Application of P22phox as drug for screening pancreatic cancer liver metastasis target molecule or NOX inhibitor in preparation of pancreatic cancer liver metastasis drug

Abstract

The invention belongs to p22phox, and particularly relates to application of P22phox as a drug for screening pancreatic cancer liver metastasis target molecules or an NOX inhibitor in preparation of pancreatic cancer liver metastasis drugs. The technical problem to be solved by the invention is to provide a new molecular target for pancreatic cancer liver metastasis. The expression level of P22phox in a pancreatic cancer liver metastasis patient sample is higher than that of a pancreatic cancer primary sample, and pancreatic cancer cell migration and pancreatic cancer liver metastasis can be effectively inhibited by inhibiting p22phox. The invention provides a new thought for further researching the pancreatic cancer liver metastasis mechanism and the medical application of p22phox.

Description

Technical Field

[0001] The present invention belongs to p22 phox , specifically involving P22 phox Application of a target molecule for pancreatic cancer liver metastasis or a NOX inhibitor in the preparation of a drug for pancreatic cancer liver metastasis Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with the lowest survival rate in the world, with less than 10% of PDAC patients having a 5-year survival rate. PDAC is usually diagnosed at an advanced stage, and current treatments have significant limitations, partly because PDAC patients have a very high rate of distant metastasis when they are first diagnosed. The most common site of distant metastasis of PDAC is the liver, and the 5-year survival rate of these patients is only 3%. The tumor microenvironment (TME) of pancreatic cancer is extremely complex, consisting of interactions between multiple types of cells (including malignant cells, immune cells, and stromal cells), which ultimately form an inflammatory fibrotic tumor microenvironment that supports distant metastasis of the tumor. Pancreatic cancer progresses insidiously, with approximately 80% of patients having metastases at the time of diagnosis, and the liver is the most common site of metastasis (accounting for 50%-70%). Liver metastases may be small in the early stages, and imaging examinations such as CT, MRI, or ultrasound may be difficult to detect, resulting in late stage diagnosis, affecting the timing of treatment. Surgical resection is the only possible cure, but when pancreatic cancer metastasizes to the liver, the metastases are often multiple or located in a location that is not suitable for surgery. In addition, pancreatic cancer has a low response rate to chemotherapy and radiotherapy, and commonly used chemotherapy regimens such as FOLFIRINOX or gemcitabine combination therapy have limited effects and large side effects. Targeted therapy and immunotherapy are not as effective in pancreatic cancer as other cancers, possibly because the tumor microenvironment suppresses the immune response or lacks effective targets. For example, gene mutations such as KRAS and TP53 are common, and the tumor microenvironment is dominated by dense fibrous stroma, which hinders drug penetration and inhibits the activity of immune cells. Even after treatment, residual cancer cells may be highly resistant and prone to recurrence. Pancreatic cancer has rich interstitial components, which may hinder drug delivery, resulting in poor treatment effects. At the same time, tumor heterogeneity is high, and different metastatic lesions may respond differently to treatment, making it difficult for a single treatment to be effective. Therefore, pancreatic cancer liver metastasis is a technical problem that needs to be solved urgently in this field.

[0003] CYBA encodes p22 phox Protein, existing research has found that p22 phox The main focus is on its role in phagocytes, where it is a key component of superoxide-producing nicotinamide adenine dinucleoside phosphate oxidases (NOXs), which are responsible for killing microorganisms during bacterial and fungal infections. Abnormal expression of p22phox is associated with a variety of inflammatory diseases and cancers, but p22phox It is not yet clear whether it plays a role in tumor metastasis. Summary of the invention

[0004] Purpose of the invention:

[0005] The technical problem to be solved by the present invention is to provide a new molecular target for pancreatic cancer liver metastasis. phox The expression level in samples of pancreatic cancer liver metastasis patients is higher than that in primary pancreatic cancer samples, inhibiting p22 phox It can effectively inhibit pancreatic cancer cell migration and pancreatic cancer liver metastasis. The present invention is to further study the mechanism of pancreatic cancer liver metastasis and p22 phox It provides new ideas for its medical use.

[0006] The present invention is intended to provide p22 phox The application of pancreatic cancer liver metastasis and NOX inhibitors in the disease can solve the problem of lack of drugs for treating pancreatic cancer liver metastasis in the prior art.

[0007] Technical Solution

[0008] The application of CYBA as a target molecule for drug screening of pancreatic cancer liver metastasis or CYBA itself in the preparation of drugs for pancreatic cancer liver metastasis can achieve anti-pancreatic cancer liver metastasis by knocking down CYBA, that is, by the interference sequence of CYBA.

[0009] P22 phox Application of NOX inhibitors as drug screening target molecules for pancreatic cancer liver metastasis or in the preparation of drugs for pancreatic cancer liver metastasis

[0010] p22 phox Application in the preparation of reagents for diagnosing pancreatic cancer liver metastasis

[0011] The application is characterized in that the drug is the NOX inhibitor GSK2795039.

[0012]

[0013] GSK2795039.

[0014] Further, the p22 phox The application of the method in the preparation of a reagent for diagnosing pancreatic cancer liver metastasis is characterized in that the p22 phox The transcription level and protein expression level of pancreatic cancer liver metastasis are increased.

[0015] Further, the p22 phox The application of the invention in the preparation of a drug for treating pancreatic cancer liver metastasis is characterized in that the drug is the NOX inhibitor GSK2795039.

[0016] Further, the p22 phox The use of the invention in the preparation of a drug for treating pancreatic cancer liver metastasis, characterized in that the p22 phox Protein as a biomarker for pancreatic cancer liver metastasis.

[0017] Beneficial Effects

[0018] Although many NOX enzyme inhibitors have shown the potential to inhibit ROS generation and cancer cell activity in the laboratory, some compounds may not be effective against cancer metastasis while inhibiting NOX enzymes. The reasons are: Complexity of cancer metastasis: Cancer metastasis involves multiple mechanisms (such as EMT, angiogenesis, immune escape, etc.), and inhibiting NOX enzymes alone may not be sufficient to block all metastatic pathways. Diverse effects of ROS: The effects of ROS may be different in different cancer types and stages, and inhibiting NOX enzymes may not completely block other sources of ROS in some cases. Drug resistance: Cancer cells may bypass NOX enzyme-dependent metastasis mechanisms by activating other signaling pathways (such as PI3K / AKT, MAPK, etc.). Insufficient compound specificity: Some NOX enzyme inhibitors may not be selective enough for specific NOX subtypes (such as NOX1, NOX4), resulting in limited effects. For example: Apocynin mechanism of action: Inhibits NOX2 assembly, reduces ROS generation, and has weak inhibitory effects on NOX1 and NOX4. In some cancer models, metastasis may not be completely blocked. DPI (diphenyliodonium) mechanism of action: Broad-spectrum NOX enzyme inhibitor, inhibits electron transfer. Lack of specificity, may affect other oxidoreductases. May produce cytotoxicity at high concentrations, limiting its clinical application. Mechanism of action of GKT137831: Selective inhibition of NOX1 and NOX4. In some cancer types, NOX2 or NOX5 may play a major role, resulting in limited inhibitory effect on metastasis. No significant anti-metastatic effect was shown in clinical trials. Mechanism of action of VAS2870: Inhibition of NOX enzyme activity. Poor stability and specificity, may affect other signaling pathways. In in vivo experiments, the anti-metastatic effect was not obvious. Some NOX enzyme inhibitors (such as GKT137831) did not show significant anti-metastatic effects in clinical trials.

[0019] CYBA codep 22phox The protein is a key subunit of the NADPH oxidase complex and a key component of superoxide-producing nicotinamide adenine dinucleoside phosphate oxidases (NOXs), which is mainly involved in the generation of reactive oxygen species (ROS). 22phox In phagocytes, p22 is responsible for killing microorganisms during bacterial and fungal infections. phox Abnormal expression is associated with a variety of inflammatory diseases and cancers, but there is no literature report on whether it is related to pancreatic cancer liver metastasis.

[0020] Pancreatic cancer is the "king of cancers", especially pancreatic cancer liver metastasis is a technical problem that needs to be solved urgently in this field, and there is currently a lack of treatment methods. The present invention first discovered CYBA or P22 phox Application of the target molecule for pancreatic cancer liver metastasis or NOX inhibitor in the preparation of drugs for pancreatic cancer liver metastasis.

[0021] Specifically:

[0022] The present invention analyzes the public database and finds and verifies p22 phox The expression abundance in pancreatic cancer liver metastasis samples was significantly increased compared with that in primary pancreatic cancer samples. phox Lentivirus knockdown and overexpression stable transfection system found that knockdown of p22 phox Overexpression of p22 can inhibit pancreatic cancer cell migration phox In nude mice, a pancreatic cancer spleen injection liver metastasis model was established and combined with luciferase imaging, it was found that p22 phox Knockdown of p22 can inhibit liver metastasis of pancreatic cancer. phox As a key subunit of NOX, we found that the NOX inhibitor GSK2795039 can inhibit the migration of pancreatic cancer cells at the cellular level in the nude mouse liver metastasis model.

[0023] Compared with existing technologies, the innovation lies in:

[0024] The present invention first discovered p22 phox Highly expressed in pancreatic cancer liver metastasis samples. P22 phox Knockout of p22 can inhibit pancreatic cancer cell migration and liver metastasis, indicating that p22 phox It is a powerful potential therapeutic target for pancreatic cancer liver metastasis. Currently, radiotherapy and chemotherapy are usually used for pancreatic cancer liver metastasis in clinic, but the improvement effect is limited and the recurrence rate is high. The inhibitor provided by the present invention can significantly improve pancreatic cancer liver metastasis, which is a good opportunity for further research on the mechanism of pancreatic cancer liver metastasis and p22 phox The function of proteins provides new ideas. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is Example 1, screening of malignant metastatic epithelial cells of pancreatic cancer, where A and B integrate multi-sample single-cell transcriptome data, C and D are UMAP maps of pancreatic cancer epithelial cells, E is the top gene and pathway enrichment analysis of each epithelial cell subpopulation, and F is the survival rate analysis of the top genes of E2 and E6 subpopulations.

[0026] Figure 2 Identification of p22 for Example 2 phoxIt is highly expressed in pancreatic cancer liver metastasis tissue and promotes pancreatic cancer cell migration. A is p22 phox Screening intersection. B is p22 based on TCGA database phox The survival rate analysis of C is the immunohistochemical results. The pathological sections were subjected to immunohistochemical experiments and the results showed that p22 phox The protein is highly expressed in pancreatic cancer liver metastasis tissue samples. D and E are p22 in pancreatic cancer cells. phox WB and immunofluorescence showed protein expression and distribution. G and H showed the expression of p22 in pancreatic cancer cells. phox Knockdown and overexpression of p22 in pancreatic cancer cells. phox Transwell migration assay of knockdown and overexpression.

[0027] Figure 3 Example 3p22 phox Knockdown can inhibit liver metastasis of pancreatic cancer, where A is the experimental design flow chart, B is the in vivo fluorescence imaging of nude mice, C is the fluorescence intensity statistics, D is the weight change of nude mice in each group, and E is the representative pictures of the liver of nude mice in each group and HE staining pictures.

[0028] Figure 4 This is Example 4: NOX inhibitors can inhibit pancreatic cancer cell migration. A shows the effect of NOX inhibitors on BxPC3 cell viability; B and C show the Transwell and cell scratch experiments of NOX inhibitors on pancreatic cancer cell migration; D and E show the WB and immunofluorescence results of NOX inhibitors on PI3K-AKT and pMLC2 signaling pathways.

[0029] Figure 5 Example 5: NOX inhibitors can inhibit pancreatic cancer liver metastasis. A is the fluorescence in vivo imaging and ex vivo imaging of the liver of each group of nude mice, B is the fluorescence intensity statistical graph of the in vivo imaging of nude mice, C is the fluorescence intensity statistical graph of the ex vivo imaging of the liver, and D is the representative images of the liver of each group of nude mice and the HE staining images and the relative area statistics of the tumor area, wherein N is the normal liver tissue area, and T is the tumor tissue area. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0031] Example 1: Screening of pancreatic cancer malignant metastatic epithelial cells

[0032] 1. Experimental Methods

[0033] 1.1 Integration of scRNA-seq data of normal pancreatic tissue (NT), primary pancreatic cancer tissue (PT), and pancreatic cancer liver metastasis tissue (HM)

[0034] In order to study the pancreatic cancer tumor microenvironment and the molecular targets associated with pancreatic cancer liver metastasis, we first downloaded the single-cell transcriptome data of GSE154778, GSE197177 and GSA: CRA001160, and integrated them through CCA, followed by PCA dimensionality reduction, cell clustering and nonlinear dimensionality reduction.

[0035] 1.2 Analysis of epithelial cell heterogeneity

[0036] PDAC originates from epithelial cells. We further clustered and reduced the dimensions of pancreatic cancer epithelial cells into 7 subgroups, and counted the proportion of cells in the three groups of NT, PT and HM in each subgroup. Heatmap was used to draw a heat map of the top 50 genes in each subgroup, combined with GO enrichment analysis and TCGA database survival analysis.

[0037] 2. Experimental results

[0038] We integrated the scRNA-seq data of 61 samples from GSE154778, GSE197177 and GSA:CRA001160 datasets ( Figure 1 A), including 12 normal pancreatic samples, 37 primary pancreatic cancer samples, and 12 pancreatic cancer liver metastasis samples, of which the number of cells in the normal group was 33005, the number of cells in the primary group was 83148, and the number of cells in the metastasis group was 40937. Subsequently, the cells were classified into 12 subgroups by dimensionality reduction cluster analysis, including epithelial cells, secretory cells, fibroblasts, endothelial cells, monocytes / macrophages, neutrophils, mast cells, T lymphocytes, NK cells, plasma cells, and B lymphocytes ( Figure 1 B).

[0039] Through re-clustering analysis, epithelial cells were divided into 7 subgroups, and cell annotation was performed based on the highly expressed differentially expressed genes in each subgroup. The annotation results included TFF1 + Epi, AMBP + Epi, MALAT + Epi, PRSS1 + Epi, SPP1 + Epi, CXCL8 + Epi, LDHA + Epi( Figure 1 C). Cell ratio analysis ( Figure 1 D), we found that the epithelial cell subpopulations in normal tissues mainly expressed AMBP + Epi and CXCL8+ Epi, pancreatic cancer primary epithelial cells mainly in TFF1 + Epi, SPP1 + Epi and LDHA + Epi, while pancreatic cancer liver metastasis group in MALAT + Epi and PRSS1 + We then selected the top 50 genes in each subgroup for GO enrichment analysis, among which TFF1 + Epi cell subsets are mainly involved in tissue homeostasis and the maintenance of epithelial cell structure, and are more inclined to normal tissue epithelial cells ( Figure 1 E). Through the survival analysis of the integrated Top50 genes, it was found that ( Figure 1 F), MALAT + Epi and LDHA + High expression of Top genes in the two epithelial cell groups indicates poor prognosis, among which MALAT + Epi accounted for a higher proportion in the metastatic group and was defined as a subpopulation of malignant epithelial cells in pancreatic cancer liver metastasis, while LDHA + Epi accounted for a higher proportion in the primary group and was defined as a subpopulation of primary malignant epithelial cells in pancreatic cancer.

[0040] Example 2: P22 phox Highly expressed in pancreatic cancer liver metastasis tissues and promotes pancreatic cancer cell migration

[0041] 1. Experimental Methods

[0042] 1.1 Immunohistochemistry

[0043] Dewaxing and hydration of tissue paraffin sections: put the sections into xylene for 30 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, and 75% ethanol for 5 minutes, and then wash with distilled water. Antigen repair was performed with citric acid repair buffer, endogenous peroxidase was blocked with 3% hydrogen peroxide solution, and 3% goat serum was blocked at room temperature for 30 minutes. Incubate the primary antibody, add the primary antibody diluted with antibody diluent to the sections, incubate at 4°C overnight, incubate HRP secondary antibody the next day, incubate at room temperature for 60 minutes, add the diluted DAB dye, incubate at room temperature for 60 seconds, then rinse with PBS, stain with hematoxylin for 5 minutes, differentiate with 1% hydrochloric acid ethanol, use ammonia water for blueing, and seal the sections with neutral gum after gradient dehydration.

[0044] 1.2 Cell scratch

[0045] The cells were uniformly seeded in a 6-well plate with a cell density of 70% to 90%. A 200 μL pipette tip was used to make straight scratches on the monolayer of cells, and photos were taken for record. After a period of time (12 h / 24 h), photos were taken for record again.

[0046] 1.3 Transwell migration assay

[0047] The cells were evenly seeded in the Transwell chamber, and 100 μL of serum-free culture medium was added to the upper chamber. 600 μL of 10% FBS culture medium was added to the lower chamber. After 24 hours, the chamber was removed, fixed with paraformaldehyde, the cells above the chamber were wiped off, and after crystal violet staining, photos were taken under a microscope.

[0048] 2. Experimental results

[0049] To further screen the molecules involved in pancreatic cancer liver metastasis, we compared the genes expressed in the top pancreatic cancer liver metastasis group with MALAT + The intersection of the top 20 genes in the Epi subgroups was taken, and 12 key genes were screened ( Figure 2 A). Many of these genes have been reported in the process of tumor metastasis, which also verifies the reliability of our screening system. Combined with the analysis of the TCGA database, we found that high expression of CYBA genes is associated with poor prognosis in pancreatic cancer ( Figure 2 B) CYBA-encoded protein p22 phox Through immunohistochemistry of pancreatic cancer tissue samples, we found that p22 phox The protein is expressed at a higher level in pancreatic cancer liver metastasis tissues ( Figure 2 C) P22 phox It is expressed in normal pancreatic epithelial cells and various pancreatic cancer cell lines, with higher expression levels in BxPC3 cell lines and lower expression levels in AsPC1 and Capan2 cells ( Figure 2 D) P22 phox The protein is mainly expressed in the cytoplasm ( Figure 2 E). We used lentiviral infection to construct the BxPC3-shCYBA cell line on the BxPC3 cell line and the AsPC1-OE-CYBA cell line on the AsPC1 cell line. Through Transwell migration and cell scratch assays, we found that knocking down p22 phox Overexpression of p22 can inhibit pancreatic cancer cell migration phox Can promote cell migration ( Figure 2 GJ).

[0050] Example 3: P22 phox Knockdown can inhibit pancreatic cancer liver metastasis

[0051] 1. Experimental Methods

[0052] 1.1 Construction of pancreatic cancer liver metastasis model

[0053] Healthy adult female nude mice (7 weeks old) were selected and anesthetized with Avertin. The abdominal cavity was opened to expose the spleen. Diluted BxPC3-Luc cell lines and BxPC3-Luc-shCYBA cell lines (2×10 7 / ml), injected into the mouse through the spleen with a microsyringe (50 μL), and the mouse was sutured with sutures. Luciferase live imaging was performed on the 7th and 21st days after surgery.

[0054] 1.2 HE staining experiment

[0055] After the paraffin sections of tissue samples were dewaxed and hydrated, they were first stained with hematoxylin, and then the sections were placed in 1% hydrochloric acid alcohol for differentiation until the cell nuclei were clearly visible, then stained with eosin, and finally dehydrated and sealed with neutral resin.

[0056] 2. Experimental results

[0057] We constructed a pancreatic cancer liver metastasis model in nude mice by injecting BxPC-Luc / BxPC3-Luc-shCYBA cell lines into the spleen ( Figure 3 A), combined with luciferase imaging, we found that p22 phox Knockout reduced the fluorescence intensity in the liver of nude mice, indicating lower levels of pancreatic cancer cell growth ( Figure 3 B and C), and alleviated the weight loss caused by liver tumors in nude mice ( Figure 3 D) Liver phenotype and HE staining also revealed that p22 phox Knockdown inhibits the growth of pancreatic cancer cells on the liver ( Figure 3 E).

[0058] Example 4: NOX inhibitors can inhibit pancreatic cancer cell migration

[0059] 1. Experimental Methods CCK8 Detection of Cell Proliferation

[0060] BxPC3 cells were seeded in 96-well plates (100 μL, 3 × 10 4 The culture plate was placed in an incubator for pre-culture for 6 h, and NOX inhibitors were added in sequence according to the concentration gradient for 24 h. Then, 10 μl of CCK-8 solution was added to each well, and the culture was continued for 2 h. The absorbance at 450 nm was measured with an ELISA reader. Five replicate wells were made for each concentration.

[0061] 2. Experimental results

[0062] P22 phox As an important component of NADPH oxidase, there is currently no direct targeting of p22 phoxWe used inhibitors of NADPH oxidase (NOX) to further explore its effect on pancreatic cancer cell migration. We found that it did not affect pancreatic cancer cell survival at 20 μM ( Figure 4 A), Transwell migration and cell scratch assays revealed that NOX inhibitors affect the migration of pancreatic cancer cells ( Figure 4 B and C), WB verification showed that the inhibitor significantly inhibited the expression of p-AKT and p-MLC2, and immunofluorescence also verified that NOX inhibitors could inhibit the expression of p-MLC2 in clusters and single BxPC3 cells ( Figure 4 D and E).

[0063] Example 5: NOX inhibitors can inhibit pancreatic cancer liver metastasis

[0064] 1. Experimental Methods

[0065] A pancreatic cancer liver metastasis model was established by injecting BxPC3-Luc cell line into the spleen. We then intraperitoneally injected 10 mg / kg of NOX inhibitor or an equal amount of saline every day, performed luciferin live imaging on day 28, and then quickly removed the nude mouse liver for luciferin ex vivo imaging.

[0066] 2. Experimental results

[0067] We then studied the effects of NOX inhibitors on pancreatic cancer liver metastasis at the animal level. We constructed a pancreatic cancer liver metastasis model by injecting pancreatic cancer cells into the spleen. We found that on day 28, NOX inhibitors could inhibit pancreatic cancer liver metastasis ( Figure 5 A to C), and liver tissue HE staining also showed that NOX inhibitors can inhibit the colonization and growth of tumor cells in the liver ( Figure 5 D).

Claims

1. The application of CYBA as a target molecule for drug screening of pancreatic cancer liver metastasis or CYBA itself in the preparation of drugs for pancreatic cancer liver metastasis. 2.P22 phox Protein as a drug screening target molecule for pancreatic cancer liver metastasis or p22 phox The protein itself is used in the preparation of drugs for treating pancreatic cancer liver metastasis. 3.p22 phox Application of the protein itself or NOX inhibitor in the preparation of drugs for treating liver metastasis of pancreatic cancer.

4. The use of a NOX inhibitor in the preparation of a drug for pancreatic cancer liver metastasis, characterized in that: The inhibitor of NOX protein is GSK2795039.

5. Application of CYBA or P22phox protein in the preparation of reagents for diagnosing pancreatic cancer liver metastasis.

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