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Int. J. Cancer: 73, 725–728 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
INTRACELLULAR Ca21 RELEASE MEDIATES URSOLIC ACID–INDUCED
APOPTOSIS IN HUMAN LEUKEMIC HL-60 CELLS
Jin Hyen BAEK1, Yong Soo LEE2, Chang Mo KANG1, Jung-Ae KIM3, Kyung Sool KWON4, Han Chul SON4 and Kyu-Won KIM1*
1Department of Molecular Biology, Pusan National University, Pusan 609-735, Korea
2Department of Physiology, College of Medicine, Kwandong University, Kangnung 210-701, Korea
3College of Pharmacy, Yeungnam University, Kyongsan 712-749, Korea
4Pusan Cancer Research Center, Pusan National University, Pusan 602-739, Korea
The effect of ursolic acid (UA) on tumor cell apoptosis was
investigated using HL-60 human promyelocytic leukemia
cells as a model cellular system. Treatment with UA resulted
in a concentration-dependent decreased cell viability assessed by MTT assay. UA also induced genomic DNA fragmentation, a hallmark of apoptosis, indicating that the mechanism by which UA induced cell death was through apoptosis.
The intracellular Ca21 level was increased by treatment with
UA. Intracellular Ca21 inhibitors, such as intracellular Ca21release blockers (dantrolene, TMB-8 and ruthenium red) and
an intracellular Ca21 chelator (BAPTA/AM), significantly
blocked the UA-induced increased intracellular Ca21 concentration. These inhibitors also blocked the effects of UA on cell
viability and apoptosis. These results suggest that enhanced
intracellular Ca21 signals may be involved in UA-induced
apoptosis in HL-60 cells. Int. J. Cancer 73:725–728, 1997.
r 1997 Wiley-Liss, Inc.
Ursolic acid (UA), a pentacyclic triterpene acid, has been
isolated from many kinds of medicinal plant, such as Eriobotrya
japonica, Rosmarinus offıcinalis and Glechoma hederaceae. UA
has been reported to produce anti-tumor activities, including
inhibition of skin tumorigenesis (Huang et al., 1994) and inhibition
of tumor promotion (Tokuda et al., 1986). It also induced tumor
cell differentiation by regulation of the expression of differentiationspecific genes in mouse F9 teratocarcinoma cells (Lee et al., 1994).
In addition, UA was shown to possess an anti-angiogenic effect in
chick chorioallantoic membrane (Sohn et al., 1993) and an
anti-invasive activity in HT1080 human fibrosarcoma cells (Cha et
al., 1996).
Apoptosis has been reported to be involved in the carcinogenic
process (Thompson, 1995; Isaacs, 1993) and in tumor therapy and
prevention (Bursch et al., 1992). Many of the presently used
chemotherapeutic agents of cancer induce apoptosis (Miyashita
and Reed, 1993). Increased intracellular Ca21 concentration has
been demonstrated to act as an important mediator of apoptosis in a
variety of cells (Dow, 1995).
Retinoic acid, a differentiation-inducing agent, has been shown
to induce tumor cell apoptosis in mouse F9 teratocarcinoma cells
(Atencia et al., 1994). Since UA was reported to have an activity of
tumor cell differentiation (Lee et al., 1994), in this study, we
investigated the possibility that UA can induce tumor cell apoptosis
using HL-60 cells as a model cellular system. We also examined the
role of intracellular Ca21 signals in the apoptosis induced by UA.
MATERIAL AND METHODS
Material
The HL-60 human promyelocytic leukemia cell line was purchased from the ATCC (Rockville, MD). The powder for RPMI
1640 medium, FBS and antibiotics (penicillin and streptomycin
mixture) were purchased from GIBCO (Grand Island, NY). UA,
dantrolene (Dant), 3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8), ruthenium red (RR), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) and diphenylamine
(DPA) were obtained from Sigma (St. Louis, MO). Bis-(o-aminophenoxy)-ethane-N,N,N8,N8-tetraacetic acid/acetoxymethyl ester
(BAPTA/AM) and 1-(2,5-carboxyoxazol-2-yl-6-aminobenzfuran-5oxyl)-2-(28-amino-58-methylphenoxy)-ethane-N,N,N8,N8-tetraace-
toxymethyl ester (Fura-2/AM) were from Molecular Probes (Eugene, OR). Unless otherwise indicated, propidium iodide (PI) and
all other chemicals were the purest grade available and were
obtained from Sigma. UA and TMB-8 were dissolved in ethanol.
Dant and BAPTA/AM were dissolved in DMSO, and other drugs
were dissolved in distilled water. These stock solutions were
sterilized by filtration through 0.2 µm disc filters (Gelman, Ann
Arbor, MI).
Cell culture
HL-60 cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml of
streptomycin at 37°C in a humidified incubator under 5% CO2/95%
air. Cells were split in a 1:5 ratio every 2 or 3 days. Cells were
treated with different concentrations of UA. UA was dissolved in
ethanol with a final concentration of 0.1% (v/v).
Cell viability test (MTT staining)
Cell viability was assessed as described by Mosmann (1983).
Briefly, cells were incubated in 100 µl of media in 96-well plates
with the indicated concentration of the drugs for 24 hr at an initial
cell density of 3 3 105 cells/ml. An appropriate volume of drug
vehicle was added to untreated cells. After each period of incubation, 10 µl of MTT solution (5 mg MTT/ml in H2O) were added and
cells incubated for a further 4 hr. One hundred microliters of
acid-isopropanol (0.04 N HCl in isopropanol) were added to each
culture and mixed by pipetting to dissolve the reduced MTT
crystals. Relative cell viability was obtained by scanning with an
ELISA reader (Molecular Devices, Menlo Park, CA) with a 570 nm
filter.
DNA isolation and electrophoresis
HL-60 cells were collected by centrifugation (200g, 5 min),
washed twice in PBS (pH 7.4) and resuspended at a density of 2 3
106 cells/200 µl in lysis buffer containing 50 mM NaCl, 5 mM
EDTA and 150 mM Tris-HCl (pH 8.0). To each 200 µl of cell
suspension, 20 µl of proteinase K (25 µg/µl) and 20 µl of 10% SDS
were added. Samples were incubated at 50°C for 48 hr. Ten
microliters of RNase A (100 U/200 µl) were added and the
incubation continued for a further 60 min. DNA was then extracted
with phenol:chloroform (24:1), precipitated by the addition of 0.1
vol of 5 M NaCl and 1 vol of isopropyl alcohol and visualized by
electrophoresis in 1.5% agarose. The pattern of DNA fragmentation was photographed under UV light, after staining the gel with
ethidium bromide (0.5 µg/ml in TAE buffer).
Quantitative analysis of fragmented DNA
For quantitative DNA analysis, HL-60 cells were collected and
washed twice with PBS. Cells were resuspended in lysis buffer
Contract grant sponsors: Korea Science and Engineering Foundation and
the Ministry of Education, Korea.
*Correspondence to: Department of Molecular Biology, Pusan National
University, Pusan 609-735, Korea. Fax: 82-51-513-9258. E-mail:
[email protected]
Received 29 April 1997; Revised 4 July 1997
BAEK ET AL.
726
FIGURE 1 – Decreased cell viability by UA in HL-60 human
promyelocytic leukemia cells. Cell viability was assessed by MTT
staining. Results are expressed as the percent change of the control
condition in which cells were grown in medium containing drug-free
vehicle. Data points represent the mean values of 4 replicates, with the
bars indicating SEM.
containing 1 mM EDTA, 10 mM Tris-HCl (pH 8.0) and 0.2%
Triton X-100 and incubated on ice for 30 min (Sentman et al.,
1991). Low and high molecular weight DNAs were separated by
centrifugation at 15,000 g at 4°C. The supernatant was collected
and the pellet resuspended in 0.5 ml of lysis buffer. DNA from both
supernatant and pellet was precipitated by addition of 1 N
perchloric acid. The DPA method (Bhalla et al., 1992) was used for
measurement of DNA content. The percent change of DNA
fragments was calculated with the following equation:
% Fragment 5 [A570 of small DNA/(A570 of small DNA
1 A570 large DNA) 3 100].
Measurement of [Ca21]i
Aliquots of HL-60 cells, cultured for 3–5 days, were washed in
Eagle’s basal salt solution (EBSS). Then, 2 µM Fura-2/AM were
added and the cells incubated for 60 min at room temperature
(22°–23°C). Unloaded Fura-2/AM was removed by centrifugation
at 150 g for 3 min. Cells were resuspended at a density of 2 3
106/ml in Ca21-free Krebs-Ringer buffer (KRB) containing 125
mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 mM
NaHCO3 , 25 mM HEPES and 6 mM glucose (pH 7.4); transferred
to a quartz cuvette; and stirred continuously. Fluorescence emission
(510 nm) was monitored with the excitation wavelength cycling
between 340 and 380 nm at 37°C using a Hitachi F2000 fluorimeter. At the end of an experiment, maximum and minimum values
of fluorescence at each excitation wavelength were obtained by
first lysing the cells with 0.1% Triton X-100 (maximum) and then
adding 10 mM EGTA (minimum). With the maximum and
minimum values, the 340:380 nm fluorescence ratios were converted into free Ca21 concentration using the Fura-2 Ca21-binding
constant (224 nM) and the formula described by Grynkiewicz et al.
(1985).
parisons; p values less than 0.05 are considered to be statistically
significant.
Data analysis
All experiments were performed 4 times. All data were displayed
as percentages of the control condition. All control experiments
were carried out in the same media containing drug-free vehicle.
Data were expressed as mean 6 SEM and analyzed using 1-way
ANOVA and the Student-Newman-Keul’s test for individual com-
Induction of apoptosis by UA
As shown in Figure 1, UA decreased cell viability of HL-60 cells
in a dose-dependent manner. Ethanol alone, at a final concentration
of 0.1% (v/v) used for the dissolution of UA, did not affect cell
FIGURE 2 – UA induces apoptotic cell death in HL-60 human
promyelocytic leukemia cells. Cells were treated for 24 hr without or
with each concentration of UA. DNA was isolated from the cells and
analyzed by 1.5% agarose gel electrophoresis (a). Lane M represents
DNA marker. Dose-dependent effect of UA on DNA fragmentation in
HL-60 human promyelocytic leukemia cells (b). Cells were treated for
24 hr without or with each concentration of UA. The amount of
fragmented DNA was measured by the DPA method. Results are
expressed as the percent change of DNA fragments compared to the
control condition in which cells were grown in medium containing
drug-free vehicle. Data points represent mean values of 4 replicates,
with the bars indicating SEM.
RESULTS
UA-INDUCED APOPTOSIS IN HL-60 CELLS
727
viability. To determine whether cell death induced by UA occurs
through an apoptotic pathway, we tested morphological changes
with PI staining. We observed that morphological changes corresponding to apoptosis, including cell shrinkage and cytoplasmic
and nuclear membrane blebbing, were elicited by 10 µM of UA,
which induced extensive decreased cell viability (data not shown).
An assay for genomic DNA fragmentation, a hallmark of apoptotic
cell death, was performed. As shown in Figure 2a, treatment with
UA produced DNA fragmentation, indicating that UA induced
apoptosis in the HL-60 cells. In addition, UA increased the quantity
of DNA fragments in a dose-dependent manner in HL-60 cells
(Fig. 2b).
Effect of UA on [Ca21]i
Increased [Ca21]i has been shown to induce Ca21/Mg21dependent endonuclease activation and apoptosis (Thompson,
1995). To investigate the interrelationship between the UA-induced
increased [Ca21]i and apoptosis, we monitored the changes of
[Ca21]i by UA in HL-60 cells. Treatment of cells with 10 µM UA
increased [Ca21]i in 2 phases, initial increase and late sustained
increase (Fig. 3a). We tested the effect of intracellular Ca21 release
blockers on the UA-induced increased intracellular Ca21. The
UA-induced increased intracellular Ca21 was inhibited significantly by treatment with Dant or TMB-8, inhibitors of intracellular
Ca21 release (Rittenhouse-Simmons and Deykin, 1978; Zhang and
Melvin, 1993), or RR, an inhibitor of intracellular Ca21 release,
specifically from the ryanodine-sensitive Ca21 pools (Ehrlich et al.,
1994) (Fig. 3b–d). BAPTA/AM, an intracellular Ca21 chelator
(Zuker and Steinhardt, 1978), showed results similar to those
depicted in Figure 3e.
FIGURE 4 – Effect of intracellular Ca21 inhibitors on UA-induced
decreased viability of HL-60 human promyelocytic leukemia cells.
Intracellular Ca21 inhibitors (25 µM Dant, 5 µM TMB-8, 0.5 µM RR
and 0.1 µM BAPT/AM) were added to the cells 4 hr before treatment
with UA. Cell viability was assessed by MTT staining. Results are
expressed as the percent change of the control condition in which cells
were grown in medium containing drug-free vehicle. We also tested the
drug-free vehicles and did not find any effects of those alone. Data
represent mean values of 4 replicates, with the bars indicating SEM.
#p , 0.05 compared to control. *p , 0.05 compared to UA alone.
Effect of intracellular Ca21 inhibitors on UA-induced apoptosis
To investigate whether these intracellular Ca21 inhibitors also
block UA-induced apoptosis in HL-60 cells, we examined the
effects of these agents on the UA-induced decreased cell viability
and DNA fragmentation. As shown in Figure 4, pre-treatment with
these intracellular Ca21 inhibitors significantly blocked the UA-
FIGURE 5 – Effect of intracellular Ca21 inhibitors on UA-induced
DNA fragmentation in HL-60 human promyelocytic leukemia cells.
Intracellular Ca21 inhibiors (25 µM Dant, 5 µM TMB-8, 0.5 µM RR
and 0.1 µM BAPTA/AM) were added to the cells 4 hr before treatment
with UA. The amount of fragmented DNA was measured by the DPA
method. Results are expressed as the percent change of DNA fragments
compared to the control condition in which cells were grown in
medium containing drug-free vehicle. We also tested the drug-free
vehicles and did not find any effects of those alone. Data points
represent mean values of 4 replicates, with the bars indicating SEM.
#p , 0.05 compared to control. *p , 0.05 compared to UA alone.
induced decreased cell viability. DNA fragmentation induced by
treatment of 10 µM UA also was decreased significantly by
pre-treatment with these intracellular Ca21 inhibitors (Fig. 5). In
both experiments, we did not find any effects of the Ca21 inhibitors
alone (data not shown).
DISCUSSION
FIGURE 3 – Effect of intracellular Ca21 inhibitors on UA-induced
increased [Ca21]i in HL-60 human promyelocytic leukemia cells. Data
represent intracellular Ca21 changes with time. Arrows show the time
points for addition of 10 µM UA. Intracellular Ca21 inhibitors were
applied 3 min before fluorescence measurements.
In this study, we found that UA induced apoptosis in HL-60 cells
(Figs. 1, 2). The apoptosis-inducing activity of UA also was found
in some other tumor cell lines, such as F9 teratocarcinoma cells and
HepG2 human hepatocellular carcinoma cells (data not shown).
Previous reports demonstrated that UA has anti-tumor activities,
such as inhibition of tumorigenesis (Huang et al., 1994), inhibition
of tumor promotion (Tokuda et al., 1986) and induction of tumor
cell differentiation (Lee et al., 1994). UA also has been shown to
728
BAEK ET AL.
inhibit effectively angiogenesis (Sohn et al., 1993) and invasion
(Cha et al., 1996), which are importantly involved in tumor
metastasis (Claffey et al., 1996; Meitar et al., 1996). Thus, these
previous and present results suggest that UA is an effective
anti-cancer agent acting at the various stages of tumor development.
Increased [Ca21]i appears to be involved in apoptosis in a variety
of cells (Dow, 1995). Although the precise mechanism by which
intracellular Ca21 mediates apoptosis is not known, the endogenous Ca21/Mg21-dependent endonuclease, which cleaves doublestrand DNAs at nucleosome linker regions, remains an attractive
target for the effect of Ca21 (Wyllie, 1980; Arends et al., 1990).
This endogenous enzyme appears to be activated by mobilization
of cytosolic Ca21 (McConkey et al., 1989). The results of the
present study show that UA increases [Ca21]i in HL-60 cells in 2
phases, initial and late sustained increases (Fig. 3). Although the
source of UA-induced increased [Ca21]i is not defined clearly in
this study, various intracellular Ca21 release blockers inhibited the
UA-induced increased [Ca21]i (Fig. 3), suggesting that UA may
mobilize intracellular Ca21 from the endoplasmic reticulum in
HL-60 cells. The UA-induced decreased cell viability and apopto-
sis were significantly decreased by several intracellular Ca21
release blockers (Figs. 4, 5), indicating that UA mobilizes intracellular Ca21, which then activates the apoptotic processes in tumor
cells. However, the precise mechanism of the apoptosis-inducing
action of UA is unknown at present. Although the results of the
present study suggest that the apoptosis-inducing activity of UA
may be mediated by intracellular Ca21 release, we cannot exclude
that the Ca21-independent mechanisms also may be involved in the
apoptosis-inducing activity of UA.
Taken together, these results suggest that UA induces apoptosis
in HL-60 cells and that enhanced intracellular Ca21 signals may be
involved in the UA-induced apoptosis. These results further
suggest that UA may be a good candidate for therapeutic studies of
human leukemia.
ACKNOWLEDGEMENTS
This work was supported by the Korea Science and Engineering
Foundation (KOSEF) through the Cancer Research Center at Seoul
National University and the Genetic Engineering Research Grant,
funded in 1995, from the Ministry of Education, Korea.
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