Exploring the impact of a naturally occurring sapogenin
diosgenin on underlying mechanisms of Ca2+ movement and
cytotoxicity in human prostate cancer cells
Gwo-Ching Sun1,2 | Chung-Ren Jan3 | Wei-Zhe Liang4
Department of Anesthesiology, Kaohsiung
Medical University Hospital, Kaohsiung,
Department of Anesthesiology, Faculty of
Medicine, College of Medicine, Kaohsiung
Medical University, Kaohsiung, Taiwan, China
Department of Medical Education and
Research, Kaohsiung Veterans General
Hospital, Kaohsiung, Taiwan, China
Department of Pharmacy, Tajen University,
Pingtung, Taiwan, China
Chung-Ren Jan, Department of Medical
Education and Research, Kaohsiung Veterans
General Hospital, Kaohsiung, Taiwan, 81362
Literature has shown that diosgenin, a naturally occurring sapogenin, inducedcytotoxic
effects in many cancer models. This study investigated the effect of diosgenin on intracellular Ca2+ concentration ([Ca2+]i) and cytotoxicity in PC3 human prostate cancer cells.
Diosgenin (250-1000 μM) caused [Ca2+]i rises which was reduced by Ca2+ removal.
Treatment with thapsigargin eliminated diosgenin-induced [Ca2+]i increases. In contrast,
incubation with diosgeninabolished thapsigargin-caused [Ca2+]i increases. Suppression
of phospholipase C with U73122 eliminated diosgenin-caused [Ca2+]i increases.
Diosgenin evoked Mn2+ influx suggesting that diosgenin induced Ca2+ entry. Diosgenininduced Ca2+influx was suppressed by PMA, GF109203X, and nifedipine, econazole,
or SKF96365. Diosgenin (250-600 μM) concentration-dependently decreased cell
viability. However, diosgenin-induced cytotoxicity was not reversed by chelation of cytosolic Ca2+ with BAPTA/AM. Together, diosgenin evoked [Ca2+]i increases via Ca2+
release and Ca2+ influx, and caused Ca2+-non-associated deathin PC3 cells. These findings reveal a newtherapeutic potential of diosgenin for human prostate cancer.
Ca2+ handling, cytotoxicity, diosgenin, endoplasmic reticulum, prostate cancer cell
1 | INTRODUCTION
Diosgenin, a naturally occurring sapogenin, is the main precursor in
manufacturing synthetic steroids.1 Diosgenin has diverse preventive
or therapeutic effects on cardiovascular disorders, diabetes, neurodisorders, allergy, cancers, and so on. Previous studies have shown
that diosgenin may improve metabolic syndrome,2 prevent bone
loss in rats,3 and protect rats with hereditary defined or D-galactose-induced accelerated senescence.4 Regarding cytotoxicity of
diosgenin in cancer cells, diosgenin was found to induce cell death
in K562 human leukemia cells,5 HEL human erythroleukemia cells,6
and Bel-7402, SMMC-7721, and HepG2 human hepatocellular carcinoma cells.7 Furthermore, diosgenin induced ROS-dependent
cytotoxicity in chronic myeloid leukemia cells,8 and inhibited the
hTERT gene expression in A549 lung cancer cell line.9 Although
diosgenin has been shown to reduce cell viability through inhibiting
migration and invasion of PC3 human prostate cancer cells,10 the
impact of diosgenin on Ca2+ homeostasis in PC3 cells needs to be
In terms of effect of diosgenin on Ca2+ movement, calcium
oxalate-induced cytotoxicity in renal cells was prevented by
diosgenin.11 Diosgenin was shown to modulate vascular smooth muscle cell function by blocking receptor-mediated Ca2+ signals in isolated
aorta,12 and stimulate Ca2+-activated K+ current in human cortical
HCN-1A neuronal cells.13 Furthermore, diosgenin was shown to
increase intracellular Ca2+ concentrations ([Ca2+]i
) in mesenteric endothelial cells,14 and to induce Ca2+-associated apoptosis in human leukemia K562 cells.5 While diosgenin can alter Ca2+ homeostasis in
various cell models, the effect of diosgenin on movement and handling of Ca2+ in PC3 cells has not been explored.
Received: 1 August 2019 Revised: 7 October 2019 Accepted: 10 October 2019
Environmental Toxicology. 2019;1–9. wileyonlinelibrary.com/journal/tox © 2019 Wiley Periodicals, Inc. 1
Oscillations in [Ca2+]i play a crucial part in cellular signaling which
modulates numerous cellular responses including fluid secretion, protein
modulation, movement, proliferation, gene expression, fertilization, receptor regulation, death, and so on.15 A [Ca2+]i rise is triggered by two pathways: Ca2+ entry through receptors and/or Ca2+ channels such as storedoperated or voltage-sensitive Ca2+ channels, and by a release of Ca2+
from intracellular stores among which the endothelium reticulum plays a
dominant role.16 Many cytosolic molecules are involved in the modulation
of Ca2+ signaling. Failing to keep [Ca2+]i under control is injurious to cell
health and death can ensue.17 Inasmuch as the importance of understanding the pharmacological action of a compound on a cell, unveiling
the pathway underlying this compound’s effect on [Ca2+]i is vital.
In in vitro research of prostate cancer, the PC3 cell was often used
because it grows rapidly in culture and produces [Ca2+]i increases upon
stimulation of different chemicals. In this cell, it has been reported that
[Ca2+]i rises and death can be evoked by chemicals including
carvacrol,18 timolol,19 and thymol.20 To this end, our study applied fura-
2 as a Ca2+-sensitive probe to measure [Ca2+]i in suspended cells in an
effort to characterize the [Ca2+]i rises, plot the concentration-response
relationship, and uncover the routes underlying diosgenin-induced Ca2+
release and Ca2+ influx. Additionally, WST-1 assays were employed to
reveal the cytotoxic effect of diosgenin on PC3 cells.
2 | MATERIALS AND METHODS
2.1 | Cell lines and cell culture
Human PC3 prostate cancer cells, OC2 oral cancer cells, and MG63 osteosarcoma cells were purchased from Bioresource Collection and Research
Center (Taiwan). PC3 and OC2 cells were cultured in Roswell Park
Memorial Institute (RPMI)-1640 medium. MG63 cells were cultured in
Minimum Essential Medium (MEM). The medium contained fetal bovine
serum (10%) and penicillin (100 U mL−1
)-streptomycin (100 μg mL−1). All
the cells were cultured at 37C in a humidified 5% CO2 atmosphere.
2.2 | Chemicals
Reagents used in cell culture were purchased from Gibco (Gaithersburg,
Maryland). 1,2-Bis(2-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid/
acetoxy methyl (BAPTA/AM) and aminopolycarboxylic acid/acetoxy
methyl (fura-2/AM) were purchased from Molecular Probes (Eugene,
Oregon). Sigma-Aldrich (St. Louis, Missouri) supplied diosgenin
(Figure 1A) and all the other reagents unless otherwise mentioned. The
purity (>98%) of diosgenin was determined by high performance liquid
2.3 | Experimental solutions for [Ca2+]i assays
Ca2+-containing medium had 5 mM KCl, 140 mM NaCl, 1 mM
MgCl2, 2 mM CaCl2, 5 mM glucose, 10 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid, and was titrated to pH 7.4 with
NaOH. Ca2+-free medium had similar reagents as Ca2+-containing
medium apart from CaCl2 was substituted with 2 mM MgCl2 and
0.3 mM ethylene glycol tetraacetic acid (EGTA). Diosgenin was
dissolved in absolute alcohol as a 0.1 M stock solution. Water,
ethanol or dimethyl sulfoxide (DMSO) was used to dissolve the
other chemicals. The level of organic solvents in the experimental
solutions was less than 0.1%, and was found not to affect viability
or the resting [Ca2+]i.
2.4 | [Ca2+]i measurements
Cells were grown on 6 cm dishes until confluence was achieved. They
were trypsinized, rinsed, and suspended in culture medium at a concentration of 1 × 106 cells mL−1
. Subsequently trypan blue exclusion
was applied to determine cell viability which was routinely >95% after
trypsinization. Suspended cells were incubated with 2 μM fura-2/AM
for 30 minutes at 25C in a shaker. Afterward, cells were rinsed with
Ca2+-containing medium twice and then made into a suspension in
Ca2+-containing medium at a concentration of 1 × 107 cells mL−1
cuvette contained 0.5 million cells and 1 mL of medium. Fura-2 fluorescence assays were conducted at 25C with stirring by using a
Shimadzu RF-5301PC spectrofluorophotometer after 0.1 mL cell suspension was added to 0.9 mL Ca2+-containing or Ca2+-free medium.
The data were acquired by recording excitation signals at 340 and
380 nm and emission signal at 510 nm at 1-second intervals. During the
recording, chemicals were administered to the cuvette by stopping the
recording for 2 seconds to open and close the chamber. For calibration
, after completion of the experiments, 0.1% of the detergent
Triton X-100 and 5 mM CaCl2 were included to the cuvette to gain the
maximal fura-2 fluorescence. The Ca2+ chelator EGTA (10 mM) was
then added to chelate Ca2+ to obtain the minimal fura-2 fluorescence.
Trypan blue exclusion assays showed that cells incubated in a cuvette
had a viability of 95% after 20 minutes of experiments. Previously published methodology was applied to calculate [Ca2+]i in our study.21
2.5 | Mn2+ quenching assays
For Mn2+ quenching experiments, fluorescence was recorded in Ca2+-
containing medium containing 50 μM MnCl2 that was added to the
cuvette 30 seconds before the recording began. Fura-2 fluorescence was
measured at excitation signal at 360 nm (Ca2+-insensitive) and emission
signal at 510 nm at 1-second intervals as published previously.22
2.6 | Cell viability measurements
As described previously,18-20 the measurement of cytotoxicity relied
on the capacity of live cells to break down tetrazolium salts by dehydrogenases. Color density will develop proportionally to the amount
of live cells. Protocols were followed according to instructions from
2 SUN ET AL.
the manufacturer (Roche Molecular Biochemical, Indianapolis, IN,
USA). Appropriate numbers of cells were cultured in 96-well plates at
a density of 1 × 104 cells per well in RPMI-1640 medium overnight
with 0 to 600 μM diosgenin. 4-[3-[4-Lodophenyl]-2-4(4-nitrophenyl)-
2H-5-tetrazolio-1,3-benzene disulfonate (WST-1; 10 μL pure solution), a chemical for measuring cell viability was added to each well
after diosgenin incubation for another 30 minutes in an incubator. For
experiments that used BAPTA/AM to bind cytosolic free Ca2+ to abolish [Ca2+]i rises, 5 μM BAPTA/AM was added to the wells for 1 hour
before diosgenin incubation. The cells were washed once with Ca2+-
containing medium and incubated with/without diosgenin for
24 hours. After completion of experiments, optical density was
recorded and normalized to the absorbance of control (unstimulated
cells) and presented as a percentage of control. An enzyme-linked
immunosorbent assay (ELISA) reader was used to process the absorbance of samples (A450).
2.7 | Statistics
Data are reported as mean ± standard error of the mean (S.E.M.) of
three independent experiments (n = 3); and were analyzed by oneway analysis of variances (ANOVA) using the Statistical Analysis System (SAS, SAS Institute Inc., Cary, North Carolina). Posthoc analysis
FIGURE 1 Effect of diosgenin on [Ca2+]i in fura-2-loaded cancer cell models. A, The chemical structure of diosgenin. B, Diosgenin was added
at 25 seconds. The concentration of diosgenin was indicated. The experiments were performed in Ca2+-containing medium. Y axis is the [Ca2+]i
induced by diosgenin in Ca2+-containing medium. C, Effect of diosgenin on [Ca2+]i in the absence of extracellular Ca2+. Diosgenin was added at
25 seconds in Ca2+-free medium. Y axis is the [Ca2+]i increase induced by diosgenin in Ca2+-free medium. (D) Concentration-response plots of
diosgenin-induced [Ca2+]i increases in the presence or absence of extracellular Ca2+. Y axis is the percentage of the net (baseline subtracted) area
under the curve (25 to 250 seconds) of the [Ca2+]i increases induced by 1000 μM diosgenin in Ca2+-containing medium. E, No alteration of
diosgenin on [Ca2+]i in MG63 and OC2 cells. Data are mean ± S.E.M. of three independent experiments. *P < .05 compared to open circles
SUN ET AL. 3
following the Tukey’s HSD (honestly significantly difference) procedure was utilized to conduct multiple comparisons between group
means and a P-value less than .05 represented significance.
3 | RESULTS
3.1 | Action of diosgenin on [Ca2+]i in PC3 cells,
OC2 cells, and MG63 cells
First, the resting [Ca2+]i concentrations in the three cells were 51
± 2 nM (Figure 1B,E). At concentrations of 250 to 1000 μM, diosgenin
induced concentration-dependent increases in [Ca2+]i
. At a concentration of 1000 μM, diosgenin induced [Ca2+]i increases of 85 ± 5 nM
(n = 3). This response saturated at 1000 μM because 1500 μM
diosgenin did not induce a larger one (not shown). In Ca2+-free
medium, diosgenin also induced concentration-dependent increases in
[Ca2+]i at concentrations of 250 to 1000 μM. At a concentration of
1000 μM, diosgenin induced [Ca2+]i increases of 23 ± 2 nM (n = 3)
(Figure 1C). Figure 1D shows the concentration-response relationship.
The EC50 value was 502 ± 3 μM in Ca2+-containing or 508 ± 2 μM in
Ca2+-free medium, respectively, by fitting to a Hill equation (P < .05).
Removal of extracellular Ca2+ suppressed the [Ca2+]i increases by
~60%. Figure 1E shows that two other human cancer cell lines (MG63
cells and OC2 cells) did not alter [Ca2+]i under similar conditions.
3.2 | Diosgenin induced Ca2+ release from the
endoplasmic reticulum in PC3 cells
Since diosgenin-induced Ca2+ response saturated at 1000 μM
(Figure 1), the response induced by 1000 μM diosgenin was used as
control in the following experiments. Efforts were conducted to investigate the Ca2+ release mechanism of diosgenin-induced [Ca2+]i
increases. Because the endoplasmic reticulum has been shown to be
the dominant Ca2+ store in most cell models,23 the role of the endoplasmic reticulum in diosgenin-evoked Ca2+ release was examined.
The experiments were conducted in Ca2+-free medium in order to
exclude the interference of Ca2+ influx. Figure 2A shows that 1 μM
thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor24 induced
[Ca2+]i increases of 25 ± 1 nM (n = 3). Diosgenin (1000 μM) added at
500 seconds did not induce a Ca2+ signal. Conversely, after diosgenin
(1000 μM) induced a Ca2+ signal, addition of 1 μM thapsigargin failed
to induce [Ca2+]i increases (Figure 2B). It seems that diosgenin
induced Ca2+ release dominantly from the endoplasmic reticulum.
3.3 | Phospholipase C (PLC) participated in
diosgenin-induced [Ca2+]i rises in PC3 cells
PLC has been shown to regulate the release of Ca2+ from the endoplasmic reticulum.15-17 Figure 2 shows that diosgenin released Ca2+
from the endoplasmic reticulum, thus the role of PLC was explored.
U73122,25 a PLC inhibitor, was used to explore whether PLC activation
was necessary for diosgenin-induced Ca2+ release. ATP, a PLCassociated agonist, induced [Ca2+]i increases in most cell models.26
Figure 3A shows that ATP (4 μM) induced [Ca2+]i increases of 26
± 2 nM (n = 3). Figure 3B shows that treatment with 2 μM U73122 did
not alter resting [Ca2+]i levels but abolished ATP-induced [Ca2+]i
increases. This suggests that PLC activation was effectively suppressed
by U73122. The data also show that incubation with U73122 and ATP
eliminated 1000 μM diosgenin-induced [Ca2+]i rises. U73343 (2 μM),
the PLC-insensitive structural analog of U73122, was used to test its
effect on ATP-induced Ca2+ signal. Our findings suggest that U73343
(2 μM) did not affect ATP-caused [Ca2+]i increases (not shown). These
data imply that diosgenin-evoked Ca2+ release from the endoplasmic
reticulum relied on PLC activity.
3.4 | Diosgenin evoked Mn2+ influx-induced
fluorescence quenching in PC3 cells
Because Mn2+ enters cells through similar mechanisms as Ca2+ but
quenches fura-2 fluorescence at all excitation wavelengths,27
quenching of fura-2 fluorescence excited at the Ca2+-insensitive excitation wavelength of 360 nm by Mn2+ suggests Ca2+ influx.
Diosgenin-evoked [Ca2+]i increases involved Ca2+ influx were performed in next experiment. Figure 4 shows that 1000 μM diosgenin
evoked a decrease in the 360 nm excitation signal that reached a maximum value of 129 ± 2 arbitrary units at 250 seconds. Therefore, it
appears that Ca2+ influx involved diosgenin-evoked [Ca2+]i increases.
3.5 | Regulation of diosgenin-caused [Ca2+]i rises
in PC3 cells
Efforts were conducted to explore the Ca2+ entry pathways of
diosgenin-induced [Ca2+]i increases. Phorbol 12-myristate 13 acetate
(PMA; 1 nM; a PKC activator), GF109203X (2 μM; a PKC inhibitor),
and three modulators of store-operated Ca2+ entry: econazole
(0.5 μM), nifedipine (1 μM), or SKF96365 (5 μM) were applied 1 minute
before diosgenin (1000 μM) in Ca2+-containing medium. All the
chemicals inhibited diosgenin-induced Ca2+ signal by 20% to 30%
(Table 1). Therefore, diosgenin-induced [Ca2+]i increases involved
PKC-regulated store-operated Ca2+ entry.
3.5.1 | Cytotoxic effect of diosgenin in PC3 cells
Given that acute incubation with diosgenin induced substantial [Ca2+]i
increases, and that unregulated [Ca2+]i rises may change cell
viability,28 experiments were performed to examine the effect of
diosgenin on viability of PC3 cells. Cells were treated with 0 to
600 μM diosgenin for 24 hours, and the tetrazolium assay was performed. In the presence of 250 to 600 μM diosgenin, cell viability
decreased concentration-dependently (Figure 5).
4 SUN ET AL.
3.5.2 | Lack of effect of BAPTA/AM on preventing
diosgenin-induced death in PC3 cells
In order to explore whether diosgenin-induced cell death was caused
by preceding [Ca2+]i rises, the intracellular Ca2+ chelator BAPTA/
AM29 was used to prevent [Ca2+]i rises during diosgenin treatment.
After treatment with 5 μM BAPTA/AM, 1000 μM diosgenin failed to
evoke [Ca2+]i rises (not shown). This suggests that cytosolic Ca2+ was
effectively chelated by BAPTA/AM. Figure 5 also shows that 5 μM
BAPTA/AM loading did not affect the control value of cell viability. In
the presence of 250 to 600 μM diosgenin, BAPTA/AM loading did
not prevent diosgenin-induced cell death. Collectively, the results
imply that diosgenin-evoked death of PC3 cells was dissociated from
preceding rises in [Ca2+]i
4 | DISCUSSION
The impact of diosgenin on prostate cancer models is noteworthy
given the multiple effects of this drug on physiology both in vivo and
in vitro. Because Ca2+ homeostasis regulates cell viability in most cell
models,30 this investigation explored if diosgenin altered Ca2+ movement and handling in PC3 cells. Our data show that diosgenin elevated [Ca2+]i in PC3 cells. The effect was not an artifact because
osteosarcoma and oral cancer cells did not respond to diosgenin’s
stimulation. The Ca2+ signal involved Ca2+ entry and Ca2+ release
because it was reduced by 60% by removing extracellular Ca2+.
The pathways of Ca2+ release underlying diosgenin-evoked [Ca2+]i
increases were examined. Ca2+ release is thought to be released upon
stimulation mainly from the endoplasmic reticulum.30 One of the mechanisms underlying Ca2+ release was via inhibiting the Ca2+-ATPase on
the membrane of the endoplasmic reticulum causing passive leak of
stored Ca2+. Thapsigargin is a chemical extracted from a plant, Thapsia
garganica, that can selectively inhibit this ATPase and induced [Ca2+]i
increases via releasing Ca2+ from the endoplasmic reticulum. The
thapsigargin-sensitive endoplasmic reticulum store seemed to be the
dominant one responsible for diosgenin-induced [Ca2+]i increases
because thapsigargin-induced depletion of the endoplasmic reticulum
Ca2+ store prevented diosgenin from releasing more Ca2+, and vice
versa, diosgenin preincubation abolished thapsigargin-induced Ca2+
A crucial pathway that underlies release of Ca2+ from the endoplasmic reticulum is via Ca2+ release through the inositol 1, 4,
5-trisphosphate (IP3) receptors on the endoplasmic reticulum membrane which are opened by IP3.
15 IP3 and diacylglycerol (DAG) are
formed upon hydrolysis of phosphatidylinositol 4,5-bisphosphate
(PIP2) in the plasma membrane by phospholipase C (PLC). IP3 and
DAG are key second messengers regulating various responses in cells.
IP3 is soluble and when it binds to IP3 receptors on the endoplasmic
reticulum, Ca2+ stored inside is released to the cytosol.16 Thus the role
of PLC in diosgenin-evoked Ca2+ release is examined. The data show
that when PLC activity was inhibited by the selective PLC inhibitor
U73122, the diosgenin-evoked Ca2+ release was inhibited. Therefore,
the Ca2+ release was via a PLC-dependent mechanism.
With regard to Ca2+ influx, quenching of fura-2 fluorescence
excited at the Ca2+-insensitive excitation wavelength of 360 nm by
Mn2+ implies Ca2+ influx. The Mn2+ quenching data support that
Ca2+ influx occurred during diosgenin incubation. In PC3 cells,
store-operated Ca2+ entry plays a critical role in Ca2+ influx.18-20
Nifedipine, econazole, and SKF96365 are often applied to modulate store-operated Ca2+ entry in cellular studies.31-34 Our data
show that 20% of diosgenin-induced [Ca2+]i increases were
inhibited by each of these three compounds. Therefore, it appears
that store-operated Ca2+ entry has a role in diosgenin-caused Ca2+
entry. Since Ca2+ influx accounts for 60% of diosgenin-induced
[Ca2+]i increases, other pathways of Ca2+ entry exist that await
FIGURE 2 Effect of thapsigargin on diosgenin-induced Ca2+ release. A,B, Thapsigargin (TG; 1 μM) and diosgenin (1000 μM) were added at
time points indicated. Experiments were performed in Ca2+-free medium. Data are mean ± S.E.M. of three independent experiments
SUN ET AL. 5
exploration. Studies have shown that in PC3 cells three are some
plasmamemmal cation channels that participate in Ca2+ signaling,
including transient receptor potential vanilloid type 2,35 transient
receptor potential cation channel subfamily M member 8,36 and
transient receptor potential cation channel subfamily C member
6.37 Since selective blockers are still lacking for these channels, the
pathways underlying diosgenin-induced Ca2+ influx in PC3 cells
deserves further investigation.
PKC belongs to the large family of protein kinases that have more
than 60 members playing pivotal regulatory roles in diverse cellular
responses, such as cell differentiation, growth, signal transduction,
survival, proliferation, and death.38 Malfunction of PKC is thought to
lead to cancers, heart diseases, mental disorders, and so on. Ca2+
homeostasis has been shown to regulate (or be regulated by) PKC
activity.39,40 The interaction of PKC and store-operated Ca2+ entry
has been reported. Steatosis was shown to inhibit liver cell storeoperated Ca2+ entry and reduce the endoplasmic reticulum Ca2+ content through a PKC-dependent mechanism.41 The results show that
FIGURE 3 Effect of U73122 on diosgenin-induced Ca2+ release.
Experiments were performed in Ca2+-free medium. A, ATP (4 μM) was
added at 25 seconds. B, First column is 1000 μM diosgenin-induced
[Ca2+]i increases. Second column shows that 2 μM U73122 did not
alter basal [Ca2+]i (*P < .05 compared to first column). Third column
shows ATP-induced [Ca2+]i increases. Fourth column shows that
U73122 pretreatment for 60 seconds abolished ATP-induced [Ca2+]i
increases (*P < .05 compared to third column). Fifth column shows
that U73122 (incubation for 60 seconds) and ATP (incubation for
30 seconds) pretreatment inhibited 1000 μM diosgenin-induced
[Ca2+]i increases (*P < .05 compared to first column). Data are
mean ± S.E.M. of three independent experiments
FIGURE 4 Effect of diosgenin on Ca2+ influx by measuring Mn2+
quenching of fura-2 fluorescence. Experiments were performed in Ca2+-containing medium. MnCl2 (50 μM) was added to cells 1 minute
before fluorescence measurements. The Y axis is fluorescence
intensity (in arbitrary units) measured at the Ca2+-insensitive
excitation wavelength of 360 nm and the emission wavelength of
510 nm. Trace a: control, without diosgenin. Trace b: diosgenin
(1000 μM) was added as indicated. Data are mean ± S.E.M. of three
TABLE 1 Exploring the effect of Ca2+ channel modulators on
diosgenin-induced [Ca2+]i increases in PC3 cells
Note: In modulator-treated group, the regulator was added 1 minute
before diosgenin (1000 μM). The concentration was 2 μM for
GF109203X, 10 nM for PMA, 0.5 μM for econazole, 1 μM for nifedipine,
and 5 μM for SKF96365. Data are presented as the percentage of control
that is the area under the curve (25-200 seconds) of 1000 μM
diosgenin-evoked [Ca2+]i rises in Ca2+-containing medium, and are mean
± S.E.M. of three independent experiments. *P < .05 compared to control.
6 SUN ET AL.
diosgenin-evoked [Ca2+]i increases were inhibited by enhancing or
inhibiting PKC activity by 30%. This suggests that diosgenin induced a
PKC-dependent [Ca2+]i signal and that a normal activity of PKC was
required for a full [Ca2+]i response.
A Ca2+ signal may or may not lead to cell death, depending on the
cell type and stimulus. For example, cell death was observed by Ca2+-
independent NOS activity after oxidative stress in rat striatum.42 In
contrast, Ras/Erk signaling was shown to mediate negative selection
of autoreactive B cells in a Ca2+-associated manner.43 Our findings
show that diosgenin-evoked cytotoxicity appears to be independent
of preceding [Ca2+]i rises because chelation of cytosolic Ca2+ failed to
Prostate cancer cells types derived from different prostate tissues
may have different cytotoxic responses, depending on the physiological function of this particular cell. Thus, the concentration responses
of diosgenin-induced cytotoxicity appear to vary among prostate cancer cell types. Previous studies have demonstrated that diosgenin
between 30 and 240 μM caused cyotoxicity for 48 hours in DU145
human prostate cancer cells.44 The DU145 cell line was derived from
a central nervous system metastasis, of primary prostate adenocarcinoma origin, removed during a parieto-occipital craniotomy.45 DU145
cells are not hormone-sensitive, do not express prostate-specific antigen (PSA) and have moderate metastatic potential compared to PC3
cells, which have high metastatic potential.45 Furthermore, diosgenin
at concentration below 20 μM for 24 or 48 hours did not affect
viability of PC3 cells significantly. Viability of PC3 cells was significantly decreased by diosgenin at 30 μM for 48 hours.10 However, our
data show that diosgenin at 250 μM induced cytotoxicity for 24 hours
in PC3 cells. Therefore, it suggests that the cytotoxic effect of
diosgenin may depend on different origin of cell lines, treatment conditions and exposure times.
Several lines of evidence showed the plasma level of diosgenin in
in vivo studies. The level of diosgenin may reach ~30 μM.46-48 In subjects taking higher doses or with liver or kidney dysfunction the level
may go considerably higher. Diosgenin at 20 to 30 μM was shown to
inhibit testosterone-induced prostate enlargement and may have a
potential for the treatment of benign prostatic hyperplasia.49 Furthermore, diosgenin was shown to inhibit high glucose-induced renal
tubular fibrosis.50 Our results show that diosgenin at a concentration
of 250 μM started to induce cell death. Therefore, this study may be
clinically relevant in some groups of patients.
5 | CONCLUSIONS
Together, in terms of Ca2+ movement, diosgenin evoked PLCdependent Ca2+ release from the endoplasmic reticulum and PKCsensitive store-operated Ca2+ entry. However, diosgenin caused Ca2+-
nonassociated cytotoxicity. Since our data may be clinically relevant in
some patients of prostate cancer, the potential use of diosgenin or its
derivatives to cope with human prostate cancer needs further exploration in in vivo studies.
This study was supported by VGHKS107-169 to C.R.J.
CONFLICT OF INTEREST
The authors report no conflict of interest.
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Diosgenin induces cell cycle arrest and apoptosis in HEL cells with
FIGURE 5 Cytotoxic effect of diosgenin. Cells were treated with
0 to 600 μM diosgenin for 24 hours, and cell viability assay was
performed. Data are mean ± S.E.M. of three independent
experiments. Each treatment had six replicates (wells). Data are
expressed as percentage of control response that is the increase in
cell numbers in diosgenin-free groups. Control had 10 125 ± 212 cells
per well before experiments, and had 13 754 ± 422 cells per well
after incubation for 24 hours. *P < .05 compared to control. In each
group, the Ca2+ chelator BAPTA/AM (5 μM) was added to cells
followed by treatment with diosgenin in medium. Cell viability assay
was subsequently performed
SUN ET AL. 7
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Additional supporting information may be found online in the
Supporting Information sectionGF109203X at the end of this article.
How to cite this article: Sun G-C, Jan C-R, Liang W-Z.
Exploring the impact of a naturally occurring sapogenin
diosgenin on underlying mechanisms of Ca2+ movement and
cytotoxicity in human prostate cancer cells.