CN104926945B - A kind of oncotherapy polypeptide and its application with FSHR targetings - Google Patents
A kind of oncotherapy polypeptide and its application with FSHR targetings Download PDFInfo
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Abstract
The present invention provides a kind of oncotherapy polypeptide and its application with FSHR targetings, and the oncotherapy that the polypeptide has FSHR targetings acts on, and the oncotherapy polypeptide of the FSHR targetings is FSH33‑53IIKK targeted fusion polypeptides, the polypeptide have the antitumor activity of enhancing IIKK polypeptides.The FSH of the present invention33‑53IIKK targeted fusions polypeptide can be applied in treatment or prevention tumour medicine is prepared.
Description
Technical field
The invention belongs to biomedical sector, is related to a kind of oncotherapy polypeptide with FSHR targetings and its application.
Technical background
Follicle-stimulating hormone receptor (FSHR) is potential prostate cancer targeting mark.FSHR is one kind by oligomerization glycoprotein
The membrane receptor of composition, category g protein coupled receptor family.FSHR is primarily present in the sertoli cell of humans and animals testis, ovary
Granulocyte, the startup of the effect of signals male puberty production of sperm by mediating follicular stimulating hormone (FSH), manhood During spermatogenesis
Maintenance and the development in female egg cell each stage.2010《New England Journal of Medicine》(Radu A,Pichon C,
CamparoP,et al.Expression of follicle-stimulating hormone receptor in tumor
blood vessels.N Engl J Med.2010Oct 21；363(17):1621-30.) report the tumour different to 1336
Sample carries out the research such as SABC, in situ hybridization, finds FSHR in prostate cancer, breast cancer, colorectal cancer, lung cancer, testis
High expression in the vascular endothelial cell of the multiple cancerous tissues such as cancer, therefore tumor vascular endothelium FSHR expression potentially contributes to tumour
The formation of new vessels.2013《BMC Cancer》(Siraj A,Desestret V,Antoine M,Fromont G,
Huerre M,Sanson M,Camparo P,Pichon C,Planeix F,Gonin J,Radu A,Ghinea
N.Expression of follicle-stimulating hormone receptor by the vascular
endothelium in tumor metastases,BMC Cancer.2013；13:246.) report including prostate cancer to existing
6 interior class tumours carry out immunohistochemical analysis in the transfer stove of major organs such as bone, liver, lymph node, brain and lung etc., find above-mentioned
FSHR altimeters reach in neoplasm metastasis, and the normal organ adjoined is without notable expression.Prompting, FSHR is prostate cancer transfer stove
One of targeting mark defined.In addition, FSHR is also in the cancer such as human ovarian cancer Caov-3, OVCAR-3, human prostate PC3
Constructive expression in cell.Further study show that after ovarian surface epithelial cell (OSE) is transfected into FSHR plasmids and overexpression,
The protein expressions such as EGFR, HER-2/neu and c-Myc of cell rise, and ERK1/2 then continues phosphorylation, and cell proliferation rate adds
It hurry up, these phenomenons are consistent with the intracellular situations of ovarian cancer cell OVCAR-3.Before speculating that being overexpressed FSHR may occur with tumour
The propagation of ovarian epithelial cell is related to the raising that oncogenic pathways are horizontal.Research is it has also been found that FSH/FSHR can be by adjusting classics
The activity of NF- κ B in PI3K/Akt signal paths strengthens ovarian cancer cell line 3AO propagation and invasive ability.Therefore,
The growth of FSHR and tumour, transfer, invasion and attack etc. are closely bound up, are an important research, therapy target.
Follicular stimulating hormone (FSH) is FSHR endogenic ligands, is made up of two subunits of α and β.Research shows on people's FSH β chains
33-53 fragments, 81-95 fragments etc. can be combined with FSHR, and wherein FSH33-53(Tyr-Thr-Arg-Asp-Leu-Val-
Tyr-Lys-Asp-Pro-Ala-Arg-Pro-Lys-Ile-Gln-Lys-Thr-Cys-Thr- Phe) with FSHR affinity most
By force, FSH and FSHR combination can be competed as antagonist.Xu Yu equalitys are to FSH33-53Carry out18F is marked, and is made18F-Al-
NOTA-MAL-FSH33-53The human prostata cancer tumor bearing nude mice imaging of the high expression of FSHR is used successfully to, is the spy of clinical prostate cancer
Specific diagnosis provides the foundation.Also research connects FSH by nano particle33-53With taxol (PTX), FSH is constructed33-53-
NP-PTX compounds, improve targetings of the PTX to tumour cell CAOV-3, treatment ability of medicine.It can be seen that FSH33-53Can be as FSHR's
Targeting vector, it is beneficial to the diagnosis and treatment of the high expression tumours of FSHR.
Polypeptide G (IIKK) nI-NH2(n=1-4), Gly- (Ile-Ile-Lys-Lys) n-Ile-NH2(n=1-4) it is, one
Class has simple repeated sequence and the cationic antibacterial peptide comprising α-helixstructure.With the increase of IIKK sequence repetition numbers,
Polypeptide IIKK alpha-helix by the connection with amphiphatic molecule film, can be strengthened, polypeptide structure is tended towards stability.In addition, more
Peptide hydroxyl terminal increase isoleucine (Ile) can further promote to maintain the ability of secondary structure, consolidate peptide molecule structure, draw
Send out the performance of simultaneously coordination function.Study by contrast, find G (IIKK)3I-NH2(IIKK) there is optimal chain length, antitumor effect
Fruit, simple in construction, toxic side effect is small, and antitumor activity is strong, is also that preferable target tumor suppresses medicine.IIKK by electrostatic with
Hydrophobic effect and tumour cell film combination, are entered in a manner of film permeabilization and accumulated in tumour cell, led to eventually through modulating apoptosis
Road or meronecrosis suppress growth of tumour cell.The good antitumor activity in IIKK inside and outsides can show that it should in clinical development
Great potential in.Lack the fused polypeptide albumen for having FSHR targetings and stronger anti-tumor function concurrently in the prior art.
The content of the invention
It is an object of the invention to have FSHR targetings and stronger anti-tumor function concurrently to solve to lack in the prior art
Fused polypeptide albumen.Based on the thinking of functional polypeptide use in conjunction, FSH is devised33-53- IIKK and IIKK-FSH33-53Targeting is melted
Polypeptide is closed, the oncotherapy polypeptide with FSHR targetings, the polypeptide has the antitumor activity of enhancing IIKK polypeptides, its ammonia
Base acid sequence is selected from following (a)-(c)：
(a), its sequence is that alpha subunit and β subunits are formed, and wherein alpha subunit is can be the same as affinity piece that FSHR is combined
Section, β subunits are the cationic antibacterial peptide with simple repeated sequence and comprising α-helixstructure,
Described affinity fragment includes 33-53 fragments or 81-95 fragments on FSH β chains,
Described cationic antibacterial peptide includes G (IIKK) nI-NH2Or Gly- (Ile-Ile-Lys-Lys) n-Ile-NH2, its
Middle n=1-4；
(b), the oncotherapy polypeptide of the FSHR targetings is FSH33-53- IIKK targeted fusion polypeptides
Tyr-Thr-Arg-Asp-Leu-Val-Tyr-Lys-Asp-Pro-Ala-Arg-Pro-Lys-Ile-Gln-Lys-
Thr-Cys-Thr-Phe-Gly-Gly-Gly-Ile-Ile-Lys-Lys-Ile-Ile-Lys-Lys-Ile-Ile-Lys-Lys-
Ile-NH2, and Gly-Ile-Ile-Lys-Lys-Ile-Ile-Lys-Lys-Ile-Ile-Lys-Lys-Ile- Gly-Gly-
Gly-Tyr-Thr-Arg-Asp-Leu-Val-Tyr-Lys-Asp-Pro-Ala-Arg-Pro-Lys-Ile-Gln-Lys-Thr-
Cys-Thr-Phe；
(c), the sequence is 1-10 substitution, missing or addition amino acid in the amino acid sequence shown in (a), (b)
Obtained from, and the amino acid sequence of the antitumor activity with enhancing IIKK polypeptides.
The recombinant fusion polypeptide also includes, and contains FSH33-53- IIKK targeted fusions polypeptide or its not loss of biological activity
Fragment, variant or derivative.
The present invention also resides in：Provide a kind of coding and weigh the FSH33-53The polynucleotides of-IIKK targeted fusion polypeptides.
Described polynucleotides also include its not fragment of loss of biological activity, variant or derivative.
The present invention also resides in：A kind of pharmaceutical composition is provided, it is used to treat or prevent tumour, comprising described
FSH33-53- IIKK targeted fusion polypeptides.Described pharmaceutical composition, also comprising pharmaceutically acceptable carrier or the composition
It is lyophilized form, unit dosage forms, solid dosage forms, liquid form.
The present invention also resides in：Provide the expression for suppressing growth of tumour cell by modulating apoptosis path or meronecrosis
Method, methods described comprise the steps：
A) nucleotide sequence, polypeptide or composition defined above are provided,
B) nucleotide sequence, polypeptide or composition are applied to or are applied to the expression system of wild tumour cell, thin
Born of the same parents, tissue or organism.
The present invention also resides in：Provide described FSH33-53- IIKK targeted fusions polypeptide, polynucleotides are preparing treatment
Or the application in prevention tumour medicine.
By described FSH33-53- IIKK targeted fusions polypeptide, described polynucleotides are preparing treatment or prevention system genitale
Application in system tumour, incidence cancer and lung cancer, malignant lymphatic tumor medicine.
By described FSH33-53- IIKK targeted fusions polypeptide, described polynucleotides are preparing treatment or prevention prostate
Application in cancer drug.
By building polypeptide FSH33-53The FSHR targeting diagnosis of mediation and the system for the treatment of simultaneously merge antineoplastic polypeptide IIKK,
It is desirable to FSH33-53- IIKK is applied to the treatment of the high expression tumours of FSHR, improves current treatment status.
Expression of the FSHR in several cells is examined by Western Blot, wherein, L929 cell FSHR radiolucent tables reach.
Mtt assay can detect the effect that medicine is bred to Carbazole alkaloid.FSH is detected using this method33-53- IIKK is right under various concentrations
PC3, Hela, SKOV3, MCF7, L929, the inhibitory action of 293 cells, and carried out with the inhibition rate of tumor cell of IIKK, cis-platinum
Compare.As a result show, FSH33-53- IIKK and IIKK has stronger inhibitory action to tumour cell, and both are to tumour cell
IC50It is close, and be in obvious dose-effect relationship.And the inhibitory action of two kinds of polypeptide normal tissue cells substantially weakens.By contrast,
Cis-platinum only reaches to the inhibitory action relative reduction of tumour cell, the particularly inhibiting rate in PC3 cells under 50 μM of concentration
(48.13 ± 3.89) %, with FSH33-53- IIKK cell inhibitory rate (96.94 ± 3.45) % compares significant difference (P ＞
0.05).And the polypeptide of same concentrations is remarkably reinforced with cisplatin effect in normal tissue cell, cis-platinum to the toxic action of cell.
It is tumorigenic to be mainly characterized by cell growth and proliferation out of control.Cell is different propagation, differentiation, apoptosis etc.
Often it has been involved in the occurrence and development of tumour.The research of common Antitumor Mechanism has：The influence of medicine cell cycle；
Inducing action of the medicine to apoptosis of tumor cells；The effect of drug-induced cell autophagy；Medicine to cell DNA influence (including
Detection, medicine with DNA binding abilities cause the influence that DNA damage, medicine are metabolized to nucleus) etc..Document report IIKK
Can be by changing cytoskeletal structure, reducing mitochondrial membrane potential, the release for promoting cromoci and activation Fas paths
Inducing cell apoptosis.Influence, the transduction of antiapoptotic signals and the autophagosome mark that the present invention is distributed from polypeptide cell cycle
Albumen LC3 three aspects of change are to fusogenic peptide FSH33-53- IIKK antitumor mechanism carries out Primary Study.
The regulation of polypeptide cell cycle and the influence of apoptosis are analyzed by flow cytomery.As a result show, it is different dense
After degree polypeptide acts on PC3 cells 24h, G0/G1, S, G2/M period profile difference of cell is not notable (P ＞ 0.05), i.e.,
FSH33-53- IIKK has no significant effect to the cycle of PC3 cells.FSH is investigated using the decoration methods of Hoechst 3325833-53-IIKK
Influence to PC3 Apoptosis, polypeptide is demonstrated from morphology can induce PC3 Apoptosis.FSH33-53- IIKK is by luring
Guided cell apoptosis plays tumor suppression function.Apoptosis is the autonomous dead process of cell by gene regulation, is autologous damage
A kind of phenomenon of wound.It is closely related that the generation of cancer and the generation of drug resistance with cell Apoptosis inhibitor occur.Hoechst 33258
7.5 μM in decoration method, 15 μM, 30 μM of FSH33-53- IIKK can induce PC3 cells typical apoptosis feature, such as cell occur
Volume-diminished, caryoplasm concentration, is presented irregular karyorhexis etc., and be in obvious concentration dependent.Under same concentrations gradient
With flow cytomery fusogenic peptide and the apoptosis rate of polypeptide IIKK inducing cell apoptosis.FSH33-53- IIKK acts on PC3 cells
Apoptosis rate be higher than apoptosis rate under IIKK respective concentrations, and the FSH of 30 μM of concentration33-53- IIKK withers to PC3 cells
The rate of dying is significantly higher than IIKK (P ＜ 0.05).Prove above, FSH33-53- IIKK can induce PC3 Apoptosis, and it is induced
The trend of Apoptosis is better than IIKK.
The occurrence and development of Apoptosis are related to the activation, expression and regulation and control of series of genes.Its main path can be divided into：
Death receptor mediated pathways, endoplasmic reticulum mediated pathways, mitochondria mediated pathways.Endogenic mitochondrial apoptotic pathway be cell by
To stimulation either internally or externally, the outer membrane permeability of mitochondria changes, and related antiapoptotic factors are discharged to cytoplasmic matrix, are triggered
The generation of apoptosis.
Bcl-2 family proteins play important regulating and controlling effect in the apoptotic pathways that mitochondria participates in, and can promote or do
The release for disturbing cromoci carrys out the apoptosis of regulating cell.The Bcl-2 homologous proteins being currently known probably have more than 20 to plant, according to it
The difference of 26S Proteasome Structure and Function can be divided into two major classes：Pro apoptotic protein Bax, Bad and Bak etc.；Anti-apoptotic proteins Bcl-2, Mcl-1 and
Bcl-xl etc..Bcl-2 interference cell pigments C release, the activation of upstream Caspase albumen is blocked, protects cell not wither
Die；And Bax/Bad promotes cromoci release, inducing cell apoptosis.
Cysteine aspartase (cysteine aspartic specific protease, Caspase) family is
The cysteine proteinase enzyme of one group of aspartic acid specific being present in endochylema, it can selectively cut some albumen
Matter, activate target protein or be allowed to inactivate, be the initiator and executor of mammalian apoptosis.In apoptosis process,
There are a variety of Caspase to participate in jointly.Under normal circumstances, Caspase with inactive precursor forms synthesis and is stored, and apoptosis
Signal can activate Caspase cascade reactions.Wherein, Caspase-8, Caspase-9 are main wither positioned at connection order reaction upstream
Die initiator；Caspase-3 etc. is located at the downstream of connection order reaction, is the executor of apoptosis.In the apoptosis that Fas or TNF is induced,
Caspase-8 is apoptosis " promoter ".After Caspase-8 receives apoptotic signal, activated by the form of self cleavage, directly or
The effector molecule Caspase-3 in indirect activation downstream etc., and Caspase-3 activated etc. decomposes Lamins, PARP (poly
ADP-ribose polymeras) etc., endonuclease is activated, causes DNA fragmentation.Caspase-3 activation is under apoptosis
Swim the key of signal transduction.
FSH is have detected using Western Blot methods33-53After-IIKK effect PC3 cells 24h, apoptosis-related protein
Procaspase-8, Procaspase-3, Bcl-2, Bad, Bax expression.As a result show, Procaspase-8,
Procaspase-3, Bcl-2 protein expression reduce, and Bad protein expressions are constant, Bax/Bcl-2 increases.Illustrate FSH33-53-IIKK
After acting on cell, Bcl-2 expression is reduced by mitochondrial apoptosis path, suppresses the release of cromoci, and then activate
Caspase cascade reactions in PC3 cells, have activated Caspase-8 and Caspase-3, have caused Apoptosis, are induced with IIKK
Apoptotic mechanism is consistent.
Cell autophagy (Autophagy) is a kind of reaction of cell reply external environment change, and generation is maintained to cell itself
Thank to balance and cell homeostasis plays an important role.On the one hand cell autophagy can provide abundant nutrition for tumour, promote
Enter tumour growth；On the other hand, cell autophagy can be to the canceration of anti-cell.Therefore, during tumor development, carefully
The effect of born of the same parents' autophagy has dual character.Most researchers think that cell autophagy is equally played the part of during to antitumor generation
Drill key player.
Cell autophagy is detected from cytomorphology using AO decoration methods.PC3 cells are through 0,7.5,15,30 μM of concentration
FSH33-53After-IIKK processing, it can be observed under fluorescence microscope, with the increase of drug concentration, the thin of autophagosome structure occur
Born of the same parents increase, and Fluorescence Increasing.Show polypeptide FSH33-53- IIKK also can induce cell and autophagy occur.
The process that autophagy occurs for cell mediates completion by a series of autophagy GAP-associated protein GAPs (Atg albumen).Microtubule associated protein
The transformation of the light chain 3 (the light chain 3, LC3 of Microtubule-associated protein 1) of matter 1 is autophagy
Mark.LC3 albumen can be cut by Atg4 protease becomes LC3- I, after autophagosome is formed, LC3- I and phosphatidyl-ethanolamine
(phosphatidylethanolamine, PE) coupling forms LC3- II.LC3- II can be stably retained on autophagosome film directly
Arrive and lysosome fusion, therefore the mark of autophagosome can be used as.
Detected using Western Blot in FSH33-53LC3- I and the protein expressions of LC3- II in the lower PC3 cells of-IIKK effects
Horizontal change, the quantity of autophagosome is reacted with this.Test result indicates that PC3 cells are after drug-treated, as medicine is dense
The increase of degree, LC3- I expression is reduced, and increase trend is presented in the protein expression levels of LC3- II under low concentration medicine, in height
Acute drug effect is lower to be reduced again.Illustrate FSH33-53- IIKK can a certain degree of inducing cell generation within the scope of debita spissitudo
Autophagy, and suppress tumor cell proliferation by other approach under the effect of high concentration polypeptide.
In view of being difficult to biochemical environment complicated in analogue body in vitro, effect of the polypeptide to solid tumor is by multiple factors
Influence, such as easily degraded in vivo by a variety of enzymes, sour environment caused by inside tumor blood supply insufficiency influences performance of polypeptide etc., institute
To need to establish the performance of targeting and therapeutic action of the internal antitumor model investigation polypeptide in solid tumor.Moved using subcutaneous cell
Plant method establishes PC3 tumor models, and tumor formation rate reaches 95%, and upgrowth situation is good in nude mouse.
In checking FSH33-53In-IIKK bodies in function of tumor inhibition experiment, physiological saline negative control group, IIKK are devised
(5mg/Kg) experimental group, FSH33-53- IIKK (5mg/Kg, 10mg/Kg) experimental group, cis-platinum (3mg/Kg) positive controls, pass through
Compare FSH33-53Influence of the medicines such as-IIKK, cis-platinum to the inhibiting rate of PC3 tumours and to nude mice changes of weight is more to investigate
The performance of the suppression tumor proliferation of peptide in vivo.Cis-platinum (DDP) is heavy metal complex, and the cell cycle for belonging to wide spectrum is non-specific
Property antineoplastic.DDP can suppress DNA of tumor cell reproduction process, destroy membrane structure, prevent tumor cell proliferation, be
The conventional chemicals of clinical treatment malignant tumour at present, is used for genital system, incidence cancer and lung cancer, malignant lymphatic
The treatment of the Several Kinds of Malignancy such as knurl.But DDP can also produce larger toxic side effect, and produce while killing tumor cell
Raw drug resistance.And prostate gland cancer cell is poor to cisplatin sensitivity, its application in prostate cancer therapy is limited.Consulting literatures
Learn, the maximum tolerated dose of cis-platinum is 9mg/kg, every 6 days single administrations.With cis-platinum 3mg/kg, administration is as sun 3 times a week
Property control group and FSH33-53- IIKK tumor-inhibiting action is compared.Experimental result shows, the FSH of same concentrations33-53- IIKK tables
Reveal the average tumor inhibiting rate higher than IIKK, FSH33-53The average tumor of-IIKK 10mg/kg experimental groups and cis-platinum group presses down
Rate processed is close, and FSH33-53Influences of-the IIKK to tumor mouse body weight is substantially less than cis-platinum group, illustrates that the toxic side effect of polypeptide is small.
The beneficial effect of invention：
FSH33-53- IIKK and IIKK has stronger inhibitory action to tumour cell, both IC to tumour cell50It is close,
And it is in obvious dose-effect relationship；FSH33-53- IIKK acts on the apoptosis rate of PC3 cells higher than the Apoptosis under IIKK respective concentrations
Rate, FSH33-53- IIKK can induce PC3 Apoptosis, and the trend of its inducing cell apoptosis is better than IIKK；FSH33-53-
After IIKK acts on cell, Bcl-2 expression is reduced by mitochondrial apoptosis path, suppresses the release of cromoci, and then swash
Caspase cascade reactions in PC3 cells living, have activated Caspase-8 and Caspase-3, have caused Apoptosis；FSH33-53-
IIKK shows the average tumor inhibiting rate higher than IIKK, 10mg/kgFSH33-53- IIKK shows similar tumour with cis-platinum
Inhibitory action, but FSH33-53Influences of-the IIKK to tumor mouse body weight is significantly lower than cis-platinum group, illustrates that the toxic side effect of polypeptide is small.
Brief description of the drawings：
Below with reference to accompanying drawing and instantiation, the present invention will be further elaborated.
Fig. 1, Western blot detect expression of the FSHR in MCF7, Hela, PC3,293, SKOV3, L929 cell.
The inhibited proliferation of Fig. 2, FSH33-53-IIKK, IIKK and cis-platinum to cell.
Fig. 3, various concentrations FSH33-53-IIKK act on fitted figure of lower PC3 cell cycles and statistical result.
A:0μM；B:7.5μM；C:15μM；D:30μM；E:Cell cycle statistical result (P ＞ 0.05)
Fig. 4, FSH33-53-IIKK act on the dyeing observation inducing cell apoptosis of PC3 cells 24h, Hoechst 33258 and made
With.
A:0μM；B:7.5μM FSH33-53-IIKK；C:15μM FSH33-53-IIKK；D:30μM FSH33-53-IIKK
The double dye methods of Fig. 5, Annexin V-FITC/PI detect FSH33-53-IIKK and IIKK to the apoptosis-induced of PC3 cells
Effect.
A:0μM；B:7.5μM FSH33-53-IIKK；C:15μM FSH33-53-IIKK；D:30μM FSH33-53-IIKK
E:7.5μM IIKK；F:15μM IIKK；G:30μM IIKK
The influence that Fig. 6, FSH33-53-IIKK are expressed PC3 cell death related proteins.
Fig. 7, FSH33-53-IIKK act on PC3 cells 24h, AO dyeing observation Induces Autophagy effect.
A:0μM；B:7.5μM FSH33-53-IIKK；C:15μM FSH33-53-IIKK；D:30μM FSH33-53-IIKK
The influence of Fig. 8, FSH33-53-IIKK to PC3 cells LC3- Ι, LC3- Π protein expressions.
The influence of Fig. 9, FSH33-53-IIKK to PC3 Nude Mouse Model tumor growth curves.
Figure 10, administration 21 days after, each experimental group mice with tumor changes of weight and tumor volume vs figure.
Embodiment：
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York：Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the condition proposed by manufacturer.
Unless otherwise defined, anticipated known to all specialties used in text and scientific words and one skilled in the art
Justice is identical.In addition, any method similar or impartial to described content and material all can be applied in the present invention.Described in text
Preferable implementation only present a demonstration and be used with material.
Embodiment 1
Western Blot detect expression of the FSHR in several cells
Human carcinoma of prostate cell line PC3, human cervical carcinoma cell lines Hela, human oophoroma cell line SKOV3, human breast carcinoma are thin
Born of the same parents' strain MCF7, human embryonic kidney cells 293, l cell L929, purchased from Chinese Academy of Sciences's Shanghai cell bank.
Male BALB/c-nu nude mices, 18~19g of body weight, this experimental animal Co., Ltd of changzhou Cavan, SPF levels, mouse
5 weeks ages.Quality certification number：NO.0026328, credit number：SCXK (Soviet Union) 2011-0003.
The cell culture processes such as PC3, MCF7, Hela, PC3,293, SKOV3, L929 are consistent used in experiment, 37 DEG C, 5%
CO2Constant incubator in subculture, with containing 10% hyclone and 100U/ml penicillin, the DMEM of 100U/ml streptomysins
Medium culture.
Cell is collected, with the RIPA reagents containing protease inhibitors, conventionally extracts cell protein and with BCA eggs
White analysis test kit measure protein concentration.After protein quantification, equal protein is taken, adds sample-loading buffer, 70 DEG C of sample
10min is denatured, SDS- Poly n alkylacrylates (SDS-PAGE), per hole loading 40-50 μ g, rear transferring film is on pvdf membrane.
With 5% skimmed milk power, shaking table is closed 1 hour at room temperature, adds primary antibody (FSHR:Rabbit-anti-FSHR, 1:1000；Actin:
Mouse-anti-Actin, m, h, 1:1000), seal, 4 DEG C of shaking tables sway overnight.TBST washes film 3 times, each 10min.Add
Secondary antibody (Anti-Rabbit-HRP, Anti-mouse-HRP, 1:1000, secondary antibody diluted), encapsulation, it is placed in shaking table upper chamber
Temperature is incubated 1h.Wash film, ECL exposure imagings as stated above again.
Expression of the FSHR in various kinds of cell
FSHR protein expression situations in cell are detected with Western Blot.Such as Fig. 1, Hela, MCF7, PC3 cell line have
High-caliber FSHR expression, L929 cells are expressed without FSHR.
Embodiment 2
Mtt assay measure polypeptide FSH33-53Influences of-the IIKK to various kinds of cell survival rate
By the cell of logarithmic phase growth according to 5 × 103The density kind that individual/hole connects is in 96 orifice plates.Overnight incubation, cell patch
After wall, it is separately added into per hole containing various concentrations FSH33-53The μ l of culture medium 100 of-IIKK, IIKK and cis-platinum (concentration gradient 0,
1,2,5,10,15,20,40,50 μM), each concentration sets 3 multiple holes.After being incubated 24h, it is 2mg/ml that 40 μ l concentration are added per hole
MTT solution, continue to cultivate, supernatant abandoned after 4h；150 μ l DMSO is added per hole, concussion 10min is fully dissolving crystallized.Enzyme mark
OD value of the instrument determination sample in 570nm wavelength.Calculate survival rate, draw survivorship curve, and obtain IC50.Survival rate=(experimental port
OD values/blank control group OD values) × 100%.
FSH33-53- IIKK vitro cytotoxicity detection
Using mtt assay measure polypeptide FSH33-53- IIKK, IIKK and cis-platinum to PC3, Hela, SKOV3, MCF7 and 293,
Cytotoxicity in L929 cells.Such as Fig. 2, FSH33-53- IIKK, IIKK and cis-platinum have more apparent to the propagation of tumour cell
Inhibitory action, and be in dose dependent.Polypeptide FSH33-53- IIKK, IIKK are compared with cis-platinum, and the above two are to tumour cell table
Reveal stronger inhibitory action, and no significant difference between two kinds of polypeptide administration groups.Polypeptide FSH33-53- IIKK, IIKK pairs
The toxicity of normal tissue cell is below cis-platinum.Compare FSH33-53The IC of-IIKK, IIKK and cis-platinum to several cells50, knot
Fruit such as table 1.
IC of the medicine of table 1 to cell line50Contrast
*p<0.05vs FSH33-53- IIKK, * * p<0.01vs FSH33-53- IIKK, * * * p<0.001vs FSH33-53-
IIKK
p-values were determined by Student’s t-test between the IC50values of
both cell lines
Embodiment 3
FSH33-53Influences of-the IIKK to the PC3 cell cycles
Cell cycle refers to cell, and once division terminates the active procedure untill division next time terminates in the past, is divided into division
Between two stages of phase and division stage.Wherein, the interkinesis is divided into G1 phases (DNA pre-synthesis phases), S phases (DNA synthesizes the phase), G2 phases again
(DNA post-synthesis phases).The M phases are m period.The G0 phases are the dormant cells phase.Propidium iodide (PI) is that one kind can be to DNA and RNA
The nuclei dyeing color reagent of dyeing, red fluorescence can be discharged after the intercalation of DNA or RNA.Although PI can not by living cells film,
But damaged cell membrane can be passed through and nucleus is dyed.Ice ethanol can increase permeability of cell membrane.Cracked with RNase
RNA, enter PI and combined into the cell with DNA selective, the fluorescence volume of intracellular dye is with endonuclear DNA content into just
Than.The DNA relative amounts of cell are can obtain by flow cytomery individual cells fluorescence intensity, further according to each phase
The difference of DNA content, cell is divided into the different cycles.
By the PC3 cells of exponential phase, with 2 × 105Individual/hole is inoculated in 6 orifice plates, and 1.5ml nutrient solution is added per hole.
Overnight incubation, after cell attachment, it is separately added into final concentration of 0 μM, 7.5 μM, 15 μM, 30 μM of FSH33-53-IIKK.Medicine acts on
After 24h, collect nutrient solution simultaneously digest attached cell, wash culture dish 3 times with PBS, collect PBS, merge cell, 1500r/min from
Heart 4min；Cell is resuspended, is added in 70% ethanol of 4 DEG C of precoolings and fixes, sealed membrane sealing, 4 DEG C overnight.2000r/min
4min is centrifuged, sucks fixer, PBS is washed 2 times.Cell is transferred in 1ml centrifuge tubes, add containing PI (50 μ g/ml),
RNaseA (50 μ g/ml) and Qula lead to the working solution of (1%), and room temperature lucifuge is incubated 30min, with flow cytomery cell
Cycle, and analyze Cell Cycle distribution.
FSH33-53Influences of-the IIKK to the PC3 cell cycles
By FSH of the PC3 cells through various concentrations33-53After-IIKK processing 24 as a child, the shadow of medicine cell cycle is detected
Ring.As a result show, 7.5,15,30 μM of FSH33-53It is poor compared with cellular control unit period profile after-IIKK acts on cell
Different not statistically significant (Fig. 3).That is, FSH33-53- IIKK suppresses the work of tumor proliferation to the period profile of cell without influence, polypeptide
With being realized by changing cell cycle distribution.
Embodiment 4
The dyeing detection Apoptosis of Hoechst 33258
Hoechst 33258 is that one kind can be with penetration cell film, the blue-fluorescence dye liquor that is combined with nucleus.Although
Hoechst can be combined with all nucleic acid, but be more likely to be combined with the DNA rich in A/T (adenine/thymidine),
350nm or so ultraviolet excitation, fluorescence intensity significantly increase.Therefore, after the dyeing of Hoechst 33258, fluorescence microscopy is used
The metamorphosis of mirror observable nucleus.
By the PC3 cells of exponential phase with 5 × 105Individual/hole is inoculated in 6 orifice plates, adds nutrient solution to be shaken to 2ml, cross
Six orifice plates are shaken, cell is uniformly scattered.Six orifice plates are placed in incubator, overnight incubation.After cell attachment, end is separately added into
Concentration is 0 μM, 7.5 μM, 15 μM, 30 μM of FSH33-53-IIKK.After acting on 24h, old nutrient solution is absorbed, is then carefully swung with PBS
Wash 2 times；The final concentration of dye liquors of 10 μ g/ml Hoechst 33258 (containing 0.2% triton x-100) 1ml is added per hole, is put into training
Foster case continues to be incubated 30min.Dye liquor is abandoned, is washed 2 times with PBS, attention action is light, cell not blown off；In fluorescence microscope
Lower observation, and photograph to record karyomorphism.
The decoration methods of Hoechst 33258 observe FSH33-53Influences of-the IIKK to PC3 Apoptosis
FSH is detected using Hoechst33258 fluorescence colours33-53- IIKK acts on the form of nucleus after PC3 cells
Change.FSH of the cell respectively through 0,7.5,15,30 μM of concentration33-53After-IIKK processing cells 24h, Hoechst dyeing, fluorescence shows
Micro- Microscopic observation.Such as Fig. 4, the nucleus of blank control group cell largely presents homogeneous, full circular or oval.Because thin
There is the process of programmed death in born of the same parents itself, have only a few cell that hyperfluorescence dyeing and heterocyst core is presented.As medicine is dense
The increase of degree, there is obvious cell volume and reduce, nuclei dyeing chromaticness shrinkage, forms semilune or hat shape invests nuclear membrane, glimmering
Light becomes fine and close, and assembled with obvious particular fluorescent body, and irregular karyorhexis phenomenon is presented, and (arrow show generation
Karyopycnosis, the typical apoptosis form of karyorrhexis).When concentration reaches 30 μM, cell attachment ability declines, cracking fragmentates.
Embodiment 5
Flow cytomery FSH33-53Apoptosis-induced effects of-the IIKK to PC3 cells
The double dye flow cytometer detection methods of AnnexinV-FITC/PI are to detect the conventional method of Apoptosis.In the morning of Apoptosis
Phase, phosphatidylserine (PS) of the distribution of specific on the inside of the lipid bilayer of cell membrane can turning up along with cell membrane
And it is exposed to cell surface.AnnexinV and PS is highly affine, and after fluorescein FITC marks, morning can be detected by fluorescence
The generation of phase Apoptosis.And be destroyed in the middle and advanced stage of Apoptosis, the integrality of cell membrane, PI can pass through cell membrane with
Nucleus combines.Two kinds of fluorescent dyes are used in combination, you can the different conditions of cell are distinguished with flow cytometer.
By the PC3 cells of exponential phase with 5 × 105Individual/hole is inoculated in 6 orifice plates, adds nutrient solution to be shaken to 2ml, cross
Six orifice plates are shaken, cell is uniformly scattered.Six orifice plates are placed in incubator, overnight incubation.After cell attachment, end is separately added into
Concentration is 0 μM, 7.5 μM, 15 μM, 30 μM of FSH33-53- IIKK, IIKK peptides.After acting on 24h, collection cell (including in nutrient solution
The dead cell of floating), then carefully swung with PBS and wash culture dish 2 times, collect PBS, cell suspension 1500r/min is centrifuged into 4min,
Supernatant discarding, cell is resuspended and goes in 1.5ml centrifuge tubes.PBS is washed 2 times, 2000r/min centrifugation 4min, supernatant discarding.Each
Sample adds binding buffer (every 1ml binding containing Annexin V-FITC and PI that 100 μ l are prepared in advance
Buffer adds Annexin each 20 μ l of V-FITC and PI, concussion, is well mixed dyeing liquor), 15-25 DEG C of lucifuge is incubated 10-
15min.Before detecting sample, often pipe sample adds appropriate binding buffer dilutions (1000 cells/microlitre dilution
Liquid).Flow cytomery Apoptosis situation, and analysis result.
FSH33-53- IIKK and comparisons of the IIKK to PC3 cell induction apoptotic effects
Flow cytometry FSH33-53- IIKK and effects of the IIKK to PC3 cell induction apoptosis.As shown in Figure 5：7.5、
15th, the FSH of 30 μM of concentration33-53After-IIKK effects 24h, PC3 apoptosis rates are followed successively by：(9.1 ± 1.35) %, (18.5 ±
1.50) %, (51.1 ± 2.00) %.Under the IIKK effects of corresponding concentration, apoptosis rate respectively (7.3 ± 1.08) %, (16.9 ±
0.81) %, (41.2 ± 2.61) %.The increase of two groups of apoptosis rates is respectively provided with significant difference (P ＜ 0.01).15th, 30 μM of concentration
In the presence of, FSH33-53The apoptosis rate of-IIKK experimental groups increased (P ＜ 0.05) compared with the apoptosis rate of IIKK control groups.
Illustrate FSH33-53- IIKK induction PC3 Apoptosis is in dose dependent, and is acted on compared with IIKK enhancings.
Embodiment 6
The expression of the albumen such as Western blot detections Procaspase-3, Procaspase-8, Bcl-2, Bax
By the PC3 cells of exponential phase with 2 × 105Individual/hole is inoculated in culture dish, treats cell growth to 80-90%
When, add 0 μM, 7.5 μM, 15 μM, 30 μM of FSH33-53- IIKK, processing cell 24h.Abandon nutrient solution and swung with PBS and washed 3 times, will
200 RIPAs of the μ l containing protease inhibitors are added in culture dish, and cold hatching 5 minutes, scraper plate collects cell protein and carries out egg
It is white quantitative.Equal protein is taken, adds sample-loading buffer, by protein sample after 70 DEG C are denatured 10min, SDS- poly amic acids coagulate
Gel electrophoresis (SDS-PAGE), are 40-50 μ g per hole applied sample amount, and electrophoresis carries out transferring film operation after terminating.With 5% skimmed milk power room temperature
Lower shaking table closing 1h, and addition primary antibody (Bcl-2, Bax, Procaspase-3, Procaspase-8, Actin, 1:1000), 4 DEG C are shaken
Bed is incubated overnight.TBST washes film 3 times, each 10min, adds 1:The secondary antibody of 1000 dilution horseradish peroxidase-labeleds, room temperature
Shaking table is incubated one hour.Wash film, ECL exposure imagings as stated above again.Internal reference is done with Actin ash is carried out to each protein band
Degree analysis, and make block diagram by ordinate of ratio.
FSH33-53Influences of-the IIKK to PC3 cell death related proteins
Western blot detect various concentrations FSH respectively33-53The table of the lower PC3 cell death related proteins of-IIKK effects
Up to change.After medicine effect 24h, the change of Procaspase-8, Procaspase-3, Bcl-2, Bad, Bax protein expression is such as
Fig. 6.With the increase of drug concentration, Procaspase-8, Procaspase-3, Bcl-2, Bax protein expression reduce, and have
There is concentration dependent.Bad protein expressions are without significant change.Although Bax protein expressions are also on a declining curve, Bax/Bcl-2 is still
It is in rising trend.As a result show, FSH33-53The Caspase that-IIKK can activate PC3 cells by mitochondria pathway is cascaded instead
Should, and final inducing cell apoptosis.
Embodiment 7
AO dyeing detection cell autophagies
Acridine orange (acridine orange, AO) is a kind of fluorescence dye liquor of permeable cell, can contaminate DNA and kytoplasm
Into bright green.When PH is reduced, AO can send red fluorescence, and fluorescence intensity and acid degree positive correlation.When cell occurs certainly
Autophagy lysosome structure is formed when biting.Autophagy lysosome is in acidity, using AO mark lysosome detection cell autophagies.
By the PC3 cells of exponential phase with 5 × 105Individual/hole is inoculated in 6 orifice plates, adds nutrient solution to be shaken to 2ml, cross
Six orifice plates are shaken, cell is uniformly scattered.Six orifice plates are placed in incubator, overnight incubation.After cell attachment, end is separately added into
Concentration is 0 μM, 7.5 μM, 15 μM, 30 μM of FSH33-53-IIKK.After acting on 24h, old nutrient solution is absorbed, is then carefully swung with PBS
Wash 2 times；Final concentration of 10 μ g/ml AO dye liquor 1ml are added per hole, 4 DEG C of refrigerators is put into and continues to be incubated 30min.Dye liquor is abandoned, uses PBS
Wash 1 time, attention action is light, cell not blown off, in fluorescence microscopy Microscopic observation, and photograph to record cell fluorescence situation.
AO dyeing detection cell autophagies
FSH is detected using AO decoration methods33-53- IIKK acts on the change of cell autophagy lysosome generation after PC3 cells.Carefully
FSH of the born of the same parents respectively through 0,7.5,15,30 μM of concentration33-53After-IIKK processing cells 24h, AO dyeing, fluorescence microscopy Microscopic observation.
Such as Fig. 7, bright green is presented in the nucleus and kytoplasm of blank control group cell.The FSH of 7.5 μM of concentration33-53- IIKK administrations are handled
Afterwards, can be observed to be distributed in cytoplasmic acid vesica presentation Chinese red in the cell of part.With the increase of drug concentration, occur
The cell quantity increase of autophagosome structure, and fluorescence intensity strengthens.When concentration reaches 30 μM, cell attachment ability declines, several
Shiny red fluorescence is distributed with the cytoplasm of all attached cells.
Embodiment 8
Western Blot detect the expression of autophagy proteins
The autophagy process of cell is to be mediated to complete by a series of autophagy GAP-associated protein GAPs (Atg albumen).Wherein, micro-pipe is related
The light chain 3 (the light chain 3, LC3/Atg8 of microtubule-associatedprotein 1) of albumen 1 is autophagosome film
On labelled protein.The intracellular LC3 albumen that II two kinds of forms of LC3- I and LC3- be present.After autophagosome is formed, LC3- I can
With phosphatidyl-ethanolamine (phosphatidylethanolamine, PE) coupling form LC3- II, be positioned at autophagosome inner membrance and
Outer membrane.By determining, comparing LC3- I and LC3- II level, the quantity of autophagosome can be reflected to a certain extent.
The step of culture of cell, agent-feeding treatment, the collection of protein sample, concentration mensuration and gel electrophoresis, as before.With
5% skimmed milk power at room temperature shaking table closing 1h after, add primary antibody (LC3,1:1000), 4 DEG C of shaking tables are incubated overnight.TBST washes film 3
It is secondary, each 10min, add 1:The secondary antibody of 1000 dilution horseradish peroxidase-labeleds, room temperature shaker are incubated 1h.Again by above-mentioned
Method washes film, ECL exposure imagings.Internal reference is done with Actin gray analysis is carried out to each protein band, and made by ordinate of ratio
Block diagram.
Cell autophagy Protein Detection
Western Blot detection various concentrations FSH33-53The expression of the lower PC3 cell autophagy GAP-associated protein GAPs of-IIKK effects becomes
Change.After medicine effect 24h, LC3- Ι, change such as Fig. 8 of LC3- Π protein expressions.With the increase of drug concentration, LC3- Ι albumen
Expression become and reveal downward trend, and increase trend is presented in the case where 7.5-15 μM of drug concentration acts on for LC3- Π albumen, and in 30 μ
Express and reduce under M concentration.As a result show, FSH33-53- IIKK can be occurred certainly under low concentration environment with induced tumor cell
Bite, and suppress cell by apoptosis or other modes in high concentration and increase.
Embodiment 9
FSH33-53Identifications of-the IIKK to PC3 transplanted tumor in nude mice inhibitory action
The foundation of PC3 Nude Mouse Models and the identification to PC3 transplanted tumor in nude mice inhibitory action.By SPF level animals
Rule raising nude mice, all drinking water, cage tool are handled through Aseptic sterilisation.PC3 cells are collected under aseptic condition, with sterile physiological salt
Water is diluted to 5 × 107Individual/ml, using subcutaneous vaccination method, inoculation PC3 cells armpit on the right side of nude mice is subcutaneous, every inoculation 1
×107Individual cell, treat it into knurl.When tumour body reaches 50~100mm3When, PC3 transplantable tumor nude mices are divided into five groups, every group 5,
Every group of mean tumour volume is more consistent.FSH is set respectively33-53- IIKK 5mg/kg and 10mg/kg administration groups, IIKK 5mg/kg give
Medicine group, cis-platinum 3mg/kg control groups and saline control group.It is administered, weekly administration 3 times, is administered using tail vein injection mode
Before weigh in, measure knurl footpath.Each group relative tumour volume (standardizing different groups of gross tumor volume) is calculated, draws knurl body
Growth curve, compare each group tumour growth situation.After being administered three weeks, cervical dislocation is put to death, and isolates tumour, weighs knurl weight, meter
Calculate tumour inhibiting rate (%).
Gross tumor volume：
V=a × b2×1/2
(a:Major diameter, b:Minor axis)
Relative tumour volume：
RTV=Vn/V0
(VnFor n-th day gross tumor volume measured, V0For the gross tumor volume measured before administration)
Tumour inhibiting rate=[(the average knurl weight of the average knurl weight-administration group of control group) the average knurl weight of/control group] × 100%
FSH33-53The function of tumor inhibition of-IIKK in vivo
By comparing FSH33-53The medicines such as-IIKK, IIKK, cis-platinum suppress the effect of PC3 transplantable tumors in vivo, with evaluation
FSH33-53Tumor killing effect inside-IIKK, IIKK.PC3 cells are inoculated in mice with tumor, and after 14 days, mean tumour volume reaches
80mm3, tumor formation rate 95%.Tumour increases to be calculated with Relative tumor appreciation rate RTV.Tumour growth curve such as Fig. 9, FSH33-53-
IIKK 5mg/kg, 10mg/kg, IIKK 5mg/kg, cis-platinum 3mg/kg, physiological saline, the comparison of five groups of tumor control rates.Administration
After 21 days, mice with tumor is put to death, isolates tumour, such as Figure 10.
By weighing in, toxicity of the monitoring medicine to nude mice.According to the change curve (see Figure 10) of tumor bearing nude mice body weight,
Cis-platinum group body weight is decreased obviously, and FSH33-53- IIKK 5mg/kg, 10mg/kg, IIKK 5mg/kg experimental groups body weight and physiology salt
Water group compares, no difference of science of statistics (P ＞ 0.05).I.e. polypeptide toxicity is less than cis-platinum.
The comparison of the PC3 transplanted tumor in nude mice of table 2 tumour inhibiting rate under several drugses effect
Obviously, above-described embodiment is only intended to clearly illustrate example, and is not the restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or
Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (7)
1. a kind of oncotherapy polypeptide with FSHR targetings, it is characterised in that the polypeptide has enhancing IIKK polypeptides
Antitumor activity, the oncotherapy polypeptide of the FSHR targetings is FSH33-53- IIKK targeted fusion peptide Ts yr-Thr-Arg-
Asp-Leu-Val-Tyr-Lys-Asp-Pro-Ala-Arg-Pro-Lys-Ile-Gln-Lys-Thr-Cys-Thr-Phe-Gly-
Gly-Gly-Ile-Ile-Lys-Lys-Ile-Ile-Lys-Lys-Ile-Ile-Lys-Lys-Ile-NH2。
2. a kind of recombinant fusion polypeptide, it is characterised in that contain the FSH described in claim 133-53- IIKK targeted fusion polypeptides.
3. one kind coding claim 1 or 2 FSH33-53The polynucleotides of-IIKK targeted fusion polypeptides.
4. polynucleotides according to claim 3, it is characterised in that contain the polynucleotides described in claim 3.
5. a kind of pharmaceutical composition, it is used to treat tumour, it is characterised in that includes the FSH of claim 1 or 233-53-
IIKK targeted fusion polypeptides.
6. pharmaceutical composition according to claim 5, it is characterised in that also comprising pharmaceutically acceptable carrier or described
Composition is unit dosage forms, solid dosage forms or liquid form.
7. according to the FSH described in claim 1-233-53- IIKK targeted fusions polypeptide is preparing treatment prostate cancer or oophoroma
Application in medicine.
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