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

• Published in: Biochimica et Biophysica Acta (BBA) - General Subjects Vol. 1864; no. 8; p. 129625
• Main Authors: Sahni, Sumit, Gillson, Josef, Park, Kyung Chan, Chiang, Shannon, Leck, Lionel Yi Wen, Jansson, Patric J., Richardson, Des R.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2020; Elsevier BV
• Subjects: acridine orange; AMP-activated protein kinase; Antineoplastic Agents; Antineoplastic Agents - pharmacology; Autophagy; Autophagy - drug effects; Cell Cycle Proteins; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Hypoxia; Cell Hypoxia - drug effects; Cell Membrane Permeability; Cell Membrane Permeability - drug effects; Cell Proliferation; Cell Proliferation - drug effects; cholesterol; confocal microscopy; energy; genes; homeostasis; Humans; Intracellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Lysosomes; Lysosomes - drug effects; Lysosomes - metabolism; membrane permeability; neoplasm cells; Pancreatic Neoplasms; Pancreatic Neoplasms - drug therapy; Pancreatic Neoplasms - metabolism; Pancreatic Neoplasms - pathology; staining; stress response; synergism; therapeutics; Thiosemicarbazones; Thiosemicarbazones - pharmacology; Tumor Cells, Cultured
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2020.129625

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. Cell culture, western analysis, confocal microscopy, acridine orange staining, cholesterol determination, cellular proliferation assessment and combination index (CI) estimation. 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). 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. [Display omitted] •NDRG1 suppresses basal and hypoxia-induced autophagy.•NDRG1 suppresses hypoxia-induced autolysosome formation.•NDRG1 increases sensitivity to Dp44mT-mediated lysosomal membrane permeabilization.•NDRG1 expression inhibits AMPK activation, promoting anabolism.•Dp44mT synergistically potentiates the anti-proliferative activity of Gemcitabine.