Inhibition of BCL-2 by antisense oligonucleotides is followed by a compensatory suppression of caspase-3 in LNCaP cells

Antisense oligonucleotides (oligos) have been evaluated for treating prostate cancer in in vivo and in vitro models. Although most oligos contain a single mRNA-binding site, our laboratory has developed bispecifics directed toward two. To evaluate specific and compensatory nonspecific effects we evaluated the growth inhibition of LNCaP cells employing mono- and bispecific oligos directed against BCL-2 [the second binding site was directed against epidermal growth factor receptor (EGFR)]; and employing reverse transcription-polymerase chain reaction (RT-PCR), expression of three apoptosis regulating proteins (targeted BCL-2 and nontargeted bax and caspase-3) were evaluated. LNCaP cells were incubated in the presence of oligos specifically directed against BCL-2 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, we found that mRNA suppression approached 100% for each oligo type: mono-specific MR4 (directed only against BCL-2), 100%; and bispecifics MR24 and MR42, 86% and 100%, respectively. Based upon the inhibition of in vitro growth and BCL-2 expression, bispecific antisense oligos directed against EGFR and BCL-2 mRNAs are at least as effective as a mono-specific directed toward BCL-2. To determine a compensatory response to evade apoptosis in the presence of BCL-2 suppression, levels of mRNA encoding nontargeted bax and caspase-3 were evaluated. Whereas suppression of the apoptosis inhibitor (BCL-2), in LNCaP cells, does not affect bax expression, nontargeted caspase-3 was suppressed, suggesting that variants arise that resist apoptosis through the diminished expression of this promoter. Should BCL-2 suppression be clinically employed with antisense oligos, similar experiments would identify genes requiring replacement (or amplification) to restore apoptosis.