Anticancer drug kit

Abstract

The anticancer drug kit comprises an anticancer agent and an anticancer drug, wherein the anticancer agent has autophagy-inhibiting activity and contains molecular hydrogen as an anticancer agent activity-enhancing substance, and the cancer cells to be applied are at least one 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 molecular hydrogen, the anticancer agent is electrolyzed hydrogen water, and the anticancer drug contains an active ingredient having an autophagy-activating activity, and the anticancer effect of the anticancer drug is enhanced by the molecular hydrogen inhibiting the autophagy-activating activity of the anticancer drug.

Description

Technology Field

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

[0002] The applicant has explored various new uses and functionalities for various types of hydrogen water, such as electrolyzed hydrogen water, and mixtures containing the same. For example, Patent Document 1 proposes a hydrogen water mixture for inhibiting alcoholic liver damage, in which an ethanol solution and hydrogen water are mixed, the concentration of ethanol is 1 to 4%, and the dissolved hydrogen concentration is 550 to 5600 ppb. This hydrogen water mixture has high safety, is easy to manufacture and inexpensive, and can effectively inhibit alcoholic liver damage. Prior art literature

[0003] Patent Document 1: Japanese Patent Publication No. 2022-126128 The problem to be solved

[0004] However, conventionally, as a treatment for cancer, drug therapy (chemotherapy) using anticancer agents that act on the DNA of cancer cells to inhibit their proliferation and thereby exhibit an anticancer effect is being used.

[0005] Conventional anticancer drugs possess excellent anticancer efficacy but have severe side effects such as nausea, vomiting, and hair loss. Furthermore, increasing the dosage increases the financial burden on patients, which poses a problem in that continuous use of the drug may be impossible. Therefore, there is a desire to reduce the dosage of anticancer drugs, even slightly, to alleviate these side effects and financial burdens on patients.

[0006] Accordingly, the present invention is made in consideration of these points and aims to provide an anticancer drug kit capable of enhancing the anticancer action of an anticancer drug without increasing the dosage of the anticancer drug. means of solving the problem

[0007] As a result of careful consideration to achieve the above objective, the inventors discovered that electrolytic hydrogen water (molecular hydrogen dissolved therein) has an activity that inhibits autophagy, and that the anticancer action of an anticancer drug can be enhanced by utilizing said activity, and thus completed the anticancer drug kit of the present invention.

[0008] The anticancer drug kit of the present invention comprises an anticancer agent and an anticancer agent having autophagy inhibitory activity and containing molecular hydrogen as an anticancer agent enhancing substance, and

[0009] ㆍ The concentration of the molecular hydrogen relative to the standard active ingredient concentration (1μM) of the above anticancer drug is 105 ppb or more and 2160 ppb or less, and

[0010] The cancer cells to which the application applies are at least one type 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 above molecular hydrogen.

[0011] The above-mentioned anticancer agent is electrolyzed hydrogen water, and

[0012] The above anticancer agent contains, as an active ingredient, at least one selected from the group consisting of doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosutinib, nilotinib, and fluorouracil, which have an autophagy-activating effect.

[0013] ㆍ The above molecular hydrogen inhibits the autophagy activation action of the above anticancer agent, thereby enhancing the anticancer effect of the above anticancer agent. However, the anticancer agent kit of the present invention may exclude an anticancer agent kit in which the cancer cells to be applied are colon cancer cells and the above anticancer agent contains fluorouracil as an active ingredient.

[0014] The anticancer drug kit of the present invention comprises an anticancer agent and an anticancer agent having autophagy inhibitory activity and containing molecular hydrogen as an anticancer agent enhancing substance, and

[0015] ㆍ The concentration of the molecular hydrogen relative to the standard active ingredient concentration (1μM) of the above anticancer drug is 105 ppb or more and 2160 ppb or less, and

[0016] The cancer cells to which the application applies are at least one type 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 above molecular hydrogen.

[0017] The above-mentioned anticancer agent is electrolyzed hydrogen water, and

[0018] The above anticancer agent contains, as an active ingredient, at least one selected from the group consisting of doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosutinib, and nilotinib, which have an autophagy-activating effect.

[0019] ㆍ It is characterized by the fact that the above molecular hydrogen inhibits the autophagy activation action of the above anticancer drug, thereby enhancing the anticancer effect of the above anticancer drug. Effects of the invention

[0020] According to the present invention, an anticancer drug kit can be provided that can enhance the anticancer action of an anticancer drug without increasing the dosage of the anticancer drug. Brief explanation of the drawing

[0021] Figure 1 is a figure showing the expression levels of LC3 protein (LC3-II) in the presence or absence of Baf.A1 using a flux assay with bafilomycin A1 (Baf.A1) in mouse embryonic fibroblasts (MEF cells). Figure 2 is a diagram showing the difference between the expression level of LC3-II in the presence of Baf.A1 and the expression level of LC3-II in the absence of Baf.A1, using MEF cells. Figure 3 is a diagram showing the results of the analysis of the autophagy activity of electrolyzed hydrogen water by fluorescence microscopy observation using an mRFP-GFP-LC3 tandem fluorescent probe with MEF cells. Figure 4 is a diagram showing the results of the analysis of the autophagy activity of electrolyzed hydrogen water by the GFP / RFP ratio using a GFP-LC3-RFP-LC3ΔG probe using human cervical cancer cells (HeLa cells). Figure 5 is a diagram showing the results of the analysis of the autophagy activity of doxorubicin (an anticancer drug) based on the GFP / RFP ratio using a GFP-LC3-RFP-LC3ΔG probe with HeLa cells. Figure 6 is a diagram showing the verification results of the cell proliferation inhibitory effect of doxorubicin using HeLa cells. Figure 7 is a diagram showing the verification results of apoptosis induction by doxorubicin using HeLa cells. Figure 8 is a diagram showing the verification results of the cell proliferation inhibitory effect of doxorubicin when used in combination with electrolyzed hydrogen water using HeLa cells (ATG7 WT). Figure 9 is a diagram showing the verification results of the cell proliferation inhibitory effect of doxorubicin when combined with electrolyzed hydrogen water using HeLa cells in which ATG7 (autophagy-related gene) was deleted (ATG7 KO). Figure 10 is a diagram showing the verification results of apoptosis induction by doxorubicin when combined with electrolyzed hydrogen water using HeLa cells. Figure 11 is a diagram showing the results of the analysis of the autophagy activity of doxorubicin when combined with electrolyzed hydrogen water, based on the GFP / RFP ratio using a GFP-LC3-RFP-LC3ΔG probe using HeLa cells. Figure 12 is a diagram showing the results of verifying the cell proliferation inhibitory effect of doxorubicin in serum-free medium or a medium containing Baf.A1 using human colon cancer cells (Caco-2 cells). Figure 13 is a diagram showing the results of verifying the cell proliferation inhibitory effect of doxorubicin in purified water medium or electrolyzed hydrogen water medium using Caco-2 cells. Figure 14 is a diagram showing the verification results of the cell proliferation inhibitory effect of electrolyzed hydrogen water using Caco-2 cells. Figure 15 is a diagram showing the results of verifying the cell proliferation inhibitory effect of doxorubicin when combined with electrolyzed hydrogen water in serum-free medium or a medium containing Baf.A1 using Caco-2 cells. Figure 16 is a diagram showing the verification results of apoptosis induction by doxorubicin when combined with electrolyzed hydrogen water using Caco-2 cells. Figure 17 is a diagram showing the results of the analysis of the autophagy activity of doxorubicin when combined with electrolyzed hydrogen water, based on the GFP / RFP ratio using a GFP-LC3-RFP probe with Caco-2 cells. Figure 18 is a diagram showing the results of the analysis of the autophagy activity of various waters based on the GFP / RFP ratio using a GFP-LC3-RFP probe with Caco-2 cells. Figure 19 is a diagram showing the verification results of the cell proliferation inhibitory effect of doxorubicin when used in combination with various waters using Caco-2 cells. Figure 20 is a diagram showing the verification results of cell proliferation of doxorubicin when combined with generated hydrogen water using HeLa cells (ATG7 WT). Figure 21 is a diagram showing the verification results of cell proliferation of doxorubicin when combined with generated hydrogen water using HeLa cells with ATG7 deficiency (ATG7 KO). Figure 22 is a diagram showing the results of the analysis of the autophagy activity of electrolyzed hydrogen water by the GFP / RFP ratio using a GFP-LC3-RFP probe with human colon cancer cells (HCT116 cells). Figure 23 is a diagram showing the results of the analysis of the autophagy activity of paclitaxel (an anticancer drug) based on the GFP / RFP ratio using a GFP-LC3-RFP-LC3ΔG probe with HeLa cells. Figure 24 is a diagram showing the results of verifying the cell proliferation inhibitory effect of paclitaxel using HeLa cells. Figure 25 is a diagram showing the results of the analysis of the autophagy activity of fluorouracil (Fluorouracil, 5-FU, anticancer drug) by the GFP / RFP ratio using a GFP-LC3-RFP probe with HCT116 cells. Figure 26 is a diagram showing the verification results of the cell proliferation inhibitory effect of paclitaxel when used in combination with electrolyzed hydrogen water using HeLa cells (ATG7 WT). Figure 27 is a diagram showing the results of verifying the cell proliferation inhibitory effect of paclitaxel when combined with electrolyzed hydrogen water using HeLa cells in which ATG7 (autophagy-related gene) was deleted (ATG7 KO). Figure 28 is a diagram showing the verification results of the cell proliferation inhibitory effect of fluorouracil when used in combination with electrolyzed hydrogen water using HCT116 cells. Specific details for implementing the invention

[0022] The anticancer drug kit according to the present embodiment comprises an anticancer action enhancer and an anticancer drug. The types of cancer targeted by the anticancer drug kit are not particularly limited, but examples include cervical cancer, colon cancer, colorectal cancer, etc.

[0023] Anticancer Action Enhancer

[0024] An anticancer agent means enhancing the effect of an anticancer agent (anticancer effect) by enhancing the action pathway of the anticancer agent itself through the activity of the anticancer agent enhancing substance, or by acting synergistically through a pathway different from the action pathway of the anticancer agent. Here, the anticancer agent according to the present embodiment contains an anticancer agent enhancing substance having an activity that inhibits autophagy (hereinafter referred to as "autophagy inhibiting activity") based on the analysis and verification results of the examples described below.

[0025] Autophagy is an intracellular recycling system (a degradation system mediated by lysosomes) that purifies intracellular components and removes harmful substances by breaking them down, thereby playing a role in the prevention of various diseases, such as carcinogenesis. Furthermore, in addition to its role as a mechanism for the degradation of intracellular components, autophagy is known to function by regulating biological mechanisms through the secretion of these components. In particular, secretory autophagy (exophagy, the extracellular release of secretions) is known to be involved in the regulation of inflammation associated with various diseases and the exchange of metabolites.

[0026] The anticancer agent according to the present embodiment contains molecular hydrogen as an anticancer agent enhancing substance having autophagy inhibitory activity. In other words, molecular hydrogen can be described as an active molecule with autophagy inhibitory activity. Furthermore, the anticancer agent has an excellent cancer cell proliferation inhibitory effect, effectively inhibiting the proliferation of cancer cells by inhibiting autophagy through molecular hydrogen.

[0027] The form of an anticancer agent containing molecular hydrogen is not particularly limited, and examples 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.

[0028] Hydrogen-containing water is a biological application solution in which molecular hydrogen is dissolved. Examples of biological application solutions include water (purified water, purified water, sterile water, etc.), physiological saline solution, buffer solution (phosphate buffer, etc., preferably identical to the anticancer drug used), intravenous fluid (infusion solution, Ringer's solution, etc., may contain therapeutic drugs), injection solution, and beverage (tea beverages such as green tea and black tea, green juice, vegetable juice, fruit juice, etc.). Specific examples of hydrogen-containing water include electrolytic hydrogen water produced by performing electrolysis treatment on water; and generated hydrogen water produced by other methods.

[0029] The hydrogen-containing gas may contain molecular hydrogen and may include other gases (oxygen, nitrogen, inert gas, etc.) to the extent that it does not impede the purpose of the present invention. Specific examples of the hydrogen-containing gas include air containing molecular hydrogen, and a mixed gas containing molecular hydrogen and other gases.

[0030] Hydrogen-containing water and hydrogen-containing gas can generally be produced using commercially available hydrogen generation devices (such as electrolytic water generation devices).

[0031] Among the anticancer activity enhancers, hydrogen-containing water (electrolyzed hydrogen water and generated hydrogen water) is preferred, and electrolyzed hydrogen water, which is safe, easy to manufacture using commercially available equipment, and inexpensive, is more preferable.

[0032] The lower limit of the molecular hydrogen concentration of the anticancer drug kit is preferably 550 ppb or higher, more preferably 640 ppb or higher, even more preferably 688 ppb or higher, even more preferably 896 ppb or higher, and even more preferably 1056 ppb or higher. In addition, the upper limit is preferably 5600 ppb or lower, more preferably 4000 ppb or lower, even more preferably 2000 ppb or lower, even more preferably 1320 ppb or lower, and even more preferably 1080 ppb or lower. Examples of the molecular hydrogen concentration ranges include 550 ppb or higher and 5600 ppb or lower, 800 ppb or higher and 1320 ppb or lower, 640 ppb or higher and 1056 ppb or lower, and 1056 ppb or higher and 1080 ppb or lower. Furthermore, the molecular hydrogen concentration refers to the dissolved hydrogen concentration when the anticancer activity enhancer is hydrogen-containing water (electrolyzed hydrogen water, etc.).

[0033] It is preferable that the concentration of molecular hydrogen (dissolved hydrogen concentration) relative to the standard active ingredient concentration (1 μM) of the anticancer drug be 105 ppb or more and 2160 ppb or less.

[0034] Anticancer drugs

[0035] Although not specifically limited, it is desirable for an anticancer drug to contain an active ingredient that has an autophagy-activating effect (hereinafter referred to as "autophagy-activating effect") as a corresponding function of action, since the anticancer agent combined as an anticancer drug kit has autophagy-inhibiting activity. By co-administering an anticancer agent having autophagy-inhibiting activity and an anticancer drug having an autophagy-activating effect as an anticancer drug kit, the anticancer agent inhibits 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, and the patient's side effects and economic burden can be alleviated.

[0036] Examples of anticancer agents having an autophagy-activating effect include those containing doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosutinib, nilotinib, 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, bosutinib, nilotinib, and fluorouracil. That is, the anticancer agent may contain each of the above active ingredients individually, or may contain two or more of them.

[0037] In addition, anticancer agents having autophagy-activating effects are described, for example, in the following paper. The following paper does not describe matters related to hydrogen.

[0038] 1) Ponatinib: Diana Corallo, et al. Autophagic flux inhibition enhances cytotoxicity of the receptor tyrosine kinase inhibitor ponatinib. J Exp Clin Cancer Res. 2020.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 5) Dasatinib: Xiao-Feng Le, et al. Dasatinib induces autophagic cell death in human ovarian cancer. Cancer. 2010.

[0043] 6) Nilotinib: Hui-Chuan Yu, et al. Nilotinib induces autophagy in hepatocellular carcinoma through AMPK activation. J Biol Chem. 2013.

[0044] As previously mentioned, electrolyzed hydrogen water (molecular hydrogen dissolved therein) inhibits autophagy. On the other hand, anticancer drugs containing, for example, doxorubicin as an active ingredient, activate autophagy, as revealed by the analysis and verification results of the examples described below. Furthermore, the inventors hypothesized that for anticancer drugs possessing an autophagy-activating effect, the activation of autophagy might attenuate the anticancer effect. Upon further investigation, they discovered the potential for utilizing the physiological action of inhibiting autophagy by molecular hydrogen. The anticancer drug kit of the present invention was developed based on these findings, and by effectively inhibiting the autophagy-activating effect of the anticancer drug using the activity of molecular hydrogen, the anticancer effect is enhanced compared to conventional methods.

[0045] The concentration of the anticancer agent (relative to the total amount of components in the anticancer agent kit) in the anticancer agent kit (the total concentration in cases containing two or more types of active ingredients) is not particularly limited and may be any 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 human cervical cancer-derived HeLa cells, the concentration of the active ingredient of the anticancer agent is approximately 0.5 μM to 2 μM. In the case of human colon cancer-derived Caco-2 cells, the concentration of the active ingredient of the anticancer agent is approximately 1 μM to 4 μM. In the case of human colorectal cancer-derived HCT116 cells, the concentration of the active ingredient of the anticancer agent is approximately 1 μM to 10 μM.

[0046] Anticancer drug kit

[0047] The form of the anticancer drug kit is not specifically limited and can be appropriately determined based on the form of the anticancer agent and the anticancer drug. For example, an anticancer drug kit equipped with hydrogen-containing water (such as electrolyzed hydrogen water) as an anticancer agent may be in the form of a liquid in which the anticancer drug is dissolved in the hydrogen-containing water. Methods of administration for the liquid anticancer drug kit may include, for example, oral administration; injections such as intravenous drips; or parenteral administration such as subcutaneous or transdermal administration. On the other hand, methods of administration for an anticancer drug kit equipped with hydrogen-containing gas as an anticancer agent may include, for example, administering the hydrogen-containing gas perforated by inhalation, followed by oral or parenteral administration of the anticancer drug. Thus, depending on their forms, the anticancer agent and the anticancer drug may be administered by different methods (they may be administered separately). In this case, the timing of administration may be simultaneous or different.

[0048] The cancer cells to which the anticancer drug kit is applied are not particularly limited, provided that they are cancer cells in which autophagy is inhibited by molecular hydrogen, from the perspective of enhancing the anticancer effect compared to conventional methods by effectively inhibiting the autophagy-activating action of the anticancer drug using the autophagy-inhibiting activity of molecular hydrogen. 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.

[0049] Preferably, the anticancer drug kit has an active ingredient concentration of 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. In addition, the active ingredient concentration of the anticancer drug is not limited to the above range, and can be appropriately determined according to the active ingredient concentration applied to the treatment of target cancer cells as described above, for example, it may be 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.

[0050] In an anticancer drug kit configured as described above, the dosage of the anticancer drug can be reduced due to an enhancement effect that boosts the anticancer effect of the drug, and as a result, problems such as patient side effects and economic burden can be resolved all at once.

[0051] Examples

[0052] The present invention will be described below based on examples. Furthermore, the present invention is not limited to these examples, and modifications and changes to these examples are possible based on the spirit of the present invention, and these are not excluded from the scope of the present invention. In the following drawings, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0053] <1. Production of Electrolyzed Hydrogen Water>

[0054] Using an electrolytic water generator (manufactured by Nihon Trim Co., Ltd., product name: TRIMION GRACE), under conditions of a temperature of 22℃ and a flow rate of 1.5L / min, electrolytic hydrogen water of different levels 1 to 4 with dissolved hydrogen concentrations was obtained, along with purified water filtered by micro carbon.

[0055] Next, the pH and dissolved hydrogen concentration of the electrolytic hydrogen water at each of the above levels were measured. In addition, the pH was measured using a pH meter (manufactured by HORIBA, Ltd., product name: LAQUA act D-71), and the dissolved hydrogen concentration was measured using a dissolved hydrogen meter DH-35A (manufactured by DKK-TOA CORPORATION).

[0056] ㆍ Level 1: 800ppb, pH 7.

[0057] ㆍ Level 2: 860 ppb, pH 8.

[0058] ㆍ Level 3: 1120 ppb, pH 9.

[0059] ㆍ Level 4: 1320ppb (∼1350ppb), pH 10.

[0060] <Production of Medium for Electrolyzed Hydrogen Water Treatment (Electrolyzed Hydrogen Water Medium)>

[0061] 5× Dulbecco Modified Eagle Medium (hereinafter referred to as "DMEM") prepared from powder was diluted five-fold with the aforementioned purified water or electrolytic hydrogen water of each level and used as a treatment medium for electrolytic hydrogen water (i.e., a treatment medium consisting of a hydrogen water mixture of 20% 5× DMEM and 80% electrolytic hydrogen water or purified water). The dissolved hydrogen concentration in the electrolytic hydrogen water medium of each level is as follows.

[0062] ㆍ Level 1: 800ppb×4 / 5=640ppb.

[0063] ㆍ Level 2: 860 ppb × 4 / 5 = 688 ppb.

[0064] ㆍ Level 3: 1120 ppb × 4 / 5 = 896 ppb.

[0065] ㆍ Level 4: 1320 ppb(∼1350 ppb)×4 / 5=1056 ppb(∼1080 ppb).

[0066] <2. Interpretation of Autophagy Inhibition in Electrolyzed Hydrogen Water (Interpretation from a Multidimensional Approach)>

[0067] Whether electrolyzed hydrogen water inhibits autophagy was interpreted from a multifaceted approach.

[0068] (2-1) Flux assay by bafilomycin A1

[0069] Mouse embryonic fibroblasts (hereinafter referred to as "MEF cells") were placed in a 6-well plate at a volume of 5.0 × 10⁶ 5 Cells were seeded into wells and cultured in DMEM at 37°C and 5% CO2 for 24 hours (hereinafter also referred to as "pre-culture"). After pre-culture, the medium was changed to various DMEMs as described below and cultured for 4 hours in the presence or absence of 200 nM Bafilomycin A1 (hereinafter referred to as "Baf.A1," an inhibitor). Subsequently, the treated cells were lysed with lysis buffer, and the lysate was Western blotted to analyze the expression level of LC3-II using a specific antibody for LC3 protein (hereinafter referred to as "LC3-II"). The results are shown in Figure 1.

[0070] Here, the expression level of LC3-II shown in Fig. 1 is correlated with the number of autophagosomes. However, the expression level of LC3-II at any given time does not represent the actual flux amount, and it is impossible to determine whether it activates or inhibits autophagy. Therefore, the expression level of LC3-II sent to lysosomes was measured by comparing the expression levels of LC3-II based on the presence or absence of Baf.A1. Specifically, the autophagy flux was analyzed by calculating the difference between the expression level of LC3-II in the presence of Baf.A1 and the expression level of LC3-II in the absence of Baf.A1. The results are shown in Fig. 2.

[0071] And, in FIGS. 1 and 2,

[0072] ㆍ MQ: In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM (ultrapure water medium) containing ultrapure water diluted only with ultrapure water,

[0073] ㆍ FW: In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM (water medium) containing purified water diluted only with purified water,

[0074] ㆍ LV1-LV4: DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with dissolved hydrogen concentration levels 1 to 4,

[0075] ㆍ +Baf.A1: Represents a medium (a medium containing Baf.A1) in which 200 nM of Baf.A1 is dissolved in the various DMEMs above.

[0076] In Figure 1, it was confirmed that in the absence of Baf.A1, the expression of LC3-II in electrolytic hydrogen water (LV1-LV4) was higher compared to ultrapure water (MQ) and purified water (FW), and increased in proportion to the dissolved hydrogen concentration. On the other hand, in the presence of Baf.A1 (+Baf.A1), it was confirmed that the expression of LC3-II in electrolytic hydrogen water was equivalent to that of ultrapure water and purified water at any dissolved hydrogen concentration.

[0077] In Figure 2, when comparing the difference between the expression levels of LC3-II in the presence of Baf.A1 and the expression levels of LC3-II in the absence of Baf.A1, it was found that electrolytic hydrogen water (LV1-LV4) is lower than ultrapure water (MQ) and purified water (FW), and decreases as the dissolved hydrogen concentration increases. From these results, it is thought that electrolytic hydrogen water inhibits degradation by autolysosomes because LC3-II accumulates as the dissolved hydrogen concentration increases (because the amount of degradation in autophagy decreases).

[0078] (2-2) Fluorescence microscopy using mRFP-GFP-LC3 tandem fluorescent probe

[0079] MEF cells expressing the mRFP-GFP-LC3 tandem fluorescent probe (hereinafter referred to as the "tf-LC3 probe") were cultured for 4 hours after changing the medium to the various DMEMs shown below. Subsequently, the MEF cells were immobilized using 2% paraformaldehyde. Intracellular fluorescence was observed using a confocal laser scanning microscope, and the number of autolysosomes was measured. The results are shown in Figure 3.

[0080] And, in Fig. 3,

[0081] ㆍ FW: In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM (water medium) containing purified water diluted only with purified water,

[0082] ㆍ EHW LV4: Represents DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4.

[0083] Furthermore, the tf-LC3 probe is a circular DNA (plasmid) containing a gene that produces a fluorescent protein capable of monitoring autophagy, as described in the following paper, for example. An MEF cell expressing the tf-LC3 probe refers to a cell that produces (expresses) a fluorescent protein capable of monitoring autophagy by delivering the above plasmid into the cell and inserting (genetically modifying) the probe gene into the genome of the MEF cell. In addition, the tf-LC3 probe utilizes the difference in sensitivity to acidity; while GFP fluorescence is quenched under acidic conditions (pH < 5.0), mRFP fluorescence is maintained relatively stably within the lysosome. In other words, the transition from the autophagosome to the acidic autolysosome can be visualized by the tf-LC3 probe.

[0084] 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.

[0085] In Figure 3, it was confirmed that the number of autolysosomes decreased in electrolytic hydrogen water (EHW LV4). From this result, it is thought that electrolytic hydrogen water inhibits degradation by autolysosomes.

[0086] (2-3) Analysis of Autophagy Activity by GFP / RFP Ratio Using GFP-LC3-RFP-LC3ΔG Probe

[0087] Human cervical cancer cells expressing the GFP-LC3-RFP-LC3ΔG probe (hereinafter referred to as "HeLa cells") were placed in a 12-well plate at a density of 1.0 x 10 5 Cells were seeded per well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was changed to the various DMEMs shown below and cultured for 4 hours. Subsequently, the cells were exfoliated and suspended, and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. Autophagy activity was quantified by determining the GFP / RFP ratio. The results are shown in Figure 4.

[0088] And, in Fig. 4,

[0089] ㆍ FW: In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM (water medium) containing purified water diluted only with purified water,

[0090] ㆍ LV1-LV4: DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with dissolved hydrogen concentration levels 1 to 4,

[0091] ㆍ T: A medium in which 1 μM Torin 1 is dissolved in DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 (water purification medium containing Torin 1),

[0092] ㆍ Baf.: Represents a medium (water purification medium containing Baf.A1) in which 200 nM of Baf.A1 is dissolved in DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4.

[0093] Furthermore, the GFP-LC3-RFP-LC3ΔG probe (or the GFP-LC3-RFP probe described below) is described, for example, in the following paper. The GFP-LC3-RFP-LC3ΔG probe (or GFP-LC3-RFP probe) is cleaved by ATG4, generating equal numbers of GFP-LC3 and RFP-LC3ΔG (or RFP) in the cytoplasm. GFP-LC3 is localized in the autophagosome and subsequently quenched and degraded within the autolysosome. RFP-LC3ΔG (or RFP) remains in the cytoplasm and functions as an internal control reflecting the amount of GFP-LC3. Additionally, the GFP / RFP ratio is obtained by quantifying the cumulative degradation of GFP-LC3 by 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.

[0094] 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.

[0095] In Figure 4, in human cervical cancer-derived HeLa cells, as the dissolved hydrogen concentration in electrolytic hydrogen water (LV1-LV4) increased, GFP accumulated relatively, and the GFP / RFP ratio increased, so electrolytic hydrogen water inhibited autophagy.

[0096] (2-4) Summary

[0097] From the results of the analysis from the multifaceted approach of (2-1) to (2-3) above, it is thought that electrolytic hydrogen water has an activity that inhibits autophagy, that is, an activity that inhibits autophagy (autophagy inhibition activity).

[0098] <3. Interpretation of Autophagy Activation by the Anticancer Drug Doxorubicin and Verification of Anticancer Effects>

[0099] We verified whether the anticancer drug doxorubicin activates autophagy and whether the activation of autophagy affects its anticancer effect.

[0100] (3-1) Analysis of Doxorubicin Autophagy Activity by GFP / RFP Ratio Using GFP-LC3-RFP-LC3ΔG Probe

[0101] HeLa cells expressing the GFP-LC3-RFP-LC3ΔG probe in a 12-well plate at a volume of 1.0×10 5Cells were seeded into wells and cultured in DMEM at 37°C and 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was changed to DMEM containing an anticancer agent containing doxorubicin (Doxorubicin, Sigma-Aldrich Co., LLC.) at a predetermined concentration (1, 2, or 4 μM), and cultured for 4 hours. Subsequently, cells were exfoliated and suspended in the same manner as in (2-3), and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. Autophagy activity was quantified by determining the GFP / RFP ratio. The results are shown in Figure 5.

[0102] In Figure 5, as the concentration of doxorubicin increases, the GFP / RFP ratio decreases, contrary to electrolytic hydrogen water (see Figure 4), so it is thought that doxorubicin has an autophagy-activating effect (autophagy-activating effect). From this result, it is predicted that autophagy activation by doxorubicin will attenuate the anticancer effect.

[0103] (3-2) Verification of the cell proliferation inhibitory effect

[0104] HeLa cells (ATG7 WT) or HeLa cells depleted of ATG7 (autophagy-related gene) (ATG7 KO) were placed in a 96-well plate at a density of 1.0 x 10 4 Cells were seeded per well and cultured (pre-culture) in DMEM at 37°C and 5% CO2 for 24 hours. After pre-culture, the medium was changed to DMEM containing an anticancer agent with a predetermined concentration (1, 2, or 4 μM) of doxorubicin, and cultured for 24 hours. Subsequently, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, incubated at 37°C for 2 hours, and the absorbance at 420 nm was measured. The results are shown in Figure 6.

[0105] (3-3) Verification of Apoptosis Induction

[0106] Apoptosis induction was evaluated according to the manual using Cell Death Detection ELISAPLUS (Roche Diagnostics, Basel, Switzerland). HeLa cells (ATG7 WT) or HeLa cells depleted of ATG7 (ATG7 KO) were placed in a 12-well plate at a density of 1.0 x 10⁻¹⁰ 5 Cells were seeded per well and cultured (pre-culture) in DMEM at 37°C and 5% CO2 for 24 hours. After pre-culture, the medium was changed to DMEM containing an anticancer agent with a predetermined concentration (1 or 2 μM) of doxorubicin, and cultured for 24 hours. Subsequently, the cells were lysed with lysis buffer, the lysate was added to each well of an ELISA plate, incubated for 2 hours with an immunoreagent buffer containing anti-histone biotin and anti-DNA POD, and washed with incubation buffer. Afterward, the cells were reacted with 100 μL of ABTS substrate buffer, and absorbance was measured at 405 nm and 490 nm. The results are shown in Figure 7.

[0107] In Figures 6 and 7, from a comparison of ATG7 WT and ATG7 KO, it was confirmed that the anticancer effect of doxorubicin (cell proliferation inhibition effect and apoptosis induction) was enhanced in proportion to the concentration due to the deletion of ATG7 (autophagy-related gene).

[0108] (3-4) Summary

[0109] Based on the results of (3-1) to (3-3) above, for anticancer agents such as doxorubicin that have an autophagy-activating effect, an enhancement of the anticancer effect is expected by combining them with electrolytic hydrogen water that inhibits autophagy.

[0110] <4. Verification of Enhancement of Anticancer Effect by Combination of Electrolyzed Hydrogen Water and Anticancer Drug Doxorubicin 1>

[0111] We verified whether the combination of electrolyzed hydrogen water and the anticancer drug doxorubicin (anticancer drug kit) enhances the anticancer effect against human cervical cancer-derived HeLa cells.

[0112] (4-1) Verification of the cell proliferation inhibitory effect

[0113] HeLa cells (ATG7 WT) or HeLa cells depleted of ATG7 (autophagy-related gene) (ATG7 KO) in a 96-well plate at a density of 1.0 × 10⁶ 4 Cells were seeded per well and cultured (pre-culture) in DMEM at 37°C and 5% CO2 for 24 hours. After pre-culture, the medium was changed to the various DMEMs shown below and cultured for 24 hours. Subsequently, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, incubated at 37°C for 2 hours, and the absorbance at 420 nm was measured. The results are shown in Figures 8 (ATG7 WT) and 9 (ATG7 KO).

[0114] And, in FIGS. 8 and 9,

[0115] ㆍ FW: In the above <Preparation of a medium for treating electrolytic hydrogen water (electrolytic hydrogen water medium)>, a DMEM (water medium) containing purified water diluted only with purified water, to which doxorubicin at a predetermined concentration (0.5, 1, or 2 μM) is added.

[0116] ㆍ EHW: Indicates that a predetermined concentration of doxorubicin has been added to DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4.

[0117] In Fig. 8 (ATG7 WT), it was confirmed that the combined use of electrolyzed hydrogen water and doxorubicin enhanced the cell proliferation inhibitory effect of doxorubicin. On the other hand, in Fig. 9 (ATG7 KO), it was confirmed that the enhancement effect of the combined use was lost due to the deletion of ATG7 (autophagy-related gene).

[0118] (4-2) Verification of Apoptosis Induction

[0119] Apoptosis induction was evaluated according to the manual using Cell Death Detection ELISAPLUS (Roche Diagnostics, Basel, Switzerland). HeLa cells (ATG7 WT) or HeLa cells depleted of ATG7 (autophagy-related gene) (ATG7 KO) were placed in a 12-well plate at a density of 1.0 × 10⁶ 5 Cells were seeded per well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was changed to the various DMEMs shown below and cultured for 24 hours. Subsequently, the cells were lysed with lysis buffer, the lysate was added to each well of an ELISA plate, incubated for 2 hours with an immunoreagent buffer containing anti-histone biotin and anti-DNA POD, and washed with incubation buffer. Afterward, the cells were reacted with 100 μL of ABTS substrate buffer, and absorbance was measured at 405 nm and 490 nm. The results are shown in Figure 10.

[0120] And, in Fig. 10,

[0121] ㆍ FW: In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM (water medium) containing purified water diluted only with purified water, with no addition (-) or 1 μM addition (+) of doxorubicin,

[0122] ㆍ EHW: Indicates that doxorubicin is added without (-) or with 1 μM (+) to DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4.

[0123] From the ATG7 WT shown in Fig. 10, it was confirmed that the combined use of electrolyzed hydrogen water and doxorubicin enhances the apoptosis-inducing effect of doxorubicin. On the other hand, from the ATG7 KO shown in Fig. 10, it was confirmed that the enhancement effect of the combined use is lost due to the deletion of ATG7 (autophagy-related gene).

[0124] (4-3) Interpretation of Autophagy Activation

[0125] HeLa cells (ATG7 WT) expressing the GFP-LC3-RFP-LC3ΔG probe were placed in a 12-well plate at a depth of 1.0×10 5 Cells were seeded into wells and cultured in DMEM at 37°C and 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was changed to various DMEMs as described below and cultured for 4 hours. Subsequently, cells were exfoliated and suspended in the same manner as in (2-3) above, and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. Autophagy activity was quantified by determining the GFP / RFP ratio. The results are shown in Fig. 11.

[0126] And, in Fig. 11,

[0127] ㆍ FW: In the above <Preparation of a medium for treating electrolytic hydrogen water (electrolytic hydrogen water medium)>, 1 μM of doxorubicin is added to DMEM (water medium) containing purified water, which is diluted only with purified water.

[0128] ㆍ EHW: Indicates that 1 μM of doxorubicin has been added to DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4.

[0129] In Figure 11, since the GFP / RFP ratio increases due to the combined use of electrolyzed hydrogen water and doxorubicin, electrolyzed hydrogen water inhibits the activation of autophagy by doxorubicin.

[0130] (4-4) Summary

[0131] Based on the results of (4-1) to (4-3) above, electrolytic hydrogen water is expected to enhance the anticancer effect in human cervical cancer cells (HeLa cells) by inhibiting the autophagy-activating action of anticancer agents such as doxorubicin.

[0132] <5. Verification of Enhancement of Anticancer Effect by Combination Use of Electrolyzed Hydrogen Water and Anticancer Drug Doxorubicin 2>

[0133] We verified whether the combination of electrolyzed hydrogen water and the anticancer drug doxorubicin (anticancer drug kit) enhances the anticancer effect against human colon cancer-derived Caco-2 cells.

[0134] (5-1) Verification of the cell proliferation inhibitory effect

[0135] Human colon cancer cells (hereinafter referred to as "Caco-2 cells") were placed in a 96-well plate at a volume of 2.0 × 10⁶ 4 Cells were seeded per well and cultured (pre-culture) in DMEM at 37°C under 5% CO2 conditions for 24 hours. After pre-culture, the medium was changed to the various DMEMs shown below and cultured for 24 hours. Subsequently, 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 to measure the absorbance at 420 nm. The results are shown in Figures 12–15.

[0136] In Fig. 12,

[0137] ㆍ DOX+serum free: Doxorubicin at a specified concentration (1, 2, or 4 μM) added to serum-free DMEM (serum-free medium),

[0138] ㆍ DOX: DMEM containing serum to which a predetermined concentration of doxorubicin has been added,

[0139] ㆍ DOX+Baf.: This indicates adding a predetermined concentration of doxorubicin to a medium (a medium containing Baf.A1) in which 200 nM of Bafilomycin A1 (Baf.A1) is dissolved in DMEM containing serum.

[0140] In Fig. 13,

[0141] ㆍ FW: In the above <Preparation of a medium for treating electrolytic hydrogen water (electrolytic hydrogen water medium)>, a DMEM (water medium) containing purified water diluted only with purified water, to which doxorubicin at a predetermined concentration (1, 2, or 4 μM) is added.

[0142] ㆍ EHW: Indicates that a predetermined concentration of doxorubicin has been added to DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4.

[0143] In FIG. 14,

[0144] ㆍ FW: In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM (water medium) containing purified water diluted only with purified water,

[0145] ㆍ EHW: Represents DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4.

[0146] In Fig. 15,

[0147] ㆍ FW(Medium+Baf.A1): A medium prepared by dissolving 200 nM of Baf.A1 in DMEM containing purified water and serum according to the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)> (a purified water medium containing Baf.A1) with no addition (-) or 2 μM addition (+) of doxorubicin,

[0148] ㆍ EHW(Medium+Baf.A1): Likewise, a medium (electrolyzed hydrogen water medium containing Baf.A1) in which 200 nM of Baf.A1 is dissolved in DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 and serum, with no doxorubicin added (-) or 2 μM added (+).

[0149] ㆍ FW (Serum-free medium): Likewise, serum-free DMEM (serum-free purified water medium) containing purified water with no addition (-) or 2 μM addition (+) of doxorubicin,

[0150] ㆍ EHW (Serum-free medium): Likewise, this refers to serum-free DMEM (serum-free electrolytic hydrogen water medium) containing electrolytic hydrogen water with a dissolved hydrogen concentration level of 4, with no doxorubicin added (-) or 2 μM added (+).

[0151] In Fig. 12, it was confirmed that the cell proliferation inhibitory effect of doxorubicin was enhanced in combination with an autophagy inhibitor (Baf.A1) (DOX+Baf.). On the other hand, it was confirmed that the cell proliferation inhibitory effect of doxorubicin was reduced under autophagy activation conditions (DOX+serum free). In Fig. 13, it was confirmed that the cell proliferation inhibitory effect was enhanced by the combination of electrolytic hydrogen water and doxorubicin (EHW). Whether this enhancement effect is due to the autophagy inhibitory activity of electrolytic hydrogen water will be further verified below.

[0152] In Figures 14 and 15, under the combined use of doxorubicin and an autophagy inhibitor (Baf.A1) ("Medium+Baf.A1", under autophagy inhibition conditions), when comparing purified water (FW) and electrolytic hydrogen water (EHW), there was no significant difference between the two, confirming that the cell proliferation inhibitory effect does not change even when electrolytic hydrogen water is used in combination. On the other hand, under autophagy activation conditions in a serum-free medium not containing doxorubicin and Baf.A1, when comparing purified water (FW) and electrolytic hydrogen water (EHW), it was confirmed that the cell proliferation inhibitory effect was enhanced by using electrolytic hydrogen water in combination. Based on these results, it is thought that the autophagy inhibitory activity of electrolytic hydrogen water is involved in the enhancement of the effect of doxorubicin.

[0153] (5-2) Verification of Apoptosis Induction

[0154] Apoptosis induction was evaluated according to the manual using Cell Death Detection ELISAPLUS (Roche Diagnostics, Basel, Switzerland). Caco-2 cells were deposited in a 12-well plate at a density of 2.0 × 10⁶ 5 Cells were seeded per well and cultured (pre-culture) in DMEM at 37°C and 5% CO2 for 24 hours. After pre-culture, the medium was changed to the various DMEMs shown below and cultured for 24 hours. Subsequently, the cells were lysed with lysis buffer, the lysate was added to each well of an ELISA plate, incubated for 2 hours with an immunoreagent buffer containing anti-histone biotin and anti-DNA POD, and washed with incubation buffer. Afterward, the cells were reacted with 100 μL of ABTS substrate buffer, and absorbance was measured at 405 nm and 490 nm. The results are shown in Figure 16.

[0155] And, in Fig. 16,

[0156] ㆍ FW: In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM (water medium) containing purified water diluted only with purified water, with no addition (-) or 2 μM addition (+) of doxorubicin,

[0157] ㆍ EHW: Indicates that doxorubicin is added without (-) or 2 μM (+) to DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4.

[0158] In Figure 16, it was confirmed that the apoptosis-inducing effect is enhanced by the combined use of doxorubicin and electrolytic hydrogen water (EHW).

[0159] (5-3) Interpretation of Autophagy Activation

[0160] 1.0 × 10⁶ Caco-2 cells expressing the GFP-LC3-RFP probe in a 12-well plate 5Cells were seeded into wells and cultured in DMEM at 37°C and 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was changed to various DMEMs as described below and cultured for 4 hours. Subsequently, cells were exfoliated and suspended in the same manner as in (2-3) above, and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. Autophagy activity was quantified by determining the GFP / RFP ratio. The results are shown in Fig. 17.

[0161] And, in Fig. 17,

[0162] ㆍ FW: In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM (water medium) containing purified water diluted only with purified water,

[0163] ㆍ EHW: DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4,

[0164] ㆍ +DOX: Indicates that 2 μM of doxorubicin was added to each of the above DMEMs.

[0165] In Figure 17, it was confirmed that electrolytic hydrogen water inhibits the autophagy activation of doxorubicin, as the GFP / RFP ratio increases with the combination of electrolytic hydrogen water and the anticancer drug doxorubicin (EHW+DOX).

[0166] (5-4) Summary

[0167] Based on the results of (5-1) to (5-3) above, it is expected that in human colon cancer cells (Caco-2 cells), as in human cervical cancer cells (HeLa cells), electrolytic hydrogen water will enhance the anticancer effect by inhibiting the autophagy-activating action of anticancer agents such as doxorubicin.

[0168] <6. Verification of substances contributing to the enhancement effect of electrolyzed hydrogen water>

[0169] The active molecules responsible for the above effects of electrolyzed hydrogen water were verified.

[0170] (6-1) Interpretation of Autophagy Activation

[0171] 1.0 × 10⁶ Caco-2 cells expressing the GFP-LC3-RFPΔG probe in a 12-well plate 5 Cells were seeded into wells and cultured in DMEM at 37°C and 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was changed to various DMEMs as described below and cultured for 4 hours. Subsequently, cells were exfoliated and suspended in the same manner as in (2-3) above, and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. Autophagy activity was quantified by determining the GFP / RFP ratio. The results are shown in Fig. 18.

[0172] And, in Fig. 18,

[0173] ㆍ FW: In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM (water medium) containing purified water diluted only with purified water,

[0174] ㆍ EHW: DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4,

[0175] ㆍ EHW(AC): In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM (Autoclave Water Medium) containing autoclave-treated water, diluted with only water obtained after two autoclave treatments of electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4,

[0176] ㆍ HW: In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, represents DMEM (generated hydrogen water medium) containing hydrogen equivalent to dissolved hydrogen concentration level 4, diluted solely with generated hydrogen water produced using the kit of TRIM SEVEN WATER (manufactured by Nihon Trim Co., Ltd.), and

[0177] ㆍ The difference between the different signs (a and b) indicates a statistically significant difference (p<0.05). Also, in the two-cycle autoclave treatment, one cycle was performed for 20 minutes at high pressure and a high temperature of 121°C, and this was repeated twice.

[0178] In FIG. 18, it was confirmed that electrolytic hydrogen water (EHW) and generated hydrogen water (HW) inhibit autophagy activity equally, as their GFP / RFP ratios are equivalent. On the other hand, when electrolytic hydrogen water is autoclaved twice (EHW(AC)), the GFP / RFP ratio becomes equivalent to that of purified water (FW), so it is thought that the autophagy-inhibiting activity is lost. From these results, it is assumed that the autophagy-inhibiting component in electrolytic hydrogen water is volatile, such as molecular hydrogen that is degassed by autoclaving.

[0179] (6-2) Verification of the cell proliferation inhibitory effect

[0180] 1.0 × 10⁶ Caco-2 cells in a 96-well plate 4 Cells were seeded per well and cultured (pre-culture) in DMEM at 37°C and 5% CO2 for 24 hours. After pre-culture, the medium was changed to the various DMEMs shown below and cultured for 24 hours. Subsequently, 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 to measure the absorbance at 420 nm. The results are shown in Fig. 19.

[0181] And, in Fig. 19,

[0182] ㆍ FW: In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM (water medium) containing purified water diluted only with purified water, with no addition (-) or 2 μM addition (+) of doxorubicin,

[0183] ㆍ EHW: DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4, with no (-) or 2 μM of doxorubicin added (+).

[0184] ㆍ EHW(AC): DMEM (autoclave water medium) containing the above autoclave-treated water with no (-) or 2 μM added (+) doxorubicin,

[0185] ㆍ HW: Indicates that doxorubicin is added without (-) or at 2 μM (+) to DMEM (generated hydrogen water medium) containing hydrogen equivalent to dissolved hydrogen concentration level 4, and

[0186] ㆍ There is a statistically significant difference (p<0.05) between the different signs (a and b).

[0187] In FIG. 19, it was confirmed that the cell proliferation inhibitory effect of doxorubicin is equally enhanced by electrolytic hydrogen water (EHW) and generated hydrogen water (HW). On the other hand, it is thought that the above enhancement effect is lost when electrolytic hydrogen water is autoclaved twice (EHW(AC)).

[0188] (6-3) Verification of cell proliferation

[0189] HeLa cells (ATG7 WT) or HeLa cells depleted of ATG7 (autophagy-related gene) (ATG7 KO) in a 96-well plate at a density of 1.0 × 10⁶ 4 Cells were seeded per well and cultured (pre-culture) in DMEM at 37°C and 5% CO2 for 24 hours. After pre-culture, the medium was changed to the various DMEMs shown below and cultured for 24 hours. Subsequently, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well and incubated at 37°C for 2 hours, after which the absorbance at 420 nm was measured. The results are shown in Figures 20 and 21.

[0190] And, in FIGS. 20 and 21,

[0191] ㆍ HW: DMEM (generated hydrogen water medium) containing generated hydrogen water with a dissolved hydrogen concentration of 0 ppm to 4 ppm,

[0192] ㆍ HW+DOX: Indicates that 0.5 μM of doxorubicin has been added to the above DMEM (generated hydrogen water medium). For example, if Sample 1 is a, Sample 2 is ab, and Sample 3 is b, it indicates that there is no significant difference between Sample 1 and Sample 2, and between Sample 2 and Sample 3, and that there is a significant difference between Sample 1 and Sample 3.

[0193] In Fig. 20 (ATG7 WT), it was confirmed that in combination with the generated hydrogen water and the anticancer drug doxorubicin (HW+DOX), cell proliferation is inhibited as the dissolved hydrogen concentration of the generated hydrogen water increases, thereby enhancing the cell proliferation inhibitory effect of doxorubicin. On the other hand, in Fig. 21 (ATG7 KO), it was confirmed that due to the deletion of ATG7 (autophagy-related gene), the cell proliferation inhibitory effect of doxorubicin is not obtained by the combination with the generated hydrogen water and the anticancer drug doxorubicin.

[0194] (6-4) Summary

[0195] Based on the results of (6-1) to (6-3) above, it was confirmed that the substance (active molecule) contributing to the enhancement of the anticancer effect (cell proliferation inhibition effect and apoptosis induction) of doxorubicin by electrolyzed hydrogen water is dissolved molecular hydrogen, and that the proliferation of cancer cells is inhibited by the inhibition of autophagy by molecular hydrogen.

[0196] <7. Interpretation of Autophagy Activation by GFP / RFP Ratio Using Different Cells>

[0197] Human colorectal cancer cells expressing the GFP-LC3-RFP probe (hereinafter referred to as "HCT116 cells") were placed in a 12-well plate at a volume of 1.0 × 10⁶ 5Cells were seeded into wells and cultured in DMEM at 37°C and 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was changed to various DMEMs as described below and cultured for 4 hours. Subsequently, cells were exfoliated and suspended in the same manner as in (2-3) above, and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. Autophagy activity was quantified by determining the GFP / RFP ratio. The results are shown in Fig. 22.

[0198] And, in Fig. 22,

[0199] ㆍ FW: In the above <Preparation of Medium for Treatment of Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM (water medium) containing purified water diluted only with purified water,

[0200] ㆍ EHW: Represents DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4.

[0201] In Fig. 22, in human colorectal cancer-derived HCT116 cells, just as in human cervical cancer-derived HeLa cells (Fig. 4), the GFP / RFP ratio increased in electrolyzed hydrogen water (EHW) compared to purified water (FW), so electrolyzed hydrogen water inhibited autophagy.

[0202] <8. Interpretation of Autophagy Activation by Other Anticancer Drugs and Verification of Anticancer Effects>

[0203] We verified whether paclitaxel or fluorouracil, as other anticancer agents, activate autophagy and whether the activation of autophagy affects the anticancer effect.

[0204] (8-1) Analysis of Paclitaxel Autophagy Activity by GFP / RFP Ratio Using GFP-LC3-RFP-LC3ΔG Probe

[0205] HeLa cells expressing the GFP-LC3-RFP-LC3ΔG probe in a 12-well plate at a volume of 1.0×10 5Cells were seeded into wells and cultured (pre-culture) in DMEM at 37°C and 5% CO2 for 24 hours. After pre-culture, the medium was changed to DMEM containing an anticancer agent (Paclitaxel, Sigma-Aldrich Co., LLC.) at a specified concentration (1, 5, or 10 nM), and cultured for 24 hours. Subsequently, cells were exfoliated and suspended in the same manner as in (2-3), and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. Autophagy activity was quantified by determining the GFP / RFP ratio. The results are shown in Fig. 23.

[0206] In Fig. 23, paclitaxel, like doxorubicin (Fig. 5), is thought to have an autophagy-activating effect (autophagy activation effect) because the GFP / RFP ratio decreases as the concentration increases, contrary to electrolytic hydrogen water (see Figs. 4, Fig. 22, etc.). From this result, it is predicted that autophagy activation with paclitaxel, like doxorubicin, will attenuate the anticancer effect.

[0207] (8-2) Verification of the cell proliferation inhibitory effect

[0208] HeLa cells (ATG7 WT) or HeLa cells depleted of ATG7 (autophagy-related gene) (ATG7 KO) in a 96-well plate at a density of 1.0 × 10⁶ 4 Cells were seeded per well and cultured (pre-culture) in DMEM at 37°C and 5% CO2 for 24 hours. After pre-culture, the medium was changed to DMEM containing an anticancer agent with paclitaxel at a specified concentration (1, 10, or 50 nM), and cultured for 24 hours. Subsequently, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, incubated at 37°C for 2 hours, and the absorbance at 420 nm was measured. The results are shown in Figure 24.

[0209] In Figure 24, from a comparison of ATG7 WT and ATG7 KO, it was confirmed that the anticancer effect (cell proliferation inhibitory effect) of paclitaxel is enhanced in proportion to the concentration due to the deletion of ATG7 (autophagy-related gene).

[0210] (8-3) Analysis of the autophagic activity of fluorouracil based on the GFP / RFP ratio using the GFP-LC3-RFP probe

[0211] HCT116 cells expressing the GFP-LC3-RFP probe in a 12-well plate at a density of 1.0 × 10 5 Cells were seeded into wells and cultured in DMEM at 37°C and 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was changed to DMEM containing an anticancer agent containing fluorouracil (Fluorouracil, 5-FU, Sigma-Aldrich Co., LLC.) at a predetermined concentration (1 or 10 μM), and cultured for 24 hours. Subsequently, cells were exfoliated and suspended in the same manner as in (2-3), and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. Autophagy activity was quantified by determining the GFP / RFP ratio. The results are shown in Fig. 25.

[0212] In Fig. 25, fluorouracil, like doxorubicin (Fig. 5), is thought to have an autophagy-activating effect (autophagy activation effect) because the GFP / RFP ratio decreases as the concentration increases, contrary to electrolytic hydrogen water (see Fig. 22). From this result, it is predicted that fluorouracil, like doxorubicin, will attenuate the anticancer effect through autophagy activation.

[0213] <9. Verification of Enhancement of Anticancer Effect by Combination Use of Electrolyzed Hydrogen Water with Anticancer Drugs Paclitaxel or Fluorouracil>

[0214] (9-1) Verification of Cell Proliferation Inhibitory Effect 1

[0215] We verified whether the combination of electrolyzed hydrogen water and the anticancer drug paclitaxel (anticancer drug kit) enhances the anticancer effect against human cervical cancer-derived HeLa cells.

[0216] HeLa cells (ATG7 WT) or HeLa cells depleted of ATG7 (autophagy-related gene) (ATG7 KO) in a 96-well plate at a density of 1.0 × 10⁶ 4 Cells were seeded per well and cultured (pre-culture) in DMEM at 37°C and 5% CO2 for 24 hours. After pre-culture, the medium was changed to the various DMEMs shown below and cultured for 24 hours. Subsequently, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, incubated at 37°C for 2 hours, and the absorbance at 420 nm was measured. The results are shown in Figures 26 (ATG7 WT) and 27 (ATG7 KO).

[0217] And, in FIGS. 26 and 27,

[0218] ㆍ FW: In the above <Preparation of a medium for treating electrolytic hydrogen water (electrolytic hydrogen water medium)>, paclitaxel at a predetermined concentration (1, 10, or 50 nM) is added to DMEM (water medium) containing purified water, which is diluted only with purified water.

[0219] ㆍ EHW: Indicates that a predetermined concentration of paclitaxel has been added to DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4.

[0220] In Fig. 26 (ATG7 WT), it was confirmed that the combination of electrolyzed hydrogen water and paclitaxel enhances the cell proliferation inhibitory effect of paclitaxel. On the other hand, in Fig. 27 (ATG7 KO), it was confirmed that the enhancement effect of the combination was lost due to the deletion of ATG7 (autophagy-related gene).

[0221] (9-2) Verification of Cell Proliferation Inhibitory Effect 2

[0222] We verified whether the combination of electrolyzed hydrogen water and the anticancer drug fluorouracil (anticancer drug kit) enhances the anticancer effect on human colorectal cancer-derived HCT116 cells.

[0223] 1.0 × 10⁶ HCT116 cells in a 12-well plate 5 Cells were seeded per well and cultured (pre-culture) in DMEM at 37°C and 5% CO2 for 24 hours. After pre-culture, the medium was changed to the various DMEMs shown below and cultured for 24 hours. Subsequently, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well, incubated at 37°C for 2 hours, and the absorbance at 420 nm was measured. The results are shown in Figure 28.

[0224] And, in Fig. 28,

[0225] ㆍ FW: In the above <Preparation of a medium for treating electrolytic hydrogen water (electrolytic hydrogen water medium)>, a DMEM (water medium) containing purified water diluted only with purified water, to which fluorouracil at a predetermined concentration (1, 5, or 10 μM) is added.

[0226] ㆍ EHW: Indicates that a predetermined concentration of fluorouracil has been added to DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4.

[0227] In Fig. 28, it was confirmed that the combined use of electrolyzed hydrogen water and fluorouracil enhances the cell proliferation inhibitory effect of fluorouracil.

[0228] Based on the results of (9-1) to (9-2) above, the two types of anticancer drug kits are expected to enhance the anticancer effect by inhibiting the autophagy activation of anticancer drugs such as paclitaxel or fluorouracil by electrolyzed hydrogen water.

[0229] [Industrial Applicability]

[0230] An example of application of the present invention is an anticancer drug kit comprising an anticancer drug activity-enhancing substance and an anticancer drug.

Claims

Claim 1 An anticancer drug kit comprising an anticancer agent and an anticancer agent, wherein the anticancer agent has autophagy-inhibiting activity and contains molecular hydrogen as an anticancer agent enhancing substance, wherein the concentration of the molecular hydrogen relative to the standard active ingredient concentration (1 μM) of the anticancer agent is 105 ppb or more and 2160 ppb or less, and the cancer cells to be applied 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, wherein the anticancer agent is electrolyzed hydrogen water, and the anticancer agent contains as an active ingredient at least one selected from the group consisting of doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosutinib, nilotinib, and fluorouracil having an autophagy-activating effect, and wherein the molecular hydrogen inhibits the autophagy-activating effect of the anticancer agent, thereby enhancing the anticancer effect of the anticancer agent. Anticancer drug kit. Claim 2 An anticancer drug kit according to claim 1, provided that the cancer cells to which the above-mentioned target is applied are colon cancer cells, and furthermore, the anticancer drug kit containing fluorouracil as an active ingredient. Claim 3 An anticancer drug kit comprising an anticancer agent and an anticancer agent, wherein the concentration of the molecular hydrogen is 105 ppb or higher and 2160 ppb or lower relative to the standard active ingredient concentration (1 μM) of the anticancer agent, and the cancer cells to be applied 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, wherein autophagy is inhibited by the molecular hydrogen; the anticancer agent is electrolyzed hydrogen water; and the anticancer agent contains as an active ingredient at least one selected from the group consisting of doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosutinib, and nilotinib, which have an autophagy-activating effect, and the anticancer agent is characterized by enhancing the anticancer effect of the anticancer agent by the molecular hydrogen inhibiting the autophagy-activating effect of the anticancer agent. Claim 4 An anticancer drug kit characterized in that, in any one of claims 1 to 3, the concentration of the active ingredient of the anticancer drug is 0.5 μM or more and 10 μM or less. Claim 5 An 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. Claim 6 An anticancer drug kit according to any one of claims 1 to 3, wherein, when the cancer cells to be applied are human cervical cancer-derived HeLa cells, the concentration of the active ingredient of the anticancer drug is 0.5μM to 2μM; when the cancer cells to be applied are human colon cancer-derived Caco-2 cells, the concentration of the active ingredient of the anticancer drug is 1μM to 4μM; and when the cancer cells to be applied are human colorectal cancer-derived HCT116 cells, the concentration of the active ingredient of the anticancer drug is 1μM to 10μM.

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