Application of 2, 3-dimethoxy-1, 4-naphthoquinone in preparation of inducer for inducing tumor cell ferroptosis and medicine for treating gastric cancer

Abstract

The invention provides application of 2, 3-dimethoxy-1, 4-naphthoquinone in preparation of an inducer for inducing tumor cell ferroptosis and a medicine for treating gastric cancer, and belongs to the technical field of biological medicine. The invention finds that DMNQ has the activity of inducing ferroptosis of gastric cancer cells and can be used as an inducer for inducing ferroptosis of tumor cells; the DMNQ is found to be capable of remarkably inhibiting the growth of gastric cancer cells and can be used for preparing the medicine for treating the gastric cancer. Theoretical support can be provided for wider application of DMNQ, and a foundation is laid for developing more effective gastric cancer treatment drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of 2,3-dimethoxy-1,4-naphthoquinone in the preparation of an inducer for inducing ferroptosis of tumor cells and a drug for treating gastric cancer. Background Art

[0002] Gastric cancer is a common digestive tract malignancy and the fourth leading cause of cancer death worldwide. Since patients with gastric cancer have no obvious specific symptoms in the early stages of the disease and there is currently a lack of specific early diagnostic indicators, most patients are already in the late stages when they are initially diagnosed, accompanied by varying degrees of distant metastasis, leading to drug resistance and poor prognosis. Therefore, there is an urgent need to find new treatments.

[0003] Ferroptosis is an iron-dependent cell death process driven by excessive accumulation of lipid peroxides. It is a newly discovered non-traditional programmed cell death method that is different from apoptosis and autophagy. On the one hand, ferroptosis can eliminate tumor cells, cut off the blood supply of tumors, stimulate the immune system to attack, and improve the response of tumor cells to other treatments. On the other hand, ferroptosis can also release some pro-inflammatory factors, trigger inflammatory responses, change the living environment of tumors, and reduce the immune susceptibility of tumor cells. It can be seen that ferroptosis inducers show great potential in cancer treatment.

[0004] DMNQ (2,3-dimethoxy-1,4-naphthoquinone) is a naphthoquinone compound extracted from the traditional Chinese medicine Lithospermum officinale. The existing technology has not reported the effect of DMNQ on ferroptosis of tumor cells. Therefore, studying the effect and regulatory mechanism of DMNQ on ferroptosis of gastric cancer cells can provide a new mode of action and theoretical support for the wider clinical application of DMNQ, and provide a new solution for clinical medication and auxiliary medication. Summary of the invention

[0005] The purpose of the present invention is to provide an inducer capable of inducing cell ferroptosis and to explore new uses of DMNQ.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of 2,3-dimethoxy-1,4-naphthoquinone in preparing an inducer for inducing ferroptosis of tumor cells.

[0008] Preferably, the tumor cells are gastric cancer cells.

[0009] The present invention also provides the use of 2,3-dimethoxy-1,4-naphthoquinone in preparing a medicine for treating gastric cancer.

[0010] Preferably, the effective concentration of the 2,3-dimethoxy-1,4-naphthoquinone on gastric cancer cells is 10-15 μM.

[0011] The present invention finds that DMNQ has the activity of inducing ferroptosis of gastric cancer cells and can be used as an inducer of ferroptosis of tumor cells. The present invention also finds that DMNQ can significantly inhibit the growth of gastric cancer cells and can be used to prepare drugs for treating gastric cancer. The present invention can provide theoretical support for the wider application of DMNQ and lay the foundation for the development of more effective drugs for treating gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 IC of human and mouse gastric cancer cells after 48 h of DMNQ treatment 50 (half maximal inhibitory concentration).

[0013] Figure 2 The figure shows the cell survival ratio of different treatment groups on human and mouse gastric cancer cell lines after 48 hours.

[0014] Figure 3 Bar graph analysis of lipid peroxidation levels in human and mouse gastric cancer cells before and after DMNQ treatment.

[0015] Figure 4 Flow cytometry results and bar graph analysis of ROS levels in human and mouse gastric cancer cells before and after DMNQ treatment.

[0016] Figure 5 Changes in mitochondrial morphology in gastric cancer cells before and after DMNQ treatment.

[0017] Figure 6 To detect the expression of phosphorylated STAT3 protein (p-STAT3) and total STAT3 protein (t-STAT3) in human and mouse gastric cancer cells before and after DMNQ treatment. DETAILED DESCRIPTION

[0018] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0019] Example 1 Effect of DMNQ on the ferroptosis of human gastric cancer cells

[0020] The CCK8 assay was used to evaluate the IC of DMNQ against human and mouse gastric cancer cells (MKN-45 and MFC, purchased from Wuhan Pronocell Life Science Co., Ltd.) 50 .

[0021] (1) Select cells that are in good growth condition and in the logarithmic growth phase for plating. Digest the cells with trypsin and resuspend them for cell counting.

[0022] (2) Inoculate cells in a 96-well plate: 5000 cells per well, final volume 100 μl. Shake the 96-well plate evenly and culture in a cell culture incubator at 37° C., 5% CO 2 .

[0023] (3) On the second day, after the cells adhered, they were replaced with 100 μL of complete culture medium (purchased from Shanghai Yuanpei Biotechnology Co., Ltd.) containing different drug concentrations (DMNQ concentration gradient: 0, 0.01, 0.1, 1, 10, 100 μM, 6 replicates for each concentration) and cultured in a cell culture incubator at 37°C, 5% CO2.

[0024] (4) After 48 h of drug action, 10% CCK8 reagent was prepared with complete culture medium, 10 μL of complete culture medium containing CCK8 reagent was added to each well, and bubbles were avoided. The cells were incubated in a 37°C cell culture incubator in the dark for 1 h.

[0025] (5) After incubation, the absorbance value of the 96-well plate was measured using an ELISA reader (wavelength: 450 nm) and the data was analyzed.

[0026] Depend on Figure 1 It can be seen that the IC of MKN-45 50 The IC value of MFC is 13.21 μM. 50 It is 3.254μM.

[0027] Example 2

[0028] (1) Take gastric cancer cells in the logarithmic growth phase, digest the cells with trypsin, resuspend them and count the cells.

[0029] (2) Inoculate cells in 96-well plates: 5000 cells per well, final volume 100 μl.

[0030] (3) Complete medium containing 15 μM DMNQ and complete medium containing 15 μM DMNQ combined with 10 μM Ferrostatin-1, 10 mΜ DFO, 10 μM Necrostatin-1 and 30 μM Z-VAD-FMK were added to the well plate as control groups, namely DMNQ group, DMNQ+Ferrostatin-1 group, DMNQ+DFO group, DMNQ+Necrostatin-1 group and DMNQ+Z-VAD-FMK group, respectively. Complete medium without DMNQ was added as blank control (NC group). Six replicate wells were set up for each condition, with 100 μL per well, and the cells were cultured at 37°C in a cell culture incubator with 5% CO2.

[0031] (4) After 48 h of drug action, 10% CCK8 reagent was prepared with complete culture medium, 10 μl of complete culture medium containing CCK8 reagent was added to each well, and the cells were incubated in a 37°C cell culture incubator in the dark for 1 hour to avoid bubbles.

[0032] (5) After incubation, the absorbance value of the 96-well plate was measured using an ELISA instrument (wavelength: 450 nm) to calculate the cell survival rate of each group. The results are as follows: Figure 2 shown.

[0033] like Figure 2 The survival rate of cells in the DMNQ group was significantly lower than that in the blank control group, while the survival rate of cells in the DMNQ and ferroptosis inhibitor (Ferrostatin-1 and DFO) groups was significantly higher than that in the DMNQ group. Necrostatin-1 and Z-VAD-FMK combined with DMNQ treatment did not affect the survival rate of cells induced by DMNQ. This result shows that ferroptosis inhibitors (Ferrostatin-1 and DFO) can prevent cell death caused by DMNQ, indicating that the type of tumor cell death caused by DMNQ is ferroptosis.

[0034] Ferrostatin-1 and DFO are specific ferroptosis inhibitors, Necrostatin-1 is a necrosis inhibitor, and Z-VAD-FMK is an apoptosis inhibitor. If DMNQ alone can cause gastric cancer cell death, and if the addition of ferroptosis inhibitors can prevent or reduce cell death, but the addition of necrosis inhibitors or apoptosis inhibitors cannot prevent DMNQ-induced cell death, it means that the type of tumor cell death caused by DMNQ is ferroptosis, not necrosis or apoptosis.

[0035] Example 3 Effects of DMNQ treatment on MDA and ROS levels and mitochondrial morphology in gastric cancer cells

[0036] After human and mouse gastric cancer cells were treated with 15 μM DMNQ for 48 h, the changes in ferroptosis-related product MDA were detected using an MDA detection kit (purchased from Wuhan Elerite Biotechnology Co., Ltd.), and the changes in ROS were detected using a flow cytometer using a ROS detection kit (purchased from Beyotime Biotechnology). The morphology of mitochondria was observed using an electron microscope and photographed. Human and mouse gastric cancer cells cultured in complete medium without DMNQ were used as blank controls (NC).

[0037] like Figure 3 As shown, DMNQ can significantly increase the level of MDA, a metabolite of ferroptosis.

[0038] like Figure 4 As shown in the results, DMNQ treatment could significantly increase the level of ROS in gastric cancer cells.

[0039] like Figure 5 As shown, DMNQ can significantly cause mitochondrial atrophy and mitochondrial membrane thickening in human gastric cancer cells (MKN-45).

[0040] Example 4 Changes in t-STAT3 and p-STAT3 protein expression after DMNQ treatment

[0041] Human and mouse gastric cancer cells were treated with 15 μM DMNQ for 48 hours, and the cells were collected and the total protein was extracted. The changes in the expression of t-STAT3 protein and p-STAT3 protein were detected by WB. The control group (NC) was human and mouse gastric cancer cells cultured in complete medium without DMNQ.

[0042] like Figure 6 As shown, WB detection found that DMNQ did not affect the expression of t-STAT3 protein, but the expression of p-STAT3 protein in the DMNQ group was significantly lower than that in the control group, indicating that DMNQ can inhibit the expression of p-STAT3 protein.

[0043] It can be seen from the above examples that DMNQ has the activity of inducing ferroptosis of gastric cancer cells and can significantly inhibit the growth of gastric cancer cells.

[0044] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of 2,3-dimethoxy-1,4-naphthoquinone in the preparation of inducers for inducing ferroptosis of tumor cells.

2. The use according to claim 1, characterized in that: The tumor cells are gastric cancer cells.

3. Application of 2,3-dimethoxy-1,4-naphthoquinone in the preparation of drugs for treating gastric cancer.

4. The use according to any one of claims 1 to 3, characterized in that: The effective concentration of the 2,3-dimethoxy-1,4-naphthoquinone on gastric cancer cells is 10-15 μM.

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