Selective inhibition of inducible cyclo‐oxygenase‐2 expression by antisense peptide nucleic acids in intact murine macrophages

Abstract

Listen

Translate article

Prostaglandins are important molecules involved in inflammation and immunomodulation. The rate‐limiting step in the synthesis of these potent mediators is the expression of the enzyme cyclo‐oxygenase (COX). The isoform responsible, COX‐2, is encoded by an immediate‐early gene induced by various pro‐inflammatory agents in macrophages. Selective blockade of COX‐2 by the use of an antisense strategy would overcome the undesirable side effects of conventional inhibitors. Here we describe cellular internalization and activity of a novel class of oligonucleotide analogues named peptide nucleic acids (PNAs) as inhibitors of COX‐2 translation. In particular, we designed two antisense murine COX‐2 PNA molecules, directed against a mRNA region spanning the AUG translation‐initiation codon and a homopurinic sequence inside the COX‐2 mRNA reading frame. These two PNA sequences, used separately or mixed together, demonstrated the capacity to inhibit the translation of murine COX‐2 enzyme in a cell‐free translation model using a rabbit retculocyte lysate model. Since PNAs display very low natural permeability across lipids bilayers, the two molecules were also re‐synthesized, modified to be used in intact cells by means of linkage to a hydrophobic peptide to obtain membrane‐diffusable PNA chimaerae. Finally, stimulated macrophages were found to be affected strongly by these two compounds, used separately or together, monitoring inhibition of COX‐2 synthesis by Western blot analysis of total lysates and enzymic activity via radioactive assay on the microsomal fractions.