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. 2007 Aug 4:7:149.
doi: 10.1186/1471-2407-7-149.

Therapeutic effects of STAT3 decoy oligodeoxynucleotide on human lung cancer in xenograft mice

Xulong Zhang  1 , Jian Zhang, Lihua Wang, Haiming Wei, Zhigang Tian
Affiliations

Therapeutic effects of STAT3 decoy oligodeoxynucleotide on human lung cancer in xenograft mice

Xulong Zhang et al. BMC Cancer. .

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
STAT3 was constitutively activated in A549 human lung cancer cell line and the efficiency of STAT3 decoy ODN transfected into A549 cells. (A) The A549 lung cancer cells were lysed in lysing buffer and the whole cell extracts (30 μg/lane) were separated by SDS-PAGE and then examined by western blotting using anti-STAT3, anti-phospho-specific STAT3 (Tyr 705, Ser727) or β-actin antibodies. The proteins were then detected by the enhanced chemiluminescence (ECL) system by exposing to X-ray film. (B) A549 cells were transfected with FITC-labeled STAT3 decoy ODN at final concentration of 12.5, 25 and 50 nmol/L in presence of lipofectamine 2000. After transfected for 6 h, the cells were examined by flow cytometry. (C) After transfected with 25 nmol/L FITC-labeled STAT3-ODN for 6 h, the cells were fixed, permeabilizated and stained with DAPI, and then fluorescent microscopy was used to observe the nucleus location of ODN. STAT3 decoy ODN (green) and nuclei (blue) were overlaid with their contrast image.
Figure 2
Figure 2
Inhibitory effect of STAT3 decoy ODN on A549 lung cancer cells in vitro. (A) A549 cells were seeded at a density of 1 × 104 cells per well in 48 well plates. After 24 h, the cells were treated with 25 nmol/L STAT3 decoy ODN, STAT3 scramble ODN or vehicle control (TE) for different time using lipofectamine 2000. The cumulative number of cells was accounted using trypan blue exclusion. Values are expressed as mean ± SD of three independent experiments. (B) A549 cells (4 × 104/well) were treated with STAT3 decoy ODN or scramble decoy ODN for 18 hours at 37°C, and then pulsed with [3H]-thymidine for an additional 6 hours. The incorporation of [3H]-thymidine was analyzed by liquid scintillation counting. (C) A549 cells were plated in six well plate, after transfected with 25 nmol/L STAT3 decoy ODN or scramble ODN, the apoptotic A549 cells were detected by flow cytometry using Annexin V-FITC and PI. The histogram represented the percentage of apoptotic cells. Values are expressed as the mean ± SD of three independent experiments. Statistical significance was determined as *p < 0.01 compared to other groups.
Figure 3
Figure 3
Inhibitory effect of STAT3-decoy-ODN on A549 tumor – bearing nude mice. (A) Human A549 tumor-bearing nude mice were established as described in materials and methods. The mice were intratumorally injected daily with STAT3 decoy ODN (25 μg), scramble ODN (25 μg) or vehicle control (25 μl PBS) for total thirty injections, and the tumor volumes were measured at the indicated day. *p < 0.01 versus PBS treatment control. (B) After daily intratumoral injection of 25 μg STAT3 decoy ODN for thirty times, the xenografts were collected, sectioned and stained for apoptotic cells by TUNEL which performed as described in materials and methods. The brown stain represented the DNA fragmentation of apoptotic cells and the blue showed the nuclei stain with hematoxylin (original magnification ×400). The cumulative results showed the average number of apoptotic cells per High Power Field. **p < 0.001 versus PBS treatment control.
Figure 4
Figure 4
STAT3 decoy ODN decreased the expression of STAT3-regulated cell growth or anti-apoptotic genes. (A) After transfected with 25 nmol/L STAT3 decoy ODN or scramble ODN for 24 h, the whole A549 cells were harvested and the total cellular RNA was isolated using Trizol Reagent, mRNA levels of STAT3-correlated genes such as mcl-1, cyclin D1, bcl-xl, cyclinE, c-myc and survivin were detected using RT-PCR method. The PCR products were electrophoresed and photographed using AlphaEaseFC. (B) After transfected with 25 nmol/L decoy ODN, the whole A549 were harvested and the whole-cell extracts were obtained. And western blotting for bcl-x1 and cyclin D1 were then examined as described in materials and methods. Histogram presented the relative expression level of each gene or protein after normalization to its corresponding internal control. Statistical significance was determined as *p < 0.01 and #p < 0.05 compared to control.
Figure 5
Figure 5
STAT3 decoy ODN decreased the expression of bcl-xl and cyclin D1 in xenografts by Immunohistochemical analysis. The harvested xenografts which treated with decoy or scramble ODN were sectioned and performed immunohistochemical analysis of bcl-xl (A) and cyclin D1 (B) as described in materials and methods. The brown showed the expression levels of bcl-xl and cycln D1; the blue showed the nuclei (original magnification ×400). Each histogram presented the number of bcl-xl and cyclin D1 positive cells per High Power Field in tumor tissues. Statistical significance was determined as #p < 0.05.
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