Application of ATGL inhibitor NG-497 in treatment of lung cancer / lung cancer metastasis

Abstract

The invention discloses an application of an ATGL inhibitor NG-497 in treatment of lung cancer / lung cancer metastasis. Researches prove that NG-497 can reduce the cell membrane fluidity and reduce the resistance of lung adenocarcinoma cells to fluid shear force by inhibiting decomposition of triglyceride, so that transfer of the lung adenocarcinoma cells is inhibited. The invention provides a potential new medicine and a new therapeutic target for lung cancer treatment or clinical treatment of lung cancer metastasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of an ATGL inhibitor, NG-497, in the treatment of lung cancer / lung cancer metastasis. Background Technology

[0002] Lung cancer is one of the most frequently diagnosed cancers worldwide, and the incidence of lung cancer metastasis is very high. Lung cancer metastasis significantly increases the morbidity and mortality rates of patients. A major reason why lung cancer cells can metastasize is that tumor cells can resist the killing effect of circulating shear stress.

[0003] Fluid shear stress (FSS) is the tangential force exerted on the cell surface by flowing fluid and plays a crucial role in tumor metastasis. Tumor cells often exhibit a "dull" response to fluid shear stress, meaning they are less sensitive than normal cells. This is primarily attributed to the downregulation of expression of cell surface mechanosensor proteins such as TRPM7. Under shear stress, tumor cells may experience mechanical damage or death, but some cells survive through adaptive mechanisms.

[0004] Metabolic reprogramming refers to the process by which cells, under specific physiological and pathological conditions, systematically adjust and transform their metabolic patterns to adapt to changes in the external environment and meet their own growth and differentiation needs. This concept is used in tumor biology, immunology, and stem cell research.

[0005] Recent studies have shown that lipids, as substrates for oxidation, play an important role in cellular redox processes, especially during metastasis and colonization. In addition, the lipid composition of the cell membrane determines the physical properties of the cell membrane, which can affect the resistance of tumor cells to fluid shear forces. However, the mechanisms by which tumor cells overcome fluid shear forces and generate distant metastases have not been fully elucidated. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned problems by providing an application of NG-497 in the treatment of lung cancer / lung cancer metastasis.

[0007] To achieve its objective, the present invention employs the following technical solution:

[0008] A first aspect of the present invention provides the use of NG-497 in the preparation of medicaments for treating lung cancer and / or lung cancer metastases.

[0009] A second aspect of the invention provides the use of NG-497 in the preparation of formulations that inhibit the migration or proliferation of lung cancer cells.

[0010] A third aspect of the invention provides the use of NG-497 in the preparation of formulations that inhibit the resistance of lung cancer cells to fluid shear forces.

[0011] In the above application technical solution, the lung cancer is lung adenocarcinoma.

[0012] In the above-mentioned application technology, NG-497 reduces the cell membrane fluidity of lung cancer cells.

[0013] In the above-mentioned application technology, NG-497 reduces the resistance of lung cancer cells to fluid shear forces.

[0014] In the above-mentioned application technology, lung cancer cells generate stronger proliferative capacity by resisting fluid shear force. Lung cancer cells improve cell membrane fluidity and resist fluid shear force by decomposing triglycerides and synthesizing more phospholipids.

[0015] In the above-mentioned application technology, NG-497 inhibits the decomposition of triglycerides in lung cancer cells, reduces the cell membrane fluidity of lung cancer cells, reduces the resistance of lung cancer cells to fluid shear forces, and thus reduces the proliferation and migration ability of lung cancer cells.

[0016] The beneficial effects of this invention are as follows: This study confirms that NG-497 can inhibit the metastasis of lung adenocarcinoma cells by inhibiting triglyceride breakdown, thereby reducing cell membrane fluidity and decreasing the resistance of lung adenocarcinoma cells to fluid shear forces. This invention provides a potential new drug and a new therapeutic target for the clinical treatment of lung cancer or lung cancer metastasis. Attached Figure Description

[0017] Figure 1 The results showed that: A. A549 cell proliferation was reduced under NG-497 treatment; B. NG-497 treatment increased triglyceride levels in A549 cells; C. NG-497 treatment reduced cell membrane fluidity in A549 cells; D. NG-497 treatment reduced the number of circulating tumor cells in an animal model; E. NG-497 treatment reduced the proliferation of A549 cells under fluid shear stress; F. NG-497 treatment increased triglyceride levels in A549 cells under fluid shear stress; G. NG-497 treatment significantly reduced cell membrane fluidity of A549 cells under fluid shear stress; H. NG-497 treatment significantly reduced the metastatic ability of A549 cells. Detailed Implementation

[0018] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0019] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0020] Example 1

[0021] Main sources of reagents and instruments:

[0022] Human lung cancer cell line A549, BALB / c nude mice, carbon dioxide incubator (MCO-18AC), clean bench (JL-CJ-HW1B), Bio-Rad vertical electrophoresis apparatus, laser confocal microscopy (LeicaDMI8), triglyceride ELISA kit (E-BC-K261-M, Sangon Biotech).

[0023] NG-497 (HY-148756, MCE): Selective human adipose triglyceride lipase (ATGL) inhibitor, CAS No.: 2598242-66-9.

[0024] 1. Experimental Methods

[0025] 1.1 Cell Culture

[0026] Our research team preserved the human lung adenocarcinoma cell line A549 in our laboratory. We cultured it in DMEM medium (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum added in an incubator at 37°C containing 5% CO2.

[0027] 1.2 NG-497 treatment of cells

[0028] NG-497 (HY-148756, MCE) was prepared into a 10 mM stock solution using DMSO and stored at -80°C. For cell treatment, a cell suspension containing 2000 A549 cells was added to a 96-well plate, followed by the addition of DMEM medium containing 10% fetal bovine serum to a volume of 100 μL. Then, 0.5 μL of NG-497 stock solution was added to each well to achieve a 50 μM NG-497 concentration. The plates were then incubated at 37°C with 5% CO2 for 12 hours before proceeding to other experiments.

[0029] 1.3 NG-497 treatment of animals

[0030] Use 1×10 6 Four-week-old female nude mice were injected intravenously with A549 cells, followed by NG-497 injections every three days starting from day one post-injection for four weeks. At injection, the NG-497 stock solution was diluted to a 0.1 mM concentration and injected according to the mouse's body weight (50 μL / g). In vivo imaging was performed on the mice four weeks later, followed by euthanasia and dissection for further tissue imaging.

[0031] 1.4 External Fluid Shear Force Treatment

[0032] Laminar fluid shear force was simulated using a peristaltic pump (DKCP-S10, Kamoer) and silicone tubing. The fluid shear force was controlled by adjusting the diameter of the silicone tubing and the power of the peristaltic pump. The fluid shear force was calculated using the Poietille equation. ,in Shear force, expressed in densities per centimeter. 2 , Let R be the dynamic viscosity of the fluid, R be the inner diameter of the silicone tube, and Q be the flow rate in ml / sec. During fluid shear stress treatment, one end of the silicone tube is placed in DMEM culture medium, and the other end is placed in a graduated cylinder. A peristaltic pump is used to pump the DMEM into the graduated cylinder within a certain time period, from which the flow rate Q can be calculated. The viscosity of the DMEM culture medium is fixed at 0.012 dynes·sec / cm³. 2 The fluid shear force can be adjusted by controlling the diameter of the silicone tube and the power of the peristaltic pump. In this invention, the inner diameter of the silicone tube is 0.1 cm, and the peristaltic pump power is adjusted to 40%, with a flow rate of 0.98125 ml / sec. At this point, the fluid shear force is 15 dynes / cm. 2 The procedure was then performed on a sterile operating table, adding 5 ml of DMEM culture medium to a sterile beaker, followed by 1–5 × 10⁻⁶ ml of [unspecified ingredient]. 6 A549 cells were obtained by placing both ends of a silicone tube into a beaker and fixing it below the liquid surface. Then, a peristaltic pump was started, and the cells were circulated for 1-12 hours to obtain cells treated with fluid shear force.

[0033] 1.5 CCK8 Experiment

[0034] The cell proliferation activity was detected using the CCK8 assay kit (C0038, Beyotime). 100 μL of 2000 A549 cells were added to each well of a 96-well plate and cultured at 37°C in an incubator containing 5% CO2 until the cells adhered. Then, 10 μL of CCK8 solution was added to each well and incubated at 37°C for 1 hour. The absorbance was then measured at 450 nm.

[0035] 1.6 Photochromic Fluorescence Recovery Technology

[0036] 10,000 A549 cells were added to a confocal microarray, and 5 μM of Dil dye (C1036, Beyotime) was added. The cells were incubated for 20 minutes to stain the cell membrane, followed by washing with PBS. Photochromism was performed using a laser confocal microarray (Leica DMI8). At 0 seconds, a fixed area of ​​the cell membrane was irradiated with strong fluorescence, followed by continuous imaging for 180 seconds, with images taken every 10 seconds. Relative cell membrane fluidity was then calculated based on the fluorescence recovery time.

[0037] 1.7 Animal Experiments with Fluid Shear Force

[0038] Before the experiment, A549 cells were pretreated with PBS or NG-497 (10 mM) for 12 hours, and then treated with 1×10⁻⁶ PBS. 6 Four-week-old female nude mice were injected with mCherry-labeled A549 cell lines via ventricular injection. Twelve hours later, blood was collected from the orbital cavity, and the mice were euthanized. Three volumes of erythrocyte lysis buffer (C3702, Beyotime) were added to the blood samples, and lysis was performed for 1-2 minutes. The samples were then centrifuged at 500g, the red supernatant was discarded, and the cells were washed twice with PBS. Cell sorting and counting were then performed using flow cytometry. Since tumor cells carry mCherry fluorescent protein, tumor cells were screened and counted based on red fluorescence.

[0039] 2. Results

[0040] 2.1 The CCK8 assay showed that NG-497 treatment significantly reduced the proliferation ability of A549 cells.

[0041] We used the CCK8 assay to determine the proliferation capacity of A549 cells treated with NG-497 under normal conditions and fluid shear stress (FSS) treatment, and found that their proliferation capacity was significantly reduced in the presence of NG-497. Figure 1 A and Figure 1 E).

[0042] 2.2 Photodecolorization and fluorescence recovery experiments showed that NG-497 reduced cell membrane fluidity in A549 cells.

[0043] We treated A549 cells with NG-497, an inhibitor of adipose triglyceride lipase (ATGL), a key enzyme in triglyceride hydrolysis. We found that NG-497 treatment significantly reduced cell membrane fluidity in A549 cells under both normal and fluid shear stress conditions. Figure 1 C and Figure 1 G).

[0044] 2.3 NG-497 significantly reduced the number of circulating tumor cells in nude mice.

[0045] We pretreated A549 cells with PBS or NG-497, and then injected the pretreated A549 cells into the ventricle of nude mice. Twelve hours later, blood was collected from the orbital fossa, and tumor cells were sorted and counted using flow cytometry. We found that NG-497 treatment significantly reduced the number of circulating tumor cells in the blood of nude mice. Figure 1 D).

[0046] 2.4 NG-497 can increase triglyceride levels in A549 cells.

[0047] We used a triglyceride ELISA kit to detect triglyceride levels in A549 cells under normal conditions and fluid shear stress, and found that triglyceride levels in A549 cells were significantly increased. Figure 1 B and Figure 1 F).

[0048] 2.5 NG-497 significantly reduced the transfer ability of A549 in nude mouse models.

[0049] To investigate the inhibitory effect of NG-497 on lung adenocarcinoma metastasis, we administered A549 cells to nude mice via intracardiac injection, followed by treatment with NG-497. The results showed that NG-497 significantly inhibited the metastatic ability of A549 cells. Figure 1 H).

[0050] The above results indicate that NG-497 can inhibit triglyceride breakdown by suppressing the function of ATGL, thereby reducing cell membrane fluidity and decreasing the resistance of lung adenocarcinoma cells to fluid shear forces, thus inhibiting metastasis.

[0051] 3. Summary and Analysis

[0052] This invention demonstrates that NG-497 can inhibit the metastasis of lung adenocarcinoma cells by reducing cell membrane fluidity and decreasing the resistance of lung adenocarcinoma cells to fluid shear forces, thereby inhibiting the breakdown of triglycerides. This invention provides a potential new drug and a new therapeutic target for the clinical treatment of lung cancer or lung cancer metastasis.

Claims

1. Use of NG-497 in the preparation of a medicament for treating lung cancer and / or lung cancer metastasis.

2. Use of NG-497 in the preparation of a preparation for inhibiting lung cancer cell migration or proliferation.

3. Use of NG-497 in the preparation of a preparation for inhibiting the resistance of lung cancer cells to fluid shear stress.

4. Use according to claim 2 or 3, characterized in that: The lung cancer is lung adenocarcinoma.

5. Use according to any one of claims 2 to 4, characterized in that: NG-497 reduces the cell membrane fluidity of lung cancer cells.

6. Use according to claim 2, characterized in that: NG-497 reduces the resistance of lung cancer cells to fluid shear stress.

7. Use according to any one of claims 1 to 3, characterized in that: Lung cancer cells produce stronger proliferation ability by resisting fluid shear stress, and lung cancer cells improve cell membrane fluidity by decomposing triglycerides and synthesizing more phospholipids, and resist fluid shear stress.

8. Use according to claim 7, characterized in that: NG-497 inhibits the decomposition of triglycerides by lung cancer cells, reduces the cell membrane fluidity of lung cancer cells, reduces the resistance of lung cancer cells to fluid shear stress, and thus reduces the proliferation and migration ability of lung cancer cells.

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