Antisense oligonucleotides (oligos) have been evaluated for treating prostate cancer in both in vivo and in vitro models. Although most oligos contain a single mRNA binding site, our laboratory evaluates bi-specific oligos directed towards two proteins. This study evaluates the growth inhibition in vitro of the LNCaP cell line employing mono- and bispecific oligos directed against BCL-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)]. These oligos were administered with lipofectin as part of a nanoparticle delivery system. Additionally, g, the expression of five apoptosis regulatory proteins, BCL-2, bax, caspase-3, clusterin and AKT-1 was evaluated by RTPCR.
LNCaP prostate tumor cells were incubated in the presence of oligos specifically directed against BCL-2 and their effect was compared to lipofectin containing controls. Significant, but comparable, growth inhibition was produced by both mono- and bi-specific forms. Employing RT-PCR to determine BCL-2 expression, we found that the greatest amount of mRNA suppression approached 100% for each type of oligo with mono-specific MR4 (directed only against BCL-2) equal 100%; and bispecifics MR24 and MR42, being 86% and 100% respectively. Based upon both inhibition of cell growth and BCL-2 expression, bi-specific antisense oligos directed against EGFR and BCL-2 mRNAs are at least as effective as a mono-specific directed solely towards BCL-2.
In an effort to determine a compensatory response by cells needed to evade apoptosis in the presence of BCL-2 suppression, the levels of mRNA encoding non-targeted bax, caspase-3, clusterin and AKT-1 were evaluated. Suppression of the apoptosis inhibitor (BCL-2) in LNCaP cells did not alter either bax or clusterin expression. However, the expression of non-targeted caspase-3 (an apoptosis promoter) was suppressed and the expression of non-targeted AKT-1 (an apoptosis inhibitor) was enhanced. This suggests that tumor variants can resist apoptosis through the altered expression of non-targeted regulators of apoptosis. If BCL-2 suppression was to be clinically targeted by antisense oligos, similar experiments may be helpful in identifying genes with a similar function.
Published on: Mar 27, 2014 Pages: 1-6