Induce polypeptide and its application of apoptosis of prostatic carcinoma cell line
Technical field
The present invention relates to technical field of pharmaceutical biotechnology, and in particular to it is a kind of induce apoptosis of prostatic carcinoma cell line polypeptide and its
Purposes in the preparation of the medicine of forefront property cancer in treating subject.
Background technology
Property cancer in forefront is that the epithelial malignancy in prostate occurs, and the incidence of disease is high, and the death rate is high, is that male is most common
One of malignant tumour.The cause of disease of prostate cancer is still not clear at present, may be relevant with many factors such as heredity, environment.And people
Class others tumour is the same, and inflammation and immunosupress play key player in forefront property carcinogenesis, evolution.
Current research is thought to participate in forming prostate cancer generation, development with the host component of tumour interaction
Immunosupress microenvironment, a large amount of evidences show that prostate cancers are immunity correlations, some existing tumour phases in prostate
Closing property antigen further demonstrate its feasibility, and these related antigens mainly have PSMA (PSMA), preceding
Row gland hepatocytic antigen (PSCA), PAP (PAP) etc..Molecular targeted medicine is designed for these related antigens
Thing, can selectivity killing or suppress tumour cell, and not injuring normal cell, thus with important Research Significance and
It is widely applied prospect.
PSMA is the type transmembrane glycoprotein of one kind II for being present in prostate epithelial cell after birth, is mainly made up of three parts:
Extracellular portion (707 amino acid), transmembrane segment (24 amino acid) and intracellular part (19 amino acid).PSMA is based on this
Transmembrane structure and participate in signal transduction so that the signal of cell surface protein can be for delivery to intracellular, and has function of receptors
Endocytosis is played, and with promotion cell migration and the characteristic of folic acid hydrolase and carboxypeptidase.
The high expression in cancerous prostate epithelial cell due to PSMA, and do not express in normal structure endothelial cell or low table
Reach, particularly in advanced prostate cancer and metastatic prostate cancer patient, PSMA rises are particularly evident, and this causes it to turn into forefront
Gland cancer diagnoses and an important target molecule for the treatment of.From first Dan Ke that have developed PSMA such as Horoszewicz in 1987
After grand antibody 7E11-C5, a series of monoclonal antibodies for PSMA such as J415, J591, J533 are separated and are purified.In early days
7E11 can only identify the intracellular antigenic determinants of PSMA, it is impossible to reference to cell living.And J591 can not only identify PSMA
Extracellular antigenic determinant, it also may incorporate the living cells of PSMA expression.This extracellular region for being primarily due to PSMA can lead to
Cross motif MXXXL cave in combination of the increase cell to J591, absorb.
The specific killing prostate gland cancer cell being derived based on these antibody is that targeted therapy prostate cancer carries
Important Research Thinking is supplied.Particularly in recent years, PSMA intracellular region and extracellular region have been found that it is multiple can be with polypeptide knot
The epitope of conjunction.The polypeptide that can be combined with epitope, the polypeptide drug particularly combined with extracellular region epitope are for prostate cancer
Treatment it is significant.
A domain that can be identified with medicine based on PSMA outside film, has researcher's design to synthesize a series of energy
The methotrexate (MTX) class conjugates activated by PSMA, promote PSMA to play exopeptidase effect, discharge methotrexate (MTX), knurl work is killed in performance
With, but may be because conjugates molecule is excessive and the reason such as methotrexate (MTX) toxicity, fall flat.
On the other hand, autophagy related gene Beclin1 missing is that tumour cell autophagocytosis reduces, anti-apoptotic ability increases
Where strong mechanism.Increase Beclin1 expression helps to strengthen the autophagocytosis of tumour cell, reduces the anti-of tumour cell and withers
Die ability.Beclin1 albumen also includes three parts, is BH3 (Bcl-2-homology-3), central spiral area respectively
(central coiled-coil domain, CCD) and evolution conservative area (evolutionary conserved district,
ECD) three domains.Wherein ECD domains play Beclin1 regulation autophagy and the function of suppression tumour is most important.Have
Research shows that the fusion protein containing Beclin1ECD domains can significantly improve the apoptosis rate of prostate gland cancer cell, and wither
Die cell gradually increases with the increase of ECD fusion protein concentration.
We are on the basis of analyzing Beclin1 amino acid sequence and with reference to J591 specific recognitions PSMA height
Become region sequence, study and design energy specific recognition PSMA, and make the polypeptide of PSMA positive cells acceleration apoptosis.
The content of the invention
It is an object of the invention to provide the polypeptide and Related Drug that apoptosis of prostatic carcinoma cell line can be induced by PSMA
Thing.
In a first aspect, the invention provides a kind of polypeptide for inducing apoptosis of prostatic carcinoma cell line, the amino of the polypeptide
Acid sequence includes SEQ ID NO:1
(WTKALKFMXXXLKWGLAWVSHFSVGS), wherein X is arbitrary amino acid.
In embodiments of the invention, the amino acid sequence of the polypeptide is SEQ ID NO:1.
In embodiments of the invention, the amino acid sequence of the polypeptide is SEQ ID NO:Any one in 2~10,
Particular sequence is as follows:
SEQ ID NO:2:WTKALKFMLTNLKWGLAWVSHFSVGS;
SEQ ID NO:3:WTKALKFMLNTLKWGLAWVSHFSVGS;
SEQ ID NO:4:WTKALKFMAHILKWGLAWVSHFSVGS;
SEQ ID NO:5:WTKALKFMGCPLKWGLAWVSHFSVGS;
SEQ ID NO:6:WTKALKFMSTMLKWGLAWVSHFSVGS;
SEQ ID NO:7:WTKALKFMDQWLKWGLAWVSHFSVGS;
SEQ ID NO:8:WTKALKFMYKKLKWGLAWVSHFSVGS;
SEQ ID NO:9:WTKALKFMFVSLKWGLAWVSHFSVGS;
SEQ ID NO:10:WTKALKFMRTELKWGLAWVSHFSVGS.
In second aspect, the invention provides the polypeptide as described in first aspect property cancer in forefront in treating subject
Purposes in the preparation of medicine.
In embodiments of the invention, the medicine can be by oral, intravenous injection, intra-arterial injection, subcutaneous note
Penetrate or intramuscular injection and apply.
In embodiments of the invention, the medicine is made into solid or physiologically acceptable liquid-carrier.
Beneficial effect:
Relative to a series of monoclonal antibodies, polypeptide and the compound that can suppress PSMA currently existed etc., sheet
The ability of the polypeptid induction apoptosis of prostatic carcinoma cell line of invention is stronger, more preferable for the therapeutic effect of prostate cancer.
Brief description of the drawings
By the description to the embodiment of the present invention referring to the drawings, above-mentioned and other purpose of the invention, feature and
Advantage will be apparent from, in the accompanying drawings:
Fig. 1 shows the SEQ ID NO of different quality concentration:2 polypeptides are lured Prostatic cancer cell lines LNcap Apoptosis
Lead result.
Fig. 2 shows the SEQ ID NO of different quality concentration:The suppression of migration of 2 polypeptides to Prostatic cancer cell lines LNcap
Effect.
Fig. 3 shows the SEQ ID NO of different quality concentration:2 polypeptides are to Apoptosis inhibitor base in Prostatic cancer cell lines LNcap
The regulating and controlling effect expressed by Bcl-2 and autophagy related gene Atg5.
Fig. 4 shows the SEQ ID NO of different quality concentration:2 polypeptides are to p-AKT in Prostatic cancer cell lines LNcap cells
The regulating and controlling effect of expression.
Fig. 5 shows the SEQ ID NO of different quality concentration:2 polypeptides and J591 monoclonal antibodies are to tumour in prostate cancer mouse
Inhibitory action.
Embodiment
Below based on embodiment, present invention is described, but the present invention is not restricted to these embodiments.
The Peptide systhesis of embodiment 1
Polypeptid acid sequence is SEQ ID NO:2:WTKALKFMLTNLKWGL AWVSHFSVGS
Entrust Nanjing Genscript Biotechnology Co., Ltd.'s synthesis.- 80 DEG C save backup after DMSO dissolvings.
Embodiment 2
By Prostatic cancer cell lines LNcap cells with 5 × 105The density in individual/hole is inoculated in 6 orifice plates, 37 DEG C, 5%CO2 bars
Different quality concentration (0,2,10,50,100 μ g/mL) polypeptide is separately added into after cultivating 24h under part.Continue to collect carefully after cultivating 48h
Born of the same parents ,-the FITC of Annexin V combine liquid and cell are resuspended after centrifugation, PBS wash 2 times, add-the FITC of Annexin V and gently mix, room
Warm lucifuge is incubated, centrifuged, abandoning supernatant, adds-the FITC of Annexin V and combines liquid resuspension cell, adds PI dyeing liquors, BD
FACSCalibur flow cytomeries.
As shown in figure 1, the apoptosis rate of LNcap cells increases with increasing for peptide concentration.Peptide concentration is 100 μ g/
During mL, LNcap apoptosis rates are more than 50%.
Embodiment 3
LNcap cells are taken, cell concentration is adjusted to 1x106Individual/ml.Connect in Cornng Transwell orifice plate upper chambers
100 μ l concentration of kind are 1x106Individual/ml LNcap cells, and it is separately added into different quality concentration (0,2,10,50,100 μ g/mL)
Polypeptide.Lower room adds RPMI1640 culture mediums of the 500 μ l containing 40%FBS, and incubator culture 18h is placed in after planting plate, takes out lower room
And culture medium in upper chamber is discarded, 4% paraformaldehyde solution is fixed, and amplifies 200 times of meters after violet staining under inverted microscope
The cell of microporous barrier lower floor is moved in number view-finder.Average each cell counts 10 visuals field.Average and analyze moving for cell
Shifting ability.
As shown in Fig. 2 the polypeptide of the present invention can effectively suppress the transfer abilities of LNcap cells, and into dose-dependant, with
The rise of peptide concentration, the transfer ability of LNcap cells gradually reduces.
Embodiment 4
By LNcap cells with 5 × 105The density in individual/hole is inoculated in 6 orifice plates, 37 DEG C, cultivate 24h under the conditions of 5%CO2 after
It is separately added into different quality concentration (0,2,10,50,100 μ g/mL) polypeptide.Continue to collect cell after cultivating 48h, extracting RNA, and
Reverse transcription is into cDNA, and using GAPDH as internal reference, qPCR detects apoptosis suppressor gene Bcl2 and autophagy related gene Atg5 expression.
Primer sequence is respectively:
GAPDH Forward:5 '-GGAGCGAGATCCCTCCAAAAT-3 ',
GAPDH Reverse:GGCTGTTGTCATACTTCTCATGG-3’;
Bcl-2Forward:GGTGGGGTCATGTGTGTGG-3 ',
Bcl-2Reverse:CGGTTCAGGTACTCAGTCATCC-3’;
Atg5Forward:5’-AAAGATGTGCTTCGAGATGTGT-3’,
Atg5Reverse:5’-CACTTTGTCAGTTACCAACGTCA-3’。
As a result as shown in figure 3, the polypeptide of the present invention can express by lowering apoptosis suppressor gene Bcl2, autophagy is raised
Related gene Atg5 is expressed to accelerate the apoptosis of LNcap cells.
Embodiment 5
PSMA can activate PI3K/AKT signal paths by raising AKT phosphorylation levels, increase prostate gland cancer cell
Propagation and transfer.
By Prostatic cancer cell lines LNcap cells with 5 × 105The density in individual/hole is inoculated in 6 orifice plates, 37 DEG C, 5%CO2 bars
Different quality concentration (0,2,10,50,100 μ g/mL) polypeptide is separately added into after cultivating 24h under part, continues to collect carefully after cultivating 48h
Born of the same parents.
After fully washing 3 times with the PBS of 4 DEG C of precoolings, add 4 DEG C containing protease inhibitors and inhibitors of phosphatases
Cell is resuspended in the μ L of RIPA lysis buffers 200, cracks 20min on ice.Supernatant protein liquid is collected by centrifugation, and takes 150 μ l protein liquids
37.5 μ 5 × albumen of l buffer are added, 100 DEG C are boiled 5min, and 4 DEG C save backup.
The protein liquid prepared is added into electrophoresis in gel, first with voltage 60V electrophoresis 5min, then with 80V electrophoresis
60min.Electrophoresis carries out transferring film, 100V, 2h after terminating.Film is taken out from electric turn trough after end, TBST is slightly rinsed, and is immersed in
Confining liquid slowly sways 1h in (containing 5% skimmed milk power).Film containing destination protein is transferred to equipped with 4ml primary antibodies respectively
In the valve bag of (Rabbit Phospho-AKT1 (Ser473) Antibody), 50rpm 1h on horizontal shaker, 4 DEG C of mistakes are gone to
Night.Take the film out within second day, rinsed four times with TBST.The film of the film and internal reference albumen that turn purposeful albumen is individually placed to No. 2
In valve bag, dilution (1 is added:5000) secondary antibody (Goat anti-Rabbit IgG (H+L) Secondary of corresponding primary antibody
Antibody), room temperature jog one hour, primary antibody and secondary antibody are purchased from eBioscience.After secondary antibody incubation terminates, floated with PBST
Wash film four times.Lucifuge adds chromogenic reagent and taken pictures.
As a result as shown in figure 4, with the rise of peptide concentration, LNcap cells p-AKT expression is gradually reduced.Say
Bright polypeptide of the invention can suppress PI3K/AKT signal paths by suppressing AKT phosphorylation, so as to suppress PSMA increasing
Grow and shift.
Embodiment 6
The tumor inhibition effect of animal model detection polypeptide in vivo.6~8 week old male C57BL/6 nude mices 42 are taken, will
LNcap cell concentrations in exponential phase are adjusted to 2x107Before individual/ml, every 100ul are inoculated in the C57BL/6 nude mices right side
Limb armpit is subcutaneous.Nude mouse tumor volume grows to 50~100mm after 1~2 week3When, nude mice is randomly divided into 6 groups, every group 7.Respectively
For 0,2,10,50,100 μ g/kg polypeptide groups and 100 μ g/kg J591 monoclonal antibody groups.0 μ g/kg groups as negative control group, with etc.
Volume DMSO is replaced.Each group polypeptide and J591 are with normal saline dilution to 200ul, through tail vein injection.2 days once, continuously
After administration 4 times, nude mice amount of survival is observed, counts survival rate.
As shown in figure 5, with the rise of peptide concentration, the nude mice time-to-live gradually extends, and peptide concentration is 100 μ g/kg
When the mouse survival time it is most long.The polypeptide of the present invention is compared with J591 monoclonal antibody groups, when being all 100 μ g/kg concentration, polypeptide
Effect is more preferable than the effect of J591 monoclonal antibody.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for those skilled in the art
For, the present invention can have various changes and change.All any modifications made within spirit and principles of the present invention, it is equal
Replace, improve etc., it should be included in the scope of the protection.
Sequence table
<110>Shenzhen Tai Hua cell engineerings Co., Ltd
<120>Induce polypeptide and its application of apoptosis of prostatic carcinoma cell line
<130> 20170806
<160> 10
<170> PatentIn version 3.5
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