Application of HDAC2 inhibitor in preparation of medicine for treating metastatic colorectal cancer with high expression of NUCKS1
Patent Information
- Application Number
- CN202411544214.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-31
- Publication Date
- 2025-01-21
Smart Images
Figure CN119326744A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of an HDAC2 inhibitor in the preparation of a drug for treating metastatic colorectal cancer with high expression of NUCKS1. Background Art
[0002] Colorectal cancer (CRC) is the third most common malignancy in the world and ranks second in cancer-related mortality, accounting for approximately 10% of global cancer morbidity and mortality (Morgan E, Gut, 2023;72:338-344). Radical tumor resection is currently the main treatment for colorectal cancer, but almost half of newly diagnosed patients have metastatic colorectal cancer (mCRC), and 80%-90% of them have unresectable liver metastases (Bray F, CA Cancer J Clin, 2018;68(6):394-424). Chemotherapy and targeted therapy are currently the main treatments. Fluorouracil, as a basic chemotherapy drug, combined with oxaliplatin or irinotecan can prolong the survival of patients with metastatic colorectal cancer (mCRC) (Cervantes A, Ann Oncol, 2023;34:10-32). The multi-targeted kinase inhibitor rapamycin has been approved for the treatment of patients with metastatic colorectal cancer (mCRC) who are resistant to standard chemotherapy (Grothey A, Cancer Treat Rev, 2020;86:101993). However, the efficacy of 5-fluorouracil combined with chemotherapy is less than 40%, and the efficacy of rapamycin is only 1.5%, and the 5-year survival rate of patients with metastatic colorectal cancer (mCRC) is still less than 20% (Grothey A, Cancer Treat Rev, 2020;86:101993; Lote H, Cancer Treat Rev, 2022;111:102480).
[0003] Therefore, for the treatment of metastatic colorectal cancer (mCRC), it is urgent to actively explore iconic tumor markers or target-specific targeted drugs to improve the anti-tumor treatment effect of colorectal cancer. At present, foreign researchers have found that lansoprazole, as a new type of HDAC2 inhibitor, can be used as a potential therapeutic drug for the treatment of colorectal cancer (CRC) (Saedeh Khadempar, Comput Biol Med, 2023:166:107518). Harbin Medical University evaluated and found that the combination of HDAC2 inhibitors (SCA) can enhance the sensitivity of colorectal cancer to anti-tumor therapy (Xin Guan, Clin Transl Med, 2024;14(6):e1692).
[0004] Santacruzamate A is a potent and selective HDAC2 inhibitor. Studies have shown that the combination of Santacruzamate A and PD-1 antibodies significantly slowed tumor growth and improved survival. However, for metastatic colorectal cancer (mCRC), no studies have shown the function and mechanism of HDAC2 inhibitors, and further studies have not been conducted on the function and effect of the potent and selective HDAC2 inhibitor Santacruzamate A in metastatic colorectal cancer (mCRC). Summary of the invention
[0005] Based on the above-mentioned research gaps in the prior art, the purpose of the present invention is to provide an application of an HDAC2 inhibitor in the preparation of a drug for treating metastatic colorectal cancer with high expression of NUCKS1.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention discloses for the first time the use of an HDAC2 inhibitor in preparing a medicine for treating metastatic colorectal cancer.
[0007] The "HDAC2 protein" described in the present invention is histone deacetylase 2, which is an important enzyme protein. The abnormal expression of HDAC2 protein is closely related to the occurrence and development of various diseases. For example, in tumors, the abnormal expression of HDAC2 may lead to abnormal proliferation of tumor cells and enhanced invasiveness.
[0008] Preferably, the drug is a drug for treating metastatic colorectal cancer with high expression of NUCKS1.
[0009] The "NUCKS1 (Nuclear ubiquitous casein and cyclin-dependentkinase substrate 1)" described in the present invention is a highly conserved DNA-binding protein associated with the cell cycle, located on human chromosome 1q32.1. NUCKS1 has multiple biological functions. For example, studies have revealed the carcinogenic mechanism of NUCKS1 in osteosarcoma and how it promotes tumor development and metastasis by upregulating the synthesis of aspartate. In addition, studies have explored the expression and mechanism of action of NUCKS1 in hepatocellular carcinoma, as well as its interaction with other non-coding small RNAs (such as miR-4461). These studies provide important clues for a deeper understanding of the biological functions and potential clinical applications of NUCKS1.
[0010] The "high expression" described in the present invention refers to the expression level of genes, proteins or other biological molecules in cells or tissues that is higher than the normal level or average expression level. High expression is closely related to the occurrence and development of various diseases. High expression of specific genes or proteins is often closely related to the malignancy, invasiveness and metastasis ability of tumors. By analyzing the expression level of specific genes or proteins in tumor tissues and drawing survival curves in combination with the patient's survival data, a strong basis can be provided for the diagnosis and treatment of tumors.
[0011] Further preferably, the drug is a drug that affects colorectal cancer metastasis by regulating HDAC2 protein expression through NUCKS1 via the lysosomal pathway.
[0012] The "lysosomal pathway" described in the present invention is one of the important pathways for the degradation of intracellular substances, and it involves a series of complex biochemical reactions and interactions between organelles. Lysosome is a kind of organelle in the cell, with a structure wrapped by a single membrane, and contains a variety of hydrolases inside, which can degrade a variety of substances inside and outside the cell. The lysosomal pathway can be divided into two major categories: non-selective lysosomal degradation and selective lysosomal degradation. The lysosomal pathway is one of the important pathways for the degradation of intracellular substances, and it involves a series of complex biochemical reactions and interactions between organelles. It plays an important role in the metabolism of intracellular substances and the maintenance of homeostasis, and is also closely related to the occurrence and development of various diseases.
[0013] Preferably, the HDAC2 inhibitor is Santacruzamate A.
[0014] The "HDAC2 inhibitor" described in the present invention is a class of compounds targeting histone deacetylase 2 (HDAC2), which can inhibit the activity of HDAC2, increase the acetylation level of histones and other proteins, and then regulate gene expression.
[0015] The "Santacruzamate A" described in the present invention, also known as CAY-10683, is a natural product and a highly effective and selective histone deacetylase (HDAC) inhibitor.
[0016] Further preferably, Santacruzamate A can significantly inhibit the metastasis of colorectal cancer caused by overexpression of NUCKS1.
[0017] More preferably, Santacruzamate A can significantly reduce liver metastasis and lung metastasis of colorectal cancer.
[0018] The present invention also discloses the application of NUCKS1 and HDAC2 in collaboration as biomarkers of metastatic colorectal cancer. NUCKS1 affects the metastasis of colorectal cancer by regulating the expression of HDAC2 protein through the lysosomal pathway.
[0019] Preferably, NUCKS1 promotes the invasion and metastasis of colorectal cancer cells, and HDAC2 inhibitors can inhibit colorectal cancer metastasis induced by NUCKS1 overexpression.
[0020] Preferably, NUCKS1 is capable of promoting the stability of HDAC2.
[0021] The present invention also discloses the application of NUCKS1 and HDAC2 in the preparation of a reagent / kit for diagnosing metastatic colorectal cancer.
[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses for the first time the use of HDAC2 inhibitors in the preparation of drugs for treating metastatic colorectal cancer (mCRC), especially HDAC2 inhibitors can treat metastatic colorectal cancer (mCRC) with high expression of NUCKS1. The present invention experimentally confirms that the group of patients with metastatic colorectal cancer (mCRC) on which the HDAC2 inhibitor Santacruzamate A works is a metastatic colorectal cancer with high expression of NUCKS1 molecules, and at the same time clarifies the new mechanism that NUCKS1 is highly expressed in metastatic colorectal cancer (mCRC), and NUCKS1 promotes the metastasis of colorectal cancer by positively regulating HDAC2. The present invention not only discloses for the first time new potential biomarkers and therapeutic targets for metastatic colorectal cancer (NUCKS1 and HDAC2 work synergistically), but also provides a new strategy for the clinical treatment of metastatic colorectal cancer (mCRC). BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1The results of NUCKS1 promoting the metastasis of colorectal cancer cells are shown in Figure 1. a is the expression level of NUCKS1 in metastatic and non-metastatic colorectal cancer analyzed by the HCMDB database; b and c are the protein expression levels detected by Western blotting when NUCKS1 is knocked down or overexpressed in colorectal cancer cells; d is the result of Transwell experiment detecting the migration and invasion of colorectal cancer cells HCT116 when NUCKS1 is knocked down; e is the result of Transwell experiment detecting the migration and invasion of colorectal cancer cells SW480 when NUCKS1 is overexpressed; Figure 2 NUCKS1 enhances the invasion and metastasis of colorectal cancer cells through HDAC2. Among them, a is a heat map showing that NUCKS1-silenced HCT116 colorectal cancer cells have 28 differentially expressed proteins compared with control cells; b is Western blotting to detect the expression of HDAC2 protein levels when colorectal cancer cells knock down or overexpress NUCKS1; c is Western blotting to detect the changes in HDAC2 protein levels when colorectal cancer cells HCT116 knock down NUCKS1 and overexpress HDAC2 or SW480 colorectal cancer cells overexpress NUCKS1 and knock down HDAC2; d is the result of Transwell experiment to detect the migration and invasion of colorectal cancer cells HCT116 under different treatment conditions; e is the result of Transwell experiment to detect the migration and invasion of colorectal cancer cells SW480 under different treatment conditions; Figure 3 The effect of HDAC2 inhibitor Santacruzamate A on colorectal cancer cells overexpressing NUCKS1. Among them, a is the result of Transwell experiment to detect cell migration and invasion when Santacruzamate A treated colorectal cancer cells SW480 overexpressing NUCKS1; b is laser confocal detection of FITC phalloidin staining of F-actin in SW480 cells overexpressing NUCKS1 treated with Santacruzamate A; Figure 4 The results of the interaction between NUCKS1 and HDAC2 are shown in Figure 1. a is the interaction between NUCKS1 and HDAC2 detected by Co-IP and Western blotting; b is the co-localization of NUCKS1 and HDAC2 by immunofluorescence analysis by laser confocal microscopy; c is the statistical result of the mean fluorescence intensity (MFI) of NUCKS1 and HDAC2; Figure 5NUCKS1 affects the stability of HDAC2 protein. Among them, a is the q-PCR detection of the expression of HDAC2 RNA level in HCT116 cells with NUCKS1 knockdown and SW480 colorectal cancer cells with NUCKS1 overexpression; b is the Western blotting detection of HDAC2 protein level in HCT116 cells with NUCKS1 knockdown and control cells after treatment with the protein synthesis inhibitor cycloheximide (CHX) for 8, 24 and 48 hours; Figure 6 NUCKS1 regulates the protein level of HDAC2 through the lysosomal pathway. Among them, a is the effect of knocking down NUCKS1 on the protein level of HDAC2 when treated with the lysosomal inhibitor chloroquine (CQ) by Western blotting; b is the effect of knocking down NUCKS1 on the expression of HDAC2 protein when treated with the proteasome inhibitor MG132 by Western blotting; c, d are the immunofluorescence co-expression of HDAC2, LC3B and LAMP2 analyzed by laser confocal microscopy when NUCKS1 is overexpressed; e is the statistical result of the mean fluorescence intensity (MFI) of HDAC2, LC3B and LAMP2; Figure 7 The results of animal-level verification that HDAC2 inhibitors can significantly reduce liver and lung metastasis of colorectal cancer. Among them, a is a display of liver and lung metastasis nests of mice in four different treatment groups; b is HE staining of liver and lung in the corresponding group; c is the statistical results of liver and lung metastasis nests of four groups of mice. DETAILED DESCRIPTION
[0024] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.
[0025] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.
[0026] The present invention is further described in detail below in conjunction with the accompanying drawings: Santacruzamate A (also known as CAY-10683) used in the following examples of the present invention is a natural product and a highly effective and selective histone deacetylase (HDAC) inhibitor. Its CAS number is 1477949-42-0; molecular formula: C 15 H 22 N2O3, molecular weight 278.35, was purchased from MCE (MedChemExpress) with the catalog number HY-N0931.
[0027] The Transwell experiment used in the following embodiments of the present invention is mainly used to study the movement mechanism of cells on porous membranes, especially the migration and invasion ability of cells. The Transwell experiment divides the culture system into two compartments, upper and lower, by placing a Transwell chamber (an insert containing a polycarbonate membrane) into a culture plate. The polycarbonate membrane has a certain pore size and permeability, so that the components in the lower culture solution can affect the cells in the upper chamber. The cells are placed in the upper chamber, and driven by external signals (such as growth factors, chemokines or extracellular matrix components), the cells will migrate through the membrane to the high-concentration culture medium or perform invasive behavior. Depending on whether matrigel is added in the chamber, the Transwell experiment can be divided into two types: migration assay and invasion assay. 1. Transwell cell migration and invasion assay To prove whether the NUCKS1 gene promotes the invasion and metastasis of colorectal cancer, the present invention first used the Human Cancer Metastasis Database (HCMDB) to analyze the expression of NUCKS1 in metastatic and non-metastatic colorectal cancer. Database GSE14095 showed that NUCKS1 was highly expressed in metastatic colorectal cancer ( Figure 1Then, colorectal cancer cells with stable knockdown or overexpression of NUCKS1 were constructed by lentiviral packaging and infection. Western blotting experiments proved that NUCKS1 was successfully knocked down or overexpressed ( Figure 1 b and c). Therefore, the present invention shows through Transwell experiments that knocking down NUCKS1 significantly reduces the invasion and metastasis of colorectal cancer cells, while overexpressing NUCKS1 significantly increases the invasion and metastasis of colorectal cancer cells ( Figure 1 The above results indicate that NUCKS1 promotes the invasion and metastasis of colorectal cancer cells. The specific experimental procedures are as follows: Transwell experiments used chambers with 8 μm pores (BD Biosciences). For cell migration experiments, cells were resuspended in 200 μL serum-free medium and inoculated into the chambers, while 500 μL medium containing 10% FBS was added to the 24-well plate on the outside of the chambers. For cell invasion experiments, matrix gel (BD Biosciences) was diluted 1:20 with serum-free medium, 50 μL was added to the chambers, and then placed in a 37°C incubator for 4 h to solidify. Cells resuspended in 200 μL serum-free medium were added to the upper layer of matrix gel, while 500 μL medium containing 10% FBS was added to the 24-well plate on the outside of the chambers. After 24 h, cells were fixed with 95% ethanol for 15 min, then stained with 1% crystal violet for 30 min, rinsed with tap water, and gently wiped with a cotton swab to remove cells on the inner surface of the chamber. The experimental results were observed using an inverted microscope, and the number of migrated or invaded cells in five randomly selected fields of view was counted using ImageJ software.
[0028] 2. Western blotting To elucidate the molecular mechanism by which NUCKS1 promotes the invasion and metastasis of colorectal cancer cells, the present invention used label-free proteomics technology to identify NUCKS1-mediated differentially expressed proteins in colorectal cancer cells in the NUCKS1 knockdown group and the control group. Through literature screening and GEPIA analysis, 28 differentially expressed proteins that were significantly correlated with NUCKS1 and highly expressed in colorectal cancer cells were identified ( Figure 2 Western blotting experiments showed that when NUCKS1 was knocked down, the HDAC2 protein level was reduced, while when NUCKS1 was overexpressed, the HDAC2 expression level was increased ( Figure 2b). Subsequently, Western blotting experiments showed that overexpression of HDAC2 in NUCKS1-knockdown colorectal cancer cells HCT116 would increase the expression level of HDAC2 again, while knockdown of HDAC2 in NUCKS1-overexpressing colorectal cancer cells SW480 would significantly reduce HDAC2 expression ( Figure 2 Transwell experiments showed that the reduction in invasion and metastasis of HCT116 cells caused by NUCKS1 knockdown was reversed by HDAC2 overexpression ( Figure 2 Middle d), NUCKS1 overexpression promoted invasion and metastasis in SW480 cells, which was inhibited by HDAC2 knockdown ( Figure 2 The specific experimental operation is as follows: RIPA lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor was prepared, cells were scraped on ice, and fully lysed for 30 min. Total protein was extracted and protein quantification was performed by BCA method.
[0029] Prepare SDS-PAGE gel and load about 40 µg protein / well. Perform electrophoresis at 80 V for stacking gel and 100 V for separation gel. Transfer to membrane at 4 ℃ and 200 mA for 2 h. Prepare 5% skim milk in TBS and block at room temperature for 2 h. Dilute the antibody with primary antibody diluent, dilute the target antibody at 1:1000, and dilute the internal reference at 1:2000, and incubate at 4 ℃ overnight. Wash the membrane 3 times with 1×TBST, 10 min / time. Dilute the secondary antibody with 1×TBST 1:4000 and incubate at room temperature for 1 h. Wash the membrane 3 times with 1×TBST, 10 min / time. Observe protein bands by chemiluminescence. Grayscale analysis of protein bands was performed by ImageJ software. The experiment was repeated at least three times.
[0030] The result graphs corresponding to this experimental method include Figure 1 b and c; Figure 2 b and c; Figure 5 Middle b and Figure 6 a and b. Figure 5 In Figure b, before harvesting cells for lysis, cycloheximide (CHX), which inhibits protein synthesis, was added at a concentration of 20 µg / mL for different time periods, i.e., 8 h, 24 h, and 48 h. Figure 6 In a, 20 µM of the autophagosome inhibitor Chloroquine (CQ) was added for 4 h before the cells were harvested for lysis. Figure 6 In center b, 5 µM proteasome inhibitor MG132 was added for 4 h before cells were harvested for lysis.
[0031] 3. Cell immunofluorescence and laser confocal microscopy Santacruzamate A is an HDAC2 inhibitor. To further demonstrate that NUCKS1 affects the invasion and metastasis of colorectal cancer by regulating HDAC2, the Transwell experiment showed that when NUCKS1-overexpressing colorectal cancer cells SW480 were treated with Santacruzamate A, the invasion and metastasis of colorectal cancer induced by NUCKS1 overexpression were significantly inhibited ( Figure 3 (a). Subsequently, immunofluorescence experiments of F-actin demonstrated that Santacruzamate A significantly reduced the distribution of cardiac myosin filaments in NUCKS1-overexpressing cells SW480 compared with untreated cells ( Figure 3 (b)
[0032] The specific experimental operations are as follows: Take 2×10 4Logarithmic phase colorectal cancer cells were plated on confocal dishes, and the supernatant was removed overnight, washed with 1×PBS for 3 min×3 times, fixed with 4% paraformaldehyde for 15 min, washed with 1×PBS for 3 min×3 times, permeabilized with 0.5% Triton X-100 for 20 min, washed with 1×PBS for 3 min×3 times, blocked with 1% BSA for 30 min, incubated with primary antibody at 4°C overnight, washed with 1×PBS for 3 min×3 times, incubated with fluorescent secondary antibody at room temperature in the dark for 1 h, washed with 1×PBS for 3 min×3 times, and added with anti-fluorescence quencher. The expression and localization of NUCKS1 and HDAC2 were observed under laser confocal microscopy. The mean fluorescence intensity (MFI) was calculated using ImageJ software. The primary antibodies used included NUCKS1 (catalog# 12023-2-AP, Proteintech) at a dilution of 1:400; HDAC2 (catalog# 67165-1-Ig, Proteintech) at a dilution of 1:800; HDAC2 (catalog# ET1607-78, HUABIO) at a dilution of 1:100; LC3B (catalog# 81004-1-RR, Proteintech) at a dilution of 1:1000; and LAMP2 (catalog# M1603-5, HUABIO) at a dilution of 1:100. The fluorescent secondary antibodies used included: anti-rabbit IgG (H+L) Alexa FluorTM 594 (catalog# A-21207, Thermo Fisher) diluted 1:500; anti-rabbit IgG (H+L) Alexa FluorTM 488 (catalog# A-21206, Thermo Fisher) diluted 1:500; anti-mouse IgG (H+L) Alexa FluorTM 594 (catalog# A-21203, ThermoFisher) diluted 1:500; anti-mouse IgG (H+L) Alexa FluorTM 488 (catalog# A-21202, Thermo Fisher) diluted 1:500. For FITC-phalloidin staining of F-actin, TraKind™ F-actin Staining Kit (Abbkine, Wuhan, China) was used, and the cells were observed under a confocal microscope.
[0033] 4. Co-immunoprecipitation (Co-IP) To demonstrate the interaction between NUCKS1 and HDAC2, total proteins of HCT116 and SW480 cells were immunoprecipitated using HDAC2 antibody. Co-IP and Western blotting experiments showed that NUCKS1 was co-immunoprecipitated ( Figure 4 Cell immunofluorescence experiments showed that NUCKS1 and HDAC2 co-localized ( Figure 4 The statistical results of mean fluorescence intensity (MFI) showed that the MFI of NUCKS1 and HDAC2 in NUCKS1 overexpressing cells were significantly higher than those in control cells ( Figure 4 (c).
[0034] The specific experimental operations are as follows: Wash the cells twice with pre-cooled PBS and remove the supernatant. Add 1mL of pre-cooled IP buffer (with protease and phosphatase inhibitors) and scrape the cells from the culture dish with a pre-cooled cell scraper, transfer the suspension to an EP tube, and rotate and mix at 4℃ for 30 min. Centrifuge at 14000 g for 15 min at 4℃, and immediately transfer the supernatant to a new EP tube. Take out 100 µL of supernatant as the input positive control. Add 30 µl of Protein A / G agarose and 1µg of IgG to the remaining supernatant, shake at 4℃ for 3 h, remove Protein A / G agarose, and transfer the supernatant to a new EP tube. Add 2 µg of rabbit IgG to the control group and 2 µg of anti-HDAC2 antibody to the experimental group, and incubate overnight at 4℃ with rotation and mixing. Add 40 μL Protein A / G agarose to capture the antigen-antibody complex, shake slowly at 4°C for 6 h, collect the agarose bead-antigen-antibody complex using a magnetic rack, remove the supernatant, wash 3 times with pre-cooled PBS, resuspend the agarose bead-antigen-antibody complex in 40 μL 2× loading buffer, and gently mix. Denature the loaded sample by boiling for 5 min, place at -20°C, and leave for electrophoresis, and boil again for 5 min before electrophoresis.
[0035] 5. Quantitative real-time PCR (q-PCR) q-PCR detection showed that when NUCKS1 was knocked down or overexpressed in colorectal cancer cells, the RNA level of HDAC2 did not change ( Figure 5 When HCT116 control or NUCKS1 knockdown cells were treated with the protein synthesis inhibitor cycloheximide (CHX), the HDAC2 protein stability expressed by the control cells was higher than that of the NUCKS1 knockdown HCT116 cells, indicating that NUCKS1 promotes the stability of HDAC2 ( Figure 5 b). The specific experimental operation is as follows: Cells were collected, washed twice with PBS, and the supernatant was removed. RNA was extracted from the cell pellet using the M5 Universal RNA Mini Kit (Mei5Bio, Beijing, China). cDNA was synthesized using the Evo M-MLV RT Mix Kit (Accurate Biotechnology, Changsha, China). q-PCR was performed using the SYBR Green PremixPro Taq HS qPCR Kit (Accurate Biotechnology, Changsha, China). All experimental procedures were performed according to the kit instructions. The primer sequences are shown in Table 1 below.
[0036] Table 1
[0037] 6. Cell immunofluorescence experiment To explore the protein degradation pathway of HDAC2 regulated by NUCKS1, Western blotting experiments showed that the lysosomal inhibitor chloroquine (CQ) inhibited the degradation of HDAC2 mediated by NUCKS1 silencing ( Figure 6 proteasome inhibitor MG132 had no effect ( Figure 6 (middle b), indicating that NUCKS1 regulates HDAC2 in a lysosome-dependent manner. Cell immunofluorescence experiments showed that NUCKS1 overexpression significantly increased the MFI of HDAC2 and reduced the distribution of autophagy and lysosome markers LC3B and LAMP2 ( Figure 6 (c, d, e).
[0038] 7. Animal Experimentation To verify the effect of NUCKS1 on colorectal cancer metastasis in vivo, a nude mouse tumor metastasis model was constructed by tail vein injection of tumor cells. Four weeks later, compared with the control group, NUCKS1 overexpression significantly promoted liver and lung metastasis, while HDAC2 knockdown significantly inhibited NUCKS1 overexpression-induced metastasis. In addition, intraperitoneal injection of the HDAC2 inhibitor Santacruzamate A led to reduced liver and lung metastasis compared with the NUCKS1 overexpression group. This reduction was observed in the liver and lung images of the four groups of nude mice ( Figure 7 (a) and the number of metastatic lesions in the liver and lungs ( Figure 7In addition, hematoxylin-eosin (HE) staining of liver and lung metastases showed that there were more and larger metastatic nests in nude mice injected with SW480 cells overexpressing NUCKS1 compared with the control group. However, the number and size of metastatic nests were significantly reduced in the NUCKS1 overexpression and HDAC2 knockdown group or the Santacruzamate A treatment group ( Figure 7 b). The specific experimental operation is as follows: Female nude mice (6-8 weeks old, 18-22 g) were provided by the Animal Center of Air Force Medical University and randomly divided into four groups (n = 6 in each group): control group, NUCKS1 overexpression group, NUCKS1 overexpression and HDAC2 knockdown group, and NUCKS1 overexpression and intraperitoneal administration of HDAC2 inhibitor Santacruzamate A group. Each mouse was injected with 1×10 6 Four weeks later, the mice were euthanized, and lung and liver tissues were collected, embedded in paraffin, and sections were stained with hematoxylin and eosin using a hematoxylin and eosin staining kit (Beyotime, Shanghai, China). The HDAC2 inhibitor Santacruzamate A (Sellechem) was administered once a day at a dose of 25 mg / kg by intraperitoneal injection for three weeks, with one day off per week. All experimental procedures were approved by the Institutional Animal Ethics and Use Committee of the Second Affiliated Hospital of Air Force Medical University (Tangdu Hospital) (No. 20230743).
[0039] In summary, the present invention illustrates from the cell and animal experimental levels that the HDAC2 inhibitor Santacruzamate A can significantly inhibit the metastasis of colorectal cancer caused by overexpression of NUCKS1. At the same time, based on the experimental results, the present invention proposes for the first time new biomarkers for metastatic colorectal cancer, namely NUCKS1 and HDAC2. More importantly, the function of the HDAC2 inhibitor Santacruzamate A in metastatic colorectal cancer with high expression of NUCKS1 is verified. The preclinical studies involved in the present invention fully demonstrate the inhibitory effect of Santacruzamate A on metastatic colorectal cancer.
[0040] The above contents are only for explaining the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.
Claims
1. Application of HDAC2 inhibitors in the preparation of drugs for the treatment of metastatic colorectal cancer.
2. The use according to claim 1, characterized in that The drug is a drug for treating metastatic colorectal cancer with high expression of NUCKS1.
3. The use according to claim 2, characterized in that The drug is a drug that affects colorectal cancer metastasis by regulating HDAC2 protein expression through NUCKS1 via the lysosomal pathway.
4. The use according to claim 1, characterized in that The HDAC2 inhibitor is Santacruzamate A.
5. The use according to claim 4, characterized in that Santacruzamate A can significantly inhibit the metastasis of colorectal cancer caused by overexpression of NUCKS1.
6. The use according to claim 4, characterized in that Santacruzamate A can significantly reduce liver and lung metastases of colorectal cancer.
7. Use of NUCKS1 and HDAC2 as biomarkers for metastatic colorectal cancer, characterized in that: NUCKS1 affects colorectal cancer metastasis by regulating HDAC2 protein expression through the lysosomal pathway.
8. The use according to claim 7, characterized in that NUCKS1 promotes the invasion and metastasis of colorectal cancer cells, while HDAC2 inhibitors can inhibit colorectal cancer metastasis induced by NUCKS1 overexpression.
9. The use according to claim 7, characterized in that NUCKS1 promotes the stability of HDAC2.
10. Use of NUCKS1 and HDAC2 in the preparation of reagents / kits for diagnosing metastatic colorectal cancer.