(A) BxPC-3 and PANC-1 cells were treated with different
concentration of DHA for 24 h in the presence or absence of 10 μmol/L Torin 1 purchase SP600125 pretreatment for 1 h. The expression levels of the LC3-I and LC3-II proteins were subsequently analyzed by immunoblotting. (B) BxPC-3 cells transfected with the GFP-LC3 plasmid, followed by 50 μmol/L DHA for 24 h with or without SP600125 (10 μmol/L). The number of GFP-LC3 dots was subsequently scored in 100 transfected cells. (C) BxPC-3 cells were treated with 50 μmol/L DHA for 24 h in the absence or presence of JNK1/2 siRNA. The expression levels of phospho-JNK and Beclin 1 protein were subsequently analyzed by immunoblotting. (D) BxPC-3 cells transfected with a non-targeting RNA or a JNK1/2-targeted siRNA were treated with 50 μmol/L DHA for 24 h. At the end of the treatment, cell viability was measured using a CCK-8 assay. *P < 0.05. To determine if JNK activation is required for Beclin 1 expression in the context of DHA-induced autophagy, JNK expression was knocked-down using a siRNA directed against JNK1/2. siRNA transient transfection down-regulated JNK (Figure 5C). More importantly, siRNA-mediated JNK down-regulation prevented Tozasertib the DHA-induced up-regulation of
Beclin 1 protein in addition to efficiently inhibiting the level of JNK phosphorylation in pancreatic cancer cells (Figure 5C). These findings suggest that JNK could be directly involved in the DHA-induced increased Beclin 1 expression. To test whether blockage of DHA-activated autophagy through JNK inhibition could enhance cytotoxicity, tumor cells were transfected with a non-targeting RNA or a siRNA targeting JNK, and were then exposed to DHA. DHA cytotoxicity was significantly increased by silencing the expression of JNK in these cells (Figure 5D). Taken together, these findings indicate that JNK could be directly involved in the DHA-induced increased Beclin
1 expression. CYC202 Furthermore, it can be concluded that the inhibition of JNK could enhance the efficacy of DHA by inhibiting autophagy. Beclin 1 siRNA knock-down blocks DHA-induced autophagy To potentially use the intrinsic role of Beclin 1 in DHA-induced autophagy, we investigated the effects of Beclin 1 knock-down on DHA-induced apoptosis. We designed Liothyronine Sodium siRNAs down-regulating Beclin 1 expression. Beclin 1 silencing significantly inhibited LC3-II induction by DHA (Figure 6A). Fewer Beclin 1-silenced cells exhibited GFP-LC3 punctae compared to the control DHA- and siRNA-treated cells (Figure 6B). These results suggest that Beclin 1 could play a crucial role in DHA-induced autophagy. Figure 6 Beclin 1 is required for DHA-induced autophagy. (A) BxPC-3 cells transfected with a non-targeting RNA or a Beclin 1-targeted siRNA were treated with 50 μmol/L DHA for 24 h. At the end of treatment, the expression levels of the Beclin 1, LC3-I, and LC3-II protein were analyzed by immunoblotting.