Application of combination of low-temperature plasma and decitabine

Abstract

The invention discloses an application of combination of low-temperature plasma and decitabine in in-vitro non-therapeutic induction of tumor cell death, and an application method comprises the following steps: firstly treating tumor cells by using low-temperature plasma, and then treating by using decitabine. According to the application scheme, breast cancer cell proliferation can be remarkably inhibited under the condition of low drug dosage, the specific killing effect on tumor cells is enhanced, tumor growth is better inhibited, and a new feasible strategy is provided for developing a breast cancer treatment scheme based on combination of plasma and chemotherapy drug decitabine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an application of low-temperature plasma combined with decitabine. Background Art

[0002] Plasma is considered to be the fourth state of matter. It is composed of a large number of active groups, high-energy electrons and ions, photons, ultraviolet rays, etc. produced after the ionization of gas. It can be divided into high-temperature plasma and low-temperature plasma (also called cold atmospheric plasma). The gas temperature of high-temperature plasma is higher than 1000°C, while the gas temperature of cold atmospheric plasma can be as low as room temperature, which can be safely in contact with the human body. It has important applications in biomedical fields such as cancer treatment, antibacterial treatment, tissue engineering, and drug / gene delivery.

[0003] Since the discovery in 2007 that low-temperature plasma can cause rapid death of melanoma cells, multiple in vitro experiments have confirmed that low-temperature plasma can effectively kill more than 20 types of tumor cells, including lung cancer, breast cancer, liver cancer, colorectal cancer, skin cancer, cervical cancer, etc. Studies have found that the killing effect of plasma on tumor cells is positively correlated with the expression level of GSDME in cells, and low-temperature plasma can mediate GSDME-mediated pyroptosis. Unlike apoptosis, pyroptosis of tumor cells occurs more quickly and easily activates the immune system in the body.

[0004] High expression of DNA methyltransferase is highly correlated with poor prognosis in breast cancer patients. Abnormal methylation of tumor suppressor genes promotes tumor proliferation and migration, and DNA methylation reduces the expression of GSDME, weakening the sensitivity of patients to chemotherapy and other treatments. The demethylating drug, decitabine, is a natural adenosine analog of 2′-deoxycytidine that inhibits DNA methyltransferase and reduces DNA methylation, hindering tumor cell proliferation and reducing the occurrence of drug resistance.

[0005] The DNA demethylation drug decitabine is the most commonly used DNA methyltransferase inhibitor, which can increase the expression of methylated genes, such as tumor suppressor genes, thereby inhibiting the proliferation and migration of tumor cells. It is mostly used clinically to treat hematological malignancies. Studies have shown that decitabine has potential anti-cancer effects in the treatment of breast cancer and has the potential to enhance the sensitivity of breast cancer to anti-cancer treatments such as doxorubicin. Combining decitabine with chemotherapy drugs to treat cancer may be a promising strategy for the treatment of breast cancer. However, the response rate of decitabine is low, and only 30%-35% of patients can improve their disease.

[0006] Low-temperature plasma can affect multiple functions of cells, such as cell cycle, cell proliferation and migration ability. Low-temperature plasma can significantly inhibit the migration and invasion ability of breast cancer cell lines, and can induce cell apoptosis, thereby killing tumor cells. Studies have shown that low-temperature plasma can induce the production of a large number of hydroxyl free radicals in cells, which will enhance the oxidative reaction signal pathway in cells, thereby inducing cell apoptosis. In normal breast cells, endothelial cells and breast cancer cells, low-temperature plasma will induce the production of hydroxyl free radicals and cause cell apoptosis. It does not have the characteristics of specifically killing tumor cells and will cause greater toxic side effects on normal tissues. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an application of low-temperature plasma combined with decitabine in view of the deficiencies in the above-mentioned prior art. The present invention aims to develop a synergistic anti-tumor solution using low-temperature plasma in combination with decitabine, using decitabine to reduce DNA methylation of tumor cells, promote the expression and activation of GSDME, and thus enhance the GSDME-dependent tumor cell pyroptosis caused by low-temperature plasma. The present invention will provide a feasible new strategy for the treatment of breast cancer.

[0008] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: to provide an application of low-temperature plasma combined with decitabine, characterized in that low-temperature plasma combined with decitabine is used to non-therapeutically induce tumor cell death in vitro.

[0009] Preferably, the application method is: first treating the tumor cells with low-temperature plasma, and then treating them with decitabine.

[0010] Preferably, the application method is: firstly treating the tumor cells with low-temperature plasma, and then culturing them with a culture medium containing decitabine.

[0011] Preferably, the low temperature plasma treatment time is 1-6 min.

[0012] Preferably, the concentration of decitabine in the medium containing decitabine is 0.1-10 μM.

[0013] Preferably, the low-temperature plasma treatment time is 3 min, and the concentration of decitabine in the medium containing decitabine is 10 μM.

[0014] Preferably, the tumor cells are breast cancer cells.

[0015] Preferably, the tumor cell is breast cancer cell MCF-7.

[0016] Preferably, the application of the low-temperature plasma combined with decitabine is specifically: the application of the low-temperature plasma combined with decitabine to non-therapeutic induce the death of breast cancer cells MCF-7 in vitro, and the application method is: S1. The breast cancer cell line MCF-7 was inoculated into a 96-well cell culture plate at a concentration of 8000-10000 cells / 100 μL / well. After culturing for 6-24 hours, the cells were treated with low-temperature plasma for 1-6 minutes. S2. Replace the culture medium with one containing 0.1-10 μM decitabine and culture for 24-96 hours.

[0017] Preferably, the application of the low-temperature plasma combined with decitabine is specifically: the application of the low-temperature plasma combined with decitabine to non-therapeutic induce the death of breast cancer cells MCF-7 in vitro, and the application method is: S1. The breast cancer cell line MCF-7 was inoculated into a 96-well cell culture plate at a concentration of 8000-10000 cells / 100 μL / well. After culturing for 12 hours, it was treated with low-temperature plasma for 3 minutes. S2. The culture medium was replaced with a culture medium containing 0.1-10 μM decitabine and cultured for 48 hours.

[0018] The beneficial effects of the present invention are: The present invention discloses an application of low-temperature plasma combined with decitabine in the treatment of breast cancer. The application scheme can significantly inhibit the proliferation of breast cancer cells at a lower drug dose, enhance the specific killing effect on tumor cells, and better inhibit tumor growth. The present invention will provide a feasible new strategy for developing a breast cancer treatment scheme based on the combination of plasma and the chemotherapy drug decitabine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The inhibition of breast cancer cells by low-temperature plasma at different action times; Figure 2 This is the change of active oxygen in cells after low-temperature plasma treatment; Figure 3 The expression of pyroptosis-related genes in breast cancer cells after low-temperature plasma treatment; Figure 4 This is the result of the synergistic anti-tumor effect of low-temperature plasma and decitabine. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0021] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0022] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0023] Example 1 1. Low-temperature plasma treatment of cells The breast cancer cell line MCF-7 was cultured in a 37°C incubator with 5% CO2. When the cell confluence reached about 90%, 4×10 5 The cells were inoculated into a 60 mm cell culture dish and cultured for about 12 hours. After the cells were completely attached to the wall, fresh culture medium was replaced in each culture dish. The culture dish was placed on the stage under the plasma nozzle, and the height of the stage was adjusted so that the distance between the culture medium liquid surface and the plasma flame nozzle was 20 mm. The cells were treated with plasma for 0 min, 1 min, 2 min, 3 min, 4 min, 5 min, 7 min, and 9 min. The cell culture dish was then placed back in the incubator for 48 hours, and the growth status of the cells under different treatment conditions was photographed and recorded using a microscope ( Figure 1 A), the results showed that as the plasma treatment time increased, the number of adherent cells decreased, and the cell morphology of cell pyroptosis was clearly observed in the plasma treatment groups of 4 min and 5 min ( Figure 1 A: Black arrows point to cells).

[0024] 2. Cell viability detection after low-temperature plasma treatment The cells treated with plasma for different time periods were inoculated into 96-well cell culture plates at a concentration of 8000-10000 cells / 100 μL / well, with 3 replicates for each concentration, and cultured in a 37°C incubator for 48 hours. The cell culture medium in each well was replaced with a culture medium containing 10 μl WST-1, incubated at 37°C for 1-4 hours, and the absorbance of each well at 450 nm was measured. At the same time, blank wells (without cells, with culture medium and WST-1 solution) and control wells (with cells, no treatment, with culture medium and WST-1 solution) were set. The inhibition rate of low-temperature plasma treatment on tumor cells was calculated using the following formula: Inhibition rate = [1-(OD 实验组 -OD 空白孔 ) / (OD 对照组 -OD空白孔 )]×100% The inhibition of low-temperature plasma treatment at different times on breast cancer cell line MCF-7 is shown in Figure 1 As shown, the results showed that as the plasma treatment time increased, the number of adherent cells decreased and the inhibitory effect on breast cancer cells increased.

[0025] 3. Detection of intracellular reactive oxygen species (ROS) levels after low-temperature plasma treatment When the cell confluence reaches 60-80%, remove the cell culture medium and wash with PBS three times. Add 2 mL of serum-free medium diluted with a final concentration of 10 μM oxidation-sensitive fluorescent probe (DCFH-DA) to each 60 mm culture dish. After culturing the cells in a 37°C incubator for 30 min, remove the culture medium, wash with serum-free medium three times, and add 3 mL of fresh culture medium. After treating the cells with low-temperature plasma and continuing to culture for 1 hour, place the cells under a fluorescence microscope to observe and record the fluorescence signals in the cells of each treatment group, and analyze the changes in the level of intracellular reactive oxygen species. The results are shown in the figure. Figure 2 As shown, it can be seen that the green fluorescence signal generated in the cells of the untreated group is low, indicating that the content of reactive oxygen in the cells is low; while the green fluorescence signal increased significantly after low-temperature plasma treatment, indicating that low-temperature plasma induced cells to produce higher levels of reactive oxygen.

[0026] 4. Total RNA extraction from cells and RT-PCR detection of gene expression changes Collect cells cultured for 48 hours after plasma treatment, wash twice with PBS, add 500 μL trizol to the cells in a 6 mm culture dish, pipette several times, transfer the cell homogenate to an RNA collection column, and extract total cell RNA according to the instructions of the RNA extraction kit. Use an RNA reverse transcription kit to transcribe and generate cDNA. Design and synthesize primers for GSDM-E, GSDM-D, and CASPASE-1 genes, and PCR amplify the expression of the target genes in cDNA ( Figure 3 ). The results showed that the expression levels of pyroptosis-related genes GSDM-E, GSDM-D, and CASPASE-1 were significantly increased in breast cancer cells treated with low-temperature plasma for 3 min, suggesting that low-temperature plasma inhibits the growth of breast cancer cells by inducing cell pyroptosis.

[0027] Example 2 Decitabine and low-temperature plasma synergistically inhibit breast cancer cell growth The breast cancer cell line MCF-7 was inoculated into a 96-well cell culture plate at a concentration of 8000-10000 cells / 100 μL / well. After the cells were cultured for about 12 hours and completely attached to the wall, they were treated with low-temperature plasma for 3 minutes. The culture medium was then replaced with a culture medium containing 0 μM, 0.1 μM, 1 μM, and 10 μM decitabine. The culture medium was cultured for 48 hours, and the growth status of the cells under different treatment conditions was observed and recorded using a microscope ( Figure 4 A) Using WST-1 solution to detect the viability of cells treated with low-temperature plasma and decitabine. The results showed that decitabine had a lower killing effect on breast cancer cells at lower concentrations, but when used in combination with low-temperature plasma, it had a synergistic effect in inhibiting tumor cell growth ( Figure 4 B).

[0028] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. An application of low-temperature plasma combined with decitabine, characterized in that: Application of low-temperature plasma combined with decitabine to non-therapeutic induction of tumor cell death in vitro.

2. The use of low-temperature plasma combined with decitabine according to claim 1, characterized in that: The application method is: tumor cells are first treated with low-temperature plasma and then with decitabine.

3. The use of low-temperature plasma in combination with decitabine according to claim 2, characterized in that: The application method is: the tumor cells are first treated with low-temperature plasma, and then cultured with a culture medium containing decitabine.

4. The use of low-temperature plasma in combination with decitabine according to claim 3, characterized in that: The low temperature plasma time is 1-6 minutes.

5. The use of low-temperature plasma in combination with decitabine according to claim 4, characterized in that: The concentration of decitabine in the medium containing decitabine is 0.1-10 μM.

6. The use of low-temperature plasma in combination with decitabine according to claim 5, characterized in that: The low-temperature plasma treatment time was 3 min, and the concentration of decitabine in the medium containing decitabine was 10 μM.

7. The use of low-temperature plasma combined with decitabine according to claim 1, characterized in that: The tumor cells are breast cancer cells.

8. The use of low-temperature plasma in combination with decitabine according to claim 7, characterized in that: The tumor cells were breast cancer cells MCF-7.

9. The use of low-temperature plasma in combination with decitabine according to claim 8, characterized in that: The application of low temperature plasma combined with decitabine in inducing non-therapeutic death of breast cancer cells MCF-7 in vitro is as follows: S1. The breast cancer cell line MCF-7 was inoculated into a 96-well cell culture plate at a concentration of 8000-10000 cells / 100 μL / well. After culturing for 6-24 hours, the cells were treated with low-temperature plasma for 1-6 minutes. S2. Replace the culture medium with one containing 0.1-10 μM decitabine and culture for 24-96 hours.

10. The use of low-temperature plasma in combination with decitabine according to claim 9, characterized in that: The application of low temperature plasma combined with decitabine in inducing non-therapeutic death of breast cancer cells MCF-7 in vitro is as follows: S1. The breast cancer cell line MCF-7 was inoculated into a 96-well cell culture plate at a concentration of 8000-10000 cells / 100 μL / well. After culturing for 12 hours, it was treated with low-temperature plasma for 3 minutes. S2. The culture medium was replaced with a culture medium containing 0.1-10 μM decitabine and cultured for 48 hours.

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