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Introduction
Liver cancer is most common in Asia. In China, about
100 000 patients die each year of primary liver cancer. To explore
efficient drugs is very helpful for those patients to overcome liver cancer.
Homoharringtonine (HHT) is an alkaloid with antileu-kemia activity against a variety of acute myeloid leukemic cells.
HHT can induce apoptosis in a variety of human myeloid leukemia cell
lines[1,2]. In HL-60 cells, HHT induced apoptosis when
the cells were exposed to 1×10-7 mol/L HHT for 4 h. DNA extracted from the treated cells showed a typical internucleosomal
DNA degradation. This effect was in a concentration- and time-dependent manner. The results suggest that the antitumor
mechanism of HHT is related to its activity to induce
apoptosis[3]. HHT is an inhibitor of protein synthesis, its effect in a
dose- and time-dependent manner. The extent of HHT-induced DNA formation correlates with the inhibition of DNA
synthesis and loss of clonogenic
survival[4]. However, so far, there have been no reports as to whether HHT can be used to treat
liver cancer.
The purpose of this study is to explore the activity of HHT on liver cancer and to examine genes regulated by HHT
treatment, so as to reveal the signal transduction pathway involved in HHT-induced apoptosis. cDNA microarrays were
utilized to investigate changes of gene transcription. In all,
4 microarrays were screened and each array represented
14 218 human genes. The 78 genes characterized are mostly
related to apoptosis, many of which are oncogenes, tumor
suppressors, enzymes, and kinases. Moreover, a few of the
identified genes are novel and may play important roles in
apoptosis.
Materials and methods
Cell culture Human hepatoma QGY-7703 cells were
cultured in RPMI-1640 medium containing 10% fetal bovine
serum in a humid atmosphere with 5% CO2 at 37 ºC. For the
experiment, the QGY-7703 cells were incubated with 36
μmol·L-1 HHT (Beijing Union Pharmaceutical Factory, Beijing,
China) or RPMI-1640 medium alone for 6, 24, and 48 h.
Detection of apoptotic DNA fragmentation
Total cellular DNA was extracted from the QGY-7703 cells according to the
method described by Slin and Stafford with some slight
modifications[5]. In brief, after washing in phosphate-buffered
saline (PBS), the QGY-7703 cells were lysed overnight at
37 ºC in lysis buffer (10 mmol/L Tris-HCl, pH 8.0, 10 mmol/L
edetic acid, 0.4% SDS, and 100 mg/L proteinase K). After
completely dissolving, saturated phenol was added to the
cell lysates and mixed entirely. The samples were then
centrifuged for 5 min. Chloroform was added to the supernatant
isolated from the previous step, mixed, and centrifuged. The
supernatant was mixed with 2.5-fold volume of absolute
ethanol and 0.2 mol/L NaCl for DNA precipitation. The DNA
pellets were obtained by centrifugation at full speed for 10
min and then air-dried, dissolved in TE buffer containing 0.5
g/L RNase (Sigma Chemical Co, St Louis, MO, USA) at 37 ºC
for 30 min. Electrophoresis was performed on 1.5% agarose
gel. The DNA was visualized by UV illumination.
Morphological observation of apoptotic cells
The QGY-7703 cells (5×106 cells, 2 mL/well) were seeded onto
standard 6-well tissue culture plates and treated with HHT for
6, 24, and 48 h. The QGY-7703 cells on the plates were
washed with PBS directly, then detached and centrifuged
and washed with PBS. All cell samples were fixed in 4%
paraformaldehyde and stained with 5 µg/mL Hoechst 33258
(Sigma Chemical Co, St Louis, MO, USA) for 15 min at room
temperature; apoptosis was detected by fluorescence
microscopy.
RNA isolation The total RNA from the QGY-7703 cells
was extracted according to the original Chomczynski method
with slight modifications[6]. The cells were collected and
homogenized in Solution D containing 1%
β-mercapto-ethanol. After centrifugation, the supernatant was extracted
with phenol:chloroform (1:1) twice and acidic
phenol:chloroform (5:1) once. The RNA in the aqueous phase was
precipitated by cold isopropanol and dissolved in deionized
H2O. Messenger RNA were purified using an Oligotex-dT mRNA
Midi Kit (Qiagen, Pudong, Shanghai, China).
Construction of microarrays and probe preparation
The microarrays were constructed according to Brown's method
(http://cmgm.stanford.edu/pbrown/protocols/index.html).
The 14 218 microarrays consisted of 14 218 full-length or
partial cDNA representing novel, known, and control genes
provided by United Gene Holdings (1111 Zhongshan Bei Er
Road, Shanghai, China). The known genes were selected
from the National Center for Biotechnology Information.
Unigenes were set and cloned into a PBS plasmid vector.
The control spots of non-human origin included the rice U2
RNA gene (8 spots), hepatitis C virus coat protein gene (8
spots), and spotting solution alone (32 spots). The cDNA
inserts were amplified by PCR using universal primers to the
plasmid vector sequences. All PCR products were examined
by agarose gel electrophoresis to ensure that the quality
and the identity of the amplified clones were as expected.
The PCR products were dissolved in the PCR buffer. The
solution was spotted onto silylated slides (CEL Associates,
Houston TX, USA) using a Cartesian PixSys 7500 motion
control robot (Cartesian Technologies, Irvine, CA, USA)
fitted with ChipMaker Micro-Spotting Technology (TeleChem
International, Sunnyvale, CA, USA). Glass slides with
spotted cDNA were then hydrated for 2 h in 70% humidity, dried
for 0.5 h at room temperature, and then UV cross-linked (65
mj/cm). They were further processed at room temperature
by soaking in 0.2% SDS for 10 min, subsequently in distilled
H2O for 10 min, and then in 0.2% sodium borohydride for 10
min. The slides were then dried and ready for hybridization.
The fluorescent cDNA probes were prepared through
reverse transcription of the isolated mRNA and then purified
according to the method described by Schena et
al[7,8]. The mRNA samples from the control cells and treated cells were
incubated with Cy5-dUTP (Amersham Pharmacia Biotech,
Piscataway, NJ, USA). The 2 color probes were then mixed,
precipitated with ethanol, and dissolved in 20 µL
hybridization solution (5× SSC, 0.4% SDS, 50% formamide, and 5×
Denhardt's solution).
Hybridization on microarrays The microarrays were
prehybridized with hybridization solution containing 0.5 g/L
denatured salmon sperm DNA at 42 ºC for 6 h. Fluorescent
probe mixtures denatured at 95 ºC for 5 min were applied
onto the prehybridized microarrays under cover glasses.
After the microarrays were hybridized at 42 ºC for 15_17 h,
they were stringently washed at 60 ºC for 10 min each in the
solution of 2×SSC and 0.2% SDS, 0.1×SSC and 0.2% SDS,
and 0.1× SSC, and then dried at room temperature.
Detection and analysis of microarrays The microarrays
were scanned with a ScanArray 3000 (GSI Lumonics, Bellerica,
MA, USA) at 2 wavelengths to detect emission from both
Cy3 and Cy5. The acquired images were analyzed using
ImaGene 3.0 software (BioDiscovery, Los Angeles, CA, USA).
The intensities of each spot at the 2 wavelengths represent
the quantity of Cy3-dUTP and Cy5-dUTP, respectively,
hybridized to each spot. The ratio of Cy5 to Cy3 was computed
for each location on each microarray. The overall intensities
were normalized with a correction coefficient obtained using
the ratio of 40 housekeeping genes (list of these genes is
available at http://www.biodoor.com/). The genes were
identified as differentially expressed if the ratio of Cy5/Cy3
was >2 or <0.5. To minimize artifacts arising from low
expression values, only genes with raw intensity values for
both Cy3 and Cy5 of >800 counts were chosen for the
differential analysis.
RT-PCR The total RNA was extracted from the cells with
TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA,
USA) and quantified by UV absorbance spectroscopy. The
reverse transcription reaction was performed using the
Superscript First-Strand Synthesis System (Invitrogen Life
Technologies, USA) in a final volume of 20 µL containing
5 µg total RNA, 200 ng random hexamers, 1×reverse
transcription buffer, 2.5 mmol/L MgCl2, 1 mmol/L deoxynucleotide
triphosphate mixture, 10 mmol/L DTT, RNase OUT
recombinant ribonuclease inhibitor (Invitrogen Life Technologies,
Carlsbad, CA, USA), 50 units Superscript reverse
transcriptase (GIBCO-BRL, Gaithersburg, MD, USA), and
diethyl-pyrocarbonate-treated water. After incubating at 42 °C for
50 min, the reverse transcription reaction was terminated by
heating at 85 °C for 5 min. The newly synthesized cDNA was
amplified by PCR. The reaction mixture contained 2 µL cDNA
template, 1.5 mmol/L MgCl2, 2.5 units Tag polymerase, and
0.5 µmol/L primer. The sequence for each primer used in this
study was as follows:
TIEG primer (5'-ACTGCGGAGGAAAGAATGGA-3'; 5'-CTGGGAGGAGTGCTGGGAAC-3');
VDUP1 primer (5'-TTGCGGAGTGGCTAAAGTG-3'; 5'-TCATCTCAGAGCTGGTTCG-3'); protein phosphatase
1 (PPP1CA) primer (5'-ACTATTTGAGTATGGCGGTTTCC-3'; 5'-TGTTCTTGTCGGCGGGCTTG-3'); NFKBIA primer
(5'-AAGACGAGGAGTACGAGCAGAT-3'; 5'-CAGCACCCAA-GGACACCAAA-3'); death-associated protein 6 (DAXX)
primer (5'-GACCCAGACTCCGCATACC-3'; 5'-GCACTGA-CCTTTGCCTTTCC-3'); insulin-like growth factor binding
protein 7 (IGFBP7) primer (5'-ATGCTGGAGAATATGAGT-GCCA-3'; 5'-CTGAAGCCTGTCCTTGGGAA-3');
inhibitor-of-differentiation (ID)3 primer
(5'-GGAGCTTTTGCCACTGA-CTC-3'; 5'-TTCAGGCCACAAGTTCACAG-3'); and G3PDH
primer (5'-ACCACAGTCCATGCCATCAC-3'; 5'-TCCACCA-CCCTGTTGCTGTA-3').
The amplication cycles were 94 °C for 3 min, then 33 cycles
at 94 °C for 1 min, 58 °C for 1 min, 72 °C for 1.5 min, followed
by 72 °C for 10 min. Aliquots of PCR product were
electrophoresed on 1.5% agarose gels and visualized by ethidium
bromide staining.
siRNA preparation and transfection The siRNA
sequence targeting ID3 was
5'-AAGGAGCTTTTGCCACT-GACT-3'. The siRNA duplex was synthesized by Shanghai
GeneChem (Zhangjiang, Shanghai). The cells in the
exponential phase of growth were seeded in 6-well plates at a
concentration of 5×105 cells/well. After incubation for 24 h,
the cells were transfected with siRNA (100 nmol/L) and
Lipofectamine 2000 (Invitrogen Life Technologies, USA)
according to the manufacturer's protocol. Silencing was
examined 48 h after transfection. The control cells were
treated with non-targeting siRNA.
Western blot analysis All cells on the plates were
collected and detached from the plates. The cells were washed
twice with PBS containing 1 mmol/L phenylmethylsulfonyl
fluoride and lysed in cold TNT buffer [20 mmol/L
Tris-HCl (pH 7.4), 200 mmol/L NaCl, 1% Triton X-100, 1
mmol/L phenylmethylsulfonyl fluoride, and 1% aprotinin]
for 45 min with occasional rocking. The lysates were
transferred to Eppendorf tubes and clarified by centrifugation
at 12 000×g for 40 min at 4 °C. Identical amounts (50 µg
of protein) of cell lysates were separated by 15%
SDS-PAGE and transferred to the nitrocellulose. The membrane
was incubated in blocking solution containing 5% powered
milk in TBST [10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L
NaCl, and 0.1% Tween 20] at room temperature for 1 h.
Then the membrane was immunoblotted with the specific
antibodies against ID3 (Santa Cruz, Santa Cruz, CA, USA),
cleaved caspase-3 (Cell Signaling Technology, Danvers,
MA, USA) and antitubulin (Sigma-Aldrich, USA).
Detection by enzyme-linked chemiluminescence was performed
according to the manufacturer's protocol Amersham Pharmacia Biotech, USA).
Statistical analysis All of the data in Table 1 represent
an average ratio of Cy5/Cy3 of 2 microarrays at each time
point. Genes were identified as significantly expressed if the
ratio of Cy5/Cy3 was >2 or <0.5.
Results
HHT induced apoptosis in QGY-7703 cells Apoptosis
induced by HHT in the QGY-7703 cells was examined
(Figure 1). DNA electrophoresis showed that the QGY-7703
cells presented the typical DNA ladder pattern of apoptosis
after treatment with 36 µmol/L HHT for 24 and 48 h.
When stained with Hoechst-33258, the control cells that
were not submitted to HHT treatment did not exhibit
chromatin condensation (Figure 2A). By
contrast, a dense and thin crown of nuclear coloration,
typical of chromatin condensation, was observed in the
HHT-treated cells at 24 h (Figure 2C). At the same time, some cells were detached from
the plates, which was typical of chromatin condensation
(Figure 2D). After treatment with HHT for 48 h, all of the cells
detached from the plates, which was
typical of chromatin condensation (Figure 2E). We found that although several
nuclei still displayed a normal morphology, most of the cells
exhibited intense staining of condensed and fragmented
chromatin. This evidence clearly showed that HHT induced
apoptosis in the QGY-7703 cells.
cDNA microarray analysis In order to identify both
early and late apoptosis-responsive genes, mRNA was
isolated from the cells treated with HHT for 6 and 24 h, and
subjected to microarrays for hybridization. RNA samples
from cells treated with RPMI-1640 medium were used as the
control. A total of 10 microarrays were screened. Two
microarrays were screened for each parallel mRNA sample at
each time point. The hybridization result was from all 10
microarrays, which were compiled and sorted on the basis of
fold change compared to the control cells. The average ratio
of the 2 microarrays screened at each time point was adopted.
The hybridized signal of a desired gene displaying 2-fold or
more changes was considered a significant change.
Seventy eight of the 14 218 genes were identified by these criteria,
53 were upregulated, and 25 were downregulated at different
time points (Table 1).
Confirmation of HHT-induced genes by RT-PCR
To confirm the results obtained from the microarray hybridiza-tion,
we used total RNA extracted from the cells and performed
RT-PCR with 6 HHT-induced genes: ID3, TIEG, NFKBIA,
VDUP1, PPP1CA, DAXX, and IGFBP7. The result showed
that the expression patterns for the 6 genes correlated with
the microarray results (Figure 3). The threshold we set for
the inclusion of HHT-induced genes in the DNA chip
analysis is therefore valid in the 6 genes examined.
Reduction of ID3 expression by siRNA attenuates the
HHT-induced activation of cleaved caspase-3 To determine
the effect of siRNA on ID3 expression, cell lysates from the
treated cells were analyzed by Western blotting. After
treatment with HHT for 48 h, the expression of ID3 was inhibited
by siRNA (Figure 4). Moreover, after the cells were treated
with HHT for 48 h, cleaved caspase-3 could not be detected
in ID3-targeting siRNA-treated cells (Figure 4).
Discussion
Most papers about HHT focus on antileukemia and
apoptosis-inducing activity in leukemia
cells[1_3]. Our experi-ments showed that HHT could also induce apoptosis in
human hepatoma QGY-7703 cells in vitro. In an attempt to
identify genes in which expression changed significantly
during HHT-induced apoptosis in the QGY-7703 cells, we
utilized cDNA microarray technology to obtain an overall
profile of gene expression.
Apoptosis-related genes Based on the data, our
analysis was focused on those genes in which expression altered
significantly during the process of cell apoptosis. Among
those genes, we found that those capable of inducing
apoptosis were upregulated, and the others protecting cells
from apoptosis were downregulated.
Among the upregulated genes, ID2 and ID3 belong to a
family of transcriptional modulators that are characterized
by a helix loop helix region. During development, ID2 is an
inducible gene during serum and potassium deprivation-
induced apoptosis of cerebellar granule neurons. Over-
expression of ID2 induces neuronal cell death, both in high
potassium and low potassium conditions. The inhibition of
ID2 expression protects neurons from
apoptosis[9]. ID2 may play a role in apoptosis-associated
atrophy of skeletal
muscles[10]. ID3 induces apoptosis through a caspase-2-
dependent mechanism that does not require p53 and is not
inhibited by bcl-2[11]. TIEG is an early growth response
product induced by transforming growth factor-beta
(TGF-β). Overexpression of TIEG mimics TGF-β action and plays a
role in TGF-β-induced apoptosis in human osteoblast cells,
pancreatic carcinoma cells, and epithelial and liver cancer
cells[12_15]. ACVRL1 is type I cell-surface receptor
recognized by the TGF-β superfamily[16]. The elevated expression
levels of TIEG and ACVRL1 indicated that the TGF-β
signaling pathways were initiated. CYR61 is cysteine-rich,
angiogenic inducer 61, which can induce
fibroblast apoptosis through its adhesion receptors, integrin
6β1, and the heparan sulfate proteoglycan syndecan-4, triggering the activation
of the transcription-independent p53-activated Bax to
render cytochrome c release and the activation of caspases-9
and -3[17]. TNFAIP3 represents TNF-a-induced
protein 3, which encodes a protein that inhibits
apoptosis[18]. NFKBIA is a nuclear factor for the kappa light chain gene enhancer in
B-cells. It inhibits NF-kB activity by binding the rel domain of
NF-kB components[19,20]. As known,
NF-kB resists apopto-sis. The upregulation of NFKBIA in our experiment would
inhibit NF-kB activity and induce apoptosis. The
upregula-tion of TNFAIP3 and NFKBIA indicated that the TNF
signaling pathways were initiated. EphA2 is a target gene of the
p53 family. P53 can bind to the promoter element of EphA2
and is responsive to wild-type p53, p73, and p63. EphA2
expression can result in an increase in apoptosis. The
activated EphA2 may serve to impair anti-apoptotic signaling,
disrupt focal adhesions, and thereby sensitize cells to
pro-apoptotic stimuli[21]. ETS2 is a v-ets erythroblastosis virus
E26 oncogene homolog 2. The overexpression of ETS2
results in apoptosis in trans-genic mice, cell lines, and in
cells from subjects with Down syndrome. In all circumstances
of ETS2 overexpression, the increased apoptosis correlated
with increased p53 and alterations in downstream factors in
the p53 pathway. The overexpression of ETS2
induces apoptosis that is dependent on p53 and involves the
activation of caspase-3[22,23]. The upregulation of EphA2 and ETS2
indicated that the p53 signaling pathways were initiated.
Vitamin D3 upregulated gene 1 (VDUP1)-mediated oxidative
stress via suppressing the thioredoxin (TRX)
function[24]. TRX has functions in defense
against oxidative stress and control of growth. VDUP1 acts as an endogenous inhibitor
of TRX by interacting with the catalytic active center of TRX
to induce apoptosis and sensitize cells to oxidative
stress-mediated
apoptosis[25]. Wee1 is a well-known cell cycle
regulator, which can interact with the Crk SH2 motif and
accelerates apoptosis. Wee1-Crk complex signaling may be
a novel apoptotic pathway[26].
Among the downregulated genes, DAXX is involved in
apoptosis and transcriptional repression. It interacts with
the death receptor FAS, promyelocytic leukemia protein
(PML), and several transcriptional repressors. It may
inhibit FAS and stress-mediated apoptosis by suppressing
pro-apoptotic gene expression outside PML
domains[27]. The inhibition of DAXX might trigger FAS signaling pathways.
RAD21 is preferentially cleaved by caspases-3 and -7 to
generate 2 major proteolytic products of 65 and 48 kDa. RAD21
is specifically proteolyzed by caspases into a subunit with a
similar 65 kDa carboxyl-terminal product in cells undergoing
apoptosis. Caspase proteolysis of RAD21
precedes apoptotic chromatin condensation and amplifies the cell
death signal[28].
Oncogenes and tumor suppressors Among the
upregul-ated genes, c-fos plays a causal role in clonal deletion of
germinal center B cells[29]. The c-fos protein was found to
induce apoptosis in 2 Syrian hamster embryo
cell lines (sup+I and sup_II) and a human colorectal carcinoma cell
line[30]. The catalytic subunit of protein phosphatase 1 (PPP1CA)
regulates mitosis and is a putative tumor
suppressor[31]. The induction of c-Myc may lead to
apoptosis[32].
Among the downregulated genes IGFBP7 is a
proto-oncogene. Rab13 is a Ras-associated small GTPase, which
is presumed to function in vesicular
traffic[33].
Enzyme/kinases Among the upregulated genes, CLK2
renders the cell hypersensitive to apoptosis triggered by
oxidative stress or DNA replication block and gradually
increases telomere length[34]. Thymidine kinase 1 (TK1) is a
cell cycle regulatory gene involved in the
G1-S phase transition during cell cycle progression. The activation of TK1
may be critical to modulate radiation-induced cell death and
cell cycle progression in irradiated K562
cells[35]. GADD34 is also known PPP1R15A, which is protein phosphatase 1,
regulatory (inhibitor) subunit 15A. GADD34 is an apoptosis-
and DNA damage-inducible gene[36,37].
Phosphoinositide-3-kinase regulatory subunit 4 (PIK3R4) is also called P150. It is
reported that the expression of PIK3R4 correlates with tumor
cell apoptosis[38], which can be cleaved by caspases both
in vitro and in vivo[39]. MAP2K3 is mitogen-activated protein
kinase 3. The activation of p38MAPK is predominantly
mediated by the 2 upstream MAPK kinases, MAP2K3 and
MAP2K6. p38 is involved in apoptosis in many cell
lines[40,41].
Among the downregulated genes, DDX1, DDX3, and DDX15 belong to the DEAD-box family. This family
contains a large group of putative RNA helicases that mediate
nucleoside triphosphate-dependent unwinding of
double-stranded RNA. In their core region, DEAD-box proteins
contain 9 conserved motifs, of which, asp-glu-ala-asp
(DEAD), was adopted for the name of the whole family.
They are implicated in a number of cellular processes
involving the alteration of RNA secondary structures such as
translation initiation, nuclear and mitochondrial splicing, and
ribosome and spliceosome assembly. Based on their
distribution patterns, some members of this family are believed to
be involved in embryogenesis, spermatogenesis, and
cellular growth and division.
Others NFIL3 is a nuclear factor and activates
inter-leukin (IL)-3 gene expression. It binds to the regulatory
sequence in the promoter region of adenovirus E4, gamma
interferon, and IL-3 genes[42]. RNA binding motif protein 4
(RBM4), with a retroviral-type zinc finger, is a putative RNA
recognition motif-type RNA-binding protein, which is
similar to the D melanogaster lark RNA-binding
protein[43].
To test whether these induced genes are related to
HTT-induced apoptosis, we chose ID3 and tested its role in
apoptosis. It is reported that the reduction of ID3
expression by siRNA abrogated the UVB-induced proteolytic
activation of caspase-3[44]. We also found that reduction of ID3
expression by siRNA attenuated the HHT-induced
activation of caspase-3 in the QGY-7703 cells, suggesting that
HHT-induced apoptosis of QGY-7703 is at least in part due to the
upregulation of ID3.
In conclusion, this was the first time that it was reported
that HHT induced apoptosis of QGY-7703 cells in
vitro. HHT is a potential drug against liver cancer. A variety of
gene transcription has changed in apoptotic QGY-7703
cells; most genes with changed transcription were related
to apoptosis, oncogenes and tumor suppressors, enzymes,
and kinases. Our results demonstrated that TGF-β, TNF,
FAS, p38MAPK, and p53 apoptosis signaling pathways were
initiated. Some specific gene-encoding factors inducing
apoptosis and tumor suppressors were upregulated.
Onco-genes and some gene-encoding factors inhibiting apoptosis
were downregulated. Other genes, such as DDX1, DDX3,
DDX15, and RBM4 were induced significantly; they may
play important roles in apoptosis signaling pathways and
deserve to be further studied.
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