CN108567981A - Application of the antagonist of interleukin-6 in preparing inhibition Notch-1 protein expressions, treating the drug of cancer of pancreas - Google Patents
Application of the antagonist of interleukin-6 in preparing inhibition Notch-1 protein expressions, treating the drug of cancer of pancreas Download PDFInfo
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- CN108567981A CN108567981A CN201810780656.8A CN201810780656A CN108567981A CN 108567981 A CN108567981 A CN 108567981A CN 201810780656 A CN201810780656 A CN 201810780656A CN 108567981 A CN108567981 A CN 108567981A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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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
Abstract
The present invention provides a kind of application of antagonist of interleukin-6 in preparing 1 protein expressions of inhibition Notch, treating the drug of cancer of pancreas.Research and inquirement cytokine interleukin element 6（IL‑6）The influence that human pancreatic cancer cell is proliferated and is migrated.The proliferation activity of cell is detected using 8 methods of CCK and draws growth curve, colony formation detects Cell colonies assay, and cell scratch experiment detects cell migration situation, the expression of protein immunoblotting experiment detection cell IL 6,1 albumen of Notch.The results show that as 6 concentration of IL increases, ability of cell proliferation significantly increases, and cloning efficiency and cell migration rate increase；The expression of IL 6, the expression of 1 albumen of Notch is inhibited also to decrease.Cell factor IL 6 may influence proliferation and the migration of pancreatic cancer cell Mia paca 2 by adjusting 1 pathway activities of Notch.Current results are that the interaction mechanism of further research IL 6 and Notch accesses is laid a good foundation.
Description
Technical field
The invention belongs to field of biomedicine technology, and in particular to a kind of antagonist of interleukin-6 inhibits preparing
Notch-1 protein expressions, treat cancer of pancreas drug in application.
Background technology
Cancer of pancreas is a kind of common malignant tumour, and death rate height, poor prognosis occupy the 7th of the global cancer cause of the death, west
The 4th of Fang Guojia, the death rate occupy the 4th in China, number of the infected compared with 10 years before the trend that is significantly increased.At present
The not diagnosis marker of early stage had been treated both for cancer of pancreas, also without effective treatment means, in addition to Operation in early stage,
Other treatment method produces little effect, but when cancer of pancreas is generally made a definite diagnosis is exactly late period.And due to the effect of postoperative adjuvant therapy
The five year survival rate of the factors such as bad, cancer of pancreas is only less than 5%.Molecular marked compound screening in chronic inflammation vicious transformation exists
Carry out in the Partial tumors that the mankind occur and successful.It is the same with other solid tumors, treatment of pancreatic cancer success or not,
It is heavily dependent on the early diagnosis and treatment of cancer of pancreas.Due to lacking the biomarker of special sign and early diagnosis
Object brings very big difficulty to the Clinics and Practices of cancer of pancreas.It was found that efficiently, sensitive, specific biomarkers to early detection,
Early diagnosis cancer of pancreas, improvement patient's prognosis are quite important.
Research shows that the chronic inflammatory state of microenvironment and the occurrence and development of cancer of pancreas are closely related, infiltrated in interstitial components
Panimmunity cell.However, under the microenvironment of tumor inducing, the normal immunological function of body is converted into suitable tumour growth
Immunosuppressive condition, hinder immune surveillance system kill tumour cell.Anti-tumor immune response and tumour cell are relevant
Balance between immunosuppressive condition generates important role for the proliferation of tumour, invasion and transfer.Therefore it is directed to cancer of pancreas
The research of immune microenvironment, it is the new hope of cancer of pancreas immunization therapy to reverse immunosuppressive condition, activation host immune function.
Interleukin-6（Interleukin-6, IL-6）It is a kind of multifunctional cytokine and complicated thin of body
Key factor in intracellular cytokine network is mainly generated by mononuclear macrophage, endothelial cell and lymphoid cell, is a kind of multiple-effect
Cell factor, IL-6 is answered mainly to play effect in a manner of autocrine or paracrine, it can adjust the effect of other cell factors,
Main inducing inflammatory reaction.
Invention content
The object of the present invention is to provide a kind of antagonist of interleukin-6 prepare inhibit Notch-1 protein expressions,
Treat the application in the drug of cancer of pancreas.
The drug is made of the antagonist and pharmaceutically acceptable carrier or excipient of interleukin-6.
Preferably, the antagonist of the interleukin-6 is purchased from Ai Bokang（Shanghai）The IL-6 of trade Co., Ltd
Antibody（Abcam, ab6672).
The beneficial effect comprise that：The present invention determines external increasings of the IL-6 to Mia-paca-2 pancreatic cancer cells
Grow capacity.As a result, it has been found that with the increase of IL-6 concentration, the appreciation rate of cell growth gradually rises.Further using clone
Experiment detection cell Proliferation variation is formed, cell scratch experiment detects cell migration capacity variation.As a result it again shows that and IL- is added
The proliferation and transfer ability of 6, Mia-paca-2 cells obviously increase, and illustrate that IL-6 makees the proliferation of pancreatic cancer cell and migration
With having a significant effect, and after IL-6 antibody is added, with the increase of IL-6 antibody dosages, cell viability is remarkably decreased.
Description of the drawings
Fig. 1 is influences of the IL-6 to pancreatic cancer cell vigor；
Fig. 2 is the influence of IL-6, DAPT to Cell Proliferation of Pancreatic Cancer Cell vigor；
Influences of Fig. 3 various concentrations IL-6 to pancreatic cancer cell clonality；
Fig. 4 is the influence that various concentration IL-6 migrates pancreatic cancer cell Mia-paca-2；
Fig. 5 is influences of the various concentration IL-6 to pancreatic cancer cell Notch1 protein expressions；
Fig. 6 is influence of the antibody of various concentration interleukin-6 to Cell Proliferation of Pancreatic Cancer Cell.
Specific implementation mode
1 materials and methods
1.1 materials and reagent
Human pancreas cancer cell strain Mia-capa-2 is purchased from Wuhan Pu Nuosai Life Sciences Co., Ltd.Fetal calf serum FBS is purchased from
Gibco companies, DMEM culture medium HyClone companies, rabbit-anti people IL-6, Notch1,3,4 antibody are purchased from abcam companies, rabbit-anti people
Myh9 antibody, β-actin antibody are purchased from Wuhan Boster Biological Technology Co., Ltd., and cell factor Human IL-6 are purchased from the U.S.
Peprotech companies.
1.2 method
1.2.1 cell culture
Mia-capa-2 is placed in 37 DEG C, 5% CO in the DMEM culture mediums containing 10% fetal calf serum2Incubator culture, every 2 d are changed
Culture medium 1 time, observation cell to exponential phase carry out next step experiment.
1.2.2 the proliferative capacity of CCK-8 methods detection pancreatic cancer cell
1.2.2.1 influences of the CCK-8 methods detection cell factor IL-6 to Cell Proliferation of Pancreatic Cancer Cell
By 0.5% trypsin digestion of the attached cell of exponential phase, 1000rpm centrifuges 5min, 1 × phosphate buffer
（PBS）Cleaning 1 time, with 2 × 103/ hole is inoculated in 96 orifice plates, per the culture medium without fetal calf serum of 100 μ L of empty access, culture
For 24 hours, the IL-6 of various concentration is added（0、12.5、25、50、100ng/mL）Effect for 24 hours, while setting blank control（Not refinement
Born of the same parents）, the CCK-8 solution of 5 μ L, CO are added per hole2Cell incubator is incubated 40min, is measured at enzyme-linked immunosorbent assay instrument 450nm
The optical density in each hole（OD）Value.3 repetitions are set per hole to be averaged, and cell Proliferation vigor is calculated according to following equation.Cell increases
Grow vigor（%）=（Processing group OD values-blank group OD values）/（Control group OD values-blank group OD values）×100%.
1.2.2.2 influences of the CCK-8 methods detection DAPT to Cell Proliferation of Pancreatic Cancer Cell
By 0.5% trypsin digestion of the attached cell of exponential phase, 1000rpm centrifuges 5min, with 2 × 103/ hole is inoculated with
In 96 orifice plates, two plates are inoculated with, the culture medium without fetal calf serum of access 100uL, culture for 24 hours, change into containing serum just per hole
The DAPT of various concentration is added in normal culture medium（0、1、10、100、1000uM/mL）, 100ng/mL is added after another plate 30min
IL-6, while setting blank control（It is not added with cell）, the 24 good rear CCK-8 solution that 5 μ L are added per hole, CO2Cell incubator is incubated
40min measures the optical density of each sky at enzyme-linked immunosorbent assay instrument 450nm（OD）Value.3 repetitions are set per hole to be averaged, root
Cell Proliferation vigor is calculated according to following equation.Cell Proliferation vigor（%）=（Processing group OD values-blank group OD values）/（Control group OD
Value-blank group OD values）×100%.
1.2.3 cell clonal formation experiment detection clonality
The cell for digesting exponential phase spreads 4 wares per about 200 cells of ware, and 10mL culture mediums containing 10%FBS are added, and makes thin
Born of the same parents are evenly dispersed.Various concentration DAPT is first added in two of which ware（5、10uM/mL）30min is acted on, is then added in each ware
100ng/mL IL-6, while setting blank control（It is added without any drug）.Set 37 DEG C, 5%CO27d is cultivated in cell incubator,
Culture is terminated when occurring macroscopic clone in culture dish.Cell is fixed using 4% paraformaldehyde solution, 0.1% after air-drying
Violet staining slowly washes away dyeing liquor with flowing water, is air-dried.It observes and counts number of cell clones.
1.2.4 the influence that cell scratch experiment detection IL-6 migrates pancreatic cancer cell
6 orifice plates are taken, and are crossed behind in 6 orifice plates, exponential phase cell is digested, is inoculated in 6 orifice plates, per hole about 5 × 105It is a
Cell is incubated overnight to aperture is paved with, and with white pipette tips perpendicular to behind horizontal line cut, 1 × PBS cleans cell 3 times, and addition contains
Different IL-6 concentration without fetal calf serum culture medium, CO2Cell incubator culture, 0h, 6h, for 24 hours observation are taken pictures.
1.2.5 Western blotting detect the expression of notch albumen
Exponential phase human pancreas cancer Mia-paca-2 cells are digested, 8 wares is spread, is divided to two groups, every group 4, divides in the first set
It Jia Ru not various concentration IL-6（0、50、100、200ng/mL）；Second group（A、B、C、D）10uM DAPT are added in two ware of A, B
30min is acted on, 100ng/mL IL-6 are then added in two ware of A, C, cell incubator culture is for 24 hours.Collect exponential phase
Cell extracts total protein of cell.After carrying out SDS-PAGE, it is transferred to nitrocellulose filter（PVDF）, 4 DEG C are closed overnight, are added
Human antibody and internal reference control β-actin antibody incubations 1h, 1 × PBS are washed 3 times, and horseradish peroxidase-labeled secondary antibody is added to be incubated
1h, 1 × PBS are washed 3 times, and the development of ECL chemiluminescence detections is taken pictures and analyzed on gel imaging system.
As a result
2.1 cell factor IL-6 influences the proliferative capacity of pancreatic cancer cell Mia-capa-2
Various concentration recombination IL-6 acts on human pancreas cancer cell strain Mia-paca-2, utilizes IL-6 pairs of CCK-8 methods detection recombination
The proliferative conditions of pancreatic cancer cell, the results are shown in Figure 1, and IL-6 is to pancreatic cancer cell Mia-paca-2 cells for Fig. 1 display recombinations
There is proliferation facilitation, difference to have statistical significance（P ＜ 0.05）.
2.2 DAPT inhibit the proliferation of pancreatic cancer cell Mia-paca-2
For effect of the detection Notch accesses in IL-6 promotes Cell Proliferation of Pancreatic Cancer Cell, experiment adds Notch pathway inhibitors
DAPT inhibits the activation of Notch-1 accesses.DAPT is added in pancreas cancer cell strain Mia-paca-2 culture solutions and acts on 30min, connects
Addition 100ng/mL recombination IL-6, CCK-8 methods detect cell Proliferation vigor.The results are shown in Figure 2, and Fig. 2, which is shown, to be individually added into
DAPT has apparent inhibiting effect to Mia-paca-2 cell Proliferations；But under the action of IL-6, cell proliferation inhibition rate declines,
Difference has statistical significance（P ＜ 0.05）.
Influences of 2.3 IL-6 to pancreatic cancer cell Mia-paca-2 clonalities
Cell clonal formation experimental result is as shown in figure 3, Fig. 3 is shown, in various concentration IL-6（0、100ng/mL）Under effect,
Mia-paca-2 cell clonal formation numbers are respectively 275 ± 9,306 ± 13；With the increase of IL-6 concentration, Mia-paca-2 is thin
Born of the same parents' clonality also improves, and difference has statistical significance（P ＜ 0.05）；As addition DAPT（5、10μM）Pretreatment
In the case of 30min, cell clonal formation significantly reduces, and respectively 227 ± 9,194 ± 7.
Influences of 2.4 IL-6 to the external transfer abilities of pancreatic cancer cell Mia-paca-2
The results are shown in Figure 4 for cell scratch experiment, and compared with blank control group, recombination IL-6 can improve cancer of pancreas Mia-paca-2
The transfer ability of cell, and concentration-dependent relation is presented, difference is statistically significant（P ＜ 0.05）.
Influences of 2.5 IL-6 to human pancreatic cancer cell Notch-1 protein expressions
Various concentration recombination IL-6 acts on people source pancreas cancer cell strain Mia-paca-2, extracts total protein of cell, western
Blot detects the expression of Notch1 albumen.The results are shown in Figure 5, with the increase of recombination IL-6 concentration, Notch1 expression
It is consequently increased.The inhibitor DAPT that Notch is added in cell strain acts on 30min, is subsequently added into recombination IL-6, and extraction cell is total
Albumen, the expression of western blot detection Notch1 albumen.The results show that recombination IL-6 dramatically increases Notch1 albumen
Expression.
Influence of the antibody of 3 interleukin-6s to Cell Proliferation of Pancreatic Cancer Cell
Normal culture human pancreas cancer cell strain Mia-paca-2 is inoculated with to exponential phase after the passage of 0.25% trypsin digestion
To 96 orifice plates（103/hole）, various concentration IL-6 antibody is added after cell is adherent, acts on human pancreas cancer cell strain Mia-
Paca-2 continues to cultivate 48h, the effect using CCK-8 methods detection IL-6 antibody to Cell Proliferation of Pancreatic Cancer Cell.As a result such as Fig. 6 institutes
Show, IL-6 antibody can significantly inhibit the proliferation of Mia-paca-2 cells, and for 10 μ g/mL inhibiting rates up to 70%, difference has statistics
Meaning（P ＜ 0.05）.
The presence of body local chronic inflammatory leads to a large amount of releases of IL-6, and then excessive activation Notch accesses, the latter
It participates in adjusting generation, the update of tumor stem cell, and ultimately forms tumour.The present invention is tested by western blotting
Show that the increase with IL-6 concentration, the content of Notch1 are consequently increased；Inhibit the expression of IL-6, the content of Notch1 also with
Reduction, further illustrate lower IL-6, Notch accesses can be inhibited, to inhibit the formation of tumour.
Claims (5)
1. application of the antagonist of interleukin-6 in preparing the drug for inhibiting Notch-1 protein expressions.
2. application of the antagonist of interleukin-6 in the drug for preparing treatment cancer of pancreas.
3. application as claimed in claim 1 or 2, it is characterised in that：The drug is the antagonist by interleukin-6
Made of pharmaceutically acceptable carrier or excipient.
4. application as claimed in claim 1 or 2, it is characterised in that：The antagonist of the interleukin-6 is IL-6 antibody.
5. application as claimed in claim 4, it is characterised in that：The IL-6 antibody is purchased from Ai Bokang（Shanghai）Trade has
The interleukin-6 antibody of limit company（Abcam, ab6672).
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103930443A (en) * | 2011-11-10 | 2014-07-16 | 瑞泽恩制药公司 | Methods of inhibiting tumor growth by antagonizing IL-6 receptor |
| CN107249637A (en) * | 2015-02-27 | 2017-10-13 | 中外制药株式会社 | Composition for treating the relevant diseases of IL 6 |
-
2018
- 2018-07-17 CN CN201810780656.8A patent/CN108567981A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103930443A (en) * | 2011-11-10 | 2014-07-16 | 瑞泽恩制药公司 | Methods of inhibiting tumor growth by antagonizing IL-6 receptor |
| CN107249637A (en) * | 2015-02-27 | 2017-10-13 | 中外制药株式会社 | Composition for treating the relevant diseases of IL 6 |
Non-Patent Citations (2)
| Title |
|---|
| GOUMAS F. A.，ET AL.: "Inhibition of IL-6 signaling significantly reduces primary tumor growth and recurrencies in orthotopic xenograft models of pancreatic cancer", 《INTERNATIONAL JOURNAL OF CANCER》 * |
| RAZIDLO G. L.，ET AL.: "Interleukin-6 Promotes Pancreatic Cancer Cell Migration by Rapidly Activating the Small GTPase CDC42", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
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