CN110101704A

CN110101704A - Application of the c-Abl kinase inhibitor in FoxM1 high expression oncotherapy

Info

Publication number: CN110101704A
Application number: CN201910449803.8A
Authority: CN
Inventors: 刘萱; 袁永倩; 董钦才; 曹诚; 靳彦文; 张部昌
Original assignee: Institute of Pharmacology and Toxicology of AMMS; Anhui University
Current assignee: Institute of Pharmacology and Toxicology of AMMS; Academy of Military Medical Sciences AMMS of PLA; Anhui University
Priority date: 2019-05-28
Filing date: 2019-05-28
Publication date: 2019-08-09

Abstract

The invention discloses application of the c-Abl kinase inhibitor in FoxM1 high expression oncotherapy.The present invention provides application of the c-Abl kinase inhibitor especially nilotinib in the product that preparation expresses tumour for treating FoxM1 high.The invention demonstrates that nilotinib, which can significantly inhibit the growth of cervical cancer cell, can significantly inhibit cervical cancer cell one-tenth knurl ability after the FoxM1 phosphorylation site that c-Abl is relied on is mutated.The present invention explores potentiality of the c-Abl kinase inhibitor nilotinib in FoxM1 high expression treatment of solid tumors, new theories integration and experimental basis are provided for its clinical application, it is expected to expand the clinical indication of nilotinib, it is made to have broad application prospects in the therapy field of tumour.

Description

Application of the c-Abl kinase inhibitor in FoxM1 high expression oncotherapy

Technical field

The present invention relates to biomedicine fields, are related to c-Abl kinase inhibitor answering in FoxM1 high expression oncotherapy With.

Background technique

C-Abl nonreceptor tyrosine kinase can pass through phosphorylated substrate protein activation associated signal paths.Normal physiological Under the conditions of, kinase activity is in from holddown, and the c-Abl kinases activated is more easily by Ubiquitin-Proteasome Pathway Degradation.In chronic myelogenous leukemia, since the Piladelphia chromosome that chromosome translocation is formed causes ABL and BCR gene to melt It closes, the BCR-ABL of formation has the kinase activity of continuous activation.Nilotinib (Nilotinib) belongs to second generation tyrosine-kinase Enzyme inhibitor inhibits the kinase activity of c-Abl by the ATP-binding site on competitive binding BCR-Abl kinases, prevents c- The phosphorylation of Abl substrate, to inhibit relevant signal path.Its inhibit range include BCR-ABL, KIT, LCK, DDR1/2, PDGFRB, ZAK, MAPK11 and EPHA3/8, clinically for treating the chronic myelogenous white of Piladelphia chromatin-positive Blood disease, especially suitable for treatment with imatinib is invalid or drug resistant patient.Nilotinib is clinically chiefly used in the blood such as leukaemia The inhibitory effect of solid tumor is still not clear in the treatment of liquid cancer.

FoxM1 is a kind of and tumour occurs and grows closely related transcription factor, a series of its controllable oncogene Transcription, and then promote the formation of tumour.Its height expression is detected in a variety of solid tumors at present, these tumours include uterine neck Cancer, breast cancer, liver cancer, lung cancer, cancer of pancreas, kidney, colon cancer and glioma etc..FoxM1 and the correlation of tumour make Its important target spot for becoming anti-tumor drug.

Summary of the invention

It is formed with tumour closely related in view of FoxM1 high is expressed, therefore, explores nilotinib applied to FoxM1 The treatment of highly expressed tumour has important meaning.

In a first aspect, claimed c-Abl kinase inhibitor is in preparation for treating FoxM1 high expression tumour Application in product.

Second aspect, claimed c-Abl kinase inhibitor is in preparing the product for inhibiting tumour growth Application；The tumour is that FoxM1 high expresses tumour.

Wherein, the inhibition tumour growth, which can be presented as, inhibits gross tumor volume to increase and/or tumor weight is inhibited to increase.

The third aspect, claimed c-Abl kinase inhibitor are preparing the production for inhibiting growth of tumour cell Application in product；The tumour cell is FoxM1 high expression tumour cell.

Fourth aspect, claimed c-Abl kinase inhibitor is in preparation for lowering FoxM1 albumen table in tumour The application in product reached；The tumour is that FoxM1 high expresses tumour.

5th aspect, claimed c-Abl kinase inhibitor is in preparation for lowering FoxM1 egg in tumour cell Application in the product of white expression；The tumour cell is FoxM1 high expression tumour cell.

In above-mentioned various aspects, the c-Abl kinase inhibitor is specially nilotinib (Nilotinib).

In above-mentioned various aspects, the FoxM1 high expression tumour can express solid tumor for FoxM1 high；The FoxM1 high table Up to tumour cell solid tumor cell can be expressed for FoxM1 high.

Further, the FoxM1 high expression solid tumor can be following any: cervical carcinoma, breast cancer, liver cancer, lung cancer, pancreas Gland cancer, kidney, colon cancer or glioma；The FoxM1 high expression solid tumor cell can be following any: cervical carcinoma is thin Born of the same parents, breast cancer cell, liver cancer cells, lung carcinoma cell, pancreatic cancer cell, kidney cancer cell, colon cancer cell or glioma are thin Born of the same parents.

In the present invention, the FoxM1 high expression solid tumor and the FoxM1 high expression solid tumor cell also meet as follows Condition: the 575th tyrosine (Y) of FoxM1 albumen is subjected to the solid tumor cell that (is such as mutated into phenylalanine F) after rite-directed mutagenesis One-tenth knurl ability reduce.

In a specific embodiment of the invention, the FoxM1 high expression solid tumor is specially cervical carcinoma；The FoxM1 Height expression solid tumor cell is specially cervical cancer cell, is more specifically Hela-S3 cell.

In above-mentioned various aspects, the product concretely drug.

In above-mentioned various aspects, the amino acid sequence of the FoxM1 albumen is specific as shown in SEQ ID No.1.

It is experimentally confirmed that nilotinib can significantly inhibit the growth of cervical cancer cell, the FoxM1 phosphorylation that c-Abl is relied on After site is mutated (Y575F), cervical cancer cell one-tenth knurl ability can be significantly inhibited.The present invention explores c-Abl kinase inhibitor Potentiality of the nilotinib in FoxM1 high expression treatment of solid tumors, new theories integration and experiment are provided for its clinical application Foundation is expected to expand the clinical indication of nilotinib, it is made to have broad application prospects in the therapy field of tumour.

Detailed description of the invention

Fig. 1 is that Control Hela-S3 cervical cancer cell and Hela-FoxM1Y575F cell colony Forming ability detect.

Fig. 2 is that Control Hela-S3 cervical cancer cell and Hela-FoxM1Y575F cell one-tenth knurl ability and Buddhist nun Lip river are replaced Buddhist nun inhibits tumor growth ability to detect.32 nude mices are randomly divided into 4 groups, every group 8, are inoculated in a manner of inoculating respectively Control Hela-S3 cervical cancer cell or Hela-FoxM1Y575F cell, first group is the inoculation palace Control Hela-S3 Neck cancer cell and stomach-filling physiological saline group, second group is inoculation Control Hela-S3 cervical cancer cell and stomach-filling nilotinib Group, third group are inoculation Hela-FoxM1Y575F cell and stomach-filling physiological saline group, and the 4th group is inoculation Hela- FoxM1Y575F cell and stomach-filling nilotinib group.A is to take the tumour of each group nude mice to take pictures and observe tumor formation effect；B is to take The tumour of each group nude mice weighs weight；C is each group nude mouse tumor volume for measuring different time points.

Fig. 3 is that immunohistochemistry detects FoxM1 expression in tumor tissues.

In each figure, Control indicates Control Hela-S3 cell；FoxM1Y575F indicates that Hela-FoxM1Y575F is thin Born of the same parents.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.

Be born 30 days nude mices: Si Beifu (Beijing) Animal Science Co., Ltd.

Nilotinib: Novartis Pharma Schweiz AG, catalog number (Cat.No.) S0045.

Hela-S3 cell: American Type Culture collection warehousing (American type culture collection), mesh Record CCL-2.2.

PSpCas9 (BB) -2A-Puro plasmid: addgene company, catalog number (Cat.No.) 62988.

DH5 α competent cell: TIANGEN Biotech (Beijing) Co., Ltd., catalog number (Cat.No.) CB101.

I enzyme of Bbs: NEB company, catalog number (Cat.No.) R0593S.T4DNA ligase: NEB company, catalog number (Cat.No.) M0202L.T4 ligase Buffer (10 ×): NEB company, catalog number (Cat.No.) B0202S.T4PNK:NEB company, catalog number (Cat.No.) M0201S.Ampicillin: sigma Company, catalog number (Cat.No.) A6140.3000 transfection reagent of Lipofectamine: invitrogen company, catalog number (Cat.No.) L3000015.It is fast Purine mycin: gibco company, catalog number (Cat.No.) A11138-03.Genome extraction kit: QIAGEN company, catalog number (Cat.No.) 51306.PCR MIX: Beijing Qing Kexin industry Bioisystech Co., Ltd, catalog number (Cat.No.) TSE101.DMEM culture medium: gibco company, catalog number (Cat.No.) 8118026.Fetal calf serum: ExCell Bio Products, catalog number (Cat.No.) FSP500.Pancreatin: SIGMA Products, catalog number (Cat.No.) T4049.PBS buffer solution: gibco Products, catalog number (Cat.No.) 8118288.Dimethyl sulfoxide: Beijing Yi Nuokai Science and Technology Ltd. Product, catalog number (Cat.No.) D3855.Giemsa stain: Beisuo Biological Technology Co., Ltd., Zhuhai's product, catalog number (Cat.No.) BA-4017.

Embodiment 1, the building of Control Hela-S3 and Hela-FoxM1Y575F cell line and inoculation nude mice

One, the building of Control Hela-S3 and Hela-FoxM1Y575F cell line

Hela-FoxM1Y575F cell line is that the 575th tyrosine residue of FoxM1 sports the homozygote of phenylalanine Cell line.

1, pSpCas9-FoxM1Y575F expression plasmid is constructed

Centered on the codon of the 575th tyrosine for encoding FoxM1, the interception total 203bp's of each 100bp of upstream and downstream FoxM1 gene order is submitted to CRISPR Photographing On-line tool (http://crispr.mit.edu/), and design generates sgRNA sequence Column 5 '-CTCCCAGCTCAGCTACTCCC-3 ', and addition CACCG, 5 ' end addition AAAC of antisense strand are held in this sequence 5 ', The annealed double-stranded DNA for forming cohesive end of the positive antisense strand primer of synthesis, by this double-stranded DNA and through I digestion of Bbs The mixing of pSpCas9 (BB) -2A-Puro plasmid, connects under T4DNA connection enzyme effect, and connection product is converted to DH5 α and is experienced State cell, correct through sequencing identification with ammonia benzyl resistance screening positive colony, i.e. acquisition pSpCas9-FoxM1Y575F expresses matter Grain.

Wherein, sgRNA annealing system and program are as follows:

PCR program: 37 DEG C 30 minutes；95 DEG C 5 minutes；Then it keeps dropping 5 DEG C in each minute, until temperature is reduced to 25 DEG C.

2, Hela-FoxM1Y575F stable cell lines and Control Hela-S3 cell line are constructed

Hela-S3 cell is cultivated in 10 centimetres of diameter of culture dish, when cell it is long to about 60% when transfected.Transfection 5 milliliters of fresh DMEM mediums are exchanged within first 2 hours, using 3000 reagent of Lipofectamine by pSpCas9-FoxM1Y575F Expression plasmid (2 microgram) and mutagenesis template Oligo-575 single stranded DNA (SEQ ID No.2) (5 receive rub) are transferred to intracellular, transfection It exchanges fresh culture after 48 hours for, and puromycin is added to 0.5 mcg/ml of final concentration, change a subculture within every two days And keeping puromycin concentration is 0.5 mcg/ml, continues to expand to 24 orifice plates by the cell clone picking of generation after about 14 days Big culture.After cell clone expands culture, about 1 × 10 is collected⁵A cell extracts genome using genome extraction kit DNA reacts amplification target fragment by PCR, segment is sequenced, until 575 tyrosine for identifying FoxM1 sport The cell strain of phenylalanine.PSpCas9 (BB) -2A-Puro empty carrier is transfected in Hela-S3 cell using identical method, The stable cell line of acquisition is Control Hela-S3 cell line.

PCR reaction amplification target fragment primer:

575-PCR-F:5'-TCTCGGAAATGCTTGTGATT-3'

575-PCR-R:5'-GGAGCCTTTGCGGTGAT-3'

Primer synthesis and sequencing transfer to Beijing Qing Kexin industry Bioisystech Co., Ltd to carry out.

PCR reaction amplification target fragment system and program:

PCR response procedures: 95 DEG C 5 minutes, (95 DEG C 30 seconds, 56 DEG C 45 seconds, 72 DEG C 30 seconds, 35 circulations), 72 DEG C 10 points Clock.

Two, Control Hela-S3 and Hela-FoxM1Y575F cell inoculation nude mice

Use DMEM culture medium culture Control Hela-S3 and Hela-FoxM1Y575F containing 10% fetal calf serum Cell, culture environment 5%CO₂37 DEG C of incubators, the culture dish used is 10 centimetres of diameter of plate.Following caution of operation Be placed in and keep low temperature on ice: a waits for that cell covers with, and cell dissociation is got off using pancreatin, and every ware cell is light using 8 milliliters of culture mediums Featheriness is beaten to neutralize pancreatin, and cell is moved into 15ml centrifuge tube, and 1000g is centrifuged 3 minutes, is outwelled culture medium and is use up with pipettor Amount exhaustion remaining medium.B gently blows and beats cell washing, 1000g using the 10ml 1xPBS buffer that prior to 4 DEG C refrigerators are pre-chilled Centrifugation 3 minutes outwells PBS buffer solution and is exhausted as far as possible residual liquid with pipettor.It is primary that c repeats step b.D uses serum-free DMEM culture medium cell suspension is gently resuspended, using cell counter calculate cell concentration, adjustment cell concentration be 10⁷cells/ml.E draws cell suspension using 1ml syringe, by pallium cell injection in nude mice forelimb oxter rich blood vessel Place, is subcutaneously injected, and every mouse injects 200 μ l of cell.

Embodiment 2, nude mice nilotinib gastric infusion

Start to be administered within the 2nd day after nude inoculation cell.Nilotinib (Nilotinib) is first dissolved in dimethyl sulfoxide by a, Concentration is 15 μ g/ μ l, then by this solution, 1:1.67 is dissolved in physiological saline by volume, is configured to the solution of 5.6 μ g/ μ l.B gives The nude mice of medicine nilotinib presses the volume of every daily 200 μ l, carries out gastric infusion using syringe and stomach-filling syringe needle.C control Group institute to solution be the physiological saline containing 37% (volume ratio) dimethyl sulfoxide, every daily 200 μ l.Each group nude mice successive administration 21 days.Every group of 8 nude mices.

Embodiment 3, the detection of soft agar clonogenic assay ability

Cell colony formation assay is usually used in detecting the division growth ability of tumour cell.Control is digested using pancreatin Hela-S3 cell and Hela-FoxM1Y575F cell, are added after the completion of digest in DMEM culture medium and pancreatin, use liquid relief Device is blown and beaten repeatedly and forms single cell suspension.Cell concentration is detected using cell counter, according to 1 × 10³The inoculum concentration of cells/well It is inoculated in six orifice plates, is put into 37 DEG C, 5%CO₂It is cultivated in incubator, every the fresh culture of replacement in 2-3 days.Cell training The visible group of the naked eyes of formation in about 10 days or so is supported, moves and abandons culture medium and 4% paraformaldehyde is added fixed cell about 20 minutes, move and abandon It is rinsed cell 2 times after 4% paraformaldehyde using 1 × PBS buffer solution, is incubated for dyeing 30 minutes using Giemsa stain room temperature, then Clear bluish violet population of cells is formed using light and slow rinse 3 times of deionized water, is placed under ordinary camera and microscope and takes pictures.

As a result as shown in Figure 1.As seen from the figure, the palace Hela-S3 after the 575th tyrosine residue mutation of FoxM1 albumen Neck cancer cell (FoxM1Y575F) clonality is obviously reduced compared with Control Hela-S3 cell.

Embodiment 4, tumor volume measurement and the detection of histogenic immunity groupization

One, tumor volume measurement

The visible tumor mass of naked eyes can be formed in inoculation position within nude inoculation tumour cell 6 days or so, use vernier caliper measurement The length and width of tumor mass, calculate gross tumor volume according to formula: V=(long × wide × wide)/2, measurement in every 3 days is primary, and weighs Nude mice weight.Inoculated tumour cell put to death nude mice after 21 days, carefully strips tumour using surgical scissors and ophthalmology tweezers and weighs weight Amount, tumor tissues are placed in fixed preservation in 4% paraformaldehyde, with the detection of pending immunohistochemistry.

Two, immunohistochemistry detects

1. tumor tissues are taken out from 4% paraformaldehyde, it is put into after being dehydrated in embedded box with paraffin embedding, dehydration Step are as follows: 70% ethyl alcohol 60 minutes, 80% ethyl alcohol 60 minutes, 90% ethyl alcohol 60 minutes, 100% ethyl alcohol 60 minutes, 100% ethyl alcohol 60 minutes, dimethylbenzene 30 minutes, dimethylbenzene 60 minutes.

2. embedded tissue is cut with slicer and obtains the suitable wax disk(-sc) of thickness, and it is fixed on glass slide.Room temperature Dewaxing, aquation, step are as follows: dimethylbenzene 10 minutes, dimethylbenzene 10 minutes, 100% ethyl alcohol 5 minutes, 90% ethyl alcohol 3 minutes, 80% Ethyl alcohol 3 minutes, 70% ethyl alcohol 3 minutes, distilled water 3 minutes, distilled water 3 minutes, PBS buffer solution 3 minutes, PBS buffer solution 3 was divided Clock.

3. slice is put into box, with distilled water flushing, 3% hydrogen peroxide at room temperature of dropwise addition incubation 10 minutes endogenous to inactivate Enzymatic activity is washed 2 times with PBS buffer solution, and 3 minutes every time, distillation was washed 2 times, every time 3 minutes.

4. configuring EDTA antigen retrieval buffers, is boiled with pot heating, slice is put into hot pot and is heated 15 minutes, it is last natural It is cooled to room temperature.PBS buffer solution is washed 3 times, every time 5 minutes.

5. lowlenthal serum confining liquid is added dropwise after slice drying, it is put into wet box, incubation at room temperature 10 minutes non-specific to close Property site.

6. get rid of confining liquid and blotted with blotting paper, primary antibody (anti-FoxM1) is added dropwise and covers tissue, is put into wet box 4 and takes the photograph Family name's degree is incubated overnight.

7. taking-up in second day is sliced, is washed 3 times with PBS buffer solution, 3 minutes every time, blot droplet with blotting paper, secondary antibody is added dropwise (anti-rabbit), it is incubated at room temperature 30 minutes.It is washed 3 times, every time 3 minutes with PBS buffer solution again.

8. about 100 microlitres of DAB solution, microscopically observation is added dropwise in every slice.Dyeing can properly terminate dyeing, with steaming Distilled water is rinsed.It uses haematoxylin dyeing 1-2 minutes again, nucleus, which becomes blue, to be terminated, and rinsed with PBS buffer solution anti-blue.

9. dehydration: after slice distilled water flushing, being successively soaked in 70% ethyl alcohol 2 minutes, 80% ethyl alcohol 2 minutes, 90% Ethyl alcohol 2 minutes, dehydrated alcohol 5 minutes, dimethylbenzene 3 minutes, dimethylbenzene 3 minutes.

10. appropriate neutral gum mounting is added dropwise after drying in slice, covered is dried to be placed under microscope and be taken pictures.

Three, result

By cell inoculation to nude mice by subcutaneous 21 days, Hela-FoxM1Y575F cell Tumor formation is markedly less than Control Hela-S3 cell；The nude mice of inoculation Control Hela-S3 cell gives nilotinib processing, compared with the control group, processing group Tumour growth be suppressed significantly (Fig. 2).

Tumor tissue pathology's slice display, nilotinib can be lowered significantly in Control Hela-S3 tumour cell The expression of FoxM1；Compared with Control Hela-S3 cell, Hela-FoxM1Y575F cell inoculation nude mice is formed swollen FoxM1 expression significantly reduces (Fig. 3) in tumor.

<110>PLA Academy of Military Sciences's military medical research institute；University of Anhui

<120>application of the c-Abl kinase inhibitor in FoxM1 high expression oncotherapy

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Claims

Application of the 1.c-Abl kinase inhibitor in the product that preparation expresses tumour for treating FoxM1 high.
2.c-Abl kinase inhibitor is preparing the application in the product for inhibiting tumour growth；The tumour is FoxM1 high table Up to tumour.
3.c-Abl kinase inhibitor is preparing the application in the product for inhibiting growth of tumour cell；The tumour cell is FoxM1 high expression tumour cell.
4.c-Abl kinase inhibitor is in preparation for lowering the application in tumour in the product of FoxM1 protein expression；The tumour Tumour is expressed for FoxM1 high.
5.c-Abl kinase inhibitor is in preparation for lowering the application in tumour cell in the product of FoxM1 protein expression；It is described Tumour cell is FoxM1 high expression tumour cell.
6. any application in -5 according to claim 1, it is characterised in that: the c-Abl kinase inhibitor is that Buddhist nun Lip river is replaced Buddhist nun.
7. any application in -6 according to claim 1, it is characterised in that: the FoxM1 high expression tumour is FoxM1 high Express solid tumor；The FoxM1 high expression tumour cell is that FoxM1 high expresses solid tumor cell.
8. application according to claim 7, it is characterised in that: the FoxM1 high expression solid tumor is following any: uterine neck Cancer, breast cancer, liver cancer, lung cancer, cancer of pancreas, kidney, colon cancer or glioma；The FoxM1 high expresses solid tumor cell It is following any: cervical cancer cell, breast cancer cell, liver cancer cells, lung carcinoma cell, pancreatic cancer cell, kidney cancer cell, colon cancer Cell or neuroglial cytoma.
9. application according to claim 8, it is characterised in that: the cervical cancer cell is Hela-S3 cell.
10. any application in -9 according to claim 1, it is characterised in that: the product is drug.