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Signal transducers and activators of transcription (STATs) were originally identified as key components of the
cytokine signaling pathways that regulate gene
expression[1,2]. Recent studies suggest that they have potential as novel molecular
targets for control of the development and survival of laryngeal carcinomas. In mammals, there are 7 members in the
stat family. Constitutive activation of one
stat family member, stat3, has been shown to play a key role in promoting proliferation,
differentiation, anti-apoptosis and cell cycle progression. Constitutive activation of
stat3 occurs in a variety of tumor cell
lines[3_7], thus suggesting that
stat3 is an important molecular target for tumor therapy. Constitutive
stat3 signaling represents one of the key molecular events in the multistep process leading to carcinogenesis.
Several recent reports demonstrate that blockade of
stat3 expression in human cancer cells suppresses proliferation
in vitro and tumorigenicity in viv
o. Attempts to block stat3 expression have been made using tyrosine kinase
inhibitors[8,9], antisense
oligonucleotides[5], decoy
oligonucleotides[10], dominant-negative
stat3 protein[11,12] and RNA interference
(RNAi)[13,14]. In vitro studies have shown that inhibition of
stat3 activity in human tumor cells induced apoptosis and/or growth
arrest. In human head and neck squamous carcinoma cells, blocking of
stat3 signaling by decoy oligonucleotides or
antisense oligonucleotides abrogates transforming growth factor and suppresses the oncogenic growth of these
cells[15,16].
In the RNAi approach, sequence-specific post-transcriptional gene silencing is achieved by small interfering RNA (siRNA):
short double-stranded RNA molecules in which the antisense strand is complementary to the target mRNA of a given
gene[17,18]. RNAi technology is currently being used not only as a powerful tool for analyzing gene function, but also for
developing highly specific therapeutics. Our previous studies have demonstrated that blockade of
stat3 expression by siRNA in Hep2 human laryngeal tumor cells suppresses proliferation and induces apoptosis
in vitro[19]. However, it has not
been determined whether blocking stat3 signaling with siRNA is sufficient to inhibit tumor growth
in vivo.
In the present study, we used a DNA-vector-based
stat3-specific RNAi approach to block
stat3 signaling and to evaluate the biological consequences of
stat3 downmodulation on tumor growth in a mouse model. The results indicate that blockade
of stat3 expression using a specific RNAi approach can significantly reduce laryngeal tumor growth and induce apoptosis
in vivo.
Material and methods
Plasmid construction pSilencer1.0-U6 (Ambion, Austin, TX, USA) was used for DNA vector-based siRNA synthesis
under the control of the U6 promoter in
vivo. The vector was constructed by first synthesizing the double-stranded DNA
template encoding the siRNA oligonucleotides (GenBank accession number for human
stat3: NM003150), which contained a sense strand of 19 nucleotides followed by a short space (TTCAAGAGA), then the reverse complement of the sense strand,
followed by five thymidines as a RNA polymerase III transcriptional stop signal. The sequences were: forward
5¡¯-GCAGCAGCTGAACAAC ATGTTCAAGAGA-CATGTTGTTCAGCTGCTGCTTTTTT-3¡¯ and reverse
5¡¯-AATTAAAAAAGCAGCAGCTGAACAACATGTCTCTTGAA-CATGTTGTTCAGCT GCTGCGGCC-3¡¯ (located in the SH2
domain). The oligonucleotides were annealed in a buffer [100 mmol/L potassium acetate, 30 mmol/L
N-2-hydroxy-ethylpiperazine-N¡¯-2-ethanesulfonic acid (HEPES)-KOH (pH 7.4), and magnesium acetate 2 mmol/L] and the mixture was
incubated at 90 °C for 3 min and then at 37 °C for 1 h. The double-stranded oligonucleotides were cloned into a
ApaI-EcoRI site in the pSilencer 1.0-U6 vector (Ambion), in which short hairpin RNAs (shRNA) were expressed under the control of the
U6 promoter. A negative control scrambled siRNA (Ambion), which had no significant homology to mouse or human gene
sequences, was designed to detect any non-specific effects.
Cell culture and establishment of animal model
Hep2 cells (2×106/150 µL) were inoculated subcutaneously into the right
flanks of nude mice, and establishment of palpable tumors was confirmed. The tumor volume
(m12×m2×0.5236, where
m1 represents the short axis, and
m2 the longer axis) was measured every 2_3 d. When tumors reached an average volume of
~50.69±11.25 mm3, 3 experimental groups (5 mice per group) were tested: (1) mock transfection (phosphate-buffered saline [PBS]
buffer alone); (2) scrambled siRNA control (20 µg/mouse); and (3) pSilencer1.0-U6-STAT3-3 siRNA (20 µg/mouse). The
samples were diluted in 50 µL of PBS buffer, and injected percutaneously into the tumor by using a syringe with a 27-gauge
needle. Immediately after injection, tumors were pulsed with an electroporation generator (ECM 830, BTX Holliston, MA,
USA). Pulses were delivered at a frequency of 1/s 150 V/cm for a duration of 50 ms. This process was repeated on day 20.
Mice were killed on d 27, the tumors treated with either scrambled siRNA or STAT3 siRNA were excised for hematoxylin and
eosin (HE) staining, and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and
fluorescence-activated cell sorting (FACS) assays.
HE staining and TUNEL assays Serial sections of tumor tissue excised from animals were fixed in formalin, stained with
HE, and processed for routine histological examination. The TUNEL assay was performed by using the
in situ Cell Death Detection Kit (Roche), which relies on fluorescent labeling of DNA strand breaks. Three-micrometer sections from
paraffin-embedded tissues were dewaxed and hydrated according to the standard protocol. After incubation with proteinase K (200
µg/mL) for 30 min at 21 °C, the TUNEL reaction mix containing BrdUTP, terminal deoxynucleotidyl transferase, and reaction
buffer was added to the slides, and they were incubated in a humidified chamber for 60 s at
37 °C, followed by washing and incubation with a fluorescein isothiocyanate-labeled anti-BrdU monoclonal antibody for 30 min at room temperature. The
reaction was visualized by fluorescence microscopy. TUNEL-positive cells exhibited green fluorescence.
Western blot analysis Anti-stat3, anti-phospho-Tyr705-stat3 (p-stat3), anti-cyclin D1, anti-survivin and
anti-b-actin antibodies were obtained from Santa Cruz Biotech. Anti-Bcl-2 antibody was obtained from Dako Biotech. For Western blot
analyses, 100 mg tumor tissue as described earlier was lysed with lysis buffer [5 mmol/L ethylenediamine tetraacetic acid
(EDTA), 300 mmol/L NaCl, 0.1% Igepal, 0.5 mmol/L NaF, 0.5 mmol/L
Na3VO4, 0.5 mmol/L phenylmethyl-sulfonyl fluoride, and
10 µg/mL each of aprotinin, pepstatin, and leupeptin; Sigma]. After centrifugation at 15
000×g for 30 min, the supernatant was analyzed for protein content using Bradford reagent (Bio-Rad, USA). For the analysis of
stat3, 50 µg of total protein was electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred onto a
PVDF membrane (Milli-pore, Bedford, MA, USA), and incubated with anti-STAT3 or
anti-p-stat3 antibody as indicated earlier. For
Bcl-2, 50 µg of total protein was resolved on a 12% SDS-PAGE gel, transferred onto PVDF membranes and then
probed with anti-Bcl-2 antibody. For cyclin D1, 50 µg of total protein was resolved on a 10% SDS-PAGE gel, transferred onto
PVDF membranes and then probed with anti-cyclin D1 antibody. For survivin, 50 µg of total protein was resolved on a 12%
SDS-PAGE gel, transferred onto PVDF membranes and then probed with anti-survivin antibody. The immunoblots were
visualized by using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, USA). The optical
density of each band in these western blots was measured by using densitometry and the results are given as the relative
expression of tumors versus normal tissue.
Northern blot analysis Total RNA was extracted from tissue with the Trizol reagent (Invitrogen) following the
manufacturer¡¯s instructions. For Northern blot analysis, 20 µg of total RNA was electrophoresed on a 1.2%
agarose-formaldehyde gel, and blotted onto
Hybond-N+ membranes (Amersham Pharmacia Biotech). Hybridization was performed
using the express Hyb buffer (BD Clontech) with
32P-labeled cDNA of survivin and
actin as probes. Blots were exposed to Kodak MS film and then quantitated using a Molecular Dynamics PhosphorImager.
Statistical analysis A c2 -analysis was performed to evaluate the significance of differences between the experimental
groups. For a single comparison of two groups, Student¡¯s
t-test was used. Two-way ANOVA using the
Student-Newman-Keuls method was used for comparisons of tumor size in mice after different treatments. For all analyses, the level of
significance was set at P<0.05. All statistical calculations were performed using the SigmaStat statistical software package
(SPSS, Chicago, IL). Data are presented as the Mean±SD.
Results
Antitumor activity of stat3 siRNA In order to evaluate the effects of the
stat3 siRNA vector on laryngeal tumor growth
in vivo, we examined its antitumor efficacy using a nude mouse model. Mice were subcutaneously inoculated with
2×106 Hep2 cells into their right flank. By d 13, palpable tumors had developed at the sites of injection (mean volume 50.69±11.25
mm3, n=5). The mice were divided into 3 groups of 5 mice each and injected intratumorally with TE Tris EDTA buffer,
scrambled-siRNA control or pSilencer1.0-U6-STAT3 siRNA. This process was repeated on d 20. Animals were killed on d 27
and tumor sizes were determined. The mean tumor volume in mice treated with buffer alone was 918.12±89.03
mm3 on d 27. The mean tumor volume in mice treated with scrambled siRNA control was 896.42±92.23
mm3, and that in the group treated with
stat3-siRNA was 306.24±28.13 mm3. The difference in tumor size between the mice treated with buffer and those treated
with siRNA did not achieve statistical significance
(P>0.05), whereas the group treated with
stat3 siRNA showed markedly suppressed tumor growth compared with the controls (Figure 1A, 1B;
P<0.01). To
determine the mechanism of tumor growth inhibition
in vivo, Hep2 tumors treated with either scrambled siRNA control or
stat3 siRNA were excised for HE staining and analyzed by using TUNEL assay. The results from both experiments showed
that stat3 siRNA-treated tumors had undergone massive apoptosis compared with the controls (Figure 1C_1H). These data
suggest that stat3 siRNA injection into the tumor can exert significant antitumor effects.
Reduction of stat3 expression siRNA-specific to the
stat3 gene can significantly suppress stat3 protein expression and
inhibit the growth of cells in
vitro[19]. To further study the molecular mechanism of growth arrest of the
tumor in vivo, stat3 and p-stat3 expression in the tumors were analyzed by Western blotting, and the results indicate that stat3
and p-stat3 expression are markedly reduced in tumors treated with siRNA stat3, whereas scrambled and buffer groups had
high levels of stat3 and p-stat3 expression
(P<0.01; Figure 2A, 2B).
Effects of downregulation of stat3 expression
Recent studies[20_27] indicate that a constitutively active stat3 induces the
expression of anti-apoptotic genes such as
Bcl-2, cyclin D1, and
survivin. In order to determine if
stat3 downregulation results in the suppression of these genes, Western blot and Northern blot analyses were performed on the extracts from
tumors transfected with stat3 siRNA. Western blotting showed that the intracellular Bcl-2, cyclin D1, and survivin levels
were significantly decreased in stat3 siRNA-transfected tumors compared with controls (Figure 2C_E). Northern blot
analysis showed that intracellular survivin mRNA was significantly decreased in the tumors (Figure 3). Thus, we concluded that
stat3 siRNA treatment downregulated the expression of Bcl-2, cyclin D1, and survivin.
Discussion
Laryngeal carcinoma, especially late-stage laryngeal carcinoma, is associated with high morbidity and poor long-term
survival because of the absence of effective treatment methods. Current therapies for advanced laryngeal cancer are only
marginally effective. Thus, better understanding of the molecular mechanisms underlying proliferation, differentiation and
survival of laryngeal carcinoma is critical for the development of optimal therapeutic methodologies.
Elevated stat3 activities have been detected in the primary tissues and cell lines of laryngeal tumors.
Stat3 activates several genes whose products promote cell cycle progres-sion, for example
cyclin D1, and
c-Myc[20_22], and prevent apoptosis, for
example Bcl-2 and
Bcl-XL[23_27]. Our previous
studies showed that stat3 plays a key role in promoting
laryngeal tumor proliferation in vitro. In the present study, we further demonstrated that STAT3 played a key role in
promoting laryngeal tumor proliferation in
vivo.
Western blot analysis with anti-STAT3 or anti-phospho-STAT3 antibodies showed
that stat3 siRNAs suppress stat3 expression in laryngeal tumors
in vivo. The expression of p-stat3 in laryngeal tumors treated with
stat3 siRNAs declined approximately 90%, indicating a good silencing efficiency (Figure 2B). More importantly, direct inhibition of
stat3 signaling was accompanied by growth inhibition and induction of apoptosis in laryngeal tumors. We observed that Bcl-2, cyclin D1,
and survivin expression were greatly diminished in tumors transfected with
stat3 siRNA (Figures 2B, 2C, 3). Additionally,
massive apoptosis of the tumor cells were detected by TUNEL and HE assays. The results of our study are consistent with
those of two recent reports, in which
stat3 siRNA was also used for the study of astrocytomas and human prostate
cancer[13,28]. The results of all 3 studies support the hypothesis that
stat3 participates in oncogenesis is by inhibiting apoptosis
through the induction of anti-apoptotic genes. Konnikova
et al reported that stat3 was required for the survival of the
anti-apoptotic genes survivin and Bcl-xL (a member of the Bcl-2 family of proteins) in astrocytoma
cells[14]. Likewise, Lee et al also
showed that inhibition of stat3 gene expression by siRNA induces apo-ptosis in human prostate
cancer[13]. Moreover, emerging evidence suggests that constitutive activation of
stat3 appears to be ubiquitous in tumors, which renders tumor
cells resistant to apoptotic death by unbalancing the expression levels of anti-apoptotic and apoptotic
genes[13,28].
Chemical synthesis of siRNAs is not cost-effective for large-scale screening projects, and simple synthetic siRNAs are
unstable in mammalian cells, especially for use in
in vivo studies. Fortunately, this problem has been addressed by using
plasmid expression vectors as a delivery
tool[11,29,30]. These vector systems produce stable amounts of siRNA by utilizing the
cellular machinery[31]. Mammalian expression vectors synthesizing siRNA-like transcripts are able to cause gene
knockdown[32]. In order to evaluate the effects of stat3 siRNA on
in vivo laryngeal tumor growth, we examined the antitumor efficacy of
STAT3 siRNA in a nude mouse tumor model. We discovered that inhibition of
stat3 by administration of appropriate
vector-based siRNAs into the tumor was an effective and feasible approach for laryngeal cancer therapy. In this study, we used
DNA injection as a tool for suppressing
stat3 and tumor growth. Further efforts to evaluate the therapeutic value of this
promising approach should be followed.
In conclusion, the data presented here show that the blockade of
stat3 signaling using the RNAi approach significantly
suppressed stat3 expression in vivo, suggesting that
stat3 signaling is a potential molecular target for laryngeal cancer
therapy. Plasmid-based siRNA therapy for tumor suppression may offer an effective and inexpensive approach for the
treatment of laryngeal tumors.
Acknowledgement
We thank Dr Su-qin PAN for providing valuable technical support.
References
1 Darnell JE Jr. STATs and gene regulation. Science 1997; 277: 1630_35.
2 Bromberg J, Darnell JE Jr. The role of STATs in transcriptional control and their impact on cellular function. Oncogene 2000; 19:
2468_73.
3 Garcia R, Yu CL, Hudnall A, Catlett R, Nelson KL, Smithgall T,
et al. Constitutive activation of STAT3 in fibroblasts transformed by
diverse oncoproteins and in breast carcinoma cells. Cell Growth Differ 1997; 8: 1267_76.
4 Garcia R, Bowman TL, Niu G, Yu H, Minton S, Muro-Cacho CA,
et al. Constitutive activation of STAT3 by the Src and JAK tyrosine
kinases participates in growth regulation of human breast carcinoma cells. Oncogene 2001; 20: 2499_513.
5 Grandis JR, Drenning SD, Chakraborty A, Zhou MY, Zeng Q, Pitt AS,
et al. Requirement of STAT3 but not stat1 activation for epidermal
growth factor receptor-mediated cell growth in
vitro. J Clin Invest 1998; 102: 1385_92.
6 Takemoto S, Mulloy JC, Cereseto A, Migone TS, Patel BK, Matsuoka M,
et al. Proliferation of adult T cell leukemia/lymphoma cells is
associated with the constitutive activation of JAK/STAT proteins. Proc Natl Acad Sci USA 1997; 94: 13897_902.
7 Gouilleux-Gruart V, Gouilleux F, Desaint C, Claisse JF, Capiod JC, Delobel J,
et al. STAT-related transcription factors are constitutively
activated in peripheral blood cells from acute leukemia patients. Blood 1996; 87: 1692_7.
8 Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z,
et al. Inhibition of acute lymphoblastic leukaemia by a jak-2 inhibitor.
Nature 1996; 379: 645_48.
9 Blaskovich MA, Sun J, Cantor A, Turkson J, Jove R, Sebti SM,
et al. Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal
transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer
cells in mice. Cancer Res 2003; 63: 1270_9.
10 Leong PL, Andrews GA, Johnson DE, Dyer KF, Xi S, Mai
JC, et al. Targeted inhibition of STAT3 with a decoy oligonucleotide abrogates
head and neck cancer cell growth. Proc Natl Acad Sci USA 2003; 100: 4138_43.
11 Ni Z, Lou W, Leman ES, Gao AC. Inhibition of constitutively activated STAT3 signaling pathway suppresses growth of prostate cancer
cells. Cancer Res 2000; 60: 1225_8.
12 Nakajima K, Yamanaka Y, Nakae K, Kojima H, Ichiba M, Kiuchi N,
et al. A central role for STAT3 in IL-6-induced regulation of growth
and differentiation in M1 leukemia cells. Embo J 1996; 15: 3651_8.
13 Lee SO, Lou W, Qureshi KM, Mehraein-Ghomi F, Trump DL, Gao AC. RNA interference targeting STAT3 inhibits growth and induces
apoptosis of human prostate cancer cells. Prostate 2004; 60: 303_9.
14 Konnikova L, Kotecki M, Kruger MM, Cochran BH. Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells.
BMC Cancer 2003; 3: 23.
15 Leong PL, Andrews GA, Johnson DE, Dyer KF, Xi SC , Mai JC,
et al. Targeted inhibition of STAT3 with a decoy oligonucleotide abrogates
head and neck cancer cell growth. Proc Natl Acad Sci USA 2003; 100: 4138_43.
16 Mora LB, Buettner R, Seigne J, Diaz J, Ahmad N, Garcia R,
et al. Constitutive activation of STAT3 in human prostate tumors and cell lines:
direct inhibition of STAT3 signaling induces apoptosis of prostate cancer cells. Cancer Res 2002; 62: 6659_66.
17 Fuchs B, Zhang K, Schabel A, Bolander ME, Sarkar G. Identification of twenty-two candidate markers for human osteogenic sarcoma.
Gene 2001; 278: 245_52.
18 Grandis JR, Zheng Q, Drenning SD. Epidermal growth factor receptor-mediated STAT3 signaling blocks apoptosis in head and neck cancer.
Laryngoscope 2000; 110: 868_74.
19 Gao LF, Xu DQ, Wen LJ, Zhang XY, Shao YT, Zhao
XJ. Inhibition of STAT3 expression by siRNA suppresses growth and induces
apoptosis in laryngeal cancer cells. Acta Pharmacol Sin 2005; 26: 377_383
20 Masuda M, Suzui M, Yasumatu R, Nakashima T, Kuratomi Y, Azuma K,
et al. Constitutive activation of signal transducers and activators
of transcription 3 correlates with cyclin D1 overexpression and may provide a novel prognostic marker in head and neck squamous cell
carcinoma. Cancer Res 2002; 62: 3351_5.
21 Sinibaldi D, Wharton W, Turkson J, Bowman T, Pledger WJ, Jove R. Induction of Survivin WAF1/CIP1 and cyclin D1 expression by the
Src oncoprotein in mouse fibroblasts: role of activated STAT3 signaling. Oncogene 2000; 19: 5419_27.
22 Hannon GJ. RNA interference. Nature 2002; 418: 244_51.
23 Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C,
et al. STAT3 as an oncogene. Cell 1999; 98: 295_303.
24 Alas S, Bonavida B. Rituximab inactivates signal transducer and activation of transcription 3 (STAT3) activity in B-non-Hodgkin's
lymphoma through inhibition of the interleukin 10 autocrine/paracrine loop and results in down-regulation of Bcl-2 and sensitization to
cytotoxic drugs. Cancer Res 2001; 61: 5137_44.
25 Puthier D, Bataille R, Amiot M. IL-6 up-regulates mcl-1 in human myeloma cells through JAK/STAT rather than ras/MAP kinase
pathway. Eur J Immunol 1999; 29: 3945_50.
26 Aoki Y, Feldman GM, Tosato G. Inhibition of STAT3 signaling induces apoptosis and decreases surviving expression in primary effusion
lymphoma. Blood 2003; 101: 1535_42.
27 Tsujimoto Y, Shimizu S. Bcl-2 family: life-or-death switch. FEBS Lett 2000; 466: 6_10.
28 Scott S, Higdon R, Beckett L, Shi XB, deVere White RW, Earle JD,
et al. Bcl 2 antisense reduces prostate cancer cell survival following
irradiation. Cancer Biother Radiopharm 2002; 17: 647_56.
29 Far KR, Sczakiel G. The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with
antisense oligonucleotides. Nucleic Acids Res 2003; 31: 4417_24.
30 Elbashir SM, Harborth J, Weber K, Tuschl T,
et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs.
Methods 2002; 26: 199_213.
31 Dechow TN, Pedranzini L, Leitch A, Leslie K, Gerald WL, Linkov I,
et al. Requirement of matrix metalloproteinase-9 for the
transformation of human mammary epithelial cells by STAT3-C. Proc Natl Acad Sci USA 2004; 101: 10602_7.
32 Lou KQ, Chang DC. The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region.
Biochem Biophys Res Commun 2004; 318: 303_10.
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