Protein kinase a riα antisense sensitization of radiation and androgen deprivation

Abstract

Protein Kinase A type I (PKA) plays a key role in cell proliferation via the control of mitogenic signals and is overexpressed in the majority of human tumors. We investigated the potential of using antisense PKA (AS) to sensitize prostate cancer cells to radiation (RT) and androgen deprivation (AD). LNCaP (wild type p53, androgen sensitive) and PC3 (p53 null, androgen insensitive) cells were cultured in vitro for 2 days in complete medium before AS was added. LNCaP cells were also cultured in AD (using charcoal-stripped serum) or AD+R1881 (synthetic androgen 10-10 M) medium. AS was added at 200 nM (apoptosis assays) or 500 nM (clonogenic assays) for 24–48 hr before RT (2–6 Gy). After RT, cells were processed at 24 hr for Annexin V staining by flow cytometric analysis and for caspases 3+7 quantification by fluorometric assay, and immediately for clonogenic survival (measured 12–14 days later). The treatment groups are shown in the Table (LC = Lipofectin control; MM = mismatch) Statistical comparisons were accomplished by one-way analysis of variance using the Bonferroni test. The Table displays the apoptosis results; the mean ± SEM is shown and the asterisk denotes significance between the group above and below, while the number sign denotes significance between AS vs. AS+RT or AS vs. AS+AD. For LNCaP cells, AS+RT resulted in significantly greater apoptosis by Annexin V staining, as compared to all other groups. In contrast, the difference between AS vs. AS+RT by caspase 3+7 was not significant. LNCaP survival by clonogenic assay was significantly reduced by AS+RT over the controls (Figure). Moreover, when AS was combined with AD, there was significantly more apoptosis over all other groups, which paralleled the clonogenic survival findings (Figure). For PC3 cells, AS+RT did not result in significantly greater apoptosis by caspase 3+7, or Annexin V. PC3 overall survival by clonogenic assay was reduced by AS+RT over the controls but the differences were not significant. Our results demonstrate the ability of AS to sensitize androgen responsive (LNCaP) prostate cancer cells to RT and AD. Although androgen insensitive (PC3) cells were not significantly sensitized by AS to RT, there was clearly an increased cell death response to AS. Antisense PKA shows promise in the treatment of high-risk prostate cancer. View Large Image Figure ViewerDownload (PPT)