Use of valproic acid in the preparation of a radiosensitizer for lung adenocarcinoma
By regulating the radiation sensitivity of lung adenocarcinoma cells with valproic acid and targeting the GLIPR2 protein to inhibit BECN1 activity, the problem of radiotherapy resistance in lung adenocarcinoma was solved, and the radiotherapy effect was significantly improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANTONG TUMOR HOSPITAL
- Filing Date
- 2025-04-14
- Publication Date
- 2026-07-24
Smart Images
Figure CN120204195B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to the application of valproic acid in the preparation of radiosensitizers for lung adenocarcinoma. Background Technology
[0002] Lung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer. Despite continuous improvements in early cancer screening and prevention in recent years, 75% of lung cancer patients are still diagnosed at an advanced stage, missing the optimal time for radical surgical treatment. Radiotherapy is a standard treatment component for patients with advanced LUAD and is widely used. However, clinical observations have revealed that 50% of LUAD patients develop radiotherapy resistance, resulting in a 5-year survival rate of less than 17%. Therefore, exploring the mechanisms of radiotherapy resistance in LUAD and discovering effective radiosensitizers are crucial steps to improve the survival rate of lung cancer patients.
[0003] Radiotherapy primarily kills tumor cells through reactive oxygen species (ROS) generation and DNA breakage. This involves complex mechanisms including abnormal DNA damage repair, changes in cell cycle and apoptosis, hypoxic environments, and metabolic alterations. These factors ultimately lead to a decrease in the overall efficacy of radiotherapy. In recent years, autophagy has been shown to play a crucial role in protecting cells from ionizing radiation damage. Post-radiotherapy, autophagy is activated, and to some extent, inhibiting autophagy can enhance the radiosensitivity of various tumor cells, including lung cancer cells. Studies have found that glioma pathogenesis-related protein 2 (GLIPR2) exhibits differential expression in various tumor cells, and its expression level is closely related to the tumor cell response to radiotherapy. GLIPR2 is a negative autophagy regulator that can bind to BECN1. By inhibiting BECN1 activity or interfering with its interaction with the PtdIns3K-C1 complex, it inhibits the activation of the autophagy pathway. However, the role of GLIPR2 in LUAD radiotherapy has not been reported in detail.
[0004] Valproic acid (VPA) is a widely used drug for the treatment of epilepsy and bipolar disorder. Recent studies have revealed its role as a histone deacetylase inhibitor (HDAC inhibitor). HDAC inhibitors, by regulating chromatin structure and gene expression, have shown potential anti-tumor activity in cancer treatment. Studies have shown that valproic acid can not only inhibit tumor cell proliferation and induce apoptosis, but may also reverse tumor cell resistance to chemotherapy drugs through multiple mechanisms. In the treatment of lung adenocarcinoma, research on valproic acid has mainly focused on the following aspects: in non-small cell lung cancer, VPA significantly increases cell sensitivity to cisplatin; in liver cancer, VPA can act as a radiosensitizer, increasing the killing effect of radiation on liver cancer cells; however, the role of VPA in LUAD radiotherapy remains unknown, and whether VPA can target GLIPR2 to inhibit autophagy and promote LUAD radiosensitization has not been reported. Summary of the Invention
[0005] In view of the above-mentioned problems in the prior art, the technical problem to be solved by the present invention is to provide the application of valproic acid in the preparation of radiosensitizers for lung adenocarcinoma, so as to enhance the effect of radiotherapy for lung adenocarcinoma.
[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0007] Application of valproic acid in the preparation of radiosensitizers for lung adenocarcinoma.
[0008] The application of valproic acid in regulating the radiation sensitivity of lung adenocarcinoma cells, wherein the lung adenocarcinoma cells are PC9 and H1299; the regulation of the radiation sensitivity of lung adenocarcinoma cells is to enhance the radiation sensitivity of lung adenocarcinoma cells; and the radiation dose is 6 Gy.
[0009] Application of valproic acid in regulating GLIPR2 protein expression.
[0010] Application of valproic acid in regulating BECN1 protein expression.
[0011] Application of valproic acid in regulating the activity of autophagy marker BECN1.
[0012] Compared with the prior art, the present invention has the following advantages:
[0013] 1) This invention discloses for the first time the application of valproic acid in the preparation of radiosensitizers for lung adenocarcinoma. The results show that at concentrations far below the half-inhibition rate of VPA, 5 μM VPA significantly enhances the sensitivity of lung adenocarcinoma cells PC9 and H1299 to radiotherapy, and the effect is more obvious with prolonged treatment time (**: P < 0.01).
[0014] 2) The present invention constructed a low-concentration VPA combined with radiotherapy group and compared it with the radiotherapy alone group. The results showed that the number and size of lung adenocarcinoma cells PC9 and H1299 were significantly reduced in the 5 μM VPA combined with radiotherapy group. The low-concentration VPA significantly promoted radiosensitization of lung adenocarcinoma.
[0015] 3) The present invention constructs a low-concentration VPA combined with radiotherapy group. Compared with the irradiation group alone, the VPA combined with irradiation group significantly promotes the binding of GLIPR2 and BECN1, thereby inhibiting the activity of the autophagy marker BECN1 and reducing the occurrence of autophagy. Attached Figure Description
[0016] Figure 1 The IC50 curves of the half-inhibition rate of VPA on lung adenocarcinoma cells PC9 and H1299 after 24 hours of treatment;
[0017] Figure 2 The graph shows the effect of 5 μM VPA concentration on significantly enhancing the killing effect of radiotherapy on lung adenocarcinoma cells PC9 and H1299;
[0018] Figure 3 A graph showing that 5 μM VPA significantly enhances the inhibitory effect of radiotherapy on the proliferation of lung adenocarcinoma cells PC9 and H1299.
[0019] Figure 4 Molecular docking diagrams of VPA (blue) with GLIPR2 (red) and BECN1 (green);
[0020] Figure 5 The diagram shows the binding interaction between GLIPR2 and BECN1 under radiotherapy promoted by 5 μM VPA.
[0021] Figure 6 A graph showing the results of VPA improving the sensitivity of lung adenocarcinoma cells to radiotherapy by targeting GLIPR2;
[0022] Figure 7 The graph shows the correlation between the expression of GLIPR2 and BECN1 in tumor tissues of patients with radiotherapy resistance and radiosensitivity in lung adenocarcinoma (A), the expression trend of GLIPR2 and BECN1 in the radiotherapy cohort of lung adenocarcinoma (B), and the prognostic survival graph of patients with radiotherapy-treated lung adenocarcinoma who express GLIPR2 in combination with BECN1 (C). Detailed Implementation
[0023] The present invention will be further described below with reference to specific embodiments. In the following embodiments, operations not described in detail are routine biological experimental procedures, which can be performed with reference to molecular biology experimental manuals and existing publicly available journal articles, or according to the instructions of the reagent kit.
[0024] The lung adenocarcinoma cells (PC9, H1299) used in this application were purchased from the ATCC cell bank.
[0025] The tumor tissue specimens used in this application from 33 patients with lung adenocarcinoma who underwent radiotherapy were obtained from paraffin tissue specimens preserved at Nantong Cancer Hospital.
[0026] Example 1
[0027] 1. VPA's half-inhibition rate (IC50) against lung adenocarcinoma cells
[0028] Lung adenocarcinoma cells (PC9 and H1299) in the logarithmic growth phase were diluted to a concentration of 5 × 10⁻⁶. 4 Cell suspension at a concentration of 100 μL / mL was seeded into 96-well plates. After cell attachment, different concentrations of VPA were added, with six replicates for each concentration. After 24 hours, 10 μL of CCK8 assay reagent was added to each well, and the plates were incubated in culture medium. Cell proliferation data were detected and analyzed using a microplate reader after 1.5 hours.
[0029] The results are as follows Figure 1 As shown, the half-inhibition rate (IC50) of VPA in lung adenocarcinoma cells PC9 was 146.1 μM, and the half-inhibition rate (IC50) of VPA in lung adenocarcinoma cells H1299 was 149.2 μM.
[0030] 2. The killing effect of 5μM VPA on lung adenocarcinoma cells during radiotherapy.
[0031] Lung adenocarcinoma cells (PC9 and H1299) in the logarithmic growth phase were diluted to a concentration of 5 × 10⁻⁶. 4 Cell suspension at a concentration of 100 μL / mL was seeded into 96-well plates. After cell attachment, the plates were divided into four groups of three: control group (normal culture), VPA group (5 μM VPA), irradiation group (IR 6 Gy, 6 Gy irradiation), and VPA combined with irradiation group (VPA + IR(6 Gy), 5 μM VPA combined with 6 Gy irradiation). Each group had six replicates. At 24, 48, and 72 hours, 10 μL of CCK8 assay reagent was added to each well, and the cells were cultured in culture medium. Cell proliferation data were detected and analyzed using a microplate reader after 1.5 hours.
[0032] The results are as follows Figure 2 As shown, at concentrations far below the VPA half-inhibition rate, 5 μM VPA significantly enhanced the sensitivity of lung adenocarcinoma cells PC9 and H1299 to radiotherapy, and the effect was more pronounced with prolonged treatment time (**: P < 0.01).
[0033] 3. Inhibitory effect of 5 μM VPA on lung adenocarcinoma cells during radiotherapy
[0034] Seed 1000 cells per well in a 6-well plate and incubate at 37°C with 5% CO2 for 24 hours. Place the 6-well plate under 6 Gy high-energy X-rays, with the construct zone set at 2 cm, to expose the cells to radiation. After radiation treatment, return the cells to the incubator and incubate for 14 days. Replace the culture medium with fresh medium every 3 days. Discard the culture medium and gently wash the cells 3 times with PBS. Fix the cells with 4% paraformaldehyde, discard after 30 minutes, wash 3 times with PBS, and finally stain with 1% crystal violet solution. Wash 3 times with PBS after staining and allow the 6-well plate to air dry.
[0035] The results are as follows Figure 3 As shown, compared with the radiotherapy alone group, the number and size of colonies of lung adenocarcinoma cells PC9 and H1299 were significantly reduced in the 5 μM VPA combined with radiotherapy group. The results indicate that low concentration VPA significantly promotes radiosensitization of lung adenocarcinoma.
[0036] 4. Molecular docking analysis
[0037] NetworkAsia database analysis indicated that VPA can bind to GLIPR2 and BECN1, and that VPA promotes GLIPR2 expression while inhibiting BECN1 expression. SDF format files of VPA were obtained from the PubChem database, and protein structures were collected from the PDB database. PyMol software was used to optimize the target site by removing water molecules and small molecule ligands, and AutoDockTools was used for hydrogenation and charge processing before saving as a pdbqt file. Using the key target as the receptor and its corresponding active ingredient as the ligand, molecular docking was performed using VIA within PyRX software, and the binding energy was calculated, resulting in the output file. Finally, PyMol software was used for result visualization.
[0038] The results are as follows Figure 4 As shown, VPA attaches to the GLIPR2 and BECN1 binding domains. The results indicate that VPA functions by promoting GLIPR2 protein expression while inhibiting BECN1 protein expression.
[0039] 5. Immunoprecipitation
[0040] PC9 cells were seeded in two 10 cm diameter round dishes. When cell confluence reached 70%, one dish was treated with 5 μM VPA, while the other dish served as a control. Both dishes were simultaneously irradiated with 6 Gy, and cells were collected after 24 hours. Cell collection and lysis: The culture medium was discarded, and cells were washed twice with pre-chilled PBS, all performed on ice. 1 mL of RIPA lysis buffer was added to a culture dish, incubated for 15 minutes, and then the protein was scraped off. 200 μL of protein lysis buffer was frozen at -80°C and designated as the input group. GLIPR2 was added to 400 μL of protein lysis buffer according to the antibody instructions, and IgG-specific antibody was added to another 400 μL of protein lysis buffer. The mixture was incubated overnight at 4°C with gentle rotation. 40 μL of Protein A / G agarose beads were washed with an appropriate amount of 0.1% Triton X-100 solution, centrifuged, and the supernatant was discarded. This process was repeated three times. Protein A / G agarose beads were added to the cell lysate incubated with the antibody, and the mixture was incubated at 4°C with slow rotation for 6-8 hours to ensure complete binding. After centrifugation, the supernatant was removed, and the agarose beads were rinsed three times. 10 μL of 2× loading buffer was added to each rinse, and the sample was heated in a dry-state thermostat at 95°C for 5 minutes to obtain a protein sample with co-precipitated target molecule and specific IgG. A 10% SDS-polyacrylamide gel was prepared, and electrophoresis was performed at 80V for 120 minutes, followed by PVDF transfer at 260mA for 90 minutes. After transfer, the PVDF membrane was blocked in 5% skim milk for 2 hours. The membrane was washed twice with TBST solution for 5 minutes each. Primary antibody incubation: Mouse anti-human BECN1 antibody was diluted 1:1000 and incubated overnight at 4°C. The membrane was washed three times with TBST solution for 10 minutes each. Secondary antibody incubation: Goat anti-mouse secondary antibody was diluted 1:1000 and incubated at room temperature for 2 hours. Wash with TBST solution for 10 minutes each time, 3 times. ECL development and result analysis.
[0041] The results are as follows Figure 5 As shown, compared with the irradiation group alone, the VPA combined irradiation group significantly promoted the binding of GLIPR2 and BECN1, thereby inhibiting the activity of the autophagy marker BECN1 and reducing the occurrence of autophagy.
[0042] 6. VPA enhances the sensitivity of lung adenocarcinoma cells to radiotherapy by targeting GLIPR2.
[0043] PC9 lung adenocarcinoma cells in the logarithmic growth phase were diluted to a concentration of 5 × 10⁻⁶. 4Cell suspensions of 100 μL per well were seeded into 96-well plates. After cell attachment, the plates were placed in an incubator with 3 wells per group. The plates were divided into a blank control group, a VPA group (with 5 μM VPA), and a VPA combined with GLIPR2 interference group (VPA+si-GLIPR2). Each treatment group was irradiated with 6 Gy and the results were measured at 24, 48 and 72 hours.
[0044] The results are as follows Figure 6 As shown, the VPA group significantly increased the efficacy of radiotherapy for lung adenocarcinoma, but interfering with GLIPR2 expression inhibited the radiosensitizing effect of VPA. The results indicate that VPA enhances the sensitivity of lung adenocarcinoma cells to radiotherapy by targeting GLIPR2.
[0045] 7. Correlation between GLIPR2 and BECN1 expression in tumor tissues of patients with radioresistant and radiosensitive lung adenocarcinoma
[0046] Immunohistochemical staining of tumor tissue specimens from 33 patients with lung adenocarcinoma who underwent radiotherapy was performed using GLIPR2 and BECN1. The method is as follows:
[0047] 1) Dewaxing of paraffin sections: Place paraffin sections in an oven at 60°C for 1 hour, then wash with xylene I and II for 10 minutes each, followed by a gradient of alcohols: 100% alcohol, 95% alcohol, 80% alcohol, and 70% alcohol for 2 minutes each, and finally wash with distilled water for 5 minutes each time.
[0048] 2) Hydrogen peroxide blocks endogenous peroxidase: Apply 3% H2O2 to the tissue and incubate at room temperature for 10 minutes in the dark. Then wash with distilled water for 5 minutes twice.
[0049] 3) Antigen retrieval: Sodium citrate buffer was used to submerge tissue sections and boil them in an autoclave. After steaming for 5 minutes, the sections were slowly cooled and then placed in PBS for 5 minutes twice.
[0050] 4) Serum blocking: Remove the slides, circle the tissue with a drawing pen, keep the tissue moist, add 100 μL of goat serum to each slide, and incubate at 37°C for 30 minutes.
[0051] 5) Add GLIPR2 and BECN1 antibodies: Absorb the serum with filter paper, do not wash, and directly add mouse anti-human BECN1 antibody (1:200) or rabbit anti-human GLIPR2 antibody (1:200), and incubate overnight at 4°C.
[0052] 6) After washing with PBS for 5 minutes × 3 times, add HRP-labeled secondary antibody and incubate at room temperature for 30 minutes.
[0053] 7) Wash with PBS for 5 minutes × 3 times, then develop with DAB, and stop the development with tap water.
[0054] 8) Counterstain with hematoxylin at room temperature for 2 minutes.
[0055] 9) Rinse with tap water for 15 minutes to restore blue color.
[0056] 10) Gradient alcohol dehydration: 5 minutes each of 70% alcohol, 80% alcohol, 95% alcohol, and 100% alcohol.
[0057] 11) Xylene clearing: Xylene I and II for 5 minutes each.
[0058] 12) Mounting: Use neutral resin for mounting.
[0059] The results are as follows Figure 7 As shown, GLIPR2 was lowly expressed and BECN1 was highly expressed in tumor tissues of patients with radiosensitive (RS) lung adenocarcinoma; while the opposite trend was observed in tumor tissues of patients with radioresistant (RR) lung adenocarcinoma. The expression trends of GLIPR2 and BECN1 in the radiotherapy cohort of lung adenocarcinoma were significantly negatively correlated (r = -0.66, P = 8.8e-5). Survival analysis results indicate that patients with radiotherapy-treated lung adenocarcinoma who showed low GLIPR2 expression and high BECN1 expression had a poorer prognosis.
Claims
1. Application of valproic acid in the preparation of radiosensitizers for lung adenocarcinoma; among which, The lung adenocarcinoma cells were PC9 and H1299. The radiation dose was 6 Gy, and the valproic acid dosage was 5 μM. The combined irradiation with valproic acid promoted the binding of GLIPR2 and BECN1, inhibited the activity of the autophagy marker BECN1, reduced the occurrence of autophagy, and improved the sensitivity of lung adenocarcinoma cells to radiotherapy.