CN107929278A - Application of the long tube Amethystoidin A in the product for suppressing esophageal squamous cell carcinoma cell Proliferation is prepared - Google Patents
Application of the long tube Amethystoidin A in the product for suppressing esophageal squamous cell carcinoma cell Proliferation is prepared Download PDFInfo
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Abstract
The invention discloses application of the long tube Amethystoidin A in the product for suppressing esophageal squamous cell carcinoma cell Proliferation is prepared.Long tube Amethystoidin A (LK A) is a kind of antKauranoids class compound extracted from glaucocalyx rabdosia herb category, mainly plays the lethal effect to esophageal squamous cell carcinoma cell by inducing cell apoptosis in vivo and in vitro.It is experimentally confirmed：LK A can suppress esophageal squamous cell carcinoma cell growth and induce the G2/M phases to block in vitro.In addition, LK A are also highly effective in Nude Mouse Model in vivo.By improving ROS levels and activation JNK and p38MAPK paths so as to cause esophageal squamous cell carcinoma Apoptosis, which has as the potentiality for the treatment of esophageal squamous cell carcinoma medicine LK A.
Description
Technical field
The invention belongs to biological technical field, and in particular to long tube Amethystoidin A is preparing the cell increasing of suppression esophageal squamous cell carcinoma
Application in the product grown.
Background technology
The cancer of the esophagus is one of most common cancer in China, its morbidity and mortality can be in any more all the time.Wherein esophageal squamous cell
Shape cell cancer (ESCC) is the main Types of China's cancer of the esophagus, accounts for more than the 90% of total case, 5 years overall survival about 15-
25%.The essential therapeutic arsenals of esophageal squamous cell carcinoma include operation, chemotherapy and radiation, but therapeutic effect is not fully up to expectations.Therefore, compel
The new medicine that can be used for treating esophageal squamous cell carcinoma will be developed by being essential.Many cancer therapy drugs are derived from plant, animal and marine organisms
Small molecule.Nature is still one of important sources of anti-cancer drugs, during from the 1940s to this section of the end of the year 2014
In, it is actually natural products or to be directed to them to have 131 in 175 small-molecule drugs of approved.
Apoptosis plays an important role in the mechanism that cancer therapy drug plays a role.Active oxygen (ROS) wide participation is various
Physiology course, and can be with inducing cell apoptosis.ROS is a handle double-edged sword, because excessive ROS increases can pass through different machines
Inducing cell apoptosis processed.Many signal paths may be influenced be subject to ROS, and including MAPK paths, its main component includes N
Terminal Kinase (JNK), p38 and extracellular signal-regulated kinase (ERK).JNK and p38 MAPK paths can be outside mediated cell
Apoptosis caused by stimulation, such as uses chemotherapeutics.
The content of the invention
The technical problem to be solved in the present invention is how to suppress esophageal squamous cell carcinoma cell Proliferation.
In order to solve the above-mentioned technical problem, present invention firstly provides the new application of long tube Amethystoidin A.
The present invention provides long tube Amethystoidin A in following A1) to A11) at least one of in application：
A1) prepare and suppress the propagation of esophageal squamous cell carcinoma cell and/or the product of growth；
A2 the product for the Clone formation for suppressing esophageal squamous cell carcinoma cell) is prepared；
A3 the product of induction esophageal squamous cell carcinoma cell-cycle arrest) is prepared；
A4) prepare and suppress the product that the DNA of esophageal squamous cell carcinoma cell is repaired；
A5) prepare induction and/or promote the product of esophageal squamous cell carcinoma Apoptosis；
A6 the product that up-regulation promotes the expression quantity of apoptosis protein) is prepared；
A7 the product for the expression quantity for lowering resistance apoptosis protein) is prepared；
A8 induction esophageal squamous cell carcinoma cytoactive oxygen (ROS) increased product) is prepared；
A9 the product of induction and/or activation esophageal squamous cell carcinoma cell JNK MAPK paths) is prepared；
A10 the product of induction and/or activation esophageal squamous cell carcinoma cell p38 MAPK paths) is prepared；
A11 the product for suppressing the growth of tumour caused by esophageal squamous cell carcinoma cell) is prepared.
Long tube Amethystoidin A is in following B1) to B11) at least one of in application fall within the protection model of the present invention
Enclose.
B1 propagation and/or the growth of esophageal squamous cell carcinoma cell) are suppressed；
B2 the Clone formation of esophageal squamous cell carcinoma cell) is suppressed；
B3 esophageal squamous cell carcinoma cell-cycle arrest) is induced；
B4 the DNA for) suppressing esophageal squamous cell carcinoma cell is repaired；
B5) induce and/or promote esophageal squamous cell carcinoma Apoptosis；
B6) up-regulation promotes the expression quantity of apoptosis protein；
B7 the expression quantity of resistance apoptosis protein) is lowered；
B8 esophageal squamous cell carcinoma cytoactive oxygen (ROS) increase) is induced；
B9) induce and/or activate esophageal squamous cell carcinoma cell JNK MAPK paths；
B10) induce and/or activate esophageal squamous cell carcinoma cell p38 MAPK paths；
B11 the growth of tumour caused by esophageal squamous cell carcinoma cell) is suppressed.
It is described to promote apoptosis protein concretely Cleaved caspase-3, Cleaved in above application
At least one of caspase-9, Cleaved PARP, γ H2AX, Bax, Bcl-2 and cytochrome c；
It is described to resist apoptosis protein concretely Bcl-2.
In above application, concretely G2/M phases cell cycle.
In above application, the esophageal squamous cell carcinoma cell concretely KYSE30 cells or KYSE450 cells.
In order to solve the above-mentioned technical problem, present invention also offers a kind of product, its active ingredient is long tube Rabdosia amethystoides first
Element；
At least one of the function of the product is following A1) to A11)：
A1 propagation and/or the growth of esophageal squamous cell carcinoma cell) are suppressed；
A2 the Clone formation of esophageal squamous cell carcinoma cell) is suppressed；
A3 esophageal squamous cell carcinoma cell-cycle arrest) is induced；
A4 the DNA for) suppressing esophageal squamous cell carcinoma cell is repaired；
A5) induce and/or promote esophageal squamous cell carcinoma Apoptosis；
A6) up-regulation promotes the expression quantity of apoptosis protein；
A7 the expression quantity of resistance apoptosis protein) is lowered；
A8 esophageal squamous cell carcinoma cytoactive oxygen (ROS) increase) is induced；
A9) induce and/or activate esophageal squamous cell carcinoma cell JNK MAPK paths；
A10) induce and/or activate esophageal squamous cell carcinoma cell p38 MAPK paths；
A11 the growth of tumour caused by esophageal squamous cell carcinoma cell) is suppressed.
It is described to promote apoptosis protein concretely Cleaved caspase-3, Cleaved in the said goods
At least one of caspase-9, Cleaved PARP, γ H2AX, Bax, Bcl-2 and cytochrome c；
It is described to resist apoptosis protein concretely Bcl-2.
In the said goods, concretely G2/M phases cell cycle.
In the said goods, the esophageal squamous cell carcinoma cell concretely KYSE30 cells or KYSE450 cells.
The product concretely medicine.
In above application or product, the long tube Amethystoidin A concretely glaucocalyx rabdosia herb extract, three leaf
Herba Rabdosiae glaucocalycis extract concretely glaucocalyx rabdosia herb ketone extract, the ketone concretely acetone.
The preparation method of the glaucocalyx rabdosia herb extract may include following steps：Using the leaf of glaucocalyx rabdosia herb as original
Material, progress acetone soak, ethyl acetate extraction, silica gel chromatographic column analysis, thin-layered chromatography detection, MCI decolorations, methanol are washed successively
De- and recrystallization.
In a specific embodiment of the present invention, the specific preparation method of the glaucocalyx rabdosia herb extract is as follows：
1) leaf of glaucocalyx rabdosia herb is dried and ground successively, obtain powdered samples；
2) powdered samples are soaked in aqueous acetone solution, is filtered after immersion, collect filtrate；
3) filtrate is evaporated under reduced pressure, removes the acetone in the filtrate, obtain crude extract；
4) ethyl acetate is added into the crude extract to be extracted, obtain acetic acid ethyl acetate extract；
5) acetic acid ethyl acetate extract described in silica gel chromatograph post separation is used, obtains ethyl acetate extract；
6) ethyl acetate extract is first detected with thin-layered chromatography, merges same section, obtain 5 cuts；So
Cut two is carried out to MCI decolorations, methanol aqueous solution elution and methanol dissolving-recrystallization successively afterwards, obtained solid matter is to grow
Pipe Amethystoidin A.The structure of long tube Amethystoidin A is as shown in Figure 1A.
In the above method, the aqueous acetone solution aqueous acetone solution that concretely volume fraction is 70%.The acetone
Aqueous solution and the proportioning of the powdered samples can be 100L：10Kg.The time of the immersion concretely 3d.
The silica gel chromatographic column is the silica gel chromatographic column filled with 200-300 mesh pore size silica gels；
The methanol aqueous solution methanol aqueous solution that concretely volume fraction is 90%.
In the ent kauranoid class compound long tube Amethystoidin A that invention finds to belong to from glaucocalyx rabdosia herb
(LK-A) there is strong inhibitory action to esophageal squamous cell carcinoma cell Proliferation in vitro, nude mice model can also be significantly inhibited in vivo
The growth of knurl.LK-A can cause intracellular ROS level to raise, and promote esophageal squamous cell carcinoma apoptosis and G2/M phases to block,
Meanwhile LK-A no obvious cytotoxic effects of performance in normal cell, in vivo also without obvious internal organs in nude mice model
Toxicity, this shows while the compound is acted on anti-esophageal squamous cell carcinoma with certain security.Above result of study shows,
LK-A can be by improving ROS levels, and activation JNK and p38MAPK paths cause the apoptosis of esophageal squamous cell carcinoma cell, have treatment oesophagus
The potentiality of squamous carcinoma medicine.
Brief description of the drawings
Fig. 1 suppresses KYSE30 and KYSE450 cell Proliferations and Clone formation for LK-A.A is the chemical structural formula of LK-A.B
To measure KYSE30, KYSE450, NIH3T3 and HEK-293 cell Proliferation by CCK-8 methods.C is LK-A pair of various dose
The influence that KYSE30 and KYSE450 cell colonies are formed.D is the mass spectral analysis of long tube Amethystoidin A.E is long tube Rabdosia amethystoides first
Element1H NMR spectrums detect.F is long tube Amethystoidin A13C NMR spectrums detect.G is long tube Rabdosia amethystoides first
The chromatography of element.
Fig. 2 induces KYSE30 the and KYSE450 cell G2/M phases to block for LK-A.A is thin in KYSE30 and KYSE450 for LK-A
The G2/M phases are induced to block in born of the same parents.B is KYSE30 and KYSE450 cell lines in cell cycle different phase percentage.C is logical
Cross the expression of Western blot measure G2/M cell cycle checkpoints GAP-associated protein GAPs (cyclin B1 and cdc2).
Fig. 3 induces KYSE30 and KYSE450 Apoptosis for LK-A.A is to be detected with Annexin V-FITC Apoptosis
KYSE30 the and KYSE450 Apoptosis of kit measurement LK-A inductions.B and C be various concentrations LK-A handle KYSE30 and
Cleaved caspase-3 after KYSE450 cells 24h, Cleaved caspase-9, Cleaved PARP, γ H2AX, Bax,
The protein expression level of Bcl-2 and cytochrome c.D is activation inducing cell apoptosis of the LK-A by caspase.
Fig. 4 is the increase that LK-A induction KYSE30 and KYSE450 Apoptosis needs ROS.A is LK-A horizontal to ROS
Influence.B is influence of the Antioxidant N-acetyl-cysteine (NAC) to the LK-A Apoptosis induced.
Fig. 5 is KYSE30 the and KYSE450 Apoptosis that JNK and p38MAPK paths mediate LK-A inductions.A is LK-A pairs
The influence of JNK/p-JNK, p38/p-p38 and Erk/p-Erk expression in KYSE30 and KYSE450 cells.B is JNK paths
The influence for the esophageal squamous cell carcinoma Apoptosis that inhibitor induces LK-A.C is the esophageal squamous cell carcinoma that p38 pathway inhibitors induce LK-A
The influence of Apoptosis.D is the influence that NAC activates JNK and p38 MAPK caused by LK-A.
Fig. 6 is the tumour growth in LK-A suppression esophageal squamous cell carcinoma mice xenograft models.A is between different disposal group
Tumor size compare.B, gross tumor volume, tumor weight and nude mice weight of the C and D between different disposal group.E is LK-A's
Treatment does not cause obvious internal organs toxicity.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, is commercially available unless otherwise specified.
Quantitative test in following embodiments, all experiments are averaged after being repeated three times, with mean ± standard deviation
Form represent.*p<0.05,**p<0.01,***p<0.001 (compared with control group).
CCK-8 reagents in following embodiments are the products of Japanese Dojindo companies.Cell cycle detection kit and
Annexiv-FITC cell apoptosis detection kits are the products of Chinese Kai base biology.Crystal violet, NAC and peroxide-sensitive type
Fluorescence probe DCFH-DA is the product in the green skies of China.Jnk inhibitor (SP600125), p38 MAPK inhibitor
(SB202190) and for all antibody of Western blot it is Cell Signaling Technologies Inc.
The product of (Danvers, MA, USA).
KYSE30 cells, KYSE450 cells, NIH3T3 cells and HEK-293 cells in following embodiments is by China
Academy of Medical Sciences institute of oncology of tumour hospital provides.Identified by STR, KYSE30 cells and KYSE450 cells meet day
The STR bit point spectrum that this cell bank (JCRB) provides.KYSE30 cells, KYSE450 cells and NIH3T3 cells are using addition tire
Cow's serum (Gibco, New York, NY, USA), penicillin and streptomysin 1640 culture mediums of RMPI (HyClone, Logan,
UT, USA) to be cultivated, HEK-293 cells use the MEM culture mediums for adding hyclone, penicillin and streptomysin
(HyClone, Logan, UT, USA) is cultivated, and hyclone, working concentration that volume fraction is 10% are added in culture medium
(100 μ g/ml) streptomysin that (100U/ml) penicillin and working concentration for 1% are 1%.All cells are at 37 DEG C, 5%CO2
Constant incubator (Thermo, USA) in culture.All cell lines that the present invention uses are examined by Short tandem repeatSTR (STR)
Survey is identified, and excludes the presence of mycoplasma.
The nude mice (SPF, 4 week old) of female BAl BIc/cA-nu no-special pathogen levels in following embodiments is Beijing Hua Fu
The product of health biotech inc.Every nude mice weight is about 18g, is raised in Cancer Hospital of Chinese Academy of Medical Sciences
Experimental Animal Center.
The statistical analysis of all data uses 6.0 softwares of GraphPad Prism in following embodiments, and data are represented
For mean+SD, and examined by one-way analysis of variance or Student's t test and compare each group experimental data.p
<0.05, which is considered difference, has the standard of statistical significance.
The preparation method of PBS (pH 7.4) in following embodiments：Weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4
With 0.24g KH2PO4800mL distilled water is dissolved in, concentrated hydrochloric acid adjusts pH value to 7.4, is settled to 1L.
The preparation and identification of embodiment 1, long tube Amethystoidin A (LK-A)
First, the preparation of long tube Amethystoidin A (LK-A)
1st, it will be dried and grind successively in the leaf of the glaucocalyx rabdosia herb of the elegant collection of Guangxi China gold, obtain powdered
Sample；10 Kg of powder shape samples are taken, it are soaked 3 days in 100 liters of volume fractions are 70% aqueous acetone solution, then mistake
Filter, collects filtrate.
2nd, the filtrate (vacuum distillation) that evaporation step 1 obtains under lower pressure, removes the acetone in filtrate, is slightly carried
Take liquid.
3rd, ethyl acetate is added in the crude extract obtained to step 2 to be extracted, extract 4 times, each ethyl acetate 15
Rise, obtain acetic acid ethyl acetate extract.
4th, the ethyl acetate obtained using the silica gel chromatographic column separating step 3 filled with 200-300 mesh pore size silica gels is extracted
Liquid (938.5g).Mobile phase is made of chloroform (A) and acetone (B), with chloroform-acetone (1:0,9:1,8:2,7:3,6:4,1:1,
0:1) gradient elution, thin-layered chromatography (TLC) detect and merge same section, obtain A, B, C, D, E totally 5 components.
5th, component B (618.5g) is decolourized on MCI gels, is then washed with the methanol aqueous solution that volume fraction is 90%
It is de-, component B1-B4 is obtained, then separated by repeating silica gel column chromatography, eluent system (petroleum ether：Acetone is (20:1)-(2:
1) component B1 (116g) and B2 (135g)), are obtained, two components are dissolved in methanol and being recrystallized, that is, obtain LK-A powder
(20g).The chemical structural formula of LK-A is as shown in Figure 1A.The structure of LK-A is a kind of ent kauranoid class compound.
6th, the LK-A powder that step 5 obtains is dissolved in dimethyl sulfoxide (DMSO) (DMSO), obtains LK-A solution, in LK-A solution
LK-A concentration is 50mM, by LK-A solution in -20 DEG C of preservations.LK-A solution is as prepared herein used in following experimental studies
LK-A solution, LK-A solution can be diluted to required working concentration by when use with 1640 culture mediums of RMPI.
2nd, the Structural Identification and purity analysis of long tube Amethystoidin A
1st, the LK-A powder obtained in the 5 of step 1 is subjected to mass spectral analysis, the detection of 1H NMR spectrums and 13C respectively
NMR spectrum detects.Detecting step bibliography " Fujita, Tetsuro, Takeda, Yoshio, Shingu,
Tetsuro,Longikaurin Aand B；new,biologically active diterpenoids from Rabdosia
longituba,J From Journal of the Chemical Society,Chemical Communications
(1980), (5), the method in 205-7. ".Testing result is successively such as Fig. 1 D, Fig. 1 E and Fig. 1 F.
Long tube Amethystoidin A：White powder；C20H28O5, mass spectrum m/z 371.1837 ([M+Na]+, calcd for
371.1829)；
1H nuclear magnetic resonance (400MHz, CDCl3)：δ H 6.54 (1H, s, OH), 6.52 (1H, s, OH), 6.18 (1H, s, H-
17a), 5.56 (1H, s, H-17b), 4.82 (1H, br s, H-14 α), 4.11 (1H, d, J=9.9Hz, H-20a), 3.82 (1H,
D, J=9.9Hz, H-20b), 3.77 (1H, m, H-6 α), 3.06 (1H, br d, J=9.4Hz, H-13), 1.10 (3H, s, H-
18), 1.09 (3, H, H-19).
13C nuclear magnetic resonance (100MHz, CDCl3)：δ C 31.0 (t, C-1), 18.5 (t, C-2), 41.1 (t, C-3), 33.6
(s, C-4), 59.2 (d, C-5), 72.6 (d, C-6), 97.3 (s, C-7), 62.0 (s, C-8), 52.8 (d, C-9), 36.3 (s, C-
10), 16.6 (t, C-11), 29.5 (t, C-12), 42.7 (d, C-13), 74.1 (d, C-14), 207.3 (s, C-15), 151.2
(s, C-16), 120.8 (t, C-17), 33.3 (q, C-18), 22.3 (q, C-19), 66.8 (t, C-20).
2nd, on 1200 liquid chromatographs of Agilent to step 15 in the LK-A powder that obtains carry out purity analysis,
Use Zorbax SB-C18 (4.6 millimeters × 250 millimeters) chromatographic column (10-100%CH3CN：H2O, 30 minutes, 238 nanometers, 1 milli
Liter/min).The result shows that：The purity of LK-A powder is more than 98% (Fig. 1 G).
The above results show that the solid matter of white powder prepared by the present invention is long tube Amethystoidin A.
The application of embodiment 2, LK-A in esophageal squamous cell carcinoma cell Proliferation and Clone formation is suppressed
First, LK-A suppresses the propagation of esophageal squamous cell carcinoma cell
In order to study influences of the LK-A to esophageal squamous cell carcinoma cell line (KYES30 and KYSE450), CCK-8 experiments have been carried out.
Comprise the following steps that：
1st, with containing 10% (v/v) hyclone, 1% (100U/mL) penicillin and 1% (100 μ g/mL) streptomysin
RPMI-1640 medium cultures cell (esophageal squamous cell carcinoma cell line KYSE30 or KYSE450, mouse embryonic fibroblasts system
NIH3T3) it is resuspended to logarithmic proliferation phase, then digestion, obtains cell suspending liquid.
2nd, 96 porocyte culture plates are taken, it is (about 3000 thin per hole that the cell suspending liquid that 100 μ L steps 1 obtain is added per hole
Born of the same parents), 12h or so, it is 100 μ L LK-A solution that liquid is changed after cell attachment, obtains cell culture system, LK-A is trained in cell
Concentration in the system of supporting is respectively 0 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, 6 μM and 8 μM, in 5%CO2, 37 DEG C of bars
24h or 48h is cultivated under part；Then 100 μ L are added per hole and contains the RPMI-1640 culture mediums of 10%CCK8 (10 μ L/1mL), and incubated
Educate 2-4h.Light absorption value at 450nm is detected using microplate reader.
Negative control hole is set in 96 porocyte culture plates, and each negative control hole adds 100 μ L step 1 gained cells
Suspension, i.e., per about 3000, hole cell.Negative control group changes liquid after cell attachment and contains 0.1%DMSO's (v/v) for 100 μ L
RPMI-1640 nutrient solutions (change liquid) with LK-A solution same time point, in 5%CO2, 37 DEG C of culture 24h or 48h；Then per hole
Add 100 μ L and contain 1640 culture mediums of RMPI of 10%CCK8 (10 μ L/mL), and be incubated 2-4h.It is negative right using microplate reader detection
According to light absorption value of the hole at 450nm.
Using concentration of the LK-A in hole as abscissa, relative activity is ordinate, compares cell to be measured after LK-A is handled
Propagation degree.Relative activity is calculated according to following formula：Relative activity (%)=(light absorption value-the moon of LK-A solution at 450nm
Property is to impinging upon the light absorption value at 450nm)/(light absorption value-negative control extinction 450nm at of the positive control at 450nm
Value) × 100%.Positive control refers to the group that LK-A concentration is 0 μM.
All experiments are averaged after being repeated three times, and are represented in the form of mean ± standard deviation.*p<0.05,**p<
0.01,***p<0.001。
Test result indicates that：LK-A passage times and dose-dependent mode suppress two kinds of esophageal squamous cell carcinoma cell lines
The propagation (Figure 1B) of (KYES-30 and KYSE-450).When LK-A is to KYSE-30 and KYSE-450 cell inhibitory effect 24h, IC50
Value is respectively 1.259 μM and 1.370 μM.In order to verify the security of LK-A, LK-A is have detected to normal cell line i.e. Development of Mouse Embryos
The toxic action of tire fibroblast (NIH3T3) and human embryonic kidney cells HEK-293 cells.Test result indicates that：In NIH3T3
The IC of LK-A in cell line50Value is about 3.941 μM, the IC of LK-A in HEK-293 cells50About 5.744 μM of value, far above two
The corresponding IC of a esophageal squamous cell carcinoma cell line50It is worth (Figure 1B).Illustrate LK-A be a kind of relative efficiency and safe drugs.
2nd, LK-A suppresses the Clone formation of esophageal squamous cell carcinoma cell
In order to study influences of the LK-A to esophageal squamous cell carcinoma cell line (KYES30 and KYSE450) Colony forming, carried out as
Lower experiment：
1st, with containing 10% (v/v) hyclone, 1% (100U/mL) penicillin and 1% (100 μ g/mL) streptomysin
Then RPMI-1640 medium culture esophageal squamous cell carcinomas cell (KYSE30 cells or KYSE450 cells) is digested to the logarithmic proliferation phase
It is resuspended, obtains cell suspending liquid.
2nd, 6 orifice plates are taken, 1950 μ L RPMI-1640 culture mediums and the cell suspending liquid of 50 μ L steps 1 acquisition are added per hole
(per about 400, hole cell), in 5%CO2, cultivate 12h under the conditions of 37 DEG C；Then LK-A solution is added, distinguishes LK-A concentration
For 0.1 μM, 0.2 μM and 0.3 μM, in 5%CO2, 37 DEG C of CMC model 48h.At the same time with the RPMI- containing 0.1% (v/v) DMSO
1640 culture mediums are as control.
3rd, after cultivating 1 week, the visible clone of naked eyes is fixed 30 minutes with paraformaldehyde, then with 0.1% violet staining,
To be visualized and be counted.
Test result indicates that：LK-A can substantially suppress the Clone formation of esophageal squamous cell carcinoma in low concentration.At LK-A
48h is managed, clone's shape in esophageal squamous cell carcinoma cell line (KYES30 and KYSE450) can be significantly inhibited by dose-dependent mode
Into (Fig. 1 C).
The application of embodiment 3, LK-A in esophageal squamous cell carcinoma cell-cycle arrest is induced
First, influences of the LK-A to the esophageal squamous cell carcinoma cell cycle
In order to study whether LK-A plays its effect by inducing cell cycle arrest, have studied LK-A to KYSE30 and
The influence of cell cycle in KYSE450 cell lines.Comprise the following steps that：
1st, with containing 10% (v/v) hyclone, 1% (100U/mL) penicillin and 1% (100 μ g/mL) streptomysin
RPMI-1640 medium cultures cell (KYSE30 cells or KYSE450 cells) to logarithmic proliferation phase, then digestion is resuspended, obtains
To cell suspending liquid.
2nd, 6 orifice plates are taken, 1950 μ L RPMI-1640 culture mediums and the cell suspending liquid of 50 μ L steps 1 acquisition are added per hole
(per hole about 1.2 × 105A cell), in 5%CO2, 37 DEG C of CMC model 12h；Then LK-A solution is added, it is 1 to make LK-A concentration
μM, 2 μM and 3 μM, in 5%CO2, 37 DEG C of CMC model 48h.At the same time with the RPMI-1640 culture mediums containing 0.1% (v/v) DMSO
As control.
3rd, cell and control cell after washing LK-A processing with PBS, were then fixed at 4 DEG C with 70% ethanol of freezing
Night.
4th, the cell of fixation is washed with PBS, is incubated with RNase A and removes within 30 minutes RNA, then dyed with propidium iodide (PI)
30 minutes.
5th, red fluorescence at record excitation wavelength 488nm is detected by flow cytometry analysis, is calculated using analysis software
And analyze different phase G2/M phases cell cycle cell, S phases cell and G0/G1 phase cell percentages.
Test result indicates that：Compared with the control, the percentage of the G2/M phase esophageal squamous cell carcinoma cells of LK-A processing dramatically increases.
Illustrate that LK-A can influence esophageal squamous cell carcinoma cell cycle progression, the induction esophageal squamous cell carcinoma cell G2/M phases block (Fig. 2A and 2B).
2nd, influences of the LK-A to the expression of esophageal squamous cell carcinoma cell cycle related proteins
In order to study expressions of the LK-A to the specific cell cycle GAP-associated protein GAP of participation regulation and control G2/M phase checkpoints
Influence.Following experiment is carried out：
1st, the total protein of cell after the LK-A in the 2 of above-mentioned steps one is handled is extracted.
2nd, Western blot are carried out to total protein of cell using DNADamage Antibody Sampler Kit.Using
Cdc2Antibody detects Cdc2, detects Cyclin B1 using Cyclin B1Antibody, is examined using GAPDH Antibody
Survey GAPDH.
Test result indicates that：Compared with control cell, the expression water of the cdc2 and Cyclin B1 after LK-A processing in cell
Pancake is low (Fig. 2 C).Illustrate LK-A by lowering CylinB1 and cdc2 expression to induce esophageal squamous cell carcinoma cell G2/M Cycle Arrests.
The application of embodiment 4, LK-A in esophageal squamous cell carcinoma Apoptosis is induced
First, LK-A induces esophageal squamous cell carcinoma Apoptosis
Apoptosis is defined in early 1970s.One of key feature of cancer cell is can to suppress cell
Apoptosis.Apoptosis is occurred by two kinds of main paths, i.e. extrinsic pathway (death receptor pathway *) and intrinsic pathway (mitochondria
Approach), these approach promote the molecular mechanism of Apoptosis to be elucidated with.Two key components of Bcl-2 albumen are：Promotion is withered
The Bcl-2 albumen (i.e. Bad, Bid and Bax) and the Bcl-2 albumen (i.e. Bcl-2, Bcl-xL and Mcl-1) of resistance apoptosis died.
Bcl-2 families include the albumen that several growth-promotings deposit and promote death.In order to study the ability of LK-A induction esophageal squamous cell carcinoma Apoptosis
And whether LK-A has apoptosis-promoting effect to esophageal squamous cell carcinoma cell line, AnnexinV/PI dyeing and flow cytometry have been carried out.
Comprise the following steps that：
1st, with containing 10% (v/v) hyclone, 1% (100U/mL) penicillin and 1% (100 μ g/mL) streptomysin
RPMI-1640 medium cultures cell (KYSE30 cells or KYSE450 cells) to logarithmic proliferation phase, then digestion is resuspended, obtains
To cell suspending liquid.
2nd, 6 orifice plates are taken, 1950 μ L RPMI-1640 culture mediums and the cell suspending liquid of 50 μ L steps 1 acquisition are added per hole
(per hole about 1.2 × 105A cell), in 5%CO2, cultivate 12h under the conditions of 37 DEG C；Then LK-A solution is added, makes LK-A concentration
For 0 μM, 1 μM, 2 μM and 3 μM, in 5%CO2, cultivate 24h or 48h under the conditions of 37 DEG C, then collect cell.Using LK-A concentration as 0
μM treatment group as a control group.
3rd, the cell being collected into is washed with PBS, is then dyed with Annexin V-FITC and PI, passes through fluidic cell afterwards
Instrument detection Level of Apoptosis (Becton Dickinson FACSCantoII, NJ, USA), and use the Annexin of triumphant base
V-FITC cell apoptosis detection kits assess Apoptosis.
Test result indicates that：Compared with control group, esophageal squamous cell carcinoma cell (the KYSE30 cells of the LK-A processing of various concentrations
With KYSE450 cells) ratio that occurs apoptosis dramatically increases, and LK-A concentration is higher, then the ratio that apoptosis occurs is higher.Explanation
LK-A can promote esophageal squamous cell carcinoma Apoptosis, and dosage and time dependence (Fig. 3 A) is presented.
2nd, influences of the LK-A to apoptosis protein expression
In order to study influences of the LK-A to apoptosis protein expression, LK-A processing is measured by Western blot
Cleaved caspase-3, Cleaved caspase-9, Cleaved PARP, γ H2AX after esophageal squamous cell carcinoma cell 24h,
The protein expression level of Bax, Bcl-2 and cytochrome c, while using GAPDH as internal reference.
Test result indicates that：With the increase of LK-A dosage, Cleaved caspase-3, Cleaved caspase-9 and
The protein expression level of Cleaved PARP gradually increases (Fig. 3 B).In addition, in KYSE30 and KYSE450 cells, γ H2AX
Protein expression level with LK-A dosage increase and raise, γ H2AX (Specific marker of double-strand break) be one kind
DNA damage mark, the rise of γ H2AX show that LK-A causes the damage of cell DNA after acting on.Bax and cromoci egg
White expression increases and raises also with the dosage of LK-A.And Bcl-2 protein expression levels with LK-A dosage increase and
Reduce.The change of Bcl-2/Bax ratios causes cromoci, Cleaved caspase-3 and Cleaved caspase-9 tables
The change (Fig. 3 C) reached.
3rd, the activation inducing cell apoptosis that LK-A passes through caspase
In order to study whether the Apoptosis of LK-A inductions needs the activation of caspase, following experiment has been carried out：
1st, with containing 10% (v/v) hyclone, 1% (100U/mL) penicillin and 1% (100 μ g/mL) streptomysin
RPMI-1640 medium cultures cell (KYSE30 cells or KYSE450 cells) to logarithmic proliferation phase, then digestion is resuspended, obtains
To cell suspending liquid.
2nd, 6 orifice plates are taken, 1950 μ L RPMI-1640 culture mediums and the cell suspending liquid of 50 μ L steps 1 acquisition are added per hole
(per hole about 1.2 × 105A cell), in 5%CO2, cultivate 12h under the conditions of 37 DEG C；One kind is added into each hole cell respectively to ooze
Inhibitor synthetic peptide Z-VAD (OMe)-FMK (CST, 60322S) of the caspase of saturating cell, makes it in cell culture system
Concentration be 10 μM；In 5%CO2, pre-process 2h under the conditions of 37 DEG C.At the same time to be not added with Z-VAD (OMe)-FMK as control.
3 and then LK-A solution is added into each hole cell, it is 3 μM to make LK-A concentration, in 5%CO2, under the conditions of 37 DEG C
24h is cultivated, then collects cell.At the same time to be not added with LK-A as control.
4th, the cell being collected into is washed with PBS, is then dyed with Annexin V-FITC and PI, passes through fluidic cell afterwards
Instrument detection Level of Apoptosis (Becton Dickinson FACSCantoII, NJ, USA), and use the Annexin of triumphant base
V-FITC cell apoptosis detection kits assess Apoptosis.
Test result indicates that：The Apoptosis of LK-A inductions can be partly reversed using Z-VAD (OMe)-FMK.Illustrate LK-
The activation inducing cell apoptosis that A passes through caspase.
The effect that embodiment 5, ROS are played in the esophageal squamous cell carcinoma Apoptosis that LK-A is induced
First, influences of the LK-A to ROS in esophageal squamous cell carcinoma cell
Research, which shows that the ROS of intracellular excess is produced, can induce polytype cancer cell-apoptosis.In order to study LK-A
Influence to ROS in esophageal squamous cell carcinoma cell, has carried out following experiment：
1st, with containing 10% (v/v) hyclone, 1% (100U/mL) penicillin and 1% (100 μ g/mL) streptomysin
RPMI-1640 medium cultures cell (KYSE30 cells or KYSE450 cells) to logarithmic proliferation phase, then digestion is resuspended, obtains
To cell suspending liquid.
2nd, 6 orifice plates are taken, 1950 μ L RPMI-1640 culture mediums and the cell suspending liquid of 50 μ L steps 1 acquisition are added per hole
(per hole about 1.2 × 105A cell), in 5%CO2, cultivate 12h under the conditions of 37 DEG C；Then LK-A solution is added, makes LK-A concentration
Respectively 1 μM, 2 μM and 3 μM, in 5%CO2, cultivate 12h under the conditions of 37 DEG C.At the same time with the RPMI- containing 0.1%DMSO (v/v)
1640 culture medium processing is as a control group.
3rd, after LK-A processing, 6 orifice plates is taken, fluorescence probe DCFH-DA (Beyotime, China) is added into each hole, makes it
Concentration in cell culture system is 10 μM, and 37 DEG C are incubated 20 minutes, collect cell.
4th, wash cell twice with PBS, be resuspended in PBS, average DCF fluorescence is measured by flow cytometer
To detect, ROS is horizontal to be changed value.
Test result indicates that：LK-A processing causes the rise of ROS in esophageal squamous cell carcinoma cell, and in dose dependent (figure
4A).Illustrate that ROS rises are probably the reason for causing esophageal squamous cell carcinoma Apoptosis.
2nd, ROS mediates LK-A induction esophageal squamous cell carcinoma Apoptosis
In order to verify whether Antioxidant N-acetyl-cysteine (NAC) can reverse the Apoptosis of LK-A inductions, into
Following experiment is gone：
1st, with containing 10% (v/v) hyclone, 1% (100U/mL) penicillin and 1% (100 μ g/mL) streptomysin
RPMI-1640 medium cultures cell (KYSE30 cells or KYSE450 cells) to logarithmic proliferation phase, then digestion is resuspended, obtains
To cell suspending liquid.
2nd, 6 orifice plates are taken, 1950 μ L RPMI-1640 culture mediums and the cell suspending liquid of 50 μ L steps 1 acquisition are added per hole
(per hole about 1.2 × 105A cell), in 5%CO2, cultivate 12h under the conditions of 37 DEG C；Then antioxidant N- second is added into each hole
Acyl cysteine (NAC), it is 10mM to make Antioxidant N-acetyl-cysteine (NAC) concentration, in 5%CO2, it is pre- under the conditions of 37 DEG C
Handle 1h.Handled as a control group with being not added with NAC at the same time.
3rd, after Antioxidant N-acetyl-cysteine (NAC) pretreatment, LK-A solution is added into each hole, makes LK-A concentration
For 3 μM, in 5%CO2, cultivate 12h under the conditions of 37 DEG C after collect cell.At the same time control is used as to be not added with LK-A processing.
4th, the cell being collected into is washed with PBS, is then dyed with Annexin V-FITC and PI, passes through fluidic cell afterwards
Instrument detection Level of Apoptosis (Becton Dickinson FACSCantoII, NJ, USA), and use the Annexin of triumphant base
V-FITC cell apoptosis detection kits assess Apoptosis.
Test result indicates that：NAC can significantly reverse the Apoptosis (Fig. 4 B) that LK-A is induced.
Result above shows：The esophageal squamous cell carcinoma Apoptosis of LK-A inductions may be mediated by ROS, while can be by antioxidant
NAC is reversed.
Embodiment 6, LK-A induce esophageal squamous cell carcinoma Apoptosis by activating JNK/p38 paths
First, influences of the LK-A to mapk kinase activity
The main component of mitogen-activated protein kinase (MAPK) path includes extracellular signal-regulated kinase (ERK), c-
Jun N- terminal Kinases or stress activated protein kinase (JNK or SAPK) and p38, it plays important in antitumor drug effect
Effect.Research shows that the activation of JNK and p38MAPK play a crucial role in the cancer cell-apoptosis that various anticancers induce.In order to
Influences of the LK-A to mapk kinase activity is studied, have detected LK-A to JNK/p-JNK, p38/ in KYSE30 and KYSE450 cells
The influence of p-p38 and Erk/p-Erk expressions.Comprise the following steps that：
1st, with containing 10% (v/v) hyclone, 1% (100U/mL) penicillin and 1% (100 μ g/mL) streptomysin
RPMI-1640 medium cultures cell (KYSE30 cells or KYSE450 cells) to logarithmic proliferation phase, then digestion is resuspended, obtains
To cell suspending liquid.
2nd, 6 orifice plates are taken, 1950 μ L RPMI-1640 culture mediums and the cell suspending liquid of 50 μ L steps 1 acquisition are added per hole
(per hole about 1.2 × 105A cell), in 5%CO2, cultivate 12h under the conditions of 37 DEG C；Then LK-A solution is added, makes LK-A concentration
For 1 μM, 2 μM and 3 μM, in 5%CO2, cultivate 24h under the conditions of 37 DEG C.At the same time with containing the RPMI-1640 of 0.1%DMSO (v/v) trainings
Nutrient solution processing is as a control group.
3rd, the total protein of cell after the LK-A in above-mentioned steps 2 is handled is extracted, is detected by Western blot at LK-A
The expression of JNK/p-JNK in cell after reason, p38/p-p38 and Erk/p-Erk.
Test result indicates that：JNK and p38 phosphorylation levels raise after LK-A processing 24h, and the expression of Erk/p-Erk
Horizontal nothing significantly sexually revises (Fig. 5 A).
2nd, the influence for the esophageal squamous cell carcinoma Apoptosis that JNK and p38 pathway inhibitors induce LK-A
It is whether most important in order to study the esophageal squamous cell carcinoma Apoptosis that JNK and p38 paths induce for LK-A, carry out
Following experiment：
1st, the influence for the esophageal squamous cell carcinoma Apoptosis that JNK pathway inhibitors induce LK-A
(1) with containing 10% (v/v) hyclone, 1% (100U/mL) penicillin and 1% (100 μ g/mL) streptomysin
RPMI-1640 medium cultures cell (KYSE30 cells or KYSE450 cells) to logarithmic proliferation phase, then digestion is resuspended, obtains
To cell suspending liquid.
(2) 6 orifice plates are taken, 1950 μ L RPMI-1640 culture mediums and the cell suspending liquid of 50 μ L steps 1 acquisition are added per hole
(per hole about 1.2 × 105A cell), in 5%CO2, cultivate 12h under the conditions of 37 DEG C；Then jnk inhibitor SP600125 is added, is made
Concentration of the SP600125 in cell culture system is 5 μM, in 5%CO2, cultivate 1h under the conditions of 37 DEG C.Pressed down at the same time with being not added with JNK
Preparation SP600125 processing is as a control group.
(3) after SP600125 processing, 6 orifice plates is taken, LK-A solution is added into each hole, it is 3 μM to make LK-A concentration, 5%
CO2, cultivate 24h under the conditions of 37 DEG C, collect cell.Handled as a control group with being not added with LK-A at the same time.
(4) cell being collected into is washed with PBS, is then dyed with Annexin V-FITC and PI, it is thin by streaming afterwards
Born of the same parents' instrument detection Level of Apoptosis (Becton Dickinson FACSCantoII, NJ, USA), and use the Annexin of triumphant base
V-FITC cell apoptosis detection kits assess Apoptosis.
2nd, the influence for the esophageal squamous cell carcinoma Apoptosis that p38 pathway inhibitors induce LK-A
According to the method in step 1, jnk inhibitor SP600125 is replaced with into p38 pathway inhibitor SB202190, and protect
Hold the influence for the esophageal squamous cell carcinoma Apoptosis that other steps are constant, and detection p38 pathway inhibitors induce LK-A.
Test result indicates that：LA-K before processings 2h is pre- with jnk inhibitor SP600125 or p38MAPK inhibitor SB202190
Processing can weaken the esophageal squamous cell carcinoma Apoptosis (Fig. 5 B and Fig. 5 C) of LK-A inductions.
3rd, the influence that NAC activates JNK caused by LK-A and p38MAPK
MAPK paths can be activated by ROS, in order to study the influence that NAC activates JNK and p38 MAPK caused by LK-A,
Following experiment is carried out：
1st, with containing 10% (v/v) hyclone, 1% (100U/mL) penicillin and 1% (100 μ g/mL) streptomysin
RPMI-1640 medium cultures cell (KYSE30 cells or KYSE450 cells) to logarithmic proliferation phase, then digestion is resuspended, obtains
To cell suspending liquid.
2nd, 6 orifice plates are taken, 1950 μ L RPMI-1640 culture mediums and the cell suspending liquid of 50 μ L steps 1 acquisition are added per hole
(per hole about 1.2 × 105A cell), in 5%CO2, cultivate 12h under the conditions of 37 DEG C；Then antioxidant N- second is added into each hole
Acyl cysteine (NAC), it is 10mM to make concentration of the Antioxidant N-acetyl-cysteine (NAC) in cell culture system,
5%CO2, pre-process 1h under the conditions of 37 DEG C.Handled as a control group with being not added with NAC at the same time.
3rd, after Antioxidant N-acetyl-cysteine (NAC) pretreatment, LK-A solution is added into each hole, makes LK-A concentration
For 3 μM, in 5%CO2, cultivate 12h under the conditions of 37 DEG C after collect cell.At the same time control is used as to be not added with LK-A processing.
4th, the total protein of cell after the LK-A in above-mentioned steps 3 is handled is extracted, is detected by Western blot at LK-A
After reason in cell JNK/p-JNK and p38/p-p38 expression.
Test result indicates that：JNK and p38 phosphorylation levels are reduced after NAC processing, illustrate that ROS causes LK-A inductions
P-JNK and p-p38 change (Fig. 5 D).In conclusion the generation of JNK and p38 MAPK phosphorylations and ROS may be to LK-A
The esophageal squamous cell carcinoma Apoptosis of induction plays synergistic effect.
Embodiment 7, LK-A suppress the tumour growth in esophageal squamous cell carcinoma mice xenograft model
In order to study the internal antitumor activity of LK-A, following experiment has been carried out：
1st, the nude mice (SPF, 4 week old) of female BAl BIc/cA-nu no-special pathogen levels is randomly divided into following four groups (often
Group 5)：LK-A (6mg/kg) group, LK-A (12mg/kg) group, cis-platinum (DDP) group and control group, are handled as follows respectively：
LK-A (6mg/kg) group：By KYSE30 cell subcutaneous injections to back on the right side of nude mice (cell infusion amount for 1.2 ×
106It is a), LK-A is injected intraperitoneally after transplanting every three days, tumour is taken out after for 3 weeks.Injection dosage is 6mg LK-A/kg naked
Mouse.
LK-A (12mg/kg) group：By KYSE30 cell subcutaneous injections to back on the right side of nude mice (cell infusion amount for 1.2 ×
106It is a), LK-A is injected intraperitoneally after transplanting every three days, tumour is taken out after for 3 weeks.Injection dosage is 12mg LK-A/kg naked
Mouse.
Cis-platinum (DDP) group：By back on the right side of KYSE30 cell subcutaneous injections to nude mice, (cell infusion amount is 1.2 × 106
It is a), LK-A is injected intraperitoneally after transplanting every three days, tumour is taken out after for 3 weeks.Injection dosage is 2mg LK-A/kg nude mices.
Control group：By back on the right side of KYSE30 cell subcutaneous injections to nude mice, (cell infusion amount is 1.2 × 106It is a), move
0.1%DMSO is injected intraperitoneally after plant every three days, tumour is taken out after for 3 weeks.Injection dosage is 200 μ L.
2nd, measure the gross tumor volume of each group mouse and be averaged by group；Weigh the tumor weight of each mouse and make even by group
Average；Every 3 days measurement gross tumor volumes and nude mice weight are once, for 3 weeks.And using injection time as abscissa, average tumor body
Product or tumor weight are ordinate, draw tumor growth curve.
Test result indicates that：Compared with control group, LK-A groups and the obvious growth for inhibiting transplantable tumor of DDP groups, and significantly
The weight and volume (Fig. 6 A, Fig. 6 B and Fig. 6 C) of transplantable tumor is reduced, illustrates that LK-A and cis-platinum can effectively suppress tumour growth,
With obvious tumors inhibition activity.
3rd, the security in order to further determine LK-A in vivo, measures nude mice weight between different groups and is averaged by group
Value, detects the change of nude mice weight between different groups.
Test result indicates that：Between different groups nude mice weight without significant difference (Fig. 6 D), while the treatment of LK-A is to naked
The general status of mouse is also without obvious harmful effect.Illustrate LK-A to nude mice non-evident effect.
4th, nude mice is put to death after testing, takes the hepatic tissue and nephridial tissue of each group mouse respectively, and be fixed in formalin
In, then carry out paraffin embedding processing and carry out HE dyeing.
Test result indicates that：The hepatic tissue and nephridial tissue of each group mouse are without obvious damage.Illustrate that the treatment of LK-A is not drawn
Play obvious internal organs toxicity (Fig. 6 E).
Claims (10)
1. long tube Amethystoidin A is in following A1) to A11) at least one of in application：
A1) prepare and suppress the propagation of esophageal squamous cell carcinoma cell and/or the product of growth；
A2 the product for the Clone formation for suppressing esophageal squamous cell carcinoma cell) is prepared；
A3 the product of induction esophageal squamous cell carcinoma cell-cycle arrest) is prepared；
A4) prepare and suppress the product that the DNA of esophageal squamous cell carcinoma cell is repaired；
A5) prepare induction and/or promote the product of esophageal squamous cell carcinoma Apoptosis；
A6 the product that up-regulation promotes the expression quantity of apoptosis protein) is prepared；
A7 the product for the expression quantity for lowering resistance apoptosis protein) is prepared；
A8 induction esophageal squamous cell carcinoma cytoactive oxygen (ROS) increased product) is prepared；
A9 the product of induction and/or activation esophageal squamous cell carcinoma cell JNK MAPK paths) is prepared；
A10 the product of induction and/or activation esophageal squamous cell carcinoma cell p38MAPK paths) is prepared；
A11 the product for suppressing the growth of tumour caused by esophageal squamous cell carcinoma cell) is prepared.
2. long tube Amethystoidin A is in following B1) to B11) at least one of in application：
B1 propagation and/or the growth of esophageal squamous cell carcinoma cell) are suppressed；
B2 the Clone formation of esophageal squamous cell carcinoma cell) is suppressed；
B3 esophageal squamous cell carcinoma cell-cycle arrest) is induced；
B4 the DNA for) suppressing esophageal squamous cell carcinoma cell is repaired；
B5) induce and/or promote esophageal squamous cell carcinoma Apoptosis；
B6) up-regulation promotes the expression quantity of apoptosis protein；
B7 the expression quantity of resistance apoptosis protein) is lowered；
B8 esophageal squamous cell carcinoma cytoactive oxygen (ROS) increase) is induced；
B9) induce and/or activate esophageal squamous cell carcinoma cell JNK MAPK paths；
B10) induce and/or activate esophageal squamous cell carcinoma cell p38MAPK paths；
B11 the growth of tumour caused by esophageal squamous cell carcinoma cell) is suppressed.
3. application according to claim 1 or 2, it is characterised in that：
The promotion apoptosis protein is Cleaved caspase-3, Cleaved caspase-9, Cleaved PARP, γ
At least one of H2AX, Bax, Bcl-2 and cytochrome c；
The resistance apoptosis protein is Bcl-2.
4. application according to claim 1 or 2, it is characterised in that：
The cell cycle is the G2/M phases.
5. application according to claim 1 or 2, it is characterised in that：
The esophageal squamous cell carcinoma cell is KYSE30 cells or KYSE450 cells.
6. a kind of product, its active ingredient is long tube Amethystoidin A；
At least one of the function of the product is following B1) to B11)：
B1 propagation and/or the growth of esophageal squamous cell carcinoma cell) are suppressed；
B2 the Clone formation of esophageal squamous cell carcinoma cell) is suppressed；
B3 esophageal squamous cell carcinoma cell-cycle arrest) is induced；
B4 the DNA for) suppressing esophageal squamous cell carcinoma cell is repaired；
B5) induce and/or promote esophageal squamous cell carcinoma Apoptosis；
B6) up-regulation promotes the expression quantity of apoptosis protein；
B7 the expression quantity of resistance apoptosis protein) is lowered；
B8 esophageal squamous cell carcinoma cytoactive oxygen (ROS) increase) is induced；
B9) induce and/or activate esophageal squamous cell carcinoma cell JNK MAPK paths；
B10) induce and/or activate esophageal squamous cell carcinoma cell p38MAPK paths；
B11 the growth of tumour caused by esophageal squamous cell carcinoma cell) is suppressed.
7. product according to claim 6, it is characterised in that：
The promotion apoptosis protein is Cleaved caspase-3, Cleaved caspase-9, Cleaved PARP, γ
At least one of H2AX, Bax, Bcl-2 and cytochrome c；
The resistance apoptosis protein is Bcl-2.
8. the product according to claim 6 or 7, it is characterised in that：
The cell cycle is the G2/M phases.
9. according to any product in claim 6-8, it is characterised in that：
The esophageal squamous cell carcinoma cell is KYSE30 cells or KYSE450 cells.
10. according to any product in claim 6-9, it is characterised in that：
The product is medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110064046A (en) * | 2019-05-16 | 2019-07-30 | 苏州大学 | Application of micro- Peptide YY 1BM in treating cancer |
| CN112522209A (en) * | 2020-12-29 | 2021-03-19 | 郑州大学 | Method for researching influence of tumor-associated antigen p62/IMP2 on esophageal squamous cell carcinoma |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749305A (en) * | 2016-11-16 | 2017-05-31 | 中国药科大学 | Oridonin derivative, the Preparation Method And The Use of A- rings transformation |
| CN107213145A (en) * | 2017-05-05 | 2017-09-29 | 中国医学科学院肿瘤医院 | Application of the Rabdocetsin B in the product for suppressing esophageal squamous cell cancer cell multiplication is prepared |
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2017
- 2017-10-30 CN CN201711041208.8A patent/CN107929278A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749305A (en) * | 2016-11-16 | 2017-05-31 | 中国药科大学 | Oridonin derivative, the Preparation Method And The Use of A- rings transformation |
| CN107213145A (en) * | 2017-05-05 | 2017-09-29 | 中国医学科学院肿瘤医院 | Application of the Rabdocetsin B in the product for suppressing esophageal squamous cell cancer cell multiplication is prepared |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110064046A (en) * | 2019-05-16 | 2019-07-30 | 苏州大学 | Application of micro- Peptide YY 1BM in treating cancer |
| CN110064046B (en) * | 2019-05-16 | 2022-11-22 | 苏州大学 | Application of mini-peptide YY1BM in treating cancer |
| CN112522209A (en) * | 2020-12-29 | 2021-03-19 | 郑州大学 | Method for researching influence of tumor-associated antigen p62/IMP2 on esophageal squamous cell carcinoma |
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Application publication date: 20180420 |