Lysosomal membrane stability plays a major role in the cytotoxic activity of the anti-proliferative agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT).
The potent and selective anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), localizes in lysosomes and forms cytotoxic copper complexes that generate reactive oxygen species (ROS), resulting in lysosomal membrane permeabilization (LMP) and cell death. Herein, the role of lysosomal membrane stability in the anti-tumor activity of Dp44mT was investigated. Studies were performed using molecules that protect lysosomal membranes against Dp44mT-induced LMP, namely heat shock protein 70 (HSP70) and cholesterol. Up-regulation or silencing of HSP70 expression did not affect Dp44mT-induced LMP in MCF7 cells. In contrast, cholesterol accumulation in lysosomes induced by the well characterized cholesterol transport inhibitor, 3-β-[2-(diethyl-amino)ethoxy]androst-5-en-17-one (U18666A), inhibited Dp44mT-induced LMP and markedly and significantly (p<0.001) reduced the ability of Dp44mT to inhibit cancer cell proliferation (i.e., increased the IC(50)) by 140-fold. On the other hand, cholesterol extraction using methyl-β-cyclodextrin enhanced Dp44mT-induced LMP and significantly (p<0.01) increased its anti-proliferative activity. The protective effect of U18666A in increasing lysosomal cholesterol and preventing the cytotoxic activity of Dp44mT was not due to induced autophagy. Instead, U18666A was found to decrease lysosomal turnover, resulting in autophagosome accumulation. Moreover, preincubation with U18666A did not prevent the ability of Dp44mT to induce autophagosome synthesis, indicating that autophagic initiation via Dp44mT occurs independently of LMP. These studies demonstrate the significance of lysosomal membrane stability in relation to the ability of Dp44mT to execute tumor cell death and overcome pro-survival autophagy. Hence, lysosomal-dependent cell death induced by Dp44mT serves as an important anti-tumor strategy. These results are important for comprehensively understanding the mechanism of action of Dp44mT.
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Elaine M
EM
Gutierrez
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Nicole A
NA
Seebacher
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Laila
L
Arzuman
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Zaklina
Z
Kovacevic
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Darius J R
DJ
Lane
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Vera
V
Richardson
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Angelica M
AM
Merlot
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Hiu
H
Lok
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Danuta S
DS
Kalinowski
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Sumit
S
Sahni
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia. Electronic address: patric.jansson@sydney.edu.au.
Patric J
PJ
Jansson
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia. Electronic address: d.richardson@sydney.edu.au.
Des R
DR
Richardson
D000736:Androstenes / Q000494:pharmacology; D000924:Anticholesteremic Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects*; D002784:Cholesterol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007425:Intracellular Membranes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D061986:MCF-7 Cells; D010539:Permeability; D034622:RNA Interference; D013882:Thiosemicarbazones / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection; D047392:beta-Cyclodextrins / Q000494:pharmacology
D000736:Androstenes; D000924:Anticholesteremic Agents; D000970:Antineoplastic Agents; D018840:HSP70 Heat-Shock Proteins; D013882:Thiosemicarbazones; D047392:beta-Cyclodextrins; C539263:di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone; C108732:methyl-beta-cyclodextrin; C006261:3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one; D002784:Cholesterol
Autophagy; Dp44mT; Lysosomal membrane permeabilization; Lysosomes