Background and Aims: The inhibition of anti-apoptotic Bcl-2 and Bcl-xL proteins is a novel strategy for cancer treatment. Two inhibitors of Bcl-2/Bcl-xL, BH3I-2’ and HA-14, have recently become available and were shown to induce death in several different cancer cells. Pancreatic cancer (PaCa) cells are notoriously resistant to apoptosis and overexpress both Bcl-2 and Bcl-xL. The aim of this study was to determine the effects of the Bcl-2/Bcl-xL on apoptosis in PaCa cells and elucidate the mechanisms underlying these effects. Methods: Bcl-2 and Bcl-xL were inhibited pharmacologically by BH3I-2’ and HA-14, or by siRNA silencing in MIA PaCa-2 and PANC-1 cells. We measured apoptosis with Cell Death ELISA and Annexin V/PI staining using flow cytometry ; cytochrome c release by Western blot; reactive oxygen species (ROS), with the fluorescent dye DCF. Mitochondria were isolated from PaCa cells by differential centrifugation. In isolated mitochondria, we measured the effects of BH3I-2’ and HA-14 on ΔΨm, respiration and ROS using, TPP+ and Clark electrodes and the fluorescent dye Amplex-Red, respectively. Results: Bcl-2/Bcl-xL inhibitors and the siRNA silencing of Bcl-2/Bcl-xL both induced apoptosis effectively. The inhibitors induced apoptosis in the range of concentrations (25-100 μM) at which they inhibited Bcl-2/Bcl-xL activities, as measured by fluorescence polarization assay. At the molecular level, inhibiting Bcl-2/Bcl-xL stimulated cytochrome c release and inhibited mitochondrial ROS production, resulting in a decrease in intracellular ROS. The observed stimulation of cytochrome c release, a key mediator of apoptosis, represents one mechanism through which the Bcl-2/Bcl-xL inhibition induces apoptosis in PaCa cells. Another mechanism whereby Bcl-2/Bcl-xL inhibition induces death responses could be through inhibition of ROS. Indeed, we recently showed that ROS play a pro-survival role in PaCa cells by activating anti-apoptotic pathways such as PI3K/Akt. ROS are also known to mediate proliferation of cancer cells. Conclusions: Bcl-2/Bcl-xL promote survival of pancreatic cancer cells at least in part by their ability to increase ROS production. The ROS, in turn, promote proliferation and inhibit apoptosis.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA