Resistance to apoptotic-induction is a major obstacle to pancreatic cancer treatment. In the search for alternative treatment strategies, one approach may be to bypass apoptosis by induction of alternative cell death pathways, such as the recently described death pathway called aponecrosis. We found that human pancreatic cancer cell lines underwent rapid cell death when treated with clinically achievable concentrations of arsenic trioxide (A), ascorbic acid (A) and disulfiram (antabuse, A). AAA had no greater toxicity than arsenic trioxide alone (48%) in normal human peripheral blood stem cells (CFU-GEMM). When pancreatic cancer cells were treated with the agents separately or in binary combinations, death was minimal, but AAA after 48h had a 12 fold greater toxicity compared to arsenic trioxide alone as measured by TUNEL analysis. Within 4h of exposure to AAA, trypan blue-positive cells doubled, accompanied by an 80% decline in intracellular ATP. Caspase-3 activity and PARP cleavage were not observed, and electron microscopy demonstrated both apoptotic and aponecrotic cells after 48h. The reactive oxygen scavenger, N-acetyl cysteine protected cells from death, and restored intracellular ATP levels. Consistent with this finding, we found that intracellular superoxide levels corresponded linearly to cell death, which was quenched by N-acetyl cysteine. Loss of lysosomal integrity followed the rise in intracellular superoxide levels closely, and cell death was partially reversed after addition of the cathepsin-D inhibitor pepstatin A. This implies that cathepsin D released from lysosomes is involved in AAA-induced cell death. We hypothesize that the mechanism of cell death is based on lysosomal damage, which triggers a caspase-independent form of apoptosis or aponecrosis. Activation of an alternative pathway to cancer cell death as demonstrated here, may lead to novel treatments for pancreatic cancer utilizing clinically achievable concentrations of AAA.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]