NDRG1 suppresses basal and hypoxia-induced autophagy at both the initiation and degradation stages and sensitizes pancreatic cancer cells to lysosomal membrane permeabilization.

BACKGROUND:N-myc downstream regulated gene 1 (NDRG1) is an established stress-response protein. This study investigated the effects of NDRG1 on autophagic degradation and how this can be therapeutically exploited. METHODS:Cell culture, western analysis, confocal microscopy, acridine orange staining, cholesterol determination, cellular proliferation assessment and combination index (CI) estimation. RESULTS:NDRG1 expression suppressed autophagic degradation and autolysosome formation, measured by increased p62 expression and reduced co-localization between the well-characterized, autophagosomal and lysosomal markers, LC3 and LAMP2, respectively. NDRG1 elicited autophagic suppression at the initiation stage of autophagy. The NDRG1-inducer and anti-cancer agent, di-2-pyridylketone 4,4,-dimethyl-3-thiosemicarbazone (Dp44mT), was able to induce lysosomal membrane permeabilization (LMP). Over-expression of NDRG1 further sensitized cells to LMP mediated by both Dp44mT, or the redox active Dp44mT‑copper complex. This sensitization may be mediated via a decrease in cholesterol levels upon NDRG1 expression, as cholesterol stabilizes lysosomal membranes. However, the effect of NDRG1 on cholesterol appeared independent of the key energy homeostasis sensor, 5' AMP-activated protein kinase (AMPK), whose activation was significantly (p < 0.001) reduced by NDRG1. Finally, Dp44mT synergistically potentiated the anti-proliferative activity of Gemcitabine that activates autophagy. In fact, Dp44mT and Gemcitabine (Combination Index (CI): 0.38 ± 0.07) demonstrated higher synergism versus the autophagy inhibitor, Bafilomycin A1 and Gemcitabine (CI: 0.64 ± 0.19). CONCLUSIONS AND GENERAL SIGNIFICANCE:Collectively, this study demonstrated a dual-inhibitory mechanism of NDRG1 on autophagic activity, and that NDRG1 expression sensitized cells to Dp44mT-induced LMP. Considering the ability of Dp44mT to inhibit autophagy, studies demonstrated the potential of combination therapy for cancer treatment of Dp44mT with Gemcitabine.

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