The present study aimed to investigate the in vitro effects of the DDW on growth rate, morphology and structure of the HT29 cells, cell cycle distribution and antioxidant enzymes. We found that the DDW significantly suppressed the proliferation of the HT29 cells in 72h. Deuterium depletion increased the cytotoxicity of 5-FU on the colon cancer cell line in a time and concentration dependent manner.
The previous in vitro studies demonstrated that the DDW could be of inhibitory effect on cancers cell lines. In the first study in this regard, Somlyai et al showed that 30 ppm DDW significantly decreased the growth rate of L929 fibroblast cells and also inhibited tumor growth in xenotransplanted mice . Subsequently, the effect of drinking water with DDW with 30ppm deuterium concentration on mice transfected with MCF-7 and MDA-MB-231 cancer cells was investigated. The results of this study indicate an increase in tumor survival time and tumor disappearance in 59% . The other studies presented results of DDW inhibitory effect on PC-3 (cancerous cell line), MDA (breast cancer), and M14 (melanoma) cancer cells lines [21, 22]. Further studies reported DDW inhibitory effect on the human lung carcinoma cell line (A-549) and human nasopharyngeal carcinoma cells established with MTT assay [22, 23].
The results in the present study are remarkably similar to those of the previous studies.
In the previous studies, the growth inhibitory effect of DDW alone and in combination with the known anti-cancer drugs: etoposide, taxol, doxorubicin and cisplatin, showed that DDW synergistically enhances the inhibitory effect of doxorubicin in almost all cancers cultures tested; such a synergistic inhibitory effect was also reported with cisplatin in the breast cancer cell lines (MDA-MB- 231), prostate (PC-3), intestine (HCT-116) and glioblastoma (U-87MG) cell cultures. In the next step, preclinical finding was supported with the clinical studies. DDW as an adjuvant therapy came into use recently. Application of the DDW as an adjutant in conventional chemotherapy in patients with some of malignancy including the prostate, lung and breast malignancies, resulted in clinical benefit in terms of noticeably prolonged median survival time (MST) in different populations under study, decreased the tumor size, the attenuated subjective symptoms and molecular responses [14, 16, 23–26].
In accordance with these previous investigations, our study showed that DDW strengthens the cytotoxic effect of 5-FU as a chemotherapeutic agent on the cancer cell lines of colon cancer. The results of the present study show that the toxic effects of less than 100 ppm concentrations of deuterium depleted water alone reflect the growth of both SW480 and HT-29 cell lines. In addition, the effect of concentrations of less than 100 ppm on DDW increased the toxicity of 5FU.
The synergistic effects of DDW and 5-FU can be explained based on their common characteristics in terms of induction of apoptosis and cell cycle arresting. As reported, 5-FU impair in the synthesis of biocompatible macromolecules and inhibitory effects on the cell cycle by inhibiting the enzyme thymidilate synthase (TS) and incorrect placement within the RNA building. Similarly, the features of stopping apoptosis and cell cycle are also described for DDW [27, 28].
Our results support the hypothesis of Laskey et al, who hypothesized that mechanisms exist in the cells that detect changes in deuterium concentration. The possible cause of the cellular growth inhibition may be due to the changes in the isotope ratio of deuterium to hydrogen, because a number of studies have shown that high concentrations of deuterium are needed for the cell growth and cellular division and thus by decreasing the amount of deuterium the growth rate of the cancer cells also declines and the time required to reach the appropriate d/h ratio is high. [29, 30].
In order to investigate the mechanisms of the cell growth inhibition under treatment with deuterium depleted water alone or in combination with 5-0FU, the
cell cycle changes of the treated and control cells were studied.
In the present study, we found that DDW 75ppm alone led to stop in cellular proliferation at the G0 / G1 stage and decreased the S phase cell population. A greater proportion of the DDW-treated HT29 cells were arrested at S phase in the cells treated with the DDW and 5-FU combinations rather than at DDW treated alone, as observed by flow cytometry. Lowe D/H ratio triggered the molecular mechanism that finally prevented the cell to enter into the S phase.
A study was conducted in 2009 on the A549 cells and the cellular changes of the treated cells at a concentration of 50ppm of deuterium water compared with the control (150 ppm). The results of this study showed that the cell population in cell cycle S of DDW treatment groups increased by 11.38% and the control group in G0/G1 and the G2/M, respectively, decreased by 88%/4% and 49/6% . However, the results presented in the study by H Wang et al in 2013 contradicted the previous research results. The study of H Wang et al on the three concentrations of 50, 75 and 100ppm of the DDW in five NPC cells, revealed that the cell cycle undergoes a change due to the decrease in deuterium concentration so that the decrease in the number of cells in the S phase and the increase in the cells in the G1 phase were significantly more than those of the control group.
The results of this study were consistent with the results of the H Wang study. As an interpretation of the cause of the changes in the cell cycle, it can be concluded that the cell division is sensitive to the changes in the concentration of intracellular deuterium and the natural concentration of deuterium is essential for the onset and progression of the cell growth. To enter the cells to stage S and start dividing the cell, the D/H ratio threshold is required (23). When the cells are cultured in a low concentration deuterium diet, the growth of the cells is inhibited by increasing the time needed to reach the proper ratio of D/H [29, 30]. D/H relative to the normal cells is faster than that of the cancer cells.
Subsequently, the changes in the activity of superoxide dismutase and catalase enzymes, which are important antioxidant enzymes, were studied. Among the active oxygen species, reference may be made to superoxide and hydrogen peroxide radicals which are produced as a product of the natural metabolism of oxygen in the cells and tissues. Due to the intrinsic activity of ROS, these compounds can easily enter into reactions that ultimately damage the cells. Most of the cancer cells exacerbate and deteriorate due to the oxidative stress induced by oncogenic stimuli, increased metabolic activity, and mitochondrial malformations . In cancer cells, the levels of the glycoltic and cellular respiration reactions are higher and lower than normal cell levels respectivly, and so this difference leads to an increased oxidative stress [32, 33]. This high oxidative state in the cell causes the tumor to grow through the cellular growth increment, and the suppression of antitumor activity can damage the DNA structure . In our study on the antioxidant system at the cell surface, the level of enzyme activity of superoxide dismutase and catalase increased in the HT-29 cells at the same concentration of DDW, which coursed the highest growth-inhibitory and cell cycle-arresting properties. Therefore, it can be concluded that the cellular degradation or cellular growth inhibition, cell cycle arresting and antioxidant enzymes activity increases ultimately prevent the growth of the cancer cells by DDW alone or together with 5-FU, with a high degree of inhibition of cell growth. This study is in accordance with the study conducted by Lucia Olarium et al. Lucia Olarium et al. In 2007, measured the changes in the activity of red blood cell antioxidant enzymes in the DDW-treated rats and showed that the DDW has the antioxidant effects .
To investigate the morphological changes induced in DDW-treated cells alone or in combination with the 5-FU, we observed the morphology and structure of HT29 cells by two optical microscopes and scanning electron microscopy. According to the optical microscope observations, HT29 control cells were spindly smooth with a flat surface, while DDW-treated cells and the 5-FU were spindle-shaped and showed irregularities. In the observations made by the scanning electron microscope, the treated cells showed a more roughness than control. The extracted microscopic cells of the treated cells confirm the membrane degradation.