CN107028935A - Application of the wogonin in treatment clear-cell carcinoma medicine is prepared - Google Patents
Application of the wogonin in treatment clear-cell carcinoma medicine is prepared Download PDFInfo
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Abstract
The present invention relates to application of the wogonin in treatment clear-cell carcinoma medicine is prepared.Present invention firstly discloses application of the wogonin as treatment clear-cell carcinoma medicine.Experimental result confirms that abnormal rapid DNA is replicated and cell propagation is one of key character of tumour, and suppressing DNA replication dna can cause DNA of tumor cell to damage, inducing apoptosis of tumour cell.Wogonin can effectively suppress renal cell carcinoma cell DNA replication dna and propagation, suppress cell invasion and migration, and inducing cell DNA damage induces Apoptosis.The mechanism of action of wogonin is relevant with the expression that specificity suppresses CDC6 albumen in renal cell carcinoma cell.Extensively, the also treatment for clear-cell carcinoma provides more medicament selections to application prospect of the wogonin in renal cell carcinoma treatment.
Description
Technical field
The present invention relates to application of the wogonin in treatment clear-cell carcinoma medicine is prepared, belong to drug development technology neck
Domain.
Background technology
With the change of people life style and living environment, the morbidity and mortality of cancer are in rising trend,
It is also global focus health problem as the major reason of human mortality is caused.Kidney is the most common evil of urinary system
One of property tumour, the incidence of disease accounts for the 3% of all malignant tumours, and its incidence of disease is in rising trend in recent years.According to world's cancer
Center data shows that the U.S. in 2015 predicts that new patients with renal cell carcinoma number is 61,560, tumour-specific Died Patients number
Also 14,080 are up to.The number of patients of China's kidney also increases year by year, as uropoiesis male genital second tumour.
Clear-cell carcinoma (renal cell carcinoma, RCC) is originating from the substantive renal cells of kidney, the incidence of disease
Height, about 90% renal cancer is RCC.RCC is for traditional radiotherapy, chemotherapy and insensitive, therefore for RCC especially middle evenings
Phase metastatic RCC, immunization therapy and targeted drug treatment have turned into the therapeutic strategy that major guides are recommended.Lost especially as molecule
The development learned is passed, increasing signaling pathway molecule, which is found in, exercises important function in RCC.On this basis, it is beautiful
State, Europe and China have approved the inhibitor medicaments such as Sutent, Sorafenib of increasing targeting EGFR-TK
Deng for treating advanced metastatic RCC.Because patient benefits extremely limited by immunization therapy, targeted therapy turns into evening
The first-line treatment means of phase kidney.However, with the application of molecular targeted agents, the drug resistance problems of targeted drug have a strong impact on
The curative effect of RCC treatments.Under the historical background of accurate medical treatment, the individuation under patient gene's detection is instructed targets medication also not
The problem of can solve advanced renal cell carcinoma patient targeting resistance.Therefore, late kidney is thin for targeted therapy and immunization therapy
Offer limited effectiveness in the treatment of born of the same parents' cancer, and toxic side effect is larger, expensive.So finding novel drugs, exploitation new treatment turns into
The primary study direction of kidney treatment.
Chinese medicine shows certain effect in the treatment of malignant tumour, and toxic side effect is smaller.From effective antitumor
Isolated active natural compounds are one of important means of anti-cancer agent research and development in traditional Chinese medicine.Labiatae
(Labiatase) plant radix scutellariae is China's conventional Chinese medicine simply.Wogonin is that radix scutellariae and belonging to together is extracted in corresponding plant roots
Flavone compound, is one of radix scutellariae effective ingredient.Research have shown wogonin there is anti-inflammatory, it is anti-oxidant, it is antiviral
Etc. many bioactivity.
In addition, wogonin can be by increasing the content of reactive oxygen species in stomach cancer, induced tumor cell withers
Die, suppress the various ways such as resistance of new vessels generation and reverse chemotherapeutics around tumour and play antineoplastic function.
But because the pathogenesis of different cancer cells is differed, whether wogonin can regulate and control clear-cell carcinoma and divide
Handset system is unclear.
The content of the invention
In view of the deficiencies of the prior art, the present invention provides application of the wogonin in treatment clear-cell carcinoma medicine is prepared,
Specifically related to wogonin prepare clear-cell carcinoma clinical medicine in medical usage.
Technical solution of the present invention is as follows：
Application of the wogonin in treatment kidney medicine is prepared.
Above-mentioned wogonin is by causing renal cell carcinoma cell DNA replication dna obstacle, radiation-indued DNA damage, inducing cell apoptosis.
According to some embodiments of the present invention, the kidney is clear-cell carcinoma.
According to some embodiments of the present invention, the administration concentration of the wogonin is 1~100 μM.
Unless otherwise defined, all technical terms or proprietary vocabulary used herein have the common of technical field
The implication that technical staff is generally understood.
Wogonin (wogonin), also known as 5,7- dihydroxy -8- methoxyl group -2- phenyl -4H-1- benzofuran -4- ketone.
It is yellow needles thing under normal temperature, it is water insoluble, organic solvent is soluble in, such as ethanol, acetone.Wogonin is from Huang
The flavone compound extracted in the root of a kind of reed mentioned in ancient books, with the effect such as anti-inflammatory, reducing blood lipid, antithrombus formation.
Kidney is one of most common malignant tumour of urinary system, including clear-cell carcinoma, the nephroblastoma and carcinoma of renal pelvis etc.
Clear-cell carcinoma (renal cell carcinoma, RCC), also known as Grawitz's tumor, are the most common pathology classes of kidney
Type, accounts for the 80%~85% of kidney.RCC is initiated by the malignant tumour of the substantive renal cells of kidney.RCC is for tradition
Radiotherapy, chemotherapy and insensitive.
Treatment by Chinese herbs tumour, which has, improves the state of an illness, extension paracmasis, Small side effects, the late result spy such as good and low-cost
Point, has shown the advantage of uniqueness.
In clear-cell carcinoma research, analyzed commonly using HK cells's JEG-3 786-O and OS-RC-2 cell gene or
Influence of the medicine to renal cell carcinoma cell tumor characteristic.And control is used as, normal human embryo kidney (HEK) epithelial cell line HEK293 cells are then
Commonly use with comparative drug to kidney and the Difference In Toxicity of normal nephrocyte, to assess the toxic side effect of tumour medicine.
According to some embodiments of the present invention, the administration concentration of the wogonin is 1-100 μM, such as 1-90 μM,
1-80 μM, 1-70 μM, 1-60 μM, 1-50 μM, 1-40 μM, 1-30 μM, 1-20 μM, 1-10 μM, 5-100 μM, 5-90 μM, 5-80 μ
M、5-70μM、5-60μM、5-50μM、5-40μM、5-30μM、5-20μM、5-10μM、10-100μM、10-90μM、10-80μM、
10-70μM、10-60μM、10-50μM、10-40μM、10-30μM、10-20μM、20-100μM、20-90μM、20-80μM、20-
70μM、20-60μM、20-50μM、20-40μM、20-30μM、30-100μM、30-90μM、30-80μM、30-70μM、30-60μ
M、30-50μM、30-40μM、40-100μM、40-90μM、40-80μM、40-70μM、40-60μM、40-50μM、50-100μM、
50-90μM、50-80μM、50-70μM、50-60μM、60-100μM、60-90μM、60-80μM、60-70μM、70-100μM、70-
90 μM, 70-80 μM, 80-100 μM, 80-90 μM or 90-100 μM.According to some embodiments of the present invention, the Chinese radix scutellariae
The administration concentration of element is 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM,
65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM or 100 μM.
Pharmaceutical composition and medicine box
Term " pharmaceutical composition " represents one or more compounds containing therapeutically effective amount and its pharmaceutically may be used
Dynamic isomer, solvate, hydrate or the salt of receiving, the mixture with other pharmaceutically acceptable carriers.Will be described
The purpose that compound is prepared into pharmaceutical composition is to be more easily administered to object.
Term " medicine box " or " kit " are used interchangeably in the application.This application discloses the institute comprising therapeutically effective amount
State therapeutic agent or the medicine box of pharmaceutical composition.According to some embodiments of the application, the medicine box is also comprising one or more
Other therapeutic agents.According to some embodiments of the application, the medicine box also includes operation instructions.According to certain of the application
A little embodiments, the medicine box also includes the device for corresponding administering mode, such as, but not limited to syringe needle.
Wogonin can be prepared into any one formulation pharmaceutically allowed, including but not limited to piece in the present invention
Agent, oral agents, electuary, injection, liposome, target administration injection, pill, capsule, granule, pulvis, suppository, dissipate
Agent, paste, patch, parenteral solution, solution, suspension, spray, lotion, drops, liniment etc..Described pharmaceutical composition can be made into
Dry powder form, and with sterilized water or buffer solution mix that solution form is made before administration.The pH of the buffer solution is usually
3-11, preferably 5-9, more preferably 7-8.
It is suitable that term " administration ", " giving " or " bestowing " refers to pass through the compound or pharmaceutical composition of doses
Administering mode gives object.
" administering mode " include but is not limited to oral administration, intravenous administration, administration in respiratory tract, sublingual administration,
Local administration, intramuscular adminstration, eye drops, Transdermal absorption, parenteral, Intraperitoneal medication, vagina administration, cheek are given
Any administering modes known in the art such as medicine, per rectum administration.Those skilled in the art should be recognized that the administering mode of object
Depending on Multiple factors, the factor includes position, the age of object, the order of severity of disease and the drug regimen of disease
Composition of thing etc..
In this application when " about " is used to modify numerical value, refer to the numerical value can fluctuate ± 10%, ± 9%,
± 8%, in the range of ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1%.
Unless be otherwise noted in this application or otherwise clearly contradicted, in the context of description the application (including
In the context of claim) term " one kind " that uses, " one ", " described ", "the" and " at least one " and similar refer to
In generation, is interpreted covering odd number and plural number.Unless be otherwise noted in this application or otherwise clearly contradicted, institute in the application
The term "comprising" that uses, " having ", " comprising " and " containing " are interpreted open-ended term (" including but is not limited to ").Remove
Non- to be otherwise noted in this application or otherwise clearly contradicted, all methods described herein can be according to art technology
The understanding of personnel, is carried out in any suitable order.
All patents, patent application and the bibliography quoted in the application are incorporated by this Shen by reference
Please, its incorporated extent is individually recited as reference just as each document.If the application and provided herein is document between
In the presence of conflict, content that should be in the application is defined.
Beneficial effect
Present invention firstly discloses application of the wogonin as treatment clear-cell carcinoma medicine.Experimental result is confirmed, abnormal
Rapid DNA is replicated and cell propagation is one of key character of tumour, and suppressing DNA replication dna can cause DNA of tumor cell to damage,
Inducing apoptosis of tumour cell.Wogonin can effectively suppress renal cell carcinoma cell DNA replication dna and propagation, suppress cell invasion and
Migration, inducing cell DNA damage induces Apoptosis.The mechanism of action of wogonin suppresses renal cell carcinoma cell with specificity
The expression of interior CDC6 albumen is relevant.Application prospect of the wogonin in renal cell carcinoma treatment is extensive, also controlling for clear-cell carcinoma
Treat and provide more medicament selections.
Brief description of the drawings
Fig. 1 shows that growth of the wogonin to clear-cell carcinoma is inhibited；
Wherein：Fig. 1 (a) shows that MTT experiment result shows that wogonin grows to the renal cell carcinoma cell of in vitro culture
It is inhibited；
Data in picture are the results of 3 parallel laboratory tests, and 5 repeating holes of experiment setting, are examined, * is represented using T every time
p≦0.05；* represents p≤0.01；
Fig. 1 (b) shows that growth of the wogonin to internal nude mice model of human colon clear-cell carcinoma transplantable tumor is inhibited；
Data in figure are 5 mouse reproducible results, are examined using T, * represents p≤0.05；* represents p≤0.01；
Fig. 2 shows Transwell test result indicates that invasion and attack of the wogonin to the renal cell carcinoma cell of in vitro culture
It is inhibited；
Data in figure are the results of 3 parallel laboratory tests, and experiment every time randomly selects 3 visual field countings, and each visual field is thin
Born of the same parents' number is more than 200；As a result examined using T, * * represent p≤0.01；
Fig. 3 shows that wogonin suppresses the DNA replication dna of renal cell carcinoma cell, induced DNA damage；
Wherein：Fig. 3 (a) shows EdU test result indicates that wogonin suppresses renal cell carcinoma cell DNA replication dna efficiency；
Fig. 3 (b) shows that comet result shows wogonin induction renal cell carcinoma cell DNA damage increase；
Fig. 4 shows that wogonin can induce the apoptosis of renal cell carcinoma cell；
Wherein：Fig. 4 (a) shows Tunel test result indicates that wogonin can induce renal cell carcinoma cell apoptosis；
Fig. 4 (b) shows that immunoblot experiment (Western blot) result shows that wogonin can substantially increase activation
The expression of caspase (caspase) 3；
Data in figure are the results of 3 parallel laboratory tests, and experiment every time randomly selects 3 visual field countings, and each visual field is thin
Born of the same parents' number is more than 200；As a result examined using T, * * represent p≤0.01.
Fig. 5 shows that wogonin can reduce the expression realization pair of CDC6 albumen in renal cell carcinoma cell by specificity
The inhibitory action of clear-cell carcinoma DNA replication dna；
Data in figure are the results of 3 parallel laboratory tests, and experiment every time randomly selects 3 visual field countings, and each visual field is thin
Born of the same parents' number is more than 200；As a result examined using T, * * represent that p≤0.01, # represents p≤0.05；
Wherein：Fig. 5 (a) shows that immunoblot experiment (Western blot) result shows that wogonin can be reduced substantially
The expression of CDC6 albumen in renal cell carcinoma cell；
Fig. 5 (b) shows that immunoblot experiment (Western blot) result shows that wogonin does not reduce human lung cancer, pancreas
The expression of CDC6 albumen in gland cancer, liver cancer and stomach cancer cell；
Fig. 5 (c) transfection external source CDC6 expression plasmids, which can effectively save wogonin, causes CDC6 eggs in renal cell carcinoma cell
The reduction expressed in vain；
Fig. 5 (d) shows EdU test result indicates that wogonin is by suppressing CDC6 protein expressions in renal cell carcinoma cell
Realize the suppression to renal cell carcinoma cell DNA replication dna efficiency.
Embodiment
Below in conjunction with the instantiation content that the present invention is furture elucidated, but protection scope of the present invention is not limited to this
A little examples.
Embodiment 1：Growth of the wogonin to the renal cell carcinoma cell of in vitro culture is inhibited
This example demonstrates that growth of the wogonin to the renal cell carcinoma cell of in vitro culture is inhibited.In this reality
Apply in example, the renal cell carcinoma cell is people's kidney clear cell adenocarcinoma cell 786-O and OS-RC-2 cell.
Wogonin is purchased from Shanghai Yuan Ye bio tech ltd (production number ZCX-00023510-005), is dissolved in
0.1M (mol/L) mother liquor is configured in DMSO and in -20 DEG C of storages.MTT cells are bred and citotoxicity detection kit purchase
From green skies Bioisystech Co., Ltd (production number C0009).Human embryonic kidney epithelial cells HEK293 (production number GNHu-43), people
Kidney clear cell adenocarcinoma cell 786-O (production number TCHu186) and OS-RC-2 (production number TCHu 40) cell are purchased from China
The American Type Culture Collection committee of academy of sciences cell bank, cell culture using RPMI-1640 culture mediums (Gibco companies of the U.S.,
Production number 11875127) 10% (V/V) hyclone of addition (Gibco companies of the U.S., (production number 1099141), and add
100U/ml penicillin and 100mg/ml streptomysins.All cells are using 37 DEG C, CO2 partial pressures as 5% (percentage by volume)
CMC model.
Tetrazolium bromide (MTT) is tested：By HEK293,786-O and OS-RC-2 cell in exponential phase with 10 × 104/
Ml cell concentrations are inoculated with 96 porocyte culture plates, per the μ L of hole 100, if 5 multiple holes, are placed in 37 DEG C, 5%CO2Cultivated in incubator
24 hours or so, then be separately added into it is final concentration of contain 5, the RPMI-1640 of 10,20,40,60,80 μM of wogonins is cultivated completely
Base, the cell for adding 0.1% (volume fraction) DMSO processing is set to control group.After being incubated 48 hours, 20 μ l 5mg/ are added per hole
Ml MTT working solutions.Culture plate is put back into 37 DEG C, 5%CO after shaking up2Continue in incubator after cultivating 4-6 hours, MTT will be contained
The supernatant of working solution is carefully removed, and 100 μ L dimethyl sulphoxide solutions are added per hole, culture plate is put into 37 DEG C, 5%CO2Culture
It is incubated in case, after purple crystal all dissolves and solution transparency is homogeneous (about 20 minutes), in multi-function microplate reader 570nm ripples
Strong point determines absorbance (OD), and cell survival rate is calculated with OD values.
Cell survival rate=(test group cell OD values-blank group cell OD values)/(cellular control unit OD values-blank group is thin
Born of the same parents OD values) × 100%；
As a result as shown in Fig. 1 (a), wogonin can significantly inhibit the growth of renal cell carcinoma cell, such as 20 μM Chinese radix scutellariaes
Element processing 786-O cells are after 48 hours, and compared with not adding wogonin processing 786-O cells, cell viability is reduced about
45%；After 40 μM of wogonins are handled 48 hours, cell viability reduces about 77%；After 60 μM of wogonins are handled 48 hours,
Cell viability reduces about 83%.Similar with 786-O cells, the vigor that wogonin processing may also lead to OS-RC-2 cells has
It is decreased obviously.Compared with not adding wogonin processing OS-RC-2 cells, after 40 μM of wogonins are handled 48 hours, cell is lived
Power reduces about 30%；After 60 μM of wogonins are handled 48 hours, cell viability reduces about 55%；80 μM of wogonin processing
After 48 hours, cell viability reduces about 70%.
It is contrasted however, can substantially suppress the characteristics of renal cell carcinoma cell grows with wogonin, wogonin is aligned
Normal kidney epithelial growth inhibitory action is weaker.As a result as shown in Fig. 1 (b), after 20-60 μM of wogonin is handled 48 hours, its cell
Vigor is compared with untreated HEK293 cells, without difference.And after 80 μM of wogonins are handled 48 hours, with untreated cell phase
Than the HEK293 cell viabilities after processing only reduce by 20%.
These results show that growth of the wogonin to the renal cell carcinoma cell of in vitro culture has specific inhibitory action,
And the inhibitory action shows significant concentration dependent.
Embodiment 2：Growth of the wogonin to internal nude mice model of human colon clear-cell carcinoma transplantable tumor is inhibited
This example demonstrates that growth of the wogonin to internal nude mice model of human colon clear-cell carcinoma transplantable tumor is inhibited.
In the present embodiment, the renal cell carcinoma cell is people's kidney clear cell adenocarcinoma cell OS-RC-2 cells.OS-RC-2 cells have
Stronger internal one-tenth knurl ability, is the important models that experiment in vivo analyzes renal cell carcinoma cell growth ability.
Nude mice is female BAl BIc/c nude mouses, limits public week old 4-6 weeks, and being purchased from Beijing magnificent experimental animal technology of dimension tonneau has
Limit company (strain coding 211).In 12 hours sunshine, temperature was 23 ± 2 DEG C, and humidity is raising under conditions of 55 ± 5%, simultaneously
Give the water and feed of rodent feeding standard.1 × 10 will be contained6100 μ l's of individual exponential phase OS-RC-2 cells
PBS is injected into flank section skin on the right side of nude mice back and subcutaneously, after being inoculated with 3 days, weighs and be grouped.
If wogonin treatment group (40mg/kg) and control treatment group (physiological saline).Every group of animal is 5, is carried out
Intraperitoneal injection, the next day injection once, successive administration 3 weeks.Mouse is put to death after being administered 3 weeks with spinal cord dislocation method, is carefully peeled
Tumor tissues, are absorbed with blotting paper after being rinsed with aseptic deionized water and show moisture, statistics tumor weight and gross tumor volume.
As a result as shown in Fig. 1 (c), compared with control treatment group, the weight and volume difference of wogonin treatment group tumour
It has dropped about 50% and 60%.
These results illustrate that wogonin can significantly inhibit the growth of renal cell carcinoma cell in Mice Body.
Embodiment 3：Invasion and attack of the wogonin to renal cell carcinoma cell are inhibited
This example demonstrates that invasion and attack of the wogonin to renal cell carcinoma cell are inhibited.In the present embodiment, institute
It is people's kidney clear cell adenocarcinoma cell 786-O and OS-RC-2 cell to state renal cell carcinoma cell.
Transwell cells containing matrigel matrix are purchased from U.S. CORNING (Corning) company (production number
354480), crystal violet dye liquor is purchased from green skies Bioisystech Co., Ltd (production number C0121).
Attack test experience：The method be the same as Example 1 of cell culture.Exponential phase cell is chosen, various concentrations are added
Wogonin is handled 24 hours, and cell precipitation is collected in pancreatin digestion, is resuspended after PBS 3 times with serum free medium, adjust to
5×105Individual/milliliter, takes 200 μ l to be seeded in the upper chamber of transwell systems, sets 3 multiple holes.Under transwell in room
The 600 μ l culture medium containing 10%FBS is added as chemotactic factor, it is to avoid produce bubble；Orifice plate is placed on 37 DEG C, 5%CO2
Cultivated 36 hours in incubator；Gently wipe the cell of transwell cells filter membrane upper surface with swab stick, then by transwell
Cell, which is put into methanol, fixes 20min, and PBS rinses 3 removal methanol, plus crystal violet dye liquor dyeing 30min, and flowing water is washed till no longer
Decolourize, normal temperature dries；Taken pictures under microscope, each transwell cells randomly select about 10 visuals field and taken pictures, then
Count staining cell.As a result as shown in Fig. 2 wogonin processing significantly reduces the cell number through the filter membrane containing matrigel,
And with the increase of wogonin concentration, reduction trend is presented in the cell number through filter membrane, and such as 20 μM wogonin processing 786-O are thin
After born of the same parents 24 hours, compared with not adding wogonin processing cell, 786-O cell invasion abilities reduce about 49%；40 μM of Chinese
After baicalein is handled 24 hours, 786-O cell invasion abilities reduce about 71%.It is similar therewith, after baicalein is handled 24 hours,
The invasive ability of OS-RC-2 cells is also decreased obviously.Compared with not adding wogonin processing OS-RC-2 cells, 40 μM of Chinese
After baicalein is handled 24 hours, OS-RC-2 cell invasion abilities reduce about 55%；After 80 μM of wogonins are handled 24 hours,
OS-RC-2 cell invasion abilities reduce about 90%.
These results show that wogonin can effectively suppress the invasive ability of renal cell carcinoma cell, and inhibitory action is presented
Concentration dependent.
Embodiment 4：Wogonin can suppress renal cell carcinoma cell DNA replication dna
This example demonstrates that wogonin can suppress renal cell carcinoma cell DNA replication dna.In the present embodiment, the kidney is thin
Born of the same parents' cancer cell is people's kidney clear cell adenocarcinoma cell 786-O and OS-RC-2 cell.
Cellular immunity dyeing fixer is purchased from green skies Bioisystech Co., Ltd (production number P0098), EdU cells propagation
Detection kit is purchased from Guangzhou Ribo Bio Co., Ltd. (production number C10310-1).Anti- quencher is purchased from
Invitrogen companies (production number P36931).Comet kit is purchased from TREVIGEN companies (production number 4250-050-
K)。
The propagation detection of EdU cells：Cell culture be the same as Example 1.Choose exponential phase cell, OS-RC-2 cells difference
40 and 80 μM of wogonins are added, 786-O cells are separately added into 20 and 40 μM of wogonins, after handling 24 hours, add after EdU
It is placed on 37 DEG C, cultivates 20 minutes in 5%CO2 incubators, PBS one time.4 DEG C of fixations of fixer are dyed using cellular immunity
Overnight, EdU dyeing is carried out.The anti-quencher containing DAPI is added, fluorescence microscope is taken pictures.
As a result as shown in Fig. 3 (a), wogonin processing significantly reduces the ratio that EdU mixes positive cell.And with the Chinese
Reduction trend is presented in baicalein concentration increase, the ratio of EdU incorporation positive cells.Such as untreated 786-O cells EdU mixed ratios
About 38%, after 20 μM of wogonins are handled 24 hours, 786-O cell EdU mixed ratios are reduced to about 24%；40 μM of Chinese radix scutellariaes
After element processing 24 hours, 786-O cell EdU mixed ratios are reduced to about 15%.It is similar therewith, after baicalein is handled 24 hours,
The EdU mixed ratios of OS-RC-2 cells are also decreased obviously.Untreated OS-RC-2 cells EdU mixed ratios are about 47%, 40 μ
After M wogonins are handled 24 hours, OS-RC-2 cell EdU mixed ratios are reduced to about 28%；80 μM of wogonin processing 24 are small
Shi Hou, OS-RC-2 cell EdU mixed ratios are reduced to about 13%.
These results show that wogonin can effectively suppress the DNA replication dna ability of renal cell carcinoma cell, and it suppresses to make
With presentation concentration dependent.
Embodiment 5：Wogonin being capable of inducing cell DNA damage
This example demonstrates that wogonin being capable of inducing cell DNA damage.In the present embodiment, the renal cell carcinoma cell
It is people's kidney clear cell adenocarcinoma cell 786-O and OS-RC-2 cell.
Comet electrophoresis is tested：Cell culture be the same as Example 1.786-O the and OS-RC-2 cells of exponential phase are chosen, plus
Enter 30 μM of wogonins to handle 24 hours, cell precipitation is collected in pancreatin digestion, is resuspended and is adjusted to 1 × 10 with PBS5Individual/milliliter.
In 37 DEG C of environment by 1 μ l blow it is even after cell suspension be mixed into 50 μ l low melting-point agarose coagulant liquid and be added dropwise to circle
Shape mark slide on and smoothen.By slide glue surface upwards, it is placed in wet box and is put at 4 DEG C of lucifuges 10-30 minutes.Glass will be carried
Piece glue surface immerses in the DNA lysates of precooling upwards, is placed at 4 DEG C of lucifuges 60 minutes.Then 50mL is immersed at 4 DEG C of lucifuges pre-
Cold neutral electrophoresis liquid 30 minutes, to elute lysate.By slide glue surface upwards, electrophoresis liquid, 4 DEG C of avoid light places 5 minutes are immersed；
4 DEG C of light protected environments, with electrode spacing (cM) × 1V constant pressure electrophoresis 1 hour；Slide is submerged into DNA precipitated liquids, room temperature is placed
30 minutes；DNA precipitated liquids are abandoned, 70% ethanol is added, room temperature is placed 30 minutes；Glass is toasted with the temperature less than 45 DEG C in an oven
Piece is completely dried cell being placed in same aspect for 10-15 minutes to it.Each sample is added dropwise about 100 μ L'sGreen
Working solution (is purchased from ThermoFisher companies, production number S7567), avoid light place 30 minutes.Slide is erect unnecessary to discard
Dye liquor, avoid light place to its drying.Fluorescence microscope is simultaneously taken pictures, and uses CASP software analysis datas.
As a result the tail length degree of cometal cells is acquired and as lesion assessment parameter.Comet head center is to comet tail
The distance of portion's center of gravity is comet tail length degree, and intracellular breakaway poing is more, and comet tail length degree will be bigger.As a result with case
Type figure represents the distribution situation of every group of cell comet tail length degree (back range).
As a result as shown in Fig. 3 (b), the comet tail length degree average of wogonin treatment group cell is obvious compared to having compared with control group
Increase.Untreated OS-RC-2 cells back range is 36 μm of %, after 30 μM of wogonins are handled 24 hours, in OS-RC-2 cell back ranges
Rise to 326 μm of %；After 30 μM of wogonins are handled 24 hours, the back range of 786-O cells is also risen by untreated 7.3 μm of preceding %
To 92 μm of %.These results show that wogonin processing can dramatically increase the DNA damage of renal cell carcinoma cell.
Embodiment 6：Wogonin can induce renal cell carcinoma cell apoptosis
This example demonstrates that wogonin can induce renal cell carcinoma cell apoptosis.Because the studies above finds wogonin
786-O killing functions of immunocytes is become apparent from, therefore in the present embodiment, renal cell carcinoma cell used is people's kidney clear cell adenocarcinoma
Cell 786-O cells.
One-step method TUNEL cell apoptosis detection kits (red fluorescence) are purchased from (the production of green skies Bioisystech Co., Ltd
Article Number C1089).Active caspase 3 (production number Ab32042), PARP (production number Ab32561) and actin
The secondary antibody of (Actin, production number Ab8227) primary antibody and HRP marks is purchased from Abcam companies (production number Ab6721).ECL is sent out
Light detection liquid (production number WBKLS0100) and pvdf membrane (production number IPVH00010) are purchased from Millipore Corp. of the U.S..Protease
Inhibitor is purchased from ThermoFisher companies (production number 88266).Protein lysate, albumen sample loading buffer (loading
Buffer) and BCA protein quantifications kit purchased from green skies Bioisystech Co., Ltd (production number is respectively P0013C,
P0015F, P0009).Protein marker (Marker) is purchased from ThermoFisher companies (production number 26623).
TUNEL methods detect Apoptosis：Cell culture be the same as Example 1.Exponential phase 786-O cells are chosen, are added respectively
Enter 20 and 40 μM of wogonins to handle 24 hours, PBS is washed one time, add immunostaining fixer room temperature and fix 20 minutes.Abandon solid
Determine liquid, PBS is washed 2 times, add 0.5%TritonX 100 PBS room temperature treatments 30 minutes.Supernatant is abandoned, PBS is washed 2 times, by 20 μ l
The TUNEL dyeing liquors of Fresh are added drop-wise on cell climbing sheet, and 37 DEG C of lucifuges are incubated 60 minutes, and add containing DAPI anti-is quenched
Agent, is taken pictures using fluorescence microscope.
As a result as shown in Fig. 4 (a), untreated 786-O Apoptosis ratio is only 2.1%, 20 μM of wogonin processing 24
Apoptotic cell ratio rises to 18.2% after hour, and apoptotic cell ratio is after 40 μM of wogonins are handled 24 hours
36.8%.The result shows that wogonin can induce renal cell carcinoma cell apoptosis, and concentration dependant is presented in apoptosis-induced effect
Property.
Western blotting (Western blot) method detects the expression of Apoptosis mark：Choose exponential phase OS-RC-
2 and 786-O cells, add various concentrations wogonin and handle 24 hours, PBS is washed one time, with the egg containing protease inhibitors
Cell is resuspended in white lysate, places on ice 20 minutes, the abundant cell lysis of suspension is shaken therebetween.By 4 DEG C of cell pyrolysis liquid
12000rpm is centrifuged 30 minutes, and supernatant is total protein of cell.Using BCA standard measure protein concentrations.By 40 μ g total protein concentrations
Handle sample and add sample loading buffer mixing, 99 DEG C of denaturation 10min carry out 10%SDS- polyacrylamide gel electrophoresises.Electrophoresis
Afterwards by protein delivery to pvdf membrane.Transferring film is finished, and is closed 1 hour with the TBS room temperatures of 5% skimmed milk power.Confining liquid is abandoned, TBST is used
By pvdf membrane rinsed clean, the 4 DEG C of overnight incubations of primary antibody diluted with TBST are added.Addition TBST after 3 times is cleaned with TBST dilute
Release secondary antibody room temperature concussion reaction 1h.Reaction abandons secondary antibody after terminating, and is cleaned with TBST 3 times, adds ECL luminescent solutions detection apoptosis related
The expression of albumen, using actin as internal reference.
As a result Fig. 4 (b) is seen, wogonin causes intracellular apoptosis mark active caspase 3, PARP expression liter
Height, and expression quantity progressively raises with the increase of wogonin concentration.These results further demonstrate that wogonin induction kidney is thin
Born of the same parents' cancer cell-apoptosis, and inducing action is in concentration dependent.
Embodiment 7：Wogonin is capable of specificity suppression renal cell carcinoma cell DNA replication dna GAP-associated protein GAP CDC6 expression
This example demonstrates that wogonin being capable of specificity suppression clear-cell carcinoma DNA replication dna GAP-associated protein GAP-cell division week
The expression of phase albumen 6 (Cell Division Cycle 6, CDC6).In the present embodiment, the renal cell carcinoma cell is people's kidney
Clear cell adenocarcinoma cell 786-O and OS-RC-2 cell, lung adenocarcinoma cell is H1299 cells, and liver cancer cells are HepG2 cells,
Stomach cancer cell is SGC-7901 cells, and human pancreatic cancer cell is PANC-1 cells.
Cell culture be the same as Example 1.Western blotting (Western blot) method detection cell be the same as Example 6.Human lung cancer is thin
Born of the same parents are H1299 (production number TCHu 160) cell, and human liver cancer cell is HepG2 (production number TCHu 72) cell, and human pancreas cancer is thin
Born of the same parents are PANC-1 (production number TCHu 98) cell, and gastric carcinoma cells are SGC-7901 (production number TCHu 46) cell, are purchased from
The American Type Culture Collection committee of Chinese Academy of Sciences cell bank.CDC6 antibody (production number ab109315) is purchased from Abcam companies.
CDC6 albumen is the key protein of DNA replication dna, in the expression increase of kinds of tumors.Suppressing CDC6 expression can suppress
The DNA replication dna of tumour cell.As a result Fig. 5 (a) is seen, wogonin processing causes people's clear cell carcinoma of kidney cell 786-O and OS-
CDC6 expressing quantities are substantially reduced in RC-2, and expression quantity is gradually reduced with the increase of wogonin concentration.Show that the Chinese is yellow
A kind of reed mentioned in ancient books element can suppress the expression of CDC6 in renal cell carcinoma cell, and inhibitory action is in concentration dependent.
The expression for suppressing CDC6 in order to further analyze wogonin whether there is tumour-specific, and inventor analyzes again
Wogonin is to human lung carcinoma cell H1299, human liver cancer cell HepG2, human pancreatic cancer cell PANC-1 and gastric carcinoma cells
The influence that CDC6 is expressed in SGC-7901.As a result Fig. 5 (b) is seen, different from renal cell carcinoma cell, 80 μM of wogonin processing are not
CDC6 albumen can be caused to be reduced in this several cell inner expression, showing wogonin suppression CDC6 expression has clear-cell carcinoma special
The opposite sex.
Embodiment 8：The inhibitory action to renal cell carcinoma cell DNA replication dna is realized in expression of the wogonin by suppressing CDC6
This example demonstrates that wogonin can by suppress CDC6 expression realize suppress renal cell carcinoma cell DNA replication dna
Function.Because the studies above finds that wogonin becomes apparent to the effect of 786-O cell DNAs inhibition of DNA replication, therefore in this implementation
In example, renal cell carcinoma cell used is people's kidney clear cell adenocarcinoma cell 786-O cells.
The CDC6 expression plasmids and control empty plasmid of HA labels give (Clare Hall by doctor Drury
Laboratories,Cancer Research UK,London,England)。LipofectamineTM2000 are purchased from Thermo
Thermo Fisher Scientific companies (production number 11668-027).
Cell culture be the same as Example 1.EdU methods detection cell DNA replicates be the same as Example 4.Western blotting (Western
Blot) method detection cell be the same as Example 6.The cell transfecting of plasmid：6 orifice plates are seeded cells into, cell density reaches after 24 hours
During to about 80~90%, former culture medium is discarded, cell is washed twice with the nutrient solution of serum-free antibiotic-free；Take 1.5ml sterile
Ep is managed, and is separately added into the culture medium of 100 μ l serum-free antibiotic-frees, and 2 μ l lipofectamin2000 transfection reagents are mixed.Plus
Enter 4 μ g plasmids to be transfected, mix, room temperature is placed 15 minutes.The transfection composite prepared is added to the culture of cell growth
In plate, 5%CO2, in 37 DEG C of constant incubators after culture 6 hours, change and continue to cultivate with the complete medium containing serum；24 is small
When after collect cell.
In order to clearly suppress effect of the CDC6 protein expressions in wogonin treats clear-cell carcinoma.Inventor is again by CDC6
Wogonin processing is reused after expression plasmid transfectional cell, CDC6 rescue experiments have been carried out.As a result such as Fig. 5 (c), external source CDC6
The transfection of plasmid can effectively reverse wogonin processing to cause intracellular CDC6 reduction.
Shown in EdU testing results such as Fig. 5 (d).Transfect empty map carrier similar with non-transfected cells, EdU incorporations are positive thin
The ratio of born of the same parents is about 51% and 53%；After 20 μM of wogonin processing, the ratio of both cells EdU incorporation positive cells is notable
It is reduced to 23% and 24%；But transfection has the cell of CDC6 expression plasmids, EdU incorporations positive cell after wogonin processing
Ratio has obvious rising, about 36%.These results illustrate that expression of the wogonin by suppressing CDC6 realizes that suppression kidney is thin
The function of born of the same parents' cancer cell DNA replication dna.
Present embodiments are exemplarily illustrated above in association with accompanying drawing.Those skilled in the art are according to this specification
Disclosure is readily apparent that, each embodiment suitably can be adjusted and reconfigured according to actual needs,
Without departing from spirit herein.The protection domain of the application is defined by following claims.
Claims (10)
1. application of the wogonin in treatment kidney medicine is prepared.
2. application as claimed in claim 1, it is characterised in that the kidney is clear-cell carcinoma.
3. application as claimed in claim 1, it is characterised in that the administration concentration of the wogonin is 1~100 μM.
4. application as claimed in claim 1, it is characterised in that the formulation of the medicine is：Tablet, oral agents, electuary, injection
Agent, liposome, target administration injection, pill, capsule, granule, pulvis, suppository, powder, paste, patch, parenteral solution,
Solution, suspension, spray, lotion, drops or liniment.
5. application as claimed in claim 1, it is characterised in that the use environment pH of the medicine is 3~11.
6. application as claimed in claim 5, it is characterised in that the use environment pH of the medicine is 5~9.
7. application as claimed in claim 6, it is characterised in that the use environment pH of the medicine is 7~8.
8. application as claimed in claim 1, it is characterised in that the administration concentration of wogonin is 1~100 μ in the medicine
M。
9. application as claimed in claim 1, it is characterised in that in the medicine administration concentration of wogonin be 1~90 μM,
1~80 μM, 1~70 μM, 1~60 μM, 1~50 μM, 1~40 μM, 1~30 μM, 1~20 μM, 1~10 μM, 5~100 μM, 5~
90 μM, 5~80 μM, 5~70 μM, 5~60 μM, 5~50 μM, 5~40 μM, 5~30 μM, 5~20 μM, 5~10 μM, 10~100 μ
M, 10~90 μM, 10~80 μM, 10~70 μM, 10~60 μM, 10~50 μM, 10~40 μM, 10~30 μM, 10~20 μM, 20
~100 μM, 20~90 μM, 20~80 μM, 20~70 μM, 20~60 μM, 20~50 μM, 20~40 μM, 20~30 μM, 30~
100 μM, 30~90 μM, 30~80 μM, 30~70 μM, 30~60 μM, 30~50 μM, 30~40 μM, 40~100 μM, 40~90 μ
M, 40~80 μM, 40~70 μM, 40~60 μM, 40~50 μM, 50~100 μM, 50~90 μM, 50~80 μM, 50~70 μM, 50
~60 μM, 60~100 μM, 60~90 μM, 60~80 μM, 60~70 μM, 70~100 μM, 70~90 μM, 70~80 μM, 80~
100 μM, 80~90 μM or 90~100 μM.
10. application as claimed in claim 9, it is characterised in that the administration concentration of wogonin is 1 μM, 5 μ in the medicine
M、10μM、15μM、20μM、25μM、30μM、35μM、40μM、45μM、50μM、55μM、60μM、65μM、70μM、75μM、80μM、
85 μM, 90 μM, 95 μM or 100 μM.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109793871A (en) * | 2017-11-16 | 2019-05-24 | 山东中医药大学附属医院 | It is a kind of for treating the Chinese medicine composition of psoriasis |
| CN115337298A (en) * | 2022-05-11 | 2022-11-15 | 中国人民解放军海军军医大学第三附属医院 | Application of scutellarin as NQO1 inhibitor in preventing or treating related diseases |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102973550A (en) * | 2011-09-05 | 2013-03-20 | 谢秀梅 | Pharmaceutical composition for inhibiting cancer stem cell growth or cancer cell metastasis by using wogonin, and application thereof |
-
2017
- 2017-06-14 CN CN201710449014.5A patent/CN107028935A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102973550A (en) * | 2011-09-05 | 2013-03-20 | 谢秀梅 | Pharmaceutical composition for inhibiting cancer stem cell growth or cancer cell metastasis by using wogonin, and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| WANG,NING ET AL.: "A chinese medicine formula gegen qinlian decoction suppresses expansion of human renal carcinoma with inhibition of matrix metallopreteinase-2", 《INTEGRATIVE CANCER THERAPIES》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109793871A (en) * | 2017-11-16 | 2019-05-24 | 山东中医药大学附属医院 | It is a kind of for treating the Chinese medicine composition of psoriasis |
| CN109793871B (en) * | 2017-11-16 | 2021-07-27 | 山东中医药大学附属医院 | A Chinese medicinal composition for treating psoriasis |
| CN115337298A (en) * | 2022-05-11 | 2022-11-15 | 中国人民解放军海军军医大学第三附属医院 | Application of scutellarin as NQO1 inhibitor in preventing or treating related diseases |
| CN115337298B (en) * | 2022-05-11 | 2024-05-03 | 中国人民解放军海军军医大学第三附属医院 | Application of baicalein as NQO1 inhibitor in preventing or treating related diseases |
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Application publication date: 20170811 |