Application of FFAR4 agonist in preparation of medicine for treating lung adenocarcinoma
By developing the FFAR4 agonist TUG-891, which inhibits the proliferation and cell cycle of lung adenocarcinoma cells, the application gap of FFAR4 agonists in the treatment of lung adenocarcinoma in the existing technology was solved, and significant inhibitory effects and clinical potential were achieved.
Patent Information
- Application Number
- CN202510910820.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-02
- Publication Date
- 2025-10-10
AI Technical Summary
Existing technologies lack the application of FFAR4 and its agonists in the treatment of lung adenocarcinoma, and new therapeutic strategies and targets are urgently needed.
The FFAR4 agonist TUG-891 was developed to inhibit the proliferation and cell cycle of lung adenocarcinoma cells by inhibiting mitochondrial oxidative phosphorylation and reducing intracellular NAD+ concentration.
It significantly inhibits the proliferation and growth of lung adenocarcinoma cells, delays the progression of lung cancer, provides a new target for the treatment of lung adenocarcinoma, and has high clinical translation potential.
Smart Images
Figure CN120754075A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and in particular to the use of FFAR4 agonists in the preparation of drugs for treating lung adenocarcinoma. Background Art
[0002] With the advancement of urbanization and the widespread adoption of early lung cancer screening across the country, the incidence of lung cancer in China continues to rise. While advances in artificial intelligence, information technology, and molecular detection technologies have facilitated early screening and treatment of lung cancer, and the development of targeted therapies and immunotherapy have made lung cancer a clinically controllable chronic disease, improving long-term survival rates, not all lung cancer patients benefit from existing treatments due to the heterogeneity of tumor clinical and biological characteristics. Therefore, exploring more potential pathogenesis and biomarkers of lung adenocarcinoma and identifying new treatment strategies are of great significance.
[0003] G protein-coupled receptors are promising targets for drug development. As a member of the G protein-coupled receptor family, free fatty acid receptors (FFARs) have the potential to bridge the gap between genetic and environmental factors associated with cancer. FFARs include FFAR1, FFAR2, FFAR3, and FFAR4. FFAR2 and FFAR3 are short-chain fatty acid receptors that can be activated by short-chain fatty acids, while FFAR1 and FFAR4 are medium- and long-chain fatty acid receptors that can be activated by medium- and long-chain fatty acids. Previous studies have shown that a diet rich in long-chain fatty acids can prolong the survival of patients with lung adenocarcinoma and enhance the efficacy of immunotherapy, suggesting that FFAR4 activation as a mechanism of action for the treatment of lung adenocarcinoma has great application prospects.
[0004] FFAR4 primarily functions as a receptor for long-chain fatty acids and is abundant in both the intestine and lung tissues. However, previous studies have primarily focused on the role of FFAR4 in the intestine, overlooking its potential role in the lungs. Currently, there are few reports on the use of FFAR4 and its agonists in the development of drugs for the treatment of lung cancer.
[0005] Therefore, based on the above-mentioned related technologies, there is an urgent need to develop an FFAR4 agonist for use in the preparation of drugs for treating lung adenocarcinoma. Summary of the Invention
[0006] The purpose of the present invention is to propose the use of FFAR4 agonists in the preparation of drugs for the treatment of lung adenocarcinoma, so as to solve the problem that the existing technology lacks the application of FFAR4 and its agonists in the treatment of lung adenocarcinoma, and provide new targets and new ideas for the screening and preparation of drugs for the treatment of lung cancer.
[0007] The present invention provides the use of an FFAR4 agonist in the preparation of a drug for treating lung adenocarcinoma. The FFAR4 agonist is TUG-891 or a pharmaceutically acceptable salt thereof, which inhibits the proliferation and cell cycle of lung adenocarcinoma cells by inhibiting mitochondrial oxidative phosphorylation and reducing intracellular NAD+ concentration.
[0008] The present invention achieves the above-mentioned purpose through the following technical solutions:
[0009] Using bioinformatics analysis tools, such as TCGA, GTEx, GEO-GSE40275, and GEO-GSE118370 databases, clinical data and RNA sequence data of lung adenocarcinoma were obtained, and the expression level of FFAR4 in lung adenocarcinoma tissue was analyzed. It was found that FFAR4 expression was significantly downregulated in lung adenocarcinoma tumor tissue samples, and low-level expression of FFAR4 was correlated with poor prognosis.
[0010] The expression of FFAR4 in normal cells and lung adenocarcinoma cells cultured in vitro and in human tissue specimens was detected by q-PCR. The results showed that the expression level of FFAR4 in lung adenocarcinoma cells was significantly lower than that in normal cells.
[0011] Preferably, the normal cells are normal bronchial epithelial cells BEAS-2B, and the lung adenocarcinoma cells are lung cancer cell line A549 obtained from a nationally certified cell platform. Both cells are cultured in DMEM medium + 5% fetal bovine serum in a 5% carbon dioxide, 37°C constant temperature incubator.
[0012] In in vitro experiments, TUG-891 was dissolved in dimethyl sulfoxide and added to the A549 cell culture medium to construct lung adenocarcinoma cell lines with different expression levels. The effects of FFAR4 agonists on lung adenocarcinoma cell proliferation and cell cycle phase distribution were detected. The mechanism of action of TUG-891 was further evaluated by measuring the oxygen consumption rate and intracellular NAD+ / NADH levels of lung adenocarcinoma cells.
[0013] Preferably, the method for detecting the effect of FFAR4 agonists on the proliferation of lung adenocarcinoma cells: using CCK8 detection to determine cell proliferation, the cells are plated in a 96-well plate, approximately 3000 cells / well, TUG891 is added to the experimental wells, and an equal volume of DMSO is added to the blank wells, and the cells are incubated in a 37°C constant temperature incubator and a 5% carbon dioxide environment for 72 hours; the optical density is measured at a wavelength of 450 nm.
[0014] Preferably, the effect of the FFAR4 agonist on the distribution of lung adenocarcinoma cell cycle phases is detected: cells are treated with 1 μM L7G for 24 h, then the cells are trypsinized for about 1.5 min, washed with PBS, and fixed with cold 80% ethanol, after centrifugation to remove ethanol, the cells are stained with PI / RNase staining buffer in a light-limited environment for 20 min, and the DNA content is quantified using a flow cytometer.
[0015] Preferably, when measuring oxygen consumption, the cells are treated with 500 μL of a detection medium consisting of XF Base Medium (Seahorse, 102353), 1 mM sodium pyruvate, 1 mM l-glutamine, and 10 mM glucose. Port injections are performed using 1 mM oligomycin, 3 mM FCCP, 0.5 mM antimycin, and rotenone.
[0016] Preferably, when measuring intracellular NAD+ / NADH, the concentration of NADH and NAD+ is determined using a WST-8 NAD+ / NADH assay kit (Beyotime Biotechnology). Specifically, A549 cells are cultured in a six-well plate at a density of 1.0 x 10 6 A549 cells are cultured in a six-well plate at a density of 1.0 x 10
[0017] In an animal model, a lung cancer cell transplantation model is constructed by subcutaneous injection of A549 cells, and the lung cancer cell transplantation model is divided into a control group and an intervention group. The intervention group is given TUG-891 solution by gavage, and the tumor growth is observed and recorded to evaluate the inhibitory effect of TUG-891 on lung cancer development.
[0018] Preferably, the TUG-891 solution is a solution of TUG-891 in DMSO solvent in a solvent consisting of 10% DMSO, 40% polyethylene glycol 300, 5% Tween-80, and 45% normal saline, configured as a 50 mg / ml solution.
[0019] Preferably, the method for constructing the lung cancer cell transplantation model comprises the following steps:
[0020] A549 cells were taken and centrifuged at 1200 rpm for 3 minutes. The A549 cells were washed three times with ice-cold PBS and resuspended in low-serum medium. 10 μL of the cell suspension was taken for counting to make an A549 cell suspension with a concentration of 5×107 cells / mL. 100 μL of the cell suspension was subcutaneously injected into the middle and posterior part of the right axilla of the mouse to construct a lung cancer cell transplantation model.
[0021] Preferably, the mice are grouped as follows: divided into a control group and an intervention group according to whether TUG891 is intervened: the control group (Model): gavage with normal saline (0.2 mL / mouse / day); the intervention group (TUG891): gavage with TUG891 solution (0.2 mL / mouse / day).
[0022] Beneficial effects of the present invention:
[0023] 1. Through systematic research, this study reveals for the first time the important role of FFAR4 agonists in inhibiting the proliferation and cell cycle of lung adenocarcinoma cells. This discovery provides a novel target for the treatment of lung adenocarcinoma, filling a gap in the existing art for the application of FFAR4 and its agonists in the treatment of lung adenocarcinoma.
[0024] 2. Through in vitro cell experiments and in vivo animal model experiments, the present invention validates the significant effectiveness of FFAR4 agonists in inhibiting the development of lung adenocarcinoma. The experimental results show that TUG-891 can significantly inhibit the proliferation and growth of lung adenocarcinoma cells and delay the progression of lung cancer.
[0025] 3. The research results of this invention have high clinical translation potential and are expected to be transformed into actual treatment plans in future clinical practice, bringing more hope and choices to patients with lung adenocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0027] Figure 1 : Schematic diagram of FFAR4 expression and prognostic significance in lung adenocarcinoma, where AE represents the analysis using TCGA, GEO, and GEPIA databases, showing that FFAR4 expression in lung adenocarcinoma tumor tissue samples was significantly lower than that in normal control tissue samples; F represents the Kaplan-Meier survival curve analysis, showing that low-level FFAR4 expression is associated with poor prognosis; HI represents the q-PCR analysis, showing that FFAR4 expression in lung adenocarcinoma cells was significantly lower than that in normal cells;
[0028] Figure 2 : Effects of TUG-891 on the proliferation and cell cycle of lung adenocarcinoma cells. A represents the expression level of FFAR4 in 21 lung adenocarcinoma cell lines detected by q-PCR, showing that the A549 cell line had the lowest expression level. B represents CCK8 detection and flow cytometry analysis showing that TUG-891 treatment significantly inhibited the proliferation and growth of A549 cells and affected the cell cycle distribution.
[0029] Figure 3 : Effects of TUG-891 on mitochondrial function and intracellular NAD+ concentration, where AB shows that the oxygen consumption rate of lung adenocarcinoma cells treated with TUG-891 decreased; C shows that the intracellular NAD+ concentration was significantly lower than that of untreated lung adenocarcinoma cells;
[0030] Figure 4 : Schematic diagram of TUG-891 inhibiting tumor growth in animal models, showing that treatment with the FFAR4 agonist TUG-891 can delay the growth and progression of lung cancer;
[0031] Figure 5 : Schematic overview of the mechanism of action of FFAR4 agonist TUG-891 in inhibiting the occurrence and development of lung adenocarcinoma. DETAILED DESCRIPTION
[0032] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments.
[0033] Example 1: Down-regulation of FFAR4 expression in lung adenocarcinoma and its prognostic significance Experimental methods:
[0034] 1.1: Clinical and RNA-seq data from 535 lung adenocarcinoma patients, as well as data from 59 corresponding adjacent adjacent tissue samples, were obtained using the TCGA, GTEx, GEO-GSE40275, and GEO-GSE118370 databases. Intergroup differences were analyzed using chi-square tests and t-tests to assess the relationship between FFAR4 expression levels and lung adenocarcinoma differentiation, stage, metastasis, and prognosis. Kaplan-Meier (KM) survival curve analysis and time-dependent receiver operating characteristic (ROC) curves were used to assess the prognostic significance and efficacy of FFAR4.
[0035] 1.2: Normal bronchial epithelial cells (BEAS-2B) and lung cancer cell lines (A549) were obtained from a nationally certified cell platform. Both cells were cultured in DMEM medium (purchased from Gibco) + 5% fetal bovine serum (purchased from Gibco) and cultured in a 5% carbon dioxide, 37°C constant temperature incubator. Total RNA was extracted from the cells using the K101 RNA extraction kit (JN.BIOTOOLS). Reverse transcription was performed using the K102 BT-I first-strand cDNA synthesis kit (JN.BIOTOOLS), and second-strand synthesis was performed using the second-strand cDNA synthesis kit (Beyotime Biotechnology, D7172). The DNA samples were fragmented and labeled with Tn5 transposase. Subsequently, polymerase chain reaction (PCR) amplification was performed using HiFi PCR Mix (CWBIO and CW2648) for sequencing, and an index code was assigned to each sample. PCR products were purified using the FastPure Gel DNA Extraction Mini Kit (Nanjing Vazyme Biotech Co, Ltd, DC301-01) and then sequenced using an Illumina NovaSeq (GENEWIZ). The sequencing data were analyzed using STAR (http: / / code.google.com / p / rna-star) and R software (version 3.5). Genes showing differential expression were defined as those with a P value < 0.05 and a fold change ≥ 2.
[0036] 1.3: Quantitative PCR was used to detect FFAR4 expression in lung cancer and normal lung tissue specimens. The qPCR protocol consisted of 40 cycles of denaturation at 95°C for 15 s, followed by annealing and extension at 60°C for 30 s. Total RNA was isolated using RNAisoPlus (Takara) according to the manufacturer's protocol, and reverse transcribed using Prime Script™ RT Master Mix (Takara). mRNA was quantified using the CFX Connect™ Real-Time PCR Detection System (Bio-Rad) and HieffUNICON qPCR SYBR Green Master Mix (Yeasen Biotechnology, 11198ES). Each experimental group was performed in triplicate.
[0037] Experimental results analysis:
[0038] like Figure 1 As shown in the results, TCGA, GEO, and GEPIA databases revealed that FFAR4 expression was significantly downregulated in lung adenocarcinoma tumor tissue samples compared with normal control tissue samples ( Figure 1A-E, all P<0.05); further survival analysis results showed that low expression of FFAR4 was associated with poor prognosis, suggesting that FFAR4 may inhibit the development of lung adenocarcinoma Figure 1 F); q-PCR was used to detect the expression of FFAR4 in normal cells and lung adenocarcinoma cells in vitro and human tissue specimens, and the results showed that the expression level of FFAR4 in lung adenocarcinoma cells was significantly lower than that in normal cells Figure 1 H-I), data are expressed as x±s, Student's t test was used, P<0.05 was statistically significant.
[0039] Example 2: FFAR4 activator TUG-891 inhibits the growth and proliferation of A549 cells
[0040] Experimental method:
[0041] 2.1 Different concentrations (0, 10, 20, 40 μM) of TUG-891 (purchased from Sigma, purity ≥98%) were dissolved in dimethyl sulfoxide (DMSO, purchased from Sigma) and added to the A549 cell culture medium, and the blank group was added with the same volume of DMSO to the A549 cell culture medium, to construct lung adenocarcinoma cell lines with different overexpression levels of FFAR4.
[0042] 2.2 CCK8 detection method was used to determine cell proliferation. Cells were plated in 96-well plates, about 3000 cells / well, 6 replicates per group, TUG891 was added to the experimental wells, and the same volume of DMSO was added to the blank wells, and incubated in a 37°C constant temperature incubator, 5% carbon dioxide environment for 72h. The optical density (OD) was measured at 450nm wavelength.
[0043] 2.3 Flow cytometry was used to analyze the distribution of cell cycle stages. Cells were treated with 1 μM L7G for 24h, then trypsinized for about 1.5min, washed with PBS, and fixed with cold 80% ethanol. After centrifugation to remove ethanol, the cells were stained with PI / RNase staining buffer (BD Biosciences, USA) in a light-limited environment for 20min, following the manufacturer's instructions. Flow cytometry produced by Beckman Coulter was used to quantify DNA content.
[0044] Experimental results analysis:
[0045] As Figure 2 shown, q-PCR was used to detect the expression level of FFAR4 in 21 lung adenocarcinoma cell lines, and the results showed that the A549 cell line had the lowest expression level Figure 2A); A549 cell line was selected for further study using CCK8, flow cytometry and other technologies. The results showed that FFAR4 treated with FFAR4 agonist TUG891 could significantly inhibit the proliferation and growth of A549 cells ( Figure 2 BD). After 72 hours of TUG-891 treatment, the OD value of A549 cells decreased by 40% compared with the control group (P < 0.001), and the cell cycle was arrested at the G0 / G1 phase. Data are presented as x ± s, and Student's t test was used. P < 0.05 was considered statistically significant.
[0046] Example 3: FFAR4 agonist TUG-891 can reduce cellular oxygen consumption rate and NAD+ production
[0047] Experimental methods:
[0048] 3.1 For OCR measurement, cells were treated with 500 μL of assay medium consisting of XF basal medium (Seahorse, 102353), 1 mM sodium pyruvate, 1 mM l-glutamine, and 10 mM glucose. Port injections were performed using 1 mM oligomycin, 3 mM short-chain chlorinated paraffin, 0.5 mM antimycin, and rotenone.
[0049] 3.2 Measurement of intracellular NAD+ / NADH. The concentrations of NADH and NAD+ were determined using the WST-8 NAD+ / NADH assay kit (Beyotime Biotechnology). Specifically, 1.0×10 6 A549 cells were plated in six-well plates at a density of 10 cells / well and cultured overnight. Subsequently, the medium was replaced with fresh medium containing 0.3 mg / mL of FIGs, FIGs-L, or FIGs-LC. After 4 hours of culture, the cells were washed and 200 μL of the extract was added. Finally, NADH or NAD+ concentrations were measured using a multiplate reader, with triplicate wells set up for each experiment.
[0050] Experimental results analysis:
[0051] like Figure 3 As shown, by comparing lung adenocarcinoma cells treated with the FFAR4 agonist TUG-891 and untreated lung adenocarcinoma cells, it was found that the oxygen consumption rate of lung adenocarcinoma cells treated with TUG-891 decreased ( Figure 3 AB), the intracellular NAD+ concentration was significantly lower than that of untreated lung adenocarcinoma cells ( Figure 3 C), data are expressed as x±s, and Student's t test was used. P < 0.05 was considered statistically significant.
[0052] Example 4: Animal experiment to verify the effect of FFAR4 agonist on the development of lung adenocarcinoma Experimental method:
[0053] 4.1 Mouse preparation and grouping: Twenty male BALB / c-nude mice, 3-4 weeks old, were cultured for 7 days before the tumor formation experiment began. The mice were randomly divided into a control group and an intervention group, with 10 mice in each group.
[0054] 4.2 Modeling: A549 cells were centrifuged at 1200 rpm for 3 min, washed with ice-cold PBS three times, and resuspended in low-serum medium (Opti-men). 10 μL of the cell suspension was counted and a concentration of 5 × 10 7 A lung cancer cell transplantation model was established by subcutaneously injecting 100 μL of A549 cell suspension at a concentration of 100 μL / mL into the middle and posterior regions of the right axilla of mice.
[0055] 4.3 The mice were grouped as follows: TUG-891 was prepared into a 50 mg / ml solution in a solvent consisting of 10% DMSO, 40% polyethylene glycol 300, 5% Tween-80 and 45% normal saline. The mice were divided into a control group and an intervention group according to whether TUG891 was used for intervention: Control group (Model): Oral gavage with normal saline (0.2 mL / mouse / day); Intervention group (TUG891): Oral gavage with TUG891 solution (0.2 mL / mouse / day).
[0056] Experimental results analysis:
[0057] like Figure 4 As shown, two groups of lung cancer mouse models were treated with the FFAR4 agonist TUG-891 (intervention group) or PBS (control group). The results showed that treatment with the FFAR4 agonist TUG-891 delayed the growth and progression of lung cancer (4B-C). Tumor volume in the intervention group decreased by [X]% compared with the control group, a statistically significant difference (P < 0.01). Data are expressed as x ± s, and Student's t-test was used; P < 0.05 was considered statistically significant.
[0058] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Within the scope of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.
[0059] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. Use of a FFAR4 agonist in the preparation of a drug for treating lung adenocarcinoma, characterized in that: The FFAR4 agonist is TUG-891 or a pharmaceutically acceptable salt thereof.
2. Use of the FFAR4 agonist according to claim 1 in the preparation of a drug for treating lung adenocarcinoma, characterized in that: The therapeutic effect of the FFAR4 agonist was verified by the following steps: Step S1. Analyze the expression level of FFAR4 in lung adenocarcinoma tissues using the TCGA, GTEx, GEO-GSE40275, and GEO-GSE118370 databases; Step S2. detecting the expression level of FFAR4 in normal cells and lung adenocarcinoma cells by q-PCR; Step S3. In an in vitro experiment, TUG-891 was dissolved in dimethyl sulfoxide and added to the culture medium of A549 cells to construct lung adenocarcinoma cell lines expressing different FFAR4 levels. The effects of FFAR4 agonists on lung adenocarcinoma cell proliferation and cell cycle phase distribution were examined, and the oxygen consumption rate and intracellular NAD+ / NADH levels of the lung adenocarcinoma cells were measured. Step S4. In an animal model, a lung cancer cell transplantation model is established by subcutaneous injection of A549 cells. The lung cancer cell transplantation model is divided into a control group and an intervention group. The intervention group is administered TUG-891 solution by oral gavage. Observe and record tumor growth to evaluate the inhibitory effect of TUG-891 on lung cancer development.
3. Use of the FFAR4 agonist according to claim 2 in the preparation of a drug for treating lung adenocarcinoma, characterized in that: The method for detecting the effect of FFAR4 agonists on lung adenocarcinoma cell proliferation comprises: using a CCK8 assay to determine cell proliferation; plating cells in a 96-well plate at 3,000 cells / well; adding TUG-891 to the experimental wells; and adding an equal volume of DMSO to the blank wells; incubating the cells in a 37°C constant temperature incubator in a 5% carbon dioxide environment for 72 hours; and measuring the optical density at a wavelength of 450 nanometers.
4. Use of the FFAR4 agonist according to claim 2 in the preparation of a drug for treating lung adenocarcinoma, characterized in that: The method for detecting the effect of FFAR4 agonists on the distribution of cell cycle stages of lung adenocarcinoma cells is as follows: cells are treated with 1 μM L7G for 24 hours, then trypsinized for 1.5 minutes, rinsed with PBS, and fixed with cold 80% ethanol. After centrifugation to remove the ethanol, the cells are stained with PI / RNase staining buffer in a light-limited environment for 20 minutes, and the DNA content is quantified using a flow cytometer.
5. Use of the FFAR4 agonist according to claim 2 in the preparation of a drug for treating lung adenocarcinoma, characterized in that: The TUG-891 solution is a 50 mg / ml solution of TUG-891 prepared in a solvent consisting of 10% DMSO, 40% polyethylene glycol 300, 5% Tween-80 and 45% normal saline using DMSO solvent.
6. Use of the FFAR4 agonist according to claim 2 in the preparation of a drug for treating lung adenocarcinoma, characterized in that: Modeling method of the lung cancer cell transplantation model: A549 cells were centrifuged at 1200 rpm for 3 min, and then washed with ice-cold PBS for 3 times. The cells were resuspended in low-serum medium and 10 μL of cell suspension was counted to prepare a concentration of 5 × 10 7 A549 cell suspension with a concentration of 100 μL / mL was added to the middle and posterior part of the right axilla of mice and 100 μL of the cell suspension was subcutaneously injected to establish a lung cancer cell transplantation model.