CN105012314B

CN105012314B - Applications of the Etonogestrel in anti-prostate cancer product is prepared

Info

Publication number: CN105012314B
Application number: CN201510385950.5A
Authority: CN
Inventors: 师咏勇; 宋智健
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2015-06-30
Filing date: 2015-06-30
Publication date: 2017-12-08
Anticipated expiration: 2035-06-30
Also published as: CN105012314A

Abstract

The invention discloses applications of the Etonogestrel in anti-prostate cancer product is prepared.The invention provides Etonogestrel to prepare the application in treating prostate cancer product.The experiment proves that, the present invention is to FDA, medicine Etonogestrel 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 this new application of Etonogestrel anti-prostate cancers, realizes that old medicine is newly used.

Description

Applications of the Etonogestrel in anti-prostate cancer product is prepared

Technical field

The present invention relates to biological technical field, more particularly to a kind of Etonogestrel is in anti-prostate cancer product is prepared Application.

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,Etonogestrel,Sunlidac,Etonogestrel, Et onogestrel,Levonorgestrel,Mesalazine,Indomethacin,Sulfasalazine,Balsalazid e,Irbesartan,Ibuprofen,Isoprenaline,Pentosan Polysulfate。

It is an object of the present invention to provide Etonogestrel new application.

Etonogestrel provided by the invention is preparing the application in treating prostate cancer product.

Second object of the present invention is to provide Etonogestrel new application.

The invention provides Etonogestrel to prepare the application in suppressing prostate gland cancer cell propagation product.

Third object of the present invention is to provide Etonogestrel new application.

The invention provides Etonogestrel to prepare the application in reducing prostate gland cancer cell IC50 value products.

In above-mentioned application, the prostate gland cancer cell is DU-145.

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 Etonogestrel；The product has following at least one work( Energy：

1) prostate cancer is treated；

2) prostate gland cancer cell propagation is suppressed；

3) prostate gland cancer cell IC50 values are reduced.

In the said goods, the prostate gland cancer cell is DU-145.

In the said goods, the product is medicine or kit.

The experiment proves that the present invention carries out tumour medicine to FDA, medicine Etonogestrel granted CFDA Thing relocates, 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 this new application of Etonogestrel anti-prostate cancers, realizes that old medicine is newly used.

Brief description of the drawings

Fig. 1 is that 96 well culture plate medicines sieve the distribution of medicine template.

Fig. 2 is Etonogestrel sensitive to prostate cancer；EC50=11.6796；IC50=16.0136；R²= 0.9788。

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 Etonogestrel in following embodiments, and its chemical structural formula is：

It is drug bank products, catalog number DB00294.

Prostate gland cancer cell DU-145, people's Lung Squamous Carcinoma Cells NCI-H1703 and human lung carcinoma cell in example below NCI-H2122 source is as follows：

DU-145 ATCC HTB-81

NCI-H1703 ATCC CRL-5889

NCI-H2122 ATCC CRL-5985

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 Etonogestrel anti-prostate cancers

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.

DU-145 cells are carried out according to the method described above, obtain DU-14596 porocyte culture plates.

DU-145 cells, complete medium RPMI-1640 is life products, A1049101, fishplate bar density (cells/ Well it is) 4000.

2nd, medicine Etonogestrel preparations and dosing (200X final concentrations) to be measured:

1) medicine Etonogestrel motherboards to be measured are prepared

A) that medicine Etonogestrel to be measured is diluted into 20mM with DMSO is stand-by.

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 to the hole of the first row second from the first hole, after mixing from 25 μ L solution are taken in second hole into the 3rd hole, the like to the 9th hole, ensure that medicine gradually carries out 3.16 times of multiple proportions gradients 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 DU-14596 porocyte culture plates, by Fig. 1 dosing template arrangement modes (Fig. 1) adds the 10 above-mentioned b of μ l into DU-14596 porocyte culture plates respectively) prepare containing various concentrations medicine cell training Base (10X final concentrations) is supported, is put into CO₂37 DEG C of culture 72h of incubator, obtain DU-145 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 DU-145 that the 3 of above-mentioned one are obtained balance 20-30 minutes at room temperature.

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.

DU-145 96 hole medicine sieve plate results are as shown in Figure 2.

IC50 values are calculated, as a result as shown in table 1.

Employment Lung Squamous Carcinoma Cells NCI-H1703 and human lung carcinoma cell are made using same method detection Etonogestrel NCI-H2122 IC50 values, as a result such as table 1.

As can be seen that Etonogestrel has Specific Inhibitory Effect to prostate gland cancer cell propagation, can be used as Treat the medicine of prostate cancer.

Table 1 is IC50 value of the different cells under the effect of Etonogestrel medicines

Cell	IC50 values
		DU-145	16.0136
NCI-H1703	100
		NCI-H2122	100

Claims

1.Etonogestrel is preparing the application in treating prostate cancer product as single-activity composition.

2.Etonogestrel is preparing the application in suppressing prostate gland cancer cell propagation product as single-activity composition.

3. application according to claim 1 or 2, it is characterised in that：The prostate gland cancer cell is DU-145.

4. application according to claim 1 or 2, it is characterised in that：The product is medicine or kit.