Method for enhancing cancer cell killing ability of natural killer cells by using electrical stimulation system

Abstract

The present invention relates to a method for activating natural killer cells, comprising a step of providing electrical stimulation to natural killer cells. Specifically, electrical stimulation under predetermined conditions is applied to natural killer cells by applying an electrical stimulation system established through a 3D-printed electrical stimulation device and a function generator. Here, cancer cell-killing ability and calcium ion-mediated mechanisms are analyzed among induced intracellular effects. In addition, with respect to the natural killer cells produced according to the present invention, a method capable of enhancing the efficacy of a cell therapeutic agent without introducing genetic modification is presented, and the cells can be used to study the correlation between the cancer cell-killing ability and the intracellular calcium ion levels of electrically stimulated natural killer cells.

Description

A method for enhancing the cancer-killing ability of natural killer cells using an electrical stimulation system.

[0001] The present invention relates to a method for enhancing the cancer cell killing ability of natural killer cells (NK cells) using an electrical stimulation system.

[0002] Natural killer (NK) cells play a crucial role in innate immunity without antigen presentation. This immune activity is regulated by the balance of signaling from activating and inhibitory receptors on the cell surface. The antitumor activity of NK cells largely involves target cell recognition, formation of an immune synapse, and ultimately cytolytic degranulation. However, the underlying mechanisms remain unclear, and NK cell activity is easily inhibited by tumor cells.

[0003] Therefore, understanding the activation mechanism of natural killer cells is essential to overcoming the immune evasion mechanism, which is one of the limitations of current cancer immunotherapy.

[0004] In addition, since calcium ions are known to be an important activating factor of NK cells in the immune process, we hypothesized that electrical stimulation could enhance the immune function of natural killer cells by inducing changes in intracellular calcium ion levels.

[0005] Accordingly, the inventors of the present invention introduced electrodes by applying platinum wires to a 3D printed plate cover, designed an electrical stimulation system capable of controlling stimulation conditions through a function generator, and observed the correlation between the influx of intracellular calcium ions induced by the electrical stimulation of this system and the activation of natural killer cells, and confirmed the effect of enhancing the efficacy of cell therapy without introducing genetic modification, leading to the present invention.

[0006] The present invention aims to provide a method for enhancing the cancer cell killing ability of natural killer cells using an electrical stimulation system, and to provide a correlation between the cancer cell killing ability of natural killer cells and the intracellular calcium ion level.

[0007] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0008] According to one embodiment of the present invention, a method for activating natural killer cells is provided, comprising the step of providing electrical stimulation to natural killer cells.

[0009] In the present invention, the electrical stimulation can be performed at a voltage of 0.3 to 1.2 V / cm for 40 to 70 minutes.

[0010] In the present invention, the electrical stimulation can increase the calcium ion concentration of the natural killer cells by 1.1 to 1.5 times.

[0011] According to another embodiment of the present invention, natural killer cells are provided, produced by a method for activating natural killer cells according to the present invention.

[0012] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer is provided, comprising natural killer cells according to the present invention as an active ingredient.

[0013] Natural killer cells that receive electrical stimulation through the electrical stimulation system according to the present invention not only exhibit 1.2 to 1.6 times higher cytotoxicity toward tumor cells compared to natural killer cells that do not receive electrical stimulation, but also can enhance the immune activity of natural killer cells by activating a calcium ion-mediated mechanism.

[0014] Accordingly, it can be used as a cell therapy agent without introducing genetic modification.

[0015] Figure 1 is a diagram showing a schematic diagram of the electrical stimulation system design process according to Manufacturing Example 2.

[0016] Figure 2 is a diagram illustrating a schematic of the experimental design process for co-cultivation of NK cells and target tumor cells according to Example 1.

[0017] Figure 3 is a diagram showing the results of analyzing the effect of electrical stimulation on the cell survival rate of NK cells according to Example 2.

[0018] Figure 4 is a diagram showing the results of confirming the effect of electrical stimulation on the cancer cell killing ability of NK cells according to Example 3.

[0019] Figure 5 is a diagram showing the results of confirming changes in gene and protein expression in NK cells after electrical stimulation, according to Experimental Example 4.

[0020] Figure 6 is a diagram showing the results of confirming changes in intracellular calcium concentration and calcium-mediated signal transmission after electrical stimulation according to Example 5.

[0021] Figure 7 is a diagram showing the results of Example 6, which confirms the effect of the BAPTA-AM calcium chelator on calcium-mediated gene expression changes and NFAT1 dephosphorylation due to electrical stimulation.

[0022] Figure 8 is a diagram schematically showing the calcineurin-NFAT signal transduction pathway according to the present invention.

[0023] Hereinafter, the present invention will be described in detail by way of examples to explain in more detail.

[0024] However, the following examples are illustrative only and the scope of the present invention is not limited thereto, and unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs.

[0025] In addition, when describing the present invention, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the present invention, the detailed description is omitted.

[0026] According to one embodiment of the present invention, a method for activating natural killer cells is provided, comprising the step of providing an electrical stimulation to natural killer cells, and more specifically, the electrical stimulation is provided through an electrical stimulation system.

[0027] The above natural killer cells are cytotoxic lymphocytes that constitute a major component of the innate immune system, and are defined as large granular lymphocytes (LGLs) and play an important role in the innate and adaptive immune systems. The above natural killer cells may include not only mature natural killer cells but also natural killer precursor cells. In addition, the above natural killer cells may be of mammalian origin, and the mammal may be a human, monkey, goat, sheep, rat, mouse, etc., and may particularly be a human, monkey, or mouse.

[0028] The increase in the cell killing capacity of the above natural killer cells may be due to an increase in the expression or secretion of substances such as perforin, granzyme, and interferon that are involved in killing target cells, or may be due to an increase in the degranulation of granules containing the above substances. In particular, the perforin may be perforin-1, perforin-2, etc., the granzyme may be granzyme A, granzyme B, granzyme H, granzyme K, granzyme M, etc., and the interferon may be type 1 interferon such as interferon-α, interferon-β, interferon-κ, interferon-ω, etc., type 2 interferon such as interferon-γ, or type 3 interferon such as interferon-L1. In addition, the above-mentioned allogeneic killing may refer to the phenomenon in which natural killer cells exhibit cell killing ability among themselves, in other words, the natural killer cells themselves act as both effector cells and target cells at the same time. The allogeneic killing between the above-mentioned natural killer cells may be increased by TGF-β, and the decrease in the allogeneic killing of the above-mentioned natural killer cells means that the degree to which the natural killer cells recognize other natural killer cells as target cells is reduced. In addition, the increase in the sensitivity of the above-mentioned natural killer cells to target cells means that the natural killer cells can more easily recognize the target cells.

[0029] The activity of the above natural killer cells may be the killing ability against target cells such as cancer cells or virus-infected cells, the degranulation phenomenon of natural killer cells, or the stimulation of activating receptors of natural killer cells or the inactivation of inhibitory receptors. In this case, the activating receptors may be NKG2D, 2B4, DNAM-1, NCRs, etc., and the inhibitory receptors may be PD-1, LAG-3, TIM-3, etc.

[0030] The enhancement of the activity of the above natural killer cells may mean an increase in the cytotoxicity of the natural killer cells against target cells, a decrease in fratricide, and an enhancement in sensitivity to target cells.

[0031] For example, the natural killer cells used in the present invention may be commonly used natural killer cells, and most suitably may be the human NK cell line KHYG-1.

[0032] In the present invention, the electrical stimulation system is designed and adjusted to fit a cell culture plate, and includes an electrical system chamber manufactured in the form of a lid that fits the cell culture plate, and a function generator connected to the electrical system chamber.

[0033] At this time, the electrical system chamber was manufactured by printing with a 3D printer (3DP-110F; Cubicon, Seongnam, Korea), and the stimulation conditions were controlled by a function generator. The electrical stimulation system manufactured accordingly was confirmed using an oscilloscope (DSOX1102A; Keysight, Santa Rosa, CA, USA).

[0034] Additionally, the electrical system chamber consists of two platinum (Pt) electrodes (cathode and anode, 99.9% Pt wire with a diameter of 0.3 mm) that are manufactured parallel to each other and spaced 20 mm apart.

[0035] For example, referring to Figure 1, an electrical system chamber manufactured to fit the size of a standard 6-well plate can be configured such that two platinum (Pt) electrodes are inserted completely across the entire plate well, such that the electrodes controlled by the function generator can influence all cells throughout the well through the medium.

[0036] At this time, the two platinum (Pt) electrodes can be inserted to a depth of 13 to 17 mm or 14 to 16 mm, and suitably inserted to a depth of 15 mm in order to affect all cells in the entire well, thereby providing electrical stimulation to the cells.

[0037] In the present invention, the electrical stimulation can be performed for 40 to 70 minutes or 50 to 65 minutes at a voltage of 0.3 to 1.2 or 0.4 to 1.1 V / cm, and suitably, in terms of enhancing cell killing ability against target cells while maintaining cell viability, it can be characterized by performing the treatment for 60 minutes at a voltage of 0.5 to 1.0 V / cm.

[0038] For example, if the electrical stimulation is applied at a voltage or time outside the scope of the present invention, problems such as a decrease in cell viability or failure to achieve the desired cell killing ability for target cells may occur.

[0039] In the present invention, the electrical stimulation may be characterized by increasing the calcium ion concentration of the natural killer cells, the expression of Granzyme B, and the dephosphorylation of nuclear factor of activated T cell 1 (NFAT 1).

[0040] In the present invention, the calcium ion concentration of the natural killer cells and the expression of Granzyme B can be increased by 1.1 to 1.5 times compared to the control group that did not receive electrical stimulation according to the present invention, and the dephosphorylation of NFAT 1 can be increased by 1.1 to 2.5 times compared to the control group.

[0041] Specifically, the electrical stimulation can enhance the gene expression level of a calcium-mediated signaling-related protein of the calcineurin-NFAT pathway, and for example, the calcium-mediated signaling-related protein can be one or more selected from the group consisting of CALM2, CALN, NFAT1, NFAT2, and NFAT4.

[0042] Additionally, it can increase the protein expression of granzyme B, a key apoptosis-inducing granule molecule, by upregulating GZMB expression.

[0043] That is, the electrical stimulation according to the present invention can enhance the immune function of natural killer cells by inducing changes in intracellular calcium ion levels by affecting calcium influx and downstream mechanisms through calcium-mediated signaling proteins.

[0044] According to another embodiment of the present invention, natural killer cells produced by a method for activating natural killer cells according to the present invention are provided.

[0045] Specifically, when the natural killer cells and target cells according to the present invention are co-cultured at a ratio of 3:1, the cytotoxic ability against target cells can be characterized by increasing by 1.2 to 1.6 times.

[0046] For example, if the ratio of natural killer cells and target cells is exceeded, a problem may arise in which cytotoxicity against the intended target cells is not exhibited, and a problem in which there is no practical benefit may arise in which a significant effect on cytotoxicity against target cells is not exhibited compared to an increase in the ratio of natural killer cells.

[0047] Therefore, in terms of enhancing the efficacy of cell therapy without introducing genetic modification, the natural killer cells can be included at a ratio three times that of target cells.

[0048] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer is provided, comprising natural killer cells according to the present invention as an active ingredient.

[0049] In the present invention, the cancer generally refers to a physiological condition of mammals characterized by uncontrolled cell growth, and refers to a condition in which a problem occurs in the control function of normal division, differentiation, and death of cells, resulting in abnormal excessive proliferation, infiltration into surrounding tissues and organs, formation of a mass, and destruction or deformation of existing structures.

[0050] For example, it may be a solid cancer or a metastatic cancer, such as colorectal cancer including colon cancer and rectal cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain tumor, head and neck carcinoma, melanoma, myeloma, leukemia, lymphoma, stomach cancer, lung cancer, pancreatic cancer, liver cancer, esophageal cancer, small intestine cancer, anal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, bone cancer, skin cancer, head and neck cancer, skin melanoma, intraocular melanoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, etc. and may be characterized by at least one selected from the group consisting of prostate cancer, uterine cancer, liver cancer, rectal cancer, lung cancer, breast cancer, and ovarian cancer.

[0051] More specifically, the cancer may refer to human breast cancer cells MDA-MB-231 or MCF-7, and most preferably MCF-7.

[0052] The above pharmaceutical composition can be formulated and used in various forms according to conventional methods. For example, it can be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can be formulated and used in the form of topical preparations, suppositories, and sterile injectable solutions.

[0053] In addition, the pharmaceutical composition of the present invention may contain one or more known effective ingredients having preventive, improving, and therapeutic effects on cancer or tumors together with natural killer cell-derived endoplasmic reticulum.

[0054] In addition, pharmaceutically acceptable additives may be further included, and pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, and white sugar.

[0055] The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight in the composition, but is not limited thereto.

[0056] In addition, it can be administered in various oral or parenteral dosage forms during actual clinical administration, and when formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used, and it is preferable to use suitable formulations known in the relevant technical field disclosed in the literature.

[0057] The above solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. In addition, the above liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included.

[0058] The above-mentioned parenteral administration formulations include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0059] The dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the method of administration, the time of administration, and / or the route of administration, and may vary depending on various factors including the type and degree of the response to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field. A person having ordinary skill in the art can easily determine and prescribe a dosage effective for the desired treatment, and therefore the dosage does not limit the scope of the present invention in any way.

[0060] That is, the route and method of administration of the pharmaceutical composition of the present invention may be independent, and are not particularly limited in their methods, and any route and method of administration may be followed as long as the pharmaceutical composition can reach the target area.

[0061] The pharmaceutical composition may be administered orally or parenterally. Parenteral administration methods include, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration.

[0062] Additionally, it can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers to prevent cancer and prevent or treat various diseases arising from cancer.

[0063] Accordingly, it can be used as a pharmaceutical composition or cell therapy agent for preventing or treating cancer, and there is no limitation thereto in terms of maintaining the effect intended by the present invention.

[0064] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0065] The contents of the present invention described above are equally applicable to each other as long as they are not mutually contradictory, and it is also included in the scope of the present invention for a person skilled in the art to make appropriate changes and implement the present invention.

[0066] The present invention will be described in more detail through the following examples. These examples are intended merely to illustrate the present invention and are not to be construed as limiting the scope of the present invention.

[0067] Example

[0068] Manufacturing Example 1: Cell Culture

[0069] Human breast cancer cells, MCF-7 and MDA-MB-231, were obtained from the Korean Cell Line Bank (KCLB; Seoul, Korea) and cultured in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C and 5% CO2.

[0070] The human NK cell line KHYG-1 was purchased from AcceGen Biotech (cat. # ABC-TC0506; Fairfield, NJ, USA). KHYG-1 cells were cultured in T-25 flasks in RPMI 1640 medium supplemented with heat-treated FBS, 1% penicillin-streptomycin, and 100 Units / mL recombinant human IL-2 (cat. # 200-02; PeproTech, Rocky Hill, NJ, USA) at 37°C and 5% CO2, with fresh medium added or subcultured every 2–3 days.

[0071] Manufacturing Example 2: Electrical Stimulation System

[0072] Among the electrical systems, the chamber was printed with a 3D printer (3DP-110F; Cubicon, Seongnam, Korea) and the lid was fabricated to fit a standard 6-well plate.

[0073] The electrical stimulation system consisted of two platinum (Pt) electrodes (cathode and anode, 99.9% Pt wires with a diameter of 0.3 mm) positioned 20 mm apart and parallel to each other. Stimulation conditions were controlled by a function generator designed to fully penetrate the electrodes throughout the plate wells, thereby influencing the cells throughout the wells through the medium.

[0074] In the present invention, the electrical stimulation system was designed and adjusted to fit a cell culture plate, and its performance was confirmed using an oscilloscope (DSOX1102A; Keysight, Santa Rosa, CA, USA), and a schematic structure of the designed electrical stimulation system is shown in Fig. 1.

[0075] Example 1. Exposure to electrical stimulation

[0076] In order to confirm the effect of electrical stimulation on the cytotoxic activity of NK cells through co-culture between NK cells and tumor cells, an experiment was designed using tumor cells cultured according to the above Preparation Example 1 and human NK cell line KHYG-1 and the electrical stimulation system of the above Preparation Example 2, and this is shown in Fig. 2.

[0077] Specifically, in the electrical stimulation system, NK cells were exposed to two voltage ranges (0.5 V / cm, 1.0 V / cm) of direct current (denoted as DC) electrical stimulation or biphasic pulse electrical stimulation (symmetric, charge-balanced, denoted as BP) at a frequency of 10 Hz for 1 h.

[0078] Next, tumor cells that reached saturation in the plate were co-cultured with NK cells that had been electrically stimulated for 1 hour in advance for 24 hours to prepare an experimental group, and cells co-cultured with tumor cells and NK cells that had not been electrically stimulated for 24 hours were set as a control group.

[0079] Additionally, in all experiments except for the cell viability assessment (CCK-8), electrical stimulation under direct current (DC) conditions was applied. That is, all subsequent references to the abbreviation 'ES' indicate electrical stimulation under direct current (DC) conditions.

[0080] Additionally, in some calcium chelation experiments, KHYG-1 cells were cultured for 30 min before electrical stimulation in medium supplemented with the cell-permeable calcium ion chelator BAPTA-AM (B6769; Invitrogen, Carlsbad, CA, USA) at a final concentration of 5 μM, or in medium containing an equivalent amount of DMSO as a control.

[0081] Example 2. Cell viability evaluation

[0082] To determine the effect of electrical stimulation on the cell viability of NK cells, the effect of electrical stimulation for 1 hour on cell viability was measured using a cell counting kit-8 (CCK-8) experiment (Dojindo Laboratories, Kumamoto, Japan), and the results are shown in Figure 3.

[0083] Specifically, KHYG-1 cells that received electrical stimulation for 1 h were transferred to a 96-well plate (100 μL, 150,000 cells), then 10 μL of CCK-8 was added, and incubated at 37°C for 3 h. The absorbance of each plate well was measured at 450 nm using a microplate reader (Thermo Fisher Scientific).

[0084] Figure 3A shows the results (n=3) after exposure to continuous direct current (DC) or bipolar (BP) square waveform (10 Hz frequency, diagonally drawn columns) for 1 hour, and it was confirmed that cell viability was slightly reduced immediately after continuous DC electrical stimulation, but this was not statistically significant.

[0085] Figure 3B shows the relative mRNA expression ratios of the apoptosis-related genes BAX (pro-apoptotic) and BCL2 (anti-apoptotic) using RT-qPCR for 1 hour after continuous DC electrical stimulation (n = 3). It was confirmed that the BAX / BCL2 ratio slightly decreased after continuous DC electrical stimulation, but this decrease was not statistically significant. In addition, it was confirmed that there were no long-term effects at 24 and 48 hours after electrical stimulation.

[0086] That is, it was confirmed that biphasic electrical stimulation (biphasic BP) significantly reduced cell survival, whereas stimulation under continuous DC conditions did not. Accordingly, subsequent experiments were conducted using continuous electrical stimulation at two different voltages (0.5 V / cm, 1.0 V / cm) under 1-h DC conditions that did not affect cell survival.

[0087] Example 3. Evaluation of cancer cell killing ability

[0088] To evaluate the effect of electrical stimulation on the cancer cell killing ability of NK cells, lactate dehydrogenase (LDH) cytotoxicity test and live / dead cell imaging test were performed, and the results are shown in Figure 4.

[0089] First, cancer cells, MDA-MB-231 and MCF-7, were cultured in 12-well plates, and NK cells (KHYG-1 cells): cancer cells (MDA-MB-231 or MCF-7) were mixed at a ratio of 3:1 and cultured as experimental and control groups, depending on whether or not they were exposed to the electrical stimulation of Example 1. Next, the medium was collected from each well of the cultured experimental and control groups, and the cells were separated by centrifugation at 600 g for 5 minutes, and then a lactate dehydrogenase (LDH) cytotoxicity test (n=3) was performed with the supernatant medium sample, which is shown in Fig. 4A. Cytotoxicity was measured and calculated according to the kit manufacturer's instructions.

[0090] Referring to Figure 4A, MDA-MB-231 cells did not show a significant increase in cytotoxicity between the control and experimental groups, but MCF-7 cells showed an increase in cytotoxicity ability, with an increase of 1.27 times (0.5 V / cm electrical stimulation) and 1.55 times (1.0 V / cm electrical stimulation) in the experimental group compared to the control group, respectively.

[0091] Next, the live / dead cell imaging experiment was conducted using control and experimental groups for MCF-7 cells. The experimental and control groups co-cultured for 6 hours were washed three times with DPBS to remove KHYG-1 cells, and then the MCF-7 cells were stained with 2 μM calcein-AM (live) and 4 μM Ethidium homodimer-1 (dead) and analyzed by fluorescence microscopy according to the manufacturer's protocol, as shown in Fig. 4B.

[0092] Referring to Figure 4B, it was confirmed that the experimental group of MCF-7 cells with electrically stimulated KHYG-1 cells showed more dead cells compared to the control group of MCF-7 cells with KHYG-1 cells that did not receive electrical stimulation. In other words, it was found that electrical stimulation could enhance the cancer cell killing ability of NK cells against MCF-7 cells.

[0093] Example 4. Changes in gene and protein expression

[0094] 1) Gene expression analysis

[0095] In order to confirm the change in gene expression related to the cancer cell killing ability of KHYG-1 cells after electrical stimulation, the change in gene expression level related to cytotoxic activity and granzyme B production was confirmed and the result is shown in Figure 5A.

[0096] Specifically, the effects of electrical stimulation on the expression of apoptotic granules, cytokine proteins, and calcium signaling proteins were analyzed using real-time reverse transcription-polymerase chain reaction (RT-qPCR). Total RNA was extracted from electrically stimulated cell samples using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's recommended protocol. The mRNA concentration of each sample was measured using a microspectrophotometer (DS-11; DeNovix, Wilmington, DE, USA), and cDNA was synthesized using the PrimeScript RT reagent kit (Takara, Shiga, Japan). In addition, real-time analysis was performed using the CFX96 analysis system (Bio-Rad) with TB Green Premix Ex Taq II (RR810A; Takara), and the target genes were perforin (PRF1), granzyme B (GZMB), interferon-gamma (IFNG), and tumor necrosis factor alpha (TNFA), which are related to the immune activity of NK cells, and other factors related to calcium signaling, including inositol trisphosphate receptor (IP3R), calmodulin (CALM2), calcineurin (CALN), calmodulin-dependent protein kinase II (CaMK II), and nuclear factor of activated T cells (NFAT), which are factors involved in calcium ion signaling. In addition, GAPDH was used as a housekeeping gene, and the relative mRNA expression level was calculated using the 2(-△△CT) method compared to the control group. The primer sequences for the target genes are listed in Table 1 below.

[0097]

[0098] Referring to Figure 5A, the relative mRNA expression levels of degranulation markers (PRF1, GZMB) and cytokines (IFNG, TNFA) were analyzed and compared with GAPDH and the unstimulated control group (n = 3). As a result, it was confirmed that GZMB gene expression significantly increased by 1.36-fold and 1.58-fold under electrical stimulation conditions of 0.5 V / cm and 1.0 V / cm, respectively.

[0099] 2) Enzyme-linked immunosorbent assay (ELISA)

[0100] Next, to determine whether the gene expression changes also affected the protein level, the results of analyzing the granzyme B protein expression level using ELISA are shown in Figure 5B.

[0101] Specifically, KHYG-1 cells were seeded in 6-well culture plates and cultured for 4 and 8 h at 37°C and 5% CO2 after stimulation at 0.5 V / cm or 1.0 V / cm for 1 h, respectively. The culture medium was then collected and centrifuged at 600 g for 5 min. Intracellular proteins were extracted using RIPA cell lysis (RIPA) buffer (Elpis Biotech, Daejeon, Korea) with the addition of a protease inhibitor cocktail (Thermo Fisher Scientific) according to the manufacturer's recommended protocol. Intracellular or cultured granzyme B protein levels were measured using a Human Granzyme B ELISA kit (3486-1H-6; MABTECH, Nacka Strand, Sweden) according to the manufacturer's instructions.

[0102] Referring to Figure 5B, the intracellular granzyme B level of KHYG-1 cells was found to be higher at 0.5 V / cm compared to the control group, and in particular, it was confirmed to increase 1.26-fold at 1.0 V / cm.

[0103] In addition, the level of granzyme B secreted into the cell medium obtained 4 hours and 8 hours after electrical stimulation was measured, and it was confirmed that the level of granzyme B released outside the cells in the medium 4 hours after electrical stimulation under 0.5 V / cm conditions was 1.20 times higher than that of the control group.

[0104] Therefore, it was found that the gene expression of granzyme B, a key apoptosis-inducing granule molecule, was significantly increased after 0.5 V / cm and 1.0 V / cm electrical stimulation, which also increased the protein level, and it was found that the increase in GZMB gene expression also caused changes in protein expression.

[0105] Example 5. Changes in intracellular calcium concentration and calcium-mediated signaling

[0106] To determine the effect of electrical stimulation on calcium influx, intracellular calcium ion measurements and Ca were performed using Fluo-4 fluorescent material that specifically binds calcium ions. 2+ -The expression levels of genes related to the NFAT pathway were evaluated and the results are shown in Figure 6.

[0107] Specifically, changes in intracellular calcium ion concentration were quantitatively detected using the Fluo-4 NW Calcium Assay Kit (F36206; Invitrogen). KHYG-1 cells were incubated with the Fluo-4 AM fluorophore at 37°C for 45 min while exposed to each voltage range of electrical stimulation, which was the maximum time recommended by the manufacturer for fluorescence staining. Next, fluorescence intensity (494 nm Ex; 516 nm Em) was measured using a Varioskan LUX multimode microplate reader (Thermo Fisher), and 100 μL was transferred to a 96-well plate to measure fluorescence intensity. Data were calculated as relative fluorescence to baseline and expressed as a percentage change relative to the untreated control. Baseline fluorescence was repeatedly established at the beginning and end of the measurements according to the manufacturer's recommended protocol. In addition, KHYG-1 cells stained with Fluo-4 AM were imaged by fluorescence microscopy independently of the quantitative analysis.

[0108] Figure 6A shows the quantitative analysis of calcium using Fluo-4 NW fluorescence and fluorescence imaging of cells stained with Fluo-4 independently performed to confirm the calcium ion influx induced by electrical stimulation (n = 3). The relative fluorescence levels of the calcium ion concentration measured for 45 minutes of electrical stimulation were calculated, and it was confirmed that they increased 1.31-fold (0.5 V / cm) and 1.11-fold (1.0 V / cm) after electrical stimulation.

[0109] Figure 6B shows the gene expression levels of calcium signaling markers after electrical stimulation for 1 hour using RT-qPCR (n = 3). When stimulated with two different voltage conditions, all calcium-related markers of the calcineurin-NFAT pathway except IP3R were significantly elevated.

[0110] Thus, we found that electrical stimulation affects calcium influx and downstream mechanisms through calcium-mediated signaling proteins.

[0111] Example 6. Confirming the effect of calcium ions

[0112] The effects of BAPTA-AM calcium chelator treatment on gene expression and NFAT1 dephosphorylation in response to electrical stimulation-induced calcium concentration increase are analyzed and shown in Figure 7.

[0113] 1) Gene expression for increased calcium concentration

[0114] To further investigate the influence of calcium signaling mechanisms on NK cell granule-mediated immune responses, we conducted experiments using BAPTA-AM, a membrane-permeable calcium chelator, and analyzed GZMB expression levels and calcineurin-NFAT signaling proteins (CALM, CALN, NFAT1) after calcium ion chelation by RT-qPCR. KHYG-1 cells were cultured with DMSO (control) or a calcium chelator (5 μM BAPTA-AM) with or without electrical stimulation (n = 3). KHYG-1 cells were treated with 5 μM BAPTA-AM for 30 minutes immediately before electrical stimulation.

[0115] Referring to Figure 7A, there was no significant difference in cell viability when 5 μM BAPTA-AM was used, regardless of whether electrical stimulation was performed. Furthermore, it was found that GZMB expression was not increased after electrical stimulation by chelating intracellular free calcium when the calcium chelator BAPTA-AM was used together.

[0116] Additionally, when BAPTA-AM was used together, the gene expression of other markers related to the calcineurin-NFAT signaling pathway was shown to be at a lower level compared to the control group.

[0117] This suggests that the transient change in intracellular calcium concentration after electrical stimulation is suppressed through chelation, resulting in inhibition of the calcium-mediated signaling mechanism.

[0118] 2) Transcriptional activity of transcription factor NFAT1

[0119] To determine the level of dephosphorylation of transcription factor NFAT1, which can lead to transcriptional activity after exposure to electrical stimulation, Western blotting of NFAT1 was performed after exposure to electrical stimulation (1.0 V / cm) or electrical stimulation with BAPTA-AM.

[0120] Specifically, after electrical stimulation, KHYG-1 cells were washed twice with cold DPBS and lysed with RIPA buffer (Elpis Biotech) containing protease and phosphatase inhibitors provided by Thermo Fisher Scientific. The cells were then stored on ice for 15 minutes and sonicated for 30 seconds. The total cell lysate was centrifuged for 15 minutes and used for further analysis. Each sample (10 μL) was diluted in Laemmli sample buffer, incubated at 95°C for 5 minutes, and then separated by 10% SDS-PAGE electrophoresis. The protein samples were then transferred to PVDF membranes using a semi-dry transfer method. The membranes were stored in Tris-buffered saline containing skim milk at 25°C for 1 hour to prevent nonspecific binding. The membrane was incubated overnight at 4°C with primary antibodies against NFAT1 (MA1-025; Invitrogen) and GAPDH (2118S; Cell Signaling Technology, Danvers, MA, USA), which served as a loading control. The membrane was specifically labeled with secondary antibodies (anti-rabbit, ab6721, Abcam; anti-mouse, 31430, Thermo Fisher). Images were acquired using a chemiluminescence substrate (W3651-012; GenDEPOT, Barker, TX, USA) and quantified using a gel imaging system (G:BOX Chemi XRQ; Syngene, Cambridge, UK) and Syngene GeneTools software. The level of dephosphorylated NFAT1 was calculated as the ratio of dephosphorylated-NFAT1 to phosphorylated NFAT1 (p-NFAT1) and compared with the control.

[0121] Referring to Figure 7B, the upper band (140 kDa) represents phosphorylated NFAT1 (p-NFAT1), and the lower band (120 kDa) represents dephosphorylated NFAT1. The dephosphorylation of NFAT1 was quantitatively expressed as the ratio of p-NFAT1 to NFAT1, and it was confirmed that the dephosphorylation ratio of NFAT1 in the experimental group treated with electrical stimulation (1.0 V / cm) increased 2.21-fold compared to the control group. In addition, it was confirmed that treatment with the calcium chelator BAPTA-AM reduced the effect of electrical stimulation on calcium ion-calcineurin-mediated dephosphorylation of NFAT1, and the ratio of dephosphorylated NFAT1 was approximately 0.17-fold lower than that of the control group.

[0122] All of the above experiments were repeated independently at least three times, and the results were expressed as the mean ± standard error of the mean. Statistical analysis was performed in Microsoft Excel 2016, and Student's t-test was used to determine the statistical significance between the results obtained in each experimental group and the control group (*P <0.05, **P <0.01, and ***P <0.001). All analyses according to Experimental Examples 1 to 6 were performed at least three times independently, and all data were expressed as the mean ± standard error. Comparisons between two groups were statistically analyzed using a two-tailed Student's t-test, and significant results were expressed as *P <0.05, **P <0.01, and ***P <0.001, depending on the P value.

[0123] Additionally, a mechanism schematic diagram for the calcineurin-NFAT signal transduction pathway according to the above embodiment is shown in Fig. 8.

[0124] In summary, when KHYG-1 cells (natural killer cells) that received electrical stimulation through the electrical stimulation system according to the present invention were treated with breast cancer MCF-7 cells and co-cultured for 24 hours (experimental group), compared to the NK cell-MCF-7 co-culture group (control group) that was not treated with stimulation, the cytotoxicity was 1.27 times (0.5 V / cm) and 1.55 times (1.0 V / cm) higher, and granzyme B (GZMB) gene expression was increased by 1.36 times (0.5 V / cm) and 1.58 times (1.0 V / cm), respectively, due to the increased activity of nuclear factor 1 (NFAT1) mediated by calcium ion activation. In addition, in the case of an experiment in which the influx of calcium ions was chelated with 5 μM BAPTA-AM, the effect of electrical stimulation was neutralized, thereby promoting gene expression of calcium ion signaling proteins and granzyme B proteins in apoptotic granules and NFAT1. It was found that dephosphorylation, i.e., inhibition of activation, was observed.

[0125] That is, the effect of electrical stimulation was shown to be mediated by a calcium ion mechanism, and calcium ions were found to affect the immune activity of natural killer cells through changes in the expression of transcription factors and apoptotic granule proteins within natural killer cells.

[0126] Therefore, this study is expected to be useful as a method to enhance the efficacy of cell therapy without introducing genetic modification.

[0127] This invention was made possible with the support of the National Research Foundation of Korea (NRF) funded by the Korean government (Ministry of Science and ICT) (RS-2023-00207801) and the Korea Foundation for Regenerative Medicine (KFRM) funded by the Korean government (Ministry of Science and ICT, Ministry of Health and Welfare) (RS-2024-00333403).

Claims

1. A method for activating natural killer cells, comprising a step of providing electrical stimulation to natural killer cells.

2. In paragraph 1, A method for activating natural killer cells, characterized in that the electrical stimulation is performed for 40 to 70 minutes at a voltage of 0.3 to 1.2 V / cm using an electrode having a depth of 13 to 17 mm.

3. In paragraph 1, A method for activating natural killer cells, characterized in that the electrical stimulation increases the calcium ion concentration of the natural killer cells, the expression of Granzyme B, and the dephosphorylation of nuclear factor of activated T cells 1 (NFAT 1).

4. Natural killer cells produced by the method for activating natural killer cells according to any one of paragraphs 1 to 3.

5. A pharmaceutical composition for preventing or treating cancer, comprising natural killer cells according to Article 4 as an active ingredient.

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