The Roles of ROS and Caspases in TRAIL-Induced Apoptosis and Necroptosis in Human Pancreatic Cancer Cells

May The Roles of ROS and Caspases in TRAIL- Induced Apoptosis and Necroptosis in Human Pancreatic Cancer Cells Min Zhang 0 1 Nanae Harashima 0 1 Tamami Moritani 0 1 Weidong Huang 0 1 Mamoru Harada 0 1 0 1 Department of Biochemistry and Molecular Biology, Ningxia Medical University , Shengli Street, Yinchuan , China , 2 Department of Immunology, Shimane University Faculty of Medicine , Izumi, Shimane , Japan 1 Academic Editor: Ajay Pratap Singh, University of South Alabama Mitchell Cancer Institute, UNITED STATES Death signaling provided by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) can induce death in cancer cells with little cytotoxicity to normal cells; this cell death has been thought to involve caspase-dependent apoptosis. Reactive oxygen species (ROS) are also mediators that induce cell death, but their roles in TRAIL-induced apoptosis have not been elucidated fully. In the current study, we investigated ROS and caspases in human pancreatic cancer cells undergoing two different types of TRAIL-induced cell death, apoptosis and necroptosis. TRAIL treatment increased ROS in two TRAIL-sensitive pancreatic cancer cell lines, MiaPaCa-2 and BxPC-3, but ROS were involved in TRAIL-induced apoptosis only in MiaPaCa-2 cells. Unexpectedly, inhibition of ROS by either N-acetyl-Lcysteine (NAC), a peroxide inhibitor, or Tempol, a superoxide inhibitor, increased the annexin V-/propidium iodide (PI)+ early necrotic population in TRAIL-treated cells. Additionally, both necrostatin-1, an inhibitor of receptor-interacting protein kinase 1 (RIP1), and siRNA-mediated knockdown of RIP3 decreased the annexin V-/PI+ early necrotic population after TRAIL treatment. Furthermore, an increase in early apoptosis was induced in TRAIL-treated cancer cells under inhibition of either caspase-2 or -9. Caspase-2 worked upstream of caspase-9, and no crosstalk was observed between ROS and caspase-2/-9 in TRAIL-treated cells. Together, these results indicate that ROS contribute to TRAIL-induced apoptosis in MiaPaCa-2 cells, and that ROS play an inhibitory role in TRAIL-induced necroptosis of MiaPaCa-2 and BxPC-3 cells, with caspase-2 and -9 playing regulatory roles in this process. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Members of the tumor necrosis factor (TNF) cytokine family, such as TNF and Fas ligand (FasL), play important roles in inflammation and immunity [1]. Although TNF-related apoptosis-inducing ligand (TRAIL) is a member of this family, this molecule can induce cancer cell death while causing almost no cytotoxicity to normal cells [2]. There are positive and Competing Interests: The authors have declared that no competing interests exist. negative receptors; death receptor (DR)4 and DR5 provide pro-apoptotic signaling, whereas decoy receptor (DcR)1 and DcR2 inhibit apoptotic signaling [3]. Normal cells are reported to show TRAIL-resistance with their preferential expression of DcRs [4]. Therefore, TRAIL and its DR are expected to be useful anti-cancer molecules and targets and have been used and targeted in several clinical trials [5, 6]. Binding of TRAIL to DR on cancer cells provides caspase8-dependent death signaling and triggers the extrinsic apoptotic pathway [7]. Activation of caspase-8 transforms Bid to tBid, thereby promoting the mitochondria-mediated caspase-9-dependent intrinsic apoptotic pathway [8]. Alternatively, ROS (reactive oxygen species) also play important roles in cell death and signaling [9]. ROS induce intrinsic apoptosis by triggering DNA damage [10]. Conversely, DNA damage induces ROS production [11]. DNA damage and/or ROS production can trigger caspase-9-dependent apoptosis. Additionally, some reports suggested that ROS is involved in apoptosis in TRAIL-treated human cancer cells [1214]. However, the roles of ROS in TRAIL-induced cancer cell death have yet to be investigated fully. In addition to apoptosis, necroptosis is now recognized as another form of programmed cell death [15, 16]. Necroptosis is programmed necrosis that can be activated upon stimulation by TNF, FasL, or TRAIL. The roles and mechanisms of apoptosis and necrosis have been well established, whereas those of necroptosis have been the focus of recent investigations [1618]. Necroptosis has received much attention as a type of cell death that induces inflammation [17]. Under caspase-8 inhibition, receptor-interacting protein kinase 1 (RIP1) and RIP3 form a complex and trigger necroptosis [19, 20]; additional recent reports have revealed that RIP3 plays a central role in the process [18, 21]. Although some reports suggest that TRAIL can induce necroptosis in cancer cells [2224], the mechanisms have not been elucidated fully. This study investigated the roles of ROS and caspases in TRAIL-induced apoptosis and necroptosis of human pancreatic cancer cells. Among four human pancreatic cancer cell lines, ROS levels were elevated in only two TRAIL-sensitive lines: MiaPaCa-2 and BxPC-3. However, ROS played a pro-apoptotic role only in MiaPaCa-2 cells, but not in BxPC-3 cells. In these experiments, we found that TRAIL treatment under ROS inhibition increased the population of annexin V-/propidium iodide (PI)+ early necrotic cells in MiaPaCa-2 and BxPC-3 cells, suggesting that ROS played an inhibitory role in TRAIL-induced necroptosis in both cell lines. In addition, necrostatin-1 (a RIP1 inhibitor) decreased the annexin V-/PI+ population in these TRAIL-treated cells, and siRNA-mediated knockdown of RIP3 showed similar results in BxPC-3 cells, implying that the cell death observed was caused by necroptosis. Finally, necroptosis was promoted by TRAIL treatment under the inhibition of either caspase-9 or -2, with the latter considered an orphan caspase whose roles in cell death have not been elucidated fully [25, 26]. Together, these findings indicate that ROS are increased in TRAIL-sensitive MiaPaCa-2 and BxPC-3 cells, but that ROS are involved in apoptosis only in TRAIL-treated MiaPaCa-2 cells. Our findings also showed that ROS and caspase-9/-2 play regulatory roles in the TRAIL-induced necroptosis of human pancreatic cancer cells. Two human pancreatic cancer cell lines (AsPC-1 and BxPC-3) were purchased from the American Type Culture Collection (Manassas, VA, USA). Two other human pancreatic cancer cell lines (MiaPaCa-2 and Panc-1) were kindly provided by Dr. K. Takenaga (Shimane University Faculty of Medicine) [27]. These cell lines were maintained in DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (Invitrogen, Grand Island, NY, USA) and 20 g/ml gentamicin (Sigma-Aldrich). PrEC is a normal prostate epithelial cell line purchased from Lonza (Walkersville, MD, USA) and was maintained in PrEBM (Lonza). Cell viability was analyzed using the WST-8 assay (Nacalai Tesque, Kyoto, Japan). At the end of the incubation period, 10 l WST-8 solution was added to each well, and the plat (...truncated)