CN114835776B - Small molecule polypeptide for targeting Smad4/PELO interaction and resisting tumor metastasis and application thereof - Google Patents
Small molecule polypeptide for targeting Smad4/PELO interaction and resisting tumor metastasis and application thereof Download PDFInfo
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- CN114835776B CN114835776B CN202210368940.0A CN202210368940A CN114835776B CN 114835776 B CN114835776 B CN 114835776B CN 202210368940 A CN202210368940 A CN 202210368940A CN 114835776 B CN114835776 B CN 114835776B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a small molecule polypeptide for inhibiting prostate cancer cell metastasis by targeting Smad4/PELO interaction, which has the amino acid sequence as follows: FCDYMFQQA. The small molecule polypeptide which can specifically block the interaction of Smad4 and PELO and inhibit the migration and invasion capacity of prostate cancer cells, and a mouse experiment also proves that the small molecule polypeptide which targets the Smad4/PELO interaction and inhibits the migration of the prostate cancer cells can inhibit the lung metastasis and the liver metastasis of the prostate cancer cells in vivo, and improve the survival rate of nude mice, so that the effect of resisting tumor metastasis is achieved, and the small molecule polypeptide can be used for preparing antitumor drugs or tumor diagnostic reagents and is used for diagnosing or treating the prostate cancer.
Description
Technical Field
The invention belongs to the field of biotechnology and medicine, and particularly relates to a small molecule polypeptide for targeting Smad4/PELO interaction and resisting tumor metastasis and application thereof.
Background
Deregulation of TGF- β signaling pathways is involved in the development, progression and resistance to treatment of many tumor types. Smad4 is a key node molecule of TGF-beta signaling pathway, mediating biological processes such as proliferation, differentiation and apoptosis of tumor cells. The TGF-beta signaling pathway has dual functions, and plays a role in inhibiting proliferation of tumor cells in early stages of tumor growth, and promotes metastasis of tumor cells when the tumor cells progress to later stages. Currently, many studies attempt to untangling the dual role of the complex signaling mechanism of TGF- β and its tumor suppression and tumor promotion in the development of prostate carcinogenesis by targeting Smad 4.
At present, small molecule inhibitors achieve the effect of treating related tumors by blocking TGF-beta signals, such as LY215799, LY364947, LY580276 and the like, but currently marketed small molecule drugs mainly aim at intracellular segments, have the defects of high toxicity, excessive targets and the like, and protein drugs can achieve the effect of inhibiting receptor signals by blocking TGF-beta signals, achieve the effect of treating tumor diseases by neutralizing TGF-beta ligands, and have good biocompatibility, such as antibodies of 2G7 (neutralizing antibodies), GC 1008 and the like. However, protein drugs have the disadvantage of having a relatively large molecular weight and are generally immunogenic when injected into humans. Because the polypeptide drugs have small molecular weight, do not have balance in human body, have small immunogenicity and good activity, the polypeptide drugs become hot spots for current research.
Disclosure of Invention
In view of the above-mentioned drawbacks or shortcomings of the prior art, an object of the present invention is to provide a small molecule polypeptide targeting Smad4/PELO interactions that is resistant to tumor metastasis.
In order to achieve the above task, the present invention adopts the following technical solutions:
a small molecule polypeptide targeting Smad4/PELO interactions that resists tumor metastasis, characterized by the amino acid sequence: FCDYMFQQA.
According to the experiments of the applicant, the small molecular polypeptide for resisting tumor metastasis, which targets the interaction of Smad4/PELO, can specifically block the interaction of Smad4 and PELO, inhibit migration and invasion capacity of prostate cancer cells, and the mouse experiments also prove that the small molecular polypeptide can inhibit lung metastasis and liver metastasis of the prostate cancer cells in vivo, improve the survival rate of nude mice and achieve the effect of resisting tumor metastasis, so that the small molecular polypeptide can be used for preparing antitumor drugs or tumor diagnostic reagents for diagnosing or treating the prostate cancer.
Drawings
FIG. 1 is a graph of the results of the effect of small molecule polypeptides on Smad4/PELO interaction affinity. Wherein: panel A shows inhibition of Smad4/PELO interactions in PC3 cells; panel B shows inhibition of Smad4 interaction with PELO in Du145 cells.
FIG. 2 is a graph of the results of the small molecule polypeptides of the invention targeting the Smad4/PELO interaction against tumor metastasis on PC3 and Du145 cell proliferation.
FIG. 3 is a graph showing the effect of small molecule polypeptides in inhibiting PC3 cell migration and invasion; wherein: FIG. A, B is a graph showing migration inhibition effect on PC3 cell lines; FIG. C, D shows the effect of inhibiting invasion of PC3 cell lines.
FIG. 4 is a graph showing the effect of small molecule polypeptides in inhibiting Du145 cell migration and invasion; fig. a and B show migration inhibition effect on the Du145 cell line, and fig. C, D shows invasion inhibition effect on the Du145 cell line.
FIG. 5 is a graph showing experimental results of small molecule polypeptides inhibiting lung and liver metastasis of PC3 cells in mice.
FIG. 6 is a graph showing the results of increasing survival rate of nude mice with small molecule polypeptides.
FIG. 7 is a graph showing the pathological effect of small molecule polypeptides in inhibiting PC3 cell metastasis in the lung and liver of nude mice. The invention is described in further detail below with reference to the drawings and examples.
Detailed Description
In the following examples, the experimental methods were carried out according to conventional experimental methods unless otherwise specified.
The experimental materials, reagents, etc. used, unless otherwise specified, are commercially available.
First, the applicant screened the Smad4 interacting protein PELO in the prostate cancer cell line PC3 in a pre-IP-MS based experiment, and designed a small peptide for the interaction site of both. The small peptide can obviously block the interaction of Smad4/PELO through IP experiments.
Secondly, the prostate cancer cell line is detected by using an XTT method, and the effect of the small peptide on the prostate cancer cells for obviously inhibiting the proliferation of the cells is found.
Subsequently, small peptides were tested for their ability to significantly inhibit migration and invasion of prostate cancer cells using the Transwell method.
Finally, animal experiments are carried out, and the small molecule polypeptide can obviously inhibit lung metastasis and liver metastasis of prostate cancer cells in the body of a nude mouse through treating the nude mouse transplanted with PC3 tumor cells, so that the survival rate of the nude mouse is improved. The designed small molecule polypeptide is proved to be a small molecule polypeptide targeting Smad4/PELO interaction and resisting tumor metastasis.
The following are specific examples given by the inventors.
Example 1: design of small molecule polypeptides
The applicant carried out an immunoprecipitation experiment in PC3 of prostate cancer cells based on a preliminary IP-MS experiment using specific antibodies to Smad4, screened a series of Smad4 interacting proteins including ribosome rescue factor PELO, and confirmed that Smad4 and PELO do have direct interactions by the methods of immunoprecipitation and GST-pull down. And further determines the interaction structural domain of the two, designs a small peptide aiming at the interaction site of the two, and uses the small peptide for further functional experimental study.
The designed small peptide sequence is as follows: FCDYMFQQA.
Example 2: the small molecule polypeptide can block the interaction of Smad4 and PELO.
(1) Experimental method
Co-immunoprecipitation experiments were performed in PC3 and Du145 cells using antibodies specific for Smad4, respectively, with IgG of the corresponding species as negative control. Western blot detection was then performed using Smad4 and PELO antibodies, respectively.
(2) Experimental results
The results are shown in FIG. 1: the small molecule polypeptide can significantly block the interaction of Smad4 and PELO in the prostate cancer cell lines PC3 and Du 145.
Example 3: cell level functional assay for small molecule polypeptides
(1) Selection of experimental cell lines
The XTT method was first performed in PC3 and Du145 cells to screen whether the synthesized small molecule polypeptides have inhibitory effects on proliferation of tumor cells.
(2) Experimental method
A. Culturing PC3 and Du145 cells in 1640 culture medium added with 10% (v/v) fetal bovine serum to logarithmic phase, digesting and collecting cells, uniformly spreading in 96-well plates with cell density of 2000/well, adding polypeptide to treat cells after adherence, and setting 5 multiple wells for each concentration gradient; wherein the final concentration of the polypeptide is 20. Mu.M, and no polypeptide is added as a control.
B. After culturing the cells for 3d and 6d, 10. Mu.l of each XTT reagent was added to each well of the medium, incubated in an incubator at 37℃for 1-2 hours, and absorbance at 450nm was measured under a microplate reader.
(3) Experimental results
The results are shown in FIG. 2: the cell proliferation experiment result shows that the small molecule polypeptide has no obvious inhibition effect on the proliferation of PC3 and Du145 cells.
Example 4: detection of small molecule polypeptides inhibiting migration of prostate cancer cells
(1) Selection of experimental cell lines
The cell lines selected were human prostate cancer cells Du145 and PC3, both of which were purchased from ATCC.
(2) Experimental method
Trypsinizing cell lines, preparing cell suspensions by resuspending cells in serum-free medium, and counting to obtain cell densities of 5×10 5 Individual/ml and 1X 10 6 Single cell suspension of 5X 10 cells/ml was added to the upper layer of the non-glued chamber 5 200 μl of single cell suspension per ml was added to the upper layer of the gel-spreading chamber at a cell density of 1×10 6 Mu.l of cells per ml were suspended and 700. Mu.l of medium containing 10% foetal calf serum was added to the lower chamber. Taking out the cell after 24h of migration experiment, taking out the cell after 48h of invasion experiment, flushing twice with PBS, fixing cells with paraformaldehyde with the concentration of 3.7%, then permeabilizing the cells with methanol, flushing twice with PBS after Giemsa staining, gently wiping off the upper cell layer of the cell with a cotton swab, drying, and photographing and counting the lower cell layer of the cell under an inverted microscope.
(3) Experimental results
The experimental results are shown in fig. 3-4: the small molecule polypeptides significantly inhibit the ability of Du145 and PC3 cells to migrate.
Example 5: detection of inhibition of prostate cancer cell invasion by small molecule polypeptides
(1) Selection of experimental cell lines
The cell lines selected were human prostate cancer cells Du145 and PC3, both of which were purchased from ATCC.
(2) The experimental method is the same as the above.
(3) Experimental results
The experimental results are shown in fig. 3-4: small molecule polypeptides can significantly inhibit the invasive capacity of Du145 and PC3 cells.
Example 6: in vivo animal experiments for inhibiting tumor metastasis by using small molecule polypeptides
15 NSG nude mice (6 weeks old, male) are purchased, SPF-grade condition is fed, the temperature is constant (22-24 ℃), the humidity is constant (50% -70%), and the feeding cage, the padding, the feed and the water are sterilized by high-pressure steam.
200. Mu.L (about 1X 10) of PC3 cell suspension was injected intravenously through the tail of the mouse using a 1ml syringe 6 And/or (c) only. Three days after inoculation, the small molecule polypeptide injection experiment was started.
Nude mice were randomly divided into 2 groups, (1) negative control group: PBS buffer 0.2ml; n= 7(2) polypeptide group: 10mg/kg; n=8, administration treatment: the tail vein injection is carried out once every three days, and the administration is carried out for 7 times continuously; mice were sacrificed for 50 days, lung and liver metastases were observed, and lungs and livers of mice were HE stained.
Starting from day 30, mice in the negative control group injected with PBS buffer began to die, and the time and number of mice to die were recorded.
The results are shown in FIGS. 5-7: according to the statistical result, the small molecule polypeptide can obviously inhibit liver metastasis and lung metastasis of the transplanted prostate cancer cells after administration, and can obviously improve the survival rate of mice.
Through the above examples 1-6, it is demonstrated that the designed small molecule polypeptide is a small molecule polypeptide targeting Smad4/PELO interaction and resisting tumor metastasis, and can be used for preparing antitumor drugs or tumor diagnostic reagents for diagnosing or treating prostate cancer.
The effective concentration of the small molecular polypeptide in the prepared antitumor drug is 20 mu M.
Of course, the prepared antitumor drug also contains one or at least two pharmaceutically acceptable carriers; the carrier is a pharmaceutically common slow release agent, excipient, filler, adhesive, wetting agent, disintegrating agent, absorption promoter, adsorption carrier, surfactant or lubricant. The preparation is injection or powder injection.
The above embodiments are preferred examples of the present invention, and the present invention is not limited to the above embodiments, and it should be understood by those skilled in the art that any modification, substitution and combination made under the spirit and principle of the technical solution of the present invention should be regarded as the protection scope of the present invention.
Claims (5)
1. A small molecule polypeptide targeting Smad4/PELO interactions that resists tumor metastasis, characterized by the amino acid sequence: FCDYMFQQA.
2. The use of a Smad 4/PELO-interaction-targeted small molecule polypeptide against tumor metastasis as claimed in claim 1 in the preparation of a medicament for inhibiting tumor metastasis, wherein the tumor is prostate cancer.
3. The use of claim 2, wherein the effective concentration of small molecule polypeptide that targets Smad4/PELO interactions in the medicament is 20 μm.
4. The use according to claim 2 or 3, wherein the medicament comprises one or at least two pharmaceutically acceptable carriers; the carrier is a slow release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption promoter, an adsorption carrier, a surfactant or a lubricant.
5. The use according to claim 2 or 3, wherein the pharmaceutical dosage form is an injection or a powder injection.
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Citations (5)
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|---|---|---|---|---|
| CN1803823A (en) * | 2006-01-05 | 2006-07-19 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | SMAD4 gene expression-inhibiting SiRNA, its encoding sequence and application |
| WO2016004387A1 (en) * | 2014-07-02 | 2016-01-07 | H. Lee Moffitt Cancer Center And Research Institute, Inc. | Gene expression signature for cancer prognosis |
| CN111254194A (en) * | 2020-01-13 | 2020-06-09 | 东南大学 | Cancer-related biomarkers based on sequencing and data analysis of cfDNA and application thereof in classification of cfDNA samples |
| CN112225821A (en) * | 2020-10-21 | 2021-01-15 | 徐州医科大学 | Polypeptide with anti-tumor effect and application thereof |
| CN112930395A (en) * | 2018-06-08 | 2021-06-08 | 洛卡纳生物股份有限公司 | RNA-targeting fusion protein compositions and methods of use |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040132642A1 (en) * | 2002-11-12 | 2004-07-08 | Government Of The U.S.A., Represented By The Secretary, Dept. Of Health & Human Services | Methods of inhibiting metastasis or growth of a tumor cell |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1803823A (en) * | 2006-01-05 | 2006-07-19 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | SMAD4 gene expression-inhibiting SiRNA, its encoding sequence and application |
| WO2016004387A1 (en) * | 2014-07-02 | 2016-01-07 | H. Lee Moffitt Cancer Center And Research Institute, Inc. | Gene expression signature for cancer prognosis |
| CN112930395A (en) * | 2018-06-08 | 2021-06-08 | 洛卡纳生物股份有限公司 | RNA-targeting fusion protein compositions and methods of use |
| CN111254194A (en) * | 2020-01-13 | 2020-06-09 | 东南大学 | Cancer-related biomarkers based on sequencing and data analysis of cfDNA and application thereof in classification of cfDNA samples |
| CN112225821A (en) * | 2020-10-21 | 2021-01-15 | 徐州医科大学 | Polypeptide with anti-tumor effect and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| PELO facilitates PLK1-induced the ubiquitination and degradation of Smad4 and promotes the progression of prostate cancer;Ping Gao et al.;《Oncogene》;第41卷;2945–2957 * |
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