S100A4 Antisense Oligodeoxynucleotide Suppresses Invasive Potential Of Neuroblastoma Cells

Background: In cancer patient disease progress is often associated with spreading of tumor cells to vital organs, known as metastasis, which is a complex cascade of events involving a finely tuned interplay between the malignant cells and host cells. A number of factors, including a cascade of genetic alterations and regulatory elements that promote or inhibit the process at one or more of the sequential steps are involved in the process of metstasis. Many genes and their products have been shown to be associated with metastasis. Neuroblastoma, arising from embryonic neural crest tissue, is a biologically heterogeneous tumors varying from spontaneous regression to rapidly progression. Recent studies shed light on the mechanisms of action of S100A4, and indicated involvement of its product in tumor invasion and metastasis. It seems that S100A4 gene product affects the expression of MMP-2, whose high expression is closely related to tumor invasion and metastasis. So far, no evidence of S100A4 involvement in human neuroblastoma metastasis has been found. Understanding the correlation between S100A4 and metastasis of neuroblastoma might give us the clues to suppress invasion and metasatasis of neuroblastoma. This study aims to investigate the effect and mechanism of S100A4 gene on invasion and metastasis of neuroblastoma through specific suppression of S100A4 expression by transfecting a S100A4-directed antisense oligodeoxy-nucleotides into human neuroblastoma cells.Methods: A 20-mer phosphorothioate oligodeoxynucleotides(asODN)targeted against the S100A4 mRNA were transfected by Lipofectamine2000 into two human neuroblastoma cell line LA-N-6 and SK-N-SH who have high expression levels of S100A4 gene. S100A4 mRNA and MMP-2 mRNA levels were quantified by reverse transcription polymerase chain reaction (RT-PCR). The effect of asODN on the vitality of neuroblastoma cells was measured with colorimetric method. The capability of invasion and migration of LA-N-6 cells was evaluated by the transwell chamber assay.Results: 1. Treated with different final concerntrations (0.3 umol/L, 0.6 umol/L, and 0.9 umol/L) of asODN, S100A4 mRNA levels in LA-N-6 and SK-N-SH cells were significantly inhibited compared to those in control(both P<0.05). The comparative expression of S100A4 decreased with the asODN's concentration increasing, and among the three-concentration groups, the 0.9 umol/L asODN inhibited S100A4 mRNA expression the most. The suppression was nearly maximal 24 h after treated with asODN and decreased thereafter. The suppression was specific to asODN because there was no significant changes in S100A4 mRNA levels in the sODN- and mODN- treated cells.2. The invasive ability of cells was significantly suppressed by asODN, and the numbers of invading LA-N-6 cells were lower in the asODN group (2.33 1.15) than those in the control group (9.00 2.65, P=0.03). However, the invasive ability of cells transfecteded with sODN and mODN was unaffected compared to that in control.3. The motile ability of cells was significantly suppressed by asODN, and the numbers of migrating LA-N-6 cells were lower in the asODN group (9.33 4.73) than those in the control group (20.67 2.89, P=0.03). The motile ability of cells transfecteded with sODN and mODN was unaffectedcompared to that in control.4. The MMP-2 mRNA levels in the asODN-treated LA-N-6 or SK-N-SH cells decreased 25.5% and 21.4% respectively. There were no significant changes in MMP-2 mRNA levels in the sODN- and mODN- treated cells.Conclusion: S100A4 asODN is capable of significantly down-regulating S100A4 mRNA expression, and suppressing the invasive and motile abilities of neuroblastoma cells, with concomitant decrease of MMP-2 mRNA levels, so the biological activity of S100A4 gene on invasion of neuroblastoma cells may realize via stimulating the motility of tumor cells and influencing the expression of MMP-2 gene.