CN106880841B - Application of aloperine in preparing lung cancer radiotherapy sensitizer - Google Patents
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- 238000001959 radiotherapy Methods 0.000 title claims abstract description 104
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- PCTYDEAPIWWHMV-UHFFFAOYSA-N aloperine Natural products C1CCN2CC3CC(CC4CCCNC34)C2C1 PCTYDEAPIWWHMV-UHFFFAOYSA-N 0.000 title claims abstract description 81
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Abstract
the aloperine is a natural compound of plant sources, and a radiotherapy sensitization mechanism of the aloperine mainly changes biomarkers Beclin1, L C3 and p62 related to the autophagy level of cells, and preferably fluorescence visualization L C3 (Ad-mRFP-GFP-L C3).
Description
Technical field
The aloperine horizontal the present invention relates to cell propagation, enhancing radiotherapy in lung cancer sensitiveness, regulating cell autophagy is suppressed, leads to
Cross and detect some specific biomarkers, lung carcinoma cell autophagy level and radiotherapy are resisted in the suppression radiotherapy for evaluating aloperine
Enhancement effect.
Background technology
Lung cancer (lung cancer), whole world tumour patient main causes of death, every year about 1,600,000 lung cancer suffer from
Person is dead.Clinically, radiotherapy is as one of primary treatments of patients with lung cancer, however, research is found recently, tumour cell
Resistant function gradually is generated to cellulotoxic effect caused by radiotherapy, causes radiotherapy in lung cancer curative effect and survival region to be significantly deteriorated.
Thus, the Regulation Mechanism of lung carcinoma cell radiotherapy radiotherapy resistance is illustrated, helps to find lung cancer therapy and the novel targets of prognosis.
Cell autophagy (autophagy) is an intracellular homeostasis process, and it is led to by fine molecular regulation
The materials such as the damaged cell device crossed in degradation of cell, misfolded protein matter, needed for cell normal activities.At present, grind
Study carefully and be found that 36 cell autophagy correlation molecules (autophagy-related genes, Atgs), including Atg6/Beclin1,
Atg8/LC3, p62 etc..The horizontal change of autophagy, can be that cell produces reply pessimal stimulation under the stimulation of stress reaction
Aversion response.Increasing research shows that cell autophagy plays important function in the occurrence and development of tumour, particularly
In the presence of radiotherapy or chemotherapeutics, autophagy shows to promote tumour, antitumor " double-edged sword " effect, thin so as to influence tumour
The treatment curative effect of born of the same parents.The exception of cell autophagy is related to sensitiveness of the tumour cell to chemicotherapy, to cell autophagy regulation mechanism
The achievement of research is being converted into the novel targets of oncotherapy.
Aloperine (Aloperine) is the natural alkaloid in kuh-seng platymiscium source, is shown in testing in vivo and in vitro good
Anti-inflammatory well, antitumor activity.Research finds that aloperine can suppress the propagation of melanoma cells, promote Apoptosis, together
When zoopery show that aloperine has the characteristics that relative safety, non-evident effect.In colon cancer cell, aloperine energy
It is enough significantly to suppress cell propagation, cause cell-cycle arrest, and then produce antitumor activity.To exist however, there has been no aloperine
Research in radiotherapy in lung cancer resistance is found.Thus, the molecular mechanism of aloperine enhancing radiotherapy in lung cancer sensitiveness is illustrated, will be had
Help aloperine as lung carcinoma cell radiotherapy hypersitization medicine is prepared.
The content of the invention
It is an object of the invention to provide aloperine lung carcinoma cell autophagy level inhibitor and radio therapy sensitization are resisted as radiotherapy
The application of medicine.The aloperine is the native compound of plant origin, and its radio therapy sensitization mechanism mainly changes cell autophagy
Level, and its related Biomarker Beclin1, LC3 and p62 change, preferably LC3 (Ad-mRFP-GFP-LC3).
By detecting these specific biomarkers, the suppression radiotherapy for evaluating aloperine is resisted lung carcinoma cell autophagy level and put
Treat enhancement effect.
The invention will be further described below：
Present invention determine that aloperine suppresses, cell autophagy is horizontal and the new molecular mechanism of enhancing radiotherapy in lung cancer sensitiveness,
The application for specifying aloperine changes cell autophagy level and lung carcinoma cell radiation sensitivity, and evaluation aloperine is as new lung cancer
The feasibility of cell autophagy level inhibitor and radiotherapeutic sensitizer.
The present invention is bitter using Beclin1, p62 detection technique and the Ad-mRFP-GFP-LC3 technologies of fluorescent visual, detection
Beans alkali resists the influence that autophagy is horizontal in cell to radiotherapy in lung cancer, as a result finds that aloperine significantly inhibits cell autophagy water
It is flat, enhance the radiation sensitivity of lung carcinoma cell.
The present invention utilizes the radiotherapy resistance of colony formation and one-hit multitarget model evaluation lung carcinoma cell, discovery and radiotherapy
Sensitive cells A549 is compared, and radiotherapy resistance cell A549/IR has significant radiotherapy Antagonism.
Using cell proliferation experiment and colony formation, the influence that aloperine is bred to radiotherapy resisting cell is analyzed, knot
Fruit finds that aloperine can significantly suppress radiotherapy resistance cell A549/IR appreciation rate；Meanwhile using flow cytometry and carefully
Born of the same parents' cycle detection point correlation molecule expression changes, and influence of the analysis aloperine to the radiotherapy resisting cell cycle, finds bitter beans
There occurs G1/S phase cell-cycle arrests by alkali induction radiotherapy resistance cell A549/IR.
By under the synergy of aloperine and radiotherapy, the detection of cell proliferation rate, it is found that aloperine can substantially increase
Radiotherapy resistance cell A549/IR radiation sensitivity.
Using the change of Western blot technology for detection cell autophagy correlation molecules, it is found that aloperine can lower radiotherapy
Resist expression, the up-regulation autophagy negative regulation molecule p62 expression of autophagy positive regulation molecule Beclin1 in cell A549/IR；
The quantity of intracellular autophagy vacuole is detected using Electron Microscopy, it is found that aloperine considerably reduces radiotherapy resistance cell
The quantity of autophagy vacuole in A549/IR；Then fluorescence microscope evaluation Ad-mRFP-GFP-LC3 fluorescence intensities, find aloperine
Endochylema Green fluorescence signal and red fluorescent can be suppressed.When aloperine and chemotherapy combined radiotherapy act on, above-mentioned autophagy phase
It is most obvious to close the changes such as molecular change, the reduction of autophagy vacuole quantity, LC3 fluorescence signals decrease, prompts aloperine to change
Under the conditions of radiotherapy, the autophagy in radiotherapy resistance cell A549/IR is horizontal, have impact on the effect of radiotherapy.
Next, positive control is used as with cell autophagy inhibitor chloroquine, Bafilomycin A1 (BafA), further
Evaluate inhibitory action of the aloperine to cell autophagy.Radio therapy sensitization effect caused by aloperine and autophagy inhibitor chloroquine, BafA
Similar, i.e., when combined radiotherapy, aloperine, chloroquine, BafA can lower Beclin1, up-regulation p62, while strengthen radiotherapy to putting
Resistance cell A549/IR cellulotoxic effect is treated, and synergy becomes apparent from.
In a word, research of the invention finds that aloperine can be by suppressing cell autophagy level, and then it is thin to enhance lung cancer
The radiation sensitivity of born of the same parents.The Ad-mRFP-GFP-LC3 of autophagy correlation molecule Beclin1, p62 and fluorescent visual changes are its works
Molecular basis, and the change such as cell propagation, cell cycle, one-hit multitarget model is the important sign of its effect.Therefore, lung
Autophagy horizontal related biomarker Beclin1, p62 and Ad-mRFP-GFP-LC3 change can be used for commenting in cancer cell
Estimating the function test such as influence of the aloperine to tumour cell autophagy, cell propagation, cell cycle, one-hit multitarget model can be used for
Evaluate the Apoptosis of aloperine.
Brief description of the drawings
Fig. 1 is that aloperine suppresses the propagation that radiotherapy in lung cancer resists cell A549/IR.A figures are shown under different radiological doses, are put
Treat sensitive cells A549, radiotherapy in lung cancer resistance cell A549/IR cell clonal formation；B figure one-hit multitargets model shows A549/
IR has obvious radiotherapy repellence；C figures show the suppression A549/IR of aloperine concentration dependent cell proliferation rate；D figures are aobvious
Show that aloperine suppresses A549/IR Clone formation；E figures are the quantitative analyses to D figures.
Fig. 2 is that aloperine causes the radiotherapy in lung cancer resistance cell A549/IR generation G1/S phases to be blocked.A figure flow cytometries show
Show that aloperine causes the A549/IR G1/S phases to be blocked, when being acted on chemotherapy combined radiotherapy, blockage effect is best；B figures are that A figures are quantified
Analysis；C figure evaluation Cell cycle checkpoint correlation molecule changes, display aloperine suppress G1/S phase Cell cycle checkpoint molecules
CDK4, CKD6, CyclinD1 expression, and the expression on G2/M phase test points CDC2 is without influence.
Fig. 3 is that aloperine enhances the radiotherapeutic sensitizer that radiotherapy in lung cancer resists cell A549/IR.A figures show that aloperine increases
Strong radiotherapy is to A549/IR cell inhibitory effect effects；B figures show that aloperine enhances radiotherapy to A549/IR cell clone shapes
Into the depression effect of ability；C figures are the quantitative analyses to B figures.
Fig. 4 is that aloperine inhibits radiotherapy in lung cancer to resist the cell autophagy signal in cell A549/IR.A figures show bitter beans
Alkali enhances inhibitory action of the radiotherapy to autophagy in A549/IR cells；B figures show that chloroquine enhances radiotherapy to A549/IR cells
The inhibitory action of middle autophagy；C figures show that BafA enhances inhibitory action of the radiotherapy to autophagy in A549/IR cells；D figures show hardship
Beans alkali enhances inhibitory action of the radiotherapy to autophagy vacuolization in A549/IR cells；E figures are the quantitative analyses to D figures；F schemes
Display aloperine enhances suppression of the radiotherapy to LC3 green florescent signals and LC3 red fluorescents in A549/IR cells and made
With；G-H figures are the quantitative analyses to F figures.
Fig. 5 is to suppress the radiation sensitivity that cell autophagy enhances lung carcinoma cell.A figures are shown when being combined with radiotherapy, bitter beans
Alkali, chloroquine have lowered the expression of autophagy positive regulation molecule Beclin1 in A549/IR cells, up-regulation autophagy negative regulation point
Sub- p62 expression；B figures are the quantitative analyses to A figures；C figures are shown when being combined with radiotherapy, and aloperine, BafA have lowered A549/
Autophagy positive regulation molecule Beclin1 expression, up-regulation autophagy negative regulation molecule p62 expression in IR cells；D figures are to C
The quantitative analysis of figure；E figures show that aloperine, chloroquine can strengthen lethal effect of the radiotherapy to A549/IR cells；F figures show hardship
Beans alkali, BafA can strengthen lethal effect of the radiotherapy to A549/IR cells.
Un represents untreated fish group (Untreated group) in figure, and ALO represents aloperine, and CQ represents chloroquine, BafA is represented
Bafilomycin A1。
Embodiment
Lung cancer cell line A549 cells used in the present invention are ATCC cell lines (CCL-185), and A549/IR cell lines are
Emory University build radiotherapy resistance cell line.
The aloperine of embodiment 1 suppresses the propagation of radiotherapy in lung cancer resistance cell line
Experimental method is as follows：The radiotherapy resistant phenotype of A549/IR cells, lung cancer cell line A549, A549/IR are analyzed first
Respectively by O, 2,4, the treatment with irradiation of 8Gy different radiological doses, cell clonal formation experiment and one-hit multitarget model are carried out
Analysis, evaluate A549/IR cells radiotherapy resistance；Then toxicity of the aloperine to lung cancer cell line A549, A549/IR is analyzed
Effect, 0,5,10,25,50mM aloperine processing cell 24h, carry out cell proliferation experiment and colony formation analysis.
As a result show, under different radiological dose effects, compared with radiotherapy sensitive cells A549, A549/IR cells
Existence fraction substantially reduces, and prompts A549/IR cells to have significant radiotherapy resistant phenotype.And aloperine can significantly suppress
The proliferation rate and cloning efficiency of A549/IR cells, and concentration dependent is presented (result is shown in Fig. 1).
The aloperine of embodiment 2 causes the G1/S phases of radiotherapy in lung cancer resistance cell line to be blocked
Cell propagation influenceed by the cell cycle, and study find, Cell cycle checkpoint molecule CDK4, CKD6 and
CyclinD1 adjusts cell and adjusts cell from the G2 phases to the conversion of M phases from the G1 phases to the conversion of S phases, CDC2.
Experimental method is as follows：A549/IR cells, 6Gy radiotherapies processing 1h, 10mM aloperine processing 24h, 6Gy radiotherapy with
Under conditions of aloperine Combined Treatment 24h, flow cytometry evaluates influence of the aloperine to A549/IR cell cycle distributions；
Western blot detection Cell cycle checkpoint molecules CDK4, CKD6, CyclinD1, CDC2 expression.
As a result show, aloperine is used alone, or is used with chemotherapy combined radiotherapy, can result in radiotherapy in lung cancer resistance cell
The A549/IR generation G1/S phases block.Meanwhile significantly inhibit G1/S phase test point molecules CDK4, CKD6, CyclinD1 albumen table
Up to level, the protein expression level without influenceing G2/M phase test point molecules CDC2 (result is shown in Fig. 2).
The radiation sensitivity of the aloperine of embodiment 3 enhancing radiotherapy in lung cancer resistance cell line
Experimental method is as follows：A549/IR cells, 6Gy radiotherapies processing 1h, 10mM aloperine processing 24h, 6Gy radiotherapy with
Under conditions of aloperine Combined Treatment 24h, 1 × 10 is taken3Cell in good condition, it is inoculated on 96 orifice plates, adds 20 μ l MTS
Reagent, incubation 0,24,48,72h, absorbance is measured under 490nm wavelength, calculate cell proliferation rate.Take 2 × 103State
Good cell, it is inoculated on six orifice plates, CO2Cultivated 10-15 days in cell culture incubator, ice methanol fixes 15min, crystal violet dye
Color, using >=50 cells as a clone, count the number of cell clones per hole.Using untreated fish group as control, gram of each group is calculated
Grand formation rate.
As a result show, aloperine is used alone, or is used with chemotherapy combined radiotherapy, can result in radiotherapy in lung cancer resistance cell
A549/IR proliferation rates and cloning efficiency lower, and the suppression of synergy is more efficient (result is shown in Fig. 3).
The cell autophagy that the aloperine of embodiment 4 suppresses radiotherapy in lung cancer resistance cell line is horizontal
Radiotherapy can cause the horizontal change of cell autophagy, so as to influence reaction of the tumour cell to radiotherapy.We connect down
To analyze whether aloperine influences the radiotherapy side effect of radiotherapy in lung cancer resistance cell.
Experimental method is as follows：A549/IR cells, 6Gy radiotherapies processing 1h, 10mM aloperine processing 24h, 6Gy radiotherapy with
Under conditions of aloperine Combined Treatment 24h, or 6Gy radiotherapies processing 1h, 10mM chloroquine processing 24h, 6Gy radiotherapy is combined with chloroquine
Under conditions of handling 24h, or 6Gy radiotherapies processing 1h, 10Mm BafA processing 24h, 6Gy radiotherapies are with BafA Combined Treatments 24h's
Under the conditions of, total protein of cell is extracted, carries out Beclin1, p62 Western Blot analyses.Take 1 × 103In good condition is thin
Born of the same parents, carry out the quantity of the intracellular autophagy vacuole of electron microscope observation.Ad-mRFP-GFP-LC3 is added to above-mentioned treatment conditions
Under cell supernatant in, act on 48h, ice methanol fixes, fluorescence microscope LC3 green/red fluorescence signal.
As a result show, aloperine is used alone, or is used with chemotherapy combined radiotherapy, can result in radiotherapy in lung cancer resistance cell
A549/IR autophagy correlation molecules change：Beclin1 is lowered, p62 up-regulations, reduces the autophagy cavitation number in A549/IR cells
Amount, suppress the LC3 green/red fluorescence signals in A549/IR cells, and the suppression of synergy is more efficient.Meanwhile bitter beans
Change of the alkali to cell autophagy correlation molecule expression, it is similar to autophagy inhibitor chloroquine, BafA effect, prompt aloperine
Can be as the inhibitor of cell autophagy (result is shown in Fig. 4).
Embodiment 5 suppresses cell autophagy and enhances toxic action of the radiotherapy to radiotherapy in lung cancer resisting cell
Experimental method is as follows：A549/IR cells, in 6Gy radiotherapies processing 1h, 10mM aloperine joint 6Gy radiotherapy processing
Under conditions of the joint 6Gy radiotherapies of 24h, 10mM chloroquine handle 24h, 6Gy radiotherapy and aloperine/chloroquine Combined Treatment 24h, Huo Zhe
6Gy radiotherapies processing 1h, 10mM aloperine joint 6Gy radiotherapy processing 24h, 10mM BafA joint 6Gy radiotherapy processing 24h, 6Gy are put
Treat with conditions of aloperine/BafA Combined Treatments 24h, extracting total protein of cell, carry out Beclin1, p62 Western
Blot is analyzed.Take 1 × 103Cell in good condition, be inoculated on 96 orifice plates, add 20 μ l MTS reagent, be incubated 0,24,48,
72h, absorbance is measured under 490nm wavelength, calculate cell proliferation rate.
As a result show, when combined radiotherapy acts on, aloperine and cell autophagy inhibitor (chloroquine, BafA), can result in
Radiotherapy in lung cancer resistance cell A549/IR autophagy correlation molecules change：Beclin1 is lowered, p62 up-regulations, simultaneously, it will be apparent that suppression
The proliferation rate of A549/IR cells processed.And during aloperine/autophagy inhibitor (chloroquine, BafA)/radiotherapy triple combination effect, it is right
A549/IR toxic effect is preferably (result is shown in Fig. 5).
Claims (5)
1. application of the aloperine on radiotherapy in lung cancer sensitizer is prepared.
2. aloperine and cell autophagy the inhibitor application on radiotherapy in lung cancer sensitizer is prepared jointly.
3. aloperine according to claim 2 and cell autophagy inhibitor jointly on radiotherapy in lung cancer sensitizer is prepared should
With described cell autophagy inhibitor includes one or both of chloroquine, Bafilomycin A1.
4. application of the aloperine on lung carcinoma cell autophagy inhibitor is prepared.
5. the one or more of reagents detected in Beclin1, LC3 and p62 are preparing the suppression radiotherapy resistance of evaluation aloperine
Application on the horizontal preparation with radio therapy sensitization effect of lung carcinoma cell autophagy.
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| US20100233730A1 (en) * | 2008-11-19 | 2010-09-16 | Rutgers, The State University Of New Jersey | Therapeutic modulation of autophagy |
| CN102008475A (en) * | 2010-09-09 | 2011-04-13 | 汕头大学医学院 | Application of quinolizidine in preparing tumor treatment drugs |
| CN104046676A (en) * | 2013-03-13 | 2014-09-17 | 中国科学院上海生命科学研究院 | Cell for removing amyloid polypeptide and use thereof |
| CN106511972A (en) * | 2016-11-17 | 2017-03-22 | 苏州大学 | Application of interleukin 33 in preparing medicine for treating epilepsy |
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| CN102008475A (en) * | 2010-09-09 | 2011-04-13 | 汕头大学医学院 | Application of quinolizidine in preparing tumor treatment drugs |
| CN104046676A (en) * | 2013-03-13 | 2014-09-17 | 中国科学院上海生命科学研究院 | Cell for removing amyloid polypeptide and use thereof |
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