Chemopreventive Effects of Magnesium Chloride Supplementation on Hormone Independent Prostate Cancer Cells

Abstract

Background: Lifestyle significantly impacts the risk factors associated with prostate cancer, out of which diet appears to be the most influential. An emerging chemopreventive approach, which involves the adequate intake of dietary constituents, has shown great potential in preventing the occurrence or progression of cancer. Magnesium is known to be an essential cofactor for more than 300 enzymatic processes, and is responsible for the regulation of various cellular reactions in the body. A plethora of studies have shown evidence that changes in the intracellular levels of magnesium could contribute to cell proliferation and apoptosis in some normal and malignant cells. The aim of the study was to investigate the effects of magnesium chloride (MgCl2) in DU-145 prostate cancer cells.

Methodology: Cultured DU-145 cells were subjected to graded concentrations or doses (50-500 µM) of MgCl2 for 48 hours. The cell viability was assessed using MTT and Resazurin reduction assays. NBT assay was also used to assess the treatment-induced intracellular ROS levels. Acridine Orange/Ethidium Bromide (AcrO/EtBr) and Rh123/EtBr fluorescent stains were used to assess the cell death type and mitochondrial membrane potential (Δψm) respectively.

Results: The results revealed a dose-dependent decrease (P < 0.05) in cell viability in treated DU-145 cells after 48 hours. The NBT assay also revealed a dose dependent biphasic response (P < 0.05) in intracellular levels of ROS. There was a drop (P < 0.05) in ROS levels in all groups except at 100 µM, where ROS level was higher than the control. Apoptosis was the primary mode of cell death as observed in the fluorescence analysis.

Conclusion: Our finding suggests that MgCl2 may be potentially chemopreventive for prostate cancer. This justifies further studies into its mechanism of action in DU-145 and other prostate cancer cell types.

Full text

Functional Foods in Health and Disease 2016; 6(1): 1-15 Page 1 of 15
Research Article Open Access
Chemopreventive Effects of Magnesium Chloride Supplementation on
Hormone Independent Prostate Cancer Cells
Saheed Oluwasina Oseni, *Elsa Quiroz, and James Kumi-Diaka
Department of Biological Sciences, Florida Atlantic University, Davie, FL 33314, USA
*Corresponding Author: Elsa Quiroz, Department of Biological Sciences, Florida Atlantic
University, Davie, FL 33314, USA
Submission Date: November 12, 2015, Acceptance date: January 12, 2016: Publication date:
January 14, 2016
ABSTRACT
Background: Lifestyle significantly impacts the risk factors associated with prostate cancer, out
of which diet appears to be the most influential. An emerging chemopreventive approach, which
involves the adequate intake of dietary constituents, has shown great potential in preventing the
occurrence or progression of cancer. Magnesium is known to be an essential cofactor for more
than 300 enzymatic processes, and is responsible for the regulation of various cellular reactions
in the body. A plethora of studies have shown evidence that changes in the intracellular levels of
magnesium could contribute to cell proliferation and apoptosis in some normal and malignant
cells. The aim of the study was to investigate the effects of magnesium chloride (MgCl2) in DU-
145 prostate cancer cells.
Methodology: Cultured DU-145 cells were subjected to graded concentrations or doses (50-500
µM) of MgCl2 for 48 hours. The cell viability was assessed using MTT and Resazurin reduction
assays. NBT assay was also used to assess the treatment-induced intracellular ROS levels.
Acridine Orange/Ethidium Bromide (AcrO/EtBr) and Rh123/EtBr fluorescent stains were used
to assess the cell death type and mitochondrial membrane potential (Δψm) respectively.
Results: The results revealed a dose-dependent decrease (P < 0.05) in cell viability in treated
DU-145 cells after 48 hours. The NBT assay also revealed a dose dependent biphasic response
(P < 0.05) in intracellular levels of ROS. There was a drop (P < 0.05) in ROS levels in all
groups except at 100 µM, where ROS level was higher than the control. Apoptosis was the
primary mode of cell death as observed in the fluorescence analysis.
Conclusion: Our finding suggests that MgCl2 may be potentially chemopreventive for prostate
cancer. This justifies further studies into its mechanism of action in DU-145 and other prostate
cancer cell types.

Functional Foods in Health and Disease 2016; 6(1): 1-15 Page 1 of 15
Keywords: Prostate cancer, Magnesium chloride, Chemoprevention, Apoptosis, Reactive
Oxygen Species.
INTRODUCTION
Prostate cancer (PCa) is the 2nd leading cause of cancer in U.S. men, and it represents 13.3 % of
all cancer cases in the U.S. [1]. In 2015 alone, an estimated 220,800 new cases and 27,540 deaths
are expected to occur due to prostate cancer, adding to the burden of the over 2.6 million men
currently living with the disease in the country [2]. Lifestyle coupled with aging has been
shown to be one of the predisposing factors that may account for about 75 percent of the
prostate cancer cases, and of all the environmental variables, diet appears to be the most
influential [3]. An emerging chemopreventive approach involving the adequate intake of dietary
constituents has shown great potential in preventing the occurrence or progression of cancer [4].
Magnesium is known to be an essential cofactor for more than 300 enzymatic processes
responsible for the regulation of various cellular reactions in the body [5]. Magnesium has been
shown to be an indispensable element in all of the body's natural self-cleansing and
detoxification responses due to its ability to chelate with intracellular ATP and competitively
bind with calcium for binding sites on proteins and cell membranes [6]. Without sufficient
magnesium, toxic waste and acid residues accumulate in cells and tissues, setting the stage
for chronic degenerative conditions, cancer, and rapid aging symptoms [7]. Plethora of studies
have shown evidences that change in the intracellular levels of minerals and micronutrients, such
as magnesium, could contribute to the modulation or regulation of cell proliferation and
apoptosis in some normal and malignant cells [8].
Magnesium occurs naturally in many forms, but is most readily assimilated and utilized for
metabolic purposes. The distribution of magnesium in the body is as such; 60 % in bone, 20 % in
skeletal muscle, 19 % other soft tissues, and less than 1 % in blood serum [9]. It has been
shown that the average diet in the U.S. is deficient in magnesium [10]. This was confirmed
through a survey that was performed to determine the daily intake of magnesium in U.S.
population, and was found to be 32 % and 20 % less than the daily recommended dietary
allowance (RDA) in women and men respectively [11]. This was also found to be similar in most
other countries.
Prostate cancer is a slow growing tumor, and has been documented to be strongly correlated
with aging, with the highest prevalence indicated in men above 60 years of age, but not below 40
[1]. A link between magnesium and aging has been a topic of discussion in literatures, with
insufficient intake of magnesium in diet or variance in magnesium metabolism associated
with aging process and susceptibility to age-related diseases [12]. For instance, magnesium
imbalance in elderly people was found to be associated with inflammation, cardiovascular
diseases, diabetes, and stress vulnerability [7]. However, metabolism in normal and abnormal
cells is markedly different, and the links between cancer and magnesium status perhaps may rely
on cellular and subcellular backgrounds. The aim of the study was to investigate the
chemopreventive effects of magnesium chloride (MgCl2) supplementation on DU-145 hormone
refractory prostate cancer cells.

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MATERIALS AND METHODS:
Cell Line and Cell Culture: DU-145 cell line was purchased from ATCC (Manassas, Virginia,
USA). It is a hormone independent prostate cancer cell line. The DU-145 cells were cultured and
maintained in complete RPMI 1640 (Sigma Aldrich Chemical Co., St Louis, Mo, USA) media
with 10 % Fetal Bovine Serum and 1 % penicillin/streptomycin.
Micronutrient: Magnesium Chloride (MgCl2) (100 % pure) was purchased from Seychelles
Organics, Inc. (Life-Flo, Bowling Green, Florida, USA) and dissolved into a stock solution (1
mM) with deionized water. Stock solution of MgCl2 was further diluted with RPMI-media to
produce aliquots ranging in concentration from 50 - 500 μM.
Treatment: The DU-145 cells were cultured in 25 cm3 flask at 37˚C, 5 % CO2 and 89 % - 90 %
humidity to achieve 80 % - 90 % confluence. The cells were harvested, centrifuged and
reconstituted into suspension with fresh RPMI 1640 media. 1 × 104 cells in 100 μL of media
were dispensed into each well of 96 well microtiter plates and cultured for 48 hours to allow
adherence and to obtain greater than 80 % confluence. The media (supernatants) for each well
were aspirated and the adhered cells were treated with graded concentrations of MgCl2 (50, 60,
70, 80, 90, 100, 200, 300, 400, 500 µM). Experiments had a control group consisting of DU-145
cells cultured in RPMI 1640 media (with 10 % FBS, 1 % penicillin/streptomycin and L-
glutamine) without treatment (0 µM). All treatments were done in quadruplicates, and cultured
for 24 - 48 hours in a humidified incubator at 37˚C and 5 % CO2. At 24 and 48 hours post-
treatment/culture, cells were subjected to bioassay analyses as follows:
Trypan Blue Exclusion Assay: Trypan blue exclusion assay was used to assess the cell viability
and initial concentration of the DU-145 cells added to the microplate wells prior to treatment,
according to standard procedures using a Neubauer counting chamber. The trypan blue exclusion
assay allows for a direct identification and enumeration of live (unstained) and dead (blue) cells
in a given population. Briefly, the trypan blue stock (0.4 %) was diluted with PBS to 0.2 % and
filtered the trypan blue with 0.2 micron filter. The cell suspension was then mixed with the 0.2 %
trypan blue at 1:1 ratio. Counting chamber slide was loaded and analyzed.
MTT Assay: MTT [3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide], a
tetrazolium dye, is used to determine metabolic status and cell viability. Since the resulting color
intensity correlates directly with the amount of metabolically active cells in each well, cell
viability can be quantitatively determined by measuring the optical density (OD) in individual
wells. After briefly treating the cells as described previously, a volume of 20 μL of MTT reagent
was added to each well at 48 hours post-treatment. The plates were further incubated at 37°C and
5% CO2 for 4 hours in the dark, after which a volume of 100 μL of DMSO was added to the cells
to solubilize the formazan. Absorbance (OD) of the resultant solution was read at 490 nm using a
BioTek microplate reader (Winooski, Vermont, USA). The relative population of live cells could
therefore be determined based on the optical absorbance (optical density, OD) of the sample. The
OD obtained was graphed against the concentrations of the MgCl2. The values of the blank wells

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were subtracted from each well of treated and control cells; and the mean percentage of post-
treatment viable cells relative to the controls was calculated as shown:
Cell viability (%) = AT – AB / AC – AB
Where AC is the absorbance of the control (mean value), AT is the absorbance of the treated
cells (mean value), and AB is the absorbance of the blank (mean value). The IC50 was
extrapolated from the graphs as a measure to determine the concentration required to obtain
expected results (potency).
Resazurin (Alamar Blue) Reduction Assay: Resazurin (Alamar blue) is a cell permeable redox
indicator that can be used to monitor viable cell number with protocols similar to those utilizing
the tetrazolium compounds, but slightly more sensitive than tetrazolium reduction assays. Viable
DU-145 cells with active metabolism can reduce resazurin into the resorufin product, which is
pink and fluorescent. This resorufin can then be quantified by measuring a change in absorbance.
Briefly, high purity resazurin was dissolved in DPBS (pH 7.4) to 0.15 mg/ml to make a stock
solution. This was then Filter-sterilized through a 0.2 μm filter into a sterile, light protected
container and stored from light at 4°C for frequent use. 20 μL resazurin solution to each well
containing MgCl2 treated DU-145 cells (in 96-wells microplate at a final volume of 100 μL/well
after 48 hours of incubation at 37°C and 5 % CO2). This was thereafter incubated for 4 hours at
37°C, and the absorbances (OD) of the resultant solution were read at 490 nm using a BioTek
microplate reader (Winooski, Vermont, USA).
Nitro-Blue Tetrazolium (NBT) Assay: On the other hand, Nitroblue tetrazolium (NBT) is a
yellow water-soluble nitro-substituted aromatic tetrazolium compound that reacts with cellular
superoxide ions to form formazan derivative that can be monitored spectrophotometrically. The
cytoplasmic NADPH, which is produced by oxidation of glucose through the hexose
monophosphate shunt, serves as an electron donor, therefore the oxidase system available in the
cytoplasm helps transfer electrons from NADPH to NBT and reduce NBT into formazan. Thus,
the NBT reaction indirectly reflects the ROS-generating activity in the cytoplasm of DU-145
cells. Briefly, DU-145 cells were seeded at 1× 104 cells per well in 96- well microplates and
allowed to attach for 24 hours at 37°C and 5 % CO2. Relevant plates were treated as discussed
previously. NBT (1 mg/mL) in HBSS medium was added to the wells 48 hours after treatment,
and incubated for 4 hours at 37°C in the dark. NBT formazan formed in each wells were then
solubilized with 100 µl of DMSO for 30 minutes. ROS production levels were quantified using
an absorbance microplate reader at 490 nm. The results were expressed as relative percentage of
ROS, and then normalized in which the control was zeroed.
Acridine Orange/Ethidium Bromide Fluorescence Assay: The acridine orange
(AcrO)/ethidium bromide (EtBr) fluorescence assay was used to differentiate between viable,
apoptotic, and necrotic cells based on fluorescence emission characteristics of acridine orange
and ethidium bromide. Acridine orange permeates both viable and nonviable cells, causing the
nuclei to emit green fluorescence. Since absorption of ethidium bromide is based on the
disruption of cell membrane integrity, ethidium bromide selectively stains the nuclei of dead
(non-viable) cells to produce orange or red fluorescence. Cells that emit orange/brown colored

Functional Foods in Health and Disease 2016; 6(1): 1-15 Page 1 of 15
fluorescence are indicative of apoptosis, while necrotic cells emit red fluorescence. Briefly,
ethidium bromide (25 μL) and acridine orange (75 μL) were mixed to make a cocktail in which 3
μL of it was added to 25 μL of the cell suspensions. Wet-mounts were prepared using 10 μL of
each cell suspension and analyzed under a fluorescent microscope with a bandpass filter.
Apoptotic cell death was quantified by counting a total of 100 cells per 2 to 3 fields of view. For
each relevant treatment regimen, fluorescence micro photographed pictures were taken directly
from under the fluorescence microscope using a digital camera (Nikon: Coolpix VR & ISO
2000, Japan).
Rhodamine 123/Ethidium Bromide Fluorescence Assay: The rhodamine 123
(Rh123)/ethidium bromide (EtBr) fluorescence assay was used to assess the mitochondrial
membrane potential (ΔΨm) in treated cells and also used to differentiate between viable,
apoptotic, and necrotic cells, as well as to determine the possible mechanism of apoptosis, based
on mitochondrial transmembrane potential. Rh123 is a cationic fluorochrome, which utilizes the
transmembrane potential of active mitochondria to diffuse into cells. The intact mitochondrial
membranes of viable DU-145 cells allow for the absorption of the dye, resulting in the emission
of bright green fluorescence. DU-145 cells, in which the integrity of the mitochondrial
membrane has been impaired (non-viable cells), stain lightly with Rh123. Meanwhile, ethidium
bromide selectively enters the disrupted membranes of dead cells and stains the nuclei to produce
orange and red fluorescence. Cell samples were briefly washed three times in PBS and re-
suspended in a final volume of 20 μL. 2 μL of the Rh123 stock solution was added to each cell
suspension and was incubated at 37˚C for 5 minutes. 20 μL of EtBr stock solution was then
added to each cell suspension and this was incubated at room temperature for 5 minutes. Finally,
10 μL of each cell mixture was transferred onto a microscope slide covered with a coverslip and
examined/analyzed under a fluorescent microscope with a band-pass filter. Green fluorescence is
indicative of viable cells; orange/brown cells are apoptotic while necrotic cells emit red
fluorescence. The percentage of apoptotic cell death was quantified from an average of 100 cells
spread across two to three regions/views on each slide.
Statistical analysis: Experiments were performed in quadruplicate and repeated twice to
confirm results. Significance of the differences in mean values was determined using Microsoft
excel (v. 2013) and the Student’s t-test. Statistical significance was defined as P < 0.05.
RESULTS:
MgCl2 supplementation inhibited growth and proliferation of DU-145 prostate cancer cells:
DU-145 human prostate cancer cell line was used to determine the chemosensitivity of hormone
independent prostate cancer to MgCl2 supplementation in vitro using MTT and Resazurin
(Alamar blue) reduction bioassays. MgCl2 was found to significantly inhibit cell growth and also
decrease cell survival or viability [Figure 2 & 3]. The data indicated that DU-145 sensitivity to
MgCl2 supplementation is dose-dependent. At lower concentrations, the DU-145 was
significantly more sensitive (P < 0.05) at 90 and 100 µM concentration with cell growth
inhibition of 41% and 47% respectively from the MTT analysis, and 49 % and 42 % from the

Functional Foods in Health and Disease 2016; 6(1): 1-15 Page 1 of 15
Alamar blue analysis. At higher concentrations, the greatest effect in this study was significantly
greatest (P < 0.05) at 500 µM concentration. From the MTT and Alamar blue cytotoxicity
results, the IC50 was determined to be 460 µM and 440 µM respectively or within 440 - 460 µM
range when put together [Figure 2 & 3]. Figure 1 A & B are showing the micrographs of the
control and MgCl2 induced cytotoxic effect at 500 µM respectively; characterized by massive
population of apoptotic cells compared to the control.
Figure 1 A & B: Phase contrast micrographs (200X) of untreated and treated DU-145 cells. Figure 1A:
Showing the micrographs of DU-145 cells on first day of culture in RPMI 1460 media. Figure 1B is
showing the micrographs of untreated viable DU-145 cells at 85 % confluence. Figure 1B is showing
micrograph of DU-145 cells following treatment with MgCl2 at 500 μM. The red arrow points to a cell
that is sensitive (apoptotic) to the treatment. Other cells showing different hallmarks of apoptosis were
observed. This micrograph was taken 48 hours post-treatment.
Figure 2: Graph showing the dose-effect curve of MgCl2-induced cytotoxicity in DU-145 PCa cells via
MTT assay. There was a dose-dependent decrease (P < 0.05) in cell viability in treated DU-145 cells with
graded concentrations from 50 to 500 μM of MgCl2 after 48 hrs. Data points represent the mean values ±
SEM of two independent experiments performed in quadruplicate (P < 0.05).
0
20
40
60
80
100
050 100 150 200 250 300 350 400 450 500
Viable Cell (%)
MgCl2 Concentration (µM)
Graph showing Cell Viability of DU-145 PCa cells to MgCl2 using MTT
Assay

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0
20
40
60
80
100
050 100 150 200 250 300 350 400 450 500
Viable Cell (%)
MgCl2 Concentration (µM)
Graph showing Cell Viability of DU-145 PCa Cells to MgCl2 using Alamar Blue
Assay
Figure 3: Graph showing the dose-effect curve of MgCl2-induced cytotoxicity in DU-145 PCa cells via
Resazurin (Alamar Blue) Reduction Assay. There was a dose-dependent decrease (P < 0.05) in cell
survival in treated DU-145 cells with graded concentrations from 50 to 500 μM of MgCl2 after 48 hrs.
Data points represent the mean values ± SEM of two independent experiments performed in quadruplicate
(P < 0.05).
MgCl2 supplementation induced a compromise in the mitochondrial membrane
potential in DU-145 human prostate cancer cells: In order to determine the effect of
different doses (0, 50, 100, 500 µM) of MgCl2 supplementation on the mitochondrial
membrane integrity, Rhodamine 123/ Ethidium bromide fluorescent stain was used. This also
gives us an assessment of the type of cell death and the mechanism by which apoptosis was
programmed. The Rh123/EtBr fluorescent revealed that about 97 % of the cells in the control
(0 µM) stained green, which demonstrate an intact mitochondrial 3 % undergoing stress
induced apoptosis. Following 48 hours incubation of DU-145 treated with MgCl2 at doses of
50, 100 and 500 µM, we observed a significant dose-dependent increase (P < 0.05) in
apoptotic cells (27 %, 48 % and 54 % respectively) with compromised mitochondria and
reduced transmembrane potential [Figure 4]. They visibly stained orange-brown. This
probably suggests that MgCl2 supplementation utilize the intrinsic-mitochondrial apoptotic
pathway and the activation of caspase proteases for its chemopreventive effects in hormone
independent prostate cancer.

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0%
25%
50%
75%
100%
Control 50 100 500
Cell Population (%)
MgCl2 Concentration (µM)
Treatment-induced Effects on Mitochondria Membrane Potential in DU-145
PCa Cells Apoptotic cells
Viable cells
Figure 4: Bar-charts showing MgCl2-induced effects on the mitochondrial membrane potential (Δψm) of
DU-145 PCa cells via Rh123/ EtBr Fluorescence Assay. There was a significant dose-dependent increase
(P < 0.05) in compromise of the Δψm. Apoptosis appears to be the major mode of cell death. At the
highest concentration (500 μM) more apoptotic cells were observed with reduced mitochondrial
transmembrane potential. Data points represent the mean values ± SEM of two independent experiments
performed in quadruplicate (P < 0.05).
MgCl2 supplementation induced apoptosis in DU-145 human prostate cancer cells
Extensive cell deaths if the treatment induced cell death occurred through cytotoxic necrosis
and / or apoptosis, cells were fluorescently stained with AcrO/EtBr after 48 hrs. of
incubation, in which live cells pick up the acridine orange dye and stain green, while the
damaged cells pick up the ethidium bromide dye and stain orange-brown, depending on the
stage of apoptosis. This can also give us a qualitative state of apoptotic cell death in different
cells exposed to different levels of treatment, especially at 0, 50, 100, and 500 µM
concentrations of MgCl2. The micrographs qualitatively show that cell death was mostly due
to apoptosis in a dose-dependent manner [Figure 7 A - F]. More apoptotic cell death (orange-
brown stained cells) was observed in micrographs containing DU-145 exposed to MgCl2 at
500 µM compared to at 50 µM.

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Figure 7 A to F: Micrographs (200X) of Acridine Orange / Ethidium Bromide fluorescently stained
DU-145 cells following treatment with MgCl2 at 50 μM, 100 μM, and 500 μM. Showing qualitatively
apoptosis as the main form of induced cell death in a dose-dependent mode. Figures 7A, 7C and 7E are
showing the micrographs of AcrO fluorescently stained DU-145 cells at 50 μM, 100 μM, and 500 μM
respectively. Figures 7B, 7D, and 7F are showing the micrographs of EtBr fluorescently stained DU-145
cells at 50 μM, 100 μM, and 500 μM correspondingly. Acridine orange stained live cells green and
Ethidium bromide stained apoptotic cells orange-red depending on the stage of apoptosis. This
micrograph was taken 48 hours post-treatment.

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-25
-10
5
20
35
50
050 60 70 80 90 100 200 300 400 500
Zeored ROS levels (%)
MgCl2 Concentration (µM)
Treatment-induced ROS levels in DU-145 PCa Cells
ROS
MgCl2 supplementation increased ROS generation in DU-145 human prostate cancer cells
Since we already elucidated the effects of MgCl2 supplementation in DU-145, our next goal was
to assess the role of MgCl2 on intracellular ROS generation. The NBT assay was used to assess
the change in intracellular ROS levels following exposure of DU-145 cells to different
concentrations of MgCl2 at 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 µM compared to the
control (0 µM). The data was zeroed to show the increase or decrease in ROS levels relative to
the control or untreated group. NBT assay ROS data also revealed a dose dependent biphasic
response (P < 0.05) in intracellular levels of ROS in DU-145 cells. There was a drop (P < 0.05)
in ROS levels in all treated groups except at 100 µM, where ROS levels were found to be higher
than the control [Figure 5]. This suggests that at 100 µM supplementation, there was a shift from
its antioxidant activity to pro-oxidant activity, but further increase in the dose of MgCl2 returns
the ROS levels to below the control again. Put together, there is the possibility that MgCl2 inhibit
DU-145 cell growth via free radical scavenging in which the ROS levels are dropped below the
normal required for the normal proliferation of the cancer cell. At 100 µM, a significant rise
(P < 0.05) in ROS generation was observed. Either way, the cancer cell viability rate was
still drastically reduced [Figure 6].
Figure 5: Bar-chart showing the MgCl2-induced ROS level in DU-145 PCa cell lines via Nitro-blue
tetrazolium (NBT) assay. There was a biphasic dose-dependent response (P < 0.05) in intracellular levels
of ROS in DU-145 PCa cells with graded concentrations from 50 to 500 μM. Significant decrease in ROS
levels below the control (0 μM) were observed except at 100 μM concentration in treated DU-145 cells
after 48 hrs, where the ROS level was significantly higher (P < 0.05) than the control (0 µM). Data points
represent the mean values ± SEM of two independent experiments performed in quadruplicate (P < 0.05).

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-20
0
20
40
60
80
100
050 100 150 200 250 300 350 400 450 500
Cell viability vs ROS (%)
MgCl2 Concentration (µM)
Correlation between MgCl2 Cell Viability vs ROS in DU-145 PCa Cells
ROS
Cell Viability (AB)
Figure 6: Graphs showing the correlation between the MgCl2-induced Cell viability vs ROS levels.
Both cell viability and ROS generation showed a dose-dependent response in DU-145 prostate cancer
cells treated with different concentrations of MgCl2. Even though at 100 μM, a significant increase (P <
0.05) in ROS generation was perceived, cell viability was positively correlated. Data points represent the
mean values ± SEM of two independent experiments performed in quadruplicate (P < 0.05).
DISCUSSION:
The role of magnesium in cancer prevention and progression is still emerging and at present
tainted with ambiguity from reports in the literature. This is due to multiple role of magnesium in
the body, and it’s overly distribution in different tissues in the body [13]. The status of
magnesium has been shown to be biphasic, in which changes in cellular magnesium can either
stimulate or inhibit cancer growth and progression. In other words, this means that it has both
anticancer effect and also carcinogenic effect. However, we believe that this variation in activity
may be related to activation of cellular transduction pathways as a result of disparity in the
concentration of magnesium in extracellular and intracellular compartments. Many studies have
focused on the extracellular serum levels of magnesium as prognostic and diagnostic tools for
hypomagnesemia or hypomagnesemia [5]; hence neglecting the intracellular regulation of
magnesium in target tissues.
This inequality in intracellular and extracellular levels of magnesium becomes challenging
when diagnosing magnesium deficiency-related disorders, and further supports the fact that
serum magnesium levels may not precisely reflect the overall magnesium status of a patient.
Moreover, tumor cells have been demonstrated to function independently of the extracellular
status of magnesium [14].

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Drugs that have the ability to scavenge for magnesium, such as Cisplatin have been shown
to lower the intracellular levels of magnesium in the body, hence causing hypomagnesemia [15
& 16]. Approximately 50 % of the testicular cancer patients treated with Cisplatin were reported
to have low levels of magnesium due to defect that may be related to magnesium absorption by
the cells. This suggests that disproportionality in the intracellular levels of magnesium in cancer
cells compared to normal cells in the body, as well as the level that can be found in the blood are
factors that should be considered, when making a prognostic and diagnostic inferences,
especially considering the role magnesium may be playing in cancer progression and inhibition.
In addition, hypomagnesaemia has also been associated with the incidence and decreased
survival rate in chronic pathologies such as diabetes, cancer, coronary heart disease and
hypertension [9]. In a histopathological study of a carcinomatous prostate gland, the prostate
cancer cells failed to show staining reactions for magnesium despite appearing morphologically
differentiated. Unlike in the cancer cells, the prostate epithelial cells showed a strong positive
staining of the prostate acini [17].
The scope of this study was to investigate the role of magnesium supplementation in the
prostate cancer development, and also assess the dose-dependent effects of magnesium chloride
in DU-145 - a hormone independent prostate cancer cell line. The cancer cells were subjected to
varied concentrations of magnesium chloride and the level of dose-response was assessed using
batteries of cell proliferation and inhibition assays. In this study, DU-145 PCa cells were found
to be responsive to MgCl2 levels dose-dependently. A significant increase (P < 0.05) in cell
growth inhibition was observed with an increase in concentration of MgCl2. This is in congruity
with prior studies, in which magnesium supplementation was found to show anti-carcinogenic
effect on 3-MC-induced fibrosarcoma [13].
The major mode of cell death was found to be apoptosis, and also found to increase with an
increase in the concentration of MgCl2 supplementation. Apoptosis has been used for many years
by researchers as a dosimeter for programmed cell death, which is controlled by the disparity in
the expression of pro-death and anti-death molecules in the body [18]. This means that the
presence of damaged or mutated cell in a tissue create a pathway geared towards the elimination
of such abnormal cell. Magnesium has also been demonstrated to be involved in membrane
transport and cellular repair [19].
In this study, DU-145 cells treated with MgCl2 were found to show more sensitivity to
magnesium levels, especially at a higher concentration of MgCl2 (100 - 500 μM). We also
observed that high concentrations of MgCl2 caused a relative decrease in the mitochondrial
membrane potential visualized from the fluorescence imaging and staining with Rh123/EtBr.
This is in concert with prior studies, in which magnesium was shown to act as a second
intracellular messenger involved in the regulation of apoptosis downstream. In addition, an
increase in magnesium levels was correlated with a rise in cytochrome C release from the
mitochondria, which is known as a driver for the post-mitochondrial, caspase-mediated apoptotic
events in cells [20]. Another study conducted on drinking water containing magnesium in
Taiwan show that a protective effect from the risk of prostate cancer was apparent in groups with
the highest levels of magnesium in their body [21].
Moreover, the normal range for serum magnesium concentration in humans is between 750
and 950 µM/L [22]. In this study, our IC50 was within 440 - 460 µM range, suggesting that the

Functional Foods in Health and Disease 2016; 6(1): 1-15 Page 1 of 15
presence of magnesium at high levels might be linked with decrease in tumor proliferation or
growth; and hence may be of clinical significance in increasing the survival rate of prostate
cancer patients.
Majority of the cell death recorded in this study were due to apoptosis, reflected by cells
showing different hallmarks of apoptosis. Some of the observed hallmarks include; cell
shrinkage, nuclear condensation, membrane blebbing, and formation of apoptotic bodies similar
to what was previously reported [23]. In a prior study, an increase in the level of magnesium
compounds to 1 mM concentration, was found to increase the apoptotic fragmentation of DNA
in cells after 15 hours of culture and reported to be more pronounced at 30 hours post-treatment
compared to when the cells were not exposed to magnesium compounds at all [24]. Another
study reported Fas-initiated apoptosis as a result of Mg mobilization in B cells [25]. They also
noticed that the increase in Magnesium mobilization correspond with an increase in DNA
fragmentation and externalization of phosphatidylserine, in which the cytosolic increase in levels
of magnesium was found to be higher in cells undergoing apoptosis [12]. Thus, emphasizing the
role of magnesium as a phase II intracellular messenger involved in downstream events in
apoptosis.
Meanwhile, in another study, co-administration of magnesium with antioxidants such as
vitamin C and vitamin E inhibited the apoptotic pathway in more than 50 % cases, indicating that
magnesium may be involved in the cellular redox reactions and extracellular signaling for
apoptosis [26]. This further buttress our finding in which dose-dependent biphasic ROS
generation responses to magnesium chloride supplementation-induced cytotoxicity was
observed. The results presented herein indicate that magnesium chloride supplementation has
dose-dependent modulatory effects - anti-proliferative and apoptotic - on DU-145 prostate cancer
cells, thus emphasizing its potential as a chemopreventive mineral for prostate cancer.
CONCLUSION:
Our finding suggests that magnesium chloride supplementation or a diet rich in magnesium may
be potentially chemopreventive for prostate cancer. This justifies further studies into its
mechanism of action in DU-145 and in other prostate cancer cell types.
Abbreviations: PCa: Prostate cancer; MgCl2: Magnesium chloride; AcrO/EtBr: Acridine
orange/Ethidium bromide; ROS: Reactive oxygen species; Rh123/EtBr: Rhodamine123/
Ethidium bromide; Δψm: Mitochondrial membrane potential; NBT: Nitroblue tetrazolium assay;
MTT: 3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide.
Competing Interests: All authors have declared that there is no conflicting interest.
Authors’ Contributions: All authors contributed equally to this study.
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50 μM 50 μM