CN108785356A - Xihuang pills are preparing the application in treating breast cancer taxol resistance drug - Google Patents
Xihuang pills are preparing the application in treating breast cancer taxol resistance drug Download PDFInfo
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Abstract
The invention discloses Xihuang pills to prepare the application in treating breast cancer taxol resistance drug.Xihuang pill drug serum can obviously inhibit the proliferation of MDA-MB-231P, and it is more notable to the proliferation inhibiting effect of MDA-MB-231P with taxol combination.In addition, Xihuang pill drug serum can be obviously promoted the apoptosis of MDA-MB-231P, and it is more notable to the apoptosis facilitation effect of MDA-MB-231P with taxol combination.Xihuang pill drug serum can raise Bax albumen in mdr cell, lower Bcl-2 protein expressions in mdr cell.So, Xihuang pills can be used in the chemotherapeutic sensitizer for preparing the taxol for treating breast cancer, it can also be used in the drug prepared for reversing breast cancer taxol resistance, can also be applied with paclitaxel plus in the pharmaceutical composition for treating breast cancer.
Description
Technical field
The invention belongs to biomedical and pharmaceutical fields, and in particular to Xihuang pills are preparing treatment breast cancer taxol resistance
Application in property drug.
Background technology
Breast cancer is one of the most common malignant tumors in women, and incidence accounts for the 25.2% of whole female malignants, mesh
Before, chemotherapy is still the essential therapeutic arsenals of breast cancer, and taxanes drug is then one of most important drug of mammary cancer chemotherapy.
However taxol drug is 32%~62% in the effective percentage of breast cancer treatment, wherein Paclitaxel Chemotherapy drug resistance is to lead to patientization
Treat the major reason of failure.
Taxol is a kind of rush microtubule polymerization and stabilization, antimitotic cell cycle specific drugs.Japanese yew refines
It is complicated to treat resistance mechanism, studies have shown that including mainly with the relevant mechanism of taxol resistance：The drugs such as P- glycoprotein pump out albumen
Expression increases；Pro-apoptotic factor gene and protein expression attenuating or anti-apoptosis factor gene and protein expression increase, and keep tumour more
Medicine mdr cell is resisted and the apoptosis of escape antitumor drug induction；The change of tubulin subtype expression or its combined with drug
The gene mutation in site causes the raising of microtubule polymerization critical concentration or micro-pipe to be combined with drug and is obstructed；Cell cycle change and
Tumor microenvironment change etc..So finding the drug for the treatment of breast cancer taxol resistance, including reverse breast cancer taxol
Drug resistance improves drug of the breast cancer cell for chemosensitivity of pacilitaxel, it appears is even more important.
Important component of the traditional Chinese medicine as combined therapy of tumour, with it safely, effectively, Small side effects the features such as pre-
In occurrence and development that are anti-and blocking tumour, there are important curative effect and unique advantage.Xihuang pills prescription from portion issue Chinese medicine at
9 page 71, standard No. WS3-B-1734-94 of square preparation, major function is clearing heat and detoxicating, and seeks detumescence, is used for ulcer malignant boil
Poison, scrofula, streamer, cancerous swelling etc..The Xihuang pills prescription is as follows：
【Prescription】Cow-bezoar 15g, Moschus 15g, frankincense (vinegar system) 550g, myrrh (vinegar system) 550g
【Preparation method】Above four taste, cow-bezoar, Moschus are finely ground, separately take glutinous millet 350g, cook drying, are ground into frankincense, myrrh
Fine powder, sieving, then with cow-bezoar, Moschus powder facing-up, sieving, mixing.With water pill, set dry in the shade at aeration-drying to get.
Have at present studies have shown that Xihuang pills can improve chemotherapeutic efficacy, and inhibited to Several Kinds of Malignancy.Such as
Xihuang pills combine CHOP chemotherapy regimens and treat non-Hodgkin lymphoma, and the 74.19% of comparison short term effect discovery observation group is apparent
Better than the 58.06% of control group；Xihuang pills joint transcatheter arterial chemoembolization can effectively improve the clinical of advanced liver cancer patient and treat
Effect.However, the research for participating in human breast carcinoma taxol resistance there has been no Xihuang pills at present is seen in report.
Invention content
In view of this, the present invention provides Xihuang pills to prepare the application in treating breast cancer taxol resistance drug.
One aspect of the present invention is that providing Xihuang pills is preparing the drug for reversing breast cancer taxol resistance
In application.
In some embodiments of the above application of the present invention, it is preferable that the Xihuang pills are by inhibiting breast cancer purple
China fir alcohol mdr cell is proliferated and induced breast cancer taxol resistance Apoptosis makes breast cancer taxol resistance sex reversal.
One aspect of the present invention is to provide Xihuang pills in the chemotherapy sensitizing for preparing the taxol for treating breast cancer
Application in agent.
In some embodiments of the above application of the present invention, it is preferable that the Xihuang pills are by inhibiting to breast cancer
Taxol resistance cell Proliferation and induced breast cancer taxol resistance Apoptosis and to chemotherapeutic drug Paclitaxel enhanced sensitivity.
One aspect of the present invention is that provide Xihuang pills is preparing the medicine for treating breast cancer with combining for taxol
Application in compositions.
In the present invention, with the human breast cancer cell line MDA-MB-231P of taxol resistance (or referred to as human breast carcinoma Japanese yew
Alcohol medicine-resistant cell line) it is used as research object, Taxol-resistant cell lines MDA-MB-231P is detected in difference using CCK-8 methods
The lower ability of cell proliferation of dosage Xihuang pill drug serum effect and detection Taxol-resistant cell lines MDA-MB-231P be not
With the lower ability of cell proliferation of dosage Xihuang pill drug serum joint taxol effect, pass through Flow cytometry various dose west
The MDA-MB-231P mdr cell apoptosis of yellow ball Contained Serum processing changes and detection various concentration taxol is independent or joins
The MDA-MB-231P mdr cell apoptosis variation of Xihuang pill drug serum processing is closed, western blot detections are in various dose
Xihuang pill drug serum acts on the change of lower cell cycle and apoptosis-related protein bcl-2 and Bax expression.
As a result it shows：Xihuang pill drug serum can obviously inhibit the increasing of Taxol-resistant cell lines MDA-MB-231P
It grows, and Xihuang pill drug serum is combined the proliferation inhibiting effect to Taxol-resistant cell lines MDA-MB-231P with taxol
Significantly.And Flow cytometry is the result shows that Xihuang pill drug serum can be obviously promoted Taxol-resistant cell lines MDA-
The apoptosis of MB-231P, and Xihuang pill drug serum withers to Taxol-resistant cell lines MDA-MB-231P with taxol combination
It is more notable to die facilitation effect.Western blot testing results show that Xihuang pill drug serum can raise Bax albumen, lower
Bcl-2 protein expressions.
It these results suggest that：Xihuang pill drug serum can activate or enhance lethal effect of the taxol to breast cancer cell,
So as to improve breast cancer cell to paclitaxel-sensitive and reverse the taxol resistance for treating breast cancer.Thus may be used
See, the Xihuang pills can be used in the chemotherapeutic sensitizer for preparing the taxol for treating breast cancer, can also be used in and be prepared
In drug for reversing breast cancer taxol resistance, it can also apply and prepared for treating breast cancer with paclitaxel plus
Pharmaceutical composition in.
Compared with the existing technology, the present invention has technique effect beneficial below：
The application that The present invention gives Xihuang pills in the chemotherapeutic sensitizer for preparing taxol for treating breast cancer,
The application and Xihuang pills prepared in the drug for reversing breast cancer taxol resistance is preparing use with combining for taxol
Application in the pharmaceutical composition for the treatment of breast cancer.
Description of the drawings
Fig. 1 is two kinds of cell strain MDA-MB-231 and MDA-MB-231P (50nmol/ under the effect of different paclitaxel concentrations
L, 100nmol/L, 200nmol/L, 300nmol/L) cell activity (cell viability) compare, correspond to Fig. 1 in, remember respectively
For：50nM-TAXOL,100nM-TAXOL,200nM-TAXOL, 300nM-TAXOL.
Fig. 2 be Taxol-resistant cell lines MDA-MB-231P various dose Xihuang pill drug serum (blank control group,
Middle dose group, high dose group) ability of cell proliferation compares down for effect, and correspond in Fig. 2, is denoted as respectively： CTRL,MID-XHW,
HIG-XHW。
Fig. 3 be Taxol-resistant cell lines MDA-MB-231P various dose Xihuang pill drug serum (blank control group,
Middle dose group, high dose group) ability of cell proliferation compares down for joint taxol (300nmol/L) effect.Corresponding in Fig. 3, divide
It is not denoted as：CTRL-TAXOL,MID-XHW-TAXOL,HIG-XHW-TAXOL.
Fig. 4 A, Fig. 4 B, Fig. 4 C are Flow cytometry respectively via various dose Xihuang pill drug serum (blank control
Group, middle dose group, high dose group) processing MDA-MB-231P mdr cell apoptosis figures.Wherein, in each figure horizontal axis indicate be
The PI dye distributions of cell are shown in the dye distribution of Annexin V, the longitudinal axis.Fig. 4 D give Flow cytometry via
The MDA-MB-231P mdr cells of various dose Xihuang pill drug serum (blank control group, middle dose group, high dose group) processing
The comparison of apoptosis rate is denoted as CTRL groups, MID-XHW groups, HIG-XHW groups respectively.
Fig. 5 A and Fig. 5 B are Flow cytometry respectively via containing 0nmol/L taxols and high dose Xihuang pills drug containing
The HIG-XHW+0Taxol groups of serum, the taxol containing same concentrations CTRL+0Taxol control groups processing MDA-MB-
231P mdr cell apoptosis figures.In each figure, what horizontal axis indicated is the dye distribution of Annexin V, and cell is shown in the longitudinal axis
PI dye distributions.
Fig. 5 C and Fig. 5 D are that Flow cytometry contains via containing 50nmol/L taxols and high dose Xihuang pills respectively
The HIG-XHW+50Taxol groups of medicine serum, the taxol containing same concentrations CTRL+50Taxol control groups processing MDA-
MB-231P mdr cell apoptosis figures.In each figure, what horizontal axis indicated is the dye distribution of Annexin V, and the longitudinal axis is shown carefully
The PI dye distributions of born of the same parents.
Fig. 5 E and Fig. 5 F are that Flow cytometry contains via containing 100nmol/L taxols and high dose Xihuang pills respectively
The HIG-XHW+100Taxol groups of medicine serum, the taxol containing same concentrations CTRL+100Taxol control groups processing
MDA-MB-231P mdr cell apoptosis figures.In each figure, what horizontal axis indicated is the dye distribution of Annexin V, what the longitudinal axis was shown
It is the PI dye distributions of cell.
Fig. 5 G and Fig. 5 H are that Flow cytometry contains via containing 300nmol/L taxols and high dose Xihuang pills respectively
The HIG-XHW+300Taxol groups of medicine serum, the taxol containing same concentrations CTRL+300Taxol control groups processing
MDA-MB-231P mdr cell apoptosis figures.In each figure, what horizontal axis indicated is the dye distribution of Annexin V, and the longitudinal axis is shown
The PI dye distributions of cell.
Fig. 5 I and Fig. 5 J are that Flow cytometry contains via containing 500nmol/L taxols and high dose Xihuang pills respectively
The HIG-XHW+500Taxol groups of medicine serum, the taxol containing same concentrations CTRL+500Taxol control groups processing
MDA-MB-231P mdr cell apoptosis figures.In each figure, what horizontal axis indicated is the dye distribution of Annexin V, what the longitudinal axis was shown
It is the PI dye distributions of cell.
Fig. 5 K be flow cytometric art detection via various concentration (0nmol/L, 50nmol/L, 100nmol/L,
200nmol/L, 300nmol/L) paclitaxel plus high dose Xihuang pill drug serum (being denoted as XHW) and be added same concentrations purple
The comparison result of the MDA-MB-231P mdr cell apoptosis rates of blank control group (the being denoted as CTRL) processing of China fir alcohol, corresponds to figure
In 5K, it is denoted as respectively：0nM-TAXOL,50nM-TAXOL, 100nM-TAXOL,300nM-TAXOL,500nM-TAXOL.
Fig. 6 A are to be handled through various dose Xihuang pill drug serum (blank control group, middle dose group, high dose group)
Immunoblotting (the Western of cell cycle and apoptosis-related protein bcl-2 and Bax expression in MDA-MB-231P cells
Blot) testing result corresponds in Fig. 6 A, is denoted as respectively：CTRL,MID-XHW, HIG-XHW.
Fig. 6 B are in fig. 6 through various dose Xihuang pill drug serum (blank control group, middle dose group, high dose group)
Bax albumen relative expression quantities compare in the MDA-MB-231P cells of processing, correspond in Fig. 6 B, are denoted as respectively：CTRL,MID-
XHW、HIG-XHW。
Fig. 6 C are in fig. 6 through various dose Xihuang pill drug serum (blank control group, middle dose group, high dose group)
Bcl-2 albumen relative expression quantities compare in the MDA-MB-231P cells of processing, correspond in Fig. 6 C, are denoted as respectively：CTRL,
MID-XHW、HIG-XHW。
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment.It will be understood by those skilled in the art that
The following examples are merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.
In the examples where no specific technique or condition is specified, according to technology or condition described in document in the art or
Person carries out according to product description.Not specified various instruments, raw materials and reagents are that this field is ripe in the following example
The commercial product known can be obtained by commercial sources.
One, material and facility explanation
DMEM culture mediums are bought from HyClone companies of the U.S.；Fetal calf serum is bought from Gibco companies of the U.S.；Antibody is bought
From Proteintech companies of the U.S.；BCA determination of protein concentration kits are bought from the green skies Bioisystech Co., Ltd in Shanghai；
Annexin V-FITC/PI cell apoptosis detection kits are bought from biouniquer companies of the U.S.；The purchase of CCK-8 reagents is certainly
Japanese colleague company.Xihuang pills are purchased from Beijing Tongrentang Technology Development Co.ltd. Pharmaceutical Factory.Taxol is purchased from Beijing rope
Lai Bao Science and Technology Ltd.s.Wistar rats are bought from this Leco Corp. of Shanghai, and every weight 200g is female, animal
Production licence number is production permit SCXK (Shanghai) 2017-0005.It raises in SPF Animal Lab, is in a good state of health.Stream
Formula cell instrument is bought from Becton Dickinson companies of the U.S.；Protein blot detecting system is bought from Bio-Rad companies of the U.S..
Xihuang pill drug serum takes following steps to prepare：
Xihuang pills middle dose group (MID-XHW) and high dose group (HIG-XHW) dosage are respectively 0.6g/kg and 1.2g/
Kg, through distilled water rinsing, dry, ultramicro grinding and ultrasonic vibration program prepare drug suspension to Xihuang pills, and suspension (gives liquid)
Volume be 10ml/kg.Distilled water group is blank control group (CTRL), dosage 10ml/kg.It is prepared by Xihuang pill drug serum
It is grouped as follows shown in table 1.
Wistar rats (4 week old 200g, be all female) are randomized into high dose group, middle dose group and blank control
Group, every group 2；Each group animal presses predetermined close gavage, once in the morning and once at night, continuous 7 days respectively.Prohibit before last 1 gavage
Food 12 hours, 1 hour after gavage, with 20% urethane (20g urethane+100ml brine), according to the dosage row abdomen of 10ml/kg
Chamber injecting anesthetic, abdominal aorta blood sampling, sterile separation serum after 30min inactivations, are set -80 DEG C of casees and are saved backup by 56 DEG C.
Table 1：Xihuang pill drug serum prepares grouping
| Group | Gavage | Frequency/period |
| Middle dose group (MID-XHW) | Xihuang pills 0.6g/kg (10ml/kg) | It is secondary daily, 14 times weekly |
| High dose group (HIG-XHW) | Xihuang pills 1.2g/kg (10ml/kg) | It is secondary daily, 14 times weekly |
| Blank control group (CTRL) | Distilled water (10ml/kg) | It is secondary daily, 14 times weekly |
Two, the foundation and identification of human breast carcinoma medicine-resistant cell line
The foundation of the breast cancer MDA-MB-231P cell strains of 2.1 taxol resistances：
Human breast cancer cell line MDA-MB-231 is purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's base in January, 2016
Plinth medical cell center (address：Three No. five, Dongcheng District, Beijing during March Dongdan), number 3111C0001CCC000014.
Human breast carcinoma medicine-resistant cell line MDA-MB-231P is to induce parent using taxol low concentration dosage successive induction method
Human breast cancer cell line MDA-MB-231 and establish.The specific method is as follows：
Human breast cancer cell line MDA-MB-231 cultures in culture medium (the DMEM culture mediums for containing 10% fetal calf serum).
Cell adherent growth, it is in culture medium (the DMEM culture mediums for containing 10% fetal calf serum) plus a concentration of when reaching exponential phase of growth
The taxol of 20nmol/L, continuous action for 24 hours abandon culture medium, washed one time, changed not with phosphate buffer (PBS) by rear withdrawal, suction
The fresh culture (the DMEM culture mediums for containing 10% fetal calf serum) of drug containing continues to cultivate.Sensitive cells is gradually dead, waits surviving
Cell restore normal growth, and merges the culture medium that the taxol containing 20nmol/L is added again up to 80% (containing 10% fetal calf serum
DMEM culture mediums)；It induces repeatedly, waits for that death no longer occurs for cell, the speed of growth is restored, then carries out next concentration continuation
Induction.Gradually increase taxol induced concentration with 20nmol/L, 40nmol/L, 60nmol/L, 80nmol/L concentration gradient to arrive
100nmol/L induces, changes liquid, passage, lasts 10 months, drug resistant taxol resistance cell strain is stablized in acquisition repeatedly
MDA-MB-231P。
2.2 cell culture condition：
MDA-MB-231 cell lines are cultivated in the DMEM culture mediums containing 10% fetal calf serum, MDA-MB-231P cell lines
The routine culture in the DMEM culture mediums containing 10% fetal calf serum and 100nmol/L taxols.In 37 DEG C, 5%CO2Constant temperature incubation
It is incubated in case, is passed on every 2~3d.Cell reaches exponential phase and is tested.
2.3 external drug sensitivity assay：Cell viability is analyzed using CCK-8 methods
The MDA-MB-231 cell dissociations of logarithmic growth phase are diluted to 2000/ at single cell suspension after cell count
Hole is inoculated in 96 orifice plates, in 37 DEG C, 5%CO2After being cultivated 24 hours in the incubator of saturated humidity, packet transaction：It replaces and contains respectively
There are culture medium (the DMEM trainings containing 10% fetal calf serum of 50nmol/L, 100nmol/L, 200nmol/L, 300nmol/L taxol
Support base), in 37 DEG C, 5%CO2(it is 1 by volume ratio by CCK-8 working solutions after being incubated 48 hours in the incubator of saturated humidity:9
CCK-8 stostes and culture medium configure, culture medium be the DMEM culture mediums containing 10% fetal calf serum) be added 96 orifice plates in,
After being incubated 1~2 hour, absorbance (OD) is read at wavelength 450nm.Every group of at least three multiple holes.
The MDA-MB-231P cell strains of logarithmic growth phase are digested to single cell suspension, are diluted to after cell count
2000/ hole is inoculated in 96 orifice plates, in 37 DEG C, 5%CO2After being cultivated 24 hours in the incubator of saturated humidity, packet transaction：Respectively
It replaces the culture medium containing 50nmol/L, 100nmol/L, 200nmol/L, 300nmol/L taxol and (contains 10% fetal calf serum
DMEM culture mediums), in 37 DEG C, 5%CO2After being incubated 48 hours in the incubator of saturated humidity, by CCK-8 working solutions (by volume
Than being 1:9 CCK-8 stostes and culture medium configures, and culture medium is the DMEM culture mediums containing 10% fetal calf serum) it is added 96
In orifice plate, after being incubated 1~2 hour, absorbance (OD) is read at wavelength 450nm.Every group of at least three multiple holes.
As shown in Figure 1：When cell is acted in the taxol of 50nmol/L, 100nmol/L, 200nmol/L, 300nmol/L
Under, after cultivating 48 hours, the cell activity of MDA-MB-231 cell strains and MDA-MB-231P cell strains is respectively 76.4% He
105.6%, 69.1% and 106.4%, 66.1% and 97.1%, 59.7% and 83.1%, each group difference has significant difference
(P<Label is * * in 0.001, Fig. 1).
As it can be seen that under different paclitaxel concentrations (50nmol/L, 100nmol/L, 200nmol/L, 300nmol/L) effect,
The cell activity of drug-resistant cell strain MDA-MB-231P is all significantly higher than parental cell strain MDA-MB-231, shows the drug resistance of induction
Cell strain MDA-MB-231P has taxol resistance.
Three, CCK-8 analyzes the lower Taxol-resistant cell lines MDA-MB-231P cell Proliferations of Xihuang pills effect
The influence that 3.1 various dose Xihuang pill drug serums are proliferated Taxol-resistant cell lines MDA-MB-231P
The MDA-MB-231P cell dissociations of logarithmic growth phase are diluted to 2000/ at single cell suspension after cell count
Hole is inoculated in 96 orifice plates, in 37 DEG C, 5%CO2After being cultivated 24 hours in the incubator of saturated humidity, packet transaction：It replaces and contains respectively
There is the culture medium of various dose Xihuang pill drug serum (being respectively MID-XHW groups, HIG-XHW groups and CTRL control groups) (to contain
The DMEM culture mediums of 10% fetal calf serum), in 37 DEG C, 5%CO2After being incubated 48 hours in the incubator of saturated humidity, by CCK-8
Working solution (is 1 by volume ratio：9 CCK-8 stostes and culture medium configures, and culture medium is the DMEM containing 10% fetal calf serum
Culture medium) it is added in 96 orifice plates, after being incubated 1~2 hour, absorbance (OD) is read at wavelength 450nm.Every group of at least three is multiple
Hole.
The results are shown in Figure 2：Compared with CTRL control groups, MDA-MB-231P cell Proliferations decline in MID-XHW groups
7.8% (P<0.05, # is denoted as in figure), MDA-MB-231P cell Proliferations decline 14.2% (P in HIG-XHW groups<0.01, in figure
It is denoted as ##)；MDA-MB-231P cell Proliferations decline 7.1% (P wherein in HIG-XHW groups ratio MID-XHW groups<0.05, remember in figure
For *).This illustrates that Xihuang pill drug serum can obviously inhibit breast carcinoma resistance cell MDA-MB-231P to be proliferated.
3.2 paclitaxel plus various dose Xihuang pill drug serums are proliferated Taxol-resistant cell lines MDA-MB-231P
Influence
The MDA-MB-231P cell dissociations of logarithmic growth phase are diluted to 2000/ at single cell suspension after cell count
Hole is inoculated in 96 orifice plates, in 37 DEG C, 5%CO2After the incubator culture 24 hours of saturated humidity, packet transaction：It replaces and contains respectively
300nmol/L taxols and various dose Xihuang pills serum (be respectively MID-XHW+Taxol groups, HIG-XHW+TAxol groups and
CTRL+Taxol control groups) the culture medium DMEM culture mediums of 10% fetal calf serum (contain), after being incubated 48 hours in incubator,
(it is 1 by volume ratio by CCK-8 working solutions:9 CCK-8 stostes and culture medium configures, and culture medium is containing 10% fetal calf serum
DMEM culture mediums) be added 96 orifice plates in, be incubated 1~2 hour after, wavelength 450nm at reading absorbance (OD).Every group extremely
Few 3 multiple holes.
The results are shown in Figure 3：Compared with CTRL+Taxol control groups, MDA-MB-231P is thin in MID-XHW+Taxol groups
Born of the same parents, which are proliferated, declines 18% (p<0.01), MDA-MB-231P cell Proliferations decline 28.5% (p in HIG-XHW+Taxol groups<
## is denoted as in 0.01, Fig. 3)；Wherein MDA-MB-231P cell Proliferations in HIG-XHW+Taxol groups ratio MID-XHW+Taxol groups
Decline 12.8% (p<* * are denoted as in 0.01, Fig. 3).This illustrates that taxol can significantly inhibit purple with Xihuang pill drug serum combination
China fir alcohol persister MDA-MB-231P cell Proliferations.
The result of Fig. 2 and Fig. 3 are compared, it is found that increase in taxol resistance strain MDA-MB-231P cell lines
In the experiment grown, compared with Xihuang pill drug serum is used alone, taxol has better inhibition with Xihuang pill drug serum combination
Cel l proliferation, this illustrates that Xihuang pill drug serum can obviously activate or enhance the lethal effect to breast cancer cell.
Four, the cell cycle changes and apoptosis is tested
Influence of the 4.1 various dose Xihuang pill drug serums to Taxol-resistant cell lines MDA-MB-231P apoptosis
The MDA-MB-231P cells of logarithmic growth phase, are digested to single cell suspension, by 2 × 107Cells/well is inoculated in 6
In porocyte culture plates, per hole 2ml culture mediums (the DMEM culture mediums for containing 10% fetal calf serum), in 37 DEG C, 5%CO2It is saturated wet
In the incubator of degree, rear progress packet transaction adherent for 24 hours is cultivated, suction abandons former culture medium, replaces contain various dose Xihuang pills respectively
The culture medium of Contained Serum (being respectively MID-XHW groups, HIG-XHW groups and CTRL control groups) (contains the DMEM of 10% fetal calf serum
Culture medium), after acting on 48 hours, cell in 6 orifice plates is collected, is placed in centrifuge tube, 800rpm centrifuges 5min, removes supernatant, presses
It is operated according to Annexin V-FITC/PI apoptosis kit specifications, washes away residual pancreatin, often 500ul bufferings are added in pipe
Liquid sequentially adds 5ul Annexin V-FITC, 5ul PI, and room temperature, which is protected from light, is incubated 10min, using flow cytomery each group
Apoptosis situation.Every group of at least three multiple holes.
As a result as shown in Fig. 4 A~Fig. 4 D.Fig. 4 A, Fig. 4 B, Fig. 4 C are Flow cytometry respectively via various dose west
The MDA-MB-231P mdr cell apoptosis figures of yellow ball Contained Serum (blank control group, middle dose group, high dose group) processing.Its
In, what horizontal axis indicated is the dye distribution of Annexin V in each figure, and the PI dye distributions of cell are shown in the longitudinal axis.In conjunction with figure
The case where 4A~Fig. 4 C, flow cytometric art are detected via various dose Xihuang pill drug serum (blank control group, middle dosage
Group, high dose group) processing MDA-MB-231P mdr cell apoptosis rates compare, comparison result is as shown in Figure 4 D, figure
Middle each group is denoted as CTRL groups, MID-XHW groups, HIG-XHW groups respectively.It can be seen that by Fig. 4 D：MID-XHW groups, HIG-XHW groups
Apoptosis rate is 1.08 times of CTRL groups, 1.42 times of (P successively<* * are denoted as in 0.01, Fig. 4 D)；Wherein HIG-XHW groups apoptosis rate ratio
MID-XHW groups improve 31.5% (P<* * * are denoted as in 0.001, Fig. 4 D).This illustrates that Xihuang pill drug serum can remarkably promote purple
China fir alcohol drug resistance MDA-MB-231P drug resistance born of the same parents system apoptosis.
4.2 Xihuang pill drug serums combine influence of the taxol to Taxol-resistant cell lines MDA-MB-231P apoptosis
The MDA-MB-231P cells of logarithmic growth phase, are digested to single cell suspension, by 2 × 107Cells/well is inoculated in 6
In porocyte culture plates, per hole 2ml culture mediums (the DMEM culture mediums for containing 10% fetal calf serum), in 37 DEG C, 5%CO2It is saturated wet
Cultivated in the incubator of degree it is adherent for 24 hours after, suction abandon former culture medium, be grouped processing：It is changed to blank control (CTRL) training respectively
Support base (the DMEM culture mediums for containing 10% fetal calf serum) and the culture medium containing high dose (HIG-XHW) Xihuang pill drug serum
The DMEM culture mediums of 10% fetal calf serum (contain), and be separately added into 0nmol/L, 50nmol/L, 100nmol/L, 300nmol/L and
The taxol of 500nmol/L, be denoted as HIG-XHW+0Taxol groups, CTRL+0Taxol control groups, HIG-XHW+ 50Taxol groups,
CTRL+50Taxol control groups, HIG-XHW+100Taxol groups, CTRL+100Taxol control groups, HIG-XHW+300Taxol groups,
CTRL+300Taxol control groups, HIG-XHW+500Taxol groups, CTRL+500Taxol control groups.After effect 48 hours, collect
Cell in 6 orifice plates, is placed in centrifuge tube, and 800rpm centrifuges 5min, removes supernatant, withers according to Annexin V-FITC/PI cells
Kit specification operation is died, residual pancreatin is washed away, often 500ul buffer solutions are added in pipe, sequentially add 5ul Annexin V-
FITC, 5ul PI, room temperature, which is protected from light, is incubated 10min, using flow cytomery each group apoptosis rate.Every group of at least three is multiple
Hole.
As a result as shown in Fig. 5 A~Fig. 5 K.
Fig. 5 A and Fig. 5 B are Flow cytometry respectively via containing 0nmol/L taxols and high dose Xihuang pills drug containing
The HIG-XHW+0Taxol groups of serum, the taxol containing same concentrations CTRL+0Taxol control groups processing MDA-MB-
231P mdr cell apoptosis figures.
Fig. 5 C and Fig. 5 D are that Flow cytometry contains via containing 50nmol/L taxols and high dose Xihuang pills respectively
The HIG-XHW+50Taxol groups of medicine serum, the taxol containing same concentrations CTRL+50Taxol control groups processing MDA-
MB-231P mdr cell apoptosis figures.
Fig. 5 E and Fig. 5 F are that Flow cytometry contains via containing 100nmol/L taxols and high dose Xihuang pills respectively
The HIG-XHW+100Taxol groups of medicine serum, the taxol containing same concentrations CTRL+100Taxol control groups processing
MDA-MB-231P mdr cell apoptosis figures.
Fig. 5 G and Fig. 5 H are that Flow cytometry contains via containing 300nmol/L taxols and high dose Xihuang pills respectively
The HIG-XHW+300Taxol groups of medicine serum, the taxol containing same concentrations CTRL+300Taxol control groups processing
MDA-MB-231P mdr cell apoptosis figures.
Fig. 5 I and Fig. 5 J are that Flow cytometry contains via containing 500nmol/L taxols and high dose Xihuang pills respectively
The HIG-XHW+500Taxol groups of medicine serum, the taxol containing same concentrations CTRL+500Taxol control groups processing
MDA-MB-231P mdr cell apoptosis figures.
In each figure, what horizontal axis indicated is the dye distribution of Annexin V, and the PI dye distributions of cell are shown in the longitudinal axis.
In conjunction with the case where above-mentioned each group picture, the detection of flow cytometric art is yellow via various concentration paclitaxel plus high dose west
Ball Contained Serum (being denoted as XHW), the MDA-MB- for thering is the blank control group (being denoted as CTRL) of the taxol of same concentrations to handle
231P mdr cell apoptosis rates compare, and comparison result is as it can be seen from figure 5k.It can be seen that by Fig. 5 K：The Japanese yew of 0nmol/L
Alcohol combines the paclitaxel plus HIG-XHW group apoptosis that HIG-XHW groups apoptosis rate improves 2.5%, 50nmol/L than blank control group
The blank control group of taxol of the rate than same concentrations are added improves the paclitaxel plus HIG-XHW groups of 8.3%, 100nmol/L
The blank control group of taxol of the apoptosis rate than same concentrations are added improves the paclitaxel plus HIG- of 10.2%, 300nmol/L
The blank control group of taxol of the XHW groups apoptosis rate than same concentrations are added improves the paclitaxel plus of 6.5%, 500nmol/L
The blank control group of taxol of the HIG-XHW groups apoptosis rate than same concentrations are added improves 24.8%.
As it can be seen that Xihuang pill drug serum promotes taxol resistance MDA-MB-231P cell line apoptosis with taxol combination group
Rate is all higher than taxol single drug group, and is all in pole significant difference (P<* * are denoted as in 0.01, Fig. 5 K), this illustrates that Xihuang pills contain
Medicine serum can effectively enhance sensibility of the drug resistant cancer cells strain to Paclitaxel Chemotherapy drug.
Five, immunoblotting (Western Blot) detects protein expression
Using tubulin as internal reference, using conventional immune protein engram technology measure in MDA-MB-231P cells the period and
The expression of apoptosis-related protein bcl-2 and Bax.Western-blot detecting steps are as follows：
By MDA-MB-231P cells according to 1 × 106A/hole is incubated at 6 porocyte culture plates, (contains per hole 2ml culture mediums
The DMEM culture mediums of 10% fetal calf serum) overnight incubation is changed to that the west MID-XHW, HIG-XHW is added is yellow respectively after adherent
The culture medium DMEM culture mediums of 10% fetal calf serum (contain) of ball Contained Serum and blank control (contain 10% fetal calf serum
DMEM culture mediums), after acting on 48 hours, the cell pyrolysis liquid that 100ul contains 1% protease inhibitors is added per hole, extracts cell
Total protein is gone in EP pipes, and 12000 × rpm centrifuges 10min, collects supernatant.Protein quantification, inspection are carried out using BCA methods
Survey protein concentration.Appropriate SDS sample-loading buffers are added to be crosslinked in 100 DEG C of metal bath solutions, record PAGE gel.Take 20ug
Protein sample loading, 10%SDS-PAGE electrophoresis are put into confining liquid by the protein electrotransfer to pvdf membrane of electrophoretic separation
Middle room temperature is closed 1 hour, and 4 DEG C of primary antibody (mouse is anti-) is added and is incubated overnight.Next day washes film, and the secondary antibody of peroxidase labelling is added
(rabbit-anti) is incubated at room temperature 1 hour, after washing film, is observed with western blot, and measuring each band absorbance value with image analysis software makees half
Quantitative analysis.
The expression of results of Western blot detection function albumen bcl-2 and Bax are as shown in Figure 6A, and concrete numerical value, which compares, asks
See Fig. 6 B and Fig. 6 C.
It can be seen that from Fig. 6 A and Fig. 6 B：It is resistance to after Xihuang pill drug serum is added compared with blank control group (CTRL groups)
The expression quantity of Bax albumen increases 40.3%, 82.3% (P respectively in MID-XHW groups, HIG-XHW groups in medicine cell<0.01, figure
* * are denoted as in 6B), it is in concentration dependent
It can be seen that from Fig. 6 A and Fig. 6 C：It is resistance to after Xihuang pill drug serum is added compared with blank control group (CTRL groups)
The expression quantity of Bcl-2 albumen reduces 36.4%, 57.8% (P respectively in MID-XHW groups, HIG-XHW groups in medicine cell<0.01,
* * are denoted as in Fig. 6 C), it is in concentration dependent.
As it can be seen that after Xihuang pill drug serum is added, Bax protein expressions increase in MDA-MB-231P cells, bcl-2 albumen
Expression reduces.
To sum up, the present invention is using the human breast cancer cell MDA-MB-231P of taxol resistance as research object, using CCK-
8 methods detect Taxol-resistant cell lines MDA-MB-231P cell Proliferation energy under the effect of various dose Xihuang pill drug serum
Power and detection Taxol-resistant cell lines MDA-MB-231P are in the joint taxol effect of various dose Xihuang pill drug serum
Lower ability of cell proliferation, the MDA-MB-231P drug resistances handled by Flow cytometry various dose Xihuang pill drug serum
Apoptosis difference and the detection various concentration taxol MDA-MB-231P that Xihuang pill drug serum is handled alone or in combination
Mdr cell apoptosis difference, the lower MDA-MB-231P drug resistances of western blot detection various dose Xihuang pill drug serum effects
Albumen bcl-2 and Bax differential expression in cell.
As a result it shows：Xihuang pill drug serum can obviously inhibit the increasing of Taxol-resistant cell lines MDA-MB-231P
It grows, and Xihuang pill drug serum is combined the proliferation inhibiting effect to Taxol-resistant cell lines MDA-MB-231P with taxol
Significantly.And Flow cytometry is the result shows that Xihuang pill drug serum can be obviously promoted Taxol-resistant cell lines MDA-
The apoptosis of MB-231P, and Xihuang pill drug serum withers to Taxol-resistant cell lines MDA-MB-231P with taxol combination
It is more notable to die inhibition.Western blot testing results show that Xihuang pill drug serum can raise Bax albumen, lower
Bcl-2 protein expressions.
It these results suggest that：Xihuang pill drug serum can activate or enhance lethal effect of the taxol to breast cancer cell,
So as to improve breast cancer cell to paclitaxel-sensitive and reverse the taxol resistance for treating breast cancer.Thus may be used
See, the Xihuang pills can be used in the chemotherapeutic sensitizer for preparing the taxol for treating breast cancer, can also be used in and be prepared
In drug for reversing breast cancer taxol resistance, it can also be combined in the pharmaceutical composition for the treatment of breast cancer with taxol
In.
It can be seen that the purpose of the present invention is achieved completely and effectively.The method and principle of the present invention is
It is shown and is illustrated in embodiment, without departing substantially from the principle, embodiment can make arbitrary modification.So
Present invention comprises all variant embodiments based on claim spirit and right.
Claims (5)
1. application of the Xihuang pills in preparing the drug for reversing breast cancer taxol resistance.
2. application as described in claim 1, which is characterized in that the Xihuang pills are by inhibiting breast cancer taxol resistance cell
Proliferation and induced breast cancer taxol resistance Apoptosis make breast cancer taxol resistance sex reversal.
3. application of the Xihuang pills in the chemotherapeutic sensitizer for preparing the taxol for treating breast cancer.
4. application as claimed in claim 3, which is characterized in that the Xihuang pills are by inhibiting breast cancer taxol resistance cell
Proliferation is with induced breast cancer taxol resistance Apoptosis and to chemotherapeutic drug Paclitaxel enhanced sensitivity.
5. the application of Xihuang pills and taxol combined in preparing the pharmaceutical composition for treating breast cancer.
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Cited By (1)
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| CN114949022A (en) * | 2022-06-06 | 2022-08-30 | 尚药局(宁夏)制药有限公司 | A Chinese medicinal composition for treating tumor |
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| CN1994353A (en) * | 2006-01-05 | 2007-07-11 | 北京凯瑞创新医药科技有限公司 | A superfine powdered Chinese medicinal composition and preparation method thereof |
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| CN114949022B (en) * | 2022-06-06 | 2023-05-05 | 尚药局(宁夏)制药有限公司 | Traditional Chinese medicine composition for treating tumors |
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