Application of P5A ATPase as a target in the preparation of drugs for the treatment of neuroblastoma

Abstract

The application relates to the field of tumor treatment drugs and discloses application of P5A ATPase as a target in preparation of a neuroblastoma treatment drug. It is found for the first time that P5A ATPase can affect the expression level of LRP8 protein and endoplasmic reticulum translocation of LRP8 signal peptide guided peptide chains, and further affect the growth and proliferation of neuroblastoma cells, so that a new target is provided for development of a neuroblastoma treatment drug. In addition, it is found that two P5A ATPase inhibitors, sodium metavanadate and DCC, can inhibit the endoplasmic reticulum translocation of LRP8 signal peptide guided peptide chains, reduce the expression level of LRP8 protein, and further inhibit the growth and proliferation of neuroblastoma cells, so that a new treatment drug for neuroblastoma is provided.

Description

Technical Field

[0001] This invention relates to the field of tumor therapeutic drugs, and more particularly to the application of P5A ATPase as a target in the preparation of neuroblastoma therapeutic drugs. Background Technology

[0002] P-type ATPases are a class of ATPase pumps widely distributed in biological membranes. They are named for the phosphorylated intermediate state they catalyze during transport, mediating the transmembrane transport of various ions or small molecules between biological membranes. Based on differences in the substrates they act on and the conservation of substrate binding site sequences, the P-type ATPase family is divided into five subfamilies: P1 to P5. Each subfamily can be further divided into multiple subgroups such as A and B. P5 is present in all eukaryotic genomes and is currently the least studied subfamily of P-type ATPases. The characteristic sequence of the fourth transmembrane domain of P5 ATPase differs from other families; compared to the conserved Pxxx (P / L) characteristic sequences of other families, P5 ATPase has an additional proline (PPxxP). Based on the differences in the PPxxP characteristic sequence, P5 ATPase is divided into two subfamilies: P5A and P5B. The P5A subfamily has only one member in human samples: ATP13A1. Unlike other P-type ATPases, such as P1-P3 ATPases, which primarily mediate Ca2+ transport, these ATPases are different transport substrates. 2+ Mg 2+ Na + K + P5A ATPase facilitates transmembrane transport of isocations, mediates lipid transport, and mediates polyamine transport. Its substrates are large proteins. P5A ATPase aids in the correct localization of tail-anchored proteins, the membrane insertion of multiple transmembrane proteins, and protein translocation to the endoplasmic reticulum (ER). However, the substrates and mechanisms by which P5A ATPase regulates ER translocation remain unclear. Investigating the transport substrates and mechanisms of P5A ATPase can provide a theoretical basis and potential drug targets for the treatment of related diseases.

[0003] Neuroblastoma (NB), originating from the neural crest cells of the sympathetic nervous system, is the most common extracranial malignant tumor in children. It is highly heterogeneous, prone to recurrence and progression, and exhibits high malignancy, easily metastasizing early and in the bone marrow, posing a serious threat to the lives and health of affected children. In addition to traditional treatments such as surgery, chemotherapy, radiotherapy, autologous stem cell transplantation, and 13-cis-retinoic acid, dipyrosine ganglioside (GD2) monoclonal antibody therapy has been included in the consensus consensus on multimodal treatment of NB. Despite this, the 5-year survival rate for high-risk NB patients remains below 50%. Some identified therapeutic targets for neuroblastoma have limitations, including difficulty in direct targeting, poor treatment efficacy, and the potential for drug resistance with long-term use. Therefore, developing more therapeutic targets is of great significance. Currently identified potential therapeutic targets for neuroblastoma include SAAL1 (patent CN119220682A), OLFM3 (patent CN119061142A), and PPEF1 (patent CN117568347A), but no studies have yet found an association between P5A ATPase and neuroblastoma. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of P5A ATPase as a target in the preparation of therapeutic drugs for neuroblastoma. This invention is the first to discover that P5A ATPase affects the expression level of LRP8 protein and the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain, thereby influencing the growth and proliferation of neuroblastoma cells, providing a new target for the development of therapeutic drugs for neuroblastoma.

[0005] The specific technical solution of this invention is as follows:

[0006] In a first aspect, the present invention provides the application of P5A ATPase as a target in the preparation of drugs for the treatment of neuroblastoma.

[0007] Neuroblastoma is highly dependent on low-density lipoprotein receptor-associated protein 8 (LRP8), and inhibition of LRP8 can induce ferroptosis in neuroblastoma cells. This invention found that knocking out P5A ATPase significantly downregulated intracellular LRP8 protein expression and reduced endoplasmic reticulum translocation of LRP8 signal peptide-guided peptide chains. Furthermore, treatment of cells with the P5A ATPase inhibitors sodium metavanadate and N,N-dicyclohexylcarbodiimide inhibited intracellular LRP8 signal peptide-guided peptide chain translocation and LRP8 protein expression, and also inhibited the growth and proliferation of neuroblastoma cells. Therefore, P5A ATPase can serve as a therapeutic target for neuroblastoma and can be used to develop therapeutic drugs for neuroblastoma.

[0008] Secondly, this invention provides the application of P5A ATPase inhibitors in the preparation of drugs for the treatment of neuroblastoma.

[0009] Preferably, the P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide (DCC).

[0010] Preferably, the P5A ATPase inhibitor is an ATP13A1 inhibitor.

[0011] Preferably, the neuroblastoma treatment drug is a drug that inhibits the growth and proliferation of neuroblastoma cells, or a drug that reduces the expression of LRP8 protein (low-density lipoprotein receptor-associated protein 8), or a drug that inhibits the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain.

[0012] Preferably, the neuroblastoma treatment includes a P5A ATPase inhibitor.

[0013] Preferably, the neuroblastoma treatment drug further includes pharmaceutically acceptable excipients; the excipients include one or more of carriers, excipients, and solvents.

[0014] Preferably, the dosage form of the neuroblastoma treatment drug is an oral liquid, capsule, tablet, pill, or injection, and the administration method is oral, intravenous, subcutaneous, or intramuscular injection; the neuroblastoma treatment drug is used on humans or animals.

[0015] Thirdly, the present invention provides a method for constructing an in vitro cell model with low expression of LRP8 and CTSA proteins, comprising the following steps: adding a P5A ATPase inhibitor to the cell culture medium, culturing the cells, and obtaining a cell model with low expression of LRP8 and CTSA proteins.

[0016] This invention reveals that treating cells with a P5A ATPase inhibitor reduces the expression levels of LRP8 and CTSA proteins. This method allows for the construction of cell models with low LRP8 and CTSA protein expression, which can be used to study the physiological functions of these proteins and develop related therapeutic drugs.

[0017] Preferably, the P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide.

[0018] Furthermore, the amount of sodium metavanadate added to the cell culture medium is 10–15 μM; the amount of N,N-dicyclohexylcarbodiimide added to the cell culture medium is 0.4–0.8 mM.

[0019] Fourthly, the present invention provides a method for constructing a cell model of abnormal endoplasmic reticulum translocation of peptide chains guided by LRP8 and CTSA signal peptides in vitro, comprising the following steps: adding a P5A ATPase inhibitor to the cell culture medium, culturing the cells, and obtaining a cell model of abnormal endoplasmic reticulum translocation of peptide chains guided by LRP8 and CTSA signal peptides.

[0020] Successful translocation of proteins in the endoplasmic reticulum (ER) is crucial for protein biosynthesis. ER translocation can be divided into three key steps: cytoplasmic targeting factors specifically recognize the protein's signal sequence; the interaction between the cytoplasmic targeting factor and its receptor on the ER membrane forms a translocation complex that targets the nascent protein to the ER; and the translocation complex on the ER membrane translocates the nascent protein into the endoplasmic lumen or inserts it into the ER membrane. As the first step in protein translocation, signal sequence recognition is essential for its proper functioning in the ER.

[0021] This invention reveals that treating cells with a P5A ATPase inhibitor inhibits the endoplasmic reticulum (ER) translocation of peptide chains guided by the LRP8 and CTSA signal peptides. A cell model of abnormal ER translocation guided by LRP8 and CTSA signal peptides can be constructed using this method, enabling the study of these ER translocation processes, their physiological functions, and the development of related therapeutic drugs.

[0022] Preferably, the P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide.

[0023] Furthermore, the amount of sodium metavanadate added to the cell culture medium is 10–15 μM; the amount of N,N-dicyclohexylcarbodiimide added to the cell culture medium is 0.4–0.8 mM.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) The present invention found that P5A ATPase affects the expression level of LRP8 protein and the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain, thereby affecting the growth and proliferation of neuroblastoma cells, providing a new target for the treatment of neuroblastoma.

[0026] (2) The present invention found that two P5A ATPase inhibitors (sodium metavanadate and N,N-dicyclohexylcarbodiimide) inhibit the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain and reduce the expression level of LRP8 protein, thereby inhibiting the growth and proliferation of neuroblastoma cells and providing new therapeutic drugs for neuroblastoma. Attached Figure Description

[0027] Figure 1The results show the effects of sodium metavanadate and DCC on the growth and proliferation of neuroblastoma cells. Among them: Figure 1 A is a microscopic image of neuroblastoma cells after treatment with DMSO, sodium metavanadate, and DCC. Figure 1 B represents the BrdU assay used to detect the effects of sodium metavanadate and DCC on the proliferation of neuroblastoma cells.

[0028] Figure 2 The results show the effects of P5A ATPase (ATP13A1) on protein translocation and expression levels. Among them: Figure 2 A represents the endoplasmic reticulum translocation of different signal peptides in wild-type cells and P5A ATPase / ATP13A1 knockout cells as detected by Western blotting. Figure 2 B represents the detection of LRP8::GFP protein levels in wild-type cells and P5A ATPase / ATP13A1 knockout cells using Western blotting. Figure 2 C represents the detection of CTSA::GFP protein levels in wild-type cells and P5A ATPase / ATP13A1 knockout cells using Western blotting.

[0029] Figure 3 The results show the effects of sodium metavanadate and DCC on LRP8 protein-related endoplasmic reticulum translocation and protein expression levels. Among them: Figure 3 A represents the effect of different drug treatments on the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain (LRP8SS::FLAG::DHFR::Opsin) as detected by Western blotting. Figure 3 B represents the effect of different drug treatments on the protein level of LRP8::GFP protein as detected by Western blotting.

[0030] Figure 4 The results show the effects of sodium metavanadate and DCC on CTSA protein-related endoplasmic reticulum translocation and protein expression levels. Among them: Figure 4 A represents the effect of different drug treatments on the endoplasmic reticulum translocation of the CTSA signal peptide-guided peptide chain (CTSASS::FLAG::DHFR::Opsin) as detected by Western blotting. Figure 4 B represents the effect of different drug treatments on the protein level of CTSA::GFP protein as detected by Western blotting. Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments.

[0032] General Implementation Examples

[0033] First, the present invention relates to the application of P5A ATPase as a target in the preparation of drugs for the treatment of neuroblastoma.

[0034] Second, the present invention relates to the use of P5A ATPase inhibitors in the preparation of drugs for the treatment of neuroblastoma.

[0035] In some specific embodiments, the P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide (DCC).

[0036] In some specific embodiments, the P5A ATPase inhibitor is an ATP13A1 inhibitor.

[0037] In some specific embodiments, the neuroblastoma treatment drug is a drug that inhibits the growth and proliferation of neuroblastoma cells, or a drug that reduces the expression level of LRP8 protein, or a drug that inhibits the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain.

[0038] In some specific embodiments, the neuroblastoma treatment drug includes a P5A ATPase inhibitor.

[0039] In some specific embodiments, the neuroblastoma treatment drug further includes pharmaceutically acceptable excipients; the excipients include one or more of a carrier, a propellant, and a solvent.

[0040] In some specific embodiments, the dosage form of the neuroblastoma treatment drug is an oral liquid, capsule, tablet, pill, or injection, and the administration method is oral, intravenous, subcutaneous, or intramuscular injection; the neuroblastoma treatment drug is applied to humans or animals.

[0041] Third, the present invention relates to a method for constructing an in vitro cell model with low expression of LRP8 and CTSA proteins, comprising the following steps: adding a P5A ATPase inhibitor to the cell culture medium, culturing the cells, and obtaining a cell model with low expression of LRP8 and CTSA proteins.

[0042] In some specific embodiments, the P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide. Specifically: in one specific embodiment, the P5A ATPase inhibitor is sodium metavanadate, and the amount added to the cell culture medium is 10–15 μM; in another specific embodiment, the P5A ATPase inhibitor is N,N-dicyclohexylcarbodiimide, and the amount added to the cell culture medium is 0.4–0.8 mM.

[0043] Fourth, the present invention relates to a method for constructing an in vitro cell model of abnormal endoplasmic reticulum translocation of peptide chains guided by LRP8 and CTSA signal peptides, comprising the following steps: adding a P5A ATPase inhibitor to the cell culture medium, culturing the cells, and obtaining a cell model of abnormal endoplasmic reticulum translocation of peptide chains guided by LRP8 and CTSA signal peptides.

[0044] In some specific embodiments, the P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide. Specifically: in one specific embodiment, the P5A ATPase inhibitor is sodium metavanadate, and the amount added to the cell culture medium is 10–15 μM; in another specific embodiment, the P5A ATPase inhibitor is N,N-dicyclohexylcarbodiimide, and the amount added to the cell culture medium is 0.4–0.8 mM. Specific Implementation

[0046] The present invention will now be described through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0047] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used in this invention are conventional in the art and can be obtained through conventional commercial means; unless otherwise specified, the methods used in this invention are conventional methods in the art.

[0048] Example 1: Effects of P5A ATPase / ATP13A1 on protein translocation and protein expression levels

[0049] 1.1 Experimental Materials and Methods

[0050] 1.1.1 Cell origin

[0051] Human embryonic kidney cells (HEK293FT) were obtained from the American Type Culture Collection (ATCC) and are adherent cells.

[0052] 1.1.2 Construction of knockout cell lines

[0053] The HEK293FT cell line with P5A ATPase / ATP13A1 knockout was constructed using CRISPR-Cas9 gene editing technology, as follows:

[0054] (1) Vector Construction: Based on the sequence information of the target gene, search for available sgRNA target sequences (located on the exons of the gene) on the sgRNA design website Sequence Scan for CRISPR, and then compare the specificity of the sgRNAs in the NCBI or UCSC database. Select 2-3 sgRNA sequences with high scores and good specificity. After determining the sgRNA target sequence, design and synthesize primers, and use overlap PCR to insert the sgRNA target sequence into the pGL3-U6-sgRNA-PGK-puromycin (Plasmid#51133) vector.

[0055] (2) Cell seeding: After the HEK293FT cell line was revived and passaged twice, it was seeded into a 6cm culture dish.

[0056] (3) Transfection: Add 8 μL of Lipofectamine 2000 (Invitrogen, 11668019) to 500 μL Opti-MEM, vortex and mix well, then let stand for 5 min. Add 1 μg of sgRNA and 3 μg of Cas9 to 500 μL Opti-MEM and mix well. Add the Lipofectamine 2000 mixture to the plasmid mixture, vortex and mix well, centrifuge for 10 seconds, and incubate at room temperature for 20 min. Take out the cells seeded the day before and replace half of the medium with fresh antibiotic-free DMEM. Label the cells. Add the incubated plasmid / Lipofectamine 2000 transfection reagent complex evenly to the cell culture dish, and gently shake the cell culture dish to distribute it evenly. 6 h after cell transfection, replace with fresh antibiotic-free DMEM medium.

[0057] (4) Drug screening: 24 h after transfection, blastin (10 μg / mL) and puromycin (1 μg / mL) were added. Because the constructed sgRNA vector contains the puromycin resistance gene and the Cas9 protein vector contains the blastin resistance gene, the simultaneous addition of blastin and puromycin can kill cells that were not successfully transfected with Cas9 and sgRNA, or those that were only successfully transfected with one of the plasmids. Cells containing both Cas9 and sgRNA plasmids are preserved.

[0058] (5) Monoclonal Cells: After cells screened with Blastidin and Puromycin reach a full size in a 6cm culture dish, remove the old culture medium using an aspirator; gently wash the cells once with 2.5mL of PBS buffer using a pipette; then add 500μL of Trypsin to detach the cells; finally, add 1mL of DMEM medium to stop the Trypsin reaction and gently disperse the cells using a pipette. Collect the cells into a 15mL centrifuge tube and centrifuge at 1000g for 3min to obtain the cell pellet. Resuspend the cells in DMEM medium containing the double antibiotics and separate them into single cells using a cell strainer. Dilute the cells to a concentration of approximately 10 cells per milliliter of culture medium using a hemocytometer and a serial dilution method. Then aliquot the cells into 96-well plates at 100μL per well (approximately 1 cell per well). Three 96-well plates are generally sufficient. Alternatively, flow cytometry can be used to separate single cells.

[0059] (6) Cell expansion: Select well-growing monoclonal cells from 96-well plates and passage them to 12-well plates after they have grown.

[0060] (7) Western Blot identification: The cell line in the 12-well plate was passaged again and excess cells were collected. The collected cells were treated with 0.5% NP40 cell lysis buffer, and the protein level of the target gene was detected by Western Blot. If the protein level was significantly reduced compared with wild-type cells or even not expressed, it indicated that the gene knockout may have been successful.

[0061] (8) PCR identification: Cell lines with significantly reduced or no protein expression in Western blotting results were screened out. PCR and next-generation sequencing were used to confirm successful knockout and to determine the cell knockout type.

[0062] (9) Cryopreservation: Select two cell lines that are correctly identified by both Western Blot and PCR, amplify them, and then cryopreserve them.

[0063] 1.1.3 Western blot analysis of the effects of p5A ATPase knockout on protein translocation and expression levels

[0064] After viable cell counting, HEK293FT wild-type cells and P5A ATPase / ATP13A1 knockout cells were seeded at 5 × 10⁶ cells per cell. 5 and 8×10 5 Cells were transferred to 35 mm diameter culture dishes and cultured at 37°C in a 5% CO2 cell culture incubator for one day, followed by transient transfection with the corresponding protein. Fresh culture medium was added 6 hours after transfection. Cells were collected the following day for Western blotting (WB) analysis.

[0065] 1.2 Experimental Results

[0066] The experimental results regarding the effects of P5A ATPase / ATP13A1 on protein translocation and protein expression levels are as follows: Figure 2 As shown. Figure 2 A shows the experimental results of Western blotting (WB) detection of the endoplasmic reticulum translocation of LRP8 and CTSA signal peptide-guided peptide chains. The Opsin tag contains N-glycosylation sites capable of glycosylation in the endoplasmic reticulum, indicating whether the protein has successfully translocated into the reticulum. "●" indicates a glycosylated band (successful translocation), and "○" indicates a non-glycosylated band (unsuccessful translocation). Figure 2 B represents the experimental results of WB detection of LRP8::GFP protein expression, where Actin is the internal control. Figure 2 C represents the experimental results of WB detection of CTSA::GFP protein expression, where Actin is the internal control.

[0067] Previous studies have found that high-risk MYCN-amplified neuroblastomas are highly dependent on LRP8, and LRP8 has been identified as a key factor in selenium / selenocysteine ​​metabolism. Inhibition of LRP8 can induce ferroptosis in neuroblastoma cells.

[0068] The experimental results in this embodiment show that knocking out P5A ATPase / ATP13A1 in human HEK293FT cells does not affect the normal growth of HEK293FT cells, but P5A ATPase knockout severely affects the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain (LRP8SS::FLAG::DHFR::Opsin) in HEK293FT cells (e.g., Figure 2 (As shown in A). To further confirm whether P5A ATPase can serve as a therapeutic target for high-risk MYCN-amplified neuroblastoma, the effect of P5A ATPase knockout on LRP8 protein levels was examined. The results showed that P5A ATPase knockout led to a significant downregulation of LRP8 protein levels (e.g., as shown in A). Figure 2 (As shown in B). The above results indicate that P5A ATPase may serve as a novel therapeutic target for neuroblastoma.

[0069] Furthermore, this embodiment also used a P5A ATPase / ATP13A1 knockout cell line to detect the effect of P5A ATPase knockout on CTSA. The results showed that P5A ATPase knockout severely affected the endoplasmic reticulum translocation of the CTSA signal peptide-guided peptide chain (CTSASS::FLAG::DHFR::Opsin) (e.g., Figure 2As shown in A), this embodiment also examined the effect of P5A ATPase knockout on CTSA protein levels. The results showed that P5A ATPase knockout led to a significant downregulation of CTSA protein levels (e.g., as shown in A). Figure 2 (as shown in C).

[0070] Example 2: Effects of sodium metavanadate and DCC on LRP8 protein-related endoplasmic reticulum translocation and protein expression levels 2.1 Experimental materials and methods

[0071] 2.1.1 Cell origin

[0072] Human embryonic kidney cells (HEK293FT) were obtained from the American Type Culture Collection (ATCC) and are adherent cells.

[0073] 2.1.2 Western blotting analysis of the effects of sodium metavanadate and DCC on LRP8 protein-related endoplasmic reticulum translocation and protein expression levels.

[0074] Live cell count, 1.5 × 10⁻⁶ 5 Live cells were seeded at a density of 1 mL / mL in 35 mm culture dishes and cultured at 37°C in a 5% CO2 cell culture incubator for 1 day, followed by transient transfection with the corresponding protein. Six hours after transfection, the culture medium was replaced with fresh medium and treated with dimethyl sulfoxide (DMSO), N,N-dicyclohexylcarbodiimide (DCC) (0.5 mM), oligomycin A (1 μM), and sodium metavanadate (10 μM), respectively. Cells were collected after 2 days for Western blotting (WB) analysis.

[0075] 2.2 Experimental Results

[0076] The effects of sodium metavanadate and DCC on LRP8 protein-related endoplasmic reticulum translocation and protein expression levels are shown in the experimental results. Figure 3 As shown, Figure 3 A shows the experimental results of Western blotting (WB) detection of the endoplasmic reticulum translocation of LRP8 signal peptide-guided peptide chains under different treatments. The Opsin tag contains N-glycosylation sites capable of glycosylation in the endoplasmic reticulum, indicating whether the protein has successfully translocated into the reticulum. "●" indicates a glycosylated band (successful translocation), and "○" indicates a non-glycosylated band (unsuccessful translocation). Figure 3 B represents the experimental results of Western blot (WB) detection of LRP8::GFP protein expression under different treatments, where Actin is the internal control.

[0077] To further demonstrate the application of P5A ATPase as a target in tumor therapy, this embodiment treated HEK293FT cells for 24 hours with control solutions DMSO, oligomycin A, sodium metavanadate, and DCC, respectively. The endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain was then examined. The results showed that both sodium metavanadate and DCC treatments led to severe defects in the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain (e.g., ...). Figure 3 (As shown in A). Furthermore, the expression level of LRP8 protein was detected in this embodiment, and the results showed that both sodium metavanadate and DCC treatment significantly reduced the amount of LRP8 protein (e.g., ...). Figure 3 (As shown in B). The above results indicate that sodium metavanadate and DCC affect the function of P5A ATPase, thereby reducing the expression level of LRP8 and inhibiting the endoplasmic reticulum translocation of the peptide chain guided by the LRP8 signal peptide.

[0078] Example 3: Effects of sodium metavanadate and DCC on CTSA protein-related endoplasmic reticulum translocation and protein expression levels 3.1 Experimental materials and methods

[0079] 3.1.1 Cell Source

[0080] Human embryonic kidney cells (HEK293FT) were obtained from the American Type Culture Collection (ATCC) and are adherent cells.

[0081] 3.1.2 Western blotting analysis of the effects of sodium metavanadate and DCC on CTSA protein-related endoplasmic reticulum translocation and protein expression levels.

[0082] Live cell count, 1.5 × 10⁻⁶ 5 Live cells were seeded at a density of / mL in 35mm diameter culture dishes and cultured at 37℃ in a 5% CO2 cell culture incubator for 1 day, followed by transient transfection with the corresponding protein. Six hours after transfection, the culture medium was replaced with fresh medium and treated with DMSO, DCC (0.5mM), oligomycin A (1μM), and sodium metavanadate (10μM), respectively. Cells were collected 2 days later for Western blotting (WB) analysis.

[0083] 3.2 Experimental Results

[0084] The experimental results of the effects of sodium metavanadate and DCC on CTSA protein-related endoplasmic reticulum translocation and protein expression levels are as follows: Figure 4 As shown, Figure 4A shows the experimental results of Western blotting (WB) detection of the endoplasmic reticulum translocation of the CTSA signal peptide-guided peptide chain under different treatments. Among them, the Opsin tag contains N-glycosylation modification sites that can undergo glycosylation modification in the endoplasmic reticulum, which are used to indicate whether the protein has successfully translocated into the endoplasmic reticulum; "●" indicates the glycosylated band (successful translocation), and "○" indicates the unglycosylated band (unsuccessful translocation). Figure 4 B represents the experimental results of Western blot (WB) detection of CTSA::GFP protein expression under different treatments, where Actin is the internal control.

[0085] In this embodiment, HEK293FT cells were treated with control solutions DMSO, oligomycin A, sodium metavanadate, and DCC for 24 hours, respectively. The endoplasmic reticulum translocation of the CTSA signal peptide-guided peptide chain was then examined. The results showed that both sodium metavanadate and DCC treatments led to severe defects in the endoplasmic reticulum translocation of the CTSA signal peptide-guided peptide chain (e.g., Figure 4 (As shown in A). Furthermore, the expression level of CTSA protein was also detected in this embodiment, and the results showed that both sodium metavanadate and DCC treatment significantly reduced the protein content of CTSA (e.g., ...). Figure 4 (As shown in B). The above results indicate that sodium metavanadate and DCC affect the function of P5A ATPase, thereby reducing the expression level of CTSA and inhibiting the endoplasmic reticulum translocation of the peptide chain guided by the CTSA signal peptide.

[0086] Example 4: Effects of sodium metavanadate and DCC on the growth and proliferation of neuroblastoma cells

[0087] 4.1 Experimental Materials and Methods

[0088] 4.1.1 Cell Source

[0089] Neuroblastoma cells (SH-SY5Y) were obtained from the American Type Culture Collection (ATCC) and are adherent cells.

[0090] 4.1.2 Photographic analysis of the effects of sodium metavanadate and DCC on neuroblastoma cell growth; viable cell count, 1.5 × 10⁻⁶. 5 / mL of live cells were seeded in 35mm diameter culture dishes and cultured at 37℃ in a 5% CO2 cell culture incubator for 1 day. Then, DMSO, DCC (0.5mM in the culture medium) and sodium metavanadate (10μM in the culture medium) were added for 2 days, and the growth status of the cells was analyzed by taking pictures.

[0091] 4.1.3 BrdU assay to detect the effects of sodium metavanadate and DCC on the proliferation of neuroblastoma cells

[0092] 1.5×105 Cells were seeded at 1 / mL in 35mm diameter culture dishes (with a coverslip placed inside), cultured for 1 day, and then synchronized for 3 days with culture medium containing 0.4% FBS, ensuring that the vast majority of cells were in the G0 phase. Synchronized cells were treated with DMSO, DCC (0.5mM in the medium), and sodium metavanadate (10μM in the medium) for 24 hours, respectively. BrdU (stock solution: 1.0mg / mL, final concentration 0.03μg / mL) was added, and the cells were incubated at 37℃ for 40 min. The culture medium was discarded, and the slides were washed three times with PBS. The cells were fixed with methanol / acetic acid for 10 min. The fixed slides were air-dried and inactivated with 0.3% H2O2-methanol for 30 min to inactivate endogenous oxidases. The cells were blocked with 5% normal rabbit serum. The nucleic acids were denatured with formamide at 100℃ for 5 min. After cooling in an ice bath, the cells were washed with PBS, and the primary antibody, anti-mouse BrdU monoclonal antibody (working concentration 1:50), was added. For the negative control, PBS or serum was added. The detection was performed using the ABC method, with hematoxylin or eosin staining. The total number of cells and the number of BrdU-positive cells were randomly counted in 10 high-power fields under a microscope, and the labeling index (LI) was calculated.

[0093] 4.2 Experimental Results

[0094] The experimental results on the effects of sodium metavanadate and DCC on the growth and proliferation of neuroblastoma cells are as follows: Figure 1 As shown. Figure 1 A shows microscopic images of neuroblastoma cells under different treatments. Figure 1 B represents the results of the BrdU assay for detecting neuroblastoma cell proliferation under different treatments. The quantification of cell proliferation was obtained from four biological replicates and is expressed as mean ± SEMs. "***" indicates p<0.001, and "****" indicates p<0.0001 (t-test).

[0095] To further explore the role of sodium metavanadate and DCC in targeting P5A ATPase in tumor treatment, this example examined the effects of DCC and sodium metavanadate on the growth of neuroblastoma cells SH-SY5Y. The results showed that both DCC and sodium metavanadate significantly affected the growth of neuroblastoma cells SH-SY5Y (e.g., ...). Figure 1 (As shown in A). Furthermore, in this embodiment, the proliferation of neuroblastoma cells SH-SY5Y was detected using the BrdU assay. The results showed that both DCC and sodium metavanadate significantly inhibited the proliferation of neuroblastoma cells SH-SY5Y (e.g., as shown in A). Figure 1 (As shown in B). This indicates that P5AATPase can serve as a drug target for the treatment of neuroblastoma, and DCC and sodium metavanadate are potential drugs.

[0096] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Application of N,N-dicyclohexylcarbodiimide in the preparation of drugs for the treatment of neuroblastoma.

2. The application according to claim 1, characterized in that, The neuroblastoma treatment drug is a drug that inhibits the growth and proliferation of neuroblastoma cells.

3. The application according to claim 1, characterized in that, The neuroblastoma treatment drug is a drug that reduces the expression level of LRP8 protein.

4. The application according to claim 1, characterized in that, The neuroblastoma treatment drug is a drug that inhibits the endoplasmic reticulum translocation of peptide chains guided by the LRP8 signal peptide.

5. The application according to claim 1, characterized in that, The neuroblastoma treatment also includes pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The auxiliary materials include a carrier.

7. The application according to claim 5, characterized in that, The excipients include excipients.

8. The application according to claim 5, characterized in that, The excipients include solvents.

9. The application according to claim 1, characterized in that, The dosage form of the neuroblastoma treatment drug is oral liquid, capsule, tablet, pill or injection.

10. The application according to claim 1, characterized in that, The neuroblastoma treatment drugs can be administered orally, intravenously, subcutaneously, or intramuscularly.

11. The application according to claim 1, characterized in that, The neuroblastoma treatment drugs are intended for use in humans or animals.

Images