Anticancer agent kit

Abstract

To provide an anticancer agent kit capable of enhancing the anticancer effect of an anticancer agent without increasing the dose of the anticancer agent.SOLUTION: An anticancer agent kit includes an anticancer action enhancer having autophagy inhibitory activity and containing molecular hydrogen as an anticancer agent action enhancing substance, and an anticancer agent.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] The applicant of the present application has been variously examining new uses and functions of various hydrogen waters such as electrolyzed hydrogen water and mixtures containing the same. For example, Patent Document 1 proposes a hydrogen water mixture for suppressing alcoholic liver injury 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 is highly safe, easy and inexpensive to prepare, and can effectively suppress alcoholic liver injury.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] By the way, conventionally, as a cancer treatment method, a drug therapy (chemotherapy) using an anticancer agent that acts on the DNA of cancer cells and exhibits an anticancer effect by suppressing the growth of cancer cells has been used.

[0005] Among conventional anticancer agents, although some have excellent anticancer effects, some have strong side effects such as nausea, vomiting, and hair loss. In addition, when the dosage of the anticancer agent increases, the economic burden on the patient also increases. Therefore, there is a problem that the continuous use of the anticancer agent may not be possible. In order to reduce the side effects and economic burden on the patient, there is a desire to reduce the dosage of the anticancer agent as much as possible.

[0006] Therefore, in view of such circumstances, the present invention has been made, and an object thereof is to provide an anticancer agent kit capable of enhancing the anticancer effect of an anticancer agent without increasing the dosage of the anticancer agent.

Means for Solving the Problems

[0007] As a result of intensive studies to achieve the above object, the present inventors have found that electrolyzed hydrogen water (molecular hydrogen dissolved therein) has an activity of suppressing autophagy, and that the anticancer effect of an anticancer agent can be enhanced by utilizing this activity, leading to the completion of the anticancer agent kit of the present invention.

[0008] The anticancer agent kit of the present invention is characterized by comprising an anticancer effect enhancer having autophagy inhibitory activity and containing molecular hydrogen as an anticancer agent effect enhancer substance, and an anticancer agent.

Effects of the Invention

[0009] According to the present invention, it is possible to provide an anticancer agent kit capable of enhancing the anticancer effect of an anticancer agent without increasing the dosage of the anticancer agent.

Brief Description of the Drawings

[0010]

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Mode for Carrying Out the Invention

[0011] 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, and examples include cervical cancer, colon cancer, colorectal cancer, etc.

[0012] <Anticancer effect enhancer> The anticancer effect enhancer means a substance that enhances the effect (anticancer effect) of an anticancer drug by enhancing the action pathway of the anticancer drug itself due to the activity of an anticancer drug action enhancing substance, or by acting synergistically through a pathway different from the action pathway of the anticancer drug. Here, the anticancer effect enhancer according to this embodiment contains an anticancer drug action enhancing substance having an activity to suppress autophagy (hereinafter referred to as "autophagy inhibitory activity") based on the analysis and verification results of the following examples.

[0013] Autophagy is an intracellular recycling system (a degradation system by lysosomes), which purifies intracellular components and removes harmful substances by degrading intracellular components, and plays a role in preventing various diseases such as cancer. In addition to its role as a mechanism for degrading intracellular components, autophagy is also known to function in regulating biological mechanisms through the secretion of intracellular components. In particular, secretory autophagy (exophagy, extracellular release of secretions) has been shown to be involved in the regulation of inflammation related to various diseases and the exchange of metabolites.

[0014] The anti-cancer action enhancer according to this embodiment contains molecular hydrogen as a substance that enhances the action of an anti-cancer agent having autophagy inhibitory activity. In other words, molecular hydrogen can be said to be an acting molecule with autophagy inhibitory activity. And the anti-cancer action enhancer has an excellent cancer cell growth inhibitory effect that the cancer cell growth is effectively suppressed by molecular hydrogen suppressing autophagy.

[0015] The form of the anti-cancer action enhancer 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"), and the like.

[0016] Hydrogen-containing water is a biologically applicable solution in which molecular hydrogen is dissolved. Examples of the biologically applicable solution include water (purified water, refined water, sterilized water, etc.), physiological saline, buffer solutions (such as phosphate buffer solutions, preferably the same as the anti-cancer agent to be used), infusion solutions (drip solutions, Ringer's solutions, etc., which may contain therapeutic drugs), injection solutions, beverages (tea beverages such as green tea and black tea, green juice, vegetable juice, fruit juice, etc.). Specific examples of hydrogen-containing water include electrolyzed hydrogen water generated by performing electrolysis treatment on water; generated hydrogen water produced by other methods.

[0017] The hydrogen-containing gas only needs to contain molecular hydrogen and may contain other gases (such as oxygen, nitrogen, inert gas, etc.) as long as it does not inhibit the object of the invention. Specific examples of the hydrogen-containing gas include air containing molecular hydrogen, a mixed gas containing molecular hydrogen and other gases, and the like.

[0018] The hydrogen-containing water and the hydrogen-containing gas can generally be produced using a commercially available hydrogen generation device (such as an electrolytic water generation device).

[0019] Among the anti-cancer effect enhancers, hydrogen-containing water (electrolyzed hydrogen water and generated hydrogen water) is preferred, and electrolyzed hydrogen water, which has high safety and can be easily prepared at low cost with a commercially available device, is more preferred.

[0020] The lower limit of the concentration of molecular hydrogen in the anti-cancer agent kit is preferably 550 ppb or more, more preferably 640 ppb or more, even more preferably 688 ppb or more, still 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, still more preferably 1320 ppb or less, and even more preferably 1080 ppb or less. Examples of the concentration range of molecular hydrogen include 550 ppb or more and 5600 ppb or less, 800 ppb or more and 1320 ppb or less, 640 ppb or more and 1056 ppb or less, 1056 ppb or more and 1080 ppb or less, and the like. It should be noted that when the anti-cancer effect enhancer is hydrogen-containing water (such as electrolyzed hydrogen water), the concentration of molecular hydrogen means the dissolved hydrogen concentration.

[0021] The concentration (dissolved hydrogen concentration) of molecular hydrogen relative to the standard active ingredient concentration (1 μM) of the anti-cancer agent is preferably 105 ppb or more and 2160 ppb or less.

[0022] <Anti-cancer agent> The anticancer agent is not particularly limited, but since the anticancer effect enhancer to be combined as an anticancer agent kit has autophagy inhibitory activity, it preferably contains an active ingredient having an action of activating autophagy (hereinafter referred to as "autophagy activation action") as an action function corresponding to the autophagy inhibitory activity. As an anticancer agent kit, by co-administering an anticancer effect enhancer having autophagy inhibitory activity and an anticancer agent having autophagy activation action, the anticancer effect enhancer suppresses the autophagy activation action of the anticancer agent, so that it becomes possible to effectively enhance the anticancer effect. As a result, it is possible to reduce the dosage of the anticancer agent and reduce the side effects and economic burden on the patient.

[0023] Examples of the anticancer agent having autophagy activation action include those containing, as an active ingredient, doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosatanib, nilotinib, fluorouracil, etc. 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, bosatanib, nilotinib, and fluorouracil. That is, the anticancer agent may contain the above active ingredients alone or in combination of two or more.

[0024] Note that the anticancer agent having autophagy activation action is described in, for example, the following papers. The following papers do not describe matters 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.。

[0025] As described above, electrolyzed hydrogen water (and molecular hydrogen dissolved therein) suppresses autophagy. On the other hand, for example, an anticancer agent containing doxorubicin or the like as an active ingredient activates autophagy, as shown by the analysis and verification results of the examples described below. Then, the present inventors hypothesized that autophagy activation by an anticancer agent having an autophagy activation effect might attenuate the anticancer effect, and as a result of further investigation, they found the possibility of utilizing the physiological effect of molecular hydrogen's autophagy suppression. The anticancer agent kit of the present invention was completed based on these findings, and by effectively suppressing the autophagy activation effect of the anticancer agent by utilizing the activity of molecular hydrogen, the anticancer effect is enhanced compared to the prior art.

[0026] The concentration of the anticancer agent (total concentration when containing two or more active ingredients) in the anticancer agent kit (relative to the total amount of the components of the anticancer agent kit) is not particularly limited, and examples 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 agent is about 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 agent is about 1 μM to 4 μM. In the case of HCT116 cells derived from human colorectal cancer, the concentration of the active ingredient of the anticancer agent is about 1 μM to 10 μM.

[0027] <Anticancer Agent Kit> The form of the anticancer agent kit is not particularly limited and may be appropriately determined according to the forms of the anticancer effect enhancer and the anticancer agent. For example, an anticancer agent kit comprising 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 agent is dissolved in the hydrogen-containing water. Examples of the administration method of the liquid anticancer agent kit include oral administration; methods of parenteral administration such as injection by drip or subcutaneous or transdermal administration. On the other hand, examples of the administration method of an anticancer agent kit comprising hydrogen-containing gas as the anticancer effect enhancer include, for example, transpulmonary administration by inhaling or aspirating the hydrogen-containing gas, and administering the anticancer agent orally or parenterally. Thus, the anticancer effect enhancer and the anticancer agent may have different administration methods according to their forms (they may be administered separately). In this case, the timing of administration may be simultaneous or different.

[0028] As for the cancer cells to which the anticancer agent kit is applied, from the viewpoint of effectively suppressing the autophagy activation effect of the anticancer agent by utilizing the autophagy inhibitory activity of molecular hydrogen and enhancing the anticancer effect more than before, there is no particular limitation as long as the autophagy is suppressed by molecular hydrogen in the cancer cells. In other words, for such cancer cells, the anticancer agent kit can be effectively applied and is not particularly limited to the cancer cells described in the examples.

[0029] It is preferable that the concentration of the active ingredient of the anticancer agent in the anticancer agent kit is 0.5 μM or more and 10 μM or less, and the concentration of molecular hydrogen in the anticancer agent kit (total amount of components) is 1056 ppb or more and 1080 ppb or less. Note that the concentration of the active ingredient of the anticancer agent is not limited to the above range, and as described above, it may be appropriately determined according to the concentration of the active ingredient applied to the treatment of the target cancer cells. 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.

[0030] In the anticancer agent kit configured as described above, due to the enhancing effect of enhancing the anticancer effect of the anticancer agent, the dosage of the anticancer agent can be reduced, and as a result, problems such as side effects and economic burden on patients can be solved all at once.

Example

[0031] The present invention will be described based on examples below. Note that the present invention is not limited to these examples, and these examples can be modified and changed based on the gist of the present invention, and they are not excluded from the scope of the present invention. In the following figures, *: p < 0.05, **: p < 0.01, ***: p < 0.001 are shown.

[0032] <1. Production of electrolyzed hydrogen water> Using an electrolyzed water generator (manufactured by Nippon Trim Co., Ltd., product name: TRIMION GRACE), under the conditions of a temperature of 22°C and a flow rate of 1.5 L / min, electrolyzed hydrogen water at different levels 1 to 4 with different dissolved hydrogen concentrations was obtained, and purified water filtered by microcarbon was obtained.

[0033] Next, the pH and dissolved hydrogen concentration of the electrolyzed hydrogen water at each of the above levels were measured. For the measurement of pH, a pH meter (manufactured by HORIBA, product name: LAQUA act D-71) was used, and for the measurement of the dissolved hydrogen concentration, a dissolved hydrogen meter DH-35A (manufactured by Toa DKK Corporation) was used. · Level 1: 800 ppb, pH 7. · Level 2: 860 ppb, pH 8. · Level 3: 1120 ppb, pH 9. · Level 4: 1320 ppb (~1350 ppb), pH 10.

[0034] <Preparation of culture medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water culture medium)> Powder-adjusted 5× Dulbecco's modified Eagle's medium (hereinafter referred to as "DMEM") was diluted 5-fold with the above purified water or electrolyzed hydrogen water at each level, and used as a culture medium for treating electrolyzed hydrogen water (that is, a hydrogen water mixture obtained by mixing 20% of 5× DMEM and 80% of electrolyzed hydrogen water or purified water). The dissolved hydrogen concentration in the electrolyzed hydrogen water culture medium at each level is as follows. · Level 1: 800 ppb × 4 / 5 = 640 ppb. · Level 2: 860 ppb × 4 / 5 = 688 ppb. · Level 3: 1120 ppb × 4 / 5 = 896 ppb. · Level 4: 1320 ppb (~1350 ppb) × 4 / 5 = 1056 ppb (~1080 ppb).

[0035] <2. Analysis of Autophagy Inhibition by Electrolyzed Hydrogen Water (Analysis from a Multifaceted Approach)> Analysis was performed from a multifaceted approach to determine whether electrolyzed hydrogen water inhibits autophagy.

[0036] (2-1) Flux Assay with Bafilomycin A1 Mouse embryonic fibroblasts (hereinafter referred to as "MEF cells") were seeded in a 6-well plate at 5.0×10 5 cells / well 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 shown below in the presence or absence of 200 nM Bafilomycin A1 (hereinafter referred to as "Baf.A1", an inhibitor), and the cells were cultured for 4 hours. Thereafter, the treated cells were lysed with lysis buffer, and the lysate was subjected to Western blotting to analyze the expression level of LC3 protein (hereinafter referred to as "LC3-II") with a specific antibody against LC3-II. The results are shown in Figure 1.

[0037] 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 certain time point does not indicate the actual flux amount, and it is not known whether autophagy is activated or inhibited. Therefore, by comparing the expression levels of LC3-II with and without Baf.A1, the expression level of LC3-II sent to lysosomes was measured. 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 Figure 2.

[0038] In Figures 1 and 2, · MQ: In the above <Preparation of Medium for Treating Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM containing ultrapure water (ultrapure water medium) diluted only with ultrapure water, · FW: In the above <Preparation of Medium for Treating Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM containing purified water (purified water medium) diluted only with purified water, · LV1 - LV4: DMEM containing electrolyzed hydrogen water with dissolved hydrogen concentration levels 1 - 4 (electrolyzed hydrogen water medium), · + Baf.A1: Indicates a medium in which 200 nM of Baf.A1 is dissolved in each of the above various DMEMs (medium containing Baf.A1).

[0039] From Figure 1, in the absence of Baf.A1, electrolyzed hydrogen water (LV1 - LV4) had a higher expression level of LC3-II compared to ultrapure water (MQ) and purified water (FW), and it was confirmed that it increased in proportion to the dissolved hydrogen concentration. On the other hand, in the presence of Baf.A1 (+ Baf.A1), it was confirmed that electrolyzed hydrogen water had an LC3-II expression level equivalent to that of ultrapure water and purified water at any dissolved hydrogen concentration.

[0040] From Figure 2, when comparing the difference between the LC3-II expression level in the presence of Baf.A1 and that in the absence of Baf.A1, it was found that electrolyzed hydrogen water (LV1 - LV4) was lower than ultrapure water (MQ) and purified water (FW), and it decreased as the dissolved hydrogen concentration increased. From this result, it is considered that electrolyzed hydrogen water suppresses the degradation by autolysosomes because LC3-II accumulates (the degradation amount in autophagy decreases) as the dissolved hydrogen concentration increases.

[0041] (2 - 2) Fluorescence Microscopy Observation Using mRFP - GFP - LC3 Tandem Fluorescent Probe In MEF cells expressing the mRFP-GFP-LC3 tandem fluorescent probe (hereinafter referred to as "tf-LC3 probe"), the medium was changed to various types of DMEM shown below and cultured for 4 hours. Then, the MEF cells were fixed using 2% paraformaldehyde. Fluorescence within the cells was observed with a confocal laser scanning microscope, and the number of autolysosomes was measured. The results are shown in Figure 3.

[0042] In Figure 3, · FW: DMEM containing purified water (purified water medium) diluted only with purified water in the above <Preparation of culture medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)>. · EHW LV4: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 (electrolyzed hydrogen water medium).

[0043] The tf-LC3 probe is a circular DNA (plasmid) having a gene that produces a fluorescent protein capable of monitoring autophagy, and is described in, for example, the following paper. MEF cells expressing the tf-LC3 probe refer to cells that can produce (express) a fluorescent protein capable of monitoring autophagy by introducing the above plasmid into the cell and integrating the probe gene into the genome of the MEF cell (performing gene recombination). In addition, the tf-LC3 probe utilizes the difference in sensitivity to acidity. While GFP fluorescence quenches under acidic conditions (pH < 5.0), mRFP fluorescence is relatively stable and retained even within lysosomes. In other words, the tf-LC3 probe enables 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.

[0044] As shown in Fig. 3, it was confirmed that the number of autolysosomes decreased in electrolyzed hydrogen water (EHW LV4). From this result, it is considered that electrolyzed hydrogen water suppresses the degradation by autolysosomes.

[0045] (2-3) Analysis of autophagy activity by GFP / RFP ratio using GFP-LC3-RFP-LC3ΔG probe Human cervical cancer cells (hereinafter referred to as "HeLa cells") expressing GFP-LC3-RFP-LC3ΔG probe were seeded in a 12-well plate at 1.0×10 5 cells / well and cultured in DMEM at 37 °C under 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was replaced with various DMEMs shown below and cultured for 4 hours. Then, the cells were detached and suspended, and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. By obtaining the GFP / RFP ratio, quantitative analysis of autophagy activity was performed. The results are shown in Fig. 4.

[0046] In Fig. 4, · FW: DMEM containing purified water (purified water medium) diluted only with purified water in the above <Preparation of culture medium for treatment with electrolyzed hydrogen water (electrolyzed hydrogen water medium)>, · LV1-LV4: DMEM containing electrolyzed hydrogen water with dissolved hydrogen concentration levels 1 to 4 of electrolyzed hydrogen water (electrolyzed hydrogen water medium), · T: Medium in which 1 μM of Torin 1 was dissolved in DMEM containing electrolyzed hydrogen water with dissolved hydrogen concentration level 4 of electrolyzed hydrogen water (electrolyzed hydrogen water medium) (purified water medium containing Torin 1), · Baf.: Medium in which 200 nM of Baf.A1 was dissolved in DMEM containing electrolyzed hydrogen water with dissolved hydrogen concentration level 4 of electrolyzed hydrogen water (electrolyzed hydrogen water medium) (purified water medium containing Baf.A1) is shown.

[0047] Note that the GFP-LC3-RFP-LC3ΔG probe (or the GFP-LC3-RFP probe described below) is described in, for example, the following paper. The GFP-LC3-RFP-LC3ΔG probe (or GFP-LC3-RFP probe) is cleaved by ATG4, generating the same number of GFP-LC3 and RFP-LC3ΔG (or RFP) in the cytoplasm. GFP-LC3 localizes to autophagosomes and is then quenched and degraded within autolysosomes. RFP-LC3ΔG (or RFP) remains in the cytoplasm and functions as an internal control reflecting the amount of GFP-LC3. Also, the GFP / RFP ratio can be obtained by quantifying the cumulative degradation amount 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 suppression. 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.

[0048] As shown in Figure 4, in HeLa cells derived from human cervical cancer, as the dissolved hydrogen concentration increased in electrolyzed hydrogen water (LV1-LV4), GFP accumulated relatively and the GFP / RFP ratio increased. Therefore, electrolyzed hydrogen water suppressed autophagy.

[0049] (2-4) Summary From the results analyzed by the above comprehensive approach (2-1) to (2-3), it is considered that electrolyzed hydrogen water suppresses autophagy, that is, it has an activity to suppress autophagy (autophagy suppression activity).

[0050] <Analysis of autophagy activation by the anticancer drug doxorubicin and verification of its anticancer effect> Verification was carried out on whether the anticancer drug doxorubicin activates autophagy and whether the activation of autophagy affects the anticancer effect.

[0051] (3-1) Analysis of the autophagy activity of doxorubicin by the GFP / RFP ratio using the GFP-LC3-RFP-LC3ΔG probe HeLa cells expressing the GFP-LC3-RFP-LC3ΔG probe were seeded in a 12-well plate at 1.0×10 5 cells / well and cultured in DMEM at 37 °C under 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was replaced with DMEM containing an anticancer agent with a predetermined concentration (1, 2, or 4 μM) of doxorubicin (Doxorubicin, Sigma-Aldrich Co., LLC.), and the cells were cultured for 4 hours. Then, in the same manner as in (2-3) above, the cells were detached and suspended, and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. Quantitative analysis of autophagy activity was performed by determining the GFP / RFP ratio. The results are shown in Figure 5.

[0052] From Figure 5, it can be considered that doxorubicin has an effect of activating autophagy (autophagy activation effect) because the GFP / RFP ratio decreases as the concentration increases, which is opposite to that of electrolyzed hydrogen water (see Figure 4). From this result, it is predicted that autophagy activation attenuates the anticancer effect in doxorubicin.

[0053] (3-2) Verification of the cell growth inhibitory effect HeLa cells (ATG7 WT) or cells with ATG7 (autophagy-related gene) deficiency in HeLa cells (ATG7 KO) were seeded in a 96-well plate at 1.0×10 4Cells were seeded at cells / well and pre-cultured in DMEM at 37 °C under 5% CO2 for 24 hours. After pre-culture, the medium was replaced with DMEM containing doxorubicin at a predetermined 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, incubated at 37 °C for 2 hours, and the absorbance at 420 nm was measured. The results are shown in Fig. 6.

[0054] (3-3) Verification of apoptosis induction Apoptosis induction was evaluated using Cell Death Detection ELISAPLUS (Roche Diagnostics, Basel, Switzerland) according to the manual. HeLa cells (ATG7 WT) or cells with ATG7 deficiency in HeLa cells (ATG7 KO) were seeded at 1.0×10 5 cells / well in a 12-well plate and pre-cultured in DMEM at 37 °C under 5% CO2 for 24 hours. After pre-culture, the medium was replaced with DMEM containing doxorubicin at a predetermined concentration (1 or 2 μM), and the cells were cultured for 24 hours. Then, the cells were lysed with lysis buffer, the lysate was added to each well of the ELISA plate, incubated with an immunoreagent buffer containing anti-histone biotin and anti-DNA POD for 2 hours, and washed with incubation buffer. Then, it was reacted with 100 μL of ABTS substrate buffer, and the absorbance was measured at 405 nm and 490 nm. The results are shown in Fig. 7.

[0055] In Figs. 6 and 7, comparison between ATG7 WT and ATG7 KO confirmed that the anti-cancer effect (cell growth inhibitory effect and apoptosis induction) of doxorubicin was enhanced proportionally to the concentration by the deficiency of ATG7 (autophagy-related gene).

[0056] (3-4) Summary Considering the above results of (3-1) to (3-3), for anticancer agents such as doxorubicin having autophagy activation effect, enhancement of the anticancer effect is expected by combination use with electrolyzed hydrogen water that suppresses autophagy.

[0057] <4. Verification of Enhancement of Anticancer Effect by Combined Use of Electrolyzed Hydrogen Water and Anticancer Agent Doxorubicin 1> Regarding the combined use of electrolyzed hydrogen water and anticancer agent doxorubicin (anticancer agent kit), verification was carried out on whether it enhances the anticancer effect on HeLa cells derived from human cervical cancer.

[0058] (4-1) Verification of Cell Growth Inhibitory Effect HeLa cells (ATG7 WT) or cells with deletion of ATG7 (autophagy-related gene) in HeLa cells (ATG7 KO) were seeded in a 96-well plate at 1.0×10 4 cells / well and cultured for 24 hours (pre-culture) at 37°C and 5% CO2 in DMEM. After pre-culture, the medium was changed to various DMEMs shown below and cultured for 24 hours. Then, 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 Fig. 8 (ATG7 WT) and Fig. 9 (ATG7 KO).

[0059] In Figs. 8 and 9, ·FW: DMEM containing purified water (purified water medium) diluted only with purified water in the above <Preparation of Medium for Treatment with Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)> with doxorubicin at a predetermined concentration ( 0 .5, 1 or 2 μM) added, ·EHW: Indicates DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 (electrolyzed hydrogen water medium) with a predetermined concentration of doxorubicin added.

[0060] From Figure 8 (ATG7 WT), it was confirmed that the combined use of electrolyzed hydrogen water and doxorubicin enhanced the inhibitory effect of doxorubicin on cell proliferation. On the other hand, from Figure 9 (ATG7 KO), it was confirmed that the deletion of ATG7 (autophagy-related gene) eliminated the enhancing effect of the above combination.

[0061] (4-2) Verification of apoptosis induction Apoptosis induction was evaluated manually using Cell Death Detection ELISAPLUS (Roche Diagnostics, Basel, Switzerland). HeLa cells (ATG7 WT) or cells with deletion of ATG7 (autophagy-related gene) in HeLa cells (ATG7 KO) were seeded in a 12-well plate at 1.0×10 5 cells / well and cultured in DMEM at 37 °C under 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was changed to various DMEMs shown below and cultured for 24 hours. Then, the cells were lysed with lysis buffer, the lysate was added to each well of the ELISA plate, incubated with an immunoreagent buffer containing anti-histone biotin and anti-DNA POD for 2 hours, and washed with incubation buffer. Then, it was 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.

[0062] In Figure 10, · FW: In the <Preparation of medium for treatment with electrolyzed hydrogen water (electrolyzed hydrogen water medium)> above, DMEM containing purified water (purified water medium) diluted only with purified water, with doxorubicin added at 0 μM (-) or 1 μM (+), · EHW: Indicates DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 (electrolyzed hydrogen water medium) with doxorubicin added at 0 μM (-) or 1 μM (+).

[0063] Compared with ATG7 WT shown in Fig. 10, it was confirmed that the combined use of electrolyzed hydrogen water and doxorubicin enhanced the apoptosis-inducing effect of doxorubicin. On the other hand, compared with ATG7 KO shown in Fig. 10, it was confirmed that the enhancement effect by the above combination disappeared due to the deficiency of ATG7 (autophagy-related gene).

[0064] (4-3) Analysis of autophagy activity HeLa cells (ATG7 WT) expressing GFP-LC3-RFP-LC3ΔG probe were seeded in a 12-well plate at 1.0×10 5 cells / well 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 shown below and cultured for 4 hours. Then, in the same manner as in (2-3) above, the cells were detached and suspended, and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. Quantitative analysis of autophagy activity was performed by calculating the GFP / RFP ratio. The results are shown in Fig. 11.

[0065] In Fig. 11, · FW: DMEM containing purified water (purified water medium) diluted only with purified water in the above <Preparation of medium for treatment with electrolyzed hydrogen water (electrolyzed hydrogen water medium)> with 1 μM doxorubicin added, · EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 (electrolyzed hydrogen water medium) with 1 μM doxorubicin added.

[0066] From Fig. 11, since the GFP / RFP ratio increases by the combined use of electrolyzed hydrogen water and doxorubicin, electrolyzed hydrogen water suppresses the activation of autophagy by doxorubicin.

[0067] (4-4) Summary Considering the results of the above (4-1) to (4-3), in human cervical cancer cells (HeLa cells), electrolyzed hydrogen water is expected to enhance the anti-cancer effect by suppressing the autophagy activation effect of anti-cancer drugs such as doxorubicin.

[0068] <5. Verification of enhanced anti-cancer effect by combination of electrolyzed hydrogen water and anti-cancer drug doxorubicin 2> The combination of electrolyzed hydrogen water and anti-cancer drug doxorubicin (anti-cancer drug kit) was verified for whether it enhances the anti-cancer effect on Caco-2 cells derived from human colon cancer.

[0069] (5-1) Verification of cell growth inhibitory effect Human colon cancer cells (hereinafter referred to as "Caco-2 cells") were seeded in a 96-well plate at 2.0×10 4 cells / well and cultured in DMEM at 37°C under 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was changed to various DMEMs shown below and cultured for 24 hours. Then, 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 12 to 15.

[0070] In Figure 12, ·DOX + serum free: A medium obtained by adding doxorubicin at a predetermined concentration (1, 2, or 4 μM) to serum-free DMEM (serum-free medium), ·DOX: A medium obtained by adding doxorubicin at a predetermined concentration to DMEM containing serum, ·DOX + Baf.: A medium obtained by adding doxorubicin at a predetermined concentration to a medium (medium containing Baf.A1) in which 200 nM of Bafilomycin A1 (Baf. A1) is dissolved in DMEM containing serum.

[0071] In Figure 13, ·FW: A medium obtained by adding doxorubicin at a predetermined concentration (1, 2, or 4 μM) to DMEM containing purified water (purified water medium) diluted only with purified water in the above <Preparation of medium for treatment with electrolyzed hydrogen water (electrolyzed hydrogen water medium)>, ·EHW: A medium obtained by adding doxorubicin at a predetermined concentration to DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 (electrolyzed hydrogen water medium).

[0072] In Figure 14, ·FW: In the above <Preparation of Medium for Treating Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, DMEM containing only purified water (purified water medium) diluted with only purified water, ·EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 (electrolyzed hydrogen water medium) is shown.

[0073] In Fig. 15, ·FW (Medium + Baf.A1): According to the above <Preparation of Medium for Treating Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, medium (purified water medium containing Baf.A1) obtained by dissolving 200 nM of Baf.A1 in DMEM containing purified water and serum, with doxorubicin added at 0 μM (-) or 2 μM (+), ·EHW (Medium + Baf.A1): Similarly, medium (electrolyzed hydrogen water medium containing Baf.A1) obtained by dissolving 200 nM of Baf.A1 in DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 and serum, with doxorubicin added at 0 μM (-) or 2 μM (+). ·FW (Serum free medium): Similarly, medium (serum-free purified water medium) obtained by adding doxorubicin at 0 μM (-) or 2 μM (+) to serum-free DMEM containing purified water, ·EHW (Serum free medium): Similarly, medium (serum-free electrolyzed hydrogen water medium) obtained by adding doxorubicin at 0 μM (-) or 2 μM (+) to serum-free DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 is shown.

[0074] From Fig. 12, it was confirmed that the combined use of doxorubicin and autophagy inhibitor (Baf.A1) (DOX + Baf.) enhanced the cell growth inhibitory effect of doxorubicin. On the other hand, under autophagy activation conditions (DOX + serum free), it was rather confirmed that the cell growth inhibitory effect of doxorubicin was reduced. From Fig. 13, it was confirmed that the combined use of electrolyzed hydrogen water and doxorubicin (EHW) enhanced the cell growth inhibitory effect. Whether this enhancement effect is due to the autophagy inhibitory activity of electrolyzed hydrogen water will be further verified below.

[0075] From Figures 14 and 15, under the combined condition of doxorubicin and autophagy inhibitor (Baf.A1) (\"Medium + Baf.A1\", under the autophagy inhibition condition), there is no significant difference between purified water (FW) and electrolyzed hydrogen water (EHW). Therefore, it was confirmed that the cell growth inhibitory effect does not change even when electrolyzed hydrogen water is combined. On the other hand, under the autophagy activation condition in a serum-free medium that does not contain doxorubicin and Baf.A1, it was confirmed that the cell growth inhibitory effect is enhanced by combining electrolyzed hydrogen water when comparing purified water (FW) and electrolyzed hydrogen water (EHW). Considering this result, it is considered that the autophagy inhibitory activity of electrolyzed hydrogen water is involved in enhancing the effect of doxorubicin.

[0076] (5-2) Verification of Apoptosis Induction Apoptosis induction was evaluated manually using Cell Death Detection ELISAPLUS (Roche Diagnostics, Basel, Switzerland). Caco-2 cells were seeded in a 12-well plate at 2.0×10 5 cells / 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 following various DMEMs and cultured for 24 hours. Then, the cells were lysed with lysis buffer, the lysate was added to each well of the ELISA plate, incubated with an immunological reagent buffer containing anti-histone biotin and anti-DNA POD for 2 hours, and washed with incubation buffer. Then, it was 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.

[0077] In Figure 16, · FW: DMEM containing purified water (purified water medium) diluted only with purified water in the above <Preparation of culture medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> with no addition (-) or 2 μM addition (+) of doxorubicin, · EHW: DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4, with or without (- or +) 2 μM doxorubicin added.

[0078] As shown in Figure 16, it was confirmed that the combined use of doxorubicin and electrolyzed hydrogen water (EHW) enhanced the apoptosis-inducing effect.

[0079] (5-3) Analysis of autophagy activity Caco-2 cells expressing GFP-LC3-RFP probe were seeded in a 12-well plate at 1.0×10 5 cells / well and cultured in DMEM at 37 °C under 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was replaced with various DMEMs shown below and cultured for 4 hours. Then, in the same manner as in (2-3) above, the cells were detached and suspended, and the fluorescence intensities of GFP and RFP 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.

[0080] In Figure 17, · FW: DMEM containing purified water (purified water medium) diluted only with purified water in the above <Preparation of culture medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)>, · EHW: DMEM (electrolyzed hydrogen water medium) containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4, · + DOX: Indicates the above DMEM with 2 μM doxorubicin added.

[0081] As shown in Figure 17, since the GFP / RFP ratio increased by the combined use of electrolyzed hydrogen water and the anticancer drug doxorubicin (EHW + DOX), it was confirmed that electrolyzed hydrogen water suppresses the autophagy activation of doxorubicin.

[0082] (5-4) Summary Considering the results of (5-1) to (5-3) above, it is expected that in human colon cancer cells (Caco-2 cells), similar to human cervical cancer cells (HeLa cells), electrolyzed hydrogen water enhances the anticancer effect by suppressing the autophagy activation effect of anticancer agents such as doxorubicin.

[0083] <6. Verification of Substances Contributing to the Enhancement Effect of Electrolyzed Hydrogen Water> Verification was carried out on the acting molecules of the above effects of electrolyzed hydrogen water.

[0084] (6-1) Analysis of Autophagy Activity Caco-2 cells expressing GFP-LC3-RFPΔG probe were seeded in a 12-well plate at 1.0×10 5 cells / well and cultured in DMEM at 37°C under 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was changed to various DMEMs shown below and cultured for 4 hours. Then, in the same manner as (2-3) above, the cells were detached and suspended, and the fluorescence intensities of GFP and RFP 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.

[0085] In Figure 18, · FW: DMEM containing purified water (purified water medium) diluted only with purified water in the above <Preparation of Medium for Treatment with Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, · EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 (electrolyzed hydrogen water medium), · EHW (AC): DMEM containing water subjected to autoclaving (autoclaved water medium) diluted only with water after subjecting electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 to autoclaving twice in the above <Preparation of Medium for Treatment with Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)>, · HW: DMEM containing hydrogen water with a hydrogen content equivalent to a dissolved hydrogen concentration level of 4 (produced hydrogen water medium) diluted only with the produced hydrogen water prepared using a kit of TRIM SEVEN WATER (manufactured by Nippon Trim Co., Ltd.) in the above <Preparation of Medium for Treatment with Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)> is shown, ·There is a statistically significant difference (p < 0.05) between different codes (a and b). Note that in the double autoclave treatment, one cycle was 20 minutes at a high pressure and a high temperature of 121 °C, and this was performed twice.

[0086] From Figure 18, it was confirmed that electrolyzed hydrogen water (EHW) and generated hydrogen water (HW) equally suppress autophagy activity because their GFP / RFP ratios are equivalent. On the other hand, when electrolyzed hydrogen water is subjected to double autoclave treatment (EHW (AC)), the GFP / RFP ratio becomes equivalent to that of purified water (FW), suggesting that its autophagy-suppressing activity is lost. From these results, it is assumed that the autophagy-suppressing component in electrolyzed hydrogen water is a volatile substance such as molecular hydrogen that is degassed by autoclave treatment.

[0087] (6-2) Verification of cell growth inhibitory effect Caco-2 cells were seeded in a 96-well plate at 1.0×10 4 cells / well and cultured for 24 hours (pre-culture) at 37 °C and 5% CO2 in DMEM. After pre-culture, the medium was changed to various DMEMs shown below and cultured for 24 hours. Then, 10 μL of Cell Counting Kit-8 (Dojindo, CK04) solution was added to each well and incubated at 37 °C for 2 hours, and the absorbance at 420 nm was measured. The results are shown in Figure 19.

[0088] In Figure 19, ·FW: DMEM containing purified water (purified water medium) diluted only with purified water in the above <Preparation of culture medium for electrolyzed hydrogen water (electrolyzed hydrogen water medium)> with doxorubicin added at 0 μM (-) or 2 μM (+), ·EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 (electrolyzed hydrogen water medium) with doxorubicin added at 0 μM (-) or 2 μM (+), ·EHW (AC): DMEM containing the water subjected to the above autoclave (autoclaved water medium) with doxorubicin added at 0 μM (-) or 2 μM (+), ·HW: DMEM (hydrogen - producing culture medium) containing hydrogen equivalent to dissolved hydrogen concentration level 4, with or without (- or +) 2 μM doxorubicin added, · A statistically significant difference (p < 0.05) exists between different symbols (a and b).

[0089] As shown in Figure 19, it was confirmed that electrolyzed hydrogen water (EHW) and hydrogen - producing water (HW) equally enhanced the inhibitory effect of doxorubicin on cell proliferation. On the other hand, when electrolyzed hydrogen water is autoclaved twice (EHW (AC)), it is considered that the above - mentioned enhancement effect disappears.

[0090] (6 - 3) Verification of cell proliferation HeLa cells (ATG7 WT) or cells with ATG7 (autophagy - related gene) deleted in HeLa cells (ATG7 KO) were seeded in a 96 - well plate at 1.0×10 4 cells / well and cultured in DMEM at 37 °C under 5% CO2 for 24 hours (pre - culture). After pre - culture, the medium was replaced with various DMEMs shown below and cultured for 24 hours. Then, 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 20 and 21.

[0091] In Figures 20 and 21, · HW: DMEM (hydrogen - producing culture medium) containing hydrogen - producing water with a dissolved hydrogen concentration of 0 ppm to 4 ppm, · HW + DOX: Indicates the above - mentioned DMEM (hydrogen - producing culture medium) with 0.5 μM doxorubicin added. For example, if Sample1 is a, Sample2 is ab, and Sample3 is b, it shows that there is no significant difference between Sample1 and Sample2, and between Sample2 and Sample3, and there is a significant difference between Sample1 and Sample3.

[0092] From Figure 20 (ATG7 WT), in the combination of generated hydrogen water and the anticancer drug doxorubicin (HW + DOX), as the dissolved hydrogen concentration of the generated hydrogen water increased, cell proliferation was suppressed, and it was confirmed that the cell growth inhibitory effect of doxorubicin was enhanced. On the other hand, from Figure 21 (ATG7 KO), it was confirmed that due to the deficiency of ATG7 (autophagy-related gene), the cell growth inhibitory effect of doxorubicin by the combination of generated hydrogen water and the anticancer drug doxorubicin could not be obtained.

[0093] (6-4) Summary Considering the above results of (6-1) to (6-3), the substance (acting molecule) that contributes to the enhancement of the anticancer effect (cell growth inhibitory effect and apoptosis induction) of doxorubicin by electrolyzed hydrogen water is dissolved molecular hydrogen. It was confirmed that molecular hydrogen suppresses autophagy and thus suppresses the cell growth of cancer cells.

[0094] <7. Analysis of autophagy activity by GFP / RFP ratio using other cells> Human colorectal cancer cells expressing the GFP-LC3-RFP probe (hereinafter referred to as "HCT116 cells") were seeded in a 12-well plate at 1.0×10 5 cells / well 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 shown below and cultured for 4 hours. Then, in the same manner as in (2-3) above, the cells were detached and suspended, and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. Quantitative analysis of autophagy activity was performed by obtaining the GFP / RFP ratio. The results are shown in Figure 22.

[0095] In Figure 22, · FW: DMEM containing purified water (purified water medium) diluted only with purified water in the above <Preparation of culture medium for treatment with electrolyzed hydrogen water (electrolyzed hydrogen water medium)>, · EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 (electrolyzed hydrogen water medium) is shown.

[0096] As shown in Fig. 22, in HCT116 cells derived from human colorectal cancer, similar to HeLa cells derived from human cervical cancer (Fig. 4), in electrolyzed hydrogen water (EHW), the GFP / RFP ratio increased compared to fresh water (FW). Therefore, electrolyzed hydrogen water suppressed autophagy.

[0097] <8. Analysis of autophagy activation of other anticancer drugs and verification of anticancer effects> Regarding other anticancer drugs, whether paclitaxel or fluorouracil activates autophagy and whether autophagy activation affects the anticancer effect were verified.

[0098] (8-1) Analysis of autophagy activity of paclitaxel by GFP / RFP ratio using GFP-LC3-RFP-LC3ΔG probe HeLa cells expressing GFP-LC3-RFP-LC3ΔG probe were seeded in a 12-well plate at 1.0×10 5 cells / well and cultured in DMEM at 37°C and 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was replaced with DMEM containing an anticancer drug with a predetermined concentration (1, 5, or 10 nM) of paclitaxel (Paclitaxel, Sigma-Aldrich Co., LLC.), and the cells were cultured for 24 hours. Then, in the same manner as in (2-3) above, the cells were detached and suspended, and the fluorescence intensities of GFP and RFP were analyzed by flow cytometry. By calculating the GFP / RFP ratio, quantitative analysis of autophagy activity was performed. The results are shown in Fig. 23.

[0099] As shown in Fig. 23, paclitaxel, similar to doxorubicin (Fig. 5), as the concentration increases, contrary to electrolyzed hydrogen water (see Fig. 4, Fig. 22, etc.), the GFP / RFP ratio decreases. Therefore, it is considered to have an effect of activating autophagy (autophagy activation effect). From these results, it is predicted that, similar to doxorubicin, autophagy activation attenuates the anticancer effect of paclitaxel.

[0100] (8-2) Verification of cell growth inhibitory effect HeLa cells (ATG7 WT) or cells with ATG7 (autophagy-related gene) deficiency in HeLa cells (ATG7 KO) were seeded in a 96-well plate at 1.0×10 4 cells / well and cultured in DMEM at 37°C under 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was replaced with DMEM containing an anticancer agent with a predetermined concentration (1, 10, or 50 nM) of paclitaxel and cultured for 24 hours. Then, 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.

[0101] From Figure 24, by comparing ATG7 WT and ATG7 KO, it was confirmed that the deficiency of ATG7 (autophagy-related gene) enhanced the anticancer effect (cell growth inhibitory effect) of paclitaxel in proportion to the concentration.

[0102] (8-3) Analysis of autophagy activity of fluorouracil by the GFP / RFP ratio using the GFP-LC3-RFP probe HCT116 cells expressing the GFP-LC3-RFP probe were seeded in a 12-well plate at 1.0×10 5 cells / well and cultured in DMEM at 37°C under 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was replaced with DMEM containing an anticancer agent with a predetermined concentration (1 or 10 μM) of fluorouracil (Fluorouracil, 5-FU, Sigma-Aldrich Co., LLC.) and cultured for 24 hours. Then, in the same manner as in (2-3) above, the cells were detached and suspended, and the fluorescence intensities of GFP and RFP 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.

[0103] As shown in Fig. 25, similar to doxorubicin (Fig. 5), the higher the concentration of fluorouracil, the more the GFP / RFP ratio decreases, which is opposite to that of electrolyzed hydrogen water (see Fig. 22). Therefore, it is considered that fluorouracil has an effect of activating autophagy (autophagy activation effect). From this result, it is predicted that, similar to doxorubicin, autophagy activation attenuates the anticancer effect of fluorouracil.

[0104] <9. Verification of the Enhancement of Anticancer Effect by the Combined Use of Electrolyzed Hydrogen Water and the Anticancer Agents Paclitaxel or Fluorouracil> (9-1) Verification of Cell Growth Inhibitory Effect 1 The combined use of electrolyzed hydrogen water and the anticancer agent paclitaxel (anticancer agent kit) was verified for whether it enhances the anticancer effect on HeLa cells derived from human cervical cancer.

[0105] HeLa cells (ATG7 WT) or cells with ATG7 (autophagy-related gene) deleted in HeLa cells (ATG7 KO) were seeded in a 96-well plate at 1.0×10 4 cells / well and cultured in DMEM at 37°C under 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was replaced with various DMEMs shown below and cultured for 24 hours. Then, 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 Figs. 26 (ATG7 WT) and 27 (ATG7 KO).

[0106] In addition, in Figs. 26 and 27, ·FW: DMEM containing purified water (purified water medium) diluted only with purified water in the above <Preparation of Medium for Treatment with Electrolyzed Hydrogen Water (Electrolyzed Hydrogen Water Medium)> and added with paclitaxel at a predetermined concentration (1, 10, or 50 nM), ·EHW: DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 (electrolyzed hydrogen water medium) added with paclitaxel at a predetermined concentration is shown.

[0107] From Figure 26 (ATG7 WT), it was confirmed that the combined use of electrolyzed hydrogen water and paclitaxel enhanced the cell growth inhibitory effect of paclitaxel. On the other hand, from Figure 27 (ATG7 KO), it was confirmed that the enhancement effect by the above combination disappeared due to the deficiency of ATG7 (autophagy-related gene).

[0108] (9-2) Verification of cell growth inhibitory effect 2 Verification was carried out on whether the combined use of electrolyzed hydrogen water and the anticancer drug fluorouracil (anticancer drug kit) enhanced the anticancer effect on HCT116 cells derived from human colorectal cancer.

[0109] HCT116 cells were seeded in a 12-well plate at 1.0×10 5 cells / well and cultured in DMEM at 37 °C under 5% CO2 for 24 hours (pre-culture). After pre-culture, the medium was changed to various DMEMs shown below and cultured for 24 hours. Then, 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.

[0110] In Figure 28, · FW: DMEM containing purified water (purified water medium) diluted only with purified water in the above <Preparation of medium for treatment with electrolyzed hydrogen water (electrolyzed hydrogen water medium)> with fluorouracil at a predetermined concentration (1, 5 or 10 μM) added, · EHW: Indicates DMEM containing electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 with a predetermined concentration of fluorouracil added.

[0111] From Figure 28, it was confirmed that the combined use of electrolyzed hydrogen water and fluorouracil enhanced the cell growth inhibitory effect of fluorouracil.

[0112] Considering the above results of (9-1) to (9-2), it is expected that the above two types of anticancer agent kits enhance the anticancer effect by suppressing the autophagy activation effect of anticancer agents such as paclitaxel and fluorouracil with electrolyzed hydrogen water.

Industrial Applicability

[0113] Examples of the utilization of the present invention include an anticancer agent kit comprising an anticancer agent action enhancer and an anticancer agent.

Claims

1. An anticancer agent kit comprising an anticancer agent enhancer having autophagy inhibitory activity and containing molecular hydrogen as an anticancer agent action enhancer, and an anticancer agent.

2. The anticancer agent kit according to claim 1, wherein the anticancer agent enhancer is electrolyzed hydrogen water.

3. The anticancer agent kit according to claim 1, wherein the active ingredient of the anticancer agent has an autophagy activating action.

4. The anticancer agent kit according to claim 1, wherein the active ingredient of the anticancer agent is at least one selected from the group consisting of doxorubicin, bosutinib, ponatinib, cisplatin, vincristine, paclitaxel, dasatinib, bosutinib, nilotinib, and fluorouracil.

5. The anticancer agent kit according to claim 1, wherein the concentration of the molecular hydrogen with respect to the reference active ingredient concentration (1 μM) of the anticancer agent is 105 ppb or more and 2160 ppb or less.

6. The anticancer agent kit according to claim 1, wherein the active ingredient concentration of the anticancer agent is about 0.5 μM or more and 10 μM or less.

7. The anticancer agent kit according to claim 1, wherein the active ingredient concentration of the anticancer agent 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.

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