- Top of page
- Material and methods
The ets-1 transcription factor plays an important role in cell proliferation, differentiation, apoptosis and tissue remodeling. Aberrant ets-1 expression correlates with aggressive tumor behavior and poorer prognosis in patients with various malignancies. This study evaluated the efficacy of double-stranded decoy oligonucleotides targeting ets-1-binding cis elements for the suppression of ets-1 in treatment of a peritoneal dissemination model of gastric cancer. In vitro, MTT assay was performed to evaluate the effect of the ets-1 decoy on cell growth. Electrophoretic mobility shift assay (EMSA) was performed to determine ets-1 activity. In vivo, the effect of the ets-1 decoy was investigated in the peritoneal dissemination nude mice model. Disseminated nodules were analyzed immunohistochemically. Ets-1 decoy, but not scrambled decoy, significantly inhibited cell growth in 2 gastric cancer cell lines, which showed overexpression of ets-1 protein by inhibiting the binding activity of ets-1. In the peritoneal dissemination model, the ets-1 decoy significantly suppressed the disseminated nodules, and tended to prolong the survival rate. PCNA index, microvessel density and VEGF expression were also reduced in peritoneal tumors treated with ets-1 decoy. Intraperitoneal injection of ets-1 decoy inhibited peritoneal dissemination of gastric cancer in a nude mice model. The results indicate that the decoy strategy for ets-1 offers a promising therapy for patients with incurable peritoneal dissemination of gastric cancer, most of which show overexpression of ets-1 protein. © 2007 Wiley-Liss, Inc.
The prognosis of advanced gastric cancer, especially in serosa-invading tumors, remains poor even after curative resection, and in these cases peritoneal dissemination originating from free cancer cells seeded from the primary gastric cancer is the most common type of recurrence.1, 2 To date, various treatments have been used for peritoneal dissemination of gastric cancer, including aggressive surgery3, 4 and intraabdominal or systemic chemotherapy.5, 6, 7, 8, 9, 10 However, the contributions of these therapies to patient survival have been unsatisfactory.
The E26 transformation-specific (ets) family, known as a transcription factor, contains a conserved winged helix-turn-helix DNA binding domain, and regulates gene expression by binding to so-called ets-binding sequences found in promoter/enhancer regions of their target genes.11 This family is involved in a diverse array of biological functions, including cellular growth, migration and differentiation.12, 13, 14, 15 Ets-1 was the first member of the ets family to be identified.11 Recently, ets-1 has been shown to play a role in tumor progression in various kinds of malignant tumors.16, 17, 18, 19, 20, 21, 22, 23 Ets-1 is induced by and is required for the activation of several genes involved in angiogenesis and remodeling of extracellular matrix (ECM), such as vascular endothelial growth factor (VEGF), angiopoietins (Ang), fibroblast growth factor (FGF), epidermal growth factor (EGF), urokinase plasminogen activator (uPA), matrix metalloproteases (MMPs) and integrin β3.24, 25, 26, 27 Overexpression of ets-1 correlates with grade of malignancy and poorer prognosis in several types of tumors, including breast,16, 17 lung,18 ovarian,19, 20 colorectal21 and gastric cancers.22, 23 The correlation between ets-1 overexpression and aggressive tumor behavior suggests that ets-1 may be an attractive molecular target for cancer therapy.
The inhibition of transcription by using short sequence oligonucleotides such as antisense DNA and RNAi is a novel approach for molecular-targeted therapies.28, 29, 30 Recently, the decoy strategy has been developed and is considered as an effective tool for transcriptional suppression of downstream genes.31, 32 The double-stranded decoy oligonucleotides closely correspond to the response element within the promoter region of downstream genes. By achieving sufficient oligonucleotide concentrations in target cells, the authentic interaction between a transcription factor and its endogenous response element in genomic DNA is impaired with subsequent suppression of gene expression.33 The decoy strategy is also applicable for a loss-of-function approach at the pretranscriptional and transcriptional levels by directly targeting a transcription factor.31 Furthermore, decoy oligonucleotides provide novel methods for global control of the expression of genes that are regulated through an enhancer, unlike antisense oligonucleotides and RNAi, which only target ets-1 the mRNA of one specific gene.
The purpose of this study was to evaluate the efficacy of double- stranded decoy oligonucleotides in suppressing the transcriptional function of ets-1 and for the treatment of peritoneal dissemination in an animal model of gastric cancer.
- Top of page
- Material and methods
Recently, oligonucleotides corresponding to the consensus binding sequence of a specific transcription factor have been explored as tools for manipulating gene expression in living cells.32 The decoy strategy using double-stranded oligonucleotides for binding sequences has been developed as a new class of antigene strategy for clinical application.31 The occupation of the DNA binding site of the transcription factor by the decoy renders the protein incapable of subsequent binding to the promoter regions of target genes. Bielinska et al.35 were the first group to describe the utility of such decoys as a tool for investigating the function of transcription factors in cell lines. To date, decoy strategies have also been applied as therapeutic agents and the potential efficacy of decoy oligonucleotides that target other transcription factors has been described in cancer treatment,36, 37, 38, 39, 40 myocardial infarction41, 42 and rheumatoid arthritis.43
The ets family consists of ∼30 genes and the ets-1 gene was the first identified as the cellular homologue of the viral oncogene v-ets of the avian transforming retrovirus E26.11 The gene product of this family is a transcription factor, controlling various cellular functions in cooperation with other families of transcription factors and cofactors.11, 12, 13, 14 All members have an activation or a repression domain for transcription and an evolutionarily conserved ets domain, which can bind to the 5′-GGAA/T-3′ core motif.15 Each member have been found to bind specifically to each 10 bp of sequences containing the core motif,11 and we designed Ets-1 decoy including this specific sequence (ACC GGAAGTA).
The target genes for the ets transcription factor include oncogenes, tumor suppressor genes, apoptosis-related genes, differentiation-related genes, angiogenesis-related genes and invasion-related genes.12, 13, 14 With regard to ets-1, previous reports indicated that it regulates the expression of MMP, urokinase type-plasminogen activator,17, 18, 43 angiopoietin24, 25, 26 and VEGF.16, 20, 23
Overexpression of ets-1 has been reported to correlate with tumor malignancy and prognosis of patients with gastric,23 breast16 and ovarian cancers.20 In particular, ets-1 overexpression in gastric tumors correlated significantly with lymph node and distant metastasis and poorer prognosis of patients.44, 45 Furthermore, reduction of cell proliferation and MMP-9 expression led by ets-1 decoy have been reported in glioma cells.46 Therefore, ets-1 appears to a promising molecular target for gastric cancer therapy, since the targeting of this protein may suppress tumor proliferation and improve patient prognosis by inhibition of invasion and tumor angiogenesis.
In the present in vitro study, we performed MTT assays and EMSA using 3 gastric cancer cell lines (MKN28, MKN45 and MKN74), treated with ets-1 or scrambled decoy to assess the hypothesis that the treatment of cells with the ets-1 decoy would interfere with the binding of ets-1 to ets-1-specific DNA response elements and lead to inhibition of cell growth. Consistent with our hypothesis, treatment with ets-1 decoy led to marked inhibition of cell growth by interfering with the binding activity of ets-1 decoy in MKN45 and MKN74 cell lines. However, the effect was different between the cell lines and ets-1 decoy had no significant effect on cell growth and binding activity in MKN28 cells, which showed low expression of ets-1 protein compared to MKN45 and MKN74 cells. Furthermore, the ets-1 expression was limited in cytoplasm of MKN28 cells by immunohistochemical analysis. These results indicate that in MKN28 cells, the ets-1 signal transduction may not play an important role in cell growth.
We have established a peritoneal dissemination model of nude mice using MKN45-EGFP cells, produced by transducing MKN45 cells with an EGFP-expressing plasmid vector.34 We applied this model to assess the therapeutic effect of ets-1 decoy against peritoneal dissemination of gastric cancer cells. The stable GFP expression in cancer cells enabled us to evaluate micrometastasis in the abdominal cavity, which was otherwise not detectable by conventional microscopy. Furthermore, we confirm that ets-1 decoy has no effect on the reduction of the fluorescent intensity in MKN45-EGFP cells. In the ets-1 decoy group, GFP nodules, which indicated peritoneal dissemination, were significantly suppressed compared to the scrambled decoy and control groups. We also evaluated peritoneal dissemination nodules by immunohistochemical staining for PCNA, PECAM-1 and VEGF to clarify the effect of ets-1 decoy. The results of staining supported the hypothesis that ets-1 decoy inhibits tumor growth and progression by inhibiting tumor angiogenesis. With regard to its effect on prognosis, ets-1 decoy tended to improve survival, although the effect was not significant compared to the scrambled decoy group (p = 0.058). In this point, we need to change the dosage, treatment schedule and/or drug delivery system to apply the ets-1 decoy therapy for clinical use. We also demonstrated the lack of toxicity of oligonucleotides used in our ets-1 decoy as determined by the amount of food intake, body weight gain and pathological findings of major organs.
In our experiments, the scrambled decoy showed the tendency, though not significant, to suppress peritoneal dissemination in the assessment of total weight and microvessel density of fluorescent nodules in the MKN45-EGFP nude mouse model. This might be caused by the nonspecific side effects with high dose of oligonucleotides; however, there are few published studies referred to the toxicity of oligonucleotides in vivo.28, 29, 40, 41 We performed invitro assay with 0.1–10 μM of decoy, whereas the therapeutic dose employed in vivo was 80 μM. Higher doses of decoy oligonucleotides might raise other possible mechanisms for the inhibitory effects of the peritoneal dissemination.
In conclusion, we have demonstrated that intraperitoneal injection of ets-1 decoy inhibited the peritoneal dissemination of gastric cancer by suppressing tumor angiogenesis in a nude mouse model. This study provides the first evidence of application of decoy as a therapeutic strategy for peritoneal dissemination invivo. These results indicate that the decoy strategy for ets-1 offers a promising therapy for patients with incurable peritoneal dissemination of gastric cancer, in most of which, ets-1 plays an important role in cell growth.