Abstract
Antisense oligonucleotides (oligos) have been administered against prostatic LNCaP cells in both in vivo and in vitro models. While most oligos consist of a single mRNA binding site targeting a single gene product, or others having sequence homology, we evaluate bispecifics directed towards two unrelated proteins. In LNCaP cells we identified bispecifics which increase the expression of prostate specific membrane antigen (PSMA) while not affecting secreted prostate specific antigen (PSA) or prostatic acid phosphatase (PAP). We then postulated that surface antigen expression is increased by bispecifics able to induce interferon (IFN-γ) through complementary intrastrand regions. To further test this hypothesis we measured the effect of oligo treatment on another secreted product, prostatic cancer antigen-3 (PCA-3). We initially evaluated the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 (the second bispecific binding site was against the epidermal growth factor receptor (EGFR)). These oligos were administered with lipofectin in a nanoparticle delivery system. Employing RT-PCR, the expression of non-targeted genes products encoding PSMA, PSA, PAP, PCA-3 and IFN-γ have now been evaluated. When LNCaP prostate tumor cells were incubated with oligos and compared to lipofectin containing controls significant growth inhibition resulted. Employing RT-PCR, the levels of mRNA encoding PSMA were unexpectedly found to be elevated following treatment with the bispecific oligos but not with a monospecific directed solely against bcl-2. No increased expression in PSA, PAP or PCA-3 following treatment with either oligo type were detected. Like PSMA, IFN-γ was significantly induced only by bispecific oligos. These data support the hypothesis that double strand forming bispecific oligos induce IFN-γ, enhancing cell surface antigen expression (PSMA) but not secretory products. Expression of tumor associated surface antigens could increase their recognition and targeting by immunologic defense mechanisms and increase the effectiveness of tumor vaccines.
Highlights
Antisense oligonucleotides have been employed in both in vivo and in vitro prostate cancer models employing LNCaP and PC-3 cell lines
We initially evaluated the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 [the second bispecific binding site was against the epidermal growth factor receptor (EGFR)]
In LNCaP cells we find that monospecific oligos targeting bcl2 and bispecifics targeting both bcl-2 and the epidermal growth factor receptor (EGFR) have comparable activities in suppressing bcl-2 expression as determined by both in vitro growth and bcl-2 expression measured by reverse transcriptase-polymerase chain reaction (RT-PCR)
Summary
Antisense oligonucleotides (oligos) have been employed in both in vivo and in vitro prostate cancer models employing LNCaP and PC-3 cell lines. In LNCaP cells we find that monospecific oligos targeting bcl and bispecifics targeting both bcl-2 and the epidermal growth factor receptor (EGFR) have comparable activities in suppressing bcl-2 expression as determined by both in vitro growth and bcl-2 expression measured by RT-PCR. [11] the presence of a second binding site did not diminish activity of the other This finding is significant because it’s naive to believe that cancer cell growth can be controlled by oligos through knockdown of a single gene product. Instead multiple genes must be down regulated, and it would be useful if more than one therapeutic activity could be combined onto a single entity
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