CN111620939A

CN111620939A - Polypeptide ZYX for treating or assisting in treating ovarian cancer36-58

Info

Publication number: CN111620939A
Application number: CN201911024001.9A
Authority: CN
Inventors: 徐娟; 贾雪梅; 王煦苏; 刘光泉; 许鹏飞; 潘新星; 贡震
Original assignee: Nanjing Maternity and Child Healthcare Hospital
Current assignee: Nanjing Maternity and Child Healthcare Hospital
Priority date: 2019-10-25
Filing date: 2019-10-25
Publication date: 2020-09-04
Anticipated expiration: 2039-10-25
Also published as: CN111620939B

Abstract

The invention discloses a polypeptide ZYX for treating or assisting in treating ovarian cancer_36‑58. Polypeptide ZYX for treating or assisting in treating ovarian cancer_36‑58The amino acid sequence is shown in SEQ ID NO. 1. The applicant screens polypeptides differentially expressed in the serum of an ovarian epithelial cancer patient and the serum of a normal examination control by using a tandem mass spectrometry method, and finds out the polypeptide ZYX with higher abundance in the serum of the normal examination control and significantly reduced abundance in the serum of the ovarian epithelial cancer patient_36‑58Can remarkably inhibit invasion and migration of ovarian cancer, promote apoptosis, and not affect proliferation. Due to ZYX_36‑58Has the advantages of small molecular weight, self-origin and toxicitySmall, and the like, and can be an important auxiliary medicine for treating ovarian cancer.

Description

Polypeptide ZYX for treating or assisting in treating ovarian cancer36-58

Technical Field

The invention belongs to the field of biological medicine, and relates to a polypeptide ZYX for treating or assisting in treating ovarian cancer_36-58。

Background

Ovarian cancer is the gynecological malignant tumor with the highest mortality rate and seriously threatens the life of women. Ovarian cancer usually has a hidden onset, metastasis is early, and abdominal metastasis or/and lymph node metastasis and distant metastasis are present in 70% of patients, which is also the main cause of high mortality of ovarian cancer [1,2 ]. With the development of molecular genetics, the understanding of the incidence mode, the typing and the cell source of ovarian cancer is deepened, the targeted therapy of ovarian cancer is continuously developed, but the overall survival rate of ovarian cancer is not obviously improved clinically [3 ]. Therefore, further analyzing the invasion and metastasis mechanism of ovarian cancer, searching new targets for treating ovarian cancer, and developing new drugs for treating ovarian cancer become urgent matters for preventing and treating gynecological tumors.

In recent years, polypeptide research has become a new focus of medicine development. Endogenous polypeptides are a class of small peptides of 3-50 amino acids in size that are produced or obtained from tissues, cells and body fluids [4 ]. The polypeptide is found to be involved in almost all life activities and closely related to immune regulation, neurohormonal transmitter regulation, tumor lesion and the like [5-7 ]. The polypeptide is also attractive in the prospect of tumor treatment, and various polypeptides capable of inhibiting tumor cell adhesion, invasion and migration have been found in previous researches, such as a peptide (peptide 1) for inhibiting VEGF expression, a peptide (MTP-NeuNT) for inhibiting ErbB2 activity, a peptide (WAHM1 and WAHM2) for inhibiting WASF3 signal path and the like. Thanks to the progress of mass spectrometry technology, tumor polypeptimics has been developed primarily, endogenous polypeptidic features of tumor tissues such as breast cancer [11], ovarian cancer [12], cholangiocellular carcinoma [13], lung cancer [14] and the like have been researched and identified, and a plurality of polypeptides which are expected to become tumor diagnosis markers, tumor treatment targets or drugs are found from the endogenous polypeptidic features, so that a new idea is provided for tumor prevention and treatment research. Therefore, it is considered necessary to search for a bioactive polypeptide having a therapeutic prospect by exploring the mechanism of occurrence and development of ovarian cancer from the perspective of the polypeptide.

Reference documents:

1Roett MA,Evans P.Ovarian cancer:an overview.AM FAM PHYSICIAN 2009；80(6):609-16.

2Bell D,Berchuck A,Birrer M,Chien J,Cramer DW,Dao F,et al..Integratedgenomic analyses of ovarian carcinoma.NATURE 2011；474(7353):609-15.

3Bhatt P,Vhora I,Patil S,Amrutiya J,Bhattacharya C,Misra A,MashruR.Role of antibodies in diagnosis and treatment of ovarian cancer:Basicapproach and clinical status.J CONTROL RELEASE 2016；226:148-67.

4 Hayakawa E,Menschaert G,De Bock PJ,Luyten W,Gevaert K,Baggerman G,et al..Improving the identification rate of endogenous peptides usingelectron transfer dissociation and collision-induced dissociation.J PROTEOMERES 2013；12(12):5410-21.

5

TA,Boes M,Wolters D,Spindeldreher S,

B,Langen H,etal..Upregulation of the CLIP self peptide on mature dendritic cellsantagonizes T helper type 1 polarization.NAT IMMUNOL 2004；5(9):909-18.

6 Nagashima H,

T,Shih HY,Davis FP,Meylan F,Huang Y,etal.Neuropeptide CGRP Limits Group 2 Innate Lymphoid Cell Responses andConstrains Type 2 Inflammation.Immunity.2019；pii:S1074-7613(19)30279-1.

7 Soragni A,Janzen DM,Johnson LM,Lindgren AG,Thai-Quynh Nguyen A,Tiourin E,et al..A Designed Inhibitor of p53 Aggregation Rescues p53 TumorSuppression in Ovarian Carcinomas.CANCER CELL 2016；29(1):90-103.

8 Zhou C,Kang J,Wang X,Wei W,Jiang W.Phage display screeningidentifies a novel peptide to suppress ovarian cancer cells in vitro and invivo in mouse models.BMC CANCER 2015；15(1):1-12.

9 Arpel A,Sawma P,Spenle C,Fritz J,Meyer L,Garnier N,etal..Transmembrane domain targeting peptide antagonizing ErbB2/Neu inhibitsbreast tumor growth and metastasis.CELL REP 2014；8(6):1714-21.

10 Teng Y,Bahassan A,Dong D,Hanold LE,Ren X,Kennedy EJ,etal..Targeting the WASF3-CYFIP1 Complex Using Stapled Peptides SuppressesCancer Cell Invasion.CANCER RES 2016；76(4):965-73.

11 Zaki A,Ramadan RA,Moez P,Ghareeb H,Elkarmouty A.Plasma PeptidomePattern of Breast Cancer Using Magnetic Beads-Based Plasma Fractionation andMALDI-TOF MS:A Case Control Study in Egypt.Asian Pac J Cancer Prev 2019；20(1):175-184.

12 Xu J,Wang X,Xu P,Liu S,Teng F,Liu X,et al.Mass spectrometry-basedpeptidome profiling of human serous ovarian cancer tissues.Int J Biochem CellBiol.2019；107:53-61.

13 Kotawong K,Thitapakorn V,Roytrakul S,Phaonakrop N,Viyanant V,Na-Bangchang K.Plasma Peptidome as a Source of Biomarkers for Diagnosis ofCholangiocarcinoma.Asian Pac J Cancer Prev.2016；17(3):1163-8.

14Wang L,Tang C,Xu B,Yang L,Qu L,Li L,et al.Mass spectrometry-basedserum peptidome profiling accurately and reliably predicts outcomes ofpemetrexed plus platinum chemotherapy in patients with advanced lungadenocarcinoma.PLoS One.2017；12(6):e0179000.

disclosure of Invention

The object of the present invention is to provide a polypeptide ZYX for the treatment or adjuvant treatment of ovarian cancer, which is against the above-mentioned subgroups of the prior art_36-58。

Another object of the present invention is to provide the use of the polypeptide.

The purpose of the invention can be realized by the following technical scheme:

polypeptide ZYX for treating or assisting in treating ovarian cancer_36-58The amino acid sequence is shown in SEQ ID NO. 1.

The polypeptide ZYX of the invention_36-58The application of the polypeptide as a therapeutic target in preparing a medicament for treating or assisting in treating ovarian cancer.

The polypeptide ZYX of the invention_36-58The application in the preparation of the medicine for treating or assisting in treating ovarian cancer.

A pharmaceutical composition for the therapeutic or adjuvant treatment of ovarian cancer comprising the polypeptide ZYX of claim 1_36-58。

The polypeptide ZYX of the invention_36-58The application of the polypeptide as a detection target in preparing a reagent for diagnosing or assisting in diagnosing ovarian cancer.

Detection of the polypeptide ZYX of the invention_36-58The use of a substance of (a) in the preparation of a reagent for the diagnosis or the aided diagnosis of ovarian cancer.

Reagent for diagnosing or assisting in diagnosing ovarian cancer, and polypeptide ZYX of the invention_36-58The substance of (1).

Has the advantages that:

the invention utilizes a tandem mass spectrometry method to screen polypeptides which are differentially expressed in the serum of an ovarian epithelial cancer patient and the serum of a normal physical examination control, and finds the polypeptide ZYX which has higher abundance in the serum of the normal physical examination control and obviously reduced abundance in the serum of the ovarian epithelial cancer patient_36-58Can obviously inhibit the invasion and migration of ovarian cancer and promote the apoptosis of ovarian cancer. Due to ZYX_36-58The compound has the characteristics of small molecular weight, self source, low toxicity and the like, and can be an important auxiliary medicament for treating ovarian cancer.

Drawings

FIG. 1 polypeptide ZYX_36-58The abundance in the serum of ovarian cancer patients is obviously reduced;

A. the mass spectrum result shows that the quantity of the polypeptide in each tissue is between 1200 and 1400; B. 93% of the polypeptides derived from the precursor protein are 1-4, and the other 6% of the polypeptides derived from the precursor protein are more than 8; C. the peptide mainly comprises 5-26 amino acids, and the molecular size is mainly concentrated on 700-3199; D. compared with the para-cancer tissues, 12 peptides with high differential expression and 36 peptides with low differential expression are contained in the cancer tissues (fold change >2, p < 0.05);

FIG. 2 polypeptide ZYX_36-58Consequences of effects on proliferation and apoptosis of ovarian cancer cells;

A. CCK8 experiment shows that polypeptide ZYX_36-58Treatment did not affect proliferation of SKOV3 cells; B. CCK8 experiment shows that polypeptide ZYX_36-58Treatment did not affect the proliferation of HO8910 cells; C. representative SKOV3 cells in control and ZYX_36-58Analyzing the experimental result of the flow cytometry after the polypeptide treatment; D. representative HO8910 cells in control and ZYX_36-58Polypeptide-treated flow cells; E. quantitative analysis finding ZYX_36-58The treatment can significantly promote apoptosis of SKOV3 cells; F. quantitative analysis finding ZYX_36-58Can remarkably promote the apoptosis of HO8910 cells.

FIG. 3: polypeptide ZYX_36-58Effects on the invasion and migration of ovarian cancer;

A. representative controls and ZYX_36-58Treating the invasion results of SKOV3 cells; B. representative controls and ZYX_36-58Treatment of the invasion results of HO8910 cells; C. quantitative analysis for finding polypeptide ZYX_36-58The treatment can obviously inhibit the invasion of SKOV3 cells; D. quantitative analysis shows that the treatment of the polypeptide ZYX36-58 can obviously inhibit the invasion of HO8910 cells; E. representative controls and ZYX_36-58Processing the scratching results of SKOV3 cells; F. control and ZYX_36-58Treating the scratch results of HO8910 cells; G. quantitative analysis for finding polypeptide ZYX_36-58The treatment can significantly inhibit migration of SKOV3 cells; H. quantitative analysis for finding polypeptide ZYX_36-58The treatment can significantly inhibit migration of HO8910 cells.

Detailed Description

Experimental materials: SKOV3 cell line was purchased from cell bank of Chinese academy of sciences, HO8910 cell was purchased from Jiangsu Kai base biotech stockDivision, Inc. Polypeptide ZYX_36-58: synthesized by Shanghai peptide Biotech, Inc.

Cell culture comprises culturing SKOV3 cells with 10% fetal calf serum, 2200mg/L sodium bicarbonate, L-glutamine, and incomplete culture medium of McCoy's 5A (1 ×) without streptomycin, culturing HO8910 cells with incomplete high-sugar culture medium of DMEM (1 ×) containing 10% fetal calf serum, 4.5g/L D-glucose, 0.584g/L L-glutamine, 3.7g/L sodium bicarbonate, 110g/L sodium pyruvate, and no streptomycin, culturing both cells at 37 deg.C and 5% CO₂The conventional conditioned incubator.

Example 1

1.1 sample Collection

We collected blood of 3 ovarian cancer patients and three age-matched physical examination control healthy persons from the affiliated women's hospital of the university of medical science of south beijing (women's child care institute of south beijing). The blood specimen is centrifuged at 3000g at 4 ℃ for 10 minutes, the supernatant is taken and added with protease inhibitor, and then the supernatant is put at-80 ℃ for standby. All experiments were approved by the ethical committee of the subsidiary gynaecological hospital of the university of medical south Beijing (women's health care institute of Nanjing) and signed patient informed consent.

1.2 polypeptide extraction and Mass Spectrometry

After thawing the samples on ice, 12000g, centrifugation at 4 ℃ for 15 minutes, then supernatant was collected, protein concentration was measured according to BCA method of kit instructions (BCA, pierce, Rockford, IL), 20% acetonitrile (v/v) was added, after gentle mixing, the mixture was left at room temperature for 30 minutes, then the sample was diluted and added to 10kDa molecular trap (Millipore, USA) pretreated with 0.5mL of MilllQ, after centrifugation, the filtrate below 10kDa was lyophilized (Scan Speed Maxi Vac, Labogen, Denmark), then the polypeptide was labeled with TMT according to the instructions of TMT-6 Standard kit (Thermo Fisher Scientific, CA, USA), and finally the labeled polypeptide was mass-spectrometrically detected using a nanoflow LC in combination with LTQ-QQTragos Mass spectrometer.

1.3 liquid chromatography-Mass Spectrometry

Separating the peptide fragment of the sample by using an Shimadzu LC-20AD liquid phase system, wherein self-contained C18 columns (75 microns in inner diameter, 3.6 microns in column material particle size and 15 cm in column length) are connected in series to separate the peptide fragment from the sampleThe 300 nl/min flow rate was separated by an effective gradient as follows: 0-8 min, 5% mobile phase B (98% ACN, 0.1% FA); from 8-43 minutes, mobile phase B increased linearly from 8% to 35%; from 43 to 48 minutes, mobile phase B rose from 35% to 60%; 48-50 minutes, mobile phase B rose from 60% to 80%; 50-55 minutes, 80% mobile phase B; 55-56 minutes, 5% mobile phase B. The peptide fragments after liquid phase separation are ionized by a nanoESI source and then enter a tandem mass spectrometer LTQerbitrap Velos (Thermo Fisher Scientific, San Jose, Calif.) for DDA (data-dependent hybridization) mode detection. The main parameters are set as follows: the nano ion source voltage is set to be 1.8V, the scanning range of the primary mass spectrum is 350-1500 m/z, and the resolution is set to be 30,000; the secondary mass spectrum starts with a fixed m/z of 100 and a resolution of 7,500. The conditions for screening the parent ions for secondary fragmentation were: precursor ions with charges above 2+, 3+ and 4+, with peak intensities above 1000, preceding the precursor 12. The HCD collision energy was set at 35 and fragment ions were detected in Orbitrap. The dynamic exclusion time was 15 s. The AGC is set as: primary 1E6, secondary 5E 4. And finally, matching the data detected by the mass spectrometry with theoretical mass spectrometry data obtained by simulating a database so as to obtain a protein identification result. Referring to FIG. 1, the mass spectrometry results show that the amount of polypeptides in each tissue is between 1200-1400; 93% of the polypeptides derived from the precursor protein are 1-4, and the other 6% of the polypeptides derived from the precursor protein are more than 8; the peptide mainly comprises 5-26 amino acids, and the molecular size is mainly concentrated on 700-3199; compared with the para-carcinoma tissues, there are 12 peptides with high differential expression in the carcinoma tissues and 36 peptides with low differential expression (fold change)>2，p<0.05); selecting one of the differentially underexpressed ZYX_36-58；VNPFRPGDSEPPPAPGAQRAQMG (SEQ ID NO. 1). 1.4 polypeptide Synthesis

The polypeptides used in the experiments were all synthesized by Shanghai peptide Biotechnology, Inc. and dissolved in water at appropriate concentrations: ZYX_36-58VNPFRPGDSEPPPAPGAQRAQMG (SEQ ID NO.1), control scrambled peptide, PPNMAVADERPQQPRSFPGGAGP.

Example 2

2.1 cell proliferation assay

Counting cells, diluting with medium containing 10% FBS to desiredAnd (3) respectively adding the polypeptide into the cell suspension to 100 mu M, adding the cell suspension into a 96-well plate, wherein each 100 mu l of suspension contains about 1000 cells, each suspension is divided into four groups of 0h, 24h, 48h and 72h, each group contains 5-6 auxiliary wells, and the auxiliary wells are placed in an incubator at 37 ℃ and 5% CO2 for 3-4h to wait for the cells to adhere to the wall. Cell activity was measured as group time after cell attachment: replacing the culture medium with a mixed solution added with 10% of CCK-8 reagent, reacting for 1 hour in a 5% CO2 incubator at 37 ℃, detecting by an enzyme-labeling instrument, and detecting the wavelength: 450 nm. The complete medium with added polypeptide was changed every 24 h. The CCK8 experiment result shows that the polypeptide ZYX_36-58Treatment did not affect proliferation of SKOV3 cells and HO8910 cells (fig. 1-2).

2.2 apoptosis assay

Control cells and ZYX were collected after 24 hours of co-culture with the peptide_36-58The treated cells were stained with PE and annexin V using a PE-annexin V apoptosis detection kit (BD Biosciences, San Diego, USA). Apoptosis was analyzed by flow cytometry (Beckman, CA, USA).

Representative SKOV3 cells and HO8910 cells in control and ZYX_36-58The experimental results of the flow cytometry after the polypeptide treatment are respectively shown in the figure 2C and the figure D, and the quantitative analysis finds that ZYX_36-58Treatment significantly promoted apoptosis in SKOV3 cells and HO8910 cells (fig. 2E-F).

Example 3 cell migration and invasion assay

1. The specific steps of the invasion experiment are as follows:

(1) subpackaging Matrigel: after the matrigel is melted on ice, the ice is subpackaged into 1.5ml of EP tubes with 60 mu l of each tube, so that repeated freezing and thawing are avoided;

(2) spreading glue: mixing with serum-free medium according to the proportion of 1: 6(v/v), spreading on the upper surface of a Transwell chamber, 60 mu l/hole, and drying in an incubator at 37 ℃; the migration experiment has no A, B steps;

2. 200ul of a cell suspension containing 5 × 10 was added to the upper surface of the Transwell chamber⁴Cells and 100. mu.M polypeptide, 600 and 800. mu.l of complete medium containing 20% FBS were added to a 24-well plate in which the chamber was placed, and the medium was placed at 37 ℃ in a 5% CO2 incubator. Collecting the migration experiment chamber after 24h of placing in the incubator, and collecting after 48hInvade the laboratory Chamber, place the Transwell Chamber in 4% paraformaldehyde to fix the cells for 60min, transfer the Chamber to 0.1% Crystal Violet dye for 30min, wash the Transwell Chamber 2 times in ddH2O, gently wipe off the residual Matrigel and dye on the upper surface of the Transwell Chamber with a cotton swab, randomly pick 3 areas per well under an inverted microscope (100 ×) to take pictures, see results in FIGS. 3A-B, add 200. mu.l of cell lysis buffer to lyse the cells on the lower surface membrane of the Transwell Chamber, then measure the light absorption at 562nm_36-58Treatment significantly inhibited the invasion of SKOV3 cells and HO8910 cells (fig. 3C-D).

Example 4 scratch test

HO8910 and SKOV3 cells were cultured in six-well plates in medium containing 10% FBS, and at 37 ℃, 5% CO₂Maintaining for 24-36h under the condition until the cell fusion degree reaches 90-100%. After maintaining the cells in serum-free medium for 6-8h, linear scratches were made on the cell layer using a 1000. mu.L pipette tip and replaced with polypeptide-supplemented serum-free medium for 24-48 h. The scratch healing process was observed under an optical microscope and analyzed using Image J (Bethesda, USA) software. The results are shown in FIGS. 3E-F, and the quantitative analysis found that the polypeptide ZYX_36-58Treatment significantly inhibited migration of SKOV3 cells and HO8910 cells (fig. 3G-H).

Sequence listing

<110> Nanjing City health care hospital for women and children

<120> a polypeptide ZYX36-58 for the treatment or co-treatment of ovarian cancer

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>23

<212>PRT

<213> human (amines)

<400>1

Val Asn Pro Phe Arg Pro Gly Asp Ser Glu Pro Pro Pro Ala Pro Gly

1 5 10 15

Ala Gln Arg Ala Gln Met Gly

20

<210>2

<211>23

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Pro Pro Asn Met Ala Val Ala Asp Glu Arg Pro Gln Gln Pro Arg Ser

1 5 10 15

Phe Pro Gly Gly Ala Gly Pro

20

Claims

1. Polypeptide ZYX for treating or assisting in treating ovarian cancer_36-58The polypeptide is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. The polypeptide ZYX of claim 1_36-58The application of the polypeptide as a therapeutic target in preparing a medicament for treating or assisting in treating ovarian cancer.

3. The polypeptide ZYX of claim 1_36-58The application in the preparation of the medicine for treating or assisting in treating ovarian cancer.

4. A pharmaceutical composition for the therapeutic or adjuvant treatment of ovarian cancer comprising the polypeptide ZYX of claim 1_36-58。

5. The polypeptide ZYX of claim 1_36-58The application of the polypeptide as a detection target in preparing a reagent for diagnosing or assisting in diagnosing ovarian cancer.

6. Detecting the polypeptide ZYX of claim 1_36-58The use of a substance of (a) in the preparation of a reagent for the diagnosis or the aided diagnosis of ovarian cancer.

7. Serodiagnosis or assistance for diagnosing ovarian cancerAuxiliary reagent, characterized by comprising the polypeptide ZYX of claim 1_36-58The substance of (1).