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Introduction
Laryngeal carcinoma especially at late-stage is associated with
high morbidity and poor long-term survival due to the absence of
effective treatment methods. Better understanding of molecular mechanisms
underlying proliferation, differentiation, and survival of laryngeal
carcinoma is critical for the development of optimal therapeutic
modalities. Recent studies suggest that signal transducers and activator
of transcription (STATs) have the potential as novel molecular targets
for the development and survival of laryngeal carcinomas.
STATs are latent cytoplasmic transcription factors that function
as intracellular effectors of cytokine and growth factor signaling
pathways . Among the STAT family, STAT3 plays a key role in promoting
proliferation, differentiation, anti-apoptosis, or cell cycle progression.
Constitutive activation of STAT3 is implicated in a variety of tumor
cell lines[1-5], thereby suggesting that STAT3 is an
important molecular target for tumor therapy.
In vitro studies have shown that inhibition of STAT3 activity
in human tumor cells induces apoptosis and/or growth arrest. In
human head and neck squamous carcinoma cells, blocking of STAT3
signaling by decoy oligonucleotide or antisense oligonucleotides
abrogates transforming growth factor and suppresses oncogenic growth
of these cells[6,7]. STAT3¦Â is a naturally accruing dominant-negative
STAT3 variant that is identical to STAT3 except for the absence
of the transactivation domain[8]. Bowman et al[2]
have reported that blockade of STAT3 signaling by STAT3¦Â in human
myeloma cells down-regulates IL-6-induced expression of the antiapoptotic
gene, Bcl-xL, resulting in a dramatic sensitization
of cells to Fas-mediated apoptosis in vitro. STAT3¦Â can promote
apoptosis in breast cancer cells, inhibit Bcl-xL expression,
and induce apoptosis[9]. Overall, these raise the possibility
that targeting STAT3 may enhance antitumor responses in vivo
in a variety of human cancers.
A relatively new technique using RNA interference (RNAi) provides
a novel approach of experimental inhibition of gene expression.
RNAi is triggered by the presence of double-stranded RNA (dsRNA)
in the cell and results in rapid degradation of the targeted mRNA
with homology to the double strand leading to potent and selective
silencing of genes. This phenomenon was first observed from studies
in Caenorhabditis elegans and Drosophila melanogaster
and subsequently, in other organisms[10]. Recent studies
have shown that short interfering (21-25 bp) RNA molecules (siRNA),
but not long dsRNA (greater than 30 bp), are key elements of RNAi.
Only recently has the use of RNAi in mammalian studies been established
by introducing siRNA[14]. At present, siRNA has been
adapted as a functional genomic tool and has potential as a therapeutic
approach in cancer.
Intriguingly, blocking of STAT3 signaling pathways by siRNA has
been shown to suppress growth and induce apoptosis in prostate cancer
cell lines and astrocytoma cells[15,16]. However, to
date no studies have studied the effect of inhibition of STAT3 gene
expression by siRNA on laryngeal cancer. Since STAT3 signaling is
critical for the regulation of proliferation, differentiation, and
apoptosis of tumor cells, we hypothesize that knockdown of STAT3
gene expression by siRNA should suppress tumor growths and induce
apoptosis in laryngeal carcinoma that observed in models of prostate
cancer and astrocytoma[16,17].
The objectives of the present study were (1) to determine the inhibitory
effect of the synthetic STAT3 siRNA on the expression of STAT3 gene
in laryngeal carcinoma cells and (2) to investigate the effect of
STAT3 siRNA on the growths and apoptosis in laryngeal carcinoma
cells.
Materials and methods
Construction of plasmids that contain DNA templates for the
synthesis of siRNAs were constructed under the control of the U6
promoter The pSilencer1.0-U6 (Ambion Inc Austin, TX, USA)
was used for DNA vector-based siRNA synthesis under the control
of U6 promoter in vivo. In brief, first, the double stranded
DNA template encoding siRNA oligonucleotides (GeneBank: access numbers
for the human STAT3: NM003150) that contained a sense strand of
19 nucleotide sequences followed by a short space (TTCAA-GAGA),
the reverse complement of the sense strand, and five thymidines
as a RNA polymerase III transcriptional stop signal were synthesized.
The sequences were forward 5´-GCAGCAGCTGAACAACATGTTCAAGAGACATGTTGT-TCAGCTGCTGCTTTTTT3´
and reverse 5´ AATTAAAAA-AGCAGCAGCTGAACAACATGTCTCTTGAACATGTTG-TTCAGCTGCTGCGGCC3´
(locate on SH2 domain). The oligo nucleotides were annealed in a
buffer (potassium actate 100 mmol/L , 30 mmol/L HEPES-KOH pH 7.4,
and magnesium acetate 2 mmol/L ) and the mixture was incubated at
90 ºC for 3 min and then at 37 ºC for 1 h. The double
stranded oligos were cloned into the ApaI-EcoR I sites
of the pSilencer 1.0-U6 vector (Ambion Inc) where short hairpin
RNAs (shRNA) were expressed under the control of the U6 promoter.
Cell culture and transfections The human laryngeal
cancer cell lines Hep2 were obtained from ATCC. Hep2 cells were
cultured in medium RPMI-1640 (Invitrogen, Inc Carlsbad, CA, USA)
supplemented with 10% fetal bovine serum (FBS) and penicillin (100
kU/L) and streptomycin (100 mg/L) at 37 ºC in a humidified
incubator with 5% CO2. For cell transfection, lipofectamine
2000 (Invitrogen) was used for transfecting the plasmids following
the manufacturer instructions. In brief, pEGFP was cotransfected
with pSilencer1.0-U6-siRNA-STAT3 or pSilencer empty vector at ratio
of 1:20 to mark the positive transfected cells, respectively. The
cells were cultured for 5-20 h and then transferred to fresh medium
with 10% FBS and lysed for 24-72 h after transfection.
Northern blot Total RNA was extracted from cell samples
with Trizol (Invitrogen, Carlsbad, CA, USA). Equal amounts of 20
µg total RNA were electrophoresed on 1.2% agarose gel with
formaldehyde 2.2 mol/L and transferred onto nylon membranes (Hybond-N,
Amersham Pharmacia Biotech). Blots were incubated with 32P-labeled
cDNA against STAT3 and actin with Hyb and washed according to the
manufacture directions. Visualization of blots was performed by
overnight exposure to Kodak MS film. Quantification of blots developed
on films was accomplished with a Molecular Dymanics phosphorimager.
Western blot Total protein was extracted from the harvested
sample cells with protein lysis buffer (5 mol/L edetic acid, 300
mmol/L NaCl, 0.1% Igepal, 0.5 mmol/L NaF, 0.5 mmol/L Na3VO4,
0.5 mmol/L PMSF, and antiprotease mixture) using sonication.
The lysates were centrifuged at 15 000×g for 30 min. Determination
of protein concentrations of the supernatants was performed by the
Bradford procedure (Bio-Rad Laboratory, Hercules, CA, USA). For
STAT3 and pTry-stat3 analysis, the supernatant with 50 µg total
protein was separated by electrophoresis on 10% SDS-Polyacrylamide
gels and transferred onto PVDF membranes (Milipore, Bedford, MA)
and blocked with 5% nonfat dry milk in PBS with 0.1% Tween-20. Blots
were incubated with specific rabbit antibodies against STAT3 and
pTry-stat3 and anti-¦Â-actin antibody (Santa Cruz Biotech, Inc, Santa
Cruz, CA, USA) and washed with TBST and subjected to corresponding
HRP-conjugated secondary antibodies as indicated. For Bcl-2 analysis,
50 mg of total protein was electrophoresed on 12% SDS/PAGE gels
and transferred onto PVDF membranes (Milipore, Bradford, MA,USA).
The membranes were probed with mouse polyclonal antibodies against
Bcl-2 antibody (Dako Biotech, Inc, Glostrup, Denmark) and washed
with TBST and subjected to corresponding HRP-conjugated secondary
antibodies as indicated. Blots were washed again with TBST and visualized
by enhanced chemiluminescence detection system (Amersham Pharmacia
Biotech, Uppsala, Sweden).
Proliferation and apoptosis assays in vitro Hep2
cells were incubated in 96-well plates. Cell proliferation was determined
by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazo-lium bromide
(MTT, Sigma) assay whereby cell numbers were counted by hemocytometer
72h after transfection. The absorbance values at 570 nm (A570)
were determined on a multiwell plate reader. Cell growth inhibition
rate was calculated according to the following formula:
Growth inhibition rate (%)=[(A570c-A570e
)/A570c]×100%
A570c: A570 in control group;
A570e: A570 in experimental
group.
For FACS analysis of apoptosis, Hep2 cells were transfected with
siRNA-STAT3 or pSilencer empty vector. After 72 h, cells were collected
and washed with cold PBS containing edetic acid 4 mmol/L. Cells
were fixed in 70% cold ethanol, collected by centrifugation, and
washed once again with PBS containing edetic acid 4 mmol/L. Cells
were resuspended in PBS containing edetic acid 4 mmol/L, 20 mL/L
of propidium iodide (Sigma), 0.2% Triton X-100, 40 mg/L RNase A,
and incubated for at least 30 min at 4 ºC. The cells were then
analyzed by flow cytometry (FACScan, Becton Dickinson, Franklin
Lakes, NJ, USA), using Cell Quest software. For fluoromicroscopic
determination of positive apoptosis cells, 95 µL floating cells
were mixed with 0.1% AO/EB [acridine orange (Sigma)/ethidium bromide
(Sigma)] and observed under microscope.
Statistical analysis Data were expressed as mean±SD.
The square c2 analysis was performed to evaluate the
significance of inter-group differences. Student's t test
was used for single comparison between two groups. Two-way ANOVA
using the Student-Newman-Keuls method was adopted for comparison
of variables after treatment. P<0.05 was considered significant.
All statistical calculations were performed using SigmaStat statistical
software package (SPSS10.0, Chicago, IL,USA).
Results
STAT3 RNAi by siRNA specifically reduces STAT3 expression in
Hep2 cells Since STAT3 levels are significantly higher in tumor
cells (including laryngeal tumor) than that in normal cells, we
attempted to determine whether the synthetic STAT3 siRNA could inhibit
the expression of STAT3 gene in Hep2 cells. Treatment of Hep2 cells
with siRNA-STAT3 resulted in a significant decrease of STAT3 expression
at both mRNA (Figure 1) and protein (Figure 2A) level compared to
the untreated Hep2 cells and the vector-treated Hep2 cells, respectively.
STAT3 expression was specifically targeted by STAT3 siRNA, since
STAT3 siRNA unchanged the expression of ¦Â-actin (Figure 1, 2A).
Of note is that pTyr-STAT3 was markedly expressed in untreated Hep2
cells and the vector-treated Hep2 cells (Figure 2B), indicating
that the detected STAT3 was indeed in the form of tyrosine-phosphorylated(ie,
activated) form. In the STAT3 siRNA Hep2 cells, Tyr-STAT3 expression
was significantly reduced, indicating that STAT3 siRNA also inhibited
the activity of STAT3. Furthermore, as shown by Figure 2C treatment
of Hep2 cells with STAT3 siRNA caused time-dependent inhibitory
effect on STAT3 expression in Hep2 cells.
STAT3 siRNA down-regulates Bcl-2 expression in Hep2 cells
Recent data indicate that constitutive activation of STAT3 induces
the expression of anti-apoptotic genes including Bcl-2[17].
In order to determine whether Bcl-2 was involved in the STAT3-mediated
apoptotic block in Hep2 cells, in this study Western blot analysis
was performed. Figure 3 showed that Bcl-2 was remarkably expressed
in the untreated Hep2 cells and the vector-treated Hep2 cells, whereas
treatment of Hep2 cells with STAT3 siRNA significantly reduced the
expression levels.
STAT3 siRNA inhibits growth and survival of Hep2 cells and induces
apoptosis of Hep2 cells in vitro To determine whether
synthetic STAT3 siRNA had an inhibitory effect on Hep2 cells growths,
we accomplished determination of cell proliferation with MTT assay.
Table 1 showed that treatment of Hep2 cells with STAT3 siRNA contributed
to dose- dependent inhibition of Hep2 cells, whereas no inhibitory
effect was observed in the untreated Hep2 cells and the vector-treated
Hep2 cells.
Two different methods including flow cytometry analysis and AO/EB
staining (nucleus condensation) were adopted for determination of
apoptosis in Hep2 cells (Table 2 and Figure 4). Flow cytometry analysis
showed that in the STAT3 siRNA treated cells the apoptosis rate
was significantly higher than that in the untreated Hep2 cells and
the vector-treated Hep2 cells, whereas the apoptosis rate in the
vector treated Hep2 was slightly higher than that in the untreated
Hep2 cells but no significance was achieved (Table 2), indicating
that siRNA-STAT3 induced apoptosis in Hep2 cells. Moreover, AO/EB
staining revealed that both early apoptotic and late apoptotic Hep2
cells were seen in the cells treated with STAT3 siRNA, suggesting
that STAT3 siRNA had not only an effect on induction of early apoptosis
but also late apoptosis in Hep2 cells (Figure 3).
Discussion
Constitutively activated STAT3 is critical to STAT3 signaling pathway-dependent
mechanism of malignancies[7,8,16]. A possible mechanism
underlying transformation by activated STAT3 is the transcriptional
upregulation of genes known to be involved in proliferation and
apoptosis in laryngeal carcinomas and other malignant carcinomas.
Under physiological conditions, STAT3 activation is transient and
lasts from several minutes to several hours due to the transient
nature of cytokine and growth factor signaling and the presence
of proteins such as suppressor of cytokine signaling (SOCS ) and
PIAS (STAT blockade) that contribute to inhibition of STAT3 signaling.
However, constitutive activation of protein tyrosine kinase (PTKs)
occurs frequently during tumorigenesis due to activated mutations
or aberrant growth factor or cytokine signaling, which results in
constitutive activation of STAT3. Therefore, it is not surprising
that constitutive activation of STAT3 is ubiquitous in human carcinomas[3,17,18].
In most instances, STAT3 that is capable of regulating growth, differentiation,
and survival of cells is characterized by growth promoting manner[19-21].
At present, STAT3 has been recognized as an important onco-gene[2,21].
STAT3 is implicated in both embryogenesis and tummorigenesis. This
may raise the question as to whether blocking STAT3 is beneficial
for malignant carcinoma cells but harmful to normal cells. Takeda
et al revealed that homozygous deletion of STAT3 was embryonically
lethal[22]. Biochemical studies have shown that disruption
of STAT3 signaling with dominant-negative approaches in murine fibroblasts
does not inhibit normal cell growth[23,24]. Also, blockade
of STAT3 signaling by decoy oligonucleotides in head and neck cancer
cells only decreases the amount of STAT3 in normal cell but has
no significant effect on cell viability.
Grandis et al[15] have reported that constitutive
activation of STAT3 signaling abrogates apoptosis in squamous cell
carcinogenesis in vivo. Treatment of tumor cells with inhibitors
of STAT signaling results in decreased cell viability and induces
apoptosis. Accumulated evidence has suggested that apoptotic regulatory
proteins are implicated in STAT3 associated-apoptosis inhibition.
It has been demonstrated that STAT3 regulates transcription from
the Bcl-x promoter. Moreover, elevated expression levels of Bcl-xL
mRNA in cells transformed by constitutively active STAT3 are
observed[25].
Although the recently established approach of applying RNAi in
mammalian studies by introducing siRNA can effectively inhibit gene
expression[14,26,27], siRNA at present has been adopted
as a promising genomic tool[17]. Such an
approach overcomes many of the shortcomings previously experienced
with approaches such as antibodies, antisense oligonucleotides,
and pharmacological inhibitors. Intriguingly, RNAi targeting STAT3
by siRNA inhibits growth and induces apoptosis of prostate cancer
and astrocytoma cells have been reported from different groups,
however to date no one has known the effect of STAT3 siRNA on laryngeal
cancer. Consequently, we have attempted to investigate the potential
use of siRNA to block the expression of gene encoding STAT3 in laryngeal
cancer.
To our knowledge we have successfully determined the inhibitory
effect of the synthetic STAT3 siRNA on STAT3 expression in Hep2
cells at both mRNA and protein level (Figure 1, 2). We found that
STAT3 was remarkably expressed in Hep2 cells, and treating
Hep2 cells with STAT3 siRNA significantly reduced STAT3 expression
levels characterized by a time-dependent inhibition of the gene
expression. Increased STAT3 activation can occur through potential
pathway of elevated constitutive levels of STAT3 protein and increased
STAT3 tyrosine phosphorylation. In this study, the use of specific
antibody of pTyr-STAT3 reveals that STAT3 siRNA inhibits not only
STAT3 expression but also the activities of STAT3 in Hep2 cells
(Figure 2), thereby confirming that the synthetic STAT3 siRNA can
effectively inhibit STAT3 gene expressions in human laryngeal carcinoma
cells. Our results are consistent with others who have shown the
potent inhibitory effect of STAT3 siRNA on STAT3 expression in the
models of prostate cancer and astrocytoma cell lines[16,17].
Moreover, other studies have shown that ubiquitous expression of
STAT3 gene exhibits in a variety of human tumors and can be effectively
inhibited by the introduction of STAT3 inhibitors, dominant negative
STAT3, and/or blockade of tyrosine kinases besides STAT3 siRNA whereby
treatment of tumors is achieved.
Recent data indicate that constitutive activation of STAT3 induces
expression of anti-apoptotic genes including Bcl-2[17].
In the present study, to determine whether STAT3-mediated
cell apoptosis in Hep2 cells exists, Western blot analysis was used
for the measurement of Bcl-2 protein expression. Additionally, quantification
of apoptotic cells by flow cytometry was also performed in this
study. We found that apoptosis in Hep2 cells was arrested as evidenced
by low percentages of apoptotic cells (0.42%±0.01% and 2.87%±1.67%)
and remarkable expression levels of Bcl-2 protein, whereas treatment
of Hep2 cells with STAT3 siRNA significantly increased the number
of apoptotic cells (18.6%±4.3%, P<0.05) and decreased
Bcl-2 protein expression levels (Table 2 and Figure 3). This suggests
that STAT3 gene expression is an important implication in the regulation
of apoptosis in Hep2 cells. Furthermore, visualization of nucleus
condensation using AO/EB dyes suggests that the synthetic STAT3
siRNA has an effect on both early and late apoptosis. Our study
is consistent with two recent intriguing reports where STAT3 siRNA
was also adopted for the study of astrocytomas and human prostate
cancer[16,17]. All are supportive for the proposed mechanism
underlying STAT3 participating in oncogenesis is by inhibiting apoptosis
through the induction of anti-apoptotic genes. Konnikova et al[17]
have demonstrated that STAT3 is required for the expression of the
anti-apoptotic genes survivin and Bcl-xL (a member of
the Bcl-2 family of proteins) in astrocytoma cells. Likewise, Lee
et al[16] have also shown that inhibition of STAT3
gene expression by siRNA induces apoptosis of human prostate cancer.
Moreover, emerging evidence suggests that constitutive activation
of STAT3 appears to be ubiquitous in tumors, which renders tumors
cells resistant to apoptotic death caused by unbalanced expression
level between anti-apoptotic genes and apoptotic genes[15,17,25].
Consistent with this, our recent work also has shown that human
breast cancer cells implanted into nude mice exhibit remarkable
expression of anti-apoptotic genes Bcl-2 but weak
expression of apoptotic gene Bax accompanied by over-expression
of STAT3 gene (Data not shown). The context is altered by
treatment of the breast cancer with STAT3 siRNA characterized by
unchanged expression of Bcl-2 genes, increased expression
of apoptotic Bax gene, and significant inhibition of expression
of STAT3 gene, and suppression of tumor growth (Data not
shown).
siRNA has been effectively used in vivo to suppress gene
expression in rats and adult mice whereby it achieves effective
treatment of various organ and/or tissue disorders, including hepatitis,
liver ischemia-reperfusion injury, allogeneic transplanted hepatocytes
rejection, and CNS disorders[27-29]. This study also
represents the first report that the synthetic STAT3 siRNA effectively
suppresses Hep2 cells growths as evidenced by elevated inhibitory
rate of Hep2 cell by STAT3 siRNA transfection (Table 1). In this
study, an unexpected finding is that treatment of Hep2 cells with
STAT3 siRNA is dose-dependent. Our results are consistent with other's
reports that STAT3 has the potential as a promising therapeutic
molecular target in tumors including laryngeal cancer, astrocytomas,
and human prostate cancer, thereby extrapolating that STAT3 siRNA
may represent a novel approach in tumor gene therapy.
Strategies for producing siRNA duplexes include direct chemical
synthesis, transcription with T7 promoter in vitro and recombinant
DNA construction by vector with U6 promoter. Our results demonstrate
that pSilencer1.0-U6 STAT3 siRNA can result in a long-term target-gene
inhibition in Hep2 cells leading to growth suppression and induction
of apoptosis in Hep2 cells. The STAT3 signaling pathway has been
shown to be critical for the survival of a number of human tumors.
This therefore raises the possibility that STAT3 siRNA could become
an effective therapeutic agent for STAT3-dependent tumors.
Conclusion
This study represents the first report that demonstrates that STAT3
siRNA effectively inhibits STAT3 gene expression in Hep2 cells leading
to growth suppression and induction of apoptosis in Hep2 cells.
The use of siRNA technique may provide a novel therapeutic approach
to treatment of laryngeal cancer and other malignant tumors expressing
constitutively activated STAT3.
Acknowledgments
We thank Dr Bao-xue YANG from UCLA, CA, USA, for Northern blot
analysis and Dr Yan MENG in the Department of Pathophysiology at
the Basic School of Medicine of Jilin University for her assistance
in siRNA design.
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