Contribution of eIF-4E inhibition to the expression and activity of heparanase in human colon adenocarcinoma cell line: LS-174T

Abstract

Aim: Heparanase degrades heparan sulfate proteoglycans (HSPGs) and is a critical mediator of tumor metastasis and angiogenesis. Recently, it has been cloned as a single gene family and found to be a potential target for antimetastasis drugs. However, the molecular basis for the regulation of heparanase expression is still not quite clear. The aim of this study was to determine whether the expression of eukaryotic initiation factor 4E (eIF-4E) correlated with the heparanase level in tumor cells and to explore the correlation between heparanase expression and metastatic potential of LS-174T cells.

Methods: A 20-mer antisense s-oligodeoxynucleotide (asODN) targeted against the translation start site of eIF-4E mRNA was introduced into LS-174T cells by lipid-mediated DNA-transfection. eIF-4E protein and mRNA levels were detected by Western blot analysis and RT-PCR, respectively. Heparanase activity was defined as the ability to degrade high molecular weight (40-100 kDa) radiolabeled HS (heparan sulfate) substrate into low molecular weight (5-15 kDa) HS fragments that could be differentiated by gel filtration chromatography. The invasive potential of tumor cell in vitro was observed by using a Matrigel invasion assay system.

Results: The 20-mer asODN against eIF-4E specifically and significantly inhibited eIF-4E expression at both transcriptional and translational levels. As a result, the expression and activity of heparanase were effectively retarded and the decreased activity of heparanase resulted in the decreased invasive potential of LS-174T.

Conclusion: eIF-4E is involved in the regulation of heparanase production in colon adenocarcinoma cell line LS-174T, and its critical function makes it a particularly interesting target for heparanase regulation. This targeting strategy in antisense chemistry may have practical applications in experimental or clinical anti-metastatic gene therapy of human colorectal carcinoma.

Figure 1

eIF-4E and GAPDH mRNA expression in oligonucle-otide-treated human colon adenocarcinoma cells. AS. anti-sense oligonucleotide-transfected cells; S. sense oligonucleotide-transfected cells; C. cells treated with liposome only as control. LS-174T cells were treated with 2 μM oligonucleotides, accord-ing to the optimal condition of this cell line in the preliminary experimental results. The numbers under each band showed the relative amount of eIF-4E fragments normalized to that of GAPDH by densitometry. The results suggest that antisense oligonucleotides against eIF-4E inhibit eIF-4E mRNA expres-sion in LS-174T cells, and sense oligonucleotides have no in-hibitory effect on eIF-4E mRNA expression.

Figure 2

eIF-4E protein expression in oligonucleotide-treated colon adenocarcinoma cells. Protein extracts were prepared from oligonucleotide-treated cells at 48 h after transfection. Cell lysates were size-fractionated by 12% SDS-PAGE, and immunodetection on the blotted membrane was performed using the eIF-4E MAb. AS, antisense oligonucle-otide-treated cells; S. sense oligonucleotide-treated cells; C, cells treated with liposome only; PC, eIF-4E standard anti-gen (derived from a human neuroblastoma cell line) as a positive control marker.

Figure 3

Western blot analysis of heparanase expression in LS-174T cells. The colon adenocarcinoma cells were lysed, and Western blot analysis was performed with an anti-hu-man heparanase polyclonal antibody. AS, antisense oligo-nucleotide-treated cells; S. sense oligonucleotide-treated cells; C, cells treated with liposome only. Cells treated with anti-sense oligonucleotides showed markedly reduced levels of heparanase protein compared to cells treated with sense oli-gonucleotides or saline in the presence of liposome. The ap-proximately 65-kDa protein represents a heparanase precursor, whereas the 50-kDa protein is a processed form, and data showed that both of them were less expressed while eIF-4E was inhibited.

Figure 4

Effects of eIF-4E down-regulation upon enzyme activity of heparanase. Cells were treated for 48 h with antisense oligonucleotide (■), sense oligonucleotide (▲) or liposome only(◆). The soluble fraction from transfected, freezed and thawed LS-174T cells was incubated with ³⁵S-la-beled HS. The incubation medium was then subjected to gel filtration over Sepharose CL-6B. Low-molecular-weight HS degradation fragments (peak II) were mainly produced dur-ing incubation with medium conditioned by sense oligonucle-otide infected cells. The cells treated with antisense oligonucle-otide exhibited low levels of heparanase activity as compared to untreated ones. Heparanase activity was also expressed as Kav×total cpm eluted in peak II.

Figure 5

Invasive profiles of LS-174T cells measured in vitro using Matrigel as ECM barrier in modified Boyden chambers. The invasiveness of the cells on Matrigel was normalized to 100% (control, untreated cells). Compared with the control, antisense oligonucleotide reduced invasion to 23%. Sense sequence didn’t have a significant influence on invasive potential of LS-174T cells. These results suggest that alteration of heparanase activity via inhibition of the expression of eIF-4E results in decreased inva-siveness in LS-174T cells. The small rings are membrane pores.