Immunoblotting revealed dose- and time-dependent increases in Beclin 1 expression in cells exposed to DHA (Figure  3B). These findings demonstrated that treatment with DHA activates JNK and Beclin 1 in both pancreatic cancer cell lines in a dose- and time-dependent manner. Up-regulation of JNK expression following DHA treatment depends on ROS JNK pathway over-activation is crucial to many processes leading to cell death, including chronic and acute oxidative stress. Although

ROS can increase JNK signaling via the activation of upstream kinases or the inactivation of phosphatases, other unknown mechanisms find more are likely to contribute to ROS-induced JNK increases in pancreatic cancer cells. To exclude the possibility that other mechanisms were responsible for our observations, we measured ROS levels in response to DHA. ROS were increased after DHA treatment and did not differ between the two tested cell lines (Figure  4A). Figure 4 JNK expression induced by DHA is dependent on ROS generation. (A) BxPC-3 and PANC-1 cells were treated with 50 μmol/L DHA for different times, and then subjected to flow cytometry to measure ROS levels, as described in the Materials and Methods section. (B, C) BxPC-3 and PANC-1 cells were treated with 50 μmol/L DHA for 24 h in the presence or absence of 10 μmol/L

SP600125 or 10 mmol/L NAC pretreatment for 1 h and then subjected to flow cytometry to Cilomilast measure the levels of ROS. (D) immunoblot analysis of the phospho-JNK levels in BxPC-3 and PANC-1 cells treated with the indicated concentrations

of DHA for 24 h in the presence or absence of 10 mmol/L NAC. *P < 0.05. To further determine whether DHA treatment requires JNK activation to generate ROS, we pre-treated BxPC-3 cells with SP600125 (a specific JNK inhibitor) for 1 h, before exposing them to DHA. In contrast to DHA treatment alone, SP600125 pretreatment prevented alterations in ROS levels (Figure  4B). To examine whether ROS inhibition impacted JNK signaling, we compared JNK activation with or without N-acetyl-L-cysteine (NAC, a ROS inhibitor). NAC pretreatment significantly lowered intracellular ROS compared with DHA-treated from cells (Figure  4C). More importantly, the degree of JNK activation after DHA treatment was decreased in the cells pretreated with NAC (Figure  4D), and this decreased JNK activation was related to the inhibition of ROS formation. These results indicate that JNK expression following DHA treatment depends on ROS. Inhibition of JNK expression down-regulates beclin 1 and reduces autophagy To further assess the role of JNK in DHA-induced autophagy, cells were pretreated with SP600125 (10 mM) for 1 h, and were then exposed to DHA. In contrast to DHA alone, SP600125 pretreatment blocked the increase in LC3-II induced by DHA (Figure  5A). Furthermore, SP600125 treatment decreased the punctate foci of LC3 in the cytoplasm (Figure  5B).