Application of ferroptosis and copper overload superimposed effect in endometrial cancer mitochondrial injury kit detection

Abstract

The invention discloses application of ferroptosis and copper overload superimposed effect in endometrial cancer mitochondrial injury kit detection, and the kit verifies the detection effect of ferroptosis and copper overload dual effect in endometrial cancer proliferation inhibition by detecting the Iron level of an endometrial cancer treatment group and LPO, ROS and GSH content indexes. The ferroptosis and copper overload superimposed effect plays an important role in preparing the endometrial cancer cell mitochondrial injury effect detection kit, provides a theoretical basis for clinical diagnosis and treatment of endometrial cancer, and has important clinical significance and research value.

Description

Technical Field

[0001] The present invention relates to the technical field of obstetrics and gynecology, and in particular to an application of the superimposed effect of ferroptosis and copper overload in a kit for detecting mitochondrial damage in endometrial cancer. Background Art

[0002] Endometrial cancer is a malignant tumor of the uterine epithelial lining. Worldwide, it has become the seventh most common malignancy, with increasing incidence and associated mortality. Endometrial cancer invades uterine fibroids, destroying the uterine structure and severely impacting fertility, leading to fertility difficulties for patients.

[0003] The causes of endometrial cancer are complex and result from the interaction of multiple factors, including estrogen metabolic imbalance, lifestyle factors, genetic factors, and environmental factors. When the body is under stress or encounters various harmful stimuli, the oxidative and antioxidant systems in the body become unbalanced, resulting in an oxidative stress response, which ultimately mediates multiple biological events such as cell apoptosis, ischemia-reperfusion injury, and inflammatory response. Ferroptosis is a new form of cell death that depends on iron ions and has been shown to play an important role in various diseases. The ferroptosis process mainly involves different pathways that directly or indirectly affect glutathione peroxidase, thereby leading to a decrease in the cell's antioxidant capacity and damage to the cell membrane through lipid peroxidation and accumulation of reactive oxygen species. Compared with normal non-cancerous cells, endometrial cancer cells show a higher iron requirement. On the other hand, copper overload triggers cellular oxidative stress through the copper death pathway.

[0004] Studies have demonstrated that targeting ferroptosis to eliminate cancer, a unique form of iron-dependent cell death, is regulated by multiple cellular metabolic pathways, including redox homeostasis, mitochondrial activity, and lipid metabolism. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of the superimposed effect of ferroptosis and copper overload in the detection of mitochondrial damage in endometrial cancer. Compared with the independent ferroptosis that inhibits the development of endometrial cancer, copper overload induced by copper chloride treatment can significantly accelerate ferroptosis to promote mitochondrial damage in endometrial cancer cells, providing a new diagnostic and treatment idea and theoretical basis for the treatment of endometrial cancer. To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The above technical objectives of the present invention are achieved through the following technical solutions:

[0007] Application of the additive effect of ferroptosis and copper overload in the detection of mitochondrial damage kit in endometrial cancer.

[0008] Preferably, the kit utilizes mitochondrial damage markers to verify that ferroptosis and copper overload promote mitochondrial damage in endometrial cancer cells.

[0009] Preferably, the mitochondrial damage markers include Iron level, LPO content, ROS content and GSH content.

[0010] Preferably, the superposition effect of ferroptosis and copper overload accelerates mitochondrial damage in endometrial cancer, significantly reduces the GSH content in endometrial cancer cells, and disrupts cellular redox homeostasis.

[0011] In summary, the present invention has the following beneficial effects: the present invention accelerates mitochondrial damage in endometrial cancer cells through the superimposed effect of ferroptosis and copper overload, and plays a significant role in the treatment and prevention of endometrial cancer: by detecting the LPO, GSH and ROS content and Iron level in the test samples, the increase of ferroptosis markers under copper overload can be effectively judged, and the superimposed effect of copper overload and ferroptosis effectively reduces the antioxidant level in endometrial cancer cells and destroys the oxidative stress homeostasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Figure 3 shows the detection of mitochondrial damage-related indicators in Ishikawa cells and KLE cells under copper overload treatment. Figure A shows the analysis results of iron content in Ishikawa cells and KLE cells under copper overload conditions; Figure B shows the analysis results of LPO content in Ishikawa cells and KLE cells under copper overload conditions; Figure C shows the analysis results of ROS content in Ishikawa cells and KLE cells under copper overload conditions; Figure D shows the analysis results of GSH content in Ishikawa cells and KLE cells under copper overload conditions.

[0013] Figure 2 The superimposed effect of ferroptosis activator Erastin and copper overload accelerated the mitochondrial damage effect; Figure A shows the iron content of the CuCl2 and Erastin treatment group (Ishokawa cells); Figure B shows the iron content of the CuCl2 and Erastin treatment group (KLE cells); Figure C shows the LPO content of the CuCl2 and Erastin treatment group (Ishokawa cells); Figure D shows the LPO content of the CuCl2 and Erastin treatment group (KLE cells); Figure E shows the ROS content of the CuCl2 and Erastin treatment group (Ishokawa cells); Figure F shows the LPO content of the CuCl2 and Erastin treatment group (KLE cells); Figure G shows the GSH content of the CuCl2 and Erastin treatment group (Ishokawa cells); Figure H shows the GSH content of the CuCl2 and Erastin treatment group (KLE cells). DETAILED DESCRIPTION

[0014] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be interpreted as limiting the present invention.

[0015] Example 1 Expression levels of ferroptosis markers in Ishikawa cells and KLE cells under copper overload conditions

[0016] Ishikawa and KLE cells were cultured in the control group (no CuCl2 treatment) and the experimental group (CuCl2 treatment). Ferroptosis-related markers such as LPO, GSH, and ROS levels, as well as iron content, were measured using a kit. The effects of copper overload on ferroptosis markers in endometrial cancer cells were observed.

[0017] (1) Cell culture

[0018] Endometrial cancer cells Ishikawa cells and KLE cells were cultured in RPMI-1640+10% FBS+1% P / S culture medium as complete culture medium at 37°C in a 5% CO2 cell culture incubator.

[0019] (2) LPO / GSH / ROS / Iron index detection

[0020] Cells in logarithmic growth phase were collected and 1×10 6 Cells were seeded into 6-well plates and cultured in a 37°C, 5% CO2 incubator. After 24 hours, CuCl2 was added, and the cells were cultured for another 48 hours before being harvested. Cells were lysed by sonication, and the supernatant was centrifuged and added to a 96-well plate. LPO / GSH / ROS / Iron detection reagents were added in that order. The absorbance of LPO, GSH, ROS, and Iron was measured using a microplate reader. The levels of LPO, GSH, ROS, and Iron were calculated using a standard curve.

[0021] The test results showed that the contents of LPO, GSH, ROS, and Iron showed a significant dependence on CuCl2 concentration. At the same time, compared with the control group, the GSH content in the treatment group was significantly decreased, and the intracellular oxidative stress homeostasis was unbalanced; the LPO, ROS, and Iron contents were significantly increased. These results indicate that copper overload induced mitochondrial damage in endometrial cancer cells. Figure 1 shown.

[0022] Example 2: The combined effects of ferroptosis and copper overload accelerate mitochondrial damage in endometrial cancer cells

[0023] Ishikawa and KLE cells were treated with either the control group (no treatment) or the experimental group (treatment with the ferroptosis activator Erastin and CuCl2). Ferroptosis-related markers such as LPO, GSH, ROS levels, and iron content were measured using a kit. The effects of the combined effects of ferroptosis and copper overload on ferroptosis markers in endometrial cancer cells were observed.

[0024] (1) Cell culture

[0025] Endometrial cancer cells Ishikawa cells and KLE cells were cultured in RPMI-1640+10% FBS+1% P / S culture medium as complete culture medium at 37°C in a 5% CO2 cell culture incubator.

[0026] (2) LPO / GSH / ROS / Iron index detection

[0027] Cells in logarithmic growth phase were collected and 1×10 6 Cells were seeded in 6-well plates and cultured in a 37°C, 5% CO2 incubator. After 24 hours, the ferroptosis activator erastin and CuCl2 were added. Culture was continued for 48 hours, and the cells were harvested. Cells were lysed by sonication, and the supernatant was collected by centrifugation and added to a 96-well plate. LPO / GSH / ROS / Iron detection reagents were added in that order. The absorbance of LPO, GSH, ROS, and Iron was measured using a microplate reader. The levels of LPO, GSH, ROS, and Iron were calculated using a standard curve.

[0028] To further verify the extent of the combined effect of the ferroptosis activator Erastin and copper overload on mitochondrial damage in endometrial cancer cells, we conducted a superposition treatment of the two drugs. The results showed that the superposition effect of the two drugs can significantly accelerate the mitochondrial damage process of endometrial cancer cells. Figure 2 shown.

[0029] The present invention accelerates mitochondrial damage in endometrial cancer cells through the superimposed effect of ferroptosis and copper overload, and plays a significant role in the treatment and prevention of endometrial cancer: by detecting the LPO, GSH and ROS content and Iron level in the test samples, the increase of ferroptosis markers under copper overload can be effectively judged. The superimposed effect of copper overload and ferroptosis effectively reduces the antioxidant level in endometrial cancer cells and destroys the oxidative stress homeostasis.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent substitutions should also be deemed to fall within the protection scope of the technical solution of the present invention.

Claims

1. Application of the superimposed effect of ferroptosis and copper overload in the detection of mitochondrial damage kit in endometrial cancer.

2. The use of the combined effect of ferroptosis and copper overload in the detection of mitochondrial damage in endometrial cancer according to claim 1, characterized in that: The kit uses mitochondrial damage markers to verify that ferroptosis and copper overload promote mitochondrial damage in endometrial cancer cells.

3. The use of the combined effect of ferroptosis and copper overload in the detection of mitochondrial damage in endometrial cancer according to claim 2, characterized in that: The mitochondrial damage markers include Iron level, LPO content, ROS content and GSH content.

4. The use of the combined effect of ferroptosis and copper overload in the detection of mitochondrial damage in endometrial cancer according to claim 1, characterized in that: The combined effect of ferroptosis and copper overload accelerates mitochondrial damage in endometrial cancer, significantly reduces the GSH content in endometrial cancer cells, and disrupts cellular redox homeostasis.

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