Bax expression remains unchanged following antisense treatment directed against BCL-2

Antisense oligonucleotides (oligos) have been evaluated in both in vivo and in vitro prostate cancer models. Although most contain a single mRNA binding site, our laboratory has also evaluated bispecific types directed toward two proteins. This study evaluates the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)]. Employing RT–PCR, the expression of two apoptosis regulating proteins, bcl-2 and non-targeted bax, was then evaluated. LNCaP prostate tumor cells were initially incubated for 24 h in the presence of oligos (6.25 μM) directed against bcl-2 and compared to lipofectin containing controls. Comparable and significant growth inhibition was produced by both mono- and bispecific forms. Employing RT–PCR to determine the expression of bcl-2, we found that the greatest amount of 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. We conclude, based upon both inhibition of in vitro growth and bcl-2 expression, that bispecific antisense oligos directed against EGFR and bcl-2 mRNAs are at least as effective as a monospecific directed solely toward bcl-2. In an effort to determine a compensatory response by cells evading apoptosis in the presence of bcl-2 suppression, the levels of mRNA encoding the non-targeted apoptosis activating protein bax were evaluated. Non-targeted protein suppression by these bispecifics has previously been demonstrated against prostate-specific membrane antigen (PSMA). However, in contrast to effects against bcl-2 and PSMA, no significant alteration in bax expression was produced by either oligo type. In LNCaP cells, bcl-2 suppression does not influence bax expression and, at least for this protein, there is no compensatory change in bax expression regulating apoptosis at this level. Identifying changes in the expression of proteins which regulate apoptosis is important if gene therapy targets bcl-2.