Application of copper death marker in preparation of product for detecting curative effect of endometrial cancer drug

Abstract

The invention provides application of a copper death marker in preparation of a product for detecting the curative effect of an endometrial cancer drug, relates to the technical field of biological diagnosis targets, and aims at providing an innovative detection method and technology aiming at the copper death marker based on a copper death approach and evaluating the treatment effect of related drugs. By deeply researching the molecular mechanism of copper death, accurate evaluation on the curative effect of the endometrial cancer related drugs is realized, the defects in the field in the prior art are overcome, and reliable reference basis is provided for clinicians. The invention provides an efficient, accurate and economic endometrial cancer curative effect detection method by detecting the change of copper death mediated mitochondrial tricarboxylic acid cycle related indexes in a sample. The method has important research significance and clinical application value, and a new tool is provided for precise treatment of endometrial cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of biological diagnostic targets, and in particular to the application of copper death markers in the preparation of products for detecting the efficacy of endometrial cancer drugs. Background Art

[0002] Endometrial cancer is a common gynecological malignancy in women, and its incidence is increasing year by year. Although surgery, radiotherapy, and chemotherapy are the main treatment options, the effectiveness of treatment varies from patient to patient. Currently, the methods for evaluating the treatment efficacy of endometrial cancer mainly include imaging examinations and pathological evaluations, but these methods have certain limitations, such as the inability to monitor treatment effects in real time and the inability to accurately assess tiny residual lesions. In addition, with the continuous deepening of medical research, some new treatment concepts and methods are constantly emerging, but the treatment of endometrial cancer still requires further research and exploration. Summary of the Invention

[0003] The purpose of the present invention is to address the lack of existing technology for validating the therapeutic effects of endometrial cancer. This application aims to provide an innovative detection method and technology for copper death markers based on the copper death pathway to evaluate the therapeutic effects of related drugs. By deeply studying the molecular mechanism of copper death, we can achieve accurate evaluation of the efficacy of drugs related to endometrial cancer, address the shortcomings of existing technology in this field, and provide a reliable reference for clinicians.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: Application of copper death marker as a detection target in the preparation of products for detecting the efficacy of endometrial cancer.

[0005] Preferably, the copper death marker is at least one of PA, CA, PDH and α-KGDH.

[0006] Preferably, by detecting cell-related copper death monitoring indicators and evaluating the occurrence of copper death in cells, the effectiveness of drugs in treating endometrial cancer is evaluated, and based on the evaluation results, the treatment plan is adjusted and optimized to achieve more precise treatment.

[0007] Preferably, the product is a detection kit.

[0008] Preferably, the detection kit detects copper death markers through CCK-8 detection, Tunel staining and related kit detection technology.

[0009] Compared with the prior art, this application has the following beneficial effects: 1. Real-time monitoring: The present invention can monitor the treatment effect in real time and adjust the treatment plan in time.

[0010] 2. High Accuracy: Provides accurate efficacy assessment by detecting the expression levels of copper metabolism-related targets.

[0011] 3. Economical: Compared with traditional imaging and pathological evaluation, this invention has higher economical and operability.

[0012] 4. Clinical application value: Provide clinicians with a reliable efficacy evaluation tool to increase treatment success rate and improve patient prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Figure 1 shows the effect of copper overload on endometrial cancer cell function in accordance with an embodiment of this example. Figures AB show the Cu content assay in Ishikawa and KLE cells; Figures CD show the CCK8 cell proliferation assay in Ishikawa and KLE cells; and Figure E shows TUNE staining in Ishikawa and KLE cells.

[0014] Figure 2 Figure 1 shows the effect of copper overload on the cellular tricarboxylic acid cycle in accordance with an embodiment of this example. Figure A shows PA content in Ishikawa and KLE cells; Figure B shows CA content in Ishikawa and KLE cells; Figure C shows PDH activity in Ishikawa and KLE cells; and Figure D shows α-KGDH activity in Ishikawa and KLE cells (Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, experiments repeated three times, with three replicate wells per concentration group). DETAILED DESCRIPTION

[0015] The present invention is further described in detail below with reference to specific embodiments.

[0016] This application provides the use of copper death markers in the preparation of products for detecting the efficacy of endometrial cancer drugs. The copper death markers are at least one of PA (pyruvate), CA (citrate), PDH (pyruvate dehydrogenase), and α-KGDH (α-ketoglutarate dehydrogenase). By detecting cell-related copper death monitoring indicators and assessing the occurrence of copper death in cells, the effectiveness of drugs for treating endometrial cancer can be evaluated. Based on the evaluation results, the treatment plan can be adjusted and optimized to achieve more precise treatment.

[0017] Among them, the copper death described above is a form of programmed cell death, and its occurrence is closely related to the metabolism and homeostasis of copper ions in cells. During the copper death process, excessive concentrations of copper ions in cells trigger a series of biochemical reactions, disrupting the normal function of cells and ultimately leading to cell death. The copper death pathway involves multiple key steps. First, after excessive copper ions enter the cell, they interact with various proteins and molecules in the cell. These interactions interfere with normal metabolic processes and signal transduction in the cell. For example, copper ions interact with proteins in mitochondria, affecting the mitochondrial oxidative phosphorylation function and leading to cellular energy metabolism disorders. At the same time, copper ions also induce excessive production of reactive oxygen species (ROS). The increase in ROS further exacerbates the oxidative stress response in cells, causing a series of chain reactions such as lipid peroxidation, protein oxidation, and DNA damage, ultimately leading to the destruction of cell structure and function.

[0018] In one embodiment, the detection product is a detection kit, and the detection product detects copper death markers through CCK-8 detection, Tunel staining and related kit detection technology.

[0019] The above contents are described below with reference to specific embodiments: Example 1: Verification of the effect of copper chloride on cell function (1) Cell culture Endometrial cancer cell lines Ishikawa and KLE were cultured in DMEM / F12+10% FBS+1% P / S medium as complete culture medium at 37°C in a 5% CO2 cell culture incubator.

[0020] (2) Copper chloride induction Endometrial cancer cells Ishikawa and KLE in the logarithmic growth phase were inoculated into six-well plates. After the cells adhered to the wall, copper chloride solutions of different concentrations, 50 μM, 100 μM, and 200 μM, were added. An equal amount of culture medium without copper chloride was added to the control group. The cells were incubated in a cell culture incubator for 48 hours, and the samples were collected to detect the intracellular copper content. The detection method was carried out according to the instructions, mainly including cell lysis and sample addition. Finally, the OD value of the samples was detected by a microplate reader, and the results were statistically analyzed.

[0021] (3) CCK8 detection of cell proliferation ability Ishikawa and KLE endometrial cancer cells in the logarithmic growth phase were seeded into 96-well plates. 100 μL of complete culture medium was added to each well and the cells were cultured in a 37°C, 5% CO2 incubator. After the cells adhered, copper chloride solutions of varying concentrations, 50 μM, 100 μM, and 200 μM, were added. For the control group, an equal volume of culture medium without copper chloride was added. 10 μL of CCK-8 solution was added to each well, and the plate was gently shaken to ensure uniform mixing. After incubation for 2 hours, the absorbance (OD) of each well was measured at a wavelength of 450 nm using a microplate reader. The cells were then cultured in the cell incubator, and cell growth curves were plotted based on the OD values ​​at different time points.

[0022] (4) Tunel detection of cell apoptosis Ishikawa and KLE endometrial cancer cells in logarithmically growing phase were seeded into 24-well plates and cultured in a 37°C, 5% CO2 incubator. After 24 hours of cell growth, different concentrations of copper chloride solution (50 μM, 100 μM, and 200 μM) were added. A control group received an equal volume of culture medium without copper chloride. The cells were incubated in the cell culture incubator for another 24 hours. The cells were fixed with 4% PFA for half an hour, washed with PBS, and treated with 0.3% Triton X-100 for 5-10 minutes. The plates were washed twice with PBS, and TUNEL assay solution was added. The plates were incubated at 37°C in the dark for 60 minutes. Care was taken to minimize evaporation of the TUNEL assay solution. The plates were washed three times with PBS, and DNA staining, mounting, and observation were performed under a fluorescence microscope.

[0023] To investigate the role of copper overload in Ishikawa and KLE cells, copper overload was induced by adding different concentrations of copper chloride, and the intracellular copper content was detected using a cell copper kit, which showed a concentration-dependent increase ( Figure 1 AB). CCK8 results showed that copper overload inhibited cell proliferation in a concentration-dependent manner ( Figure 1 CD). Tunel experiments showed that copper overload promoted cell apoptosis in a concentration-dependent manner ( Figure 1 E). These results suggest that copper overload can affect cell proliferation and apoptosis.

[0024] Example 2: Cell-related copper death monitoring indicators and verification (1) Cell culture Endometrial cancer cell lines Ishikawa and KLE cells were cultured according to the aforementioned method.

[0025] (2) PA, CA, PDH, and α-KGDH detection Endometrial cancer cells Ishikawa and KLE in the logarithmic growth phase were seeded into six-well plates. After the cells adhered to the wall, copper chloride solutions of different concentrations (50 μM, 100 μM, and 200 μM) were added. The control group was treated with an equal amount of culture medium without copper chloride. The cells were incubated in a cell culture incubator for 48 hours and the samples were collected to detect the contents of mitochondrial tricarboxylic acid cycle-related indicators PA, CA, PDH, and α-KGDH. The detection method was carried out according to the instructions. Finally, the OD value of the samples was detected using a microplate reader. Statistical analysis results showed that treatment with copper chloride at different concentrations significantly increased PA, CA, PDH, and α-KGDH in endometrial cancer cells, and the levels were concentration-dependent. ( Figure 2 AD).

[0026] In summary, this application screens markers related to the treatment response of endometrial cancer based on the copper death pathway-related pathways and reference to copper metabolism-related targets. The expression levels of these markers change significantly in samples after treatment with copper death pathway-dependent drugs, which can be used to determine whether the treatment is successful. In addition, for detection methods, relevant kits, Tunel staining and other related technologies can be used to detect the expression levels of relevant targets in patient samples.

[0027] The detection samples targeted in this application are tumor tissues or other related biological samples, and the treatment effect is evaluated by comparing the expression levels of markers before and after treatment. A significant decrease in the expression level of the marker indicates that the treatment is effective, otherwise it indicates that the treatment effect is poor.

[0028] This study provides an efficient, accurate, and economical method for evaluating the efficacy of endometrial cancer treatment by detecting changes in copper-mediated mitochondrial tricarboxylic acid cycle markers PA, CA, PDH, and α-KGDH in samples. This method has significant research significance and clinical application value, providing a new tool for the precise treatment of endometrial cancer.

Claims

1. Application of copper death marker as a detection target in the preparation of products for detecting the efficacy of endometrial cancer.

2. The use of the copper death marker according to claim 1 as a detection target in the preparation of a product for detecting the efficacy of endometrial cancer drugs, characterized in that: The copper death marker is at least one of PA, CA, PDH and α-KGDH.

3. The use of the copper death marker according to claim 2 as a detection target in the preparation of a product for detecting the efficacy of endometrial cancer drugs, characterized in that: By detecting cell-related copper death monitoring indicators and evaluating the occurrence of copper death in cells, the effectiveness of drugs in treating endometrial cancer can be evaluated. Based on the evaluation results, the treatment plan can be adjusted and optimized to achieve more precise treatment.

4. The use of the copper death marker according to claim 3 as a detection target in the preparation of a product for detecting the efficacy of endometrial cancer drugs, characterized in that: The product is a detection kit.

5. The use of the copper death marker according to claim 4 as a detection target in the preparation of a product for detecting the efficacy of endometrial cancer drugs, characterized in that: The detection kit detects copper death markers through CCK-8 detection, Tunel staining and related kit detection technology.

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