CN109385459A - A kind of method that basic element of cell division inhibits cell Proliferation by inhibition DNA polymerase activity - Google Patents
A kind of method that basic element of cell division inhibits cell Proliferation by inhibition DNA polymerase activity Download PDFInfo
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- CN109385459A CN109385459A CN201811213728.7A CN201811213728A CN109385459A CN 109385459 A CN109385459 A CN 109385459A CN 201811213728 A CN201811213728 A CN 201811213728A CN 109385459 A CN109385459 A CN 109385459A
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Abstract
The present invention provides a kind of basic elements of cell division by inhibiting DNA polymerase activity to inhibit the method for cell Proliferation, belongs to Biochemistry and Molecular Biology technical field.The archaeal dna polymerase used in us is found in PDB lane database, setting parameter optimizes the structure of archaeal dna polymerase；Then look for the active site of archaeal dna polymerase；Prepare BAR structural formula and optimizes；The method of LibDock carries out molecular docking, can successively check the docking structure of each ligand, be ranked up according to scoring functions.Data of the invention, which are established, may interfere with archaeal dna polymerase biocatalysis synthetic DNA in tumour cell in the basic element of cell division to inhibit on the survival of tumour cell and the basis of proliferation.
Description
Technical field
The present invention relates to ucleosides basic element of cell division N6- Benzyladenosine (BAR) is to the anti-swollen of human lung carcinoma cell line
Tumor effect, belongs to Biochemistry and Molecular Biology technical field.
Background technique
At present more and more the study found that the basic element of cell division not only adjusts the growth and development of plant simultaneously to zooblast
Proliferation and growth also play a significant role.Research finds some nucleoside form ortho-Topolin of the basic element of cell division
Riboside(oTR)、Kinetin riboside(KR)、N6-Benzyladenosine(BAR)、N6-
Isopentenyladenosine (iPR) has very strong toxicity and apoptosis-promoting effect to cancer cell in vitro.When thin with these
Cause after born of the same parents' mitogen processing cancer cell the cell cycle it is different degrees of be obstructed and (or) apoptosis, this depends primarily on different
Susceptibility of the cell line to the different basic elements of cell division.Although natural CK and their analog to the effect of zooblast
Through repeatedly being reported, but there is presently no the researchs to basic element of cell division apoptosis-promoting effect of a system.
Polymerase (polymerase) is also known as archaeal dna polymerase, be special biocatalysis synthesis DNA (DNA) and
The general designation of the class of enzymes of ribonucleic acid (RNA).12 kinds of archaeal dna polymerases, including polymerase are at least had found in eukaryocyte
α, β, γ, δ, ε, ζ, η, θ, ι, κ, λ and μ, they are repaired by the proofreading effect or DNA for participating in DNA polymerization reaction exonuclease
Multiple process, plays an important role to the maintenance of DNA replication dna fidelity, directly affects the stability of cytogenetics, therefore DNA polymerize
Enzyme plays a significant role in DNA synthesis process.DNA synthesis process is the important link in proliferation process, therefore DNA is poly-
The activity of synthase may influence proliferation process.
Lung cancer is currently disease incidence and the highest malignant tumour of the death rate in global range.In the past 20 years, due to pushing greatly
The disease incidence of row smoking cessation, western countries' male lung cancer such as Europe and the U.S. has been begun to decline, but the disease incidence of female lung cancer is held
It is continuous to rise.China is that cigarette produces and sells big country, no matter male or women, the disease incidence of lung cancer is in that lasting rise becomes
Gesture is risen faster with women disease incidence especially.Clinical research shows carcinoma in situ cure rate close to 5 years of 100%, I phase patients with lung cancer
Survival rate is up to 60%~90%, and 5 years survival rates of III b and IV patient only 5%~20%.Clinically for treating the normal of lung cancer
Although rule drug plays an important role to treatment, there is apparent toxic side effects.Through a large amount of it was verified that with modern
The active constituent of technology, the natural active matter obtained from natural products can treat lung cancer, while the poison for mitigating chemicotherapy is secondary
Effect.Therefore, it finds and effectively improves therapeutic efficiency, the small natural drug of toxic side effect is come to treat lung cancer be very necessary.
LibDock is one of interconnection method in Discovery Studio.The interconnection method first can for by
Hot-zone figure is calculated in body active site, which includes polarity and nonpolar moiety；Then the ligand molecular of tripe systems elephant
It is rigidly overlapped respectively to hot-zone figure and forms proper interaction；Then it carries out energy-optimised；Finally retain marking
Higher docking conformation.
Summary of the invention
The present invention is directed to the above-mentioned prior art, and the present invention provides N6- Benzyladenosine (BAR) is thin in human lung cancer
A kind of mechanism of apoptosis-promoting effect in born of the same parents' strain A549.
Technical solution of the present invention:
A kind of method that basic element of cell division inhibits cell Proliferation by inhibition DNA polymerase activity, steps are as follows:
(1) cell culture: human lung carcinoma cell line A549 is suspended in the sterile DMEM culture solution containing inactivated serum,
Being placed in concentration is 5%CO2, relative humidity 90%, cultivate in the incubator that temperature is 37 DEG C, to the adherent length of cell to culture bottle
70%~80% passage of floor space 1 time；
(2) drug is prepared: BAR 0.1%DMSO dissolution respectively continues the oTR that will have been dissolved with sterile DMEM media
Being diluted to concentration is 0.1 μM -30 μM, filtration sterilization, room temperature preservation；
(3) it handles cell: the cell for being in logarithmic growth phase in step (1) is digested with pancreatin, it is dilute after cell count
It releases, diluting cells density is 3 × 104~5 × 104A/ml, piping and druming mix, and by cell inoculation in 96 orifice plates, every hole takes 100 μ l
After human lung carcinoma cell line A549,12h, after cell is adherent, 7 groups of experiments of setting, every group of 4 multiple holes, the 1st group: blank control group；
2nd group: 100 μ Μ BAR group；3rd group: 20 μM BAR group；4th group: 10 μ Μ BAR group；5th group: 2 μ Μ BAR group；6th group: 1 μ
Μ BAR group；7th group: 0.2 μ Μ BAR group；The corresponding drug of 10 μ l is added into each group respectively, after mixing gently, is placed in concentration
For 5%CO2, relative humidity 90%, be incubated for 2 days in the incubator that temperature is 37 DEG C, detection cell inhibitory effect activity；
(4) influence of the bioinformatics detection BAR to archaeal dna polymerase: firstly, being found used in us in PDB lane database
Archaeal dna polymerase, setting parameter the structure of archaeal dna polymerase is optimized；Then look for the active site of archaeal dna polymerase；It is quasi-
Standby BAR structural formula simultaneously optimizes；The method of LibDock carries out molecular docking, can successively check the docking structure of each ligand,
It is ranked up according to scoring functions.
Beneficial effects of the present invention: data of the invention, which are established, may interfere with DNA polymerization in tumour cell in the basic element of cell division
Enzymes biocatalysis synthetic DNA is to inhibit on the survival of tumour cell and the basis of proliferation.
Detailed description of the invention
Fig. 1 is inhibited proliferation of the BAR to A549.
Specific embodiment
Below in conjunction with attached drawing and technical solution, a specific embodiment of the invention is further illustrated.
Cell, kit and the reagent that the present invention uses: A549 cell, fetal calf serum, dual anti-(penicillin/strepto-
Element), DMEM culture medium, BAR.
Embodiment 1
BAR inhibits A549 cell Proliferation: the cell in logarithmic growth phase digested with pancreatin, after cell count, and dilution,
Diluting cells density is 3 × 104~5 × 104A/ml, piping and druming mix, and by cell inoculation in 96 orifice plates, every hole takes 100 μ l people
After 12h, after cell is adherent, 7 groups of experiments are arranged in lung cancer cell types, every group of 4 multiple holes, and the 1st group: blank control group；The
2 groups: 100 μM of BAR groups；3rd group: 20 μM BAR group；4th group: 10 μM BAR group；5th group: 2 μ Μ BAR group；6th group: 1 μ Μ
BAR group；7th group: 0.2 μ Μ BAR group；The corresponding drug of 10 μ l is added into each group respectively, after mixing gently, being placed in concentration is
5%CO2, relative humidity 90%, be incubated for 2 days in the incubator that temperature is 37 DEG C, detect the cell in 96 orifice plates with CCK-8 method
Light absorption value, BAR significantly inhibits the proliferation of A549 as the result is shown, wherein IC50=1.21 μM.
Embodiment 2
Bioinformatics detects influence of the BAR to archaeal dna polymerase: firstly, finding used in us in PDB lane database
Archaeal dna polymerase, setting parameter optimize the structure of archaeal dna polymerase；Then look for the active site of archaeal dna polymerase；Prepare
BAR structural formula simultaneously optimizes；The method of LibDock carries out molecular docking, can successively check the docking structure of each ligand, press
It is ranked up according to scoring functions.It is simulated and is found by bioinformatics molecular docking, BAR and archaeal dna polymerase are subjected to molecule mould
Quasi- docking, BAR can be very good to be entrenched in the active pocket of archaeal dna polymerase crystal model, small molecule compatibility scoring functions
Libdockscore value is analysis shows BAR can inhibit the activity of archaeal dna polymerase multiple to influence DNA in conjunction with archaeal dna polymerase
Process processed inhibits cell Proliferation.
Claims (1)
1. a kind of basic element of cell division is by inhibiting DNA polymerase activity to inhibit the method for cell Proliferation, which is characterized in that step is such as
Under:
(1) cell culture: human lung carcinoma cell line A549 is suspended in the sterile DMEM culture solution containing inactivated serum, is placed in
Concentration is 5%CO2, relative humidity 90%, cultivate in the incubator that temperature is 37 DEG C, to the adherent length of cell to culture bottle bottom surface
70%~80% long-pending passage 1 time；
(2) drug is prepared: BAR 0.1%DMSO dissolution respectively continues to be diluted the oTR dissolved with sterile DMEM media
It is 0.1 μM -30 μM to concentration, filtration sterilization, room temperature preservation；
(3) it handles cell: the cell for being in logarithmic growth phase in step (1) is digested with pancreatin, after cell count, dilution is dilute
Releasing cell density is 3 × 104~5 × 104A/ml, piping and druming mix, and by cell inoculation in 96 orifice plates, every hole takes 100 μ l people's lungs
After cancer cell line A549,12h, after cell is adherent, 7 groups of experiments of setting, every group of 4 multiple holes, the 1st group: blank control group；2nd
Group: 100 μ Μ BAR groups；3rd group: 20 μM BAR group；4th group: 10 μ Μ BAR group；5th group: 2 μ Μ BAR group；6th group: 1 μ Μ
BAR group；7th group: 0.2 μ Μ BAR group；The corresponding drug of 10 μ l is added into each group respectively, after mixing gently, being placed in concentration is
5%CO2, relative humidity 90%, be incubated for 2 days in the incubator that temperature is 37 DEG C, detection cell inhibitory effect activity；
(4) influence of the bioinformatics detection BAR to archaeal dna polymerase: firstly, finding the DNA used in us in PDB lane database
Polymerase, setting parameter optimize the structure of archaeal dna polymerase；Then look for the active site of archaeal dna polymerase；Prepare BAR
Structural formula simultaneously optimizes；The method of LibDock carries out molecular docking, can successively check the docking structure of each ligand, according to
Scoring functions are ranked up.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030236216A1 (en) * | 2001-06-12 | 2003-12-25 | Devos Rene Robert | 4'-substituted nucleoside derivatives as inhibitors of HCV RNA replication |
| TW201124141A (en) * | 2011-04-09 | 2011-07-16 | Medivir Ab | HCV nucleoside inhibitor |
| CN103635576A (en) * | 2011-06-30 | 2014-03-12 | 箭头研究公司 | Compositions and methods for inhibiting gene expression of Hepatitis B Virus |
| CN107142298A (en) * | 2017-06-15 | 2017-09-08 | 大连理工大学 | A kind of applications of cell-cycle arrest agent 6BAR in human lung carcinoma cell |
| CN107326010A (en) * | 2017-06-15 | 2017-11-07 | 大连理工大学 | A kind of applications of cell-cycle arrest agent 6BAR in human breast cancer cell |
-
2018
- 2018-10-18 CN CN201811213728.7A patent/CN109385459A/en not_active Withdrawn
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030236216A1 (en) * | 2001-06-12 | 2003-12-25 | Devos Rene Robert | 4'-substituted nucleoside derivatives as inhibitors of HCV RNA replication |
| CN1516590A (en) * | 2001-06-12 | 2004-07-28 | - | 4'-substituted nucleosides |
| TW201124141A (en) * | 2011-04-09 | 2011-07-16 | Medivir Ab | HCV nucleoside inhibitor |
| CN103635576A (en) * | 2011-06-30 | 2014-03-12 | 箭头研究公司 | Compositions and methods for inhibiting gene expression of Hepatitis B Virus |
| CN107142298A (en) * | 2017-06-15 | 2017-09-08 | 大连理工大学 | A kind of applications of cell-cycle arrest agent 6BAR in human lung carcinoma cell |
| CN107326010A (en) * | 2017-06-15 | 2017-11-07 | 大连理工大学 | A kind of applications of cell-cycle arrest agent 6BAR in human breast cancer cell |
Non-Patent Citations (4)
| Title |
|---|
| SARA CASTIGLIONI等: "N6-Isopentenyladenosine and its analogue N6-Benzyladenosine induce cell cycle arrest and apoptosis in bladder carcinoma T24 cells", 《ANTI-CANCER AGENTS MEDICINAL CHEMISTRY》 * |
| 周仲楼等: "腺苷对人肺腺癌细胞A549增殖和细胞周期的影响", 《温州医学院学报》 * |
| 李燕: "《精编分子生物学实验技术》", 30 September 2017, 世界图书出版公司 * |
| 魏来: "《肝脏疾病》", 31 July 2006, 中国医药科技出版社 * |
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