Use of beta-glucuronidase, its coding nucleic acid, expression vector and genetically engineered host cell in preparation of drug for preventing and treating breast cancer

Abstract

This invention discloses the application of β-glucuronidase, its encoding nucleic acid, expression vector, and genetically engineered host cells in the preparation of drugs for the prevention and treatment of breast cancer, belonging to the pharmaceutical field. This invention seeks protection for the application of β-glucuronidase, recombinant β-glucuronidase, and their upstream materials in the preparation of drugs for the prevention or treatment of breast cancer. This invention is the first to discover that administration of β-glucuronidase can effectively inhibit the growth of primary breast cancer tumors and lung metastases of breast cancer. Therefore, β-glucuronidase, recombinant β-glucuronidase, the encoding nucleic acid of recombinant β-glucuronidase or an expression vector containing the encoding nucleic acid, and genetically engineered host cells expressing and secreting β-glucuronidase have the potential to be developed into drugs for the prevention or treatment of breast cancer (preventing or inhibiting the growth of primary breast cancer tumors and / or preventing or inhibiting lung metastases of breast cancer).

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to new uses of known proteins, specifically the application of β-glucuronidase, its encoded nucleic acid, expression vector, and genetically engineered host cells in the preparation of drugs for the prevention and treatment of breast cancer. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide and one of the leading causes of cancer death in women. Statistics show that in 2023, there were approximately 2.3 million new cases of breast cancer and 764,000 deaths worldwide. In my country, the incidence of breast cancer is also on the rise, seriously threatening women's health. The development of breast cancer is related to multiple factors. Besides genetic susceptibility (accounting for only about 10%), lifestyle factors such as a Western diet, obesity, alcohol consumption, and a sedentary lifestyle are all recognized as important risk factors.

[0003] Distant metastasis is the leading cause of death in breast cancer patients. Among distant metastases, lung metastasis has the highest incidence and the worst prognosis. The 5-year survival rate for metastatic breast cancer patients is only about 20%, with particularly poor outcomes for those with lung metastases. Breast cancer cell metastasis to the lungs is a complex biological process involving multiple steps and factors, including tumor cell invasion, infiltration, circulation, extravasation, and colonization and growth in the lung microenvironment. Current treatments for breast cancer lung metastases include surgical resection, radiotherapy, radiofrequency ablation, and systemic drug therapy, but their efficacy remains limited, and patient survival benefits are poor. Therefore, in-depth research into the molecular mechanisms of breast cancer lung metastasis and the development of novel and effective treatment strategies have become core issues urgently needing to be addressed in this field.

[0004] β-glucuronidase (GUSB) is an important glycoside hydrolase widely distributed in various tissues, body fluids, and intestinal microorganisms of the human body. The human GUSB gene is located on the long arm of chromosome 7 (7q21.11), and the enzyme protein it encodes is a glycoprotein composed of four identical subunits, mainly found in lysosomes of mammalian cells (Huang Xixi et al. Research progress on β-glucuronidase [J]. Chinese Pharmacist, 2017). The crystal structure of GUSB consists of three distinct domains: a jelly roll barrel, an immunoglobulin constant domain, and a TIM barrel domain. The TIM barrel domain contains the enzyme's active site and is a key site for catalytic activity (Jain S, Drendel WB, Chen ZW, et al. Structure of human beta-glucuronidase reveals candidate lysosomal targeting and active-site motifs [J]. Nat Struct Biol, 1996). The core function of GUSB is to catalyze the hydrolysis of β-D-glucuronide bonds, releasing glucuronic acid and the corresponding ligands (Huang Xixi et al. Research progress on β-glucuronidase [J]. Chinese Pharmacist, 2017). In human physiological processes, GUSB participates in various important metabolic pathways, including steroid metabolism, bilirubin metabolism, and the degradation of mucopolysaccharides (such as dermatin sulfate and keratin sulfate). Mutations in the GUSB gene (most commonly missense mutations) can lead to altered or lost enzyme activity, which in turn can cause serious diseases such as mucopolysaccharidosis type VII (Sly syndrome) (Huang Xixi et al. Research progress on β-glucuronidase [J]. Chinese Pharmacist, 2017).

[0005] GUSB is closely related to the occurrence, development, diagnosis, and treatment of tumors. As early as the mid-20th century, studies found that the activity of GUSB in various human tumor tissues (including gastric cancer, breast cancer, colon cancer, uterine cancer, and ovarian cancer) was significantly higher than in adjacent normal tissues (Cao Jinghe. The Relationship between β-glucuronidase and Tumors [J]. Journal of Bengbu Medical College, 1964; Yao Tianming, Han Bing. Application of β-glucuronidase in Tumor Diagnosis and Treatment [J]. Journal of the Fourth Military Medical University, 2001). In tumor diagnosis, detecting GUSB activity in gastric juice has auxiliary diagnostic value for gastric cancer, and detecting GUSB activity in urine is helpful in the diagnosis of bladder cancer (Yao Tianming, Han Bing. Application of β-glucuronidase in Tumor Diagnosis and Treatment [J]. Journal of the Fourth Military Medical University, 2001). GUSB is also considered an important tumor marker. In the field of tumor treatment, GUSB also demonstrates significant application value. Based on the "antibody-guided enzymatic prodrug therapy" (ADEPT) strategy, GUSB is used to specifically hydrolyze glucuronidated prodrugs at the tumor site, releasing active chemotherapeutic drugs, thereby enhancing antitumor effects and reducing systemic toxicity (Yao Tianming, Han Bing. Application of β-glucuronidase in tumor diagnosis and treatment [J]. Journal of the Fourth Military Medical University, 2001). Furthermore, studies have found that GUSB inhibitors (such as nojirimycin A) can effectively inhibit experimental lung metastasis of mouse melanoma cells, suggesting that GUSB activity also plays an important role in tumor metastasis (Yao Tianming, Han Bing. Application of β-glucuronidase in tumor diagnosis and treatment [J]. Journal of the Fourth Military Medical University, 2001).

[0006] In recent years, an increasing number of studies have shown that the gut microbiota plays an important role in the development and progression of hormone receptor-positive (HR+) breast cancer (Arnone AA, Cook KL. Gut and Breast Microbiota as Endocrine Regulators of Hormone Receptor-positive Breast Cancer Risk and Therapy Response [J]. Endocrinology, 2023; Fernandez-Murga ML, Gil-Ortiz F, Serrano-Garcia L, et al. A New Paradigm in the Relationship between Gut Microbiota and Breast Cancer: β-glucuronidase Enzyme Identified as Potential Therapeutic Target [J]. Pathogens, 2023). Among these, gut microbiota-derived GUSB has received widespread attention.Its mechanism of action mainly involves the enterohepatic circulation of estrogen: the liver uses uridine diphosphate glucuronyl transferase (UGT) to bind estrogen with glucuronic acid, generating highly water-soluble estrogen-glucuronide (EG), which is then excreted into the intestine via bile (Arnone AA, Cook KL. Gut and Breast Microbiota as Endocrine Regulators of Hormone Receptor-positive Breast Cancer Risk and Therapy Response [J]. Endocrinology, 2023); some bacteria in the intestine (such as Bacteroides, Clostridium, Escherichia coli, etc.) express GUSB enzymes. These bacterial GUSBs can hydrolyze estrogen-glucuronide conjugates, removing the glucuronic acid group and releasing active free estrogen (Fernandez-Murga ML, Gil-Ortiz F, Serrano-Garcia L, et al. A New Paradigm in the Relationship between Gut Microbiota and Breast Cancer: β-glucuronidase Enzyme). Identified as Potential Therapeutic Target [J]. Pathogens, 2023); Reactivated free estrogen can be reabsorbed through the intestinal mucosa and re-enter the systemic circulation via the portal vein, leading to an increase in the level of biologically active estrogen in the circulation (Arnone AA, Cook KL. Gut and Breast Microbiota as Endocrine Regulators of HormoneReceptor-positive Breast Cancer Risk and Therapy Response [J]. Endocrinology, 2023). Given that estrogen is a key driver of HR+ breast cancer cell proliferation, angiogenesis, and metastasis, intestinal GUSB-mediated estrogen reactivation is thought to increase the risk of HR+ breast cancer. Clinical studies have also provided evidence for this mechanism. In a 2015 case-control study of postmenopausal women, Goedert et al. found that the gut microbiota composition (β-diversity) of breast cancer patients differed significantly from that of healthy controls. Furthermore, the fecal microbiota α-diversity of healthy women was positively correlated with the total estrogen level in urine, while no such correlation was found in breast cancer patients. This suggests that breast cancer patients may have reduced gut microbiota diversity and abnormal estrogen metabolism.Another study also showed that GUSB-positive bacteria were more abundant in the gut of breast cancer patients compared with healthy controls (Zhu J, Liao M, Yao Z, et al. Breast cancer inpostmenopausal women is associated with an altered gut metagenome [J]. Microbiome, 2018). Therefore, gut microbiota GUSB has become a potential new target in the prevention and treatment of hormone receptor-positive breast cancer.

[0007] Based on existing technology, GUSB may promote the occurrence and development of breast cancer, and there are no reports of GUSB inhibiting breast cancer tumor growth and lung metastasis. Based on the applicant's findings, this invention is hereby proposed. Summary of the Invention

[0008] The first objective of this invention is to provide the application of β-glucuronidase in the preparation of drugs for the prevention or treatment of breast cancer; the second objective is to provide the application of recombinant β-glucuronidase in the preparation of drugs for the prevention or treatment of breast cancer; the third objective is to provide the application of the encoding nucleic acid of recombinant β-glucuronidase or an expression vector containing the encoding nucleic acid in the preparation of drugs for the prevention or treatment of breast cancer; and the fourth objective is to provide the application of genetically engineered host cells that express and secrete β-glucuronidase in the preparation of drugs for the prevention or treatment of breast cancer.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides the application of β-glucuronidase in the preparation of drugs for the prevention or treatment of breast cancer. Pharmacodynamic tests conducted according to specific embodiments of this invention have verified that administration of β-glucuronidase can effectively inhibit the growth of primary breast cancer tumors and lung metastases, providing a novel and effective active pharmaceutical component for the prevention and clinical treatment of breast cancer.

[0010] In one specific embodiment, the prevention or treatment is to prevent or inhibit the growth of primary breast cancer tumors, and / or to prevent or inhibit lung metastasis of breast cancer.

[0011] This invention provides the application of recombinant β-glucuronidase in the preparation of drugs for the prevention or treatment of breast cancer. Pharmacodynamic tests of specific embodiments of this invention have verified that administration of a recombinant β-glucuronidase can effectively inhibit the growth of primary breast cancer tumors and lung metastases, providing a novel and effective active pharmaceutical component for the prevention and clinical treatment of breast cancer.

[0012] In one specific embodiment, the amino acid sequence of the recombinant β-glucuronidase is shown in SEQ ID NO. 2.

[0013] In one specific embodiment, the prevention or treatment is to prevent or inhibit the growth of primary breast cancer tumors, and / or to prevent or inhibit lung metastasis of breast cancer.

[0014] This invention provides the application of a recombinant β-glucuronidase encoding nucleic acid or an expression vector containing such encoding nucleic acid in the preparation of drugs for the prevention or treatment of breast cancer. Through molecular biological experiments and in vivo efficacy verification, this invention confirms that the nucleic acid sequence encoding a specific recombinant β-glucuronidase and the expression vector containing this sequence can efficiently express a functional protein with anti-breast cancer activity in host cells. The expressed recombinant enzyme can effectively inhibit the growth of primary breast cancer tumors and lung metastases of breast cancer, and can be used as a core biomaterial for the preparation of gene-based drugs and protein-based drugs for the prevention and treatment of breast cancer.

[0015] In one specific embodiment, the nucleotide sequence encoding the nucleic acid of the recombinant β-glucuronidase is shown in SEQ ID NO. 1.

[0016] In one specific embodiment, the prevention or treatment is to prevent or inhibit the growth of primary breast cancer tumors, and / or to prevent or inhibit lung metastasis of breast cancer.

[0017] This invention provides the application of a genetically engineered host cell that expresses and secretes β-glucuronidase in the preparation of a drug for the prevention or treatment of breast cancer; wherein the prevention or treatment is to prevent or inhibit the growth of primary breast cancer tumors, and / or to prevent or inhibit lung metastasis of breast cancer. This genetically engineered host cell can stably express and secrete β-glucuronidase with specific functions. Pharmacological experiments in specific embodiments of this invention fully demonstrate that the active enzyme protein secreted by this engineered cell can effectively inhibit the growth of primary breast cancer tumors and lung metastasis of breast cancer.

[0018] In one specific embodiment, the amino acid sequence of the β-glucuronidase is shown in SEQ ID NO. 2.

[0019] Beneficial effects: This invention is the first to discover that administration of β-glucuronidase can effectively inhibit the growth of primary breast cancer tumors and lung metastases. Therefore, β-glucuronidase, recombinant β-glucuronidase, the encoding nucleic acid of recombinant β-glucuronidase or an expression vector containing such encoding nucleic acid, and genetically engineered host cells that express and secrete β-glucuronidase hold promise for development into drugs for the prevention or treatment of breast cancer (preventing or inhibiting the growth of primary breast cancer tumors and / or preventing or inhibiting lung metastases). Attached Figure Description

[0020] Figure 1 Graphs showing changes in GUSB levels in breast cancer metastasis and non-metastasis groups detected by ELISA. (A) Serum GUSB levels in patients with and without breast cancer lung metastasis; (B) GUSB levels in bronchoalveolar lavage fluid of mice with and without adenocarcinoma lung metastasis; (C) Serum GUSB levels in mice with and without breast cancer lung metastasis.

[0021] Figure 2 GUSB knockout accelerates lung metastasis in mice with breast cancer. (A) Mouse survival rate graph; (B) In vivo imaging of mouse lung tumors; (C) Imaging of mouse lung tissue and statistical analysis results; (D) HE staining of mouse lung tumors.

[0022] Figure 3 The image shows GUSB inhibiting lung metastasis in breast cancer. (A) Mouse survival rate; (B) In vivo imaging of mouse lung tumors; (C) Mouse lung tissue imaging and statistical analysis results; (D) HE staining of mouse lung tumors.

[0023] Figure 4 The images show GUSB inhibiting tumor growth. (A) Size and volume of mouse tumors; (B) Fluorescence of CD8+ T immune cells in mouse tumor sites; (C) Flow cytometry analysis of CD8+ T cells in mouse tumor sites.

[0024] Figure 5 To investigate the effects of GUSB on CD8+ T cell migration and enhanced cytotoxicity. (A) CD8+ T cell migration map and corresponding statistical analysis; (B) ELISA detection of GZMB and IFN-γ expression levels in CD8+ T cells after stimulation with PBS and GUSB. Detailed Implementation

[0025] The substantive content of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art should know that the scope of protection of the present invention should not be limited to these specific embodiments.

[0026] Example 1: β-Glucuronidase (GUSB) may be involved in lung metastasis of breast cancer. 1. GUSB was reduced in breast cancer patients with lung metastases and mice. Serum samples were collected from breast cancer patients with and without lung metastases. The GUSB levels were measured using an ELISA kit (Thermo Fisher Scientific, catalog number EH43RB) according to the manufacturer's instructions. The results showed that the GUSB levels in the serum of breast cancer patients with lung metastases were significantly lower than those in the non-metastatic group. Figure 1 (A)

[0027] Simultaneously, we injected 5×10-10 of the mammary fat pads into 8-week-old female C57BL / 6 mice. 5 Two months after the onset of breast cancer, E0771 cells (a spontaneous breast cancer cell line derived from C57BL / 6 mice) were used to detect lung metastasis in mice. The levels of GUSB in bronchoalveolar lavage fluid and serum of mice with and without breast cancer lung metastasis were measured using an ELISA kit. The results showed that GUSB levels in the bronchoalveolar lavage fluid and serum of mice without metastasis were significantly higher than those in the metastasis group. These results suggest that GUSB may be involved in the lung metastasis process of breast cancer. Figure 1 (B and C in the middle).

[0028] 2. GUSB knockout accelerates lung metastasis in mice with breast cancer. We commissioned Saiye Suzhou Biotechnology Co., Ltd. to construct GUSB systemic knockout mice. We administered 1×10⁻⁶ GUSB systemic knockout mice via tail vein infusion to 8-week-old female mice from both wild-type and GUSB systemic knockout groups. 6 We used E0771 cells to observe the survival time (40 days) and lung metastasis of breast cancer in mice (21 days). We found that the survival time of GUSB knockout mice was significantly reduced. Figure 2 (A) The condition of breast cancer with lung metastasis worsened ( Figure 2 (BD). This result indicates that GUSB knockout accelerates lung metastasis in mice with breast cancer.

[0029] Example 2: Recombinant protein GUSB (rGUSB) for the treatment of lung metastases in breast cancer I. Preparation of rGUSB The coding region of the mouse GUSB gene (ID: 110006) was found in the NCBI database. The recombinant expression fragment of the GUSB gene was optimized to form the recombinant GUSB gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0030] 1 gtcaggcata tggggaaacg atccaggact tcaagcgact tcctctttct gcagagtcca 61 gccttggggc ctctagatag ccttgagcca gcagctgtac aagcgcttgc cctcaaccaa 121 gttggcgcag gcggcttctc tcgagaacat gtgactcttc caggtcccgc ctctgccctc 181 ccagagagga ggctaaaaag cccagccgct ggtcggtggg cagcctggcc agtatgtccc 241 taaaatggag tgcgtgttgg gtcgcgctgg gccagctgct gtgcagctgc gcgctggctc 301 tgaagggcgg gatgctgttc ccgaaggaga gcccgtcgcg ggagctcaag gcgctggacg 361 gactgtggca cttccgcgcc gacctctcga acaaccggct gcagggtttc gagcagcaat 421 ggtaccggca gccgctacgg gagtcgggcc cagtcttgga catgcctgtc cttctagct 481 tcaatgacat cacccaagaa gcagcccttc gggactttat tggctgggtg tggtatgaac 541 gggaagcaat cctgccacgg cgatggaccc aagataccga catgagagtg gtgttgagga 601 tcaacagtgc ccattattat gcagttgtgg tatcccaagg gttactttgt ccaggacaca 661 agctttgact tcttcaacta tgcgggactg catcgatctg tggtcccta taccacccct 721 accacttaca tcgatgatat cactgtgatc actaatgtgg agcaagacat cggtgaggaa 781 tcccggcggg aggctctgtg ctaggctggg tccatttta tctcaattcc caagaggaaa 841 aaaaatccct aaggagaaaa aggcttcaag catcttttag ccctcccctg cctcattatc 901 ctcagaagat aagactcaga ctgcccaagc tgacaccttt ctggtgggtc ttcctttgca 961 aggaagtgag gaccaatgac aggtggggtc ttgtctaaag tgacatcact caaaacattg 1021 ggtcacttcc ttcctccttg taactcctaa tctctgcaca gtaagatttaaaaaaaacta 1081 aataaataaa aggacaagc (SEQ ID NO.1) The amino acid sequence of the recombinant GUSB protein (rGUSB) expressed by the recombinant GUSB gene is shown in SEQ ID NO. 2.

[0031] 1 mslkwsacwv algqllcsca lalkggmlfp kespsrelka ldglwhfrad lsnnrlqgfe 61 qqwyrqplre sgpvldmpvp ssfnditqea alrdfigwvw yereailprr wtqdtdmrvv 121 lrinsahyya vvvsqgllcp ghkl (SEQ ID NO .2) All reagents used in this experiment are commercially available and can be purchased normally.

[0032] Using cDNA from C57BL / 6 mouse bone marrow-derived macrophages as a template, PCR amplification was performed using Q5 High-Fidelity 2X Master Mix (New England Biolabs, NEB, catalog number M0492). The primer sequences (F-terminus: GCAGTTTGTGGTATCCCAAGG; R-terminus: CAAAGGAAGACCCACCAGAAAG) yielded the target gene fragment encoding the mGUSB truncated protein shown in SEQ ID NO. 2. After confirmation of the amplified product by agarose gel electrophoresis, the target band was excised and purified using the TIANgel Midi Purification Kit (TIANGen Biotech, catalog number DP209-02).

[0033] The purified mGUSB target fragment was recombined with the pET-28a(+) expression vector (Novagen / Merck, catalog number 69864). The recombination reaction was performed using the ClonExpress Ultra One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd., Vazyme, catalog number C115-01 or C115-02). After the reaction, the recombinant product was transformed into DH5α chemocompetent cells (Vazyme, catalog number C502-02), and the revived bacterial culture was plated on LB agar containing kanamycin for screening. Kanamycin sulfate was purchased from Solarbio (Catalyme K8020), and the final concentration in the culture medium was 50 μg / mL.

[0034] After overnight culture, single colonies were picked and preliminarily identified using culture PCR. Clones with amplified bands matching expectations were selected for further culture and plasmids were extracted using the TIANprep Mini Plasmid Kit (Tiangen Biotech (Beijing) Co., Ltd., catalog number DP103-02). The obtained candidate plasmids were sent to Genewiz Biotechnology Co., Ltd. for Sanger sequencing. The sequencing results were compared with the theoretical sequence to confirm that the insertion direction and open reading frame of the target fragment were correct, and no abnormal sequences affecting the expression of the target protein were found. The recombinant expression plasmid pET28a-mGUSB was then obtained.

[0035] Subsequently, the correctly identified pET28a-mGUSB plasmid was transformed into BL21(DE3) chemically competent cells (Vazyme, catalog number C504-02). After kanamycin resistance screening and bacterial PCR identification, an engineered strain carrying the mGUSB recombinant expression plasmid was obtained and named pET28a-mGUSB-DE3.

[0036] The correctly identified pET28a-mGUSB-DE3 engineered strain was entrusted to Beijing Yiqiao Shenzhou Technology Co., Ltd. for scale-up culture, induced expression, and rGUSB protein preparation. For recombinant proteins with a His tag, affinity purification was performed using HisPurNi-NTA Resin (Thermo Scientific, catalog number 88221). The purified rGUSB protein concentration was determined using the PierceBCA Protein Assay Kit (Thermo Scientific, catalog number 23225), and its apparent molecular weight was analyzed by gel electrophoresis; the protein size was approximately 16 kDa.

[0037] II. Activity determination of rGUSB 1. GUSB improves lung metastasis in breast cancer (1) Experimental materials Eight-week-old female C57BL / 6 mice weighing approximately 19g were used in the experiment. Mouse breast cancer cells E0771 (Yuanjing Biotechnology, catalog number YC-C034-Luc-P) were cultured under standard conditions, and cells in the logarithmic growth phase and in good growth condition were selected for animal experiments. Recombinant GUSB protein (rGUSB) was prepared using the aforementioned method and diluted to an appropriate concentration with sterile PBS, prepared fresh before administration or stored according to protein stability requirements. The control group received an equal volume of PBS.

[0038] (2) Experimental methods Eight-week-old female C57BL / 6 mice were randomly divided into a PBS control group and an rGUSB treatment group. E0771 cells in the logarithmic growth phase were collected, digested, centrifuged, washed with PBS, and resuspended to adjust cell concentration. One × 10^6 E0771 cells were injected into each mouse via the tail vein to establish an experimental lung metastasis model of breast cancer. After modeling, the rGUSB treatment group received rGUSB via the tail vein at a dose of 1.5 mg / kg every 3 days; rGUSB was dissolved in sterile PBS. The PBS control group received an equal volume of PBS via the same route and frequency. To evaluate the effect of rGUSB on the survival of tumor-bearing mice, the mice were observed continuously for 40 days from the date of E0771 cell inoculation, and their survival status was recorded daily. Kaplan-Meier survival curves were plotted to compare the survival of the two groups. Mice that underwent modeling and administration using the same method were also used, and lung metastasis burden was evaluated on day 21 after modeling. Tumor-related signals in the mice were detected using an in vivo imaging system, and the signal intensity was quantitatively analyzed. Mice were euthanized after imaging, and lung tissue was completely isolated for further in vitro lung tissue imaging to evaluate the tumor metastasis burden in the lungs. Subsequently, the lung tissue was fixed, embedded in paraffin, and sectioned for hematoxylin and eosin (H&E) staining. The formation of breast cancer metastases within the lung tissue was observed under a microscope. Based on the actual experimental design, the number of lung metastases, the area of ​​metastases, and / or the proportion of metastases to the lung tissue area were statistically analyzed to evaluate the effect of rGUSB on breast cancer lung metastases.

[0039] (3) Experimental results Survival analysis showed that, compared with the PBS control group, the survival of mice in the rGUSB treatment group was significantly improved and their survival time was prolonged, suggesting that exogenous supplementation with rGUSB can improve the survival rate of mice with breast cancer lung metastasis. Figure 3 The survival rate of mice treated with rGUSB was 50% higher than that of the PBS control group. In vivo imaging analysis was performed on day 21 post-modeling, showing a significant decrease in lung tumor-related imaging signals in the rGUSB-treated group compared to the PBS control group. Further imaging of ex vivo lung tissue revealed a significant reduction in lung tumor burden in the rGUSB-treated group. Figure 3 (B, C). H&E staining results of lung tissue showed that, compared with the PBS control group, the number of breast cancer metastases in the lung tissue of mice treated with rGUSB was significantly reduced, and the degree of lung metastasis was decreased. Figure 3 (D). The above results indicate that in the E0771 breast cancer experimental lung metastasis model, exogenous administration of rGUSB can reduce the lung tumor metastasis burden and improve the survival of tumor-bearing mice, suggesting that rGUSB has a significant inhibitory effect on breast cancer lung metastasis and has potential application value for the prevention and / or treatment of breast cancer lung metastasis.

[0040] 2. rGUSB inhibits breast cancer growth (1) Experimental materials Eight-week-old female C57BL / 6 mice were used in the experiment, and mouse breast cancer cells E0771 were used to establish an orthotopic xenograft model of breast cancer. E0771 cells were cultured under standard conditions, and cells in the logarithmic growth phase and in good growth condition were selected for animal experiments.

[0041] rGUSB was prepared using the aforementioned method and diluted to an appropriate concentration using sterile PBS. The control group was treated with an equal volume of PBS. CD8+ T cells in tumor tissue were detected using immunofluorescence staining (Thermo Fisher Scientific, catalog number 740029T) and flow cytometry (Live / Dead, CD3, CD8a, CD4; BioLegend catalog numbers 423105, 300430, 301014, 300512).

[0042] (2) Experimental methods Eight-week-old female C57BL / 6 mice were randomly divided into a PBS control group and an rGUSB treatment group. E0771 cells in the logarithmic growth phase were collected, digested, centrifuged, washed with PBS, resuspended, and the cell concentration was adjusted. Five × 10⁵ E0771 cells were inoculated into the mammary fat pad of each mouse to establish an orthotopic xenograft model of breast cancer. Local drug administration began on day 1 after E0771 cell inoculation. The rGUSB treatment group received a local injection of 30 μg rGUSB at the tumor inoculation site. The rGUSB was dissolved in sterile PBS and administered every 3 days for 21 consecutive days. The PBS control group received an equal volume of PBS via the same route and frequency of administration.

[0043] During the experiment, the mice's condition was observed regularly, and the long and short diameters of the tumors were measured and the changes in tumor volume were recorded. The tumor volume was calculated as V = (long diameter × short diameter²) / 2, and tumor growth curves for different treatment groups were plotted.

[0044] At the end of the experiment, mice were sacrificed and the tumor tissue was completely dissected. A portion of the tumor tissue was fixed, embedded, and sectioned for immunofluorescence staining, and CD8-specific antibodies were used to label the tumor tissue. Cells were observed and counted using a fluorescence microscope. Cell infiltration within tumor tissue was assessed. Another portion of fresh tumor tissue was minced and digested to prepare a single-cell suspension. After filtration to remove tissue debris, immunolabeling of cell surfaces was performed. Flow cytometry was used to detect the presence of cells in the tumor tissue. Cell proportion, and tumor invasion based on markers such as CD3, CD4, and CD8. Cells were analyzed to compare the differences in the tumor immune microenvironment between the PBS control group and the rGUSB treatment group.

[0045] (3) Experimental results Tumor growth monitoring results showed that, compared with the PBS control group, local administration of rGUSB significantly slowed the growth rate of mouse mammary tumors, and the tumor volume at the experimental endpoint was significantly reduced (the tumor in the rGUSB group shrank by approximately 27.3% compared to the PBS control group), indicating that rGUSB can inhibit the growth of E0771 mammary tumors. Figure 4 (A). Immunofluorescence staining results of tumor tissue showed that in the rGUSB-treated group of tumor tissue... Cell infiltration increased significantly ( Figure 4 (B). Further analysis using flow cytometry also showed that, compared with the PBS control group, the rGUSB treatment group had significantly lower levels of [unclear - likely referring to a specific type of tumor tissue]. The proportion and / or number of cells are significantly increased ( Figure 4 (C). The above results indicate that local supplementation with rGUSB can not only inhibit the growth of breast tumors, but also increase the intracellular content of rGUSB in tumor tissue. Cell infiltration suggests that rGUSB may exert its tumor-suppressive effect by improving the tumor immune microenvironment in breast cancer and enhancing anti-tumor immune responses.

[0046] 3. rGUSB induces CD8+ cell recruitment and enhances their immune function. (1) Experimental materials Experimental use Cells were isolated from mouse spleen or peripheral immune tissues and separated by flow cytometry (Live / Dead, CD3, CD8a, CD4; BioLegend catalog numbers 423105, 300430, 301014, 300512). rGUSB was prepared using the aforementioned method and diluted to the required concentration using sterile PBS. Cell migration ability was detected using Transwell chambers; the secretion levels of granzyme B (GZMB) (Thermo Fisher Scientific, catalog number 88-8022-88) and interferon-γ (IFN-γ) (Thermo Fisher Scientific, catalog number BMS609) were detected using ELISA.

[0047] (2) Experimental methods To evaluate rGUSB The effect on cell migration ability was detected using a Transwell migration assay. Cells were resuspended in a suitable cell culture medium, and the upper chamber of each Transwell was supplemented with... indivual Cells. In the rGUSB treatment group, rGUSB was added to the lower chamber culture medium to a final concentration of 100 ng / mL, while no rGUSB was added to the upper chamber culture medium, thus creating an rGUSB concentration gradient from the upper to the lower chamber. In the control group, no rGUSB was added to either chamber. After 24 hours of culture, cells that migrated to the lower chamber of the Transwell were collected, and the number of migrated cells was quantitatively analyzed by cell counting, flow cytometry, or other suitable methods. The number of cells migrating to the lower chamber was compared between the rGUSB treatment group and the control group. Cell count to evaluate rGUSB's effect Effects on cell migration. Further evaluation of rGUSB's effects. Effects on cellular effector function. The isolated... Cells were seeded in culture plates and in vitro stimulated with rGUSB to a final concentration of 100–150 ng / mL; the control group received an equal volume of PBS. After an appropriate culture time, the culture supernatant was collected and analyzed by ELISA. Expression levels of GZMB and IFN-γ in cells.

[0048] (3) Experimental results Transwell migration assay results showed that, compared with the control group, the addition of 100 ng / mL rGUSB to the lower chamber resulted in a significant increase in the number of cells migrating to the lower chamber. The number of cells increased significantly. Figure 5 (A) indicates that rGUSB can promote Cells migrated along their concentration gradient, suggesting that rGUSB... Cells have certain recruitment or chemotaxis-promoting effects.

[0049] Further After in vitro stimulation of cells with rGUSB, it was found that treatment with 100–150 ng / mL rGUSB significantly improved cell function compared with the PBS control group. Expression and / or secretion levels of GZMB and IFN-γ in cells ( Figure 5 (B). GZMB and IFN-γ are both... The above results indicate that rGUSB is an important effector molecule for cells to exert cytotoxic and anti-tumor immune effects. Cell migration can also enhance its effector functions.

[0050] Combining the experimental results from the aforementioned orthotopic xenograft and lung metastasis models of breast cancer, it is evident that rGUSB can inhibit breast tumor growth and lung metastasis, and promote the growth of tumor cells in tumor tissue. Cell recruitment and infiltration, while enhancing The expression of effector molecules such as GZMB and IFN-γ in cells was monitored. These results indicate that rGUSB can regulate... Cell migration and antitumor effects are involved in the regulation of breast cancer progression and lung metastasis. Therefore, rGUSB can serve as a candidate active ingredient for the prevention, delay, or inhibition of breast cancer lung metastasis, and has potential application value in the preparation of drugs for the prevention and treatment of breast cancer and its lung metastasis.

[0051] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. Application of β-glucuronidase in the preparation of drugs for the prevention or treatment of breast cancer.

2. The application according to claim 1, characterized in that: The prevention or treatment is to prevent or inhibit the growth of primary breast cancer tumors, and / or to prevent or inhibit lung metastasis of breast cancer.

3. Application of recombinant β-glucuronidase in the preparation of drugs for the prevention or treatment of breast cancer.

4. The application according to claim 3, characterized in that: The amino acid sequence of the recombinant β-glucuronidase is shown in SEQ ID NO.

2.

5. The application according to claim 3 or 4, characterized in that: The prevention or treatment is to prevent or inhibit the growth of primary breast cancer tumors, and / or to prevent or inhibit lung metastasis of breast cancer.

6. The use of a recombinant β-glucuronidase encoding nucleic acid or an expression vector containing the encoding nucleic acid in the preparation of a drug for the prevention or treatment of breast cancer.

7. The application according to claim 6, characterized in that: The nucleotide sequence encoding the recombinant β-glucuronidase is shown in SEQ ID NO.

1.

8. The application according to claim 6 or 7, characterized in that: The prevention or treatment is to prevent or inhibit the growth of primary breast cancer tumors, and / or to prevent or inhibit lung metastasis of breast cancer.

9. The application of a genetically engineered host cell that expresses and secretes β-glucuronidase in the preparation of a drug for the prevention or treatment of breast cancer; wherein, The prevention or treatment is to prevent or inhibit the growth of primary breast cancer tumors, and / or to prevent or inhibit lung metastasis of breast cancer.

10. The application according to claim 9, characterized in that: The amino acid sequence of the β-glucuronidase is shown in SEQ ID NO. 2.

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