Application of 2-indolecarboxylic acid in preparation of tumor targeted therapy drugs

Abstract

The invention provides an application of 2-indolecarboxylic acid in preparation of a tumor targeted therapy drug. Through computer-aided drug design and molecular docking optimization, 2-indolecarboxylic acid shows high binding energy to an HER2 kinase structural domain. In HER2 positive NCI-N87 gastric cancer cells, the 2-indolecarboxylic acid significantly inhibits cell proliferation and induces G0 / G1 phase arrest and apoptosis. The mechanism research shows that the 2-indoleformic acid can play a role by regulating and controlling the HER2 / Akt / beta-catenin pathway (Western blot verification). In an NCI-N87 cell tumor-bearing nude mouse model, 2-indolecarboxylic acid (15 mg / kg) significantly inhibits tumor growth, and does not cause obvious weight loss or organ toxicity. Immunohistochemical analysis shows that the positive rate of a tumor tissue proliferation marker Ki-67 in a treatment group is remarkably reduced, and apoptosis detection shows that the proportion of TUNEL positive cells is remarkably increased, which indicates that 2-indolecarboxylic acid inhibits tumor cell proliferation and promotes apoptosis at the same time. The invention provides a lead compound with clinical transformation potential for HER2 targeted therapy.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to the application of 2-indolecarboxylic acid in the preparation of tumor-targeted therapy drugs. Background Technology

[0002] Human epidermal growth factor receptor 2 (HER2 / ErbB2) is an important member of the epidermal growth factor receptor (EGFR) family. As a transmembrane tyrosine kinase, it regulates cell proliferation, differentiation, and survival by forming dimers with other EGFR family members and activating downstream MAPK and PI3K / AKT pathways. Numerous studies have shown that HER2 overexpression is prevalent in various solid tumors, including breast cancer, gastric cancer, and ovarian cancer, and is closely associated with high tumor invasiveness, metastatic potential, and poor prognosis. Targeted therapies against HER2, such as monoclonal antibodies (trastuzumab), small molecule tyrosine kinase inhibitors (lapatinib), and antibody-drug conjugates, have shown some efficacy in clinical applications. However, existing drugs still suffer from problems such as drug resistance, significant toxic side effects, and high costs. There is an urgent need to develop next-generation small molecule HER2 inhibitors with high selectivity and low toxicity to further improve patient treatment outcomes and quality of life.

[0003] Natural products derived from traditional Chinese medicine (TCM) have attracted widespread attention in the development of new anti-tumor drugs due to their diverse structures, novel targets, significant biological activities, and relatively high safety. In recent years, with the modernization of TCM and the development of precision medicine, research on small-molecule targeted drugs based on the active ingredients of TCM has become increasingly popular. The ZINC database, as a leading international database of natural compounds, contains a large number of monomeric components of TCM and their structurally optimized derivatives, providing valuable resources for drug screening and mechanism of action research in the context of TCM modernization. With the increasing maturity of computer-aided drug design technology, structure-based virtual screening (SBVS) provides an efficient strategy for target discovery and optimization of the active ingredients of natural products.

[0004] 2-Indole-2-carboxylic acid is an indole-type natural product with chemical structural characteristics characteristic of traditional Chinese medicine. It is widely found in some legumes, solanaceae, and umbelliferae medicinal herbs, and some derivatives have been identified as secondary metabolites in traditional Chinese medicines such as Scutellaria baicalensis and Coptis chinensis. Existing literature reports that 2-indole-2-carboxylic acid and its derivatives possess multiple biological activities, including anti-inflammatory, antioxidant, neuromodulatory, and protein kinase inhibitory activities. Some of its structures have been applied to the development of small molecule drugs targeting kinases, showing promising drug development prospects and a solid pharmacological basis. Summary of the Invention

[0005] The purpose of this invention is to provide new pharmaceutical uses for 2-indolecarboxylic acid.

[0006] The novel pharmaceutical use of 2-indolecarboxylic acid provided by this invention is the application of 2-indolecarboxylic acid or a pharmaceutically acceptable salt thereof in the preparation of tumor-targeted therapeutic drugs.

[0007] In this application, the tumor is a tumor that overexpresses the HER2 protein.

[0008] The tumor is a solid tumor, specifically stomach cancer.

[0009] In this application, 2-indolecarboxylic acid or a pharmaceutically acceptable salt thereof simultaneously inhibits tumor cell proliferation and promotes tumor cell apoptosis.

[0010] In the aforementioned applications, 2-indolecarboxylic acid or a pharmaceutically acceptable salt thereof may function by regulating the HER2 / Akt / β-catenin pathway.

[0011] The present invention also provides the use of 2-indolecarboxylic acid or a pharmaceutically acceptable salt thereof in the preparation of small molecule HER2 inhibitors.

[0012] This invention, through computer-aided drug design (CADD) and molecular docking optimization, revealed that 2-indolecarboxylic acid (2-indolecarboxylic acid) exhibits a high binding energy to the HER2 kinase domain. Cellular experiments showed that 2-indolecarboxylic acid significantly inhibited the proliferation of HER2-positive NCI-N87 gastric cancer cells and induced G0 / G1 phase arrest and apoptosis. Mechanistic studies indicated that 2-indolecarboxylic acid may exert its effects by regulating the HER2 / Akt / β-catenin signaling pathway. In an NCI-N87 tumor-bearing nude mouse model, 15 mg / kg of 2-indolecarboxylic acid significantly inhibited tumor growth without causing significant weight loss or organ toxicity. Immunohistochemical analysis showed a significant decrease in the Ki-67 positivity rate in the treatment group, while apoptosis detection showed a significant increase in the proportion of TUNEL-positive cells, indicating that 2-indolecarboxylic acid simultaneously inhibits tumor cell proliferation and promotes apoptosis. This invention provides a lead compound with clinical translational potential for HER2-targeted therapy. Attached Figure Description

[0013] Figure 1 This study demonstrates the ability to detect HER2 expression levels in different gastric cancer cells using Western blotting. A. Western blotting was used to detect HER2 expression levels in gastric cancer cell lines (NCI-N87, HGC-27) and normal gastric mucosal cells (GES-1); B. HER2 expression was quantified (HER2 / β-actin), with β-actin used as an internal control. The HER2 expression ratio in GES-1 cells was set at 1.0. Data are expressed as mean ± SD.n =3 (*** p <0.001 vs. GES-1 cells).

[0014] Figure 2 This study demonstrates the expression localization of HER2 in different gastric cancer cells using immunofluorescence. A. Immunofluorescence detection of HER2 expression localization in gastric cancer cell lines (NCI-N87, HGC-27) and normal gastric mucosal cells (GES-1); B. Optical density analysis of HER2 expression levels. n =3(**** p <0.0001 vs. GES-1 cells).

[0015] Figure 3 This study investigated the effect of 2-indolecarboxylic acid (2-indolecarboxylic acid) on the viability of HER2-positive gastric cancer cells NCI-N87. A. Cell viability of NCI-N87 cells after 24 h of 2-indolecarboxylic acid treatment; B. Cell proliferation inhibition curve of NCI-N87 cells after 24 h of 2-indolecarboxylic acid treatment. Data are expressed as mean ± SD. n =3 (*** p <0.001 vs. (Control group).

[0016] Figure 4 This study demonstrates the inhibitory effect of 2-indolecarboxylic acid on the colony formation of gastric cancer cells. A. Colony formation observation of NCI-N87 and HGC-27 cells after treatment with different concentrations (0.05, 0.25, 0.5, 1 mM) of 2-indolecarboxylic acid for 24 h; B, C. Statistical analysis of colony formation. Data are expressed as mean ± SD. n =3 (** p <0.01, *** p <0.001, **** p <0.0001 vs. (Control group).

[0017] Figure 5 This indicates that 2-indolecarboxylic acid (2-indolecarboxylic acid) induces apoptosis in NCI-N87 and HGC-27 cells. A. NCI-N87 and HGC-27 cells were treated with different concentrations of 2-indolecarboxylic acid (0.05, 0.25, 0.5, 1 mM) for 24 h, and apoptosis was detected by Annexin V-FITC / PI double staining. B, C. Statistical analysis of apoptosis rate. Data are expressed as mean ± SD. n =3(**** p <0.0001 vs. (Control group).

[0018] Figure 6This indicates that 2-indolecarboxylic acid (2-indolecarboxylic acid) induces cell cycle arrest in NCI-N87 and HGC-27 cells. A. Cell cycle distribution of NCI-N87 and HGC-27 cells after treatment with different concentrations of 2-indolecarboxylic acid (0.5, 1 mM) for 24 h; B, C. Statistical analysis of cell cycle proportions. Data are expressed as mean ± SD. n =3 (* p <0.05, *** p <0.001, **** p <0.0001 vs. (Control group).

[0019] Figure 7 This indicates that 2-indolecarboxylic acid (2-indolecarboxylic acid) regulates the HER2 / Akt / β-catenin signaling pathway to induce apoptosis in NCI-N87 gastric cancer cells. A. Western blotting analysis of the expression of key proteins in the HER2 / Akt / β-catenin signaling pathway in NCI-N87 cells after 24 h of 2-indolecarboxylic acid treatment; B. Quantitative analysis of protein expression (target protein / β-actin), with β-actin as an internal control. Data are expressed as mean ± SD. n =3 (* p <0.05,** p <0.01 vs. (Control group).

[0020] Figure 8 To quantitatively analyze the uptake and pharmacokinetic characteristics of 2-indolecarboxylic acid (2-indolecarboxylic acid) in gastric cancer cells and animals using mass spectrometry. A. Uptake analysis of 2-indolecarboxylic acid in NCI-N87 cells after treatment with 2-indolecarboxylic acid; B. Enrichment analysis of 2-indolecarboxylic acid in nude mice with NCI-N87 cell xenografts after tail vein injection of 5 mg / kg 2-indolecarboxylic acid at different time points; C. Pharmacokinetic characteristics of 2-indolecarboxylic acid in rats after tail vein injection of 10 mg / kg 2-indolecarboxylic acid at different time points. Data are expressed as mean ± SD. n =3 (** p <0.01 vs. Group 0 h).

[0021] Figure 9 This study demonstrates the effects of different doses of 2-indolecarboxylic acid on the growth of NCI-N87 cell xenografts, body weight in tumor-bearing mice, and survival rate. A. Tumor volume change curves during treatment. n =5; B. Weight monitoring curve of tumor-bearing mice; C. Kaplan-Meier survival curve (Log-rank test); D. Tumor images at the end of treatment in the PBS and 15 mg / kg groups. Data are expressed as mean ± SD. n =5.

[0022] Figure 10 This study investigated the effects of 2-indolecarboxylic acid on apoptosis and proliferation of NCI-N87 cell xenografts. A. Tumor cell apoptosis was detected by co-staining with TUNEL (red) and DAPI (blue) at 200x magnification and a scale bar of 50 μm. B. Tumor cell proliferation was indicated by Ki67 (red) immunofluorescence at 200x magnification and a scale bar of 50 μm. C and D. Tumors and major organs were stained with HE at 200x magnification and a scale bar of 50 μm.

[0023] Figure 11 This study investigated the effects of 2-indolecarboxylic acid on liver and kidney function in tumor-bearing mice. Serum liver function (UA, BUN) and kidney function (CREA) indicators were measured. Data are expressed as mean ± SD. n =3. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0026] Structure-based virtual screening revealed that 2-indolecarboxylic acid (2-ICD) stably binds to the HER2 kinase domain (PDB:3RCD), with a CDocker binding energy of -80.79 kcal / mol, significantly superior to other molecules in the screening library. Key molecular interactions include π-π stacking with His878 and hydrogen bond networks formed with Lys753 and Leu796, directly blocking the HER2 dimerization active pocket and thus inhibiting receptor autophosphorylation signal transduction.

[0027] Example Experimental procedure: Cell culture Cell lines: NCI-N87 (#TCH-C504), HGC-27 (#TCH-C202), and GES-1 (#TCH-C410) were all purchased from Haixing Biotechnology Co., Ltd. (China). Culture medium: RPMI-1640 MA0215 (Meilunbio), containing 10% fetal bovine serum (#FSP500, ExCell) and 1% penicillin / streptomycin (#P1400, Solarbio), cultured in an incubator at 37°C and 5% CO2. Consumables: Corning® T25 culture flasks (#13112, LABSELECT). Resuscitation: Cryopreserved tubes were removed from liquid nitrogen, incubated at 37°C for 1 min, centrifuged (800 rpm, 4 min), resuspended, seeded, and cultured at 37°C and 5% CO2. Passaging: Wash with PBS, digest with 0.25% trypsin (containing EDTA) (#BL512A, Biosharp), terminate digestion, and passage at a 1:3 ratio. Observe cell morphology daily. All operations are performed in a clean bench, and mycoplasma contamination is checked regularly.

[0028] Western Blotting Lysis buffer: RIPA (#R0010, Solarbio) + 1% protease inhibitor PMSF (#FJP0100, Solarbio). Antibodies: β-catenin antibody (#ab68183, abcam), HER2 / ErbB2 antibody (#18299-1-AP, Proteintech), Phospho-HER2 / ErbB2 antibody (Tyr1221 / 1222, #2243T, CST), HRP-secondary antibody (#RS0002, Immunoway), β-actin (#GB15003, Servicebio), AKT antibody (#GTX121937, GeneTex), phospho-AKT antibody (Ser473, #GTX128414, GeneTex). Cells were treated with 2-indolecarboxylic acid (#HY-10096, MCE) for 24 h, then lysed on ice for 30 min with RIPA lysis buffer (containing protease / phosphatase inhibitors), centrifuged (12000 rpm, 15 min, 4℃), and total protein was extracted and quantified using the BCA method (#PC0020, Solarbio). 10–30 μg of protein was separated by SDS-PAGE (10% separating gel) and transferred to a PVDF membrane (#IPVH00010, Millipore). Blocked with 5% skim milk (#LP0033B, Solarbio) or 5% BSA (#A8020, Solarbio) for 1 h, incubated overnight at 4°C with primary antibodies (anti-β-actin 1:2000, anti-HER2 1:2000, anti-phospho-HER2 1:1000, anti-AKT 1:2000, anti-phospho-AKT 1:2000, anti-β-catenin 1:1000), washed 3 times with TBST (#T1081, Solarbio), incubated at room temperature with HRP-labeled secondary antibody (1:20000) for 1 h, developed with ECL chemiluminescence (#P1050, APPLYGEN), and protein expression levels were quantitatively analyzed using ImageJ.

[0029] Apoptosis and cell cycle Apoptosis detection kit: Annexin V-FITC / PI Kit (#40302ES60, YEASEN). Cycle detection kit: PI staining solution (#40301ES50, YEASEN). Apoptosis detection: Annexin V-FITC / PI double staining, flow cytometry to distinguish early apoptosis (Annexin V...). + / PI - ) and late apoptosis (Annexin V) + / PI +Cell cycle detection: Cells were digested and collected, washed with PBS, and fixed overnight with 70% ethanol (-20℃). RNase A was treated for 30 min, followed by incubation with PI staining solution (50 μg / mL) in the dark for 30 min, and then analyzed by flow cytometry.

[0030] Cell proliferation CCK-8 reagent (#CK001-01, SUNVIEW) and 96-well plates (#11510, LABSELECT) were used. Tumor cells in logarithmic growth phase, NCI-N87 at 5000 cells / well and HGC-27 at 3000 cells / well, were seeded in 96-well plates and pre-cultured for 24 h. Then, graded concentrations of the test drug were added for 24 h. 10 μL of CCK-8 working solution was added to each well, and the plates were incubated at 37°C for 2 h. The absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the formula [(OD experimental group - OD blank) / (OD negative control - OD blank)] to assess the inhibitory effect of the drug on tumor cells. A dose-response curve was plotted to calculate the IC50. 50 value.

[0031] Cloning Six-well plates (#11110, LABSELECT), 4% formaldehyde fixative (#P1110, Solarbio), and crystal violet (#P1110, Solarbio) were used. Single-cell suspensions treated with different concentrations of the drug for 24 h were seeded into six-well plates (10,000 cells / well for NCI-N87, 10,000 cells / well for HGC-27), and cultured at 37°C and 5% CO2 for 14 days. Cells were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 30 min, and washed with PBS. The number of clones formed (clones with more than 50 cells) was photographed and counted. The colony formation rate was calculated as an indicator of cell proliferation capacity: colony formation rate = (number of clones in experimental group / number of clones in control group) × 100%.

[0032] Cellular immunofluorescence Preparation of cell culture slides: Place sterile coverslips (or dedicated cell culture slides) into the wells of the culture plate. Cell seeding: Digest cells in logarithmic growth phase and adjust the density to 1×10⁻⁶ cells / well. 5 –3×10 5Add 2 mL of cell suspension to each well and gently shake to mix. Culture: Incubate at 37°C in a 5% CO2 incubator until the target density is reached. Fixation: Discard the culture medium, gently wash twice with PBS; add 4% paraformaldehyde (or pre-cooled methanol) and fix for 20 min at room temperature. Permeabilization (optional): If intracellular staining is required, wash with PBS and then treat with 0.2% Triton X-100 (#T8200, Solarbio) for 10 min. Blocking: Wash three times with PBS, add 3% BSA blocking solution, and incubate at 37°C for 30 min. Staining: Discard the blocking solution, directly add primary antibody working solution (to cover the slide), and incubate in a humidified chamber at 37°C for 1 h or 4°C overnight; wash three times with PBS, then add fluorescent secondary antibody (#RS3211, Immunoway, 1:500) and incubate in the dark for 1 h. Mounting: Wash thoroughly with PBS; add anti-quenching mounting medium containing DAPI, cover with a coverslip, and store in the dark. Observation: Images were observed and acquired under a 10× confocal fluorescence microscope.

[0033] Animal experiments Animals: BALB / c nude mice (female, 4 weeks old), housed in an SPF-grade animal facility. Inoculation: Subcutaneous injection of suspended NCI-N87 cells (5 × 10⁻⁶ cells) into the right axilla. 6 (e.g., tumor cells / 100 μL PBS), until tumor volume ≈ 100 mm² 3 (When tumor volume = major diameter × minor diameter² / 2) random grouping n =5): PBS control group, 2-indolecarboxylic acid treatment group (1 mg / kg, 5 mg / kg, 15 mg / kg, tail vein injection, once every 3 days, for a total of 5 times), and the control group received an equal volume of PBS. Monitoring: Tumor volume (tumor volume = major diameter × minor diameter² / 2) and body weight were measured every 3 days to plot tumor growth curves. Sample collection: Uric acid (#S03035, Rayto), blood urea nitrogen (#S03036, Rayto), and creatinine (#S03076, Rayto) were measured in nude mouse plasma using a kit. Whole blood samples were collected in heparinized tubes, plasma was separated by centrifugation, and uric acid, blood urea nitrogen, and creatinine were automatically measured using a fully automated biochemical analyzer (Chemray 800, Rayto). Results are expressed in μmol / L to assess the effects of the drug on liver and kidney function. Nude mice were euthanized by cervical dislocation, and tumor tissue and other major organs, including heart, liver, spleen, lungs, kidneys, and stomach, were collected.

[0034] NCI-N87 cells were treated with 0.25 mM 2-indolecarboxylic acid at different time points (0, 2, 4, 24 h). Cells were scraped off directly with a cell scraper, and the cell pellet was collected. The drug was extracted by grinding with liquid nitrogen, and the supernatant was collected by centrifugation (12000 rpm, 12 min). Tumor-bearing nude mice were administered 5 mg / kg of 2-indolecarboxylic acid via tail vein. The transplanted tumors were isolated at different time points (0.5, 1, 4, 8, 16, 24, 48, 72 h). The tumors were lysed with high-efficiency RIPA cell / tissue lysis buffer (containing PMSF), and the supernatant was collected by centrifugation (12000 rpm, 12 min). Pharmacokinetics (Rat Plasma Assay): After rats were administered 10 mg / kg of 2-indolecarboxylic acid via tail vein, blood samples were collected from the orbital cavity at different time points (5, 10, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min). The blood was anticoagulated with heparin, and the supernatant was collected by centrifugation (4000 rpm, 10 min). All samples were subjected to methanol precipitation for protein, and the supernatant was analyzed by liquid chromatography-mass spectrometry (LC-MS / MS). A standard curve was established to quantify drug concentration, and uptake and pharmacokinetic parameters (such as AUC and Cmax) were calculated.

[0035] Immunofluorescence of tumor tissue After fixation with 4% paraformaldehyde, the transplanted tumor tissue was dehydrated, paraffin-impregnated, and sectioned (4 μm thick). TUNEL staining (#G1502, Servicebio): Sections were treated with proteinase K and incubated with TUNEL reaction mixture for 1 h to detect apoptotic cell nuclei (fluorescent labeling). Ki67 staining (#GB151499, Servicebio): Sections were incubated overnight with Ki67 primary antibody, followed by the addition of fluorescent secondary antibody (#G1231, Servicebio). After DAPI counterstaining, apoptosis and proliferation were observed under an upright fluorescence microscope (Nikon Eclipse C1, Nikon), and the entire section was scanned using an imaging scanner (Pannoramic MIDI, 3DHISTECH, HU).

[0036] HE-stained tissue sections After dehydration and paraffin embedding, tissue sections (4 μm thick) were prepared. After dewaxing and hydration, the sections were stained with hematoxylin and eosin (#G1076, Servicebio), dehydrated, and mounted. The tissue structure, necrosis, and inflammatory infiltration were observed under an upright optical microscope (Nikon Eclipse E100, Nikon), and the complete sections were scanned using an imaging system (Nikon DS-U3, Nikon).

[0037] Experimental results: Figure 1This study demonstrates the effectiveness of Western blotting in detecting HER2 expression in different gastric cancer cells. A. Western blotting was used to detect HER2 expression in gastric cancer cell lines (NCI-N87, HGC-27) and normal gastric mucosal cells (GES-1). B. Protein expression was quantified, with β-actin as an internal control. The HER2 expression ratio (HER2 / β-actin) in GES-1 cells was set at 1.0. Data are presented as mean ± SD. n =3 (*** p <0.001 vs. (GES-1 cells). The results showed that HER2 expression was highest in NCI-N87 cells.

[0038] Figure 2 This study demonstrates the expression localization of HER2 in different gastric cancer cells using immunofluorescence. A. Immunofluorescence detection of HER2 expression localization in gastric cancer cell lines (NCI-N87, HGC-27) and normal gastric mucosal cells (GES-1), magnification 100x, scale bar = 100 μm; B. Optical density analysis of HER2 expression levels. n =3(**** p <0.0001 vs. (GES-1 cells). The results showed that the HER2 fluorescence signal was strongest in NCI-N87 cells. Based on Western blotting and immunofluorescence experiments, NCI-N87 cells were selected for subsequent experiments.

[0039] Figure 3 This study investigated the effect of 2-indolecarboxylic acid (2-indolecarboxylic acid) on the viability of HER2-positive gastric cancer cells NCI-N87. A. Cell viability of NCI-N87 cells after 24 h of 2-indolecarboxylic acid treatment; B. Cell proliferation inhibition curve of NCI-N87 cells after 24 h of 2-indolecarboxylic acid treatment. Data are expressed as mean ± SD. n =3 (*** p <0.001 vs. (Control group). Results showed that after 24 h of treatment with 2-indolecarboxylic acid, cell viability decreased in a concentration-dependent manner, and the IC50 value was [not specified]. 50 =1 mM.

[0040] Figure 4 This study demonstrates the inhibitory effect of 2-indolecarboxylic acid on the colony formation of gastric cancer cells. A. Colony formation observation of NCI-N87 and HGC-27 cells after treatment with different concentrations (0.05, 0.25, 0.5, 1 mM) of 2-indolecarboxylic acid for 24 h; B, C. Statistical analysis of colony formation. Data are expressed as mean ± SD. n =3 (** p <0.01, ***p <0.001, **** p <0.0001 vs. (Control group). The results showed that, at the same concentration, 2-indolecarboxylic acid had a stronger inhibitory effect on colony formation in NCI-N87 cells than in HGC-27 cells.

[0041] Figure 5 This indicates that 2-indolecarboxylic acid (2-indolecarboxylic acid) induces apoptosis in NCI-N87 and HGC-27 cells. A. NCI-N87 and HGC-27 cells were treated with different concentrations of 2-indolecarboxylic acid (0.05, 0.25, 0.5, 1 mM) for 24 h, and apoptosis was detected by Annexin V-FITC / PI double staining. B, C. Statistical analysis of apoptosis rate. Data are expressed as mean ± SD. n =3(**** p <0.0001 vs. (Control group).

[0042] Figure 6 This indicates that 2-indolecarboxylic acid (2-indolecarboxylic acid) induces cell cycle arrest in NCI-N87 and HGC-27 cells. A. Cell cycle distribution of NCI-N87 and HGC-27 cells after treatment with different concentrations of 2-indolecarboxylic acid (0.5, 1 mM) for 24 h; B, C. Statistical analysis of cell cycle proportions. Data are expressed as mean ± SD. n =3 (* p <0.05, *** p <0.001, **** p <0.0001 vs. (Control group).

[0043] The effects of different concentrations (0.5, 1 mM) of 2-indolecarboxylic acid on apoptosis and cell cycle arrest in NCI-N87 and HGC-27 cells were detected by flow cytometry, with HGC-27 cells expressing low levels of HER2 as a control. Figure 6 The results showed that, at the same concentration, 2-indolecarboxylic acid had a stronger pro-apoptotic effect on NCI-N87 cells than on HGC-27 cells, and caused a higher G0 / G1 phase arrest ratio.

[0044] Figure 7 This indicates that 2-indolecarboxylic acid (2-indolecarboxylic acid) regulates the HER2 / Akt / β-catenin signaling pathway to induce apoptosis in NCI-N87 gastric cancer cells. A. Western blotting analysis of the expression of key proteins in the HER2 / Akt / β-catenin signaling pathway in NCI-N87 cells after 24 h of 2-indolecarboxylic acid treatment; B. Quantitative analysis of protein expression (target protein / β-actin), with β-actin as an internal control. Data are expressed as mean ± SD.n =3 (* p <0.05,** p <0.01 vs. (Control group). The results showed that 2-indolecarboxylic acid could inhibit HER2 and AKT phosphorylation in NCI-N87 cells and inhibit β-catenin expression, suggesting that 2-indolecarboxylic acid may inhibit NCI-N87 cell proliferation by inhibiting the HER2 / Akt / β-catenin signaling pathway.

[0045] Figure 8 To quantitatively analyze the uptake and pharmacokinetic characteristics of 2-indolecarboxylic acid (2-indolecarboxylic acid) in gastric cancer cells and animals using mass spectrometry. A. Uptake analysis of 2-indolecarboxylic acid in NCI-N87 cells; B. Enrichment analysis of 2-indolecarboxylic acid in nude mice with NCI-N87 cell xenografts after tail vein injection of 5 mg / kg 2-indolecarboxylic acid at different time points; C. Pharmacokinetic characteristics of 2-indolecarboxylic acid in rats after tail vein injection of 10 mg / kg 2-indolecarboxylic acid at different time points. Data are expressed as mean ± SD. n =3 (** p <0.01 vs. (0 h group). Intracellular uptake results showed that the drug content in NCI-N87 cells increased over time. In the tumor-bearing nude mouse model, tumor tissue uptake was significant (peak concentration at 0.5 h post-injection: ng / g tissue). After administering 10 mg / kg of 2-indolecarboxylic acid to rats, the plasma pharmacokinetics were fitted using Phoenix WinNonlin software, yielding key parameters: Cmax = 20596.14 ng / mL, Tmax = 10 min, T1 / 2 = 40.48 h, and AUC(0-t) = 1157824.69 h*ng / mL, indicating that 2-indolecarboxylic acid has rapid tissue penetration and tumor targeting capabilities. The above data summarize that the efficient accumulation of 2-indolecarboxylic acid in cells and animal models contributes to its antitumor effect.

[0046] Figure 9 This study demonstrates the effects of 2-indolecarboxylic acid on the growth of NCI-N87 cell xenografts, body weight in tumor-bearing mice, and survival rate. A. Tumor volume change curve during treatment. n =5; B. Weight monitoring curve of tumor-bearing mice; C. Kaplan-Meier survival curve (Log-rank test); D. Tumor images at the end of treatment in the PBS and 15 mg / kg groups. Data are expressed as mean ± SD. n =5.

[0047] The therapeutic effects of different doses of 2-indolecarboxylic acid (1 mg / kg, 5 mg / kg, and 15 mg / kg) were evaluated using a nude mouse NCI-N87 xenograft model and compared with a PBS control group. Results showed that the treatment groups exhibited dose-dependent growth inhibition of tumors, with the 15 mg / kg group showing significantly smaller tumor volume than the PBS control group on day 25. At the end of treatment, the tumor volume in the 15 mg / kg group was significantly smaller than that in the control group. There was no significant difference in body weight between the mouse groups and the control group, indicating that 2-indolecarboxylic acid did not cause significant toxicity within the 15 mg / kg range. Furthermore, log-rank trend analysis (…) p <0.05) indicates that the median survival time of mice in the four different dose groups was 30 days, 32 days, 34 days and 42 days, respectively. The median survival time of the 15 mg / kg group was extended by about 40% compared with the PBS group. The longest survival time of mice in the four different dose groups was 39, 42, 43 and 76 days, respectively, indicating that the 15 mg / kg dose can significantly prolong the survival time of mice.

[0048] Figure 10 This study investigated the effects of 2-indolecarboxylic acid (2-ICD) on apoptosis and proliferation of NCI-N87 cell xenografts. A. Tumor cell apoptosis was detected by co-staining with TUNEL (red) and DAPI (blue) at 200x magnification and a scale bar of 50 μm. B. Tumor cell proliferation was visualized by Ki67 (red) immunofluorescence at 200x magnification and a scale bar of 50 μm. C and D. Tumor and major organs were stained with hematoxylin and eosin (HE) at 200x magnification and a scale bar of 50 μm. TUNEL immunofluorescence staining showed a significant increase in the number of apoptotic cells in the tumor tissue of the 2-indolecarboxylic acid treatment group compared to the PBS control group (enhanced red fluorescence signal). Simultaneously, Ki67 immunofluorescence detection indicated a significant inhibition of tumor cell proliferation activity (weakened red fluorescence signal). Histopathological HE staining further confirmed that no significant pathological changes were observed in the heart, liver, spleen, lungs, kidneys, and stomach of tumor-bearing mice, indicating that 2-indolecarboxylic acid has a selective antitumor effect.

[0049] Figure 11 This study investigated the effects of 2-indolecarboxylic acid on liver and kidney function in tumor-bearing mice. Serum liver function (UA, BUN) and kidney function (CREA) indicators were measured. Data are expressed as mean ± SD. n =3. From Figure 11 It can be seen that serum biochemical tests showed that the levels of uric acid (UA), blood urea nitrogen (BUN), and creatinine (CREA) in the 2-indolecarboxylic acid treatment group mice were not statistically different from those in the control group, and all indicators remained within the normal physiological range, indicating that the treatment regimen did not cause significant liver and kidney function damage within the experimental dose range.

[0050] These results indicate that 2-indolecarboxylic acid can exert significant anti-tumor effects by inducing apoptosis and inhibiting proliferation, while exhibiting good organ safety characteristics, providing important preclinical experimental evidence for its further clinical translation research.

[0051] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

Use of 1,2-indolecarboxylic acid or a pharmaceutically acceptable salt thereof in the preparation of tumor-targeted therapeutic agents.

2. The application according to claim 1, characterized in that, In this application, the tumor is a tumor that overexpresses the HER2 protein.

3. The application according to claim 2, characterized in that, The tumor is a solid tumor, specifically stomach cancer.

4. The application according to claim 1, characterized in that, In this application, 2-indolecarboxylic acid or a pharmaceutically acceptable salt thereof simultaneously inhibits tumor cell proliferation and promotes tumor cell apoptosis.

5. The application according to claim 1, characterized in that, In the aforementioned applications, 2-indolecarboxylic acid or a pharmaceutically acceptable salt thereof exerts its effects by regulating the HER2 / Akt / β-catenin pathway. 6,2-Indolecarboxylic acid or a pharmaceutically acceptable salt thereof in the preparation of small molecule HER2 inhibitors.

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