Antisense inhibition of the p65 subunit of NF-kappa B blocks tumorigenicity and causes tumor regression.

Abstract

The NF-KB transcription factor, composed of two proteins, p50 and p65, is a pleiotropic activator that participates in the induction of a wide variety of cellular genes.Various cell adhesion molecules have NF-w.B binding sites and may play an important role in inflammatory response, tumorigenicity, and metastasis. In an earlier study, we demonstrated that adhesion of diverse transformed cells was blocked by antisense inhibition of the p65 subunit of NF-cB. Since cell-substratum interactions play an important role in tumorigenicity, we reasoned that antisense p65 could inhibit tumor- Cell-cell and cell-substratum adhesion plays an important role in the regulation of normal and neoplastic cell growth (1). These adhesion events are mediated by diverse cell adhesion molecules (CAMs) and integrins (1,2). Cell transformation is often associated with qualitative alteration in the integrin repertoire (3). The process of tumor progression is complex and requires malignant cells to modulate their adhesion properties at various points of tumor development (4). Inhibition of NF-KB function by antisense technology elicits a strong block in the adhesion of diverse cell types; if the p65 subunit is inhibited this effect can be observed in most cell types, but if the p50 subunit is inhibited the effect is dependent on the differentiative status of the cells (5). The cellular adhesion of differentiated HL-60 cells stimulated by phorbol 12-myristate 13-acetate is significantly altered, an effect associated with a marked reduction in CD llb integrin expression (6). These results suggested to us that antisense inhibition of p65 function could have profound effects on cellular adhesion.Utilizing diverse tumor-derived cell lines, we demonstrate a pronounced inhibition of adhesion and in vitro growth after treatment with p65 antisense oligonucleotides. Expression of dexamethasone (Dex)-inducible antisense RNA to p65 in a fibrosarcoma cell line inhibited tumorigenicity and caused regression of tumors in nude mice in a Dex-dependentThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.manner. Based on these results, we have attempted to establish the in vivo therapeutic efficacy of antisense p65 oligonucleotides utilizing nude mouse tumor models. MATERIALS AND METHODSAntisense Oligonucleotides. The antisense and sense phosphorothioate analogs of oligonucleotides to the 5' end of the different subunits of NF-KB, including the ATG initiation codon (18-to 24-mer), were synthesized using an automated synthesizer (model 394, Applied Biosystems) as described (5,6) following the procedure of Matsukura et al. (7).Cell Lines and Growth Assay. The K-BALB, B-16, SW-480, HT-29, and T-47D cell lines were obtained from the American Type Culture Collection; Rat-1 ras (8), HOS-MNNG (9), and Rat-1 p65A (10) have been described. In four independent experiments, cells (3 x 106) were trypsinized ...

Down-regulation of NF-κB activity and NF-κB p65 subunit expression by ras and polyoma middle T oncogenes in human colonic Caco-2 cells

“…This ®nding contrasts with that of Higgins et al (1993) who reported that inhibition of p65 expression reduced the growth and tumorigenicity of various transformed cells. The reason for these di erent results is not clear at the moment but may involve di erences in the cell types studied.…”

NF‐κB as a potential molecular target for cancer therapy

“…On the other hand, the NF-κB targeting strategy includes: (a) blocking the DNA binding of NF-κB using decoy oligodeoxynucleotides (ODNs) [28]; (b) blocking the transactivation of NF-κB using glucocorticoids [93]; or (c) interfering with NF-κB mRNA using NF-κB antisense oligonucleotide (ASO) [43].…”

NF-κB is required for H-ras oncogene induced abnormal cell proliferation and tumorigenesis

“…These results suggest that NF-kB activity is not necessary for normal cell growth, but required for Ras-activated abnormal cell proliferation, which contributes to Ras-mediated cell transformation. Earlier studies suggest that NF-kB activation may contribute to cell transformation and tumorigenicity of Ras transformed cells by regulating the expression of genes encoding cell adhesion molecules and by inhibiting transformation-associated cell death (Mayo et al, 1997;Higgins et al, 1993). It has been demonstrated that p65 antisense oligonucleotide inhibited growth and tumorigenicity of variously transformed cell lines including Ras transformed 3T3 cell, presumably by inhibiting cell adhesion processes Narayanan et al, 1993).…”