Download Materials and Methods

Effects of Bufalin and Cinobufagin on the Androgen Dependent and
Androgen Independent Prostate Cancer Cell Lines
Ching-Han Yu, Hui-Wen Hsu, Pei-Chi Chen, and Paulus S. Wang.
Department of Physiology, School of Medicine, National Yang-Ming University, Taipei (11221),
Taiwan, Republic of China.
Correspondence: Paulus S. Wang. Ph. D.
Department of Physiology
National Yang-Ming University
Shih-Pai, Taipei, R. O. C.
Tel: 886-2-28267082
Fax: 886-2-28264049
Email: [email protected]
Running Title: BF & CB Inhibits Proliferation of Prostate Cancer Cells
Key Words: Chan su, topoisomerase Ⅱ, Na+-K+-ATPase, prostate cancer, apoptosis
ABSTRACT
Purpose: It has been well known that the extracts of Chan-Su are cardiac glycosides, and were
used as treatments for heart failure. Chan-Su has been known to induce cytostatic or oncolytic effects
in several cancers, but few studies about Chan-Su on prostate cancer cells have been reported. This
study was to evaluate the anti-proliferation effects and the underlying mechanisms of bufalin and
cinobufagin, the bufadienolides extracts from Chan-Su, on the androgen-dependent and -independent
prostate cancer cell lines.
Experimental Design: Cell proliferation of three prostate cancer cell lines, LNCaP, DU145 and
PC3, was measured by MTT assay, and calculated for 50% inhibition concentration (IC50) and
doubling time (tD). Both western blot and colorimetric assay were used to analyze the protein
expressions and activities of caspases. Other protein expressions of apoptosis modulators, such as
mitochondrial Bax, and cytosolic cytochrome c, were also analyzed by western blot.
Results: Bufalin and cinobufagin inhibited cell proliferation of all three prostate cancer cell lines
in a dose-dependent manner. The IC50 of bufalin on prostate cancer cell lines was less than that of
cinobufagin. Therefore, the cytotoxicity of bufalin was more serious than that of cinobufagin. In
addition, bufalin and cinobufagin caused cell growth doubling time changes in three prostate cancer
cell lines, which was greater than those in human glomerular cells (human mesangial cells, HMC).
An increase of the active form of caspases expression was observed in the bufalin or
cinobufagin-treated cells, while the caspase activities were also elevated. Protein expressions of
upstream apoptosis modulators, Bax and cytochrome c, were increased.
Conclusion: These results suggest that bufalin and cinobufagin suppress the proliferation and
cause cell apoptosis in androgen-dependent and -independent prostate cancer cell lines via an
increase of the expressions of a sequence of apoptosis modulators and effectors including Bax,
cytochrome c, and a cascade of caspases.
INTRODUCTION
Prostate cancer has the highest incidence, and is the second most common cause of cancer death of
men in the United States (1). In addition, the incidence and mortality of prostate cancer also increase
in Asia countries for the past decade. Traditionally, radiation therapy and surgery are curative
treatments for prostate cancer in situ. Androgen ablation is the mainstay treatment for metastatic
prostate cancer; however, it results in the recurrence of androgen-independent cancer cells in 80% of
patients, which has the median survival for six to nine months (2). There are still no effective
chemotherapy drugs for hormone-refractory prostate cancer. Therefore, it is important to investigate
a novel and effectual treatment for hormone-insensitive prostate cancer.
Bufalin, one of the prominent components of Chan su extracts from the venom of Bufo bufo
gargarizan, is reported as a Na+-K+-ATPase inhibitor which results in an elevation of intracellular
calcium concentration (3). On the basis of this mechanism, bufalin may increase vasoconstriction and
blood pressure, and be used as a treatment for heart failure in Chinese medicine. Disruption of
intracellular calcium homeostasis induces apoptosis in diverse cell types (4). Our previous results
have illustrated that bufalin, with digitalis-like activity, is able to induce the increase of intracellular
calcium and cell apoptosis in prostate cancer cells, but the detail mechanism remains unclear (5). On
the other hand, bufalin is also shown as a topoisomerase Ⅱ inhibitor. Topoisomerase Ⅱ is a nuclear
enzyme that relaxes supercoiled DNA at the time of DNA replication. Topoisomerase Ⅱ inhibitors,
such as etoposide and adriamycin, maintain the complex of topoisomerase Ⅱ and the 5'-cleaved ends
of the DNA resulting in protein-linked DNA double strand breaks (6). Owing to the mechanism
described above, bufalin may induce leukemia cell differentiation (7) and apoptosis (8). Furthermore,
inhibition of solid tumor growth (9), and endothelial cell proliferation and angiogenesis caused by
bufalin in vitro has also been reported (10). Since inhibitors of topoisomerase and Na+-K+-ATPase
have been demonstrated to induce apoptosis in some cancer cell lines, it is interested to investigate
the effects of bufalin and cinobufagin, another major component of Chan su, in androgen-dependet,
LNCaP, and androgen-independent, DU145 and PC3, prostate cancer cell lines.
Inducing cell apoptosis has been the target mechanism for chemotherapy drugs to treat a variety of
cancer. Caspase cascade is a well known key pathway in the apoptotic signal transduction. Caspases
are normally present in the cell as zymogens (procaspases), and could be divided into two types of
subfamilies: upstream initiator caspases (caspase 8 and 9), which are involved in regulatory events,
and downstream effector caspases (caspase 3, 6, and 7), which are directly response for the change of
cell morphological events (11). There are two major apoptotic pathways on the upstream of caspase
cascade to date, which are either the intrinsic pathway (the mitochondria) or the extrinsic pathway
(the cell surface receptors) (12). Because of the character of bufalin as an inhibitor of topoisomerase
Ⅱ, it may induce DNA damage and activate the mitochondrial pathway to regulate cell apoptosis.
Bcl-2 family may be divided into two functional subfamilies such as pro-apoptotic proteins (Bax and
Bid) and anti-apoptotic proteins (Bcl-2 and Bcl-xL). Bcl-2 family members translocate to
mitochondria and mediate the membrane potential to induce cytochrome c release. The cytosolic
cytochrome c is further involved in the signal transduction of caspase activation and finally cause
cell apoptosis.
The rationale to develop bufalin as a potential therapy for prostate cancer is based on its ability to
increase intracellular calcium concentration and to activate mitochondria-mediated cell apoptosis in a
variety of cancer cells. The principal objectives of the present study were to: (a) characterize the
morphological transformation induced by bufalin or cinobufagin, as well as the IC 50 (50% of
inhibition concentration) of both treatments in three prostate cancer cell lines; (b) determine the
alteration of the doubling time (tD) generated by bufalin or cinobufagin during the time of cell
proliferation in three kinds of prostate cancer cells and a normal human cell (human mesangial cells,
HMC) for comparing the toxicity effects in normal cells with that in cancer cells; (c) explore the
mechanism of antiproliferative effects occasioned by both bufadienolides depended on the
measurement of caspase activation and protein expressions of caspases as well as the upstream
molecules, Bax and cytochrome c. Such investigations may, in part, illustrate the antitumor effects of
bufalin and cinobufagin in prostate cancer, and help to introduce a new treatment in cancer patients.
MATERIALS AND METHODS
Cells and Culture Conditions.
Androgen dependent human prostate carcinoma cell line LNCaP was purchased from Culture
Collection and Research Center (CCRC) of Food Industry Research and Development Institute
(FIRDI), Taiwan, ROC. Androgen independent prostate cancer cell lines DU145 and PC3 were
provided by Dr. C. R. Jan (Department of Medical Education and Research, Veterans General
Hospital-Kaohsiung, Kaohsiung, Taiwan, ROC). The primary cultured human mesangial cells (HMC)
were generously given by Dr. L. Y. Yang (Department of Pediatrics, Taipei Veterans General Hospital,
Taipei, Taiwan, ROC). Cell lines were maintained in RPMI 1640 (Gibco Laboratories, Buffalo,
Grand Island, NY, USA)(LNCaP) or in Dulbecco’s Modified Eagle’s Media (DMEM, Gibco
Laboratories, Buffalo, Grand Island, NY, USA)(DU145 and PC3) with 50 IU/ml potassium penicillin
G (Sigma, St. Louis, MO, USA), 50 IU/ml streptomycin sulfate (Sigma, St. Louis, MO, USA) and
10% fetal calf serum (FCS, PAA, Pasching, Austria) as standard media in an atmosphere of 5 % CO2
at 37℃. HMC were cultured in RPMI 1640 media with the presence of 200 mM glutamine, 100 mM
sodium-pyruvate, 10 mM nonessential amino acid, 100 IU/ml insulin, 4 mg/ml transferrin, 50 IU/ml
potassium penicillin G, 50 IU/ml streptomycin sulfate, 250 μg/ml fungizone, and 10% FCS at 37℃
in an atmosphere of 5 % CO2.
Cell Morphology Microscopy.
Cancer cells grown in 10-cm dishes for 24 h (DU145 and PC3) or 48 h (LNCaP) were challenged
with different concentration of drug or a drug-free medium (control condition). At certain time points,
cultures were photographed with a phase contrast microscope (Nikon, Chiyoda-Ku, Tokyo, Japan).
Cell Proliferation Assay.
Cell proliferation was determined by the modified colorimetric 3-(4,5-dimethylthiazol-2-yle)-2,5-
diphenyltetrazolium bromide (MTT) assay. Cells were dispensed at a density of 2000/well of 96-well
microplates in 100 μl medium supplemented with 10% FBS. After cell attachment, the media were
replaced by the new media containing different concentration of bufalin or cinobufagin (day 0). The
MTT (Sigma, St. Louis, MO, USA) assay was performed on day 1, 2, 3, and 4 modified from
Janssen, et al., 1996. Briefly, the media were supplanted by 50 l MTT solution (1 mg/ml in RPMI
1640 or DMEM). After 4-h incubation, the MTT solution was displaced by 50 l DMSO, and the
plates were shaken for 1 min. The optical density of each plate was quantified for absorbance at a
wavelength of 570 nm with a reference wavelength of 630 nm using a microplate reader (Dynatech
Laboratories, Chantilly, VA, USA). Proliferation index of each day referred to the optical density of
that day divided by the optical density of day 0. Each experimental condition was performed in three
preparations and repeated by four times.
Proliferation indexes of different concentration at day 1 to day 4 were calculated and plotted by
fitting to the sigmoidal logistic four parameter nonlinear regression formula (SigmaPlot, Jandel
Scientific Software, San Rafael, CA, USA) (14). IC50 value of each day was determined from plot of
percent of untreated control cell growth versus the logarithm of the drug concentration.
To find out the change of doubling time caused by bufalin or cinobufagin at the concentration of
0.1 M in HMC and three prostate cancer cell lines, the proliferation index were plotted on the
common log scale and fit by linear regression using the equation: y = log(ODt/OD0) = (log2/tD)t,
where ODt and OD0 represent the optical density at the day t or that at the day 0, respectively; tD
represents for doubling time. The doubling time was calculated by tD = Log2/S (days), where S is
the slope of the regression line (15).
Caspase Activity Analysis.
Caspase activation was measured in cytosolic extracts using the peptide substrates, DEVD-pNA,
IETD-pNA, and LEHD-pNA, for caspase 3, 8, and 9, respectively (R&D System, Minneapolis, MN,
USA). Concisely, the drug treated prostate cancer cells were harvested and washed by PBS for three
times. Cell pellet was lysed on ice for 10 min, and then samples were centrifuged at 10,000 x g for 5
min. Protein content in the supernatants was measured using the Bradford reagent. Extracts were
diluted to 4 g/l with dilution buffer, and loaded with 50 l for each reaction well of 96-well
microplate. Each reaction also required 50 l of 2 x reaction buffer containing 10 mM of
dithiothreitol and 5 l of caspase colorimetric substrate. After 2-h incubation at 37℃, absorbance
was measured at 405 nm on a microplate reader. Results are expressed as absorbance percent relative
to control group (100%). Each experimental condition was repeated by three times.
Immunoblotting Assessment
After culture under the treatment of bufalin or cinobufagin at certain time points, cells were
harvested and washed twice by ice-cold PBS. Cells were lysed in RIPA buffer (50 mM Tri-HCl, pH
7.4, 1 % NP-40, 0.25 % Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, l mM phenylmethylsulfonyl
fluoride, 1 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml pepstatin, 1mM Na-orthovanadate, 1 mM
NaF) for 30-min on ice. The lysate was centrifuged at 14,000×g in 4℃ for 15 min, and the
supernatant was collected. Equal amount of cell extract proteins (50-100 g) were subjected to 10%
(caspase 9) or 15% (caspase 3 and 8) SDS- polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH, USA). Membranes were
incubated in blocking solution (5% dry milk in TBST containing 20 mM Tris-HCl, 135 mM NaCl,
0.1 % Tween 20, pH 7.6) followed by incubation with primary antibody overnight. The following
primary antibodies were used as 1 g/ml: caspase 3 from Imgenex (San Diego, CA, USA); caspase 8
from BioVion (Mountain View, CA, USA), caspase 9 from Medical and Biological Labortories
(Nagoya, Japan); or β-actin from Sigma (St. Louis, MO, USA). After washing for three times by
TBST, the blot was incubated with horseradish peroxidase-conjugated goat anti-mouse secondary
antibody (1:20,000, Promega Corporation, Madison, WI, USA), and proteins were visualized using
enhanced chemiluminescence detection (ECL, Western blotting reagents, Amersham International,
Bucks, UK).
Isolation of Cytosol and Mitochondrial Fractions
Release of cytochrome c from mitochondria and translocation of bax to mitochondria was
measured by immunoblotting essentially as described previously (16). Briefly, drug treated cancer
cells were collected and washed twice by ice-cold PBS. The cell pellet was resuspended in 1 ml of
extraction buffer (20 mM K+-Hepes, 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2 · 6H2O, 0.1 mM
EDTA, 1 mM EGTA, and protease inhibitors). Cells were lysed by 40 passages through a 26-gauge
needle, and homogenates were centrifuged at 1000 x g for 5 min. The supernatant was again
centrifuged at 10,000 x g for 15 min following the separation of supernatant (cytosolic fraction) from
pellet (mitochondrial fraction). Cytosolic fraction was then concentrated to 50-100 l using 10-kDa
molecular mass centrifugal concentration device (microcon YM-10, Millipore Co., Bedford, MA,
USA) according to the manufacturer’s instructions. The resulting mitochondrial pellets were
resuspended in 50 l of cell lysis buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton, 1 mM
phenylmethylsulfonyl fluride, and protease inhibitor mixture). These fractions were separated on
15% SDS-polyacrylamide electrophoresis gels with an equal amount of protein loaded onto each lane
as determined by Bradford assay reagent. Cytochrome c and bax were detected by mouse
monoclonal antibody at dilution of 1:400 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Each experimental condition was replicated by three times.
Statistics
All values are given as the mean  standard error of the mean (SEM). In some cases, Student’s t
test was used. Others, means were tested for homogeneity by one-way analysis of variance
(ANOVA), and the difference between specific means was tested for significance by Duncan’s
multiple-range test (17). The difference between two means was considered statistically significant
when P<0.05.
RESULTS
Antiproliferative Effects of Bufalin and Cinobufagin on Human Prostate Cancer Cells
Our previous results indicated that bufalin and cinobufagin inhibit the proliferation of human
prostate cancer cells (5). In the present study, the detail mechanisms of antiproliferative effects of
bufalin and cinobufagin were investigated. The change of cell morphology caused by bufalin or
cinobufaogn was shown in figure 1 using phase contrast microscope with two hundred times of
magnification. After a treatment with 10 M bufalin or cinobufagin for 24-h, the cell number of
LNCaP and DU145 cells decreased, and the cells began to draw back their membranes and became a
round shape. Some of these cells even rounded up from the dishes and floated in media, especially
for the weak attachable LNCaP cells. However, the above phenomenon was found in PC3 cells after
treatment for 48-h. In addition, bufalin is more effective than cinobufagin to inhibit the proliferation
in these three prostate cancer cell lines.
According to the different responses of these three prostate cancer cell lines, bufalin was used at
the concentration of 0.01 to 1 M for LNCaP cells and 0.001 to 0.1 M for DU145 and PC3 cells.
Concentration of 0.01 to 1 M of cinobufagin was used for three of the prostate cancer cells.
Proliferation status was measured by MTT assay. Proliferation indexes of different concentrations at
day 1 to day 4 were calculated and plotted to fit by linear regression analysis. IC50 value of each day
was calculated from nonlinear regression equation of that specific day. (Table 1). In three prostate
cancer cell lines, IC50 values of bufalin were less than that of cinobufagin from day 1 to day 4
(P<0.01). As the day of treatment increased, the IC50 values of both bufadienolides were decreased
significantly as compared with day 1 in all three prostate cancer cells (P<0.01). Determination of
IC50 values indicated that the androgen-independent prostate cancer cells, DU145 and PC3, were
more sensitive to both bufadienolides, whereas those for the LNCaP cells were approximately 5 to 10
times more than that in DU145 and PC3 cells.
The Change of Doubling Time Caused by Bufalin and Cinobufagin in HMC and Prostate
Cancer Cells
Previous results indicated that bufalin and cinobufagin have antiproliferative effects on prostate
cacner cells. Thus, it is important to examine whether inhibition of proliferation could reveal in
bufadienolides-treated normal human cells. In this study, the change of doubling time caused by
bufalin or cinobufagin at the concentration of 0.1 M in HMC and three prostate cancer cell lines
was determined by MTT assay. Proliferation indexes of day 1 to day 4 with or without treatment
were fitted by linear regression equation (Fig. 2). Doubling times of control or drug-treated groups
were computed from the regression equations. After the treatment of bufalin, the doubling time of
LNCaP cells increased from 31 h to 105 h, and the similar effects could be found in cinobufagin
treated LNCaP cells. The increase of doubling time by bufalin or cinobufagin in LNCaP cells was
approximately 3.1 times (P<0.01). Nevertheless, bufalin and cinobufagin were more effective on
prolonging the doubling time in DU145 and PC3 cells for 5.8 or 8.6 times, respectively (P<0.01).
However, the lengthening of doubling times in HMC cells of bufalin or cinobufagin is slightly and
not significantly. Consistent with our previous observations, the doubling time of bufalin-treated
prostate cancer cells increased more than that of cinobufagin treatment.
Effects of Bufalin and Cinobufagin on Proteolytic Processing of Caspases
Our recent studies suggested that apoptosis mechanism was involved in the antiproliferation
effects caused by bufalin and cinobufagin (5). To examine whether bufalin and cinobufagin activated
the caspases cascade, proteases of the apoptotic pathway, we measured caspase 3, 8, and 9 activities
at different time points in three types of prostate cancer cell lines (Fig. 3). Owing to the distinct
nature of three prostate cancer cells, colorimetric caspase activity assay were performed after
treatment in LNCaP and DU145 cells for 8-, 12-, 18-, and 24-h (Fig. 3a and 3b), whereas in PC3
cells were for 18-, 24-, 36-, and 48-h (Fig. 3c). In LNCaP cells, bufalin at the concentration of 10 M
increased the activities of caspase 3, 8, and 9 from 200% to 250% (P<0.01) since treatment for 12-h,
and the activity of effector caspase 3 was much than that in initiator caspase 8 and 9. The peak
response appeared at the time point about treatment for 18-h. Moreover, at the concentration of 1 M
of bufalin, elevated caspase activities from 150% to 250% (P<0.05) during the 12- and 24-h time
points. Cinobufagin had similar effects as bufalin in LNCaP cells; only the increasing ranges were
not as much as that caused by bufalin. Bufalin and cinobufagin induced resembling results in DU145
cells. In PC3 cells, bufalin at concentration of 1 and 10 M increased caspase 3 activity from 150%
to 250% (P<0.01) since treatment for 18-h, and activities of caspase 8, and 9 were slightly increased.
The peak response showed at the time point about treatment for 24-h. Cinobufagin had similar
effects as bufalin in PC3 cells. In summary, bufalin was more effective on increasing caspase activity
in LNCaP and DU145 cells than that of cinobufagin, whereas these two bufadienolides made no
much different in PC3 cells.
On the basis of these results described above, immunoblot analyses of whole cell lysates obtained
from three kinds of prostate cancer cells undergoing apoptosis during treatment with bufalin or
cinobufagin were performed using antibodies for caspase 3, 8, and 9 at the time point in peak
response of the increasing of caspase activity (Fig. 4). Caspase exist as inactive zymogens in resting
cells, and activation of a particular caspase will result in a reduction in its proenzyme form (18). We
found that protein levels of procaspase 3, 8, and 9 were down regulated as increasing the
concentration of bufalin or cinobufagin, meanwhile, the proteolytic cleavage of these caspases was
detectable in cell lysates obtained after 18-, 18-, 24-h of treatment in LNCaP, DU145, and PC3 cells,
respectively. Active forms of these caspases were elevated while concentration of treatment
increased.
Effects of Bufalin and Cinobufagin on Bax Translocation and Cytochrome c Release
As noted above, caspase activation involved in apoptotic effects induced by the treatment of
bufalin and cinobufagin. Alterations of mitochondria leading to the release of cytochrome c are
thought to mediate caspase activation in many models of cell death programs. The role of
mitochondria is crucial participants in cell apoptosis by targeting Bcl-2 family members to the outer
membrane of mitochondrial (19). We used Western blotting to analyze the protein levels of
mitochondrial bax, a proapoptotic molecule in Bcl-2 family, and cytosolic cytochrome c. Consistent
with this mechanism, treatment of bufalin or cinobufagin resulted in an increase of bax in
mitochondrial fraction, and in release of cytochrome c into cytosolic fraction of three prostate cancer
cell lines (fig. 5). The first noted time points in LNCaP (fig. 5a), DU145 (fig. 5b), and PC3 (fig. 5c)
cells were 8-, 8-, and 18-h, respectively. The increase of cytochrome c release and bax translocation
is in a dose- and time-dependent manner. These results mentioned above appeared to precede
activation of caspase 3, 8, and 9 (compare Figs. 3 and 5).
DISCUSSION
We have demonstrated the following phenomena: (a) bufalin and cinobufagin cause the
morphological change (cell rounds up) and reducing cell number in androgen-dependent and
-independent prostate cancer cell lines. (b) IC50 of treatment is relatively high in androgen-dependent,
LNCaP, cells than in androgen-independent, DU145 and PC3, cells. Besides, prolongating the time
of treatment declines the IC50 in three prostate cell lines. (c) bufalin and cinobufagin elongate the
doubling time of cell proliferation selectively in prostate cancer cells rather than in HMC cells; (d)
both bufadienolides induce prostate cancer cell apoptosis through caspase activation, and the peak
responses are at the time points of 18-, 18-, and 24-h in LNCaP, DU145, and PC3 cells, respectively;
(e) mitochondrial pathway is involved in the upstream of caspase cascade by releasing cytochrome c
from mitochondria to cytosol, and the proapoptotic Bcl-2 family member, Bax, manipulates this
phenomena.
Nowadays, prostate cancer is a worldwide malignant and age-related cancer in men. Although PSA
detection assay is used widespread to help the early diagnosis of prostate cancer, metastasis still
occurs in about 30% of patients within 3 years of treatment (20). The nature history of prostate
cancer follows a formation of progression from localized disease with androgen-dependent character
to more accelerated, invasive and metastatic disease which often connects with gain of
androgen-refractory capacity (21). Men with metastatic prostate cancer are prospected to survive for
six to nine months by means. Therefore, it is essential to develop an efficacious therapy targeting to
the character of prostate cancer and to eliminate the ravage cause by prostate cancer in men.
Bufalin is a digitalis-like molecule of animal source and is also able to inhibit Na+-K+-ATPase by
binding to this enzyme on the cellular membrane. The capacity of bufalin to inhibit Na+-K+-ATPase
is much more than that in ouabain which is the most effective molecule among digitalis to induce the
antiproliferative effects in prostate cancer cells (22, 23). On the basis of this mechanism, the influx of
extracellular calcium results in an elevation of intracellular calcium concentration. Role of
intracellular calcium have been well studied in prostate cells. Androgen ablation leads to apoptosis
associated with calcium influx and calcium-regulated endonuclease activation in normal prostate
cells (24). Besides, thapsigargin, a calcium ionophore, directly stimulates an increase of intracellular
calcium, which triggers apoptosis in androgen-dependent and androgen-refractory prostate cancer
cells (25). Importantly, prostate cancer cells express the Na+-K+-ATPase, which presents as the target
for digitalis-like drugs (26). In addition to be an inhibitor of Na+-K+-ATPase, bufalin also functions
as an inhibitor of topoisomerase Ⅱ. Topoisomerase Ⅱ is the target of several antitumor drugs, such
as etoposide, adriamycin, genistein, and ICRF-193 (27). By blocking the enzyme function of
topoisomerase Ⅱ, DNA double strand breaks remain, and DNA damage occurs. Cells are obviously
able to recognize such DNA damage and to eliminate the over injured cells by apoptosis.
On the basis of either increase intracellular calcium or stimulating DNA damage, bufalin may
cause cell apoptosis. In this study, we use bufalin and another active component of Chan su,
cinobufagin, to inspect the effects induced by both bufadienolides in androgen-dependent and
-independent prostate cancer cells. Our result shown that bufalin and cinobufagin caused cell round
up from the culture dish (Fig. 1). Because of the light-attached character in LNCaP cells, the
appearance of LNCaP cells was totally unattached after treating with bufalin or cinobufagin. With the
nature of tight attachment, longer time of treatment was demanded for PC3 cells to observe round up
cells. Besides, the mechanism of caspase 3-mediated apoptosis was reported to process focal
adhesion kinase (FAK), in turn, results in floating cells (28, 29). This signal transduction might be
partly involved in the unattached cells treated by bufalin or cinobufagin.
Digitalis, a drug family for congestive heart failure and supraventricular arrhythmias, has a narrow
therapeutic range with plasma level from 0.8-2.0 ng/ml in patients (30). Bufalin, a digitalis-like
molecule from the animal source, has a potential of fatal toxicity similar to digitalis, and can be
eliminated with digitalis specific antibody fragment (Fab) (31). It is important to estimate the
effective drug concentration on the antiproliferative efficacy in prostate cancer cells. Results of IC50
after treatment for 1 to 4 days in three prostate cancer cell lines were shown in Table 1. Although
values of IC50 from day 1 to 4 were higher than the therapeutic range of digitalis (calculated as 2.1 to
5.2 nM for bufalin; 1.8 to 4.5 nM for cinobufagin), IC50 values declined significantly as prolonging
the time of treatment. By means of this event, it is possible that bufalin and cinobufagin in the
therapeutic range may cause prostate cancer cell death without poisoning the patient by prolonging
the time of treatment. After a 24-h treatment, bufalin and cinobufagin at the concentration of 10 μM
caused LNCaP cells round up from the dish completely. It seems that LNCaP cells are more sensitive
to both bufadienolides compared with DU145 and PC3 cells. However, results of IC50 reflected that
high doses of treatment of digitalis were required to reduce cell numbers to 50% of control cells in
LNCaP cells. Combination of both phenomena, LNCaP cells were less sensitive to bufalin or
cinobufagin, and the cell floating caused by treatments was probably owing to the nature of
light-attached character.
Since bufalin and cinobufagin might induce cell apoptosis in prostate cancer cells, the cytotoxic
effects of treatments in normal human cells was expected to explore. The doubling time of cell
proliferation was used to monitor the cytotoxicity in three prostate cancer cell lines and in normal
human cells (HMC). Observations shown in figure 2 indicated that increase of doubling time was
significantly in prostate cancer cells rather than in HMC cells with bufadienolides at the
concentration of 0.1 μM. Moreover, elongation of doubling time in androgen-independent prostate
cancer cells was much longer than that in androgen-dependent cells. After acting by a topoisomerase
Ⅱ inhibitor, DNA breaks were formed in bufalin-treated cells at the time of DNA replication (6).
With relatively short growth period, the formation of DNA damage in DU145 and PC3 cells would
be more than that in LNCaP cells. Most of normal cells stay in the cell cycle at Go period, which will
be less sensitive to proliferating cells-targeted chemotherapeutic agents. In human mesangial cells,
the doubling time prolonged slightly with no significance. Similar results found previously
demonstrated that digitalis had no particular effects in the normal glomerular epithelial cells (23).
Although human mesangial and glomerular epithelial cells can not be represented for each kind of
normal human cells, it is apparently that the antiproliferative effects of bufalin targets to proliferate
cancer cells other than to normal cells with slow growth period.
Caspase activation was involved in the apoptotic signal transduction induced by digitalis or bufalin
(5, 32). We treated cancer cells with bufalin or cinobufagin at the concentration of 0, 0.1, 1, and 10
μM in order to examine the signal transduction of treatments, immediately. Figure 3 and 4 illustrated
that the time point of the peak response of caspase procession differed in three kinds of prostate
cancer cells. In LNCaP and DU145 cells, the peak response occurred around 18-h after treatment,
whereas, in PC3 cells, it was slightly late (about 24-h). It have been reported that DNA damage
might induce p53 activation and Bax translocation to mitochondria, in turn, resulted in release of
cytochrome c (6, 33). We also investigated the effects of bufalin or cinobufagin on the upstream
molecules of Bax and cytochrome c in mitochondrial pathway. The increase of protein expressions of
Bax and cytochrome c in LNCaP and DU145 cells were observed after treatment for 8-h, whereas,
those for PC3 cells were at 18-h. To conclude the circumstances found in bufalin treated LNCaP,
DU145, and PC3 cells, it was easier to induce apoptotic signal transduction in LNCaP and DU145
cells than in PC3 cells. However, the results of IC50 and the change of doubling time indicated that
bufalin is more effective in PC3 cells as prolonging the time of treatment. It might be resulted from
the nature of these three prostate cancer cell lines. The fact that LNCaP cells which have wild type
p53 and the character of releasing prostate specific antigen (PSA) are thought to be more sensitive to
chemotherapeutic drugs than the androgen-independent prostate cancer cells with mutant type p53
(34, 35). It was also found that topoisomerase Ⅱ was expressed at the highest level in rapidly
proliferating DU145 cells (36). Loss the enzyme activity by bufalin treatment resulted in dramatic
DNA damage and cell apoptosis. These results in part explain the different effects caused by bufalin
in three types of prostate cancer cell lines.
Bufalin induce DNA double strand breaks with the function of topoisomerase Ⅱ inhibitor. DNA
damage caused by topoisomerase Ⅱ inhibitor might induce the phosphorylation of p53. The active
form of p53 prompted Bax translocation to mitochondria and cytochrome c release to cytosol (6, 33).
The mechanism described above might be the signal transduction caused by bufalin in LNCaP cells
with wild-type p53. We also found the protein expression of topoisomerase Ⅱ declined and that of
p53 elevated as increase the concentration of bufalin and cinobufagin in LNCaP cells (data not
shown). In DU145 and PC3 cells, which has mutant and null p53, c-Jun NH2-terminal kinas
(JNK)-dependent Fas activation apoptosis may in place of the p53-dependent apoptotic pathway (26,
37). In Fas-manipulated apoptotic pathway, activated caspase 8 can directly activate the downstream
caspase 3, and it can also cross talk with mitochondrial pathway through cleavage of Bid, a Bcl-2
family protein. Truncated Bid (tBid) translocates to mitochondria, inducing cytochrome c release,
which sequentially activates caspase 9 and 3 (38). These investigations concluded that bufalin
induced apoptosis, either via p53 or Fas, might results in Bax translocation, cytochrome c release,
and caspase 3, 8, and 9 activation.
It is known that bufalin induces cell apoptosis by blocking topoisomerase Ⅱ (8, 9) or by inhibiting
Na+-K+-ATPase (5). However, Pastor et al. shown that inhibition of topoisomerase Ⅱ were not
sufficient for bufalin to cause cell apoptosis. In addition, ouabain resistant cells would not go on
apoptosis by blocking the Na+-K+-ATPase (40). It also demonstrated that intracellular calcium ions
play an essential role in induction of apoptosis by inhibitors of topoisomerase (41). On the basis of
those results, the induction of apoptosis by bufalin may attribute to the inhibition of both
topoisomerase Ⅱ and Na+-K+-ATPase. Besides, lowering the level of topoisomerase Ⅱ sensitized
cancer cells to other chemotherapeutic agents, such as cisplatin (42). It suggested that a combination
of anticancer agents with bufalin might elevate the efficacy and lower the side effects of both
treatments.
In this present observations, bufalin and cinobufagin had antiproliferative effects in
androgen-sensitive and -refractory prostate cancer cells through an increase of mitochondrial Bax,
cytosolic cytochrome c, and a cascade of caspases.
REFERENCES
1.
Jemal, A., Tiwari, R. C., Murray, T., Ghafoor, A., Samuels, A., Ward, E., Feuer, E. J., and Thun,
M. J. Cancer statistics, 2004. CA Cancer J. Clin., 54: 8-29, 2004.
2.
Kantoff, P. W. New agents in the therapy of hormone-refractory patients with prostate cancer.
Semin. Oncol., 22: 32-34, 1995.
3.
Bagrov, A. V., Roukoyatkina, N. I., Fedorova, O. V., Pinaev, A. G., and Ukhanova, M. V.
Digitalis-like and vasoconstrictor effects of endogenous digoxin-like factor(s) from the venom
of Bufo matinus toad. Eur. J. Pharmacol., 234: 165-172, 1993
4.
McConkey, D. J., and Orrenius, S. The role of calcium in the regulation of apoptosis. Biochem.
Biophys. Res. Commun., 239: 357-366, 1997.
5.
Yeh, J. Y., Huang, W. J., Kan, S. F., and Wang, P. S. Effects of bufalin and cinobufagin on the
proliferation of androgen dependent and independent prostate cancer cells. Prostate, 54:
112-124, 2003.
6.
Karpinich, N. O., Tafani, M., Rothman, R. J., Russo, M. A., and Farber, J. L. The course of
etoposide-induced apoptosis from damage to DNA and p53 activation to mitochondrial release
of cytochrome c. J. Biol. Chem., 277: 16547-16552, 2002.
7.
Zhang, L., Nakaya, K., Yoshida, T., and Kuroiwa, T. Induction by bufalin of differentiation of
human leukemia cells HL60, U937, and ML1 toward macrophage/monocyte-like cells and its
potent synergistic effect on the differentiation of human leukemia cells in combination with
other inducers. Cancer Res., 52: 4634–4641, 1992.
8.
Watabe, M., Masuda, Y., Nakajo, S., Yoshida, T., Kuroiwa, Y., and Nakaya, K. The cooperative
interaction of two different signaling pathways in response to bufalin induces apoptosis in
human leukemia U937 cells. J. Biol. Chem., 271: 14067–14072, 1996.
9.
Yoshida, T. The presence of a common antigen between human gastric cancer cells and OK-432.
Biotherapy, 7: 1682-1688, 1993.
10. Lee, D. Y., Yasuda, M., Yamamoto, T., Yoshida, T., and Kuroiwa, Y. Bufalin inhibits endothelial
cell proliferation and angiogenesis in vitro. Life Sci., 60: 127-134, 1997.
11. Ranger, A. M., Malynn, B. A., and Korsmeyer, S. J. Mouse models of cell death. Nature Genet.,
28: 113-118, 2001.
12. Shi, Y. A structural view of mitochondria-mediated apoptosis. Nature Struct. Biol., 8: 394-401,
2001.
13. Janssen, T., Darro, F., Petein, M., Raviv, G., Pasteels, J. L., Kiss, R., and Schulman, C. C. In
vitro characterization of prolactin-induced effects on proliferation in the neoplastic LNCaP,
DU145, and PC3 models of the human prostate. Cancer, 77: 144-149, 1996.
14. Barbosa, M. D. F. S., Ross, H. O., Hillman, M. C., Meade, R. P., Kurilla, M. G., and Pompliano,
D. L. A multitarget assay for inhibitors of membrane-associated steps of peptidoglycan
biosynthesis. Anal. Biochem., 280: 315-319, 2002.
15. Zhuang, S. H., and Burnstein, K. L. Antiproliferative effects of 1α,25-dihydroxyvitamin D3 in
human prostate cancer cell line LNCaP involves reduction of cyclin-dependent kinas 2 activity
and persistent G1 accumulation. Endocrinology, 139: 1197-1207, 1998.
16. Gottlieb, R. A., and Granville, D. J. Analyzing mitochondrial changes during apoptosis.
Methods, 26: 341-347, 2002.
17. Steel, R. G.. D., and Torrie, J. H. Principles and procedures of statistics. McGraw-Hill, New
York, 1960.
18. Mesner, P. W. Jr., Bible, K. C., Martins, L. M., Kottke, T. J., Srinivasula, S. M., Svingen, P. A.,
Chilcote, T. J., Basi, G. S., Tung, T. S., Krajewski, S., Reed, J. C., Alnemri, E. S., Earnshaw, W.
C., and Kaufmann, S. H. Characterization of caspase processing and activation in HL-60 cell
cytosol under cell-free conditions. Nucleotide requirement and inhibitor profile. J. Biol. Chem.,
274: 22635-22645, 1999.
19. Bernardi, P., Petronilli, V., Di Lisa, F., and Forte, M. A mitochondrial perspective on cell death.
Trends Biochem. Sci., 26: 112-117, 2001.
20. Isaacs, J. T., Lundmo, P. I., Berges, R., Martikainen, P., Kyprianou, N., and English, H. F.
Androgen regulation of programmed death of normal and malignant prostate cells. J. Androl.,
13: 457-464, 1992.
21. Small, E. J., Reese, D. M., and Vogelzang, N. J. Hormone-refractory prostate cancer: An
evolving standard of care. Semin. Oncol., 26: 61-67, 1999.
22. Bick, R. J., Poindexter, B. J., Sweney, R. R., and Dasgupta, A. Effects of Chan Su, a traditional
Chinese medicine, on the calcium transients of isolated cardiomyocytes: cardiotoxicity due to
more than Na, K-ATPase blocking. Life Sci., 72: 699-709, 2002.
23. Yeh, J. Y., Huang, W. J., Kan, S. F., and Wang, P. S. Inhibitory effects of digitalis on the
proliferation of androgen dependent and independent prostate cancer cells. J. Urol., 166:
1937-1942, 2001.
24. Kyprianou, N., English, H. F., and Isaacs, J. T. Activation of a Ca2+-Mg2+-dependent
endonuclease as an early event in castration-induced prostatic cell death. Prostate, 13: 103-117,
1988.
25. Furuya, Y., Lundmo, P., Short, A. D., Gill, D. L., and Isaacs, J. T. The role of calcium, pH, and
cell proliferation in the programmed (apoptotic) death of androgen-independent prostate cancer
cells induced by thapsigargin. Cancer Res., 54: 6167-6175, 1994.
26. McConkey, D. J., Lin, Y., Nutt, L. K., Ozel, H. Z., and Newman, R. A. Cardiac glycosides
stimulate Ca2+ increases and apoptosis in androgen-independent, metastatic human prostate
adenocarcinoma cells. Cancer Res., 60: 3807-3812, 2000.
27. Chen, A. Y., and Lin, L. F. DNA topoisomerase: essential enzymes and lethal targets. Annu. Rev.
Pharmacol. Toxicol., 34: 191-218, 1994.
28. van de Water, B., Nagelkerke, J. F., and Stevens, J. L. Dephosphorylation of focal adhesion
kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during
apoptosis in renal epithelial cells. J. Biol. Chem., 274: 13328-13337, 1999.
29. Sasaki, H., Kotsuji, F., and Tsang, B. K. Caspase 3-mediated focal adhesion kinase processing in
human ovarian cancer cells: possible regulation by X-linked inhibitor of apoptosis protein.
Gynecol. Oncol., 85: 339-350, 2002.
30. Shrestha, S., Paeng, I. R., Deo, S. K., and Daunert, S. Cysteine-free mutant of aequorin as a
photolabel in immunoassay development. Bioconjugate Chem., 13: 269-275, 2002.
31. Brubacher, J. R., Lachmanen, D., Ravikumar, P. R., and Hoffman, R. S. Efficacy of digoxin
specific Fab fragments (Digibind) in the treatment of toad venom poisoning. Toxicon, 37:
931-942, 1999.
32. Thatte, U., Bagadev, S., and Dahanukar, S. Modulation of programmed cell death by medicinal
plants. Cell. Mol. Bio., 46: 199-214, 2000.
33. Goping, I. S., Gross, A., Lavoie, J. N., Nguyen, M., Jemmerson, R., Roth, K., Korsmeyer, S. J.,
and Shore, G. C. Regulated targeting of BAX to mitochondria. J. Cell Biol., 143: 207-215, 1998.
34. Wible, B. A., Wang, L., Kuryshev, Y. A., Basu, A., Haldar, S., and Brown, A. M. Increased K+
efflux and apoptosis induced by the potassium channel modulatory protein KChAP/PIAS3beta
in prostate cancer cells. J. Biol. Chem., 277: 17852-17862, 2002.
35. Li, Y., Raffo, A. J., Drew, L., Mao Y., Tran, A., Petrylak, D. P., Fine, R. L. Fas-mediated
apoptosis is dependent on wild-type p53 status in human cancer cells expressing a
temperature-sensitive p53 mutant alanine-143. Cancer Res., 63: 1527-1533, 2003.
36. van Brussel, J. P., van Steenbrugge, G. J., Romijn, J. C., Schroder, F. H., and Mickisch, G. H.
Chemosensitivity of prostate cancer cell lines and expression of multidrug resistance-related
proteins. Eur. J. Cancer, 35: 664-671, 1999.
37. Shimada, K., Nakamura, M., Ishida, E., Kishi, M., Yonehara, S., and Konishi, N. c-Jun
NH2-terminal kinase-dependent Fas activation contributes to etoposide-induced apoptosis in
p53-mutated prostate cancer cells. Prostate, 55: 265-280, 2003.
38. Li, H., Zhu, H., Xu, C. J., and Yuan, J. Cleavage of BID by caspase 8 mediates the
mitochondrial damage in the Fas pathway of apoptosis. Cell, 94: 491-501, 1998.
39. Pastor, N., Dommguez, I., Mateos, S., and Cortes, F. A comparative study of genotoxic effects
of anti-topoisomerase Ⅱ drugs ICRF-193 and bufalin in Chinese hamster ovary cells. Mutat.
Res., 515: 171-180, 2002.
40. Ho, C. K., Chen, S. M., and Wang, S. Y. Ouabain resistance of a human trophoblast cell line is
not related to its reactivity to ouabain. Eur. J. Cell Bio., 79: 765-770, 2000.
41. Yoshida, A., Ueda, T., and Nakamura, T. DNA damage and cell killing by camptothecin and its
derivative in human leukemia HL-60 cells. Jpn. J. Cancer Res., 84: 566-573, 1993.
42. Hashimoto, S., Jing, Y., Kawazoe, N., Masuda, Y., Nakajo, S., Yoshida, T., Kuroiwa, Y., and
Nakaya, K. Bufalin reduces the level of topoisomerase Ⅱ in human leukemia cells and affects
the cytotoxicity of anticancer drugs. Leuk. Res., 21: 875-883, 1997.
FIGURE LEGENDS
Fig. 1 Phase contrast microscopic photographs of LNCaP, DU145, and PC3 cells with bufalin or
cinobufagin treatment. Cells were treated with bufalin or cinobufagin for 24-h (LNCaP and
DU145) or 48-h (PC3) at the concentration of 10 μM, and were observed under a phase
contrast microscope. Magnificaiton, 200X.
Fig. 2 Effects of bufalin or cinobufagin on the change of doubling time in LNCaP, DU145, PC3 and
human mesangial cells (HMC) cells following an incubation period from 1 to 4 days. The
doubling time was calculated from the regression equation of the proliferation index (see
Material and Method). Each value represents mean ± SEM.
Fig. 3 Activities of caspase 3, 8, and 9 in LNCaP, DU145, and PC3 cell lines after administration of
bufalin or cinobufagin. Caspase activities were assayed by a colorimetric kit (see Material
and Method). a and b, represent for LNCaP and DU145 cells, respectively. To determine the
caspase activation in both of the cell lines were after the treatment for 8-, 12-, 18-, and 24-h. c,
caspase activities in PC3 cells were analyzed after the treatment for 18-, 24-, 36-, and 48-h.
Each value represents mean ± SEM.
Fig. 4 Effects of bufalin or cinobufagin on the protein expression of caspases in LNCaP, DU145, and
PC3 cell lines. Cells were incubated with bufalin or cinobufagin for 18- (LNCaP and DU145)
or 24-h (PC3). Cell lysates were resolved on 10% (caspase 9) or 15% (caspase 3, and 8)
SDS-PAGE and analyzed by Western blotting. a, LNCaP cells; b, DU145 cells, c, PC3 cells.
Each lane was loaded with 30 g protein of samples. Similar results were obtained in three
other experiments.
Fig. 5 Effects of bufalin or cinobufagin on the protein expressions of bax and cytochrome c in
LNCaP, DU145, and PC3 cell lines. a, and b, LNCaP and DU145 cells were incubated with
bufalin or cinobufagin for 8- and 12-h, whereas, c, PC3 cells were treated for 18- and 24-h.
Cell lysates were separated as cytosolic and mitochondrial fractions. Samples were resolved
on 15% SDS-PAGE and analyzed by Western blotting. Each lane was loaded with 40 μg
protein of samples. Similar results were obtained in three other experiments.
Table. 1. The IC50 of bufalin or cinobufagin on LNCaP, DU145 and PC3 cells. The incubation
period was from 1 to 4 days. IC50 was calculated from the regression equation of the
proliferation index each incubation day. a Mean ± SEM, b P<0.05 compared with
cinobufagin treatment, c P<0.01 compared with incubation of time day 1.
Incubation
Time (day)
LNCaP
1
2
3
4
DU145
1
2
3
4
PC3
1
2
3
4
a
Bufalin,
IC50 (nM)
832.22 ± 28.49a, b
298.46 ± 24.49c
90.51 ± 19.74c
63.71 ± 8.43c
85.76 ± 2.41b
15.12 ± 2.71b, c
14.28 ± 1.51b, c
13.59 ± 2.32b, c
92.68 ± 4.60
12.73 ± 2.22b, c
10.10 ± 0.79b, c
8.96 ± 1.24b,c
Cinobufagin,
IC50 (nM)
1061.71 ± 44.81
299.80 ± 28.22c
128.74 ± 16.86c
74.90 ± 7.61c
108.76 ± 1.67
30.86 ± 1.54c
27.88 ± 1.47c
25.25 ± 1.20c
100.11 ± 2.01
21.56 ± 1.39c
18.92 ± 1.16c
17.46 ± 1.53c
Mean ± SEM
b
P<0.01 compared with cinobufagin treatment
c
P<0.01 compared with incubation time of day 1
Fig. 1
Bufalin
0.1 M
Vehicle
Cinobufagin
Vehicle
0.1 M
LNCaP (n=4)
LNCaP (n=4)
10
tD(Control)=32 hr
Value of Cell Proliferation Index to Control Cells at Day 0
tD(Control)=31 hr
tD(0.1 M)=91 hr
tD(0.1 M)=105 hr
1
DU145 (n=4)
DU145 (n=4)
10
tD(Control)=25 hr
tD(Control)=25 hr
tD(0.1 M)=156 hr
tD(0.1 M)=134 hr
1
PC3 (n=4)
PC3 (n=4)
10
tD(Control)=26 hr
tD(Control)=26 hr
tD(0.1 M)=216 hr
tD(0.1 M)=231 hr
1
HMC (n=3)
HMC (n=3)
10
tD(Control)=58 hr
tD(Control)=56 hr
tD(0.1 M)=79 hr
1
1
2
3
4
tD(0.1 M)=65 hr
1
Incubation Time (day)
Fig. 2
2
3
4
% of Caspase Activity Relative to Control Group
a
LNCaP
0M
300
Cinobufagin
Bufalin
0.1 M
1 M
10 M
0M
0.1 M
Caspase 3 (n=3)
Caspase 3 (n=3)
Caspase 8 (n=3)
Caspase 8 (n=3)
Caspase 9 (n=3)
Caspase 9 (n=3)
1 M
10 M
200
100
0
300
200
100
0
300
200
100
0
8
12
18
24
8
12
18
24
Incubation Time (h)
% of Caspase Activity Relative to Control Group
b
DU145
Bufalin
0M
300
0.1 M
Cinobufagin
1 M
10 M
0M
0.1 M
Caspase 3 (n=3)
Caspase 3 (n=3)
Caspase 8 (n=3)
Caspase 8 (n=3)
Caspase 9 (n=3)
Caspase 9 (n=3)
1 M
10 M
200
100
0
300
200
100
0
300
200
100
0
8
12
18
24
8
12
18
24
Incubation Time (h)
% of Caspase Activity Relative to Control Group
c
PC3
Bufalin
0M
0.1 M
Cinobufagin
1 M
10 M
0M
Caspase 3 (n=3)
Caspase 3 (n=3)
Caspase 8 (n=3)
Caspase 8 (n=3)
Caspase 9 (n=3)
Caspase 9 (n=3)
10 M
10 M
200
100
0
200
100
0
200
100
0
18
24
36
48
18
Incubation Time (h)
Fig. 3
0.1 M
24
36
48
Fig. 4
Fig. 5