PKA knockdown enhances cell killing in response to radiation and androgen deprivation

Abstract

The therapeutic efficacy of Gem®231, a second generation antisense molecule targeted to the RIα subunit of PKA(RIα) (AS-PKA), administered in combination with androgen deprivation (AD) and radiation therapy (RT), was examined in androgen sensitive (LNCaP) and insensitive (PC3) cell lines. Apoptosis was assayed by Caspase 3 + 7 activity and Annexin V binding. AS-PKA significantly increased apoptosis in vitro from RT (both lines), with further increases in LNCaP cells grown in AD medium. In LNCaP cells, AD increased phosphorylated mitogen activated protein-kinase (pMAPK), which was reduced by AS-PKA relative to the mismatch (MM) controls. AS-PKA also reduced pMAPK levels in PC3 cells. Cell death was measured by clonogenic survival assays. In vivo, LNCaP cells were grown orthotopically in nude mice. Tumor kinetics were measured by magnetic resonance imaging and serum prostate-specific antigen. PC3 cells were grown subcutaneously and tumor volume assessed by caliper measurements. In PC3 xenografts, AS-PKA caused a significant increase in tumor doubling time relative to MM controls as a monotherapy or in combination with RT. In orthotopic LNCaP tumors, AS-PKA was ineffective as a monotherapy; however, it caused a statistically significant increase in tumor doubling time relative to MM controls when used in combination with AD, with or without RT. PKA(RIα) levels in tumors were quantified via immunohistochemical (IHC) staining and image analysis. IHC measurements in LNCaP cells exhibited that AS-PKA reduced PKA(RIα) levels in vivo. We demonstrate for the first time that AS-PKA enhances cell killing androgen sensitive prostate cancer cells to AD ± RT and androgen insensitive cells to RT.

Figure 1

Western blot analysis of protein levels in LNCaP cells (A); protein levels in PC3 cells (B); immunohistochemical staining of PKA expression from LNCaP tumors grown in vivo (C; the error bars indicate the standard deviation); and clonogenic survival assay results (D–F). The p-values on the charts in D–F are for the AS-PKA groups vs controls. The AS-PKA vs MM comparisons were: 2 Gy, p = 0.063; 4 Gy, p = 0.005; 6 Gy, p = 0.0183 (D); 2 Gy, p = 0.063; 4 Gy, p = 0.05; 6 Gy, p = 0.049 (E); 2 Gy, p = 0.102; 4 Gy, p = 0.884; 6 Gy, p = 0.356 (F). The Bonferroni test was used for all comparisons.

Figure 2

Growth curves for LNCaP (A) and PC3 (B) tumors in nude mice. The curves represent the treatment group average of values fitted by a kinetic model for individual animals (n=8–13). For LNCaP tumors (A), combination therapies were performed with mismatch control (MM - left), and AS-PKA oligos (right), and tumor size estimated by MRI measurements (top) and serum PSA levels (bottom). For PC3 tumors (B), tumor size was estimated by caliper measurements.