CN104926945A - FSHR (follicle-stimulating hormone receptor)-targeted tumor treatment polypeptide and application thereof - Google Patents
FSHR (follicle-stimulating hormone receptor)-targeted tumor treatment polypeptide and application thereof Download PDFInfo
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Abstract
The invention provides FSHR (follicle-stimulating hormone receptor)-targeted tumor treatment polypeptide and an application thereof. Polypeptide has an FSHR-targeted tumor treatment effect, the FSHR-targeted tumor treatment polypeptide is FSH33-53-IIKK-targeted fusion polypeptide, and the polypeptide has anti-tumor activity of enhanced IIKK polypeptide. The FSH33-53-IIKK-targeted fusion polypeptide can be applied to the preparation of drugs for treating or preventing tumors.
Description
Technical field
The invention belongs to biomedical sector, relate to a kind of oncotherapy polypeptide and the application thereof with FSHR targeting.
Technical background
Follicle-stimulating hormone receptor (FSHR) is potential prostate cancer targeting mark.FSHR is a kind of membrane receptor be made up of oligosaccharide albumen, belongs to g protein coupled receptor family.FSHR is mainly present in sustenticular cell, the gonad granulocyte of humans and animals testis, by the startup of effect of signals production of sperm in male pubescence, the maintenance of Adulthood During spermatogenesis and the growth in female ovum each stage of mediation follicular stimulating hormone (FSH)." New England Journal of Medicine " (Radu A in 2010, Pichon C, CamparoP, et al.Expression of follicle-stimulating hormone receptor in tumorblood vessels.N Engl J Med.2010Oct 21; 363 (17): 1621-30.) report carries out the research such as immunohistochemical methods, in situ hybridization to the different tumor specimen of 1336 example, find FSHR high expression level in the vascular endothelial cell of the multiple cancerous tissues such as prostate cancer, mammary cancer, colorectal cancer, lung cancer, carcinoma of testis, therefore the expression of tumor vascular endothelium FSHR may contribute to tumor neovasculature formation." BMC Cancer " (Siraj A, Desestret V, Antoine M in 2013, 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 endotheliumin tumor metastases, BMC Cancer.2013; 13:246.) report carries out immunohistochemical analysis to the 6 class tumours comprising prostate cancer in the metastasis of major organs as bone, liver, lymphoglandula, brain and lung etc., find that in above-mentioned neoplasm metastasis, FSHR highly expresses, and the normal organ adjoined is without remarkable expression.Prompting, FSHR is one of target mark of defining of prostate cancer metastasis.In addition, FSHR also at human ovarian cancer Caov-3, OVCAR-3, constructive expression in the cancer cells such as human prostate PC3.Further research finds ovarian surface epithelial cell (OSE) transfection FSHR plasmid and after process LAN, the protein expressions such as EGFR, HER-2/neu and c-Myc of cell rise, ERK1/2 then continues phosphorylation, cell proliferation rate is accelerated, and these phenomenons are consistent with situation in ovarian cancer cell OVCAR-3 cell.Infer that process LAN FSHR may that the raising of the propagation of front ovarian epithelial cell and oncogenic pathways level occurs be relevant to tumour.Research also finds that FSH/FSHR is by regulating the activity of the NF-κ B in classical PI3K/Akt signal path to strengthen propagation and the invasive ability of ovarian cancer cell line 3AO.Therefore, the growth, transfer, invasion and attack etc. of FSHR and tumour are closely bound up, are an important research, therapy target.
Follicular stimulating hormone (FSH) is FSHR endogenic ligand, is made up of α and β two subunits.Research shows 33-53 fragment on people FSH β chain, and 81-95 fragment etc. all can combine with FSHR, and wherein FSH
33-53(Tyr-Thr-Arg-Asp-Leu-Val-Tyr-Lys-Asp-Pro-Ala-Arg-Pro-Lys-Ile-Gln-Lys-Thr-Cys-Thr-Phe) is the strongest with the avidity of FSHR, can be used as the combination of antagonist competition FSH and FSHR.Xu Yu equality is to FSH
33-53carry out
18f marks, and makes
18f-Al-NOTA-MAL-FSH
33-53be used successfully to the human prostata cancer tumor bearing nude mice video picture of FSHR high expression level, for the specific diagnosis of clinical prostate cancer provides the foundation.Also research is had to connect FSH by nano particle
33-53with taxol (PTX), construct FSH
33-53-NP-PTX mixture, improves target, the treatment ability of PTX to tumour cell CAOV-3.Visible, FSH
33-53can be used as the targeting vector of FSHR, be of value to the Treatment and diagnosis of FSHR high expression level tumour.
Polypeptide G (IIKK) nI-NH
2(n=1-4), Gly-(Ile-Ile-Lys-Lys) n-Ile-NH
2(n=1-4) be, that a class has simple repeated sequence and comprises the cationic antibacterial peptide of α-helixstructure.Along with the increase of IIKK sequence repetition number, by the connection with amphipathic molecule film, the alpha-helix of polypeptide IIKK can be strengthened, polypeptide structure tended towards stability.In addition, increase at polypeptide hydroxyl terminal the ability that Isoleucine (Ile) can promote to maintain secondary structure further, firm peptide molecule structure, cause and the performance of coordination function.Through comparative study, find G (IIKK)
3i-NH
2(IIKK) have best chain length, antitumous effect, structure is simple, and toxic side effect is little, and anti-tumor activity is strong, is also that desirable target tumor suppresses medicine.IIKK is combined with tumor cell membrane with hydrophobic interaction by electrostatic, thoroughly changes mode enter and accumulate in tumour cell with film, carrys out inhibition tumor cell growth eventually through modulating apoptosis path or necrocytosis.The good antitumor performance in IIKK inside and outside shows its great potential in clinical development application.The fusion polypeptide albumen having FSHR target and more powerful antitumor function concurrently is lacked in prior art.
Summary of the invention
The object of the invention is to solve in prior art the fusion polypeptide albumen lacking and have FSHR target and more powerful antitumor function concurrently.Based on the thinking of functional polypeptide combined utilization, devise FSH
33-53-IIKK and IIKK-FSH
33-53targeted fusion polypeptide, has the oncotherapy polypeptide of FSHR targeting, and described polypeptide has the anti-tumor activity strengthening IIKK polypeptide, and its aminoacid sequence is selected from following (a)-(c):
A (), its sequence are that alpha subunit and β subunit are formed, wherein alpha subunit is the affinity fragment that can combine with FSHR, and β subunit is the cationic antibacterial peptide having simple repeated sequence and comprise α-helixstructure,
Described affinity fragment comprises 33-53 fragment or 81-95 fragment on FSH β chain,
Described cationic antibacterial peptide comprises G (IIKK) nI-NH
2or Gly-(Ile-Ile-Lys-Lys) n-Ile-NH
2, wherein n=1-4;
B the oncotherapy polypeptide of (), described FSHR targeting is FSH
33-53-IIKK targeted fusion polypeptide
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-NH
2, 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 (), described sequence, in the aminoacid sequence shown in (a), (b), replace, lack or add 1-10 amino acid and obtain, and have the aminoacid sequence of the anti-tumor activity strengthening IIKK polypeptide.
Described recombinant fusion polypeptide also comprises, containing FSH
33-53-IIKK targeted fusion polypeptide or its not fragment of loss of biological activity, variant or derivative.
The present invention is also: provide the described FSH of a kind of coding power
33-53the polynucleotide of-IIKK targeted fusion polypeptide.
Described polynucleotide also comprise its not fragment of loss of biological activity, variant or derivative.
The present invention is also: provide a kind of pharmaceutical composition, and it is used for the treatment of or prophylaxis of tumours, comprises described FSH
33-53-IIKK targeted fusion polypeptide.Described pharmaceutical composition, also comprises pharmaceutically acceptable carrier or described composition is lyophilized form, unit dosage, solid dosage, liquid form.
The present invention is also: the method providing the expression grown by modulating apoptosis path or necrocytosis inhibition tumor cell, described method comprises the steps:
A) nucleotide sequence defined above, polypeptide or composition is provided,
B) described nucleotide sequence, polypeptide or composition are applied to or are applied to the expression system of wild tumour cell, cell, tissue or organism.
The present invention is also: provide described FSH
33-53the application in preparation treatment or prophylaxis of tumours medicine of-IIKK targeted fusion polypeptide, polynucleotide.
By described FSH
33-53-IIKK targeted fusion polypeptide, described polynucleotide are treated in preparation or are prevented the application in genital system tumor, incidence cancer and lung cancer, malignant lymphatic tumor medicine.
By described FSH
33-53the application in preparation treatment or prevention prostate cancer medicine of-IIKK targeted fusion polypeptide, described polynucleotide.
By building polypeptide FSH
33-53mediation FSHR targeting diagnosis and treatment system and merge tumor protein p53 IIKK, expecting can by FSH
33-53-IIKK is applied to the treatment of FSHR high expression level tumour, improves current treatment status.
Check the expression of FSHR in several cell by Western Blot, wherein, L929 cell FSHR feminine gender is expressed.Mtt assay can the detection of drugs effect of breeding Carbazole alkaloid.The method is adopted to detect FSH
33-53-IIKK to the restraining effect of PC3, Hela, SKOV3, MCF7, L929,293 cells, and to compare with the inhibition rate of tumor cell of IIKK, cis-platinum under different concns.Result shows, FSH
33-53-IIKK and IIKK have stronger restraining effect to tumour cell, and both are to the IC of tumour cell
50close, and in pronounced amount effect relationship.And the restraining effect of two peptide species normal tissue cells obviously weakens.By contrast, the restraining effect of cis-platinum to tumour cell reduces relatively, particularly at the inhibiting rate of PC3 cell, only reaches (48.13 ± 3.89) %, with FSH under 50 μMs of concentration
33-53cell inhibitory rate (96.94 ± 3.45) % of-IIKK compares significant difference (P > 0.05).And the polypeptide of same concentrations and cisplatin effect are in normal tissue cell, the toxic action of cis-platinum to cell obviously strengthens.
Tumorigenic principal character is Growth of Cells and proliferation out of control.The exception of cell in propagation, differentiation, apoptosis etc. all take part in generation and the development of tumour.The research of common Antitumor Mechanism has: the impact of medicine cell cycle; The apoptotic inducing action of drug on tumor; The effect of drug-induced cell autophagy; Medicine is on the impact (comprise and cause DNA damage, medicine to the impact of nucleus metabolism with the detection of DNA binding ability, medicine) etc. of cell DNA.Bibliographical information IIKK can by changing cytoskeletal structure, reducing mitochondrial membrane potential, promote the release of cytochrome C and activating Fas path cell death inducing.Change three aspects of the impact that the present invention distributes from polypeptide cell cycle, the transduction of antiapoptotic signals and autophagosome labelled protein LC3 are to fusogenic peptide FSH
33-53the antitumor mechanism of-IIKK carries out Primary Study.
The impact of the adjustment of polypeptide cell cycle and apoptosis is analyzed by flow cytomery.Result shows, after different concns polypeptide acts on PC3 cell 24h, and G0/G1, S, G2/M period profile difference remarkable (P > 0.05) of cell, i.e. FSH
33-53the cycle of-IIKK to PC3 cell has no significant effect.Hoechst 33258 staining is utilized to investigate FSH
33-53-IIKK, on the apoptotic impact of PC3, demonstrates polypeptide from morphology and can induce PC3 apoptosis.FSH
33-53-IIKK plays tumor suppression function by cell death inducing.Apoptosis is by the autonomous dead process of the cell of gene regulating, is a kind of phenomenon from bulk damage.The generation of cancer and the generation of resistance and cell occur that Apoptosis inhibitor is closely related.The FSH of 7.5 μMs, 15 μMs, 30 μMs in Hoechst 33258 staining
33-53-IIKK can induce PC3 cell to occur typical apoptosis feature, and as cell volume reduces, caryoplasm concentrates, and presents irregular karyorhexis etc., and in significant concn dependency.The apoptosis rate of flow cytomery fusogenic peptide and polypeptide IIKK cell death inducing is used under same concentrations gradient.FSH
33-53-IIKK acts on the apoptosis rate of PC3 cell higher than the apoptosis rate under IIKK respective concentration, and the FSH of 30 μMs of concentration
33-53the apoptosis rate of-IIKK to PC3 cell is significantly higher than IIKK (P < 0.05).More than prove, FSH
33-53-IIKK can induce PC3 apoptosis, and the trend of its cell death inducing is better than IIKK.
Apoptotic generation and development relate to the activation of series of genes, expression and regulation and control.Its main path can be divided into: death receptor mediated pathways, endoplasmic reticulum mediated pathways, plastosome mediated pathways.Endogenic mitochondrial apoptotic pathway is the stimulation that cell is subject to inside or outside, and mitochondrial outer membrane permeability changes, and to the antiapoptotic factors that cytoplasmic matrix release is relevant, causes the generation of apoptosis.
Play important regulating and controlling effect in the apoptotic pathways that Bcl-2 family protein participates at plastosome, to promote or the release of interference cell pigment C carrys out the apoptosis of regulating cell.Bcl-2 homologous protein known at present probably has more than 20 to plant, and can be divided into two large classes: pro apoptotic protein Bax, Bad and Bak etc. according to the difference of its structure and function; Inhibitor of apoptosis protein Bcl-2, Mcl-1 and Bcl-xl etc.The release of Bcl-2 interference cell pigment C, blocking the activation of upstream Caspase albumen, there is not apoptosis in Cell protection; And Bax/Bad promotes cytochrome C release, cell death inducing.
Cysteine aspartase (cysteine aspartic specific protease, Caspase) family is one group of cysteine proteinase enzyme being present in the aspartic acid specific in endochylema, selectivity can cut some protein, activating target protein or make it inactivation, is initiator and the executive of mammalian apoptosis.In apoptosis process, multiple Caspase is had jointly to participate in.Under normal circumstances, Caspase is with the synthesis of the precursor forms of non-activity and storage, and apoptotic signal can activate Caspase cascade reaction.Wherein, Caspase-8, Caspase-9 are positioned at connection order reaction upstream, are main apoptosis initiators; Caspase-3 etc. are positioned at the downstream of connection order reaction, are the executives of apoptosis.In the apoptosis of Fas or TNF induction, Caspase-8 is apoptosis " promoter ".After Caspase-8 receives apoptotic signal, activated by the form of self cleavage, the effector molecule Caspase-3 etc. in direct or indirect activation downstream, and the Caspase-3 activated etc. decompose Lamins, PARP (poly ADP-ribose polymeras) etc., activate endonuclease, cause DNA fragmentation.The activation of Caspase-3 is the key of apoptosis downstream signal conduction.
Western Blot method is utilized to have detected FSH
33-53after-IIKK acts on PC3 cell 24h, the expression level of apoptosis-related protein Procaspase-8, Procaspase-3, Bcl-2, Bad, Bax.Result shows, Procaspase-8, Procaspase-3, Bcl-2 protein expression reduces, and Bad protein expression is constant, and Bax/Bcl-2 increases.FSH is described
33-53after-IIKK acts on cell, reduced the expression of Bcl-2 by mitochondrial apoptosis path, the release of T suppression cell pigment C, and then the Caspase cascade reaction activated in PC3 cell, activate Caspase-8 and Caspase-3, cause apoptosis, consistent with IIKK cell death inducing mechanism.
Cell autophagy (Autophagy) is the one reaction of cell reply external environment change, plays an important role to cell self maintenance metabolism balance and cell homeostasis.Cell autophagy can provide abundant nutrition for tumour on the one hand, promotes tumor growth; On the other hand, cell autophagy can to the canceration of anti-cell.Therefore, in the process of tumor development, the effect of cell autophagy has dual character.Most researchers is thought, cell autophagy is played an important role equally in the process to antitumor generation.
AO staining is adopted to detect cell autophagy from morphocytology.PC3 cell is through the FSH of 0,7.5,15,30 μM of concentration
33-53after-IIKK process, can be observed under fluorescent microscope, along with the increase of drug level, occur the cytosis of autophagosome structure, and Fluorescence Increasing.Show polypeptide FSH
33-53-IIKK also can inducing cell generation autophagy.
The process of cell generation autophagy has been mediated by a series of autophagy associated protein (Atg albumen).The transformation of microtubule-associated proteins 1 light chain 3 (Microtubule-associated protein 1 light chain 3, LC3) is the mark that autophagy occurs.LC3 albumen can be become LC3-I by the cutting of Atg4 proteolytic enzyme, and after autophagosome is formed, LC3-I and phosphatidylethanolamine (phosphatidylethanolamine, PE) coupling form LC3-II.To be retained on autophagosome film LC3-II Absorbable organic halogens until and lysosome fusion, therefore can be used as the mark of autophagosome.
Western Blot is utilized to detect at FSH
33-53in the lower PC3 cell of-IIKK effect, the change of LC3-I and LC3-II protein expression level, reacts the quantity of autophagosome with this.Experimental result shows, PC3 cell is after drug treating, and along with the increase of drug level, the expression of LC3-I reduces, and LC3-II protein expression level presents increase trend under lower concentration medicine, reduces again under high concentration medicine effect.FSH is described
33-53-IIKK can inducing cell generation autophagy to a certain degree within the scope of proper concn, and by other approach inhibition tumor cells propagation under the effect of high density polypeptide.
Consider the external biochemical environment being difficult to complexity in analogue body, the effect of polypeptide on solid tumor is subject to the impact of multiple factors, as in vivo easily by multiple enzyme liberating, the sour environment that inside tumor blood supply insufficiency causes affects the performance etc. of polypeptide, so need to set up anti-tumor in vivo model to investigate the target of polypeptide at solid tumor and the performance of therapeutic action.Adopt subcutaneous cell transfer methods to set up PC3 tumor model, tumor formation rate reaches 95%, and in nude mouse, upgrowth situation is good.
At checking FSH
33-53in-IIKK body in function of tumor inhibition experiment, devise physiological saline negative control group, IIKK (5mg/Kg) experimental group, FSH
33-53-IIKK (5mg/Kg, 10mg/Kg) experimental group, cis-platinum (3mg/Kg) positive controls, by comparing FSH
33-53the medicine such as-IIKK, cis-platinum is on the inhibiting rate of PC3 tumour and the performance impact of nude mice body weight change being investigated to polypeptide Tumor suppression propagation in vivo.Cis-platinum (DDP) is heavy metal complex, belongs to the CCNS antitumor drug of wide spectrum.DDP can inhibition tumor cell DNA replication dna process, destroy membrane structure, stop tumor cell proliferation, be the chemicals that current clinical treatment malignant tumour is commonly used, be used for the treatment of genital system tumor, incidence cancer and the Several Kinds of Malignancy such as lung cancer, malignant lymphoma.But DDP is while killing tumor cell, also can produces larger toxic side effect, and produce resistance.And prostate cancer cell is poor to cisplatin sensitivity, limit its application in prostate cancer therapy.By consulting literatures is learnt, the maximum tolerated dose of cis-platinum is 9mg/kg, every 6 days single administrations.With cis-platinum 3mg/kg, 3 administrations are as positive controls and FSH weekly
33-53the tumor-inhibiting action of-IIKK compares.Experimental result shows, the FSH of same concentrations
33-53-IIKK shows the average tumor inhibiting rate higher than IIKK, FSH
33-53the average tumor inhibiting rate of-IIKK 10mg/kg experimental group and cis-platinum group is close, and FSH
33-53-IIKK significantly lower than cis-platinum group, illustrates that the toxic side effect of polypeptide is little on the impact of tumor mouse body weight.
The beneficial effect of the invention:
FSH
33-53-IIKK and IIKK have stronger restraining effect to tumour cell, and both are to the IC of tumour cell
50close, and in pronounced amount effect relationship; FSH
33-53-IIKK acts on the apoptosis rate of PC3 cell higher than the apoptosis rate under IIKK respective concentration, FSH
33-53-IIKK can induce PC3 apoptosis, and the trend of its cell death inducing is better than IIKK; FSH
33-53after-IIKK acts on cell, reduced the expression of Bcl-2 by mitochondrial apoptosis path, the release of T suppression cell pigment C, and then activate the Caspase cascade reaction in PC3 cell, activated Caspase-8 and Caspase-3, caused apoptosis; FSH
33-53-IIKK shows the average tumor inhibiting rate higher than IIKK, 10mg/kgFSH
33-53-IIKK and cis-platinum show close tumor inhibition effect, but FSH
33-53the impact of-IIKK on tumor mouse body weight is starkly lower than cis-platinum group, illustrates that the toxic side effect of polypeptide is little.
Accompanying drawing illustrates:
Below with reference to accompanying drawing and specific examples, the present invention will be further elaborated.
Fig. 1, Western blot detect FSHR MCF7, Hela, PC3,293, expression in SKOV3, L929 cell.
Fig. 2, FSH33-53-IIKK, IIKK and cis-platinum are to the inhibited proliferation of cell.
Fig. 3, the lower PC3 cell cycle fitted figure of different concns FSH33-53-IIKK effect and statistics.
A:0 μM; B:7.5 μM; C:15 μM; D:30 μM; E: cell cycle statistics (P > 0.05)
Fig. 4, FSH33-53-IIKK act on PC3 cell 24h, and Hoechst 33258 dyes and observes cell death inducing effect.
A:0μM；B:7.5μM FSH
33-53-IIKK；C:15μM FSH
33-53-IIKK；D:30μMFSH
33-53-IIKK
The two dye method of Fig. 5, Annexin V-FITC/PI detects FSH33-53-IIKK and IIKK to the apoptosis-induced effect of PC3 cell.
A:0μM；B:7.5μM FSH
33-53-IIKK；C:15μM FSH
33-53-IIKK；D:30μMFSH
33-53-IIKK
E:7.5μM IIKK；F:15μM IIKK；G:30μM IIKK
The impact that Fig. 6, FSH33-53-IIKK express PC3 cell death related protein.
Fig. 7, FSH33-53-IIKK act on PC3 cell 24h, and Induces Autophagy effect is observed in AO dyeing.
A:0μM；B:7.5μM FSH
33-53-IIKK；C:15μM FSH
33-53-IIKK；D:30μMFSH
33-53-IIKK
Fig. 8, FSH33-53-IIKK are on the impact of PC3 cell LC3-Ι, LC3-Π protein expression.
Fig. 9, FSH33-53-IIKK are on the impact of PC3 Nude Mouse Model tumor growth curve.
Figure 10, administration are after 21 days, and each experimental group mice with tumor body weight change and tumor volume vs scheme.
Embodiment:
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
Embodiment 1
Western Blot detects the expression of FSHR in several cell
Human carcinoma of prostate cell line PC3, human cervical carcinoma cell lines Hela, human oophoroma cell line SKOV3, Breast cancer lines MCF7, HEKC 293, l cell L929, purchased from Chinese Academy of Sciences's Shanghai cell bank.
Male BALB/c-nu nude mice, body weight 18 ~ 19g, this laboratory animal company limited of Cavan, changzhou, SPF level, 5 weeks ages of mouse.Conformity certification number: NO.0026328, credit number: SCXK (Soviet Union) 2011-0003.
Test PC3 used, MCF7, Hela, PC3,293, the cell culture processes such as SKOV3, L929 is consistent, at 37 DEG C, 5%CO
2constant incubator in subculture, by the DMEM culture medium culturing containing 10% foetal calf serum and 100U/ml penicillin, 100U/ml Streptomycin sulphate.
Collecting cell, with the RIPA reagent containing proteinase inhibitor, conventionally extracts cell protein and measures protein concentration by BCA analysis of protein test kit.After protein quantification, get equal protein, add sample-loading buffer, sample 70 DEG C of sex change 10min, SDS-Poly n alkylacrylates (SDS-PAGE), every hole loading 40-50 μ g, rear transferring film is on pvdf membrane.Close 1 hour with shaking table under 5% skim-milk room temperature, add primary antibodie (FSHR:Rabbit-anti-FSHR, 1:1000; Actin:mouse-anti-Actin, m, h, 1:1000), sealing, 4 DEG C of shaking tables sway and spend the night.TBST washes film 3 times, each 10min.Add two anti-(Anti-Rabbit-HRP, Anti-mouse-HRP, 1:1000, two anti-diluted), encapsulation, is placed in incubated at room 1h on shaking table.Again wash film as stated above, ECL exposure imaging.
The expression of FSHR in various kinds of cell
FSHR protein expression situation in cell is detected with Western Blot.As Fig. 1, Hela, MCF7, PC3 cell strain have high-caliber FSHR to express, L929 cell is expressed without FSHR.
Embodiment 2
Mtt assay measures polypeptide FSH
33-53-IIKK is on the impact of various kinds of cell survival rate
Cell logarithmic phase grown is according to 5 × 10
3the density kind that individual/hole connects is in 96 orifice plates.Overnight incubation, after cell attachment, every hole adds respectively containing different concns FSH
33-53the substratum 100 μ l of-IIKK, IIKK and cis-platinum (concentration gradient is 0,1,2,5,10,15,20,40, and 50 μMs), each concentration establishes 3 multiple holes.After hatching 24h, every hole adds the MTT solution that 40 μ l concentration are 2mg/ml, continues to cultivate, and abandons supernatant after 4h; Every hole adds the DMSO of 150 μ l, and 10min is fully dissolving crystallized in concussion.Microplate reader working sample is in the OD value of 570nm wavelength.Calculate survival rate, draw survival curve, and obtain IC
50.Survival rate=(experimental port OD value/blank group OD value) × 100%.
FSH
33-53the vitro cytotoxicity of-IIKK detects
Mtt assay is adopted to measure polypeptide FSH
33-53-IIKK, IIKK and cis-platinum are to the cytotoxicity in PC3, Hela, SKOV3, MCF7 and 293, L929 cell.As Fig. 2, FSH
33-53-IIKK, IIKK and cis-platinum all have obvious restraining effect to the propagation of tumour cell, and in dose-dependently.Polypeptide FSH
33-53-IIKK, IIKK compare with cis-platinum, and the above two show stronger restraining effect to tumour cell, and two peptide species administration group difference not statistically significants.Polypeptide FSH
33-53the toxicity of-IIKK, IIKK normal tissue cell is all lower than cis-platinum.Relatively FSH
33-53-IIKK, IIKK and cis-platinum are to the IC of several cell
50, result is as table 1.
Table 1 medicine is to the IC of cell strain
50contrast
*p<0.05vs FSH
33-53-IIKK，**p<0.01vs FSH
33-53-IIKK，***p<0.001vs FSH
33-53-IIKK
p-values were determined by Student’s t-test between the IC
50values of both cell lines
Embodiment 3
FSH
33-53-IIKK is on the impact of PC3 cell cycle
Cell cycle phalangeal cell once divides the active procedure terminated to division next time terminates in the past, is divided into interkinesis and two stages of division stage.Wherein, the interkinesis is divided into again G1 phase (pre-synthesis phase of DNA), S phase (DNA synthesis phase), G2 phase (DNA post-synthesis phase).The M phase is m period.The G0 phase is the dormant cells phase.Propidium iodide (PI) is a kind of nuclei dyeing color reagent that can dye to DNA and RNA, can discharge red fluorescence after the intercalation of DNA or RNA.Although PI is not by membrane, can dye to nucleus through damaged cytolemma.Ice ethanol can increase permeability of cell membrane.Use RNase cleaving rna, PI is entered in cell and is combined with DNA selective, the fluorescence volume of intracellular dye is directly proportional to endonuclear DNA content.The DNA relative content of cell can be obtained by flow cytomery individual cells fluorescence intensity, then according to the difference of each phase DNA content, cell is divided into the different cycles.
By the PC3 cell of logarithmic phase, with 2 × 10
5individual/hole is inoculated in 6 orifice plates, and every hole adds the nutrient solution of 1.5ml.Overnight incubation, after cell attachment, adds the FSH that final concentration is 0 μM, 7.5 μMs, 15 μMs, 30 μMs respectively
33-53-IIKK.After drug effect 24h, collect nutrient solution and digest attached cell, washing culture dish 3 times with PBS, collecting PBS, merging cell, the centrifugal 4min of 1500r/min; Cell is resuspended, and join in 70% ethanol of 4 DEG C of precoolings fixing, sealed membrane seals, and 4 DEG C are spent the night.The centrifugal 4min of 2000r/min, sucks stationary liquid, and PBS washes 2 times.Cell is transferred in 1ml centrifuge tube, add the working solution containing PI (50 μ g/ml), RNaseA (50 μ g/ml) and Triton (1%), room temperature lucifuge hatches 30min, with the flow cytomery cell cycle, and Entropy density deviation during the analysis of cells cycle.
FSH
33-53-IIKK is on the impact of PC3 cell cycle
By the FSH of PC3 cell through different concns
33-53after-IIKK process 24 as a child, the impact of detection of drugs cell cycle.Result shows, the FSH of 7.5,15,30 μMs
33-53after-IIKK acts on cell, compared with cellular control unit period profile, no significant difference (Fig. 3).That is, FSH
33-53-IIKK is on the period profile of cell without impact, and the effect of polypeptide Tumor suppression propagation is not realize by changing cell cycle distribution.
Embodiment 4
Hoechst 33258 staining examine apoptosis
Hoechst 33258 is that one can permeates cell membranes, the blue-fluorescence dye liquor be combined with nucleus.Although Hoechst can be combined with all nucleic acid, be more prone to and the DNA chain combination of being rich in A/T (VITAMIN B4/thymus pyrimidine), at the ultraviolet excitation of about 350nm, fluorescence intensity significantly strengthens.Therefore, after Hoechst 33258 dyes, with the nuclear metamorphosis of fluorescent microscope observable.
By the PC3 cell of logarithmic phase with 5 × 10
5individual/hole is inoculated in 6 orifice plates, adds nutrient solution to 2ml, and cross rocks six orifice plates, and cell is evenly scattered.Six orifice plates are placed in incubator, overnight incubation.After cell attachment, adding final concentration is respectively 0 μM, 7.5 μMs, 15 μMs, 30 μMs FSH
33-53-IIKK.After effect 24h, absorb old nutrient solution, then carefully swing with PBS and wash 2 times; It is 10 μ g/ml Hoechst 33258 dye liquor (containing 0.2% triton x-100) 1ml that every hole adds final concentration, puts into incubator and continues to hatch 30min.Abandon dye liquor, wash 2 times with PBS, attention action wants light, is not blown off by cell; At fluorescence microscopy Microscopic observation, and Taking Pictures recording karyomorphism.
Hoechst 33258 staining observes FSH
33-53-IIKK is on the apoptotic impact of PC3
Hoechst33258 fluorescence colour is adopted to detect FSH
33-53-IIKK is nuclear metamorphosis after acting on PC3 cell.Cell is respectively through the FSH of 0,7.5,15,30 μM of concentration
33-53after-IIKK processes cell 24h, Hoechst dyes, fluorescence microscopy Microscopic observation.As Fig. 4, the nucleus major part of blank group cell presents homogeneous, full circle or ellipse.Because cell self exists the process of programmed death, only a few cell is had to present hyperfluorescenceZeng Yongminggaoyingguang dyeing and heterocyst core.Along with the increase of drug level, occur that obvious cell volume reduces, the shrinkage of nuclei dyeing chromaticness, formation lune or hat shape invest nuclear membrane, fluorescence becomes fine and close, and assemble with obvious particular fluorescent body, present irregular karyorhexis phenomenon (arrow is depicted as the typical apoptosis form that pyknosis, nuclear fragmentation occur).When concentration reaches 30 μMs, cell attachment ability declines, is cracked into fragment.
Embodiment 5
Flow cytomery FSH
33-53-IIKK is to the apoptosis-induced effect of PC3 cell
AnnexinV-FITC/PI two dye flow cytometer detection method detects apoptotic ordinary method.In early days apoptotic, the phosphatidylserine of distribution of specific inside the lipid bilayer of cytolemma (PS) can be exposed to cell surface along with turning up of cytolemma.AnnexinV and PS is highly affine, after fluorescein FITC marks, can detect early stage apoptotic generation by fluorescence.And at apoptotic middle and advanced stage, the destroy integrity of cytolemma, PI can permeate through cell membranes be combined with nucleus.Two kinds of fluorescence dye conbined usage, namely available flow cytometer distinguishes the different states of cell.
By the PC3 cell of logarithmic phase with 5 × 10
5individual/hole is inoculated in 6 orifice plates, adds nutrient solution to 2ml, and cross rocks six orifice plates, and cell is evenly scattered.Six orifice plates are placed in incubator, overnight incubation.After cell attachment, add the FSH that final concentration is 0 μM, 7.5 μMs, 15 μMs, 30 μMs respectively
33-53-IIKK, IIKK peptide.After effect 24h, collecting cell (comprising dead cell floating in nutrient solution), then carefully swings with PBS and washes culture dish 2 times, and collect PBS, by centrifugal for cell suspension 1500r/min 4min, supernatant discarded, re-suspended cell also goes in 1.5ml centrifuge tube.PBS washes 2 times, the centrifugal 4min of 2000r/min, supernatant discarded.Each sample adds the binding buffer containing Annexin V-FITC and PI that 100 μ l prepare in advance, and (every 1ml binding buffer adds each 20 μ l of AnnexinV-FITC and PI, concussion, staining fluid is mixed), 15-25 DEG C of lucifuge hatches 10-15min.Before detecting sample, often pipe sample adds appropriate binding buffer dilution (1000 cell/j diluent) again.Flow cytomery apoptosis situation, and analytical results.
FSH
33-53-IIKK and IIKK comparing PC3 cell induction apoptotic effect
Flow cytometry FSH
33-53-IIKK and IIKK are to the effect of PC3 cell induction apoptosis.As shown in Figure 5: 7.5, the FSH of 15,30 μMs of concentration
33-53after-IIKK acts on 24h, PC3 apoptosis rate is followed successively by: (9.1 ± 1.35) %, (18.5 ± 1.50) %, (51.1 ± 2.00) %.Under the IIKK effect of corresponding concentration, apoptosis rate is respectively (7.3 ± 1.08) %, (16.9 ± 0.81) %, (41.2 ± 2.61) %.The increase of two groups of apoptosis rates all has significant difference (P < 0.01).Under the effect of 15,30 μMs of concentration, FSH
33-53the apoptosis rate of-IIKK experimental group increases (P < 0.05) to some extent compared with the apoptosis rate of IIKK control group.FSH is described
33-53-IIKK induces PC3 apoptosis to be dose-dependently, and acts on comparatively IIKK enhancing.
Embodiment 6
Western blot detects the expression of the albumen such as Procaspase-3, Procaspase-8, Bcl-2, Bax
By the PC3 cell of logarithmic phase with 2 × 10
5individual/hole is inoculated in culture dish, when Growth of Cells is to 80-90%, adds the FSH of 0 μM, 7.5 μMs, 15 μMs, 30 μMs
33-53-IIKK, process cell 24h.Abandon nutrient solution PBS to swing and wash 3 times, join in culture dish by 200 μ l containing the RIPA of proteinase inhibitor, cold hatching 5 minutes, scraper plate collecting cell albumen also carries out protein quantification.Get equal protein, add sample-loading buffer, by protein sample after 70 DEG C of sex change 10min, SDS-Poly n alkylacrylates (SDS-PAGE), every hole applied sample amount is 40-50 μ g, carries out transferring film operation after electrophoresis terminates.Close 1h with shaking table under 5% skim-milk room temperature, add primary antibodie (Bcl-2, Bax, Procaspase-3, Procaspase-8, Actin, 1:1000), 4 DEG C of shaking table overnight incubation.TBST washes film 3 times, each 10min, and add 1:1000 dilution horseradish peroxidase-labeled two resist, and room temperature shaker hatches one hour.Again wash film as stated above, ECL exposure imaging.Do internal reference with Actin and gray analysis is carried out to each protein band, and be that ordinate zou makes histogram with ratio.
FSH
33-53-IIKK is on the impact of PC3 cell death related protein
Western blot detects different concns FSH respectively
33-53the expression change of the lower PC3 cell death related protein of-IIKK effect.After drug effect 24h, the change of Procaspase-8, Procaspase-3, Bcl-2, Bad, Bax protein expression is as Fig. 6.Along with the increase of drug level, Procaspase-8, Procaspase-3, Bcl-2, Bax protein expression all reduces, and has concentration dependent.Bad protein expression is without considerable change.Although Bax protein expression is also on a declining curve, Bax/Bcl-2 is still in rising trend.Result shows, FSH
33-53-IIKK can activate the Caspase cascade reaction of PC3 cell by mitochondria pathway, and final cell death inducing.
Embodiment 7
AO staining examine cell autophagy
Acridine orange (acridine orange, AO) is a kind of fluorescence dye liquor of permeable cell, DNA and kytoplasm can be dyed bright green.When PH reduces, AO can send red fluorescence, and fluorescence intensity and acid degree positive correlation.Autophagy lysosome structure is formed when cell generation autophagy.Autophagy lysosome, in acid, can utilize AO to mark lysosome and detect cell autophagy.
By the PC3 cell of logarithmic phase with 5 × 10
5individual/hole is inoculated in 6 orifice plates, adds nutrient solution to 2ml, and cross rocks six orifice plates, and cell is evenly scattered.Six orifice plates are placed in incubator, overnight incubation.After cell attachment, add the FSH that final concentration is 0 μM, 7.5 μMs, 15 μMs, 30 μMs respectively
33-53-IIKK.After effect 24h, absorb old nutrient solution, then carefully swing with PBS and wash 2 times; It is 10 μ g/ml AO dye liquor 1ml that every hole adds final concentration, puts into 4 DEG C of refrigerators and continues to hatch 30min.Abandon dye liquor, wash 1 time with PBS, attention action wants light, is not blown off by cell, at fluorescence microscopy Microscopic observation, and Taking Pictures recording cell fluorescence situation.
AO staining examine cell autophagy
AO staining is adopted to detect FSH
33-53the change that after-IIKK acts on PC3 cell, cell autophagy lysosome occurs.Cell is respectively through the FSH of 0,7.5,15,30 μM of concentration
33-53after-IIKK processes cell 24h, AO dyes, fluorescence microscopy Microscopic observation.As Fig. 7, nucleus and the kytoplasm of blank group cell present bright green.The FSH of 7.5 μMs of concentration
33-53after-IIKK administration process, can be observed in part cell, to be distributed in cytoplasmic acid vesica and present orange.Along with the increase of drug level, occur that the cell quantity of autophagosome structure increases, and fluorescence intensity strengthens.When concentration reaches 30 μMs, cell attachment ability declines, and is distributed with shiny red fluorescence in the tenuigenin of nearly all attached cell.
Embodiment 8
Western Blot detects the expression of autophagy proteins
The autophagy process of cell has been mediated by a series of autophagy associated protein (Atg albumen).Wherein, microtubule-associated protein 1 light chain 3 (microtubule-associatedprotein 1 light chain 3, LC3/Atg8) is the labelled protein on autophagosome film.The LC3 albumen of LC3-I and LC3-II two kinds of forms is there is in cell.After autophagosome is formed, LC3-I can form LC3-II with phosphatidylethanolamine (phosphatidylethanolamine, PE) coupling, is positioned autophagosome inner membrance and adventitia.By measuring, comparing the level of LC3-I and LC3-II, the quantity of autophagosome can be reflected to a certain extent.
The step of the collection of the cultivation of cell, agent-feeding treatment, protein sample, concentration determination and gel electrophoresis, as front.After 1h closed by shaking table under 5% skim-milk room temperature, add primary antibodie (LC3,1:1000), 4 DEG C of shaking table overnight incubation.TBST washes film 3 times, each 10min, and add 1:1000 dilution horseradish peroxidase-labeled two resist, and room temperature shaker hatches 1h.Again wash film as stated above, ECL exposure imaging.Do internal reference with Actin and gray analysis is carried out to each protein band, and be that ordinate zou makes histogram with ratio.
Cell autophagy Protein Detection
Western Blot detects different concns FSH
33-53the expression change of the lower PC3 cell autophagy associated protein of-IIKK effect.After drug effect 24h, the change of LC3-Ι, LC3-Π protein expression is as Fig. 8.Along with the increase of drug level, downtrending is cashed out in the expression of LC3-Ι albumen, and LC3-Π albumen presents increase trend under 7.5-15 μM of drug level effect, and expresses minimizing under 30 μMs of concentration.Result shows, FSH
33-53-IIKK, can inducing tumor cell generation autophagy under lower concentration environment, and is increased by apoptosis or other mode T suppression cell when high density.
Embodiment 9
FSH
33-53-IIKK is to the inhibiting qualification of PC3 transplanted tumor in nude mice
The foundation of PC3 Nude Mouse Model and to the inhibiting qualification of PC3 transplanted tumor in nude mice.Raise nude mice by SPF level animal routine, all tap water, cage tool are all through Aseptic sterilisation process.Collect PC3 cell under aseptic condition, be diluted to 5 × 10 by stroke-physiological saline solution
7individual/ml, adopts subcutaneous vaccination method, and inoculation PC3 cell armpit on the right side of nude mice is subcutaneous, every only inoculation 1 × 10
7individual cell, treats that it becomes knurl.When tumour body reaches 50 ~ 100mm
3time, PC3 transplanted tumor nude mice is divided into five groups, often organizes 5, often organize mean tumour volume more consistent.Establish FSH respectively
33-53-IIKK 5mg/kg and 10mg/kg administration group, IIKK 5mg/kg administration group, cis-platinum 3mg/kg control group and saline control group.Adopt the administration of tail vein injection mode, Per-Hop behavior 3 times, weighs in before administration, measures knurl footpath.Calculate each group of relative tumour volume (the gross tumor volume stdn by difference group), draw the growth curve of knurl body, respectively organize tumor growth situation.Administration is after three weeks, and cervical dislocation is put to death, and isolates tumour, weighs knurl weight, calculates tumour inhibiting rate (%).
Gross tumor volume:
V＝a×b
2×1/2
(a: major diameter, b: minor axis)
Relative tumour volume:
RTV＝V
n/V
0
(V
nbe the gross tumor volume recorded for n-th day, V
0gross tumor volume for recording before administration)
Tumour inhibiting rate=[(control group average knurl weight-administration group average knurl weight) the average knurl weight of/control group] × 100%
FSH
33-53-IIKK function of tumor inhibition in vivo
By comparing FSH
33-53the medicines such as-IIKK, IIKK, cis-platinum suppress the effect of PC3 transplanted tumor in vivo, to evaluate FSH
33-53tumor killing effect in the body of-IIKK, IIKK.PC3 cell is inoculated in mice with tumor, and after 14 days, mean tumour volume reaches 80mm
3, tumor formation rate 95%.Tumor propagation calculates with Relative tumor appreciation rate RTV.Tumor propagation curve is as Fig. 9, FSH
33-53-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, is put to death mice with tumor, is isolated tumour, as Figure 10.
By weighing in, monitoring medicine is to the toxicity of nude mice.According to the change curve (see Figure 10) of tumor bearing nude mice body weight, cis-platinum group body weight obviously declines, and FSH
33-53-IIKK 5mg/kg, 10mg/kg, IIKK 5mg/kg experimental group body weight compares with physiological saline group, no difference of science of statistics (P > 0.05).Namely polypeptide toxicity is less than cis-platinum.
The comparison of table 2 PC3 transplanted tumor in nude mice tumour inhibiting rate under several drugs effect
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (10)
1. have an oncotherapy polypeptide for FSHR targeting, it is characterized in that, described polypeptide has the anti-tumor activity strengthening IIKK polypeptide, and its aminoacid sequence is selected from following (a)-(c):
A (), its sequence are that alpha subunit and β subunit are formed, wherein alpha subunit is the affinity fragment that can combine with FSHR, and β subunit is the cationic antibacterial peptide having simple repeated sequence and comprise α-helixstructure,
Described affinity fragment comprises 33-53 fragment or 81-95 fragment on FSH β chain,
Described cationic antibacterial peptide comprises G (IIKK) nI-NH
2or Gly-(Ile-Ile-Lys-Lys) n-Ile-NH
2, wherein n=1-4;
B the oncotherapy polypeptide of (), described FSHR targeting is FSH
33-53-IIKK targeted fusion peptide T yr-Thr-Arg-Asp-Leu-Val-Tyr-Lys-Asp-Pro-Ala-Arg-Pro-Lys-I le-Gln-Lys-Thr-Cys-Thr-Phe-Gly-Gly-Gly-Ile-Ile-Lys-Lys-I le-Ile-Lys-Lys-Ile-Ile-Lys-Lys-Ile-NH
2; Or IIKK-FSH
33-53targeted fusion polypeptide 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 (), described sequence, in the aminoacid sequence shown in (a), (b), replace, lack or add 1-10 amino acid and obtain, and have the aminoacid sequence of the anti-tumor activity strengthening IIKK polypeptide.
2. a recombinant fusion polypeptide, is characterized in that, containing FSH according to claim 1
33-53-IIKK targeted fusion polypeptide or its not fragment of loss of biological activity, variant or derivative.
3. FSH described in a coding claim 1 or 2
33-53the polynucleotide of-IIKK targeted fusion polypeptide.
4. polynucleotide according to claim 3, is characterized in that, containing polynucleotide according to claim 3 or its not fragment of loss of biological activity, variant or derivative.
5. a pharmaceutical composition, it is used for the treatment of or prophylaxis of tumours, it is characterized in that, comprises FSH described in claim 1 or 2
33-53-IIKK targeted fusion polypeptide.
6. pharmaceutical composition according to claim 5, is characterized in that, also comprises pharmaceutically acceptable carrier or described composition is lyophilized form, unit dosage, solid dosage, liquid form.
7., for a method for the expression by modulating apoptosis path or necrocytosis growth of tumour cell, described method comprises the steps:
A) nucleotide sequence, polypeptide or the composition that define any one of claim 1-6 are provided,
B) described nucleotide sequence, polypeptide or composition are applied to or are applied to the expression system of wild tumour cell, cell, tissue or organism.
8. the FSH according to claim 1-2
33-53the application in preparation treatment or prophylaxis of tumours medicine of-IIKK targeted fusion polypeptide, polynucleotide described in claim 3-4.
9. application according to claim 8, is characterized in that: the FSH according to claim 1-2
33-53the application in preparation treatment or prevention genital system tumor, incidence cancer and lung cancer, malignant lymphatic tumor medicine of-IIKK targeted fusion polypeptide, polynucleotide described in claim 3-4.
10. application according to claim 9, is characterized in that: the FSH according to claim 1-2
33-53the application in preparation treatment or prevention prostate cancer medicine of-IIKK targeted fusion polypeptide, polynucleotide described in claim 3-4.
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