Application of glutamine in preparation of medicine for enhancing radiotherapy and killing wound pharyngeal squamous cell carcinoma

Abstract

The invention discloses application of glutamine in preparation of drugs for enhancing radiotherapy and killing wound pharyngeal squamous cell carcinoma, and relates to the technical field of biological medicines. According to the application disclosed by the invention, the tumor radiosensitivity is improved by taking glutamine as a target spot for the first time, a mechanism for improving the radiosensitivity of oropharyngeal squamous cell carcinoma by HPV infection is analyzed, the effectiveness of regulating the OPSCC radiosensitivity by glutamine is proved, and a new scheme is provided for developing tumor sensitization treatment medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of glutamine in the preparation of a drug for enhancing radiotherapy and killing pharyngeal squamous cell carcinoma. Background Art

[0002] Head and neck cancer is the sixth most common malignancy worldwide, of which oropharyngeal squamous cell carcinoma (OPSCC) is a key component, and radiotherapy is a key treatment option. Human papillomavirus (HPV), a key factor in the development and progression of squamous cell carcinoma, has a high positive detection rate in OPSCC, with an increasing trend annually. However, clinical findings indicate that HPV-positive OPSCC patients have better survival and prognosis, a favorable prognosis closely associated with the tumor's higher radiosensitivity. Therefore, exploring the underlying mechanisms of radiosensitization by HPV infection and identifying key molecules involved in this process are crucial for the development of radiosensitizing drugs, focusing on HPV-positive OPSCC.

[0003] In light of this, the research team conducted metabolomics analysis of HPV-positive and -negative OPSCC cells and found that glutamine was significantly downregulated in HPV-positive tumors. Glutamine is an important nitrogen source for the de novo synthesis of purine and pyrimidine nucleotides and a substrate for glutathione synthesis. Nucleotides are crucial substrates for the repair of DNA double-strand breaks (DSBs), and glutathione is a key molecule regulating intracellular oxidative stress. DSBs and oxidative stress are the core pathways for radiation-induced tumor cell destruction. A decrease in glutamine levels can lead to a decrease in both nucleotide levels, substrates for DNA damage repair, and glutathione, a reducing substance that responds to oxidative stress. Therefore, using a cell model treated with culture medium containing varying concentrations of glutamine, the research team investigated the effects of glutamine levels on the radiosensitivity of oropharyngeal squamous cell carcinoma in vitro and its mechanism of action, providing new avenues for the development of sensitizing drugs for radiation-resistant tumors.

[0004] Currently, clinical approaches to enhance the efficacy of radiotherapy by targeting factors that influence cellular radiosensitivity include combining it with chemotherapy drugs (such as cisplatin), hyperthermia, anti-angiogenesis inhibitors (such as recombinant human endostatin and bevacizumab), and nanotechnology. However, these approaches are associated with adverse reactions such as liver and kidney damage and hematologic abnormalities. Furthermore, no studies have demonstrated how changes in glutamine levels regulate the radiosensitivity of OPSCC cells. Summary of the Invention

[0005] In view of the above problems, the present invention provides the use of glutamine in the preparation of a drug for enhancing radiotherapy and killing pharyngeal squamous cell carcinoma, such as Figure 1 This study aims to investigate the effectiveness of glutamine in regulating radiosensitivity in OPSCC and its molecular mechanism by which it potentiates radiation-induced tumor damage by inhibiting DNA damage repair and increasing redox levels. This study aims to clarify the key role of glutamine in tumor radiosensitization and provide a new molecular target for sensitizing radiation-resistant tumors.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides an application of glutamine in preparing a drug for enhancing radiotherapy and killing pharyngeal squamous cell carcinoma.

[0007] Furthermore, reducing glutamine levels can enhance the radiation sensitivity of FaDu cells.

[0008] Furthermore, reducing glutamine levels can increase the level of DNA damage in cells after irradiation.

[0009] Furthermore, reducing glutamine levels can increase the level of intracellular oxidative stress after irradiation.

[0010] Compared with the prior art, the present invention has the following beneficial effects: This invention proposes for the first time to use glutamine as a target to improve tumor radiosensitivity, analyzes the mechanism by which HPV infection increases the radiosensitivity of oropharyngeal squamous cell carcinoma, confirms the effectiveness of glutamine in regulating the radiosensitivity of OPSCC, and provides a new solution for the development of tumor sensitization therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a diagram of the molecular mechanism by which low glutamine levels increase cellular radiation sensitivity in an embodiment of the present invention; Figure 2 Radiation-induced cellular metabolic reprogramming: A comparative study of FaDu and FaDu-E6E7 cells. Using LC-MS-based metabolomics, we systematically analyzed the metabolic profiles of FaDu and FaDu-E6E7 cells after 4 Gy of X-ray irradiation. Figure 3 Systematic metabolomics analysis and glutamine metabolism verification of irradiated FaDu and FaDu-E6E7 cells. A: Data analysis based on LC-MS metabolomics screening results; B: Intracellular glutamine levels in FaDu and FaDu-E6E7 cells after 0 or 4 Gy X-ray irradiation were measured using a glutamine kit. Figure 4Lowering glutamine levels can improve the radiation sensitivity of FaDu cells. A: Glutamine kit detects intracellular glutamine levels in cells treated with different concentrations of glutamine medium; B: CCK-8 assay detects different glutamine levels and cell viability after irradiation; CD: Clonogenic assay detects cell colony formation and survival ability; Figure 5 To demonstrate that reducing glutamine levels can increase the level of DNA damage in cells after irradiation, AB: Western blot analysis of γ-H2AX expression; CD: Comet assay analysis of DSBs by measuring the tail length of each group before and after irradiation and after different glutamine treatments. Figure 6 To reduce the glutamine level, the intracellular oxidative stress level can be increased after irradiation. A: ROS kit was used to detect the intracellular oxidative stress level; BE: Western blot was used to detect the expression levels of 3NT and 4-HNE; Note: Quantitative data are expressed as X±SD. P <0.05 is defined as a significant difference. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. DETAILED DESCRIPTION

[0012] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0013] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0014] Example 1 Glutamine exhibits radiation-induced low expression in HPV-positive tissues Experimental Design: The FaDu-E6E7 cell line harboring HPV16 E6E7 was established using lipofectamine transfection. The LV5-HPV16E6 / E7 plasmid was synthesized by Higen Genetics, using the LV5 vector. The target sequence is as follows, as shown in SEQ ID NO: 1: .

[0015] Using LC-MS-based metabolomics, we systematically profiled the metabolic activity of FaDu and FaDu-E6E7 cells following 4 Gy of X-ray irradiation. Using a fold-difference of 1.5 as a screening criterion, we identified glutamine as a significantly downregulated metabolite. To validate this finding, we further measured basal glutamine levels in both cell types using a glutamine assay.

[0016] The specific experimental groups are as follows: Control group (FaDu): FaDu cells were cultured in 10 cm dishes and treated with DMEM complete medium containing 10% fetal bovine serum + 1% cell culture double antibody.

[0017] Irradiation group (FaDu-IR): When the cells treated with complete medium grew to 70-80%, a single cell irradiation was performed with a dose rate of 1.02 Gy / min and a total dose of 4 Gy.

[0018] HPV infection group (FaDu-E6E7): FaDu-E6E7 cells were cultured in 10 cm dishes and treated with DMEM complete medium containing 10% fetal bovine serum + 1% cell culture double antibody.

[0019] HPV infection combined with irradiation group (FaDu-E6E7-IR): FaDu-E6E7 cells treated with complete medium were irradiated once when they grew to 70-80% of their normal growth rate, with a dose rate of 1.02 Gy / min and a total dose of 4 Gy.

[0020] Experimental results: Metabolomics analysis showed that glutamine expression was downregulated by 0.71 times compared with the control group ( Figure 2 、 Figure 3 A). Glutamine detection kit showed that the glutamine content in FaDu-E6E7 cells was indeed significantly lower than that in FaDu cells ( Figure 3 B).

[0021] Example 2: Reducing glutamine levels can increase the radiation sensitivity of FaDu cells Experimental Design: In vitro experiments were conducted using the human OPSCC cell line FaDu, which was irradiated with 4 Gy. Six hours later, cells were harvested using a cell scraper. Glutamine levels were assessed using a glutamine assay kit to preliminarily demonstrate that exogenous addition or deprivation of culture medium glutamine alters intracellular glutamine levels. Finally, CCK-8 and colony formation assays confirmed that glutamine inhibited cell death and reduced radiation sensitivity in irradiated FaDu cells.

[0022] The specific experimental groups are as follows: Control group (FaDu): FaDu cells were cultured in 10 cm dishes and treated with DMEM complete medium (4 mM glutamine) containing 10% fetal bovine serum and 1% cell culture double antibody.

[0023] Irradiation group (FaDu-IR): When the cells treated with complete medium (4 mM glutamine) grew to 70-80%, a single cell irradiation was performed at a dose rate of 1.02 Gy / min and a total dose of 4 Gy.

[0024] Low-glutamine group (FaDu-glutamine (-)): When the cells treated with complete medium (4 mM glutamine) grew to 60-70%, they were treated with low-glutamine medium (2 mM glutamine) for 12 h.

[0025] Low glutamine + irradiation group (FaDu-glutamine (-) -IR): The cells were treated with low glutamine medium (2 mM glutamine) for 12 h, and then irradiated once with a dose rate of 1.02 Gy / min and a total dose of 4 Gy.

[0026] High-glutamine group (FaDu-glutamine (+)): When the cells treated with complete medium (4 mM glutamine) grew to 60-70%, they were treated with high-glutamine medium (8 mM glutamine) for 12 h.

[0027] High glutamine + irradiation group (FaDu-glutamine (+) -IR): The cells were treated with high glutamine medium (8 mM glutamine) for 12 h, and then irradiated once with a dose rate of 1.02 Gy / min and a total dose of 4 Gy.

[0028] Experimental results: like Figure 4 As shown, the intracellular glutamine level can be altered by adding or depriving the culture medium of glutamine ( Figure 4 A). The results of CCK-8 and clone formation assays showed that the glutamine-added group exhibited higher survival and clone formation abilities after irradiation compared to the control group, while glutamine deprivation showed the opposite results ( Figure 4 BD).

[0029] Example 3: Reducing glutamine levels can increase the level of DNA damage in cells after irradiation Experimental Design and Grouping: FaDu cells were treated with different glutamine-containing media (2, 4, and 8 mM glutamine) and then irradiated (4 Gy) or unirradiated. Six hours later, cells were harvested by scraping. Western blot analysis of γ-H2AX, a marker of DNA double-strand breaks, initially demonstrated that decreased glutamine levels increased the accumulation of DNA damage in irradiated cells, while increased glutamine levels exhibited the opposite effect. Subsequently, a neutral comet assay was performed using the same cell model to further confirm the correlation between intracellular glutamine levels and the level of DNA damage in irradiated cells.

[0030] Experimental results: like Figure 5 As shown, after irradiation, the level of DNA double-strand break marker γ-H2AX decreased in the glutamine-added group compared with the control group ( Figure 5A), while the deprivation group showed a significant increase compared with the control group after irradiation ( Figure 5 B). Neutral comet assay showed that cells treated with low-glutamine medium and co-treated with IR exhibited longer comet tails compared to cells treated with IR alone ( Figure 5 D), while adding groups does the opposite ( Figure 5 C).

[0031] Example 4: Reducing glutamine levels can increase oxidative stress levels in cells after irradiation Experimental Design and Grouping: X-rays can directly cause DNA breakage and damage, but they can also indirectly kill tumor cells by promoting the production of ROS. We have demonstrated that glutamine deprivation can increase intracellular DNA damage and enhance cellular radiation sensitivity. Next, using the same cell model as in Example 2, we examined the oxidative stress markers 3NT and 4HNE using a ROS assay and Western blot analysis to further investigate whether changes in glutamine levels can induce changes in intracellular ROS levels.

[0032] Experimental results: Intracellular oxidative stress levels Figure 6 A, Western Blot showed that the glutamine-added group could significantly reduce the levels of oxidative damage markers 3NT and 4HNE after irradiation ( Figure 6 B and C), but the glutamine deprivation group showed the opposite phenomenon ( Figure 6 D and E).

[0033] In summary, this example proposes for the first time the use of glutamine as a target to improve tumor radiosensitivity, analyzes the mechanism by which HPV infection increases the radiosensitivity of oropharyngeal squamous cell carcinoma, confirms the effectiveness of glutamine in regulating the radiosensitivity of OPSCC, and provides a new solution for the development of tumor sensitization therapeutic drugs.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of glutamine in the preparation of drugs for enhancing radiotherapy to kill pharyngeal squamous cell carcinoma.

2. The use according to claim 1, characterized in that: Reducing the glutamine level can increase the radiation sensitivity of FaDu cells.

3. The use according to claim 1, characterized in that: Reducing the glutamine level can increase the level of cellular DNA damage after irradiation.

4. The use according to claim 1, characterized in that: Reducing the glutamine level can increase the level of intracellular oxidative stress after irradiation.

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