Cancer Letters 206 (2004) 43–50

www.elsevier.com/locate/canlet

Brusatol-induced HL-60 cell differentiation

involves NF-kB activation
Muriel Cuendeta, Joell J. Gillsa, John M. Pezzutoa,b,*
a
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL, USA
b
Department of Surgical Oncology, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA
Received 22 June 2003; received in revised form 4 November 2003; accepted 5 November 2003

Abstract
Brusatol, a quassinoid isolated from the fruit of Brucea javanica, induces cell differentiation with various leukemic cell lines
in the concentration range of 5 – 25 ng/ml. To investigate its mechanism of action, cultured HL-60 cells were treated with
brusatol (25 ng/ml) for various periods of time and qualitatively analyzed for differential gene expression using a cDNA
macroarray. As suggested by these preliminary data, we investigated the effect of brusatol on the Rel/nuclear factor-kappa B
(NF-kB) family members and their role in HL-60 cell differentiation. When cells were treated with brusatol (25 ng/ml),
p100/p52, p105/p50 and p65 mRNA were found to be up-regulated with a maximum after 8 h. As determined by electrophoretic
mobility shift assays (EMSA), NF-kB was activated, involving p50 and p65. Moreover, immunoblots showed a decrease of
IkBa and generation of the phosphorylated form of IkBa in whole cell lysates. Cell differentiation induced by brusatol was
inhibited by SN50, a NF-kB translocation inhibitor. These results strongly suggest that brusatol induces activation of NF-kB
and the activation and translocation of NF-kB into the nucleus is responsible for promoting HL-60 cell differentiation.
q 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Brusatol; Cell differentiation; NF-kB

1. Introduction apoptosis. In leukemic cells, genetic changes

The process of neoplastic cell growth can be
depicted as a dysfunctional balance between control
of cell proliferation, apoptosis and terminal differen-
tiation. In normal cells, activation of specific path-
ways leads to cellular differentiation, which typically
is accompanied by cell growth arrest followed by
* Corresponding author. Address: Heine Pharmacy Building,
Purdue University, Room 104, 575 Stadium Mall Drive, West
Lafayette, IN, 47907, USA. Tel.: þ1-765-494-1368; fax: þ 1-765-
494-7880.
E-mail address: jpezzuto@purdue.edu (J.M. Pezzuto).
0304-3835/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2003.11.011
(chromosomal translocations, point mutations, gene
amplifications or deletions) block the normal differ-
entiation program [1]. Conventional cytotoxic
chemotherapy focuses on cell killing to achieve
complete hematological remissions (i.e. less than
5% blasts). In the past few years, however, several
non-conventional selective anti-leukemic agents
have been developed that function by targeting
molecules involved directly in the pathogenesis of
the disease [2,3].
Nuclear factor kappa B (NF-kB) is a potent
transcription factor that is found in a great variety of
44 M. Cuendet et al. / Cancer Letters 206 (2004) 43–50
Fig. 1. Structure of brusatol.
immune cells and is known to participate in the
regulation of multiple genes involved in development
and immunity. NF-kB was originally identified as a
protein binding to a specific decameric DNA
sequence within the intronic enhancer of the immuno-
globulin kappa light chain gene in mature B- and
plasma cells [4]. To date, five structurally related
proteins of the Rel/NF-kB family have been identified
in mammals: p50, p52, p65 (RelA), RelB and c-Rel.
They all contain a conserved domain of 300 amino
acids in length, the Rel homology domain, which is
responsible for dimerization and DNA binding [5].
Various dimer combinations have been described and
Rel/NF-kB proteins have been classified into two
groups. The first is comprised of p65, RelB and c-Rel,
which contain transactivation domains in their
C-terminal sequences [6]. In contrast, the members
of the second group, p50 and p52, lack C-terminal
transactivating regions and therefore are not con-
sidered to be transcriptionally active. Cell stimulation
leads to a phosphorylation of the inhibitors of Rel/NF-
kB (IkBs) [7] through specific IkB kinases (IKK) and
dissociation of IkB from the complex. The resulting
free NF-kB dimers translocate into the nucleus and
induce gene transcription. NF-kB not only partici-
pates in the induction of the expression of many
genes, including those encoding proteins that fulfill
important roles in the processes of immunity and
inflammation [8], but also contributes to the regu-
lation of apoptosis [9]. Studies on B-cells from
p50/NF-kB knockout mice and on B-cells and
thymocytes transformed with IkBaM, a strong
dominant-negative inhibitor of the activation of
different Rel/NF-kB complexes that bind IkB pro-
teins, have indicated that NF-kB is required for or
participates in immune cell differentiation and deve-
lopment [10,11]. In addition, with a neuroblastoma
cell line, it has been shown that NF-kB is activated
prior to neuronal differentiation, and both its acti-
vation and differentiation into neurons are blocked by
a dominant-negative inhibitor of NF-kB [12].
Brusatol (Fig. 1) is a quassinoid obtained from
Brucea species (Simaroubaceae). This compound and
analogues are capable of inducing an array of
biological responses including antiinflammatory and
antileukemic effects with murine models [13]. A
major mechanism responsible for antineoplastic
activity at the molecular level has been attributed to
inhibition of protein synthesis [14]. Such inhibition
has been shown to occur via interference at the
peptidyltransferase site, thus preventing peptide bond
formation [15].
In our program for the procurement of novel plant-
derived chemotherapeutic/chemopreventive agents,
HL-60 cell differentiation activity has been used as
one marker of activity [16]. This led to the
identification of brusatol as a potent inducer of
HL-60 cell differentiation [17]. The effect of brusatol
was evaluated with a panel of leukemic cells with
representative chromosomal translocations and other
gene mutations; the compound induced cell death
events in some cell lines and terminal differentiation
in others. A significant finding was potent down-
regulation of c-MYC oncoproteins. Cell lines expres-
sing high levels of c-MYC oncoprotein were most
sensitive to brusatol- and bruceantin (a brusatol
analogue)-mediated effects [18]. With multiple
myeloma cells, bruceantin induced apoptosis, and
this involved caspase and mitochondrial pathways.
Moreover, with in vivo experiments using a xenograft
model, bruceantin treatment resulted in multiple
myeloma regression without overt toxicity [19]. In
the present study, we investigated the expression and
DNA binding activity of Rel/NF-kB family members,
as well as their participation in HL-60 cell differen-
tiation after treatment of the cells with brusatol.
2. Materials and methods
2.1. Chemicals
Brusatol was isolated from Brucea javanica [20].
All other compounds were purchased from Sigma
Chemical Co. (St Louis, MO).
 M. Cuendet et al. / Cancer Letters 206 (2004) 43–50
2.2. Cell culture
HL-60 cells were obtained from the American
Type Culture Collection (Rockville, MO). The cell
line was maintained in suspension culture using RPMI
1640 medium (Invitrogen, Carlsbad, CA) sup-
plemented with 10% heat-inactivated fetal bovine
serum, 100 units of penicillin/ml and 100 mg of
streptomycin/ml at 37 8C in a humidified atmosphere
of 5% CO2 in air. Cells were routinely tested for
mycoplasma contamination. Brusatol was dissolved
in DMSO and the final DMSO concentration in the
media was 0.1%.
2.3. Northern blot analysis
Cells were treated with brusatol (25 ng/ml) for 2, 4,
8, 12, 18, 24 or 30 h, and RNA was isolated with
TRIZOLw (Invitrogen, Carlsbad, CA). RNA samples
(10 mg/lane) were separated by electrophoresis on 1%
agarose/formaldehyde gels, transferred to nylon
membranes (Zeta-Probe, Bio-Rad, Hercules, CA)
and UV cross-linked. To make probes, insert DNA
was restriction digested from plasmids isolated from
I.M.A.G.E. clones, then random prime labeled with
a32P-dATP using a Prime-it II kit (Stratagene, La
Jolla, CA). Prehybridization was performed for 1 h at
65 8C in hybridization buffer (0.5 M Na2HPO4,
pH 7.2, 7% SDS, 1% blocking reagent [Roche,
Indianapolis, IN] and 1 mM EDTA) containing 10%
denatured salmon sperm DNA, and hybridization was
performed overnight at 65 8C in hybridization buffer
containing the probe. The membrane was washed for
1 h at 65 8C with the first buffer (0.5 M Na2HPO4, pH
7.2, 1% SDS and 1 mM EDTA), then for 30 min at
65 8C with the second buffer (0.25 M Na2HPO4, pH
7.2, 1% SDS and 1 mM EDTA), and finally for 30 min
at 65 8C with the third buffer (0.1 M Na2HPO4, pH
7.2, 1% SDS and 1 mM EDTA). Autoradiography
was performed at 2 70 8C. To provide an internal
control, membranes were stripped and reprobed for
GAPDH.
2.4. Preparation of nuclear extracts
Nuclear extracts were generated using the follow-
ing protocol. Cells (20 £ 106) were synchronized by
FBS starvation for 48 h, treated with brusatol
45
(25 ng/ml) for 2, 4, 8, 12, 18 or 24 h, washed twice
in ice – cold PBS and suspended in 200 ml ice-cold
lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl,
0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM
phenylmethylsulfonyl fluoride [PMSF], 2.0 mg/ml
leupeptin and 0.5 mg/ml benzamidine). Cells were
allowed to swell on ice for 15 min, 10% Nonidetw P40
(12.5 ml) was added, and the mixture vortexed for
10 s. Nuclei were pelleted and the supernatant was
discarded. Isolated nuclei were suspended in 25 ml
ice-cold nuclear extraction buffer (20 mM HEPES,
pH 7.9, 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1 mM DTT, 1 mM PMSF, 2.0 mg/ml leupeptin and
0.5 mg/ml benzamidine). After a 30 min incubation
on ice, the samples were centrifuged and the
supernatant (nuclear extract) was collected and frozen
at 2 70 8C. Protein concentrations were determined
by the bicinchoninic acid method.
2.5. Electrophoretic mobility shift assay (EMSA)
The EMSA was performed using a Nushifte kit for
Rel/NF-kB family (Active Motif, Carlsbad, CA)
following the manufacturer’s protocol. In brief, the
unlabeled wild-type oligonucleotide probe was
labeled with 32P using a T4 polynucleotide kinase
(Invitrogen, Carlsbad, CA). Nuclear extracts (10 mg)
were mixed gently with the antibody premix and
incubated for 20 min at 4 8C. During the incubation,
the probe premix was prepared and then added to the
pre-incubated sample/antibody premix, which were
mixed gently and incubated for 20 min at 4 8C. The
entire content of each reaction was loaded onto a 5%
polyacrylamide (38:2) gel, pre-cooled to 4 8C in 1 £
Tris – Glycine electrophoresis buffer (30.28 g
Tris – base, 142.7 g glycine, 3.92 g EDTA in 1 l
H2O, pH 8.5) and run for 2 h at 180 V at 4 8C. At
the end of the electrophoresis, the gel was equilibrated
in H2O for 5 min, dried for 2 h using the Bio-Rad gel
dryer (Hercules, CA) and exposed to Kodak film
(Rochester, NY) for 15 h at 2 70 8C.
2.6. Measure of NF-kB activation
The TransAM NF-kB Family kit (Active Motif,
Carlsbad, CA) was used and the manufacturer’s
protocol was followed. In brief, nuclear extracts and
binding buffer were incubated at room temperature for
46 M. Cuendet et al. / Cancer Letters 206 (2004) 43–50

1 h in a 96-well plate coated with an oligonucleotide the incubation with brusatol (25 ng/ml), cells were
that contained the NF-kB consensus binding site analyzed to determine the percentage exhibiting
under mild agitation, then the plate was washed. p65, functional nitroblue tetrazolium (NBT) reduction.
p50, p52, c-Rel or RelB antibodies were added to the Evaluation of NBT reduction was used to assess the
wells, the plate was incubated for 1 h at room ability of sample-treated cells to produce superoxide
temperature, and then washed. Following this, the when challenged with TPA. A 1:1 (v/v) mixture of a
secondary antibody was added to the wells, the plate cell suspension (106 cells) and TPA/NBT solution
was incubated for 1 h at room temperature, and then (2 mg/ml NBT and 1 mg/ml TPA in PBS) was
washed. Finally, the developing solution was added. incubated for 1 h at 37 8C. Then, cells were smeared
After 5 –10 min incubation at room temperature, the on glass slides, and counterstained with 0.3% (w/v)
stop solution was added and the absorbance was read safranin O in methanol. Positive cells reduce NBT
at 450 nm. yielding intracellular black-blue formazan deposits.
The same experiment was performed, adding a NF-kB
2.7. Immunoblot analysis specific inhibitor (SN50, 50 mg/ml, Biomol, Plymouth
Meeting) or a control peptide (SN50M, 50 mg/ml,
The levels of IkBa and pIkBa were assessed by Biomol) 1 h prior to treatment with brusatol.
immunoblots as previously described [21]. In brief,
cells ð20 £ 106 Þ were treated with brusatol (25 ng/ml)
and harvested after 0.5, 2, 4, 8, 12, 18 or 24 h. Whole-
cell pellets were treated with detergent lysis buffer
(1 ml/107 cells, 50 mM Tris –HCl buffer, pH 8.0,
150 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 1%
Nonidetw P40, 0.5% sodium deoxycholate, 0.1%
sodium dodecylsulfate, 100 mg/ml PMSF, 1 mg/ml
aprotinin, 2 mg/ml leupeptin and 100 mM sodium
vanadate) to obtain protein lysates, and protein
concentrations were quantified using a bicinchoninic
acid kit. Total protein (30 mg) was separated by
12.5% SDS-PAGE, electroblotted to PVDF mem-
branes, and blocked overnight with 5% non-fat dry
milk. The membrane was incubated with a 1:500
(IkBa and pIkBa, Santa Cruz Biotechnology, Santa
Cruz, CA) solution of the primary antibody, prepared
in 1% blocking solution, for 2 h at room temperature,
washed three-times for 15 min with PBS-T (PBS with
0.1%, v/v, Tween 20), and incubated with a 1:2500
dilution of horseradish peroxidase-conjugated
secondary antibody for 30 min at 37 8C. Blots were
again washed three-times for 10 min each in PBS-T
and developed by enhanced chemiluminescence
(Amersham, Piscataway, NJ). Membranes were exposed Fig. 2. Expression of p100/p52, p105/p50 and p65 mRNA in HL-60
to Kodak Biomax film (Rochester, NY), then stripped cells treated with brusatol (25 mg/ml). (a) Total RNA was prepared
and reprobed for b-actin (Sigma, St Louis, MO). from cells treated with brusatol for 2, 4, 8, 12, 18, 24 or 30 h,
10 mg/lane were loaded, and p100/p52, p105/p50 and p65 mRNA
expression was analyzed by northern blot analysis. As an internal
2.8. Cell differentiation assay
control, the membrane was reprobed for GAPDH. (b) Densitometric
analyses. Results are shown as fold differences of p100/p52,
Cells were tested using a 4-day incubation p105/p50 and p65 mRNA expression, relative to the levels observed
protocol as previously described [18]. At the end of in cells treated with solvent (0.1%, v/v, DMSO) only.
 M. Cuendet et al. / Cancer Letters 206 (2004) 43–50 47

Fig. 3. DNA binding assays with nuclear extracts derived from cells treated with brusatol (25 mg/ml). (a) DNA binding assays were carried out
with nuclear extracts of cells treated with brusatol for 2, 4, 8, 12, 18 or 24 h. The specificity of binding was determined by the use of
50 £ unlabeled probe. (b) The presence of p65, p50, p52, c-Rel or RelB in the complex was identified by an ELISA assay.

3. Results Therefore, we determined the effect of brusatol on the

Analysis of differentially expressed genes in
HL-60 cells treated with brusatol by a macroarray
experiment showed 87 differentially-expressed genes
(15%), among which 35 showed lower expression in
the treated cells and 51 showed higher expression
[22]. Based on the qualitative results obtained from
this analysis, expression of mRNA for some members
of the Rel/NF-kB family was analyzed in HL-60 cells
treated with brusatol for different periods of time.
Significant up-regulation of p100/p52 mRNA was
observed, with a maximum after 8 h of treatment, and
reduction to basal levels between 12 and 30 h.
Up-regulation of p65 and p105/p50 was also observed
with maxima around 8 h, but these levels remained
up-regulated until 30 h (Fig. 2).
Although the presence of NF-kB proteins is a pre-
requisite for action as transcription factors, bio-
logical activity is regulated by subcellular localization.
NF-kB binding activity in nuclear preparations by
EMSA, using a 32P-labeled oligonucleotide with a
NF-kB binding site as a probe. EMSA performed with
nuclear extracts derived from HL-60 cells, in the
absence of brusatol, showed low constitutive NF-kB.
Over a period of 4 h, treatment with brusatol
(25 ng/ml) significantly activated NF-kB. From 8 to
24 h, the activation decreased, but the level was still
higher than the control (untreated) cells (Fig. 3).
Specific antibodies were used to determine which
Rel/NF-kB family members were induced using an
ELISA assay. The majority of the inducible NF-kB
binding activity reacted with p50 and p65 specific
antibodies, suggesting the p50/p65 heterodimer repre-
sented the main inducible complex. In contrast, the
amount of p52-, c-Rel-, and RelB-containing com-
plexes remained low and unaltered (Fig. 3).
The presence of IkBa and pIkBa in the whole cell
lysate was analyzed by western blots. Levels of IkBa
48 M. Cuendet et al. / Cancer Letters 206 (2004) 43–50
Fig. 4. Expression of IkBa and pIkBa in cells treated with brusatol.
(a) Protein lysate was prepared from cells treated with brusatol for
0.5, 2, 4, 8, 12, 18 or 24 h, 30 mg/lane were loaded and analyzed by
western blotting. As an internal control, the membrane was
reprobed with b-actin. (b) Densitometric analyses. Results are
shown as fold differences of IkBa and pIkBa protein expression,
relative to the levels observed in cells treated with solvent (0.1%,
v/v, DMSO) only. The experiment was repeated twice and similar
results were obtained in both cases.
were reduced after an 8 h treatment with brusatol, and
remained lower than control (untreated) cells until
24 h treatment. To the contrary, pIkBa levels
increased, inducing IkBa degradation and liberating
p65, which could migrate to the nucleus (Fig. 4).
Previous studies performed in our laboratory
demonstrated that brusatol was able to induce
differentiation of HL-60 cells in a concentration-
dependent fashion [18]. Thus, an important issue to
resolve was whether the observed increase in NF-kB
activity by brusatol was associated with the differen-
tiation program. In the current study, cells were
treated with solvent, SN50, a NF-kB translocation
inhibitor (100 mg/ml), or a control peptide, SN50M
(50 mg/ml), 1 h prior to treatment with brusatol
(25 ng/ml). After four days, the cells were harvested
and evaluated for markers of differentiation. Analysis
of NBT reduction for assessment of superoxide
formation demonstrated a statistically significant
reduced myeloid maturation in the cells pretreated
with SN50 (9.8 ^ 1.6%) compared with the cells
pretreated with solvent (22.7 ^ 0.9%) or SN50M
(22.4 ^ 1.2%) (Fig. 5).
4. Discussion
Brusatol, a quassinoid isolated from Brucea
species, is known to inhibit protein synthesis [23].
Previously, we found that brusatol down-regulated
c-MYC mRNA and protein in 10 different leukemic
cell lines, and the extent of activity correlated with
this response, based on cell death or terminal
differentiation [18]. Further, a significant antitumor
response was mediated in athymic mice carrying
human multiple myeloma [19]. In order to investigate
the mechanism of apoptosis and terminal differen-
tiation induced by brusatol, the effect on gene
expression was determined using DNA macroarrays.
With HL-60 cells treated with brusatol (25 ng/ml),
results showed up-regulation of the NF-kB gene
family, p100/p52 and p105/p50, after 6 and 12 h or 6
and 18 h, respectively (data not shown). Guided by
these preliminary observations, the current study
showed that treatment of HL-60 cells with brusatol
caused up-regulation of p100/p52, p105/p50 and p65
mRNA, activation of NF-kB (p50/p65 heterodimer),
and phosphorylation of the NF-kB inhibitor, IkBa,
resulting in NF-kB translocation from the cytoplasm
to the nucleus.
Fig. 5. HL-60 cells were treated with DMSO, the NF-kB inhibitor
SN50 (50 mg/ml in ddH2O), the control peptide SN50M (50 mg/ml
in ddH2O), brusatol (25 mg/ml) (B25), brusatol and SN50, or
brusatol and SN50M. The percentage of NBT-positive cells induced
by brusatol was determined. Data points are the mean of
quadruplicate samples ^ standard deviation. *Significantly differ-
ent from control values, determined by Student’s t-test ðP , 0:005Þ:
 M. Cuendet et al. / Cancer Letters 206 (2004) 43–50
The mechanism of action facilitated by various
differentiation and apoptosis inducers remains largely
unknown, but the participation of some key genes,
such as NF-kB, has been demonstrated. Activation of
NF-kB proteins has been observed during neuronal
differentiation in brain and neuroblastoma cells [12].
Increases in the DNA binding activities of different
NF-kB homo- and heterodimers correlate with
differentiation stages of monocytes, macrophages
and dendritic cells [24]. Also, tumor necrosis factor
a (TNFa) induced activation of NF-kB, promoting
the differentiation of anaplastic thyroid carcinoma
cells, and this response was inhibited by SN50 [23].
In the current study, HL-60 cell differentiation
induced by brusatol was closely related to NF-kB
activation, since myeloid maturation was significantly
reduced by SN50, a NF-kB translocation inhibitor.
Induction of apoptosis by compounds such as cyanide or
ajoene is also dependent on NF-kB activation [25,26]. In
a previous study, we found that bruceantin, an analog of
brusatol, induced apoptosis in a multiple myeloma cell
line [19]. At higher concentrations, rather than cell
differentiation, brusatol can induce apoptosis in HL-60
cells. For example, after a 24 h treatment, the IC50 for
apoptosis was 18 ng/ml (33 nM), as observed by the
formation of apoptotic bodies with DAPI staining.
Further, incorporation of the DiOC6 fluorescent probe
decreased in a dose-dependent manner, indicating
disruption of mitochondrial trans-membrane potential,
and proteolytic degradation of the caspase-3 substrate
PARP was observed in HL-60 cells treated by brusatol
(data not shown). However, apoptosis appears to be
independent of NF-kB activation, as there was no
significant change in induction when SN50 was added to
the culture medium before treatment with brusatol (data
not shown). In summary, with the experimental
conditions described herein, brusatol induces activation
of NF-kB and this activation and translocation of NF-kB
into the nucleus appears to play a role in promoting HL-
60 cell differentiation.
Acknowledgements
This work was supported by program project P01
CA48112 funded by the National Cancer Institute,
NIH, Bethesda, MD.
49
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