CN108236622A - A kind of application of substance for lowering 36 expression of ER- α in the drug for treating disease is prepared - Google Patents
A kind of application of substance for lowering 36 expression of ER- α in the drug for treating disease is prepared Download PDFInfo
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Abstract
The present invention relates to field of medicaments, more particularly to a kind of application of substance for lowering new estrogenic receptor ER α 36 in the drug for treating disease is prepared, in a preferred scheme, the substance is baicalein, it can effectively inhibit triple negative breast cancer cell and other growth of tumour cell by lowering the expression of ER α 36 using baicalein, and sensibility of the triple negative breast cancer cell to antiestrogen can be improved, baicalein and antiestrogen combination have synergistic effect, there is huge prospect in clinical practice by lowering the diseases such as 36 expression treatment tumours of ER α.
Description
Technical field
The present invention relates to field of medicaments, and in particular to a kind of substance for lowering 36 expression of ER- α is preparing for treating disease
Drug in application.
Background technology
Estrogen is the general name of sterid mainly formed in ovary, testis and possible adrenal cortex.
Estrogen and the example of compound with estrogen active include that diethyl diethylstilbestrol, fosfestrol, oneself is female
Phenol, Polyestradiol Phosphate, B. D. P. E, Chlorotrianisene, dienestrol, diethyl diethylstilbestrol, meprane, estradiol,
Estriol, oestrone, ethinylestradiol, mestranol, estriol cyclopentyl ether, quinestrol, coumestrol, 4',5,7-trihydroxyflavone, 4 ', 5,7-, tri- hydroxyls
Base flavones, citrus aglycon, coumestrol, siskin isoflavonoid, Daidzein and female horse alcohol etc..Estrogen adjusts germinal tissue and breast
Various physiological processes in room, angiocarpy, bone, liver and brain tissue.Estrogen is also used in oral contraceptive.Estrogen it is other
Purposes includes, and alleviates the discomfort of menopause, inhibits lactation and treatment osteoporosis, threatened abortion and various functions oophorpathy
Disease.Antiestrogen can be used for treatment metastatic breast cancer and advanced prostate cancer.
The effect of estrogen is completed by Mediated by Estrogen Receptor, has been cloned and first female has been swashed in Greene in 1986 et al.
Plain receptor (ER) (Greene et al., Nature, 320:134,1986 and Greene et al., Science, 231:1150,
1986), this receptor is now referred to as ER- α (molecular weight 66kDa, ER- α 66), by 3 independent but can interact
Functional domain forms：N- ends A/B structural domains, C or DNA- binding structural domains and D/E/F or ligand-binding domain.ER-α
N- terminal domains coding independently of ligand activation function (AF-1), both participated in the interaction with coactivator and target base
The region of the transcription activating of cause.2 zinc fingers are contained in DNA- binding structural domains or C-structure domain, Receptor dimerization and with
It plays an important role in the combination of specific DNA sequences.C- ends D/E/F structural domains are a kind of ligand-binding domains, energy
Mediating ligand combination, Receptor dimerization, nuclear translocation and ligand-independent trans-activation function (AF-2).AF-1 and AF-2 pairs
Transcribe control Relative Contribution by cell-specific and DNA promoters specificity in a manner of change (Berry etc., EMBO J.,
9：2811(1990)).
Wang Zhaoyi in 2006 teaches a kind of splice variant of ER- α 66 of successful clone, and the ER- α that molecular weight is 36kDa are sub-
Type is named as ER- α 36, and research confirms that the function of ER- α 36 is different from ER- α 66, and ER- α 36 are primarily present on cell membrane, phase
Than lacking AF-1 the and AF-2 domains with transcription activating in ER- α 66,36 structures of ER- α, but maintain DNA binding domain
And its part dimerization and ligand binding domain (Figure 12).ER- α 36 can mediate quick estrogen receptor access such as to activate MAPK/
ERK and PI3K/AKT signal transductions (Wang Zhaoyi et al., Identification, cloning, and expression of
human estrogen receptor-α36,a novel variant of human estrogen receptor-α66,
Biochemical and biophysical research communications, 336 (2005) 1023-1027. Wang Zhao mono-
Et al., A variant of estrogen receptor- α, hER- α 36:transduction of estrogen-and
antiestrogen-dependent membrane-initiated mitogenic signaling,Proceedings of
the National Academy of Sciences,103(2006)9063-9068.)。
It is expressed due to lacking ER- α 66, it is well known that triple negative breast cancer (TNBC) cell can not use antiestrogen
The treatments such as object such as tamoxifen.But have been reported that find high concentration tamoxifen can inhibit TNBC cells increase (Obrero,
Et al. M. (2002) Estrogen receptor-dependent and estrogen receptor-independnet
pathways for Tamoxifen and 4-hydroxytamoxifen-induced programmed cell
Death.J.Biol.Chem., 277,45695-45703.Mandleker, S. et al. (2000) Activation of
caspase-3 and cJun NH2-terminal Kinase-1 signaling pathways in tamoxifen-
induced apoptosis of human cancer cells.Cancer Res.60,5995-6000.).Tamoxifen
The main problem of long-term treatment is its negative interaction in endometrium, can increase the risk of carcinoma of endometrium, in addition high dose
Tamoxifen can generate other toxic side effects of bigger.In addition it is reported that the activation of different growth factor signal accesses also may be used
Tamoxifen resistance (Mandleker, S.and Kong, T.A.N. (2001) can be caused by bypassing estrogen receptor transmission
Mechanisms of tamoxifen-induced apoptosis.Apoptosis, 6,469-477), triple negative breast cancer is
The aobvious feminine gender of estrogen, progestational hormone and hEGF 2, is one of breast cancer hypotype, accounts for about the 15% of breast cancer, compared with
The breast cancer of its alloytype, with higher recurrence rate, the pernicious features such as Overall survival is shorter.Due to lacking corresponding target spot such as ER
And HER2, TNBC, which is difficult to use, is based on HER2With the therapy that hormone receptor is target spot, such as Herceptin, tamoxifen and fragrance
Enzyme inhibitor.It is more effective more there is an urgent need to one kind at present in view of the serious cytotoxicity of classic chemotherapy and its adverse reaction
The therapy of hypotoxicity.Therefore seeking the therapeutic agent of safer and more effective treatment triple negative breast cancer urgently needs to solve
Problem.
Baicalein (Baicalein, Noroxylin,5,6,7-Trihydroxy flavone), alias baicalein, baicalein is Labiatae
(Labiatase) dry root of plant radix scutellariae (ScutellariabaicalensisGeorgi), record earliest in《Legendary god of farming's book on Chinese herbal medicine
Through》, the middle product that grass roots is wanted are classified as, there is such as Figure 1A structures：
One of the principle active component of baicalein for radix scutellariae has anti-oxidant, anti-inflammatory, resistance state, antibacterial, anticancer, disease-resistant
The effects that poison, heat-clearing, diuresis, calmness, decompression, liver protection, cholagogic (Journal of Agricultural Engineering, 2003,11 (19), 218-221).But
It is that the mechanism of action of these activity is not illustrated thoroughly or correctly also, only gos deep into thorough research mechanism of drug action, ability
It is enough precisely effectively to select suitable drug, it can preferably reach using existing drug in the prior art and cure disease
Purpose.
Invention content
In order to overcome technical problem in the prior art, the present invention provides a kind of new applications of baicalein：Radix scutellariae
Element is used to lower the purposes of the expression of ER- α 36.
Further, the present invention provides a kind of substance for lowering 36 expression of ER- α in the drug for treating disease is prepared
Application, the substance for lowering 36 expression of ER- α is from Baical Skullcap root P.E, for example, labiate radix scutellariae can be derived from
The extracts such as root, Yunnan scutellariae,radix, sun plant root, apocynaceae plant eel rattan.
The disease mainly includes：Liver cancer, lung cancer, oophoroma, breast cancer, carcinoma of endometrium, prostate cancer.
In a preferred embodiment, the disease is breast cancer.
In a preferred embodiment, the disease is triple negative breast cancer.
In a preferred embodiment, the substance for lowering 36 expression of ER- α is baicalein.
In a specific embodiment, the present invention has detected baicalein and liver cancer cells, stomach cancer cell, cancer of pancreas are thin
Born of the same parents, lung carcinoma cell, neuroglial cytoma growth influence, find baicalein can lower liver cancer cells, stomach cancer cell, pancreas
36 protein levels of adenocarcinoma cell ER- α, but unobvious are lowered to, lung carcinoma cell and 36 albumen of neuroglial cytoma ER- α.
The liver cancer cells illustrative example be Huh-7, stomach cancer cell illustrative example be AGS or MKN-28, cancer of pancreas
Cell illustrative example is Panc-1, and the expression quantity of ER- α 36 is reduced with the increase of baicalein concentration, the preferred Huang
The effective dose of a kind of reed mentioned in ancient books element is 50-100 μM.
Illustratively, the lung carcinoma cell is H460, and the neuroglial cytoma is U87, the expression quantity of ER- α 36 with
Increasing and increasing for baicalein concentration, is had significant change at a concentration of 10 μM, raising is apparent at 50 μM and 100 μM.
In a preferred embodiment, the present invention has detected baicalein and the shadow to triple negative breast cancer cell growth
It rings, it is found that baicalein can significantly inhibit triple negative breast cancer cell growth and effectively lower 36 protein levels of ER- α.We
Observe that baicalein can weaken the cell-stimulating activity of the estrogen in triple negative breast cancer cell.Illustratively, described three is cloudy
Property breast cancer cell be MDA-MB-231 or MDA-MB-436.
In a preferred embodiment, baicalein downward ER- α 36 are added in express, triple negative breast cancer can be improved
Cell is to the sensibility of antiestrogen.The drug that estrogen receptor positive breast cancer is selected includes selective estrogen receptor
Conditioning agent (Henke BR et al., Recent advances in estrogen receptor modulators, CurrOpin
Drug Devel, 2005,8 (4)：437-448, pure antiestrogen (Elkak AE et al., Pure antiestrogens and
Breast cancer.Curr Med Res Opin, 2001,17 (4), 282-289) and aromatization enzyme inhibitor (Younus J
Et al., A practical overview of aromatase inhibitors in postmenopausal women
with hormone receptor positive breast cancer.Bull Cancer,2005,92(4),E39-E44)
Three categories.Preceding two classes drug can be affine with ER height, by eliminating or interfering the interaction of estrogen and ER, promotes cancer cell
Apoptosis.Latter class drug is reduced internal estrogen level, is played similar anti-female by the inhibiting effect to cytochrome P 450 enzymes
The effect of hormone.Antiestrogen as described herein is mainly selected from first two antiestrogen such as tamoxifen, fluorine dimension department
Group etc., is commonly used for tamoxifen.
Therefore baicalein can enhance and restore sensibility of the triple negative breast cancer cell to antiestrogen.So as to reduce
Antiestrogenic usage amount.
Present invention discover that baicalein can lower the stable state expression of 36 albumen of ER- α.AutoDock has also been made in we
Experiment finds that baicalein can combine ER- α 36, and form hydrogen bond with ER- α 36Glu180.
Research report high concentration baicalein can show strong rush cells apoptosis by weakening anti-apoptotic proteins in the past.
In the cell of the baicalein processing of concentration used it is observed that there is not significant apoptotic cell, still in researcher of the present invention
MAPK/ERK signals can effectively be inhibited.Therefore researcher of the present invention speculates that the effect for being possible to baicalein is dose-dependent.
In a preferred embodiment, the effective dose of the baicalein is 1-20 μM.
In a preferred embodiment, the effective dose of the baicalein is 1-10 μM.
In a preferred embodiment, the effective dose of the baicalein is 1-5 μM.
Such as tamoxifen is widely used to the hormonotherapy of ER positive breast cancers as antiestrogen.These are anti-
Estrogen has been determined that recurrence, pernicious rate and Metastasis in Breast Cancer risk can be mitigated.Unfortunately, since triple negative breast cancer lacks
ER- α 66 are expressed, these antiestrogens cannot inhibit triple negative breast cancer cell growth.High concentration has been had been reported that before
(>15 μM) tamoxifen can cause ER negative breast cancer cells apoptosis (Obrero, M. et al. (2002) Estrogen
receptor-dependent and estrogen receptor-independnet pathways for Tamoxifen
and 4-hydroxytamoxifen-induced programmed cell death.J.Biol.Chem.,277,45695-
45703.Mandleker, S. et al. (2000) Activation of caspase-3 and cJun NH2-terminal
Kinase-1 signaling pathways in tamoxifen-induced apoptosis of human cancer
Cells.Cancer Res.60,5995-6000.Mandleker, S. et al., (2001) Mechanisms of
tamoxifen-induced apoptosis. Apoptosis,6,469-477).If the researcher of the present invention has found to reduce
36 expressions of ER- α, 0.5 μM of tamoxifen then can effectively inhibit triple negative breast cancer cell growth, this discovery shows
ER- α 36 participate in the tamoxifen drug resistance of triple negative breast cancer cell.Baicalein can lower ER- α 36 and express, and can make three feminine genders
Breast cancer cell is to tamoxifen sensitivity, it is meant that baicalein and tamoxifen combination have synergistic effect.
Further, on the basis of above-mentioned discovery, researcher of the invention is further tested using under baicalein
Adjust whether ER- α 36 and EGFR expression can improve sensibility of the triple negative breast cancer cell to other antiestrogens, such as fluorine
Dimension department group, result of the test show that baicalein can improve the fulvestrant sensibility of triple negative breast cancer cell pair, it is meant that yellow
A kind of reed mentioned in ancient books element and the combination of this kind of antiestrogen have synergistic effect.
In the present invention, cited antiestrogen is only exemplary, the antiestrogen that the present invention is covered
Object in addition to the exemplary antiestrogen enumerated, can also include the pro-drug of these antiestrogens, and variant spreads out
Biology or metabolite, such as the metabolite of tamoxifen：4-hydroxytamoxifen.Mechanism of action disclosed by the invention
It is inferred that can be affine with ER height, by eliminating or interfering the interaction of estrogen and ER, promote the anti-of cancer cell-apoptosis
Estrogenic can improve sensibility of the triple negative breast cancer cell to it with baicalein combination.
In a preferred embodiment, the substance for lowering 36 expression of ER- α is baicalein and antiestrogen
Composition.
The antiestrogen is selected from tamoxifen, fluorine Wei Siqiong.
In a preferred embodiment, the effective dose of the baicalein is 0.5-10 μM.
Particularly preferred, the effective dose of the baicalein is 1-5 μM.
In a preferred embodiment, the effective dose of the antiestrogen is 0.1-10 μM.
Particularly preferred, the effective dose of the antiestrogen is 0.5-5 μM.
MAPK/ERK signal paths are signal of interest (K.Knudsen et al. Cyclin D1 of cell growth:
polymorphism,aberrant splicing and cancer risk,Oncogene,25(2006)1620-1628).This
Disclosure of the invention baicalein can weaken what estrogen in triple negative breast cancer MDA-MB-231 and MDA-MB-436 cell induced
The activation of MAPK/ERK signals, thus it is speculated that be the expression by lowering ER- α 36 in triple negative breast cancer cell.
Cyclin D_1 gene plays an important role in cell growth, can be adjusted by estrogen receptor access.
Estrogen can induce Cyclin D_1 gene expression, promote G1 phases cell cycle progression and stimulating cellular growth.It is the invention demonstrates that yellow
A kind of reed mentioned in ancient books element can weaken the Cyclin D_1 gene expression of estrogen induction, this research has shown that baicalein acts on triple negative breast cancer cell
Another molecular events for lower Cyclin D_1 gene expression.
When triple negative breast cancer is treated, it is clinical preferred to reduce side reaction and heighten the effect of a treatment simultaneously.We are research shows that radix scutellariae
Element can lower the expression of ER- α 36 in triple negative breast cancer cell, so as to inhibit to be stimulated in triple negative breast cancer cell by estrogen
Cell increase.Our research also indicates that baicalein and antiestrogen use in conjunction can effectively inhibit three negative breasts
Cancer cell increases, it was demonstrated that has huge potentiality using baicalein clinical treatment triple negative breast cancer.It can be carried using baicalein
The effect of high antiestrogen treatment triple negative breast cancer, can reduce the dosage of antiestrogen, should there are huge
Use prospect.
In one aspect of the invention, the composition of baicalein and antiestrogen, the antiestrogenic are provided
The specific example of drug, such as can be tamoxifen, fulvestrant etc., which can be made commonly used in the art
Dosage form, such as injection, tablet, capsule, pill, powder, paste, granule, syrup, oral liquid, freeze dried powder.
In the present invention in the combined pharmaceutical formulation of baicalein and antiestrogen, advised according to different dosage forms and preparation
Pharmaceutical field customary adjuvant can be used in lattice, the auxiliary material that preparation uses, and is reacted with the present composition of getting along well or does not influence this
Premised on the effect of invention drug.Such as customary adjuvant includes filler, disintegrant, lubricant, suspending agent, adhesive, sweet taste
Agent, corrigent, preservative, matrix etc..Filler includes：Starch, pregelatinized starch, lactose, mannitol, chitin, crystallite are fine
Tie up element, sucrose etc.；Disintegrant includes：Starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, crosslinked polyethylene pyrroles
Alkanone, low-substituted hydroxypropyl cellulose, croscarmellose sodium etc.；Lubricant includes：Magnesium stearate lauryl sodium sulfate,
Talcum powder, silica etc.；Suspending agent includes：Polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methyl are fine
Tie up element etc.；Adhesive includes, starch slurry, polyvinylpyrrolidone, hydroxypropyl methyl cellulose etc.；Sweetener includes：Saccharin sodium,
Aspartame, sucrose, honey element, enoxolone etc.；Corrigent includes：Sweetener and various essence；Preservative includes：Nipalgin
Class, benzoic acid, sodium benzoate, sorbic acid, sorbic acid and its esters, benzalkonium bromide, acetic acid chlorhexidine, eucalyptus oil etc.；Matrix packet
It includes：PEG6000, PEG4000, insect wax etc..For above-mentioned dosage form is enable to realize pharmacy of Chinese materia medica, must add when preparing these dosage forms
Enter pharmaceutically acceptable other auxiliary materials.
In a preferred embodiment, in pharmaceutical composition of the present invention, the baicalein rubs with antiestrogen
You are than being (2-10)：1.
In a preferred embodiment, in pharmaceutical composition of the present invention, the baicalein rubs with antiestrogen
You are than being (2-5)：1.
Preferably, the antiestrogen is tamoxifen.
The dosage of the pharmaceutical composition of the present invention kind can according to the dosage form difference of administration object, administration route or drug
To carry out appropriate variation but premised on ensureing that the pharmaceutical composition can reach effective blood concentration in vivo.
Term：
Cyclin D_1 gene：Cyclin D1, major function be promote cell Proliferation.Cyclin D1 are by combining and swashing
G1 periods distinctive cyclin-dependent kinase CDK4 living, G1 periods phase inhibit albumen (Rb) to be phosphorylated, the Rb of phosphorylation
Albumen is dissociated from the E2F transcription factors that it is combined, the gene in E2F transcription factors starting transcription living cells period, so as to push away
The kinetocyte period enters S periods by G1 periods.In addition, the study found that in addition to promote cell into line splitting other than, D types cyclin
Also there are certain promotion genetic transcriptions independently of CDK kinase activities.Cyclin D1 can remove acetyl with bonding histone
Base enzyme P/CAF can also combine transcription factor TF II D etc., and by the combination with these transcription factors, cyclin D1 are played
Promote the function of genetic transcription.At present, cyclin D1 have been acknowledged as a kind of proto-oncogene, and overexpression can cause cell
Proliferation out of control and malignization.Research shows that being found that Cyclin D1 gene overexpressions and gene magnification in kinds of tumors, wrap
Include breast cancer, carcinoma of urinary bladder, parathyroidoma, lymthoma, melanoma/lung cancer and cell centre type lymthoma etc..
Tamoxifen：Tamoxifen, Chinese name：(Z) -2- [4- (1,2- diphenyl -1- butylene) phenoxy group]-N, N- bis-
Methyl ethyl-amine, structural formula are as follows：
There are Z-type and E-isomer, Z-type has antiestrogenic sample effect, and estrogen is prevented to play effect, and E types then have
There is weak estrogenic activity (Ding Yuli et al., tamoxifen clinical application research progress, Inpharm research magazine in April, 2016
The 2nd phase of volume 43,275-279).Tamoxifen of the present invention is mainly Z-type.For treating advanced breast cancer and ovary
Cancer.Clinical treatment breast cancer, efficient generally in 30% effect, estrogen receptor positive patient curative effect is preferable (49%), negative
Patient's weak curative effect (7%).Premenopausal and postmenopausal patients can be used, and the post menopausal and people of 60 years old or more is more premenopausal and year
The effect of light patient is preferably.From the point of view of lesions position, the tumor efficiency of skin, lymph node and soft tissue is preferable, and bone and internal organ turn
The effect for moving tumour is poor.Triple negative breast cancer：Triple negative breast cancer refers to that cancerous tissue immunohistochemical detection result is female
Hormone receptor (ER), progesterone receptor (PR) and proto-oncogene hEGF 2 are negative breast cancer.This kind of breast
Gland cancer accounts for the 10.0%~20.8% of all breast cancer histological types, multiple studies have shown that, triple negative breast cancer mostly occurs in absolutely
Premenstrual young woman, especially African American women.Triple negative breast cancer clinical manifestation is a kind of aggressive course of disease, distant place
It shifts risk higher, visceral metastases chance is high compared with Bone tumour, and brain metastes probability is also higher.The DISTANT METASTASES IN wind of triple negative breast cancer
Danger was peaked at 3 years, may be declined later.The median tumor size of triple negative breast cancer is 2cm, and 50% has leaching
Fawn on transfer.
Fulvestrant：Fulvestrant is a kind of intramuscular injection drug researched and developed by Astrazeneca AB, in April, 2002 U.S.
State's Food and Drug Administration approval listing, suitable for treating the postmenopausal women for the deterioration that still becomes by anti-estrogen therapy disease
The metastatic breast cancer for the estrogen receptor positive suffered from.There are estrogen receptor (ER), estrogen in many breast cancer cells
The growth of such tumour can be stimulated.Fulvestrant is the estrogen receptor antagon of a kind of " pure ", does not have partial estrogen sample
Stirring effect, it by combining, blocking and lower ER so as to inhibit estrogen receptor access, can with ER competitive bindings, with ER
Affinity close to estrogen, chemical name for (7a, 17b) -7- [9- (4,4,5,5,5- five fluorine, penta sulfinyl) nonyl] -
Female steroid -1,3,5- (10)-triolefin -3,17- glycol, structural formula are as follows：
G1 cell cycles phase：The G1 phases (first gap) from mitosis to DNA replication dna before one period, also known as synthesize
Early period, this phase mainly synthesize RNA and ribosomes.The phase feature is that metabolism enlivens, rapid to synthesize RNA and protein, cell
Volume significantly increases.The major significance of this phase is that the DNA replication dna for phase next stage S performs the preparation of matter and energy.
Description of the drawings
In Fig. 1,1A represents baicalein chemical structural formula；1B, 1C, 1D, 1E represent that baicalein can lower the albumen of ER- α 36
The protein expression of ER- α 36 in expression, Western blot MDA-MB-231 (1B) and MDA-MB-436 (1D) cell；Icon
Show the concentration of baicalein processing, each pillar represents the average plus-minus standard deviation of three independent experiments, and with empty carrier (V)
The control cell comparison of transfection, * P ＜ 0.05.
Fig. 2 represents that baicalein can inhibit MAPK/ERK phosphorylations, reduces Cyclin D_1 gene expression and slows down three negative breasts
Adenocarcinoma cell is grown；ERK phosphorylations (2A) and Cyclin D_1 gene expression in Western blot MDA-MB-231
(2C)；ERK phosphorylations (2B) and Cyclin D_1 gene expression (2D) in MDA-MB-436；2E represents baicalein to MDA-MB-231
With the line chart that delays of MDA-MB-436 cell growths, every average plus-minus standard deviation for representing three independent experiments.
Fig. 3 represents that baicalein can weaken in triple negative breast cancer cell EGF signals to cell growth stimulating effect；(3A,
3B) represent MAPK/ERK phosphorylations in Western blot triple negative breast cancer cell；(3C, 3D) represents Western blot point
Cyclin D1 protein expressions in triple negative breast cancer cell are analysed, (3E, 3F) expression is individually handled with EGF or with indicating concentration
The triple negative breast cancer cell growth status that baicalein was jointly processed by, every average plus-minus mark for representing three independent experiments
It is accurate poor, compared with the control group handled with solvent (V), * P ＜ 0.05.
Fig. 4 represents that baicalein can inhibit the estrogen receptor for leading to triple negative breast cancer cell growth, and (4A, 4B) is represented
MAPK/ERK phosphorylations in Western blot triple negative breast cancer cell, wherein triple negative breast cancer cell 17 β-female two
Individually processing or the baicalein with indicating concentration are jointly processed by alcohol (E2).(4C, 4D) represents the negative breast of Western blot three
Cyclin D_1 gene is expressed in adenocarcinoma cell, wherein triple negative breast cancer cell (E2) independent processing or the Huang with indicating concentration
A kind of reed mentioned in ancient books element is jointly processed by.Three feminine genders that (4E, 4F) is represented individually to be handled with (E2) or be jointly processed by with indicating the baicalein of concentration
Breast cancer cell growth situation, every mean plus-minus standard deviation for representing independent experiment three times, compares with what is handled with solvent (V)
Group is compared, * P ＜ 0.05.
Fig. 5 represents that baicalein can enhance triple negative breast cancer cell to tamosifen sensibility, and 5A, 5B represent ER- α 36
Weak triple negative breast cancer cell growing state in 0.5 μM of tamoxifen, * P ＜ 0.05 are struck in expression.With tamoxifen list
Reason of staying alone or the triple negative breast cancer cell growth status being jointly processed by with indicating the baicalein of concentration, every representative are only three times
The average plus-minus standard deviation of vertical experiment, compared with the control group handled with solvent (V), * P ＜ 0.05.
In Fig. 6,6A represents expression quantity of the Western blot baicalein concentration to ER- α 36 in liver cancer cells Huh-7
Influence, 6B represent baicalein individually handle, the liver cancer cells that the baicalein of various concentration ratio and fulvestrant are jointly processed by
Huh-7 growing states, 6C represent the liver that baicalein is individually handled, the baicalein of various concentration ratio and tamoxifen are jointly processed by
Cancer cell Huh-7 growing states.
In Fig. 7,7A represents Western blot baicalein concentration to the expression quantity of ER- α 36 in lung carcinoma cell H460
It influences, 7B expression baicaleins are individually handled, the lung carcinoma cell that the baicalein of various concentration ratio and fulvestrant are jointly processed by
H460 growing states, 7C represent the lung that baicalein is individually handled, the baicalein of various concentration ratio and tamoxifen are jointly processed by
Cancer cell H460 growing states.
In Fig. 8,8A represents table of the Western blot baicalein concentration to ER- α 36 in neuroglial cytoma U87
Up to the influence of amount, 8B expression baicaleins are individually handled, the nerve that the baicalein of various concentration ratio and fulvestrant are jointly processed by
Glioma cell U87 growing states, 8C expression baicaleins are individually handled, the baicalein of various concentration ratio and tamoxifen are common
The neuroglial cytoma U87 growing states of processing.
In Fig. 9,9A represents Western blot baicalein concentration to the expression quantity of ER- α 36 in cancer of pancreas Panc-1
It influences, 9B represents the cancer of pancreas Panc- that baicalein is individually handled, the baicalein of various concentration ratio and fulvestrant are jointly processed by
1 growing state, 9C represent the cancer of pancreas that baicalein is individually handled, the baicalein of various concentration ratio and tamoxifen are jointly processed by
Panc-1 growing states.
In Figure 10,10A represents expression quantity of the Western blot baicalein concentration to ER- α 36 in stomach cancer cell AGS
Influence, 10B represent baicalein individually handle, the stomach cancer cell that the baicalein of various concentration ratio and fulvestrant are jointly processed by
AGS growing states, 10C represent the stomach that baicalein is individually handled, the baicalein of various concentration ratio and tamoxifen are jointly processed by
Cancer cell AGS growing states.
In Figure 11,11A represents expression of the Western blot baicalein concentration to ER- α 36 in stomach cancer cell MKN-28
The influence of amount, 11B represent that the gastric cancer that baicalein is individually handled, the baicalein of various concentration ratio and fulvestrant are jointly processed by is thin
Born of the same parents' MKN-28 growing states, 11C represent that baicalein is individually handled, the baicalein of various concentration ratio and tamoxifen are jointly processed by
Stomach cancer cell MKN-28 growing states.
Figure 12 shows the protein structure of ER- α 66 and ER- α 36, and structural domain (is marked) with A-F, amino acid sequence number,
AF-1 and AF-2, DNA binding structural domain, ligand-binding domain and dimeric structure domain have also indicated that phosphorylation site and every
The function of a structural domain.
In order to which those skilled in the art is made to be easier to understand technical scheme of the present invention, with reference to specific embodiment
It elaborates to present disclosure, but specific embodiment is not the limitation to the content of present invention.
Specific embodiment
Materials and methods
1 cell strain
MDA-MB-231 and MDA-MB-436 cells are that this laboratory preserves.
2 drugs, antibody and reagent
Baicalein (98% purity) is bought in Aladdin (Aladdin) (W101155).Fetal calf serum (FBS, lot number
1581731), carbon adsorption fetal calf serum (CS-FBS, lot number 1598176), DMEM culture mediums (lot number 8416246), without phenol red
DMEM culture mediums (lot number 1802669), trypsase (containing 0.25%EDTA, lot number 1816869), buffer solution PBS (lot numbers
8116481) purchased from Gibco companies of the U.S., dimethyl sulfoxide (DMSO) (DMSO) estrogen (E2), tamoxifen,FulvestrantIt is purchased from
In Sigma Co., USA, BCA kits (lot number P0009), purchased from the green skies bioengineering Co., Ltd in Beijing, primary antibody：
P-ERK lot numbers #9106S, ERK lot number 4695S, purchased from CST companies of the U.S., Cyclin D1 lot numbers Sc-8396 is purchased from the U.S.
Santa Cruz Technology companies, ER- α 36 are by Pacific Immunology Corp.It prepares.Secondary antibody (goat-anti rabbit, batch
Number A0208；Sheep anti mouse, lot number A0216) purchased from the green skies bioengineering Co., Ltd in Beijing.
1.3 instrument
CO2Cell incubator, superclean bench, Thermo Fisher companies of the U.S.；Mini-Protean electrophoresis and electricity turn
Shifting system, microplate reader, Bio-Rad companies of the U.S.；Inverted microscope, Japanese Olympus companies；BP-211D ten a ten thousandths point
Analyse balance, German Sartorius companies；4 DEG C of ultracentrifuges, Sigma Co., USA；Cell counter, U.S. Life
Technologies Corporation companies.
1.4 cell culture and processing
It is primary every passage in 2-3 days with the DMEM medium cultures containing 10%FBS after cell recovery；With containing before experiment
2.5%CS-FBS without phenol red DMEN culture mediums Nature enemy for 24 hours.
1.5 Western blot
It takes the logarithm the cell in growth period, with 105A to be passed in 6 orifice plates per hole, Nature enemy for 24 hours, adds in corresponding afterwards for 24 hours
Drug-treated 16h when P-PERK (detection handle 30min), total protein of cell is extracted, with the amount of every 40 μ g of hole after measured concentration
SDS-PAGE electrophoresis is carried out, and by protein delivery to pvdf membrane, 5%BSA closing 2h, TBST buffer solutions wash film 3 times every time
10min, primary antibody (ER- α 36 1:10000, EGFR 1：2000, ER- α 66 1：2000, P-ERK 1：2000, CyclinD1 1:
2000) 4 ° of overnight incubations, TBST buffer solutions wash 3 each 10min of film, secondary antibody (1:2000) 1h is incubated, TBST buffer solutions wash film
It 3 times, using β-actin and ERK as control, adds in luminescent solution and is exposed.
1.6 cell growth assay
By cell with every ware 104A cell is passed in 60mm culture dishes, is cultivated with without phenol red DMEM to addition is corresponding afterwards for 24 hours
The baicalein of concentration, and with the DMSO of same concentrations as a control group.It is used after 7 daysII Automated Cell
Counter carries out cell count.
1.7CCK-8 detects cell activity
By cell with 104It is passed per hole and adds in the baicalein for designing concentration after being cultivated 12 hours, 12 hours in 96 orifice plates,
Each concentration sets 3 repetitions, while set control group and zeroing group.After cell is continued culture to corresponding number of days, per hole
10 μ L CCK-8 of middle addition continue to cultivate 1h, and light absorption value calculates cell survivaling number at microplate reader detection 450nm.
1.8 data analyses and statistics
On stated experiment independently be repeated 3 times, using 19 softwares of SPSS progress statistical analysis, all experimental datas
It is represented by mean ± standard deviation (± s) mode, using one-way analysis of variance, p between each group<0.05 (*) has system for difference
Meter learns meaning.
Experimental procedure and interpretation of result
Embodiment 1：Baicalein can lower 36 protein expressions of ER- α
For effect of the research baicalein to triple negative breast cancer cell growth, we use triple negative breast cancer cell
MDA-MB-436 and MDA-MB-231 are tested.We are by MDA-MB-436 and MDA-MB-231 cells with every hole 105A biography
In 6 orifice plates, made with 2.5%CS-FBS without adding in baicalein after phenol red DMEM culture mediums Nature enemy 24 hours, 24 hours
Its final concentration is respectively (0.1 μM, 1 μM, 5 μM and 10 μM), and to add the DMSO of same concentrations as a control group.At drug
Reason collects cell after 16 hours, extract total protein, carries out Western blot detection.As a result it can be in dose-dependant to show baicalein
Mode reduce the expression of ER- α 36 in triple negative breast cancer cell.
Embodiment 2：Baicalein can inhibit the phosphorylation of MAPK/ERK, the expression for reducing cyclinD1 and inhibit three feminine genders
The growth of breast cancer cell
Because after ER- α 36 are most important to the growth of triple negative breast cancer cell, therefore we have detected baicalein processing
To the influence that cell MAPK/ERK phosphorylations and cyclinD1 are expressed, and observe its work to triple negative breast cancer cell growth
With.By MDA-MB-436 and MDA-MB-231 cells with every hole 105A biography is in 6 orifice plates, with 2.5%CS-FBS without phenol red
DMEM culture mediums Nature enemy adds in baicalein after 24 hours, 24 hours make its final concentration of (0.1 μM, 1 μM, 5 μM and 10 μM),
And add the DMSO of same concentrations as a control group.Drug-treated 16 hours (detection phosphorylation level processing 30min) is collected afterwards
Cell extracts total protein, carries out carrying out Western blot detection.As a result phosphorylation-ERK in cell is shown after baicalein processing
It decreases with cyclinD1 expressions.It is found by the statistics of cell quantity after handling baicalein, baicalein processing 7
The growth of triple negative breast cancer cell can be significantly inhibited after it.The above results show that baicalein can inhibit triple negative breast cancer
The growth of cell.
Embodiment 3：Baicalein can inhibit the triple negative breast cancer cell growth that estrogen receptor access stimulates due to ER-
α 36 can adjust quick estrogen receptor access and stimulate triple negative breast cancer cell growth, therefore we have detected baicalein
Whether estrogen (E2) stimulation to triple negative breast cancer cell growth can be inhibited.Plating cells are simultaneously 24 hours hungry
Afterwards, independent estrogen (1nM) processing group and estrogen and baicalein (1 μM, 5 μM) while processing group or addition same volume are set
Long-pending DMSO is as a control group.The table of Western blot detection phosphorylation-ERK and cyclinD1 is carried out after drug-treated 30min
It is counted up to level, and to the living cells quantity after handling 7 days.The results show that the estrogen of 1nM can induce MAPK/
The phosphorylation of ERK, the expression for stimulating cyclinD1, but addition baicalein then can effectively inhibit it.In addition, radix scutellariae
Element can also reduce stimulation of the estrogen to triple negative breast cancer cell growth.Illustrate that baicalein can be by lowering ER- α
36 expression inhibits stimulation of the estrogen receptor access to triple negative breast cancer cell growth.
Embodiment 4：Baicalein increases triple negative breast cancer cell to antiestrogenic sensibility
Due to lacking ER- alpha expressions, triple negative breast cancer cell typically exhibits insensitive to antiestrogenic, can not use anti-
Estrogenic is treated.Early-stage study finds that ER- α 36 participate in resistance of the breast cancer cell to antiestrogenic tamoxifen,
And the breast cancer cell of anti-tamoxifen can be allowed again to tamoxifen sensitivity by lowering the expression of ER- α 36.Therefore, we guess
Whether baicalein can allow triple negative breast cancer cell to tamoxifen sensitivity.First, we have detected tamoxifen to expression
The influence of the triple negative breast cancer cell growth of different level ER- α 36.By 104A cell is passed in 60mm culture dishes, 24 hours
0.5 μM of tamoxifen is added afterwards to continue to cultivate, and quantity detection is carried out to cell after 7 days.As a result, it has been found that 0.5 μ compared with the control group
The tamoxifen of M can significantly inhibit the growth for the triple negative breast cancer cell for striking drop ER- α 36, illustrate ER- α 36 in three feminine genders
It plays an important role in the anti-tamoxifen of breast cancer cell.Then we are had detected again at baicalein and tamoxifen collaboration
Manage the influence to triple negative breast cancer cell growth.By 104A cell is laid in 60mm culture dishes, wherein two after 24 hours
Baicalein or tamoxifen are individually added in group, it is respectively 1 μM and 0.5 μM to make its final concentration, same in another group of cell
The baicalein and tamoxifen of Shi Tianjia same concentrations, and using DMSO groups as control, processing detects living cells quantity after 7 days.Knot
Fruit shows, compared with control group and individually addition tamoxifen group, while adds baicalein and tamoxifen and can improve three the moon
Property breast cancer cell to the sensibility of tamoxifen, inhibit cell growth.
Embodiment 5
Baicalein, baicalein and tamoxifen combination, baicalein are examined using test procedure same as Example 1 respectively
It is combined with fulvestrant respectively to ER- in liver cancer cells, stomach cancer cell, pancreatic cancer cell, lung carcinoma cell, neuroglial cytoma
The influence of 36 protein expressions of α, it is specific as follows：
After baicalein processing in Huh-7 cells ER- α 36 expression
By Huh-7 cells with 106/ hole is passed in 6 orifice plates, with without phenol red DMEM medium cultures, is added after cell is adherent
The baicalein of respective concentration, it is respectively 0.1 μM, 1 μM, 10 μM, 50 μM and 100 μM to make its final concentration, adds same concentrations
DMSO as a control group (NC), carries total protein of cell and is WesternBlot, detect ER- α 36 in cell after drug-treated 16H
(8586,1:10000) expression, and with Actin (1:5000) it is analyzed (see Fig. 6 A) as control.
Huh-7 cell quantities detect after the processing of the fulvestrant (Flu) of baicalein (Bai) and various concentration
By Huh-7 cells with 104/ hole is passed in 6 orifice plates, with without phenol red DMEM medium cultures, is added after cell is adherent
The baicalein and fulvestrant of upper figure respective concentration, processing detect living cells quantity after 7 days and do figure (see Fig. 6 B).
The detection of Huh-7 cell quantities is thin by Huh-7 after the processing of the tamoxifen (TAM) of baicalein (Bai) and various concentration
Born of the same parents are with 104/ hole is passed in 6 orifice plates, and with without phenol red DMEM medium cultures, the radix scutellariae of upper figure respective concentration is added after cell is adherent
Element and tamoxifen, processing detect living cells quantity after 7 days and do figure (see Fig. 6 C).
After baicalein processing in H460 cells ER- α 36 expression
By H460 cells with 106/ hole is passed in 6 orifice plates, and with without phenol red DMEM medium cultures, phase is added after cell is adherent
The baicalein of concentration is answered, it is respectively 0.1 μM, 1 μM, 10 μM, 50 μM and 100 μM to make its final concentration, adds the DMSO of same concentrations
(NC) as a control group carries total protein of cell after drug-treated 16h and is WesternBlot, detect the expression of ER- α 36 in cell
Situation, and analyzed (see Fig. 7 A) using Actin as control.
H460 cell quantities detect after the processing of the fulvestrant (Flu) of baicalein (Bai) and various concentration.H460 is thin
Born of the same parents are with 104/ hole is passed in 6 orifice plates, and with without phenol red DMEM medium cultures, the radix scutellariae of upper figure respective concentration is added after cell is adherent
Element and fulvestrant detect living cells quantity and do figure after 7 days (see Fig. 7 B).
H460 cell quantities are detected H460 cells after the processing of the tamoxifen (TAM) of baicalein (Bai) and various concentration
With 104/ hole is passed in 6 orifice plates, and with without phenol red DMEM medium cultures, the baicalein of upper figure respective concentration is added after cell is adherent
And tamoxifen, living cells quantity is detected after 7 days and does figure (see Fig. 7 C).
After baicalein processing in U87 cells ER- α 36 expression
By U87 cells with 106/ hole is passed in 6 orifice plates, and with without phenol red DMEM medium cultures, phase is added after cell is adherent
The baicalein of concentration is answered, it is respectively 0.1 μM, 1 μM, 10 μM, 50 μM and 100 μM to make its final concentration, adds the DMSO of same concentrations
(NC) as a control group carries total protein of cell after drug-treated 16H and is WesternBlot, detect the expression of ER- α 36 in cell
Situation, and analyzed (see Fig. 8 A) using Actin as control.
U87 cell quantities detect after the processing of the fulvestrant (Flu) of baicalein (Bai) and various concentration
By U87 cells with 104/ hole is passed in 6 orifice plates, with without phenol red DMEM medium cultures, is added after cell is adherent
The baicalein and fulvestrant of figure respective concentration detect living cells quantity and do figure after 7 days (see Fig. 8 B).Baicalein (Bai) and
U87 cell quantities detect after tamoxifen (TAM) processing of various concentration
By U87 cells with 104/ hole is passed in 6 orifice plates, with without phenol red DMEM medium cultures, is added after cell is adherent
The baicalein and tamoxifen of figure respective concentration detect living cells quantity and do figure after 7 days (see Fig. 8 C).
After baicalein processing in Panc-1 cells ER- α 36 expression
By Panc-1 cells with 106/ hole is passed in 6 orifice plates, with without phenol red DMEM medium cultures, is added after cell is adherent
The baicalein of respective concentration, it is respectively 0.1 μM, 1 μM, 10 μM, 50 μM and 100 μM to make its final concentration, adds same concentrations
DMSO as a control group (NC), carries total protein of cell and is WesternBlot after drug-treated 16H, detect ER- α 36 in cell
Expression, and analyzed (see Fig. 9 A) using Actin as control.
Panc-1 cell quantities detect after the processing of the fulvestrant (Flu) of baicalein (Bai) and various concentration
By Panc-1 cells with 104/ hole is passed in 6 orifice plates, with without phenol red DMEM medium cultures, is added after cell is adherent
The baicalein and fulvestrant of upper figure respective concentration detect living cells quantity and do figure after 7 days (see Fig. 9 B).
Panc-1 cell quantities are detected Panc-1 after the processing of the tamoxifen (TAM) of baicalein (Bai) and various concentration
Cell is with 104/ hole is passed in 6 orifice plates, and with without phenol red DMEM medium cultures, the Huang of upper figure respective concentration is added after cell is adherent
A kind of reed mentioned in ancient books element and tamoxifen detect living cells quantity and do figure after 7 days (see Fig. 9 C).
After baicalein processing in ags cell ER- α 36 expression
By ags cell with 106/ hole is passed in 6 orifice plates, and with without phenol red DMEM medium cultures, phase is added after cell is adherent
The baicalein of concentration is answered, it is respectively 0.1 μM, 1 μM, 10 μM, 50 μM and 100 μM to make its final concentration, adds the DMSO of same concentrations
(NC) as a control group carries total protein of cell after drug-treated 16H and is WesternBlot, detect the expression of ER- α 36 in cell
Situation, and analyzed (see Figure 10 A) using Actin as control.
Ags cell quantity detects after the processing of the fulvestrant (Flu) of baicalein (Bai) and various concentration
By ags cell with 104/ hole is passed in 6 orifice plates, with without phenol red DMEM medium cultures, is added after cell is adherent
The baicalein and fulvestrant of figure respective concentration detect living cells quantity and do figure after 7 days (see Figure 10 B).
Ags cell quantity detects after the processing of the tamoxifen (TAM) of baicalein (Bai) and various concentration
By ags cell with 104/ hole is passed in 6 orifice plates, with without phenol red DMEM medium cultures, is added after cell is adherent
The baicalein and tamoxifen of figure respective concentration detect living cells quantity and do figure after 7 days (see Figure 10 C).
After baicalein processing in MKN-28 cells ER- α 36 expression
By MKN-28 cells with 106/ hole is passed in 6 orifice plates, with without phenol red DMEM medium cultures, is added after cell is adherent
The baicalein of respective concentration, it is respectively 0.1 μM, 1 μM, 10 μM, 50 μM and 100 μM to make its final concentration, adds same concentrations
DMSO as a control group (NC), carries total protein of cell and is WesternBlot after drug-treated 16H, detect ER- α 36 in cell
Expression, and analyzed (see Figure 11 A) using Actin as control.
MKN-28 cell quantities detect after the processing of the fulvestrant (Flu) of baicalein (Bai) and various concentration
By MKN-28 cells with 104/ hole is passed in 6 orifice plates, with without phenol red DMEM medium cultures, is added after cell is adherent
The baicalein and fulvestrant of upper figure respective concentration detect living cells quantity and do figure after 7 days (see Figure 11 B).
MKN-28 cell quantities detect after the processing of the tamoxifen (TAM) of baicalein (Bai) and various concentration
By MKN-28 cells with 104/ hole is passed in 6 orifice plates, with without phenol red DMEM medium cultures, is added after cell is adherent
The baicalein and tamoxifen of upper figure respective concentration detect living cells quantity and do figure after 7 days (see Figure 11 C).
Result of the test shows：
In liver cancer cells Huh-7, stomach cancer cell AGS, MKN-28, pancreatic cancer cell Panc-1 the expression quantity of ER- α 36 with
The increase of baicalein concentration and reduce, but reduce all under high concentration group (50 μM, 100 μM) apparent, low concentration is almost without change
Change.
In lung carcinoma cell H460 and neuroglial cytoma U87 the expression quantity of ER- α 36 with increasing for baicalein concentration and
Raising, has significant change at a concentration of 10 μM, and raising is significantly at 50 μM and 100 μM.
Each cancer cell after cell quantity aspect 0.5 μM of baicalein of addition and various concentration fulvestrant or tamoxifen
Quantity changes unobvious, and wherein Huh-7, AGS and MKN-28 have certain reduction, and H460 and U87 have faint raising, but
There is no significant difference.
Embodiment 6：Influence of the baicalein and tamoxifen of various concentration combination to triple negative breast cancer cell growth
We are by cell with 104The Huang that various combination is added in after being cultivated 12 hours, 12 hours in 96 orifice plates is passed per hole
A kind of reed mentioned in ancient books element and tamoxifen, each concentration sets 3 repetitions, while sets control group and zeroing group.Cell is continued into culture three days
Afterwards, 10 μ L CCK-8 are added in every hole, continues culture 1 hour, light absorption value calculates cell survivaling number at microplate reader detection 450nm.
Group 1:Baicalein (0.5 μM)+tamoxifen (0.1 μM)；Group 2:Baicalein (0.5 μM)+tamoxifen (0.2 μM)；Group 3:Radix scutellariae
Plain (0.5 μM)+tamoxifen (0.5 μM)；Group 4:Baicalein (0.5 μM)+tamoxifen (1 μM)；Group 5:Baicalein (1 μM)+he
Former times is not fragrant (0.1 μM)；Group 6:Baicalein (1 μM)+tamoxifen (0.2 μM)；Group 7:Baicalein (1 μM)+tamoxifen (0.5 μ
M) for we have found that baicalein can all generate synergistic effect in various concentration with the tamoxifen of various concentration, effectively inhibition three is cloudy
Property breast cancer cell growth.And baicalein (1 μM)+tamoxifen (0.5 μM) concentration versus cell rejection ability is set dense at us
It is most strong in degree group.Result of the test is shown in Tables 1 and 2.
Baicalein and the combination synergy of tamoxifen promote MDA-MB-231 cell death assays (CCK-8 results)
Survival rate=medicine group OD values/control group OD values
Table 1
| Group | Usage amount (μM) | Cell survival rate (%) | |
| Control group | –– | 100 | |
| Group 1 | Baicalein | 0.5 | 58% |
| Tamoxifen | 0.1 | ||
| Group 2 | Baicalein | 0.5 | 42% |
| Tamoxifen | 0.2 | ||
| Group 3 | Baicalein | 0.5 | 35% |
| Tamoxifen | 0.5 | ||
| Group 4 | Baicalein | 0.5 | 26% |
| Tamoxifen | 1 | ||
| Group 5 | Baicalein | 1 | 49% |
| Tamoxifen | 0.1 | ||
| Group 6 | Baicalein | 1 | 33% |
| Tamoxifen | 0.2 | ||
| Group 7 | Baicalein | 1 | 16% |
| Tamoxifen | 0.5 |
Baicalein and the combination synergy of tamoxifen promote MDA-MB-436 cell death assays
Survival rate=medicine group OD values/control group OD values
Table 2
| Group | Usage amount (μM) | Cell survival rate (%) | |
| Control group | –– | 100 | |
| Group 1 | Baicalein | 0.5 | 56% |
| Tamoxifen | 0.1 | ||
| Group 2 | Baicalein | 0.5 | 43% |
| Tamoxifen | 0.2 | ||
| Group 3 | Baicalein | 0.5 | 26% |
| Tamoxifen | 0.5 | ||
| Group 4 | Baicalein | 0.5 | 14% |
| Tamoxifen | 1 | ||
| Group 5 | Baicalein | 1 | 38% |
| Tamoxifen | 0.1 | ||
| Group 6 | Baicalein | 1 | 22% |
| Tamoxifen | 0.2 | ||
| Group 7 | Baicalein | 1 | 8% |
| Tamoxifen | 0.5 |
Embodiment 7：Influence of the baicalein and fulvestrant of various concentration combination to triple negative breast cancer cell growth
We are by cell with 104The radix scutellariae that various combination is added in after being cultivated 12 hours, 12 hours in 96 orifice plates is passed per hole
Element and fulvestrant, each concentration sets 3 repetitions, while sets control group and zeroing group.After cell is continued culture three days,
10 μ L CCK-8 are added in per hole, continue culture 1 hour, light absorption value calculates cell survivaling number at microplate reader detection 450nm.Group
1:Baicalein (0.5 μM)+fulvestrant (0.1 μM)；Group 2:Baicalein (0.5 μM)+fulvestrant (0.2 μM)；Group 3:Baicalein
(0.5 μM)+fulvestrant (0.5 μM)；Group 4:Baicalein (0.5 μM)+fulvestrant (1 μM)；Group 5:Baicalein (1 μM)+fluorine is tieed up
Take charge of group's (0.1 μM)；Group 6:Baicalein (1 μM)+fulvestrant (0.2 μM)；Group 7:Baicalein (1 μM)+fulvestrant (0.5 μM)
We have found that baicalein can all generate synergistic effect in various concentration with the fulvestrant of various concentration, effectively inhibit three negative breasts
Adenocarcinoma cell is grown.And baicalein (1 μM)+fulvestrant (0.5 μM) concentration versus cell rejection ability is in our set concentration groups
In it is most strong.Result of the test is shown in Table 3 and table 4.
The combination synergy of baicalein and fulvestrant promotes MDA-MB-231 cell death assays (CCK-8 results)
Survival rate=medicine group OD values/control group OD values
Table 3
| Group | Usage amount (μM) | Cell survival rate (%) | |
| Control group | –– | 100 | |
| Group 1 | Baicalein | 0.5 | 72% |
| Fulvestrant | 0.1 | ||
| Group 2 | Baicalein | 0.5 | 63% |
| Fulvestrant | 0.2 | ||
| Group 3 | Baicalein | 0.5 | 55% |
| Fulvestrant | 0.5 | ||
| Group 4 | Baicalein | 0.5 | 46% |
| Fulvestrant | 1 | ||
| Group 5 | Baicalein | 1 | 66% |
| Fulvestrant | 0.1 | ||
| Group 6 | Baicalein | 1 | 51% |
| Fulvestrant | 0.2 | ||
| Group 7 | Baicalein | 1 | 40% |
| Fulvestrant | 0.5 |
The combination synergy of baicalein and fulvestrant promotes MDA-MB-436 cell death assays survival rate=drug
Group OD values/control group OD values
Table 4
| Group | Usage amount (μM) | Cell survival rate (%) | |
| Control group | –– | 100 | |
| Group 1 | Baicalein | 0.5 | 61% |
| Fulvestrant | 0.1 | ||
| Group 2 | Baicalein | 0.5 | 48% |
| Fulvestrant | 0.2 | ||
| Group 3 | Baicalein | 0.5 | 40% |
| Fulvestrant | 0.5 | ||
| Group 4 | Baicalein | 0.5 | 32% |
| Fulvestrant | 1 | ||
| Group 5 | Baicalein | 1 | 55% |
| Fulvestrant | 0.1 | ||
| Group 6 | Baicalein | 1 | 37% |
| Fulvestrant | 0.2 | ||
| Group 7 | Baicalein | 1 | 20% |
| Fulvestrant | 0.5 |
Claims (10)
1. a kind of application of substance for lowering 36 expression of ER- α in the drug for treating disease is prepared, the substance are selected from Huang
A kind of reed mentioned in ancient books extract, labiate scutellariae,radix, Yunnan scutellariae,radix, sun plant root, apocynaceae plant eel boisiana extract.
2. application according to claim 1, the disease is selected from：Liver cancer, gastric cancer, breast cancer, carcinoma of endometrium, prostate
Cancer, cancer of pancreas.
3. application according to claim 1, the disease is breast cancer.
4. application according to claim 3, the disease is triple negative breast cancer.
5. according to the application described in claim 1-4 any claims, the substance for lowering 36 expression of ER- α is baicalein.
6. according to the application described in claim 1-4 any claims, the substance for lowering 36 expression of ER- α is baicalein
With the composition of antiestrogen.
7. application according to claim 6, the antiestrogen is selected from tamoxifen, fluorine Wei Siqiong.
8. application according to claim 6, the effective dose of the baicalein is 1-20 μM.
9. application according to claim 6, the effective dose of the baicalein is 0.5-10 μM.
10. application according to claim 6, the effective dose of the antiestrogen is 0.1-10 μM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810105438.4A CN108236622B (en) | 2018-02-02 | 2018-02-02 | Application of composition for down-regulating ER-alpha 36 expression in preparation of medicine for treating triple negative breast cancer |
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| WO2022134433A1 (en) * | 2020-12-21 | 2022-06-30 | 苏州普瑞森基因科技有限公司 | Composition and application thereof in treatment of tumors |
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| CN103655600A (en) * | 2013-12-18 | 2014-03-26 | 成都中医药大学 | Drug composition for treating cancers, preparation method and applications |
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| CN103655600A (en) * | 2013-12-18 | 2014-03-26 | 成都中医药大学 | Drug composition for treating cancers, preparation method and applications |
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| 徐维恒，肖雷，马宜明: "黄岑素对乳腺癌细胞MCF-7和MDA-MB-231增殖的影响", 《中国药师》 * |
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| WO2022134433A1 (en) * | 2020-12-21 | 2022-06-30 | 苏州普瑞森基因科技有限公司 | Composition and application thereof in treatment of tumors |
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