signal paths have not
With the suppression of degree, and there is dose-dependent relation in inhibition level with the increase of AF38469 concentration.
4th, AF38469 has on the nude mice model of subcutaneous vaccination and in-situ inoculating suppresses glioma growth and migration
Function
The nude mice of 4 week old is randomly divided into two groups, every group 5, every mouse bare subcutaneous injection 1 × 105Ten thousand U87 cells, 7
Start to give tumor-bearing mice intraperitoneal injection after it, experimental group gives 0.2mg/kg AF38469 (solvent is DMSO), compare
Group gives isometric solvent DMSO, and during which successive administration 21 days observes the animation of two groups of nude mices, vernier is used weekly daily
Kind of calliper and the subcutaneous tumor volumes size for recording nude mice, to after 28 days, spinal cord detachment puts to death nude mice, subcutaneous to peel off tumour simultaneously
Film recording, collects making gross tumor volume size change curve by volume size.As a result see Fig. 4 A and 4B, experimental group nude mice it is swollen
Knurl volume size is obviously reduced compared with control group.
The nude mice of 4 week old is randomly divided into two groups, every group 8, every nude mice encephalic in-situ injection 1 × 105Ten thousand U87 are thin
Born of the same parents, start to give tumor-bearing mice intraperitoneal injection, experimental group gives 0.2mg/kg AF38469 (solvent is DMSO) after 3 days,
Control group gives isometric solvent DMSO, and successive administration 10 days observes the animation of two groups of nude mices, records naked daily afterwards
The mouse death time, untill last 1 nude mice is dead, nude mice survivorship curve is drawn, is then dissected every dead nude mice,
Monoblock brain is taken out, outward appearance is visually observed, then carry out HE dyeing.The brain outward appearance of dead nude mice shows, nude mice of control group it is big
There is oedema in brain, and the brain distinct of experimental group nude mice.HE coloration results show, glioma in experimental group nude mice brain
Migration is substantially suppressed (Fig. 4 C).Survivorship curve shows that, relative to control group, the 50% survival rate time of experimental group nude mice is bright
Aobvious extension (Fig. 4 D).
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
Cross above preferred embodiment the present invention is described in detail, it is to be understood by those skilled in the art that can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (3)
- Applications of the 1.AF38469 in anti-glioma drugs or health products are prepared.
- 2. application according to claim 1, it is characterised in that the anti-glioma be suppress glioma cell propagation and Migration.
- 3. application according to claim 2, it is characterised in that the anti-glioma be by block GSK-3 β/β- Catenin signal paths suppress propagation and the migration of glioma cell.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710213815.1A CN107007601A (en) | 2017-04-01 | 2017-04-01 | Applications of the AF38469 in anti-glioma drugs or health products are prepared |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710213815.1A CN107007601A (en) | 2017-04-01 | 2017-04-01 | Applications of the AF38469 in anti-glioma drugs or health products are prepared |
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| CN107007601A true CN107007601A (en) | 2017-08-04 |
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| CN201710213815.1A Pending CN107007601A (en) | 2017-04-01 | 2017-04-01 | Applications of the AF38469 in anti-glioma drugs or health products are prepared |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20160222100A1 (en) * | 2015-01-30 | 2016-08-04 | The Board Of Trustees Of The Leland Stanford Junior University | Method of Treating Glioma |
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