CN107157981B - Application of xanthenone compound CCE9 - Google Patents

Abstract

The invention discloses an application of a xanthone compound CCE9, wherein the xanthone compound CCE9 is used for preparing a medicament for treating cancer, and p38MAPK is activated to ensure that Bcl-2 is phosphorylated, so that Bcl-2 conformation is changed, Bax is activated, and apoptosis of tumor cells is induced. Therefore, the xanthenone compound CCE9 can be used for preparing a medicine which treats cancer and takes Bcl-2 as an action target. In addition, Bcl-2 phosphorylation can be used as an action target for screening xanthenone compounds.

Description

Application of xanthenone compound CCE9

Technical Field

The invention belongs to the application field of traditional Chinese medicine extracts, and particularly relates to an application of xanthenone compounds CCE 9.

Background

The p38 mitogen-activated protein kinases (p38 mitogen-activated protein kinases (p38MAPK)) can be activated by a variety of extracellular stresses including UV, radiation, heat shock, proinflammatory molecules, specific antigens and other stress responses and play a central role in apoptosis, cytokine production, transcriptional regulation and seizures.

Recent studies have shown that p38MAPK plays an important role in apoptosis. For example, activation of the p38MAPK pathway can lead to apoptosis of nerve cells. In tumor cells, p38MAPK activity is elevated and involved in the regulation of apoptosis. Recent studies have shown that p38MAPK can induce apoptosis by inducing Bcl-2 phosphorylation.

Bcl-2 is one of the most important regulators of the apoptosis process. Recently, there is increasing evidence that phosphorylation of structurally disordered chain regions of Bcl-2 regulates Bcl-2 survival, and that Bcl-2 phosphorylation inactivates its survival. Under the action of environmental factors, inflammatory cytokines, Lipopolysaccharide (LPS) and other factors, p38MAPK can mediate phosphorylation of Ser87 and Thr56 on a disordered chain of a Bcl-2 structure, thereby mediating the processes of NGF withdrawal, tumor necrosis factor and NO-induced apoptosis. In the above cases, p38MAPK mediated phosphorylation of Bcl-2 was shown to be a pro-apoptotic event, inducing the release of cytochrome c from mitochondria, either in cells or in cell-free experiments.

Phosphorylation of the structurally disordered chain region of Bcl-2 may inhibit its anti-apoptotic function by affecting its interaction with a Bcl-2 family pro-apoptotic protein, such as Bax. In memory B lymphocytes, NGF withdrawal induces p38 MAPK-dependent phosphorylation, inhibits its anti-apoptotic function, and induces cytochrome c release. Similarly, H2O2 mediated p38MAPK dependent Bcl-2 phosphorylation in adult rat heart cells induced apoptosis. Evidence has shown that Ser87 and Thr56 on the structurally disordered chain of Bcl-2 are sites for phosphorylation of Bcl-2 by p38MAPK, and phosphorylation of this residue reduces the anti-apoptotic effect of Bcl-2.

CCE9 is a xanthone compound extracted from Chinese medicinal material Boerhavia diffusa. The yellow bull (Cratoxylonochinchennsis Bl) named as Huangniu tea, Huangya wood, Candida, Bromus alata, Cerbera manghas, Francolina pintadeana, Equisetum, Manchurian red (Guangxi), Chilo caogu, Meikui, Shandong Mao, etc. is a plant of Bos (Cratoxylum) of Guttiferae. Has the functions of clearing away summer-heat, eliminating dampness, eliminating stagnation, relieving swelling and arresting bleeding, and is mainly used for treating cold and fever, enteritis and diarrhea, cough and hoarseness, jaundice and the like; the main chemical component of the yellow bull wood is xanthone, and in addition, the yellow bull wood also contains anthraquinone benzophenone, triterpenes and flavonoid components; modern pharmacological research shows that the yellow cattle wood has the functions of resisting oxidation, cytotoxicity and nerve growth factor synergism.

Although the literature reports that the yellow cattle wood has anticancer activity and some active ingredients are separated, most of researches are limited to in vitro cytotoxic activity tests of 7 pyridone compounds separated from C.cochinchinense roots in more basic MTT experimental stages such as Laphokhieo and the like, and the results of the in vitro cytotoxic activity tests are compared with the anticancer activity of camptothecin to show that the compound cochinenone A has stronger growth inhibition activity on breast cancer, cervical cancer, intestinal cancer and KB cell lines; the Boonnak Nawong and the like find that the compound gerantoxanthone I has stronger cytotoxic activity and the like on breast cancer, cervical cancer, intestinal cancer and KB cell lines, but the simple activity researches cannot define the anti-tumor action target and action mechanism of active ingredients, which is the main reason for restricting the further development of the compound gerantoxanthone I into anti-cancer drugs.

Disclosure of Invention

The first purpose of the invention is to provide the application of the xanthenone compound CCE9 in preparing the medicines for treating the cancers.

The second purpose of the invention is to provide an application of screening xanthenone compounds by taking Bcl-2 as an action target.

In order to achieve the purpose of the invention, the invention provides application of a xanthenone compound CCE9 in preparing a medicament for treating cancer.

Furthermore, the xanthenone compound CCE9 takes Bcl-2 as an action target.

Further, the xanthenone compound CCE9 has a use for inducing tumor cell apoptosis.

Further, the xanthenone compound CCE9 has the purpose of inducing tumor cell apoptosis by activating p38 MAPK.

Further, the xanthenone compound CCE9 has the application of inducing tumor cell apoptosis by inducing Bcl-2 phosphorylation.

Further, the xanthenone compound CCE9 has the application of inducing tumor cell apoptosis by inducing Bcl-2 conformational change.

Further, the xanthenone compound CCE9 has the purpose of inducing tumor cell apoptosis through Bax activation.

Further, the cancer is cervical cancer, lung cancer and liver cancer.

The xanthenone compound CCE9(1,3,7-trihydroxy-2,4-diprenylxanthone) is a traditional Chinese medicine yellow bull wood extract, and has the following structure:

according to the invention, Bcl-2 phosphorylation is taken as a target point, and the method can be used for screening xanthenone compounds so as to further develop and treat various cancers.

The apoptosis of the tumor cells induced by Bcl-2 provided by the invention can be used for guiding and screening xanthenone compounds to obtain the compounds with anti-tumor activity, and the obtained drugs can be further used for preparing the drugs with the activity and function for the Bcl-2 induced apoptosis of the tumor cells.

Drawings

FIG. 1A is a graph of fluorescence staining to detect apoptosis of CCE 9-induced tumor cells;

figure 1B is a graph of the results of the percentage of CCE9 induced apoptosis in tumor cells;

figure 1C is a graph of CCE9 induced PARP protein cleavage in tumor cells;

FIG. 2 is a graph showing the result of flow cytometry analysis of the apoptosis of HeLa229 cervical cancer cells induced by the double staining detection of CCE9 by Annexin-V/PI;

FIG. 3 is a graph of the activation effect of CCE9 on p38MAPK in HeLa229 cells;

fig. 4A is a graph of inhibition of tumor cell apoptosis induced by CCE9 by p38MAPK inhibitors;

fig. 4B is a graph of knockdown of p38 expression inhibiting CCE 9-induced tumor cell apoptosis;

FIG. 5 is a graph of flow cytometry analysis of Annexin-V/PI double staining to detect Bcl-2 inhibition of CCE 9-induced tumor cell apoptosis in knockout cells;

FIG. 6 is a graph of the effect of CCE9 on the phosphorylation of Bcl-2 in HeLa229 cells and the inhibition of Bcl-2 phosphorylation by a p38MAPK inhibitor;

FIG. 7 is a graph of CCE9 induced conformational changes of Bcl-2 in HeLa229 cells;

fig. 8 is a graph of CCE9 induced Bax activation in HeLa229 cells;

FIG. 9A is a graph of the conformational change of inhibition of Bcl-2 by p38MAPK inhibitors;

FIG. 9B is a graph of the conformational changes of Bcl-2 induced by suppressing CCE9 by knocking down p38 expression.

Detailed Description

The following detailed description of embodiments of the invention is intended to be illustrative of the invention and is not to be construed as limiting the invention. The experimental methods in the examples, in which specific conditions are not specified, are generally carried out according to conventional conditions such as molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

Reagents used in the present invention:

the structure of the adopted xanthenone compound CCE9 is as follows:

the 10. mu.M CCE9 solution described in examples 1-4, 6-9 was prepared by dissolving CCE9 in 99.9% DMSO solution (DMSO final concentration < 0.1%), preparing 10mM CCE9 solution, and adding 1. mu.L CCE9 solution to 1mL MEM medium.

The 10 μ M CCE9 solution described in example 5 was prepared by dissolving CCE9 in 99.9% DMSO solution (DMSO final concentration < 0.1%), preparing 10mM CCE9 solution, and adding 1 μ L CCE9 solution to 1mL DMEM medium.

Example 1 CCE9 Induction of tumor cell apoptosis

1. Cell culture

Selecting cervical cancer cell line HeLa229(ATCC) using 10% calf serum in MEM medium at constant temperature of 37 deg.C and 5% CO₂The cultures were removed from the incubator and plated in 24-well tissue culture plates, and 24 hours later, the plates were changed and treated with chemicals (without serum). CCE9 was dissolved in 99.9% DMSO solution (DMSO final concentration)<0.1%), 10mM CCE9 solution was prepared, and 1. mu.L of CCE9 solution was added to 1mL MEM medium, with a final concentration of 10. mu.M CCE9, and the cells were treated for 3 hours and 6 hours, and the control group was treated with the same concentration of DMSO solution for 6 hours.

2. DAPI staining of cell nuclei

The total volume of the solution in each well of the reaction plate was 1 mL. After cell treatment, the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde, membrane-crossed with 1% Triton, and finally stained for cell nuclei with 50. mu.g/mL DAPI and 100. mu.g/mL DNase-free RNaseA at 37 ℃ for 20 minutes. The morphology of the nuclei was visualized by glycerol mounting and fluorescence microscopy, and the results are shown in FIG. 1A. At least 300 cells, more than 5 random visual fields, were counted by two different observers and the percentage of apoptosis was calculated, the results are shown in figure 1B.

As can be seen from fig. 1A, HeLa229 cells exposed to 10 μ M CCE9 solution for 3 hours and 6 hours were significantly apoptotic, HeLa229 cells exposed to 3 hours were less numerous than HeLa229 cells exposed to 3 hours and had more wrinkled cytoplasm, and apoptotic cells were identified by morphologically typical shrinkage of cytoplasm, membrane blebbing, and nuclear condensation, fragmentation.

As shown in fig. 1B, in the case of few apoptosis of HeLa229 cells in the control group (4.7%), the apoptosis rate reached 17% after 3 hours of the CCE9 solution treatment, and as the treatment time was prolonged, 34.3% of the HeLa229 cells were apoptotic after 6 hours, and the HeLa229 cells were significantly increased in apoptosis.

3. CCE9 induces PAPR cleavage in HeLa229, A549 and HepG2 cells

Cleavage by Poly (ADP-ribose) transferase (PARP) is a marker of early apoptosis. Cleavage of PARP can be recognized by its antibody. Selecting cervical cancer cell line HeLa229(ATCC), MEM culture medium using 10% calf serum, lung cancer cell line A549(ATCC) and liver cancer cell line HepG2(ATCC), DMEM culture medium using 10% calf serum, constant temperature 37 deg.C, 5% CO₂The cultures were removed from the incubator in 12-well tissue culture plates and 24 hours later, replaced and treated with a drug (serum free). CCE9 was dissolved in 99.9% DMSO solution (DMSO final concentration)<0.1%), 10mM CCE9 solution was prepared, added to 1mL MEM medium with 1. mu.L of CCE9 solution at a final concentration of 10. mu.M CCE9 for 0.25, 0.5, 1,3 and 6 hours, and the control was treated with DMSO at the same concentration for 6 hours. Cells were lysed in RIPA cell lysate (50mM Tris-HclPH7.4, 1% NP-40, 0.25% Na-deoxyholate, 150mM NaCl) for 30 min, run on the same load, 8% SDS-PAGE, blotted, blocked with 5% skim milk TBST (50mM Tris-HCL (pH 7.4), 150mM NaCland 0.1% Tween-20) for 1h at room temperature, incubated primary anti-PARP (1:1000 dilution) overnight at 4 ℃, incubated secondary antibody for 1h at room temperature, developed in ECL, and exposed. The results are shown in the figure1C.

As shown in FIG. 1C, the 10 μ M CCE9 solution was exposed for 3 hours to cause the cleavage of PARP in the cervical cancer cell line HeLa 229; the A549 lung cancer cells are acted for 6 hours in 10 mu M CCE9 solution, and PARP cleavage occurs; the same PARP cleavage effect was observed in HepG2 liver cancer cells in 10. mu.MCCE 9 solution for 1 hour.

As shown in fig. 1A, 1B, and 1C, CCE9 induces apoptosis in cervical cancer cells, liver cancer cells, and lung cancer cells.

Example 2 Annexin-V/PI double staining assay CCE9 induces apoptosis in HeLa229 cervical cancer cells

HeLa229 cells were exposed to 10. mu.M CCE9 solution in serum-free MEM (purchased from Hyclone) for 6 hours, as compared to cells without CCE9 solution. Cells were stained for PI and Annexin-V according to the Vybrant Apoptosis Assay Kit #2 operating manual and analyzed by flow cytometry (Beckman Coulter Cytoflex). The results are shown in FIG. 2, using Beckman CoultCytExpert software for analysis.

As can be seen from fig. 2, only 1.87% of HeLa229 cells in the control group were early apoptotic, while 19.85% of HeLa229 cells in the experimental group treated with 10 μ M CCE9 solution were early apoptotic. Thus, CCE9 can induce HeLa229 cells to undergo significant apoptosis.

Example 3 activation of p38MAPK in HeLa229 cells by CCE9

1. Cell culture

Cervical cancer cell line HeLa229(ATCC) was selected, cultured in 12-well tissue culture plates in MEM medium containing 10% calf serum at 37 ℃ in a 5% CO2 incubator at constant temperature, and then subjected to liquid exchange and drug addition (without serum) treatment after 24 hours. CCE9 was dissolved in 99.9% DMSO solution (final DMSO concentration < 0.1%), 10mM CCE9 solution was prepared, added to 1mL MEM medium with 1 μ L CCE9 solution such that the final CCE9 concentration was 10 μ M, treated with 10 μ M CCE9 solution for 0.25, 0.5, 1,3 and 6 hours, and the control group was treated with the same concentration DMSO for 6 hours.

2. Western blotting analysis of activation of p38MAPK

Cells were lysed in RIPA cell lysate (50mM Tris-HclPH7.4, 1% NP-40, 0.25% Na-deoxyholate, 150mM NaCl) for 30 min, run on the same load, 10% SDS-PAGE, blotted, blocked with 5% skim milk TBST (50mM Tris-HCL (pH 7.4), 150mM NaCl and 0.1% Tween-20) for 1 hr at room temperature, incubated primary anti-P-P38(1:1000 dilution) overnight at 4 deg.C, incubated secondary antibody for 1 hr at room temperature, ECL developed and exposed. The results are shown in FIG. 3.

As shown in FIG. 3, the lanes from left to right represent the time for treating HeLa229 cells with CCE9 solution at 0h, 0.25h, 0.5h, 1h, 3h and 6h, respectively. The band appeared in the lane corresponding to 0.25h and the color of the band became darker with the increase of time, which indicates that the 10 μ M CCE9 solution is acted for 25 minutes to activate p38 in the cervical cancer cell strain HeLa229, and the activation degree is gradually increased with the increase of action time. Therefore, CCE9 enables rapid and efficient activation of the p38MAPK, with time-dependent regularity.

Example 4 apoptotic Effect of CCE9 on HeLa229 cells was dependent on the activation of p38MAPK

1. P38MAPK inhibitor SB203580 inhibits tumor cell apoptosis induced by CCE9

HeLa229 cells were treated with 10. mu.M CCE9 solution in serum-free MEM (purchased from Hyclone) medium in the presence and absence of the p38MAPK inhibitors SB203580 and SP600125 for 3 hours, as compared to cells without CCE9 solution. Western blotting analysis of cleavage of PARP was carried out in the same manner as in experiment 3 in example 1, and the results are shown in FIG. 4A.

As shown in fig. 4A: no band appeared in the lanes corresponding to the control group without any treatment, the test group with only SB203580 added, and the test group with both SB203580 and CCE9 added, the lanes corresponding to P-P38 in the test group with only SP600125 added, the lanes corresponding to P-P38 in the test group with only SP600125 added, and the lanes corresponding to P-P38 in the test group with only CCE9 added, and the CCE9 added, and the SP600125 added, were long and thick. This can be achieved. Many PARP cleavages in the experimental group with addition of CCE9 solution alone and CCE9 solution simultaneously with SP 600125; while the amount of PARP cleavage was very small for the experimental group with SB203580 added only to SB203580 and CCE9 solution added simultaneously; the amount of PARP cleavage was greater in the experimental group with SP600125 alone than SB203580 alone. As can be seen from fig. 4A, SB203580 can largely inhibit the CCE 9-induced PARP cleavage, whereas SP600125(JNK inhibitor) has only a weak effect.

2. Knocking down p38 expression inhibits CCE 9-induced tumor cell apoptosis

HeLa229 cells were transfected with p38MAPK specific siRNA (5 '-CUGAAACAUAUUGUGAUdTdT-3'; 5 '-CUUGUAAGAUCACUCUUAA dTdT-3'; 5 '-GAAGCAAUGGGAAUUUACA dTdT-3', purchased from Sigma) for 48 hours, and HeLa229 cells were treated with 10. mu.M CCE9 solution for 3 hours, while cells without CCE9 solution were used as controls. Westernblotting analysis of the cleavage of PARP, which was performed in the same manner as in 3 of example 1, gave the results shown in FIG. 4B.

As shown in fig. 4B, p38MAPK was not expressed without PARP cleavage in the experimental group into which specific siRNA of p38MAPK was transferred in HeLa229 cells. Therefore, the reduction of the expression of p38MAPK in the cells can remarkably inhibit PARP cleavage in tumor cells induced by CCE 9.

The above results all indicate that p38MAPK plays an important role in CCE 9-induced tumor cell apoptosis.

Example 5 flow cytometry analysis of Annexin-V/PI double staining to detect Bcl-2 inhibition of CCE 9-induced tumor cell apoptosis in knockout cells

MEF cells and MEF Bcl-2 knockout cells (Bcl-2-/-MEF) were treated with 10. mu.M CCE9 solution in serum-free DMEM (purchased from Hyclone) medium for 6 hours, respectively, with cells without CCE9 solution as a control. Cells were stained for PI and Annexin-V according to the Vybrant Apoptosis Assay Kit #2 operating manual and analyzed by flow cytometry (BeckmanCoulter Cytoflex). The results of analysis by Beckman Coulter CytExpert software are shown in FIG. 5, the control group of MEF cells is 2.8% of cells in early apoptosis, and the experimental group of MEF cells treated by CCE9 solution is 16.51% of cells in early apoptosis; MEF Bcl-2 knockout cells in the control group 3.62% of early apoptosis, MEF Bcl-2 knockout cells plus CCE9 solution treatment of the experimental group of 3.73% of early apoptosis.

Thus, as shown in FIG. 5, CCE9 did not induce apoptosis in MEF Bcl-2 knock-out cells (Bcl-2-/-MEF).

Example 6 phosphorylation of Bcl-2 by CCE9 in HeLa229 cells and inhibition of Bcl-2 phosphorylation by p38MAPK inhibitors

HeLa229 cells were treated with 10. mu.M CCE9 solution in serum-free MEM (purchased from Hyclone) medium in the presence and absence of the p38MAPK inhibitor SB203580 for 3 hours, as compared to cells without CCE9 solution. Cells were lysed in RIPA cell lysate (50mM Tris-HclPH7.4, 1% NP-40, 0.25% Na-deoxyholate, 150mM NaCl) for 30 min, 10. mu.L of antibody to Bcl-2 (purchased from Santa Cruz) was added, incubated at 4 ℃ for 2 h in a shaker, then 30. mu.l of protein A/G agarose was added, incubated at 4 ℃ for 1-2 h in a shaker; then centrifugation at 2000 rpm for 2 minutes at 4 ℃ was performed, the supernatant was carefully washed off, and then 1mL of ice-cold cell lysate was washed on a4 ℃ shaker for at least 3 times, 5 minutes each, and finally centrifugation at 2000 rpm for 2 minutes at 4 ℃ was performed to collect the beads. An appropriate amount of 2 xSDS loading buffer was added to the centrifuge tubes and denatured at 100 ℃ for 10 minutes. The samples were loaded at the same volume, subjected to 12% SDS-PAGE, transferred to a membrane, blocked with 5% skimmed milk powder TBST (50mM Tris-HCl (pH 7.4), 150mM NaCl and 0.1% Tween-20) for 1 hour at room temperature, incubated overnight at 4 ℃ with primary anti-P-Thr (1:1000 dilution) or anti-P-Ser (1:1000 dilution), incubated with secondary antibody for 1 hour at room temperature, developed with ECL, and exposed. The results are shown in FIG. 6.

As shown in fig. 6, CCE9 can induce Bcl-2-dependent serine and threonine phosphorylation, while the inhibitor SB203580 of p38MAPK can inhibit CCE 9-induced Bcl-2 phosphorylation. Thus, CCE9 is able to induce p38MAPK dependent Bcl-2 phosphorylation.

Example 7 CCE9 induces conformational changes in Bcl-2 in HeLa229 cells

HeLa229 cells were exposed to 10. mu.M CCE9 solution for 2 hours in serum-free MEM medium, and cells without CCE9 solution were used as a control. Cells were fixed with 4% formaldehyde (PBS) for 10 minutes at room temperature, 0.1% triton X-100+0.1 MGcycle was permeabilized on ice for 30 minutes, 5mg/mL BSA was blocked for 1 hour at room temperature, anti-Bcl-2(BH3), (diluted 1: 50-100 with 5 mg/mLBSA) (purchased from Abcom) an antibody specifically recognizing the BH3 domain of Bcl-2, only after the change in the conformation of Bcl-2 could be recognized by the Bcl-2(BH3) antibody, incubated for 1-3 hours at room temperature in a wet box, a secondary antibody (diluted 1: 50-100 mg/mLBSA) incubated for 1-3 hours at room temperature in the dark in a wet box, DAPI (1. mu.g/mL in PBS) was incubated for 1-5 minutes at room temperature, mounted, photographed in an LSM-510 confocal microscope (Carl Zeiss, Germany). The results are shown in FIG. 7.

As shown in FIG. 7, although there was highly expressed Bcl-2 protein in HeLa229 cells, Bcl-2 in control cells could not be stained by Bcl-2(BH3) antibody, indicating that the BH3 domain of Bcl-2 in HeLa229 cells is hidden inside the protein molecule. After the cells are treated by CCE9, the cells are stained positive by Bcl-2(BH3) antibody. It is suggested that CCE9 may induce Bcl-2 conformational changes.

Example 8 CCE9 induces Bax activation in HeLa229 cells

HeLa229 cells were exposed to 10. mu.M CCE9 solution for 2 hours in serum-free MEM medium, and HeLa229 cells without CCE9 solution were used as a control. Staining was performed as described in example 7 with anti-Bax (6A7), an antibody specifically recognizing activated Bax. Observed in a LSM-510 confocal microscope (Carl Zeiss, Oberkochen, Germany) and photographed. The results are shown in FIG. 8.

As shown in fig. 8, although there was a high expression of Bax protein in HeLa229 cells, Bax in control HeLa229 cells could not be stained by Bax (6a7) antibody, and CCE9 treatment caused strong staining of Bax (6a7) in HeLa229 cells, suggesting that CCE9 could activate Bax.

Example 9 CCE 9-induced conformational changes of Bcl-2 are dependent on activation of the p38MAPK

1. P38MAPK inhibitors inhibit conformational changes of Bcl-2

HeLa229 cells were treated with 10. mu.M CCE9 solution in serum-free MEM (purchased from Hyclone) medium in the presence and absence of the p38MAPK inhibitor SB203580 for 2 hours, as compared to cells without CCE9 solution. The experimental procedure was the same as in example 7, and the results of the immunofluorescent staining for the conformational change of Bcl-2 are shown in FIG. 9A.

As shown in fig. 9A: although there is highly expressed Bcl-2 protein in HeLa229 cells, Bcl-2 in control HeLa229 cells could not be stained by Bcl-2(BH3) antibody, and HeLa229 cells treated with CCE9 showed positive staining for Bcl-2(BH3) antibody, while HeLa229 cells treated with CCE9 and SB203580 showed negative staining for Bcl-2(BH3) antibody, thus SB203580 could largely inhibit Bcl-2 conformational change induced by CCE 9.

2. Knocking down p38 expression inhibits CCE 9-induced conformational change of Bcl-2

The specific siRNA of p38MAPK was transferred into HeLa229 cells for 48 hours, and HeLa229 cells were treated with 10. mu.M CCE9 solution for 3 hours, while cells without CCE9 solution were used as a control. The conformational change of Bcl-2 was detected by immunofluorescence staining as described in example 7, and the results are shown in FIG. 9B.

As shown in fig. 9B: although the HeLa229 cells have high expression of Bcl-2 protein, the control group of HeLa229 cells cannot be stained by Bcl-2(BH3) antibody, the HeLa229 cells are treated by CCE9 and show positive staining of Bcl-2(BH3) antibody, and the cells treated by CCE9 and transferred with p38MAPK specific siRNA show negative staining of Bcl-2(BH3) antibody. Therefore, the reduction of the expression of p38MAPK in HeLa229 cells can remarkably inhibit the conformational change of Bcl-2 induced by CCE 9.

The above results all indicate that p38MAPK plays an important role in CCE 9-induced conformational changes of Bcl-2.

In conclusion, the xanthenone compound CCE9 disclosed by the invention can activate p38MAPK to phosphorylate Bcl-2, so that the conformation change of the Bcl-2 is caused, Bax is activated, and the apoptosis of tumor cells is induced. Therefore, the xanthenone compound CCE9 can be used for preparing a medicine which treats cancer and takes Bcl-2 as an action target. In addition, Bcl-2 phosphorylation can be used as an action target for screening xanthenone compounds.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims (7)

1. Application of xanthenone compound CCE9 in preparation of cervical cancer treatment drugThe structure of the xanthenone compound CCE9 is shown as follows:

。

2. the use according to claim 1, characterized in that the xanthenone compounds CCE9 target Bcl-2.

3. The use according to claim 1, characterized in that said xanthenone compound CCE9 has the use to induce apoptosis of cervical cancer cells.

4. The use according to claim 2, characterized in that said xanthenone compounds CCE9 have the use to induce apoptosis in cervical cancer cells by activating p38 MAPK.

5. The use according to claim 2, characterized in that said xanthenone compound CCE9 has the use to induce apoptosis of cervical cancer cells by inducing Bcl-2 phosphorylation.

6. The use according to claim 2, characterized in that said xanthenone compound CCE9 has the use to induce apoptosis of cervical cancer cells by inducing conformational changes of Bcl-2.

7. The use according to claim 2, characterized in that said xanthenone compound CCE9 has a use for inducing apoptosis of cervical cancer cells by Bax activation.

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