1. ¹Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA
2. ²Burnham Institute for Medical Research, La Jolla, CA, USA
3. ³Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

Correspondence: Dr J An, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: jingan@burnham.org

Received 28 July 2004; Revised 22 May 2006; Accepted 9 June 2006; Published online 7 August 2006.

Abstract

Bcl-2 overexpression is an important mechanism underlying the aggressive behavior of prostate cancer cells and their resistance to radio- or chemotherapy. HA14-1, a recently discovered organic Bcl-2 inhibitor, potently induces apoptosis in various human cancer cells. Sequential exposure of radioresistant LNCaP (wild-type (wt) p53), LNCaP/Bcl-2 (wt p53) and PC3 (mutant p53) prostate cancer cells to a minimally cytotoxic concentration of 10 M HA14-1 for 1 h followed by 1–6 Gy gamma radiation, resulted in a highly synergistic (combination index <1.0) induction of cell death as determined by an apoptosis assay at 72 h, and a clonogenicity assay at 12 days, after the initial treatment. The reverse treatment sequence did not cause a synergistic induction of cell death. When compared to individual treatments, cell death induced by the combined treatment was associated with dramatically increased reactive oxygen species (ROS) generation, c-Jun N-terminal kinase (JNK) activation, Bcl-2 phosphorylation, cytochrome c release, caspase-3 activation and DNA fragmentation. Exposure to either 200 g/ml of the antioxidant alpha-tocopherol or 10 M JNK inhibitor SP600125 before the combined treatment resulted in decreased activation of JNK and caspase-3 as well as decreased DNA fragmentation. However, treatment with the pancaspase inhibitor carbobenzoxyl-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone before the combined treatment inhibited apoptosis without affecting JNK activation, and this inhibitory effect was enhanced in the presence of alpha-tocopherol or SP600125. Taken together, our results indicate that HA14-1 potently sensitizes radioresistant LNCaP and PC3 cells to gamma radiation, regardless of the status of p53. ROS and JNK are important early signals that trigger both caspase-dependent and -independent cell death pathways and contribute to the apoptotic synergy induced by the combined treatments.