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

Sumit Sahni; Josef Gillson; Kyung Chan Park; Shannon Chiang; Lionel Yi Wen Leck; Patric J Jansson; Des R Richardson

doi:10.1016/j.bbagen.2020.129625

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

Biochim Biophys Acta Gen Subj. 2020 Aug;1864(8):129625. doi: 10.1016/j.bbagen.2020.129625. Epub 2020 Apr 23.

Authors

Sumit Sahni¹, Josef Gillson¹, Kyung Chan Park², Shannon Chiang², Lionel Yi Wen Leck³, Patric J Jansson³, Des R Richardson⁴

Affiliations

¹ Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Medical Foundation Building (K25), University of Sydney, Sydney, New South Wales 2006, Australia; Northern Clinical School, Faculty of Medicine and Health, University of Sydney, NSW, Australia; Kolling Institute of Medical Research, St Leonards, NSW, Australia.
² Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Medical Foundation Building (K25), University of Sydney, Sydney, New South Wales 2006, Australia.
³ Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Medical Foundation Building (K25), University of Sydney, Sydney, New South Wales 2006, Australia; Cancer Drug Resistance Program, University of Sydney, Sydney, New South Wales 2006, Australia.
⁴ Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Medical Foundation Building (K25), University of Sydney, Sydney, New South Wales 2006, Australia; Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; Centre for Cancer Cell Biology, Griffith Institute for Drug Discovery, Griffith University, Nathan, Brisbane, Queensland, Australia. Electronic address: d.richardson@griffith.edu.au.

PMID: 32335136
DOI: 10.1016/j.bbagen.2020.129625

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antineoplastic Agents / pharmacology
Autophagy / drug effects
Cell Cycle Proteins / genetics
Cell Cycle Proteins / metabolism*
Cell Hypoxia / drug effects*
Cell Membrane Permeability* / drug effects
Cell Proliferation / drug effects
Humans
Intracellular Signaling Peptides and Proteins / genetics
Intracellular Signaling Peptides and Proteins / metabolism*
Lysosomes / drug effects
Lysosomes / metabolism
Pancreatic Neoplasms / drug therapy
Pancreatic Neoplasms / metabolism*
Pancreatic Neoplasms / pathology
Thiosemicarbazones / pharmacology
Tumor Cells, Cultured

Substances

Antineoplastic Agents
Cell Cycle Proteins
Intracellular Signaling Peptides and Proteins
N-myc downstream-regulated gene 1 protein
Thiosemicarbazones
di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone