CN104983736A - Application of Levonorgestrel in preparation of ovarian cancer resistant product - Google Patents

Abstract

The invention discloses an application of Levonorgestrel in preparation of an ovarian cancer resistant product, and provides an application of the Levonorgestrel in preparation of an ovarian cancer curing product. An experiment of the application proves that relocation of an anti-tumor drug is carried out on the Levonorgestrel which is approved by the FDA and the CFDA, according to different cell lines (histopathological types) and mutation sites of a tumor, screening is carried out to screen out drugs showing indications which are not resistant to tumors, a novel use of being resistant to ovarian cancer of the Levonorgestrel is found, and the novel use for the approved drug is achieved.

Description

Levonorgestrel is preparing the application in ovarian cancer resistance product

Technical field

The present invention relates to biological technical field, particularly relate to a kind of Levonorgestrel and preparing the application in ovarian cancer resistance product.

Background technology

Tumor threatens the most common of human health to be also the most serious a kind of disease, and research and development high efficiency anti-tumor medicine is most important to the life cycle extending patient.In recent years, along with the develop rapidly of cancer genomics and molecular pharmacology, the research and development of new type antineoplastic medicine achieve good achievement, but because new drug development has high input, cycle, the long bottleneck that waits cannot overcome, and the feature such as tumor individual inheritance variation is large, caused many traditional anti-tumor medicine effects not add, new drug is expensive, and side effect is not bright.

The researcheres such as Barabasi AL were published in " Nature Reviews Genetics " opinion in 2011 the article pointed out, the molecular network analysis carried out based on GWAS result of study and mutual work group (interactome) strategy is expected to disclose the new drug target of complex disease and molecular marker, and the understanding that final formation is machine-processed to disease incidence and therapeutic scheme is brand-new.Even more noteworthy, medicine reorientation (drug repositioning) research finds, GWAS studies the tumor susceptibility gene of locking and has protein-protein interaction (protein-protein interaction with it, PPI) gene more easily becomes medicine indirectly target spot, and this finds that there is and helps explain that the mechanism of action of existing medicine and guides the research and development of new drug.2014, show in 101 tumor susceptibility genes of the rheumatoid arthritis that GWAS result of study confluence analysis obtains in the paper that the researcheres such as Okada Y are delivered on " Nature " and have 98 direct or indirect targets being used for treatment medicine for treating rheumatoid arthritis at present, but also studied by medicine reorientation, they have also found that the granted medicine for other indications of dozens of also can be used for treating rheumatoid arthritis.

Summary of the invention

This research is just by integrating the drug data base that in the cancer gene spectrum CancerGene Census of cancer group Cosmic version72 data base and protein interaction STRING version 10 data base and DrugBankversion4.2, FDA ratifies, the candidate's reorientation medicine obtained, examination experiment is carried out to tumor cell line, screens the antitumor drug made new advances.The candidate tumor doing tumor cell line screening suppresses medicine to see as follows:

Nicardipine,Promethazine,Estrone,Levonorgestrel,Sunlidac,Levonorgestrel,Etonogestrel,Levonorgestrel,Mesalazine,Indomethacin,Sulfasalazine,Balsalazide,Irbesartan,Ibuprofen,Isoprenaline,Pentosan Polysulfate。

An object of the present invention is to provide the novelty teabag of Levonorgestrel.

The application of Levonorgestrel provided by the invention in preparation treatment ovarian cancer product.

Second object of the present invention is to provide the novelty teabag of Levonorgestrel.

The invention provides the application of Levonorgestrel in preparation suppression human epithelial ovarian carcinoma cells proliferation product.

3rd object of the present invention is to provide the novelty teabag of Levonorgestrel.

The invention provides the application of Levonorgestrel in preparation reduction ovarian cancer cell IC50 value product.

In above-mentioned application, described ovarian cancer cell is SKOV-3.

In above-mentioned application, described product is medicine or test kit.

4th object of the present invention is to provide a kind of product.

Product provided by the invention, its active component is Levonorgestrel; Described product has following at least one function:

1) ovarian cancer is treated;

2) human epithelial ovarian carcinoma cells proliferation is suppressed;

3) ovarian cancer cell IC50 value is reduced.

In the said goods, described ovarian cancer cell is SKOV-3.

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

Experiment of the present invention proves, the present invention is to FDA, the granted medicine Levonorgestrel of CFDA carries out tumour medicine reorientation, the cell line (organization type) different according to tumor and mutational site are not that anti-tumor drug screens to indication, find this novelty teabag of Levonorgestrel ovarian cancer resistance, realize old medicine and newly use.

Accompanying drawing explanation

Fig. 1 is 96 well culture plate medicine sieve medicine template distributions.

Fig. 2 is that Levonorgestrel is responsive to ovarian cancer; EC50=3.0820; IC50=3.3366; R ²=0.9311.

Detailed description of the invention

The experimental technique used in following embodiment if no special instructions, is conventional method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

In following embodiment, medicine to be measured is Levonorgestrel, and its chemical structural formula is as follows:

it is drug bank product, and catalog number is DB00367.

The source of the ovarian cancer cell SKOV-3 below in embodiment, people's pulmonary carcinoma non-small cell NCI-H1993 and human lung adenocarcinoma cell NCI-H441 is as follows:

SKOV-3 ATCC HTB-77

NCI-H1993 ATCC CRL-5909

NCI-H441 ATCC HTB-174

Main instrument below in embodiment and consumptive material

DMSO(from Sigma,Cat.No.D4540)

The 96-well white saturating Tissue Culture Plate in the end (from Corning, Cat.No.3610)

CellTiter Glo test kit (from Promega, Cat.No.G7573)

Doxorubicin positive drug (from MCE, Cat.No.HY-15142)

Fetal Bovine Serum(from Gibco,Cat#10099141)

100mm culture dish (from Corning, Cat#430167)

RPMI-1640 medium(from Gibco,Cat#A1049101)

DMEM medium(from Gibco,Cat#11995081)

DMEM/F12 medium(from Gibco,Cat#11330057)

EMEM medium(from Gibco,Cat#10370021)

Mutidrop 384 cell liquor separator (Thermo, Cat#5840150)

The multi-functional plate reading machine of EnSpire (Perkin Elmer, Cat#2300-001M)

Embodiment 1, CELLTITER-GLO detect Levonorgestrel ovarian cancer resistance

One, the preparation of orifice plate to be measured

1, plating cells

A) complete medium needed for each cell is prepared.

B) before experiment starts, confirm the medicine sieve cell name be marked on 100mm culture dish, culture medium and generation time, the information such as generation, guarantee that experiment is errorless.

C) attached cell operation refer step d) to i), suspension cell operation refer step j) to l).

D) vacuum pump is utilized to draw cell culture medium during sterile working.

E) with the aseptic PBS solution rinse cell surface layer of 2ml, then vacuum pump sucking-off PBS waste liquid is used.

F) 1ml 0.25% (w/v) Trypsin-0.038% (w/v) EDTA solution digestion cell is added gently to culture dish, mix gently several under, solution covers cell surface layer, under inverted microscope, observation of cell digestion situation, stops trypsinization effect when cell will come off.

G) in culture dish, add the preheated complete medium of 5ml 37 DEG C, blow and beat cell gently with pipet, make it split away off bottom culture dish.

H) this cell suspension is transferred in 15ml or 50ml sterile centrifugation tube, the centrifugal 5min of 1000rpm.

I) vacuum pump sterile working sucking-off supernatant culture medium is utilized.Add the complete medium re-suspended cell precipitation that 5ml 37 DEG C is preheated, blow and beat mixing gently.

J) blow and beat cell gently with pipet, make it split away off completely bottom culture dish.

K) this cell suspension is transferred in 15ml or 50ml sterile centrifugation tube, the centrifugal 5min of 1000rpm.

L) vacuum pump sterile working sucking-off supernatant culture medium is utilized.Add the complete medium re-suspended cell precipitation that 5ml 37 DEG C is preheated, blow and beat mixing gently.

M) count cell suspension with cell counter, adjustment cell suspension carries out plating cells experiment to proper density fishplate bar.

N) SKOV-3 cell is carried out according to the method described above, obtain SKOV-396 porocyte culture plate.

SKOV-3 cell, complete medium is McCOY'S 5A, is life product, 16600082, fishplate bar density (cells/well) is 12000.

2, medicine Levonorgestrel to be measured prepares and dosing (200X final concentration):

1) medicine Levonorgestrel motherboard preparation to be measured

A) with DMSO, medicine Levonorgestrel to be measured is diluted to 20mM stand-by.

B) getting the 20mM medicine 79 to be measured μ L configured in a) step adds in dilution plate the first row first hole, add the DMSO solvent of 54 μ L subsequently to the first row second hole in the 9th hole, 25 μ L solution are got in the second hole from the first hole, from the second hole, 25 μ L solution are got in the 3rd hole after mixing, the like to the 9th hole, ensure medicine successively carry out 3.16 times of multiple proportions gradient dilutions.

2) positive drug Doxorubicin (MCE, Cat.No.HY-15142) motherboard preparation

A) with DMSO, positive drug Doxorubicin is diluted to 6mM stand-by.

B) 6mM positive drug Doxorubicin solution is added in dilution plate, this medicine to be measured of DMSO solution 1:3.16 times of multiple proportions gradient dilution.

3, the preparation of medicine working plate and dosing

A) medicine to be measured and positive drug application of sample template are illustrated in fig. 1 shown below, and wherein, S1208: positive drug Doxorubicin, DMSO: Positive control wells, Cpd 1,2,3: medicine to be measured, DMSO final concentration is 0.5% (DMSO is compatible).

B) in working plate, add the specific complete medium of 95 μ l cell, corresponding 9 holes of each medicine, with multiple tracks volley of rifle fire difference transferase 45 μ l a series of (9 holes) solution (10X final concentration) that doubling dilution is good successively from medicine to be measured and positive drug doxorubicin motherboard, obtain the cell culture medium containing variable concentrations medicine.

C) from incubator, take out step 1 prepare SKOV-396 porocyte culture plate, by Fig. 1 dosing template arrangement mode (Fig. 1) respectively to adding the above-mentioned cell culture medium (10X final concentration) containing variable concentrations medicine b) prepared of 10 μ l in SKOV-396 porocyte culture plate, put into CO ₂incubator 37 DEG C cultivates 72h, obtains the 96 hole medicine sieve plates of SKOV-3.

Not add the hole in contrast of any medicine.

Final concentration in 96 orifice plates of above-mentioned medicine to be measured, positive drug Doxorubicin and control wells and dosing situation as follows:

The final concentration (μM) of medicine to be measured in the 2-10 hole of Fig. 1 is followed successively by: 100,31.64557,10.01442,3.16912,1.002886,0.317369,0.100433,0.031783,0.010058;

The final concentration (μM) of positive drug Doxorubicin in the 2-10 hole of Fig. 1 is followed successively by: 30,9.493671,3.004326,0.950736,0.300866,0.095211,0.03013,0.009535,0.003017;

In addition, in 96 orifice plates S1208 hole (E1-H1 and A12-D12): 10 μ l final concentrations, 100 μMs of Doxorubicin solution (solvent is the complete medium solution comprising 0.5%DMSO), DMSO hole (A1-D1, E12-H12 and A11-H11): 10 μ l comprise the complete medium solution of 0.5%DMSO.

Two, CELLTITER-GLO fluorecyte activity monitor system detects

1, CellTiter-Glo preparation of reagents

A) before using, CellTiter-Glo reagent Buffer is melted, equilibrate to room temperature and use.

B) before using, CellTiter-Glo reagent agar substrate is melted, equilibrate to room temperature and use.

C) get the CellTiter-Glo Buffer that 100ml balances to join in bottled CellTiter-Glo reagent agar substrate, make its abundant resuspended formation enzyme/substrate mixture, namely so-called CellTiter-Glo detectable.

D) mix vortex gently, and be repeatedly inverted the uniform solution of acquisition.In general, in 1 minute, CellTiter-Glo substrate reagent will fully dissolve, subpackage, keeps in Dark Place for subsequent use at-20 DEG C, avoids multigelation.

2, detect

A), before detecting, the 96 hole medicine sieve plates of above-mentioned one 3 SKOV-3 obtained are balanced 20-30 minute at room temperature.

B) cellular morphology and the death condition of every block culture plate observed by inverted microscope, mark abnormal conditions and repetition measurement once.

C) in all medicine sieve apertures, the CellTiter-Glo reagent of above-mentioned 1 preparation of 100 μ l is added, mixing.

D) on 96 hole microwell plate agitators, fully shake mixing 2 minutes, make the complete cracking of cell.

E) lucifuge room temperature is placed after 15 minutes and is carried out luminescence signal detection, ensures the stability of signal.

F) use during the multi-functional plate reading machine 570nm of EnSpire and read luminescence signal.

G) analyzing and processing data.

The 96 hole medicine sieve plate results of SKOV-3 as shown in Figure 2.

Calculate IC50 value, result is as shown in table 1.

Adopting the detection Levonorgestrel that uses the same method and making employment pulmonary carcinoma non-small cell NCI-H1993 and human lung adenocarcinoma cell NCI-H441, the IC50 value of cell, result is as table 1.

Can find out, Levonorgestrel has Specific Inhibitory Effect to human epithelial ovarian carcinoma cells proliferation, can be used as the medicine for the treatment of ovarian cancer.

Table 1 is the IC50 value of different cell under Levonorgestrel drug effect

Cell	IC50 value
		SKOV-3	3.3366

NCI-H441	100
		NCI-H1993	100

Claims (7)

1. The application of 1.Levonorgestrel in preparation treatment ovarian cancer product.
2. 2.Levonorgestrel suppresses the application in human epithelial ovarian carcinoma cells proliferation product in preparation.
3. 3. application according to claim 1 and 2, is characterized in that: described ovarian cancer cell is SKOV-3.
4. 4., according to described application arbitrary in claim 1-3, it is characterized in that: described product is medicine or test kit.
5. 5. a product, its active component is Levonorgestrel; Described product has following 1) and/or 2) function:

   1) ovarian cancer is treated;

   2) human epithelial ovarian carcinoma cells proliferation is suppressed.
6. 6. product according to claim 5, is characterized in that: described ovarian cancer cell is SKOV-3.
7. 7. the product according to claim 5 or 6, is characterized in that: described product is medicine or test kit.