CN104983734B - Applications of the Estradiol in anti-small cell lung carcinoma and/or oophoroma and/or osteosarcoma product is prepared - Google Patents
Applications of the Estradiol in anti-small cell lung carcinoma and/or oophoroma and/or osteosarcoma product is prepared Download PDFInfo
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- CN104983734B CN104983734B CN201510386611.9A CN201510386611A CN104983734B CN 104983734 B CN104983734 B CN 104983734B CN 201510386611 A CN201510386611 A CN 201510386611A CN 104983734 B CN104983734 B CN 104983734B
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Abstract
The invention discloses applications of the Estradiol in anti-small cell lung carcinoma and/or oophoroma and/or osteosarcoma product is prepared.The invention provides applications of the Estradiol in treatment ED-SCLC and/or oophoroma and/or osteosarcoma product is prepared.The experiment proves that, the present invention is to FDA, medicine Estradiol granted CFDA carries out tumour medicine reorientation, it is not that anti-tumor drug screens to indication according to the different cell line of tumour (organization type) and mutational site, it was found that Estradiol anti-small cell lung carcinomas and/or this new application of oophoroma and/or osteosarcoma, realize that old medicine is newly used.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of Estradiol to prepare anti-small cell lung carcinoma and/or ovum
Application in nest cancer and/or osteosarcoma product.
Background technology
Tumour is a kind of disease for the most common and most serious for threatening human health, and research and development high efficiency anti-tumor medicine is to prolonging
The life cycle of long patient is most important.In recent years, it is new with the rapid development of cancer genomics and molecular pharmacology
The research and development of antineoplastic achieve good achievement, but because new drug development input is big, the bottleneck such as cycle length can not overcome, with
And the features such as tumour individual inheritance variation is big, cause many traditional anti-tumor medicine effects to be not added with, new drug is expensive, side effect
It is unknown.
The researchers such as Barabasi AL were published in 2011《Nature Reviews Genetics》Paper middle finger
Go out, the molecular network analysis carried out based on GWAS results of study and interaction group (interactome) strategy is expected to disclose complexity
Disease new drug target and molecular marker, and ultimately form the understanding brand-new to disease incidence mechanism and therapeutic scheme.More
It is worth noting that, medicine reorientation (drug repositioning) research find, GWAS research locking tumor susceptibility gene and
The gene for having protein-protein interaction (protein-protein interaction, PPI) with it is easier to turn into medicine
Indirect target spot, this discovery help to explain that the mechanism of action of existing medicine and guides the research and development of new drug.2014, Okada
The researchers such as Y exist《Nature》On show the rheumatoid arthritis that GWAS result of study confluence analysises obtain in the paper delivered
101 tumor susceptibility genes in have 98 at present be used for treat medicine for treating rheumatoid arthritis direct or indirect targets, and
Also relocated and studied by medicine, they also found that the granted medicine for other indications of dozens of can also be used for controlling
Treat rheumatoid arthritis.
The content of the invention
This research is exactly based on the cancer gene spectrum Cancer for integrating cancer group Cosmic version72 databases
FDA in Gene Census and the databases of protein interaction STRING version 10 and DrugBankversion4.2
The drug data base of approval, the candidate of acquisition relocate medicine, and examination experiment is carried out to tumor cell line, filter out new resisting and swell
Tumor medicine.Do tumor cell line screening candidate tumor suppress medicine see it is as follows:
Nicardipine,Promethazine,Estrone,Estradiol,Sunlidac,Estradiol,
Etonoges trel,Levonorgestrel,Mesalazine,Indomethacin,Sulfasalazine,
Balsalazide,Irbe sartan,Ibuprofen,Isoprenaline,Pentosan Polysulfate。
It is an object of the present invention to provide Estradiol new application.
Estradiol provided by the invention is in treatment ED-SCLC and/or oophoroma and/or osteosarcoma product is prepared
Application.
Second object of the present invention is to provide Estradiol new application.
Suppress ED-SCLC and/or ovarian cancer cell in preparation the invention provides Estradiol and/or osteosarcoma is thin
Born of the same parents breed the application in product.
Third object of the present invention is to provide Estradiol new application.
ED-SCLC and/or ovarian cancer cell are reduced in preparation and/or osteosarcoma is thin the invention provides Estradiol
Application in born of the same parents' IC50 value products.
In above-mentioned application, the small cell lung cancer cell is NCI-H82, and the ovarian cancer cell is SKOV-3, the bone
Sarcoma cell is U2OS.
In above-mentioned application, the product is medicine or kit.
Fourth object of the present invention is to provide a kind of product.
Product provided by the invention, its active component are Estradiol；The product has following at least one function：
1) ED-SCLC and/or oophoroma and/or osteosarcoma are treated；
2) small cell lung cancer cell and/or ovarian cancer cell and/or human osteosarcoma cell proliferation are suppressed；
3) small cell lung cancer cell and/or ovarian cancer cell and/or osteosarcoma cell IC50 values are reduced.
In the said goods, the small cell lung cancer cell is NCI-H82, and the ovarian cancer cell is SKOV-3, the bone
Sarcoma cell is U2OS.
In the said goods, the product is medicine or kit.
The experiment proves that the present invention carries out tumour medicine to FDA, medicine Estradiol granted CFDA
Reorientation, it is not that anti-tumor drug is carried out to indication according to the different cell line of tumour (organization type) and mutational site
Screening, Estradiol anti-small cell lung carcinomas and/or oophoroma/this new application of osteosarcoma cell carcinoma are found, realizes old medicine
It is new to use.
Brief description of the drawings
Fig. 1 is that 96 well culture plate medicines sieve the distribution of medicine template.
Fig. 2 is Estradiol sensitive to oophoroma；EC50=3.0280；IC50=3.0662；R2=0.9990.
Fig. 3 is Estradiol sensitive to ED-SCLC；EC50=15.2786；IC50=16.2549；R2=
0.9971。
Fig. 4 is Estradiol sensitive to osteosarcoma cell；EC50=28.7837；IC50=32.6945；R2=
0.9983。
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Medicine to be measured is Estradiol in following embodiments, its chemical structural formula：
It is drug bank products, and catalog number is
DB00783。
Small cell lung cancer cell NCI-H82, ovarian cancer cell SKOV-3, human liver cancer cell SNU- in example below
475th, the source of human lung carcinoma cell NCI-H810 and osteosarcoma U 2OS is as follows：
Main instrument and consumptive material in example below
DMSO(from Sigma,Cat.No.D4540)
The saturating Tissue Culture Plate in 96-well whites bottom (from Corning, Cat.No.3610)
CellTiter Glo kits (from Promega, Cat.No.G7573)
Doxorubicin positive drugs (from MCE, Cat.No.HY-15142)
Fetal Bovine Serum(from Gibco,Cat#10099141)
100mm culture dishes (from Corning, Cat#430167)
RPMI-1640medium(from Gibco,Cat#A1049101)
DMEM medium(from Gibco,Cat#11995081)
DMEM/F12medium(from Gibco,Cat#11330057)
EMEM medium(from Gibco,Cat#10370021)
The cell knockouts (Thermo, Cat#5840150) of Mutidrop 384
The multi-functional plate reading machines of EnSpire (Perkin Elmer, Cat#2300-001M)
Embodiment 1, CELLTITER-GLO detection Estradiol anti-small cell lung carcinomas and/or oophoroma/osteosarcoma epithelium
Cell cancer
First, the preparation of orifice plate to be measured
1st, plating cells
A) complete medium needed for each cell is prepared.
B) experiment start before, medicine sieve cell name of the confirmation flag on 100mm culture dishes, culture medium and passage when
Between, the information such as generation, it is ensured that experiment is errorless.
C) attached cell operation is with reference to step d) to i), and suspension cell operation is with reference to step j) to l).
D) cell culture medium is drawn using vavuum pump during sterile working.
E) 2ml sterile PBS solution rinse cell surface layer is used, then PBS waste liquids are pumped out with vacuum.
F) it is thin that 1ml 0.25% (w/v) Trypsin-0.038% (w/v) EDTA solution digestions are gently added to culture dish
Born of the same parents, gently mix it is several under, solution covers cell surface layer, under inverted microscope observe cell dissociation situation, will in cell
Pancreatin digestion is terminated when coming off.
G) 37 DEG C of preheated complete mediums of 5ml are added into culture dish, cell is gently blown and beaten with pipette, makes it
Split away off from culture dish bottom.
H) this cell suspension is transferred in 15ml or 50ml sterile centrifugation tubes, 1000rpm centrifugations 5min.
I) supernatant culture medium is suctioned out using vavuum pump sterile working.The preheated complete mediums of 37 DEG C of 5ml are added to be resuspended
Cell precipitation, gently piping and druming mix.
J) cell is gently blown and beaten with pipette, makes it completely fall off from culture dish bottom.
K) this cell suspension is transferred in 15ml or 50ml sterile centrifugation tubes, 1000rpm centrifugations 5min.
L) supernatant culture medium is suctioned out using vavuum pump sterile working.The preheated complete mediums of 37 DEG C of 5ml are added to be resuspended
Cell precipitation, gently piping and druming mix.
M) cell suspension is counted with cell counter, adjustment cell suspension to proper density fishplate bar carries out cell paving
Plate is tested.
NCI-H82 cells and SKOV-3 cells are carried out according to the method described above, respectively obtain the cell culture of NCI-H8296 holes
Plate and SKOV-396 porocyte culture plates.
NCI-H82 cells, complete medium RPMI-1640 is life products, A1049101, fishplate bar density (cells/
Well it is) 12000.
SKOV-3 cells, complete medium are McCOY'S 5A, are life products, 16600082, fishplate bar density (cells/
Well it is) 12000.
2nd, medicine Estradiol preparations and dosing (200X final concentrations) to be measured:
1) medicine Estradiol motherboards to be measured are prepared
A) that medicine Estradiol to be measured is diluted into 20mM with DMSO is stand-by.
B) take the 20mM configured in a) step the μ L of medicine 79 to be measured to add in the first hole of dilution plate the first row, then add
54 μ L DMSO solvents, into the 9th hole, take 25 μ L solution into the second hole, after mixing to the hole of the first row second from the first hole
25 μ L solution are taken from the second hole into the 3rd hole, the like to the 9th hole, ensure that medicine gradually carries out 3.16 times of multiple proportions ladders
Degree dilution.
2) positive drug Doxorubicin (MCE, Cat.No.HY-15142) motherboard is prepared
A) that positive drug Doxorubicin is diluted into 6mM with DMSO is stand-by.
B) 6mM positive drug Doxorubicin solution is added in dilution plate, DMSO solution 1:3.16 times of multiple proportions gradient dilutions
The medicine to be measured.
3rd, the preparation of medicine working plate and dosing
A) medicine to be measured and positive drug sample-adding template are illustrated in fig. 1 shown below, wherein, S1208:Positive drug Doxorubicin,
DMSO:Positive control wells, Cpd 1,2,3:Medicine to be measured, DMSO final concentration of 0.5% (DMSO compatibility).
B) the 95 specific complete mediums of μ l cells, corresponding 9 holes of each medicine, with the multiple tracks volley of rifle fire are added into working plate
A series of good molten of (9 holes) doubling dilutions of transferase 45 μ l successively respectively from medicine to be measured and positive drug doxorubicin motherboards
Liquid (10X final concentrations), obtains the cell culture medium containing various concentrations medicine.
C) step 1 is taken out from incubator and prepares NCI-H8296 porocyte culture plates and SKOV-396 porocyte culture plates,
By Fig. 1 dosing template arrangement modes (Fig. 1) respectively into NCI-H8296 porocyte culture plates and SKOV-396 porocyte culture plates
The above-mentioned cell culture mediums (10X final concentrations) containing various concentrations medicine b) prepared of 10 μ l are added, are put into CO2Incubator 37
DEG C culture 72h, obtain NCI-H82 96 hole medicine sieve plates and SKOV-3 96 hole medicine sieve plates.
Control wells are used as using be not added with any medicine.
The final concentration and dosing situation of above-mentioned medicine to be measured, positive drug Doxorubicin and control wells in 96 orifice plates are such as
Under：
Final concentration (μM) of the medicine to be measured in Fig. 1 2-10 holes is followed successively by:100、31.64557、10.01442、
3.16912、1.002886、0.317369、0.100433、0.031783、0.010058；
Final concentrations (μM) of the positive drug Doxorubicin in Fig. 1 2-10 holes is followed successively by:30、9.493671、
3.004326、0.950736、0.300866、0.095211、0.03013、0.009535、0.003017；
In addition, S1208 holes (E1-H1 and A12-D12) in 96 orifice plates:10 100 μM of Doxorubicin solution of μ l final concentrations
(solvent is the complete medium solution comprising 0.5%DMSO), DMSO holes (A1-D1, E12-H12 and A11-H11):10 μ l are included
0.5%DMSO complete medium solution.
2nd, CELLTITER-GLO fluorecytes activity monitor system detects
1st, CellTiter-Glo preparation of reagents
A) before use, CellTiter-Glo reagents Buffer is melted, room temperature use is equilibrated to.
B) before use, CellTiter-Glo reagent agars substrate is melted, room temperature use is equilibrated to.
C) the CellTiter-Glo Buffer for taking 100ml to balance are added to bottled CellTiter-Glo reagents agar
In substrate, it is set fully to be resuspended to form enzyme/substrate mixture, that is, so-called CellTiter-Glo detection reagents.
D) gently mix and be vortexed, and be inverted repeatedly and obtain uniform solution.In general, CellTiter-Glo in 1 minute
Substrate reagent will fully dissolve, and packing, be kept in dark place standby at -20 DEG C, avoid multigelation.
2nd, detect
A) before detecting, 96 hole medicine sieve plates for the NCI-H82 that the 3 of above-mentioned one are obtained and SKOV-3 96 hole medicine sieve plates are in room
Middle benefit gas balances 20-30 minutes.
B) inverted microscope observes the cellular morphology and death condition of every piece of culture plate, marks abnormal conditions and repetition measurement one
It is secondary.
C) CellTiter-Glo reagents prepared by 100 μ l above-mentioned 1 are added into all medicine sieve apertures, are mixed.
D) fully shaking mixes 2 minutes in 96 hole micropore plate oscillators, cell is cracked completely.
E) luminescence signal detection is carried out after lucifuge room temperature is placed 15 minutes, ensures the stability of signal.
F) luminescence signal is read during plate reading machine 570nm multi-functional using EnSpire.
G) analyzing and processing data.
NCI-H82 96 hole medicine sieve plate results are as shown in Figure 2.
SKOV-3 96 hole medicine sieve plate results are as shown in Figure 3.
IC50 values are calculated, as a result as shown in table 1.
Using same method detection Estradiol effect osteosarcoma U 2OSs, the IC50 values of cell, as a result such as table 1
And Fig. 4.U2OS cells, complete medium DMEM is life products, 11995081, fishplate bar density (cells/well) is
3000。
The human liver cancer cell SNU-475 and thin NCI-H810 of human lung cancer is acted on using same method detection Estradiol, carefully
The IC50 values of born of the same parents, as a result such as table 1.
As can be seen that Estradiol has specifically to ED-SCLC and/or ovarian cancer cell and/or osteosarcoma propagation
Inhibitory action, it can be used as treating ED-SCLC and/or the medicine of oophoroma and/or osteosarcoma.
Table 1 is IC50 value of the different cells under the effect of Estradiol medicines
Claims (3)
1.Estradiol is preparing treatment ED-SCLC and/or oophoroma and/or osteosarcoma product as single-activity composition
In application；The small cell lung cancer cell is NCI-H82, and the ovarian cancer cell is SKOV-3, and the osteosarcoma cell is
U2OS。
2.Estradiol is preparing suppression ED-SCLC and/or ovarian cancer cell and/or osteosarcoma as single-activity composition
Application in cell propagation product；The small cell lung cancer cell is NCI-H82, and the ovarian cancer cell is SKOV-3, described
Osteosarcoma cell is U2OS.
3. application according to claim 1 or 2, it is characterised in that：The product is medicine or kit.
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| CN201510386611.9A CN104983734B (en) | 2015-06-30 | 2015-06-30 | Applications of the Estradiol in anti-small cell lung carcinoma and/or oophoroma and/or osteosarcoma product is prepared |
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| CN201510386611.9A CN104983734B (en) | 2015-06-30 | 2015-06-30 | Applications of the Estradiol in anti-small cell lung carcinoma and/or oophoroma and/or osteosarcoma product is prepared |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1660437A (en) * | 2004-12-29 | 2005-08-31 | 山东蓝金生物工程有限公司 | Combination of slow released anticancer medication |
| CN101181230A (en) * | 2007-12-19 | 2008-05-21 | 山东蓝金生物工程有限公司 | Estramustine sustained-release implantation agent for curing entity tumour |
-
2015
- 2015-06-30 CN CN201510386611.9A patent/CN104983734B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1660437A (en) * | 2004-12-29 | 2005-08-31 | 山东蓝金生物工程有限公司 | Combination of slow released anticancer medication |
| CN101181230A (en) * | 2007-12-19 | 2008-05-21 | 山东蓝金生物工程有限公司 | Estramustine sustained-release implantation agent for curing entity tumour |
Non-Patent Citations (1)
| Title |
|---|
| 雌二醇对苯并(a)芘致雄性小鼠肺癌抑制作用;李海峰等;《中国公共卫生》;20080531;第24卷(第5期);第548页摘要 * |
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