CN105879033B - II type antagonist of GnRH is inhibiting the application in progestational hormone drug resistance endometrial carcinoma cell proliferation - Google Patents
II type antagonist of GnRH is inhibiting the application in progestational hormone drug resistance endometrial carcinoma cell proliferation Download PDFInfo
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- 206010014759 Endometrial neoplasm Diseases 0.000 title claims abstract description 61
- 206010014733 Endometrial cancer Diseases 0.000 title claims abstract description 47
- NMJREATYWWNIKX-UHFFFAOYSA-N GnRH Chemical compound C1CCC(C(=O)NCC(N)=O)N1C(=O)C(CC(C)C)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)CNC(=O)C(NC(=O)C(CO)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C(CC=1NC=NC=1)NC(=O)C1NC(=O)CC1)CC1=CC=C(O)C=C1 NMJREATYWWNIKX-UHFFFAOYSA-N 0.000 title claims abstract description 23
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 title claims abstract description 23
- 239000005557 antagonist Substances 0.000 title claims abstract description 23
- 201000003914 endometrial carcinoma Diseases 0.000 title abstract description 47
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- 230000002401 inhibitory effect Effects 0.000 title description 18
- 230000014509 gene expression Effects 0.000 claims abstract description 17
- PSGAAPLEWMOORI-PEINSRQWSA-N medroxyprogesterone acetate Chemical compound C([C@@]12C)CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2CC[C@]2(C)[C@@](OC(C)=O)(C(C)=O)CC[C@H]21 PSGAAPLEWMOORI-PEINSRQWSA-N 0.000 claims description 49
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
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- Pharmacology & Pharmacy (AREA)
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Abstract
The invention discloses a kind of II type antagonists of GnRH to inhibit the application in progestational hormone drug resistance endometrial carcinoma cell proliferation.Pass through test prove: II type antagonist of GnRH be used alone or with MPA use in conjunction, it can inhibit the growth of endometrial carcinoma cell especially progestational hormone drug resistance endometrial carcinoma cell, and up-regulation PR expression can be passed through, inhibit the mechanism such as PI3K signal path, progestational hormone drug resistance is reversed, increases progestational hormone drug resistance Endometrial carcinoma cell line to the sensibility of progestational hormone.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to II type antagonist of GnRH is inhibiting progestational hormone drug resistance intrauterine
Application in film cancer cell multiplication.
Background technique
Carcinoma of endometrium is one of big malignant tumour of female genital tract three, accounts for the 7% of women whole body malignant tumour, genital tract
The 25~30% of malignant tumour.Carcinoma of endometrium incidence is about twice of 1970s early stage in nearly 10~20 years, and
And morbidity has rejuvenation trend.About 75% is early stage, and lesion is still confined to uterus when diagnosis.
The treatment of carcinoma of endometrium, other than conventional operative treatment, radiation and chemotherapy, endocrine therapy also becomes one
The important auxiliary treatment means of kind.At present at home and abroad endocrine therapy more be used for advanced stage, recurrent endometrial carcinoma patient with
And it requires to retain in the treatment of the Early endometrial carcinoma patient of fecundity.Clinical most common endocrine therapeutic agents are pregnant
Hormone.But long-term progestogen therapy can cause PR expression to lower so as to cause carcinoma of endometrium progestational hormone drug resistance.
Summary of the invention
It is an object of the present invention to provide the new applications of II type antagonist of GnRH.
The present invention provides II type antagonist of GnRH answering in the product of preparation prevention and/or treatment carcinoma of endometrium
With.Carcinoma of endometrium therein can be the carcinoma of endometrium having no drug resistance, and be also possible to the carcinoma of endometrium for having drug resistance, resistance to
Medicine concretely resistance to progestational hormone.
The present invention also provides II type antagonists of GnRH to have the production of at least one of following (1)-(5) function in preparation
Application in product:
(1) promote the expression of endometrial carcinoma cell Progesterone receptor mRNA；
(2) inhibit endometrial carcinoma cell proliferation；
(3) inhibit the phosphorylation level of endometrial carcinoma cell AKT；
(4) inhibit the activation of the PI3K/AKT signal path of endometrial carcinoma cell；
(5) inhibit the one-tenth knurl ability of endometrial carcinoma cell.
In above-mentioned application,
The one-tenth knurl ability for inhibiting endometrial carcinoma cell, which is embodied in, reduces endometrial carcinoma cell gross tumor volume
And/or reduce endometrial carcinoma cell tumor weight.
In above-mentioned application,
The II type antagonist of GnRH is Trptorelix-1.
It is a further object to provide a kind of prevention and/or the substances for the treatment of carcinoma of endometrium.
The active constituent of the substance of prevention and/or treatment carcinoma of endometrium provided by the invention be it is following 1) or 2):
1) II type antagonist of GnRH；
2) composition being made of II type antagonist of GnRH and megestrol acetate.
In above-mentioned substance
The II type antagonist of GnRH is Trptorelix-1.
In above-mentioned substance,
It is described 2) in, the molar ratio of the II type antagonist of GnRH and the megestrol acetate is 1:1.
Application of the above-mentioned substance in the product that preparation has at least one of following (1)-(6) function:
(1) prevent and/or treat carcinoma of endometrium；
(2) promote the expression of endometrial carcinoma cell Progesterone receptor mRNA；
(3) inhibit endometrial carcinoma cell proliferation；
(4) inhibit the phosphorylation level of endometrial carcinoma cell AKT；
(5) inhibit the activation of the PI3K/AKT signal path of endometrial carcinoma cell；
(6) inhibit the one-tenth knurl ability of endometrial carcinoma cell.
In above-mentioned application or above-mentioned substance or above-mentioned application, the endometrial carcinoma cell can be the uterus having no drug resistance
Inner membrance cancer cell, is also possible to the endometrial carcinoma cell for having drug resistance, drug resistance concretely resistance to progestational hormone.The endometrium
Cancer cell is specially progestational hormone drug resistance endometrial carcinoma cell.
In above-mentioned application or above-mentioned substance or above-mentioned application, the product or substance are drug.
Pass through test prove: II antagonist of GnRH (Trptorelix-1) be used alone or with MPA use in conjunction, can
Effectively inhibit the growth of progestational hormone drug resistance endometrial carcinoma cell, and II antagonist of GnRH (Trptorelix-1) is combined with MPA
The inhibitory effect of application becomes apparent from.II antagonist of GnRH (Trptorelix-1) can pass through up-regulation PR expression and inhibition PI3K
The mechanism such as signal path reverse progestational hormone drug resistance, increase progestational hormone drug resistance Endometrial carcinoma cell line to the sensibility of progestational hormone.
Detailed description of the invention
Fig. 1 is Cetrorelix and Trptorelix-1 (contains in culture medium/increases without MPA) to Ishikawa-MPA cell
The influence grown.Figure 1A is Cetrorelix and Trptorelix-1 (in culture medium containing/be free of MPA) to Ishikawa cell Proliferation
Influence (growth curve chart)；Figure 1B is that Cetrorelix and Trptorelix-1 (in culture medium containing/be free of MPA) are right
The influence (growth curve chart) of Ishikawa-MPA cell Proliferation；Fig. 1 C is Cetrorelix and Trptorelix-1 (culture medium
In containing/be free of MPA) influence (histogram corresponding with growth curve chart) to two kinds of Cells Cell Proliferations；Fig. 1 D is
The comparison of Cetrorelix and Trptorelix-1 (in culture medium containing/be free of MPA) to two kinds of cell inhibitory rates.Wherein
Control is without any medicine group；M is to add MPA group；C is Cetrorelix group；C+M is that Cetrorelix and MPA joint is used
Medicine group；T is Trptorelix-1 group；T+M is Trptorelix-1 and MPA drug combination group.
Fig. 2 is the growth curve of Xenografts in nude mice.Wherein, Fig. 2A is the Xenografts in nude mice of Ishikawa cell
Growth curve；Fig. 2 B is the growth curve of the Xenografts in nude mice of Ishikawa-MPA cell；Fig. 2 C is at different pharmaceutical
Tumor weight (*: P < 0.05) of each cell after 21 days after reason.
Fig. 3 is that MPA uses the volume to people's Endometrial Carcinoma in Nude Mice subcutaneous transplantation tumor to GnRH antagonist alone or in combination
Influence.
Fig. 4 is the PR mRNA expression that Realtime-PCR detects different endometrial carcinoma cells.Fig. 4 A is
Realtime-PCR (real-time fluorescence quantitative PCR) detects the PR mRNA expression of different endometrial carcinoma cells；Fig. 4 B is
After Cetrorelix and Trptorelix-1 acts on Ishikawa-MPA cell different time, the change of PR mRNA expression
Change (*: P < 0.05 compared with the control group；*: P < 0.01).
Fig. 5 is shadow of the GnRH antagonist in different time effect Ishikawa-MPA cell to p-AKT/AKT signal path
It rings.Wherein, Fig. 5 A is that Cetrorelix (10 μM) are logical to p-AKT/AKT signal in different time effect Ishikawa-MPA cell
The influence on road；Fig. 5 B is that Trptorelix-1 (10 μM) believe p-AKT/AKT in different time effect Ishikawa-MPA cell
The influence of number access.
Fig. 6 is Cetrorelix and Trptorelix-1 (in culture medium containing/be free of MPA) to Ishikawa-MPA cell
The influence of PI3K/AKT signal path after 30min.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Endometrial adenocarcinoma cells Ishikawa (ISK) in following embodiments is well-differentiated adenocarcinoma, ER α, β and PR
It is the positive, from Japan, in document " Zhao Lijun, Wei Lihui, Li Xiaoping, Wang Jianliu gonadotropin-releasing hormone (GRH) I type
The effect China journal of obstetrics and gynecology .2009,44 of agonist and II type to different PTEN gene expression status endometrial carcinoma cells
(1): being disclosed in 45-49 ", saved by gynemetrics, The People's Hospital of Peking University laboratory.
Ishikawa-MPA cell strain (ISK-MPA) in following embodiments is that induction early period obtains the pregnant of PR expression downward
Steroid-Resistance Ishikawa cell strain, long-term cultivation are in the DMEM-F12 high glucose medium (U.S. Hyclone for containing MPA (10 μM)
Company) in, document " foundation of people's carcinoma of endometrium cell strain of resistance to medroxyprogesterone acetate, Chinese Journal of Clinical Obstetrics and Gynecology,
2014,15 (4) phases: being disclosed in 341-344 ", and the public can obtain from The People's Hospital of Peking University.
GnRH I type antagonist in following embodiments: Cetrorelix Cetrorelix (Cet), amino acid sequence are as follows:
Ac-D-Nal(2)-DPhe(4Cl)-D-Pal(3)-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2；By Shanghai purple domain
Co., Ltd's synthesis.
II type antagonist of GnRH in following embodiments: Trptorelix-1 (Trp-1), amino acid sequence are as follows: Ac-
D-Nal(2)-DPhe(4Cl)-D-Pal(3)-Ser-Tyr-D-Cit-Trp-Tyr-Pro-D-Ala-NH2；Had by Shanghai purple domain
The synthesis of limit company.
Megestrol acetate (MPA) in following embodiments is the product of Qingdao Guohai Biopharmaceutical Co., Ltd.；Chemistry
Title: 6- methyl-17 a- monohydric pregnant -4,6- diene -3,20- diketone 17- acetate；Molecular formula: C24H32O4；Molecular weight:
384.52。
Cetrorelix, Trptorelix-1 and MPA in following embodiments are first dissolved into DMSO when in use
10-2The dry liquid of M, then 1000 times are diluted with complete medium (the DMEM-F12 high glucose medium containing 10%FBS), make
Final concentration of the 10 of Cetrorelix, Trptorelix-1 or MPA-5M(10μM)。
DMEM-F12 high glucose medium is the product of Hyclone company, the U.S. in following embodiments.
Embodiment 1, GnRH antagonist are inhibiting the application in Ishikawa and Ishikawa-MPA cell Proliferation
Cell strain in the present embodiment: Ishikawa-MPA cell and Ishikawa cell.
GnRH antagonist in the present embodiment: Cetrorelix and Trptorelix-1.
1, bed board
(1) when Ishikawa (ISK) and progestational hormone drug-resistant cell strain (ISK-MPA) cell grow to logarithmic phase, inoculation
96 orifice plates；
(2) with mass fraction be 0.25% trypsin solution (0.25g trypsase and 0.02gEDTA are dissolved into
100mlPBS solution) each cell in digestion process step (1) respectively, then cultivated with the DMEM-F12 high sugar containing 10%FBS
Base terminates digestion, and blows and beats and single cell suspension is made, and 10 μ l single cell suspensions is taken to be counted with cell counting board, with containing 10%FBS
The dilution of DMEM-F12 high glucose medium, making the final concentration of each cell is 5000/ml, and 100 μ l are added in every hole；
(3) 5 multiple holes of every kind of cell, while being inoculated with 7 96 orifice plates；Zeroing hole i.e. blank group is arranged simultaneously (only plus to cultivate
Base), lateral opening addition sterilizing PBS, to guarantee the water saturation of intermediate cell；37 DEG C, 5%CO2Culture in incubator.
(4) culture changes liquid in incubation to adherent timing of cell every other day, one plate of termination daily respectively at the 1-7 days,
Cell quantity is measured with MTS method.
2, drug-treated
After cell inoculation, culture is for 24 hours, different according to the drug of addition, is divided into following six groups:
First group (abbreviation Control): DMSO is added into each cell culture system in step 1, as control；Make
Volume fraction of the DMSO in cell culture system is 0.1%.
Second group (abbreviation M): into each cell culture system in step 1, (solvent is DMSO, MPA to addition MPA solution
Solution concentration is 10 μM), make 10 μM of concentration of the MPA in each cell culture system；
Third group (abbreviation C): into each cell culture system in step 1 Cetrorelix solution being added, (solvent is
DMSO, Cetrorelix solution concentration are 10 μM), making concentration of the Cetrorelix in each cell culture system is 10 μ
M；
4th group (abbreviation C+M): MPA solution and Cetrorelix being added into each cell culture system in step 1
Solution, making the concentration of MPA and Cetrorelix in each cell culture system is 10 μM；
5th group (abbreviation T): Trptorelix-1 solution being added into each cell culture system in step 1, makes
Concentration of the Trptorelix-1 in each cell culture system is 10 μM；
6th group (abbreviation T+M): MPA solution and Trptorelix- being added into each cell culture system in step 1
The concentration of 1 solution, MPA and Trptorelix-1 in each cell culture system is 10 μM.
Every kind of cell sets up 5 parallel controls, changes liquid after dosing every other day, takes out within the 1-7 days a plate respectively, and every hole is added
20 μ l of MTS solution, selects 490nm wavelength after continuing culture 4 hours, measures in enzyme-linked immunosorbent assay instrument (96 hole microplate reader) each
Hole absorbance value (OD490nm)。
3, the testing result of Ishikawa-MPA cell Proliferation
As a result MPA (M) is used alone as shown in Figure 1: does not have inhibiting effect to Ishikawa-MPA cell Proliferation, right
Ishikawa cell Proliferation has inhibiting effect.GnRH antagonist Cetrorelix (C) or Trptorelix-1 (T) exclusive use can
Inhibit the proliferation of Ishikawa and Ishikawa-MPA cell.In Ishikawa cell, Cetrorelix (C) or
The inhibition that cell proliferation is used alone in Trptorelix-1 (T) is (P stronger than inhibiting effect associated with both drugs and MPA (M)
< 0.05).And in Ishikawa-MPA cell, Cetrorelix (C) and Trptorelix-1 (T) and MPA (M) combination are this
Inhibiting effect is stronger (P < 0.05), illustrates that GnRH antagonist can make Ishikawa-MPA cell restore the sensitivity to progestational hormone
Property, to inhibit cell Proliferation.
Embodiment 2, GnRH antagonist are inhibiting the application in transplanted tumor in nude mice
One, experimental material
Ishikawa (ISK), progestational hormone drug-resistant cell strain (Ishikawa-MPA), receptor 5-6 week old BALB/C lose
Pass background Female nude mice.
Two, the foundation of Nude Mouse Model
1, the endometrial carcinoma cell (Ishikawa, Ishikawa-MPA) of logarithmic growth phase is prepared into unicellular respectively
Suspension (5 × 106A cell is suspended in the physiological saline of 0.5ml), respectively obtain Ishikawa single cell suspension and
Ishikawa-MPA single cell suspension；
2, the Ishikawa single cell suspension and Ishikawa-MPA single cell suspension for preparing step 1 under aseptic condition
It is inoculated in subcutaneous (the every kind of cell inoculation 5 of 5 5-6 week old BALB/C genetic background Female nude mice bilateral forelimb shoulder backs respectively
Only)；
3,100% tumor formation in 10-20 days after being inoculated with, cervical dislocation is lethal, sterilizing operation area skin, cuts skin, removing
Tumor mass out is placed on plate (plate should be placed on ice cube, the built-in PBS to sterilize a little), rejects nonneoplastic tissue and downright bad group
It knits, chooses well-grown without downright bad red, flesh of fish shape tumor tissue, and be cut into 2mm3Fritter；
4, before the tumor tissue that step 3 obtains being inoculated in 5 5-6 week old BALB/C genetic background Female nude mice bilaterals respectively
Limb shoulder back is subcutaneous (totally 60).
5, to tumor volume about 75mm3When, the nude mice of every kind of cell tumor formation is randomized into following 6 groups (n=5/group)
Carry out drug therapy processing (drug treatment regimes are intraperitoneal injection):
First group (Con): blank solvent (DMSO)；
Second group of (M): MPA (100mg/kg weight)；
Third group (C): Cetrorelix (100 μ g/ are only)；
4th group (C+M): MPA and Cetrorelix combination therapy group；
5th group of (T): Trptorelix-1 (100 μ g/ are only)；
6th group (T+M): MPA and Trptorelix-1 combination therapy group；
Drug-treated concrete mode is as follows: MPA is handled, abdomen by MPA solution (solvent DMSO, concentration are 10 μM)
Chamber injects 200 μ l 3 times a week (totally 9 times)；By Cetrorelix solution, (solvent is Cetrorelix and Trptorelix-1
DMSO, concentration are 10 μM) 200 μ l of daily intraperitoneal injection are (altogether with Trptorelix-1 solution (solvent DMSO, concentration are 10 μM)
21 times).Nude mice of control group only injects same volume drug solvent.
6, gross tumor volume (volume calculation formula=1/2ab is measured after tumor formation 2 times a week2, wherein a, b are respectively transplantable tumor
Long and short diameter).Tumour cell the 21st day execution nude mice from after going and buy Chinese medicine takes tumor to measure three groups of tumor average volumes, knurl weights, acquisition
Tumor specimen.Cleaning is fixed.And monitor nude mice weight during the administration to assess drug side-effect.
Three, endometrial carcinomas Xenografts in nude mice model experiment results
Endometrial carcinomas Xenografts in nude mice model experiment results are as shown in Figures 2 and 3: with the control group for only giving drug solvent
(Con) it compares, the growth of C group knurl is most slow (Fig. 2A) in Ishikawa cell transplantation tumor, in Ishikawa-MPA cell, (C+
M) group tumor killing effect is most obvious (Fig. 2 B).
The transplantable tumor size and weight result of different dosing group is as shown in Figure 2 C: (C+M) group is in progestational hormone drug resistance intrauterine
Tumor killing effect in film cancer cell (Ishikawa-MPA) is the most significant；Cetrorelix independent medication group (C) is to Ishikawa
It is 71% and 38% with Ishikawa-MPA gross tumor volume inhibiting rate, and tumor weight is reduced to 48% and 25% respectively.
Cetrorelix and MPA drug combination group (C+M) is 66% He to Ishikawa and Ishikawa-MPA gross tumor volume inhibiting rate
78%, tumor weight reduces by 46% and 80% respectively.Illustrate that Cetrorelix and MPA drug combination can enhance the sensibility of MPA,
It is obvious for progestational hormone mdr cell (Ishikawa-MPA) transplantable tumor inhibitory effect.
Trptorelix-1 independent medication group (T) is 32% to Ishikawa and Ishikawa-MPA gross tumor volume inhibiting rate
With 27%, and tumor weight is reduced to 36% and 16% respectively.Trptorelix-1 and MPA drug combination group (T+M) is right
Ishikawa and Ishikawa-MPA gross tumor volume inhibiting rate is 62% and 42%, and tumor weight is reduced to 41% He respectively
40%.Illustrate that Trptorelix-1 and MPA drug combination can enhance the sensibility of MPA, for progestational hormone mdr cell
(Ishikawa-MPA) transplantable tumor inhibitory effect is obvious.
The above results, which illustrate Cetrorelix and Trptorelix-1, can inhibit endometrial carcinoma cell repressor palace
Inner membrance cancer cell one-tenth knurl ability, and the sensibility of MPA can be enhanced with MPA drug combination, for progestational hormone mdr cell
(Ishikawa-MPA) transplantable tumor inhibitory effect is more obvious
The influence of embodiment 3, GnRH antagonist to PR mRNA and GnRHR mRNA expression in different cell strains
Cell strain used in the present embodiment: Ishikawa (ISK), progestational hormone drug-resistant cell strain (Ishikawa-MPA,
ISK-MPA).GnRH antagonist used in the present embodiment: Cetrorelix and Trptorelix-1.
1, bed board
(1) when Ishikawa (ISK) and progestational hormone drug-resistant cell strain (ISK-MPA) cell grow to logarithmic phase, inoculation
96 orifice plates；
(2) with mass fraction be 0.25% trypsin solution (0.25g trypsase and 0.02gEDTA are dissolved into 100ml
PBS solution) each cell in digestion process step (1) respectively, then terminated with the DMEM-F12 high glucose medium containing 10%FBS
Digestion, and blow and beat and single cell suspension is made, take 10 μ l single cell suspensions to be counted with cell counting board, with the DMEM- containing 10%FBS
The dilution of F12 high glucose medium, making the final concentration of each cell is 5000/ml, and 100 μ l are added in every hole；
(3) 5 multiple holes of every kind of cell, while being inoculated with 7 96 orifice plates；Zeroing hole i.e. blank group is arranged simultaneously (only plus to cultivate
Base), lateral opening addition sterilizing PBS, to guarantee the water saturation of intermediate cell；37 DEG C, 5%CO2Culture in incubator.
(4) culture changes liquid in incubation to adherent timing of cell every other day, one plate of termination daily respectively at the 1-7 days,
Cell quantity is measured with MTS method.
2, drug-treated
After 96 orifice plate of cell inoculation, culture for 24 hours, is divided into following 3 groups according to the difference of drug-treated type:
(1) into each cell culture system in step 1 be added GnRH I type antagonist Cetrorelix (drug it is molten
Agent is DMSO), making final concentration of the Cetrorelix in cell culture system is 10 μM, and DMSO is in cell culture system
Volume fraction is 0.1%.
(2) II type antagonist Trptorelix-1 (drug of GnRH is added into each cell culture system in step 1
Solvent be DMSO), making final concentration of the Trptorelix-1 in cell culture system is 10 μM, and DMSO is in cell culture body
Volume fraction in system is 0.1%.
(3) DMSO is added into each cell culture system in step 1, as negative control (control), makes DMSO
Volume fraction in cell culture system is 0.1%.
3, growth curve measures
After two kinds of cell Ishikawa (ISK) and progestational hormone drug-resistant cell strain (ISK-MPA) cell are inoculated with 96 orifice plates respectively,
The fresh training containing said medicine is replaced every other day according to the drug treatment regimes dosing of above-mentioned steps 2 after culture for 24 hours, in incubation
Nutrient solution, terminated a plate respectively at the 1-7 days daily, measured cell quantity with MTS method.Draw cell growth curve.
4, RT-PCR is detected
Ishikawa (ISK) and progestational hormone mdr cell after RT-PCR detection drug-treated for 24 hours, after 48h, 72h and 96h
The expression of PR (progesterone receptor) mRNA in strain (ISK-MPA) cell.The pcr amplification reaction primer of PR and internal control primer
Sequence is as shown in table 1, and pcr amplification reaction condition is as shown in table 2.
Table 1, PCR primer
Table 2, amplification condition
Read plate
72 DEG C 5 minutes
Melting curve is from 60 DEG C -92 DEG C, plate of every 0.2 DEG C of reading, and every PCR reaction is at least in triplicate.
5, RT-PCR testing result
The RT-PCR testing result of PR mRNA expression before drug-treated is as shown in Figure 4 A: it can be seen from the figure that
PR mRNA expression is very high in Ishikawa cell, and induces the PR mRNA expression of the Ishikawa-MPA cell strain obtained
Level is well below Ishikawa cell, and endometrial carcinoma cell PR is lowered during illustrating long-term progestational hormone inducible resistance；
Different pharmaceutical Cetrorelix and Trptorelix-1 handles Ishikawa-MPA cell the RT- after different time
PCR testing result is as shown in Figure 4 B: it can be seen from the figure that when Cetrorelix and Trptorelix-1 is to Ishikawa-MPA
After cytosis, with extended durations of action, PR mRNA expression is gradually risen, and when 96h significantly increases.
The influence of embodiment 4, GnRH antagonist to PI3K/AKT signal path in Ishikawa-MPA cell
Cell strain in the present embodiment: Ishikawa (ISK), progestational hormone drug-resistant cell strain (Ishikawa-MPA).
GnRH antagonist in the present embodiment: Cetrorelix and Trptorelix-1.
One, phosphorylation of the Cetrorelix and Trptorelix-1 different role time to Ishikawa-MPA cell AKT
Horizontal influence
1, bed board
(1) when Ishikawa (ISK) and progestational hormone drug-resistant cell strain (ISK-MPA) cell grow to logarithmic phase, inoculation
96 orifice plates；
(2) with mass fraction be 0.25% trypsin solution (0.25g trypsase and 0.02gEDTA are dissolved into 100ml
PBS solution) each cell in digestion process step (1) respectively, then terminated with the DMEM-F12 high glucose medium containing 10%FBS
Digestion, and blow and beat and single cell suspension is made, take 10 μ l single cell suspensions to be counted with cell counting board, with the DMEM- containing 10%FBS
The dilution of F12 high glucose medium, making the final concentration of each cell is 5000/ml, and 100 μ l are added in every hole；
(3) 5 multiple holes of every kind of cell, while being inoculated with 7 96 orifice plates；Zeroing hole i.e. blank group is arranged simultaneously (only plus to cultivate
Base), lateral opening addition sterilizing PBS, to guarantee the water saturation of intermediate cell；37 DEG C, 5%CO2Culture in incubator.
(4) culture changes liquid in incubation to adherent timing of cell every other day, one plate of termination daily respectively at the 1-7 days,
Cell quantity is measured with MTS method.
2, drug effect
96 orifice plate of cell inoculation, culture are separately added into 10 μM of Cetrorelix and 10 μM of Trptorelix-1 afterwards for 24 hours,
0,15min, 30min, 1h, 3h, 6h and 12h are acted on respectively.
3, In cell Western method detects PI3K/AKT protein level
In cell Western method detects each cell PI3K/AKT protein level after 2 drug-treated of above-mentioned steps
The specific method is as follows:
(1) culture medium is removed, the fixed cell of the fixed buffer of fresh 3.7% formaldehyde for being rapidly added 150 μ l (is fixed slow
When fliud flushing, carefully it is added with pipettor along tube wall, avoids making cell detachment), exempt from shake and is incubated for 20min at room temperature.
(2) fixed buffer is removed, divides 4 elution cells with Triton elution buffer, every time 200 μ l, it can with guarantee
With permeation cell, (elution can carry out on shaking table every time, under room temperature 5min)
(3) Triton elution buffer is removed, each hole is carefully added 150 μ l, 5% skimmed milk power from tube wall and blockades
Liquid slowly shakes on shaking table and is incubated for 1.5h at room temperature.
(4) liquid is blockaded in removal, every hole be added with blockade liquid dilute the anti-human pAKT monoclonal antibody of mouse and rabbit-anti people AKT monoclonal antibody (1:
100) 50ul blockades liquid with 50 μ l, and as the secondary antibody marked due to nir dye, there may be the controls of background.4 DEG C are not
It shakes and is incubated overnight
(5) be added 200 μ l of 0.1%Tween eluent, slowly shake at room temperature, elute 5min, be repeated 4 times with
On.
(6) every hole is added with blockading the diluted fluorescence secondary antibody of liquid, goat-anti rabbit IRDyeTM800 (1:200), sheep anti mouse
IRDyeTM50ul is added after being sufficiently mixed in 680 (1:800) each 25ml, while 50ul secondary antibody is also added in background hole.It is protected from light at room temperature
It slowly shakes, is incubated for 60min
(7) Tween eluent is added along tube wall, repeats step E4 times or more.
(8) after elution terminates, eluent is completely removed out of hole.It is bottom-up to overturn microwell plate, is gently struck in paper handkerchief
It beats, removes remaining eluent.
(9) it is scanned simultaneously with two channels 700nm and 800nm, selection mean quality, 169 μm of resolution ratio, 3.0mm
Focal length, brightness 5.
The testing result of PI3K/AKT protein level is as shown in Figure 5: Ishikawa-MPA cell is acted in Cetrorelix
Obviously inhibit the phosphorylation level of AKT after 15min, and nothing substantially change (Fig. 5 A) in effect 12 hours；Ishikawa-MPA
Cell then obviously activates the phosphorylation level of AKT after Trptorelix-1 acts on 15min and is in continue affective state, and 12 hours
After decrease (Fig. 5 B).The phosphorus of endometrial carcinoma cell AKT can be inhibited by illustrating Cetrorelix and Trptorelix-1
Acidification is horizontal.
Two, influence of the Cetrorelix and Trptorelix-1 to PI3K/AKT signal path in Ishikawa-MPA cell
1, bed board
(1) when progestational hormone drug-resistant cell strain (ISK-MPA) cell grows to logarithmic phase, 96 orifice plates are inoculated with；
(2) with mass fraction be 0.25% trypsin solution (0.25g trypsase and 0.02gEDTA are dissolved into
100mlPBS solution) each cell in digestion process step (1) respectively, then cultivated with the DMEM-F12 high sugar containing 10%FBS
Base terminates digestion, and blows and beats and single cell suspension is made, and 10 μ l single cell suspensions is taken to be counted with cell counting board, with containing 10%FBS
The dilution of DMEM-F12 high glucose medium, making the final concentration of each cell is 5000/ml, and 100 μ l are added in every hole；
(3) 5 multiple holes of every kind of cell, while being inoculated with 7 96 orifice plates；Zeroing hole i.e. blank group is arranged simultaneously (only plus to cultivate
Base), lateral opening addition sterilizing PBS, to guarantee the water saturation of intermediate cell；37 DEG C, 5%CO2Culture in incubator.
(4) culture changes liquid in incubation to adherent timing of cell every other day, one plate of termination daily respectively at the 1-7 days,
Cell quantity is measured with MTS method.
2, drug effect
By Ishikawa-MPA cell with 4 × 105After the density of a/ml is inoculated in 96 orifice plates, culture for 24 hours, be divided into
Different drugs is added in lower five groups, every group and acts on 30min:
First group (abbreviation Control): DMSO is added into each cell culture system in step 1, as control；Make
Volume fraction of the DMSO in cell culture system is 0.1%.
Second group (abbreviation M): into each cell culture system in step 1, (solvent is DMSO, MPA to addition MPA solution
Solution concentration is 10 μM), make 10 μM of concentration of the MPA in each cell culture system；
Third group (abbreviation C): into each cell culture system in step 1 Cetrorelix solution being added, (solvent is
DMSO, Cetrorelix solution concentration are 10 μM), making concentration of the Cetrorelix in each cell culture system is 10 μ
M；
4th group (abbreviation C+M): MPA solution and Cetrorelix being added into each cell culture system in step 1
Solution, making the concentration of MPA and Cetrorelix in each cell culture system is 10 μM；
5th group (abbreviation T): Trptorelix-1 solution being added into each cell culture system in step 1, makes
Concentration of the Trptorelix-1 in each cell culture system is 10 μM；
6th group (abbreviation T+M): MPA solution and Trptorelix- being added into each cell culture system in step 1
The concentration of 1 solution, MPA and Trptorelix-1 in each cell culture system is 10 μM.
3, In Cell Western method detects PI3K/AKT protein level
Detection method with step 13.
The testing result of PI3K/AKT protein level is as shown in Figure 6: inhibiting after the Ishikawa-MPA cell 30min of C group
PAKT level is apparently higher than Control group, and inhibits pAKT horizontal obvious strong after the Ishikawa-MPA cell 30min of C+M group
In C group, illustrate that Cetrorelix can inhibit the activation of PI3K/AKT signal path of endometrial carcinoma cell, is combined with MPA
Inhibitory effect is more more aobvious afterwards.
Claims (7)
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