Deuterium-depleted water inhibits the malignant progression of colorectal cancer cells by modulating oxidative stress

Abstract

Colorectal cancer (CRC) is one of the most common types of cancer worldwide. Alternative therapy has been widely used in CRC treatment, with deuterium-depleted water (DDW) demonstrating promising anticancer effects in a number of cancer types. The aim of the present study was to assess the anticancer effects of DDW in CRC cells and the possible mechanism involved. HT-29 and DLD-1 cells were cultured in conditioned medium prepared with DDW. Cell malignant behaviors were assessed using EdU, colony formation, tumor-sphere formation, wound-healing and Transwell assays. Stemness-related proteins, Nanog and octamer-binding transcription factor-4, were assessed using western blotting. Intracellular reactive oxygen species (ROS) levels were determined using 2',7'-dichlorodihydrofluorescein diacetate fluorescent probes. Reverse transcription-quantitative PCR and western blotting were used to assess the expression of forkhead box protein M1 (FoxM1), cyclin D1 (CCND1) and matrix metalloproteinase 9 (MMP9). The results indicated that treatment with DDW significantly inhibited the proliferation, tumor-sphere formation, migration and invasion of HT-29 and DLD-1 cells, as well as the expression of stemness-related proteins. In the mechanistic analysis, DDW treatment was revealed to decrease ROS production and downregulate the expression of FoxM1. As the downstream targets of FoxM1, the expression levels of CCND1 and MMP9 were also shown to be decreased. Moreover, H₂O₂-induced oxidative stress rescued FoxM1 expression in the presence of DDW treatment, and overexpression of FoxM1 was demonstrated to abolish the DDW-mediated tumor suppressive effects. The findings from the present study indicate that the anticancer effects of DDW in CRC cells occur by inactivating the ROS/FoxM1 signaling pathway. Moreover, the results provide a possible agent for CRC treatment.

Keywords: colorectal cancer; deuterium-depleted water; forkhead box protein M1; metastasis; oxidative stress; proliferation; stemness.

Figure 1.

DDW inhibits the proliferation and stemness of colorectal cancer cells. (A) EdU incorporation and (B) colony formation assays were performed using HT-29 and DLD-1 cells to evaluate the effects of DDW on cell proliferation. (C) Tumor-sphere formation assays were used to assess the cancer stem cell-like property of HT-29 and DLD-1 cells following treatment with DDW. (D) Western blotting demonstrated the protein expression levels of Nanog and OCT-4 in HT-29 and DLD-1 cells treated with DDW. *P<0.05, **P<0.01. DDW, deuterium-depleted water; OCT-4, octamer-binding transcription factor-4; Ctrl, control.

Figure 2.

DDW restrains the migration and invasion of colorectal cancer cells. The effects of DDW on HT-29 cell (A) migration and (B) invasion were assessed using wound healing and Transwell assays, respectively. The effects of DDW on DLD-1 cell (C) migration and (D) invasion were assessed using wound healing and Transwell assays, respectively. *P<0.05, **P<0.01, ***P<0.001. DDW, deuterium-depleted water; Ctrl, control.

Figure 3.

DDW downregulates FoxM1 signaling through suppressing ROS production in colorectal cancer cells. (A) Levels of ROS in HT-29 and DLD-1 cells treated with DDW or DDW + H₂O₂ were assessed using 2′,7′-dichlorodihydrofluorescein diacetate fluorescent analysis. The mRNA and protein levels of FoxM1, CCND1 and MMP9 in HT-29 and DLD-1 cells treated with DDW were analyzed using (B) reverse transcription-quantitative PCR and (C) western blotting. (D) Protein levels of FoxM1 in HT-29 and DLD-1 cells treated with DDW or DDW + H₂O₂ were analyzed using western blotting. *P<0.05 vs. Ctrl group, **P<0.01 vs. Ctrl group, ^#P<0.05 vs. DDW group, ^###P<0.001 vs. DDW group. DDW, deuterium-depleted water; FoxM1, forkhead box protein M1; ROS, reactive oxygen species; CCND1, cyclin D1; MMP9, matrix metalloproteinase 9; Ctrl, control.

Figure 4.

Overexpression of FoxM1 in colorectal cancer cells. FoxM1 was successfully overexpressed in both HT-29 and DLD-1 cells, and the transduction efficiency was assessed using (A) reverse transcription-quantitative PCR and (B) western blotting. *P<0.05, ***P<0.001. FoxM1, forkhead box protein M1; OE, overexpression; NC, negative control.

Figure 5.

Overexpression of FoxM1 abolishes the inhibitory effects of DDW on proliferation and stemness in colorectal cancer cells. (A) EdU incorporation and (B) colony formation assays were performed to assess the proliferation of HT-29 and DLD-1 cells with FoxM1 overexpression in the presence of DDW treatment. (C) The cancer stem cell-like property of HT-29 and DLD-1 cells was evaluated using the tumor-sphere formation assay. (D) Protein levels of Nanog and OCT-4 in HT-29 and DLD-1 cells were assessed using western blotting. **P<0.01 vs. Ctrl + OE-NC group, ***P<0.001 vs. Ctrl + OE-NC group, ^#P<0.05 vs. DDW + OE-NC group, ^##P<0.01 vs. DDW + OE-NC group. FoxM1, forkhead box protein M1; DDW, deuterium-depleted water; OCT-4, octamer-binding transcription factor-4; OE, overexpression; Ctrl, control; NC, negative control.

Figure 6.

Overexpression of FoxM1 abrogates the effects of DDW on the migration and invasion of colorectal cancer cells. Migration was assessed using wound healing assays in (A) HT-29 and (B) DLD-1 cells with FoxM1 overexpression in the presence of DDW treatment. The invasion of (C) HT-29 and (D) DLD-1 cells was evaluated using Transwell assays. *P<0.05 vs. Ctrl + OE-NC group, **P<0.01 vs. Ctrl + OE-NC group, ^#P<0.05 vs. DDW + OE-NC group, ^##P<0.01 vs. DDW + OE-NC group. FoxM1, forkhead box protein M1; DDW, deuterium-depleted water; OE, overexpression; Ctrl, control; NC, negative control.

Figure 7.

Overexpression of FoxM1 rescues the DDW-mediated inhibition of CCND1 and MMP9 in colorectal cancer cells. The (A) mRNA and (B) protein expression levels of CCND1 and MMP9 in HT-29 cells were assessed using RT-qPCR and western blotting, respectively. The (C) mRNA and (D) protein expression levels of CCND1 and MMP9 in DLD-1 cells were assessed using RT-qPCR and western blotting, respectively. *P<0.05 vs. Ctrl + OE-NC group, **P<0.01 vs. Ctrl + OE-NC group, ***P<0.001 vs. Ctrl + OE-NC group, ^#P<0.05 vs. DDW + OE-NC group, ^##P<0.01 vs. DDW + OE-NC group. FoxM1, forkhead box protein M1; DDW, deuterium-depleted water; CCND1, cyclin D1; MMP9, matrix metalloproteinase 9; OE, overexpression; Ctrl, control; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR.