Effects of BCL-2 suppression by antisense oligonucleotides on additional regulators of apoptosis compensatory change in non-targeted protein expression.

Antisense oligonucleotides (oligos) have been employed against prostate cancer in both in vivo and in vivo models. While most oligos contain a single mRNA binding site, our laboratory has developed bispecifics directed towards two. Previous work has determined that when oligos are used to suppress the expression of individual proteins in highly regulated physiologic processes, additional proteins can be affected. These non-targeted (non-specific effects) regulators can compensate for proteins specifically targeted, reverse the intended effect and promote tumor growth. To evaluate specific and compensatory non-specific effects on growth inhibition of LNCaP cells employing mono- and bispecific oligos directed against BCL-2, LNCaP cells were incubated in the presence of oligos specifically directed against BCL-2 [the second binding site was directed against epidermal growth factor receptor (EGFR)] and compared to lipofectin containing controls. Significant, but comparable, growth inhibition was produced by mono- and bispecific forms. Employing RT-PCR to determine BCL-2 expression, mRNA suppression approached 100% for each oligo type: monospecific MR4 (directed only against BCL-2), 100%; and bispecifics MR24 and MR42, 86% and 100% respectively. Based upon inhibition of in vivo growth and BCL-2 expression, bispecific antisense oligos directed against EGFR and BCL-2 mRNAs are at least as effective as a monospecific directed towards BCL-2. To identify a compensatory response to evade apoptosis in the presence of BCL-2 suppression, levels of mRNA encoding non-targeted BAX, caspase-3 and clusterin were evaluated. We initially found that specific suppression of the apoptosis inhibitor BCL-2 in LNCaP cells does not affect (non-targeted) BAX expression and (non-targeted) caspase-3 expression was suppressed. This suggested that tumor cell variants develop which resist apoptosis through diminished expression of this promoter. This study suggests that compensatory changes in the regulation of apoptosis may not be widespread or be limited to apoptosis promoters (caspase-3), since the expression of the non-targeted apoptosis inhibitor clusterin is not affected. Should BCL-2 suppression be clinically employed with antisense oligos, it may only require maintainance (or replacement) of caspase-3 activity.