Artemisinin has been shown to contain an endoperoxide bridge, which reacts with iron to form ROS. Interestingly, we observed that DHA also activates autophagy in pancreatic cancer cells, and various findings indicate that a number of antineoplastic therapies induce a type of protective, Ixazomib datasheet pro-survival autophagy [29–31]. Moreover, ROS-mediated JNK activation is required for the formation of autophagosomes . However, the mechanism by which JNK induces autophagy and the association with anticancer therapy remains mostly unknown. Therefore, in this present study, we explored the involvement of JNK
activation and Beclin 1 expression in DHA-induced autophagy. The aim of the present study was to assess the exact relationships between Beclin 1 expression, JNK pathway activation, and autophagy. We demonstrated that DHA-induced autophagy involved the JNK pathway in pancreatic cancer cell lines, resulting in increased expression of Beclin 1. SP600125 or small interfering RNAs (siRNAs) targeting JNK1/2 inhibited the up-regulation of Beclin 1, as well as autophagy. Results from the present study provide further clues explaining Beclin 1 regulation click here in autophagy induced by cancer treatments. Materials and methods Cell lines and culture The human pancreatic cancer cell lines BxPC-3 (CRL-1687) and PANC-1 (CRL-1469) were
purchased from the American Type Culture Collection (ATCC, Manassas, USA). The cells were cultured in RPMI 1640 (SH30809.01B, Hyclone, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (16000, GIBCO, Invitrogen Inc., Carlsbad,
CA, USA), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen, 15070–063). Cells were maintained at 37°C in a humidified P-type ATPase atmosphere containing 5% CO2. Reagents and antibodies The following reagents were purchased from Sigma-Aldrich (St-Louis, MO, USA): DHA (D7439), NAC (A7250), 3MA (M9281), rapamycin (R0395), and SP600125 (S5567). The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): JNK (sc-7345), p-JNK (sc-6254), and β-actin (sc-130301). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): caspase-3 (9665), LC3 (2775), and Beclin 1 (3738). Cell proliferation assay Cells were plated in 96-well or 6-well cell culture plates (5 × 103 cells per well) and treated with various compounds, as indicated in the figure legends. At the end of treatments, cell proliferation was evaluated using a Cell Counting Kit-8 (CCK-8, CK04-13, Dojindo Molecular Technologies, Kimamoto, Japan) or Crystal Violet (C6158, Sigma) staining according to the instructions of the manufacturer, or by photometric measurements to determine cell viability. Three or four independent experiments were performed for each assay condition. Electron microscopy Cells were harvested by trypsinization, fixed in 2.5% glutaraldehyde/4% paraformaldehyde in 0.