CN109646669A - The use in conjunction and combination medicine of 9R-P201 polypeptide and 5FU - Google Patents
The use in conjunction and combination medicine of 9R-P201 polypeptide and 5FU Download PDFInfo
- Publication number
- CN109646669A CN109646669A CN201910086295.1A CN201910086295A CN109646669A CN 109646669 A CN109646669 A CN 109646669A CN 201910086295 A CN201910086295 A CN 201910086295A CN 109646669 A CN109646669 A CN 109646669A
- Authority
- CN
- China
- Prior art keywords
- cell
- hepg2
- drug
- polypeptide
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- A61K38/1761—Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Marine Sciences & Fisheries (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses the use in conjunction methods and combination medicine of a kind of 9R-P201 polypeptide and 5FU, application of the 9R-P201 polypeptide joint 5FU in the drug of preparation treatment liver cancer is specifically referred to, quality proportioning 9:15~20 of 9R-P201 polypeptide and 5FU in prepared combination medicine.Experiment in vitro discovery, combination medicine can reverse HepG2 cell to the resistance of 5FU, cell is improved to the concentration of the sensibility of 5FU and the intracellular 5FU drug of increase, the lethal effect to liver cancer cells can be significantly increased, there is the huge potentiality to be exploited and application prospect for pushing clinical therapy of tumor.
Description
Technical field
The invention belongs to field of medicine preparing technology, and in particular to 9R-P201 polypeptide and the use in conjunction of 5FU and combine use
Drug.
Background technique
Liver cancer is second-biggest-in-the-world cancer mortality reason, and the annual new cases 400,000 of China increase death newly every year
400000, and morbidity and mortality are all rising year by year.5 FU 5 fluorouracil (5FU) is a kind of common chemotherapeutics, can
Effectively treatment Several Kinds of Malignancy.However, for a long time using 5FU meeting because of acquired or endogenous drug resistance, and substantially reducing
Therapeutic effect.A variety of resistance mechanisms are likely to form using 5FU, such as Apoptosis inhibits, drug is excessive and proliferation increases, to drop
The validity of low drug.Wherein, the excessive mechanism of tumour cell drug is attributable to the imbalance of MDR1 gene, leads to the albumen
Overexpression.MDR1, also known as p- glycoprotein (P-gp), P-gp belong to ATP-binding boxlike (ABC) transport protein family,
It is a kind of cross-film pump, for removing a variety of toxic compounds, including main cancer chemotherapeutic drug.
In recent years, with the gradually understanding of biology and molecular signal transduction system to liver cancer cells, molecular targeted medicine
Object becomes the hot spot in the Therapy study field anti-hepatocellular carcinoma (Heptocellular cancer, HCC).FoxM1 is the cell cycle
Progress, DNA replication dna, angiogenesis, transfer, drug resistant key transcription factor belong to a plug cassette family, have the conservative wing
Helical dna binding domain.Multiple studies have shown that FoxM1 takes part in the drug resistance to cis-platinum, taxol, docetaxel and epirubicin,
Illustrate that FoxM1 plays a significant role in drug resistance.Binding peptide P201 disclosed in the prior art has FoxM1c-DBD high affine
Power, and the polypeptide 9R-P201 after modification can inhibit the development processes such as proliferation, migration and the invasion of hepatocellular carcinoma H22 and lure
Guided cell tune is died.
It is had not been reported at present using 9R-P201 polypeptide preparation joint other drugs treatment liver cancer.
Summary of the invention
The object of the present invention is to provide the application of 9R-P201 polypeptide and 5FU in the combination medicine of preparation treatment liver cancer
And the combination medicine for the treatment of liver cancer.
Technical scheme is as follows:
The present invention proposes a kind of application of 9R-P201 polypeptide and 5FU in the combination medicine of preparation treatment liver cancer, to reach
To the purpose of better liver cancer treatment effect.The realization mechanism of liver cancer treatment drug combination is: 9R-P201 is in induction liver cancer cells
While apoptosis, the MDR1 in liver cancer HepG2-R cell can be reversed to express by the inhibition of targeting FOXM1, thus Partial Inverse
The 5FU resistance for turning HepG2 cell, promotes the therapeutic effect of 5FU.In this application, 9R-P201 polypeptide can be by successive with 5FU
Sequence is given, but in order to reach better effect, 9R-P201 polypeptide is with 5FU preferably while giving.
The present invention also provides a kind of combination medicine for treating liver cancer, which includes 9R-P201 polypeptide and 5FU, and
And the quality proportioning of 9R-P201 polypeptide and 5FU are 9:15~20 in drug.Further, the quality of 9R-P201 polypeptide and 5FU are matched
Than being preferably 9:20.
The amino acid sequence of 9R-P201 polypeptide used in the present invention is to be prepared by the following:
Pass through the DNA binding structural domain albumen (FoxM1c-DBD) from the closely related transcription factor FoxM1c of tumour
Phage random dodecapeptide library high flux screening, acquisition sequence are the polypeptide of WHLDYPSMWYLD (SEQ ID No:1), referred to as
P201.9 D- type arginine and two polyglycine serines are added at the end N- of P201 again, being formed has 25 amino acid
Polypeptide sequence, as 9R-P201, the sequence of 9R-P201 are as follows: RRRRRRRRRGSGSWHLDYPSMWYLD (SEQ ID No:2).
The beneficial effects of the present invention are:
Combination medicine in the present invention includes 9R-P201 polypeptide and 5FU, and wherein on the one hand 9R-P201 can inhibit resistance to
The expression of medicine gene FoxM1, thus direct killing cell；On the other hand, 9R-P201 is by the inhibition to MDR1 signal path,
The expression of drug resistant gene MDR1 can be significantly lowered, i.e. 9R-P201 can reverse liver cancer HepG2 thin by targeting the inhibition of FoxM1
MDR1 expression in born of the same parents and 5FU drug resistance hepatoma Hep G 2 cells HepG2/R improves thin to reverse the 5FU resistance of HepG2 cell
Born of the same parents are to the sensibility of 5FU and the concentration of the intracellular 5FU drug of increase, to enhance the lethal effect of cell.Two kinds medication combined
It uses, each single medicine group is significantly stronger than to the inhibition lethal effect of HepG2 cell, more preferably to the therapeutic effect of liver cancer.
Detailed description of the invention
Fig. 1 is the variation of HepG2 cellular morphology in 5FU Induction Process；
Fig. 2 be HepG2 cell through drug-treated for 24 hours after cellular morphology variation；
Fig. 3 is the variation of HepG2 cell cellular morphology after drug-treated 48h；
Fig. 4 is the variation of 5FU mdr cell (HepG2/R) cellular morphology after drug-treated 48h of HepG2；
Fig. 5 be HepG2 cell through drug-treated for 24 hours after CCK-8 testing result；
Fig. 6 is CCK-8 testing result of the HepG2 cell after drug-treated 48h；
Fig. 7 is CCK-8 testing result of the HepG2/R cell after drug-treated 48h；
Fig. 8 is qRT-PCR testing result of the HepG2 cell after drug-treated 48h；
Fig. 9 is the mrna expression amount of HepG2 cell and HepG2/R cell FoxM1 after drug-treated；
Figure 10 is the mrna expression amount of HepG2 cell and HepG2/R cell MDR1 after drug-treated；
Figure 11 is qRT-PCR testing result of the HepG2/R cell after drug-treated 48h；
Figure 12 is HepG2 cell and AO/EB double fluorescent staining result of the HepG2/R cell after drug-treated；
Figure 13 is HepG2 cell and Flow cytometry result of the HepG2/R cell after drug-treated；
Figure 14 is cell scratch experiment result of the HepG2 cell after drug-treated；
Figure 15 is cell scratch experiment result of the HepG2/R cell after drug-treated；
Figure 16 is that drug acts on the inhibition of metastasis of HepG2 cell；
Figure 17 is that drug acts on the inhibition of metastasis of HepG2/R cell；
Figure 18 is that Transwell cell of the HepG2 cell after drug-treated migrates experimental result；
Figure 19 is that Transwell cell of the HepG2/R cell after drug-treated migrates experimental result.
Figure 20 is that the cell Transwell of HepG2 cell and HepG2/R cell after drug-treated migrates experimental result.
Specific embodiment
One, the present invention is tested the material and reagent to be used
Source of people HepG2 cell, source of people liver cancer 5FU mdr cell HepG2/R, 9R-P201 polypeptide, 5 FU 5 fluorouracil
(5FU), DMEM (Gibco), fetal calf serum, Trypsin-EDTA (Gibco), CCK-8 kit (the triumphant base biology in Jiangsu), AO-
EB double fluorescent staining kit, Annexin V-FITC cell apoptosis detection kit (the triumphant limited public affairs of base biotechnology share in Jiangsu
Department), Trizol@Reagent, Reverse Transcriptase kit (Thermo Fischer Scient Inc.), PCR kit for fluorescence quantitative (examine by Roche
Stopping pregnancy product Shanghai Co., Ltd), anti-FoxM1, MDR1 mouse monoclonal antibody, sheep anti-mouse igg-Biotin secondary antibody (Wuhan doctor's moral
Company), ECL chemical luminescence reagent kit (Thermo Fischer Scient Inc.), PCR amplification instrument (BIO-RAD 96), DYY6B type pressure stabilizing
Warm flow electrophoresis instrument (Liuyi Instruments Plant, Beijing), 3001 type microplate reader (Thermo Fisher).
Two, experimental method
1. cell culture
Source of people hepatocellular carcinoma H22 cell strain is by this laboratory passage conservation, mdr cell HepG2/R (this laboratory structure
Build) 37 DEG C, 5%CO are incubated at using DMEM culture medium (100U/mL penicillin, 100U/mL streptomysin, 10%FBS)2, saturation
In the cell incubator of humidity.
2. experimental group
Blank control group (only plus culture solution), cell controls group, the mono- medicine control group of 9R-P201 (45~50 μ g/ are set up in experiment
ML), the mono- medicine group of 5FU (80~100 μ g/mL), drug combination group (45 μ g/mL+5FU of 9R-P201,80 μ g/mL；9R-P201 45
μg/mL+5FU 100μg/mL；9R-P201 50μg/mL+5FU 80μg/mL).
3. morphological observation and the detection of CCK-8 method cell viability
HepG2 cell and mdr cell HepG2/R are reached the third generation to be used to test.It is digested through pancreatin, with 5.0 × 103
A/hole is inoculated with 100 μ L in 96 orifice plates, different drug-treateds is carried out after being incubated overnight, every group of concentration sets 6 repeating holes.Culture
In 37 DEG C, 5%CO2, saturated humidity cell incubator in, observe and cellular morphology and take pictures after 48h, then 10 μ L are added in every hole
CCK-8 solution is protected from light in incubator and is incubated for 1.5h, measured under 450nm wavelength using microplate reader.Cell survival rate calculation formula
It is as follows:
In formula, A0: zeroing group absorbance value, A1: experimental group absorbance value, A: blank group absorbance value.
4. cell scratch (Wound healing) is tested
Human liver cancer cell HepG2 in good condition, mdr cell HepG2/R are pressed 2.5 × 105Cells/well is spread to 6
Orifice plate, 37 DEG C of incubations to cell fusion reach 80-90%.Then with the sterile pipette tips scratch of 200 μ L, culture medium is sucked, (4 DEG C pre- by PBS
It is cold) cell under drawing is washed away, each experimental group handles HepG2 respectively, and HepG2/R cell is for 24 hours and 48h.After reaching action time,
It sets microscopically observation, take pictures.Finally use Image J quantitative comparison cell migration rates.Test 3 repetitions.
5.Transwell Cell migration assay
HepG2 cell is adjusted after blank control group, each drug-treated group effect 48h are set and HepG2/R mdr cell is dense
It spends to 2.5 × 104Cell suspension is added in the upper chamber of the cell Transwell by a/mL by 200 holes μ L/, while in lower room
Middle addition DMEM culture solution of the 800 μ L containing 10%FBS.Continue after cultivating 12h in incubator, PBS is washed 2 times, and 4% poly is added
Formaldehyde fixes 15min, 1% violet staining 15min, and micro- sem observation is taken pictures.
Acridine orange/ethidium bromide 6. (AO-EB) double fluorescent staining
HepG2, mdr cell HepG2/R cell press 2.5 × 104Cells/well is spread to six orifice plates for being put into coverslip
In, it is incubated overnight rear each dosing group and handles 48h respectively, take out coverslip after PBS washing, it is preparatory that 25 μ L are dripped on every coverslip
The AO-EB working solution (+100 μ L PBS of+20 μ L EB liquid storage of 20 μ L AO liquid storage, storage is protected from light after mixing) of configuration, by coverslip
It is placed on glass slide, fluorescence microscopy is taken pictures under the microscope.
The detection of 7.Annexin V-FITC/PI Apoptosis
HepG2, mdr cell HepG2/R are respectively after each medicine group acts on 48h, and experiment is collected in grouping and control group is thin
Born of the same parents therefrom take 1 × 10 after PBS washing5-5×105A cell removes supernatant after centrifugation, 500 μ L Annexin V-FITC/PI are added
Binding Buffer is resuspended, and sequentially adds 5 μ L Annexin V-FITC, 5 μ L PI mixing, and room temperature, which is protected from light, is incubated for 15min,
So analyzed recklessly with flow cytomery.
8. plate clone forms experiment
HepG2 in good condition, mdr cell HepG2/R are pressed 1.0 × 103Cells/dish is spread respectively to culture dish
(Φ=6.0cm), 37 DEG C are incubated for rear each group agent-feeding treatment for 24 hours, and the culture solution containing each processing medicine, coexistence were then changed every 3 days
Reason 2 weeks after reaching action time, sucks culture medium, 4% poly formic acid fixed 20min, violet staining 20min, PBS elution 3
Secondary, camera is taken pictures, and is counted as a clone to clone number > 50.
9.qRT-PCR
HepG2, mdr cell HepG2/R are pressed 2.5 × 105Cells/well is incubated overnight in 6 orifice plates, then each group
Agent-feeding treatment 48h.Sample total serum IgE is extracted referring to Trizol method, further according to Invitrogen M-MLV the first chain synthetic agent box
Specification carries out the synthesis of total cDNA.Exist referring to FastStart DNA Green Master (Roche) kit specification
QRT-PCR is carried out on LightCycler96 instrument (Roche company, Switzerland).Experiment detection gene and internal reference GAPDH primer information are such as
Shown in table 1.
1 qRT-PCR of table detects gene and internal reference GAPDH primer information
10. statistical analysis
Using SPSS19.0 software to experiment the data obtained carry out statistical analysis, all chart numerical value with mean ±
S.D. it indicates, using the significant difference between two groups of data of t check analysis, P < 0.05 thinks with significant difference.
Below with reference to embodiment, specific embodiments of the present invention will be described in detail.
Embodiment one: building mdr cell HepG2/R
Using obtaining mdr cell HepG2/R, morphology after chemotherapeutics 5FU repeated action hepatoma H22 cells
Significant change occurs, as shown in Figure 1.When initial low concentration 5FU induction parental cell HepG2 volume become larger and be rounded (Fig. 1 (a),
Induce 12h), as in uneven antenniferous shuttle shape, (Fig. 1 (b) induces 48h to the increase cell of induced concentration and induction time；
Fig. 1 (c) induces 72h).Mdr cell HepG2/R and parent HepG2 cell are respectively 628 μ g/mL and 282 μ to the IC50 of 5FU
g/mL；Mdr cell HepG2/R cell is reduced compared with sensibility of the parent HepG2 cell to 5FU inhibited proliferation, when 5FU is dense
When degree is 100 μ g/mL or less, with the increase of action time, HepG2 cell is to 5FU inhibited proliferation compared with mdr cell
HepG2/R is also obviously increased, and so far mdr cell constructs successfully.
Embodiment two: suppression of the 9R-P201 polypeptide joint 5FU to hepatocellular carcinoma H22 and its mdr cell HepG2/R is investigated
Lethal effect processed
1. respectively with 5FU (70~100 μ g/mL) and 9R-P201 (45~50 μ g/mL) individually to HepG2 cell at
Reason, and combined with 9R-P201+5FU and HepG2 cell is handled, as a result respectively such as Fig. 2 (processing is for 24 hours) and Fig. 3 (processing
Shown in 48h).As can be seen that processing group is compared with the HepG2 cell of blank control group, significant change is had occurred in cellular morphology,
The cell of HepG2 blank control group (Control) is in uniform HepG2 cell that is round, and handling by 5FU, with drug
The increase cell volume of concentration and action time, which become larger, to be rounded and largely floats；The HepG2 cell of 9R-P201 processing, with
Drug concentration and the increase of action time show as intracellular granular and increase and shrinkage, broken；As drug combination (9R-P201+
When 5FU) concentration for the treatment of reaches 9R-P201 (45 μ g/mL)+5FU (100 μ g/mL), can obviously it be observed under optical microscopy
The death of HepG2 cell.
2. individually being carried out respectively to HepG2/R cell with 5FU (70~100 μ g/mL) and 9R-P201 (45~50 μ g/mL)
Processing, and combined with 9R-P201+5FU and HepG2/R cell is handled, as a result as shown in Figure 4.It can be seen that processing group
Compared with the HepG2/R cell of blank control group, significant change, HepG2/R blank control group is equally had occurred in cellular morphology
(Control) cell is in uneven antenniferous shuttle shape, and passes through the HepG2/R cell of 5FU processing, and initial cell volume becomes
It is rounded greatly, with drug concentration and the increase cell of action time is in unevenly antenniferous shuttle shape but floating cells are seldom, 9R-
P201 handle HepG2/R cell, when 9R-P201 concentration equally reaches 45 μ g/mL compared with HepG2/R cells show be intracellular
Grain significantly increases and shrinkage, broken；When drug combination (9R-P201+5FU) concentration for the treatment of reaches P201 (45 μ g/mL)+5FU
When (100 μ g/mL), it is able to observe that HepG2/R cell is significantly dead under optical microscopy.
3. being shown simultaneously by CCK-8 testing result, drug combination group (the 9R-P201/45 μ g/mL+5FU/ of various concentration
80μg/mL；9R-P201/45μg/mL+5FU/100μg/mL；9R-P201/50 μ g/mL+5FU/80 μ g/mL) intervene HepG2 it is thin
After born of the same parents and HepG2/R mdr cell 48h, compared with the control group, two plants of Apoptosis can be significantly induced, with drug concentration
Increase, induces two plants of cells apoptosis to gradually increase, the 9R-P201 (45 μ g/mL) with same concentrations, 5FU (100 μ g/
ML) single medicine group compares, and drug combination group (9R-P201/45 μ g/mL+5FU/100 μ g/mL) induces the effect of two plants of Apoptosis
It is stronger, and difference is extremely significant (P < 0.001), has synergistic effect between two medicines.As can be seen that drug combination reaches from Fig. 5~7
The optimal processing time acted on to maximum suppression is 48h, and optimal joint concentration is 9R-P201/45 μ g/mL+5FU/100 μ g/mL.
Therefore subsequent experimental controls 9R-P201 (45 μ g/mL), 5FU (100 μ g/mL), and the processing time is 48h.
Embodiment three: the correlation of FoxM1 and MDR1 expression quantity in two plants of cells is investigated
Pass through the expression of related gene and albumen after fluorescence quantitative PCR detection drug combination two plants of cells of processing.Such as
Shown in Fig. 8, compared with control group HepG2, after 5FU individually handles 48h, FoxM1 is lowered HepG2 cell, MDR1 up-regulation；And it passes through
After 9R-P201 is individually handled, FoxM1 up-regulation, MDR1 is lowered；Drug combination group, FoxM1 and MDR1 are significantly lowered, FOXO3a
Up-regulation, compared with single medicine group, drug combination group (9R-P201+5FU) group inhibits FoxM1, and the mRNA expressional function of MDR1 is stronger,
With significant difference (P < 0.01).And the mrna expression amount of FoxM1 and MDR1 is significantly higher than in mdr cell HepG2/R
HepG2 cell, difference is extremely significant (P < 0.01), sees Fig. 9 and Figure 10, and compared with control group HepG2/R, mdr cell HepG2/
R can significantly lower drug resistance after 9R-P201,5FU are individually handled and drug combination handles HepG2/R mdr cell 48h
The mrna expression amount of FoxM1 and MDR1, such as Figure 11 in cell HepG2/R.And find out from CCK-8 detection, it is in 9R-P201 concentration
When 45 μ g/mL, 9R-P201 is significantly stronger than it to HepG2 (43%) to the inhibition lethal effect (60%) of mdr cell HepG2/R
Lethal effect.It can explain are as follows: HepG2/R cell is HepG2 cell by obtaining after 5FU repeated action, although to chemotherapeutic
5FU generates drug resistance, but its sensibility to 5FU chemotherapeutic is increased after 9R-P201 is handled.Compared with single medicine group, connection
Sharing medicine group (9R-P201+5FU) inhibits the mRNA expressional function of FoxM1 and MDR1 stronger, between drug combination group and two single medicine groups
Difference has statistical significance (P < 0.01).
Example IV: influence of the drug combination to HepG2 and its mdr cell HepG2/R apoptosis is investigated
Pass through the double dye experiment discoveries of AO-EB cell fluorescence, under fluorescence microscope, HepG2 and its mdr cell HepG2/R
Blank control group cell size form is uniform, uniform green fluorescence occurs；And treated that cell shape is then presented for each medication group
There is the different pyknosis and fluorescent orange of degree in irregular variation, nuclear chromatin, typical Apoptosis feature are presented, and join
Share cell proportion of the medicine group with apoptosis feature in significant ascendant trend, as shown in figure 12.
Further pass through Apoptosis by Flow Cytometry situation, discovery HepG2 cell 5FU group, 9R-P201 group and connection
Sharing medicine group (9R-P201+5FU) early apoptosis rate is 8.04%, 7.75% and 19.73% respectively, and late apoptic rate is respectively
11.37%, 17.41% and 23.68%.And HepG2/R mdr cell 5FU group, 9R-P201 group and drug combination group (9R-P201
+ 5FU) early apoptosis rate is respectively 6.36%, 5.69% and 12.34%, and late apoptic rate is respectively 7.25%, 17.68% and
24.69%, show that 5FU compared with 5FU and 9R-P201 is used alone, can dramatically increase cell and wither with 9R-P201 use in conjunction
It dies；As a result as shown in figure 13.
Embodiment five: proliferation and migration that drug combination inhibits cancer cell HepG2 and its mdr cell HepG2/R are investigated
By scratch experiment and the migration experiment of the cell Transwell as can be seen that drug combination handles HepG2 and its drug resistance
After cell HepG2/R, its clonality, migration distance are decreased obviously compared with control group and each single medicine processing group.From
It is thin that Figure 14 and Figure 15 can be seen that drug combination group HepG2 and its mdr cell HepG2/R after handling 48h in scratch experiment
The amplitude that born of the same parents' scratch reduces is reduced, and the healing ability of scratch weakens；It can be seen that drug combination group is opposite from Figure 16 and Figure 17 to move
Shifting is zero, and a large amount of cell deaths occurs, and significant difference (P < 0.01) is presented compared with control group and each single medicine group.From Figure 18,
Figure 19 and Figure 20 can be seen that in the migration experiment of the cell Transwell, drug combination group HepG2 and its mdr cell HepG2/R
Migrating cell rate is respectively 10% and 9%.
Although in conjunction with the embodiments and attached drawing is described in detail a specific embodiment of the invention, should not be understood
For the restriction of the protection scope to this patent.In range described by claims, those skilled in the art are without creation
Property the various modifications that can make of labour and deformation still belong to the protection scope of this patent.
Sequence table
<110>Southwest Jiaotong University
<120>use in conjunction and combination medicine of 9R-P201 polypeptide and 5FU
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Trp His Leu Asp Tyr Pro Ser Met Trp Tyr Leu Asp
1 5 10
<210> 2
<211> 25
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Ser Gly Ser Trp His Leu
1 5 10 15
Asp Tyr Pro Ser Met Trp Tyr Leu Asp
20 25
Claims (5)
- The application of 1.9R-P201 polypeptide and 5FU in the combination medicine of preparation treatment liver cancer.
- 2. application according to claim 1, it is characterised in that: 9R-P201 polypeptide is given simultaneously with 5FU.
- 3. a kind of combination medicine for treating liver cancer, it is characterised in that: including 9R-P201 polypeptide and 5FU.
- 4. combination medicine according to claim 3, it is characterised in that: in the combination medicine 9R-P201 polypeptide with The quality proportioning of 5FU is 9:15~20.
- 5. combination medicine according to claim 4, it is characterised in that: the quality proportioning of 9R-P201 polypeptide and 5FU are 9: 20。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910086295.1A CN109646669A (en) | 2019-01-29 | 2019-01-29 | The use in conjunction and combination medicine of 9R-P201 polypeptide and 5FU |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910086295.1A CN109646669A (en) | 2019-01-29 | 2019-01-29 | The use in conjunction and combination medicine of 9R-P201 polypeptide and 5FU |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109646669A true CN109646669A (en) | 2019-04-19 |
Family
ID=66120857
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910086295.1A Pending CN109646669A (en) | 2019-01-29 | 2019-01-29 | The use in conjunction and combination medicine of 9R-P201 polypeptide and 5FU |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109646669A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111320671A (en) * | 2020-03-06 | 2020-06-23 | 西南交通大学 | P201 optimized peptide, anti-tumor polypeptide, medicine prepared from same and targeted inhibitor |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH031950B2 (en) * | 1984-03-24 | 1991-01-11 | Kyomi Sato | |
| CN104267188A (en) * | 2014-08-28 | 2015-01-07 | 汪建平 | Applications of related preparations aiming at MSK1 gene in preparation of 5-FU drug resistance detection reagent and 5-FU drug resistance reversal agent |
| CN105524159A (en) * | 2015-11-16 | 2016-04-27 | 西南交通大学 | Polypeptide molecule exerting selective killing and migration inhibiting effect on cancer cells, and design method and application thereof |
| CN109125333A (en) * | 2018-08-18 | 2019-01-04 | 内蒙古大学 | Penta-O-galloyl-D-glucopyranose and 5 FU 5 fluorouracil are combined inhibiting effect and its application to HepG2 cancer cell invasion phenotype |
-
2019
- 2019-01-29 CN CN201910086295.1A patent/CN109646669A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH031950B2 (en) * | 1984-03-24 | 1991-01-11 | Kyomi Sato | |
| CN104267188A (en) * | 2014-08-28 | 2015-01-07 | 汪建平 | Applications of related preparations aiming at MSK1 gene in preparation of 5-FU drug resistance detection reagent and 5-FU drug resistance reversal agent |
| CN105524159A (en) * | 2015-11-16 | 2016-04-27 | 西南交通大学 | Polypeptide molecule exerting selective killing and migration inhibiting effect on cancer cells, and design method and application thereof |
| CN109125333A (en) * | 2018-08-18 | 2019-01-04 | 内蒙古大学 | Penta-O-galloyl-D-glucopyranose and 5 FU 5 fluorouracil are combined inhibiting effect and its application to HepG2 cancer cell invasion phenotype |
Non-Patent Citations (3)
| Title |
|---|
| KAORU OKADA等: "Overexpression of Forkhead Box M1 Transcription Factor (FOXM1) is a Potential Prognostic Marker and Enhances Chemoresistance for Docetaxel in Gastric Cancer", 《ANNALS OF SURGICAL ONCOLOGY》 * |
| 毕振飞: "多肽9R-P201对肝癌细胞HepG2的抑制及其作用机制研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 雷越等: "下调 FoxM1 通过激活 JNK /线粒体通路增加人鼻咽癌细胞对紫杉醇的敏感性", 《第三军医大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111320671A (en) * | 2020-03-06 | 2020-06-23 | 西南交通大学 | P201 optimized peptide, anti-tumor polypeptide, medicine prepared from same and targeted inhibitor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20210228573A1 (en) | Novel application of gzd824 and pharmaceutically acceptable salts thereof in treating diseases | |
| Liu et al. | Involvement of the mitochondrial pathway in bruceine D-induced apoptosis in Capan-2 human pancreatic adenocarcinoma cells | |
| Chen et al. | Baicalin inhibits EMT through PDK1/AKT signaling in human nonsmall cell lung cancer | |
| Song et al. | Ad-apoptin inhibits glycolysis, migration and invasion in lung cancer cells targeting AMPK/mTOR signaling pathway | |
| Xiao et al. | Flavokawain A suppresses the vasculogenic mimicry of HCC by inhibiting CXCL12 mediated EMT | |
| Yang et al. | Ruyiping extract reduces lung metastasis in triple negative breast cancer by regulating macrophage polarization | |
| Karsch-Bluman et al. | High mobility group box 1 antagonist limits metastatic seeding in the lungs via reduction of cell–cell adhesion | |
| CN109568299A (en) | Ambroxol purposes in preparing tumor chemotherapeutic drug Synergistic preparations | |
| Kuo et al. | Antiangiogenic and antihepatocellular carcinoma activities of the Juniperus chinensis extract | |
| CN109646669A (en) | The use in conjunction and combination medicine of 9R-P201 polypeptide and 5FU | |
| Pamuk et al. | Effects of tacrolimus and nifedipine, alone or in combination, on gingival tissues | |
| CN108303552A (en) | Detection method of the cucoline to pancreatic carcinoma SW1990 Proliferation and apoptosis | |
| CN113230259A (en) | Pharmaceutical application of compound istradefylline capable of reversing drug resistance of paclitaxel | |
| CN107823644A (en) | A kind of application of NO compound donators in the invasion and attack and transfer ability medicine that suppress the tumour cell rich in thiol molecule are prepared | |
| Zhang et al. | Portal vein tumor thrombus is more sensitive to irradiation | |
| CN115414465B (en) | Application of JWA polypeptide in the preparation of anti-tumor drug synergist | |
| NL2030464B1 (en) | Aralia armata (wall.) seem-derived total saponin and use thereof in preparation of drug for treating post-vascular injury restenosis | |
| CN116808018A (en) | Application of arnica lactone C in preparing tumor chemosensitization medicines | |
| CN111035752B (en) | Application of silkworm antimicrobial peptide Cecropin A in the treatment of esophageal cancer | |
| Wang et al. | Effects and mechanism of siomycin A on the growth and apoptosis of MiaPaCa-2 cancer cells | |
| CN104459151B (en) | Diagnosis of colon cancer reagent, kit | |
| CN113476466A (en) | Application of baicalin in preparation of drug resistant to chronic myeloid leukemia | |
| CN110664821A (en) | Application of Panaxadiol in the Preparation of Drugs for Inhibiting the Expression of PD-L1 and Tumor Cell Proliferation Proteins | |
| Shen et al. | Extract of Astragali Radix and Solanum nigrum Linne regulates microglia and macrophage polarization and inhibits the growth and infiltration of C6 glioblastoma | |
| CN113116917B (en) | Application of 8-Br-cGMP as PKG I activator in the preparation of medicaments for preventing or treating epithelial ovarian cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |