CN111467478A - Application of polypeptide Kp-10 in preparation of medicine for treating and preventing osteosarcoma - Google Patents

Application of polypeptide Kp-10 in preparation of medicine for treating and preventing osteosarcoma Download PDF

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CN111467478A
CN111467478A CN202010291237.5A CN202010291237A CN111467478A CN 111467478 A CN111467478 A CN 111467478A CN 202010291237 A CN202010291237 A CN 202010291237A CN 111467478 A CN111467478 A CN 111467478A
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osteosarcoma
polypeptide
medicament
gpr54
preparation
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肖建如
李珍惜
王冬生
何韶辉
茹炳华
孙海涛
魏海峰
吴志鹏
赵剑
钱明
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Shanghai Changzheng Hospital
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/08Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • A61P35/04Antineoplastic agents specific for metastasis

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Abstract

The invention discloses an application of a polypeptide Kp-10 in preparing a medicine for treating and preventing osteosarcoma, wherein the amino acid sequence of the polypeptide Kp-10 is DSSDSSDSSDSSDSSDSSYNWNSFG L RF., researches show that the expression of GPR54/KiSS-1 in osteosarcoma tissues is obviously lower than that of paraneoplastic normal tissues, and the polypeptide Kp-10 and a receptor GPR54 thereof are highly related to clinical prognosis, and can inhibit the proliferation and migration capacity and lung transfer capacity of osteosarcoma cells, so that the polypeptide Kp-10 can be used for preparing the medicine for treating and preventing osteosarcoma, and a new thought is provided for the search of targeted medicines for osteosarcoma.

Description

Application of polypeptide Kp-10 in preparation of medicine for treating and preventing osteosarcoma
Technical Field
The invention relates to the field of biological medicine, in particular to application of a polypeptide Kp-10 in preparing a medicine for treating and preventing osteosarcoma.
Background
Osteosarcoma is the most common one of bone malignancies, a malignant osteogenic tumor derived from mesenchymal cells. The patients are adolescents and males. It is best seen in the skull, where the frontal bone is the most, followed by the parietal, temporal, occipital bones, and in the facial bones, most located in the maxilla, mandible, zygomatic, nasal bones, followed by the frontal sinus, orbit, etc., and 1/3 in the anterior side of the tibia. The tumor grows slowly and has light symptoms, and the tumor mostly appears in children, the development of the body gradually grows, and the tumor usually attracts attention due to the occurrence of lumps after years of 10-20 years of age and the course of disease of decades or decades. Moreover, lung metastases usually occur in 80% of cases when osteosarcoma is diagnosed.
At present, the treatment of osteosarcoma is mainly based on large-dose individualized neoadjuvant chemotherapy (preoperative chemotherapy) and operative treatment, and is assisted by drug treatment. The survival rate of the patient in 5 years is greatly improved by combining the development of the new auxiliary chemotherapy with the postoperative chemotherapy, and the limb protection operation is carried out on the patient with limb protection finger syndrome under the cooperation of a scientific and reasonable chemotherapy scheme, so that the life cycle of the patient is prolonged, and the life quality of the patient is also greatly improved. In addition, the development and research of new effective targeted drugs are also very necessary for the generation and development of osteosarcoma and the inhibition of lung metastasis, so that the pain of patients can be reduced, and the reasonable allocation of medical resources can be realized.
The KiSS-1 gene is found as a tumor suppressor in melanoma and encodes a protein of 145 amino acids, which is cleaved by proteasomes to yield a biologically active secreted peptide consisting of 54 amino acids, KiSSpeptin-54, which is further cleaved to KiSSpeptin-14, KiSSpeptin-13, KiSSpeptin-10, which specifically binds to GPR54 and exerts a biological effect, which is a natural ligand of GPR54, wherein KiSSpeptin-10 is abbreviated Kp-10, which is the shortest KiSSpeptin in active form, which is the smallest of these biological peptides, which consists of 10 amino acids, and clinical analysis of patients shows that, by gene knockout, mouse and clinical analysis, KiSS-1 is a natural ligand of GPR54, the hypothalamic erythropoietin (Kp) regulates the secretion of various bioactive peptides, and thus the whole-cycle secretion of GnH 2 can be regulated and regulated in the same species.
The previous research proves that GPR54 is deficient to activate osteoclast and cause osteoporosis, and the natural ligand Kp-10 of GPR54 has an anti-osteoporosis effect by inhibiting the formation and the function of osteoclast. However, whether KiSS-1/GPR54 plays a role in inhibiting osteosarcoma has not been reported, and development and research of the role in preparing drugs for inhibiting the development of osteosarcoma and lung metastasis are also urgently needed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the application of the polypeptide Kp-10 in preparing the medicine for treating and preventing osteosarcoma.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a use of a polypeptide Kp-10 in the preparation of a medicament for treating and preventing osteosarcoma, wherein the amino acid sequence of the polypeptide Kp-10 is DSSDSSDSSDSSDSSDSSYNWNSFG L R F, specifically (Asp-Ser-Ser)6- (D-Tyr) -Asn- (D-Trp) -Asn-Ser-Phe- (azaGly) -L eu-Arg (Me) -Phe-NH2 (abbreviated as (AspSerSer) 6-Kp-10). The (AspSerSer)6-Kp-10 in the invention is formed by connecting 6 repeated serial aspartic acid-serine sequences (AspSerSer)6 to the N-terminal of Kp-10.
Further, the medicine also comprises a pharmaceutically acceptable carrier or excipient.
Further, the administration route of the drug is oral, transdermal, intramuscular, subcutaneous or intravenous injection.
Further, the medicine can be tablets, pills, granules, capsules or injections.
Further, the medicine can inhibit proliferation ability and migration ability of osteosarcoma cells.
Further, the drug is dose-dependent on the proliferative and migratory capacity of osteosarcoma cells.
Further, the medicine can inhibit lung metastasis ability of osteosarcoma cells.
Further, the osteosarcoma cell line is MNNG/HOS, ZOS or 143B cell line.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the application of the polypeptide Kp-10 in the preparation of the medicine for treating and preventing osteosarcoma, provided by the invention, discovers that the expression of GPR54/KiSS-1 in osteosarcoma tissues is obviously lower than that of paraneoplastic normal tissues and is highly negatively related to clinical prognosis through research, and the genes Kp-10 and GPR54 can inhibit the proliferation and migration capacity and lung transfer capacity of osteosarcoma cells, so that the polypeptide Kp-10 can be used for preparing the medicine for treating and preventing osteosarcoma, and a new thought is provided for the search of targeted medicines for osteosarcoma.
Drawings
FIG. 1 is a graph showing the expression results of GPR54/KiSS-1 in one embodiment of the present invention; wherein, the graph A is a graph of the immunohistochemistry results of GPR54/KiSS-1 in osteosarcoma patient tissues and paraneoplastic normal tissues, and the graph B is a graph of the expression WB results in a plurality of osteosarcoma cell lines;
FIG. 2 is a graph showing the relationship between the expression level of GPR54/KiSS-1 and the clinical prognosis of osteosarcoma patients in accordance with one embodiment of the present invention;
FIG. 3 is a graph of Kp-10 polypeptide and its concentration as a function of proliferation and migration capacity of the osteosarcoma cell lines MNNG/HOS and ZOS cells in accordance with an embodiment of the present invention; wherein, the graph A is a graph of the relationship between the Kp-10 polypeptide and the proliferation and migration capacity of an osteosarcoma cell line MNNG/HOS, and the graph B is a graph of the relationship between the Kp-10 polypeptide and the proliferation and migration capacity of an osteosarcoma cell line ZOS;
FIG. 4 is a graph showing the results of changes in the proliferation and migration abilities of MNNG/HOS and ZOS cells after knocking down the GPR54 gene in accordance with one embodiment of the present invention; wherein, the graph A is a graph of expression WB of GPR54 in ZOS cells and MNNG/HOS cells of the control group and the knockdown group, and the graph B is a graph of the influence of the control group and the knockdown group on the proliferation and migration capacity of the ZOS cells and the MNNG/HOS cells;
FIG. 5 is a graph showing the results of lung metastasis of MNNG/HOS cells in mice after knocking down the GPR54 gene according to one embodiment of the present invention;
FIG. 6 is a graph showing the results of lung metastasis of 143B cells stimulated with Kp-10(100nM/Kg) exogenously administered in one embodiment of the present invention.
Detailed Description
The invention provides application of a polypeptide Kp-10 in preparing a medicine for treating and preventing osteosarcoma, wherein the amino acid sequence of the polypeptide Kp-10 is DSSDSSDSSDSSDSSDSSYNWNSFG L RF, in particular to (Asp-Ser-Ser)6- (D-Tyr) -Asn- (D-Trp) -Asn-Ser-Phe- (azaGly) -L eu-Arg (Me) -Phe-NH2 (short for (AspSer) 6-Kp-10), and the (AspSerSer)6-Kp-10 is formed by connecting 6 repeated aspartic acid-serine serial sequences (AspSerSer)6 to the N end of the Kp-10.
The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.
Example one
This example demonstrates that GPR54/KiSS-1 is expressed in osteosarcoma tissue significantly lower than paraneoplastic normal tissue, and the specific experimental contents and results are as follows:
the collected human osteosarcoma tissue specimens were stained by immunohistochemical staining using human osteoblast line HFOB1.19 as a normal control, and the results are shown in FIG. 1. Wherein, 143B and MNNG/HOS cell line are from osteosarcoma TE85 cell line, 143B cell has stronger transfer activity by transferring ki-ras gene; ZOS and ZOSM cell lines are derived from the same patient from both in situ and distant metastatic lesions. As can be seen from FIG. 1, GPR54/KiSS-1 was expressed in the tissues of osteosarcoma patients significantly lower than in paraneoplastic normal tissues; and the expression level of KiSS-1 and GPR54 in an osteosarcoma cell line is reduced by taking a human osteoblast cell line HFOB1.19 as a normal control, wherein 143B cells have stronger transfer activity by introducing a ki-ras gene, the expression level of KiSS-1 and GPR54 is lower, the expression level of ZOSM has stronger transfer capability, and the expression level of KiSS-1 and GPR54 is lower.
Example two
This example demonstrates that GPR54/KiSS-1 expression is highly correlated with the clinical prognosis of osteosarcoma patients, and the specific experimental contents and results are as follows:
by counting the expression of GPR54/KiSS-1, the results shown in FIG. 2 were obtained in combination with the clinical follow-up data of patients. As can be seen from FIG. 2, the expression of GPR54/KiSS-1 is highly correlated with the clinical prognosis of osteosarcoma patients, i.e., a lower expression of GPR54/KiSS-1 is indicative of a shorter survival of the patients.
EXAMPLE III
This example demonstrates the inhibitory effect of GPR54/KiSS-1 on the proliferation and migration ability of the osteosarcoma cell line MNNG/HOS and ZOS cells, and the specific experimental contents and results are as follows:
the effect of Kp-10, the shortest polypeptide with activity in the KiSS protein, on the proliferation and migration capacity of the osteosarcoma cell line MNNG/HOS and ZOS cells was investigated by colony formation and transwell experiments and the results are shown in FIG. 3. The GPR54 gene was knocked down to study changes in the proliferation and migration abilities of MNNG/HOS and ZOS cells, and the results are shown in FIG. 4.
As can be seen from FIG. 3, Kp-10 was able to significantly inhibit the proliferation and migration ability of the MNNG/HOS and ZOS cells, which are osteosarcoma cell lines, and both were dose-dependent. As seen in FIG. 4A, the expression level of GPR54 was decreased in the knockdown group; as can be seen from FIG. 4B, the clone number and cell number in the knock-down group (shGPR54-1 and shGPR54-2) were significantly higher than those in the blank control group (Ctrl) and the empty control group (shCtrl), i.e., the proliferation and migration ability of MNNG/HOS and ZOS cells were significantly enhanced after knocking down the GPR54 gene.
Example four
This example demonstrates the inhibitory effect of GPR54/KiSS-1 on lung metastatic capacity of osteosarcoma cells, and the specific experimental contents and results are as follows:
an osteosarcoma tail vein lung metastasis model is constructed by injecting MNNG/HOS and a GPR54 knockout cell strain into the tail vein of a nude mouse, and the metastasis condition of the osteosarcoma in the mouse is detected by IVIS (in vivo animal imaging system) on 21 th, 28 th, 35 th and 42 th days respectively, and the result is shown in figure 5. 143B cells were injected into the tail vein of nude mice and stimulated with Kp-10(100nM/Kg) exogenously, and the transfer of osteosarcoma in mice was examined by IVIS (in vivo animal imaging System) on days 14, 21, and 28, respectively, and the results are shown in FIG. 6.
As shown in fig. 5, compared to the control group (nude mice injected with MNNG/HOS cell strain alone), the nude mice injected with GPR54 knockout cell have stronger in vivo fluorescence signal and significantly stronger and wider range of lung metastasis foci, i.e., the GPR54 gene knockout group has significantly enhanced lung metastasis ability. As can be seen from FIG. 6, the fluorescence signal of lung was significantly reduced in the Kp-10 group of mice as compared with the control group, and thus Kp-10 inhibited the lung-metastatic ability of 143B cells.
In conclusion, the expression of GPR54/KiSS-1 in osteosarcoma tissues is obviously lower than that of paraneoplastic normal tissues, the tissue is highly negatively related to clinical prognosis, and the polypeptide Kp-10 and the receptor GPR54 thereof can inhibit the proliferation and migration of tumor cells and the lung metastasis capacity, so that a target is expected to be provided for preparing a medicament for treating osteosarcoma.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims (8)

1. The application of the polypeptide Kp-10 in preparing the medicine for treating and preventing osteosarcoma is characterized in that the amino acid sequence of the polypeptide Kp-10 is DSSDSSDSSDSSDSSDSSYNWNSFG L RF.
2. The use of the polypeptide Kp-10 according to claim 1 for the preparation of a medicament for the treatment and prevention of osteosarcoma, wherein said medicament further comprises a pharmaceutically acceptable carrier or excipient.
3. The use of the polypeptide Kp-10 according to claim 1 for the preparation of a medicament for the treatment and prevention of osteosarcoma, wherein said medicament is administered orally, transdermally, intramuscularly, subcutaneously or intravenously.
4. The use of the polypeptide Kp-10 according to claim 1 for the preparation of a medicament for the treatment and prevention of osteosarcoma, wherein said medicament can be in the form of tablets, pills, granules, capsules or injections.
5. The use of the polypeptide Kp-10 according to claim 1 for the preparation of a medicament for the treatment and prevention of osteosarcoma, wherein said medicament is capable of inhibiting the proliferative and migratory capacity of osteosarcoma cells.
6. The use of the polypeptide Kp-10 according to claim 5 for the preparation of a medicament for the treatment and prevention of osteosarcoma, wherein said medicament is dose-dependent on the proliferative and migratory capacity of osteosarcoma cells.
7. The use of the polypeptide Kp-10 according to claim 1 for the preparation of a medicament for the treatment and prevention of osteosarcoma, wherein said medicament is capable of inhibiting the pulmonary metastatic capacity of osteosarcoma cells.
8. The use of the polypeptide Kp-10 according to any of claims 5 to 7 for the preparation of a medicament for the treatment and prevention of osteosarcoma, wherein said osteosarcoma cell is a MNNG/HOS, ZOS or 143B cell line.
CN202010291237.5A 2020-04-14 2020-04-14 Application of polypeptide Kp-10 in preparation of medicine for treating and preventing osteosarcoma Pending CN111467478A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110172461A (en) * 2019-06-03 2019-08-27 上海长征医院 A kind of construction method of novel osteosarcoma Lung metastases model and its application
CN112457371A (en) * 2020-11-29 2021-03-09 北京泽勤生物医药有限公司 Bone formation promoting polypeptide and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100160241A1 (en) * 2006-04-26 2010-06-24 Kyoto University Novel compound having gpr54 agonistic activity
CN109111506A (en) * 2018-01-17 2019-01-01 上海长征医院 A kind of polypeptide and its preparation method and application for treating osteoporosis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100160241A1 (en) * 2006-04-26 2010-06-24 Kyoto University Novel compound having gpr54 agonistic activity
CN109111506A (en) * 2018-01-17 2019-01-01 上海长征医院 A kind of polypeptide and its preparation method and application for treating osteoporosis

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
王冬生: "KiSSl/GPR54在骨肉瘤转移和增殖中的作用及机制研究", 中国博士学位论文全文数据库医药卫生科技辑 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110172461A (en) * 2019-06-03 2019-08-27 上海长征医院 A kind of construction method of novel osteosarcoma Lung metastases model and its application
CN112457371A (en) * 2020-11-29 2021-03-09 北京泽勤生物医药有限公司 Bone formation promoting polypeptide and application thereof
CN112457371B (en) * 2020-11-29 2021-12-10 北京泽勤生物医药有限公司 Bone formation promoting polypeptide and application thereof

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Application publication date: 20200731