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Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 [Y. I., P. P., S. K., D. K.], and Norris Cotton Cancer Center and Department of Pharmacology, Dartmouth Medical School [A. P., M. B. S.], Department of Chemistry, Dartmouth College [G. W. G., T. H.], Hanover, New Hampshire 03755
Abstract
The oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a multifunctional molecule that induces growth inhibition and differentiation of human myeloid leukemia cells. The present studies demonstrate that CDDO treatment results in apoptosis of U-937 and HL-60 myeloid leukemia cells. Similar to 1-ß-D-arabinofuranosylcytosine (ara-C), another agent that inhibits growth and induces apoptosis of these cells, CDDO induced the release of mitochondrial cytochrome c and activation of caspase-3. Overexpression of Bcl-xL blocked cytochrome c release, caspase-3 activation, and apoptosis in ara-C-treated cells. By contrast, CDDO-induced release of cytochrome c, and activation of caspase-3 were diminished only in part by Bcl-xL. In concert with these findings, we demonstrate that CDDO, but not ara-C, activates caspase-8 and thereby caspase-3 by a cytochrome c-independent mechanism. The results also show that CDDO-induced cytochrome c release is mediated by caspase-8-dependent cleavage of Bid. These findings demonstrate that CDDO induces apoptosis of myeloid leukemia cells and that this novel agent activates an apoptotic signaling cascade distinct from that induced by the cytotoxic agent ara-C.
Introduction
The new synthetic oleanane triterpenoid
CDDO3
induces monocytic differentiation of human myeloid leukemia cells
(1)
. This agent also induces adipogenic differentiation of
mouse 3T3-L1 fibroblasts and contributes to nerve growth factor-induced
neuronal differentiation of rat PC12 cells (1)
. The
mechanisms responsible for the differentiating effects of CDDO remain
unclear. CDDO inhibits the proliferation of diverse types of human
tumor cell lines. In addition, the finding that CDDO inhibits the
induction of iNOS and COX-2 in macrophages, microglia, and fibroblasts
has supported a wide range of actions (1)
. The structure
of CDDO resembles that of steroids and other isoprenoids, and recent
studies (unpublished) have suggested that interaction of CDDO with the
nuclear receptor, peroxisome proliferator-activated receptor-
, may
account for some of its actions.
ara-C incorporates into replicating DNA and inhibits proliferation by
functioning as a relative chain terminator (2, 3, 4, 5)
. Other
studies have also demonstrated that treatment of myeloid leukemia cells
with ara-C is associated with induction of a differentiated phenotype
(6, 7, 8)
. The cellular response to ara-C and other agents
that inhibit DNA replication also includes the induction of apoptosis
(9)
. ara-C induced apoptosis is associated with
internucleosomal DNA fragmentation (10)
and proteolytic
activation of protein kinase C
(11)
. In addition,
expression of the antiapoptotic Bcl-2 or Bcl-xL
proteins blocks ara-C-induced apoptosis (12, 13, 14)
. By
contrast, although expression of CrmA inhibits apoptosis induced by
activation of the tumor necrosis factor or Fas receptors (15
, 16)
, this protein has no detectable effect on ara-C-induced
apoptotic cell death. These findings have indicated that inhibitors of
DNA replication activate a protease cascade that differs at least in
part from that induced by proapoptotic agents, which act at the cell
membrane.
Studies have demonstrated that mitochondria transduce proapoptotic signals by release of cytochrome c into the cytoplasm (17, 18, 19) . Cytochrome c associates with cytoplasmic Apaf-1 and thereby activates procaspase-9 (20 , 21) . In turn, caspase-9 cleaves and activates caspase-3 (20 , 21) . A central role for caspase-3 in cell death is supported by involvement of this executioner caspase in the apoptotic response to diverse stimuli (22 , 23) . Because cytochrome c release and activation of caspase 9 represent one pathway for cleavage of caspase-3, other studies have shown that caspase-8 can directly activate caspase-3 (24) . For example, caspase-8 is activated by stimulation of the Fas receptor, recruitment of FADD/Mort-1 to the receptor, and thereby oligomerization and autoprocessing of caspase-8 (25 , 26) . These findings have indicated that receptor-mediated apoptosis can be induced by a mitochondria-independent mechanism. Caspase-8, however, also cleaves Bid, a proapoptotic member of the Bcl-2 family that induces the release of cytochrome c (27 , 28) . Thus, Bid-induced release of cytochrome c can amplify caspase-8-initiated induction of apoptosis.
The present studies demonstrate that CDDO induces apoptosis of myeloid leukemia cells. The results also show that CDDO-induced cell death is mediated by caspase-8dependent cleavage of caspase-3 and amplified by cytochrome c release. By contrast, ara-C-induced apoptosis is mediated by cytochrome c-dependent activation of caspase-3. The finding that Bcl-xL blocks ara-C-induced cell death but only attenuates CDDO-induced cell death provides further support for two distinct pathways in the apoptotic response to these agents.
Results
The available evidence indicates that CDDO is a potent
multifunctional molecule (1)
. To determine whether CDDO
induces apoptosis, we treated human U-937 myeloid leukemia cells with
this agent and then assayed for internucleosomal DNA fragmentation. The
results demonstrate that exposure to 5 µM CDDO results in
endonucleolytic DNA cleavage (Fig. 1A)
. Similar results were obtained after CDDO treatment of
HL-60 myeloid leukemia cells (Fig. 1A)
. In concert with
induction of apoptosis, U-937 and HL-60 cells also responded to CDDO
with cleavage of PARP (Fig. 1B)
.
|
. U-937 cells that overexpress Bcl-xL
(U-937/Bcl-xL) are resistant to ara-C-induced
apoptosis (Fig. 2B)
. By contrast, CDDO-induced
apoptotic death was attenuated only in part by
Bcl-xL overexpression (Fig. 2B)
. These
findings indicated that CDDO induces apoptosis by a mechanism distinct
from that activated by ara-C.
|
. The release of cytochrome c was diminished in
part in CDDO-treated U-937/Bcl-xL cells (Fig. 3A)
. By contrast, overexpression of
Bcl-xL substantially blocked ara-C-induced
cytochrome c release (Fig. 3B)
. To assess whether
CDDO activates caspase-3, lysates were subjected to immunoblotting with
anti-caspase-3. Treatment of U-937 and
U-937/Bcl-xL cells with CDDO resulted in cleavage
of caspase-3, although this effect was partially diminished by
Bcl-xL overexpression (Fig. 4A)
. In concert with the more complete block in cytochrome
c release associated with Bcl-xL
overexpression, ara-C treatment of U-937, but not
U-937/Bcl-xL, cells exhibited activation of
caspase-3 (Fig. 4B)
. In addition, treatment of both U-937
and U-937/Bcl-xL cells with CDDO was associated
with cleavage of the caspase-3 substrate PKC (Fig. 4C)
.
These findings indicated that CDDO induces caspase-3 activation and
apoptosis by mechanism that is predominantly independent of cytochrome
c release.
|
|
. By contrast, treatment with ara-C resulted in
a 1.5-fold increase (Fig. 5A, left panel)
. Similar results
were obtained when HL-60 cells were treated with CDDO or ara-C (Fig. 5A, middle panel
and data not shown). In addition,
overexpression of Bcl-xL had no detectable effect
on CDDO-induced increases in caspase 8 activity (Fig. 5A, right panel)
. Because caspase-8 cleaves Bid (27
, 28)
, we
subjected cell lysates to immunoblot analysis with an anti-Bid
antibody. The results demonstrate that Bid is cleaved in CDDO-treated,
but not ara-C-treated, U-937 and HL-60 cells (Fig. 5B
and
data not shown). To define the temporal relationships among the
CDDO-induced events, U-937 cells were treated with CDDO and then
harvested at various intervals. Induction of caspase-8 (1.5–1.8-fold)
and caspase-3 activities was detectable at 2–4 h of CDDO treatment
(Fig. 6)
. By contrast, significant induction of Bid cleavage and release of
cytochrome c was not observed until 4–6 h (Fig. 6B)
. These findings indicated that, in contrast to ara-C,
CDDO induces caspase-8 activation and thereby direct cleavage of
caspase-3. Moreover, CDDO-induced cleavage of Bid and the subsequent
release of cytochrome c at 4–6 h amplified caspase-3
activation, which was maximal at 6 h (Fig. 6B, bottom panel)
.
|
|
. Moreover, CrmA inhibited CDDO-induced
apoptosis (Fig. 7C)
. By contrast, CrmA expression had no
detectable effect on ara-C-induced caspase-3 activation or apoptosis
(data not shown). These findings provided further support for a model
in which CDDO induces apoptosis by a caspase-8-mediated mechanism.
|
Triterpenoids are biosynthesized in plants by the cyclization of squalene. These agents are known to exhibit anti-inflammatory and anticarcinogenic activity (30 , 31) . CDDO is a synthetic triterpenoid analogue that suppresses the formation of iNOS and COX-2 in various cell types stimulated with inflammatory cytokines (1) . Suppression of iNOS and COX-2 production has been a focus of chemoprevention because of the role of these enzymes as enhancers of carcinogenesis (32, 33, 34, 35) . CDDO has also been found to function as an inducer of differentiation of human myeloid leukemia cells and certain other cell types (1) . The present studies extend the analysis of CDDO-mediated effects by demonstrating that CDDO also induces apoptosis of myeloid leukemia cells.
Despite having a wide range of biological activities, little is known
about the molecular mechanisms of action of CDDO. Nonetheless, the
finding that CDDO induces apoptosis provided an opportunity to assess
the effects of this agent on signaling pathways associated with cell
death mechanisms. Other work has demonstrated that ara-C induces
differentiation and apoptosis of myeloid leukemia cells
(8, 9, 10)
. Moreover, similar to ara-C, CDDO inhibits
cellular proliferation (1)
. As such, one expectation was
that CDDO and ara-C might induce apoptosis of myeloid leukemia cells by
similar mechanisms. The results demonstrate that both CDDO and ara-C
induce cleavage of caspase-3. Both agents also induced cleavage of the
caspase-3 substrates, PARP and PKC. However, although certain
downstream mechanisms associated with CDDO- and ara-C-induced apoptosis
are similar, the results support distinct upstream cascades.
Previous studies have demonstrated that IR and other genotoxic agents induce the release of mitochondrial cytochrome c (12) . Although the upstream signals responsible for DNA damage-induced cytochrome c release are unclear, the downstream events involve interaction of cytochrome c with Apaf-1 and the activation of caspase-9 (20 , 21) . ara-C functions as an inhibitor of DNA replication (2 , 4) as compared with agents, such as IR, that induce DNA lesions. Nonetheless, the present results demonstrate that ara-C, like IR (12) , induces the release of cytochrome c. The results also demonstrate that overexpression of Bcl-xL blocks ara-C-induced cytochrome c release. The finding that Bcl-xL inhibits ara-C induced caspase-3 activation and apoptosis further support a cytochrome c-dependent signaling pathway. In addition, ara-C treatment is associated with activation of caspase-9 (data not shown). Thus, the results demonstrate that ara-C induces cytochrome c release and thereby activation of caspase-3 and induction of apoptosis.
In contrast to the findings with ara-C, the results support a
mechanism in which CDDO induces apoptosis by caspase-8-mediated
cleavage of caspase-3. In this context, CDDO-induced activation of
caspase-8 and caspase-3 preceded Bid cleavage and release of cytochrome
c. Moreover, CDDO-induced cytochrome c release,
caspase-3 activation, and apoptosis were diminished only in part by
overexpression of Bcl-xL. The finding that CDDO
treatment is associated with induction of caspase-8 activity further
supported activation of a caspase 8
caspase-3 pathway (Fig. 8)
. Also in support of a caspase-8-dependent pathway is the finding that
CrmA overexpression blocks CDDO-induced, but not ara-C-induced,
activation of caspase-3 and apoptosis. These results collectively
indicate that CDDO, in contrast to ara-C, activates a cell death
pathway in which caspase-8 is the initiator caspase. The finding that
the response to CDDO is diminished only in part by
Bcl-xL overexpression supports a model in which
the caspase-8-initiated cascade is amplified by mitochondrial signaling
(Fig. 8)
.
|
Cell Culture and Reagents.
Human U-937 and HL-60 myeloid leukemia cells (American Type Culture
Collection, Rockville, MD) were grown in RPMI 1640 (Sigma) supplemented
with 10% heat-inactivated fetal bovine serum (Sigma), 100 units/ml
penicillin, 100 µg/ml streptomycin, and 2 mM
L-glutamine. U-937/Bcl-xL
(36)
and U-937/CrmA (29)
cells were cultured
in medium containing 500 µg/ml geneticin sulfate (Life Technologies,
Inc.). Stock solutions of 10 mM CDDO were made in DMSO, and
aliquots were frozen at -20°C. Cells were seeded at a density of
2.5 x 105/ml 24 h before treating with
5 µM CDDO or 10 µM ara-C (Sigma).
Isolation of the Cytosolic Fraction.
Cytosolic fractions were prepared as described (12)
. Cells
were washed twice with PBS and then suspended in ice-cold buffer [20
mM HEPES (pH 7.5), 1.5 mM
MgCl2, 10 mM KCl, 1 mM
EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin, aprotinin, and
pepstatin A] containing 250 mM sucrose. The cells were
disrupted by five strokes in a Dounce homogenizer. After centrifugation
of the lysate at 10,000 x g for 5 min at 4°C, the
supernatant fraction was centrifuged at 105,000 x g
for 30 min at 4°C. The resulting supernatant was used as the soluble
cytosolic fraction.
Immunoblot Analyses.
Total cell lysates were prepared as described in lysis buffer
containing 1% NP40 (37)
. Proteins were separated by
SDS-10, 12.5 or 15% PAGE, and then transferred to nitrocellulose
filters. After blocking with 5% dried milk in PBS-Tween, the filters
were incubated with anti-cytochrome c (38)
,
anti-Bid (27)
, anti-caspase-9 (PharMingen), anti-caspase 3
(anti-CPP32; Transduction Laboratories), anti-PKC (Santa Cruz
Biotechnology), anti-PARP (39)
,
anti-Bcl-xL (Novartis, East Hanover, NJ),
or anti-CrmA. After washing and incubation with horseradish
peroxidase-conjugated anti-rabbit (Amersham) or antimouse (Amersham),
the antigen-antibody complexes were visualized by enhanced
chemiluminescence (Amersham).
Assays of Caspase-8 Activity.
Caspase-8 activity was measured by spectrophotometric detection (405
nm) of the chromophore pNA after cleavage from the labeled
substrate IETD-pNA (FLICE/Caspase-8 Colorimetric Assay kit;
BioVision Research Products, Palo Alto, CA).
Analysis of DNA Fragmentation.
Cells (1 x 106) were washed with PBS and
incubated in 20 µl of 50 mM Tris-HCl (pH 8.0), 10
mM EDTA, 0.5% SDS, and 0.5 µg/ml proteinase K (Sigma) at
50°C for 30 min. Ten µl of 0.5 mg/ml RNase A (Boehringer Mannheim)
were added, and the mixture was incubated for an additional 1 h.
The digested samples were incubated with 10 ml of 10 mM
EDTA (pH 8.0), containing 1% (w/v) low-melting-point agarose, 0.25%
bromphenol blue, and 40% sucrose at 70°C. The DNA was separated in
gels containing 2% agarose/TAE (40 mM Tris-acetate and 10
mM EDTA, pH 8.0) buffer at 23 V for 16 h and
visualized by UV illumination after ethidium bromide staining.
Flow Cytometry.
DNA content was assessed by staining ethanol-fixed cells with propidium
iodide and monitoring by FACScan (Becton Dickinson). Numbers of cells
with sub-G1 DNA content were determined with the
MODFIT LT program (Verity Software House, Topsham, ME).
Acknowledgments
We thank Drs. Lawrence Prochaska for providing antibody to cytochrome c and Xiadong Wang for anti-Bid antibody.
Footnotes
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.
1 This investigation was supported by PHS Grants
CA-42802 and CA-29431 awarded by the National Cancer Institute,
Department of Health and Human Services. 
2 To whom requests for reprints should be
addressed, at Department of Medicine, Dana-Farber Cancer Institute,
Harvard Medical School, 44 Binney Street, Boston, MA 02115.
3 The abbreviations used are: CDDO,
2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; ara-C,
1-ß-D-arabinofuranosylcytosine; iNOS, inducible nitric
oxide synthase; COX-2, cyclooxygenase 2; PKC, protein kinase C; PARP,
poly(ADP-ribose) polymerase; IR, ionizing radiation;
pNA, p-nitroanilide.
Received for publication 1/ 5/00. Revision received 4/ 6/00. Accepted for publication 4/ 7/00.
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) and
U-937/Bcl-xL (
) cells were treated with 5
µM CDDO or 10 µM ara-C. Cells were fixed in
70% ethanol, stained with propidium iodide, and analyzed for DNA
content by flow cytometry. The results are expressed as the percentage
of cells with sub-G1 DNA (mean percentage apoptosis of
three independent experiments; bars, SD).
), and U-937/Bcl-xL (