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

Abstract

Oxidative stress is involved in cancer development. Hydrogen (H2) is a potent antioxidant and exhibits anti-inflammatory and potentially anticancer-like activities. This study aimed to investigate the role of H2 incombination with 5-fluorouracil (5-FU) in cancer treatment both in vitro and in vivo using the colon 26 cell line. The survival rate was determined using the Kaplan-Meier survival test, and cell viability was assessed using cell viability imaging kit and the MTT assay, and activation of the cell apoptosis pathway (Phosphorylated adenosine monophosphate activated protein kinase (p-AMPK), Apoptosis-inducing factor (AIF) and Caspase 3) were characterized by western blots. Hydrogen water administration improved the survival of mice with colon 26-induced cancer. Furthermore, hydrogen water enhanced cell apoptosis in cancer cells, resulting in a marked increase in the expression of p-AMPK, AIF and Caspase 3 in colon 26 cells. Hydrogen water also increased the inhibitory effect of 5-FU on colon 26 cells with spect to cell survival rate and anticancer functions. Additionally, high-content hydrogen water exhibited stronger antioxidative and anticancer activity than did the natural hydrogen water. In conclusion, high-content hydrogen water can inhibit colon cancer, particularly in combination with 5-fluorouracil.

Keywords: 5-fluorouracil; Antioxidant; Apoptosis; Cancer; Hydrogen.

Figure 1. Kaplan–Meier.

The survival rate of the HHW-treated group was slightly higher than the NHW group. The addition of 5-FU on day 14 significantly prolonged the survival rates (P < 0.05) of colon 26-bearing mice. Combination therapy of HHW and 5-FU produced the longest survival rate, followed by the combination NHW with 5-FU and 5-FU therapy alone. The result of log rank test analysis: for Colon26 and HHW, P < 0.05 (Fig. 1A); colon26 and Colon26 + 5FU, P < 0.001; Colon26 and Colon26 + 5FU + HHW, P < 0.001; Colon26 and Colon26 + 5FU + NHW, P < 0.001; and Colon26 + 5FU and Colon26 + 5FU + HHW, P < 0.05. The tumor size and weight are shown in Fig. 1C and 1D. The 5-FU group was found to be significantly decreased compared to the control group in both tumor weight and size (n = 7, ^∗p < 0.05). Similarly the tumor weight and size was further decreased in H₂ treated groups of 5FU + HHW and 5FU + NHW (n = 7, ^#p < 0.05).

Figure 2. The hydrogen (H₂) concentration of three treatments (HHW, NHW, and control) over an 8-h period.

(A) Direct examination of H₂ concentrations in each container, (B) H₂ concentrations in the in vitro experiment, and (C) H₂ concentrations in the in vivo experiment as measured in the blood after direct catheterization of hydrogen water into the animal’s stomach.

Figure 3. The cell viability of colon 26 cells 4 h after treatment with 5-FU, HHW, NHW, 5-FU+ HHW, and 5-FU+ NHW.

The administration of 5-FU decreased the viability of colon 26 cells; H₂ water (HHW and NHW) also decreased viability but to a lesser extent. Combined treatment of 5-FU+ HHW exhibited the greatest cell apoptosis in colon 26 cells, followed by a combination of 5-FU and NHW.

Figure 4. Cell viability of colon 26 (A) and HepG2 (B) were measured by MTT assay.

The cell viability of colon 26 cells was significantly reduced (p < 0.05) in all of the treatment groups (5-FU, HHW, NHW) compared with the control group. 5-FU decreased the cell viability of colon 26 cells most significantly, followed by HHW and NHW, respectively. Combined therapy of 5-FU and HHW or NHW decreased cell viability (p < 0.05) relative to HHW alone. 5-FU but not H₂ decreased cell viability of HepG2.

Figure 5. The free-radical (DPPH) scavenging activity of HHW and NHW in comparison with Vitamin C at several ranges of the doses.

^∗p < 0.05 for the difference between Vitamin C and HHW. ^#p < 0.05 for the difference between Vitamin C and NHW.

Figure 6. Western blot analyses of p-AMPK, AIF and Caspase 3 for cell apoptosis in colon 26 cells after treatment with HHW or NHW and in combination with 5-FU.

The expression of p-AMPK, AIF and Caspase 3 were significantly increased after administration of both H₂ water treatments (HHW and NHW). The levels of all examined proteins were increased by the addition of 5-FU. Higher expression was observed after treatment with HHW than after treated with NHW.