Application of reagent for detecting epigenetically modified H3T3 and application of epigenetically modified H3T3 inhibitor

Abstract

The invention provides application of a reagent for detecting epigenetic modification H3T3 and application of an epigenetic modification H3T3 inhibitor, and belongs to the technical field of medicines. Different from the fact that H3pT45 is almost not expressed in tumors and normal tissues, H3pT3 is almost not expressed in the normal tissues but is highly expressed in the tumor tissues, so that high expression of H3pT3 in the colorectal cancer can be used as a screening index, the screening accuracy and the early detection rate are remarkably improved, and a new marker is provided for early detection of the colorectal cancer. In addition, the invention also finds that the H3pT3 inhibitor can significantly inhibit proliferation of colorectal cancer cell lines and significantly delay tumor progression in a mouse subcutaneous tumor model, and shows good anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to uses of reagents for detecting epigenetically modified H3T3 and uses of epigenetically modified H3T3 inhibitors. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, with high morbidity and mortality rates. Early diagnosis is crucial to improving the survival rate of colorectal cancer patients, but current diagnostic methods still have certain limitations. Traditional diagnostic methods mainly include colonoscopy, imaging examinations, and blood tumor marker detection. Although colonoscopy is the "gold standard" for diagnosing colorectal cancer, its invasiveness and patient tolerance limit its widespread application. Although blood tumor markers such as CEA and CA19-9 are relatively common, their sensitivity and specificity are low, which makes it difficult to meet the needs of early diagnosis.

[0003] In recent years, with in-depth research into the pathogenesis of colorectal cancer, a variety of biomarkers have been discovered and applied in clinical diagnosis. For example, non-invasive diagnostic methods such as circulating tumor DNA (ctDNA) testing, fecal genetic testing, and fecal microbial marker testing have gradually gained attention. Among them, fecal genetic testing, which detects gene mutations and methylation status in stool, has demonstrated high sensitivity and specificity. In addition, fecal microbial markers such as Fusobacterium nucleatum have also been shown to be closely associated with the development and progression of colorectal cancer. Therefore, the exploration of new biomarkers has important clinical significance for the early diagnosis and treatment of colorectal cancer.

[0004] H3T3 phosphorylation (H3pT3) refers to the phosphorylation of threonine 3 (T3) of histone H3, a modification that plays an important regulatory role in cell biology. However, there are currently no reports of histone H3T3 phosphorylation as a diagnostic biomarker for colorectal cancer. Summary of the Invention

[0005] The present invention aims to provide uses of reagents for detecting epigenetically modified H3T3 and uses of inhibitors of epigenetically modified H3T3.

[0006] The technical solutions of the present invention are as follows: The present invention provides use of a reagent for detecting epigenetically modified H3T3 in preparing a colorectal cancer screening kit.

[0007] Furthermore, the reagent for detecting epigenetically modified H3T3 is an immunohistochemical detection reagent or an immunoblotting detection reagent.

[0008] Furthermore, the epigenetically modified H3T3 is H3pT3.

[0009] The present invention also provides use of an epigenetically modified H3T3 inhibitor in the preparation of a drug for preventing and / or treating colorectal cancer.

[0010] Furthermore, the epigenetically modified H3T3 is H3pT3.

[0011] Furthermore, the epigenetic modification H3T3 inhibitor is CX6258 or a salt thereof.

[0012] Furthermore, the salt is hydrochloride.

[0013] Furthermore, the drug is a drug that inhibits the proliferation of cancer cells.

[0014] Furthermore, the drug is a pharmaceutical preparation prepared with the epigenetically modified H3T3 inhibitor as the active ingredient and pharmaceutically acceptable excipients.

[0015] Furthermore, the pharmaceutical preparation is an oral preparation or an injectable preparation.

[0016] Furthermore, the oral preparation is a tablet, granule, or capsule; and the injectable preparation is a powder injection.

[0017] Definitions of terms in this invention: The term "colorectal cancer" as used herein refers to cancer that occurs in the colon and rectum.

[0018] As used herein, "biomarkers" refer to biochemical markers that can identify changes or potential changes in the structure or function of systems, organs, tissues, cells, and subcellular structures. These markers have a wide range of applications. Biomarkers can be used to diagnose and stage diseases, or to evaluate the safety and efficacy of new drugs or therapies in target populations.

[0019] The present invention has achieved the following beneficial effects: (1) The present invention discovered that high expression of H3pT3 can be used as a screening indicator for colorectal cancer, providing a new biomarker for the early detection of colorectal cancer. Existing colorectal cancer diagnostic markers such as CEA and CA19-9 have the disadvantages of insufficient sensitivity and specificity. H3pT3, as a new marker, is expected to make up for the shortcomings of existing markers, improve screening accuracy and early detection rate, and thus improve patient prognosis. Using tumor tissue as a test sample is minimally harmful to patients. The present invention has good application prospects.

[0020] (2) The key to the present invention is that it has been determined that the content of H3pT3 in tumor tissue is significantly correlated with the risk of colorectal cancer. Therefore, the risk of colorectal cancer can be determined by detecting the content of H3pT3 in human tumor tissue. As for the specific means of detecting H3pT3 in human tumor tissue, various means disclosed in the prior art can be used. The embodiments of the present invention specifically use immunohistochemistry and immunoblotting for detection, but are not limited to these means. Any method that can detect the content of H3pT3 can be used for colorectal cancer screening.

[0021] (3) Through in vitro and in vivo experiments, the present invention found that the use of the H3pT3 inhibitor CX6258.HCl provides a new target and drug for the treatment of colorectal cancer, which is expected to develop more effective treatment plans, reduce dependence on traditional chemotherapy drugs, and reduce toxic side effects during treatment.

[0022] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0023] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Figure 4 shows the immunohistochemical detection results of Ki67, H3T3 phosphorylation (H3pT3), and H3T45 phosphorylation (H3pT45) in colorectal cancer patients and normal controls (10 representative cases).

[0025] Figure 2 is the expression level of H3pT3 in colorectal cancer patients and normal subjects.

[0026] Figure 3 The expression location statistics of H3pT3 and Ki67 in colorectal cancer.

[0027] Figure 4 Figure 2 is the ROC statistics of H3pT3.

[0028] Figure 5 The compound CX6258.HCl inhibits H3pT3 in colorectal cancer cell lines HCT116 and HT29.

[0029] Figure 6 The compound CX6258.HCl inhibits the proliferation of colorectal cancer cell lines HCT116 and HT29.

[0030] Figure 7 Compound CX6258.HCl inhibits the volume of subcutaneous tumors in mice.

[0031] Figure 8 Compound CX6258.HCl inhibits the weight of subcutaneous tumors in mice.

[0032] Figure 9 These are the results of HE staining and immunohistochemistry testing of compound CX6258.HCl in inhibiting the malignancy of subcutaneous tumors in mice.

[0033] Figure 10 The treatment with compound CX6258.HCl caused the downregulation of H3pT3 expression.

[0034] Figure 11 Treatment with compound CX6258.HCl had no effect on the body weight of mice.

[0035] Figure 12 The results of HE staining of the compound CX6258.HCl on the mouse heart, liver, spleen, lung, and kidney. DETAILED DESCRIPTION

[0036] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0037] The experimental methods used in the embodiments of the present invention are as follows: 1. Immunohistochemistry (1) Place the paraffin tissue sections in a drying machine and bake for 20 minutes; (2) Rehydration: xylene I for 5 min, xylene II for 5 min, 100% ethanol I for 5 min, 100% ethanol II for 5 min, 95% ethanol for 5 min, 75% ethanol for 5 min, 50% ethanol for 5 min, pure water for 5 min; (3) Antigen retrieval: Place the slices in diluted antigen retrieval solution, microwave on high for 2 minutes, then on medium-low for 10 minutes, and cool naturally at room temperature; (4) Punching: Place the cooled slide in 0.3% Triton-X PBS solution and soak for 20 min. Wash with PBS three times, 3 min each time. (5) Use an oil pen to draw a circle around the tissue, add endogenous catalase blocker and react for 10 minutes. After the reaction, wash with PBS three times, each time for 3 minutes. (6) Add primary antibodies and incubate overnight at 4°C; H3pT3 (abcam, ab78351); H3pT45 (abconal, AP0898); Ki67 (abclonal, A20018).

[0038] (7) Wash with PBS three times, 3 minutes each time, add reaction enhancement solution dropwise, and incubate at room temperature for 20 minutes; (8) Wash with PBS three times, 3 minutes each time; (9) Add secondary antibody (IgG polymer) at room temperature for 1 hour, and wash with PBS three times, each time for 3 minutes; (10) Develop with DAB colorimetric solution for about 2 minutes (depending on the situation), then wash three times with PBS, each time for 3 minutes; (11) Hematoxylin for 5 minutes, then rinse with tap water for 5 minutes; (12) 95% ethanol for 3 min, 3 times; 100% ethanol for 3 min, 3 times; xylene for 3 min, 3 times; (13) Seal the slides.

[0039] 2. Immunoblotting (1) Protein sample preparation Collect cells into 1.5 mL EP tubes and wash twice with pre-chilled PBS, centrifuging at 1000 rpm for 3 minutes each time. Remove the supernatant and place on ice. Add RIPA lysis buffer (Servicebio, G2002) (containing protease inhibitors) based on the cell number to thoroughly resuspend the cells. Sonicate the lysate twice using a 3 mm diameter ultrasonic probe, sonicating for 5 seconds with 5 seconds between bursts at 25% output power. Centrifuge at 12000 rpm at 4°C for 10 minutes. Transfer the supernatant to a fresh EP tube and place on ice. Boil the sample at 95°C for 10 minutes, aliquot, and store frozen at -20°C.

[0040] (2) SDS-PAGE gel preparation Select the appropriate separation gel concentration according to the molecular weight of the target protein to be separated.

[0041] (3) Electrophoresis Remove the protein sample from the -20°C freezer, boil at 100°C for 5 minutes, and then centrifuge for 1 minute. Carefully remove the gel slab comb, rinse with ultrapure water, and mount it on the electrophoresis stand. Place it in the electrophoresis tank. Pour running buffer over the gel slab, add the protein marker and sample, and connect the power supply. Run the electrophoresis at 80V. Once the protein marker is relatively separated and the sample has run below the plane of the separating gel, adjust the voltage to 120V and continue running until the electrophoresis is complete.

[0042] (4) Transfer In the transfer solution, arrange the sponge, three sheets of filter paper, gel, NC membrane, three sheets of filter paper, and sponge in a "sandwich" pattern, from negative electrode to positive electrode. Be careful to avoid creating air bubbles. Place the assembled transfer clamps into the transfer tank, pour in the transfer solution, add an ice pack, and place in an ice bath. Transfer at a constant current of 250 mA for 2 hours.

[0043] (5) Immunohistochemical hybridization After transfer, block the membrane in 5% skim milk with gentle shaking at room temperature for 1 hour. Cut the membrane according to the size of the target protein and place it in a hybridization bag. Add the proportionally diluted primary antibody and incubate overnight at 4°C.

[0044] (6) Development Remove the PVDF membrane from the overnight incubation, recover the primary antibody from the incubation bag, and wash three times with 1× TBST buffer for 10 minutes each. After washing, place the membrane in the secondary antibody and incubate on a shaker at room temperature for 1–2 hours. Wash twice with TBST for 10 minutes each, then develop.

[0045] 3. 5-Ethynyl-2'-deoxyuridine (EdU) detection (Beyotime, C0071S) (1) Cell culture Cells in logarithmic phase were taken and 7×10 cells were seeded into each well of a 24-well plate. 3 cells and culture them to the normal growth stage.

[0046] (2) Drug treatment Subsequent experiments were performed after adding CX6258.HCl (Selleck, S7041) and treating for 48 hours. The structure of the compound CX6258.HCl is as follows: .

[0047] (3) Prepare 2X EdU working solution: dilute EdU (10 mM) at a final concentration of 10 μM (1X) with cell culture medium at a ratio of 1:500 to obtain 2X EdU working solution (20 μM).

[0048] (4) Add an equal volume of 2X EdU working solution (20 μM) preheated at 37°C to a 24-well plate to make the final EdU concentration in the 24-well plate 1X. For example, if the final concentration is designed to be 10 μM, make the final EdU concentration in the culture medium 10 μM. Continue incubating the cells for 2 hours.

[0049] (5) After EdU labeling of cells, remove the culture medium and add 1 ml of 4% paraformaldehyde and fix at room temperature for 15 minutes.

[0050] (6) Remove the fixative and wash the cells three times with 1 ml PBS per well, each time for 3-5 minutes.

[0051] (7) Incubate in PBS with 0.3% Triton X-100 at room temperature for 10-15 minutes.

[0052] (8) Prepare the Click Additive Solution by adding 0.5 ml of Click reaction solution to each well. Gently shake the culture plate to ensure that the reaction mixture evenly covers the sample. Incubate at room temperature in the dark for 30 minutes.

[0053] (9) Aspirate the Click reaction solution and wash three times with washing solution, each time for 3-5 minutes.

[0054] (10) DNA staining: Prepare an appropriate amount of 1×Hoechst 33342 reaction solution and store it in the dark. Then, add 100 μL of 1×Hoechst 33342 reaction solution to each well, place it on a decolorization shaker, incubate at room temperature in the dark for 30 min, and then discard the reaction solution. Finally, add 100 μL of PBS to each well and wash 1 to 3 times, 10 min each time.

[0055] (11) Image acquisition and analysis.

[0056] 4. Subcutaneous Tumor Administration in Mice Six nude mice were subcutaneously inoculated with 10 7 HCT116 cells (day 1). After tumors grew to a tumor volume (TV) of approximately 200-300 mm³, mice were randomly divided into two groups: a control group and a CX-6258.HCl group, which received daily abdominal injections (50 mg / kg). Tumors were measured daily using a vernier caliper. Antitumor activity was assessed using TV, estimated according to the following formula: TV = AB 2 = / 2 (where A and B are tumor length and width, respectively). At the end of the experiment, the mice were euthanized by cervical dislocation. All experiments involving animals were performed in accordance with the guidelines of the Arrivals and approved by the ethics committee (ethics number: 20250611006).

[0057] 5. HE staining (1) Place the tissue slide in xylene for 3 minutes each time, 3 times.

[0058] (2) Place in 100% ethanol for 3 minutes each time, 3 times.

[0059] (3) Place in 95% ethanol solution, 3 minutes each time, 3 times.

[0060] (4) Use hematoxylin to drop on the tissue until it is completely covered and stain for 3 minutes.

[0061] (5) Place the slide in running tap water for 10-15 minutes.

[0062] (6) Soak in eosin solution for 3 minutes.

[0063] (7) Soak in 95% ethanol three times, 3 minutes each time.

[0064] (8) Soak in 100% ethanol three times, 3 minutes each time.

[0065] (9) Soak the slides in xylene three times, 3 min each time, and dry them in a fume hood.

[0066] (10) Drop neutral resin on a glass slide and cover it with a coverslip. After the neutral resin dries, place it in a scanner and take a picture.

[0067] Example 1: High expression of H3pT3 can be used as a diagnostic indicator for colorectal cancer Ki-67 is a nuclear antigen associated with cell proliferation. Its expression level is closely related to tumor development, progression, invasion, metastasis, and prognosis. Ki-67 expression level can be used as a potential biomarker to assess the treatment response of patients with colorectal cancer.

[0068] The present invention collected normal tissues and tumor tissues from 40 patients (25 males and 15 females, aged 35 to 89 years) (the above normal tissues and tumor tissues were obtained from colorectal surgical specimens of patients with colorectal adenocarcinoma identified by pathological sections at West China Hospital of Sichuan University) and randomly divided them into a test set and a validation set (1:1). The present invention performed immunohistochemical testing on normal tissues and tumor tissues and counted the number of positive cells (the number of H3pT3-positive cells per 100 cells). It was found that H3T3 phosphorylation (H3pT3) was almost not expressed in normal tissues, but highly expressed in tumor tissues ( Figure 1 , Figure 2 H3T45 phosphorylation is almost not expressed in both tumors and normal tissues ( Figure 1 By counting the expression positions of H3pT3 and Ki67 (the number of cells with the same expression position of Ki67-positive and H3pT3-positive cells per 100 cells), it was found that the expression positions of H3pT3 and Ki67 were almost the same ( Figure 1 , Figure 3 ), indicating that H3pT3 can be used as a tumor-specific marker. The present invention further performed ROC statistics on the expression level of tissue H3pT3 (the number of H3pT3 positive cells per 100 cells), AUC = 0.9363, p = 0.0001 ( Figure 4 ).

[0069] These experimental results demonstrate that elevated H3pT3 expression can serve as a diagnostic marker for colorectal cancer. By measuring H3pT3 expression in tissues, the risk of colorectal cancer in individuals can be screened: high H3pT3 levels indicate a higher risk of colorectal cancer. This approach can be used as an auxiliary diagnosis for lung cancer, providing effective evidence for patient treatment decisions and promising clinical applications.

[0070] Example 2: H3pT3 inhibitor CX6258.HCl significantly inhibits the proliferation of colorectal cancer cell lines in vitro Compound CX6258.HCl, as a HASPIN inhibitor, can specifically inhibit the H3pT3 level in colorectal cancer cell lines HCT116 and HT29 ( Figure 5 The present invention uses the EdU method to detect the inhibitory level of CX6258.HCl on colorectal cancer cell lines and finds that CX6258.HCl has a concentration-dependent inhibitory effect on colorectal cell lines ( Figure 6 ).

[0071] Example 3: H3pT3 inhibitor CX6258.HCl significantly inhibits subcutaneous tumor progression in colorectal cancer mice Mice were injected subcutaneously with HCT116 cells and then intraperitoneally with 50 mg / kg CX6258.HCl. The tumor volume was measured daily. CX6258.HCl significantly inhibited the subcutaneous tumor volume in mice ( Figure 7 ) and weight ( Figure 8 HE staining and immunohistochemical analysis of tumor sections revealed that CX6258.HCl could significantly inhibit the malignancy of tumors ( Figure 9 ), and the expression of H3pT3 was downregulated after CX6258.HCl treatment ( Figure 10 At the same time, the present invention conducted statistics on the weight of mice and found that CX6258.HCl had no effect on the weight of mice ( Figure 11 HE staining showed that CX6258.HCl had no toxicity to the heart, liver, spleen, lung and kidney of mice ( Figure 12 ).

[0072] In summary, the present invention provides the use of reagents for detecting epigenetically modified H3T3 and the use of epigenetically modified H3T3 inhibitors. Unlike H3pT45, which is hardly expressed in both tumors and normal tissues, the present invention found that H3pT3 is hardly expressed in normal tissues but highly expressed in tumor tissues. Therefore, the high expression of H3pT3 in colorectal cancer can be used as a screening indicator, significantly improving the accuracy and early detection rate of screening, and providing a new marker for the early detection of colorectal cancer. In addition, the present invention also found that H3pT3 inhibitors can significantly inhibit the proliferation of colorectal cancer cell lines and significantly delay tumor progression in a mouse subcutaneous tumor model, showing good anti-tumor activity. These findings not only provide new biomarkers for the diagnosis of colorectal cancer, but also provide a strong basis for the development of new therapeutic drugs, which are expected to improve patients' prognosis and survival, and have important clinical application prospects.

Claims

1. Use of a reagent for detecting epigenetically modified H3T3 in preparing a colorectal cancer screening kit; the epigenetically modified H3T3 is H3pT3.

2. The use according to claim 1, characterized in that The reagent for detecting epigenetically modified H3T3 is an immunohistochemical detection reagent or an immunoblotting detection reagent.

3. Use of an epigenetically modified H3T3 inhibitor in the preparation of a medicament for preventing and / or treating colorectal cancer; the epigenetically modified H3T3 is H3pT3.

4. The use according to claim 3, characterized in that The epigenetic modification H3T3 inhibitor is CX6258 or a salt thereof.

5. The use according to claim 4, characterized in that The salt is hydrochloride.

6. The use according to claim 3, characterized in that The drug is a drug that inhibits the proliferation of cancer cells.

7. The use according to any one of claims 3 to 6, characterized in that The drug is a pharmaceutical preparation prepared by using an epigenetically modified H3T3 inhibitor as an active ingredient and adding pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that The pharmaceutical preparation is an oral preparation or an injection preparation.

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