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NEOPLASIA
From the Department of Blood and Marrow
Transplantation, Section of Molecular Hematology and Therapy, and the
Departments of Bioimmunotherapy and Biostatistics, The University
of Texas M. D. Anderson Cancer Center, Houston, TX; The University
of Florida, Gainesville; Department of Medicine, Dartmouth College,
Department of Chemistry and Dartmouth Medical School, Department of
Pharmacology, Hanover, NH.
It has been shown that the novel synthetic triterpenoid CDDO
inhibits proliferation and induces differentiation and apoptosis in
myeloid leukemia cells. In the current study the effects of the C-28
methyl ester of CDDO, CDDO-Me, were analyzed on cell growth and
apoptosis of leukemic cell lines and primary acute myelogenous leukemia
(AML). CDDO-Me decreased the viability of leukemic cell lines,
including multidrug resistant (MDR)-1-overexpressing, p53null HL-60-Dox and of primary AML cells, and it
was 3- to 5-fold more active than CDDO. CDDO-Me induced a loss of
mitochondrial membrane potential, induction of caspase-3
cleavage, increase in annexin V binding and DNA fragmentation,
suggesting the induction of apoptosis. CDDO-Me induced pro-apoptotic
Bax protein that preceded caspase activation. Furthermore,
CDDO-Me inhibited the activation of ERK1/2, as determined by the
inhibition of mitochondrial ERK1/2 phosphorylation, and it blocked
Bcl-2 phosphorylation, rendering Bcl-2 less anti-apoptotic. CDDO-Me
induced granulo-monocytic differentiation in HL-60 cells and monocytic
differentiation in primary cells. Of significance, colony formation of
AML progenitors was significantly inhibited in a dose-dependent
fashion, whereas normal CD34+ progenitor cells were less
affected. Combinations with ATRA or the RXR-specific ligand LG100268
enhanced the effects of CDDO-Me on cell viability and terminal
differentiation of myeloid leukemic cell lines. In conclusion, CDDO-Me
is an MDR-1- and a p53-independent compound that exerts strong
antiproliferative, apoptotic, and differentiating effects in myeloid
leukemic cell lines and in primary AML samples when given in
submicromolar concentrations. Differential effects of CDDO-Me on
leukemic and normal progenitor cells suggest that CDDO-Me has potential
as a novel compound in the treatment of hematologic malignancies.
(Blood. 2002;99:326-335) Acute myelogenous leukemia (AML) remains
incurable in most patients, largely because of its resistance to
chemotherapy. The therapeutic regimens used have not changed in the
past 3 decades and usually include cytosine arabinoside (ara-C) and
anthracycline analogs.1,2 Recently, topoisomerase
inhibitors, cytokines, and multidrug resistant (MDR)-1 blockers have
been evaluated, but they failed to have major impact on patient
survival.3-7
Most chemotherapy agents used in the treatment of hematologic
malignancies eliminate cells by inducing apoptosis, and many factors
that inhibit chemotherapy-induced apoptosis have been identified. The
initiation of a cascade of cysteine proteases of the ICE/ced3 family
(caspases) plays a pivotal role in apoptosis.8 The
extrinsic death receptor pathway, triggered by members of the tumor necrosis factor family, is activated when the proximal regulator caspase-8 is recruited into the death receptor complex. The
Bcl-2 family of proteins, on the other hand, seems to play a central
role in the regulation of the mitochondrial (intrinsic) apoptotic pathway. In particular, Bcl-2 and Bcl-XL
overexpression prevent the mitochondrial release of cytochrome
c, caspase activation, and apoptosis (for review, see
9-11). Bax is a pro-apoptotic member of the Bcl-2 family
that dimerizes with itself or with Bcl-2/Bcl-XL, and an
increase in the levels of free Bax, or Bax homodimers, promotes
apoptosis.12,13 Bcl-2 has been the subject of intense study as a mechanism of chemoresistance because of its ability to
suppress chemotherapy-induced apoptosis.12,14,15 Recent studies have demonstrated that phosphorylation of Bcl-2 at the serine
70 site is required to inhibit apoptosis.16,17 Protein kinase (PKC)- Triterpenoids, together with their close chemical relatives, steroids,
are members of a larger family of structurally related compounds called
cyclosqualenoids.21 Oleanolic and ursolic acids are both
derived from squalene and have definite, though weak, anti-inflammatory
and anticarcinogenic properties.22,23
2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a novel
synthetic triterpenoid that is more than 10 000-fold more potent than
its parent compound, oleanolic acid, in suppressing the
Reagents
Cell lines
Patients Samples of bone marrow or peripheral blood were obtained for in vitro studies from patients with newly diagnosed or recurrent AML with high (more than 70%) blast count and from patients with myeloid transformation of chronic myeloid leukemia (CML). Informed consent was obtained following institutional guidelines. Mononuclear cells were separated by Ficoll-Hypaque (Sigma Chemical) density-gradient centrifugation.Suspension culture of leukemic cells Leukemic cell lines were cultured at a density of 3.0 × 105 cells/mL, and AML mononuclear cells were cultured at 5 × 105 cells/mL in the presence or absence of indicated concentrations of CDDO-Me. Appropriate amounts of DMSO (final concentration less than 0.05%) were included as control. For cytotoxicity studies, 1 µM ara-C was added to the cultures. After 24 to 72 hours, viable cells were counted with the trypan blue dye exclusion method using a hematocytometer.Cell kinetic and DNA fragmentation studies Cell cycle kinetics was determined by staining cells with acridine orange for cellular DNA and RNA content and was followed by flow cytometric analysis as described.33 Samples were measured in a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using the 488-nm line of a 15-nm argon laser and filter settings for green (530 nm) (DNA) and red (585 nm) (RNA) fluorescence. Ten thousand events were stored in list mode for analysis. The percentage of cells in the sub-G1 peak defined the proportion of apoptotic cells in the tested populations. Cell debris was defined as events in the lowest 10% range of fluorescence, and these results were eliminated from analysis. Cell-cycle kinetics was analyzed using ModFit software (Verity Software House, Topsham, ME).Acute myelogenous leukemia blast colony assay A previously described method was used to measure AML blast colony formation.34,35 Briefly, 1 × 105 T-cell-depleted, nonadherent, low-density bone marrow cells were plated in 0.8% methylcellulose in Iscoves modified Dulbecco medium (IMDM; Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum and 15 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF). CDDO-Me was added at the initiation of cultures at concentrations ranging from 0.05 to 0.5 µM. AML blast colonies were evaluated under a microscope on day 7 of culture in duplicate dishes.Granulocyte-erythroid-macrophage-megakaryocyte colony-forming unit assay In 3 experiments, 2 × 105 CD34+ cells isolated from normal bone marrow (n = 1) or G-CSF-stimulated peripheral blood (n = 2) were plated in 0.8% methylcellulose with IMDM, 1 U/mL human erythropoietin (Terry Fox Laboratories, Vancouver, BC, Canada), and 50 ng/mL recombinant hGM-CSF. CDDO-Me was added at the initiation of cultures at concentrations ranging from 0.05 to 0.5 µM. All cultures were evaluated after 14 days for the number of erythroid burst-forming unit (BFU-E) colonies, defined as aggregates of more than 500 hemoglobinized cells or 3 or more erythroid subcolonies, and granulocyte-macrophage colony-forming unit (CFU-GM) colonies, defined as a cluster of 40 or more granulocytes, monocyte-macrophages, or both.Western blot analysis An equal amount of protein lysate was placed on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2 hours at 100 V, followed by transfer of the protein to a Nytran membrane (Schleicher & Schuell, Keene, NH) and immunoblotting. Polyclonal rabbit antibodies to Bcl-2, Bcl-XL, and Bax36,37 were kindly provided by Dr J. C. Reed. Antibodies against poly (ADP-ribose) polymerase (PARP) was obtained from PharMingen (San Diego, CA), DFF-45 was from Oncogene (Cambridge, MA), XIAP was from Transduction Laboratories (Lexington, KY), caspase-3 was from PharMingen, and phospho-specific anti-pERK1/2 antibodies were from Calbiochem. A specific antibody recognizing only the p20-processed caspase-3 band was provided by Idun Pharmaceutical (La Jolla, CA).Cell fractionation and Bax immunolocalization studies Subcellular fractionation of cells was performed by a previously described method.38 Briefly, cells were swollen in ice-cold hypotonic HEPES buffer (10 mM HEPES, pH 7.4, 5 mM MgCl2, 40 mM KCl, 1 mM PMSF, 10 µg/mL aprotinin, 10 µg/mL leupeptin) for 30 minutes, aspirated repeatedly through a 25-gauge needle (25 strokes), and centrifuged at 200g to pellet the nuclei. The resultant supernatant was then centrifuged at 10 000g to pellet the heavy-membrane fraction containing the mitochondria. The heavy-membrane supernatant was centrifuged at 150 000g to pellet the plasma membranes, and the supernatant represented the cytosol (Cyt). Subcellular fractions were subjected to denaturing electrophoresis in a 12% acrylamide-0.1% SDS gel and transferred to nitrocellulose for Bax Western blot analysis.Northern blot analysis The Bax probe was obtained by cloning the polymerase chain reaction products of amplified cDNA. The sequence was compared with GenBank data to ensure that the correct cDNA was cloned. Twenty micrograms total RNA was denatured and run overnight on a 1% formamide agarose gel at 30 V. After staining in ethidium bromide, RNA was transferred to a Nitran filter and left overnight in 10× sodium chloride-sodium citrate, followed by drying at 80°C. Hybridization was carried out at 42°C for 20 hours, and the filters were washed under highly stringent conditions. Signals were analyzed with a Betascope 603 (Betagen, Waltham, MA).Metabolic labeling, immunoprecipitation, and immunoblot analysis Cells were labeled with [32P]orthophosphoric acid and then treated with 0.1 µM CDDO-Me, after which Bcl-2 was analyzed by immunoprecipitation, as previously described.17,39 Samples were electrophoresed in a 12% acrylamide-0.1% SDS gel, transferred to nitrocellulose, and exposed to Hyperfilm (Amersham Pharmacia Biotech, United Kingdom) at 80°C. The same blot was used
for Western blot analysis with anti-Bcl-2 antisera.
In vitro ERK assay The effect of CDDO-Me was determined using an in vitro MAP kinase assay kit from Upstate Biotechnology (Lake Placid, NY) and ERK1/2 antibody from Santa Cruz Biotechnology (Santa Cruz, CA). For each sample, ERK 1/2 was immunoprecipitated from 2 × 107 K562 or 1 × 107 HL60 cells using a specific anti-ERK 1/2 antibody and Protein A agarose (Life Technologies, Rockville, MD). The ERK-containing agarose pellet was resuspended in assay buffer containing an inhibitor cocktail (PKC inhibitor peptide, PKA inhibitor peptide, and compound R24571) to block possible contaminating non-ERK kinases. Where appropriate, varying concentrations (0.1, 1, and 10 µM) of CDDO-Me were added. Dephosphorylated myelin basic protein (MBP; 25 µg) was used as substrate. Phosphorylation of MBP was observed by using an anti-phospho-MBP antibody. As a negative control, a lysate containing inactive ERK (obtained from K562 cells treated for 4 hours in vivo with 10 µM MEK inhibitor PD98059) was used in the assay. The amount of ERK2 immunoprecipitated from each sample was determined by using anti-ERK2 antibody.For K562 cells, a control experiment was performed to determine that CDDO-Me could at least inhibit ERK upstream, if not directly. K562 cells were treated in vivo for 4 hours with 1 µM CDDO-Me, and lysate from these cells was used in the in vitro kinase assay. Immunophenotyping Phycoerythrin-conjugated anti-CD11b, fluorescein isothiocyanate (FITC)-conjugated anti-CD14 monoclonal antibody (mAb) (Becton Dickinson) and phycoerythrin-conjugated anti-CD95 mAb (PharMingen) were used at a 1:10 dilution. Percentage positive cells was calculated by subtracting the percentage of cells with a fluorescence intensity greater than the set marker using the isotype control (background) from the percentage of cells with a fluorescence intensity greater than the same marker using the specific antibody.40Annexin V staining Cells were washed in phosphate-buffered saline and resuspended in 100 µL binding buffer containing annexin V (Roche Diagnostic, Indianapolis, IN). Cells were analyzed by flow cytometry after the addition of propidium iodide (PI).41 Annexin V binds to those cells that express phosphatidylserine on the outer layer of the cell membrane, and PI stains the cellular DNA of those cells with a compromised cell membrane. This allows for live cells (unstained with either fluorochrome) to be discriminated from apoptotic cells (stained only with annexin V) and necrotic cells (stained with both annexin and PI).42Cytofluorometric analysis of the   m, cells were incubated
with the cationic lipophilic dye chlorophenyl-X-rosamine (CMXRos; 150 nM; Molecular Probes, Eugene, OR).43 CMXRos is
incorporated into mitochondria driven by the m and
reacts with thiol residues to form covalent thiol ester bonds. CMXRos
fluorescence was recorded by flow cytometry in fluorescence channel 3 (FL-3). Background values of the apoptosis of control cells
cultured without the CDDO-Me or in DMSO-solvent control (less than 10%
CMXRos-low) were subtracted from the values obtained under
experimental conditions.
In a series of experiments, cells were pretreated for 1 hour with 10 µM CyA or 50 µM BA before CDDO-Me was added. These agents prevent a
reduction in Detection of active caspases Cell-permeable fluorigenic substrate Phi-Phi-Lux-G1D2 was administered to monitor caspase activity according to the manufacturer's recommendations (OncoImmunin, Kensington, MD).46 Briefly, 106 cells were resuspended in 5 µL substrate solution and incubated for 1 hour at 37°C in the dark. After incubation, cells were washed, and the fluorescence emission was determined using the FL-1 channel of a Becton Dickinson FACScan flow cytometer.Statistics Results are expressed as means ± SEM. Levels of significance were evaluated by a 2-tailed paired Student t test, and P < .05 was considered significant.
CDDO-Me decreases viability and induces apoptosis in leukemic cell lines CDDO-Me decreased the viability in leukemic HL-60, KG-1, and NB4 cells, with respective IC50 values of 0.4, 0.4, and 0.27 µM, as determined by cell counts at 48 hours. To study the mechanism of growth inhibition, we analyzed the effect of CDDO-Me on cell cycle and apoptosis in HL-60 cells. CDDO-Me at 0.05 and 0.1 µM inhibited cell growth in a dose- and time-dependent fashion, and no viable cells were recovered at 0.5 µM (Figure 1A). Although cell-cycle measurements revealed no significant differences in cell-cycle distribution, a dose-dependent increase in annexin V binding in CDDO-Me-treated cells was seen (Figure 1B), suggesting that apoptosis contributed to CDDO-Me-induced growth arrest. HL-60 cells are p53null, suggesting that the cytotoxic effect of CDDO-Me is p53 independent. MDR-1 overexpressing HL-60-Dox cells were also sensitive to CDDO-Me, and blocking MDR-1 using the specific inhibitor PSC-833 did not enhance CDDO-Me cytotoxicity (data not shown). Hence, CDDO-Me appears to be p53 and MDR-1 independent.
CDDO-Me decreases viability, induces apoptosis, and inhibits colony formation in primary acute myelogenous leukemia cells In primary AML cells, CDDO-Me induced a dose-dependent increase in the percentage of apoptotic cells, as determined by DNA flow cytometry (n = 4, Figure 2A). The paired mean difference between 0.1 µM CDDO-Me and DMSO-treated cells was 26.9% ± 10.8% (CDDO-Me-DMSO) and 43.2% ± 6.0% at 0.5 µM CDDO-Me (P < .01). In an additional series of 7 patient samples, apoptosis was induced in 6 of 7 AML samples at 1 µM CDDO-Me, and 5 µM induced apoptosis in all samples with more than 70% apoptotic cells in 6 of 7 samples (Figure 2B). CDDO-Me also induced apoptosis in CML blast crisis samples in vitro (in 3 of 4 samples at 1 µM, in all 4 samples at 5 µM).
We then examined the effect of CDDO-Me on clonogenic AML cells. Colony
formation of AML progenitors was significantly inhibited in a
dose-dependent fashion, with 46.7% ± 6.6% surviving colonies at
0.1 µM CDDO-Me and only 8.8% ± 3.8% at 0.5 µM (Figure
3). CDDO-Me at 0.5 µM also inhibited
more than 50% of colonies in 2 CML myeloid blast crisis samples. In
contrast, 64.5% ± 1.1% and 60.8% ± 2.5% CFU-GM and CFU-E from
normal CD34+ cells survived treatment with 0.5 µM
CDDO-Me. The difference between AML and normal progenitors was highly
significant (P < .02).
CDDO-Me induces changes in the apoptotic machinery To determine the sequence of molecular changes during CDDO-Me-induced cell death, we performed time-course studies of apoptosis in U937 and HL-60 cells. Loss of mitochondrial membrane potential (m) was described in a number of different
models of apoptosis. CDDO-Me-treated U937 cells exhibited a
time-dependent decrease in m (Figure
4A), with complete loss of mitochondrial
membrane potential at 6 hours. Similarly, exposure of HL-60 to 1 µM
CDDO-Me for 2 and 4 hours decreased the percentage of cells with intact m by 46% and 57%. We next used pharmacologic
inhibitors of permeability transition (CyA44 and
BA45) to validate the role of mitochondrial disruption. CyA
partially inhibited CDDO-Me-triggered m loss, providing further evidence that CDDO-Me affects m
(Figure 4C). CyA also prevented CDDO-Me-induced cell killing at 4 hours (HL-60 control, 4.8 × 105 cells/mL; 1 µM
CDDO-Me, 2.8 × 105 cells/mL; CDDO-Me + CyA,
4.1 × 105 cells/mL). Similarly, BA reduced mitochondrial
depolarization by CDDO-Me (77% vs 48% CMXRos-high cells) and
decreased the percentage of cells with activated caspases by
60%.
Because caspase-3 has a pivotal role in the intrinsic apoptosis
pathway,8,47,48 we examined the effect of CDDO-Me on cleavage of caspase-3 by Western blot analysis. CDDO-Me-induced activation of caspase-3 resulted in the appearance of the 17-kd proteolytic product of caspase-3 at 2 hours and complete disappearance of uncleaved 32-kd caspase-3 after 6 hours (Figure 4D). Using the
fluorigenic caspase-3 substrate Phi-Phi-Lux, 24% and 67% of U937
cells were Phi-Phi-Lux-positive at 4 and 6 hours after CDDO-Me (1 µM;
Figure 4C). We also observed cleavage of the caspase-3 substrates PARP
and DFF-45 starting at 4 hours (data not shown). Finally, translocation
of phosphatidylserine to the cell surface was detected at 4 and 6 hours. Pretreatment of U937 and HL-60 cells with the caspase-3
inhibitor Z-DEVD-fmk (25 µM) significantly reduced annexin V
positivity (Figure 5A), diminished the
cleavage of Phi-Phi-Lux, and prevented CDDO-Me-induced cytotoxicity,
as determined by cell counts. Western blot analysis using an antibody specific for cleaved caspase-3 demonstrated disappearance of the cleavage product (Figure 5B). Notably, pretreatment with Z-DEVD-fmk prevented mitochondrial depolarization (4% vs 40%) in U937 cells, suggesting the existence of a caspase-mitochondrial amplification loop. These data establish a key role of caspase-3 in CDDO-Me-induced apoptosis. Collectively, results demonstrate that CDDO-Me induces apoptosis by decreasing
We also investigated the relationship between the sensitivity of
leukemic cells to CDDO-Me and Fas signaling. No induction of Fas was
observed by flow cytometry in several cell types tested. Anti-Fas
blocking antibody ZB4 did not prevent CDDO-Me-induced killing in NB4
or Jurkat cells, and Fas-activating antibody CH11 did not sensitize
cells to CDDO-Me cytotoxicity (not shown). In addition, no changes in
caspase-8 were detected by Western blot analysis in HL-60 and U937
cells despite massive cell death. These data suggest that the induction
of the FasL-Fas-caspase-8 pathway is not essential in the execution
of CDDO-Me-induced cell death. Because Bcl-2/Bax is known to regulate
mitochondrial membrane integrity, we examined the effects of CDDO-Me on
Bcl-2 and Bax levels. By Northern blot analysis, CDDO-Me induced Bax
mRNA levels in both HL-60 and U937 cells (Figure
6A). These changes paralleled the
increase in Bax protein levels, whereas Bcl-2 protein was only
minimally affected in HL-60 and NB4 cells, despite substantial cell
killing (Figure 6B). At high concentrations of CDDO-Me, Bcl-2 cleavage
was observed when cell viability decreased by more than 50% (data not
shown), presumably as a consequence of caspase-3 activation.49 Induction of apoptosis was shown to shift
Bax from the cytosol to mitochondrial membranes, where it directly induces cytochrome c release.50 We therefore
performed subcellular fractionation studies to isolate mitochondrial
and cytosolic fractions of control and CDDO-Me-treated cells. As shown
in Figure 6C, CDDO-Me induced Bax protein levels, coincident with a Bax
decrease in the cytosolic and with an increase in the mitochondrial
(heavy membrane) fraction. The new band at 18 kd detected in the total lysate and in the mitochondrial but not in the cytosolic fraction likely represents a cleavage product of Bax.51
We next examined the effect of Bcl-2 overexpression on CDDO-Me-induced
cytotoxicity. Surprisingly, U937/Bcl-2 cells were not protected from
CDDO-Me-induced cytotoxicity as detected by cell growth and
CDDO-Me abrogates Bcl-2 phosphorylation We then determined the effect of CDDO-Me on Bcl-2 phosphorylation by performing metabolic labeling studies with 32P-orthophosphoric acid. After treatment with 0.1 µM CDDO-Me (a concentration that induces apoptosis), Bcl-2 phosphorylation was virtually abrogated (Figure 8).52 To further investigate mechanisms of inhibition of Bcl-2 phosphorylation that could play a role in CDDO-Me-induced cell death, we selected U937 cells that were stably transfected with a serine 70 alanine, Bcl-2 mutant (S70A). Previous studies demonstrated that the S70A mutant was unable to be
phosphorylated and that this mutation was also incapable of protecting
cells from chemotherapy-induced apoptosis.17 The U937/S70A
Bcl-2 cells were found to be sensitive to CDDO-Me (60% decrease in
viability after 0.1 µM CDDO-Me). In contrast, a SerGlu mutant of
Bcl-2, S70E, which may mimic a phosphate charge and was shown to
potently suppress apoptosis, was found to be much more resistant to
CDDO-Me-induced apoptosis compared with wild-type Bcl-2 (75% viable
cells after 0.1 µM CDDO-Me compared with 30% for U937/wt; Figure
9). These data indicate that the
phosphorylation status of Bcl-2 contributes to cell sensitivity to
CDDO-Me-induced killing. Of note, U937/wt and the S70E and S70A Bcl-2
transfectants express roughly equivalent levels of Bcl-2 protein as
determined by densitometry (data not shown), suggesting that the
observed differential effects are not related to differences in
Bcl-2 expression.
Because PKC-
To determine specific effects of CDDO-Me on ERK activity, we used an in vitro MAP kinase assay kit. ERK was immunoprecipitated from K562 cells because these cells contain the Bcr-Abl kinase and activated ERK present in these cells under basal conditions.53 CDDO-Me inhibited MBP phosphorylation in a dose-dependent manner (Figure 10B, lanes 4-7). Similar effects were observed in HL-60 cells (Figure 10C). These data suggest that there may be a direct effect of the compound on ERK kinase activity. CDDO-Me induces differentiation and enhances the effects of ara-C and retinoids in leukemic cell lines Although CDDO induces differentiation,24 our data demonstrated that its methyl ester is a more potent inducer of granulomonocytic differentiation. At 0.1 µM CDDO-Me, 86.6% of HL-60 cells expressed CD11b, whereas 1 µM CDDO was needed to exert a similar effect. Monocytic differentiation was induced in 2 of the 5 AML samples, as shown by induction of the monocytic differentiation marker CD14. CDDO-Me also enhanced ara-C-induced cell killing in primary AML (DMSO control, 24.9% ± 7.4%; 1 µM CDDO-Me, 50.5% ± 15%; 1 µM ara-C, 39.8% ± 8.2%; CDDO-Me + ara-C, 65.4% ± 10.2%; n = 6) (P < .01). In CML blast crisis samples, CDDO-Me also enhanced ara-C-induced cell death but induced differentiation in only 1 of 4 samples.We then tested the combined effect of CDDO-Me and ATRA in leukemic cell lines. In HL-60, 59.4% and 21.2% of cells remained viable at 0.1 and 1 µM CDDO-Me; combining this with 0.5 µM ATRA further diminished the number of viable cells (31.6% and 9.6%, respectively). This decrease in viability was associated with increased DNA fragmentation. The combination decreased the numbers of viable cells in all cell lines tested (NB4, KG-1, HL-60) more than either CDDO-Me or ATRA alone, again suggesting that the combination of CDDO-Me and ATRA activates apoptotic pathways. In primary AML cells, ATRA enhanced CDDO-Me-induced apoptosis in 3 of 8 samples tested. Others and we have previously demonstrated that ATRA down-regulates Bcl-2 mRNA transcription and protein expression.54,55 ATRA alone decreased Bcl-2 protein levels; however, there was no additive effect on Bcl-2 levels by combinations of ATRA and CDDO-Me. At the highest concentrations (0.5 µM CDDO-Me and 1 µM ATRA), Bcl-2 was cleaved (data not shown). Of interest, in the same experiment we also observed cleavage of XIAP, an IAP family member, a finding that correlates with a marked decrease of viability. XIAP cleavage product (30 kd) was detected in HL-60 cells at 0.5 µM CDDO-Me alone or at 0.3 µM CDDO-Me combined with 1 µM ATRA. However, cleavage of both Bcl-2 and XIAP occurred only after a pronounced decrease in cell viability and was likely to be a consequence of caspase activation, not the initiating event. We then examined whether CDDO-Me in combination with ATRA enhances differentiation. HL-60 cells were cultured with 0.1 µM CDDO-Me for 72 hours, alone or in combination with 1 µM ATRA. CDDO-Me and ATRA were additive in inducing granulomonocytic differentiation: 25.8% ± 1.7% CDDO-Me- and 21% ± 2.5% ATRA-treated cells were positive for CD11b, whereas 52.6% ± 7.6% of cells treated with both agents expressed for CD11b (after subtraction of CD11b positivity in DMSO-treated controls). We next tested whether combinations of CDDO-Me with the RXR-specific
ligand LG100268 significantly potentiated the antileukemic effect of
CDDO-Me. LG100268 at 10 nM, 100 nM, and 1 µM enhanced the killing of
HL-60 cells in a dose-dependent manner (Table
1). At 1 µM LG100268, cell growth was
markedly inhibited, and the percentage of cells in S+G2M
decreased by 50%. Collectively, our data show that combinations of
CDDO-Me with either retinoids or rexinoids markedly decreased cell
viability and induced terminal differentiation in myeloid leukemic cell
lines.
In this study, we observed that myeloid leukemia cell lines and primary AML blasts underwent rapid apoptosis when incubated with submicromolar concentrations of the novel triterpenoid, CDDO-Me. CDDO-Me was consistently more active than the parental compound, CDDO.25 Because HL-60 and U937 cells lack functional p53 protein, it appears that CDDO-Me triggers apoptosis independent of p53. HL-60-Dox cells overexpressing MDR-1 are sensitive to CDDO-Me-induced cell death, suggesting that CDDO-Me can induce apoptosis independent of the MDR-1 protein. This was further confirmed by the finding that the MDR-1 inhibitor, PSC 833, did not enhance CDDO-Me-induced killing. CDDO-Me profoundly inhibited clonogenic cell growth in AML and in blast crisis CML, known to be resistant to most chemotherapeutic agents. In contrast, more than 50% of normal CFU-GM progenitor cells survived treatment with 0.5 µM CDDO-Me, a concentration that killed more than 90% of leukemic colonies in most AML. CDDO-Me also sensitized leukemic cells to apoptosis induced by ara-C. In vitro studies have shown that ATRA alone or in combination with
chemotherapeutic agents is effective in AML cells.56,57 ATRA binds to the 3 RAR nuclear receptors, whereas LG100268
specifically binds to RXR receptors.58 RXRs play a central
role in the cellular physiology of the nuclear receptor superfamily by
virtue of their ability to modulate the activity of many other
receptors. Therefore, we investigated the combined effects of CDDO-Me
and retinoids on the viability, apoptosis, and differentiation of
leukemic cells. Intriguingly, the combination of CDDO-Me with either
ATRA or LG100268 exerted a potent cytotoxic effect in leukemic cells at
concentrations that were ineffective when the agents were given singly.
The primary mechanism of decreased leukemic cell survival appears to be
the induction of apoptosis, as shown by DNA fragmentation and annexin V
staining. ATRA also enhanced CDDO-Me-induced apoptosis in primary AML
samples. Whether these effects can be attributed to the recent finding
that CDDO-Me is an antagonist for the PPAR Our findings further demonstrated that CDDO-Me induced the translocation of phosphatidylserine to the cell surface and caspase activation. Caspase-3 is the likely caspase that mediates CDDO-Me-induced apoptosis, as shown by Western blot analysis, flow cytometry using a florigenic substrate, and cleavage of the known caspase substrates PARP and DFF-45. Significantly, caspase-3 inhibition prevented the apoptosis of leukemic cells, establishing an essential role for caspase activation in CDDO-Me-induced apoptosis. In contrast, caspase-8 activation in Fas-Fas-ligand pathway does not play a critical role in the execution of CDDO-Me-induced cell death. Early changes in mitochondrial membrane integrity point to involvement
of the intrinsic cell death pathway. We studied whether apoptosis
induced by CDDO-Me is the result of mitochondrial permeability transition changes by testing the effects of specific inhibitors of
permeability transition pores, BA, and CyA on CDDO-Me-induced cell
death. In addition to abolishing the apoptotic The response of cells to apoptotic stimuli critically depends on the balance between pro-apoptotic and anti-apoptotic members of the Bcl-2 family. Bax promotes cell death, and its up-regulation has been associated with enhanced apoptosis.59,60 Although CDDO-Me did not affect the levels of Bcl-2 and Bcl-XL proteins, it induced Bax mRNA with a resulting increase in Bax protein levels. Results of subcellular fractionation studies demonstrated that CDDO-Me decreased the cytosolic and increased the mitochondrial Bax fraction. This translocation of Bax from the cytosol to mitochondrial membranes is known to directly induce cytochrome c release.50,61 The critical role of Bax in apoptosis was recently demonstrated by the complete abrogation of the apoptotic response in cells that lacked functional Bax (Bax knockouts).62 The resistance of leukemic cells to chemotherapy-induced
apoptosis remains the most significant problem in the treatment of AML.
Specifically, high intracellular levels of Bcl-2 inhibit the apoptosis
typically induced by chemotherapeutic agents.63,64 Furthermore, posttranslational modifications of Bcl-2 appear to be
involved in the regulation of apoptosis. In particular, Bcl-2 phosphorylation was associated with enhanced Bcl-2 function in some
studies17,18,39,65 and with the inactivation of Bcl-2 in
others.66-69 Phosphorylation at serine 70 was also found
to be required for the anti-apoptotic function of Bcl-2 to be mediated by mitochondrial PKC- Bcl-2 must be targeted to mitochondrial membranes to efficiently
suppress apoptosis.72,73 Subcellular fractionation studies revealed that ERK1/2, but not PKC- In conclusion, our data provide the first evidence that exposure to the
novel triterpenoid, CDDO-Me, induces apoptosis and inhibits colony
formation of myeloid leukemic cells. CDDO-Me also directly affects
We thank Dr Steven Grant for Bcl-2-transfected leukemic cell lines, Tena Horton and Rosemarie Lauzon for their help in the preparation of the manuscript, and Dr Edward Sausville and Kenneth Snader and the RAID program (Nation |
Top
Abstract
Introduction
and the mitogen-activated protein (MAP) kinases ERK1
(p44) and ERK2 (p42) were identified as physiologic Bcl-2 kinases,
-interferon-induced synthesis of nitric oxide by mouse
macrophages.
