Epithelial-to-mesenchymal transition (EMT) facilitates the escape of pancreatic cancer cells from the primary tumor site, which is a key early event in metastasis. In the present study, we examined if intermittent hypoxia facilitates the invasiveness of human pancreatic cancer cell lines (Panc-1 and BxPC-3) by Transwell assay. We used western blotting and flow cytometry analysis to quantify stem-like cells in the migratory cells during intermittent hypoxia in the human pancreatic cancer cells. Under normoxia or intermittent hypoxia, the expression of autophagy-related proteins (LC3-II and Beclin), hypoxia-inducible factor-1α (HIF-1α) and EMT-related markers (E-cadherin, Vimentin and N-cadherin) was examined by western blotting. siRNA and the autophagic inhibitor were used to access the role of HIF-1α and autophagy in promoting metastasis and EMT. Under intermittent hypoxia, pancreatic cancer cells demonstrated enhanced invasive ability and enriched stem-like cells. The migratory cells displayed stem-like cell characteristics and elevated the expression of LC3-II and Beclin-1, HIF-1α, E-cadherin, Vimentin and N-cadherin under intermittent hypoxia conditions. Moreover, enhanced autophagy was induced by the elevated level of HIF-1α. The metastatic ability and EMT of pancreatic cancer stem cells was associated with HIF-1α and autophagy. This novel finding may indicate the specific role of HIF-1α and autophagy in promoting the metastatic ability of pancreatic cancer stem cells. Additionally, it emphasizes the importance of developing therapeutic strategies targeting cancer stem cells and autophagy to reduce metastasis.

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This study was designed to quantify the role of reactive oxygen metabolites (ROMs) in two distinct components of murine peritoneal macrophage activity, phagocytosis and killing, and to discriminate quantitatively the degree to which each component is dependent on NADPH oxidase and/or xanthine oxidase. A fluorochromatic vital staining technique was modified to simultaneously quantify phagocytosis and microbicidal activity of macrophages incubated with Candida parapsilosis targets. To determine the role of ROMs, macrophages were preincubated with free radical scavengers [superoxide dismutase (SOD) and/or catalase] or with selective inhibitors of xanthine oxidase (XO, e.g., allopurinol) or NADPH oxidase [diphenyleneiodonium (DPI)]. Phagocytosis was not affected by treatment of macrophages with SOD, catalase, allopurinol, or DPI. Candidacidal activity, however, was inhibited by SOD, allopurinol, or DPI. The inhibitory effects of DPI and allopurinol were additive. Histochemical and biochemical assays demonstrated substantial quantities of XO in murine peritoneal macrophages. The findings suggest that the generation of ROMs by XO- and NADPH oxidase-dependent pathways are each important for phagocytic killing by murine peritoneal macrophages.