Application of selenium-containing preparation in medicine for antagonizing NK cell injury caused by chemotherapy and medicine

Abstract

The invention provides an application of a selenium-containing preparation in a drug for antagonizing NK cell injury caused by chemotherapy and a drug for antagonizing cisplatin or adriamycin induced NK cell injury. At present, common cancer treatment methods include operative treatment, chemotherapy, radiotherapy and immunotherapy, and chemotherapy is still a main treatment means for most advanced malignant tumors. Meanwhile, chemotherapy has an inhibition effect on an immune system, which is mainly manifested in that the immune system of a patient is damaged and T cells and NK cells are reduced due to direct killing of chemotherapy drugs on immune cells, so that the normal physiological function of the immune system is influenced, tumor cells can be induced to generate immune tolerance, and the metastasis of the tumor cells is accelerated. The selenium-containing preparation is applied to antagonism of induced damage of cis-platinum or adriamycin to NK cells, damage of cis-platinum or adriamycin to NK cells can be antagonized, and effectiveness of chemotherapy and immunotherapy is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of synthetic drug chemistry, and particularly relates to an application of a selenium-containing preparation in a drug for antagonizing cisplatin or doxorubicin-induced NK cell damage, and a drug for antagonizing cisplatin or doxorubicin-induced NK cell damage. Background Art

[0002] It is reported that one sixth of deaths worldwide are caused by cancer. Currently, common methods for treating cancer include surgery, chemotherapy, radiotherapy and immunotherapy. Chemotherapy is still the main treatment for most advanced malignant tumors. Chemotherapy exerts its cytotoxic mechanism by interfering with the DNA transcription process and various proteins that affect DNA synthesis and replication. Most chemotherapy drugs lack tumor-specific targeting ability, causing serious side effects on normal cells and tissues, killing white blood cells and reducing immune function, leading to a state of systemic immunosuppression. The inhibitory effect of chemotherapy on the immune system is mainly manifested in the direct killing of immune cells by chemotherapy drugs, which damages the patient's immune system, reduces T cells and NK cells, and then affects the normal physiological function of the immune system. Secondly, it can also induce tumor cells to produce immune tolerance and accelerate the metastasis of tumor cells. Some literature reports point out that chemotherapy may overcome some obstacles that hinder effective anti-tumor immune response by stimulating the production of cytokines, chemokines and other damage signals. Therefore, improving systemic immunosuppression after chemotherapy is a necessary condition for improving anti-tumor immunity after chemotherapy.

[0003] Natural killer (NK) cells are important players in the innate immune system and the first line of defense against pathogens and cancer cells. They play an indispensable role in immunotherapy. NK cells kill target cells by secreting lytic granules containing pore-forming perforins and apoptosis-inducing granzymes. NK cells can spontaneously kill syngeneic and allogeneic tumor cells without prior sensitization or affinity maturation, which provides great potential for clinical trials of NK cell-based immunotherapy. However, chemotherapeutic drugs may induce NK cell dysfunction and exhaustion while killing tumor cells due to their lack of targeting. Therefore, there is a need for a drug that can effectively reverse the immunosuppressive effects of chemotherapeutic drugs on NK cells. Summary of the invention

[0004] The present invention aims to solve the deficiencies in the prior art and creatively provide an application of a selenium-containing preparation, especially in antagonizing NK cell damage induced by chemotherapeutic drugs (such as cisplatin and doxorubicin), thereby effectively protecting NK cell function and improving the safety and effectiveness of the chemotherapy process.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The invention discloses a medicine for antagonizing cisplatin or doxorubicin-induced NK cell damage, comprising a selenium-containing preparation.

[0007] Specifically, cisplatin or doxorubicin is used as an anticancer drug to induce damage to NK cells.

[0008] Specifically, the anticancer drug is a drug for treating breast cancer.

[0009] Specifically, the drug is a pretreatment drug.

[0010] Specifically, the concentration of the selenium-containing preparation in the drug is 0.1uM-2uM.

[0011] Specifically, the selenium-containing preparation comprises one or a combination of several of SeNPs, CS·SeNPs, SeMet, Me·SeCys, SeCyS2, EbSelen, Na2SeO3, and Na2SeO4.

[0012] Use of a selenium-containing preparation in any of the above-mentioned medicines.

[0013] The advantages of this application compared to the prior art are:

[0014] While killing tumor cells, chemotherapy drugs also damage NK cells. The present invention provides a selenium preparation for use in drugs that antagonize cisplatin or doxorubicin damage to NK cells, enhance the immune killing effect of NK cells inhibited by cisplatin or doxorubicin, inhibit cisplatin from damaging the mitochondrial function of NK cells, increase the glucose uptake of NK cells, and improve the effectiveness of chemotherapy immunotherapy. After NK cells are pretreated with selenium preparations, the growth inhibitory effect of cisplatin on them is significantly reduced. Compared with NK cells that have not been pretreated, their immune killing effect is significantly enhanced, mitochondrial function damage is reduced, and glucose uptake is significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is an evaluation of the effects of cisplatin and doxorubicin on the cell survival rates of MCF-7 cells and NK92 cells, as well as the effect of cisplatin on the immunocytotoxic effect of NK92 cells.

[0016] in( Figure 1 -A) is the evaluation of the survival rate of breast cancer MCF-7 cells by cisplatin; ( Figure 1 -B) is the evaluation of the survival rate of breast cancer MCF-7 cells by doxorubicin; ( Figure 1 -C) is the evaluation of cisplatin on the survival rate of NK92 cells; ( Figure 1 -D) is the evaluation of the survival rate of NK92 cells by doxorubicin; ( Figure 1 -E, F) Cisplatin treatment inhibited the killing effect of NK92 cells on MCF-7 cells at different effector-target ratios; ( Figure 1 -G, H) are the expressions of cytokines TNF-α and IFN-γ in NK92 cells after NK92 cells were treated with different concentrations of cisplatin and then co-incubated with tumor MCF-7 cells.

[0017] Figure 2 The activity of different forms of selenium drugs in reversing cisplatin- or doxorubicin-induced growth inhibition of NK92 cells was evaluated.

[0018] in( Figure 2 -A, B) are the cell survival rates of NK92 cells after being pretreated with different selenium drugs and then co-treated with cisplatin for 24h or 48h; ( Figure 2 -C, D) are the cell survival rates of NK92 cells after being pretreated with different selenium drugs for 24 hours and then co-treated with doxorubicin for 24 hours or 48 hours.

[0019] Figure 3 The purpose is to evaluate the effectiveness of different forms of selenium drugs in reversing the inhibitory effect of cisplatin-induced NK92 cell immune killing.

[0020] in( Figure 3 -A, B) Pretreatment with different forms of selenium drugs can effectively antagonize the immunolytic effect of cisplatin on NK92 cells against MCF-7 cells; ( Figure 3 -C, D) NK92 cells pretreated with different selenium drugs and then co-treated with cisplatin for 24 hours before co-incubation with tumor cells. The expression of IFN-γ and TNF-α in NK92 cells; Selenium drugs alone ( Figure 3 -E), cisplatin alone ( Figure 3 -F), selenium drugs combined with cisplatin ( Figure 3 -G) Effect on the survival rate of MCF-7 cells.

[0021] Figure 4 The effect of pretreatment with different selenium drugs on cisplatin-induced mitochondrial membrane potential dissipation in NK cells.

[0022] Figure 5 The effect of different selenium drug pretreatment on cisplatin-induced NK cell mitochondrial function damage.

[0023] in( Figure 5 -A, B) are the effects of pretreatment with different selenium drugs on cisplatin-induced mitochondrial superoxide production in NK cells; ( Figure 5 -C, D) are the effects of pretreatment with different selenium drugs on the reduction of mitochondrial mass of NK cells induced by cisplatin. ( Figure 5 -E, F) are the fluorescence photos and fluorescence quantification of the reduction of mitochondrial mass of NK cells induced by cisplatin after pretreatment with different selenium drugs. ( Figure 5-G) is the effect of pretreatment with different selenium drugs on the total ROS production of NK cells induced by cisplatin.

[0024] Figure 6 The effect of pretreatment with different selenium drugs on cisplatin-induced inhibition of NK cell glucose uptake. DETAILED DESCRIPTION

[0025] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0026] Experiment 1 Evaluation of cell survival rate of MCF-7 cells and NK92 cells with different concentrations of cisplatin and doxorubicin.

[0027] 1. Cultivation of MCF-7 cells and NK-92 cells: MCF-7 cells and NK-92 cells were purchased from the American Type Culture Collection (ATCC). MCF-7 cells were cultured with complete medium (DMEM, Gibco), and NK-92 cells were cultured with minimal essential medium (MEM, Gibco). 10% (v / v) fetal bovine serum (FBS, Gibco) and 1% (v / v) double antibody (penicillin-streptomycin mixture, Gibco) were added to the culture medium, and the cells were cultured at 37°C in a humidified incubator with 5% (v / v) carbon dioxide.

[0028] 2. First, the killing effects of chemotherapy drugs cisplatin and doxorubicin on breast cancer MCF-7 cells and immune cells NK92 cells were tested. The cell concentration was 3×10 4 MCF-7 cells were added to 96-well culture plates, and 100 μL of complete medium was added to each well. After culturing for 24 h, different concentrations of cisplatin or doxorubicin were added. After culturing for 24 h or 48 h at 37 °C and 5% CO2, 20 μL of MTT (5 mg / mL) was added to each well. After 4 h, the supernatant was discarded, and 100 μL of DMSO was added to each well. The plates were shaken for about 10 min, and the OD was measured by a microplate reader. 570 The result is as follows Figure 1 -A. Figure 1 -B shows that cisplatin and doxorubicin showed a concentration gradient effect on the toxicity of breast cancer MCF-7 cells. When cisplatin was treated for 24 hours, the cell survival rates were 93%, 82% and 79% at the concentrations of 2μM, 4μM and 8μM, respectively; when doxorubicin was treated for 24 hours, the cell survival rates were 78%, 67% and 49% at the concentrations of 0.5μM, 1μM and 2μM, respectively.

[0029] 3. Next, test the killing effect of chemotherapy drugs cisplatin and doxorubicin on immune cell NK92 cells. 4 NK92 cells / well were added to a 96-well culture plate, and 100 μL of complete medium was added to each well. After culturing for 24 h, different concentrations of cisplatin or doxorubicin were given. After culturing for 24 h or 48 h at 37°C and 5% CO2, 20 μL / well of CCK-8 (5 mg / mL) was added. After 4 h, OD was measured by microplate reader. 570 The result is as follows Figure 1 -C. Figure 1 -D shows that cisplatin and doxorubicin showed a concentration gradient effect on the toxicity of breast cancer NK92 cells. When cisplatin was treated for 48 hours, the cell survival rates were 72%, 66% and 62% at the concentrations of 2μM, 4μM and 8μM, respectively; when doxorubicin was treated for 48 hours, the cell survival rates were 69%, 63% and 59% at the concentrations of 0.25μM, 0.5μM and 1μM, respectively.

[0030] Based on the above results, it can be found that both cisplatin and doxorubicin can inhibit the growth of tumors and NK cells. Doxorubicin is more sensitive to tumor killing, while cisplatin is more sensitive to NK cell killing. Therefore, cisplatin was selected as a chemotherapy drug in subsequent experiments.

[0031] 4. Next, the effect of the chemotherapy drug cisplatin on NK92 cells killing breast cancer MCF-7 cells was detected. First, different concentrations of cisplatin were added to NK92 cells for 24 hours, and then the treated NK92 cells were added to the MCF-7 cell plate. After incubation for 4 hours, the tumor cell supernatant was collected for subsequent cytokine detection, and the attached MCF-7 cells were washed twice with PBS to remove the floating NK92 cells, and 100 μL of complete medium was added again, and MTT (5 mg / mL) 20 μL / well was added. After incubation for 4 hours, the supernatant was discarded, DMSO 100 μL / well was added, and oscillation was performed for about 10 minutes. The OD was measured by a microplate reader. 570 The result is as follows Figure 1 -E, F, and Figure 1 -G, H, cisplatin showed a concentration-dependent inhibition of NK92 cells' killing of tumors. The supernatant collected from the above experiment was used to detect the secretion of cytokines TNF-α and IFN-γ by NK92 cells using an Elisa kit. The results showed that cisplatin showed a concentration-dependent inhibition of NK92 cells' secretion of TNF-α and IFN-γ.

[0032] Based on the above results, we found that cisplatin and doxorubicin have growth inhibitory effects on tumors and NK cells. At the same time, cisplatin showed a concentration-dependent inhibition of the killing effect of NK cells on tumors and reduced the ability of NK92 cells to secrete IFN-γ and TNF-α.

[0033] Experiment 2 Evaluation of different concentrations of different forms of selenium drugs in antagonizing chemotherapy-induced NK92 cell growth inhibition.

[0034] In order to test whether eight selenium drugs can improve the cell survival rate of NK-92 cells inhibited by cisplatin or doxorubicin, the CCK8 kit was used to detect the effect of selenium drugs in reversing the cytotoxicity of NK-92 cells induced by chemotherapy drugs. Figure 2 -A, B) NK92 cells were pretreated with different selenium drugs for 24 hours and then cisplatin was added for co-treatment for 24 hours or 48 hours, and then the cell survival rate of NK92 cells was detected; ( Figure 2 -C, D) NK92 cells were pretreated with different selenium drugs for 24 hours and then treated with doxorubicin for 24 hours or 48 hours, and then the cell survival rate of NK92 cells was detected.

[0035] The experimental results showed that selenium drug pretreatment could antagonize cisplatin and doxorubicin-induced NK cell growth inhibition and increase the survival rate of NK cells.

[0036] Experiment 3: Evaluation of the effect of selenium drugs on antagonizing cisplatin in inhibiting the immune killing of NK92 cells.

[0037] The killing effects of chemotherapy drugs cisplatin and doxorubicin on breast cancer MCF-7 cells and immune cells NK92 cells were detected. First, NK92 cells were pretreated, and SeNPs / SeMet / SeCyS2 with different concentration gradients were added to NK92 cells for 24 hours, and then 4μM cisplatin was added for 24 hours. At the same time, a single 4μM cisplatin reaction group for 24 hours was set up. MCF-7 cells were added to a 96-well culture plate to adhere to the wall, and the NK92 cells treated as above were added to the MCF-7 cell plate. After 4 hours of reaction, the tumor cell supernatant was collected for subsequent cytokine detection, and the attached MCF-7 cells were washed twice with PBS, and 100μL complete culture medium was added again, and MTT (5mg / mL) 20μL / well was added. After 4 hours, the supernatant was discarded, DMSO 100μL / well was added, and oscillation was performed for about 10 minutes. The OD was measured by a microplate reader. 570 The supernatant collected from the above experiment was used to detect the secretion of cytokines TNF-α and IFN-γ by NK92 cells using an Elisa kit. Figure 3 As shown, low concentrations of selenium drugs pretreatment can enhance the killing effect of NK cells inhibited by cisplatin on tumors (MCF-7), and increase the secretion of cytokines TNF-α and IFN-γ by NK cells. In order to exclude the combined effect of cisplatin, selenium and NK cells in the killing effect, the killing effect of selenium and cisplatin alone and the combination of the two on tumors was detected. Under the same treatment conditions, the results showed that NK cells played a leading role, while low concentrations of selenium drugs (such as below 2uM) and cisplatin had little damage to tumors.

[0038] Experiment 4 Effect of selenium drug pretreatment on cisplatin-induced mitochondrial membrane potential dissipation in NK cells.

[0039] NK92 cells were pre-treated with 0.5 μM SeNPs / SeMet / SeCyS2 for 24 h, and then 4 μM cisplatin was added for 24 h. A single 4 μM cisplatin reaction group was set up for 24 h. The cells were collected into a 2 mL Eppendorf tube, washed twice with PBS buffer, and then stained with JC-1 probe for 30 min in the dark. Finally, the cells were analyzed by flow cytometer GytoFLEXS (Beckman Coulter, USA).

[0040] like Figure 4 As shown, pretreatment with low concentrations of selenium drugs can reduce cisplatin-induced mitochondrial membrane potential dissipation in NK cells.

[0041] Experiment 5: Damage to mitochondrial function of NK cells induced by cisplatin after pretreatment with selenium drugs.

[0042] NK92 cells were treated with the same drug treatment method as in Experiment 4. The mtSOX Deep Red probe was used to detect the production of mitochondrial superoxide in NK92 cells. MitoTracker Red CMXRos and MitoTracker Green FM were used to detect the mitochondrial mass of NK92 cells. The flow cytometry results and mitochondrial mass fluorescence images showed that low concentrations of selenium drugs pretreatment could reduce the production of mitochondrial superoxide in cisplatin-induced NK cells and increase the mitochondrial mass of cisplatin-induced NK cells. The DCF probe was used to detect the total ROS production in NK92 cells, as shown in Figure 2. Figure 5 As shown, pretreatment with low concentrations of selenium drugs can reduce the production of total ROS in NK cells induced by cisplatin.

[0043] Experiment 6: Effect of selenium drug pretreatment on cisplatin-induced inhibition of NK cell glucose uptake.

[0044] To examine the metabolic capacity of NK cells, the ability of 2-NBDG to uptake glucose was analyzed by flow cytometry staining, as shown in Figure 6 As shown, low concentration of selenium drug pretreatment can antagonize cisplatin-induced inhibition of NK cell glucose uptake.

[0045] Since cisplatin or doxorubicin can effectively kill tumor cells, they will inevitably damage immune cells such as NK cells. The present invention innovatively provides an application of a selenium-containing preparation for antagonizing the toxic effects of cisplatin or doxorubicin on immune cells such as NK cells. The selenium-containing preparation can effectively inhibit the damage of cisplatin to the mitochondrial function of NK cells, thereby promoting the glucose uptake and energy metabolism of NK cells, thereby significantly enhancing the immune killing ability of NK cells inhibited by cisplatin. This application will bring important technological breakthroughs in the field of cancer treatment.

[0046] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A drug for antagonizing cisplatin or doxorubicin-induced NK cell damage, characterized in that: Including selenium-containing preparations.

2. The drug according to claim 1, characterized in that The cisplatin or doxorubicin inducing NK cell damage specifically refers to: using cisplatin or doxorubicin as an anticancer drug to induce NK cell damage.

3. The drug according to claim 2, characterized in that The anticancer drug is specifically a drug for treating breast cancer.

4. The drug according to any one of claims 1 to 3, characterized in that This drug is a pretreatment drug.

5. The drug according to any one of claims 1 to 3, characterized in that The concentration of the selenium-containing preparation in the drug is 0.1uM-2uM.

6. The use according to any one of claims 1 to 3, characterized in that: The selenium-containing preparation comprises one or a combination of several of SeNPs, CS·SeNPs, SeMet, Me·SeCys, SeCyS2, EbSelen, Na2SeO3, and Na2SeO4.

7. Use of a selenium-containing preparation in the medicine according to any one of claims 1 to 6.

Images