CN105879032A - Application of GnRH I type antagonist in inhibiting proliferation of progesterone-resistance endometrial cancer cells - Google Patents

Application of GnRH I type antagonist in inhibiting proliferation of progesterone-resistance endometrial cancer cells Download PDF

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CN105879032A
CN105879032A CN201610191443.2A CN201610191443A CN105879032A CN 105879032 A CN105879032 A CN 105879032A CN 201610191443 A CN201610191443 A CN 201610191443A CN 105879032 A CN105879032 A CN 105879032A
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cell
mpa
gnrh
endometrial carcinoma
ishikawa
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CN105879032B (en
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赵丽君
魏丽惠
李明珠
李小平
王建六
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Peking University Peoples Hospital
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Peking University Peoples Hospital
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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
    • 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
    • A61K38/09—Luteinising hormone-releasing hormone [LHRH], i.e. Gonadotropin-releasing hormone [GnRH]; Related peptides

Abstract

The invention discloses an application of a GnRH I type antagonist in inhibiting proliferation of progesterone-resistance endometrial cancer cells. Tests prove that the GnRH I type antagonist, when used independently or used in a mode of being combined with MPA (mercaptopropionic acid), can be used for inhibiting the growth of the endometrial cancer cells, in particular the growth of the progesterone-resistance endometrial cancer cells; and by up-regulating PR expression, inhibiting a PI3K signal path and other mechanisms and by reversing progesterone resistance, the sensitivity of a progesterone-resistance endometrial cancer cell line to progesterone is enhanced.

Description

The application in suppression progestogen drug resistance endometrial carcinoma cell propagation of the GnRH I type antagonist
Technical field
The invention belongs to biological technical field, be specifically related to the application in suppression progestogen drug resistance endometrial carcinoma cell propagation of the GnRH I type antagonist.
Background technology
Carcinoma of endometrium is one of big malignant tumor of female genital tract three, accounts for the 7% of women whole body malignant tumor, the 25～30% of reproductive tract malignant tumor.Within nearly 10～20 years, endometrial incidence rate is about the seventies in last century of twice in early days, and morbidity has rejuvenation trend.About 75% is that during diagnosis, pathological changes is still confined to uterus in early days.
The treatment of carcinoma of endometrium, in addition to conventional operative treatment, radiation and chemotherapy, endocrine therapy also becomes a kind of important auxiliary treatment means.During the most at home and abroad endocrine therapy is used for the treatment of the Early endometrial carcinoma patient that late period, recurrent endometrial carcinoma patient and requirement retain fertility more.Clinical the most frequently used endocrine therapeutic agents is progestogen.But long-term progestogen therapy can cause PR down-regulated expression thus cause carcinoma of endometrium progestogen drug resistance.
Summary of the invention
It is an object of the present invention to provide the new application of GnRH I type antagonist.
The invention provides the application in the product of preparation prevention and/or treatment carcinoma of endometrium of the GnRH I type antagonist.Carcinoma of endometrium therein can be the carcinoma of endometrium having no drug resistance, it is also possible to be the carcinoma of endometrium having drug resistance, the most resistance to progestogen of drug resistance.
Present invention also offers the application in preparation has (1)-(5) as follows in the product of at least one function of the GnRH I type antagonist:
(1) expression of endometrial carcinoma cell Progesterone receptor mRNA is promoted；
(2) suppression endometrial carcinoma cell propagation；
(3) phosphorylation level of endometrial carcinoma cell AKT is suppressed；
(4) activation of the PI3K/AKT signal path of suppression endometrial carcinoma cell；
(5) the one-tenth tumor ability of endometrial carcinoma cell is suppressed.
In above-mentioned application,
The one-tenth tumor ability of described suppression endometrial carcinoma cell is embodied in reduction endometrial carcinoma cell gross tumor volume and/or reduces endometrial carcinoma cell tumor weight.
In above-mentioned application,
Described GnRH I type antagonist is cetrorelix.
It is a further object to provide a kind of prevention and/or the material for the treatment of carcinoma of endometrium.
Prevention and/or the active component of the material for the treatment of carcinoma of endometrium that the present invention provides are following 1) or 2):
1) GnRH I type antagonist；
2) compositions being made up of GnRH I type antagonist and megestrol acetate.
In above-mentioned substance
Described GnRH I type antagonist is cetrorelix.
In above-mentioned substance,
Described 2), in, the mol ratio of described GnRH I type antagonist and described megestrol acetate is 1:1.
Above-mentioned substance is the application in the product of at least one function in preparation has (1)-(6) as follows:
(1) prevent and/or treat carcinoma of endometrium；
(2) expression of endometrial carcinoma cell Progesterone receptor mRNA is promoted；
(3) suppression endometrial carcinoma cell propagation；
(4) phosphorylation level of endometrial carcinoma cell AKT is suppressed；
(5) activation of the PI3K/AKT signal path of suppression endometrial carcinoma cell；
(6) the one-tenth tumor ability of endometrial carcinoma cell is suppressed.
In above-mentioned application or above-mentioned substance or above-mentioned application, described endometrial carcinoma cell can be the endometrial carcinoma cell having no drug resistance, it is also possible to be the endometrial carcinoma cell having drug resistance, the most resistance to progestogen of drug resistance.Described endometrial carcinoma cell is specially progestogen drug resistance endometrial carcinoma cell.
In above-mentioned application or above-mentioned substance or above-mentioned application, described product or material are medicine.
Proved by test: GnRH I type antagonist (cetrorelix) be used alone or with MPA use in conjunction, can effectively suppress the growth of progestogen drug resistance endometrial carcinoma cell, and the inhibition of GnRH I type antagonist (cetrorelix) and MPA use in conjunction becomes apparent from.GnRH I type antagonist (cetrorelix) can express by raising PR and suppress the mechanism such as PI3K signal path to reverse progestogen drug resistance, increases the progestogen drug resistance Endometrial carcinoma cell line sensitivity to progestogen.
Accompanying drawing explanation
Fig. 1 is the Cetrorelix and Trptorelix-1 (in culture medium containing/without the MPA) impact on Ishikawa-MPA cell proliferation.Figure 1A is the Cetrorelix and Trptorelix-1 (in culture medium containing/without the MPA) impact (growth curve chart) on Ishikawa cell proliferation；Figure 1B is the Cetrorelix and Trptorelix-1 (in culture medium containing/without the MPA) impact (growth curve chart) on Ishikawa-MPA cell proliferation；Fig. 1 C is the Cetrorelix and Trptorelix-1 (in culture medium containing/without the MPA) impact (block diagram corresponding with growth curve chart) on two kinds of Cells Cell Proliferation；Fig. 1 D is the Cetrorelix and Trptorelix-1 (in culture medium containing/without the MPA) comparison to two kinds of cell inhibitory rates.Wherein Control is without any medicine group；M is for adding MPA group；C is Cetrorelix group；C+M is Cetrorelix and MPA drug combination 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. 2 A is the growth curve of Xenografts in nude mice of Ishikawa cell；Fig. 2 B is the growth curve of the Xenografts in nude mice of Ishikawa-MPA cell；Fig. 2 C is each cell tumor weight (*: P ＜ 0.05) after 21 days after different pharmaceutical processes.
Fig. 3 is the impact that GnRH antagonist MPA alone or in combination uses the volume on people's Endometrial Carcinoma in Nude Mice subcutaneous transplantation tumor.
Fig. 4 is the PR mrna expression level of the different endometrial carcinoma cell of Realtime-PCR detection.Fig. 4 A is the PR mrna expression level of the different endometrial carcinoma cell of Realtime-PCR (real-time fluorescence quantitative PCR) detection；Fig. 4 B is after Cetrorelix and Trptorelix-1 acts on Ishikawa-MPA cell different time, the change of PR mrna expression level (compared with matched group *: P ＜ 0.05；*: P ＜ 0.01).
Fig. 5 is GnRH antagonist in the impact on p-AKT/AKT signal path of the different time effect Ishikawa-MPA cell.Wherein, Fig. 5 A is Cetrorelix (10 μMs) in the impact on p-AKT/AKT signal path of the different time effect Ishikawa-MPA cell；Fig. 5 B is Trptorelix-1 (10 μMs) in the impact on p-AKT/AKT signal path of the different time effect Ishikawa-MPA cell.
Fig. 6 is that Cetrorelix and Trptorelix-1 (in culture medium containing/without MPA) is on the impact of PI3K/AKT signal path after Ishikawa-MPA cell 30min.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Endometrial adenocarcinoma cells Ishikawa (ISK) in following embodiment is well-differentiated adenocarcinoma, ER α, β and PR are the positive, derive from Japan, document " Zhao Lijun; Wei Lihui, Li Xiaoping, Wang Jianliu. the effect to different PTEN gene expression status endometrial carcinoma cells of gonadotropin releasing hormone I type agonist and II type. China journal of obstetrics and gynecology .2009; 44 (1): 45-49 " disclosed in mistake, The People's Hospital of Peking University's department of obstetrics and gynecology laboratory preserve.
Ishikawa-MPA cell strain (ISK-MPA) in following embodiment is that early stage induction obtains the progestogen drug resistance Ishikawa cell strain of PR down-regulated expression, long-term cultivation in the DMEM-F12 high glucose medium (Hyclone company of the U.S.) containing MPA (10 μMs), mistake disclosed 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) phase: 341-344 ", the public can obtain from The People's Hospital of Peking University.
GnRH I type antagonist in following embodiment: cetrorelix Cetrorelix (Cet), its structural formula is: Ac-D-Nal (2)-DPhe (4Cl)-D-Pal (3)-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2；Synthesized by Shanghai Zi Yu company limited.
GnRH II type antagonist in following embodiment: Trptorelix-1 (Trp-1), its structural formula is: Ac-D-Nal (2)-DPhe (4Cl)-D-Pal (3)-Ser-Tyr-D-Cit-Trp-Tyr-Pro-D-Ala-NH2；Synthesized by Shanghai Zi Yu company limited.
Megestrol acetate (MPA) in following embodiment is the product of Qingdao Guohai Biopharmaceutical Co., Ltd.；Chemical name: 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 embodiment, in use, is the most first dissolved into 10 with DMSO-2The dry liquid of M, then dilute 1000 times 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)。
In following embodiment, DMEM-F12 high glucose medium is the product of Hyclone company of the U.S..
The application in suppression Ishikawa and Ishikawa-MPA cell proliferation of embodiment 1, GnRH antagonist
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 progestogen drug-resistant cell strain (ISK-MPA) cell grow to logarithmic (log) phase, 96 orifice plates are inoculated；
(2) with each cell in the trypsin solution (0.25g trypsin and 0.02gEDTA are dissolved into 100ml PBS solution) that mass fraction is 0.25% respectively digestion process step (1), digestion is terminated again with the DMEM-F12 high glucose medium containing 10%FBS, and blow and beat make single cell suspension, take 10 μ l single cell suspension cell counting count boards to count, dilute with the DMEM-F12 high glucose medium containing 10%FBS, the final concentration making each cell is 5000/ml, and every hole adds 100 μ l；
(3) 5 multiple holes, simultaneously 7 96 orifice plates of inoculation of every kind of cell；Arranging the most blank group (only adding culture medium) in zeroing hole, lateral opening addition sterilizing PBS, to ensure the water saturation of intermediate cell simultaneously；37 DEG C, 5%CO2Cultivate in incubator.
(4) cultivation plays timing to cell attachment, changes liquid every other day, terminated a plate respectively at 1-7 days every days in incubation, measures cell quantity by MTS method.
2, drug treating
After cell inoculation, cultivate 24h, different according to the medicine added, it is divided into following six groups:
First group (being called for short Control): each cell culture system in step 1 adds DMSO, as comparison；DMSO volume fraction in cell culture system is made to be 0.1%.
Second group (being called for short M): adding MPA solution (solvent is DMSO, and MPA solution concentration is 10 μMs) in each cell culture system in step 1, making MPA concentration in each cell culture system is 10 μMs；
3rd group (being called for short C): (solvent is DMSO to add Cetrorelix solution in each cell culture system in step 1, Cetrorelix solution concentration is 10 μMs), make Cetrorelix concentration in each cell culture system be 10 μMs；
4th group (being called for short C+M): each cell culture system in step 1 adds MPA solution and Cetrorelix solution, makes MPA and Cetrorelix concentration in each cell culture system be 10 μMs；
5th group (being called for short T): each cell culture system in step 1 adds Trptorelix-1 solution, makes Trptorelix-1 concentration in each cell culture system be 10 μMs；
6th group (being called for short T+M): adding MPA solution and Trptorelix-1 solution in each cell culture system in step 1, MPA and Trptorelix-1 concentration in each cell culture system is 10 μMs.
Every kind of cell sets up 5 parallel controls, changing liquid after dosing every other day, within the 1-7 days, take out a plate respectively, every hole adds MTS solution 20 μ l, 490nm wavelength is selected, at enzyme-linked immunosorbent assay instrument (96 hole microplate reader) each hole absorbance (OD of upper mensuration after continuing to cultivate 4 hours490nm)。
3, the testing result of Ishikawa-MPA cell proliferation
Result is as shown in Figure 1: being used alone MPA (M) does not has inhibitory action to Ishikawa-MPA cell proliferation, and Ishikawa cell proliferation is had inhibitory action.GnRH antagonist Cetrorelix (C) or Trptorelix-1 (T) is used alone the propagation that can suppress Ishikawa and Ishikawa-MPA cell.In Ishikawa cell, it is strong (P ＜ 0.05) that Cetrorelix (C) or Trptorelix-1 (T) is used alone inhibitory action associated with rejection ratio both medicines of cell proliferation and MPA (M).And in Ishikawa-MPA cell, Cetrorelix (C) and Trptorelix-1 (T) and MPA (M) is combined this inhibitory action higher (P ＜ 0.05), illustrate that GnRH antagonist can make the Ishikawa-MPA cellular-restoring sensitivity to progestogen, to suppression cell proliferation.
The application in suppression transplanted tumor in nude mice of embodiment 2, GnRH antagonist
One, experiment material
Ishikawa (ISK), progestogen drug-resistant cell strain (Ishikawa-MPA), receptor 5-6 week old BALB/C genetic background Female nude mice.
Two, the foundation of Nude Mouse Model
1, the endometrial carcinoma cell (Ishikawa, Ishikawa-MPA) of trophophase of taking the logarithm is prepared as single cell suspension (5 × 10 respectively6Individual cell suspension is in the normal saline of 0.5ml), respectively obtain Ishikawa single cell suspension and Ishikawa-MPA single cell suspension；
2, Ishikawa single cell suspension step 1 prepared under aseptic condition and Ishikawa-MPA single cell suspension are inoculated in 5 5-6 week old BALB/C genetic background Female nude mice bilateral forelimb shoulder dorsal sc (every kind of cell inoculates 5) respectively；
3, within 10-20 days 100%, tumor is become after inoculation, cervical dislocation is lethal, sterilizing operation area skin, cutting skin, separate tumor mass, (plate should be placed on ice cube to be placed in plate, the PBS of built-in a little sterilizing) on, reject nonneoplastic tissue and slough, choose well-grown without downright bad redness, flesh of fish shape tumor tissue, and be cut into 2mm3Fritter；
4, tumor tissue step 3 obtained is inoculated in 5 5-6 week old BALB/C genetic background Female nude mice bilateral forelimb shoulder dorsal sc (totally 60) respectively.
5, tumor volume about 75mm is treated3Time, every kind of cell becomes the nude mice of tumor to be randomized into following 6 groups (n=5 only/group) to carry out Drug therapy process (drug treatment regimes is lumbar injection):
First group (Con): blank solvent (DMSO)；
Second group of (M): MPA (100mg/kg body weight)；
3rd group of (C): Cetrorelix (100 μ g/ are only)；
4th group (C+M): MPA and Cetrorelix therapeutic alliance group；
5th group of (T): Trptorelix-1 (100 μ g/ are only)；
6th group (T+M): MPA and Trptorelix-1 therapeutic alliance group；
The concrete mode of drug treating is as follows: MPA is processed by MPA solution (solvent is DMSO, and concentration is 10 μMs), lumbar injection 200 μ l (totally 9 times) 3 times a week；Cetrorelix Yu Trptorelix-1 is by Cetrorelix solution (solvent is DMSO, and concentration is 10 μMs) and Trptorelix-1 solution (solvent is DMSO, and concentration is 10 μMs) lumbar injection 200 μ l every day (totally 21 times).Nude mice of control group only injects same volume drug solvent.
6, gross tumor volume (volume computing formula=1/2ab is measured 2 times a week after becoming tumor2, wherein, a, b are respectively the long and short footpath of transplanted tumor).Tumor cell puts to death nude mice in the 21st day after laxative, takes tumor and measures three groups of tumor average volumes, tumor weight, gathers tumor specimen.Clean, fix.And in administration process, monitor nude mice body weight to assess drug side effect.
Three, endometrial carcinomas Xenografts in nude mice model experiment results
Endometrial carcinomas Xenografts in nude mice model experiment results is as shown in Figures 2 and 3: compared with the matched group (Con) of only drug solvent, in Ishikawa cell transplantation tumor, C group tumor bulk-growth is the most slowly (Fig. 2 A), in Ishikawa-MPA cell, (C+M) group tumor killing effect is the most substantially (Fig. 2 B).
The transplanted tumor size of different dosing group and weight result be as shown in Figure 2 C: (C+M) group tumor killing effect in progestogen drug resistance endometrial carcinoma cell (Ishikawa-MPA) is the most notable；Cetrorelix independent medication group (C) is 71% and 38% to Ishikawa and Ishikawa-MPA gross tumor volume suppression ratio, and tumor weight is reduced to 48% and 25% respectively.Cetrorelix and MPA drug combination group (C+M) is 66% and 78% to Ishikawa and Ishikawa-MPA gross tumor volume suppression ratio, and tumor weight reduces 46% and 80% respectively.Illustrate that Cetrorelix and MPA drug combination can strengthen the sensitivity of MPA, obvious for progestogen mdr cell (Ishikawa-MPA) transplanted tumor inhibition.
Trptorelix-1 independent medication group (T) is 32% and 27% to Ishikawa and Ishikawa-MPA gross tumor volume suppression ratio, and tumor weight is reduced to 36% and 16% respectively.Trptorelix-1 and MPA drug combination group (T+M) is 62% and 42% to Ishikawa and Ishikawa-MPA gross tumor volume suppression ratio, and tumor weight is reduced to 41% and 40% respectively.Illustrate that Trptorelix-1 and MPA drug combination can strengthen the sensitivity of MPA, obvious for progestogen mdr cell (Ishikawa-MPA) transplanted tumor inhibition.
The above results explanation Cetrorelix with Trptorelix-1 all can suppress endometrial carcinoma cell suppression endometrial carcinoma cell to become tumor ability, and the sensitivity of MPA can be strengthened with MPA drug combination, progestogen mdr cell (Ishikawa-MPA) transplanted tumor inhibition is become apparent from
Embodiment 3, GnRH antagonist are on PR mRNA and the impact of GnRHR mrna expression level in different cell strains
Cell strain used in the present embodiment: Ishikawa (ISK), progestogen 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 progestogen drug-resistant cell strain (ISK-MPA) cell grow to logarithmic (log) phase, 96 orifice plates are inoculated；
(2) with each cell in the trypsin solution (0.25g trypsin and 0.02gEDTA are dissolved into 100ml PBS solution) that mass fraction is 0.25% respectively digestion process step (1), digestion is terminated again with the DMEM-F12 high glucose medium containing 10%FBS, and blow and beat make single cell suspension, take 10 μ l single cell suspension cell counting count boards to count, dilute with the DMEM-F12 high glucose medium containing 10%FBS, the final concentration making each cell is 5000/ml, and every hole adds 100 μ l；
(3) 5 multiple holes, simultaneously 7 96 orifice plates of inoculation of every kind of cell；Arranging the most blank group (only adding culture medium) in zeroing hole, lateral opening addition sterilizing PBS, to ensure the water saturation of intermediate cell simultaneously；37 DEG C, 5%CO2Cultivate in incubator.
(4) cultivation plays timing to cell attachment, changes liquid every other day, terminated a plate respectively at 1-7 days every days in incubation, measures cell quantity by MTS method.
2, drug treating
After cell inoculates 96 orifice plates, cultivate 24h, be divided into following 3 groups according to the difference of drug treating kind:
(1) cell culture system of each in step 1 adds GnRH I type antagonist Cetrorelix (solvent of medicine is DMSO), making Cetrorelix final concentration in cell culture system be 10 μMs, DMSO volume fraction in cell culture system is 0.1%.
(2) cell culture system of each in step 1 adds GnRH II type antagonist Trptorelix-1 (solvent of medicine is DMSO), making Trptorelix-1 final concentration in cell culture system be 10 μMs, DMSO volume fraction in cell culture system is 0.1%.
(3) cell culture system of each in step 1 adds DMSO, as negative control (control), make DMSO volume fraction in cell culture system be 0.1%.
3, growth curve measures
After two kinds of cell Ishikawa (ISK) and progestogen drug-resistant cell strain (ISK-MPA) cell inoculate 96 orifice plates respectively, according to the drug treatment regimes dosing of above-mentioned steps 2 after cultivation 24h, incubation is changed fresh medium containing said medicine every other day, terminate a plate respectively at 1-7 days every days, measure cell quantity by MTS method.Draw cell growth curve.
4, RT-PCR detection
The expression of PR (progesterone receptor) mRNA in Ishikawa (ISK) after 24h, 48h, 72h and 96h and progestogen drug-resistant cell strain (ISK-MPA) cell after RT-PCR detection drug treating.The pcr amplification reaction primer of PR and the sequence of internal reference primer are as shown in table 1, and pcr amplification reaction condition is as shown in table 2.
Table 1, PCR primer
Table 2, amplification condition
5, RT-PCR testing result
The RT-PCR testing result of the PR mrna expression level before drug treating is as shown in Figure 4 A: as can be seen from the figure, in Ishikawa cell, PR mrna expression level is the highest, and induce the PR mrna expression level of the Ishikawa-MPA cell strain of acquisition well below Ishikawa cell, during long-term progestogen inducible resistance is described, endometrial carcinoma cell PR lowers；
Different pharmaceutical Cetrorelix and Trptorelix-1 processes the RT-PCR testing result after different time as shown in Figure 4 B to Ishikawa-MPA cell: as can be seen from the figure, after Cetrorelix and Trptorelix-1 is to Ishikawa-MPA cytosis, along with extended durations of action, PR mrna expression level gradually rises, and significantly increases during 96h.
Embodiment 4, GnRH antagonist are on the impact of PI3K/AKT signal path in Ishikawa-MPA cell
Cell strain in the present embodiment: Ishikawa (ISK), progestogen drug-resistant cell strain (Ishikawa-MPA).
GnRH antagonist in the present embodiment: Cetrorelix and Trptorelix-1.
One, Cetrorelix with the Trptorelix-1 different action time of the impact on the phosphorylation level of Ishikawa-MPA cell AKT
1, bed board
(1) when Ishikawa (ISK) and progestogen drug-resistant cell strain (ISK-MPA) cell grow to logarithmic (log) phase, 96 orifice plates are inoculated；
(2) with each cell in the trypsin solution (0.25g trypsin and 0.02gEDTA are dissolved into 100ml PBS solution) that mass fraction is 0.25% respectively digestion process step (1), digestion is terminated again with the DMEM-F12 high glucose medium containing 10%FBS, and blow and beat make single cell suspension, take 10 μ l single cell suspension cell counting count boards to count, dilute with the DMEM-F12 high glucose medium containing 10%FBS, the final concentration making each cell is 5000/ml, and every hole adds 100 μ l；
(3) 5 multiple holes, simultaneously 7 96 orifice plates of inoculation of every kind of cell；Arranging the most blank group (only adding culture medium) in zeroing hole, lateral opening addition sterilizing PBS, to ensure the water saturation of intermediate cell simultaneously；37 DEG C, 5%CO2Cultivate in incubator.
(4) cultivation plays timing to cell attachment, changes liquid every other day, terminated a plate respectively at 1-7 days every days in incubation, measures cell quantity by MTS method.
2, medicine effect
Cell inoculates 96 orifice plates, is separately added into the Cetrorelix and the Trptorelix-1 of 10 μMs of 10 μMs after cultivating 24h, acts on 0 respectively, 15min, 30min, 1h, 3h, 6h and 12h.
3, In cell Western method detection PI3K/AKT protein level
The concrete grammar of each cell PI3K/AKT protein level after In cell Western method detection above-mentioned steps 2 drug treating is as follows:
(1) culture medium is removed, it is rapidly added fresh 3.7% formaldehyde of 150 μ l to fix buffer and fix cell (when being fixed buffer, add along tube wall with pipettor carefully, it is to avoid make cell detachment), exempt to shake and hatch 20min at ambient temperature.
(2) remove fixing buffer, divide 4 eluting cells with Triton elution buffer, each 200 μ l, so that ensure can be with permeation cell, (each eluting can be carried out on shaking table, 5min under room temperature condition)
(3) removing Triton elution buffer, each hole adds 150 μ l 5% defatted milk powder from tube wall carefully and blockades liquid, slow shake incubated at room temperature 1.5h on shaking table.
(4) removing liquid of blockading, every hole adds with liquid dilution mouse-anti people's pAKT monoclonal antibody and rabbit anti-human AKT monoclonal antibody (1:100) 50ul of blockading, with the liquid of blockading of 50 μ l, as due to the two anti-comparisons that may produce background of nir dye labelling.4 DEG C are not shaken overnight incubation
(5) add 0.1%Tween eluent 200 μ l, the most slowly shake, eluting 5min, be repeated 4 times above.
(6) addition fluorescence two of liquid dilution of blockading in every hole resists, goat-anti rabbit IRDyeTM800 (1:200), sheep anti mouse IRDyeTM680 (1:800) each 25ml adds 50ul after being sufficiently mixed, background hole also adds 50ul bis-and resists simultaneously.Under room temperature, lucifuge is slowly shaken, and hatches 60min
(7) add Tween eluent along tube wall, repeat step more than E4 time.
(8), after eluting terminates, in hole, eluent is removed completely.Upset microwell plate is bottom-up, beats gently at napkin, removes the eluent of residual.
(9) scanning with two passages of 700nm and 800nm, all selecting mean quality, the resolution of 169 μm, the focal length of 3.0mm, brightness is 5 simultaneously.
The testing result of PI3K/AKT protein level is as shown in Figure 5: Ishikawa-MPA cell substantially suppresses the phosphorylation level of AKT after Cetrorelix effect 15min, and without substantially changing (Fig. 5 A) in effect 12 hours；Ishikawa-MPA cell the most substantially activates the phosphorylation level of AKT and in lasting affective state after Trptorelix-1 effect 15min, and decreased after 12 hours (Fig. 5 B).Illustrate that Cetrorelix and Trptorelix-1 all can suppress the phosphorylation level of endometrial carcinoma cell AKT.
Two, Cetrorelix and Trptorelix-1 is on the impact of PI3K/AKT signal path in Ishikawa-MPA cell
1, bed board
(1) when progestogen drug-resistant cell strain (ISK-MPA) cell grows to logarithmic (log) phase, 96 orifice plates are inoculated；
(2) with each cell in the trypsin solution (0.25g trypsin and 0.02gEDTA are dissolved into 100ml PBS solution) that mass fraction is 0.25% respectively digestion process step (1), digestion is terminated again with the DMEM-F12 high glucose medium containing 10%FBS, and blow and beat make single cell suspension, take 10 μ l single cell suspension cell counting count boards to count, dilute with the DMEM-F12 high glucose medium containing 10%FBS, the final concentration making each cell is 5000/ml, and every hole adds 100 μ l；
(3) 5 multiple holes, simultaneously 7 96 orifice plates of inoculation of every kind of cell；Arranging the most blank group (only adding culture medium) in zeroing hole, lateral opening addition sterilizing PBS, to ensure the water saturation of intermediate cell simultaneously；37 DEG C, 5%CO2Cultivate in incubator.
(4) cultivation plays timing to cell attachment, changes liquid every other day, terminated a plate respectively at 1-7 days every days in incubation, measures cell quantity by MTS method.
2, medicine effect
By Ishikawa-MPA cell with 4 × 105After the density of individual/ml is inoculated in 96 orifice plates, cultivates 24h, be divided into following five groups, group often adds different medicines and acts on 30min:
First group (being called for short Control): each cell culture system in step 1 adds DMSO, as comparison；DMSO volume fraction in cell culture system is made to be 01%.
Second group (being called for short M): adding MPA solution (solvent is DMSO, and MPA solution concentration is 10 μMs) in each cell culture system in step 1, making MPA concentration in each cell culture system is 10 μMs；
3rd group (being called for short C): (solvent is DMSO to add Cetrorelix solution in each cell culture system in step 1, Cetrorelix solution concentration is 10 μMs), make Cetrorelix concentration in each cell culture system be 10 μMs；
4th group (being called for short C+M): each cell culture system in step 1 adds MPA solution and Cetrorelix solution, makes MPA and Cetrorelix concentration in each cell culture system be 10 μMs；
5th group (being called for short T): each cell culture system in step 1 adds Trptorelix-1 solution, makes Trptorelix-1 concentration in each cell culture system be 10 μMs；
6th group (being called for short T+M): adding MPA solution and Trptorelix-1 solution in each cell culture system in step 1, MPA and Trptorelix-1 concentration in each cell culture system is 10 μMs.
3, In Cell Western method detection PI3K/AKT protein level
Detection method is with the 3 of step one.
The testing result of PI3K/AKT protein level is as shown in Figure 6: suppress pAKT level apparently higher than Control group after the Ishikawa-MPA cell 30min of C group, and after the Ishikawa-MPA cell 30min of C+M group, suppress pAKT level to be significantly stronger than C group, illustrating that Cetrorelix can suppress the activation of the PI3K/AKT signal path of endometrial carcinoma cell, after being combined with MPA, inhibition is the most aobvious.

Claims (9)

  1. The application in the product of preparation prevention and/or treatment carcinoma of endometrium of the 1.GnRH I type antagonist.
  2. 2.GnRH I type antagonist is the application in the product of at least one function in preparation has (1)-(5) as follows:
    (1) expression of endometrial carcinoma cell Progesterone receptor mRNA is promoted；
    (2) suppression endometrial carcinoma cell propagation；
    (3) phosphorylation level of endometrial carcinoma cell AKT is suppressed；
    (4) activation of the PI3K/AKT signal path of suppression endometrial carcinoma cell；
    (5) the one-tenth tumor ability of endometrial carcinoma cell is suppressed.
  3. Application the most according to claim 1 and 2, it is characterised in that:
    Described GnRH I type antagonist is cetrorelix.
  4. 4. prevention and/or a material for treatment carcinoma of endometrium, its active component is following 1) or 2):
    1) GnRH I type antagonist；
    2) compositions being made up of GnRH I type antagonist and megestrol acetate.
  5. Material the most according to claim 4, it is characterised in that:
    Described GnRH I type antagonist is cetrorelix.
  6. 6. according to the material described in claim 4 or 5, it is characterised in that:
    Described 2), in, the mol ratio of described GnRH I type antagonist and described megestrol acetate is 1:1.
  7. 7. in claim 4-6, arbitrary described material has at least one function in (1)-(6) as follows in preparation Product in application:
    (1) prevent and/or treat carcinoma of endometrium；
    (2) expression of endometrial carcinoma cell Progesterone receptor mRNA is promoted；
    (3) suppression endometrial carcinoma cell propagation；
    (4) phosphorylation level of endometrial carcinoma cell AKT is suppressed；
    (5) activation of the PI3K/AKT signal path of suppression endometrial carcinoma cell；
    (6) the one-tenth tumor ability of endometrial carcinoma cell is suppressed.
  8. 8. according to arbitrary described material or right in described application arbitrary in claim 1-3 or claim 4-6 Require the application described in 7, it is characterised in that: described endometrial carcinoma cell is progestogen drug resistance endometrial carcinoma cells.
  9. 9. according to arbitrary described material or right in described application arbitrary in claim 1-3 or claim 4-6 Require the application described in 7, it is characterised in that: described product or material are medicine.
CN201610191443.2A 2015-04-01 2016-03-30 GnRH I type antagonist is inhibiting the application in progestational hormone drug resistance endometrial carcinoma cell proliferation Active CN105879032B (en)

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