Recent Research in LNCaP Cells Inhibition of BCL-2 by Antisense Oligonucleotides Results in Compensatory Changes Involving Apoptosis, Transcription and Immunoregulation

Antisense oligonucleotides (oligos) have been evaluated for treating prostate cancer in both in vivo and in vitro models. This study evaluates the growth inhibition of in vitro propagating LNCaP cells employing mono- and bi-specific oligos directed against BCL-2 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 (PDL1) 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 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: Mono-specific MR4 (directed only against BCL-2), 100%; and bispecifics MR24 and MR42, 86% and 100% respectively. 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, nontargeted 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.