CN115300493A - Application of nuclear receptor RXR alpha ligand and antitumor drug - Google Patents

Application of nuclear receptor RXR alpha ligand and antitumor drug Download PDF

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CN115300493A
CN115300493A CN202210814561.XA CN202210814561A CN115300493A CN 115300493 A CN115300493 A CN 115300493A CN 202210814561 A CN202210814561 A CN 202210814561A CN 115300493 A CN115300493 A CN 115300493A
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methoxybenzophenone
hydroxy
pharmaceutically acceptable
acceptable salt
cells
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蒋福全
张晓坤
胡贤雯
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Xiamen University
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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Abstract

The invention relates to an application of a nuclear receptor RXR alpha ligand and an antitumor drug, belonging to the fields of biotechnology and biomedicine. The application of 1, 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof in preparing an antitumor drug is disclosed, wherein the result of the 2-hydroxy-4-methoxybenzophenone is shown as a formula 1:
Figure DDA0003741762130000011
the 1.2-hydroxy-4-methoxybenzophenone or the solvent compound or the pharmaceutically acceptable salt thereof provided by the invention can be used as a nuclear receptor RXR alpha ligand to specifically target the interaction of specific p-RXR alpha/PLK 1 in tumor cells, so that the positioning of PLK1 in the tumor cells in a central body and the assembly of a spindle body are influenced, the tumor cells are specifically induced to generate M-phase cycle block, and meanwhile, no obvious toxic effect is generated on normal organ tissues, 1.2-The hydroxyl-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is an antitumor drug with great potential development value.

Description

Application of nuclear receptor RXR alpha ligand and antitumor drug
Technical Field
The invention belongs to the fields of biotechnology and biomedicine, and particularly relates to application of a nuclear receptor RXR alpha ligand and an antitumor drug.
Background
The occurrence and development of tumor are the result of normal cells undergoing a series of transformation, breaking through normal limits of survival and proliferation, becoming tumor cells and metastasizing and spreading from the primary site. Tumors have specific functions such as unstable cell genome, death resistance, continuous maintenance of cell proliferation signals, escape of tumor suppressor genes, immortalization and enabling of cell replication.
Polo-like kinase 1 (PLK 1), an important member of the Polo-like kinase family, belongs to the serine/threonine protein kinases, which are highly conserved in function and structure and are involved in the regulation of various intracellular processes such as DNA replication, mitosis and stress response. Centrosome maturation, segregation and microtubule attachment can be regulated to ensure accurate replication of genetic material.
PLK1 plays an important role in initiating entry into the mitotic phase and throughout the M phase, and is closely associated with cell proliferation. PLK1 is not expressed or is expressed very low in most static tissue cells, but is highly expressed in cells with vigorous proliferation and tumor tissues, such as various malignant tumors, such as ovarian cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, head and neck cancer, melanoma and the like; thus, PLK1 is widely recognized as an oncogene and an important target for cancer intervention.
Mitosis-related kinase inhibitors show very excellent inhibition effect on tumors, wherein PLK1 inhibitors are the key direction for the development of antitumor drugs. At present, more than 10 PLK1 specific inhibitors are commercially available, and four PLK1 specific inhibitors, namely BI2536, BI6727, GSK461364 and NMS-1286937, are ATP competitive inhibitors, and inhibit recruitment of PLK1 at the centrosome in the mitotic M phase by competitive binding with PLK1 ATP, so that microtubules disaggregate and separate from centromere, thereby arresting cell mitosis and inducing apoptosis. However, the poor specificity of the inhibitor on mitotic regulation can influence both tumor cells and PLK1 recruitment to centrosomes in normal cell M phase in vivo, so that serious toxic and side effects exist, and the practical clinical application of the inhibitor is limited.
Disclosure of Invention
The invention aims to provide an anti-tumor compound which can remarkably induce the tumor tissue of a nude mouse with transplanted tumor to generate G2/M cycle retardation so as to cause apoptosis and remarkably inhibit the growth of the transplanted tumor, and has no obvious toxic effect on normal organ tissues, and provides an application of a nuclear receptor RXR alpha ligand and an anti-tumor medicament.
In a first aspect, the invention provides an application of 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof in preparing an antitumor drug, which adopts the following technical scheme:
the application of 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof in preparing an antitumor drug is disclosed, wherein the result of the 2-hydroxy-4-methoxybenzophenone is shown as a formula 1:
Figure BDA0003741762110000021
retinol X receptor α (RXR α) is a novel nuclear receptor specifically responsive to vitamin a metabolites, which is expressed in highly abundant form in the liver, kidney, spleen, placenta, epithelium and other various visceral tissues. RXR alpha is used as a ligand-activated transcription regulation factor, participates in regulation and control of various physiological processes of body metabolism, embryonic development, cell proliferation, differentiation, apoptosis, immunity and the like, and expression, positioning or function abnormality of RXR alpha is closely related to a plurality of human diseases, such as metabolic syndrome, cardiovascular diseases, tumors and the like. In the mitotic M phase, serine at positions 56 and 70 on RXR alpha can be specifically phosphorylated by CDK1, and the phosphorylated RXR alpha can be positioned to a centrosome to interact with PLK1, so that activation of PLK1 and maturation of the centrosome are promoted, and the mitotic process is further regulated and controlled.
The 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof provided by the invention can be used as a ligand of RXR alpha, can be specifically combined with RXR alpha, and can inhibit the co-localization of RXR alpha and PLK1 in a centrosome, so as to inhibit the phosphorylation of PLK1 and the kinase activity thereof, finally induce the mitotic arrest of tumor cells in a RXR alpha dependent manner and trigger cycle specific apoptosis, and can be used as an antitumor drug targeting mitosis.
Further, the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used as a ligand of a nuclear receptor RXR alpha and is used for preparing a medicine combined with the RXR alpha;
preferably, the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used for preparing a medicament which binds to a surface binding site of a co-regulatory factor of RXR alpha. Further, the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used for preparing a medicament for inhibiting the generation of p-RXR alpha by tumor cells;
preferably, the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used for the preparation of a medicament for inhibiting the interaction and co-localization of PLK1 and rxra.
Further, the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used for preparing a medicament for inducing mitotic arrest of tumor cells and triggering cycle-specific apoptosis.
Further, the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used for preparing a medicament for inducing tumor cells to generate G2/M cycle block;
preferably, the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used for preparing a medicament for inducing tumor cells to generate G2/M cycle block which is dependent on RXR alpha.
Further, the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used for preparing a medicament for inducing high expression of tumor cell cleared Caspase3 protein to trigger cycle-specific apoptosis.
In a second aspect, the present invention provides an anti-tumor drug, which adopts the following technical scheme:
an antitumor drug contains benzophenone compound or its solvent compound or its pharmaceutically acceptable salt with pharmaceutically effective dose;
preferably, the result of the benzophenone compound is shown as formula 2 or formula 3:
Figure BDA0003741762110000041
wherein R is 1 = H or SO 3 H，R 2 H or OH, R 3 ＝CH 3 H or OCH 3 ；
Further, the benzophenone compound is 2-hydroxy-4-methoxybenzophenone.
Further, the composition also comprises an active component; preferably, the active ingredient is a substance having anti-tumor activity.
Further, a pharmaceutically acceptable carrier is also included; preferably, the carrier is one or more of a diluent, an excipient, a filler, a binder, an absorption enhancer, a surface agent or a lubricant.
Has the beneficial effects that:
(1) The nuclear receptor RXR alpha ligand provided by the invention can be specifically combined with RXR alpha, so that RXR alpha-dependent G2/M cycle arrest of tumor cells is induced, and tumor cell apoptosis is caused, thereby obviously inhibiting the growth of transplanted tumor;
(2) The nuclear receptor RXR alpha ligand provided by the invention specifically targets the specific interaction of p-RXR alpha/PLK 1 in tumor cells, thereby influencing the positioning of PLK1 in the tumor cells in a central body and the assembly of a spindle body, further specifically inducing the tumor cells to generate M-phase cycle block, simultaneously having no obvious toxic effect on normal organ tissues, and being an antitumor drug with great potential development value;
(3) The invention also provides an anti-tumor medicament which contains the benzophenone compound as an anti-cancer active ingredient, and the benzophenone compound is specifically combined with RXR alpha to further induce tumor cells to generate G2/M phase cycle block, so that the growth of transplanted tumors is obviously inhibited, a good anti-cancer effect is realized, and the anti-tumor medicament has a good clinical application prospect.
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In order to illustrate the technical solution of the present invention more clearly, the drawings will be briefly described below, and it is apparent that the drawings in the following description relate only to some embodiments of the present invention and are not intended to limit the present invention.
Fig. 1 is a specific structural formula of a benzophenone compound provided in example 1 of the present invention;
FIG. 2 is a graph showing the results of statistical analysis by flow cytometry after treating MDA-MB-231 cells for 24h with 0, 20. Mu.M, 40. Mu.M and 80. Mu.M concentrations of epothilone provided in example 2 of this invention;
FIG. 3 (a) is a graph showing the results of a fluorescence titration experiment of purified His-RXR α -LBD protein provided in example 3 of the present invention with Mexenone; fig. 3 (b) is a graph of the results of the reporter gene experiments provided in example 3 of the present invention, # P <0.05, # P <0.01;
FIG. 3 (c) is a graph showing the results of the molecular docking simulation provided in example 3 of the present invention (PDB: 3 FUG);
FIG. 4 is a graph showing the results of experiments on the formation of crystal violet-stained cell clones of HeLa, MDA-MB-231, 786-O, A549, hepG2 and A431 cell lines after 14 days of incubation with Mexenone (0,30. Mu.M, 60. Mu.M) as provided in example 4 of the present invention;
FIG. 5 (a) is a graph showing the results of experiments performed by flow cytometry analysis after treating cells with Mexenone at concentrations of 0, 25. Mu.M and 50. Mu.M in MDA-MB-231, heLa and 786-O cells for 24h, as provided in example 5 of the present invention;
FIG. 5 (b) is a graph showing the results of experiments in which MDA-MB-231 cells were cultured in complete media containing 0 and 25. Mu.M Mexenone and statistically analyzed by flow cytometry after the synchronization treatment according to example 5 of the present invention;
FIG. 6 (a) is a graph showing the results of experiments in which the expression of G2/M phase-related proteins was detected by immunoblotting after MDA-MB-231 cells were treated with Mexenone at concentrations of 0, 15. Mu.M, 25. Mu.M, and 40. Mu.M, according to example 5 of the present invention;
FIG. 6 (b) is a graph showing the results of an experiment for detecting the expression of G2/M phase-related proteins by immunoblotting after HeLa cells were treated with Mexenone at concentrations of 0, 15. Mu.M, 25. Mu.M, and 40. Mu.M, as provided in example 5 of the present invention;
FIG. 7 (a) shows that the RXR alpha gene in HeLa cells is knocked out by Crasper technology by using 0, 25. Mu.M and 50. Mu.M Mexenone treated by PX330 as a vector provided in example 5 of the invention -/- After 24 hours, carrying out statistical analysis on the cell and the wild HeLa cell by a flow cytometer;
FIG. 7 (b) shows that the RXR alpha gene in A549 cells is knocked out by Crasper technology by using 0, 25. Mu.M and 50. Mu.M Mexenone treated by PX330 as a vector provided by example 5 of the invention -/- After 24 hours, the cells and the wild type A549 cells are subjected to statistical analysis by a flow cytometer to obtain an experimental result chart;
FIG. 7 (c) shows that the RXR α gene in HepG2 cells was knocked out by Crasper technology to construct the RXR α gene by treating PX330 as a vector with Mexenone at concentrations of 0, 25 μ M and 50 μ M, which is provided in example 5 of the present invention -/- After 24 hours, carrying out statistical analysis on the cell and the wild HepG2 cell by a flow cytometer;
FIG. 8 is a graph showing the results of flow cytometry analysis of HeLa cells transfected with Myc-Vector and Myc-RXR α for 24h treated with Mexenone at concentrations of 0, 25 μ M and 50 μ M as provided in example 5 of the present invention;
FIG. 9 (a) is a gel electrophoresis chart obtained by electrophoresis after releasing HeLa cells subjected to synchronization treatment using Mexenone at concentrations of 0 and 25. Mu.M for 10 hours according to example 6 of the present invention and subjecting to co-immunoprecipitation treatment;
FIG. 9 (b) is a gel electrophoresis chart obtained by performing immunofluorescence staining electrophoresis on HeLa cells subjected to release of synchronized treatment for 10h by Mexenone with concentrations of 0 and 25 μ M provided in example 6 of the present invention, wherein red represents RXR α -rabbit antibody, green represents PLK 1-mouse antibody, blue represents DAPI, and Scale bar is 5 μ M;
FIG. 10 is a volume growth curve of a transplanted tumor in a nude mouse, provided in example 7 of the present invention;
fig. 11 (a) is a photograph of axillary subcutaneous transplantation tumor of nude mice provided in example 7 of the present invention, n =6;
FIG. 11 (b) is a weight statistical chart of transplanted tumor in nude mouse, provided in example 7 of the present invention;
fig. 11 (c) is a nude mouse graft tumor volume growth curve provided in example 7 of the present invention, P <0.05;
FIG. 12 is an immunofluorescent staining graph of frozen sections of nude mouse transplantable tumors provided in example 7 of the present invention, primary antibody ratio 1:200, wherein cleared Caspase3 (red), blue is DAPI, and Scale bar 50 μm;
FIG. 13 is a H & E staining pattern of paraffin sections of heart, liver, spleen and kidney tissues (left to right) of nude mice, scale bar 50 μm, provided in example 7 of the present invention;
FIG. 14 shows that paraffin sections of nude mouse liver tissues, which are provided in example 7 of the present invention, were IHC-stained with p-Histone H3, cyclin B1 and p-PLK1, and the primary antibody ratio was 1.
Detailed Description
Example 1 antitumor drug
The embodiment provides an anti-tumor drug, which comprises one or more of benzophenone compounds with the following structures, and the specific structures are shown in fig. 1. Cell clone formation experiments on MDA-MB-231 cells by using the compound provided by the figure 1 show that the substance can induce G2/M cycle block of the MDA-MD-231 cells in a concentration-dependent manner, and further realize the effect of inhibiting the growth and proliferation of tumor cells.
Example 2 Exiphenone induces G2/M cycle arrest in tumor cells
This example 1X 10 statistical analysis of DAPI-stained nuclei by flow cytometry after 24h treatment of cells with 0, 20. Mu.M, 40. Mu.M and 80. Mu.M concentration of epothilone in MDA-MB-231 cells 4 The results are shown in FIG. 2.
As can be seen from FIG. 2, the ratio of MDA-MB-231 cells treated with epothilone to G2/M phase is greater than that of MDA-MB-231 cells cultured without epothilone, and increases with the increase of the concentration of epothilone treated, indicating that epothilone has the effect of inducing the G2/M cycle arrest in tumor cells.
Example 3 application of 2-hydroxy-4-methoxybenzophenone as nuclear receptor RXR alpha ligand
This example provides the use of 2-hydroxy-4-methoxybenzophenone (Mexenone) as a nuclear receptor RXR α ligand, the structure of Mexenone is shown below:
Figure BDA0003741762110000091
at present, mexenone is a benzophenone type ultraviolet absorbent, and is often added into cosmetics as a functional agent to play a role of sun protection. The inventor creatively selects Mexenone as the nuclear receptor RXR alpha ligand, and carries out the following experiments and calculations:
first, mexenone and a positive drug CD3254 were prepared into a solution using DMSO as a solvent, and a fluorescence titration experiment was performed using purified His-RXR α -LBD protein, and the results are shown in fig. 3 (a). The dissociation constant K of Mexenone and RXR alpha-LBD protein is obtained by Origin software fitting d =27.61 μ M, mexenone has greater affinity for RXR α -LBD protein in vitro;
next, reporter gene experiments were performed using HEK-293T cells to examine the effect of Mexenone on RXR α transcriptional activity, and the results are shown in fig. 3 (b). As shown in FIG. 3 (b), mexenone can significantly inhibit 9-cis-RA-induced RXR alpha transcriptional activation, and can be used as an antagonist of RXR alpha;
then, use
Figure BDA0003741762110000092
The software performed molecular docking simulations of Mexenone with RXR α (PDB: 3 FUG), the results of which are shown in FIG. 3 (c). As can be seen from fig. 3 (c), mexenone can bind to the co-regulatory factor binding site on the surface of RXR α protein, where Mexenone binding at the PHE450 site of the RXR α surface region is critical.
From the experimental results, mexenone can be combined with a co-regulatory factor binding site on the surface of RXR alpha protein in vitro to be used as an antagonist of RXR alpha, so that the growth and proliferation of tumor cells are inhibited, and the Mexenone can be used for preparing antitumor drugs.
Example 4 Nuclear receptor RXR alpha ligands inhibit tumor cell growth and proliferation
In the embodiment, mexenone is used for carrying out cell clone formation experiments on six tumor cells, namely HeLa, MDA-MB-231, 786-O, A549, hepG2 and A431, and the method specifically comprises the following steps:
a. preparing a crystal violet solution: firstly, dissolving crystal violet solid by using methanol as a solvent to prepare 0.5wt% of crystal violet solution; then diluting the solution into 0.1wt% crystal violet solution by PBS;
b. digesting and collecting cells, and gently blowing and beating cell suspensions of six tumor cells, namely HeLa, MDA-MB-231, 786-O, A549, hepG2 and A431 by using a culture medium to form a single cell state;
c. paving a plate: after counting the cells, the cells were seeded in a six-well plate at a cell density of 100 cells/well, the plate was tapped around to distribute them uniformly and then placed in a volume fraction of 5% CO 2, Culturing in a cell culture box at 37 ℃;
d. adding medicine: after at least 12h of culture, 0,30 and 60 μm of Mexenone was added to each tumor cell culture well, respectively, taking care that the volume of the medium to be added was 4 mL/well, preventing evaporation during the culture and avoiding errors in the experimental results due to the medium added midway, and then continuously placing at a volume fraction of 5% 2, Culturing in a cell culture box at 37 ℃ for 14 days;
e. collecting and fixing: discarding the culture medium, washing with PBS for three times, and fixing 4wt% paraformaldehyde at 1 mL/hole for 30min at room temperature;
f. crystal violet staining: washing with PBS for three times, dyeing with 0.1wt% crystal violet solution 1 mL/hole at room temperature for 30min, and washing with slow running water for 10min;
g. after the moisture on the pore plate is dried, the six pore plates are uncovered and placed on a piece of clean A4 paper for photographing.
The results of the cell clone formation experiments are shown in FIG. 4, the addition of Mexenone in the cell culture process can inhibit the growth of six tumor cells, namely HeLa, MDA-MB-231, 786-O, A549, hepG2 and A431, and the inhibition effect is more remarkable with the increase of the input amount, which indicates that Mexenone can inhibit the growth and proliferation of six tumor cells, namely HeLa, MDA-MB-231, 786-O, A549, hepG2 and A431, in a concentration gradient manner.
Example 5 Effect of Nuclear receptor RXR α ligands on mitosis of tumor cells
This example explores the effect of adding Mexenone during culture on the mitosis of tumor cells by the following experiments, including specifically the following:
1. mexenone induces tumor cells to generate G2/M cycle block
(1) After 24h treatment of the cells with Mexenone at concentrations of 0, 25. Mu.M and 50. Mu.M in MDA-MB-231, heLa and 786-O cells, the DAPI-stained nuclei were statistically analyzed 1X 10 by flow cytometry 4 The results are shown in FIG. 5 (a).
As can be seen from FIG. 5 (a), the proportion of tumor cells in the G2/M cycle increased after treatment with Mexenone, and the proportion of cells in the G2/M cycle increased with the increase in the concentration of Mexenone treatment, indicating that Mexenone has the effect of inducing the tumor cells to develop G2/M cycle arrest.
(2) The MDA-MB-231 cells were cultured for 16h using Thymidine containing medium containing 2mM thymus (Thymidine) for blocking so that MDA-MB-231 cells were synchronized to the G0 phase, followed by discarding the Thymidine containing medium, addition of complete medium containing 25. Mu.M Mexenone or no Mexenone for release, and collection analysis at 0h, 6h, 8h, and 10h of release, as shown in FIG. 5 (b).
FIG. 5 (b) shows the first behavior of the negative control group without Mexenone, in which the percentage of G2/M phase is 27.9% after 10 hours of release, indicating that the group of MDA-MB-231 cells is about to exit the G2/M phase; the ratio of G2/M phase in the Mexenone drug adding group in the second row is 42.2 percent, which indicates that MDA-MB-231 cells are blocked in the G2/M phase, and further proves that the Mexenone drug adding group can induce tumor cells to generate G2/M cycle block.
2. Mexenone induces the expression of G2/M cycle-associated protein of tumor cells to be increased
After treating the cells with Mexenone at concentrations of 0, 15. Mu.M, 25. Mu.M, and 40. Mu.M in MDA-MB-231 and HeLa cells for 24h, the expression of G2/M phase-related protein was detected by immunoblotting, and the results are shown in FIG. 6 (a) and FIG. 6 (b).
FIG. 6 (a) shows the expression of G2/M phase-related protein in MDA-MB-231 cells, and FIG. 6 (B) shows the expression of G2/M phase-related protein in HeLa cells, and it can be seen that the expression levels of proteins such as p-Histone H3, cyclin B1, p-PLK1 and PLK1 in two tumor cells are significantly up-regulated, and it is proved from the protein level that Mexenone can induce the tumor cells to generate G2/M cycle block.
3. Mexenone induces tumor cells to generate RXR alpha-dependent G2/M cycle block
(1) Knock out RXR alpha genes in HeLa, A549 and HepG2 cells by using PX330 as a vector and utilizing the Crasper technology to construct the RXR alpha -/- Cells, wild-type HeLa, A549 and HepG2 cells as controls, 1X 10 cells stained with DAPI and statistically analyzed by flow cytometry after treating the cells with Mexenone at concentrations of 0, 25. Mu.M and 50. Mu.M for 24 hours 4 The results of the individual cells are shown in FIGS. 7 (a), (b) and (c).
As is clear from FIGS. 7 (a), (b) and (c), RXR. Alpha. Is knocked out by the RXR. Alpha. Gene -/- The cells do not express RXR α protein; RXR alpha as compared to wild type tumor cells -/- The decrease in the cell proportion of cells in the G2/M phase indicates that RXR α is present -/- Mexenone in cells induces fewer numbers of G2/M cycle blocks in cells.
(2) After transfection of HeLa cells with Myc-Vector and Myc-RXR α for 24h, respectively, followed by treatment of the cells with 0, 25 μ M and 50 μ M Mexenone for 24h, DAPI stained nuclei were statistically analyzed by flow cytometry at 1X 10 4 The results are shown in FIG. 8.
As can be seen from FIG. 8, when Myc-Vector transfected HeLa cells were used as a control, RXR α was overexpressed in the Myc-RXR α transfected HeLa cells, and the percentage of G2/M phase cells in the Myc-RXR α group was significantly increased compared to the Myc-Vector group, indicating that overexpression of RXR α in tumor cells increases the number of cells in which Mexenone induces G2/M cycle arrest in cells.
In conclusion, it was found that the RXR α -dependent G2/M cycle block induced by Mexenone in tumor cells was induced by changing the expression level of RXR α in tumor cells (making tumor cells non-expressed or over-expressed) and analyzing by flow cytometry.
Example 6 Nuclear receptor RXR alpha ligands affect cellular M-phase p-RXR alpha interaction with PLK1
RXR alpha is phosphorylated by CDK1 in the mitotic stage of tumor cells, and then is positioned to a centrosome to interact with PLK1, so that activation and mitotic process of PLK1 are promoted. And Mexenone can be used as a ligand of RXR alpha, so that whether Mexenone influences the interaction of p-RXR alpha and PLK1 or not is verified through co-immunoprecipitation and immunofluorescence staining in the embodiment, so that the activation and mitosis of PLK1 are inhibited, and G2/M cycle block is induced finally.
HeLa cells were synchronized to G0 using 2mM thymus, followed by 10h to M phase release using Mexenone at concentrations of 0 and 25. Mu.M, co-immunoprecipitation and immunofluorescent staining were performed, and the results are shown in FIGS. 9 (a) and 9 (b). As is clear from FIGS. 9 (a) and 9 (b), the expression level of p-RXR α was decreased in HeLa cells treated with 25. Mu.M mexene as compared with cells not treated with mexene. FIG. 9 (b) shows RXR α -rabbit antibody in red, PLK 1-rat antibody in green, and DAPI in blue, and analysis revealed that in cells not treated with Mexenone, bright green spots were seen in the middle of the two sides of the chromosomes in alignment, and red foci were also seen on the two sides of the chromosomes, indicating that PLK1 was also well recruited to the centrosome, while both RXR α and PLK1 were well co-localized; whereas in HeLa cells treated with 25 μ M Mexenone, the red foci were less well dispersed and the green spots were scattered, indicating that PLK1 was not recruited to the centrosome and could not co-localize with RXR α.
From the results of co-immunoprecipitation and immunofluorescence staining, mexenone can reduce the expression level of p-RXR alpha of tumor cells, and further inhibit the interaction and co-localization of RXR alpha and PLK1, thereby influencing the recruitment of the central body to PLK1 protein.
Example 7 pharmacodynamic evaluation of Nuclear receptor RX α ligands in nude mouse model of Breast cancer transplantable tumors
1. Nude mouse model establishment and experiment scheme
(1) Model establishment: an MDA-MB-231 cell line with high tumor formation rate is selected to establish a nude mouse subcutaneous transplantation tumor model to further study the in-vivo anti-tumor effect of Mexenone. 1 × 10 of each of the nude mice is subcutaneously administered in the lateral middle of axilla of 7-week-old female nude mice according to pharmacodynamic guidelines for antitumor drugs 6 Inoculating the cells until the tumor volume is increased to 100mm 3 Randomly, and begin dosing.
(2) The experimental scheme is as follows: nude mice were randomly divided into three groups of 6 mice each: negative control group (DMSO +90% corn oil 10%), mexenone low dose group (60 mg/kg), mexenone high dose group (120 mg/kg). Intraperitoneal injection administration is carried out every day according to the body weight of the mice, meanwhile, the nude mice are weighed once every two days, a graph 10 is obtained by statistical drawing, and the tumor volume is measured and recorded. The mice were sacrificed 16 days after administration, and the tumors and tissues of heart, liver, spleen, kidney, etc. were removed for subsequent experiments.
2. Effect of Mexenone on growth of nude mouse transplanted tumor of breast cancer cell
(1) Mexenone for inhibiting growth of breast cancer cell nude mouse transplanted tumor
After the sampling, the size of the tumor tissue was measured and the weight of the tumor tissue was weighed, and as shown in fig. 11 (a), (b) and (c), mexenone was effective in inhibiting the growth of MDA-MB-231 cell nude mouse transplanted tumor, and the tumor inhibition rates of the low dose group and the high dose group were 23.17% and 32.57%, respectively, compared to the negative control group.
(2) Mexenone induced apoptosis of nude mice transplantation tumor cells
To further examine whether Mexenone can induce apoptosis in transplanted tumor cells, the inventors examined the expression of clear Caspase3 protein in transplanted tumor tissues, and the results are shown in fig. 12, in which red indicates the clear Caspase3 protein and blue indicates DAPI in fig. 12, compared to the negative control group. As can be seen from FIG. 12, clear Caspase3 is highly expressed in the Mexenone administration group, and the expression level of clear Caspase3 is increased along with the increase of the administration concentration, which indicates that Mexenone can induce apoptosis of breast cancer transplantation tumor cells under the endothelium of nude mice.
3. Mexenone has no obvious toxicity and side effect on nude mice with breast cancer transplantable tumor
Firstly, as can be seen from fig. 10, the weight curves of the nude mice of the negative control group, the low dose group and the high dose group have no obvious difference, which indicates that Mexenone has no obvious side effect on the growth of the nude mice and has no obvious toxicity in the nude mice;
secondly, after the heart, liver, spleen and kidney in each group of mice are taken down and stained by paraffin section H & E, the result is shown in FIG. 13, and no obvious change of cell morphology and arrangement rule is observed, which indicates that Mexenone has low systemic toxicity and high safety to nude mice;
moreover, the liver is a metabolic organ in the body of a nude mouse, and the division of liver cells is more vigorous compared with other organs; therefore, paraffin sections of the liver of a nude mouse are selected for immunohistochemical staining of p-Histone H3, cyclin B1 and p-PLK1, and whether Mexenone induces the liver cells to generate G2/M cycle block is detected, and the result is shown in FIG. 14, wherein brown is p-Histone H3, cyclin B1 and p-PLK1 antibody expression, and blue is cell nucleus. As can be seen from FIG. 14, there was no significant difference in the expression levels of p-Histone H3, cyclin B1 and p-PLK1 in the liver tissues of the negative control group and the administration group, indicating that Mexenone did not induce G2/M cycle arrest of liver cells of nude mice with MDA-MB-231 cell transplantation tumor.
In the embodiment, experimental research on an animal level proves that Mexenone can obviously inhibit the growth of the transplanted tumor in a transplanted tumor nude mouse body and cause the growth of tumor cells, and meanwhile, the Mexenone has no obvious toxic effect on normal organ tissues of the transplanted tumor nude mouse and is an anti-tumor compound with potential development value.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention can be made, and the same should be considered as the disclosure of the present invention as long as the idea of the present invention is not violated.

Claims (10)

  1. The application of 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof in preparing an antitumor drug is disclosed, wherein the result of the 2-hydroxy-4-methoxybenzophenone is shown as a formula 1:
    Figure FDA0003741762100000011
  2. 2. the use according to claim 1, wherein the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used as a ligand of a nuclear receptor RXR α for the preparation of a medicament which binds to RXR α;
    preferably, the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used for preparing a medicament which binds to a surface binding site of a co-regulatory factor of RXR alpha.
  3. 3. The use according to claim 1, wherein the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used for the preparation of a medicament for inhibiting the production of p-rxra by tumor cells;
    preferably, the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used for the preparation of a medicament for inhibiting the interaction and co-localization of PLK1 and RXR α.
  4. 4. The use according to claim 1, wherein the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used in the manufacture of a medicament for inducing mitotic arrest and triggering cycle-specific apoptosis in tumour cells.
  5. 5. The use according to claim 4, wherein the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used in the manufacture of a medicament for inducing G2/M cycle arrest in tumour cells;
    preferably, the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used for preparing a medicament for inducing tumor cells to generate G2/M cycle block which is dependent on RXR alpha.
  6. 6. The use according to claim 4, wherein the 2-hydroxy-4-methoxybenzophenone or a solvent compound thereof or a pharmaceutically acceptable salt thereof is used in the preparation of a medicament for inducing high expression of a tumor cell clear Caspase3 protein to trigger cycle-specific apoptosis.
  7. 7. An antitumor drug is characterized by comprising benzophenone compounds or solvent compounds thereof or pharmaceutically acceptable salts thereof with pharmaceutically effective dose;
    preferably, the result of the benzophenone compound is shown as formula 2 or formula 3:
    Figure FDA0003741762100000021
    wherein R is 1 = H or SO 3 H，R 2 H or OH, R 3 ＝CH 3 H or OCH 3 。
  8. 8. The antitumor drug according to claim 7, wherein the benzophenone compound is 2-hydroxy-4-methoxybenzophenone.
  9. 9. The antitumor agent as claimed in claim 7, further comprising an active ingredient; preferably, the active ingredient is a substance having anti-tumor activity.
  10. 10. The antitumor agent as claimed in claim 7, further comprising a pharmaceutically acceptable carrier; preferably, the carrier is one or more of a diluent, an excipient, a filler, a binder, an absorption enhancer, a surface agent or a lubricant.
CN202210814561.XA 2022-07-12 2022-07-12 Application of nuclear receptor RXR alpha ligand and antitumor drug Pending CN115300493A (en)

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