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Introduction
The percentage of long-term remitters and survivors in
adult acute lymphoblastic leukemia has not improved
significantly during recent decades, although several trials have
attempted to intensify the induction and post-remission
strategy, including early bone marrow transplantation. New
drug exploitation is one of the effective options. Gambogic
acid (GA), an extraction from the resin of Garcinia
hanburyi, was used as folk medicine and a colorant in China. Recently,
it has been demonstrated that GA has strong antitumor
activities in many solid tumors, such as human
hepatoma[1,2], gastric
adenocarcinoma[3], lung
carcinoma[4], and breast
cancer[5]. Although the mechanism is not fully understood, it has
been found to block the cell cycle[7], induce
apoptosis[1_6], and inhibit the gene expression of human telomerase reverse
transcriptase[4,6,8,9].
Death inducer-obliterator 1 (DIO-1) is identified as a gene
upregulated early in apoptosis by several
stimuli. The overexpression of DIO-1 in cells induced massive apoptosis
without any apoptotic stimuli[10]. Its predicted amino acid
sequence consists of a glutamine-rich region, an acidic
sequence, and a canonical bipartite nuclear localization
signal (NLS) in the N-terminal region, 2 Zn-finger motifs in the
central region, and a C-terminal lysine-rich
sequence[10]. In healthy cells, DIO-1 was located in the cytoplasm. After
overexpression or apoptosis induction, DIO-1 translocates
to the nucleus and induces apoptosis by caspase activation.
A NLS deletion mutant of DIO-1 (DIO-1ΔNLS) is unable to
translocate to the nucleus and upregulates pro-caspase
levels or triggers cell death[11]. The human
Dio-1 gene is mapped to the long arm of chromosome 20. DIO-1 expression
abnormalities have been found in 100% of human
myelodysplastic/myeloproliferative diseases (MDS/MPD) and other myeloid
neoplasm patients, but not in lymphoid neoplasm patients
or healthy donors. Here, we explored the inhibition effect of
GA on Jurkat cells and the reasonable
mechanism[12].
Materials and methods
Cells and reagents The Jurkat cells were obtained from
the China Center for Typical Culture Collection (Wuhan,
China) and cultured in RPMI-1640 medium (Gibco, Grand
Island, NY, USA) supplemented with 10% fetal bovine
serum (Gibco, USA) at 37 °C in a humidified atmosphere of 5%
CO2 and 95% air. GA (Figure 1) was provided by Calbiochem
(Darmstadt, Germany).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was bought from Janssen
Chimica (New Brunswick, NJ, USA). Hoechst33258, DMSO,
and monoclonal and mouse anti-g-tubulin antiserum were
purchased from Sigma-Aldrich (St Louis, MO, USA).
Monoclonal rabbit anti-DIO-1 and anticaspase 3 antiserum,
monoclonal mouse anti-Bcl-2, and NF-κB antiserum were the
products of Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Enhanced chemiluminescence reagent kits were purchased
from Pierce Biotechnology (Rockford, IL, USA). Annexin
V-fluorescein-isothiocyanate (FITC)/propidium iodide (PI) was
from Keygen (Nanjing, China). The Genomic DNA
purification kit was bought from Fermentas (Burlington, Canada).
Cytotoxicity assay of GA Cell proliferation was assessed
by MTT assay. Briefly, logarithmically-growing Jurkat cells
were seeded in triplicate at a concentration of
5×104 cells/mL in culture medium, and then exposed to various
concentrations (0, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 3.0, and
6.0 μmol/L) and exposure intervals (up to 3 d) of GA. Then,
the MTT working solution was added to each well and the cells were
incubated for 4 h. The water-insoluble formazan was formed
during incubation and was solubilized by adding DMSO to
each well. Suspension culture growth inhibition and the
50% inhibitory concentration values
(IC50) for GA were determined as previously
described[12].
Apoptosis assessment For the Annexin V-FITC/PI assay,
after GA treatment (0, 1, 2, and 3 μmol/L),
5×105 cells were washed in phosphate-buffered saline
(PBS) and resuspended in 500 μL 1×binding buffer. In total,
5 μL Annexin V-FITC and 5 μL PI were then added into the buffer. After 15-min
incubation at room temperature in the dark, the cells were
analyzed by flow cytometry. Annexin V-FITC and PI
fluorescence was detected in the FL-1 (green) and FL-2 (red)
channels, respectively, after the correction to the spectral
overlap between the 2 channels was made. Data were
analyzed by CellQuest software (Becton Dickinson, San Jose,
CA, USA).
For the DNA fragmentation assay, the Jurkat cells were
treated with 0, 1, 2, 3, and 4 μmol/L GA for 24 h, as well as 0
and 2 μmol/L GA for 24 h and 48 h, respectively. DNA was
extracted by the Genomic DNA purification kit, and then the
extraction was electrophoresed on a 1.5% agarose gel and
observed by ethidium bromide (EB) staining using the
Gel-Pro analyzer (Peiqing Technology, Shanghai, China). For
the comet assay, the Jurkat cells were exposed to 0 or 2
μmol/L GA for 24 h, and then embedded in agarose on microscope
slides, lysed, and electrophoresed. DNA fragments caused
by single- or double-stranded breaks migrated faster than
intact DNA. DNA were stained with PI. The DNA fragments
were visible as comet tails by fluorescence microscopy.
Western blotting The Jurkat cells were treated by 0, 0.5,
1.0, 2.0, and 4.0 μmol/L GA for 24 h or 0, 2, and 4
μmol/L GA for 2 h. The harvested cells were homogenized respectively
in the homogenization buffer, containing protease inhibitor
cocktail (1:1000) in 50 mmol/L Tris, pH 7.0, 1 mmol/L EDTA,
and 1 mmol/L phenylmethylsulfonyl fluoride. The raw
homogenate was centrifuged at 4 °C for 20 min at 13
400×g. The supernatants (20 μg) with 5× loading buffer were heated for
10 min at 100 °C and then loaded onto 10%_15% SDS-PAGE
for proteins with different molecule weights. The proteins
were electrophoresed and then transferred onto a
nitrocellulose membrane. The membrane was blocked with 5% non-fat
dry milk for 1 h and subsequently incubated overnight with
monoclonal rabbit anti-DIO-1 and anticaspase 3 antiserum
(1:500), monoclonal mouse anti-Bcl-2, and NF-κB antiserum
(1:500), as well as anti-γ-tubulin antiserum (1:10
000). The proteins were detected by using horseradish
peroxidase-conjugated goat antirabbit and antimouse secondary
antibodies (1:5000), visualized by using a chemiluminescent
substrate kit, and exposed to medical X-ray film. The intensity
of the blots was quantified with a gel image analyzer (JS380,
Peiqing Science and Technology, Shanghai, China).
DIO-1 immunofluorescence and Hoechst33258 double
staining For detecting the nucleus translocation of DIO-1,
5×106 Jurkat cells were treated with 4
μmol/L GA for 24 h. After fixing in 4% paraformaldehyde in 0.1 mol/L phosphate
buffer (PB) (pH 7.3) at 4 °C, the cells were plated on glass
coverslips in a 24-well plate on the day before the experiment.
Briefly, the staining procedure was performed at room
temperature in the dark: (1) blocked and permeabilized in the
blocking solution containing 3% bovine serum albumin, 2%
normal goat serum, and 1% Triton X-100 in PBS for 30 min;
(2) incubated with rabbit anti-DIO-1 antiserum (1:100) in
blocking solution for 24 h; (3) incubated with
FITC-conjugated donkey antirabbit immunoglobulin G antibody (1:500)
in blocking solution for 3 h; and (4) incubated with 10
μg/L Hoechst33258 by the end of step 3 for 15 min. The
immunofluorescence was finally visualized in
a laser scanning confocal microscope (FV500, Olympus, Tokyo, Japan).
Results
GA declined proliferation of Jurkat cells The effects of
GA on Jurkat cell proliferation were assessed by MTT assay
(Figure 2). GA was able to suppress the proliferation of
Jurkat cell in a dose- and time-dependent manner. The
IC50 values for GA at each interval (24, 48, and 72 h) were
1.51±0.09, 0.98±0.13, and 0.67±0.12
μmol/L (P<0.01), respectively. However, the time dependence was not significant when the
GA concentration was very low or very high.
GA-induced apoptosis of Jurkat cells To clarify the
mechanism of GA-induced proliferation inhibition, we
examined the GA-induced apoptosis in Jurkat cells. In the Annexin
V-FITC/PI assay (Figure 3A-3D), we found that a lower dose
of GA (less than 1 μmol/L) made no difference. The
percentages of apoptosis and necrosis cells were less than 4%.
However, a higher dose (2 and 3 μmol/L) of GA treatment in
the Jurkat cells resulted in a markedly increased
accumulation of apoptotic cells. DNA defragmentation (Figure 3E,3F)
and comet assay (Figure 3G, 3H) was designated to analyze
the DNA changes in late apoptosis. After incubation with
GA, the Jurkat cells induced apoptosis, as shown by the
formation of distinct internucleosomal DNA fragmentation,
in a dose- and time-dependent manner. In the comet assay,
the cells with DNA damage exhibited an oval comet tail.
GA-triggered caspase activation associated with DIO-1
upregulation and translocation DIO-1 was reported to be
able to trigger the apoptotic process by caspase activation
in vitro and was involved in hematological myeloid neoplasm.
However, in our research, we also found DIO-1 expression in
lymphoid cells by Western blotting and immunofluorescence.
By Western blotting, we detected a dose- and
time-dependent increase of DIO-1 expression in Jurkat cells (Figure
4A-4D). The dose-dependent increase of DIO-1 expression was
accompanied by the activation of pro-caspase. Pro-caspase
was cleaved into 2 activated subunits: p17 and p20 (Figure
4A).
The subcellular localization of DIO-1 was examined by
immunofluorescence. Although the nucleus of control cells
also slightly exhibited green fluorescence of DIO-1, DIO-1 in
the nucleus, if any, was scattered and the nucleus per se was
intact (Figure 5A, 5C, 5E, 5G). Treated with 4 μmol/L
GA for 24 h, DIO-1 formed a nuclear aggregate in each early apoptotic
cell, which showed relatively intact but condensed
chromatins. In late apoptotic cells, the condensation started
to disintegrate into similarly dense, smaller particles, and
DIO-1 aggregation could not be seen (Figure 5B, 5D, 5F, 5H).
GA weakened inhibition of caspase activity related to
NF-κB and Bcl-2 downregulation We then detected the
Bcl-2 and NF-κB expressions in GA-treated Jurkat cells and
demonstrated the dose-dependent decline in both Bcl-2 and
NF-κB expressions (Figure 4A).
Discussion
DIO-1-induced apoptosis was able to be inhibited by
benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone
(z-VAD-fmk), a pan caspase inhibitor[10,
11], suggesting that DIO-1-induced apoptosis was caspase dependent. In the present
study, we reported that the DIO-1 upregulation and
translocation was accompanied by the cleavage of pro-caspase 3
into 2 activated subunits: p17 and p20, so GA-triggered caspase
activation may be associated with DIO-1
upregulation and translocation. However, the underlying molecular
mechanisms on nuclear translocation and caspase activation
remain unknown. DIO-1 forms homo-oligomers in
vivo. The cytosolic form of DIO-1 is phosphorylated on
serine/threonine and is predominant in healthy cells, whereas the
unphosphorylated nuclear form is found under apoptotic
conditions. However, phosphorylation alone is not the
basis of the mobility change. Additional modifications may
thus be demanded for the DIO-1
translocation[11].
Although the function of DIO-1 was not fully clear,
several studies evidenced the transcriptional activities of some
domains in the DIO-1 sequence. DIO-1 contains a plant
homeodomain finger and a transcription factor S-II domain;
both domains are usually associated with transcription. It
has been also reported that the spen paralog and ortholog C
terminal (SPOC) is present in the DIO-1 family of proteins
and is essential for normal
function[14]. The SPOC domain of DIO-1 adopts a similar fold than the SPOC domain of the
silencing mediator for retinoid and thyroid receptors
(SMRT)/histone deacetylase 1 (HDAC1)-associated repressor protein
(SHARP), and the 7 strands β-barrel core is
maintained[14]. Thus, DIO-1 may share a common function and molecular
mechanism with SHARP, which has been identified as a
component of transcriptional repression complexes in both
nuclear receptor and Notch/ human recombination signal
sequence-binding protein (RBP-Jκ) signaling
pathways[15_17]. The conserved residues Arg 3552 in the SPOC domain may
be important for the protein-protein interaction. Arginine
methylation is a common post-translational modification in
transcription regulation proteins and is involved in a wide
range of cellular processes, including pre-mRNA splicing,
polyadenylation, transcription, signal transduction, and
cytoskeleton and DNA repair[14]. Arginine is also frequently
found at the active sites of enzymes, and the SPOC domain may
contribute to a specific catalytic
function[18]. DIO-1ΔNLS was unable to translocate to the nucleus or trigger
apoptosis[11]. The 2 NLS sequences may play an important role in nucleus
translocation, recognized by the nucleus transduction
components.
In our study, GA induced the downregulation of
NF-κB and Bcl-2. After the activation of NF-κB, its p65 subunit is
phosphorylated, leading to nucleus translocation and
binding to a specific sequence in DNA, which in turn results in
gene transcription, including anti-apoptotic genes (eg
Bcl-2, cellular inhibitor of apoptosis protein (cIAP), survivin,
and tumor necrosis factor receptor-associated factor
(TRAF)); cyclooxygenase-2; matrix metalloproteinase-9;
genes encoding adhesion molecules, chemokines, and
inflammatory cytokines; and cell cycle regulatory genes (eg
cyclin D and c-Myc)[19]. In Jurkat cells,
NF-κB is highly expressed and may result in the overexpression of Bcl-2.
Bcl-2 is one of the dominant regulators in apoptosis, whose
downregulation releases cells from anti-apoptotic status. The
overexpression of Bcl-2 blocks DIO-1-induced
apoptosis[10]. This indicates that DIO-1 is upstream of the caspase
cascade and the induction of apoptosis driven by this gene
proceeds through the main apoptotic route. The
dose-dependent decline in Bcl-2 expression may be mediated by
NF-κB downregulation. In normal tissues, there must be a
balance between the NF-κB-regulated cell proliferation and the
DIO-1-mediated apoptosis. However, in tumor tissues, the
high expression of NF-κB expression inevitably suppresses
the caspase activity and inhibits DIO-1-mediated apoptosis.
We found that GA not only triggered DIO-1-mediated caspase
activation, but also decreased or eliminated the inhibition of
caspase activity in Jurkat T cells, suggesting that GA
anti-leukemia effect was mediated by DIO-1 upregulation and
translocation and associated with the caspase-dependent
signaling pathway. In summary, it appears that GA as an
apoptotic inducer is a potential drug, and DIO-1 as a caspase
upstream regulator is a potential target for lymphoblastic
leukemia treatment.
Acknowledgements
We thank Dr He LI, Department of Anatomy, Tongji
Medical College, Huazhong University of Science and
Technology, for the provision of experimental facilities and
technical support in the research work. We also thank
Wei-xi WANG and Yi-nong ZHANG, technicians from the
Division of Histology and Embryology, Tongji Medical College,
Huazhong University of Science and Technology, for their
technical support in laser confocal microscopy.
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