Thus, our results indicate that the inhibition mechanisms of PFT on DHA-induced cytotoxicity and autophagy depend on mitochondrial damage. It selleck has not yet been shown

that mitochondria are selected for autophagy depending on the level of oxidative damage to their membranes, but some evidence suggests that mitochondrial permeability plays a role in the initiation of autophagy (Lemasters et al., 2002 and Mijaljica et al., 2007). As shown in Fig. 7, single incubation with DHA showed concentration- and time-dependent decreases in ΔΨM after incubation for 12 h. Fig. 3 and our previous report (Kanno et al., 2011) show that DHA-induced oxidative stress significantly increases after incubation, and release of cytochrome c increases after incubation with DHA ( Fig. 6). Interestingly, changes in ΔΨM by DHA were not observed before the detection of oxidative stress and release of cytochrome c; changes in ΔΨM occur in a comparatively later stage of DHA treatment.

JC-1 (prototype of JC-10) is reported to be a more reliable indicator of ΔΨM than other dyes ( Mathur et al., 2000), and it has been indicated that J-aggregate-forming lipophilic cations might be useful for probing ΔΨM in living cells ( selleck products Reers et al., 1995). In this study, pretreatment with PFT increased in J-aggregate formation under basal cellular conditions ( Fig. 7). It has been demonstrated that ΔΨM controls ROS production ( Sanderson et al., 2013). Several reports have shown that chemical reagent-induced elevation STK38 of ΔΨM reduces ROS production and indicates a cytoprotective effect. (−) Deprenyl is an irreversible inhibitor of monoamine oxidase-B, which protects cells from hypoxia/re-oxygenization, maintains ΔΨM and prevents

increases in ROS induced by hypoxia/re-oxygenation in a dose-dependent manner ( Simon et al., 2005). 1,2-Dimethylhydrazine treatment increases the formation of J-aggregate at higher ΔΨM, decreases ROS function and restricts cell death ( Saini and Sanyal, 2012). These reports suggest that higher ΔΨM protects ROS production and results in the prevention of ROS-mediated cytotoxicity. We speculate that PFT activates ΔΨM in living cells, thereby increasing the threshold of sensitivity produced by DHA-induced oxidative stress. Thus, PFT may protect against mitochondrial damage by DHA. It is conceivable that increases in J-aggregate represent respiration or energy synthesis hot spots in the cells and may protect against cellular injury by DHA. It is unclear how PFT affects mitochondria and increases J-aggregate, and we are therefore studying this issue further. Based on the present results, we propose the following mechanism for the effects of PFT against DHA-induced cytotoxicity. First, pretreatment with PFT protects against DHA-induced mitochondria damage by increasing ΔΨM in living cells.