Biological Trace Element Research (2023) 201:2895–2903

https://doi.org/10.1007/s12011-022-03393-2

The Supplement of Magnesium Element to Inhibit Colorectal Tumor

Cells
Heng Li1 · Xiaonan Feng2 · Hai Li1 · Shuo Ma1 · Wei Song1 · Bao Yang1 · Tao Jiang1 · Chun Yang1

Received: 2 May 2022 / Accepted: 12 August 2022 / Published online: 25 August 2022
© The Author(s) 2022

Abstract
Magnesium ions are essential elements to the human body, with a daily intake of about 350 mg for an adult. Recently, a
meta-analysis reported that magnesium ion intake is related to a reduced risk of colorectal tumors. In addition, implantation
of biodegradable magnesium pins after colorectal tumor resection could potentially inhibit the residual tumor cells. These
impressive results implied that magnesium ions possess inhibitory properties against colorectal carcinoma. However, this
hypothesis has yet to be confirmed by experimental results. In this work, different concentrations of magnesium ions were
modulated to investigate their inhibitory effects on cell viability through cell cycle arrest, subsequently inducing apoptosis
by activating the caspase-3 pathway. The animal experiments revealed that magnesium injection restricted tumor growth
after 3 weeks of treatment compared to the control group. According to the immunohistochemistry and transmission electron
microscopy results, the remarkable effect may be attributed to promoting the apoptotic rate of tumor cells. The evidence
highlights the potential for the clinical use of magnesium implants to inhibit the growth of residual cells after colorectal
tumor surgery.

Keywords Magnesium ions · Colorectal tumor · Tumor inhibition · In vitro · In vivo

Introduction have indicated that magnesium (Mg) intake may decrease

the risk of CRC, although this observation is not universal
Colorectal carcinoma (CRC) is the most widespread cancer [4, 5]. Zan et al. [6] reported that high-purity magnesium
of the intestinal tract system and is a major public health staples inhibit the viability and migration of CRC cells
issue. In 2020, CSC had a 10.0% incidence with a 9.4% mainly due to the released hydrogen inducing tumor cells
mortality rate and was one of the three most common apoptosis through the p53-lysosome-mitochondria pathway
oncological diseases, gradually affecting younger patients [7]. However, further research is required to investigate the
[1–3]. Combined surgical removal and chemotherapy is a effects of magnesium ions ­(Mg2+) on CRC. M ­ g2+ is the
typical treatment for CRC. However, postoperative recur- second most plentiful divalent cation in the human body
rence and metastases remain a challenge for surgeons and and is involved in hundreds of physiologic processes asso-
patients, with the risk of secondary surgery or even life- ciated with energy metabolism and nucleic acid synthesis
threatening consequences. Recently, epidemiologic studies [8]. The “Dietary Reference Intakes (DRIs)” recommends
a daily intake of 220–350 mg of M ­ g2+ for adults [9]. As a
nutritional element in the body, ­Mg2+ supplementation is
Heng Li and Xiaonan Feng contributed equally to this work. beneficial for several medical conditions, such as inflam-
* Tao Jiang mation [10], bone regeneration [11, 12], arrhythmia [13],
[email protected] and depression [14]. The homeostasis of M ­ g2+ is regulated
* Chun Yang through the circulatory system and excessive M ­ g2+ can be
[email protected] excreted via the feces and urine [15]. Therefore, adverse
­ g2+ accumulation are rare. Recent studies
effects due to M
1
Department of Colorectal Surgery, General Hospital reported that serum M ­ g2+ levels are associated with the
of Ningxia Medical University, Yinchuan 750004, China
immune system, and an M ­ g2+-rich environment promotes
2
Department of Nephrology, General Hospital of Ningxia CD 8 + T cells to eliminate abnormal or infected cells [16].
Medical University, Yinchuan 750004, China

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2896 H. Li et al.

Furthermore, ­Mg2+ inhibits the proliferation and migration incubator for 20 min. Finally, the cells were washed once
of bone tumor cells through the parallel pathways snail1- and photographed.
microRNA181c-NLK and snail1-microRNA181d-TIMP3
[17]. Moreover, M ­ g2+ may alleviate the side effects caused Cell Apoptosis and Cell Cycle Analysis
by platinum-based chemotherapeutic agents via the tran-
sient receptor potential melastatin 7 channel [18]. Two groups were set up (Mg-supplemented group and nor-
Magnesium chloride ­(MgCl2) is an inorganic compound mal medium group) to study the influence of Mg on tumor
consisting of one magnesium and two chloride ions. It has apoptosis. After 72 h of culture, the DLD-1 and RKO cells
high water solubility, low toxicity, and is used as a source were harvested by 0.25% trypsin without ethylenediamine-
of ­Mg2+ in medicine, which is essential for many cellular tetraacetic acid. After being washed twice with PBS solu-
activities. In previous in vitro studies, ­MgCl2 was used as tion, the cells were stained by FITC annexin V and propid-
an ­Mg2+ supplement to investigate its influence on bacte- ium iodide solution from the apoptosis detection kit (Sony
ria, normal osteoblast cells, tumor cells, etc. [19–21]. Here, Biotechnology, Japan). Finally, the levels of apoptotic cells
­MgCl2 was added to the RPMI-1640 medium to study the were tested by a flow cytometer (Beckman Coulter, USA).
efficacy of ­Mg2+ on the viability, apoptosis, and cell cycle The DLD-1 cells were cultured for 72 h in either the
of colorectal tumor cells in vitro. Additionally, the effect RPMI-1640 culture medium with M ­ gCl2 or normal RPMI-
of ­Mg2+ on tumor tissue was investigated in animal experi- 1640 medium without M ­ gCl2. The cells were washed twice
ments, by establishing a cell-derived xenograft model in with PBS and fixed in 75% ethanol overnight in a − 20 °C
nude mice. Magnesium may play an invaluable role in the fridge. After being stained by a cell cycle kit (BD bio-
post-surgical treatment of CRC. sciences, USA), the cell cycle distribution was tested.

The Immunofluorescence Image of Cleaved Caspase

Experimental Section 3

Cell Culture After ­Mg2+ treatment for 72 h, DLD-1 tumor cells were
fixed and washed twice. Then, the cells were permeabilized
The human colorectal adenocarcinoma cell line DLD-1 and blocked for 1 h. Subsequently, the cells were stained
and RKO was purchased from the Cell Bank of the Chi- overnight by the cleaved caspase-3 antibody (1:200, Abcam,
nese Academy of Sciences. The RPMI-1640 medium sup- UK) and counterstained with a goat anti-rabbit IgG (H&L)
plemented with 10% fetal bovine serum (FBS, Gibco, USA) secondary antibody (Alexa Fluor® 488, 1:500, Abcam).
was used to culture cells according to the cell culture guide- Confocal laser scanning microscopy (Leica, Germany) was
lines. The cells were passaged twice a week using trypsin used to capture immunofluorescence images.
(Gibco, USA).
The Effect of Mg on Tumor Model in Vivo
Cell Viability
The animal study was approved by the Approval of the Medi-
The number of 3000 DLD-1 or RKO cells was seeded in cal Research Ethics Review Committee of General Hospital of
each well of 96-well plates. After 24 h of attachment, the Ningxia Medical University. The cell line-derived xenograft
culture medium was exchanged for a culture medium supple- (CDX) model was used to construct tumors in BALB/c nude
mented with different concentrations (15 mM and 30 mM) of mice (8 weeks, male). The initial concentration of magnesium
­MgCl2 (Aladdin, China) powder. Cell viability was tested at ions in the serum of BALB/c nude mice was about 2.5 mg%
24, 48, and 72 h by CCK8 assay (DOJINDO, Japan). About [6]. In brief, 2 × ­106 DLD-1 cells were subcutaneously injected
100 μL of the medium-CCK8 solution was added and left into the back of the mice. When the size of the tumor grew over
for 4 h. The absorbance at 450 nm was then measured by a 125 ­mm3, 10 mice were randomly assigned to the Mg injection
microplate reader (Spark, Tescan, Czech Republic). group (namely Mg group) and control group. The tumor-bearing
mice in the control group were injected with 10 μL saline. The
Live/Dead Staining Mg group got a 500 mM M ­ gCl2 injection every 3 days. On the
last day, all mice were sacrificed and the tumor tissue, normal
After ­MgCl2 addition for 72 h, the DLD-1 cells were con- organs were collected and stained by H&E. The tumor weight
ducted live/dead staining. The cells were washed with PBS was recorded and the volume (V) of the tumor tissue was cal-
and stained by calcein-AM (green, live) and ethidiumho- culated. The tumor tissues from each group were harvested for
modimer III (red, dead) according to the live/dead staining H&E staining, Ki67 immunohistochemical analysis and trans-
kit (Biotium, USA). Then, the plates were placed into a cell mission electron microscope observation were performed.

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The Supplement of Magnesium Element to Inhibit Colorectal Tumor Cells 2897

­ g2+ on the viability of DLD-1 (a) and RKO (b) cells after 24, 48, and 72 h of exposure, respectively. n = 12, *p < 0.05,
Fig. 1  The effect of M
**p < 0.01, ***p < 0.001

Statistical Analysis ANOVA analysis of variance using GraphPad Prism 8 software

(GraphPad, USA). All the figures shown in this article were
All the data were presented as mean ± SD, and statistical anal- obtained from experiments that were independently repeated
ysis between multiple groups was performed by the one-way at least three times. Statistical significance is represented by
*p < 0.05, **p < 0.01, and ***p < 0.001.
Fig. 2  The live/dead staining
of DLD-1 cells with different
concentrations of M­ g2+ treat-
ments for 72 h

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2898 H. Li et al.

Fig. 3  The apoptosis rate and corresponding statistics of DLD-1 (a) and RKO (b) cells with different treatments after 72 h. n = 4

Fig. 4  The expression of

cleaved caspase 3 of DLD-1
cells for 72 h treatment

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The Supplement of Magnesium Element to Inhibit Colorectal Tumor Cells 2899

Results and Discussion The live/dead staining also showed that M­ g2+ suppressed
the viability of DLD-1 cells in a dose-dependent manner.
The Cell Viability As shown in Fig. 2, a high proportion of dead cells (red) to
live cells (green) are found in the group supplemented with
To investigate the effect of ­Mg2+ on the viability of colo- 30 mM ­Mg2+, while more live cells and fewer dead cells
rectal adenocarcinoma cells, the culture medium was sup- were found in the control group. In addition, the supple-
plemented with 15 mM or 30 mM of M ­ gCl2. Meanwhile, the ment of 15 mM ­Mg2+ increases cell death by a small amount
culture medium without M ­ g2+ addition was set as the control compared to the control group, which is in accordance with
group. The M ­ g2+ concentration was kept within the normal the results of Fig. 1.
osmolality range of the cells so that no additional osmotic
pressure was exerted [17, 22]. As shown in Fig. 1a, the cell Apoptosis Analysis
viability of the DLD-1 cells was significantly inhibited after
48 h of ­Mg2+ treatment and showed a gradual decrease as Apoptosis is a programmed cell death process, which is an
the ­Mg2+ concentration was increased. After 72 h treatment, essential factor in clinical oncological therapy to eliminate
the cell viability decreased by 71.7% in the 30 mM ­Mg2+ the tumor cells [23, 24]. In recent studies, compelling evi-
group and by 11.7% in the 15 mM ­Mg2+ group compared dence has indicated that the initiation of apoptosis effectively
to the control group. The RKO cells showed less sensitivity inhibits tumor cell growth and subsequently influences pro-
to ­Mg2+ at the 15 mM concentration. After 72-h treatment, liferation and differentiation [25]. Therefore, the interaction
the cell viability decreased by 85.8% in the 30 mM ­Mg2+ between ­Mg2+ and the incidence of apoptosis in DLD-1 cells
group and by 5.1% in the 15 mM ­Mg2+ group compared to and RKO cells was investigated. After 72 h of treatment, the
the control group (Fig. 1b). apoptosis rate of DLD-1 cells was proportionally increased
with the dose of ­Mg2+, yielding apoptosis rates of 13.2 ± 0.6%

Fig. 5  The cell cycle of DLD-1 cells in different groups after treatment with 0, 15, and 30 mM Mg.2+ for 72 h. n = 4

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2900 H. Li et al.

and 33.0 ± 3.0% in the 15 mM and 30 mM groups, respec- (Fig. 5). Compared to the control group, both the M ­ g2+ sup-
tively, which were significantly greater than the control group plemented groups showed a significant increase in the per-
(10.7 ± 2.8%) (Fig. 3a). Meanwhile, the apoptosis rate of centage of cells arrested in the G0/G1 phase (p ˂ 0.001). This
RKO cells in the 15 mM ­Mg2+ medium was 8.4 ± 0.9% and finding reveals that ­Mg2+ inhibits proliferation and promotes
98.2 ± 0.5% in the 30 mM medium, respectively (Fig. 3b). the apoptosis of colorectal adenocarcinoma cells by inducing
­Mg2+ exhibited encouraging apoptotic efficacy on RKO cells. cell cycle arrest at the G0/G1 phase [32].
The caspase family, especially caspase-3, holds a critical Based on the above results, M ­ g2+ supplementation may
function in programmed cell death and activated protease in promote apoptosis in a dose-dependent manner through reg-
apoptosis [26]. FITC-conjugated cleaved caspase-3 was used ulating the cell cycle of the DLD-1 tumor cells, subsequently
to evaluate the expression in DLD-1 cells after M ­ g2+ therapy. inhibiting the proliferation in vitro.
Among all the groups, the expression of cleaved caspase-3
(green) was the highest in the 30 mM M ­ g2+ group (Fig. 4), indi- The Effect of Mg on the Tumor Model in Mice
cating increased pro-apoptotic protein levels [27]. The results
of the corresponding apoptosis assay and caspase-3 expression To evaluate the therapeutic effect of ­Mg2+ in vivo, a subcu-
jointly indicated that ­Mg2+ could induce apoptosis in colorectal taneous tumor model was established in BALB/c nude mice.
adenocarcinoma DLD-1 cells in vitro. ­ g2+ injection were
Furthermore, the therapeutic effects of the M
compared with the control group (Fig. 6a). Furthermore, the
The Cell Cycle dose of ­Mg2+ injection was based on previous studies [33].
After treatment for 21 days, all the tumor tissues of mice are
The link between proliferation and apoptosis is regulated harvested, and the volume and weight are measured. As dem-
by cell cycle proteins, such as p21, and cdk2 [28–30]. It onstrated in Fig. 6, the volume and weight of tumor tissue were
has been reported that anti-tumor agents inhibit the rapid significantly decreased in the Mg group in contrast to the con-
duplication of tumor cells and arrest tumor cells in the G0/ trol group. Hence, the results suggested that sufficient M ­ g2+
G1 phase to induce apoptosis [31]. This study found that could efficiently decrease the growth of the tumor tissue in vivo.
­ g2+ might induce DLD-1 cell arrest in G0/
the addition of M As displayed by the H&E staining images, the purple-stained
G1 phase. The percentage of cells in the G0/G1 phase in the cells indicate that the majority of the tumor cells were alive
control group, 15 mM ­Mg2+ group, and 30 mM ­Mg2+ group in the control group. On the contrary, the Mg group induced
were 45.7 ± 1.7%, 51.6 ± 1.8%, and 60.6 ± 1.4%, respectively more tumor apoptosis, resulting in some necrotic areas without

Fig. 6  (a) Schematic diagram of the Mg ion treatment protocol for tumor therapy in vivo. (b) The photograph of the isolated tumor tissue after
21 days of treatment. Scale bar: 1 mm. (c, d) The volume (c) and weight (d) of tumors in different groups after 21 days of treatment. n = 6

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The Supplement of Magnesium Element to Inhibit Colorectal Tumor Cells 2901

Fig. 7  H&E, Ki67, and TEM

analysis of the mouse tumor
tissues on day 21

abundant inflammatory cell infiltration. In addition, the Ki67 through cell cycle G0/G1 arresting, thus inhibiting the pro-
staining was consistent with the H&E staining, showing a liferation. Intra-articular injection of Mg ions inhibits the
higher degree of apoptosis after Mg treatment. Furthermore, growth of tumor tissue in the nude mice. The Mg ions induce
examination under the transmission electron microscope (TEM) tumor apoptosis in the tumor-bearing mice. This study pro-
revealed karyorrhexis and karyolysis of many tumor cell nuclei vides additional evidence supporting the use of Mg implants
in the Mg group, which is a typical apoptotic feature (Fig. 7). in future surgery.
These results indicated that Mg effectively inhibits the growth
and induces apoptosis of colorectal adenocarcinoma DLD-1 Acknowledgements We would like to thank the General Hospital of
Ningxia Medical University for supporting this study.
cells in vivo, supported by the cell experiments results.
Author Contribution Heng Li and Xiaonan Feng designed this study. Hai
Li conducted the cell experiments. Shuo Ma did the animal experiments.
Conclusion Wei Song wrote the first version of the manuscript. Bao Yang did the data
analysis. Jiang Tao and Chun Yang reviewed the manuscript.
In summary, this study demonstrated the anti-tumor prop-
Funding Dr. Yang Chun was supported by the First-Class Discipline
erty of Mg ions in colorectal adenocarcinoma. The Mg ions Construction Founded Project of Ningxia Medical University and the
induce tumor cells apoptosis in a dose-dependent manner

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2902 H. Li et al.

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