Guggulsterones induce apoptosis and differentiation in acute

Transcription

1982
Guggulsterones induce apoptosis and differentiation
in acute myeloid leukemia: identification of
isomer-specific antileukemic activities of
the pregnadienedione structure
Ismael Samudio,1 Marina Konopleva,1
Stephen Safe,2,3 Teresa McQueen,1
and Michael Andreeff1
1
Section of Molecular Hematology and Therapy, Department of
Blood and Marrow Transplantation, The University of Texas
M.D. Anderson Cancer Center; 2Institute of Biosciences and
Technology, Texas A&M University, Houston, Texas and
3
Department of Veterinary Physiology and Pharmacology, Texas
A&M University, College Station, Texas
phosphatidylserine externalization of CD34-positive
blasts from primary leukemic samples. This study is the
first to show that guggulsterones and 16-dehydroprogesterone exert antileukemic effects via the induction of
apoptosis and differentiation and, more importantly,
identifies the pregnadienedione structure as a potential
chemotherapeutic scaffold. [Mol Cancer Ther 2005;
4(12):1982 – 92]
Introduction
Abstract
In this study, the antileukemic effects of three isomeric
pregnadienedione steroids [i.e., cis -guggulsterone,
trans -guggulsterone, and 16-dehydroprogesterone]
were investigated in HL60 and U937 cells as well as
in primary leukemic blasts in culture. Our results show
that all three compounds inhibited the proliferation of
HL60 and U937 cells, with IC50s ranging from 3.6 to
10.9 Mmol/L after treatment for 6 days. These growth
inhibitory effects correlated with externalization of
phosphatidylserine and loss of mitochondrial membrane
potential, suggesting that these isomeric steroids induce
apoptosis in leukemia cells. z-VAD-fmk prevented phosphatidylserine externalization but not mitochondrial
membrane potential loss, indicating that mitochondrial
dysfunction occurred in the absence of caspase activation. Interestingly, although all three compounds
increased the generation of reactive oxygen species
and decreased phosphorylation of extracellular signalregulated kinase, only cis-guggulsterone induced a rapid
depletion of reduced glutathione levels and oxidation of
the mitochondrial phospholipid cardiolipin. 16-Dehydroprogesterone and trans-guggulsterone induced differentiation of HL60 and NB4 cells as evidenced by increased
surface expression of CD11b and/or CD14, and all three
steroids rapidly induced mitochondrial dysfunction and
Received 7/18/05; revised 9/14/05; accepted 9/23/05.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Michael Andreeff, Section of Molecular Hematology
and Therapy, Department of Blood and Marrow Transplantation, The
University of Texas M.D. Anderson Cancer Center, Unit 448, 1400
Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-7260;
Fax 713-794-474. E-mail: [email protected]
Copyright C 2005 American Association for Cancer Research.
doi:10.1158/1535-7163.MCT-05-0247
Acute myeloid leukemias (AML) are clonal malignancies
characterized by increased numbers of immature myeloid
progenitor cells arrested at different stages of granulocytic
and monocytic differentiation. First-line treatment of AML
consists of a combination of cytarabine and an anthracycline, and although this combination results in 60% to 80%
complete remissions in newly diagnosed patients, most
patients will relapse with resistant disease (1). Because
achievement of complete remission is a prerequisite for
long-term survival (2), several novel therapeutic modalities
have been investigated, including the use of different
anthracycline formulations, different nucleoside analogues,
and the combination of the antiangiogenic agent thalidomide with cytarabine/anthracycline or topotecan/anthracycline (3 – 5). However, overall improvement in survival
rates has been marginal at best underlining the need for
development of more effective therapies. The most striking
increase of complete remission and survival has been
achieved by ligation of the nuclear retinoic acid receptor a
in acute promyelocytic leukemias with all-trans retinoic
acid (6, 7).
The gum resin from the guggul tree Commiphora mukul
has been used in Ayurvedic medicine for centuries to treat
inflammatory and lipid disorders (8), and an ethylacetate
extract of the resin, termed guggulipid, has been reported
to have an antiobesity and antilipidemic effect in clinical
trials with no significant toxicity (9 – 12). The active
substances in guggulipid are the pregnane plant sterols
cis-guggulsterone and trans-guggulsterone, which have
been shown to lower cholesterol and triglycerides in
normal and high-fat-fed rats (9). The antilipidemic effects
of guggulsterone may be mediated by antagonism of the
orphan receptor FXR (13) as well as promiscuous interactions with other nuclear receptors (14). Notably, although
most studies on guggulsterone have focused on their
antilipidemic activity, these compounds have also shown
potent anti-inflammatory effects, such as preventing
oxidative damage during isoproterenol-induced myocardial necrosis in rats (15, 16) and decreasing inflammation
Mol Cancer Ther 2005;4(12). December 2005
Molecular Cancer Therapeutics
associated with nodulocystic acne (17). These observations
suggest that in addition to its lipid-lowering activity
guggulsterone may modulate anti-inflammatory and antioxidant responses.
A variety of naturally occurring compounds exhibit
chemopreventive and anti-inflammatory effects, including resveratrol, betulinic acid, saikosaponin, and curcumin. Some of the chemotherapeutic activities of these
compounds may be related to their inhibition of nuclear
factor-nB signaling (18 – 22), and a recent study reported
that cis-guggulsterone inhibited tumor necrosis factor-a –
induced nuclear factor-nB signaling and sensitized
cancer cells to apoptosis induced by taxol, doxorubicin,
and tumor necrosis factor-a (23). Surprisingly, there are
no studies to date investigating the direct antiproliferative and proapoptotic effects of guggulsterone in cancer
cell lines in culture. We therefore hypothesized that
guggulsterone, like other anti-inflammatory and chemopreventive agents, may decrease the proliferation of
cancer cells in culture. Here, we report that both isomers
of guggulsterone, cis-guggulsterone and trans-guggulsterone, effectively inhibit the proliferation of leukemic
cancer cell lines and induce apoptosis and differentiation. Interestingly, a mammalian steroid metabolite and
chemical isomer of guggulsterone, 16-dehydroprogesterone, also induced a comparable pattern of differentiation,
growth inhibition, and apoptosis, suggesting that the
pregnadienedione structure of these steroids (Fig. 1A)
offers the potential for development of novel chemotherapeutics. Our results are the first to show the
antileukemic effects of guggulsterone isomers and 16dehydroprogesterone, and current studies are investigating their mechanism of action and development of more
potent novel steroidal analogues.
Figure 1. Guggulsterone isomers and 16-dehydroprogesterone prevent the proliferation of HL60 and U937 cells in long-term culture. A, structure of the
pregnadienedione isomers used in this study. B, HL60 cells were cultured in the presence of increasing concentrations of the guggulsterone isomers and
16-dehydroprogesterone (10 – 20 Amol/L) for 72 and 144 h. Viable cells were counted using a hemocytometer after trypan blue staining. C, U937 cells
were treated with the guggulsterone isomers and 16-dehydroprogesterone as for HL60 cells above. All experiments were done in duplicate and repeated at
least thrice. cGS, cis -guggulsterone; tGS, trans -guggulsterone; P, 16-dehydroprogesterone. Points, mean of three independent experiments; bars, SE.
1983
1984 Pregnadienediones Induce Apoptosis in AML
Materials and Methods
Cell Lines, Chemicals, and Biochemicals
U937 and HL60 cells were maintained in RPMI supplemented with 10% FCS, 1% glutamine, and 100 units/mL
penicillin in a 37jC incubator containing 5% CO2. 16Dehydroprogesterone, cis-guggulsterone, and trans-guggulsterone were purchased from Steraloids, Inc. (Newport,
RI). TMRM, dihydroethidine, and Cell Tracker Green were
all obtained from Molecular Probes (Eugene, OR). z-VADfmk was purchased from Alexis Biochemicals (Axxora
LLC, San Diego, CA). Phospho – extracellular signal-regulated kinase (ERK) and total ERK antibodies were
purchased from Cell Signaling Technologies, Inc. (Beverly,
MA). Heme oxygenase-1 antibody was purchased from BD
Biosciences (San Jose, CA) and a-tubulin was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). All other
chemicals used were of the highest purity available.
Human Subjects
Bone marrow or peripheral blood samples were obtained
for in vitro studies from patients with AML. Samples were
collected during routine diagnostic procedures after informed consent was obtained in accordance with regulations and protocols approved by the Institutional Review
Board of The University of Texas M. D. Anderson Cancer
Center (Houston, TX). Mononuclear cells were separated
by Ficoll-Hypaque (Sigma Chemical, St. Louis, MO) density
gradient centrifugation. Patient sample 1 was a bone
marrow aspirate containing 85% blasts from an AML-M1
relapse patient (7del). Patient sample 2 was a bone
relapse patient [t(12,17)]. Patient sample 3 was a bone
relapse patient (normal cytogenetics). Patient sample 4 was
a peripheral blood sample containing 92% blasts from an
AML-M0 relapse patient [7del; t(11,19)]. To investigate
the effects of the guggulsterone and 16-dehydroprogesterone on normal cells, blood samples from three healthy
volunteers (A-C) were obtained and peripheral blood
mononuclear cells (PBMC) were separated by FicollHypaque density gradient centrifugation. PBMC samples
were then exposed to 100 Amol/L guggulsterone and 16dehydroprogesterone for 20 hours, and phosphatidylserine
externalization was quantitated by flow cytometry.
Measurement of Intracellular Glutathione by Flow
Cytometry
Cells (3 105/mL; 0.5 mL) were treated with compounds
as indicated or with 2 mmol/L diethylmaleate for 30
minutes. Cells were then collected by centrifugation,
washed in PBS once, and resuspended in 0.2 mL PBS
containing 400 Amol/L Cell Tracker Green and incubated at
20jC protected from light for 10 minutes. Cells were then
washed in PBS several times, and Cell Tracker Green
fluorescence was quantitated by flow cytometry. The mean
Cell Tracker Green fluorescence from diethylmaleatetreated samples was considered to be background and
subtracted accordingly. All experiments were done in
duplicate and repeated at least thrice.
Measurement of Phosphatidylserine Externalization
and Mitochondrial Membrane Potential
After appropriate treatments, cells were washed twice in
PBS and then resuspended in 100 AL Annexin binding
buffer [140 mmol/L NaCl, 10 mmol/L KH2PO4, 5 mmol/L
CaCl2 (pH 7.4)] containing 25 nmol/L TMRM and 1:100
dilution of Annexin V-FLUOS (Roche Diagnostics,
Mannheim, Germany) incubated at 37jC for 30 minutes.
Cells were then analyzed by flow cytometry in a
FACSCalibur flow cytometer using a 488 nm argon excitation laser.
Measurement of Reactive Oxygen Species Generation
After appropriate treatments, cells were harvested
by centrifugation, washed in PBS, and loaded with the
O
2 -sensitive probe dihydroethidine. Cells were incubated at
37jC for 10 minutes and washed in PBS, and FL2 fluorescence was examined by flow cytometry. Results presented are means F SE of three independent experiments.
Western Blot Analysis
Cells where harvested by centrifugation, washed twice in
PBS, and resuspended in ice cold lysis buffer [1% Triton
X-100, 45 mmol/L KCl, 10 mmol/L Tris (pH 7.5)] supplemented with protease and phosphatase inhibitors and then
subjected to SDS-PAGE in 10% or 12% polyacrylamide gels
followed by protein transfer to a Hybond-P membrane
(Amersham Pharmacia Biotech, Little Chalfont, United
Kingdom) and immunoblotting. Signals were detected by
a PhosphorImager (Storm 860, version 4.0, Molecular
Dynamics, Sunnyvale, CA).
Measurement of Cardiolipin Content
After appropriate treatments, cells were harvested by
centrifugation, washed once in PBS, and resuspended in
PBS containing 10 nmol/L nonyl acridine orange, a
probe that binds with high affinity to reduced but not
oxidized cardiolipin (24). Cells were incubated 37jC for
30 minutes and FL1 fluorescence was quantitated by
flow cytometry.
HL60 and NB4 Cell Differentiation
HL60 and NB4 cells were treated with 10 Amol/L
guggulsterone and 16-dehydroprogesterone, and after
120 hours, the cells were collected, washed once in
PBS, and resuspended in PBS containing 1:100 dilution
of CD11b-phycoerythrin and CD14-FITC (both form BD
Biosciences). Cells were incubated at room temperature
for 15 minutes, washed in PBS, and analyzed by flow
cytometry gating on viable cells as determined by
forward and side scatter. Cells stained with mouse IgGphycoerythrin and mouse IgG-FITC served as negative
controls. In parallel, viable cells were counted in a
hemocytometer after trypan blue exclusion. The absolute
number of differentiated cells was calculated from the
equation:
ð½Viable cells ð1 10 4 Þ ½%CD14 or CD11bþ cellsÞ=100%
Results were expressed as the total number of viable cells
positive for CD11b or CD14 surface expression.
Results
Guggulsterone Isomers and 16-Dehydroprogesterone
Prevent the Growth of Leukemic Cells in Culture by
Inducing Apoptosis
Because there are no reports in the literature investigating the antiproliferative effects of guggulsterone or related
pregnadienediones, we cultured HL60 and U937 cells with
increasing concentrations of both guggulsterone isomers
and 16-dehydroprogesterone for 72 and 144 hours and
quantitated the number of viable cells remaining after
treatment. The results in Fig. 1B show that cis-guggulsterone, trans-guggulsterone, and 16-dehydroprogesterone
inhibited the proliferation of HL60 cells in a time- and
dose-dependent manner displaying 144-hour IC50s ranging from 8.3 to 10.9 Amol/L. Similarly, all three
compounds inhibited the proliferation of U937 cells with
slightly higher potencies displaying IC50s varying from 3.6
to 8.7 Amol/L (Fig. 1C). To investigate whether apoptosis
contributed to the antiproliferative effects of the guggulsterone and 16-dehydroprogesterone, we quantitated the
percentage of HL60 and U937 cells that externalized
phosphatidylserine after treatment with these agents for
72 hours. We found that all three compounds significantly
increased phosphatidylserine externalization, albeit cisguggulsterone was the most potent compound displaying
72-hour IC50s of 16.1 and 19.8 Amol/L after treatment of
HL60 and U937 cells, respectively, for 72 hours (Fig. 2A).
Furthermore, the increase in phosphatidylserine externalization correlated with a marked loss in mitochondrial
membrane potential (DCm) as evidenced by reduced
accumulation of the potentiometric probe TMRM,
which was more pronounced in cells treated with cisguggulsterone (Fig. 2B). Taken together, these results
show that the isomeric pregnadienedione steroids guggulsterone and 16-dehydroprogesterone prevent the proliferation of leukemic cells in culture partly by inducing
mitochondrial dysfunction and apoptosis.
Induce Differentiation of HL60 and NB4 Cells in Culture
HL60 cells have been shown to differentiate in culture
after treatment with several anticancer drugs and apoptosis
inducers with lipophilic and/or steroidal structure, and
Figure 2. Guggulsterone isomers and 16-dehydroprogesterone induce apoptosis in HL60 and U937 cells and promote differentiation of HL60 and
NB4 cells in long-term culture. A, HL60 cells were cultured in the presence of increasing concentrations of the guggulsterone isomers and
16-dehydroprogesterone (10 – 20 Amol/L) for 72 h. Phosphatidylserine externalization and DCm were quantitated as described in Materials and Methods. B,
U937 cells were treated with the guggulsterone isomers and 16-dehydroprogesterone as for HL60 cells above. C, HL60 cells were treated with 10 Amol/L
cis -guggulsterone, trans -guggulsterone, or 16-dehydroprogesterone for 120 h and the number of cells expressing surface CD11b and CD14 was evaluated
by flow cytometry as described in Materials and Methods. D, NB4 cells were treated with 10 Amol/L cis -guggulsterone, trans -guggulsterone, or 16dehydroprogesterone for 96 h and the number of cells expressing surface CD14 was evaluated by flow cytometry as above. All experiments were done in
duplicate and repeated at least thrice. Columns, mean of three independent experiments; bars, SE. *, P < 0.05; **, P < 0.005.
1985
these include the synthetic triterpenoid CDDO-Me, 12-Otetradecanoylphorbol-13-acetate, oxysterols, and 1,25-dihydroxyvitamin D3 (25 – 30). Because one mechanism that
may contribute to the antiproliferative effects of the
guggulsterone and 16-dehydroprogesterone is the induction of differentiation, we examined the surface expression
of the monocytic and myelomonocytic markers CD14 and
CD11b, respectively, in HL60 cells treated with 10 Amol/L
16-dehydroprogesterone, cis-guggulsterone, and trans-guggulsterone for 120 hours. Of note, because the selective
killing of immature cells by these steroids would increase
the relative numbers of differentiated cells, we calculated
the absolute number of cells expressing surface CD14 or
CD11b as described in Materials and Methods. These
results presented in Fig. 2C illustrate that trans-guggulsterone was the more potent inducer of myelomonocytic
differentiation, promoting a 2.5-fold increase in HL60 cells
expressing surface CD11b (P < 0.004) followed by 16dehydroprogesterone, which induced a modest but not
statistically significant 1.7-fold increase (P > 0.05). transGuggulsterone also significantly increased the number of
Figure 3.
Guggulsterone isomers and 16-dehydroprogesterone induced
the generation of O
2 , decrease the activation of ERK, and induce
expression of the stress response protein heme oxygenase-1. A, U937
cells were treated with 20 Amol/L cis -guggulsterone, trans -guggulsterone,
and 16-dehydroprogesterone for 24 h and the levels of O
2 were
quantitated by flow cytometry as described in Materials and Methods.
B, U937 cells were treated with 15 Amol/L cis -guggulsterone, trans guggulsterone, or 16-dehydroprogesterone for 48 h and the levels of
pERK, total ERK, heme oxygenase-1 (HO-1 ), and a-tubulin were examined
by Western blot as described in Materials and Methods. Superoxide
measurements were done in triplicate and repeated at least twice.
Columns, mean of a representative experiment; bars, SD. *, P < 0.05;
**, P < 0.0005.
HL60 cells expressing surface CD14 (2.2-fold; P < 0.03),
whereas neither 16-dehydroprogesterone nor cis-guggulsterone promoted an increase in cells expressing this
monocytic marker. Interestingly, cis-guggulsterone seemed
to decrease the numbers of cells expressing both surface
markers probably owing to its higher cytotoxicity at
10 Amol/L compared with 16-dehydroprogesterone or
trans-guggulsterone. Finally, we also investigated if these
steroids would promote differentiation in a different cell
context. For these experiments, we treated the acute
promyelocytic leukemia cell line NB4, which has been
shown to undergo monocytic differentiation after treatment
with retinoic acid and 1,25-dihydroxyvitamin D3 (28 – 30),
with 10 Amol/L guggulsterone and 16-dehydroprogesterone for 96 hours and examined the cell surface expression of
CD14 (Fig. 2D). 16-Dehydroprogesterone and trans-guggulsterone induced a robust increase in NB4 cells expressing
surface CD14 (3.8- and 3.5-fold, respectively; P < 0.008),
whereas cis-guggulsterone induced a more modest but
significant 1.7-fold increase (P < 0.03). Under these
conditions, 16-dehydroprogesterone and trans-guggulsterone decrease the number of viable NB4 cells by f50%,
whereas cis-guggulsterone induced a more pronounced
f80% decrease (data not shown). Notably, we have
observed that lower concentrations of cis-guggulsterone
(<10 Amol/L), which are not markedly cytotoxic, do not
induce an increase in differentiated HL60 or NB4 cells (data
not shown), suggesting that the mild differentiating effects
of this agent are closely associated with its cytotoxicity.
Together, our findings indicate that all three steroids can
promote differentiation of AML cell lines in culture, albeit
the higher cytotoxicity of cis-guggulsterone seems to mask
this effect.
Increase Generation of Reactive Oxygen Species,
Decrease the Phosphorylation of ERK, and Induce
the Expression of Heme Oxygenase-1 in U937 Cells in
Culture
Because reactive oxygen species (ROS) have been
implicated in triggering apoptosis, we investigated the
effects of 15 Amol/L of the guggulsterone isomers and 16dehydroprogesterone on the levels of superoxide radicals
in U937 cells after treatment for 48 hours. The results show
that cis-guggulsterone and trans-guggulsterone induced a
significant 16% and 19% increase in levels of superoxide
radicals (O
2 ), respectively, compared with cells treated
with DMSO (P < 0.0001), whereas 16-dehydroprogesterone
promoted a weaker albeit significant (P < 0.0001) 8%
increase in O
2 (Fig. 3A). Under these conditions, there was
minimal apoptosis (data not shown), suggesting that the
observed increase in O
2 was not a consequence of cell
death. Previous reports have shown that induction of
apoptosis in U937 cells by agents, such as CDDO-Me,
bortezomib, adaphostin, and arsenic trioxide, which induce
oxidative stress, is accompanied by inhibition of ERK phosphorylation (31 – 34). We therefore investigated if the effect
of guggulsterone isomers and 16-dehydroprogesterone
on the levels of phosphorylated ERK (pERK) in U937 cells
by Western blot. Guggulsterone isomers induced a
f90% decrease in the levels of pERK after 48 hours
compared with cells treated with vehicle (DMSO). In
contrast, U937 cells treated with 16-dehydroprogesterone
only exhibited a f 20% inhibition of ERK phosphorylation.
In addition, the enhanced formation of O
2 promoted by the
guggulsterone isomers and 16-dehydroprogesterone was
associated with increased expression of the oxidative stress
responsive gene heme oxygenase-1 (Fig. 3B), and this
increase was greater in cells treated with cis-guggulsterone
(38-fold) and trans-guggulsterone (32-fold) than in cells
treated with 16-dehydroprogesterone (3-fold). Thus, guggulsterone isomers and, to a lesser extent, 16-dehydroprogesterone induce oxidative stress in U937 cells that is
accompanied by decreased activation of ERK and increased
levels of heme oxygenase-1.
Rapid Cytotoxicity of Higher Concentrations of
Guggulsterone Isomers and 16-Dehydroprogesterone Is
Associated with ROS Generation, Inactivation of ERK,
and Induction of Apoptosis
To further investigate the short-term cytotoxic effects of
the guggulsterone isomers and 16-dehydroprogesterone,
we treated U937 cells with higher concentrations of these
agents (25– 75 Amol/L) for 20 hours. Our results show that,
as observed for lower concentrations (10 – 20 Amol/L) of
guggulsterone isomers and 16-dehydroprogesterone, higher
concentrations of these agents also induced a dosedependent externalization of phosphatidylserine, loss of
DCm, and increased generation of O
2 (Fig. 4A), suggesting
a priori that at higher concentrations these compounds elicit
similar cytotoxic responses albeit displaying faster kinetics.
Moreover, 16-dehydroprogesterone was as effective as
guggulsterone isomers in decreasing the levels of pERK
after treatment with 50 Amol/L for 20 hours and this
correlates with its comparable ability to induce ROS at these
concentrations. The 50 Amol/L concentrations of all compounds induced heme oxygenase-1 expression and cleavage
of caspase-3, suggesting that the observed oxidative stress
correlates with caspase activation. To further investigate the
kinetics of oxidative stress induced by 16-dehydroprogesterone and the guggulsterone, we quantitated the generation of
O
2 in cells treated with these compounds for 3 and 6 hours.
Interestingly, at 3 hours, 16-dehydroprogesterone and transguggulsterone induced a significant (P < 0.05) accumulation
of O
2 (6.6- and 5.3-fold, respectively), whereas cis-guggulsterone failed to significantly increase the levels of
O
2 (P > 0.05; Fig. 4B). Increased generation of ROS by 16dehydroprogesterone and trans-guggulsterone was sustained
for 6 hours, and at this time, cis-guggulsterone showed a
significant (P < 0.003) 7.8-fold increase in the O
2 levels,
suggesting that, although all three agents provoke ROS
generation, they induce this response with different kinetics.
Cytotoxic Concentrations of the Guggulsterone Isomers and 16-Dehydroprogesterone Uncover a Selective
Depletion of Reduced Glutathione and Oxidation of
Cardiolipin Induced by cis-Guggulsterone
Because reduced glutathione levels are critical determinants of intracellular redox homeostasis (35), we also
investigated the effects of guggulsterone isomers and 16dehydroprogesterone on the levels of this intracellular
antioxidant in U937 cells. Interestingly, at 3 hours, cisguggulsterone induced a significant (P < 0.0002) 24%
decrease in the levels of intracellular glutathione, whereas
trans-guggulsterone, which induced significant increases
in ROS at this time point, failed to affect the levels of
glutathione; U937 cells treated with 16-dehydroprogesterone for 3 hours only displayed a slight albeit significant
(P < 0.003) 7% decrease in glutathione (Fig. 4C). The
decrease in glutathione induced by cis-guggulsterone was
maintained 6 hours after treatment, and at this time, neither
16-dehydroprogesterone nor trans-guggulsterone elicited a
significant decrease (P > 0.05) in the levels of this antioxidant in U937 cells. Because cis-guggulsterone decreased
the levels of intracellular glutathione, we investigated if
this pregnadienedione would promote oxidation of the
mitochondrial phospholipid cardiolipin. Cardiolipin is
essential for mitochondrial function and for preventing
apoptosis by sequestering cytochrome c (36, 37). Glutathione is required for maintaining appropriate levels of
reduced cardiolipin via the action of a glutathionedependent peroxidase that is antiapoptotic (38). Indeed,
consistent with the effects of cis-guggulsterone on glutathione, the results presented in Fig. 4D show that after
20 hours treatment cis-guggulsterone induced a dramatic
increase in the percentage of U937 cells with low levels of
cardiolipin, whereas 16-dehydroprogesterone and transguggulsterone failed to elicit a similar response. Taken
together, these results show that, although all three pregnadienediones induce rapid generation of ROS in U937 cells,
they display different kinetics and only cis-guggulsterone
provokes marked decreases in glutathione and substantial
loss of cardiolipin.
Mitochondrial Dysfunction Induced by the Guggulsterone Isomers and 16-Dehydroprogesterone Is Independent of Caspase Activation
Because it has been reported recently that caspases
mediate loss of DCm induced by a variety of proapoptotic
stimuli (39, 40), we investigated if mitochondrial dysfunction and apoptosis induced by 50 Amol/L guggulsterone
and 16-dehydroprogesterone after 24 hours was dependent
on the activity of these proteases. In addition, we also
investigated if the potent antioxidant N-acetylcysteine
could prevent cytotoxicity induced by guggulsterone and
16-dehydroprogesterone. The results presented in Fig. 5A
show that loss of DCm induced by all three compounds was
not affected by pharmacologic inhibition of caspases using
the pancaspase inhibitor z-VAD-fmk. In contrast, z-VADfmk significantly (P < 0.001) prevented the externalization
of phosphatidylserine induced by all three compounds,
suggesting that caspase inhibition in cells treated with
guggulsterone and 16-dehydroprogesterone switches the
mode of cell death from apoptosis to necrosis (Fig. 5B).
Interestingly, the antioxidant N-acetylcysteine completely
prevented the cytotoxicity induced by 16-dehydroprogesterone but not that induced by cis-guggulsterone or transguggulsterone, suggesting that 16-dehydroprogesterone
1987
Figure 4. Cytotoxicity of higher concentrations of
the guggulsterone isomers and 16-dehydroprogesterone
is still mediated by the generation of O
2 and the
induction of apoptosis but differentiates cis -guggulsterone from trans -guggulsterone and 16-dehydroprogesterone. A, U937 cells were treated with increasing
concentrations of theguggulsterone isomers and
16-dehydroprogesterone (25 – 75 Amol/L) for 20 h, and
phosphatidylserine externalization, DCm , and O 2
generation were quantitated as described in Materials
and Methods. In addition, the levels of pERK,
total ERK, heme oxygenase-1, and a-tubulin were
examined. B, U937 cells were treated with 75 Amol/L
cis -guggulsterone, trans -guggulsterone, and 16dehydroprogesterone for 3 and 6 h and O
2 generation
was quantitated as above. C, U937 cells were
treated with 75 Amol/L cis -guggulsterone, trans guggulsterone, and 16-dehydroprogesterone for 3
and 6 h and intracellular glutathione was quantitated
as described in Materials and Methods. D, U937
cells were treated with increasing concentrations
of the guggulsterone and 16-dehydroprogesterone
(25 – 75 Amol/L) for 20 h and the oxidation of cardiolipin
was examined by flow cytometry as described in
Materials and Methods. Flow cytometry experiments
were done in triplicate and repeated at least twice.
Points, mean of a representative experiment; bars, SD.
*, P < 0.05; **, P < 0.0005.
may depend solely on ROS to induce cell death. These
data illustrate that the cis-guggulsterone and transguggulsterone, but not 16-dehydroprogesterone, induce
cytotoxicity independent of the generation of ROS and
that all three compounds induce mitochondrial dysfunction in the absence of caspase activation, but caspases
contribute to the onset of apoptosis in cells treated with
these agents.
Induce Mitochondrial Dysfunction and Apoptosis in
CD34-Positive Cells from Primary Leukemia Samples
To determine if the guggulsterone and 16-dehydroprogesterone would induce apoptosis in CD34-positive cells
from primary leukemia samples, we exposed ex vivo four
primary leukemic samples to increasing concentrations
(25 – 100 Amol/L) of these agents for 15 hours and
examined externalization of phosphatidylserine in CD34positive cells by flow cytometry. For three samples, we also
investigated the loss of DCm in CD34-positive cells. Our
results illustrated in Fig. 6A show that 25 Amol/L
guggulsterone isomers and 16-dehydroprogesterone induced phosphatidylserine externalization in CD34-positive
blasts from all samples tested (P < 0.02), albeit sample 3
was markedly more resistant to the cytotoxicity of all three
compounds. Furthermore, all three compounds induced
significant (P < 0.03) decreases in DCm in CD34-positive
blasts from patients 2 to 4 (Fig. 6B). Finally, we investigated
the cytotoxicity of 100 Amol/L guggulsterone and
16-dehydroprogesterone in normal PBMCs obtained from
healthy volunteers. As shown in Fig. 6C, none of the three
agents induced apoptosis in normal PBMC to the same
extent as in leukemia blasts. Notably, trans-guggulsterone
was the least cytotoxic pregnadienedione to PBMC minimally increasing phosphatidylserine externalization above
DMSO-treated cells by an average of 2.1 F 5.4% compared
with 50.7 F 21.6% in leukemia blasts (P < 0.02). Similarly,
16-dehydroprogesterone induced significantly less apoptosis in normal PBMC than in leukemia blasts (11.5 F 6%
versus 47.9 F 20.7%; P < 0.04). cis-Guggulsterone exhibited
the highest cytotoxicity in PBMC increasing phosphatidylserine externalization by 22.5 F 7.7%, albeit this increase
was significantly lower (P < 0.05) than the observed 60.7 F
23.5% increase in leukemia blasts treated with this agent.
Taken together, these findings show that guggulsterone
isomers and 16-dehydroprogesterone effectively induce
apoptosis in CD34-positive cells from primary leukemia
samples but not in normal PBMC and that apoptosis
induced in leukemia blasts is associated with marked
mitochondrial dysfunction.
Discussion
Natural products have provided a large number of
currently used chemotherapeutics and will continue to be
Figure 5.
Mitochondrial dysfunction induced by the guggulsterone
isomers and 16-dehydroprogesterone is independent of caspase activation
and the antioxidant N -acetylcysteine (NAC ) only prevents cell death
induced by 16-dehydroprogesterone but not by cis -guggulsterone or
trans -guggulsterone. Briefly, U937 cells were treated with 50 Amol/L cis guggulsterone, trans -guggulsterone, or 16-dehydroprogesterone alone or
in combination with the pancaspase inhibitor z-VAD-fmk (50 Amol/L) or
the potent antioxidant N -acetylcysteine (5 mmol/L), and DCm (A) and
phosphatidylserine externalization (B) were quantitated after 24 h.
an important component of drug discovery (41, 42). In fact,
there are >1,000 species of plants that possess anticancer
properties (43), and many active biological components
have been chemically modified to generate promising new
chemotherapeutic drugs (44 – 46). We have shown previously that synthetic derivatives of oleanolic acid and
diindolylmethane, found in the oleander tree and cruciferous vegetables, respectively, potently induced apoptosis
in leukemic cell lines and primary leukemic samples
(26, 47, 48). The naturally occurring plant sterols, the
guggulsterones, are the active components of the antilipidemic extract of the guggul tree C. mukul that are currently
being evaluated for treatment of hypercholesterolemia and
obesity (8, 10, 11). Interestingly, these pregnane sterols also
possess anti-inflammatory activity that may be in part
dependent on their ability to inhibit nuclear factor-nB
signaling (15 – 17, 23). We thus hypothesized a priori that
like other nuclear factor-nB inhibitors, such as curcumin
and betulinic acid, the guggulsterone isomers would also
display antiproliferative activities against leukemic cells
in culture.
We first investigated the effects of both cis-guggulsterone and trans-guggulsterone isomers as well as 16dehydroprogesterone, a steroidal isomer of guggulsterone,
on the long-term proliferation of U937 and HL60 cells in
culture. Our results show that the guggulsterone isomers
as well as 16-dehydroprogesterone similarly inhibited the
proliferation of both cell lines in culture, suggesting that
the pregnadienedione scaffold of these agents may be an
important structural feature required for their antiproliferative activity. In addition, our results indicate that
apoptosis contributes, at least in part, to the antiproliferative effects of all three compounds, and this is
associated with marked mitochondrial dysfunction in
both cell lines. We also investigated the ability of the
guggulsterone isomers and 16-dehydroprogesterone to
induce expression of differentiation markers on the
surface of HL60 cells and found that at a concentration
of 10 Amol/L trans-guggulsterone, but not cis-guggulsterone or 16-dehydroprogesterone, induced a significant
increase in the number of cells expressing CD11b and
CD14 after treatment for 120 hours, suggesting that
myelomonocytic and monocytic differentiation contribute
to the antiproliferative effects of trans-guggulsterone in
HL60 cells. Of note, because cis-guggulsterone was
observed to be more cytotoxic than trans-guggulsterone
or 16-dehydroprogesterone under these conditions, we
hypothesize that any differentiating effects of cis-guggulsterone in HL60 cells may be masked by its increased
cytotoxicity. Further investigation of the differentiating
activity of these steroids revealed that all three compounds significantly increased the number of NB4 cells
expressing CD14, suggesting that these steroids can
promote monocytic differentiation in a cell context –
dependent manner. These are the first data that show
the antiproliferative, proapoptotic, and differentiating
effects of the guggulsterone isomers and 16-dehydroprogesterone in leukemic cells in culture.
1989
Figure 6.
Guggulsterone isomers and 16-dehydroprogesterone
induced mitochondrial dysfunction and apoptosis in CD34-positive
cells from primary leukemic samples. Briefly, patient samples were
collected as described in Materials and Methods and cultured in the
presence of increasing concentrations (25 – 100 Amol/L) of cis guggulsterone, trans -guggulsterone, or 16-dehydroprogesterone
for 15 h. Phosphatidylserine externalization (A) and DCm (B) were
quantitated in CD34-positive cells. C, PBMC samples from three
healthy volunteers (A – C) were exposed to 100 Amol/L guggulsterone and 16-dehydroprogesterone for 20 h and phosphatidylserine externalization was quantitated by flow cytometry.
Because the generation of O
2 is an early event in many
forms of cell death and is an indicator of mitochondrial
dysfunction (49, 50), we also examined if the cytotoxicity
of low concentrations (<20 Amol/L) of the guggulsterone
isomers and 16-dehydroprogesterone was associated with
increased generation of this ROS. We observed that the
guggulsterone isomers and, to a lesser extent, 16-dehydroprogesterone indeed generated increased levels of O
2
before the onset of apoptosis, suggesting that the long-term
cytotoxicity of these agents is associated with oxidative
stress. Interestingly, our observations also indicate that at
low concentrations (<20 Amol/L) cis-guggulsterone and
trans -guggulsterone, but not 16-dehydroprogesterone,
markedly decreased the pERK expression after treatment
for 48 hours, suggesting that the increased ROS generated
by these compounds may contribute to the inactivation of
ERK signaling (32, 33). Finally, we observed that cytotoxicity induced by cis-guggulsterone, trans-guggulsterone,
and 16-dehydroprogesterone was accompanied by increased expression of the oxidative stress response protein
heme oxygenase-1, and cis -guggulsterone and trans guggulsterone were more effective than 16-dehydroprogesterone in inducing this response probably due to their
increased ability to generate ROS. These data indicate
that oxidative stress is associated with the cytotoxicity
of the guggulsterone and 16-dehydroprogesterone and
that the guggulsterone can abrogate the activation of ERK
in leukemic cells.
To further investigate the mechanism of action of the
guggulsterone isomers and 16-dehydroprogesterone, we
evaluated the short-term effects of higher concentrations
(25 – 75 Amol/L) of these agents. Our results indicate that at
higher concentrations the cytotoxicity of these agents is still
associated with apoptosis, mitochondrial dysfunction, and
ROS generation. In addition, at higher concentrations, 16dehydroprogesterone was as effective as the guggulsterone
isomers in decreasing pERK levels and this correlated with
its increased ability to induce ROS at these concentrations.
However, although all three agents induced a dose- and
time-dependent increase in the generation of ROS, only cisguggulsterone significantly decreased glutathione levels,
and this occurred before the increase in O2 levels,
suggesting that cis-guggulsterone may act through a
different mechanism to induce oxidative stress. Notably,
only cis-guggulsterone markedly decreased the levels of
cardiolipin, suggesting that the decrease in glutathione
induced by this agent may lead to oxidation of this critical
mitochondrial phospholipid. The use of higher concentrations of the guggulsterone and 16-dehydroprogesterone
uncovered a cis-guggulsterone-specific effect on the levels
of glutathione and cardiolipin in U937 cells, suggesting that
the cytotoxicity of cis-guggulsterone may be mediated by a
different mechanism from that of trans-guggulsterone or
16-dehydroprogesterone.
We also investigated if caspases were involved in the
cytotoxicity of the guggulsterone and 16-dehydroprogesterone and found that pharmacologic inhibition of these
proteases with z-VAD-fmk prevented phosphatidylserine
externalization but not loss of DCm, suggesting that
mitochondrial dysfunction induced by these agents occurs
before caspase activation but caspases contribute to the
induction of apoptosis. The potent antioxidant N-acetylcysteine completely prevented the cytotoxicity of 16dehydroprogesterone but not that of the guggulsterone
isomers. This observation suggests that either (a) the
oxidative stress induced by the guggulsterone cannot be
reversed by 5 mmol/L N-acetylcysteine cotreatment or (b)
the cytotoxicity of 16-dehydroprogesterone depends solely
on the generation of ROS.
Finally, we investigated if the guggulsterone and 16dehydroprogesterone could induce apoptosis and mitochondrial dysfunction in primary CD34-positive leukemia
cells in culture. Our results show that all three agents
induced rapid (f15 hours) apoptosis in CD34-positive cells
from primary leukemia samples and that this was
associated with mitochondrial dysfunction, although one
sample seemed to be more resistant to the cytotoxic effects
of these compounds. Most notably, the guggulsterone and
16-dehydroprogesterone were more cytotoxic to leukemia
blasts than to normal PBMC, suggesting that the pregnadienedione structure of these agents may display a
therapeutic window. These results are the first to show
the antileukemic activity of the guggulsterone isomers and
16-dehydroprogesterone in CD34-positive primary leukemic cells.
In conclusion, the guggulsterone isomers and 16-dehydroprogesterone represent a novel class of naturally
occurring compounds that exhibit antileukemic activity
by inducing apoptosis and differentiation. Our results
indicate that the pregnadienedione structure of these
steroid isomers has inherent antiproliferative, proapoptotic,
and differentiating activities and may display some
selectivity for leukemia blasts over normal PBMC. We are
currently investigating the antileukemic effect of synthetic
derivatives of these agents as well as their pharmacokinetic
properties in animal models with the goal of developing
novel and more effective treatments for AML. The
understanding of the mechanism of action of this novel
class of steroidal compounds will offer additional targets
for the treatment of human leukemias.
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