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

The therapeutic efficacy of Gem (R) 231, a second generation antisense molecule targeted to the RI alpha subunit of PKA(RI alpha) (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(RI alpha), levels in tumors were quantified via immunohistochemical (IHC) staining and image analysis. IHC measurements in LNCaP cells exhibited that AS-PKA reduced PKAR(RI alpha), 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.