3.1 The cell viability
To investigate the effect of Mg2+ on the viability of colorectal adenocarcinoma cells, we adjusted the culture medium with 15 mM or 30 mM of MgCl2 and cultured the DLD-1 cells and RKO cells. Meanwhile, the culture medium without Mg2+ addition was set as the control group. The Mg2+ concentration was chosen to be within the optimal osmolality range of the cells which does not directly disrupt cell growth due to the increase in osmotic pressure [17, 19]. As shown in Fig. 1a, the cell viability of the DLD-1 cells was significantly alleviated after 48 h treatment of the Mg2+ and gradually decreased with the increase of Mg2+ concentration. After 72 h treatment, the cell viability decreased 71.7% in the 30 mM Mg2+ group and 11.7% in the 15 mM Mg2+ group compare to the control group. The RKO cells showed less sensitivity to Mg2+ when its concentration is 15 mM. After 72 h treatment, the cell viability decreased 85.8% in the 30 mM Mg2+ group and 5.1% in the 15 mM Mg2+ group compare to the control group.
The live/dead staining also showed that Mg2+ suppressed the viability of DLD-1 cells in a dosed manner. As shown in Fig. 2, many dead cells (red) with few live cells are found in the group supplement with 30 mM Mg2+, while more live cells and fewer dead cells were found in the control group.
3.2 The apoptosis
Apoptosis is a programmed cell death process, which has been an essential factor in clinical oncological therapy to eliminate the tumor cells [20, 21]. In recent studies, compelling evidence indicates that the initiation of apoptosis effectively inhibits tumor cell recurrence, subsequently influences proliferation and differentiation . Therefore, we investigated the interaction between Mg2+ and the incidence of apoptosis in DLD-1 cells and RKO cells. After 72 h treatment, the apoptosis rate of DLD-1 cells was increased with the enhancement of the dose of Mg2+, and the apoptosis rates in the 15 mM and 30 mM groups were 13.2 ± 0.6% and 33.0 ± 3.0%, respectively, which shows significant difference from the control group (10.7 ± 2.8%) (Fig. 3a). Meanwhile, the apoptosis rates of RKO cells in the 15 mM was 8.4 ± 0.9%, and 30 mM groups was and 98.2 ± 0.5%, respectively. Mg2+ exhibit encouraging apoptotic efficacy on RKO cells.
The family of caspase, especially caspase-3, holds a critical function in programmed cell death and activated protease in apoptosis . We used FITC-conjugated cleaved caspase-3 to evaluate the expression in DLD-1 cells after Mg2+ therapy. Among all groups, the expression of cleaved caspase-3 (green) is the highest in the 30 mM Mg2+ group (Fig. 4), indicating an increased pro-apoptotic protein to amplify the apoptosis of tumor cells . The results of the corresponding apoptosis assay and caspase-3 expression jointly indicate that Mg2+ can induce apoptosis in colorectal adenocarcinoma DLD-1 cells in vitro.
3.3 The cell cycle
The link between proliferation and apoptosis is regulated by the cell cycle proteins, such as p21, cdk2 [25–27]. It has been reported that antitumor agents target the predisposition of tumor cells to rapidly duplicate their DNA, and arrest tumor cells in the G0/G1 phase to induce apoptosis . Here, we found that the addition of Mg2+ is a feasibility to induce DLD-1 cells arrest in G0/G1 phase. The percentages of G0/G1 phase in the control group, 15 mM Mg2+ added group, and 30 mM Mg2+ added group are 45.7 ± 1.7%, 51.6 ± 1.8% and 60.6 ± 1.4%, respectively (Fig. 5). In comparison to the control group, both the Mg2+ added groups markedly arrested in the G0/G1 phase (P ˂ 0.001). This finding reveals that Mg2+ inhibits proliferation and promotes apoptosis of colorectal adenocarcinoma cells by increasing the percentage of G0/G1 phase .
Based on the above results, the supplement Mg2+ can promote apoptosis in a dosed manner through the regulation of the cell cycle of the DLD-1 tumor cells, subsequently inhibiting the proliferation in vitro.
3.4 The effect of Mg on the tumor model in mice
To evaluate the therapeutic effect of Mg2+ in vivo, we establish a subcutaneous tumor model in BALB/c nude mice. Furthermore, administration of the Mg2+ injection group with the control group is assessed the therapeutic performances, respectively, which received an injection of saline (control group) or MgCl2 solution (Fig. 6a). Furthermore, the dose of Mg2+ injection was referred according to the ref. . After treatment for 21 days, all tumor tissues of mice were harvested for volume measurement and weighting. As demonstrated in Fig. 6, the volume and weight of tumor tissue are significantly decreased in the Mg group in contrast to the control group. Hence, it was found that sufficient Mg2+ addition could efficiently decrease the growth of the tumor tissue in vivo.
As shown in the H&E staining images, the purple staining cells indicate that the majority of the tumor cells were alive in the control group. On the contrary, the Mg group induced more tumor cells apoptosis, performing some necrotic areas occurred without abundant inflammatory cell infiltration. In addition, the analysis of Ki67 staining was consistent with the H&E staining, showing fewer proliferate cells after Mg treatment. We also found many nuclei of tumor cells in the Mg group have undergone karyorrhexis and karyolysis by transmission electron microscope (TEM), which is typical apoptotic performance (Fig. 7). These results indicated that Mg effectively inhibits the growth and induces apoptosis of colorectal adenocarcinoma DLD-1 cells in vivo, in agreement with the results found in cell experiments.