CN105030792B - Applications of the Desogestrel in the positive breast cancer product of the negative AhRs of inhibitor against colon carcinoma cells/ER is prepared - Google Patents
Applications of the Desogestrel in the positive breast cancer product of the negative AhRs of inhibitor against colon carcinoma cells/ER is prepared Download PDFInfo
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- CN105030792B CN105030792B CN201510386614.2A CN201510386614A CN105030792B CN 105030792 B CN105030792 B CN 105030792B CN 201510386614 A CN201510386614 A CN 201510386614A CN 105030792 B CN105030792 B CN 105030792B
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Abstract
The invention discloses applications of the Desogestrel in the positive breast cancer product of the negative AhRs of inhibitor against colon carcinoma cells/ER is prepared.The invention provides applications of the Desogestrel in treatment colon cancer and/or breast cancer product is prepared.The experiment proves that, the present invention is to FDA, medicine Desogestrel granted CFDA carries out tumour medicine reorientation, it is not that anti-tumor drug is screened to indication according to the different cell line of tumour (organization type) and mutational site, it was found that this new application of Desogestrel inhibitor against colon carcinoma cells, realizes that old medicine is newly used.
Description
Technical field
Inhibitor against colon carcinoma cells/ER feminine genders are being prepared the present invention relates to biological technical field, more particularly to a kind of Desogestrel
Application in the positive breast cancer product of AhR.
Background technology
Tumour be threaten human health it is most common be also most serious a kind of disease, research and development high efficiency anti-tumor medicine to prolonging
The life cycle of long patient is most important.In recent years, developing rapidly with cancer genomics and molecular pharmacology, new
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 feature 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 complicated
Disease new drug target and molecular marker, and ultimately form the understanding to disease incidence mechanism and therapeutic scheme completely newly.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 contributes to the mechanism of action for explaining existing medicine to and guide the research and development of new drug., Okada in 2014
The researchers such as Y exist《Nature》On show the rheumatoid arthritis that GWAS result of study confluence analysises are obtained 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, candidate's reorientation medicine of acquisition, examination experiment is carried out to tumor cell line, is filtered out new resisting and is swollen
Tumor medicine.The candidate tumor suppression medicine for doing tumor cell line screening is shown in as follows:
Nicardipine,Promethazine,Estrone,Desogestrel,Sunlidac,Estradiol,
Etonogestrel,Levonorgestrel,Mesalazine,Indomethacin,Sulfasalazine,
Balsalazide,Irbesartan,Ibuprofen,Isoprenaline,Pentosan Polysulfate。
It is an object of the present invention to provide Desogestrel new application.
Applications of the Desogestrel that the present invention is provided in treatment colon cancer and/or breast cancer product is prepared.
Second object of the present invention is to provide Desogestrel new application.
The invention provides Desogestrel in suppression colon cancer cell and/or Cells Proliferation of Human Breast Cancer product is prepared
Application.
Third object of the present invention is to provide Desogestrel new application.
Reduction colon cancer cell and/or breast cancer cell IC50 value products are being prepared the invention provides Desogestrel
In application.
In above-mentioned application, the colon cancer cell is LOVO or RKO；The breast cancer is the positive breast of the negative AhRs of ER
Gland cancer, the breast cancer cell is specially MDA-MB-468.
In above-mentioned application, the product is medicine or kit.
Fourth object of the present invention is to provide a kind of product.
The product that the present invention is provided, its active component is Desogestrel；The product has following at least one work(
Energy：
1) treatment colon cancer and/or breast cancer；
2) colon cancer cell and/or Cells Proliferation of Human Breast Cancer are suppressed；
3) reduction colon cancer cell and/or breast cancer cell IC50 values.
In the said goods, the colon cancer cell is LOVO or RKO；The breast cancer is the positive breast of the negative AhRs of ER
Gland cancer, the breast cancer cell is specially MDA-MB-46.
In the said goods, the product is medicine or kit.
The experiment proves that, the present invention is to FDA, and medicine Desogestrel granted CFDA carries out tumour medicine
Thing is relocated, and is not that anti-tumor drug enters to indication according to the different cell line of tumour (organization type) and mutational site
Row screening, finds the positive this new application of breast cancer of the negative AhRs of Desogestrel inhibitor against colon carcinoma cells/ER, realizes old medicine
It is new to use.
Brief description of the drawings
Fig. 1 is the sieve medicine template distribution of 96 well culture plate medicines.
Fig. 2 is Desogestrel sensitive to colon cancer；EC50=136.2267；IC50=32.0768；R2=0.9921.
Fig. 3 is Desogestrel sensitive to colon cancer；EC50=26.3045；IC50=26.2417；R2=0.9988.
Fig. 4 is Desogestrel sensitive to the positive breast cancer of AhR negative ER；EC50=11.3284；IC50=
12.4694；R2=0.9872.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Medicine to be measured is Desogestrel in following embodiments, and its chemical structural formula is：
It is drug bank products, and catalog number is DB00304.
The AhR positive breast cancer cells MDA-MB- of colon cancer cell LOVO and RKO, ER feminine gender in example below
468th, breast duct cancer cell BT-474 product source：
LOVO ATCC CCL-229
RKO ATCC CRL-2577
MDA-MB-468 ATCC HTB-132
BT-474 ATCC HTB-20
Main instrument and consumptive material in example below
DMSO(from Sigma,Cat.No.D4540)
The 96-well white saturating Tissue Culture Plates in 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)
Breast duct cancer cell BT-474, complete medium is RPMI-1640, is life products, and A1049101, fishplate bar is close
It is 4000 to spend (cells/well).
Embodiment 1, CELLTITER-GLO detection Desogestrel inhibitor against colon carcinoma cells
First, the preparation of orifice plate to be measured
1st, plating cells
A) complete medium needed for a cell is prepared.
B) before experiment starts, medicine sieve cell name of the confirmation flag on 100mm culture dishes, when culture medium and passage
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) with 2ml sterile PBS solution rinse cell surface layer, then PBS waste liquids are pumped out with vacuum.
F) 1ml 0.25% (w/v) Trypsin-0.038% (w/v) EDTA solution digestions are gently added to culture dish thin
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 is mixed.
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 is mixed.
M) cell suspension is counted with cell counter, adjustment cell suspension to proper density fishplate bar carries out cell paving
Plate is tested.
RKO cells, LOVO cells and breast duct cancer cell MDA-MB-468 are carried out according to the method described above, respectively obtained
96 porocyte culture plates of RKO 96 porocyte culture plates, LOVO 96 porocyte culture plates and MDA-MB-468.
RKO cells, complete medium is DMEM, is life products, 11995081, fishplate bar density (cells/well) is
12000。
LOVO cells, complete medium is MEM, is life products, 10370021, fishplate bar density (cells/well) is
8000。
AhR positive breast cancer cells MDA-MB-468 negative ER, complete medium is Leibovitz's L-15,
For life products, 11415064, fishplate bar density (cells/well) is 12000.
2nd, medicine Desogestrel to be measured is prepared and dosing (200X final concentrations):
1) medicine Desogestrel motherboards to be measured are prepared
A) that medicine Desogestrel 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, be subsequently added
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, it is ensured 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, medicine working plate is prepared 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 are added into working plate, the multiple tracks volley of rifle fire is used in each 9 holes of medicine correspondence
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) taken out from incubator step 1 prepare RKORKO96 porocyte culture plates, LOVO96 porocyte culture plates and
MDA-MB-468 96 porocyte culture plates, by Fig. 1 dosing template arrangement modes (Fig. 1) respectively to the cell culture of RKORKO96 holes
Containing not for the above-mentioned b) preparations of 10 μ l is added in 96 porocyte culture plates of plate, LOVO96 porocyte culture plates and MDA-MB-468
With the cell culture medium (10X final concentrations) of acute drug, CO is put into237 DEG C of culture 72h of incubator, obtain RKO 96 hole medicines
96 hole medicine sieve plates of sieve plate, LOVO 96 hole medicine sieve plates and MDA-MB-468.
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 is detected
1st, CellTiter-Glo preparation of reagents
A) before use, CellTiter-Glo reagents Buffer is melted, equilibrate to room temperature and use.
B) before use, CellTiter-Glo reagent agars substrate is melted, equilibrate to room temperature and use.
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 the uniform solution of acquisition repeatedly.In general, CellTiter-Glo in 1 minute
Substrate reagent will fully dissolve, packing, be kept in dark place standby at -20 DEG C, it is to avoid multigelation.
2nd, detect
A) before detecting, 96 hole medicine sieve plates for the RKO that the 3 of above-mentioned one are obtained and LOVO 96 hole medicine sieve plates are put down at room temperature
Weighing apparatus 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 is mixed 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, it is ensured that the stability of signal.
F) luminescence signal is read during plate reading machine 570nm multi-functional using EnSpire.
G) analyzing and processing data.
RKO 96 hole medicine sieve plate results are as shown in Figure 2.
LOVO 96 hole medicine sieve plate results are as shown in Figure 3.
MDA-MB-468 96 hole medicine sieve plate results are as shown in Figure 4.
IC50 values are calculated, as a result as shown in table 1.
Breast duct cancer cell BT-474 IC50 values are acted on using same method detection Desogestrel, as a result such as
Table 1.
As can be seen that Desogestrel has Specific Inhibitory Effect to Colon Cancer Cells, treatment can be used as
The medicine of colon cancer.
Table 1 is IC50 value of the different cells under the effect of Desogestrel medicines
| Cell | IC50 values |
| LOVO | 32.0768 |
| MDA-MB-468 | 12.4694 |
| RKO | 26.2417 |
| BT-474 | 100 |
Claims (4)
- Applications of the 1.Desogestrel as single-activity composition in treatment colon cancer and/or breast cancer product is prepared；The breast cancer is the negative AhR positive breast cancers of ER.
- 2.Desogestrel is preparing suppression colon cancer cell and/or Cells Proliferation of Human Breast Cancer product as single-activity composition In application；The breast cancer is the negative AhR positive breast cancers of ER.
- 3. application according to claim 1 or 2, it is characterised in that：The colon cancer cell is LOVO or RKO；The breast Adenocarcinoma cell is MDA-MB-468.
- 4. application according to claim 1 or 2, it is characterised in that：The product is medicine or kit.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510386614.2A CN105030792B (en) | 2015-06-30 | 2015-06-30 | Applications of the Desogestrel in the positive breast cancer product of the negative AhRs of inhibitor against colon carcinoma cells/ER is prepared |
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| CN201510386614.2A CN105030792B (en) | 2015-06-30 | 2015-06-30 | Applications of the Desogestrel in the positive breast cancer product of the negative AhRs of inhibitor against colon carcinoma cells/ER is prepared |
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| CN105030792A CN105030792A (en) | 2015-11-11 |
| CN105030792B true CN105030792B (en) | 2017-08-25 |
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| CN111936152A (en) * | 2017-12-22 | 2020-11-13 | 阿托莎医疗公司 | Method for intraductal treatment of mammary disorders |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1122226A (en) * | 1994-06-08 | 1996-05-15 | 阿克佐诺贝尔公司 | Tablet, capsule, or granule comprising desogestrel |
| CN1349411A (en) * | 1999-05-04 | 2002-05-15 | 美国家庭用品有限公司 | Compositions containing benzimidazolones and progestogens |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19739916C2 (en) * | 1997-09-11 | 2001-09-13 | Hesch Rolf Dieter | Use of a combination of a progestogen and an estrogen for the continuous inhibition of ovulation and possibly simultaneous treatment and / or prophylaxis of tumors of the mammary glands |
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- 2015-06-30 CN CN201510386614.2A patent/CN105030792B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1122226A (en) * | 1994-06-08 | 1996-05-15 | 阿克佐诺贝尔公司 | Tablet, capsule, or granule comprising desogestrel |
| CN1349411A (en) * | 1999-05-04 | 2002-05-15 | 美国家庭用品有限公司 | Compositions containing benzimidazolones and progestogens |
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