CN107157981A - A kind of Xanthene ketone compound CCE9 purposes - Google Patents
A kind of Xanthene ketone compound CCE9 purposes Download PDFInfo
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Abstract
The invention discloses a kind of Xanthene ketone compound CCE9 purposes, the Xanthene ketone compound CCE9 of the present invention is used to prepare treating cancer medicine, by activating p38MAPK, so that phosphorylation occurs for Bcl 2, cause the conformational changes of Bcl 2, Bax is activated, so that elicitor apoptosis of tumor cells.Therefore, Xanthene ketone compound CCE9 can be used for prepare treating cancer with medicines of the Bcl 2 for action target spot.In addition, the phosphorylations of Bcl 2 can be used to screen Xanthene ketone compound as action target spot.
Description
Technical field
The invention belongs to the application field of Chinese medical extract, and in particular to a kind of Xanthene ketone compound CCE9 use
On the way.
Background technology
P38 MAPKs (p38 mitogen-activated protein kinases (p38MAPK))
UV can be included by extracellular various emergency, radioactive ray, heat shock promotees inflammatory molecule, specific antigen and other stress reactions
Activation, and in apoptosis, cell factor is produced, its important effect in transcriptional regulatory and epileptic attack.
Recent studies suggest that p38 MAPK play an important role in Apoptosis.Activation such as p38 MAPK paths can cause
Nerve cell apoptosis.In tumour cell, p38 MAPK activity rises, and participate in modulating apoptosis.Recent studies have shown that p38
MAPK can be by inducing Bcl-2 phosphorylations, inducing cell apoptosis.
Bcl-2 is one of most important regulatory factor of apoptosis process.Recently, increasing evidence shows, Bcl-2
The phosphorylation in structural disorder chain region Bcl-2 existence function, its function of surviving of Bcl-2 inactivating phosphorylations.In ring
In the presence of the border factor, inflammatory cytokine, lipopolysaccharides (LPS) and other factors, p38 MAPK can mediate Bcl-2 structures without
Ser87 and Thr56 phosphorylation on sequence chain, thus mediate NGF to remove, the Apoptosis mistake that TNF and NO are induced
Journey.In these cases, either in cell or in cell-free assays, the equal table of Bcl-2 hyperphosphorylations of p38 MAPK mediations
Now to promote apoptotic event, induced cytochrome c is from mitochondrial release process.
The phosphorylation in Bcl-2 structural disorder chains region may be by influenceing it with Bcl-2 families pro apoptotic proteins such as Bax
Interaction suppresses its anti-apoptotic function.In Memorability bone-marrow-derived lymphocyte, NGF removes the phosphorylation that induction relies on p38MAPK,
Suppress its anti-apoptotic function, induced cytochrome c release.Equally, H2O2 mediations are relied in adult rat heart cell
P38MAPK Bcl-2 phosphorylations, inducing cell apoptosis.It has been demonstrated that, Ser87 on Bcl-2 structural disorder chains and
Thr56 is p38 MAPK phosphorylations Bcl-2 site, and the phosphorylation of this this residue reduces Bcl-2 Anti-G value.
CCE9 is a kind of Xanthene ketone compound extracted from Chinese medicine cow-bezoar in wooden.Ox wood (Cratoxylon
Cochinchinensis Bl) nickname shortstyle cratoxylum leaf root or bark, yellow bud wood, dog bud wood, sparrow cage wood, Shui Mango, francolin wood, sessile alternanthera herb, over the sky
It is Guttiferae (Guttiferae) ox wood category that red (Guangxi), tea, which cough up the low excellent, U.S.A of table, plum and opens strong, culpeo bud etc.,
(Cratoxylum) plant.Function clearing away summerheat, dampness elimination disappear it is stagnant, detumescence hemostasis, cure mainly cold, fever, enteritis, diarrhea, cough
Neigh, jaundice etc.；The main chemical compositions of ox wood are Xanthene ketone, additionally containing anthraquinone Benzophenone class, triterpenes
And flavones ingredient；Modern pharmacological research shows that ox wood has anti-oxidant, cytotoxicity and nerve growth factor synergistic effect.
Although existing document report ox wood has an active anticancer, and some isolated active components, research is big
The MTT experiment stage such as Laphookhieo being only limited to compared with based on to be separated in C.cochinchinense roots 7 more
Pyrrones compound carries out in vitro cytotoxic effect test, is as a result compared with the active anticancer of camptothecine and shows compound
Cochinchinone A have stronger growth inhibitory activity to breast cancer, cervical carcinoma, intestinal cancer and KB cell lines；Boonnak
Nawong etc. has found compound gerontoxanthone I to breast cancer, cervical carcinoma, and intestinal cancer and KB cell lines have stronger thin
Born of the same parents' cytotoxic activity etc., but these simple activity research can not specify the antitumor action target spot of active component, the mechanism of action, this
It is to restrict it to be developed further into main reason for cancer therapy drug.
The content of the invention
First purpose of the present invention is preparing treating cancer medicine there is provided a kind of Xanthene ketone compound CCE9
Purposes in thing.
Second object of the present invention is to screen Xanthene ketone using Bcl-2 as action target spot there is provided one kind
The application of compound.
In order to realize the object of the invention, it is used to prepare the invention provides a kind of Xanthene ketone compound CCE9 and treats
The purposes of cancer drug.
Further, the Xanthene ketone compound CCE9 is using Bcl-2 as action target spot.
Further, the Xanthene ketone compound CCE9 has the purposes for inducing apoptosis of tumour cell.
Further, the Xanthene ketone compound CCE9 has by activating p38 MAPK inducing apoptosis of tumour cell
Purposes.
Further, the Xanthene ketone compound CCE9 has by inducing Bcl-2 phosphorylation induced tumor thin
The purposes of born of the same parents' apoptosis.
Further, the Xanthene ketone compound CCE9 has the conformation change induced tumor by inducing Bcl-2
The purposes of Apoptosis.
Further, the Xanthene ketone compound CCE9 is with the activation-inducing apoptosis of tumor cells by Bax
Purposes.
Further, the cancer is cervix cancer, lung cancer and liver cancer.
During the Xanthene ketone compound CCE9 (1,3,7-trihydroxy-2,4-diprenylxanthone) is
Medicine ox wood extract, its structure is as follows：
According to the present invention, target spot is turned to Bcl-2 phosphoric acid, can be used for screening Xanthene ketone compound, to enter
One step is developed for treating various cancers.
What the present invention was provided act on, and Bcl-2 inducing apoptosis of tumour cell can be used for instructing screening Xanthene ketone
Compound, therefrom obtains the compound with antitumor activity, and by the medicine of acquisition and then can prepare swollen available for Bcl-2 inductions
The medicine of the activity and function of apoptosis of tumor.
Brief description of the drawings
Figure 1A is the apoptosis figure that fluorescent staining detects CCE9 induced tumor cells；
Figure 1B is the percentage result figure of CCE9 inducing apoptosis of tumour cell；
Fig. 1 C are the PARP Protein cleavage figures in CCE9 induced tumor cells；
Fig. 2 is the double dye detection CCE9 induction HeLa229 cervical cancer cells of flow cytometry analysis Annexin-V/PI
Apoptosis result figure；
Fig. 3 is activation figures of the CCE9 to p38 MAPK in HeLa229 cells；
Fig. 4 A are the apoptosis of tumor cells figure that p38 MAPK inhibitor suppresses CCE9 inductions；
Fig. 4 B are the apoptosis of tumor cells figure for striking low p38 expression inhibitings CCE9 inductions；
Fig. 5 is that the Bcl-2 that the double dye detections of flow cytometry analysis Annexin-V/PI are knocked out in cell suppresses CCE9 inductions
Apoptosis of tumor cells figure；
Fig. 6 is phosphorylations and p38 MAPK inhibitor of the CCE9 to Bcl-2 in HeLa229 cells to Bcl-2's
The inhibitory action figure of phosphorylation；
Fig. 7 is the conformation change figure that CCE9 induces Bcl-2 in HeLa229 cells；
Fig. 8 is the activation figure that CCE9 induces Bax in HeLa229 cells；
Fig. 9 A are the conformation change figure that p38 MAPK inhibitor suppresses Bcl-2；
Fig. 9 B are the conformation change figure for the Bcl-2 for striking low p38 expression inhibitings CCE9 inductions.
Embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is intended to be used to explain the present invention, and can not
It is interpreted as limitation of the present invention.The experimental method of unreceipted actual conditions in embodiment, generally according to normal condition such as《Molecule
Clone：Laboratory manual》(New York:Cold Spring Harbor Laboratory Press, 1989) described in bar
Part, or according to the condition proposed by manufacturer.
The reagent used in the present invention：
Xanthene ketone compound CCE9 its structure used is as follows：
10 μM of CCE9 solution are dissolved in 99.9%DMSO solution (DMSO final concentrations by CCE9 described in embodiment 1-4,6-9
<0.1%) 10mM CCE9 solution, is prepared, is obtained with being prepared in 1 μ L CCE9 solution addition 1mL MEM culture mediums.
10 μM of CCE9 solution are dissolved in 99.9%DMSO solution (DMSO final concentrations by CCE9 described in embodiment 5<
0.1%) 10mM CCE9 solution, is prepared, is obtained with being prepared in 1 μ L CCE9 solution addition 1mL DMEM culture mediums.
The CCE9 inducing apoptosis of tumour cell of embodiment 1
1st, cell culture
Cervical cancer cell strain HeLa229 (ATCC) is selected, using the MEM culture mediums of 10% calf serum, in constant temperature 37
DEG C, 5%CO2Take out and be incubated in 24 hole tissue culturing plates in incubator, liquid is changed after 24 hours and dosing (being free of serum) is carried out
Processing.CCE9 is dissolved in 99.9%DMSO solution (DMSO final concentrations<0.1%) 10mM CCE9 solution, is prepared, with 1 μ L CCE9
Solution is added in 1mL MEM culture mediums, final concentration of 10 μM of CCE9, and processing cell 3 hours and 6 hours, control group is used same dense
DMSO solution is spent to handle 6 hours.
2nd, DAPI staining cells core
The cumulative volume of each hole solution is 1mL in reaction plate.Cell is after processing, and PBS is washed 3 times, and 4% paraformaldehyde is consolidated
Fixed, 1%Triton wears film, is eventually adding 50 μ g/mL DAPI and adds 100 μ g/mL DNase-free RNaseA, 37 DEG C of dyes carefully
Karyon 20 minutes.Glycerine mounting, fluorescence microscopy Microscopic observation karyomorphism, as a result as shown in Figure 1A.At least 300 cells,
It is random more than 5 to regard so wild, counted by two different observers, calculate the percentage of apoptosis, as a result as shown in Figure 1B.
From Figure 1A, with 10 μM of CCE9 solution effects HeLa229 cells 3 hours and 6 hours, effect 3 hours
Apoptosis substantially occurs for HeLa229 cells, and the HeLa229 cell quantities of effect 6 hours are compared to the HeLa229 cells of 3 hours more
Few, and cytoplasm more wrinkles, apoptotic cell is according to typical cytoplasm shrinkage in form, and film foaming and core are condensing, crush and be subject to
Identification.
By Figure 1B statistical result showeds, in the case where apoptosis seldom occurs for control group HeLa229 cells (4.7%), make
17% is reached with apoptosis rate after CCE9 solution 3 hours, and with the extension of action time, apoptosis HeLa229 cells reach after 6 hours
Dramatically increased to 34.3%, HeLa229 Apoptosis.
3rd, CCE9 induces the PAPR cuttings of HeLa229 cells, A549 cells and HepG2 cells
The cutting of polyadenylic acid diphosphonic acid phosphoribosynltransferase (Poly (ADP-ribose) polymerase, PARP) is early stage
The mark of apoptosis.PARP cutting can be recognized by its antibody.Cervical cancer cell strain HeLa229 (ATCC) is selected,
With the MEM culture mediums of 10% calf serum, lung cancer cell types (ATCC) and hepatoma H22 cells (ATCC), with 10%
The DMEM culture mediums of calf serum, 37 DEG C of constant temperature, 5%CO2Take out and be incubated in 12 hole tissue culturing plates in incubator, 24 hours
After change liquid and carry out dosing (be free of serum) processing.CCE9 is dissolved in 99.9%DMSO solution (DMSO final concentrations<0.1%), match somebody with somebody
10mM CCE9 solution processed, is added in 1mL MEM culture mediums, final concentration of 10 μM of CCE9 with 1 μ L CCE9 solution, processing 0.25,
0.5th, 1,3 hours and 6 hours, control group was handled 6 hours with same concentration DMSO.Cell is with RIPA cell pyrolysis liquids (50mM
Tris-HclPH7.4,1%NP-40,0.25%Na-deoxycholate, 150mM NaCl) crack 30 minutes, with identical loading
Amount, 8%SDS-PAGE electrophoresis, transferring film, with TBST (the 50mM Tris-HCL (pH 7.4), 150mM NaCl of 5% skimmed milk power
And 0.1%Tween-20) room temperature close 1 hour, 4 DEG C of night incubation primary antibody anti-PARP (1:1000 dilutions), incubation at room temperature
Secondary antibody 1 hour, ECL colour developings, exposure.As a result as shown in Figure 1 C.
As shown in Figure 1 C, 10 μM of CCE9 solution effects 3 hours, so that it may cause PARP in cervical cancer cell strain HeLa229
Cutting；In 10 μM of CCE9 solution effects 6 hours PARP cuttings occur for A549 lung carcinoma cells；HepG2 liver cancer cells are at 10 μM
Same PARP dissections have can be observed in CCE9 solution effects in 1 hour.
It can be obtained by Figure 1A, Figure 1B, Fig. 1 C, CCE9 can induce cervical cancer cell, liver cancer cells and lung carcinoma cell and substantially send out
Raw apoptosis.
The double dye detection CCE9 of the Annexin-V/PI of embodiment 2 induce the apoptosis of HeLa229 cervical cancer cells
It is small with 10 μM of CCE9 solution effects HeLa229 cells 6 in serum-free MEM (buys from Hyclone) culture medium
When, to be not added with the cell of CCE9 solution as control.Cell contaminates according to Vybrant Apoptosis Assay Kit#2 operation manuals
Color PI and Annexin-V, are analyzed with flow cytometer (Beckman Coulter CytoFLEX).Use Beckman Coulter
CytExpert software analysis, as a result as shown in Figure 2.
As seen from Figure 2, there was only 1.87% HeLa229 early apoptosis of cells in control group, and it is molten with 10 μM of CCE9
The experimental group of liquid processing has 19.85% HeLa229 early apoptosis of cells.Therefore it can obtain, it is bright that CCE9 can induce HeLa229 cells
It is aobvious to occur apoptosis.
Activations of the CCE9 of embodiment 3 to p38 MAPK in HeLa229 cells
1st, cell culture
Cervical cancer cell strain HeLa229 (ATCC) is selected, with the MEM culture mediums of 10% calf serum, 37 DEG C of constant temperature,
Take out and be incubated in 12 hole tissue culturing plates in 5%CO2 incubators, liquid is changed after 24 hours and dosing (being free of serum) place is carried out
Reason.CCE9 is dissolved in 99.9%DMSO solution (DMSO final concentrations<0.1%) 10mM CCE9 solution, is prepared, it is molten with 1 μ L CCE9
Liquid is added in 1mL MEM culture mediums so that final concentration of 10 μM of CCE9, and it is small to handle 0.25,0.5,1,3 with 10 μM of CCE9 solution
When and 6 hours, control group with same concentration DMSO handle 6 hours.
2nd, Western blotting analyze p38 MAPK activation
Cell is with RIPA cell pyrolysis liquids (50mM Tris-HclPH7.4,1%NP-40,0.25%Na-
Deoxycholate, 150mM NaCl) crack 30 minutes, with identical applied sample amount, 10%SDS-PAGE electrophoresis, transferring film is de- with 5%
TBST (50mM Tris-HCL (pH 7.4), 150mM NaCl and 0.1%Tween-20) room temperature closing 1 of fat milk powder is small
When, 4 DEG C of night incubation primary antibody anti-P-p38 (1:1000 dilutions), incubation at room temperature secondary antibody 1 hour, ECL colour developings, exposure.As a result
As shown in Figure 3.
As shown in figure 3, the time that swimming lane from left to right represents CCE9 solution processing HeLa229 cells successively is respectively
0h, 0.25h, 0.5h, 1h, 3h, 6h.Occur band by the corresponding swimming lanes of 0.25h, and increase over time band color
It is increasingly deeper, illustrate 10 μM of CCE9 solution effects 25 minutes, so that it may cause p38's in cervical cancer cell strain HeLa229 sharp
It is living, and with the increase of action time, activation degree gradually strengthens.Therefore, CCE9 can quickly and effectively activate p38
MAPK, and this activation is all in the rule of time-dependent.
The CCE9 of embodiment 4 depends on p38 MAPK activation to the apoptotic effect of HeLa229 cells
1st, p38 MAPK inhibitor SB203580 suppresses the apoptosis of tumor cells of CCE9 inductions
In serum-free MEM (buys from Hyclone) culture medium, in p38 MAPK inhibitor SB203580, SP600125
In the presence of, lack in the case of, with 10 μM of CCE9 solution effects HeLa229 cells 3 hours, using be not added with the cell of CCE9 solution as
Control.Western blotting analyze in PARP cutting, method be the same as Example 13 experimental method, as a result such as Fig. 4 A institutes
Show.
As shown in Figure 4 A：Without the control group of any processing, only plus SB203580 experimental groups, while add SB203580
Swimming lane corresponding with the experimental group P-p38 of CCE9 solution processing does not occur band, only adds P-p38 in SP600125 experimental group
There is a bit of band in corresponding swimming lane, only adds CCE9 solution and adds simultaneously in CCE9 solution and SP600125 experimental group
There is long and thick band in the corresponding swimming lanes of P-p38.It can thus be concluded that.Only add CCE9 solution and simultaneously add CCE9 solution and
Many PARP cuttings in SP600125 experimental group；And only add SB203580 and while add CCE9 solution and SB203580
The amount of experimental group PARP cuttings is seldom；The amount of PARP cuttings in SP600125 experimental group is only added than only adding SB203580
It is many.It can be obtained by Fig. 4 A, SB203580 can significantly suppress the PARP cuttings of CCE9 inductions, and (JNK suppresses SP600125
Agent) only there is faint influence.
2nd, the apoptosis of tumor cells of low p38 expression inhibitings CCE9 inductions is struck
P38 MAPK special siRNA (5 '-CUGAGAAACAUAUUGUGAUdTdT-3 ' are transferred in HeLa229 cells；
5’-CUUGUAAGAUCACUCUUAA dTdT-3’；5 '-GAAGCAAUGGGAAUUUACA dTdT-3 ', purchased from Sigma) it is 48 small
When, with 10 μM of CCE9 solution effects HeLa229 cells 3 hours, to be not added with the cell of CCE9 solution as control.Western
Blotting analyzes in PARP cutting, method be the same as Example 13 experimental method, as a result as shown in Figure 4 B.
As shown in Figure 4 B, p38 MAPK in p38 MAPK special siRNA experimental group are transferred in HeLa229 cells
Do not express, no PARP cuttings.Therefore can must reduce the expression of p38 MAPK in cell can significantly suppress the swollen of CCE9 inductions
PARP cuttings in oncocyte.
Result above illustrates that p38 MAPK play an important role in the apoptosis of tumor cells that CCE9 is induced.
The Bcl-2 that the double dye detections of the flow cytometry analysis Annexin-V/PI of embodiment 5 are knocked out in cell suppresses CCE9 and lured
The apoptosis of tumor cells led
In serum-free DMEM (purchase from Hyclone) culture medium, acted on respectively with 10 μM of CCE9 solution MEF cells and
MEF Bcl-2 knock out cell (Bcl-2-/- MEF) 6 hours, to be not added with the cell of CCE9 solution as control.Cell according to
Vybrant Apoptosis Assay Kit#2 operation manuals dye PI and Annexin-V, with flow cytometer (Beckman
Coulter CytoFLEX) analysis.With Beckman Coulter CytExpert software analysis, as a result as shown in figure 5, MEF is thin
The early apoptosis of cells of control group 2.8% of born of the same parents, MEF cells add the cell early stage of the experimental group 16.51% of CCE9 solution processing to wither
Die；MEF Bcl-2 knock out the early apoptosis of cells of control group 3.62% of cell, and MEF Bcl-2 knock out cell and added at CCE9 solution
The early apoptosis of cells of the experimental group 3.73% of reason.
Therefore it can be obtained by Fig. 5, in MEF Bcl-2 knock out cell (Bcl-2-/- MEF), CCE9 can not inducing cell hair
Raw apoptosis.
Phosphorylations and p38 MAPK inhibitor of the CCE9 of embodiment 6 to Bcl-2 in HeLa229 cells are to Bcl-2
Phosphorylation inhibitory action
In serum-free MEM (buys from Hyclone) culture medium, in p38 MAPK inhibitor SB203580 presence, lack
In the case of, with 10 μM of CCE9 solution effects HeLa229 cells 3 hours, to be not added with the cell of CCE9 solution as control.Cell
With RIPA cell pyrolysis liquids (50mM Tris-HclPH7.4,1%NP-40,0.25%Na-deoxycholate, 150mM
NaCl) crack 30 minutes, add Bcl-2 antibody (purchase from Santa Cruz) 10 μ L, be incubated 2 hours in 4 DEG C of shaking tables, then
30 μ l protein A/G agarose are added, 4 DEG C of shaking tables are incubated 1-2 hours；Then the heart is left 2 minutes for 4 DEG C 2000, careful
Supernatant is washed away, then washs with ice-cold cell pyrolysis liquid 1mL at least 3 times on 4 DEG C of shaking tables, 5 minutes every time, finally at 4 DEG C
2000 leave the heart 2 minutes, collect pearl.Appropriate 2 × SDS loading buffer are added into centrifuge tube in change in 100 DEG C
Property 10 minutes.With identical applied sample amount, 12%SDS-PAGE electrophoresis, transferring film, with TBST (the 50mM Tris-HCL of 5% skimmed milk power
(pH 7.4), 150mM NaCl and 0.1%Tween-20) room temperature close 1 hour, 4 DEG C of night incubation primary antibody anti-P-Thr
(1:1000 dilutions) or anti-P-Ser (1:1000 dilutions), incubation at room temperature secondary antibody 1 hour, ECL colour developings, exposure.As a result such as Fig. 6
It is shown.
As shown in fig. 6, CCE9 can induce phosphorylations of the Bcl-2 dependent on serine and threonine, and p38 MAPK
Inhibitor SB203580 can suppress the Bcl-2 phosphorylations of CCE9 inductions.So as to which CCE9 can be induced dependent on p38 MAPK's
Bcl-2 phosphorylations.
Bcl-2 conformation change in the CCE9 of embodiment 7 induction HeLa229 cells
It is molten to be not added with CCE9 with 10 μM of CCE9 solution effects HeLa229 cells 2 hours in serum-free MEM culture mediums
The cell of liquid is control.Cell fixes 10 minutes with 4% formaldehyde (in PBS) room temperature, 0.1%triton X-100+0.1M
Gycine wears film 30 minutes on ice, and 5mg/mL BSA room temperatures are closed 1 hour, and primary antibody anti-Bcl-2 (BH3) (uses 5mg/mL
BSA dilutions 1：50-100) antibody of (be purchased from Abcom) specific recognition Bcl-2 BH3 domains, only Bcl-2 conformation
It can be recognized, be incubated at room temperature 1-3 hours in wet box, secondary antibody (5mg/mL by Bcl-2 (BH3) antibody after changing
BSA dilutions 1：It is incubated 1-3 hours during 50-100) room temperature is dark in wet box, 1-5 points of DAPI (1 μ g/mL in PBS) incubations at room temperature
Clock, mounting in LSM-510 Laser Scanning Confocal Microscopes (Carl Zeiss, Oberkochen, Germany) observation, and is taken pictures.As a result
As shown in Figure 7.
As shown in Figure 7, although have the high Bcl-2 albumen expressed, but the Bcl-2 in cellular control unit in HeLa229 cells
It can not illustrate that the BH3 domains of Bcl-2 in HeLa229 born of the same parents are hidden in the inside of protein molecular by Bcl-2 (BH3) antibody staining.
After cell is handled through CCE9, Bcl-2 (BH3) antibody staining is manifested positive.Illustrate that CCE9 can induce Bcl-2 conformational changes.
Bax activation in the CCE9 of embodiment 8 induction HeLa229 cells
It is molten to be not added with CCE9 with 10 μM of CCE9 solution effects HeLa229 cells 2 hours in serum-free MEM culture mediums
The HeLa229 cells of liquid are control.Method as described in Example 7, with anti-Bax (6A7), specific recognition activation Bax's
Antibody, dyeing.In LSM-510 Laser Scanning Confocal Microscopes (Carl Zeiss, Oberkochen, Germany) observation, and take pictures.Knot
Fruit is as shown in Figure 8.
As shown in Figure 8, although have the high Bax albumen expressed in HeLa229 cells, but in control group HeLa229 cells
Bax can not be caused the strong dyeing of Bax (6A7) in HeLa229 cells, be illustrated CCE9 by Bax (6A7) antibody staining, CCE9 processing
Bax can be activated.
The Bcl-2 of the CCE9 of embodiment 9 inductions conformation change depends on p38 MAPK activation
1st, p38 MAPK inhibitor suppresses Bcl-2 conformation change
In serum-free MEM (buys from Hyclone) culture medium, in p38 MAPK inhibitor SB203580 presence, lack
In the case of, with 10 μM of CCE9 solution effects HeLa229 cells 2 hours, to be not added with the cell of CCE9 solution as control.Experiment
Method be the same as Example 7, immunofluorescence staining detects Bcl-2 conformational change situation, as a result as shown in Figure 9 A.
As shown in Figure 9 A：Although having in HeLa229 cells in the high Bcl-2 albumen expressed, control group HeLa229 cells
Bcl-2 can not by Bcl-2 (BH3) antibody staining, after HeLa229 cells are handled through CCE9, manifest Bcl-2 (BH3) antibody dye
Color is positive, and manifests Bcl-2 (BH3) antibody staining feminine gender through the CCE9 and SB203580 HeLa229 cells handled simultaneously, thus
SB203580, which can be obtained, can significantly suppress the Bcl-2 conformation change of CCE9 inductions.
2nd, the Bcl-2 of low p38 expression inhibitings CCE9 inductions conformation change is struck
It is transferred in HeLa229 cells special siRNA48 hours of p38 MAPK, with 10 μM of CCE9 solution effects
HeLa229 cells 3 hours, to be not added with the cell of CCE9 solution as control.With the methods described of embodiment 7, immunofluorescence staining
Bcl-2 conformational change situation is detected, as a result as shown in Figure 9 B.
As shown in Figure 9 B：Although having in HeLa229 cells in the high Bcl-2 albumen expressed, control group HeLa229 cells
Bcl-2 can not by Bcl-2 (BH3) antibody staining, after HeLa229 cells are handled through CCE9, manifest Bcl-2 (BH3) antibody dye
Color is positive, and the cell for handling and being transferred to simultaneously p38 MAPK special siRNA through CCE9 manifests Bcl-2 (BH3) antibody staining
It is negative.It can thus be concluded that, p38 MAPK expression can significantly suppress the Bcl-2's of CCE9 inductions in reduction HeLa229 cells
Conformation change.
Result above illustrates that p38 MAPK play an important role in the CCE9 Bcl-2 induced conformation change.
In summary, Xanthene ketone compound CCE9 of the invention is by activating p38 MAPK so that Bcl-2 occurs
Phosphorylation, causes Bcl-2 conformational changes, Bax is activated, so that the apoptosis of induced tumor cell.Therefore, Xanthene ketone chemical combination
Thing CCE9 can be used for the medicine using Bcl-2 as action target spot for preparing treating cancer.In addition, Bcl-2 phosphorylations can conduct
Action target spot is used to screen Xanthene ketone compound.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art is not departing from the principle and objective of the present invention
In the case of above-described embodiment can be changed within the scope of the invention, change, replace and modification.
Claims (8)
1. a kind of Xanthene ketone compound CCE9 is used for the purposes for preparing treating cancer medicine.
2. purposes according to claim 1, it is characterised in that the Xanthene ketone compound CCE9 using Bcl-2 as
Action target spot.
3. purposes according to claim 1, it is characterised in that the Xanthene ketone compound CCE9 has induction swollen
The purposes of apoptosis of tumor.
4. purposes according to claim 2, it is characterised in that the Xanthene ketone compound CCE9 has by swashing
The purposes of p38MAPK inducing apoptosis of tumour cell living.
5. purposes according to claim 2, it is characterised in that the Xanthene ketone compound CCE9 has by luring
Lead the purposes of Bcl-2 phosphorylation inducing apoptosis of tumour cell.
6. purposes according to claim 2, it is characterised in that the Xanthene ketone compound CCE9 has by luring
Lead the purposes of Bcl-2 conformation change inducing apoptosis of tumour cell.
7. purposes according to claim 2, it is characterised in that the Xanthene ketone compound CCE9, which has, to be passed through
The purposes of Bax activation-inducing apoptosis of tumor cells.
8. purposes according to claim 1, it is characterised in that the cancer is cervix cancer, lung cancer and liver cancer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107698550A (en) * | 2017-09-26 | 2018-02-16 | 莆田学院 | A kind of xanthone compound of 2,4 difluorophenyl substitution and its preparation method and application |
| CN107759558A (en) * | 2017-09-26 | 2018-03-06 | 莆田学院 | A kind of xanthone compound of trifluoromethyl substitution and its preparation method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101904880A (en) * | 2010-07-20 | 2010-12-08 | 暨南大学 | Mangosteen total xanthone extract and application thereof in preparation of TR3 receptor inducer |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101904880A (en) * | 2010-07-20 | 2010-12-08 | 暨南大学 | Mangosteen total xanthone extract and application thereof in preparation of TR3 receptor inducer |
Non-Patent Citations (2)
| Title |
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| XIAOJIN ZHANG ET AL: ""Synthesis, SAR and Biological Evaluation of Natural and Non-natural Hydroxylated and Prenylated Xanthones as Antitumor Agents"", 《MEDICINAL CHEMISTRY》 * |
| 古晓娜等: ""MAPKs在Bcl-2家族调控细胞凋亡中的作用"", 《国外医学 卫生学分册》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107698550A (en) * | 2017-09-26 | 2018-02-16 | 莆田学院 | A kind of xanthone compound of 2,4 difluorophenyl substitution and its preparation method and application |
| CN107759558A (en) * | 2017-09-26 | 2018-03-06 | 莆田学院 | A kind of xanthone compound of trifluoromethyl substitution and its preparation method and application |
| CN107759558B (en) * | 2017-09-26 | 2021-08-13 | 莆田学院 | Trifluoromethyl substituted xanthone compound and preparation method and application thereof |
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| CN107157981B (en) | 2020-11-03 |
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