Piperlongumine rapidly induces the death of human pancreatic cancer cells mainly through the induction of ferroptosis

Pancreatic cancer is one of the most lethal types of cancer with a mortality rate of almost 95%. Treatment with current chemotherapeutic drugs has limited success due to poor responses. Therefore, the development of novel drugs or effective combination therapies is urgently required. Piperlongumine (PL) is a natural product with cytotoxic properties restricted to cancer cells by significantly increasing intracellular reactive oxygen species (ROS) levels. In the present study, we demonstrated that PL induced cancer cell death through, at least in part, the induction of ferroptosis, as the cancer cell-killing activity was inhibited by the antioxidant, N-acetylcysteine, ferroptosis inhibitors (ferrostatin-1 and liproxstatin-1) and the iron chelator, deferoxamine (DFO), but not by the apoptosis inhibitor, Z-VAD-FMK, or the necrosis inhibitor, necrostatin-1. Cotylenin A (CN-A; a plant growth regulator) exhibits potent antitumor activities in several cancer cell lines, including pancreatic cancer cell lines. We found that CN-A and PL synergistically induced the death of pancreatic cancer MIAPaCa-2 and PANC-1 cells for 16 h. CN-A enhanced the induction of ROS by PL for 4 h. The synergistic induction of cell death was also abrogated by the ferroptosis inhibitors and DFO. The present results revealed that clinically approved sulfasalazine (SSZ), a ferroptosis inducer, enhanced the death of pancreatic cancer cells induced by PL and the combined effects were abrogated by the ferroptosis inhibitors and DFO. SSZ further enhanced the cancer cell-killing activities induced by combined treatment with PL plus CN-A. On the other hand, the synergistic induction of cell death by PL and CN-A was not observed in mouse embryonic fibroblasts (MEFs), and SSZ did not enhance the death of MEFs induced by PL plus CN-A. These results suggest that the triple combined treatment with PL, CN-A and SSZ is highly effective against pancreatic cancer.