To further characterize the mechanism by which inhibition of GRP78 induces cell death in MIA PaCa-2 and S2-VP10 cells, two different markers of apoptosis were monitored: caspase-3 activation and annexin V staining. shown to be an effective compound against pancreatic cancer. Our results show that triptolide induces the UPR by activating the PKR-like ER kinase-eukaryotic initiation factor 2 axis and the inositol-requiring enzyme 1-X-box-binding protein 1 axis of the UPR and leads to chronic ER stress in pancreatic cancer. Our results further show that glucose-regulated protein 78 (GRP78), one of the major regulators of ER stress, is downregulated by triptolide, leading to cell death by apoptosis in MIA PaCa-2 cells and autophagy in S2-VP10 cells. using siRNA also kills pancreatic cancer cells by activating apoptosis in MIA PaCa-2 cells and autophagy in S2-VP10 cells, which is in accordance with our earlier study with triptolide. Furthermore, we also show that triptolide-induced ER stress is GSK256066 important in cell death, since inhibition of ER stress by knockdown of shows a significant rescue of triptolide-mediated cell death. EXPERIMENTAL PROCEDURES Reagents. Triptolide was purchased from Calbiochem (San Diego, CA); siRNA pool, siRNA pool, and nonsilencing small interfering RNA (siRNA) from Dharmacon (Lafayette, CO); and Opti-MEM I, DMEM, and RPMI 1640 tissue culture medium from Invitrogen (Carlsbad, CA). The WST-8 viability assay was purchased from Dojindo Molecular Technologies (Gaithersburg, MD), the Caspase-Glo 3/7 assay kit from Promega (San Luis Obispo, CA), and the bicinchoninic acid protein assay kit from Pierce (Rockford, IL). All other reagents were obtained from Sigma Aldrich (St. Louis, MO). Cell culture. The pancreatic cancer cell line MIA PaCa-2 [American Type Culture Collection (ATCC)] was grown and propagated in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 g/ml streptomycin; S2-013 and S2-VP10 cell lines (kind gift from Prof. D. Buschbaum, University of Alabama) were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 g/ml streptomycin, while AsPC1 cells (ATCC) were cultured in RPMI 1640 medium supplemented with INSL4 antibody 20% FBS. The human pancreatic ductal epithelial cells (ATCC) were cultured in keratinocyte medium supplemented with bovine pituitary hormone and EGF. All cells were maintained at 37C in a humidified air atmosphere with 5% CO2. ON-TARGETplus SMARTpool human siRNA, human siRNA, and heat shock protein 70 (and was carried out using primers procured from Qiagen (Valencia, CA). RNA was isolated from the different cell lines and from the tumor samples according to the manufacturer’s instruction using TRIzol (Life Technologies, Carlsbad, CA). Total RNA (1 g) was used to perform real-time PCR (Applied Biosystems 7300 real-time PCR system) using the QuantiTect SYBR Green PCR kit (Qiagen) according to the manufacturer’s instructions. All data were normalized to the housekeeping gene 18S (18S QuantiTect primer assay, Qiagen). Western blotting. Cell lysates for Western blotting were prepared as described previously (28). Equal amounts of protein samples were resolved by SDS-PAGE using precast 10% or 4C15% TrisHCl gels GSK256066 (Bio-Rad), transferred onto nitrocellulose membranes (Bio-Rad), processed for immunoblotting with specific antibodies, and detected using the enhanced chemiluminescence system. Anti-LC3B, anti-Grp78, anti-phosphorylated (Ser51) eIF2, anti-total eIF2, and Ire1 antibodies were purchased from Cell Signaling Technology. Anti–actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunofluorescence. Pancreatic cancer cells were plated in chamber slides and incubated for 24 h at 37C. The slides were treated with triptolide for 24 h, fixed with 3.7% paraformaldehyde, and permeabilized with 0.1% Triton X-100. The slides were incubated with a 1:200 dilution of rabbit polyclonal anti-LC3B antibody (Cell signaling Technologies) and a 1:800 dilution of Alexa 488-conjugated donkey anti-mouse IgG (Molecular Probes) for LC3 staining. The slides were mounted using Prolong Gold antifade with 4,6-diaminido-2-phenylindole (Molecular Probes). Immunofluorescence images were obtained on a confocal microscope (Nikon Eclipse Ti) with a 60 oil-immersion objective. EZ-C software version 3.80 was used to obtain test; < 0.05 was considered statistically significant. GSK256066 RESULTS Triptolide induces ER stress in pancreatic cancer cells via activation of the PERK-eIF2 and Ire1-XBP1 arms of the UPR GSK256066 cascade. Previous results from our laboratory showed that triptolide downregulated HSP70, one of the major chaperones in a cancer. GSK256066