Hydrogen–water enhances 5-fluorouracil-induced inhibition of colon cancer

Cell line

The mouse colon carcinoma cell line (Colon 26) was purchased from the American Type Culture Collection (ATCC) (Manassas, Virginia, USA) through Summit Pharmaceuticals Intl. Corp. (Tokyo, Japan). Cells were cultured in ATCC-formulated RPMI-1640 medium supplemented with fetal bovine serum to a final concentration of 10%. The cells were maintained at 37 °C in a humidified 5% CO₂ environment until approximately 60% confluence. The medium was then replaced with serum-free cell medium containing assorted 5-FU, HHW, NHW, 5-FU + HHW, and 5-FU + NHW treatment (Kyowa Hakko Kogyo Co. Ltd, Tokyo, Japan). After incubation for 24 h, the cells were harvested for further studies.

Animals

Six-week old female BALB/c mice, weighing 19 to 24 g, were maintained in a pathogen-free environment under a 12 h light/12 h dark cycle with a controlled room temperature at the animal center of Kagoshima University (Kagoshima, Japan). Food and tap water were provided ad libitum. To develop cancers, 5 × 10⁵ viable Colon 26 cells in 100 µl RPMI-1640 medium were injected into the abdominal cavity by intraperitoneal injection (Perboni et al., 2008). All procedures in this study were approved by the Ethics Committee for Animal Care and Use of Kagoshima University (IRB approval number MD13062) and conducted by following the Japanese national standardized guideline for animal experiments of Kagoshima University.

High-content hydrogen water

High-content hydrogen water was prepared in our laboratory by dissolving H₂ in pure water under high pressure (0.4 MPa) for 24 h to achieve overall 0.8 mM hydrogen saturation. For comparison, natural H₂ water (NHW) drawn from Mount Fuji (Yamanashi, Japan) containing 0.125 mM of hydrogen (VanaH Co., Ltd., Yamanashi, Japan) was also used in this study.

Measurement of hydrogen concentration

In animals

The animals were seperated into 5 groups (5-FU, HHW, NHW, 5-FU + HHW, and 5-FU + NHW). Hydrogen waters 0.6 ml (HHW and NHW) was force-fed into the mice’s stomach using gavage. At each predetermined timepoint (0, 2, 4, 6, 8 h), 150 µl of venous blood was withdrawn, and the hydrogen concentration was measured by hydrogen electrode.

Cell viability assay

The Live/Dead Cell Imaging Kit (Life Technologies, Carlsbad, California, USA) was qualitatively used to examine cell viability. Live green (1 ml of 1 µM) solution was dissolved into dead red solution (1 mM lyophilized stock). Colon 26 cells and HepG2 cells were treated with a 2× equal volume of testing solution for 1 h. After incubation for 15 min at room temperature, cell images were captured by microscopy.

Cell viability was also determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Five treatments (5-FU, HHW, NHW, 5-FU and HHW, and 5-FU and NHW) were applied to 10⁴ colon 26 cells in 96-well plates for 24 h. The medium was then substituted with 200 µl/well free medium in each well (5-FU group final dose: 50 µM). After incubation for 4 h at 37 °C, dark blue formazan crystals were formed by the viable cells, and 0.01 M HCl/10% SDS was added. Synergy HT Multi-mode Microplate Reader (Bio-Rad, Hercules, California, USA) was then used at 570 and 600 nm to quantify the absorbance of each well as described previously (Yu et al., 2012). The data were collected from triplicate experiments.

In vivo and in vitro experiments

In vivo: after the confirmation of tumor induction in all groups except the cancer-free mice drinking hydrogen water, mice were treated every 8 h with MQ water (Milli-Q Integral per produce), HHW or NHW by oral administration (250 µl), and 5-FU dissolved in a 5% glucose solution was administered (100 mg/kg) by intraperitoneal injection weekly (Perboni et al., 2008). At ten days after tumor induction, the mice were sacrificed, and the tumors were excised and scaled. Tumor size is measured with caliper and calculated using the following equation: Length × width² × 0.5, as previously described (Lee et al., 2012).

In vitro A solution of 50 µl MQ, HHW (0.8 mM) or NHW (0.125 mM) was added to each slide chamber (IWAKI, Japan), and the results were compared with slides chambers administered only 5-FU at a final dose of 50 µM.

Western blot analysis

The expression of p-AMPK, AIF, and Caspase 3 was examined using western blotting analysis. Ice-cold radio-immuno-precipitation assay (RIPA) buffer supplemented with phosphatase and protease inhibitors (50 mM sodium vanadate, 0.5 mM phenylmethylsulphonyl fluoride, 2 mg/ml aprotinin, and 0.5 mg/ml leupeptin) was used for protein extraction and elimination of homogenates and cell lysate. The protein concentrations were then measured by BCA protein assay (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). Total protein samples (30 µg) were filtered via SDS-PAGE (Polyacrylamide Gel Electrophoresis) (10% acrylamide gel) using the Bio-Rad Trans-Blot system and were transferred to membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), incubated for one hour, washed in TBS-T and hybridized with primary antibodies diluted to a suitable concentration in TBS for 16 h. Afterwards, specific antibodies for AMPK and p-AMPK (Cell Signaling, Danvers, Massachusetts, USA), AIF (Merck Millipore, Billerica, Massachusetts, USA), and Caspase 3 (Merck Millipore, Billerica, Massachusetts, USA) were used. Additionally, membranes were incubated with secondary antibody to bind the β-actin (Merck Millipore, Billerica, Massachusetts, USA) (1:5,000 dilution), which served as the internal control. Incubation with secondary antibodies and the detection of the antigen-antibody complex was performed using an ECL kit (Amersham Biosciences, Amersham, UK). After comparing with the marker for specificity, the immunoblots of p-AMPK (62 kDa), AMPK (62 kDa), β-actin (43 kDa), AIF (57 kDa), and Caspase 3 (17 kDa) were quantified with a laser densitometer.

Free radical scavenging assay

The free radical scavenging activity of H₂ was determined using a 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging photometric assay and compared to the activity of vitamin C. Twenty microliters of each sample solution [0.01-1 mM of vitamin C (Sigma-Aldrich, Seelze, Germany), 0.01–0.8 mM of H₂ (HHW), and 0.0001–0.1 mM of H₂ (NHW)] and 200 ml of DPPH solution [2 mM of 2, 2 diphenyl-1-picryl-hydrazyl (Sigma-Aldrich, Seelze, Germany) prepared in methanol] were added to each micro plate. Samples were then incubated in the dark at room temperature for 10 min, and the absorbance (Ab) was measured at 540 nm using a microplate reader. Radical scavenging activity was calculated using the following equation: % antioxidant activity = [12(Ab of sample/Ab of blank)] ×100, as described previously (Amitani et al., 2013).

Statistical analysis

The data are presented as the means ±SEM with respect to the number of samples (n) in each group. Statistical analysis wa s conducted using repeated measures one-way analysis of variance and two-way analysis of variance (ANOVA). Tukey’s test was used to determine significant differences. Results with p < 0.05 were considered statistically significant. The survival rates were analyzed using the Kaplan–Meier survival test and the statistical analysis is performed using log rank test with p < 0.05 being considered as significant.

Hydrogen water increases the survival rate of colon 26-bearing mice

The survival rate in the groups treated with only H₂ water (HHW and NHW) was slightly increased relative to the group of cancer-bearing mice that did not receive treatment (Fig. 1A). Moreover, the survival rate in the HHW-treated group was significantly greater than that in the NHW group (Fig. 1A). After treatment with 5-FU, the survival rate significantly increased (Fig. 1B). The combination of HHW with 5-FU exhibited a marked increase in survival rate (p < 0.05) , whereas the combination of NHW with 5-FU was less effective (Fig. 1B). Water intake was monitored throughout the experimental period and was nearly the same among the groups. Body weight and food intake were also monitored and were significantly lower in the colon 26 tumor-bearing mice group than in the control group (data not shown). However, no significant difference was observed in body mass recovery between the hydrogen water treated group and the control group (data not shown). The tumor size and weight of the 5-FU group is significantly decreased compared to the control group (Figs. 1C and 1D).

Hydrogen attenuation rate in vitro and in vivo

Hydrogen concentrations were monitored for up to 8 h (Figs. 2A–2C). Fresh preparations of HHW exhibited maximum H₂ concentrations of nearly 0.8 mM, whereas NHW maintained an H₂ concentration of 0.1 mM (Fig. 2A). In cell culture media, the H₂ concentration of HHW reached a maximum concentration of 0.3 mM, whereas NHW reached a maximum concentration of approximately 70 µM (Fig. 2B). In mice, the hydrogen concentration in the blood peaked at 6 µM for HHW and 4 µM for NHW (Fig. 2C).

Hydrogen water enhances the cancer cell apoptotic effect of 5-FU

The cell viability of colon 26 cells was assessed after a 24-h incubation with 5-FU, hydrogen water (HHW, NHW) or a combination of 5-FU with hydrogen water. As shown in Fig. 3, the combination of 5-FU and HHW produced the greatest induction of cell apoptosis in colon 26 cells, followed by the combination of 5-FU and NHW. The administration of H₂ water (HHW and NHW) resulted in a decrease in viability of colon 26 cells, but the effectiveness was lower than the treatment of 5-FU.

The cell viability of colon 26 cells was then examined by MTT assay (Fig. 4A). All treatments by each agent (5-FU, HHW, NHW) in this experiment were effective in reducing the cell viability of colon 26 cells compared with the control. In isolation, treatment with 5-FU was most effective, followed by HHW and NHW. In combination, HHW with 5-FU resulted in marked cell apoptosis in colon 26 cells, and NHW with 5-FU water produced a similar result. However, H₂ did not show any statistical significant effects in HepG2 cells (Fig. 4B)

HHW exhibits a stronger anti-oxidative effect than NHW

The anti-oxidative assay, which measures free radical (DPPH) scavenging activity, indicated that both HHW (0.01 and 0.8 mM) and NHW (0.01 and 0.1 mM) exhibited antioxidant activity (Fig. 5). Additionally, HHW exhibited a stronger anti-oxidative effect than NHW, approaching the anti-oxidative action of Vitamin C (1 mM). At each maximum dose Vitamin C (1.0 mM) has highest anti-oxidative effect followed by HHW (0.8 mM ) and NHW (0.1 mM ) respectively. However, at mole/mole comparison, HHW (0.1 mM ) and NHW (0.1 mM ) have the same level of anti-oxidative effect.

Identification of cell apoptosis in colon 26 cells via p-AMPK, AIF and Caspase 3 expression

The expression of p-AMPK, AIF and Caspase 3 in colon 26 cells was elevated after treatment with both hydrogen waters (HHW and NHW). The expressions of these proteins were further increased in colon 26 cells after the combined treatment with 5-FU, as shown in Fig. 6.