Therapeutic effects of STAT3 decoy oligodeoxynucleotide on human lung cancer in xenograft mice
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Signal transducer and activator of transcription 3 (STAT3) is usually constitutively activated in a variety of malignancies. Therefore, STAT3 may be a promising target for treatment of tumor cells. To explore the possibility of a double-stranded decoy oligodeoxynucleotide (ODN) targeted blocking STAT3 over-activated tumor cells, we, here, evaluate the efficacy of STAT3 decoy ODN on human lung cancer cells in vitro and in vivo.
A STAT3 decoy ODN was transfected into A549 lung cancer cell line in vitro by using lipofectamine. The flow cytometry and fluorescent microscopy were used to detect the transfection efficiency and the sub-cellular localization of STAT3 decoy ODN in A549 cells. Cell proliferation was determined by counting cell numbers and [3H]-thymidine uptake. Cell apoptosis was examined with Annexin V and propidum iodide by flow cytometry. The expression levels of STAT3 target genes were identified by RT-PCR and immunoblot. For in vivo experiment, A549 lung carcinoma-nude mice xenograft was used as a model to examine the effect of the STAT3 decoy by intratumoral injection. At the end of treatment, TUNEL and immunohistochemistry were used to examine the apoptosis and the expression levels of bcl-xl and cyclin D1 in tumor tissues.
STAT3 decoy ODN was effectively transfected into A549 lung cancer cells and mainly located in nucleus. STAT3-decoy ODN significantly induced apoptosis and reduced [3H]-thymidine incorporation of A549 cells as well as down-regulated STAT3-target genes in vitro. STAT3 decoy ODN also dramatically inhibited the lung tumor growth in xenografted nude mice and decreased gene expression of bcl-xl and cyclin D1.
STAT3 decoy ODN significantly suppressed lung cancer cells in vitro and in vivo, indicating that STAT3 decoy ODN may be a potential therapeutic approach for treatment of lung cancer.
KeywordsLung Cancer Cell A549 Cell Lung Cancer Cell Line Human Lung Cancer Cell A549 Lung Cancer Cell
signal transducer and activator of transcription
transcription factor decoy
vascular endothelial growth factor
transforming growth factor-β.
The signal transducers and activators of transcription 3 (STAT3), a member of a transcription factor family of seven proteins (STAT1, 2, 3, 4, 5a, 5b, and 6), plays important roles in regulating cell growth, differentiation, apoptosis, angiogenesis, and immune responses [1, 2]. STAT3 transduces extracellular signals made by numerous cytokines and growth factors to nucleus and directly regulate gene transcription . Cytokines (e.g. interleukin-6) and growth factors (e.g. epidermal growth factor) may bind to their cognate receptors and then activate tyrosine kinases, which phosphorylates the tyrosine residue of STAT3. Upon activation, STAT3 dimers translocate into the nucleus, where they bind to STAT3 specific DNA response elements and activate their transcription [4, 5].
Dysregulation and constitutive activation of STAT3 have been found in numerous primary cancers such as lymphomas, leukemias, multiple myelomas, prostate, breast, lung, head and neck, melanoma, pancreas, ovary and gastric cancer cells [6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16]. Lung cancer is the leading cause of death in cancer in the United States. In 2006, an estimated 174,470 new cases, accounting for about 12% of cancer diagnoses and an estimated 162,460 deaths, accounting for about 29% of all cancer deaths, are expected to occur (The American Cancer Society). Constitutive activation of STAT3 correlates with cell proliferation in non-small-cell lung cancer (NSCLC) and also inhibits apoptosis [11, 17]. Restoration of suppressors of cytokine signalling-3 (SOCS-3), which is frequently silenced by hypermethylation in lung cancer cells, resulted in the down-regulation of activated STAT3, leading to induction of apoptosis and growth suppression .
The constantly activated STAT3 contributes to oncogenesis by up-regulation of genes encoding bcl-xl, bcl-2, c-myc, cyclin D1, survivin, mcl-1 [7, 8, 16, 19], and VEGF, IL-10, TGF-β et al [20, 21, 22], which can protect apoptosis, enhance cell proliferation, promote angiogenesis and evade immune surveillance . Though there are multiple oncogenic signaling pathways in each individual tumor, blockade of STAT3 signaling is often sufficient to induce growth arrest and apoptosis in many different tumors [8, 9, 10, 11, 12, 16]. Therefore, the association of STAT3 activation with tumor progression suggests that STAT3 may be an attractive molecular target for cancer therapy.
Many methods to block STAT3 activation were developed including RNA antisense, RNA interference (RNAi) and dominant negative mutants [9, 23, 24]. A double-stranded decoy oligodeoxynucleotide (dsODN) against transcription factor  is based on the competition between the endogenous cis-elements within the regulatory regions of target genes and the exogenously added molecules mimicking the specific cis-elements . Transfection of dsODN will result in the attenuation of the authentic interactions of STAT3 with their cis-elements and subsequent alteration of gene expression. This strategy has been successfully used for inhibition of STAT3 in head and neck cancer and also for inactivation of STAT6 in shifting IL-4-driven Th2 cell activity [27, 28]. Recently, we have found that STAT3 decoy ODN could inhibit cell growth of a pulmonary giant cell carcinoma cell line PG in vitro .
In the present study, we demonstrate that STAT3 decoy ODN can suppress lung cancer cells in vitro and in vivo by efficiently blocking STAT3 signaling, suggesting the therapeutic potential of STAT3 decoy ODN in treatment of human lung carcinoma.
Cell line and cell culture
The human non-small-cell-lung cancer line, A549, were grown in RPMI 1640 medium (GIBCO/BRL) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin in 5% CO2 at 37°C.
Antibodies and reagents
Anti-STAT3, anti-phospho-specific STAT3 (Tyr705, Ser727), anti-bcl-xl, anti-cyclinD1, anti-β-actin antibodies and horseradish peroxidase-conjugated second antibody were purchased from Cell Signaling Technology (New England BioLabs Inc.). Annexin V-FITC was obtained from BD Biosciences (BD PharMingen, San Diego, CA).
STAT3 decoy and scramble ODN
Phosphorothioated sense and antisense strands of STAT3 decoy or scramble ODNs were synthesized by Expedite™ Nucletic Acid Synthesis System (TaKaRa Biotechnology, Dalian). The STAT3 decoy ODN sequence was 5'-CATTTCCCGTAAATC-3', 3'-GTAAAGGGCATTTAG-5' and the scramble ODN sequence was 5'-CATCTTGCCAATATC-3', 3'-GTAGAACGGTTATAG-5' [27, 30]. The sense and antisense strands were annealed and purified by HPLC.
Transfection of ODN
Transfections were carried out by lipofectamine 2000 (Invitrogen life technologies, Carlsbad, CA) according to the manufacturer's instructions. Briefly, cells were seeded at a density of 7 × 103 cells/well (400 μl) in 48 well plates (Costar, Corning, NY) for 24 h. After washing once with PBS, cells were transfected with lipofectamine 2000/control, lipofectamine 2000/decoy ODN, or lipofectamine 2000/scramble ODN complexes. The final concentration of ODN was 50, 25 and 12.5 nmol/L separately.
Flow cytometry and fluorescent microscope
The decoy and scramble ODN were labeled with FITC at 5' of ODNs (TaKaRa Biotechnology, Dalian). After transfection of FITC-ODN, cells were detached, washed with PBS extensively and detected by flow cytometry (FACSCalibur, BD Biosciences, CA). For ODN location analysis, transfected cells were washed three times, fixed and permeabilized, nuclei were stained with DAPI (Jingmei Biotechnologies, China) for 10 min at room temperature. After washing, the cells were observed under a fluorescent microscope.
Cell proliferation assay
Cells were plated and transfected with decoy or scramble ODN as described above. Cell number was determined by counting with a hemocytometer using trypan blue exclusion. Above 90% viability was regarded as no toxicity. Cell proliferation was examined by measuring DNA synthesis using tritiated thymidine (3H-dThd) uptake. Before transfection, cells (4 × 104/well) were cultured in a 24-well plate in growth media for 24 h. STAT3 decoy or scramble ODN were transfected for 18 h separately, and then pulsed for the remaining 6 h with [3H]-thymidine (3.75 μCi/ml). [3H]-thymidine incorporation was analyzed by liquid scintillation counting.
Cell apoptosis assay
Twelve hours after transfection of 25 nmol/L STAT3 decoy or scramble ODN, cells were detached, washed and resuspended in 100 μl Annexin V binding buffer, and subsequently stained with Annexin V-FITC and propidum iodide (Sigma) according to the manufacturer's recommendation. Data acquisition and analysis were performed by a flow cytometry using CellQuest software.
Isolation of Total RNA and reverse transcription (RT)-polymerase chain reaction (PCR)
RT-PCR primers for STAT3 target genes
5'-CTC CTT AAT GTC ACG CAC GAT TT-3'
5'-GTG GGG CGC CCC AGG CAC CA-3'
5'-GAG ACC ATC CCC CTG ACG GC-3'
5'-CTC TTC CTC CTC CTC GGC GGC-3'
5'-GGA AAG CGT AGA CAA GGA GAT GC-3'
5'-GGT GGG AGG GTA GAG TGG ATG GT-3'
5'-GGT CTT CCC CTA CCC TCT CAA CGA-3'
5'-GGC AGC AGG ATA GTC CTT CCG AGT-3'
5'-GTG GTG GTG GTG GTT GGT TA-3'
5'-CGG CAG TCG CTG GAG ATT AT-3'
5'-GCA TGG GTG CCC CGA CGT TG-3'
5'-GCT CCG GCC AGA GGC CTC AA-3'
5'-ATA CAG ACC CAC AGA GAC AG-3'
5'-TGC CAT CCA CAG AAA TAC TT-3'
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were processed as described previously . Briefly, the whole cells were lysed in lysing buffer after transfected with STAT3 decoy ODN for 24 h. The whole cell extracts (30 μg/lane) were separated by SDS-PAGE and transferred to nitrocellulose membrane. After blocked in Tris-buffered saline with 5% (w/v) nonfat dry milk, membranes were incubated with primary antibodies (1:1000 dilution) according to the manufacturer's instructions and then incubated with horseradish peroxidase conjugated secondary antibody. The proteins were detected by the enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL) using X-ray film. The bands were examined by densitometry using AlphaEaseFC software (Version 4.0.0, Alpha Innotech Corporation) with normalization of each band to their corresponding loading control.
Human tumor-bearing nude mouse
Female athymic nude mice (Balb-nu/nu, 4 weeks old, weighing 18 ± 2 g) were purchased from the Shanghai Experimental Animal Center (Chinese Academy of Sciences, Shanghai), and maintained at an animal facility under pathogen-free conditions. The handling of mice and experimental procedures were conducted in accordance with experimental animal guidelines. A549 cells were harvested and re-suspended in RPMI 1640 media. 8 × 106 cells (150 μl) were injected into the right flank of the mice subcutaneously. About 7 days later, when the tumors reached about 35 mm3, the mice were randomly assigned to three groups with six mice in each group (PBS treatment control, STAT3 scramble ODN treatment control and STAT3 decoy ODN treatment group). ODN (25 μg in 25 μl PBS) was intratumorally injected every day for totally 30 days. During this period, the tumor volume was determined by measuring the length (l) and the width (w) every 5 days, and calculated by using the function (V = l w2/2).
Terminal Deoxynucleotidyl Transferase-mediated Nick End Labeling (TUNEL)
A549 xenografts were collected on the termination day of antitumor experiment, sectioned and stained for apoptotic cells by TUNEL using the In Situ Cell Death Detection Kit, POD (Roche Applied Science, Germany) according to manufacturer's guides. Briefly, frozen sections were fixed in 4% paraformaldehyde, incubated in blocking solution and in permeabilisation solution, and incubated in TUNEL reaction mixture for 60 min at 37°C. After rinsing, the sections were incubated with Converter-POD for 30 min at 37°C and developed with DAB Substate Kit (Boster, Wuhan). The slides were lightly counterstained with hematoxylin and then dehydrated and mounted. For each group, 4 samples were evaluated and 5–10 fields of view were quantitated on each section. The number of apoptotic cells per high power field (HPF) was counted.
The immunohistochemical staining was performed using peroxidase labeled streptavidin-biotin method (Histostain plus kits, ZYMED, CA) according to the manufacturer's protocol. Briefly, five-μm frozen sections were fixed in acetone. Endogenous peroxidase was blocked by 3% hydrogen peroxide. After blocked with normal goat serum, diluted bcl-xl and cyclin D1 primary antibodies (at a dilution of 1:300) were added to completely cover tissue and incubated in humidified chamber overnight at 4°C, and then incubated with biotinylated second antibody, and finally incubated with streptavidin-peroxidase conjugate (S-A/HRP). The immune complex was visualized with 3, 3-diaminobenzidine (DAB substate kit, Boster, Wuhan). The slides were lightly counterstained with hematoxylin and then dehydrated and mounted. As negative controls, rabbit immunoglobulin was used to replace primary antibody.
Statistical analysis was performed with SPSS software (version 10.0, SPSS Inc). Data were presented as mean ± SD. p < 0.05 were considered statistically significant.
STAT3 was constitutively activated in A549 human lung cancer cell line and STAT3 decoy ODN was efficiently transfected into A549 cell lines
To assay the transfection efficiency of decoy ODN into cells, we used a FITC-labeled decoy ODN to verify whether the decoy ODN could be introduced into A549 cells. The results measured by a flow cytometry showed the decoy ODN could be efficiently transfected into A549 cells with a dose-dependent manner after incubating for 6 h (Fig. 1B). The transfection efficiency was 93%, and the mean fluorescence intensity (MFI) was 53.36 at 25 nmol/L, and 92.31 at 50 nmol/L of ODN, respectively. Similar results were observed with scramble ODN. Moreover, the sub-cellular localization of decoy ODN was determined by a fluorescent microscopy. As shown in Fig. 1C, most of decoy ODN were located in nucleus. These data indicated that decoy ODN could be efficiently transfected into cells and located in the nucleus.
STAT3 decoy ODN inhibited the growth of A549 cells in vitro
STAT3 decoy ODN inhibited tumor growth and increased apoptosis in xenografts
STAT3 decoy ODN decreased the expression of STAT3-regulated cell growth or anti-apoptotic genes of lung cancer cells in culture
STAT3 decoy ODN decreased the expression of bcl-xl and cyclin D1 in xenografts by Immunohistochemical analysis
Cumulative evidence support that STAT3 activation and the novel decoy ODN approach may be used as an antitumor strategy. Unlike in normal cells and tissues, the constitutively activated STAT3 has been observed in a wide variety of human cancer cell lines and primary tumors. Therefore, STAT3 has also been recognized as an oncogene [2, 19]. By using the specific antibody against phosphorylated STAT3 and western blot, we, here, show that STAT3 is constitutively activated in A549 human non-small-cell lung cancer cell line (Fig. 1A), which was consistent with our previous observations  in human pulmonary giant cell carcinoma PG cell line. These results further support the idea that the activated STAT3 may be a good molecular target for lung cancer treatment.
Decoy ODNs have been proposed as a useful approach to block the function of transcription factors. There are several attractive advantages of decoy method over the other gene therapeutic approaches [32, 33]. First, decoy ODN, as a small DNA molecule, can be easily delivered to specific tissues and transfected into cells, and directly abrogate the activated transcript factors. Second, a growing number of transcript factors with their promoter sequences have been found, which made the potential drug targets plentiful and readily identifiable. Third, the synthesis, storage and transportation of decoy ODNs are much simpler than other approaches. Finally, decoy ODNs have been documented to be more effective than antisense ODN in blocking constitutively activated transcript factors . Moreover, the clinical application of decoy ODN against E2F was approved by the Food and Drug Administration (FDA) to treat neointimal hyperplasia in vein bypass grafts. A clinical trial using NF-κB decoy ODN was also started in Japan, and positive results of phase I/II trial for the treatment of atopic dermatitis was made in 2006.
In present study, we used STAT3 decoy ODN to turn down the constitutively activated STAT3 in lung cancer cells. The results showed that STAT3 decoy ODN significantly decreased viable cell number by inducing apoptosis and reducing [3H]-thymidine incorporation of A549 non-small-cell lung cancer cell line in vitro (Fig. 2), which was in parallel with our recent findings in a different type of lung cancer cell line PG , suggesting that STAT3 decoy could effectively suppress cell growth of lung cancer. Since either cells that are dead or undergoing apoptosis, like cells that are stuck in G0/G1, may also show reduced [3H]-thymidine incorporation compared with control cells, further studies are necessary to clarify what the relative contributions of apoptosis vs. reduced proliferation are to the growth inhibition observed in the lung cancer cells.
To confirm the above inhibitory effect of STAT3 decoy ODN on human lung cancer cells in culture, we used human A549 lung tumor xenograft mice to assay the in vivo efficacy of STAT3 decoy ODN. Compared to PBS vehicle control and scramble control, STAT3 decoy ODN significantly inhibited the tumor growth (Fig. 3A) in mice as well as remarkably increased the number of TUNEL-positive apoptotic cells in xenograft tumor tissues (Fig. 3B). Both in vitro and in vivo experimental results demonstrated the potential values of STAT3 decoy ODN approach in the treatment of lung cancer.
We next investigated the molecular mechanisms by which STAT3 decoy ODN inhibited growth of lung cancer cells. Blockade of STAT3 signaling by STAT3 decoy ODN could down-regulate the cell growth genes (such as cyclin D1) or anti-apoptotic genes (such as bcl-xl) at both mRNA and protein levels (Fig.4 &5). Inhibition of STAT3 signalling resulted in the decreased survivin and c-myc expression in many tumor cell lines, however, in A549 cells, the transcription levels of survivin and c-myc were not down-regulated by STAT3 decoy ODN treatment. One of the reasons was that the expressions of c-myc and survivin may also be controlled by other transcription factors such as STAT5 and STAT1, which may compensate it when the STAT3 was blocked [34, 35]. Therefore, the inhibition of tumor growth by STAT3 decoy ODN was significantly associated with blockade of the STAT3-regulated cell cycle and anti-apoptotic proteins.
In summary, we have provided evidence that STAT3 decoy ODN suppresses growth of lung cancer cells in vitro and in vivo through affecting the balance between cell proliferation and rate of apoptosis. This inhibitory effect may be correlated to blockade of STAT3-regulated cell cycle genes or anti-apoptotic genes. Our findings highlight that STAT3 may be a good candidate molecular target, and STAT3 decoy ODN may potentially be used as an anti-lung cancer therapeutic small bio-molecule.
This work was supported by Natural Science Foundation of China (#30628014; #30571696) and National 973 Science Program by Ministry of Science and Technology of China (#2004CB518807; #2004CB520804; #2006CB504303).
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