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-specifics directed towards two proteins. This study evaluates the growth inhibition of in vitro
propagating LNCaP cells employing mono- and bi-specific oligos directed against BCL-2 [the second binding site was directed against the epidermal
growth factor receptor (EGFR)]; and employing RT-PCR, the expression of five apoptosis regulatory proteins (BCL-2, bax, caspase-3, clusterin, AKT-
1), a tumor associated transcription factor (MED-12) and an immune blockade associated regulatory marker (PD-L1) were evaluated.
LNCaP prostate tumor cells were incubated in the presence of oligos specifically directed against BCL-2 (entering the cells through a form of
nanodelivery) and compared to lipofectin containing controls. Significant, but comparable, growth inhibition was produced by both mono- and bispecific
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: mono-specific MR4 (directed only against BCL-2), 100%; and bispecifics MR24 and MR42, 86% and 100% respectively. Based upon
both inhibition of in vitro 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.
The objective of these experiments was to determine a compensatory response by cells to (again) evade apoptosis in the presence of BCL-2 suppression. Levels of mRNA encoding non-targeted bax, caspase-3, clusterin and AKT-1 were initially evaluated, while additional experiments sought to identify additional mechanisms of tumor adaptability and resistance. Suppression of the apoptosis inhibitor (BCL-2) in LNCaP cells did not alter either bax or clusterin expression. However, non-targeted caspase-3 (an apoptosis promoter) was suppressed and 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. Additional
experiments identified other mechanisms of compensation involving transcription and immune regulation suggesting further studies are needed.