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Introduction
The nuclear pore complex (NPC) comprises a central
eightfold symmetrical ring and spoke assembly, cytoplasmic fibers,
and a filamentous nuclear basket[1]. Molecular trafficking
between the nucleus and the cytoplasm of interphase cells
occurs via the NPC, which are large molecular assemblies
that are embedded in the double-membraned nuclear
envelope (NE)[2]. The NPC provide peripheral channels of
approximately 9 nm in diameter, which allow the diffusion of
ions and small molecules, and mediate the selective
transport of nuclear proteins, RNA, and ribonucleoprotein (RNP)
particles by energy-dependent mechanisms. Several
interactions between individual FG (Phe-Gly) repeat-containing
nucleoporins and transport factors have been reported,
leading to the idea that such interactions may play a pivotal role
in the docking, translocation, and/or termination steps of
the transport process[3]. Recent research has shown that
Nup98 can dynamically associate with the nuclear pore and
shuttle between the NPC and intranuclear bodies, and
additionally between the nucleus and the cytoplasm in a
transcription-dependent manner. The most common oncogenic
fusions involve a segment of the gene encoding the
FG-repeat domain of Nup98, which, in turn, becomes linked to
genes of the homeobox family of transcription
factors[4]. Nucleoporins are involved in several types of acute myeloid
leukemia and a few other hematological malignancies, as well
as rare cases of other tumors. Overexpression of nucleoporin
88 (Nup88) is associated with malignant tumors, whereas in
most other cases the role of the Nup proteins in
tumorigenesis stems from chromosomal rearrangements that results in
oncogenic fusion proteins[5]. Nup98 and Nup88 play
important roles in nucleocytoplasmic shuttling activity in
carcinoma cells.
Several natural compounds, in particular plant products
and dietary constituents, have been found to have
chemo-preventive activities both in vitro and
in vivo[6]. Deguelin has been isolated from several plant species, including
Mundulea sericea (Leguminosae). Recent experiments have
verified that deguelin can lead the cell cycle to block and
induce apoptosis; however, the mechanism by which it acts
is not yet completely clear[7-9]. In our previous studies, we
found that deguelin was able to inhibit the proliferation of
BurkittĄ¯s lymphoma cell line Daudi cells by regulating the
cell cycle such that cells were arrested at the
G0/G1 phase, and apoptosis was induced. Moreover, deguelin has low
toxicity in human peripheral blood monocular cells (PBMC),
but selectively induces the apoptosis of Daudi cells.
Deguelin has antitumor effects because it downregulates
the expression of cyclin D1 and the pRb
protein[10]. In the present study, we chose human myeloid precursor cell line
U937 cells as the target. This study was designed to explore
the mechanism by which deguelin regulates Nup98 and
Nup88 in U937 cells. We focused on changes in the
expression of Nup98 and Nup88, and analyzed the underlying
mechanism by which molecular trafficking between the nucleus
and the cytoplasm is carried out.
Materials and methods
Drugs and reagents Deguelin was purchased from the
Sigma (St Louis, MO, USA) and was initially dissolved in
dimethylsulfoxide (Me2SO), and stored at -20 °C, and was
then thawed before use. 3-(4,5-dimethyl-2
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from
Janssen Chimica Company (New Brunswick, NJ, USA).
RPMI-1640 medium, propidium iodide (PI), Hoechst 33258,
and Me2SO were purchased from Sigma. Fetal calf serum
(FCS), anti-Nup88 and anti-Nup98 antibodies were purchased
from Santa Cruz (California, USA). Fluorescein isothiocyanate
(FITC)-labeled secondary antibodies were purchased from
Zhongshan Company (Beijing, China). Chemiluminescence
(ECL) reagent kits were purchased from Pierce
Biotechnology (Rockford, IL, USA). The U937 cell line was obtained
from the China Center for Typical Culture Collection (Wuhan,
China). All cell groups were grown in an RPMI-1640 culture
medium containing 10% FCS and 2 mmol/L L-glutamine at
37 ºC in a 5% CO2 incubator.
MTT assay The antiproliferative effect of deguelin
against different group cells was determined by using the
MTT dye uptake method as described
previously[11]. Briefly, the final concentrations of deguelin were 0, 5, 10, 20, 40, 80,
and 160 nmol/L. Each concentration of deguelin was added
to 6 wells, respectively. The plates were in the presence or
absence of the indicated test samples for 0 h, 12 h, 24 h, 36 h,
48 h, 60 h, and 72 h. Thereafter, 20 µL MTT solution [5
mg/mL in phosphate-buffered saline (PBS)]was added to each
well. After incubation for 4 h at 37 °C, the supernatant was
removed and 150 µL Me2SO was added. When the blue
crystals were dissolved, the optical density
(OD) was detected in a microplate reader at a wavelength of 570 nm using
a 96-well multiscanner autoreader (Biotech Instruments
µQuant, NY, USA). The following formula was used: cell
proliferation inhibited (%)=[1-(OD of the experimental
samples/OD of the control)]×100%
(n=6. Mean±SD).
DNA content and cell cycle analysis Untreated and
treated cells were collected, after being cultured in the
presence or absence of deguelin for the indicated time, rinsed
with PBS, and suspended in 75% ethanol at -20 ºC overnight.
Fixed cells were centrifuged at 1200×g and washed twice
with PBS. For detecting DNA content, cells were incubated
in the dark with 50 mg/L PI and 0.1% RNase A in 400 µL PBS
at room temperature for 30 min. Stained cells were analyzed
using FACSort (Becton Dickinson, New Jersey, USA). The
percentage of cells was determined using the CellQuest
software program (Becton Dickinson, New Jersey, USA). Cells
were grouped as follows: the control group; and those treated
with deguelin at concentrations of 5, 10, 20, 40, and 80
nmol/L for 24 h, respectively (n=3).
Immunofluorescence with confocal microscopy
For the immunofluorescence experiments, cells were fixed in 4%
paraformaldehyde for 10 min and permeabilized with 0.2%
Triton X-100 on ice for 10 min. Samples were blocked with
3% bovine serum albumin plus 0.02% Triton X-100 in PBS
for 30 min, incubated with anti-Nup88 (1:200) and anti-Nup98
(1:100) antibodies overnight at 4 ºC, and washed 4 times with
1.5 % bovine serum albumin plus 0.02% Triton X-100 in PBS.
FITC-labeled secondary antibodies diluted in PBS were
applied for 30 min, and cells were washed 3 times every 15 min.
Hoechst 33258 (1 µg/mL) and PI (50 mg/L) were included in
the penultimate wash step to visualize the DNA. Coverslips
were mounted with 3-amino propyltriethoxy silane (APES).
Images were captured using a FV500 confocal microscope
(Olympus, Tokyo, Japan).
Nup98 and Nup88 protein analysis using flow
cytometry Flow cytometry was performed to determine the
expression of Nup98 and Nup88 in cells by
using primary antibodies to the peptide-binding domain. A total of
1×106 cells were collected, washed with PBS, and anti-Nup88 antibody (1:50)
and anti-Nup98 antibody (1:50) were added, then the mixture
was kept at 4 ºC overnight. Mouse IgG1 (1:50) antibody was
the isotype control group. FITC-labeled secondary
antibody diluted in PBS (1:100) was applied for 30 min at room
temperature. Stained cells were analyzed by using FACSort.
A total of 10 000 cells were analyzed from each cell group.
The percentage of cells was determined using the CellQuest
software program. Cells were grouped as follows: the
negative group; the blank group; those treated with 10 nmol/L
deguelin for 24 h; those treated with 20 nmol/L deguelin for
24 h.
Immunoelectron microscopy U937 cells on coverslips
were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde
and washed with 0.1 mol/L phosphate buffer (PB; pH 7.4),
followed with PB containing 0.1% sodium borohydride to
inactivate residual aldehyde groups. The cells were
permeabilized with PB containing 0.05% Triton X-100 for 20
min at room temperature and washed with PB. The blocking
solution was PBS (pH 7.4) containing 4% normal goat serum
(NGS). After blocking, cells were incubated with
affinity-purified goat anti-Nup98 antibody (1:100) and
affinity-purified mouse anti-Nup88 antibody (1:100) in PBS containing
1% NGS at 4 ºC overnight. After 6 washes with PBS, cells
were incubated with a biotinylated respondent secondary
antibody (1:200) in PBS and 1% NGS. Immunoreactivity was
visualized by incubation with 0.05% diamino-benzidine (DAB;
Sigma) and 0.003% hydrogen peroxide in 0.05 mol/L Tris (pH
7) for 2 min. Cells were washed, postfixed with 2.5%
glutaraldehyde in PB, washed again, fixed with 0.5% osmium
tetroxide for 15 min, dehydrated, and embedded in Leica ultracut
UCT (Wetzlar, Germany) for sectioning. Sections were
observed on an electron microscope (Tecnai F12; FEI,
Eindhoven, the Netherlands).
Western blot analysis Lysates were prepared from
1×107 cells by dissolving cell pellets in 100 µL of lysis buffer [20
mmol/L Na2PO4 (pH 7.4), 150 mmol/L NaCl, 1% Triton X-100,
1% aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 10
g/L leupeptin, 100 mmol/L NaF, and 2 mmol/L
Na3VO4]. Lysates were centrifuged at 18
000×g for 15 min and the supernatant was collected. Protein content was determined using
a Bio-Rad protein assay (Bio-Rad laboratories, Hercules, CA,
USA). Sodium dodecylsulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer [10 mmol/L Tris-HCl (pH
6.8) 2% SDS, 10% glycerol, 0.2 mol/L 1,4-Dithiothreitol,
(DTT)] was added to the lysates. Lysates were heated to
100 ºC for 5 min, and 100 µg of protein was loaded into each well of
a 10% SDS-PAGE gel. Resolved proteins were
electrophoretically transferred to nitrocellulose and blocked with 5%
non-fat milk. After incubation with the Nup88 antibody (at a
1:2500 dilution) and the Nup98 antibody (dilution
1:1000) at 4 °C overnight, the blots were washed 3 times with
TBS/Tween [TBST; 25 mmol/L Tris-HCl (pH 8.0), 125 mmol/L
NaCl, 0.1% Tween 20], and exposed to horseradish
peroxidase (HRP)-conjugated corresponding secondary
antibodies for 1 h, and finally detected by using ECL. Quantification
of the bands was carried out using the Quantity One
densitometric analysis software (Bio-Rad).
Statistical analysis All data are expressed as mean±SD,
and analyzed using SPSS 10.0 for Windows 98. Linear
t-test were used for statistical analyses, and
P<0.05 was considered to be statistically significant.
Results
Effects of deguelin on proliferation of U937
cells U937 cells treated with different concentrations of deguelin (0, 5,
10, 20, 40, 80, or 160 nmol/L) for 0 h, 12 h, 24 h, 36 h, 48 h, 60
h, and 72 h, respectively, resulted in the inhibition of cell
proliferation in a dose- and time-dependent manner. The
OD value of the deguelin-treated group was significantly lower
than that of the untreated group (Figure 1). The
IC50 value for 24 h for the U937 cells was 21.61
nmol/L, whereas the IC50 value for 36 h was 17.07 nmol/L.
Effects of deguelin on the cell cycle of human leukemia
U937 cells Figure 2 illustrates the changes in DNA content
distribution in cells treated with 0, 5, 10, 20, 40, or 80 nmol/L
deguelin for 24 h. As the treatment dose increased, the
percentage of cells in S phase and G2/M phase increased,
whereas the number in the G1/G0
phase decreased accordingly. After treatment with 0, 5, 10, 20, 40, or 80 nmol/L deguelin for
24 h, the proportion of cells in the
G1/G0 phase were 73.01%, 71.15%, 68.42%, 53.83%, 43.99%, and 22.82%, respectively;
the proportion decreased in a dose-dependent manner. The
proportion of S phase cells were 17.18%, 16.30%, 18.09%,
27.56%, 31.21%, and 46.85%, respectively; the proportion
increased in a dose-dependent manner. The proportion in
the G2/M phase was 9.75%, 12.31%, 13.99%, 18.99%, 24.83%,
and 27.79%, respectively. These results show that deguelin
arrested the U937 cells at the S phase and the
G2/M phase, and decreased the number of cells in the
G1/G0 phase in vitro.
Effects of deguelin on Nup98 and Nup88 in U937
cells In this study, FITC-labeled secondary antibodies marked
Nup98 and Nup88 with green fluorescence, and Hoechst
33258 and PI labeled the DNA of U937 cells with blue and red
fluorescence, respectively. In Figure 3, part Ad is merged
parts Ab and Ac, and part Bd is merged parts Bb and Bc
using confocal microscopy. We found that Nup98 had low
fluorescence intensity, with an OD value of 50.23 in U937
cells, and the fluorescence was located both within the
nucleoplasm and nucleus in intensely fluorescent dots.
After treatment with 20 nmol/L deguelin for 24 h, Nup98 had
greater fluorescence intensity, with an OD value of 252.28,
and its distribution was mainly within the nucleus. In U937
cells, Nup88 had high fluorescence intensity, with an
OD value of 215.16. After treatment with 20 nmol/L deguelin for
24 h, Nup88 had lower fluorescence intensity, with an
OD value of 63.24. Compared with the control group, expression
of Nup98 and Nup88 after treatment with 20 nmol/L deguelin
for 24 h was significantly different (P<0.05). When these
data are considered together, we conclude that Nup98 and
Nup88 are regulated by deguelin, but the mechanism by
which this occurs is not completely clear.
Nup98 and Nup88 protein analysis in U937 cells
We used flow cytometry to measure the expression of Nup98
and Nup88 in untreated U937 cells, and in U937 cells treated
with deguelin. In untreated cells, the average fluorescence
intensity of Nup98 in the blank controls was 23.55. In cells
treated with 10 nmol/L and 20 nmol/L deguelin for 24 h, the
average fluorescence intensity of Nup98 was 189.58 and
249.32, respectively. The average fluorescence intensity of
Nup88 in the blank controls was 53.92. When cells were
treated with 10 nmol/L and 20 nmol/L deguelin for 24 h, the
average fluorescence intensity of Nup88 was 11.12 and
10.03, respectively (Figure 4; n=3).
Location of Nup98 and Nup88 in U937 cells
To visualize the subcellular location of Nup98 and Nup88, untreated and
treated U937 cells were detected by using immunoelectron
microscopy. In Figure 5, the nuclear pore proteins are
indicated by arrowheads and are mainly located within the
nuclear membrane. The density of the nuclear pore protein
is based on DAB staining. In Figure 5, the electronic density
of Nup98 in untreated cells is lower than that in
deguelin-treated U937 cells. The electronic density of Nup88 in
untreated cells was higher than that in deguelin-treated U937
cells.
Expression of Nup98 and Nup88 in U937 cells
Our
results reveal that deguelin can induce antiproliferation and
apoptosis in U937 cells. However, it is unclear how U937
induces these effects. U937 cells treated with 10 nmol/L and
20 nmol/L of deguelin for 24 h were lysed and resolved using
10% SDS-PAGE, and then Western blot analysis was carried
out using anti-Nup98 and anti-Nup88. Figure 6 shows the
marked change in Nup98 and Nup88 expression following
deguelin treatment. Deguelin is related to upregulating the
expression of Nup98 and downregulating the expression of
the Nup88 protein. This indicates that Nup98 and Nup88 are
related to the deguelin-mediated nucleocytoplasm shuttling
activity, which is related to cell proliferation and apoptosis.
Discussion
Deguelin, a natural plant extract, is commonly used as an
insecticide in Africa, South America and China. Deguelin
belongs to a class of compounds called rotenoids, which
have chemopreventive activity[6]. In fact, deguelin has
already been shown to prevent skin and breast tumors in
experimental models[7]. It has chemopreventive activity, and
acts by inhibiting NADH:ubiquinone oxidoreductase activity,
and by regulating the cell cycle and inducing
apoptosis[7]. Chun et al found that deguelin inhibited the growth of and
promoted apoptosis of premalignant and malignant
cells[8]. In contrast, the compound had little effect on normal HBE
cells. The drugĄ¯s antineoplastic effects and specificity
appeared to be due to its ability to inhibit phosphatidylinositol
3-kinase (PI3K)/Akt-mediated signaling
pathways[9,10]. Bortul
et al found that deguelin enhanced the sensitivity of U937
leukemia cells and acute myeloid leukaemia (AML) blasts to
chemotherapeutic drugs with an activated PI3K/Akt
network[10]. In the present experiment we found that deguelin suppressed
the proliferation of U937 cells, and that deguelin may have
potential as an anti-tumor medicine. After treatment with
different doses of deguelin, U937 cells accumulated in the S
and G2/M phases, whereas the number of cells in the
G0/G1 phase decreased in a dose-dependent manner. We also found
that deguelin upregulated the expression of Nup98 and
downregulated the expression of Nup88 in U937 cells. This
suggests that changes in the ratio of Nup98 and
Nup88 might contribute to the apoptosis-promoting activity of
deguelin in these cells. The changes effected in nucleoporin in U937
cells by deguelin offer new possibilities for exploring the
underlying anti-tumor mechanism of deguelin.
The various events of nuclear division, cytoplasmic
division, cell growth, and cell maturation are repeated in each
generation of cells[12]. The periods of time and the sequence
of events from one cell division to the next are collectively
referred to as the cell cycle. Severe defects in chromosomes
block progression through the cell cycle, and can lead to cell
suicide or apoptosis[13]. Deguelin plays an important
pharmacological role by acting on different stages of the cell
cycle in tumor cells. In this study, U937 cells were arrested
mainly in the S and G2/M phases by deguelin, whereas the
proportion of cells in the
G0/G1 phase gradually declined.
After treatment with different doses of deguelin for 24 h,
cells in the G0/G1 phase were earliest influenced, and the
proportion of cells in this phase decreased gradually in a
dose-dependent manner. At the same time, the proportion
of cells in the S phase increased gradually, with treatment
with 80 nmol/L deguelin resulting in the highest value. The
proportion of cells in the G2/M phase increased in a
dose-dependent manner. These data show that deguelin usually
regulates the G1/S and G2/M checkpoints in U937 cells. Cell
cycle checkpoint controls at the G1 to S transition and the G2
to M transition prevent the cell cycle from progressing when
DNA is damaged[14]. That deguelin regulates the cell cycle
checkpoints has been verified in other tumor cells. Chun
et al found that after treatment with deguelin, the proportion of
premalignant cells in the
G0/G1 phase increased and the
proportion of malignant HBE cells in the
G2/M phase increased from 9.6% to
40.2%[8]. Bortul et al found that deguelin (10
nmol/L) induced S phase arrest by interrfering with the
progression to G2/M[10]. In our previous
studies [11], we found that deguelin was able to inhibit the proliferation of Daudi
cells by regulating the cell cycle that arrests cells at the
G0/G1 phase, and had no effect on the
G2/M phase. The mechanism by which deguelin causes these different cell cycle
changes is not yet completely clear, but it is mainly related to
the sensitivity of tumor cells to deguelin.
Transport between the nucleus and the cytoplasm
occurs through NPC embedded in the nuclear envelope.
Nucleoporins are involved in several types of acute myeloid
leukemia and a few other hematological malignancies, as well
as rare cases of other tumors[15]. An expanding subgroup of
chromosomal translocation-generated oncoproteins in
human acute myeloid leukemias (AML) involve the FG
repeat-containing NPC proteins Nup98 and
CAN/Nup214[16]. The Nup98 gene is
found at the breakpoints of two distinct chromosomal rearrangements: t (7;11)(p15;p15) and
inv(11)(p15;q22), which link Nup98 to the class I homeotic transcription
factor HOXA9 and the putative RNA helicase DDX10, respectively. The most common oncogenic fusions involve
a segment of the gene encoding the FG-repeat domain of
Nup98, which, in turn, becomes linked to genes of the
homeobox family of transcription factors. The
Nup98-derived FG-repeat segments of the resulting oncogene interact
with the transcriptional coactivators CBP (CREB binding
protein) and P300, thereby leading to increased gene
transcription[17]. FG-repeat segments of two other nucleoporins,
Nup153 and CAN/Nup214, can substitute for the Nup98
segment in the oncogenic fusion protein. When Nup98 is
disrupted, it selectively impairs discrete protein import
pathways, which supports the idea that transport of distinct
import complexes through the NCP is mediated by specific
subsets of nucleoporins. Because Nup98 plays a role in
RNA export, its mobility suggests that Nup98 might
associate with RNA close to its transcription site and then further
accompany the processed RNA through the NPC into the
cytoplasm[18]. Using confocal microscopy, we found that
Nup98 was present on both sides of the NPC in U937 cells,
and was localized inside the nucleus. When cells were treated
with 10 nmol/L and 20 nmol/L deguelin for 24 h, the average
fluorescence intensity of Nup98 was 189.58 and 249.32.
Deguelin can regulate the expression of Nup98, and this
shows that deguelin participates in Nup98 leading
nucleocytoplasmic traffic.
Nup88 is a NCP protein; the Nup88 gene is localized at
17p13 and the Nup88 protein is involved in
nuclear-cytoplasmic transport and cell
growth[19]. Overexpression of Nup88 has been found in human tumors of the stomach,
colon, liver, pancreas, breast, lung, ovary, uterus, prostate
and kidney[20]. The Nup88 protein is also overexpressed in
malignant tumor tissue relative to normal surrounding tissue.
Recent studies have found that the Nup88 protein is
enhanced in most metastatic melanomas relative to their corre
sponding primary tumors[21]. Both RNA transcription and
protein expression levels are higher in malignant tumor cell
lines compared with non-transformed cells. The Nup88
protein is strongly expressed in the invasive margins of both
primary and metastatic breast, endometrial and colorectal
carcinomas[22]. Overexpression of Nup88 in malignant
tumors is probably due to the enhanced nucleocytoplasmic
transport required to meet the increased demand for
proteins in the tumor cells. Nup88 is considered to be a tumor
growth factor, and is a positive regulator in the cell cycle.
For this reason, we devised the present study on Nup88
expression in U937 cells and studied the regulative effects of
deguelin on Nup88. Our data support the idea that Nup88
might be involved in the tumor progression of U937 cells,
and these findings show that deguelin participates in Nup88
leading nucleocytoplasmic traffic.
In summary, our results show that deguelin can inhibit
U937 cell proliferation in a dose- and time-dependent manner,
with an IC50 value for 24 h of 21.61 nmol/L and an
IC50 value for 36 h of 17.07 nmol/L. Deguelin usually regulates the
G1/S and G2/M checkpoints. In addition, deguelin causes
upregulation of the expression of Nup98, and downregulation
of the Nup88 protein in U937 cells. The reasons for this
effect, and the mechanisms for this regulation of Nup98 and
Nup88, and whether these mechanisms are linked to
inhibited cell proliferation, are unknown, but our findings
suggest that Nup98 and Nup88 may be involved in
nucleocytoplasmic shuttling in carcinoma cells.
Acknowledgements
We thank the Tumour Biology Laboratory, Center of
Gynaecology, Tongji Hospital, Huazhong University of
Science and Technology, China, for offering relevant
experimental facilities and technical support. We wish to
particularly thank Prof Yun-ping LU for guidance and help with the
experiment.
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