the LC3-II/LC3-I ratio, an indicator of autophagosome formation, in a dose- and time-dependent manner. SSHE also decreased SQSTM1/ p62 protein levels, one of the specific substrates degraded through the autophagy-lysosomal pathway, in Panc-1 cells. SSHE activated AMPK, a positive regulator of autophagy, and inhibited mTOR, a negative autophagic regulator. The autophagy inhibitors 3-methyladenine and chloroquine partially prevented cell death. Morphologically, SSHE-get Dehydroxymethylepoxyquinomicin treated cells showed massive vacuolization PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19666584 of the cytoplasm approximately 24 h after treatment. These cytoplasmic vacuoles were likely autophagosomes because GFP-LC3 puncta appeared after treatment with SSHE. Some LC3 puncta were co-localized with MitoTracker-positive mitochondria in the cells treated PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19666601 with 100 g/ml SSHE for 20 h, suggesting the occurrence of mitophagy. Thus, SSHE-induced cell death seemed to be autophagic cell death. However, TEM analyses 28 h after SSHE treatment showed electron-dense mitochondria, empty vacuoles and focal perinuclear swelling, which are not observed in classical type autophagy. Thus, we concluded that SSHE-induced cell death was caspase-independent and resembled autophagy cell death. This form of cell death coincides well with autosis, a recently characterized Na+,K+-ATPase-regulated form of cell death. Unfortunately, because the cardiac glycoside digoxin, an 
antagonist of Na+,K+-ATPase that is reported to inhibit autosis, was too cytotoxic to Panc-1 cells, we could not evaluate its effect on SSHE-induced cell death. Mitochondrial ROS generation is involved in SSHE-induced cell death Changes in ROS generation, as assessed by H2DCFDA staining, after treatment of Panc-1 cells with SSHE showed a biphasic pattern. At the early stages, ROS generation was inhibited by SSHE. However, prolonged treatment resulted in a robust increase in ROS generation. MitoSOX Red staining also showed the increased production of mitochondrial superoxide. The antioxidant N-acetylcystein significantly attenuated the cell death induced by SSHE as evaluated by the trypan blue dye exclusion test. These results suggested ROS generation as a cause of SSHE-induced cell death. Mitochondria are reported to be the primary source of ROS required for autophagy induction. To examine whether mitochondrial ROS play a role in SSHE-induced autotic cell 8 / 22 Antitumor Activity of Ginger Extract against Pancreatic Cancer Fig 2. Effect of SSHE on the features of apoptosis in Panc-1 cells. Mitochondrial membrane potential. Panc-1 cells were treated with vehicle alone, 100 g/ml SSHE or 200 g/ml SSHE for 20 h and then subjected to the JC-1 assay. Healthy cells with functional mitochondria that contain red JC-1 Jaggregates and apoptotic or unhealthy cells with collapsed mitochondria that contain mainly green JC-1 monomers were detected by cytometry. Annexin V/PI staining. Panc-1 cells were treated with vehicle alone, 100 g/ml SSHE or 200 g/ml SSHE for 24 h and then subjected to annexin V/PI staining. Effect of zVAD-fmk on SSHE-induced cell death. Panc-1 cells were incubated for 42 h. Cell viability was assessed by the MTT assay. Bars; SD. Caspase-3 activation. Panc-1 cells were incubated with 200 g/ml SSHE for various time periods or treated with 10 M pifithrin- and 100 ng/ml TRAIL for 60 h. The cell lysates were subjected to Western blot analysis with anti-caspase 3 antibody. doi:10.1371/journal.pone.0126605.g002 9 / 22 Antitumor Activity of Ginger Extract against Pancreatic Cancer Fig 3