In LNCaP Cells Inhibition of BCL-2 by Antisense Oligonucleonucleotides Results in Compensatory Changes in Apoptosis

• Main Authors: Marvin Rubenstein (Author), Courtney M.P. Hollowell (Author), Patrick Guinan (Author)
• Format: Book
• Published: Scientific Journal of Genetics and Gene Therapy - Peertechz Publications, 2014-03-27.
• Subjects: Research Article
• Online Access: Connect to this object online.

Summary

<p>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.</p><p>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.</p><p>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.</p>

DOI

10.17352/sjggt.000001