CN106963765B

CN106963765B - Application of EZH2 inhibitor compound in preparation of medicine for treating ocular melanoma

Info

Publication number: CN106963765B
Application number: CN201710193414.4A
Authority: CN
Inventors: 范先群; 张赫; 柴佩韦; 文旭洋; 葛盛芳; 贾仁兵
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2017-03-28
Filing date: 2017-03-28
Publication date: 2020-04-07
Anticipated expiration: 2037-03-28
Also published as: CN106963765A

Abstract

The invention discloses an application of an EZH2 inhibitor compound in preparing a medicament for treating ocular melanoma, wherein the EZH2 inhibitor compound comprises GSK503, GSK343, EPZ005687 and EPZ-6438, and the molecular formulas of the compounds are respectively C₃₁H₃₈N₆O₂、C₃₁H₃₉N₇O2、C₃₄H₄₄N₄O4、C₃₂H₃₇N₅O₃The molecular structural formulas are respectively:

the invention aims to provide a new target spot and a treatment medicine for clinical treatment of the melanoma on the eyes, improve the treatment effectiveness, reduce the removal rate of the eyeballs, improve the vision prognosis of patients and develop a new field unprecedentedly.

Description

Application of EZH2 inhibitor compound in preparation of medicine for treating ocular melanoma

Technical Field

The invention relates to the field of medicine, in particular to application of an EZH2 inhibitor compound in preparing a medicine for treating ocular melanoma.

Background

The eye melanoma includes Uveal Melanoma (UM) and Conjunctival Melanoma (CM). Wherein UM is the intraocular malignant tumor with the highest adult incidence, originates in the uvea, has high malignancy degree, is easy to generate early metastasis, has poor prognosis and high mortality rate, and seriously threatens the vision and the life of patients. CM is hidden when getting ill, does not affect vision in early stage, often has diffusion transfer when being found, and seriously affects life safety of patients. Therefore, the research on the generation mechanism of the ocular melanoma has very important clinical guidance and scientific significance for searching new targets for early diagnosis and treatment of the ocular melanoma, improving the treatment effectiveness, reducing the removal rate and transfer rate of eyeballs, and improving the visual prognosis and life span of patients.

At present, the treatment aiming at the tumor is mainly non-etiological treatment such as operation, radiotherapy, chemotherapy and the like, and a treatment means aiming at the etiological factor is lacked. Enhancer of Zeste homolog 2(EZH2) is one of the members of the polycombin complex (PcG) family, and is the catalytically active subunit of polycombin inhibitory complex 2(PRC 2). EZH2 is a intracellular histone methyltransferase that promotes methylation of the trimethyl at amino acid 27 of the intracellular histone H3 (H3K27me3) and has been shown to be a protooncogene, associated with the growth and metastasis of a variety of tumors. EZH2 can inhibit the expression of tumor suppressor genes by increasing the level of H3K27me3 methylation in cells, leading to tumorigenesis. The EZH2 small-molecule inhibitor can be found to have better effect by screening a small-molecule compound library at high flux, but no report of the application of the EZH2 inhibitor in the choroidal melanoma exists so far, and the curative effect and the action mechanism of the EZH2 inhibitor on the choroidal melanoma and the conjunctival melanoma are not clear.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides an application of an EZH2 inhibitor compound in preparing a medicament for treating ocular melanoma, which is characterized in that the EZH2 inhibitor compound is GSK503, GSK343, EPZ005687 or EPZ-6438, and the molecular formulas are respectively C₃₁H₃₈N₆O₂、C₃₁H₃₉N₇O2、 C₃₄H₄₄N₄O4、C₃₂H₃₇N₅O₃The molecular structural formulas are respectively:

preferably, the concentration of the EZH2 inhibitor compound is at least 10 μm.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers for the first time that GSK503, GSK343, EPZ-6438 and EPZ005687 can obviously inhibit the growth of choroidal melanoma and conjunctival melanoma, and the methylation degree of H3K27me3 in cells is reduced by inhibiting EZH2, so that the cancer suppressor gene is re-expressed, and the effect of inhibiting the growth of tumors is achieved. The invention aims to provide a new target spot and a treatment medicine for clinical treatment of the melanoma on the eyes, improve the treatment effectiveness, reduce the removal rate of the eyeballs, improve the vision prognosis of patients and develop a new field unprecedentedly.

Description of the drawings:

fig. 1 is a graph showing the effect of the methylation degree of H3K27me3 in the choroidal melanoma and conjunctival melanoma, which is significantly increased in the present example.

FIG. 2 is a schematic diagram showing the effect of GSK503, GSK343, EPZ005687 and EPZ-6438 in inhibiting the proliferation of melanoma cells in the eye region according to the present invention.

Fig. 3 is a graph showing that EZH2 inhibitor significantly inhibited the proliferation of retinoblastoma in the examples of the present invention.

FIG. 4 is a schematic diagram showing that 10uM EZH2 inhibitor (GSK503, GSK343, EPZ-6438, EPZ005687) can significantly inhibit the migration of ocular melanoma cells in the present invention.

Fig. 5 is a schematic diagram of the effect of EZH2 inhibitor on reducing ocular melanoma H3K27me3 levels in an embodiment of the invention.

Detailed Description

The invention will be further understood by reference to the following examples.

The EZH2 inhibitor compounds of the invention are GSK503, GSK343, EPZ005687 and EPZ-6438, the molecular formulas of which are respectively C₃₁H₃₈N₆O₂、C₃₁H₃₉N₇O2、C₃₄H₄₄N₄O4、C₃₂H₃₇N₅O₃The molecular structural formulas are respectively:

example 1

Immunofluorescence assay

Experimental materials: a choroidal melanoma tissue chip (Me1004a) available from sienna alice biotechnology limited; H3K27me3 antibody and a second antibody, purchased from Abcam (eboantibody (shanghai) trade ltd).

The tissue chips were placed in a wet box and PBS was spread over the tissue chips. The diluted H3K27me3 antibody was centrifuged for 2min at 13500g and 4 ℃. The PBS buffer on the slide was aspirated off at one end of the tissue chip and the antibody was added from the other end, covered with the humidified box and incubated for 1h at room temperature. The slide was washed 3 times with PBS (5 min/time), fresh PBS buffer was added from one end of the section, and old buffer was removed from the other end. Diluted secondary antibody was centrifuged at 13500g for 2min at 4 ℃. Secondary antibody was applied to the tissue chip, incubated in a humidified box for 1h at room temperature, and the slide was washed 3 times with PBS (5 min/time). The cover glass was covered with a glass slide, and the result was observed under a microscope, and the result is shown in FIG. 1. It can be seen that the methylation degree of H3K27me3 is remarkably increased in choroidal melanoma and conjunctival melanoma

Example 2

Plate clone formation experiment

Experimental materials: human choroidal melanoma OCM1, OM431 cells, human conjunctival melanoma CRMM1, CM2005.1 cells, 37 deg.C, 5% CO₂Conventional culture in dmem (gibco) with 10% Fetal Bovine Serum (FBS); GSK503, GSK343, EPZ005687, EPZ-6438 from MedChemexpress (MCE, China); six-well plates were purchased from Thermo Fisher (semer fly, usa); crystal violet was purchased from bio-chemical company (bio-china);

OCM1, OM431, CRMM1 and CM2005.1 were cultured to a density of 70% -80%, grown well, digested and centrifuged at a high refractive index, and resuspended in 10% FBS DMEM. The cells were counted and 1000 cells/well plated on a six-well plate containing 2 ml of 10% DMEM medium per well. After 2 weeks, the cells were stained with crystal violet, washed 2 times with PBS, and the number of cell colonies was counted by photographing, and the results are shown in FIG. 2. It can be seen that GSK503, GSK343, EPZ005687 and EPZ-6438 can inhibit the proliferation ability of ocular melanoma cells

Example 3:

cell plate clone proliferation assay

Experimental materials: CCK8 from Homophilus Japan

OCM1, CM2005.1 cells retinal pigment epithelial cells ARPE-19 and melanocyte PIG-1 were used as normal controls as described previously. OCM1 and CM2005.1 cells were inoculated into 96-well plates at 37 deg.C with 2000 cells per well, 100ul culture medium, ARPE19 and PIG-1 cells at 4000 cells per well, and 5% CO₂Culturing, adding 10ul CCK8 at 0h, 24h, 48h and 72h, incubating at 37 deg.C for 4h, and measuring absorbance value with computer OD450nm, the result is shown in FIG. 3. The EZH2 inhibitor can remarkably inhibit the proliferation of retinoblastoma, and the inhibition effect is enhanced along with the increase of concentration. The same concentration of drug is less toxic to normal cells.

Example 4

The name of the experiment: transwell cell migration experiment

Experimental materials: 37 ℃ and 5% CO₂Conventional culture in dmem (gibco) with 10% Fetal Bovine Serum (FBS); GSK503, GSK343, EPZ005687, EPZ-6438 from MedChemexpress (MCE, China); six well plates were purchased from ThermoFisher (Sammerfei, USA); crystal violet was purchased from bio-chemical company (bio-china); transwell dishes were purchased from millipore (millipore, usa).

The experimental steps are as follows: an 8 μm 24-well Transwell chamber was used. The 24-well plate was loaded with 900. mu.l of 10% FBS DMEM medium per well, and the cells were suspended in 24 wells, and 10000 cells were seeded in 200. mu.l of 2% FBS DMEM medium per cell. After 2-3 days of culture, the culture medium in the wells and the chamber was aspirated and carefully washed once with PBS. Add crystal violet to stain for 30min and wash carefully once with PBS. The cells tested in the chamber were wiped clean. The outside cells of the chamber were photographed for observation. The crystal violet is completely dissolved by using 33% acetic acid solution, and the OD value is measured by 630nm light absorption value of an enzyme-labeling instrument for statistical analysis, and the result is shown in figure 4. The 10uM EZH2 inhibitor (GSK503, GSK343, EPZ-6438, EPZ005687) can remarkably inhibit the migration capacity of the melanoma cells of eyes.

Example 5:

the name of the experiment: western blot

Preparing materials: the nucleoprotein extraction kit is purchased from Thermo Fisher (Saimer fly, USA), the protein loading and ladder are purchased from Biyunnan Bio Inc., the H3K27me3, EZH2, H3, beta-actin, rabbit anti-antibody are purchased from Abcam, USA, and the Western transfer membrane and the electrophoresis reagent are purchased from Bio-rad, USA.

The experimental steps are as follows: discarding the original culture solution, washing off the exfoliated cell debris with cold PBS, completely sucking PBS, adding appropriate amount of lysis solution RIPA containing PMSF protein, covering the cell surface, and lysing for 30min on ice. Cell debris was scraped off using a cell scraper, and the whole liquid was transferred to a clean EP tube, centrifuged at 12000rpm at 4 ℃ for 30min, and then the supernatant liquid was transferred to another clean EP tube. The BSA method detects protein concentration. Adding 4 xSDS protein loading according to the protein volume, and performing protein denaturation in boiling water for 10 min. Storing at-80 ℃ for later use. The nuclear protein was extracted separately using Thermo Fisher Bioreagents cytoplasm and nuclear protein extraction kit. Electrophoresis was performed using 10% SDS-polyacrylamide gel electrophoresis. 4 mul of protein Marker and denatured protein with equal mass are added into the pore channel. Placing in 800ml electrophoresis buffer solution [ 0.1% SDS, 250mM glycine (pH8.3)25mM Tris base ], performing constant voltage electrophoresis at 80V for about 30min until the protein sample enters the lower layer gel, changing the voltage to 120V, and continuing electrophoresis for 40 min. The gel was removed and the excess was cut off, thoroughly soaked in pre-cooled spin-on buffer (39mM glycine, 10% methanol, 48mM Tris base) along with the sponge and filter paper. An appropriately sized PVDF membrane was cut and placed in methanol for activation. The power anode, the sponge, the filter paper, the PVDF membrane, the PAGE gel, the filter paper, the sponge and the electrophoresis cathode are sequentially placed in sequence and clamped into a sandwich structure. Add ice-cream and put in 1L of membrane-transfer buffer. The membrane is constantly transferred by using 200 mA current, and the membrane transferring time is adjusted according to the size of protein molecules. The PVDF membrane successfully subjected to membrane transfer is placed in BSA (bovine serum albumin) prepared in 5% TBS (TBS) and blocked for 1h at room temperature. Add appropriate primary antibody formulated with 5% TBS, seal in hybridization bag, incubate overnight at 4 ℃. The PVDF membrane was removed and washed 3 times with 5% TBST at room temperature for 10min each time. Adding a proper amount of secondary antibody, sealing in a hybridization bag, and incubating for 1h at room temperature in a dark place. The PVDF membrane was removed and washed with 5% TBST 3 times in the dark at room temperature for 10min each time. The film is ready for scanning use. PVDF membrane was scanned and analyzed by spreading on an Odyssey laser imaging system. The results are shown in FIG. 5. The 10uM EZH2 inhibitor can effectively reduce the level of the eye melanoma H3K27me 3.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. An application of an EZH2 inhibitor compound in preparing a medicament for treating conjunctival melanoma is characterized in that the EZH2 inhibitor compound is GSK503, GSK343, EPZ005687 and EPZ-6438, and the molecular formulas are respectively C₃₁H₃₈N₆O₂、C₃₁H₃₉N₇O2、C₃₄H₄₄N₄O4、C₃₂H₃₇N₅O₃The molecular structural formula is respectively shown as formula I, formula II, formula III and formula IV:

2. the use of an EZH2 inhibitor compound for the preparation of a medicament for the treatment of conjunctival melanoma according to claim 1, wherein the EZH2 inhibitor compound is present at a concentration of at least 10 μ Μ.