CN110478339A - Butein targets the application restored in Mutation p53 conformation drug in preparation - Google Patents
Butein targets the application restored in Mutation p53 conformation drug in preparation Download PDFInfo
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- CN110478339A CN110478339A CN201910739593.6A CN201910739593A CN110478339A CN 110478339 A CN110478339 A CN 110478339A CN 201910739593 A CN201910739593 A CN 201910739593A CN 110478339 A CN110478339 A CN 110478339A
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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
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
-
- 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
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Abstract
The invention discloses buteins, and the application restored in Mutation p53 conformation drug is targeted in preparation, experimental result of the present invention is shown: compound Butein can reduce the expression of Mutation p53, the expression for increasing wild type p53 in tumour cell under Mutation p53 background, raises p53 downstream target genepuma、p21、noxa；Therefore compound Butein can restore Mutation p53 conformation, play antitumor action；Meanwhile butein can in selective degradation Mutation p53 cell p53 expression；Native compound butein is disclosed in targeting Mutation p53 and restores its conformation, performance anti-tumor function and its stemness for maintaining stem cell, these features show that butein has the application potential for the personalized treatment drug for being directed to Mutation p53 function of restoring and degrade.
Description
Technical field
The invention belongs to field of medicaments, are related to butein and are restoring the application in Mutation p53 conformation drug.
Background technique
P53 confirms that Mutation p53 can be used as the target spot of drug as existing a large amount of research, and main scheme includes induction
The degradation of mutant p53 albumen reactivates wild type p53 in tumour cell, and Mutation p53 is made to restore wild type p53 suppression cancer
Function, another strategy for targeting Mutation p53 are to develop the small molecule compound that p53 protein function can be rebuild in tumour cell,
So that mutain is changed conformation, restores its transcriptional activation function, to restore its cancer suppressing function.
Some natural products are found to have the effect of targeting Mutation p53, and butein (Butein) is also known as butein,
Chemical name is 2,3,4,4 '-tetrahydroxy chalcones (2,3,4,4 '-tetrahydroxychaleo), is a chalcones
Close object, belong to plant polyphenol, be cashew nut tree (Semecarpus anacardium) stem skin, Dalbergia
(Dalbergia odorifera) carpel, Chinese traditional herbs book on Chinese herbal medicine Chrysolophus (Caragana jubata) and lacquer tree
(Rhus verniciflua) stem main active.Currently, studies have shown that: butein can inhibit diabetes comprehensive
It seeks peace blood pressure lowering；The generation of the relevant glomerulitis of antibody can be reduced；It can inhibit the liver fibrosis by tetrachloro-methane induction；
The acute renal failure by cisplatin induction can be improved, improve the concentrating function of kidney.It is reported in test in vivo and in vitro: butein
It is able to suppress the proliferation of a variety of human tumor cells, including breast cancer, lung cancer, lymph cancer, osteosarcoma and melanoma etc. have
Extensive biological activity.Such as: promoting osteosarcoma cell that apoptosis occurs by autophagy approach；Pass through activation AMPK/FOXO3a letter
Number access inhibits the proliferation of esophagus carcinoma cell line EC 109, transfer ability, activates oxidative stress, promotes Apoptosis；Pass through inhibition
Akt-mTOR-S6 signal path and the phosphorylation modification of albumen such as FAK, ERK1/2 that vegf receptor 2 (VEGFR2) mediates and press down
Angiogenesis processed, and then inhibit tumour growth.Currently, there is not been reported in the effect of targeting Mutation p53 for butein.
Summary of the invention
In order to expand the frontier of chalcone compounds application, it is an object of the invention to develop chalcone compounds
Butein restores the natural drug of Mutation p53 as a kind of targeting, and effect is mainly: 1) Mutation p53 recovery being changed into open country
Raw type conformation, to restore its cancer suppressing function；2) by degradation Mutation p53 to inhibit growth of tumour cell process.
It is an object of the present invention to provide a kind of pharmaceutical compositions, contain a effective amount of chalcone compounds butein and pharmacy
Upper acceptable carrier.
The present invention is another object is that applying butein in preparing anti-tumor drugs targeting.
The compounds of this invention can be applied to patient in need for the treatment of by way of oral or injection；When for taking orally,
Can be made into tablet, sustained release tablets, controlled release tablet, pastille, hard or soft capsule, dripping pill, pellet, aqueous or oil suspension, emulsion, can
The powder or granule of dispersion, oral administration solution, syrup or elixir；When for injecting, the aqueous or oiliness that can be made into sterilizing is molten
Liquid, aseptic powdery, liposome, emulsion, microemulsion, nanoemulsion or micro-capsule.
The present invention is that oral or injection can further be made using chalcone compounds butein as effective active composition
Preparation, wherein one or more pharmaceutically acceptable auxiliary materials can be added to improve the assimilation effect of derived product or convenient for clothes
With the auxiliary material includes filler, diluent, adhesive, excipient, the sorbefacient, surface work of pharmaceutical field routine
Property agent and stabilizer etc., can also be added if necessary flavouring agent, pigment and sweetener etc..
The present invention targets screening system by DNA recombinant technique basis, and PUMA promoter sequence is inserted into double reporter genes
The GLuc reporter gene upstream of carrier PEZX-GA01, and obtain stablizing expression PUMA promoter fluorescein through lipofection
The H1299 P53 R175H/ P53 R273H cell of enzyme Reporter System, by detection compound to being reported in the cell
The expression of gene influences, and can characterize the combination situation of transcription factor p53 and target gene PUMA promoter, and then as mutp53
The foundation of reactivation agent primary dcreening operation, for high-throughput, the quick targeting of tumour medicine is screened；Preliminary discovery butein
(Butein) similar with PRIMA-1 (it is verified that wild type function that Mutation p53 can be restored) effect, it can be increased
The relative fluorescence element enzyme activity of PUMA promoter；Then, we pass through western blotting, it was demonstrated that, Butein can be reduced
The protein expression of p53, using immunofluorescence and immunoprecipitation assay, respectively with PAb1620 (identification wild type p53) and PAb240
(identification mutant p53), experimental result is shown: after drug-treated, Mutation p53 is reduced, and wild type p53 increases；From above
As the result is shown: prompt Butein can restore Mutation p53 conformation and degradation Mutation p53, this is mentioned for natural products oncotherapy
For new action target spot, new strategy is also provided for oncotherapy.
Detailed description of the invention
Fig. 1 is compound Butein processing H1299 p53R175H/p53R273H-PUMA promoter BS2 cell 24
Relative fluorescence element enzyme activity expression of results after hour；Wherein A figure is H1299 p53R175H-PUMA promoter BS2 cell, B
Figure is H1299 p53R273H-PUMA promoter BS2 cell；
Fig. 2 is the egg of p53 and downstream target gene after compound Butein is handled HT29, SK-BR-3 cell 12,24,48 hours
White expression of results；Wherein A figure is HT29, and B figure is SK-BR-3 cell；
Fig. 3 is the expression of wild type p53 and mutant p53 after compound Butein is handled HT29, SK-BR-3 cell 48 hours
As a result.Figure is immunoprecipitation assay result；Wherein upper figure is HT29, and the following figure is SK-BR-3 cell；
Fig. 4 is the expression of wild type p53 and mutant p53 after compound Butein is handled HT29, SK-BR-3 cell 48 hours
As a result.Figure is immunofluorescent test as a result, wherein A figure is HT29, and B figure is SK-BR-3 cell.
Specific embodiment
Below by embodiment, invention is further described in detail, but protection scope of the present invention be not limited to it is described
Content, it is conventional commercial unless otherwise specified using reagent that method is all made of conventional method unless otherwise specified in embodiment
Reagent or the reagent configured using conventional method.
Embodiment 1:mutp53 reactivation agent screening
1, well-grown, the H1299 p53R175H/ p53R273H-PUMA promoter BS2 cell of convergence degree 80-90% are taken
(stablizing expression PUMA promoter luciferase reporting gene), is seeded in 6 orifice plates by 1.5 × 105 cells/wells, and rolling is even, puts
It is complete to cell patch wall for 24 hours to enter 37 DEG C of incubators incubations；
2, the complete H1299 p53R175H/ p53R273H-PUMA promoter BS2 cell of wall will be pasted to take out from incubator,
Old culture medium is sopped up, 1640 culture medium of fresh RPMI of 2mL concentration containing relevant work compound is added, rolling is even, is put into 37 DEG C of trainings
It supports case and is incubated for 15h；
3, it after GLuc, SEAP belong to the albumen of secreting type, therefore compound is disposed, can gently collect in cell culture
Clearly, the activity of GLuc, SEAP are detected immediately；
A, the activity of GLuc is detected
(1) the 100 μ L cells and supernatants that collection compound is disposed are subsequently placed in room temperature to 1.5mL centrifuge tube；
(2) by 10 × GL-S buffer take out and thaw at normal temperature, take appropriate 10 after mixing well × GL-S buffer
1 × GL-S buffer is diluted to ultrapure water.1 × GL-S buffer dosage is 100 μ L/ reaction；
(3) under light protected environment, the Substrate GL (100 ×) of 1/100 total volume is added toward 1 × GL-S buffer, it is sufficiently mixed
It is even, match to obtain GLuc working solution；
(4) supernatant to be detected and GLuc working solution are moved into respectively in 25 DEG C of hybrid heaters and is incubated 25 minutes；
(5) clean opaque 96 hole elisa Plates are taken, the supernatant finished will be incubated and working solution taking-up pipettes 10 respectively,
100 μ L are mixed evenly into hole with liquid-transfering gun；
(6) after mixing, 96 orifice plates are put into 25 DEG C of hybrid heaters and are incubated for 1 minute, are then by the read plate mode setting of microplate reader
" chemiluminescence " detects the biological fluorescent that GLuc oxyluciferin is occurred in 96 orifice plates.(it should strive for after incubation at room temperature at 5 points
Read plate in clock)
B, the activity of SEAP is detected
(1) 50 μ L cells and supernatants are drawn to new 1.5mL centrifuge tube, then 15min is heated in 65 DEG C of hybrid heaters, takes
Under to be placed in ice bath spare；
(2) by 10 × AP buffer take out and natural thaw at normal temperature, take suitable 10 after mixing well × AP buffer
Liquid is diluted to 1 × AP buffer with ultrapure water.The dosage of 1 × GL-S buffer is 100 μ L/ reaction；
(3) under light protected environment, the Substrate AP (100 ×) of 1/100 total volume is added toward 1 × AP buffer, it is sufficiently mixed
It is even, match to obtain SEAP working solution；
(4) supernatant to be detected and SEAP working solution are moved into respectively in 25 DEG C of hybrid heaters and is incubated 10 minutes；
(5) clean opaque 96 hole elisa Plates are taken, the supernatant finished will be incubated and the taking-up of SEAP working solution pipettes respectively
10,100 μ L are mildly mixed into hole with liquid-transfering gun；
(6) after mixing, 96 orifice plates are put into 25 DEG C of hybrid heaters and are incubated for 10 minutes, are then detected in 96 orifice plates by microplate reader
The luminous intensity of SEAP and substrate reactions.
The result is shown in Figure 1, it can be seen that can be increased after Butien processing with the expression of PUMA reporter gene, i.e., in Cong Tuzhong 1
Its relative fluorescence element enzyme activity can be increased.
Embodiment 2: immunoblot experiment
(1) HT29, SK-BR-3 cell of logarithmic growth phase, using (20/10 μM) processing 12h of butein compound, for 24 hours,
48h, with cell scraper by under cell scraper, 4 DEG C of centrifugations cleans one time with 1 × PBS, are moved in 1.5mL centrifuge tube and are obtained cell and sink
It forms sediment；
(2) suitable cell pyrolysis liquid is added, cell precipitation is ultrasonically treated, condition 25-30Hz, super 10s stop 5s, and totally 10
It is secondary, if precipitating can thoroughly not increase ultrasonic number suitably by complete ultrasound.The albumen extracted, which utilizes, examines blue or BCA method
It is quantified；
(3) albumen loading system: 15 μ L, 20 μ g is configured, adds bromophenol blue (plus beta -mercaptoethanol) dense eventually in albumen loading system
Degree is 1 × loading system；After system prepares, boiling water boiling 7min or so, cooling centrifugation；
(4) run glue: configuring 12% separation gel and 4.5% concentration glue, configuration 1 × Running Buffer (SDS containing 1%),
Prepare loading；
(5) transferring film: configuration transferring film liquid, 10 × Running Buffer pure water constant volume of 200mL methanol+100mL to 1L, pvdf membrane
With being activated before with methanol；It is white according to plank black flour-- three layers of sponge filter paper-three layers of pvdf membrane of-glue-filter paper-sponge-plank
Face sequence prepares membrane-transferring device.Transferring film condition: 180mA, 300V, 2h30min or 3h；
(6) 5% milk closing, first clean the transferring film liquid on pvdf membrane with 1 × PBST, then with 5% 37 DEG C of closing 2h of milk or
It is that 4 DEG C of shaking table closings are stayed overnight；
(7) mark primary antibody: with 1 × PBST clean close milk, then use prepare p53 (Do-1) (1:500), PUMA (1:100),
4 DEG C of p21 (1:1000), Noxa (1:500) antibody shaking tables are stayed overnight；
P53 (Do-1) (SC-126, Santa), PUMA (7467, CST),
P21 (556430, BD), Noxa (SC-30209, Santa) 1:10000 dilution proportion carry out secondary antibody closing, room temperature
2h；
(8) develop: according to 1:1 proportional arrangement developer solution, developing；
As a result see in Fig. 2: Cong Tuzhong 2 it can be seen that Butien makes the table of Mutation p53 as time increases after Butien processing
Up to reduction, while the protein expression of p53 downstream target gene PUMA, p21, Noxa being induced to raise.
Embodiment 3: immunoprecipitation assay
(1) well-grown, HT29, SK-BR-3 cell of convergence degree 80-90%, by 2 × 10 cell inoculation: are taken6A cell/ware connects
For kind to 10cm culture dish (big ware), rolling is even, and it is complete to cell patch wall for 24 hours to be put into 37 DEG C of incubators incubations；
(2) compound is handled: will be pasted complete HT29, SK-BR-3 cell of wall and is taken out from incubator, exhaust old culture medium, is added
Fresh 1640 culture medium of 10mL (Butein containing 10 μM, 20 μM), rolling is even, is put into 37 DEG C of incubators and is incubated for corresponding time point；
(3) it collects cell: collecting cell with cell scraper and move into cell suspension in the centrifuge tube of 15mL, 4 DEG C, 1000g centrifugation
5mins is discarded supernatant, and 1 × PBS is pre-chilled with 1mL is resuspended suspension is gone to the centrifuge tube of 1.5mL after cell, and 4 DEG C, with 1000g
It is centrifuged 5mins, discard supernatant and stayed precipitating is stored in -80 DEG C for use；
(4) after cell precipitation is resuspended with appropriate RIPA lysate, ultrasonication, function protein extraction: are carried out to sample in ice bath
Rate is set as 25%, ultrasonic 10s, is spaced 6s, ultrasound 10 times；Then, sample is placed in 4 DEG C of 360 ° of mute vortex mixer cracking 2-3h.4
DEG C, 10000g be centrifuged 30mins, the careful supernatant that shifts is to new 1.5mL centrifuge tube；
(5) prewashing: 20 μ L Protein A+G Sepharose beads is taken to manage and be resuspended with 1 × PBS of 1mL clear to new 1.5mLEP
After washing sepharose 4B, 4 DEG C, 8000rpm is centrifuged 1min, abandons supernatant, is repeated twice, by the IgG antibody of (2) protein sample and 1 μ g
It moves into EP pipe and is placed in 360 ° of mute blending instruments, 4 DEG C of prewashing 2h to removal and the albumen of pearl non-specific binding, at 4 DEG C,
After being centrifuged 30mins, it is spare to be transferred to the new 1.5mL of pre-cooling EP pipe by 5000rpm for supernatant；
(6) immune precipitation: according to determination of protein concentration as a result, taking the protein sample of 100-500 μ g to new 1.5mL EP
The antibody (or IgG) of 1 μ g mesh is added in pipe, moves in 4 DEG C of 360 ° of mute blending instruments and is immunoreacted overnight, next day, will be immune anti-
Answer sample to be completely transferred in the Protein A+G sepharose 4B that 20 μ L are sufficiently cleaned with PBS, add 600 μ L pre-cooling 1 ×
PBS continues to be incubated overnight in 4 DEG C of 360 ° of mute blending instruments；
(7) clean: sample takes out from 4 DEG C, and after ice bath stands 2mins, 6000g, 4 DEG C are centrifuged 1min, abandons supernatant；Along wall plus
Enter 1mL and 1 × PBS is pre-chilled, after gently cleaning pearl, 4 DEG C, 6000g are centrifuged 1min, abandon supernatant, clean 8-10 times；
(8) denaturation and Western blotting: 16 1 × PBS of μ L and 4 μ L 5 × Loading dye being added into cleaning sample pipe,
After of short duration low-speed centrifugal, being put into boiling water bath and boiling 7-10mins will be completely dissociated the immune complex for being incorporated into sepharose 4B；
After denatured sample condensation, it is centrifuged and carries out SDS-PAGE electrophoresis；
Preparation of reagents needed for this experiment
(1) 0.5M EDTA(pH7.4) solution preparation:
(2) preparation of RIPA lysate:
(3) preparation of antibody:
As a result see in Fig. 3: Cong Tuzhong 3 after can be seen that Butein processing, Butein reduces the expression of Mutation p53, while wild
The expression of raw type p53 increases.
Embodiment 4: immunofluorescent test
(1) well-grown, HT29, SK-BR-3 cell of convergence degree 80-90%, by 2 × 10 cell inoculation: are taken5A cell/ware connects
For kind to the 6cm culture dish (middle ware) for being placed with sterilized slide, rolling is even, and it is complete to cell patch wall for 24 hours to be put into 37 DEG C of incubators incubations
Entirely；
(2) compound is handled: will be pasted complete HT29, SK-BR-3 cell of wall and is taken out from incubator, sop up old culture medium, is added
Fresh 1640 culture medium of 2mL (Butein containing 10 μM, 20 μM), rolling is even, is put into 37 DEG C of incubators and is incubated for corresponding time point；
(3) cell cleans: six orifice plates of creep plate are taken out, sop up old culture medium, use 1 × PBS by compound after treatment
2min is cleaned and stands, in triplicate；
(4) 1mL fixer, 27 DEG C of fixed 10min fixed cell: are added in every hole.After the completion of fixation, fixation is sopped up
Liquid cleans using 1 × PBS and stands 2min, in triplicate；
(5) Cell-transmission model: being added 800 μ L, 1%NP-40 processing 5min in every hole, permeability of cell membranes can be improved,
Conducive to the entrance of antibody, after the completion of punching, exhaust NP-40, and 2min is cleaned and stood using 1 × PBS, repeats three
It is secondary；
(6) it closes: 1mL is added in every hole, 5%BSA closes 2h, the specific binding of purpose antibody can be improved.Closing is completed
Afterwards, exhaust BSA, 2min is cleaned and stood using 1 × PBS, in triplicate.It is significant to note that subsequent experimental needs
It is carried out in the dark；
(7) it marks primary antibody: antibody (being prepared using 2%BSA), every 100 μ L of hole, 4 DEG C of layers is proportionally added into according to requirement of experiment
It analyses cabinet to be incubated overnight, after the completion of incubation, exhaust antibody, 2min is cleaned and stood using 1 × PBS, in triplicate；
(8) it marks secondary antibody: corresponding fluorescence secondary antibody is selected according to primary antibody, in proportion dilution (being prepared using 2%BSA), every hole
100 μ L, 27 DEG C of incubation 2h.After the completion of incubation, exhaust antibody, 5min is cleaned and stood using 1 × PBS, in triplicate；
(9) core dye nuclear targeting: is carried out using DAPI.Dilution (being prepared using 2%BSA) in proportion, every 100 μ L of hole,
27 DEG C of incubation 30min.After the completion of incubation, exhaust DAPI, 5min is cleaned and stood using 1 × PBS, in triplicate；
(10) film-making: preparing glass slide in advance and mark, then will lid in suitable anti-quencher is added dropwise on glass slide
Slide is gently placed on glass slide (have cell one is encapsulated in down in anti-quencher), avoids the generation of bubble.Film-making is completed
Afterwards will in flakes be placed in magazine can carry out fluorescence microscope take pictures or 4 DEG C preservation；It is significant to note that in experimentation
When middle addition liquid inlet handhole plate, movement wants light and slow, avoids the cell directly impacted on lid fragmentation, in order to avoid cause cell detachment；
Preparation of reagents needed for this experiment
(1) 20% sucrose solution is prepared: being weighed 10g sucrose powder and is dissolved in suitable 1 × PBS, mix dissolution, constant volume arrives
50mL, room temperature storage are spare；
(2) 5% paraformaldehyde solutions are prepared: it weighs 2.5g paraformaldehyde powder and is dissolved in suitable 1 × PBS, mix dissolution,
To 50mL, room stores for future use constant volume；
(3) 1%NP-40 solution is prepared: being taken 0.5mL NP-40 to be dissolved in suitable 1 × PBS, is mixed dissolution, constant volume arrives
50mL, room store for future use；
(4) fixer is prepared:
As a result see in Fig. 4: Cong Tuzhong 4 after can be seen that Butien processing, Butien reduces the expression of Mutation p53, while wild
The expression of raw type p53 increases.
Claims (3)
- Restore to apply in Mutation p53 conformation drug 1. butein is targeted in preparation.
- 2. application according to claim 1, it is characterised in that: butein restores Mutation p53 conformation drug as targeting, makes Mutation p53 restore wild type function, while in selective degradation Mutation p53 cell p53 expression.
- 3. butein restores Mutation p53 conformation drug application in preparation of anti-tumor drugs as targeting.
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