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EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 31, No. 4, 174-178, December 1999
Tanshinone IIA, an ingredient of
Salvia miltiorrhiza
BUNGE,
induces apoptosis in human leukemia cell lines through the
activation of caspase-3
Hyun Jea Sung1,3, Sun Mi Choi1, Yoosik Yoon1
and Kyu Suk An2
1Korea Institute of Oriental Medicine, Seoul 135-100, Korea
2School of Oriental Medicine, Kyung Hee University,
Seoul 130-701, Korea
3Corresponding author: Tel, 82-2-3442-1994(256);
Fax, 82-2-3442-0220; E-mail, ysyoon66@hanmail.net
Accepted 27 September 1999
Abbreviations: PI, propidium iodide; SM, size marker; PARP,
poly(ADP-ribose)polymerase; DMSO, dimethylsulfoxide; UV, ultravi-
olet
Abstract
Tanshinone II-A is a derivative of phenanthrene-
quinone isolated from
Salvia miltiorrhiza
BUNGE, a
traditional herbal medicine that is known to induce
antiinflammatory, anti-oxidative and cytotoxic activ-
ity. We have examined cellular effects of Tanshione
II-A on HL60 human promyelocytic leukemic cells
and K562 human erythroleukemic cells. Tanshione
II-A induced a dose- and time-dependent DNA frag-
mentation into the multiples of 180 bp and specific
proteolytic cleavage of poly(ADP-ribose) polymer-
ase in both cell lines. PI-staining and flow cytometry
analysis of K562 cells following Tanshione II-A treat-
ment showed an increase of the cells possessing
hypodiploid DNA indicative of apoptotic state of cells.
Caspase-3 activity was significantly increased dur-
ing Tanshinone II-A treatment of both HL60 and K562
cells, whereas caspase-1 activity was not changed.
These results suggest that Tanshione II-A induced
HL60 and K562 cellular apoptosis that may be asso-
ciated with the selective members of caspase family.
Keywords: apoptosis, caspase-3, leukemia,
Salvia mil-
tiorrhiza
BUNGE, tanshinone II-A
Introduction
A correlation between induction of apoptosis and neoplasia
has been documented in some carcinogenesis model.
Increasing evidences suggest that the process of neo-
plastic transformation, progression and metastasis involve
alteration of normal apoptotic pathways (Bold
et al
., 1997).
Apoptosis also provides some important clues on effective
anticancer therapy and many chemotherapeutic agents
were reported to exert their anti-tumor effects by inducing
apoptosis on the chemosensitive cancer cells (Kamesaki,
1998).
Salvia miltiorrhiza
BUNGE
is a traditional oriental medi-
cinal herb, the root of which has been traditionally used
for multiple therapeutic remedies. According to phytochemi-
cal reports, ingredients of the root of
Salvia miltiorrhiza
BUNGE can be classified into two groups (Tang and
Eisenbrand, 1992). The first group is phenolics such as
salvianolic acid and lithospermate B, and the other is a
group of abietane type-diterpene quinone pigments such
as tanshinone I, tanshinone IIA, tanshinone IIB and
cryptotanshinone. Lithospermate B was shown to be
effective on renal failure in rats (Yokozawa
et al
., 1989).
Diterpene quinones have been reported to have an anti-
platelet aggregation effect (Wang
et al
., 1989). Recently,
the growth inhibitory effects of various diterpene quinones
on five tumor cell lines were reported (Ryu
et al
., 1997).
In this study, it was found that Tanshinone IIA, the
most abundant and structurally representative diterpene
quinone of
Salvia miltiorrhiza
BUNGE, induced apoptosis
in human leukemic cell lines.
Materials and Methods
Chemicals
Caspase-3 substrate (Ac-DEVD-pNA) and caspase-1
substrate (Ac-YVAD-pNA) were purchased from BACHEM
AG, Hauptstrasse, Switzerland. Anti-poly(ADP-ribose)
polymerase antibody was from Boeringer Mannheim,
Mannheim, Germany. Tanshinone IIA was kindly provided
by Dr. Hee-Juhn Park, Sangji University, Wonju 220-
702, Korea. All other chemicals were purchased from
Sigma Chemical Co., St. Louis, MO, USA.
Cell culture
HL60 human promyelocytic leukemic cell line and K562
human erythroleukemic cell line were cultured in RPMI
1640 medium containing 10% fetal bovine serum, 100
U/ml penicillin and 100 µg/ml streptomycin.
Internucleosomal DNA fragmentation
Cells were harvested and suspended in 500 µl of lysis
buffer containing 20 mM Tris-HCl (pH 7.4), 4 mM EDTA,
Tanshinone IIA induces apoptosis in leukemia cell lines
175
0.4% (v/v) Triton X100 and 10 µg/ml digitonin, and incu-
bated on ice for 30 min. After centrifugation for 5 min at
13,000 rpm using Eppendorf tube centrifuge, supernat-
ants were collected. Each supernatant was extracted
with phenol three times and once with chloroform. Then,
DNA was precipitated by incubating at -80oC for 30 min
after the addition of 1 µg of glycogen, 100 µl of 5 M
NaCl, and 700 ul of isopropanol to each sample. DNA
was collected by centrifuging at 13,000 rpm for 5 min,
and washed once with 70% ethanol. DNA pellets were
dissolved in 30 µl of TE buffer containing 10 µg/ml
RNase A, and incubated at 37oC for 30 min. 10 µl of
each DNA samples were loaded on 1.8% agarose gel.
Propidium iodide staining and flow cytometry
Cells were harvested and washed once with cold PBS.
Then cell pellets were suspended in 500 µl of Propidium
iodide (PI) solution containing 50 µg/ml of PI, 0.1% (w/
v) sodium citrate, and 0.1% (v/v) NP-40. Cell samples
were incubated at 4oC in the dark for at least 15 min,
and analyzed using flow cytometer (FACSCalibur, Beckton
Dickinson) and Cell Quest software.
Proteolytic cleavage of poly(ADP-ribose) polymer-
ase (PARP)
Cells were harvested and washed once with cold PBS.
Then cell pellets were lysed in 100 µl of lysis buffer
containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1% (v/
v) Triton X100, and 1 mM PMSF for 30 min on ice. After
centrifugation at 13,000 rpm at 4oC for 5 min, supernat-
ants were collected and mixed with SDS sample buffer.
Then, each samples were loaded on 8% polyacrylamide
gel and transferred to nitrocellulose membrane. Western
blotting experiment was done using rabbit polyclonal
anti-PARP antibody and alkaline phosphatase-conjugat-
ed secondary antibody.
Measurement of caspase-1 and caspase-3 activity
Cells were harvested and washed once with cold PBS.
Then cell pellets were lysed using lysis buffer containing
20 mM Hepes (pH 7.4), 100 mM NaCl, 0.5% (v/v) NP-40
and 10 mM DTT on ice for 30 min. After centrifugation
at 13,000 rpm at 4oC for 5 min using Eppendorf centrifuge,
supernatants were collected and added with Ac-YVAD-
pNA (caspase-1 substrate) or Ac-DEVD-pNA (caspase-3
substrate) to the final concentration of 100 µM. Each
samples were incubated at 37oC for 1 h and optical
density at 405 nm was measured. A standard curve was
made by measuring A405 of various amounts of pNA, and
A405 value was converted to the amounts of pNA produced.
Results
Recently, growth inhibitory effect of the ingredients of
Salvia miltiorrhiza
BUNGE on tumor cell line was report-
ed. Tanshinone IIA, the most abundant and structurally
representative ingredient of
Salvia miltiorrhiza
BUNGE,
inhibited the proliferation of several human tumor cell
lines (Ryu
et al
., 1997). Since many anticancer drugs
are apoptotic, the apoptosis-inducing activity of tanshinone
IIA was investigated. Figure 1 shows the chemical
structure of tanshinone IIA. Because tanshinone IIA can
not be dissolved in water, DMSO was used. The final
concentration of DMSO was adjusted to 0.1% (v/v) in
cell culture medium, and 0.1% DMSO-treated cells were
used as controls for all experiments. DMSO 0.1% itself
had no effect on cells. Human promyelocytic leukemic
cell line, HL60, was treated with various concentrations
of tanshinone IIA for 4 h, and it was found that tanshi-
none IIA induced internucleosomal DNA fragmentation
into the multiples of 180 bp at the concentration of 1 µg/
ml (Figure 2). UV radiation was used as a positive
control for the induction of apoptosis (Yoon
et al
., 1996).
In time course experiment in which 3 µg/ml of Tanshinone
IIA was treated, 180 bp DNA ladder was generated 2 h
after the treatment (Figure 3). Tanshinone IIA induced
internucleosomal DNA fragmentation in K562 human
Figure 1. Chemical Structure of tanshinone IIA.
Figure 2. Tanshinone IIA-induced internucleosomal DNA fragmentation i
n
HL60 cell line. Cells were treated with various concentrations of tanshinone
IIA for 4 h. Ultraviolet (UV) treatment was used as a positive inducer o
f
apoptotic cell death. SM represents DNA size marker.
176
Exp. Mol. Med. Vol. 31(4), 174-178, 1999
erythroleukemic cell line at the concentration of 3 µg/ml
(data not shown). Flow cytometry analysis of Tanshi-
none IIA-treated K562 cells showed the increase of
hypodiploid apoptotic cells and G2/M phase of cell cycle,
while the cell populations at G1 phase was decreased,
suggesting that Tanshinone IIA induced an arrest of G2/
M phase cell cycle and apoptosis (Figure 4). Apoptotic
cell poputations were 4.34%, 26.95%, 26.53% and 34.53%
in cells treated with 1, 3, 10, and 30 µg/ml of tanshinone
IIA respectively, showing that apoptosis-inducing concent-
ration was between 1 and 3 µg/ml. In control cells treated
with 0.1% DMSO alone, 5.03% of apoptotic cell population
was detected (data not shown).
PARP is a nuclear enzyme which is involved in DNA
repair process, and recently, it was found that 113 kD
PARP protein is cleaved into 89 kD and 24 kD fragments
by the action of CPP32, a protease recently named as
Figure 3. Time course experiment for tanshinone IIA-induced internucleo-
somal DNA fragmentation in HL60 cell line. Cells were treated with 3 µg/m
l
of tanshinone IIA for various time periods. SM represents DNA size marker
.
Figure 4. PI staining and flow cytometry analysis of tanshinone IIA-induced
apoptosis in K562 cell line. Cells were treated with tanshinone IIA for 24 h
at the concentration of 1 µg/ml (A), 3 µg/ml (B), 10 µg/ml (C) and 30 µg
/
ml (D). Apoptotic cell population which has hypodiploid DNA content wa
s
marked by M1.
Figure 5. Proteolytic cleavage of PARP during the time course of tanshi
-
none IIA-induced apoptosis. HL60 cells were treated with 3 µg/ml o
f
tanshinone IIA for various time periods, and analyzed by western blotting
using anti-PARP antibody. 113 kD PARP protein is specifically cleaved into
89 kD fragment after 4 h.
Figure 6. Change of caspase-1 (A) and caspase-3 (B) activities durin
g
tanshinone IIA-induced apoptosis in HL60 cells. Cells were treated with 3
µg/ml tanshinone IIA for various time periods, and each enzyme activity wa
s
measured using specific tetra-peptide substrate. Each data represent the
mean and standard deviation of 4 experiments.
Tanshinone IIA induces apoptosis in leukemia cell lines
177
caspase-3 (Nicholson
et al
., 1995). Since the specific
proteolytic cleavage of PARP is considered to be a
biochemical characteristic of apoptosis (Nicholson
et al
.,
1995), we did western blotting experiment using the
antibody against PARP. Figure 5 shows that PARP is
cleaved into 89 kD fragment by the treatment of
Tanshinone IIA in HL60 cells, indicating that caspase-3
was activated. PARP cleavage was also detected in
K562 cells after 3 µg/ml of tanshinone IIA treatment
(data not shown). To measure the caspase-3 activity
directly and quantitatively, we used Ac-DEVD-pNA, a
specific colorimetric substrate of caspase-3 (Datta
et al
.,
1996). Figure 6 shows that caspase-3 activity began to
increase 2 h after the Tanshinone IIA treatment in HL60
cells. Caspase-1 activity was also measured using a
specific tetrapeptide substrate, Ac-YVAD-pNA, and it was
found that there was no change in its activity during
tanshinone IIA-induced apoptosis, suggesting that there
is a selective involvement among the caspase family
members. The selective activation of caspase-3 was
also detected in K562 cells after tanshinone IIA treat-
ment (Figure 7).
Discussion
In this study, it was found that HL60 and K562 human
leukemic cell lines showed the features of apoptotic cell
death after tanshinone IIA treatment. Among the various
features of apoptosis, a ladder-like pattern of DNA frag-
mentation into the multiples of 180 bp has been consid-
ered as a biochemical hallmark of apoptosis. Recently,
however, there are some controversial reports that inter-
nucleosomal DNA fragmentation also occurs in some
necrotic cell death, suggesting the possibility that inter-
nucleosomal DNA fragmentation may not be an essen-
tial indicator of apoptotic cell death (Cohen
et al
., 1992;
Schulze-Osthoff
et al
., 1994).
Caspase activation is now considered as a most
reasonable criterion for a distinction between apoptosis
and necrosis, because the central mechanism of apoptosis
is evolutionarily conserved from nematode to mammals
and caspase activation is an essential step in this com-
plicated pathways (Thornberry and Lazebnik, 1998). The
data shown in Figure 6 and Figure 7, therefore, represent
the most important evidence that tanshinone IIA-induced
cell death is apoptosis.
Tanshinone IIA treatment induced apoptotic cell death
both in HL60 and K562 cell lines at concentrations rang-
ing 1~3 µg/ml with a different responding time. Tanshi-
none IIA-treated HL60 and K562 cells became apoptotic
after 2 h (Figure 3 and Figure 6) and 24 h (data not
shown) respectively. A similar time difference between
HL60 and K562 cell lines, 2-3 h and 24-48 h respectively,
was also reported in etoposide-induced apoptosis (Martins
et al
., 1997), suggesting that there is some intrinsic
difference between two leukemic cell lines.
Acknowledgement
Authors are grateful to Dr. Hee-Juhn Park, Sangji Univer-
sity for providing tanshinone IIA isolated in his laboratory.
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