New application of lansoprazole

Abstract

The invention relates to the field of biological medicine, in particular to novel application of lansoprazole. The invention discloses application of lansoprazole to preparation of a medicine for treating breast cancer by inhibiting an FASN target spot to act on breast cancer stem cells. The lansoprazole is applied to preparation of the anti-breast cancer medicine by inhibiting expression of FASN and depletion of CD8 + T cells and remodeling a breast cancer immune microenvironment. And a basis is provided for clinical transformation of lansoprazole'new use of old medicine '.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a new use of lansoprazole. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women worldwide. Its recurrence, metastasis and treatment resistance are closely related to breast cancer stem cells (BCSCs). BCSCs have the characteristics of self-renewal, multidirectional differentiation and high drug resistance, making them difficult to eradicate with traditional therapies. Our previous studies have found that BCSCs can induce CD8 + T cell exhaustion, which creates a suppressive immune microenvironment, leads to poor clinical outcomes in breast cancer patients, but the underlying mechanisms remain unclear. Furthermore, BCSCs are known to express multiple transporters (such as multidrug resistance protein 1 (MDR1), MRP1, and BCRP1) as well as proteins involved in DNA repair (such as Ung, Uhrf1, and Xrcc5). These proteins confer resistance to chemotherapy and radiotherapy in BCSCs, reducing the effectiveness of conventional treatments for this subpopulation. Breast cancer stem cells (BCSCs) play a crucial role in breast cancer recurrence, metastasis, and treatment resistance, and are a major cause of breast cancer recurrence and metastasis. Therefore, targeting BCSCs for treatment holds promise for addressing current clinical bottlenecks in breast cancer treatment. Fatty acid synthase (FASN), a key enzyme in lipid metabolism, is highly expressed in breast cancer cells, but its role and mechanism in BCSCs remain unclear. In a patent application filed on the same day, the inventors discovered and validated the role of FASN in targeting BCSCs, providing guidance for the screening and development of inhibitors targeting FASN in BCSCs. Summary of the Invention

[0003] In view of the problems in the prior art, the present invention aims to provide a new use of lansoprazole, specifically:

[0004] Use of lansoprazole in the preparation of a drug for inhibiting the activity of breast cancer stem cells.

[0005] The lansoprazole is used in preparing a drug for inhibiting the activity of breast cancer stem cells by acting on the FASN target.

[0006] The lansoprazole is used in preparing a drug for treating breast cancer by inhibiting the FASN target point and acting on breast cancer stem cells.

[0007] Furthermore, lansoprazole inhibited the expression of FASN and CD8 + The application of T cell depletion and reshaping of the breast cancer immune microenvironment in the preparation of anti-breast cancer drugs.

[0008] Beneficial effects: It was verified that the FASN inhibitor lansoprazole weakened the characteristics of BCSCs by acting on the FASN target to activate the Wnt / β-catenin signaling pathway. Lansoprazole inhibited the expression of FASN and CD8 + The exhaustion of T cells produces an anti-breast cancer effect by reshaping the immune microenvironment of breast cancer, providing a basis for the clinical transformation of lansoprazole into a new use of an old drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Results show the effects of the proton pump inhibitor lansoprazole on the expression, activity and characteristics of FASN in BCSCs: (A) Molecular docking diagram of lansoprazole and FASN; (B) Lansoprazole significantly inhibited the expression of FASN in BCSCs; (C) Lansoprazole significantly inhibited the activity of FASN in BCSCs; (D) Lansoprazole significantly inhibited the proliferation of BCSCs; (E) Lansoprazole significantly inhibited the colony-forming ability of BCSCs; (F) Lansoprazole significantly inhibited the sphere-forming ability of BCSCs.

[0010] Figure 2 The results of the effects of lansoprazole on the Wnt / β-catenin signaling pathway in BCSCs: (AB) Lansoprazole significantly inhibited the nuclear translocation of β-catenin in BCSCs; (C) Lansoprazole significantly regulated key signaling proteins in the Wnt / β-catenin signaling pathway in BCSCs; (D) Lansoprazole significantly inhibited the secretion of Wnt3a in BCSCs.

[0011] Note: Figure 1-Figure 2 The data are expressed as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.

[0012] Figure 3 Results of the effects of the proton pump inhibitor lansoprazole on BCSC tumor formation in animal models: (A) Example of an animal model in which lansoprazole inhibits BCSC tumor formation; (B) Lansoprazole inhibits the tumor weight of BCSC; (C) Lansoprazole inhibits the tumor size of BCSC; (D) Lansoprazole inhibits the expression of Ki-67, Wnt3a, and β-catenin in neoplastic BCSC.

[0013] Figure 4 FASN high PANCK +Results of the effects of cells on the exhaustion of CD8+ T cells: A. Multicolor immunofluorescence labeling of whole-mount in situ breast cancer tissue of animals; B. Example of single-color labeling; C. Spatial distribution of FASNhigh PANCK+ cells and CD8+ T cells with and without exhaustion; D. Comparison of the number of exhausted CD8+ T cells between FASNhigh PANCK+ cells and FASNlow PANCK+ cells within a spatial range of 20μm.

[0014] Figure 5 Results of the effects of lansoprazole on 4T1 orthotopic tumor growth and immune microenvironment: A. Tumor formation in the orthotopic breast cancer model of BALB / C mice treated with lansoprazole, blank control; B. Effect of lansoprazole on tumor weight in the orthotopic breast cancer model; C. Effect of lansoprazole on tumor size in the orthotopic breast cancer model; D. Example of multicolor immunofluorescence staining; E. Inhibitory effect of lansoprazole on FASN in cancer cells of the orthotopic cancer model; F. Effect of lansoprazole on the number of CD8+ T cells in the cancer tissue of the orthotopic cancer model; G. Lansoprazole inhibits the exhaustion of CD8+ T cells in the cancer tissue of the orthotopic cancer model.

[0015] Note: Figure 3-Figure 5 The data are expressed as mean ± SD, n = 6, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0016] Given the critical role of FASN in maintaining the stemness of breast cancer stem cells (BCSCs) and its involvement in activation of the Wnt / β-catenin signaling pathway, we further explored potential inhibitors targeting FASN.

[0017] 1. Research Methods

[0018] 1. Cell Culture and Drug Treatment: The human breast cancer cell lines MCF-7 and MDA-MB-468 used in this study were obtained from the American Type Culture Collection (ATCC). M3k is a 3000-fold doxorubicin-resistant cell line induced in MCF-7 parent cells. MCF-7 and MDA-MB-468 cells were cultured in Dulbecco's Methionine Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 atmosphere. M3k cells were cultured in RPMI 1640 medium supplemented with 10% FBS under the same conditions. All cell lines were passaged no more than eight times after thawing. Lansoprazole (Lot No. PHR1390) used in this study was purchased from Supelco.

[0019] 2. Generation of FASN-modified cell lines: To construct FASN overexpression and knockout cell lines, we used genetic engineering techniques.

[0020] FASN Overexpression: Human FASN cDNA was cloned into the pcDNA3 vector. MCF-7 breast cancer cells were transfected with either FASN-pcDNA3 or empty pcDNA3 vector using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.). 48 hours later, transfected cells were selected with 800 μg / mL G418 for two weeks to establish stable cell lines. Successfully transfected cells were then expanded for further experiments.

[0021] FASN knockdown: Design a shRNA sequence targeting FASN mRNA (AACCCTGAGATCC CAGCGCTG) and clone it into an appropriate plasmid. Transfect M3k cells with high FASN expression with either the FASN-targeting shRNA plasmid or a scrambled shRNA control plasmid using Lipofectamine 3000. After 24 hours, select the transfected cells with 800 μg / mL G418 for two weeks to establish stable cell lines. Identify clones with efficient FASN knockdown and expand them for subsequent use.

[0022] The FASN-overexpressing MCF-7 cell line and its control, as well as the FASN-knockdown M3k cell line and its control were generated by the above method.

[0023] 3. Colony formation assay: The colony formation ability of M3k and MCF-7 cell lines with specific genetic modifications was evaluated.

[0024] The M3k cell line included the M3k / Scr (scrambled shRNA control) and M3k / ShFASN (FASN knockdown) groups; the MCF-7 cell line included the MCF-7 / Vec (vector control) and MCF-7 / FASN (FASN overexpression) groups.

[0025] 300 viable cells from each group were inoculated into 6-well plates (Corning) and cultured at 37°C, 5% CO2 for 24 hours, repeated three times. After incubation, the cells were cultured in complete medium for 10 days. The colonies were then fixed for 5 minutes using a solution of acetic acid and methanol in a 1:7 ratio. After fixation, the colonies were stained with 0.5% crystal violet for 20 minutes at room temperature and then washed three times with PBS to remove excess dye. Colonies containing more than 50 cells were counted using an inverted phase contrast microscope (Nikon Corporation). Representative images of the stained colonies were captured using a Canon scanner (CanoScan 5600F) for visualization and recording.

[0026] 4. Spheroid Formation Assay: Evaluate the sphere-forming ability of M3k and MCF-7 cell lines with specific genetic modifications.

[0027] The M3k cell line included the M3k / Scr (scrambled shRNA control) and M3k / ShFASN (FASN knockdown) groups; the MCF-7 cell line included the MCF-7 / Vec (vector control) and MCF-7 / FASN (FASN overexpression) groups.

[0028] To evaluate spheroid formation ability, we seeded 5000 cells per well in a 6-well ultra-low attachment plate (Corning, Inc.). Cells were cultured at 37°C in a 5% CO2 atmosphere using cancer stem cell (CSC) enrichment medium. The medium consisted of serum-free DMEM / F12 medium supplemented with 2% B27, 20 ng / mL epidermal growth factor (EGF), and 20 ng / mL recombinant human basic fibroblast growth factor (RH-bFGF) (all from Gibco, Thermo Fisher Scientific, Inc.). After incubation for 7 days, the tumor spheres formed were analyzed. Spheroids with a diameter of at least 50 μm were imaged and counted using an inverted phase contrast microscope (Nikon Corporation) at 100x magnification.

[0029] 5. Western Blot Analysis: Cells were lysed in ice-cold RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1% Triton-X-100, and 0.1% sodium deoxycholate) supplemented with phosphatase inhibitors (1 mM Na3VO4) and protease inhibitors (1 mM phenylmethylsulfonyl fluoride). Cell lysates were centrifuged at 12,000 x g for 10 minutes at 4°C to remove debris. Protein concentration was measured using the Pierce Fast Gold Biuret Acid (BCA) Kit (ThermoFisher Scientific, Inc.). 20-50 μg of protein per lane was separated on a 6% or 12% SDS-PAGE gel and transferred to a Sequi-Blot PVDF membrane (Bio-Rad Laboratories, Inc.). The membrane was blocked with 5% skim milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 2 hours at room temperature. Primary antibodies were incubated with the membrane overnight at 4°C. Specific antibodies used included FASN (Affinity, Product No. DF6106), APC (Proteintech, Product No. 19782-1-AP), Axin-1 (Bioss, Product No. bs-21732R), GSK-3β (Abcam, Product No. ab93926), TCF-4 (Proteintech, Product No. 13838-1-AP), and TCF-7 (Proteintech, Product No. 14464-1-AP). β-catenin and GAPDH were used as internal controls. After primary antibody incubation, the membrane was washed three times with TBST and then incubated with the appropriate primary antibody (anti-mouse, product number A2429, or anti-rabbit, product number A3937, from Sigma-Aldrich, Merck KGaA) for 2 hours at room temperature. Protein bands were visualized using enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) and detected using an X-ray film system (Ece Scientific Co., Inc.). Band intensities were quantified using ImageJ software (version 1.52; National Institutes of Health).

[0030] 6. Enzyme-linked immunosorbent assay: Coat a microplate with purified anti-WNT3a antibody and incubate overnight at 4°C. The next day, wash the wells with PBS containing 0.05% Tween-20 to remove unbound antibody. Subsequently, add 100 μL of standard or sample to each well and incubate at 37°C for 2 hours. After incubation, discard the liquid and pat the wells dry on absorbent paper. Then, add 100 μL of biotinylated anti-WNT3a antibody to each well and incubate at 37°C for 1 hour. After washing with PBS-T, add 100 μL of horseradish peroxidase (HRP)-conjugated streptavidin to each well and incubate at 37°C for 1 hour. Then, wash the wells five times with PBS-T. For color development, add 90 μL of TMB (3,3',5,5'-tetramethylbenzidine) substrate solution to each well and incubate at 37°C in the dark for 15-30 minutes. The reaction was terminated by adding 50 μL of 2N sulfuric acid to each well, causing the color to change from blue to yellow. The optical density (OD) of each well was measured at 450 nm using a microplate reader. The OD value is proportional to the concentration of WNT3a in the sample, and a standard curve was generated to determine the concentration of WNT3a in the test samples.

[0031] 7. Analysis of tumor microenvironment and immune status

[0032] To explore the differences in biological functions between high-risk and low-risk groups in the model, we first performed gene set enrichment analysis (GSEA). We then used the ESTIMATE package to specifically analyze the composition of the tumor microenvironment (TME), including the immune score, stromal score, and ESTIMATE score in tumor purity. The CIBERSORT algorithm was then used to obtain the expression levels of 22 immune cells in patients with different risk scores. Furthermore, we used the Wilcoxon test to explore differences in immune scores, immune checkpoint expression, and immune cell infiltration between different risk groups in the model and to analyze the correlation between immune cells and immune cell infiltration.

[0033] 8. Multicolor Immunofluorescence Encoding Detection

[0034] Multiplex immunofluorescence staining of breast cancer tissue samples was performed using the AlphaTSA Multicolor Fluorescent Staining Kit from Beijing Opson Biotechnology. Briefly, sections were deparaffinized with xylene and absolute ethanol to remove paraffin residues and then rehydrated with graded ethanol. Subsequently, sections were rinsed twice with distilled water and retrieval was performed using the antigen retrieval solution provided by the kit. After cooling, sections were rinsed three times with PBST and immersed in blocking solution for 15 minutes at room temperature. Diluted primary antibodies were incubated at 37°C for 1 hour, followed by three rinses with PBST. The samples were then incubated with secondary antibodies at 37°C for 10 minutes, rinsed three times again with PBST, and treated with fluorescent dyes for 5 minutes at room temperature. The staining process was repeated to ensure complete labeling of all relevant markers. Finally, sections were treated with a nuclear dye (DAPI) for 8 minutes at room temperature. After rinsing with PBST, sections were mounted for subsequent image scanning. Images were acquired using a ZEISS Axioscan7 whole-slice imaging system and analyzed using ZEN 3.3 software. Quantitative analysis was performed using StrataQuest software (TissueGnostics), including calculation of parameters such as nuclear area, fluorescence intensity, and cell density per cell region, to identify positive cells.

[0035] 9. Mouse Mammary Fat Pad Orthotopic Tumor Formation Model

[0036] 1×10 5 4T1 cells were inoculated into the fat pad next to the mammary gland of BALB / C mice and the BALB / C mice were kept. When the tumor reached about 50 mm, 3 Around 3:00 a.m., start medication.

[0037] 10. Statistical Analysis: Data were analyzed using R software (version 4.3.1). Unless otherwise specified, all data are expressed as mean ± SD (n = 3). Differences were considered statistically significant when P < 0.05.

[0038] 2. Results

[0039] 1. Lansoprazole kills BCSCs by inhibiting FASN

[0040] We used computer-aided drug design (CADD) and high-throughput screening to identify potential FASN inhibitors and ultimately selected lansoprazole as a promising candidate ( Figure 1A). To evaluate the effect of lansoprazole on FASN in BCSCs, we treated isolated and purified BCSCs (MCF-7 (CD44+ / CD24-) and MDA-MB-468 (CD44+ / CD24-)) with lansoprazole. Western blot analysis confirmed that lansoprazole significantly downregulated FASN protein expression and significantly reduced FASN enzyme activity in BCSCs, indicating effective inhibition of FASN function ( Figure 1 BC). Subsequent functional experiments showed that lansoprazole treatment significantly reduced the proliferation capacity of BCSCs ( Figure 1 D). The clone formation experiment further showed that lansoprazole significantly inhibited the clone formation ability of these cells ( Figure 1 E). In addition, lansoprazole significantly impaired the sphere-forming ability of BCSCs, indicating a significant inhibitory effect on their self-renewal properties ( Figure 1 F). These findings indicate that lansoprazole significantly reduces the proliferation and self-renewal capacity of BCSCs by effectively inhibiting the expression and activity of FASN in them. This highlights lansoprazole as a potential therapeutic agent to target FASN to inhibit the stem cell properties of BCSCs.

[0041] 2. Mechanism of Lansoprazole Inhibiting FASN-Mediated BCSCs Death

[0042] The results showed that lansoprazole treatment significantly reduced the nuclear translocation of β-catenin in BCSCs ( Figure 2 AB). This was accompanied by a significant upregulation of APC, Axin-1, and GSK-3β, and a significant downregulation of TCF-4, TCF-7, and Wnt3a secretion ( Figure 2 CD). These findings suggest that the anti-BCSC effect of lansoprazole may be mediated by its targeting of FASN to inhibit the Wnt / β-catenin signaling pathway.

[0043] 3. Verification of Lansoprazole's Inhibitory Effect on BCSCs Growth in Vivo

[0044] To further verify the inhibitory effect of lansoprazole on BCSCs and elucidate its potential mechanism in vivo, we used BCSCs derived from MCF-7 (CD44+ / CD24-) and MDA-MB-468 (CD44+ / CD24-) cell lines to establish subcutaneous xenograft tumors in nude mice. 3 Mice were given lansoprazole (treated group) or an equal volume of DMSO (control group) every other day by oral gavage at a dose of 30 mg / kg. After 21 days of treatment, the mice were sacrificed and the tumors were removed for analysis. The results showed that the tumor growth in the lansoprazole-treated group was significantly reduced compared with the control group ( Figure 3A). In MCF-7 and MDA-MB-468 xenograft tumors treated with lansoprazole, tumor weight and volume were significantly reduced, indicating that lansoprazole effectively inhibited the tumorigenic potential of BCSCs ( Figure 3 BC). Histopathological examination using hematoxylin-eosin (H&E) staining revealed extensive necrosis and loose cellularity in the tumors of the lansoprazole-treated group, indicating that tumor proliferation was inhibited. Immunohistochemical analysis further supported these findings, showing that the expression of Ki-67, Wnt3a, and β-catenin in tumor tissues was significantly downregulated after lansoprazole treatment ( Figure 3 D). These results suggest that the antitumor effect of lansoprazole is mediated by inhibiting cell proliferation and suppressing the Wnt / β-catenin signaling pathway.

[0045] 4.FASN high PANCK + CD8 + T cells have the potential to induce exhaustion

[0046] We used multicolor immunofluorescence and TissueFAXS cell technology to perform panoramic tissue quantitative analysis ( Figure 4 A, B) It is well known that CD8 + The activation or exhaustion of T cells plays an important role in shaping the tumor immune microenvironment. Programmed death 1 (PD-1) is a protein that exhausts CD8 + The co-inhibitory receptor on the surface of T cells is highly expressed in CD8 + Our analysis showed that FASN low PANCK + Compared to cells, FASN high PANCK + Cells within 20 μm significantly increased the number of depleted CD8 + The number of T cells ( Figure 4 C, D), the results show that FASN high Breast cancer cells have potential CD8 + The induction of T cell exhaustion is crucial for the formation of an immunosuppressive microenvironment. This further suggests that high FASN expression leading to poor prognosis may be due to the tumor immunosuppressive microenvironment shaped by them.

[0047] 5. The proton pump inhibitor lansoprazole significantly inhibited the growth of orthotopic mammary tumors in mice and CD8 + T cell depletion

[0048] Previous studies have shown that the proton pump inhibitor lansoprazole specifically targets and inhibits FASN and has shown exciting anti-breast cancer effects in cell experiments and animal models. However, no study has yet reported whether lansoprazole affects the immune microenvironment of breast cancer. To investigate the above scientific questions, we used the mouse tumor cell line 4T1 to form orthotopic tumors in BALB / C mice. When the tumors were approximately 50 mm 3 At the same time, blank control and 30mg / kg lansoprazole were given every other day, and the living conditions of the mice were observed. Then, the mice were killed after 4 weeks according to the tumor size and the progression and deterioration of the in situ tumor. The tumor was peeled off and the weight and volume were measured. The results showed that the tumor formation in the lansoprazole group was significantly inhibited ( Figure 5 A), tumor weight was significantly lighter ( Figure 5 B), the tumor volume was significantly smaller ( Figure 5 C). Previous multicolor immunofluorescence results showed that FASN high PANCK + CD8 + Next, we used multicolor immunofluorescence labeling to explore the effects of the FASN inhibitor lansoprazole on CD8 T cells in the immune microenvironment of in situ tumor formation. + The effect of T cell status ( Figure 5 D) We found that FASN in the lansoprazole-treated group + The number of breast cancer cells was significantly reduced ( Figure 5 E), and CD8 + The number of T cells increased significantly ( Figure 5 F), depletion of CD8 + The proportion of T cells was significantly reduced ( Figure 5 G). The above results showed that lansoprazole significantly inhibited the expression of FASN and CD8 in the orthotopic breast cancer model. + T cell depletion produces anti-breast cancer effects by reshaping the breast cancer immune microenvironment.

[0049] The above embodiments are preferred implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of lansoprazole in the preparation of drugs for inhibiting the activity of breast cancer stem cells.

2. The use according to claim 1, characterized in that: Application of lansoprazole in the preparation of drugs for inhibiting the activity of breast cancer stem cells by acting on the FASN target.

3. The use according to claim 2, characterized in that: The application of lansoprazole in the preparation of drugs for treating breast cancer by inhibiting FASN target on breast cancer stem cells.

4. The use according to claim 1, characterized in that: Lansoprazole inhibits the expression of FASN and CD8 + The application of T cell depletion and reshaping of the breast cancer immune microenvironment in the preparation of anti-breast cancer drugs.

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