Anticancer agent kit

Abstract

This anticancer agent kit comprises an anticancer agent and an anticancer action enhancer having autophagy inhibitory action and containing molecular hydrogen as an anticancer agent action enhancing substance, wherein targeted cancer cells are at least one kind of human cervical cancer-derived HeLa cells, human colon cancer-derived Caco-2 cells, and human large bowel cancer-derived HCT116 cells in which autophagy is inhibited by molecular hydrogen, the anticancer action enhancer is electrolyzed hydrogen water, the anticancer agent contains an active ingredient having autophagy activating action, and the anticancer effect of the anticancer agent is enhanced by molecular hydrogen inhibiting the autophagy activating action of the anticancer agent.

Description

Anticancer drug kit

[0001] The present invention relates to an anticancer drug kit.

[0002] The present applicant has been conducting various studies into new uses and functions of various hydrogen waters, such as electrolyzed hydrogen water, and mixtures containing such hydrogen waters. For example, Patent Document 1 proposes a hydrogen water mixture for suppressing alcoholic liver damage, which is a mixture of an ethanol solution and hydrogen water, with an ethanol concentration of 1-4% and a dissolved hydrogen concentration of 550-5600 ppb. This hydrogen water mixture is highly safe, easy to prepare, and inexpensive, and can effectively suppress alcoholic liver damage.

[0003] Japanese Patent Application Laid-Open No. 2022-126128

[0004] Conventionally, cancer treatments have been carried out using drug therapies (chemotherapy) that use anticancer agents that act on the DNA of cancer cells and inhibit the proliferation of cancer cells, thereby exerting an anticancer effect.

[0005] Although conventional anticancer drugs have excellent anticancer effects, some of them have strong side effects such as nausea, vomiting, and alopecia, and as the dosage of the anticancer drug increases, the economic burden on patients also increases, which can lead to problems such as the inability to continue using the anticancer drug. In order to reduce the side effects and economic burden on patients, there is a demand to reduce the dosage of anticancer drugs even as much as possible.

[0006] Therefore, the present invention has been made in consideration of these points, and aims to provide an anticancer drug kit that can enhance the anticancer effect of an anticancer drug without increasing the dosage of the anticancer drug.

[0007] As a result of extensive research to achieve the above-mentioned object, the inventors discovered that electrolyzed hydrogen water (the molecular hydrogen dissolved in it) has the activity of suppressing autophagy, and that this activity can be utilized to enhance the anticancer effect of anticancer drugs, leading to the completion of the anticancer drug kit of the present invention.

[0008] The anticancer drug kit of the present invention comprises an anticancer drug and an anticancer effect enhancer having autophagy-inhibiting activity and containing molecular hydrogen as an anticancer drug effect enhancer, wherein the concentration of the molecular hydrogen relative to the reference active ingredient concentration (1 μM) of the anticancer drug is 105 ppb or more and 2160 ppb or less, the cancer cells to be treated are at least one selected from the group consisting of human cervical cancer-derived HeLa cells, human colon cancer-derived Caco-2 cells, and human colorectal cancer-derived HCT116 cells, in which autophagy is inhibited by the molecular hydrogen, the anticancer effect enhancer is electrolyzed hydrogen water, and the anticancer drug contains at least one active ingredient selected from the group consisting of doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosatinib, nilotenib, and fluorouracil, which have an autophagy-activating effect, The molecular hydrogen suppresses the autophagy-activating action of the anticancer drug, thereby enhancing the anticancer effect of the anticancer drug. However, the anticancer drug kit of the present invention may exclude an anticancer drug kit in which the cancer cells to be treated are colon cancer cells and the anticancer drug contains fluorouracil as an active ingredient.

[0009] The anticancer drug kit of the present invention comprises an anticancer drug and an anticancer effect enhancer having autophagy-inhibiting activity and containing molecular hydrogen as an anticancer drug effect enhancer, wherein: the concentration of the molecular hydrogen relative to the reference active ingredient concentration (1 μM) of the anticancer drug is 105 ppb or more and 2160 ppb or less, the cancer cells to be treated are at least one selected from the group consisting of human cervical cancer-derived HeLa cells, human colon cancer-derived Caco-2 cells, and human colorectal cancer-derived HCT116 cells, in which autophagy is inhibited by the molecular hydrogen, the anticancer effect enhancer is electrolyzed hydrogen water, and the anticancer drug contains at least one active ingredient selected from the group consisting of doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosatinib, and nilotenib, which have an autophagy-activating effect, The molecular hydrogen suppresses the autophagy-activating action of the anticancer drug, thereby enhancing the anticancer effect of the anticancer drug.

[0010] According to the present invention, it is possible to provide an anticancer drug kit that can enhance the anticancer effect of an anticancer drug without increasing the dose of the anticancer drug.

[0011] Figure 1 shows the expression level of LC3 protein (LC3-II) in the presence or absence of Bafilomycin A1 (Baf.A1) using a flux assay with mouse embryonic fibroblasts (MEF cells). Figure 2 shows the difference between the expression level of LC3-II in the presence of Baf.A1 and the expression level in the absence of Baf.A1 using MEF cells. Figure 3 shows the results of analyzing the autophagy activity of electrolyzed hydrogen water using MEF cells and fluorescence microscopy with an mRFP-GFP-LC3 tandem fluorescent probe. Figure 4 shows the results of analyzing the autophagy activity of electrolyzed hydrogen water using human cervical cancer cells (HeLa cells) and measuring the GFP / RFP ratio with a GFP-LC3-RFP-LC3ΔG probe. Figure 5 shows the results of analyzing the autophagy activity of doxorubicin (an anticancer drug) using HeLa cells and measuring the GFP / RFP ratio with a GFP-LC3-RFP-LC3ΔG probe. Figure 6 shows the results of an investigation into the cell proliferation inhibitory effect of doxorubicin using HeLa cells. Figure 7 shows the results of an investigation into the apoptosis induction of doxorubicin using HeLa cells. Figure 8 shows the results of an investigation into the cell proliferation inhibitory effect of doxorubicin when used in combination with electrolyzed hydrogen water using HeLa cells (ATG7 WT). Figure 9 shows the results of an investigation into the cell proliferation inhibitory effect of doxorubicin when used in combination with electrolyzed hydrogen water using HeLa cells (ATG7 KO) lacking ATG7 (an autophagy-related gene). Figure 10 shows the results of an investigation into the apoptosis induction of doxorubicin when used in combination with electrolyzed hydrogen water using HeLa cells. Figure 11 shows the results of an analysis of the autophagy activity of doxorubicin when used in combination with electrolyzed hydrogen water, using HeLa cells and measuring the GFP / RFP ratio using the GFP-LC3-RFP-LC3ΔG probe. FIG. 12 shows the results of verifying the cell growth inhibitory effect of doxorubicin on human colon cancer cells (Caco-2 cells) in a serum-free medium or a medium containing Baf.A1.Figure 13 shows the results of an investigation into the cell proliferation inhibitory effect of doxorubicin in purified water medium or electrolyzed hydrogen water medium using Caco-2 cells. Figure 14 shows the results of an investigation into the cell proliferation inhibitory effect of electrolyzed hydrogen water using Caco-2 cells. Figure 15 shows the results of an investigation into the cell proliferation inhibitory effect of doxorubicin when used in combination with electrolyzed hydrogen water in serum-free medium or medium containing Baf.A1 using Caco-2 cells. Figure 16 shows the results of an investigation into the apoptosis induction of doxorubicin when used in combination with electrolyzed hydrogen water using Caco-2 cells. Figure 17 shows the results of an analysis of the autophagy activity of doxorubicin when used in combination with electrolyzed hydrogen water using Caco-2 cells, based on the GFP / RFP ratio using a GFP-LC3-RFP probe. Figure 18 shows the results of an analysis of the autophagy activity of various waters using Caco-2 cells, based on the GFP / RFP ratio using a GFP-LC3-RFP probe. Figure 19 shows the results of an investigation into the cell proliferation inhibitory effect of doxorubicin when used in combination with various types of water, using Caco-2 cells. Figure 20 shows the results of an investigation into the cell proliferation of doxorubicin when used in combination with hydrogen-generated water, using HeLa cells (ATG7 WT). Figure 21 shows the results of an investigation into the cell proliferation of doxorubicin when used in combination with hydrogen-generated water, using HeLa cells with ATG7 deleted (ATG7 KO). Figure 22 shows the results of an analysis of the autophagy activity of electrolyzed hydrogen water using human colon cancer cells (HCT116 cells) based on the GFP / RFP ratio using a GFP-LC3-RFP probe. Figure 23 shows the results of an analysis of the autophagy activity of paclitaxel (an anticancer drug) using a GFP-LC3-RFP-LC3ΔG probe based on the GFP / RFP ratio using HeLa cells. Figure 24 shows the results of an investigation into the cell proliferation inhibitory effect of paclitaxel using HeLa cells. FIG. 25 shows the results of analyzing the autophagy activity of fluorouracil (5-FU, an anticancer drug) in HCT116 cells based on the GFP / RFP ratio using a GFP-LC3-RFP probe.Figure 26 shows the results of an investigation into the cell proliferation inhibitory effect of paclitaxel when used in combination with electrolyzed hydrogen water, using HeLa cells (ATG7 WT). Figure 27 shows the results of an investigation into the cell proliferation inhibitory effect of paclitaxel when used in combination with electrolyzed hydrogen water, using HeLa cells (ATG7 KO) in which ATG7 (an autophagy-related gene) had been deleted. Figure 28 shows the results of an investigation into the cell proliferation inhibitory effect of fluorouracil when used in combination with electrolyzed hydrogen water, using HCT116 cells.

[0012] The anticancer drug kit according to this embodiment includes an anticancer effect enhancer and an anticancer drug. The type of cancer targeted by the anticancer drug kit is not particularly limited, but examples include cervical cancer, colon cancer, and colorectal cancer.

[0013] <Anti-cancer effect enhancer> An anti-cancer effect enhancer refers to a substance that enhances the effect of an anti-cancer agent (anti-cancer effect) by enhancing the action pathway of the anti-cancer agent itself through the activity of the anti-cancer agent effect enhancer, or by acting synergistically via a pathway different from the action pathway of the anti-cancer agent. Here, the anti-cancer effect enhancer according to the present embodiment contains an anti-cancer agent effect enhancer that has the activity of suppressing autophagy (hereinafter referred to as "autophagy suppression activity"), based on the results of analysis and verification in the examples described below.

[0014] Autophagy is an intracellular recycling system (a degradation system using lysosomes) that breaks down intracellular components, purifying them and removing harmful substances, thereby preventing various diseases such as cancer. In addition to its role as a mechanism for the breakdown of intracellular components, autophagy is also known to function in regulating biological mechanisms through the secretion of intracellular components. In particular, secretory autophagy (exophagy, the extracellular release of secreted products) has been shown to be involved in the regulation of inflammation and the exchange of metabolites, which are associated with various diseases.

[0015] The anticancer effect enhancer according to this embodiment contains molecular hydrogen as an anticancer agent effect enhancer having autophagy-inhibiting activity. In other words, molecular hydrogen can be said to be the active molecule for autophagy-inhibiting activity. Furthermore, the anticancer effect enhancer has an excellent cancer cell proliferation inhibitory effect in that molecular hydrogen inhibits autophagy, thereby effectively suppressing the proliferation of cancer cells.

[0016] The form of the anticancer effect enhancer containing molecular hydrogen is not particularly limited, and examples thereof include a liquid containing molecular hydrogen (hereinafter also referred to as "hydrogen-containing water"), a gas containing molecular hydrogen (hereinafter also referred to as "hydrogen-containing gas"), etc.

[0017] Hydrogen-containing water is a biologically applicable liquid that has molecular hydrogen dissolved in it. Examples of biologically applicable liquids include water (purified water, purified water, sterilized water, etc.), physiological saline, buffer solutions (phosphate buffer solutions, etc., preferably the same as the anticancer agent used), infusion solutions (infusion solutions, Ringer's solution, etc., which may contain a therapeutic agent), injection solutions, beverages (tea beverages such as green tea and black tea, green juice, vegetable juice, fruit juice, etc.), etc. Specific examples of hydrogen-containing water include electrolyzed hydrogen water produced by electrolyzing water; hydrogen water produced by other methods, etc.

[0018] The hydrogen-containing gas may contain other gases (oxygen, nitrogen, inert gas, etc.) as long as it contains molecular hydrogen, and may contain other gases (oxygen, nitrogen, inert gas, etc.) as long as the object of the invention is not impaired. Specific examples of the hydrogen-containing gas include air containing molecular hydrogen and a mixed gas containing molecular hydrogen and other gases.

[0019] The hydrogen-containing water and hydrogen-containing gas can be produced using a commercially available hydrogen generator (such as an electrolytic water generator).

[0020] Among anticancer effect enhancers, hydrogen-containing water (electrolyzed hydrogen water and generated hydrogen water) is preferred, and electrolyzed hydrogen water is more preferred because it is highly safe, easy to prepare using commercially available equipment, and inexpensive.

[0021] The lower limit of the molecular hydrogen concentration in the anticancer drug kit is preferably 550 ppb or more, more preferably 640 ppb or more, even more preferably 688 ppb or more, even more preferably 896 ppb or more, and even more preferably 1056 ppb or more. The upper limit is preferably 5600 ppb or less, more preferably 4000 ppb or less, even more preferably 2000 ppb or less, even more preferably 1320 ppb or less, and even more preferably 1080 ppb or less. Examples of molecular hydrogen concentration ranges include 550 ppb to 5600 ppb, 800 ppb to 1320 ppb, 640 ppb to 1056 ppb, and 1056 ppb to 1080 ppb. When the anticancer effect enhancer is hydrogen-containing water (such as electrolyzed hydrogen water), the molecular hydrogen concentration refers to the dissolved hydrogen concentration.

[0022] The concentration of molecular hydrogen (dissolved hydrogen concentration) relative to the standard active ingredient concentration (1 μM) of the anticancer drug is preferably 105 ppb or more and 2160 ppb or less.

[0023] <Anticancer Drug> The anticancer drug is not particularly limited, but since the anticancer effect enhancer combined in the anticancer drug kit has autophagy-inhibiting activity, it is preferable that the anticancer drug contains an active ingredient that has the effect of activating autophagy (hereinafter referred to as "autophagy-activating effect") as a function corresponding to the autophagy-inhibiting activity. By co-administering an anticancer effect enhancer with autophagy-inhibiting activity and an anticancer drug with autophagy-activating effect in the anticancer drug kit, the anticancer effect enhancer suppresses the autophagy-activating effect of the anticancer drug, thereby effectively enhancing the anticancer effect. As a result, the dosage of the anticancer drug can be reduced, thereby alleviating side effects and the economic burden on patients.

[0024] Examples of anticancer agents that have an autophagy activating effect include those containing doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosatinib, nilotenib, fluorouracil, etc. as active ingredients. The active ingredient of the anticancer agent may be at least one selected from the group consisting of doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosatinib, nilotenib, and fluorouracil. That is, the anticancer agent may contain each of the above active ingredients alone, or may contain two or more of them.

[0025] Anticancer agents with autophagy activating properties are described in the following paper, for example: The paper does not mention anything related to hydrogen.1) Ponatinib: Diana Corallo, et al. Autophagic flux inhibition enhances cytotoxicity of the receptor tyrosine kinase inhibitor ponatinib. J Exp Clin Cancer Res. 2020. 2) Cisplatin: Ji-Fan Lin, et al. Cisplatin induces protective autophagy through activation of BECN1 in human bladder cancer cells. Drug Des Devel Ther. 2017. 3) Vincristine: ZhenZhen Zhan, et al. Autophagy-mediated HMGB1 release antagonizes apoptosis of gastric cancer cells induced by vincristine via transcriptional regulation of Mcl-1. Autophagy. 2012. 4) Paclitaxel: Jian Wen, et al. Autophagy inhibition re-sensitizes pulse stimulation-selected paclitaxel-resistant triple negative breast cancer cells to chemotherapy-induced apoptosis. Breast Cancer Res Treat. 2015. 5) Dasatinib: Xiao-Feng Le, et al. Dasatinib induces autophagic cell death in human ovarian cancer. Cancer. 2010. 6) Nilotinib: Hui-Chuan Yu, et al. Nilotinib induces autophagy in hepatocellular carcinoma through AMPK activation. J Biol Chem. 2013.。

[0026] As described above, electrolyzed hydrogen water (and the molecular hydrogen dissolved in it) inhibits autophagy. On the other hand, anticancer drugs containing, for example, doxorubicin as an active ingredient activate autophagy, as shown in the analytical and verification results of the Examples described below. The inventors hypothesized that autophagy activation might attenuate the anticancer effects of anticancer drugs that activate autophagy. Further investigation led them to discover the potential for utilizing the physiological effect of molecular hydrogen, which inhibits autophagy. The anticancer drug kit of the present invention was developed based on these findings. By utilizing the activity of molecular hydrogen to effectively inhibit the autophagy-activating effect of anticancer drugs, the kit achieves enhanced anticancer effects compared to conventional methods.

[0027] The concentration of the anticancer drug in the anticancer drug kit (relative to the total amount of the components in the anticancer drug kit) (total concentration when two or more active ingredients are contained) is not particularly limited, and examples thereof include the concentration of the active ingredient applied to the treatment of the target cancer cells. For example, as described in the Examples below, in the case of HeLa cells derived from human cervical cancer, the concentration of the active ingredient of the anticancer drug is approximately 0.5 μM to 2 μM. In the case of Caco-2 cells derived from human colon cancer, the concentration of the active ingredient of the anticancer drug is approximately 1 μM to 4 μM. In the case of HCT116 cells derived from human colon cancer, the concentration of the active ingredient of the anticancer drug is approximately 1 μM to 10 μM.

[0028] <Anticancer Drug Kit> The form of the anticancer drug kit is not particularly limited and may be determined appropriately depending on the form of the anticancer effect enhancer and the anticancer drug. For example, an anticancer drug kit including hydrogen-containing water (such as electrolyzed hydrogen water) as the anticancer effect enhancer may be in the form of a liquid in which the anticancer drug is dissolved in the hydrogen-containing water. Examples of administration methods for a liquid anticancer drug kit include oral administration; injection such as intravenous drip; and parenteral administration such as subcutaneous or transdermal administration. On the other hand, examples of administration methods for an anticancer drug kit including a hydrogen-containing gas as the anticancer effect enhancer include pulmonary administration of the hydrogen-containing gas via inhalation, aspiration, or the like, and oral or parenteral administration of the anticancer drug. Thus, the anticancer effect enhancer and the anticancer drug may be administered by different methods (or separately) depending on their forms. In this case, the administration times may be simultaneous or different.

[0029] The cancer cells to which the anticancer drug kit can be applied are not particularly limited as long as they are cancer cells in which autophagy is suppressed by molecular hydrogen, from the viewpoint of utilizing the autophagy-inhibiting activity of molecular hydrogen to effectively suppress the autophagy-activating action of anticancer drugs and thereby enhance the anticancer effect more than ever before. In other words, the anticancer drug kit can be effectively applied to such cancer cells, and is not particularly limited to the cancer cells described in the examples.

[0030] In the anticancer drug kit, it is preferable that the concentration of the active ingredient of the anticancer drug is 0.5 μM or more and 10 μM or less, and the concentration of molecular hydrogen in the anticancer drug kit (total amount of ingredients) is 1056 ppb or more and 1080 ppb or less. Note that the concentration of the active ingredient of the anticancer drug is not limited to the above range and, as described above, may be appropriately determined depending on the active ingredient concentration applied to the treatment of the target cancer cells, and may be, for example, 0.5 μM or more and 5 μM or less, 0.5 μM or more and 4 μM or less, 0.5 μM or more and 2 μM or less, or 0.5 μM or more and 1 μM or less.

[0031] In the anticancer drug kit configured as described above, the anticancer effect of the anticancer drug is enhanced, which allows the dosage of the anticancer drug to be reduced, thereby solving problems such as side effects and economic burden on patients at once.

[0032] The present invention will be described below based on examples. Note that the present invention is not limited to these examples, and these examples can be modified or changed based on the spirit of the present invention, and such modifications are not excluded from the scope of the present invention. In the following figures, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

[0033] <1. Production of electrolyzed hydrogen water> Using an electrolyzed water generator (manufactured by Nippon Trim Co., Ltd., product name: TRIMION GRACE), electrolyzed hydrogen water with different dissolved hydrogen concentrations of levels 1 to 4 was produced at a temperature of 22°C and a flow rate of 1.5 L / min, and purified water was also obtained by filtering using microcarbon.

[0034] Next, the pH and dissolved hydrogen concentration of the electrolyzed hydrogen water at each of the above levels were measured. The pH was measured using a pH meter (manufactured by HORIBA, product name: LAQUA act D-71), and the dissolved hydrogen concentration was measured using a dissolved hydrogen meter DH-35A (manufactured by Toa DKK). Level 1: 800 ppb, pH 7. Level 2: 860 ppb, pH 8. Level 3: 1120 ppb, pH 9. Level 4: 1320 ppb (up to 1350 ppb), pH 10.

[0035] <Preparation of culture medium for electrolyzed hydrogen water treatment (electrolyzed hydrogen water culture medium)> 5x Dulbecco's modified Eagle's medium (hereinafter referred to as "DMEM") prepared from the powder was diluted 5 times with the above-mentioned purified water or each level of electrolyzed hydrogen water, and used as a culture medium for electrolyzed hydrogen water treatment (i.e., a treatment medium consisting of a hydrogen water mixture of 20% 5x DMEM and 80% electrolyzed hydrogen water or purified water). The dissolved hydrogen concentrations in the electrolyzed hydrogen water culture medium for each level were as follows: Level 1: 800 ppb x 4 / 5 = 640 ppb; Level 2: 860 ppb x 4 / 5 = 688 ppb; Level 3: 1120 ppb x 4 / 5 = 896 ppb; Level 4: 1320 ppb (up to 1350 ppb) x 4 / 5 = 1056 ppb (up to 1080 ppb).

[0036] <2. Analysis of the autophagy inhibition effect of electrolyzed hydrogen water (analysis from multiple angles)> We analyzed whether electrolyzed hydrogen water inhibits autophagy from multiple angles.

[0037] (2-1) Bafilomycin A1 Flux Assay Mouse embryonic fibroblasts (hereinafter referred to as "MEF cells") were cultured in a 6-well plate at a concentration of 5.0 × 10 5Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (hereinafter referred to as "preculture"). After preculture, the medium was replaced with the various DMEMs listed below in the presence or absence of 200 nM Bafilomycin A1 (hereinafter referred to as "Baf.A1", an inhibitor), and cultured for 4 hours. The treated cells were then lysed in lysis buffer, and the lysates were subjected to Western blotting to analyze the expression level of LC3 protein (hereinafter referred to as "LC3-II") using a specific antibody against LC3 protein (hereinafter referred to as "LC3-II"). The results are shown in Figure 1.

[0038] Here, the expression level of LC3-II shown in Figure 1 correlates with the number of autophagosomes. However, the expression level of LC3-II at a given time point alone does not indicate the actual flux, and it is unclear whether it activates or inhibits autophagy. Therefore, we measured the amount of LC3-II delivered to lysosomes by comparing the expression levels of LC3-II in the presence or absence of Baf.A1. Specifically, we analyzed autophagic flux by calculating the difference between the expression levels of LC3-II in the presence of Baf.A1 and in the absence of Baf.A1. The results are shown in Figure 2.

[0039] In Figures 1 and 2, MQ: DMEM containing ultrapure water diluted with only ultrapure water in the above <Preparation of a medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> (ultrapure water medium), FW: DMEM containing purified water diluted with only purified water in the above <Preparation of a medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> (purified water medium), LV1-LV4: DMEM containing electrolyzed hydrogen water with dissolved hydrogen concentration levels 1 to 4 (electrolyzed hydrogen water medium), + Baf.A1: Medium in which 200 nM Baf.A1 has been dissolved in each of the above DMEMs (medium containing Baf.A1).

[0040] As shown in Figure 1, in the absence of Baf.A1, electrolyzed hydrogen water (LV1-LV4) showed higher LC3-II expression levels than ultrapure water (MQ) and purified water (FW), and the expression increased in proportion to the dissolved hydrogen concentration. On the other hand, in the presence of Baf.A1 (+ Baf.A1), electrolyzed hydrogen water showed the same LC3-II expression levels as ultrapure water and purified water, regardless of the dissolved hydrogen concentration.

[0041] As shown in Figure 2, when comparing the difference in LC3-II expression levels in the presence and absence of Baf.A1, electrolyzed hydrogen water (LV1-LV4) showed lower levels than ultrapure water (MQ) and purified water (FW), and the expression levels decreased as the dissolved hydrogen concentration increased. These results suggest that electrolyzed hydrogen water inhibits autolysosomal degradation because the higher the dissolved hydrogen concentration, the more LC3-II accumulates (reducing the amount of degradation in autophagy).

[0042] (2-2) Fluorescence microscopy using the mRFP-GFP-LC3 tandem fluorescent probe. MEF cells expressing the mRFP-GFP-LC3 tandem fluorescent probe (hereafter referred to as "tf-LC3 probe") were cultured for 4 hours after medium replacement with the various DMEMs listed below. The MEF cells were then fixed with 2% paraformaldehyde. Intracellular fluorescence was observed using a confocal laser scanning microscope, and the number of autolysosomes was counted. The results are shown in Figure 3.

[0043] In Figure 3, FW: DMEM containing purified water (purified water medium) diluted with purified water only in the above <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)>, EHW LV4: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration of level 4 (electrolyzed hydrogen water medium).

[0044] The tf-LC3 probe is a circular DNA (plasmid) carrying a gene encoding a fluorescent protein capable of monitoring autophagy. It is described, for example, in the following paper. MEF cells expressing the tf-LC3 probe are cells that produce (express) a fluorescent protein capable of monitoring autophagy by introducing the above plasmid into the cells and incorporating the probe gene into the genome of the MEF cells (genetic modification). The tf-LC3 probe also utilizes the difference in sensitivity to acidity: GFP fluorescence is quenched under acidic conditions (pH < 5.0), whereas mRFP fluorescence is relatively stable and is retained within lysosomes. In other words, the tf-LC3 probe allows visualization of the transition from autophagosomes to acidic autolysosomes. Shunsuke Kimura, Takeshi Noda, Tamotsu Yoshimori. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy. 2007.3(5):452-60. doi: 10.4161 / auto.4451.

[0045] Figure 3 confirms that electrolyzed hydrogen water (EHW LV4) reduces the number of autolysosomes. This result suggests that electrolyzed hydrogen water inhibits decomposition by autolysosomes.

[0046] (2-3) Analysis of autophagy activity based on the GFP / RFP ratio using the GFP-LC3-RFP-LC3ΔG probe. Human cervical cancer cells (hereafter referred to as "HeLa cells") expressing the GFP-LC3-RFP-LC3ΔG probe were plated in a 12-well plate at 1.0 × 10 5Cells were seeded at 1000 x g / well and cultured in DMEM at 37°C with 5% CO2 for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 4 hours. The cells were then detached and suspended, and the GFP and RFP fluorescence intensities were analyzed by flow cytometry. Autophagy activity was quantitatively analyzed by calculating the GFP / RFP ratio. The results are shown in Figure 4.

[0047] In Figure 4, FW: DMEM containing purified water diluted with purified water only in the above <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> (purified water medium); LV1-LV4: DMEM containing electrolyzed hydrogen water with dissolved hydrogen concentration levels 1 to 4 (electrolyzed hydrogen water medium); T: Medium in which 1 μM Torin 1 has been dissolved in DMEM containing electrolyzed hydrogen water with dissolved hydrogen concentration level 4 (electrolyzed hydrogen water medium) (purified water medium containing Torin 1); Baf.: Medium in which 200 nM Baf.A1 has been dissolved in DMEM containing electrolyzed hydrogen water with dissolved hydrogen concentration level 4 (electrolyzed hydrogen water medium) (purified water medium containing Baf.A1).

[0048] The GFP-LC3-RFP-LC3ΔG probe (or the GFP-LC3-RFP probe described below) is described in the following paper, for example. The GFP-LC3-RFP-LC3ΔG probe (or GFP-LC3-RFP probe) is cleaved by ATG4, generating equal amounts of GFP-LC3 and RFP-LC3ΔG (or RFP) in the cytoplasm. GFP-LC3 localizes to autophagosomes and is subsequently quenched and degraded within autolysosomes. RFP-LC3ΔG (or RFP) remains in the cytoplasm and serves as an internal control reflecting the amount of GFP-LC3. The GFP / RFP ratio can be determined by quantifying the cumulative amount of GFP-LC3 degradation due to autophagy. A decrease in the GFP / RFP ratio indicates autophagy activation. On the other hand, an increase in the GFP / RFP ratio indicates autophagy inhibition. Takeshi Kaizuka, Hideaki Morishita, Yutaro Hama, Satoshi Tsukamoto, Takahide Matsui, Yuichiro Toyota, Akihiko Kodama, Tomoaki Ishihara, Tohru Mizushima, Noboru Mizushima. An Autophagic Flux Probe that Releases an Internal Control. Mol Cell. 2016;64(4):835-849. doi: 10.1016 / j.molcel.2016.09.037.

[0049] As shown in Figure 4, in HeLa cells derived from human cervical cancer, the higher the dissolved hydrogen concentration in electrolyzed hydrogen water (LV1-LV4), the greater the relative accumulation of GFP and the increased GFP / RFP ratio, indicating that electrolyzed hydrogen water suppressed autophagy.

[0050] (2-4) Summary Based on the results of the analysis using the multifaceted approaches described above in (2-1) to (2-3), it is believed that electrolyzed hydrogen water inhibits autophagy, that is, it is believed to have the activity of inhibiting autophagy (autophagy-inhibiting activity).

[0051] 3. Analysis of autophagy activation by the anticancer drug doxorubicin and verification of its anticancer effect We investigated whether the anticancer drug doxorubicin activates autophagy and whether autophagy activation affects its anticancer effect.

[0052] (3-1) Analysis of doxorubicin-induced autophagy activity based on the GFP / RFP ratio using the GFP-LC3-RFP-LC3ΔG probe. 1.0 × 10 HeLa cells expressing the GFP-LC3-RFP-LC3ΔG probe were plated in a 12-well plate. 5 Cells were seeded at 1000 x g / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (preculture). After preculture, the medium was replaced with DMEM supplemented with an anticancer drug (Doxorubicin, Sigma-Aldrich Co., LLC.) at a predetermined concentration (1, 2, or 4 μM), and the cells were cultured for 4 hours. The cells were then detached and suspended as described above (2-3), and the GFP and RFP fluorescence intensities were analyzed by flow cytometry. Quantitative analysis of autophagy activity was performed by calculating the GFP / RFP ratio. The results are shown in Figure 5.

[0053] As shown in Figure 5, the GFP / RFP ratio decreases as the concentration of doxorubicin increases, in contrast to electrolyzed hydrogen water (see Figure 4), and so it is thought to have the effect of activating autophagy (autophagy activation effect). Based on these results, it is predicted that autophagy activation attenuates the anticancer effect of doxorubicin.

[0054] (3-2) Verification of cell proliferation inhibitory effect HeLa cells (ATG7 WT) or HeLa cells lacking ATG7 (autophagy-related gene) (ATG7 KO) were plated in a 96-well plate at 1.0 × 10 4Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C in 5% CO for 24 hours (preculture). After preculture, the medium was replaced with DMEM supplemented with an anticancer drug containing doxorubicin at a specified concentration (1, 2, or 4 μM), and the cells were cultured for 24 hours. Then, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 420 nm was measured. The results are shown in Figure 6.

[0055] (3-3) Verification of apoptosis induction Apoptosis induction was evaluated using Cell Death Detection ELISAPLUS (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. HeLa cells (ATG7 WT) or HeLa cells lacking ATG7 (ATG7 KO) were plated in a 12-well plate at 1.0 × 10 5 Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (preculture). After preculture, the medium was replaced with DMEM supplemented with an anticancer drug containing doxorubicin at a specified concentration (1 or 2 μM), followed by 24 hours of culture. The cells were then lysed in lysis buffer, and the lysate was added to each well of an ELISA plate. The plate was then incubated for 2 hours in immunoreagent buffer containing anti-histone biotin and anti-DNA POD, and washed with incubation buffer. The plate was then reacted with 100 μL of ABTS substrate buffer, and the absorbance was measured at 405 nm and 490 nm. The results are shown in Figure 7.

[0056] 6 and 7 , a comparison between ATG7 WT and ATG7 KO confirmed that the anticancer effects of doxorubicin (cell proliferation inhibitory effect and apoptosis induction) were enhanced in proportion to the concentration by deficiency of ATG7 (an autophagy-related gene).

[0057] (3-4) Summary Considering the results of (3-1) to (3-3) above, it is expected that the anticancer effects of anticancer drugs such as doxorubicin, which have autophagy-activating properties, will be enhanced when used in combination with electrolyzed hydrogen water, which suppresses autophagy.

[0058] <4. Verification of the Enhancement of Anticancer Effects by the Combination of Electrolyzed Hydrogen Water and the Anticancer Drug Doxorubicin 1> We verified whether the combination of electrolyzed hydrogen water and the anticancer drug doxorubicin (anticancer drug kit) enhances the anticancer effects on HeLa cells derived from human cervical cancer.

[0059] (4-1) Verification of cell proliferation inhibitory effect HeLa cells (ATG7 WT) or HeLa cells lacking ATG7 (autophagy-related gene) (ATG7 KO) were plated in a 96-well plate at 1.0 × 10 4 Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C in 5% CO for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 24 hours. Then, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 420 nm was measured. The results are shown in Figures 8 (ATG7 WT) and 9 (ATG7 KO).

[0060] 8 and 9, FW: DMEM (purified water medium) containing purified water diluted with purified water only as described in the above <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> to which a predetermined concentration (0.5, 1, or 2 μM) of doxorubicin was added; EHW: DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration of Level 4 to which a predetermined concentration of doxorubicin was added.

[0061] Figure 8 (ATG7 WT) confirms that the combined use of electrolyzed hydrogen water and doxorubicin enhances the cell proliferation inhibitory effect of doxorubicin. On the other hand, Figure 9 (ATG7 KO) confirms that the enhanced effect of the combined use is lost due to the deficiency of ATG7 (an autophagy-related gene).

[0062] (4-2) Verification of apoptosis induction Apoptosis induction was evaluated using Cell Death Detection ELISAPLUS (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. HeLa cells (ATG7 WT) or HeLa cells lacking ATG7 (an autophagy-related gene) (ATG7 KO) were plated in a 12-well plate at 1.0 × 10 5 Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 24 hours. The cells were then lysed in lysis buffer, and the lysates were added to each well of an ELISA plate. The plate was then incubated for 2 hours in immunoreagent buffer containing anti-histone biotin and anti-DNA POD, and washed with incubation buffer. The plate was then reacted with 100 μL of ABTS substrate buffer, and the absorbance was measured at 405 nm and 490 nm. The results are shown in Figure 10.

[0063] In Figure 10, FW: DMEM containing purified water (purified water medium) diluted with purified water only in the above <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)>, to which doxorubicin was added (-) or 1 μM (+), and EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level 4 (electrolyzed hydrogen water medium) to which doxorubicin was added (-) or 1 μM (+).

[0064] The ATG7 WT shown in Figure 10 confirmed that the combined use of electrolyzed hydrogen water and doxorubicin enhanced the apoptosis-inducing effect of doxorubicin. On the other hand, the ATG7 KO shown in Figure 10 confirmed that the enhanced effect of the combined use was eliminated due to the deficiency of ATG7 (an autophagy-related gene).

[0065] (4-3) Analysis of autophagy activity. HeLa cells (ATG7 WT) expressing the GFP-LC3-RFP-LC3ΔG probe were plated in a 12-well plate at 1.0 × 10 5Cells were seeded at 1000 x g / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 4 hours. The cells were then detached and suspended as described above (2-3), and the GFP and RFP fluorescence intensities were analyzed by flow cytometry. Quantitative analysis of autophagy activity was performed by calculating the GFP / RFP ratio. The results are shown in Figure 11.

[0066] In Figure 11, FW: DMEM (purified water medium) containing purified water diluted with purified water only as described in <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> above, to which 1 μM doxorubicin was added; EHW: DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration of Level 4, to which 1 μM doxorubicin was added.

[0067] Figure 11 shows that the combined use of electrolyzed hydrogen water and doxorubicin increases the GFP / RFP ratio, indicating that electrolyzed hydrogen water suppresses the activation of autophagy by doxorubicin.

[0068] (4-4) Summary Considering the results of (4-1) to (4-3) above, it is expected that electrolyzed hydrogen water will enhance the anti-cancer effect of anti-cancer drugs such as doxorubicin by suppressing the autophagy-activating action in human cervical cancer cells (HeLa cells).

[0069] <5. Verification of the Enhanced Anti-Cancer Effect of the Combined Use of Electrolyzed Hydrogen Water and the Anti-Cancer Drug Doxorubicin 2> We verified whether the combined use of electrolyzed hydrogen water and the anti-cancer drug doxorubicin (anti-cancer drug kit) enhances the anti-cancer effect on human colon cancer-derived Caco-2 cells.

[0070] (5-1) Verification of cell proliferation inhibitory effect Human colon cancer cells (hereinafter referred to as "Caco-2 cells") were placed in a 96-well plate at 2.0 × 10 4Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 24 hours. Then, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 420 nm was measured. The results are shown in Figures 12 to 15.

[0071] In Figure 12, the following conditions are shown: DOX + serum free: serum-free DMEM (serum-free medium) to which a predetermined concentration of doxorubicin (1, 2, or 4 μM) was added; DOX: serum-containing DMEM to which a predetermined concentration of doxorubicin was added; and DOX + Baf.: serum-containing DMEM to which 200 nM Bafilomycin A1 (Baf. A1) was dissolved (medium containing Baf. A1) to which a predetermined concentration of doxorubicin was added.

[0072] In Figure 13, FW: DMEM (purified water medium) containing purified water diluted with purified water only as described in <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> above, to which a predetermined concentration (1, 2 or 4 μM) of doxorubicin was added; EHW: DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration of Level 4, to which a predetermined concentration of doxorubicin was added.

[0073] In Figure 14, FW: DMEM containing purified water (purified water medium) diluted with purified water only as described in <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> above, EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration of Level 4 (electrolyzed hydrogen water medium).

[0074] In Figure 15, -FW (Medium + Baf.A1): A medium prepared by dissolving 200 nM Baf.A1 in DMEM containing purified water and serum (purified water medium containing Baf.A1) according to the above <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)>, to which doxorubicin was added at 2 μM (-) or 2 μM (+); -EHW (Medium + Baf.A1): A medium prepared by similarly dissolving 200 nM Baf.A1 in DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level 4 and serum (electrolyzed hydrogen water medium containing Baf.A1) to which doxorubicin was added at 2 μM (-) or 2 μM (+).・FW (Serum free medium): Similarly, serum-free DMEM (serum-free purified water medium) containing purified water with no addition (-) or with 2 μM doxorubicin added (+). ・EHW (Serum free medium): Similarly, serum-free DMEM (serum-free electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration of level 4 with no addition (-) or with 2 μM doxorubicin added (+).

[0075] Figure 12 confirms that the combined use of doxorubicin and the autophagy inhibitor (Baf.A1) (DOX + Baf.) enhances the cell proliferation inhibitory effect of doxorubicin. On the other hand, under autophagy-activating conditions (DOX + serum-free), the cell proliferation inhibitory effect of doxorubicin is actually reduced. Figure 13 confirms that the combined use of electrolyzed hydrogen water and doxorubicin (EHW) enhances the cell proliferation inhibitory effect. We will further examine below whether this enhancement is due to the autophagy-inhibiting activity of electrolyzed hydrogen water.

[0076] As shown in Figures 14 and 15, under the combined use of doxorubicin and the autophagy inhibitor (Baf.A1) ("Medium + Baf.A1," autophagy-inhibiting conditions), there was no significant difference between purified water (FW) and electrolyzed hydrogen water (EHW), confirming that the cell proliferation inhibitory effect did not change even when EHW was used. On the other hand, under autophagy-activating conditions in serum-free medium containing doxorubicin and Baf.A1, the cell proliferation inhibitory effect was enhanced by the combined use of EHW compared to purified water (FW) and electrolyzed hydrogen water (EHW). Considering these results, it is thought that the autophagy-inhibiting activity of EHW is involved in the enhanced effect of doxorubicin.

[0077] (5-2) Verification of apoptosis induction Apoptosis induction was evaluated using Cell Death Detection ELISAPLUS (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. Caco-2 cells were plated in a 12-well plate at 2.0 × 10 5 Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 24 hours. The cells were then lysed in lysis buffer, and the lysates were added to each well of an ELISA plate. The wells were incubated for 2 hours in immunoreagent buffer containing anti-histone biotin and anti-DNA POD, and washed with incubation buffer. The cells were then reacted with 100 μL of ABTS substrate buffer, and the absorbance was measured at 405 nm and 490 nm. The results are shown in Figure 16.

[0078] In Figure 16, FW: DMEM containing purified water (purified water medium) diluted with purified water only in the above <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)>, to which doxorubicin was added (-) or 2 μM (+), and EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level 4 (electrolyzed hydrogen water medium) to which doxorubicin was added (-) or 2 μM (+).

[0079] From FIG. 16, it was confirmed that the apoptosis-inducing effect was enhanced by the combined use of doxorubicin and electrolyzed hydrogen water (EHW).

[0080] (5-3) Analysis of autophagy activity Caco-2 cells expressing the GFP-LC3-RFP probe were plated in a 12-well plate at 1.0 × 10 5 Cells were seeded at 1000 x g / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 4 hours. The cells were then detached and suspended as described above (2-3), and the GFP and RFP fluorescence intensities were analyzed by flow cytometry. Quantitative analysis of autophagy activity was performed by calculating the GFP / RFP ratio. The results are shown in Figure 17.

[0081] In Figure 17, FW: DMEM containing purified water diluted with purified water only in the above <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> (purified water medium), EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration of level 4 (electrolyzed hydrogen water medium), + DOX: Each of the above DMEMs to which 2 μM doxorubicin has been added.

[0082] As shown in Figure 17, the combined use of electrolyzed hydrogen water and the anticancer drug doxorubicin (EHW + DOX) increased the GFP / RFP ratio, confirming that electrolyzed hydrogen water suppresses the autophagy activation induced by doxorubicin.

[0083] (5-4) Summary Considering the results of (5-1) to (5-3) above, it is expected that electrolyzed hydrogen water will enhance the anti-cancer effects of human colon cancer cells (Caco-2 cells) as well as human cervical cancer cells (HeLa cells) by suppressing the autophagy-activating action of anti-cancer drugs such as doxorubicin.

[0084] 6. Verification of substances that contribute to the enhancing effect of electrolyzed hydrogen water The molecules that act on the above-mentioned effects of electrolyzed hydrogen water were verified.

[0085] (6-1) Analysis of autophagy activity. Caco-2 cells expressing the GFP-LC3-RFPΔG probe were plated in a 12-well plate at 1.0 × 105 Cells were seeded at 1000 x g / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 4 hours. The cells were then detached and suspended as described above (2-3), and the GFP and RFP fluorescence intensities were analyzed by flow cytometry. Quantitative analysis of autophagy activity was performed by calculating the GFP / RFP ratio. The results are shown in Figure 18.

[0086] 18, FW: DMEM containing purified water diluted with only purified water in the above <Preparation of a medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> (purified water medium); EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration of level 4 (electrolyzed hydrogen water medium); EHW (AC): DMEM containing autoclaved water obtained by diluting electrolyzed hydrogen water with dissolved hydrogen concentration level 4 with only water after autoclaving twice in the above <Preparation of a medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> (autoclave water medium); HW: DMEM containing hydrogen equivalent to a dissolved hydrogen concentration of level 4, diluted with only produced hydrogen water produced using a TRIM SEVEN WATER kit (manufactured by Nippon Trim Co., Ltd.) in the above <Preparation of a medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> (produced hydrogen water medium); Different symbols (a and b) indicate a statistically significant difference (p<0.05). In the double autoclave treatment, one cycle consisted of 20 minutes at high pressure and a high temperature of 121°C, and this treatment was carried out twice.

[0087] As shown in Figure 18, electrolyzed hydrogen water (EHW) and generated hydrogen water (HW) have similar GFP / RFP ratios, confirming that they equally inhibit autophagy activity. On the other hand, when electrolyzed hydrogen water is autoclaved twice (EHW (AC)), the GFP / RFP ratio becomes equivalent to that of purified water (FW), suggesting that its autophagy-inhibiting activity is lost. Based on these results, it is assumed that the autophagy-inhibiting component in electrolyzed hydrogen water is a volatile compound, such as molecular hydrogen, which is degassed by autoclaving.

[0088] (6-2) Verification of cell proliferation inhibitory effect Caco-2 cells were cultured in a 96-well plate at 1.0 × 104 Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C and 5% CO for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 24 hours. Then, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 420 nm was measured. The results are shown in Figure 19.

[0089] In Figure 19, FW: DMEM containing purified water diluted with purified water only in the above <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)>, to which doxorubicin was either added (-) or 2 μM (+), EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration of level 4 (electrolyzed hydrogen water medium), to which doxorubicin was either added (-) or 2 μM (+), EHW (AC): DMEM containing autoclaved water as described above (autoclave water medium), to which doxorubicin was either added (-) or 2 μM (+), HW: DMEM containing hydrogen equivalent to dissolved hydrogen concentration level 4 (generated hydrogen water medium), to which doxorubicin was either added (-) or 2 μM (+), Different symbols (a and b) indicate a statistically significant difference (p<0.05).

[0090] 19, it was confirmed that electrolyzed hydrogen water (EHW) and generated hydrogen water (HW) equally enhanced the cell proliferation inhibitory effect of doxorubicin. On the other hand, autoclaving electrolyzed hydrogen water twice (EHW (AC)) is thought to eliminate this enhanced effect.

[0091] (6-3) Verification of cell proliferation HeLa cells (ATG7 WT) or HeLa cells lacking ATG7 (an autophagy-related gene) (ATG7 KO) were plated in a 96-well plate at 1.0 × 10 4Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C and 5% CO for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 24 hours. Then, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 420 nm was measured. The results are shown in Figures 20 and 21.

[0092] 20 and 21, HW: DMEM containing hydrogen-generated water with a dissolved hydrogen concentration of 0 ppm to 4 ppm (hydrogen-generated water medium), HW + DOX: The above DMEM (hydrogen-generated water medium) to which 0.5 μM doxorubicin has been added. For example, if Sample 1 is a, Sample 2 is ab, and Sample 3 is b, this indicates that there is no significant difference between Sample 1 and Sample 2, or between Sample 2 and Sample 3, but that there is a significant difference between Sample 1 and Sample 3.

[0093] Figure 20 (ATG7 WT) confirms that the combined use of generated hydrogen water and the anticancer drug doxorubicin (HW + DOX) inhibits cell proliferation as the dissolved hydrogen concentration in the generated hydrogen water increases, thereby enhancing the cell proliferation inhibitory effect of doxorubicin. On the other hand, Figure 21 (ATG7 KO) confirms that the deficiency of ATG7 (an autophagy-related gene) prevents the combined use of generated hydrogen water and the anticancer drug doxorubicin from achieving the cell proliferation inhibitory effect of doxorubicin.

[0094] (6-4) Summary Considering the results of (6-1) to (6-3) above, it was confirmed that the substance (active molecule) that contributes to the enhancement of the anti-cancer effects of doxorubicin (cell proliferation inhibitory effect and apoptosis induction) by electrolyzed hydrogen water is dissolved molecular hydrogen, and that molecular hydrogen inhibits autophagy, thereby inhibiting cell proliferation of cancer cells.

[0095] 7. Analysis of autophagy activity based on the GFP / RFP ratio using other cells Human colon cancer cells expressing the GFP-LC3-RFP probe (hereafter referred to as "HCT116 cells") were cultured in a 12-well plate at 1.0 x 10 5Cells were seeded at 1000 x g / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 4 hours. The cells were then detached and suspended as described above (2-3), and the GFP and RFP fluorescence intensities were analyzed by flow cytometry. Quantitative analysis of autophagy activity was performed by calculating the GFP / RFP ratio. The results are shown in Figure 22.

[0096] In Figure 22, FW: DMEM containing purified water (purified water medium) diluted with purified water only in the above <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)>, EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration of level 4 (electrolyzed hydrogen water medium).

[0097] As shown in Figure 22, in human colon cancer-derived HCT116 cells, as in human cervical cancer-derived HeLa cells (Figure 4), the GFP / RFP ratio increased in electrolyzed hydrogen water (EHW) compared to purified water (FW), indicating that electrolyzed hydrogen water inhibited autophagy.

[0098] 8. Analysis of autophagy activation by other anticancer drugs and verification of their anticancer effects We investigated whether paclitaxel or fluorouracil, other anticancer drugs, activate autophagy and whether autophagy activation affects their anticancer effects.

[0099] (8-1) Analysis of paclitaxel-induced autophagy activity based on the GFP / RFP ratio using the GFP-LC3-RFP-LC3ΔG probe. 1.0 × 10 HeLa cells expressing the GFP-LC3-RFP-LC3ΔG probe were plated in a 12-well plate. 5Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (preculture). After preculture, the medium was replaced with DMEM supplemented with an anticancer drug (Paclitaxel, Sigma-Aldrich Co., LLC.) at a predetermined concentration (1, 5, or 10 nM), and the cells were cultured for 24 hours. The cells were then detached and suspended as described above (2-3), and the GFP and RFP fluorescence intensities were analyzed by flow cytometry. Quantitative analysis of autophagy activity was performed by calculating the GFP / RFP ratio. The results are shown in Figure 23.

[0100] As shown in Figure 23, paclitaxel, like doxorubicin (Figure 5), is thought to have the effect of activating autophagy (autophagy activation effect), as the GFP / RFP ratio decreases as the concentration increases, in contrast to electrolyzed hydrogen water (see Figures 4, 22, etc.). Based on these results, it is predicted that, like doxorubicin, autophagy activation attenuates the anticancer effect of paclitaxel.

[0101] (8-2) Verification of cell proliferation inhibitory effect HeLa cells (ATG7 WT) or HeLa cells lacking ATG7 (autophagy-related gene) (ATG7 KO) were plated in a 96-well plate at 1.0 × 10 4 Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C and 5% CO for 24 hours (preculture). After preculture, the medium was replaced with DMEM supplemented with an anticancer drug containing paclitaxel at a predetermined concentration (1, 10, or 50 nM), and the cells were cultured for 24 hours. Then, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 420 nm was measured. The results are shown in Figure 24.

[0102] As shown in Figure 24, a comparison between ATG7 WT and ATG7 KO confirmed that the anticancer effect (cell proliferation inhibitory effect) of paclitaxel was enhanced in proportion to the concentration by the deficiency of ATG7 (autophagy-related gene).

[0103] (8-3) Analysis of fluorouracil-induced autophagy activity by measuring the GFP / RFP ratio using the GFP-LC3-RFP probe. HCT116 cells expressing the GFP-LC3-RFP probe were plated in a 12-well plate at 1.0 × 10 5 Cells were seeded at 1000 x g / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (preculture). After preculture, the medium was replaced with DMEM supplemented with an anticancer drug containing a predetermined concentration (1 or 10 μM) of fluorouracil (5-FU, Sigma-Aldrich Co., LLC.), and the cells were cultured for 24 hours. The cells were then detached and suspended as described above (2-3), and the GFP and RFP fluorescence intensities were analyzed by flow cytometry. Quantitative analysis of autophagy activity was performed by calculating the GFP / RFP ratio. The results are shown in Figure 25.

[0104] As shown in Figure 25, similar to doxorubicin (Figure 5), the GFP / RFP ratio decreases as the concentration of fluorouracil increases, in contrast to electrolyzed hydrogen water (see Figure 22), suggesting that fluorouracil has the effect of activating autophagy (autophagy activation effect). Based on these results, it is predicted that, similar to doxorubicin, autophagy activation attenuates the anticancer effect of fluorouracil.

[0105] 9. Verification of the enhanced anti-cancer effect of combined use of electrolyzed hydrogen water and the anti-cancer drug paclitaxel or fluorouracil (9-1) Verification of cell proliferation inhibitory effect 1 We verified whether the combined use of electrolyzed hydrogen water and the anti-cancer drug paclitaxel (anti-cancer drug kit) enhances the anti-cancer effect on HeLa cells derived from human cervical cancer.

[0106] HeLa cells (ATG7 WT) or HeLa cells lacking ATG7 (an autophagy-related gene) (ATG7 KO) were plated in a 96-well plate at 1.0 × 10 4Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C and 5% CO for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 24 hours. Then, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 420 nm was measured. The results are shown in Figures 26 (ATG7 WT) and 27 (ATG7 KO).

[0107] 26 and 27, FW: DMEM (purified water medium) containing purified water diluted with purified water only as described in the above <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)>, to which a predetermined concentration (1, 10 or 50 nM) of paclitaxel was added; EHW: DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration of level 4, to which a predetermined concentration of paclitaxel was added.

[0108] Figure 26 (ATG7 WT) confirms that the combined use of electrolyzed hydrogen water and paclitaxel enhances the cell proliferation inhibitory effect of paclitaxel. On the other hand, Figure 27 (ATG7 KO) confirms that the enhancement effect of the combined use is lost due to the deficiency of ATG7 (an autophagy-related gene).

[0109] (9-2) Verification of cell proliferation inhibitory effect 2 We verified whether the combined use of electrolyzed hydrogen water and the anticancer drug fluorouracil (anticancer drug kit) enhances the anticancer effect on HCT116 cells derived from human colon cancer.

[0110] HCT116 cells were plated in a 12-well plate at 1.0 × 10 5 Cells were seeded at 1000 cells / well and cultured in DMEM at 37°C and 5% CO for 24 hours (preculture). After preculture, the medium was replaced with the various DMEMs listed below and cultured for 24 hours. Then, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 420 nm was measured. The results are shown in Figure 28.

[0111] In Figure 28, FW: DMEM (purified water medium) containing purified water diluted with purified water only as described in the above <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> to which a predetermined concentration (1, 5 or 10 μM) of fluorouracil was added; EHW: DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration of level 4 to which a predetermined concentration of fluorouracil was added.

[0112] From FIG. 28, it was confirmed that the combined use of electrolyzed hydrogen water and fluorouracil enhanced the cell proliferation inhibitory effect of fluorouracil.

[0113] Considering the results of (9-1) and (9-2) above, the two types of anticancer drug kits are expected to enhance the anticancer effects by suppressing the autophagy-activating action of anticancer drugs such as paclitaxel and fluorouracil using electrolyzed hydrogen water.

[0114] An example of the use of the present invention is an anticancer drug kit comprising an anticancer drug activity enhancer and an anticancer drug.

Claims

1. An anticancer drug kit comprising an anticancer drug and an anticancer effect enhancer having autophagy-inhibiting activity and containing molecular hydrogen as an anticancer drug effect enhancer, wherein the concentration of the molecular hydrogen relative to the standard active ingredient concentration (1 μM) of the anticancer drug is 105 ppb or more and 2160 ppb or less, the cancer cells to be treated are at least one selected from the group consisting of human cervical cancer-derived HeLa cells, human colon cancer-derived Caco-2 cells, and human colorectal cancer-derived HCT116 cells, in which autophagy is inhibited by the molecular hydrogen, the anticancer effect enhancer is electrolyzed hydrogen water, and the anticancer drug contains at least one active ingredient selected from the group consisting of doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosatinib, nilotenib, and fluorouracil, which have an autophagy-activating effect, The molecular hydrogen suppresses the autophagy activating action of the anticancer drug, thereby enhancing the anticancer effect of the anticancer drug.

2. The anticancer drug kit according to claim 1, characterized in that the cancer cells to which the anticancer drug is to be applied are colon cancer cells and the anticancer drug does not include fluorouracil as an active ingredient.

3. An anticancer drug kit comprising an anticancer drug and an anticancer effect enhancer having autophagy-inhibiting activity and containing molecular hydrogen as an anticancer drug effect enhancer, wherein the concentration of the molecular hydrogen relative to the standard active ingredient concentration (1 μM) of the anticancer drug is 105 ppb or more and 2160 ppb or less, the cancer cells to be treated are at least one selected from the group consisting of human cervical cancer-derived HeLa cells, human colon cancer-derived Caco-2 cells, and human colorectal cancer-derived HCT116 cells, in which autophagy is inhibited by the molecular hydrogen, the anticancer effect enhancer is electrolyzed hydrogen water, and the anticancer drug contains at least one active ingredient selected from the group consisting of doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosatinib, and nilotenib, which have an autophagy-activating effect, The molecular hydrogen suppresses the autophagy activating action of the anticancer drug, thereby enhancing the anticancer effect of the anticancer drug.

4. The anticancer drug kit according to any one of claims 1 to 3, characterized in that the concentration of the active ingredient of the anticancer drug is 0.5 μM or more and 10 μM or less.

5. The anticancer drug kit according to any one of claims 1 to 3, characterized in that the concentration of the active ingredient of the anticancer drug is 0.5 μM or more and 10 μM or less, and the concentration of the molecular hydrogen is 1056 ppb or more and 1080 ppb or less.

6. The anticancer drug kit according to any one of claims 1 to 3, characterized in that when the cancer cells to be treated are HeLa cells derived from human cervical cancer, the concentration of the active ingredient of the anticancer drug is 0.5 μM to 2 μM, when the cancer cells to be treated are Caco-2 cells derived from human colon cancer, the concentration of the active ingredient of the anticancer drug is 1 μM to 4 μM, and when the cancer cells to be treated are HCT116 cells derived from human colon cancer, the concentration of the active ingredient of the anticancer drug is 1 μM to 10 μM.

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