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Differentiated prostatic antigen expression in LNCaP cells following treatment with bispecific antisense
Marvin Rubenstein1, Courtney M P Hollowell, Patrick Guinan
1Division of Cellular Biology, Hektoen Institute for Medical Research, 2240 West Ogden Avenue, 2'nd Floor, Chicago, IL 60612, USA. DrMarv@Prodigy.net
Abstract:
Antisense oligonucleotides (oligos) have been administered against in vivo and in vitro prostate cancer models employing LNCaP and PC-3 cell lines. While most oligos consist of a single mRNA binding site targeting a single gene product or those with sequence homology, our lab has developed bispecific oligos directed toward two unrelated proteins. In LNCaP cells, we initially identified bispecifics that increased the expression of prostate-specific membrane antigen (PSMA) while not affecting secreted prostate-specific antigen (PSA). We postulated that surface antigen expression is increased by bispecifics able to form double-stranded regions, acting as interferon (IFN-γ) inducers. In other systems, when induced, IFN-γ promotes cell surface antigen expression, including HLA and receptors for tumor necrosis factor. To test this hypothesis, we measured the effect of oligo treatment on both IFN-γ induction and the expression of another secreted product of differentiated prostate cells, prostatic acid phosphatase (PAP). This study 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). Employing RT-PCR, the expression of non-targeted proteins encoded by mRNA for PSMA, PSA, PAP, and IFN-γ was subsequently valuated. 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 differences were detected in mRNA levels encoding PSA following treatment with either oligo type. IFN-γ was significantly induced only by bispecific oligos, and PAP expression was similar to PSA. These data support the hypothesis that double strand-forming bispecific oligos induce IFN-γ that enhances cell surface PSMA expression. Expression of tumor-associated surface antigens could increase their recognition and targeting by immunologic defense mechanisms and increase the effectiveness of tumor vaccines.
Insights
Bispecific antisense oligonucleotides targeting two proteins were found to inhibit prostate cancer cell growth. These oligos also induced interferon-gamma, enhancing prostate-specific membrane antigen expression for potential immune targeting.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- Antisense oligonucleotides (oligos) are investigated for prostate cancer therapy.
- Bispecific oligos targeting two proteins represent a novel approach.
- The role of interferon-gamma (IFN-γ) in modulating cell surface antigens is known.
Purpose of the Study:
- To evaluate the efficacy of bispecific oligos against prostate cancer cell lines.
- To investigate the mechanism of action, including IFN-γ induction and antigen expression.
- To assess the impact on prostate-specific membrane antigen (PSMA) and other markers.
Main Methods:
- In vitro studies using LNCaP and PC-3 prostate cancer cell lines.
- Administration of mono- and bispecific antisense oligos.
- RT-PCR analysis to quantify mRNA levels of PSMA, PSA, PAP, and IFN-γ.
- Assessment of cell growth inhibition.
Main Results:
- Bispecific oligos significantly inhibited LNCaP cell growth compared to controls.
- PSMA mRNA levels were unexpectedly elevated by bispecific oligos.
- IFN-γ was significantly induced only by bispecific oligos.
- PSA and PAP mRNA levels showed no significant changes.
Conclusions:
- Double-strand forming bispecific oligos induce IFN-γ.
- Induced IFN-γ enhances cell surface PSMA expression.
- Targeting tumor-associated antigens may improve immunologic recognition and vaccine efficacy.
