Similarly, single incubation with DHA showed concentration-dependent reductions in cell survival, and PFT significantly

inhibited the cytotoxic effects of DHA in both cell types ( Fig. 2). Thus, PFT abrogated DHA-induced cytotoxicity Angiogenesis inhibitor independently of p53 expression. We examined the effects of PFT on DHA-induced oxidative stress, as indicated by DCF fluorescence (Fig. 3). Induction of oxidative stress by DHA at 120 μM was significantly elevated after 1 h of incubation (126.8 ± 12.8%; p < 0.05), and increased further at 2, 4 and 6 h (154.2 ± 8.1%, 196.6 ± 32.8% and 229.8 ± 20.3%, respectively), as compared to controls (p < 0.01). These DHA-induced increase in oxidative stress were abrogated by pretreatment with PFT after incubation for 1 h (110.8 ± 3.6%; p < 0.05), selleck products and were further blocked by longer incubation for 2, 4 and 6 h (113.8 ± 12.4%, 106.5 ± 2.3% and 103.9 ± 12.2%, respectively; p < 0.01). To confirm the inhibitory effects of PFT on DHA-induced oxidative stress and whether PFT has antioxidant capacity, we performed TAC assay.

As shown in Fig. 4, PFT does not show antioxidant capacity when compared with Trolox, even at 2000 μM. In order to explore the inhibition mechanisms of PFT on DHA-induced cytotoxicity, we focused on the induction of autophagy (Fig. 5). Levels of LC3A-II, which is an LC3-phosphatidyl-ethanolamine conjugate and a promising autophagosomal marker (Asanuma et al., 2003), showed concentration-dependent increases in incubation with DHA on Western blotting (Fig. 5A and B). Expression was completely blocked by PFT. This inhibitory effect of PFT was also observed in both Hep3B and Huh7 by incubation with high concentrations of DHA at 200 μM (Fig. 5C and D). On immunofluorescence, PFT incubation for 24 h Aurora Kinase showed no changes when compared with control groups, but the DHA-treated group showed increased numbers of LC3-positive cells, and this effect

was apparently blocked by pretreatment with PFT (Fig. 5E). Similarly, after transfection with pAcGFP-LC3 in HepG2 cells, PFT blocked the formation of LC3 puncta in cells on incubation with DHA (see Supplementary data 1). Next, we examined the release of cytochrome c from mitochondria to cytosol by DHA ( Fig. 6). Cytochrome c is a critical mediator of mitochondrial cell death. COX IV, a specific mitochondrial marker, was detected in mitochondrial fractions, indicating good-quality mitochondrial preparations ( Fig. 6A). Cytochrome c decreased in the mitochondrial fraction and increased in the cytosol fraction after incubation with DHA. On densitometric measurement of bands on Western blotting (ratio is expressed as cytosol/mitochondria fraction), single incubation with DHA for 1 or 4 h gave ratios of 0.95 ± 0.15 or 1.33 ± 0.29 when compared with controls, and this release of cytochrome c was significantly suppressed by pretreatment with PFT (0.56 ± 0.